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WO2025240670A2 - Anti-pd-1 antibodies and related binding molecules and methods and uses thereof - Google Patents

Anti-pd-1 antibodies and related binding molecules and methods and uses thereof

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Publication number
WO2025240670A2
WO2025240670A2 PCT/US2025/029446 US2025029446W WO2025240670A2 WO 2025240670 A2 WO2025240670 A2 WO 2025240670A2 US 2025029446 W US2025029446 W US 2025029446W WO 2025240670 A2 WO2025240670 A2 WO 2025240670A2
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cdr
seq
set forth
region
nos
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French (fr)
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WO2025240670A3 (en
Inventor
Akshay PAINTAL
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Abalytics Oncology Inc
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Abalytics Oncology Inc
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Publication of WO2025240670A2 publication Critical patent/WO2025240670A2/en
Publication of WO2025240670A3 publication Critical patent/WO2025240670A3/en
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C07K2317/524—CH2 domain
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/64—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising a combination of variable region and constant region components
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/66—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising a swap of domains, e.g. CH3-CH2, VH-CL or VL-CH1
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/72—Increased effector function due to an Fc-modification
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance

Definitions

  • the present disclosure provides novel monoclonal antibodies or antibody fragments against protein programmed cell death 1 (PD-1).
  • the antibodies or antibody fragments disclosed can block the binding of the PD-L1 ligand to PD-1 and provide potent agents for the treatment of cancers via the modulation of human immune function.
  • the present disclosure also provides binding molecules, such as bispecific antibodies, directed against PD-1 and also cytotoxic T-lymphocyte-associated protein 4 (CTLA-4).
  • CTLA-4 cytotoxic T-lymphocyte-associated protein 4
  • the present disclosure further relates to pharmaceutical composition and methods and uses of such antibodies and binding molecules for treatment of disease in which specific targeting of cells that express PD-1, or PD-1 and CTLA-4, is desired.
  • Cancer therapies comprise a wide range of therapeutic approaches, including surgery, radiation, and chemotherapy. While the various approaches offer a broad selection of options for treating cancer, many therapeutics suffer from disadvantages, such as a lack of selectivity of targeting cancer cells over normal, healthy cells, and the development of resistance by the cancer to the treatment. Immunotherapy targeting checkpoint molecules has emerged as one of the most compelling approaches for treating patients with cancers, due to promising results from preclinical and clinical trials.
  • PD-1 a type I cell surface receptor
  • PD-L1 or PD-L2 two ligands
  • the ligands for PD-1 are differentially expressed on various tissues and cell types, including antigen-presenting cells of the immune system, and are upregulated on many types of tumor cells. Upregulation of PD-L1 within the tumor microenvironment is a proposed mechanism of tumors to subvert protective anti-tumor immune responses by the host.
  • Antibodies directed at PD-1 that block the interaction of the receptor with either of its ligands hold promise in the treatment of many cancers, and many such molecules are in the clinic for use as monotherapy or in combination with other biologies.
  • antibodies against PD1 are known and commercially available, it is desirable to find novel anti-PDl antibodies suitable for cancer treatments with improved efficacy and reduced side effects. Embodiments provided herein address these needs.
  • an anti-programmed cell death protein 1 (PD-1) antibody or antigen-binding fragment thereof comprising a heavy chain variable (VH) region and a light chain variable (VL) region
  • the VH region comprises a heavy chain complementarity determining region 1 (CDR-H1), a heavy chain complementarity determining region 2 (CDR-H2), and a heavy chain complementarity determining region 3 (CDR-H3) contained within any one of SEQ ID NOs: 1-45, 157, and 165-170
  • the VL region comprises a light chain complementarity determining region 1 (CDR-L1), a light chain complementarity determining region 2 (CDR-L2) and a light chain complementarity determining region 3 (CDR-L3) contained within any one of SEQ ID NOs: 46-90, 158, and 171-172.
  • the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO:3, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 158;
  • the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO:4, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 158;
  • the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO:5, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 158;
  • the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within
  • an anti-PD-1 antibody or antigen-binding fragment thereof comprising a heavy chain variable (VH) region and a light chain variable (VL) region
  • the VH region comprises a heavy chain complementarity determining region 1 (CDR-H1) comprising the sequence set forth in any one of SEQ ID NOs: 91-101, a heavy chain complementarity determining region 2 (CDR-H2) comprising the sequence set forth in any one of SEQ ID NOs: 102- 112, and 173, and a heavy chain complementarity determining region 3 (CDR-H3) comprising the sequence set forth in any one of SEQ ID NOs: 113-123; and the VL region comprises a light chain complementarity determining region 1 (CDR-L1) comprising the sequence set forth in any one of SEQ ID NOs: 124-134, a light chain complementarity determining region 2 (CDR-L2) comprising the sequence set forth in any one of SEQ ID NOs:
  • the VH region comprises a heavy chain complementarity determining region 1 (CDR-H1) comprising the sequence set forth in any one of SEQ ID NOs: 92-101, a heavy chain complementarity determining region 2 (CDR-H2) comprising the sequence set forth in any one of SEQ ID NOs: 103-112, and 173, and a heavy chain complementarity determining region 3 (CDR-H3) comprising the sequence set forth in any one of SEQ ID NOs: 114-123.
  • CDR-H1 heavy chain complementarity determining region 1
  • CDR-H2 heavy chain complementarity determining region 2
  • CDR-H3 heavy chain complementarity determining region 3
  • the VL region comprises a light chain complementarity determining region 1 (CDR-L1) comprising the sequence set forth in any one of SEQ ID NOs: 125-134, a light chain complementarity determining region 2 (CDR-L2) comprising the sequence set forth in any one of SEQ ID NOs: 136-145, and a light chain complementarity determining region 3 (CDR-L3) comprising the sequence set forth in any one of SEQ ID NOs: 147-156.
  • CDR-L1 light chain complementarity determining region 1
  • CDR-L2 comprising the sequence set forth in any one of SEQ ID NOs: 136-145
  • CDR-L3 comprising the sequence set forth in any one of SEQ ID NOs: 147-156.
  • the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 92, 102, and 113, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 124, 135, and 146, respectively;
  • the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 93, 102, and 113, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 124, 135, and 146, respectively;
  • the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 1 and SEQ ID NO: 158, respectively;
  • the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 2 and SEQ ID NO: 158, respectively;
  • the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 3 and
  • the VH region and the VL region can be or can comprise: the sequence set forth in SEQ ID NO: 1 and 158, respectively; the sequence set forth in SEQ ID NO: 2 and 158, respectively; the sequence set forth in SEQ ID NO: 3 and 158, respectively; the sequence set forth in SEQ ID NO: 4 and 158, respectively; the sequence set forth in SEQ ID NO: 5 and 158, respectively; the sequence set forth in SEQ ID NO: 6 and 158, respectively; the sequence set forth in SEQ ID NO: 7 and 158, respectively; the sequence set forth in SEQ ID NO: 8 and 158, respectively; the sequence set forth in SEQ ID NO: 9 and 158, respectively; the sequence set forth in SEQ ID NO: 10 and 158, respectively; the sequence set forth in SEQ ID NO: 11 and 158, respectively; the sequence set forth in SEQ ID NO: 12 and 158, respectively; the sequence set forth in SEQ ID NO: 13 and 158, respectively; the sequence set forth in SEQ ID NO: 1 and 158, respectively; the
  • an anti-programmed cell death protein 1 (PD-1) antibody or antigen-binding fragment thereof comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and the VL region are or comprise: the sequence set forth in SEQ ID NO: 1 and 158, respectively; the sequence set forth in SEQ ID NO: 2 and 158, respectively; the sequence set forth in SEQ ID NO: 3 and 158, respectively; the sequence set forth in SEQ ID NO: 4 and 158, respectively; the sequence set forth in SEQ ID NO: 5 and 158, respectively; the sequence set forth in SEQ ID NO: 6 and 158, respectively; the sequence set forth in SEQ ID NO: 7 and 158, respectively; the sequence set forth in SEQ ID NO: 8 and 158, respectively; the sequence set forth in SEQ ID NO: 9 and 158, respectively; the sequence set forth in SEQ ID NO: 10 and 158, respectively; the sequence set forth in SEQ ID NO: 1 and 158, respectively; the sequence set forth in
  • the antibody can be a full length antibody.
  • the full length antibody comprises a constant region (Fc) from an IgAl, IgA2, IgD, IgE, IgGl, IgG2, IgG3, IgG4, or IgM.
  • the antibody can be an antigen-binding fragment.
  • the antigen-binding fragment is selected from the group consisting of a single domain antibody, a single chain antibody, an unibody, a single chain variable fragment (scFv), a Fab fragment, and a F(ab')2 fragment.
  • the anti-PD-1 antibody or antigen-binding fragment thereof can be recombinant.
  • the VH region and the VL region can be human or can be from a human protein.
  • the antibody or antigen-binding fragment thereof can bind to human PD-1 protein with an equilibrium dissociation constant (KD) of from about 7 x 10 10 M to about 2 x 10 8 M at pH 7.35 to 7.45, optionally at pH 7.40. In some embodiments, the antibody or antigen-binding fragment thereof binds to human PD-1 protein with a KD of lower than 1 x 10 9 M, optionally from about 7 x 10 10 M to about 1 x 10 9 M.
  • KD equilibrium dissociation constant
  • the antibody or antigen-binding fragment thereof can bind to human PD-1 protein with an equilibrium dissociation constant (KD) of from about 3 x 10 10 M to about 4 x 10 8 M at pH 5.0 to 6.8, optionally at pH 6.0 or pH 5.0. In some embodiments, the antibody or antigen-binding fragment thereof binds to human PD-1 protein with a KD of lower than 1 x 10 9 M, optionally from about 3 x 10 10 to about 1 x 10 9 M.
  • KD equilibrium dissociation constant
  • the antibody or antigen-binding fragment thereof can bind to human PD-1 protein with a lower KD at pH 5.0 to 6.8, optionally pH 6.0 or pH 5.0, than pH 7.35 to 7.45, optionally pH 7.4. In any of the embodiments herein, the antibody or antigen-binding fragment thereof can bind to human PD-1 protein with a lower KD at pH 7.35 to 7.45, optionally pH 7.4, than at pH 5.0 to 6.8, optionally pH 6.0 or pH 5.0.
  • the antibody or antigenbinding fragment thereof can bind to human PD-1 protein with a KD at pH 7.35 to 7.45, optionally pH 7.4, and at pH 5.6 to 6.8, optionally pH 6.0 or pH 5.0, that is substantially the same.
  • the antibody or antigen-binding fragment thereof can bind to human PD-1 protein with an association rate constant (K on ) of 4 x 10 5 1/Ms to about 2 x 10 6 1/Ms at pH 7.35 to 7.45, optionally at pH 7.40.
  • the antibody or antigen-binding fragment thereof can bind to human PD-1 protein with an dissociation rate constant (Koff) of 4 x 10 4 1/s to about 9 x 10 3 1/s at pH 7.35 to 7.45, optionally at pH 7.40.
  • the antibody or antigen-binding fragment thereof can bind to human PD-1 protein with an association rate constant (K on ) of 4 x 10 5 1/Ms to about 5 x 10 6 1/Ms at pH 5.0 to 6.8, optionally at pH 6.0 pH 6.0 or pH 5.0.
  • the antibody or antigen-binding fragment thereof can bind to human PD-1 protein with an dissociation rate constant (Koff) of 4 x 10 4 1/s to about 2 x 10 2 1/s at pH 5.0 to 6.8, optionally at pH 6.0 or pH 5.0.
  • a conjugate comprising the anti-PD-1 antibody or antigen-binding fragment thereof of any of the embodiments herein and a heterologous molecule or moiety.
  • the heterologous molecule or moiety is a cytotoxic agent, a drug, enzymatically active toxin or fragment thereof, or a radioactive atom.
  • a bispecific antibody comprising the anti-PD-1 antibody or antigen-binding fragment thereof of any of any of the embodiments herein.
  • a PD-1 directed chimeric antigen receptor comprising an extracellular antigen-binding domain comprising the anti-PD-1 antibody or antigenbinding fragment thereof of any of the embodiments herein, a transmembrane region and an intracellular signaling region.
  • a polynucleotide comprising a nucleic acid encoding the anti-PD-1 antibody or antigen-binding domain thereof of any of the embodiments herein.
  • a polynucleotide comprising a nucleic acid encoding the anti-PD-1 chimeric antigen receptor of some embodiments.
  • a vector comprising the polynucleotide of any of the embodiments herein.
  • the vector is a viral vector.
  • the viral vector is a retroviral vector or a lentiviral vector.
  • a engineered cell comprising the anti-PD-1 antibody or antigen-binding fragment thereof of any of the embodiments herein, the bispecific antibody of some embodiments, or the conjugate of any of the embodiments herein.
  • an engineered cell comprising the PD-1 directed chimeric antigen receptor of some embodiments.
  • the cell can be a lymphocyte.
  • the cell is a natural killer (NK) cell or a T cell.
  • the cell can be a T cell and the T cell can be a CD4+ T cell or a CD8+ T cell.
  • a cell comprising the polynucleotide of any of the embodiments herein, or the vector of any of the embodiments herein.
  • the cell is a mammalian cell.
  • disclosed herein is a method of producing an antibody comprising culturing the cell of any of the embodiments herein under a condition that produces the antibody. In some embodiments, the method further comprises recovering the antibody produced by the cell.
  • an antibody or antigen-binding fragment thereof produced by the method of any of the embodiments herein.
  • composition comprising the cell of any of the embodiments herein.
  • compositions comprising the anti-PD-1 antibody or antigen-binding fragment thereof of any of the embodiments herein, the conjugate of any of the embodiments herein, the bispecific antibody of some embodiments, or the PD-1 directed chimeric antigen receptor of some embodiments.
  • the composition can further comprise a pharmaceutically acceptable excipient.
  • a method of treatment comprising administering the cell of any of the embodiments herein or the composition of any of the embodiments herein to a subject having a disease or disorder associated with PD-1.
  • disclosed herein is a method of increasing the activity of an immune cell, comprising contacting an immune cell with the anti-PD-1 antibody or antigen-binding fragment thereof of any of the embodiments herein, the conjugate of any of the embodiments herein, or the bispecific antibody of some embodiments.
  • a method of increasing the activity of an immune cell comprising administering to a subject in need thereof a therapeutically effective amount of the anti-PD-1 antibody or antigen-binding fragment thereof of any of the embodiments herein, the conjugate of any of the embodiments herein, the bispecific antibody of some embodiments.
  • the method can be used for an ex vivo treatment in a subject.
  • the treatment can be the treatment of cancer, the treatment of an infection or infectious disease or as a vaccine adjuvant.
  • a binding molecule comprising a PD-1 binding domain that binds PD- 1, and a cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) binding domain that binds CTLA-4, wherein: (a) the PD-1 binding domain comprises a heavy chain and a light chain comprising the heavy chain variable (VH) region and the light chain variable (VL ) region, respectively, of the anti- PD-1 antibody or antigen-binding fragment thereof of any of claims 1- 26, and (b) the CTLA-4 binding domain comprises a heavy chain comprising a heavy chain variable (CTLA-4 VH) region and a light chain comprising a light chain variable (CTLA-4 VL ) region, wherein: (i) the CTLA-4 VH region comprises a heavy chain complementarity determining region 1 (CDR-H1) comprising the sequence set forth in any one of SEQ ID NOs: 278-285, a heavy chain complementarity determining region 2 (CDR-H2) comprising the sequence
  • the CTLA-4 VH region comprises a CDR-H1 comprising the sequence set forth in any one of SEQ ID NOs: 278-285, a CDR-H2 comprising the sequence set forth in any one of SEQ ID NOs: 286-296, and a CDR-H3 comprising the sequence set forth in any one of SEQ ID NOs: 297-302; and the CTLA-4 VL region comprises a CDR-L1 comprising the sequence set forth in any one of SEQ ID NOs: 303-317, a CDR-L2 comprising the sequence set forth in any one of SEQ ID NOs: 318-326, and a CDR-L3 comprising the sequence set forth in any one of SEQ ID NOs: 327-329, wherein the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR- L2 and CDR-L3 are not set forth in SEQ ID NOS: 278, 286, 297,303, 318
  • the CTLA-4 VH region comprises a CDR-H1, a CDR- H2, and a CDR-H3 contained within SEQ ID NO: 230
  • the CTLA-4 VL region comprises a CDR- Ll, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 347
  • the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 231
  • the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 347
  • the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 232
  • the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 347
  • the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 287, and 297, respectively, and the CDR-L1, the CDR- L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 303, 318, and 327, respectively;
  • the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 286, and 298, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 303, 318, 327, respectively;
  • the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 280, 286, and 297, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth
  • the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 230 and SEQ ID NO: 347, respectively;
  • the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 231 and SEQ ID NO: 347, respectively;
  • the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 95%,
  • CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 234 and SEQ ID NO: 277, respectively.
  • the CTLA-4 VH region and the CTLA-4 VL region are or comprise: the sequence set forth in SEQ ID NO: 230 and 347, respectively; the sequence set forth in SEQ ID NO: 231 and 347, respectively; the sequence set forth in SEQ ID NO: 232 and 347, respectively; the sequence set forth in SEQ ID NO: 233 and 347, respectively; the sequence set forth in SEQ ID NO: 234 and 347, respectively; the sequence set forth in SEQ ID NO: 235 and 347, respectively; the sequence set forth in SEQ ID NO: 236 and 347, respectively; the sequence set forth in SEQ ID NO: 237 and 347, respectively; the sequence set forth in SEQ ID NO: 238 and 347, respectively; the sequence set forth in SEQ ID NO: 239 and 347, respectively; the sequence set forth in SEQ ID NO: 240 and 347, respectively; the sequence set forth in SEQ ID NO: 241 and 347, respectively; the sequence set forth in SEQ ID NO:
  • a binding molecule comprising a cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) binding domain that binds CTLA-4, and a PD-1 binding domain that binds PD-1, wherein: (a) the PD-1 binding domain comprises a heavy chain and a light chain comprising the heavy chain variable (VH) region (PD-1 VH ) and the light chain variable (VL) region (PD-1 VL), respectively, of the anti-PD-1 antibody or antigen-binding fragment thereof of some of any embodiments, and (b) the CTLA-4 binding domain comprises a heavy chain variable (CTLA-4 VH) region and a light chain variable (CTLA-4 VL ) region, wherein: the CTLA-4 VH region comprises the sequence set forth in:
  • the CTLA-4 VH region comprises a CDR-H1 comprising the sequence set forth in SEQ ID NO: 278 or SEQ ID NO: 281, a CDR-H2 comprising the sequence set forth in SEQ ID NO: 289 or SEQ ID NO: 296, and a CDR-H3 comprising the sequence set forth in SEQ ID NO: 297 or SEQ ID NO: 301; and the CTLA-4 VL region comprises a CDR-L1 comprising the sequence set forth in SEQ ID NO: 305 or SEQ ID NO: 313; a CDR-L2 comprising the sequence set forth in SEQ ID NO: 318, and a CDR-L3 comprising the sequence set forth in SEQ ID NO: 327 or SEQ ID NO: 328.
  • the CTLA-4 VH region comprises: (a) a serine (S) at position 31, a glutamic acid (E) at position 54, a histidine (H) at position 59, a tryptophan (W) at position 101, and a leucine (L) at position 102, wherein the numbering is relative to SEQ ID NO: 407, or (b) a arginine (R) at position 31, a glutamic acid (E) at position 54, a tyrosine (Y) at position 59, a tryptophan (W) at position 101, and a histidine (H) at position 102, wherein the numbering is relative to SEQ ID NO: 407.
  • the CTLA-4 VL region comprises: (a) a histidine (H) at position 28, a serine (S) at position 32, and a glutamine (Q) at position 91, wherein the numbering is relative to SEQ ID NO: 408, or (b) a serine (S) at position 28, a serine (S) at position 32, and a histidine (H) at position 91, wherein the numbering is relative to SEQ ID NO: 408.
  • the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 254 and SEQ ID NO: 258, respectively;
  • the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 254 and SEQ ID NO: 268, respectively;
  • the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 96%, 97%, 9
  • the CTLA-4 VH region and the CTLA-4 VL region are or comprise: the sequence set forth in SEQ ID NO: 254 and 258, respectively; the sequence set forth in SEQ ID NO: 254 and 268, respectively; the sequence set forth in SEQ ID NO: 252 and 268, respectively.
  • the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 254 and SEQ ID NO: 268, respectively.
  • the PD-1 VH region and the PD-1 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 168 and SEQ ID NO: 172, respectively.
  • the PD-1 VH region is or comprises an amino acid sequence set forth in SEQ ID NO: 168
  • the PD-1 VL region is or comprises an amino acid sequence set forth in SEQ ID NO: 172.
  • the CTLA-4 binding domain and the PD-1 binding domain are Fab fragments each comprising a VH region and a VL region, wherein one of the Fab fragments is a crossover Fab such that the VH region and the VL region of such Fab are replaced by each other.
  • the CTLA-4 binding domain is a crossover CTLA-4 Fab and the PD-1 binding domain is a Fab
  • the crossover CTLA-4 Fab comprises: (a) a heavy chain (HC1) comprising the CTLA-4 VL region and a heavy chain constant region 1 (CHI), designated CTLA-4 VL-CH1 ; and (b) a light chain (LC1) comprising the CTLA-4 VH region and a light chain constant region (CL), designated CTLA-4 VH-CL
  • the PD-1 binding domain comprises (a) a heavy chain 2 (HC2) comprising the PD-1 VH region and a heavy chain constant region 1 (CHI), designated PD-1 VH-CH1 ; and (b) a light chain 2 (LC2) comprising the PD-1 VL region and a light chain constant region (CL), designated PD-1 VL-CL.
  • the CHI of the CTLA-4 VL-CH1 comprises the amino acid sequence set forth in SEQ ID NO: 363 and the CL of the CTLA-4 VH-CL comprises the amino acid sequence set forth in SEQ ID NO: 367.
  • the CHI of the PD-1 VH-CH1 comprises the amino acid sequence set forth in SEQ ID NO: 371 and the CL of the PD-1 VL-CL comprises the amino acid sequence set forth in SEQ ID NO: 175.
  • the CTLA-4 binding domain is a CTLA-4 Fab and the PD-1 binding domain is a crossover Fab, wherein: the CTLA-4 Fab comprises: (a) a heavy chain (HC1) comprising the CTLA-4 VH region and a heavy chain constant region 1 (CHI), designated CTLA-4 VH-CH1 ; and (b) a light chain (LC1) comprising the CTLA-4 VL region and a light chain constant region (CL), designated CTLA-4 VL-CL; and the PD-1 binding domain comprises (a) a heavy chain 2 (HC2) comprising the PD-1 VL region and a heavy chain constant region 1 (CHI), designated PD-1 VL-CH1 ; and (b) a light chain 2 (LC2) comprising the PD-1 VH region and a light chain constant region (CL), designated PD-1 VH-CL.
  • the CTLA-4 Fab comprises: (a) a heavy chain (HC1) comprising the CTLA-4 VH region and a heavy chain constant region 1 (CHI), designated CTLA-4 V
  • the CHI of the PD-1 VL-CH1 comprises the amino acid sequence set forth in SEQ ID NO: 363 and the CL of the PD-1 VH-CL comprises the amino acid sequence set forth in SEQ ID NO: 367.
  • the CHI of the CTLA-4 VH-CH1 comprises the amino acid sequence set forth in SEQ ID NO: 371 and the CL of the CTLA-4 VL-CL comprises the amino acid sequence set forth in SEQ ID NO: 175.
  • the CTLA-4 VH comprises the sequence of amino acids set forth in SEQ ID NO: 254 and the CTLA-4 VL comprises the sequence of amino acids set forth in SEQ ID NO: 268; and the PD-1 VH comprises the sequence of amino acids set forth in SEQ ID NO: 168 and the PD-1 VL comprise the sequence of amino acids set forth in SEQ ID NO: 172.
  • the provided binding molecule further comprises an Fc region comprising a first and second polypeptide each comprising a hinge-CH2-CH3, wherein one of the first polypeptide and second polypeptide is linked to the CHI of the CTLA-4 Fab and the other of the first polypeptide and second polypeptide is linked to the CHI of the PD-1 Fab.
  • the Fc region is a heterodimeric Fc region.
  • each of the first and second polypeptide of the heterodimeric Fc comprises one or more amino acid substitutions in a wild-type Fc polypeptide region to effect heterodimer formation between the first polypeptide and the second polypeptide.
  • the wild-type Fc region is an IgGl Fc region.
  • the wild-type Fc region comprises the sequence set forth in SEQ ID NO:407 or SEQ ID NO: 408.
  • the one more amino acid substitutions are a knob-into-hole modification, a charge mutation to reduce or prevent self-association due to charge repulsion, or disulfide bonding pairing mutation.
  • the one or more amino acid substitutions are: (i) amino acid substitution T366W in the first polypeptide of the heterodimeric Fc and amino acid substitutions T366S, L368A and Y407V in the second polypeptide of the heterodimeric Fc; (ii) amino acid substitution E356K, E357K and D399K in the first polypeptide of the heterodimeric Fc and amino acid substitutions K439E, K370E and K409D in the second polypeptide of the heterodimeric Fc; or (iii) amino acid substitution S354C on the first polypeptide of the heterodimeric Fc and amino acid substitution Y349C on the second polypeptide of the heterodimeric Fc.
  • the one or more substitutions are a knob-into- hole modification.
  • the one or more amino acid substitutions of the first Fc polypeptide comprises Thr366Trp and the one or more amino acid substitutions of the second Fc polypeptide of the heterodimeric Fc comprises the Thr366Ser, Leu368Ala and Tyr407Val.
  • the first and/or second Fc polypeptide further comprises the amino acid substitution. H435R.
  • the heterodimeric Fc region comprises one or more amino acid substitutions to reduce binding affinity to an Fc receptor and/or to reduce effector function, optionally as compared to a wild-type IgGl Fc domain.
  • the one or more amino acid substitutions are selected from L234A, L234V, L235A, L235E, G237A, D265S, S267K, R292C, N297G, V302C, and P329G by EU numbering.
  • the one or more amino acid substitution comprises L234A and L235A.
  • the one or more amino acid substitution comprises L234A, L235A, and D265S. In any of the embodiments herein, the one or more amino acid substitution comprises L234A, L235A, and P329G. In any of the embodiments herein, one or both polypeptides of the heterodimeric Fc lacks Lys447.
  • one of the first polypeptide and the second polypeptide of the heterodimeric Fc region comprise an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity SEQ ID NO: 364; and the other of the first polypeptide and second polypeptide of the heterodimeric Fc region comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity SEQ ID NO: 373.
  • one of the first polypeptide and the second polypeptide of the heterodimeric Fc region comprise the amino acid sequence set forth in SEQ ID NO: 364; and the other of the first polypeptide and the second polypeptide of the heterodimeric Fc region comprises the amino acid sequence set forth in SEQ ID NO: 373.
  • the first polypeptide of the heterodimeric Fc region comprise the amino acid sequence set forth in SEQ ID NO: 364 and the second polypeptide of the heterodimeric Fc region comprises the amino acid sequence set forth in SEQ ID NO:373.
  • the first polypeptide of the heterodimeric Fc region comprise the amino acid sequence set forth in SEQ ID NO: 373 and the second polypeptide of the heterodimeric Fc region comprises the amino acid sequence set forth in SEQ ID NO:364.
  • HC1 polypeptide comprising a VL sequence set forth in SEQ ID NO:268, a CHI sequence set forth in SEQ ID NO:363, and a hinge-CH2-CH3 sequence set forth in SEQ ID NO: 364
  • LC1 polypeptide comprising a VH sequence set forth in SEQ ID NO:254 and a CL sequence set forth in SEQ ID NO: 367
  • HC2 polypeptide comprising a Vn sequence set forth in SEQ ID NO:168,a CHI sequence set forth in SEQ ID NO:371, and a hinge-CH2-CH3 sequence set forth in SEQ ID NO:373
  • LC2 polypeptide comprising a VL sequence set forth in SEQ ID NO: 172 and a CL sequence set forth in SEQ ID NO: 175.
  • the binding molecule comprises a) a HC1 polypeptide that is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 378; (b) a HC2 polypeptide that is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 387; (c) a LC1 polypeptide that is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 381; and (d) a HC1 polypeptide that is or
  • the provided binding molecule comprises (a) a HC1 polypeptide that is or comprises the amino acid sequence set forth in SEQ ID NO: 378; (b) a HC2 polypeptide that is or comprises the amino acid sequence set forth in SEQ ID NO: 387; (c) a LC1 polypeptide that is or comprises the amino acid sequence set forth in SEQ ID NO: 381; and (d) a LC2 polypeptide that is or comprises the amino acid sequence set forth in SEQ ID NO: 389.
  • the provided binding molecule comprises (a) a heavy chain 1 (HC1) polypeptide comprising a VL sequence set forth in SEQ ID NO: 172, a CHI sequence set forth in SEQ ID NO:363, and a hinge-CH2-CH3 sequence set forth in SEQ ID NO: 364; (b) a LC1 polypeptide comprising a VH sequence set forth in SEQ ID NO: 168, and a CL sequence set forth in SEQ ID NO:367; and (c) a HC2 polypeptide comprising a VH sequence set forth in SEQ ID NO:254, a CHI sequence set forth in SEQ ID NO:371, and a hinge-CH2-CH3 sequence set forth in SEQ ID NO: 373; and (d) a LC2 polypeptide comprising a VL sequence set forth in SEQ ID NO:268, and a CL sequence set forth in SEQ ID NO: 175.
  • HC1 heavy chain 1 polypeptide comprising a VL sequence set forth in SEQ ID NO:
  • the provided binding molecule comprises (a) a HC1 polypeptide that is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 360; (b) a HC2 polypeptide that is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 370; (c) a LC1 polypeptide that is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 366 and (d)
  • the provided binding molecule comprises (a) a HC1 polypeptide that is or comprises an amino acid sequence set forth in SEQ ID NO: 360; (b) a HC2 polypeptide that is or comprises an amino acid sequence set forth in SEQ ID NO: 370; (c) a LC1 polypeptide that is or comprises an amino acid sequence set forth in SEQ ID NO: 366 and (d) a LC2 polypeptide that is or comprises an amino acid sequence set forth in SEQ ID NO: 375.
  • the heavy chain and/or the light chain further comprising a signal peptide.
  • a polynucleotide or polynucleotides comprising a nucleic acid sequence encoding any of the provided binding molecules or portion thereof, including a heavy or light chain thereof.
  • the binding molecule is a bispecific antibody directed against PD-1 and CTLA-4.
  • a polynucleotide comprising a nucleic acid comprising a nucleic acid sequence encoding any of the provided the bispecific antibodies or portion thereof.
  • a vector comprising any of the provided polynucleotides. In some embodiments, the vector is useful for expressing a provided binding molecule, such as a bispecific antibody.
  • the vector is a viral vector.
  • the viral vector is a retroviral vector or a lentiviral vector.
  • a cell comprising any of the provided polynucleotides or vectors.
  • the cell is a mammalian cell.
  • a method of producing a binding molecule, such as a bispecific antibody comprising culturing any of the provided cells under a condition that produces the binding molecule, wherein optionally the binding molecule is an antibody.
  • any of the provided methods further comprising recovering the binding molecule, such as a bispecific antibody, produced by the cell.
  • a binding molecule such as a bispecific antibody, produced by any of the provided methods.
  • a composition comprising any of the binding molecules, such as bispecific antibodies, provided herein.
  • the composition further comprises a pharmaceutically acceptable excipient.
  • a method for treating a subject with a disease or condition comprising administering a therapeutically effective amount of any of the provided binding molecules, such as bispecific antibodies, or compositions.
  • the disease or condition is a cancer.
  • the cancer is a solid tumor.
  • the cancer is a melanoma, breast carcinoma, HCC, Renal cell carcinoma, Colorectal carcinoma, non-small-cell lung carcinoma, Cervical cancer or Mesothelioma.
  • administering the binding molecules, such as bispecific antibody, to the subject results in higher T e ff-to-suppressor cells (myeloid-derived suppressor cell and T reg ) ratios.
  • administering the binding molecule, such as bispecific antibody, to the subject results in increased pro-inflammatory cytokines, optionally interferon-gamma (IFN-y) and/or tumor necrosis factor-alpha (TNF-a).
  • IFN-y interferon-gamma
  • TNF-a tumor necrosis factor-alpha
  • the method comprises administering to the subject an effective amount of any of the provided binding molecules, such as bispecific antibodies, or the compositions prior to a surgery or after a surgery to remove a solid tumor in the subject.
  • the provided binding molecules such as bispecific antibodies
  • FIG.l depicts the EC50 values for exemplary provided antibodies for binding to human PD-1 at normal pH about 7.4.
  • FIG.2 depicts the IC50 values for exemplary provided antibodies for inhibition of PD-1 binding to human PD-L1 in a standard reporter assay.
  • FIG. 3 shows a schematic drawing of a standard reporter assay to test for inhibition of PD- 1 binding to human PD-L1.
  • FIGs. 4A-4F show the thermal melt curves measured by Differential Scanning Fluorimetry (DSF).
  • FIG. 4A shows the thermal melt curve of PDl-abl l3.
  • FIG. 4B shows the thermal melt curve of PDl-abl 16.
  • FIG. 4C shows the thermal melt curve of PDl-abl 19.
  • FIG. 4D shows the thermal melt curve of PDl-abl 22.
  • FIG. 4E shows the thermal melt curve of PDl-abl 25.
  • FIG. 4F shows the thermal melt curve of PDl-abl29.
  • FIGs. 5A-5S show results of binding tests of exemplary provided antibodies for binding to human CTLA-4 at pH 5 and at pH 7.4.
  • FIG. 5A shows results of binding tests of anti-CTLA-4 antibodies (CTLA-4-AB-7, CTLA-4-AB-9, CTLA-4-AB-10, and CTLA-4-AB-11) and reference antibodies for binding to human CTLA-4 at pH 5 and at pH 7.4.
  • FIG. 5B shows results of binding tests of anti-CTLA-4 antibodies (CTLA-4-AB-13, CTLA-4-AB-14, CTLA-4-AB-15, and CTLA-4-AB-16) and reference antibodies for binding to human CTLA-4 at pH 5 and at pH 7.4.
  • FIG. 5C shows results of binding tests of anti-CTLA-4 antibodies (CTLA-4-AB-17, CTLA-4-AB-18, CTLA-4-AB-19, and CTLA-4- AB-20) and reference antibodies for binding to human CTLA-4 at pH 5 and at pH 7.4.
  • FIG. 5D shows results of binding tests of anti-CTLA-4 antibodies (CTLA-4-AB-21, CTLA-4- AB-22, CTLA-4- AB-23, and CTLA-4- AB-24) and reference antibodies for binding to human CTLA-4 at pH 5 and at pH 7.4.
  • FIG. 5E shows results of binding tests of anti-CTLA-4 antibodies (CTLA-4- AB -25, CTLA-4- AB-26, CTLA-4- AB-27, and CTLA-4- AB-28) and reference antibodies for binding to human CTLA-4 at pH 5 and at pH 7.4.
  • FIG. 5F shows results of binding tests of anti-CTLA-4 antibodies (CTLA-4- AB-29, CTLA-4- AB-30, CTLA-4-AB-31, and CTLA-4- AB-32) and reference antibodies for binding to human CTLA-4 at pH 5 and at pH 7.4.
  • 5G shows results of binding tests of anti-CTLA- 4 antibodies (CTLA-4- AB -34, CTLA-4-AB-35, CTLA-4-AB-36, CTLA-4-AB-37, CTLA-4-AB-38 and CTLA-4- AB-39) for binding to human CTLA-4 at pH 5 and at pH 7.4.
  • FIG. 5H shows results of binding tests of anti-CTLA-4 antibodies (CTLA-4- AB -42, CTLA-4- AB -43, CTLA-4- AB -44, and CTLA-4- AB-45) and reference antibodies for binding to human CTLA-4 at pH 5 and at pH 7.4.
  • FIG. 51 shows results of binding tests of anti-CTLA-4 antibodies (CTLA-4-AB-8, CTLA-4-AB-12, CTLA- 4- AB-33, and CTLA-4- AB-46) and reference antibodies for binding to human CTLA-4 at pH 5 and at pH 7.4.
  • FIG. 5J shows results of binding tests of anti-CTLA-4 antibody CTLA-4- AB-47 and reference antibodies for binding to human CTLA-4 at pH 5 and at pH 7.4.
  • FIG. 5K shows results of binding tests of anti-CTLA-4 antibody CTLA-4- AB -48 and reference antibodies for binding to human CTLA-4 at pH 5 and at pH 7.4.
  • 5L shows results of binding tests of anti-CTLA-4 antibodies (CTLA-4- AB- 49, CTLA-4- AB-50, and CTLA-4- AB-51) and reference antibodies for binding to human CTLA-4 at pH 5 and at pH 7.4.
  • FIG. 5M shows results of binding tests of anti-CTLA-4 antibodies (CTLA-4- AB- 52, CTLA-4-AB-53, and CTLA-4-AB-54) and reference antibodies for binding to human CTLA-4 at pH 5 and at pH 7.4.
  • FIG. 5N shows results of binding tests of anti-CTLA-4 antibodies (CTLA-4- AB- 55, CTLA-4-AB-56, and CTLA-4-AB-57) and reference antibodies for binding to human CTLA-4 at pH 5 and at pH 7.4.
  • FIG. 50 shows results of binding tests of anti-CTLA-4 antibodies (CTLA-4- AB- 58, CTLA-4-AB-59, and CTLA-4-AB-60) and reference antibodies for binding to human CTLA-4 at pH 5 and at pH 7.4.
  • FIG. 5P shows results of binding tests of anti-CTLA-4 antibodies and reference antibodies (CTLA-4-AB-61, CTLA-4-AB-62, and CTLA-4- AB-63) for binding to human CTLA-4 at pH 5 and at pH 7.4.
  • FIG. 5Q shows results of binding tests of anti-CTLA-4 antibodies (CTLA-4- AB- 64, CTLA-4-AB-65, and CTLA-4-AB-66) and reference antibodies for binding to human CTLA-4 at pH 5 and at pH 7.4.
  • FIG. 5R shows results of binding tests of anti-CTLA-4 antibodies (CTLA-4- AB- 67, CTLA-4-AB-68, and CTLA-4-AB-69) and reference antibodies for binding to human CTLA-4 at pH 5 and at pH 7.4.
  • FIG. 5S shows results of binding tests of anti-CTLA-4 antibodies (CTLA-4- AB- 70, CTLA-4-AB-71, and CTLA-4- AB-72) and reference antibodies for binding to human CTLA-4 at pH 5 and at pH 7.4.
  • FIGs. 6A-6C show results of binding tests of exemplary provided antibodies for binding to human CTLA-4 at different pH’s.
  • FIG. 6A shows results of binding tests of a reference antibody for binding to human CTLA-4 at pH 5.5, pH 6, pH 6.5, pH 7 and pH 7.4.
  • FIG. 6B shows results of binding tests of CTLA-4- AB-62 for binding to human CTLA-4 at pH 5.5, pH 6, pH 6.5, pH 7 and pH 7.4.
  • FIG. 6C shows results of binding tests of CTLA-4-AB-68 for binding to human CTLA-4 at pH 5.5, pH 6, pH 6.5, pH 7 and pH 7.4.
  • FIGs. 7A-7C show effects of anti-CTLA-4 antibodies on CTLA-4 expression in treated human-CTLA-4 expressing Cells.
  • FIG. 7A shows hCTLA-4 expression levels in cells treated with anti- CTLA-4 antibodies.
  • FIG. 7B shows a schematic drawing of the workflow of confocal microscopy.
  • FIG. 7C shows results of confocal microscopy.
  • HEK293-hCTLA-4 cells were incubated with lOpg/mL antibody
  • FIGs. 8A-8B show effect of anti-CTLA-4 antibodies on antibody dependent cellular cytotoxicity (ADCC).
  • FIG. 8A shows selection of NK cells.
  • FIG. 8B shows target cell lysis was evaluated at various concentrations of four different antibodies and EC50 values calculated from doseresponse curves.
  • FIGs. 9A-9E show in vivo anti-tumor efficacy of anti-CTLA-4 antibodies.
  • FIG. 9A shows a schematic drawing of the experimental procedure.
  • FIGs. 10A-10H show effect of anti-CTLA-4 antibodies on immune cell subsets in the tumor and blood of hCTLA4 knock-in mice.
  • FIG. 10A shows gating strategy for tumor samples to analyze. This panel was designed to analyze absolute cell numbers of CD45+, Myeloid, T cells, CD4 T, CD8 T and Treg in tumor of CT26 model in hCTLA4 knock-in mice.
  • FIG. 10A shows gating strategy for tumor samples to analyze. This panel was designed to analyze absolute cell numbers of CD45+, Myeloid, T cells, CD4 T, CD8 T and Treg in tumor of CT26 model in hCTLA4 knock-in mice.
  • FIG. 10B shows gating strategy for blood samples to analyze absolute cell numbers of CD45+, Myeloid, T cells, CD4 T, CD8 T and Treg in blood of CT26 model in hCTLA4 knock-in mice, and the expression level of hCTLA-4 on Treg cells in the blood of CT26 model in hCTLA4 knock-in mice.
  • FIG. 10C shows results of the FCM analysis for tumor samples. One-way ANOVA was used for statistical analysis. When a significant F- statistics (a ratio of treatment variance to the error variance) was obtained, comparisons between group 1 and other groups were carried out with Games-Howell (equal variances not assumed) or Tukey (equal variances assumed) test. * p ⁇ 0.05; ** p ⁇ 0.01; *** p ⁇ 0.001.
  • FIG. 10D shows results of the FCM analysis for blood samples showing percentages of myeloid, T cells and T cell subpopulations in CD45+ cells.
  • FIG. 10E shows results of the FCM analysis for blood samples showing percentages of T cell subpopulations in T cells.
  • FIG. 10F shows results of the FCM analysis for blood samples showing cell numbers of CD45+, myeloid, T cells and T cell subpopulations per 100 pL blood.
  • FIG. 10G shows results of the FCM analysis for blood samples showing ratios between T cell subpopulations.
  • CD4Teff refers to CD4T cells other than Treg.
  • FIG. 10H shows mean fluorescence intensity (MFI) of hCTLA4 of Treg cells.
  • FIG.ll depicts an exemplary format (“Crossmab”) of a PD-l/CTLA-4 bispecifc antibody binding molecule.
  • the binding molecule includes a variable heavy (VH) chain and a variable light (VL) chain of an anti- PD-1 antibody, and a variable heavy (VH) chain and a variable light (VL) chain of an anti-CTLA-4 antibody, each configured as Fabs such that for one the VH and a VL region are interchanged in one binding arm (“crossmab” format).
  • VH variable heavy
  • VL variable light
  • crossmab variable light
  • the Fc region of the bispecific antibody is formed with a heterodimeric Fc containing knob-into-hole heterodimeric connections to form two different heavy chains.
  • an antibody designated PC_abl in which the Targetl is CTLA4 and the CTLA4 Fab is a crossmab format and the Target2 is PD-1 with a conventional PD-1 Fab.
  • an antibody designated PC_ab2 in which the Targetl is PD-1 and the PD-1 Fab is a crossmab format, and the Target2 is CTLA-4 with a conventional CTLA-4 Fab.
  • FIGs 12A-12B show reducing and non-reducing SDS-PAGE gels showing the heavy and light chains of the bispecific antibodies.
  • FIGs. 13A-13B show SEC-HPLC results for CTLA-4/PD-l-abl and CTLA-4/PD-l-ab2.
  • FIGs. 14A-14C are binding results of the engineered bispecific PD-l/CTLA-4 antibodies from an Enzyme-Linked Immuno-Sorbent Assay at different pH 5.0 and 7.4. Detailed Description
  • the present disclosure concerns anti-PD-1 antibodies or antibody fragments suitable for therapeutic use.
  • the present disclosure additionally concerns binding molecules, such as multispecific antibodies (e.g., bispecific antibodies), that contain a PD1 binding domain (e.g., antibody fragment, such as a Fab) that binds PD-1 and a CTLA-4 binding domain (e.g., antibody fragment, such as a Fab) for therapeutic use.
  • a PD1 binding domain e.g., antibody fragment, such as a Fab
  • CTLA-4 binding domain e.g., antibody fragment, such as a Fab
  • PD1 Programmed cell death 1; also known as CD279 (cluster of differentiation 279)
  • CD279 cluster of differentiation 279
  • the PD-1 pathway is a regulator in the induction and maintenance of immunological tolerance.
  • the protein functions as an "immune checkpoint" inhibitor, i.e. it acts to modulate the activity of cells in the immune system so as to regulate and limit autoimmune diseases.
  • PD-1 has two ligands, PD-L1 and PD-L2, which interact with the cell surface receptor. On binding, PD-1 induces an intracellular signal, which negatively regulates T-cell responses. On the surface of activated T cells, PD-
  • PD-1 expression is upregulated after the recognition of peripheral antigens by T cells; subsequently, the elevated binding of PD-1 to PD-L1 and PD-L2 becomes a key step for downstream inhibitory signaling.
  • PD-1 is also associated with increased Treg-cell proliferation and enhanced immunosuppressive function.
  • provided anti-PD-1 antibodies or antibody fragments act as PD-1 inhibitors to activate the immune system to attack tumors, including in methods for treating cancer.
  • anti-PD-1 antibodies or antibody fragments may exhibit improved binding kinetics to PD-1 in a tumor environment versus PD-1 present in normal tissue.
  • the disclosed anti-PD-1 antibodies or antibody fragments may also exhibit physiochemical or biological properties that lead to improved efficacy and safety to anti-PD-1 antibodies known in the art.
  • provided anti-PD-1 antibodies or antibody fragments have a higher binding affinity to PD-1 in a tumor in comparison with PD-1 present in normal tissue.
  • these anti-PD-1 antibodies or antibody fragments have at least comparable efficacy as well as a longer half-life, but reduced side-effects, in comparison with monoclonal anti-PD-1 antibodies known in the art. This may permit use of higher dosages of these anti-PD-1 antibodies or antibody fragments, thus providing a more effective therapeutic option without a corresponding significant increase in side effects.
  • a binding molecule such as a bispecific antibody, that is directed against PD-1 and also CTLA-4 can simultaneously block the PD-1 and also block CTLA-4 to thus reduce immune responses to the tumor site.
  • a binding molecules that comprise a CTLA-4 binding domain that confers specific binding properties to human CTLA-4 under different pH conditions.
  • the disclosed binding molecules also exhibit physiochemical or biological properties that lead to improved efficacy and safety as compared to CTLA-4 binding domains known in the art.
  • An altered pH microenvironment is the most common microenvironment found in disease states such as tumor microenvironments, and it is the most uniform within the disease microenvironment compared to other properties such as hypoxia (see e.g. Fogh Andersen et al. (1995) Clin. Chem., 41:1522-1525; Bhujwalla et al. (2002) NMR Biomed., 15:114-119; Helmlinger et al. (1997) Nature Med., 3:177; Gerweck and Seetharaman (1996), Cancer Res. 56(6): 1194-1198).
  • hypoxia see e.g. Fogh Andersen et al. (1995) Clin. Chem., 41:1522-1525; Bhujwalla et al. (2002) NMR Biomed., 15:114-119; Helmlinger et al. (1997) Nature Med., 3:177; Gerweck and Seetharaman (1996), Cancer Res. 56(6): 1194-1198.
  • the ‘Warburg effect’ creates a microenviron
  • the immune system is responsible for defending the body against foreign substances, such as pathogens and abnormal cells.
  • T cells Central to the immune response are T cells, a type of white blood cell, which act as key orchestrators of immune reactions. T cell activation is tightly regulated to prevent excessive immune responses that could lead to tissue damage and autoimmune disorders.
  • CTLA-4 a protein predominantly expressed on the surface of activated T cells, serves as an important checkpoint molecule for downregulating immune responses.
  • CTLA-4 acts by binding to its ligands, CD80 (B7-1) and CD86 (B7-2), on antigen- presenting cells (APCs), thereby attenuating T cell activation and proliferation.
  • the engagement of CTLA-4 with its ligands delivers inhibitory signals to T cells, leading to suppression of immune reactions.
  • Dysregulation of the CTLA-4 pathway has been implicated in several pathological conditions, including autoimmune diseases as well as cancer, where tumor cells can exploit the immune system’s regulatory mechanisms to evade destruction.
  • CTLA-4 a specific anti-CTLA-4 antibody (Ipilimumab, marketed as Yervoy) has demonstrated efficacy in treating melanoma
  • treatment and targeting of CTLA-4 is associated with autoimmune like toxicities.
  • Characteristic side effects from inhibition of CTLA-4 are generally called immune-related adverse events (ir AEs) and the most common irAEs are skin rash, hepatitis, colitis and endocrinopathies, particularly hypopituitarism.
  • CTLA-4 is recycled between the plasma membrane and endosomes by binding to lipopolysaccharide -responsive and beige-like anchor protein (LRBA).
  • LRBA lipopolysaccharide -responsive and beige-like anchor protein
  • Antibodies that bind CTLA-4 disrupt the recycling process. Consequently, CTLA-4 is systemically directed to lysosomal degradation, and autoimmunity-related adverse effects are developed due to the unstoppable action of cytotoxic T cells. Therefore, there is a desire to improve the therapeutic potential of CTLA-4 binding domains by increasing efficacy while reducing the associated irAEs.
  • the provided binding molecules comprise a CTLA-4 binding domain that possesses pH dependent binding properties to human CTLA-4 that facilitates the dissociation of antibody from the antibody: CTLA-4 complex upon internalization into a cell, allowing either the antibody, CTLA-4, or both to be recycled instead of being degraded in the lysosome.
  • the provided binding molecules preserve high affinity for CTLA-4 in neutral pH environments (such as at pH>7), lower affinity in acidic environments (such as at pH 5.5- 7 found in early endosomes), and significantly reduced affinity for CTLA-4 at more acidic environments (such as at pH ⁇ 5.5 found in late endosomes and lysosomes).
  • provided binding molecules comprise a CTLA-4 binding domain that has a lower binding affinity (i.e. greater dissociation constant) to CTLA-4 at an acidic pH as present in endosomes of a cell in comparison with the binding affinity of CTLA-4 present in normal tissue.
  • the dissociation constant (KD) for binding at acidic pH e.g., pH 5.0 to 6.0
  • the dissociation constant for binding at physiological pH e.g., e.g. pH 7.35 to 7.45.
  • these binding molecules have reduced side-effects, in comparison with monoclonal anti- CTLA-4 antibodies known in the art. This may permit use of higher dosages of these binding molecules, thus providing a more effective therapeutic option without a corresponding significant increase in side effects.
  • the provided binding molecules comprise a CTLA-4 binding domain that can be used to block inhibitory activity of CTLA-4 by CTLA-4-expressing cells including regulatory T cells (Tregs), and in some cases may also mediate depletion of such cells by an ADCC mechanism.
  • Tregs regulatory T cells
  • depletion of Tregs in the tumor contributes to anti-tumor efficacy of the provided antibodies.
  • the provided binding molecules comprise a CTLA- 4 binding domain that reduces CTLA-4 mediated signaling in effector T cells and exhibit activity to deplete Tregs in the tumor microenvironment that might otherwise suppress an anti-tumor immune response.
  • the binding molecule or a pharmaceutical composition comprising the binding molecule induces strong deletion of Treg and local T cell activation in tumor microenvironment but minimal systemic T cell activation. In some embodiments, the binding molecule or a pharmaceutical composition comprising the binding molecule induces a favorable ratio of Treg cells to T effector cells in the tumor microenvironment.
  • anti-PD-1 antibodies including full-length antibodies or antigenbinding fragments, that contain a heavy chain variable region (VH) sequence and a light chain variable region (VL) sequence as described, or a sufficient antigen-binding portion thereof.
  • VH is the region of the anti-PD-1 antibody that comprises the three heavy chain complementarity determining regions (CDRs) and the VL chain is the region of the anti-PD-1 antibody that comprises the three light chain CDRs.
  • provided anti-PD-1 antibodies including full-length antibodies or antigen-binding fragments, are antibodies that contains a VH region sequence that contains a CDR-H1, a CDR-H2 and a CDR-H3 as described and contains a VL region sequence that contains a CDR-L1, a CDR-L2 and a CDR-L3 as described.
  • the antibodies include antibodies that specifically bind to PD-1, e.g., human PD-1.
  • human antibodies or antibodies that are modified from or variant of human antibodies.
  • the antibodies include isolated antibodies.
  • PD-1 -binding molecules containing such antibodies, such as single-chain proteins, fusion proteins, conjugates and/or recombinant receptors such as chimeric antigen receptors.
  • an PD-1 -targeted antibodydrug conjugate (ADC) comprising an anti-PD-1 antibody or antigen binding fragment thereof described herein.
  • ADC antibodydrug conjugate
  • a bispecific immune cell engager that is a fusion protein comprising the anti-PD-1 antibody or antigen binding fragment thereof described herein.
  • the bispecific immune cell engager is a bispecific T cell engager.
  • the PD-1 -binding molecules include isolated molecules.
  • polynucleotides containing nucleic acids sequences encoding all or a portion of such antibodies, including an antigen-binding fragment, or binding molecule can be incorporated into constructs, such as deoxyribonucleic acid (DNA) or RNA constructs, such as those that can be introduced into cells for expression of the encoded anti-PD-1 antibodies or binding molecules.
  • DNA deoxyribonucleic acid
  • RNA constructs such as those that can be introduced into cells for expression of the encoded anti-PD-1 antibodies or binding molecules.
  • antibody herein is used in the broadest sense and includes polyclonal and monoclonal antibodies, including intact antibodies and functional (antigen-binding) antibody fragments, including fragment antigen binding (Fab) fragments, F(ab’)2 fragments, Fab’ fragments, Fv fragments, recombinant IgG (rlgG) fragments, heavy chain variable (VH) regions capable of specifically binding the antigen, single chain antibody fragments, including single chain variable fragments (scFv), and single domain antibodies (e.g., sdAb, sdFv, nanobody, VHH) fragments.
  • Fab fragment antigen binding
  • rlgG fragment antigen binding
  • VH heavy chain variable regions capable of specifically binding the antigen
  • single chain antibody fragments including single chain variable fragments (scFv)
  • single domain antibodies e.g., sdAb, sdFv, nanobody, VHH fragments.
  • the term encompasses genetically engineered and/or otherwise modified forms of immunoglobulins, such as intrabodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies, and heteroconjugate antibodies, multispecific, e.g., bispecific or trispecific, antibodies, diabodies, triabodies, and tetrabodies, tandem di-scFv, tandem tri-scFv.
  • antibody should be understood to encompass functional antibody fragments thereof also referred to herein as “antigenbinding fragments.”
  • the term also encompasses intact or full-length antibodies, including antibodies of any class or sub-class, including IgG and sub-classes thereof, IgM, IgE, IgA, and IgD.
  • CDR complementarity determining region
  • HVR hypervariable region
  • CDR-H1, CDR-H2, CDR-H3 three CDRs in each heavy chain variable region
  • CDR-L1, CDR-L2, CDR-L3 three CDRs in each light chain variable region
  • “Framework regions” and “FR” are known to refer to the non-CDR portions of the variable regions of the heavy and light chains.
  • FR-H1, FR-H2, FR-H3, and FR-H4 there are four FRs in each full-length heavy chain variable region (FR-H1, FR-H2, FR-H3, and FR-H4), and four FRs in each full-length light chain variable region (FR-L1, FR-L2, FR-L3, and FR-L4).
  • Numbering for both the Kabat and Chothia schemes is based upon the most common antibody region sequence lengths, in some cases with insertions. Insertions in the sequence relative to the standard numbering scheme are indicated using insertion letter codes. For example, residues that are inserted between residues L30 and L31 are indicated as L31A, L31B, etc. Deletions in the sequence relative to the standard scheme are accommodated by skipping numbers. The two schemes place certain insertions and deletions (“indels”) at different positions, resulting in differential numbering. For instance, the Chothia numbering scheme is nearly identical to the Kabat numbering scheme, except that insertions are placed at structural positions and topologically equivalents residues do get assigned the same numbers.
  • the Contact scheme is based on analysis of complex crystal structures and is similar in many respects to the Chothia numbering scheme.
  • the AbM scheme is a compromise between Kabat and Chothia definitions based on that used by Oxford Molecular’s AbM antibody modeling software.
  • the IgBLAST scheme is based on matching to germline V, D and J genes, and can be determined using National Center for Biotechnology Information (NCBI)’s IgBLAST tool.
  • Kabat numbering can be determined by known sequence rules as described in, for example, Kabat et al. (1991), “Sequences of Proteins of Immunological Interest,” 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD.
  • the Kabat numbering scheme in some aspects can include any of the following rules to designate CDRs: CDR-L1 starts at approximately residue 24 of the light chain, always has a preceding C residue, and always has a following W residue; the end of CDR-L1 is defined by a stretch of 3 residues, where the W residue can be followed by Y, L, or F, followed by Q or L; CDR-1 has a length of 10 to 17 residues; CDR-L2 always starts 16 residues after the end of CDR-L1; the two residues before CDR-L2 are I and Y but can also be V and Y, I and K, or I and F; CDR-L2 is always 7 residues long; CDR-L3 always starts 33 residues after the end of CDR-L2, always has a preceding C residue, and is strictly followed by a F-G-X-G sequence motif, where X is any amino acid; CDR-L3 has a length of 7 to 11 residues; CDR-H1
  • boundary positions of certain CDRs can differ based on different definitions for the CDRs (See e.g., Martin, ACR, Antibody Information: How to identify the CDRs by looking at a sequence [online] bioinf.org.uk/abs/info.html).
  • the boundary positions for CDR-L1 according to Chothia numbering can be L26— L32 (Chothia et al., Science, 1986; 233(4765):755-8 and Chothia C. and Lesk A.M. J Mol Biol, 1987; 196(4):901-17).
  • the boundary positions for CDR-L1 can be L25— L32 (Al-Lazikani et al., J Mol Biol, 1997; 273(4):927-48).
  • the boundary positions for CDR-L2 can be L50— L52 and for CDR-L3 can be L91— L96 (Chothia et al., Science, 1986; 233(4765):755-8; Chothia C. and Lesk A.M. J Mol Biol, 1987; 196(4):901-17; and Al-Lazikani et al., J Mol Biol, 1997; 273(4):927-48).
  • the boundary positions for CDR-H1 according to Chothia numbering can be H26— H32 (Chothia et al., Science, 1986; 233(4765):755-8; Chothia C. and Lesk A.M. J Mol Biol, 1987; 196(4):901-17; and Al-Lazikani et al., J Mol Biol, 1997; 273(4):927-48).
  • the boundary positions for CDR-H2 can be H53— H55 (Chothia et al., Science, 1986; 233(4765):755-8 and Chothia C. and Lesk A.M.
  • the boundary positions for CDR-H3 can be H96— H101 (Chothia et al., Science, 1986; 233(4765):755-8 and Chothia C. and Lesk A.M. J Mol Biol., 1987; 196(4):901-17).
  • the boundary positions for CDR-H3 can be H92— H104 (Morea et al., Biophys Chem, 1997; 68(1-3): 9-16 and Morea et al., J Mol Biol., 1998; 275(2): 269-94).
  • Table 1 exemplifies exemplary numbering and lists exemplary position boundaries of CDR-L1, CDR-L2, CDR-L3 and CDR-H1, CDR-H2, CDR-H3 as identified by Kabat, Chothia, AbM, and Contact schemes, respectively.
  • residue numbering is listed using both the Kabat and Chothia numbering schemes.
  • FRs are located between CDRs, for example, with FR-L1 located before CDR-L1, FR-L2 located between CDR-L1 and CDR-L2, FR-L3 located between CDR- L2 and CDR-L3 and so forth.
  • CDR complementary determining region
  • individual specified CDRs e.g., CDR-H1, CDR-H2, CDR-H3
  • CDR-H1, CDR-H2, CDR-H3 individual specified CDRs
  • a particular CDR e.g., a CDR-H3
  • a CDR-H3 contains the amino acid sequence of a corresponding CDR in a given VH or VL region amino acid sequence
  • a CDR has a sequence of the corresponding CDR (e.g., CDR-H3) within the variable region, as defined by any of the aforementioned schemes, or other known schemes.
  • an antibody or antigen-binding fragment thereof comprises a CDR-H1, a CDR-H2, and a CDR-H3 as contained within a given VH region amino acid sequence and a CDR-L1, a CDR-L2, and a CDR-L3 as contained within a given VL region amino acid sequence
  • the CDRs can be defined by any of the aforementioned schemes, such as Kabat, Chothia, AbM, IgBLAST, IMGT, or Contact method, or other known scheme.
  • specific CDR sequences are specified. Exemplary CDR sequences of provided antibodies are described using various numbering schemes, although it is understood that a provided antibody can include CDRs as described according to any of the other aforementioned numbering schemes or other known numbering schemes.
  • FR or individual specified FR(s) e.g., FR-H1, FR- H2, FR-H3, FR-H4, FR-E1, FR-E2, FR-E3, and/or FR-E4
  • FR-H1, FR- H2, FR-H3, FR-H4, FR-E1, FR-E2, FR-E3, and/or FR-E4 should be understood to encompass a (or the specific) framework region as defined by any of the known schemes.
  • the scheme for identification of a particular CDR, FR, or FRs or CDRs is specified, such as the CDR as defined by the Kabat, Chothia, AbM, IgBEAST, IMGT, or Contact method, or other known schemes.
  • the particular amino acid sequence of a CDR or FR is given.
  • an antibody or antigen-binding fragment thereof comprises a FR-H1, a FR-H2, a FR-H3, and a FR-H4 as contained within a given VH region amino acid sequence and a FR-E1, a FR-E2, a FR-E3, and a FR-E4 as contained within a given VL region amino acid sequence
  • the FRs can be defined by any of the aforementioned schemes, such as Kabat, Chothia, AbM, IgBLAST, IMGT, or Contact method, or other known scheme.
  • variable region refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to antigen.
  • the variable regions of the heavy chain and light chain (VH and VL, respectively) of a native antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three CDRs (See, e.g. , Kindt et al. Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007)).
  • FRs conserved framework regions
  • antibodies that bind a particular antigen may be isolated using a VH or VL domain from an antibody that binds the antigen to screen a library of complementary VL or VH domains, respectively. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).
  • an “antibody fragment” or “antigen-binding fragment” refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds.
  • an antigen binding fragment includes all CDRs of a variable heavy chain (VH) and variable light chain (VL) sequence from antibodies that bind PD-1 set forth herein.
  • antibody fragments include but are not limited to Fv, Fab, Fab’, Fab’-SH, F(ab’)2; diabodies; linear antibodies; heavy chain variable (VH) regions, single-chain antibody molecules such as scFvs and single-domain antibodies comprising only the VH region; and multispecific antibodies formed from antibody fragments.
  • Papain digestion of antibodies produce two identical antigen-binding fragments, called "Fab” fragments, and a residual "Fc” fragment, a designation reflecting the ability to crystallize readily.
  • the Fab fragment is composed of an entire L chain along with the variable region domain of the H chain (VH), and the first constant domain of one heavy chain (CHI).
  • Each Fab fragment is monovalent with respect to antigen binding, i.e., it has a single antigen-binding site.
  • Pepsin treatment of an antibody yields a single large F(ab')2 fragment which roughly corresponds to two disulfide linked Fab fragments having different antigen-binding activity and is still capable of cross-linking antigen.
  • Fab' fragments differ from Fab fragments by having a few additional residues at the carboxy terminus of the CHI domain including one or more cysteines from the antibody hinge region.
  • Fab'-SH is the designation herein for Fab' in which the cysteine residue(s) of the constant domains bear a free thiol group.
  • the antibody is or comprises an antibody fragment comprising a variable heavy chain (VH) and a variable light chain (VL) region.
  • the antibodies are single-chain antibody fragments comprising a heavy chain variable (VH) region and/or a light chain variable (VL) region, such as scFvs.
  • Antibody fragments can be made by various techniques, including but not limited to proteolytic digestion of an intact antibody as well as production by recombinant host cells.
  • the antibodies are recombinantly-produced fragments, such as fragments comprising arrangements that do not occur naturally, such as those with two or more antibody regions or chains joined by synthetic linkers, e.g., peptide linkers, and/or that are may not be produced by enzyme digestion of a naturally-occurring intact antibody.
  • the antibody fragments are scFvs.
  • F(ab) refers to two of the protein fragments resulting from proteolytic cleavage of immunoglobulin G (IgG) molecules by the enzyme papain. Each F(ab) comprises a covalent heterodimer of the VH chain and VL chain and includes an intact antigen-binding site.
  • F(ab)2 refers to a protein fragment of IgG generated by proteolytic cleavage by the enzyme pepsin. Each F(ab')2 fragment comprises two F(ab) fragments, thus comprising both antigen-binding sites.
  • An “Fv fragment” for use according to certain embodiments of the present invention can be produced by preferential proteolytic cleavage of an IgM, and on rare occasions of an IgG or IgA immunoglobulin molecule. Fv fragments are, however, more commonly derived using recombinant techniques known in the art.
  • the Fv fragment includes a non-covalent VH::VL heterodimer including an antigen-binding site which retains much of the antigen recognition and binding capabilities of the native antibody molecule, but lacking the CHI and CL domains contained within a Fab.
  • single chain Fv (scFv) antibodies are contemplated and may be prepared using standard molecular biology techniques following the teachings of the present application with regard to selecting antibodies having the desired specificity.
  • chimeric antibodies may be made.
  • a chimeric antibody may comprise CDRs and framework regions from different antibodies. These antibodies may be produced through recombinant molecular biological techniques or may be physically conjugated together.
  • a scFv polypeptide is a covalently linked VH::VL heterodimer which is expressed from a gene fusion including VH- and V
  • a number of methods have been described to discern chemical structures for converting the naturally aggregated — but chemically separated — light and heavy polypeptide chains from an antibody V region into an scFv molecule which will fold into a three dimensional structure substantially similar to the structure of an antigen-binding site. See, e.g., U.S. Pat. Nos. 5,091,513 and 5,132,405, to Huston et al. ', and U.S. Pat. No. 4,946,778, to Ladner et al.
  • the antibodies described herein may be provided in the form of a UniBody®.
  • a UniBody® is an IgG4 antibody with the hinge region removed (see GenMab Utrecht, The Netherlands; see also, e.g., US20090226421). This antibody technology creates a stable, smaller antibody format with an anticipated longer therapeutic window than current small antibody formats. IgG4 antibodies are considered inert and thus do not interact with the immune system. Fully human IgG4 antibodies may be modified by eliminating the hinge region of the antibody to obtain halfmolecule fragments having distinct stability properties relative to the corresponding intact IgG4 (GenMab, Utrecht).
  • the UniBody® Halving the IgG4 molecule leaves only one area on the UniBody® that can bind to cognate antigens (e.g., disease targets) and the UniBody® therefore binds univalently to only one site on target cells. For certain cancer cell surface antigens, this univalent binding may not stimulate the cancer cells to grow as may be seen using bivalent antibodies having the same antigen specificity, and hence UniBody® technology may afford treatment options for some types of cancer that may be refractory to treatment with conventional antibodies.
  • the small size of the UniBody® can be a great benefit when treating some forms of cancer, allowing for better distribution of the molecule over larger solid tumors and potentially increasing efficacy.
  • the antibodies of the present disclosure may be chimeric antibodies.
  • a chimeric antibody is comprised of an antigen-binding fragment of an anti- PD-1 antibody operably linked or otherwise fused to a heterologous Fc portion of a different antibody.
  • the heterologous Fc domain is of human origin.
  • the heterologous Fc domain may be from a different Ig class from the parent antibody, including IgA (including subclasses IgAl and IgA2), IgD, IgE, IgG (including subclasses IgGl, IgG2, IgG3, and IgG4), and IgM.
  • the heterologous Fc domain may be comprised of CH2 and CH3 domains from one or more of the different Ig classes.
  • the antibodies of the present disclosure may be “non-naturally occurring” antibodies.
  • Non-naturally occurring antibodies can refer to antibodies that comprise one or more amino acid modifications, such that the resultant antibody is substantially non-naturally occurring (e.g., does not exist in nature). These amino acid modifications can include point mutations, wherein a naturally occurring amino acid is substituted for another naturally occurring amino acid. In some embodiments, the amino acid modifications can include point mutations wherein a non-naturally occurring amino acid is substituted for a naturally occurring amino acid.
  • Non-naturally occurring antibodies can also refer to antibodies that are conjugated to a heterologous protein or compound, such as a detectable marker.
  • human antibodies are human antibodies.
  • a “human antibody” is an antibody with an amino acid sequence corresponding to that of an antibody produced by a human or a human cell, or non-human source that utilizes human antibody repertoires or other human antibodyencoding sequences, including human antibody libraries.
  • the term excludes humanized forms of non- human antibodies comprising non-human antigen-binding regions, such as those in which all or substantially all CDRs are non-human.
  • the term includes antigen-binding fragments of human antibodies.
  • Human antibodies may be prepared by administering an immunogen to a transgenic animal that has been modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigenic challenge.
  • Such animals typically contain all or a portion of the human immunoglobulin loci, which replace the endogenous immunoglobulin loci, or which are present extrachromosomally or integrated randomly into the animal’s chromosomes. In such transgenic animals, the endogenous immunoglobulin loci have generally been inactivated.
  • Human antibodies also may be derived from human antibody libraries, including phage display and cell-free libraries, containing antibody-encoding sequences derived from a human repertoire.
  • monoclonal antibodies including monoclonal antibody fragments.
  • the term “monoclonal antibody” as used herein refers to an antibody obtained from or within a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical, except for possible variants containing naturally occurring mutations or arising during production of a monoclonal antibody preparation, such variants generally being present in minor amounts.
  • polyclonal antibody preparations which typically include different antibodies directed against different epitopes
  • each monoclonal antibody of a monoclonal antibody preparation is directed against a single epitope on an antigen.
  • a monoclonal antibody may be made by a variety of techniques, including but not limited to generation from a hybridoma, recombinant DNA methods, phage-display and other antibody display methods.
  • the antibody or the antigen-binding fragments of the antibody is isolated.
  • variants of the antibodies disclosed herein contemplates variants of the antibodies disclosed herein.
  • such variant antibodies or antigen-binding fragments, or CDRs thereof bind to PD-1 at least about 50%, at least about 70%, at least about 80%, at least about 85%, at least about 90% and in certain embodiments, at least about 95% as well as an antibody sequence specifically set forth herein.
  • such variant antibodies or antigen-binding fragments, or CDRs thereof bind to PD-1 with greater affinity than the antibodies set forth herein, for example, that bind quantitatively at least about 105%, 106%, 107%, 108%, 109%, or 110% as well as an antibody sequence specifically set forth herein.
  • a subject antibody may have: a) a heavy chain variable region having an amino acid sequence that is at least 80% identical, at least 85% identical, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or 99% or 100% identical, to a heavy chain variable region of an anti-PD-1 antibody described herein; and b) a light chain variable region having an amino acid sequence that is at least 80% identical, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or 99% or 100% identical, to a light chain variable region of an anti-PD-1 antibody described herein.
  • the anti-PD-1 antibody contains a VH region and a VL region containing a combination of six CDRs as described below.
  • the anti-PD-1 antibody contains a VH region and a VL region as described below.
  • a VH region sequence can be any of the VH region sequence described herein.
  • a VL region sequence can be any of the VL region sequence described herein.
  • any of the VH region sequence and any of the VL region sequence described herein can be used in combination.
  • the antibody is an antigen-binding fragment, such as a Fab or an scFv.
  • the antibody or antigen-binding fragment further comprises at least a portion of an immunoglobulin constant region or a variant thereof.
  • the antibody is a full-length antibody that also contains a constant region.
  • an anti- PD-1 antibody comprises at least one heavy chain comprising a VH region and at least a portion of a heavy chain constant region, and at least one light chain comprising a VL region and at least a portion of a light chain constant region.
  • an anti-PD-1 antibody comprises two heavy chains, wherein each heavy chain comprises a VH region and at least a portion of a heavy chain constant region, and two light chains, wherein each light chain comprises a VL region and at least a portion of a light chain constant region. In some embodiments, an anti-PD-1 antibody comprises two heavy chains, wherein each heavy chain comprises a VH region and a heavy chain constant region, and two light chains, wherein each light chain comprises a VL region and a light chain constant region.
  • Anti-PDl antibodies including antigen-binding fragments thereof, include any combination of the heavy chain and light chain complementarity-determining regions (CDRs) discussed herein.
  • the anti-PD-1 antibody or antigen-binding fragment thereof comprises any one of the CDR-H1 as described herein, any one of the CDR-H2 as described herein, any one of the CDR-H3 as described herein, any one of the CDR-L1 as described herein, any one of the CDR-L2 as described herein and any one of the CDR-L3 as described here.
  • any one or more of the CDR-H1, the CDR-H2 and the CDR-H3 sequences described herein, and any one or more of the CDR-L1, the CDR-L2 and the CDR-L3 sequences described herein can be used in combination.
  • a provided anti-PD-1 antibody or an antigen-binding fragment thereof has a CDR-H1, a CDR-H2 and a CDR-H3 present in a VH region amino acid sequence set forth in any one of SEQ ID NOs: 1-45, 157, and 165-170, or an amino acid sequence that has at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the VH region amino acid sequence set forth in any one of SEQ ID NOs: 1-45, 157, and 165- 170.
  • a provided anti-PD-1 antibody or an antigen-binding fragment thereof has a CDR-L1, a CDR-L2 and a CDR-L3 present in a VL region amino acid sequence set forth in any one of SEQ ID NOs: 46-90, 158, and 171-172, or an amino acid sequence that has at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the VL region amino acid sequence set forth in any one of SEQ ID NOs: 46-90, 158, and 171-172.
  • the provided anti-PD-1 antibody contains a combination of any of such six CDRs (a CDR-H1, a CDR-H2, a CDR-H3, a CDR-L1, a CDR-L2 and a CDR-L3) described above.
  • a provided anti-PD-1 antibody or an antigen-binding fragment thereof has a CDR-H1, a CDR-H2 and a CDR-H3 present in a VH region amino acid sequence set forth in any one of SEQ ID NOs: 1-45, 157, 165-170, and a CDR-L1, a CDR-L2 and a CDR-L3 present in a VL region amino acid sequence set forth in any one of SEQ ID NOs: 46-90, 158, and 171-172.
  • the combination of six CDRs (a CDR-H1, a CDR-H2, a CDR-H3, a CDR-L1, a CDR-L2 and a CDR-L3) is according to Kabat numbering. In some embodiments, the combination of six CDRs (a CDR-H1, a CDR-H2, a CDR-H3, a CDR-L1, a CDR-L2 and a CDR-L3) is according to Chothia numbering.
  • the combination of six CDRs (a CDR-H1, a CDR-H2, a CDR-H3, a CDR-L1, a CDR-L2 and a CDR-L3) is according to AbM numbering, In some embodiments, the combination of six CDRs (a CDR-H1, a CDR-H2, a CDR-H3, a CDR-L1, a CDR-L2 and a CDR-L3) is according to IMGT numbering. In some embodiments, the combination of six CDRs (a CDR-H1, a CDR-H2, a CDR- H3, a CDR-L1, a CDR-L2 and a CDR-L3) is according to IgBLAST numbering.
  • the VH region contains a CDR-H1 set forth in any one of SEQ ID NOs: 91-101, a CDR-H2 set forth in any one of SEQ ID NOs: 102-112, and 173, and a CDR-H3 set forth in any one of SEQ ID NOs: 113-123; and the VL region contains a CDR-L1 set forth in any one of SEQ ID NOs: 124-134, a CDR-L2 set forth in any one of SEQ ID NOs: 135-145, and a CDR-L3 set forth in any one of SEQ ID NOs: 146-156.
  • the anti-PD-1 antibody or antigen-binding fragment does not contain the combination of CDRs set forth in SEQ ID NO: 91, SEQ ID NO: 102, SEQ ID NO: 113, SEQ ID NO: 124, SEQ ID NO: 135 and SEQ ID NO: 146.
  • the VH region comprises a CDR-H1 comprising the sequence set forth in any one of SEQ ID NOs: 92-101, a CDR-H2 comprising the sequence set forth in any one of SEQ ID NOs: 103-112, and 173, and a CDR-H3 comprising the sequence set forth in any one of SEQ ID NOs: 114-123
  • the VL region comprises a CDR-L1 comprising the sequence set forth in any one of SEQ ID NOs: 124-134, a CDR-L2 comprising the sequence set forth in any one of SEQ ID NOs: 135-145; and a CDR-L3 comprising the sequence set forth in any one of SEQ ID NOs: 146-156.
  • the VH region comprises a CDR-H1 comprising the sequence set forth in any one of SEQ ID NOs: 91-101, a CDR-H2 comprising the sequence set forth in any one of SEQ ID NOs: 102-112, and 173, and a CDR- H3 comprising the sequence set forth in any one of SEQ ID NOs: 113-123
  • the VL region comprises a CDR-L1 comprising the sequence set forth in any one of SEQ ID NOs: 125-134, a CDR-L2 comprising the sequence set forth in any one of SEQ ID NOs: 136-145; and a CDR-L3 comprising the sequence set forth in any one of SEQ ID NOs: 147-156.
  • antibodies are those having sequences at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% identical to any such CDR sequence, e.g., any of the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, CDR-L3.
  • among the antibodies are those in which a CDR contained therein has no more than 2 amino acid difference compared to any such above CDR sequence, e.g., any of the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, CDR-L3. In some embodiments, among the antibodies are those in which a CDR contained therein has no more than 1 amino acid difference compared to any such above CDR sequence, e.g., any of the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, CDR-L3.
  • the VH region contains a CDR-H1 set forth in any one of SEQ ID NOs: 92-101, a CDR-H2 set forth in any one of SEQ ID NOs: 103-112, and 173, and a CDR-H3 set forth in any one of SEQ ID NOs: 114-123.; and the VL region contains a CDR-L1 set forth in any one of SEQ ID NOs: 125-134, a CDR-L2 set forth in any one of SEQ ID NOs: 136-145, and a CDR-L3 set forth in any one of SEQ ID NOs: 147-156.
  • the antibody or antigen-binding fragment has a VH region that contains a CDR-H1, a CDR-H2 and a CDR-H3 present in SEQ ID NO:1. In some embodiments, the antibody or antigen-binding fragment has a VH region that contains a CDR-H1, a CDR-H2 and a CDR- H3 present in SEQ ID NO:2. In some embodiments, the antibody or antigen-binding fragment has a VH region that contains a CDR-H1, a CDR-H2 and a CDR-H3 present in SEQ ID NO:3.
  • the antibody or antigen-binding fragment has a VH region that contains a CDR-H1, a CDR-H2 and a CDR-H3 present in SEQ ID NO:4. In some embodiments, the antibody or antigenbinding fragment has a VH region that contains a CDR-H1, a CDR-H2 and a CDR-H3 present in SEQ ID NO:5. In some embodiments, the antibody or antigen-binding fragment has a VH region that contains a CDR-H1, a CDR-H2 and a CDR-H3 present in SEQ ID NO:6.
  • the antibody or antigen-binding fragment has a VH region that contains a CDR-H1, a CDR-H2 and a CDR-H3 present in SEQ ID NO: 7. In some embodiments, the antibody or antigen-binding fragment has a VH region that contains a CDR-H1, a CDR-H2 and a CDR-H3 present in SEQ ID NO:8. In some embodiments, the antibody or antigen-binding fragment has a VH region that contains a CDR-H1, a CDR-H2 and a CDR- H3 present in SEQ ID NO: 9.
  • the antibody or antigen-binding fragment has a VH region that contains a CDR-H1, a CDR-H2 and a CDR-H3 present in SEQ ID NO: 10. In some embodiments, the antibody or antigen-binding fragment has a VH region that contains a CDR-H1, a CDR-H2 and a CDR-H3 present in SEQ ID NO: 11. In some embodiments, the antibody or antigenbinding fragment has a VH region that contains a CDR-H1, a CDR-H2 and a CDR-H3 present in SEQ ID NO: 12.
  • the antibody or antigen-binding fragment has a VH region that contains a CDR-H1, a CDR-H2 and a CDR-H3 present in SEQ ID NO:13. In some embodiments, the antibody or antigen-binding fragment has a VH region that contains a CDR-H1, a CDR-H2 and a CDR- H3 present in SEQ ID NO: 14. In some embodiments, the antibody or antigen-binding fragment has a VH region that contains a CDR-H1, a CDR-H2 and a CDR-H3 present in SEQ ID NO: 15.
  • the antibody or antigen-binding fragment has a VH region that contains a CDR-H1, a CDR-H2 and a CDR-H3 present in SEQ ID NO: 16. In some embodiments, the antibody or antigenbinding fragment has a VH region that contains a CDR-H1, a CDR-H2 and a CDR-H3 present in SEQ ID NO: 17. In some embodiments, the antibody or antigen-binding fragment has a VH region that contains a CDR-H1, a CDR-H2 and a CDR-H3 present in SEQ ID NO: 18.
  • the antibody or antigen-binding fragment has a VH region that contains a CDR-H1, a CDR-H2 and a CDR- H3 present in SEQ ID NO: 19. In some embodiments, the antibody or antigen-binding fragment has a VH region that contains a CDR-H1, a CDR-H2 and a CDR-H3 present in SEQ ID NO:20. In some embodiments, the antibody or antigen-binding fragment has a VH region that contains a CDR-H1, a CDR-H2 and a CDR-H3 present in SEQ ID NO:21.
  • the antibody or antigenbinding fragment has a VH region that contains a CDR-H1, a CDR-H2 and a CDR-H3 present in SEQ ID NO:22. In some embodiments, the antibody or antigen-binding fragment has a VH region that contains a CDR-H1, a CDR-H2 and a CDR-H3 present in SEQ ID NO:23. In some embodiments, the antibody or antigen-binding fragment has a VH region that contains a CDR-H1, a CDR-H2 and a CDR- H3 present in SEQ ID NO:24.
  • the antibody or antigen-binding fragment has a VH region that contains a CDR-H1, a CDR-H2 and a CDR-H3 present in SEQ ID NO:25. In some embodiments, the antibody or antigen-binding fragment has a VH region that contains a CDR-H1, a CDR-H2 and a CDR-H3 present in SEQ ID NO:26. In some embodiments, the antibody or antigenbinding fragment has a VH region that contains a CDR-H1, a CDR-H2 and a CDR-H3 present in SEQ ID NO:27.
  • the antibody or antigen-binding fragment has a VH region that contains a CDR-H1, a CDR-H2 and a CDR-H3 present in SEQ ID NO:28. In some embodiments, the antibody or antigen-binding fragment has a VH region that contains a CDR-H1, a CDR-H2 and a CDR- H3 present in SEQ ID NO:29. In some embodiments, the antibody or antigen-binding fragment has a VH region that contains a CDR-H1, a CDR-H2 and a CDR-H3 present in SEQ ID NO:30.
  • the antibody or antigen-binding fragment has a VH region that contains a CDR-H1, a CDR-H2 and a CDR-H3 present in SEQ ID NO:31. In some embodiments, the antibody or antigenbinding fragment has a VH region that contains a CDR-H1, a CDR-H2 and a CDR-H3 present in SEQ ID NO:32. In some embodiments, the antibody or antigen-binding fragment has a VH region that contains a CDR-H1, a CDR-H2 and a CDR-H3 present in SEQ ID NO:33.
  • the antibody or antigen-binding fragment has a VH region that contains a CDR-H1, a CDR-H2 and a CDR- H3 present in SEQ ID NO:34. In some embodiments, the antibody or antigen-binding fragment has a VH region that contains a CDR-H1, a CDR-H2 and a CDR-H3 present in SEQ ID NO:35. In some embodiments, the antibody or antigen-binding fragment has a VH region that contains a CDR-H1, a CDR-H2 and a CDR-H3 present in SEQ ID NO:36.
  • the antibody or antigenbinding fragment has a VH region that contains a CDR-H1, a CDR-H2 and a CDR-H3 present in SEQ ID NO:37. In some embodiments, the antibody or antigen-binding fragment has a VH region that contains a CDR-H1, a CDR-H2 and a CDR-H3 present in SEQ ID NO:38. In some embodiments, the antibody or antigen-binding fragment has a VH region that contains a CDR-H1, a CDR-H2 and a CDR- H3 present in SEQ ID NO:39.
  • the antibody or antigen-binding fragment has a VH region that contains a CDR-H1, a CDR-H2 and a CDR-H3 present in SEQ ID NO:40. In some embodiments, the antibody or antigen-binding fragment has a VH region that contains a CDR-H1, a CDR-H2 and a CDR-H3 present in SEQ ID NO:41. In some embodiments, the antibody or antigenbinding fragment has a VH region that contains a CDR-H1, a CDR-H2 and a CDR-H3 present in SEQ ID NO:42.
  • the antibody or antigen-binding fragment has a VH region that contains a CDR-H1, a CDR-H2 and a CDR-H3 present in SEQ ID NO:43. In some embodiments, the antibody or antigen-binding fragment has a VH region that contains a CDR-H1, a CDR-H2 and a CDR- H3 present in SEQ ID NO:44. In some embodiments, the antibody or antigen-binding fragment has a VH region that contains a CDR-H1, a CDR-H2 and a CDR-H3 present in SEQ ID NO:45.
  • the antibody or antigen-binding fragment has a VH region that contains a CDR-H1, a CDR-H2 and a CDR-H3 present in SEQ ID NO: 157. In some embodiments, the antibody or antigenbinding fragment has a VH region that contains a CDR-H1, a CDR-H2 and a CDR-H3 present in SEQ ID NO: 165. In some embodiments, the antibody or antigen-binding fragment has a VH region that contains a CDR-H1, a CDR-H2 and a CDR-H3 present in SEQ ID NO:166.
  • the antibody or antigen-binding fragment has a VH region that contains a CDR-H1, a CDR-H2 and a CDR- H3 present in SEQ ID NO: 167. In some embodiments, the antibody or antigen-binding fragment has a VH region that contains a CDR-H1, a CDR-H2 and a CDR-H3 present in SEQ ID NO:168. In some embodiments, the antibody or antigen-binding fragment has a VH region that contains a CDR-H1, a CDR-H2 and a CDR-H3 present in SEQ ID NO: 169.
  • the antibody or antigenbinding fragment has a VH region that contains a CDR-H1, a CDR-H2 and a CDR-H3 present in SEQ ID NO: 170.
  • the CDR sequence is according to Kabat numbering. In some embodiments, the CDR sequence is according to Chothia numbering. In some embodiments, the CDR sequence is according to AbM numbering. In some embodiments, the CDR sequence is according to IMGT numbering. In some embodiments, the CDR sequence is according to IgBLAST numbering.
  • the antibody or antigen-binding fragment has a VL region that contains a CDR-L1, a CDR-L2 and a CDR-L3 present in SEQ ID NO:46. In some embodiments, the antibody or antigen-binding fragment has a VL region that contains a CDR-L1, a CDR-L2 and a CDR- L3 present in SEQ ID NO: 47. In some embodiments, the antibody or antigen-binding fragment has a VL region that contains a CDR-L1, a CDR-L2 and a CDR-L3 present in SEQ ID NO:48.
  • the antibody or antigen-binding fragment has a VL region that contains a CDR-L1, a CDR-L2 and a CDR-L3 present in SEQ ID NO:49. In some embodiments, the antibody or antigenbinding fragment has a VL region that contains a CDR-L1, a CDR-L2 and a CDR-L3 present in SEQ ID NO:50. In some embodiments, the antibody or antigen-binding fragment has a VL region that contains a CDR-L1, a CDR-L2 and a CDR-L3 present in SEQ ID NO:51.
  • the antibody or antigen-binding fragment has a VL region that contains a CDR-L1, a CDR-L2 and a CDR- L3 present in SEQ ID NO:52. In some embodiments, the antibody or antigen-binding fragment has a VL region that contains a CDR-L1, a CDR-L2 and a CDR-L3 present in SEQ ID NO:53. In some embodiments, the antibody or antigen-binding fragment has a VL region that contains a CDR-L1, a CDR-L2 and a CDR-L3 present in SEQ ID NO:54.
  • the antibody or antigenbinding fragment has a VL region that contains a CDR-L1, a CDR-L2 and a CDR-L3 present in SEQ ID NO:55. In some embodiments, the antibody or antigen-binding fragment has a VL region that contains a CDR-L1, a CDR-L2 and a CDR-L3 present in SEQ ID NO:56. n some embodiments of the antibody or antigen-binding fragment has a VL region that contains a CDR-L1, a CDR-L2 and a CDR- L3 present in SEQ ID NO:57.
  • the antibody or antigen-binding fragment has a VL region that contains a CDR-L1, a CDR-L2 and a CDR-L3 present in SEQ ID NO:58. In some embodiments, the antibody or antigen-binding fragment has a VL region that contains a CDR-L1, a CDR-L2 and a CDR-L3 present in SEQ ID NO:59. In some embodiments, the antibody or antigenbinding fragment has a VL region that contains a CDR-L1, a CDR-L2 and a CDR-L3 present in SEQ ID NO:60.
  • the antibody or antigen-binding fragment has a VL region that contains a CDR-L1, a CDR-L2 and a CDR-L3 present in SEQ ID NO:61. In some embodiments, the antibody or antigen-binding fragment has a VL region that contains a CDR-L1, a CDR-L2 and a CDR- L3 present in SEQ ID NO:62. In some embodiments, the antibody or antigen-binding fragment has a VL region that contains a CDR-L1, a CDR-L2 and a CDR-L3 present in SEQ ID NO:63.
  • the antibody or antigen-binding fragment has a VL region that contains a CDR-L1, a CDR-L2 and a CDR-L3 present in SEQ ID NO:64. In some embodiments, the antibody or antigenbinding fragment has a VL region that contains a CDR-L1, a CDR-L2 and a CDR-L3 present in SEQ ID NO: 65. In some embodiments, the antibody or antigen-binding fragment has a VL region that contains a CDR-L1, a CDR-L2 and a CDR-L3 present in SEQ ID NO:66.
  • the antibody or antigen-binding fragment has a VL region that contains a CDR-L1, a CDR-L2 and a CDR- L3 present in SEQ ID NO: 67. In some embodiments, the antibody or antigen-binding fragment has a VL region that contains a CDR-L1, a CDR-L2 and a CDR-L3 present in SEQ ID NO:68. In some embodiments, the antibody or antigen-binding fragment has a VL region that contains a CDR-L1, a CDR-L2 and a CDR-L3 present in SEQ ID NO:69.
  • the antibody or antigenbinding fragment has a VL region that contains a CDR-L1, a CDR-L2 and a CDR-L3 present in SEQ ID NO:70. In some embodiments, the antibody or antigen-binding fragment has a VL region that contains a CDR-L1, a CDR-L2 and a CDR-L3 present in SEQ ID NO:71. In some embodiments, the antibody or antigen-binding fragment has a VL region that contains a CDR-L1, a CDR-L2 and a CDR- L3 present in SEQ ID NO:72.
  • the antibody or antigen-binding fragment has a VL region that contains a CDR-L1, a CDR-L2 and a CDR-L3 present in SEQ ID NO:73. In some embodiments, the antibody or antigen-binding fragment has a VL region that contains a CDR-L1, a CDR-L2 and a CDR-L3 present in SEQ ID NO:74. In some embodiments, the antibody or antigenbinding fragment has a VL region that contains a CDR-L1, a CDR-L2 and a CDR-L3 present in SEQ ID NO:75.
  • the antibody or antigen-binding fragment has a VL region that contains a CDR-L1, a CDR-L2 and a CDR-L3 present in SEQ ID NO:76. In some embodiments, the antibody or antigen-binding fragment has a VL region that contains a CDR-L1, a CDR-L2 and a CDR- L3 present in SEQ ID NO: 77. In some embodiments, the antibody or antigen-binding fragment has a VL region that contains a CDR-L1, a CDR-L2 and a CDR-L3 present in SEQ ID NO:78.
  • the antibody or antigen-binding fragment has a VL region that contains a CDR-L1, a CDR-L2 and a CDR-L3 present in SEQ ID NO:79. In some embodiments, the antibody or antigenbinding fragment has a VL region that contains a CDR-L1, a CDR-L2 and a CDR-L3 present in SEQ ID NO:80. In some embodiments, the antibody or antigen-binding fragment has a VL region that contains a CDR-L1, a CDR-L2 and a CDR-L3 present in SEQ ID NO:81.
  • the antibody or antigen-binding fragment has a VL region that contains a CDR-L1, a CDR-L2 and a CDR- L3 present in SEQ ID NO: 82. In some embodiments, the antibody or antigen-binding fragment has a VL region that contains a CDR-L1, a CDR-L2 and a CDR-L3 present in SEQ ID NO:83. In some embodiments, the antibody or antigen-binding fragment has a VL region that contains a CDR-L1, a CDR-L2 and a CDR-L3 present in SEQ ID NO: 84.
  • the antibody or antigenbinding fragment has a VL region that contains a CDR-L1, a CDR-L2 and a CDR-L3 present in SEQ ID NO:85. In some embodiments, the antibody or antigen-binding fragment has a VL region that contains a CDR-L1, a CDR-L2 and a CDR-L3 present in SEQ ID NO:86. In some embodiments, the antibody or antigen-binding fragment has a VL region that contains a CDR-L1, a CDR-L2 and a CDR- L3 present in SEQ ID NO: 87.
  • the antibody or antigen-binding fragment has a VL region that contains a CDR-L1, a CDR-L2 and a CDR-L3 present in SEQ ID NO:88. In some embodiments, the antibody or antigen-binding fragment has a VL region that contains a CDR-L1, a CDR-L2 and a CDR-L3 present in SEQ ID NO: 89. In some embodiments, the antibody or antigenbinding fragment has a VL region that contains a CDR-L1, a CDR-L2 and a CDR-L3 present in SEQ ID NO:90.
  • the antibody or antigen-binding fragment has a VL region that contains a CDR-L1, a CDR-L2 and a CDR-L3 present in SEQ ID NO: 158. In some embodiments, the antibody or antigen-binding fragment has a VL region that contains a CDR-L1, a CDR-L2 and a CDR- L3 present in SEQ ID NO: 171. In some embodiments, the antibody or antigen-binding fragment has a VL region that contains a CDR-L1, a CDR-L2 and a CDR-L3 present in SEQ ID NO: 172. In some embodiments, the CDR sequence is according to Kabat numbering.
  • the CDR sequence is according to Chothia numbering. In some embodiments, the CDR sequence is according to AbM numbering. In some embodiments, the CDR sequence is according to IMGT numbering. In some embodiments, the CDR sequence is according to IgBLAST numbering.
  • the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 92, 102, and 113, respectively; SEQ ID NOS: 93, 102, and 113, respectively; SEQ ID NOS: 94, 102, and 113, respectively; SEQ ID NOS: 95, 102, and 113, respectively; SEQ ID NOS: 96, 102, and 113, respectively; SEQ ID NOS: 97, 102, and 113, respectively; SEQ ID NOS: 98, 102, and 113, respectively; SEQ ID NOS: 99, 102, and 113, respectively; SEQ ID NOS: 100, 102, and 113, respectively; SEQ ID NOS: 101, 102, and 113, respectively; SEQ ID NOS: 91, 103, and 113, respectively; SEQ ID NOS: 91, 103, and 113, respectively; SEQ ID NOS: 91
  • the VL region comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS:
  • the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 92, 102, and 113, respectively
  • the VL region comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 124, 135, and 146, respectively.
  • the VH region comprises a CDR- Hl, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 93, 102, and 113, respectively
  • the VL region comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 124, 135, and 146, respectively.
  • the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 94, 102, and 113, respectively
  • the VL region comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 124, 135 and 146, respectively.
  • the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 95, 102, and 113, respectively
  • the VL region comprises a CDR- Ll, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 124, 135, and 146, respectively.
  • the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 96, 102, and 113, respectively
  • the VL region comprises a CDR-L1, a CDR-L2 and a CDR- L3 comprising the sequence set forth in SEQ ID NOS: 124, 135 and 146, respectively.
  • the VH region comprises a CDR- Hl, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 97, 102, and 113, respectively
  • the VL region comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 124, 135 and 146, respectively.
  • the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 98, 102, and 113, respectively
  • the VL region comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 124, 135 and 146, respectively.
  • the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 99, 102, and 113, respectively
  • the VL region comprises a CDR- Ll, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 124, 135 and 146, respectively.
  • the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 100, 102, and 113, respectively
  • the VL region comprises a CDR-L1, a CDR-L2 and a CDR- L3 comprising the sequence set forth in SEQ ID NOS: 124, 135 and 146, respectively.
  • the VH region comprises a CDR- Hl, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 101, 102, and 113, respectively
  • the VL region comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 124, 135 and 146, respectively.
  • the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 91, 103, and 113, respectively
  • the VL region comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 125, 135 and 146, respectively.
  • the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 91, 104, and 113, respectively
  • the VL region comprises a CDR- Ll, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 124, 135 and 146, respectively.
  • the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 91, 105, and 113, respectively
  • the VL region comprises a CDR-L1, a CDR-L2 and a CDR- L3 comprising the sequence set forth in SEQ ID NOS: 124, 135 and 146, respectively.
  • the VH region comprises a CDR- Hl, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 91, 106, and 113, respectively
  • the VL region comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 125, 135 and 146, respectively.
  • the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 91, 107, and 113, respectively
  • the VL region comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 124, 135 and 146, respectively.
  • the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 91, 108, and 113, respectively
  • the VL region comprises a CDR- Ll, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 124, 135 and 146, respectively.
  • the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 91, 109, and 113, respectively
  • the VL region comprises a CDR-L1, a CDR-L2 and a CDR- L3 comprising the sequence set forth in SEQ ID NOS: 124, 135 and 146, respectively.
  • the VH region comprises a CDR- Hl, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 91, 110, and 113, respectively
  • the VL region comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 124, 135 and 146, respectively.
  • the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 91, 111, and 113, respectively
  • the VL region comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 124, 135 and 146, respectively.
  • the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 91, 112, and 113, respectively
  • the VL region comprises a CDR- Ll, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 124, 135, 146, respectively.
  • the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 91, 102, and 114, respectively
  • the VL region comprises a CDR-L1, a CDR-L2 and a CDR- L3 comprising the sequence set forth in SEQ ID NOS: 124, 135 and 146, respectively.
  • the VH region comprises a CDR- Hl, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 91, 102, and 115, respectively
  • the VL region comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 124, 135 and 146, respectively.
  • the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 91, 102, and 116, respectively
  • the VL region comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 124, 135 and 146, respectively.
  • the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 91, 102, and 117, respectively
  • the VL region comprises a CDR- Ll, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 124, 135 and 146, respectively.
  • the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 91, 102, and 118, respectively
  • the VL region comprises a CDR-L1, a CDR-L2 and a CDR- L3 comprising the sequence set forth in SEQ ID NOS: 124, 135 and 146, respectively.
  • the VH region comprises a CDR- Hl, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 91, 102, and 119, respectively
  • the VL region comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 124, 135 and 146, respectively.
  • the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 91, 102, and 120, respectively
  • the VL region comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 124, 135 and 146, respectively.
  • the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 91, 102, and 121, respectively
  • the VL region comprises a CDR- Ll, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 124, 135 and 146, respectively.
  • the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 91, 102, and 122, respectively
  • the VL region comprises a CDR-L1, a CDR-L2 and a CDR- L3 comprising the sequence set forth in SEQ ID NOS: 124, 135 and 146, respectively.
  • the VH region comprises a CDR- Hl, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 91, 102, and 123, respectively
  • the VL region comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 124, 135 and 146, respectively.
  • the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 101, 103, and 114, respectively
  • the VL region comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 124, 135 and 146, respectively.
  • the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 97, 103, and 117, respectively
  • the VL region comprises a CDR- Ll, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 124, 135, and 146, respectively.
  • the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 99, 105, and 115, respectively
  • the VL region comprises a CDR-L1, a CDR-L2 and a CDR- L3 comprising the sequence set forth in SEQ ID NOS: 124, 135 and 146, respectively.
  • the VH region comprises a CDR- Hl, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 96, 104, and 116, respectively
  • the VL region comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 124, 135 and 146, respectively.
  • the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 98, 106, and 114, respectively
  • the VL region comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 124, 135 and 146, respectively.
  • the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 96, 107, and 117, respectively
  • the VL region comprises a CDR- LI, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 124, 135 and 146, respectively.
  • the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 97, 105, and 117, respectively
  • the VL region comprises a CDR-L1, a CDR-L2 and a CDR- L3 comprising the sequence set forth in SEQ ID NOS: 124, 135 and 146, respectively.
  • the VH region comprises a CDR- Hl, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 99, 111, and 115, respectively
  • the VL region comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 124, 135, and 146, respectively.
  • the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 97, 111, and 116, respectively
  • the VL region comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 124, 135 and 146, respectively.
  • the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 100, 105, and 121, respectively
  • the VL region comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 124, 135 and 146, respectively.
  • the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 93, 103, and 115, respectively
  • the VL region comprises a CDR-L1, a CDR-L2 and a CDR- L3 comprising the sequence set forth in SEQ ID NOS: 124, 135 and 146, respectively.
  • the VH region comprises a CDR- Hl, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 100, 108, and 119, respectively
  • the VL region comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 124, 135 and 146, respectively.
  • the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 99, 111, and 116, respectively
  • the VL region comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 124, 135 and 146, respectively.
  • the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 91, 102, and 113, respectively
  • the VL region comprises a CDR- Ll, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 125, 135 and 146, respectively.
  • the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 91, 102, and 113, respectively
  • the VL region comprises a CDR-L1, a CDR-L2 and a CDR- L3 comprising the sequence set forth in SEQ ID NOS: 126, 135 and 146, respectively.
  • the VH region comprises a CDR- Hl, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 91, 102, and 113, respectively
  • the VL region comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 127, 135 and 146, respectively.
  • the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 91, 102, and 113, respectively
  • the VL region comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 128, 135 and 146, respectively.
  • the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 91, 102, and 113, respectively
  • the VL region comprises a CDR- Ll, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 129, 135 and 146, respectively.
  • the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 91, 102, and 113, respectively
  • the VL region comprises a CDR-L1, a CDR-L2 and a CDR- L3 comprising the sequence set forth in SEQ ID NOS: 130, 135 and 146, respectively.
  • the VH region comprises a CDR- Hl, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 91, 102, and 113, respectively
  • the VL region comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 131, 135 and 146, respectively.
  • the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 91, 102, and 113, respectively
  • the VL region comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 132, 135 and 146, respectively.
  • the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 91, 102, and 113, respectively
  • the VL region comprises a CDR- Ll, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 133, 135 and 146, respectively.
  • the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 91, 102, and 113, respectively
  • the VL region comprises a CDR-L1, a CDR-L2 and a CDR- L3 comprising the sequence set forth in SEQ ID NOS: 134, 135 and 146, respectively.
  • the VH region comprises a CDR- Hl, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 91, 102, and 113, respectively
  • the VL region comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 124, 136 and 146, respectively.
  • the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 91, 102, and 113, respectively
  • the VL region comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 124, 137 and 146, respectively.
  • the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 91, 102, and 113, respectively
  • the VL region comprises a CDR- Ll, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 124, 138 and 146, respectively.
  • the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 91, 102, and 113, respectively
  • the VL region comprises a CDR-L1, a CDR-L2 and a CDR- L3 comprising the sequence set forth in SEQ ID NOS: 124, 139 and 146, respectively.
  • the VH region comprises a CDR- Hl, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 91, 102, and 113, respectively
  • the VL region comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 124, 140 and 146, respectively.
  • the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 91, 102, and 113, respectively
  • the VL region comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 124, 141 and 146, respectively.
  • the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 91, 102, and 113, respectively
  • the VL region comprises a CDR- Ll, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 124, 142 and 146, respectively.
  • the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 91, 102, and 113, respectively
  • the VL region comprises a CDR-L1, a CDR-L2 and a CDR- L3 comprising the sequence set forth in SEQ ID NOS: 124, 143 and 146, respectively.
  • the VH region comprises a CDR- Hl, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 91, 102, and 113, respectively
  • the VL region comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 124, 144 and 146, respectively.
  • the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 91, 102, and 113, respectively
  • the VL region comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 124, 145 and 146, respectively.
  • the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 91, 102, and 113, respectively
  • the VL region comprises a CDR- Ll, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 124, 135 and 147, respectively.
  • the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 91, 102, and 113, respectively
  • the VL region comprises a CDR-L1, a CDR-L2 and a CDR- L3 comprising the sequence set forth in SEQ ID NOS: 124, 135 and 148, respectively.
  • the VH region comprises a CDR- Hl, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 91, 102, and 113, respectively
  • the VL region comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 124, 135 and 149, respectively.
  • the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 91, 102, and 113, respectively
  • the VL region comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 124, 135 and 150, respectively.
  • the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 91, 102, and 113, respectively
  • the VL region comprises a CDR- Ll, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 124, 135 and 151, respectively.
  • the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 91, 102, and 113, respectively
  • the VL region comprises a CDR-L1, a CDR-L2 and a CDR- L3 comprising the sequence set forth in SEQ ID NOS: 124, 135 and 152, respectively.
  • the VH region comprises a CDR- Hl, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 91, 102, and 113, respectively
  • the VL region comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 124, 135 and 153, respectively.
  • the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 91, 102, and 113, respectively
  • the VL region comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 124, 135 and 154, respectively.
  • the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 91, 102, and 113, respectively
  • the VL region comprises a CDR- Ll, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 124, 135 and 155, respectively.
  • the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 91, 102, and 113, respectively
  • the VL region comprises a CDR-L1, a CDR-L2 and a CDR- L3 comprising the sequence set forth in SEQ ID NOS: 124, 135 and 156, respectively.
  • the VH region comprises a CDR- Hl, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 91, 102, and 113, respectively
  • the VL region comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 131, 142 and 149, respectively.
  • the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 91, 102, and 113, respectively
  • the VL region comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 131, 139 and 150, respectively.
  • the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 91, 102, and 113, respectively
  • the VL region comprises a CDR- Ll, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 128, 138, and 148, respectively.
  • the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 91, 102, and 113, respectively
  • the VL region comprises a CDR-L1, a CDR-L2 and a CDR- L3 comprising the sequence set forth in SEQ ID NOS: 126, 145 and 153, respectively.
  • the VH region comprises a CDR- Hl, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 91, 102, and 113, respectively
  • the VL region comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 126, 144 and 154, respectively.
  • the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 91, 102, and 113, respectively
  • the VL region comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 125, 140 and 153, respectively.
  • the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 91, 102, and 113, respectively
  • the VL region comprises a CDR- Ll, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 132, 137 and 152, respectively.
  • the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 91, 102, and 113, respectively
  • the VL region comprises a CDR-L1, a CDR-L2 and a CDR- L3 comprising the sequence set forth in SEQ ID NOS: 131, 142 and 152, respectively.
  • the VH region comprises a CDR- Hl, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 91, 102, and 113, respectively
  • the VL region comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 132, 141 and 154, respectively.
  • the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 91, 102, and 113, respectively
  • the VL region comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 133, 144 and 150, respectively.
  • the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 91, 102, and 113, respectively
  • the VL region comprises a CDR- LI, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 134, 138 and 149, respectively.
  • the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 91, 102, and 113, respectively
  • the VL region comprises a CDR-L1, a CDR-L2 and a CDR- L3 comprising the sequence set forth in SEQ ID NOS: 126, 137 and 149, respectively.
  • the VH region comprises a CDR- Hl, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 91, 102, and 113, respectively
  • the VL region comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 126, 137 and 152, respectively.
  • the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 91, 102, and 113, respectively
  • the VL region comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 132, 142 and 154, respectively.
  • the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 91, 102, and 113, respectively
  • the VL region comprises a CDR- Ll, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 127, 140 and 151, respectively.
  • the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 91, 106, and 113, respectively
  • the VL region comprises a CDR-L1, a CDR-L2 and a CDR- L3 comprising the sequence set forth in SEQ ID NOS: 126, 135 and 146, respectively.
  • the VH region comprises a CDR- Hl, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 91, 102, and 114, respectively
  • the VL region comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 126, 135 and 146, respectively.
  • the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 99, 105, and 115, respectively
  • the VL region comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 131, 142 and 149, respectively.
  • the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 99, 105, and 115, respectively
  • the VL region comprises a CDR- Ll, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 126, 145 and 153, respectively.
  • the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 99, 105, and 115, respectively
  • the VL region comprises a CDR-L1, a CDR-L2 and a CDR- L3 comprising the sequence set forth in SEQ ID NOS: 125, 140, and 153, respectively.
  • the VH region comprises a CDR- Hl, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 99, 105, and 115, respectively
  • the VL region comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 132, 141 and 154, respectively.
  • the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 99, 105, and 115, respectively
  • the VL region comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 132, 142 and 154, respectively.
  • the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 98, 106, and 114, respectively
  • the VL region comprises a CDR- Ll, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 131, 142 and 149, respectively.
  • the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 98, 106, and 114, respectively
  • the VL region comprises a CDR-L1, a CDR-L2 and a CDR- L3 comprising the sequence set forth in SEQ ID NOS: 126, 145 and 153, respectively.
  • the VH region comprises a CDR- Hl, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 98, 106, and 114, respectively
  • the VL region comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 125, 140 and 153, respectively.
  • the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 98, 106, and 114, respectively
  • the VL region comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 132, 141 and 154, respectively.
  • the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 98, 106, and 114, respectively
  • the VL region comprises a CDR- Ll, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 132, 142 and 154, respectively.
  • the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 99, 111, and 115, respectively
  • the VL region comprises a CDR-L1, a CDR-L2 and a CDR- L3 comprising the sequence set forth in SEQ ID NOS: 131, 142 and 149, respectively.
  • the VH region comprises a CDR- Hl, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 99, 111, and 115, respectively
  • the VL region comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 126, 145 and 153, respectively.
  • the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 99, 111, and 115, respectively
  • the VL region comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 125, 140 and 153, respectively.
  • the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 99, 111, and 115, respectively
  • the VL region comprises a CDR- Ll, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 132, 141 and 154, respectively.
  • the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 99, 111, and 115, respectively
  • the VL region comprises a CDR-L1, a CDR-L2 and a CDR- L3 comprising the sequence set forth in SEQ ID NOS: 132, 142 and 154, respectively.
  • the VH region comprises a CDR- Hl, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 100, 105 and 121, respectively
  • the VL region comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 131, 142 and 149, respectively.
  • the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 100, 105 and 121, respectively
  • the VL region comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 126, 145 and 153, respectively.
  • the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 100, 105 and 121, respectively
  • the VL region comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 125, 140 and 153, respectively.
  • the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 100, 105 and 121, respectively
  • the VL region comprises a CDR-L1, a CDR-L2 and a CDR- L3 comprising the sequence set forth in SEQ ID NOS: 132, 141 and 154, respectively.
  • the VH region comprises a CDR- Hl, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 100, 105 and 121, respectively
  • the VL region comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 132, 142 and 154, respectively.
  • the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 100, 173 and 121, respectively
  • the VL region comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 126, 145 and 153, respectively.
  • the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 100, 173 and 121, respectively
  • the VL region comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 132, 145 and 153, respectively.
  • the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 100, 173 and 121, respectively
  • the VL region comprises a CDR-L1, a CDR-L2 and a CDR- L3 comprising the sequence set forth in SEQ ID NOS: 126, 143 and 153, respectively.
  • the VH region comprises a CDR- Hl, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 95, 105 and 121, respectively
  • the VL region comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 126, 145 and 153, respectively.
  • the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 95, 105 and 121, respectively
  • the VL region comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 132, 145 and 153, respectively.
  • the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 95, 105 and 121, respectively
  • the VL region comprises a CDR- Ll, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 126, 143 and 153, respectively.
  • the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 95, 173 and 121, respectively
  • the VL region comprises a CDR-L1, a CDR-L2 and a CDR- L3 comprising the sequence set forth in SEQ ID NOS: 126, 145 and 153, respectively.
  • the VH region comprises a CDR- Hl, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 95, 173 and 121, respectively
  • the VL region comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 132, 145 and 153, respectively.
  • the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 95, 173 and 121, respectively
  • the VL region comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 126, 143 and 153, respectively.
  • the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 95, 106 and 114, respectively
  • the VL region comprises a CDR- Ll, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 126, 145 and 153, respectively.
  • the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 95, 106 and 114, respectively
  • the VL region comprises a CDR-L1, a CDR-L2 and a CDR- L3 comprising the sequence set forth in SEQ ID NOS: 132, 145 and 153, respectively.
  • the VH region comprises a CDR- Hl, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 95, 106 and 114, respectively
  • the VL region comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 126, 143 and 153, respectively.
  • the provided antibody is a human antibody or antigen-binding fragment thereof that contains a framework region that contains human germline gene segment sequences.
  • the human antibody contains a VH region in which the framework region, e.g.FRl, FR2, FR3 and FR4, has at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a framework region encoded by a human germline antibody segment, such as a V segment and/or J segment.
  • the human antibody contains a VL region in which the framework region e.g.FRl, FR2, FR3 and FR4, has at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a framework region encoded by a human germline antibody segment, such as a V segment and/or J segment.
  • the framework region sequence contained within the VH region and/or VL region differs by no more than 10 amino acids, such as no more than 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid, compared to the framework region sequence encoded by a human germline antibody segment.
  • the VH region comprises any of the CDR-H1, CDR-H2 and CDR-H3 as described and comprises a framework region 1 (FR1), a FR2, a FR3 and/or a FR4 having at least at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% sequence identity, respectively, to a FR1, a FR2, a FR3 and/or a FR4 contained within the VH region amino acid sequence set forth in any one of SEQ ID NOs: 1-45, 157, and 165-170.
  • the VH region comprises any of the CDR-H1, CDR-H2 and CDR-H3 as described and comprises a framework region 1 (FR1), a FR2, a FR3 and/or a FR4 contained within the VH region amino acid sequence set forth in any one of SEQ ID NOs: 1-45, 157, and 165-170.
  • FR1 framework region 1
  • FR2 FR3
  • FR4 FR4 contained within the VH region amino acid sequence set forth in any one of SEQ ID NOs: 1-45, 157, and 165-170.
  • the VL region comprises any of the CDR-L1, CDR-L2 and CDR-L3 as described and comprises a framework region 1 (FR1), a FR2, a FR3 and/or a FR4 having at least at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% sequence identity, respectively, to a FR1, a FR2, a FR3 and/or a FR4 contained within the VL region amino acid sequence set forth in any one of SEQ ID NOs: 46-90, 158, and 171-172.
  • FR1 framework region 1
  • the VL region comprises any of the CDR-H1, CDR-H2 and CDR-H3 as described and comprises a framework region 1 (FR1), a FR2, a FR3 and/or a FR4 contained within the VL region amino acid sequence set forth in any one of SEQ ID NOs: 46-90, 158, and 171-172.
  • FR1 framework region 1
  • FR2 framework region 2
  • FR3 framework region 1
  • FR4 contained within the VL region amino acid sequence set forth in any one of SEQ ID NOs: 46-90, 158, and 171-172.
  • any of the provided anti-PD-1 antibodies or antigen binding fragments has a VH region having an amino acid sequence that has at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the VH region amino acid sequence set forth in any one of SEQ ID NOs: 1-45, 157, and 165-170, and has a VL region having an amino acid sequence that has at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the VL region amino acid sequence set forth in any one of SEQ ID NOs: 46-90, 158, and 171-172.
  • the antibody or antigen-binding fragment has the VH region amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the antibody or antigen-binding fragment has the VH region amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, the antibody or antigen-binding fragment the VH region amino acid sequence set forth in SEQ ID NO: 3. In some embodiment, the antibody or antigen-binding fragment has the VH region amino acid sequence set forth in SEQ ID NO: 4. In some embodiments, the antibody or antigen-binding fragment has the VH region amino acid sequence set forth in SEQ ID NO: 5. In some embodiment, the antibody or antigen-binding fragment has the VH region amino acid sequence set forth in SEQ ID NO:6.
  • the antibody or antigen-binding fragment has the VH region amino acid sequence set forth in SEQ ID NO: 7. In some embodiments, the antibody or antigen-binding fragment has the VH region amino acid sequence set forth in SEQ ID NO: 8. In some embodiment, the antibody or antigenbinding fragment the VH region amino acid sequence set forth in SEQ ID NO: 9. In some embodiments, the antibody or antigen-binding fragment has the VH region amino acid sequence set forth in SEQ ID NO: 10. In some embodiments, the antibody or antigen-binding fragment has the VH region amino acid sequence set forth in SEQ ID NO: 11. In some embodiments, the antibody or antigen-binding fragment has the VH region amino acid sequence set forth in SEQ ID NO: 12.
  • the antibody or antigen-binding fragment has the VH region amino acid sequence set forth in SEQ ID NO: 13. In some embodiments, the antibody or antigen-binding fragment has the VH region amino acid sequence set forth in SEQ ID NO: 14. In some embodiments, the antibody or antigen-binding fragment has the VH region amino acid sequence set forth in SEQ ID NO: 15. In some embodiments, the antibody or antigen-binding fragment has the VH region amino acid sequence set forth in SEQ ID NO: 16. In some embodiments, the antibody or antigen-binding fragment has the VH region amino acid sequence set forth in SEQ ID NO: 17. In some embodiment, the antibody or antigen-binding fragment has the VH region amino acid sequence set forth in SEQ ID NO: 18.
  • the antibody or antigenbinding fragment has the VH region amino acid sequence set forth in SEQ ID NO: 19. In some embodiments, the antibody or antigen-binding fragment has the VH region amino acid sequence set forth in SEQ ID NO: 20. In some embodiments, the antibody or antigen-binding fragment has the VH region amino acid sequence set forth in SEQ ID NO: 21. In some embodiments, the antibody or antigenbinding fragment has the VH region amino acid sequence set forth in SEQ ID NO: 22. In some embodiments, the antibody or antigen-binding fragment has the VH region amino acid sequence set forth in SEQ ID NO: 23. In some embodiments, the antibody or antigen-binding fragment has the VH region amino acid sequence set forth in SEQ ID NO: 24.
  • the antibody or antigenbinding fragment has the VH region amino acid sequence set forth in SEQ ID NO: 25. In some embodiments, the antibody or antigen-binding fragment has the VH region amino acid sequence set forth in SEQ ID NO: 26. In some embodiments, the antibody or antigen-binding fragment has the VH region amino acid sequence set forth in SEQ ID NO: 27. In some embodiments, the antibody or antigenbinding fragment has the VH region amino acid sequence set forth in SEQ ID NO: 28. In some embodiments, the antibody or antigen-binding fragment has the VH region amino acid sequence set forth in SEQ ID NO: 29. In some embodiments, the antibody or antigen-binding fragment has the VH region amino acid sequence set forth in SEQ ID NO: 30.
  • the antibody or antigenbinding fragment has the VH region amino acid sequence set forth in SEQ ID NO: 31. In some embodiments, the antibody or antigen-binding fragment has the VH region amino acid sequence set forth in SEQ ID NO: 32. In some embodiments, the antibody or antigen-binding fragment has the VH region amino acid sequence set forth in SEQ ID NO: 33. In some embodiments, the antibody or antigenbinding fragment has the VH region amino acid sequence set forth in SEQ ID NO: 34. In some embodiments, the antibody or antigen-binding fragment has the VH region amino acid sequence set forth in SEQ ID NO: 35. In some embodiments, the antibody or antigen-binding fragment has the VH region amino acid sequence set forth in SEQ ID NO: 36.
  • the antibody or antigenbinding fragment has the VH region amino acid sequence set forth in SEQ ID NO: 37. In some embodiments, the antibody or antigen-binding fragment has the VH region amino acid sequence set forth in SEQ ID NO: 38. In some embodiments, the antibody or antigen-binding fragment has the VH region amino acid sequence set forth in SEQ ID NO: 39. In some embodiments, the antibody or antigenbinding fragment has the VH region amino acid sequence set forth in SEQ ID NO: 40. In some embodiments, the antibody or antigen-binding fragment has the VH region amino acid sequence set forth in SEQ ID NO: 41. In some embodiments, the antibody or antigen-binding fragment has the VH region amino acid sequence set forth in SEQ ID NO: 42.
  • the antibody or antigenbinding fragment has the VH region amino acid sequence set forth in SEQ ID NO: 43. In some embodiments, the antibody or antigen-binding fragment has the VH region amino acid sequence set forth in SEQ ID NO: 44. In some embodiments, the antibody or antigen-binding fragment has the VH region amino acid sequence set forth in SEQ ID NO: 45. In some embodiments, the antibody or antigenbinding fragment has the VH region amino acid sequence set forth in SEQ ID NO: 157. In some embodiments, the antibody or antigen-binding fragment has the VH region amino acid sequence set forth in SEQ ID NO: 165.
  • the antibody or antigen-binding fragment has the VH region amino acid sequence set forth in SEQ ID NO: 166. In some embodiments, the antibody or antigen-binding fragment has the VH region amino acid sequence set forth in SEQ ID NO: 167. In some embodiments, the antibody or antigen-binding fragment has the VH region amino acid sequence set forth in SEQ ID NO: 168. In some embodiments, the antibody or antigen-binding fragment has the VH region amino acid sequence set forth in SEQ ID NO: 169. In some embodiments, the antibody or antigen-binding fragment has the VH region amino acid sequence set forth in SEQ ID NO: 170.
  • the antibody or antigen-binding fragment has a VH region having an amino acid sequence that has at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the SEQ ID NO.
  • the antibody or antigen-binding fragment has the VL region amino acid sequence set forth in SEQ ID NO: 46. In some embodiments, the antibody or antigen-binding fragment has the VL region amino acid sequence set forth in SEQ ID NO: 47. In some embodiments, the antibody or antigen-binding fragment has the VL region amino acid sequence set forth in SEQ ID NO: 48. In some embodiments, the antibody or antigen-binding fragment has the VL region amino acid sequence set forth in SEQ ID NO: 49. In some embodiments, the antibody or antigen-binding fragment has the VL region amino acid sequence set forth in SEQ ID NO: 50.
  • the antibody or antigen-binding fragment has the VL region amino acid sequence set forth in SEQ ID NO:51. In some embodiments, the antibody or antigen-binding fragment has the VL region amino acid sequence set forth in SEQ ID NO: 52. In some embodiment, the antibody or antigen-binding fragment has the VL region amino acid sequence set forth in SEQ ID NO: 53. In some embodiment, the antibody or antigen-binding fragment has the VL region amino acid sequence set forth in SEQ ID NO: 54. In some embodiments, the antibody or antigen-binding fragment has the VL region amino acid sequence set forth in SEQ ID NO: 55. In some embodiments, the antibody or antigen-binding fragment has the VL region amino acid sequence set forth in SEQ ID NO: 56.
  • the antibody or antigenbinding fragment has the VL region amino acid sequence set forth in SEQ ID NO: 57. In some embodiments, the antibody or antigen-binding fragment has the VL region amino acid sequence set forth in SEQ ID NO: 58. In some embodiments, the antibody or antigen-binding fragment has the VL region amino acid sequence set forth in SEQ ID NO: 59. In some embodiments, the antibody or antigenbinding fragment has the VL region amino acid sequence set forth in SEQ ID NO: 60. In some embodiments, the antibody or antigen-binding fragment has the VL region amino acid sequence set forth in SEQ ID NO: 61.
  • the antibody or antigen-binding fragment has the VL region amino acid sequence set forth in SEQ ID NO: 62. In some embodiment, the antibody or antigen-binding fragment has the VL region amino acid sequence set forth in SEQ ID NO: 63. In some embodiments, the antibody or antigen-binding fragment has the VL region amino acid sequence set forth in SEQ ID NO: 64. In some embodiments, the antibody or antigen-binding fragment has the VL region amino acid sequence set forth in SEQ ID NO: 65. In some embodiments, the antibody or antigen-binding fragment has the VL region amino acid sequence set forth in SEQ ID NO: 66.
  • the antibody or antigen-binding fragment has the VL region amino acid sequence set forth in SEQ ID NO: 67. In some embodiments, the antibody or antigen-binding fragment has the VL region amino acid sequence set forth in SEQ ID NO: 68. In some embodiments, the antibody or antigen-binding fragment has the VL region amino acid sequence set forth in SEQ ID NO: 69. In some embodiments, the antibody or antigen-binding fragment has the VL region amino acid sequence set forth in SEQ ID NO: 70. In some embodiments, the antibody or antigen-binding fragment has the VL region amino acid sequence set forth in SEQ ID NO: 71.
  • the antibody or antigen-binding fragment has the VL region amino acid sequence set forth in SEQ ID NO: 72. In some embodiments, the antibody or antigenbinding fragment has the VL region amino acid sequence set forth in SEQ ID NO: 73. In some embodiments, the antibody or antigen-binding fragment has the VL region amino acid sequence set forth in SEQ ID NO: 74. In some embodiments, the antibody or antigen-binding fragment has the VL region amino acid sequence set forth in SEQ ID NO: 75. In some embodiments, the antibody or antigenbinding fragment has the VL region amino acid sequence set forth in SEQ ID NO: 76.
  • the antibody or antigen-binding fragment has the VL region amino acid sequence set forth in SEQ ID NO: 77. In some embodiments, the antibody or antigen-binding fragment has the VL region amino acid sequence set forth in SEQ ID NO: 78. In some embodiments, the antibody or antigenbinding fragment has the VL region amino acid sequence set forth in SEQ ID NO: 79. In some embodiments, the antibody or antigen-binding fragment has the VL region amino acid sequence set forth in SEQ ID NO: 80. In some embodiments, the antibody or antigen-binding fragment has the VL region amino acid sequence set forth in SEQ ID NO: 81.
  • the antibody or antigenbinding fragment has the VL region amino acid sequence set forth in SEQ ID NO: 82. In some embodiments, the antibody or antigen-binding fragment has the VL region amino acid sequence set forth in SEQ ID NO: 83. In some embodiments, the antibody or antigen-binding fragment has the VL region amino acid sequence set forth in SEQ ID NO: 84. In some embodiments, the antibody or antigenbinding fragment has a VL region having an amino acid sequence that has the VL region amino acid sequence set forth in SEQ ID NO: 85. In some embodiments, the antibody or antigen-binding fragment has the VL region amino acid sequence set forth in SEQ ID NO: 86.
  • the antibody or antigen-binding fragment has the VL region amino acid sequence set forth in SEQ ID NO: 87. In some embodiments, the antibody or antigen-binding fragment has the VL region amino acid sequence set forth in SEQ ID NO: 88. In some embodiments, the antibody or antigen-binding fragment has the VL region amino acid sequence set forth in SEQ ID NO: 89. In some embodiment, the antibody or antigen-binding fragment has the VL region amino acid sequence set forth in SEQ ID NO: 90. In some embodiments, the antibody or antigen-binding fragment has the VL region amino acid sequence set forth in SEQ ID NO: 158.
  • the antibody or antigen-binding fragment has the VL region amino acid sequence set forth in SEQ ID NO: 171. In some embodiments, the antibody or antigenbinding fragment has the VL region amino acid sequence set forth in SEQ ID NO: 172. In some of any of the above embodiments, the antibody or antigen-binding fragment has a VL region having an amino acid sequence that has at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the SEQ ID NO.
  • the antibody or antigen-binding fragment has a VH region having an amino acid sequence that is less than 100% identical to SEQ ID NO: 157 and has at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the VH region amino acid sequence set forth in SEQ ID NO: 157.
  • the antibody or antigen-binding fragment has a VL region having an amino acid sequence that is less than 100% identical to SEQ ID NO: 158 and has at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the VL region amino acid sequence set forth in SEQ ID NO: 158.
  • the VH region does not have the amino acid sequence set forth in SEQ ID NO: 157.
  • the VL region does not have the amino acid sequence set forth in SEQ ID NO: 158.
  • the VH region and the VL region of the antibody are not set forth SEQ ID NO: 157 and 158, respectively.
  • the heavy chain of the constant region does not have the amino acid sequence set forth in SEQ ID NO: 178.
  • the light chain of the constant region does not have the amino acid sequence set forth in SEQ ID NO: 179.
  • the heavy chain and the light chain of the antibody are not set forth SEQ ID NO: 178 and 179, respectively.
  • the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 1 and SEQ ID NO: 158, respectively.
  • the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 2 and SEQ ID NO: 158, respectively.
  • the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 3 and SEQ ID NO: 158, respectively.
  • the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 4 and SEQ ID NO: 158, respectively.
  • the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 5 and SEQ ID NO: 158, respectively.
  • the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 6 and SEQ ID NO: 158, respectively.
  • the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 7 and SEQ ID NO: 158, respectively.
  • the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 8 and SEQ ID NO: 158, respectively.
  • the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 9 and SEQ ID NO: 158, respectively.
  • the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 10 and SEQ ID NO: 158, respectively.
  • the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 11 and SEQ ID NO: 158, respectively.
  • the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 12 and SEQ ID NO: 158, respectively.
  • the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 13 and SEQ ID NO: 158, respectively.
  • the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 14 and SEQ ID NO: 158, respectively.
  • the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO and SEQ ID NO: 158, respectively.
  • the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 16 and SEQ ID NO: 158, respectively.
  • the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 17 and SEQ ID NO: 158, respectively.
  • the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 18 and SEQ ID NO: 158, respectively.
  • the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 19 and SEQ ID NO: 158, respectively.
  • the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 20 and SEQ ID NO: 158, respectively.
  • the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 21 and SEQ ID NO: 158, respectively.
  • the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 22 and SEQ ID NO: 158, respectively.
  • the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 23 and SEQ ID NO: 158, respectively.
  • the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 24 and SEQ ID NO: 158, respectively.
  • the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 25 and SEQ ID NO: 158, respectively.
  • the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 26 and SEQ ID NO: 158, respectively.
  • the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 27 and SEQ ID NO: 158, respectively.
  • the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 28 and SEQ ID NO: 158, respectively.
  • the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 29 and SEQ ID NO: 158, respectively.
  • the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 30 and SEQ ID NO: 158, respectively.
  • the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 31 and SEQ ID NO: 158, respectively.
  • the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 32 and SEQ ID NO: 158, respectively.
  • the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 33 and SEQ ID NO: 158, respectively.
  • the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 34 and SEQ ID NO: 158, respectively.
  • the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 35 and SEQ ID NO: 158, respectively.
  • the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 36 and SEQ ID NO: 158, respectively.
  • the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 37 and SEQ ID NO: 158, respectively.
  • the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 38 and SEQ ID NO: 158, respectively.
  • the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 39 and SEQ ID NO: 158, respectively.
  • the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 40 and SEQ ID NO: 158, respectively.
  • the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 41 or ID NO: 158, respectively.
  • the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 42 and SEQ ID NO: 158, respectively.
  • the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 43 and SEQ ID NO: 158, respectively.
  • the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 44 and SEQ ID NO: 158, respectively.
  • the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 45 and SEQ ID NO: 158, respectively.
  • the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 46, respectively.
  • the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 47, respectively.
  • the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 48, respectively.
  • the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 49, respectively.
  • the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 50, respectively.
  • the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 51, respectively.
  • the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 52, respectively.
  • the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 53, respectively.
  • the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 54, respectively.
  • the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 55, respectively.
  • the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 56, respectively.
  • the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 57, respectively.
  • the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 58, respectively.
  • the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 59, respectively.
  • the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 60, respectively.
  • the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 61, respectively.
  • the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 62, respectively.
  • the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 63, respectively.
  • the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 64, respectively.
  • the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 65, respectively.
  • the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 66, respectively.
  • the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 or ID NO: 67, respectively.
  • the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 68, respectively.
  • the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 69, respectively.
  • the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 70, respectively.
  • the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 71, respectively.
  • the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 72, respectively.
  • the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 73, respectively.
  • the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 74, respectively.
  • the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 75, respectively.
  • the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 76, respectively.
  • the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 77, respectively.
  • the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 78, respectively.
  • the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 79, respectively.
  • the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 80, respectively.
  • the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 81, respectively.
  • the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 82, respectively.
  • the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 83, respectively.
  • the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 84, respectively.
  • the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 85, respectively.
  • the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 86, respectively.
  • the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 87, respectively.
  • the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 88, respectively.
  • the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 81%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 89, respectively.
  • the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 90, respectively.
  • the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 16 and SEQ ID NO: 47, respectively.
  • the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 23 and SEQ ID NO: 47, respectively.
  • the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 35 and SEQ ID NO: 76, respectively.
  • the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 35 and SEQ ID NO: 79, respectively.
  • the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 35 and SEQ ID NO: 81, respectively.
  • the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 35 and SEQ ID NO: 84, respectively.
  • the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 35 and SEQ ID NO: 89, respectively.
  • the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 37 and SEQ ID NO: 76, respectively.
  • the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 37 and SEQ ID NO: 79, respectively.
  • the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 37 and SEQ ID NO: 81, respectively.
  • the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 37 and SEQ ID NO: 84, respectively.
  • the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 37 and SEQ ID NO: 89, respectively.
  • the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 40 and SEQ ID NO: 76, respectively.
  • the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 40 and SEQ ID NO: 79, respectively.
  • the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 40 and SEQ ID NO: 81, respectively.
  • the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 40 and SEQ ID NO: 84, respectively.
  • the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 40 and SEQ ID NO: 89, respectively.
  • the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 42 and SEQ ID NO: 76, respectively.
  • the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 42 and SEQ ID NO: 79, respectively.
  • the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 42 and SEQ ID NO: 81, respectively.
  • the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 42 and SEQ ID NO: 84, respectively.
  • the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 42 and SEQ ID NO: 89, respectively.
  • the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 165 and SEQ ID NO: 79, respectively.
  • the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 165 and SEQ ID NO: 171, respectively.
  • the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 165 and SEQ ID NO: 172, respectively.
  • the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 166 and SEQ ID NO: 79, respectively.
  • the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 166 and SEQ ID NO: 171, respectively.
  • the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 166 and SEQ ID NO: 172, respectively.
  • the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 167 and SEQ ID NO: 79, respectively.
  • the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 167 and SEQ ID NO: 171, respectively.
  • the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 167 and SEQ ID NO: 172, respectively.
  • the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 168 and SEQ ID NO: 79, respectively.
  • the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 168 and SEQ ID NO: 171, respectively.
  • the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 168 and SEQ ID NO: 172, respectively.
  • the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 169 and SEQ ID NO: 79, respectively.
  • the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 169 and SEQ ID NO: 171, respectively.
  • the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 169 and SEQ ID NO: 172, respectively.
  • the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 170 and SEQ ID NO: 79, respectively.
  • the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 170 and SEQ ID NO: 171, respectively.
  • the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 170 and SEQ ID NO: 172, respectively.
  • the VH region of the antibody or antigen-binding fragment thereof comprises the amino acid sequence of any one of SEQ ID NOs: 1-45, 157, and 165-170
  • the VL region of the antibody or antigen-binding fragment comprises the amino acid sequence of any one of SEQ ID NOs: 46-90, 158, and 171-172.
  • the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 1 and 158, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 2 and 158, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 3 and 158, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 4 and 158, respectively.
  • the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 5 and 158, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 6 and 158, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 7 and 158, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 8 and 158, respectively.
  • the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 9 and 158, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 10 and 158, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 11 and 158, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 12 and 158, respectively.
  • the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 13 and 158, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 14 and 158, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 15 and 158, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 16 and 158, respectively.
  • the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 17 and 158, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 18 and 158, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 19 and 158, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 20 and 158, respectively.
  • the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 21 and 158, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 22 and 158, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 23 and 158, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 24 and 158, respectively.
  • the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 25 and 158, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 26 and 158, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 27 and 158, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 28 and 158, respectively.
  • the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 29 and 158, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 30 and 158, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 31 and 158, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 32 and 158, respectively.
  • the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 33 and 158, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 34 and 158, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 35 and 158, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 36 and 158, respectively.
  • the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 37 and 158, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 38 and 158, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 39 and 158, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 40 and 158, respectively.
  • the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 41 and 158, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 42 and 158, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 43 and 158, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 44 and 158, respectively.
  • the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 45 and 158, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 157 and 46, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 157 and 47, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 157 and 48, respectively.
  • the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 157 and 49, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 157 and 50, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 157 and 51, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 157 and 52, respectively.
  • the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 157 and 53, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 157 and 54, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 157 and 55, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 157 and 56, respectively.
  • the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 157 and 57, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 157 and 58, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 157 and 59, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 157 and 60, respectively.
  • the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 157 and 61, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 157 and 62, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 157 and 63, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 157 and 64, respectively.
  • the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 157 and 65, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 157 and 66, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 157 and 67, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 157 and 68, respectively.
  • the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 157 and 69, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 157 and 70, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 157 and 71, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 157 and 72, respectively.
  • the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 157 and 73, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 157 and 74, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 157 and 75, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 157 and 76, respectively.
  • the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 157 and 77, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 157 and 78, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 157 and 79, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 157 and 80, respectively.
  • the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 157 and 81, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 157 and 82, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 157 and 83, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 157 and 84, respectively.
  • the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 157 and 85, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 157 and 86, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 157 and 87, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 157 and 88, respectively.
  • the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 157 and 89, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 157 and 90, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 16 and 47, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 23 and 47, respectively.
  • the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 35 and 76, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 35 and 79, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 35 and 81, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 35 and 84, respectively.
  • the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 35 and 89, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 37 and 76, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 37 and 79, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 37 and 81, respectively.
  • the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 37 and 84, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 37 and 89, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 40 and 76, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 40 and 79, respectively.
  • the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 40 and 81, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 40 and 84, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 40 and 89, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 42 and 76, respectively.
  • the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 42 and 79, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 42 and 81, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 42 and 84, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 42 and 89, respectively.
  • the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 165 and 79, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 165 and 171, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 165 and 172, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 166 and 79, respectively.
  • the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 166 and 171, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 166 and 172, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 167 and 79, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 167 and 171, respectively.
  • the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 167 and 172, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 168 and 79, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 168 and 171, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 168 and 172, respectively.
  • the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 169 and 79, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 169 and 171, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 169 and 172, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 170 and 79, respectively.
  • the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 170 and 171, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 170 and 172, respectively.
  • Table El provides the SEQ ID NOS: of exemplary provided antibody or antigen-binding fragment thereof.
  • the PD-1 -binding antibody or fragment thereof comprises a VH region that comprises the CDR-H1, the CDR-H2 and the CDR-H3 sequence and a VL region that comprises the CDR-L1, the CDR-L2 and the CDR-L3 sequence set forth in the SEQ ID NOS: listed in each row of Table El below (by Kabat numbering scheme).
  • the PD-l-binding antibody or fragment thereof comprises a VH region sequence and a VL region sequence set forth in the SEQ ID NOS: listed in each row of Table El below, or an antibody comprising a VH region and a VL region amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the VH region sequence and the VL region sequence set forth in the SEQ ID NOS: listed in each row of Table El below.
  • the PD-l-binding antibody or fragment thereof comprises a VH region sequence and a VL region sequence set forth in the SEQ ID NOS: listed in each row of Table El below.
  • the provided antibody or antigen-binding fragment thereof comprises a VH region and a VL region, wherein the VH region of the antibody or antigen-binding fragment thereof can contain a combination of any of the CDR-H1, the CDR-H2 and the CDR-H3 amino acid sequences set forth in Table El, and the VL region of the antibody or antigen-binding fragment thereof can contain a combination of any of the CDR-L1, the CDR-L2 and the CDR-L3 amino acid sequences set forth in Table El.
  • the provided antibody or antigen-binding fragment thereof comprises a VH region and/or a VL region set forth in Table El, in any combination, orientation or containing a different linker.
  • the antibody or antigen-binding fragment thereof comprises a VH region described in Table El.
  • the antibody or antigen-binding fragment thereof comprises a VL region described in Table El.
  • antibody variable domains as described are fused to immunoglobulin constant domain sequences.
  • the fusion is with an Ig heavy chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions.
  • the antibody further comprises a heavy chain constant domain and/or a light chain constant domain.
  • the heavy chain and/or light constant domain is murine or human.
  • a “constant region” or “constant domain” is a domain in an antibody heavy or light chain that contains a sequence of amino acids that is comparatively more conserved among antibodies than the variable region domain.
  • the constant regions of immunoglobulins show less sequence diversity than the variable regions, and are responsible for binding a number of natural proteins to elicit important biochemical events.
  • Each light chain has a single light chain constant region (CL) domain, which is either of the kappa or lambda type.
  • Each heavy chain contains one or more heavy chain constant region (CH) domains that can be assigned to different classes or isotypes.
  • IgA, IgD, IgE, IgG and IgM having heavy chains designated a, 8, e, y and p. respectively.
  • Full-length IgA, IgD and IgG isotypes contain CHI, CH2, CH3 and a hinge region, while IgE and IgM contain CHI , CH2, CH3 and CH4.
  • the y and a classes are further divided into subclasses on the basis of relatively minor differences in the CH sequence and function, e.g., humans express the following subclasses: IgGl, IgG2, IgG3, IgG4, IgAl and IgA2.
  • IgGl antibodies can exist in multiple polymorphic variants termed allotypes (reviewed in Jefferis and Lefranc 2009. mAbs Vol 1 Issue 4 1- 7) any of which are suitable. In humans there are five different classes of antibodies including IgA (which includes subclasses IgAl and IgA2), IgD, IgE, IgG (which includes subclasses IgGl, IgG2, IgG3, and IgG4), and IgM. The distinguishing features between these antibody classes are their constant, Fc regions, although subtler differences may exist in the V region. An antibody constant region can include an Fc portion.
  • the human IgG heavy-chain Fc region usually includes the region containing the CH2 and CH3 domains and the hinge region, such as an amino acid residue at position Cys226, or from Pro230, to the carboxyl-terminus thereof.
  • a provided antibody contains an Ig heavy chain constant domain comprising at least part of the hinge, CH2, and CH3 regions. Certain embodiments have the first heavychain constant region (CHI) containing the site necessary for light chain bonding, present in at least one of the fusions. DNAs encoding the immunoglobulin heavy chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are co-transfected into a suitable host cell. This provides for greater flexibility in adjusting the mutual proportions of the three polypeptide fragments in embodiments when unequal ratios of the three polypeptide chains used in the construction provide the optimum yield of the desired fusion antibody.
  • CHI first heavychain constant region
  • the antibody or antigen-binding fragment thereof may contain at least a portion of an immunoglobulin constant region, such as one or more constant region domain.
  • the constant regions include a light chain constant region and/or a heavy chain constant region 1 (CHI).
  • the antibody includes at least a portion of a hinge region or a variant thereof.
  • the antibody includes a CH2 and/or CH3 domain, such as an Fc region.
  • the Fc region is an Fc region from an IgAl, IgA2, IgD, IgE, IgGl, IgG2, IgG3, IgG4, or IgM.
  • the heavy chain constant sequence includes an IgG4 containing the S228P mutation, which has been shown to prevent recombination between a therapeutic antibody and an endogenous IgG4 by Fab-arm exchange (see e.g. Labrijin et al. (2009) Nat. Biotechnol., 27(8): 767- 71.)
  • the heavy chain constant domain comprises the sequence set forth in SEQ ID NO: 174.
  • the light chain constant domain comprises the sequence set forth in SEQ ID NO: 175.
  • the heavy chain further comprises a signal peptide.
  • the signal peptide can be cleaved upon expression from a cell.
  • the signal peptide is set forth in SEQ ID NO: 176.
  • the light chain constant region is a kappa light chain.
  • the kappa light chain comprises the sequence set forth in SEQ ID NO: 175.
  • the light chain further comprises a signal peptide.
  • the signal peptide can be cleaved upon expression from a cell.
  • the signal peptide is set forth in SEQ ID NO: 177.
  • the antibody or antigen-binding fragment thereof is a single-chain antibody fragment, such as a single chain variable fragment (scFv).
  • the antibody or antigen binding fragment is a multi-domain antibody, such as an scFv comprising a heavy chain variable (VH) region and a light chain variable (VL) region.
  • the single-chain antibody fragment e.g., scFv
  • the linker typically is a peptide linker, e.g., a flexible and/or soluble peptide linker.
  • the linkers are those rich in glycine and serine and/or in some cases threonine.
  • the linkers further include charged residues such as lysine and/or glutamate, which can improve solubility.
  • the linkers further include one or more proline.
  • the linkers rich in glycine and serine (and/or threonine) include at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% such amino acid(s). In some embodiments, they include at least at or about 50%, 55%, 60%, 70%, or 75%, glycine, serine, and/or threonine. In some embodiments, the linker is comprised substantially entirely of glycine, serine, and/or threonine.
  • the linkers generally are between about 5 and about 50 amino acids in length, typically between at or about 10 and at or about 30, e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30, and in some examples between 10 and 25 amino acids in length.
  • Exemplary linkers include linkers having various numbers of repeats of the sequence GGGGS (4GS; SEQ ID NO: 159) or GGGS (3GS; SEQ ID NO: 160), such as between 2, 3, 4 and 5 repeats of such a sequence.
  • Exemplary linkers include those having or consisting of an sequence set forth in SEQ ID NO: 161 (GGGGSGGGGSGGGGS).
  • Exemplary linkers further include those having or consisting of the sequence set forth in SEQ ID NO: 162 (GSTSGSGKPGSGEGSTKG). Exemplary linkers further include those having or consisting of the sequence set forth in SEQ ID NO: 163 (SRGGGGSGGGGSGGGGSLEMA). An exemplary linker includes those having or consisting of the sequence set forth in SEQ ID NO; 164 (GSRGGGGSGGGGSGGGGSLEMA).
  • the provided antibodies have one or more specified functional features, such as binding properties, including binding to particular epitopes or exhibiting lower or reduced binding to a related but non-specific antigen.
  • the provided antibodies can bind to an epitope that is similar to or overlaps with epitopes of other antibodies, such as reference antibodies, and/or exhibit particular binding affinities.
  • the provided antibodies can bind to an epitope that is different from epitopes of other antibodies, e.g., binding a conformational epitope.
  • the provided antibodies or antigen-binding fragment thereof specifically bind to a programmed cell death protein 1 (PD-1) protein.
  • PD-1 refers to human PD-1.
  • the provided antibodies and antibody fragments specifically bind to human PD-1.
  • a molecule is said to exhibit "specific binding” or “preferential binding” if it reacts or associates more frequently, more rapidly, with greater duration and/or with greater affinity with a particular cell or substance (e.g. a cell expressing a PD-1 antigen or a recombinant PD-1 antigen) than it does with alternative cells or substances.
  • An antibody specifically binds or preferentially binds to a target (i.e.
  • PD-1) if it binds with greater affinity, avidity, more readily, and/or with greater duration than it binds to other substances.
  • an antibody that specifically or preferentially binds to PD-1 is an antibody that binds PD-1 with greater affinity, avidity, more readily, and/or with greater duration than it binds to a different protein antigen. It is also understood by reading this definition that specific binding or preferential binding does not necessarily require (although it can include) exclusive binding. Methods to determine such specific or preferential binding are also well known in the art, e.g., an immunoassay.
  • an antibody or other binding molecule binds to PD-1 or specifically binds to PD-1 does not necessarily mean that it binds to PD-1 from every species.
  • features of binding to PD-1 such as the ability to specifically bind thereto and/or to compete for binding thereto with a reference antibody, and/or to bind with a particular affinity or compete to a particular degree, in some embodiments, refers to the ability with respect to a human PD- 1 protein and the antibody may not have this feature with respect to a PD-1 of another species such as mouse.
  • the provided antibodies are capable of binding PD-1, such as human PD-1, with at least a certain affinity, as measured by any of a number of known methods.
  • the affinity is represented by an equilibrium dissociation constant (KD).
  • a variety of assays are known for assessing binding affinity, equilibrium dissociation constant (KD), equilibrium association constant (KA), ECSO, on-rate (association rate constant; k on or k a ; units of 1/Ms or M ’s 1 ) and the off-rate (dissociation rate constant; k o ff or k ; units of 1/s or s ’) and/or determining whether a binding molecule (e.g., an antibody or fragment thereof) specifically binds to a particular ligand e.g., an antigen, such as a PD-1 protein).
  • KD equilibrium dissociation constant
  • KA equilibrium association constant
  • ECSO on-rate (association rate constant; k on or k a ; units of 1/Ms or M ’s 1 ) and the off-rate (dissociation rate constant; k o ff or k ; units of 1/s or s ’) and/or determining whether a binding molecule
  • a BIAcore® instrument can be used to determine the binding kinetics and constants of a complex between two proteins (e.g. , an antibody or fragment thereof, and an antigen, such as a PD-1 protein), using surface plasmon resonance (SPR) analysis (see, e.g., Scatchard et al., Ann. N.Y. Acad. Sci. 51:660, 1949; Wilson, Science 295:2103, 2002; Wolff etal., Cancer Res. 53:2560, 1993; and U.S. Patent Nos. 5,283,173, 5,468,614, or the equivalent).
  • SPR surface plasmon resonance
  • SPR measures changes in the concentration of molecules at a sensor surface as molecules bind to or dissociate from the surface.
  • the change in the SPR signal is directly proportional to the change in mass concentration close to the surface, thereby allowing measurement of binding kinetics between two molecules.
  • the dissociation rate constant (k o ff or kd), the association rate constant (k on or k a ) and/or equilibrium dissociation constant (KD) and/or equilibrium association constant (KA) for the complex can be determined by monitoring changes in the refractive index with respect to time as buffer is passed over the chip.
  • suitable assays for measuring the binding of one protein to another include, for example, immunoassays such as enzyme linked immunosorbent assays (ELISA) and radioimmunoassays (RIA), or determination of binding by monitoring the change in the spectroscopic or optical properties of the proteins through fluorescence, UV absorption, circular dichroism, or nuclear magnetic resonance (NMR).
  • immunoassays such as enzyme linked immunosorbent assays (ELISA) and radioimmunoassays (RIA), or determination of binding by monitoring the change in the spectroscopic or optical properties of the proteins through fluorescence, UV absorption, circular dichroism, or nuclear magnetic resonance (NMR).
  • exemplary assays include, but are not limited to, Western blot, ELISA, analytical ultracentrifugation, spectroscopy, flow cytometry, sequencing, genetic reporter assays, flow cytometry, and other methods for detection of expressed nucleic acids or binding of proteins.
  • binding can be determined under conditions that mimic those that exist in natural environments in the body of a subject. For instance, acid pH of about 5.6 to 6.8 such as pH about 6.0 mimics more closely the acidic tumor microenvironment. In contrast, physiological pH is a more neutral pH 7.35 to 7.45, such as pH about 7.4 and more closely mimics the environment of normal tissues or cells in the blood.
  • the antibody or fragment thereof exhibits a binding affinity to PD- 1 at pH 7.35 to 7.45 (e.g. pH about 7.4), such as present in normal pH of blood or a tissue environment, with a KD of equal to or less than 2 x 10 8 M.
  • the equilibrium dissociation constant KD at pH 7.35 to 7.45 e.g. pH about 7.4
  • a provided antibody or antibody fragment an off-rate (dissociation rate constant; k o ff or kdj) at pH 7.35 to 7.45 (e.g.
  • a provided antibody or antibody fragment has an off-rate (dissociation rate constant; k o ff or kdj) at pH 7.35 to 7.45 (e.g. pH about 7.4), such as present in normal pH of blood or a tissue environment, of 4 x 10 5 1/Ms to about 2 x 10 6 1/Ms.
  • the antibody or fragment thereof exhibits a binding affinity to PD- 1 at an acidic pH, such as at pH 5.0 to 6.8 (e.g. pH about 6.0 or pH about 5.0) with a KD of equal to or less than 4 x 10 8 M.
  • the pH is a pH for example as present in a microenvironment of tumor.
  • the equilibrium dissociation constant KD at an acidic microenvironment pH 5.0 to 6.8 e.g. pH about 6.0 or about pH 5.0
  • the antibody or fragment thereof exhibits a binding affinity to PD-1 at an acidic pH, such as at pH 5.6 to 6.8 (e.g. pH about 6.0), for example as present in a microenvironment of tumor, with a KD of equal to or less than 4 x 10 8 M.
  • the equilibrium dissociation constant KD at an acidic microenvironment pH 5.6 to 6.8 e.g. pH about 6.0
  • the antibody or fragment thereof exhibits a binding affinity to PD-1 at an acidic pH, such as at pH 5.0 to 6.0 (e.g.
  • pH about 5.0 for example as present in a microenvironment of tumor, with a KD of equal to or less than 8 x 10 9 M.
  • the equilibrium dissociation constant KD at an acidic microenvironment pH 5.0 to 6.0 can range from about 3 x 10 10 M to about 8 x 10 9 M, such as about 6 x 10 10 to about 8 x 10 9 M.
  • the antibody or fragment thereof has an off-rate at an acidic pH, such as at pH 5.0 to 6.8 (e.g. pH about 6.0 or about 5.0), for example as present in a microenvironment of tumor, of 4 x 10 4 1/s to about 2 x 10 2 1/s.
  • the antibody or fragment thereof has an off-rate at an acidic pH, such as at pH 5.6 to 6.8 (e.g. pH about 6.0), for example as present in a microenvironment of tumor, of 6 x 10 4 1/s to about 2 x 10 2 1/s.
  • the antibody or fragment thereof has an off-rate at an acidic pH, such as at pH 5.0 to 6.0 (e.g. pH about 5.0), for example as present in a microenvironment of tumor, of 4 x 10 4 1/s to about 2 x 10 2 1/s.
  • the antibody or fragment thereof has an on-rate at an acidic pH, such as at pH 5.0 to 6.8 (e.g. pH about 6.0 or about 5.0), for example as present in a microenvironment of tumor, of 4 x 10 5 1/Ms to about 5 x 10 6 1/Ms.
  • the antibody or fragment thereof has an on-rate at an acidic pH, such as at pH 5.6 to 6.8 (e.g. pH about 6.0), for example as present in a microenvironment of tumor, of 4 x 10 5 1/Ms to about 3 x 10 6 1/Ms.
  • the antibody or fragment thereof has an on-rate at an acidic pH, such as at pH 5.0 to 6.0 (e.g. pH about 5.0), for example as present in a microenvironment of tumor, of 4 x 10 5 1/Ms to about 5 x 10 6 1/Ms.
  • an acidic pH such as at pH 5.0 to 6.0 (e.g. pH about 5.0)
  • pH about 5.0 e.g. pH about 5.0
  • antibodies and antibody fragments are antibodies or antibody fragments that have higher binding affinity to PD-1 at a pH in the tumor microenvironment than at a pH in a non-tumor microenvironment.
  • the antibodies and antibody fragments have a higher binding affinity to PD-1 at pH 5.0 to 6.8 (e.g. pH 6.0 or pH 5.0) than at pH 7.35 to 7.45 (e.g. pH 7.4).
  • the anti-PD-1 antibodies or antibody fragments have a higher binding affinity (e.g. lower KD) to PD-1 in a tumor in comparison with their binding affinity to PD-1 in a normal tissue or to a cell in the blood.
  • these anti-PD-1 antibodies or antibody fragments have a longer half-life and reduced side-effects, as well as comparable efficacy, in comparison with reference monoclonal anti-PD-1 antibodies known in the art. These features permit use of a higher dosage of these anti-PD-1 antibodies or antibody fragments to be delivered to a patient thus being a more effective therapeutic option.
  • the antibody or fragment thereof exhibits a higher binding affinity (e.g. lower KD) to PD-1 at an acidic pH as present in the microenvironment of tumor (such as at pH 5.0 to 6.8, e.g. pH 6.0 or pH 5.0) than at pH of a non-tumor microenvironment (such as at pH 7.35 to 7.45, e.g.
  • the PD-1 binding at an acidic pH as present in the microenvironment of tumor (such as at pH 5.0 to 6.8, e.g. pH 6.0 or pH 5.0) compared to PD-1 binding at pH of a non-tumor microenvironment (such as at pH 7.35 to 7.45, e.g. 7.4) as present at normal pH of blood or a normal tissue is higher by a ratio of at least or 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50 or more.
  • the PD-1 binding is higher by at least 5%, 10%, 15%, 20%, 25%, 35%, 50%, 100%, 2-fold, 5-fold, 10-fold, 20-fold or more.
  • the KD at an acidic pH as present in the microenvironment of tumor (such as at pH 5.0 to 6.8, e.g. pH 6.0 or pH 5.0) is about 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 25-fold, 30-fold, 40-fold, 50-fold or 100-fold lower than the KD at pH of a non-tumor microenvironment (such as at pH 7.35 to 7.45, e.g. 7.4) as present at normal pH of blood or a normal tissue.
  • a non-tumor microenvironment such as at pH 7.35 to 7.45, e.g. 7.4
  • the antibody or fragment thereof exhibits a higher binding affinity (e.g. lower KD) for binding PD-1 at normal pH of blood or a normal tissue (such as at pH 7.35 to 7.45, e.g. pH 7.4) than that at an acidic pH as present in the microenvironment of tumor (such as at pH 5.0 to
  • the PD-1 binding at pH of a non-tumor microenvironment (such as at pH 7.35 to 7.45, e.g. 7.4) as present at normal pH of blood or a normal tissue compared to PD-1 binding at an acidic pH as present in the microenvironment of tumor (such as at pH 5.0 to 6.8, e.g. pH 6.0 or pH 5.0) is higher by a ratio of at least or 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8,
  • the PD-1 binding is higher by at least 5%, 10%, 15%, 20%, 25%, 35%, 50%, 100%, 2-fold, 5-fold, 10- fold, 20-fold or more.
  • the KD at pH of a non-tumor microenvironment such as at pH 7.35 to 7.45, e.g.
  • the antibody or fragment exhibits a similar PD-1 binding at an acidic pH as present in the microenvironment of tumor (such as at pH 5.0 to 6.8, e.g. pH 6.0 or pH 5.0) compared to PD-1 binding at normal pH as present in blood or a normal tissue (such as at pH 7.35 to 7.45, e.g. pH 7.4).
  • the KD at an acidic pH as present in the microenvironment of tumor (such as at pH 5.0 to 6.8, e.g. pH 6.0 or pH 5.0) is about the same (e.g. 0.7-fold to 1.3-fold) the KD at pH of a non-tumor microenvironment (such as at pH 7.35 to 7.45, e.g. 7.4) as present at normal pH of blood or a normal tissue.
  • properties or features of the provided antibodies are described in relation to properties observed for another antibody, e.g., a reference antibody.
  • the reference antibody is a human or humanized anti-PD-1 antibody.
  • Exemplary reference anti-PD-1 antibodies include pembrolizumab (SEQ ID NOs: 178 and 179, VH and VL set forth in SEQ ID Nos: 157 and 158, respectively); e.g. described in U.S. Patent No. 8,354,509 and PCT publ. No.
  • a provided antibody or fragment exhibits a higher binding affinity (e.g. lower KD) than the reference antibody to PD-1 at an acidic pH as present in the microenvironment of tumor (such as at pH 5.0 to 6.8, e.g. pH 6.0 or pH 5.0).
  • the PD-1 binding of a provided antibody or fragment at an acidic pH as present in the microenvironment of tumor (such as at pH 5.0 to 6.8, e.g.
  • pH 6.0 or pH 5.0 is higher by at least or 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50 or more compared to the reference antibody for PD-1 binding at the same pH or condition.
  • the PD-1 binding is higher by at least 5%, 10%, 15%, 20%, 25%, 35%, 50%, 100%, 2-fold, 5-fold, 10-fold, 20-fold or more.
  • the KD of a provided antibody for PD-1 binding at an acidic pH as present in the microenvironment of tumor such as at pH 5. Oto 6.8, e.g.
  • pH 6.0 or pH 5.0 is about 1.5-fold, 2-fold, 3- fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 25-fold, 30-fold, 40-fold, 50-fold or 100-fold lower than the KD of the reference antibody for PD-1 binding at the same pH or condition.
  • a provided antibody or fragment exhibits a similar PD-1 binding compared to a reference antibody to PD-1 at a pH of a non-tumor microenvironment (such as at pH 7.35 to 7.45, e.g. 7.4) as present at normal pH of blood or a normal tissue.
  • the KD of a provided antibody for PD-1 at a pH of a non-tumor microenvironment (such as at pH 7.35 to 7.45, e.g. 7.4) as present at normal pH of blood or a normal tissue is about the same (e.g. 0.7-fold to 1.3-fold) as a reference antibody at the same pH.
  • amino acid sequence modification(s) of the antibodies described herein are contemplated. For example, it may be desirable to improve the binding affinity and/or other biological properties of the antibody.
  • amino acid sequence variants of an antibody may be prepared by introducing appropriate nucleotide changes into a polynucleotide that encodes the antibody, or a chain thereof, or by peptide synthesis. Such modifications include, for example, deletions from, and/or insertions into and/or substitutions of, residues within the amino acid sequences of the antibody. Any combination of deletion, insertion, and substitution may be made to arrive at the final antibody, provided that the final construct possesses the desired characteristics (e.g., high affinity binding to PD-1).
  • the amino acid changes may alter post-translational processes of the antibody, such as changing the number or position of glycosylation sites. Any of the variations and modifications described above for polypeptides of the present invention may be included in antibodies of the present invention.
  • the antibodies include one or more amino acid variations, e.g., substitutions, deletions, insertions, and/or mutations, compared to the sequence of an antibody described herein.
  • Exemplary variants include those designed to improve the binding affinity and/or other biological properties of the antibody.
  • Amino acid sequence variants of an antibody may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions from, and/or insertions into and/or substitutions of residues within the amino acid sequences of the antibody. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g., antigen-binding.
  • the antibodies include one or more amino acid substitutions, e.g., as compared to an antibody sequence described herein and/or compared to a sequence of a natural repertoire, e.g., human repertoire.
  • Sites of interest for substitutional mutagenesis include the CDRs and FRs.
  • Amino acid substitutions may be introduced into an antibody of interest and the products screened for a desired activity, e.g., retained/improved antigen binding, decreased immunogenicity, improved half-life, and/or improved effector function, such as the ability to promote antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC).
  • ADCC antibody-dependent cellular cytotoxicity
  • CDC complement-dependent cytotoxicity
  • one or more residues within a CDR of a parent antibody is/are substituted.
  • the substitution is made to revert a sequence or position in the sequence to a germline sequence, such as an antibody sequence found in the germline (e.g., human germline), for example, to reduce the likelihood of immunogenicity, e.g., upon administration to a human subject.
  • alterations are made in CDR “hotspots,” residues encoded by codons that undergo mutation at high frequency during the somatic maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and/or residues that contact antigen, with the resulting variant VH or VL being tested for binding affinity.
  • Affinity maturation by constructing and reselecting from secondary libraries has been described, e.g., in Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (O’Brien et al., ed., Human Press, Totowa, NJ, (2001)).
  • affinity maturation diversity is introduced into the variable genes chosen for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide - directed mutagenesis).
  • a secondary library is then created. The library is then screened to identify any antibody variants with the desired affinity.
  • Another method to introduce diversity involves CDR- directed approaches, in which several CDR residues (e.g., 4-6 residues at a time) are randomized.
  • CDR residues involved in antigen binding may be specifically identified, e.g., using alanine scanning mutagenesis or modeling.
  • CDR-H3 and CDR-L3 in particular are often targeted.
  • substitutions, insertions, or deletions may occur within one or more CDRs so long as such alterations do not substantially reduce the ability of the antibody to bind antigen.
  • conservative alterations e.g., conservative substitutions as provided herein
  • Such alterations may, for example, be outside of antigen contacting residues in the CDRs.
  • each CDR either is unaltered, or contains no more than one, two or three amino acid substitutions.
  • Amino acid sequence insertions include amino- and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues.
  • terminal insertions include an antibody with an N-terminal methionyl residue.
  • Other insertional variants of the antibody molecule include the fusion to the N- or C-terminus of the antibody to an enzyme or a polypeptide which increases the serum half-life of the antibody.
  • the antibody is altered to increase or decrease the extent to which the antibody is glycosylated, for example, by removing or inserting one or more glycosylation sites by altering the amino acid sequence and/or by modifying the oligosaccharide(s) attached to the glycosylation sites, e.g., using certain cell lines.
  • an N-linked glycosylation which is a glycosylation site that occurs at asparagines in the consensus sequence -Asn-Xaa-Ser/Thr is removed or inserted. In some embodiments, one or more re replaced with another amino acid to remove the glycosylation site.
  • modified antibodies are those having one or more amino acid modifications in the Fc region, such as those having a human Fc region sequence or other portion of a constant region (e.g., a human IgGl, IgG2, IgG3 or IgG4 Fc region) comprising an amino acid modification (e.g. a substitution) at one or more amino acid positions.
  • a human Fc region sequence or other portion of a constant region e.g., a human IgGl, IgG2, IgG3 or IgG4 Fc region
  • an amino acid modification e.g. a substitution
  • Such modifications can be made, e.g., to improve half-life, alter binding to one or more types of Fc receptors, and/or alter effector functions.
  • cysteine engineered antibodies such as “thioMAbs” and other cysteine engineered variants, in which one or more residues of an antibody are substituted with cysteine residues, in order to generate reactive thiol groups at accessible sites, e.g., for use in conjugation of agents and linker-agents, to produce immunoconjugates.
  • Cysteine engineered antibodies are described, e.g., in U.S. Patent Nos. 7,855,275 and 7,521,541.
  • the antibodies are modified to contain additional nonproteinaceous moieties, including water soluble polymers.
  • exemplary polymers include, but are not limited to, polyethylene glycol (PEG), copolymers of ethylene glycol/propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1, 3-dioxolane, poly-1, 3, 6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids (either homopolymers or random copolymers), and dextran or poly(n-vinyl pyrrolidone)polyethylene glycol, propropylene glycol homopolymers, prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.
  • PEG polyethylene glycol
  • Polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in water.
  • the polymer may be of any molecular weight, and may be branched or unbranched.
  • the number of polymers attached to the antibody may vary, and if more than one polymer is attached, they can be the same or different molecules. In general, the number and/or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the particular properties or functions of the antibody to be improved, whether the antibody derivative will be used in a therapy under defined conditions, etc.
  • Antibodies, and antigen-binding fragments and variants thereof, of the present invention may also be modified to include a detectable label, e.g., an epitope tag or label, e.g., for use in purification or diagnostic applications. These may be conjugated to the antibody as a fusion protein or conjugate, e.g., using a linker or linking group.
  • a linker or linking group There are many linking groups known in the art for making antibody conjugates, including, for example, those disclosed in U.S. Pat. No. 5,208,020 or EP Patent 0 425 235 Bl, and Chari et al., Cancer Research 52: 127-131 (1992).
  • Linking groups include disulfide groups, thioester groups, acid labile groups, photolabile groups, peptidase labile groups, or esterase labile groups, as disclosed in the above-identified patents.
  • tags and/or labels can include, but are not limited to, FLAG tags, poly-histidine tags (e.g. 6xHis), cMyc tags, glutathione-S- transferase tags, avidin, fluorescent labels, polymer particles, metal particles, haptens, enzyme labels, luminescent labels, electrochemiluminescent labels, bioluminescent labels, radioisotopes, or oligonucleotides .
  • conjugates containing an anti-PD-1 antibody or antigen-binding fragment provided herein and one or more further moiety can be an effector moiety, which provides at least one additional function or property of the conjugate as compared to the unconjugated antibody.
  • an effector moiety can be any substance having biological or detectable activity, for example, therapeutic agents, detectable labels, binding agents, or prodrugs, which are metabolized to an active agent in vivo.
  • the moiety can be a targeting moiety, a small molecule drug (non-polypeptide drug of less than 500 Daltons molar mass), a toxin, a cytostatic agent, a cytotoxic agent, an immunosuppressive agent, a radioactive agent suitable for diagnostic purposes, a radioactive metal ion for therapeutic purposes, a prodrug-activating enzyme, an agent that increases biological half-life, or a diagnostic or detectable agent.
  • the effector moiety is a protein, peptide or a nucleic acid molecule, which can be synthesized or produced by recombinant means.
  • the effector moiety is a drug moiety, which may be synthesized artificially or purified from a natural source.
  • the drug moiety has an intracellular activity.
  • the anti-PD-1 antibody or antigen-binding fragment of the conjugate is internalized and the drug moiety is a cytotoxin that blocks the protein synthesis of the cell, therein leading to cell death.
  • the conjugate can be used for inhibiting the multiplication of a tumor cell or cancer cell, causing apoptosis in a tumor or cancer cell, or for treating cancer in a patient.
  • the conjugate can be used accordingly in a variety of settings for the treatment of animal cancers.
  • the conjugate can be used to deliver a drug to a tumor cell or cancer cell.
  • the conjugate and/or drug can be taken up inside a tumor cell or cancer cell through receptor-mediated endocytosis.
  • the conjugate is an antibody drug conjugate (ADC, also called immunoconjugates) containing an anti-PD-1 antibody or antigen-binding fragment provided herein conjugated to a drug moiety that acts as a therapeutic agent, which is either cytotoxic, cytostatic or otherwise provides some therapeutic benefit.
  • ADC antibody drug conjugate
  • the cytotoxic agent is a chemotherapeutic agent, a drug, a growth inhibitory agent, a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate).
  • the drug moiety is an auristatin, such as auristatin E (also known in the art as a derivative of dolastatin-10) or a derivative thereof.
  • the auristatin can be, for example, an ester formed between auristatin E and a keto acid.
  • auristatin E can be reacted with paraacetyl benzoic acid or benzoylvaleric acid to produce AEB and AEVB, respectively.
  • Other typical auristatins include AFP, MMAF, and MMAE.
  • Auristatins have been shown to interfere with microtubule dynamics and nuclear and cellular division and have anticancer activity.
  • Auristatins of the present invention bind tubulin and can exert a cytotoxic or cytostatic effect on a 5T4 expressing cell or cell line.
  • Assays known in the art, that can be used for determining whether an auristatin or resultant antibodydrug conjugate exerts a cytostatic or cytotoxic effect on a desired cell or cell line.
  • Methods for determining whether a compound binds tubulin are known in the art. See, for example, Muller et al., Anal. Chem 2006, 78, 4390-4397; Hamel et al., Molecular Pharmacology, 1995 47: 965-976; and Hamel et al., The Journal of Biological Chemistry, 1990 265:28, 17141-17149.
  • Examples of drugs or pay loads are selected from the group consisting of DM1 (may tansine, N2'-deacetyl-N2'-(3-mercapto-l -oxopropyl)- or N2'-deacetyl-N2'-(3-mercapto-l-oxopropyl)- maytansine), mc-MMAD (6-maleimidocaproyl-monomethylauristatin-D or N-methyl-L-valyl-N- [(lS,2R)-2-methoxy-4-[(2S)-2-[(lR,2R)-l-methoxy-2-methyl-3-oxo-3-[[(lS)-2-phenyl-l-(2- th iazol y l)cthy 11 amino] propyl] - 1 -pyrrolidinyl] - 1 - [( 1 S)- 1 -methylpropyl] -4-oxo
  • DM1 is a derivative of the tubulin inhibitor maytansine while MMAD, MMAE, and MMAF are auristatin derivatives.
  • the preferred payloads of the present invention are selected from the group consisting of mc-MMAF and mc-Val-Cit-PABA-MMAE.
  • the drug moiety is an anti-cancer agent.
  • anti-cancer agents include, but are not limited to, cytostatics, enzyme inhibitors, gene regulators, cytotoxic nucleosides, tubulin binding agents or tubulin inhibitors, proteasome inhibitors, hormones and hormone antagonists, anti-angiogenesis agents, and the like.
  • cytostatic anti-cancer agents include alkylating agents such as the anthracycline family of drugs (e.g.
  • adriamycin carminomycin, cyclosporin-A, chloroquine, methopterin, mithramycin, porfiromycin, streptonigrin, porfiromycin, anthracenediones, and aziridines).
  • cytostatic anti-cancer agents include DNA synthesis inhibitors (e.g., methotrexate and dichloromethotrexate, 3-amino-l,2,4-benzotriazine 1,4-dioxide, aminopterin, cytosine P-D-arabinofuranoside, 5-fluoro-5'-deoxyuridine, 5-fluorouracil, ganciclovir, hydroxyurea, actinomycin-D, and mitomycin C), DNA-intercalators or cross-linkers (e.g., bleomycin, carboplatin, carmustine, chlorambucil, cyclophosphamide, cis-diammineplatinum(II) dichloride (cisplatin), melphalan, mitoxantrone, and oxaliplatin), and DNA-RNA transcription regulators (e.g., actinomycin D, daunorubicin, doxorubicin, homoharringtonine, and idarubi
  • quinonoid derivatives e.g. quinolones, genistein, bactacyclin
  • busulfan ifosfamide, mechlorethamine
  • triaziquone diaziquone
  • carbazilquinone indoloquinone EO9
  • diaziridinyl-benzoquinone methyl DZQ triethylene
  • a conjugate comprising anti-PD-1 antibody or antigen-binding fragment provided herein conjugated with a toxin.
  • the therapeutic agent is a cytotoxin comprising a polypeptide having ribosome-inactivating activity including, for example, gelonin, bouganin, saporin, ricin, ricin A chain, bryodin, diphtheria toxin, restrictocin, Pseudomonas exotoxin A and variants thereof.
  • the effector moiety is a radionuclide (e.g., a peptide receptor radionuclide) or a radiolabel.
  • the effector moiety is a radionuclide or radiolabel with high-energy ionizing radiation that are capable of causing multiple strand breaks in nuclear DNA, leading to cell death.
  • high-energy radionuclides include: 90Y, 1251, 1311, 1231, U lin, 105Rh, 153Sm, 67Cu, 67Ga, 166Ho, 177Lu, 186Re and 188Re. These isotopes typically produce high energy a- or P-particles which have a short path length.
  • radionuclides kill cells to which they are in close proximity, for example neoplastic cells to which the conjugate has attached or has entered. They have little or no effect on non-localized cells and are essentially non-immunogenic.
  • high-energy isotopes may be generated by thermal irradiation of an otherwise stable isotope, for example as in boron neutron-capture therapy (Guan et al., PNAS, 95: 13206-10, 1998).
  • conjugate comprising an anti-PD-1 antibody or antigen-binding fragment provided herein conjugated with a label, which can generate a detectable signal, indirectly or directly.
  • conjugates can be used for research or diagnostic applications, such as for the in vivo detection of cancer.
  • the label is preferably capable of producing, either directly or indirectly, a detectable signal.
  • the label may be radio-opaque or a radioisotope, such as 3H, 14C, 32P, 35S, 1231, 1251, 1311; a fluorescent (fluorophore) or chemiluminescent (chromophore) compound, such as fluorescein isothiocyanate, rhodamine or luciferin; an enzyme, such as alkaline phosphatase, P-galactosidase or horseradish peroxidase; an imaging agent; or a metal ion.
  • a radioisotope such as 3H, 14C, 32P, 35S, 1231, 1251, 1311
  • a fluorescent (fluorophore) or chemiluminescent (chromophore) compound such as fluorescein isothiocyanate, rhodamine or luciferin
  • an enzyme such as alkaline phosphatase, P-galactosidase or horseradish peroxidase
  • the label is a radioactive atom for scintigraphic studies, for example 99Tc or 1231, or a spin label for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, MRI), such as zirconium-89, iodine-123, iodine-131, indium-i l l, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron.
  • NMR nuclear magnetic resonance
  • Zirconium-89 may be complexed to various metal chelating agents and conjugated to antibodies, e.g., for PET imaging (WO 2011/056983).
  • the conjugates may be prepared using any methods known in the art. See, e.g., WO 2009/067800, WO 2011/133886, and U.S. Patent Application Publication No. 2014322129, incorporated by reference herein in their entirety.
  • the linkage of an antibody or antigen binding fragment to a moiety is a direct or indirect linkage.
  • the attachment can be covalent or non-covalent, e.g., via a biotin-streptavidin non- covalent interaction.
  • a moiety can be attached by alkylation (e.g., at the epsilon-amino group lysines or the N-terminus of antibodies), reductive amination of oxidized carbohydrate, transesterification between hydroxyl and carboxyl groups, amidation at amino groups or carboxyl groups, and conjugation to thiols.
  • the attachment of a moiety can be by chemical conjugation and linkage methods known in the art.
  • the moiety can be linked to an antibody by a linker.
  • linkers such as peptide linkers, cleavable linkers, non-cleavable linkers or linkers that aid in the conjugation reaction, can be used to link or conjugate the effector moieties to the antibody or antigen-binding fragment. Attachment of a linker to an antibody can be accomplished in a variety of ways, such as through surface lysines, reductive-coupling to oxidized carbohydrates, and through cysteine residues liberated by reducing interchain disulfide linkages. A variety of antibody drug conjugate linkage systems are known in the art, including hydrazone-, disulfide- and peptide-based linkages.
  • an anti-PD-1 antibody or antigen-binding fragment provided herein is conjugated to one or more moieties, e.g. about 1 to about 20 drug moieties, through a linker (L).
  • the conjugate comprises the following components: (antibody or antigen-binding fragment), (L) q and (moiety) m , wherein the antibody or antigen-binding fragment is any of the described capable of specifically binding PD-1 as described; L is a linker for linking the protein or polypeptide to the moiety; m is at least 1; q is 0 or more; and the resulting conjugate binds to PD-1.
  • m is 1 to 4 and q is 0 to 8.
  • the linker may be composed of one or more linker components.
  • the linker typically has two reactive functional groups, i.e. bivalency in a reactive sense.
  • Bivalent linker reagents which are useful to attach two or more functional or biologically active moieties, such as peptides, nucleic acids, drugs, toxins, antibodies, haptens, and reporter groups are known, and methods have been described their resulting conjugates (Hermanson, G. T. (1996) Bioconjugate Techniques; Academic Press: New York, p 234-242).
  • the linker may comprise amino acid residues.
  • Exemplary amino acid linker components include a dipeptide, a tripeptide, a tetrapeptide or a pentapeptide.
  • Exemplary dipeptides include: valine-citrulline (vc or val-cit), alanine-phenylalanine (af or ala-phe).
  • Exemplary tripeptides include: glycine-valine-citrulline (gly-val-cit) and glycine-glycine-glycine (gly-gly-gly).
  • Amino acid residues which comprise an amino acid linker component include those occurring naturally, as well as minor amino acids and non-naturally occurring amino acid analogs, such as citrulline.
  • Amino acid linker components can be designed and optimized in their selectivity for enzymatic cleavage by a particular enzymes, for example, a tumor-associated protease, cathepsin B, C and D, at a plasmin protease.
  • Exemplary linker components include 6-maleimidocaproyl (“MC”), maleimidopropanoyl (“MP”), valine-citrulline (“val-cit”), a alanine-phenylalanine (“ala-phe”), p-aminobenzyloxycarbonyl (“PAB”), N-Succinimidyl 4-(2-pyridylthio)pentanoate (“SPP”), N-Succinimidyl 4-(N- maleimidomethyl)cyclohexane-I carboxylate (“SMCC”), and N-Succinimidyl (4-iodo- acetyl) aminobenzoate (“SIAB”).
  • MC 6-maleimidocaproyl
  • MP maleimidopropanoyl
  • val-cit valine-citrulline
  • ala-phe a alanine-phenylalanine
  • PAB p-aminobenzyloxycarbonyl
  • Conjugates of an anti-PD-1 antibody or antigen-binding fragment provided herein and cytotoxic agent can be made using a variety of bifunctional protein-coupling agents such as N- succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HC1), active esters (such as disuccinimidyl substrate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5- difluoro-2,4-dinitrobenzene).
  • SPDP
  • the antibody drug conjugate can be prepared by a variety of methods, such as organic chemistry reactions, conditions, and reagents known to those skilled in the art.
  • a fusion protein containing an antibody or antigen-binding fragment and cytotoxic agent may be made, e.g., by recombinant techniques or peptide synthesis.
  • the length of DNA may comprise respective regions encoding the two portions of the conjugate either adjacent one another or separated by a region encoding a linker peptide which does not destroy the desired properties of the conjugate
  • PD-1 -binding polypeptides that are multispecific containing at least one antibody or antigen-binding fragment that binds PD-1 and one or more additional binding domains.
  • the one or more additional domains bind to a second antigen or protein other than PD-1.
  • the further antigen or protein may be an antigen expressed on a tumor, a molecule or receptor expressed on an immune cell, such as a T cell, e.g. a CD3, or an additional inhibitory receptor (e.g. CTLA-4, LAG3, TIM3, VISTA, TIGIT, SIRPa, NKG2A, B7H3, B7H4) or an activating receptor (e.g.
  • the one or more additional domain is an antibody or antigen-binding fragment specific for the second antigen or protein.
  • a provided binding molecule is a bispecific T cell engager that is composed of a PD-1 antibody or antigen-binding fragment as described herein and at least one additional binding molecule capable of binding to a surface molecule expressed on a T cell.
  • the surface molecule is an activating component of a T cell, such as a component of the T cell receptor complex.
  • the surface molecule is an activating T cell antigen that is expressed on a T cell and is capable of inducing T cell activation upon interaction with an antigen binding molecule.
  • interaction of an antigen binding molecule with an activating T cell antigen may induce T cell activation by triggering the signaling cascade of the T cell receptor complex.
  • Suitable assays to measure T cell activation are known, and include any assay to measure or assess proliferation, differentiation, cytokine secretion, cytotoxic activity and/or expression of one or more activation marker.
  • the simultaneous or near simultaneous binding of such a PD-1 -binding polypeptide to both of its targets, PD-1 expressed on target cell and a T cell molecule expressed on a T cell, e.g. activating T cell antigen can result in a temporary interaction between the target cell and T cell, thereby resulting in activation, e.g. cytotoxic activity, of the T cell and subsequent lysis of the target cell.
  • the T surface molecule such as activating T cell antigen
  • a provided bispecific PD-1 -binding polypeptide is capable of specifically binding an activating T cell antigen expressed on a human T cell, such as human CD3 or human CD3.
  • the additional binding domain that is specific to the activating T cell antigen is an antibody or antigen-binding fragment.
  • bispecific antibody T cell-engagers are bispecific T cell engager (BiTE) molecules, which contain tandem scFv molecules fused by a flexible linker (see e.g. Nagorsen and Bauerle, Exp Cell Res 317, 1255-1260 (2011); tandem scFv molecules fused to each other via, e.g.
  • a flexible linker and that further contain an Fc domain composed of a first and a second subunit capable of stable association
  • diabodies and derivatives thereof including tandem diabodies (Holliger et al, Prot Eng 9, 299-305 (1996); Kipriyanov et al, J Mol Biol 293, 41-66 (1999)); dual affinity retargeting (DART) molecules that can include the diabody format with a C-terminal disulfide bridge; or triomabs that include whole hybrid mouse/rat IgG molecules (Seimetz et al, Cancer Treat Rev 36, 458-467 (2010). Similar formats of any of the above molecules can be generated using any of the anti- PD-1 antibodies or antigen binding fragments provided herein.
  • the additional binding domain specific to an activating T cell antigen is an antigen-binding fragment selected from a Fab fragment, a F(ab')2 fragment, an Fv fragment, a scFv, disulfide stabilized Fv fragment (dsFv), a scAb, a dAb, a single domain heavy chain antibody (VHH), or a single domain light chain antibody.
  • the additional binding domain is monovalent for binding the activating T cell antigen, such as CD2 or CD3.
  • the additional binding domain is capable of binding to CD3 or a CD3 complex.
  • a CD3 complex is a complex of at least five membrane-bound polypeptides in mature T-lymphocytes that are non-covalently associated with one another and with the T-cell receptor.
  • the CD3 complex includes the gamma, delta, epsilon, zeta, and eta chains (also referred to as subunits).
  • the additional binding molecule is an antibody or antigen-binding fragment capable of specifically binding to CD3 or a CD3 complex, also called a CD3-binding domain.
  • the CD3-binding domain capable of binding CD3 or a CD3 complex includes one or more copies of an anti-CD3 Fab fragment, an anti-CD3 F(ab')2 fragment, an anti-CD3 Fv fragment, an anti-CD3 scFv, an anti-CD3 dsFv, an anti-CD3 scAb, an anti-CD3 dAb, an anti-CD3 single domain heavy chain antibody (VHH), and an anti-CD3 single domain light chain antibody.
  • the anti-CD3 binding domain is monovalent for binding CD3.
  • the CD3-binding domain recognizes the CD3s-chain.
  • the anti-CD3s binding domain includes one or more copies of an anti-CD3s Fab fragment, an anti-CD3s F(ab')2 fragment, an anti-CD3s Fv fragment, an anti-CD3s scFv, an anti-CD3s dsFv, an anti-CD3s scAb, an anti-CD3s dAb, an anti-CD3s single domain heavy chain antibody (VHH), and an anti-CD3s single domain light chain antibody.
  • the anti-CD3s binding domain is monovalent for binding CD3s.
  • Exemplary monoclonal antibodies against CD3 or a CD3 complex include, but are not limited to, OKT3, SP34, UCHT1 or 64.1, or an antigen-binding fragment thereof (See e.g., June, et al., J. Immunol. 136:3945-3952 (1986); Yang, et al., J. Immunol. 137:1097-1100 (1986); and Hayward, et al., Immunol. 64:87-92 (1988)).
  • the CD3- binding domain monovalently and specifically binds a CD3 antigen, and is derived from OKT3 (ORTHOCLONE-OKT3TM (muromonab-CD3); humanized OKT3 (U.S. Pat. No. 7,635,475 and published international application No. W02005040220); SP34 (Pessano et al. The EMBO Journal.
  • the PD-1 -binding polypeptide is a bispecific construct that is or comprises at least one anti-PD-1 antibody or antigen binding fragment provided herein and at least one additional binding molecule capable of binding to a surface molecule expressed on a Natural Killer (NK) cells and/or recruiting NK cells.
  • the multispecific antibody is bispecific for PD-1 and the NK cell surface molecule.
  • the surface molecule is CD16 (FcyRIII).
  • CD 16 a low affinity receptor for the Fc portion of some IgGs known to be involved in antibody-dependent cellular cytotoxicity (ADCC), is the best-characterized membrane receptor responsible for triggering of target cell lysis by NK cells (Mandelboim et al., 1999, PNAS 96:5640- 5644). Generally, a large majority (approximately 90%) of human NK cells express CD56 at low density (CD56dim) and FcyRIII (CD16) at a high level (Cooper et al., 2001, Trends Immunol. 22:633-640).
  • the additional binding molecule is capable of specifically binding CD16a.
  • the alpha chain of CD16a associates with the immunoreceptor tyrosine-based activation motif (ITAM) containing FceRI y-chain and/or the T-cell receptor (TCR)/CD3 ⁇ -chain to mediate signaling (Wirthmueller et al., 1992, J. Exp. Med.
  • ITAM immunoreceptor tyrosine-based activation motif
  • TCR T-cell receptor
  • Engagement of CD16a can result in activating of NK cells expressing CD16a, thereby eliciting a biological response to trigger cell killing of target cells in a manner analogous to antibody-dependent cellular cytotoxicity (ADCC).
  • ADCC antibody-dependent cellular cytotoxicity
  • a binding molecule that specifically binds PD-1 expressed on a tumor cell may target NK-cells to the tumor cell.
  • activation of the NK cell caused by the binding molecule binding to CD 16a can lead to killing of the tumor cells.
  • the additional binding domain specific to an activating NK cell receptor is an antigen-binding fragment selected from a Fab fragment, a F(ab')2 fragment, an Fv fragment, a scFv, disulfide stabilized Fv fragment (dsFv), a scAb, a dAb, a single domain heavy chain antibody (VHH), or a single domain light chain antibody.
  • the additional binding domain is monovalent for binding the activating T NK cell receptor, such as CD 16a.
  • Antibodies and antigen-binding fragments thereof specific for CD 16a are known and include, for example, NM3E2 (McCall et al. (1999) Mol.
  • anti-CD16a antibodies also can be used in the constructs provided herein, including any described in published U.S. patent application Ser. No. 10/160,280,795; U.S. Pat. No. 9,701,750; Behar et al. (2008) Protein Eng Des Sei. 21:1-10; Arndt et al., (1999) Blood 94:2562-2568.
  • the anti-CD16a is an anti- CD16a scFv.
  • the anti-CD16a is an anti-CD16a antibody included in a TandAb molecule (see e.g. Reush et al. (2014) Mabs, 6:727-738).
  • the anti-CD16a is an anti- CD16a or antigen binding fragment, such as an scFv, described in U.S. Pat. No. 9,035,026.
  • Single domain antibodies, including VHH domains that bind to CD16a are known, see e.g. published U.S. patent application No. US20160280795.
  • a chimeric antigen receptor that contains an extracellular antigen-binding domain that includes an any of the provided anti-PD-1 antibodies or antigen binding fragments, a transmembrane domain and an intracellular signaling region composed of one or more signaling domains.
  • the extracellular antigen binding domain is an scFv comprising a variable heavy chain and a variable light chain of any of the provided antibodies.
  • engineered cells in which the CAR is expressed on the surface of a cell.
  • provided herein are engineered cells that express any of the provided antibodies or antigen-binding fragments or PD-1 binding molecules described herein.
  • the provided antibodies or antigen binding fragments or PD-1 bind molecules thereof are secreted from the cell.
  • such an antibody or antigen binding fragment or PD-1 binding molecule comprises a signal peptide, e.g., an antibody signal peptide or other efficient signal sequence to get domains outside of cell, such as to cause the protein to be secreted by the engineered cell.
  • the signal peptide, or a portion of the signal peptide is cleaved from the binding molecule with secretion.
  • the antibody or antigen binding fragment or PD-1 binding molecule can be encoded by a nucleic acid (which can be part of an expression vector).
  • the antibody or antigen binding fragment or a PD-1 binding molecule thereof is expressed and secreted by a cell (such as an immune cell, for example a primary immune cell).
  • such provided engineered cells that are engineered to express a a antibody or antigen-binding fragment or PD-1 binding molecules described herein further contain a chimeric antigen receptor (CARs).
  • the CAR contains an extracellular domain comprising one or more antigen binding domain specific to a tumor antigen (TAA).
  • TAA tumor antigen
  • CAR constructs include an extracellular domain containing the one or more extracellular antigen binding domain, a transmembrane domain and an intracellular signaling region.
  • the extracellular antigen binding domain is an scFv or a single domain antibody (VHH).
  • the extracellular antigen binding domain which form the antigen binding unit of the CAR “binds” or is “capable of binding”, i.e. targets, a target antigen with sufficient affinity such the CAR is useful in therapy in targeting a cell or tissue expressing the target antigen.
  • Non-limiting examples of a tumor antigen include, but are not limited to, carbonic anhydrase IX (CAIX), carcinoembryonic antigen (CEA), CD8, CD7, CD10, CD19, CD20, CD22, CD30, CD33, CLL1, CD34, CD38, CD41, CD44, CD49c, CD49f, CD56, CD66c, CD73, CD74, CD104, CD133, CD138, CD123, CD142, CD44V6, an antigen of a cytomegalovirus (CMV) infected cell (e.g., a cell surface antigen), cutaneous lymphocyte- associated antigen (CLA; a specialized glycoform of P-selectin glycoprotein ligand-1 (PSGL-1)), epithelial glycoprotein-2 (EGP- 2), epithelial glycoprotein-40 (EGP-40), epithelial cell adhesion molecule (EpCAM), receptor tyrosine-protein kinases erb-B2,3,4 (er
  • the CAR is the CAR present in any of a variety of known engineered cell products.
  • the tumor antigen is CD19.
  • the tumor antigen is BCMA.
  • the CAR may include, but is not limited to a CAR engineered into cells of ABECMA®, JCARH125, CARVYKTITM (NJ-68284528; Janssen/Legend), P-BCMA-101 (Poseida), PBCAR269A (Poseida), P-BCMA- Allol (Poseida), Allo- 715 (Pfizer/ Allogene), CT053 (Carsgen), Descartes-08 (Cartesian), PHE885 (Novartis), CTX120 (CRISPR Therapeutics); YESCARTA®, KYMRIAH®, TECARTUS®, or BREYANZI®.
  • the CAR comprises a CAR of a commercial CAR cell therapy.
  • a CAR in commercial cell based therapies include the CAR engineered in cells of brexucabtagene autoleucel (TECARTUS®), axicabtagene ciloleucel (YESCARTA®), idecabtagene vicleucel (ABECMA®), ciltacabtagene autoleucel (CARVYKTITM), lisocabtagene maraleucel (BREYANZI®), tisagenlecleucel (KYMRIAH®).
  • TECARTUS® brexucabtagene autoleucel
  • YESCARTA® axicabtagene ciloleucel
  • ABECMA® idecabtagene vicleucel
  • CARVYKTITM ciltacabtagene autoleucel
  • BREYANZI® lisocabtagene maraleucel
  • KYMRIAH® tisagen
  • the transmembrane domain of a CAR is a domain that typically crosses or is capable of crossing or spanning the plasma membrane and is connected, directly or indirectly (e.g. via a spacer, such as an immunoglobulin hinge sequence) to the extracellular antigen binding domain and the endoplasmic portion containing the intracellular signaling domain.
  • the transmembrane domain of the CAR is a transmembrane region of a transmembrane protein (for example Type I transmembrane proteins), an artificial hydrophobic sequence or a combination thereof.
  • the transmembrane domain comprises the CD3zeta domain or CD28 transmembrane domain.
  • Other transmembrane domains will be apparent to those of skill in the art and may be used in connection with embodiments of a CAR provided herein.
  • the intracellular signaling region of a CAR provided herein contains one or more intracellular signaling domain that transmits a signal to a T cell upon engagement of the antigen binding domain of the CAR, such as upon binding antigen.
  • the intracellular region contains an intracellular signaling domain that is or contains an IT AM signaling domain.
  • Exemplary intracellular signaling domains include, for example, a signaling domain derived from chain of the T-cell receptor complex or any of its homologs (e.g., r
  • the intracellular signaling region contains an intracellular signaling domain derived from the human CD3 zeta chain.
  • the intracellular signaling region of a CAR can further contain an intracellular signaling domain derived from a costimulatory molecule.
  • a signaling domain may enhance CAR-T cell activity, such as via enhancement of proliferation, survival and/or development of memory cells, after antigen specific engagement, for example, compared to a CAR that only contains an ITAM containing signaling domain, e.g. CD3 zeta.
  • the co-stimulatory domain is a functional signaling domain obtained from a protein selected from: CD28, CD137 (4-IBB), CD134 (0X40), DapIO, CD27, CD2, CD5, ICAM-1, EFA-1 (CD1 la/CD18), Eck, TNFR-I, TNFR-II, Fas, CD30, CD40 or combinations thereof.
  • the costimulatory signaling domain is derived or obtained from a human protein.
  • the costimulatory signaling domain is derived or obtained from human CD28 or human CD137 (4-IBB).
  • the CAR further comprises a hinge or spacer region which connects the extracellular antigen binding domain and the transmembrane domain.
  • This hinge or spacer region can be used to achieve different lengths and flexibility of the resulting CAR.
  • Examples of the hinge or spacer region that can be used include, but are not limited to, Fc fragments of antibodies or fragments or derivatives thereof, hinge regions of antibodies, or fragments or derivatives thereof, CH2 regions of antibodies, CH3 regions of antibodies, artificial spacer sequences, for example peptide sequences, or combinations thereof.
  • Other hinge or spacer region will be apparent to those of skill in the art and may be used.
  • the hinge is an lgG4 hinge or a CD8A hinge.
  • the cell is selected from the group consisting of a T cell, a Natural Killer (NK) cell, a cytotoxic T lymphocyte (CTE), a regulatory T cell, hematopoietic stem cells and/or pluripotent embryonic/induced stem cells.
  • the cell is a T cell, such as a CD4 and/or CD8 T cell.
  • the cells are autologous to the subject.
  • T cells may be isolated from a patient (also called primary T cells) for engineering, e.g. transfection or transduction, with a CAR nucleic acid construct.
  • primary T-cells can be purified ex vivo (CD4 cells or CD8 cells or both) and stimulated with a TCR/CD28 agonists, such as anti-CD3/anti-CD28 coated beads.
  • a recombinant expression vector encoding the antibody or binding molecule and/or CAR can be stably introduced into the primary T cells through standard lentiviral or retroviral transduction protocols or plasmid electroporation strategies.
  • Cells can be monitored for secretion of an antibody or PD-1 binding molecule and/or CAR expression by, for example, flow cytometry using anti-epitope tag or antibodies that cross-react with native parental molecule.
  • the antibody or PD-1 binding molecule and/or CAR engineered T-cells can be assayed for appropriate function by a variety of means.
  • in vitro cytotoxicity, proliferation, or cytokine assays e.g., IFN-gamma expression
  • Exemplary standard endpoints are percent lysis of a tumor line, proliferation of the engineered T-cell, or IFN-gamma protein expression in culture supernatant.
  • the ability to stimulate activation of T cells upon stimulation of the CAR, e.g. via antigen can be assessed, such as by monitoring expression of activation markers such as CD69, CD44, or CD62L, proliferation and/or cytokine production.
  • a subject is in need of treatment for the disease or condition, pharmaceutically active amount of a cell and/or of a pharmaceutical composition of the invention.
  • multispecific binding molecules that comprise any of the provided PD-1 binding domains and also further comprise a CTLA-4 binding domain that binds CTLA-4, e.g., human CTLA-4.
  • the binding domains are antibody fragments, such as a Fab fragment or an scFv.
  • the binding molecule is composed of at least one CTEA- 4 binding domain e.g., anti-CTEA-4 Fab) directed against CTEA-4, and at least one PD-1 binding domain (e.g. anti-PDl Fab) directed against PD-1.
  • the binding molecule has binding specificity for both CTEA-4 and PD-1.
  • the binding molecule is a bispecific antibody that is specific for CTLA-4 and PD-1 (also termed an anti-PD-l/anti-CTLA-4 bispecific antibody).
  • a bispecific antibody comprising a CTLA-4 binding domain that binds CTLA-4 and a PD-1 binding domain that binds PD-1.
  • the PD-1 binding domain includes an antibody or an antigenbinding fragment thereof selected from the group consisting of a Fab fragment, a F(ab')2 fragment, an Fv fragment, an scFv such as a dsFv, a scAb, a dAb, a single domain heavy chain antibody, and a single domain light chain antibody.
  • the PD-1 binding domain is a Fab.
  • the PD-1 binding domain can include any of the antibodies or antigen-binding fragments as described in Section I.
  • the CTLA-4 binding domain includes an antibody or an antigen-binding fragment thereof selected from the group consisting of a Fab fragment, a F(ab')2 fragment, an Fv fragment, an scFv such as a dsFv, a scAb, a dAb, a single domain heavy chain antibody, and a single domain light chain antibody.
  • the CTLA-4 binding domain is a Fab.
  • the CTLA-4 binding domain can include any of the antibodies or antigen-binding fragments as described in Section II.B.
  • the binding domains contains a Vnregion and a VL region that together form an antigen-binding site.
  • VH is the region of the CTLA-4 binding domain or the PD-1 binding domain that comprises the three heavy chain complementarity determining regions (CDRs) and the VL region is the region of the CTLA-4 binding domain or the PD-1 binding domain that comprises the three light chain CDRs.
  • the CTLA-4 binding domain and PD-1 binding domain each contain a VH region sequence that contains a CDR-H1, a CDR-H2 and a CDR-H3 as described and a VL region sequence that contains a CDR-L1, a CDR-L2 and a CDR-L3 as described.
  • Table 1 above lists exemplary position boundaries of CDR-L1, CDR-L2, CDR-L3 and CDR-H1, CDR-H2, CDR-H3 as identified by Kabat, Chothia, AbM, and Contact schemes, respectively.
  • the binding domains of the provided binding molecules are antibody fragments that retain the ability to specifically bind to an antigen (e.g., human CTLA-4 and human PD-1).
  • the antibody fragments are Fab composed of an entire light chain (i.e. VL region and a constant light chain (CL)) and the VH region and the first constant domain of one heavy chain (CHI).
  • the binding molecules further contains a heavy chain constant region including a CHI, hinge, CH2 and CH3, such as from an IgGl.
  • the binding domains such as the CTLA-4 binding domain and the PD-1 binding domain, as described are fused to immunoglobulin constant domain sequences.
  • the fusion is with an Ig heavy chain constant domain, comprising a Fc region, such as at least part of the hinge, CH2, and CH3 regions.
  • the binding molecule provided herein is composed of the CTLA-4 binding domain, the PD-1 binding domain, and a Fc region (hinge-CH2-CH3).
  • the CTLA-4 binding domain and the PD-1 binding domain are linked via their C-termini to the hinge region of the Fc region.
  • the Fc region acts to multimerize two heavy chain polypeptides of the binding molecule, such a via a disulfide bond formed between identical Fc polypeptides.
  • the Fc region is a multimerization domain.
  • the human IgG heavy-chain Fc region usually includes the region containing the CH2 and CH3 domains and the hinge region, such as an amino acid residue at position Cys226, or from Pro230, to the carboxyl-terminus thereof.
  • Exemplary Fc regions are provided in Section II.C.
  • the CTLA-4 binding domain and/or the PD-1 binding domain are linked via their C-termini to the hinge region of the Fc region.
  • the CTEA-4 binding domain and/or the PD-1 binding domain are linked via their N-termini to the CH3 region of the Fc region.
  • the antibody further comprises a light chain constant domain.
  • the Fc region is a homodimeric Fc region that facilitates formation of a homodimer containing the same heavy chain polypeptides in the bispecific binding molecule.
  • the provided binding molecules such as bispecific antibodies, are binding molecules that include two identical copies of the CTEA-4 binding domain, two identical copies of the PD-1 binding domain, and a Fc region composed of two identical polypeptide chains linked by a disulfide bond.
  • the bispecific antibody is a 2 + 2 tetravalent bispecific antibody that exhibits bivalent binding for CTEA-4 and bivalent binding for PD-1.
  • the bispecific antibody is a 1 + 1 bivalent bispecific antibody that exhibits monovalent binding for CTLA-4 and monovalent binding for PD-1.
  • the binding molecule such as a bispecific antibody, contains two identical heavy chains and two identical light chains.
  • the Fc region is a heterodimeric Fc region that facilitates formation of a heterodimer containing two different heavy chain polypeptides in the bispecific binding molecule.
  • such binding molecules may contain different heavy chain polypeptides.
  • the binding molecule, such as a bispecific antibody is a 1 + 1 bivalent bispecific antibody that exhibits monovalent binding for CTEA-4 and monovalent binding for PD-1.
  • the binding molecule, such as a bispecific antibody is a 2 +1 tri valent bispecific antibody that exhibits bivalent binding for CTEA-4 and monovalent binding for PD-1.
  • the binding molecule, such as a bispecific antibody is a 2 + 1 bivalent bispecific antibody that exhibits bivalent binding for PD-1 and monovalent binding for CTLA-4.
  • the CTLA-4 binding domain and the PD-1 binding domain each is a Fab, comprising a VH-CH1 and a VL-CE of the respective antibody fragment.
  • the Fab has an IgGl isotype, IgG2 isotype, IgG3 isotype or IgG4 isotype, and the CHI of the CTEA-4-Fab or PD-l-Fab is the CHI of the immunoglobulin isotype.
  • the CHI is of the IgGl isotype, IgG2 isotype, or IgG4 isotype.
  • the CHI is of the IgGl isotype.
  • the CHI is from an IgGl.
  • the CHI comprises an amino acid sequence having at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 409.
  • the CHI comprises the amino acid sequence set forth in SEQ ID NO: 409.
  • the CL is a X (lambda) light chain.
  • the light chain is a K (kappa) light chain.
  • the CL comprises an amino acid sequence having at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 410.
  • the CL comprises an amino acid sequence set forth in SEQ ID NO: 410.
  • the multispecific antibody (e.g., a bispecific antibody described herein) includes a heterodimeric Fc region for dimerizing the heavy chains of the antibody.
  • the Fc region comprises a first and second polypeptide each comprising a hinge-CH2- CH3.
  • one of the first polypeptide and second polypeptide is linked to the CHI of the CTLA-4 Fab and the other of the first polypeptide and second polypeptide is linked to the CHI of the PD-1 Fab.
  • the first and second polypeptide of the heterodimeric Fc comprises one or more amino acid substitutions (e.g., knob-into-hole modification) in a wild-type Fc polypeptide region to effect heterodimer formation between the first polypeptide and the second polypeptide.
  • amino acid substitutions e.g., knob-into-hole modification
  • the heavy chain further comprises a signal peptide.
  • the signal peptide can be cleaved upon expression from a cell.
  • the heavy chain signal peptide is set forth in SEQ ID NO: 176 or 361.
  • the light chain signal peptide is set forth in SEQ ID NO: 177.
  • the binding molecule provided herein is composed of a CTEA- 4 binding domain that is a Fab fragment for specifically binding to CTEA-4 (further named also as “CTEA-4-Fab”), a PD-1 binding domain that is Fab fragment for specifically binding PD-1 (further named also as “PD-1 -Fab), and a Fc region.
  • CTEA-4 binding domain and PD-1 binding domain are each N-terminal to the Fc region an.
  • the binding molecule comprises the CTEA-4 binding domain that is a CTLA-4 Fab comprising the CTEA-4 VH region and a heavy chain constant region 1 (CHI), and a light chain (EC) comprising the CTLA-4 VL region and a light chain constant region (CL); a PD-1 Fab comprising the PD-1 VH region and a heavy chain constant region 1 (CHI), and a light chain (EC) comprising the PD-1 VL region and a light chain constant region (CE), and an Fc region.
  • a CTLA-4 Fab comprising the CTEA-4 VH region and a heavy chain constant region 1 (CHI)
  • EC light chain
  • CE light chain constant region
  • biological activity or functional activity of a provided bispecific antibody can be measured using any of a number of known methods.
  • the activity can be assessed or determined either in vitro or in vivo.
  • activity can be assessed once the bispecific antibody is administered to the subject (e.g., human).
  • Parameters to assess include specific binding of the bispecific antibody to a T cell (e.g. natural T cell) or target antigenexpressing cell, e.g., in vivo, e.g., by imaging, or ex vivo, e.g., by ELISA or flow cytometry.
  • the ability of the bispecific antibody to destroy target cells can be measured using any suitable known methods, such as cytotoxicity assays described in, for example, Kochenderfer et al., J. Immunotherapy, 32(7): 689-702 (2009), and Herman et al. J.
  • the biological activity can be measured using an animal model of the disease or condition, such as a tumor xenograft model, and assessing the reduction in tumor burden or load and/or survival. In some aspects the biological activity is measured by assessing clinical outcome, such as reduction in tumor burden or load.
  • a binding molecule provided herein is not limited to any particular bispecific format or method of producing it. Binding molecules can be prepared as full length antibodies or antibody fragments. Among the multispecific antibodies are multispecific single-chain antibodies, e.g., diabodies, triabodies, and tetrabodies, scFv-IgG bispecific antibodies, tandem di-scFvs, and tandem tri-scFvs. In some embodiments, the binding molecule is formatted as a bivalent antibody. In some embodiments, the binding molecule is formatted as a trivalent antibody. In some embodiments, the binding molecule is formatted as a tetravalent antibody.
  • the at least two binding domains and the variable domains (VH/VL) of the antibody construct of the present invention may or may not comprise peptide linkers (spacer peptides).
  • the term “peptide linker” comprises in accordance with the present invention an amino acid sequence by which the amino acid sequences of one (variable and/or binding) domain and another (variable and/or binding) domain of the antibody construct of the invention are linked with each other.
  • the peptide linkers can also be used to fuse the third domain to the other domains of the antibody construct of the invention.
  • An essential technical feature of such peptide linker is that it does not comprise any polymerization activity.
  • suitable peptide linkers are those described in U.S. Pat. Nos. 4,751,180 and 4,935,233 or WO 88/09344.
  • the peptide linkers can also be used to attach other domains or modules or regions (such as half-life extending domains) to the antibody construct of the invention.
  • any format or technology for generating a multispecific antibody such as a bispecific antibody, may be used to make the binding molecules of the present invention.
  • a binding domain such as an antibody or antigen-binding fragment thereof, having a first antigen binding specificity can be functionally linked (e.g., by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other molecular entities, such as another binding domain, such as an antibody or antibody fragment having a second antigen-binding specificity to produce a bispecific binding molecule.
  • bispecific formats that can be used in the context of the present invention include, without limitation, e.g., scFv-based or diabody bispecific formats, IgG-scFv fusions, dual variable domain (DVD)-Ig, Quadroma, knobs- into-holes, common light chain (e.g., common light chain with knobs-into-holes,etc.), CrossMab, CrossFab, (SEED) body, leucine zipper, Duobody, IgGl/IgG2, dual acting Fab (DAF)-IgG, and Mabe bispecific formats (See, for example, Spiess et al. (2015) Mol Immunol 67(2 Pt A):95-106 and Brinkmann et al. (2017) MAbs 9(2): 182-212, and the references cited therein, for a review of the foregoing formats).
  • bispecific antibodies which contain tandem scFv molecules fused by a flexible linker (see e.g. Nagorsen and Bauerle, Exp Cell Res 317, 1255-1260 (2011); tandem scFv molecules fused to each other via, e.g.
  • a flexible linker and that further contain an Fc domain composed of a first and a second subunit capable of stable association
  • diabodies and derivatives thereof including tandem diabodies (Holliger et al, Prot Eng 9, 299-305 (1996); Kipriyanov et al, J Mol Biol 293, 41-66 (1999)); dual affinity retargeting (DART) molecules that can include the diabody format with a C-terminal disulfide bridge; or triomabs that include whole hybrid mouse/rat IgG molecules (Seimetz et al, Cancer Treat Rev 36, 458-467 (2010); and trivalent bispecific antibodies (e.g. Mayer et al. 2015 Int. J Mol Sci, 16:27497-27507 and U.S. patent publication No. US20200062826).
  • bispecific antibody molecules formats which may be used include (i) a single antibody that has two arms comprising different antigen-binding regions; (ii) a single chain antibody that has specificity to two different epitopes, e.g., via two scFvs linked in tandem by an extra peptide linker; (iii) a dual-variable-domain antibody (DVD-Ig), where each light chain and heavy chain contains two variable domains in tandem through a short peptide linkage (Wu et al., Generation and Characterization of a Dual Variable Domain Immunoglobulin (DVD-IgTM) Molecule, In: Antibody Engineering, Springer Berlin Heidelberg (2010)); (iv) a chemically-linked bispecific (Fab')2 fragment; (v) a Tandab, which is a fusion of two single chain diabodies resulting in a tetravalent bispecific antibody that has two binding sites for each of the target antigens; (vi) a flexi
  • Various strategies for preparing multi-specific antibodies may also be provided in an asymmetric form with a domain crossover in one or more binding arms of the same antigen specificity (so-called “CrossMab” technology), i.e. by exchanging the VH/VL domains (see e.g., WO 2009/080252 and WO 2015/150447), the CHI/CL domains (see e.g., WO 2009/080253) or the complete Fab arms (see e.g., WO 2009/080251, WO 2016/016299, also see Schaefer et al, PNAS, 108 (2011) 1187-1191, and Klein at al., MAbs 8 (2016) 1010-20).
  • Asymmetrical Fab arms can also be engineered by introducing charged or non-charged amino acid mutations into domain interfaces to direct correct Fab pairing. See e g., WO 2016/172485.
  • provided binding molecules include at least one crossover Fab, in which the variable domains or the constant domains of the Fab heavy and light chain are exchanged/replaced by each other.
  • the variable domains of the Fab heavy and light chains of one binding molecule e.g., PD-1 or CTLA-4
  • a bispecific antibody as described herein comprises a polypeptide comprising a light chain variable domain VL of CTLA-4 and a heavy chain constant domain 1 (CHI) (e.g., VL-CH1) and a second polypeptide comprising a heavy chain variable domain VH of CTLA-4 and the light chain constant domain CL (e.g., VH-CL).
  • CHI heavy chain constant domain 1
  • a conventional Fab molecule is a Fab molecule in its natural state.
  • a conventional Fab molecule comprises for example, a heavy chain variable and a constant domain (e.g., VH-CH1) and a light chain variable and a constant domain (e.g., VL-CL).
  • a bispecific antibody described herein comprises at least one crossover Fab.
  • the CTLA4 binding domain is a crossover Fab and the PD-1 binding domain is a conventional Fab.
  • the PD-1 binding domain is a crossover Fab and the CTLA-4 binding domain is a conventional Fab.
  • the binding molecule including the CTLA-4 binding domain (e.g., anti-CTLA-4 antibody fragment) and/or the PD-1 binding domain (e.g., anti- PD-1 antibody fragment), comprises at least one post-translational modification of the amino acid sequence.
  • Post-translational modifications can include, e.g., glycosylation, phosphorylation, citrullination, isomerization, ubiquitination, acetylation, hydroxylation, methylation, AMPylation, prenylation, deamidation, eliminylation, carbamylation and carbamoylation.
  • the post-translational modification is the modification of an amino acid side chain, such as conversion of an N-terminal amino acid (e.g., glutamate or glutamine) to pyroglutamate. In some such embodiments, this modification occurs at the N-terminus of the heavy chain.
  • the post-translational modification includes a post-translational modification of a heavy chain N-terminal glutamine (Q) to a pyroglutamate.
  • such a post- translational modification to pyroglutamate is a dominant post-translational modification; in some such embodiments, the antibody or antigen binding fragment thereof in which an N-terminal pyroglutamate is present represents greater than 50%, greater than 60%, greater than 70%, greater than 80%, greater than 90%, or greater than 95% (w/w) of the antibody or antigen binding fragment thereof in the composition.
  • the post-translational modification is the cleavage of one or more, particularly one or two, amino acids from the C-terminus of the heavy chain.
  • an antibody produced by expression of a specific nucleic acid molecule encoding a full-length heavy chain may include the full-length heavy chain, or it may include a cleaved variant of the full-length heavy chain. This may be the case where the final two C-terminal amino acids of the heavy chain are glycine and lysine, respectively. Therefore, the C- terminal lysine, or the C-terminal glycine and lysine, of the Fc region may or may not be present.
  • an antibody may include a heavy chain constant domain with both a C-terminal glycine and lysine, without the C-terminal lysine, or without both the C-terminal glycine and lysine.
  • glycine cleavage is not a dominant post-translational modification; in some such embodiments, the binding molecule in which the C-terminal lysine is cleaved from the heavy chain constant domain represents less than 10%, less than 5%, or less than 4% (w/w) of the antibody or antigen binding fragment thereof in the composition.
  • the provided polynucleotides can be incorporated into constructs, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) constructs, such as those that can be introduced into cells for expression thereof.
  • a provided binding molecule contains an Fc region comprising at least part of the hinge, CH2, and CH3 regions.
  • Certain embodiments have the first heavy-chain constant region (CHI) containing the site necessary for light chain bonding, present in at least one of the fusions.
  • DNAs encoding the immunoglobulin heavy chain fusions and, if desired, the immunoglobulin light chain are inserted into separate expression vectors, and are co-transfected into a suitable host cell. This provides for greater flexibility in adjusting the mutual proportions of the three polypeptide fragments in embodiments when unequal ratios of the three polypeptide chains used in the construction provide the optimum yield of the desired fusion antibody.
  • binding molecules such as bispecific antibodies, contain a PD-1 binding domain, including antigen-binding fragments as described in Section I.
  • the binding molecules include antibodies that specifically bind to PD-1, e.g., human PD-1. Exemplary antibodies are described in Section I.
  • the PD-1 binding domain e.g., the anti- PD-1 antibody fragment, contains a PD-1 heavy chain variable region (VH) region sequence and a PD- 1 light chain variable region (VL) region sequence as described.
  • the PD-1 binding domain contains a VH region and a VL region containing a combination of six CDRs as described herein, such as in Section I above.
  • the PD-1 binding domain contains a PD-1 VH region and a PD-1 VL region as described herein, such as in Section I above.
  • a PD-1 VH region sequence can be any of the PD-1 VH region sequences described herein.
  • a PD-1 VL region sequence can be any of the PD-1 VL region sequence described herein.
  • any of the PD-1 VH region sequence and any of the PD-1 VL region sequence described herein can be used in combination.
  • PD-1 binding domains include any combination of the heavy chain and light chain complementarity-determining regions (CDRs) discussed herein.
  • the PD-1 binding domain comprises any one of the CDR-H1 as described herein, any one of the CDR-H2 as described herein, any one of the CDR-H3 as described herein, any one of the CDR-L1 as described herein, any one of the CDR-L2 as described herein and any one of the CDR-L3 as described herein.
  • any one or more of the CDR-H1, the CDR-H2 and the CDR-H3 sequences described herein, and any one or more of the CDR-L1, the CDR-L2 and the CDR-L3 sequences described herein can be used in combination.
  • the PD-1 VH region and the PD-1 VL region of the PD-1 binding domain is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 168 and SEQ ID NO: 172, respectively.
  • the PD-1 VH region is or comprises an amino acid sequence set forth in SEQ ID NO: 168
  • the PD-1 VL region is or comprises an amino acid sequence set forth in SEQ ID NO: 172.
  • the PD-1 binding domain is a Fab.
  • the Fab comprises at least a portion of an immunoglobulin constant region or a variant thereof, such as a CHI.
  • an PD-1 binding domain comprises one heavy chain or portion thereof comprising a PD-1 VH region and at least a portion of a heavy chain constant region (e.g., CHI), and at least one light chain comprising a PD-1 VL region and at least a portion of a light chain constant region.
  • the PD-lbinding domain is a crossover Fab (also termed “Crossfab”).
  • the PD-lcrossover Fab is a Fab molecule in which the variable domains of the PD-1 Fab heavy and light chain are exchanged (i.e., replaced by each other).
  • the crossover PD-1 Fab comprises a peptide chain composed of the light chain variable domain VL and the heavy chain constant domain 1 CHI (e.g., VL-CH1, in N- to C-terminal direction), and a peptide chain composed of the heavy chain variable domain VH and the light chain constant domain CL (e.g., VH-CL, in N- to C-terminal direction).
  • binding molecules such as bispecific antibodies, contain a CTLA-4 binding domain, including antigen-binding fragments.
  • the binding molecules include antibodies that specifically bind to CTLA-4, e.g., human CTLA-4. Exemplary antibodies are described below.
  • the CTLA-4 binding domain, e.g., the anti-CTLA-4 antibody fragment contains a CTLA-4 heavy chain variable region (VH) region sequence and a CTLA-4 light chain variable region (VL) region sequence as described.
  • VH CTLA-4 heavy chain variable region
  • VL CTLA-4 light chain variable region
  • the CTLA-4 binding domain includes one or more copies of an antibody or an antigen-binding fragment thereof selected from the group consisting of a Fab fragment, a F(ab')2 fragment, an Fv fragment, or an a scFv.
  • the CTLA-4 binding domain is monovalent for binding CTLA-4.
  • the CTLA-4 binding domain is a Fab.
  • the CTLA-4 binding domain contains a VH region and a VL region containing a combination of six CDRs as described below.
  • the CTLA-4 binding domain contains a CTLA-4 VH region and a CTLA-4 VL region as described below.
  • a CTLA-4 VH region sequence can be any of the CTLA-4 VH region sequences described herein.
  • a CTLA-4 VL region sequence can be any of the CTLA-4 VL region sequence described herein.
  • any of the CTLA-4 VH region sequence and any of the CTLA-4 VL region sequence described herein can be used in combination.
  • the CTLA-4 binding domain is a Fab.
  • the Fab comprises at least a portion of an immunoglobulin constant region or a variant thereof, such as a CHI.
  • an CTLA-4 binding domain comprises one heavy chain or portion thereof comprising a CTLA-4 VH region and at least a portion of a heavy chain constant region (e.g., CHI), and at least one light chain comprising a CTLA-4 VL region and at least a portion of a light chain constant region.
  • the CTLA-4 binding domain is a crossover Fab (also termed “Crossfab”).
  • the CTLA-4 crossover Fab is a Fab molecule in which the variable domains of the CTLA-4 Fab heavy and light chain are exchanged (i.e., replaced by each other).
  • the crossover CTLA-4 Fab comprises a peptide chain composed of the light chain variable domain VL and the heavy chain constant domain 1 CHI (e.g., VL-CH1, in N- to C- terminal direction), and a peptide chain composed of the heavy chain variable domain VH and the light chain constant domain CL (e.g., VH-CL, in N- to C-terminal direction).
  • CTLA-4 binding domains include any combination of the heavy chain and light chain complementarity-determining regions (CDRs) discussed herein.
  • the CTLA-4 binding domain comprises any one of the CDR-H1 as described herein, any one of the CDR-H2 as described herein, any one of the CDR-H3 as described herein, any one of the CDR-L1 as described herein, any one of the CDR-L2 as described herein and any one of the CDR-L3 as described herein.
  • any one or more of the CDR-H1, the CDR- H2 and the CDR-H3 sequences described herein, and any one or more of the CDR-L1, the CDR- L2 and the CDR-L3 sequences described herein can be used in combination.
  • CTLA-4 binding domains are provided in Tables 3A and 3B.
  • a provided CTLA-4 binding domain has a CDR-H1, a CDR-H2 and a CDR-H3 present in a CTLA-4 VH region amino acid sequence set forth in any one of SEQ ID NOs: 230-256, or 341, or an amino acid sequence that has at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the CTLA-4 VH region amino acid sequence set forth in any one of SEQ ID NOs: 230-256, or 341.
  • a provided CTLA-4 binding domain has a CDR-L1, a CDR-L2 and a CDR-L3 present in a CTLA-4 VL region amino acid sequence set forth in any one of SEQ ID NOs: 257- 277, 347, or an amino acid sequence that has at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the CTLA-4 VL region amino acid sequence set forth in any one of SEQ ID NOs: 257-277, or 347.
  • the provided CTLA-4 binding domain contains a combination of any of such six CDRs (a CDR- Hl, a CDR-H2, a CDR-H3, a CDR-L1, a CDR-L2 and a CDR-L3) described above.
  • a provided CTLA-4 binding domain has a CDR-H1, a CDR-H2 and a CDR-H3 present in a CTLA-4 VH region amino acid sequence set forth in any one of SEQ ID NOs: 230- 256, 341, and a CDR-L1, a CDR-L2 and a CDR-L3 present in a CTLA-4 VL region amino acid sequence set forth in any one of SEQ ID NOs: 257-277, 347, wherein the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 are not set forth in SEQ ID NOs: 278-286, 297, 303, 318 and 327, respectively.
  • the combination of six CDRs (a CDR-H1, a CDR-H2, a CDR-H3, a CDR-L1, a CDR-L2 and a CDR-L3) is according to Kabat numbering. In some embodiments, the combination of six CDRs (a CDR-H1, a CDR-H2, a CDR-H3, a CDR-L1, a CDR-L2 and a CDR-L3) is according to Chothia numbering.
  • the combination of six CDRs (a CDR-H1, a CDR-H2, a CDR-H3, a CDR-L1, a CDR-L2 and a CDR- L3) is according to AbM numbering, In some embodiments, the combination of six CDRs (a CDR-H1, a CDR-H2, a CDR-H3, a CDR-L1, a CDR-L2 and a CDR-L3) is according to IMGT numbering. In some embodiments, the combination of six CDRs (a CDR-H1, a CDR-H2, a CDR- H3, a CDR-L1, a CDR-L2 and a CDR-L3) is according to IgBLAST numbering.
  • the CTLA-4 VH region comprises a heavy chain complementarity determining region 1 (CDR-H1) comprising the sequence set forth in any one of SEQ ID NOs: 278- 285, a heavy chain complementarity determining region 2 (CDR-H2) comprising the sequence set forth in any one of SEQ ID NOs: 286-296, and a heavy chain complementarity determining region 3 (CDR-H3) comprising the sequence set forth in any one of SEQ ID NOs: 297-302; and the CTLA-4 VL region comprises a light chain complementarity determining region 1 (CDR-L1) comprising the sequence set forth in any one of SEQ ID NOs: 303-317, a light chain complementarity determining region 2 (CDR-L2) comprising the sequence set forth in any one of SEQ ID NOs: 318-326, and a light chain complementarity determining region
  • the CTLA-4 VH region comprises a heavy chain complementarity determining region 1 (CDR-H1), a heavy chain complementarity determining region 2 (CDR-H2), and a heavy chain complementarity determining region 3 (CDR-H3) contained within any one of SEQ ID NOs: 230-256, and 341
  • the CTLA-4 VL region comprises a light chain complementarity determining region 1 (CDR-L1), a light chain complementarity determining region 2 (CDR-L2), and a light chain complementarity determining region 3 (CDR-L3) contained within any one of SEQ ID NOs:257, 277, and 347.
  • the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 are not set forth in SEQ ID NOS: 278, 286, 297,303, 318, and 327, respectively.
  • the CTLA-4 VH region comprises a CDR-H1 comprising the sequence set forth in any one of SEQ ID NOs: 278-285, a CDR-H2 comprising the sequence set forth in any one of SEQ ID NOs: 286-296, and a CDR-H3 comprising the sequence set forth in any one of SEQ ID NOs: 297-302; and the CTLA-4 VL region comprises a CDR-L1 comprising the sequence set forth in any one of SEQ ID NOs: 303-317, a CDR-L2 comprising the sequence set forth in any one of SEQ ID NOs: 318-326, and a CDR-L3 comprising the sequence set forth in any one of SEQ ID NOs: 327-329.
  • the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 are not set forth in SEQ ID NOS: 278, 286, 297,303, 318, and 327, respectively.
  • the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 230
  • the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 347
  • the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 231
  • the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 347.
  • the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 232
  • the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 347
  • the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO:233
  • the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 347.
  • the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO:234, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 347.
  • the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO:235
  • the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 347.
  • the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO:236, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 347.
  • the CTLA-4 VH region comprises a CDR- Hl, a CDR-H2, and a CDR-H3 contained within SEQ ID NO:237
  • the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 347.
  • the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO:238, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 347.
  • the CTLA-4 VH region comprises a CDR- Hl, a CDR-H2, and a CDR-H3 contained within SEQ ID NO:239
  • the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 347.
  • the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO:240
  • the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 347
  • the CTLA-4 VH region comprises a CDR- Hl, a CDR-H2, and a CDR-H3 contained within SEQ ID NO:241
  • the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 347.
  • the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO:242, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 347.
  • the CTLA-4 VH region comprises a CDR- Hl, a CDR-H2, and a CDR-H3 contained within SEQ ID NO:243
  • the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 347.
  • the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO:244, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 347.
  • the CTLA-4 VH region comprises a CDR- Hl, a CDR-H2, and a CDR-H3 contained within SEQ ID NO:245, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 347.
  • the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO:246, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 347.
  • the CTLA-4 VH region comprises a CDR- Hl, a CDR-H2, and a CDR-H3 contained within SEQ ID NO:247
  • the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 347.
  • the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO:248, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 347.
  • the CTLA-4 VH region comprises a CDR- Hl, a CDR-H2, and a CDR-H3 contained within SEQ ID NO:249
  • the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 347.
  • the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO:250
  • the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 347
  • the CTLA-4 VH region comprises a CDR- Hl, a CDR-H2, and a CDR-H3 contained within SEQ ID NO:251
  • the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 347.
  • the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 341
  • the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 257.
  • the CTLA-4 VH region comprises a CDR- Hl, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 341
  • the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 258.
  • the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 341
  • the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 259.
  • the CTLA-4 VH region comprises a CDR- Hl, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 341
  • the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 260.
  • the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 341
  • the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 261.
  • the CTLA-4 VH region comprises a CDR- Hl, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 341
  • the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 262.
  • the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 34
  • the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 263.
  • the CTLA-4 VH region comprises a CDR- Hl, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 341
  • the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 264.1n some embodiments, the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 341
  • the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 265.
  • the CTLA-4 VH region comprises a CDR- Hl, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 341
  • the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 266.
  • the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 341
  • the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 267.
  • the CTLA-4 VH region comprises a CDR- Hl, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 341
  • the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 268.
  • the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 341
  • the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 269.
  • the CTLA-4 VH region comprises a CDR- Hl, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 341
  • the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 270.
  • the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 341
  • the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 271.
  • the CTLA-4 VH region comprises a CDR- Hl, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 341
  • the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 272.
  • the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 341
  • the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 273.
  • the CTLA-4 VH region comprises a CDR- Hl, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 341
  • the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 274.
  • the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 341
  • the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 275.
  • the CTLA-4 VH region comprises a CDR- Hl, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 252, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 270.
  • the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 253, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 270.
  • the CTLA-4 VH region comprises a CDR- Hl, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 254, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 270.
  • the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 255
  • the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 270.
  • the CTLA-4 VH region comprises a CDR- Hl, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 251, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 270.
  • the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 256, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 270.
  • the CTLA-4 VH region comprises a CDR- Hl, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 252, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 276.
  • the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 253, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 276.
  • the CTLA-4 VH region comprises a CDR- Hl, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 254, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 276.
  • the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 255
  • the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 276.
  • the CTLA-4 VH region comprises a CDR- Hl, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 251, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 276.
  • the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 256, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 276.
  • the CTLA-4 VH region comprises a CDR- Hl, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 252, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 258 In some embodiments, the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 253, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 258.
  • the CTLA-4 VH region comprises a CDR- Hl, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 254, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 258.
  • the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 255
  • the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 258.
  • the CTLA-4 VH region comprises a CDR- Hl, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 251, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 258.
  • the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 256
  • the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 258.
  • the CTLA-4 VH region comprises a CDR- Hl, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 252, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 268.
  • the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 253
  • the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 268.
  • the CTLA-4 VH region comprises a CDR- Hl, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 254, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 268.
  • the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 255
  • the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 268.
  • the CTLA-4 VH region comprises a CDR- Hl, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 251, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 268.
  • the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 256
  • the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 268.
  • the CTLA-4 VH region comprises a CDR- Hl, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 234, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 277.
  • the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 287, and 297, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 303, 318, and 327, respectively.
  • the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 286, and 298, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 303, 318, 327, respectively.
  • the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 280, 286, and 297, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 303, 318, and 327, respectively.
  • the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 279, 288, and 297, respectively, and the -LI, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 303, 318, and 327, respectively.
  • the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 281, 289, and 297, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 303, 318, and 327, respectively.
  • the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 281, 290, and 297, respectively, and the CDR-L1, the CDR- L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 303, 318, and 327, respectively.
  • the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 280, 291, 297, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 303, 318, and 327, respectively.
  • the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 279, 292, and 297, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 303, 318, and 327, respectively.
  • the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 279, 286, and 298, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 303, 318, and 327, respectively.
  • the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 282, 286, and 297, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 303, 318, and 327, respectively.
  • the CDR-H1, the CDR- H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 282, 286, and 299, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 303, 318, and 327, respectively.
  • the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 283, 286, and 300, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 303, 318, and 327, respectively.
  • the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 284, 286, and 297, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 303, 318, and 327, respectively.
  • the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 285, 286, and 301, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 303, 318, and 327, respectively.
  • the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 293, 297, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 303, 318, and 327, respectively.
  • the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 289, and 297, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 303, 318, and 327, respectively.
  • the CDR-H1, the CDR- H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 294, 297, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 303, 318, and 327, respectively.
  • the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 295, and 297, respectively, and the CDR-L1, a CDR-L2, and a CDR-L3 comprise the sequences set forth in SEQ ID NOS: 303, 318, and 327, respectively.
  • the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 286, and 300, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 303, 318, and 327, respectively.
  • the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 286, and 302, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 303, 318, and 327, respectively.
  • the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 286, and 301, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 303, 318, and 327, respectively.
  • the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 286, and 297, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 304, 318, and 327, respectively.
  • the CDR-H1, the CDR- H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 286, and 297, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 305, 318, and 327, respectively.
  • the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 286, and 297, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 306, 318, 327, respectively.
  • the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 286, and 297, respectively, and the CDR-L1, the CDR- L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 307, 319, and 327, respectively.
  • the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 286, and 297, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 307, 319, and 327, respectively.
  • the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 286, and 297, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 310, 320, and 327, respectively.
  • the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 286, and 297, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 307, 318, and 328, respectively.
  • the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 286, and 297, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 310, 321, and 327, respectively.
  • the CDR-H1, the CDR- H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 286, and 297, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 311, 318, and 327, respectively.
  • the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 286, and 297, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 311, 322, and 327, respectively.
  • the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 286, and 297, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 312, 321, and 327, respectively.
  • the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 286, and 297, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 313, 318, and 328, respectively.
  • the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 286, and 297, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 313, 318, and 329, respectively.
  • the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 286, and 297, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 314, 318, and 327, respectively.
  • the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 286, and 297, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 315, 323, and 327, respectively.
  • the CDR-H1, the CDR- H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 286, and 297, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 316, 324, and 327, respectively.
  • the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 286, and 297, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 314, 325, and 327, respectively.
  • the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 286, and 297, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 317, 318, and 327, respectively.
  • the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 286, and 297, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 303, 326, and 327, respectively.
  • the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 296, and 297, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 314, 318, and 327respectively.
  • the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 294, and 302, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 314, 318, and 327, respectively.
  • the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 281, 289, and 301, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 314, 318, and 327, respectively.
  • the CDR-H1, the CDR- H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 281, 296, and 297, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 314, 318, and 327, respectively.
  • the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 286, and 301, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 314, 318, and 327, respectively.
  • the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 294, and 301, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 314, 318, and 327, respectively.
  • the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 296, and 297, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 313, 318, and 327, respectively.
  • the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 294, and 302, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 313, 318, and 327, respectively.
  • the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 281, 289, and 301, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 313, 318, and 327, respectively.
  • the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 281, 296, and 297, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 313, 318, and 327, respectively.
  • the CDR-H1, the CDR- H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 286, and 301, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 313, 318, and 327, respectively.
  • the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 294, and 301, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 313, 318, and 327, respectively.
  • the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 296, and 297, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 305, 318, and 327, respectively.
  • the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 294, and 302, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 305, 318, and 327, respectively.
  • the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 281, 289, and 301, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 305, 318, and 327, respectively.
  • the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 281, 296, and 297, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 305, 318, and 327, respectively.
  • the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 286, and 301, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 305, 318, and 327, respectively.
  • the CDR-H1, the CDR- H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 294, 301, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 305, 318, 327, respectively.
  • the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 296, and 297, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 313, 318, and 328, respectively.
  • the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 294, and 302, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 313, 318, and 328, respectively.
  • the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 281, 289, and 301, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 313, 318, and 328, respectively.
  • the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 281, 296, and 297, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 313, 318, and 328, respectively.
  • the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 286, and 301, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 313, 318, and 328, respectively.
  • the CDR-H1, the CDR- H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 294, and 301, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 313, 318, and 328, respectively.
  • the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 281, 289, and 297, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 317, 318, 329, respectively.
  • the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 230 and SEQ ID NO: 347, respectively.
  • the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 231 and SEQ ID NO: 347, respectively.
  • the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 232 and SEQ ID NO: 347, respectively.
  • the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 233 and SEQ ID NO: 347, respectively.
  • the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 234 and SEQ ID NO: 347, respectively.
  • the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 235 and SEQ ID NO: 347, respectively.
  • the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 236 and SEQ ID NO: 347, respectively.
  • the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 237 and SEQ ID NO: 347, respectively.
  • the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 238 and SEQ ID NO: 347, respectively.
  • the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 239 and SEQ ID NO: 347, respectively.
  • the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 240 and SEQ ID NO: 347, respectively.
  • the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 241 and SEQ ID NO: 347, respectively.
  • the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 242 and SEQ ID NO: 347, respectively.
  • the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 243 and SEQ ID NO: 347, respectively.
  • the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 244 and SEQ ID NO: 347, respectively.
  • the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 245 and SEQ ID NO: 347, respectively.
  • the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 246 and SEQ ID NO: 347, respectively.
  • the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 247 and SEQ ID NO: 347, respectively.
  • the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 248 and SEQ ID NO: 347, respectively.
  • the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 249 and SEQ ID NO: 347, respectively.
  • the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 250 and SEQ ID NO: 347, respectively.
  • the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 251 and SEQ ID NO: 347, respectively.
  • the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 341 and SEQ ID NO: 257, respectively.
  • the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 341 and SEQ ID NO: 258, respectively.
  • the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 341 and SEQ ID NO: 259, respectively.
  • the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 341 and SEQ ID NO: 260, respectively.
  • the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 341 and SEQ ID NO: 261, respectively.
  • the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 341 and SEQ ID NO: 262, respectively.
  • the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 341 and SEQ ID NO: 263, respectively.
  • the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 341 and SEQ ID NO: 264, respectively.
  • the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 341 and SEQ ID NO: 265, respectively.
  • the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 341 and SEQ ID NO: 266, respectively.
  • the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 341 and SEQ ID NO: 267, respectively.
  • the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 341 and SEQ ID NO: 268, respectively.
  • the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 341 and SEQ ID NO: 269, respectively.
  • the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 341 and SEQ ID NO: 270, respectively.
  • the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 341 and SEQ ID NO: 271, respectively.
  • the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 341 and SEQ ID NO: 272, respectively.
  • the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 341 and SEQ ID NO: 273, respectively.
  • the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 341 and SEQ ID NO: 274, respectively.
  • the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 341 and SEQ ID NO: 275, respectively.
  • the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 252 and SEQ ID NO: 270, respectively.
  • the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 253 and SEQ ID NO: 270, respectively.
  • the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 254 and SEQ ID NO: 270, respectively.
  • the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 255 and SEQ ID NO: 270, respectively.
  • the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 251 and SEQ ID NO: 270, respectively.
  • the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 256 and SEQ ID NO: 270, respectively.
  • the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 252 and SEQ ID NO: 276, respectively.
  • the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 253 and SEQ ID NO: 276, respectively.
  • the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 254 and SEQ ID NO: 276, respectively.
  • the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 255 and SEQ ID NO: 276, respectively.
  • the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 251 and SEQ ID NO: 276, respectively.
  • the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 256 and SEQ ID NO: 276, respectively.
  • the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 252 and SEQ ID NO: 258, respectively.
  • the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 253 and SEQ ID NO: 258, respectively.
  • the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 254 and SEQ ID NO: 258, respectively.
  • the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 255 and SEQ ID NO: 258, respectively.
  • the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 251 and SEQ ID NO: 258, respectively.
  • the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 256 and SEQ ID NO: 258, respectively.
  • the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 252 and SEQ ID NO: 268, respectively.
  • the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 253 and SEQ ID NO: 268, respectively.
  • the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 254 and SEQ ID NO: 268, respectively.
  • the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 255 and SEQ ID NO: 268, respectively.
  • the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 251 and SEQ ID NO: 268, respectively.
  • the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 256 and SEQ ID NO: 268, respectively.
  • CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 234 and SEQ ID NO: 277, respectively.
  • the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 254 and SEQ ID NO: 268, respectively.
  • the CTLA-4 VH region comprises a CDR-H1 comprising the sequence set forth in SEQ ID NO: 278 or SEQ ID NO: 281, a CDR-H2 comprising the sequence set forth in SEQ ID NO: 289 or SEQ ID NO: 296, and a CDR-H3 comprising the sequence set forth in SEQ ID NO: 297 or SEQ ID NO: 301; and the CTLA-4 VL region comprises a CDR-L1 comprising the sequence set forth in SEQ ID NO: 305 or SEQ ID NO: 313; a CDR-L2 comprising the sequence set forth in SEQ ID NO: 318, and a CDR-L3 comprising the sequence set forth in SEQ ID NO: 327 or SEQ ID NO: 328.
  • the CTLA-4 VH region and the CTLA-4 VL region are or comprise: the sequence set forth in SEQ ID NO: 230 and 347, respectively; the sequence set forth in SEQ ID NO: 231 and 347, respectively; the sequence set forth in SEQ ID NO: 232 and 347, respectively; the sequence set forth in SEQ ID NO: 233 and 347, respectively; the sequence set forth in SEQ ID NO: 234 and 347, respectively; the sequence set forth in SEQ ID NO: 235 and 347, respectively; the sequence set forth in SEQ ID NO: 236 and 347, respectively; the sequence set forth in SEQ ID NO: 237 and 347, respectively; the sequence set forth in SEQ ID NO: 238 and 347, respectively; the sequence set forth in SEQ ID NO: 239 and 347, respectively; the sequence set forth in SEQ ID NO: 240 and 347, respectively; the sequence set forth in SEQ ID NO: 241 and 347, respectively; the sequence set forth in SEQ ID NO: 24
  • Table E5 provides the SEQ ID NOS: of exemplary provided CTLA-4 binding domains.
  • the CTLA-4-binding domain comprises a CTLA-4 VH region that comprises the CDR-H1, the CDR-H2 and the CDR-H3 sequence and a CTLA-4 VL region that comprises the CDR-L1, the CDR-L2 and the CDR-L3 sequence set forth in the SEQ ID NOS: listed in each row of Table E5 below (by Kabat numbering scheme).
  • the CTLA-4-binding domain comprises a CTLA-4 VH region sequence and a CTLA-4 VL region sequence set forth in the SEQ ID NOS: listed in each row of Table E5 below, or an antibody comprising a CTLA-4 VH region and a CTLA-4 VL region amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the CTLA-4 VH region sequence and the CTLA-4 VL region sequence set forth in the SEQ ID NOS: listed in each row of Table E5 below.
  • the CTLA-4-binding domain comprises a CTLA-4 VH region sequence and a CTLA-4 VL region sequence set forth in the SEQ ID NOS: listed in each row of Table E5 below.
  • the CTLA-4 binding domain comprises a CTLA-4 VH region and a CTLA-4 VL region, wherein the CTLA-4 VH region of the CTLA-4 binding domain can contain a combination of any of the CDR-H1, the CDR-H2 and the CDR-H3 amino acid sequences set forth in Table E5, and the CTLA-4 VL region of the CTLA-4 binding domain can contain a combination of any of the CDR-L1, the CDR-L2 and the CDR-L3 amino acid sequences set forth in Table E5.
  • the provided CTLA-4 binding domain comprises a CTLA-4 VH region and/or a CTLA-4 VL region set forth in Table E5, in any combination, orientation or containing a different linker.
  • the CTLA-4 binding domain comprises a CTLA-4 VH region described in Table E5.
  • the CTLA-4 binding domain comprises a CTLA-4 VL region described in Table E5.
  • provided CTLA-4 binding domains include those that include amino acid residues (such as due to mutation compared to a reference sequence) that alter pH binding such that the CTLA-4 binding domains exhibit better binding at neutral pH (e.g., pH 7.4) compared to acidic pH (e.g., pH 5.0).
  • neutral pH e.g., pH 7.4
  • acidic pH e.g., pH 5.0
  • CTLA-4 binding domains e.g., anti-CTLA-4 antibodies, including antibody fragments
  • provided CTLA-4 binding domains e.g., anti-CTLA-4 antibodies, including antibody fragments
  • CTLA-4 VH region that is S31H, D54H, D54E, Y59H, W101H, or L102H, which correspond to S31R, D53H, D53E, Y58H, W97H, or L98H based on Kabat numbering, respectively, or any combination thereof, compared to the CTLA-4 VH region set forth in SEQ ID NO:341; and/or one or more amino acid mutation in the CTLA-4 VL region that is S28H, S31H, S32H, or Q91H, which correspond to S27aH, S30H, S31H, or Q90H based on Kabat numbering, respectively, or any combination thereof, compared to the CTLA-4 VL region set forth in SEQ ID NO:347.
  • the provided CTLA-4 binding domains comprises one or more mutations selected from the group consisting of: S31R, D54H, D54E, Y59H, W101H, and L102H (S31R, D53H, D53E, Y58H, W97H, and L98H, respectively, based on Kabat numbering) in the CTLA-4 VH region, such as compared to a reference CTLA-4 binding domain, such as an anti-CTLA-4 antibody VH region, e.g. set forth in SEQ ID NO: 341.
  • the provided CTLA-4 binding domains (e.g., anti-CTLA-4 antibodies, including antibody fragments) comprise a histidine (H) at position 98 based on Kabat numbering (e.g., L102H mutation compared to SEQ ID NO:341) in the CTLA-4 VH region.
  • the provided CTLA-4 binding domains (e.g., anti-CTLA-4 antibodies, including antibody fragments) comprises an arginine (R) at position 31 based on Kabat numbering) (e.g., S31R mutation compared to SEQ ID NO:341) in the CTLA-4 VH region.
  • the provided anti-CTLA-4 antibody or antigen-binding fragment thereof comprises a glutamic acid (E) at position 53 based on Kabat numbering) (e.g., D54E mutation compared to SEQ ID NO:341) in the CTLA-4 VH region.
  • the provided CTLA-4 binding domains e.g., anti-CTLA-4 antibodies, including antibody fragments
  • the provided CTLA-4 binding domains comprises a histidine (H) at position 58 based on Kabat numbering (e.g., Y59H mutation compared to SEQ ID NO:341) in the CTLA-4 VH region.
  • the provided CTLA-4 binding domains comprises a histidine (H) at position 97 based on Kabat numbering (e.g., W101H mutation compared to SEQ ID NO:341) in the CTLA-4 VH region.
  • the provided CTLA-4 binding domains (e.g., anti-CTLA-4 antibodies, including antibody fragments) comprise a histidine (H) at position 98 based on Kabat numbering and a arginine (R) at position 31 based on Kabat numbering (e.g., L102H and S31R mutation compared to SEQ ID NO: 341) in the CTLA-4 VH region.
  • H histidine
  • R arginine
  • the provided CTLA-4 binding domains comprises a histidine (H) at position 98 based on Kabat numbering and a glutamic acid (E) at position 53 based on Kabat numbering (e.g., L102H and D54E mutations compared to SEQ ID NO:341) in the CTLA-4 VH region.
  • H histidine
  • E glutamic acid
  • the provided CTLA-4 binding domains comprises a histidine (H) at position 98 based on Kabat numbering and a histidine (H) at position 53 based on Kabat numbering (e.g., L102H and D54H mutations compared to SEQ ID NO:341) in the CTLA-4 VH region.
  • H histidine
  • H histidine
  • the provided CTLA-4 binding domains comprises a histidine (H) at position 98 based on Kabat numbering and a histidine (H) at position 58 based on Kabat numbering (e.g., L102H and Y59H mutations compared to SEQ ID NO:341) in the CTLA-4 VH region.
  • H histidine
  • H histidine
  • the provided CTLA-4 binding domains comprises a histidine (H) at position 98 based on Kabat numbering and a histidine (H) at position 97 based on Kabat numbering (e.g., L102H and W101H mutations compared to SEQ ID NO:341) in the CTLA-4 VH region.
  • H histidine
  • H histidine
  • the provided CTLA-4 binding domains comprises an arginine (R) at position 31 based on Kabat numbering) and a glutamic acid (E) at position 53 based on Kabat numbering (e.g., S31R and D54E mutations compared to SEQ ID NO: 341) in the CTLA-4 VH region.
  • R arginine
  • E glutamic acid
  • the provided CTLA-4 binding domains (e.g., anti-CTLA-4 antibodies, including antibody fragments) comprise an arginine (R) at position 31 based on Kabat numbering and a histidine (H) at position 53 based on Kabat numbering (e.g., S31R and D54H mutations compared to SEQ ID NO: 341) in the CTLA-4 VH region.
  • R arginine
  • H histidine
  • the provided CTLA-4 binding domains comprises an arginine (R) at position 31 based on Kabat numbering and a histidine (H) at position 58 based on Kabat numbering (e.g., S31R and Y59H mutations compared to SEQ ID NO: 341) in the CTLA-4 VH region.
  • R arginine
  • H histidine
  • the provided CTLA-4 binding domains comprises an arginine (R) at position 31 based on Kabat numbering and a histidine (H) at position 97 based on Kabat numbering (e.g., S31R and W101H mutations compared to SEQ ID NO: 341) in the CTLA-4 VH region.
  • R arginine
  • H histidine
  • the provided CTLA-4 binding domains comprises a glutamic acid (E) at position 53 based on Kabat numbering and a histidine (H) at position 58 based on Kabat numbering (e.g., D54E and Y59H mutations compared to SEQ ID NO: 341) in the CTLA-4 VH region.
  • E glutamic acid
  • H histidine
  • the provided CTLA-4 binding domains comprises a glutamic acid (E) at position 53 based on Kabat numbering and a histidine (H) at position 97 based on Kabat numbering (e.g., D54E and W101H mutations compared to SEQ ID NO: 341) in the CTLA-4 VH region.
  • E glutamic acid
  • H histidine
  • the provided CTLA-4 binding domains comprises a histidine (H) at position 53 based on Kabat numbering and a histidine (H) at position 58 based on Kabat numbering (e.g., D54H and Y59H mutations compared to SEQ ID NO: 341) in the CTLA-4 VH region.
  • H histidine
  • H histidine
  • the provided CTLA-4 binding domains comprises a histidine (H) at position 53 based on Kabat numbering and a histidine (H) at position 97 based on Kabat numbering (e.g., D54H and W101H mutations compared to SEQ ID NO: 341) in the CTLA-4 VH region.
  • H histidine
  • H histidine
  • the provided CTLA-4 binding domains comprises a histidine (H) at position 58 based on Kabat numbering and a histidine (H) at position 97 based on Kabat numbering (e.g., Y59H and W101H mutations compared to SEQ ID NO: 341) in the CTLA-4 VH region.
  • the provided CTLA-4 binding domains comprises a histidine (H) at position 98 based on Kabat numbering, an arginine (R) at position 31 based on Kabat numbering and a glutamic acid (E) at position 53 based on Kabat numbering (e.g., L102H, S31R, and D54E mutations compared to SEQ ID NO: 341) in the CTLA-4 VH region.
  • H histidine
  • R arginine
  • E glutamic acid
  • the provided CTLA-4 binding domains comprises a histidine (H) at position 98 based on Kabat numbering, an arginine (R) at position 31 based on Kabat numbering and a histidine (H) at position 53 based on Kabat numbering (e.g., L102H, S31R, and D54H mutations compared to SEQ ID NO: 341) in the CTLA-4 VH region.
  • H histidine
  • R arginine
  • H histidine
  • the provided CTLA-4 binding domains comprises a histidine (H) at position 98 based on Kabat numbering, an arginine (R) at position 31 based on Kabat numbering and a histidine (H) at position 58 based on Kabat numbering (e.g., L102H, S31R, and Y59H mutations compared to SEQ ID NO: 341) in the CTLA-4 VH region.
  • H histidine
  • R arginine
  • H histidine
  • the provided CTLA-4 binding domains comprises a histidine (H) at position 98 based on Kabat numbering, an arginine (R) at position 31 based on Kabat numbering and a histidine (H) at position 97 based on Kabat numbering (e.g., L102H, S31R, and W101H mutations compared to SEQ ID NO: 341) in the CTLA-4 VH region.
  • H histidine
  • R arginine
  • H histidine
  • the provided CTLA-4 binding domains comprises a histidine (H) at position 98 based on Kabat numbering, a glutamic acid (E) at position 53 based on Kabat numbering and a histidine (H) at position 58 based on Kabat numbering (e.g., L102H, D54E, and Y59H mutations compared to SEQ ID NO: 341) in the CTLA-4 VH region.
  • H histidine
  • E glutamic acid
  • H histidine
  • the provided CTLA-4 binding domains comprises a histidine (H) at position 98 based on Kabat numbering, a glutamic acid (E) at position 53 based on Kabat numbering and a histidine (H) at position 97 based on Kabat numbering (e.g., L102H, D54E, and W101H mutations compared to SEQ ID NO: 341) in the CTLA-4 VH region.
  • H histidine
  • E glutamic acid
  • H histidine
  • the provided CTLA-4 binding domains comprises a histidine (H) at position 98 based on Kabat numbering, a histidine (H) at position 53 based on Kabat numbering and a histidine (H) at position 58 based on Kabat numbering (e.g., L102H, D54H, and Y59H mutations compared to SEQ ID NO: 341) in the CTLA-4 VH region.
  • the provided CTLA-4 binding domains comprises a histidine (H) at position 98 based on Kabat numbering, a histidine (H) at position 53 based on Kabat numbering and a histidine (H) at position 97 based on Kabat numbering (e.g., L102H, D54H, and W101H mutations compared to SEQ ID NO: 341) in the CTLA-4 VH region.
  • the provided CTLA-4 binding domains comprises a histidine (H) at position 98 based on Kabat numbering, a histidine (H) at position 58 based on Kabat numbering and a histidine (H) at position 97 based on Kabat numbering (e.g., L102H, Y59H, and W101H mutations compared to SEQ ID NO: 341) in the CTLA-4 VH region.
  • the provided CTLA-4 binding domains comprises an arginine (R) at position 31 based on Kabat numbering), a glutamic acid (E) at position based on Kabat numbering, and a histidine (H) at position 58 based on Kabat numbering (e.g., S31R, D54E, and Y59H mutations compared to SEQ ID NO: 341) in the CTLA-4 VH region.
  • R arginine
  • E glutamic acid
  • H histidine
  • the provided CTLA-4 binding domains comprises an arginine (R) at position 31 based on Kabat numbering), a glutamic acid (E) at position 53 based on Kabat numbering, and a histidine (H) at position 97 based on Kabat numbering (e.g., S31R, D54E, and W101H mutations compared to SEQ ID NO: 341) in the CTLA-4 VH region.
  • R arginine
  • E glutamic acid
  • H histidine
  • the CTLA-4 binding domains comprises an arginine (R) at position 31 based on Kabat numbering), a histidine (H) at position 53 based on Kabat numbering, and a histidine (H) at position 58 based on Kabat numbering (e.g., S31R, D54H, and Y59H mutations compared to SEQ ID NO: 341) in the CTLA-4 VH region.
  • R arginine
  • H histidine
  • H histidine
  • the provided CTLA-4 binding domains comprises an arginine (R) at position 31 based on Kabat numbering, a histidine (H) at position 53 based on Kabat numbering, and a histidine (H) at position 97 based on Kabat numbering (e.g., S31R, D54H, and W101H mutations compared to SEQ ID NO: 341) in the CTLA-4 VH region.
  • R arginine
  • H histidine
  • H histidine
  • the provided CTLA-4 binding domains comprises an arginine (R) at position 31 based on Kabat numbering), a histidine (H) at position 58 based on Kabat numbering, and a histidine (H) at position 97 based on Kabat numbering (e.g., S31R, Y59H, and W101H mutations compared to SEQ ID NO: 341) in the CTLA-4 VH region.
  • R arginine
  • H histidine
  • H histidine
  • the provided CTLA-4 binding domains comprises a glutamic acid (E) at position 53 based on Kabat numbering, a histidine (H) at position 58 based on Kabat numbering, and a histidine (H) at position 97 based on Kabat numbering (e.g., D54E, Y59H, and W101H mutations compared to SEQ ID NO: 341) in the CTLA-4 VH region.
  • E glutamic acid
  • H histidine
  • H histidine
  • H histidine
  • the provided CTLA-4 binding domains comprises a histidine (H) at position 53 based on Kabat numbering, a histidine (H) at position 58 based on Kabat numbering, and a histidine (H) at position 97 based on Kabat numbering (e.g., D54H, Y59H, and W101H mutations compared to SEQ ID NO: 341) in the CTLA-4 VH region.
  • the provided CTLA-4 binding domains comprises a histidine (H) at position 98 based on Kabat numbering, an arginine (R) at position 31 based on Kabat numbering, a glutamic acid (E) at position 53 based on Kabat numbering, and a histidine (H) at position 58 based on Kabat numbering (e.g., L102H, S31R, D54E, and Y59H mutations compared to SEQ ID NO: 341) in the CTLA-4 VH region.
  • H histidine
  • H histidine
  • the provided CTLA-4 binding domains comprises a histidine (H) at position 98 based on Kabat numbering, an arginine (R) at position 31 based on Kabat numbering, a glutamic acid (E) at position 53 based on Kabat numbering, and a histidine (H) at position 97 based on Kabat numbering (e.g., L102H, S31R, D54E, and W101H mutations compared to SEQ ID NO: 341) in the CTLA-4 VH region.
  • H histidine
  • the provided CTLA-4 binding domains comprises a histidine (H) at position 98 based on Kabat numbering, an arginine (R) at position 31 based on Kabat numbering, a histidine (H) at position 53 based on Kabat numbering, and a histidine (H) at position 58 based on Kabat numbering (e.g., L102H, S31R, D54H, and Y59H mutations compared to SEQ ID NO: 341) in the CTLA-4 VH region.
  • the provided CTLA-4 binding domains comprises a histidine (H) at position 98 based on Kabat numbering, an arginine (R) at position 31 based on Kabat numbering, a histidine (H) at position 53 based on Kabat numbering, and a histidine (H) at position 97 based on Kabat numbering (e.g., L102H, S31R, D54H, and W101H mutations compared to SEQ ID NO: 341) in the CTLA-4 VH region.
  • the provided CTLA-4 binding domains comprises a histidine (H) at position 98 based on Kabat numbering, an arginine (R) at position 31 based on Kabat numbering, a histidine (H) at position 58 based on Kabat numbering, and a histidine (H) at position 97 based on Kabat numbering (e.g., L102H, S31R, Y59H, and W101H mutations compared to SEQ ID NO: 341) in the CTLA-4 VH region.
  • the provided CTLA-4 binding domains comprises a histidine (H) at position 98 based on Kabat numbering, a glutamic acid (E) at position 53 based on Kabat numbering, a histidine (H) at position 58 based on Kabat numbering, and a histidine (H) at position 97 based on Kabat numbering (e.g., L102H, D54E, Y59H, and W101H mutations compared to SEQ ID NO: 341) in the CTLA-4 VH region.
  • H histidine
  • E glutamic acid
  • H histidine
  • H histidine
  • the provided CTLA-4 binding domains comprises a histidine (H) at position 98 based on Kabat numbering, a histidine (H) at position 53, a histidine (H) at position 58 based on Kabat numbering, and a histidine (H) at position 97 based on Kabat numbering (e.g., L102H, D54H, Y59H, and W101H mutations compared to SEQ ID NO: 341) in the CTLA-4 VH region.
  • H histidine
  • the provided CTLA-4 binding domains comprises an arginine (R) at position 31 based on Kabat numbering, a glutamic acid (E) at position 53 based on Kabat numbering, a histidine (H) at position 58 based on Kabat numbering, and a histidine (H) at position 97 based on Kabat numbering (e.g., S31R, D54E, Y59H, and W101H mutations compared to SEQ ID NO: 341) in the CTLA-4 VH region.
  • R arginine
  • E glutamic acid
  • H histidine
  • H histidine
  • the provided CTLA-4 binding domains comprises a histidine (H) at position 98 based on Kabat numbering, an arginine (R) at position 31 based on Kabat numbering, a glutamic acid (E) at position 53 based on Kabat numbering, a histidine (H) at position 58 based on Kabat numbering, and a histidine (H) at position 97 based on Kabat numbering (e.g., L102H, S31R, D54E, Y59H, and W101H mutations compared to SEQ ID NO: 341) in the CTLA-4 VH region.
  • H histidine
  • the provided CTLA-4 binding domains comprises a histidine (H) at position 98 based on Kabat numbering, an arginine (R) at position 31 based on Kabat numbering, a histidine (H) at position 53 based on Kabat numbering, a histidine (H) at position 58 based on Kabat numbering, and a histidine (H) at position 97 based on Kabat numbering (e.g., L102H, S31R, D54H, Y59H, and W101H mutations compared to SEQ ID NO: 341) in the CTLA-4 VH region.
  • the provided CTLA-4 binding domains comprises one or more mutations selected from the group consisting of: S28H, S31H, S32H, and Q91H (S27aH, S30H, S31H, and Q90H, respectively, based on Kabat numbering) in the CTLA-4 VL region, such as compared to a reference anti-CTLA-4 antibody VL, e.g. set forth in SEQ ID NO: 347.
  • the provided CTLA-4 binding domains comprises a histidine (H) at position 27a based on Kabat numbering (e.g., S28H mutation compared to SEQ ID NO:347) in the CTLA-4 VL region.
  • the provided CTLA-4 binding domains comprises a histidine (H) at position 30 based on Kabat numbering (e.g., S31H mutation compared to SEQ ID NO:347) in the CTLA-4 VL region.
  • the provided CTLA-4 binding domains comprises a histidine (H) at position 31 based on Kabat numbering (e.g., S32H mutation compared to SEQ ID NO:347) in the CTLA-4 VL region.
  • the provided CTLA-4 binding domains comprises a histidine (H) at position 90 based on Kabat numbering (e.g., Q91H mutation compared to SEQ ID NO:347) in the CTLA-4 VL region.
  • the provided CTLA-4 binding domains comprises a histidine (H) at position 27a based on Kabat numbering and a histidine (H) at position 30 based on Kabat numbering (e.g., S28H and S31H mutations compared to SEQ ID NO: 347) in the CTLA-4 VL region.
  • H histidine
  • H histidine
  • the provided CTLA-4 binding domains comprises a histidine (H) at position 27a based on Kabat numbering and a histidine (H) at position 31 based on Kabat numbering (e.g., S28H and S32H mutations compared to SEQ ID NO: 347) in the CTLA-4 VL region.
  • H histidine
  • H histidine
  • the provided CTLA-4 binding domains comprises a histidine (H) at position 27a based on Kabat numbering and a histidine (H) at position 90 based on Kabat numbering (e.g., S28H and Q91H mutations compared to SEQ ID NO: 347) in the CTLA-4 VL region.
  • H histidine
  • H histidine
  • the provided CTLA-4 binding domains comprises a histidine (H) at position 30 based on Kabat numbering and a histidine (H) at position 31 based on Kabat numbering (e.g., S31H and S32H mutations compared to SEQ ID NO: 347) in the CTLA-4 VL region.
  • the provided CTLA-4 binding domains comprises a histidine (H) at position 30 based on Kabat numbering and a histidine (H) at position 31 based on Kabat numbering (e.g., S31H and S32H mutations compared to SEQ ID NO: 347) in the CTLA-4 VL region.
  • the provided CTLA-4 binding domains comprises a histidine (H) at position
  • the provided CTLA-4 binding domains comprises a histidine (H) at position 31 based on Kabat numbering and a histidine (H) at position 90 based on Kabat numbering (e.g., S32H and Q91H mutations compared to SEQ ID NO: 347) in the CTLA-4 VL region.
  • the provided CTLA-4 binding domains comprises a histidine (H) at position 27a based on Kabat numbering, a histidine (H) at position 30 based on Kabat numbering, and a histidine (H) at position
  • the provided CTLA-4 binding domains comprises a histidine (H) at position 28 (position 27a based on Kabat numbering), a histidine (H) at position 30 based on Kabat numbering, and a histidine (H) at position 90 based on Kabat numbering (e.g., S28H, S31H, Q91H mutations compared to SEQ ID NO: 347) in the CTLA-4 VL region.
  • the provided CTLA-4 binding domains comprises a histidine (H) at position 27a based on Kabat numbering, a histidine (H) at position 31 based on Kabat numbering, and a histidine (H) at position 90 based on Kabat numbering (e.g., S28H, S32H, Q91H mutations compared to SEQ ID NO: 347) in the CTLA-4 VL region.
  • the provided CTLA-4 binding domains comprises a histidine (H) at position 30 based on Kabat numbering, a histidine (H) at position 31 based on Kabat numbering, and a histidine (H) at position 90 based on Kabat numbering (e.g., S31H, S32H, Q91H mutations compared to SEQ ID NO: 347) in the CTLA-4 VL region.
  • the provided CTLA-4 binding domains comprises a histidine (H) at position 27a based on Kabat numbering, a histidine (H) at position 30 based on Kabat numbering, a histidine (H) at position 31 based on Kabat numbering, and a histidine (H) at position 91 (position 90 based on Kabat numbering) (e.g., S28H, S31H, S32H, Q91H mutations compared to SEQ ID NO: 347) in the CTLA-4 VL region.
  • the CTLA-4 VH region comprises an arginine (R) at position 31, glutamic acid (E) or histidine (H) at position 53, histidine (H) at position 58, histidine (H) at position 97, or histidine (H) at position 98, respectively, based on Kabat numbering, or any combination thereof (e.g., S31R, D54H, D54E, Y59H, W101H, or L102H mutations, or any combination thereof, compared to SEQ ID NO:341), and the CTLA-4 VL region comprises a histidine (H) at position 27a, histidine (H) at position 30, histidine (H) at position 31, or histidine (H) at position 90, respectively, based on Kabat numbering), or any combination thereof (e.g., S28H, S31H, S32H, or Q91H mutations, or any combination thereof, compared to SEQ ID NO:347).
  • R arginine
  • E glutamic
  • the CTLA-4 VH region comprises the sequence set forth in: QVQLVESGGGVVQPGRSLRLSCAASGFTFSX , YTMHW VRQAPGKGLEWVTFIS YX 2 GNNKX 3 YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAIYYCARTGX4X5GPFDYWGQGTLVTVSS, wherein X, is S or R; X 2 is E or H; X 3 is H or Y; X 4 is W or H; and X 5 is L or H (SEQ ID NO: 407); and the CTLA-4 VL region comprises the sequence set forth in: EIVLTQSPGTLSLSPGERATLSCRASQX1VGHX2YLAWYQQKPGQAPRLLIYGAFSRATGIPD RFSGSGSGTDFTLTISRLEPEDFAVYYCQX3YGSSPWTFGQGTKVEIK, wherein X, is H or S;
  • the CTLA-4 VH region comprises: a serine (S) at position 31, a glutamic acid (E) at position 54, a histidine (H) at position 59, a tryptophan (W) at position 101, and a leucine (L) at position 102, wherein the numbering is relative to SEQ ID NO: 407.
  • the CTLA-4 VH region comprises: an arginine (R) at position 31, a glutamic acid (E) at position 54, a tyrosine (Y) at position 59, a tryptophan (W) at position 101, and a histidine (H) at position 102, wherein the numbering is relative to SEQ ID NO: 407.
  • the CTLA-4 VL region comprises: a histidine (H) at position 28, a serine (S) at position 32, and a glutamine (Q) at position 91, wherein the numbering is relative to SEQ ID NO: 408.
  • the CTLA-4 VL region comprises: a serine (S) at position 28, a serine (S) at position 32, and a histidine (H) at position 91, wherein the numbering is relative to SEQ ID NO: 408.
  • the CTLA-4 VH region comprises a serine (S) at position 31, a glutamic acid (E) at position 54, a histidine (H) at position 59, a tryptophan (W) at position 101, and a leucine (L) at position 102, wherein the numbering is relative to SEQ ID NO: 407; and the CTLA-4 VL region comprises a histidine (H) at position 28, a serine (S) at position 32, and a glutamine (Q) at position 91, wherein the numbering is relative to SEQ ID NO: 408.
  • the CTLA- 4 VH region comprises an arginine (R) at position 31, a glutamic acid (E) at position 54, a tyrosine (Y) at position 59, a tryptophan (W) at position 101, and a histidine (H) at position 102, wherein the numbering is relative to SEQ ID NO: 407; and the CTLA-4 VL region comprises a serine (S) at position 28, a serine (S) at position 32, and a histidine (H) at position 91, wherein the numbering is relative to SEQ ID NO: 408.
  • CTLA-4 VH and CTLA-4 VL regions with one or more of the above residues are set forth in Tables 4A and Table 4B.
  • any CTLA-4 binding domains comprising at least one of such CTLA-4 VH or CTLA-4 VL region set forth in Table 4A or Table 4B, paired with any respective other CTLA-4 VH and CTLA-4 VL described herein.
  • the CTLA-4 binding domain comprises a CTLA-4 VH set forth in any one of SEQ ID NOs: 234, 235, 243, 246- 248, and 250-256, and a CTLA-4 VL set forth in any one of SEQ ID NOs: 258, 259, 261, 263, 268- 270, 273, and 276.
  • the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 234 and a CTLA-4 VL set forth in SEQ ID NO: 258. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 234 and a CTLA-4 VL set forth in SEQ ID NO: 259. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 234 and a CTLA-4 VL set forth in SEQ ID NO: 261.
  • the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 234 and a CTLA-4 VL set forth in SEQ ID NO: 263. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 324 and a CTLA-4 VL set forth in SEQ ID NO: 268. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 234 and a CTLA-4 VL set forth in SEQ ID NO: 269. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 234 and a CTLA-4 VL set forth in SEQ ID NO: 270.
  • the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 234 and a CTLA-4 VL set forth in SEQ ID NO: 273. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 234 and a CTLA-4 VL set forth in SEQ ID NO: 276. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 235 and a CTLA-4 VL set forth in SEQ ID NO: 258. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 235 and a CTLA-4 VL set forth in SEQ ID NO: 259.
  • the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 235 and a CTLA-4 VL set forth in SEQ ID NO: 261. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 235 and a CTLA-4 VL set forth in SEQ ID NO: 263. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 235 and a CTLA-4 VL set forth in SEQ ID NO: 268. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 235 and a CTLA-4 VL set forth in SEQ ID NO: 269.
  • the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 235 and a CTLA-4 VL set forth in SEQ ID NO: 270. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 235 and a CTLA-4 VL set forth in SEQ ID NO: 273. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 235 and a CTLA-4 VL set forth in SEQ ID NO: 276. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 243 and a CTLA-4 VL set forth in SEQ ID NO: 258.
  • the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 243 and a CTLA-4 VL set forth in SEQ ID NO: 259. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 243 and a CTLA-4 VL set forth in SEQ ID NO: 261. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 243 and a CTLA-4 VL set forth in SEQ ID NO: 263. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 243 and a CTLA-4 VL set forth in SEQ ID NO: 268.
  • the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 243 and a CTLA-4 VL set forth in SEQ ID NO: 269. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 243 and a CTLA-4 VL set forth in SEQ ID NO: 270. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 243 and a CTLA-4 VL set forth in SEQ ID NO: 273. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 243 and a CTLA-4 VL set forth in SEQ ID NO: 276.
  • the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 246 and a CTLA-4 VL set forth in SEQ ID NO: 258. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 246 and a CTLA-4 VL set forth in SEQ ID NO: 259. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 246 and a CTLA-4 VL set forth in SEQ ID NO: 261. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 246 and a CTLA-4 VL set forth in SEQ ID NO: 263.
  • the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 246 and a CTLA-4 VL set forth in SEQ ID NO: 268. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 246 and a CTLA-4 VL set forth in SEQ ID NO: 269. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 246 and a CTLA-4 VL set forth in SEQ ID NO: 270. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 246 and a CTLA-4 VL set forth in SEQ ID NO: 273.
  • the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 246 and a CTLA-4 VL set forth in SEQ ID NO: 276. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 247 and a CTLA-4 VL set forth in SEQ ID NO: 258. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 247 and a CTLA-4 VL set forth in SEQ ID NO: 259. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 247 and a CTLA-4 VL set forth in SEQ ID NO: 261.
  • the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 247 and a CTLA-4 VL set forth in SEQ ID NO: 263. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 247 and a CTLA-4 VL set forth in SEQ ID NO: 268. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 247 and a CTLA-4 VL set forth in SEQ ID NO: 269. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 247 and a CTLA-4 VL set forth in SEQ ID NO: 270.
  • the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 247 and a CTLA-4 VL set forth in SEQ ID NO: 273. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 247 and a CTLA-4 VL set forth in SEQ ID NO: 276. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 248 and a CTLA-4 VL set forth in SEQ ID NO: 258. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 248 and a CTLA-4 VL set forth in SEQ ID NO: 30.
  • the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 248 and a CTLA-4 VL set forth in SEQ ID NO: 261. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 248 and a CTLA-4 VL set forth in SEQ ID NO: 263. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 248 and a CTLA-4 VL set forth in SEQ ID NO: 268. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 248 and a CTLA-4 VL set forth in SEQ ID NO: 269.
  • the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 248 and a CTLA-4 VL set forth in SEQ ID NO: 270. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 248 and a CTLA-4 VL set forth in SEQ ID NO: 273. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 248 and a CTLA-4 VL set forth in SEQ ID NO: 276. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 250 and a CTLA-4 VL set forth in SEQ ID NO: 258.
  • the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 250 and a CTLA-4 VL set forth in SEQ ID NO: 259. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 250 and a CTLA-4 VL set forth in SEQ ID NO: 261. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 250 and a CTLA-4 VL set forth in SEQ ID NO: 263. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 250 and a CTLA-4 VL set forth in SEQ ID NO: 268.
  • the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 250 and a CTLA-4 VL set forth in SEQ ID NO: 269. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 250 and a CTLA-4 VL set forth in SEQ ID NO: 270. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 250 and a CTLA-4 VL set forth in SEQ ID NO: 273. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 250 and a CTLA-4 VL set forth in SEQ ID NO: 276.
  • the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 251 and a CTLA-4 VL set forth in SEQ ID NO: 258. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 251 and a CTLA-4 VL set forth in SEQ ID NO: 259. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 251 and a CTLA-4 VL set forth in SEQ ID NO: 261. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 251 and a CTLA-4 VL set forth in SEQ ID NO: 263.
  • the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 251 and a CTLA-4 VL set forth in SEQ ID NO: 268. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 251 and a CTLA-4 VL set forth in SEQ ID NO: 269. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 251 and a CTLA-4 VL set forth in SEQ ID NO: 270. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 251 and a CTLA-4 VL set forth in SEQ ID NO: 273.
  • the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 251 and a CTLA-4 VL set forth in SEQ ID NO: 276. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 252 and a CTLA-4 VL set forth in SEQ ID NO: 258. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 252 and a CTLA-4 VL set forth in SEQ ID NO: 259. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 252 and a CTLA-4 VL set forth in SEQ ID NO: 261.
  • the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 252 and a CTLA-4 VL set forth in SEQ ID NO: 263. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 252 and a CTLA-4 VL set forth in SEQ ID NO: 268. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 252 and a CTLA-4 VL set forth in SEQ ID NO: 269. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 252 and a CTLA-4 VL set forth in SEQ ID NO: 270.
  • the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 252 and a CTLA-4 VL set forth in SEQ ID NO: 273. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 252 and a CTLA-4 VL set forth in SEQ ID NO: 276. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 253 and a CTLA-4 VL set forth in SEQ ID NO: 258. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 253 and a CTLA-4 VL set forth in SEQ ID NO: 259.
  • the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 253 and a CTLA-4 VL set forth in SEQ ID NO: 261. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 253 and a CTLA-4 VL set forth in SEQ ID NO: 263. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 253 and a CTLA-4 VL set forth in SEQ ID NO: 268. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 253 and a CTLA-4 VL set forth in SEQ ID NO: 269.
  • the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 253 and a CTLA-4 VL set forth in SEQ ID NO: 270. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 253 and a CTLA-4 VL set forth in SEQ ID NO: 273. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 253 and a CTLA-4 VL set forth in SEQ ID NO: 276. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 254 and a CTLA-4 VL set forth in SEQ ID NO: 258.
  • the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 254 and a CTLA-4 VL set forth in SEQ ID NO: 259. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 254 and a CTLA-4 VL set forth in SEQ ID NO: 261. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 254 and a CTLA-4 VL set forth in SEQ ID NO: 263. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 254 and a CTLA-4 VL set forth in SEQ ID NO: 268.
  • the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 254 and a CTLA-4 VL set forth in SEQ ID NO: 269. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 254 and a CTLA-4 VL set forth in SEQ ID NO: 270. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 254 and a CTLA-4 VL set forth in SEQ ID NO: 273. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 254 and a CTLA-4 VL set forth in SEQ ID NO: 276.
  • the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 255 and a CTLA-4 VL set forth in SEQ ID NO: 258. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 255 and a CTLA-4 VL set forth in SEQ ID NO: 259. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 255 and a CTLA-4 VL set forth in SEQ ID NO: 261. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 255 and a CTLA-4 VL set forth in SEQ ID NO: 263.
  • the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 255 and a CTLA-4 VL set forth in SEQ ID NO: 268. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 255 and a CTLA-4 VL set forth in SEQ ID NO: 269. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 255 and a CTLA-4 VL set forth in SEQ ID NO: 270. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 255 and a CTLA-4 VL set forth in SEQ ID NO: 273.
  • the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 255 and a CTLA-4 VL set forth in SEQ ID NO: 276. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 256 and a CTLA-4 VL set forth in SEQ ID NO: 258. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 256 and a CTLA-4 VL set forth in SEQ ID NO: 259. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 256 and a CTLA-4 VL set forth in SEQ ID NO: 261.
  • the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 256 and a CTLA-4 VL set forth in SEQ ID NO: 263. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 256 and a CTLA-4 VL set forth in SEQ ID NO: 268. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 256 and a CTLA-4 VL set forth in SEQ ID NO: 269. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 256 and a CTLA-4 VL set forth in SEQ ID NO: 270.
  • the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 256 and a CTLA-4 VL set forth in SEQ ID NO: 273. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 256 and a CTLA-4 VL set forth in SEQ ID NO: 276.
  • the above examples are non-limiting and any combinations are contemplated and provided by the present disclosure.
  • CTLA-4 binding domains that contain a CTLA-4 VH region sequence that exhibits a sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% to any of such CTLA-4 VH region sequences in Table 4A, in which one of the pH residues in Table 4A is present.
  • CTLA-4 binding domains that contain a CTLA-4 VL region sequence that exhibits a sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% to any of such CTLA-4 VL region sequences in Table 4B, in which one of the pH residues in Table 4B is present.
  • CTLA-4 binding domains that contain a CTLA-4 VH region sequence that exhibits a sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% to any of such CTLA-4 VH region sequences in Table 4A, in which one of the pH residues in Table 4A is present; and a CTLA-4 VL region sequence that exhibits a sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% to any of such
  • CTLA-4 VL region sequences in Table 4B in which one of the pH residues in Table 4B is present.
  • the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 comprising the sequence set forth in SEQ ID NOs: 281,289, and 301, respectively
  • the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 comprising the sequence set forth in SEQ ID NOs: 305, 318, and 327, respectively.
  • the CTLA-4 VH region comprises the sequence set forth in SEQ ID NO: 254 and the CTLA-4 VL region comprises the sequence set forth in SEQ ID NO: 258.
  • the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 comprising the sequence set forth in SEQ ID NOs: 278, 296, and 297, respectively
  • the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 comprising the sequence set forth in SEQ ID NOs: 313, 318, and 328, respectively.
  • the CTLA-4 VH region comprises the sequence set forth in SEQ ID NO: 252 and the CTLA-4 VL comprises the sequence set forth in SEQ ID NO: 268.
  • the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 comprising the sequence set forth in SEQ ID NOs: 281, 289, and 301, respectively
  • the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 comprising the sequence set forth in SEQ ID NOs: 313, 318, and 328, respectively.
  • the CTLA-4 VH region comprises the sequence set forth in SEQ ID NO: 254 and the CTLA-4 VL comprises the sequence set forth in SEQ ID NO: 268.
  • the provided binding molecules comprise a Fc region (also referred to as Fc domain).
  • the CTLA-4 binding domain is N-terminal to the Fc region and the PD-1 binding domain is C-terminal to the Fc region.
  • the PD-1 binding domain is N-terminal to the Fc region and the CTLA-4 binding domain is C-terminal to the Fc region.
  • the binding molecule is a dimer formed by polypeptides, each containing an Fc region.
  • the polypeptides are each the same and the polypeptides can be dimerized to yield a homodimer.
  • the dimer is a homodimer in which two polypeptide chains of the multispecific polypeptide construct, e.g., bispecific antibody, are the same.
  • the Fc region is an Fc region from an IgAl, IgA2, IgD, IgE, IgGl, IgG2, IgG3, IgG4, or IgM.
  • the Fc region is human IgGl or is derived from human IgGl, or is or is derived from a naturally occurring variant thereof.
  • the Fc region is a wild-type IgGl Fc polypeptide.
  • an IgGl Fc polypeptide (or a variant thereof such as any described below) can be made in a G1 ml or G1 m3 allotype.
  • the Fc region can contain amino acids of the human G1 ml allotype, such as residues containing Asp (D) and Feu (E) at positions 356 and 358, e.g. as set forth in SEQ ID NO: 411.
  • an Fc polypeptide can contain amino acid substitutions E356D and M358E to reconstitute residues of allotype G1 ml.
  • the Fc region can contain amino acids of the human G1 m3 allotype, such as residues Glu (E) and Met (M) at positions 356 and 358 by EU numbering.
  • an Fc polypeptide can contain amino acid substitutions D356E and E358M to reconstitute residues of allotype G1 m3.
  • a bispecific antibody is formatted with an Fc region that lacks a C- terminal Lys (K) residue.
  • the human IgGl Fc region lacks Lys447 (EU index of Kabat et al 1991 Sequences of Proteins of Immunological Interest).
  • the Fc region comprises a hinge-CH2-CH3 amino acid sequence. In some embodiments, the Fc region comprises the sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 411. In some embodiments, the Fc region comprises the sequence set forth in SEQ ID NO: 411.
  • the binding molecule or each polypeptide of an immunoglobulin Fc region comprises a human IgGl polypeptide sequence including a hinge-CH2- CH3 amino acid sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:
  • the Fc region comprises the sequence set forth in SEQ ID NO: 411.
  • each polypeptide of an immunoglobulin Fc region comprises a human IgGl polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:
  • the Fc region comprises the sequence set forth in SEQ ID NO: 412.
  • the binding molecule includes two CTLA-4 binding domains, two PD-1 binding domains, and the homodimeric Fc composed of two identical Fc polypeptides (e.g., each set forth in SEQ ID NO:411).
  • the binding molecule is composed of two identical heavy chain polypeptides and two identical light chain polypeptides that form the two CTLA-4 binding domains, the two PD-1 binding domains and the homodimeric Fc region in which the heavy chain polypeptides of the Fc region are connected by a disulfide bond.
  • the Fc region is a modified human IgG Fc region.
  • the multimerizing domains e.g., Fc regions, may comprise one or more amino acid changes (e.g., insertions, deletions or substitutions) as compared to the wild- type, naturally occurring version of the Fc region.
  • the Fc region is formed by Fc domains that are mutated or modified to promote heterodimerization in which different polypeptides can be dimerized to yield a heterodimer.
  • the dimer is a heterodimer in which two polypeptide chains of the multispecific polypeptide construct, e.g., bispecific antibody, are different.
  • a binding molecule provided herein is composed of a heterodimeric Fc region that facilitates interactions of two different heavy chain polypeptides in which each heavy chain polypeptide contains at least one different binding arm of the binding molecule.
  • the bispecific antibody includes a first heavy chain containing at least one anti- CTLA-4 antibody or antigen-binding fragment as described herein and a first polypeptide chain of the heterodimeric Fc and a second heavy chain containing one anti-PD-1 antibody or antigen binding fragment as described in Section I, and the second polypeptide chain of the heterodimeric Fc.
  • the first polypeptide chain of the heterodimeric Fc-region comprises a first CH3 region
  • the second polypeptide chain of the heterodimeric Fc region comprises a second CH3 region
  • the sequences of the first and second CH3 regions are different and are such that the heterodimeric interaction between said first and second CH3 regions is stronger than each of the homodimeric interactions of said first and second CH3 regions.
  • the human IgG Fc region is modified to induce heterodimerization.
  • Various methods are known for promoting heterodimerization of complementary Fc polypeptides, see e.g. Ridgway et al, Protein Eng. 9:617- 621 (1996); Merchant et al, Nat. Biotechnol. 16(7): 677-81 (1998); Moore et al. (2011) MAbs, 3:546-57; Von Kreudenstein et al. MAbs, (2013) 5:646-54; Gunasekaran et al. (2010) J. Biol. Chem., 285:19637-46; Leaver-Fay et al.
  • Methods to promote heterodimerization of Fc chains include mutagenesis of the Fc region, such as by including a set of “knob-into-hole” mutations or including mutations to effect electrostatic steering of the Fc to favor attractive interactions among different polypeptide chains.
  • the Fc polypeptides of a heterodimer includes a mutation to alter charge polarity across the Fc dimer interface such that co-expression of electrostatically matched Fc chains support favorable attractive interactions thereby promoting desired Fc heterodimer formation, whereas unfavorable repulsive charge interactions suppress unwanted Fc homodimer formation (Guneskaran et al. (2010) JBC, 285: 19637-19646).
  • heterodimeric Fc When co-expressed in a cell, association between the chains is possible but the chains do not substantially self-associate due to charge repulsion.
  • Other strategies for generating a heterodimeric Fc include mixing human IgG and IgA CH3 domain segments to create a complementary CH3 heterodimer, which is referred to as a SEED Fc.
  • Methods and variants for heterodimerization also include those described in published international PCT App. WO2014/145806, including “knobs and holes” mutations (also called “skew” variants), mutations that relate to “electrostatic steering” or “charge pairs,” and pl variants.
  • Heterodimeric variants also include any as described in U.S. published Appl. No.
  • modifications include introduction of a protuberance (knob) into a first Fc polypeptide and a cavity (hole) into a second Fc polypeptide such that the protuberance is positionable in the cavity to promote complexing of the first and second Fc- containing polypeptides.
  • Amino acids targeted for replacement and/or modification to create protuberances or cavities in a polypeptide are typically interface amino acids that interact or contact with one or more amino acids in the interface of a second polypeptide.
  • a first Fc polypeptide that is modified to contain protuberance (hole) amino acids include replacement of a native or original amino acid with an amino acid that has at least one side chain which projects from the interface of the first Fc polypeptide and is therefore positionable in a compensatory cavity (hole) in an adjacent interface of a second polypeptide.
  • the replacement amino acid is one which has a larger side chain volume than the original amino acid residue.
  • the replacement residues for the formation of a protuberance are naturally occurring amino acid residues and include, for example, arginine (R), phenylalanine (F), tyrosine (Y), or tryptophan (W).
  • the original residue identified for replacement is an amino acid residue that has a small side chain such as, for example, alanine, asparagine, aspartic acid, glycine, serine, threonine, or valine.
  • a second Fc polypeptide that is modified to contain a cavity is one that includes replacement of a native or original amino acid with an amino acid that has at least one side chain that is recessed from the interface of the second polypeptide and thus is able to accommodate a corresponding protuberance from the interface of a first polypeptide.
  • the replacement amino acid is one which has a smaller side chain volume than the original amino acid residue.
  • the replacement residues for the formation of a cavity are naturally occurring amino acids and include, for example, alanine (A), serine (S), threonine (T) and valine (V).
  • the original amino acid identified for replacement is an amino acid that has a large side chain such as, for example, tyrosine, arginine, phenylalanine, or tryptophan.
  • the CH3 interface of human IgGl involves sixteen residues on each domain located on four anti-parallel -strands which buries 1090 A2 from each surface (see e.g., Deisenhofer et al. (1981) Biochemistry, 20:2361-2370; Miller et al., (1990) J Mol. Biol., 216, 965- 973; Ridgway et al., (1996) Prot. Engin., 9: 617-621; U.S. Pat. No. 5,731,168). Modifications of a CH3 domain to create protuberances or cavities are described, for example, in U.S. Pat. No.
  • modifications of a CH3 domain to create protuberances or cavities are typically targeted to residues located on the two central anti-parallel P-strands. The aim is to minimize the risk that the protuberances which are created can be accommodated by protruding into the surrounding solvent rather than being accommodated by a compensatory cavity in the partner CH3 domain.
  • both polypeptides of the Fc heterodimer contain paired or complementary amino acid modifications.
  • Exemplary paired amino acid modification of polypeptides of an Fc fusion, such as an IgGl Fc, are set forth in Table 5.
  • the binding molecule contains a heterodimeric Fc region in which the first Fc polypeptide of the heterodimeric Fc has at least 85%, 90%, 95% or 98% sequence identity to the Fc polypeptide set forth in SEQ ID NO: 412 and contains the mutations S354C, T366W and E234A, E235E, G237A; and the second Fc polypeptide of the heterodimeric Fc has at least 85%, 90%, 95% or 98% sequence identity to the Fc polypeptide set forth in SEQ ID NO:413 and contains the mutations Y349C, T366S, E368A, Y407V and E234A, E235E, G237A.
  • the binding molecule contains a heterodimeric Fc region in which the first Fc polypeptide of the heterodimeric Fc is set forth in SEQ ID NO:412 and the second Fc polypeptide of the heterodimeric Fc is set forth in SEQ ID NO:413.
  • the binding molecule contains a heterodimeric Fc region in which the first Fc polypeptide of the heterodimeric Fc has at least 85%, 90%, 95% or 98% sequence identity to the Fc polypeptide set forth in SEQ ID NO:414 and contains the mutations S354C, T366W and E234A, E235A, D265S; and the second Fc polypeptide of the heterodimeric Fc has at least 85%, 90%, 95% or 98% sequence identity to the Fc polypeptide set forth in SEQ ID NO:415 and contains the mutations Y349C, T366S, E368A, Y407V and E234A, E235A, D265S.
  • the binding molecule contains a heterodimeric Fc region in which the first Fc polypeptide of the heterodimeric Fc is set forth in SEQ ID NO:414 and the second Fc polypeptide of the heterodimeric Fc is set forth in SEQ ID NO:415.
  • the binding molecule contains a heterodimeric Fc region in which the first Fc polypeptide of the heterodimeric Fc has at least 85%, 90%, 95% or 98% sequence identity to the Fc polypeptide set forth in SEQ ID NO: 416 and contains the mutations T350V, L351Y, F405A and Y407V; and the second Fc polypeptide of the heterodimeric Fc has at least 85%, 90%, 95% or 98% sequence identity to the Fc polypeptide set forth in SEQ ID NO: 417 and contains the mutations T350V, T366L, K392L and T394W.
  • the binding molecule contains a heterodimeric Fc region in which the first Fc polypeptide is set forth in SEQ ID NO:416 and the second Fc polypeptide is set forth in SEQ ID NO:417.
  • the binding molecule contains a heterodimeric Fc region in which the first Fc polypeptide of the heterodimeric Fc has at least 85%, 90%, 95% or 98% sequence identity to the Fc polypeptide set forth in SEQ ID NO: 418 and contains the mutations T350V, L351Y, F405A and Y407V and the mutations L234A, L235A and D265S; and the second Fc polypeptide of the heterodimeric Fc has at least 85%, 90%, 95% or 98% sequence identity to the Fc polypeptide set forth in SEQ ID NO:419and contains the mutations T350V, T366L, K392L and T394W and the mutations L234A, L235A and D265S.
  • the binding molecule contains a heterodimeric Fc region in which the first Fc polypeptide is set forth in SEQ ID NO: 418 and the second Fc polypeptide is set forth in SEQ ID NO: 418 and the second Fc
  • the binding molecule contains a heterodimeric Fc region in which the first Fc polypeptide of the heterodimeric Fc has at least 85%, 90%, 95% or 98% sequence identity to the Fc polypeptide set forth in SEQ ID NO: 420 and contains the mutations T350V, L351Y, F405A and Y407V and the mutations L234A, L235A and P329G; and the second Fc polypeptide of the heterodimeric Fc region has at least 85%, 90%, 95% or 98% sequence identity to the Fc polypeptide set forth in SEQ ID NO: 421 and contains the mutations T350V, T366L, K392L and T394W and the mutations L234A, L235A and P329G.
  • the binding molecule contains a heterodimeric Fc region in which the first Fc polypeptide is set forth in SEQ ID NO:420and the second Fc polypeptide is set forth in SEQ ID NO:420 and the second Fc poly
  • the bispecific antibody contains a heterodimeric Fc region in which the first Fc polypeptide of the heterodimeric Fc has at least 85%, 90%, 95% or 98% sequence identity to the Fc polypeptide set forth in SEQ ID NO:422 and contains the mutations S354C, T366W and P238K; and the second Fc polypeptide of the heterodimeric Fc has at least 85%, 90%, 95% or 98% sequence identity to the Fc polypeptide set forth in SEQ ID NO:423 and contains the mutations Y349C, T366S, L368A, Y407V and P238K.
  • the bispecific antibody contains a heterodimeric Fc region in which the first Fc polypeptide of the heterodimeric Fc is set forth in SEQ ID NO:422 and the second Fc polypeptide of the heterodimeric Fc is set forth in SEQ ID NO:423.
  • the Fc region is responsible for effector functions, such as complementdependent cytotoxicity (CDC) and antibody-dependent cell cytotoxicity (ADCC), in addition to the antigen-binding capacity, which is the main function of immunoglobulins.
  • the FcRn sequence present in the Fc region plays the role of regulating the IgG level in serum by increasing the in vivo half-life by conjugation to an in vivo FcRn receptor.
  • such functions can be altered, such as reduced or enhanced, in an Fc for use with the provided multispecific polypeptide constructs.
  • the Fc region includes an Fc polypeptide that is mutated or modified to alter one or more effector functions.
  • Fc polypeptides to alter, such as reduce, effector function are known, including any as described below.
  • reference to amino acid substitutions in an Fc region is by EU numbering by Kabat (also called Kabat numbering) unless described with reference to a specific SEQ ID NO.
  • EU numbering is known and is according to the most recently updated IMGT Scientific Chart (IMGT®, the international ImMunoGeneTics information system®, http://www.imgt.org/IMGTScientificChart/Numbering/Hu_IGHGnber.html (created: 17 May 2001, last updated: 10 Jan 2013) and the EU index as reported in Kabat, E.A. et al. Sequences of Proteins of Immunological interest. 5th ed. US Department of Health and Human Services, NIH publication No. 91-3242 (1991).
  • the human IgGl Fc region is modified to alter antibodydependent cellular cytotoxicity (ADCC) and/or complement-dependent cytotoxicity (CDC), e.g., the amino acid modifications described in Natsume et al., 2008 Cancer Res, 68(10): 3863-72; Idusogie et al., 2001 J Immunol, 166(4): 2571-5; Moore et al., 2010 mAbs, 2(2): 181-189; Lazar et al., 2006 PNAS, 103(11): 4005-4010, Shields et al., 2001 JBC, 276(9): 6591-6604; Stavenhagen et al., 2007 Cancer Res, 67(18): 8882-8890; Stavenhagen et al., 2008 Advan.
  • ADCC antibodydependent cellular cytotoxicity
  • CDC complement-dependent cytotoxicity
  • the Fc region such as the human IgGl Fc region is modified to enhance ADCC activity or CDC activity.
  • mutations that enhance ADCC include modification at Ser239 and Ile332, for example Ser239Asp and Ile332Glu (S239D, I332E) (SEQ ID NO: 336).
  • mutations that enhance CDC include modifications at Lys326 and Glu333.
  • the Fc region is modified at one or both of these positions, for example Lys326Ala and/or Glu333Ala (K326A and E333A) using the Kabat numbering system.
  • the Fc region is altered to provide reduced Fc-mediated effector functions, such as via reduced Fc receptor binding, e.g. binding to FcyR binding but generally not FcRn binding.
  • the human IgGl Fc region fusion proteins of the present disclosure lack or have reduced Fucose attached to the N-linked glycan-chain at N297.
  • fucosylation including but not limited to production in a FUT8 deficient cell line; addition inhibitors to the mammalian cell culture media, for example Castanospermine; and metabolic engineering of the production cell line.
  • the human IgGl Fc region is modified at amino acid Asn297 to prevent glycosylation of the fusion protein, e.g., Asn297Ala (N297A) or Asn297Asp (N297D).
  • the Fc region of the fusion protein is altered at one or more of the following positions to reduce Fc receptor binding: Leu 234 (L234), Leu235 (L235), Asp265 (D265), Asp270 (D270), Ser298 (S298), Asn297 (N297), Asn325 (N325) or Ala327 (A327).
  • Leu 234Ala (L234A), Leu235Ala (L235A), Asp265Asn (D265N), Asp270Asn (D270N), Ser298Asn (S298N), Asn297Ala (N297A), Asn325Glu (N325E) orAla327Ser (A327S).
  • the Fc region of the fusion protein is modified at amino acid Leu235 to alter Fc receptor interactions, e.g., Leu235Glu (L235E) or Leu235Ala (L235A).
  • the Fc region of the fusion protein is modified at amino acid Leu234 to alter Fc receptor interactions, e.g., Leu234Ala (L234A).
  • the Fc region of the fusion protein is altered at both amino acid 234 and 235, e.g., Leu234Ala and Leu235Ala (L234A/L235A) or Leu234Val and Leu235Ala (L234V/L235A).
  • the Fc region contains LALA mutations Leu234Ala and Leu235Ala (L234A/L235A).
  • modifications within the Fc region reduce binding to Fc-receptor-gamma receptors while have minimal impact on binding to the neonatal Fc receptor (FcRn).
  • the Fc comprises amino acid modification of at least one of L234, L235, or D265. In another embodiment, the Fc comprises amino acid modification of at least L234, L235 and D265. In another embodiment, the Fc comprises the amino acid modification L234A, L235A and D265S.
  • the IgGl has been modified to comprise or consist of an L234A, an L235A, and a D265S mutation as identified using Kabat numbering to render the Fc effector-less.
  • the Fc comprises a P329G mutation in the Fc together with L234A-L235A (LALA) mutations.
  • the IgGl has been modified to comprise or consist of an L234A, an L235A, and a P329G mutation as identified using Kabat numbering to render the Fc effector-less.
  • P329G is able to disrupt the interaction between hlgG and hFcyR, which combined with the effectorless mutations L234A/L235A mutations create a triple mutant L234A/L235A/P329G with no detectable binding to Clq or FcyRs, resulting in abrogated ADCC.
  • such an Fc disrupts the interaction with Fcy-receptors (FcyRs) while maintaining the binding to neonatal Fc-receptor (nFcR) and therefore wildtype- antibody-like pharmacokinetics in subjects.
  • FcyRs Fcy-receptors
  • nFcR neonatal Fc-receptor
  • the binding molecules comprising one or more modifications in the Fc domain that results in a modified Fc domain having a modified binding interaction (e.g., enhanced or diminished) between Fc and FcRn.
  • the human IgG Fc region is modified to enhance FcRn binding.
  • the bispecific antigen-binding molecule comprises a modification in a CH2 or a CH3 region, wherein the modification increases the affinity of the Fc domain to FcRn in an acidic environment e.g., in an endosome where pH ranges from about 5.5 to about 6.0).
  • Fc mutations that enhance binding to FcRn are Met252Tyr, Ser254Thr, Thr256Glu (M252Y, S254T, T256E, respectively)(e.g fashion SEQ ID NO: 339) (Kabat numbering, Dall’Acqua et al 2006, J. Biol Chem Vol. 281(33) 23514-23524), Met428Leu and Asn434Ser (M428L, N434S)(SEQ ID NO: 340) (Zalevsky et al 2010 Nature Biotech, Vol. 28(2) 157-159) (EU index of Kabat et al 1991 Sequences of Proteins of Immunological Interest).
  • Non-limiting examples of such Fc modifications include, e.g., a modification at position 250 (e.g., E or Q); 250 and 428 (e.g., L or F);252 (e.g., L/Y/F/W or T), 254 (e.g., S or T), and 256 (e.g., S/R/Q/E/D orT); or a modification at position 428 and/or 433 (e.g., L/R/S/P/Q or K)and/or 434 (e.g., H/F or Y); or a modification at position 250 and/or 428; or a modification at position 307 or 308 (e.g., 308F, V308F), and434.
  • a modification at position 250 e.g., E or Q
  • 250 and 428 e.g., L or F
  • 252 e.g., L/Y/F/W or T
  • 254 e.g., S or T
  • the mutated or modified Fc polypeptide includes the following mutations: Met252Tyr and Met428Leu or Met252Tyr and Met428Val (M252Y, M428L, or M252Y, M428V) using the Kabat numbering system.
  • the modification comprises a 428L (e.g., M428L)and 434S (e.g., N434S) modification; a 428L, 2591 (e.g., V259I), and 308F(e.g., V308F) modification; a 433K (e.g., H433K) and a 434 (e.g., 434Y)modification; a 252, 254, and 256 (e.g., 252Y, 254T, and 256E)modification; a 250Q and 428L modification (e.g., T250Q and M428L); and a307 and/or 308 modification (e.g., 308F or 308P).
  • a 428L e.g., M428L
  • 434S e.g., N434S
  • 428L, 2591 e.g., V259I
  • the Fc region is mutated in one or more of the following positions to reduce Fc receptor binding: Glu233 (E233), Leu234 (L234), or Leu235 (L235).
  • the one or more mutations can include E233P, L234V and/or L235A.
  • the Fc region of the fusion protein is altered at Gly236 to reduce Fc receptor binding.
  • Gly236 is deleted from the fusion protein.
  • the human IgGl Fc region is modified at amino acid Gly236 to enhance the interaction with CD32A, e.g., Gly236Ala (G236A).
  • the Fc comprises amino acid modification of at least one of G236A, A330L, or I332E (SEQ ID NO: 337). In another embodiment, the Fc comprises amino acid modification of at least one of G236A, S239D, A330L, or I332E (SEQ ID NO: 338). [0360] In particular embodiments, the mutations of the Fc region to reduce Fc effector function, e.g.
  • Fc receptor binding to FcyR include mutations from among any of G236R/L328R, E233P/L234V/L235A/G236del/S239K, E233P/L234V/L235A/G236del/S267K, E233P/L234V/L235A/G236del/S239K/A327G, E233P/L234V/L235A/G236del/S267K/A327G or E233P/L234V/L235A/G236del.
  • any of the provided Fc regions may include a mutation to eliminate or reduce the effector function of any antibody by mutating P238, e.g. to P238K, such as in an IgGl or IgG2 Fc.
  • P238, e.g. to P238K such as in an IgGl or IgG2 Fc.
  • further mutations can be made, e.g., to the CHI, hinge, CH2 or CH3 domain, e.g., to further reduce the effector function, binding to FcyRs, and/or the stability of the antibodies.
  • any of the mutations described herein may be combined with a P238, e.g., P238K, mutation, such as in an IgGl or IgG2 Fc.
  • Fc regions with P238K mutation demonstrate essentially no detectable binding signal towards the low affinity FcyRs hCD32a-H131, hCD32a-R131, hCD32b, hCD16a-V158 or hCD16b-NA2 and/or reduced binding affinity and a faster off-rate to the high affinity FcyR CD64 (see e.g., U.S. published patent application No. US20240141059).
  • bispecific antibodies comprising a first binding region that binds CTLA-4 and a second binding region that binds PD-1.
  • the provided binding molecules are bispecific antibodies that include two antigen binding sites, namely CTLA-4 binding domain and a PD-1 binding domains.
  • provided bispecific binding molecules are bivalent bispecific antibodies that are monovalent for binding to CTLA-4 and monovalent for binding to PD-1.
  • binding molecule comprising a cytotoxic T- lymphocyte-associated protein 4 (CTLA-4) binding domain that binds CTLA-4, and a PD-1 binding domain that binds PD-1
  • CTLA-4 binding domain comprises a heavy chain variable (CTLA-4 VH) region and a light chain variable (CTLA-4 VL) region
  • CTLA-4 VH heavy chain variable
  • CTLA-4 VL light chain variable
  • PD-1 binding domain comprises a heavy chain variable (PD-1 VH) region and a light chain variable (PD-1 VL) region that binds PD-1.
  • the binding molecule comprises: (a) a first heavy chain (HC1) comprising from N- to C-terminal order: (i) the CTLA-4 VH region or PD-1 Vnregion and the heavy chain constant region 1 (CHI) comprised in the Fab, and (ii) a first polypeptide of a heterodimeric Fc ; (b) a second heavy chain (HC2) comprising from N- to C-terminal order: (i) the other of the CTLA-4 VH region or PD-1 Vnregion and the heavy chain constant region 1 (CHI) comprised in the Fab, (ii) a second polypeptide of a heterodimeric Fc; (c) a first light chain (LC1) comprising the CTLA-4 VL region or the PD-1 VL region and the CL comprised in the Fab; and (d) a second light chain (LC2) comprising the other of the CTLA-4 VL region or the PD-1 VL region and the CL comprised in the Fab.
  • HC1 comprising from N
  • the binding molecule comprises: (a) a heavy chain 1 (HC1) polypeptide comprising a VL sequence set forth in SEQ ID NO:268, a CHI sequence set forth in SEQ ID NO:363, and a hinge-CH2-CH3 sequence set forth in SEQ ID NO: 364; (b) a light chain 1 (LC1) polypeptide comprising a VH sequence set forth in SEQ ID NO:254 and a CL sequence set forth in SEQ ID NO: 367; (c) a HC2 polypeptide comprising a VH sequence set forth in SEQ ID NO:168,a CHI sequence set forth in SEQ ID NO:371, and a hinge-CH2-CH3 sequence set forth in SEQ ID NO:373; and (d) a LC2 polypeptide comprising a VL sequence set forth in SEQ ID NO: 172 and a CL sequence set forth in SEQ ID NO: 175.
  • HC1 heavy chain 1
  • LC1 light chain 1
  • LC1 polypeptide comprising
  • the binding molecule comprises: (a) a HC1 polypeptide that is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 378; (b) a HC2 polypeptide that is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 387; (c) a LC1 polypeptide that is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 381; and (d) a LC2 is
  • the binding molecule comprises: (a) a HC1 polypeptide that is or comprises the amino acid sequence set forth in SEQ ID NO: 378; (b) a HC2 polypeptide that is or comprises the amino acid sequence set forth in SEQ ID NO: 387; (c) a LC1 polypeptide that is or comprises the amino acid sequence set forth in SEQ ID NO: 381; and (d) a LC2 polypeptide that is or comprises the amino acid sequence set forth in SEQ ID NO: 389.
  • the binding molecule comprises: (a) a heavy chain 1 (HC1) polypeptide comprising a VL sequence set forth in SEQ ID NO: 172, a CHI sequence set forth in SEQ ID NO:363, and a hinge-CH2-CH3 sequence set forth in SEQ ID NO: 364; (b) a LC1 polypeptide comprising a VH sequence set forth in SEQ ID NO: 168, and a CL sequence set forth in SEQ ID NO:367; (c) a HC2 polypeptide comprising a VH sequence set forth in SEQ ID NO:254, a CHI sequence set forth in SEQ ID NO:371, and a hinge-CH2-CH3 sequence set forth in SEQ ID NO: 373; and (d) a LC2 polypeptide comprising a VL sequence set forth in SEQ ID NO:268, and a CL sequence set forth in SEQ ID NO: 175.
  • HC1 polypeptide comprising a VH sequence set forth in SEQ ID NO: 172,
  • the binding molecule comprises: (a) a HC1 polypeptide that is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 360; (b) a HC2 polypeptide that is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 370; (c) a LC1 polypeptide that is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 366 and (d) a LC2 that is or comprises an amino acid
  • the binding molecule comprises: (a) a HC1 polypeptide that is or comprises an amino acid sequence set forth in SEQ ID NO: 360; (b) a HC2 polypeptide that is or comprises an amino acid sequence set forth in SEQ ID NO: 370; (c) a LC1 polypeptide that is or comprises an amino acid sequence set forth in SEQ ID NO: 366 and (d) a LC2 polypeptide that is or comprises an amino acid sequence set forth in SEQ ID NO: 375.
  • the provided binding molecules have one or more specified functional features, such as binding properties, including binding to particular epitopes or exhibiting lower or reduced binding to a related but non-specific antigen.
  • the provided binding molecules can bind to an epitope that is similar to or overlaps with epitopes of other binding molecules, such as reference antibodies, and/or exhibit particular binding affinities.
  • the provided binding molecules can bind to an epitope that is different from epitopes of other binding molecules, e.g., binding a conformational epitope.
  • the provided binding molecules specifically bind to a cytotoxic T- lymphocyte-associated protein 4 (CTLA-4) protein.
  • CTLA-4 refers to human CTLA-4.
  • the provided binding molecules specifically bind to human CTLA-4.
  • the provided binding molecules specifically bind to PD-1 protein.
  • PD-1 refers to human PD-1.
  • the provided binding molecules specifically bind to human PD-1.
  • a molecule is said to exhibit "specific binding” or “preferential binding” if it reacts or associates more frequently, more rapidly, with greater duration and/or with greater affinity with a particular cell or substance (e.g. a cell expressing a CTLA-4 or PD-1 antigen or a recombinant CTLA- 4 or PD-1 antigen) than it does with alternative cells or substances.
  • a binding molecule specifically binds or preferentially binds to a target (i.e. CTLA-4 or PD-1) if it binds with greater affinity, avidity, more readily, and/or with greater duration than it binds to other substances.
  • a binding molecule that specifically or preferentially binds to CTLA-4 or PD-1 is a binding molecule that binds CTLA-4 or PD-1 with greater affinity, avidity, more readily, and/or with greater duration than it binds to a different protein antigen. It is also understood by reading this definition that specific binding or preferential binding does not necessarily require (although it can include) exclusive binding. Methods to determine such specific or preferential binding are also well known in the art, e.g., an immunoassay.
  • a binding molecule binds to a target antigen (e.g., CTLA-4 or PD-1) or specifically binds to a target antigen does not necessarily mean that it binds to the target antigen from every species.
  • features of binding to the target antigen such as the ability to specifically bind thereto and/or to compete for binding thereto with a reference antibody, and/or to bind with a particular affinity or compete to a particular degree, in some embodiments, refers to the ability with respect to a human target antigen protein and the binding molecule may not have this feature with respect to a target antigen of another species such as mouse.
  • the provided binding molecules are capable of binding CTLA-4, such as human CTLA-4, with at least a certain affinity, as measured by any of a number of known methods.
  • the affinity is represented by absorbance.
  • the affinity is represented by equilibrium dissociation constant (KD).
  • the provided binding molecules are capable of binding PD-1, such as human PD-1, with at least a certain affinity, as measured by any of a number of known methods.
  • the affinity is represented by absorbance.
  • the affinity is represented by equilibrium dissociation constant (KD).
  • ELISA enzyme linked immunosorbent assays
  • ELISA enzyme linked immunosorbent assays
  • the antigen target macromolecule
  • a solid surface microplate
  • Detection is accomplished by measuring the activity of the reporter enzyme via incubation with the appropriate substrate to produce a measurable product.
  • a variety of assays are known for assessing binding affinity, equilibrium dissociation constant (KD), equilibrium association constant (KA), ECSO, on-rate (association rate constant; k on or k a ; units of 1/Ms or M ’s 1 ) and the off-rate (dissociation rate constant; k o ff or k ; units of 1/s or s' 1 ) and/or determining whether a binding molecule (e.g., an antibody or fragment thereof) specifically binds to a particular ligand e.g., an antigen, such as a CTLA-4 or PD-1 protein).
  • KD equilibrium dissociation constant
  • KA equilibrium association constant
  • ECSO on-rate (association rate constant; k on or k a ; units of 1/Ms or M ’s 1 ) and the off-rate (dissociation rate constant; k o ff or k ; units of 1/s or s' 1 ) and/or determining
  • a BIAcore® instrument can be used to determine the binding kinetics and constants of a complex between two proteins (e.g., an antibody or fragment thereof, and an antigen, such as a CTLA-4 protein), using surface plasmon resonance (SPR) analysis (see, e.g., Scatchard et al. , Am. N.Y. Acad. Sci.
  • SPR surface plasmon resonance
  • SPR measures changes in the concentration of molecules at a sensor surface as molecules bind to or dissociate from the surface.
  • the change in the SPR signal is directly proportional to the change in mass concentration close to the surface, thereby allowing measurement of binding kinetics between two molecules.
  • the dissociation rate constant (k o ff or kd), the association rate constant (k on or k a ) and/or equilibrium dissociation constant (KD) and/or equilibrium association constant (KA) for the complex can be determined by monitoring changes in the refractive index with respect to time as buffer is passed over the chip.
  • suitable assays for measuring the binding of one protein to another include, for example, immunoassays such as enzyme linked immunosorbent assays (ELISA) and radioimmunoassays (RIA), or determination of binding by monitoring the change in the spectroscopic or optical properties of the proteins through fluorescence, UV absorption, circular dichroism, or nuclear magnetic resonance (NMR).

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Abstract

Provided are novel monoclonal antibodies or antibody fragments against protein programmed cell death 1 (PD-1). The antibodies or antibody fragments disclosed can block the binding of the PD- L1 ligand to PD-1 and provide potent agents for the treatment of cancers via the modulation of human immune function. Also provided are novel multispecific antibodies directed against PD-1 and cytotoxic T-lymphocyte-associated protein 4 CTLA-4.

Description

ANTI-PD-1 ANTIBODIES AND RELATED BINDING MOLECULES AND METHODS AND USES THEREOF
Cross-Reference to Related Applications
[0001] This application claims priority to U.S. Provisional Patent Application No. 63/648,094 filed on May 15, 2024, and to U.S. Provisional Patent Application No. 63/757,775 filed on February 12, 2025, the contents of each of which are incorporated by reference in their entirety.
Reference to Electronic Sequence Listing
[0002] The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled 281632000340SeqList.xml created May 14, 2025, which is 542,096 bytes in size. The information in the electronic format of the Sequence Listing is incorporated by reference in its entirety.
Field
[0003] The present disclosure provides novel monoclonal antibodies or antibody fragments against protein programmed cell death 1 (PD-1). The antibodies or antibody fragments disclosed can block the binding of the PD-L1 ligand to PD-1 and provide potent agents for the treatment of cancers via the modulation of human immune function. The present disclosure also provides binding molecules, such as bispecific antibodies, directed against PD-1 and also cytotoxic T-lymphocyte-associated protein 4 (CTLA-4). The present disclosure further relates to pharmaceutical composition and methods and uses of such antibodies and binding molecules for treatment of disease in which specific targeting of cells that express PD-1, or PD-1 and CTLA-4, is desired.
Background
[0004] Cancer therapies comprise a wide range of therapeutic approaches, including surgery, radiation, and chemotherapy. While the various approaches offer a broad selection of options for treating cancer, many therapeutics suffer from disadvantages, such as a lack of selectivity of targeting cancer cells over normal, healthy cells, and the development of resistance by the cancer to the treatment. Immunotherapy targeting checkpoint molecules has emerged as one of the most compelling approaches for treating patients with cancers, due to promising results from preclinical and clinical trials.
[0005] Interaction of PD-1, a type I cell surface receptor, with either of its two ligands, PD-L1 or PD-L2, results in a dominant negative checkpoint signal that limits subsequent antigen receptor-driven cellular activation. The ligands for PD-1 are differentially expressed on various tissues and cell types, including antigen-presenting cells of the immune system, and are upregulated on many types of tumor cells. Upregulation of PD-L1 within the tumor microenvironment is a proposed mechanism of tumors to subvert protective anti-tumor immune responses by the host. Antibodies directed at PD-1 that block the interaction of the receptor with either of its ligands hold promise in the treatment of many cancers, and many such molecules are in the clinic for use as monotherapy or in combination with other biologies. Though antibodies against PD1 are known and commercially available, it is desirable to find novel anti-PDl antibodies suitable for cancer treatments with improved efficacy and reduced side effects. Embodiments provided herein address these needs.
Summary
[0006] In some embodiments, provided herein is an anti-programmed cell death protein 1 (PD-1) antibody or antigen-binding fragment thereof, comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein: the VH region comprises a heavy chain complementarity determining region 1 (CDR-H1), a heavy chain complementarity determining region 2 (CDR-H2), and a heavy chain complementarity determining region 3 (CDR-H3) contained within any one of SEQ ID NOs: 1-45, 157, and 165-170, and the VL region comprises a light chain complementarity determining region 1 (CDR-L1), a light chain complementarity determining region 2 (CDR-L2) and a light chain complementarity determining region 3 (CDR-L3) contained within any one of SEQ ID NOs: 46-90, 158, and 171-172.
[0007] In some embodiments, the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO:3, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 158; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO:4, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 158; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO:5, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 158; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO:6, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 158; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO:7, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 158; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO:8, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 158; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO:9, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 158; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 10, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 158; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 11, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 158; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 12, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 158; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO:13, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 158; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 14, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 158; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 15, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 158; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO:16, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 158; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 17, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 158; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 18, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 158; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 19, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 158; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO:20, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 158; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO:21, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 158; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO:22, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 158; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO:23, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 158; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO:24, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 158; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO:25, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 158; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO:26, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 158; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO:27, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 158; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO:28, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 158; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO:29, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 158; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO:30, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 158; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO:31, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 158; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO:32, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 158; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO:33, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 158; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO:34, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 158; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO:35, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 158; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO:36, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 158; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO:37, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 158; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO:38, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 158; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO:39, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 158; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO:40, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 158; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO:41, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 158; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO:42, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 158; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO:43, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 158; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO:44, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 158; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO:45, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 158; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 157, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 46; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 157, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 47; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 157, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 48; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 157, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 49; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 157, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 50; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 157, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 51; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 157, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 52; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 157, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 53; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 157, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 54; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 157, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 55; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 157, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 56; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 157, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 57; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 157, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 58; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 157, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 59; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 157, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 60; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 157, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 61; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 157, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 62; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 157, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 63; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 157, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 64; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 157, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 65; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 157, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 66; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 157, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 67; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 157, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 68; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 157, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 69; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 157, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 70; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 157, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 71; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 157, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 72; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 157, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 73; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 157, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 74; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 157, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 75; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 157, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 76; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 157, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 77; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 157, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 78; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 157, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 79; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 157, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 80; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 157, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 81; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 157, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 82; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 157, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 83; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 157, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 84; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 157, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 85; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 157, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 86; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 157, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 87; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 157, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 88; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 157, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 89; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 157, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 90; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 16, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 47; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 23, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 47; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 35, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 76; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 35, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 79; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 35, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 81; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 35, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 84; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 35, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 89; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 37, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 76; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 37, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 79; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 37, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 81; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 37, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 84; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 37, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 89; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 40, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 76; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 40, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 79; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 40, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 81; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 40, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 84; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 40, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 89; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 42, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 76; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 42, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 79; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 42, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 81; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 42, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 84; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 42, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 89; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 165, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 79; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 165, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 171; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 165, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 172; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 166, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 79; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 166, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 171; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 166, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 172; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 167, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 79; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 167, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 171; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 167, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 172; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 168, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 79; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 168, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 171; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 168, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 172; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 169, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 79; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 169, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 171; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 169, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 172; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 170, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 79; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 170, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 171; or the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 170, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 172.
[0008] In some embodiments, provided herein is an anti-PD-1 antibody or antigen-binding fragment thereof, comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein: the VH region comprises a heavy chain complementarity determining region 1 (CDR-H1) comprising the sequence set forth in any one of SEQ ID NOs: 91-101, a heavy chain complementarity determining region 2 (CDR-H2) comprising the sequence set forth in any one of SEQ ID NOs: 102- 112, and 173, and a heavy chain complementarity determining region 3 (CDR-H3) comprising the sequence set forth in any one of SEQ ID NOs: 113-123; and the VL region comprises a light chain complementarity determining region 1 (CDR-L1) comprising the sequence set forth in any one of SEQ ID NOs: 124-134, a light chain complementarity determining region 2 (CDR-L2) comprising the sequence set forth in any one of SEQ ID NOs: 135-145, and a light chain complementarity determining region 3 (CDR-L3) comprising the sequence set forth in any one of SEQ ID NOs:146-156. [0009] In some embodiments, the VH region comprises a heavy chain complementarity determining region 1 (CDR-H1) comprising the sequence set forth in any one of SEQ ID NOs: 92-101, a heavy chain complementarity determining region 2 (CDR-H2) comprising the sequence set forth in any one of SEQ ID NOs: 103-112, and 173, and a heavy chain complementarity determining region 3 (CDR-H3) comprising the sequence set forth in any one of SEQ ID NOs: 114-123.
[0010] In some embodiments, the VL region comprises a light chain complementarity determining region 1 (CDR-L1) comprising the sequence set forth in any one of SEQ ID NOs: 125-134, a light chain complementarity determining region 2 (CDR-L2) comprising the sequence set forth in any one of SEQ ID NOs: 136-145, and a light chain complementarity determining region 3 (CDR-L3) comprising the sequence set forth in any one of SEQ ID NOs: 147-156.
[0011] In any of the embodiments herein, the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 92, 102, and 113, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 124, 135, and 146, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 93, 102, and 113, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 124, 135, and 146, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 94, 102, and 113, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 124, 135, and 146, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 95, 102, and 113, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 124, 135, and 146, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 96, 102, and 113, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 124, 135, and 146, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 97, 102, and 113, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 124, 135, and 146, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 98, 102, and 113, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 124, 135, and 146, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 99, 102, and 113, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 124, 135, and 146, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 100, 102, and 113, respectively, and the CDR-L1, the CDR- L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 124, 135, and 146, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 101, 102, and 113, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 124, 135, and 146, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 91, 103, and 113, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 124, 135, and 146, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 91, 104, and 113, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 124, 135, and 146, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 91, 105, and 113, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 124, 135, and 146, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 91, 106, and 113, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 124, 135, and 146, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 91, 107, and 113, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 124, 135, and 146, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 91, 108, and 113, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 124, 135, and 146, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 91, 109, and 113, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 124, 135, and 146, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 91, 110, and 113, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 124, 135, and 146, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 91, 111, and 113, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 124, 135, and 146, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 91, 112, and 113, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 124, 135, and 146, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 91, 102, and 114, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 124, 135, and 146, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 91, 102, and 115, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 124, 135, and 146, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 91, 102, and 116, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 124, 135, and 146, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 91, 102, and 117, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 124, 135, and 146, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 91, 102, and 118, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 124, 135, and 146, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 91, 102, and 119, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 124, 135, and 146, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 91, 102, and 120, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 124, 135, and 146, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 91, 102, and 121, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 124, 135, and 146, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 91, 102, and 122, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 124, 135, and 146, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 91, 102, and 123, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 124, 135, and 146, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 101, 103, and 114, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 124, 135, and 146, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 97, 103, and 117, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 124, 135, and 146, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 99, 105, and 115, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 124, 135, and 146, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 96, 104, and 116, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 124, 135, and 146, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 98, 106, and 114, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 124, 135, and 146, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 96, 107, and 117, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 124, 135, and 146, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 97, 105, and 117, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 124, 135, and 146, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 99, 111, and 115, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 124, 135, and 146, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 97, 111, and 116, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 124, 135, and 146, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 100, 105, and 121, respectively, and the CDR-L1, the CDR- L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 124, 135, and 146, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 93, 103, and 115, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 124, 135, and 146, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 100, 108, and 119, respectively, and the CDR-L1, the CDR- L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 124, 135, and 146, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 99, 111, and 116, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 124, 135, and 146, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 91, 102, and 113, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 125, 135, and 146, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 91, 102, and 113, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 126, 135, and 146, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 91, 102, and 113, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 127, 135, and 146, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 91, 102, and 113, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 128, 135, and 146, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 91, 102, and 113, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 129, 135, and 146, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 91, 102, and 113, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 130, 135, and 146, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 91, 102, and 113, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 131, 135, and 146, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 91, 102, and 113, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 132, 135, and 146, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 91, 102, and 113, respectively, and the CDR-L1, theCDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 133, 135, and 146, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 91, 102, and 113, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 134, 135, and 146, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 91, 102, and 113, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 124, 136, and 146, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 91, 102, and 113, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 124, 137, and 146, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 91, 102, and 113, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 124, 138, and 146, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 91, 102, and 113, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 124, 139, and 146, respectively; the CDR-H1, theCDR-H2, and theCDR-H3 comprise the sequences set forth in SEQ ID NOS: 91, 102, and 113, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 124, 140, and 146, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 91, 102, and 113, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 124, 141, and 146, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 91, 102, and 113, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 124, 142, and 146, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 91, 102, and 113, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 124, 143, and 146, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 91, 102, and 113, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 124, 144, and 146, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 91, 102, and 113, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 124, 145, and 146, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 91, 102, and 113, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 124, 135, and 147, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 91, 102, and 113, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 124, 135, and 148, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 91, 102, and 113, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 124, 135, and 149, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 91, 102, and 113, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 124, 135, and 150, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 91, 102, and 113, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 124, 135, and 151, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 91, 102, and 113, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 124, 135, and 152, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 91, 102, and 113, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 124, 135, and 153, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 91, 102, and 113, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 124, 135, and 154, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 91, 102, and 113, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 124, 135, and 155, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 91, 102, and 113, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 124, 135, and 156, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 91, 102, and 113, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 131, 142, and 149, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 91, 102, and 113, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 131, 139, and 150, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 91, 102, and 113, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 128, 138, and 148, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 91, 102, and 113, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 126, 145, and 153, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 91, 102, and 113, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 126, 144, and 154, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 91, 102, and 113, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 125, 140, and 153, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 91, 102, and 113, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 132, 137, and 152, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 91, 102, and 113, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 131, 142, and 152, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 91, 102, and 113, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 132, 141, and 154, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 91, 102, and 113, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 133, 144, and 150, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 91, 102, and 113, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 134, 138, and 149, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 91, 102, and 113, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 126, 137, and 149, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 91, 102, and 113, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 126, 137, and 152, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 91, 102, and 113, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 132, 142, and 154, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 91, 102, and 113, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 127, 140, and 151, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 91, 106, and 113, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 126, 135, and 146, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 91, 102, and 114, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 126, 135, and 146, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 99, 105, and 115, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 131, 142, and 149, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 99, 105, and 115, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 126, 145, and 153, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 99, 105, and 115, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 125, 140, and 153, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 99, 105, and 115, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 132, 141, and 154, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 99, 105, and 115, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 132, 142, and 154, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 98, 106, and 114, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 131, 142, and 149, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 98, 106, and 114, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 126, 145, and 153, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 98, 106, and 114, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 125, 140, and 153, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 98, 106, and 114, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 132, 141, and 154, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 98, 106, and 114, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 132, 142, and 154, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 99, 111, and 115, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 131, 142, and 149, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 99, 111, and 115, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 126, 145, and 153, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 99, 111, and 115, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 125, 140, and 153, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 99, 111, and 115, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 132, 141, and 154, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 99, 111, and 115, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 132, 142, and 154, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 100, 105, and 121, respectively, and the CDR-L1, the CDR- L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 131, 142, and 149, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 100, 105, and 121, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 126, 145, and 153, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 100, 105, and 121, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 125, 140, and 153, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 100, 105, and 121, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 132, 141, and 154, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 100, 105, and 121, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 132, 142, and 154, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 100, 173 and 121, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 126, 145 and 153, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 100, 173 and 121, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 132, 145 and 153, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 100, 173 and 121, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 126, 143 and 153, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 95, 105 and 121, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 126, 145 and 153, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 95, 105 and 121, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 132, 145 and 153, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 95, 105 and 121, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 126, 143 and 153, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 95, 173 and 121, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 126, 145 and 153, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 95, 173 and 121, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 132, 145 and 153, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 95, 173 and 121, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 126, 143 and 153, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 95, 106 and 114, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 126, 145 and 153, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 95, 106 and 114, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 132, 145 and 153, respectively; or the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 95, 106 and 114, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 126, 143 and 153, respectively.
[0012] In any of the embodiments herein, the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 1 and SEQ ID NO: 158, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 2 and SEQ ID NO: 158, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 3 and SEQ ID NO: 158, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 4 and SEQ ID NO: 158, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 5 and SEQ ID NO: 158, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 6 and SEQ ID NO: 158, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 7 and SEQ ID NO: 158, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 8 and SEQ ID NO: 158, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 9 and SEQ ID NO: 158, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 10 and SEQ ID NO: 158, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 11 and SEQ ID NO: 158, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 12 and SEQ ID NO: 158, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 13 and SEQ ID NO: 158, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 14 and SEQ ID NO: 158, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO and SEQ ID NO: 158, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 16 and SEQ ID NO: 158, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 17 and SEQ ID NO: 158, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 18 and SEQ ID NO: 158, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 19 and SEQ ID NO: 158, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 20 and SEQ ID NO: 158, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 21 and SEQ ID NO: 158, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 22 and SEQ ID NO: 158, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 23 and SEQ ID NO: 158, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 24 and SEQ ID NO: 158, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 25 and SEQ ID NO: 158, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 26 and SEQ ID NO: 158, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 27 and SEQ ID NO: 158, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 28 and SEQ ID NO: 158, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 29 and SEQ ID NO: 158, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 30 and SEQ ID NO: 158, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 31 and SEQ ID NO: 158, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 32 and SEQ ID NO: 158, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 33 and SEQ ID NO: 158, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 34 and SEQ ID NO: 158, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 35 and SEQ ID NO: 158, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 36 and SEQ ID NO: 158, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 37 and SEQ ID NO: 158, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 38 and SEQ ID NO: 158, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 39 and SEQ ID NO: 158, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 40 and SEQ ID NO: 158, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 41 or ID NO: 158, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 42 and SEQ ID NO: 158, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 43 and SEQ ID NO: 158, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 44 and SEQ ID NO: 158, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 45 and SEQ ID NO: 158, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 46, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 47, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 48, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 49, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 50, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 51, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 52, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 53, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 54, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 55, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 56, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 57, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 58, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 59, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 60, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 61, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 62, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 63, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 64, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 65, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 66, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 or ID NO: 67, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 68, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 69, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 70, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 71, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 72, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 73, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 74, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 75, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 76, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 77, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 78, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 79, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 80, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 81, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 82, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 83, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 84, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 85, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 86, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 87, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 88, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 89, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 90, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 16 and SEQ ID NO: 47, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 23 and SEQ ID NO: 47, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 35 and SEQ ID NO: 76, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 35 and SEQ ID NO: 79, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 35 and SEQ ID NO: 81, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 35 and SEQ ID NO: 84, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 35 and SEQ ID NO: 89, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 37 and SEQ ID NO: 76, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 37 and SEQ ID NO: 79, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 37 and SEQ ID NO: 81, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 37 and SEQ ID NO: 84, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 37 and SEQ ID NO: 89, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 40 and SEQ ID NO: 76, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 40 and SEQ ID NO: 79, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 40 and SEQ ID NO: 81, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 40 and SEQ ID NO: 84, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 40 and SEQ ID NO: 89, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 42 and SEQ ID NO: 76, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 42 and SEQ ID NO: 79, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 42 and SEQ ID NO: 81, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 42 and SEQ ID NO: 84, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 42 and SEQ ID NO: 89, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 165 and SEQ ID NO: 79, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 165 and SEQ ID NO: 171, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 165 and SEQ ID NO: 172, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 166 and SEQ ID NO: 79, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 166 and SEQ ID NO: 171, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 166 and SEQ ID NO: 172, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 167 and SEQ ID NO: 79, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 167 and SEQ ID NO: 171, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 167 and SEQ ID NO: 172, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 168 and SEQ ID NO: 79, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 168 and SEQ ID NO: 171, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 168 and SEQ ID NO: 172, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 169 and SEQ ID NO: 79, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 169 and SEQ ID NO: 171, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 169 and SEQ ID NO: 172, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 170 and SEQ ID NO: 79, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 170 and SEQ ID NO: 171, respectively; or the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 170 and SEQ ID NO: 172, respectively.
[0013] In any of the embodiments herein, the VH region and the VL region can be or can comprise: the sequence set forth in SEQ ID NO: 1 and 158, respectively; the sequence set forth in SEQ ID NO: 2 and 158, respectively; the sequence set forth in SEQ ID NO: 3 and 158, respectively; the sequence set forth in SEQ ID NO: 4 and 158, respectively; the sequence set forth in SEQ ID NO: 5 and 158, respectively; the sequence set forth in SEQ ID NO: 6 and 158, respectively; the sequence set forth in SEQ ID NO: 7 and 158, respectively; the sequence set forth in SEQ ID NO: 8 and 158, respectively; the sequence set forth in SEQ ID NO: 9 and 158, respectively; the sequence set forth in SEQ ID NO: 10 and 158, respectively; the sequence set forth in SEQ ID NO: 11 and 158, respectively; the sequence set forth in SEQ ID NO: 12 and 158, respectively; the sequence set forth in SEQ ID NO: 13 and 158, respectively; the sequence set forth in SEQ ID NO: 14 and 158, respectively; the sequence set forth in SEQ ID NO: 15 and 158, respectively; the sequence set forth in SEQ ID NO: 16 and 158, respectively; the sequence set forth in SEQ ID NO: 17 and 158, respectively; the sequence set forth in SEQ ID NO: 18 and 158, respectively; the sequence set forth in SEQ ID NO: 19 and 158, respectively; the sequence set forth in SEQ ID NO: 20 and 158, respectively; the sequence set forth in SEQ ID NO: 21 and 158, respectively; the sequence set forth in SEQ ID NO: 22 and 158, respectively; the sequence set forth in SEQ ID NO: 23 and 158, respectively; the sequence set forth in SEQ ID NO: 24 and 158, respectively; the sequence set forth in SEQ ID NO: 25 and 158, respectively; the sequence set forth in SEQ ID NO: 26 and 158, respectively; the sequence set forth in SEQ ID NO: 27 and 158, respectively; the sequence set forth in SEQ ID NO: 28 and 158, respectively; the sequence set forth in SEQ ID NO: 29 and 158, respectively; the sequence set forth in SEQ ID NO: 30 and 158, respectively; the sequence set forth in SEQ ID NO: 31 and 158, respectively; the sequence set forth in SEQ ID NO: 32 and 158, respectively; the sequence set forth in SEQ ID NO: 33 and 158, respectively; the sequence set forth in SEQ ID NO: 34 and 158, respectively; the sequence set forth in SEQ ID NO: 35 and 158, respectively; the sequence set forth in SEQ ID NO: 36 and 158, respectively; the sequence set forth in SEQ ID NO: 37 and 158, respectively; the sequence set forth in SEQ ID NO: 38 and 158, respectively; the sequence set forth in SEQ ID NO: 39 and 158, respectively; the sequence set forth in SEQ ID NO: 40 and 158, respectively; the sequence set forth in SEQ ID NO: 41 and 158, respectively; the sequence set forth in SEQ ID NO: 42 and 158, respectively; the sequence set forth in SEQ ID NO: 43 and 158, respectively; the sequence set forth in SEQ ID NO: 44 and 158, respectively; the sequence set forth in SEQ ID NO: 45 and 158, respectively; the sequence set forth in SEQ ID NO: 157 and 46, respectively; the sequence set forth in SEQ ID NO: 157 and 47, respectively; the sequence set forth in SEQ ID NO: 157 and 48, respectively; the sequence set forth in SEQ ID NO: 157 and 49, respectively; the sequence set forth in SEQ ID NO: 157 and 50, respectively; the sequence set forth in SEQ ID NO: 157 and 51, respectively; the sequence set forth in SEQ ID NO: 157 and 52, respectively; the sequence set forth in SEQ ID NO: 157 and 53, respectively; the sequence set forth in SEQ ID NO: 157 and 54, respectively; the sequence set forth in SEQ ID NO: 157 and 55, respectively; the sequence set forth in SEQ ID NO: 157 and 56, respectively; the sequence set forth in SEQ ID NO: 157 and 57, respectively; the sequence set forth in SEQ ID NO: 157 and 58, respectively; the sequence set forth in SEQ ID NO: 157 and 59, respectively; the sequence set forth in SEQ ID NO: 157 and 60, respectively; the sequence set forth in SEQ ID NO: 157 and 61, respectively; the sequence set forth in SEQ ID NO: 157 and 62, respectively; the sequence set forth in SEQ ID NO: 157 and 63, respectively; the sequence set forth in SEQ ID NO: 157 and 64, respectively; the sequence set forth in SEQ ID NO: 157 and 65, respectively; the sequence set forth in SEQ ID NO: 157 and 66, respectively; the sequence set forth in SEQ ID NO: 157 and 67, respectively; the sequence set forth in SEQ ID NO: 157 and 68, respectively; the sequence set forth in SEQ ID NO: 157 and 69, respectively; the sequence set forth in SEQ ID NO: 157 and 70, respectively; the sequence set forth in SEQ ID NO: 157 and 71, respectively; the sequence set forth in SEQ ID NO: 157 and 72, respectively; the sequence set forth in SEQ ID NO: 157 and 73, respectively; the sequence set forth in SEQ ID NO: 157 and 74, respectively; the sequence set forth in SEQ ID NO: 157 and 75, respectively; the sequence set forth in SEQ ID NO: 157 and 76, respectively; the sequence set forth in SEQ ID NO: 157 and 77, respectively; the sequence set forth in SEQ ID NO: 157 and 78, respectively; the sequence set forth in SEQ ID NO: 157 and 79, respectively; the sequence set forth in SEQ ID NO: 157 and 80, respectively; the sequence set forth in SEQ ID NO: 157 and 81, respectively; the sequence set forth in SEQ ID NO: 157 and 82, respectively; the sequence set forth in SEQ ID NO: 157 and 83, respectively; the sequence set forth in SEQ ID NO: 157 and 84, respectively; the sequence set forth in SEQ ID NO: 157 and 85, respectively; the sequence set forth in SEQ ID NO: 157 and 86, respectively; the sequence set forth in SEQ ID NO: 157 and 87, respectively; the sequence set forth in SEQ ID NO: 157 and 88, respectively; the sequence set forth in SEQ ID NO: 157 and 89, respectively; the sequence set forth in SEQ ID NO: 157 and 90, respectively; the sequence set forth in SEQ ID NO: 16 and 47, respectively; the sequence set forth in SEQ ID NO: 23 and 47, respectively; the sequence set forth in SEQ ID NO: 35 and 76, respectively; the sequence set forth in SEQ ID NO: 35 and 79, respectively; the sequence set forth in SEQ ID NO: 35 and 81, respectively; the sequence set forth in SEQ ID NO: 35 and 84, respectively; the sequence set forth in SEQ ID NO: 35 and 89, respectively; the sequence set forth in SEQ ID NO: 37 and 76, respectively; the sequence set forth in SEQ ID NO: 37 and 79, respectively; the sequence set forth in SEQ ID NO: 37 and 81, respectively; the sequence set forth in SEQ ID NO: 37 and 84, respectively; the sequence set forth in SEQ ID NO: 37 and 89, respectively; the sequence set forth in SEQ ID NO: 40 and 76, respectively; the sequence set forth in SEQ ID NO: 40 and 79, respectively; the sequence set forth in SEQ ID NO: 40 and 81, respectively; the sequence set forth in SEQ ID NO: 40 and 84, respectively; the sequence set forth in SEQ ID NO: 40 and 89, respectively; the sequence set forth in SEQ ID NO: 42 and 76, respectively; the sequence set forth in SEQ ID NO: 42 and 79, respectively; the sequence set forth in SEQ ID NO: 42 and 81, respectively; the sequence set forth in SEQ ID NO: 42 and 84, respectively; the sequence set forth in SEQ ID NO: 42 and 89, respectively; the sequence set forth in SEQ ID NO: 165 and 79, respectively; the sequence set forth in SEQ ID NO: 165 and 171, respectively; the sequence set forth in SEQ ID NO: 165 and 172, respectively; the sequence set forth in SEQ ID NO: 166 and 79, respectively; the sequence set forth in SEQ ID NO: 166 and 171, respectively; the sequence set forth in SEQ ID NO: 166 and 172, respectively; the sequence set forth in SEQ ID NO: 167 and 79, respectively; the sequence set forth in SEQ ID NO: 167 and 171, respectively; the sequence set forth in SEQ ID NO: 167 and 172, respectively; the sequence set forth in SEQ ID NO: 168 and 79, respectively; the sequence set forth in SEQ ID NO: 168 and 171, respectively; the sequence set forth in SEQ ID NO: 168 and 172, respectively; the sequence set forth in SEQ ID NO: 169 and 79, respectively; the sequence set forth in SEQ ID NO: 169 and 171, respectively; the sequence set forth in SEQ ID NO: 169 and 172, respectively; the sequence set forth in SEQ ID NO: 170 and 79, respectively; the sequence set forth in SEQ ID NO: 170 and 171, respectively; or the sequence set forth in SEQ ID NO: 170 and 172, respectively.
[0014] In some embodiments, disclosed herein is an anti-programmed cell death protein 1 (PD-1) antibody or antigen-binding fragment thereof, comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and the VL region are or comprise: the sequence set forth in SEQ ID NO: 1 and 158, respectively; the sequence set forth in SEQ ID NO: 2 and 158, respectively; the sequence set forth in SEQ ID NO: 3 and 158, respectively; the sequence set forth in SEQ ID NO: 4 and 158, respectively; the sequence set forth in SEQ ID NO: 5 and 158, respectively; the sequence set forth in SEQ ID NO: 6 and 158, respectively; the sequence set forth in SEQ ID NO: 7 and 158, respectively; the sequence set forth in SEQ ID NO: 8 and 158, respectively; the sequence set forth in SEQ ID NO: 9 and 158, respectively; the sequence set forth in SEQ ID NO: 10 and 158, respectively; the sequence set forth in SEQ ID NO: 11 and 158, respectively; the sequence set forth in SEQ ID NO: 12 and 158, respectively; the sequence set forth in SEQ ID NO: 13 and 158, respectively; the sequence set forth in SEQ ID NO: 14 and 158, respectively; the sequence set forth in SEQ ID NO: 15 and 158, respectively; the sequence set forth in SEQ ID NO: 16 and 158, respectively; the sequence set forth in SEQ ID NO: 17 and 158, respectively; the sequence set forth in SEQ ID NO: 18 and 158, respectively; the sequence set forth in SEQ ID NO: 19 and 158, respectively; the sequence set forth in SEQ ID NO: 20 and 158, respectively; the sequence set forth in SEQ ID NO: 21 and 158, respectively; the sequence set forth in SEQ ID NO: 22 and 158, respectively; the sequence set forth in SEQ ID NO: 23 and 158, respectively; the sequence set forth in SEQ ID NO: 24 and 158, respectively; the sequence set forth in SEQ ID NO: 25 and 158, respectively; the sequence set forth in SEQ ID NO: 26 and 158, respectively; the sequence set forth in SEQ ID NO: 27 and 158, respectively; the sequence set forth in SEQ ID NO: 28 and 158, respectively; the sequence set forth in SEQ ID NO: 29 and 158, respectively; the sequence set forth in SEQ ID NO: 30 and 158, respectively; the sequence set forth in SEQ ID NO: 31 and 158, respectively; the sequence set forth in SEQ ID NO: 32 and 158, respectively; the sequence set forth in SEQ ID NO: 33 and 158, respectively; the sequence set forth in SEQ ID NO: 34 and 158, respectively; the sequence set forth in SEQ ID NO: 35 and 158, respectively; the sequence set forth in SEQ ID NO: 36 and 158, respectively; the sequence set forth in SEQ ID NO: 37 and 158, respectively; the sequence set forth in SEQ ID NO: 38 and 158, respectively; the sequence set forth in SEQ ID NO: 39 and 158, respectively; the sequence set forth in SEQ ID NO: 40 and 158, respectively; the sequence set forth in SEQ ID NO: 41 and 158, respectively; the sequence set forth in SEQ ID NO: 42 and 158, respectively; the sequence set forth in SEQ ID NO: 43 and 158, respectively; the sequence set forth in SEQ ID NO: 44 and 158, respectively; the sequence set forth in SEQ ID NO: 45 and 158, respectively; the sequence set forth in SEQ ID NO: 157 and 46, respectively; the sequence set forth in SEQ ID NO: 157 and 47, respectively; the sequence set forth in SEQ ID NO: 157 and 48, respectively; the sequence set forth in SEQ ID NO: 157 and 49, respectively; the sequence set forth in SEQ ID NO: 157 and 50, respectively; the sequence set forth in SEQ ID NO: 157 and 51, respectively; the sequence set forth in SEQ ID NO: 157 and 52, respectively; the sequence set forth in SEQ ID NO: 157 and 53, respectively; the sequence set forth in SEQ ID NO: 157 and 54, respectively; the sequence set forth in SEQ ID NO: 157 and 55, respectively; the sequence set forth in SEQ ID NO: 157 and 56, respectively; the sequence set forth in SEQ ID NO: 157 and 57, respectively; the sequence set forth in SEQ ID NO: 157 and 58, respectively; the sequence set forth in SEQ ID NO: 157 and 59, respectively; the sequence set forth in SEQ ID NO: 157 and 60, respectively; the sequence set forth in SEQ ID NO: 157 and 61, respectively; the sequence set forth in SEQ ID NO: 157 and 62, respectively; the sequence set forth in SEQ ID NO: 157 and 63, respectively; the sequence set forth in SEQ ID NO: 157 and 64, respectively; the sequence set forth in SEQ ID NO: 157 and 65, respectively; the sequence set forth in SEQ ID NO: 157 and 66, respectively; the sequence set forth in SEQ ID NO: 157 and 67, respectively; the sequence set forth in SEQ ID NO: 157 and 68, respectively; the sequence set forth in SEQ ID NO: 157 and 69, respectively; the sequence set forth in SEQ ID NO: 157 and 70, respectively; the sequence set forth in SEQ ID NO: 157 and 71, respectively; the sequence set forth in SEQ ID NO: 157 and 72, respectively; the sequence set forth in SEQ ID NO: 157 and 73, respectively; the sequence set forth in SEQ ID NO: 157 and 74, respectively; the sequence set forth in SEQ ID NO: 157 and 75, respectively; the sequence set forth in SEQ ID NO: 157 and 76, respectively; the sequence set forth in SEQ ID NO: 157 and 77, respectively; the sequence set forth in SEQ ID NO: 157 and 78, respectively; the sequence set forth in SEQ ID NO: 157 and 79, respectively; the sequence set forth in SEQ ID NO: 157 and 80, respectively; the sequence set forth in SEQ ID NO: 157 and 81, respectively; the sequence set forth in SEQ ID NO: 157 and 82, respectively; the sequence set forth in SEQ ID NO: 157 and 83, respectively; the sequence set forth in SEQ ID NO: 157 and 84, respectively; the sequence set forth in SEQ ID NO: 157 and 85, respectively; the sequence set forth in SEQ ID NO: 157 and 86, respectively; the sequence set forth in SEQ ID NO: 157 and 87, respectively; the sequence set forth in SEQ ID NO: 157 and 88, respectively; the sequence set forth in SEQ ID NO: 157 and 89, respectively; the sequence set forth in SEQ ID NO: 157 and 90, respectively; the sequence set forth in SEQ ID NO: 16 and 47, respectively; the sequence set forth in SEQ ID NO: 23 and 47, respectively; the sequence set forth in SEQ ID NO: 35 and 76, respectively; the sequence set forth in SEQ ID NO: 35 and 79, respectively; the sequence set forth in SEQ ID NO: 35 and 81, respectively; the sequence set forth in SEQ ID NO: 35 and 84, respectively; the sequence set forth in SEQ ID NO: 35 and 89, respectively; the sequence set forth in SEQ ID NO: 37 and 76, respectively; the sequence set forth in SEQ ID NO: 37 and 79, respectively; the sequence set forth in SEQ ID NO: 37 and 81, respectively; the sequence set forth in SEQ ID NO: 37 and 84, respectively; the sequence set forth in SEQ ID NO: 37 and 89, respectively; the sequence set forth in SEQ ID NO: 40 and 76, respectively; the sequence set forth in SEQ ID NO: 40 and 79, respectively; the sequence set forth in SEQ ID NO: 40 and 81, respectively; the sequence set forth in SEQ ID NO: 40 and 84, respectively; the sequence set forth in SEQ ID NO: 40 and 89, respectively; the sequence set forth in SEQ ID NO: 42 and 76, respectively; the sequence set forth in SEQ ID NO: 42 and 79, respectively; the sequence set forth in SEQ ID NO: 42 and 81, respectively; the sequence set forth in SEQ ID NO: 42 and 84, respectively; the sequence set forth in SEQ ID NO: 42 and 89, respectively; the sequence set forth in SEQ ID NO: 165 and 79, respectively; the sequence set forth in SEQ ID NO: 165 and 171, respectively; the sequence set forth in SEQ ID NO: 165 and 172, respectively; the sequence set forth in SEQ ID NO: 166 and 79, respectively; the sequence set forth in SEQ ID NO: 166 and 171, respectively; the sequence set forth in SEQ ID NO: 166 and 172, respectively; the sequence set forth in SEQ ID NO: 167 and 79, respectively; the sequence set forth in SEQ ID NO: 167 and 171, respectively; the sequence set forth in SEQ ID NO: 167 and 172, respectively; the sequence set forth in SEQ ID NO: 168 and 79, respectively; the sequence set forth in SEQ ID NO: 168 and 171, respectively; the sequence set forth in SEQ ID NO: 168 and 172, respectively; the sequence set forth in SEQ ID NO: 169 and 79, respectively; the sequence set forth in SEQ ID NO: 169 and 171, respectively; the sequence set forth in SEQ ID NO: 169 and 172, respectively; the sequence set forth in SEQ ID NO: 170 and 79, respectively; the sequence set forth in SEQ ID NO: 170 and 171, respectively; or the sequence set forth in SEQ ID NO: 170 and 172, respectively.
[0015] In any of the embodiments herein, the antibody can be a full length antibody. In some embodiments, the full length antibody comprises a constant region (Fc) from an IgAl, IgA2, IgD, IgE, IgGl, IgG2, IgG3, IgG4, or IgM.
[0016] In any of the embodiments herein, the antibody can be an antigen-binding fragment. In some embodiments, the antigen-binding fragment is selected from the group consisting of a single domain antibody, a single chain antibody, an unibody, a single chain variable fragment (scFv), a Fab fragment, and a F(ab')2 fragment.
[0017] In any of the embodiments herein, the anti-PD-1 antibody or antigen-binding fragment thereof can be recombinant.
[0018] In any of the embodiments herein, the VH region and the VL region can be human or can be from a human protein.
[0019] In any of the embodiments herein, the antibody or antigen-binding fragment thereof can bind to human PD-1 protein with an equilibrium dissociation constant (KD) of from about 7 x 10 10 M to about 2 x 10 8 M at pH 7.35 to 7.45, optionally at pH 7.40. In some embodiments, the antibody or antigen-binding fragment thereof binds to human PD-1 protein with a KD of lower than 1 x 109 M, optionally from about 7 x 10 10 M to about 1 x 109 M. [0020] In any of the embodiments herein, the antibody or antigen-binding fragment thereof can bind to human PD-1 protein with an equilibrium dissociation constant (KD) of from about 3 x 10 10 M to about 4 x 10 8 M at pH 5.0 to 6.8, optionally at pH 6.0 or pH 5.0. In some embodiments, the antibody or antigen-binding fragment thereof binds to human PD-1 protein with a KD of lower than 1 x 109 M, optionally from about 3 x 10 10 to about 1 x 109 M.
[0021] In any of the embodiments herein, the antibody or antigen-binding fragment thereof can bind to human PD-1 protein with a lower KD at pH 5.0 to 6.8, optionally pH 6.0 or pH 5.0, than pH 7.35 to 7.45, optionally pH 7.4. In any of the embodiments herein, the antibody or antigen-binding fragment thereof can bind to human PD-1 protein with a lower KD at pH 7.35 to 7.45, optionally pH 7.4, than at pH 5.0 to 6.8, optionally pH 6.0 or pH 5.0. In any of the embodiments herein, the antibody or antigenbinding fragment thereof can bind to human PD-1 protein with a KD at pH 7.35 to 7.45, optionally pH 7.4, and at pH 5.6 to 6.8, optionally pH 6.0 or pH 5.0, that is substantially the same.
[0022] In any of the embodiments herein, the antibody or antigen-binding fragment thereof can bind to human PD-1 protein with an association rate constant (Kon) of 4 x 105 1/Ms to about 2 x 106 1/Ms at pH 7.35 to 7.45, optionally at pH 7.40. In any of the embodiments herein, the antibody or antigen-binding fragment thereof can bind to human PD-1 protein with an dissociation rate constant (Koff) of 4 x 104 1/s to about 9 x 103 1/s at pH 7.35 to 7.45, optionally at pH 7.40.
[0023] In any of the embodiments herein, the antibody or antigen-binding fragment thereof can bind to human PD-1 protein with an association rate constant (Kon) of 4 x 105 1/Ms to about 5 x 106 1/Ms at pH 5.0 to 6.8, optionally at pH 6.0 pH 6.0 or pH 5.0. In any of the embodiments herein, the antibody or antigen-binding fragment thereof can bind to human PD-1 protein with an dissociation rate constant (Koff) of 4 x 104 1/s to about 2 x 102 1/s at pH 5.0 to 6.8, optionally at pH 6.0 or pH 5.0.
[0024] In some embodiments, disclosed herein is a conjugate, comprising the anti-PD-1 antibody or antigen-binding fragment thereof of any of the embodiments herein and a heterologous molecule or moiety. In some embodiments, the heterologous molecule or moiety is a cytotoxic agent, a drug, enzymatically active toxin or fragment thereof, or a radioactive atom.
[0025] In some embodiments, disclosed herein is a bispecific antibody, comprising the anti-PD-1 antibody or antigen-binding fragment thereof of any of any of the embodiments herein.
[0026] In some embodiments, disclosed herein is a PD-1 directed chimeric antigen receptor (CAR) comprising an extracellular antigen-binding domain comprising the anti-PD-1 antibody or antigenbinding fragment thereof of any of the embodiments herein, a transmembrane region and an intracellular signaling region.
[0027] In some embodiments, disclosed herein is a polynucleotide comprising a nucleic acid encoding the anti-PD-1 antibody or antigen-binding domain thereof of any of the embodiments herein. [0028] In some embodiments, disclosed herein is a polynucleotide comprising a nucleic acid encoding the anti-PD-1 chimeric antigen receptor of some embodiments.
[0029] In some embodiments, disclosed herein is a vector, comprising the polynucleotide of any of the embodiments herein. In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is a retroviral vector or a lentiviral vector.
[0030] In some embodiments, disclosed herein is a engineered cell comprising the anti-PD-1 antibody or antigen-binding fragment thereof of any of the embodiments herein, the bispecific antibody of some embodiments, or the conjugate of any of the embodiments herein.
[0031] In some embodiments, disclosed herein is an engineered cell comprising the PD-1 directed chimeric antigen receptor of some embodiments. In any of the embodiments herein, the cell can be a lymphocyte. In some embodiments, the cell is a natural killer (NK) cell or a T cell. In any of the embodiments herein, the cell can be a T cell and the T cell can be a CD4+ T cell or a CD8+ T cell.
[0032] In some embodiments, disclosed herein is a cell comprising the polynucleotide of any of the embodiments herein, or the vector of any of the embodiments herein. In some embodiments, the cell is a mammalian cell.
[0033] In some embodiments, disclosed herein is a method of producing an antibody comprising culturing the cell of any of the embodiments herein under a condition that produces the antibody. In some embodiments, the method further comprises recovering the antibody produced by the cell.
[0034] In some embodiments, disclosed herein is an antibody or antigen-binding fragment thereof produced by the method of any of the embodiments herein.
[0035] In some embodiments, disclosed herein is a composition comprising the cell of any of the embodiments herein.
[0036] In some embodiments, disclosed herein is a composition comprising the anti-PD-1 antibody or antigen-binding fragment thereof of any of the embodiments herein, the conjugate of any of the embodiments herein, the bispecific antibody of some embodiments, or the PD-1 directed chimeric antigen receptor of some embodiments. In any of the embodiments herein, the composition can further comprise a pharmaceutically acceptable excipient.
[0037] In some embodiments, disclosed herein is a method of treatment, comprising administering the cell of any of the embodiments herein or the composition of any of the embodiments herein to a subject having a disease or disorder associated with PD-1.
[0038] In some embodiments, disclosed herein is a method of increasing the activity of an immune cell, comprising contacting an immune cell with the anti-PD-1 antibody or antigen-binding fragment thereof of any of the embodiments herein, the conjugate of any of the embodiments herein, or the bispecific antibody of some embodiments. [0039] In some embodiments, disclosed herein is a method of increasing the activity of an immune cell, comprising administering to a subject in need thereof a therapeutically effective amount of the anti-PD-1 antibody or antigen-binding fragment thereof of any of the embodiments herein, the conjugate of any of the embodiments herein, the bispecific antibody of some embodiments.
[0040] In any of the embodiments herein, the method can be used for an ex vivo treatment in a subject.
[0041] In any of the embodiments herein, the treatment can be the treatment of cancer, the treatment of an infection or infectious disease or as a vaccine adjuvant.
[0042] Provided herein is a binding molecule comprising a PD-1 binding domain that binds PD- 1, and a cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) binding domain that binds CTLA-4, wherein: (a) the PD-1 binding domain comprises a heavy chain and a light chain comprising the heavy chain variable (VH) region and the light chain variable (VL ) region, respectively, of the anti- PD-1 antibody or antigen-binding fragment thereof of any of claims 1- 26, and (b) the CTLA-4 binding domain comprises a heavy chain comprising a heavy chain variable (CTLA-4 VH) region and a light chain comprising a light chain variable (CTLA-4 VL ) region, wherein: (i) the CTLA-4 VH region comprises a heavy chain complementarity determining region 1 (CDR-H1) comprising the sequence set forth in any one of SEQ ID NOs: 278-285, a heavy chain complementarity determining region 2 (CDR-H2) comprising the sequence set forth in any one of SEQ ID NOs: 286-296, and a heavy chain complementarity determining region 3 (CDR-H3) comprising the sequence set forth in any one of SEQ ID NOs: 297-302; and the CTLA-4 VL region comprises a light chain complementarity determining region 1 (CDR-L1) comprising the sequence set forth in any one of SEQ ID NOs: 303-317, a light chain complementarity determining region 2 (CDR-L2) comprising the sequence set forth in any one of SEQ ID NOs: 318-326, and a light chain complementarity determining region 3 (CDR-L3) comprising the sequence set forth in any one of SEQ ID NOs: 327- 329, wherein the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 are not set forth in SEQ ID NOS: 278, 286, 297,303, 318, and 327, respectively; or (ii) the CTLA-4 VH region comprises a heavy chain complementarity determining region 1 (CDR-H1), a heavy chain complementarity determining region 2 (CDR-H2), and a heavy chain complementarity determining region 3 (CDR-H3) contained within any one of SEQ ID NOs: 230-256, and 341, and the CTLA-4 VL region comprises a light chain complementarity determining region 1 (CDR-L1), a light chain complementarity determining region 2 (CDR-L2), and a light chain complementarity determining region 3 (CDR-L3) contained within any one of SEQ ID NOs:257, 277, and 347, wherein the CDR- Hl, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 are not set forth in SEQ ID NOS: 278, 286, 297,303, 318, and 327, respectively. [0043] In any of the embodiments herein, the CTLA-4 VH region comprises a CDR-H1 comprising the sequence set forth in any one of SEQ ID NOs: 278-285, a CDR-H2 comprising the sequence set forth in any one of SEQ ID NOs: 286-296, and a CDR-H3 comprising the sequence set forth in any one of SEQ ID NOs: 297-302; and the CTLA-4 VL region comprises a CDR-L1 comprising the sequence set forth in any one of SEQ ID NOs: 303-317, a CDR-L2 comprising the sequence set forth in any one of SEQ ID NOs: 318-326, and a CDR-L3 comprising the sequence set forth in any one of SEQ ID NOs: 327-329, wherein the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR- L2 and CDR-L3 are not set forth in SEQ ID NOS: 278, 286, 297,303, 318, and 327, respectively.
[0044] In any of the embodiments herein, the CTLA-4 VH region comprises a CDR-H1, a CDR- H2, and a CDR-H3 contained within SEQ ID NO: 230, and the CTLA-4 VL region comprises a CDR- Ll, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 347; the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 231, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 347; the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 232, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 347; the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO:233, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 347; the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO:234, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 347; the CTLA-4 VH region comprises a CDR-H1, a CDR- H2, and a CDR-H3 contained within SEQ ID NO:235, and the CTLA-4 VL region comprises a CDR- Ll, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 347; the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO:236, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 347; the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO:237, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 347; the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO:238, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 347; the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO:239, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 347; the CTLA-4 VH region comprises a CDR-H1, a CDR- H2, and a CDR-H3 contained within SEQ ID NO:240, and the CTLA-4 VL region comprises a CDR- Ll, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 347; the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO:241, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 347; the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO:242, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 347; the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO:243, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 347; the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO:244, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 347; the CTLA-4 VH region comprises a CDR-H1, a CDR- H2, and a CDR-H3 contained within SEQ ID NO:245, and the CTLA-4 VL region comprises a CDR- Ll, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 347; the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO:246, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 347; the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO:247, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 347; the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO:248, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 347; the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO:249, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 347; the CTLA-4 VH region comprises a CDR-H1, a CDR- H2, and a CDR-H3 contained within SEQ ID NO:250, and the CTLA-4 VL region comprises a CDR- Ll, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 347; the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO:251, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 347; the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 341, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 257; the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 341, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 258; the CTLA-4 Vnregion comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 341, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 259; the CTLA-4 Vnregion comprises a CDR-H1, a CDR- H2, and a CDR-H3 contained within SEQ ID NO: 341, and the CTLA-4 VL region comprises a CDR- Ll, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 260; the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 341, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 261; the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 341, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 262; the CTLA-4 Vnregion comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 341, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 263; the CTLA-4 Vnregion comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 341, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 264; the CTLA-4 Vnregion comprises a CDR-H1, a CDR- H2, and a CDR-H3 contained within SEQ ID NO: 341, and the CTLA-4 VL region comprises a CDR- Ll, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 265; the CTLA-4 Vnregion comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 341, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 266; the CTLA-4 Vn region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 341, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 267; the CTLA-4 Vnregion comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 341, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 268; the CTLA-4 Vnregion comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 341, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 269; the CTLA-4 Vnregion comprises a CDR-H1, a CDR- H2, and a CDR-H3 contained within SEQ ID NO: 341, and the CTLA-4 VL region comprises a CDR- Ll, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 270; the CTLA-4 Vnregion comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 341, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 271; the CTLA-4 Vn region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 341, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 272; the CTLA-4 Vnregion comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 341, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 273; the CTLA-4 Vnregion comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 341, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 274; the CTLA-4 Vnregion comprises a CDR-H1, a CDR- H2, and a CDR-H3 contained within SEQ ID NO: 341, and the CTLA-4 VL region comprises a CDR- Ll, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 275; the CTLA-4 Vnregion comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 252, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 270; the CTLA-4 Vn region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 253, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 270; the CTLA-4 Vnregion comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 254, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 270; the CTLA-4 Vnregion comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 255, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 270;the CTLA-4 Vnregion comprises a CDR-H1, a CDR- H2, and a CDR-H3 contained within SEQ ID NO: 251, and the CTLA-4 VL region comprises a CDR- Ll, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 270; the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 256, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 270; the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 252, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 276; the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 253, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 276; the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 254, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 276; the CTLA-4 VH region comprises a CDR-H1, a CDR- H2, and a CDR-H3 contained within SEQ ID NO: 255, and the CTLA-4 VL region comprises a CDR- Ll, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 276; the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 251, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 276; the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 256, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 276; the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 252, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 258; the CTLA-4 Vnregion comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 253, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 258; the CTLA-4 Vnregion comprises a CDR-H1, a CDR- H2, and a CDR-H3 contained within SEQ ID NO: 254, and the CTLA-4 VL region comprises a CDR- Ll, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 258; the CTLA-4 Vnregion comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 255, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 258; the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 251, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 258; the CTLA-4 Vnregion comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 256, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 258; the CTLA-4 Vnregion comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 252, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 268; the CTLA-4 Vnregion comprises a CDR-H1, a CDR- H2, and a CDR-H3 contained within SEQ ID NO: 253, and the CTLA-4 VL region comprises a CDR- Ll, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 268; the CTLA-4 Vnregion comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 254, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 268; the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 255, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 268; the CTLA-4 Vnregion comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 251, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 268; the CTLA-4 Vnregion comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 256, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 268; or the CTLA-4 Vnregion comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 234, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 277.
[0045] In any of the embodiments herein, the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 287, and 297, respectively, and the CDR-L1, the CDR- L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 303, 318, and 327, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 286, and 298, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 303, 318, 327, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 280, 286, and 297, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 303, 318, and 327, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 279, 288, and 297, respectively, and the -LI, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 303, 318, and 327, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 281, 289, and 297, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 303, 318, and 327, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 281, 290, and 297, respectively, and the CDR-L1, the CDR- L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 303, 318, and 327, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 280, 291, 297, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 303, 318, and 327, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 279, 292, and 297, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 303, 318, and 327, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 279, 286, and 298, respectively, and the CDR-L1, the CDR-L2, and the CDR- L3 comprise the sequences set forth in SEQ ID NOS: 303, 318, and 327, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 282, 286, and 297, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 303, 318, and 327, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 282, 286, and 299, respectively, and the CDR-L1, the CDR- L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 303, 318, and 327, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 283, 286, and 300, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 303, 318, and 327, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 284, 286, and 297, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 303, 318, and 327, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 285, 286, and 301, respectively, and the CDR-L1, the CDR-L2, and the CDR- L3 comprise the sequences set forth in SEQ ID NOS: 303, 318, and 327, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 293, 297, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 303, 318, and 327, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 289, and 297, respectively, and the CDR-L1, the CDR- L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 303, 318, and 327, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 294, 297, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 303, 318, and 327, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 295, and 297, respectively, and the CDR-L1, a CDR-L2, and a CDR-L3 comprise the sequences set forth in SEQ ID NOS: 303, 318, and 327, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 286, and 300, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 303, 318, and 327, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 286, and 302, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 303, 318, and 327, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 286, and 301, respectively, and the CDR-L1, the CDR- L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 303, 318, and 327, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 286, and 297, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 304, 318, and 327, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 286, and 297, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 305, 318, and 327, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 286, and 297, respectively, and the CDR-L1, the CDR-L2, and the CDR- L3 comprise the sequences set forth in SEQ ID NOS: 306, 318, 327, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 286, and 297, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 307, 319, and 327, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 286, and 297, respectively, and the CDR-L1, the CDR- L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 307, 319, and 327, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 286, and 297, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 310, 320, and 327, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 286, and 297, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 307, 318, and 328, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 286, and 297, respectively, and the CDR-L1, the CDR-L2, and the CDR- L3 comprise the sequences set forth in SEQ ID NOS: 310, 321, and 327, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 286, and 297, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 311, 318, and 327, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 286, and 297, respectively, and the CDR-L1, the CDR- L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 311, 322, and 327, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 286, and 297, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 312, 321, and 327, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 286, and 297, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 313, 318, and 328, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 286, and 297, respectively, and the CDR-L1, the CDR-L2, and the CDR- L3 comprise the sequences set forth in SEQ ID NOS: 313, 318, and 329, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 286, and 297, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 314, 318, and 327, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 286, and 297, respectively, and the CDR-L1, the CDR- L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 315, 323, and 327, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 286, and 297, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 316, 324, and 327, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 286, and 297, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 314, 325, and 327, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 286, and 297, respectively, and the CDR-L1, the CDR-L2, and the CDR- L3 comprise the sequences set forth in SEQ ID NOS: 317, 318, and 327, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 286, and 297, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 303, 326, and 327, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 296, and 297, respectively, and the CDR-L1, the CDR- L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 314, 318, and 327, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 294, and 302, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 314, 318, and 327, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 281, 289, and 301, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 314, 318, and 327, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 281, 296, and 297, respectively, and the CDR-L1, the CDR-L2, and the CDR- L3 comprise the sequences set forth in SEQ ID NOS: 314, 318, and 327, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 286, and 301, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 314, 318, and 327, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 294, and 301, respectively, and the CDR-L1, the CDR- L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 314, 318, and 327, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 296, and 297, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 313, 318, and 327, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 294, and 302, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 313, 318, and 327, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 281, 289, and 301, respectively, and the CDR-L1, the CDR-L2, and the CDR- L3 comprise the sequences set forth in SEQ ID NOS: 313, 318, and 327, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 281, 296, and 297, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 313, 318, and 327, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 286, and 301, respectively, and the CDR-L1, the CDR- L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 313, 318, and 327, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 294, and 301, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 313, 318, and 327, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 296, and 297, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 305, 318, and 327, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 294, and 302, respectively, and the CDR-L1, the CDR-L2, and the CDR- L3 comprise the sequences set forth in SEQ ID NOS: 305, 318, and 327, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 281, 289, and 301, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 305, 318, and 327, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 281, 296, and 297, respectively, and the CDR-L1, the CDR- L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 305, 318, and 327, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 286, and 301, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 305, 318, and 327, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 294, 301, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 305, 318, 327, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 296, and 297, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 313, 318, and 328, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 294, and 302, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 313, 318, and 328, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 281, 289, and 301, respectively, and the CDR-L1, the CDR- L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 313, 318, and 328, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 281, 296, and 297, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 313, 318, and 328, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 286, and 301, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 313, 318, and 328, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 294, and 301, respectively, and the CDR-L1, the CDR-L2, and the CDR- L3 comprise the sequences set forth in SEQ ID NOS: 313, 318, and 328, respectively; or the CDR- Hl, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 281, 289, and 297, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 317, 318, 329, respectively. [0046] In any of the embodiments herein, the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 230 and SEQ ID NO: 347, respectively; the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 231 and SEQ ID NO: 347, respectively; the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 232 and SEQ ID NO: 347, respectively; the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 233 and SEQ ID NO: 347, respectively; the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 234 and SEQ ID NO: 347, respectively; the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 235 and SEQ ID NO: 347, respectively; the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 236 and SEQ ID NO: 347, respectively; the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 237 and SEQ ID NO: 347, respectively; the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 238 and SEQ ID NO: 347, respectively; the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 239 and SEQ ID NO: 347, respectively; the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 240 and SEQ ID NO: 347, respectively; the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 241 and SEQ ID NO: 347, respectively; the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 242 and SEQ ID NO: 347, respectively; the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 243 and SEQ ID NO: 347, respectively; the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 244 and SEQ ID NO: 347, respectively; the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 245 and SEQ ID NO: 347, respectively; the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 246 and SEQ ID NO: 347, respectively; the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 247 and SEQ ID NO: 347, respectively; the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 248 and SEQ ID NO: 347, respectively; the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 249 and SEQ ID NO: 347, respectively; the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 250 and SEQ ID NO: 347, respectively; the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 251 and SEQ ID NO: 347, respectively; the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 341 and SEQ ID NO: 257, respectively; the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 341 and SEQ ID NO: 258, respectively; the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 341 and SEQ ID NO: 259, respectively; the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 341 and SEQ ID NO: 260, respectively; the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 341 and SEQ ID NO: 261, respectively; the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 341 and SEQ ID NO: 262, respectively; the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 341 and SEQ ID NO: 263, respectively; the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 341 and SEQ ID NO: 264, respectively; the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 341 and SEQ ID NO: 265, respectively; the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 341 and SEQ ID NO: 266, respectively; the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 341 and SEQ ID NO: 267, respectively; the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 341 and SEQ ID NO: 268, respectively; the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 341 and SEQ ID NO: 26, respectively; the CTLA-4 VH region and the CTLA- 4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 341 and SEQ ID NO: 270, respectively; the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 341 and SEQ ID NO: 271, respectively; the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 341 and SEQ ID NO: 272, respectively; the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 341 and SEQ ID NO: 273, respectively; the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 341 and SEQ ID NO: 274, respectively; the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 341 and SEQ ID NO: 275, respectively; the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 252 and SEQ ID NO: 270, respectively; the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 253 and SEQ ID NO: 270, respectively; the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 254 and SEQ ID NO: 270, respectively; the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 255 and SEQ ID NO: 270, respectively; the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 251 and SEQ ID NO: 270, respectively; the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 256 and SEQ ID NO: 270, respectively; the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 252 and SEQ ID NO: 276, respectively; the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 253 and SEQ ID NO: 276, respectively; the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 254 and SEQ ID NO: 276, respectively; the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 255 and SEQ ID NO: 276, respectively; the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 251 and SEQ ID NO: 276, respectively; the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 256 and SEQ ID NO: 276, respectively; the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 252 and SEQ ID NO: 258, respectively; the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 253 and SEQ ID NO: 258, respectively; the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 254 and SEQ ID NO: 258, respectively; the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 255 and SEQ ID NO: 258, respectively; the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 251 and SEQ ID NO: 258, respectively; the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 256 and SEQ ID NO: 258, respectively; the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 252 and SEQ ID NO: 268, respectively; the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 253 and SEQ ID NO: 268, respectively; the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 254 and SEQ ID NO: 268, respectively; the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 255 and SEQ ID NO: 268, respectively; the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 251 and SEQ ID NO: 268, respectively; the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 256 and SEQ ID NO: 268, respectively; or the
CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 234 and SEQ ID NO: 277, respectively.
[0047] In any of the embodiments herein, the CTLA-4 VH region and the CTLA-4 VL region are or comprise: the sequence set forth in SEQ ID NO: 230 and 347, respectively; the sequence set forth in SEQ ID NO: 231 and 347, respectively; the sequence set forth in SEQ ID NO: 232 and 347, respectively; the sequence set forth in SEQ ID NO: 233 and 347, respectively; the sequence set forth in SEQ ID NO: 234 and 347, respectively; the sequence set forth in SEQ ID NO: 235 and 347, respectively; the sequence set forth in SEQ ID NO: 236 and 347, respectively; the sequence set forth in SEQ ID NO: 237 and 347, respectively; the sequence set forth in SEQ ID NO: 238 and 347, respectively; the sequence set forth in SEQ ID NO: 239 and 347, respectively; the sequence set forth in SEQ ID NO: 240 and 347, respectively; the sequence set forth in SEQ ID NO: 241 and 347, respectively; the sequence set forth in SEQ ID NO: 242 and 347, respectively; the sequence set forth in SEQ ID NO: 243 and 347, respectively; the sequence set forth in SEQ ID NO: 244 and 347, respectively; the sequence set forth in SEQ ID NO: 245 and 347, respectively; the sequence set forth in SEQ ID NO: 246 and 347, respectively; the sequence set forth in SEQ ID NO: 247 and 347, respectively; the sequence set forth in SEQ ID NO: 248 and 347, respectively; the sequence set forth in SEQ ID NO: 249 and 347, respectively; the sequence set forth in SEQ ID NO: 250 and 347, respectively; the sequence set forth in SEQ ID NO: 251 and 347, respectively; the sequence set forth in SEQ ID NO: 341 and 257, respectively; the sequence set forth in SEQ ID NO: 341 and 258, respectively; the sequence set forth in SEQ ID NO: 341 and 259, respectively; the sequence set forth in SEQ ID NO: 341 and 260, respectively; the sequence set forth in SEQ ID NO: 341 and 261, respectively; the sequence set forth in SEQ ID NO: 341 and 262, respectively; the sequence set forth in SEQ ID NO: 341 and 263, respectively; the sequence set forth in SEQ ID NO: 341 and 264, respectively; the sequence set forth in SEQ ID NO: 341 and 265, respectively; the sequence set forth in SEQ ID NO: 341 and 266, respectively; the sequence set forth in SEQ ID NO: 341 and 267, respectively; the sequence set forth in SEQ ID NO: 341and 268, respectively; the sequence set forth in SEQ ID NO: 341 and 269, respectively; the sequence set forth in SEQ ID NO: 341and 270, respectively; the sequence set forth in SEQ ID NO: 341and 271, respectively; the sequence set forth in SEQ ID NO: 341and 272, respectively; the sequence set forth in SEQ ID NO: 341and 273, respectively; the sequence set forth in SEQ ID NO: 341and 274, respectively; the sequence set forth in SEQ ID NO: 341and 275, respectively; the sequence set forth in SEQ ID NO: 252 and 270, respectively; the sequence set forth in SEQ ID NO: 253 and 270, respectively; the sequence set forth in SEQ ID NO: 254 and 270, respectively; the sequence set forth in SEQ ID NO: 255 and 270, respectively; the sequence set forth in SEQ ID NO: 251 and 270, respectively; the sequence set forth in SEQ ID NO: 256 and 270, respectively; the sequence set forth in SEQ ID NO: 252 and 276, respectively; the sequence set forth in SEQ ID NO: 253 and 276, respectively; the sequence set forth in SEQ ID NO: 254 and 276, respectively; the sequence set forth in SEQ ID NO: 255 and 276, respectively; the sequence set forth in SEQ ID NO: 251 and 276, respectively; the sequence set forth in SEQ ID NO: 256 and 276, respectively; the sequence set forth in SEQ ID NO: 252 and 258, respectively; the sequence set forth in SEQ ID NO: 253 and 258, respectively; the sequence set forth in SEQ ID NO: 254 and 258, respectively; the sequence set forth in SEQ ID NO: 255 and 258, respectively; the sequence set forth in SEQ ID NO: 251 and 258, respectively; the sequence set forth in SEQ ID NO: 256 and 258, respectively; the sequence set forth in SEQ ID NO: 252 and 268, respectively; the sequence set forth in SEQ ID NO: 253 and 268, respectively; the sequence set forth in SEQ ID NO: 254 and 268, respectively; the sequence set forth in SEQ ID NO: 255 and 268, respectively; the sequence set forth in SEQ ID NO: 251 and 268, respectively; the sequence set forth in SEQ ID NO: 256 and 268, respectively; the sequence set forth in SEQ ID NO: 234 and 277, respectively.
[0048] Provided herein is a binding molecule comprising a cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) binding domain that binds CTLA-4, and a PD-1 binding domain that binds PD-1, wherein: (a) the PD-1 binding domain comprises a heavy chain and a light chain comprising the heavy chain variable (VH) region (PD-1 VH ) and the light chain variable (VL) region (PD-1 VL), respectively, of the anti-PD-1 antibody or antigen-binding fragment thereof of some of any embodiments, and (b) the CTLA-4 binding domain comprises a heavy chain variable (CTLA-4 VH) region and a light chain variable (CTLA-4 VL ) region, wherein: the CTLA-4 VH region comprises the sequence set forth in:
QVQLVESGGGVVQPGRSLRLSCAASGFTFSX 1 YTMHWVRQAPGKGLEW VTFIS YX2GNNKX 3YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAIYYCARTGX4X5GPFDYWGQGTLVTVSS, wherein XI is S or R; X2 is E or H; X3 is H or Y; X4 is W or H; and X5 is L or H (SEQ ID NO: 407); and the CTLA-4 VL region comprises the sequence set forth in: EIVLTQSPGTLSLSPGERATLSCRASQX1VGHX2YLAWYQQKPGQAPRLLIYGAFSRATGIPD RFSGSGSGTDFTLTISRLEPEDFAVYYCQX3YGSSPWTFGQGTKVEIK, wherein XI is H or S; X2 is H or S; and X3 is Q or H (SEQ ID NO: 408).
[0049] In any of the embodiments herein, the CTLA-4 VH region comprises a CDR-H1 comprising the sequence set forth in SEQ ID NO: 278 or SEQ ID NO: 281, a CDR-H2 comprising the sequence set forth in SEQ ID NO: 289 or SEQ ID NO: 296, and a CDR-H3 comprising the sequence set forth in SEQ ID NO: 297 or SEQ ID NO: 301; and the CTLA-4 VL region comprises a CDR-L1 comprising the sequence set forth in SEQ ID NO: 305 or SEQ ID NO: 313; a CDR-L2 comprising the sequence set forth in SEQ ID NO: 318, and a CDR-L3 comprising the sequence set forth in SEQ ID NO: 327 or SEQ ID NO: 328.
[0050] In any of the embodiments herein, the CTLA-4 VH region comprises: (a) a serine (S) at position 31, a glutamic acid (E) at position 54, a histidine (H) at position 59, a tryptophan (W) at position 101, and a leucine (L) at position 102, wherein the numbering is relative to SEQ ID NO: 407, or (b) a arginine (R) at position 31, a glutamic acid (E) at position 54, a tyrosine (Y) at position 59, a tryptophan (W) at position 101, and a histidine (H) at position 102, wherein the numbering is relative to SEQ ID NO: 407.
[0051] In any of the embodiments herein, the CTLA-4 VL region comprises: (a) a histidine (H) at position 28, a serine (S) at position 32, and a glutamine (Q) at position 91, wherein the numbering is relative to SEQ ID NO: 408, or (b) a serine (S) at position 28, a serine (S) at position 32, and a histidine (H) at position 91, wherein the numbering is relative to SEQ ID NO: 408.
[0052] In any of the embodiments herein, the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 254 and SEQ ID NO: 258, respectively; the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 254 and SEQ ID NO: 268, respectively; the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 252 and SEQ ID NO: 268, respectively.
[0053] In any of the embodiments herein, the CTLA-4 VH region and the CTLA-4 VL region are or comprise: the sequence set forth in SEQ ID NO: 254 and 258, respectively; the sequence set forth in SEQ ID NO: 254 and 268, respectively; the sequence set forth in SEQ ID NO: 252 and 268, respectively.
[0054] In any of the embodiments herein, the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 254 and SEQ ID NO: 268, respectively.
[0055] In any of the embodiments herein, the PD-1 VH region and the PD-1 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 168 and SEQ ID NO: 172, respectively.
[0056] In any of the embodiments herein, the PD-1 VH region is or comprises an amino acid sequence set forth in SEQ ID NO: 168, and the PD-1 VL region is or comprises an amino acid sequence set forth in SEQ ID NO: 172.
[0057] In any of the embodiments herein, the CTLA-4 binding domain and the PD-1 binding domain are Fab fragments each comprising a VH region and a VL region, wherein one of the Fab fragments is a crossover Fab such that the VH region and the VL region of such Fab are replaced by each other.
[0058] In any of the embodiments herein, the CTLA-4 binding domain is a crossover CTLA-4 Fab and the PD-1 binding domain is a Fab, wherein: the crossover CTLA-4 Fab comprises: (a) a heavy chain (HC1) comprising the CTLA-4 VL region and a heavy chain constant region 1 (CHI), designated CTLA-4 VL-CH1 ; and (b) a light chain (LC1) comprising the CTLA-4 VH region and a light chain constant region (CL), designated CTLA-4 VH-CL; and the PD-1 binding domain comprises (a) a heavy chain 2 (HC2) comprising the PD-1 VH region and a heavy chain constant region 1 (CHI), designated PD-1 VH-CH1 ; and (b) a light chain 2 (LC2) comprising the PD-1 VL region and a light chain constant region (CL), designated PD-1 VL-CL.
[0059] In any of the embodiments herein, the CHI of the CTLA-4 VL-CH1 comprises the amino acid sequence set forth in SEQ ID NO: 363 and the CL of the CTLA-4 VH-CL comprises the amino acid sequence set forth in SEQ ID NO: 367.
[0060] In any of the embodiments herein, the CHI of the PD-1 VH-CH1 comprises the amino acid sequence set forth in SEQ ID NO: 371 and the CL of the PD-1 VL-CL comprises the amino acid sequence set forth in SEQ ID NO: 175.
[0061] In any of the embodiments herein, the CTLA-4 binding domain is a CTLA-4 Fab and the PD-1 binding domain is a crossover Fab, wherein: the CTLA-4 Fab comprises: (a) a heavy chain (HC1) comprising the CTLA-4 VH region and a heavy chain constant region 1 (CHI), designated CTLA-4 VH-CH1 ; and (b) a light chain (LC1) comprising the CTLA-4 VL region and a light chain constant region (CL), designated CTLA-4 VL-CL; and the PD-1 binding domain comprises (a) a heavy chain 2 (HC2) comprising the PD-1 VL region and a heavy chain constant region 1 (CHI), designated PD-1 VL-CH1 ; and (b) a light chain 2 (LC2) comprising the PD-1 VH region and a light chain constant region (CL), designated PD-1 VH-CL.
[0062] In any of the embodiments herein, the CHI of the PD-1 VL-CH1 comprises the amino acid sequence set forth in SEQ ID NO: 363 and the CL of the PD-1 VH-CL comprises the amino acid sequence set forth in SEQ ID NO: 367.
[0063] In any of the embodiments herein, the CHI of the CTLA-4 VH-CH1 comprises the amino acid sequence set forth in SEQ ID NO: 371 and the CL of the CTLA-4 VL-CL comprises the amino acid sequence set forth in SEQ ID NO: 175.
[0064] In any of the embodiments herein, the CTLA-4 VH comprises the sequence of amino acids set forth in SEQ ID NO: 254 and the CTLA-4 VL comprises the sequence of amino acids set forth in SEQ ID NO: 268; and the PD-1 VH comprises the sequence of amino acids set forth in SEQ ID NO: 168 and the PD-1 VL comprise the sequence of amino acids set forth in SEQ ID NO: 172. [0065] In any of the embodiments herein, the provided binding molecule further comprises an Fc region comprising a first and second polypeptide each comprising a hinge-CH2-CH3, wherein one of the first polypeptide and second polypeptide is linked to the CHI of the CTLA-4 Fab and the other of the first polypeptide and second polypeptide is linked to the CHI of the PD-1 Fab. In any of the embodiments herein, the Fc region is a heterodimeric Fc region. In any of the embodiments herein, each of the first and second polypeptide of the heterodimeric Fc comprises one or more amino acid substitutions in a wild-type Fc polypeptide region to effect heterodimer formation between the first polypeptide and the second polypeptide.
[0066] In any of the embodiments herein, the wild-type Fc region is an IgGl Fc region. In any of the embodiments herein, the wild-type Fc region comprises the sequence set forth in SEQ ID NO:407 or SEQ ID NO: 408. In any of the embodiments herein, the one more amino acid substitutions are a knob-into-hole modification, a charge mutation to reduce or prevent self-association due to charge repulsion, or disulfide bonding pairing mutation. In any of the embodiments herein, the one or more amino acid substitutions are: (i) amino acid substitution T366W in the first polypeptide of the heterodimeric Fc and amino acid substitutions T366S, L368A and Y407V in the second polypeptide of the heterodimeric Fc; (ii) amino acid substitution E356K, E357K and D399K in the first polypeptide of the heterodimeric Fc and amino acid substitutions K439E, K370E and K409D in the second polypeptide of the heterodimeric Fc; or (iii) amino acid substitution S354C on the first polypeptide of the heterodimeric Fc and amino acid substitution Y349C on the second polypeptide of the heterodimeric Fc. In any of the embodiments herein, the one or more substitutions are a knob-into- hole modification. In any of the embodiments herein, the one or more amino acid substitutions of the first Fc polypeptide comprises Thr366Trp and the one or more amino acid substitutions of the second Fc polypeptide of the heterodimeric Fc comprises the Thr366Ser, Leu368Ala and Tyr407Val. In any of the embodiments herein, the first and/or second Fc polypeptide further comprises the amino acid substitution. H435R. In any of the embodiments herein, the heterodimeric Fc region comprises one or more amino acid substitutions to reduce binding affinity to an Fc receptor and/or to reduce effector function, optionally as compared to a wild-type IgGl Fc domain. In any of the embodiments herein, the one or more amino acid substitutions are selected from L234A, L234V, L235A, L235E, G237A, D265S, S267K, R292C, N297G, V302C, and P329G by EU numbering. In any of the embodiments herein, the one or more amino acid substitution comprises L234A and L235A. In any of the embodiments herein, the one or more amino acid substitution comprises L234A, L235A, and D265S. In any of the embodiments herein, the one or more amino acid substitution comprises L234A, L235A, and P329G. In any of the embodiments herein, one or both polypeptides of the heterodimeric Fc lacks Lys447.
[0067] In any of the embodiments herein, one of the first polypeptide and the second polypeptide of the heterodimeric Fc region comprise an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity SEQ ID NO: 364; and the other of the first polypeptide and second polypeptide of the heterodimeric Fc region comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity SEQ ID NO: 373.
[0068] In any of the embodiments herein, one of the first polypeptide and the second polypeptide of the heterodimeric Fc region comprise the amino acid sequence set forth in SEQ ID NO: 364; and the other of the first polypeptide and the second polypeptide of the heterodimeric Fc region comprises the amino acid sequence set forth in SEQ ID NO: 373. In any of the embodiments herein, the first polypeptide of the heterodimeric Fc region comprise the amino acid sequence set forth in SEQ ID NO: 364 and the second polypeptide of the heterodimeric Fc region comprises the amino acid sequence set forth in SEQ ID NO:373. In any of the embodiments herein, the first polypeptide of the heterodimeric Fc region comprise the amino acid sequence set forth in SEQ ID NO: 373 and the second polypeptide of the heterodimeric Fc region comprises the amino acid sequence set forth in SEQ ID NO:364. In any of the embodiments herein, (a) a heavy chain 1 (HC1) polypeptide comprising a VL sequence set forth in SEQ ID NO:268, a CHI sequence set forth in SEQ ID NO:363, and a hinge-CH2-CH3 sequence set forth in SEQ ID NO: 364; (b) a light chain 1 (LC1) polypeptide comprising a VH sequence set forth in SEQ ID NO:254 and a CL sequence set forth in SEQ ID NO: 367; (c) a HC2 polypeptide comprising a Vn sequence set forth in SEQ ID NO:168,a CHI sequence set forth in SEQ ID NO:371, and a hinge-CH2-CH3 sequence set forth in SEQ ID NO:373; and (d) a LC2 polypeptide comprising a VL sequence set forth in SEQ ID NO: 172 and a CL sequence set forth in SEQ ID NO: 175. In any of the embodiments herein, the binding molecule provided comprises a) a HC1 polypeptide that is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 378; (b) a HC2 polypeptide that is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 387; (c) a LC1 polypeptide that is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 381; and (d) a LC2 is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 389.
[0069] In any of the embodiments herein, the provided binding molecule comprises (a) a HC1 polypeptide that is or comprises the amino acid sequence set forth in SEQ ID NO: 378; (b) a HC2 polypeptide that is or comprises the amino acid sequence set forth in SEQ ID NO: 387; (c) a LC1 polypeptide that is or comprises the amino acid sequence set forth in SEQ ID NO: 381; and (d) a LC2 polypeptide that is or comprises the amino acid sequence set forth in SEQ ID NO: 389. In any of the embodiments herein, the provided binding molecule comprises (a) a heavy chain 1 (HC1) polypeptide comprising a VL sequence set forth in SEQ ID NO: 172, a CHI sequence set forth in SEQ ID NO:363, and a hinge-CH2-CH3 sequence set forth in SEQ ID NO: 364; (b) a LC1 polypeptide comprising a VH sequence set forth in SEQ ID NO: 168, and a CL sequence set forth in SEQ ID NO:367; and (c) a HC2 polypeptide comprising a VH sequence set forth in SEQ ID NO:254, a CHI sequence set forth in SEQ ID NO:371, and a hinge-CH2-CH3 sequence set forth in SEQ ID NO: 373; and (d) a LC2 polypeptide comprising a VL sequence set forth in SEQ ID NO:268, and a CL sequence set forth in SEQ ID NO: 175.
[0070] In any of the embodiments herein, the provided binding molecule comprises (a) a HC1 polypeptide that is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 360; (b) a HC2 polypeptide that is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 370; (c) a LC1 polypeptide that is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 366 and (d) a LC2 that is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 375.
[0071] In any of the embodiments herein, the provided binding molecule comprises (a) a HC1 polypeptide that is or comprises an amino acid sequence set forth in SEQ ID NO: 360; (b) a HC2 polypeptide that is or comprises an amino acid sequence set forth in SEQ ID NO: 370; (c) a LC1 polypeptide that is or comprises an amino acid sequence set forth in SEQ ID NO: 366 and (d) a LC2 polypeptide that is or comprises an amino acid sequence set forth in SEQ ID NO: 375.
[0072] In any of the embodiments herein, the heavy chain and/or the light chain further comprising a signal peptide.
[0073] Provided herein is a polynucleotide or polynucleotides comprising a nucleic acid sequence encoding any of the provided binding molecules or portion thereof, including a heavy or light chain thereof. In some embodiments, the binding molecule is a bispecific antibody directed against PD-1 and CTLA-4. Provided herein is a polynucleotide comprising a nucleic acid comprising a nucleic acid sequence encoding any of the provided the bispecific antibodies or portion thereof. Provided herein is a vector comprising any of the provided polynucleotides. In some embodiments, the vector is useful for expressing a provided binding molecule, such as a bispecific antibody.
[0074] In any of the embodiments herein, the vector is a viral vector. In any of the embodiments herein, the viral vector is a retroviral vector or a lentiviral vector.
[0075] Provided herein is a cell comprising any of the provided polynucleotides or vectors. In any of the embodiments herein, the cell is a mammalian cell. [0076] Provided herein is a method of producing a binding molecule, such as a bispecific antibody, comprising culturing any of the provided cells under a condition that produces the binding molecule, wherein optionally the binding molecule is an antibody. In any of the embodiments herein, any of the provided methods further comprising recovering the binding molecule, such as a bispecific antibody, produced by the cell.
[0077] Provided herein is a binding molecule, such as a bispecific antibody, produced by any of the provided methods. Provided herein is a composition comprising any of the binding molecules, such as bispecific antibodies, provided herein. In any of the embodiments herein, the composition further comprises a pharmaceutically acceptable excipient.
[0078] Provided herein is a method for treating a subject with a disease or condition, the method comprising administering a therapeutically effective amount of any of the provided binding molecules, such as bispecific antibodies, or compositions. In any of the embodiments herein, the disease or condition is a cancer.
[0079] Provided herein is a method of cancer immunotherapy, the method comprising administering to a subject in need thereof a therapeutically effective amount of any of the provided binding molecules, such as bispecific antibodies, or compositions. In any of the embodiments herein, the cancer is a solid tumor. In any of the embodiments herein, the cancer is a melanoma, breast carcinoma, HCC, Renal cell carcinoma, Colorectal carcinoma, non-small-cell lung carcinoma, Cervical cancer or Mesothelioma. In any of the embodiments herein, administering the binding molecules, such as bispecific antibody, to the subject results in higher Teff-to-suppressor cells (myeloid-derived suppressor cell and Treg) ratios. In any of the embodiments herein, administering the binding molecule, such as bispecific antibody, to the subject results in increased pro-inflammatory cytokines, optionally interferon-gamma (IFN-y) and/or tumor necrosis factor-alpha (TNF-a).
[0080] In any of the embodiments herein, the method comprises administering to the subject an effective amount of any of the provided binding molecules, such as bispecific antibodies, or the compositions prior to a surgery or after a surgery to remove a solid tumor in the subject.
Brief Description of the Drawings
[0081] FIG.l depicts the EC50 values for exemplary provided antibodies for binding to human PD-1 at normal pH about 7.4.
[0082] FIG.2 depicts the IC50 values for exemplary provided antibodies for inhibition of PD-1 binding to human PD-L1 in a standard reporter assay.
[0083] FIG. 3 shows a schematic drawing of a standard reporter assay to test for inhibition of PD- 1 binding to human PD-L1. [0084] FIGs. 4A-4F show the thermal melt curves measured by Differential Scanning Fluorimetry (DSF). FIG. 4A shows the thermal melt curve of PDl-abl l3. FIG. 4B shows the thermal melt curve of PDl-abl 16. FIG. 4C shows the thermal melt curve of PDl-abl 19. FIG. 4D shows the thermal melt curve of PDl-abl 22. FIG. 4E shows the thermal melt curve of PDl-abl 25. FIG. 4F shows the thermal melt curve of PDl-abl29.
[0085] FIGs. 5A-5S show results of binding tests of exemplary provided antibodies for binding to human CTLA-4 at pH 5 and at pH 7.4. FIG. 5A shows results of binding tests of anti-CTLA-4 antibodies (CTLA-4-AB-7, CTLA-4-AB-9, CTLA-4-AB-10, and CTLA-4-AB-11) and reference antibodies for binding to human CTLA-4 at pH 5 and at pH 7.4. FIG. 5B shows results of binding tests of anti-CTLA-4 antibodies (CTLA-4-AB-13, CTLA-4-AB-14, CTLA-4-AB-15, and CTLA-4-AB-16) and reference antibodies for binding to human CTLA-4 at pH 5 and at pH 7.4. FIG. 5C shows results of binding tests of anti-CTLA-4 antibodies (CTLA-4-AB-17, CTLA-4-AB-18, CTLA-4-AB-19, and CTLA-4- AB-20) and reference antibodies for binding to human CTLA-4 at pH 5 and at pH 7.4. FIG. 5D shows results of binding tests of anti-CTLA-4 antibodies (CTLA-4-AB-21, CTLA-4- AB-22, CTLA-4- AB-23, and CTLA-4- AB-24) and reference antibodies for binding to human CTLA-4 at pH 5 and at pH 7.4. FIG. 5E shows results of binding tests of anti-CTLA-4 antibodies (CTLA-4- AB -25, CTLA-4- AB-26, CTLA-4- AB-27, and CTLA-4- AB-28) and reference antibodies for binding to human CTLA-4 at pH 5 and at pH 7.4. FIG. 5F shows results of binding tests of anti-CTLA-4 antibodies (CTLA-4- AB-29, CTLA-4- AB-30, CTLA-4-AB-31, and CTLA-4- AB-32) and reference antibodies for binding to human CTLA-4 at pH 5 and at pH 7.4. FIG. 5G shows results of binding tests of anti-CTLA- 4 antibodies (CTLA-4- AB -34, CTLA-4-AB-35, CTLA-4-AB-36, CTLA-4-AB-37, CTLA-4-AB-38 and CTLA-4- AB-39) for binding to human CTLA-4 at pH 5 and at pH 7.4. FIG. 5H shows results of binding tests of anti-CTLA-4 antibodies (CTLA-4- AB -42, CTLA-4- AB -43, CTLA-4- AB -44, and CTLA-4- AB-45) and reference antibodies for binding to human CTLA-4 at pH 5 and at pH 7.4. FIG. 51 shows results of binding tests of anti-CTLA-4 antibodies (CTLA-4-AB-8, CTLA-4-AB-12, CTLA- 4- AB-33, and CTLA-4- AB-46) and reference antibodies for binding to human CTLA-4 at pH 5 and at pH 7.4. FIG. 5J shows results of binding tests of anti-CTLA-4 antibody CTLA-4- AB-47 and reference antibodies for binding to human CTLA-4 at pH 5 and at pH 7.4. FIG. 5K shows results of binding tests of anti-CTLA-4 antibody CTLA-4- AB -48 and reference antibodies for binding to human CTLA-4 at pH 5 and at pH 7.4. FIG. 5L shows results of binding tests of anti-CTLA-4 antibodies (CTLA-4- AB- 49, CTLA-4- AB-50, and CTLA-4- AB-51) and reference antibodies for binding to human CTLA-4 at pH 5 and at pH 7.4. FIG. 5M shows results of binding tests of anti-CTLA-4 antibodies (CTLA-4- AB- 52, CTLA-4-AB-53, and CTLA-4-AB-54) and reference antibodies for binding to human CTLA-4 at pH 5 and at pH 7.4. FIG. 5N shows results of binding tests of anti-CTLA-4 antibodies (CTLA-4- AB- 55, CTLA-4-AB-56, and CTLA-4-AB-57) and reference antibodies for binding to human CTLA-4 at pH 5 and at pH 7.4. FIG. 50 shows results of binding tests of anti-CTLA-4 antibodies (CTLA-4- AB- 58, CTLA-4-AB-59, and CTLA-4-AB-60) and reference antibodies for binding to human CTLA-4 at pH 5 and at pH 7.4. FIG. 5P shows results of binding tests of anti-CTLA-4 antibodies and reference antibodies (CTLA-4-AB-61, CTLA-4-AB-62, and CTLA-4- AB-63) for binding to human CTLA-4 at pH 5 and at pH 7.4. FIG. 5Q shows results of binding tests of anti-CTLA-4 antibodies (CTLA-4- AB- 64, CTLA-4-AB-65, and CTLA-4-AB-66) and reference antibodies for binding to human CTLA-4 at pH 5 and at pH 7.4. FIG. 5R shows results of binding tests of anti-CTLA-4 antibodies (CTLA-4- AB- 67, CTLA-4-AB-68, and CTLA-4-AB-69) and reference antibodies for binding to human CTLA-4 at pH 5 and at pH 7.4. FIG. 5S shows results of binding tests of anti-CTLA-4 antibodies (CTLA-4- AB- 70, CTLA-4-AB-71, and CTLA-4- AB-72) and reference antibodies for binding to human CTLA-4 at pH 5 and at pH 7.4.
[0086] FIGs. 6A-6C show results of binding tests of exemplary provided antibodies for binding to human CTLA-4 at different pH’s. FIG. 6A shows results of binding tests of a reference antibody for binding to human CTLA-4 at pH 5.5, pH 6, pH 6.5, pH 7 and pH 7.4. FIG. 6B shows results of binding tests of CTLA-4- AB-62 for binding to human CTLA-4 at pH 5.5, pH 6, pH 6.5, pH 7 and pH 7.4. FIG. 6C shows results of binding tests of CTLA-4-AB-68 for binding to human CTLA-4 at pH 5.5, pH 6, pH 6.5, pH 7 and pH 7.4.
[0087] FIGs. 7A-7C show effects of anti-CTLA-4 antibodies on CTLA-4 expression in treated human-CTLA-4 expressing Cells. FIG. 7A shows hCTLA-4 expression levels in cells treated with anti- CTLA-4 antibodies. FIG. 7B shows a schematic drawing of the workflow of confocal microscopy. FIG. 7C shows results of confocal microscopy. HEK293-hCTLA-4 cells were incubated with lOpg/mL antibody
[0088] FIGs. 8A-8B show effect of anti-CTLA-4 antibodies on antibody dependent cellular cytotoxicity (ADCC). FIG. 8A shows selection of NK cells. FIG. 8B shows target cell lysis was evaluated at various concentrations of four different antibodies and EC50 values calculated from doseresponse curves.
[0089] FIGs. 9A-9E show in vivo anti-tumor efficacy of anti-CTLA-4 antibodies. FIG. 9A shows a schematic drawing of the experimental procedure. FIG.9B shows tumor volume results of the vehicle, biweekly for 3 weeks, n=8. FIG. 9C shows tumor volume results of reference 1 at 1 mg/kg, biweekly for 3 weeks, n=8. FIG. 9D shows tumor volume results of CTLA-4- AB-68.1 at 1 mg/kg, biweekly for 3 weeks, n=8. FIG. 9E shows tumor volume results of CTLA-4- AB -68.1 at 10 mg/kg, biweekly for 3 weeks, n=8.
[0090] FIGs. 10A-10H show effect of anti-CTLA-4 antibodies on immune cell subsets in the tumor and blood of hCTLA4 knock-in mice. FIG. 10A shows gating strategy for tumor samples to analyze. This panel was designed to analyze absolute cell numbers of CD45+, Myeloid, T cells, CD4 T, CD8 T and Treg in tumor of CT26 model in hCTLA4 knock-in mice. FIG. 10B shows gating strategy for blood samples to analyze absolute cell numbers of CD45+, Myeloid, T cells, CD4 T, CD8 T and Treg in blood of CT26 model in hCTLA4 knock-in mice, and the expression level of hCTLA-4 on Treg cells in the blood of CT26 model in hCTLA4 knock-in mice. FIG. 10C shows results of the FCM analysis for tumor samples. One-way ANOVA was used for statistical analysis. When a significant F- statistics (a ratio of treatment variance to the error variance) was obtained, comparisons between group 1 and other groups were carried out with Games-Howell (equal variances not assumed) or Tukey (equal variances assumed) test. * p<0.05; ** p<0.01; *** p<0.001. Error bars represented Standard Error of Mean (SEM). FIG. 10D shows results of the FCM analysis for blood samples showing percentages of myeloid, T cells and T cell subpopulations in CD45+ cells. FIG. 10E shows results of the FCM analysis for blood samples showing percentages of T cell subpopulations in T cells. FIG. 10F shows results of the FCM analysis for blood samples showing cell numbers of CD45+, myeloid, T cells and T cell subpopulations per 100 pL blood. FIG. 10G shows results of the FCM analysis for blood samples showing ratios between T cell subpopulations. CD4Teff refers to CD4T cells other than Treg. FIG. 10H shows mean fluorescence intensity (MFI) of hCTLA4 of Treg cells. One-way ANOVA was used for statistical analysis. When a significant F-statistics (a ratio of treatment variance to the error variance) was obtained, comparisons between group 1 and other groups were carried out with Games-Howell (equal variances not assumed) or Tukey (equal variances assumed) test. * p<0.05; ** p<0.01; *** p<0.001. Error bars represented Standard Error of Mean (SEM).
[0091] FIG.ll depicts an exemplary format (“Crossmab”) of a PD-l/CTLA-4 bispecifc antibody binding molecule. The binding molecule includes a variable heavy (VH) chain and a variable light (VL) chain of an anti- PD-1 antibody, and a variable heavy (VH) chain and a variable light (VL) chain of an anti-CTLA-4 antibody, each configured as Fabs such that for one the VH and a VL region are interchanged in one binding arm (“crossmab” format). As depicted, the Fc region of the bispecific antibody is formed with a heterodimeric Fc containing knob-into-hole heterodimeric connections to form two different heavy chains. In one configuration is an antibody designated PC_abl in which the Targetl is CTLA4 and the CTLA4 Fab is a crossmab format and the Target2 is PD-1 with a conventional PD-1 Fab. In another configuration is an antibody designated PC_ab2 in which the Targetl is PD-1 and the PD-1 Fab is a crossmab format, and the Target2 is CTLA-4 with a conventional CTLA-4 Fab.
[0092] FIGs 12A-12B show reducing and non-reducing SDS-PAGE gels showing the heavy and light chains of the bispecific antibodies.
[0093] FIGs. 13A-13B show SEC-HPLC results for CTLA-4/PD-l-abl and CTLA-4/PD-l-ab2.
[0094] FIGs. 14A-14C are binding results of the engineered bispecific PD-l/CTLA-4 antibodies from an Enzyme-Linked Immuno-Sorbent Assay at different pH 5.0 and 7.4. Detailed Description
[0095] The present disclosure concerns anti-PD-1 antibodies or antibody fragments suitable for therapeutic use. The present disclosure additionally concerns binding molecules, such as multispecific antibodies (e.g., bispecific antibodies), that contain a PD1 binding domain (e.g., antibody fragment, such as a Fab) that binds PD-1 and a CTLA-4 binding domain (e.g., antibody fragment, such as a Fab) for therapeutic use.
[0096] PD1 (Programmed cell death 1; also known as CD279 (cluster of differentiation 279)), is a cell surface receptor protein expressed primarily on T-cells, but also on other immune cells.
The PD-1 pathway is a regulator in the induction and maintenance of immunological tolerance. The protein functions as an "immune checkpoint" inhibitor, i.e. it acts to modulate the activity of cells in the immune system so as to regulate and limit autoimmune diseases. PD-1 has two ligands, PD-L1 and PD-L2, which interact with the cell surface receptor. On binding, PD-1 induces an intracellular signal, which negatively regulates T-cell responses. On the surface of activated T cells, PD-
1 expression is upregulated after the recognition of peripheral antigens by T cells; subsequently, the elevated binding of PD-1 to PD-L1 and PD-L2 becomes a key step for downstream inhibitory signaling. PD-1 is also associated with increased Treg-cell proliferation and enhanced immunosuppressive function. In some embodiments, provided anti-PD-1 antibodies or antibody fragments act as PD-1 inhibitors to activate the immune system to attack tumors, including in methods for treating cancer.
[0097] Relative to existing known therapies, some of these anti-PD-1 antibodies or antibody fragments may exhibit improved binding kinetics to PD-1 in a tumor environment versus PD-1 present in normal tissue. The disclosed anti-PD-1 antibodies or antibody fragments may also exhibit physiochemical or biological properties that lead to improved efficacy and safety to anti-PD-1 antibodies known in the art.
[0098] In some embodiments, provided anti-PD-1 antibodies or antibody fragments have a higher binding affinity to PD-1 in a tumor in comparison with PD-1 present in normal tissue. In some such embodiments, these anti-PD-1 antibodies or antibody fragments have at least comparable efficacy as well as a longer half-life, but reduced side-effects, in comparison with monoclonal anti-PD-1 antibodies known in the art. This may permit use of higher dosages of these anti-PD-1 antibodies or antibody fragments, thus providing a more effective therapeutic option without a corresponding significant increase in side effects.
[0099] In provided embodiments, a binding molecule, such as a bispecific antibody, that is directed against PD-1 and also CTLA-4 can simultaneously block the PD-1 and also block CTLA-4 to thus reduce immune responses to the tumor site. Likewise, among the provided binding molecules containing a PD-1 binding domain and a CTLA-4 binding domain are binding molecules that comprise a CTLA-4 binding domain that confers specific binding properties to human CTLA-4 under different pH conditions. In some embodiments, the disclosed binding molecules also exhibit physiochemical or biological properties that lead to improved efficacy and safety as compared to CTLA-4 binding domains known in the art.
[0100] An altered pH microenvironment is the most common microenvironment found in disease states such as tumor microenvironments, and it is the most uniform within the disease microenvironment compared to other properties such as hypoxia (see e.g. Fogh Andersen et al. (1995) Clin. Chem., 41:1522-1525; Bhujwalla et al. (2002) NMR Biomed., 15:114-119; Helmlinger et al. (1997) Nature Med., 3:177; Gerweck and Seetharaman (1996), Cancer Res. 56(6): 1194-1198). For example, in many tumors the ‘Warburg effect’ creates a microenvironment with a pH ranging from 5.6 to 6.8.
[0101] The immune system is responsible for defending the body against foreign substances, such as pathogens and abnormal cells. Central to the immune response are T cells, a type of white blood cell, which act as key orchestrators of immune reactions. T cell activation is tightly regulated to prevent excessive immune responses that could lead to tissue damage and autoimmune disorders. CTLA-4, a protein predominantly expressed on the surface of activated T cells, serves as an important checkpoint molecule for downregulating immune responses.
[0102] CTLA-4 acts by binding to its ligands, CD80 (B7-1) and CD86 (B7-2), on antigen- presenting cells (APCs), thereby attenuating T cell activation and proliferation. The engagement of CTLA-4 with its ligands delivers inhibitory signals to T cells, leading to suppression of immune reactions. Dysregulation of the CTLA-4 pathway has been implicated in several pathological conditions, including autoimmune diseases as well as cancer, where tumor cells can exploit the immune system’s regulatory mechanisms to evade destruction.
[0103] Based on promising preclinical tumor model studies, the clinical potential of antibodies against CTLA-4 has been explored in different human malignancies. Although a specific anti-CTLA-4 antibody (Ipilimumab, marketed as Yervoy) has demonstrated efficacy in treating melanoma, treatment and targeting of CTLA-4 is associated with autoimmune like toxicities. Characteristic side effects from inhibition of CTLA-4 are generally called immune-related adverse events (ir AEs) and the most common irAEs are skin rash, hepatitis, colitis and endocrinopathies, particularly hypopituitarism. Without wishing to be bound by any theory, CTLA-4 is recycled between the plasma membrane and endosomes by binding to lipopolysaccharide -responsive and beige-like anchor protein (LRBA). Antibodies that bind CTLA-4 disrupt the recycling process. Consequently, CTLA-4 is systemically directed to lysosomal degradation, and autoimmunity-related adverse effects are developed due to the unstoppable action of cytotoxic T cells. Therefore, there is a desire to improve the therapeutic potential of CTLA-4 binding domains by increasing efficacy while reducing the associated irAEs. [0104] In some embodiments, the provided binding molecules comprise a CTLA-4 binding domain that possesses pH dependent binding properties to human CTLA-4 that facilitates the dissociation of antibody from the antibody: CTLA-4 complex upon internalization into a cell, allowing either the antibody, CTLA-4, or both to be recycled instead of being degraded in the lysosome.
[0105] In some embodiments, the provided binding molecules preserve high affinity for CTLA-4 in neutral pH environments (such as at pH>7), lower affinity in acidic environments (such as at pH 5.5- 7 found in early endosomes), and significantly reduced affinity for CTLA-4 at more acidic environments (such as at pH<5.5 found in late endosomes and lysosomes).
[0106] In some embodiments, provided binding molecules comprise a CTLA-4 binding domain that has a lower binding affinity (i.e. greater dissociation constant) to CTLA-4 at an acidic pH as present in endosomes of a cell in comparison with the binding affinity of CTLA-4 present in normal tissue. In some embodiments, the dissociation constant (KD) for binding at acidic pH (e.g., pH 5.0 to 6.0) is higher than the dissociation constant for binding at physiological pH (e.g., e.g. pH 7.35 to 7.45). In some such embodiments, these binding molecules have reduced side-effects, in comparison with monoclonal anti- CTLA-4 antibodies known in the art. This may permit use of higher dosages of these binding molecules, thus providing a more effective therapeutic option without a corresponding significant increase in side effects.
[0107] In some embodiments, the provided binding molecules comprise a CTLA-4 binding domain that can be used to block inhibitory activity of CTLA-4 by CTLA-4-expressing cells including regulatory T cells (Tregs), and in some cases may also mediate depletion of such cells by an ADCC mechanism. In some embodiments, depletion of Tregs in the tumor contributes to anti-tumor efficacy of the provided antibodies. In some embodiments, the provided binding molecules comprise a CTLA- 4 binding domain that reduces CTLA-4 mediated signaling in effector T cells and exhibit activity to deplete Tregs in the tumor microenvironment that might otherwise suppress an anti-tumor immune response.
[0108] In some embodiments, the binding molecule or a pharmaceutical composition comprising the binding molecule induces strong deletion of Treg and local T cell activation in tumor microenvironment but minimal systemic T cell activation. In some embodiments, the binding molecule or a pharmaceutical composition comprising the binding molecule induces a favorable ratio of Treg cells to T effector cells in the tumor microenvironment.
[0109] All publications, including patent documents, scientific articles and databases, referred to in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication were individually incorporated by reference. If a definition set forth herein is contrary to or otherwise inconsistent with a definition set forth in the patents, applications, published applications and other publications that are herein incorporated by reference, the definition set forth herein prevails over the definition that is incorporated herein by reference.
[0110] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.
I. ANTI-PD-1 ANTIBODIES AND ANTIGEN-BINDING FRAGMENTS AND BINDING MOLECULES THEREOF
[0111] Provided herein are anti-PD-1 antibodies, including full-length antibodies or antigenbinding fragments, that contain a heavy chain variable region (VH) sequence and a light chain variable region (VL) sequence as described, or a sufficient antigen-binding portion thereof. In some such embodiments, the VH is the region of the anti-PD-1 antibody that comprises the three heavy chain complementarity determining regions (CDRs) and the VL chain is the region of the anti-PD-1 antibody that comprises the three light chain CDRs. Among provided anti-PD-1 antibodies, including full-length antibodies or antigen-binding fragments, are antibodies that contains a VH region sequence that contains a CDR-H1, a CDR-H2 and a CDR-H3 as described and contains a VL region sequence that contains a CDR-L1, a CDR-L2 and a CDR-L3 as described. In some embodiments, the antibodies include antibodies that specifically bind to PD-1, e.g., human PD-1. Among the provided anti-PD-1 antibodies are human antibodies, or antibodies that are modified from or variant of human antibodies. The antibodies include isolated antibodies. Also provided are PD-1 -binding molecules containing such antibodies, such as single-chain proteins, fusion proteins, conjugates and/or recombinant receptors such as chimeric antigen receptors. In some embodiments, provided herein is an PD-1 -targeted antibodydrug conjugate (ADC) comprising an anti-PD-1 antibody or antigen binding fragment thereof described herein. In some embodiments, provided herein is a bispecific immune cell engager that is a fusion protein comprising the anti-PD-1 antibody or antigen binding fragment thereof described herein. In some embodiments, the bispecific immune cell engager is a bispecific T cell engager. In some aspects, the PD-1 -binding molecules include isolated molecules.
[0112] Also provided are polynucleotides containing nucleic acids sequences encoding all or a portion of such antibodies, including an antigen-binding fragment, or binding molecule. The provided polynucleotides can be incorporated into constructs, such as deoxyribonucleic acid (DNA) or RNA constructs, such as those that can be introduced into cells for expression of the encoded anti-PD-1 antibodies or binding molecules.
[0113] The term “antibody” herein is used in the broadest sense and includes polyclonal and monoclonal antibodies, including intact antibodies and functional (antigen-binding) antibody fragments, including fragment antigen binding (Fab) fragments, F(ab’)2 fragments, Fab’ fragments, Fv fragments, recombinant IgG (rlgG) fragments, heavy chain variable (VH) regions capable of specifically binding the antigen, single chain antibody fragments, including single chain variable fragments (scFv), and single domain antibodies (e.g., sdAb, sdFv, nanobody, VHH) fragments. The term encompasses genetically engineered and/or otherwise modified forms of immunoglobulins, such as intrabodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies, and heteroconjugate antibodies, multispecific, e.g., bispecific or trispecific, antibodies, diabodies, triabodies, and tetrabodies, tandem di-scFv, tandem tri-scFv. Unless otherwise stated, the term “antibody” should be understood to encompass functional antibody fragments thereof also referred to herein as “antigenbinding fragments.” The term also encompasses intact or full-length antibodies, including antibodies of any class or sub-class, including IgG and sub-classes thereof, IgM, IgE, IgA, and IgD.
[0114] The terms “complementarity determining region,” and “CDR,” synonymous with “hypervariable region” or “HVR,” are known to refer to non-contiguous sequences of amino acids within antibody variable regions, which confer antigen specificity and/or binding affinity. In general, there are three CDRs in each heavy chain variable region (CDR-H1, CDR-H2, CDR-H3) and three CDRs in each light chain variable region (CDR-L1, CDR-L2, CDR-L3). “Framework regions” and “FR” are known to refer to the non-CDR portions of the variable regions of the heavy and light chains. In general, there are four FRs in each full-length heavy chain variable region (FR-H1, FR-H2, FR-H3, and FR-H4), and four FRs in each full-length light chain variable region (FR-L1, FR-L2, FR-L3, and FR-L4).
[0115] The precise amino acid sequence boundaries of a given CDR or FR can be readily determined using any of a number of well-known schemes, including those described by Kabat et al. (1991), “Sequences of Proteins of Immunological Interest,” 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (“Kabat” numbering scheme); Al-Lazikani et al., J Mol Biol, 1997; 273(4):927-48 (“Chothia” numbering scheme); MacCallum et al., J. Mol. Biol, 1996; 262:732-745.” (“Contact” numbering scheme); Lefranc MP et al., Dev Comp Immunol, 2003; 27(l):55-77 (“IMGT” numbering scheme); Honegger A and Pliickthun A, J Mol Biol, 2001; 309(3):657-70, (“Aho” numbering scheme); Martin et al., PNAS, 1989; 86(23):9268-9272, (“AbM” numbering scheme); and Ye et al., Nucleic Acids Res. 2013; 41(Web Server issue):W34-40, (“IgBLAST numbering scheme). Details regarding various numbering schemes are also described in, for example, Jarasch et al., Proteins, 2017; 85(1):65-71; Martin et al., Bioinformatics tools for antibody engineering. In: Diibel, S. (editor) Handbook of Therapeutic Antibodies, Vol. 1. Wiley-VCH, Weinheim, Germany; Martin, A.C.R. (2010). Protein Sequence and Structure Analysis of Antibody Variable Domains. In: Kontermann, R., Diibel, S. (eds) Antibody Engineering. Springer Protocols Handbooks. Springer, Berlin, Heidelberg; and Martin, ACR, Antibody Information: How to identify the CDRs by looking at a sequence [online] bioinf.org.uk/abs/info.html, all of which are incorporated by reference in their entireties. Various prediction algorithm tools are available and known for numbering antibody residues and CDRs (e.g., AbYsis, Abnum, AbYmod, AbRSA, IgBLAST, IMGT, or ANARCI). [0116] The boundaries of a given CDR or FR may vary depending on the scheme used for identification. For example, the Kabat scheme is based on structural alignments, while the Chothia scheme is based on structural information. Numbering for both the Kabat and Chothia schemes is based upon the most common antibody region sequence lengths, in some cases with insertions. Insertions in the sequence relative to the standard numbering scheme are indicated using insertion letter codes. For example, residues that are inserted between residues L30 and L31 are indicated as L31A, L31B, etc. Deletions in the sequence relative to the standard scheme are accommodated by skipping numbers. The two schemes place certain insertions and deletions (“indels”) at different positions, resulting in differential numbering. For instance, the Chothia numbering scheme is nearly identical to the Kabat numbering scheme, except that insertions are placed at structural positions and topologically equivalents residues do get assigned the same numbers. The Contact scheme is based on analysis of complex crystal structures and is similar in many respects to the Chothia numbering scheme. The AbM scheme is a compromise between Kabat and Chothia definitions based on that used by Oxford Molecular’s AbM antibody modeling software. The IgBLAST scheme is based on matching to germline V, D and J genes, and can be determined using National Center for Biotechnology Information (NCBI)’s IgBLAST tool.
[0117] In some embodiments, Kabat numbering can be determined by known sequence rules as described in, for example, Kabat et al. (1991), “Sequences of Proteins of Immunological Interest,” 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. In some embodiments, the Kabat numbering scheme in some aspects can include any of the following rules to designate CDRs: CDR-L1 starts at approximately residue 24 of the light chain, always has a preceding C residue, and always has a following W residue; the end of CDR-L1 is defined by a stretch of 3 residues, where the W residue can be followed by Y, L, or F, followed by Q or L; CDR-1 has a length of 10 to 17 residues; CDR-L2 always starts 16 residues after the end of CDR-L1; the two residues before CDR-L2 are I and Y but can also be V and Y, I and K, or I and F; CDR-L2 is always 7 residues long; CDR-L3 always starts 33 residues after the end of CDR-L2, always has a preceding C residue, and is strictly followed by a F-G-X-G sequence motif, where X is any amino acid; CDR-L3 has a length of 7 to 11 residues; CDR-H1 starts at approximately position 26 of the heavy chain; the first amino acid in CDR-H1 is always 9 residues after a conserved C residue; CDR-H1 is followed by an invariant W residue followed by typically V, but also can be I or A; CDR-H1 has a length of 5 to 7 residues; CDR-H2 always starts at 15 residues after the end of CDR-H1 ; the first residue in CDR-H2 is usually preceded by the sequence motif L-E-W-I-G but a number of variations exist; the end of CDR-H2 is defined by a motif of 3 residues - the first residue of the motif of 3 residues can be either K or R, the second residue of the motif of 3 residues can be L, I, V, F, T, or A, the third residue of the motif of 3 residues can be T, S, I, or A; CDR- H2 has a length of 16 to 19 residues; CDR-H3 always starts 33 residues after the end of CDR-H2 and is always 3 residues after a C residue - the first residue of CDR-H3 is preceded by the conserved C residue followed by two residues, which are usually A-R; the residues following CDR-H3 is strictly followed by a W-G-X-G sequence motif, where the X is any amino acid; CDR-H3 typically has a length of 3 to 25 residues; CDR-H3 can be much longer than 25 residues.
[0118] In some cases, according to the Chothia numbering scheme, exact boundary positions of certain CDRs can differ based on different definitions for the CDRs (See e.g., Martin, ACR, Antibody Information: How to identify the CDRs by looking at a sequence [online] bioinf.org.uk/abs/info.html). For example, in some instances, the boundary positions for CDR-L1 according to Chothia numbering can be L26— L32 (Chothia et al., Science, 1986; 233(4765):755-8 and Chothia C. and Lesk A.M. J Mol Biol, 1987; 196(4):901-17). In some instances, the boundary positions for CDR-L1 can be L25— L32 (Al-Lazikani et al., J Mol Biol, 1997; 273(4):927-48). In some instances, the boundary positions for CDR-L2 can be L50— L52 and for CDR-L3 can be L91— L96 (Chothia et al., Science, 1986; 233(4765):755-8; Chothia C. and Lesk A.M. J Mol Biol, 1987; 196(4):901-17; and Al-Lazikani et al., J Mol Biol, 1997; 273(4):927-48). In some instances, the boundary positions for CDR-H1 according to Chothia numbering can be H26— H32 (Chothia et al., Science, 1986; 233(4765):755-8; Chothia C. and Lesk A.M. J Mol Biol, 1987; 196(4):901-17; and Al-Lazikani et al., J Mol Biol, 1997; 273(4):927-48). In some instances, the boundary positions for CDR-H2 can be H53— H55 (Chothia et al., Science, 1986; 233(4765):755-8 and Chothia C. and Lesk A.M. J Mol Biol, 1987, 196(4):901-17); H52a-H55 (Tramontane et al., J Mol Biol, 1990, 215(1): 175-82), or H52— H56 (Al-Lazikani et al., J Mol Biol., 1997; 273(4):927-48). In some instances, the boundary positions for CDR-H3 can be H96— H101 (Chothia et al., Science, 1986; 233(4765):755-8 and Chothia C. and Lesk A.M. J Mol Biol., 1987; 196(4):901-17). In some instances, the boundary positions for CDR-H3 can be H92— H104 (Morea et al., Biophys Chem, 1997; 68(1-3): 9-16 and Morea et al., J Mol Biol., 1998; 275(2): 269-94).
[0119] Table 1, below, exemplifies exemplary numbering and lists exemplary position boundaries of CDR-L1, CDR-L2, CDR-L3 and CDR-H1, CDR-H2, CDR-H3 as identified by Kabat, Chothia, AbM, and Contact schemes, respectively. For CDR-H1, residue numbering is listed using both the Kabat and Chothia numbering schemes. FRs are located between CDRs, for example, with FR-L1 located before CDR-L1, FR-L2 located between CDR-L1 and CDR-L2, FR-L3 located between CDR- L2 and CDR-L3 and so forth. It is noted that because the shown Kabat numbering scheme places insertions at H35A and H35B, the end of the Chothia CDR-H1 loop when numbered using the shown Kabat numbering convention varies between H32 and H34, depending on the length of the loop.
1 - Kabat et al. (1991), “Sequences of Proteins of Immunological Interest,” 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD
2 - Al-Lazikani et al., J Mol Biol., 1997; 273(4):927-48).
[0120] Thus, unless otherwise specified, a “CDR” or “complementary determining region,” or individual specified CDRs (e.g., CDR-H1, CDR-H2, CDR-H3), of a given antibody or region thereof, such as a variable region thereof, should be understood to encompass a (or the specific) complementary determining region as defined by any of the aforementioned schemes, or other known schemes. For example, where it is stated that a particular CDR (e.g., a CDR-H3) contains the amino acid sequence of a corresponding CDR in a given VH or VL region amino acid sequence, it is understood that such a CDR has a sequence of the corresponding CDR (e.g., CDR-H3) within the variable region, as defined by any of the aforementioned schemes, or other known schemes. In some embodiments, where it is stated that an antibody or antigen-binding fragment thereof comprises a CDR-H1, a CDR-H2, and a CDR-H3 as contained within a given VH region amino acid sequence and a CDR-L1, a CDR-L2, and a CDR-L3 as contained within a given VL region amino acid sequence, the CDRs can be defined by any of the aforementioned schemes, such as Kabat, Chothia, AbM, IgBLAST, IMGT, or Contact method, or other known scheme. In some embodiments, specific CDR sequences are specified. Exemplary CDR sequences of provided antibodies are described using various numbering schemes, although it is understood that a provided antibody can include CDRs as described according to any of the other aforementioned numbering schemes or other known numbering schemes.
[0121] Likewise, unless otherwise specified, a FR or individual specified FR(s) (e.g., FR-H1, FR- H2, FR-H3, FR-H4, FR-E1, FR-E2, FR-E3, and/or FR-E4), of a given antibody or region thereof, such as a variable region thereof, should be understood to encompass a (or the specific) framework region as defined by any of the known schemes. In some instances, the scheme for identification of a particular CDR, FR, or FRs or CDRs is specified, such as the CDR as defined by the Kabat, Chothia, AbM, IgBEAST, IMGT, or Contact method, or other known schemes. In other cases, the particular amino acid sequence of a CDR or FR is given. In some embodiments, where it is stated that an antibody or antigen-binding fragment thereof comprises a FR-H1, a FR-H2, a FR-H3, and a FR-H4 as contained within a given VH region amino acid sequence and a FR-E1, a FR-E2, a FR-E3, and a FR-E4 as contained within a given VL region amino acid sequence, the FRs can be defined by any of the aforementioned schemes, such as Kabat, Chothia, AbM, IgBLAST, IMGT, or Contact method, or other known scheme.
[0122] The term “variable region” or “variable domain” refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to antigen. The variable regions of the heavy chain and light chain (VH and VL, respectively) of a native antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three CDRs (See, e.g. , Kindt et al. Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007)). A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind a particular antigen may be isolated using a VH or VL domain from an antibody that binds the antigen to screen a library of complementary VL or VH domains, respectively. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).
[0123] Among the provided antibodies are antibody fragments. An “antibody fragment” or “antigen-binding fragment” refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds. Typically, an antigen binding fragment includes all CDRs of a variable heavy chain (VH) and variable light chain (VL) sequence from antibodies that bind PD-1 set forth herein. Examples of antibody fragments include but are not limited to Fv, Fab, Fab’, Fab’-SH, F(ab’)2; diabodies; linear antibodies; heavy chain variable (VH) regions, single-chain antibody molecules such as scFvs and single-domain antibodies comprising only the VH region; and multispecific antibodies formed from antibody fragments. Papain digestion of antibodies produce two identical antigen-binding fragments, called "Fab" fragments, and a residual "Fc" fragment, a designation reflecting the ability to crystallize readily. The Fab fragment is composed of an entire L chain along with the variable region domain of the H chain (VH), and the first constant domain of one heavy chain (CHI). Each Fab fragment is monovalent with respect to antigen binding, i.e., it has a single antigen-binding site. Pepsin treatment of an antibody yields a single large F(ab')2 fragment which roughly corresponds to two disulfide linked Fab fragments having different antigen-binding activity and is still capable of cross-linking antigen. Fab' fragments differ from Fab fragments by having a few additional residues at the carboxy terminus of the CHI domain including one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine residue(s) of the constant domains bear a free thiol group. F(ab')2 antibody fragments originally were produced as pairs of Fab' fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known. In some embodiments, the antibody is or comprises an antibody fragment comprising a variable heavy chain (VH) and a variable light chain (VL) region. In particular embodiments, the antibodies are single-chain antibody fragments comprising a heavy chain variable (VH) region and/or a light chain variable (VL) region, such as scFvs.
[0124] Antibody fragments can be made by various techniques, including but not limited to proteolytic digestion of an intact antibody as well as production by recombinant host cells. In some embodiments, the antibodies are recombinantly-produced fragments, such as fragments comprising arrangements that do not occur naturally, such as those with two or more antibody regions or chains joined by synthetic linkers, e.g., peptide linkers, and/or that are may not be produced by enzyme digestion of a naturally-occurring intact antibody. In some aspects, the antibody fragments are scFvs.
[0125] The term “F(ab)” refers to two of the protein fragments resulting from proteolytic cleavage of immunoglobulin G (IgG) molecules by the enzyme papain. Each F(ab) comprises a covalent heterodimer of the VH chain and VL chain and includes an intact antigen-binding site.
[0126] The term “F(ab)2” refers to a protein fragment of IgG generated by proteolytic cleavage by the enzyme pepsin. Each F(ab')2 fragment comprises two F(ab) fragments, thus comprising both antigen-binding sites.
[0127] An “Fv fragment” for use according to certain embodiments of the present invention can be produced by preferential proteolytic cleavage of an IgM, and on rare occasions of an IgG or IgA immunoglobulin molecule. Fv fragments are, however, more commonly derived using recombinant techniques known in the art. The Fv fragment includes a non-covalent VH::VL heterodimer including an antigen-binding site which retains much of the antigen recognition and binding capabilities of the native antibody molecule, but lacking the CHI and CL domains contained within a Fab. Inbar et al. (1972) Proc. Nat. Acad. Sci. USA 69:2659-2662; Hochman et al. (1976) Biochem / 5'.2106-21 \ 0 and Ehrlich et al. (1980) Biochem 79:4091-4096.
[0128] In certain embodiments, single chain Fv (scFv) antibodies are contemplated and may be prepared using standard molecular biology techniques following the teachings of the present application with regard to selecting antibodies having the desired specificity.
[0129] In still further embodiments, chimeric antibodies may be made. For example, a chimeric antibody may comprise CDRs and framework regions from different antibodies. These antibodies may be produced through recombinant molecular biological techniques or may be physically conjugated together.
[0130] A scFv polypeptide is a covalently linked VH::VL heterodimer which is expressed from a gene fusion including VH- and V| -cncoding genes linked by a peptide-encoding linker. Huston et al. (1988) Proc. Nat. Acad. Sci. USA S5(16):5879-5883. A number of methods have been described to discern chemical structures for converting the naturally aggregated — but chemically separated — light and heavy polypeptide chains from an antibody V region into an scFv molecule which will fold into a three dimensional structure substantially similar to the structure of an antigen-binding site. See, e.g., U.S. Pat. Nos. 5,091,513 and 5,132,405, to Huston et al. ', and U.S. Pat. No. 4,946,778, to Ladner et al.
[0131] In certain embodiments, the antibodies described herein may be provided in the form of a UniBody®. A UniBody® is an IgG4 antibody with the hinge region removed (see GenMab Utrecht, The Netherlands; see also, e.g., US20090226421). This antibody technology creates a stable, smaller antibody format with an anticipated longer therapeutic window than current small antibody formats. IgG4 antibodies are considered inert and thus do not interact with the immune system. Fully human IgG4 antibodies may be modified by eliminating the hinge region of the antibody to obtain halfmolecule fragments having distinct stability properties relative to the corresponding intact IgG4 (GenMab, Utrecht). Halving the IgG4 molecule leaves only one area on the UniBody® that can bind to cognate antigens (e.g., disease targets) and the UniBody® therefore binds univalently to only one site on target cells. For certain cancer cell surface antigens, this univalent binding may not stimulate the cancer cells to grow as may be seen using bivalent antibodies having the same antigen specificity, and hence UniBody® technology may afford treatment options for some types of cancer that may be refractory to treatment with conventional antibodies. The small size of the UniBody® can be a great benefit when treating some forms of cancer, allowing for better distribution of the molecule over larger solid tumors and potentially increasing efficacy.
[0132] In certain embodiments, the antibodies of the present disclosure may be chimeric antibodies. In this regard, a chimeric antibody is comprised of an antigen-binding fragment of an anti- PD-1 antibody operably linked or otherwise fused to a heterologous Fc portion of a different antibody. In certain embodiments, the heterologous Fc domain is of human origin. In further embodiments, the heterologous Fc domain may be from a different Ig class from the parent antibody, including IgA (including subclasses IgAl and IgA2), IgD, IgE, IgG (including subclasses IgGl, IgG2, IgG3, and IgG4), and IgM. In further embodiments, the heterologous Fc domain may be comprised of CH2 and CH3 domains from one or more of the different Ig classes.
[0133] In certain embodiments, the antibodies of the present disclosure may be “non-naturally occurring” antibodies. Non-naturally occurring antibodies can refer to antibodies that comprise one or more amino acid modifications, such that the resultant antibody is substantially non-naturally occurring (e.g., does not exist in nature). These amino acid modifications can include point mutations, wherein a naturally occurring amino acid is substituted for another naturally occurring amino acid. In some embodiments, the amino acid modifications can include point mutations wherein a non-naturally occurring amino acid is substituted for a naturally occurring amino acid. Non-naturally occurring antibodies can also refer to antibodies that are conjugated to a heterologous protein or compound, such as a detectable marker.
[0134] Among the provided antibodies are human antibodies. A “human antibody” is an antibody with an amino acid sequence corresponding to that of an antibody produced by a human or a human cell, or non-human source that utilizes human antibody repertoires or other human antibodyencoding sequences, including human antibody libraries. The term excludes humanized forms of non- human antibodies comprising non-human antigen-binding regions, such as those in which all or substantially all CDRs are non-human. The term includes antigen-binding fragments of human antibodies. [0135] Human antibodies may be prepared by administering an immunogen to a transgenic animal that has been modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigenic challenge. Such animals typically contain all or a portion of the human immunoglobulin loci, which replace the endogenous immunoglobulin loci, or which are present extrachromosomally or integrated randomly into the animal’s chromosomes. In such transgenic animals, the endogenous immunoglobulin loci have generally been inactivated. Human antibodies also may be derived from human antibody libraries, including phage display and cell-free libraries, containing antibody-encoding sequences derived from a human repertoire.
[0136] Among the provided antibodies are monoclonal antibodies, including monoclonal antibody fragments. The term “monoclonal antibody” as used herein refers to an antibody obtained from or within a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical, except for possible variants containing naturally occurring mutations or arising during production of a monoclonal antibody preparation, such variants generally being present in minor amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different epitopes, each monoclonal antibody of a monoclonal antibody preparation is directed against a single epitope on an antigen. The term is not to be construed as requiring production of the antibody by any particular method. A monoclonal antibody may be made by a variety of techniques, including but not limited to generation from a hybridoma, recombinant DNA methods, phage-display and other antibody display methods.
[0137] In some aspects, the antibody or the antigen-binding fragments of the antibody is isolated.
[0138] The present disclosure contemplates variants of the antibodies disclosed herein. In certain embodiments, such variant antibodies or antigen-binding fragments, or CDRs thereof, bind to PD-1 at least about 50%, at least about 70%, at least about 80%, at least about 85%, at least about 90% and in certain embodiments, at least about 95% as well as an antibody sequence specifically set forth herein. In further embodiments, such variant antibodies or antigen-binding fragments, or CDRs thereof, bind to PD-1 with greater affinity than the antibodies set forth herein, for example, that bind quantitatively at least about 105%, 106%, 107%, 108%, 109%, or 110% as well as an antibody sequence specifically set forth herein.
[0139] In particular embodiments, a subject antibody may have: a) a heavy chain variable region having an amino acid sequence that is at least 80% identical, at least 85% identical, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or 99% or 100% identical, to a heavy chain variable region of an anti-PD-1 antibody described herein; and b) a light chain variable region having an amino acid sequence that is at least 80% identical, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or 99% or 100% identical, to a light chain variable region of an anti-PD-1 antibody described herein. A. Exemplary Antibodies
[0140] In some embodiments, the anti-PD-1 antibody contains a VH region and a VL region containing a combination of six CDRs as described below. In some embodiments, the anti-PD-1 antibody contains a VH region and a VL region as described below. In any such embodiments, a VH region sequence can be any of the VH region sequence described herein. In any such embodiments, a VL region sequence can be any of the VL region sequence described herein. In any such embodiments, any of the VH region sequence and any of the VL region sequence described herein can be used in combination. In some such embodiments, the antibody is an antigen-binding fragment, such as a Fab or an scFv. In some embodiments, the antibody or antigen-binding fragment further comprises at least a portion of an immunoglobulin constant region or a variant thereof. In some such embodiments, the antibody is a full-length antibody that also contains a constant region. In some embodiments, an anti- PD-1 antibody comprises at least one heavy chain comprising a VH region and at least a portion of a heavy chain constant region, and at least one light chain comprising a VL region and at least a portion of a light chain constant region. In some embodiments, an anti-PD-1 antibody comprises two heavy chains, wherein each heavy chain comprises a VH region and at least a portion of a heavy chain constant region, and two light chains, wherein each light chain comprises a VL region and at least a portion of a light chain constant region. In some embodiments, an anti-PD-1 antibody comprises two heavy chains, wherein each heavy chain comprises a VH region and a heavy chain constant region, and two light chains, wherein each light chain comprises a VL region and a light chain constant region.
[0141] Anti-PDl antibodies, including antigen-binding fragments thereof, include any combination of the heavy chain and light chain complementarity-determining regions (CDRs) discussed herein. In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises any one of the CDR-H1 as described herein, any one of the CDR-H2 as described herein, any one of the CDR-H3 as described herein, any one of the CDR-L1 as described herein, any one of the CDR-L2 as described herein and any one of the CDR-L3 as described here. In some of any such embodiments, any one or more of the CDR-H1, the CDR-H2 and the CDR-H3 sequences described herein, and any one or more of the CDR-L1, the CDR-L2 and the CDR-L3 sequences described herein can be used in combination.
[0142] In some embodiments, a provided anti-PD-1 antibody or an antigen-binding fragment thereof has a CDR-H1, a CDR-H2 and a CDR-H3 present in a VH region amino acid sequence set forth in any one of SEQ ID NOs: 1-45, 157, and 165-170, or an amino acid sequence that has at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the VH region amino acid sequence set forth in any one of SEQ ID NOs: 1-45, 157, and 165- 170. In some embodiments, a provided anti-PD-1 antibody or an antigen-binding fragment thereof has a CDR-L1, a CDR-L2 and a CDR-L3 present in a VL region amino acid sequence set forth in any one of SEQ ID NOs: 46-90, 158, and 171-172, or an amino acid sequence that has at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the VL region amino acid sequence set forth in any one of SEQ ID NOs: 46-90, 158, and 171-172. In some embodiments, the provided anti-PD-1 antibody contains a combination of any of such six CDRs (a CDR-H1, a CDR-H2, a CDR-H3, a CDR-L1, a CDR-L2 and a CDR-L3) described above. In some embodiments, a provided anti-PD-1 antibody or an antigen-binding fragment thereof has a CDR-H1, a CDR-H2 and a CDR-H3 present in a VH region amino acid sequence set forth in any one of SEQ ID NOs: 1-45, 157, 165-170, and a CDR-L1, a CDR-L2 and a CDR-L3 present in a VL region amino acid sequence set forth in any one of SEQ ID NOs: 46-90, 158, and 171-172. In some embodiments, the combination of six CDRs (a CDR-H1, a CDR-H2, a CDR-H3, a CDR-L1, a CDR-L2 and a CDR-L3) is according to Kabat numbering. In some embodiments, the combination of six CDRs (a CDR-H1, a CDR-H2, a CDR-H3, a CDR-L1, a CDR-L2 and a CDR-L3) is according to Chothia numbering. In some embodiments, the combination of six CDRs (a CDR-H1, a CDR-H2, a CDR-H3, a CDR-L1, a CDR-L2 and a CDR-L3) is according to AbM numbering, In some embodiments, the combination of six CDRs (a CDR-H1, a CDR-H2, a CDR-H3, a CDR-L1, a CDR-L2 and a CDR-L3) is according to IMGT numbering. In some embodiments, the combination of six CDRs (a CDR-H1, a CDR-H2, a CDR- H3, a CDR-L1, a CDR-L2 and a CDR-L3) is according to IgBLAST numbering.
[0143] Exemplary heavy and light chain CDR sequences of the anti-PD-1 antibodies and antigenbinding fragments thereof are provided in Tables 2A and 2B.
[0144] In some of any of the provided embodiments, the VH region contains a CDR-H1 set forth in any one of SEQ ID NOs: 91-101, a CDR-H2 set forth in any one of SEQ ID NOs: 102-112, and 173, and a CDR-H3 set forth in any one of SEQ ID NOs: 113-123; and the VL region contains a CDR-L1 set forth in any one of SEQ ID NOs: 124-134, a CDR-L2 set forth in any one of SEQ ID NOs: 135-145, and a CDR-L3 set forth in any one of SEQ ID NOs: 146-156. In some embodiments, the anti-PD-1 antibody or antigen-binding fragment does not contain the combination of CDRs set forth in SEQ ID NO: 91, SEQ ID NO: 102, SEQ ID NO: 113, SEQ ID NO: 124, SEQ ID NO: 135 and SEQ ID NO: 146. In some embodiments, the VH region comprises a CDR-H1 comprising the sequence set forth in any one of SEQ ID NOs: 92-101, a CDR-H2 comprising the sequence set forth in any one of SEQ ID NOs: 103-112, and 173, and a CDR-H3 comprising the sequence set forth in any one of SEQ ID NOs: 114-123, and the VL region comprises a CDR-L1 comprising the sequence set forth in any one of SEQ ID NOs: 124-134, a CDR-L2 comprising the sequence set forth in any one of SEQ ID NOs: 135-145; and a CDR-L3 comprising the sequence set forth in any one of SEQ ID NOs: 146-156. In some embodiments, the VH region comprises a CDR-H1 comprising the sequence set forth in any one of SEQ ID NOs: 91-101, a CDR-H2 comprising the sequence set forth in any one of SEQ ID NOs: 102-112, and 173, and a CDR- H3 comprising the sequence set forth in any one of SEQ ID NOs: 113-123, and the VL region comprises a CDR-L1 comprising the sequence set forth in any one of SEQ ID NOs: 125-134, a CDR-L2 comprising the sequence set forth in any one of SEQ ID NOs: 136-145; and a CDR-L3 comprising the sequence set forth in any one of SEQ ID NOs: 147-156. Also among the antibodies are those having sequences at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% identical to any such CDR sequence, e.g., any of the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, CDR-L3. In some embodiments, among the antibodies are those in which a CDR contained therein has no more than 2 amino acid difference compared to any such above CDR sequence, e.g., any of the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, CDR-L3. In some embodiments, among the antibodies are those in which a CDR contained therein has no more than 1 amino acid difference compared to any such above CDR sequence, e.g., any of the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, CDR-L3. [0145] In some of any of the provided embodiments, the VH region contains a CDR-H1 set forth in any one of SEQ ID NOs: 92-101, a CDR-H2 set forth in any one of SEQ ID NOs: 103-112, and 173, and a CDR-H3 set forth in any one of SEQ ID NOs: 114-123.; and the VL region contains a CDR-L1 set forth in any one of SEQ ID NOs: 125-134, a CDR-L2 set forth in any one of SEQ ID NOs: 136-145, and a CDR-L3 set forth in any one of SEQ ID NOs: 147-156.
[0146] In some embodiments, the antibody or antigen-binding fragment has a VH region that contains a CDR-H1, a CDR-H2 and a CDR-H3 present in SEQ ID NO:1. In some embodiments, the antibody or antigen-binding fragment has a VH region that contains a CDR-H1, a CDR-H2 and a CDR- H3 present in SEQ ID NO:2. In some embodiments, the antibody or antigen-binding fragment has a VH region that contains a CDR-H1, a CDR-H2 and a CDR-H3 present in SEQ ID NO:3. In some embodiments, the antibody or antigen-binding fragment has a VH region that contains a CDR-H1, a CDR-H2 and a CDR-H3 present in SEQ ID NO:4. In some embodiments, the antibody or antigenbinding fragment has a VH region that contains a CDR-H1, a CDR-H2 and a CDR-H3 present in SEQ ID NO:5. In some embodiments, the antibody or antigen-binding fragment has a VH region that contains a CDR-H1, a CDR-H2 and a CDR-H3 present in SEQ ID NO:6. In some embodiments, the antibody or antigen-binding fragment has a VH region that contains a CDR-H1, a CDR-H2 and a CDR-H3 present in SEQ ID NO: 7. In some embodiments, the antibody or antigen-binding fragment has a VH region that contains a CDR-H1, a CDR-H2 and a CDR-H3 present in SEQ ID NO:8. In some embodiments, the antibody or antigen-binding fragment has a VH region that contains a CDR-H1, a CDR-H2 and a CDR- H3 present in SEQ ID NO: 9. In some embodiments, the antibody or antigen-binding fragment has a VH region that contains a CDR-H1, a CDR-H2 and a CDR-H3 present in SEQ ID NO: 10. In some embodiments, the antibody or antigen-binding fragment has a VH region that contains a CDR-H1, a CDR-H2 and a CDR-H3 present in SEQ ID NO: 11. In some embodiments, , the antibody or antigenbinding fragment has a VH region that contains a CDR-H1, a CDR-H2 and a CDR-H3 present in SEQ ID NO: 12. In some embodiments, , the antibody or antigen-binding fragment has a VH region that contains a CDR-H1, a CDR-H2 and a CDR-H3 present in SEQ ID NO:13. In some embodiments, the antibody or antigen-binding fragment has a VH region that contains a CDR-H1, a CDR-H2 and a CDR- H3 present in SEQ ID NO: 14. In some embodiments, the antibody or antigen-binding fragment has a VH region that contains a CDR-H1, a CDR-H2 and a CDR-H3 present in SEQ ID NO: 15. In some embodiments, the antibody or antigen-binding fragment has a VH region that contains a CDR-H1, a CDR-H2 and a CDR-H3 present in SEQ ID NO: 16. In some embodiments, the antibody or antigenbinding fragment has a VH region that contains a CDR-H1, a CDR-H2 and a CDR-H3 present in SEQ ID NO: 17. In some embodiments, the antibody or antigen-binding fragment has a VH region that contains a CDR-H1, a CDR-H2 and a CDR-H3 present in SEQ ID NO: 18. In some embodiments, the antibody or antigen-binding fragment has a VH region that contains a CDR-H1, a CDR-H2 and a CDR- H3 present in SEQ ID NO: 19. In some embodiments, the antibody or antigen-binding fragment has a VH region that contains a CDR-H1, a CDR-H2 and a CDR-H3 present in SEQ ID NO:20. In some embodiments, the antibody or antigen-binding fragment has a VH region that contains a CDR-H1, a CDR-H2 and a CDR-H3 present in SEQ ID NO:21. In some embodiments, the antibody or antigenbinding fragment has a VH region that contains a CDR-H1, a CDR-H2 and a CDR-H3 present in SEQ ID NO:22. In some embodiments, the antibody or antigen-binding fragment has a VH region that contains a CDR-H1, a CDR-H2 and a CDR-H3 present in SEQ ID NO:23. In some embodiments, the antibody or antigen-binding fragment has a VH region that contains a CDR-H1, a CDR-H2 and a CDR- H3 present in SEQ ID NO:24. In some embodiments, the antibody or antigen-binding fragment has a VH region that contains a CDR-H1, a CDR-H2 and a CDR-H3 present in SEQ ID NO:25. In some embodiments, the antibody or antigen-binding fragment has a VH region that contains a CDR-H1, a CDR-H2 and a CDR-H3 present in SEQ ID NO:26. In some embodiments, the antibody or antigenbinding fragment has a VH region that contains a CDR-H1, a CDR-H2 and a CDR-H3 present in SEQ ID NO:27. In some embodiments, the antibody or antigen-binding fragment has a VH region that contains a CDR-H1, a CDR-H2 and a CDR-H3 present in SEQ ID NO:28. In some embodiments, the antibody or antigen-binding fragment has a VH region that contains a CDR-H1, a CDR-H2 and a CDR- H3 present in SEQ ID NO:29. In some embodiments, the antibody or antigen-binding fragment has a VH region that contains a CDR-H1, a CDR-H2 and a CDR-H3 present in SEQ ID NO:30. In some embodiments, the antibody or antigen-binding fragment has a VH region that contains a CDR-H1, a CDR-H2 and a CDR-H3 present in SEQ ID NO:31. In some embodiments, the antibody or antigenbinding fragment has a VH region that contains a CDR-H1, a CDR-H2 and a CDR-H3 present in SEQ ID NO:32. In some embodiments, the antibody or antigen-binding fragment has a VH region that contains a CDR-H1, a CDR-H2 and a CDR-H3 present in SEQ ID NO:33. In some embodiments, the antibody or antigen-binding fragment has a VH region that contains a CDR-H1, a CDR-H2 and a CDR- H3 present in SEQ ID NO:34. In some embodiments, the antibody or antigen-binding fragment has a VH region that contains a CDR-H1, a CDR-H2 and a CDR-H3 present in SEQ ID NO:35. In some embodiments, the antibody or antigen-binding fragment has a VH region that contains a CDR-H1, a CDR-H2 and a CDR-H3 present in SEQ ID NO:36. In some embodiments, the antibody or antigenbinding fragment has a VH region that contains a CDR-H1, a CDR-H2 and a CDR-H3 present in SEQ ID NO:37. In some embodiments, the antibody or antigen-binding fragment has a VH region that contains a CDR-H1, a CDR-H2 and a CDR-H3 present in SEQ ID NO:38. In some embodiments, the antibody or antigen-binding fragment has a VH region that contains a CDR-H1, a CDR-H2 and a CDR- H3 present in SEQ ID NO:39. In some embodiments, the antibody or antigen-binding fragment has a VH region that contains a CDR-H1, a CDR-H2 and a CDR-H3 present in SEQ ID NO:40. In some embodiments, the antibody or antigen-binding fragment has a VH region that contains a CDR-H1, a CDR-H2 and a CDR-H3 present in SEQ ID NO:41. In some embodiments, the antibody or antigenbinding fragment has a VH region that contains a CDR-H1, a CDR-H2 and a CDR-H3 present in SEQ ID NO:42. In some embodiments, the antibody or antigen-binding fragment has a VH region that contains a CDR-H1, a CDR-H2 and a CDR-H3 present in SEQ ID NO:43. In some embodiments, the antibody or antigen-binding fragment has a VH region that contains a CDR-H1, a CDR-H2 and a CDR- H3 present in SEQ ID NO:44. In some embodiments, the antibody or antigen-binding fragment has a VH region that contains a CDR-H1, a CDR-H2 and a CDR-H3 present in SEQ ID NO:45. In some embodiments, the antibody or antigen-binding fragment has a VH region that contains a CDR-H1, a CDR-H2 and a CDR-H3 present in SEQ ID NO: 157. In some embodiments, the antibody or antigenbinding fragment has a VH region that contains a CDR-H1, a CDR-H2 and a CDR-H3 present in SEQ ID NO: 165. In some embodiments, the antibody or antigen-binding fragment has a VH region that contains a CDR-H1, a CDR-H2 and a CDR-H3 present in SEQ ID NO:166. In some embodiments, the antibody or antigen-binding fragment has a VH region that contains a CDR-H1, a CDR-H2 and a CDR- H3 present in SEQ ID NO: 167. In some embodiments, the antibody or antigen-binding fragment has a VH region that contains a CDR-H1, a CDR-H2 and a CDR-H3 present in SEQ ID NO:168. In some embodiments, the antibody or antigen-binding fragment has a VH region that contains a CDR-H1, a CDR-H2 and a CDR-H3 present in SEQ ID NO: 169. In some embodiments, the antibody or antigenbinding fragment has a VH region that contains a CDR-H1, a CDR-H2 and a CDR-H3 present in SEQ ID NO: 170. In some embodiments, the CDR sequence is according to Kabat numbering. In some embodiments, the CDR sequence is according to Chothia numbering. In some embodiments, the CDR sequence is according to AbM numbering. In some embodiments, the CDR sequence is according to IMGT numbering. In some embodiments, the CDR sequence is according to IgBLAST numbering.
[0147] In some embodiments, the antibody or antigen-binding fragment has a VL region that contains a CDR-L1, a CDR-L2 and a CDR-L3 present in SEQ ID NO:46. In some embodiments, the antibody or antigen-binding fragment has a VL region that contains a CDR-L1, a CDR-L2 and a CDR- L3 present in SEQ ID NO: 47. In some embodiments, the antibody or antigen-binding fragment has a VL region that contains a CDR-L1, a CDR-L2 and a CDR-L3 present in SEQ ID NO:48. In some embodiments, the antibody or antigen-binding fragment has a VL region that contains a CDR-L1, a CDR-L2 and a CDR-L3 present in SEQ ID NO:49. In some embodiments, the antibody or antigenbinding fragment has a VL region that contains a CDR-L1, a CDR-L2 and a CDR-L3 present in SEQ ID NO:50. In some embodiments, the antibody or antigen-binding fragment has a VL region that contains a CDR-L1, a CDR-L2 and a CDR-L3 present in SEQ ID NO:51. In some embodiments, the antibody or antigen-binding fragment has a VL region that contains a CDR-L1, a CDR-L2 and a CDR- L3 present in SEQ ID NO:52. In some embodiments, the antibody or antigen-binding fragment has a VL region that contains a CDR-L1, a CDR-L2 and a CDR-L3 present in SEQ ID NO:53. In some embodiments, the antibody or antigen-binding fragment has a VL region that contains a CDR-L1, a CDR-L2 and a CDR-L3 present in SEQ ID NO:54. In some embodiments, the antibody or antigenbinding fragment has a VL region that contains a CDR-L1, a CDR-L2 and a CDR-L3 present in SEQ ID NO:55. In some embodiments, the antibody or antigen-binding fragment has a VL region that contains a CDR-L1, a CDR-L2 and a CDR-L3 present in SEQ ID NO:56. n some embodiments of the antibody or antigen-binding fragment has a VL region that contains a CDR-L1, a CDR-L2 and a CDR- L3 present in SEQ ID NO:57. In some embodiments, the antibody or antigen-binding fragment has a VL region that contains a CDR-L1, a CDR-L2 and a CDR-L3 present in SEQ ID NO:58. In some embodiments, the antibody or antigen-binding fragment has a VL region that contains a CDR-L1, a CDR-L2 and a CDR-L3 present in SEQ ID NO:59. In some embodiments, the antibody or antigenbinding fragment has a VL region that contains a CDR-L1, a CDR-L2 and a CDR-L3 present in SEQ ID NO:60. In some embodiments, the antibody or antigen-binding fragment has a VL region that contains a CDR-L1, a CDR-L2 and a CDR-L3 present in SEQ ID NO:61. In some embodiments, the antibody or antigen-binding fragment has a VL region that contains a CDR-L1, a CDR-L2 and a CDR- L3 present in SEQ ID NO:62. In some embodiments, the antibody or antigen-binding fragment has a VL region that contains a CDR-L1, a CDR-L2 and a CDR-L3 present in SEQ ID NO:63. In some embodiments, the antibody or antigen-binding fragment has a VL region that contains a CDR-L1, a CDR-L2 and a CDR-L3 present in SEQ ID NO:64. In some embodiments, the antibody or antigenbinding fragment has a VL region that contains a CDR-L1, a CDR-L2 and a CDR-L3 present in SEQ ID NO: 65. In some embodiments, the antibody or antigen-binding fragment has a VL region that contains a CDR-L1, a CDR-L2 and a CDR-L3 present in SEQ ID NO:66. In some embodiments, the antibody or antigen-binding fragment has a VL region that contains a CDR-L1, a CDR-L2 and a CDR- L3 present in SEQ ID NO: 67. In some embodiments, the antibody or antigen-binding fragment has a VL region that contains a CDR-L1, a CDR-L2 and a CDR-L3 present in SEQ ID NO:68. In some embodiments, the antibody or antigen-binding fragment has a VL region that contains a CDR-L1, a CDR-L2 and a CDR-L3 present in SEQ ID NO:69. In some embodiments, the antibody or antigenbinding fragment has a VL region that contains a CDR-L1, a CDR-L2 and a CDR-L3 present in SEQ ID NO:70. In some embodiments, the antibody or antigen-binding fragment has a VL region that contains a CDR-L1, a CDR-L2 and a CDR-L3 present in SEQ ID NO:71. In some embodiments, the antibody or antigen-binding fragment has a VL region that contains a CDR-L1, a CDR-L2 and a CDR- L3 present in SEQ ID NO:72. In some embodiments, the antibody or antigen-binding fragment has a VL region that contains a CDR-L1, a CDR-L2 and a CDR-L3 present in SEQ ID NO:73. In some embodiments, the antibody or antigen-binding fragment has a VL region that contains a CDR-L1, a CDR-L2 and a CDR-L3 present in SEQ ID NO:74. In some embodiments, the antibody or antigenbinding fragment has a VL region that contains a CDR-L1, a CDR-L2 and a CDR-L3 present in SEQ ID NO:75. In some embodiments, the antibody or antigen-binding fragment has a VL region that contains a CDR-L1, a CDR-L2 and a CDR-L3 present in SEQ ID NO:76. In some embodiments, the antibody or antigen-binding fragment has a VL region that contains a CDR-L1, a CDR-L2 and a CDR- L3 present in SEQ ID NO: 77. In some embodiments, the antibody or antigen-binding fragment has a VL region that contains a CDR-L1, a CDR-L2 and a CDR-L3 present in SEQ ID NO:78. In some embodiments, the antibody or antigen-binding fragment has a VL region that contains a CDR-L1, a CDR-L2 and a CDR-L3 present in SEQ ID NO:79. In some embodiments, the antibody or antigenbinding fragment has a VL region that contains a CDR-L1, a CDR-L2 and a CDR-L3 present in SEQ ID NO:80. In some embodiments, the antibody or antigen-binding fragment has a VL region that contains a CDR-L1, a CDR-L2 and a CDR-L3 present in SEQ ID NO:81. In some embodiments, the antibody or antigen-binding fragment has a VL region that contains a CDR-L1, a CDR-L2 and a CDR- L3 present in SEQ ID NO: 82. In some embodiments, the antibody or antigen-binding fragment has a VL region that contains a CDR-L1, a CDR-L2 and a CDR-L3 present in SEQ ID NO:83. In some embodiments, the antibody or antigen-binding fragment has a VL region that contains a CDR-L1, a CDR-L2 and a CDR-L3 present in SEQ ID NO: 84. In some embodiments, the antibody or antigenbinding fragment has a VL region that contains a CDR-L1, a CDR-L2 and a CDR-L3 present in SEQ ID NO:85. In some embodiments, the antibody or antigen-binding fragment has a VL region that contains a CDR-L1, a CDR-L2 and a CDR-L3 present in SEQ ID NO:86. In some embodiments, the antibody or antigen-binding fragment has a VL region that contains a CDR-L1, a CDR-L2 and a CDR- L3 present in SEQ ID NO: 87. In some embodiments, the antibody or antigen-binding fragment has a VL region that contains a CDR-L1, a CDR-L2 and a CDR-L3 present in SEQ ID NO:88. In some embodiments, the antibody or antigen-binding fragment has a VL region that contains a CDR-L1, a CDR-L2 and a CDR-L3 present in SEQ ID NO: 89. In some embodiments, the antibody or antigenbinding fragment has a VL region that contains a CDR-L1, a CDR-L2 and a CDR-L3 present in SEQ ID NO:90. In some embodiments, the antibody or antigen-binding fragment has a VL region that contains a CDR-L1, a CDR-L2 and a CDR-L3 present in SEQ ID NO: 158. In some embodiments, the antibody or antigen-binding fragment has a VL region that contains a CDR-L1, a CDR-L2 and a CDR- L3 present in SEQ ID NO: 171. In some embodiments, the antibody or antigen-binding fragment has a VL region that contains a CDR-L1, a CDR-L2 and a CDR-L3 present in SEQ ID NO: 172. In some embodiments, the CDR sequence is according to Kabat numbering. In some embodiments, the CDR sequence is according to Chothia numbering. In some embodiments, the CDR sequence is according to AbM numbering. In some embodiments, the CDR sequence is according to IMGT numbering. In some embodiments, the CDR sequence is according to IgBLAST numbering.
[0148] In some embodiments of the anti-PD-1 antibody or antigen-binding fragment, the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 92, 102, and 113, respectively; SEQ ID NOS: 93, 102, and 113, respectively; SEQ ID NOS: 94, 102, and 113, respectively; SEQ ID NOS: 95, 102, and 113, respectively; SEQ ID NOS: 96, 102, and 113, respectively; SEQ ID NOS: 97, 102, and 113, respectively; SEQ ID NOS: 98, 102, and 113, respectively; SEQ ID NOS: 99, 102, and 113, respectively; SEQ ID NOS: 100, 102, and 113, respectively; SEQ ID NOS: 101, 102, and 113, respectively; SEQ ID NOS: 91, 103, and 113, respectively; SEQ ID NOS: 91, 104, and 113, respectively; SEQ ID NOS: 91, 105, and 113, respectively; SEQ ID NOS: 91, 106, and 113, respectively; SEQ ID NOS: 91, 107, and 113, respectively; SEQ ID NOS: 91, 108, and 113, respectively; SEQ ID NOS: 91, 109, and 113, respectively; SEQ ID NOS: 91, 110, and 113, respectively; SEQ ID NOS: 91, 111, and 113, respectively; SEQ ID NOS: 91, 112, and 113, respectively; SEQ ID NOS: 91, 102, and 114, respectively; SEQ ID NOS: 91, 102, and 115, respectively; SEQ ID NOS: 91, 102, and 116, respectively; SEQ ID NOS: 91, 102, and 117, respectively; SEQ ID NOS: 91, 102, and 118, respectively; SEQ ID NOS: 91, 102, and 119, respectively; SEQ ID NOS: 91, 102, and 120, respectively; SEQ ID NOS: 91, 102, and 121, respectively; SEQ ID NOS: 91, 102, and 122, respectively; SEQ ID NOS: 91, 102, and 123, respectively; SEQ ID NOS: 101, 103, and 114, respectively; SEQ ID NOS: 97, 103, and 117, respectively; SEQ ID NOS: 99, 105, and 115, respectively; SEQ ID NOS: 96, 104, and 116, respectively; SEQ ID NOS: 98, 106, and 114, respectively; SEQ ID NOS: 96, 107, and 117, respectively; SEQ ID NOS: 97, 105, and 117, respectively; SEQ ID NOS: 99, 111, and 115, respectively; SEQ ID NOS: 97, 111, and 116, respectively; SEQ ID NOS: 100, 105, and 121, respectively; SEQ ID NOS: 93, 103, and 115, respectively; SEQ ID NOS: 100, 108, and 119, respectively; SEQ ID NOS: 99, 111, and 116, respectively; SEQ ID NOS: 91, 102, and 113, respectively; SEQ ID NOS: 91, 106, and 113, respectively; SEQ ID NOS: 100, 173, and 121, respectively; SEQ ID NOS: 95, 105, and 121, respectively; SEQ ID NOS: 95, 173, and 121, respectively; SEQ ID NOS: 95, 106, and 114, respectively; or SEQ ID NOS: 91, 102, and 114, respectively.
[0149] In some embodiments of the anti-PD-1 antibody or antigen-binding fragment, the VL region comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS:
124, 135, and 146, respectively; SEQ ID NOS: 125, 135, and 146, respectively; SEQ ID NOS: 126,
135, and 146, respectively; SEQ ID NOS: 127, 135, and 146, respectively; SEQ ID NOS: 128, 135, and
146, respectively; SEQ ID NOS: 129, 135, and 146, respectively; SEQ ID NOS: 130, 135, and 146, respectively; SEQ ID NOS: 131, 135, and 146, respectively; SEQ ID NOS: 132, 135, and 146, respectively; SEQ ID NOS: 133, 135, and 146, respectively; SEQ ID NOS: 134, 135, and 146, respectively; SEQ ID NOS: 124, 136, and 146, respectively; SEQ ID NOS: 124, 137, and 146, respectively; SEQ ID NOS: 124, 138, and 146, respectively; SEQ ID NOS: 124, 139, and 146, respectively; SEQ ID NOS: 124, 140, and 146, respectively; SEQ ID NOS: 124, 141, and 146, respectively; SEQ ID NOS: 124, 142, and 146, respectively; SEQ ID NOS: 124, 143, and 146, respectively; SEQ ID NOS: 124, 144, and 146, respectively; SEQ ID NOS: 124, 145, and 146, respectively; SEQ ID NOS: 124, 135, and 147, respectively; SEQ ID NOS: 124, 135, and 148, respectively; SEQ ID NOS: 124, 135, and 149, respectively; SEQ ID NOS: 124, 135, and 150, respectively; SEQ ID NOS: 124, 135, and 151, respectively; SEQ ID NOS: 124, 135, and 152, respectively; SEQ ID NOS: 124, 135, and 153, respectively; SEQ ID NOS: 124, 135, and 154, respectively; SEQ ID NOS: 124, 135, and 155, respectively; SEQ ID NOS: 124, 135, and 156, respectively; SEQ ID NOS: 131, 142, and 149, respectively; SEQ ID NOS: 131, 139, and 150, respectively; SEQ ID NOS: 128, 138, and 148, respectively; SEQ ID NOS: 126, 145, and 153, respectively; SEQ ID NOS: 126, 144, and 154, respectively; SEQ ID NOS: 125, 140, and 153, respectively; SEQ ID NOS: 132, 137, and 152, respectively; SEQ ID NOS: 131, 142, and 152, respectively; SEQ ID NOS: 132, 141, and 154, respectively; SEQ ID NOS: 133, 144, and 150, respectively; SEQ ID NOS: 134, 138, and 149, respectively; SEQ ID NOS: 126, 137, and 149, respectively; SEQ ID NOS: 126, 137, and 152, respectively; SEQ ID NOS: 132, 142, and 154, respectively; SEQ ID NOS: 127, 140, and 151, respectively; SEQ ID NOS: 132, 145, and 153, respectively; SEQ ID NOS: or 126, 143, and 153, respectively.
[0150] In some embodiments of the anti-PD-1 antibody or antigen-binding fragment, the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 92, 102, and 113, respectively, and the VL region comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 124, 135, and 146, respectively. In some embodiments of the anti-PD-1 antibody or antigen-binding fragment, the VH region comprises a CDR- Hl, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 93, 102, and 113, respectively, and the VL region comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 124, 135, and 146, respectively. In some embodiments of the anti- PD-1 antibody or antigen-binding fragment, the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 94, 102, and 113, respectively, and the VL region comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 124, 135 and 146, respectively. In some embodiments of the anti-PD-1 antibody or antigenbinding fragment, the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 95, 102, and 113, respectively, and the VL region comprises a CDR- Ll, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 124, 135, and 146, respectively. In some embodiments of the anti-PD-1 antibody or antigen-binding fragment, the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 96, 102, and 113, respectively, and the VL region comprises a CDR-L1, a CDR-L2 and a CDR- L3 comprising the sequence set forth in SEQ ID NOS: 124, 135 and 146, respectively. In some embodiments of the anti-PD-1 antibody or antigen-binding fragment, the VH region comprises a CDR- Hl, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 97, 102, and 113, respectively, and the VL region comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 124, 135 and 146, respectively. In some embodiments of the anti- PD-1 antibody or antigen-binding fragment, the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 98, 102, and 113, respectively, and the VL region comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 124, 135 and 146, respectively. In some embodiments of the anti-PD-1 antibody or antigenbinding fragment, the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 99, 102, and 113, respectively, and the VL region comprises a CDR- Ll, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 124, 135 and 146, respectively. In some embodiments of the anti-PD-1 antibody or antigen-binding fragment, the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 100, 102, and 113, respectively, and the VL region comprises a CDR-L1, a CDR-L2 and a CDR- L3 comprising the sequence set forth in SEQ ID NOS: 124, 135 and 146, respectively. In some embodiments of the anti-PD-1 antibody or antigen-binding fragment, the VH region comprises a CDR- Hl, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 101, 102, and 113, respectively, and the VL region comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 124, 135 and 146, respectively. In some embodiments of the anti- PD-1 antibody or antigen-binding fragment, the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 91, 103, and 113, respectively, and the VL region comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 125, 135 and 146, respectively. In some embodiments of the anti-PD-1 antibody or antigenbinding fragment, the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 91, 104, and 113, respectively, and the VL region comprises a CDR- Ll, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 124, 135 and 146, respectively. In some embodiments of the anti-PD-1 antibody or antigen-binding fragment, the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 91, 105, and 113, respectively, and the VL region comprises a CDR-L1, a CDR-L2 and a CDR- L3 comprising the sequence set forth in SEQ ID NOS: 124, 135 and 146, respectively. In some embodiments of the anti-PD-1 antibody or antigen-binding fragment, the VH region comprises a CDR- Hl, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 91, 106, and 113, respectively, and the VL region comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 125, 135 and 146, respectively. In some embodiments of the anti- PD-1 antibody or antigen-binding fragment, the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 91, 107, and 113, respectively, and the VL region comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 124, 135 and 146, respectively. In some embodiments of the anti-PD-1 antibody or antigenbinding fragment, the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 91, 108, and 113, respectively, and the VL region comprises a CDR- Ll, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 124, 135 and 146, respectively. In some embodiments of the anti-PD-1 antibody or antigen-binding fragment, the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 91, 109, and 113, respectively, and the VL region comprises a CDR-L1, a CDR-L2 and a CDR- L3 comprising the sequence set forth in SEQ ID NOS: 124, 135 and 146, respectively. In some embodiments of the anti-PD-1 antibody or antigen-binding fragment, the VH region comprises a CDR- Hl, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 91, 110, and 113, respectively, and the VL region comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 124, 135 and 146, respectively. In some embodiments of the anti- PD-1 antibody or antigen-binding fragment, the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 91, 111, and 113, respectively, and the VL region comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 124, 135 and 146, respectively. In some embodiments of the anti-PD-1 antibody or antigenbinding fragment, the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 91, 112, and 113, respectively, and the VL region comprises a CDR- Ll, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 124, 135, 146, respectively. In some embodiments of the anti-PD-1 antibody or antigen-binding fragment, the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 91, 102, and 114, respectively, and the VL region comprises a CDR-L1, a CDR-L2 and a CDR- L3 comprising the sequence set forth in SEQ ID NOS: 124, 135 and 146, respectively. In some embodiments of the anti-PD-1 antibody or antigen-binding fragment, the VH region comprises a CDR- Hl, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 91, 102, and 115, respectively, and the VL region comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 124, 135 and 146, respectively. In some embodiments of the anti- PD-1 antibody or antigen-binding fragment, the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 91, 102, and 116, respectively, and the VL region comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 124, 135 and 146, respectively. In some embodiments of the anti-PD-1 antibody or antigenbinding fragment, the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 91, 102, and 117, respectively, and the VL region comprises a CDR- Ll, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 124, 135 and 146, respectively. In some embodiments of the anti-PD-1 antibody or antigen-binding fragment, the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 91, 102, and 118, respectively, and the VL region comprises a CDR-L1, a CDR-L2 and a CDR- L3 comprising the sequence set forth in SEQ ID NOS: 124, 135 and 146, respectively. In some embodiments of the anti-PD-1 antibody or antigen-binding fragment, the VH region comprises a CDR- Hl, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 91, 102, and 119, respectively, and the VL region comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 124, 135 and 146, respectively. In some embodiments of the anti- PD-1 antibody or antigen-binding fragment, the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 91, 102, and 120, respectively, and the VL region comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 124, 135 and 146, respectively. In some embodiments of the anti-PD-1 antibody or antigenbinding fragment, the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 91, 102, and 121, respectively, and the VL region comprises a CDR- Ll, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 124, 135 and 146, respectively. In some embodiments of the anti-PD-1 antibody or antigen-binding fragment, the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 91, 102, and 122, respectively, and the VL region comprises a CDR-L1, a CDR-L2 and a CDR- L3 comprising the sequence set forth in SEQ ID NOS: 124, 135 and 146, respectively. In some embodiments of the anti-PD-1 antibody or antigen-binding fragment, the VH region comprises a CDR- Hl, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 91, 102, and 123, respectively, and the VL region comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 124, 135 and 146, respectively. In some embodiments of the anti- PD-1 antibody or antigen-binding fragment, the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 101, 103, and 114, respectively, and the VL region comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 124, 135 and 146, respectively. In some embodiments of the anti-PD-1 antibody or antigenbinding fragment, the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 97, 103, and 117, respectively, and the VL region comprises a CDR- Ll, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 124, 135, and 146, respectively. In some embodiments of the anti-PD-1 antibody or antigen-binding fragment, the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 99, 105, and 115, respectively, and the VL region comprises a CDR-L1, a CDR-L2 and a CDR- L3 comprising the sequence set forth in SEQ ID NOS: 124, 135 and 146, respectively. In some embodiments of the anti-PD-1 antibody or antigen-binding fragment, the VH region comprises a CDR- Hl, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 96, 104, and 116, respectively, and the VL region comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 124, 135 and 146, respectively. In some embodiments of the anti- PD-1 antibody or antigen-binding fragment, the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 98, 106, and 114, respectively, and the VL region comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 124, 135 and 146, respectively. In some embodiments of the anti-PD-1 antibody or antigenbinding fragment, the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 96, 107, and 117, respectively, and the VL region comprises a CDR- LI, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 124, 135 and 146, respectively. In some embodiments of the anti-PD-1 antibody or antigen-binding fragment, the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 97, 105, and 117, respectively, and the VL region comprises a CDR-L1, a CDR-L2 and a CDR- L3 comprising the sequence set forth in SEQ ID NOS: 124, 135 and 146, respectively. In some embodiments of the anti-PD-1 antibody or antigen-binding fragment, the VH region comprises a CDR- Hl, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 99, 111, and 115, respectively, and the VL region comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 124, 135, and 146, respectively. In some embodiments of the anti- PD-1 antibody or antigen-binding fragment, the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 97, 111, and 116, respectively, and the VL region comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 124, 135 and 146, respectively. In some embodiments of the anti-PD-1 antibody or antigenbinding fragment, the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 100, 105, and 121, respectively, and the VL region comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 124, 135 and 146, respectively. In some embodiments of the anti-PD-1 antibody or antigen-binding fragment, the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 93, 103, and 115, respectively, and the VL region comprises a CDR-L1, a CDR-L2 and a CDR- L3 comprising the sequence set forth in SEQ ID NOS: 124, 135 and 146, respectively. In some embodiments of the anti-PD-1 antibody or antigen-binding fragment, the VH region comprises a CDR- Hl, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 100, 108, and 119, respectively, and the VL region comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 124, 135 and 146, respectively. In some embodiments of the anti- PD-1 antibody or antigen-binding fragment, the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 99, 111, and 116, respectively, and the VL region comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 124, 135 and 146, respectively. In some embodiments of the anti-PD-1 antibody or antigenbinding fragment, the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 91, 102, and 113, respectively, and the VL region comprises a CDR- Ll, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 125, 135 and 146, respectively. In some embodiments of the anti-PD-1 antibody or antigen-binding fragment, the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 91, 102, and 113, respectively, and the VL region comprises a CDR-L1, a CDR-L2 and a CDR- L3 comprising the sequence set forth in SEQ ID NOS: 126, 135 and 146, respectively. In some embodiments of the anti-PD-1 antibody or antigen-binding fragment, the VH region comprises a CDR- Hl, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 91, 102, and 113, respectively, and the VL region comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 127, 135 and 146, respectively. In some embodiments of the anti- PD-1 antibody or antigen-binding fragment, the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 91, 102, and 113, respectively, and the VL region comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 128, 135 and 146, respectively. In some embodiments of the anti-PD-1 antibody or antigenbinding fragment, the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 91, 102, and 113, respectively, and the VL region comprises a CDR- Ll, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 129, 135 and 146, respectively. In some embodiments of the anti-PD-1 antibody or antigen-binding fragment, the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 91, 102, and 113, respectively, and the VL region comprises a CDR-L1, a CDR-L2 and a CDR- L3 comprising the sequence set forth in SEQ ID NOS: 130, 135 and 146, respectively. In some embodiments of the anti-PD-1 antibody or antigen-binding fragment, the VH region comprises a CDR- Hl, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 91, 102, and 113, respectively, and the VL region comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 131, 135 and 146, respectively. In some embodiments of the anti- PD-1 antibody or antigen-binding fragment, the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 91, 102, and 113, respectively, and the VL region comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 132, 135 and 146, respectively. In some embodiments of the anti-PD-1 antibody or antigenbinding fragment, the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 91, 102, and 113, respectively, and the VL region comprises a CDR- Ll, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 133, 135 and 146, respectively. In some embodiments of the anti-PD-1 antibody or antigen-binding fragment, the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 91, 102, and 113, respectively, and the VL region comprises a CDR-L1, a CDR-L2 and a CDR- L3 comprising the sequence set forth in SEQ ID NOS: 134, 135 and 146, respectively. In some embodiments of the anti-PD-1 antibody or antigen-binding fragment, the VH region comprises a CDR- Hl, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 91, 102, and 113, respectively, and the VL region comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 124, 136 and 146, respectively. In some embodiments of the anti- PD-1 antibody or antigen-binding fragment, the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 91, 102, and 113, respectively, and the VL region comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 124, 137 and 146, respectively. In some embodiments of the anti-PD-1 antibody or antigenbinding fragment, the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 91, 102, and 113, respectively, and the VL region comprises a CDR- Ll, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 124, 138 and 146, respectively. In some embodiments of the anti-PD-1 antibody or antigen-binding fragment, the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 91, 102, and 113, respectively, and the VL region comprises a CDR-L1, a CDR-L2 and a CDR- L3 comprising the sequence set forth in SEQ ID NOS: 124, 139 and 146, respectively. In some embodiments of the anti-PD-1 antibody or antigen-binding fragment, the VH region comprises a CDR- Hl, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 91, 102, and 113, respectively, and the VL region comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 124, 140 and 146, respectively. In some embodiments of the anti- PD-1 antibody or antigen-binding fragment, the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 91, 102, and 113, respectively, and the VL region comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 124, 141 and 146, respectively. In some embodiments of the anti-PD-1 antibody or antigenbinding fragment, the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 91, 102, and 113, respectively, and the VL region comprises a CDR- Ll, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 124, 142 and 146, respectively. In some embodiments of the anti-PD-1 antibody or antigen-binding fragment, the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 91, 102, and 113, respectively, and the VL region comprises a CDR-L1, a CDR-L2 and a CDR- L3 comprising the sequence set forth in SEQ ID NOS: 124, 143 and 146, respectively. In some embodiments of the anti-PD-1 antibody or antigen-binding fragment, the VH region comprises a CDR- Hl, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 91, 102, and 113, respectively, and the VL region comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 124, 144 and 146, respectively. In some embodiments of the anti- PD-1 antibody or antigen-binding fragment, the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 91, 102, and 113, respectively, and the VL region comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 124, 145 and 146, respectively. In some embodiments of the anti-PD-1 antibody or antigenbinding fragment, the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 91, 102, and 113, respectively, and the VL region comprises a CDR- Ll, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 124, 135 and 147, respectively. In some embodiments of the anti-PD-1 antibody or antigen-binding fragment, the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 91, 102, and 113, respectively, and the VL region comprises a CDR-L1, a CDR-L2 and a CDR- L3 comprising the sequence set forth in SEQ ID NOS: 124, 135 and 148, respectively. In some embodiments of the anti-PD-1 antibody or antigen-binding fragment, the VH region comprises a CDR- Hl, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 91, 102, and 113, respectively, and the VL region comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 124, 135 and 149, respectively. In some embodiments of the anti- PD-1 antibody or antigen-binding fragment, the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 91, 102, and 113, respectively, and the VL region comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 124, 135 and 150, respectively. In some embodiments of the anti-PD-1 antibody or antigenbinding fragment, the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 91, 102, and 113, respectively, and the VL region comprises a CDR- Ll, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 124, 135 and 151, respectively. In some embodiments of the anti-PD-1 antibody or antigen-binding fragment, the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 91, 102, and 113, respectively, and the VL region comprises a CDR-L1, a CDR-L2 and a CDR- L3 comprising the sequence set forth in SEQ ID NOS: 124, 135 and 152, respectively. In some embodiments of the anti-PD-1 antibody or antigen-binding fragment, the VH region comprises a CDR- Hl, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 91, 102, and 113, respectively, and the VL region comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 124, 135 and 153, respectively. In some embodiments of the anti- PD-1 antibody or antigen-binding fragment, the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 91, 102, and 113, respectively, and the VL region comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 124, 135 and 154, respectively. In some embodiments of the anti-PD-1 antibody or antigenbinding fragment, the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 91, 102, and 113, respectively, and the VL region comprises a CDR- Ll, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 124, 135 and 155, respectively. In some embodiments of the anti-PD-1 antibody or antigen-binding fragment, the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 91, 102, and 113, respectively, and the VL region comprises a CDR-L1, a CDR-L2 and a CDR- L3 comprising the sequence set forth in SEQ ID NOS: 124, 135 and 156, respectively. In some embodiments of the anti-PD-1 antibody or antigen-binding fragment, the VH region comprises a CDR- Hl, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 91, 102, and 113, respectively, and the VL region comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 131, 142 and 149, respectively. In some embodiments of the anti- PD-1 antibody or antigen-binding fragment, the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 91, 102, and 113, respectively, and the VL region comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 131, 139 and 150, respectively. In some embodiments of the anti-PD-1 antibody or antigenbinding fragment, the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 91, 102, and 113, respectively, and the VL region comprises a CDR- Ll, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 128, 138, and 148, respectively. In some embodiments of the anti-PD-1 antibody or antigen-binding fragment, the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 91, 102, and 113, respectively, and the VL region comprises a CDR-L1, a CDR-L2 and a CDR- L3 comprising the sequence set forth in SEQ ID NOS: 126, 145 and 153, respectively. In some embodiments of the anti-PD-1 antibody or antigen-binding fragment, the VH region comprises a CDR- Hl, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 91, 102, and 113, respectively, and the VL region comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 126, 144 and 154, respectively. In some embodiments of the anti- PD-1 antibody or antigen-binding fragment, the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 91, 102, and 113, respectively, and the VL region comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 125, 140 and 153, respectively. In some embodiments of the anti-PD-1 antibody or antigenbinding fragment, the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 91, 102, and 113, respectively, and the VL region comprises a CDR- Ll, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 132, 137 and 152, respectively. In some embodiments of the anti-PD-1 antibody or antigen-binding fragment, the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 91, 102, and 113, respectively, and the VL region comprises a CDR-L1, a CDR-L2 and a CDR- L3 comprising the sequence set forth in SEQ ID NOS: 131, 142 and 152, respectively. In some embodiments of the anti-PD-1 antibody or antigen-binding fragment, the VH region comprises a CDR- Hl, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 91, 102, and 113, respectively, and the VL region comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 132, 141 and 154, respectively. In some embodiments of the anti- PD-1 antibody or antigen-binding fragment, the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 91, 102, and 113, respectively, and the VL region comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 133, 144 and 150, respectively. In some embodiments of the anti-PD-1 antibody or antigenbinding fragment, the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 91, 102, and 113, respectively, and the VL region comprises a CDR- LI, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 134, 138 and 149, respectively. In some embodiments of the anti-PD-1 antibody or antigen-binding fragment, the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 91, 102, and 113, respectively, and the VL region comprises a CDR-L1, a CDR-L2 and a CDR- L3 comprising the sequence set forth in SEQ ID NOS: 126, 137 and 149, respectively. In some embodiments of the anti-PD-1 antibody or antigen-binding fragment, the VH region comprises a CDR- Hl, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 91, 102, and 113, respectively, and the VL region comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 126, 137 and 152, respectively. In some embodiments of the anti- PD-1 antibody or antigen-binding fragment, the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 91, 102, and 113, respectively, and the VL region comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 132, 142 and 154, respectively. In some embodiments of the anti-PD-1 antibody or antigenbinding fragment, the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 91, 102, and 113, respectively, and the VL region comprises a CDR- Ll, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 127, 140 and 151, respectively. In some embodiments of the anti-PD-1 antibody or antigen-binding fragment, the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 91, 106, and 113, respectively, and the VL region comprises a CDR-L1, a CDR-L2 and a CDR- L3 comprising the sequence set forth in SEQ ID NOS: 126, 135 and 146, respectively. In some embodiments of the anti-PD-1 antibody or antigen-binding fragment, the VH region comprises a CDR- Hl, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 91, 102, and 114, respectively, and the VL region comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 126, 135 and 146, respectively. In some embodiments of the anti- PD-1 antibody or antigen-binding fragment, the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 99, 105, and 115, respectively, and the VL region comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 131, 142 and 149, respectively. In some embodiments of the anti-PD-1 antibody or antigenbinding fragment, the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 99, 105, and 115, respectively, and the VL region comprises a CDR- Ll, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 126, 145 and 153, respectively. In some embodiments of the anti-PD-1 antibody or antigen-binding fragment, the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 99, 105, and 115, respectively, and the VL region comprises a CDR-L1, a CDR-L2 and a CDR- L3 comprising the sequence set forth in SEQ ID NOS: 125, 140, and 153, respectively. In some embodiments of the anti-PD-1 antibody or antigen-binding fragment, the VH region comprises a CDR- Hl, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 99, 105, and 115, respectively, and the VL region comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 132, 141 and 154, respectively. In some embodiments of the anti- PD-1 antibody or antigen-binding fragment, the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 99, 105, and 115, respectively, and the VL region comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 132, 142 and 154, respectively. In some embodiments of the anti-PD-1 antibody or antigenbinding fragment, the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 98, 106, and 114, respectively, and the VL region comprises a CDR- Ll, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 131, 142 and 149, respectively. In some embodiments of the anti-PD-1 antibody or antigen-binding fragment, the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 98, 106, and 114, respectively, and the VL region comprises a CDR-L1, a CDR-L2 and a CDR- L3 comprising the sequence set forth in SEQ ID NOS: 126, 145 and 153, respectively. In some embodiments of the anti-PD-1 antibody or antigen-binding fragment, the VH region comprises a CDR- Hl, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 98, 106, and 114, respectively, and the VL region comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 125, 140 and 153, respectively. In some embodiments of the anti- PD-1 antibody or antigen-binding fragment, the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 98, 106, and 114, respectively, and the VL region comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 132, 141 and 154, respectively. In some embodiments of the anti-PD-1 antibody or antigenbinding fragment, the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 98, 106, and 114, respectively, and the VL region comprises a CDR- Ll, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 132, 142 and 154, respectively. In some embodiments of the anti-PD-1 antibody or antigen-binding fragment, the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 99, 111, and 115, respectively, and the VL region comprises a CDR-L1, a CDR-L2 and a CDR- L3 comprising the sequence set forth in SEQ ID NOS: 131, 142 and 149, respectively. In some embodiments of the anti-PD-1 antibody or antigen-binding fragment, the VH region comprises a CDR- Hl, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 99, 111, and 115, respectively, and the VL region comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 126, 145 and 153, respectively. In some embodiments of the anti- PD-1 antibody or antigen-binding fragment, the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 99, 111, and 115, respectively, and the VL region comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 125, 140 and 153, respectively. In some embodiments of the anti-PD-1 antibody or antigenbinding fragment, the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 99, 111, and 115, respectively, and the VL region comprises a CDR- Ll, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 132, 141 and 154, respectively. In some embodiments of the anti-PD-1 antibody or antigen-binding fragment, the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 99, 111, and 115, respectively, and the VL region comprises a CDR-L1, a CDR-L2 and a CDR- L3 comprising the sequence set forth in SEQ ID NOS: 132, 142 and 154, respectively. In some embodiments of the anti-PD-1 antibody or antigen-binding fragment, the VH region comprises a CDR- Hl, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 100, 105 and 121, respectively, and the VL region comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 131, 142 and 149, respectively. In some embodiments of the anti- PD-1 antibody or antigen-binding fragment, the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 100, 105 and 121, respectively, and the VL region comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 126, 145 and 153, respectively. In some embodiments of the anti-PD-1 antibody or antigenbinding fragment, the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 100, 105 and 121, respectively, and the VL region comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 125, 140 and 153, respectively. In some embodiments of the anti-PD-1 antibody or antigen-binding fragment, the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 100, 105 and 121, respectively, and the VL region comprises a CDR-L1, a CDR-L2 and a CDR- L3 comprising the sequence set forth in SEQ ID NOS: 132, 141 and 154, respectively. In some embodiments of the anti-PD-1 antibody or antigen-binding fragment, the VH region comprises a CDR- Hl, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 100, 105 and 121, respectively, and the VL region comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 132, 142 and 154, respectively. In some embodiments of the anti- PD-1 antibody or antigen-binding fragment, the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 100, 173 and 121, respectively, and the VL region comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 126, 145 and 153, respectively. In some embodiments of the anti-PD-1 antibody or antigenbinding fragment, the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 100, 173 and 121, respectively, and the VL region comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 132, 145 and 153, respectively. In some embodiments of the anti-PD-1 antibody or antigen-binding fragment, the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 100, 173 and 121, respectively, and the VL region comprises a CDR-L1, a CDR-L2 and a CDR- L3 comprising the sequence set forth in SEQ ID NOS: 126, 143 and 153, respectively. In some embodiments of the anti-PD-1 antibody or antigen-binding fragment, the VH region comprises a CDR- Hl, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 95, 105 and 121, respectively, and the VL region comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 126, 145 and 153, respectively. In some embodiments of the anti- PD-1 antibody or antigen-binding fragment, the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 95, 105 and 121, respectively, and the VL region comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 132, 145 and 153, respectively. In some embodiments of the anti-PD-1 antibody or antigenbinding fragment, the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 95, 105 and 121, respectively, and the VL region comprises a CDR- Ll, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 126, 143 and 153, respectively. In some embodiments of the anti-PD-1 antibody or antigen-binding fragment, the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 95, 173 and 121, respectively, and the VL region comprises a CDR-L1, a CDR-L2 and a CDR- L3 comprising the sequence set forth in SEQ ID NOS: 126, 145 and 153, respectively. In some embodiments of the anti-PD-1 antibody or antigen-binding fragment, the VH region comprises a CDR- Hl, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 95, 173 and 121, respectively, and the VL region comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 132, 145 and 153, respectively. In some embodiments of the anti- PD-1 antibody or antigen-binding fragment, the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 95, 173 and 121, respectively, and the VL region comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 126, 143 and 153, respectively. In some embodiments of the anti-PD-1 antibody or antigenbinding fragment, the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 95, 106 and 114, respectively, and the VL region comprises a CDR- Ll, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 126, 145 and 153, respectively. In some embodiments of the anti-PD-1 antibody or antigen-binding fragment, the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 95, 106 and 114, respectively, and the VL region comprises a CDR-L1, a CDR-L2 and a CDR- L3 comprising the sequence set forth in SEQ ID NOS: 132, 145 and 153, respectively. In some embodiments of the anti-PD-1 antibody or antigen-binding fragment, the VH region comprises a CDR- Hl, a CDR-H2 and a CDR-H3 comprising the sequence set forth in SEQ ID NOS: 95, 106 and 114, respectively, and the VL region comprises a CDR-L1, a CDR-L2 and a CDR-L3 comprising the sequence set forth in SEQ ID NOS: 126, 143 and 153, respectively. [0151] In some embodiments, the provided antibody is a human antibody or antigen-binding fragment thereof that contains a framework region that contains human germline gene segment sequences. For example, in some embodiments, the human antibody contains a VH region in which the framework region, e.g.FRl, FR2, FR3 and FR4, has at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a framework region encoded by a human germline antibody segment, such as a V segment and/or J segment. In some embodiments, the human antibody contains a VL region in which the framework region e.g.FRl, FR2, FR3 and FR4, has at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a framework region encoded by a human germline antibody segment, such as a V segment and/or J segment. For example, in some such embodiments, the framework region sequence contained within the VH region and/or VL region differs by no more than 10 amino acids, such as no more than 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid, compared to the framework region sequence encoded by a human germline antibody segment.
[0152] In some embodiments of the antibody or antigen-binding fragment thereof provided herein, the VH region comprises any of the CDR-H1, CDR-H2 and CDR-H3 as described and comprises a framework region 1 (FR1), a FR2, a FR3 and/or a FR4 having at least at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% sequence identity, respectively, to a FR1, a FR2, a FR3 and/or a FR4 contained within the VH region amino acid sequence set forth in any one of SEQ ID NOs: 1-45, 157, and 165-170. In some embodiments of the antibody or antigen-binding fragment thereof provided herein, the VH region comprises any of the CDR-H1, CDR-H2 and CDR-H3 as described and comprises a framework region 1 (FR1), a FR2, a FR3 and/or a FR4 contained within the VH region amino acid sequence set forth in any one of SEQ ID NOs: 1-45, 157, and 165-170.
[0153] In some embodiments of the antibody or antigen-binding fragment thereof provided herein, the VL region comprises any of the CDR-L1, CDR-L2 and CDR-L3 as described and comprises a framework region 1 (FR1), a FR2, a FR3 and/or a FR4 having at least at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% sequence identity, respectively, to a FR1, a FR2, a FR3 and/or a FR4 contained within the VL region amino acid sequence set forth in any one of SEQ ID NOs: 46-90, 158, and 171-172. In some embodiments of the antibody or antigen-binding fragment thereof provided herein, the VL region comprises any of the CDR-H1, CDR-H2 and CDR-H3 as described and comprises a framework region 1 (FR1), a FR2, a FR3 and/or a FR4 contained within the VL region amino acid sequence set forth in any one of SEQ ID NOs: 46-90, 158, and 171-172.
[0154] In some embodiments, any of the provided anti-PD-1 antibodies or antigen binding fragments has a VH region having an amino acid sequence that has at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the VH region amino acid sequence set forth in any one of SEQ ID NOs: 1-45, 157, and 165-170, and has a VL region having an amino acid sequence that has at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the VL region amino acid sequence set forth in any one of SEQ ID NOs: 46-90, 158, and 171-172.
[0155] In some embodiments, the antibody or antigen-binding fragment has the VH region amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the antibody or antigen-binding fragment has the VH region amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, the antibody or antigen-binding fragment the VH region amino acid sequence set forth in SEQ ID NO: 3. In some embodiment, the antibody or antigen-binding fragment has the VH region amino acid sequence set forth in SEQ ID NO: 4. In some embodiments, the antibody or antigen-binding fragment has the VH region amino acid sequence set forth in SEQ ID NO: 5. In some embodiment, the antibody or antigen-binding fragment has the VH region amino acid sequence set forth in SEQ ID NO:6. In some embodiments, the antibody or antigen-binding fragment has the VH region amino acid sequence set forth in SEQ ID NO: 7. In some embodiments, the antibody or antigen-binding fragment has the VH region amino acid sequence set forth in SEQ ID NO: 8. In some embodiment, the antibody or antigenbinding fragment the VH region amino acid sequence set forth in SEQ ID NO: 9. In some embodiments, the antibody or antigen-binding fragment has the VH region amino acid sequence set forth in SEQ ID NO: 10. In some embodiments, the antibody or antigen-binding fragment has the VH region amino acid sequence set forth in SEQ ID NO: 11. In some embodiments, the antibody or antigen-binding fragment has the VH region amino acid sequence set forth in SEQ ID NO: 12. In some embodiments, the antibody or antigen-binding fragment has the VH region amino acid sequence set forth in SEQ ID NO: 13. In some embodiments, the antibody or antigen-binding fragment has the VH region amino acid sequence set forth in SEQ ID NO: 14. In some embodiments, the antibody or antigen-binding fragment has the VH region amino acid sequence set forth in SEQ ID NO: 15. In some embodiments, the antibody or antigen-binding fragment has the VH region amino acid sequence set forth in SEQ ID NO: 16. In some embodiments, the antibody or antigen-binding fragment has the VH region amino acid sequence set forth in SEQ ID NO: 17. In some embodiment, the antibody or antigen-binding fragment has the VH region amino acid sequence set forth in SEQ ID NO: 18. In some embodiments, the antibody or antigenbinding fragment has the VH region amino acid sequence set forth in SEQ ID NO: 19. In some embodiments, the antibody or antigen-binding fragment has the VH region amino acid sequence set forth in SEQ ID NO: 20. In some embodiments, the antibody or antigen-binding fragment has the VH region amino acid sequence set forth in SEQ ID NO: 21. In some embodiments, the antibody or antigenbinding fragment has the VH region amino acid sequence set forth in SEQ ID NO: 22. In some embodiments, the antibody or antigen-binding fragment has the VH region amino acid sequence set forth in SEQ ID NO: 23. In some embodiments, the antibody or antigen-binding fragment has the VH region amino acid sequence set forth in SEQ ID NO: 24. In some embodiments, the antibody or antigenbinding fragment has the VH region amino acid sequence set forth in SEQ ID NO: 25. In some embodiments, the antibody or antigen-binding fragment has the VH region amino acid sequence set forth in SEQ ID NO: 26. In some embodiments, the antibody or antigen-binding fragment has the VH region amino acid sequence set forth in SEQ ID NO: 27. In some embodiments, the antibody or antigenbinding fragment has the VH region amino acid sequence set forth in SEQ ID NO: 28. In some embodiments, the antibody or antigen-binding fragment has the VH region amino acid sequence set forth in SEQ ID NO: 29. In some embodiments, the antibody or antigen-binding fragment has the VH region amino acid sequence set forth in SEQ ID NO: 30. In some embodiments, the antibody or antigenbinding fragment has the VH region amino acid sequence set forth in SEQ ID NO: 31. In some embodiments, the antibody or antigen-binding fragment has the VH region amino acid sequence set forth in SEQ ID NO: 32. In some embodiments, the antibody or antigen-binding fragment has the VH region amino acid sequence set forth in SEQ ID NO: 33. In some embodiments, the antibody or antigenbinding fragment has the VH region amino acid sequence set forth in SEQ ID NO: 34. In some embodiments, the antibody or antigen-binding fragment has the VH region amino acid sequence set forth in SEQ ID NO: 35. In some embodiments, the antibody or antigen-binding fragment has the VH region amino acid sequence set forth in SEQ ID NO: 36. In some embodiments, the antibody or antigenbinding fragment has the VH region amino acid sequence set forth in SEQ ID NO: 37. In some embodiments, the antibody or antigen-binding fragment has the VH region amino acid sequence set forth in SEQ ID NO: 38. In some embodiments, the antibody or antigen-binding fragment has the VH region amino acid sequence set forth in SEQ ID NO: 39. In some embodiments, the antibody or antigenbinding fragment has the VH region amino acid sequence set forth in SEQ ID NO: 40. In some embodiments, the antibody or antigen-binding fragment has the VH region amino acid sequence set forth in SEQ ID NO: 41. In some embodiments, the antibody or antigen-binding fragment has the VH region amino acid sequence set forth in SEQ ID NO: 42. In some embodiments, the antibody or antigenbinding fragment has the VH region amino acid sequence set forth in SEQ ID NO: 43. In some embodiments, the antibody or antigen-binding fragment has the VH region amino acid sequence set forth in SEQ ID NO: 44. In some embodiments, the antibody or antigen-binding fragment has the VH region amino acid sequence set forth in SEQ ID NO: 45. In some embodiments, the antibody or antigenbinding fragment has the VH region amino acid sequence set forth in SEQ ID NO: 157. In some embodiments, the antibody or antigen-binding fragment has the VH region amino acid sequence set forth in SEQ ID NO: 165. In some embodiments, the antibody or antigen-binding fragment has the VH region amino acid sequence set forth in SEQ ID NO: 166. In some embodiments, the antibody or antigen-binding fragment has the VH region amino acid sequence set forth in SEQ ID NO: 167. In some embodiments, the antibody or antigen-binding fragment has the VH region amino acid sequence set forth in SEQ ID NO: 168. In some embodiments, the antibody or antigen-binding fragment has the VH region amino acid sequence set forth in SEQ ID NO: 169. In some embodiments, the antibody or antigen-binding fragment has the VH region amino acid sequence set forth in SEQ ID NO: 170. In some of any of the above embodiments, the antibody or antigen-binding fragment has a VH region having an amino acid sequence that has at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the SEQ ID NO.
[0156] In some embodiments, the antibody or antigen-binding fragment has the VL region amino acid sequence set forth in SEQ ID NO: 46. In some embodiments, the antibody or antigen-binding fragment has the VL region amino acid sequence set forth in SEQ ID NO: 47. In some embodiments, the antibody or antigen-binding fragment has the VL region amino acid sequence set forth in SEQ ID NO: 48. In some embodiments, the antibody or antigen-binding fragment has the VL region amino acid sequence set forth in SEQ ID NO: 49. In some embodiments, the antibody or antigen-binding fragment has the VL region amino acid sequence set forth in SEQ ID NO: 50. In some embodiment, the antibody or antigen-binding fragment has the VL region amino acid sequence set forth in SEQ ID NO:51. In some embodiments, the antibody or antigen-binding fragment has the VL region amino acid sequence set forth in SEQ ID NO: 52. In some embodiment, the antibody or antigen-binding fragment has the VL region amino acid sequence set forth in SEQ ID NO: 53. In some embodiment, the antibody or antigen-binding fragment has the VL region amino acid sequence set forth in SEQ ID NO: 54. In some embodiments, the antibody or antigen-binding fragment has the VL region amino acid sequence set forth in SEQ ID NO: 55. In some embodiments, the antibody or antigen-binding fragment has the VL region amino acid sequence set forth in SEQ ID NO: 56. In some embodiments, the antibody or antigenbinding fragment has the VL region amino acid sequence set forth in SEQ ID NO: 57. In some embodiments, the antibody or antigen-binding fragment has the VL region amino acid sequence set forth in SEQ ID NO: 58. In some embodiments, the antibody or antigen-binding fragment has the VL region amino acid sequence set forth in SEQ ID NO: 59. In some embodiments, the antibody or antigenbinding fragment has the VL region amino acid sequence set forth in SEQ ID NO: 60. In some embodiments, the antibody or antigen-binding fragment has the VL region amino acid sequence set forth in SEQ ID NO: 61. In some embodiments, the antibody or antigen-binding fragment has the VL region amino acid sequence set forth in SEQ ID NO: 62. In some embodiment, the antibody or antigen-binding fragment has the VL region amino acid sequence set forth in SEQ ID NO: 63. In some embodiments, the antibody or antigen-binding fragment has the VL region amino acid sequence set forth in SEQ ID NO: 64. In some embodiments, the antibody or antigen-binding fragment has the VL region amino acid sequence set forth in SEQ ID NO: 65. In some embodiments, the antibody or antigen-binding fragment has the VL region amino acid sequence set forth in SEQ ID NO: 66. In some embodiments, the antibody or antigen-binding fragment has the VL region amino acid sequence set forth in SEQ ID NO: 67. In some embodiments, the antibody or antigen-binding fragment has the VL region amino acid sequence set forth in SEQ ID NO: 68. In some embodiments, the antibody or antigen-binding fragment has the VL region amino acid sequence set forth in SEQ ID NO: 69. In some embodiments, the antibody or antigen-binding fragment has the VL region amino acid sequence set forth in SEQ ID NO: 70. In some embodiments, the antibody or antigen-binding fragment has the VL region amino acid sequence set forth in SEQ ID NO: 71. In some embodiments, the antibody or antigen-binding fragment has the VL region amino acid sequence set forth in SEQ ID NO: 72. In some embodiments, the antibody or antigenbinding fragment has the VL region amino acid sequence set forth in SEQ ID NO: 73. In some embodiments, the antibody or antigen-binding fragment has the VL region amino acid sequence set forth in SEQ ID NO: 74. In some embodiments, the antibody or antigen-binding fragment has the VL region amino acid sequence set forth in SEQ ID NO: 75. In some embodiments, the antibody or antigenbinding fragment has the VL region amino acid sequence set forth in SEQ ID NO: 76. In some embodiments, the antibody or antigen-binding fragment has the VL region amino acid sequence set forth in SEQ ID NO: 77. In some embodiments, the antibody or antigen-binding fragment has the VL region amino acid sequence set forth in SEQ ID NO: 78. In some embodiments, the antibody or antigenbinding fragment has the VL region amino acid sequence set forth in SEQ ID NO: 79. In some embodiments, the antibody or antigen-binding fragment has the VL region amino acid sequence set forth in SEQ ID NO: 80. In some embodiments, the antibody or antigen-binding fragment has the VL region amino acid sequence set forth in SEQ ID NO: 81. In some embodiments, the antibody or antigenbinding fragment has the VL region amino acid sequence set forth in SEQ ID NO: 82. In some embodiments, the antibody or antigen-binding fragment has the VL region amino acid sequence set forth in SEQ ID NO: 83. In some embodiments, the antibody or antigen-binding fragment has the VL region amino acid sequence set forth in SEQ ID NO: 84. In some embodiments, the antibody or antigenbinding fragment has a VL region having an amino acid sequence that has the VL region amino acid sequence set forth in SEQ ID NO: 85. In some embodiments, the antibody or antigen-binding fragment has the VL region amino acid sequence set forth in SEQ ID NO: 86. In some embodiments, the antibody or antigen-binding fragment has the VL region amino acid sequence set forth in SEQ ID NO: 87. In some embodiments, the antibody or antigen-binding fragment has the VL region amino acid sequence set forth in SEQ ID NO: 88. In some embodiments, the antibody or antigen-binding fragment has the VL region amino acid sequence set forth in SEQ ID NO: 89. In some embodiment, the antibody or antigen-binding fragment has the VL region amino acid sequence set forth in SEQ ID NO: 90. In some embodiments, the antibody or antigen-binding fragment has the VL region amino acid sequence set forth in SEQ ID NO: 158. In some embodiments, the antibody or antigen-binding fragment has the VL region amino acid sequence set forth in SEQ ID NO: 171. In some embodiments, the antibody or antigenbinding fragment has the VL region amino acid sequence set forth in SEQ ID NO: 172. In some of any of the above embodiments, the antibody or antigen-binding fragment has a VL region having an amino acid sequence that has at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the SEQ ID NO.
[0157] In some embodiments, the antibody or antigen-binding fragment has a VH region having an amino acid sequence that is less than 100% identical to SEQ ID NO: 157 and has at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the VH region amino acid sequence set forth in SEQ ID NO: 157. In some embodiments, the antibody or antigen-binding fragment has a VL region having an amino acid sequence that is less than 100% identical to SEQ ID NO: 158 and has at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the VL region amino acid sequence set forth in SEQ ID NO: 158. In some embodiments, the VH region does not have the amino acid sequence set forth in SEQ ID NO: 157. In some embodiments, the VL region does not have the amino acid sequence set forth in SEQ ID NO: 158. In some embodiments, the VH region and the VL region of the antibody are not set forth SEQ ID NO: 157 and 158, respectively. In some embodiments, the heavy chain of the constant region does not have the amino acid sequence set forth in SEQ ID NO: 178. In some embodiments, the light chain of the constant region does not have the amino acid sequence set forth in SEQ ID NO: 179. In some embodiments, the heavy chain and the light chain of the antibody are not set forth SEQ ID NO: 178 and 179, respectively.
[0158] In some embodiments, the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 1 and SEQ ID NO: 158, respectively. In some embodiments, the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 2 and SEQ ID NO: 158, respectively. In some embodiments, the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 3 and SEQ ID NO: 158, respectively. In some embodiments, the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 4 and SEQ ID NO: 158, respectively. In some embodiments, the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 5 and SEQ ID NO: 158, respectively. In some embodiments, the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 6 and SEQ ID NO: 158, respectively. In some embodiments, the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 7 and SEQ ID NO: 158, respectively. In some embodiments, the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 8 and SEQ ID NO: 158, respectively. In some embodiments, the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 9 and SEQ ID NO: 158, respectively. In some embodiments, the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 10 and SEQ ID NO: 158, respectively. In some embodiments, the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 11 and SEQ ID NO: 158, respectively. In some embodiments, the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 12 and SEQ ID NO: 158, respectively. In some embodiments, the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 13 and SEQ ID NO: 158, respectively. In some embodiments, the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 14 and SEQ ID NO: 158, respectively. In some embodiments, the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO and SEQ ID NO: 158, respectively. In some embodiments, the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 16 and SEQ ID NO: 158, respectively. In some embodiments, the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 17 and SEQ ID NO: 158, respectively. In some embodiments, the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 18 and SEQ ID NO: 158, respectively. In some embodiments, the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 19 and SEQ ID NO: 158, respectively. In some embodiments, the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 20 and SEQ ID NO: 158, respectively. In some embodiments, the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 21 and SEQ ID NO: 158, respectively. In some embodiments, the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 22 and SEQ ID NO: 158, respectively. In some embodiments, the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 23 and SEQ ID NO: 158, respectively. In some embodiments, the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 24 and SEQ ID NO: 158, respectively. In some embodiments, the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 25 and SEQ ID NO: 158, respectively. In some embodiments, the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 26 and SEQ ID NO: 158, respectively. In some embodiments, the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 27 and SEQ ID NO: 158, respectively. In some embodiments, the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 28 and SEQ ID NO: 158, respectively. In some embodiments, the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 29 and SEQ ID NO: 158, respectively. In some embodiments, the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 30 and SEQ ID NO: 158, respectively. In some embodiments, the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 31 and SEQ ID NO: 158, respectively. In some embodiments, the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 32 and SEQ ID NO: 158, respectively. In some embodiments, the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 33 and SEQ ID NO: 158, respectively. In some embodiments, the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 34 and SEQ ID NO: 158, respectively. In some embodiments, the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 35 and SEQ ID NO: 158, respectively. In some embodiments, the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 36 and SEQ ID NO: 158, respectively. In some embodiments, the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 37 and SEQ ID NO: 158, respectively. In some embodiments, the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 38 and SEQ ID NO: 158, respectively. In some embodiments, the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 39 and SEQ ID NO: 158, respectively. In some embodiments, the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 40 and SEQ ID NO: 158, respectively. In some embodiments, the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 41 or ID NO: 158, respectively. In some embodiments, the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 42 and SEQ ID NO: 158, respectively. In some embodiments, the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 43 and SEQ ID NO: 158, respectively. In some embodiments, the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 44 and SEQ ID NO: 158, respectively. In some embodiments, the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 45 and SEQ ID NO: 158, respectively. In some embodiments, the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 46, respectively. In some embodiments, the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 47, respectively. In some embodiments, the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 48, respectively. In some embodiments, the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 49, respectively. In some embodiments, the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 50, respectively. In some embodiments, the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 51, respectively. In some embodiments, the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 52, respectively. In some embodiments, the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 53, respectively. In some embodiments, the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 54, respectively. In some embodiments, the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 55, respectively. In some embodiments, the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 56, respectively. In some embodiments, the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 57, respectively. In some embodiments, the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 58, respectively. In some embodiments, the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 59, respectively. In some embodiments, the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 60, respectively. In some embodiments, the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 61, respectively. In some embodiments, the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 62, respectively. In some embodiments, the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 63, respectively. In some embodiments, the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 64, respectively. In some embodiments, the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 65, respectively. In some embodiments, the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 66, respectively. In some embodiments, the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 or ID NO: 67, respectively. In some embodiments, the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 68, respectively. In some embodiments, the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 69, respectively. In some embodiments, the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 70, respectively. In some embodiments, the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 71, respectively. In some embodiments, the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 72, respectively. In some embodiments, the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 73, respectively. In some embodiments, the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 74, respectively. In some embodiments, the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 75, respectively. In some embodiments, the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 76, respectively. In some embodiments, the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 77, respectively. In some embodiments, the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 78, respectively. In some embodiments, the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 79, respectively. In some embodiments, the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 80, respectively. In some embodiments, the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 81, respectively. In some embodiments, the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 82, respectively. In some embodiments, the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 83, respectively. In some embodiments, the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 84, respectively. In some embodiments, the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 85, respectively. In some embodiments, the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 86, respectively. In some embodiments, the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 87, respectively. In some embodiments, the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 88, respectively. In some embodiments, the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 81%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 89, respectively. In some embodiments, the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 90, respectively. In some embodiments, the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 16 and SEQ ID NO: 47, respectively. In some embodiments, the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 23 and SEQ ID NO: 47, respectively. In some embodiments, the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 35 and SEQ ID NO: 76, respectively. In some embodiments, the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 35 and SEQ ID NO: 79, respectively. In some embodiments, the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 35 and SEQ ID NO: 81, respectively. In some embodiments, the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 35 and SEQ ID NO: 84, respectively. In some embodiments, the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 35 and SEQ ID NO: 89, respectively. In some embodiments, the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 37 and SEQ ID NO: 76, respectively. In some embodiments, the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 37 and SEQ ID NO: 79, respectively. In some embodiments, the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 37 and SEQ ID NO: 81, respectively. In some embodiments, the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 37 and SEQ ID NO: 84, respectively. In some embodiments, the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 37 and SEQ ID NO: 89, respectively. In some embodiments, the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 40 and SEQ ID NO: 76, respectively. In some embodiments, the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 40 and SEQ ID NO: 79, respectively. In some embodiments, the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 40 and SEQ ID NO: 81, respectively. In some embodiments, the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 40 and SEQ ID NO: 84, respectively. In some embodiments, the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 40 and SEQ ID NO: 89, respectively. In some embodiments, the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 42 and SEQ ID NO: 76, respectively. In some embodiments, the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 42 and SEQ ID NO: 79, respectively. In some embodiments, the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 42 and SEQ ID NO: 81, respectively. In some embodiments, the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 42 and SEQ ID NO: 84, respectively. In some embodiments, the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 42 and SEQ ID NO: 89, respectively. In some embodiments, the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 165 and SEQ ID NO: 79, respectively. In some embodiments, the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 165 and SEQ ID NO: 171, respectively. In some embodiments, the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 165 and SEQ ID NO: 172, respectively. In some embodiments, the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 166 and SEQ ID NO: 79, respectively. In some embodiments, the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 166 and SEQ ID NO: 171, respectively. In some embodiments, the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 166 and SEQ ID NO: 172, respectively. In some embodiments, the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 167 and SEQ ID NO: 79, respectively. In some embodiments, the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 167 and SEQ ID NO: 171, respectively. In some embodiments, the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 167 and SEQ ID NO: 172, respectively. In some embodiments, the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 168 and SEQ ID NO: 79, respectively. In some embodiments, the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 168 and SEQ ID NO: 171, respectively. In some embodiments, the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 168 and SEQ ID NO: 172, respectively. In some embodiments, the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 169 and SEQ ID NO: 79, respectively. In some embodiments, the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 169 and SEQ ID NO: 171, respectively. In some embodiments, the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 169 and SEQ ID NO: 172, respectively. In some embodiments, the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 170 and SEQ ID NO: 79, respectively. In some embodiments, the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 170 and SEQ ID NO: 171, respectively. In some embodiments, the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 170 and SEQ ID NO: 172, respectively.
[0159] In some embodiments, the VH region of the antibody or antigen-binding fragment thereof comprises the amino acid sequence of any one of SEQ ID NOs: 1-45, 157, and 165-170, and the VL region of the antibody or antigen-binding fragment comprises the amino acid sequence of any one of SEQ ID NOs: 46-90, 158, and 171-172.
[0160] In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 1 and 158, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 2 and 158, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 3 and 158, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 4 and 158, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 5 and 158, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 6 and 158, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 7 and 158, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 8 and 158, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 9 and 158, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 10 and 158, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 11 and 158, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 12 and 158, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 13 and 158, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 14 and 158, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 15 and 158, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 16 and 158, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 17 and 158, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 18 and 158, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 19 and 158, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 20 and 158, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 21 and 158, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 22 and 158, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 23 and 158, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 24 and 158, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 25 and 158, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 26 and 158, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 27 and 158, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 28 and 158, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 29 and 158, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 30 and 158, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 31 and 158, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 32 and 158, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 33 and 158, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 34 and 158, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 35 and 158, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 36 and 158, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 37 and 158, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 38 and 158, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 39 and 158, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 40 and 158, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 41 and 158, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 42 and 158, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 43 and 158, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 44 and 158, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 45 and 158, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 157 and 46, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 157 and 47, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 157 and 48, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 157 and 49, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 157 and 50, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 157 and 51, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 157 and 52, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 157 and 53, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 157 and 54, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 157 and 55, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 157 and 56, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 157 and 57, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 157 and 58, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 157 and 59, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 157 and 60, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 157 and 61, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 157 and 62, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 157 and 63, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 157 and 64, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 157 and 65, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 157 and 66, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 157 and 67, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 157 and 68, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 157 and 69, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 157 and 70, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 157 and 71, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 157 and 72, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 157 and 73, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 157 and 74, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 157 and 75, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 157 and 76, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 157 and 77, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 157 and 78, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 157 and 79, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 157 and 80, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 157 and 81, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 157 and 82, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 157 and 83, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 157 and 84, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 157 and 85, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 157 and 86, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 157 and 87, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 157 and 88, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 157 and 89, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 157 and 90, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 16 and 47, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 23 and 47, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 35 and 76, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 35 and 79, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 35 and 81, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 35 and 84, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 35 and 89, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 37 and 76, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 37 and 79, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 37 and 81, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 37 and 84, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 37 and 89, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 40 and 76, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 40 and 79, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 40 and 81, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 40 and 84, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 40 and 89, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 42 and 76, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 42 and 79, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 42 and 81, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 42 and 84, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 42 and 89, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 165 and 79, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 165 and 171, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 165 and 172, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 166 and 79, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 166 and 171, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 166 and 172, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 167 and 79, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 167 and 171, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 167 and 172, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 168 and 79, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 168 and 171, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 168 and 172, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 169 and 79, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 169 and 171, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 169 and 172, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 170 and 79, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 170 and 171, respectively. In some embodiments of the antibody or antigen-binding fragment provided herein, the VH region and the VL region are or comprise the sequence set forth in SEQ ID NO: 170 and 172, respectively.
[0161] Table El provides the SEQ ID NOS: of exemplary provided antibody or antigen-binding fragment thereof. In some embodiments, the PD-1 -binding antibody or fragment thereof comprises a VH region that comprises the CDR-H1, the CDR-H2 and the CDR-H3 sequence and a VL region that comprises the CDR-L1, the CDR-L2 and the CDR-L3 sequence set forth in the SEQ ID NOS: listed in each row of Table El below (by Kabat numbering scheme). In some embodiments, the PD-l-binding antibody or fragment thereof comprises a VH region sequence and a VL region sequence set forth in the SEQ ID NOS: listed in each row of Table El below, or an antibody comprising a VH region and a VL region amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the VH region sequence and the VL region sequence set forth in the SEQ ID NOS: listed in each row of Table El below. In some embodiments, the PD-l-binding antibody or fragment thereof comprises a VH region sequence and a VL region sequence set forth in the SEQ ID NOS: listed in each row of Table El below.
[0162] In some embodiments, the provided antibody or antigen-binding fragment thereof comprises a VH region and a VL region, wherein the VH region of the antibody or antigen-binding fragment thereof can contain a combination of any of the CDR-H1, the CDR-H2 and the CDR-H3 amino acid sequences set forth in Table El, and the VL region of the antibody or antigen-binding fragment thereof can contain a combination of any of the CDR-L1, the CDR-L2 and the CDR-L3 amino acid sequences set forth in Table El. In some embodiments, the provided antibody or antigen-binding fragment thereof comprises a VH region and/or a VL region set forth in Table El, in any combination, orientation or containing a different linker. In some aspects, the antibody or antigen-binding fragment thereof comprises a VH region described in Table El. In some aspects, the antibody or antigen-binding fragment thereof comprises a VL region described in Table El.
[0163] In certain embodiments, antibody variable domains as described are fused to immunoglobulin constant domain sequences. In certain embodiments, the fusion is with an Ig heavy chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. In some of any of the embodiments herein, the antibody further comprises a heavy chain constant domain and/or a light chain constant domain. In some embodiments, the heavy chain and/or light constant domain is murine or human.
[0164] A “constant region” or “constant domain” is a domain in an antibody heavy or light chain that contains a sequence of amino acids that is comparatively more conserved among antibodies than the variable region domain. The constant regions of immunoglobulins show less sequence diversity than the variable regions, and are responsible for binding a number of natural proteins to elicit important biochemical events. Each light chain has a single light chain constant region (CL) domain, which is either of the kappa or lambda type. Each heavy chain contains one or more heavy chain constant region (CH) domains that can be assigned to different classes or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, having heavy chains designated a, 8, e, y and p. respectively. Full-length IgA, IgD and IgG isotypes contain CHI, CH2, CH3 and a hinge region, while IgE and IgM contain CHI , CH2, CH3 and CH4. The y and a classes are further divided into subclasses on the basis of relatively minor differences in the CH sequence and function, e.g., humans express the following subclasses: IgGl, IgG2, IgG3, IgG4, IgAl and IgA2. IgGl antibodies can exist in multiple polymorphic variants termed allotypes (reviewed in Jefferis and Lefranc 2009. mAbs Vol 1 Issue 4 1- 7) any of which are suitable. In humans there are five different classes of antibodies including IgA (which includes subclasses IgAl and IgA2), IgD, IgE, IgG (which includes subclasses IgGl, IgG2, IgG3, and IgG4), and IgM. The distinguishing features between these antibody classes are their constant, Fc regions, although subtler differences may exist in the V region. An antibody constant region can include an Fc portion. Although the boundaries of the Fc region of an immunoglobulin heavy chain might vary, the human IgG heavy-chain Fc region usually includes the region containing the CH2 and CH3 domains and the hinge region, such as an amino acid residue at position Cys226, or from Pro230, to the carboxyl-terminus thereof.
[0165] In certain embodiments, a provided antibody contains an Ig heavy chain constant domain comprising at least part of the hinge, CH2, and CH3 regions. Certain embodiments have the first heavychain constant region (CHI) containing the site necessary for light chain bonding, present in at least one of the fusions. DNAs encoding the immunoglobulin heavy chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are co-transfected into a suitable host cell. This provides for greater flexibility in adjusting the mutual proportions of the three polypeptide fragments in embodiments when unequal ratios of the three polypeptide chains used in the construction provide the optimum yield of the desired fusion antibody. It is, however, possible to insert the coding sequences for two or all three polypeptide chains into a single expression vector when the expression of at least two polypeptide chains in equal ratios results in high yields or when the ratios have no significant effect on the yield of the desired chain combination.
[0166] In some embodiments, the antibody or antigen-binding fragment thereof, may contain at least a portion of an immunoglobulin constant region, such as one or more constant region domain. In some embodiments, the constant regions include a light chain constant region and/or a heavy chain constant region 1 (CHI). In some embodiments, the antibody includes at least a portion of a hinge region or a variant thereof. In some embodiments, the antibody includes a CH2 and/or CH3 domain, such as an Fc region. In some embodiments, the Fc region is an Fc region from an IgAl, IgA2, IgD, IgE, IgGl, IgG2, IgG3, IgG4, or IgM.
[0167] In some embodiments, the heavy chain constant sequence includes an IgG4 containing the S228P mutation, which has been shown to prevent recombination between a therapeutic antibody and an endogenous IgG4 by Fab-arm exchange (see e.g. Labrijin et al. (2009) Nat. Biotechnol., 27(8): 767- 71.)
[0168] In some embodiments, the heavy chain constant domain comprises the sequence set forth in SEQ ID NO: 174. In some embodiments, the light chain constant domain comprises the sequence set forth in SEQ ID NO: 175. In some embodiments, the heavy chain further comprises a signal peptide. In some embodiments, the signal peptide can be cleaved upon expression from a cell. In some embodiments, the signal peptide is set forth in SEQ ID NO: 176.
[0169] In some embodiments, the light chain constant region is a kappa light chain. In some embodiments, the kappa light chain comprises the sequence set forth in SEQ ID NO: 175. In some embodiments, the light chain further comprises a signal peptide. In some embodiments, the signal peptide can be cleaved upon expression from a cell. In some embodiments, the signal peptide is set forth in SEQ ID NO: 177.
[0170] In some embodiments, the antibody or antigen-binding fragment thereof is a single-chain antibody fragment, such as a single chain variable fragment (scFv). In some embodiments, the antibody or antigen binding fragment is a multi-domain antibody, such as an scFv comprising a heavy chain variable (VH) region and a light chain variable (VL) region. In some embodiments, the single-chain antibody fragment e.g., scFv) includes one or more linkers joining two antibody domains or regions, such as a heavy chain variable (VH) region and a light chain variable (VL) region. The linker typically is a peptide linker, e.g., a flexible and/or soluble peptide linker. Among the linkers are those rich in glycine and serine and/or in some cases threonine. In some embodiments, the linkers further include charged residues such as lysine and/or glutamate, which can improve solubility. In some embodiments, the linkers further include one or more proline.
[0171] In some aspects, the linkers rich in glycine and serine (and/or threonine) include at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% such amino acid(s). In some embodiments, they include at least at or about 50%, 55%, 60%, 70%, or 75%, glycine, serine, and/or threonine. In some embodiments, the linker is comprised substantially entirely of glycine, serine, and/or threonine. The linkers generally are between about 5 and about 50 amino acids in length, typically between at or about 10 and at or about 30, e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30, and in some examples between 10 and 25 amino acids in length. Exemplary linkers include linkers having various numbers of repeats of the sequence GGGGS (4GS; SEQ ID NO: 159) or GGGS (3GS; SEQ ID NO: 160), such as between 2, 3, 4 and 5 repeats of such a sequence. Exemplary linkers include those having or consisting of an sequence set forth in SEQ ID NO: 161 (GGGGSGGGGSGGGGS). Exemplary linkers further include those having or consisting of the sequence set forth in SEQ ID NO: 162 (GSTSGSGKPGSGEGSTKG). Exemplary linkers further include those having or consisting of the sequence set forth in SEQ ID NO: 163 (SRGGGGSGGGGSGGGGSLEMA). An exemplary linker includes those having or consisting of the sequence set forth in SEQ ID NO; 164 (GSRGGGGSGGGGSGGGGSLEMA).
B. Exemplary Features
[0172] In some aspects, the provided antibodies have one or more specified functional features, such as binding properties, including binding to particular epitopes or exhibiting lower or reduced binding to a related but non-specific antigen. In some aspects, the provided antibodies can bind to an epitope that is similar to or overlaps with epitopes of other antibodies, such as reference antibodies, and/or exhibit particular binding affinities. In some aspects, the provided antibodies can bind to an epitope that is different from epitopes of other antibodies, e.g., binding a conformational epitope.
[0173] In some embodiments, the provided antibodies or antigen-binding fragment thereof specifically bind to a programmed cell death protein 1 (PD-1) protein. In some of any of the embodiments provided herein, PD-1 refers to human PD-1. In particular embodiments, the provided antibodies and antibody fragments specifically bind to human PD-1. A molecule is said to exhibit "specific binding" or "preferential binding" if it reacts or associates more frequently, more rapidly, with greater duration and/or with greater affinity with a particular cell or substance (e.g. a cell expressing a PD-1 antigen or a recombinant PD-1 antigen) than it does with alternative cells or substances. An antibody specifically binds or preferentially binds to a target (i.e. PD-1) if it binds with greater affinity, avidity, more readily, and/or with greater duration than it binds to other substances. For example, an antibody that specifically or preferentially binds to PD-1 is an antibody that binds PD-1 with greater affinity, avidity, more readily, and/or with greater duration than it binds to a different protein antigen. It is also understood by reading this definition that specific binding or preferential binding does not necessarily require (although it can include) exclusive binding. Methods to determine such specific or preferential binding are also well known in the art, e.g., an immunoassay.
[0174] The observation that an antibody or other binding molecule binds to PD-1 or specifically binds to PD-1 does not necessarily mean that it binds to PD-1 from every species. For example, in some embodiments, features of binding to PD-1, such as the ability to specifically bind thereto and/or to compete for binding thereto with a reference antibody, and/or to bind with a particular affinity or compete to a particular degree, in some embodiments, refers to the ability with respect to a human PD- 1 protein and the antibody may not have this feature with respect to a PD-1 of another species such as mouse.
[0175] In some embodiments, the provided antibodies are capable of binding PD-1, such as human PD-1, with at least a certain affinity, as measured by any of a number of known methods. In some embodiments, the affinity is represented by an equilibrium dissociation constant (KD).
[0176] A variety of assays are known for assessing binding affinity, equilibrium dissociation constant (KD), equilibrium association constant (KA), ECSO, on-rate (association rate constant; konor ka; units of 1/Ms or M ’s 1) and the off-rate (dissociation rate constant; koff or k ; units of 1/s or s ’) and/or determining whether a binding molecule (e.g., an antibody or fragment thereof) specifically binds to a particular ligand e.g., an antigen, such as a PD-1 protein). One can determine the binding affinity of a binding molecule, e.g., an antibody or an antigen-binding fragment thereof, for an antigen, e.g., PD-1, such as human PD-1, such as by using any of a number of binding assays that are well known. For example, in some embodiments, a BIAcore® instrument can be used to determine the binding kinetics and constants of a complex between two proteins (e.g. , an antibody or fragment thereof, and an antigen, such as a PD-1 protein), using surface plasmon resonance (SPR) analysis (see, e.g., Scatchard et al., Ann. N.Y. Acad. Sci. 51:660, 1949; Wilson, Science 295:2103, 2002; Wolff etal., Cancer Res. 53:2560, 1993; and U.S. Patent Nos. 5,283,173, 5,468,614, or the equivalent).
[0177] SPR measures changes in the concentration of molecules at a sensor surface as molecules bind to or dissociate from the surface. The change in the SPR signal is directly proportional to the change in mass concentration close to the surface, thereby allowing measurement of binding kinetics between two molecules. The dissociation rate constant (koff or kd), the association rate constant (kon or ka) and/or equilibrium dissociation constant (KD) and/or equilibrium association constant (KA) for the complex can be determined by monitoring changes in the refractive index with respect to time as buffer is passed over the chip. Other suitable assays for measuring the binding of one protein to another include, for example, immunoassays such as enzyme linked immunosorbent assays (ELISA) and radioimmunoassays (RIA), or determination of binding by monitoring the change in the spectroscopic or optical properties of the proteins through fluorescence, UV absorption, circular dichroism, or nuclear magnetic resonance (NMR). Other exemplary assays include, but are not limited to, Western blot, ELISA, analytical ultracentrifugation, spectroscopy, flow cytometry, sequencing, genetic reporter assays, flow cytometry, and other methods for detection of expressed nucleic acids or binding of proteins.
[0178] In some binding can be determined under conditions that mimic those that exist in natural environments in the body of a subject. For instance, acid pH of about 5.6 to 6.8 such as pH about 6.0 mimics more closely the acidic tumor microenvironment. In contrast, physiological pH is a more neutral pH 7.35 to 7.45, such as pH about 7.4 and more closely mimics the environment of normal tissues or cells in the blood.
[0179] In some embodiments, the antibody or fragment thereof exhibits a binding affinity to PD- 1 at pH 7.35 to 7.45 (e.g. pH about 7.4), such as present in normal pH of blood or a tissue environment, with a KD of equal to or less than 2 x 108 M. For example, the equilibrium dissociation constant KD at pH 7.35 to 7.45 (e.g. pH about 7.4) can range from 7 x 10 10 M to 2 x 108 M, such as about 7 x 10 10 M to about 1 x 109 M. In some embodiments, a provided antibody or antibody fragment an off-rate (dissociation rate constant; koff or kdj) at pH 7.35 to 7.45 (e.g. pH about 7.4), such as present in normal pH of blood or a tissue environment, of 4 x 104 1/s to about 9 x 103 1/s. In some embodiments, a provided antibody or antibody fragment has an off-rate (dissociation rate constant; koff or kdj) at pH 7.35 to 7.45 (e.g. pH about 7.4), such as present in normal pH of blood or a tissue environment, of 4 x 105 1/Ms to about 2 x 106 1/Ms.
[0180] In some embodiments, the antibody or fragment thereof exhibits a binding affinity to PD- 1 at an acidic pH, such as at pH 5.0 to 6.8 (e.g. pH about 6.0 or pH about 5.0) with a KD of equal to or less than 4 x 10 8 M. In some embodiments, the pH is a pH for example as present in a microenvironment of tumor. In some embodiments, the equilibrium dissociation constant KD at an acidic microenvironment pH 5.0 to 6.8 (e.g. pH about 6.0 or about pH 5.0), can range from about 3 x 10 10 M to about 4 x 108 M, such as about 3 x 10 10 to about 1 x 10 8 M. In some embodiments, the antibody or fragment thereof exhibits a binding affinity to PD-1 at an acidic pH, such as at pH 5.6 to 6.8 (e.g. pH about 6.0), for example as present in a microenvironment of tumor, with a KD of equal to or less than 4 x 10 8 M. For example, the equilibrium dissociation constant KD at an acidic microenvironment pH 5.6 to 6.8 (e.g. pH about 6.0), can range from about 3 x 10 10 M to about 4 x 10 8 M, such as about 3 x 10 10 to about 1 x 108 M. In some embodiments, the antibody or fragment thereof exhibits a binding affinity to PD-1 at an acidic pH, such as at pH 5.0 to 6.0 (e.g. pH about 5.0), for example as present in a microenvironment of tumor, with a KD of equal to or less than 8 x 109 M. For example, the equilibrium dissociation constant KD at an acidic microenvironment pH 5.0 to 6.0 (e.g. pH about 5.0), can range from about 3 x 10 10 M to about 8 x 109 M, such as about 6 x 10 10 to about 8 x 109 M.
[0181] In some embodiments, the antibody or fragment thereof has an off-rate at an acidic pH, such as at pH 5.0 to 6.8 (e.g. pH about 6.0 or about 5.0), for example as present in a microenvironment of tumor, of 4 x 104 1/s to about 2 x 102 1/s. In some embodiments, the antibody or fragment thereof has an off-rate at an acidic pH, such as at pH 5.6 to 6.8 (e.g. pH about 6.0), for example as present in a microenvironment of tumor, of 6 x 104 1/s to about 2 x 102 1/s. In some embodiments, the antibody or fragment thereof has an off-rate at an acidic pH, such as at pH 5.0 to 6.0 (e.g. pH about 5.0), for example as present in a microenvironment of tumor, of 4 x 104 1/s to about 2 x 102 1/s.
[0182] In some embodiments, the antibody or fragment thereof has an on-rate at an acidic pH, such as at pH 5.0 to 6.8 (e.g. pH about 6.0 or about 5.0), for example as present in a microenvironment of tumor, of 4 x 105 1/Ms to about 5 x 106 1/Ms. In some embodiments, the antibody or fragment thereof has an on-rate at an acidic pH, such as at pH 5.6 to 6.8 (e.g. pH about 6.0), for example as present in a microenvironment of tumor, of 4 x 105 1/Ms to about 3 x 1061/Ms. In some embodiments, the antibody or fragment thereof has an on-rate at an acidic pH, such as at pH 5.0 to 6.0 (e.g. pH about 5.0), for example as present in a microenvironment of tumor, of 4 x 105 1/Ms to about 5 x 106 1/Ms.
[0183] Among such provided antibodies and antibody fragments are antibodies or antibody fragments that have higher binding affinity to PD-1 at a pH in the tumor microenvironment than at a pH in a non-tumor microenvironment. For example, the antibodies and antibody fragments have a higher binding affinity to PD-1 at pH 5.0 to 6.8 (e.g. pH 6.0 or pH 5.0) than at pH 7.35 to 7.45 (e.g. pH 7.4). In some embodiments, the anti-PD-1 antibodies or antibody fragments have a higher binding affinity (e.g. lower KD) to PD-1 in a tumor in comparison with their binding affinity to PD-1 in a normal tissue or to a cell in the blood. In some embodiments, these anti-PD-1 antibodies or antibody fragments have a longer half-life and reduced side-effects, as well as comparable efficacy, in comparison with reference monoclonal anti-PD-1 antibodies known in the art. These features permit use of a higher dosage of these anti-PD-1 antibodies or antibody fragments to be delivered to a patient thus being a more effective therapeutic option. In some embodiments, the antibody or fragment thereof exhibits a higher binding affinity (e.g. lower KD) to PD-1 at an acidic pH as present in the microenvironment of tumor (such as at pH 5.0 to 6.8, e.g. pH 6.0 or pH 5.0) than at pH of a non-tumor microenvironment (such as at pH 7.35 to 7.45, e.g. 7.4) as present at normal pH of blood or a normal tissue. In some embodiments, the PD-1 binding at an acidic pH as present in the microenvironment of tumor (such as at pH 5.0 to 6.8, e.g. pH 6.0 or pH 5.0) compared to PD-1 binding at pH of a non-tumor microenvironment (such as at pH 7.35 to 7.45, e.g. 7.4) as present at normal pH of blood or a normal tissue is higher by a ratio of at least or 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50 or more. In some examples, the PD-1 binding is higher by at least 5%, 10%, 15%, 20%, 25%, 35%, 50%, 100%, 2-fold, 5-fold, 10-fold, 20-fold or more. In some embodiments, the KD at an acidic pH as present in the microenvironment of tumor (such as at pH 5.0 to 6.8, e.g. pH 6.0 or pH 5.0) is about 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 25-fold, 30-fold, 40-fold, 50-fold or 100-fold lower than the KD at pH of a non-tumor microenvironment (such as at pH 7.35 to 7.45, e.g. 7.4) as present at normal pH of blood or a normal tissue.
[0184] In some embodiments, the antibody or fragment thereof exhibits a higher binding affinity (e.g. lower KD) for binding PD-1 at normal pH of blood or a normal tissue (such as at pH 7.35 to 7.45, e.g. pH 7.4) than that at an acidic pH as present in the microenvironment of tumor (such as at pH 5.0 to
6.8, e.g. pH 6.0 or pH 5.0). In some embodiments, the PD-1 binding at pH of a non-tumor microenvironment (such as at pH 7.35 to 7.45, e.g. 7.4) as present at normal pH of blood or a normal tissue compared to PD-1 binding at an acidic pH as present in the microenvironment of tumor (such as at pH 5.0 to 6.8, e.g. pH 6.0 or pH 5.0) is higher by a ratio of at least or 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8,
1.9, 2.0, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50 or more. In some examples, the PD-1 binding is higher by at least 5%, 10%, 15%, 20%, 25%, 35%, 50%, 100%, 2-fold, 5-fold, 10- fold, 20-fold or more. In some embodiments, the KD at pH of a non-tumor microenvironment (such as at pH 7.35 to 7.45, e.g. 7.4) as present at normal pH of blood or a normal tissue is about 1.5-fold, 2- fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 25-fold, 30-fold, 40-fold, 50- fold or 100-fold lower than the KD at an acidic pH as present in the microenvironment of tumor (such as at pH 5.0 to 6.8, e.g. pH 6.0 or pH 5.0).
[0185] In some embodiments, the antibody or fragment exhibits a similar PD-1 binding at an acidic pH as present in the microenvironment of tumor (such as at pH 5.0 to 6.8, e.g. pH 6.0 or pH 5.0) compared to PD-1 binding at normal pH as present in blood or a normal tissue (such as at pH 7.35 to 7.45, e.g. pH 7.4). In some embodiments, the KD at an acidic pH as present in the microenvironment of tumor (such as at pH 5.0 to 6.8, e.g. pH 6.0 or pH 5.0) is about the same (e.g. 0.7-fold to 1.3-fold) the KD at pH of a non-tumor microenvironment (such as at pH 7.35 to 7.45, e.g. 7.4) as present at normal pH of blood or a normal tissue.
[0186] In some embodiments, properties or features of the provided antibodies are described in relation to properties observed for another antibody, e.g., a reference antibody. In some embodiments, the reference antibody is a human or humanized anti-PD-1 antibody. Exemplary reference anti-PD-1 antibodies include pembrolizumab (SEQ ID NOs: 178 and 179, VH and VL set forth in SEQ ID Nos: 157 and 158, respectively); e.g. described in U.S. Patent No. 8,354,509 and PCT publ. No. WO2008156712A1), Camrelizumab (VH and VL set forth in SEQ ID NOs: 182 and 183), Cemiplimab (VH and VL set forth in SEQ ID NOs: 184 and 185), Dostarlimab (VH and VL set forth in SEQ ID NOs: 186 and 187), Penpulimab (VH and VL set forth in SEQ ID NOs: 188 and 189), Pucotenlimab (VH and VL set forth in SEQ ID NOs: 190 and 191), Serplulimab (VH and VL set forth in SEQ ID NOs: 192 and 193), Sintilimab (VH and VL set forth in SEQ ID NOs: 194 and 195), Tislelizumab (VH and VL set forth in SEQ ID NOs: 196 and 197), Toripalimab (VH and VL set forth in SEQ ID NOs: 198 and 199), Zimberelimab (VH and VL set forth in SEQ ID NOs: 200 and 201), Budigalimab (VH and VL set forth in SEQ ID NOs: 202 and 203), Ezabenlimab (VH and VL set forth in SEQ ID NOs: 204 and 205), Peresolimab (VH and VL set forth in SEQ ID NOs: 206 and 207), Pidilizumab (VH and VL set forth in SEQ ID NOs: 208 and 209), Pimivalimab (VH and VL set forth in SEQ ID NOs: 210 and 211), Rosnilimab (VH and VL set forth in SEQ ID NOs: 212 and 213), Spartalizumab (VH and VL set forth in SEQ ID NOs: 214 and 215), Rulonilimab (VH and VL set forth in SEQ ID NOs: 216 and 217), Balstilimab (VH and VL set forth in SEQ ID NOs: 218 and 219), Cetrelimab (VH and VL set forth in SEQ ID NOs: 220 and 221), Finotonlimab (VH and VL set forth in SEQ ID NOs: 222 and 223), Nofazinlimab (VH and VL set forth in SEQ ID NOs: 224 and 225), Prolgolimab (VH and VL set forth in SEQ ID NOs:226 and 227), Sasanlimab (VH and VL set forth in SEQ ID NOs: 228 and 229), and Nivolumab (VH and VL set forth in SEQ ID NOs: 180 and 181; e.g. described in U.S. Patent No. 8,008,449 and PCT publ. No. W02006121168 ). In some embodiments, the provided antibody contains heavy and light chain CDRs that are distinct from the CDRs present in the reference antibody or antibodies.
[0187] In some embodiments, a provided antibody or fragment exhibits a higher binding affinity (e.g. lower KD) than the reference antibody to PD-1 at an acidic pH as present in the microenvironment of tumor (such as at pH 5.0 to 6.8, e.g. pH 6.0 or pH 5.0). In some embodiments, the PD-1 binding of a provided antibody or fragment at an acidic pH as present in the microenvironment of tumor (such as at pH 5.0 to 6.8, e.g. pH 6.0 or pH 5.0) is higher by at least or 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50 or more compared to the reference antibody for PD-1 binding at the same pH or condition. In some examples, the PD-1 binding is higher by at least 5%, 10%, 15%, 20%, 25%, 35%, 50%, 100%, 2-fold, 5-fold, 10-fold, 20-fold or more. In some embodiments, the KD of a provided antibody for PD-1 binding at an acidic pH as present in the microenvironment of tumor (such as at pH 5. Oto 6.8, e.g. pH 6.0 or pH 5.0) is about 1.5-fold, 2-fold, 3- fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 25-fold, 30-fold, 40-fold, 50-fold or 100-fold lower than the KD of the reference antibody for PD-1 binding at the same pH or condition.
[0188] In some embodiments, a provided antibody or fragment exhibits a similar PD-1 binding compared to a reference antibody to PD-1 at a pH of a non-tumor microenvironment (such as at pH 7.35 to 7.45, e.g. 7.4) as present at normal pH of blood or a normal tissue. In some embodiments, the KD of a provided antibody for PD-1 at a pH of a non-tumor microenvironment (such as at pH 7.35 to 7.45, e.g. 7.4) as present at normal pH of blood or a normal tissue is about the same (e.g. 0.7-fold to 1.3-fold) as a reference antibody at the same pH.
C. Variants and Modifications
[0189] Amino acid sequence modification(s) of the antibodies described herein are contemplated. For example, it may be desirable to improve the binding affinity and/or other biological properties of the antibody. For example, amino acid sequence variants of an antibody may be prepared by introducing appropriate nucleotide changes into a polynucleotide that encodes the antibody, or a chain thereof, or by peptide synthesis. Such modifications include, for example, deletions from, and/or insertions into and/or substitutions of, residues within the amino acid sequences of the antibody. Any combination of deletion, insertion, and substitution may be made to arrive at the final antibody, provided that the final construct possesses the desired characteristics (e.g., high affinity binding to PD-1). The amino acid changes may alter post-translational processes of the antibody, such as changing the number or position of glycosylation sites. Any of the variations and modifications described above for polypeptides of the present invention may be included in antibodies of the present invention.
[0190] In certain embodiments, the antibodies include one or more amino acid variations, e.g., substitutions, deletions, insertions, and/or mutations, compared to the sequence of an antibody described herein. Exemplary variants include those designed to improve the binding affinity and/or other biological properties of the antibody. Amino acid sequence variants of an antibody may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions from, and/or insertions into and/or substitutions of residues within the amino acid sequences of the antibody. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g., antigen-binding.
[0191] In certain embodiments, the antibodies include one or more amino acid substitutions, e.g., as compared to an antibody sequence described herein and/or compared to a sequence of a natural repertoire, e.g., human repertoire. Sites of interest for substitutional mutagenesis include the CDRs and FRs. Amino acid substitutions may be introduced into an antibody of interest and the products screened for a desired activity, e.g., retained/improved antigen binding, decreased immunogenicity, improved half-life, and/or improved effector function, such as the ability to promote antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC).
[0192] In some embodiments, one or more residues within a CDR of a parent antibody (e.g. a humanized or human antibody) is/are substituted. In some embodiments, the substitution is made to revert a sequence or position in the sequence to a germline sequence, such as an antibody sequence found in the germline (e.g., human germline), for example, to reduce the likelihood of immunogenicity, e.g., upon administration to a human subject.
[0193] In some embodiments, alterations are made in CDR “hotspots,” residues encoded by codons that undergo mutation at high frequency during the somatic maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and/or residues that contact antigen, with the resulting variant VH or VL being tested for binding affinity. Affinity maturation by constructing and reselecting from secondary libraries has been described, e.g., in Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (O’Brien et al., ed., Human Press, Totowa, NJ, (2001)). In some embodiments of affinity maturation, diversity is introduced into the variable genes chosen for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide - directed mutagenesis). A secondary library is then created. The library is then screened to identify any antibody variants with the desired affinity. Another method to introduce diversity involves CDR- directed approaches, in which several CDR residues (e.g., 4-6 residues at a time) are randomized. CDR residues involved in antigen binding may be specifically identified, e.g., using alanine scanning mutagenesis or modeling. CDR-H3 and CDR-L3 in particular are often targeted.
[0194] In certain embodiments, substitutions, insertions, or deletions may occur within one or more CDRs so long as such alterations do not substantially reduce the ability of the antibody to bind antigen. For example, conservative alterations (e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity may be made in CDRs. Such alterations may, for example, be outside of antigen contacting residues in the CDRs. In certain embodiments of the variant VH and VL sequences provided above, each CDR either is unaltered, or contains no more than one, two or three amino acid substitutions.
[0195] Amino acid sequence insertions include amino- and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include an antibody with an N-terminal methionyl residue. Other insertional variants of the antibody molecule include the fusion to the N- or C-terminus of the antibody to an enzyme or a polypeptide which increases the serum half-life of the antibody.
[0196] In certain embodiments, the antibody is altered to increase or decrease the extent to which the antibody is glycosylated, for example, by removing or inserting one or more glycosylation sites by altering the amino acid sequence and/or by modifying the oligosaccharide(s) attached to the glycosylation sites, e.g., using certain cell lines.
[0197] In some embodiments, an N-linked glycosylation, which is a glycosylation site that occurs at asparagines in the consensus sequence -Asn-Xaa-Ser/Thr is removed or inserted. In some embodiments, one or more re replaced with another amino acid to remove the glycosylation site.
[0198] Exemplary modifications, variants, and cell lines are described, e.g., in Patent Publication Nos. US 2003/0157108, US 2004/0093621, US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614; US 2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US 2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO 2005/035586; WO 2005/035778; W02005/053742; W02002/031140; Okazaki et al. J. Mol. Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004). Ripka et al. Arch. Biochem. Biophys. 249:533- 545 (1986); US Pat Appl No US 2003/0157108 Al, Presta, L; and WO 2004/056312 Al, Yamane- Ohnuki et al. Biotech. Bioeng. 87: 614 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4): 680-688 (2006); and W02003/085107); W02003/011878 (Jean-Mairet et al.); US Patent No. 6,602,684 (Umana et al.); and US 2005/0123546 (Umana et al.); WO 1997/30087 (Patel et al.); WO 1998/58964 (Raju, S.); and WO 1999/22764 (Raju, S.).
[0199] Among the modified antibodies are those having one or more amino acid modifications in the Fc region, such as those having a human Fc region sequence or other portion of a constant region (e.g., a human IgGl, IgG2, IgG3 or IgG4 Fc region) comprising an amino acid modification (e.g. a substitution) at one or more amino acid positions.
[0200] Such modifications can be made, e.g., to improve half-life, alter binding to one or more types of Fc receptors, and/or alter effector functions.
[0201] Also among the variants are cysteine engineered antibodies such as “thioMAbs” and other cysteine engineered variants, in which one or more residues of an antibody are substituted with cysteine residues, in order to generate reactive thiol groups at accessible sites, e.g., for use in conjugation of agents and linker-agents, to produce immunoconjugates. Cysteine engineered antibodies are described, e.g., in U.S. Patent Nos. 7,855,275 and 7,521,541.
[0202] In some embodiments, the antibodies are modified to contain additional nonproteinaceous moieties, including water soluble polymers. Exemplary polymers include, but are not limited to, polyethylene glycol (PEG), copolymers of ethylene glycol/propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1, 3-dioxolane, poly-1, 3, 6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids (either homopolymers or random copolymers), and dextran or poly(n-vinyl pyrrolidone)polyethylene glycol, propropylene glycol homopolymers, prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in water. The polymer may be of any molecular weight, and may be branched or unbranched. The number of polymers attached to the antibody may vary, and if more than one polymer is attached, they can be the same or different molecules. In general, the number and/or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the particular properties or functions of the antibody to be improved, whether the antibody derivative will be used in a therapy under defined conditions, etc.
[0203] Antibodies, and antigen-binding fragments and variants thereof, of the present invention may also be modified to include a detectable label, e.g., an epitope tag or label, e.g., for use in purification or diagnostic applications. These may be conjugated to the antibody as a fusion protein or conjugate, e.g., using a linker or linking group. There are many linking groups known in the art for making antibody conjugates, including, for example, those disclosed in U.S. Pat. No. 5,208,020 or EP Patent 0 425 235 Bl, and Chari et al., Cancer Research 52: 127-131 (1992). Linking groups include disulfide groups, thioester groups, acid labile groups, photolabile groups, peptidase labile groups, or esterase labile groups, as disclosed in the above-identified patents. Examples of tags and/or labels can include, but are not limited to, FLAG tags, poly-histidine tags (e.g. 6xHis), cMyc tags, glutathione-S- transferase tags, avidin, fluorescent labels, polymer particles, metal particles, haptens, enzyme labels, luminescent labels, electrochemiluminescent labels, bioluminescent labels, radioisotopes, or oligonucleotides .
D. Conjugates
[0204] Provided herein are conjugates containing an anti-PD-1 antibody or antigen-binding fragment provided herein and one or more further moiety. The further moiety can be an effector moiety, which provides at least one additional function or property of the conjugate as compared to the unconjugated antibody. In some embodiments, an effector moiety can be any substance having biological or detectable activity, for example, therapeutic agents, detectable labels, binding agents, or prodrugs, which are metabolized to an active agent in vivo. In some embodiments, the moiety can be a targeting moiety, a small molecule drug (non-polypeptide drug of less than 500 Daltons molar mass), a toxin, a cytostatic agent, a cytotoxic agent, an immunosuppressive agent, a radioactive agent suitable for diagnostic purposes, a radioactive metal ion for therapeutic purposes, a prodrug-activating enzyme, an agent that increases biological half-life, or a diagnostic or detectable agent. In some embodiments, the effector moiety is a protein, peptide or a nucleic acid molecule, which can be synthesized or produced by recombinant means. In some embodiments, the effector moiety is a drug moiety, which may be synthesized artificially or purified from a natural source.
[0205] In some embodiments, the drug moiety has an intracellular activity. In some embodiments, the anti-PD-1 antibody or antigen-binding fragment of the conjugate is internalized and the drug moiety is a cytotoxin that blocks the protein synthesis of the cell, therein leading to cell death. The conjugate can be used for inhibiting the multiplication of a tumor cell or cancer cell, causing apoptosis in a tumor or cancer cell, or for treating cancer in a patient. The conjugate can be used accordingly in a variety of settings for the treatment of animal cancers. The conjugate can be used to deliver a drug to a tumor cell or cancer cell. In some embodiments, upon binding to PD-1 on a tumor cell, the conjugate and/or drug can be taken up inside a tumor cell or cancer cell through receptor-mediated endocytosis.
[0206] In some embodiments, the conjugate is an antibody drug conjugate (ADC, also called immunoconjugates) containing an anti-PD-1 antibody or antigen-binding fragment provided herein conjugated to a drug moiety that acts as a therapeutic agent, which is either cytotoxic, cytostatic or otherwise provides some therapeutic benefit. In some embodiments, the cytotoxic agent is a chemotherapeutic agent, a drug, a growth inhibitory agent, a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate). In some embodiments, provided antibody drug conjugates of the present disclosure allow targeted-delivery of the drug moiety to tumors. In some cases, this can result in targeted killing of the tumor cell. [0207] In some embodiments, the drug moiety is an auristatin, such as auristatin E (also known in the art as a derivative of dolastatin-10) or a derivative thereof. The auristatin can be, for example, an ester formed between auristatin E and a keto acid. For example, auristatin E can be reacted with paraacetyl benzoic acid or benzoylvaleric acid to produce AEB and AEVB, respectively. Other typical auristatins include AFP, MMAF, and MMAE. The synthesis and structure of exemplary auristatins are described in U.S. Pat. Nos. 6,884,869, 7,098,308, 7,256,257, 7,423,116, 7,498,298 and 7,745,394, each of which is incorporated by reference herein in its entirety and for all purposes.
[0208] Auristatins have been shown to interfere with microtubule dynamics and nuclear and cellular division and have anticancer activity. Auristatins of the present invention bind tubulin and can exert a cytotoxic or cytostatic effect on a 5T4 expressing cell or cell line. There are a number of different assays, known in the art, that can be used for determining whether an auristatin or resultant antibodydrug conjugate exerts a cytostatic or cytotoxic effect on a desired cell or cell line. Methods for determining whether a compound binds tubulin are known in the art. See, for example, Muller et al., Anal. Chem 2006, 78, 4390-4397; Hamel et al., Molecular Pharmacology, 1995 47: 965-976; and Hamel et al., The Journal of Biological Chemistry, 1990 265:28, 17141-17149.
[0209] Examples of drugs or pay loads are selected from the group consisting of DM1 (may tansine, N2'-deacetyl-N2'-(3-mercapto-l -oxopropyl)- or N2'-deacetyl-N2'-(3-mercapto-l-oxopropyl)- maytansine), mc-MMAD (6-maleimidocaproyl-monomethylauristatin-D or N-methyl-L-valyl-N- [(lS,2R)-2-methoxy-4-[(2S)-2-[(lR,2R)-l-methoxy-2-methyl-3-oxo-3-[[(lS)-2-phenyl-l-(2- th iazol y l)cthy 11 amino] propyl] - 1 -pyrrolidinyl] - 1 - [( 1 S)- 1 -methylpropyl] -4-oxobutyl] -N-methyl-(9Cl)- L-valinamide), mc-MMAF (maleimidocaproyl-monomethylauristatin F or N-[6-(2,5-dihydro-2,5- dioxo- IH-pyrrol- 1 -yl) - 1 -oxohexyl] -N-methyl-L-valyl-L-valyl-(3R,4S,5S)-3-methoxy-5-methyl-4- (methylamino)heptanoyl-(aR,pR,2S)-P-methoxy-a-methyl-2-pyrrolidinepropanoyl-L-phenylalanine) and mc-Val-Cit-PABA-MMAE (6-maleimidocaproyl-ValcCit-(p-aminobenzyloxycarbonyl)- monomethylauristatin E or N-[[[4-[[N-[6-(2,5-dihydro-2,5-dioxo-lH-pyrrol-l-yl)-l-oxohexyl]-L- valyl-N5 -(aminocarbonyl) -L-ornithyl] amino] phenyl] methoxy] carbonyl] -N -methyl-L- valyl-N - [( 1 S,2R)-4- [ (2S ) -2- [( 1 R,2R)-3- [ [( 1 R,2S)-2-hydroxy- 1 -methyl-2-phenylethyl] amino] - 1 -methoxy-2- methyl-3 -oxopropyl] - 1 -pyrrolidinyl] -2-methoxy- 1 - [( 1 S) - 1 -methylpropyl] -4-oxobutyl] -N -methyl-L- valinamide). DM1 is a derivative of the tubulin inhibitor maytansine while MMAD, MMAE, and MMAF are auristatin derivatives. The preferred payloads of the present invention are selected from the group consisting of mc-MMAF and mc-Val-Cit-PABA-MMAE.
[0210] In a further embodiment, the drug moiety is an anti-cancer agent. Exemplary anti-cancer agents include, but are not limited to, cytostatics, enzyme inhibitors, gene regulators, cytotoxic nucleosides, tubulin binding agents or tubulin inhibitors, proteasome inhibitors, hormones and hormone antagonists, anti-angiogenesis agents, and the like. Exemplary cytostatic anti-cancer agents include alkylating agents such as the anthracycline family of drugs (e.g. adriamycin, carminomycin, cyclosporin-A, chloroquine, methopterin, mithramycin, porfiromycin, streptonigrin, porfiromycin, anthracenediones, and aziridines). Other cytostatic anti-cancer agents include DNA synthesis inhibitors (e.g., methotrexate and dichloromethotrexate, 3-amino-l,2,4-benzotriazine 1,4-dioxide, aminopterin, cytosine P-D-arabinofuranoside, 5-fluoro-5'-deoxyuridine, 5-fluorouracil, ganciclovir, hydroxyurea, actinomycin-D, and mitomycin C), DNA-intercalators or cross-linkers (e.g., bleomycin, carboplatin, carmustine, chlorambucil, cyclophosphamide, cis-diammineplatinum(II) dichloride (cisplatin), melphalan, mitoxantrone, and oxaliplatin), and DNA-RNA transcription regulators (e.g., actinomycin D, daunorubicin, doxorubicin, homoharringtonine, and idarubicin). Other exemplary cytostatic agents that are compatible with the present disclosure include ansamycin benzoquinones, quinonoid derivatives (e.g. quinolones, genistein, bactacyclin), busulfan, ifosfamide, mechlorethamine, triaziquone, diaziquone, carbazilquinone, indoloquinone EO9, diaziridinyl-benzoquinone methyl DZQ, triethylenephosphor amide, and nitrosourea compounds (e.g. carmustine, lomustine, semustine).
[0211] In some embodiments, there is provided a conjugate comprising anti-PD-1 antibody or antigen-binding fragment provided herein conjugated with a toxin. In some embodiments, the therapeutic agent is a cytotoxin comprising a polypeptide having ribosome-inactivating activity including, for example, gelonin, bouganin, saporin, ricin, ricin A chain, bryodin, diphtheria toxin, restrictocin, Pseudomonas exotoxin A and variants thereof.
[0212] In some embodiments, the effector moiety is a radionuclide (e.g., a peptide receptor radionuclide) or a radiolabel. In some embodiments, the effector moiety is a radionuclide or radiolabel with high-energy ionizing radiation that are capable of causing multiple strand breaks in nuclear DNA, leading to cell death. Exemplary high-energy radionuclides include: 90Y, 1251, 1311, 1231, U lin, 105Rh, 153Sm, 67Cu, 67Ga, 166Ho, 177Lu, 186Re and 188Re. These isotopes typically produce high energy a- or P-particles which have a short path length. Such radionuclides kill cells to which they are in close proximity, for example neoplastic cells to which the conjugate has attached or has entered. They have little or no effect on non-localized cells and are essentially non-immunogenic. Alternatively, high-energy isotopes may be generated by thermal irradiation of an otherwise stable isotope, for example as in boron neutron-capture therapy (Guan et al., PNAS, 95: 13206-10, 1998).
[0213] In some embodiments, there is provided a conjugate comprising an anti-PD-1 antibody or antigen-binding fragment provided herein conjugated with a label, which can generate a detectable signal, indirectly or directly. These conjugates can be used for research or diagnostic applications, such as for the in vivo detection of cancer. The label is preferably capable of producing, either directly or indirectly, a detectable signal. For example, the label may be radio-opaque or a radioisotope, such as 3H, 14C, 32P, 35S, 1231, 1251, 1311; a fluorescent (fluorophore) or chemiluminescent (chromophore) compound, such as fluorescein isothiocyanate, rhodamine or luciferin; an enzyme, such as alkaline phosphatase, P-galactosidase or horseradish peroxidase; an imaging agent; or a metal ion. In some embodiments, the label is a radioactive atom for scintigraphic studies, for example 99Tc or 1231, or a spin label for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, MRI), such as zirconium-89, iodine-123, iodine-131, indium-i l l, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron. Zirconium-89 may be complexed to various metal chelating agents and conjugated to antibodies, e.g., for PET imaging (WO 2011/056983).
[0214] The conjugates may be prepared using any methods known in the art. See, e.g., WO 2009/067800, WO 2011/133886, and U.S. Patent Application Publication No. 2014322129, incorporated by reference herein in their entirety.
[0215] In some embodiments, the linkage of an antibody or antigen binding fragment to a moiety, such as a drug or toxin or other heterologous moiety, is a direct or indirect linkage. In some embodiments, the attachment can be covalent or non-covalent, e.g., via a biotin-streptavidin non- covalent interaction. For example, a moiety can be attached by alkylation (e.g., at the epsilon-amino group lysines or the N-terminus of antibodies), reductive amination of oxidized carbohydrate, transesterification between hydroxyl and carboxyl groups, amidation at amino groups or carboxyl groups, and conjugation to thiols. In some embodiments, the attachment of a moiety, such as a therapeutic agent, can be by chemical conjugation and linkage methods known in the art. In some embodiments, the moiety can be linked to an antibody by a linker. In some embodiments, linkers such as peptide linkers, cleavable linkers, non-cleavable linkers or linkers that aid in the conjugation reaction, can be used to link or conjugate the effector moieties to the antibody or antigen-binding fragment. Attachment of a linker to an antibody can be accomplished in a variety of ways, such as through surface lysines, reductive-coupling to oxidized carbohydrates, and through cysteine residues liberated by reducing interchain disulfide linkages. A variety of antibody drug conjugate linkage systems are known in the art, including hydrazone-, disulfide- and peptide-based linkages.
[0216] In some embodiments, an anti-PD-1 antibody or antigen-binding fragment provided herein is conjugated to one or more moieties, e.g. about 1 to about 20 drug moieties, through a linker (L). In some embodiments, the conjugate comprises the following components: (antibody or antigen-binding fragment), (L)q and (moiety)m, wherein the antibody or antigen-binding fragment is any of the described capable of specifically binding PD-1 as described; L is a linker for linking the protein or polypeptide to the moiety; m is at least 1; q is 0 or more; and the resulting conjugate binds to PD-1. In particular embodiments, m is 1 to 4 and q is 0 to 8.
[0217] The linker may be composed of one or more linker components. For covalent attachment of the antibody and the drug moiety the linker typically has two reactive functional groups, i.e. bivalency in a reactive sense. Bivalent linker reagents which are useful to attach two or more functional or biologically active moieties, such as peptides, nucleic acids, drugs, toxins, antibodies, haptens, and reporter groups are known, and methods have been described their resulting conjugates (Hermanson, G. T. (1996) Bioconjugate Techniques; Academic Press: New York, p 234-242). [0218] In some embodiments, the linker may comprise amino acid residues. Exemplary amino acid linker components include a dipeptide, a tripeptide, a tetrapeptide or a pentapeptide. Exemplary dipeptides include: valine-citrulline (vc or val-cit), alanine-phenylalanine (af or ala-phe). Exemplary tripeptides include: glycine-valine-citrulline (gly-val-cit) and glycine-glycine-glycine (gly-gly-gly). Amino acid residues which comprise an amino acid linker component include those occurring naturally, as well as minor amino acids and non-naturally occurring amino acid analogs, such as citrulline. Amino acid linker components can be designed and optimized in their selectivity for enzymatic cleavage by a particular enzymes, for example, a tumor-associated protease, cathepsin B, C and D, at a plasmin protease.
[0219] Exemplary linker components include 6-maleimidocaproyl (“MC”), maleimidopropanoyl (“MP”), valine-citrulline (“val-cit”), a alanine-phenylalanine (“ala-phe”), p-aminobenzyloxycarbonyl (“PAB”), N-Succinimidyl 4-(2-pyridylthio)pentanoate (“SPP”), N-Succinimidyl 4-(N- maleimidomethyl)cyclohexane-I carboxylate (“SMCC”), and N-Succinimidyl (4-iodo- acetyl) aminobenzoate (“SIAB”).
[0220] Conjugates of an anti-PD-1 antibody or antigen-binding fragment provided herein and cytotoxic agent can be made using a variety of bifunctional protein-coupling agents such as N- succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HC1), active esters (such as disuccinimidyl substrate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5- difluoro-2,4-dinitrobenzene).
[0221] The antibody drug conjugate can be prepared by a variety of methods, such as organic chemistry reactions, conditions, and reagents known to those skilled in the art. Alternatively, a fusion protein containing an antibody or antigen-binding fragment and cytotoxic agent may be made, e.g., by recombinant techniques or peptide synthesis. The length of DNA may comprise respective regions encoding the two portions of the conjugate either adjacent one another or separated by a region encoding a linker peptide which does not destroy the desired properties of the conjugate
E. Multispecific Antibodies
[0222] Provided herein are PD-1 -binding polypeptides that are multispecific containing at least one antibody or antigen-binding fragment that binds PD-1 and one or more additional binding domains. Typically, the one or more additional domains bind to a second antigen or protein other than PD-1. In some aspects, the further antigen or protein may be an antigen expressed on a tumor, a molecule or receptor expressed on an immune cell, such as a T cell, e.g. a CD3, or an additional inhibitory receptor (e.g. CTLA-4, LAG3, TIM3, VISTA, TIGIT, SIRPa, NKG2A, B7H3, B7H4) or an activating receptor (e.g. 0X40, GITR, 41BB, CD40, CD27, CD28 or ICOS) or to confer an additional specificity to a target cell (e.g. CD8 or CD4). In some embodiments, the one or more additional domain is an antibody or antigen-binding fragment specific for the second antigen or protein.
[0223] In some embodiments, a provided binding molecule is a bispecific T cell engager that is composed of a PD-1 antibody or antigen-binding fragment as described herein and at least one additional binding molecule capable of binding to a surface molecule expressed on a T cell. In some embodiments, the surface molecule is an activating component of a T cell, such as a component of the T cell receptor complex. In particular aspects, the surface molecule is an activating T cell antigen that is expressed on a T cell and is capable of inducing T cell activation upon interaction with an antigen binding molecule. For example, in some aspects, interaction of an antigen binding molecule with an activating T cell antigen may induce T cell activation by triggering the signaling cascade of the T cell receptor complex. Suitable assays to measure T cell activation are known, and include any assay to measure or assess proliferation, differentiation, cytokine secretion, cytotoxic activity and/or expression of one or more activation marker. In some embodiments, the simultaneous or near simultaneous binding of such a PD-1 -binding polypeptide to both of its targets, PD-1 expressed on target cell and a T cell molecule expressed on a T cell, e.g. activating T cell antigen, can result in a temporary interaction between the target cell and T cell, thereby resulting in activation, e.g. cytotoxic activity, of the T cell and subsequent lysis of the target cell.
[0224] In some embodiments, the T surface molecule, such as activating T cell antigen, is CD3 or is CD2. Specifically, a provided bispecific PD-1 -binding polypeptide is capable of specifically binding an activating T cell antigen expressed on a human T cell, such as human CD3 or human CD3. In particular aspects, the additional binding domain that is specific to the activating T cell antigen (e.g. CD3 or CD2) is an antibody or antigen-binding fragment.
[0225] Among bispecific antibody T cell-engagers are bispecific T cell engager (BiTE) molecules, which contain tandem scFv molecules fused by a flexible linker (see e.g. Nagorsen and Bauerle, Exp Cell Res 317, 1255-1260 (2011); tandem scFv molecules fused to each other via, e.g. a flexible linker, and that further contain an Fc domain composed of a first and a second subunit capable of stable association (WO2013026837); diabodies and derivatives thereof, including tandem diabodies (Holliger et al, Prot Eng 9, 299-305 (1996); Kipriyanov et al, J Mol Biol 293, 41-66 (1999)); dual affinity retargeting (DART) molecules that can include the diabody format with a C-terminal disulfide bridge; or triomabs that include whole hybrid mouse/rat IgG molecules (Seimetz et al, Cancer Treat Rev 36, 458-467 (2010). Similar formats of any of the above molecules can be generated using any of the anti- PD-1 antibodies or antigen binding fragments provided herein.
[0226] In some embodiments, the additional binding domain specific to an activating T cell antigen is an antigen-binding fragment selected from a Fab fragment, a F(ab')2 fragment, an Fv fragment, a scFv, disulfide stabilized Fv fragment (dsFv), a scAb, a dAb, a single domain heavy chain antibody (VHH), or a single domain light chain antibody. In some embodiments, the additional binding domain is monovalent for binding the activating T cell antigen, such as CD2 or CD3.
[0227] In some embodiments, the additional binding domain is capable of binding to CD3 or a CD3 complex. A CD3 complex is a complex of at least five membrane-bound polypeptides in mature T-lymphocytes that are non-covalently associated with one another and with the T-cell receptor. The CD3 complex includes the gamma, delta, epsilon, zeta, and eta chains (also referred to as subunits). In some embodiments, the additional binding molecule is an antibody or antigen-binding fragment capable of specifically binding to CD3 or a CD3 complex, also called a CD3-binding domain. In some embodiments, the CD3-binding domain capable of binding CD3 or a CD3 complex includes one or more copies of an anti-CD3 Fab fragment, an anti-CD3 F(ab')2 fragment, an anti-CD3 Fv fragment, an anti-CD3 scFv, an anti-CD3 dsFv, an anti-CD3 scAb, an anti-CD3 dAb, an anti-CD3 single domain heavy chain antibody (VHH), and an anti-CD3 single domain light chain antibody. In some embodiments, the anti-CD3 binding domain is monovalent for binding CD3.
[0228] In some cases, the CD3-binding domain recognizes the CD3s-chain. In some embodiments, the anti-CD3s binding domain includes one or more copies of an anti-CD3s Fab fragment, an anti-CD3s F(ab')2 fragment, an anti-CD3s Fv fragment, an anti-CD3s scFv, an anti-CD3s dsFv, an anti-CD3s scAb, an anti-CD3s dAb, an anti-CD3s single domain heavy chain antibody (VHH), and an anti-CD3s single domain light chain antibody. In some embodiments, the anti-CD3s binding domain is monovalent for binding CD3s.
[0229] Exemplary monoclonal antibodies against CD3 or a CD3 complex include, but are not limited to, OKT3, SP34, UCHT1 or 64.1, or an antigen-binding fragment thereof (See e.g., June, et al., J. Immunol. 136:3945-3952 (1986); Yang, et al., J. Immunol. 137:1097-1100 (1986); and Hayward, et al., Immunol. 64:87-92 (1988)). In some aspects, clustering of CD3 on T cells, e.g., by immobilized or cell-localized or tethered anti-CD3-antibodies, leads to T cell activation similar to the engagement of the T cell receptor but independent from its clone typical specificity. In one embodiment, the CD3- binding domain monovalently and specifically binds a CD3 antigen, and is derived from OKT3 (ORTHOCLONE-OKT3™ (muromonab-CD3); humanized OKT3 (U.S. Pat. No. 7,635,475 and published international application No. W02005040220); SP34 (Pessano et al. The EMBO Journal. 4: 337-344, 1985); humanized variant of SP34 (WO2015001085); Teplizumab™ (MGA031, Eli Lilly); an anti-CD3 binding molecule described in US2011/0275787; UCHT1 (Pollard et al. 1987 J Histochem Cytochem. 35(11): 1329-38; W02000041474); NI0401 (W02007/033230); visilizumab (U.S. Pat. No. 5,834,597); BC-3 (Anasetti et al., Transplantation 54: 844 (1992); H2C (described in PCT publication no. W02008/119567); V9 (described in Rodrigues et al., Int J Cancer Suppl 7, 45-50 (1992) and U.S. Pat. No. 6,054,297)). Other anti-CD3 antibodies also can be used in the constructs provided herein, including any described in International published PCT application Nos. WO199404679, WO2008119567, WO2015095392, WO2016204966, WO2019133761; published patent application Nos. US20170369563, US20180194842, US20180355038; U.S. Pat. Nos. 7,728,114, 7,381,803,
7,994,289.
[0230] In some embodiments, the PD-1 -binding polypeptide is a bispecific construct that is or comprises at least one anti-PD-1 antibody or antigen binding fragment provided herein and at least one additional binding molecule capable of binding to a surface molecule expressed on a Natural Killer (NK) cells and/or recruiting NK cells. In particular aspects, the multispecific antibody is bispecific for PD-1 and the NK cell surface molecule. In some embodiments, the surface molecule is CD16 (FcyRIII).
[0231] CD 16, a low affinity receptor for the Fc portion of some IgGs known to be involved in antibody-dependent cellular cytotoxicity (ADCC), is the best-characterized membrane receptor responsible for triggering of target cell lysis by NK cells (Mandelboim et al., 1999, PNAS 96:5640- 5644). Generally, a large majority (approximately 90%) of human NK cells express CD56 at low density (CD56dim) and FcyRIII (CD16) at a high level (Cooper et al., 2001, Trends Immunol. 22:633-640). Human FcyRIII exists as two isoforms, CD16a (FcyRIIIA) and CD16b (FcyRIIIB), that share 96% sequence identity in their extracellular immunoglobulin-binding regions (van de Winkel and Capel, 1993, Immunol. Today 14(5):215-221). In particular embodiments, the additional binding molecule is capable of specifically binding CD16a. On NK cells, the alpha chain of CD16a associates with the immunoreceptor tyrosine-based activation motif (ITAM) containing FceRI y-chain and/or the T-cell receptor (TCR)/CD3^-chain to mediate signaling (Wirthmueller et al., 1992, J. Exp. Med. 175:1381- 1390). Engagement of CD16a can result in activating of NK cells expressing CD16a, thereby eliciting a biological response to trigger cell killing of target cells in a manner analogous to antibody-dependent cellular cytotoxicity (ADCC). For example, a binding molecule that specifically binds PD-1 expressed on a tumor cell may target NK-cells to the tumor cell. In some cases, activation of the NK cell caused by the binding molecule binding to CD 16a can lead to killing of the tumor cells.
[0232] In some embodiments, the additional binding domain specific to an activating NK cell receptor, such as CD 16a, is an antigen-binding fragment selected from a Fab fragment, a F(ab')2 fragment, an Fv fragment, a scFv, disulfide stabilized Fv fragment (dsFv), a scAb, a dAb, a single domain heavy chain antibody (VHH), or a single domain light chain antibody. In some embodiments, the additional binding domain is monovalent for binding the activating T NK cell receptor, such as CD 16a. Antibodies and antigen-binding fragments thereof specific for CD 16a are known and include, for example, NM3E2 (McCall et al. (1999) Mol. Immunol., 36:433-045. Other anti-CD16a antibodies also can be used in the constructs provided herein, including any described in published U.S. patent application Ser. No. 10/160,280,795; U.S. Pat. No. 9,701,750; Behar et al. (2008) Protein Eng Des Sei. 21:1-10; Arndt et al., (1999) Blood 94:2562-2568. In particular examples, the anti-CD16a is an anti- CD16a scFv. In some embodiments, the anti-CD16a is an anti-CD16a antibody included in a TandAb molecule (see e.g. Reush et al. (2014) Mabs, 6:727-738). In some aspects, the anti-CD16a is an anti- CD16a or antigen binding fragment, such as an scFv, described in U.S. Pat. No. 9,035,026. Single domain antibodies, including VHH domains that bind to CD16a are known, see e.g. published U.S. patent application No. US20160280795.
F. Engineered Cells and Chimeric Antigen Receptor
[0233] In some embodiments, provided herein is a chimeric antigen receptor (CAR) that contains an extracellular antigen-binding domain that includes an any of the provided anti-PD-1 antibodies or antigen binding fragments, a transmembrane domain and an intracellular signaling region composed of one or more signaling domains. In particular embodiments, the extracellular antigen binding domain is an scFv comprising a variable heavy chain and a variable light chain of any of the provided antibodies. Also provided herein are engineered cells in which the CAR is expressed on the surface of a cell.
[0234] In other embodiments, provided herein are engineered cells that express any of the provided antibodies or antigen-binding fragments or PD-1 binding molecules described herein. In particular example, the provided antibodies or antigen binding fragments or PD-1 bind molecules thereof are secreted from the cell. In some embodiments, such an antibody or antigen binding fragment or PD-1 binding molecule comprises a signal peptide, e.g., an antibody signal peptide or other efficient signal sequence to get domains outside of cell, such as to cause the protein to be secreted by the engineered cell. Generally, the signal peptide, or a portion of the signal peptide, is cleaved from the binding molecule with secretion. The antibody or antigen binding fragment or PD-1 binding molecule can be encoded by a nucleic acid (which can be part of an expression vector). In some embodiments, the antibody or antigen binding fragment or a PD-1 binding molecule thereof is expressed and secreted by a cell (such as an immune cell, for example a primary immune cell).
[0235] In some embodiments, such provided engineered cells that are engineered to express a a antibody or antigen-binding fragment or PD-1 binding molecules described herein further contain a chimeric antigen receptor (CARs). In some embodiments, the CAR contains an extracellular domain comprising one or more antigen binding domain specific to a tumor antigen (TAA). CAR constructs include an extracellular domain containing the one or more extracellular antigen binding domain, a transmembrane domain and an intracellular signaling region. In some cases, the extracellular antigen binding domain is an scFv or a single domain antibody (VHH).
[0236] In general, the extracellular antigen binding domain which form the antigen binding unit of the CAR “binds” or is “capable of binding”, i.e. targets, a target antigen with sufficient affinity such the CAR is useful in therapy in targeting a cell or tissue expressing the target antigen. Non-limiting examples of a tumor antigen include, but are not limited to, carbonic anhydrase IX (CAIX), carcinoembryonic antigen (CEA), CD8, CD7, CD10, CD19, CD20, CD22, CD30, CD33, CLL1, CD34, CD38, CD41, CD44, CD49c, CD49f, CD56, CD66c, CD73, CD74, CD104, CD133, CD138, CD123, CD142, CD44V6, an antigen of a cytomegalovirus (CMV) infected cell (e.g., a cell surface antigen), cutaneous lymphocyte- associated antigen (CLA; a specialized glycoform of P-selectin glycoprotein ligand-1 (PSGL-1)), epithelial glycoprotein-2 (EGP- 2), epithelial glycoprotein-40 (EGP-40), epithelial cell adhesion molecule (EpCAM), receptor tyrosine-protein kinases erb-B2,3,4 (erb-B2,3,4), folate- binding protein (EBP), fetal acetylcholine receptor (AChR), folate receptor- alpha, Ganglioside G2 (GD2), Ganglioside G3 (GD3), human Epidermal Growth Factor Receptor 2 (HER2), human telomerase reverse transcriptase (hTERT), Interleukin- 13 receptor subunit alpha-2 (IL- 13Ralpha2), kappa-light chain, kinase insert domain receptor (KDR), Lewis Y (LeY), LI cell adhesion molecule (L1CAM), melanoma antigen family A, 1 (MAGE-A1), Mucin 16 (MUC16), Mucin 1 (MUC1), Mesothelin (MSLN), ERBB2, MAGE A3, p53, MARTI, GP100, Proteinase3 (PR1), Tyrosinase, Survivin, hTERT, EphA2, an NKG2D ligand, cancer-testis antigen NY-ESO-1, oncofetal antigen (h5T4), prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), R0R1, tetraspanin 8 (TSPAN8), tumor-associated glycoprotein 72 (TAG-72), vascular endothelial growth factor R2 (VEGF-R2), Wilms tumor protein (WT-1), cytokine receptor-like factor 2 (CRLF2), BCMA, GPC3, NKCS1, EGF1R, EGFR-VIII, and ERBB. In some embodiments, the CAR is the CAR present in any of a variety of known engineered cell products. In some embodiments, the tumor antigen is CD19. In some embodiments, the tumor antigen is BCMA. The CAR may include, but is not limited to a CAR engineered into cells of ABECMA®, JCARH125, CARVYKTI™ (NJ-68284528; Janssen/Legend), P-BCMA-101 (Poseida), PBCAR269A (Poseida), P-BCMA- Allol (Poseida), Allo- 715 (Pfizer/ Allogene), CT053 (Carsgen), Descartes-08 (Cartesian), PHE885 (Novartis), CTX120 (CRISPR Therapeutics); YESCARTA®, KYMRIAH®, TECARTUS®, or BREYANZI®. In some embodiments, the CAR comprises a CAR of a commercial CAR cell therapy. Non-limiting examples of a CAR in commercial cell based therapies include the CAR engineered in cells of brexucabtagene autoleucel (TECARTUS®), axicabtagene ciloleucel (YESCARTA®), idecabtagene vicleucel (ABECMA®), ciltacabtagene autoleucel (CARVYKTI™), lisocabtagene maraleucel (BREYANZI®), tisagenlecleucel (KYMRIAH®).
[0237] In provided embodiments, the transmembrane domain of a CAR is a domain that typically crosses or is capable of crossing or spanning the plasma membrane and is connected, directly or indirectly (e.g. via a spacer, such as an immunoglobulin hinge sequence) to the extracellular antigen binding domain and the endoplasmic portion containing the intracellular signaling domain. In one embodiment, the transmembrane domain of the CAR is a transmembrane region of a transmembrane protein (for example Type I transmembrane proteins), an artificial hydrophobic sequence or a combination thereof. In one embodiment, the transmembrane domain comprises the CD3zeta domain or CD28 transmembrane domain. Other transmembrane domains will be apparent to those of skill in the art and may be used in connection with embodiments of a CAR provided herein.
[0238] In provided embodiments, the intracellular signaling region of a CAR provided herein contains one or more intracellular signaling domain that transmits a signal to a T cell upon engagement of the antigen binding domain of the CAR, such as upon binding antigen. In some embodiments, the intracellular region contains an intracellular signaling domain that is or contains an IT AM signaling domain. Exemplary intracellular signaling domains include, for example, a signaling domain derived from chain of the T-cell receptor complex or any of its homologs (e.g., r| chain, FcsRIy and P chains, MB 1 (Iga) chain, B29 (Ig) chain, etc.), human CD3zeta chain, CD3 polypeptides (A, 5 and s). syk family tyrosine kinases (Syk, ZAP 70, etc.), src family tyrosine kinases (Lek, Fyn, Eyn, etc.) and other molecules involved in T-cell transduction, such as CD2, CD5, 0X40 and CD28. In particular embodiments, the intracellular signaling region contains an intracellular signaling domain derived from the human CD3 zeta chain.
[0239] In some embodiments, the intracellular signaling region of a CAR can further contain an intracellular signaling domain derived from a costimulatory molecule. In such examples, such a signaling domain may enhance CAR-T cell activity, such as via enhancement of proliferation, survival and/or development of memory cells, after antigen specific engagement, for example, compared to a CAR that only contains an ITAM containing signaling domain, e.g. CD3 zeta. In some embodiments, the co-stimulatory domain is a functional signaling domain obtained from a protein selected from: CD28, CD137 (4-IBB), CD134 (0X40), DapIO, CD27, CD2, CD5, ICAM-1, EFA-1 (CD1 la/CD18), Eck, TNFR-I, TNFR-II, Fas, CD30, CD40 or combinations thereof. In particular embodiments, the costimulatory signaling domain is derived or obtained from a human protein. In some aspects, the costimulatory signaling domain is derived or obtained from human CD28 or human CD137 (4-IBB).
[0240] In particular embodiments, the CAR further comprises a hinge or spacer region which connects the extracellular antigen binding domain and the transmembrane domain. This hinge or spacer region can be used to achieve different lengths and flexibility of the resulting CAR. Examples of the hinge or spacer region that can be used include, but are not limited to, Fc fragments of antibodies or fragments or derivatives thereof, hinge regions of antibodies, or fragments or derivatives thereof, CH2 regions of antibodies, CH3 regions of antibodies, artificial spacer sequences, for example peptide sequences, or combinations thereof. Other hinge or spacer region will be apparent to those of skill in the art and may be used. In one embodiment, the hinge is an lgG4 hinge or a CD8A hinge.
[0241] Also provided is an isolated cell or cell population that has been genetically modified to express an anti-PD-1 antibody or antigen binding fragment or PD1 -binding molecule thereof and/or CAR provided herein. In one embodiment, the cell is selected from the group consisting of a T cell, a Natural Killer (NK) cell, a cytotoxic T lymphocyte (CTE), a regulatory T cell, hematopoietic stem cells and/or pluripotent embryonic/induced stem cells. In some cases, the cell is a T cell, such as a CD4 and/or CD8 T cell. In some embodiments, the cells are autologous to the subject. For example, in some embodiments, T cells may be isolated from a patient (also called primary T cells) for engineering, e.g. transfection or transduction, with a CAR nucleic acid construct.
[0242] In an exemplary example, primary T-cells can be purified ex vivo (CD4 cells or CD8 cells or both) and stimulated with a TCR/CD28 agonists, such as anti-CD3/anti-CD28 coated beads. After a 2 or 3 day activation process, a recombinant expression vector encoding the antibody or binding molecule and/or CAR can be stably introduced into the primary T cells through standard lentiviral or retroviral transduction protocols or plasmid electroporation strategies. Cells can be monitored for secretion of an antibody or PD-1 binding molecule and/or CAR expression by, for example, flow cytometry using anti-epitope tag or antibodies that cross-react with native parental molecule.
[0243] The antibody or PD-1 binding molecule and/or CAR engineered T-cells can be assayed for appropriate function by a variety of means. In some cases, in vitro cytotoxicity, proliferation, or cytokine assays (e.g., IFN-gamma expression) can be used to assess the function of engineered T-cells. Exemplary standard endpoints are percent lysis of a tumor line, proliferation of the engineered T-cell, or IFN-gamma protein expression in culture supernatant. In some cases, the ability to stimulate activation of T cells upon stimulation of the CAR, e.g. via antigen, can be assessed, such as by monitoring expression of activation markers such as CD69, CD44, or CD62L, proliferation and/or cytokine production.
[0244] Also provided herein are methods for the prevention and/or treatment of a disease or condition in a subject, such as a cancer, that includes administering to a subject engineered cells provided herein. Generally, the subject is in need of treatment for the disease or condition, pharmaceutically active amount of a cell and/or of a pharmaceutical composition of the invention.
IL MULTISPECIFIC BINDING MOLECULES
[0245] Also provided herein are multispecific binding molecules that comprise any of the provided PD-1 binding domains and also further comprise a CTLA-4 binding domain that binds CTLA-4, e.g., human CTLA-4. In some embodiments, the binding domains are antibody fragments, such as a Fab fragment or an scFv. In some embodiments, the binding molecule is composed of at least one CTEA- 4 binding domain e.g., anti-CTEA-4 Fab) directed against CTEA-4, and at least one PD-1 binding domain (e.g. anti-PDl Fab) directed against PD-1. In any of such embodiments, the binding molecule has binding specificity for both CTEA-4 and PD-1. In some embodiments, the binding molecule is a bispecific antibody that is specific for CTLA-4 and PD-1 (also termed an anti-PD-l/anti-CTLA-4 bispecific antibody). Among provided embodiments is a bispecific antibody comprising a CTLA-4 binding domain that binds CTLA-4 and a PD-1 binding domain that binds PD-1.
[0246] In some embodiments, the PD-1 binding domain includes an antibody or an antigenbinding fragment thereof selected from the group consisting of a Fab fragment, a F(ab')2 fragment, an Fv fragment, an scFv such as a dsFv, a scAb, a dAb, a single domain heavy chain antibody, and a single domain light chain antibody. In some embodiments, the PD-1 binding domain is a Fab. In some embodiments, the PD-1 binding domain can include any of the antibodies or antigen-binding fragments as described in Section I. [0247] In some embodiments, the CTLA-4 binding domain includes an antibody or an antigen-binding fragment thereof selected from the group consisting of a Fab fragment, a F(ab')2 fragment, an Fv fragment, an scFv such as a dsFv, a scAb, a dAb, a single domain heavy chain antibody, and a single domain light chain antibody. In some embodiments, the CTLA-4 binding domain is a Fab. In some embodiments, the CTLA-4 binding domain can include any of the antibodies or antigen-binding fragments as described in Section II.B.
[0248] In some such embodiments, the binding domains (e.g., Fab) contains a Vnregion and a VL region that together form an antigen-binding site. VH is the region of the CTLA-4 binding domain or the PD-1 binding domain that comprises the three heavy chain complementarity determining regions (CDRs) and the VL region is the region of the CTLA-4 binding domain or the PD-1 binding domain that comprises the three light chain CDRs. Among provided binding molecules, the CTLA-4 binding domain and PD-1 binding domain each contain a VH region sequence that contains a CDR-H1, a CDR-H2 and a CDR-H3 as described and a VL region sequence that contains a CDR-L1, a CDR-L2 and a CDR-L3 as described. Table 1 above lists exemplary position boundaries of CDR-L1, CDR-L2, CDR-L3 and CDR-H1, CDR-H2, CDR-H3 as identified by Kabat, Chothia, AbM, and Contact schemes, respectively.
[0249] In some embodiments, the binding domains of the provided binding molecules, such as bispecific antibodies, are antibody fragments that retain the ability to specifically bind to an antigen (e.g., human CTLA-4 and human PD-1). In some embodiments, the antibody fragments are Fab composed of an entire light chain (i.e. VL region and a constant light chain (CL)) and the VH region and the first constant domain of one heavy chain (CHI).
[0250] In provided embodiments, the binding molecules further contains a heavy chain constant region including a CHI, hinge, CH2 and CH3, such as from an IgGl.
[0251] In some embodiments, the binding domains, such as the CTLA-4 binding domain and the PD-1 binding domain, as described are fused to immunoglobulin constant domain sequences. In some embodiments, the fusion is with an Ig heavy chain constant domain, comprising a Fc region, such as at least part of the hinge, CH2, and CH3 regions. In some embodiments, the binding molecule provided herein is composed of the CTLA-4 binding domain, the PD-1 binding domain, and a Fc region (hinge-CH2-CH3). In some embodiments, the CTLA-4 binding domain and the PD-1 binding domain are linked via their C-termini to the hinge region of the Fc region. In some embodiments, the Fc region acts to multimerize two heavy chain polypeptides of the binding molecule, such a via a disulfide bond formed between identical Fc polypeptides. Hence, the Fc region is a multimerization domain. Although the boundaries of the Fc region of an immunoglobulin heavy chain might vary, the human IgG heavy-chain Fc region usually includes the region containing the CH2 and CH3 domains and the hinge region, such as an amino acid residue at position Cys226, or from Pro230, to the carboxyl-terminus thereof. Exemplary Fc regions are provided in Section II.C.
[0252] In some embodiments, the CTLA-4 binding domain and/or the PD-1 binding domain are linked via their C-termini to the hinge region of the Fc region. In some embodiments, the CTEA-4 binding domain and/or the PD-1 binding domain are linked via their N-termini to the CH3 region of the Fc region.
[0253] In some of any of the embodiments herein, the antibody further comprises a light chain constant domain.
[0254] In some embodiments, the Fc region is a homodimeric Fc region that facilitates formation of a homodimer containing the same heavy chain polypeptides in the bispecific binding molecule. Among the provided binding molecules, such as bispecific antibodies, are binding molecules that include two identical copies of the CTEA-4 binding domain, two identical copies of the PD-1 binding domain, and a Fc region composed of two identical polypeptide chains linked by a disulfide bond. In some embodiments, the bispecific antibody is a 2 + 2 tetravalent bispecific antibody that exhibits bivalent binding for CTEA-4 and bivalent binding for PD-1. In some embodiments, the bispecific antibody is a 1 + 1 bivalent bispecific antibody that exhibits monovalent binding for CTLA-4 and monovalent binding for PD-1. In some of any of the provided embodiments, the binding molecule, such as a bispecific antibody, contains two identical heavy chains and two identical light chains.
[0255] In some embodiments, the Fc region is a heterodimeric Fc region that facilitates formation of a heterodimer containing two different heavy chain polypeptides in the bispecific binding molecule. In some embodiments, such binding molecules may contain different heavy chain polypeptides. In some embodiments, the binding molecule, such as a bispecific antibody, is a 1 + 1 bivalent bispecific antibody that exhibits monovalent binding for CTEA-4 and monovalent binding for PD-1. In some embodiments, the binding molecule, such as a bispecific antibody, is a 2 +1 tri valent bispecific antibody that exhibits bivalent binding for CTEA-4 and monovalent binding for PD-1. In some embodiments, the binding molecule, such as a bispecific antibody is a 2 + 1 bivalent bispecific antibody that exhibits bivalent binding for PD-1 and monovalent binding for CTLA-4.
[0256] In some embodiments, the CTLA-4 binding domain and the PD-1 binding domain each is a Fab, comprising a VH-CH1 and a VL-CE of the respective antibody fragment.
[0257] In some embodiments, the Fab has an IgGl isotype, IgG2 isotype, IgG3 isotype or IgG4 isotype, and the CHI of the CTEA-4-Fab or PD-l-Fab is the CHI of the immunoglobulin isotype. In some embodiments, the CHI is of the IgGl isotype, IgG2 isotype, or IgG4 isotype. In some embodiments, the CHI is of the IgGl isotype. In some embodiments, the CHI is from an IgGl. In some embodiments, the CHI comprises an amino acid sequence having at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 409. In some embodiments, the CHI comprises the amino acid sequence set forth in SEQ ID NO: 409.
[0258] In some embodiments, the CL is a X (lambda) light chain. In some embodiments, the light chain is a K (kappa) light chain. In some embodiments, the CL comprises an amino acid sequence having at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 410. In some embodiments, the CL comprises an amino acid sequence set forth in SEQ ID NO: 410.
[0259] In some embodiments, the multispecific antibody (e.g., a bispecific antibody described herein) includes a heterodimeric Fc region for dimerizing the heavy chains of the antibody. In some embodiments, the Fc region comprises a first and second polypeptide each comprising a hinge-CH2- CH3. In some aspects, one of the first polypeptide and second polypeptide is linked to the CHI of the CTLA-4 Fab and the other of the first polypeptide and second polypeptide is linked to the CHI of the PD-1 Fab. The first and second polypeptide of the heterodimeric Fc comprises one or more amino acid substitutions (e.g., knob-into-hole modification) in a wild-type Fc polypeptide region to effect heterodimer formation between the first polypeptide and the second polypeptide.
[0260] In some embodiments, the heavy chain further comprises a signal peptide. In some embodiments, the signal peptide can be cleaved upon expression from a cell. In some embodiments, the heavy chain signal peptide is set forth in SEQ ID NO: 176 or 361. In some embodiments, the light chain signal peptide is set forth in SEQ ID NO: 177.
[0261] In some embodiments, the binding molecule provided herein is composed of a CTEA- 4 binding domain that is a Fab fragment for specifically binding to CTEA-4 (further named also as “CTEA-4-Fab”), a PD-1 binding domain that is Fab fragment for specifically binding PD-1 (further named also as “PD-1 -Fab), and a Fc region. In some embodiments, the CTEA-4 binding domain and PD-1 binding domain are each N-terminal to the Fc region an.
[0262] In some embodiments, the binding molecule comprises the CTEA-4 binding domain that is a CTLA-4 Fab comprising the CTEA-4 VH region and a heavy chain constant region 1 (CHI), and a light chain (EC) comprising the CTLA-4 VL region and a light chain constant region (CL); a PD-1 Fab comprising the PD-1 VH region and a heavy chain constant region 1 (CHI), and a light chain (EC) comprising the PD-1 VL region and a light chain constant region (CE), and an Fc region.
[0263] In some embodiments, biological activity or functional activity of a provided bispecific antibody, can be measured using any of a number of known methods. The activity can be assessed or determined either in vitro or in vivo. In some embodiments, activity can be assessed once the bispecific antibody is administered to the subject (e.g., human). Parameters to assess include specific binding of the bispecific antibody to a T cell (e.g. natural T cell) or target antigenexpressing cell, e.g., in vivo, e.g., by imaging, or ex vivo, e.g., by ELISA or flow cytometry. In certain embodiments, the ability of the bispecific antibody to destroy target cells can be measured using any suitable known methods, such as cytotoxicity assays described in, for example, Kochenderfer et al., J. Immunotherapy, 32(7): 689-702 (2009), and Herman et al. J.
Immunological Methods, 285(1): 25-40 (2004). In some aspects the biological activity can be measured using an animal model of the disease or condition, such as a tumor xenograft model, and assessing the reduction in tumor burden or load and/or survival. In some aspects the biological activity is measured by assessing clinical outcome, such as reduction in tumor burden or load.
[0264] A binding molecule provided herein is not limited to any particular bispecific format or method of producing it. Binding molecules can be prepared as full length antibodies or antibody fragments. Among the multispecific antibodies are multispecific single-chain antibodies, e.g., diabodies, triabodies, and tetrabodies, scFv-IgG bispecific antibodies, tandem di-scFvs, and tandem tri-scFvs. In some embodiments, the binding molecule is formatted as a bivalent antibody. In some embodiments, the binding molecule is formatted as a trivalent antibody. In some embodiments, the binding molecule is formatted as a tetravalent antibody.
[0265] The at least two binding domains and the variable domains (VH/VL) of the antibody construct of the present invention may or may not comprise peptide linkers (spacer peptides). The term “peptide linker” comprises in accordance with the present invention an amino acid sequence by which the amino acid sequences of one (variable and/or binding) domain and another (variable and/or binding) domain of the antibody construct of the invention are linked with each other. The peptide linkers can also be used to fuse the third domain to the other domains of the antibody construct of the invention. An essential technical feature of such peptide linker is that it does not comprise any polymerization activity. Among the suitable peptide linkers are those described in U.S. Pat. Nos. 4,751,180 and 4,935,233 or WO 88/09344. The peptide linkers can also be used to attach other domains or modules or regions (such as half-life extending domains) to the antibody construct of the invention.
[0266] Any format or technology for generating a multispecific antibody, such as a bispecific antibody, may be used to make the binding molecules of the present invention. For example, a binding domain, such as an antibody or antigen-binding fragment thereof, having a first antigen binding specificity can be functionally linked (e.g., by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other molecular entities, such as another binding domain, such as an antibody or antibody fragment having a second antigen-binding specificity to produce a bispecific binding molecule. Specific exemplary bispecific formats that can be used in the context of the present invention include, without limitation, e.g., scFv-based or diabody bispecific formats, IgG-scFv fusions, dual variable domain (DVD)-Ig, Quadroma, knobs- into-holes, common light chain (e.g., common light chain with knobs-into-holes,etc.), CrossMab, CrossFab, (SEED) body, leucine zipper, Duobody, IgGl/IgG2, dual acting Fab (DAF)-IgG, and Mabe bispecific formats (See, for example, Spiess et al. (2015) Mol Immunol 67(2 Pt A):95-106 and Brinkmann et al. (2017) MAbs 9(2): 182-212, and the references cited therein, for a review of the foregoing formats).
[0267] Methods of producing bispecific antibodies are known, including fusion of two different hybridomas (Milstein and Cuello, Nature 1983;305:537-540), chemical tethering though heterobifunctional cross linkers (Staerz et al. Nature 1985; 314:628-631), manufacturing of scFv- based agents of multivalency and multispecificity by varying the linker length (e.g. U.S. Pat. No. 5,844,094, U.S. Pat. No. 5,837,242 and WO 98/44001), diabodies including Dual-Affinity ReTargeting (DART®) (see, e.g., Moore et al., 2011, Blood 117:4542-51; Veri et al, 2010, Arthritis Rheum 62: 1933-43), and tetravalent and bispecific antibody-like proteins known as DVD-Igs which are engineered from two monoclonal antibodies (Wu, C. et al., Nature Biotechnology, 25, p 1290- 1297, 2007). Exemplary bispecific formats are known, see e.g. Brinkmann et al. MAbs 2017, 9:182-212.
[0268] For instance, among such exemplary binding molecules are bispecific antibodies, which contain tandem scFv molecules fused by a flexible linker (see e.g. Nagorsen and Bauerle, Exp Cell Res 317, 1255-1260 (2011); tandem scFv molecules fused to each other via, e.g. a flexible linker, and that further contain an Fc domain composed of a first and a second subunit capable of stable association (WO2013026837); diabodies and derivatives thereof, including tandem diabodies (Holliger et al, Prot Eng 9, 299-305 (1996); Kipriyanov et al, J Mol Biol 293, 41-66 (1999)); dual affinity retargeting (DART) molecules that can include the diabody format with a C-terminal disulfide bridge; or triomabs that include whole hybrid mouse/rat IgG molecules (Seimetz et al, Cancer Treat Rev 36, 458-467 (2010); and trivalent bispecific antibodies (e.g. Mayer et al. 2015 Int. J Mol Sci, 16:27497-27507 and U.S. patent publication No. US20200062826).
[0269] Examples of bispecific antibody molecules formats which may be used include (i) a single antibody that has two arms comprising different antigen-binding regions; (ii) a single chain antibody that has specificity to two different epitopes, e.g., via two scFvs linked in tandem by an extra peptide linker; (iii) a dual-variable-domain antibody (DVD-Ig), where each light chain and heavy chain contains two variable domains in tandem through a short peptide linkage (Wu et al., Generation and Characterization of a Dual Variable Domain Immunoglobulin (DVD-Ig™) Molecule, In: Antibody Engineering, Springer Berlin Heidelberg (2010)); (iv) a chemically-linked bispecific (Fab')2 fragment; (v) a Tandab, which is a fusion of two single chain diabodies resulting in a tetravalent bispecific antibody that has two binding sites for each of the target antigens; (vi) a flexibody, which is a combination of scFvs with a diabody resulting in a multivalent molecule; (vii) a so-called “dock and lock” molecule, based on the “dimerization and docking domain” in Protein Kinase A, which, when applied to Fabs, can yield a trivalent bispecific binding protein consisting of two identical Fab fragments linked to a different Fab fragment; (viii) a so-called Scorpion molecule, comprising, e.g., two scFvs fused to both termini of a human Fab-arm; and (ix) a diabody.In some embodiments, at least one Fab fragment is a crossover Fab (also termed “Crossfab”), such as a CrossMab format. Various strategies for preparing multi-specific antibodies may also be provided in an asymmetric form with a domain crossover in one or more binding arms of the same antigen specificity (so-called “CrossMab” technology), i.e. by exchanging the VH/VL domains (see e.g., WO 2009/080252 and WO 2015/150447), the CHI/CL domains (see e.g., WO 2009/080253) or the complete Fab arms (see e.g., WO 2009/080251, WO 2016/016299, also see Schaefer et al, PNAS, 108 (2011) 1187-1191, and Klein at al., MAbs 8 (2016) 1010-20). Asymmetrical Fab arms can also be engineered by introducing charged or non-charged amino acid mutations into domain interfaces to direct correct Fab pairing. See e g., WO 2016/172485.
[0270] In some embodiments, provided binding molecules include at least one crossover Fab, in which the variable domains or the constant domains of the Fab heavy and light chain are exchanged/replaced by each other. In some embodiments, the variable domains of the Fab heavy and light chains of one binding molecule (e.g., PD-1 or CTLA-4) are replaced by each other. For example, a bispecific antibody as described herein comprises a polypeptide comprising a light chain variable domain VL of CTLA-4 and a heavy chain constant domain 1 (CHI) (e.g., VL-CH1) and a second polypeptide comprising a heavy chain variable domain VH of CTLA-4 and the light chain constant domain CL (e.g., VH-CL).
[0271] In contrast, a conventional Fab molecule is a Fab molecule in its natural state. A conventional Fab molecule comprises for example, a heavy chain variable and a constant domain (e.g., VH-CH1) and a light chain variable and a constant domain (e.g., VL-CL).
[0272] In some aspects, a bispecific antibody described herein comprises at least one crossover Fab. In some embodiments, the CTLA4 binding domain is a crossover Fab and the PD-1 binding domain is a conventional Fab. In some embodiments, the PD-1 binding domain is a crossover Fab and the CTLA-4 binding domain is a conventional Fab.
[0273] In some of any of such embodiments, the binding molecule, including the CTLA-4 binding domain (e.g., anti-CTLA-4 antibody fragment) and/or the PD-1 binding domain (e.g., anti- PD-1 antibody fragment), comprises at least one post-translational modification of the amino acid sequence. Post-translational modifications can include, e.g., glycosylation, phosphorylation, citrullination, isomerization, ubiquitination, acetylation, hydroxylation, methylation, AMPylation, prenylation, deamidation, eliminylation, carbamylation and carbamoylation. In some embodiments, the post-translational modification is the modification of an amino acid side chain, such as conversion of an N-terminal amino acid (e.g., glutamate or glutamine) to pyroglutamate. In some such embodiments, this modification occurs at the N-terminus of the heavy chain. In some embodiments, the post-translational modification includes a post-translational modification of a heavy chain N-terminal glutamine (Q) to a pyroglutamate. In some embodiments, in a composition of an antibody or antigen-binding fragment thereof as disclosed herein, such a post- translational modification to pyroglutamate (e.g., at the N-terminus of the heavy chain) is a dominant post-translational modification; in some such embodiments, the antibody or antigen binding fragment thereof in which an N-terminal pyroglutamate is present represents greater than 50%, greater than 60%, greater than 70%, greater than 80%, greater than 90%, or greater than 95% (w/w) of the antibody or antigen binding fragment thereof in the composition.
[0274] In some embodiments, the post-translational modification is the cleavage of one or more, particularly one or two, amino acids from the C-terminus of the heavy chain. For instance, an antibody produced by expression of a specific nucleic acid molecule encoding a full-length heavy chain (e.g., by expression in a host cell) may include the full-length heavy chain, or it may include a cleaved variant of the full-length heavy chain. This may be the case where the final two C-terminal amino acids of the heavy chain are glycine and lysine, respectively. Therefore, the C- terminal lysine, or the C-terminal glycine and lysine, of the Fc region may or may not be present. Thus, an antibody may include a heavy chain constant domain with both a C-terminal glycine and lysine, without the C-terminal lysine, or without both the C-terminal glycine and lysine. In some embodiments, in a composition of a binding molecule as disclosed herein, glycine cleavage is not a dominant post-translational modification; in some such embodiments, the binding molecule in which the C-terminal lysine is cleaved from the heavy chain constant domain represents less than 10%, less than 5%, or less than 4% (w/w) of the antibody or antigen binding fragment thereof in the composition.
[0275] Also provided are polynucleotides containing nucleic acids sequences encoding all or a portion of any of the provided binding molecules, including one or more of the polypeptide chains of the bispecific antibodies. The provided polynucleotides can be incorporated into constructs, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) constructs, such as those that can be introduced into cells for expression thereof.
[0276] In certain embodiments, a provided binding molecule contains an Fc region comprising at least part of the hinge, CH2, and CH3 regions. Certain embodiments have the first heavy-chain constant region (CHI) containing the site necessary for light chain bonding, present in at least one of the fusions. DNAs encoding the immunoglobulin heavy chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are co-transfected into a suitable host cell. This provides for greater flexibility in adjusting the mutual proportions of the three polypeptide fragments in embodiments when unequal ratios of the three polypeptide chains used in the construction provide the optimum yield of the desired fusion antibody. It is, however, possible to insert the coding sequences for two or all three polypeptide chains into a single expression vector when the expression of at least two polypeptide chains in equal ratios results in high yields or when the ratios have no significant effect on the yield of the desired chain combination.
A. PD-1 binding domain
[0277] Among provided binding molecules, such as bispecific antibodies, contain a PD-1 binding domain, including antigen-binding fragments as described in Section I. In some embodiments, the binding molecules include antibodies that specifically bind to PD-1, e.g., human PD-1. Exemplary antibodies are described in Section I. In some embodiments, the PD-1 binding domain, e.g., the anti- PD-1 antibody fragment, contains a PD-1 heavy chain variable region (VH) region sequence and a PD- 1 light chain variable region (VL) region sequence as described.
[0278] In some embodiments, the PD-1 binding domain contains a VH region and a VL region containing a combination of six CDRs as described herein, such as in Section I above. In some embodiments, the PD-1 binding domain contains a PD-1 VH region and a PD-1 VL region as described herein, such as in Section I above. In any such embodiments, a PD-1 VH region sequence can be any of the PD-1 VH region sequences described herein. In any such embodiments, a PD-1 VL region sequence can be any of the PD-1 VL region sequence described herein. In any such embodiments, any of the PD-1 VH region sequence and any of the PD-1 VL region sequence described herein can be used in combination.
[0279] PD-1 binding domains include any combination of the heavy chain and light chain complementarity-determining regions (CDRs) discussed herein. In some embodiments, the PD-1 binding domain comprises any one of the CDR-H1 as described herein, any one of the CDR-H2 as described herein, any one of the CDR-H3 as described herein, any one of the CDR-L1 as described herein, any one of the CDR-L2 as described herein and any one of the CDR-L3 as described herein. In some of any such embodiments, any one or more of the CDR-H1, the CDR-H2 and the CDR-H3 sequences described herein, and any one or more of the CDR-L1, the CDR-L2 and the CDR-L3 sequences described herein can be used in combination.
[0280] In some embodiments, the PD-1 VH region and the PD-1 VL region of the PD-1 binding domain is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 168 and SEQ ID NO: 172, respectively. In some embodiments, the PD-1 VH region is or comprises an amino acid sequence set forth in SEQ ID NO: 168, and the PD-1 VL region is or comprises an amino acid sequence set forth in SEQ ID NO: 172.
[0281] In some embodiments, the PD-1 binding domain is a Fab. In some embodiments, the Fab comprises at least a portion of an immunoglobulin constant region or a variant thereof, such as a CHI. In some embodiments, an PD-1 binding domain comprises one heavy chain or portion thereof comprising a PD-1 VH region and at least a portion of a heavy chain constant region (e.g., CHI), and at least one light chain comprising a PD-1 VL region and at least a portion of a light chain constant region.
[0282] In some embodiments, the PD-lbinding domain is a crossover Fab (also termed “Crossfab”). In some embodiments, the PD-lcrossover Fab is a Fab molecule in which the variable domains of the PD-1 Fab heavy and light chain are exchanged (i.e., replaced by each other). In some embodiments, the crossover PD-1 Fab comprises a peptide chain composed of the light chain variable domain VL and the heavy chain constant domain 1 CHI (e.g., VL-CH1, in N- to C-terminal direction), and a peptide chain composed of the heavy chain variable domain VH and the light chain constant domain CL (e.g., VH-CL, in N- to C-terminal direction).
B. CTLA-4
[0283] Among provided binding molecules, such as bispecific antibodies, contain a CTLA-4 binding domain, including antigen-binding fragments. In some embodiments, the binding molecules include antibodies that specifically bind to CTLA-4, e.g., human CTLA-4. Exemplary antibodies are described below. In some embodiments, the CTLA-4 binding domain, e.g., the anti-CTLA-4 antibody fragment contains a CTLA-4 heavy chain variable region (VH) region sequence and a CTLA-4 light chain variable region (VL) region sequence as described.
[0284] In some embodiments, the CTLA-4 binding domain includes one or more copies of an antibody or an antigen-binding fragment thereof selected from the group consisting of a Fab fragment, a F(ab')2 fragment, an Fv fragment, or an a scFv. In some embodiments, the CTLA-4 binding domain is monovalent for binding CTLA-4. In some embodiments, the CTLA-4 binding domain is a Fab.
[0285] In some embodiments, the CTLA-4 binding domain contains a VH region and a VL region containing a combination of six CDRs as described below. In some embodiments, the CTLA-4 binding domain contains a CTLA-4 VH region and a CTLA-4 VL region as described below. In any such embodiments, a CTLA-4 VH region sequence can be any of the CTLA-4 VH region sequences described herein. In any such embodiments, a CTLA-4 VL region sequence can be any of the CTLA-4 VL region sequence described herein. In any such embodiments, any of the CTLA-4 VH region sequence and any of the CTLA-4 VL region sequence described herein can be used in combination.
[0286] In some embodiments, the CTLA-4 binding domain is a Fab. In some embodiments, the Fab comprises at least a portion of an immunoglobulin constant region or a variant thereof, such as a CHI. In some embodiments, an CTLA-4 binding domain comprises one heavy chain or portion thereof comprising a CTLA-4 VH region and at least a portion of a heavy chain constant region (e.g., CHI), and at least one light chain comprising a CTLA-4 VL region and at least a portion of a light chain constant region. [0287] In some embodiments, the CTLA-4 binding domain is a crossover Fab (also termed “Crossfab”). In some embodiments, the CTLA-4 crossover Fab is a Fab molecule in which the variable domains of the CTLA-4 Fab heavy and light chain are exchanged (i.e., replaced by each other). In some embodiments, the crossover CTLA-4 Fab comprises a peptide chain composed of the light chain variable domain VL and the heavy chain constant domain 1 CHI (e.g., VL-CH1, in N- to C- terminal direction), and a peptide chain composed of the heavy chain variable domain VH and the light chain constant domain CL (e.g., VH-CL, in N- to C-terminal direction).
[0288] CTLA-4 binding domains include any combination of the heavy chain and light chain complementarity-determining regions (CDRs) discussed herein. In some embodiments, the CTLA-4 binding domain comprises any one of the CDR-H1 as described herein, any one of the CDR-H2 as described herein, any one of the CDR-H3 as described herein, any one of the CDR-L1 as described herein, any one of the CDR-L2 as described herein and any one of the CDR-L3 as described herein. In some of any such embodiments, any one or more of the CDR-H1, the CDR- H2 and the CDR-H3 sequences described herein, and any one or more of the CDR-L1, the CDR- L2 and the CDR-L3 sequences described herein can be used in combination.
[0289] Exemplary heavy and light chain CDR sequences of the CTLA-4 binding domains are provided in Tables 3A and 3B.
[0290] In some embodiments, a provided CTLA-4 binding domain has a CDR-H1, a CDR-H2 and a CDR-H3 present in a CTLA-4 VH region amino acid sequence set forth in any one of SEQ ID NOs: 230-256, or 341, or an amino acid sequence that has at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the CTLA-4 VH region amino acid sequence set forth in any one of SEQ ID NOs: 230-256, or 341. In some embodiments, a provided CTLA-4 binding domain has a CDR-L1, a CDR-L2 and a CDR-L3 present in a CTLA-4 VL region amino acid sequence set forth in any one of SEQ ID NOs: 257- 277, 347, or an amino acid sequence that has at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the CTLA-4 VL region amino acid sequence set forth in any one of SEQ ID NOs: 257-277, or 347. In some embodiments, the provided CTLA-4 binding domain contains a combination of any of such six CDRs (a CDR- Hl, a CDR-H2, a CDR-H3, a CDR-L1, a CDR-L2 and a CDR-L3) described above. In some embodiments, a provided CTLA-4 binding domain has a CDR-H1, a CDR-H2 and a CDR-H3 present in a CTLA-4 VH region amino acid sequence set forth in any one of SEQ ID NOs: 230- 256, 341, and a CDR-L1, a CDR-L2 and a CDR-L3 present in a CTLA-4 VL region amino acid sequence set forth in any one of SEQ ID NOs: 257-277, 347, wherein the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 are not set forth in SEQ ID NOs: 278-286, 297, 303, 318 and 327, respectively. In some embodiments, the combination of six CDRs (a CDR-H1, a CDR-H2, a CDR-H3, a CDR-L1, a CDR-L2 and a CDR-L3) is according to Kabat numbering. In some embodiments, the combination of six CDRs (a CDR-H1, a CDR-H2, a CDR-H3, a CDR-L1, a CDR-L2 and a CDR-L3) is according to Chothia numbering. In some embodiments, the combination of six CDRs (a CDR-H1, a CDR-H2, a CDR-H3, a CDR-L1, a CDR-L2 and a CDR- L3) is according to AbM numbering, In some embodiments, the combination of six CDRs (a CDR-H1, a CDR-H2, a CDR-H3, a CDR-L1, a CDR-L2 and a CDR-L3) is according to IMGT numbering. In some embodiments, the combination of six CDRs (a CDR-H1, a CDR-H2, a CDR- H3, a CDR-L1, a CDR-L2 and a CDR-L3) is according to IgBLAST numbering.
[0291] Exemplary heavy and light chain CDR sequences of the CTLA-4 binding domains are provided in Table E5. [0292] In some embodiments, the CTLA-4 VH region comprises a heavy chain complementarity determining region 1 (CDR-H1) comprising the sequence set forth in any one of SEQ ID NOs: 278- 285, a heavy chain complementarity determining region 2 (CDR-H2) comprising the sequence set forth in any one of SEQ ID NOs: 286-296, and a heavy chain complementarity determining region 3 (CDR-H3) comprising the sequence set forth in any one of SEQ ID NOs: 297-302; and the CTLA-4 VL region comprises a light chain complementarity determining region 1 (CDR-L1) comprising the sequence set forth in any one of SEQ ID NOs: 303-317, a light chain complementarity determining region 2 (CDR-L2) comprising the sequence set forth in any one of SEQ ID NOs: 318-326, and a light chain complementarity determining region 3 (CDR-L3) comprising the sequence set forth in any one of SEQ ID NOs: 327-329, wherein the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 are not set forth in SEQ ID NOS: 278, 286, 297,303, 318, and 327, respectively.
[0293] In some embodiments, the CTLA-4 VH region comprises a heavy chain complementarity determining region 1 (CDR-H1), a heavy chain complementarity determining region 2 (CDR-H2), and a heavy chain complementarity determining region 3 (CDR-H3) contained within any one of SEQ ID NOs: 230-256, and 341, and the CTLA-4 VL region comprises a light chain complementarity determining region 1 (CDR-L1), a light chain complementarity determining region 2 (CDR-L2), and a light chain complementarity determining region 3 (CDR-L3) contained within any one of SEQ ID NOs:257, 277, and 347. In some embodiments, the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 are not set forth in SEQ ID NOS: 278, 286, 297,303, 318, and 327, respectively.
[0294] In some embodiments, the CTLA-4 VH region comprises a CDR-H1 comprising the sequence set forth in any one of SEQ ID NOs: 278-285, a CDR-H2 comprising the sequence set forth in any one of SEQ ID NOs: 286-296, and a CDR-H3 comprising the sequence set forth in any one of SEQ ID NOs: 297-302; and the CTLA-4 VL region comprises a CDR-L1 comprising the sequence set forth in any one of SEQ ID NOs: 303-317, a CDR-L2 comprising the sequence set forth in any one of SEQ ID NOs: 318-326, and a CDR-L3 comprising the sequence set forth in any one of SEQ ID NOs: 327-329. In some embodiments, the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 are not set forth in SEQ ID NOS: 278, 286, 297,303, 318, and 327, respectively.
[0295] In some embodiments, the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 230, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 347. In some embodiments, the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 231, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 347. In some embodiments, the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 232, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 347. In some embodiments, the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO:233, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 347. In some embodiments, the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO:234, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 347. In some embodiments, the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO:235, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 347. In some embodiments, the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO:236, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 347. In some embodiments, the CTLA-4 VH region comprises a CDR- Hl, a CDR-H2, and a CDR-H3 contained within SEQ ID NO:237, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 347. In some embodiments, the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO:238, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 347. In some embodiments, the CTLA-4 VH region comprises a CDR- Hl, a CDR-H2, and a CDR-H3 contained within SEQ ID NO:239, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 347. In some embodiments, the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO:240, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 347. In some embodiments, the CTLA-4 VH region comprises a CDR- Hl, a CDR-H2, and a CDR-H3 contained within SEQ ID NO:241, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 347. In some embodiments, the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO:242, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 347. In some embodiments, the CTLA-4 VH region comprises a CDR- Hl, a CDR-H2, and a CDR-H3 contained within SEQ ID NO:243, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 347. In some embodiments, the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO:244, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 347. In some embodiments, the CTLA-4 VH region comprises a CDR- Hl, a CDR-H2, and a CDR-H3 contained within SEQ ID NO:245, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 347. In some embodiments, the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO:246, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 347. In some embodiments, the CTLA-4 VH region comprises a CDR- Hl, a CDR-H2, and a CDR-H3 contained within SEQ ID NO:247, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 347. In some embodiments, the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO:248, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 347. In some embodiments, the CTLA-4 VH region comprises a CDR- Hl, a CDR-H2, and a CDR-H3 contained within SEQ ID NO:249, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 347. In some embodiments, the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO:250, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 347. In some embodiments, the CTLA-4 VH region comprises a CDR- Hl, a CDR-H2, and a CDR-H3 contained within SEQ ID NO:251, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 347. In some embodiments, the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 341, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 257. In some embodiments, the CTLA-4 VH region comprises a CDR- Hl, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 341, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 258. In some embodiments, the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 341, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 259. In some embodiments, the CTLA-4 VH region comprises a CDR- Hl, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 341, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 260. In some embodiments, the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 341, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 261. In some embodiments, the CTLA-4 VH region comprises a CDR- Hl, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 341, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 262. In some embodiments, the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 341, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 263. In some embodiments, the CTLA-4 VH region comprises a CDR- Hl, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 341, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 264.1n some embodiments, the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 341, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 265. In some embodiments, the CTLA-4 VH region comprises a CDR- Hl, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 341, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 266. In some embodiments, the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 341, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 267. In some embodiments, the CTLA-4 VH region comprises a CDR- Hl, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 341, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 268. In some embodiments, the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 341, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 269. In some embodiments, the CTLA-4 VH region comprises a CDR- Hl, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 341, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 270. In some embodiments, the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 341, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 271. In some embodiments, the CTLA-4 VH region comprises a CDR- Hl, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 341, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 272. In some embodiments, the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 341, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 273. In some embodiments, the CTLA-4 VH region comprises a CDR- Hl, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 341, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 274. In some embodiments, the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 341, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 275. In some embodiments, the CTLA-4 VH region comprises a CDR- Hl, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 252, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 270. In some embodiments, the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 253, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 270. In some embodiments, the CTLA-4 VH region comprises a CDR- Hl, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 254, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 270. In some embodiments, the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 255, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 270. In some embodiments, the CTLA-4 VH region comprises a CDR- Hl, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 251, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 270. In some embodiments, the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 256, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 270. In some embodiments, the CTLA-4 VH region comprises a CDR- Hl, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 252, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 276. In some embodiments, the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 253, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 276. In some embodiments, the CTLA-4 VH region comprises a CDR- Hl, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 254, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 276. In some embodiments, the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 255, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 276. In some embodiments, the CTLA-4 VH region comprises a CDR- Hl, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 251, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 276. In some embodiments, the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 256, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 276. In some embodiments, the CTLA-4 VH region comprises a CDR- Hl, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 252, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 258 In some embodiments, the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 253, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 258. In some embodiments, the CTLA-4 VH region comprises a CDR- Hl, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 254, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 258. In some embodiments, the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 255, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 258. In some embodiments, the CTLA-4 VH region comprises a CDR- Hl, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 251, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 258. In some embodiments, the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 256, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 258. In some embodiments, the CTLA-4 VH region comprises a CDR- Hl, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 252, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 268. In some embodiments, the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 253, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 268. In some embodiments, the CTLA-4 VH region comprises a CDR- Hl, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 254, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 268. In some embodiments, the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 255, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 268. In some embodiments, the CTLA-4 VH region comprises a CDR- Hl, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 251, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 268. In some embodiments, the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 256, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 268. In some embodiments, the CTLA-4 VH region comprises a CDR- Hl, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 234, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 277.
[0296] In some embodiments, the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 287, and 297, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 303, 318, and 327, respectively. In some embodiments, the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 286, and 298, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 303, 318, 327, respectively. In some embodiments, the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 280, 286, and 297, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 303, 318, and 327, respectively. In some embodiments, the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 279, 288, and 297, respectively, and the -LI, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 303, 318, and 327, respectively. In some embodiments, the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 281, 289, and 297, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 303, 318, and 327, respectively. In some embodiments, the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 281, 290, and 297, respectively, and the CDR-L1, the CDR- L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 303, 318, and 327, respectively. In some embodiments, the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 280, 291, 297, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 303, 318, and 327, respectively. In some embodiments, the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 279, 292, and 297, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 303, 318, and 327, respectively. In some embodiments, the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 279, 286, and 298, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 303, 318, and 327, respectively. In some embodiments, the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 282, 286, and 297, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 303, 318, and 327, respectively. In some embodiments, the CDR-H1, the CDR- H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 282, 286, and 299, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 303, 318, and 327, respectively. In some embodiments, the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 283, 286, and 300, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 303, 318, and 327, respectively. In some embodiments, the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 284, 286, and 297, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 303, 318, and 327, respectively. In some embodiments, the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 285, 286, and 301, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 303, 318, and 327, respectively. In some embodiments, the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 293, 297, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 303, 318, and 327, respectively. In some embodiments, the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 289, and 297, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 303, 318, and 327, respectively. In some embodiments, the CDR-H1, the CDR- H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 294, 297, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 303, 318, and 327, respectively. In some embodiments, the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 295, and 297, respectively, and the CDR-L1, a CDR-L2, and a CDR-L3 comprise the sequences set forth in SEQ ID NOS: 303, 318, and 327, respectively. In some embodiments, the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 286, and 300, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 303, 318, and 327, respectively. In some embodiments, the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 286, and 302, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 303, 318, and 327, respectively. In some embodiments, the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 286, and 301, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 303, 318, and 327, respectively. In some embodiments, the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 286, and 297, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 304, 318, and 327, respectively. In some embodiments, the CDR-H1, the CDR- H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 286, and 297, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 305, 318, and 327, respectively. In some embodiments, the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 286, and 297, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 306, 318, 327, respectively. In some embodiments, the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 286, and 297, respectively, and the CDR-L1, the CDR- L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 307, 319, and 327, respectively. In some embodiments, the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 286, and 297, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 307, 319, and 327, respectively. In some embodiments, the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 286, and 297, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 310, 320, and 327, respectively. In some embodiments, the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 286, and 297, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 307, 318, and 328, respectively. In some embodiments, the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 286, and 297, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 310, 321, and 327, respectively. In some embodiments, the CDR-H1, the CDR- H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 286, and 297, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 311, 318, and 327, respectively. In some embodiments, the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 286, and 297, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 311, 322, and 327, respectively. In some embodiments, the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 286, and 297, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 312, 321, and 327, respectively. In some embodiments, the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 286, and 297, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 313, 318, and 328, respectively. In some embodiments, the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 286, and 297, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 313, 318, and 329, respectively. In some embodiments, the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 286, and 297, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 314, 318, and 327, respectively. In some embodiments, the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 286, and 297, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 315, 323, and 327, respectively. In some embodiments, the CDR-H1, the CDR- H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 286, and 297, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 316, 324, and 327, respectively. In some embodiments, the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 286, and 297, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 314, 325, and 327, respectively. In some embodiments, the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 286, and 297, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 317, 318, and 327, respectively. In some embodiments, the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 286, and 297, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 303, 326, and 327, respectively. In some embodiments, the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 296, and 297, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 314, 318, and 327respectively. In some embodiments, the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 294, and 302, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 314, 318, and 327, respectively. In some embodiments, the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 281, 289, and 301, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 314, 318, and 327, respectively. In some embodiments, the CDR-H1, the CDR- H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 281, 296, and 297, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 314, 318, and 327, respectively. In some embodiments, the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 286, and 301, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 314, 318, and 327, respectively. In some embodiments, the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 294, and 301, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 314, 318, and 327, respectively. In some embodiments, the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 296, and 297, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 313, 318, and 327, respectively. In some embodiments, the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 294, and 302, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 313, 318, and 327, respectively. In some embodiments, the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 281, 289, and 301, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 313, 318, and 327, respectively. In some embodiments, the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 281, 296, and 297, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 313, 318, and 327, respectively. In some embodiments, the CDR-H1, the CDR- H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 286, and 301, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 313, 318, and 327, respectively. In some embodiments, the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 294, and 301, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 313, 318, and 327, respectively. In some embodiments, the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 296, and 297, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 305, 318, and 327, respectively. In some embodiments, the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 294, and 302, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 305, 318, and 327, respectively. In some embodiments, the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 281, 289, and 301, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 305, 318, and 327, respectively. In some embodiments, the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 281, 296, and 297, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 305, 318, and 327, respectively. In some embodiments, the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 286, and 301, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 305, 318, and 327, respectively. In some embodiments, the CDR-H1, the CDR- H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 294, 301, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 305, 318, 327, respectively. In some embodiments, the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 296, and 297, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 313, 318, and 328, respectively. In some embodiments, the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 294, and 302, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 313, 318, and 328, respectively. In some embodiments, the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 281, 289, and 301, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 313, 318, and 328, respectively. In some embodiments, the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 281, 296, and 297, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 313, 318, and 328, respectively. In some embodiments, the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 286, and 301, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 313, 318, and 328, respectively. In some embodiments, the CDR-H1, the CDR- H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 294, and 301, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 313, 318, and 328, respectively. In some embodiments, the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 281, 289, and 297, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 317, 318, 329, respectively.
[0297] In some embodiments, the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 230 and SEQ ID NO: 347, respectively. In some embodiments, the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 231 and SEQ ID NO: 347, respectively. In some embodiments, the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 232 and SEQ ID NO: 347, respectively. In some embodiments, the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 233 and SEQ ID NO: 347, respectively. In some embodiments, the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 234 and SEQ ID NO: 347, respectively. In some embodiments, the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 235 and SEQ ID NO: 347, respectively. In some embodiments, the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 236 and SEQ ID NO: 347, respectively. In some embodiments, the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 237 and SEQ ID NO: 347, respectively. In some embodiments, the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 238 and SEQ ID NO: 347, respectively. In some embodiments, the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 239 and SEQ ID NO: 347, respectively. In some embodiments, the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 240 and SEQ ID NO: 347, respectively. In some embodiments, the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 241 and SEQ ID NO: 347, respectively. In some embodiments, the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 242 and SEQ ID NO: 347, respectively. In some embodiments, the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 243 and SEQ ID NO: 347, respectively. In some embodiments, the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 244 and SEQ ID NO: 347, respectively. In some embodiments, the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 245 and SEQ ID NO: 347, respectively. In some embodiments, the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 246 and SEQ ID NO: 347, respectively. In some embodiments, the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 247 and SEQ ID NO: 347, respectively. In some embodiments, the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 248 and SEQ ID NO: 347, respectively. In some embodiments, the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 249 and SEQ ID NO: 347, respectively. In some embodiments, the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 250 and SEQ ID NO: 347, respectively. In some embodiments, the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 251 and SEQ ID NO: 347, respectively. In some embodiments, the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 341 and SEQ ID NO: 257, respectively. In some embodiments, the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 341 and SEQ ID NO: 258, respectively. In some embodiments, the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 341 and SEQ ID NO: 259, respectively. In some embodiments, the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 341 and SEQ ID NO: 260, respectively. In some embodiments, the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 341 and SEQ ID NO: 261, respectively. In some embodiments, the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 341 and SEQ ID NO: 262, respectively. In some embodiments, the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 341 and SEQ ID NO: 263, respectively. In some embodiments, the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 341 and SEQ ID NO: 264, respectively. In some embodiments, the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 341 and SEQ ID NO: 265, respectively. In some embodiments, the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 341 and SEQ ID NO: 266, respectively. In some embodiments, the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 341 and SEQ ID NO: 267, respectively. In some embodiments, the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 341 and SEQ ID NO: 268, respectively. In some embodiments, the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 341 and SEQ ID NO: 269, respectively. In some embodiments, the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 341 and SEQ ID NO: 270, respectively. In some embodiments, the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 341 and SEQ ID NO: 271, respectively. In some embodiments, the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 341 and SEQ ID NO: 272, respectively. In some embodiments, the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 341 and SEQ ID NO: 273, respectively. In some embodiments, the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 341 and SEQ ID NO: 274, respectively. In some embodiments, the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 341 and SEQ ID NO: 275, respectively. In some embodiments, the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 252 and SEQ ID NO: 270, respectively. In some embodiments, the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 253 and SEQ ID NO: 270, respectively. In some embodiments, the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 254 and SEQ ID NO: 270, respectively. In some embodiments, the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 255 and SEQ ID NO: 270, respectively. In some embodiments, the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 251 and SEQ ID NO: 270, respectively. In some embodiments, the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 256 and SEQ ID NO: 270, respectively. In some embodiments, the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 252 and SEQ ID NO: 276, respectively. In some embodiments, the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 253 and SEQ ID NO: 276, respectively. In some embodiments, the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 254 and SEQ ID NO: 276, respectively. In some embodiments, the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 255 and SEQ ID NO: 276, respectively. In some embodiments, the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 251 and SEQ ID NO: 276, respectively. In some embodiments, the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 256 and SEQ ID NO: 276, respectively. In some embodiments, the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 252 and SEQ ID NO: 258, respectively. In some embodiments, the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 253 and SEQ ID NO: 258, respectively. In some embodiments, the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 254 and SEQ ID NO: 258, respectively. In some embodiments, the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 255 and SEQ ID NO: 258, respectively. In some embodiments, the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 251 and SEQ ID NO: 258, respectively. In some embodiments, the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 256 and SEQ ID NO: 258, respectively. In some embodiments, the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 252 and SEQ ID NO: 268, respectively. In some embodiments, the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 253 and SEQ ID NO: 268, respectively. In some embodiments, the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 254 and SEQ ID NO: 268, respectively. In some embodiments, the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 255 and SEQ ID NO: 268, respectively. In some embodiments, the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 251 and SEQ ID NO: 268, respectively. In some embodiments, the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 256 and SEQ ID NO: 268, respectively. In some embodiments, or the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 234 and SEQ ID NO: 277, respectively.
[0298] In some embodiments, the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 254 and SEQ ID NO: 268, respectively.
[0299] In some embodiments, the CTLA-4 VH region comprises a CDR-H1 comprising the sequence set forth in SEQ ID NO: 278 or SEQ ID NO: 281, a CDR-H2 comprising the sequence set forth in SEQ ID NO: 289 or SEQ ID NO: 296, and a CDR-H3 comprising the sequence set forth in SEQ ID NO: 297 or SEQ ID NO: 301; and the CTLA-4 VL region comprises a CDR-L1 comprising the sequence set forth in SEQ ID NO: 305 or SEQ ID NO: 313; a CDR-L2 comprising the sequence set forth in SEQ ID NO: 318, and a CDR-L3 comprising the sequence set forth in SEQ ID NO: 327 or SEQ ID NO: 328.
[0300] In some embodiments, the CTLA-4 VH region and the CTLA-4 VL region are or comprise: the sequence set forth in SEQ ID NO: 230 and 347, respectively; the sequence set forth in SEQ ID NO: 231 and 347, respectively; the sequence set forth in SEQ ID NO: 232 and 347, respectively; the sequence set forth in SEQ ID NO: 233 and 347, respectively; the sequence set forth in SEQ ID NO: 234 and 347, respectively; the sequence set forth in SEQ ID NO: 235 and 347, respectively; the sequence set forth in SEQ ID NO: 236 and 347, respectively; the sequence set forth in SEQ ID NO: 237 and 347, respectively; the sequence set forth in SEQ ID NO: 238 and 347, respectively; the sequence set forth in SEQ ID NO: 239 and 347, respectively; the sequence set forth in SEQ ID NO: 240 and 347, respectively; the sequence set forth in SEQ ID NO: 241 and 347, respectively; the sequence set forth in SEQ ID NO: 242 and 347, respectively; the sequence set forth in SEQ ID NO: 243 and 347, respectively; the sequence set forth in SEQ ID NO: 244 and 347, respectively; the sequence set forth in SEQ ID NO: 245 and 347, respectively; the sequence set forth in SEQ ID NO: 246 and 347, respectively; the sequence set forth in SEQ ID NO: 247 and 347, respectively; the sequence set forth in SEQ ID NO: 248 and 347, respectively; the sequence set forth in SEQ ID NO: 249 and 347, respectively; the sequence set forth in SEQ ID NO: 250 and 347, respectively; the sequence set forth in SEQ ID NO: 251 and 347, respectively; the sequence set forth in SEQ ID NO: 341 and 257, respectively; the sequence set forth in SEQ ID NO: 341 and 258, respectively; the sequence set forth in SEQ ID NO: 341 and 259, respectively; the sequence set forth in SEQ ID NO: 341 and 260, respectively; the sequence set forth in SEQ ID NO: 341 and 261, respectively; the sequence set forth in SEQ ID NO: 341 and 262, respectively; the sequence set forth in SEQ ID NO: 341 and 263, respectively; the sequence set forth in SEQ ID NO: 341 and 264, respectively; the sequence set forth in SEQ ID NO: 341 and 265, respectively; the sequence set forth in SEQ ID NO: 341 and 266, respectively; the sequence set forth in SEQ ID NO: 341 and 267, respectively; the sequence set forth in SEQ ID NO: 341and 268, respectively; the sequence set forth in SEQ ID NO: 341 and 269, respectively; the sequence set forth in SEQ ID NO: 341and 270, respectively; the sequence set forth in SEQ ID NO: 341and 271, respectively; the sequence set forth in SEQ ID NO: 341and 272, respectively; the sequence set forth in SEQ ID NO: 341and 273, respectively; the sequence set forth in SEQ ID NO: 341and 274, respectively; the sequence set forth in SEQ ID NO: 341and 275, respectively; the sequence set forth in SEQ ID NO: 252 and 270, respectively; the sequence set forth in SEQ ID NO: 253 and 270, respectively; the sequence set forth in SEQ ID NO: 254 and 270, respectively; the sequence set forth in SEQ ID NO: 255 and 270, respectively; the sequence set forth in SEQ ID NO: 251 and 270, respectively; the sequence set forth in SEQ ID NO: 256 and 270, respectively; the sequence set forth in SEQ ID NO: 252 and 276, respectively; the sequence set forth in SEQ ID NO: 253 and 276, respectively; the sequence set forth in SEQ ID NO: 254 and 276, respectively; the sequence set forth in SEQ ID NO: 255 and 276, respectively; the sequence set forth in SEQ ID NO: 251 and 276, respectively; the sequence set forth in SEQ ID NO: 256 and 276, respectively; the sequence set forth in SEQ ID NO: 252 and 258, respectively; the sequence set forth in SEQ ID NO: 253 and 258, respectively; the sequence set forth in SEQ ID NO: 254 and 258, respectively; the sequence set forth in SEQ ID NO: 255 and 258, respectively; the sequence set forth in SEQ ID NO: 251 and 258, respectively; the sequence set forth in SEQ ID NO: 256 and 258, respectively; the sequence set forth in SEQ ID NO: 252 and 268, respectively; the sequence set forth in SEQ ID NO: 253 and 268, respectively; the sequence set forth in SEQ ID NO: 254 and 268, respectively; the sequence set forth in SEQ ID NO: 255 and 268, respectively; the sequence set forth in SEQ ID NO: 251 and 268, respectively; the sequence set forth in SEQ ID NO: 256 and 268, respectively; the sequence set forth in SEQ ID NO: 234 and 277, respectively.
[0301] Table E5 provides the SEQ ID NOS: of exemplary provided CTLA-4 binding domains. In some embodiments, the CTLA-4-binding domain comprises a CTLA-4 VH region that comprises the CDR-H1, the CDR-H2 and the CDR-H3 sequence and a CTLA-4 VL region that comprises the CDR-L1, the CDR-L2 and the CDR-L3 sequence set forth in the SEQ ID NOS: listed in each row of Table E5 below (by Kabat numbering scheme). In some embodiments, the CTLA-4-binding domain comprises a CTLA-4 VH region sequence and a CTLA-4 VL region sequence set forth in the SEQ ID NOS: listed in each row of Table E5 below, or an antibody comprising a CTLA-4 VH region and a CTLA-4 VL region amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the CTLA-4 VH region sequence and the CTLA-4 VL region sequence set forth in the SEQ ID NOS: listed in each row of Table E5 below. In some embodiments, the CTLA-4-binding domain comprises a CTLA-4 VH region sequence and a CTLA-4 VL region sequence set forth in the SEQ ID NOS: listed in each row of Table E5 below. [0302] In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH region and a CTLA-4 VL region, wherein the CTLA-4 VH region of the CTLA-4 binding domain can contain a combination of any of the CDR-H1, the CDR-H2 and the CDR-H3 amino acid sequences set forth in Table E5, and the CTLA-4 VL region of the CTLA-4 binding domain can contain a combination of any of the CDR-L1, the CDR-L2 and the CDR-L3 amino acid sequences set forth in Table E5. In some embodiments, the provided CTLA-4 binding domain comprises a CTLA-4 VH region and/or a CTLA-4 VL region set forth in Table E5, in any combination, orientation or containing a different linker. In some aspects, the CTLA-4 binding domain comprises a CTLA-4 VH region described in Table E5. In some aspects, the CTLA-4 binding domain comprises a CTLA-4 VL region described in Table E5.
[0303] In some embodiments, provided CTLA-4 binding domains include those that include amino acid residues (such as due to mutation compared to a reference sequence) that alter pH binding such that the CTLA-4 binding domains exhibit better binding at neutral pH (e.g., pH 7.4) compared to acidic pH (e.g., pH 5.0). Results herein surprisingly found enrichment of a number of CTLA-4 binding domains (e.g., anti-CTLA-4 antibodies, including antibody fragments) with such activity that contain one or more of the following amino acids in the CTLA-4 VH region: arginine (R) at position 31, glutamic acid (E) or histidine (H) at position 54, histidine (H) at position 59, histidine (H) at position 101, or histidine (H) at position 102 (corresponding to arginine (R) at position 31, glutamic acid (E) or histidine (H) at position 53, histidine (H) at position 58, histidine (H) at position 97, or histidine (H) at position 98, respectively, based on Kabat numbering), or any combination thereof; and/or in the CTLA-4 VL region: histidine (H) at position 28, histidine (H) at position 31, histidine (H) at position 32, or a histidine (H) at position 91 (corresponding to histidine (H) at position 27a, histidine (H) at position 30, histidine (H) at position 31, or histidine (H) at position 90, respectively, based on Kabat numbering), or any combination thereof. In some embodiments, provided CTLA-4 binding domains (e.g., anti-CTLA-4 antibodies, including antibody fragments) herein contain one or more amino acid mutation in the CTLA-4 VH region that is S31H, D54H, D54E, Y59H, W101H, or L102H, which correspond to S31R, D53H, D53E, Y58H, W97H, or L98H based on Kabat numbering, respectively, or any combination thereof, compared to the CTLA-4 VH region set forth in SEQ ID NO:341; and/or one or more amino acid mutation in the CTLA-4 VL region that is S28H, S31H, S32H, or Q91H, which correspond to S27aH, S30H, S31H, or Q90H based on Kabat numbering, respectively, or any combination thereof, compared to the CTLA-4 VL region set forth in SEQ ID NO:347.
[0304] In some embodiments, the provided CTLA-4 binding domains (e.g., anti-CTLA-4 antibodies, including antibody fragments) comprises one or more mutations selected from the group consisting of: S31R, D54H, D54E, Y59H, W101H, and L102H (S31R, D53H, D53E, Y58H, W97H, and L98H, respectively, based on Kabat numbering) in the CTLA-4 VH region, such as compared to a reference CTLA-4 binding domain, such as an anti-CTLA-4 antibody VH region, e.g. set forth in SEQ ID NO: 341.
[0305] In some embodiments, the provided CTLA-4 binding domains (e.g., anti-CTLA-4 antibodies, including antibody fragments) comprise a histidine (H) at position 98 based on Kabat numbering (e.g., L102H mutation compared to SEQ ID NO:341) in the CTLA-4 VH region. In some embodiments, the provided CTLA-4 binding domains (e.g., anti-CTLA-4 antibodies, including antibody fragments) comprises an arginine (R) at position 31 based on Kabat numbering) (e.g., S31R mutation compared to SEQ ID NO:341) in the CTLA-4 VH region. In some embodiments, the provided anti-CTLA-4 antibody or antigen-binding fragment thereof comprises a glutamic acid (E) at position 53 based on Kabat numbering) (e.g., D54E mutation compared to SEQ ID NO:341) in the CTLA-4 VH region. In some embodiments, the provided CTLA-4 binding domains (e.g., anti-CTLA-4 antibodies, including antibody fragments) comprises a histidine (H) at position 53 based on Kabat numbering (e.g., D54H mutation compared to SEQ ID NO:341) in the CTLA-4 VH region. In some embodiments, the provided CTLA-4 binding domains (e.g., anti-CTLA-4 antibodies, including antibody fragments) comprises a histidine (H) at position 58 based on Kabat numbering (e.g., Y59H mutation compared to SEQ ID NO:341) in the CTLA-4 VH region. In some embodiments, the provided CTLA-4 binding domains (e.g., anti- CTLA-4 antibodies, including antibody fragments) comprises a histidine (H) at position 97 based on Kabat numbering (e.g., W101H mutation compared to SEQ ID NO:341) in the CTLA-4 VH region.
[0306] In some embodiments, the provided CTLA-4 binding domains (e.g., anti-CTLA-4 antibodies, including antibody fragments) comprise a histidine (H) at position 98 based on Kabat numbering and a arginine (R) at position 31 based on Kabat numbering (e.g., L102H and S31R mutation compared to SEQ ID NO: 341) in the CTLA-4 VH region. In some embodiments, the provided CTLA-4 binding domains (e.g., anti-CTLA-4 antibodies, including antibody fragments) comprises a histidine (H) at position 98 based on Kabat numbering and a glutamic acid (E) at position 53 based on Kabat numbering (e.g., L102H and D54E mutations compared to SEQ ID NO:341) in the CTLA-4 VH region. In some embodiments, the provided CTLA-4 binding domains (e.g., anti-CTLA-4 antibodies, including antibody fragments) comprises a histidine (H) at position 98 based on Kabat numbering and a histidine (H) at position 53 based on Kabat numbering (e.g., L102H and D54H mutations compared to SEQ ID NO:341) in the CTLA-4 VH region. In some embodiments, the provided CTLA-4 binding domains (e.g., anti-CTLA-4 antibodies, including antibody fragments) comprises a histidine (H) at position 98 based on Kabat numbering and a histidine (H) at position 58 based on Kabat numbering (e.g., L102H and Y59H mutations compared to SEQ ID NO:341) in the CTLA-4 VH region. In some embodiments, the provided CTLA-4 binding domains (e.g., anti-CTLA-4 antibodies, including antibody fragments) comprises a histidine (H) at position 98 based on Kabat numbering and a histidine (H) at position 97 based on Kabat numbering (e.g., L102H and W101H mutations compared to SEQ ID NO:341) in the CTLA-4 VH region. In some embodiments, the provided CTLA-4 binding domains (e.g., anti- CTLA-4 antibodies, including antibody fragments) comprises an arginine (R) at position 31 based on Kabat numbering) and a glutamic acid (E) at position 53 based on Kabat numbering (e.g., S31R and D54E mutations compared to SEQ ID NO: 341) in the CTLA-4 VH region. In some embodiments, the provided CTLA-4 binding domains (e.g., anti-CTLA-4 antibodies, including antibody fragments) comprise an arginine (R) at position 31 based on Kabat numbering and a histidine (H) at position 53 based on Kabat numbering (e.g., S31R and D54H mutations compared to SEQ ID NO: 341) in the CTLA-4 VH region. In some embodiments, the provided CTLA-4 binding domains (e.g., anti-CTLA-4 antibodies, including antibody fragments) comprises an arginine (R) at position 31 based on Kabat numbering and a histidine (H) at position 58 based on Kabat numbering (e.g., S31R and Y59H mutations compared to SEQ ID NO: 341) in the CTLA-4 VH region. In some embodiments, the provided CTLA-4 binding domains (e.g., anti-CTLA-4 antibodies, including antibody fragments) comprises an arginine (R) at position 31 based on Kabat numbering and a histidine (H) at position 97 based on Kabat numbering (e.g., S31R and W101H mutations compared to SEQ ID NO: 341) in the CTLA-4 VH region. In some embodiments, the provided CTLA-4 binding domains (e.g., anti-CTLA-4 antibodies, including antibody fragments) comprises a glutamic acid (E) at position 53 based on Kabat numbering and a histidine (H) at position 58 based on Kabat numbering (e.g., D54E and Y59H mutations compared to SEQ ID NO: 341) in the CTLA-4 VH region. In some embodiments, the provided CTLA-4 binding domains (e.g., anti-CTLA-4 antibodies, including antibody fragments) comprises a glutamic acid (E) at position 53 based on Kabat numbering and a histidine (H) at position 97 based on Kabat numbering (e.g., D54E and W101H mutations compared to SEQ ID NO: 341) in the CTLA-4 VH region. In some embodiments, the provided CTLA-4 binding domains (e.g., anti-CTLA-4 antibodies, including antibody fragments) comprises a histidine (H) at position 53 based on Kabat numbering and a histidine (H) at position 58 based on Kabat numbering (e.g., D54H and Y59H mutations compared to SEQ ID NO: 341) in the CTLA-4 VH region. In some embodiments, the provided CTLA-4 binding domains (e.g., anti-CTLA-4 antibodies, including antibody fragments) comprises a histidine (H) at position 53 based on Kabat numbering and a histidine (H) at position 97 based on Kabat numbering (e.g., D54H and W101H mutations compared to SEQ ID NO: 341) in the CTLA-4 VH region. In some embodiments, the provided CTLA-4 binding domains (e.g., anti-CTLA-4 antibodies, including antibody fragments) comprises a histidine (H) at position 58 based on Kabat numbering and a histidine (H) at position 97 based on Kabat numbering (e.g., Y59H and W101H mutations compared to SEQ ID NO: 341) in the CTLA-4 VH region. [0307] In some embodiments, the provided CTLA-4 binding domains (e.g., anti-CTLA-4 antibodies, including antibody fragments) comprises a histidine (H) at position 98 based on Kabat numbering, an arginine (R) at position 31 based on Kabat numbering and a glutamic acid (E) at position 53 based on Kabat numbering (e.g., L102H, S31R, and D54E mutations compared to SEQ ID NO: 341) in the CTLA-4 VH region. In some embodiments, the provided CTLA-4 binding domains (e.g., anti-CTLA-4 antibodies, including antibody fragments) comprises a histidine (H) at position 98 based on Kabat numbering, an arginine (R) at position 31 based on Kabat numbering and a histidine (H) at position 53 based on Kabat numbering (e.g., L102H, S31R, and D54H mutations compared to SEQ ID NO: 341) in the CTLA-4 VH region. In some embodiments, the provided CTLA-4 binding domains (e.g., anti-CTLA-4 antibodies, including antibody fragments) comprises a histidine (H) at position 98 based on Kabat numbering, an arginine (R) at position 31 based on Kabat numbering and a histidine (H) at position 58 based on Kabat numbering (e.g., L102H, S31R, and Y59H mutations compared to SEQ ID NO: 341) in the CTLA-4 VH region. In some embodiments, the provided CTLA-4 binding domains (e.g., anti-CTLA-4 antibodies, including antibody fragments) comprises a histidine (H) at position 98 based on Kabat numbering, an arginine (R) at position 31 based on Kabat numbering and a histidine (H) at position 97 based on Kabat numbering (e.g., L102H, S31R, and W101H mutations compared to SEQ ID NO: 341) in the CTLA-4 VH region. In some embodiments, the provided CTLA-4 binding domains (e.g., anti- CTLA-4 antibodies, including antibody fragments) comprises a histidine (H) at position 98 based on Kabat numbering, a glutamic acid (E) at position 53 based on Kabat numbering and a histidine (H) at position 58 based on Kabat numbering (e.g., L102H, D54E, and Y59H mutations compared to SEQ ID NO: 341) in the CTLA-4 VH region. In some embodiments, the provided CTLA-4 binding domains (e.g., anti-CTLA-4 antibodies, including antibody fragments) comprises a histidine (H) at position 98 based on Kabat numbering, a glutamic acid (E) at position 53 based on Kabat numbering and a histidine (H) at position 97 based on Kabat numbering (e.g., L102H, D54E, and W101H mutations compared to SEQ ID NO: 341) in the CTLA-4 VH region. In some embodiments, the provided CTLA-4 binding domains (e.g., anti-CTLA-4 antibodies, including antibody fragments) comprises a histidine (H) at position 98 based on Kabat numbering, a histidine (H) at position 53 based on Kabat numbering and a histidine (H) at position 58 based on Kabat numbering (e.g., L102H, D54H, and Y59H mutations compared to SEQ ID NO: 341) in the CTLA-4 VH region. In some embodiments, the provided CTLA-4 binding domains (e.g., anti- CTLA-4 antibodies, including antibody fragments) comprises a histidine (H) at position 98 based on Kabat numbering, a histidine (H) at position 53 based on Kabat numbering and a histidine (H) at position 97 based on Kabat numbering (e.g., L102H, D54H, and W101H mutations compared to SEQ ID NO: 341) in the CTLA-4 VH region. In some embodiments, the provided CTLA-4 binding domains (e.g., anti-CTLA-4 antibodies, including antibody fragments) comprises a histidine (H) at position 98 based on Kabat numbering, a histidine (H) at position 58 based on Kabat numbering and a histidine (H) at position 97 based on Kabat numbering (e.g., L102H, Y59H, and W101H mutations compared to SEQ ID NO: 341) in the CTLA-4 VH region. In some embodiments, the provided CTLA-4 binding domains (e.g., anti-CTLA-4 antibodies, including antibody fragments) comprises an arginine (R) at position 31 based on Kabat numbering), a glutamic acid (E) at position based on Kabat numbering, and a histidine (H) at position 58 based on Kabat numbering (e.g., S31R, D54E, and Y59H mutations compared to SEQ ID NO: 341) in the CTLA-4 VH region. In some embodiments, the provided CTLA-4 binding domains (e.g., anti-CTLA-4 antibodies, including antibody fragments) comprises an arginine (R) at position 31 based on Kabat numbering), a glutamic acid (E) at position 53 based on Kabat numbering, and a histidine (H) at position 97 based on Kabat numbering (e.g., S31R, D54E, and W101H mutations compared to SEQ ID NO: 341) in the CTLA-4 VH region. In some embodiments, the CTLA-4 binding domains (e.g., anti-CTLA-4 antibodies, including antibody fragments) comprises an arginine (R) at position 31 based on Kabat numbering), a histidine (H) at position 53 based on Kabat numbering, and a histidine (H) at position 58 based on Kabat numbering (e.g., S31R, D54H, and Y59H mutations compared to SEQ ID NO: 341) in the CTLA-4 VH region. In some embodiments, the provided CTLA-4 binding domains (e.g., anti-CTLA-4 antibodies, including antibody fragments) comprises an arginine (R) at position 31 based on Kabat numbering, a histidine (H) at position 53 based on Kabat numbering, and a histidine (H) at position 97 based on Kabat numbering (e.g., S31R, D54H, and W101H mutations compared to SEQ ID NO: 341) in the CTLA-4 VH region. In some embodiments, the provided CTLA-4 binding domains (e.g., anti-CTLA-4 antibodies, including antibody fragments) comprises an arginine (R) at position 31 based on Kabat numbering), a histidine (H) at position 58 based on Kabat numbering, and a histidine (H) at position 97 based on Kabat numbering (e.g., S31R, Y59H, and W101H mutations compared to SEQ ID NO: 341) in the CTLA-4 VH region. In some embodiments, the provided CTLA-4 binding domains (e.g., anti- CTLA-4 antibodies, including antibody fragments) comprises a glutamic acid (E) at position 53 based on Kabat numbering, a histidine (H) at position 58 based on Kabat numbering, and a histidine (H) at position 97 based on Kabat numbering (e.g., D54E, Y59H, and W101H mutations compared to SEQ ID NO: 341) in the CTLA-4 VH region. In some embodiments, the provided CTLA-4 binding domains (e.g., anti-CTLA-4 antibodies, including antibody fragments) comprises a histidine (H) at position 53 based on Kabat numbering, a histidine (H) at position 58 based on Kabat numbering, and a histidine (H) at position 97 based on Kabat numbering (e.g., D54H, Y59H, and W101H mutations compared to SEQ ID NO: 341) in the CTLA-4 VH region.
[0308] In some embodiments, the provided CTLA-4 binding domains (e.g., anti-CTLA-4 antibodies, including antibody fragments) comprises a histidine (H) at position 98 based on Kabat numbering, an arginine (R) at position 31 based on Kabat numbering, a glutamic acid (E) at position 53 based on Kabat numbering, and a histidine (H) at position 58 based on Kabat numbering (e.g., L102H, S31R, D54E, and Y59H mutations compared to SEQ ID NO: 341) in the CTLA-4 VH region. In some embodiments, the provided CTLA-4 binding domains (e.g., anti- CTLA-4 antibodies, including antibody fragments) comprises a histidine (H) at position 98 based on Kabat numbering, an arginine (R) at position 31 based on Kabat numbering, a glutamic acid (E) at position 53 based on Kabat numbering, and a histidine (H) at position 97 based on Kabat numbering (e.g., L102H, S31R, D54E, and W101H mutations compared to SEQ ID NO: 341) in the CTLA-4 VH region. In some embodiments, the provided CTLA-4 binding domains (e.g., anti- CTLA-4 antibodies, including antibody fragments) comprises a histidine (H) at position 98 based on Kabat numbering, an arginine (R) at position 31 based on Kabat numbering, a histidine (H) at position 53 based on Kabat numbering, and a histidine (H) at position 58 based on Kabat numbering (e.g., L102H, S31R, D54H, and Y59H mutations compared to SEQ ID NO: 341) in the CTLA-4 VH region. In some embodiments, the provided CTLA-4 binding domains (e.g., anti- CTLA-4 antibodies, including antibody fragments) comprises a histidine (H) at position 98 based on Kabat numbering, an arginine (R) at position 31 based on Kabat numbering, a histidine (H) at position 53 based on Kabat numbering, and a histidine (H) at position 97 based on Kabat numbering (e.g., L102H, S31R, D54H, and W101H mutations compared to SEQ ID NO: 341) in the CTLA-4 VH region. In some embodiments, the provided CTLA-4 binding domains (e.g., anti- CTLA-4 antibodies, including antibody fragments) comprises a histidine (H) at position 98 based on Kabat numbering, an arginine (R) at position 31 based on Kabat numbering, a histidine (H) at position 58 based on Kabat numbering, and a histidine (H) at position 97 based on Kabat numbering (e.g., L102H, S31R, Y59H, and W101H mutations compared to SEQ ID NO: 341) in the CTLA-4 VH region. In some embodiments, the provided CTLA-4 binding domains (e.g., anti- CTLA-4 antibodies, including antibody fragments) comprises a histidine (H) at position 98 based on Kabat numbering, a glutamic acid (E) at position 53 based on Kabat numbering, a histidine (H) at position 58 based on Kabat numbering, and a histidine (H) at position 97 based on Kabat numbering (e.g., L102H, D54E, Y59H, and W101H mutations compared to SEQ ID NO: 341) in the CTLA-4 VH region. In some embodiments, the provided CTLA-4 binding domains (e.g., anti- CTLA-4 antibodies, including antibody fragments) comprises a histidine (H) at position 98 based on Kabat numbering, a histidine (H) at position 53, a histidine (H) at position 58 based on Kabat numbering, and a histidine (H) at position 97 based on Kabat numbering (e.g., L102H, D54H, Y59H, and W101H mutations compared to SEQ ID NO: 341) in the CTLA-4 VH region. In some embodiments, the provided CTLA-4 binding domains (e.g., anti-CTLA-4 antibodies, including antibody fragments) comprises an arginine (R) at position 31 based on Kabat numbering, a glutamic acid (E) at position 53 based on Kabat numbering, a histidine (H) at position 58 based on Kabat numbering, and a histidine (H) at position 97 based on Kabat numbering (e.g., S31R, D54E, Y59H, and W101H mutations compared to SEQ ID NO: 341) in the CTLA-4 VH region. In some embodiments, the provided CTLA-4 binding domains (e.g., anti-CTLA-4 antibodies, including antibody fragments) comprises a histidine (H) at position 98 based on Kabat numbering, an arginine (R) at position 31 based on Kabat numbering, a glutamic acid (E) at position 53 based on Kabat numbering, a histidine (H) at position 58 based on Kabat numbering, and a histidine (H) at position 97 based on Kabat numbering (e.g., L102H, S31R, D54E, Y59H, and W101H mutations compared to SEQ ID NO: 341) in the CTLA-4 VH region. In some embodiments, the provided CTLA-4 binding domains (e.g., anti-CTLA-4 antibodies, including antibody fragments) comprises a histidine (H) at position 98 based on Kabat numbering, an arginine (R) at position 31 based on Kabat numbering, a histidine (H) at position 53 based on Kabat numbering, a histidine (H) at position 58 based on Kabat numbering, and a histidine (H) at position 97 based on Kabat numbering (e.g., L102H, S31R, D54H, Y59H, and W101H mutations compared to SEQ ID NO: 341) in the CTLA-4 VH region.
[0309] In some embodiments, the provided CTLA-4 binding domains (e.g., anti-CTLA-4 antibodies, including antibody fragments) comprises one or more mutations selected from the group consisting of: S28H, S31H, S32H, and Q91H (S27aH, S30H, S31H, and Q90H, respectively, based on Kabat numbering) in the CTLA-4 VL region, such as compared to a reference anti-CTLA-4 antibody VL, e.g. set forth in SEQ ID NO: 347. In some embodiments, the provided CTLA-4 binding domains (e.g., anti-CTLA-4 antibodies, including antibody fragments) comprises a histidine (H) at position 27a based on Kabat numbering (e.g., S28H mutation compared to SEQ ID NO:347) in the CTLA-4 VL region. In some embodiments, the provided CTLA-4 binding domains (e.g., anti-CTLA-4 antibodies, including antibody fragments) comprises a histidine (H) at position 30 based on Kabat numbering (e.g., S31H mutation compared to SEQ ID NO:347) in the CTLA-4 VL region. In some embodiments, the provided CTLA-4 binding domains (e.g., anti-CTLA-4 antibodies, including antibody fragments) comprises a histidine (H) at position 31 based on Kabat numbering (e.g., S32H mutation compared to SEQ ID NO:347) in the CTLA-4 VL region. In some embodiments, the provided CTLA-4 binding domains (e.g., anti- CTLA-4 antibodies, including antibody fragments) comprises a histidine (H) at position 90 based on Kabat numbering (e.g., Q91H mutation compared to SEQ ID NO:347) in the CTLA-4 VL region.
[0310] In some embodiments, the provided CTLA-4 binding domains (e.g., anti-CTLA-4 antibodies, including antibody fragments) comprises a histidine (H) at position 27a based on Kabat numbering and a histidine (H) at position 30 based on Kabat numbering (e.g., S28H and S31H mutations compared to SEQ ID NO: 347) in the CTLA-4 VL region. In some embodiments, the provided CTLA-4 binding domains (e.g., anti-CTLA-4 antibodies, including antibody fragments) comprises a histidine (H) at position 27a based on Kabat numbering and a histidine (H) at position 31 based on Kabat numbering (e.g., S28H and S32H mutations compared to SEQ ID NO: 347) in the CTLA-4 VL region. In some embodiments, the provided CTLA-4 binding domains (e.g., anti- CTLA-4 antibodies, including antibody fragments) comprises a histidine (H) at position 27a based on Kabat numbering and a histidine (H) at position 90 based on Kabat numbering (e.g., S28H and Q91H mutations compared to SEQ ID NO: 347) in the CTLA-4 VL region. In some embodiments, the provided CTLA-4 binding domains (e.g., anti-CTLA-4 antibodies, including antibody fragments) comprises a histidine (H) at position 30 based on Kabat numbering and a histidine (H) at position 31 based on Kabat numbering (e.g., S31H and S32H mutations compared to SEQ ID NO: 347) in the CTLA-4 VL region. In some embodiments, the provided CTLA-4 binding domains (e.g., anti-CTLA-4 antibodies, including antibody fragments) comprises a histidine (H) at position
30 based on Kabat numbering and a histidine (H) at position 90 based on Kabat numbering (e.g., S31H and Q91H mutations compared to SEQ ID NO: 347) in the CTLA-4 VL region. In some embodiments, the provided CTLA-4 binding domains (e.g., anti-CTLA-4 antibodies, including antibody fragments) comprises a histidine (H) at position 31 based on Kabat numbering and a histidine (H) at position 90 based on Kabat numbering (e.g., S32H and Q91H mutations compared to SEQ ID NO: 347) in the CTLA-4 VL region.
[0311] In some embodiments, the provided CTLA-4 binding domains (e.g., anti-CTLA-4 antibodies, including antibody fragments) comprises a histidine (H) at position 27a based on Kabat numbering, a histidine (H) at position 30 based on Kabat numbering, and a histidine (H) at position
31 based on Kabat numbering) (e.g., S28H, S31H, S32H mutations compared to SEQ ID NO: 347 (S27aH, S30H, S31H mutations compared to SEQ ID NO: 347, based on Kabat numbering)) in the CTLA-4 VL region. In some embodiments, the provided CTLA-4 binding domains (e.g., anti- CTLA-4 antibodies, including antibody fragments) comprises a histidine (H) at position 28 (position 27a based on Kabat numbering), a histidine (H) at position 30 based on Kabat numbering, and a histidine (H) at position 90 based on Kabat numbering (e.g., S28H, S31H, Q91H mutations compared to SEQ ID NO: 347) in the CTLA-4 VL region. In some embodiments, the provided CTLA-4 binding domains (e.g., anti-CTLA-4 antibodies, including antibody fragments) comprises a histidine (H) at position 27a based on Kabat numbering, a histidine (H) at position 31 based on Kabat numbering, and a histidine (H) at position 90 based on Kabat numbering (e.g., S28H, S32H, Q91H mutations compared to SEQ ID NO: 347) in the CTLA-4 VL region. In some embodiments, the provided CTLA-4 binding domains (e.g., anti-CTLA-4 antibodies, including antibody fragments) comprises a histidine (H) at position 30 based on Kabat numbering, a histidine (H) at position 31 based on Kabat numbering, and a histidine (H) at position 90 based on Kabat numbering (e.g., S31H, S32H, Q91H mutations compared to SEQ ID NO: 347) in the CTLA-4 VL region. [0312] In some embodiments, the provided CTLA-4 binding domains (e.g., anti-CTLA-4 antibodies, including antibody fragments) comprises a histidine (H) at position 27a based on Kabat numbering, a histidine (H) at position 30 based on Kabat numbering, a histidine (H) at position 31 based on Kabat numbering, and a histidine (H) at position 91 (position 90 based on Kabat numbering) (e.g., S28H, S31H, S32H, Q91H mutations compared to SEQ ID NO: 347) in the CTLA-4 VL region.
[0313] In some embodiments, the CTLA-4 VH region comprises an arginine (R) at position 31, glutamic acid (E) or histidine (H) at position 53, histidine (H) at position 58, histidine (H) at position 97, or histidine (H) at position 98, respectively, based on Kabat numbering, or any combination thereof (e.g., S31R, D54H, D54E, Y59H, W101H, or L102H mutations, or any combination thereof, compared to SEQ ID NO:341), and the CTLA-4 VL region comprises a histidine (H) at position 27a, histidine (H) at position 30, histidine (H) at position 31, or histidine (H) at position 90, respectively, based on Kabat numbering), or any combination thereof (e.g., S28H, S31H, S32H, or Q91H mutations, or any combination thereof, compared to SEQ ID NO:347).
[0314] In some embodiments, the CTLA-4 VH region comprises the sequence set forth in: QVQLVESGGGVVQPGRSLRLSCAASGFTFSX , YTMHW VRQAPGKGLEWVTFIS YX2GNNKX3 YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAIYYCARTGX4X5GPFDYWGQGTLVTVSS, wherein X, is S or R; X2 is E or H; X3 is H or Y; X4 is W or H; and X5 is L or H (SEQ ID NO: 407); and the CTLA-4 VL region comprises the sequence set forth in: EIVLTQSPGTLSLSPGERATLSCRASQX1VGHX2YLAWYQQKPGQAPRLLIYGAFSRATGIPD RFSGSGSGTDFTLTISRLEPEDFAVYYCQX3YGSSPWTFGQGTKVEIK, wherein X, is H or S; X2 is H or S; and X3 is Q or H (SEQ ID NO: 408).
[0315] In some embodiments, the CTLA-4 VH region comprises: a serine (S) at position 31, a glutamic acid (E) at position 54, a histidine (H) at position 59, a tryptophan (W) at position 101, and a leucine (L) at position 102, wherein the numbering is relative to SEQ ID NO: 407. In some embodiments, the CTLA-4 VH region comprises: an arginine (R) at position 31, a glutamic acid (E) at position 54, a tyrosine (Y) at position 59, a tryptophan (W) at position 101, and a histidine (H) at position 102, wherein the numbering is relative to SEQ ID NO: 407.
[0316] In some embodiments, the CTLA-4 VL region comprises: a histidine (H) at position 28, a serine (S) at position 32, and a glutamine (Q) at position 91, wherein the numbering is relative to SEQ ID NO: 408. In some embodiments, the CTLA-4 VL region comprises: a serine (S) at position 28, a serine (S) at position 32, and a histidine (H) at position 91, wherein the numbering is relative to SEQ ID NO: 408. [0317] In some embodiments, the CTLA-4 VH region comprises a serine (S) at position 31, a glutamic acid (E) at position 54, a histidine (H) at position 59, a tryptophan (W) at position 101, and a leucine (L) at position 102, wherein the numbering is relative to SEQ ID NO: 407; and the CTLA-4 VL region comprises a histidine (H) at position 28, a serine (S) at position 32, and a glutamine (Q) at position 91, wherein the numbering is relative to SEQ ID NO: 408. In some embodiments, the CTLA- 4 VH region comprises an arginine (R) at position 31, a glutamic acid (E) at position 54, a tyrosine (Y) at position 59, a tryptophan (W) at position 101, and a histidine (H) at position 102, wherein the numbering is relative to SEQ ID NO: 407; and the CTLA-4 VL region comprises a serine (S) at position 28, a serine (S) at position 32, and a histidine (H) at position 91, wherein the numbering is relative to SEQ ID NO: 408.
[0318] Exemplary CTLA-4 VH and CTLA-4 VL regions with one or more of the above residues are set forth in Tables 4A and Table 4B. Provided herein are any CTLA-4 binding domains comprising at least one of such CTLA-4 VH or CTLA-4 VL region set forth in Table 4A or Table 4B, paired with any respective other CTLA-4 VH and CTLA-4 VL described herein. Also provided are CTLA-4 binding domain containing a paired combination of any CTLA-4 VH set forth in Table 4A and any CTLA-4 VL set forth in Table 4B. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in any one of SEQ ID NOs: 234, 235, 243, 246- 248, and 250-256, and a CTLA-4 VL set forth in any one of SEQ ID NOs: 258, 259, 261, 263, 268- 270, 273, and 276.
[0319] In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 234 and a CTLA-4 VL set forth in SEQ ID NO: 258. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 234 and a CTLA-4 VL set forth in SEQ ID NO: 259. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 234 and a CTLA-4 VL set forth in SEQ ID NO: 261. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 234 and a CTLA-4 VL set forth in SEQ ID NO: 263. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 324 and a CTLA-4 VL set forth in SEQ ID NO: 268. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 234 and a CTLA-4 VL set forth in SEQ ID NO: 269. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 234 and a CTLA-4 VL set forth in SEQ ID NO: 270. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 234 and a CTLA-4 VL set forth in SEQ ID NO: 273. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 234 and a CTLA-4 VL set forth in SEQ ID NO: 276. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 235 and a CTLA-4 VL set forth in SEQ ID NO: 258. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 235 and a CTLA-4 VL set forth in SEQ ID NO: 259. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 235 and a CTLA-4 VL set forth in SEQ ID NO: 261. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 235 and a CTLA-4 VL set forth in SEQ ID NO: 263. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 235 and a CTLA-4 VL set forth in SEQ ID NO: 268. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 235 and a CTLA-4 VL set forth in SEQ ID NO: 269. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 235 and a CTLA-4 VL set forth in SEQ ID NO: 270. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 235 and a CTLA-4 VL set forth in SEQ ID NO: 273. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 235 and a CTLA-4 VL set forth in SEQ ID NO: 276. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 243 and a CTLA-4 VL set forth in SEQ ID NO: 258. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 243 and a CTLA-4 VL set forth in SEQ ID NO: 259. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 243 and a CTLA-4 VL set forth in SEQ ID NO: 261. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 243 and a CTLA-4 VL set forth in SEQ ID NO: 263. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 243 and a CTLA-4 VL set forth in SEQ ID NO: 268. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 243 and a CTLA-4 VL set forth in SEQ ID NO: 269. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 243 and a CTLA-4 VL set forth in SEQ ID NO: 270. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 243 and a CTLA-4 VL set forth in SEQ ID NO: 273. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 243 and a CTLA-4 VL set forth in SEQ ID NO: 276. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 246 and a CTLA-4 VL set forth in SEQ ID NO: 258. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 246 and a CTLA-4 VL set forth in SEQ ID NO: 259. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 246 and a CTLA-4 VL set forth in SEQ ID NO: 261. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 246 and a CTLA-4 VL set forth in SEQ ID NO: 263. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 246 and a CTLA-4 VL set forth in SEQ ID NO: 268. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 246 and a CTLA-4 VL set forth in SEQ ID NO: 269. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 246 and a CTLA-4 VL set forth in SEQ ID NO: 270. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 246 and a CTLA-4 VL set forth in SEQ ID NO: 273. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 246 and a CTLA-4 VL set forth in SEQ ID NO: 276. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 247 and a CTLA-4 VL set forth in SEQ ID NO: 258. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 247 and a CTLA-4 VL set forth in SEQ ID NO: 259. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 247 and a CTLA-4 VL set forth in SEQ ID NO: 261. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 247 and a CTLA-4 VL set forth in SEQ ID NO: 263. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 247 and a CTLA-4 VL set forth in SEQ ID NO: 268. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 247 and a CTLA-4 VL set forth in SEQ ID NO: 269. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 247 and a CTLA-4 VL set forth in SEQ ID NO: 270. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 247 and a CTLA-4 VL set forth in SEQ ID NO: 273. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 247 and a CTLA-4 VL set forth in SEQ ID NO: 276. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 248 and a CTLA-4 VL set forth in SEQ ID NO: 258. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 248 and a CTLA-4 VL set forth in SEQ ID NO: 30. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 248 and a CTLA-4 VL set forth in SEQ ID NO: 261. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 248 and a CTLA-4 VL set forth in SEQ ID NO: 263. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 248 and a CTLA-4 VL set forth in SEQ ID NO: 268. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 248 and a CTLA-4 VL set forth in SEQ ID NO: 269. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 248 and a CTLA-4 VL set forth in SEQ ID NO: 270. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 248 and a CTLA-4 VL set forth in SEQ ID NO: 273. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 248 and a CTLA-4 VL set forth in SEQ ID NO: 276. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 250 and a CTLA-4 VL set forth in SEQ ID NO: 258. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 250 and a CTLA-4 VL set forth in SEQ ID NO: 259. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 250 and a CTLA-4 VL set forth in SEQ ID NO: 261. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 250 and a CTLA-4 VL set forth in SEQ ID NO: 263. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 250 and a CTLA-4 VL set forth in SEQ ID NO: 268. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 250 and a CTLA-4 VL set forth in SEQ ID NO: 269. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 250 and a CTLA-4 VL set forth in SEQ ID NO: 270. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 250 and a CTLA-4 VL set forth in SEQ ID NO: 273. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 250 and a CTLA-4 VL set forth in SEQ ID NO: 276. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 251 and a CTLA-4 VL set forth in SEQ ID NO: 258. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 251 and a CTLA-4 VL set forth in SEQ ID NO: 259. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 251 and a CTLA-4 VL set forth in SEQ ID NO: 261. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 251 and a CTLA-4 VL set forth in SEQ ID NO: 263. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 251 and a CTLA-4 VL set forth in SEQ ID NO: 268. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 251 and a CTLA-4 VL set forth in SEQ ID NO: 269. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 251 and a CTLA-4 VL set forth in SEQ ID NO: 270. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 251 and a CTLA-4 VL set forth in SEQ ID NO: 273. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 251 and a CTLA-4 VL set forth in SEQ ID NO: 276. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 252 and a CTLA-4 VL set forth in SEQ ID NO: 258. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 252 and a CTLA-4 VL set forth in SEQ ID NO: 259. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 252 and a CTLA-4 VL set forth in SEQ ID NO: 261. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 252 and a CTLA-4 VL set forth in SEQ ID NO: 263. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 252 and a CTLA-4 VL set forth in SEQ ID NO: 268. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 252 and a CTLA-4 VL set forth in SEQ ID NO: 269. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 252 and a CTLA-4 VL set forth in SEQ ID NO: 270. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 252 and a CTLA-4 VL set forth in SEQ ID NO: 273. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 252 and a CTLA-4 VL set forth in SEQ ID NO: 276. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 253 and a CTLA-4 VL set forth in SEQ ID NO: 258. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 253 and a CTLA-4 VL set forth in SEQ ID NO: 259. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 253 and a CTLA-4 VL set forth in SEQ ID NO: 261. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 253 and a CTLA-4 VL set forth in SEQ ID NO: 263. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 253 and a CTLA-4 VL set forth in SEQ ID NO: 268. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 253 and a CTLA-4 VL set forth in SEQ ID NO: 269. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 253 and a CTLA-4 VL set forth in SEQ ID NO: 270. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 253 and a CTLA-4 VL set forth in SEQ ID NO: 273. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 253 and a CTLA-4 VL set forth in SEQ ID NO: 276. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 254 and a CTLA-4 VL set forth in SEQ ID NO: 258. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 254 and a CTLA-4 VL set forth in SEQ ID NO: 259. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 254 and a CTLA-4 VL set forth in SEQ ID NO: 261. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 254 and a CTLA-4 VL set forth in SEQ ID NO: 263. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 254 and a CTLA-4 VL set forth in SEQ ID NO: 268. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 254 and a CTLA-4 VL set forth in SEQ ID NO: 269. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 254 and a CTLA-4 VL set forth in SEQ ID NO: 270. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 254 and a CTLA-4 VL set forth in SEQ ID NO: 273. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 254 and a CTLA-4 VL set forth in SEQ ID NO: 276. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 255 and a CTLA-4 VL set forth in SEQ ID NO: 258. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 255 and a CTLA-4 VL set forth in SEQ ID NO: 259. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 255 and a CTLA-4 VL set forth in SEQ ID NO: 261. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 255 and a CTLA-4 VL set forth in SEQ ID NO: 263. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 255 and a CTLA-4 VL set forth in SEQ ID NO: 268. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 255 and a CTLA-4 VL set forth in SEQ ID NO: 269. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 255 and a CTLA-4 VL set forth in SEQ ID NO: 270. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 255 and a CTLA-4 VL set forth in SEQ ID NO: 273. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 255 and a CTLA-4 VL set forth in SEQ ID NO: 276. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 256 and a CTLA-4 VL set forth in SEQ ID NO: 258. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 256 and a CTLA-4 VL set forth in SEQ ID NO: 259. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 256 and a CTLA-4 VL set forth in SEQ ID NO: 261. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 256 and a CTLA-4 VL set forth in SEQ ID NO: 263. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 256 and a CTLA-4 VL set forth in SEQ ID NO: 268. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 256 and a CTLA-4 VL set forth in SEQ ID NO: 269. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 256 and a CTLA-4 VL set forth in SEQ ID NO: 270. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 256 and a CTLA-4 VL set forth in SEQ ID NO: 273. In some embodiments, the CTLA-4 binding domain comprises a CTLA-4 VH set forth in SEQ ID NO: 256 and a CTLA-4 VL set forth in SEQ ID NO: 276. The above examples are non-limiting and any combinations are contemplated and provided by the present disclosure.
[0320] Also provided herein are CTLA-4 binding domains that contain a CTLA-4 VH region sequence that exhibits a sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% to any of such CTLA-4 VH region sequences in Table 4A, in which one of the pH residues in Table 4A is present. Also provided herein are CTLA-4 binding domains that contain a CTLA-4 VL region sequence that exhibits a sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% to any of such CTLA-4 VL region sequences in Table 4B, in which one of the pH residues in Table 4B is present. Also provided herein are CTLA-4 binding domains that contain a CTLA-4 VH region sequence that exhibits a sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% to any of such CTLA-4 VH region sequences in Table 4A, in which one of the pH residues in Table 4A is present; and a CTLA-4 VL region sequence that exhibits a sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% to any of such
CTLA-4 VL region sequences in Table 4B, in which one of the pH residues in Table 4B is present.
[0321] In some embodiments of the CTLA-4 binding domain, the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 comprising the sequence set forth in SEQ ID NOs: 281,289, and 301, respectively, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 comprising the sequence set forth in SEQ ID NOs: 305, 318, and 327, respectively. In some embodiments of the CTLA-4 binding domain, the CTLA-4 VH region comprises the sequence set forth in SEQ ID NO: 254 and the CTLA-4 VL region comprises the sequence set forth in SEQ ID NO: 258. [0322] In some embodiments of the CTLA-4 binding domain, the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 comprising the sequence set forth in SEQ ID NOs: 278, 296, and 297, respectively, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 comprising the sequence set forth in SEQ ID NOs: 313, 318, and 328, respectively. In some embodiments of the CTLA-4 binding domain, the CTLA-4 VH region comprises the sequence set forth in SEQ ID NO: 252 and the CTLA-4 VL comprises the sequence set forth in SEQ ID NO: 268.
[0323] In some embodiments of the CTLA-4 binding domain, the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 comprising the sequence set forth in SEQ ID NOs: 281, 289, and 301, respectively, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 comprising the sequence set forth in SEQ ID NOs: 313, 318, and 328, respectively. In some embodiments of the CTLA-4 binding domain, the CTLA-4 VH region comprises the sequence set forth in SEQ ID NO: 254 and the CTLA-4 VL comprises the sequence set forth in SEQ ID NO: 268.
C. Multimerizing domain (Fc region)
[0324] In some embodiments, the provided binding molecules comprise a Fc region (also referred to as Fc domain). In some embodiments, the CTLA-4 binding domain is N-terminal to the Fc region and the PD-1 binding domain is C-terminal to the Fc region. In some embodiments, the PD-1 binding domain is N-terminal to the Fc region and the CTLA-4 binding domain is C-terminal to the Fc region.
[0325] In some embodiments, the binding molecule is a dimer formed by polypeptides, each containing an Fc region. In some embodiments, the polypeptides are each the same and the polypeptides can be dimerized to yield a homodimer. Thus, in some embodiments, the dimer is a homodimer in which two polypeptide chains of the multispecific polypeptide construct, e.g., bispecific antibody, are the same.
[0326] In some embodiments, the Fc region is an Fc region from an IgAl, IgA2, IgD, IgE, IgGl, IgG2, IgG3, IgG4, or IgM. In some embodiments, the Fc region is human IgGl or is derived from human IgGl, or is or is derived from a naturally occurring variant thereof.
[0327] In some embodiments, the Fc region is a wild-type IgGl Fc polypeptide. In some embodiments, an IgGl Fc polypeptide (or a variant thereof such as any described below) can be made in a G1 ml or G1 m3 allotype. In some embodiments, the Fc region can contain amino acids of the human G1 ml allotype, such as residues containing Asp (D) and Feu (E) at positions 356 and 358, e.g. as set forth in SEQ ID NO: 411. In some cases, an Fc polypeptide can contain amino acid substitutions E356D and M358E to reconstitute residues of allotype G1 ml. In other embodiments, the Fc region can contain amino acids of the human G1 m3 allotype, such as residues Glu (E) and Met (M) at positions 356 and 358 by EU numbering. In some cases, an Fc polypeptide can contain amino acid substitutions D356E and E358M to reconstitute residues of allotype G1 m3. [0328] In some embodiments, a bispecific antibody is formatted with an Fc region that lacks a C- terminal Lys (K) residue. In some embodiments, the human IgGl Fc region lacks Lys447 (EU index of Kabat et al 1991 Sequences of Proteins of Immunological Interest).
[0329] In some embodiments, the Fc region comprises a hinge-CH2-CH3 amino acid sequence. In some embodiments, the Fc region comprises the sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 411. In some embodiments, the Fc region comprises the sequence set forth in SEQ ID NO: 411.
[0330] In some embodiments, the binding molecule or each polypeptide of an immunoglobulin Fc region comprises a human IgGl polypeptide sequence including a hinge-CH2- CH3 amino acid sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:
411. In some embodiments, the Fc region comprises the sequence set forth in SEQ ID NO: 411. In some embodiments, each polypeptide of an immunoglobulin Fc region comprises a human IgGl polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:
412. In some embodiments, the Fc region comprises the sequence set forth in SEQ ID NO: 412.
[0331] In some embodiments, the binding molecule includes two CTLA-4 binding domains, two PD-1 binding domains, and the homodimeric Fc composed of two identical Fc polypeptides (e.g., each set forth in SEQ ID NO:411). In such embodiments, the binding molecule is composed of two identical heavy chain polypeptides and two identical light chain polypeptides that form the two CTLA-4 binding domains, the two PD-1 binding domains and the homodimeric Fc region in which the heavy chain polypeptides of the Fc region are connected by a disulfide bond.
[0332] In some embodiments, the Fc region is a modified human IgG Fc region. In the context of binding molecules, such as bispecific antibodies, of the present invention, the multimerizing domains, e.g., Fc regions, may comprise one or more amino acid changes (e.g., insertions, deletions or substitutions) as compared to the wild- type, naturally occurring version of the Fc region.
[0333] In some embodiments, the Fc region is formed by Fc domains that are mutated or modified to promote heterodimerization in which different polypeptides can be dimerized to yield a heterodimer. Thus, in some embodiments, the dimer is a heterodimer in which two polypeptide chains of the multispecific polypeptide construct, e.g., bispecific antibody, are different.
Exemplary modifications to promote heterodimerization are known, including any as described below. [0334] In one aspect, a binding molecule provided herein is composed of a heterodimeric Fc region that facilitates interactions of two different heavy chain polypeptides in which each heavy chain polypeptide contains at least one different binding arm of the binding molecule. In some embodiments, the bispecific antibody includes a first heavy chain containing at least one anti- CTLA-4 antibody or antigen-binding fragment as described herein and a first polypeptide chain of the heterodimeric Fc and a second heavy chain containing one anti-PD-1 antibody or antigen binding fragment as described in Section I, and the second polypeptide chain of the heterodimeric Fc. In some embodiments the first polypeptide chain of the heterodimeric Fc-region comprises a first CH3 region, and the second polypeptide chain of the heterodimeric Fc region comprises a second CH3 region, wherein the sequences of the first and second CH3 regions are different and are such that the heterodimeric interaction between said first and second CH3 regions is stronger than each of the homodimeric interactions of said first and second CH3 regions.
[0335] In particular embodiments of binding molecules provided herein, the human IgG Fc region is modified to induce heterodimerization. Various methods are known for promoting heterodimerization of complementary Fc polypeptides, see e.g. Ridgway et al, Protein Eng. 9:617- 621 (1996); Merchant et al, Nat. Biotechnol. 16(7): 677-81 (1998); Moore et al. (2011) MAbs, 3:546-57; Von Kreudenstein et al. MAbs, (2013) 5:646-54; Gunasekaran et al. (2010) J. Biol. Chem., 285:19637-46; Leaver-Fay et al. (2016) Structure, 24:641-51; Ha et al. (2016) Frontiers in Immunology, 7:1; Davis et al. (2010) Protein Eng Des Sei, 23:195-202; published international PCT Appl. No. WO 1998/050431, WO 2009/089004, WO2011143545 WO 2014/067011, WO 2012/058768, W02018027025; published U.S. patent Appl. No. US20140363426, US20150307628, US20180016354, US20150239991; and U.S. patent Nos. US5731168, US7183076, US9701759, US9605084, and US9650446. Methods to promote heterodimerization of Fc chains include mutagenesis of the Fc region, such as by including a set of “knob-into-hole” mutations or including mutations to effect electrostatic steering of the Fc to favor attractive interactions among different polypeptide chains. For example, in some embodiments, the Fc polypeptides of a heterodimer includes a mutation to alter charge polarity across the Fc dimer interface such that co-expression of electrostatically matched Fc chains support favorable attractive interactions thereby promoting desired Fc heterodimer formation, whereas unfavorable repulsive charge interactions suppress unwanted Fc homodimer formation (Guneskaran et al. (2010) JBC, 285: 19637-19646). When co-expressed in a cell, association between the chains is possible but the chains do not substantially self-associate due to charge repulsion. Other strategies for generating a heterodimeric Fc include mixing human IgG and IgA CH3 domain segments to create a complementary CH3 heterodimer, which is referred to as a SEED Fc.
[0336] Methods and variants for heterodimerization also include those described in published international PCT App. WO2014/145806, including “knobs and holes” mutations (also called “skew” variants), mutations that relate to “electrostatic steering” or “charge pairs,” and pl variants.
Heterodimeric variants also include any as described in U.S. published Appl. No.
US2012/0149876 or US2018/011883.
[0337] In some embodiments, modifications include introduction of a protuberance (knob) into a first Fc polypeptide and a cavity (hole) into a second Fc polypeptide such that the protuberance is positionable in the cavity to promote complexing of the first and second Fc- containing polypeptides. Amino acids targeted for replacement and/or modification to create protuberances or cavities in a polypeptide are typically interface amino acids that interact or contact with one or more amino acids in the interface of a second polypeptide.
[0338] In some embodiments, a first Fc polypeptide that is modified to contain protuberance (hole) amino acids include replacement of a native or original amino acid with an amino acid that has at least one side chain which projects from the interface of the first Fc polypeptide and is therefore positionable in a compensatory cavity (hole) in an adjacent interface of a second polypeptide. Most often, the replacement amino acid is one which has a larger side chain volume than the original amino acid residue. One of skill in the art knows how to determine and/or assess the properties of amino acid residues to identify those that are ideal replacement amino acids to create a protuberance. In some embodiments, the replacement residues for the formation of a protuberance are naturally occurring amino acid residues and include, for example, arginine (R), phenylalanine (F), tyrosine (Y), or tryptophan (W). In some examples, the original residue identified for replacement is an amino acid residue that has a small side chain such as, for example, alanine, asparagine, aspartic acid, glycine, serine, threonine, or valine.
[0339] In some embodiments, a second Fc polypeptide that is modified to contain a cavity (hole) is one that includes replacement of a native or original amino acid with an amino acid that has at least one side chain that is recessed from the interface of the second polypeptide and thus is able to accommodate a corresponding protuberance from the interface of a first polypeptide. Most often, the replacement amino acid is one which has a smaller side chain volume than the original amino acid residue. One of skill in the art knows how to determine and/or assess the properties of amino acid residues to identify those that are ideal replacement residues for the formation of a cavity. Generally, the replacement residues for the formation of a cavity are naturally occurring amino acids and include, for example, alanine (A), serine (S), threonine (T) and valine (V). In some examples, the original amino acid identified for replacement is an amino acid that has a large side chain such as, for example, tyrosine, arginine, phenylalanine, or tryptophan.
[0340] The CH3 interface of human IgGl, for example, involves sixteen residues on each domain located on four anti-parallel -strands which buries 1090 A2 from each surface (see e.g., Deisenhofer et al. (1981) Biochemistry, 20:2361-2370; Miller et al., (1990) J Mol. Biol., 216, 965- 973; Ridgway et al., (1996) Prot. Engin., 9: 617-621; U.S. Pat. No. 5,731,168). Modifications of a CH3 domain to create protuberances or cavities are described, for example, in U.S. Pat. No. 5,731,168; International Patent Applications WO98/50431 and WO 2005/063816; and Ridgway et al., (1996) Prot. Engin., 9: 617-621. In some examples, modifications of a CH3 domain to create protuberances or cavities are typically targeted to residues located on the two central anti-parallel P-strands. The aim is to minimize the risk that the protuberances which are created can be accommodated by protruding into the surrounding solvent rather than being accommodated by a compensatory cavity in the partner CH3 domain.
[0341] In some embodiments, to promote heterodimerization both polypeptides of the Fc heterodimer contain paired or complementary amino acid modifications. Exemplary paired amino acid modification of polypeptides of an Fc fusion, such as an IgGl Fc, are set forth in Table 5.
[0342] In some embodiments, the binding molecule contains a heterodimeric Fc region in which the first Fc polypeptide of the heterodimeric Fc has at least 85%, 90%, 95% or 98% sequence identity to the Fc polypeptide set forth in SEQ ID NO: 412 and contains the mutations S354C, T366W and E234A, E235E, G237A; and the second Fc polypeptide of the heterodimeric Fc has at least 85%, 90%, 95% or 98% sequence identity to the Fc polypeptide set forth in SEQ ID NO:413 and contains the mutations Y349C, T366S, E368A, Y407V and E234A, E235E, G237A. In some embodiments, the binding molecule contains a heterodimeric Fc region in which the first Fc polypeptide of the heterodimeric Fc is set forth in SEQ ID NO:412 and the second Fc polypeptide of the heterodimeric Fc is set forth in SEQ ID NO:413.
[0343] In some embodiments, the binding molecule contains a heterodimeric Fc region in which the first Fc polypeptide of the heterodimeric Fc has at least 85%, 90%, 95% or 98% sequence identity to the Fc polypeptide set forth in SEQ ID NO:414 and contains the mutations S354C, T366W and E234A, E235A, D265S; and the second Fc polypeptide of the heterodimeric Fc has at least 85%, 90%, 95% or 98% sequence identity to the Fc polypeptide set forth in SEQ ID NO:415 and contains the mutations Y349C, T366S, E368A, Y407V and E234A, E235A, D265S. In some embodiments, the binding molecule contains a heterodimeric Fc region in which the first Fc polypeptide of the heterodimeric Fc is set forth in SEQ ID NO:414 and the second Fc polypeptide of the heterodimeric Fc is set forth in SEQ ID NO:415. [0344] In some embodiments, the binding molecule contains a heterodimeric Fc region in which the first Fc polypeptide of the heterodimeric Fc has at least 85%, 90%, 95% or 98% sequence identity to the Fc polypeptide set forth in SEQ ID NO: 416 and contains the mutations T350V, L351Y, F405A and Y407V; and the second Fc polypeptide of the heterodimeric Fc has at least 85%, 90%, 95% or 98% sequence identity to the Fc polypeptide set forth in SEQ ID NO: 417 and contains the mutations T350V, T366L, K392L and T394W. In some embodiments, the binding molecule contains a heterodimeric Fc region in which the first Fc polypeptide is set forth in SEQ ID NO:416 and the second Fc polypeptide is set forth in SEQ ID NO:417.
[0345] In some embodiments, the binding molecule contains a heterodimeric Fc region in which the first Fc polypeptide of the heterodimeric Fc has at least 85%, 90%, 95% or 98% sequence identity to the Fc polypeptide set forth in SEQ ID NO: 418 and contains the mutations T350V, L351Y, F405A and Y407V and the mutations L234A, L235A and D265S; and the second Fc polypeptide of the heterodimeric Fc has at least 85%, 90%, 95% or 98% sequence identity to the Fc polypeptide set forth in SEQ ID NO:419and contains the mutations T350V, T366L, K392L and T394W and the mutations L234A, L235A and D265S. In some embodiments, the binding molecule contains a heterodimeric Fc region in which the first Fc polypeptide is set forth in SEQ ID NO: 418 and the second Fc polypeptide is set forth in SEQ ID NO: 419.
[0346] In some embodiments, the binding molecule contains a heterodimeric Fc region in which the first Fc polypeptide of the heterodimeric Fc has at least 85%, 90%, 95% or 98% sequence identity to the Fc polypeptide set forth in SEQ ID NO: 420 and contains the mutations T350V, L351Y, F405A and Y407V and the mutations L234A, L235A and P329G; and the second Fc polypeptide of the heterodimeric Fc region has at least 85%, 90%, 95% or 98% sequence identity to the Fc polypeptide set forth in SEQ ID NO: 421 and contains the mutations T350V, T366L, K392L and T394W and the mutations L234A, L235A and P329G. In some embodiments, the binding molecule contains a heterodimeric Fc region in which the first Fc polypeptide is set forth in SEQ ID NO:420and the second Fc polypeptide is set forth in SEQ ID NO: 421.
[0347] In some embodiments, the bispecific antibody contains a heterodimeric Fc region in which the first Fc polypeptide of the heterodimeric Fc has at least 85%, 90%, 95% or 98% sequence identity to the Fc polypeptide set forth in SEQ ID NO:422 and contains the mutations S354C, T366W and P238K; and the second Fc polypeptide of the heterodimeric Fc has at least 85%, 90%, 95% or 98% sequence identity to the Fc polypeptide set forth in SEQ ID NO:423 and contains the mutations Y349C, T366S, L368A, Y407V and P238K. In some embodiments, the bispecific antibody contains a heterodimeric Fc region in which the first Fc polypeptide of the heterodimeric Fc is set forth in SEQ ID NO:422 and the second Fc polypeptide of the heterodimeric Fc is set forth in SEQ ID NO:423. [0348] In general, the Fc region is responsible for effector functions, such as complementdependent cytotoxicity (CDC) and antibody-dependent cell cytotoxicity (ADCC), in addition to the antigen-binding capacity, which is the main function of immunoglobulins. Additionally, the FcRn sequence present in the Fc region plays the role of regulating the IgG level in serum by increasing the in vivo half-life by conjugation to an in vivo FcRn receptor. In some embodiments, such functions can be altered, such as reduced or enhanced, in an Fc for use with the provided multispecific polypeptide constructs.
[0349] In some embodiments, the Fc region includes an Fc polypeptide that is mutated or modified to alter one or more effector functions. Various examples of mutations to Fc polypeptides to alter, such as reduce, effector function are known, including any as described below. In some embodiments, reference to amino acid substitutions in an Fc region is by EU numbering by Kabat (also called Kabat numbering) unless described with reference to a specific SEQ ID NO. EU numbering is known and is according to the most recently updated IMGT Scientific Chart (IMGT®, the international ImMunoGeneTics information system®, http://www.imgt.org/IMGTScientificChart/Numbering/Hu_IGHGnber.html (created: 17 May 2001, last updated: 10 Jan 2013) and the EU index as reported in Kabat, E.A. et al. Sequences of Proteins of Immunological interest. 5th ed. US Department of Health and Human Services, NIH publication No. 91-3242 (1991).
[0350] In some embodiments, the human IgGl Fc region is modified to alter antibodydependent cellular cytotoxicity (ADCC) and/or complement-dependent cytotoxicity (CDC), e.g., the amino acid modifications described in Natsume et al., 2008 Cancer Res, 68(10): 3863-72; Idusogie et al., 2001 J Immunol, 166(4): 2571-5; Moore et al., 2010 mAbs, 2(2): 181-189; Lazar et al., 2006 PNAS, 103(11): 4005-4010, Shields et al., 2001 JBC, 276(9): 6591-6604; Stavenhagen et al., 2007 Cancer Res, 67(18): 8882-8890; Stavenhagen et al., 2008 Advan. Enzyme Regul., 48: 152-164; Alegre et al, 1992 J Immunol, 148: 3461-3468; Reviewed in Kaneko and Niwa, 2011 Biodrugs, 25(1): 1-11 ; U.S. Patent No. 8101720; U.S. Patent No. 9475881; and U.S. Patent No. 10851164, the contents of each of which are hereby incorporated by reference in their entireties.
[0351] In some embodiments, the Fc region, such as the human IgGl Fc region is modified to enhance ADCC activity or CDC activity. Examples of mutations that enhance ADCC include modification at Ser239 and Ile332, for example Ser239Asp and Ile332Glu (S239D, I332E) (SEQ ID NO: 336). Examples of mutations that enhance CDC include modifications at Lys326 and Glu333. In some embodiments, the Fc region is modified at one or both of these positions, for example Lys326Ala and/or Glu333Ala (K326A and E333A) using the Kabat numbering system.
[0352] In some embodiments, the Fc region is altered to provide reduced Fc-mediated effector functions, such as via reduced Fc receptor binding, e.g. binding to FcyR binding but generally not FcRn binding. In some embodiments, the human IgGl Fc region fusion proteins of the present disclosure lack or have reduced Fucose attached to the N-linked glycan-chain at N297. There are numerous ways to prevent fucosylation, including but not limited to production in a FUT8 deficient cell line; addition inhibitors to the mammalian cell culture media, for example Castanospermine; and metabolic engineering of the production cell line. In some embodiments, the human IgGl Fc region is modified at amino acid Asn297 to prevent glycosylation of the fusion protein, e.g., Asn297Ala (N297A) or Asn297Asp (N297D).
[0353] In some embodiments, the Fc region of the fusion protein is altered at one or more of the following positions to reduce Fc receptor binding: Leu 234 (L234), Leu235 (L235), Asp265 (D265), Asp270 (D270), Ser298 (S298), Asn297 (N297), Asn325 (N325) or Ala327 (A327). For example, Leu 234Ala (L234A), Leu235Ala (L235A), Asp265Asn (D265N), Asp270Asn (D270N), Ser298Asn (S298N), Asn297Ala (N297A), Asn325Glu (N325E) orAla327Ser (A327S). In some embodiments, the Fc region of the fusion protein is modified at amino acid Leu235 to alter Fc receptor interactions, e.g., Leu235Glu (L235E) or Leu235Ala (L235A). In some embodiments, the Fc region of the fusion protein is modified at amino acid Leu234 to alter Fc receptor interactions, e.g., Leu234Ala (L234A). In some embodiments, the Fc region of the fusion protein is altered at both amino acid 234 and 235, e.g., Leu234Ala and Leu235Ala (L234A/L235A) or Leu234Val and Leu235Ala (L234V/L235A). In particular embodiments, the Fc region contains LALA mutations Leu234Ala and Leu235Ala (L234A/L235A). In some embodiments, modifications within the Fc region reduce binding to Fc-receptor-gamma receptors while have minimal impact on binding to the neonatal Fc receptor (FcRn).
[0354] In some embodiments, the Fc comprises amino acid modification of at least one of L234, L235, or D265. In another embodiment, the Fc comprises amino acid modification of at least L234, L235 and D265. In another embodiment, the Fc comprises the amino acid modification L234A, L235A and D265S. In particular aspects, the IgGl has been modified to comprise or consist of an L234A, an L235A, and a D265S mutation as identified using Kabat numbering to render the Fc effector-less.
[0355] In some embodiments, the Fc comprises a P329G mutation in the Fc together with L234A-L235A (LALA) mutations. In particular aspects, the IgGl has been modified to comprise or consist of an L234A, an L235A, and a P329G mutation as identified using Kabat numbering to render the Fc effector-less. In some embodiments, P329G is able to disrupt the interaction between hlgG and hFcyR, which combined with the effectorless mutations L234A/L235A mutations create a triple mutant L234A/L235A/P329G with no detectable binding to Clq or FcyRs, resulting in abrogated ADCC. In some embodiments, such an Fc disrupts the interaction with Fcy-receptors (FcyRs) while maintaining the binding to neonatal Fc-receptor (nFcR) and therefore wildtype- antibody-like pharmacokinetics in subjects.
[0356] In some embodiments, the binding molecules comprising one or more modifications in the Fc domain that results in a modified Fc domain having a modified binding interaction (e.g., enhanced or diminished) between Fc and FcRn. In some embodiments, the human IgG Fc region is modified to enhance FcRn binding. In one embodiment, the bispecific antigen-binding molecule comprises a modification in a CH2 or a CH3 region, wherein the modification increases the affinity of the Fc domain to FcRn in an acidic environment e.g., in an endosome where pH ranges from about 5.5 to about 6.0). Examples of Fc mutations that enhance binding to FcRn are Met252Tyr, Ser254Thr, Thr256Glu (M252Y, S254T, T256E, respectively)(e.g„ SEQ ID NO: 339) (Kabat numbering, Dall’Acqua et al 2006, J. Biol Chem Vol. 281(33) 23514-23524), Met428Leu and Asn434Ser (M428L, N434S)(SEQ ID NO: 340) (Zalevsky et al 2010 Nature Biotech, Vol. 28(2) 157-159) (EU index of Kabat et al 1991 Sequences of Proteins of Immunological Interest). Non-limiting examples of such Fc modifications include, e.g., a modification at position 250 (e.g., E or Q); 250 and 428 (e.g., L or F);252 (e.g., L/Y/F/W or T), 254 (e.g., S or T), and 256 (e.g., S/R/Q/E/D orT); or a modification at position 428 and/or 433 (e.g., L/R/S/P/Q or K)and/or 434 (e.g., H/F or Y); or a modification at position 250 and/or 428; or a modification at position 307 or 308 (e.g., 308F, V308F), and434. In some embodiments, the mutated or modified Fc polypeptide includes the following mutations: Met252Tyr and Met428Leu or Met252Tyr and Met428Val (M252Y, M428L, or M252Y, M428V) using the Kabat numbering system. In one embodiment, the modification comprises a 428L (e.g., M428L)and 434S (e.g., N434S) modification; a 428L, 2591 (e.g., V259I), and 308F(e.g., V308F) modification; a 433K (e.g., H433K) and a 434 (e.g., 434Y)modification; a 252, 254, and 256 (e.g., 252Y, 254T, and 256E)modification; a 250Q and 428L modification (e.g., T250Q and M428L); and a307 and/or 308 modification (e.g., 308F or 308P).
[0357] In some embodiments, the Fc region is mutated in one or more of the following positions to reduce Fc receptor binding: Glu233 (E233), Leu234 (L234), or Leu235 (L235). The one or more mutations can include E233P, L234V and/or L235A.
[0358] In some embodiments, the Fc region of the fusion protein is altered at Gly236 to reduce Fc receptor binding. For example, wherein Gly236 is deleted from the fusion protein. In some embodiments, the human IgGl Fc region is modified at amino acid Gly236 to enhance the interaction with CD32A, e.g., Gly236Ala (G236A).
[0359] In some embodiments, the Fc comprises amino acid modification of at least one of G236A, A330L, or I332E (SEQ ID NO: 337). In another embodiment, the Fc comprises amino acid modification of at least one of G236A, S239D, A330L, or I332E (SEQ ID NO: 338). [0360] In particular embodiments, the mutations of the Fc region to reduce Fc effector function, e.g. via reducing Fc receptor binding to FcyR, include mutations from among any of G236R/L328R, E233P/L234V/L235A/G236del/S239K, E233P/L234V/L235A/G236del/S267K, E233P/L234V/L235A/G236del/S239K/A327G, E233P/L234V/L235A/G236del/S267K/A327G or E233P/L234V/L235A/G236del.
[0361] In some embodiments, any of the provided Fc regions may include a mutation to eliminate or reduce the effector function of any antibody by mutating P238, e.g. to P238K, such as in an IgGl or IgG2 Fc. In some embodiments, in addition to the modifications such as mutations described herein to antibodies to enhance their biological activity or reduce effector function, further mutations can be made, e.g., to the CHI, hinge, CH2 or CH3 domain, e.g., to further reduce the effector function, binding to FcyRs, and/or the stability of the antibodies. For example, any of the mutations described herein may be combined with a P238, e.g., P238K, mutation, such as in an IgGl or IgG2 Fc. In some embodiments, Fc regions with P238K mutation demonstrate essentially no detectable binding signal towards the low affinity FcyRs hCD32a-H131, hCD32a-R131, hCD32b, hCD16a-V158 or hCD16b-NA2 and/or reduced binding affinity and a faster off-rate to the high affinity FcyR CD64 (see e.g., U.S. published patent application No. US20240141059).
D. Exemplary Binding Molecules
[0362] Provided herein are exemplary bispecific antibodies comprising a first binding region that binds CTLA-4 and a second binding region that binds PD-1.
[0363] In some embodiments, the provided binding molecules are bispecific antibodies that include two antigen binding sites, namely CTLA-4 binding domain and a PD-1 binding domains. In some embodiments, provided bispecific binding molecules are bivalent bispecific antibodies that are monovalent for binding to CTLA-4 and monovalent for binding to PD-1.
[0364] In some embodiments, there is provided binding molecule comprising a cytotoxic T- lymphocyte-associated protein 4 (CTLA-4) binding domain that binds CTLA-4, and a PD-1 binding domain that binds PD-1, wherein the CTLA-4 binding domain comprises a heavy chain variable (CTLA-4 VH) region and a light chain variable (CTLA-4 VL) region and the PD-1 binding domain comprises a heavy chain variable (PD-1 VH) region and a light chain variable (PD-1 VL) region that binds PD-1.
[0365] In some embodiments, the binding molecule comprises: (a) a first heavy chain (HC1) comprising from N- to C-terminal order: (i) the CTLA-4 VH region or PD-1 Vnregion and the heavy chain constant region 1 (CHI) comprised in the Fab, and (ii) a first polypeptide of a heterodimeric Fc ; (b) a second heavy chain (HC2) comprising from N- to C-terminal order: (i) the other of the CTLA-4 VH region or PD-1 Vnregion and the heavy chain constant region 1 (CHI) comprised in the Fab, (ii) a second polypeptide of a heterodimeric Fc; (c) a first light chain (LC1) comprising the CTLA-4 VL region or the PD-1 VL region and the CL comprised in the Fab; and (d) a second light chain (LC2) comprising the other of the CTLA-4 VL region or the PD-1 VL region and the CL comprised in the Fab.
[0366] In some embodiments, the binding molecule comprises: (a) a heavy chain 1 (HC1) polypeptide comprising a VL sequence set forth in SEQ ID NO:268, a CHI sequence set forth in SEQ ID NO:363, and a hinge-CH2-CH3 sequence set forth in SEQ ID NO: 364; (b) a light chain 1 (LC1) polypeptide comprising a VH sequence set forth in SEQ ID NO:254 and a CL sequence set forth in SEQ ID NO: 367; (c) a HC2 polypeptide comprising a VH sequence set forth in SEQ ID NO:168,a CHI sequence set forth in SEQ ID NO:371, and a hinge-CH2-CH3 sequence set forth in SEQ ID NO:373; and (d) a LC2 polypeptide comprising a VL sequence set forth in SEQ ID NO: 172 and a CL sequence set forth in SEQ ID NO: 175. In some embodiments, the binding molecule comprises: (a) a HC1 polypeptide that is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 378; (b) a HC2 polypeptide that is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 387; (c) a LC1 polypeptide that is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 381; and (d) a LC2 is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 389. In some embodiments, the binding molecule comprises: (a) a HC1 polypeptide that is or comprises the amino acid sequence set forth in SEQ ID NO: 378; (b) a HC2 polypeptide that is or comprises the amino acid sequence set forth in SEQ ID NO: 387; (c) a LC1 polypeptide that is or comprises the amino acid sequence set forth in SEQ ID NO: 381; and (d) a LC2 polypeptide that is or comprises the amino acid sequence set forth in SEQ ID NO: 389.
[0367] In some embodiments, the binding molecule comprises: (a) a heavy chain 1 (HC1) polypeptide comprising a VL sequence set forth in SEQ ID NO: 172, a CHI sequence set forth in SEQ ID NO:363, and a hinge-CH2-CH3 sequence set forth in SEQ ID NO: 364; (b) a LC1 polypeptide comprising a VH sequence set forth in SEQ ID NO: 168, and a CL sequence set forth in SEQ ID NO:367; (c) a HC2 polypeptide comprising a VH sequence set forth in SEQ ID NO:254, a CHI sequence set forth in SEQ ID NO:371, and a hinge-CH2-CH3 sequence set forth in SEQ ID NO: 373; and (d) a LC2 polypeptide comprising a VL sequence set forth in SEQ ID NO:268, and a CL sequence set forth in SEQ ID NO: 175. In some embodiments, the binding molecule comprises: (a) a HC1 polypeptide that is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 360; (b) a HC2 polypeptide that is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 370; (c) a LC1 polypeptide that is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 366 and (d) a LC2 that is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 375. In some embodiments, the binding molecule comprises: (a) a HC1 polypeptide that is or comprises an amino acid sequence set forth in SEQ ID NO: 360; (b) a HC2 polypeptide that is or comprises an amino acid sequence set forth in SEQ ID NO: 370; (c) a LC1 polypeptide that is or comprises an amino acid sequence set forth in SEQ ID NO: 366 and (d) a LC2 polypeptide that is or comprises an amino acid sequence set forth in SEQ ID NO: 375.
E. Exemplary Feature of Bispecific Binding Molecules
[0368] In some aspects, the provided binding molecules have one or more specified functional features, such as binding properties, including binding to particular epitopes or exhibiting lower or reduced binding to a related but non-specific antigen. In some aspects, the provided binding molecules can bind to an epitope that is similar to or overlaps with epitopes of other binding molecules, such as reference antibodies, and/or exhibit particular binding affinities. In some aspects, the provided binding molecules can bind to an epitope that is different from epitopes of other binding molecules, e.g., binding a conformational epitope.
[0369] In some embodiments, the provided binding molecules specifically bind to a cytotoxic T- lymphocyte-associated protein 4 (CTLA-4) protein. In some of any of the embodiments provided herein, CTLA-4 refers to human CTLA-4. In particular embodiments, the provided binding molecules specifically bind to human CTLA-4.
[0370] In some embodiments, the provided binding molecules specifically bind to PD-1 protein. In some of any of the embodiments provided herein, PD-1 refers to human PD-1. In particular embodiments, the provided binding molecules specifically bind to human PD-1.
[0371] A molecule is said to exhibit "specific binding" or "preferential binding" if it reacts or associates more frequently, more rapidly, with greater duration and/or with greater affinity with a particular cell or substance (e.g. a cell expressing a CTLA-4 or PD-1 antigen or a recombinant CTLA- 4 or PD-1 antigen) than it does with alternative cells or substances. A binding molecule specifically binds or preferentially binds to a target (i.e. CTLA-4 or PD-1) if it binds with greater affinity, avidity, more readily, and/or with greater duration than it binds to other substances. For example, a binding molecule that specifically or preferentially binds to CTLA-4 or PD-1 is a binding molecule that binds CTLA-4 or PD-1 with greater affinity, avidity, more readily, and/or with greater duration than it binds to a different protein antigen. It is also understood by reading this definition that specific binding or preferential binding does not necessarily require (although it can include) exclusive binding. Methods to determine such specific or preferential binding are also well known in the art, e.g., an immunoassay.
[0372] The observation that a binding molecule binds to a target antigen (e.g., CTLA-4 or PD-1) or specifically binds to a target antigen does not necessarily mean that it binds to the target antigen from every species. For example, in some embodiments, features of binding to the target antigen, such as the ability to specifically bind thereto and/or to compete for binding thereto with a reference antibody, and/or to bind with a particular affinity or compete to a particular degree, in some embodiments, refers to the ability with respect to a human target antigen protein and the binding molecule may not have this feature with respect to a target antigen of another species such as mouse.
[0373] In some embodiments, the provided binding molecules are capable of binding CTLA-4, such as human CTLA-4, with at least a certain affinity, as measured by any of a number of known methods. In some embodiments, the affinity is represented by absorbance. In some embodiments, the affinity is represented by equilibrium dissociation constant (KD).
[0374] In some embodiments, the provided binding molecules are capable of binding PD-1, such as human PD-1, with at least a certain affinity, as measured by any of a number of known methods. In some embodiments, the affinity is represented by absorbance. In some embodiments, the affinity is represented by equilibrium dissociation constant (KD).
[0375] A variety of assays can be used for assessing absorbance. For example, enzyme linked immunosorbent assays (ELISA) is a plate-based assay technique designed for detecting and quantifying soluble substances. In an ELISA, the antigen (target macromolecule) is immobilized on a solid surface (microplate) and then complexed with an antibody that is linked to a reporter enzyme. Detection is accomplished by measuring the activity of the reporter enzyme via incubation with the appropriate substrate to produce a measurable product.
[0376] A variety of assays are known for assessing binding affinity, equilibrium dissociation constant (KD), equilibrium association constant (KA), ECSO, on-rate (association rate constant; konor ka; units of 1/Ms or M ’s 1) and the off-rate (dissociation rate constant; koff or k ; units of 1/s or s'1) and/or determining whether a binding molecule (e.g., an antibody or fragment thereof) specifically binds to a particular ligand e.g., an antigen, such as a CTLA-4 or PD-1 protein). One can determine the binding affinity of a binding molecule, e.g., an antibody or an antigen-binding fragment thereof, for an antigen, e.g., CTLA-4 or PD-1, such as human CTLA-4 or PD-1, such as by using any of a number of binding assays that are well known. For example, in some embodiments, a BIAcore® instrument can be used to determine the binding kinetics and constants of a complex between two proteins (e.g., an antibody or fragment thereof, and an antigen, such as a CTLA-4 protein), using surface plasmon resonance (SPR) analysis (see, e.g., Scatchard et al. , Am. N.Y. Acad. Sci. 51:660, 1949; Wilson, Science 295:2103, 2002; Wolff et al., Cancer Res. 53:2560, 1993; and U.S. Patent Nos. 5,283,173, 5,468,614, or the equivalent). [0377] SPR measures changes in the concentration of molecules at a sensor surface as molecules bind to or dissociate from the surface. The change in the SPR signal is directly proportional to the change in mass concentration close to the surface, thereby allowing measurement of binding kinetics between two molecules. The dissociation rate constant (koff or kd), the association rate constant (kon or ka) and/or equilibrium dissociation constant (KD) and/or equilibrium association constant (KA) for the complex can be determined by monitoring changes in the refractive index with respect to time as buffer is passed over the chip. Other suitable assays for measuring the binding of one protein to another include, for example, immunoassays such as enzyme linked immunosorbent assays (ELISA) and radioimmunoassays (RIA), or determination of binding by monitoring the change in the spectroscopic or optical properties of the proteins through fluorescence, UV absorption, circular dichroism, or nuclear magnetic resonance (NMR). Other exemplary assays include, but are not limited to, Western blot, analytical ultracentrifugation, spectroscopy, flow cytometry, sequencing, genetic reporter assays, flow cytometry, and other methods for detection of expressed nucleic acids or binding of proteins.
[0378] In some embodiments, binding can be determined under conditions that mimic those that exist in natural environments in the body of a subject. For instance, acidic pH such as pH about 5.0 mimics more closely the endosomes in a cell. In contrast, physiological pH is a more neutral pH 7.35 to 7.45, such as pH about 7.4 and more closely mimics the environment of normal tissues or cells in the blood.
[0379] In some embodiments, the binding molecule exhibits a similar CTLA-4 binding at an acidic pH as present in the microenvironment of tumor (such as at pH 5.0 to 6.8, e.g. pH 6.0 or pH 5.0) compared to CTLA-4 binding at normal pH as present in blood or a normal tissue (such as at pH 7.35 to 7.45, e.g. pH 7.4). In some embodiments, the KD at an acidic pH as present in the microenvironment of tumor (such as at pH 5.0 to 6.8, e.g. pH 6.0 or pH 5.0) is about the same (e.g. 0.7-fold to 1.3-fold) the KD at pH of a non-tumor microenvironment (such as at pH 7.35 to 7.45, e.g. 7.4) as present at normal pH of blood or a normal tissue.
[0380] In some embodiments, properties or features of the provided binding molecules are described in relation to properties observed for another binding molecule, e.g., a reference antibody. In some embodiments, the reference antibody is a human or humanized anti-CTLA-4 antibody. Exemplary reference anti-CTLA-4 antibodies include ipilimumab (VH and VL set forth in SEQ ID Nos: 341 and 347, respectively), tremelimumab (VH and VL set forth in SEQ ID NOs: 342 and 348, respectively), botensilimab (VH and VL set forth in SEQ ID NOs: 343 and 349, respectively), gotistobart (VH and VL set forth in SEQ ID NOs: 344 and 350, respectively), quavonlimab (VH and VL set forth in SEQ ID NOs: 345 and 351, respectively), ipi.101 (VH and VL set forth in SEQ ID NOs: 346 and 352, respectively). Exemplary reference anti-PD-1 antibodies include any described above. In some embodiments, the provided antibody contains heavy and light chain CDRs that are distinct from the
CDRs present in the reference antibody or antibodies. [0381] In some embodiments, a provided binding molecule exhibits a lower or similar binding affinity (e.g. lower absorbance) than the reference antibody to CTLA-4 at an acidic pH as present in the endosomes of a cell (such as at pH 5.0).
[0382] In some embodiments, a provided binding molecule exhibits a higher CTLA-4 binding compared to a reference antibody to CTLA-4 at a neutral pH (such as at pH 7.4) as present at normal pH of blood or a normal tissue. In some embodiments, the CTLA-4 binding of a provided binding molecule at a neutral pH as present in the blood or a normal tissue (such as at pH 7.4) is higher by at least or 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50 or more compared to the reference antibody for CTLA-4 binding at the same pH or condition. In some examples, the CTLA-4 binding is higher by at least 5%, 10%, 15%, 20%, 25%, 35%, 50%, 100%, 2-fold, 5-fold, 10-fold, 20-fold or more. In some embodiments, the absorbance of a provided binding molecule for CTLA-4 binding in an ELISA or other binding assay at a neutral pH as present in the blood or a normal tissue (such as at pH 7.4) is about 1.5-fold, 2-fold, 3-fold, 4-fold, 5- fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 25-fold, 30-fold, 40-fold, 50-fold or 100-fold higher than the absorbance of the reference antibody for CTLA-4 binding at the same pH or condition. In some embodiments, the EC50 of a provided antibody for CTLA-4 binding at an acidic pH as present within a tumor microenvironment or endosome of a cell (such as at pH 5.0) is about 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 25-fold, 30-fold, 40-fold, 50-fold or 100- fold higher than the EC50 of the reference antibody for CTLA-4 binding at the same pH or condition. In some embodiments, the KD of provided antibody for CTLA-4 binding at an acidic pH as present within a tumor microenvironment or endosome of a cell (such as at pH 5.0) is about 1.5-fold, 2-fold, 3- fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 25-fold, 30-fold, 40-fold, 50-fold or 100-fold higher than the KD of the reference antibody for CTLA-4 binding at the same pH or condition.
[0383] In some embodiments, the provided binding molecule can impact safety and efficacy through involvement with the broader immune system. In some embodiments, the provided binding molecule thereof induces a favorable ratio of immune cell subsets in the tumor or blood, such as T cells, CD4+ T cells, CD8+ T cells, and/or Treg cells. In some embodiments, the provided binding molecule induces a favorable ratio of CTLA-4 expression on immune cell subsets in the tumor or blood, such as T cells, CD4+ T cells, CD8+ T cells, and/or Treg cells.
III. NUCLEIC ACIDS AND EXPRESSION VECTORS AND METHODS OF PRODUCING ANTIBODIES
[0384] The present disclosure provides a nucleic acid or nucleic acids encoding an antibody or antigen-binding fragment thereof, such as any described in Section I. The nucleic acid(s) of the present disclosure may comprise a polynucleotide sequence encoding any one of the anti-PD-1 antibodies or antigen-binding fragments thereof disclosed herein. The polypeptide sequences may be used to determine appropriate nucleic acid sequences encoding the particular antibody disclosed thereby. The nucleic acid sequence may be optimized to reflect particular codon "preferences" for various expression systems according to standard methods well known to those of skill in the art.
[0385] Among the provided nucleic acid molecules are those that comprise polynucleotides that encode one or more polypeptides (e.g., an immunoglobulin heavy chain or light chain) of a provided anti-PD-1 antibody. In some embodiments, a nucleic acid molecule comprises a polynucleotide that encodes a heavy chain or a light chain of an anti-PD-1 antibody. In some embodiments, a nucleic acid molecule comprises both a polynucleotide that encodes a heavy chain and a polynucleotide that encodes a light chain of an anti-PD-1 antibody. In some embodiments, a first nucleic acid molecule comprises a first polynucleotide that encodes a heavy chain and a second nucleic acid molecule comprises a second polynucleotide that encodes a light chain. Also provided herein are nucleic acid molecules comprising polynucleotides that encode one or more polypeptides of a binding molecule containing any of the provided anti-PD-1 antibodies, such as a PD-1 -targeted antibody-drug conjugate (ADC) or a bispecific immune cell engager comprising the anti-PD-1 antibody or antigen binding fragment thereof as described herein.
[0386] The present disclosure also provides a nucleic acid or nucleic acids encoding any one of the binding molecules described herein, such as any bispecific binding molecule described herein in Section II. The polypeptide sequences may be used to determine appropriate nucleic acid sequences encoding the binding domains disclosed thereby. The nucleic acid sequence may be optimized to reflect particular codon "preferences" for various expression systems according to standard methods well known to those of skill in the art.
[0387] Among the provided nucleic acid molecules are those that comprise polynucleotides that encode one or more polypeptides (e.g., an immunoglobulin heavy chain or light chain) of a provided binding molecule or binding domain, such as a CTLA-4 binding domain or PD-1 binding domain, and including full-length antibodies and functional antigen-binding fragments. In some embodiments, a nucleic acid molecule comprises a polynucleotide that encodes a heavy chain or a light chain of a binding molecule, including full-length antibodies and functional antigen-binding fragments. In some embodiments, a nucleic acid molecule comprises both a polynucleotide that encodes a heavy chain and a polynucleotide that encodes a light chain of any of the described binding molecules. In some embodiments, a first nucleic acid molecule comprises a first polynucleotide that encodes a heavy chain and a second nucleic acid molecule comprises a second polynucleotide that encodes a light chain. Among the provided nucleic acid molecules are nucleic acid molecules comprising polynucleotides that encode one or more polypeptides of a binding molecules containing any of the provided CTLA-4 binding domains as described herein in Section II.B or any of the provided PD-1 binding domains as described herein in Section I or Section II. A [0388] In some embodiments, the nucleic acid(s) is an isolated nucleic acid(s). The polynucleotides may include those encompassing natural and/or non-naturally occurring nucleotides and bases, e.g., including those with backbone modifications. The terms “nucleic acid molecule”, “nucleic acid”, “sequence of nucleotides”, and “polynucleotide” may be used interchangeably, and refer to a polymer of nucleotides. Such polymers of nucleotides may contain natural and/or non-natural nucleotides, and include, but are not limited to, DNA, RNA, and PNA. “Nucleic acid sequence” refers to the linear sequence of nucleotides that comprise the nucleic acid molecule or polynucleotide.
[0389] In some such embodiments, the heavy chain and the light chain are expressed from one nucleic acid molecule, or from two separate nucleic acid molecules, as two separate polypeptides. In some embodiments, such as when an antibody is an scFv, a single polynucleotide encodes a single polypeptide comprising both a heavy chain variable domain fragment and a light chain variable domain fragment linked together.
[0390] This disclosure is not intended to be limited with regard to the source of the antibody or the manner in which it is made. Various procedures known within the art may be used for the production of monoclonal antibodies directed against PD-1 (See, for example, Antibodies: A Laboratory Manual, Harlow E, and Lane D, 1988, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, incorporated herein by reference). In some embodiments, the anti-PD-1 antibodies, and antigen binding fragments thereof, are recombinantly expressed and produced.
[0391] In some embodiments, a nucleic acid of the present disclosure may be operably linked to a transcriptional control element, e.g., a promoter, and enhancer, etc. Suitable promoter and enhancer elements are known to those of skill in the art.
[0392] In some embodiments, a polynucleotide encoding a heavy chain or light chain of an anti- PD-1 antibody comprises a nucleotide sequence that encodes a leader sequence, which, when translated, is located at the N terminus of the heavy chain or light chain. The leader sequence may be the native heavy or light chain leader sequence, or may be another heterologous leader sequence.
[0393] Nucleic acid molecules may be constructed using conventional recombinant DNA techniques well-known in the art. In some embodiments, a nucleic acid molecule is an expression vector that is suitable for expression in a selected host cell.
[0394] Vectors comprising polynucleotides that encode anti-PD-1 heavy chains and/or anti-PD-1 light chains are provided herein. Vectors comprising polynucleotides that encode anti-PD-1 heavy chains and/or anti-PD-1 light chains are also provided herein. In certain embodiments, the vector is selected from the group consisting of a DNA vector, an RNA vector, a plasmid, a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, and a retroviral vector. In certain embodiments, the vector is an expression vector. A nucleic acid of the present disclosure may be present within an expression vector and/or a cloning vector. An expression vector can include a selectable marker, an origin of replication, and other features that provide for replication and/or maintenance of the vector. Suitable expression vectors include, e.g., plasmids, viral vectors, and the like. Large numbers of suitable vectors and promoters are known to those of skill in the art; many are commercially available for generating a subject recombinant construct. The following vectors are provided by way of example and should not be construed in anyway as limiting: Bacterial: pBs, phagescript, PsiX174, pBluescript SK, pBs KS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene, La Jolla, Calif., USA); pTrc99A, pKK223- 3, pKK233-3, pDR540, and pRIT5 (Pharmacia, Uppsala, Sweden). Eukaryotic: pWLneo, pSV2cat, pOG44, PXR1, pSG (Stratagene) pSVK3, pBPV, pMSG and pSVL (Pharmacia).
[0395] Expression vectors generally have convenient restriction sites located near the promoter sequence to provide for the insertion of nucleic acid sequences encoding heterologous proteins. A selectable marker operative in the expression host may be present. Suitable expression vectors include, but are not limited to, viral vectors (e.g. viral vectors based on vaccinia virus; poliovirus; adenovirus (see, e.g., Li et al., Invest. Opthalmol. Vis. Sci. (1994) 35: 2543-2549; Borras et al., Gene Ther. (1999) 6: 515-524; Li and Davidson, Proc. Natl. Acad. Sci. USA (1995) 92: 7700-7704; Sakamoto et al., H. Gene Ther. (1999) 5: 1088-1097; WO 94/12649, WO 93/03769; WO 93/19191; WO 94/28938; WO 95/11984 and WO 95/00655); adeno-associated virus (see, e.g., Ali et al., Hum. Gene Ther. (1998) 9: 81-86, Flannery et al., Proc. Natl. Acad. Sci. USA (1997) 94: 6916-6921; Bennett et al., Invest. Opthalmol. Vis. Sci. (1997) 38: 2857-2863; Jomary et al., Gene Ther. (1997) 4:683 690, Rolling et al., Hum. Gene Ther. (1999) 10: 641-648; Ali et al., Hum. Mol. Genet. (1996) 5: 591-594; Srivastava in WO 93/09239, Samulski et al., J. Vir. (1989) 63: 3822-3828; Mendelson et al., Virol. (1988) 166: 154- 165; and Flotte et al., Proc. Natl. Acad. Sci. USA (1993) 90: 10613-10617); SV40; herpes simplex virus; human immunodeficiency virus (see, e.g., Miyoshi et al., Proc. Natl. Acad. Sci. USA (1997) 94: 10319-23; Takahashi et al., J. Virol. (1999) 73: 7812-7816); a retroviral vector e.g., Murine Leukemia Virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus); and the like.
[0396] Additional expression vectors suitable for use are, e.g., without limitation, a lentivirus vector, a gamma retrovirus vector, a foamy virus vector, an adeno-associated virus vector, an adenovirus vector, a pox virus vector, a herpes virus vector, an engineered hybrid virus vector, a transposon mediated vector, and the like. Viral vector technology is well known in the art and is described, for example, in Sambrook et al., 2012, Molecular Cloning: A Laboratory Manual, volumes 1-4, Cold Spring Harbor Press, NY), and in other virology and molecular biology manuals. Viruses, which are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno- associated viruses, herpes viruses, and lenti viruses. [0397] In general, a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers, (e.g., WO 01/96584; WO 01/29058; and U.S. Pat. No. 6,326,193).
[0398] In some embodiments, a vector comprises a first polynucleotide sequence encoding a heavy chain and a second polynucleotide sequence encoding a light chain. In some embodiments, the heavy chain and light chain are expressed from the vector as two separate polypeptides. In some embodiments, the heavy chain variable domain fragment and light chain variable domain fragment are expressed as part of a single polypeptide, such as, for example, when the antibody is an scFv.
[0399] In some embodiments, a first vector comprises a polynucleotide that encodes a heavy chain and a second vector comprises a polynucleotide that encodes a light chain. In some embodiments, the first vector and second vector are transfected into host cells in similar amounts (such as similar molar amounts or similar mass amounts). In some embodiments, a mole- or mass-ratio of between 5:1 and 1:5 of the first vector and the second vector is transfected into host cells. In some embodiments, a mass ratio of between 1 : 1 and 1 :5 for the vector encoding the heavy chain and the vector encoding the light chain is used. In some embodiments, a mass ratio of 1:2 for the vector encoding the heavy chain and the vector encoding the light chain is used.
[0400] Also provided is a host cell comprising any of the vectors or nucleic acids disclosed herein. The host cell may be of eukaryotic, prokaryotic, mammalian, or bacterial origin. In various embodiments, anti-PD-1 heavy chains and/or anti-PD-1 light chains may be expressed in prokaryotic cells, such as bacterial cells; or in eukaryotic cells, such as fungal cells (such as yeast), plant cells, insect cells, and mammalian cells. Such expression may be carried out, for example, according to procedures known in the art. Exemplary eukaryotic cells that may be used to express polypeptides include, but are not limited to, COS cells, including COS 7 cells; HEK293 cells, including HEK293-6E cells; CHO cells, including CHO-S, DG44. Lecl3 CHO cells, and FUT8 CHO cells; PER.C6® cells (Crucell); and NSO cells. In some embodiments, anti-PD-1 heavy chains and/or anti-PD-1 light chains may be expressed in yeast. See, e.g., U.S. Publication No. US 2006/0270045 Al. In some embodiments, a particular eukaryotic host cell is selected based on its ability to make desired post-translational modifications to the anti-PD-1 heavy chains and/or anti-PD-1 light chains. For example, in some embodiments, CHO cells produce polypeptides that have a higher level of sialylation than the same polypeptide produced in HEK293 cells.
[0401] Introduction of one or more nucleic acids into a desired host cell may be accomplished by any method, including but not limited to, calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, etc. Nonlimiting exemplary methods are described, e.g., in Sambrook et al., Molecular Cloning, A Laboratory Manual, 3rd ed. Cold Spring Harbor Laboratory Press (2001). Nucleic acids may be transiently or stably transfected in the desired host cells, according to any suitable method.
[0402] Also provided herein are host cells comprising any of the polynucleotides or vectors described herein. In some embodiments host cells are capable of over-expressing heterologous DNAs can be used for the purpose of isolating the genes encoding the antibody, polypeptide or protein of interest. Non-limiting examples of mammalian host cells include but not limited to COS, HeLa, and CHO cells. See also PCT Publication No. WO 87/04462. Suitable non-mammalian host cells include prokaryotes (such as E. coli or B. subtilis) and yeast (such as S. cerevisiae, S. pombe; or K. lactis).
[0403] A method of producing an antibody or antigen-binding fragment thereof that binds to PD- 1 is also provided herein, wherein the method comprises culturing the host cell. In some embodiments, the nucleic acids encoding a subject antibody are introduced directly into a host cell, and the cell incubated under conditions sufficient to induce expression of the encoded antibody. Expression may conveniently be achieved by culturing under appropriate conditions recombinant host cells containing the nucleic acid. Following production by expression, an antibody or antigen-binding fragment thereof, may be isolated and/or purified using any suitable technique, and then used as desired.
[0404] Anti-PD-1 antibodies may be purified by any suitable method. Such methods include, but are not limited to, the use of affinity matrices or hydrophobic interaction chromatography. Suitable affinity ligands such as ligands that bind antibody constant regions. For example, a Protein A, Protein G, Protein A/G, or an antibody affinity column may be used to bind the constant region and to purify an anti-PD-1 antibody. Hydrophobic interactive chromatography, for example, a butyl or phenyl column, may also suitable for purifying some polypeptides such as antibodies. Ion exchange chromatography (e.g., anion exchange chromatography and/or cation exchange chromatography) may also suitable for purifying some polypeptides such as antibodies. Mixed-mode chromatography (e.g., reversed phase/anion exchange, reversed phase/cation exchange, hydrophilic interaction/anion exchange, hydrophilic interaction/cation exchange, etc.) may also suitable for purifying some polypeptides such as antibodies. Many methods of purifying polypeptides are known in the art.
[0405] Antibodies or antigen-binding fragments thereof as provided herein, and encoding nucleic acid molecules and vectors, may be isolated and/or purified, e.g. from their natural environment, in substantially pure or homogeneous form, or, in the case of nucleic acid, free or substantially free of nucleic acid or genes of origin other than the sequence encoding a polypeptide with the desired function.
IV. PHARMACEUTICAL COMPOSITIONS
[0406] Provided herein are compositions containing any of the provided antibodies or antigenbinding fragments or binding molecules containing same as described herein. The pharmaceutical composition can further comprise a pharmaceutically acceptable excipient. For example, the pharmaceutical composition can contain one or more excipients for modifying, maintaining or preserving, for example, the pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption, or penetration of the composition. Such compositions may comprise buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives.
[0407] In some embodiments, the pharmaceutical composition is a solid, such as a powder, capsule, or tablet. For example, the components of the pharmaceutical composition can be lyophilized. In some embodiments, the solid pharmaceutical composition is reconstituted or dissolved in a liquid prior to administration.
[0408] In some embodiments, the pharmaceutical composition is a liquid, for example a provided protein dissolved in an aqueous solution (such as physiological saline or Ringer’s solution). In some embodiments, the pH of the pharmaceutical composition is between about 4.0 and about 8.5 (such as between about 4.0 and about 5.0, between about 4.5 and about 5.5, between about 5.0 and about 6.0, between about 5.5 and about 6.5, between about 6.0 and about 7.0, between about 6.5 and about 7.5, between about 7.0 and about 8.0, or between about 7.5 and about 8.5).
[0409] In some embodiments, the pharmaceutical composition comprises a pharmaceutically- acceptable excipient, for example a filler, binder, coating, preservative, lubricant, flavoring agent, sweetening agent, coloring agent, a solvent, a buffering agent, a chelating agent, or stabilizer. Examples of pharmaceutically-acceptable fillers include cellulose, dibasic calcium phosphate, calcium carbonate, microcrystalline cellulose, sucrose, lactose, glucose, mannitol, sorbitol, maltol, pregelatinized starch, corn starch, or potato starch. Examples of pharmaceutically-acceptable binders include polyvinylpyrrolidone, starch, lactose, xylitol, sorbitol, maltitol, gelatin, sucrose, polyethylene glycol, methyl cellulose, or cellulose. Examples of pharmaceutically-acceptable coatings include hydroxypropyl methylcellulose (HPMC), shellac, corn protein zein, or gelatin. Examples of pharmaceutically-acceptable disintegrants include polyvinylpyrrolidone, carboxymethyl cellulose, or sodium starch glycolate. Examples of pharmaceutically-acceptable lubricants include polyethylene glycol, magnesium stearate, or stearic acid. Examples of pharmaceutically-acceptable preservatives include methyl parabens, ethyl parabens, propyl paraben, benzoic acid, or sorbic acid. Examples of pharmaceutically-acceptable sweetening agents include sucrose, saccharine, aspartame, or sorbitol. Examples of pharmaceutically-acceptable buffering agents include carbonates, citrates, gluconates, acetates, phosphates, or tartrates.
[0410] In some embodiments, the pharmaceutical composition further comprises an agent for the controlled or sustained release of the product, such as injectable microspheres, bio-erodible particles, polymeric compounds (polylactic acid, polyglycolic acid), beads, or liposomes. [0411] In some embodiments, the pharmaceutical composition is sterile. Sterilization may be accomplished by filtration through sterile filtration membranes or radiation. Where the composition is lyophilized, sterilization using this method may be conducted either prior to or following lyophilization and reconstitution. The composition for parenteral administration may be stored in lyophilized form or in solution. In addition, parenteral compositions generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.
[0412] A pharmaceutically acceptable carrier may be a pharmaceutically acceptable material, composition, or vehicle. For example, the carrier may be a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, or some combination thereof. Each component of the carrier must be “pharmaceutically acceptable” in that it must be compatible with the other ingredients of the formulation. It also must be suitable for contact with any tissue, organ, or portion of the body that it may encounter, meaning that it must not carry a risk of toxicity, irritation, allergic response, immunogenicity, or any other complication that excessively outweighs its therapeutic benefits.
[0413] In some embodiments, the pharmaceutical composition is for administration to a subject. Generally, dosages and routes of administration of the pharmaceutical composition are determined according to the size and condition of the subject, according to standard pharmaceutical practice. For example, the therapeutically effective dose can be estimated initially either in cell culture assays or in animal models such as mice, rats, rabbits, dogs, pigs, or monkeys. An animal model may also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans. The exact dosage will be determined in light of factors related to the subject requiring treatment. Dosage and administration are adjusted to provide sufficient levels of the active compound or to maintain the desired effect. Factors that may be taken into account include the severity of the disease state, the general health of the subject, the age, weight, and gender of the subject, time and frequency of administration, drug combination(s), reaction sensitivities, and response to therapy.
[0414] Fong -acting pharmaceutical compositions may be administered every 3 to 4 days, every week, or biweekly depending on the half-life and clearance rate of the particular formulation. The frequency of dosing will depend upon the pharmacokinetic parameters of the molecule in the formulation used. Typically, a composition is administered until a dosage is reached that achieves the desired effect. The composition may therefore be administered as a single dose, or as multiple doses (at the same or different concentrations/dosages) over time, or as a continuous infusion. Further refinement of the appropriate dosage is routinely made. Appropriate dosages may be ascertained through use of appropriate dose-response data. [0415] In some embodiments, the pharmaceutical composition is for administration to a subject through any route, including orally, transdermally, by inhalation, intravenously, intra-arterially, intramuscularly, direct application to a wound site, application to a surgical site, intraperitoneally, by suppository, subcutaneously, intradermally, transcutaneously, by nebulization, intrapleurally, intraventricularly, intra-articularly, intraocularly, or intraspinally.
[0416] A provided pharmaceutical formulation may, for example, be in a form suitable for intravenous infusion.
[0417] In some embodiments, the dosage of the pharmaceutical composition is a single dose or a repeated dose. In some embodiments, the doses are given to a subject once per day, twice per day, three times per day, or four or more times per day. In some embodiments, about 1 or more (such as about 2 or more, about 3 or more, about 4 or more, about 5 or more, about 6 or more, or about 7 or more) doses are given in a week. In some embodiments, multiple doses are given over the course of days, weeks, months, or years. In some embodiments, a course of treatment is about 1 or more doses (such as about 2 or more does, about 3 or more doses, about 4 or more doses, about 5 or more doses, about 7 or more doses, about 10 or more doses, about 15 or more doses, about 25 or more doses, about 40 or more doses, about 50 or more doses, or about 100 or more doses).
[0418] In some embodiments, an administered dose of the pharmaceutical composition is about 1 pg of protein per kg subject body mass or more (such as about 2 pg of protein per kg subject body mass or more, about 5 pg of protein per kg subject body mass or more, about 10 pg of protein per kg subject body mass or more, about 25 pg of protein per kg subject body mass or more, about 50 pg of protein per kg subject body mass or more, about 100 pg of protein per kg subject body mass or more, about 250 pg of protein per kg subject body mass or more, about 500 pg of protein per kg subject body mass or more, about 1 mg of protein per kg subject body mass or more, about 2 mg of protein per kg subject body mass or more, or about 5 mg of protein per kg subject body mass or more).
V. METHODS OF USE
[0419] The antibodies and antigen-binding fragments or PD-1 binding molecules containing the same described herein, including anti-PD-l/anti-CTLA-4 bispecific antibodies containing the same as described herein or any of the multispecific binding molecules described herein, including pharmaceutical compositions thereof, can be used in a variety of therapeutic applications, such as the treatment of a disease. Such methods and uses include therapeutic methods and uses, for example, involving administration of the molecules or compositions containing the same, to a subject having a disease or disorder. In some embodiments, the molecule or engineered cell is administered in an effective amount to effect treatment of the disease or disorder. Uses include uses of the antibodies and antigen-binding fragments, PD-1 binding molecules, including anti-PD-l/anti-CTLA-4 bispecific antibodies containing the same as described herein or any of the multispecific binding molecules described herein, including pharmaceutical compositions thereof, in such methods and treatments, and in the preparation of a medicament in order to carry out such therapeutic methods. In some embodiments, the methods are carried out by administering the antibodies and antigen-binding fragments or PD-1 binding molecules, including anti-PD-l/anti-CTLA-4 bispecific antibodies containing the same as described herein or any of the multispecific binding molecules described herein, including pharmaceutical compositions thereof, or compositions comprising the same, to the subject having or suspected of having the disease or disorder. In some embodiments, the methods thereby treat the disease or disorder in the subject. Also provided are any methods or uses involving administering a provided engineered cell as described, or pharmaceutical compositions containing the same, for treating a disease or disorder.
[0420] In some embodiments, the disease or disorder is a tumor or cancer.
[0421] Illustrative subjects include mammalian subjects, such as farm animals, domestic animals, and human patients. In particular embodiments, the subject is a human subject. The terms subject and patient are used interchangeably herein.
[0422] In some cases, a subject is selected that is known, suspected or that has been identified as having a tumor with cells expressing PD-1. In some cases, a subject is selected that is known, suspected or that has been identified as having a tumor with cells expressing PD-1 and CTLA-4. A provided antibody or antigen-binding fragment, PD-1 binding molecule (including anti-PD-l/anti-CTLA-4 bispecific antibodies containing the same as described herein or any of the multispecific binding molecules described herein), or engineered cell as described herein, or pharmaceutical composition comprising the same, is administered to the subject. The administration to the subject will generally have an effect due to its binding with the PD-1 target. In some cases in which a bispecific binding molecule targeting PD-1 and CTLA-4 is administered, the administration to the subject will generally have an effect due to its binding with the PD-1 target and the CTLA-4 target.
[0423] Generally, alleviation or treatment of a disease or disorder involves the lessening of one or more symptoms or medical problems associated with the disease or disorder. For example, in the case of cancer, the therapeutically effective amount of the drug can accomplish one or a combination of the following: reduce the number of cancer cells; reduce the tumor size; inhibit (i.e., to decrease to some extent and/or stop) cancer cell infiltration into peripheral organs; inhibit tumor metastasis; inhibit, to some extent, tumor growth; and/or relieve to some extent one or more of the symptoms associated with the cancer. In some embodiments, a composition of this disclosure can be used to prevent the onset or reoccurrence of the disease or disorder in the subject.
[0424] In some embodiments, the antibody or antigen-binding fragment, PD-1 binding molecule (including anti-PD-l/anti-CTLA-4 bispecific antibodies containing the same as described herein or any of the multispecific binding molecules described herein) or engineered cell as described herein, or pharmaceutical composition comprising the same, can be used to inhibit growth of mammalian cancer cells (such as human cancer cells). A method of treating cancer can include administering an effective amount of any of the pharmaceutical compositions described herein to a subject with cancer. The effective amount of the pharmaceutical composition can be administered to inhibit, halt, or reverse progression of cancers. Human cancer cells can be treated in vivo, or ex vivo. In ex vivo treatment of a human patient, tissue or fluids containing cancer cells are treated outside the body and then the tissue or fluids are reintroduced back into the patient. In some embodiments, the cancer is treated in a human patient in vivo by administration of the therapeutic composition into the patient.
[0425] In some embodiments, the antibody or antigen-binding fragment, PD-1 binding molecule (including anti-PD-l/anti-CTLA-4 bispecific antibodies containing the same as described herein or any of the multispecific binding molecules described herein) or engineered cell as described herein, or pharmaceutical composition comprising the same, are useful in treating, alleviating a symptom of, ameliorating and/or delaying the progression of a cancer or other neoplastic condition. In some embodiments, the cancer is bladder cancer, breast cancer, uterine/cervical cancer, ovarian cancer, prostate cancer, testicular cancer, esophageal cancer, gastrointestinal cancer, pancreatic cancer, colorectal cancer, colon cancer, kidney cancer, head and neck cancer, lung cancer, stomach cancer, germ cell cancer, bone cancer, liver cancer, thyroid cancer, skin cancer, neoplasm of the central nervous system, lymphoma, leukemia, myeloma, sarcoma, and virus-related cancer. In certain embodiments, the cancer is a metastatic cancer, refractory cancer, or recurrent cancer.
[0426] Compositions of the invention can be administered in dosages and routes and at times to be determined in appropriate pre-clinical and clinical experimentation and trials. Compositions may be administered multiple times at dosages within these ranges. Administration of the compositions may be combined with other methods useful to treat the desired disease or condition as determined by those of skill in the art. Typically, precise amount of the compositions of the present disclosure to be administered can be determined by a physician with consideration of individual differences in age, weight, tumor size, extent of infection or metastasis, and condition of the patient (subject).
[0427] In some embodiments, a therapeutically effective dose may be, by way of nonlimiting example, from about 0.01 pg/kg body weight to about 10 mg/kg body weight. In some embodiments, the therapeutically effective dose may be, by way of nonlimiting example, from about 0.01 mg/kg body weight to about 5-10 mg/kg body weight. Common dosing frequencies may range, for example, from twice daily to once a week.
[0428] In some embodiments, a therapeutic amount of an engineered cell composition of the present disclosure is administered. It can generally be stated that a pharmaceutical composition comprising engineered cells, e.g., T cells, as described herein may be administered at a dosage of 104 to 109 cells/kg body weight, such as 105 to 106 cells/kg body weight, including all integer values within those ranges. Engineered cell compositions, such as T cell compositions, may also be administered multiple times at these dosages. The cells can be administered by using infusion techniques that are commonly known in immunotherapy (see, e.g., Rosenberg et al, New Eng. J. of Med. 319: 1676, 1988). The optimal dosage and treatment regime for a particular patient can readily be determined by one skilled in the art of medicine by monitoring the patient for signs of disease and adjusting the treatment accordingly.
[0429] The antibody or antigen-binding fragment, PD-1 binding molecule (including anti-PD- l/anti-CTLA-4 bispecific antibodies containing the same as described herein or any of the multispecific binding molecules described herein), or engineered cell, or pharmaceutical composition comprising the same, of the present disclosure can be administered alone or in combination with other modes of treatment, such as other anti-cancer agents. They can be provided before, substantially contemporaneous with, or after other modes of treatment (i.e., concurrently or sequentially). In some embodiments, the method of treatment described herein can further include administering: radiation therapy, chemotherapy, vaccination, targeted tumor therapy, CAR-T therapy, oncolytic virus therapy, cancer immunotherapy, cytokine therapy, surgical resection, chromatin modification, ablation, cryotherapy, an antisense agent against a tumor target, a siRNA agent against a tumor target, a microRNA agent against a tumor target or an anti-cancer/tumor agent, or a biologic, such as an antibody, cytokine, or receptor extracellular domain-Fc fusion.
VI. ARTICLES OF MANUFACTURE OR KITS
[0430] Provided herein are articles of manufacture and kits comprising the provided compositions In some embodiments, the kit comprises any of the provided compositions and instructions for administering the composition to a subject.
[0431] Kits can optionally include one or more components such as instructions for use, devices and additional reagents e.g., sterilized water or saline solutions for dilution of the compositions and/or reconstitution of lyophilized protein), and components, such as tubes, containers and syringes for practice of the methods. In some embodiments, the kits can further contain reagents for collection of samples, preparation and processing of samples, and/or reagents for quantitating the amount of one or more surface markers in a sample, such as, but not limited to, detection reagents, such as antibodies, buffers, substrates for enzymatic staining, chromagens or other materials, such as slides, containers, microtiter plates, and optionally, instructions for performing the methods. Those of skill in the art will recognize many other possible containers and plates and reagents that can be used in accord with the provided methods.
[0432] In some embodiments, the kits can be provided as articles of manufacture that include packing materials for the packaging of the cells, antibodies or reagents, or compositions thereof, or one or more other components. For example, the kits can contain containers, bottles, tubes, vial and any packaging material suitable for separating or organizing the components of the kit. The one or more containers may be formed from a variety of materials such as glass or plastic. In some embodiments, the one or more containers hold a composition comprising cells or an antibody or other reagents for use in the methods. The article of manufacture or kit herein may comprise the cells, antibodies or reagents in separate containers or in the same container.
[0433] In some embodiments, the one or more containers holding the composition may be a singleuse vial or a multi-use vial, which, in some cases, may allow for repeat use of the composition. In some embodiments, the article of manufacture or kit may further comprise a second container comprising a suitable diluent. The article of manufacture or kit may further include other materials desirable from a commercial, therapeutic, and user standpoint, including other buffers, diluents, filters, needles, syringes, therapeutic agents and/or package inserts with instructions for use.
[0434] In some embodiments, the kit can, optionally, include instructions. Instructions typically include a tangible expression describing the cell composition, reagents and/or antibodies and, optionally, other components included in the kit, and methods for using such. In some embodiments, the instructions indicate methods for using the cell compositions and antibodies for administration to a subject for treating a disease or condition, such as in accord with any of the provided embodiments. In some embodiments, the instructions are provided as a label or a package insert, which is on or associated with the container. In some embodiments, the instructions may indicate directions for reconstitution and/or use of the composition.
VII. DEFINITIONS
[0435] Unless defined otherwise, all terms of art, notations and other technical and scientific terms or terminology used herein are intended to have the same meaning as is commonly understood by one of ordinary skill in the art to which the claimed subject matter pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art.
[0436] It is understood that embodiments of the invention described herein include “consisting” and/or “consisting essentially of’ embodiments. As used herein, the singular form “a”, “an”, and “the” includes plural references unless indicated otherwise. Use of the term “or” herein is not meant to imply that alternatives are mutually exclusive.
[0437] In this application, the use of “or” means “and/or” unless expressly stated or understood by one skilled in the art. In the context of a multiple dependent claim, the use of “or” refers back to more than one preceding independent or dependent claim.
[0438] The term “about” as used herein refers to the usual error range for the respective value readily known to the skilled person in this technical field. Reference to “about” a value or parameter herein includes (and describes) embodiments that are directed to that value or parameter per se. For example, description referring to “about X” includes description of “X”.
[0439] As used herein, “and/or” is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term “and/or” as used in a phrase such as “A and/or B” includes “A and B,” “A or B,” “A” alone, and “B” alone. Likewise, the term “and/or” as used in a phrase such as “A, B, and/or C” encompasses each of the following: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A alone; B alone; and C alone. I ma
[0440] As used herein, recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. For example, if a concentration range is stated as 1% to 50%, it is intended that values such as 2% to 40%, 10% to 30%, or 1% to 3% are expressly enumerated in this specification. These are only examples of what is specifically intended, and all possible combinations of numerical values between and including the lowest value and the highest value enumerated are to be considered to be expressly stated in this disclosure.
[0441] As used herein, "optional" or "optionally" means that the subsequently described event or circumstance does or does not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not. For example, an optionally substituted group means that the group is unsubstituted or is substituted.
[0442] The term “bispecific” as used herein refers to an antibody construct which comprises at least a first binding domain and a second binding domain, wherein the first binding domain binds to one antigen or target (here: CTLA-4), and the second binding domain binds to another antigen or target (here: PD1). Accordingly, antibody constructs according to the invention comprise specificities for at least two different antigens or targets. A bispecific antibody can be a monoclonal antibody that has binding specificity for two different epitopes. Given that the binding molecules are (at least) bispecific, they do not occur naturally and they are markedly different from naturally occurring products. A “bispecific” antibody construct or immunoglobulin is hence an artificial hybrid antibody or immunoglobulin having at least two distinct binding sides with different specificities. Bispecific antibody constructs can be produced by a variety of methods including fusion of hybridomas or linking of Fab' fragments. See, e.g., Songsivilai & Lachmann, Clin. Exp. Immunol. 79:315-321 (1990).
[0443] The term "Fc region" herein is used to define a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. In one embodiment, a human IgG heavy chain Fc region extends from Cys226, or from Pro230, to the carboxyl-terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise specified herein, numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also called the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991.
[0444] “Effector functions” refer to those biological activities attributable to the Fc region of an antibody, which vary with the antibody isotype. Examples of antibody effector functions include: Clq binding and complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g. B cell receptor); and B cell activation.
[0445] The terms “nucleic acid molecule”, “nucleic acid” and “polynucleotide” may be used interchangeably, and refer to a polymer of nucleotides. Such polymers of nucleotides may contain natural and/or non-natural nucleotides, and include, but are not limited to, DNA, RNA, and PNA. “Nucleic acid sequence” refers to the linear sequence of nucleotides comprised in the nucleic acid molecule or polynucleotide.
[0446] The term “isolated” as used herein refers to a molecule that has been separated from at least some of the components with which it is typically found in nature or produced. For example, a polypeptide is referred to as “isolated” when it is separated from at least some of the components of the cell in which it was produced. Where a polypeptide is secreted by a cell after expression, physically separating the supernatant containing the polypeptide from the cell that produced it is considered to be “isolating” the polypeptide. Similarly, a polynucleotide is referred to as “isolated” when it is not part of the larger polynucleotide (such as, for example, genomic DNA or mitochondrial DNA, in the case of a DNA polynucleotide) in which it is typically found in nature, or is separated from at least some of the components of the cell in which it was produced, for example, in the case of an RNA polynucleotide. Thus, a DNA polynucleotide that is contained in a vector inside a host cell may be referred to as “isolated”.
[0447] The terms “polypeptide” and “protein” are used interchangeably to refer to a polymer of amino acid residues, and are not limited to a minimum length. Such polymers of amino acid residues may contain natural or non-natural amino acid residues, and include, but are not limited to, peptides, oligopeptides, dimers, trimers, and multimers of amino acid residues. Both full-length proteins and fragments thereof are encompassed by the definition. The terms also include post-expression modifications of the polypeptide, for example, glycosylation, sialylation, acetylation, phosphorylation, and the like. Furthermore, for purposes of the present disclosure, a “polypeptide” refers to a protein which includes modifications, such as deletions, additions, and substitutions (generally conservative in nature), to the native sequence, as long as the protein maintains the desired activity. These modifications may be deliberate, as through site-directed mutagenesis, or may be accidental, such as through mutations of hosts which produce the proteins or errors due to PCR amplification. [0448] The term "isolated protein" referred to herein means that a subject protein (1) is free of at least some other proteins with which it would typically be found in nature, (2) is essentially free of other proteins from the same source, e.g., from the same species, (3) is expressed by a cell from a different species, (4) has been separated from at least about 50 percent of polynucleotides, lipids, carbohydrates, or other materials with which it is associated in nature, (5) is not associated (by covalent or noncovalent interaction) with portions of a protein with which the "isolated protein" is associated in nature, (6) is operably associated (by covalent or noncovalent interaction) with a polypeptide with which it is not associated in nature, or (7) does not occur in nature. Such an isolated protein can be encoded by genomic DNA, cDNA, mRNA or other RNA, of may be of synthetic origin, or any combination thereof. In certain embodiments, the isolated protein is substantially pure or substantially free from proteins or polypeptides or other contaminants that are found in its natural environment that would interfere with its use (therapeutic, diagnostic, prophylactic, research or otherwise).
[0449] The term “purified” as applied to nucleic acids or proteins generally denotes a nucleic acid or polypeptide that is substantially free from other components as determined by analytical techniques well known in the art (e.g., a purified polypeptide or polynucleotide forms a discrete band in an electrophoretic gel, chromatographic eluate, and/or a media subjected to density gradient centrifugation). For example, a nucleic acid or polypeptide that gives rise to essentially one band in an electrophoretic gel is “purified.” A purified nucleic acid or protein is at least about 50% pure, usually at least about 75%, 80%, 85%, 90%, 95%, 96%, 99% or more pure (e.g., percent by weight or on a molar basis).
[0450] The term “recombinant” indicates that the material (e.g., a nucleic acid or a polypeptide) has been artificially (i.e., non-naturally) altered by human intervention. The alteration can be performed on the material within, or removed from, its natural environment or state. For example, a “recombinant nucleic acid” is one that is made by recombining nucleic acids, e.g., during cloning, affinity modification, DNA shuffling or other well-known molecular biological procedures. A “recombinant DNA molecule,” is comprised of segments of DNA joined together by means of such molecular biological techniques. The term “recombinant protein” or “recombinant polypeptide” as used herein refers to a protein molecule which is expressed using a recombinant DNA molecule. A “recombinant host cell” is a cell that contains and/or expresses a recombinant nucleic acid or that is otherwise altered by genetic engineering, such as by introducing into the cell a nucleic acid molecule encoding a recombinant protein, such as a immunomodulatory protein provided herein. Transcriptional control signals in eukaryotes comprise “promoter” and “enhancer” elements. Promoters and enhancers consist of short arrays of DNA sequences that interact specifically with cellular proteins involved in transcription. Promoter and enhancer elements have been isolated from a variety of eukaryotic sources including genes in yeast, insect and mammalian cells and viruses (analogous control elements, i.e., promoters, are also found in prokaryotes). The selection of a particular promoter and enhancer depends on what cell type is to be used to express the protein of interest.
[0451] As used herein, “substantially pure” means an object species is the predominant species present (i.e., on a molar basis it is more abundant than any other individual species in the composition), and a substantially purified fraction is a composition wherein the object species comprises at least about 50 percent (on a molar basis) of all macromolecular species present. Generally, a substantially pure composition will comprise more than about 80 percent of all macromolecular species present in the composition, for example, in some embodiments, more than about 85%, 90%, 95%, and 99%. In some embodiments, the object species is purified to essential homogeneity (contaminant species cannot be detected in the composition by conventional detection methods) wherein the composition consists essentially of a single macromolecular species.
[0452] The term “substantially similar” or “substantially the same,” as used herein, denotes a sufficiently high degree of similarity between two or more numeric values such that one of skill in the art would consider the difference between the two or more values to be of little or no biological and/or statistical significance within the context of the biological characteristic measured by said value. In some embodiments the two or more substantially similar values differ by no more than about any one of 5%, 10%, 15%, 20%, 25%, or 50%.
[0453] A polypeptide “variant” means a biologically active polypeptide having at least about 80% amino acid sequence identity with the native sequence polypeptide after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Such variants include, for instance, polypeptides wherein one or more amino acid residues are added, or deleted, at the N- or C-terminus of the polypeptide. In some embodiments, a variant will have at least about 80% amino acid sequence identity. In some embodiments, a variant will have at least about 90% amino acid sequence identity. In some embodiments, a variant will have at least about 95% amino acid sequence identity with the native sequence polypeptide.
[0454] As used herein, “percent (%) amino acid sequence identity” and “homology” with respect to a peptide, polypeptide or antibody sequence are defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the specific peptide or polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or MEGALIGNTM (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. [0455] An amino acid substitution may include but are not limited to the replacement of one amino acid in a polypeptide with another amino acid. Exemplary substitutions are shown in Table 6. Amino acid substitutions may be introduced into an antibody of interest and the products screened for a desired activity, for example, retained/improved antigen binding, decreased immunogenicity, or improved ADCC or CDC.
[0456] Amino acids may be grouped according to common side-chain properties:
(1) hydrophobic: Norleucine, Met, Ala, Vai, Leu, He;
(2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin;
(3) acidic: Asp, Glu;
(4) basic: His, Lys, Arg;
(5) residues that influence chain orientation: Gly, Pro;
(6) aromatic: Trp, Tyr, Phe.
[0457] Non-conservative substitutions will entail exchanging a member of one of these classes for another class.
[0458] The term “conservative amino acid substitution” as used herein means an amino acid substitution in which an amino acid residue is substituted by another amino acid residue having a side chain R group with similar chemical properties (e.g., charge or hydrophobicity). Examples of groups of amino acids that have side chains with similar chemical properties include 1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; 2) aliphatic -hydroxyl side chains: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains: lysine, arginine, and histidine; 6) acidic side chains: aspartic acid and glutamic acid; and 7) sulfur-containing side chains: cysteine and methionine. Conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine- valine, glutamate-aspartate, and asparagine-glutamine.
[0459] The term, “corresponding to” with reference to positions of a protein, such as recitation that nucleotides or amino acid positions “correspond to” nucleotides or amino acid positions in a disclosed sequence, such as set forth in the Sequence Listing, refers to nucleotides or amino acid positions identified upon alignment with the disclosed sequence based on structural sequence alignment or using a standard alignment algorithm, such as the GAP algorithm. By aligning the sequences, one skilled in the art can identify corresponding residues, for example, using conserved and identical amino acid residues as guides.
[0460] The term “specifically binds” as used herein means the ability of a protein, under specific binding conditions, to bind to a target protein such that its affinity or avidity is at least 10 times as great, but optionally 50, 100, 250 or 500 times as great, or even at least 1000 times as great as the average affinity or avidity of the same protein to a collection of random peptides or polypeptides of sufficient statistical size. A specifically binding protein need not bind exclusively to a single target molecule but may specifically bind to more than one target molecule. In some cases, a specifically binding protein may bind to a protein that has similarity in structural conformation with the target protein (e.g., paralogs or orthologs). Those of skill will recognize that specific binding to a molecule having the same function in a different species of animal (i.e., ortholog) or to a molecule having a substantially similar epitope as the target molecule (e.g., paralog) is possible and does not detract from the specificity of binding which is determined relative to a statistically valid collection of unique non-targets (e.g., random polypeptides). Thus, a protein of the invention may specifically bind to more than one distinct species of target molecule due to cross-reactivity. Solid-phase ELISA immunoassays, ForteBio Octet or Biacore measurements can be used to determine specific binding between two proteins. Generally, interactions between two binding proteins have dissociation constants (Kd) less than about 1x105 M, and often as low as about 1 x 10 12 M. In certain aspects of the present disclosure, interactions between two binding proteins have dissociation constants of less than about 1x106 M, 1x107 M, 1x10 8 M, 1x10 9 M, 1x10 10 M, or 1x10 11 M or less.
[0461] The term “affinity” with reference to binding refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule (for example, an antibody) and its binding partner (for example, an antigen). The affinity or the apparent affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (KD) or the Ko-apparent, respectively. Affinity can be measured by common methods known in the art (such as, for example, ELISA KD, KinExA, flow cytometry, and/or surface plasmon resonance devices), including those described herein. Such methods include, but are not limited to, methods involving BIAcore®, Octet®, or flow cytometry. In some embodiments, the KD of the antigen-binding molecule is measured by flow cytometry using an antigen-expressing cell line and fitting the mean fluorescence measured at each antibody concentration to a non-linear one-site binding equation (Prism Software graphpad).
[0462] The term “vector” is used to describe a polynucleotide that can be engineered to contain a cloned polynucleotide or polynucleotides that can be propagated in a host cell. A vector can include one or more of the following elements: an origin of replication, one or more regulatory sequences (such as, for example, promoters and/or enhancers) that regulate the expression of the polypeptide of interest, and/or one or more selectable marker genes (such as, for example, antibiotic resistance genes and genes that can be used in colorimetric assays, for example, P-galactosidase). The term “expression vector” refers to a vector that is used to express a polypeptide of interest in a host cell.
[0463] A “host cell” refers to a cell that may be or has been a recipient of a vector or isolated polynucleotide. Host cells may be prokaryotic cells or eukaryotic cells. Exemplary eukaryotic cells include mammalian cells, such as primate or non-primate animal cells; fungal cells, such as yeast; plant cells; and insect cells. Nonlimiting exemplary mammalian cells include, but are not limited to, NSO cells, PER.C6® cells (Crucell), and 293 and CHO cells, and their derivatives, such as 293-6E, CHO- DG44, CHO-K1, CHO-S, and CHO-DS cells. Host cells include progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or in genomic DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation. A host cell includes cells transfected in vivo with a polynucleotide(s) a provided herein.
[0464] The term “expression” refers to the process by which a polynucleotide is transcribed from a DNA template (such as into an mRNA or other RNA transcript) and/or the process by which a transcribed mRNA is subsequently translated into peptide, polypeptides or proteins. Transcripts and encoded polypeptides may be collectively referred to as “gene product.” If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell.
[0465] The term “tumor microenvironment” as used herein refers to the environment in which a tumor exists, which is the non-cellular area within the tumor and the area directly outside the tumorous tissue but does not pertain to the intracellular compartment of the cancer cell itself. The tumor and the tumor microenvironment are closely related and interact constantly. A tumor can change its microenvironment, and the microenvironment can affect how a tumor grows and spreads. Typically, the tumor microenvironment has a low pH in the range of 5.6 to 6.8, more commonly in the range of 6.0 to 6.8, in the range of 6.2-6.8. On the other hand, a normal physiological pH is in the range of 7.2- 7.8, more commonly 7.35 to 7.45, such as about 7.4. The tumor microenvironment is also known to have lower concentration of glucose and other nutrients, but higher concentration of lactic acid, in comparison with blood plasma. Furthermore, the tumor microenvironment can have a temperature that is 0.3 to 1° C. higher than the normal physiological temperature. The tumor microenvironment has been discussed in Gillies et al., “MRI of the Tumor Microenvironment,” Journal of Magnetic Resonance Imaging, vol. 16, pp. 430-450, 2002, hereby incorporated by reference herein its entirety. The term “non-tumor microenvironment” refers to a microenvironment at a site other than a tumor.
[0466] The terms “individual” and “subject” are used interchangeably herein to refer to an animal; for example a mammal. The term patient includes human and veterinary subjects. In some embodiments, methods of treating mammals, including, but not limited to, humans, rodents, simians, felines, canines, equines, bovines, porcines, ovines, caprines, mammalian laboratory animals, mammalian farm animals, mammalian sport animals, and mammalian pets, are provided. The subject can be male or female and can be any suitable age, including infant, juvenile, adolescent, adult, and geriatric subjects. In some embodiments, the subject to receive the treatment can be a patient, designating the fact that the subject has been identified as having a disorder of relevance to the treatment, or being at adequate risk of contracting the disorder. In particular embodiments, the subject is a human, such as a human patient.
[0467] The term “composition” refers to any mixture of two or more products, substances, or compounds, including cells or antibodies. It may be a solution, a suspension, liquid, powder, a paste, aqueous, non-aqueous or any combination thereof. The preparation is generally in such form as to permit the biological activity of the active ingredient (e.g. antibody) to be effective.
[0468] A “pharmaceutically acceptable carrier” refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, which is nontoxic to a subject. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative.
[0469] As used herein, the term “treatment” or “treating” refers to clinical intervention designed to alter the natural course of the individual or cell being treated during the course of clinical pathology. Desirable effects of treatment include decreasing the rate of disease progression, ameliorating or palliating the disease state, and remission or improved prognosis. An individual is successfully “treated”, for example, if one or more symptoms associated with a disorder (e.g., an eosinophil- mediated disease) are mitigated or eliminated. For example, an individual is successfully “treated” if treatment results in increasing the quality of life of those suffering from a disease, decreasing the dose of other medications required for treating the disease, reducing the frequency of recurrence of the disease, lessening severity of the disease, delaying the development or progression of the disease, and/or prolonging survival of individuals.
[0470] An “effective amount” refers to at least an amount effective, at dosages and for periods of time necessary, to achieve the desired or indicated effect, including a therapeutic or prophylactic result. An effective amount can be provided in one or more administrations. A “therapeutically effective amount” is at least the minimum dose of cells required to effect a measurable improvement of a particular disorder. In some embodiments, a therapeutically effective amount is the amount of a composition that reduces the severity, the duration and/or the symptoms associated with cancer, viral infection, microbial infection, or septic shock in an animal. A therapeutically effective amount herein may vary according to factors such as the disease state, age, sex, and weight of the patient. A therapeutically effective amount may also be one in which any toxic or detrimental effects of the antibody are outweighed by the therapeutically beneficial effects. A “prophylactically effective amount” refers to an amount effective, at the dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically but not necessarily, since a prophylactic dose is used in subjects prior to or at the earlier stage of disease, the prophylactically effective amount can be less than the therapeutically effective amount.
[0471] A “disease” or “disorder” as used herein refers to a condition where treatment is needed and/or desired.
[0472] The term “tumor cell”, “cancer cell”, “cancer”, “tumor”, and/or “neoplasm”, unless otherwise designated, are used herein interchangeably and refer to a cell (or cells) exhibiting an uncontrolled growth and/or abnormal increased cell survival and/or inhibition of apoptosis which interferes with the normal functioning of bodily organs and systems. Included in this definition are benign and malignant cancers, polyps, hyperplasia, as well as dormant tumors or micrometastases.
[0473] The terms “cancer” and “tumor” encompass solid and hematological/lymphatic cancers and also encompass malignant, pre-malignant, and benign growth, such as dysplasia. Also, included in this definition are cells having abnormal proliferation that is not impeded (e.g. immune evasion and immune escape mechanisms) by the immune system (e.g. virus infected cells). Exemplary cancers include, but are not limited to: basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; brain and central nervous system cancer; breast cancer; cancer of the peritoneum; cervical cancer; choriocarcinoma; colon and rectum cancer; connective tissue cancer; cancer of the digestive system; endometrial cancer; esophageal cancer; eye cancer; cancer of the head and neck; gastric cancer (including gastrointestinal cancer); glioblastoma; hepatic carcinoma; hepatoma; intra-epithelial neoplasm; kidney or renal cancer; larynx cancer; leukemia; liver cancer; lung cancer e.g., small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung); melanoma; myeloma; neuroblastoma; oral cavity cancer (lip, tongue, mouth, and pharynx); ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; cancer of the respiratory system; salivary gland carcinoma; sarcoma; skin cancer; squamous cell cancer; stomach cancer; testicular cancer; thyroid cancer; uterine or endometrial cancer; cancer of the urinary system; vulval cancer; lymphoma including Hodgkin's and non-Hodgkin's lymphoma, as well as B-cell lymphoma (including low grade/follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediate grade/follicular NHL; intermediate grade diffuse NHL; high grade immunobias tic NHL; high grade lymphoblastic NHL; high grade small non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's Macroglobulinemia; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); Hairy cell leukemia; chronic myeloblastic leukemia; as well as other carcinomas and sarcomas; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal vascular proliferation associated with phakomatoses, edema (such as that associated with brain tumors), and Meigs' syndrome.
[0474] As used herein, combination refers to any association between or among two or more items. The combination can be two or more separate items, such as two compositions or two collections, can be a mixture thereof, such as a single mixture of the two or more items, or any variation thereof. The elements of a combination are generally functionally associated or related.
[0475] As used herein, a kit is a packaged combination that optionally includes other elements, such as additional agents and instructions for use of the combination or elements thereof, for a purpose including, but not limited to, therapeutic uses.
[0476] The term “wild-type” or “natural” or “native,” which are used interchangeably, as used herein is used in connection with biological materials such as nucleic acid molecules, proteins, host cells, and the like, that are found in nature and not modified by human intervention.
VIII. EXAMPLES
[0477] The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention.
Example 1: Generation of Anti-PD-1 Antibodies
[0478] Anti-PD-1 antibodies were computationally designed via a series of steps to identify novel antibodies with potentially desirable properties. The process included: a) Analyzing the structure, function, and relevant biological context of the PD1 antigen and existing antibodies that may have potential for PD1 targeting. b) Generating in silico models of candidate antibodies using molecular modeling techniques and predicting their properties, such as binding affinity and stability, using computational methods, such as molecular dynamics simulations. c) Using protein engineering algorithms to introduce mutations, deletions, or insertions in the antibody sequences to optimize their properties, such as affinity, specificity, stability, immunogenicity, and isoelectric point. d) Assessing the designed antibody sequences using computational methods, such as molecular docking and molecular dynamics simulations, to validate their binding interactions with PD1 and stability in different environments. e) Refining and optimizing the antibody designs iteratively based on the computational and experimental results, using a feedback loop between computational predictions and experimental validation. [0479] Coding sequences for the computationally designed antibodies were ligated into a plasmid and expressed in HEK293 cells or CHO cells with nucleic acid encoding a constant heavy chain (SEQ ID NO: 174) with a heavy chain signal peptide (SEQ ID NO: 176), and nucleic acid encoding a constant light chain (SEQ ID NO: 175) with a light chain signal peptide (SEQ ID NO: 177). The expressed antibodies were analyzed for purity using SDS-PAGE under reducing and non-reducing conditions.
[0480] Table El lists sequence identifiers (SEQ ID NO:) corresponding to amino acid (aa) sequences of the variable heavy (VH) chain and variable light (VL) chain of the designed antibodies, including complementarity determining regions (CDRs, by Kabat numbering) of each chain.
Example 2: Functional Analyses of Anti-PD-1 Antibodies
[0481] The engineered antibodies generated in HEK293 cells as described in Example 1 were tested for binding to human PD-1 using Enzyme-Linked Immuno-Sorbent Assay (ELISA) and compared to a reference antibody (the tested concentrations of antibodies ranged from 0.002 ng/mL to 1000 ng/mL). As shown in FIG. 1, the EC50 values of the engineered antibodies were comparable to that of the reference antibody, indicating a strong affinity of the engineered antibodies towards human PD-1.
[0482] The engineered antibodies were tested for inhibition of PD-1 binding to human PD-L1 a standard reporter assay (FIG. 3). In this functional ELISA, wells were coated with recombinant Human PD-l/Fc Chimera (Sino Biological, Inc., Catalog # 10377-H02H) at 2 pg/mL in the plate. The antibody constructs were tested for their ability to block the binding of 3 pg/mL of recombinant Human PD-L1- His (Sino Biological, Inc., Catalog # 10084-H08H) to human PD-1 as detected via 0.1 pg/mL Anti- His/HRP antibody after washing.
[0483] As shown in FIG. 2 (also set forth in Table E2), the half maximal inhibitory concentration (IC50) of the antibodies were comparable to that of the reference anti-PD-1 antibody, indicating suppression of PD-Ll activity.
[0484] Table E2: inhibition of PD-1 binding to human PD-L1
Example 3: Binding Kinetics to PD-1 at Different pH Conditions
[0485] The engineered antibodies generated in HEK293 cells as described in Example 1 were measured for binding kinetics to PD-1 at different pH conditions. Biolayer interferometry (BLI) was used to measure equilibrium dissociation constant (KD), association rate constant (kon), and dissociation rate constant (kdis) of the interaction between the anti-PD-1 antibodies as described above and PD-1 at pH 6.0 or pH 7.4, and compared to the reference antibody. Briefly, BLI is an optical analytical technique that analyzes the interference pattern of white light reflected from two surfaces: a layer of immobilized protein on the biosensor tip, and an internal reference layer. Any change in the number of molecules bound to the biosensor tip causes a shift in the interference pattern that can be measured in real-time. As shown in Table E3A and Table E3B, the computationally designed antibodies exhibited differences in kinetics relative to the reference antibody and to each other.
Example 4: Thermal Stability of Additional Antibodies
[0486] Additional engineered antibodies were generated in CHO cells as described in Example 1 and their thermal stability was measured using Differential Scanning Fluorimetry.
[0487] Differential Scanning Fluorimetry (DSF) is a widely used biophysical technique to study the thermal stability of proteins, including antibodies. It measures the change in fluorescence intensity of a protein-specific fluorophore as a function of temperature, which can provide information about the protein’s melting temperature Tm and stability.
[0488] Temperature was increased from a starting point of 20 °C to a final temperature of 90 °C at a rate of 1.0 °C/min, with continuous monitoring of the fluorescence signal at the excitation and emission wavelengths of the protein-specific fluorophore.
[0489] The change in fluorescence intensity was plotted as a function of temperature to obtain the thermal melt curve for the antibody sample. The Tm values correspond to the temperature at the midpoint of an unfolding transition and can be determined from the inflection point of the thermal melt curve.
• Tonset: The temperature at which the protein structure begins to unfold, that is, the temperature at which the protein structure begins to change
• Tml : The temperature at which half of the protein's first domain is opened
• Tm2: The temperature at which half of the protein’s second domain is opened
• Tm3: The temperature at which half of the protein’s third domain is opened
• N/A means that we detected it, but the change value was too small to be recognized by the analysis software.
[0490] The thermal melt for tested antibodies are shown in Table E4A, which was comparable to that of the reference anti-PD-1 antibody. Thermal melt curves for exemplary antibodies are further shown in FIGs. 4A-4F.
[0491] The binding kinetics were then measured using BLI as described in Example 3. This study was performed to measure the binding affinity of the anti-PD-1 antibodies for human PD-1 using Octet RFD 384. The assay was performed at 30°C and at 1000 rpm. The antibody construct was firstly immobilized onto AHC2 biosensor. Human PD-1 was applied as analyte for association and dissociation steps.
[0492] As shown in Table E4B, Table E4C, and Table E4D the computationally designed antibodies exhibited differences in kinetics relative to the reference antibody and to each other. Example 5: Generation of Anti-CTLA-4 Antibodies
[0493] Anti-CTLA-4 antibodies were computationally designed via a series of steps to identify novel antibodies with potentially desirable properties. The process included:
1) Analyzing the structure, function, and relevant biological context of the CTLA-4 antigen and existing antibodies that may have potential for CTLA-4 targeting.
2) Generating in silico models of candidate antibodies using molecular modeling techniques and predicting their properties, such as binding affinity and stability, using computational methods, such as molecular dynamics simulations.
3) Using protein engineering algorithms to introduce mutations, deletions, or insertions in the antibody sequences to optimize their properties, such as affinity, specificity, stability, immunogenicity, and isoelectric point.
4) Assessing the designed antibody sequences using computational methods, such as molecular docking and molecular dynamics simulations, to validate their binding interactions with CTLA- 4 and stability in different environments.
5) Refining and optimizing the antibody designs iteratively based on the computational and experimental results, using a feedback loop between computational predictions and experimental validation.
[0494] Coding sequences for the computationally designed antibodies were recombinantly expressed in HEK293 cells. Briefly, the gene encoding the protein-of-interest was synthesized via homologous recombination amplification, and the target gene was inserted into the an expression vector via restriction enzyme digestion and ligation with nucleic acid encoding a constant heavy chain (SEQ ID NO: 330 or SEQ ID NO: 335) with a heavy chain signal peptide (SEQ ID NO: 333), and nucleic acid encoding a constant light chain (SEQ ID NO: 332) with a light chain signal peptide (SEQ ID NO: 334). The vectors were sequenced to confirm the correct insertion of the target gene. The vectors were transiently transfected into host cells and after culture, protein was purified from the culture supernatant via affinity chromatography or a combination of chromatography methods. The final purified product was subjected to buffer exchange into the storage buffer. Methods such as SDS-PAGE, UV (OD280), and SEC were used to assess the purity and quantity of the crude samples after each purification step as well as the final protein product.
[0495] Table E5 lists sequence identifiers (SEQ ID NO:) corresponding to amino acid (aa) sequences of the variable heavy (VH) chain and variable light (VL) chain of the designed antibodies, including complementarity determining regions (CDRs, by Kabat numbering) of each chain.
Example 6: Functional Analyses of Anti-CTLA-4 Antibodies
[0496] The engineered antibodies generated in HEK293 cells as described in Example 5 were tested for binding to recombinant human CTLA-4 using Enzyme-Linked Immuno-Sorbent Assay (ELISA) at different pH’s.
[0497] The tested concentrations of antibodies ranged from 0.1152 ng/mL to 1800 ng/mL and results were compared to reference antibodies (e.g., Reference 1, ipilimumab; Reference2, tremelimumab; Referenced gotistobart; and Referenced, ipi.101). As shown in FIGs. 5A-5S, the computationally designed antibodies exhibited differences in binding relative to the reference antibodies and to each other at pH 5.0 and pH 7.4.
[0498] Additionally, antibodies were tested for binding to human CTLA-4 using ELISA at different pH conditions, and EC50 and absorbance were measured. As shown in FIGs. 6A-6C and Table E6, the computationally designed antibodies exhibited differences in binding relative to the reference antibodies and to each other at different pH’s.
[0499] Among the antibodies are antibodies that exhibit better binding at neutral pH than acidic pH. In many cases, the ratio of binding at neutral pH (e.g., pH 7.4) than acidic pH (e.g., pH 5.0) was substantially increased compared to reference antibodies, while retaining similar or better binding at pH 7.4 than reference antibodies. In particular, it is found herein that antibodies with such a pH-dependent binding feature are enriched in antibodies that contain one or more of the following amino acids in the VH region: a histidine (H) at position 98, an arginine (R) at position 31, a glutamic acid (E) or a histidine (H) at position 53, a histidine (H) at position 58, and a histidine (H) at position 97, or any combination thereof; and/or in the VL region: a histidine (H) at position 27a, a histidine (H) at position 30, a histidine (H) at position 31 , and a histidine (H) at position 90, or any combination thereof, each based on Kabat numbering. Without wishing to be bound by theory, it is believed that an antibody with higher activity at neutral pH (e.g., pH 7.4) than at acidic pH (e.g., pH 5.0) will exhibit less risk of toxic effect because the antibody can dissociate from CTLA-4 upon internalization of the antibody-CTLA-4 complex, thereby allowing CTLA-4 and the anti-CTLA-4 antibody to recycle to the cell surface. The concept that recycling of CTLA-4 is important in human immune function is supported by the observation that defects in this pathway due to lipopolysaccharide (LPS)-responsive and beige-like anchor protein (LRBA) deficiency are associated with autoimmunity. Immunotherapy-related adverse effect (irAE) can also be caused by loss of CTLA-4 at the cell surface. For anti-CTLA-4 antibodies that display pH sensitive binding to CTLA-4, as the pH drops in early endosomes, it is more likely the anti-CTLA4 antibodies will dissociate from CTLA-4, enabling CTLA-4 and the anti-CTLA-4 antibodies to traffic back to the cell surface via interaction with LBRA and the neonatal Fc receptor (FcRn) respectively. Contrarily, CTEA-4 interaction with anti-CTEA4 antibodies that bind better at a lower pH can cause depletion of both CTEA-4 and the anti-CTEA-4 antibody via lysosomal degradation. This property is critical for proper CTEA-4 function and balance between immune system activation and self-immune tolerance. Thus, the provided antibodies are expected to maintain therapeutic efficacy while also being safe due to critical properties enabling proper CTLA-4 function and balance between immune system activation and self-immune tolerance.
[0500] Among the antibodies are antibodies that exhibit better binding to the FcGRIIIA receptor than antibodies comprising an IgGl constant region domain. Without wishing to be bound by theory, it is believed that a significant contributor to the anti-tumor response of anti-CTLA-4 antibodies in the clinic involves depletion of Tregs via Fc gamma receptor engagement and mediated antibody-dependent cellular cytotoxicity & phagocytosis. Priming of the immune response via FcR engagement is also a suggested contributor to the mechanism of anti-CTEA4 antibodies in cancer treatment. Thus the provided antibodies are expected to alter the ratio of effector T cells to regulatory T cells via Fc gamma receptor interaction in the tumor microenvironment in a favorable way.
Example 7: Binding Kinetics to Human CTLA-4 at Different pH Conditions
[0501] The engineered antibodies generated in HEK293 cells as described in Example 1 were measured for binding kinetics to human CTEA-4 at different pH conditions. Biolayer interferometry (BEI) was used to measure equilibrium dissociation constant (KD), association rate constant (kon), and dissociation rate constant (kdis) of the interaction between the anti-CTEA-4 antibodies as described above and CTLA-4 at pH 5.0 or pH 7.4, and compared to a reference antibody. Briefly, BLI is an optical analytical technique that analyzes the interference pattern of white light reflected from two surfaces: a layer of immobilized protein on the biosensor tip, and an internal reference layer. Any change in the number of molecules bound to the biosensor tip causes a shift in the interference pattern that can be measured in real-time. As shown in Table E7A and Table E7B the computationally designed antibodies exhibited differences in kinetics relative to the reference antibody and to each other.
Example 8: Effect of Anti-CTLA-4 Antibodies on CTLA-4 Expression in Treated Human-CTLA-4 Expressing Cells
[0502] Functional experiments were conducted to evaluate the impact of anti-CTLA-4 antibodies on CTLA-4 expression levels in CTLA-4 expressing cells. Human embryonic kidney (HEK) 293T cells were transiently transfected with CTLA-4 cDNA and incubated for four hours with various antibodies, including a control hlgG-Fc, Referencel, Reference4, and CTLA-4- AB-68. The CTLA-4 protein levels were assessed using western blot analysis, with ACTB (beta-actin) serving as the loading control.
[0503] Quantitative analysis of the western blot bands was performed using image analysis software, which calculated the sum, area, average, and median pixel intensities for each band. The CTLA-4 levels were normalized to the beta-actin levels to account for variations in sample loading. As depicted in FIG. 7A and shown in Tables E8A-E8C, cells treated with CTLA-4- AB-68 exhibited hCTLA-4 levels similar to those of the negative control. In contrast, cells treated with Referencel, known for depleting CTLA-4, showed reduced hCTLA-4 levels. Reference4, an anti-CTLA-4 antibody with a pH-dependent binding profile reported to reduce CTLA-4 degradation, also displayed reduced hCTLA-4 levels but to a lesser extent than Referencel. These findings suggest that Referencel promotes the depletion of CTLA-4 via trafficking to the lysosome and subsequent degradation, while Reference4, despite its pH-dependent binding, still leads to some CTLA-4 degradation. On the other hand, CTLA-4- AB-68 effectively maintained CTLA-4 levels, likely due to its unique pH-depending binding properties that facilitate CTLA-4 recycling.
[0504] Additionally, confocal microscopy experiments were performed to visualize the trafficking of CTLA-4 and the antibodies. Although not a quantitative readout, this qualitative analysis provided visual evidence supporting the western blot findings, demonstrating the distinct intracellular localization patterns of both CTLA-4 and anti-CTLA-4 antibodies upon treatment with CTLA-4- AB-68 vs. Referencel. A simplified workflow is provided in FIG. 7B. HEK293-hCTLA-4 cells were incubated with lOpg/mL antibody. Results in FIG. 7C further underscores the unique property of CTLA-4- AB -68 in preserving CTLA-4 recycling to reduce CTLA-4 depletion.
Example 9: Effect of Anti-CTLA-4 Antibodies on Antibody Dependent Cellular
Cytotoxicity
[0505] The ability of anti-CTLA-4 antibodies to induce antibody dependent cellular cytotoxicity (ADCC) was assessed by co-culturing human CTLA-4-expressing target cells with NK cells isolated from the peripheral blood mononuclear cells PBMCs of healthy donors. Briefly, NK cells were purified using negative selection techniques and verified by flow cytometry, gating out CD3-positive cells and selecting CD56-positive cells as shown in FIG. 8A. HEK293 cells engineered to overexpress human CTLA-4 (293T-hCTLA-4) were used as target cells. These cells were labeled with 50 nM CellTrace Far-Red for identification. Multiple Effector:Target cell ratios were tested to identify the optimal condition for differentiating ADCC activity. The ratios tested included 0:1, 1:1, 2:1, 4:1, and 8:1. Based on the percentage of target cell lysis, the optimal E:T ratio of 2:1 was selected for further characterization. Purified NK cells and labeled target cells were co-incubated for 6 hours at the optimized E:T ratio of 2:1. Target cell lysis was measured by the reduction in CellTrace Far-Red fluorescence. The percentage of target cell lysis was evaluated at various concentrations of four different antibodies, with EC50 values calculated from dose-response curves. As depicted in FIG. 8B and shown in Table E9, CTLA-4- AB-68 (IgGl) showed slightly higher ADCC compared to Referencel (IgGl), and the isotype control antibody (IgGl) exhibited minimal ADCC activity. Notably, CTLA-4- AB -68.1 (IgGl S239D/A330L/I332E), an Fc variant of CTLA-4- AB -68 (IgGl), demonstrated substantially greater ADCC compared to the other molecules tested. Example 10: Evaluation of In Vivo Anti-Tumor Efficacy of Anti-CTLA-4
Antibodies in the Subcutaneous CT-26 Murine Colorectal Carcinoma Syngeneic
Model Using hCTLA-4 Knock-In Mice
[0506] In this study, the antitumor activity of anti-CTLA-4 antibodies was assessed using a syngeneic mouse model for colon cancer (CT26 tumor-bearing mice). This model was specifically selected because reference anti-human-CTLA-4 antibodies do not cross-react with murine CTLA-4, necessitating the use of human CTLA-4 (hCTLA-4) knock-in mice to accurately evaluate the therapeutic potential of the antibodies. These mice were developed on a BALB/c background, providing an immune-competent, genetically consistent environment that closely mimics the human immune response. The study utilized female hCTLA-4 knock-in BALB/c mice, aged 6-8 weeks and weighing approximately 18-22g.
[0507] CT-26 tumor cells were cultured in vitro as a monolayer in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum, 100 U/ml penicillin, and 100 pg/ml streptomycin. Cultures were maintained at 37°C in a 5% CO2 atmosphere and subcultured twice weekly using trypsin-EDTA treatment. Cells in the exponential growth phase were harvested and counted for tumor inoculation.
[0508] For tumor development, each mouse was inoculated subcutaneously on the right lower flank with 3 x 10A5 CT-26 tumor cells in 0.1 mL of serum-free PBS. To ensure accurate subcutaneous delivery, the skin fold was lifted with one hand while the cells were injected with the other. To minimize tumor ulceration, a new syringe and needle were used for each mouse, and any droplets of cells were wiped from the skin with KimWipes post-injection. The animals were randomized, and treatment commenced when the average tumor volume reached 80 mmA3. Treatment was administered intraperitoneally at a fixed mg/kg dose, with the dosing volume adjusted based on body weight (10 pL/g). Treatment was delivered bi-weekly for 3 weeks, totaling six doses, as shown in FIG. 9A.
[0509] Tumor dimensions were measured three times per week during both the treatment and observation periods. Measurements were taken in two dimensions using calipers, and tumor volume was calculated using the formula: V = 0.5 * a * bA2, where “a” and “b” represent the long and short diameters of the tumor, respectively.
[0510] Tumor volume results are summarized in FIGs. 9B-9E. While tumor growth progressed in all vehicle control-treated mice, treatment with CTLA-4- AB -68.1 led to complete tumor rejection in several mice and significantly slowed tumor growth in others. Notably, mice treated with CTLA-4- AB-68.1 appeared to demonstrate improved tumor rejection at increased doses and superior tumor rejection compared to those treated with Referencel at a low (1 mg/kg) dose. Example 11: Effect of Anti-CTLA-4 Antibodies on Immune Cell Subsets in the Tumor and Blood of hCTLA4 Knock-In Mice
[0511] hCTLA4 knock-in mice bearing CT26 colorectal carcinoma tumors were prepared as described in Example 10. Multi-parameter flow cytometry assays were designed to evaluate the absolute cell numbers of various immune cell subsets in tumor and blood samples 5 days after a single 5 mg/kg dose treatment of Vehicle control, CTLA-4-AB-68.1, or Referencel. Each group included four mice. The animals were randomized, and treatment commenced when the average tumor volume reached 80 mmA3. Treatment was administered intraperitoneally.
[0512] Five days after administration of the single dose, tumor and blood samples were collected from the mice, and single-cell suspensions were prepared. Red blood cells were lysed from the blood samples to isolate leukocytes. The flow cytometry panels with fluorophore-conjugated-antibodies were designed as shown in Table E10A and Table E10B for tumor and blood samples respectively. Cells were stained with antibodies according to the manufacturer's instructions. After staining, cells were washed and resuspended in an appropriate buffer for flow cytometry analysis.
[0513] Data acquisition was performed using multi-parameter flow cytometry, incorporating compensation controls and single-stained controls to ensure accuracy. The flow cytometry data were analyzed to gate and quantify the following cell populations: CD45+ leukocytes, CD4 T cells (CD3+, CD4+), CD8 T cells (CD3+, CD8+), regulatory T cells (CD4+, CD25+, FoxP3+), and myeloid cells (CDl lb+). [0514] The gating strategy for tumor and blood samples are detailed in FIG. 10A and FIG. 10B, respectively. Absolute cell numbers were calculated per 100 mg of tumor tissue and per 100 microliters of blood.
[0515] The results of the FCM analysis for tumor are presented in FIG. 10C, and the results of the FCM analysis for blood samples are shown in FIGs. 10D-10H, respectively. In FIG. 10C, cell numbers of CD45+, myeloid, T cells and T cell subpopulations per 100 mg tumor are shown. CD4Teff refers to CD4T cells other than Treg. Treatment of hCTLA4 knock-in mice bearing CT26 colorectal carcinoma tumors with CTLA-4-AB-68.1 led to significant differences in immune cell subsets in both the tumor and the blood compared to the vehicle control and Reference 1.
[0516] Notably, CTLA-4-AB-68.1 treatment increased the ratio of CD4Teff/TReg and the ratio of CD8T/TReg cells in the tumor relative to the vehicle control and Reference 1. Additionally, while treatment with Reference 1 resulted in depleted levels of CTLA-4 expression on TReg cells in the blood compared to the vehicle control, treatment with CTLA-4- AB -68.1 did not deplete CTLA-4 expression on TReg cells in the blood.
Example 12: Generation of bispecific antibodies that bind to human CTLA-4 and human PD-1
[0517] Bispecific antibodies that bind to human CTLA-4 and human PD-1 in the crossmab (also referred to as crossover) format (FIG. 11) were produced. The bispecific antibodies included a variable heavy (VH) chain and a variable light (VL) chain of exemplary PD-1 antibodies described above, and a variable heavy (VH) chain and a variable light (VL) chain of exemplary CTLA-4 antibodies described above. The anti-human CTLA-4 binding domain and the anti-human PD-1 binding domain were configured as Fabs each containing a heavy chain with the VH and a CHI and a light chain with a VL and a CL. The bispecific antibodies were configured such that a VH and a VL region were interchanged in one binding arm (“crossmab” format). The Fc region of the bispecific antibody was formed with a heterodimeric Fc containing knob-into-hole heterodimeric connections.
[0518] Table Ell lists sequence identifiers (SEQ ID NO:) corresponding to amino acid (aa) sequences of the designed bispecific PD-1 /CTLA-4 antibodies, including of the heavy chains (HC) and light chains (LC) of the designed bispecific antibodies, including individual binders and the heterodimeric Fc regions. The full heavy or full light chains may additionally express a signal peptide (such as SEQ ID NOS: 361 or 177) for secretion.
Table Ell. Sequence Identifier (SEQ ID NO) Summary of the constructs
*crossover Fab
[0519] DNA sequences encoding amino acids in Table Ell were codon optimized, synthesized, and cloned into a proprietary production vector. The construct was sequenced for verification. Plasmids were then prepared to obtain DNA for transfection. The plasmids were transfected into CHO-K1 cells at 20mL followed by a two-step purification process.The purified proteins were formulated in a designated buffer that included 20 mM Histidine Acetate, 150 mM NaCl, pH 5.5. Analytical testing, including A280, SDS-PAGE / Caliper-SDS R&NR, SEC-HPLC and LC-MS R/NR was performed to verify the formation of the bispecific antibodies. The expressed antibodies were analyzed for purity using SDS-PAGE under reducing and non-reducing conditions. FIGs 12A-12B reveal reducing and non-reducing SDS-PAGE gels showing the heavy and light chains. FIG 13A and FIG 13B show SEC-HPLC results for CTLA-4/PD-l-abl and CTLA-4/PD-l-ab2. Both tests showed the LC and HC at the right sizes for each bispecific antibody, indicating that the two bispecific antibodies were produced.
Example 13: ELISA Binding of Bispecific Antibodies to Human CTLA-4 at different pH conditions
[0520] The engineered bispecific antibodies generated in CHO-K1 cells as described in Example 12 were tested for binding to recombinant human CTLA-4 using Enzyme-Linked Immuno-Sorbent Assay (ELISA) at different pHs. As shown in FIGs. 14A-14C and Table E12, the engineered bispecific antibodies exhibited differences in binding relative to the reference antibodies and to each other at pH 5.0 and pH 7.4. Example 14: ELISA Binding of Bispecific Antibodies to Human PD-1
[0521] The engineered bispecific antibodies generated in CH0-K1 cells as described in Example 12 were tested for binding to recombinant human PD-1 using Enzyme-Linked Immuno-Sorbent Assay (ELISA) at different pHs. As shown in Table E13, the engineered bispecific antibodies exhibited differences in binding relative to the reference antibodies and to each other:
Example 15: Binding Kinetics of Bispecific Antibodies to Human CTLA-4 and Human PD-1
[0522] The engineered antibodies generated in CHO-K1 cells as described in Example 12 were measured for binding kinetics to human CTLA-4 and human PD-1. Biolayer interferometry (BLI) was used to measure equilibrium dissociation constant (KD), association rate constant (kon), and dissociation rate constant (kdis) of the interaction between the anti-CTLA-4 and anti-PD-1 bispecific antibodies as described above and compared to a reference antibody. Briefly, BLI is an optical analytical technique that analyzes the interference pattern of white light reflected from two surfaces: a layer of immobilized protein on the biosensor tip, and an internal reference layer. Any change in the number of molecules bound to the biosensor tip causes a shift in the interference pattern that can be measured in real-time. As shown in Table E14A and E14B, the engineered bispecific antibodies exhibited unique binding kinetics to human CTLA-4, and human PD-1. Example 16: Surface Plasmon Resonance (SPR) Analysis of Bispecific Antibodies binding to Human CTLA-4, Human PD-1, Cyno CTLA-4, and Cyno PD-1
[0523] Surface plasmon resonance (SPR) experiments were conducted to evaluate the binding kinetics and affinities of bispecific antibodies targeting programmed cell death protein 1 (PD-1) and cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4). The analysis assessed binding to both human and cynomolgus monkey (cyno) orthologs of PD- 1 and CTLA-4.
[0524] SPR measurements were performed using a Biacore 8K system (Cytiva) with CM5 sensor chips (Cytiva). The assays were carried out at 25 °C using lx HBS-EP+ running buffer (Teknova) as the mobile phase. Anti-human IgG Fc antibodies (Jackson ImmunoResearch) were immobilized onto the CM5 sensor chip via standard amine coupling. Bispecific antibodies were captured through their Fc regions at a flow rate of 10 pL/min for 30 seconds.
[0525] Following capture, recombinant human and cynomolgus PD-1 and CTLA-4 proteins were injected as analytes to assess binding. Association and dissociation phases were performed at a flow rate of 30 pL/min. Association and dissociation times were selected based on kinetic range requirements. Analyte concentrations were prepared in serial dilutions for accurate kinetic fitting. Between binding cycles, the sensor surface was regenerated using 10 mM glycine at pH 1.5 to remove residual bound analyte and restore baseline response levels. Sensorgrams were analyzed using Biacore evaluation software.
[0526] Kinetic parameters (association rate constant ka, dissociation rate constant kd) and equilibrium dissociation constant (KD) values were derived using a 1 : 1 Langmuir binding model, with results displayed in Table E15. This SPR method enabled characterization of binding interactions between bispecific antibodies and PD-1 /CTLA-4 orthologs across species demonstrating optimization of candidates for cross-reactivity and therapeutic potential.
[0527] The present invention is not intended to be limited in scope to the particular disclosed embodiments, which are provided, for example, to illustrate various aspects of the invention. Various modifications to the compositions and methods described will become apparent from the description and teachings herein. Such variations may be practiced without departing from the true scope and spirit of the disclosure and are intended to fall within the scope of the present disclosure.
SEQUENCES

Claims

1. An anti-programmed cell death protein 1 (PD-1) antibody or antigen-binding fragment thereof, comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein: the VH region comprises a heavy chain complementarity determining region 1 (CDR-H1), a heavy chain complementarity determining region 2 (CDR-H2), and a heavy chain complementarity determining region 3 (CDR-H3) contained within any one of SEQ ID NOs: 1-45, 157, and 165-170, and the VL region comprises a light chain complementarity determining region 1 (CDR-L1), a light chain complementarity determining region 2 (CDR-L2) and a light chain complementarity determining region 3 (CDR-L3) contained within any one of SEQ ID NOs: 46-90, 158, and 171-172.
2. The anti-PD-1 antibody or antigen-binding fragment thereof of claim 1, wherein: the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO:3, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 158; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO:4, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 158; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO:5, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 158; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO:6, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 158; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO:7, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 158; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO:8, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 158; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO:9, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 158; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 10, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 158; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 11, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 158; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 12, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 158; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO:13, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 158; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 14, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 158; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 15, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 158; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO:16, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 158; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 17, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 158; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 18, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 158; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 19, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 158; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO:20, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 158; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO:21, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 158; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO:22, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 158; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO:23, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 158; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO:24, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 158; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO:25, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 158; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO:26, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 158; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO:27, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 158; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO:28, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 158; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO:29, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 158; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO:30, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 158; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO:31, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 158; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO:32, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 158; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO:33, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 158; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO:34, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 158; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO:35, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 158; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO:36, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 158; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO:37, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 158; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO:38, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 158; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO:39, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 158; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO:40, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 158; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO:41, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 158; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO:42, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 158; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO:43, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 158; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO:44, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 158; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO:45, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 158; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 157, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 46; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 157, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 47; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 157, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 48; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 157, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 49; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 157, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 50; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 157, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 51; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 157, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 52; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 157, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 53; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 157, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 54; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 157, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 55; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 157, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 56; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 157, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 57; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 157, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 58; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 157, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 59; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 157, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 60; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 157, and the VL region comprises a CDR-L1 , a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 61 ; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 157, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 62; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 157, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 63; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 157, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 64; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 157, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 65; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 157, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 66; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 157, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 67; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 157, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 68; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 157, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 69; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 157, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 70; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 157, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 71; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 157, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 72; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 157, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 73; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 157, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 74; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 157, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 75; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 157, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 76; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 157, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 77; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 157, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 78; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 157, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 79; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 157, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 80; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 157, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 81; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 157, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 82; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 157, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 83; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 157, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 84; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 157, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 85; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 157, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 86; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 157, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 87; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 157, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 88; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 157, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 89; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 157, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 90; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 16, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 47; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 23, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 47; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 35, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 76; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 35, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 79; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 35, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 81; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 35, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 84; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 35, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 89; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 37, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 76; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 37, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 79; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 37, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 81; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 37, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 84; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 37, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 89; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 40, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 76; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 40, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 79; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 40, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 81; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 40, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 84; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 40, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 89; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 42, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 76; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 42, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 79; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 42, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 81; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 42, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 84; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 42, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 89; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 165, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 79; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 165, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 171; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 165, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 172; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 166, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 79; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 166, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 171; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 166, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 172; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 167, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 79; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 167, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 171; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 167, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 172; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 168, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 79; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 168, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 171; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 168, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 172; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 169, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 79; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 169, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 171; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 169, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 172; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 170, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 79; the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 170, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 171; or the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 170, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 172.
3. An anti-PD-1 antibody or antigen-binding fragment thereof, comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein: the VH region comprises a heavy chain complementarity determining region 1 (CDR-H1) comprising the sequence set forth in any one of SEQ ID NOs: 91-101, a heavy chain complementarity determining region 2 (CDR-H2) comprising the sequence set forth in any one of SEQ ID NOs: 102- 112, and 173, and a heavy chain complementarity determining region 3 (CDR-H3) comprising the sequence set forth in any one of SEQ ID NOs: 113-123; and the VL region comprises a light chain complementarity determining region 1 (CDR-L1) comprising the sequence set forth in any one of SEQ ID NOs: 124-134, a light chain complementarity determining region 2 (CDR-L2) comprising the sequence set forth in any one of SEQ ID NOs: 135-145, and a light chain complementarity determining region 3 (CDR-L3) comprising the sequence set forth in any one of SEQ ID NOs:146-156.
4. The anti-PD-1 antibody or antigen-binding fragment thereof of claim 3, wherein the VH region comprises a heavy chain complementarity determining region 1 (CDR-H1) comprising the sequence set forth in any one of SEQ ID NOs: 92-101, a heavy chain complementarity determining region 2 (CDR-H2) comprising the sequence set forth in any one of SEQ ID NOs: 103-112, and 173, and a heavy chain complementarity determining region 3 (CDR-H3) comprising the sequence set forth in any one of SEQ ID NOs:l 14-123.
5. The anti-PD-1 antibody or antigen-binding fragment thereof of claim 3, wherein the VL region comprises a light chain complementarity determining region 1 (CDR-L1) comprising the sequence set forth in any one of SEQ ID NOs: 125-134, a light chain complementarity determining region 2 (CDR-L2) comprising the sequence set forth in any one of SEQ ID NOs: 136-145, and a light chain complementarity determining region 3 (CDR-L3) comprising the sequence set forth in any one of SEQ ID NOs:147-156.
6. The anti-PD-1 antibody or antigen-binding fragment thereof of any of claims 1-5, wherein: the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 92, 102, and 113, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 124, 135, and 146, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 93, 102, and 113, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 124, 135, and 146, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 94, 102, and 113, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 124, 135, and 146, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 95, 102, and 113, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 124, 135, and 146, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 96, 102, and 113, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 124, 135, and 146, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 97, 102, and 113, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 124, 135, and 146, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 98, 102, and 113, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 124, 135, and 146, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 99, 102, and 113, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 124, 135, and 146, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 100, 102, and 113, respectively, and the CDR-L1, the CDR- L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 124, 135, and 146, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 101, 102, and 113, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 124, 135, and 146, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 91, 103, and 113, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 124, 135, and 146, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 91, 104, and 113, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 124, 135, and 146, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 91, 105, and 113, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 124, 135, and 146, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 91, 106, and 113, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 124, 135, and 146, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 91, 107, and 113, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 124, 135, and 146, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 91, 108, and 113, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 124, 135, and 146, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 91, 109, and 113, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 124, 135, and 146, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 91, 110, and 113, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 124, 135, and 146, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 91, 111, and 113, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 124, 135, and 146, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 91, 112, and 113, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 124, 135, and 146, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 91, 102, and 114, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 124, 135, and 146, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 91, 102, and 115, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 124, 135, and 146, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 91, 102, and 116, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 124, 135, and 146, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 91, 102, and 117, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 124, 135, and 146, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 91, 102, and 118, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 124, 135, and 146, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 91, 102, and 119, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 124, 135, and 146, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 91, 102, and 120, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 124, 135, and 146, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 91, 102, and 121, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 124, 135, and 146, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 91, 102, and 122, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 124, 135, and 146, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 91, 102, and 123, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 124, 135, and 146, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 101, 103, and 114, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 124, 135, and 146, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 97, 103, and 117, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 124, 135, and 146, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 99, 105, and 115, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 124, 135, and 146, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 96, 104, and 116, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 124, 135, and 146, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 98, 106, and 114, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 124, 135, and 146, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 96, 107, and 117, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 124, 135, and 146, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 97, 105, and 117, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 124, 135, and 146, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 99, 111, and 115, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 124, 135, and 146, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 97, 111, and 116, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 124, 135, and 146, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 100, 105, and 121, respectively, and the CDR-L1, the CDR- L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 124, 135, and 146, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 93, 103, and 115, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 124, 135, and 146, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 100, 108, and 119, respectively, and the CDR-L1, the CDR- L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 124, 135, and 146, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 99, 111, and 116, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 124, 135, and 146, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 91, 102, and 113, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 125, 135, and 146, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 91, 102, and 113, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 126, 135, and 146, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 91, 102, and 113, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 127, 135, and 146, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 91, 102, and 113, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 128, 135, and 146, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 91, 102, and 113, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 129, 135, and 146, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 91, 102, and 113, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 130, 135, and 146, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 91, 102, and 113, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 131, 135, and 146, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 91, 102, and 113, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 132, 135, and 146, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 91, 102, and 113, respectively, and the CDR-L1, theCDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 133, 135, and 146, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 91, 102, and 113, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 134, 135, and 146, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 91, 102, and 113, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 124, 136, and 146, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 91, 102, and 113, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 124, 137, and 146, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 91, 102, and 113, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 124, 138, and 146, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 91, 102, and 113, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 124, 139, and 146, respectively; the CDR-H1, theCDR-H2, and theCDR-H3 comprise the sequences set forth in SEQ ID NOS: 91, 102, and 113, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 124, 140, and 146, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 91, 102, and 113, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 124, 141, and 146, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 91, 102, and 113, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 124, 142, and 146, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 91, 102, and 113, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 124, 143, and 146, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 91, 102, and 113, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 124, 144, and 146, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 91, 102, and 113, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 124, 145, and 146, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 91, 102, and 113, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 124, 135, and 147, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 91, 102, and 113, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 124, 135, and 148, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 91, 102, and 113, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 124, 135, and 149, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 91, 102, and 113, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 124, 135, and 150, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 91, 102, and 113, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 124, 135, and 151, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 91, 102, and 113, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 124, 135, and 152, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 91, 102, and 113, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 124, 135, and 153, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 91, 102, and 113, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 124, 135, and 154, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 91, 102, and 113, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 124, 135, and 155, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 91, 102, and 113, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 124, 135, and 156, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 91, 102, and 113, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 131, 142, and 149, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 91, 102, and 113, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 131, 139, and 150, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 91, 102, and 113, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 128, 138, and 148, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 91, 102, and 113, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 126, 145, and 153, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 91, 102, and 113, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 126, 144, and 154, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 91, 102, and 113, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 125, 140, and 153, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 91, 102, and 113, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 132, 137, and 152, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 91, 102, and 113, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 131, 142, and 152, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 91, 102, and 113, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 132, 141, and 154, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 91, 102, and 113, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 133, 144, and 150, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 91, 102, and 113, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 134, 138, and 149, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 91, 102, and 113, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 126, 137, and 149, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 91, 102, and 113, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 126, 137, and 152, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 91, 102, and 113, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 132, 142, and 154, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 91, 102, and 113, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 127, 140, and 151, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 91, 106, and 113, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 126, 135, and 146, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 91, 102, and 114, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 126, 135, and 146, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 99, 105, and 115, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 131, 142, and 149, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 99, 105, and 115, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 126, 145, and 153, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 99, 105, and 115, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 125, 140, and 153, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 99, 105, and 115, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 132, 141, and 154, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 99, 105, and 115, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 132, 142, and 154, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 98, 106, and 114, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 131, 142, and 149, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 98, 106, and 114, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 126, 145, and 153, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 98, 106, and 114, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 125, 140, and 153, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 98, 106, and 114, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 132, 141, and 154, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 98, 106, and 114, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 132, 142, and 154, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 99, 111, and 115, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 131, 142, and 149, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 99, 111, and 115, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 126, 145, and 153, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 99, 111, and 115, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 125, 140, and 153, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 99, 111, and 115, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 132, 141, and 154, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 99, 111, and 115, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 132, 142, and 154, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 100, 105, and 121, respectively, and the CDR-L1, the CDR- L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 131, 142, and 149, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 100, 105, and 121, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 126, 145, and 153, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 100, 105, and 121, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 125, 140, and 153, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 100, 105, and 121, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 132, 141, and 154, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 100, 105, and 121, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 132, 142, and 154, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 100, 173 and 121, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 126, 145 and 153, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 100, 173 and 121, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 132, 145 and 153, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 100, 173 and 121, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 126, 143 and 153, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 95, 105 and 121, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 126, 145 and 153, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 95, 105 and 121, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 132, 145 and 153, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 95, 105 and 121, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 126, 143 and 153, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 95, 173 and 121, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 126, 145 and 153, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 95, 173 and 121, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 132, 145 and 153, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 95, 173 and 121, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 126, 143 and 153, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 95, 106 and 114, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 126, 145 and 153, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 95, 106 and 114, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 132, 145 and 153, respectively; or the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 95, 106 and 114, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 126, 143 and 153, respectively.
7. The anti-PD-1 antibody or antigen-binding fragment thereof of any of claims 1-6, wherein: the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 1 and SEQ ID NO: 158, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 2 and SEQ ID NO: 158, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 3 and SEQ ID NO: 158, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 4 and SEQ ID NO: 158, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 5 and SEQ ID NO: 158, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 6 and SEQ ID NO: 158, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 7 and SEQ ID NO: 158, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 8 and SEQ ID NO: 158, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 9 and SEQ ID NO: 158, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 10 and SEQ ID NO: 158, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 11 and SEQ ID NO: 158, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 12 and SEQ ID NO: 158, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 13 and SEQ ID NO: 158, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 14 and SEQ ID NO: 158, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO and SEQ ID NO: 158, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 16 and SEQ ID NO: 158, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 17 and SEQ ID NO: 158, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 18 and SEQ ID NO: 158, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 19 and SEQ ID NO: 158, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 20 and SEQ ID NO: 158, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 21 and SEQ ID NO: 158, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 22 and SEQ ID NO: 158, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 23 and SEQ ID NO: 158, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 24 and SEQ ID NO: 158, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 25 and SEQ ID NO: 158, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 26 and SEQ ID NO: 158, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 27 and SEQ ID NO: 158, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 28 and SEQ ID NO: 158, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 29 and SEQ ID NO: 158, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 30 and SEQ ID NO: 158, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 31 and SEQ ID NO: 158, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 32 and SEQ ID NO: 158, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 33 and SEQ ID NO: 158, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 34 and SEQ ID NO: 158, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 35 and SEQ ID NO: 158, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 36 and SEQ ID NO: 158, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 37 and SEQ ID NO: 158, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 38 and SEQ ID NO: 158, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 39 and SEQ ID NO: 158, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 40 and SEQ ID NO: 158, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 41 or ID NO: 158, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 42 and SEQ ID NO: 158, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 43 and SEQ ID NO: 158, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 44 and SEQ ID NO: 158, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 45 and SEQ ID NO: 158, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 46, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 47, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 48, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 49, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 50, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 51, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 52, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 53, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 54, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 55, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 56, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 57, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 58, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 59, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 60, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 61, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 62, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 63, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 64, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 65, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 66, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 or ID NO: 67, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 68, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 69, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 70, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 71, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 72, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 73, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 74, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 75, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 76, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 77, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 78, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 79, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 80, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 81, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 82, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 83, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 84, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 85, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 86, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 87, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 88, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 89, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157 and SEQ ID NO: 90, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 16 and SEQ ID NO: 47, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 23 and SEQ ID NO: 47, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 35 and SEQ ID NO: 76, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 35 and SEQ ID NO: 79, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 35 and SEQ ID NO: 81, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 35 and SEQ ID NO: 84, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 35 and SEQ ID NO: 89, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 37 and SEQ ID NO: 76, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 37 and SEQ ID NO: 79, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 37 and SEQ ID NO: 81, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 37 and SEQ ID NO: 84, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 37 and SEQ ID NO: 89, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 40 and SEQ ID NO: 76, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 40 and SEQ ID NO: 79, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 40 and SEQ ID NO: 81, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 40 and SEQ ID NO: 84, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 40 and SEQ ID NO: 89, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 42 and SEQ ID NO: 76, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 42 and SEQ ID NO: 79, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 42 and SEQ ID NO: 81, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 42 and SEQ ID NO: 84, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 42 and SEQ ID NO: 89, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 165 and SEQ ID NO: 79, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 165 and SEQ ID NO: 171, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 165 and SEQ ID NO: 172, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 166 and SEQ ID NO: 79, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 166 and SEQ ID NO: 171, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 166 and SEQ ID NO: 172, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 167 and SEQ ID NO: 79, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 167 and SEQ ID NO: 171, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 167 and SEQ ID NO: 172, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 168 and SEQ ID NO: 79, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 168 and SEQ ID NO: 171, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 168 and SEQ ID NO: 172, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 169 and SEQ ID NO: 79, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 169 and SEQ ID NO: 171, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 169 and SEQ ID NO: 172, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 170 and SEQ ID NO: 79, respectively; the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 170 and SEQ ID NO: 171, respectively; or the VH region and the VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 170 and SEQ ID NO: 172, respectively.
8. The anti-PD-1 antibody or antigen-binding fragment thereof of any of claims 1-7, wherein the VH region and the VL region are or comprise: the sequence set forth in SEQ ID NO: 1 and 158, respectively; the sequence set forth in SEQ ID NO: 2 and 158, respectively; the sequence set forth in SEQ ID NO: 3 and 158, respectively; the sequence set forth in SEQ ID NO: 4 and 158, respectively; the sequence set forth in SEQ ID NO: 5 and 158, respectively; the sequence set forth in SEQ ID NO: 6 and 158, respectively; the sequence set forth in SEQ ID NO: 7 and 158, respectively; the sequence set forth in SEQ ID NO: 8 and 158, respectively; the sequence set forth in SEQ ID NO: 9 and 158, respectively; the sequence set forth in SEQ ID NO: 10 and 158, respectively; the sequence set forth in SEQ ID NO: 11 and 158, respectively; the sequence set forth in SEQ ID NO: 12 and 158, respectively; the sequence set forth in SEQ ID NO: 13 and 158, respectively; the sequence set forth in SEQ ID NO: 14 and 158, respectively; the sequence set forth in SEQ ID NO: 15 and 158, respectively; the sequence set forth in SEQ ID NO: 16 and 158, respectively; the sequence set forth in SEQ ID NO: 17 and 158, respectively; the sequence set forth in SEQ ID NO: 18 and 158, respectively; the sequence set forth in SEQ ID NO: 19 and 158, respectively; the sequence set forth in SEQ ID NO: 20 and 158, respectively; the sequence set forth in SEQ ID NO: 21 and 158, respectively; the sequence set forth in SEQ ID NO: 22 and 158, respectively; the sequence set forth in SEQ ID NO: 23 and 158, respectively; the sequence set forth in SEQ ID NO: 24 and 158, respectively; the sequence set forth in SEQ ID NO: 25 and 158, respectively; the sequence set forth in SEQ ID NO: 26 and 158, respectively; the sequence set forth in SEQ ID NO: 27 and 158, respectively; the sequence set forth in SEQ ID NO: 28 and 158, respectively; the sequence set forth in SEQ ID NO: 29 and 158, respectively; the sequence set forth in SEQ ID NO: 30 and 158, respectively; the sequence set forth in SEQ ID NO: 31 and 158, respectively; the sequence set forth in SEQ ID NO: 32 and 158, respectively; the sequence set forth in SEQ ID NO: 33 and 158, respectively; the sequence set forth in SEQ ID NO: 34 and 158, respectively; the sequence set forth in SEQ ID NO: 35 and 158, respectively; the sequence set forth in SEQ ID NO: 36 and 158, respectively; the sequence set forth in SEQ ID NO: 37 and 158, respectively; the sequence set forth in SEQ ID NO: 38 and 158, respectively; the sequence set forth in SEQ ID NO: 39 and 158, respectively; the sequence set forth in SEQ ID NO: 40 and 158, respectively; the sequence set forth in SEQ ID NO: 41 and 158, respectively; the sequence set forth in SEQ ID NO: 42 and 158, respectively; the sequence set forth in SEQ ID NO: 43 and 158, respectively; the sequence set forth in SEQ ID NO: 44 and 158, respectively; the sequence set forth in SEQ ID NO: 45 and 158, respectively; the sequence set forth in SEQ ID NO: 157 and 46, respectively; the sequence set forth in SEQ ID NO: 157 and 47, respectively; the sequence set forth in SEQ ID NO: 157 and 48, respectively; the sequence set forth in SEQ ID NO: 157 and 49, respectively; the sequence set forth in SEQ ID NO: 157 and 50, respectively; the sequence set forth in SEQ ID NO: 157 and 51, respectively; the sequence set forth in SEQ ID NO: 157 and 52, respectively; the sequence set forth in SEQ ID NO: 157 and 53, respectively; the sequence set forth in SEQ ID NO: 157 and 54, respectively; the sequence set forth in SEQ ID NO: 157 and 55, respectively; the sequence set forth in SEQ ID NO: 157 and 56, respectively; the sequence set forth in SEQ ID NO: 157 and 57, respectively; the sequence set forth in SEQ ID NO: 157 and 58, respectively; the sequence set forth in SEQ ID NO: 157 and 59, respectively; the sequence set forth in SEQ ID NO: 157 and 60, respectively; the sequence set forth in SEQ ID NO: 157 and 61, respectively; the sequence set forth in SEQ ID NO: 157 and 62, respectively; the sequence set forth in SEQ ID NO: 157 and 63, respectively; the sequence set forth in SEQ ID NO: 157 and 64, respectively; the sequence set forth in SEQ ID NO: 157 and 65, respectively; the sequence set forth in SEQ ID NO: 157 and 66, respectively; the sequence set forth in SEQ ID NO: 157 and 67, respectively; the sequence set forth in SEQ ID NO: 157 and 68, respectively; the sequence set forth in SEQ ID NO: 157 and 69, respectively; the sequence set forth in SEQ ID NO: 157 and 70, respectively; the sequence set forth in SEQ ID NO: 157 and 71, respectively; the sequence set forth in SEQ ID NO: 157 and 72, respectively; the sequence set forth in SEQ ID NO: 157 and 73, respectively; the sequence set forth in SEQ ID NO: 157 and 74, respectively; the sequence set forth in SEQ ID NO: 157 and 75, respectively; the sequence set forth in SEQ ID NO: 157 and 76, respectively; the sequence set forth in SEQ ID NO: 157 and 77, respectively; the sequence set forth in SEQ ID NO: 157 and 78, respectively; the sequence set forth in SEQ ID NO: 157 and 79, respectively; the sequence set forth in SEQ ID NO: 157 and 80, respectively; the sequence set forth in SEQ ID NO: 157 and 81, respectively; the sequence set forth in SEQ ID NO: 157 and 82, respectively; the sequence set forth in SEQ ID NO: 157 and 83, respectively; the sequence set forth in SEQ ID NO: 157 and 84, respectively; the sequence set forth in SEQ ID NO: 157 and 85, respectively; the sequence set forth in SEQ ID NO: 157 and 86, respectively; the sequence set forth in SEQ ID NO: 157 and 87, respectively; the sequence set forth in SEQ ID NO: 157 and 88, respectively; the sequence set forth in SEQ ID NO: 157 and 89, respectively; the sequence set forth in SEQ ID NO: 157 and 90, respectively; the sequence set forth in SEQ ID NO: 16 and 47, respectively; the sequence set forth in SEQ ID NO: 23 and 47, respectively; the sequence set forth in SEQ ID NO: 35 and 76, respectively; the sequence set forth in SEQ ID NO: 35 and 79, respectively; the sequence set forth in SEQ ID NO: 35 and 81, respectively; the sequence set forth in SEQ ID NO: 35 and 84, respectively; the sequence set forth in SEQ ID NO: 35 and 89, respectively; the sequence set forth in SEQ ID NO: 37 and 76, respectively; the sequence set forth in SEQ ID NO: 37 and 79, respectively; the sequence set forth in SEQ ID NO: 37 and 81, respectively; the sequence set forth in SEQ ID NO: 37 and 84, respectively; the sequence set forth in SEQ ID NO: 37 and 89, respectively; the sequence set forth in SEQ ID NO: 40 and 76, respectively; the sequence set forth in SEQ ID NO: 40 and 79, respectively; the sequence set forth in SEQ ID NO: 40 and 81, respectively; the sequence set forth in SEQ ID NO: 40 and 84, respectively; the sequence set forth in SEQ ID NO: 40 and 89, respectively; the sequence set forth in SEQ ID NO: 42 and 76, respectively; the sequence set forth in SEQ ID NO: 42 and 79, respectively; the sequence set forth in SEQ ID NO: 42 and 81, respectively; the sequence set forth in SEQ ID NO: 42 and 84, respectively; the sequence set forth in SEQ ID NO: 42 and 89, respectively; the sequence set forth in SEQ ID NO: 165 and 79, respectively; the sequence set forth in SEQ ID NO: 165 and 171, respectively; the sequence set forth in SEQ ID NO: 165 and 172, respectively; the sequence set forth in SEQ ID NO: 166 and 79, respectively; the sequence set forth in SEQ ID NO: 166 and 171, respectively; the sequence set forth in SEQ ID NO: 166 and 172, respectively; the sequence set forth in SEQ ID NO: 167 and 79, respectively; the sequence set forth in SEQ ID NO: 167 and 171, respectively; the sequence set forth in SEQ ID NO: 167 and 172, respectively; the sequence set forth in SEQ ID NO: 168 and 79, respectively; the sequence set forth in SEQ ID NO: 168 and 171, respectively; the sequence set forth in SEQ ID NO: 168 and 172, respectively; the sequence set forth in SEQ ID NO: 169 and 79, respectively; the sequence set forth in SEQ ID NO: 169 and 171, respectively; the sequence set forth in SEQ ID NO: 169 and 172, respectively; the sequence set forth in SEQ ID NO: 170 and 79, respectively; the sequence set forth in SEQ ID NO: 170 and 171, respectively; or the sequence set forth in SEQ ID NO: 170 and 172, respectively.
9. An anti-programmed cell death protein 1 (PD-1) antibody or antigen-binding fragment thereof, comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region and the VL region are or comprise: the sequence set forth in SEQ ID NO: 1 and 158, respectively; the sequence set forth in SEQ ID NO: 2 and 158, respectively; the sequence set forth in SEQ ID NO: 3 and 158, respectively; the sequence set forth in SEQ ID NO: 4 and 158, respectively; the sequence set forth in SEQ ID NO: 5 and 158, respectively; the sequence set forth in SEQ ID NO: 6 and 158, respectively; the sequence set forth in SEQ ID NO: 7 and 158, respectively; the sequence set forth in SEQ ID NO: 8 and 158, respectively; the sequence set forth in SEQ ID NO: 9 and 158, respectively; the sequence set forth in SEQ ID NO: 10 and 158, respectively; the sequence set forth in SEQ ID NO: 11 and 158, respectively; the sequence set forth in SEQ ID NO: 12 and 158, respectively; the sequence set forth in SEQ ID NO: 13 and 158, respectively; the sequence set forth in SEQ ID NO: 14 and 158, respectively; the sequence set forth in SEQ ID NO: 15 and 158, respectively; the sequence set forth in SEQ ID NO: 16 and 158, respectively; the sequence set forth in SEQ ID NO: 17 and 158, respectively; the sequence set forth in SEQ ID NO: 18 and 158, respectively; the sequence set forth in SEQ ID NO: 19 and 158, respectively; the sequence set forth in SEQ ID NO: 20 and 158, respectively; the sequence set forth in SEQ ID NO: 21 and 158, respectively; the sequence set forth in SEQ ID NO: 22 and 158, respectively; the sequence set forth in SEQ ID NO: 23 and 158, respectively; the sequence set forth in SEQ ID NO: 24 and 158, respectively; the sequence set forth in SEQ ID NO: 25 and 158, respectively; the sequence set forth in SEQ ID NO: 26 and 158, respectively; the sequence set forth in SEQ ID NO: 27 and 158, respectively; the sequence set forth in SEQ ID NO: 28 and 158, respectively; the sequence set forth in SEQ ID NO: 29 and 158, respectively; the sequence set forth in SEQ ID NO: 30 and 158, respectively; the sequence set forth in SEQ ID NO: 31 and 158, respectively; the sequence set forth in SEQ ID NO: 32 and 158, respectively; the sequence set forth in SEQ ID NO: 33 and 158, respectively; the sequence set forth in SEQ ID NO: 34 and 158, respectively; the sequence set forth in SEQ ID NO: 35 and 158, respectively; the sequence set forth in SEQ ID NO: 36 and 158, respectively; the sequence set forth in SEQ ID NO: 37 and 158, respectively; the sequence set forth in SEQ ID NO: 38 and 158, respectively; the sequence set forth in SEQ ID NO: 39 and 158, respectively; the sequence set forth in SEQ ID NO: 40 and 158, respectively; the sequence set forth in SEQ ID NO: 41 and 158, respectively; the sequence set forth in SEQ ID NO: 42 and 158, respectively; the sequence set forth in SEQ ID NO: 43 and 158, respectively; the sequence set forth in SEQ ID NO: 44 and 158, respectively; the sequence set forth in SEQ ID NO: 45 and 158, respectively; the sequence set forth in SEQ ID NO: 157 and 46, respectively; the sequence set forth in SEQ ID NO: 157 and 47, respectively; the sequence set forth in SEQ ID NO: 157 and 48, respectively; the sequence set forth in SEQ ID NO: 157 and 49, respectively; the sequence set forth in SEQ ID NO: 157 and 50, respectively; the sequence set forth in SEQ ID NO: 157 and 51, respectively; the sequence set forth in SEQ ID NO: 157 and 52, respectively; the sequence set forth in SEQ ID NO: 157 and 53, respectively; the sequence set forth in SEQ ID NO: 157 and 54, respectively; the sequence set forth in SEQ ID NO: 157 and 55, respectively; the sequence set forth in SEQ ID NO: 157 and 56, respectively; the sequence set forth in SEQ ID NO: 157 and 57, respectively; the sequence set forth in SEQ ID NO: 157 and 58, respectively; the sequence set forth in SEQ ID NO: 157 and 59, respectively; the sequence set forth in SEQ ID NO: 157 and 60, respectively; the sequence set forth in SEQ ID NO: 157 and 61, respectively; the sequence set forth in SEQ ID NO: 157 and 62, respectively; the sequence set forth in SEQ ID NO: 157 and 63, respectively; the sequence set forth in SEQ ID NO: 157 and 64, respectively; the sequence set forth in SEQ ID NO: 157 and 65, respectively; the sequence set forth in SEQ ID NO: 157 and 66, respectively; the sequence set forth in SEQ ID NO: 157 and 67, respectively; the sequence set forth in SEQ ID NO: 157 and 68, respectively; the sequence set forth in SEQ ID NO: 157 and 69, respectively; the sequence set forth in SEQ ID NO: 157 and 70, respectively; the sequence set forth in SEQ ID NO: 157 and 71, respectively; the sequence set forth in SEQ ID NO: 157 and 72, respectively; the sequence set forth in SEQ ID NO: 157 and 73, respectively; the sequence set forth in SEQ ID NO: 157 and 74, respectively; the sequence set forth in SEQ ID NO: 157 and 75, respectively; the sequence set forth in SEQ ID NO: 157 and 76, respectively; the sequence set forth in SEQ ID NO: 157 and 77, respectively; the sequence set forth in SEQ ID NO: 157 and 78, respectively; the sequence set forth in SEQ ID NO: 157 and 79, respectively; the sequence set forth in SEQ ID NO: 157 and 80, respectively; the sequence set forth in SEQ ID NO: 157 and 81, respectively; the sequence set forth in SEQ ID NO: 157 and 82, respectively; the sequence set forth in SEQ ID NO: 157 and 83, respectively; the sequence set forth in SEQ ID NO: 157 and 84, respectively; the sequence set forth in SEQ ID NO: 157 and 85, respectively; the sequence set forth in SEQ ID NO: 157 and 86, respectively; the sequence set forth in SEQ ID NO: 157 and 87, respectively; the sequence set forth in SEQ ID NO: 157 and 88, respectively; the sequence set forth in SEQ ID NO: 157 and 89, respectively; the sequence set forth in SEQ ID NO: 157 and 90, respectively; the sequence set forth in SEQ ID NO: 16 and 47, respectively; the sequence set forth in SEQ ID NO: 23 and 47, respectively; the sequence set forth in SEQ ID NO: 35 and 76, respectively; the sequence set forth in SEQ ID NO: 35 and 79, respectively; the sequence set forth in SEQ ID NO: 35 and 81, respectively; the sequence set forth in SEQ ID NO: 35 and 84, respectively; the sequence set forth in SEQ ID NO: 35 and 89, respectively; the sequence set forth in SEQ ID NO: 37 and 76, respectively; the sequence set forth in SEQ ID NO: 37 and 79, respectively; the sequence set forth in SEQ ID NO: 37 and 81, respectively; the sequence set forth in SEQ ID NO: 37 and 84, respectively; the sequence set forth in SEQ ID NO: 37 and 89, respectively; the sequence set forth in SEQ ID NO: 40 and 76, respectively; the sequence set forth in SEQ ID NO: 40 and 79, respectively; the sequence set forth in SEQ ID NO: 40 and 81, respectively; the sequence set forth in SEQ ID NO: 40 and 84, respectively; the sequence set forth in SEQ ID NO: 40 and 89, respectively; the sequence set forth in SEQ ID NO: 42 and 76, respectively; the sequence set forth in SEQ ID NO: 42 and 79, respectively; the sequence set forth in SEQ ID NO: 42 and 81, respectively; the sequence set forth in SEQ ID NO: 42 and 84, respectively; the sequence set forth in SEQ ID NO: 42 and 89, respectively; the sequence set forth in SEQ ID NO: 165 and 79, respectively; the sequence set forth in SEQ ID NO: 165 and 171, respectively; the sequence set forth in SEQ ID NO: 165 and 172, respectively; the sequence set forth in SEQ ID NO: 166 and 79, respectively; the sequence set forth in SEQ ID NO: 166 and 171, respectively; the sequence set forth in SEQ ID NO: 166 and 172, respectively; the sequence set forth in SEQ ID NO: 167 and 79, respectively; the sequence set forth in SEQ ID NO: 167 and 171, respectively; the sequence set forth in SEQ ID NO: 167 and 172, respectively; the sequence set forth in SEQ ID NO: 168 and 79, respectively; the sequence set forth in SEQ ID NO: 168 and 171, respectively; the sequence set forth in SEQ ID NO: 168 and 172, respectively; the sequence set forth in SEQ ID NO: 169 and 79, respectively; the sequence set forth in SEQ ID NO: 169 and 171, respectively; the sequence set forth in SEQ ID NO: 169 and 172, respectively; the sequence set forth in SEQ ID NO: 170 and 79, respectively; the sequence set forth in SEQ ID NO: 170 and 171, respectively; or the sequence set forth in SEQ ID NO: 170 and 172, respectively.
10. The anti-PD-1 antibody or antigen-binding fragment thereof of any of claims 1-9, wherein the antibody is a full length antibody.
11. The anti-PD-1 antibody or antigen-binding fragment thereof of claim 10, wherein the full length antibody comprises a constant region (Fc) from an IgAl, IgA2, IgD, IgE, IgGl, IgG2, IgG3, IgG4, or IgM.
12. The anti-PD-1 antibody or antigen-binding fragment thereof of any of claims 1-9, wherein the antibody is an antigen-binding fragment.
13. The anti-PD-1 antibody or antigen-binding fragment thereof of claim 12, wherein the antigen-binding fragment is selected from the group consisting of a single domain antibody, a single chain antibody, an unibody, a single chain variable fragment (scFv), a Fab fragment, and a F(ab')2 fragment.
14. The anti-PD-1 antibody or antigen-binding fragment thereof of any of claims 1-13, wherein the anti-PD-1 antibody or antigen-binding fragment thereof is recombinant.
15. The anti-PD-1 antibody or antigen-binding fragment thereof of any of claims 1-14, wherein the VH region and the VL region is human or is from a human protein.
16. The anti-PD-1 antibody or antigen-binding fragment thereof of any of claims 1-15, wherein the antibody or antigen-binding fragment thereof binds to human PD-1 protein with an equilibrium dissociation constant (KD) of from about 7 x 10 10 M to about 2 x 108 M at pH 7.35 to 7.45, optionally at pH 7.40.
17. The anti-PD-1 antibody or antigen-binding fragment thereof of claim 16, wherein the antibody or antigen-binding fragment thereof binds to human PD-1 protein with a KD of lower than 1 x 109 M, optionally from about 7 x 10 10 M to about 1 x 109 M.
18. The anti-PD-1 antibody or antigen-binding fragment thereof of any of claims 1-17, wherein the antibody or antigen-binding fragment thereof binds to human PD-1 protein with an equilibrium dissociation constant (KD) of from about 3 x 10 10 M to about 4 x 108 M at pH 5.0 to 6.8, optionally at pH 6.0 or pH 5.0.
19. The anti-PD-1 antibody or antigen-binding fragment thereof of claim 18, wherein the antibody or antigen-binding fragment thereof binds to human PD-1 protein with a KD of lower than 1 x 109 M, optionally from about 3 x 10 10 to about 1 x 109 M.
20. The anti-PD-1 antibody or antigen-binding fragment thereof of any of claims 1-19, wherein the antibody or antigen-binding fragment thereof binds to human PD-1 protein with a lower KD at pH 5.0 to 6.8, optionally pH 6.0 or pH 5.0, than pH 7.35 to 7.45, optionally pH 7.4.
21. The anti-PD-1 antibody or antigen-binding fragment thereof of any of claims 1-19, wherein the antibody or antigen-binding fragment thereof binds to human PD-1 protein with a lower KD at pH 7.35 to 7.45, optionally pH 7.4, than at pH 5.0 to 6.8, optionally pH 6.0 or pH 5.0.
22. The anti-PD-1 antibody or antigen-binding fragment thereof of any of claims 1-19, wherein the antibody or antigen-binding fragment thereof binds to human PD-1 protein with a KD at pH 7.35 to 7.45, optionally pH 7.4, and at pH 5.6 to 6.8, optionally pH 6.0 or pH 5.0, that is substantially the same.
23. The anti-PD-1 antibody or antigen-binding fragment thereof of any of claims 1-22, wherein the antibody or antigen-binding fragment thereof binds to human PD-1 protein with an association rate constant (Kon) of 4 x 105 1/Ms to about 2 x 106 1/Ms at pH 7.35 to 7.45, optionally at pH 7.40.
24. The anti-PD-1 antibody or antigen-binding fragment thereof of any of claims 1-23, wherein the antibody or antigen-binding fragment thereof binds to human PD-1 protein with an dissociation rate constant (Koff) of 4 x 104 1/s to about 9 x 103 1/s at pH 7.35 to 7.45, optionally at pH 7.40.
25. The anti-PD-1 antibody or antigen-binding fragment thereof of any of claims 1-24, wherein the antibody or antigen-binding fragment thereof binds to human PD-1 protein with an association rate constant (Kon) of 4 x 105 1/Ms to about 5 x 106 1/Ms at pH 5.0 to 6.8, optionally at pH 6.0 or pH 5.0.
26. The anti-PD-1 antibody or antigen-binding fragment thereof of any of claims 1-25, wherein the antibody or antigen-binding fragment thereof binds to human PD-1 protein with an dissociation rate constant (Koff) of 4 x 104 1/s to about 2 x 102 1/s at pH 5.0 to 6.8, optionally at pH 6.0 or pH 5.0.
27. A conjugate, comprising the anti-PD-1 antibody or antigen-binding fragment thereof of any of claims 1-26 and a heterologous molecule or moiety.
28. The conjugate of claim 27, wherein the heterologous molecule or moiety is a cytotoxic agent, a drug, enzymatically active toxin or fragment thereof, or a radioactive atom.
29. A bispecific antibody, comprising the anti-PD-1 antibody or antigen-binding fragment thereof of any of claims 1-26.
30. A PD-1 directed chimeric antigen receptor (CAR) comprising an extracellular antigenbinding domain comprising the anti-PD-1 antibody or antigen-binding fragment thereof of any of claims 1-26, a transmembrane region and an intracellular signaling region.
31. A polynucleotide or polynucleotides comprising a nucleic acid sequence encoding the anti-PD-1 antibody or antigen-binding domain thereof of any of claims 1-26 or a heavy or light chain thereof.
32. A polynucleotide comprising a nucleic acid encoding the PD-1 directed chimeric antigen receptor of claim 30.
33. A vector, comprising the polynucleotide of claim 31 or 32.
34. The vector of claim 33, wherein the vector is a viral vector.
35. The vector of claim 34, wherein the viral vector is a retroviral vector or a lentiviral vector.
36. An engineered cell comprising the anti-PD-1 antibody or antigen-binding fragment thereof of any of claims 1-26, the bispecific antibody of claim 29, or the conjugate of claim 27 or 28.
37. An engineered cell comprising the PD-1 directed chimeric antigen receptor of claim 30.
38. The engineered cell of claim 36 or 37, wherein the cell is a lymphocyte.
39. The engineered cell of claim 38, wherein the lymphocyte is a natural killer (NK) cell or a T cell.
40. The engineered cell of any of claims 36-39, wherein the cell is a T cell and the T cell is a CD4+ T cell or a CD8+ T cell.
41. A cell comprising the polynucleotide of claim 31 or 32, or the vector of any of claims 33-35.
42. The cell of claim 41, wherein the cell is a mammalian cell.
43. A method of producing an antibody comprising culturing the cell of claim 41 or 42 under a condition that produces the antibody.
44. The method of claim 43, further comprising recovering the antibody produced by the cell.
45. An antibody or antigen-binding fragment thereof produced by the method of claim 43 or claim 44.
46. A composition comprising the cell of any of claims 36-42.
47. A composition comprising the anti-PD-1 antibody or antigen-binding fragment thereof of any of claims 1-26 and 45, the conjugate of claim 27 or 28, the bispecific antibody of claim 29, or the PD-1 directed chimeric antigen receptor of claim 30.
48. The composition of claim 46 or claim 47, further comprising a pharmaceutically acceptable excipient.
49. A method of treatment, comprising administering the cell of any of claims 36-42 or the composition of any of claims 46-48 to a subject having a disease or disorder associated with PD-1.
50. A method of increasing the activity of an immune cell, comprising contacting an immune cell with the anti-PD-1 antibody or antigen-binding fragment thereof of any of claims 1-26 and 42, the conjugate of claim 27 or 28, or the bispecific antibody of claim 29.
51. A method of increasing the activity of an immune cell, comprising administering to a subject in need thereof a therapeutically effective amount of the anti-PD-1 antibody or antigen-binding fragment thereof of any of claims 1-26 and 42, the conjugate of claim 27 or 28, the bispecific antibody of claim 29.
52. The method of claim 50 or claim 51, wherein the method is used for an ex vivo treatment of a disease or disorder in a subject.
53. The method of claim 49 or claim 52, wherein the disease or disorder is a cancer or an infection or infectious disease.
54. A binding molecule comprising a PD-1 binding domain that binds PD-1, and a cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) binding domain that binds CTLA-4, wherein:
(a) the PD-1 binding domain comprises a heavy chain and a light chain comprising the heavy chain variable (VH) region (PD-1 VH ) and the light chain variable (VL) region (PD-1 VL), respectively, of the anti-PD-1 antibody or antigen-binding fragment thereof of any of claims 1- 26, and
(b) the CTLA-4 binding domain comprises a heavy chain comprising a heavy chain variable (CTLA-4 VH) region and a light chain comprising a light chain variable (CTLA-4 VL) region, wherein:
(i) the CTLA-4 VH region comprises a heavy chain complementarity determining region 1 (CDR-H1) comprising the sequence set forth in any one of SEQ ID NOs: 278-285, a heavy chain complementarity determining region 2 (CDR-H2) comprising the sequence set forth in any one of SEQ ID NOs: 286-296, and a heavy chain complementarity determining region 3 (CDR-H3) comprising the sequence set forth in any one of SEQ ID NOs: 297-302; and the CTLA-4 VL region comprises a light chain complementarity determining region 1 (CDR-L1) comprising the sequence set forth in any one of SEQ ID NOs: 303-317, a light chain complementarity determining region 2 (CDR-L2) comprising the sequence set forth in any one of SEQ ID NOs: 318-326, and a light chain complementarity determining region 3 (CDR-L3) comprising the sequence set forth in any one of SEQ ID NOs: 327- 329, wherein the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 are not set forth in SEQ ID NOS: 278, 286, 297,303, 318, and 327, respectively; or (ii) the CTLA-4 VH region comprises a heavy chain complementarity determining region 1 (CDR-H1), a heavy chain complementarity determining region 2 (CDR-H2), and a heavy chain complementarity determining region 3 (CDR-H3) contained within any one of SEQ ID NOs: 230-256, and 341, and the CTLA-4 VL region comprises a light chain complementarity determining region 1 (CDR-L1), a light chain complementarity determining region 2 (CDR-L2), and a light chain complementarity determining region 3 (CDR-L3) contained within any one of SEQ ID NOs: 257, 277, and 347, wherein the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 are not set forth in SEQ ID NOS: 278, 286, 297,303, 318, and 327, respectively.
55. The binding molecule of claim 54, wherein: the CTLA-4 VH region comprises a CDR-H1 comprising the sequence set forth in any one of SEQ ID NOs: 278-285, a CDR-H2 comprising the sequence set forth in any one of SEQ ID NOs: 286- 296, and a CDR-H3 comprising the sequence set forth in any one of SEQ ID NOs: 297-302; and the CTLA-4 VL region comprises a CDR-L1 comprising the sequence set forth in any one of SEQ ID NOs: 303-317, a CDR-L2 comprising the sequence set forth in any one of SEQ ID NOs: 318- 326, and a CDR-L3 comprising the sequence set forth in any one of SEQ ID NOs: 327-329, wherein the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 are not set forth in SEQ ID NOS: 278, 286, 297,303, 318, and 327, respectively.
56. The binding molecule of claim 54 or claim 55, wherein: the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 230, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 347; the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 231, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 347; the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 232, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 347; the CTLA-4 VH region comprises a CDR- H1 , a CDR-H2, and a CDR-H3 contained within SEQ ID NO:233, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 347; the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO:234, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 347; the CTLA-4 VH region comprises a CDR-H1 , a CDR-H2, and a CDR-H3 contained within SEQ ID NO:235, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 347; the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO:236, and the CTLA-4 VL region comprises a CDR-L1 , a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 347; the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO:237, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 347; the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO:238, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 347; the CTLA-4 VH region comprises a CDR- H1 , a CDR-H2, and a CDR-H3 contained within SEQ ID NO:239, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 347; the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO:240, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 347; the CTLA-4 VH region comprises a CDR-H1 , a CDR-H2, and a CDR-H3 contained within SEQ ID NO:241 , and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 347; the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO:242, and the CTLA-4 VL region comprises a CDR-L1 , a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 347; the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO:243, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 347; the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO:244, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 347; the CTLA-4 VH region comprises a CDR- H1 , a CDR-H2, and a CDR-H3 contained within SEQ ID NO:245, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 347; the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO:246, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 347; the CTLA-4 VH region comprises a CDR-H1 , a CDR-H2, and a CDR-H3 contained within SEQ ID NO:247, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 347; the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO:248, and the CTLA-4 VL region comprises a CDR-L1 , a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 347; the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO:249, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 347; the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO:250, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 347; the CTLA-4 VH region comprises a CDR- H1 , a CDR-H2, and a CDR-H3 contained within SEQ ID NO:251 , and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 347; the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 341, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 257; the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 341, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 258; the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 341, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 259; the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 341, and the CTLA-4 VL region comprises a CDR-L1, a CDR- L2, and a CDR-L3 contained within SEQ ID NO: 260; the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 341, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 261; the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 341, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 262; the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 341, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 263; the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 341, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 264; the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 341, and the CTLA-4 VL region comprises a CDR-L1, a CDR- L2, and a CDR-L3 contained within SEQ ID NO: 265; the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 341, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 266; the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 341, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 267; the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 341, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 268; the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 341, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 269; the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 341, and the CTLA-4 VL region comprises a CDR-L1, a CDR- L2, and a CDR-L3 contained within SEQ ID NO: 270; the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 341, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 271; the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 341, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 272; the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 341, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 273; the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 341, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 274; the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 341, and the CTLA-4 VL region comprises a CDR-L1, a CDR- L2, and a CDR-L3 contained within SEQ ID NO: 275 the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 252, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 270; the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 253, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 270; the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 254, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 270; the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 255, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 270; the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 251, and the CTLA-4 VL region comprises a CDR-L1, a CDR- L2, and a CDR-L3 contained within SEQ ID NO: 270; the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 256, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 270; the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 252, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 276; the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 253, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 276; the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 254, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 276; the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 255, and the CTLA-4 VL region comprises a CDR-L1, a CDR- L2, and a CDR-L3 contained within SEQ ID NO: 276; the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 251, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 276; the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 256, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 276; the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 252, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 258 the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 253, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 258; the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 254, and the CTLA-4 VL region comprises a CDR-L1, a CDR- L2, and a CDR-L3 contained within SEQ ID NO: 258; the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 255, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 258; the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 251, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 258; the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 256, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 258; the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 252, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 268; the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 253, and the CTLA-4 VL region comprises a CDR-L1, a CDR- L2, and a CDR-L3 contained within SEQ ID NO: 268; the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 254, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 268; the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 255, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 268; the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 251, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 268; the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 256, and the CTLA-4 VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 contained within SEQ ID NO: 268; or the CTLA-4 VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 contained within SEQ ID NO: 234, and the CTLA-4 VL region comprises a CDR-L1, a CDR- L2, and a CDR-L3 contained within SEQ ID NO: 277.
57. The binding molecule of any one of claims 54-56, wherein: the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 287, and 297, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 303, 318, and 327, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 286, and 298, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 303, 318, 327, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 280, 286, and 297, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 303, 318, and 327, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 279, 288, and 297, respectively, and the -LI, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 303, 318, and 327, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 281, 289, and 297, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 303, 318, and 327, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 281, 290, and 297, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 303, 318, and 327, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 280, 291, 297, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 303, 318, and 327, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 279, 292, and 297, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 303, 318, and 327, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 279, 286, and 298, respectively, and the CDR-L1, the CDR- L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 303, 318, and 327, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 282, 286, and 297, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 303, 318, and 327, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 282, 286, and 299, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 303, 318, and 327, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 283, 286, and 300, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 303, 318, and 327, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 284, 286, and 297, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 303, 318, and 327, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 285, 286, and 301, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 303, 318, and 327, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 293, 297, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 303, 318, and 327, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 289, and 297, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 303, 318, and 327, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 294, 297, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 303, 318, and 327, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 295, and 297, respectively, and the CDR-L1, a CDR-L2, and a CDR-L3 comprise the sequences set forth in SEQ ID NOS: 303, 318, and 327, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 286, and 300, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 303, 318, and 327, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 286, and 302, respectively, and the CDR-L1, the CDR- L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 303, 318, and 327, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 286, and 301, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 303, 318, and 327, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 286, and 297, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 304, 318, and 327, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 286, and 297, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 305, 318, and 327, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 286, and 297, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 306, 318, 327, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 286, and 297, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 307, 319, and 327, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 286, and 297, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 307, 319, and 327, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 286, and 297, respectively, and the CDR-L1, the CDR-L2, and the CDR- L3 comprise the sequences set forth in SEQ ID NOS: 310, 320, and 327, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 286, and 297, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 307, 318, and 328, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 286, and 297, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 310, 321, and 327, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 286, and 297, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 311, 318, and 327, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 286, and 297, respectively, and the CDR-L1, the CDR- L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 311, 322, and 327, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 286, and 297, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 312, 321, and 327, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 286, and 297, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 313, 318, and 328, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 286, and 297, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 313, 318, and 329, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 286, and 297, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 314, 318, and 327, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 286, and 297, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 315, 323, and 327, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 286, and 297, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 316, 324, and 327, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 286, and 297, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 314, 325, and 327, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 286, and 297, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 317, 318, and 327, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 286, and 297, respectively, and the CDR-L1, the CDR- L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 303, 326, and 327, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 296, and 297, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 314, 318, and 327, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 294, and 302, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 314, 318, and 327, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 281, 289, and 301, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 314, 318, and 327, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 281, 296, and 297, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 314, 318, and 327, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 286, and 301, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 314, 318, and 327, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 294, and 301, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 314, 318, and 327, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 296, and 297, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 313, 318, and 327, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 294, and 302, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 313, 318, and 327, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 281, 289, and 301, respectively, and the CDR-L1, the CDR- L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 313, 318, and 327, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 281, 296, and 297, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 313, 318, and 327, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 286, and 301, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 313, 318, and 327, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 294, and 301, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 313, 318, and 327, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 296, and 297, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 305, 318, and 327, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 294, and 302, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 305, 318, and 327, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 281, 289, and 301, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 305, 318, and 327, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 281, 296, and 297, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 305, 318, and 327, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 286, and 301, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 305, 318, and 327, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 294, 301, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 305, 318, 327, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 296, and 297, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 313, 318, and 328, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 294, and 302, respectively, and the CDR-L1, the CDR- L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 313, 318, and 328, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 281, 289, and 301, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 313, 318, and 328, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 281, 296, and 297, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 313, 318, and 328, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 286, and 301, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 313, 318, and 328, respectively; the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 278, 294, and 301, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 313, 318, and 328, respectively; or the CDR-H1, the CDR-H2, and the CDR-H3 comprise the sequences set forth in SEQ ID NOS: 281, 289, and 297, respectively, and the CDR-L1, the CDR-L2, and the CDR-L3 comprise the sequences set forth in SEQ ID NOS: 317, 318, 329, respectively.
58. The binding molecule of any one of claims 54-57, wherein: the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 230 and SEQ ID NO: 347, respectively; the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 231 and SEQ ID NO: 347, respectively; the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 232 and SEQ ID NO: 347, respectively; the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 233 and SEQ ID NO: 347, respectively; the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 234 and SEQ ID NO: 347, respectively; the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 235 and SEQ ID NO: 347, respectively; the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 236 and SEQ ID NO: 347, respectively; the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 237 and SEQ ID NO: 347, respectively; the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 238 and SEQ ID NO: 347, respectively; the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 239 and SEQ ID NO: 347, respectively; the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 240 and SEQ ID NO: 347, respectively; the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 241 and SEQ ID NO: 347, respectively; the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 242 and SEQ ID NO: 347, respectively; the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 243 and SEQ ID NO: 347, respectively; the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 244 and SEQ ID NO: 347, respectively; the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 245 and SEQ ID NO: 347, respectively; the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 246 and SEQ ID NO: 347, respectively; the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 247 and SEQ ID NO: 347, respectively; the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 248 and SEQ ID NO: 347, respectively; the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 249 and SEQ ID NO: 347, respectively; the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 250 and SEQ ID NO: 347, respectively; the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 251 and SEQ ID NO: 347, respectively; the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 341 and SEQ ID NO: 257, respectively; the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 341 and SEQ ID NO: 258, respectively; the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 341 and SEQ ID NO: 259, respectively; the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 341 and SEQ ID NO: 260, respectively; the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 341 and SEQ ID NO: 261, respectively; the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 341 and SEQ ID NO: 262, respectively; the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 341 and SEQ ID NO: 263, respectively; the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 341 and SEQ ID NO: 264, respectively; the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 341 and SEQ ID NO: 265, respectively; the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 341 and SEQ ID NO: 266, respectively; the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 341 and SEQ ID NO: 267, respectively; the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 341 and SEQ ID NO: 268, respectively; the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 341 and SEQ ID NO: 26, respectively; the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 341 and SEQ ID NO: 270, respectively; the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 341 and SEQ ID NO: 271, respectively; the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 341 and SEQ ID NO: 272, respectively; the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 341 and SEQ ID NO: 273, respectively; the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 341 and SEQ ID NO: 274, respectively; the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 341 and SEQ ID NO: 275, respectively; the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 252 and SEQ ID NO: 270, respectively; the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 253 and SEQ ID NO: 270, respectively; the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 254 and SEQ ID NO: 270, respectively; the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 255 and SEQ ID NO: 270, respectively; the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 251 and SEQ ID NO: 270, respectively; the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 256 and SEQ ID NO: 270, respectively; the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 252 and SEQ ID NO: 276, respectively; the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 253 and SEQ ID NO: 276, respectively; the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 254 and SEQ ID NO: 276, respectively; the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 255 and SEQ ID NO: 276, respectively; the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 251 and SEQ ID NO: 276, respectively; the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 256 and SEQ ID NO: 276, respectively; the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 252 and SEQ ID NO: 258, respectively; the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 253 and SEQ ID NO: 258, respectively; the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 254 and SEQ ID NO: 258, respectively; the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 255 and SEQ ID NO: 258, respectively; the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 251 and SEQ ID NO: 258, respectively; the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 256 and SEQ ID NO: 258, respectively; the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 252 and SEQ ID NO: 268, respectively; the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 253 and SEQ ID NO: 268, respectively; the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 254 and SEQ ID NO: 268, respectively; the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 255 and SEQ ID NO: 268, respectively; the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 251 and SEQ ID NO: 268, respectively; the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 256 and SEQ ID NO: 268, respectively; or the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 234 and SEQ ID NO: 277, respectively.
59. The binding molecule of any one of claims 54-58, wherein: the CTLA-4 VH region and the CTLA-4 VL region are or comprise: the sequence set forth in SEQ ID NO: 230 and 347, respectively; the sequence set forth in SEQ ID NO: 231 and 347, respectively; the sequence set forth in SEQ ID NO: 232 and 347, respectively; the sequence set forth in SEQ ID NO: 233 and 347, respectively; the sequence set forth in SEQ ID NO: 234 and 347, respectively; the sequence set forth in SEQ ID NO: 235 and 347, respectively; the sequence set forth in SEQ ID NO: 236 and 347, respectively; the sequence set forth in SEQ ID NO: 237 and 347, respectively; the sequence set forth in SEQ ID NO: 238 and 347, respectively; the sequence set forth in SEQ ID NO: 239 and 347, respectively; the sequence set forth in SEQ ID NO: 240 and 347, respectively; the sequence set forth in SEQ ID NO: 241 and 347, respectively; the sequence set forth in SEQ ID NO: 242 and 347, respectively; the sequence set forth in SEQ ID NO: 243 and 347, respectively; the sequence set forth in SEQ ID NO: 244 and 347, respectively; the sequence set forth in SEQ ID NO: 245 and 347, respectively; the sequence set forth in SEQ ID NO: 246 and 347, respectively; the sequence set forth in SEQ ID NO: 247 and 347, respectively; the sequence set forth in SEQ ID NO: 248 and 347, respectively; the sequence set forth in SEQ ID NO: 249 and 347, respectively; the sequence set forth in SEQ ID NO: 250 and 347, respectively; the sequence set forth in SEQ ID NO: 251 and 347, respectively; the sequence set forth in SEQ ID NO: 341 and 257, respectively; the sequence set forth in SEQ ID NO: 341 and 258, respectively; the sequence set forth in SEQ ID NO: 341 and 259, respectively; the sequence set forth in SEQ ID NO: 341 and 260, respectively; the sequence set forth in SEQ ID NO: 341 and 261, respectively; the sequence set forth in SEQ ID NO: 341 and 262, respectively; the sequence set forth in SEQ ID NO: 341 and 263, respectively; the sequence set forth in SEQ ID NO: 341 and 264, respectively; the sequence set forth in SEQ ID NO: 341 and 265, respectively; the sequence set forth in SEQ ID NO: 341 and 266, respectively; the sequence set forth in SEQ ID NO: 341 and 267, respectively; the sequence set forth in SEQ ID NO: 341and 268, respectively; the sequence set forth in SEQ ID NO: 341 and 269, respectively; the sequence set forth in SEQ ID NO: 341and 270, respectively; the sequence set forth in SEQ ID NO: 341and 271, respectively; the sequence set forth in SEQ ID NO: 341and 272, respectively; the sequence set forth in SEQ ID NO: 341and 273, respectively; the sequence set forth in SEQ ID NO: 341and 274, respectively; the sequence set forth in SEQ ID NO: 341and 275, respectively; the sequence set forth in SEQ ID NO: 252 and 270, respectively; the sequence set forth in SEQ ID NO: 253 and 270, respectively; the sequence set forth in SEQ ID NO: 254 and 270, respectively; the sequence set forth in SEQ ID NO: 255 and 270, respectively; the sequence set forth in SEQ ID NO: 251 and 270, respectively; the sequence set forth in SEQ ID NO: 256 and 270, respectively; the sequence set forth in SEQ ID NO: 252 and 276, respectively; the sequence set forth in SEQ ID NO: 253 and 276, respectively; the sequence set forth in SEQ ID NO: 254 and 276, respectively; the sequence set forth in SEQ ID NO: 255 and 276, respectively; the sequence set forth in SEQ ID NO: 251 and 276, respectively; the sequence set forth in SEQ ID NO: 256 and 276, respectively; the sequence set forth in SEQ ID NO: 252 and 258, respectively; the sequence set forth in SEQ ID NO: 253 and 258, respectively; the sequence set forth in SEQ ID NO: 254 and 258, respectively; the sequence set forth in SEQ ID NO: 255 and 258, respectively; the sequence set forth in SEQ ID NO: 251 and 258, respectively; the sequence set forth in SEQ ID NO: 256 and 258, respectively; the sequence set forth in SEQ ID NO: 252 and 268, respectively; the sequence set forth in SEQ ID NO: 253 and 268, respectively; the sequence set forth in SEQ ID NO: 254 and 268, respectively; the sequence set forth in SEQ ID NO: 255 and 268, respectively; the sequence set forth in SEQ ID NO: 251 and 268, respectively; the sequence set forth in SEQ ID NO: 256 and 268, respectively; the sequence set forth in SEQ ID NO: 234 and 277, respectively.
60. A binding molecule comprising a cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) binding domain that binds CTLA-4, and a PD-1 binding domain that binds PD-1, wherein:
(a) the PD-1 binding domain comprises a heavy chain and a light chain comprising the heavy chain variable (VH) region (PD-1 VH ) and the light chain variable (VL) region (PD-1 VL), respectively, of the anti-PD-1 antibody or antigen-binding fragment thereof of any of claims 1- 26; and
(b) the CTLA-4 binding domain comprises a heavy chain variable (CTLA-4 VH) region and a light chain variable (CTLA-4 VL) region, wherein: the CTLA-4 VH region comprises the sequence set forth in: QVQLVESGGGVVQPGRSLRLSCAASGFTFSX , YTMHW VRQAPGKGLEWVTFIS YX2GNNKX3 YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAIYYCARTGX4X5GPFDYWGQGTLVTVSS, wherein X, is S or R; X2 is E or H; X3 is H or Y; X4 is W or H; and X5 is L or H (SEQ ID NO: 407); and the CTLA-4 VL region comprises the sequence set forth in: EIVLTQSPGTLSLSPGERATLSCRASQX1VGHX2YLAWYQQKPGQAPRLLIYGAFSRATGIPD RFSGSGSGTDFTLTISRLEPEDFAVYYCQX3YGSSPWTFGQGTKVEIK, wherein X, is H or S; X2 is H or S; and X3 is Q or H (SEQ ID NO: 408).
61. The binding molecule of claim 54-60, wherein: the CTLA-4 VH region comprises a CDR-H1 comprising the sequence set forth in SEQ ID NO: 278 or SEQ ID NO: 281, a CDR-H2 comprising the sequence set forth in SEQ ID NO: 289 or SEQ ID NO: 296, and a CDR-H3 comprising the sequence set forth in SEQ ID NO: 297 or SEQ ID NO: 301; and the CTLA-4 VL region comprises a CDR-L1 comprising the sequence set forth in SEQ ID NO: 305 or SEQ ID NO: 313; a CDR-L2 comprising the sequence set forth in SEQ ID NO: 318, and a CDR- L3 comprising the sequence set forth in SEQ ID NO: 327 or SEQ ID NO: 328.
62. The binding molecule of any one of claims 54-61, wherein the CTLA-4 VH region comprises:
(a) a serine (S) at position 31, a glutamic acid (E) at position 54, a histidine (H) at position 59, a tryptophan (W) at position 101, and a leucine (L) at position 102, wherein the numbering is relative to SEQ ID NO: 407, or (b) a arginine (R) at position 31, a glutamic acid (E) at position 54, a tyrosine (Y) at position 59, a tryptophan (W) at position 101, and a histidine (H) at position 102, wherein the numbering is relative to SEQ ID NO: 408.
63. The binding molecule of any one of claims 54-62, wherein the CTLA-4 VL region comprises:
(a) a histidine (H) at position 28, a serine (S) at position 32, and a glutamine (Q) at position 91, wherein the numbering is relative to SEQ ID NO: 407, or(b) a serine (S) at position 28, a serine (S) at position 32, and a histidine (H) at position 91, wherein the numbering is relative to SEQ ID NO: 408.
64. The binding molecule of any one of claims 54-62, wherein: the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 254 and SEQ ID NO: 258, respectively; the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 254 and SEQ ID NO: 268, respectively; the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 252 and SEQ ID NO: 268, respectively.
65. The binding molecule of any one of claims 54-64, wherein: the CTLA-4 VH region and the CTLA-4 VL region are or comprise: the sequence set forth in SEQ ID NO: 254 and 258, respectively; the sequence set forth in SEQ ID NO: 254 and 268, respectively; the sequence set forth in SEQ ID NO: 252 and 268, respectively.
66. The binding molecule of any one of claims 54-65, wherein: the CTLA-4 VH region and the CTLA-4 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98% or 99% identity to SEQ ID NO: 254 and SEQ ID NO: 268, respectively.
67. The binding molecule of any one of claims 54-66, wherein: the PD-1 VH region and the PD-1 VL region is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 168 and SEQ ID NO: 172, respectively.
68. The binding molecule of any one of claims 54-67, wherein: the PD-1 VH region is or comprises an amino acid sequence set forth in SEQ ID NO: 168, and the PD-1 VL region is or comprises an amino acid sequence set forth in SEQ ID NO: 172.
69. The binding molecule of any one of claims 54-68 , wherein the CTLA-4 binding domain and the PD-1 binding domain are Fab fragments each comprising a VH region and a VL region, wherein one of the Fab fragments is a crossover Fab such that the VH region and the VL region of such Fab are replaced by each other.
70. The binding molecule of any one of claims 54-69, wherein the CTLA-4 binding domain is a crossover CTLA-4 Fab and the PD-1 binding domain is a Fab, wherein: the crossover CTEA-4 Fab comprises: (a) a heavy chain (HC1) comprising the CTEA-4 VL region and a heavy chain constant region 1 (CHI), designated CTEA-4 VL-CH1 ; and (b) a light chain (ECI) comprising the CTLA-4 VH region and a light chain constant region (CL), designated CTLA-4 VH-CL; and the PD-1 binding domain comprises (a) a heavy chain 2 (HC2) comprising the PD-1 VH region and a heavy chain constant region 1 (CHI), designated PD-1 VH-CH1 ; and (b) a light chain 2 (LC2) comprising the PD-1 VL region and a light chain constant region (CL), designated PD-1 VL-CL.
71. The binding molecule of claim 70, wherein the CHI of the CTLA-4 VL-CH1 comprises the amino acid sequence set forth in SEQ ID NO: 363 and the CE of the CTEA-4 VH-CL comprises the amino acid sequence set forth in SEQ ID NO: 367.
72. The binding molecule of claim 70 and claim 71, wherein the CHI of the PD-1 VH-CH1 comprises the amino acid sequence set forth in SEQ ID NO: 371 and the CE of the PD-1 VL-CE comprises the amino acid sequence set forth in SEQ ID NO: 175.
73. The binding molecule of any one of claims 54-69, wherein the CTLA-4 binding domain is a CTLA-4 Fab and the PD-1 binding domain is a crossover Fab, wherein: the CTEA-4 Fab comprises: (a) a heavy chain (HC1) comprising the CTEA-4 VH region and a heavy chain constant region 1 (CHI), designated CTLA-4 VH-CH1 ; and (b) a light chain (LC1) comprising the CTLA-4 VL region and a light chain constant region (CL), designated CTLA-4 VL-CL; and the PD-1 binding domain comprises (a) a heavy chain 2 (HC2) comprising the PD-1 VL region and a heavy chain constant region 1 (CHI), designated PD-1 VL-CH1 ; and (b) a light chain 2 (LC2) comprising the PD-1 VH region and a light chain constant region (CL), designated PD-1 VH-CL.
74. The binding molecule of claim 73, wherein the CHI of the PD-1 VL-CH1 comprises the amino acid sequence set forth in SEQ ID NO: 363 and the CL of the PD-1 VH-CL comprises the amino acid sequence set forth in SEQ ID NO: 367.
75. The binding molecule of claim 73 and claim 74, wherein the CHI of the CTLA-4 VH- CH1 comprises the amino acid sequence set forth in SEQ ID NO: 371 and the CL of the CTLA-4 VL- CL comprises the amino acid sequence set forth in SEQ ID NO: 175.
76. The binding molecule of any of claims 69-75, wherein: the CTLA-4 VH comprises the sequence of amino acids set forth in SEQ ID NO: 254 and the CTLA-4 VL comprises the sequence of amino acids set forth in SEQ ID NO: 268; and the PD-1 VH comprises the sequence of amino acids set forth in SEQ ID NO: 168 and the PD- 1 VL comprise the sequence of amino acids set forth in SEQ ID NO: 172.
77. The binding molecule of any one of claims 54-76, further comprising an Fc region comprising a first and second polypeptide each comprising a hinge-CH2-CH3, wherein one of the first polypeptide and second polypeptide is linked to the CHI of the CTLA-4 Fab and the other of the first polypeptide and second polypeptide is linked to the CHI of the PD-1 Fab.
78. The binding molecule of claim 77, wherein the Fc region is a heterodimeric Fc region.
79. The binding molecule of claim 78, wherein each of the first and second polypeptide of the heterodimeric Fc comprises one or more amino acid substitutions in a wild-type Fc polypeptide region to effect heterodimer formation between the first polypeptide and the second polypeptide.
80. The binding molecule of claim 79, wherein the wild-type Fc region is an IgGl Fc region.
81. The binding molecule of claim 79 or claim 80, wherein the wild-type Fc region comprises the sequence set forth in SEQ ID NO:407 or SEQ ID NO: 408.
82. The binding molecule of any of claims 79-81, wherein the one more amino acid substitutions are a knob-into-hole modification, a charge mutation to reduce or prevent selfassociation due to charge repulsion, or disulfide bonding pairing mutation.
83. The binding molecule of any of claims 79-82, wherein the one or more amino acid substitutions are: (i) amino acid substitution T366W in the first polypeptide of the heterodimeric Fc and amino acid substitutions T366S, E368A and Y407V in the second polypeptide of the heterodimeric Fc; (ii) amino acid substitution E356K, E357K and D399K in the first polypeptide of the heterodimeric Fc and amino acid substitutions K439E, K370E and K409D in the second polypeptide of the heterodimeric Fc; or (iii) amino acid substitution S354C on the first polypeptide of the heterodimeric Fc and amino acid substitution Y349C on the second polypeptide of the heterodimeric Fc.
84. The binding molecule of any of claims 79-82, wherein the one or more substitutions are a knob-into-hole modification.
85. The binding molecule of any of claims 79-84, wherein the one or more amino acid substitutions of the first Fc polypeptide comprises Thr366Trp and the one or more amino acid substitutions of the second Fc polypeptide of the heterodimeric Fc comprises the Thr366Ser, Leu368Ala and Tyr407Val.
86. The binding molecule of any of claims 79-85, wherein the first and/or second Fc polypeptide further comprises the amino acid substitution H435R.
87. The binding molecule of any of claims 79-86, wherein the heterodimeric Fc region comprises one or more amino acid substitutions to reduce binding affinity to an Fc receptor and/or to reduce effector function, optionally as compared to a wild-type IgGl Fc domain.
88. The binding molecule of claim 87, wherein the one or more amino acid substitutions are selected from L234A, L234V, L235A, L235E, G237A, D265S, S267K, R292C, N297G, V302C, and P329G by EU numbering.
89. The binding molecule of claim 87 or claim 88, wherein the one or more amino acid substitutions comprises L234A and L235A.
90. The binding molecule of any of claims 87-89, wherein the one or more amino acid substitutions comprises L234A, L235A, and D265S.
91. The binding molecule of any of claims 87-90, wherein the one or more amino acid substitutions comprises L234A, L235A, and P329G.
92. The binding molecule of any of claims 79-91, wherein one or both polypeptides of the heterodimeric Fc lacks Lys447.
93. The binding molecule of any of claims 79-92, wherein: one of the first polypeptide and the second polypeptide of the heterodimeric Fc region comprise an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity SEQ ID NO:364; and the other of the first polypeptide and second polypeptide of the heterodimeric Fc region comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity SEQ ID NO: 373.
94. The binding molecule of any of claims 79-93, wherein: one of the first polypeptide and the second polypeptide of the heterodimeric Fc region comprise the amino acid sequence set forth in SEQ ID NO: 364; and the other of the first polypeptide and the second polypeptide of the heterodimeric Fc region comprises the amino acid sequence set forth in SEQ ID NO: 373.
95. The binding molecule of any of claims 79-94, wherein the first polypeptide of the heterodimeric Fc region comprise the amino acid sequence set forth in SEQ ID NO: 364 and the second polypeptide of the heterodimeric Fc region comprises the amino acid sequence set forth in SEQ ID NO:373.
96. The binding molecule of any of claims 79-94, wherein the first polypeptide of the heterodimeric Fc region comprise the amino acid sequence set forth in SEQ ID NO: 373 and the second polypeptide of the heterodimeric Fc region comprises the amino acid sequence set forth in SEQ ID
NO:364.
97. The binding molecule of any of claims 54-96, comprising:
(a) a heavy chain 1 (HC1) polypeptide comprising a VL sequence set forth in SEQ ID NO:268, a CHI sequence set forth in SEQ ID NO: 363, and a hinge-CH2-CH3 sequence set forth in SEQ ID NO: 364; (b) a light chain 1 (LC1) polypeptide comprising a VH sequence set forth in SEQ ID NO:254 and a CL sequence set forth in SEQ ID NO: 367; (c) a HC2 polypeptide comprising a VH sequence set forth in SEQ ID NO: 168, a CHI sequence set forth in SEQ ID NO:371, and a hinge-CH2-CH3 sequence set forth in SEQ ID NO:373; and (d) a LC2 polypeptide comprising a VL sequence set forth in SEQ ID NO: 172 and a CL sequence set forth in SEQ ID NO: 175.
98. The binding molecule of claim 97, comprising:
(a) a HC1 polypeptide that is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 378; (b) a HC2 polypeptide that is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 387; (c) a LC1 polypeptide that is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 381; and (d) a LC2 is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 389.
99. The binding molecule of claim 97 or 98, comprising:
(a) a HC1 polypeptide that is or comprises the amino acid sequence set forth in SEQ ID NO: 378; (b) a HC2 polypeptide that is or comprises the amino acid sequence set forth in SEQ ID NO: 387;
(c) a LC1 polypeptide that is or comprises the amino acid sequence set forth in SEQ ID NO: 381; and
(d) a LC2 polypeptide that is or comprises the amino acid sequence set forth in SEQ ID NO: 389.
100. The binding molecule of any of claims 54-96, comprising:
(a) a heavy chain 1 (HC1) polypeptide comprising a VL sequence set forth in SEQ ID NO: 172, a CHI sequence set forth in SEQ ID NO: 363, and a hinge-CH2-CH3 sequence set forth in SEQ ID NO: 364; (b) a LC1 polypeptide comprising a VH sequence set forth in SEQ ID NO: 168, and a CL sequence set forth in SEQ ID NO:367; and (c) a HC2 polypeptide comprising a VH sequence set forth in SEQ ID NO:254, a CHI sequence set forth in SEQ ID NO:371, and a hinge-CH2-CH3 sequence set forth in SEQ ID NO: 373; and (d) a LC2 polypeptide comprising a VL sequence set forth in SEQ ID NO:268, and a CL sequence set forth in SEQ ID NO: 175.
101. The binding molecule of claim 100, comprising: (a) a HC1 polypeptide that is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 360; (b) a HC2 polypeptide that is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 370; (c) a LC1 polypeptide that is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 366; and (d) a LC2 that is or comprises an amino acid sequence having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 375.
102. The binding molecule of claim 100-101, comprising:
(a) a HC1 polypeptide that is or comprises an amino acid sequence set forth in SEQ ID NO: 360; (b) a HC2 polypeptide that is or comprises an amino acid sequence set forth in SEQ ID NO: 370;
(c) a LC1 polypeptide that is or comprises an amino acid sequence set forth in SEQ ID NO: 366; and
(d) a LC2 polypeptide that is or comprises an amino acid sequence set forth in SEQ ID NO: 375.
103. The binding molecule of any of claims 54-102, wherein the heavy chain and/or the light chain further comprising a signal peptide.
104. A polynucleotide or polynucleotides comprising a nucleic acid sequence encoding the binding molecule of any one of claims 54-103 or a heavy or light chain thereof.
105. A vector comprising the polynucleotide of claim 104.
106. The vector of claim 105, wherein the vector is a viral vector.
107. The vector of claim 106, wherein the viral vector is a retroviral vector or a lentiviral vector.
108. A cell comprising the polynucleotide or polynucleotides of claim 104, or the vector of any of claims 105-107.
109. The cell of claim 108, wherein the cell is a mammalian cell.
110. A method of producing a binding molecule comprising culturing the cell of claim 108 or claim 109 under a condition that produces the binding molecule, optionally wherein the binding molecule is bispecific antibody binding molecule.
111. The method of claim 110, further comprising recovering the binding molecule produced by the cell.
112. A binding molecule produced by the method of claim 110 or claim 111.
113. A composition comprising the binding molecule of any one of claims 54-103 and claim 112.
114. The composition of claim 113, further comprising a pharmaceutically acceptable excipient.
115. A method for treating a subject with a disease or condition, the method comprising administering a therapeutically effective amount of the binding molecule of any one of claims 54-103 and 112 or the composition of claim 113 or claim 114.
116. The method of claim 115, wherein the disease or condition is a cancer.
117. A method of cancer immunotherapy, the method comprising administering to a subject in need thereof a therapeutically effective amount of the binding molecule of any one of claims 54-103 and 112 or composition of claim 113 or claim 114.
118. The method of claim 117, wherein the cancer is a solid tumor.
119. The method of any of claims 116-118, wherein the cancer is a melanoma, breast carcinoma, HCC, Renal cell carcinoma, Colorectal carcinoma, non-small-cell lung carcinoma, Cervical cancer or Mesothelioma.
120. The method of any of claims 115-119, wherein administering the binding molecule to the subject results in higher Teff-to-suppressor cells (myeloid-derived suppressor cell and Treg) ratios.
121. The method of any of claims 115-120, wherein administering the binding molecule to the subject results in increased pro-inflammatory cytokines, optionally interferon-gamma (IFN-y) and/or tumor necrosis factor-alpha (TNF-a).
122. The method of any one of claims 115-121, wherein the method comprises administering to the subject an effective amount of the binding molecule of any one of claims 54-103 and 112 or the composition of claim 113 or claim 114 prior to a surgery or after a surgery to remove a solid tumor in the subject.
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