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WO2024199269A9 - Immunoconjugués contenant du tnf-alpha et procédés associés et compositions associées - Google Patents

Immunoconjugués contenant du tnf-alpha et procédés associés et compositions associées

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Publication number
WO2024199269A9
WO2024199269A9 PCT/CN2024/084035 CN2024084035W WO2024199269A9 WO 2024199269 A9 WO2024199269 A9 WO 2024199269A9 CN 2024084035 W CN2024084035 W CN 2024084035W WO 2024199269 A9 WO2024199269 A9 WO 2024199269A9
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Prior art keywords
amino acid
antibody
seq
tnf
acid sequence
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Pending
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WO2024199269A1 (fr
Inventor
Lucas Bailey
Qufei LI
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Suzhou Fuse Biosciences Ltd
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Suzhou Fuse Biosciences Ltd
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Publication of WO2024199269A1 publication Critical patent/WO2024199269A1/fr
Anticipated expiration legal-status Critical
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Pending legal-status Critical Current

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    • 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
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
    • 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
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/39558—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against tumor tissues, cells, antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/525—Tumour necrosis factor [TNF]
    • 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
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C07K16/241—Tumor Necrosis Factors
    • 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
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/55—Fab or Fab'
    • 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
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • 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
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • 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
    • C07K2317/70—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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present disclosure generally relates to tumor necrosis factor alpha (TNF- ⁇ ) containing immunoconjugate molecules. More particularly, the present disclosure concerns immunoconjugate molecules exhibiting improved properties for use as immunotherapeutic agents due to the ability of modulating the immune system. The present disclosure further relates to therapeutic uses and pharmaceutical compositions of the immunoconjugate molecules for treating diseases such as cancer and other chronic infectious diseases.
  • TNF- ⁇ tumor necrosis factor alpha
  • Tumor necrosis factor alpha is a 17-kDa protein consisting of 157 amino acids that is a homotrimer in solution.
  • TNF- ⁇ is a potent, pleiotropic cytokine capable of triggering apoptosis of tumor endothelial cells, and consequently tumor necrosis.
  • TNF- ⁇ has gained approval in Europe for use in the treatment of soft tissue sarcoma in combination with chemotherapy. However, due to its severe systemic toxicities, TNF- ⁇ has a low maximum tolerated total systemic dose of ⁇ 0.4 mg.
  • the systemic toxicities include respiratory failure, coagulopathies, hypotension, thrombocytopenia, leukopenia, neurotoxicity, fever, headache, nausea/vomiting, hepatopathy, as well as general symptoms of malaise and weakness.
  • authorized use of TNF- ⁇ in soft tissue sarcoma is limited to isolated limb perfusion to reduce systemic exposure. Therefore, the full potential of TNF- ⁇ has not been realized, and there remains a need in the art to further enhance the therapeutic usefulness of TNF- ⁇ . The present disclosure meets this need.
  • the present disclosure provides immunoconjugate molecules comprising a TNF- ⁇ polypeptide.
  • the present disclosure also provides, in certain embodiments, polynucleotides and vectors comprising sequences encoding such immunoconjugate molecules, and compositions, reagents, and kits comprising such immunoconjugate molecules.
  • provided herein are also methods for delivery and/or activation of a TNF- ⁇ activity at a target site, or reduced toxicity and/or other side-effects associated with systemic exposure to the TNF- ⁇ activity in a subject through the use of the immunoconjugate molecules according to the present disclosure.
  • a two-in-one antibody or antigen-binding fragment thereof that binds to TNF- ⁇ and FAP, wherein the antibody or antigen-binding fragment thereof comprises: a light chain variable region (VL) comprising VL complementarity determining region 1 (CDR1) , VL CDR2, and VL CDR3 of antibody FN15 as set forth in Table 15; and/or a heavy chain variable region (VH) comprising VH complementarity determining region 1 (CDR1) , VH CDR2, and VH CDR3 of antibody FN15 as set forth in Table 16; or a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3 having an amino acid sequence of a VL CDR1, a VL CDR2, and a VL CDR3, respectively, of a VL having an amino acid sequence of SEQ ID NO: 27; and/or a VH comprising a VH CDR1, a VH CDR2, and
  • the anti-TNF- ⁇ /anti-FAP two-in-one antibody or antigen-binding fragment thereof (a) the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 amino acid sequences are according to the Kabat numbering system; (b) the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 amino acid sequences are according to the Chothia numbering system; (c) the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 amino acid sequences are according to the ABM numbering system; (d) the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 amino acid sequences are according to the Contact numbering system; or (e) the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and
  • the VL CDR1, VL CDR2, and VL CDR3 comprise amino acid sequences of SEQ ID NOS: 15, 16, and 17, respectively, and the VH CDR1, VH CDR2, and VH CDR3 comprise amino acid sequences of SEQ ID NOS: 18, 19, and 20, respectively.
  • the antibody or antigen-binding fragment thereof comprises a VL comprising an amino acid sequence having at least 95%sequence identity to SEQ ID NO: 27.
  • the antibody or antigen-binding fragment thereof comprises a VL comprising an amino acid sequence of SEQ ID NO: 27.
  • the antibody or antigen-binding fragment thereof comprises a VH comprising an amino acid sequence having at least 95%sequence identity to SEQ ID NO: 28.
  • the antibody or antigen-binding fragment thereof comprises a VH comprising an amino acid sequence of SEQ ID NO: 28.
  • the antibody or antigen-binding fragment thereof comprises a VL comprising an amino acid sequence having at least 95%sequence identity to SEQ ID NO: 27 and a VH comprising an amino acid sequence having at least 95%sequence identity to SEQ ID NO: 28.
  • the antibody or antigen-binding fragment thereof comprises: a VL comprising an amino acid sequence of SEQ ID NO: 27; and a VH comprising an amino acid sequence of SEQ ID NO: 28.
  • an immunoconjugate molecule comprising a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises a TNF- ⁇ moiety fused to a heavy chain variable region (VH) of a first antibody, wherein the second polypeptide chain comprises a light chain variable region (VL) of the first antibody fused to a second antibody capable of binding to FAP; and wherein the VH and VL forms a two-in-one binding domain capable of binding to TNF- ⁇ and FAP.
  • VH heavy chain variable region
  • VL light chain variable region
  • the VL comprises VL CDR1, VL CDR2, and VL CDR3 of antibody FN15 as set forth in Table 15; and/or wherein the VH comprises VH CDR1, VH CDR2, and VH CDR3 of antibody FN15 as set forth in Table 16; or (b) the VL comprises a VL CDR1, a VL CDR2, and a VL CDR3 having an amino acid sequence of a VL CDR1, a VL CDR2, and a VL CDR3, respectively, of a VL having an amino acid sequence of SEQ ID NO: 27; and/or the VH comprises a VH CDR1, a VH CDR2, and a VH CDR3 having an amino acid sequence of a VH CDR1, a VH CDR2, and a VH CDR3, respectively, of a VH having an amino acid sequence of SEQ ID NO: 28.
  • the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 amino acid sequences are according to the Kabat numbering system; (b) the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 amino acid sequences are according to the Chothia numbering system; (c) the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 amino acid sequences are according to the ABM numbering system; (d) the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 amino acid sequences are according to the Contact numbering system; or (e) the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 amino acid sequences are according to the IM
  • the VL CDR1, VL CDR2, and VL CDR3 comprise amino acid sequences of SEQ ID NOS: 15, 16, and 17, respectively, and the VH CDR1, VH CDR2, and VH CDR3 comprise amino acid sequences of SEQ ID NOS: 18, 19, and 200, respectively.
  • the antibody or antigen-binding fragment thereof comprises a VL comprising an amino acid sequence having at least 95%sequence identity to SEQ ID NO: 27.
  • the antibody or antigen-binding fragment thereof comprises a VL comprising an amino acid sequence of SEQ ID NO: 27.
  • the antibody or antigen-binding fragment thereof comprises a VH comprising an amino acid sequence having at least 95%sequence identity to SEQ ID NO: 28.
  • the antibody or antigen-binding fragment thereof comprises a VH comprising an amino acid sequence of SEQ ID NO: 28.
  • the antibody or antigen-binding fragment thereof comprises a VL comprising an amino acid sequence having at least 95%sequence identity to SEQ ID NO: 27 and a VH comprising an amino acid sequence having at least 95%sequence identity to SEQ ID NO: 28.
  • the antibody or antigen-binding fragment thereof comprises: a VL comprising an amino acid sequence of SEQ ID NO: 27; and a VH comprising an amino acid sequence of SEQ ID NO: 28.
  • the TNF- ⁇ moiety comprises an amino acid sequence of SEQ ID NO: 1 or a mutant thereof.
  • the mutant comprises a S95A substitution, a N34H substitution, a Y87A substitution, or a Y87F substitution in the amino acid sequence of SEQ ID NO: 1.
  • the second antibody is a scFv or VHH capable of binding to FAP.
  • the scFv comprises the sequence of SEQ ID NO: 51, or wherein the VHH comprises the sequence of SEQ ID NO: 50.
  • the TNF- ⁇ moiety in the first polypeptide chain, is fused to the N terminus of the VH via a first peptidic linker.
  • the first peptidic linker is about 50 amino acids in length; wherein optionally the first peptidic linker is a 10X (G4S) linker.
  • the second antibody in the second polypeptide chain, is fused to the N terminus of the VL via a second peptidic linker.
  • the second peptidic linker is about 30 amino acids in length or about 70 amino acids in length, wherein optionally the second peptidic linker is a 6X (G4S) linker or a 14X (G4S) linker.
  • the first polypeptide chain further comprises a CH1 domain of the first antibody; optionally wherein the CH1 domain is fused to the C terminus of the VH.
  • the second polypeptide chain further comprises a CL domain of the first antibody; optionally wherein the CL domain is fused to the C terminus of the VL.
  • an immunoconjugate molecule comprising two polypeptide chains, wherein (a) the first polypeptide chain comprises an amino acid sequence of SEQ ID NO: 53, and the second polypeptide chain comprises an amino acid sequence of SEQ ID NO: 54; (b) the first polypeptide chain comprises an amino acid sequence of SEQ ID NO: 55, and the second polypeptide chain comprises an amino acid sequence of SEQ ID NO: 54; (c) the first polypeptide chain comprises an amino acid sequence of SEQ ID NO: 56, and the second polypeptide chain comprises an amino acid sequence of SEQ ID NO: 54; (d) the first polypeptide chain comprises an amino acid sequence of SEQ ID NO: 57, and the second polypeptide chain comprises an amino acid sequence of SEQ ID NO: 54; (e) the first polypeptide chain comprises an amino acid sequence of SEQ ID NO: 58, and the second polypeptide chain comprises an amino acid sequence of SEQ ID NO: 59; (f) the first polypeptide chain comprises an amino acid sequence of SEQ
  • a two-in-one antibody or antigen-binding fragment thereof that binds to TNF- ⁇ and Trop-2, wherein the antibody or antigen-binding fragment thereof comprises: a light chain variable region (VL) comprising VL complementarity determining region 1 (CDR1) , VL CDR2, and VL CDR3 of antibody TN01 as set forth in Table 19; and/or a heavy chain variable region (VH) comprising VH complementarity determining region 1 (CDR1) , VH CDR2, and VH CDR3 of antibody TN01 as set forth in Table 20; or a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3 having an amino acid sequence of a VL CDR1, a VL CDR2, and a VL CDR3, respectively, of a VL having an amino acid sequence of SEQ ID NO: 29; and/or a VH comprising a VH CDR1, a VH CDR2, and
  • the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 amino acid sequences are according to the Kabat numbering system; (b) the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 amino acid sequences are according to the Chothia numbering system; (c) the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 amino acid sequences are according to the ABM numbering system; (d) the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 amino acid sequences are according to the Contact numbering system; or (e) the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 amino acid sequences are according to the Contact numbering system; or (e) the VH CDR1, VH
  • the VL CDR1, VL CDR2, and VL CDR3 comprise amino acid sequences of SEQ ID NOS: 21, 22, and 23, respectively, and the VH CDR1, VH CDR2, and VH CDR3 comprise amino acid sequences of SEQ ID NOS: 24, 25, and 26, respectively.
  • the antibody or antigen-binding fragment thereof comprises a VL comprising an amino acid sequence having at least 95%sequence identity to SEQ ID NO: 29.
  • the antibody or antigen-binding fragment thereof comprises a VL comprising an amino acid sequence of SEQ ID NO: 29.
  • the antibody or antigen-binding fragment thereof comprises a VH comprising an amino acid sequence having at least 95%sequence identity to SEQ ID NO: 30.
  • the antibody or antigen-binding fragment thereof comprises a VH comprising an amino acid sequence of SEQ ID NO: 30.
  • the antibody or antigen-binding fragment thereof comprises a VL comprising an amino acid sequence having at least 95%sequence identity to SEQ ID NO: 29 and a VH comprising an amino acid sequence having at least 95%sequence identity to SEQ ID NO: 30.
  • the antibody or antigen-binding fragment thereof comprises: a VL comprising an amino acid sequence of SEQ ID NO: 29; and a VH comprising an amino acid sequence of SEQ ID NO: 30.
  • an immunoconjugate molecule comprising a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises a TNF- ⁇ moiety fused to a heavy chain variable region (VH) of a first antibody, wherein the second polypeptide chain comprises a light chain variable region (VL) of the first antibody fused to a second antibody capable of binding to Trop-2; and wherein the VH and VL forms a two-in-one binding domain capable of binding to TNF- ⁇ and Trop-2.
  • VH heavy chain variable region
  • VL light chain variable region
  • the VL comprises VL CDR1, VL CDR2, and VL CDR3 of antibody TN01 as set forth in Table 19; and/or wherein the VH comprises VH CDR1, VH CDR2, and VH CDR3 of antibody TN01 as set forth in Table 20; or (b) the VL comprises a VL CDR1, a VL CDR2, and a VL CDR3 having an amino acid sequence of a VL CDR1, a VL CDR2, and a VL CDR3, respectively, of a VL having an amino acid sequence of SEQ ID NO: 29; and/or the VH comprises a VH CDR1, a VH CDR2, and a VH CDR3 having an amino acid sequence of a VH CDR1, a VH CDR2, and a VH CDR3, respectively, of a VH having an amino acid sequence of SEQ ID NO: 30.
  • the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 amino acid sequences are according to the Kabat numbering system; (b) the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 amino acid sequences are according to the Chothia numbering system; (c) the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 amino acid sequences are according to the ABM numbering system; (d) the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 amino acid sequences are according to the Contact numbering system; or (e) the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 amino acid sequences are according to the IMGT
  • the VL CDR1, VL CDR2, and VL CDR3 comprise amino acid sequences of SEQ ID NOS: 21, 22, and 23, respectively, and the VH CDR1, VH CDR2, and VH CDR3 comprise amino acid sequences of SEQ ID NOS: 24, 25, and 26, respectively.
  • the antibody or antigen-binding fragment thereof comprises a VL comprising an amino acid sequence having at least 95%sequence identity to SEQ ID NO: 29.
  • the antibody or antigen-binding fragment thereof comprises a VL comprising an amino acid sequence of SEQ ID NO: 29.
  • the antibody or antigen-binding fragment thereof comprises a VH comprising an amino acid sequence having at least 95%sequence identity to SEQ ID NO: 30.
  • the antibody or antigen-binding fragment thereof comprises a VH comprising an amino acid sequence of SEQ ID NO: 30.
  • the antibody or antigen-binding fragment thereof comprises a VL comprising an amino acid sequence having at least 95%sequence identity to SEQ ID NO: 29 and a VH comprising an amino acid sequence having at least 95%sequence identity to SEQ ID NO: 30.
  • the antibody or antigen-binding fragment thereof comprises: a VL comprising an amino acid sequence of SEQ ID NO: 29; and a VH comprising an amino acid sequence of SEQ ID NO: 30.
  • the TNF- ⁇ moiety comprises an amino acid sequence of SEQ ID NO: 1 or a mutant thereof.
  • the mutant comprises a S95A substitution, a N34H substitution, a Y87A substitution, or a Y87F substitution in the amino acid sequence of SEQ ID NO: 1.
  • the second antibody is a scFv or VHH capable of binding to Trop-2.
  • the scFv comprises the sequence of SEQ ID NO: 52.
  • the TNF- ⁇ moiety in the first polypeptide chain, is fused to the N terminus of the VH via a first peptidic linker.
  • the first peptidic linker is about 30 amino acids in length; wherein optionally the first peptidic linker is a 6X (G4S) linker.
  • the second antibody in the second polypeptide chain, is fused to the N terminus of the VL via a second peptidic linker.
  • the second peptidic linker is about 50 amino acids in length, wherein optionally the second peptidic linker comprises the amino acid sequence of SEQ ID NO: 12.
  • the first polypeptide chain further comprises a CH1 domain of the first antibody; optionally wherein the CH1 domain is fused to the C terminus of the VH.
  • the second polypeptide chain further comprises a CL domain of the first antibody; optionally wherein the CL domain is fused to the C terminus of the VL.
  • an immunoconjugate molecule comprising two polypeptide chains, wherein (a) the first polypeptide chain comprises an amino acid sequence of SEQ ID NO: 63, and the second polypeptide chain comprises an amino acid sequence of SEQ ID NO: 64; (b) the first polypeptide chain comprises an amino acid sequence of SEQ ID NO: 65, and the second polypeptide chain comprises an amino acid sequence of SEQ ID NO: 64; (c) the first polypeptide chain comprises an amino acid sequence of SEQ ID NO: 66, and the second polypeptide chain comprises an amino acid sequence of SEQ ID NO: 64; or (d) the first polypeptide chain comprises an amino acid sequence of SEQ ID NO: 67, and the second polypeptide chain comprises an amino acid sequence of SEQ ID NO: 64.
  • a complex comprising a homotrimer of the immunoconjugate molecule as described herein, and wherein the trimerization is through the TNF- ⁇ moiety of the
  • composition comprising the immunoconjugate molecule described herein, and a pharmaceutical acceptable carrier.
  • provided herein is a polynucleotide encoding the immunoconjugate molecule described herein or a fragment thereof.
  • the polynucleotide is operably linked to a promoter.
  • a vector comprising the polynucleotide described herein.
  • a cell comprising the polynucleotide described herein or the vector described herein.
  • kit comprising the immunoconjugate molecule or complex of immunoconjugate molecules as described herein.
  • an immunoconjugate molecule comprising culturing the cell described herein to express the immunoconjugate molecule, wherein the immunoconjugate molecule is in a monomeric, dimeric, or trimeric form.
  • an immunoconjugate molecule comprising expressing the polynucleotide described herein, wherein the immunoconjugate molecule is in a monomeric, dimeric, or trimeric form.
  • a method for activating a TNF- ⁇ mediated effect at a target site comprising delivering to the target site the immunoconjugate molecule or the complex of the immunoconjugate molecules as described herein.
  • delivering the immunoconjugate molecule to the target site comprises administering the immunoconjugate molecule to a subject.
  • the TNF- ⁇ mediated effect is at least about 10%, 20%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98%lower at a non-target site as compared to the TNF- ⁇ mediated effect at the target site after administering the immunoconjugate molecule to the subject.
  • the TNF- ⁇ mediated effect is cell apoptosis.
  • provided herein is a method for enriching a TNF- ⁇ polypeptide at a target site, the method comprising delivering to the target site the immunoconjugate molecule or complex of the immunoconjugate molecules as described herein.
  • delivering the immunoconjugate molecule to the target site comprises administering the immunoconjugate molecule to a subject.
  • the concentration of the TNF- ⁇ polypeptide is at least about 10%, 20%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98%lower at a non-target site as compared to the concentration of the TNF- ⁇ polypeptide at the target site after administering the immunoconjugate molecule to the subject.
  • a toxicity or side-effect associated with TNF- ⁇ in the subject is reduced.
  • cytokine toxicity or side-effect is reduced at least about 10%, 20%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98%as compared to administering to the subject an equivalent amount of TNF- ⁇ in an unconjugated form.
  • the reduction in toxicity or side-effect is measured as the elongation of life span of the administered subject.
  • the reduction in toxicity or side-effect is measured as reduction in loss of body weight of the administered subject.
  • the reduction in toxicity or side-effect is measured as change in the level of an immune response in the administered subject.
  • the reduction in toxicity or side-effect is measured as a change in an inflammatory response in the administered subject.
  • the target site is a tumor microenvironment.
  • the target site is a cancerous cell expressing FAP and/or Trop-2.
  • the cancerous cell also expresses a TNF- ⁇ receptor.
  • the cancerous cell does not express a TNF- ⁇ receptor.
  • provided herein is a method for inhibiting growth or induce apoptosis of a cancer cell comprising contacting the cancer cell with an effective amount of the immunoconjugate molecule or complex of the immunoconjugate molecules as described herein.
  • provided herein is a method for treating cancer in a subject in need thereof, wherein the method comprising administering an effective amount of the immunoconjugate molecule or complex of the immunoconjugate molecules as described herein.
  • FIG. 1A is a schematic illustration of a soluble cytokine polypeptide in the form of homotrimer.
  • FIGS. 1B to 1L are schematic illustrations of complexes containing multimerized antibody-cytokine immunoconjugates having different molecular configurations according to the present disclosure.
  • the multimerization between the immunoconjugate molecules can be via covalent (e.g., disulfide bond or peptidic linker) or non-covalent interactions.
  • FIGS. 1B to 1L illustrate the configurations in the timer form, immunoconjugates in the monomer, dimer, or higher-order multimer (e.g., greater than 3) forms are also within the scope of the present disclosure.
  • FIG. 1B shows an immunoconjugate containing an anti-cytokine /anti-tumor associated antigen (TAA) two-in-one single chain Fv (scFv) antibody fused to a cytokine.
  • TAA anti-cytokine /anti-tumor associated antigen
  • scFv two-in-one single chain Fv
  • the trimeric complex as shown in the figure can be a homotrimer containing three copies of the immunoconjugate molecules of the same configuration, or alternatively a heterotrimer containing three copies of the immunoconjugate molecules having two or three different configurations as described herein.
  • the cytokine is wild-type or mutant TNF- ⁇ .
  • the TAA is transmembrane glycoprotein encoded by the Tacstd2 gene (Trop-2) or fibroblast activation protein alpha (FAP) .
  • FIG. 1C shows an immunoconjugate containing an anti-cytokine /anti-TAA two-in-one Fab antibody fused to a cytokine.
  • cytokine-VH-CH1 x VL-CL there are four possible configurations: (i) cytokine-VH-CH1 x VL-CL, (ii) cytokine-VL-CL x VH-CH1, (iii) VH-CH1-cytokine x VL-CL, and (iv) VL-CL-cytokine x VH-CH1, where “x” separates different peptidic chains forming part of an immunoconjugate molecule, “-” denotes a linker or direct fusion between different moieties, and the different moieties in a peptidic chain are listed according to the N to C orientation in the peptidic chain.
  • the trimeric complex as shown in the figure can be a homotrimer containing three copies of the immunoconjugate molecules of the same configuration, or alternatively a heterotrimer containing three copies of the immunoconjugate molecules having two or three different configurations as described herein.
  • the cytokine is wild-type or mutant TNF- ⁇ .
  • the TAA is Trop-2 or FAP.
  • FIG. 1D shows an immunoconjugate containing an anti-cytokine /anti-TAA two-in-one single chain fragment antigen binding (scFab) antibody fused to a cytokine.
  • scFab anti-cytokine /anti-TAA two-in-one single chain fragment antigen binding
  • the trimeric complex as shown in the figure can be a homotrimer containing three copies of the immunoconjugate molecules of the same configuration, or alternatively a heterotrimer containing three copies of the immunoconjugate molecules having two or three different configurations as described herein.
  • the cytokine is wild-type or mutant TNF- ⁇ .
  • the TAA is Trop-2 or FAP.
  • FIG. 1E shows an immunoconjugate containing (a) an anti-cytokine /anti-TAA two-in-one scFv antibody, (b) a cytokine polypeptide, and (c) an anti-TAA scFv antibody.
  • the trimeric complex as shown in the figure can be a homotrimer containing three copies of the immunoconjugate molecules of the same configuration, or alternatively a heterotrimer containing three copies of the immunoconjugate molecules having two or three different configurations as described herein.
  • the cytokine is wild-type or mutant TNF- ⁇ .
  • the TAA is Trop-2 or FAP.
  • FIG. 1F shows an immunoconjugate containing (a) an anti-cytokine /anti-TAA two-in-one scFv antibody, (b) a cytokine polypeptide, and (c) an anti-TAA VHH antibody.
  • cytokine-scFv VH-VL) -VHH
  • cytokine-scFv VL-VH
  • scFv VH-VL
  • scFv VL-VH-VHH-cytokine
  • the trimeric complex as shown in the figure can be a homotrimer containing three copies of the immunoconjugate molecules of the same configuration, or alternatively a heterotrimer containing three copies of the immunoconjugate molecules having two or three different configurations as described herein.
  • the cytokine is wild-type or mutant TNF- ⁇ .
  • the TAA is Trop-2 or FAP.
  • FIG. 1G shows an immunoconjugate containing (a) an anti-cytokine /anti-TAA two-in-one Fab antibody, (b) a cytokine polypeptide, and (c) an anti-TAA scFv antibody.
  • the trimeric complex as shown in the figure can be a homotrimer containing three copies of the immunoconjugate molecules of the same configuration, or alternatively a heterotrimer containing three copies of the immunoconjugate molecules having two or three different configurations as described herein.
  • the cytokine is wild-type or mutant TNF- ⁇ .
  • the TAA is Trop-2 or FAP.
  • FIG. 1H shows an immunoconjugate containing (a) an anti-cytokine /anti-TAA two-in-one Fab antibody, (b) a cytokine polypeptide, and (c) an anti-TAA VHH antibody.
  • cytokine-VH-CH1 x VHH-VL-CL There are four possible configurations: (i) cytokine-VH-CH1 x VHH-VL-CL, (ii) cytokine-VL-CL x VHH-VH-CH1, (iii) VH-CH1-cytokine x VL-CL-VHH, (iv) VL-CL-cytokine x VH-CH1-VHH, where “x” separates different peptidic chains forming part of a immunoconjugate molecule, “-” denotes a linker or direct fusion between different moieties, and the different moieties in a peptidic chain are listed according to the N to C orientation in the peptidic
  • the trimeric complex as shown in the figure can be a homotrimer containing three copies of the immunoconjugate molecules of the same configuration, or alternatively a heterotrimer containing three copies of the immunoconjugate molecules having two or three different configurations as described herein.
  • the cytokine is wild-type or mutant TNF- ⁇ .
  • the TAA is Trop-2 or FAP.
  • FIG. 1I shows an immunoconjugate containing (a) an anti-cytokine /anti-TAA two-in-one Fab antibody, (b) a cytokine polypeptide, and (c) an anti-TAA scFv antibody.
  • the trimeric complex as shown in the figure can be a homotrimer containing three copies of the immunoconjugate molecules of the same configuration, or alternatively a heterotrimer containing three copies of the immunoconjugate molecules having two or three different configurations as described herein.
  • the cytokine is wild-type or mutant TNF- ⁇ .
  • the TAA is Trop-2 or FAP.
  • FIG. 1J shows an immunoconjugate containing (a) an anti-cytokine /anti-TAA two-in-one Fab antibody, (b) a cytokine polypeptide, and (c) an anti-TAA scFv antibody.
  • the trimeric complex as shown in the figure can be a homotrimer containing three copies of the immunoconjugate molecules of the same configuration, or alternatively a heterotrimer containing three copies of the immunoconjugate molecules having two or three different configurations as described herein.
  • the cytokine is wild-type or mutant TNF- ⁇ .
  • the TAA is Trop-2 or FAP.
  • FIG. 1K shows an immunoconjugate containing (a) an anti-cytokine /anti-TAA two-in-one Fab antibody, (b) a cytokine polypeptide, and (c) an anti-TAA VHH antibody.
  • cytokine-VH-CH1 x VL-CL-VHH There are four possible configurations: (i) cytokine-VH-CH1 x VL-CL-VHH, (ii) cytokine-VL-CL x VH-CH1-VHH, (iii) VH-CH1-cytokine x VHH-VL-CL, and (iv) VL-CL-cytokine x VHH-VH-CH1, where “x” separates different peptidic chains forming part of a immunoconjugate molecule, “-” denotes a linker or direct fusion between different moieties, and the different moieties in a peptidic chain are listed according to the N to C orientation in the peptid
  • the trimeric complex as shown in the figure can be a homotrimer containing three copies of the immunoconjugate molecules of the same configuration, or alternatively a heterotrimer containing three copies of the immunoconjugate molecules having two or three different configurations as described herein.
  • the cytokine is wild-type or mutant TNF- ⁇ .
  • the TAA is Trop-2 or FAP.
  • FIG. 1L shows an immunoconjugate containing (a) an anti-cytokine /anti-TAA two-in-one Fab antibody, (b) a cytokine polypeptide, and (c) an anti-TAA VHH antibody.
  • cytokine-VH-CH1-VHH x VL-CL There are four possible configurations: (i) cytokine-VH-CH1-VHH x VL-CL, (ii) cytokine-VL-CL-VHH x VH-CH1, (iii) VHH-VH-CH1-cytokine x VL-CL, and (iv) VHH-VL-CL-cytokine x VH-CH1, where “x” separates different peptidic chains forming part of a immunoconjugate molecule, “-” denotes a linker or direct fusion between different moieties, and the different moieties in a peptidic chain are listed according to the N to C orientation in the peptidic
  • the trimeric complex as shown in the figure can be a homotrimer containing three copies of the immunoconjugate molecules of the same configuration, or alternatively a heterotrimer containing three copies of the immunoconjugate molecules having two or three different configurations as described herein.
  • the cytokine is wild-type or mutant TNF- ⁇ .
  • the TAA is Trop-2 or FAP.
  • FIG. 2A is a schematic illustration of a soluble cytokine polypeptide.
  • FIGS. 2B to 2U are schematic illustrations of antibody-cytokine immunoconjugates of different molecular configurations according to the present disclosure. Particularly, FIG. 2B shows an immunoconjugate containing a cytokine polypeptide fused to the C-terminus of one of the two heavy chain fragments in an immunoglobulin Fc domain (e.g., Fc-knob) .
  • an immunoglobulin Fc domain e.g., Fc-knob
  • FIG. 2C shows an immunoconjugate containing (a) anti-cytokine /anti-TAA two-in-one Fab antibody fused at the N-terminus of its heavy chain fragment to the C-terminus of one of the heavy chain fragments of the immunoglobulin Fc domain (e.g., Fc-hole) , and (b) a cytokine polypeptide fused to the C-terminus of the other heavy chain fragment in the immunoglobulin Fc domains (e.g., Fc-knob) , and.
  • a cytokine polypeptide fused to the C-terminus of the other heavy chain fragment in the immunoglobulin Fc domains
  • FIG. 2D shows an immunoconjugate containing (a) an anti-cytokine /anti-TAA two-in-one Fab antibody fused at the N-terminus of its heavy chain fragment to the C-terminus of one of the heavy chain fragments of the immunoglobulin Fc domain (e.g., the Fc-hole) , (b) a cytokine polypeptide fused to the C-terminus of the other one of the two heavy chain fragments of an immunoglobulin Fc domain (e.g., the Fc-knob) , and (c) an anti-TAA scFv antibody fused to the N terminus of one of the two heavy chain fragments in the immunoglobulin Fc domain (e.g., the Fc-knob) .
  • an anti-cytokine /anti-TAA two-in-one Fab antibody fused at the N-terminus of its heavy chain fragment to the C-terminus of one of the heavy chain fragments of the immunoglobulin Fc domain (
  • FIG. 2E shows an immunoconjugate containing (a) an anti-cytokine /anti-TAA two-in-one Fab antibody fused at the N-terminus of its heavy chain fragment to the C-terminus of one of the two heavy chain fragments of the immunoglobulin Fc domain (e.g., Fc-hole) , and (b) a cytokine polypeptide fused to the N terminus of the light chain fragment of the Fab antibody.
  • an immunoconjugate containing (a) an anti-cytokine /anti-TAA two-in-one Fab antibody fused at the N-terminus of its heavy chain fragment to the C-terminus of one of the two heavy chain fragments of the immunoglobulin Fc domain (e.g., Fc-hole) , and (b) a cytokine polypeptide fused to the N terminus of the light chain fragment of the Fab antibody.
  • FIG. 2F shows an immunoconjugate containing (a) an anti-cytokine/anti-TAA two-in-one Fab antibody fused at the N-terminus of its heavy chain fragment to the C-terminus of one of the heavy chain fragments of an immunoglobulin Fc domain (e.g., Fc-knob) ; (b) a cytokine polypeptide fused to the C-terminus of the other heavy chain fragment of the immunoglobulin Fc domain (e.g., Fc-hole) , and (c) an anti-TAA single domain antibody fused to the N-terminus of one of the two heavy chain fragments in the immunoglobulin Fc domain (e.g., Fc-knob) .
  • an immunoglobulin Fc domain e.g., Fc-knob
  • FIG. 2G shows an immunoconjugate containing (a) an anti-cytokine /anti-TAA two-in-one scFv antibody fused at the C-terminus of its heavy chain fragment to the N-terminus of one of the heavy chain fragments of an immunoglobulin Fc domain (e.g., Fc-hole) , (b) a cytokine polypeptide fused to the C-terminus of the other heavy chain fragment of the immunoglobulin Fc domain (e.g., Fc-knob) , and (c) an anti-TAA Fab antibody fused to the N-terminus of one of the two heavy chain fragments in the immunoglobulin Fc domain (e.g., Fc-knob) .
  • an immunoglobulin Fc domain e.g., Fc-hole
  • a cytokine polypeptide fused to the C-terminus of the other heavy chain fragment of the immunoglobulin Fc domain
  • an anti-TAA Fab antibody fused
  • FIG. 2H shows an immunoconjugate containing (a) an anti-cytokine /anti-TAA two-in-one Fab antibody fused at the N-terminus of its heavy chain fragment to the C-terminus of one of the two heavy chain fragments of the immunoglobulin Fc domain (e.g., the Fc-knob) , (b) a cytokine polypeptide fused to the N-terminus of the light chain fragment of the Fab antibody, and (c) an anti-TAA scFv antibody fused to the N-terminus of one of the two heavy chain fragments in the immunoglobulin Fc domain (e.g., Fc-hole) .
  • an anti-cytokine /anti-TAA two-in-one Fab antibody fused at the N-terminus of its heavy chain fragment to the C-terminus of one of the two heavy chain fragments of the immunoglobulin Fc domain (e.g., the Fc-knob)
  • FIG. 2I shows an immunoconjugate containing (a) an anti-cytokine /anti-TAA two-in-one Fab antibody fused at the N-terminus of its heavy chain fragment to the C-terminus of one of the two heavy chain fragments of the immunoglobulin Fc domain (e.g., Fc-hole) , (b) a cytokine peptide fused to the C-terminus of the other heavy chain fragments in an immunoglobulin Fc domains (e.g., Fc-knob) , and (c) an anti-TAA Fab fused to the N-terminus of one of the two heavy chain fragments in the immunoglobulin Fc domain (e.g., Fc-knob) .
  • an anti-TAA Fab fused to the N-terminus of one of the two heavy chain fragments in the immunoglobulin Fc domain (e.g., Fc-knob) .
  • FIG. 2J shows an immunoconjugate containing (a) an anti-cytokine/anti-TAA two-in-one Fab antibody fused at the N-terminus of its heavy chain fragment to the C-terminus of one of the two heavy chain fragments of the immunoglobulin Fc domain (e.g., the Fc-hole) , (b) a cytokine peptide fused to the N-terminus of the light chain fragment of the Fab antibody, and (c) an anti-TAA scFv antibody fused to the N-terminus of one of the two heavy chain fragments in the immunoglobulin Fc domain (e.g., Fc-knob) .
  • an anti-cytokine/anti-TAA two-in-one Fab antibody fused at the N-terminus of its heavy chain fragment to the C-terminus of one of the two heavy chain fragments of the immunoglobulin Fc domain (e.g., the Fc-hole)
  • a cytokine peptide fused
  • FIG. 2K shows an immunoconjugate containing (a) an anti-cytokine/anti-TAA two-in-one Fab antibody fused at the C-terminus of its heavy chain fragment to the N-terminus of one of the heavy chain fragments of the immunoglobulin Fc domain (e.g., the Fc-knob) , (b) a cytokine peptide fused to the N-terminus of the heavy chain fragment of a Fab antibody, and (c) an anti-TAA Fab antibody fused at the C-terminus of its heavy chain fragment to the N-terminus of the other heavy chain fragment of the immunoglobulin Fc domain (e.g., the Fc-hole) .
  • an anti-cytokine/anti-TAA two-in-one Fab antibody fused at the C-terminus of its heavy chain fragment to the N-terminus of one of the heavy chain fragments of the immunoglobulin Fc domain (e.g., the Fc-knob)
  • FIG. 2L shows an immunoconjugate containing (a) an anti-cytokine /anti-TAA two-in-one Fab antibody fused at the N-terminus of its heavy chain fragment to the C-terminus of one of the heavy chain fragments of the immunoglobulin Fc domain (e.g., the Fc-hole) , (b) a cytokine polypeptide fused to the C-terminus of the other heavy chain fragment of the immunoglobulin Fc domain (e.g., the Fc-knob) , and (c) an anti-TAA scFv antibody fused to the N-terminus of one of the two heavy chain fragments in the immunoglobulin Fc domain (e.g., the Fc-hole) .
  • an anti-cytokine /anti-TAA two-in-one Fab antibody fused at the N-terminus of its heavy chain fragment to the C-terminus of one of the heavy chain fragments of the immunoglobulin Fc domain (e.g.,
  • FIG. 2M shows an immunoconjugate containing (a) an anti-cytokine/anti-TAA two-in-one Fab antibody fused at the N-terminus of its heavy chain fragment to the C-terminus of one of the heavy chain fragments of the immunoglobulin Fc domain (e.g., the Fc-hole) , (b) a cytokine peptide fused to the C-terminus of the other heavy chain fragment of the immunoglobulin Fc domain (Fc-knob) , and (c) and anti-TAA scFv fused to the C-terminus of the heavy chain fragment of the anti-cytokine/anti-TAA two-in-one Fab antibody.
  • an anti-cytokine/anti-TAA two-in-one Fab antibody fused at the N-terminus of its heavy chain fragment to the C-terminus of one of the heavy chain fragments of the immunoglobulin Fc domain (e.g., the Fc-hole)
  • FIG. 2N shows an immunoconjugate containing (a) an anti-cytokine/anti-TAA two-in-one Fab antibody fused at the C-terminus of its heavy chain fragment to the N-terminus of one of the heavy chain fragments of the immunoglobulin Fc domain (e.g., the Fc-hole) , (b) a cytokine peptide fused to the N-terminus of the heavy chain fragment of a Fab antibody, and (c) an anti-TAA Fab antibody fused at the C-terminus of its heavy chain to the N-terminus of the other heavy chain fragment of the immunoglobulin Fc domain (e.g., the Fc-knob) .
  • an anti-cytokine/anti-TAA two-in-one Fab antibody fused at the C-terminus of its heavy chain fragment to the N-terminus of one of the heavy chain fragments of the immunoglobulin Fc domain (e.g., the Fc-hole)
  • FIG. 2O shows an immunoconjugate containing (a) an anti-cytokine/anti-TAA two-in-one Fab antibody fused at the C-terminus of its heavy chain fragment to the N-terminus of one of the heavy chain fragments of the immunoglobulin Fc domain (e.g., Fc-knob) , (b) a cytokine peptide fused to the N-terminus of the heavy chain fragment of the Fab antibody, and (c) an anti-TAA single domain antibody fused to the N-terminus of the other one of the two heavy chain fragments in the immunoglobulin Fc domain (e.g., Fc-hole) .
  • an anti-cytokine/anti-TAA two-in-one Fab antibody fused at the C-terminus of its heavy chain fragment to the N-terminus of one of the heavy chain fragments of the immunoglobulin Fc domain (e.g., Fc-knob)
  • a cytokine peptide fused to the N-
  • FIG. 2P shows an immunoconjugate containing (a) an anti-cytokine/anti-TAA two-in-one Fab antibody fused at the C-terminus of its heavy chain fragment to the N-terminus of one of the heavy chain fragments of the immunoglobulin Fc domain (e.g., Fc-knob) , (b) a cytokine peptide fused to the N-terminus of the heavy chain fragment of the Fab antibody, and (c) an anti-TAA scFv antibody fused to the N-terminus of the other one of the two heavy chain fragments in the immunoglobulin Fc domain (e.g., Fc-hole) .
  • an anti-cytokine/anti-TAA two-in-one Fab antibody fused at the C-terminus of its heavy chain fragment to the N-terminus of one of the heavy chain fragments of the immunoglobulin Fc domain (e.g., Fc-knob)
  • a cytokine peptide fused to
  • FIG. 2Q shows an immunoconjugate containing (a) an anti-cytokine/anti-TAA two-in-one Fab antibody fused at the C-terminus of its heavy chain fragment to the N-terminus of one of the heavy chain fragments of the immunoglobulin Fc domain (e.g., Fc-hole) , (b) a cytokine peptide fused to the N-terminus of the heavy chain fragment of the Fab antibody, and (c) an anti-TAA scFv antibody fused to the N-terminus of the other one of the two heavy chain fragments in the immunoglobulin Fc domain (e.g., Fc-knob) .
  • an anti-cytokine/anti-TAA two-in-one Fab antibody fused at the C-terminus of its heavy chain fragment to the N-terminus of one of the heavy chain fragments of the immunoglobulin Fc domain (e.g., Fc-hole)
  • a cytokine peptide fused to the
  • FIG. 2R shows an immunoconjugate containing (a) an anti-cytokine/anti-TAA two-in-one Fab antibody fused at the C-terminus of its heavy chain fragment to the N-terminus of one of the heavy chain fragments of the immunoglobulin Fc domain (e.g., Fc-hole) , (b) a cytokine peptide fused to the N-terminus of the light chain fragment of the Fab antibody, and (c) an anti-TAA scFv antibody fused to the N-terminus of the other one of the two heavy chain fragments in the immunoglobulin Fc domain (e.g., Fc-knob) .
  • an anti-cytokine/anti-TAA two-in-one Fab antibody fused at the C-terminus of its heavy chain fragment to the N-terminus of one of the heavy chain fragments of the immunoglobulin Fc domain (e.g., Fc-hole)
  • a cytokine peptide fused to the
  • FIG. 2S shows an immunoconjugate containing (a) an anti-cytokine/anti-TAA two-in-one Fab antibody fused at the C-terminus of its heavy chain fragment to the N-terminus of one of the heavy chain fragments of the immunoglobulin Fc domain (e.g., Fc-knob) , (b) a cytokine peptide fused to the N-terminus of the light chain fragment of the Fab antibody, and (c) an anti-TAA scFv antibody fused to the N-terminus of the other one of the two heavy chain fragments in the immunoglobulin Fc domain (e.g., Fc-hole) .
  • an anti-cytokine/anti-TAA two-in-one Fab antibody fused at the C-terminus of its heavy chain fragment to the N-terminus of one of the heavy chain fragments of the immunoglobulin Fc domain (e.g., Fc-knob)
  • a cytokine peptide fused to
  • FIG. 2T shows an immunoconjugate containing (a) an anti-cytokine/anti-TAA two-in-one Fab antibody fused at the C-terminus of its heavy chain fragment to the N-terminus of one of the heavy chain fragments of the immunoglobulin Fc domain (e.g., Fc-knob) , (b) an anti-TAA Fab antibody fused at the C-terminus of its heavy chain fragment to the N-terminus of the other heavy chain fragment in the immunoglobulin Fc domain (e.g., Fc-hole) , and (c) a cytokine peptide fused to the N-terminus of the heavy chain fragment of the Fab antibody.
  • an anti-cytokine/anti-TAA two-in-one Fab antibody fused at the C-terminus of its heavy chain fragment to the N-terminus of one of the heavy chain fragments of the immunoglobulin Fc domain (e.g., Fc-knob)
  • FIG. 2U shows an immunoconjugate containing (a) an anti-cytokine/anti-TAA two-in-one Fab antibody fused at the C-terminus of its heavy chain fragment to the N-terminus of one of the heavy chain fragments of the immunoglobulin Fc domain (e.g., Fc-knob) , and (b) a cytokine peptide fused to the N-terminus of the heavy chain fragment of the Fab antibody.
  • an immunoconjugate containing (a) an anti-cytokine/anti-TAA two-in-one Fab antibody fused at the C-terminus of its heavy chain fragment to the N-terminus of one of the heavy chain fragments of the immunoglobulin Fc domain (e.g., Fc-knob) , and (b) a cytokine peptide fused to the N-terminus of the heavy chain fragment of the Fab antibody.
  • FIGS. 3A to 3L show binding affinities of two-in-one antibody variants to both TNF- ⁇ and tumor associated antigen ( “TAA” ) through biolayer interferometry analysis. Particularly, FIGS. 3A to 3F show binding affinities of six two-in-one Fab-Fc capable of binding to both TNF- ⁇ and FAP; FIGS. 3G to 3L show binding affinities of six two-in-one Fab-Fc capable of binding to both TNF- ⁇ and Trop2. The numeric values of the binding affinities are summarized in Table 3 and Table 4.
  • FIG. 4A is a schematic illustration of a trimer of TNF- ⁇ containing immunoconjugates according to one embodiment of the present disclosure.
  • each of the immunoconjugates comprises (i) a wild-type TNF- ⁇ polypeptide capable of mediating a cellular effect, (ii) a masking moiety capable of binding to TNF- ⁇ and inhibiting the cellular effect of TNF- ⁇ , and (iii) a peptidic linker connecting the portions described in (i) and (ii) of the immunoconjugate.
  • FIG. 4B shows TNF- ⁇ activities of immunoconjugates having the configuration in FIG. 4A measured using the HEK Blue TNF- ⁇ reporter cell line.
  • X-axis shows the concentration (pM) of the TNF- ⁇ containing immunoconjugates;
  • Y-axis shows the absorbance at 635 nm (A 635 ) determined using a TECAN plate reader, which reflects the level of secreted embryonic alkaline phosphatase (SEAP) and thus TNF- ⁇ activity.
  • SEAP secreted embryonic alkaline phosphatase
  • a commercially available wild-type TNF- ⁇ polypeptide in the non-conjugated form was also included in the assay as a control (Sino Biological; square) .
  • TNF- ⁇ activities of both tested immunoconjugates were inhibited by anti-TNF- ⁇ scFvs in these immunoconjugates as compared to the unconjugated TNF- ⁇ control.
  • FIG. 4C shows TNF- ⁇ activities of immunoconjugates having the configuration in FIG. 4A measured using the murine L929 cell line.
  • X-axis shows the concentration (pM) of the TNF- ⁇ containing immunoconjugates in the presence of 1 ⁇ g/mL actinomycin D;
  • Y-axis shows the intensity of the Alamar blue fluorescence, which reflects the amount of live cells and thus TNF- ⁇ activity.
  • Two immunoconjugates were tested: TNF- ⁇ -adascFv (up triangle) and TNF- ⁇ -certvhvl (diamond) .
  • FIG. 5A is a schematic illustration of a trimer of immunoconjugates containing mutated TNF- ⁇ according to one embodiment of the present disclosure.
  • each of the immunoconjugates comprises (i) a mutated TNF- ⁇ polypeptide with further diminished activity under the shielded condition but capable of mediating a cellular effect under the de-shielded condition, (ii) a masking moiety capable of binding to TNF- ⁇ and inhibiting the cellular effect of TNF- ⁇ , and (iii) a peptidic linker connecting the portions described in (i) and (ii) of the immunoconjugate.
  • FIGS. 5B to 5D show TNF- ⁇ activities of immunoconjugates having the configuration in FIG. 5A measured using the HEK Blue TNF- ⁇ reporter cell line.
  • X-axis shows the concentration (pM) of the immunoconjugates containing the mutated TNF- ⁇ ;
  • Y-axis shows the absorbance at 635 nm (A 635 ) determined using a TECAN plate reader, which reflects the level of secreted embryonic alkaline phosphatase (SEAP) and thus TNF- ⁇ activity.
  • the bottom panels of FIGS. 5B to 5D show TNF- ⁇ activities of immunoconjugates having the configuration in FIG. 5A measured using the murine L929 cell line.
  • X-axis shows the concentration (pM) of the immunoconjugates containing the mutated TNF- ⁇ ; Y-axis shows the intensity of the Alamar blue fluorescence, which reflects the amount of live cells and thus TNF- ⁇ activity.
  • TNF- ⁇ S147A in FIG. 5B TNF- ⁇ N34H in FIG. 5C
  • TNF- ⁇ S95A in FIG. 5D TNF- ⁇ activities of the unconjugated mutant TNF- ⁇ (solid circle in FIG. 5B, solid diamond in FIG. 5C, solid circle in FIG. 5D) were comparable to that of the unconjugated wild-type TNF- ⁇ (solid square and open square) .
  • TNF- ⁇ activities of immunoconjugates containing the mutant TNF- ⁇ polypeptide conjugated to a scFv derived from anti-TNF- ⁇ adalimumab were significantly inhibited.
  • FIG. 6A is a schematic illustration of a trimer of immunoconjugates containing mutated TNF- ⁇ according to one embodiment of the present disclosure.
  • each of the immunoconjugates comprises (i) a mutated TNF- ⁇ polypeptide with further diminished activity under the shielded condition but capable of mediating a cellular effect in the unconjugated form, (ii) a masking moiety capable of binding to TNF- ⁇ and inhibiting the cellular effect of TNF- ⁇ , (iii) an anchoring moiety capable of binding to a tumor associated antigen: fibroblast activation protein alpha (FAP) , thereby immobilizing the immunoconjugate in an environment enriched of FAP, and (iv) peptidic linkers connecting the portions described in (i) , (ii) , and (iii) of the immunoconjugate.
  • FAP fibroblast activation protein alpha
  • FIGS. 6B and 6C show TNF- ⁇ activities of immunoconjugates having the configuration in FIG. 6A measured using the HEK Blue TNF- ⁇ reporter cell line.
  • the assays were performed in the absence (solid circle and triangle) or presence (open circle and triangle) of FAP expressed on the surface of HEK Blue TNF- ⁇ reporter cells.
  • X-axis shows the concentration (pM) of the immunoconjugates containing both the mutated TNF- ⁇ and the anchoring moiety;
  • Y-axis shows the absorbance at 635 nm (A 635 ) determined using a TECAN plate reader, which reflects the level of secreted embryonic alkaline phosphatase (SEAP) and thus TNF- ⁇ activity.
  • SEAP secreted embryonic alkaline phosphatase
  • TNF- ⁇ S147G plus linker (sc70) in Protein 17 TNF- ⁇ Y87F plus linker (sc70) in Protein 18.
  • Unconjugated wild-type TNF- ⁇ polypeptides in the absence (solid circle) or presence of FAP-expressing cells (open circle) were included as controls. As shown, in the absence of FAP-expressing cells, TNF- ⁇ activities of the tested immunoconjugates (solid triangles) were significantly inhibited. In contrast, in the presence of FAP expression, TNF- ⁇ activities of the tested immunoconjugates (open triangles) were partially restored as compared to the control groups.
  • FIG. 7A is a schematic illustration of a trimer of immunoconjugates containing mutant TNF- ⁇ according to one embodiment of the present disclosure.
  • each of the immunoconjugates comprises (i) a mutant TNF- ⁇ polypeptide with further diminished activity under the shielded condition but capable of mediating a cellular effect under the naked condition; (ii) a two-in-one masking moiety capable of (a) binding to TNF- ⁇ and inhibiting the cellular effect of TNF- ⁇ , and (b) binding to FAP; (iii) an anchoring moiety capable of binding to FAP, thereby immobilizing the immunoconjugate in an environment enriched of FAP, and (iv) peptidic linkers connecting the portions described in (i) , (ii) , and (iii) of the immunoconjugate.
  • FIGS. 7B to 7G show TNF- ⁇ activities of immunoconjugates having the configuration in FIG. 7A measured using the HEK Blue TNF- ⁇ reporter cell line.
  • the assays were performed in the absence (solid circle and triangle) or presence (open circle and triangle) of FAP expressed on the surface of HEK Blue TNF- ⁇ reporter cells.
  • X-axis shows the concentration (pM) of the immunoconjugates;
  • Y-axis shows the absorbance at 635 nm (A 635 ) determined using a TECAN plate reader, which reflects the level of secreted embryonic alkaline phosphatase (SEAP) and thus TNF- ⁇ activity.
  • SEAP secreted embryonic alkaline phosphatase
  • Each of the six tested immunoconjugates contained a Y87F or S147G point mutation of TNF- ⁇ , a different two-in-one masking moiety, and a different anchoring moiety.
  • Unconjugated wild-type TNF- ⁇ polypeptides in the absence (solid circle) or presence of FAP-expressing cells (open circle) were included as controls.
  • TNF- ⁇ activities of the tested immunoconjugates (solid triangle) were significantly inhibited.
  • TNF- ⁇ activities of the tested immunoconjugates open triangle were at least partially restored as compared to the control groups.
  • FIGS. 8A to 8F show TNF- ⁇ activities of immunoconjugates having the configuration in FIG. 7A measured using the HEK Blue TNF- ⁇ reporter cell line.
  • the assays were performed in the absence (solid circle and triangle) or presence (open circle and triangle) of FAP-expressing cells co-cultured with HEK Blue TNF- ⁇ reporter cells.
  • X-axis shows the concentration (pM) of the immunoconjugates;
  • Y-axis shows the absorbance at 635 nm (A 635 ) determined using a TECAN plate reader, which reflects the level of secreted embryonic alkaline phosphatase (SEAP) and thus TNF- ⁇ activity.
  • SEAP secreted embryonic alkaline phosphatase
  • Each of the six immunoconjugates contained a Y87F or S147G point mutation of TNF- ⁇ , a different two-in-one masking moiety, and a different anchoring moiety.
  • Unconjugated wild-type TNF- ⁇ polypeptides in the absence (solid circle) or presence of FAP-expressing cells (open circle) were included as controls. As shown, in the absence of FAP-expressing cells, TNF- ⁇ activities of the tested immunoconjugates (solid triangle) were significantly inhibited. In contrast, in the presence of FAP-expressing cells, TNF- ⁇ activities of the tested immunoconjugates (open triangle) were at least partially restored as compared to the control groups.
  • FIG. 9A is a schematic illustration of a trimer of immunoconjugates containing mutated TNF- ⁇ according to one embodiment of the present disclosure.
  • each of the immunoconjugates comprises (i) a mutated TNF- ⁇ polypeptide with further diminished activity under the shielded condition but capable of mediating a cellular effect under the unconjugated form; (ii) a two-in-one masking moiety capable of (a) binding to TNF- ⁇ and inhibiting the cellular effect of TNF- ⁇ , and (b) binding to FAP; and (iii) peptidic linkers connecting the portions described in (i) and (ii) of the immunoconjugate.
  • FIG. 9B shows TNF- ⁇ activities of immunoconjugates having the configuration in FIG. 9A measured using the HEK Blue TNF- ⁇ reporter cell line.
  • the assays were performed in the absence (circle and up triangle) or presence (down triangle and diamond) of FAP expressed on the surface of HEK Blue TNF- ⁇ reporter cells.
  • X-axis shows the concentration (pM) of the immunoconjugates;
  • Y-axis shows the absorbance at 635 nm (A 635 ) determined using a TECAN plate reader, which reflects the level of secreted embryonic alkaline phosphatase (SEAP) and thus TNF- ⁇ activity.
  • SEAP secreted embryonic alkaline phosphatase
  • the two-in-one masking moiety is FN06 or FN15 in the two tested immunoconjugates.
  • TNF- ⁇ activities of the tested immunoconjugates (circle and up triangle) were significantly inhibited.
  • TNF- ⁇ activities of the tested immunoconjugates down triangle and diamond were at least partially restored.
  • FIGS. 10A to 10D show TNF- ⁇ activities of immunoconjugates having the configuration in FIG. 7A and with different lengths of the linker between mutant TNF- ⁇ polypeptide and the two-in-one masking moiety (50, 70, or 160 amino acids long) or the linker between the anchoring moiety and the two-in-one masking moiety (70 or 160 amino acids long) .
  • TNF- ⁇ activities of the tested immunoconjugates were measured using the HEK Blue TNF- ⁇ reporter cell line. The assays were performed in the absence (solid circle and triangles) or presence (open circle and triangles) of FAP expressed on the surface of HEK Blue TNF- ⁇ reporter cells.
  • X-axis shows the concentration (pM) of the immunoconjugates
  • Y-axis shows the absorbance at 635 nm (A 635 ) determined using a TECAN plate reader, which reflects the level of secreted embryonic alkaline phosphatase (SEAP) and thus TNF- ⁇ activity.
  • SEAP embryonic alkaline phosphatase
  • FIG. 10A contained a S147G point mutation in the TNF- ⁇ polypeptide and FN06 as the two-in-one masking moiety
  • the immunoconjugates tested in FIG. 10B contained a S147G point mutation in the TNF- ⁇ polypeptide and FN15 as the two-in-one masking moiety
  • 10C contained a Y87F point mutation in the TNF- ⁇ polypeptide and FN06 as the two-in-one masking moiety; the immunoconjugates tested in FIG. 10D contained a Y87F point mutation in the TNF- ⁇ polypeptide and FN15 as the two-in-one masking moiety; the anchoring moiety of all tested immunoconjugates is scFv70.
  • the commercially available wild-type TNF- ⁇ polypeptide in the non-conjugated form was included in the assay as a control (Sino Biological; solid square) .
  • TNF- ⁇ activities of the tested immunoconjugates were all significantly inhibited; in the presence of FAP-expressing cells, TNF- ⁇ activities of the tested immunoconjugates (open circle and triangles) were at least partially restored as compared to the control groups.
  • FIG. 11A shows the SDS-PAGE results of purified protein samples from constructs designed to express covalent disulfide-linked TNF- ⁇ variants.
  • Five of the eleven purified protein samples expressed at comparable level as wild type TNF- ⁇ , and appeared at expected size of a TNF- ⁇ trimer (51-kDa) (top panel of FIG. 11A) .
  • the same five purified protein samples appeared at the size of a TNF- ⁇ monomer (17-kDa) (bottom panel of FIG. 11A) .
  • FIG. 11B shows TNF- ⁇ activities of the five confirmed covalent disulfide-linked TNF- ⁇ variants (ID NOs: 2, 7, 9, 10, and 11) , as well as the wild-type TNF- ⁇ .
  • Three covalent disulfide-linked TNF- ⁇ variants (Protein ID NOs: 2, 9, and 11; open circle, open and solid triangles) maintained or exceeded the potency of wild-type non-covalent TNF- ⁇ (solid circle) .
  • FIG. 12A to FIG. 12D show different configurations of TNF- ⁇ containing immunoconjugates screened for cell killing activity using the WEHI-164 co-culture assay.
  • FIGS. 13A to 13J show cell killing activities of different TNF- ⁇ containing immunoconjugate molecules screened using the WEHI-164 co-culture assay.
  • FIGS. 13A and 13B show TNF- ⁇ activities of an immunoconjugate having the configuration in FIG. 12B.
  • the assays were performed in the absence (solid circles and up triangles) or presence (open circles and up triangles) of hFAP-M2C2 cells.
  • the TNF- ⁇ activity was analyzed at a proliferation time of 24 hours (FIG. 21A) and 48 hours (FIG. 21B) .
  • the immunoconjugate contained a N34H point mutation of TNF- ⁇ , a scFV70 anchoring moiety, and an FN15 two-in-one masking moiety. Unconjugated wild-type TNF- ⁇ polypeptides were included as controls.
  • FIGS. 13C and 13D show TNF- ⁇ activities of an immunoconjugate having the configuration in FIG. 12B measuring using the WEHI-164 co-culture assay.
  • the assays were performed in the absence (solid circles and up triangles) or presence (open circles and up triangles) of hFAP-M2C2 cells.
  • the TNF- ⁇ activity was analyzed at a proliferation time of 24 hours (FIG. 21C) and 48 hours (FIG. 21D) .
  • the immunoconjugate contained a S95A point mutation of TNF- ⁇ , a scFV70 anchoring moiety, and an FN15 two-in-one masking moiety. Unconjugated wild-type TNF- ⁇ polypeptides were included as controls..
  • FIGS. 13E and 13F show TNF- ⁇ activities of an immunoconjugate having the configuration in FIG. 12B measuring using the WEHI-164 co-culture assay.
  • the assays were performed in the absence (solid circles and up triangles) or presence (open circles and up triangles) of hFAP-M2C2 cells.
  • the TNF- ⁇ activity was analyzed at a proliferation time of 24 hours (FIG. 21E) and 48 hours (FIG. 21F) .
  • the immunoconjugate contained a A145G point mutation of TNF- ⁇ , a scFV70 anchoring moiety, and an FN15 two-in-one masking moiety. Unconjugated wild-type TNF- ⁇ polypeptides were included as controls.
  • FIGS. 13G and 13H show TNF- ⁇ activities of an immunoconjugate having the configuration in FIG. 12B measuring using the WEHI-164 co-culture assay.
  • the assays were performed in the absence (solid circles and up triangles) or presence (open circles and up triangles) of hFAP-M2C2 cells.
  • the TNF- ⁇ activity was analyzed at a proliferation time of 24 hours (FIG. 21G) and 48 hours (FIG. 21H) .
  • the immunoconjugate contained a S147A point mutation of TNF- ⁇ , a scFV70 anchoring moiety, and an FN15 two-in-one masking moiety. Unconjugated wild-type TNF- ⁇ polypeptides were included as controls.
  • FIGS. 13I and 13J show TNF- ⁇ activities of an immunoconjugate having the configuration in FIG. 12B measuring using the WEHI-164 co-culture assay.
  • the assays were performed in the absence (solid circles and up triangles) or presence (open circles and up triangles) of hFAP-M2C2 cells.
  • the TNF- ⁇ activity was analyzed at a proliferation time of 24 hours (FIG. 21I) and 48 hours (FIG. 21J) .
  • the immunoconjugate contained a N34H point mutation of TNF- ⁇ , a FAP1SCFV anchoring moiety, and an FN15 two-in-one masking moiety. Unconjugated wild-type TNF- ⁇ polypeptides were included as controls.
  • FIGS. 14A to 14N show cell killing activities of different TNF- ⁇ containing immunoconjugate molecules screened using the WEHI-164 co-culture assay. Unconjugated wild-type TNF- ⁇ polypeptides were included as controls. Particularly, FIGS. 14A to 14G show TNF- ⁇ activities of an immunoconjugate having the configuration in FIG. 12D measured using the WEHI-164 co-culture assay. The assays were performed in the absence (solid circles and up triangles) or presence (open circles and up triangles) of hFAP-M2C2 cells.
  • the immunoconjugates contained either a N34H, S147A, or S95A point mutation of TNF- ⁇ , a variant VHH anchoring moiety, and an FN15 two-in-one masking moiety.
  • FIGS. 14H to 14N show TNF- ⁇ activities of an immunoconjugate having the configuration in FIG. 12B measured using the WEHI-164 co-culture assay.
  • the assays were performed in the absence (solid circles and up triangles) or presence (open circles and up triangles) of hFAP-M2C2 cells.
  • the immunoconjugates contained either a N34H/A33T, N34H/A145G, N34H/A145V, A33T/A145G, A33T/A145V, S147A, or S95A point mutation of TNF- ⁇ , a FAP1-SCFV anchoring moiety, and an FN15 two-in-one masking moiety.
  • FIG. 15A shows TNF- ⁇ activities of unconjugated wild-type TNF- ⁇ polypeptides measured using the WEHI-164 co-culture assay.
  • the assays were performed in the absence (solid circles) or presence (open circles) of hFAP-M2C2 cells.
  • the assays were also performed in the presence of 1.0 ⁇ g/mL ActD or 2.0 ⁇ g/mL ActD.
  • X-axis shows the concentration (pM) of the immunoconjugate.
  • Y-axis shows the absorbance of the immunoconjugate using a TECAN plate reader, which reflects the levels of secreted embryonic alkaline phosphatase (SEAP) and thus TNF- ⁇ activity.
  • SEAP secreted embryonic alkaline phosphatase
  • FIG. 15B shows TNF- ⁇ activities of an immunoconjugate having the configuration in FIG. 12B measured using the WEHI-164 co-culture assay.
  • the assays were performed in the absence (solid circles) or presence (open circles) of hFAP-M2C2 cells.
  • the assays were also performed in the presence of 1.0 ⁇ g/mL ActD or 2.0 ⁇ g/mL ActD.
  • the immunoconjugates contained a N34H point mutation of TNF- ⁇ , an scFV70 anchoring moiety, and an FN15 two-in-one masking moiety. Unconjugated wild-type TNF- ⁇ polypeptides were included as controls.
  • FIGS. 16A to 16C show TNF- ⁇ activities of immunoconjugates having the configuration in FIG. 12B measured using the WEHI-164 co-culture assay.
  • the assays were performed in the absence (solid circles and up triangles) or presence (open circles and up triangles) of hFAP-M2C2 cells.
  • X-axis shows the concentration (pM) of the immunoconjugate.
  • Y-axis shows the activity of the immunoconjugate using a TECAN plate reader, which reflects the levels of secreted embryonic alkaline phosphatase (SEAP) and thus TNF- ⁇ activity.
  • SEAP secreted embryonic alkaline phosphatase
  • the immunoconjugates contained either a S95A or A33T point mutation of TNF- ⁇ , an FAP1SCFV anchoring moiety, and a variant FN15 two-in-one masking moiety. Unconjugated wild-type TNF- ⁇ polypeptides were included as controls.
  • FIGS. 16D to 16F show TNF- ⁇ activities of immunoconjugates having the configuration in FIG. 12D measured using the WEHI-164 co-culture assay.
  • the assays were performed in the absence (solid circles and up triangles) or presence (open circles and up triangles) of hFAP-M2C2 cells.
  • X-axis shows the concentration (pM) of the immunoconjugate.
  • Y-axis shows the activity of the immunoconjugate using a TECAN plate reader, which reflects the levels of secreted embryonic alkaline phosphatase (SEAP) and thus TNF- ⁇ activity.
  • SEAP secreted embryonic alkaline phosphatase
  • the immunoconjugates contained either a S95A or A33T point mutation of TNF- ⁇ , a VHH anchoring moiety, and a variant FN15 two-in-one masking moiety. Unconjugated wild-type TNF- ⁇ polypeptides were included as controls.
  • FIGS. 17A to 17E show TNF- ⁇ activities of different immunoconjugates measured using the WEHI-164 co-culture assay.
  • the immunoconjugate T116 ( “full” immunoconjugate) shown in FIG. 17A has a configuration as shown in FIG. 7A.
  • the immunoconjugate T117 (lacking the two-in-one masking moiety capable of binding to FAP) shown in FIG. 17B has a configuration as shown in FIG. 17C.
  • the immunoconjugate T114 (lacking the anchoring moiety capable of binding to FAP) has a configuration shown in FIG. 17D has a configuration as shown in FIG. 17E.
  • the assays were performed in the absence (solid circles and up triangles) or presence (open circles and up triangles) of FAP-expressing cell lines. Unconjugated wild-type TNF- ⁇ polypeptides were included as controls. These results demonstrates the impact of FAP-binding capability of an immunoconjugate molecule on the activation of cell killing activity of TNF- ⁇ containing immunoconjugate.
  • FIGS. 18A to 18L show screening of various TNF- ⁇ containing immunoconjugate molecules constructed with different anchoring moieties and/or two-in-one masking moiety derived from FAP/TNF- ⁇ two-in-one antibody FN15 using the WEHI-164 co-culture assay.
  • FIGS. 19A to 19E show TNF- ⁇ activities of the immunoconjugates having the configuration in FIG. 12C (T102 containing a S95A point mutation of TNF- ⁇ , a VHH anchoring moiety and the same FN15 two-in-one masking moiety) . measured using the WEHI-164 co-culture assay.
  • the assays were performed in the absence (solid circles, up triangles, diamonds, down triangles, and squares) or presence (open circles, up triangles, diamonds, down triangles, and squares) of FAP.
  • the assays were performed with the following concentrations of Actinomycin D (ActD) : 0.0 ⁇ g/mL ActD (FIG.
  • FIGS. 20A to 20C show TNF- ⁇ activities of the T102 immunoconjugate measured using the WEHI-164 co-culture assay.
  • the assays were performed in the absence (solid circles and up triangles) or presence (open circles, up triangles, and squares) of soluble FAP. Unconjugated wild-type TNF- ⁇ polypeptides were included as controls. As shown, 10 nM soluble FAP was unable to induce T102 to kill cells without FAP expression (FIG. 20B, open squares) , suggesting this immunoconjugate molecule requires cell-surface anchorage (via expressed FAP) to unleash full activity.
  • FIGS. 21A to 21E show TNF- ⁇ activities of the T102 immunoconjugate in Tumor cell line WEHI-164 were co-cultured in the presence or absence of a WEHI-164 cell line ectopically expressing FAP (WEHI-164 Clone M2C2) .
  • Cell viability was determined with the alamarBlue assay (Invitrogen, Waltham, Massachusetts) monitoring fluorescence (540 nm excitation, 590 nm emission) .
  • Data were plotted as relative fluorescence (y-axis, relative fluorescence units) vs. immunoconjugate concentration (x-axis, in picomolar) .
  • the signal of the y-axis is proportional to the number of viable cells in the assay.
  • ActD Actinomycin D
  • 0.0 ⁇ g/mL ActD FIG. 21A
  • 0.1 ⁇ g/mL ActD FIG. 21B
  • 0.2 ⁇ g/mL ActD FIG. 21C
  • 0.4 ⁇ g/mL ActD FIG. 21D
  • 0.6 ⁇ g/mL ActD FIG. 21E
  • FIG. 22A shows TNF- ⁇ activities of measured using the HEK Blue TNF- ⁇ and HEK Blue TNF- ⁇ FAP reporter cell lines.
  • the results show that the potency of TNF- ⁇ immunoconjugate T017 in HEK Blue TNF- ⁇ FAP assay (solid down triangle) in the presence of HEK Blue TNF- ⁇ with FAP in cis-presentation was comparable to unconjugated TNF- ⁇ (solid circle) in HEK Blue TNF- ⁇ assay.
  • the immunoconjugate T017 was almost silent (open down triangle) .
  • FIG. 22B shows TNF- ⁇ activities of measured using the HEK Blue TNF- ⁇ reporter cell line co-cultured with either HEK 293T cells or HEK 293T-FAP cells.
  • the results show that TNF- ⁇ immunoconjugate T017 was silent when co-cultured with HEK 293T cells (open down triangle) .
  • T017 was activated but its potency was ⁇ 100 folds less than the TNF- ⁇ (solid circle) .
  • FIG. 22C shows TNF- ⁇ activities of an immunoconjugate having the configuration in FIG. 12C measured using the WEHI-164 and WEHI-164-FAP apoptotic assays.
  • the results show that TNF- ⁇ immunoconjugate T102 in WEHI-164-FAP assay (solid down triangle) in the presence of surface FAP at cis-presentation was more potent than unconjugated TNF- ⁇ (solid circle) in WEHI-164 assay. In the absence of surface FAP in WEHI-164 assay, the immunoconjugate T102 was silent (open down triangle) .
  • FIG. 22D shows TNF- ⁇ activities of measured using the WEHI-164 and WEHI-164-FAP co-culture assays.
  • the results show that increasing amount of Actinomycin D increased WEHI-164’s apoptotic sensitivity to TNF- ⁇ (open and solid circles) .
  • T102 caused similar apoptotic response as TNF- ⁇ (solid down triangle) which indicates the 50%WEHI-164 cells were killed too likely due to the trans-presentation of FAP from the rest 50%WEHI-164-FAP cells.
  • X-axis shows the concentration (pM) of the immunoconjugate.
  • Y-axis shows normalized cell viability.
  • the immunoconjugate contained a S95A point mutation of TNF- ⁇ , a VHH anchoring moiety, and an FN15 two-in-one masking moiety. Unconjugated wild-type TNF- ⁇ polypeptides were included as controls.
  • FIGS. 23A and 23B show the clinical potential of the TNF- ⁇ immunoconjugate molecules described herein.
  • FIG. 23A shows that human soft tissue sarcoma cells (SA3831, CrownBio) express high levels of FAP ( ( ⁇ 1x10 6 FAP/cell) .
  • FIG. 23B shows a TNF- ⁇ containing immunoconjugate molecule as described herein is synergistic with chemotherapeutic agent Actinomycin D on human primary tumor cells within therapeutically relevant concentrations.
  • FIG. 24A left panel shows the sizes of the purified immunoconjugate protein and components thereof confirmed via SDS-PAGE.
  • Right panel shows accelerated stability of an TNF- ⁇ containing immunoconjugate as described herein measured using size-exclusion chromatography (SEC) .
  • SEC size-exclusion chromatography
  • FIG. 24B shows serum concentration (ng/mL) of the TNF- ⁇ containing immunoconjugate or a non-conjugated TNF ⁇ polypeptide days after administration to a test subject, and the measurement of half life (hour) , which demonstrating that the immunoconjugate molecule had more than 100 folds increased half life compared to unconjugated TNF- ⁇ polypeptide.
  • FIG. 24C shows body weight change in mice administered PBS, or 4 ⁇ g unconjugated TNF- ⁇ polypeptide or 200 ⁇ g immunoconjugate molecule having the configuration in FIG. 12B (T098 containing a S95A point mutation of TNF- ⁇ , a scFV anchoring moiety derived from FAP1 and FN15 two-in-one masking moiety) . As shown T098 was less toxic compared to unconjugated TNF- ⁇ polypeptide as measured by body weight reduction following administration.
  • FIGS. 25A to 25C show tumor regression and efficacy in various animal models following administration of T098 immunoconjugate.
  • FIG. 25A shows that the administration of the immunoconjugate was able to inhibit tumor growth in Balb/c mouse cancer model.
  • FIG. 25B shows that the administration of the immunoconjugate did not change body weight in the mice.
  • FIG. 25C shows that the administration of the immunoconjugate did not change tumor volume in M-NSG immunocompromised mice.
  • FIG. 26A shows tumor growth inhibition and body weight change in Balb/c mice following administration of 4 ⁇ g of unconjugated TNF- ⁇ polypeptide, 1 ⁇ g Actinomycin D, or 100 ⁇ g TNF- ⁇ containing immunoconjugate (T102) in combination with 1 ⁇ g Actinomycin D.
  • the result shows that the administration of the combination treatment inhibited tumor growth comparable to unconjugated TNF- ⁇ polypeptide, but significantly reduced toxicity of the treatment as measured by body weight reduction following administration.
  • FIG. 26B shows Hematoxylin and Eosin (H&E) staining 48 hours after first dosing of PBS (control) or 25 ⁇ g TNF- ⁇ containing immunoconjugate as described herein, indicting the administered immunoconjugate molecule induced destruction and lymphocyte infiltration in tumor.
  • H&E Hematoxylin and Eosin
  • FIG. 26C shows the tumor inhibition efficacy of an example of a TNF- ⁇ containing immunoconjugates molecule as described herein.
  • Two animal models were used: WEHI-164 WT and WEHI-164-FAP.
  • the WEHI-164-FAP was engineered to express FAP, while the WEHI-164 WT model did not express FAP. This result demonstrates that tumor inhibition efficacy of this TNF- ⁇ containing immunoconjugates depends on the presence of FAP to unshield TNF- ⁇ activity.
  • FIGS. 27A to 27R show screening data of various TNF- ⁇ -containing immunoconjugate molecules having Configuration 6 as shown in FIG. 1G.
  • the immunoconjugate molecule contains two amino acid chains.
  • the first chain contains, from N to C, a cytokine moiety containing a wild-type or mutant TNF- ⁇ , which is fused via a linker peptide to the VH domain of a two-in-one anti-TNF- ⁇ /anti-FAP mab (masking moiety) , and a CH domain of the two-in-one masking moiety;
  • the second chain contains, from N to C, an anti- FAP anchoring moiety either in the scFv format that is fused via a linker peptide to the VL domain of the two-in-one masking moiety, and a CL domain of the two-in-one masking moiety.
  • TNF- ⁇ -containing immunoconjugate molecule having this configuration may multimiezie via the TNF-amoiety (e.g., forming a trimer as shown in FIG. 1G) .
  • TNF-amoiety e.g., forming a trimer as shown in FIG. 1G
  • Various cytokine domain having different TNF- ⁇ mutations e.g., A33T, N34H, Y87A, S95A, A145G, A147A
  • various two-in-one masking moiety having different VH sequences e.g., FN15 VH, FN15 H2var1, FN15 H2var2, FN15 H2var3, FN15 H2var4, FN15 H2Rev1 and/or different VL sequences (e.g., FN15 VL, or FN15 L1 Rev3)
  • the screening assay measured TNF- ⁇ activities of the different immunoconjugate molecules using the WEHI-164 co-culture assay.
  • the assays were performed with WEHI-164 cells or a WEHI-164 cell line ectopically expressing hFAP (hFAP-M2C2 cells) .
  • X-axis shows the concentration (pM) of the immunoconjugate.
  • Y-axis shows the activity of the immunoconjugate using a TECAN plate reader where the cell viability was determined with the alamarBlue assay (Invitrogen, Waltham, Massachusetts) monitoring fluorescence (540 nm excitation, 590 nm emission) .
  • Unconjugated wild-type TNF- ⁇ polypeptides were included as controls.
  • the present disclosure provides immunoconjugate molecules comprising a cytokine polypeptide.
  • the present disclosure also provides, in certain embodiments, polynucleotides and vectors comprising sequences encoding such immunoconjugate molecules, and compositions, reagents, and kits comprising such immunoconjugate molecules.
  • polynucleotides and vectors comprising sequences encoding such immunoconjugate molecules
  • compositions, reagents, and kits comprising such immunoconjugate molecules.
  • methods for delivery and/or activation of a cytokine activity at a target site or reduce toxicity and/or other side-effects associated with systemic exposure to the cytokine activity in a subject through the use of the immunoconjugate molecules according to the present disclosure.
  • oligonucleotides and “nucleic acids” are used interchangeably and are written left to right in 5’ to 3’ orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively. Therefore, in general, the codon at the 5’ -terminus of an oligonucleotide will correspond to the N-terminal amino acid residue that is incorporated into a translated protein or peptide product. Similarly, in general, the codon at the 3’ -terminus of an oligonucleotide will correspond to the C-terminal amino acid residue that is incorporated into a translated protein or peptide product. It is to be understood that this present disclosure is not limited to the particular methodology, protocols, and reagents described, as these may vary, depending upon the context they are used by those of skill in the art.
  • soluble when used in connection with a peptide or polypeptide (e.g., a cytokine or antigen) indicates that such peptide or polypeptide is not associated with solid surface or attached to the surface of a cell.
  • a soluble antigen in contrast to a cell surface antigen, is secreted to the extracellular space.
  • a tumor associated antigen can be expressed on the cell surface or soluble.
  • TNF- ⁇ refers to any native TNF- ⁇ from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats) , unless otherwise indicated.
  • the term encompasses unprocessed TNF- ⁇ as well as any form of TNF- ⁇ (e.g., protein complex containing multimerized TNF- ⁇ ) that results from processing in the cell.
  • the term also encompasses naturally occurring variants of TNF- ⁇ , e.g., splice variants or allelic variants.
  • the amino acid sequence of an exemplary human TNF- ⁇ is
  • TNF- ⁇ polypeptides can form a homotrimer complex through the extensive trimer interface of the TNF- ⁇ polypeptides. Eck and Sprang, J Biol Chem. 1989 Oct 15; 264 (29) : 17595-605.
  • TNF- ⁇ mutant or “mutant TNF- ⁇ ” as used herein is intended to encompass any mutant forms of various forms of the TNF- ⁇ molecule including full-length TNF- ⁇ , truncated forms of TNF- ⁇ and forms where TNF- ⁇ polypeptide containing one or more amino acid mutations in its sequence.
  • Full-length when used in reference to TNF- ⁇ is intended to mean the mature, natural length TNF- ⁇ molecule.
  • full-length human TNF- ⁇ refers to a molecule that has 157 amino acids (see e.g., SEQ ID NO: 1) .
  • TNF- ⁇ mutants are characterized in having at least one amino acid mutation affecting the interaction of TNF- ⁇ with one or both of its two receptors: TNFR1 and TNFR2. This mutation may involve substitution, deletion, truncation or modification of the wild-type amino acid residue normally located at that position.
  • a TNF- ⁇ mutant may be referred to herein as a TNF- ⁇ mutant peptide sequence, TNF- ⁇ mutant polypeptide, TNF- ⁇ mutant protein or TNF- ⁇ mutant analog.
  • Designation of various forms of TNF- ⁇ is herein made with respect to the sequence shown in SEQ ID NO: 1. Various designations may be used herein to indicate the same mutation.
  • a mutation from serine at position 147 to alanine can be indicated as 147A, A147, A 147 , S147A, or Ser147Ala.
  • the numbering of the positions of mutated amino acid residues is according to the wild-type human TNF- ⁇ sequence (SEQ ID NO: 1) .
  • single mutations A33T, N34A, N34H, Y87A, Y87F, S95A, A145G, A145V, S147A, and S147G each can reduce binding affinity between TNF- ⁇ and one or both of its receptors TNF- ⁇ receptor 1 (TNFR1) and TNF- ⁇ receptor 2 (TNFR2) .
  • the wild-type TNF- ⁇ can be mutated to modulate self-interaction affinity between TNF- ⁇ polypeptides during oligomerization.
  • wild type TNF- ⁇ can be mutated to include one or more cysteine residues, such that multimerization between TNF- ⁇ or TNF- ⁇ containing immunoconjugate molecules can be stabilized through the formation of the covalent disulfide bond between the added cysteine residues.
  • Exemplary mutations that can facilitate multimerization of TNF- ⁇ or TNF- ⁇ contain immunoconjugate molecules include P08C, L55C, Q102C, E104C, S95C, G148C, I97C, Y115C, H73C, and P113C.
  • a mutant TNF- ⁇ contains one or more mutations selected from P08C, L55C, Q102C, E104C, S95C, G148C, I97C, Y115C, H73C, and P113C. In specific embodiments, a mutant TNF- ⁇ contains double mutations selected from P08C, L55C, Q102C, E104C, S95C, G148C, I97C, Y115C, H73C, and P113C.
  • a mutant TNF- ⁇ contains double mutations of (i) P08C and L55C, (ii) Q102C and E104C, (iii) S95C and G148C, (iv) I97C and Y115C, (v) H73C and P113C, or (vi) any combination of (i) to (v) .
  • Additional mutant TNF- ⁇ polypeptides that can be used in connection with the present disclosure include those described in, for example, Loetscher et al., Journal of Biological Chemistry, 268 (35) : 26350-26357, which is hereby incorporated by reference in its entirety.
  • a “wild-type” form of TNF- ⁇ is a form of TNF- ⁇ that is otherwise the same as the mutant TNF- ⁇ polypeptide except that the wild-type form has a wild-type amino acid at each amino acid position of the mutant TNF- ⁇ polypeptide.
  • the wild-type form of this mutant is full-length native TNF- ⁇ .
  • the wild-type form of this TNF- ⁇ mutant is TNF- ⁇ with a wild-type amino acid sequence fused to the same downstream polypeptide.
  • the TNF- ⁇ mutant is a truncated form of TNF- ⁇ (the mutated or modified sequence within the non-truncated portion of TNF- ⁇ ) then the wild-type form of this TNF- ⁇ mutant is a similarly truncated TNF- ⁇ that has a wild-type sequence.
  • wild-type encompasses forms of TNF- ⁇ comprising one or more amino acid mutation that does not affect TNF- ⁇ receptor binding compared to the naturally occurring, native TNF- ⁇ .
  • the wild-type TNF- ⁇ polypeptide to which the mutant TNF- ⁇ polypeptide is compared comprises the amino acid sequence of SEQ ID NO: 1.
  • a “corresponding site” in a polypeptide with respect to a reference polypeptide sequence can be determined by aligning and comparing the sequences. After aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, those pairs of sites in the two sequences that are considered aligned with one another are corresponding sites. Alignment for purposes of determining corresponding sites in two amino acid sequences 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 MEGALIGN (DNAStar, Inc. ) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.
  • TNFR1 refers to any native TNFR1 from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats) , unless otherwise indicated.
  • the term encompasses “full-length” , unprocessed TNFR1 as well as any form of TNFR1 that results from processing in the cell.
  • the term also encompasses naturally occurring variants of TNFR1, e.g., splice variants or allelic variants.
  • TNFR1 is human TNFR1.
  • the amino acid sequence of an exemplary human TNFR1 is shown below:
  • TNFR2 refers to any native TNFR2 from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats) , unless otherwise indicated.
  • the term encompasses “full-length” , unprocessed TNFR2 as well as any form of TNFR2 that results from processing in the cell.
  • the term also encompasses naturally occurring variants of TNFR2, e.g., splice variants or allelic variants.
  • TNFR2 is human TNFR2.
  • the amino acid sequence of an exemplary human TNFR2 is shown below:
  • identity refers to a relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules, as determined by aligning and comparing the sequences. “Percent (%) amino acid sequence identity” with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference 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 MEGALIGN (DNAStar, Inc. ) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.
  • TAA tumor associated antigen
  • the term “tumor associated antigen” or “TAA” refers to an antigen expressed by a cancer cell or in the stroma of a solid tumor.
  • the TAA can be a protein, nucleic acid, lipid or other antigen.
  • the TAA can be a cell-surface expressed TAA.
  • the TAA can be expressed in the stroma of a solid tumor mass.
  • stroma refers to components in a solid tumor mass other than a cancer cell.
  • the stroma can include fibroblasts, epithelial cells, other blood vessel components or extracellular matrix components.
  • stroma does not include components of the immune system, such as immune cells (e.g., B-cells, T-cells, dendritic cells, macrophages, natural killer cells, and the like) .
  • immune cells e.g., B-cells, T-cells, dendritic cells, macrophages, natural killer cells, and the like.
  • TAAs are known in the art. Identifying TAA can be performed using methods known in the art, such as disclosed in Zhang et al., Methods Mol. Biol., 520: 1-10 (2009) ; the content of which is enclosed herein by reference.
  • fibroblast activation protein refers to any native FAP from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats) , unless otherwise indicated.
  • the term encompasses unprocessed FAP as well as any form of FAP that results from processing in the cell.
  • the term also encompasses naturally occurring variants of FAP, e.g., splice variants or allelic variants.
  • the amino acid sequence of an exemplary human FAP is shown below
  • Trop-2 Tumor-associated calcium signal transducer 2
  • Trop-2 refers to any native Trop-2 from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats) , unless otherwise indicated.
  • the term encompasses unprocessed Trop-2 as well as any form of Trop-2 that results from processing in the cell.
  • the term also encompasses naturally occurring variants of Trop-2, e.g., splice variants or allelic variants.
  • the amino acid sequence of an exemplary human Trop-2 is shown below
  • tumor microenvironment refers to any and all elements of the neoplasia milieu that creates a structural and/or functional environment for the neoplastic process to survive, expand, and/or spread.
  • a tumor microenvironment is constituted by the cells, molecules, fibroblasts, extracellular matrix and/or blood vessels that surround and/or feed one or more neoplastic cells, such as a solid tumor.
  • the neoplastic disease is a solid tumor.
  • Exemplary cells or tissue within the tumor microenvironment include, but are not limited to, tumor vasculature, tumor infiltrating lymphocytes, fibroblast reticular cells, endothelial progenitor cells (EPC) , cancer-associated fibroblasts, pericytes, other stromal cells, components of the extracellular matrix (ECM) , dendritic cells, antigen presenting cells, T-cells, regulatory T-cells, macrophages, neutrophils, and other immune cells located proximal to a tumor.
  • ECM extracellular matrix
  • Exemplary cellular functions affecting the tumor microenvironment include, but are not limited to, production of cytokines and/or chemokines, response to cytokines, antigen processing and presentation of peptide antigen, regulation of leukocyte chemotaxis and migration, regulation of gene expression, complement activation, regulation of signaling pathways, cell-mediated cytotoxicity, cell-mediated immunity, humoral immune responses, and innate immune responses, etc.
  • antibody immunoglobulin, ” or “Ig” is used interchangeably herein, and is used in the broadest sense and specifically encompasses, for example, individual monoclonal antibodies (including agonist, antagonist, neutralizing antibodies, full length or intact monoclonal antibodies) , antibody compositions with polyepitopic or monoepitopic specificity, polyclonal or monovalent antibodies, multivalent antibodies, multispecific antibodies (e.g., bispecific antibodies so long as they exhibit the desired biological activity) , formed from at least two intact antibodies, single chain antibodies, and fragments of antibodies, as described below.
  • An antibody can be human, humanized, chimeric and/or affinity matured, as well as an antibody from other species, for example, mouse and rabbit, etc.
  • antibody is intended to include a polypeptide product of B cells within the immunoglobulin class of polypeptides that is able to bind to a specific molecular antigen and is composed of two identical pairs of polypeptidic chains, wherein each pair has one heavy chain (about 50-70 kDa) and one light chain (about 25 kDa) , each amino-terminal portion of each chain includes a variable region of about 100 to about 130 or more amino acids, and each carboxy-terminal portion of each chain includes a constant region. See, e.g., Antibody Engineering (Borrebaeck ed., 2d ed. 1995) ; and Kuby, Immunology (3d ed. 1997) .
  • the specific molecular antigen can be bound by an antibody provided herein, such as a TNF- ⁇ polypeptide, a TNF- ⁇ fragment, or a TNF- ⁇ epitope.
  • Antibodies also include, but are not limited to, synthetic antibodies, recombinantly produced antibodies, camelized antibodies, intrabodies, anti-idiotypic (anti-Id) antibodies, and functional fragments (e.g., antigen-binding fragments such as TNF- ⁇ -binding fragments) of any of the above, which refers to a portion of an antibody heavy or light chain polypeptide that retains some or all of the binding activity of the antibody from which the fragment was derived.
  • Non-limiting examples of functional fragments include single-chain Fvs (scFv) (e.g., including monospecific, bispecific, etc. ) , Fab fragments (e.g., including monospecific, bispecific, etc. ) , F (ab’ ) fragments, F (ab) 2 fragments, F (ab’ ) 2 fragments, disulfide-linked Fvs (dsFv) , Fd fragments, Fv fragments, diabody, triabody, tetrabody, minibody, and single domain antibody (VHH or nanobody) .
  • scFv single-chain Fvs
  • Fab fragments e.g., including monospecific, bispecific, etc.
  • antibodies provided herein include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, for example, antigen-binding domains or molecules that contain an antigen-binding site that binds to an TNF- ⁇ antigen (e.g., one or more CDRs of an anti-TNF- ⁇ antibody) .
  • TNF- ⁇ antigen e.g., one or more CDRs of an anti-TNF- ⁇ antibody
  • Such antibody fragments can be found in, for example, Harlow and Lane, Antibodies: A Laboratory Manual (1989) ; Mol. Biology and Biotechnology: A Comprehensive Desk Reference (Myers ed., 1995) ; Huston et al., 1993, Cell Biophysics 22: 189-224; Plückthun and Skerra, 1989, Meth. Enzymol.
  • the antibodies provided herein can be of any class (e.g., IgG, IgE, IgM, IgD, and IgA) or any subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) of immunoglobulin molecule.
  • a “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, e.g., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts, and each monoclonal antibody will typically recognize a single epitope on the antigen.
  • a “monoclonal antibody, ” as used herein is an antibody produced by a single hybridoma or other cell, wherein the antibody binds to only one epitope as determined, for example, by ELISA or other antigen-binding or competitive binding assay known in the art.
  • the term “monoclonal” is not limited to any particular method for making the antibody.
  • the monoclonal antibodies useful in the present disclosure may be prepared by the hybridoma methodology first described by Kohler et al., 1975, Nature 256: 495, or may be made using recombinant DNA methods in bacterial or eukaryotic animal or plant cells (see, e.g., U.S. Pat. No. 4,816,567) .
  • the “monoclonal antibodies” may also be isolated from phage antibody libraries using the techniques described in Clackson et al., 1991, Nature 352: 624-28 and Marks et al., 1991, J. Mol. Biol. 222: 581-97, for example.
  • Polyclonal antibodies refer to an antibody population generated in an immunogenic response to a protein having many epitopes and thus includes a variety of different antibodies directed to the same or different epitopes within the protein. Methods for producing polyclonal antibodies are known in the art (See, e.g., Short Protocols in Molecular Biology (Ausubel et al. eds., 5th ed. 2002) ) .
  • an “antigen” is a predetermined antigen to which an antibody can selectively bind.
  • a target antigen may be a polypeptide, carbohydrate, nucleic acid, lipid, hapten, or other naturally occurring or synthetic compound. In some embodiments, the target antigen is a polypeptide.
  • antigen-binding fragment refers to that portion of an antibody, which comprises the amino acid residues that interact with an antigen and confer on the binding agent its specificity and affinity for the antigen (e.g., the CDRs) .
  • a “bispecific antibody” as used herein refers to an antibody or antigen binding fragment thereof that is capable of binding with two different target antigens.
  • a “two-in-one antibody” as used herein refers to a bispecific antibody that is capable of binding with two different target antigens via a single antigen binding domain.
  • the target antigens compete with one another for binding with the single antigen binding domain of the two-in-one antibody, such that the two-in-one antibody, upon binding with one target antigen, dissociates from the other target antigen.
  • An “epitope” is the site on the surface of an antigen molecule to which a single antibody molecule binds, such as a localized region on the surface of an antigen, such as a TNF- ⁇ polypeptide or a TNF- ⁇ polypeptide fragment, that is capable of being bound to one or more antigen binding regions of an antibody, and that has antigenic or immunogenic activity in an animal, such as a mammal (e.g., a human) , that is capable of eliciting an immune response.
  • An epitope having immunogenic activity is a portion of a polypeptide that elicits an antibody response in an animal.
  • An epitope having antigenic activity is a portion of a polypeptide to which an antibody binds as determined by any method well known in the art, including, for example, by an immunoassay.
  • Antigenic epitopes need not necessarily be immunogenic. Epitopes often consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and have specific three dimensional structural characteristics as well as specific charge characteristics.
  • Antibody epitopes may be linear epitopes or conformational epitopes. Linear epitopes are formed by a continuous sequence of amino acids in a protein. Conformational epitopes are formed of amino acids that are discontinuous in the protein sequence, but which are brought together upon folding of the protein into its three-dimensional structure.
  • Induced epitopes are formed when the three dimensional structure of the protein is in an altered conformation, such as following activation or binding of another protein or ligand.
  • an antigen has several or many different epitopes and may react with many different antibodies.
  • an antigen e.g., FAP
  • FAP can have more than one epitopes that are recognized and bound by different anti-FAP antibodies.
  • different anti-FAP antibodies compete with one another for binding with the same epitope of FAP.
  • an antibody binds “an epitope, ” “essentially the same epitope, ” or “the same epitope” as a reference antibody, when the two antibodies recognize identical, overlapping, or adjacent epitopes in a three-dimensional space.
  • the most widely used and rapid methods for determining whether two antibodies bind to identical, overlapping, or adjacent epitopes in a three-dimensional space are competition assays, which can be configured in a number of different formats, for example, using either labeled antigen or labeled antibody.
  • the antigen is immobilized on a 96-well plate, or expressed on a cell surface, and the ability of unlabeled antibodies to block the binding of labeled antibodies is measured using radioactive, fluorescent, or enzyme labels.
  • Epitope mapping is the process of identifying the binding sites, or epitopes, of antibodies on their target antigens.
  • Epitope binning is the process of grouping antibodies based on the epitopes they recognize. More particularly, epitope binning comprises methods and systems for discriminating the epitope recognition properties of different antibodies, using competition assays combined with computational processes for clustering antibodies based on their epitope recognition properties and identifying antibodies having distinct binding specificities.
  • binding refers to an interaction between molecules including, for example, to form a complex. Interactions can be, for example, non-covalent interactions including hydrogen bonds, ionic bonds, hydrophobic interactions, and/or van der Waals interactions. A complex can also include the binding of two or more molecules held together by covalent or non-covalent bonds, interactions, or forces. The strength of the total non-covalent interactions between a single antigen-binding site on an antibody and a single epitope of a target molecule, such as TNF- ⁇ , is the affinity of the antibody or functional fragment for that epitope.
  • the ratio of dissociation rate (k off ) to association rate (k on ) of an antibody to a monovalent antigen (k off /k on ) is the dissociation constant K D , which is inversely related to affinity.
  • K D the dissociation constant
  • the value of K D varies for different complexes of antibody and antigen and depends on both k on and k off .
  • the dissociation constant K D for an antibody provided herein can be determined using any method provided herein or any other method well known to those skilled in the art.
  • the affinity at one binding site does not always reflect the true strength of the interaction between an antibody and an antigen.
  • the avidity of an antibody can be a better measure of its binding capacity than is the affinity of its individual binding sites. For example, high avidity can compensate for low affinity as is sometimes found for pentameric IgM antibodies, which can have a lower affinity than IgG, but the high avidity of IgM, resulting from its multivalence, enables it to bind antigen effectively.
  • antibodies that specifically bind to an antigen bind to an antigen
  • antibodies that specifically bind to an epitope are also used interchangeably herein and refer to antibodies that specifically bind to the antigen, or fragment, or epitope of the antigen.
  • An antibody that specifically binds to an antigen can be identified, for example, by immunoassays, or other techniques known to those of skill in the art.
  • An antibody binds specifically to an antigen when it binds to the antigen with higher affinity than to any cross-reactive antigen as determined using experimental techniques, such as radioimmunoassays (RIA) and enzyme linked immunosorbent assays (ELISAs) .
  • RIA radioimmunoassays
  • ELISAs enzyme linked immunosorbent assays
  • a specific or selective reaction will be at least twice background signal or noise and may be more than 10 times background. See, e.g., Fundamental Immunology 332-36 (Paul ed., 2d ed. 1989) for a discussion regarding antibody specificity.
  • An antibody which “binds an antigen of interest” e.g., a target antigen such as TNF- ⁇
  • a target antigen such as TNF- ⁇
  • the extent of binding of the antibody to a “non-target” protein will be less than about 10%of the binding of the antibody to its particular target protein, for example, as determined by fluorescence activated cell sorting (FACS) analysis or RIA.
  • FACS fluorescence activated cell sorting
  • the term “specific binding, ” “specifically binds to, ” or “is specific for” a particular polypeptide or an epitope on a particular polypeptide target means binding that is measurably different from a non-specific interaction. Specific binding can be measured, for example, by determining binding of a molecule compared to binding of a control molecule, which generally is a molecule of similar structure that does not have binding activity.
  • specific binding can be determined by competition with a control molecule that is similar to the target, for example, an excess of non-labeled target. In this case, specific binding is indicated if the binding of the labeled target to a probe is competitively inhibited by excess unlabeled target.
  • an antibody that binds to an antigen of the present disclosure has a dissociation constant (K D ) of less than or equal to 10 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, or 0.1 nM.
  • K D dissociation constant
  • Compet when used in the context of antibodies (e.g., antibodies and binding proteins that bind to a cell surface antigen and compete for the same epitope or binding site on a target) means competition as determined by an assay in which the antibody (or binding fragment) thereof under study prevents or inhibits the specific binding of a reference molecule (e.g., a reference ligand or reference antigen-binding protein, such as a reference antibody) to a common antigen (e.g., FAP or a fragment thereof) .
  • a reference molecule e.g., a reference ligand or reference antigen-binding protein, such as a reference antibody
  • a common antigen e.g., FAP or a fragment thereof
  • Numerous types of competitive binding assays can be used to determine if a test antibody competes with a reference antibody for binding to an antigen (e.g., human FAP) .
  • assays examples include solid phase direct or indirect RIA, solid phase direct or indirect enzyme immunoassay (EIA) , sandwich competition assay (see, e.g., Stahli et al., 1983, Methods in Enzymology 9: 242-53) , solid phase direct biotin-avidin EIA (see, e.g., Kirkland et al., 1986, J. Immunol.
  • solid phase direct labeled assay solid phase direct labeled sandwich assay (see, e.g., Harlow and Lane, Antibodies, A Laboratory Manual (1988) )
  • solid phase direct label RIA using I-125 label see, e.g., Morel et al., 1988, Mol. Immunol. 25: 7-15
  • direct labeled RIA Mimetic et al., 1990, Scand. J. Immunol. 32: 77-82
  • such an assay involves the use of a purified antigen (e.g., TNF- ⁇ ) bound to a solid surface, or cells bearing either of an unlabelled test antigen-binding protein (e.g., test anti-TNF- ⁇ antibody) or a labeled reference antigen-binding protein (e.g., reference anti-TNF- ⁇ antibody) .
  • a purified antigen e.g., TNF- ⁇
  • an unlabelled test antigen-binding protein e.g., test anti-TNF- ⁇ antibody
  • a labeled reference antigen-binding protein e.g., reference anti-TNF- ⁇ antibody
  • Antibodies identified by competition assay include antibodies binding to the same epitope as the reference antibody and/or antibodies binding to an adjacent epitope sufficiently proximal to the epitope bound by the reference for antibodies steric hindrance to occur. Additional details regarding methods for determining competitive binding are described herein. Usually, when a competing antibody protein is present in excess, it will inhibit specific binding of a reference antibody to a common antigen by at least 30%, for example 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75%. In some instance, binding is inhibited by at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more.
  • the term “heavy chain” when used in reference to an antibody refers to a polypeptidic chain of about 50-70 kDa, wherein the amino-terminal portion includes a variable region of about 120 to 130 or more amino acids, and a carboxy-terminal portion includes a constant region.
  • the constant region can be one of five distinct types, (e.g., isotypes) referred to as alpha ( ⁇ ) , delta ( ⁇ ) , epsilon ( ⁇ ) , gamma ( ⁇ ) , and mu ( ⁇ ) , based on the amino acid sequence of the heavy chain constant region.
  • the distinct heavy chains differ in size: ⁇ , ⁇ , and ⁇ contain approximately 450 amino acids, while ⁇ and ⁇ contain approximately 550 amino acids.
  • heavy chains When combined with a light chain, these distinct types of heavy chains give rise to five well known classes (e.g., isotypes) of antibodies, IgA, IgD, IgE, IgG, and IgM, respectively, including four subclasses of IgG, namely IgG1, IgG2, IgG3, and IgG4.
  • a heavy chain can be a human heavy chain.
  • light chain when used in reference to an antibody refers to a polypeptidic chain of about 25 kDa, wherein the amino-terminal portion includes a variable region of about 100 to about 110 or more amino acids, and a carboxy-terminal portion includes a constant region.
  • the approximate length of a light chain is 211 to 217 amino acids.
  • Light chain amino acid sequences are well known in the art.
  • a light chain can be a human light chain.
  • variable region refers to a portion of the light or heavy chains of an antibody that is generally located at the amino-terminal of the light or heavy chain and has a length of about 120 to 130 amino acids in the heavy chain and about 100 to 110 amino acids in the light chain, and are used in the binding and specificity of each particular antibody for its particular antigen.
  • the variable region of the heavy chain may be referred to as “VH. ”
  • the variable region of the light chain may be referred to as “VL. ”
  • variable refers to the fact that certain segments of the variable regions differ extensively in sequence among antibodies. The V region mediates antigen binding and defines specificity of a particular antibody for its particular antigen.
  • variable regions consist of less variable (e.g., relatively invariant) stretches called framework regions (FRs) of about 15-30 amino acids separated by shorter regions of greater variability (e.g., extreme variability) called “hypervariable regions” that are each about 9-12 amino acids long.
  • FRs framework regions
  • hypervariable regions that are each about 9-12 amino acids long.
  • the variable regions of heavy and light chains each comprise four FRs, largely adopting a ⁇ sheet configuration, connected by three hypervariable regions, which form loops connecting, and in some cases form part of, the ⁇ sheet structure.
  • the hypervariable regions in each chain are held together in close proximity by the FRs and, with the hypervariable regions from the other chain, contribute to the formation of the antigen-binding site of antibodies (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest (5th ed. 1991) ) .
  • the constant regions are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody dependent cellular cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC) .
  • the variable regions differ extensively in sequence between different antibodies.
  • the variable region is a human variable region.
  • variable region residue numbering as in Kabat or “amino acid position numbering as in Kabat” , and variations thereof, refer to the numbering system used for heavy chain variable regions or light chain variable regions of the compilation of antibodies in Kabat et al., supra. Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to a shortening of, or insertion into, an FR or CDR of the variable domain.
  • a heavy chain variable domain may include a single amino acid insert (residue 52a according to Kabat) after residue 52 and three inserted residues (e.g., residues 82a, 82b, and 82c, etc. according to Kabat) after residue 82.
  • the Kabat numbering of residues may be determined for a given antibody by alignment at regions of homology of the sequence of the antibody with a “standard” Kabat numbered sequence.
  • the Kabat numbering system is generally used when referring to a residue in the variable domain (approximately residues 1-107 of the light chain and residues 1-113 of the heavy chain) (e.g., Kabat et al., supra) .
  • the “EU numbering system” or “EU index” is generally used when referring to a residue in an immunoglobulin heavy chain constant region (e.g., the EU index reported in Kabat et al., supra) .
  • the “EU index as in Kabat” refers to the residue numbering of the human IgG 1 EU antibody. Other numbering systems have been described, for example, by AbM, Chothia, Contact, IMGT, and AHon.
  • CDR refers to one of three hypervariable regions (H1, H2 or H3) within the non-framework region of the immunoglobulin (Ig or antibody) VH ⁇ -sheet framework, or one of three hypervariable regions (L1, L2 or L3) within the non-framework region of the antibody VL ⁇ -sheet framework. Accordingly, CDRs are variable region sequences interspersed within the framework region sequences. CDR regions are well known to those skilled in the art and have been defined by, for example, Kabat as the regions of most hypervariability within the antibody variable (V) domains (Kabat et al., 1997, J. Biol. Chem. 252: 6609-16; Kabat, 1978, Adv. Prot. Chem.
  • CDR region sequences also have been defined structurally by Chothia as those residues that are not part of the conserved ⁇ -sheet framework, and thus are able to adapt different conformations (Chothia and Lesk, 1987, J. Mol. Biol. 196: 901-17) . Both terminologies are well recognized in the art. CDR region sequences have also been defined by AbM, Contact, and IMGT. The positions of CDRs within a canonical antibody variable region have been determined by comparison of numerous structures (Al-Lazikani et al., 1997, J. Mol. Biol. 273: 927-48; Morea et al., 2000, Methods 20: 267-79) .
  • hypervariable region refers to the regions of an antibody variable region that are hypervariable in sequence and/or form structurally defined loops.
  • antibodies comprise six hypervariable regions, three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3) .
  • a number of hypervariable region delineations are in use and are encompassed herein.
  • the Kabat Complementarity Determining Regions are based on sequence variability and are the most commonly used (see, e.g., Kabat et al., supra) .
  • Chothia refers instead to the location of the structural loops (see, e.g., Chothia and Lesk, 1987, J. Mol. Biol. 196: 901-17) .
  • the end of the Chothia CDR-H1 loop when numbered using the Kabat numbering convention varies between H32 and H34 depending on the length of the loop (this is because the Kabat numbering scheme places the insertions at H35A and H35B; if neither 35A nor 35B is present, the loop ends at 32; if only 35A is present, the loop ends at 33; if both 35A and 35B are present, the loop ends at 34) .
  • the AbM hypervariable regions represent a compromise between the Kabat CDRs and Chothia structural loops, and are used by Oxford Molecular’s AbM antibody modeling software (see, e.g., Antibody Engineering Vol. 2 (Kontermann and Dübel eds., 2d ed. 2010) ) .
  • the “contact” hypervariable regions are based on an analysis of the available complex crystal structures. The residues from each of these hypervariable regions or CDRs are noted below.
  • IMGT ImMunoGeneTics
  • IG immunoglobulins
  • TCR T cell receptors
  • MHC major histocompatibility complex
  • the CDRs are as defined by the IMGT numbering system. In other embodiments, the CDRs are as defined by the Kabat numbering system. In certain embodiments, the CDRs are as defined by the AbM numbering system. In other embodiments, the CDRs are as defined by the Chothia system. In yet other embodiments, the CDRs are as defined by the Contact numbering system.
  • Hypervariable regions may comprise “extended hypervariable regions” as follows: 24-36 or 24-34 (L1) , 46-56 or 50-56 (L2) , and 89-97 or 89-96 (L3) in the VL, and 26-35 or 26-35A (H1) , 50-65 or 49-65 (H2) , and 93-102, 94-102, or 95-102 (H3) in the VH.
  • HVR extended hypervariable regions
  • constant region refers to a carboxyl terminal portion of the light and heavy chain which is not directly involved in binding of the antibody to antigen but exhibits various effector function, such as interaction with the Fc receptor.
  • the term refers to the portion of an immunoglobulin molecule having a more conserved amino acid sequence relative to the other portion of the immunoglobulin, the variable region, which contains the antigen binding site.
  • the constant region may contain the CH1, CH2, and CH3 regions of the heavy chain and the CL region of the light chain.
  • FR refers to those variable region residues flanking the CDRs. FR residues are present, for example, in chimeric, humanized, human, domain antibodies, diabodies, linear antibodies, and bispecific antibodies. FR residues are those variable domain residues other than the hypervariable region residues or CDR residues.
  • Fc region herein is used to define a C-terminal region of an immunoglobulin heavy chain, including, for example, native sequence Fc regions, recombinant Fc regions, and variant Fc regions. Although the boundaries of the Fc region of an immunoglobulin heavy chain might vary, the human IgG heavy chain Fc region is often defined to stretch from an amino acid residue at position Cys226, or from Pro230, to the carboxyl-terminus thereof.
  • the C-terminal lysine (residue 447 according to the EU numbering system) of the Fc region may be removed, for example, during production or purification of the antibody, or by recombinantly engineering the nucleic acid encoding a heavy chain of the antibody. Accordingly, a composition of intact antibodies may comprise antibody populations with all K447 residues removed, antibody populations with no K447 residues removed, and antibody populations having a mixture of antibodies with and without the K447 residue.
  • a “functional Fc region” possesses an “effector function” of a native sequence Fc region.
  • effector functions include C1q binding; complement dependent cytotoxicity (CDC) ; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC) ; antibody-dependent cellular phagocytosis (ADCP) ; cytokine secretion, downregulation of cell surface receptors (e.g., B cell receptor) , and B cell activation, etc.
  • Such effector functions generally require the Fc region to be combined with a binding region or binding domain (e.g., an antibody variable region or domain) and can be assessed using various assays as disclosed.
  • an “activating Fc receptor” is an Fc receptor that following engagement by an Fc region of an antibody elicits signaling events that stimulate the receptor-bearing cell to perform effector functions.
  • exemplary activating Fc receptors include Fc ⁇ RIII ⁇ (CD16 ⁇ ) , Fc ⁇ RI (CD64) , Fc ⁇ RII ⁇ (CD32) , and Fc ⁇ RI (CD89) .
  • a “native sequence Fc region” comprises an amino acid sequence identical to the amino acid sequence of an Fc region found in nature, and not manipulated, modified, and/or changed (e.g., isolated, purified, selected, including or combining with other sequences such as variable region sequences) by a human.
  • Native sequence human IgG1 Fc regions include a native sequence human IgG1 Fc region (non-Aand A allotypes) ; native sequence human IgG2 Fc region; native sequence human IgG3 Fc region; and native sequence human IgG4 Fc region as well as naturally occurring variants thereof.
  • a native human IgG1 Fc region amino acid sequence is provided below:
  • a “variant Fc region” comprises an amino acid sequence which differs from that of a native sequence Fc region by virtue of at least one amino acid modification (e.g., substituting, addition, or deletion) .
  • the variant Fc region has at least one amino acid substitution compared to a native sequence Fc region or to the Fc region of a parent polypeptide, for example, from about one to about ten amino acid substitutions, or from about one to about five amino acid substitutions in a native sequence Fc region or in the Fc region of a parent polypeptide.
  • the variant Fc region herein can possess at least about 80%homology with a native sequence Fc region and/or with an Fc region of a parent polypeptide, or at least about 90%homology therewith, for example, at least about 95%homology therewith.
  • a variant can possess at least about 80%homology with a native sequence Fc region and/or with an Fc region of a parent polypeptide, or at least about 90%homology therewith, for example, at least about 95%homology therewith.
  • a “modification” of an amino acid residue/position refers to a change of a primary amino acid sequence as compared to a starting amino acid sequence, wherein the change results from a sequence alteration involving said amino acid residue/position.
  • typical modifications include substitution of the residue with another amino acid (e.g., a conservative or non-conservative substitution) , insertion of one or more (e.g., generally fewer than 5, 4, or 3) amino acids adjacent to said residue/position, and/or deletion of said residue/position.
  • a “modification promoting heterodimerization” is a manipulation of the peptide backbone or the post-translational modifications of a polypeptide, e.g., an immunoglobulin heavy chain, that reduces or prevents the association of the polypeptide with an identical polypeptide to form a homodimer.
  • a modification promoting heterodimerization as used herein particularly includes separate modifications made to each of two polypeptides desired to form a dimer, wherein the modifications are complementary to each other so as to promote association of the two polypeptides.
  • a modification promoting heterodimerization may alter the structure or charge of one or both of the polypeptides desired to form a dimer so as to make their association sterically or electrostatically favorable, respectively.
  • Heterodimerization occurs between two non-identical polypeptides, such as two immunoglobulin heavy chains wherein further immunoconjugate components fused to each of the heavy chains (e.g., TNF- ⁇ polypeptide) are not the same.
  • the modification promoting heterodimerization is in the heavy chain (s) , specifically in the Fc domain, of an immunoglobulin molecule.
  • the modification promoting heterodimerization comprises an amino acid mutation, specifically an amino acid substitution.
  • the modification promoting heterodimerization comprises a separate amino acid mutation, specifically an amino acid substitution, in each of the two immunoglobulin heavy chains.
  • Fc domain herein is used to define the C-terminal portion of an immunoglobulin composed of the Fc regions of both heavy chains of the immunoglobulin.
  • Each heavy chain Fc region in an Fc domain is herein referred to as a subunit of the Fc domain.
  • the two subunits of a Fc domain can be both native sequence Fc regions, or both variant Fc regions, or one native sequence Fc region and one variant Fc region.
  • the Fc domain comprises a modification promoting hetero-dimerization of two non-identical immunoglobulin heavy chains.
  • the site of most extensive protein-protein interaction between the two polypeptidic chains of a human IgG Fc domain is in the CH3 domain of the Fc regions.
  • said modification is in the CH3 domain of the Fc regions.
  • said modification is a knob-into-hole modification, comprising a knob modification in one of the Fc subunits, referred to as “Fc-Knob, ” and a hole modification in the other one of the Fc subunits, referred to as “Fc-hole. ”
  • the knob-into-hole technology is described e.g., in U.S. Pat. No. 5,731,168; U.S. Pat. No. 7,695,936; Ridgway et al., Prat Eng 9, 617-621 (1996) and Carter, J Immunol Meth 248, 7-15 (2001) .
  • the method involves introducing a protuberance ( “knob” ) at the interface of a first polypeptide and a corresponding cavity ( “hole” ) in the interface of a second polypeptide, such that the protuberance can be positioned in the cavity so as to promote heterodimer formation and hinder homodimer formation.
  • Protuberances are constructed by replacing small amino acid side chains from the interface of the first polypeptide with larger side chains (e.g., tyrosine or tryptophan) .
  • Compensatory cavities of identical or similar size to the protuberances are created in the interface of the second polypeptide by replacing large amino acid side chains with smaller ones (e.g., alanine or threonine) .
  • the protuberance and cavity can be made by altering the nucleic acid encoding the polypeptides, e.g., by site- specific mutagenesis, or by peptide synthesis.
  • a knob modification comprises the amino acid substitution T366W in one of the two Fc subunits
  • the hole modification comprises the amino acid substitutions T366S, L368A and Y407V in the other one of the two Fc subunits.
  • the Fc subunit comprising the knob modification additionally comprises the amino acid substitution S354C
  • the immunoglobulin heavy chain comprising the hole modification additionally comprises the amino acid substitution Y349C. Introduction of these two cysteine residues results in formation of a disulfide bridge between the two heavy chains, further stabilizing the dimer (Carter, J. Immunol Methods 248, 7-15 (2001) ) .
  • variants when used in relation to a peptide or polypeptide, to an antibody may refer to a peptide or polypeptide comprising one or more (such as, for example, about 1 to about 25, about 1 to about 20, about 1 to about 15, about 1 to about 10, or about 1 to about 5) amino acid sequence substitutions, deletions, and/or additions as compared to a native or unmodified sequence.
  • a TNF- ⁇ variant may result from one or more (such as, for example, about 1 to about 25, about 1 to about 20, about 1 to about 15, about 1 to about 10, or about 1 to about 5) changes to an amino acid sequence of a native TNF- ⁇ .
  • a variant of an anti-TNF- ⁇ antibody may result from one or more (such as, for example, about 1 to about 25, about 1 to about 20, about 1 to about 15, about 1 to about 10, or about 1 to about 5) changes to an amino acid sequence of a native or previously unmodified anti-TNF- ⁇ antibody.
  • Variants may be naturally occurring, such as allelic or splice variants, or may be artificially constructed.
  • Polypeptide variants may be prepared from the corresponding nucleic acid molecules encoding the variants.
  • the TNF- ⁇ variant or anti-TNF- ⁇ antibody variant at least retains TNF- ⁇ or anti-TNF- ⁇ antibody functional activity, respectively.
  • an anti-TNF- ⁇ antibody variant is a bispecific antibody that binds to both FAP and TNF- ⁇ .
  • an anti-TNF- ⁇ antibody variant is a bispecific antibody that binds to both Trop-2 and TNF- ⁇ .
  • the variant is encoded by a single nucleotide polymorphism (SNP) variant of a nucleic acid molecule that encodes TNF- ⁇ or anti-TNF- ⁇ antibody VH or VL regions or subregions, such as one or more CDRs.
  • SNP single nucleotide polymorphism
  • an “intact” antibody is one comprising an antigen-binding site as well as a CL and at least heavy chain constant regions, CH1, CH2 and CH3.
  • the constant regions may include human constant regions or amino acid sequence variants thereof.
  • an intact antibody has one or more effector functions.
  • Antibody fragments comprise a portion of an intact antibody, such as the antigen-binding or variable region of the intact antibody.
  • antibody fragments include, without limitation, Fab, Fab’ , F (ab’ ) 2 , and Fv fragments; diabodies and di-diabodies (see, e.g., Holliger et al., 1993, Proc. Natl. Acad. Sci. 90: 6444-48; Lu et al., 2005, J. Biol. Chem. 280: 19665-72; Hudson et al., 2003, Nat. Med. 9: 129-34; WO 93/11161; and U.S. Pat. Nos.
  • single-chain antibody molecules see, e.g., U.S. Pat. Nos. 4,946,778; 5,260,203; 5,482,858; and 5,476,786) ; dual variable domain antibodies (see, e.g., U.S. Pat. No. 7,612,181) ; single domain antibodies (sdAbs) (see, e.g., Woolven et al., 1999, Immunogenetics 50: 98-101; and Streltsov et al., 2004, Proc Natl Acad Sci USA. 101: 12444-49) ; and multispecific antibodies formed from antibody fragments.
  • a “functional fragment, ” “binding fragment, ” or “antigen-binding fragment” of a therapeutic antibody will exhibit at least one if not some or all of the biological functions attributed to the intact antibody, the function comprising at least binding to the target antigen (e.g., an TNF- ⁇ binding fragment or fragment that binds to TNF- ⁇ ) .
  • the target antigen e.g., an TNF- ⁇ binding fragment or fragment that binds to TNF- ⁇
  • the term “immunoconjugate” refers to a polypeptide molecule that includes at least one cytokine moiety and at least one antigen binding moiety.
  • the immunoconjugate comprises at least one cytokine moiety (e.g., TNF- ⁇ ) , and at least two antigen binding moieties (e.g., a masking moiety and an anchoring moiety as described herein) .
  • immunoconjugates according to the present disclosure comprise one cytokine moiety and two antigen binding moieties joined by one or more linker sequences.
  • immunoconjugates according to the present disclosure comprises one cytokine moiety and two antigen binding moieties joined by an Fc domain of immunoglobulin.
  • the antigen binding moiety can be joined to the cytokine moiety by a variety of interactions and in a variety of configurations as described herein.
  • fusion, ” “fuse” or other grammatical variants thereof when used in relation to a peptide or polypeptide, or to an antibody refers to the joining of a peptide or polypeptide, or fragment, variant, and/or derivative thereof, with a heterologous peptide or polypeptide.
  • an “affinity matured” antibody is one with one or more alterations (e.g., amino acid sequence variations, including changes, additions, and/or deletions) in one or more HVRs thereof which result in an improvement in the affinity of the antibody for antigen, compared to a parent antibody which does not possess those alteration (s) .
  • Affinity matured antibodies can have nanomolar or even picomolar affinities for the target antigen. Affinity matured antibodies are produced by procedures known in the art. For review, see Hudson and Souriau, 2003, Nature Medicine 9: 129-34; Hoogenboom, 2005, Nature Biotechnol. 23: 1105-16; Quiroz and Sinclair, 2010, Revista Ingeneria Biomedia 4: 39-51.
  • Binding affinity generally refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule (e.g., a binding protein such as an antibody) and its binding partner (e.g., an antigen) .
  • binding affinity refers to intrinsic binding affinity which reflects a 1: 1 interaction between members of a binding pair (e.g., antibody and antigen) .
  • the affinity of a binding molecule X for its binding partner Y can generally be represented by the dissociation constant (K D ) . Affinity can be measured by common methods known in the art, including those described herein.
  • the “K D ” or “K D value” may be measured by assays known in the art, for example by a binding assay.
  • the K D may be measured in a RIA, for example, performed with the Fab version of an antibody of interest and its antigen (Chen et al., 1999, J. Mol Biol 293: 865-81) .
  • the K D or K D value may also be measured by using surface plasmon resonance assays by using, for example, a or a or by biolayer interferometry using, for example, a or Gator TM system.
  • An “on-rate” or “rate of association” or “association rate” or “k on ” may also be determined with the same surface plasmon resonance or biolayer interferometry techniques described above using, for example, a or a or a or Gator TM system.
  • inhibitor refers to partial (such as, 1%, 2%, 5%, 10%, 20%, 25%, 50%, 75%, 90%, 95%, 99%) or complete (i.e., 100%) inhibition.
  • Fc receptor or “FcR” describes a receptor that binds to the Fc region of an antibody.
  • An exemplary FcR is a native sequence human FcR.
  • an exemplary FcR is one that binds an IgG antibody (e.g., a gamma receptor) and includes receptors of the Fc ⁇ RI, Fc ⁇ RII, and Fc ⁇ RIII subclasses, including allelic variants and alternatively spliced forms of these receptors.
  • Fc ⁇ RII receptors include Fc ⁇ RIIA (an “activating receptor” ) and Fc ⁇ RIIB (an “inhibiting receptor” ) , which have similar amino acid sequences that differ primarily in the cytoplasmic domains thereof (see, e.g., 1997, Annu. Rev. Immunol. 15: 203-34) .
  • Various FcRs are known (see, e.g., Ravetch and Kinet, 1991, Annu. Rev. Immunol. 9: 457-92; Capel et al., 1994, Immunomethods 4: 25-34; and de Haas et al., 1995, J. Lab. Clin. Med. 126: 330-41) .
  • FcR FcR
  • the term also includes the neonatal receptor, FcRn, which is responsible for the transfer of maternal IgGs to the fetus (see, e.g., Guyer et al., 1976, J. Immunol. 117: 587-93; and Kim et al., 1994, Eu. J. Immunol. 24: 2429-34) .
  • Antibody variants with improved or diminished binding to FcRs have been described (see, e.g., WO 2000/42072; U.S. Pat. Nos. 7,183,387; 7,332,581; and 7.335,742; Shields et al. 2001, J. Biol. Chem. 9 (2) : 6591-604) .
  • vector refers to a substance that is used to carry or include a nucleic acid sequence, including for example, a nucleic acid sequence encoding an antibody or a cytokine polypeptide as described herein, in order to introduce a nucleic acid sequence into a host cell.
  • Vectors applicable for use include, for example, expression vectors, plasmids, phage vectors, viral vectors, episomes, and artificial chromosomes, which can include selection sequences or markers operable for stable integration into a host cell’s chromosome. Additionally, the vectors can include one or more selectable marker genes and appropriate expression control sequences.
  • Selection control sequences can include constitutive and inducible promoters, transcription enhancers, transcription terminators, and the like, which are well known in the art.
  • both nucleic acid molecules can be inserted, for example, into a single expression vector or in separate expression vectors.
  • the encoding nucleic acids can be operationally linked to one common expression control sequence or linked to different expression control sequences, such as one inducible promoter and one constitutive promoter.
  • nucleic acid molecules into a host cell can be confirmed using methods well known in the art. Such methods include, for example, nucleic acid analysis such as Northern blots or polymerase chain reaction (PCR) amplification of mRNA, immunoblotting for expression of gene products, or other suitable analytical methods to test the expression of an introduced nucleic acid sequence or its corresponding gene product. It is understood by those skilled in the art that the nucleic acid molecules are expressed in a sufficient amount to produce a desired product (e.g., an anti-FAP antibody as described herein) , and it is further understood that expression levels can be optimized to obtain sufficient expression using methods well known in the art.
  • a desired product e.g., an anti-FAP antibody as described herein
  • an “isolated nucleic acid” is a nucleic acid, for example, an RNA, DNA, or a mixed nucleic acids, which is substantially separated from other genome DNA sequences as well as proteins or complexes such as ribosomes and polymerases, which naturally accompany a native sequence.
  • An “isolated” nucleic acid molecule is one which is separated from other nucleic acid molecules which are present in the natural source of the nucleic acid molecule.
  • an “isolated” nucleic acid molecule, such as a cDNA molecule can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.
  • nucleic acid molecules encoding an antibody as described herein are isolated or purified.
  • the term embraces nucleic acid sequences that have been removed from their naturally occurring environment, and includes recombinant or cloned DNA isolates and chemically synthesized analogues or analogues biologically synthesized by heterologous systems.
  • a substantially pure molecule may include isolated forms of the molecule.
  • Polynucleotide or “nucleic acid, ” as used interchangeably herein, refers to polymers of nucleotides of any length and includes DNA and RNA.
  • the nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase or by a synthetic reaction.
  • a polynucleotide may comprise modified nucleotides, such as methylated nucleotides and their analogs.
  • Oligonucleotide refers to short, generally single-stranded, synthetic polynucleotides that are generally, but not necessarily, fewer than about 200 nucleotides in length.
  • oligonucleotide and “polynucleotide” are not mutually exclusive. The description above for polynucleotides is equally and fully applicable to oligonucleotides.
  • a cell that produces an antibody of the present disclosure may include a parent hybridoma cell, as well as bacterial and eukaryotic host cells into which nucleic acids encoding the antibodies have been introduced. Suitable host cells are disclosed below.
  • the left-hand end of any single-stranded polynucleotide sequence disclosed herein is the 5’ end; the left-hand direction of double-stranded polynucleotide sequences is referred to as the 5’ direction.
  • the direction of 5’ to 3’ addition of nascent RNA transcripts is referred to as the transcription direction; sequence regions on the DNA strand having the same sequence as the RNA transcript that are 5’ to the 5’ end of the RNA transcript are referred to as “upstream sequences” ; sequence regions on the DNA strand having the same sequence as the RNA transcript that are 3’ to the 3’ end of the RNA transcript are referred to as “downstream sequences. ”
  • nucleic acid or grammatical equivalents thereof as it is used in reference to nucleic acid molecule refers to a nucleic acid molecule in its native state or when manipulated by methods well known to those skilled in the art that can be transcribed to produce mRNA, which is then translated into a polypeptide and/or a fragment thereof.
  • the antisense strand is the complement of such a nucleic acid molecule, and the encoding sequence can be deduced therefrom.
  • recombinant antibody refers to an antibody that is prepared, expressed, created, or isolated by recombinant means.
  • Recombinant antibodies can be antibodies expressed using a recombinant expression vector transfected into a host cell, antibodies isolated from a recombinant, combinatorial antibody library, antibodies isolated from an animal (e.g., a mouse or cow) that is transgenic and/or transchromosomal for human immunoglobulin genes (see, e.g., Taylor et al., 1992, Nucl. Acids Res. 20: 6287-95) , or antibodies prepared, expressed, created, or isolated by any other means that involves splicing of immunoglobulin gene sequences to other DNA sequences.
  • Such recombinant antibodies can have variable and constant regions, including those derived from human germline immunoglobulin sequences (See Kabat et al., supra) .
  • such recombinant antibodies may be subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) , thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo.
  • composition is intended to encompass a product containing the specified ingredients (e.g., an immunoconjugate molecule provided herein) in, optionally, the specified amounts.
  • Carriers as used herein include pharmaceutically acceptable carriers, excipients, or stabilizers that are nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations employed. Often the physiologically acceptable carrier is an aqueous pH buffered solution.
  • physiologically acceptable carriers include buffers, such as phosphate, citrate, and other organic acids; antioxidants, including ascorbic acid; low molecular weight (e.g., fewer than about 10 amino acid residues) polypeptide; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrins; chelating agents, such as EDTA; sugar alcohols, such as mannitol or sorbitol; salt-forming counterions, such as sodium; and/or nonionic surfactants, such as TWEEN TM , polyethylene glycol (PEG) , and PLURONICS TM .
  • buffers such as phosphate, citrate, and other organic acids
  • antioxidants including ascorbic acid
  • carrier can also refer to a diluent, adjuvant (e.g., Freund’s adjuvant (complete or incomplete) ) , excipient, or vehicle.
  • adjuvant e.g., Freund’s adjuvant (complete or incomplete)
  • Such carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Water is an exemplary carrier when a composition (e.g., a pharmaceutical composition) is administered intravenously.
  • Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions.
  • Suitable excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, and the like.
  • the composition if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.
  • Compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations, and the like.
  • compositions can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in Remington and Gennaro, Remington’s Pharmaceutical Sciences (18th ed. 1990) .
  • Compositions, including pharmaceutical compounds may contain an antibody, for example, in isolated or purified form, together with a suitable amount of carriers.
  • pharmaceutically acceptable means being approved by a regulatory agency of the Federal or a state government, or listed in United States Pharmacopeia, European Pharmacopeia , or other generally recognized Pharmacopeia for use in animals, and more particularly in humans.
  • excipient refers to an inert substance which is commonly used as a diluent, vehicle, preservative, binder, or stabilizing agent, and includes, but is not limited to, proteins (e.g., serum albumin, etc. ) , amino acids (e.g., aspartic acid, glutamic acid, lysine, arginine, glycine, histidine, etc. ) , fatty acids and phospholipids (e.g., alkyl sulfonates, caprylate, etc. ) , surfactants (e.g., SDS, polysorbate, nonionic surfactant, etc.
  • proteins e.g., serum albumin, etc.
  • amino acids e.g., aspartic acid, glutamic acid, lysine, arginine, glycine, histidine, etc.
  • fatty acids and phospholipids e.g., alkyl sulfonates, caprylate, etc.
  • saccharides e.g., sucrose, maltose, trehalose, etc.
  • polyols e.g., mannitol, sorbitol, etc.
  • a subject is a mammal, such as a non-primate (e.g., cow, pig, horse, cat, dog, rat, etc. ) or a primate (e.g., monkey and human) .
  • a primate e.g., monkey and human
  • the subject is a human.
  • administering refers to the act of injecting or otherwise physically delivering a substance as it exists outside the body (e.g., an immunoconjugate molecule as described herein) into a patient, such as by mucosal, intradermal, intravenous, intramuscular delivery, and/or any other method of physical delivery described herein or known in the art.
  • a substance as it exists outside the body (e.g., an immunoconjugate molecule as described herein) into a patient, such as by mucosal, intradermal, intravenous, intramuscular delivery, and/or any other method of physical delivery described herein or known in the art.
  • substantially all refers to at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or about 100%.
  • the phrase “substantially similar” or “substantially the same” denotes a sufficiently high degree of similarity between two numeric values (e.g., one associated with an antibody of the present disclosure and the other associated with a reference antibody) such that one of skill in the art would consider the difference between the two values to be of little or no biological and/or statistical significance within the context of the biological characteristic measured by the values (e.g., K D values) .
  • the difference between the two values may be less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10%, or less than about 5%, as a function of the value for the reference antibody.
  • the phrase “substantially increased, ” “substantially reduced, ” or “substantially different, ” as used herein, denotes a sufficiently high degree of difference between two numeric values (e.g., one associated with an antibody of the present disclosure and the other associated with a reference antibody) such that one of skill in the art would consider the difference between the two values to be of statistical significance within the context of the biological characteristic measured by the values. For example, the difference between said two values can be greater than about 10%, greater than about 20%, greater than about 30%, greater than about 40%, or greater than about 50%, as a function of the value for the reference antibody.
  • cytokine-containing immunoconjugate molecules are fusion proteins comprising a cytokine moiety and a non-cytokine portion operably linked to one another.
  • the cytokine-containing immunoconjugate molecules are capable of delivery and activation of cellular activities of the cytokine at particular tissue or cellular location in a subject. For example, in some embodiments, the cytokine activity is reduced or blocked when the immunoconjugate molecules are present in an environment lacking an activation signal for the cytokine.
  • the cytokine activity is activated or enhanced when the immunoconjugate molecule are present in an environment containing or enriched of the activation signal for the cytokine.
  • the immunoconjugate molecules are configured for tissue-specific distribution upon administration to a subject.
  • the immunoconjugate molecules are capable of being enriched in certain tissue or cellular environment providing the activation signal for the cytokine, thereby activating the cytokine activity specifically in such tissue or cellular environment.
  • the activation signal for the cytokine is the presence of a signal molecule in the target tissue or cellular environment where the cytokine activity is activated.
  • the signal molecule is enriched in the target tissue or cellular environment, while present at other non-target tissue or cellular environment at a lower amount or concentration.
  • the activation signal for the cytokine is the presence of a signal molecule in the target tissue or cellular environment at a concentration above a threshold.
  • the signal molecule is capable of interacting with the immunoconjugate molecule, thereby activates the cytokine activity.
  • the signal molecule is a peptide molecule.
  • the immunoconjugate molecules are configured for the targeted delivery and activation of the cytokine activity in cancerous tissues, such as a tumor.
  • the signal molecule for activating the cytokine can be an antigen that is expressed or enriched in the cancerous tissue, such as in the tumor microenvironment.
  • the activation signal for the cytokine is an antigen expressed on the tumor cells.
  • the activation signal for the cytokine is an antigen expressed on the cells in the tumor microenvironment, such as tumor stromal cells.
  • the activation signal for the cytokine is a tumor associated antigen.
  • the non-cytokine portion of the immunoconjugate molecule comprises a masking moiety capable of binding with the cytokine moiety, and upon the binding, the masking moiety reduces or blocks the cytokine activity.
  • the immunoconjugate molecule comprises an antibody or antigen binding fragment thereof that is fused to a cytokine polypeptide, and the antibody or antigen binding fragment thereof is capable of binding with the cytokine polypeptide and reduces or blocks the cytokine activity.
  • the intramolecular binding between the cytokine moiety and the masking moiety of an immunoconjugate molecule is reversible. Accordingly, in some embodiments, the immunoconjugate molecules can switch between cytokine active and inactive states, through the reversible binding and disassociation between the cytokine moiety and the masking moiety.
  • the masking moiety is a bispecific two-in-one antibody or a binding fragment thereof, which is capable of binding to the cytokine moiety and a second target antigen that is different from the cytokine.
  • the masking moiety when the immunoconjugate molecule is in an environment where the second target antigen is absent, the masking moiety comprising the two-in-one antibody or antigen binding fragment thereof binds with the cytokine moiety of the immunoconjugate molecule, thereby inhibiting the cytokine activity.
  • the masking moiety comprising the two-in-one antibody or antigen binding fragment thereof binds with the cytokine moiety of the immunoconjugate molecule, thereby inhibiting the cytokine activity.
  • the environment is a cellular environment or a tissue-specific environment.
  • the environment is a cancerous tissue or a tumor microenvironment.
  • the second target antigen is an antigen expressed by the cancer cells.
  • the second target antigen is an antigen expressed by the cells in the tumor microenvironment, such as tumor stromal cells.
  • the second target antigen is a tumor associated antigen.
  • the masking moiety is a bispecific two-in-one antibody or a binding fragment thereof, which is capable of binding to the cytokine moiety and a second target antigen that is different from the cytokine.
  • the masking moiety comprising the two-in-one antibody or antigen binding fragment thereof binds with the second antigen and disassociates from the cytokine moiety of the immunoconjugate molecule, thereby activating the cytokine activity.
  • the masking moiety comprising the two-in-one antibody or antigen binding fragment thereof binds with the second antigen and disassociates from the cytokine moiety of the immunoconjugate molecule, thereby activating the cytokine activity.
  • the environment is a cellular environment or a tissue-specific environment.
  • the environment is a cancerous tissue or a tumor microenvironment.
  • the second target antigen is an antigen expressed by the tumor cells.
  • the second target antigen is an antigen expressed by the cells in the tumor microenvironment, such as tumor stromal cells.
  • the second target antigen is a tumor associated antigen.
  • the masking moiety is a bispecific two-in-one antibody or a binding fragment thereof, which is capable of binding to the cytokine moiety and a second target antigen that is different from the cytokine.
  • the masking moiety comprising the two-in-one antibody or antigen binding fragment thereof binds with the second antigen and disassociates from the cytokine moiety of the immunoconjugate molecule, thereby activating the cytokine activity.
  • the masking moiety comprising the two-in-one antibody or antigen binding fragment thereof binds with the second antigen and disassociates from the cytokine moiety of the immunoconjugate molecule, thereby activating the cytokine activity.
  • the second target antigen is expressed by a cell that also expresses a receptor for the cytokine forming part of the immunoconjugate molecule.
  • the second target antigen is expressed by a cell that does not itself express a receptor for the cytokine forming part of the immunoconjugate molecule and is nearby another cell expressing a receptor for the cytokine.
  • the immunoconjugate molecules of the present disclosure comprises a cytokine moiety and a non-cytokine portion, where the cytokine moiety comprises an TNF- ⁇ polypeptide, and the non-cytokine portion comprises a bispecific two-in-one antibody capable of binding to both the TNF- ⁇ polypeptide in the immunoconjugate molecule and a second target antigen that is not TNF- ⁇ .
  • the second target antigen is an antigen expressed by the tumor cells.
  • the second target antigen is an antigen expressed by the cells in the tumor microenvironment, such as tumor stromal cells.
  • the second target antigen is a tumor associated antigen.
  • the second target antigen is fibroblast activation protein (FAP) .
  • the second target antigen is Tumor-associated calcium signal transducer 2 (Trop-2) .
  • the TNF- ⁇ polypeptide is wild-type TNF- ⁇ polypeptide.
  • the TNF- ⁇ polypeptide is a mutant TNF- ⁇ polypeptide.
  • the TNF- ⁇ polypeptide is a human TNF- ⁇ polypeptide.
  • the TNF- ⁇ polypeptide is a monkey TNF- ⁇ polypeptide.
  • the TNF- ⁇ polypeptide is a mouse TNF- ⁇ polypeptide.
  • the TNF- ⁇ polypeptide is a mutant TNF- ⁇ polypeptide as described herein. In some embodiments, the TNF- ⁇ polypeptide is a mutant TNF- ⁇ polypeptide as described herein. In specific embodiments, the mutant TNF- ⁇ polypeptide is human TNF- ⁇ having one or more point mutation (s) selected from A33T, N34A, N34H, Y87A, Y87F, S95A, A145G, A145V, S147A, and S147G.
  • point mutation selected from A33T, N34A, N34H, Y87A, Y87F, S95A, A145G, A145V, S147A, and S147G.
  • the mutant TNF- ⁇ polypeptide is human TNF- ⁇ having one or more point mutation (s) selected from P08C, L55C, Q102C, E104C, S95C, G148C, I97C, Y115C, H73C, and P113C.
  • a mutant TNF- ⁇ is human TNF- ⁇ containing (i) double mutations of P08C and L55C; (ii) double mutations of Q102C and E104C, (iii) double mutations of S95C and G148C, (iv) double mutations of I97C and Y115C; (v) double mutations of H73C and P113C; or (vi) any combinations of (i) to (v) .
  • the mutant TNF- ⁇ polypeptide is a non-human TNF- ⁇ having one or more point mutation (s) at corresponding sites of N34, Y87, S95, and S147 of human TNF- ⁇ sequence as provided herein.
  • the mutant TNF- ⁇ polypeptide is a non-human TNF- ⁇ having one or more point mutation (s) corresponding to A33T, N34A, N34H, Y87A, Y87F, S95A, A145G, A145V, S147A, and S147G of human TNF- ⁇ sequence as provided herein.
  • the mutant TNF- ⁇ polypeptide is a non-human TNF- ⁇ having one or more point mutation (s) at corresponding sites of P08C, L55C, Q102C, E104C, S95C, G148C, I97C, Y115C, H73C, and P113C of human TNF- ⁇ sequence as provided herein.
  • the mutant TNF- ⁇ polypeptide is a non-human TNF- ⁇ having one or more point mutation (s) corresponding to P08C, L55C, Q102C, E104C, S95C, G148C, I97C, Y115C, H73C, and P113C of human TNF- ⁇ sequence as provided herein.
  • the mutant TNF- ⁇ polypeptide is a non-human TNF- ⁇ having one or more point mutation (s) at corresponding sites of (i) double mutations of P08C and L55C; (ii) double mutations of Q102C and E104C, (iii) double mutations of S95C and G148C, (iv) double mutations of I97C and Y115C; (v) double mutations of H73C and P113C; or (vi) any combinations of (i) to (v) of human TNF- ⁇ sequence as provided herein.
  • the mutant TNF- ⁇ polypeptide is a non-human TNF- ⁇ having one or more point mutation (s) corresponding to (i) double mutations of P08C and L55C; (ii) double mutations of Q102C and E104C, (iii) double mutations of S95C and G148C, (iv) double mutations of I97C and Y115C; (v) double mutations of H73C and P113C; or (vi) any combinations of (i) to (v) of human TNF- ⁇ sequence as provided herein.
  • point mutation corresponding to (i) double mutations of P08C and L55C; (ii) double mutations of Q102C and E104C, (iii) double mutations of S95C and G148C, (iv) double mutations of I97C and Y115C; (v) double mutations of H73C and P113C; or (vi) any combinations of (i) to (v) of human TNF- ⁇ sequence as provided herein.
  • TNF- ⁇ polypeptides that can be used in connection with the present disclosure can be found in Loetscher et al., Journal of Biological Chemistry, 268 (35) : 26350-26357, which is hereby incorporated by reference in its entirety.
  • the non-cytokine portion of the immunoconjugate molecule comprises an anchoring moiety configured to tether the immunoconjugate molecule to a target location of delivery.
  • immunoconjugate molecules of the present disclosure having the anchoring moiety can achieve tissue-specific distribution after being administered to a subject, such as after systemic administration to a subject.
  • the anchoring moiety of the immunoconjugate molecule is capable of specific binding to a target molecule that is present in the target location of delivery.
  • the anchoring moiety of the immunoconjugate molecule comprises an antibody or antigen binding fragment thereof capable of binding to an antigen present in the target location of delivery, thereby tethering the immunoconjugate molecule to the target location of delivery.
  • the target location of delivery is a cellular environment, or a tissue-specific environment.
  • the target location of delivery also contains an activation signal for the cytokine of the immunoconjugate molecule, such that the cytokine activity can be activated in the target location.
  • the target location of delivery is a cancerous tissue or a tumor microenvironment. In some embodiments, the target location of delivery is a particular type of tissue or population of cells in a subject.
  • the anchoring moiety of the immunoconjugate molecule comprises an antibody or antigen binding fragment thereof that bind to an antigen expressed on a target cell.
  • the target cell also expresses a receptor for the cytokine forming part of the immunoconjugate molecule. In alternative embodiments, the target cell does not itself express a receptor for the cytokine forming part of the immunoconjugate molecule, and is nearby another cell expressing a receptor for the cytokine.
  • the target location of delivery is a cancerous tissue or a tumor microenvironment.
  • the target location of delivery is a particular type of tissue or population of cells in a subject.
  • the anchoring moiety of the immunoconjugate molecule comprises an antibody or antigen binding fragment thereof that bind to an antigen expressed on cancer cells. Accordingly, in those embodiments, the immunoconjugate molecule, upon administration to a subject having cancer, can bind to a population of cancer cells in the subject.
  • the anchoring moiety of the immunoconjugate molecule comprises an antibody or antigen binding fragment thereof that bind to an antigen present in the tumor microenvironment, such as an antigen expressed on surface of a tumor cells or antigen secreted by cells in the tumor microenvironment, such as tumor stromal cells. Accordingly, in those embodiments, the immunoconjugate molecule, upon administration to a subject having a solid tumor, can enrich in the tumor microenvironment in the subject.
  • the immunoconjugate molecules of the present disclosure comprises a cytokine moiety, a masking moiety and an anchoring moiety that are operably connected with one another.
  • the masking moiety is a bispecific two-in-one antibody or antigen binding fragment thereof capable of binding to both the cytokine moiety and a second target antigen that is not the cytokine.
  • the anchoring moiety is an antibody or antigen binding fragment thereof capable of binding to a third target antigen, such as an antigen present in a target location of delivery for the immunoconjugate molecule.
  • the target location of delivery also contains the second target antigen in a sufficient amount to compete with the cytokine for binding with the masking moiety, resulting in disassociation of the masking moiety from the cytokine and activation of cytokine activity at the target location of delivery.
  • the immunoconjugate molecules upon administration to a subject, can achieve tissue-specific distribution and enrich in a target tissue or cellular environment in the subject that contains sufficient amount of the third antigen.
  • the target tissue or cellular environment also contains the second target antigen in a sufficient amount to compete with the cytokine for binding with the masking moiety, resulting in disassociation of the masking moiety from the cytokine and activation of cytokine activity in the target tissue or cellular environment.
  • the second and the third target antigens respectively recognized by the masking moiety and the anchoring moiety of the immunoconjugate are the same antigen. In alternative embodiments, the second and the third target antigens respectively recognized by the masking moiety and the anchoring moiety of the immunoconjugate are different antigens.
  • the second and the third target antigens respectively recognized by the masking moiety and the anchoring moiety of the immunoconjugate are the same antigen, and such antigen is expressed by a cell that also expresses a receptor for the cytokine forming part of the immunoconjugate molecule.
  • the second and the third target antigens respectively recognized by the masking moiety and the anchoring moiety of the immunoconjugate are the same antigen, and such antigen is expressed by a cell that does not itself expresses a receptor for the cytokine forming part of the immunoconjugate molecule and is nearby another cell expressing the receptor for the cytokine.
  • the second and the third target antigens respectively recognized by the masking moiety and the anchoring moiety of the immunoconjugate are different antigens, and the second target antigen is expressed by a cell that also expresses a receptor for the cytokine forming part of the immunoconjugate molecule.
  • the cell can also express the third target antigen.
  • the cell does not itself express the third target antigen and is nearby another cell expressing the third target antigen.
  • the second and the third target antigens respectively recognized by the masking moiety and the anchoring moiety of the immunoconjugate are different antigens
  • the third target antigen is expressed by a cell that also expresses a receptor for the cytokine forming part of the immunoconjugate molecule.
  • the cell can also express the second target antigen.
  • the cell does not itself express the second target antigen and is nearby another cell expressing the second target antigen.
  • the second and the third target antigens respectively recognized by the masking moiety and the anchoring moiety of the immunoconjugate are different antigens, and the second target antigen is expressed by a cell that does not itself expresses a receptor for the cytokine forming part of the immunoconjugate molecule and is nearby another cell expressing the receptor for the cytokine.
  • the cell expressing the second target antigen can also express the third target antigen.
  • the cell expressing the receptor for the cytokine can also express the third target antigen.
  • the third target antigen is express a cell nearby the cell expressing the second target antigen and/or the cell expressing the receptor for the cytokine.
  • the second and the third target antigens respectively recognized by the masking moiety and the anchoring moiety of the immunoconjugate are different antigens
  • the third target antigen is expressed by a cell that does not itself expresses a receptor for the cytokine forming part of the immunoconjugate molecule and is nearby another cell expressing the receptor for the cytokine.
  • the cell expressing the third target antigen can also express the second target antigen.
  • the cell expressing the receptor for the cytokine can also express the second target antigen.
  • the second target antigen is express a cell nearby the cell expressing the third target antigen and/or the cell expressing the receptor for the cytokine.
  • the cytokine moiety comprises a TNF- ⁇ polypeptide
  • the non-cytokine portion of the immunoconjugate molecule comprises a masking moiety comprising a bispecific two-in-one antibody capable of binding to both the TNF- ⁇ polypeptide in the immunoconjugate molecule and a second target antigen that is not TNF- ⁇ .
  • the second target antigen is an antigen expressed by the tumor cells.
  • the second target antigen is an antigen expressed by the cells in the tumor microenvironment, such as tumor stromal cells.
  • the second target antigen is a tumor associated antigen.
  • the second target antigen is fibroblast activation protein (FAP) .
  • the bispecific two-in-one antibody capable of binding to both the TNF- ⁇ polypeptide in the immunoconjugate molecule and a second target antigen FAP is an anti-TNF- ⁇ /anti-FAP two-in-one antibody or antigen binding fragment thereof as described in Section 5.3.1 (Anti-TNF- ⁇ /Anti-FAP Two-In-One Antibodies) .
  • the second target antigen is tumor-associated calcium signal transducer 2 (Trop-2) .
  • the bispecific two-in-one antibody capable of binding to both the TNF- ⁇ polypeptide in the immunoconjugate molecule and a second target antigen Trop-2 is an anti-TNF- ⁇ /anti-Trop2 two-in-one antibody or antigen binding fragment thereof as described in Section 5.3.2 (Anti-TNF- ⁇ /Anti-Trop2 Two-In-One Antibodies) .
  • the non-cytokine portion of the immunoconjugate molecule further comprises an anchoring moiety comprising an antibody or antigen binding fragment capable of binding to a third target antigen that is not TNF- ⁇ .
  • the third target antigen is an antigen expressed by the tumor cells.
  • the third target antigen is an antigen expressed by the cells in the tumor microenvironment, such as tumor stromal cells. In some embodiments, the third target antigen is a tumor associated antigen. In specific embodiments, the third target antigen is fibroblast activation protein (FAP) . In specific embodiments, the non-cytokine portion of the immunoconjugate molecule further comprises an anchoring moiety that binds FAP, wherein the anchoring moiety comprises anti-FAP antibody or antigen binding fragment thereof as described in Section 5.3.3 (Anti-FAP Antibodies) . In specific embodiments, the third target antigen is tumor-associated calcium signal transducer 2 (Trop-2) .
  • Trop-2 tumor-associated calcium signal transducer 2
  • the non-cytokine portion of the immunoconjugate molecule further comprises an anchoring moiety that binds Trop-2, wherein the anchoring moiety comprises anti-Trop2 antibody or antigen binding fragment thereof as described in Section 5.3.4 (Anti-Trop2 Antibodies) .
  • the TNF- ⁇ polypeptide is wild-type TNF- ⁇ polypeptide.
  • the TNF- ⁇ polypeptide is a mutant TNF- ⁇ polypeptide.
  • the TNF- ⁇ polypeptide is a human TNF- ⁇ polypeptide.
  • the TNF- ⁇ polypeptide is a monkey TNF- ⁇ polypeptide.
  • the TNF- ⁇ polypeptide is a mouse TNF- ⁇ polypeptide. In some embodiments, the TNF- ⁇ polypeptide is a mutant TNF- ⁇ polypeptide as described herein. In specific embodiments, the mutant TNF- ⁇ polypeptide is human TNF- ⁇ having one or more point mutation (s) selected from A33T, N34A, N34H, Y87A, Y87F, S95A, A145G, A145V, S147A, and S147G.
  • point mutation selected from A33T, N34A, N34H, Y87A, Y87F, S95A, A145G, A145V, S147A, and S147G.
  • the mutant TNF- ⁇ polypeptide is human TNF- ⁇ having one or more point mutation (s) selected from P08C, L55C, Q102C, E104C, S95C, G148C, I97C, Y115C, H73C, and P113C.
  • a mutant TNF- ⁇ is human TNF- ⁇ containing (i) double mutations of P08C and L55C; (ii) double mutations of Q102C and E104C, (iii) double mutations of S95C and G148C, (iv) double mutations of I97C and Y115C; (v) double mutations of H73C and P113C; or (vi) any combinations of (i) to (v) .
  • the mutant TNF- ⁇ polypeptide is a non-human TNF- ⁇ having one or more point mutation (s) at corresponding sites of N34, Y87, S95, and S147 of human TNF- ⁇ sequence as provided herein.
  • the mutant TNF- ⁇ polypeptide is a non-human TNF- ⁇ having one or more point mutation (s) corresponding to A33T, N34A, N34H, Y87A, Y87F, S95A, A145G, A145V, S147A, and S147G of human TNF- ⁇ sequence as provided herein.
  • the mutant TNF- ⁇ polypeptide is a non-human TNF- ⁇ having one or more point mutation (s) at corresponding sites of P08C, L55C, Q102C, E104C, S95C, G148C, I97C, Y115C, H73C, and P113C of human TNF- ⁇ sequence as provided herein.
  • the mutant TNF- ⁇ polypeptide is a non-human TNF- ⁇ having one or more point mutation (s) corresponding to P08C, L55C, Q102C, E104C, S95C, G148C, I97C, Y115C, H73C, and P113C of human TNF- ⁇ sequence as provided herein.
  • the mutant TNF- ⁇ polypeptide is a non-human TNF- ⁇ having one or more point mutation (s) at corresponding sites of (i) double mutations of P08C and L55C; (ii) double mutations of Q102C and E104C, (iii) double mutations of S95C and G148C, (iv) double mutations of I97C and Y115C; (v) double mutations of H73C and P113C; or (vi) any combinations of (i) to (v) of human TNF- ⁇ sequence as provided herein.
  • the mutant TNF- ⁇ polypeptide is a non-human TNF- ⁇ having one or more point mutation (s) corresponding to (i) double mutations of P08C and L55C; (ii) double mutations of Q102C and E104C, (iii) double mutations of S95C and G148C, (iv) double mutations of I97C and Y115C; (v) double mutations of H73C and P113C; or (vi) any combinations of (i) to (v) of human TNF- ⁇ sequence as provided herein.
  • point mutation corresponding to (i) double mutations of P08C and L55C; (ii) double mutations of Q102C and E104C, (iii) double mutations of S95C and G148C, (iv) double mutations of I97C and Y115C; (v) double mutations of H73C and P113C; or (vi) any combinations of (i) to (v) of human TNF- ⁇ sequence as provided herein.
  • TNF- ⁇ polypeptides that can be used in connection with the present disclosure can be found in Loetscher et al., Journal of Biological Chemistry, 268 (35) : 26350-26357, which is hereby incorporated by reference in its entirety.
  • the present immunoconjugate molecule comprises an anchoring moiety, a masking moiety and a cytokine moiety that are operably linked to one another via peptidic linkers.
  • the peptidic linker has at least 5 amino acid residues. In some embodiments, the peptidic linker has at least 7 amino acid residues. In some embodiments, the peptidic linker has at least 10 amino acid residues. In some embodiments, the peptidic linker has at least 15 amino acid residues. In some embodiments, the peptidic linker has at least 18 amino acid residues. In some embodiments, the peptidic linker has at least 20 amino acid residues. In some embodiments, the peptidic linker has at least 30 amino acid residues.
  • the peptidic linker has at least 40 amino acid residues. In some embodiments, the peptidic linker has at least 50 amino acid residues. In some embodiments, the peptidic linker has at least 60 amino acid residues. In some embodiments, the peptidic linker has at least 70 amino acid residues. In some embodiments, the peptidic linker has at least 80 amino acid residues. In some embodiments, the peptidic linker has at least 90 amino acid residues. In some embodiments, the peptidic linker has at least 100 amino acid residues. In some embodiments, the peptidic linker has at least 110 amino acid residues. In some embodiments, the peptidic linker has at least 120 amino acid residues.
  • the peptidic linker has at least 130 amino acid residues. In some embodiments, the peptidic linker has at least 140 amino acid residues. In some embodiments, the peptidic linker has at least 150 amino acid residues. In some embodiments, the peptidic linker has at least 160 amino acid residues. In some embodiments, the peptidic linker has at least 170 amino acid residues. In some embodiments, the peptidic linker has at least 180 amino acid residues. In some embodiments, the peptidic linker has at least 190 amino acid residues. In some embodiments, the peptidic linker has at least 200 amino acid residues. In some embodiments, the peptidic linker has at least 250 amino acid residues.
  • the peptidic linker has at least 300 amino acid residues.
  • the peptidic linker comprises an amino acid fragment GGGGS (i.e., G4S) (SEQ ID NO: 80) .
  • the peptidic linker comprises multiple tandem copies of the G4S fragments of varied length, such as 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 150, or 300 amino acids (SEQ ID NO: 81) .
  • n ⁇ (G4S) or (G4S) n means a peptide having n units of G4S, where n is a positive integer (SEQ ID NO: 81) .
  • 5 ⁇ (G4S) and (G4S) 5 both refer a peptide having the sequence of GGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 82) .
  • the peptidic linker connecting the masking moiety and cytokine can have the same length as the peptidic linker connecting the masking moiety and the anchoring moiety. In alternative embodiments, the peptidic linker connecting the masking moiety and cytokine can have a different length from the peptidic linker connecting the masking moiety and the anchoring moiety. In specific embodiments, the peptidic linker connecting the masking moiety and cytokine can be at least 5, 7, 10, 15, 18, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, or 300 amino acids.
  • the peptidic linker connecting the masking moiety and anchoring moiety can be at least 5, 7, 10, 15, 18, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, or 300 amino acids.
  • the linker sequence is selected from the sequences of Table 12.
  • FIGS. 1B to 1L are schematic illustrations of complexes containing multimerized antibody-cytokine immunoconjugates having different molecular configurations according to the present disclosure.
  • the multimerization between the immunoconjugate molecules can be via covalent (e.g., disulfide bond) or non-covalent interactions.
  • FIGS. 1B to 1L illustrate the configurations in the timer form, based on these illustrations, those of ordinary skill in the art could also envision immunoconjugates in the monomer, dimer, or higher-order multimer (e.g., greater than 3) forms, which are also within the scope of the present disclosure.
  • the present immunoconjugate comprises an anti-cytokine /anti-TAA two-in-one scFv antibody fused to a cytokine.
  • the immunoconjugate monomer can assume any one of the four different configurations: (i) cytokine-scFv (VH-VL) , (ii) cytokine-scFv (VL-VH) , (iii) scFv (VH-VL) -cytokine, and (iv) scFv (VL-VH) -cytokine, where “-” denotes a linker or direct fusion between different moieties, and the different moieties in a peptidic chain are listed according to the N to C orientation in the peptidic chain.
  • the monomer immunoconjugate molecule can multimerize into a complex, such as a trimer.
  • multimerization is through interaction between the cytokine domains of two or more immunoconjugate molecules.
  • immunoconjugate molecules forming the multimer can each assume a configuration selected from (i) to (iv) of this paragraph, which configurations can be the same (homo-multimer complex) as, or different (hetero-multimer complex) from, one another.
  • the cytokine is wild-type or mutant TNF- ⁇ .
  • the multimerization is through interaction between the extensive trimer interfaces of the TNF- ⁇ polypeptides contained in the immunoconjugate molecules.
  • the TAA is Trop-2 or FAP.
  • the present immunoconjugate comprises an anti-cytokine /anti-TAA two-in-one Fab antibody fused to a cytokine.
  • the immunoconjugate monomer can assume any one of the four possible configurations: (i) cytokine-VH-CH1 x VL-CL, (ii) cytokine-VL-CL x VH-CH1, (iii) VH-CH1-cytokine x VL-CL, and (iv) VL-CL-cytokine x VH-CH1, where “-” denotes a linker or direct fusion between different moieties, and the different moieties in a peptidic chain are listed according to the N to C orientation in the peptidic chain.
  • the monomer immunoconjugate molecule can multimerize into a complex, such as a trimer.
  • multimerization is through interaction between the cytokine domains of two or more immunoconjugate molecules.
  • immunoconjugate molecules forming the multimer can each assume a configuration selected from (i) to (iv) of this paragraph, which configurations can be the same (homo-multimer complex) as, or different (hetero-multimer complex) from, one another.
  • the cytokine is wild-type or mutant TNF- ⁇ .
  • the multimerization is through interaction between the extensive trimer interfaces of the TNF- ⁇ polypeptides contained in the immunoconjugate molecules.
  • the TAA is Trop-2 or FAP.
  • the present immunoconjugate comprises an anti-cytokine /anti-TAA two-in-one scFab antibody fused to a cytokine.
  • the immunoconjugate monomer can assume any one of the four possible configurations: (i) cytokine-scFab (VH-CH1-VL-CL) , (ii) cytokine-scFab (VL-CL-VH-CH1) , (iii) scFab (VH-CH1-VL-CL) -cytokine, and (iv) scFab (VL-CL-VH-CH1) -cytokine, where “-” denotes a linker or direct fusion between different moieties, and the different moieties in a peptidic chain are listed according to the N to C orientation in the peptidic chain.
  • the monomer immunoconjugate molecule can multimerize into a complex, such as a trimer.
  • multimerization is through interaction between the cytokine domains of two or more immunoconjugate molecules.
  • immunoconjugate molecules forming the multimer can each assume a configuration selected from (i) to (iv) of this paragraph, which configurations can be the same (homo-multimer complex) as, or different (hetero-multimer complex) from, one another.
  • the cytokine is wild-type or mutant TNF- ⁇ .
  • the multimerization is through interaction between the extensive trimer interfaces of the TNF- ⁇ polypeptides contained in the immunoconjugate molecules.
  • the TAA is Trop-2 or FAP.
  • the present immunoconjugate comprises (a) an anti-cytokine /anti-TAA two-in-one scFv antibody, (b) a cytokine polypeptide, and (c) an anti-TAA scFv antibody.
  • the immunoconjugate monomer can assume any one of the eight possible configurations: (i) cytokine-scFv (VH-VL) -scFv (VH-VL) , (ii) cytokine-scFv (VL-VH) -scFv (VH-VL) , (iii) cytokine-scFv (VH-VL) -scFv (VL-VH) , (iv) cytokine-scFv (VL-VH) -scFv (VL-VH) , (v) scFv (VH-VL) -scFv (VH-VL) -cytokine, (vi) scFv (VL-VH) -scFv (VH-VL) -cytokine, (vii) scFv (VH-VL) -scFv (VL) -s
  • the monomer immunoconjugate molecule can multimerize into a complex, such as a trimer.
  • multimerization is through interaction between the cytokine domains of two or more immunoconjugate molecules.
  • immunoconjugate molecules forming the multimer can each assume a configuration selected from (i) to (viii) of this paragraph, which configurations can be the same (homo-multimer complex) as, or different (hetero-multimer complex) from, one another.
  • the cytokine is wild-type or mutant TNF- ⁇ .
  • the multimerization is through interaction between the extensive trimer interfaces of the TNF- ⁇ polypeptides contained in the immunoconjugate molecules.
  • the TAA is Trop-2 or FAP.
  • the present immunoconjugate comprises (a) an anti-cytokine /anti-TAA two-in-one scFv antibody, (b) a cytokine polypeptide, and (c) an anti-TAA VHH antibody.
  • the immunoconjugate monomer can assume any one of the four possible configurations: (i) cytokine-scFv (VH-VL) -VHH, (ii) cytokine-scFv (VL-VH) -VHH, (iii) scFv (VH-VL) -VHH-cytokine, and (iv) scFv (VL-VH) -VHH-cytokine, where “-” denotes a linker or direct fusion between different moieties, and the different moieties in a peptidic chain are listed according to the N to C orientation in the peptidic chain.
  • the monomer immunoconjugate molecule can multimerize into a complex, such as a trimer.
  • multimerization is through interaction between the cytokine domains of two or more immunoconjugate molecules.
  • immunoconjugate molecules forming the multimer can each assume a configuration selected from (i) to (iv) of this paragraph, which configurations can be the same (homo-multimer complex) as, or different (hetero-multimer complex) from, one another.
  • the cytokine is wild-type or mutant TNF- ⁇ .
  • the multimerization is through interaction between the extensive trimer interfaces of the TNF- ⁇ polypeptides contained in the immunoconjugate molecules.
  • the TAA is Trop-2 or FAP.
  • the present immunoconjugate comprises (a) an anti-cytokine /anti-TAA two-in-one Fab antibody, (b) a cytokine polypeptide, and (c) an anti-TAA scFv antibody.
  • the immunoconjugate monomer can assume any one of the eight possible configurations: (i) cytokine-VH-CH1 x scFv (VH-VL) -VL-CL, (ii) cytokine-VL-CL x scFv (VH-VL) -VH-CH1, (iii) cytokine-VH-CH1 x scFv (VL-VH) -VL-CL, (iv) cytokine-VL-CL x scFv (VL-VH) -VH-CH1, (v) VH-CH1-cytokine x scFv (VH-VL) -VL-CL, (vi) VL-CL-cytokine x scFv (VH-VL) -VH-CH1, (vii) VH-CH1-cytokine x scFv (VL-VH) -V
  • the monomer immunoconjugate molecule can multimerize into a complex, such as a trimer.
  • multimerization is through interaction between the cytokine domains of two or more immunoconjugate molecules.
  • immunoconjugate molecules forming the multimer can each assume a configuration selected from (i) to (viii) of this paragraph, which configurations can be the same (homo-multimer complex) as, or different (hetero-multimer complex) from, one another.
  • the cytokine is wild-type or mutant TNF- ⁇ .
  • the multimerization is through interaction between the extensive trimer interfaces of the TNF- ⁇ polypeptides contained in the immunoconjugate molecules.
  • the TAA is Trop-2 or FAP.
  • the present immunoconjugate comprises (a) an anti-cytokine /anti-TAA two-in-one Fab antibody, (b) a cytokine polypeptide, and (c) an anti-TAA VHH antibody.
  • the immunoconjugate monomer can assume any one of the four possible configurations: (i) cytokine-VH-CH1 x VHH-VL-CL, (ii) cytokine-VL-CL x VHH-VH-CH1, (iii) VH-CH1-cytokine x VHH-VL-CL, and (iv) VL-CL-cytokine x VHH-VH-CH1, where “-” denotes a linker or direct fusion between different moieties, and the different moieties in a peptidic chain are listed according to the N to C orientation in the peptidic chain.
  • the monomer immunoconjugate molecule can multimerize into a complex, such as a trimer.
  • multimerization is through interaction between the cytokine domains of two or more immunoconjugate molecules.
  • immunoconjugate molecules forming the multimer can each assume a configuration selected from (i) to (iv) of this paragraph, which configurations can be the same (homo-multimer complex) as, or different (hetero-multimer complex) from, one another.
  • the cytokine is wild-type or mutant TNF- ⁇ .
  • the multimerization is through interaction between the extensive trimer interfaces of the TNF- ⁇ polypeptides contained in the immunoconjugate molecules.
  • the TAA is Trop-2 or FAP.
  • the present immunoconjugate comprises (a) an anti-cytokine /anti-TAA two-in-one Fab antibody, (b) a cytokine polypeptide, and (c) an anti-TAA scFv antibody.
  • the immunoconjugate monomer can assume any one of the eight possible configurations: (i) cytokine-VH-CH1 x VL-CL-scFv (VH-VL) , (ii) cytokine-VL-CL x VH-CH1-scFv (VH-VL) , (iii) cytokine-VH-CH1 x VL-CL- scFv (VL-VH) , (iv) cytokine-VL-CL x VH-CH1-scFv (VL-VH) , (v) VH-CH1-cytokine x VL-CL-scFv (VH-VL) , (vi) VL-CL-cytokine x VH-CH1-scFv (VH-VL) , (vii) VH-CH1-cytokine x VL-CL-scFv (VL-VH)
  • the monomer immunoconjugate molecule can multimerize into a complex, such as a trimer.
  • multimerization is through interaction between the cytokine domains of two or more immunoconjugate molecules.
  • immunoconjugate molecules forming the multimer can each assume a configuration selected from (i) to (viii) of this paragraph, which configurations can be the same (homo-multimer complex) as, or different (hetero-multimer complex) from, one another.
  • the cytokine is wild-type or mutant TNF- ⁇ .
  • the multimerization is through interaction between the extensive trimer interfaces of the TNF- ⁇ polypeptides contained in the immunoconjugate molecules.
  • the TAA is Trop-2 or FAP.
  • the present immunoconjugate comprises (a) an anti-cytokine /anti-TAA two-in-one Fab antibody, (b) a cytokine polypeptide, and (c) an anti-TAA scFv antibody.
  • the immunoconjugate monomer can assume any one of the eight possible configurations: (i) cytokine-VH-CH1-scFv (VH-VL) x VL-CL, (ii) cytokine-VL-CL-scFv (VH-VL) x VH-CH1, (iii) cytokine-VH-CH1-scFv (VL-VH) x VL-CL, (iv) cytokine-VL-CL-scFv (VL-VH) x VH-CH1, (v) VH-CH1-scFv (VH-VL) -cytokine x VL-CL, (vi) VL-CL-scFv (VH-VL) -cytokine x VH-CH1, (vii) VH-CH1-scFv (VL-VH) -cytokine x VL-CL, and (viii) VL
  • the monomer immunoconjugate molecule can multimerize into a complex, such as a trimer.
  • multimerization is through interaction between the cytokine domains of two or more immunoconjugate molecules.
  • immunoconjugate molecules forming the multimer can each assume a configuration selected from (i) to (viii) of this paragraph, which configurations can be the same (homo-multimer complex) as, or different (hetero-multimer complex) from, one another.
  • the cytokine is wild-type or mutant TNF- ⁇ .
  • the multimerization is through interaction between the extensive trimer interfaces of the TNF- ⁇ polypeptides contained in the immunoconjugate molecules.
  • the TAA is Trop-2 or FAP.
  • the present immunoconjugate comprises (a) an anti-cytokine /anti-TAA two-in-one Fab antibody, (b) a cytokine polypeptide, and (c) an anti-TAA VHH antibody.
  • the immunoconjugate monomer can assume any one of the four possible configurations: (i) cytokine-VH-CH1-VHH x VL-CL, (ii) cytokine-VL-CL-VHH x VH-CH1, (iii) VH-CH1-VHH-cytokine x VL-CL, and (iv) VL-CL-VHH-cytokine x VH-CH1, where “-” denotes a linker or direct fusion between different moieties, and the different moieties in a peptidic chain are listed according to the N to C orientation in the peptidic chain.
  • the monomer immunoconjugate molecule can multimerize into a complex, such as a trimer.
  • multimerization is through interaction between the cytokine domains of two or more immunoconjugate molecules.
  • immunoconjugate molecules forming the multimer can each assume a configuration selected from (i) to (iv) of this paragraph, which configurations can be the same (homo-multimer complex) as, or different (hetero-multimer complex) from, one another.
  • the cytokine is wild-type or mutant TNF- ⁇ .
  • the multimerization is through interaction between the extensive trimer interfaces of the TNF- ⁇ polypeptides contained in the immunoconjugate molecules.
  • the TAA is Trop-2 or FAP.
  • the present immunoconjugate comprises (a) an anti-cytokine /anti-TAA two-in-one Fab antibody, (b) a cytokine polypeptide, and (c) an anti-TAA VHH antibody.
  • the immunoconjugate monomer can assume any one of the four possible configurations: (i) cytokine-VH-CH1 x VL-CL-VHH, (i) cytokine-VL-CL x VH-CH1-VHH, (i) VH-CH1-cytokine x VL-CL-VHH, and (i) VL-CL-cytokine x VH-CH1-VHH, where “-” denotes a linker or direct fusion between different moieties, and the different moieties in a peptidic chain are listed according to the N to C orientation in the peptidic chain.
  • the monomer immunoconjugate molecule can multimerize into a complex, such as a trimer.
  • multimerization is through interaction between the cytokine domains of two or more immunoconjugate molecules.
  • immunoconjugate molecules forming the multimer can each assume a configuration selected from (i) to (iv) of this paragraph, which configurations can be the same (homo-multimer complex) as, or different (hetero-multimer complex) from, one another.
  • the cytokine is wild-type or mutant TNF- ⁇ .
  • the multimerization is through interaction between the extensive trimer interfaces of the TNF- ⁇ polypeptides contained in the immunoconjugate molecules.
  • the TAA is Trop-2 or FAP.
  • the present immunoconjugate molecule comprises an anchoring moiety, a masking moiety and a cytokine moiety that are operably linked to one another via a conjugating moiety.
  • the conjugating moiety comprises an immunoglobulin Fc domain composed of the Fc regions of both heavy chains of the immunoglobulin (each a subunit of the Fc domain) .
  • the Fc domain is the Fc domain of an IgG molecule (e.g., IgG1, IgG2, IgG3, or IgG4) .
  • the two subunits of the Fc domain can be both native sequence Fc regions. In some embodiments, the two subunits of the Fc domain can be both variant Fc regions. In some embodiments, the two subunits of the Fc domain can be one native sequence Fc region and one variant Fc region. In certain embodiments, the Fc domain comprises a modification promoting hetero-dimerization of two non-identical immunoglobulin heavy chains. The site of most extensive protein-protein interaction between the two polypeptidic chains of a human IgG Fc domain is in the CH3 domain of the Fc regions. Thus, in one embodiment, said modification is in the CH3 domain of the Fc regions.
  • said modification is a knob-into-hole modification, comprising a knob modification in one of the Fc subunits and a hole modification in the other one of the Fc subunits.
  • the knob-into-hole technology is described e.g., in U.S. Pat. No. 5,731,168; U.S. Pat. No. 7,695,936; Ridgway et al., Prat Eng 9, 617-621 (1996) and Carter, J Immunol Meth 248, 7-15 (2001) .
  • the method involves introducing a protuberance ( “knob” ) at the interface of a first polypeptide and a corresponding cavity ( “hole” ) in the interface of a second polypeptide, such that the protuberance can be positioned in the cavity so as to promote heterodimer formation and hinder homodimer formation.
  • Protuberances are constructed by replacing small amino acid side chains from the interface of the first polypeptide with larger side chains (e.g., tyrosine or tryptophan) .
  • Compensatory cavities of identical or similar size to the protuberances are created in the interface of the second polypeptide by replacing large amino acid side chains with smaller ones (e.g., alanine or threonine) .
  • the protuberance and cavity can be made by altering the nucleic acid encoding the polypeptides, e.g., by site-specific mutagenesis, or by peptide synthesis.
  • a knob modification comprises the amino acid substitution T366W in one of the two Fc subunits
  • the hole modification comprises the amino acid substitutions T366S, L368A and Y407V in the other one of the two Fc subunits.
  • the Fc subunit comprising the knob modification additionally comprises the amino acid substitution S354C
  • the immunoglobulin heavy chain comprising the hole modification additionally comprises the amino acid substitution Y349C. Introduction of these two cysteine residues results in formation of a disulfide bridge between the two heavy chains, further stabilizing the dimer (Carter, J. Immunol Methods 248, 7-15 (2001) ) .
  • a modification promoting heterodimerization of two non-identical polypeptidic chains comprises a modification mediating electrostatic steering effects, e.g., as described in PCT publication WO 2009/089004.
  • this method involves replacement of one or more amino acid residues at the interface of the two polypeptidic chains by charged amino acid residues so that homodimer formation becomes electro statically unfavorable but heterodimerization electrostatically favorable.
  • an Fc domain confers to the immunoconjugate molecule favorable pharmacokinetic properties, including a long serum half-life which contributes to good accumulation in the target tissue and a favorable tissue-blood distribution ratio.
  • an Fc domain may lead to undesirable targeting of the immunoconjugate molecules to cells expressing Fc receptors rather than to the target antigen-bearing cells.
  • the co-activation of Fc receptor signaling pathways may lead to cytokine release which, in combination with the cytokine polypeptide in the immunoconjugate molecule and the long half-life of the immunoconjugate, results in excessive activation of cytokine receptors and severe side effects upon systemic administration.
  • the modification to the Fc region of the antibody results in the decrease or elimination of an effector function of the antibody.
  • the effector function is ADCC, ADCP, and/or CDC. In some embodiments, the effector function is ADCC. In other embodiments, the effector function is ADCP. In other embodiments, the effector function is CDC. In one embodiment, the effector function is ADCC and ADCP. In one embodiment, the effector function is ADCC and CDC. In one embodiment, the effector function is ADCP and CDC. In one embodiment, the effector function is ADCC, ADCP and CDC. This may be achieved by introducing one or more amino acid substitutions in an Fc region of the antibody.
  • a salvage receptor binding epitope refers to an epitope of the Fc region of an IgG molecule (e.g., IgG1, IgG2, IgG3, or IgG4) that is responsible for increasing the in vivo serum half-life of the IgG molecule.
  • the Fc domain forming part of the immunoconjugate molecule according to the present disclosure is engineered to have reduced binding affinity to an Fc receptor.
  • the Fc domain comprises one or more amino acid mutation that reduces the binding affinity of the Fc domain to an Fc receptor.
  • the one or more such amino acid mutations are present in one of the two Fc subunits of the Fc domain.
  • the one or more such amino acid mutations are present in both of the two Fc subunits of the Fc domain.
  • such amino acid mutations reduce the binding affinity of the immunoconjugate to the Fc receptor by at least 2-fold, at least 5-fold, or at least 10-fold.
  • the combination of these amino acid mutations can reduce the binding affinity of the Fc domain to the Fc receptor by at least 10-fold, at least 20-fold, or even at least 50-fold.
  • the immunoconjugate comprising an engineered immunoglobulin molecule exhibits less than 20%, particularly less than 10%, more particularly less than 5%of the binding affinity to an Fc receptor as compared to an immunoconjugate comprising a non-engineered immunoglobulin molecule.
  • the Fc receptor is an activating Fc receptor.
  • the Fc receptor is an Fc ⁇ receptor.
  • the Fc receptor is an Fc ⁇ RIII ⁇ , Fc ⁇ RI or Fc ⁇ RII ⁇ receptor.
  • binding of the Fc domain to each of these exemplary receptors is reduced.
  • binding affinity of the Fc domain to a complement component is reduced.
  • binding affinity of the Fc domain to C1q is reduced.
  • binding affinity to neonatal Fc receptor (FcRn) is not reduced.
  • Substantially similar binding to FcRn i.e., preservation of the binding affinity of the Fc domain to said receptor, is achieved when the immunoconjugate comprising said Fc domain exhibits greater than about 70%of the binding affinity of a non-engineered form of the immunoconjugate molecule comprising said non-engineered form of the Fc to FcRn.
  • Immunoglobulins, or immunoconjugates comprising said immunoglobulins may exhibit greater than about 80%and even greater than about 90%of such affinity.
  • the Fc domain forming part of the present immunoconjugate molecule is not a native sequence Fc domain and has at least one amino acid mutation in one of its Fc subunits. In some embodiments, the Fc domain forming part of the present immunoconjugate molecule is not a native sequence Fc domain and has at least one amino acid mutation in both of its Fc subunits. In some embodiments, the amino acid mutations in both Fc subunits of an Fc domain are the same mutations. In some embodiments, the amino acid mutations in the two Fc subunits of an Fc domain are different mutations. In some embodiments, the amino acid mutation is selected from amino acid substitution, amino acid deletion and amino acid insertion.
  • one or both of the Fc subunits in the Fc domain of the immunoconjugate molecule comprise one or more amino acid mutations at any one or more amino acid positions 228, 233, 234, 235, 236, 265, 297, 329, 330, and 331 of the Fc subunit, where the number of the residues in the Fc subunit is that of the EU index as in Kabat.
  • such one or more amino acid substitutions comprise S228P.
  • such one or more amino acid substitutions comprise E233P.
  • such one or more amino acid substitutions comprise L234V or L234A.
  • such one or more amino acid substitutions comprise L235A or L235E.
  • such one or more amino acid deletion comprises ⁇ G236.
  • such one or more amino acid substitutions comprise D265G.
  • such one or more amino acid substitutions comprise N297A or N297D.
  • such one or more amino acid substitutions comprise P329E, P329A or P329G, particularly P329E.
  • such one or more amino acid substitutions comprise A330S.
  • such one or more amino acid substitutions comprise P331S.
  • the Fc domain comprises amino acid mutations at positions E233, L234, L235, G236, A330, and P331.
  • both of the two Fc subunits comprises amino acid mutations at positions E233, L234, L235, G236, A330, and P331.
  • the Fc domain comprises amino acid mutations of E233P, L234V, L235A, ⁇ G236, A330S, and P331S.
  • both of the two Fc subunits comprises amino acid mutations of E233P, L234V, L235A, ⁇ G236, P329S, A330S, and P331S.
  • the Fc domain comprises amino acid mutations at positions L234, L235, A330, and P331. In specific embodiments, both of the two Fc subunits comprise amino acid mutations at positions L234, L235, A330, and P331. In particular embodiments, the Fc domain comprises amino acid mutations of L234A, L235A, A330S, and P331S. In specific embodiments, both of the two Fc subunits comprise amino acid mutations of L234A, L235A, A330S, and P331S.
  • the Fc domain comprises amino acid mutations at positions E233, L234, L235, G236, P329, A330, and P331.
  • both of the two Fc subunits comprise amino acid mutations at positions E233, L234, L235, G236, P329, A330, and P331.
  • the Fc domain comprises amino acid mutations of E233P, L234V, L235A, ⁇ G236, P329E, A330S, and P331S.
  • both of the two Fc subunits comprise amino acid mutations of E233P, L234V, L235A, ⁇ G236, P329E, A330S, and P331S.
  • the Fc domain comprises amino acid mutations at positions L234, L235, P329, A330, and P331. In specific embodiments, both of the two Fc subunits comprise amino acid mutations at positions L234, L235, P329, A330, and P331. In particular embodiments, the Fc domain comprises amino acid mutations of L234A, L235A, P329E, A330S, and P331S. In specific embodiments, both of the two Fc subunits comprise amino acid mutations of L234A, L235A, P329E, A330S, and P331S.
  • the Fc domain comprises amino acid mutations at positions E233, L234, L235, G236, and P329. In specific embodiments, both of the two Fc subunits comprise amino acid mutations at positions E233, L234, L235, G236, and P329. In particular embodiments, the Fc domain comprises amino acid mutations of E233P, L234V, L235A, ⁇ G236, and P329E. In specific embodiments, both of the two Fc subunits comprise amino acid mutations of E233P, L234V, L235A, ⁇ G236, and P329E.
  • the Fc domain comprises amino acid mutations at positions L234, L235, P329. In specific embodiments, both of the two Fc subunits comprise amino acid mutations at positions L234, L235, P329. In particular embodiments, the Fc domain comprises amino acid mutations of L234A, L235A, and P329E. In specific embodiments, both of the two Fc subunits comprise amino acid mutations of L234A, L235A, and P329E.
  • the Fc domain comprises amino acid mutations at positions E233, L234, L235, G236, D265, A330, and P331.
  • both of the two Fc subunits comprise amino acid mutations at positions E233, L234, L235, G236, D265, A330, and P331.
  • the Fc domain comprises amino acid mutations of E233P, L234V, L235A, ⁇ G236, D265G, A330S, and P331S.
  • both of the two Fc subunits comprise amino acid mutations of E233P, L234V, L235A, ⁇ G236, D265G, A330S, and P331S.
  • the Fc domain has reduced binding affinity to the Fc ⁇ receptor.
  • the Fc domain comprises amino acid mutations at positions L234, L235, D265, A330, and P331. In specific embodiments, both of the two Fc subunits comprise amino acid mutations at positions L234, L235, D265, A330, and P331. In particular embodiments, the Fc domain comprises amino acid mutations of L234A, L235A, D265G, A330S, and P331S. In specific embodiments, both of the two Fc subunits comprise amino acid mutations of L234A, L235A, D265G, A330S, and P331S.
  • the Fc domain comprises amino acid mutations at positions E233, L234, L235, G236, D265, P329, A330, and P331.
  • both of the two Fc subunits comprise amino acid mutations at positions E233, L234, L235, G236, D265, P329, A330, and P331.
  • the Fc domain comprises amino acid mutations of E233P, L234V, L235A, ⁇ G236, D265G, P329E, A330S, and P331S.
  • both of the two Fc subunits comprise amino acid mutations of E233P, L234V, L235A, ⁇ G236, D265G, P329E, A330S, and P331S.
  • the Fc domain comprises amino acid mutations at positions L234, L235, D265, P329, A330, and P331. In specific embodiments, both of the two Fc subunits comprise amino acid mutations at positions L234, L235, D265, P329, A330, and P331. In particular embodiments, the Fc domain comprises amino acid mutations of L234A, L235A, D265G, P329E, A330S, and P331S. In specific embodiments, both of the two Fc subunits comprise amino acid mutations of L234A, L235A, D265G, P329E, A330S, and P331S.
  • the Fc domain comprises amino acid mutations at positions E233, L234, L235, G236, D265, and P329.
  • both of the two Fc subunits comprise amino acid mutations at positions E233, L234, L235, G236, D265, and P329.
  • the Fc domain comprises amino acid mutations of E233P, L234V, L235A, ⁇ G236, D265G, and P329E.
  • both of the two Fc subunits comprise amino acid mutations of E233P, L234V, L235A, ⁇ G236, D265G, and P329E.
  • the Fc domain comprises amino acid mutations at positions L234, L235, D265, and P329. In specific embodiments, both of the two Fc subunits comprise amino acid mutations at positions L234, L235, D265, and P329. In particular embodiments, the Fc domain comprises amino acid mutations of L234A, L235A, D265G, and P329E. In specific embodiments, both of the two Fc subunits comprise amino acid mutations of L234A, L235A, D265G, and P329E.
  • the Fc domain comprises amino acid mutations at positions L234, L235, and P329. In specific embodiments, both of the two Fc subunits comprise amino acid mutations at positions L234, L235, and P329. In particular embodiments, the Fc domain comprises amino acid mutations of L234A, L235A, and P329G. In specific embodiments, both of the two Fc subunits comprise amino acid mutations of L234A, L235A, and P329G.
  • the present immunoconjugate molecule comprises an anchoring moiety, a masking moiety and a cytokine moiety that are operably linked to one another via a conjugating moiety.
  • the cytokine moiety comprises a cytokine polypeptide.
  • the masking moiety comprises a bispecific two-in-one antibody or antigen binding fragment capable of binding to the cytokine polypeptide and a second target antigen.
  • the anchoring moiety comprises an antibody or antigen binding fragment thereof capable of binding to a third target antigen.
  • the conjugating moiety comprises an immunoglobulin Fc domain composed of two Fc regions of immunoglobulin heavy chains (each Fc region is referred to as a subunit of the Fc domain or “Fc subunit” ) .
  • the Fc domain comprises a modification promoting hetero-dimerization of the two Fc subunits.
  • said modification is a knob-into-hole modification, comprising a knob modification in one of the Fc subunits (Fc-knob) and a hole modification in the other one of the Fc subunits (Fc-hole) .
  • the cytokine moiety, the masking moiety, and the anchoring moiety of the immunoconjugate molecule can be operably linked to one another via the conjugating moiety in a variety of different configurations.
  • FIGS. 2B to 2U are schematic illustrations of antibody-cytokine immunoconjugates of different molecular configurations according to the present disclosure.
  • the cytokine moiety comprises a cytokine polypeptide that is fused to the C-terminus of one Fc subunit.
  • the masking moiety comprises an antibody or antigen binding fragment thereof that is fused to the C-terminus of one Fc subunit.
  • the cytokine moiety comprises a cytokine polypeptide that is fused to the C-terminus of one subunit of the Fc domain, and the masking moiety comprises an antibody or antigen binding fragment thereof that is fused to the C-terminus of the other Fc subunit.
  • the masking moiety is fused to the C-terminus of the Fc subunit.
  • the Fc domain comprises a modification promoting hetero-dimerization of the two Fc subunits.
  • said modification is a knob-into-hole modification, comprising a knob modification in one of the Fc subunits (Fc-knob subunit) and a hole modification in the other one of the Fc subunits (Fc-hole subunit) .
  • the cytokine moiety comprises a cytokine polypeptide that is fused to the C-terminus of one Fc subunit.
  • the masking moiety comprises a bispecific two-in-one antibody or antigen binding fragment thereof that is fused to the C-terminus of one Fc subunit.
  • the anchoring moiety comprises an antibody or antigen binding fragment thereof that is fused to the N-terminus of one Fc subunit.
  • the cytokine moiety comprises a cytokine polypeptide that is fused to the C-terminus of one subunit of the Fc domain, and the masking moiety comprises a bispecific two-in-one antibody or antigen binding fragment thereof that is fused to the C-terminus of the other Fc subunit.
  • the cytokine moiety comprises a cytokine polypeptide that is fused to the C-terminus of one subunit of the Fc domain
  • the masking moiety comprises a bispecific two-in-one antibody or antigen binding fragment thereof that is fused to the C-terminus of the other Fc subunit
  • the anchoring moiety comprises an antibody or antigen binding fragment thereof that is fused to the N-terminus of one subunit of the Fc domain.
  • the anchoring moiety and the cytokine moiety are fused to the N-and C-terminus of the same Fc subunit, respectively.
  • the masking moiety and the cytokine moiety are fused to the N-and C-terminus of the same Fc subunit, respectively.
  • the Fc domain comprises a modification promoting hetero-dimerization of the two Fc subunits.
  • said modification is a knob-into-hole modification, comprising a knob modification in one of the Fc subunits (Fc-knob subunit) and a hole modification in the other one of the Fc subunits (Fc-hole subunit) .
  • the masking moiety comprises a bispecific two-in-one antibody or antigen binding fragment thereof that is fused to the N-terminus of one Fc subunit.
  • the cytokine moiety comprises a cytokine polypeptide that is fused to the masking moiety.
  • the masking moiety comprises a bispecific two-in-one antibody or antigen binding fragment thereof that is fused to the N-terminus of one Fc subunit, and the cytokine moiety comprises a cytokine polypeptide that is fused to the masking moiety.
  • the anchoring moiety comprises an antibody or antigen binding fragment thereof that is fused to the N-terminus of one Fc subunit.
  • the cytokine moiety comprises a cytokine polypeptide that is fused to the anchoring moiety.
  • the masking moiety comprises a bispecific two-in-one antibody or antigen binding fragment thereof that is fused to the N-terminus of one Fc subunit
  • the anchoring moiety comprises an antibody or antigen binding fragment thereof that is fused to the N-terminus of the other Fc subunit
  • the cytokine moiety comprises a cytokine polypeptide that is fused to the masking moiety.
  • the masking moiety comprises a bispecific two-in-one antibody or antigen binding fragment thereof that is fused to the N-terminus of one Fc subunit
  • the anchoring moiety comprises an antibody or antigen binding fragment thereof that is fused to the N-terminus of the other Fc subunit
  • the cytokine moiety comprises a cytokine polypeptide that is fused to the anchoring moiety.
  • the Fc domain comprises a modification promoting hetero-dimerization of the two Fc subunits.
  • said modification is a knob-into-hole modification, comprising a knob modification in one of the Fc subunits (Fc-knob subunit) and a hole modification in the other one of the Fc subunits (Fc-hole subunit) .
  • the masking moiety comprises a bispecific two-in-one antibody or an antigen binding fragment thereof that is fused to the C-terminus of one Fc subunit.
  • the cytokine moiety comprises a cytokine polypeptide that is fused to the masking moiety.
  • the masking moiety comprises a bispecific two-in-one antibody or an antigen binding fragment thereof that is fused to the C-terminus of one Fc subunit, and the cytokine moiety comprises a cytokine polypeptide that is fused to the masking moiety.
  • the anchoring moiety comprising an antibody or antigen binding fragment thereof that is fused to the N terminus of one Fc subunit.
  • the masking moiety comprises a bispecific two-in-one antibody or an antigen binding fragment thereof that is fused to the C-terminus of one Fc subunit
  • the anchoring moiety comprises an antibody or antigen binding fragment thereof that is fused to the N-terminus of the other Fc subunit
  • the cytokine moiety comprises a cytokine polypeptide fused to the masking moiety.
  • the masking moiety and the anchoring moiety bind to the same Fc subunit.
  • the masking moiety and the anchoring moiety bind to different Fc subunits.
  • the Fc domain comprises a modification promoting hetero-dimerization of the two Fc subunits.
  • said modification is a knob-into-hole modification, comprising a knob modification in one of the Fc subunits (Fc-knob subunit) and a hole modification in the other one of the Fc subunits (Fc-hole subunit) .
  • the masking moiety comprises a bispecific two-in-one antibody or an antigen binding fragment thereof that is fused to the C-terminus of one Fc subunit.
  • the cytokine moiety comprises a cytokine polypeptide that is fused to the C-terminus of one Fc subunit.
  • the anchoring moiety comprises an antibody or antigen binding fragment thereof that is fused to the masking moiety.
  • the Fc domain comprises a modification promoting hetero-dimerization of the two Fc subunits.
  • said modification is a knob-into-hole modification, comprising a knob modification in one of the Fc subunits (Fc-knob subunit) and a hole modification in the other one of the Fc subunits (Fc-hole subunit) .
  • the masking moiety comprises a bispecific two-in-one antibody or an antigen binding fragment thereof that is fused to the N-terminus of one Fc subunit.
  • the cytokine moiety comprises a cytokine polypeptide that is fused to the N-terminus of one Fc subunit.
  • the anchoring moiety comprises an antibody or antigen binding fragment thereof that is fused to the masking moiety.
  • the Fc domain comprises a modification promoting hetero-dimerization of the two Fc subunits.
  • said modification is a knob-into-hole modification, comprising a knob modification in one of the Fc subunits (Fc-knob subunit) and a hole modification in the other one of the Fc subunits (Fc-hole subunit) .
  • the different moieties of the immunoconjugate molecule can be connected with a peptidic linker sequence.
  • the peptidic linker has at least 5 amino acid residues. In some embodiments, the peptidic linker has at least 7 amino acid residues. In some embodiments, the peptidic linker has at least 10 amino acid residues. In some embodiments, the peptidic linker has at least 15 amino acid residues. In some embodiments, the peptidic linker has at least 18 amino acid residues. In some embodiments, the peptidic linker has at least 20 amino acid residues. In some embodiments, the peptidic linker has at least 30 amino acid residues. In some embodiments, the peptidic linker has at least 50 amino acid residues. In some embodiments, the peptidic linker has at least 70 amino acid residues. In some embodiments, the linker is selected from the sequences of Table 12.
  • an antibody forming part of the immunoconjugate molecule can be synthetic antibodies, recombinantly produced antibodies, camelized antibodies, intrabodies, anti-idiotypic (anti-Id) antibodies.
  • an antibody forming part of the immunoconjugate molecule is a monoclonal antibody.
  • an antigen binding fragment forming part of the immunoconjugate molecule can be functional fragments of an antibody that retains some or all of the binding activity of the antibody from which the fragment was derived.
  • Non-limiting examples of functional fragments include single-chain Fvs (scFv) (e.g., including monospecific, bispecific, etc. ) , Fab fragments (e.g., including monospecific, bispecific, etc. ) , F (ab’ ) fragments, F (ab) 2 fragments, F (ab’ ) 2 fragments, disulfide-linked Fvs (dsFv) , Fd fragments, Fv fragments, diabody, triabody, tetrabody, minibody, and single domain antibody (VHH or nanobody) .
  • the immunoconjugate molecule can have any of the configurations as shown in FIGS. 1 and 2.
  • the bispecific two-in-one antibody in the masking moiety of the present immunoconjugate molecule is a Fab fragment.
  • the bispecific two-in-one antibody in the masking moiety of the present immunoconjugate molecule is a ScFv fragment.
  • the bispecific two-in-one antibody in the masking moiety of the present immunoconjugate molecule is a single domain (VHH) antibody.
  • the antibody in the anchoring moiety of the present immunoconjugate molecule is a Fab fragment.
  • the antibody in the anchoring moiety of the present immunoconjugate molecule is a ScFv fragment.
  • the antibody in the anchoring moiety of the present immunoconjugate molecule is a single domain (VHH) antibody.
  • the bispecific two-in-one antibody in the masking moiety of the present immunoconjugate molecule is a Fab fragment
  • the antibody in the anchoring moiety of the immunoconjugate molecule is also a Fab fragment
  • the bispecific two-in-one antibody in the masking moiety of the present immunoconjugate molecule is a Fab fragment
  • the antibody in the anchoring moiety of the immunoconjugate molecule is a ScFv fragment.
  • the bispecific two-in-one antibody in the masking moiety of the present immunoconjugate molecule is a Fab fragment
  • the antibody in the anchoring moiety of the immunoconjugate molecule is a single domain (VHH) fragment.
  • the bispecific two-in-one antibody in the masking moiety of the present immunoconjugate molecule is a ScFv fragment
  • the antibody in the anchoring moiety of the immunoconjugate molecule is a Fab fragment
  • the bispecific two-in-one antibody in the masking moiety of the present immunoconjugate molecule is a ScFv fragment
  • the antibody in the anchoring moiety of the immunoconjugate molecule is also ScFv fragment.
  • the bispecific two-in-one antibody in the masking moiety of the present immunoconjugate molecule is a ScFv fragment
  • the antibody in the anchoring moiety of the immunoconjugate molecule is a single domain (VHH) fragment.
  • the bispecific two-in-one antibody in the masking moiety of the present immunoconjugate molecule is a single domain (VHH) antibody
  • the antibody in the anchoring moiety of the immunoconjugate molecule is a Fab fragment
  • the bispecific two-in-one antibody in the masking moiety of the present immunoconjugate molecule is a single domain (VHH) antibody
  • the antibody in the anchoring moiety of the immunoconjugate molecule is ScFv fragment.
  • the bispecific two-in-one antibody in the masking moiety of the present immunoconjugate molecule is a single domain (VHH) antibody
  • the antibody in the anchoring moiety of the immunoconjugate molecule is also a single domain (VHH) fragment.
  • the present disclosure provides two-in-one antibodies that can find use herein for generating the present immunoconjugate molecules, e.g., as a masking moiety.
  • Exemplary antibodies include polyclonal, monoclonal, humanized, human, bispecific, and heteroconjugate antibodies, as well as variants thereof having improved affinity or other properties.
  • an anti-TNF- ⁇ /anti-FAP two-in-one antibody or antigen binding fragment thereof comprises a VH region, VL region, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and/or VL CDR3 of any one of the two-in-one antibodies provided herein, such as an amino acid sequence depicted in Tables 13-14.
  • the two-in-one antibody or functional fragment thereof provided herein comprises one, two, and/or three heavy chain CDRs and/or one, two, and/or three light chain CDRs from the antibody FN15 as shown in Tables 13-14.
  • a two-in-one antibody provided herein comprises or consists of six CDRs, for example, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and/or VL CDR3 identified in Tables 13-14.
  • the two-in-one antibody comprises or consists of one, two, three, four, or five CDRs selected from the group consisting of VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and/or VL CDR3 identified in Tables 13-14.
  • the two-in-one antibody comprises or consists of one, two, three, four, or five CDRs of anyone of the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and/or VL CDR3 identified in Tables 13-14.
  • the two-in-one antibodies provided herein comprise one or more (e.g., one, two, or three) VL CDRs listed in Table 13. In some embodiments, the two-in-one antibodies provided herein comprise one or more (e.g., one, two, or three) VH CDRs listed in Table 14. In yet other embodiments, the two-in-one antibodies provided herein comprise one or more (e.g., one, two, or three) VL CDRs listed in Table 13 and one or more (e.g., one, two, or three) VH CDRs listed in Table 14.
  • the two-in-one antibodies comprise a VL CDR1 having an amino acid sequence of any one of SEQ ID NO: 15.
  • the two-in-one antibodies comprise a VL CDR2 having an amino acid sequence of SEQ ID NO: 16.
  • the two-in-one antibodies comprise a VL CDR3 having an amino acid sequence of SEQ ID NO: 17.
  • the two-in-one antibodies comprise a VL CDR3 having an amino acid sequence of SEQ ID NO: 68.
  • the two-in-one antibodies comprise a VL CDR1 and/or a VL CDR2 and/or a VL CDR3 independently selected from any one of the VL CDR1, VL CDR2, VL CDR3 amino acid sequences as depicted in Table 13.
  • the two-in-one antibodies comprise a VH CDR1 having an amino acid sequence of SEQ ID NO: 18. In some embodiments, the two-in-one antibodies comprise a VH CDR2 having an amino acid sequence of SEQ ID NO: 19. In some embodiments, the two-in-one antibodies comprise a VH CDR3 having an amino acid sequence of SEQ ID NO: 20. In some embodiments, the two-in-one antibodies comprise a VH CDR3 having an amino acid sequence of SEQ ID NO: 69. In some embodiments, the two-in-one antibodies comprise a VH CDR3 having an amino acid sequence of SEQ ID NO: 70.
  • the two-in-one antibodies comprise a VH CDR3 having an amino acid sequence of SEQ ID NO: 71. In some embodiments, the two-in-one antibodies comprise a VH CDR3 having an amino acid sequence of SEQ ID NO: 72. In some embodiments, the two-in-one antibodies comprise a VH CDR3 having an amino acid sequence of SEQ ID NO: 73. In some embodiments, the two-in-one antibodies comprise a VH CDR1 and/or a VH CDR2 and/or a VH CDR3 independently selected from any one of the VH CDR1, VH CDR2, VH CDR3 amino acid sequence (s) as depicted in Table 14.
  • two-in-one antibodies comprising one or more (e.g., one, two, or three) VH CDRs and one or more (e.g., one, two, or three) VL CDRs listed in Tables 13-14.
  • a two-in-one antibody comprising a VH CDR1 (SEQ ID NO: 18) and a VL CDR1 (SEQ ID NO: 15) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 19) and a VL CDR1 (SEQ ID NO: 15) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 19) and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 19) and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR3 (SEQ ID NO: 20) and a VL CDR1 (SEQ ID NO: 15) .
  • the two-in-one antibody comprises a VH CDR3 (SEQ ID NO: 20) and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR3 (SEQ ID NO: 20) and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR2 (SEQ ID NO: 19) , and a VL CDR1 (SEQ ID NO: 15) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR2 (SEQ ID NO: 19) , and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR2 (SEQ ID NO: 19) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 19) , a VH CDR3 (SEQ ID NO: 20) , and a VL CDR1 (SEQ ID NO: 15) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 19) , a VH CDR3 (SEQ ID NO: 20) , and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 19) , a VH CDR3 (SEQ ID NO: 20) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR3 (SEQ ID NO: 20) , and a VL CDR1 (SEQ ID NO: 15) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR3 (SEQ ID NO: 20) , and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR3 (SEQ ID NO: 20) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VL CDR1 (SEQ ID NO: 15) , and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VL CDR1 (SEQ ID NO: 15) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VL CDR2 (SEQ ID NO: 16) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 19) , a VL CDR1 (SEQ ID NO: 15) , and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 19) , a VL CDR1 (SEQ ID NO: 15) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 19) , a VL CDR2 (SEQ ID NO: 16) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR3 (SEQ ID NO: 20) , a VL CDR1 (SEQ ID NO: 15) , and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR3 (SEQ ID NO: 20) , a VL CDR1 (SEQ ID NO: 15) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR3 (SEQ ID NO: 20) , a VL CDR2 (SEQ ID NO: 16) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR2 (SEQ ID NO: 19) , a VH CDR3 (SEQ ID NO: 20) , and a VL CDR1 (SEQ ID NO: 15) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR2 (SEQ ID NO: 19) , a VH CDR3 (SEQ ID NO: 20) , and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR2 (SEQ ID NO: 19) , a VH CDR3 (SEQ ID NO: 20) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR2 (SEQ ID NO: 19) , a VL CDR1 (SEQ ID NO: 15) , and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR2 (SEQ ID NO: 19) , a VL CDR1 (SEQ ID NO: 15) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR2 (SEQ ID NO: 19) , a VL CDR2 (SEQ ID NO: 16) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR3 (SEQ ID NO: 20) , a VL CDR1 (SEQ ID NO: 15) , and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR3 (SEQ ID NO: 20) , a VL CDR1 (SEQ ID NO: 15) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR3 (SEQ ID NO: 20) , a VL CDR2 (SEQ ID NO: 16) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 19) , a VH CDR3 (SEQ ID NO: 20) , a VL CDR1 (SEQ ID NO: 15) , and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 19) , a VH CDR3 (SEQ ID NO: 20) , a VL CDR1 (SEQ ID NO: 15) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 19) , a VH CDR3 (SEQ ID NO: 20) , a VL CDR2 (SEQ ID NO: 16) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR2 (SEQ ID NO: 19) , a VH CDR3 (SEQ ID NO: 20) , a VL CDR1 (SEQ ID NO: 15) , and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR2 (SEQ ID NO: 19) , a VH CDR3 (SEQ ID NO: 20) , a VL CDR1 (SEQ ID NO: 15) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR2 (SEQ ID NO: 19) , a VH CDR3 (SEQ ID NO: 20) , a VL CDR2 (SEQ ID NO: 16) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR2 (SEQ ID NO: 19) , a VL CDR1 (SEQ ID NO: 15) , a VL CDR2 (SEQ ID NO: 16) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR3 (SEQ ID NO: 20) , a VL CDR1 (SEQ ID NO: 15) , a VL CDR2 (SEQ ID NO: 16) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 19) , a VH CDR3 (SEQ ID NO: 20) , a VL CDR1 (SEQ ID NO: 15) , a VL CDR2 (SEQ ID NO: 16) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VL CDR1 (SEQ ID NO: 15) , a VL CDR2 (SEQ ID NO: 16) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 19) , a VL CDR1 (SEQ ID NO: 15) , a VL CDR2 (SEQ ID NO: 16) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR3 (SEQ ID NO: 20) , a VL CDR1 (SEQ ID NO: 15) , a VL CDR2 (SEQ ID NO: 16) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises any combination thereof of the VH CDRs and VL CDRs listed in Tables 13-14.
  • two-in-one antibodies comprising one or more (e.g., one, two, or three) VH CDRs and one or more (e.g., one, two, or three) VL CDRs listed in Tables 13-14.
  • a two-in-one antibody comprising a VH CDR1 (SEQ ID NO: 18) and a VL CDR1 (SEQ ID NO: 68) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 19) and a VL CDR1 (SEQ ID NO: 68) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 19) and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 19) and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR3 (SEQ ID NO: 20) and a VL CDR1 (SEQ ID NO: 68) . In other embodiments, the two-in-one antibody comprises a VH CDR3 (SEQ ID NO: 20) and a VL CDR2 (SEQ ID NO: 16) . In some embodiments, the two-in-one antibody comprises a VH CDR3 (SEQ ID NO: 20) and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR2 (SEQ ID NO: 19) , and a VL CDR1 (SEQ ID NO: 68) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR2 (SEQ ID NO: 19) , and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR2 (SEQ ID NO: 19) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 19) , a VH CDR3 (SEQ ID NO: 20) , and a VL CDR1 (SEQ ID NO: 68) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 19) , a VH CDR3 (SEQ ID NO: 20) , and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 19) , a VH CDR3 (SEQ ID NO: 20) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR3 (SEQ ID NO: 20) , and a VL CDR1 (SEQ ID NO: 68) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR3 (SEQ ID NO: 20) , and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR3 (SEQ ID NO: 20) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VL CDR1 (SEQ ID NO: 68) , and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VL CDR1 (SEQ ID NO: 68) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VL CDR2 (SEQ ID NO: 16) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 19) , a VL CDR1 (SEQ ID NO: 68) , and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 19) , a VL CDR1 (SEQ ID NO: 68) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 19) , a VL CDR2 (SEQ ID NO: 16) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR3 (SEQ ID NO: 20) , a VL CDR1 (SEQ ID NO: 68) , and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR3 (SEQ ID NO: 20) , a VL CDR1 (SEQ ID NO: 68) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR3 (SEQ ID NO: 20) , a VL CDR2 (SEQ ID NO: 16) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR2 (SEQ ID NO: 19) , a VH CDR3 (SEQ ID NO: 20) , and a VL CDR1 (SEQ ID NO: 68) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR2 (SEQ ID NO: 19) , a VH CDR3 (SEQ ID NO: 20) , and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR2 (SEQ ID NO: 19) , a VH CDR3 (SEQ ID NO: 20) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR2 (SEQ ID NO: 19) , a VL CDR1 (SEQ ID NO: 68) , and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR2 (SEQ ID NO: 19) , a VL CDR1 (SEQ ID NO: 68) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR2 (SEQ ID NO: 19) , a VL CDR2 (SEQ ID NO: 16) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR3 (SEQ ID NO: 20) , a VL CDR1 (SEQ ID NO: 68) , and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR3 (SEQ ID NO: 20) , a VL CDR1 (SEQ ID NO: 68) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR3 (SEQ ID NO: 20) , a VL CDR2 (SEQ ID NO: 16) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 19) , a VH CDR3 (SEQ ID NO: 20) , a VL CDR1 (SEQ ID NO: 68) , and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 19) , a VH CDR3 (SEQ ID NO: 20) , a VL CDR1 (SEQ ID NO: 68) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 19) , a VH CDR3 (SEQ ID NO: 20) , a VL CDR2 (SEQ ID NO: 16) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR2 (SEQ ID NO: 19) , a VH CDR3 (SEQ ID NO: 20) , a VL CDR1 (SEQ ID NO: 68) , and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR2 (SEQ ID NO: 19) , a VH CDR3 (SEQ ID NO: 20) , a VL CDR1 (SEQ ID NO: 68) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR2 (SEQ ID NO: 19) , a VH CDR3 (SEQ ID NO: 20) , a VL CDR2 (SEQ ID NO: 16) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR2 (SEQ ID NO: 19) , a VL CDR1 (SEQ ID NO: 68) , a VL CDR2 (SEQ ID NO: 16) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR3 (SEQ ID NO: 20) , a VL CDR1 (SEQ ID NO: 68) , a VL CDR2 (SEQ ID NO: 16) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 19) , a VH CDR3 (SEQ ID NO: 20) , a VL CDR1 (SEQ ID NO: 68) , a VL CDR2 (SEQ ID NO: 16) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VL CDR1 (SEQ ID NO: 68) , a VL CDR2 (SEQ ID NO: 16) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 19) , a VL CDR1 (SEQ ID NO: 68) , a VL CDR2 (SEQ ID NO: 16) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR3 (SEQ ID NO: 20) , a VL CDR1 (SEQ ID NO: 68) , a VL CDR2 (SEQ ID NO: 16) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises any combination thereof of the VH CDRs and VL CDRs listed in Tables 13-14.
  • two-in-one antibodies comprising one or more (e.g., one, two, or three) VH CDRs and one or more (e.g., one, two, or three) VL CDRs listed in Tables 13-14.
  • a two-in-one antibody comprising a VH CDR1 (SEQ ID NO: 18) and a VL CDR1 (SEQ ID NO: 15) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 69) and a VL CDR1 (SEQ ID NO: 15) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 69) and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 69) and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR3 (SEQ ID NO: 20) and a VL CDR1 (SEQ ID NO: 15) .
  • the two-in-one antibody comprises a VH CDR3 (SEQ ID NO: 20) and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR3 (SEQ ID NO: 20) and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR2 (SEQ ID NO: 69) , and a VL CDR1 (SEQ ID NO: 15) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR2 (SEQ ID NO: 69) , and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR2 (SEQ ID NO: 69) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 69) , a VH CDR3 (SEQ ID NO: 20) , and a VL CDR1 (SEQ ID NO: 15) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 69) , a VH CDR3 (SEQ ID NO: 20) , and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 69) , a VH CDR3 (SEQ ID NO: 20) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR3 (SEQ ID NO: 20) , and a VL CDR1 (SEQ ID NO: 15) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR3 (SEQ ID NO: 20) , and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR3 (SEQ ID NO: 20) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VL CDR1 (SEQ ID NO: 15) , and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VL CDR1 (SEQ ID NO: 15) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VL CDR2 (SEQ ID NO: 16) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 69) , a VL CDR1 (SEQ ID NO: 15) , and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 69) , a VL CDR1 (SEQ ID NO: 15) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 69) , a VL CDR2 (SEQ ID NO: 16) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR3 (SEQ ID NO: 20) , a VL CDR1 (SEQ ID NO: 15) , and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR3 (SEQ ID NO: 20) , a VL CDR1 (SEQ ID NO: 15) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR3 (SEQ ID NO: 20) , a VL CDR2 (SEQ ID NO: 16) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR2 (SEQ ID NO: 69) , a VH CDR3 (SEQ ID NO: 20) , and a VL CDR1 (SEQ ID NO: 15) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR2 (SEQ ID NO: 69) , a VH CDR3 (SEQ ID NO: 20) , and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR2 (SEQ ID NO: 69) , a VH CDR3 (SEQ ID NO: 20) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR2 (SEQ ID NO: 69) , a VL CDR1 (SEQ ID NO: 15) , and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR2 (SEQ ID NO: 69) , a VL CDR1 (SEQ ID NO: 15) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR2 (SEQ ID NO: 69) , a VL CDR2 (SEQ ID NO: 16) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR3 (SEQ ID NO: 20) , a VL CDR1 (SEQ ID NO: 15) , and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR3 (SEQ ID NO: 20) , a VL CDR1 (SEQ ID NO: 15) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR3 (SEQ ID NO: 20) , a VL CDR2 (SEQ ID NO: 16) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 69) , a VH CDR3 (SEQ ID NO: 20) , a VL CDR1 (SEQ ID NO: 15) , and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 69) , a VH CDR3 (SEQ ID NO: 20) , a VL CDR1 (SEQ ID NO: 15) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 69) , a VH CDR3 (SEQ ID NO: 20) , a VL CDR2 (SEQ ID NO: 16) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR2 (SEQ ID NO: 69) , a VH CDR3 (SEQ ID NO: 20) , a VL CDR1 (SEQ ID NO: 15) , and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR2 (SEQ ID NO: 69) , a VH CDR3 (SEQ ID NO: 20) , a VL CDR1 (SEQ ID NO: 15) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR2 (SEQ ID NO: 69) , a VH CDR3 (SEQ ID NO: 20) , a VL CDR2 (SEQ ID NO: 16) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR2 (SEQ ID NO: 69) , a VL CDR1 (SEQ ID NO: 15) , a VL CDR2 (SEQ ID NO: 16) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR3 (SEQ ID NO: 20) , a VL CDR1 (SEQ ID NO: 15) , a VL CDR2 (SEQ ID NO: 16) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 69) , a VH CDR3 (SEQ ID NO: 20) , a VL CDR1 (SEQ ID NO: 15) , a VL CDR2 (SEQ ID NO: 16) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VL CDR1 (SEQ ID NO: 15) , a VL CDR2 (SEQ ID NO: 16) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 69) , a VL CDR1 (SEQ ID NO: 15) , a VL CDR2 (SEQ ID NO: 16) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR3 (SEQ ID NO: 20) , a VL CDR1 (SEQ ID NO: 15) , a VL CDR2 (SEQ ID NO: 16) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises any combination thereof of the VH CDRs and VL CDRs listed in Tables 13-14.
  • two-in-one antibodies comprising one or more (e.g., one, two, or three) VH CDRs and one or more (e.g., one, two, or three) VL CDRs listed in Tables 13-14.
  • a two-in-one antibody comprising a VH CDR1 (SEQ ID NO: 18) and a VL CDR1 (SEQ ID NO: 68) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 69) and a VL CDR1 (SEQ ID NO: 68) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 69) and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 69) and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in- one antibody comprises a VH CDR3 (SEQ ID NO: 20) and a VL CDR1 (SEQ ID NO: 68) .
  • the two-in-one antibody comprises a VH CDR3 (SEQ ID NO: 20) and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR3 (SEQ ID NO: 20) and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR2 (SEQ ID NO: 69) , and a VL CDR1 (SEQ ID NO: 68) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR2 (SEQ ID NO: 69) , and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR2 (SEQ ID NO: 69) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 69) , a VH CDR3 (SEQ ID NO: 20) , and a VL CDR1 (SEQ ID NO: 68) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 69) , a VH CDR3 (SEQ ID NO: 20) , and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 69) , a VH CDR3 (SEQ ID NO: 20) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR3 (SEQ ID NO: 20) , and a VL CDR1 (SEQ ID NO: 68) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR3 (SEQ ID NO: 20) , and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR3 (SEQ ID NO: 20) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VL CDR1 (SEQ ID NO: 68) , and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VL CDR1 (SEQ ID NO: 68) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VL CDR2 (SEQ ID NO: 16) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 69) , a VL CDR1 (SEQ ID NO: 68) , and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 69) , a VL CDR1 (SEQ ID NO: 68) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 69) , a VL CDR2 (SEQ ID NO: 16) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR3 (SEQ ID NO: 20) , a VL CDR1 (SEQ ID NO: 68) , and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR3 (SEQ ID NO: 20) , a VL CDR1 (SEQ ID NO: 68) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR3 (SEQ ID NO: 20) , a VL CDR2 (SEQ ID NO: 16) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR2 (SEQ ID NO: 69) , a VH CDR3 (SEQ ID NO: 20) , and a VL CDR1 (SEQ ID NO: 68) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR2 (SEQ ID NO: 69) , a VH CDR3 (SEQ ID NO: 20) , and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR2 (SEQ ID NO: 69) , a VH CDR3 (SEQ ID NO: 20) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR2 (SEQ ID NO: 69) , a VL CDR1 (SEQ ID NO: 68) , and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR2 (SEQ ID NO: 69) , a VL CDR1 (SEQ ID NO: 68) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR2 (SEQ ID NO: 69) , a VL CDR2 (SEQ ID NO: 16) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR3 (SEQ ID NO: 20) , a VL CDR1 (SEQ ID NO: 68) , and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR3 (SEQ ID NO: 20) , a VL CDR1 (SEQ ID NO: 68) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR3 (SEQ ID NO: 20) , a VL CDR2 (SEQ ID NO: 16) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 69) , a VH CDR3 (SEQ ID NO: 20) , a VL CDR1 (SEQ ID NO: 68) , and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 69) , a VH CDR3 (SEQ ID NO: 20) , a VL CDR1 (SEQ ID NO: 68) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 69) , a VH CDR3 (SEQ ID NO: 20) , a VL CDR2 (SEQ ID NO: 16) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR2 (SEQ ID NO: 69) , a VH CDR3 (SEQ ID NO: 20) , a VL CDR1 (SEQ ID NO: 68) , and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR2 (SEQ ID NO: 69) , a VH CDR3 (SEQ ID NO: 20) , a VL CDR1 (SEQ ID NO: 68) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR2 (SEQ ID NO: 69) , a VH CDR3 (SEQ ID NO: 20) , a VL CDR2 (SEQ ID NO: 16) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR2 (SEQ ID NO: 69) , a VL CDR1 (SEQ ID NO: 68) , a VL CDR2 (SEQ ID NO: 16) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR3 (SEQ ID NO: 20) , a VL CDR1 (SEQ ID NO: 68) , a VL CDR2 (SEQ ID NO: 16) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 69) , a VH CDR3 (SEQ ID NO: 20) , a VL CDR1 (SEQ ID NO: 68) , a VL CDR2 (SEQ ID NO: 16) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VL CDR1 (SEQ ID NO: 68) , a VL CDR2 (SEQ ID NO: 16) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 69) , a VL CDR1 (SEQ ID NO: 68) , a VL CDR2 (SEQ ID NO: 16) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR3 (SEQ ID NO: 20) , a VL CDR1 (SEQ ID NO: 68) , a VL CDR2 (SEQ ID NO: 16) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises any combination thereof of the VH CDRs and VL CDRs listed in Tables 13-14.
  • two-in-one antibodies comprising one or more (e.g., one, two, or three) VH CDRs and one or more (e.g., one, two, or three) VL CDRs listed in Tables 13-14.
  • a two-in-one antibody comprising a VH CDR1 (SEQ ID NO: 18) and a VL CDR1 (SEQ ID NO: 15) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 70) and a VL CDR1 (SEQ ID NO: 15) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 70) and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 70) and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR3 (SEQ ID NO: 20) and a VL CDR1 (SEQ ID NO: 15) .
  • the two-in-one antibody comprises a VH CDR3 (SEQ ID NO: 20) and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR3 (SEQ ID NO: 20) and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR2 (SEQ ID NO: 70) , and a VL CDR1 (SEQ ID NO: 15) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR2 (SEQ ID NO: 70) , and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR2 (SEQ ID NO: 70) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 70) , a VH CDR3 (SEQ ID NO: 20) , and a VL CDR1 (SEQ ID NO: 15) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 70) , a VH CDR3 (SEQ ID NO: 20) , and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 70) , a VH CDR3 (SEQ ID NO: 20) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR3 (SEQ ID NO: 20) , and a VL CDR1 (SEQ ID NO: 15) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR3 (SEQ ID NO: 20) , and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR3 (SEQ ID NO: 20) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VL CDR1 (SEQ ID NO: 15) , and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VL CDR1 (SEQ ID NO: 15) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VL CDR2 (SEQ ID NO: 16) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 70) , a VL CDR1 (SEQ ID NO: 15) , and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 70) , a VL CDR1 (SEQ ID NO: 15) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 70) , a VL CDR2 (SEQ ID NO: 16) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR3 (SEQ ID NO: 20) , a VL CDR1 (SEQ ID NO: 15) , and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR3 (SEQ ID NO: 20) , a VL CDR1 (SEQ ID NO: 15) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR3 (SEQ ID NO: 20) , a VL CDR2 (SEQ ID NO: 16) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR2 (SEQ ID NO: 70) , a VH CDR3 (SEQ ID NO: 20) , and a VL CDR1 (SEQ ID NO: 15) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR2 (SEQ ID NO: 70) , a VH CDR3 (SEQ ID NO: 20) , and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR2 (SEQ ID NO: 70) , a VH CDR3 (SEQ ID NO: 20) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR2 (SEQ ID NO: 70) , a VL CDR1 (SEQ ID NO: 15) , and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR2 (SEQ ID NO: 70) , a VL CDR1 (SEQ ID NO: 15) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR2 (SEQ ID NO: 70) , a VL CDR2 (SEQ ID NO: 16) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR3 (SEQ ID NO: 20) , a VL CDR1 (SEQ ID NO: 15) , and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR3 (SEQ ID NO: 20) , a VL CDR1 (SEQ ID NO: 15) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR3 (SEQ ID NO: 20) , a VL CDR2 (SEQ ID NO: 16) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 70) , a VH CDR3 (SEQ ID NO: 20) , a VL CDR1 (SEQ ID NO: 15) , and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 70) , a VH CDR3 (SEQ ID NO: 20) , a VL CDR1 (SEQ ID NO: 15) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 70) , a VH CDR3 (SEQ ID NO: 20) , a VL CDR2 (SEQ ID NO: 16) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR2 (SEQ ID NO: 70) , a VH CDR3 (SEQ ID NO: 20) , a VL CDR1 (SEQ ID NO: 15) , and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR2 (SEQ ID NO: 70) , a VH CDR3 (SEQ ID NO: 20) , a VL CDR1 (SEQ ID NO: 15) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR2 (SEQ ID NO: 70) , a VH CDR3 (SEQ ID NO: 20) , a VL CDR2 (SEQ ID NO: 16) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR2 (SEQ ID NO: 70) , a VL CDR1 (SEQ ID NO: 15) , a VL CDR2 (SEQ ID NO: 16) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR3 (SEQ ID NO: 20) , a VL CDR1 (SEQ ID NO: 15) , a VL CDR2 (SEQ ID NO: 16) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 70) , a VH CDR3 (SEQ ID NO: 20) , a VL CDR1 (SEQ ID NO: 15) , a VL CDR2 (SEQ ID NO: 16) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VL CDR1 (SEQ ID NO: 15) , a VL CDR2 (SEQ ID NO: 16) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 70) , a VL CDR1 (SEQ ID NO: 15) , a VL CDR2 (SEQ ID NO: 16) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR3 (SEQ ID NO: 20) , a VL CDR1 (SEQ ID NO: 15) , a VL CDR2 (SEQ ID NO: 16) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises any combination thereof of the VH CDRs and VL CDRs listed in Tables 13-14.
  • two-in-one antibodies comprising one or more (e.g., one, two, or three) VH CDRs and one or more (e.g., one, two, or three) VL CDRs listed in Tables 13-14.
  • a two-in-one antibody comprising a VH CDR1 (SEQ ID NO: 18) and a VL CDR1 (SEQ ID NO: 68) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) and a VL CDR2 (SEQ ID NO: 16) . In other embodiments, the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) and a VL CDR3 (SEQ ID NO: 17) . In another embodiment, the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 70) and a VL CDR1 (SEQ ID NO: 68) . In some embodiments, the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 70) and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 70) and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR3 (SEQ ID NO: 20) and a VL CDR1 (SEQ ID NO: 68) .
  • the two-in-one antibody comprises a VH CDR3 (SEQ ID NO: 20) and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR3 (SEQ ID NO: 20) and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR2 (SEQ ID NO: 70) , and a VL CDR1 (SEQ ID NO: 68) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR2 (SEQ ID NO: 70) , and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR2 (SEQ ID NO: 70) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 70) , a VH CDR3 (SEQ ID NO: 20) , and a VL CDR1 (SEQ ID NO: 68) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 70) , a VH CDR3 (SEQ ID NO: 20) , and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 70) , a VH CDR3 (SEQ ID NO: 20) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR3 (SEQ ID NO: 20) , and a VL CDR1 (SEQ ID NO: 68) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR3 (SEQ ID NO: 20) , and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR3 (SEQ ID NO: 20) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VL CDR1 (SEQ ID NO: 68) , and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VL CDR1 (SEQ ID NO: 68) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VL CDR2 (SEQ ID NO: 16) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 70) , a VL CDR1 (SEQ ID NO: 68) , and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 70) , a VL CDR1 (SEQ ID NO: 68) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 70) , a VL CDR2 (SEQ ID NO: 16) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR3 (SEQ ID NO: 20) , a VL CDR1 (SEQ ID NO: 68) , and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR3 (SEQ ID NO: 20) , a VL CDR1 (SEQ ID NO: 68) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR3 (SEQ ID NO: 20) , a VL CDR2 (SEQ ID NO: 16) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR2 (SEQ ID NO: 70) , a VH CDR3 (SEQ ID NO: 20) , and a VL CDR1 (SEQ ID NO: 68) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR2 (SEQ ID NO: 70) , a VH CDR3 (SEQ ID NO: 20) , and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR2 (SEQ ID NO: 70) , a VH CDR3 (SEQ ID NO: 20) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR2 (SEQ ID NO: 70) , a VL CDR1 (SEQ ID NO: 68) , and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR2 (SEQ ID NO: 70) , a VL CDR1 (SEQ ID NO: 68) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR2 (SEQ ID NO: 70) , a VL CDR2 (SEQ ID NO: 16) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR3 (SEQ ID NO: 20) , a VL CDR1 (SEQ ID NO: 68) , and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR3 (SEQ ID NO: 20) , a VL CDR1 (SEQ ID NO: 68) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR3 (SEQ ID NO: 20) , a VL CDR2 (SEQ ID NO: 16) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 70) , a VH CDR3 (SEQ ID NO: 20) , a VL CDR1 (SEQ ID NO: 68) , and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 70) , a VH CDR3 (SEQ ID NO: 20) , a VL CDR1 (SEQ ID NO: 68) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 70) , a VH CDR3 (SEQ ID NO: 20) , a VL CDR2 (SEQ ID NO: 16) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR2 (SEQ ID NO: 70) , a VH CDR3 (SEQ ID NO: 20) , a VL CDR1 (SEQ ID NO: 68) , and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR2 (SEQ ID NO: 70) , a VH CDR3 (SEQ ID NO: 20) , a VL CDR1 (SEQ ID NO: 68) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR2 (SEQ ID NO: 70) , a VH CDR3 (SEQ ID NO: 20) , a VL CDR2 (SEQ ID NO: 16) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR2 (SEQ ID NO: 70) , a VL CDR1 (SEQ ID NO: 68) , a VL CDR2 (SEQ ID NO: 16) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR3 (SEQ ID NO: 20) , a VL CDR1 (SEQ ID NO: 68) , a VL CDR2 (SEQ ID NO: 16) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 70) , a VH CDR3 (SEQ ID NO: 20) , a VL CDR1 (SEQ ID NO: 68) , a VL CDR2 (SEQ ID NO: 16) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VL CDR1 (SEQ ID NO: 68) , a VL CDR2 (SEQ ID NO: 16) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 70) , a VL CDR1 (SEQ ID NO: 68) , a VL CDR2 (SEQ ID NO: 16) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR3 (SEQ ID NO: 20) , a VL CDR1 (SEQ ID NO: 68) , a VL CDR2 (SEQ ID NO: 16) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises any combination thereof of the VH CDRs and VL CDRs listed in Tables 13-14.
  • two-in-one antibodies comprising one or more (e.g., one, two, or three) VH CDRs and one or more (e.g., one, two, or three) VL CDRs listed in Tables 13-14.
  • a two-in-one antibody comprising a VH CDR1 (SEQ ID NO: 18) and a VL CDR1 (SEQ ID NO: 15) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 71) and a VL CDR1 (SEQ ID NO: 15) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 71) and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 71) and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR3 (SEQ ID NO: 20) and a VL CDR1 (SEQ ID NO: 15) .
  • the two-in-one antibody comprises a VH CDR3 (SEQ ID NO: 20) and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR3 (SEQ ID NO: 20) and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR2 (SEQ ID NO: 71) , and a VL CDR1 (SEQ ID NO: 15) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR2 (SEQ ID NO: 71) , and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR2 (SEQ ID NO: 71) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 71) , a VH CDR3 (SEQ ID NO: 20) , and a VL CDR1 (SEQ ID NO: 15) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 71) , a VH CDR3 (SEQ ID NO: 20) , and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 71) , a VH CDR3 (SEQ ID NO: 20) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR3 (SEQ ID NO: 20) , and a VL CDR1 (SEQ ID NO: 15) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR3 (SEQ ID NO: 20) , and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR3 (SEQ ID NO: 20) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VL CDR1 (SEQ ID NO: 15) , and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VL CDR1 (SEQ ID NO: 15) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VL CDR2 (SEQ ID NO: 16) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 71) , a VL CDR1 (SEQ ID NO: 15) , and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 71) , a VL CDR1 (SEQ ID NO: 15) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 71) , a VL CDR2 (SEQ ID NO: 16) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR3 (SEQ ID NO: 20) , a VL CDR1 (SEQ ID NO: 15) , and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR3 (SEQ ID NO: 20) , a VL CDR1 (SEQ ID NO: 15) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR3 (SEQ ID NO: 20) , a VL CDR2 (SEQ ID NO: 16) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR2 (SEQ ID NO: 71) , a VH CDR3 (SEQ ID NO: 20) , and a VL CDR1 (SEQ ID NO: 15) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR2 (SEQ ID NO: 71) , a VH CDR3 (SEQ ID NO: 20) , and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR2 (SEQ ID NO: 71) , a VH CDR3 (SEQ ID NO: 20) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR2 (SEQ ID NO: 71) , a VL CDR1 (SEQ ID NO: 15) , and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR2 (SEQ ID NO: 71) , a VL CDR1 (SEQ ID NO: 15) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR2 (SEQ ID NO: 71) , a VL CDR2 (SEQ ID NO: 16) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR3 (SEQ ID NO: 20) , a VL CDR1 (SEQ ID NO: 15) , and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR3 (SEQ ID NO: 20) , a VL CDR1 (SEQ ID NO: 15) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR3 (SEQ ID NO: 20) , a VL CDR2 (SEQ ID NO: 16) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 71) , a VH CDR3 (SEQ ID NO: 20) , a VL CDR1 (SEQ ID NO: 15) , and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 71) , a VH CDR3 (SEQ ID NO: 20) , a VL CDR1 (SEQ ID NO: 15) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 71) , a VH CDR3 (SEQ ID NO: 20) , a VL CDR2 (SEQ ID NO: 16) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR2 (SEQ ID NO: 71) , a VH CDR3 (SEQ ID NO: 20) , a VL CDR1 (SEQ ID NO: 15) , and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR2 (SEQ ID NO: 71) , a VH CDR3 (SEQ ID NO: 20) , a VL CDR1 (SEQ ID NO: 15) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR2 (SEQ ID NO: 71) , a VH CDR3 (SEQ ID NO: 20) , a VL CDR2 (SEQ ID NO: 16) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR2 (SEQ ID NO: 71) , a VL CDR1 (SEQ ID NO: 15) , a VL CDR2 (SEQ ID NO: 16) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR3 (SEQ ID NO: 20) , a VL CDR1 (SEQ ID NO: 15) , a VL CDR2 (SEQ ID NO: 16) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 71) , a VH CDR3 (SEQ ID NO: 20) , a VL CDR1 (SEQ ID NO: 15) , a VL CDR2 (SEQ ID NO: 16) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VL CDR1 (SEQ ID NO: 15) , a VL CDR2 (SEQ ID NO: 16) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 71) , a VL CDR1 (SEQ ID NO: 15) , a VL CDR2 (SEQ ID NO: 16) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR3 (SEQ ID NO: 20) , a VL CDR1 (SEQ ID NO: 15) , a VL CDR2 (SEQ ID NO: 16) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises any combination thereof of the VH CDRs and VL CDRs listed in Tables 13-14.
  • two-in-one antibodies comprising one or more (e.g., one, two, or three) VH CDRs and one or more (e.g., one, two, or three) VL CDRs listed in Tables 13-14.
  • a two-in-one antibody comprising a VH CDR1 (SEQ ID NO: 18) and a VL CDR1 (SEQ ID NO: 68) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 71) and a VL CDR1 (SEQ ID NO: 68) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 71) and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 71) and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR3 (SEQ ID NO: 20) and a VL CDR1 (SEQ ID NO: 68) . In other embodiments, the two-in-one antibody comprises a VH CDR3 (SEQ ID NO: 20) and a VL CDR2 (SEQ ID NO: 16) . In some embodiments, the two-in-one antibody comprises a VH CDR3 (SEQ ID NO: 20) and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR2 (SEQ ID NO: 71) , and a VL CDR1 (SEQ ID NO: 68) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR2 (SEQ ID NO: 71) , and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR2 (SEQ ID NO: 71) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 71) , a VH CDR3 (SEQ ID NO: 20) , and a VL CDR1 (SEQ ID NO: 68) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 71) , a VH CDR3 (SEQ ID NO: 20) , and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 71) , a VH CDR3 (SEQ ID NO: 20) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR3 (SEQ ID NO: 20) , and a VL CDR1 (SEQ ID NO: 68) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR3 (SEQ ID NO: 20) , and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR3 (SEQ ID NO: 20) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VL CDR1 (SEQ ID NO: 68) , and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VL CDR1 (SEQ ID NO: 68) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VL CDR2 (SEQ ID NO: 16) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 71) , a VL CDR1 (SEQ ID NO: 68) , and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 71) , a VL CDR1 (SEQ ID NO: 68) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 71) , a VL CDR2 (SEQ ID NO: 16) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR3 (SEQ ID NO: 20) , a VL CDR1 (SEQ ID NO: 68) , and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR3 (SEQ ID NO: 20) , a VL CDR1 (SEQ ID NO: 68) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR3 (SEQ ID NO: 20) , a VL CDR2 (SEQ ID NO: 16) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR2 (SEQ ID NO: 71) , a VH CDR3 (SEQ ID NO: 20) , and a VL CDR1 (SEQ ID NO: 68) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR2 (SEQ ID NO: 71) , a VH CDR3 (SEQ ID NO: 20) , and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR2 (SEQ ID NO: 71) , a VH CDR3 (SEQ ID NO: 20) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR2 (SEQ ID NO: 71) , a VL CDR1 (SEQ ID NO: 68) , and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR2 (SEQ ID NO: 71) , a VL CDR1 (SEQ ID NO: 68) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR2 (SEQ ID NO: 71) , a VL CDR2 (SEQ ID NO: 16) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR3 (SEQ ID NO: 20) , a VL CDR1 (SEQ ID NO: 68) , and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR3 (SEQ ID NO: 20) , a VL CDR1 (SEQ ID NO: 68) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR3 (SEQ ID NO: 20) , a VL CDR2 (SEQ ID NO: 16) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 71) , a VH CDR3 (SEQ ID NO: 20) , a VL CDR1 (SEQ ID NO: 68) , and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 71) , a VH CDR3 (SEQ ID NO: 20) , a VL CDR1 (SEQ ID NO: 68) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 71) , a VH CDR3 (SEQ ID NO: 20) , a VL CDR2 (SEQ ID NO: 16) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR2 (SEQ ID NO: 71) , a VH CDR3 (SEQ ID NO: 20) , a VL CDR1 (SEQ ID NO: 68) , and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR2 (SEQ ID NO: 71) , a VH CDR3 (SEQ ID NO: 20) , a VL CDR1 (SEQ ID NO: 68) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR2 (SEQ ID NO: 71) , a VH CDR3 (SEQ ID NO: 20) , a VL CDR2 (SEQ ID NO: 16) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR2 (SEQ ID NO: 71) , a VL CDR1 (SEQ ID NO: 68) , a VL CDR2 (SEQ ID NO: 16) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR3 (SEQ ID NO: 20) , a VL CDR1 (SEQ ID NO: 68) , a VL CDR2 (SEQ ID NO: 16) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 71) , a VH CDR3 (SEQ ID NO: 20) , a VL CDR1 (SEQ ID NO: 68) , a VL CDR2 (SEQ ID NO: 16) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VL CDR1 (SEQ ID NO: 68) , a VL CDR2 (SEQ ID NO: 16) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 71) , a VL CDR1 (SEQ ID NO: 68) , a VL CDR2 (SEQ ID NO: 16) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR3 (SEQ ID NO: 20) , a VL CDR1 (SEQ ID NO: 68) , a VL CDR2 (SEQ ID NO: 16) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises any combination thereof of the VH CDRs and VL CDRs listed in Tables 13-14.
  • two-in-one antibodies comprising one or more (e.g., one, two, or three) VH CDRs and one or more (e.g., one, two, or three) VL CDRs listed in Tables 13-14.
  • a two-in-one antibody comprising a VH CDR1 (SEQ ID NO: 18) and a VL CDR1 (SEQ ID NO: 15) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 72) and a VL CDR1 (SEQ ID NO: 15) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 72) and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 72) and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR3 (SEQ ID NO: 20) and a VL CDR1 (SEQ ID NO: 15) .
  • the two-in-one antibody comprises a VH CDR3 (SEQ ID NO: 20) and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR3 (SEQ ID NO: 20) and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR2 (SEQ ID NO: 72) , and a VL CDR1 (SEQ ID NO: 15) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR2 (SEQ ID NO: 72) , and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR2 (SEQ ID NO: 72) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 72) , a VH CDR3 (SEQ ID NO: 20) , and a VL CDR1 (SEQ ID NO: 15) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 72) , a VH CDR3 (SEQ ID NO: 20) , and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 72) , a VH CDR3 (SEQ ID NO: 20) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR3 (SEQ ID NO: 20) , and a VL CDR1 (SEQ ID NO: 15) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR3 (SEQ ID NO: 20) , and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR3 (SEQ ID NO: 20) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VL CDR1 (SEQ ID NO: 15) , and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VL CDR1 (SEQ ID NO: 15) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VL CDR2 (SEQ ID NO: 16) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 72) , a VL CDR1 (SEQ ID NO: 15) , and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 72) , a VL CDR1 (SEQ ID NO: 15) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 72) , a VL CDR2 (SEQ ID NO: 16) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR3 (SEQ ID NO: 20) , a VL CDR1 (SEQ ID NO: 15) , and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR3 (SEQ ID NO: 20) , a VL CDR1 (SEQ ID NO: 15) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR3 (SEQ ID NO: 20) , a VL CDR2 (SEQ ID NO: 16) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR2 (SEQ ID NO: 72) , a VH CDR3 (SEQ ID NO: 20) , and a VL CDR1 (SEQ ID NO: 15) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR2 (SEQ ID NO: 72) , a VH CDR3 (SEQ ID NO: 20) , and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR2 (SEQ ID NO: 72) , a VH CDR3 (SEQ ID NO: 20) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR2 (SEQ ID NO: 72) , a VL CDR1 (SEQ ID NO: 15) , and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR2 (SEQ ID NO: 72) , a VL CDR1 (SEQ ID NO: 15) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR2 (SEQ ID NO: 72) , a VL CDR2 (SEQ ID NO: 16) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR3 (SEQ ID NO: 20) , a VL CDR1 (SEQ ID NO: 15) , and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR3 (SEQ ID NO: 20) , a VL CDR1 (SEQ ID NO: 15) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR3 (SEQ ID NO: 20) , a VL CDR2 (SEQ ID NO: 16) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 72) , a VH CDR3 (SEQ ID NO: 20) , a VL CDR1 (SEQ ID NO: 15) , and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 72) , a VH CDR3 (SEQ ID NO: 20) , a VL CDR1 (SEQ ID NO: 15) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 72) , a VH CDR3 (SEQ ID NO: 20) , a VL CDR2 (SEQ ID NO: 16) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR2 (SEQ ID NO: 72) , a VH CDR3 (SEQ ID NO: 20) , a VL CDR1 (SEQ ID NO: 15) , and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in- one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR2 (SEQ ID NO: 72) , a VH CDR3 (SEQ ID NO: 20) , a VL CDR1 (SEQ ID NO: 15) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR2 (SEQ ID NO: 72) , a VH CDR3 (SEQ ID NO: 20) , a VL CDR2 (SEQ ID NO: 16) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR2 (SEQ ID NO: 72) , a VL CDR1 (SEQ ID NO: 15) , a VL CDR2 (SEQ ID NO: 16) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR3 (SEQ ID NO: 20) , a VL CDR1 (SEQ ID NO: 15) , a VL CDR2 (SEQ ID NO: 16) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 72) , a VH CDR3 (SEQ ID NO: 20) , a VL CDR1 (SEQ ID NO: 15) , a VL CDR2 (SEQ ID NO: 16) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VL CDR1 (SEQ ID NO: 15) , a VL CDR2 (SEQ ID NO: 16) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 72) , a VL CDR1 (SEQ ID NO: 15) , a VL CDR2 (SEQ ID NO: 16) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR3 (SEQ ID NO: 20) , a VL CDR1 (SEQ ID NO: 15) , a VL CDR2 (SEQ ID NO: 16) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises any combination thereof of the VH CDRs and VL CDRs listed in Tables 13-14.
  • two-in-one antibodies comprising one or more (e.g., one, two, or three) VH CDRs and one or more (e.g., one, two, or three) VL CDRs listed in Tables 13-14.
  • a two-in-one antibody comprising a VH CDR1 (SEQ ID NO: 18) and a VL CDR1 (SEQ ID NO: 68) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 72) and a VL CDR1 (SEQ ID NO: 68) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 72) and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 72) and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR3 (SEQ ID NO: 20) and a VL CDR1 (SEQ ID NO: 68) . In other embodiments, the two-in-one antibody comprises a VH CDR3 (SEQ ID NO: 20) and a VL CDR2 (SEQ ID NO: 16) . In some embodiments, the two-in-one antibody comprises a VH CDR3 (SEQ ID NO: 20) and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR2 (SEQ ID NO: 72) , and a VL CDR1 (SEQ ID NO: 68) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR2 (SEQ ID NO: 72) , and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR2 (SEQ ID NO: 72) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 72) , a VH CDR3 (SEQ ID NO: 20) , and a VL CDR1 (SEQ ID NO: 68) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 72) , a VH CDR3 (SEQ ID NO: 20) , and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 72) , a VH CDR3 (SEQ ID NO: 20) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR3 (SEQ ID NO: 20) , and a VL CDR1 (SEQ ID NO: 68) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR3 (SEQ ID NO: 20) , and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR3 (SEQ ID NO: 20) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VL CDR1 (SEQ ID NO: 68) , and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VL CDR1 (SEQ ID NO: 68) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VL CDR2 (SEQ ID NO: 16) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 72) , a VL CDR1 (SEQ ID NO: 68) , and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 72) , a VL CDR1 (SEQ ID NO: 68) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 72) , a VL CDR2 (SEQ ID NO: 16) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR3 (SEQ ID NO: 20) , a VL CDR1 (SEQ ID NO: 68) , and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR3 (SEQ ID NO: 20) , a VL CDR1 (SEQ ID NO: 68) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR3 (SEQ ID NO: 20) , a VL CDR2 (SEQ ID NO: 16) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR2 (SEQ ID NO: 72) , a VH CDR3 (SEQ ID NO: 20) , and a VL CDR1 (SEQ ID NO: 68) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR2 (SEQ ID NO: 72) , a VH CDR3 (SEQ ID NO: 20) , and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR2 (SEQ ID NO: 72) , a VH CDR3 (SEQ ID NO: 20) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR2 (SEQ ID NO: 72) , a VL CDR1 (SEQ ID NO: 68) , and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR2 (SEQ ID NO: 72) , a VL CDR1 (SEQ ID NO: 68) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR2 (SEQ ID NO: 72) , a VL CDR2 (SEQ ID NO: 16) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR3 (SEQ ID NO: 20) , a VL CDR1 (SEQ ID NO: 68) , and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR3 (SEQ ID NO: 20) , a VL CDR1 (SEQ ID NO: 68) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR3 (SEQ ID NO: 20) , a VL CDR2 (SEQ ID NO: 16) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 72) , a VH CDR3 (SEQ ID NO: 20) , a VL CDR1 (SEQ ID NO: 68) , and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 72) , a VH CDR3 (SEQ ID NO: 20) , a VL CDR1 (SEQ ID NO: 68) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 72) , a VH CDR3 (SEQ ID NO: 20) , a VL CDR2 (SEQ ID NO: 16) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR2 (SEQ ID NO: 72) , a VH CDR3 (SEQ ID NO: 20) , a VL CDR1 (SEQ ID NO: 68) , and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR2 (SEQ ID NO: 72) , a VH CDR3 (SEQ ID NO: 20) , a VL CDR1 (SEQ ID NO: 68) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR2 (SEQ ID NO: 72) , a VH CDR3 (SEQ ID NO: 20) , a VL CDR2 (SEQ ID NO: 16) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR2 (SEQ ID NO: 72) , a VL CDR1 (SEQ ID NO: 68) , a VL CDR2 (SEQ ID NO: 16) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR3 (SEQ ID NO: 20) , a VL CDR1 (SEQ ID NO: 68) , a VL CDR2 (SEQ ID NO: 16) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 72) , a VH CDR3 (SEQ ID NO: 20) , a VL CDR1 (SEQ ID NO: 68) , a VL CDR2 (SEQ ID NO: 16) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VL CDR1 (SEQ ID NO: 68) , a VL CDR2 (SEQ ID NO: 16) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 72) , a VL CDR1 (SEQ ID NO: 68) , a VL CDR2 (SEQ ID NO: 16) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR3 (SEQ ID NO: 20) , a VL CDR1 (SEQ ID NO: 68) , a VL CDR2 (SEQ ID NO: 16) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises any combination thereof of the VH CDRs and VL CDRs listed in Tables 13-14.
  • two-in-one antibodies comprising one or more (e.g., one, two, or three) VH CDRs and one or more (e.g., one, two, or three) VL CDRs listed in Tables 13-14.
  • a two-in-one antibody comprising a VH CDR1 (SEQ ID NO: 18) and a VL CDR1 (SEQ ID NO: 15) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 73) and a VL CDR1 (SEQ ID NO: 15) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 73) and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 73) and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR3 (SEQ ID NO: 20) and a VL CDR1 (SEQ ID NO: 15) .
  • the two-in-one antibody comprises a VH CDR3 (SEQ ID NO: 20) and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR3 (SEQ ID NO: 20) and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR2 (SEQ ID NO: 73) , and a VL CDR1 (SEQ ID NO: 15) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR2 (SEQ ID NO: 73) , and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR2 (SEQ ID NO: 73) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 73) , a VH CDR3 (SEQ ID NO: 20) , and a VL CDR1 (SEQ ID NO: 15) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 73) , a VH CDR3 (SEQ ID NO: 20) , and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 73) , a VH CDR3 (SEQ ID NO: 20) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR3 (SEQ ID NO: 20) , and a VL CDR1 (SEQ ID NO: 15) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR3 (SEQ ID NO: 20) , and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR3 (SEQ ID NO: 20) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VL CDR1 (SEQ ID NO: 15) , and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VL CDR1 (SEQ ID NO: 15) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VL CDR2 (SEQ ID NO: 16) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 73) , a VL CDR1 (SEQ ID NO: 15) , and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 73) , a VL CDR1 (SEQ ID NO: 15) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 73) , a VL CDR2 (SEQ ID NO: 16) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR3 (SEQ ID NO: 20) , a VL CDR1 (SEQ ID NO: 15) , and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR3 (SEQ ID NO: 20) , a VL CDR1 (SEQ ID NO: 15) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR3 (SEQ ID NO: 20) , a VL CDR2 (SEQ ID NO: 16) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR2 (SEQ ID NO: 73) , a VH CDR3 (SEQ ID NO: 20) , and a VL CDR1 (SEQ ID NO: 15) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR2 (SEQ ID NO: 73) , a VH CDR3 (SEQ ID NO: 20) , and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR2 (SEQ ID NO: 73) , a VH CDR3 (SEQ ID NO: 20) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR2 (SEQ ID NO: 73) , a VL CDR1 (SEQ ID NO: 15) , and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR2 (SEQ ID NO: 73) , a VL CDR1 (SEQ ID NO: 15) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR2 (SEQ ID NO: 73) , a VL CDR2 (SEQ ID NO: 16) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR3 (SEQ ID NO: 20) , a VL CDR1 (SEQ ID NO: 15) , and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR3 (SEQ ID NO: 20) , a VL CDR1 (SEQ ID NO: 15) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR3 (SEQ ID NO: 20) , a VL CDR2 (SEQ ID NO: 16) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 73) , a VH CDR3 (SEQ ID NO: 20) , a VL CDR1 (SEQ ID NO: 15) , and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 73) , a VH CDR3 (SEQ ID NO: 20) , a VL CDR1 (SEQ ID NO: 15) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 73) , a VH CDR3 (SEQ ID NO: 20) , a VL CDR2 (SEQ ID NO: 16) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR2 (SEQ ID NO: 73) , a VH CDR3 (SEQ ID NO: 20) , a VL CDR1 (SEQ ID NO: 15) , and a VL CDR2 (SEQ ID NO: 16) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR2 (SEQ ID NO: 73) , a VH CDR3 (SEQ ID NO: 20) , a VL CDR1 (SEQ ID NO: 15) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR2 (SEQ ID NO: 73) , a VH CDR3 (SEQ ID NO: 20) , a VL CDR2 (SEQ ID NO: 16) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR2 (SEQ ID NO: 73) , a VL CDR1 (SEQ ID NO: 15) , a VL CDR2 (SEQ ID NO: 16) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VH CDR3 (SEQ ID NO: 20) , a VL CDR1 (SEQ ID NO: 15) , a VL CDR2 (SEQ ID NO: 16) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 73) , a VH CDR3 (SEQ ID NO: 20) , a VL CDR1 (SEQ ID NO: 15) , a VL CDR2 (SEQ ID NO: 16) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 18) , a VL CDR1 (SEQ ID NO: 15) , a VL CDR2 (SEQ ID NO: 16) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 73) , a VL CDR1 (SEQ ID NO: 15) , a VL CDR2 (SEQ ID NO: 16) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises a VH CDR3 (SEQ ID NO: 20) , a VL CDR1 (SEQ ID NO: 15) , a VL CDR2 (SEQ ID NO: 16) , and a VL CDR3 (SEQ ID NO: 17) .
  • the two-in-one antibody comprises any combination thereof of the VH CDRs and VL CDRs listed in Tables 13-14.
  • the two-in-one antibodies provided herein comprise a VH region comprising (1) a VH CDR1 having an amino acid sequence of SEQ ID NO: 18; (2) a VH CDR2 having an amino acid sequence of SEQ ID NO: 19; and (3) a VH CDR3 having an amino acid sequence of SEQ ID NO: 20.
  • the two-in-one antibodies provided herein comprise a VL region comprising: (1) a VL CDR1 having an amino acid sequence of SEQ ID NO: 15; (2) a VL CDR2 having an amino acid sequence of SEQ ID NO: 16; and (3) a VL CDR3 having an amino acid sequence of SEQ ID NO: 17.
  • the two-in-one antibodies provided herein comprise a VH region comprising (1) a VH CDR1 having an amino acid sequence of SEQ ID NO: 18; (2) a VH CDR2 having an amino acid sequence of SEQ ID NO: 19; and (3) a VH CDR3 having an amino acid sequence of SEQ ID NO: 20; and a VL region comprising: (1) a VL CDR1 having an amino acid sequence of SEQ ID NO: 15; (2) a VL CDR2 having an amino acid sequence of SEQ ID NO: 16; and (3) a VL CDR3 having an amino acid sequence of SEQ ID NO: 17.
  • the two-in-one antibodies provided herein comprise a VH region comprising (1) a VH CDR1 having an amino acid sequence of SEQ ID NO: 18; (2) a VH CDR2 having an amino acid sequence of SEQ ID NO: 19; and (3) a VH CDR3 having an amino acid sequence of SEQ ID NO: 20.
  • the two-in-one antibodies provided herein comprise a VL region comprising: (1) a VL CDR1 having an amino acid sequence of SEQ ID NO: 68; (2) a VL CDR2 having an amino acid sequence of SEQ ID NO: 16; and (3) a VL CDR3 having an amino acid sequence of SEQ ID NO: 17.
  • the two-in-one antibodies provided herein comprise a VH region comprising (1) a VH CDR1 having an amino acid sequence of SEQ ID NO: 18; (2) a VH CDR2 having an amino acid sequence of SEQ ID NO: 19; and (3) a VH CDR3 having an amino acid sequence of SEQ ID NO: 20; and a VL region comprising: (1) a VL CDR1 having an amino acid sequence of SEQ ID NO: 68; (2) a VL CDR2 having an amino acid sequence of SEQ ID NO: 16; and (3) a VL CDR3 having an amino acid sequence of SEQ ID NO: 17.
  • the two-in-one antibodies provided herein comprise a VH region comprising (1) a VH CDR1 having an amino acid sequence of SEQ ID NO: 18; (2) a VH CDR2 having an amino acid sequence of SEQ ID NO: 69; and (3) a VH CDR3 having an amino acid sequence of SEQ ID NO: 20.
  • the two-in-one antibodies provided herein comprise a VL region comprising: (1) a VL CDR1 having an amino acid sequence of SEQ ID NO: 15; (2) a VL CDR2 having an amino acid sequence of SEQ ID NO: 16; and (3) a VL CDR3 having an amino acid sequence of SEQ ID NO: 17.
  • the two-in-one antibodies provided herein comprise a VH region comprising (1) a VH CDR1 having an amino acid sequence of SEQ ID NO: 18; (2) a VH CDR2 having an amino acid sequence of SEQ ID NO: 69; and (3) a VH CDR3 having an amino acid sequence of SEQ ID NO: 20; and a VL region comprising: (1) a VL CDR1 having an amino acid sequence of SEQ ID NO: 15; (2) a VL CDR2 having an amino acid sequence of SEQ ID NO: 16; and (3) a VL CDR3 having an amino acid sequence of SEQ ID NO: 17.
  • the two-in-one antibodies provided herein comprise a VH region comprising (1) a VH CDR1 having an amino acid sequence of SEQ ID NO: 18; (2) a VH CDR2 having an amino acid sequence of SEQ ID NO: 69; and (3) a VH CDR3 having an amino acid sequence of SEQ ID NO: 20.
  • the two-in-one antibodies provided herein comprise a VL region comprising: (1) a VL CDR1 having an amino acid sequence of SEQ ID NO: 68; (2) a VL CDR2 having an amino acid sequence of SEQ ID NO: 16; and (3) a VL CDR3 having an amino acid sequence of SEQ ID NO: 17.
  • the two-in-one antibodies provided herein comprise a VH region comprising (1) a VH CDR1 having an amino acid sequence of SEQ ID NO: 18; (2) a VH CDR2 having an amino acid sequence of SEQ ID NO: 69; and (3) a VH CDR3 having an amino acid sequence of SEQ ID NO: 20; and a VL region comprising: (1) a VL CDR1 having an amino acid sequence of SEQ ID NO: 68; (2) a VL CDR2 having an amino acid sequence of SEQ ID NO: 16; and (3) a VL CDR3 having an amino acid sequence of SEQ ID NO: 17.
  • the two-in-one antibodies provided herein comprise a VH region comprising (1) a VH CDR1 having an amino acid sequence of SEQ ID NO: 18; (2) a VH CDR2 having an amino acid sequence of SEQ ID NO: 70; and (3) a VH CDR3 having an amino acid sequence of SEQ ID NO: 20.
  • the two-in-one antibodies provided herein comprise a VL region comprising: (1) a VL CDR1 having an amino acid sequence of SEQ ID NO: 15; (2) a VL CDR2 having an amino acid sequence of SEQ ID NO: 16; and (3) a VL CDR3 having an amino acid sequence of SEQ ID NO: 17.
  • the two-in-one antibodies provided herein comprise a VH region comprising (1) a VH CDR1 having an amino acid sequence of SEQ ID NO: 18; (2) a VH CDR2 having an amino acid sequence of SEQ ID NO: 70; and (3) a VH CDR3 having an amino acid sequence of SEQ ID NO: 20; and a VL region comprising: (1) a VL CDR1 having an amino acid sequence of SEQ ID NO: 15; (2) a VL CDR2 having an amino acid sequence of SEQ ID NO: 16; and (3) a VL CDR3 having an amino acid sequence of SEQ ID NO: 17.
  • the two-in-one antibodies provided herein comprise a VH region comprising (1) a VH CDR1 having an amino acid sequence of SEQ ID NO: 18; (2) a VH CDR2 having an amino acid sequence of SEQ ID NO: 70; and (3) a VH CDR3 having an amino acid sequence of SEQ ID NO: 20.
  • the two-in-one antibodies provided herein comprise a VL region comprising: (1) a VL CDR1 having an amino acid sequence of SEQ ID NO: 68; (2) a VL CDR2 having an amino acid sequence of SEQ ID NO: 16; and (3) a VL CDR3 having an amino acid sequence of SEQ ID NO: 17.
  • the two-in-one antibodies provided herein comprise a VH region comprising (1) a VH CDR1 having an amino acid sequence of SEQ ID NO: 18; (2) a VH CDR2 having an amino acid sequence of SEQ ID NO: 70; and (3) a VH CDR3 having an amino acid sequence of SEQ ID NO: 20; and a VL region comprising: (1) a VL CDR1 having an amino acid sequence of SEQ ID NO: 68; (2) a VL CDR2 having an amino acid sequence of SEQ ID NO: 16; and (3) a VL CDR3 having an amino acid sequence of SEQ ID NO: 17.
  • the two-in-one antibodies provided herein comprise a VH region comprising (1) a VH CDR1 having an amino acid sequence of SEQ ID NO: 18; (2) a VH CDR2 having an amino acid sequence of SEQ ID NO: 71; and (3) a VH CDR3 having an amino acid sequence of SEQ ID NO: 20.
  • the two-in-one antibodies provided herein comprise a VL region comprising: (1) a VL CDR1 having an amino acid sequence of SEQ ID NO: 15; (2) a VL CDR2 having an amino acid sequence of SEQ ID NO: 16; and (3) a VL CDR3 having an amino acid sequence of SEQ ID NO: 17.
  • the two-in-one antibodies provided herein comprise a VH region comprising (1) a VH CDR1 having an amino acid sequence of SEQ ID NO: 18; (2) a VH CDR2 having an amino acid sequence of SEQ ID NO: 71; and (3) a VH CDR3 having an amino acid sequence of SEQ ID NO: 20; and a VL region comprising: (1) a VL CDR1 having an amino acid sequence of SEQ ID NO: 15; (2) a VL CDR2 having an amino acid sequence of SEQ ID NO: 16; and (3) a VL CDR3 having an amino acid sequence of SEQ ID NO: 17.
  • the two-in-one antibodies provided herein comprise a VH region comprising (1) a VH CDR1 having an amino acid sequence of SEQ ID NO: 18; (2) a VH CDR2 having an amino acid sequence of SEQ ID NO: 71; and (3) a VH CDR3 having an amino acid sequence of SEQ ID NO: 20.
  • the two-in-one antibodies provided herein comprise a VL region comprising: (1) a VL CDR1 having an amino acid sequence of SEQ ID NO: 68; (2) a VL CDR2 having an amino acid sequence of SEQ ID NO: 16; and (3) a VL CDR3 having an amino acid sequence of SEQ ID NO: 17.
  • the two-in-one antibodies provided herein comprise a VH region comprising (1) a VH CDR1 having an amino acid sequence of SEQ ID NO: 18; (2) a VH CDR2 having an amino acid sequence of SEQ ID NO: 71; and (3) a VH CDR3 having an amino acid sequence of SEQ ID NO: 20; and a VL region comprising: (1) a VL CDR1 having an amino acid sequence of SEQ ID NO: 68; (2) a VL CDR2 having an amino acid sequence of SEQ ID NO: 16; and (3) a VL CDR3 having an amino acid sequence of SEQ ID NO: 17.
  • the two-in-one antibodies provided herein comprise a VH region comprising (1) a VH CDR1 having an amino acid sequence of SEQ ID NO: 18; (2) a VH CDR2 having an amino acid sequence of SEQ ID NO: 72; and (3) a VH CDR3 having an amino acid sequence of SEQ ID NO: 20.
  • the two-in-one antibodies provided herein comprise a VL region comprising: (1) a VL CDR1 having an amino acid sequence of SEQ ID NO: 15; (2) a VL CDR2 having an amino acid sequence of SEQ ID NO: 16; and (3) a VL CDR3 having an amino acid sequence of SEQ ID NO: 17.
  • the two-in-one antibodies provided herein comprise a VH region comprising (1) a VH CDR1 having an amino acid sequence of SEQ ID NO: 18; (2) a VH CDR2 having an amino acid sequence of SEQ ID NO: 72; and (3) a VH CDR3 having an amino acid sequence of SEQ ID NO: 20; and a VL region comprising: (1) a VL CDR1 having an amino acid sequence of SEQ ID NO: 15; (2) a VL CDR2 having an amino acid sequence of SEQ ID NO: 16; and (3) a VL CDR3 having an amino acid sequence of SEQ ID NO: 17.
  • the two-in-one antibodies provided herein comprise a VH region comprising (1) a VH CDR1 having an amino acid sequence of SEQ ID NO: 18; (2) a VH CDR2 having an amino acid sequence of SEQ ID NO: 72; and (3) a VH CDR3 having an amino acid sequence of SEQ ID NO: 20.
  • the two-in-one antibodies provided herein comprise a VL region comprising: (1) a VL CDR1 having an amino acid sequence of SEQ ID NO: 68; (2) a VL CDR2 having an amino acid sequence of SEQ ID NO: 16; and (3) a VL CDR3 having an amino acid sequence of SEQ ID NO: 17.
  • the two-in-one antibodies provided herein comprise a VH region comprising (1) a VH CDR1 having an amino acid sequence of SEQ ID NO: 18; (2) a VH CDR2 having an amino acid sequence of SEQ ID NO: 72; and (3) a VH CDR3 having an amino acid sequence of SEQ ID NO: 20; and a VL region comprising: (1) a VL CDR1 having an amino acid sequence of SEQ ID NO: 68; (2) a VL CDR2 having an amino acid sequence of SEQ ID NO: 16; and (3) a VL CDR3 having an amino acid sequence of SEQ ID NO: 17.
  • the two-in-one antibodies provided herein comprise a VH region comprising (1) a VH CDR1 having an amino acid sequence of SEQ ID NO: 18; (2) a VH CDR2 having an amino acid sequence of SEQ ID NO: 73; and (3) a VH CDR3 having an amino acid sequence of SEQ ID NO: 20.
  • the two-in-one antibodies provided herein comprise a VL region comprising: (1) a VL CDR1 having an amino acid sequence of SEQ ID NO: 15; (2) a VL CDR2 having an amino acid sequence of SEQ ID NO: 16; and (3) a VL CDR3 having an amino acid sequence of SEQ ID NO: 17.
  • the two-in-one antibodies provided herein comprise a VH region comprising (1) a VH CDR1 having an amino acid sequence of SEQ ID NO: 18; (2) a VH CDR2 having an amino acid sequence of SEQ ID NO: 73; and (3) a VH CDR3 having an amino acid sequence of SEQ ID NO: 20; and a VL region comprising: (1) a VL CDR1 having an amino acid sequence of SEQ ID NO: 15; (2) a VL CDR2 having an amino acid sequence of SEQ ID NO: 16; and (3) a VL CDR3 having an amino acid sequence of SEQ ID NO: 17.
  • the two-in-one antibodies provided herein comprise a VH region comprising (1) a VH CDR1 having an amino acid sequence of SEQ ID NO: 18; (2) a VH CDR2 having an amino acid sequence of SEQ ID NO: 73; and (3) a VH CDR3 having an amino acid sequence of SEQ ID NO: 20.
  • the two-in-one antibodies provided herein comprise a VL region comprising: (1) a VL CDR1 having an amino acid sequence of SEQ ID NO: 68; (2) a VL CDR2 having an amino acid sequence of SEQ ID NO: 16; and (3) a VL CDR3 having an amino acid sequence of SEQ ID NO: 17.
  • the two-in-one antibodies provided herein comprise a VH region comprising (1) a VH CDR1 having an amino acid sequence of SEQ ID NO: 18; (2) a VH CDR2 having an amino acid sequence of SEQ ID NO: 73; and (3) a VH CDR3 having an amino acid sequence of SEQ ID NO: 20; and a VL region comprising: (1) a VL CDR1 having an amino acid sequence of SEQ ID NO: 68; (2) a VL CDR2 having an amino acid sequence of SEQ ID NO: 16; and (3) a VL CDR3 having an amino acid sequence of SEQ ID NO: 17.
  • the two-in-one antibodies provided herein comprise a VH region or VH domain. In other embodiments, the two-in-one antibodies provided herein comprise a VL region or VL domain. In certain embodiments, the two-in-one antibodies provided herein have a combination of (i) a VH domain or VH region; and/or (ii) a VL domain or VL region.
  • the two-in-one antibodies provided herein have a combination of (i) a VH domain or VH region selected from the group consisting of SEQ ID NOS: 28, 75, 76, 77, 78, and 79, as set forth in Table 16; and/or (ii) a VL domain or VL region selected from the group consisting of SEQ ID NOS: 27 and 74 as set forth in Table 15.
  • the two-in-one antibodies provided herein comprise a VH region comprising (1) a VH CDR1 having an amino acid sequence of SEQ ID NO: 18; (2) a VH CDR2 having an amino acid sequence of SEQ ID NO: 19; and (3) a VH CDR3 having an amino acid sequence of SEQ ID NO: 20; and a VL region as set forth in Table 15.
  • the VL region has an amino acid sequence of SEQ ID NO: 27.
  • the VL region has an amino acid sequence of SEQ ID NO: 74.
  • the two-in-one antibodies provided herein comprise a VH region comprising (1) a VH CDR1 having an amino acid sequence of SEQ ID NO: 18; (2) a VH CDR2 having an amino acid sequence of SEQ ID NO: 69; and (3) a VH CDR3 having an amino acid sequence of SEQ ID NO: 20; and a VL region as set forth in Table 15.
  • the VL region has an amino acid sequence of SEQ ID NO: 27.
  • the VL region has an amino acid sequence of SEQ ID NO: 74.
  • the two-in-one antibodies provided herein comprise a VH region comprising (1) a VH CDR1 having an amino acid sequence of SEQ ID NO: 18; (2) a VH CDR2 having an amino acid sequence of SEQ ID NO: 70; and (3) a VH CDR3 having an amino acid sequence of SEQ ID NO: 20; and a VL region as set forth in Table 15.
  • the VL region has an amino acid sequence of SEQ ID NO: 27.
  • the VL region has an amino acid sequence of SEQ ID NO: 74.
  • the two-in-one antibodies provided herein comprise a VH region comprising (1) a VH CDR1 having an amino acid sequence of SEQ ID NO: 18; (2) a VH CDR2 having an amino acid sequence of SEQ ID NO: 71; and (3) a VH CDR3 having an amino acid sequence of SEQ ID NO: 20; and a VL region as set forth in Table 15.
  • the VL region has an amino acid sequence of SEQ ID NO: 27.
  • the VL region has an amino acid sequence of SEQ ID NO: 74.
  • the two-in-one antibodies provided herein comprise a VH region comprising (1) a VH CDR1 having an amino acid sequence of SEQ ID NO: 18; (2) a VH CDR2 having an amino acid sequence of SEQ ID NO: 72; and (3) a VH CDR3 having an amino acid sequence of SEQ ID NO: 20; and a VL region as set forth in Table 15.
  • the VL region has an amino acid sequence of SEQ ID NO: 27.
  • the VL region has an amino acid sequence of SEQ ID NO: 74.
  • the two-in-one antibodies provided herein comprise a VH region comprising (1) a VH CDR1 having an amino acid sequence of SEQ ID NO: 18; (2) a VH CDR2 having an amino acid sequence of SEQ ID NO: 73; and (3) a VH CDR3 having an amino acid sequence of SEQ ID NO: 20; and a VL region as set forth in Table 15.
  • the VL region has an amino acid sequence of SEQ ID NO: 27.
  • the VL region has an amino acid sequence of SEQ ID NO: 74.
  • the two-in-one antibodies provided herein comprise a VH consisting of SEQ ID NO: 28 as set forth in Table 16; and a VL region comprising (1) a VL CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NO: 15; (2) a VL CDR2 having an amino acid sequence of SEQ ID NO: 16; and (3) a VL CDR3 having an amino acid sequence of SEQ ID NO: 17.
  • the two-in-one antibodies provided herein comprise a VH consisting of SEQ ID NO: 75 as set forth in Table 16; and a VL region comprising (1) a VL CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NO: 15; (2) a VL CDR2 having an amino acid sequence of SEQ ID NO: 16; and (3) a VL CDR3 having an amino acid sequence of SEQ ID NO: 17.
  • the two-in-one antibodies provided herein comprise a VH consisting of SEQ ID NO: 76 as set forth in Table 16; and a VL region comprising (1) a VL CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NO: 15; (2) a VL CDR2 having an amino acid sequence of SEQ ID NO: 16; and (3) a VL CDR3 having an amino acid sequence of SEQ ID NO: 17.
  • the two-in-one antibodies provided herein comprise a VH consisting of SEQ ID NO: 77 as set forth in Table 16; and a VL region comprising (1) a VL CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NO: 15; (2) a VL CDR2 having an amino acid sequence of SEQ ID NO: 16; and (3) a VL CDR3 having an amino acid sequence of SEQ ID NO: 17.
  • the two-in-one antibodies provided herein comprise a VH consisting of SEQ ID NO: 78 as set forth in Table 16; and a VL region comprising (1) a VL CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NO: 15; (2) a VL CDR2 having an amino acid sequence of SEQ ID NO: 16; and (3) a VL CDR3 having an amino acid sequence of SEQ ID NO: 17.
  • the two-in-one antibodies provided herein comprise a VH consisting of SEQ ID NO: 79 as set forth in Table 16; and a VL region comprising (1) a VL CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NO: 15; (2) a VL CDR2 having an amino acid sequence of SEQ ID NO: 16; and (3) a VL CDR3 having an amino acid sequence of SEQ ID NO: 17.
  • the two-in-one antibodies provided herein comprise a VH consisting of SEQ ID NO: 75 as set forth in Table 16; and a VL region comprising (1) a VL CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NO: 68; (2) a VL CDR2 having an amino acid sequence of SEQ ID NO: 16; and (3) a VL CDR3 having an amino acid sequence of SEQ ID NO: 17.
  • the two-in-one antibodies provided herein comprise a VH consisting of SEQ ID NO: 76 as set forth in Table 16; and a VL region comprising (1) a VL CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NO: 68; (2) a VL CDR2 having an amino acid sequence of SEQ ID NO: 16; and (3) a VL CDR3 having an amino acid sequence of SEQ ID NO: 17.
  • the two-in-one antibodies provided herein comprise a VH consisting of SEQ ID NO: 77 as set forth in Table 16; and a VL region comprising (1) a VL CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NO: 68; (2) a VL CDR2 having an amino acid sequence of SEQ ID NO: 16; and (3) a VL CDR3 having an amino acid sequence of SEQ ID NO: 17.
  • the two-in-one antibodies provided herein comprise a VH consisting of SEQ ID NO: 78 as set forth in Table 16; and a VL region comprising (1) a VL CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NO: 68; (2) a VL CDR2 having an amino acid sequence of SEQ ID NO: 16; and (3) a VL CDR3 having an amino acid sequence of SEQ ID NO: 17.
  • the two-in-one antibodies provided herein comprise a VH consisting of SEQ ID NO: 79 as set forth in Table 16; and a VL region comprising (1) a VL CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NO: 68; (2) a VL CDR2 having an amino acid sequence of SEQ ID NO: 16; and (3) a VL CDR3 having an amino acid sequence of SEQ ID NO: 17.
  • the two-in-one antibody or antigen-binding fragment thereof comprises a VL comprising an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%sequence identity, or at least about 99%sequence identity to SEQ ID NO: 27. In some embodiments, the two-in-one antibody or antigen-binding fragment thereof comprises a VL comprising an amino acid sequence having of SEQ ID NO: 27.
  • the two-in-one antibody or antigen-binding fragment thereof comprises a VL comprising an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%sequence identity, or at least about 99%sequence identity to SEQ ID NO: 74. In some embodiments, the two-in-one antibody or antigen-binding fragment thereof comprises a VL comprising an amino acid sequence having of SEQ ID NO: 74.
  • the two-in-one antibody or antigen-binding fragment thereof comprises a VH comprising an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%sequence identity, or at least about 99%sequence identity to SEQ ID NO: 28. In some embodiments, the two-in-one antibody or antigen-binding fragment thereof comprises a VH comprising an amino acid sequence having of SEQ ID NO: 28.
  • the two-in-one antibody or antigen-binding fragment thereof comprises a VH comprising an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%sequence identity, or at least about 99%sequence identity to SEQ ID NO: 75. In some embodiments, the two-in-one antibody or antigen-binding fragment thereof comprises a VH comprising an amino acid sequence having of SEQ ID NO: 75.
  • the two-in-one antibody or antigen-binding fragment thereof comprises a VH comprising an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%sequence identity, or at least about 99%sequence identity to SEQ ID NO: 76. In some embodiments, the two-in-one antibody or antigen-binding fragment thereof comprises a VH comprising an amino acid sequence having of SEQ ID NO: 76.
  • the two-in-one antibody or antigen-binding fragment thereof comprises a VH comprising an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%sequence identity, or at least about 99%sequence identity to SEQ ID NO: 77. In some embodiments, the two-in-one antibody or antigen-binding fragment thereof comprises a VH comprising an amino acid sequence having of SEQ ID NO: 77.
  • the two-in-one antibody or antigen-binding fragment thereof comprises a VH comprising an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%sequence identity, or at least about 99%sequence identity to SEQ ID NO: 78. In some embodiments, the two-in-one antibody or antigen-binding fragment thereof comprises a VH comprising an amino acid sequence having of SEQ ID NO: 78.
  • the two-in-one antibody or antigen-binding fragment thereof comprises a VH comprising an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%sequence identity, or at least about 99%sequence identity to SEQ ID NO: 79. In some embodiments, the two-in-one antibody or antigen-binding fragment thereof comprises a VH comprising an amino acid sequence having of SEQ ID NO: 79.
  • the two-in-one antibody or antigen-binding fragment thereof comprises a VL comprising an amino acid sequence having at least about 95%sequence identity to SEQ ID NO: 27, and a VH comprising an amino acid sequence having at least about 95%sequence identity to SEQ ID NO: 28.
  • the two-in-one antibody or antigen-binding fragment thereof comprises a VL comprising an amino acid sequence having of SEQ ID NO: 27 and a VL comprising an amino acid sequence having of SEQ ID NO: 28.
  • the two-in-one antibody or antigen-binding fragment thereof comprises a VL comprising an amino acid sequence having at least about 95%sequence identity to SEQ ID NO: 74, and a VH comprising an amino acid sequence having at least about 95%sequence identity to SEQ ID NO: 28.
  • the two-in-one antibody or antigen-binding fragment thereof comprises a VL comprising an amino acid sequence having of SEQ ID NO: 74 and a VL comprising an amino acid sequence having of SEQ ID NO: 28.
  • the two-in-one antibody or antigen-binding fragment thereof comprises a VL comprising an amino acid sequence having at least about 95%sequence identity to SEQ ID NO: 27, and a VH comprising an amino acid sequence having at least about 95%sequence identity to SEQ ID NO: 75.
  • the two-in-one antibody or antigen-binding fragment thereof comprises a VL comprising an amino acid sequence having of SEQ ID NO: 27 and a VL comprising an amino acid sequence having of SEQ ID NO: 75.
  • the two-in-one antibody or antigen-binding fragment thereof comprises a VL comprising an amino acid sequence having at least about 95%sequence identity to SEQ ID NO: 74, and a VH comprising an amino acid sequence having at least about 95%sequence identity to SEQ ID NO: 75.
  • the two-in-one antibody or antigen-binding fragment thereof comprises a VL comprising an amino acid sequence having of SEQ ID NO: 74 and a VL comprising an amino acid sequence having of SEQ ID NO: 75.
  • the two-in-one antibody or antigen-binding fragment thereof comprises a VL comprising an amino acid sequence having at least about 95%sequence identity to SEQ ID NO: 27, and a VH comprising an amino acid sequence having at least about 95%sequence identity to SEQ ID NO: 76.
  • the two-in-one antibody or antigen-binding fragment thereof comprises a VL comprising an amino acid sequence having of SEQ ID NO: 27 and a VL comprising an amino acid sequence having of SEQ ID NO: 76.
  • the two-in-one antibody or antigen-binding fragment thereof comprises a VL comprising an amino acid sequence having at least about 95%sequence identity to SEQ ID NO: 74, and a VH comprising an amino acid sequence having at least about 95%sequence identity to SEQ ID NO: 76.
  • the two-in-one antibody or antigen-binding fragment thereof comprises a VL comprising an amino acid sequence having of SEQ ID NO: 74 and a VL comprising an amino acid sequence having of SEQ ID NO: 76.
  • the two-in-one antibody or antigen-binding fragment thereof comprises a VL comprising an amino acid sequence having at least about 95%sequence identity to SEQ ID NO: 27, and a VH comprising an amino acid sequence having at least about 95%sequence identity to SEQ ID NO: 77.
  • the two-in-one antibody or antigen-binding fragment thereof comprises a VL comprising an amino acid sequence having of SEQ ID NO: 27 and a VL comprising an amino acid sequence having of SEQ ID NO: 77.
  • the two-in-one antibody or antigen-binding fragment thereof comprises a VL comprising an amino acid sequence having at least about 95%sequence identity to SEQ ID NO: 74, and a VH comprising an amino acid sequence having at least about 95%sequence identity to SEQ ID NO: 77.
  • the two-in-one antibody or antigen-binding fragment thereof comprises a VL comprising an amino acid sequence having of SEQ ID NO: 74 and a VL comprising an amino acid sequence having of SEQ ID NO: 77.
  • the two-in-one antibody or antigen-binding fragment thereof comprises a VL comprising an amino acid sequence having at least about 95%sequence identity to SEQ ID NO: 27, and a VH comprising an amino acid sequence having at least about 95%sequence identity to SEQ ID NO: 78.
  • the two-in-one antibody or antigen-binding fragment thereof comprises a VL comprising an amino acid sequence having of SEQ ID NO: 27 and a VL comprising an amino acid sequence having of SEQ ID NO: 78.
  • the two-in-one antibody or antigen-binding fragment thereof comprises a VL comprising an amino acid sequence having at least about 95%sequence identity to SEQ ID NO: 74, and a VH comprising an amino acid sequence having at least about 95%sequence identity to SEQ ID NO: 78.
  • the two-in-one antibody or antigen-binding fragment thereof comprises a VL comprising an amino acid sequence having of SEQ ID NO: 74 and a VL comprising an amino acid sequence having of SEQ ID NO: 78.
  • the two-in-one antibody or antigen-binding fragment thereof comprises a VL comprising an amino acid sequence having at least about 95%sequence identity to SEQ ID NO: 27, and a VH comprising an amino acid sequence having at least about 95%sequence identity to SEQ ID NO: 79.
  • the two-in-one antibody or antigen-binding fragment thereof comprises a VL comprising an amino acid sequence having of SEQ ID NO: 27 and a VL comprising an amino acid sequence having of SEQ ID NO: 79.
  • the two-in-one antibody or antigen-binding fragment thereof comprises a VL comprising an amino acid sequence having at least about 95%sequence identity to SEQ ID NO: 74, and a VH comprising an amino acid sequence having at least about 95%sequence identity to SEQ ID NO: 79.
  • the two-in-one antibody or antigen-binding fragment thereof comprises a VL comprising an amino acid sequence having of SEQ ID NO: 74 and a VL comprising an amino acid sequence having of SEQ ID NO: 79.
  • Table 15 Two-In-One Antibody VL Domain Amino Acid Sequences.
  • a two-in-one antibody or antigen binding fragment described in this Section 5.3.1 can be used as a masking moiety in a TNF- ⁇ containing immunoconjugate molecule described herein.
  • an anti-TNF- ⁇ /anti-Trop2 two-in-one antibody or antigen binding fragment thereof comprises a VH region, VL region, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and/or VL CDR3 of any one of the two-in-one antibodies provided herein, such as an amino acid sequence depicted in Tables 17-18.
  • the two-in-one antibody or functional fragment thereof provided herein comprises one, two, and/or three heavy chain CDRs and/or one, two, and/or three light chain CDRs from the antibody TN01 as shown in Tables 17-18.
  • a two-in-one antibody provided herein comprises or consists of six CDRs, for example, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and/or VL CDR3 identified in Tables 17-18.
  • the two-in-one antibody comprises or consists of one, two, three, four, or five CDRs selected from the group consisting of VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and/or VL CDR3 identified in Tables 17-18.
  • the two-in-one antibody comprises or consists of one, two, three, four, or five CDRs of anyone of the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and/or VL CDR3 identified in Tables 17-18.
  • the two-in-one antibodies provided herein comprise one or more (e.g., one, two, or three) VL CDRs listed in Table 17. In some embodiments, the two-in-one antibodies provided herein comprise one or more (e.g., one, two, or three) VH CDRs listed in Table 18. In yet other embodiments, the two-in-one antibodies provided herein comprise one or more (e.g., one, two, or three) VL CDRs listed in Table 17 and one or more (e.g., one, two, or three) VH CDRs listed in Table 18.
  • the two-in-one antibodies comprise a VL CDR1 having an amino acid sequence of any one of SEQ ID NO: 21.
  • the two-in-one antibodies comprise a VL CDR2 having an amino acid sequence of SEQ ID NO: 22.
  • the two-in-one antibodies comprise a VL CDR3 having an amino acid sequence of SEQ ID NO: 23.
  • the two-in-one antibodies comprise a VL CDR1 and/or a VL CDR2 and/or a VL CDR3 independently selected from any one of the VL CDR1, VL CDR2, VL CDR3 amino acid sequences as depicted in Table 17.
  • the two-in-one antibodies comprise a VH CDR1 having an amino acid sequence of SEQ ID NO: 24. In some embodiments, the two-in-one antibodies comprise a VH CDR2 having an amino acid sequence of SEQ ID NO: 25. In some embodiments, the two-in-one antibodies comprise a VH CDR3 having an amino acid sequence of SEQ ID NO: 26. In some embodiments, the two-in-one antibodies comprise a VH CDR1 and/or a VH CDR2 and/or a VH CDR3 independently selected from any one of the VH CDR1, VH CDR2, VH CDR3 amino acid sequence (s) as depicted in Table 18.
  • two-in-one antibodies comprising one or more (e.g., one, two, or three) VH CDRs and one or more (e.g., one, two, or three) VL CDRs listed in Tables 17-18.
  • a two-in-one antibody comprising a VH CDR1 (SEQ ID NO: 24) and a VL CDR1 (SEQ ID NO: 21) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 24) and a VL CDR2 (SEQ ID NO: 22) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 24) and a VL CDR3 (SEQ ID NO: 23) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 25) and a VL CDR1 (SEQ ID NO: 21) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 25) and a VL CDR2 (SEQ ID NO: 22) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 25) and a VL CDR3 (SEQ ID NO: 23) .
  • the two-in-one antibody comprises a VH CDR3 (SEQ ID NO: 26) and a VL CDR1 (SEQ ID NO: 21) . In other embodiments, the two-in-one antibody comprises a VH CDR3 (SEQ ID NO: 26) and a VL CDR2 (SEQ ID NO: 22) . In some embodiments, the two-in-one antibody comprises a VH CDR3 (SEQ ID NO: 26) and a VL CDR3 (SEQ ID NO: 23) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 24) , a VH CDR2 (SEQ ID NO: 25) , and a VL CDR1 (SEQ ID NO: 21) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 24) , a VH CDR2 (SEQ ID NO: 25) , and a VL CDR2 (SEQ ID NO: 22) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 24) , a VH CDR2 (SEQ ID NO: 25) , and a VL CDR3 (SEQ ID NO: 23) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 25) , a VH CDR3 (SEQ ID NO: 26) , and a VL CDR1 (SEQ ID NO: 21) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 25) , a VH CDR3 (SEQ ID NO: 26) , and a VL CDR2 (SEQ ID NO: 22) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 25) , a VH CDR3 (SEQ ID NO: 26) , and a VL CDR3 (SEQ ID NO: 23) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 24) , a VH CDR3 (SEQ ID NO: 26) , and a VL CDR1 (SEQ ID NO: 21) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 24) , a VH CDR3 (SEQ ID NO: 26) , and a VL CDR2 (SEQ ID NO: 22) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 24) , a VH CDR3 (SEQ ID NO: 26) , and a VL CDR3 (SEQ ID NO: 23) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 24) , a VL CDR1 (SEQ ID NO: 21) , and a VL CDR2 (SEQ ID NO: 22) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 24) , a VL CDR1 (SEQ ID NO: 21) , and a VL CDR3 (SEQ ID NO: 23) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 24) , a VL CDR2 (SEQ ID NO: 22) , and a VL CDR3 (SEQ ID NO: 23) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 25) , a VL CDR1 (SEQ ID NO: 21) , and a VL CDR2 (SEQ ID NO: 22) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 25) , a VL CDR1 (SEQ ID NO: 21) , and a VL CDR3 (SEQ ID NO: 23) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 25) , a VL CDR2 (SEQ ID NO: 22) , and a VL CDR3 (SEQ ID NO: 23) .
  • the two-in-one antibody comprises a VH CDR3 (SEQ ID NO: 26) , a VL CDR1 (SEQ ID NO: 21) , and a VL CDR2 (SEQ ID NO: 22) .
  • the two-in-one antibody comprises a VH CDR3 (SEQ ID NO: 26) , a VL CDR1 (SEQ ID NO: 21) , and a VL CDR3 (SEQ ID NO: 23) .
  • the two-in-one antibody comprises a VH CDR3 (SEQ ID NO: 26) , a VL CDR2 (SEQ ID NO: 22) , and a VL CDR3 (SEQ ID NO: 23) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 24) , a VH CDR2 (SEQ ID NO: 25) , a VH CDR3 (SEQ ID NO: 26) , and a VL CDR1 (SEQ ID NO: 21) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 24) , a VH CDR2 (SEQ ID NO: 25) , a VH CDR3 (SEQ ID NO: 26) , and a VL CDR2 (SEQ ID NO: 22) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 24) , a VH CDR2 (SEQ ID NO: 25) , a VH CDR3 (SEQ ID NO: 26) , and a VL CDR3 (SEQ ID NO: 23) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 24) , a VH CDR2 (SEQ ID NO: 25) , a VL CDR1 (SEQ ID NO: 21) , and a VL CDR2 (SEQ ID NO: 22) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 24) , a VH CDR2 (SEQ ID NO: 25) , a VL CDR1 (SEQ ID NO: 21) , and a VL CDR3 (SEQ ID NO: 23) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 24) , a VH CDR2 (SEQ ID NO: 25) , a VL CDR2 (SEQ ID NO: 22) , and a VL CDR3 (SEQ ID NO: 23) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 24) , a VH CDR3 (SEQ ID NO: 26) , a VL CDR1 (SEQ ID NO: 21) , and a VL CDR2 (SEQ ID NO: 22) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 24) , a VH CDR3 (SEQ ID NO: 26) , a VL CDR1 (SEQ ID NO: 21) , and a VL CDR3 (SEQ ID NO: 23) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 24) , a VH CDR3 (SEQ ID NO: 26) , a VL CDR2 (SEQ ID NO: 22) , and a VL CDR3 (SEQ ID NO: 23) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 25) , a VH CDR3 (SEQ ID NO: 26) , a VL CDR1 (SEQ ID NO: 21) , and a VL CDR2 (SEQ ID NO: 22) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 25) , a VH CDR3 (SEQ ID NO: 26) , a VL CDR1 (SEQ ID NO: 21) , and a VL CDR3 (SEQ ID NO: 23) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 25) , a VH CDR3 (SEQ ID NO: 26) , a VL CDR2 (SEQ ID NO: 22) , and a VL CDR3 (SEQ ID NO: 23) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 24) , a VH CDR2 (SEQ ID NO: 25) , a VH CDR3 (SEQ ID NO: 26) , a VL CDR1 (SEQ ID NO: 21) , and a VL CDR2 (SEQ ID NO: 22) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 24) , a VH CDR2 (SEQ ID NO: 25) , a VH CDR3 (SEQ ID NO: 26) , a VL CDR1 (SEQ ID NO: 21) , and a VL CDR3 (SEQ ID NO: 23) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 24) , a VH CDR2 (SEQ ID NO: 25) , a VH CDR3 (SEQ ID NO: 26) , a VL CDR2 (SEQ ID NO: 22) , and a VL CDR3 (SEQ ID NO: 23) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 24) , a VH CDR2 (SEQ ID NO: 25) , a VL CDR1 (SEQ ID NO: 21) , a VL CDR2 (SEQ ID NO: 22) , and a VL CDR3 (SEQ ID NO: 23) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 24) , a VH CDR3 (SEQ ID NO: 26) , a VL CDR1 (SEQ ID NO: 21) , a VL CDR2 (SEQ ID NO: 22) , and a VL CDR3 (SEQ ID NO: 23) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 25) , a VH CDR3 (SEQ ID NO: 26) , a VL CDR1 (SEQ ID NO: 21) , a VL CDR2 (SEQ ID NO: 22) , and a VL CDR3 (SEQ ID NO: 23) .
  • the two-in-one antibody comprises a VH CDR1 (SEQ ID NO: 24) , a VL CDR1 (SEQ ID NO: 21) , a VL CDR2 (SEQ ID NO: 22) , and a VL CDR3 (SEQ ID NO: 23) .
  • the two-in-one antibody comprises a VH CDR2 (SEQ ID NO: 25) , a VL CDR1 (SEQ ID NO: 21) , a VL CDR2 (SEQ ID NO: 22) , and a VL CDR3 (SEQ ID NO: 23) .
  • the two-in-one antibody comprises a VH CDR3 (SEQ ID NO: 26) , a VL CDR1 (SEQ ID NO: 21) , a VL CDR2 (SEQ ID NO: 22) , and a VL CDR3 (SEQ ID NO: 23) .
  • the two-in-one antibody comprises any combination thereof of the VH CDRs and VL CDRs listed in Tables 17-18.
  • the two-in-one antibodies provided herein comprise a VH region comprising (1) a VH CDR1 having an amino acid sequence of SEQ ID NO: 24; (2) a VH CDR2 having an amino acid sequence of SEQ ID NO: 25; and (3) a VH CDR3 having an amino acid sequence of SEQ ID NO: 26.
  • the two-in-one antibodies provided herein comprise a VL region comprising: (1) a VL CDR1 having an amino acid sequence of SEQ ID NO: 21; (2) a VL CDR2 having an amino acid sequence of SEQ ID NO: 22; and (3) a VL CDR3 having an amino acid sequence of SEQ ID NO: 23.
  • the two-in-one antibodies provided herein comprise a VH region comprising (1) a VH CDR1 having an amino acid sequence of SEQ ID NO: 24; (2) a VH CDR2 having an amino acid sequence of SEQ ID NO: 25; and (3) a VH CDR3 having an amino acid sequence of SEQ ID NO: 26; and a VL region comprising: (1) a VL CDR1 having an amino acid sequence of SEQ ID NO: 21; (2) a VL CDR2 having an amino acid sequence of SEQ ID NO: 22; and (3) a VL CDR3 having an amino acid sequence of SEQ ID NO: 23.
  • the two-in-one antibodies provided herein comprise a VH region or VH domain. In other embodiments, the two-in-one antibodies provided herein comprise a VL region or VL domain. In certain embodiments, the two-in-one antibodies provided herein have a combination of (i) a VH domain or VH region; and/or (ii) a VL domain or VL region. In yet other embodiments, the two-in-one antibodies provided herein have a combination of (i) a VH domain or VH region; and/or (ii) a VL domain or VL region selected from the group consisting of SEQ ID NOS: 29 and 30 as set forth in Tables 19-20.
  • the two-in-one antibodies provided herein comprise a VH region comprising (1) a VH CDR1 having an amino acid sequence of SEQ ID NO: 24; (2) a VH CDR2 having an amino acid sequence of SEQ ID NO: 25; and (3) a VH CDR3 having an amino acid sequence of SEQ ID NO: 26; and a VL region as set forth in Table 19.
  • the VL region has an amino acid sequence of SEQ ID NO: 29.
  • the two-in-one antibodies provided herein comprise a VH consisting of SEQ ID NO: 30 as set forth in Table 20; and a VL region comprising (1) a VL CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NO: 21; (2) a VL CDR2 having an amino acid sequence of SEQ ID NO: 22; and (3) a VL CDR3 having an amino acid sequence of SEQ ID NO: 23.
  • the two-in-one antibody or antigen-binding fragment thereof comprises a VL comprising an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%sequence identity, or at least about 99%sequence identity to SEQ ID NO: 29. In some embodiments, the two-in-one antibody or antigen-binding fragment thereof comprises a VL comprising an amino acid sequence having of SEQ ID NO: 29.
  • the two-in-one antibody or antigen-binding fragment thereof comprises a VH comprising an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%sequence identity, or at least about 99%sequence identity to SEQ ID NO: 30. In some embodiments, the two-in-one antibody or antigen-binding fragment thereof comprises a VH comprising an amino acid sequence having of SEQ ID NO: 30.
  • the two-in-one antibody or antigen-binding fragment thereof comprises a VL comprising an amino acid sequence having at least about 95%sequence identity to SEQ ID NO: 29, and a VH comprising an amino acid sequence having at least about 95%sequence identity to SEQ ID NO: 30.
  • the two-in-one antibody or antigen-binding fragment thereof comprises a VL comprising an amino acid sequence having of SEQ ID NO: 29 and a VL comprising an amino acid sequence having of SEQ ID NO: 30.
  • a two-in-one antibody or antigen binding fragment described in this Section 5.3.2 can be used as a masking moiety in a TNF- ⁇ containing immunoconjugate molecule described herein.
  • the present disclosure provides antibodies or antigen binding fragment thereof that specifically binds to a tumor-associated antigen (TAA) , which in some embodiments, can find use herein for generating the present immunoconjugate molecules, e.g., as an anchoring moiety.
  • TAA tumor-associated antigen
  • Exemplary antibodies include polyclonal, monoclonal, humanized, human, bispecific, and heteroconjugate antibodies, as well as variants thereof having improved affinity or other properties.
  • the antibody or antigen binding fragment thereof specifically binds to FAP.
  • the anti-FAP antibody comprises a VH region, VL region, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and/or VL CDR3 of any one of the anti-FAP antibodies provided herein, such as an amino acid sequence depicted in Tables 21-22.
  • the anti-FAP antibody or antigen binding fragment thereof provided herein comprises one, two, and/or three heavy chain CDRs and/or one, two, and/or three light chain CDRs from the antibodies FAP1, or E21 as shown in Tables 21-22.
  • an anti-FAP antibody provided herein comprises or consists of six CDRs, for example, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and/or VL CDR3 identified in Tables 21-22.
  • the anti-FAP antibody comprises or consists of one, two, three, four, or five CDRs selected from the group consisting of VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and/or VL CDR3 identified in Tables 21-22.
  • the anti-FAP antibody comprises or consists of one, two, three, four, or five CDRs selected from the group consisting of VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and/or VL CDR3 of the anti-FAP described herein. Accordingly, in some embodiments, the anti-FAP antibody comprises or consists of one, two, three, four, or five CDRs of anyone of the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and/or VL CDR3 identified in Tables 21-22.
  • the anti-FAP antibodies provided herein comprise one or more (e.g., one, two, or three) VH CDRs listed in Table 22. In other embodiments, the anti-FAP antibodies provided herein comprise one or more (e.g., one, two, or three) VL CDRs listed in Table 21. In yet other embodiments, the anti-FAP antibodies provided herein comprise one or more (e.g., one, two, or three) VH CDRs listed in Table 22 and one or more VL CDRs listed in Table 21.
  • the anti-FAP antibodies comprise a VH CDR1 having an amino acid sequence of SEQ ID NO: 37.
  • the anti-FAP antibodies comprise a VH CDR2 having an amino acid sequence of SEQ ID NO: 38.
  • the anti-FAP antibodies comprise a VH CDR3 having an amino acid sequence of SEQ ID NO: 39.
  • the anti-FAP antibodies comprise a VH CDR1 and/or a VH CDR2 and/or a VH CDR3 independently selected from any one of the VH CDR1, VH CDR2, VH CDR3 amino acid sequence (s) as depicted in Table 22.
  • the anti-FAP antibodies comprise a VL CDR1 having an amino acid sequence of any one of SEQ ID NO: 31. In another embodiment, the anti-FAP antibodies comprise a VL CDR2 having an amino acid sequence of SEQ ID NO: 32. In some embodiments, the anti-FAP antibodies comprise a VL CDR3 having an amino acid sequence of SEQ ID NO: 33. In some embodiments, the anti-FAP antibodies comprise a VL CDR1 and/or a VL CDR2 and/or a VL CDR3 independently selected from any one of the VL CDR1, VL CDR2, VL CDR3 amino acid sequences as depicted in Table 21.
  • anti-FAP antibodies comprising one or more (e.g., one, two, or three) VH CDRs and one or more (e.g., one, two, or three) VL CDRs listed in Tables 21-22.
  • an anti-FAP antibody comprising a VH CDR1 (SEQ ID NO: 37) and a VL CDR1 (SEQ ID NO: 31) .
  • the anti-FAP antibody comprises a VH CDR1 (SEQ ID NO: 37) and a VL CDR2 (SEQ ID NO: 32) .
  • the anti-FAP antibody comprises a VH CDR1 (SEQ ID NO: 37) and a VL CDR3 (SEQ ID NO: 33) .
  • the anti-FAP antibody comprises a VH CDR2 (SEQ ID NO: 38) and a VL CDR1 (SEQ ID NO: 31) .
  • the anti-FAP antibody comprises a VH CDR2 (SEQ ID NO: 38) and a VL CDR2 (SEQ ID NO: 32) .
  • the anti-FAP antibody comprises a VH CDR2 (SEQ ID NO: 38) and a VL CDR3 (SEQ ID NO: 33) .
  • the anti-FAP antibody comprises a VH CDR3 (SEQ ID NO: 39) and a VL CDR1 (SEQ ID NO: 31) . In other embodiments, the anti-FAP antibody comprises a VH CDR3 (SEQ ID NO: 39) and a VL CDR2 (SEQ ID NO: 32) . In some embodiments, the anti-FAP antibody comprises a VH CDR3 (SEQ ID NO: 39) and a VL CDR3 (SEQ ID NO: 33) .
  • the anti-FAP antibody comprises a VH CDR1 (SEQ ID NO: 37) , a VH CDR2 (SEQ ID NO: 38) , and a VL CDR1 (SEQ ID NO: 31) .
  • the anti-FAP antibody comprises a VH CDR1 (SEQ ID NO: 37) , a VH CDR2 (SEQ ID NO: 38) , and a VL CDR2 (SEQ ID NO: 32) .
  • the anti-FAP antibody comprises a VH CDR1 (SEQ ID NO: 37) , a VH CDR2 (SEQ ID NO: 38) , and a VL CDR3 (SEQ ID NO: 33) .
  • the anti-FAP antibody comprises a VH CDR2 (SEQ ID NO: 38) , a VH CDR3 (SEQ ID NO: 39) , and a VL CDR1 (SEQ ID NO: 31) .
  • the anti-FAP antibody comprises a VH CDR2 (SEQ ID NO: 38) , a VH CDR3 (SEQ ID NO: 39) , and a VL CDR2 (SEQ ID NO: 32) .
  • the anti-FAP antibody comprises a VH CDR2 (SEQ ID NO: 38) , a VH CDR3 (SEQ ID NO: 39) , and a VL CDR3 (SEQ ID NO: 33) .
  • the anti-FAP antibody comprises a VH CDR1 (SEQ ID NO: 37) , a VH CDR3 (SEQ ID NO: 39) , and a VL CDR1 (SEQ ID NO: 31) .
  • the anti-FAP antibody comprises a VH CDR1 (SEQ ID NO: 37) , a VH CDR3 (SEQ ID NO: 39) , and a VL CDR2 (SEQ ID NO: 32) .
  • the anti-FAP antibody comprises a VH CDR1 (SEQ ID NO: 37) , a VH CDR3 (SEQ ID NO: 39) , and a VL CDR3 (SEQ ID NO: 33) .
  • the anti-FAP antibody comprises a VH CDR1 (SEQ ID NO: 37) , a VL CDR1 (SEQ ID NO: 31) , and a VL CDR2 (SEQ ID NO: 32) .
  • the anti-FAP antibody comprises a VH CDR1 (SEQ ID NO: 37) , a VL CDR1 (SEQ ID NO: 31) , and a VL CDR3 (SEQ ID NO: 33) .
  • the anti-FAP antibody comprises a VH CDR1 (SEQ ID NO: 37) , a VL CDR2 (SEQ ID NO: 32) , and a VL CDR3 (SEQ ID NO: 33) .
  • the anti-FAP antibody comprises a VH CDR2 (SEQ ID NO: 38) , a VL CDR1 (SEQ ID NO: 31) , and a VL CDR2 (SEQ ID NO: 32) .
  • the anti-FAP antibody comprises a VH CDR2 (SEQ ID NO: 38) , a VL CDR1 (SEQ ID NO: 31) , and a VL CDR3 (SEQ ID NO: 33) .
  • the anti-FAP antibody comprises a VH CDR2 (SEQ ID NO: 38) , a VL CDR2 (SEQ ID NO: 32) , and a VL CDR3 (SEQ ID NO: 33) .
  • the anti-FAP antibody comprises a VH CDR3 (SEQ ID NO: 39) , a VL CDR1 (SEQ ID NO: 31) , and a VL CDR2 (SEQ ID NO: 32) .
  • the anti-FAP antibody comprises a VH CDR3 (SEQ ID NO: 39) , a VL CDR1 (SEQ ID NO: 31) , and a VL CDR3 (SEQ ID NO: 33) .
  • the anti-FAP antibody comprises a VH CDR3 (SEQ ID NO: 39) , a VL CDR2 (SEQ ID NO: 32) , and a VL CDR3 (SEQ ID NO: 33) .
  • the anti-FAP antibody comprises a VH CDR1 (SEQ ID NO: 37) , a VH CDR2 (SEQ ID NO: 38) , a VH CDR3 (SEQ ID NO: 39) , and a VL CDR1 (SEQ ID NO: 31) .
  • the anti-FAP antibody comprises a VH CDR1 (SEQ ID NO: 37) , a VH CDR2 (SEQ ID NO: 38) , a VH CDR3 (SEQ ID NO: 39) , and a VL CDR2 (SEQ ID NO: 32) .
  • the anti-FAP antibody comprises a VH CDR1 (SEQ ID NO: 37) , a VH CDR2 (SEQ ID NO: 38) , a VH CDR3 (SEQ ID NO: 39) , and a VL CDR3 (SEQ ID NO: 33) .
  • the anti-FAP antibody comprises a VH CDR1 (SEQ ID NO: 37) , a VH CDR2 (SEQ ID NO: 38) , a VL CDR1 (SEQ ID NO: 31) , and a VL CDR2 (SEQ ID NO: 32) .
  • the anti-FAP antibody comprises a VH CDR1 (SEQ ID NO: 37) , a VH CDR2 (SEQ ID NO: 38) , a VL CDR1 (SEQ ID NO: 31) , and a VL CDR3 (SEQ ID NO: 33) .
  • the anti-FAP antibody comprises a VH CDR1 (SEQ ID NO: 37) , a VH CDR2 (SEQ ID NO: 38, a VL CDR2 (SEQ ID NO: 32) , and a VL CDR3 (SEQ ID NO: 33) .
  • the anti-FAP antibody comprises a VH CDR1 (SEQ ID NO: 37) , a VH CDR3 (SEQ ID NO: 39) , a VL CDR1 (SEQ ID NO: 31) , and a VL CDR2 (SEQ ID NO: 32) .
  • the anti-FAP antibody comprises a VH CDR1 (SEQ ID NO: 37) , a VH CDR3 (SEQ ID NO: 39) , a VL CDR1 (SEQ ID NO: 31) , and a VL CDR3 (SEQ ID NO: 33) .
  • the anti-FAP antibody comprises a VH CDR1 (SEQ ID NO: 37) , a VH CDR3 (SEQ ID NO: 39) , a VL CDR2 (SEQ ID NO: 32) , and a VL CDR3 (SEQ ID NO: 33) .
  • the anti-FAP antibody comprises a VH CDR2 (SEQ ID NO: 38) , a VH CDR3 (SEQ ID NO: 39) , a VL CDR1 (SEQ ID NO: 31) , and a VL CDR2 (SEQ ID NO: 32) .
  • the anti-FAP antibody comprises a VH CDR2 (SEQ ID NO: 38) , a VH CDR3 (SEQ ID NO: 39) , a VL CDR1 (SEQ ID NO: 31) , and a VL CDR3 (SEQ ID NO: 33) .
  • the anti-FAP antibody comprises a VH CDR2 (SEQ ID NO: 38) , a VH CDR3 (SEQ ID NO: 39) , a VL CDR2 (SEQ ID NO: 32) , and a VL CDR3 (SEQ ID NO: 33) .
  • the anti-FAP antibody comprises a VH CDR1 (SEQ ID NO: 37) , a VH CDR2 (SEQ ID NO: 38) , a VH CDR3 (SEQ ID NO: 39) , a VL CDR1 (SEQ ID NO: 31) , and a VL CDR2 (SEQ ID NO: 32) .
  • the anti-FAP antibody comprises a VH CDR1 (SEQ ID NO: 37) , a VH CDR2 (SEQ ID NO: 38) , a VH CDR3 (SEQ ID NO: 39) , a VL CDR1 (SEQ ID NO: 31) , and a VL CDR3 (SEQ ID NO: 33) .
  • the anti-FAP antibody comprises a VH CDR1 (SEQ ID NO: 37) , a VH CDR2 (SEQ ID NO: 38) , a VH CDR3 (SEQ ID NO: 39) , a VL CDR2 (SEQ ID NO: 32) , and a VL CDR3 (SEQ ID NO: 33) .
  • the anti-FAP antibody comprises a VH CDR1 (SEQ ID NO: 37) , a VH CDR2 (SEQ ID NO: 38) , a VL CDR1 (SEQ ID NO: 31) , a VL CDR2 (SEQ ID NO: 32) , and a VL CDR3 (SEQ ID NO: 33) .
  • the anti-FAP antibody comprises a VH CDR1 (SEQ ID NO: 37) , a VH CDR3 (SEQ ID NO: 39) , a VL CDR1 (SEQ ID NO: 31) , a VL CDR2 (SEQ ID NO: 32) , and a VL CDR3 (SEQ ID NO: 33) .
  • the anti-FAP antibody comprises a VH CDR2 (SEQ ID NO: 38) , a VH CDR3 (SEQ ID NO: 39) , a VL CDR1 (SEQ ID NO: 31) , a VL CDR2 (SEQ ID NO: 32) , and a VL CDR3 (SEQ ID NO: 33) .
  • the anti-FAP antibody comprises a VH CDR1 (SEQ ID NO: 37) , a VL CDR1 (SEQ ID NO: 31) , a VL CDR2 (SEQ ID NO: 32) , and a VL CDR3 (SEQ ID NO: 33) .
  • the anti-FAP antibody comprises a VH CDR2 (SEQ ID NO: 38) , a VL CDR1 (SEQ ID NO: 31) , a VL CDR2 (SEQ ID NO: 32) , and a VL CDR3 (SEQ ID NO: 33) .
  • the anti-FAP antibody comprises a VH CDR3 (SEQ ID NO: 39) , a VL CDR1 (SEQ ID NO: 31) , a VL CDR2 (SEQ ID NO: 32) , and a VL CDR3 (SEQ ID NO: 33) .
  • the anti-FAP antibody comprises any combination thereof of the VH CDRs and VL CDRs listed in Tables 21-22.
  • the anti-FAP antibodies provided herein comprise a VH region comprising: (1) a VH CDR1 having an amino acid sequence of SEQ ID NO: 37; (2) a VH CDR2 having an amino acid sequence of SEQ ID NO: 38; and (3) a VH CDR3 having an amino acid sequence of SEQ ID NO: 39.
  • the anti-FAP antibodies provided herein comprise a VL region comprising: (1) a VL CDR1 having an amino acid sequence of SEQ ID NO: 31; (2) a VL CDR2 having an amino acid sequence of SEQ ID NO: 32; and (3) a VL CDR3 having an amino acid sequence of SEQ ID NO: 33.
  • the anti-FAP antibodies provided herein comprise a VH region comprising (1) a VH CDR1 having an amino acid sequence of SEQ ID NO: 37; (2) a VH CDR2 having an amino acid sequence of SEQ ID NO: 38; and (3) a VH CDR3 having an amino acid sequence of SEQ ID NO: 39; and a VL region comprising: (1) a VL CDR1 having an amino acid sequence of SEQ ID NO: 31; (2) a VL CDR2 having an amino acid sequence of SEQ ID NO: 32; and (3) a VL CDR3 having an amino acid sequence of SEQ ID NO: 33.
  • the anti-FAP antibodies provided herein comprise a VH region or VH domain. In other embodiments, the anti-FAP antibodies provided herein comprise a VL region or VL domain. In certain embodiments, the anti-FAP antibodies provided herein have a combination of (i) a VH domain or VH region; and/or (ii) a VL domain or VL region. In yet other embodiments, the anti-FAP antibodies provided herein have a combination of (i) a VH domain or VH region consisting of SEQ ID NO: 47; and/or (ii) a VL domain or VL region consisting of SEQ ID NO: 46 as set forth in Tables 23-24.
  • the anti-FAP antibodies provided herein comprise a VH region comprising (1) a VH CDR1 having an amino acid sequence of SEQ ID NO: 37; (2) a VH CDR2 having an amino acid sequence of SEQ ID NO: 38; and (3) a VH CDR3 having an amino acid sequence of SEQ ID NO: 39; and a VL region as set forth in Table 23.
  • the VL region has an amino acid sequence of SEQ ID NO: 46.
  • the anti-FAP antibodies provided herein comprise a VH consisting of SEQ ID NO: 47 as set forth in Table 24; and a VL region comprising (1) a VL CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NO: 31; (2) a VL CDR2 having an amino acid sequence of SEQ ID NO: 32; and (3) a VL CDR3 having an amino acid sequence of SEQ ID NO: 33.
  • the anti-FAP antibody or antigen-binding fragment thereof comprises a VL comprising an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%sequence identity, or at least about 99%sequence identity to SEQ ID NO: 46. In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises a VL comprising an amino acid sequence having of SEQ ID NO: 46.
  • the anti-FAP antibody or antigen-binding fragment thereof comprises a VH comprising an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%sequence identity, or at least about 99%sequence identity to SEQ ID NO: 47. In some embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises a VH comprising an amino acid sequence having of SEQ ID NO: 47.
  • the anti-FAP antibody or antigen-binding fragment thereof comprises a VL comprising an amino acid sequence having at least about 95%sequence identity to SEQ ID NO: 46, and a VH comprising an amino acid sequence having at least about 95%sequence identity to SEQ ID NO: 47.
  • the anti-FAP antibody is a scFv comprising a VL comprising VL comprising an amino acid sequence having at least about 95%sequence identity to SEQ ID NO: 46 fused to a VH comprising an amino acid sequence having at least about 95%sequence identity to SEQ ID NO: 47 via a linker peptide.
  • the linker peptide connecting the VH and VL comprises an amino acid sequence of GSTSGSGKPGSGEGSTKG (SEQ ID NO: 13) .
  • the linker peptide connecting the VH and VL comprises an amino acid sequence of GGGGSGGGGSGGGGS (SEQ ID NO: 14) .
  • the anti-FAP antibody or antigen-binding fragment thereof comprises a VL comprising an amino acid sequence having of SEQ ID NO: 46 and a VL comprising an amino acid sequence having of SEQ ID NO: 47.
  • the anti-FAP antibody is a scFv comprising a VL comprising an amino acid sequence of SEQ ID NO: 46 fused to a VH comprising an amino acid sequence of SEQ ID NO: 47 via a linker peptide comprising an amino acid sequence of SEQ ID NO: 13 or SEQ ID NO: 14.
  • the anti-FAP antibody is a scFv comprising the sequence of SEQ ID NO: 51 as set forth in Table 24A.
  • the anti-FAP antibody is a VHH antibody comprises or consists of three CDRs, for example, VH CDR1, VH CDR2, and/or VH CDR3 of antibody E21 identified in Table 22. In some embodiments, the anti-FAP antibody comprises or consists of three CDRs VH CDR1, VH CDR2, and VH CDR3 of antibody E21 identified in Table 22. In some embodiments, the anti-FAP antibody comprises or consists of two CDRs, for example, VH CDR1 and VH CDR2, or VH CDR1 and VH CDR3, or VH CDR2 and VH CDR3, of antibody E21 identified in Table 22. In some embodiments, the anti-FAP antibody comprises or consists of one CDR, for example, VH CDR1 2, or VH CDR2, or VH CDR3 of antibody E21 identified in Table 22.
  • the anti-FAP antibody is a VHH antibody comprising a VH CDR1 amino acid sequence of SEQ ID NO: 43.
  • the anti-FAP antibody is a VHH antibody comprising a VH CDR2 amino acid sequence of SEQ ID NO: 44.
  • the anti-FAP antibody is a VHH antibody comprising a VH CDR3 amino acid sequence of SEQ ID NO: 45.
  • the anti-FAP antibody is a VHH antibody comprising a VH CDR1 amino acid sequence of SEQ ID NO: 43, and a VH CDR2 amino acid sequence of SEQ ID NO: 44.
  • the anti-FAP antibody is a VHH antibody comprising a VH CDR1 amino acid sequence of SEQ ID NO: 43, and a VH CDR2 amino acid sequence of SEQ ID NO: 45.
  • the anti-FAP antibody is a VHH antibody comprising a VH CDR1 amino acid sequence of SEQ ID NO: 44, and a VH CDR2 amino acid sequence of SEQ ID NO: 45.
  • the anti-FAP antibody is a VHH antibody comprising a VH CDR1 amino acid sequence of SEQ ID NO: 43, a VH CDR2 amino acid sequence of SEQ ID NO: 44, and a VH CDR3 amino acid sequence of SEQ ID NO: 45.
  • the anti-FAP antibodies provided herein is a VHH antibody comprising a VH region or VH domain comprising an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%sequence identity, or at least about 99%sequence identity to SEQ ID NO: 50.
  • the anti-FAP antibodies provided herein is a VHH antibody comprising a VH region or VH domain comprising an amino acid sequence of SEQ ID NO: 50.
  • an anti-FAP antibody or antigen binding fragment described in this Section 5.3.3 can be used as an anchoring moiety in a TNF- ⁇ containing immunoconjugate molecule described herein.
  • the antibody or antigen binding fragment thereof specifically binds to FAP.
  • the anti-Trop2 antibody comprises a VH region, VL region, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and/or VL CDR3 of any one of the anti-FAP antibodies provided herein, such as an amino acid sequence depicted in Tables 25-26.
  • the anti-Trop-2 antibody or antigen binding fragment thereof provided herein comprises one, two, and/or three heavy chain CDRs and/or one, two, and/or three light chain CDRs from the antibodies FAP1, or E21 as shown in Tables 25-26.
  • the present disclosure provides antibodies or antigen binding fragment thereof that specifically binds to a tumor-associated antigen (TAA) , which in some embodiments, can find use herein for generating the present immunoconjugate molecules, e.g., as an anchoring moiety.
  • TAA tumor-associated antigen
  • Exemplary antibodies include polyclonal, monoclonal, humanized, human, bispecific, and heteroconjugate antibodies, as well as variants thereof having improved affinity or other properties.
  • the antibody or antigen binding fragment thereof specifically binds to FAP.
  • the anti-Trop2 antibody comprises a VH region, VL region, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and/or VL CDR3 of any one of the anti-Trop2 antibodies provided herein, such as an amino acid sequence depicted in Tables 25-26.
  • the anti-Trop2 antibody or antigen binding fragment thereof provided herein comprises one, two, and/or three heavy chain CDRs and/or one, two, and/or three light chain CDRs from the anti-Trop2 antibody as shown in Tables 25-26.
  • an anti-Trop2 antibody provided herein comprises or consists of six CDRs, for example, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and/or VL CDR3 identified in Tables 25-26.
  • the anti-Trop2 antibody comprises or consists of one, two, three, four, or five CDRs selected from the group consisting of VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and/or VL CDR3 identified in Tables 25-26.
  • the anti-Trop2 antibody comprises or consists of one, two, three, four, or five CDRs selected from the group consisting of VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and/or VL CDR3 of the anti-Trop2 described herein. Accordingly, in some embodiments, the anti-Trop2 antibody comprises or consists of one, two, three, four, or five CDRs of anyone of the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and/or VL CDR3 identified in Tables 25-26.
  • the anti-Trop2 antibodies provided herein comprise one or more (e.g., one, two, or three) VH CDRs listed in Table 26. In other embodiments, the anti-Trop2 antibodies provided herein comprise one or more (e.g., one, two, or three) VL CDRs listed in Table 25. In yet other embodiments, the anti-Trop2 antibodies provided herein comprise one or more (e.g., one, two, or three) VH CDRs listed in Table 26 and one or more VL CDRs listed in Table 25.
  • the anti-Trop2 antibodies comprise a VH CDR1 having an amino acid sequence of SEQ ID NO: 40.
  • the anti-Trop2 antibodies comprise a VH CDR2 having an amino acid sequence of SEQ ID NO: 41.
  • the anti-Trop2 antibodies comprise a VH CDR3 having an amino acid sequence of SEQ ID NO: 42.
  • the anti-Trop2 antibodies comprise a VH CDR1 and/or a VH CDR2 and/or a VH CDR3 independently selected from any one of the VH CDR1, VH CDR2, VH CDR3 amino acid sequence (s) as depicted in Table 26.
  • the anti-Trop2 antibodies comprise a VL CDR1 having an amino acid sequence of any one of SEQ ID NO: 34. In another embodiment, the anti-Trop2 antibodies comprise a VL CDR2 having an amino acid sequence of SEQ ID NO: 35. In some embodiments, the anti-Trop2 antibodies comprise a VL CDR3 having an amino acid sequence of SEQ ID NO: 36. In some embodiments, the anti-Trop2 antibodies comprise a VL CDR1 and/or a VL CDR2 and/or a VL CDR3 independently selected from any one of the VL CDR1, VL CDR2, VL CDR3 amino acid sequences as depicted in Table 25.
  • anti-Trop2 antibodies comprising one or more (e.g., one, two, or three) VH CDRs and one or more (e.g., one, two, or three) VL CDRs listed in Tables 25-26.
  • an anti-Trop2 antibody comprising a VH CDR1 (SEQ ID NO: 40) and a VL CDR1 (SEQ ID NO: 34) .
  • the anti-Trop2 antibody comprises a VH CDR1 (SEQ ID NO: 40) and a VL CDR2 (SEQ ID NO: 35) .
  • the anti-Trop2 antibody comprises a VH CDR1 (SEQ ID NO: 40) and a VL CDR3 (SEQ ID NO: 36) .
  • the anti-Trop2 antibody comprises a VH CDR2 (SEQ ID NO: 41) and a VL CDR1 (SEQ ID NO: 34) .
  • the anti-Trop2 antibody comprises a VH CDR2 (SEQ ID NO: 41) and a VL CDR2 (SEQ ID NO: 35) .
  • the anti-Trop2 antibody comprises a VH CDR2 (SEQ ID NO: 41) and a VL CDR3 (SEQ ID NO: 36) .
  • the anti-Trop2 antibody comprises a VH CDR3 (SEQ ID NO: 42) and a VL CDR1 (SEQ ID NO: 34) .
  • the anti-Trop2 antibody comprises a VH CDR3 (SEQ ID NO: 42) and a VL CDR2 (SEQ ID NO: 35) .
  • the anti-Trop2 antibody comprises a VH CDR3 (SEQ ID NO: 42) and a VL CDR3 (SEQ ID NO: 36) .
  • the anti-Trop2 antibody comprises a VH CDR1 (SEQ ID NO: 40) , a VH CDR2 (SEQ ID NO: 41) , and a VL CDR1 (SEQ ID NO: 34) .
  • the anti-Trop2 antibody comprises a VH CDR1 (SEQ ID NO: 40) , a VH CDR2 (SEQ ID NO: 41) , and a VL CDR2 (SEQ ID NO: 35) .
  • the anti-Trop2 antibody comprises a VH CDR1 (SEQ ID NO: 40) , a VH CDR2 (SEQ ID NO: 41) , and a VL CDR3 (SEQ ID NO: 36) .
  • the anti-Trop2 antibody comprises a VH CDR2 (SEQ ID NO: 41) , a VH CDR3 (SEQ ID NO: 42) , and a VL CDR1 (SEQ ID NO: 34) .
  • the anti-Trop2 antibody comprises a VH CDR2 (SEQ ID NO: 41) , a VH CDR3 (SEQ ID NO: 42) , and a VL CDR2 (SEQ ID NO: 35) .
  • the anti-Trop2 antibody comprises a VH CDR2 (SEQ ID NO: 41) , a VH CDR3 (SEQ ID NO: 42) , and a VL CDR3 (SEQ ID NO: 36) .
  • the anti-Trop2 antibody comprises a VH CDR1 (SEQ ID NO: 40) , a VH CDR3 (SEQ ID NO: 42) , and a VL CDR1 (SEQ ID NO: 34) .
  • the anti-Trop2 antibody comprises a VH CDR1 (SEQ ID NO: 40) , a VH CDR3 (SEQ ID NO: 42) , and a VL CDR2 (SEQ ID NO: 35) .
  • the anti-Trop2 antibody comprises a VH CDR1 (SEQ ID NO: 40) , a VH CDR3 (SEQ ID NO: 42) , and a VL CDR3 (SEQ ID NO: 36) .
  • the anti-Trop2 antibody comprises a VH CDR1 (SEQ ID NO: 40) , a VL CDR1 (SEQ ID NO: 34) , and a VL CDR2 (SEQ ID NO: 35) .
  • the anti-Trop2 antibody comprises a VH CDR1 (SEQ ID NO: 40) , a VL CDR1 (SEQ ID NO: 34) , and a VL CDR3 (SEQ ID NO: 36) .
  • the anti-Trop2 antibody comprises a VH CDR1 (SEQ ID NO: 40) , a VL CDR2 (SEQ ID NO: 35) , and a VL CDR3 (SEQ ID NO: 36) .
  • the anti-Trop2 antibody comprises a VH CDR2 (SEQ ID NO: 41) , a VL CDR1 (SEQ ID NO: 34) , and a VL CDR2 (SEQ ID NO: 35) .
  • the anti-Trop2 antibody comprises a VH CDR2 (SEQ ID NO: 41) , a VL CDR1 (SEQ ID NO: 34) , and a VL CDR3 (SEQ ID NO: 36) .
  • the anti-Trop2 antibody comprises a VH CDR2 (SEQ ID NO: 41) , a VL CDR2 (SEQ ID NO: 35) , and a VL CDR3 (SEQ ID NO: 36) .
  • the anti-Trop2 antibody comprises a VH CDR3 (SEQ ID NO: 42) , a VL CDR1 (SEQ ID NO: 34) , and a VL CDR2 (SEQ ID NO: 35) .
  • the anti-Trop2 antibody comprises a VH CDR3 (SEQ ID NO: 42) , a VL CDR1 (SEQ ID NO: 34) , and a VL CDR3 (SEQ ID NO: 36) .
  • the anti-Trop2 antibody comprises a VH CDR3 (SEQ ID NO: 42) , a VL CDR2 (SEQ ID NO: 35) , and a VL CDR3 (SEQ ID NO: 36) .
  • the anti-Trop2 antibody comprises a VH CDR1 (SEQ ID NO: 40) , a VH CDR2 (SEQ ID NO: 41) , a VH CDR3 (SEQ ID NO: 42) , and a VL CDR1 (SEQ ID NO: 34) .
  • the anti-Trop2 antibody comprises a VH CDR1 (SEQ ID NO: 40) , a VH CDR2 (SEQ ID NO: 41) , a VH CDR3 (SEQ ID NO: 42) , and a VL CDR2 (SEQ ID NO: 35) .
  • the anti-Trop2 antibody comprises a VH CDR1 (SEQ ID NO: 40) , a VH CDR2 (SEQ ID NO: 41) , a VH CDR3 (SEQ ID NO: 42) , and a VL CDR3 (SEQ ID NO: 36) .
  • the anti-Trop2 antibody comprises a VH CDR1 (SEQ ID NO: 40) , a VH CDR2 (SEQ ID NO: 41) , a VL CDR1 (SEQ ID NO: 34) , and a VL CDR2 (SEQ ID NO: 35) .
  • the anti-Trop2 antibody comprises a VH CDR1 (SEQ ID NO: 40) , a VH CDR2 (SEQ ID NO: 41) , a VL CDR1 (SEQ ID NO: 34) , and a VL CDR3 (SEQ ID NO: 36) .
  • the anti-Trop2 antibody comprises a VH CDR1 (SEQ ID NO: 40) , a VH CDR2 (SEQ ID NO: 41, a VL CDR2 (SEQ ID NO: 35) , and a VL CDR3 (SEQ ID NO: 36) .
  • the anti-Trop2 antibody comprises a VH CDR1 (SEQ ID NO: 40) , a VH CDR3 (SEQ ID NO: 42) , a VL CDR1 (SEQ ID NO: 34) , and a VL CDR2 (SEQ ID NO: 35) .
  • the anti-Trop2 antibody comprises a VH CDR1 (SEQ ID NO: 40) , a VH CDR3 (SEQ ID NO: 42) , a VL CDR1 (SEQ ID NO: 34) , and a VL CDR3 (SEQ ID NO: 36) .
  • the anti-Trop2 antibody comprises a VH CDR1 (SEQ ID NO: 40) , a VH CDR3 (SEQ ID NO: 42) , a VL CDR2 (SEQ ID NO: 35) , and a VL CDR3 (SEQ ID NO: 36) .
  • the anti-Trop2 antibody comprises a VH CDR2 (SEQ ID NO: 41) , a VH CDR3 (SEQ ID NO: 42) , a VL CDR1 (SEQ ID NO: 34) , and a VL CDR2 (SEQ ID NO: 35) .
  • the anti-Trop2 antibody comprises a VH CDR2 (SEQ ID NO: 41) , a VH CDR3 (SEQ ID NO: 42) , a VL CDR1 (SEQ ID NO: 34) , and a VL CDR3 (SEQ ID NO: 36) .
  • the anti-Trop2 antibody comprises a VH CDR2 (SEQ ID NO: 41) , a VH CDR3 (SEQ ID NO: 42) , a VL CDR2 (SEQ ID NO: 35) , and a VL CDR3 (SEQ ID NO: 36) .
  • the anti-Trop2 antibody comprises a VH CDR1 (SEQ ID NO: 40) , a VH CDR2 (SEQ ID NO: 41) , a VH CDR3 (SEQ ID NO: 42) , a VL CDR1 (SEQ ID NO: 34) , and a VL CDR2 (SEQ ID NO: 35) .
  • the anti-Trop2 antibody comprises a VH CDR1 (SEQ ID NO: 40) , a VH CDR2 (SEQ ID NO: 41) , a VH CDR3 (SEQ ID NO: 42) , a VL CDR1 (SEQ ID NO: 34) , and a VL CDR3 (SEQ ID NO: 36) .
  • the anti-Trop2 antibody comprises a VH CDR1 (SEQ ID NO: 40) , a VH CDR2 (SEQ ID NO: 41) , a VH CDR3 (SEQ ID NO: 42) , a VL CDR2 (SEQ ID NO: 35) , and a VL CDR3 (SEQ ID NO: 36) .
  • the anti-Trop2 antibody comprises a VH CDR1 (SEQ ID NO: 40) , a VH CDR2 (SEQ ID NO: 41) , a VL CDR1 (SEQ ID NO: 34) , a VL CDR2 (SEQ ID NO: 35) , and a VL CDR3 (SEQ ID NO: 36) .
  • the anti-Trop2 antibody comprises a VH CDR1 (SEQ ID NO: 40) , a VH CDR3 (SEQ ID NO: 42) , a VL CDR1 (SEQ ID NO: 34) , a VL CDR2 (SEQ ID NO: 35) , and a VL CDR3 (SEQ ID NO: 36) .
  • the anti-Trop2 antibody comprises a VH CDR2 (SEQ ID NO: 41) , a VH CDR3 (SEQ ID NO: 42) , a VL CDR1 (SEQ ID NO: 34) , a VL CDR2 (SEQ ID NO: 35) , and a VL CDR3 (SEQ ID NO: 36) .
  • the anti-Trop2 antibody comprises a VH CDR1 (SEQ ID NO: 40) , a VL CDR1 (SEQ ID NO: 34) , a VL CDR2 (SEQ ID NO: 35) , and a VL CDR3 (SEQ ID NO: 36) .
  • the anti-Trop2 antibody comprises a VH CDR2 (SEQ ID NO: 41) , a VL CDR1 (SEQ ID NO: 34) , a VL CDR2 (SEQ ID NO: 35) , and a VL CDR3 (SEQ ID NO: 36) .
  • the anti-Trop2 antibody comprises a VH CDR3 (SEQ ID NO: 42) , a VL CDR1 (SEQ ID NO: 34) , a VL CDR2 (SEQ ID NO: 35) , and a VL CDR3 (SEQ ID NO: 36) .
  • the anti-Trop2 antibody comprises any combination thereof of the VH CDRs and VL CDRs listed in Tables 25-26.
  • the anti-Trop2 antibodies provided herein comprise a VH region comprising: (1) a VH CDR1 having an amino acid sequence of SEQ ID NO: 40; (2) a VH CDR2 having an amino acid sequence of SEQ ID NO: 41; and (3) a VH CDR3 having an amino acid sequence of SEQ ID NO: 42.
  • the anti-Trop2 antibodies provided herein comprise a VL region comprising: (1) a VL CDR1 having an amino acid sequence of SEQ ID NO: 34; (2) a VL CDR2 having an amino acid sequence of SEQ ID NO: 35; and (3) a VL CDR3 having an amino acid sequence of SEQ ID NO: 36.
  • the anti-Trop2 antibodies provided herein comprise a VH region comprising (1) a VH CDR1 having an amino acid sequence of SEQ ID NO: 40; (2) a VH CDR2 having an amino acid sequence of SEQ ID NO: 41; and (3) a VH CDR3 having an amino acid sequence of SEQ ID NO: 42; and a VL region comprising: (1) a VL CDR1 having an amino acid sequence of SEQ ID NO: 34; (2) a VL CDR2 having an amino acid sequence of SEQ ID NO: 35; and (3) a VL CDR3 having an amino acid sequence of SEQ ID NO: 36.
  • the anti-Trop2 antibodies provided herein comprise a VH region or VH domain. In other embodiments, the anti-Trop2 antibodies provided herein comprise a VL region or VL domain. In certain embodiments, the anti-Trop2 antibodies provided herein have a combination of (i) a VH domain or VH region; and/or (ii) a VL domain or VL region. In yet other embodiments, the anti-Trop2 antibodies provided herein have a combination of (i) a VH domain or VH region consisting of SEQ ID NO: 49; and/or (ii) a VL domain or VL region consisting of SEQ ID NO: 48 as set forth in Tables 27-28.
  • the anti-Trop2 antibodies provided herein comprise a VH region comprising (1) a VH CDR1 having an amino acid sequence of SEQ ID NO: 40; (2) a VH CDR2 having an amino acid sequence of SEQ ID NO: 41; and (3) a VH CDR3 having an amino acid sequence of SEQ ID NO: 42; and a VL region as set forth in Table 27.
  • the VL region has an amino acid sequence of SEQ ID NO: 48.
  • the anti-Trop2 antibodies provided herein comprise a VH consisting of SEQ ID NO: 49 as set forth in Table 28; and a VL region comprising (1) a VL CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NO: 34; (2) a VL CDR2 having an amino acid sequence of SEQ ID NO: 35; and (3) a VL CDR3 having an amino acid sequence of SEQ ID NO: 36.
  • the anti-Trop2 antibody or antigen-binding fragment thereof comprises a VL comprising an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%sequence identity, or at least about 99%sequence identity to SEQ ID NO: 48. In some embodiments, the anti-Trop2 antibody or antigen-binding fragment thereof comprises a VL comprising an amino acid sequence having of SEQ ID NO: 48.
  • the anti-Trop2 antibody or antigen-binding fragment thereof comprises a VH comprising an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%sequence identity, or at least about 99%sequence identity to SEQ ID NO: 49. In some embodiments, the anti-Trop2 antibody or antigen-binding fragment thereof comprises a VH comprising an amino acid sequence having of SEQ ID NO: 49.
  • the anti-Trop2 antibody or antigen-binding fragment thereof comprises a VL comprising an amino acid sequence having at least about 95%sequence identity to SEQ ID NO: 48, and a VH comprising an amino acid sequence having at least about 95%sequence identity to SEQ ID NO: 49.
  • the anti-Trop2 antibody is a scFv comprising a VL comprising an amino acid sequence having at least about 95%sequence identity to SEQ ID NO: 48 fused to a VH comprising an amino acid sequence having at least about 95%sequence identity to SEQ ID NO: 49.
  • the linker peptide connecting the VH and VL comprises an amino acid sequence of GSTSGSGKPGSGEGSTKG (SEQ ID NO: 13) . In some embodiments, the linker peptide connecting the VH and VL comprises an amino acid sequence of GGGGSGGGGSGGGGS (SEQ ID NO: 14) .
  • the anti-Trop2 antibody or antigen-binding fragment thereof comprises a VL comprising an amino acid sequence having of SEQ ID NO: 48 and a VL comprising an amino acid sequence having of SEQ ID NO: 49.
  • the anti-Trop2 antibody is a scFv comprising a VL comprising an amino acid sequence of SEQ ID NO: 48 fused to a VH comprising an amino acid sequence of SEQ ID NO: 49 via a linker peptide comprising an amino acid sequence of SEQ ID NO: 13 or SEQ ID NO: 14.
  • the anti-Trop2 antibody is a scFv comprising the sequence of SEQ ID NO: 52 as set forth in Table 28A
  • an anti-Trop2 antibody or antigen binding fragment described in this Section 5.3.4 can be used as an anchoring moiety in a TNF- ⁇ containing immunoconjugate molecule described herein
  • the TNF- ⁇ containing immunocytokine molecule comprises two amino acid chains, where the first chain contains from the N-to-C orientation, a cytokine moiety containing a wild-type or mutant TNF- ⁇ , which is fused via a linker peptide to the VH domain of a two-in-one anti-TNF- ⁇ /anti-FAP mab (masking moiety) , and a CH domain of the two-in-one masking moiety; the second chain contains, from the N to C orientation, an anti-FAP anchoring moiety either in the scFv format (configuration 6 in FIG. 1G) or VHH format (configuration 7 in FIG.
  • the two polypeptide chains of the immunoconjugate molecule have the sequences listed in Table 29.
  • the TNF- ⁇ containing immunoconjugate comprises two polypeptide chains comprising the amino acid sequences of SEQ ID NO: 53 and SEQ ID NO: 54, respectively.
  • the TNF- ⁇ containing immunoconjugate comprises two polypeptide chains comprising the amino acid sequences of SEQ ID NO: 55 and SEQ ID NO: 54, respectively.
  • the TNF- ⁇ containing immunoconjugate comprises two polypeptide chains comprising the amino acid sequences of SEQ ID NO: 56 and SEQ ID NO: 54, respectively.
  • the TNF- ⁇ containing immunoconjugate comprises two polypeptide chains comprising the amino acid sequences of SEQ ID NO: 57 and SEQ ID NO: 54, respectively.
  • the TNF- ⁇ containing immunoconjugate comprises two polypeptide chains comprising the amino acid sequences of SEQ ID NO: 58 and SEQ ID NO: 59, respectively.
  • the TNF- ⁇ containing immunoconjugate comprises two polypeptide chains comprising the amino acid sequences of SEQ ID NO: 61 and SEQ ID NO: 59, respectively.
  • the TNF- ⁇ containing immunoconjugate comprises two polypeptide chains comprising the amino acid sequences of SEQ ID NO: 62 and SEQ ID NO: 59, respectively.
  • the TNF- ⁇ containing immunoconjugate comprises two polypeptide chains comprising the amino acid sequences of SEQ ID NO: 63 and SEQ ID NO: 64, respectively.
  • the TNF- ⁇ containing immunoconjugate comprises two polypeptide chains comprising the amino acid sequences of SEQ ID NO: 65 and SEQ ID NO: 64, respectively.
  • the TNF- ⁇ containing immunoconjugate comprises two polypeptide chains comprising the amino acid sequences of SEQ ID NO: 66 and SEQ ID NO: 64, respectively.
  • the TNF- ⁇ containing immunoconjugate comprises two polypeptide chains comprising the amino acid sequences of SEQ ID NO: 67 and SEQ ID NO: 64, respectively.
  • the antibodies forming part of the immunoconjugate molecules of the present disclosure may comprise polyclonal antibodies. Methods of preparing polyclonal antibodies are known to the skilled artisan. Polyclonal antibodies can be raised in a mammal, for example, by one or more injections of an immunizing agent and, if desired, an adjuvant. Typically, the immunizing agent and/or adjuvant will be injected in the mammal by multiple subcutaneous or intraperitoneal injections.
  • the immunizing agent may include a polypeptide or a fusion protein thereof (e.g., TNF- ⁇ polypeptide or FAP polypeptide) .
  • the immunizing agent may be useful to conjugate the immunizing agent to a protein known to be immunogenic in the mammal being immunized or to immunize the mammal with the protein and one or more adjuvants.
  • immunogenic proteins include, but are not limited to, keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor.
  • adjuvants which may be employed include Ribi, CpG, Poly 1C, Freund’s complete adjuvant, and MPL-TDM adjuvant (monophosphoryl Lipid A, synthetic trehalose dicorynomycolate) .
  • the immunization protocol may be selected by one skilled in the art without undue experimentation.
  • lymphocytes may be obtained from the immunized animal for fusion and preparation of monoclonal antibodies from hybridoma as described below.
  • the antibodies forming part of the immunoconjugate molecules of the present disclosure may alternatively be monoclonal antibodies.
  • Monoclonal antibodies may be made using the hybridoma method first described by Kohler et al., 1975, Nature 256: 495-97, or may be made by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567) .
  • a mouse or other appropriate host animal such as a hamster
  • lymphocytes may be immunized in vitro.
  • lymphocytes are isolated and then fused with a myeloma cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice 59-103 (1986) ) .
  • the hybridoma cells thus prepared are seeded and grown in a suitable culture medium which, in certain embodiments, contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells (also referred to as fusion partner) .
  • a suitable culture medium which, in certain embodiments, contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells (also referred to as fusion partner) .
  • the parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT)
  • HGPRT or HPRT the selective culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (HAT medium) , which prevent the growth of HGPRT-deficient cells.
  • Exemplary fusion partner myeloma cells are those that fuse efficiently, support stable high-level production of antibody by the selected antibody-producing cells, and are sensitive to a selective medium that selects against the unfused parental cells.
  • Exemplary myeloma cell lines are murine myeloma lines, such as SP-2 and derivatives, for example, X63-Ag8-653 cells available from the American Type Culture Collection (Manassas, VA) , and those derived from MOPC-21 and MPC-11 mouse tumors available from the Salk Institute Cell Distribution Center (San Diego, CA) .
  • Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies (Kozbor, 1984, Immunol. 133: 3001-05; and Brodeur et al., Monoclonal Antibody Production Techniques and Applications 51-63 (1987) ) .
  • Culture medium in which hybridoma cells are growing is assayed for production of monoclonal antibodies directed against the antigen.
  • the binding specificity of monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as RIA or ELISA.
  • the binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis described in Munson et al., 1980, Anal. Biochem. 107: 220-39.

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Abstract

L'invention concerne des molécules immunoconjuguées contenant un polypeptide TNF-α et une fraction de masquage capable d'inhiber et d'activer l'activité TNF-α dans des conditions appropriées. L'invention concerne également des procédés de production des molécules immunoconjuguées. Enfin, l'invention concerne les utilisations thérapeutiques des molécules immunoconjugués en raison de leurs effets de modulation sur le système immunitaire pour le traitement de maladies telles que le cancer et d'autres maladies infectieuses chroniques.
PCT/CN2024/084035 2023-03-27 2024-03-27 Immunoconjugués contenant du tnf-alpha et procédés associés et compositions associées Pending WO2024199269A1 (fr)

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