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WO2025064498A1 - Anticorps bispécifiques anti-cd45 x pd-1 et anti-cd43 x pd-1 modulés par affinité pour traiter le cancer et l'auto-immunité - Google Patents

Anticorps bispécifiques anti-cd45 x pd-1 et anti-cd43 x pd-1 modulés par affinité pour traiter le cancer et l'auto-immunité Download PDF

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Publication number
WO2025064498A1
WO2025064498A1 PCT/US2024/047201 US2024047201W WO2025064498A1 WO 2025064498 A1 WO2025064498 A1 WO 2025064498A1 US 2024047201 W US2024047201 W US 2024047201W WO 2025064498 A1 WO2025064498 A1 WO 2025064498A1
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Prior art keywords
seq
protein
bispecific antibody
acid sequence
arm
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Shalom LERRER
Adam Mor
Ruijiang Song
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Columbia University in the City of New York
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Columbia University in the City of New York
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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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
    • 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
    • C07K16/289—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against CD45
    • 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
    • C07K16/2896—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
    • 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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70589—CD45
    • 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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70596—Molecules with a "CD"-designation not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • 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
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present invention relates generally to antibodies and expression systems for producing antibodies. More particularly, described herein are methods of relocalizing proteins of the immune synapse to modulate T cell activation and the use of such methods to treat a disease in a subject in need thereof. Further described herein are anti-CD45 antibodies with reduced affinity for CD45 as well as anti-CD45xPD-l and anti-CD43xPD-l bispecific antibodies.
  • Targeting immune checkpoint receptors on T cells is a common cancer treatment strategy. Frequently, this is accomplished through monoclonal antibodies targeting the ligand binding sites of inhibitory co-receptors. Blocking the immune checkpoint PD-1 binding to its ligands PD-L1 and PD-L2 prevents downstream signaling and enhances specific T functions. Since 2013, the FDA has approved seven monoclonal antibodies to inhibit PD-1 signaling. This therapeutic approach improved the care of patients with cancers. However, many patients are unresponsive, and some develop immune-related adverse events (irAEs). There is a need for prophylactic and/or therapeutic agents with improved specificity and efficacy, and reduced side effects.
  • compositions for relocalizing a protein to a compartment of the immune synapse in a subject comprising: a molecule capable of binding the protein at the immune synapse, wherein the protein is relocalized to the distal compartment of the immune synapse or wherein the protein is relocalized to the core of the immune synapse.
  • the protein is relocalized to the distal compartment of the immune synapse and wherein the molecule is an antibody to the protein capable of localizing the protein to the distal compartment of the immune synapse.
  • the antibody is a bispecific antibody to the protein and to another protein at the distal compartment of the immune synapse, wherein the bispecific antibody is capable of localizing the protein away from the immune synapse.
  • the protein is relocalized to the core of the immune synapse and wherein the molecule is an antibody to the protein capable of localizing the protein to the core of the immune synapse.
  • the antibody is a bispecific antibody to the protein and to another protein at the core of the immune synapse, wherein the bispecific antibody is capable of localizing the protein to the core of the immune synapse.
  • the protein at the immune synapse is PD-1 (CD279), CD352
  • SLAMF6 CD6, CD28, CD3E, CD80, CD86, CD276, CD70, CD46, CD209, CD40, CD247, CD4, CD8A, CD3G, TRBC1, TRBC2, CD3D, CD8B, Thyl, LAT, PAG1, CD276, PTPRC (CD45), TCR alpha/beta/gamma/delta, CD294, PD-L1 (CD274), PD-L2 (PDCD1LG2), CTLA- 4, BTN3A1, HLA-DRB1, HLA-DRB3, ICOSL (B7h), TIGIT, LAG3 (CD223), CD196 (CCR6), CD56 (NCAM), CD366 (TIM3), CD45RA, CD 154 (CD40L), CD278 (ICOS), CD25 (IL2R), CD152 (CTLA4), CXCR1, HLA-DR, CD16, CD5, CD69, CD183 (CXCR3), CD127 (IL17RA), CD254
  • the bispecific antibody is any of the bi specific antibodies described herein.
  • a method for treating cancer in a subject in need thereof comprising: administering to the subject a therapy comprising an effective amount of a molecule capable of localizing a protein to a distal compartment of an immune synapse.
  • the molecule is an antibody or an antigen binding fragment thereof capable of localizing the protein to a distal compartment of the immune synapse.
  • the antibody is a bispecific antibody to the protein and to another protein at the distal compartment of the immune synapse, wherein the bispecific antibody is capable of localizing the protein to the distal compartment from the immune synapse.
  • a monoclonal antibody or antigen binding fragment thereof comprising: a first arm comprising a first variable heavy chain domain and a first variable light chain domain, wherein a portion of the first arm is capable of binding to a portion of a CD45 protein; and a second arm comprising a second variable heavy chain domain and a second variable light chain domain, wherein a portion of the second arm is capable of binding to a portion of a CD45 protein.
  • the first variable heavy chain domain comprises an amino acid sequence of the variable heavy chain portion of SEQ ID NO: 2, 4, 6, 8, 10, 29, 30, 31, 32, 33, or 34, wherein the first variable light chain domain comprises an amino acid sequence of the variable light chain portion of SEQ ID NO: 2, 4, 6, 8, 10, 29, 30, 31, 32, 33, or 34.
  • the first arm comprises an amino acid sequence selected from SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, or SEQ ID NO: 34
  • the second arm comprises an amino acid sequence selected from SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, or SEQ ID NO: 34.
  • the portion of the first arm is capable of binding to the portion of the CD45 protein with an affinity between about 5.6 E-7 KD (M) and about 3.6 E-l 1 KD (M).
  • the first arm comprises an amino acid sequence selected from SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, or SEQ ID NO: 34
  • the second arm comprises an amino acid sequence selected from SEQ ID NO: 26, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, or SEQ ID NO: 43.
  • the portion of the first arm is capable of binding to the portion of the CD45 protein with an affinity between about 5.6 E-7 KD (M) and about 3.6 E-l 1 KD (M).
  • the first arm comprises an amino acid sequence selected from SEQ ID NO: 4 or SEQ ID NO: 6, and wherein the second arm comprises an amino acid sequence selected from SEQ ID NO: 26, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, or SEQ ID NO: 43.
  • the portion of the first arm is capable of binding to the portion of the CD45 protein, and wherein the bispecific antibody is capable of localizing a PD-1 protein away from a core of an immune synapse.
  • the bispecific antibody is capable of disrupting downstream signaling of a PD-1 mediated response in a T cell. In some embodiments, the bispecific antibody is capable of enhancing T cell function. In some embodiments, the bispecific antibody is capable of binding to the CD45 portion of the CD45 protein and to the PD-1 portion on the PD-1 protein, wherein the CD45 protein and PD-1 protein are located on a same T cell. In some embodiments, the portion of the first arm is capable of binding to the portion of the CD43 protein, and wherein the bispecific antibody is capable of localizing a PD-1 protein away from a core of an immune synapse. In some embodiments, the bispecific antibody is capable of disrupting a downstream signaling of a PD-1 mediated response in a T cell. In some embodiments, the bispecific antibody is capable of enhancing T cell function.
  • the bispecific antibody is capable of binding to the CD43 portion of the CD43 protein and to the PD-1 portion on the PD-1 protein wherein the CD43 protein and PD-1 protein are located on a same T cell.
  • the PD-1 protein is located on a T cell, and wherein the bispecific antibody is capable of preventing the PD-1 protein from binding to a PDL-1 protein or a PDL-2 protein on a tumor cell.
  • the bispecific antibody is capable of dephosphorylating a tail of the PD-1 protein.
  • the bispecific antibody is capable of inducing a cytokine secretion in a T cell.
  • the cytokine secretion is a secretion of IL-2.
  • described herein is a pharmaceutical composition comprising: the bispecific antibody described herein and a pharmaceutically acceptable carrier.
  • described herein is a method of preventing or treating cancer in a subject comprising administering to the subject an effective amount of the composition described herein.
  • the cancer is selected from colorectal cancer, lung cancer, bladder cancer, breast cancer, cervical cancer, kidney cancer, leukemia, Hodgkin lymphoma, non-Hodgkin lymphoma, prostate cancer, skin cancer (e.g., melanoma), head and neck cancer, endometrial cancer, colon cancer, rectal cancer, liver cancer, thyroids cancer, esophageal cancer, renal cell cancer, and a combination thereof.
  • described herein is a method of preventing or treating a viral infection in a subject comprising administering to the subject an effective amount of the composition described herein.
  • the viral infection is HIV.
  • described herein is a pharmaceutical composition
  • a pharmaceutical composition comprising: the bispecific antibody described herein and a pharmaceutically acceptable carrier.
  • described herein is a method of preventing or treating inflammation and autoimmunity in a subject comprising administering to the subject an effective amount of the composition described herein.
  • the diseases is selected from rheumatoid arthritis, systemic lupus erythematosus, psoriasis and psoriatic arthritis, spondyloarthropathy, type 1 diabetes, and multiple sclerosis.
  • a variable heavy chain domain of the first arm comprises an amino acid sequence of the variable heavy chain portion of SEQ ID NO: 2, 4, 6, 8, 10, 22, 29, 30, 31, 32, 33, or 34
  • a first variable light chain domain of the first arm comprises an amino acid sequence of the variable light chain portion of SEQ ID NO: 2, 4, 6, 8, 10, 22, 29, 30, 31, 32, 33, or 34
  • a variable heavy chain domain of the second arm comprises an amino acid sequence of the variable heavy chain portion of SEQ ID NO: 26, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, or SEQ ID NO: 43
  • a variable light chain domain of the second arm comprises an amino acid sequence selected from SEQ ID NO: 26, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ
  • described herein is one or more host cells comprising: one or more vectors comprising a polynucleotide sequence encoding any of the bispecific antibodies described herein.
  • described herein is one or more host cells comprising: a first vector comprising a polynucleotide sequence encoding a first arm of a bispecific antibody or fragment thereof, wherein a portion of the first arm is capable of binding to a portion of a CD45 protein or a portion of a CD43 protein; and a second vector comprising a polynucleotide sequence encoding a second arm of the bispecific antibody or fragment thereof, wherein a portion of the second arm is capable of binding to a portion of a PD-1 protein.
  • the first vector and the second vector are the same vector. In some embodiments, the first vector and the second vector are two different vectors.
  • the first arm comprises a first variable heavy chain domain and a first variable light chain domain
  • the second arm comprises a second variable heavy chain domain and a second variable light chain domain
  • the first variable heavy chain domain comprises an amino acid sequence of the variable heavy chain portion of SEQ ID NO: 2, 4, 6, 8, 10, 29, 30, 31, 32, 33, or 34
  • the first variable light chain domain comprises an amino acid sequence of the variable light chain portion of SEQ ID NO: 2, 4, 6, 8, 10, 29, 30, 31, 32, 33, or 34.
  • described herein is a method of making a bispecific antibody or fragment thereof comprising: culturing the one or more host cells described herein under conditions suitable for an expression of the one or more vectors; and recovering the bispecific antibody or fragment thereof.
  • described herein is a method of making a bispecific antibody or fragment thereof comprising: culturing the one or more host cells described herein under conditions suitable for an expression of the first vector and the second vector; and recovering the bispecific antibody or fragment thereof.
  • compositions comprising: one or more vectors comprising a polynucleotide sequence encoding any of the bispecific antibodies described herein.
  • compositions comprising: a first vector comprising a polynucleotide sequence encoding a first arm of the bispecific antibody or fragment thereof, wherein a portion of the first arm is capable of binding to a portion of a CD45 protein or a portion of a CD43 protein; and a second vector comprising a polynucleotide sequence encoding a second arm of the bispecific antibody or fragment thereof, wherein a portion of the second arm is capable of binding to a portion of a PD-1 protein.
  • the first vector and the second vector are the same vector. In some embodiments, the first vector and the second vector are two different vectors.
  • the first arm comprises a first variable heavy chain domain and a first variable light chain domain
  • the second arm comprises a second variable heavy chain domain and a second variable light chain domain
  • the first variable heavy chain domain comprises an amino acid sequence of the variable heavy chain portion of SEQ ID NO: 2, 4, 6, 8, 10, 29, 30, 31, 32, 33, or 34
  • the first variable light chain domain comprises an amino acid sequence of the variable light chain portion of SEQ ID NO: 2, 4, 6, 8, 10, 29, 30, 31, 32, 33, or 34.
  • the first arm comprises an amino acid sequence selected from SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 22, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, or SEQ ID NO: 34
  • the second arm comprises an amino acid sequence selected from SEQ ID NO: 26, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, or SEQ ID NO: 43.
  • the first arm comprises SEQ ID NO: 4.
  • the first arm comprises SEQ ID NO: 6. In some embodiments, the first arm comprises SEQ ID NO: 22. [0031] In certain aspects, described herein is a means for binding: a portion of a CD45 protein or a portion of a CD43 protein; and a portion of a PD-1 protein. In some embodiments, the means comprises a bispecific antibody or fragment thereof.
  • the bispecific antibody or a fragment thereof comprises: a first arm comprising a first variable heavy chain domain and a first variable light chain domain, wherein a portion of the first arm is capable of binding to the portion of the CD45 protein or the portion of the CD43 protein; and a second arm comprising a second variable heavy chain domain and a second variable light chain domain, wherein a portion of the second arm is capable of binding to the portion of the PD-1 protein.
  • the first variable heavy chain domain comprises an amino acid sequence of the variable heavy chain portion of SEQ ID NO: 2, 4, 6, 8, 10, 29, 30, 31, 32, 33, or 34
  • the first variable light chain domain comprises an amino acid sequence of the variable light chain portion of SEQ ID NO: 2, 4, 6, 8, 10, 29, 30, 31, 32, 33, or 34
  • the portion of the first arm is capable of binding to the portion of the CD45 protein.
  • the first arm comprises an amino acid sequence selected from SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, or SEQ ID NO: 34
  • the second arm comprises SEQ ID NO: 26, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, or SEQ ID NO: 43
  • the portion of the first arm is capable of binding to the portion of the CD45 protein with an affinity between about 5.6 E-7 KD (M) and about 3.6 E- 11 KD (M).
  • the first arm comprises SEQ ID NO: 22, wherein the second arm comprises SEQ ID NO: 26, and wherein the portion of the second arm is capable of binding to the portion of the CD43 protein.
  • the portion of the first arm is capable of binding to the portion of the CD43 protein with an affinity between about 1.0 E-7 KD (M) and about 1.0 E-9 7 KD (M).
  • the first arm comprises an amino acid sequence selected from SEQ ID NO: 4 or SEQ ID NO: 6, and wherein the second arm comprises SEQ ID NO: 26.
  • the portion of the first arm is capable of binding to the portion of the CD45 protein, and wherein the bispecific antibody is capable of localizing a PD-1 protein away from a core of an immune synapse.
  • the bispecific antibody is capable of disrupting downstream signaling of a PD-1 mediated response in a T cell.
  • the bispecific antibody is capable of enhancing T cell function.
  • the bispecific antibody is capable of binding to the CD45 portion of the CD45 protein and to the PD-1 portion on the PD-1 protein, wherein the CD45 protein and PD-1 protein are located on a same T cell.
  • the portion of the first arm is capable of binding to the portion of the CD43 protein, and wherein the bispecific antibody is capable of localizing a PD-1 protein away from a core of an immune synapse.
  • the bispecific antibody is capable of disrupting a downstream signaling of a PD-1 mediated response in a T cell.
  • the bispecific antibody is capable of enhancing T cell function.
  • the bispecific antibody is capable of binding to the CD43 portion of the CD43 protein and to the PD-1 portion on the PD-1 protein wherein the CD43 protein and PD-1 protein are located on a same T cell.
  • the PD-1 protein is located on a T cell, and wherein the bispecific antibody is capable of preventing the PD-1 protein from binding to a PDL-1 protein or a PDL-2 protein on a tumor cell. In some embodiments, the bispecific antibody is capable of dephosphorylating a tail of the PD-1 protein. In some embodiments, the bispecific antibody is capable of inducing a cytokine secretion in a T cell. In some embodiments, the cytokine secretion is a secretion of IL-2.
  • the term “antibody” includes synthetic antibodies, monoclonal antibodies, oligoclonal or polyclonal antibodies, multiclonal antibodies, recombinantly produced antibodies, intrabodies, monospecific antibodies, monovalent antibodies, multispecific antibodies, multivalent antibodies, bispecific antibodies, bivalent antibodies, human antibodies, humanized antibodies, chimeric antibodies, CDR-grafted antibodies, primatized antibodies, Fab fragments, F(ab’) fragments, F(ab’)2 fragments, Fv fragments, single-chain FvFcs (scFv-Fc), single-chain Fvs (scFv), Dabs, nanobodies, anti-idiotypic (anti-Id) antibodies, and any other immunologically-reactive/antigen-binding fragments thereof.
  • immunoglobulin Ig
  • the antibody binds to CD45. In some embodiments, the antibody binds to CD45. In some embodiments to CD45. In some embodiments, the antibody binds to CD45. In some embodiments, the
  • the term “bispecific antibody” refers to an antibody having specificities for at least two different, typically non-overlapping, epitopes. Such epitopes may be on the same or different targets. If the epitopes are on different targets, such targets may be on the same cell or different cells or cell types.
  • the bispecifc antibody comprises a first and second chain.
  • the first chain comprises an scFv with specificity for a first epitope and the second chain comprises an scFv with specificity for a second epitope.
  • the first and second chains each further comprise a Fc domain.
  • the bispecific antibody comprises a first and a second heavy chain.
  • the bispecific antibody comprises a first and a second heavy chain and a first and a second light chain. In some embodiments, the bispecific antibody comprises any of the designs described in Figure 2 of Brinkmann U, Kontermann RE, The making of bispecific antibodies, MAbs, 2017 Feb/Mar, 9(2): 182-212, PMID: 28071970 the content of which is hereby incorporated by reference in its entirety. In some embodiments, at least one of the specificities of the bispecific antibody is for CD45. In some embodiments, the bispecific antibody binds to CD45 and PD-1 or binds to CD43 and PD-1. In some embodiments, the bispecific antibody binds to CD45 with reduced affinity.
  • FIG. 1 shows pVaxl vector (ThermoFisher, V26020, Kanamycin-resistant) into which antibody constructs were cloned at HindHI/Xbal enzyme sites.
  • FIG. 2 shows ELISA binding for an anti-CD45xPD-l bispecific antibody to CD45 and PD-1.
  • FIGs. 3A-B show imaging of live cocultured cells.
  • Jurkat T cells stably expressing GFP-PD-1 were cocultured with Raji B cells expressing mCherry-PD-Ll.
  • the Raji B cells were pretreated with SEE (50 pg/mL), and the Jurakt T cells were pretreated with anti-CD45xPD-l, anti-PD-1, and control antibodies (1 pg/mL), for 45 minutes.
  • Live cocultured cells were imaged with confocal microscopy (Zeiss) for 30 minutes, and the number of the conjugates where PD-1 was enriched at the immune synapse was calculated using ImageJ.
  • FIG. 3 A shows representative images and
  • FIG. 4 shows IL-2 levels (pg/mL) in PBMC that were isolated from healthy volunteers and then pretreated with anti-CD43xPD-l bispecific antibody, anti-CD45MlxPD-l bispecific antibody, anti-CD45M2xPD-l bispecific antibody, anti-PD-1 antibody, and control antibody (1 ug/ml) and SEE (50 ug/ml).
  • FIG. 6 shows ELISA binding curves for binding of four anti-PD-lxCD45 bispecific antibodies (wild-type anti-PD-lxCD45 and three mutant anti-PD-lxCD45) to human-PD-l-His- coated plates.
  • the three mutant antibodies anti-PD-lxCD45-mutl, anti-PD-lxCD45-mut3, and anti-PD-lxCD45-mut4 had reduced affinity to CD45.
  • FIG. 7 shows IL-2 concentration in Jurkat-Raji co-culture in the presence of 10 pg/ml of 100 pg/ml SEE (Staphylococcal Enterotoxin E) and an anti-PD-1 antibody, wild-type anti-PD-lxCD45 antibody, or anti-PD-lxCD45-mut4 antibody compared to SEE alone with no antibody and no SEE no antibody controls.
  • SEE Staphylococcal Enterotoxin E
  • FIGs. 8A-B show binding curves to human-CD45RO-ECD-His-coated plates was quantified by ELISA.
  • FIG. 8 A shows binding curves for anti-CD45 antibody clones 023, 026, 027, and 028, CD45RO Monoclonal Antibody (UCHL1) (eBioscienceTM), anti-HEL-human IgGl isotype control, and blank.
  • FIG. 8B shows binding curves for anti-CD45 antibody clones 031, and 042, CD45RO Monoclonal Antibody (UCHL1) (eBioscienceTM), anti-HEL-human IgGl isotype control, and blank.
  • EC50 values were calculated with GraphPad Prism (vl0.2.1).
  • FIGs. 9A-F shows SRP analysis of binding of anti-CD45 antibodies to captured human-CD45RO-ECD-His.
  • FIG. 9A shows binding of CD45RO Monoclonal Antibody (UCHL1) to captured human-CD45RO-ECD-His.
  • FIG. 9B shows binding of anti-CD45 antibody clone 23 to captured human-CD45RO-ECD-His.
  • FIG. 9C shows binding of anti-CD45 antibody clone 26 to captured human-CD45RO-ECD-His.
  • FIG. 9D shows binding of anti-CD45 antibody clone 27 to captured human-CD45RO-ECD-His.
  • FIG. 9E shows binding of anti-CD45 antibody clone 28 to captured human-CD45RO-ECD-His.
  • FIG. 9F shows binding of anti-CD45 antibody clone 31 to captured human-CD45RO-ECD-His.
  • FIG. 10 shows binding of anti-CD45 antibodies to cell-expressed CD45 in Jurkat cells by flow cytometry.
  • FIG. 10 shows binding for anti-CD45 antibody clones 027, 042, 028, 031, 026, and 023 compared to unstained, sec-only, and non-relevant control.
  • FIG. 11 shows binding of anti-CD45 antibodies to cell-expressed CD45 in Raji cells by flow cytometry.
  • FIG. 11 shows binding for anti-CD45 antibody clones 027, 042, 028, 031, 026, and 023 compared to unstained, sec-only, and non-relevant control.
  • FIGs. 12A-B show binding curves to human-PD-l-His-coated plates quantified by ELISA.
  • FIG. 12A shows binding curves for anti-PD-1 antibody clones 01, 02, 03, and 07, antihuman PD-1 antibody Penbio (pembrolizumab), anti-HEL-human IgGl isotype control, and blank.
  • FIG. 12B shows binding curves for anti-PD-1 antibody clones 09, 51, 55, 79, and 80, anti-human PD-1 antibody Penbio (pembrolizumab), anti-HEL-human IgGl isotype control, and blank.
  • EC50 values were calculated with GraphPad Prism (vl0.2.1).
  • FIG. 13 shows binding of anti-PD-1 clones 01, 02, 03, 07, 09, 51, 55, 79, and 80 to cell-expressed PD-1 by flow cytometry.
  • FIG. 14 shows anti-PD-1 clones 01, 02, 03, 07, 09, 51, 55, 79, and 80 blocking the binding of rhPD-L2 to cell-expressed PD-1.
  • FIG. 15 shows IL-2 concentrations determined by ELISA following addition of anti- PD-1 clones 01, 02, 03, 07, 09, 51, 55, 79, or 80 and SEE (Staphylococcal Enterotoxin E) in Jurkat-Raji co-culture compared to no SEE and no antibody control and SEE and no antibody control.
  • SEE Staphylococcal Enterotoxin E
  • FIG. 16 shows IL-2 concentrations determined by ELISA following addition of anti- PD-1 clones 01, 02, 03, 07, 09, 51, 55, 79, or 80 at different concentrations (10, 2, 0.5 or 0.1 pg/ml) and SEE (Staphylococcal Enterotoxin E) in Jurkat-Raji co-culture compared to no SEE and no antibody control and SEE and no antibody control.
  • SEE Staphylococcal Enterotoxin E
  • FIG. 17 shows concentrations of IL-2, IFNy, IL-6, IL-4 and IL-ip determined by ELISA in PBMCs in the presence of anti-PD-1 clones 01, 02, 03, 07, 09, 51, 55, 79, or 80 and SEE (Staphylococcal Enterotoxin E).
  • FIG. 18 shows the amino acid sequences for variable heavy chain and variable light chain portions of anti-PD-1 antibody clones 01, 02, 03, 07, 09, 51, 55, 79, and 80. Bolded and underlined amino acids indicates CDRs.
  • the target protein of the immune synapse is relocalized to the distal compartment away from the immune synapse.
  • the target protein is a stimulatory receptor (for example, CD2 or CD28)
  • the target protein is an inhibitory receptor (for example, PD-1)
  • such relocalization can lead to T cell activation and is useful in treating disease, such as cancers.
  • novel monoclonal and bispecific antibodies that in some embodiments are useful for preventing or treating disease (e.g., cancer) or infection (e.g., human immunodeficiency virus (HIV)).
  • disease e.g., cancer
  • infection e.g., human immunodeficiency virus (HIV)
  • Described herein are antibodies, including, but not limited to, bispecific antibodies that bind to CD45.
  • the antibodies or bispecific antibodies disclosed herein bind to CD45 with reduced affinity.
  • novel anti-CD45xPD-l and anti-CD43xPD-l bispecific antibodies that in some embodiments are advantageous over existing anti -PD-1 monospecific antibodies for prevention or treatment of one or more cancers.
  • the novel anti-CD45xPD-l and anti-CD43xPD-l bispecific antibodies inhibit PD-1 signaling with enhanced specificity.
  • the bispecific antibodies disclosed herein bind to CD45 with reduced affinity.
  • T cells have surface receptors that can act as immune checkpoint receptors, such as PD-1.
  • immune checkpoint receptors such as PD-1.
  • These receptors act as “checkpoints” to prevent excessive immune activation.
  • cancer cells can exploit these checkpoint pathways to evade immune detection and attack.
  • PD-1 is expressed on the surface of activated T cells while its ligands PD-L1 and PD-L2 are expressed on various cancer cells. See Liu, et al. (2021).
  • the immune synapse is the interface between T cells and tumor cells. This interface, or microenvironment, includes the checkpoint receptors (e.g., PD-1, PDL-1, PDL-2) and other immune receptors and ligands needed for T cells to function.
  • the immune synapse is organized into three compartments; every protein has a specific location.
  • T cell receptor TCR
  • PD-1 and CD28 are located in the peripheral synapse
  • LFA-1, CD43, and CD45 are found in the distal compartment of the synapse.
  • Exclusion of CD43 from the immunological synapse is mediated by phosphorylation-regulated relocation of the cytoskeletal adaptor moesin. Immunity. 2001 Nov;15(5):691-701. doi: 10.1016/sl074-7613(01)00231-x.
  • Cancer immunotherapy drugs work by blocking these checkpoint receptors on T cells, thereby unleashing the immune system to recognize and destroy cancer cells more effectively.
  • PD-1 inhibitors prevent PD-1 on T cells from binding to its ligands PDL-1 and PDL-2 on tumor cells, thereby preventing the tumor cells from evading the T cell immune response.
  • At least seven monoclonal antibodies targeting PD-1 have been approved by the FDA for cancer treatment. However, some individuals do not respond to this treatment, and some experience immune-related adverse events (irAEs).
  • the design of antibodies for the prevention or treatment of cancer is improved by taking into account the localization of proteins (such as PD-1 (CD279), CD352 (SLAMF6), CD6, CD28, CD3E, CD80, CD86, CD276, CD70, CD46, CD209, CD40, CD247, CD4, CD8A, CD3G, TRBC1, TRBC2, CD3D, CD8B, Thyl, LAT, PAG1, CD276, PTPRC (CD45), TCR alpha/beta/gamma/delta, CD294, PD-L1 (CD274), PD-L2 (PDCD1LG2), CTLA-4, BTN3A1, HLA-DRB1, HLA-DRB3, ICOSL (B7h), TIGIT, LAG3 (CD223), CD196 (CCR6), CD56 (NCAM), CD366 (TIM3), CD45RA, CD154 (CD40
  • proteins such as PD-1 (CD279), CD352 (SLAMF6),
  • compositions or methods for relocalizing proteins of the immune synapse to the different compartments of the immune synapse are compositions or methods for relocalizing proteins of the immune synapse to the different compartments of the immune synapse.
  • relocalizing proteins at the immune synapse promotes T cell activation, and localizing proteins away from the immune synapse draws with it proteins necessary for inhibition of T cell activation.
  • localizing proteins away from the immune synapse can avoid activating all T cells and instead only activate those T cells forming a synapse with a cancer cell, reducing the risk of nonspecific T cell activation and immune related adverse events.
  • localizing proteins away from the immune synapse relocalizes proteins to the distal compartment of the immune synapse.
  • changing the location of proteins within the different compartments of the immune synapse could serve as an alternative, efficient, and safer approach to treating cancer patients.
  • an advantage of removing a protein from the synapse with a molecule is its ability to prevent said protein ligand-independent downstream tonic signaling. While certain synaptic proteins are localized to the core of the immune synapse (for example, TCR, CD28, CTLA-4, PD-1, PKC-teta), others, due to their size, are localized to the distal synapse (for example, CD45, CD43, CD44, LFA-1, Talin, CD2).
  • a molecule that binds distal synaptic proteins and core synaptic proteins could allow the distal synaptic protein to serve as an anchor, outside the synapse, and pull the core synaptic protein away from the core of the synapse.
  • one or more of the molecules described herein function by binding to a protein at the core of the synapse and a distal synaptic protein pulling the core synaptic protein away from the core of the synapse, disrupting downstream signaling and effector pathways, and/or enhancing T-cell function.
  • Described herein are inhibitors of proteins at the immune synapse capable of relocalizing the protein.
  • the inhibitors are antibodies that bind to the proteins at the immune synapse and relocalize the protein.
  • Several monoclonal antibodies targeting a synaptic protein have been approved by the FDA for cancer treatment. However, the response to these treatments is suboptimal, and some patients experience irAEs and other toxicities. Without intending to be bound by any particular theory, it is hypothesized that antibodies that bind and inhibit synaptic proteins pull the synaptic protein away from the immune synapse.
  • one or more of the antibodies described herein function by binding to a protein at the immune synapse and pulling the synaptic protein away from the immune synapse, disrupting downstream signaling and effector pathways, and/or enhancing and/or inhibiting T-cell function.
  • the proteins at the immune synapse could be PD-1 (CD279), CD352 (SLAMF6), CD6, CD28, CD3E, CD80, CD86, CD276, CD70, CD46, CD209, CD40, CD247, CD4, CD8A, CD3G, TRBC1, TRBC2, CD3D, CD8B, Thyl, LAT, PAG1, CD276, PTPRC (CD45), TCR alpha/beta/gamma/delta, CD294, PD-L1 (CD274), PD-L2 (PDCD1LG2), CTLA-4, BTN3A1, HLA-DRB1, HLA-DRB3, ICOSL (B7h), TIGIT, LAG3 (CD223), CD196 (CCR6), CD56 (NCAM), CD366 (TIM3), CD45RA, CD154 (CD40L), CD278 (ICOS), CD25 (IL2R), CD152 (CTLA4), CX
  • one or more of the antibodies described herein function by binding to one of these proteinsat the immune synapse and pulling the protein away from the immune synapse, disrupting downstream signaling and effector pathways, and/or enhancing T-cell function.
  • localizing proteins away from the immune synapse relocalizes proteins to the distal compartment of the immune synapse.
  • the proteins at the immune synapse could be PD-1 (CD279), CD352 (SLAMF6), CD6, CD28, CD3E, CD80, CD86, CD276, CD70, CD46, CD209, CD40, CD247, CD4, CD8A, CD3G, TRBC1, TRBC2, CD3D, CD8B, Thyl, LAT, PAG1, CD276, PTPRC (CD45), TCR alpha/beta/gamma/delta, CD294, PD-L1 (CD274), PD-L2 (PDCD1LG2), CTLA-4, BTN3A1, HLA-DRB1, HLA-DRB3, ICOSL (B7h), TIGIT, LAG3 (CD223), CD196 (CCR6), CD56 (NCAM), CD366 (TIM3), CD45RA, CD154 (CD40L), CD278 (ICOS), CD25 (IL2R), CD152 (CTLA4), CX
  • the novel bispecific antibodies disclosed herein bind to a protein of the immune synapse for relocalization and CD43 on the same cells (binding in cis) and not across cells (binding in trans, i.e., where one arm of the antibody binds to the protein of the immune synapse for relocalization on one cell, and the other arm of the antibody binds to CD43 on another cell).
  • the method of treating cancer comprises administering to the subject in need thereof a composition comprising a molecule that binds to a protein at the immune synapse and exclude said protein from the immune synapse.
  • the composition comprises an antibody against the protein at the immune synapse.
  • the method of treating cancer comprises administering to a subject in need thereof a composition comprising an antibody (including without limitation a bispecific antibody) against PD-1 (CD279), CD352 (SLAMF6), CD6, CD28, CD3E, CD80, CD86, CD276, CD70, CD46, CD209, CD40, CD247, CD4, CD8A, CD3G, TRBC1, TRBC2, CD3D, CD8B, Thyl, LAT, PAG1, CD276, PTPRC (CD45), TCR alpha/beta/gamma/delta, CD294, PD-L1 (CD274), PD-L2 (PDCD1LG2), CTLA-4, BTN3A1, HLA-DRB1, HLA-DRB3, ICOSL (B7h), TIGIT, LAG3 (CD223), CD196 (CCR6), CD56 (NCAM), CD366 (TIM3), CD45RA, CD154 (CD40L), CD278 (ICOS), CD
  • an antibody
  • any of the above proteins are relocalized to the distal compartment of the immune synapse.
  • the bispecific antibody binds the protein to be relocalized and a protein that is localized to the core of the immune synapse. In some embodiments, the bispecific antibody binds the protein to be relocalized and a protein that is localized to the distal synapse.
  • the composition comprises a bispecific antibody that binds to PD-1 and CD45 on the same cells and not across cells. In some embodiments, the composition comprises a bispecific antibody that binds to PD-1 and CD43 on the same cells and not across cells.
  • the composition comprises a bispecific antibody that binds a protein of the immune synapse for relocalization and CD45 on the same cells and not across cells. In some embodiments, the composition comprises a bispecific antibody that binds a protein of the immune synapse for relocalization and CD43 on the same cells and not across cells.
  • the compositions as disclosed herein, (e.g., composition comprising a molecule that binds to a protein at the immune synapse and excludes said protein from the immune synapse), is used to prevent or treat cancer in a subject.
  • the cancer is selected from colorectal cancer, lung cancer, bladder cancer, breast cancer, cervical cancer, kidney cancer, leukemia, Hodgkin lymphoma, non-Hodgkin lymphoma, prostate cancer, skin cancer (e.g., melanoma), head and neck cancer, endometrial cancer, colon cancer, rectal cancer, liver cancer, thyroids cancer, esophageal cancer, renal cell cancer, and a combination thereof.
  • compositions as disclosed herein, (e.g., composition comprising a molecule that binds to a protein at the immune synapse and excludes said protein from the immune synapse), is used to prevent or treat disease caused by any solid tumor that is not able to repair errors in its DNA that occur when the DNA is copied.
  • compositions as disclosed herein, (e.g., composition comprising a molecule that binds to a protein at the immune synapse and excludes said protein from the immune synapse), is used to prevent or treat a viral infection.
  • the viral infection is caused by HIV in a subject.
  • an advantage of removing PD-1 from the synapse with bispecific antibodies is its ability to prevent PD-1 ligandindependent downstream tonic signaling.
  • PD-1 is localized to the core of the immune synapse, and CD45 and CD43, due to their size, are localized to the distal synapse.
  • the novel bispecific antibodies disclosed herein bind to PD-1 and CD43 on the same cells (binding in cis) and not across cells (binding in trans, i.e., where one arm of the antibody binds to PD-1 on one cell, and the other arm of the antibody binds to CD43 on another cell).
  • anti-CD45xPD-l bispecific antibodies that bind to CD45 and localize PD-1 away from the core of the synapse.
  • anti-CD43xPD-l bispecific antibodies that bind to CD43 and localize PD-1 away from the core of the synapse.
  • altering the location of PD-1 disrupts downstream signaling and effector pathways, and enhances T-cell function.
  • the anti-CD45xPD-l bispecific antibody has a modified affinity for CD45 relative to a preselected affinity threshold.
  • the affinity of the anti-CD45xPD-l bispecific antibody to CD45 is lower than an anti-CD45 antibody known in the art.
  • the affinity of the anti-CD45xPD-l bispecific antibody to CD45 is lower than an anti-CD45 antibody with variable heavy and light chains of SEQ ID NO: 2.
  • the affinity (e.g., measured as a dissociation constant (KD)) of the anti-CD45xPD-l bispecific antibody to CD45 is between about 5.6 E-7 KD (M) and about 3.6 E-l 1 KD (M). In some embodiments, the affinity (e.g., measured as a dissociation constant (KD)) of the anti-CD45xPD-l bispecific antibody to CD45 is between 5.6 E-7 KD (M) and 3.6 E-l 1 KD (M).
  • the anti-CD43xPD-l bispecific antibody has a modified affinity for CD43 relative to a preselected affinity threshold.
  • the affinity of the anti-CD43xPD-l bispecific antibody to CD43 is lower than an anti-CD43 antibody known in the art.
  • the affinity (e.g., measured as a dissociation constant (KD)) of the anti-CD43xPD-l bispecific antibody to CD43 is between about 1.0 E-7 KD (M) and about 1.0 E-9 7 KD (M).
  • the affinity (e.g., measured as a dissociation constant (KD)) of the anti-CD43xPD-l bispecific antibody to CD43 is between 1.0 E-7 KD (M) and 1.0 E-9 7 KD (M).
  • the use of anti-CD45xPD-l and/or anti-CD43xPD-l bispecific antibodies disclosed herein have one or more of the following advantages over monospecific antibodies: 1) they prevent PD-1 interaction with its ligands, 2) the reduced affinity to CD45 or CD43 can ensure binding in cis as well as cell type specificity targeting, 3) they localize PD-1 away from the synapse and prevent it from interacting with its downstream effectors, and 4) the proximity of PD-1 to CD45 or CD43 facilitates dephosphorylation of the tail of PD-1, further neutralizing its activity.
  • using one or more of the innovative bispecific antibodies describes herein offer a more potent and safer technology to treat cancer patients resistant to current immunotherapies.
  • an anti-CD45xPD-l and/or an anti-CD43xPD-l bispecific antibody is used to prevent or treat cancer in a subject.
  • the cancer is selected from colorectal cancer, lung cancer, bladder cancer, breast cancer, cervical cancer, kidney cancer, leukemia, Hodgkin lymphoma, non-Hodgkin lymphoma, prostate cancer, skin cancer (e.g., melanoma), head and neck cancer, endometrial cancer, colon cancer, rectal cancer, liver cancer, thyroids cancer, esophageal cancer, renal cell cancer, and a combination thereof.
  • an anti-CD45xPD-l and/or an anti-CD43xPD-l bispecific antibody is used to prevent or treat disease caused by any solid tumor that is not able to repair errors in its DNA that occur when the DNA is copied.
  • an anti-CD45xPD-l and/or an anti-CD43xPD-l bispecific antibody, as disclosed herein, is used to prevent or treat a viral infection.
  • the viral infection is caused by HIV in a subject.
  • IgA immunoglobulin-like antibodies
  • IgG immunoglobulin-like antibodies
  • IgG immunoglobulin-like antibodies
  • the IgG immunoglobulin molecule consists of four polypeptide chains, two identical light (L) chains and two identical heavy (H) chains.
  • the four chains are joined by disulfide bonds in a “Y” configuration wherein the light chains bracket the heavy chains starting at the mouth of the “Y” and continuing through the variable region to the dual ends of the “Y”.
  • Each L chain is linked to an H chain by one covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype.
  • Each H and L chain also has regularly spaced intrachain disulfide bridges.
  • Each heavy chain consists of an N-terminal variable domain (VH) and three constant domains (CHI, CH2, CH3), with an additional “hinge region” between CHI and CH2.
  • the light chains consist of an N- terminal variable domain (VL) and a constant domain (CL).
  • the variable domains of the heavy chain and light chain may be referred to as “VH” and “VL”, respectively. These domains are generally the most variable parts of the antibody (relative to other antibodies of the same class) and contain the antigen binding sites.
  • the VL is aligned with the VH and the CL is aligned with the first constant domain of the heavy chain (CHI). The pairing of a VH and VL together forms a single antigen-binding site.
  • Fc fragment crystalline
  • CDRs complementarity determining regions
  • HVRs hypervariable regions
  • FR framework regions
  • the variable domains of native heavy and light chains each comprise four FR regions, largely adopting a beta-sheet configuration, connected by three CDRs, which form loops connecting, and in some cases forming part of, the beta-sheet structure.
  • the CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the other chain, contribute to the formation of the antigen binding site of antibodies.
  • CDRs may be defined using the nomenclature described by Kabat et al. (1991, NIH Publication 91-3242, National Technical Information Service, Springfield, Va.), incorporated by reference in its entirety herein. Specifically, residues 31-35 (CDR-H1), 50-65 (CDR-H2), and 95-102 (CDR-H3) in the heavy chain variable region and residues 24-34 (CDR-L1), 50-56 (CDR-L2), and 89-97 (CDR-L3) in the light chain variable region.
  • the antibody is a monoclonal antibody.
  • the monoclonal antibody comprises a first, second, third and fourth chain.
  • the first and third chains each comprise a VH domain and the second and fourth chains each comprise a VL domain.
  • the first and third chains each further comprises a CHI domain, a hinge domain, and a Fc domain.
  • the second and fourth chains each further comprises a CL domain. The pairing of the VH and VL of the first and second chains together forms a single antigen-binding site specific for an epitope on and the pairing of the VH and VL of the third and fourth chains together forms a single antigenbinding site specific for the same epitope.
  • the first and second chains are linked by one or more covalent disulfide bonds and the third and fourth chains are linked by one or more covalent disulfide bonds. In some embodiments, the first and third chains are linked by one or more disulfide bonds.
  • the antigen-binding site is specific for CD45. In some embodiments, the antigen-binding site is specific for CD45 and binds to CD45 with reduced affinity.
  • the antibodies disclosed herein are not limited to full-length antibodies.
  • the antibodies of the various embodiments disclosed herein can include one or more of synthetic antibodies, monoclonal antibodies, oligoclonal or polyclonal antibodies, multiclonal antibodies, recombinantly produced antibodies, intrabodies, monospecific antibodies, monovalent antibodies, multispecific antibodies, multivalent antibodies, bispecific antibodies, bivalent antibodies, human antibodies, humanized antibodies, chimeric antibodies, CDR-grafted antibodies, primatized antibodies, Fab fragments, F(ab’) fragments, F(ab’)2 fragments, Fv fragments, single-chain FvFcs (scFv-Fc), single-chain Fvs (scFv), Dabs, nanobodies, anti-idiotypic (anti-Id) antibodies, and any other immunologically-reactive/antigen-binding fragments thereof.
  • the monoclonal antibody comprises a first and second chain that associate together.
  • the first chain and second chain each comprises an scFv with specificity for an epitope on CD-45 and the first and second chains each further comprise a Fc domain.
  • An scFv comprises a variable heavy domain and variable light chain domain separated by a linker.
  • the linker is a glycine-serine linker.
  • the Fc domain of the first chain comprises knob mutations and the Fc domain of the second chain comprise hole mutations, or vice versa.
  • the antibody is a scFv-Fc antibody comprising a first and second chain that associate together, each chain comprising a variable heavy chain (VH) domain, a linker, a variable light chain (VL) domain, an Fc domain.
  • the antibody is a bispecific antibody.
  • the bispecific antibody comprises a first and second chain.
  • the first chain comprises an scFv with specificity for a first epitope and the second chain comprises an scFv with specificity for a second epitope.
  • the first and second chains each further comprise a Fc domain.
  • the bispecific antibody comprises a first and a second heavy chain and a first and a second light chain.
  • the pairing of the first VH and first VL together forms a single antigen-binding site specific for a first epitope and the pairing of the second VH and second VL together forms a single antigen-binding site specific for a second epitope.
  • Such epitopes may be on the same or different targets. If the epitopes are on different targets, such targets may be on the same cell or different cells or cell types.
  • the bispecific antibody comprises a first and a second heavy chain and does not comprise any light chains, wherein the first VH forms a single antigen-binding site specific for a first epitope and the second VH forms a single antigen-binding site specific for a second epitope.
  • Such epitopes may be on the same or different targets.
  • the bispecific antibody comprises any of the designs described in Figure 2 of Brinkmann U, Kontermann RE, The making of bispecific antibodies, MAbs, 2017 Feb/Mar, 9(2): 182-212, PMID: 28071970 the content of which is hereby incorporated by reference in its entirety.
  • the monoclonal antibodies, or antigen binding fragments thereof, disclosed herein contain various modifications, substitutions, additions, or deletions to the variable or binding regions of the ant-CD45 binding arm disclosed herein.
  • the bispecific antibodies disclosed herein contain various modifications, substitutions, additions, or deletions to the variable or binding regions of one or more arms of an anti-CD45xPD-l antibody or an anti-CD43xPD-l antibody disclosed herein.
  • the monoclonal antibodies, or antigen binding fragments thereof, or the bispecific antibodies disclosed herein may contain substitutions or modifications of the constant region (i.e., the Fc region).
  • the antibodies disclosed herein may contain one or more additional amino acid residue substitutions, mutations and/or modifications, which result in a compound with preferred characteristics including, but not limited to: altered pharmacokinetics, increased serum half-life, increase binding affinity, reduced binding affinity, reduced immunogenicity, increased production, altered Fc ligand binding, enhanced or reduced ADCC or CDC activity, altered glycosylation and/or disulfide bonds and modified binding specificity.
  • Amino acids shown inside single square brackets represent one or more amino acid positions that, in some embodiments, can be substituted relative to the sequence shown. In some embodiments, the substitution(s) reduce(s) the affinity of the anti-CD45 portion of the CD45xPD-l bispecific antibody to CD45 relative to an anti-CD45 antibody known in the art. Amino acids shown inside double square brackets represent amino acids that in some embodiments were substituted to reduce the affinity of the anti-CD45 portion of the CD45xPD-l bispecific antibody to CD45 relative to an anti-CD45 antibody known in the art. Italicized amino acids represent the Fc portion. Bolded and italicized amino acids represent the “hole” mutations for the production of knob-in-hole antibodies.
  • the amino acid sequence comprising the anti-CD45 portion of an anti- CD45xPD-l bispecific antibody can comprise the “knob” mutations, while the “hole” mutations are present on an anti-PD-1 portion of an anti-CD45xPD-l bispecific antibody.
  • the amino acid sequence of the anti-CD45 portion (scFv-Fc) of the anti-CD45xPD-l bispecific antibody comprises a signal peptide comprising SEQ ID NO: 1.
  • MGWSCIILFLVATATGVHS SEQ ID NO: 1
  • amino acid sequence of the anti-CD45 portion (scFv-Fc) of an anti-CD45xPD-l bispecific antibody (without the signal peptide) comprises SEQ ID NO: 2.
  • the amino acid sequence of the anti-CD45 portion (scFv-Fc) of an anti-CD45xPD-l bispecific antibody comprises SEQ ID NO: 1 immediately followed by SEQ ID NO: 2.
  • the signal peptide is cleaved during post- translational modifications that occur in vitro or in vivo.
  • the signal peptide of the anti-CD45 portion (scFv-Fc) of the anti-CD45xPD-l bispecific antibody comprises an amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to SEQ ID NO: 1.
  • the anti-CD45 portion (scFv-Fc) of the anti-CD45xPD-l bispecific antibody comprises an amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to SEQ ID NO: 2.
  • the anti-CD45 portion of an anti-CD45xPD-l bispecific antibody comprises an amino acid sequence comprising the CDRs of the variable heavy chain domain and the CDRs of the variable light chain domain of the anti-CD45 portion of the anti-CD45xPD-l bispecific antibody wherein the CDR sequences are indicated above (in bold and underline).
  • the framework regions (FRs) of the variable heavy chain domain and the FRs of the variable light chain domain of the anti-CD45 portion of the anti-CD45xPD-l bispecific antibody comprise an amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to the FRs of SEQ ID NO: 2.
  • the linker between the VH and VL domains comprises a glycine-serine linker.
  • any combination of glycine and serine residues can be used.
  • the glycine-serine linker is between about 2 and 25 amino acids in length. In some embodiments, the glycine-serine linker is about 15 amino acids in length.
  • one or more amino acids of an amino acid sequence encoding one or more CDRs of SEQ ID NO: 2 is substituted. In some embodiments, one or more amino acids of an amino acid sequence encoding one or more variable heavy chain CDRs of SEQ ID NO: 2 is substituted. In some embodiments, one or more amino acids of an amino acid sequence encoding one or more variable light chain CDRs of SEQ ID NO: 2 is substituted.
  • the one or more amino acids of an amino acid sequence encoding one or more CDRs of SEQ ID NO: 2 is substituted such that the anti-CD45 arm of the anti-CD45xPD-l bispecific antibody has a reduced binding affinity to CD45 compared to the binding affinity of the anti-CD45 arm encoded by SEQ ID NO: 2 to CD45.
  • the amino acid sequence of the anti-CD45 portion (scFv-Fc) of the anti-CD45xPD-l bispecific antibody comprises a signal peptide comprising SEQ ID NO: 1.
  • MGWSCIILFLVATATGVHS SEQ ID NO: 1
  • the amino acid sequence of the anti-CD45 portion (scFv-Fc) of an anti-CD45xPD-l bispecific antibody (without the signal peptide) comprises SEQ ID NO: 4.
  • SEQ ID NO: 4 depicts the amino acid sequence of the anti-CD45 portion (scFv-Fc) of an anti-CD45xPD-l bispecific antibody (without the signal peptide) with a W to A mutation in the third CDR of the VH domain.
  • one or more amino acids shown inside single square brackets can also be substituted relative to the sequence shown.
  • the amino acid sequence of the anti-CD45 portion (scFv-Fc) of an anti- CD45xPD-l bispecific antibody comprises SEQ ID NO: 1 immediately followed by SEQ ID NO: 4.
  • the signal peptide is cleaved during post-translational modifications that occur in vitro or in vivo.
  • the anti-CD45 portion (scFv-Fc) of an anti-CD45xPD-l bispecific antibody comprising SEQ ID: NO 4 has lower affinity for CD45 relative to the anti- CD45 portion (scFv-Fc) of an anti-CD45xPD-l bispecific antibody comprising SEQ ID: NO 2.
  • the signal peptide of the anti-CD45 portion (scFv-Fc) of the anti-CD45xPD-l bispecific antibody comprises an amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to SEQ ID NO: 1.
  • the anti-CD45 portion (scFv-Fc) of the anti-CD45xPD-l bispecific antibody comprises an amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to SEQ ID NO: 4.
  • the anti-CD45 portion of an anti-CD45xPD-l bispecific antibody comprises an amino acid sequence comprising the CDRs of the variable heavy chain domain and the CDRs of the variable light chain domain of the anti-CD45 portion (scFv-Fc) of the anti-CD45xPD-l bispecific antibody wherein the CDR sequences are indicated above (in bold and underline).
  • the framework regions (FRs) of the variable heavy chain domain and the FRs of the variable light chain domain of the anti-CD45 portion of the anti-CD45xPD-l bispecific antibody comprise an amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to the FRs of SEQ ID NO: 4.
  • the linker between the VH and VL domains comprises a glycine-serine linker.
  • any combination of glycine and serine residues can be used.
  • the glycine-serine linker is between about 2 and 25 amino acids in length. In some embodiments, the glycine-serine linker is about 15 amino acids in length.
  • the amino acid sequence of the anti-CD45 portion (scFv-Fc) of the anti-CD45xPD-l bispecific antibody comprises a signal peptide comprising SEQ ID NO: 1.
  • MGWSCIILFLVATATGVHS SEQ ID NO: 1
  • the amino acid sequence of the anti-CD45 portion (scFv-Fc) of an anti-CD45xPD-l bispecific antibody (without the signal peptide) comprises SEQ ID NO: 6.
  • SEQ ID NO: 6 depicts the amino acid sequence of the anti-CD45 portion (scFv-Fc) of an anti-CD45xPD-l bispecific antibody (without the signal peptide) with a W to A mutation in the first CDR of the VH domain.
  • one or more amino acids shown inside single square brackets can also be substituted relative to the sequence shown.
  • the amino acid sequence of the anti-CD45 portion (scFv-Fc) of an anti- CD45xPD-l bispecific antibody comprises SEQ ID NO: 1 immediately followed by SEQ ID NO: 6.
  • the signal peptide is cleaved during post-translational modifications that occur in vitro or in vivo.
  • the anti-CD45 portion (scFv-Fc) of an anti-CD45xPD-l bispecific antibody comprising SEQ ID: NO 6 has lower affinity for CD45 relative to the anti- CD45 portion (scFv-Fc) of an anti-CD45xPD-l bispecific antibody comprising SEQ ID: NO 2.
  • the signal peptide of the anti-CD45 portion (scFv-Fc) of the anti-CD45xPD-l bispecific antibody comprises an amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to SEQ ID NO: 1.
  • the anti-CD45 portion (scFv-Fc) of the anti-CD45xPD-l bispecific antibody comprises an amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to SEQ ID NO: 6.
  • the anti-CD45 portion of an anti-CD45xPD-l bispecific antibody comprises an amino acid sequence comprising the CDRs of the variable heavy chain domain and the CDRs of the variable light chain domain of the anti-CD45 portion (scFv-Fc) of the anti-CD45xPD-l bispecific antibody wherein the CDR sequences are indicated above (in bold and underline).
  • the framework regions (FRs) of the variable heavy chain domain and the FRs of the variable light chain domain of the anti-CD45 portion of the anti-CD45xPD-l bispecific antibody comprise an amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to the FRs of SEQ ID NO: 6.
  • the linker between the VH and VL domains comprises a glycine-serine linker.
  • any combination of glycine and serine residues can be used.
  • the glycine-serine linker is between about 2 and 25 amino acids in length. In some embodiments, the glycine-serine linker is about 15 amino acids in length.
  • the amino acid sequence of the anti-CD45 portion (scFv-Fc) of the anti-CD45xPD-l bispecific antibody comprises a signal peptide comprising SEQ ID NO: 1.
  • MGWSCIILFLVATATGVHS SEQ ID NO: 1
  • amino acid sequence of the anti-CD45 portion (scFv-Fc) of an anti-CD45xPD-l bispecific antibody (without the signal peptide) comprises SEQ ID NO: 8.
  • SEQ ID NO: 8 depicts the amino acid sequence of the anti-CD45 portion (scFv-Fc) of an anti-CD45xPD-l bispecific antibody (without the signal peptide) with a Y to A mutation in the second CDR of the VH domain.
  • one or more amino acids shown inside single square brackets can also be substituted relative to the sequence shown.
  • the amino acid sequence of the anti-CD45 portion (scFv-Fc) of an anti- CD45xPD-l bispecific antibody comprises SEQ ID NO: 1 immediately followed by SEQ ID NO: 8.
  • the signal peptide is cleaved during post-translational modifications that occur in vitro or in vivo.
  • the anti-CD45 portion (scFv-Fc) of an anti-CD45xPD-l bispecific antibody comprising SEQ ID: NO 8 has lower affinity for CD45 relative to the anti- CD45 portion (scFv-Fc) of an anti-CD45xPD-l bispecific antibody comprising SEQ ID: NO 2.
  • the signal peptide of the anti-CD45 portion (scFv-Fc) of the anti-CD45xPD-l bispecific antibody comprises an amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to SEQ ID NO: 1.
  • the anti-CD45 portion (scFv-Fc) of the anti-CD45xPD-l bispecific antibody comprises an amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to SEQ ID NO: 8.
  • the anti-CD45 portion of an anti-CD45xPD-l bispecific antibody comprises an amino acid sequence comprising the CDRs of the variable heavy chain domain and the CDRs of the variable light chain domain of the anti-CD45 portion (scFv-Fc) of the anti-CD45xPD-l bispecific antibody wherein the CDR sequences are indicated above (in bold and underline).
  • the framework regions (FRs) of the variable heavy chain domain and the FRs of the variable light chain domain of the anti-CD45 portion of the anti-CD45xPD-l bispecific antibody comprise an amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to the FRs of SEQ ID NO: 8.
  • the linker between the VH and VL domains comprises a glycine-serine linker.
  • any combination of glycine and serine residues can be used.
  • the glycine-serine linker is between about 2 and 25 amino acids in length. In some embodiments, the glycine-serine linker is about 15 amino acids in length.
  • the amino acid sequence of the anti-CD45 portion (scFv-Fc) of the anti-CD45xPD-l bispecific antibody comprises a signal peptide comprising SEQ ID NO: 1.
  • MGWSCIILFLVATATGVHS SEQ ID NO: 1
  • the amino acid sequence of the anti-CD45 portion (scFv-Fc) of an anti-CD45xPD-l bispecific antibody (without the signal peptide) comprises SEQ ID NO: 10.
  • SEQ ID NO: 10 depicts the amino acid sequence of the anti-CD45 portion (scFv-Fc) of an anti-CD45xPD-l bispecific antibody (without the signal peptide) with a Y to A mutation in the second CDR of the VH domain and a V to A mutation in the second CDR of the VH domain.
  • one or more amino acids shown inside single square brackets can also be substituted relative to the sequence shown.
  • the amino acid sequence of the anti-CD45 portion (scFv-Fc) of an anti-CD45xPD-l bispecific antibody comprises SEQ ID NO: 1 immediately followed by SEQ ID NO: 10.
  • the signal peptide is cleaved during post-translational modifications that occur in vitro or in vivo.
  • the anti-CD45 portion (scFv-Fc) of an anti-CD45xPD-l bispecific antibody comprising SEQ ID: NO 10 has lower affinity for CD45 relative to the anti- CD45 portion (scFv-Fc) of an anti-CD45xPD-l bispecific antibody comprising SEQ ID: NO 2.
  • the signal peptide of the anti-CD45 portion (scFv-Fc) of the anti-CD45xPD-l bispecific antibody comprises an amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to SEQ ID NO: 1.
  • the anti-CD45 portion (scFv-Fc) of the anti-CD45xPD-l bispecific antibody comprises an amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to SEQ ID NO: 10.
  • the anti-CD45 portion of an anti-CD45xPD-l bispecific antibody comprises an amino acid sequence comprising the CDRs of the variable heavy chain domain and the CDRs of the variable light chain domain of the anti-CD45 portion (scFv-Fc) of the anti-CD45xPD-l bispecific antibody wherein the CDR sequences are indicated above (in bold and underline).
  • the framework regions (FRs) of the variable heavy chain domain and the FRs of the variable light chain domain of the anti-CD45 portion of the anti-CD45xPD-l bispecific antibody comprise an amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to the FRs of SEQ ID NO: 10.
  • the linker between the VH and VL domains comprises a glycine-serine linker.
  • any combination of glycine and serine residues can be used.
  • the glycine-serine linker is between about 2 and 25 amino acids in length. In some embodiments, the glycine-serine linker is about 15 amino acids in length.
  • the amino acid sequence of the anti-CD45 portion (scFv-Fc) of the anti-CD45xPD-l bispecific antibody comprises a signal peptide comprising SEQ ID NO: 1.
  • MGWSCIILFLVATATGVHS SEQ ID NO: 1
  • the amino acid sequence of the anti-CD45 portion (scFv-Fc) of an anti-CD45xPD-l bispecific antibody (without the signal peptide) comprises SEQ ID NO: 29.
  • SEQ ID NO: 29 depicts the amino acid sequence of the anti-CD45 portion (scFv-Fc) of an anti-CD45xPD-l bispecific antibody (without the signal peptide).
  • the amino acid sequence of the anti-CD45 portion (scFv-Fc) of an anti- CD45xPD-l bispecific antibody comprises SEQ ID NO: 1 immediately followed by SEQ ID NO: 29.
  • the signal peptide is cleaved during post-translational modifications that occur in vitro or in vivo.
  • the anti-CD45 portion (scFv-Fc) of an anti-CD45xPD-l bispecific antibody comprising SEQ ID: NO 29 has lower affinity for CD45 relative to the anti- CD45 portion (scFv-Fc) of an anti-CD45xPD-l bispecific antibody comprising SEQ ID: NO 2.
  • the signal peptide of the anti-CD45 portion (scFv-Fc) of the anti-CD45xPD-l bispecific antibody comprises an amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to SEQ ID NO: 1.
  • the anti-CD45 portion (scFv-Fc) of the anti-CD45xPD-l bispecific antibody comprises an amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to SEQ ID NO: 29.
  • the anti-CD45 portion of an anti-CD45xPD-l bispecific antibody comprises an amino acid sequence comprising the CDRs of the variable heavy chain domain and the CDRs of the variable light chain domain of the anti-CD45 portion (scFv-Fc) of the anti-CD45xPD-l bispecific antibody wherein the CDR sequences are indicated above (in bold and underline).
  • the framework regions (FRs) of the variable heavy chain domain and the FRs of the variable light chain domain of the anti-CD45 portion of the anti-CD45xPD-l bispecific antibody comprise an amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to the FRs of SEQ ID NO: 29.
  • the linker between the VH and VL domains comprises a glycine-serine linker.
  • any combination of glycine and serine residues can be used.
  • the glycine-serine linker is between about 2 and 25 amino acids in length. In some embodiments, the glycine-serine linker is about 15 amino acids in length.
  • the amino acid sequence of the anti-CD45 portion (scFv-Fc) of the anti-CD45xPD-l bispecific antibody comprises a signal peptide comprising SEQ ID NO: 1.
  • MGWSCIILFLVATATGVHS SEQ ID NO: 1
  • the amino acid sequence of the anti-CD45 portion (scFv-Fc) of an anti-CD45xPD-l bispecific antibody (without the signal peptide) comprises SEQ ID NO: 30.
  • SEQ ID NO: 30 depicts the amino acid sequence of the anti-CD45 portion (scFv-Fc) of an anti-CD45xPD-l bispecific antibody (without the signal peptide).
  • the amino acid sequence of the anti-CD45 portion (scFv-Fc) of an anti- CD45xPD-l bispecific antibody comprises SEQ ID NO: 1 immediately followed by SEQ ID NO: 30.
  • the signal peptide is cleaved during post-translational modifications that occur in vitro or in vivo.
  • the anti-CD45 portion (scFv-Fc) of an anti-CD45xPD-l bispecific antibody comprising SEQ ID: NO 30 has lower affinity for CD45 relative to the anti- CD45 portion (scFv-Fc) of an anti-CD45xPD-l bispecific antibody comprising SEQ ID: NO 2.
  • the signal peptide of the anti-CD45 portion (scFv-Fc) of the anti-CD45xPD-l bispecific antibody comprises an amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to SEQ ID NO: 1.
  • the anti-CD45 portion (scFv-Fc) of the anti-CD45xPD-l bispecific antibody comprises an amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to SEQ ID NO: 30.
  • the anti-CD45 portion of an anti-CD45xPD-l bispecific antibody comprises an amino acid sequence comprising the CDRs of the variable heavy chain domain and the CDRs of the variable light chain domain of the anti-CD45 portion (scFv-Fc) of the anti-CD45xPD-l bispecific antibody wherein the CDR sequences are indicated above (in bold and underline).
  • the framework regions (FRs) of the variable heavy chain domain and the FRs of the variable light chain domain of the anti-CD45 portion of the anti-CD45xPD-l bispecific antibody comprise an amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to the FRs of SEQ ID NO: 30.
  • the linker between the VH and VL domains comprises a glycine-serine linker.
  • any combination of glycine and serine residues can be used.
  • the glycine-serine linker is between about 2 and 25 amino acids in length. In some embodiments, the glycine-serine linker is about 15 amino acids in length.
  • the amino acid sequence of the anti-CD45 portion (scFv-Fc) of the anti-CD45xPD-l bispecific antibody comprises a signal peptide comprising SEQ ID NO: 1.
  • MGWSCIILFLVATATGVHS SEQ ID NO: 1
  • the amino acid sequence of the anti-CD45 portion (scFv-Fc) of an anti-CD45xPD-l bispecific antibody (without the signal peptide) comprises SEQ ID NO: 31.
  • SEQ ID NO: 31 depicts the amino acid sequence of the anti-CD45 portion (scFv-Fc) of an anti-CD45xPD-l bispecific antibody (without the signal peptide).
  • the amino acid sequence of the anti-CD45 portion (scFv-Fc) of an anti-CD45xPD-l bispecific antibody comprises SEQ ID NO: 1 immediately followed by SEQ ID NO: 31.
  • the signal peptide is cleaved during post-translational modifications that occur in vitro or in vivo.
  • the anti-CD45 portion (scFv-Fc) of an anti-CD45xPD-l bispecific antibody comprising SEQ ID: NO 31 has lower affinity for CD45 relative to the anti- CD45 portion (scFv-Fc) of an anti-CD45xPD-l bispecific antibody comprising SEQ ID: NO 2.
  • the signal peptide of the anti-CD45 portion (scFv-Fc) of the anti-CD45xPD-l bispecific antibody comprises an amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to SEQ ID NO: 1.
  • the anti-CD45 portion (scFv-Fc) of the anti-CD45xPD-l bispecific antibody comprises an amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to SEQ ID NO: 31.
  • the anti-CD45 portion of an anti-CD45xPD-l bispecific antibody comprises an amino acid sequence comprising the CDRs of the variable heavy chain domain and the CDRs of the variable light chain domain of the anti-CD45 portion (scFv-Fc) of the anti-CD45xPD-l bispecific antibody wherein the CDR sequences are indicated above (in bold and underline).
  • the framework regions (FRs) of the variable heavy chain domain and the FRs of the variable light chain domain of the anti-CD45 portion of the anti-CD45xPD-l bispecific antibody comprise an amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to the FRs of SEQ ID NO: 31.
  • the linker between the VH and VL domains comprises a glycine-serine linker.
  • any combination of glycine and serine residues can be used.
  • the glycine-serine linker is between about 2 and 25 amino acids in length. In some embodiments, the glycine-serine linker is about 15 amino acids in length.
  • the amino acid sequence of the anti-CD45 portion (scFv-Fc) of the anti-CD45xPD-l bispecific antibody comprises a signal peptide comprising SEQ ID NO: 1.
  • MGWSCIILFLVATATGVHS SEQ ID NO: 1
  • the amino acid sequence of the anti-CD45 portion (scFv-Fc) of an anti-CD45xPD-l bispecific antibody (without the signal peptide) comprises SEQ ID NO: 32.
  • SEQ ID NO: 32 depicts the amino acid sequence of the anti-CD45 portion (scFv-Fc) of an anti-CD45xPD-l bispecific antibody (without the signal peptide).
  • the amino acid sequence of the anti-CD45 portion (scFv-Fc) of an anti-CD45xPD-l bispecific antibody comprises SEQ ID NO: 1 immediately followed by SEQ ID NO: 32.
  • the signal peptide is cleaved during post-translational modifications that occur in vitro or in vivo.
  • the anti-CD45 portion (scFv-Fc) of an anti-CD45xPD-l bispecific antibody comprising SEQ ID: NO 32 has lower affinity for CD45 relative to the anti- CD45 portion (scFv-Fc) of an anti-CD45xPD-l bispecific antibody comprising SEQ ID: NO 2.
  • the signal peptide of the anti-CD45 portion (scFv-Fc) of the anti-CD45xPD-l bispecific antibody comprises an amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to SEQ ID NO: 1.
  • the anti-CD45 portion (scFv-Fc) of the anti-CD45xPD-l bispecific antibody comprises an amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to SEQ ID NO: 32.
  • the anti-CD45 portion of an anti-CD45xPD-l bispecific antibody comprises an amino acid sequence comprising the CDRs of the variable heavy chain domain and the CDRs of the variable light chain domain of the anti-CD45 portion (scFv-Fc) of the anti-CD45xPD-l bispecific antibody wherein the CDR sequences are indicated above (in bold and underline).
  • the framework regions (FRs) of the variable heavy chain domain and the FRs of the variable light chain domain of the anti-CD45 portion of the anti-CD45xPD-l bispecific antibody comprise an amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to the FRs of SEQ ID NO: 32.
  • the linker between the VH and VL domains comprises a glycine-serine linker.
  • any combination of glycine and serine residues can be used.
  • the glycine-serine linker is between about 2 and 25 amino acids in length. In some embodiments, the glycine-serine linker is about 15 amino acids in length.
  • the amino acid sequence of the anti-CD45 portion (scFv-Fc) of the anti-CD45xPD-l bispecific antibody comprises a signal peptide comprising SEQ ID NO: 1.
  • MGWSCIILFLVATATGVHS SEQ ID NO: 1
  • the amino acid sequence of the anti-CD45 portion (scFv-Fc) of an anti-CD45xPD-l bispecific antibody (without the signal peptide) comprises SEQ ID NO: 33.
  • SEQ ID NO: 33 depicts the amino acid sequence of the anti-CD45 portion (scFv-Fc) of an anti-CD45xPD-l bispecific antibody (without the signal peptide).
  • the amino acid sequence of the anti-CD45 portion (scFv-Fc) of an anti- CD45xPD-l bispecific antibody comprises SEQ ID NO: 1 immediately followed by SEQ ID NO: 33.
  • the signal peptide is cleaved during post-translational modifications that occur in vitro or in vivo.
  • the anti-CD45 portion (scFv-Fc) of an anti-CD45xPD-l bispecific antibody comprising SEQ ID: NO 33 has lower affinity for CD45 relative to the anti- CD45 portion (scFv-Fc) of an anti-CD45xPD-l bispecific antibody comprising SEQ ID: NO 2.
  • the signal peptide of the anti-CD45 portion (scFv-Fc) of the anti-CD45xPD-l bispecific antibody comprises an amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to SEQ ID NO: 1.
  • the anti-CD45 portion (scFv-Fc) of the anti-CD45xPD-l bispecific antibody comprises an amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to SEQ ID NO: 33.
  • the anti-CD45 portion of an anti-CD45xPD-l bispecific antibody comprises an amino acid sequence comprising the CDRs of the variable heavy chain domain and the CDRs of the variable light chain domain of the anti-CD45 portion (scFv-Fc) of the anti-CD45xPD-l bispecific antibody wherein the CDR sequences are indicated above (in bold and underline).
  • the framework regions (FRs) of the variable heavy chain domain and the FRs of the variable light chain domain of the anti-CD45 portion of the anti-CD45xPD-l bispecific antibody comprise an amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to the FRs of SEQ ID NO: 33.
  • the linker between the VH and VL domains comprises a glycine-serine linker.
  • any combination of glycine and serine residues can be used.
  • the glycine-serine linker is between about 2 and 25 amino acids in length. In some embodiments, the glycine-serine linker is about 15 amino acids in length.
  • the amino acid sequence of the anti-CD45 portion (scFv-Fc) of the anti-CD45xPD-l bispecific antibody comprises a signal peptide comprising SEQ ID NO: 1.
  • MGWSCIILFLVATATGVHS SEQ ID NO: 1
  • the amino acid sequence of the anti-CD45 portion (scFv-Fc) of an anti-CD45xPD-l bispecific antibody (without the signal peptide) comprises SEQ ID NO: 34.
  • SEQ ID NO: 34 depicts the amino acid sequence of the anti-CD45 portion (scFv-Fc) of an anti-CD45xPD-l bispecific antibody (without the signal peptide).
  • the amino acid sequence of the anti-CD45 portion (scFv-Fc) of an anti-CD45xPD-l bispecific antibody comprises SEQ ID NO: 1 immediately followed by SEQ ID NO: 34.
  • the signal peptide is cleaved during post-translational modifications that occur in vitro or in vivo.
  • the anti-CD45 portion (scFv-Fc) of an anti-CD45xPD-l bispecific antibody comprising SEQ ID: NO 34 has lower affinity for CD45 relative to the anti- CD45 portion (scFv-Fc) of an anti-CD45xPD-l bispecific antibody comprising SEQ ID: NO 2.
  • the signal peptide of the anti-CD45 portion (scFv-Fc) of the anti-CD45xPD-l bispecific antibody comprises an amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to SEQ ID NO: 1.
  • the anti-CD45 portion (scFv-Fc) of the anti-CD45xPD-l bispecific antibody comprises an amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to SEQ ID NO: 34.
  • the anti-CD45 portion of an anti-CD45xPD-l bispecific antibody comprises an amino acid sequence comprising the CDRs of the variable heavy chain domain and the CDRs of the variable light chain domain of the anti-CD45 portion (scFv-Fc) of the anti-CD45xPD-l bispecific antibody wherein the CDR sequences are indicated above (in bold and underline).
  • the framework regions (FRs) of the variable heavy chain domain and the FRs of the variable light chain domain of the anti-CD45 portion of the anti-CD45xPD-l bispecific antibody comprise an amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to the FRs of SEQ ID NO: 34.
  • the linker between the VH and VL domains comprises a glycine-serine linker.
  • any combination of glycine and serine residues can be used.
  • the glycine-serine linker is between about 2 and 25 amino acids in length. In some embodiments, the glycine-serine linker is about 15 amino acids in length.
  • nucleic acid sequences encoding the features identified in the corresponding amino acid sequences (e.g., CDRs, linker sequence, variable heavy chain and variable light chain domains, Fc portion, knob/hole regions, and any amino acids that are associated with reduced binding affinity) by translating the nucleic acid sequences into amino acid sequences.
  • the nucleic acid sequence comprising the anti-CD45 portion of an anti-CD45xPD-l bispecific antibody can comprise a nucleic acid sequence encoding the “knob” mutations, while the “hole” mutations are present on an anti-PD-1 portion of an anti-CD45xPD-l bispecific antibody.
  • the nucleic acid sequences may be codon optimized.
  • the nucleic acid sequence of the anti-CD45 portion (scFv-Fc) of the anti-CD45xPD-l bispecific antibody comprises a nucleic acid sequence encoding a signal peptide comprising SEQ ID NO: 11.
  • nucleic acid sequence encoding the anti-CD45 portion (scFv-Fc) of an anti-CD45xPD-l bispecific antibody (without the signal peptide) comprises SEQ ID NO: 12.
  • nucleic acid sequence encoding the anti-CD45 portion (scFv-Fc) of an anti-CD45xPD-l bispecific antibody comprises SEQ ID NO: 11 immediately followed by SEQ ID NO: 12.
  • the signal peptide is cleaved during post-translational modifications that occur in vitro or in vivo.
  • Nucleic acid sequences encoding the respective CDRs, linker portions, VH portion, VL portion, Fc portion and knob/hole mutations in SEQ ID NO: 12 can be determined from the amino acid sequence of SEQ ID NO: 2 as identified herein by a person of skill in the art.
  • the nucleic acid sequence encoding a signal peptide of the anti-CD45 portion (scFv-Fc) of the anti-CD45xPD-l bispecific antibody comprise a nucleic acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to SEQ ID NO: 11.
  • the nucleic acid sequence encoding a anti- CD45 portion (scFv-Fc) of the anti-CD45xPD-l bispecific antibody comprise a nucleic acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to SEQ ID NO: 12.
  • the nucleic acid sequence encodes CDRs of the variable heavy chain domain and the CDRs of the variable light chain domain of the anti-CD45 portion (scFv-Fc) of the anti-CD45xPD-l bispecific antibody wherein the CDR sequences are indicated above (in bold and underline).
  • the nucleic acid sequence encoding framework regions (FRs) of the variable heavy chain domain and the FRs of the variable light chain domain of the anti-CD45 portion of the anti-CD45xPD-l bispecific antibody comprise a nucleic acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to the FRs of SEQ ID NO: 12.
  • the linker between the VH and VL domains comprises a glycine-serine linker.
  • any combination of glycine and serine residues can be used.
  • the glycine-serine linker comprises a nucleic acid sequence encoding a linker that is between about 2 and 25 amino acids in length.
  • the nucleic acid sequence encodes a glycine-serine linker that is about 15 amino acids in length.
  • one or more nucleic acids of a nucleic acid sequence encoding one or more CDRs of SEQ ID NO: 12 is substituted. In some embodiments, one or more nucleic acids of a nucleic acid sequence encoding one or more variable heavy chain CDRs of SEQ ID NO: 12 is substituted. In some embodiments, one or more nucleic acids of a nucleic acid sequence encoding one or more variable light chain CDRs of SEQ ID NO: 12 is substituted.
  • the one or more nucleic acids of a nucleic acid sequence encoding one or more CDRs of SEQ ID NO: 12 is substituted such that the anti-CD45 arm of the anti-CD45xPD- 1 bispecific antibody has a reduced binding affinity to CD45 compared to the binding affinity of the anti-CD45 arm encoded by SEQ ID NO: 12 to CD45.
  • the nucleic acid sequence of the anti-CD45 portion (scFv-Fc) of the anti-CD45xPD-l bispecific antibody comprises a signal peptide comprising SEQ ID NO: 11.
  • nucleic acid sequence of the anti-CD45 portion (scFv-Fc) of an anti-CD45xPD-l bispecific antibody comprises SEQ ID NO: 14.
  • the nucleic acid sequence encoding the anti-CD45 portion (scFv-Fc) of an anti-CD45xPD-l bispecific antibody comprises SEQ ID NO: 11 immediately followed by SEQ ID NO: 14.
  • the signal peptide is cleaved during post-translational modifications that occur in vitro or in vivo.
  • Nucleic acid sequences encoding the respective CDRs, linker portions, VH portion, VL portion, Fc portion and knob/hole mutations in SEQ ID NO: 14 can be determined from the amino acid sequence of SEQ ID NO: 4 as identified herein by a person of skill in the art.
  • the nucleic acid sequence encoding a signal peptide of the anti-CD45 portion (scFv-Fc) of the anti-CD45xPD-l bispecific antibody comprises a nucleic acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to SEQ ID NO: 11.
  • the nucleic acid sequence encoding an anti- CD45 portion (scFv-Fc) of the anti-CD45xPD-l bispecific antibody comprises a nucleic acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to SEQ ID NO: 14.
  • the nucleic acid sequence encodes a CDRs of the variable heavy chain domain and the CDRs of the variable light chain domain of the anti-CD45 portion (scFv-Fc) of the anti-CD45xPD-l bispecific antibody wherein the CDR sequences are indicated above (in bold and underline).
  • the nucleic acid sequence encoding framework regions (FRs) of the variable heavy chain domain and the FRs of the variable light chain domain of the anti-CD45 portion of the anti-CD45xPD-l bispecific antibody comprise a nucleic acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to the FRs of SEQ ID NO: 14.
  • the linker between the VH and VL domains comprises a glycine-serine linker.
  • any combination of glycine and serine residues can be used.
  • the glycine-serine linker comprises a nucleic acid sequence encoding a linker that is between about 2 and 25 amino acids in length.
  • the nucleic acid sequence encodes a glycine-serine linker that is about 15 amino acids in length.
  • the nucleic acid sequence encoding the anti-CD45 portion (scFv-Fc) of the anti- CD45xPD-l bispecific antibody comprises a nucleic acid sequence encoding a signal peptide comprising SEQ ID NO: 11.
  • nucleic acid sequence encoding the anti- CD45 portion (scFv-Fc) of an anti-CD45xPD-l bispecific antibody (without the signal peptide) comprises SEQ ID NO: 16.
  • nucleic acid sequence encoding the anti-CD45 portion (scFv-Fc) of an anti-CD45xPD-l bispecific antibody comprises SEQ ID NO: 11 immediately followed by SEQ ID NO: 16.
  • the signal peptide is cleaved during post-translational modifications that occur in vitro or in vivo.
  • Nucleic acid sequences encoding the respective CDRs, linker portions, VH portion, VL portion, Fc portion and knob/hole mutations in SEQ ID NO: 16 can be determined from the amino acid sequence of SEQ ID NO: 6 as identified herein by a person of skill in the art.
  • the nucleic acid sequence encoding the signal peptide of the anti-CD45 portion (scFv-Fc) of the anti-CD45xPD-l bispecific antibody comprise a nucleic acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to SEQ ID NO: 11.
  • the nucleci acid sequence encoding the anti-CD45 portion (scFv-Fc) of the anti-CD45xPD-l bispecific antibody comprise a nucleic acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to SEQ ID NO: 16.
  • the nucleci acid sequence encodes the CDRs of the variable heavy chain domain and the CDRs of the variable light chain domain of the anti-CD45 portion (scFv-Fc) of the anti-CD45xPD-l bispecific antibody wherein the CDR sequences are indicated above (in bold and underline).
  • the nucleic acid sequence encoding the framework regions (FRs) of the variable heavy chain domain and the FRs of the variable light chain domain of the anti-CD45 portion of the anti-CD45xPD-l bispecific antibody comprise a nucleic acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to the FRs of SEQ ID NO: 16.
  • the linker between the VH and VL domains comprises a glycine-serine linker.
  • any combination of glycine and serine residues can be used.
  • the glycine-serine linker comprises a nucleic acid sequence encoding a linker that is between about 2 and 25 amino acids in length.
  • the nucleic acid sequence encodes a glycine-serine linker that is about 15 amino acids in length.
  • the nucleic acid sequence encoding the anti-CD45 portion (scFv-Fc) of the anti-CD45xPD-l bispecific antibody comprises a nucleic acid sequence encoding a signal peptide comprising SEQ ID NO: 11.
  • nucleic acid sequence encoding the anti- CD45 portion (scFv-Fc) of an anti-CD45xPD-l bispecific antibody (without the signal peptide) comprises SEQ ID NO: 18.
  • nucleic acid sequence encoding the anti-CD45 portion (scFv-Fc) of an anti-CD45xPD-l bispecific antibody comprises SEQ ID NO: 11 immediately followed by SEQ ID NO: 18.
  • the signal peptide is cleaved during post-translational modifications that occur in vitro or in vivo.
  • Nucleic acid sequences encoding the respective CDRs, linker portions, VH portion, VL portion, Fc portion and knob/hole mutations in SEQ ID NO: 18 can be determined from the amino acid sequence of SEQ ID NO: 8 as identified herein by a person of skill in the art.
  • the nucleic acid sequence encoding the signal peptide of the anti-CD45 portion (scFv-Fc) of the anti-CD45xPD-l bispecific antibody comprise a nucleic acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to SEQ ID NO: 11.
  • the nucleic acid sequence encoding the anti-CD45 portion (scFv-Fc) of the anti-CD45xPD-l bispecific antibody comprise a nucleic acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to SEQ ID NO: 18.
  • the nucleic acid sequence encodes CDRs of the variable heavy chain domain and the CDRs of the variable light chain domain of the anti-CD45 portion (scFv-Fc) of the anti-CD45xPD-l bispecific antibody wherein the CDR sequences are indicated above (in bold and underline).
  • the framework regions (FRs) of the variable heavy chain domain and the FRs of the variable light chain domain of the anti-CD45 portion of the anti-CD45xPD-l bispecific antibody comprise a nucleic acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to the FRs of SEQ ID NO: 18.
  • the linker between the VH and VL domains comprises a glycine-serine linker.
  • any combination of glycine and serine residues can be used.
  • the glycine-serine linker comprises a nucleic acid sequence encoding a linker that is between about 2 and 25 amino acids in length.
  • the nucleic acid sequence encodes a glycine-serine linker that is about 15 amino acids in length.
  • the nucleic acid sequence encoding the anti-CD45 portion (scFv-Fc) of the anti-CD45xPD-l bispecific antibody comprises a nucleic acid sequence encoding a signal peptide comprising SEQ ID NO: 11.
  • nucleic acid sequence encoding the anti- CD45 portion (scFv-Fc) of an anti-CD45xPD-l bispecific antibody (without the signal peptide) comprises SEQ ID NO: 20.
  • the nucleic acid sequence encoding the anti-CD45 portion (scFv-Fc) of an anti-CD45xPD-l bispecific antibody comprises SEQ ID NO: 19 immediately followed by SEQ ID NO: 20.
  • the signal peptide is cleaved during post-translational modifications that occur in vitro or in vivo.
  • Nucleic acid sequences encoding the respective CDRs, linker portions, VH portion, VL portion, Fc portion and knob/hole mutations in SEQ ID NO: 20 can be determined from the amino acid sequence of SEQ ID NO: 10 as identified herein by a person of skill in the art.
  • the nucleic acid sequence encoding the signal peptide of the anti-CD45 portion (scFv-Fc) of the anti-CD45xPD-l bispecific antibody comprise a nucleic acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to SEQ ID NO: 11.
  • the nucleic acid sequence encoding the anti-CD45 portion (scFv-Fc) of the anti-CD45xPD-l bispecific antibody (without the signal peptide) comprise a nucleic acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to SEQ ID NO: 20.
  • the nucleic acid sequence encodes CDRs of the variable heavy chain domain and the CDRs of the variable light chain domain of the anti-CD45 portion (scFv-Fc) of the anti-CD45xPD-l bispecific antibody wherein the CDR sequences are indicated above (in bold and underline).
  • the framework regions (FRs) of the variable heavy chain domain and the FRs of the variable light chain domain of the anti-CD45 portion of the anti-CD45xPD-l bispecific antibody comprise a nucleic acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to the FRs of SEQ ID NO: 20.
  • the linker between the VH and VL domains comprises a glycine-serine linker.
  • any combination of glycine and serine residues can be used.
  • the glycine-serine linker comprises a nucleic acid sequence encoding a linker that is between about 2 and 25 amino acids in length.
  • the nucleic acid sequence encodes a glycine-serine linker that is about 15 amino acids in length.
  • the anti-CD43 portion of the anti-CD43xPD-l bispecific antibody a single-chain variable fragment (scFv) with a fragment crystallizable (Fc) region (i.e., scFv-Fc antibody) comprising a signal peptide, a variable heavy chain domain, a linker, a variable light chain domain, and an Fc portion.
  • scFv-Fc antibody fragment crystallizable region
  • Underlined and bolded amino acids represent the respective complementarity determining regions (CDRs).
  • Double underlined amino acids represent a linker sequence between the variable heavy chain and the variable light chain portions. Italicized amino acids represent the Fc portion.
  • Bolded and italicized amino acids represent the “hole” mutations for the production of knob-in-hole antibodies.
  • the amino acid sequence comprising the anti- CD43 portion of an anti-CD43xPD-l bispecific antibody can comprise the “knob” mutations, while the “hole” mutations are present on an anti-PD-1 portion of an anti-CD43xPD-l bispecific antibody.
  • the amino acid sequence of the anti-CD43 portion (scFv-Fc) of the anti-CD43xPD-l bispecific antibody comprises a signal peptide comprising SEQ ID NO: 17. MGWSCIILFLVATATGVHS (SEQ ID NO: 1). In some embodiments, the amino acid sequence of the anti-CD43 portion (scFv-Fc) of an anti-CD43xPD-l bispecific antibody (without the signal peptide) comprises SEQ ID NO: 22.
  • the amino acid sequence of the anti-CD43 portion (scFv-Fc) of an anti-CD43xPD-l bispecific antibody comprises SEQ ID NO: 1 immediately followed by SEQ ID NO: 22.
  • the signal peptide is cleaved during post- translational modifications that occur in vitro or in vivo.
  • the signal peptide of the anti-CD43 portion (scFv-Fc) of the anti-CD43xPD-l bispecific antibody comprises an amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to SEQ ID NO: 1.
  • the anti-CD43 portion (scFv-Fc) of the anti-CD43xPD-l bispecific antibody comprises an amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to SEQ ID NO: 22.
  • the anti-CD43 portion of an anti-CD43xPD-l bispecific antibody comprises an amino acid sequence comprising the CDRs of the variable heavy chain domain and the CDRs of the variable light chain domain of the anti-CD43 portion (scFv-Fc) of the anti-CD43xPD-l bispecific antibody wherein the CDR sequences are indicated above (in bold and underline).
  • the framework regions (FRs) of the variable heavy chain domain and the FRs of the variable light chain domain of the anti-CD43 portion of the anti-CD43xPD-l bispecific antibody comprise an amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to the FRs of SEQ ID NO: 22.
  • the linker between the VH and VL domains comprises a glycine-serine linker.
  • any combination of glycine and serine residues can be used.
  • the glycine-serine linker is between about 2 and 25 amino acids in length. In some embodiments, the glycine-serine linker is about 15 amino acids in length.
  • one or more amino acids of an amino acid sequence encoding one or more CDRs of SEQ ID NO: 22 is substituted. In some embodiments, one or more amino acids of an amino acid sequence encoding one or more variable heavy chain CDRs of SEQ ID NO: 22 is substituted. In some embodiments, one or more amino acids of an amino acid sequence encoding one or more variable light chain CDRs of SEQ ID NO: 22 is substituted.
  • the one or more amino acids of an amino acid sequence encoding one or more CDRs of SEQ ID NO: 22 is substituted such that the anti-CD43 arm of the anti-CD43xPD- 1 bispecific antibody has a reduced binding affinity to CD43 compared to the binding affinity of the anti-CD43 arm encoded by SEQ ID NO: 22 to CD43.
  • nucleic acid sequences encoding the features identified in the corresponding amino acid sequences (e.g., CDRs, linker sequence, variable heavy chain and variable light chain domains, Fc portion, knob/hole regions, and any amino acids that are associated with reduced binding affinity) by translating the nucleic acid sequences into amino acid sequences.
  • the nucleic acid sequence comprising the anti-CD43 portion of an anti-CD43xPD-l bispecific antibody can comprise a nucleic acid sequence encoding the “knob” mutations, while the “hole” mutations are present on an anti-PD-1 portion of an anti-CD43xPD-l bispecific antibody.
  • the nucleic acid sequences may be codon optimized.
  • nucleic acid sequence of the anti-CD43 portion (scFv-Fc) of an anti-CD43xPD-l bispecific antibody comprises SEQ ID NO: 24.
  • the nucleic acid sequence encoding a signal peptide of the anti-CD43 portion (scFv-Fc) of the anti-CD43xPD-l bispecific antibody comprises a nucleic acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to SEQ ID NO: 11.
  • the nucleic acid sequence encoding an anti- CD43 portion (scFv-Fc) of the anti-CD43xPD-l bispecific antibody comprises a nucleic acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to SEQ ID NO: 24.
  • the nucleic acid sequence encodes CDRs of the variable heavy chain domain and the CDRs of the variable light chain domain of the anti-CD43 portion (scFv-Fc) of the anti-CD43xPD-l bispecific antibody wherein the CDR sequences are indicated above (in bold and underline).
  • the nucleic acid sequence encoding framework regions (FRs) of the variable heavy chain domain and the FRs of the variable light chain domain of the anti-CD43 portion of the anti-CD43xPD-l bispecific antibody comprise a nucleic acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to the FRs of SEQ ID NO: 24.
  • the linker between the VH and VL domains comprises a glycine-serine linker.
  • any combination of glycine and serine residues can be used.
  • the glycine-serine linker comprises a nucleic acid sequence encoding a linker that is between about 2 and 25 amino acids in length.
  • the nucleic acid sequence encodes a glycine-serine linker that is about 15 amino acids in length.
  • one or more nucleic acids of a nucleic acid sequence encoding one or more CDRs of SEQ ID NO: 24 is substituted. In some embodiments, one or more nucleic acids of a nucleic acid sequence encoding one or more variable heavy chain CDRs of SEQ ID NO: 24 is substituted. In some embodiments, one or more nucleic acids encoding one or more variable light chain CDRs of SEQ ID NO: 24 is substituted.
  • the one or more nucleic acids of a nucleic acid sequence encoding one or more CDRs of SEQ ID NO: 24 is substituted such that the anti-CD43 arm of the anti-CD43xPD-l bispecific antibody has a reduced binding affinity to CD43 compared to the binding affinity of the anti-CD43 arm encoded by SEQ ID NO: 24 to CD43.
  • the anti-PD-1 portion of the anti-CD45xPD-l or anti- CD43xPD-l bispecific antibody a single-chain variable fragment (scFv) with a fragment crystallizable (Fc) region (i.e., scFv-Fc antibody) comprising a signal peptide, a variable heavy chain domain, a linker, a variable light chain domain, and an Fc portion.
  • scFv-Fc antibody fragment crystallizable region
  • scFv-Fc antibody comprising a signal peptide, a variable heavy chain domain, a linker, a variable light chain domain, and an Fc portion.
  • Underlined and bolded amino acids shown in Figure 18 represent the respective complementarity determining regions (CDRs).
  • Double underlined amino acids represent a linker sequence between the variable heavy chain and the variable light chain portions. Italicized amino acids represent the Fc portion.
  • Bolded and italicized amino acids represent the “knob” mutations for the production of knob-in- hole antibodies.
  • the amino acid sequence comprising the anti-PD-1 portion of an anti-CD45xPD-l bispecific antibody can comprise the “hole” mutations, while the “knob” mutations are present on an anti-CD45 portion of an anti-CD45xPD-l bispecific antibody or an anti-CD43 portion of an anti-CD43xPD-l bispecific antibody.
  • the amino acid sequence of the anti-PD-1 portion (scFv-Fc) of an anti-CD45xPD-l or an anti-CD43xPD-l bispecific antibody comprises a signal peptide comprising SEQ ID NO: 1.
  • MGWSCIILFLVATATGVHS SEQ ID NO: 1
  • the amino acid sequence of the anti-PD-1 portion (scFv-Fc) of an anti-CD45xPD- 1 or anti-CD43xPD-l bispecific antibody (without the signal peptide) comprises SEQ ID NO: 26.
  • the amino acid sequence of the anti-PD-1 portion (scFv-Fc) of an anti-CD45xPD-l or anti-CD43xPD-l bispecific antibody comprises SEQ ID NO: 1 immediately followed by SEQ ID NO: 26.
  • the signal peptide is cleaved during post-translational modifications that occur in vitro or in vivo.
  • the signal peptide of the anti-PD-1 portion (scFv-Fc) of the anti-CD45xPD-l or the anti-CD43xPD-l bispecific antibody comprises an amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to SEQ ID NO: 1.
  • the anti-PD-1 portion (scFv-Fc) of the anti-CD45xPD-l or the anti-CD43xPD-l bispecific antibody comprises an amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to SEQ ID NO: 26.
  • the anti-PD-1 portion of an anti-CD45xPD- 1 bispecific antibody comprises an amino acid sequence comprising the CDRs of the variable heavy chain domain and the CDRs of the variable light chain domain of the anti-PD-1 portion (scFv-Fc) of the anti-CD45xPD-l or the anti-CD43xPD-l bispecific antibody wherein the CDR sequences indicated above (in bold and underline).
  • the framework regions (FRs) of the variable heavy chain domain and the FRs of the variable light chain domain of the anti-PD-1 portion of the anti-CD45xPD-l or anti-CD43xPD-l bispecific antibody comprise an amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to the FRs of SEQ ID NO: 26.
  • the linker between the VH and VL domains comprises a glycine-serine linker.
  • any combination of glycine and serine residues can be used.
  • the glycine-serine linker is between about 2 and 25 amino acids in length. In some embodiments, the glycine-serine linker is about 15 amino acids in length.
  • the amino acid sequence of the anti-PD-1 portion (scFv-Fc) of an anti-CD45xPD-l or an anti-CD43xPD-l bispecific antibody comprises a signal peptide comprising SEQ ID NO: 1.
  • MGWSCIILFLVATATGVHS SEQ ID NO: 1
  • the amino acid sequence of the anti-PD-1 portion (scFv-Fc) of an anti-CD45xPD- 1 or anti-CD43xPD-l bispecific antibody (without the signal peptide) comprises SEQ ID NO: 35.
  • the amino acid sequence of the anti-PD-1 portion (scFv-Fc) of an anti-CD45xPD-l or anti-CD43xPD-l bispecific antibody comprises SEQ ID NO: 1 immediately followed by SEQ ID NO: 35.
  • the signal peptide is cleaved during post-translational modifications that occur in vitro or in vivo.
  • the signal peptide of the anti-PD-1 portion (scFv-Fc) of the anti-CD45xPD-l or the anti-CD43xPD-l bispecific antibody comprises an amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to SEQ ID NO: 1.
  • the anti-PD-1 portion (scFv-Fc) of the anti-CD45xPD-l or the anti-CD43xPD-l bispecific antibody comprises an amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to SEQ ID NO: 35.
  • the anti-PD-1 portion of an anti-CD45xPD- 1 bispecific antibody comprises an amino acid sequence comprising the CDRs of the variable heavy chain domain and the CDRs of the variable light chain domain of the anti-PD-1 portion (scFv-Fc) of the anti-CD45xPD-l or the anti-CD43xPD-l bispecific antibody wherein the CDR sequences indicated above (in bold and underline).
  • the framework regions (FRs) of the variable heavy chain domain and the FRs of the variable light chain domain of the anti-PD-1 portion of the anti-CD45xPD-l or anti-CD43xPD-l bispecific antibody comprise an amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to the FRs of SEQ ID NO: 35.
  • the linker between the VH and VL domains comprises a glycine-serine linker.
  • any combination of glycine and serine residues can be used.
  • the glycine-serine linker is between about 2 and 25 amino acids in length. In some embodiments, the glycine-serine linker is about 15 amino acids in length.
  • the amino acid sequence of the anti-PD-1 portion (scFv-Fc) of an anti-CD45xPD-l or an anti-CD43xPD-l bispecific antibody comprises a signal peptide comprising SEQ ID NO: 1.
  • MGWSCIILFLVATATGVHS SEQ ID NO: 1
  • the amino acid sequence of the anti-PD-1 portion (scFv-Fc) of an anti-CD45xPD- 1 or anti-CD43xPD-l bispecific antibody (without the signal peptide) comprises SEQ ID NO: 36.
  • the amino acid sequence of the anti-PD-1 portion (scFv-Fc) of an anti-CD45xPD-l or anti-CD43xPD-l bispecific antibody comprises SEQ ID NO: 1 immediately followed by SEQ ID NO: 36.
  • the signal peptide is cleaved during post-translational modifications that occur in vitro or in vivo.
  • the signal peptide of the anti-PD-1 portion (scFv-Fc) of the anti-CD45xPD-l or the anti-CD43xPD-l bispecific antibody comprises an amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to SEQ ID NO: 1.
  • the anti-PD-1 portion (scFv-Fc) of the anti-CD45xPD-l or the anti-CD43xPD-l bispecific antibody comprises an amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to SEQ ID NO: 36.
  • the anti-PD-1 portion of an anti-CD45xPD- 1 bispecific antibody comprises an amino acid sequence comprising the CDRs of the variable heavy chain domain and the CDRs of the variable light chain domain of the anti-PD-1 portion (scFv-Fc) of the anti-CD45xPD-l or the anti-CD43xPD-l bispecific antibody wherein the CDR sequences indicated above (in bold and underline).
  • the framework regions (FRs) of the variable heavy chain domain and the FRs of the variable light chain domain of the anti-PD-1 portion of the anti-CD45xPD-l or anti-CD43xPD-l bispecific antibody comprise an amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to the FRs of SEQ ID NO: 36.
  • the linker between the VH and VL domains comprises a glycine-serine linker.
  • any combination of glycine and serine residues can be used.
  • the glycine-serine linker is between about 2 and 25 amino acids in length. In some embodiments, the glycine-serine linker is about 15 amino acids in length.
  • the amino acid sequence of the anti-PD-1 portion (scFv-Fc) of an anti-CD45xPD-l or an anti-CD43xPD-l bispecific antibody comprises a signal peptide comprising SEQ ID NO: 1.
  • MGWSCIILFLVATATGVHS SEQ ID NO: 1
  • the amino acid sequence of the anti-PD-1 portion (scFv-Fc) of an anti-CD45xPD- 1 or anti-CD43xPD-l bispecific antibody (without the signal peptide) comprises SEQ ID NO: 37.
  • the signal peptide of the anti-PD-1 portion (scFv-Fc) of the anti-CD45xPD-l or the anti-CD43xPD-l bispecific antibody comprises an amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to SEQ ID NO: 1.
  • the anti-PD-1 portion (scFv-Fc) of the anti-CD45xPD-l or the anti-CD43xPD-l bispecific antibody comprises an amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to SEQ ID NO: 37.
  • the anti-PD-1 portion of an anti-CD45xPD- 1 bispecific antibody comprises an amino acid sequence comprising the CDRs of the variable heavy chain domain and the CDRs of the variable light chain domain of the anti-PD-1 portion (scFv-Fc) of the anti-CD45xPD-l or the anti-CD43xPD-l bispecific antibody wherein the CDR sequences indicated above (in bold and underline).
  • the framework regions (FRs) of the variable heavy chain domain and the FRs of the variable light chain domain of the anti-PD-1 portion of the anti-CD45xPD-l or anti-CD43xPD-l bispecific antibody comprise an amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to the FRs of SEQ ID NO: 37.
  • the linker between the VH and VL domains comprises a glycine-serine linker.
  • any combination of glycine and serine residues can be used.
  • the glycine-serine linker is between about 2 and 25 amino acids in length. In some embodiments, the glycine-serine linker is about 15 amino acids in length.
  • the amino acid sequence of the anti-PD-1 portion (scFv-Fc) of an anti-CD45xPD-l or an anti-CD43xPD-l bispecific antibody comprises a signal peptide comprising SEQ ID NO: 1.
  • MGWSCIILFLVATATGVHS SEQ ID NO: 1
  • the amino acid sequence of the anti-PD-1 portion (scFv-Fc) of an anti-CD45xPD- 1 or anti-CD43xPD-l bispecific antibody (without the signal peptide) comprises SEQ ID NO: 38.
  • the signal peptide of the anti-PD-1 portion (scFv-Fc) of the anti-CD45xPD-l or the anti-CD43xPD-l bispecific antibody comprises an amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to SEQ ID NO: 1.
  • the anti-PD-1 portion (scFv-Fc) of the anti-CD45xPD-l or the anti-CD43xPD-l bispecific antibody comprises an amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to SEQ ID NO: 38.
  • the anti-PD-1 portion of an anti-CD45xPD- 1 bispecific antibody comprises an amino acid sequence comprising the CDRs of the variable heavy chain domain and the CDRs of the variable light chain domain of the anti-PD-1 portion (scFv-Fc) of the anti-CD45xPD-l or the anti-CD43xPD-l bispecific antibody wherein the CDR sequences indicated above (in bold and underline).
  • the framework regions (FRs) of the variable heavy chain domain and the FRs of the variable light chain domain of the anti-PD-1 portion of the anti-CD45xPD-l or anti-CD43xPD-l bispecific antibody comprise an amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to the FRs of SEQ ID NO: 38.
  • the linker between the VH and VL domains comprises a glycine-serine linker.
  • any combination of glycine and serine residues can be used.
  • the glycine-serine linker is between about 2 and 25 amino acids in length. In some embodiments, the glycine-serine linker is about 15 amino acids in length.
  • the amino acid sequence of the anti-PD-1 portion (scFv-Fc) of an anti-CD45xPD-l or an anti-CD43xPD-l bispecific antibody comprises a signal peptide comprising SEQ ID NO: 1.
  • MGWSCIILFLVATATGVHS SEQ ID NO: 1
  • the amino acid sequence of the anti-PD-1 portion (scFv-Fc) of an anti-CD45xPD- 1 or anti-CD43xPD-l bispecific antibody (without the signal peptide) comprises SEQ ID NO: 39.
  • the amino acid sequence of the anti-PD-1 portion (scFv-Fc) of an anti-CD45xPD-l or anti-CD43xPD-l bispecific antibody comprises SEQ ID NO: 1 immediately followed by SEQ ID NO: 39.
  • the signal peptide is cleaved during post-translational modifications that occur in vitro or in vivo.
  • the signal peptide of the anti-PD-1 portion (scFv-Fc) of the anti-CD45xPD-l or the anti-CD43xPD-l bispecific antibody comprises an amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to SEQ ID NO: 1.
  • the anti-PD-1 portion (scFv-Fc) of the anti-CD45xPD-l or the anti-CD43xPD-l bispecific antibody comprises an amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to SEQ ID NO: 39.
  • the anti-PD-1 portion of an anti-CD45xPD- 1 bispecific antibody comprises an amino acid sequence comprising the CDRs of the variable heavy chain domain and the CDRs of the variable light chain domain of the anti-PD-1 portion (scFv-Fc) of the anti-CD45xPD-l or the anti-CD43xPD-l bispecific antibody wherein the CDR sequences indicated above (in bold and underline).
  • the framework regions (FRs) of the variable heavy chain domain and the FRs of the variable light chain domain of the anti-PD-1 portion of the anti-CD45xPD-l or anti-CD43xPD-l bispecific antibody comprise an amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to the FRs of SEQ ID NO: 39.
  • the linker between the VH and VL domains comprises a glycine-serine linker.
  • any combination of glycine and serine residues can be used.
  • the glycine-serine linker is between about 2 and 25 amino acids in length. In some embodiments, the glycine-serine linker is about 15 amino acids in length.
  • the amino acid sequence of the anti-PD-1 portion (scFv-Fc) of an anti-CD45xPD-l or an anti-CD43xPD-l bispecific antibody comprises a signal peptide comprising SEQ ID NO: 1.
  • MGWSCIILFLVATATGVHS SEQ ID NO: 1
  • the amino acid sequence of the anti-PD-1 portion (scFv-Fc) of an anti-CD45xPD- 1 or anti-CD43xPD-l bispecific antibody (without the signal peptide) comprises SEQ ID NO: 40.
  • the amino acid sequence of the anti-PD-1 portion (scFv-Fc) of an anti-CD45xPD-l or anti-CD43xPD-l bispecific antibody comprises SEQ ID NO: 1 immediately followed by SEQ ID NO: 40.
  • the signal peptide is cleaved during post-translational modifications that occur in vitro or in vivo.
  • the signal peptide of the anti-PD-1 portion (scFv-Fc) of the anti-CD45xPD-l or the anti-CD43xPD-l bispecific antibody comprises an amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to SEQ ID NO: 1.
  • the anti-PD-1 portion (scFv-Fc) of the anti-CD45xPD-l or the anti-CD43xPD-l bispecific antibody comprises an amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to SEQ ID NO: 40.
  • the anti-PD-1 portion of an anti-CD45xPD- 1 bispecific antibody comprises an amino acid sequence comprising the CDRs of the variable heavy chain domain and the CDRs of the variable light chain domain of the anti-PD-1 portion (scFv-Fc) of the anti-CD45xPD-l or the anti-CD43xPD-l bispecific antibody wherein the CDR sequences indicated above (in bold and underline).
  • the framework regions (FRs) of the variable heavy chain domain and the FRs of the variable light chain domain of the anti-PD-1 portion of the anti-CD45xPD-l or anti-CD43xPD-l bispecific antibody comprise an amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to the FRs of SEQ ID NO: 40.
  • the linker between the VH and VL domains comprises a glycine-serine linker.
  • any combination of glycine and serine residues can be used.
  • the glycine-serine linker is between about 2 and 25 amino acids in length. In some embodiments, the glycine-serine linker is about 15 amino acids in length.
  • the amino acid sequence of the anti-PD-1 portion (scFv-Fc) of an anti-CD45xPD-l or an anti-CD43xPD-l bispecific antibody comprises a signal peptide comprising SEQ ID NO: 1.
  • MGWSCIILFLVATATGVHS SEQ ID NO: 1
  • the amino acid sequence of the anti-PD-1 portion (scFv-Fc) of an anti-CD45xPD- 1 or anti-CD43xPD-l bispecific antibody (without the signal peptide) comprises SEQ ID NO: 41.
  • the amino acid sequence of the anti-PD-1 portion (scFv-Fc) of an anti- CD45xPD-l or anti-CD43xPD-l bispecific antibody comprises SEQ ID NO: 1 immediately followed by SEQ ID NO: 41.
  • the signal peptide is cleaved during post- translational modifications that occur in vitro or in vivo.
  • the signal peptide of the anti-PD-1 portion (scFv-Fc) of the anti-CD45xPD-l or the anti-CD43xPD-l bispecific antibody comprises an amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to SEQ ID NO: 1.
  • the anti-PD-1 portion (scFv-Fc) of the anti-CD45xPD-l or the anti-CD43xPD-l bispecific antibody comprises an amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to SEQ ID NO: 41.
  • the anti-PD-1 portion of an anti-CD45xPD- 1 bispecific antibody comprises an amino acid sequence comprising the CDRs of the variable heavy chain domain and the CDRs of the variable light chain domain of the anti-PD-1 portion (scFv-Fc) of the anti-CD45xPD-l or the anti-CD43xPD-l bispecific antibody wherein the CDR sequences indicated above (in bold and underline).
  • the framework regions (FRs) of the variable heavy chain domain and the FRs of the variable light chain domain of the anti-PD-1 portion of the anti-CD45xPD-l or anti-CD43xPD-l bispecific antibody comprise an amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to the FRs of SEQ ID NO: 41.
  • the linker between the VH and VL domains comprises a glycine-serine linker.
  • any combination of glycine and serine residues can be used.
  • the glycine-serine linker is between about 2 and 25 amino acids in length. In some embodiments, the glycine-serine linker is about 15 amino acids in length.
  • the amino acid sequence of the anti-PD-1 portion (scFv-Fc) of an anti-CD45xPD-l or an anti-CD43xPD-l bispecific antibody comprises a signal peptide comprising SEQ ID NO: 1.
  • MGWSCIILFLVATATGVHS SEQ ID NO: 1
  • the amino acid sequence of the anti-PD-1 portion (scFv-Fc) of an anti-CD45xPD- 1 or anti-CD43xPD-l bispecific antibody (without the signal peptide) comprises SEQ ID NO: 42.
  • the amino acid sequence of the anti-PD-1 portion (scFv-Fc) of an anti- CD45xPD-l or anti-CD43xPD-l bispecific antibody comprises SEQ ID NO: 1 immediately followed by SEQ ID NO: 42.
  • the signal peptide is cleaved during post- translational modifications that occur in vitro or in vivo.
  • the signal peptide of the anti-PD-1 portion (scFv-Fc) of the anti-CD45xPD-l or the anti-CD43xPD-l bispecific antibody comprises an amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to SEQ ID NO: 1.
  • the anti-PD-1 portion (scFv-Fc) of the anti-CD45xPD-l or the anti-CD43xPD-l bispecific antibody comprises an amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to SEQ ID NO: 42.
  • the anti-PD-1 portion of an anti-CD45xPD- 1 bispecific antibody comprises an amino acid sequence comprising the CDRs of the variable heavy chain domain and the CDRs of the variable light chain domain of the anti-PD-1 portion (scFv-Fc) of the anti-CD45xPD-l or the anti-CD43xPD-l bispecific antibody wherein the CDR sequences indicated above (in bold and underline).
  • the framework regions (FRs) of the variable heavy chain domain and the FRs of the variable light chain domain of the anti-PD-1 portion of the anti-CD45xPD-l or anti-CD43xPD-l bispecific antibody comprise an amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to the FRs of SEQ ID NO: 42.
  • the linker between the VH and VL domains comprises a glycine-serine linker.
  • any combination of glycine and serine residues can be used.
  • the glycine-serine linker is between about 2 and 25 amino acids in length. In some embodiments, the glycine-serine linker is about 15 amino acids in length.
  • the amino acid sequence of the anti-PD-1 portion (scFv-Fc) of an anti-CD45xPD-l or an anti-CD43xPD-l bispecific antibody comprises a signal peptide comprising SEQ ID NO: 1.
  • MGWSCIILFLVATATGVHS SEQ ID NO: 1
  • the amino acid sequence of the anti-PD-1 portion (scFv-Fc) of an anti-CD45xPD- 1 or anti-CD43xPD-l bispecific antibody (without the signal peptide) comprises SEQ ID NO: 43.
  • the amino acid sequence of the anti-PD-1 portion (scFv-Fc) of an anti- CD45xPD-l or anti-CD43xPD-l bispecific antibody comprises SEQ ID NO: 1 immediately followed by SEQ ID NO: 43.
  • the signal peptide is cleaved during post- translational modifications that occur in vitro or in vivo.
  • the signal peptide of the anti-PD-1 portion (scFv-Fc) of the anti-CD45xPD-l or the anti-CD43xPD-l bispecific antibody comprises an amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to SEQ ID NO: 1.
  • the anti-PD-1 portion (scFv-Fc) of the anti-CD45xPD-l or the anti-CD43xPD-l bispecific antibody comprises an amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to SEQ ID NO: 43.
  • the anti-PD-1 portion of an anti-CD45xPD- 1 bispecific antibody comprises an amino acid sequence comprising the CDRs of the variable heavy chain domain and the CDRs of the variable light chain domain of the anti-PD-1 portion (scFv-Fc) of the anti-CD45xPD-l or the anti-CD43xPD-l bispecific antibody wherein the CDR sequences indicated above (in bold and underline).
  • the framework regions (FRs) of the variable heavy chain domain and the FRs of the variable light chain domain of the anti-PD-1 portion of the anti-CD45xPD-l or anti-CD43xPD-l bispecific antibody comprise an amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to the FRs of SEQ ID NO: 43.
  • the linker between the VH and VL domains comprises a glycine-serine linker.
  • any combination of glycine and serine residues can be used.
  • the glycine-serine linker is between about 2 and 25 amino acids in length. In some embodiments, the glycine-serine linker is about 15 amino acids in length.
  • a person skilled in the art can identify the nucleic acid sequences encoding the features identified in the corresponding amino acid sequences (e.g., CDRs, linker sequence, variable heavy chain and variable light chain domains, Fc portion, knob/hole regions, and any amino acids that are associated with reduced binding affinity) by translating the nucleic acid sequences into amino acid sequences.
  • amino acid sequences e.g., CDRs, linker sequence, variable heavy chain and variable light chain domains, Fc portion, knob/hole regions, and any amino acids that are associated with reduced binding affinity
  • the nucleic acid sequence comprising the anti-PD-1 portion of an anti-CD45xPD-l bispecific antibody can comprise the “hole” mutations, while the “knob” mutations are present on an anti- CD45 portion of an anti-CD45xPD-l bispecific antibody or an anti-CD43 portion of an anti- CD43xPD-l bispecific antibody.
  • the nucleic acid sequences may be codon optimized.
  • the nucleic acid sequence encoding the anti-PD-1 portion (scFv-Fc) of the anti-CD45xPD-l or the anti-CD43xPD-l bispecific antibody comprises a signal peptide comprising SEQ ID NO: 11.
  • nucleic acid sequence encoding the anti-PD-1 portion (scFv-Fc) of an anti-CD45xPD-l or an anti-CD43xPD-l bispecific antibody (without the signal peptide) comprises SEQ ID NO: 28.
  • the nucleic acid sequence encoding the anti-PD-1 portion (scFv-Fc) of an anti- CD45xPD-l or anti-CD43xPD-l bispecific antibody comprises SEQ ID NO: 11 immediately followed by SEQ ID NO: 28.
  • the signal peptide is cleaved during post- translational modifications that occur in vitro or in vivo.
  • Nucleic acid sequences encoding the respective CDRs, linker portions, VH portion, VL portion, Fc portion and knob/hole mutations in SEQ ID NO:28 can be determined from the amino acid sequence of SEQ ID NO: 26 as identified herein by a person of skill in the art.
  • the nucleic acid sequence encoding a signal peptide of the anti-PD-1 portion (scFv-Fc) of the anti-CD45xPD-l or anti-CD43xPD-l bispecific antibody comprises an nucleic acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to SEQ ID NO: 11.
  • the nucleic acid sequence encoding an anti-PD-1 portion (scFv-Fc) of the anti-CD45xPD-l or anti-CD43xPD-l bispecific antibody comprises an nucleic acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to SEQ ID NO: 28.
  • the nucleic acid sequence encodes CDRs of the variable heavy chain domain and the CDRs of the variable light chain domain of the anti-PD-1 portion (scFv-Fc) of the anti-CD45xPD-l or anti-CD43xPD-l bispecific antibody wherein the CDR sequences are indicated above (in bold and underline).
  • the nucleic acid sequence encoding framework regions (FRs) of the variable heavy chain domain and the FRs of the variable light chain domain of the anti-PD-1 portion of the anti-CD45xPD-l or anti-CD43xPD-l bispecific antibody comprise an nucleic acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to the FRs of SEQ ID NO: 28.
  • the linker between the VH and VL domains comprises a glycine-serine linker.
  • any combination of glycine and serine residues can be used.
  • the glycine-serine linker comprises a nucleic acid sequence encoding a linker that is between about 2 and 25 amino acids in length.
  • the nucleic acid sequence encodes a glycine-serine linker that is about 15 amino acids in length.
  • the anti-PD-1 portion of the anti-CD45xPD-l or anti- CD43xPD-l bispecific antibody comprises a single-chain variable fragment (scFv) with a fragment crystallizable (Fc) region (i.e., scFv-Fc antibody) comprising a signal peptide, a variable heavy chain domain, a linker, a variable light chain domain, and an Fc portion.
  • scFv single-chain variable fragment
  • Fc fragment crystallizable region
  • the amino acid sequence of the anti-PD-1 portion of the anti-CD45xPD-l or anti- CD43xPD-l bispecific antibody comprises at least a portion of the amino acid sequence of Nivolumab, Pembrolizumab, Cemiplimab, Retifanlimab, Dostarlimab, Zimberelimab, Tiselelizumab, Camrelizumab, Sintilimab, Penpulimab, or any other anti-PD-1 antibody known in the art.
  • the amino acid sequence of the anti-PD-1 portion of the anti- CD45xPD-l or anti-CD43xPD-l bispecific antibody comprises at least a variable heavy and variable light chain portions of the amino acid sequence of Nivolumab, Pembrolizumab, Cemiplimab, Retifanlimab, Dostarlimab, Zimberelimab, Tiselelizumab, Camrelizumab, Sintilimab, Penpulimab, or any other anti-PD-1 antibody known in the art
  • the amino acid sequence of the anti-PD-1 portion of the anti-CD45xPD-l or anti- CD43xPD-l bispecific antibody comprises at least the CDRs of the variable heavy chain and the CDRs of the variable light chain portions of the amino acid sequence of Nivolumab, Pembrolizumab, Cemiplimab, Retifanlimab, Dostarlimab, Zimberelimab, Tiselelizumab
  • the nucleic acid sequence of the anti-PD-1 portion of the anti-CD45xPD-l or anti-CD43xPD-l bispecific antibody codes for an amino acid sequence that comprises at least a variable heavy and variable light chain portions of the amino acid sequence of Nivolumab, Pembrolizumab, Cemiplimab, Retifanlimab, Dostarlimab, Zimberelimab, Tiselelizumab, Camrelizumab, Sintilimab, Penpulimab, or any other anti-PD-1 antibody known in the art.
  • the nucleic acid sequence encoding the anti-PD-1 portion of the anti-CD45xPD-l or anti-CD43xPD-l bispecific antibody codes for an amino acid sequence that comprises at least the CDRs of the variable heavy chain and the CDRs of the variable light chain portions of the amino acid sequence of Nivolumab, Pembrolizumab, Cemiplimab, Retifanlimab, Dostarlimab, Zimberelimab, Tiselelizumab, Camrelizumab, Sintilimab, Penpulimab, or any other anti-PD-1 antibody known in the art.
  • the antibody comprising SEQ ID NO: 2 of the anti-CD45 portion and SEQ ID NO: 26 the anti-PD-1 portion is represented by antibody “wild-type anti-CD45xPD-l” in embodiments described and depicted in this disclosure.
  • the antibody comprising SEQ ID NO: 6 of the anti-CD45 portion and SEQ ID NO: 26 the anti-PD-1 portion is represented by antibody “anti-CD45MlPD-l” or “anti-PD-lxCD45- mutl” in embodiments described and depicted in this disclosure.
  • the antibody comprising SEQ ID NO: 8 of the anti-CD45 portion and SEQ ID NO: 26 of the anti-PD-1 portion is represented by antibody “anti-CD45M2PD-l” in embodiments described and depicted in this disclosure.
  • the antibody comprising SEQ ID NO: 10 of the anti-CD45 portion and SEQ ID NO: 26 of the anti-PD-1 portion is represented by antibody “anti-CD45M3PD-l” or “anti-PD- lxCD45-mut3” in embodiments described and depicted in this disclosure.
  • the antibody comprising SEQ ID NO: 4 of the anti-CD45 portion and SEQ ID NO: 26 of the anti-PD-1 portion is represented by antibody “anti-CD45M4PD-l” or “anti-PD- lxCD45-mut4” in embodiments described and depicted in this disclosure.
  • An anti-CD45 monoclonal antibody comprising the VH and VL of SEQ ID NO: 29 is represented by antibody clone “23” or “023” in embodiments described and depicted in this disclosure.
  • a bispecific antibody comprises the anti-CD45 portion comprising SEQ ID NO: 29 and an anti-PD-1 portion comprising SEQ ID NO: 26.
  • An anti-CD45 monoclonal antibody comprising the VH and VL of SEQ ID NO: 30 is represented by antibody clone “26” or “026” in embodiments described and depicted in this disclosure.
  • a bispecific antibody comprises the anti-CD45 portion comprising SEQ ID NO: 30 and an anti-PD-1 portion comprising SEQ ID NO: 26.
  • An anti-CD45 monoclonal antibody comprising the VH and VL of SEQ ID NO: 31 is represented by antibody clone “27” or “027” in embodiments described and depicted in this disclosure.
  • a bispecific antibody comprises the anti-CD45 portion comprising SEQ ID NO: 31 and an anti-PD-1 portion comprising SEQ ID NO: 26.
  • An anti-CD45 monoclonal antibody comprising the VH and VL of SEQ ID NO: 32 is represented by antibody clone “28” or “028” in embodiments described and depicted in this disclosure.
  • a bispecific antibody comprises the anti-CD45 portion comprising SEQ ID NO: 32 and an anti-PD-1 portion comprising SEQ ID NO: 26.
  • An anti-CD45 monoclonal antibody comprising the VH and VL of SEQ ID NO: 33 is represented by antibody clone “31” or “031” in embodiments described and depicted in this disclosure.
  • a bispecific antibody comprises the anti-CD45 portion comprising SEQ ID NO: 33 and an anti-PD-1 portion comprising SEQ ID NO: 26.
  • An anti-CD45 monoclonal antibody comprising the VH and VL of SEQ ID NO: 34 is represented by antibody clone “42” or “042” in embodiments described and depicted in this disclosure.
  • a bispecific antibody comprises the anti-CD45 portion comprising SEQ ID NO: 34 and an anti-PD-1 portion comprising SEQ ID NO: 26.
  • the anti-CD45xPD-l bispecific antibody comprises an anti- CD45 portion selected from SEQ ID NOs: 2, 4, 6, 8, 10, 29, 30, 31, 32, 33, or 34 and an anti- PD-1 portion selected from SEQ ID NOs: 26, 35, 36, 37, 38, 39, 40, 41, 42, or 43.
  • the antibody comprising SEQ ID NO: 12 of the anti-CD45 portion and SEQ ID NO: 28 of the anti-PD-1 portion is represented by antibody “wild-type anti-CD45xPD-l” in embodiments described and depicted in this disclosure.
  • the antibody comprising SEQ ID NO: 16 of the anti-CD45 portion and SEQ ID NO: 28 of the anti-PD-1 portion is represented by antibody “anti-CD45MlPD-l” in embodiments described and depicted in this disclosure.
  • the antibody comprising SEQ ID NO: 18 of the anti-CD45 portion and SEQ ID NO: 28 of the anti-PD-1 portion is represented by antibody “anti-CD45M2PD-l” in embodiments described and depicted in this disclosure.
  • the antibody comprising SEQ ID NO: 20 of the anti-CD45 portion and SEQ ID NO: 28 of the anti-PD-1 portion is represented by antibody “anti-CD45M3PD-l” in embodiments described and depicted in this disclosure.
  • the antibody comprising SEQ ID NO: 14 of the anti-CD45 portion and SEQ ID NO: 28 of the anti-PD-1 portion is represented by antibody “anti-CD45M4PD-l” in embodiments described and depicted in this disclosure.
  • the antibody comprising SEQ ID NO: 22 of the anti-CD43 portion and SEQ ID NO: 26 of the anti-PD-1 portion is represented by antibody “anti-CD43MlPD-l” in embodiments described and depicted in this disclosure.
  • the anti-CD43xPD-l bispecific antibody comprises an anti- CD43 portion comprising SEQ ID NO: 22 and an anti-PD-1 portion selected from SEQ ID NOs: 26, 35, 36, 37, 38, 39, 40, 41, 42, or 43.
  • the antibody comprising SEQ ID NO: 24 of the anti-CD43 portion and SEQ ID NO: 28 of the anti-PD-1 portion is represented by antibody “anti-CD43xPD-l” in embodiments described and depicted in this disclosure.
  • the molecular three-dimensional structure of an anti- CD45xPD-l or an anti-CD43xPD-l bispecific antibody can be predicted based on X-ray crystallography, and/or cryo-EM, and/or using structure prediction algorithms (e.g., machine learning algorithms) known in the art, such as AlphaFold or RaptorX.
  • the structure prediction algorithm is a computational method that is used to predict three- dimensional (3D) antibody structures based on a given nucleic acid or amino acid sequence.
  • the structure prediction algorithm predicts the 3D coordinates of all heavy atoms for a given antibody using a nucleic acid or amino acid sequence and/or aligned sequences of homologues as inputs.
  • the structure of an anti-CD45xPD-l or an anti-CD43xPD-l bispecific antibody is predicted using a combination of methods, e.g., using a combination of AlphaFold (or any other structure prediction algorithm known in the art) and X-ray crystallography or cryo-EM.
  • the structure prediction is improved by combining the use of AlphaFold (or any other structure prediction algorithm known in the art) and X-ray crystallography or cryo-EM.
  • the structure of an anti-CD45xPD-l or an anti-CD43xPD-l bispecific antibody is predicted by using a computational structure prediction algorithm (e.g., AlphaFold or RaptorX) and the structure prediction of the anti-CD45xPD-l or the anti-CD43xPD-l bispecific antibody is then refined by using X-ray crystallography or cryo-EM.
  • the anti-CD45xPD-l or the anti-CD43xPD-l bispecific antibody comprises a three-dimensional structure that is similar to the three-dimensional structure of an anti-CD45xPD-l or an anti-CD43xPD-l bispecific antibody that comprises SEQ ID NO: 2, 4, 6, 8, 18, 22, 26, 29-43, or a combination thereof.
  • the structure prediction algorithm can be used to model the structure of a first bispecific antibody (e.g., an anti-CD45xPD-l or an anti-CD43xPD-l bispecific antibody) (e.g., a reference an anti-CD45xPD-l or an anti-CD43xPD-l bispecific antibody comprising SEQ ID NO: 2, 4, 6, 8, 18, 22, 26, 29-43, or a combination thereof) and compare a predicted structure of second bispecific antibody (e.g., an anti-CD45xPD-l or an anti- CD43xPD-l bispecific antibody) against the predicted structure of the first antibody such that the second antibody can be categorized in the same class as the first bispecific antibody based on its structural similarity to the first bispecific antibody.
  • a metric of structural similarity between two antibodies can be obtained based on the output of a structure prediction algorithm known in the art.
  • the metric of structural similarity between two antibodies is based on a similarity distance.
  • the structure of the anti-CD45xPD-l bispecific antibody allows the anti-CD45xPD-l bispecific antibody to bind to CD45 and PD-1.
  • disclosed herein is a new class of anti-CD45xPD-l bispecific antibodies which comprise structural similarity to one another such that the new class of anti-CD45xPD-l bispecific antibodies are capable of binding to CD45 and PD-1.
  • the anti- CD45xPD-l bispecific antibody comprises means for binding CD45 and PD-1.
  • means for binding CD45 and PD-1 comprises an anti-CD45xPD-l bispecific antibody that comprises SEQ ID NOs: 2 and 26, 4 and 26, 6 and 26, 8 and 26, 10 and 26, 29 and 26, 30 and 26, 31 and 26, 32 and 26, 33 and 26, 34 and 26, 2 and any of 35-43, 4 and any of 35- 43, 6 and any of 35-43, 8 and any of 35-43, 10 and any of 35-43, 29 and any of 35-43, 30 and any of 35-43, 31 and any of 35-43, 32 and any of 35-43, 33 and any of 35-43, or 34 and any of 35-43.
  • the structure of the anti-CD43xPD-l bispecific antibody allows the anti-CD43xPD-l bispecific antibody to bind to CD43 and PD-1.
  • disclosed herein is a new class of anti-CD43xPD-l bispecific antibodies which comprise structural similarity to one another such that the new class of anti-CD43xPD-l bispecific antibodies are capable of binding to CD43 and PD-1.
  • the anti- CD43xPD-l bispecific antibody comprises means for binding CD43 and PD-1.
  • means for binding CD43 and PD-1 comprises an anti-CD43xPD-l bispecific antibody that comprises SEQ ID NO: 22 and 26, 22 and 35, 22 and 36, 22 and 37, 22 and 38, 22 and 39, 22 and 40, 22 and 41, 22 and 42, or 22 and 43.
  • Described herein are monoclonal antibodies or antigen binding fragment thereof comprising an antigen binding site against CD45.
  • the antibodies have reduced affinity to CD45.
  • the reduced affinity anti-CD45 monoclonal antibodies or antigen binding fragment thereof can be used for enhanced cancer immunotherapy, improved understanding of immune synapse dynamics, and/or the development of combination therapies to overcome cancer treatment resistance.
  • amino acid sequences and nucleic acid sequences of anti-CD45 monoclonal antibodies or antigen binding fragment thereof can comprise an antigen binding site against CD45 comprising an amino acid sequence of the anti-CD45 portions described herein.
  • the monoclonal antibodies or antigen binding fragment thereof contemplated herein can comprise an antigen binding site against CD45 with reduced affinity to CD45 comprising an amino acid sequence of the anti-CD45 portions described herein.
  • the amino acid sequences of the anti-CD45 portion is encoded by the nucleic acid sequences disclosed herein.
  • the anti-CD45 antibody comprises two polypeptide heavy chains each comprising a variable heavy chain (VH) domain, CHI domain, a hinge domain, a Fc domain (CH2-CH3) and two polypeptide light chains comprising a variable light chain (VL) domain and a CL domain.
  • the first and second chains are linked by one or more covalent disulfide bonds and the third and fourth chains are linked by one or more covalent disulfide bonds.
  • the first and third chains are linked by one or more disulfide bonds.
  • the VH domain comprises the VH domain sequence of SEQ ID NO: 2 and the VL domain comprises the VL domain sequence of SEQ ID NO: 2.
  • the Fc domain comprises the Fc domain sequence of SEQ ID NO: 2.
  • the substitution(s) reduce(s) the affinity of the anti-CD45 antibody to CD45 relative to an anti-CD45 antibody known in the art.
  • Amino acids shown inside double square brackets of SEQ ID NO: 2 represent amino acids that in some embodiments were substituted to reduce the affinity of the anti-CD45 antibody to CD45 relative to an anti-CD45 antibody known in the art.
  • the anti-CD45 antibody heavy chain comprises a VH domain with an amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to the VH domain sequence of SEQ ID NO: 2.
  • the anti-CD45 antibody light chain comprises a VL domain with amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to the VL domain sequence of SEQ ID NO: 2.
  • the anti-CD45 antibody comprises an amino acid sequence comprising the CDRs of the variable heavy chain domain and the CDRs of the variable light chain domain of the anti-CD45 antibody, wherein the CDR sequences are indicated above (in bold and underline) in SEQ ID NO: 2.
  • the framework regions (FRs) of the variable heavy chain domain and the FRs of the variable light chain domain of the anti-CD45 antibody comprise an amino acid sequence 80, 81, 82, 83, 84,
  • the VH domain comprises the VH domain sequence of SEQ ID NO: 4 and the VL domain comprises the VL domain sequence of SEQ ID NO: 4.
  • the Fc domain comprises the VL domain sequence of SEQ ID NO: 4.
  • the anti-CD45 antibody heavy chain comprises a VH domain with an amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to the VH domain sequence of SEQ ID NO: 4.
  • the anti-CD45 antibody light chain comprises a VL domain with amino acid sequence 80, 81, 82, 83, 84, 85,
  • the anti-CD45 antibody comprises an amino acid sequence comprising the CDRs of the variable heavy chain domain and the CDRs of the variable light chain domain of the anti-CD45 antibody, wherein the CDR sequences are indicated above (in bold and underline) in SEQ ID NO: 4.
  • the framework regions (FRs) of the variable heavy chain domain and the FRs of the variable light chain domain of the anti- CD45 antibody comprise an amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to the FRs of SEQ ID NO: 4.
  • the VH domain comprises the VH domain sequence of SEQ ID NO: 6 and the VL domain comprises the VL domain sequence of SEQ ID NO: 6.
  • the Fc domain comprises the VL domain sequence of SEQ ID NO: 6.
  • the anti-CD45 antibody heavy chain comprises a VH domain with an amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to the VH domain sequence of SEQ ID NO: 6.
  • the anti-CD45 antibody light chain comprises a VL domain with amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to the VL domain sequence of SEQ ID NO: 6.
  • the anti-CD45 antibody comprises an amino acid sequence comprising the CDRs of the variable heavy chain domain and the CDRs of the variable light chain domain of the anti-CD45 antibody, wherein the CDR sequences are indicated above (in bold and underline) in SEQ ID NO: 6.
  • the framework regions (FRs) of the variable heavy chain domain and the FRs of the variable light chain domain of the anti- CD45 antibody comprise an amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to the FRs of SEQ ID NO: 6.
  • the VH domain comprises the VH domain sequence of SEQ ID NO: 8 and the VL domain comprises the VL domain sequence of SEQ ID NO: 8.
  • the Fc domain comprises the VL domain sequence of SEQ ID NO: 8.
  • the anti-CD45 antibody heavy chain comprises a VH domain with an amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to the VH domain sequence of SEQ ID NO: 8.
  • the anti-CD45 antibody light chain comprises a VL domain with amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to the VL domain sequence of SEQ ID NO: 8.
  • the anti-CD45 antibody comprises an amino acid sequence comprising the CDRs of the variable heavy chain domain and the CDRs of the variable light chain domain of the anti-CD45 antibody, wherein the CDR sequences are indicated above (in bold and underline) in SEQ ID NO: 8.
  • the framework regions (FRs) of the variable heavy chain domain and the FRs of the variable light chain domain of the anti- CD45 antibody comprise an amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to the FRs of SEQ ID NO: 8.
  • the VH domain comprises the VH domain sequence of SEQ ID NO: 10 and the VL domain comprises the VL domain sequence of SEQ ID NO: 10.
  • the Fc domain comprises the VL domain sequence of SEQ ID NO: 10.
  • SEQ ID NO: 10 depicts the amino acid sequence of the anti-CD45 antiobdy with a Y to A mutation in the second CDR of the VH domain and a V to A mutation in the second CDR of the VH domain.
  • one or more amino acids shown inside single square brackets of SEQ ID NO: 10 can also be substituted relative to the sequence shown.
  • the anti- CD45 antibody heavy chain comprises a VH domain with an amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to the VH domain sequence of SEQ ID NO: 10.
  • the anti-CD45 antibody light chain comprises a VL domain with amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to the VL domain sequence of SEQ ID NO: 10.
  • the anti-CD45 antibody comprises an amino acid sequence comprising the CDRs of the variable heavy chain domain and the CDRs of the variable light chain domain of the anti-CD45 antibody, wherein the CDR sequences are indicated above (in bold and underline) in SEQ ID NO: 10.
  • the framework regions (FRs) of the variable heavy chain domain and the FRs of the variable light chain domain of the anti-CD45 antibody comprise an amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to the FRs of SEQ ID NO: 10.
  • the VH domain comprises the VH domain sequence of SEQ ID NO: 29 and the VL domain comprises the VL domain sequence of SEQ ID NO: 29.
  • the Fc domain comprises the VL domain sequence of SEQ ID NO: 29.
  • the anti-CD45 antibody heavy chain comprises a VH domain with an amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to the VH domain sequence of SEQ ID NO: 29.
  • the anti-CD45 antibody light chain comprises a VL domain with amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to the VL domain sequence of SEQ ID NO: 29.
  • the anti-CD45 antibody comprises an amino acid sequence comprising the CDRs of the variable heavy chain domain and the CDRs of the variable light chain domain of the anti-CD45 antibody, wherein the CDR sequences are indicated above (in bold and underline) in SEQ ID NO: 29.
  • the framework regions (FRs) of the variable heavy chain domain and the FRs of the variable light chain domain of the anti- CD45 antibody comprise an amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to the FRs of SEQ ID NO: 29.
  • the VH domain comprises the VH domain sequence of SEQ ID NO: 30 and the VL domain comprises the VL domain sequence of SEQ ID NO: 30.
  • the Fc domain comprises the VL domain sequence of SEQ ID NO: 30.
  • the anti-CD45 antibody heavy chain comprises a VH domain with an amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to the VH domain sequence of SEQ ID NO: 30.
  • the anti-CD45 antibody light chain comprises a VL domain with amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to the VL domain sequence of SEQ ID NO: 30.
  • the anti-CD45 antibody comprises an amino acid sequence comprising the CDRs of the variable heavy chain domain and the CDRs of the variable light chain domain of the anti-CD45 antibody, wherein the CDR sequences are indicated above (in bold and underline) in SEQ ID NO: 30.
  • the framework regions (FRs) of the variable heavy chain domain and the FRs of the variable light chain domain of the anti- CD45 antibody comprise an amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to the FRs of SEQ ID NO: 30.
  • the VH domain comprises the VH domain sequence of SEQ ID NO: 31 and the VL domain comprises the VL domain sequence of SEQ ID NO: 31.
  • the Fc domain comprises the VL domain sequence of SEQ ID NO: 31.
  • the anti-CD45 antibody heavy chain comprises a VH domain with an amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to the VH domain sequence of SEQ ID NO: 31.
  • the anti-CD45 antibody light chain comprises a VL domain with amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to the VL domain sequence of SEQ ID NO: 31.
  • the anti-CD45 antibody comprises an amino acid sequence comprising the CDRs of the variable heavy chain domain and the CDRs of the variable light chain domain of the anti-CD45 antibody, wherein the CDR sequences are indicated above (in bold and underline) in SEQ ID NO: 31.
  • the framework regions (FRs) of the variable heavy chain domain and the FRs of the variable light chain domain of the anti- CD45 antibody comprise an amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to the FRs of SEQ ID NO: 31.
  • the VH domain comprises the VH domain sequence of SEQ ID NO: 32 and the VL domain comprises the VL domain sequence of SEQ ID NO: 32.
  • the Fc domain comprises the VL domain sequence of SEQ ID NO: 32.
  • the anti-CD45 antibody heavy chain comprises a VH domain with an amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to the VH domain sequence of SEQ ID NO: 32.
  • the anti-CD45 antibody light chain comprises a VL domain with amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to the VL domain sequence of SEQ ID NO: 32.
  • the anti-CD45 antibody comprises an amino acid sequence comprising the CDRs of the variable heavy chain domain and the CDRs of the variable light chain domain of the anti-CD45 antibody, wherein the CDR sequences are indicated above (in bold and underline) in SEQ ID NO: 32.
  • the framework regions (FRs) of the variable heavy chain domain and the FRs of the variable light chain domain of the anti- CD45 antibody comprise an amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to the FRs of SEQ ID NO: 32.
  • the VH domain comprises the VH domain sequence of SEQ ID NO: 33 and the VL domain comprises the VL domain sequence of SEQ ID NO: 33.
  • the Fc domain comprises the VL domain sequence of SEQ ID NO: 33.
  • the anti-CD45 antibody heavy chain comprises a VH domain with an amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to the VH domain sequence of SEQ ID NO: 33.
  • the anti-CD45 antibody light chain comprises a VL domain with amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to the VL domain sequence of SEQ ID NO: 33.
  • the anti-CD45 antibody comprises an amino acid sequence comprising the CDRs of the variable heavy chain domain and the CDRs of the variable light chain domain of the anti-CD45 antibody, wherein the CDR sequences are indicated above (in bold and underline) in SEQ ID NO: 33.
  • the framework regions (FRs) of the variable heavy chain domain and the FRs of the variable light chain domain of the anti- CD45 antibody comprise an amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to the FRs of SEQ ID NO: 33.
  • the VH domain comprises the VH domain sequence of SEQ ID NO: 34 and the VL domain comprises the VL domain sequence of SEQ ID NO: 34.
  • the Fc domain comprises the VL domain sequence of SEQ ID NO: 34.
  • the anti-CD45 antibody heavy chain comprises a VH domain with an amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to the VH domain sequence of SEQ ID NO: 34.
  • the anti-CD45 antibody light chain comprises a VL domain with amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to the VL domain sequence of SEQ ID NO: 34.
  • the anti-CD45 antibody comprises an amino acid sequence comprising the CDRs of the variable heavy chain domain and the CDRs of the variable light chain domain of the anti-CD45 antibody, wherein the CDR sequences are indicated above (in bold and underline) in SEQ ID NO: 34.
  • the framework regions (FRs) of the variable heavy chain domain and the FRs of the variable light chain domain of the anti- CD45 antibody comprise an amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to the FRs of SEQ ID NO: 34.
  • the amino acid sequence of the anti-CD45 antibody heavy and light chain comprises SEQ ID NO: 1 immediately followed by the sequences of the heavy or light chains.
  • the signal peptide is cleaved during post-translational modifications that occur in vitro or in vivo.
  • the monoclonal antibody is a scFv-Fc antibody comprising a first and second chain that associate together, each chain comprising a variable heavy chain (VH) domain of SEQ ID NO: 2, a linker (e g., GGGGSGGGGSGGGGS (SEQ ID NO: 3)), a variable light chain (VL) domain of SEQ ID NO: 2, an Fc domain of SEQ ID NO: 2.
  • VH variable heavy chain
  • VL variable light chain domain of SEQ ID NO: 2
  • Fc domain of SEQ ID NO: 2 an Fc domain of SEQ ID NO: 2.
  • the monoclonal antibody is a scFv-Fc antibody comprising SEQ ID NO: 2.
  • the monoclonal antibody is a scFv-Fc antibody comprising a first and second chain that associate together, each chain comprising a variable heavy chain (VH) domain of SEQ ID NO: 4, a linker (e g., GGGGSGGGGSGGGGS (SEQ ID NO: 3)), a variable light chain (VL) domain of SEQ ID NO: 4, an Fc domain of SEQ ID NO: 4.
  • VH variable heavy chain
  • VL variable light chain
  • the monoclonal antibody is a scFv-Fc antibody comprising SEQ ID NO: 4.
  • the monoclonal antibody is a scFv-Fc antibody comprising a first and second chain that associate together, each chain comprising a variable heavy chain (VH) domain of SEQ ID NO: 6, a linker (e g., GGGGSGGGGSGGGGS (SEQ ID NO: 3)), a variable light chain (VL) domain of SEQ ID NO: 6, an Fc domain of SEQ ID NO: 6.
  • VH variable heavy chain
  • VL variable light chain
  • the monoclonal antibody is a scFv-Fc antibody comprising SEQ ID NO: 6.
  • the monoclonal antibody is a scFv-Fc antibody comprising a first and second chain that associate together, each chain comprising a variable heavy chain (VH) domain of SEQ ID NO: 8, a linker (e g., GGGGSGGGGSGGGGS (SEQ ID NO: 3)), a variable light chain (VL) domain of SEQ ID NO: 8, an Fc domain of SEQ ID NO: 8.
  • VH variable heavy chain
  • VL variable light chain domain of SEQ ID NO: 8
  • Fc domain of SEQ ID NO: 8 an Fc domain of SEQ ID NO: 8.
  • the monoclonal antibody is a scFv-Fc antibody comprising SEQ ID NO: 8.
  • the monoclonal antibody is a scFv-Fc antibody comprising a first and second chain that associate together, each chain comprising a variable heavy chain (VH) domain of SEQ ID NO: 10, a linker (e g., GGGGSGGGGSGGGGS (SEQ ID NO: 3)), a variable light chain (VL) domain of SEQ ID NO: 10, an Fc domain of SEQ ID NO: 10.
  • VH variable heavy chain
  • VL variable light chain
  • the monoclonal antibody is a scFv-Fc antibody comprising SEQ ID NO: 10.
  • the monoclonal antibody is a scFv-Fc antibody comprising a first and second chain that associate together, each chain comprising a variable heavy chain (VH) domain of SEQ ID NO: 29, a linker (e g., GGGGSGGGGSGGGGS (SEQ ID NO: 3)), a variable light chain (VL) domain of SEQ ID NO: 29, an Fc domain of SEQ ID NO: 29.
  • the monoclonal antibody is a scFv-Fc antibody comprising SEQ ID NO: 29.
  • the monoclonal antibody is a scFv-Fc antibody comprising a first and second chain that associate together, each chain comprising a variable heavy chain (VH) domain of SEQ ID NO: 30, a linker (e g., GGGGSGGGGSGGGGS (SEQ ID NO: 3)), a variable light chain (VL) domain of SEQ ID NO: 30, an Fc domain of SEQ ID NO: 30.
  • the monoclonal antibody is a scFv-Fc antibody comprising SEQ ID NO: 30.
  • the monoclonal antibody is a scFv-Fc antibody comprising a first and second chain that associate together, each chain comprising a variable heavy chain (VH) domain of SEQ ID NO: 31, a linker (e g., GGGGSGGGGSGGGGS (SEQ ID NO: 3)), a variable light chain (VL) domain of SEQ ID NO: 31, an Fc domain of SEQ ID NO: 31.
  • the monoclonal antibody is a scFv-Fc antibody comprising SEQ ID NO: 31.
  • the monoclonal antibody is a scFv-Fc antibody comprising a first and second chain that associate together, each chain comprising a variable heavy chain (VH) domain of SEQ ID NO: 32, a linker (e g., GGGGSGGGGSGGGGS (SEQ ID NO: 3)), a variable light chain (VL) domain of SEQ ID NO: 32, an Fc domain of SEQ ID NO: 32.
  • VH variable heavy chain
  • VL variable light chain domain of SEQ ID NO: 32
  • the monoclonal antibody is a scFv-Fc antibody comprising SEQ ID NO: 32.
  • the monoclonal antibody is a scFv-Fc antibody comprising a first and second chain that associate together, each chain comprising a variable heavy chain (VH) domain of SEQ ID NO: 33, a linker (e g., GGGGSGGGGSGGGGS (SEQ ID NO: 3)), a variable light chain (VL) domain of SEQ ID NO: 33, an Fc domain of SEQ ID NO: 33.
  • the monoclonal antibody is a scFv-Fc antibody comprising SEQ ID NO: 33.
  • the monoclonal antibody is a scFv-Fc antibody comprising a first and second chain that associate together, each chain comprising a variable heavy chain (VH) domain of SEQ ID NO: 34, a linker (e g., GGGGSGGGGSGGGGS (SEQ ID NO: 3)), a variable light chain (VL) domain of SEQ ID NO: 34, an Fc domain of SEQ ID NO: 34.
  • the monoclonal antibody is a scFv-Fc antibody comprising SEQ ID NO: 34.
  • the amino acid sequence of the anti-CD45 (scFv-Fc) antibody comprises a signal peptide comprising SEQ ID NO: 1. MGWSCIILFLVATATGVHS (SEQ ID NO: 1). In some embodiments, the amino acid sequence of the anti-CD45 (scFv-Fc) antibody (without the signal peptide) comprises SEQ ID NO: 2, 4, 6, 8, 10, 29, 30, 31, 32, 33, or 34. In some embodiments, the amino acid sequence of the anti-CD45 (scFv-Fc) antibody comprises SEQ ID NO: 1 immediately followed by SEQ ID NO: 2, 4, 6, 8, 10, 29, 30, 31, 32, 33, or 34. In some embodiments, the signal peptide is cleaved during post-translational modifications that occur in vitro or in vivo.
  • nucleic acid sequences encoding the features identified in the corresponding amino acid sequences e.g., CDRs, variable heavy chain and variable light chain domains, Fc portion, and any amino acids that are associated with reduced binding affinity
  • the nucleic acid sequence encoding the VH domain, VL domain, and/or Fc portion of an anti-CD45 antibody comprises the VH, VL, and/or Fc encoding portions of SEQ ID NO: 12, 14, 16, 18, 20.
  • the monoclonal anti-CD45 antibody, or antigen binding fragment thereof has a modified affinity for CD45 relative to a preselected affinity threshold.
  • the affinity of the monoclonal anti-CD45 antibody, or antigen binding fragment thereof, to CD45 is lower than an anti-CD45 antibody known in the art.
  • the affinity of the monoclonal anti-CD45 antibody, or antigen binding fragment thereof, to CD45 is lower than an anti-CD45 antibody with variable heavy and light chains of SEQ ID NO: 2.
  • the affinity (e.g., measured as a dissociation constant (KD)) of the anti-CD45 antibody to CD45 is between about 5.6 E-7 KD (M) and about 3.6 E-l 1 KD (M).
  • an anti-CD45 antibody, or antigen binding fragment thereof, as disclosed herein is used to prevent or treat cancer in a subject.
  • the cancer is selected from colorectal cancer, lung cancer, bladder cancer, breast cancer, cervical cancer, kidney cancer, leukemia, Hodgkin lymphoma, non-Hodgkin lymphoma, prostate cancer, skin cancer (e.g., melanoma), head and neck cancer, endometrial cancer, colon cancer, rectal cancer, liver cancer, thyroids cancer, esophageal cancer, renal cell cancer, and a combination thereof.
  • an anti-CD45 antibody, or antigen binding fragment thereof, as disclosed herein is used to prevent or treat disease caused by any solid tumor that is not able to repair errors in its DNA that occur when the DNA is copied.
  • an anti-CD45 antibody, or antigen binding fragment thereof, as disclosed herein is used to prevent or treat a viral infection.
  • the viral infection is caused by HIV in a subject.
  • compositions for relocalizing a protein to a compartment of the immune synapse in a subject comprising: a molecule capable of binding the protein at the immune synapse, wherein the protein is relocalized to the distal compartment of the immune synapse or wherein the protein is relocalized to the core of the immune synapse.
  • the protein is relocalized to the distal compartment of the immune synapse and wherein the molecule is an antibody to the protein capable of localizing the protein to the distal compartment of the immune synapse.
  • the antibody is a bispecific antibody to the protein and to another protein at the distal compartment of the immune synapse, wherein the bispecific antibody is capable of localizing the protein away from the immune synapse.
  • the protein is relocalized to the core of the immune synapse and wherein the molecule is an antibody to the protein capable of localizing the protein to the core of the immune synapse.
  • the antibody is a bispecific antibody to the protein and to another protein at the core of the immune synapse, wherein the bispecific antibody is capable of localizing the protein to the core of the immune synapse.
  • the protein at the immune synapse is PD-1 (CD279), CD352 (SLAMF6), CD6, CD28, CD3E, CD80, CD86, CD276, CD70, CD46, CD209, CD40, CD247, CD4, CD8A, CD3G, TRBC1, TRBC2, CD3D, CD8B, Thyl, LAT, PAG1, CD276, PTPRC (CD45), TCR alpha/beta/gamma/delta, CD294, PD-L1 (CD274), PD-L2 (PDCD1LG2), CTLA- 4, BTN3A1, HLA-DRB1, HLA-DRB3, ICOSL (B7h), TIGIT, LAG3 (CD223), CD196 (CCR6), CD56 (NCAM), CD366 (TIM3), CD45RA, CD 154 (CD40L), CD278 (ICOS), CD25 (IL2R), CD152 (CTLA4), CXCR1,
  • the bispecific antibody is any of the bi specific antibodies described herein.
  • a method for treating cancer in a subject in need thereof comprising: administering to the subject a therapy comprising an effective amount of a molecule capable of localizing a protein to a distal compartment of an immune synapse.
  • the molecule is an antibody or an antigen binding fragment thereof capable of localizing the protein to a distal compartment of the immune synapse.
  • the antibody is a bispecific antibody to the protein and to another protein at the distal compartment of the immune synapse, wherein the bispecific antibody is capable of localizing the protein to the distal compartment from the immune synapse.
  • the protein at the immune synapse is CD6, CD28, CD3E, CD80, CD86, CD276, CD70, CD46, CD209, CD40, CD247, CD4, CD8A, CD3G, TRBC1, TRBC2, CD3D, CD8B, Thyl, LAT, PAG1, CD276, PTPRC (CD45), PD-1 (CD279), TCR alpha/beta/gamma/delta, CD294, PD-L1 (CD274), PD-L2 (PDCD1LG2), CTLA-4, BTN3A1, HLA-DRB1, HLA-DRB3, ICOSL (B7h), TIGIT, LAG3 (CD223), CD196 (CCR6), CD56 (NCAM), CD366 (TIM3), CD45RA, CD154 (CD40L), CD278 (ICOS), CD25 (IL2R), CD152 (CTLA4), CXCR1, HLA-DR, CD16, CD6, CD28, CD3E, CD
  • an antibody or antigen binding fragment thereof which binds epitope on CD45 comprising three light chain CDRs and three heavy chain CDRs portions of the SEQ ID NO: 4, 6, 8, 10, 29, 30, 31, 32, 33, or 34, wherein said antibody or antigen binding fragment thereof has at least one of the following characteristics: a. binds CD45 with an affinity between about 5.6 E-7 KD (M) and about 3.6 E-l 1 KD (M); b. binds CD45 with about the same KD as an antibody having SEQ ID NO: 2; or c. binds CD45 with the KD lower than as an antibody having SEQ ID NO: 2.
  • a monoclonal antibody or antigen binding fragment thereof comprising: a first arm comprising a first variable heavy chain domain and a first variable light chain domain, wherein a portion of the first arm is capable of binding to a portion of a CD45 protein; and a second arm comprising a second variable heavy chain domain and a second variable light chain domain, wherein a portion of the second arm is capable of binding to a portion of a CD45 protein.
  • the first arm is encoded by a first polypeptide chain and the second arm is encoded by a second polypeptide chain that associate together.
  • the first arm comprises a linker between the first variable heavy domain and first variable light chain domain.
  • the second arm comprises a linker between the first variable heavy domain and first variable light chain domain.
  • the linker is a glycine-serine linker.
  • the first and second arms each further comprise a fragment, crystallizable (Fc) region.
  • the Fc region of the first arm comprises knob mutations and the Fc region of the second arm comprise hole mutations, or vice versa.
  • the first variable heavy chain domain comprises an amino acid sequence of the variable heavy chain portion of SEQ ID NO: 2, 4, 6, 8, 10, 29, 30, 31, 32, 33, or 34, wherein the first variable light chain domain comprises an amino acid sequence of the variable light chain portion of SEQ ID NO: 2, 4, 6, 8, 10, 29, 30, 31, 32, 33, or 34.
  • the first arm comprises an amino acid sequence selected from SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, or SEQ ID NO: 34
  • the second arm comprises an amino acid sequence selected from SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, or SEQ ID NO: 34.
  • the portion of the first arm is capable of binding to the portion of the CD45 protein with an affinity between about 5.6 E-7 KD (M) and about 3.6 E-l 1 KD (M).
  • a bispecific antibody or a fragment thereof comprising: a first arm comprising a first variable heavy chain domain and a first variable light chain domain, wherein a portion of the first arm is capable of binding to a portion of a CD45 protein or a portion of a CD43 protein; and a second arm comprising a second variable heavy chain domain and a second variable light chain domain, wherein a portion of the second arm is capable of binding to a portion of a PD-1 protein.
  • the first arm is encoded by a first polypeptide chain and the second arm is encoded by a second polypeptide chain that associate together.
  • the first arm comprises a linker between the first variable heavy domain and first variable light chain domain.
  • the second arm comprises a linker between the first variable heavy domain and first variable light chain domain.
  • the linker is a glycine-serine linker.
  • the first and second arms each further comprise a fragment, crystallizable (Fc) region.
  • the Fc region of the first arm comprises knob mutations and the Fc region of the second arm comprise hole mutations, or vice versa.
  • the first variable heavy chain domain comprises an amino acid sequence of the variable heavy chain portion of SEQ ID NO: 2, 4, 6, 8, 10, 29, 30, 31, 32, 33, or 34, wherein the first variable light chain domain comprises an amino acid sequence of the variable light chain portion of SEQ ID NO: 2, 4, 6, 8, 10, 29, 30, 31, 32, 33, or 34.
  • the first arm comprises an amino acid sequence selected from SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, or SEQ ID NO: 34
  • the second arm comprises an amino acid sequence selected from SEQ ID NO: 26, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, or SEQ ID NO: 43.
  • the portion of the first arm is capable of binding to the portion of the CD45 protein with an affinity between about 5.6 E-7 KD (M) and about 3.6 E-l 1 KD (M).
  • the first variable heavy chain domain comprises an amino acid sequence of the variable heavy chain portion of SEQ ID NO: 22, wherein the first variable light chain domain comprises an amino acid sequence of the variable light chain portion of SEQ ID NO: 22.
  • the first arm comprises SEQ ID NO: 22, wherein the second arm comprises SEQ ID NO: 26.
  • the portion of the first arm is capable of binding to the portion of the CD43 protein with an affinity between about 1.0 E-7 KD (M) and about 1.0 E-9 7 KD (M).
  • the second variable heavy chain domain comprises an amino acid sequence of the variable heavy chain portion of SEQ ID NO: 22, wherein the first variable light chain domain comprises an amino acid sequence of the variable light chain portion of SEQ ID NO: 26, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, or SEQ ID NO: 43.
  • the first arm comprises an amino acid sequence selected from SEQ ID NO: 4 or SEQ ID NO: 6, and wherein the second arm comprises an amino acid sequence selected from SEQ ID NO: 26, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, or SEQ ID NO: 43.
  • the portion of the first arm is capable of binding to the portion of the CD45 protein, and wherein the bispecific antibody is capable of localizing a PD-1 protein away from a core of an immune synapse.
  • the bispecific antibody is capable of disrupting downstream signaling of a PD-1 mediated response in a T cell. In some embodiments, the bispecific antibody is capable of enhancing T cell function. In some embodiments, the bispecific antibody is capable of binding to the CD45 portion of the CD45 protein and to the PD-1 portion on the PD-1 protein, wherein the CD45 protein and PD-1 protein are located on a same T cell. In some embodiments, the portion of the first arm is capable of binding to the portion of the CD43 protein, and wherein the bispecific antibody is capable of localizing a PD-1 protein away from a core of an immune synapse. In some embodiments, the bispecific antibody is capable of disrupting a downstream signaling of a PD-1 mediated response in a T cell. In some embodiments, the bispecific antibody is capable of enhancing T cell function.
  • the bispecific antibody is capable of binding to the CD43 portion of the CD43 protein and to the PD-1 portion on the PD-1 protein wherein the CD43 protein and PD-1 protein are located on a same T cell.
  • the PD-1 protein is located on a T cell, and wherein the bispecific antibody is capable of preventing the PD-1 protein from binding to a PDL-1 protein or a PDL-2 protein on a tumor cell.
  • the bispecific antibody is capable of dephosphorylating a tail of the PD-1 protein.
  • the bispecific antibody is capable of inducing a cytokine secretion in a T cell.
  • the cytokine secretion is a secretion of IL-2.
  • described herein is a pharmaceutical composition comprising: the bispecific antibody described herein and a pharmaceutically acceptable carrier.
  • described herein is a method of preventing or treating cancer in a subject comprising administering to the subject an effective amount of the composition described herein.
  • the cancer is selected from colorectal cancer, lung cancer, bladder cancer, breast cancer, cervical cancer, kidney cancer, leukemia, Hodgkin lymphoma, non-Hodgkin lymphoma, prostate cancer, skin cancer (e.g., melanoma), head and neck cancer, endometrial cancer, colon cancer, rectal cancer, liver cancer, thyroids cancer, esophageal cancer, renal cell cancer, and a combination thereof.
  • described herein is a method of preventing or treating a viral infection in a subject comprising administering to the subject an effective amount of the composition described herein.
  • the viral infection is HIV.
  • described herein is a pharmaceutical composition
  • a pharmaceutical composition comprising: the bispecific antibody described herein and a pharmaceutically acceptable carrier.
  • described herein is a method of preventing or treating inflammation and autoimmunity in a subject comprising administering to the subject an effective amount of the composition described herein.
  • the diseases is selected from rheumatoid arthritis, systemic lupus erythematosus, psoriasis and psoriatic arthritis, spondyloarthropathy, type 1 diabetes, and multiple sclerosis.
  • kits for generating a bispecific antibody or fragment thereof comprising one or more vectors comprising a polynucleotide sequence encoding any of the bispecific antibodies described herein.
  • a kit for generating a bispecific antibody or fragment thereof comprising: a first vector comprising a polynucleotide sequence encoding a first arm of the bispecific antibody or fragment thereof, wherein a portion of the first arm is capable of binding to a portion of a CD45 protein or a portion of a CD43 protein; and a second vector comprising a polynucleotide sequence encoding a second arm of the bispecific antibody or fragment thereof, wherein a portion of the second arm is capable of binding to a portion of a PD-1 protein.
  • the first vector and the second vector are the same vector.
  • the first vector and the second vector are two different vectors.
  • a variable heavy chain domain of the first arm comprises an amino acid sequence of the variable heavy chain portion of SEQ ID NO: 2, 4, 6, 8, 10, 22, 29, 30, 31, 32, 33, or 34
  • a first variable light chain domain of the first arm comprises an amino acid sequence of the variable light chain portion of SEQ ID NO: 2, 4, 6, 8, 10, 22, 29, 30, 31, 32, 33, or 34
  • a variable heavy chain domain of the second arm comprises an amino acid sequence of the variable heavy chain portion of SEQ ID NO: 26, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, or SEQ ID NO: 43
  • a variable light chain domain of the second arm comprises an amino acid sequence selected from SEQ ID NO: 26, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, S
  • the first arm comprises an amino acid sequence selected from SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 22, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, or SEQ ID NO: 34, wherein the second arm comprises SEQ ID NO: 22.
  • the first arm comprises SEQ ID NO: 4.
  • the first arm comprises SEQ ID NO: 6.
  • the first arm comprises SEQ ID NO: 22.
  • described herein is one or more host cells comprising: one or more vectors comprising a polynucleotide sequence encoding any of the bispecific antibodies described herein.
  • described herein is one or more host cells comprising: a first vector comprising a polynucleotide sequence encoding a first arm of a bispecific antibody or fragment thereof, wherein a portion of the first arm is capable of binding to a portion of a CD45 protein or a portion of a CD43 protein; and a second vector comprising a polynucleotide sequence encoding a second arm of the bispecific antibody or fragment thereof, wherein a portion of the second arm is capable of binding to a portion of a PD-1 protein.
  • the first vector and the second vector are the same vector. In some embodiments, the first vector and the second vector are two different vectors.
  • the first arm comprises a first variable heavy chain domain and a first variable light chain domain
  • the second arm comprises a second variable heavy chain domain and a second variable light chain domain
  • the first variable heavy chain domain comprises an amino acid sequence of the variable heavy chain portion of SEQ ID NO: 2, 4, 6, 8, 10, 29, 30, 31, 32, 33, or 34
  • the first variable light chain domain comprises an amino acid sequence of the variable light chain portion of SEQ ID NO: 2, 4, 6, 8, 10, 29, 30, 31, 32, 33, or 34.
  • the first arm comprises an amino acid sequence selected from SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 22, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, or SEQ ID NO: 34
  • the second arm comprises an amino acid sequence selected from SEQ ID NO: 26, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, or SEQ ID NO: 43.
  • the first arm comprises SEQ ID NO: 4.
  • the first arm comprises SEQ ID NO: 6.
  • the first arm comprises SEQ ID NO: 22.
  • described herein is a method of making a bispecific antibody or fragment thereof comprising: culturing the one or more host cells described herein under conditions suitable for an expression of the one or more vectors; and recovering the bispecific antibody or fragment thereof.
  • described herein is a method of making a bispecific antibody or fragment thereof comprising: culturing the one or more host cells described herein under conditions suitable for an expression of the first vector and the second vector; and recovering the bispecific antibody or fragment thereof.
  • compositions comprising: one or more vectors comprising a polynucleotide sequence encoding any of the bispecific antibodies described herein.
  • compositions comprising: a first vector comprising a polynucleotide sequence encoding a first arm of the bispecific antibody or fragment thereof, wherein a portion of the first arm is capable of binding to a portion of a CD45 protein or a portion of a CD43 protein; and a second vector comprising a polynucleotide sequence encoding a second arm of the bispecific antibody or fragment thereof, wherein a portion of the second arm is capable of binding to a portion of a PD-1 protein.
  • the first vector and the second vector are the same vector. In some embodiments, the first vector and the second vector are two different vectors.
  • the first arm comprises a first variable heavy chain domain and a first variable light chain domain
  • the second arm comprises a second variable heavy chain domain and a second variable light chain domain
  • the first variable heavy chain domain comprises an amino acid sequence of the variable heavy chain portion of SEQ ID NO: 2, 4, 6, 8, 10, 29, 30, 31, 32, 33, or 34
  • the first variable light chain domain comprises an amino acid sequence of the variable light chain portion of SEQ ID NO: 2, 4, 6, 8, 10, 29, 30, 31, 32, 33, or 34.
  • the first arm comprises an amino acid sequence selected from SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 22, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, or SEQ ID NO: 34
  • the second arm comprises an amino acid sequence selected from SEQ ID NO: 26, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, or SEQ ID NO: 43.
  • the first arm comprises SEQ ID NO: 4.
  • the first arm comprises SEQ ID NO: 6. In some embodiments, the first arm comprises SEQ ID NO: 22. [00279] In certain aspects, described herein is a means for binding: a portion of a CD45 protein or a portion of a CD43 protein; and a portion of a PD-1 protein. In some embodiments, the means comprises a bispecific antibody or fragment thereof.
  • the bispecific antibody or a fragment thereof comprises: a first arm comprising a first variable heavy chain domain and a first variable light chain domain, wherein a portion of the first arm is capable of binding to the portion of the CD45 protein or the portion of the CD43 protein; and a second arm comprising a second variable heavy chain domain and a second variable light chain domain, wherein a portion of the second arm is capable of binding to the portion of the PD-1 protein.
  • the first variable heavy chain domain comprises an amino acid sequence of the variable heavy chain portion of SEQ ID NO: 2, 4, 6, 8, 10, 29, 30, 31, 32, 33, or 34
  • the first variable light chain domain comprises an amino acid sequence of the variable light chain portion of SEQ ID NO: 2, 4, 6, 8, 10, 29, 30, 31, 32, 33, or 34
  • the portion of the first arm is capable of binding to the portion of the CD45 protein.
  • the first arm comprises an amino acid sequence selected from SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, or SEQ ID NO: 34
  • the second arm comprises SEQ ID NO: 26, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, or SEQ ID NO: 43
  • the portion of the first arm is capable of binding to the portion of the CD45 protein with an affinity between about 5.6 E-7 KD (M) and about 3.6 E- 11 KD (M).
  • the first arm comprises SEQ ID NO: 22, wherein the second arm comprises SEQ ID NO: 26, and wherein the portion of the second arm is capable of binding to the portion of the CD43 protein.
  • the portion of the first arm is capable of binding to the portion of the CD43 protein with an affinity between about 1.0 E-7 KD (M) and about 1.0 E-9 7 KD (M).
  • the first arm comprises an amino acid sequence selected from SEQ ID NO: 4 or SEQ ID NO: 6, and wherein the second arm comprises SEQ ID NO: 26.
  • the portion of the first arm is capable of binding to the portion of the CD45 protein, and wherein the bispecific antibody is capable of localizing a PD-1 protein away from a core of an immune synapse.
  • the bispecific antibody is capable of disrupting downstream signaling of a PD-1 mediated response in a T cell.
  • the bispecific antibody is capable of enhancing T cell function.
  • the bispecific antibody is capable of binding to the CD45 portion of the CD45 protein and to the PD-1 portion on the PD-1 protein, wherein the CD45 protein and PD-1 protein are located on a same T cell.
  • the portion of the first arm is capable of binding to the portion of the CD43 protein, and wherein the bispecific antibody is capable of localizing a PD-1 protein away from a core of an immune synapse.
  • the bispecific antibody is capable of disrupting a downstream signaling of a PD-1 mediated response in a T cell.
  • the bispecific antibody is capable of enhancing T cell function.
  • the bispecific antibody is capable of binding to the CD43 portion of the CD43 protein and to the PD-1 portion on the PD-1 protein wherein the CD43 protein and PD-1 protein are located on a same T cell.
  • the PD-1 protein is located on a T cell, and wherein the bispecific antibody is capable of preventing the PD-1 protein from binding to a PDL-1 protein or a PDL-2 protein on a tumor cell. In some embodiments, the bispecific antibody is capable of dephosphorylating a tail of the PD-1 protein. In some embodiments, the bispecific antibody is capable of inducing a cytokine secretion in a T cell. In some embodiments, the cytokine secretion is a secretion of IL-2.
  • a prophylactic or therapeutic composition of this disclosure comprises one or more antibodies (or one or more polynucleotides encoding one or more antibodies) and is administered in a pharmaceutical composition that includes a pharmaceutically acceptable carrier.
  • the prophylactic or therapeutic composition is comprised of one or more antibodies (or one or more polynucleotides encoding one or more antibodies) comprising SEQ ID NOs: 2 and 26 (e.g., antibody “anti-CD45MlPD- 1”), SEQ ID NOs: 4 and 26, (e.g., “anti-CD45M2PD-l”), comprising SEQ ID NOs: 10 and 26 (“anti-CD43MlPD-l”), comprising SEQ ID NOs: 29 and 26, comprising SEQ ID NOs: 30 and 26, comprising SEQ ID NOs: 31 and 26, comprising SEQ ID NOs: 32 and 26, comprising SEQ ID NOs: 33 and 26, comprising SEQ ID NOs: 34 and 26, comprising SEQ ID NOs: 2 and 26 (e.g., antibody
  • the prophylactic or therapeutic composition is comprised of one or more antibodies (or one or more polynucleotides encoding one or more antibodies) comprising the VH domain and VL domain of SEQ ID NOs: 4, 6, 8, 10, or 29-34.
  • the pharmaceutical composition is in the form of a spray, aerosol, gel, solution, emulsion, nanoparticle (e.g., lipid nanoparticle), or suspension.
  • composition is preferably administered to a subject with a pharmaceutically acceptable carrier.
  • a pharmaceutically acceptable carrier typically, in some embodiments, an appropriate amount of a pharmaceutically acceptable salt is used in the formulation, which in some embodiments can render the formulation isotonic.
  • the one or more antibodies are provided as a composition comprising any one of the antibodies described herein (e.g., “anti-CD45MlPD-l”, “anti-CD45M2PD-l”, “anti- CD45M3PD-1”, or “anti-CD45M4PD-l” bispecific antibody, bispecific antibody comprising SEQ ID NOs: 29 and 26, comprising SEQ ID NOs: 30 and 26, comprising SEQ ID NOs: 31 and 26, comprising SEQ ID NOs: 32 and 26, comprising SEQ ID NOs: 33 and 26, comprising SEQ ID NOs: 34 and 26, comprising SEQ ID NOs: 2 and any of 35-43, comprising SEQ ID NOs: 4 and any of 35-43, comprising SEQ ID NOs: 6 and any of 35-43, comprising SEQ ID NOs: 8 and any of 35-43, comprising SEQ ID NOs: 10 and any of 35-43, comprising
  • the pharmaceutically acceptable carrier is selected from the group consisting of saline, Ringer's solution, dextrose solution, and a combination thereof.
  • suitable pharmaceutically acceptable carriers known in the art are contemplated. Suitable carriers and their formulations are described in Remington's Pharmaceutical Sciences, 2005, Mack Publishing Co.
  • the pH of the solution is preferably from about 5 to about 8, and more preferably from about 7 to about 7.5.
  • the formulation may also comprise a lyophilized powder.
  • Further carriers include sustained release preparations such as semipermeable matrices of solid hydrophobic polymers, which matrices are in the form of shaped articles, e.g., films, liposomes or microparticles. It will be apparent to those persons skilled in the art that certain carriers may be more preferable depending upon, for instance, the route of administration and concentration of antibodies being administered.
  • pharmaceutically acceptable carrier means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting the subject pharmaceutical agent from one organ, or portion of the body, to another organ, or portion of the body.
  • a pharmaceutically acceptable material, composition or vehicle such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting the subject pharmaceutical agent from one organ, or portion of the body, to another organ, or portion of the body.
  • Each carrier is acceptable in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient.
  • materials which can serve as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as butylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer’
  • carrier denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application.
  • the components of the pharmaceutical compositions also are capable of being comingled with the compounds of the present invention, and with each other, in a manner such that there is no interaction which would substantially impair the desired pharmaceutical efficiency.
  • the composition may also include additional agents such as an isotonicity agent, a preservative, a surfactant, and, a divalent cation, preferably, zinc.
  • the composition can also include an excipient, or an agent for stabilization of an antibody composition, such as a buffer, a reducing agent, a bulk protein, amino acids (such as e.g., glycine or praline) or a carbohydrate.
  • an excipient or an agent for stabilization of an antibody composition
  • a buffer such as a buffer, a reducing agent, a bulk protein, amino acids (such as e.g., glycine or praline) or a carbohydrate.
  • amino acids such as e.g., glycine or praline
  • carbohydrate such as a carbohydrate.
  • Typical carbohydrates useful in formulating compositions include but are not limited to sucrose, mannitol, lactose, trehalose, or glucose.
  • Surfactants may also be used to prevent soluble and insoluble aggregation and/or precipitation of antibodies included in the composition.
  • Suitable surfactants include but are not limited to sorbitan trioleate, soya lecithin, and oleic acid.
  • solution aerosols are preferred using solvents such as ethanol.
  • formulations including antibodies can also include a surfactant that can reduce or prevent surface-induced aggregation of antibodies by atomization of the solution in forming an aerosol.
  • Various conventional surfactants can be employed, such as polyoxyethylene fatty acid esters and alcohols, and polyoxyethylene sorbitol fatty acid esters. Amounts will generally range between 0.001% and 4% by weight of the formulation.
  • surfactants used with the present disclosure are polyoxyethylene sorbitan mono-oleate, polysorbate 80, polysorbate 20. Additional agents known in the art can also be included in the composition.
  • the pharmaceutical compositions and dosage forms further comprise one or more compounds that reduce the rate by which an active ingredient will decay, or the composition will change in character.
  • stabilizers or preservatives may include, but are not limited to, amino acids, antioxidants, pH buffers, or salt buffers.
  • antioxidants include butylated hydroxy anisole (BHA), ascorbic acid and derivatives thereof, tocopherol and derivatives thereof, butylated hydroxy anisole and cysteine.
  • preservatives include parabens, such as methyl or propyl p- hydroxybenzoate and benzalkonium chloride.
  • Additional nonlimiting examples of amino acids include glycine or proline.
  • the present invention also teaches the stabilization (preventing or minimizing thermally or mechanically induced soluble or insoluble aggregation and/or precipitation of an inhibitor protein) of liquid solutions containing antibodies at neutral pH or less than neutral pH by the use of amino acids including proline or glycine, with or without divalent cations resulting in clear or nearly clear solutions that are stable at room temperature or preferred for pharmaceutical administration.
  • the composition is a pharmaceutical composition of single unit or multiple unit dosage forms.
  • Pharmaceutical compositions of single unit or multiple unit dosage forms of the invention comprise a prophylactically or therapeutically effective amount of one or more compositions (e.g., a compound of the invention, or other prophylactic or therapeutic agent), typically, one or more vehicles, carriers, or excipients, stabilizing agents, and/or preservatives.
  • the vehicles, carriers, excipients, stabilizing agents and preservatives are pharmaceutically acceptable.
  • the pharmaceutical compositions and dosage forms comprise anhydrous pharmaceutical compositions and dosage forms.
  • Anhydrous pharmaceutical compositions and dosage forms of the invention can be prepared using anhydrous or low moisture containing ingredients and low moisture or low humidity conditions.
  • Pharmaceutical compositions and dosage forms that comprise lactose and at least one active ingredient that comprise a primary or secondary amine are preferably anhydrous if substantial contact with moisture and/or humidity during manufacturing, packaging, and/or storage is expected.
  • An anhydrous pharmaceutical composition should be prepared and stored such that its anhydrous nature is maintained.
  • anhydrous compositions are preferably packaged using materials known to prevent exposure to water such that they can be included in suitable formulary kits. Examples of suitable packaging include, but are not limited to, hermetically sealed foils, plastics, unit dose containers (e.g., vials), blister packs, and strip packs.
  • Suitable vehicles are well known to those skilled in the art of pharmacy, and nonlimiting examples of suitable vehicles include glucose, sucrose, starch, lactose, gelatin, rice, silica gel, glycerol, talc, sodium chloride, dried skim milk, propylene glycol, water, sodium stearate, ethanol, and similar substances well known in the art.
  • Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid vehicles. Whether a particular vehicle is suitable for incorporation into a pharmaceutical composition or dosage form depends on a variety of factors well known in the art including, but not limited to, the way in which the dosage form will be administered to a patient and the specific active ingredients in the dosage form.
  • Pharmaceutical vehicles 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.
  • a pharmaceutical composition can be packaged in a hermetically sealed container such as an ampoule or sachette indicating the quantity.
  • the pharmaceutical composition can be supplied as a dry sterilized lyophilized powder in a delivery device suitable for administration to the lower airways of a patient.
  • the pharmaceutical compositions can, if desired, be presented in a pack or dispenser device that can contain one or more unit dosage forms containing the active ingredient.
  • the pack can for example comprise metal or plastic foil, such as a blister pack.
  • the pack or dispenser device can be accompanied by instructions for administration.
  • Methods of preparing these formulations or compositions include the step of bringing into association a compound of the present invention with the carrier and, optionally, one or more accessory ingredients.
  • the formulations are prepared by uniformly and intimately bringing into association a compound of the present invention with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.
  • Formulations of the invention suitable for administration may be in the form of powders, granules, or as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and/or as mouthwashes and the like, each containing a predetermined amount of a compound of the present invention (e.g., antibodies) as an active ingredient.
  • a compound of the present invention e.g., antibodies
  • a liquid composition herein can be used as such with a delivery device, or they can be used for the preparation of pharmaceutically acceptable formulations comprising antibodies that are prepared for example by the method of spray drying.
  • the liquid solutions herein are freeze spray dried and the spray-dried product is collected as a dispersible peptide-containing powder that is therapeutically effective when administered to an individual.
  • the compounds and pharmaceutical compositions of the present invention can be employed in combination therapies, that is, the compounds and pharmaceutical compositions can be administered concurrently with, prior to, or subsequent to, one or more other desired therapeutics or medical procedures (e.g., antibodies can be used in combination treatment with another treatment such as antivirals or with a vaccine, and/or another treatment).
  • desired therapeutics or medical procedures e.g., antibodies can be used in combination treatment with another treatment such as antivirals or with a vaccine, and/or another treatment.
  • the particular combination of therapies (therapeutics or procedures) to employ in a combination regimen will take into account compatibility of the desired therapeutics and/or procedures and the desired therapeutic effect to be achieved. It will also be appreciated that the therapies employed may achieve a desired effect for the same disorder (for example, the compound of the present invention may be administered concurrently with another therapeutic or prophylactic).
  • the invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention.
  • Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.
  • the current invention provides for dosage forms comprising peptides suitable for treating cancer or other diseases.
  • the dosage forms can be formulated, e.g., as sprays, aerosols, nanoparticles, liposomes, or other forms known to one of skill in the art.
  • a dosage form used in the acute treatment of a disease may contain larger amounts of one or more of the active ingredients it comprises than a dosage form used in the chronic treatment of the same disease.
  • the prophylactically and therapeutically effective dosage form may vary among different conditions.
  • a therapeutically effective dosage form may contain one or more antibodies that have an appropriate therapeutic action when intending to treat cancer or a viral infection such as HIV.
  • a different effective dosage may contain one or more antibodies that have an appropriate prophylactic action when intending to prevent cancer or an infection caused by a virus (e.g, HIV).
  • the pH of a pharmaceutical composition or dosage form may also be adjusted to improve delivery and/or stability of one or more active ingredients.
  • the polarity of a solvent carrier, its ionic strength, or tonicity can be adjusted to improve delivery.
  • Compounds such as stearates can also be added to pharmaceutical compositions or dosage forms to alter advantageously the hydrophilicity or lipophilicity of one or more active ingredients to improve delivery.
  • stearates can also serve as a lipid vehicle for the formulation, as an emulsifying agent or surfactant, and as a delivery enhancing or penetration-enhancing agent.
  • Different salts, hydrates, or solvates of the active ingredients can be used to adjust further the properties of the resulting composition.
  • compositions can be formulated with appropriate carriers and adjuvants using techniques to yield compositions suitable for prophylaxis or treatment.
  • the compositions can include an adjuvant, such as, for example but not limited to, alum, poly IC, MF-59, squalene- based adjuvants, or liposomal based adjuvants suitable for prophylaxis or treatment.
  • the antibodies described herein are encoded by nucleic acids which are prepared in a mRNA-LNP or a DNA-LNP formulation for administration to a subject.
  • the antibodies e.g., monoclonal antibodies, bispecific antibodies
  • the antibodies disclosed herein can be produced by any method known in the art.
  • the antibodies disclosed herein are produced by culturing a cell transfected or transformed with a vector comprising nucleic acid sequences encoding an antibody described herein and isolating the antibody.
  • the bispecific antibodies can be produced using chemical cross-linking of two IgG molecules, via fusion of two hybridomas, or via recombinant methods, e.g., via “knobs-into-holes” heterodimerization technology. See Marvin, Jonathan S., and Zhenping Zhu, Recombinant Approaches to IgG-like Bispecific Antibodies, Acta Pharmacologica Sinica 26.6 (2005): 649-658, incorporated by reference in its entirety herein.
  • antibodies are synthesized by the hybridoma culture method which results in antibodies that are not contaminated by other immunoglobulins.
  • the modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.
  • the monoclonal antibodies to be used in accordance with the present invention may be made by a variety of techniques known in the art, including, for example, the hybridoma method (e.g., Kohler and Milstein., Nature, 256:495-97 (1975); Hongo et al, Hybridoma, 14 (3): 253-260 (1995), Harlow et al, Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling et al, in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.
  • the hybridoma method e.g., Kohler and Milstein., Nature, 256:495-97 (1975); Hongo et al, Hybridoma, 14 (3): 253-260 (1995), Harlow et al, Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling et al, in: Monoclonal Anti
  • Methods 284(1-2): 119-132 (2004) and technologies for producing human or humanlike antibodies in animals that have parts or all of the human immunoglobulin loci or genes encoding human immunoglobulin sequences (see, e.g., Lonberg et al, Nature 368: 856-859 (1994); Morrison, Nature 368: 812-813 (1994); Fishwild et al, Nature Biotechnol 14: 845-851 (1996); Neuberger, Nature Biotechnol. 14: 826 (1996); and Lonberg and Huszar, Intern. Rev. Immunol. 13: 65-93 (1995).
  • expression of an antibody comprises expression vector(s) containing a polynucleotide that encodes an anti-CD45xlPD-l or an anti-CD43xPD-l bispecific antibody.
  • expression of an antibody comprises expression vector(s) containing a polynucleotide that encodes an anti-CD45 antibody.
  • replicable vectors comprising a nucleotide sequence encoding an anti-CD45xlPD-l or an anti-CD43xPD-l bispecific antibody or an anti-CD45 antibody disclosed herein operably linked to a promoter.
  • such vectors may include a nucleotide sequence encoding the heavy chain of an antibody molecule (or fragment thereof), a nucleotide sequence encoding the light chain of an antibody (or fragment thereof), or both the heavy and light chain.
  • a bispecific antibody described herein is made using the “knob-into-hole” or “KnH” technology.
  • This method involves directing the pairing of two polypeptides together in vitro or in vivo by introducing a protuberance (knob) into one polypeptide and a cavity (hole) into the other polypeptide at an interface in which they interact.
  • KnHs have been introduced in the Fc:Fc binding interfaces, CL:CH1 interfaces or VH/VL interfaces of antibodies. See, e.g., US2007/0178552; WO 96/027011; WO 98/050431; Zhu et al.
  • bispecific antibodies with the KnHs strategy can be based on a single amino acid substitution in the opposite CH3 domains that promotes heavy chain heterodimerization.
  • a small amino acid has been replaced with a larger one in the CH3 domain.
  • a large amino acid has been replaced with a smaller one.
  • a “hole” is formed, permitting the interaction with the “knobs.” See Ridgway JBB, Presta LG, Carter P., “Knobs-into-Holes ” Engineering of Antibody CH3 Domains for Heavy Chain Heterodimerization. Protein Eng. 1996, 9:617-621, incorporated in its entirety herein.
  • This method can be used to pair two different heavy chains together during the manufacture of multispecific antibodies such as bispecific antibodies.
  • multispecific antibodies having KnH in their Fc regions can further comprise single variable domains linked to each Fc region, or further comprise different heavy chain variable domains that pair with similar or different light chain variable domains.
  • the bispecific antibodies described herein are scFv-Fc antibodies.
  • the scFv-Fc is a bispecific, bivalent molecule which is developed by fusing scFvs with different specificity to each Fc chain. In an scFv-Fc, the knobs- into-holes mutations in Fc force the formation of heterodimer.
  • the polynucleotide encoding the antibody may be modified, for example, by substituting the coding sequence for human heavy- and light-chain constant domains in place of the homologous murine sequences (U.S. Patent No. 4,816,567; Morrison, et al, Proc. Natl Acad. ScL USA, 81 :6851 (1984)), or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide.
  • nonimmunoglobulin polypeptides are substituted for the constant domains of an antibody, or they are substituted for the variable domains of one antigen-combining site of an antibody to create a chimeric bivalent antibody comprising one antigen-combining site having specificity for an antigen and another antigen-combining site having specificity for a different antigen.
  • the monoclonal antibodies described herein may by monovalent, the preparation of which is well known in the art. For example, one method involves recombinant expression of immunoglobulin light chain and a modified heavy chain. The heavy chain is truncated generally at any point in the Fc region so as to prevent heavy chain crosslinking.
  • cysteine residues may be substituted with another amino acid residue or are deleted so as to prevent crosslinking.
  • in vitro methods are also suitable for preparing monovalent antibodies. Digestion of antibodies to produce fragments thereof, particularly Fab fragments, can be accomplished using routine techniques known in the art. Chimeric or hybrid antibodies also may be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents.
  • Various expression systems for producing antibodies are known in the art, and include, prokaryotic (e.g., bacteria), plant, insect, yeast, and mammalian expression systems. Suitable cell lines, can be transformed, transduced, or transfected with nucleic acids containing coding sequences for antibodies or portions of antibodies disclosed herein in order to produce the antibody of interest.
  • Expression vectors containing such nucleic acid sequences which can be linked to at least one regulatory sequence in a manner that allows expression of the nucleotide sequence in a host cell, can be introduced via methods known in the art. Practitioners in the art understand that designing an expression vector can depend on factors, such as the choice of host cell to be transfected and/or the type and/or amount of desired protein to be expressed.
  • Enhancer regions which are those sequences found upstream or downstream of the promoter region in non-coding DNA regions, are also known in the art to be important in optimizing expression. If needed, origins of replication from viral sources can be employed, such as if a prokaryotic host is utilized for introduction of plasmid DNA. However, in eukaryotic organisms, chromosome integration is a common mechanism for DNA replication. For stable transfection of mammalian cells, a small fraction of cells can integrate introduced DNA into their genomes. The expression vector and transfection method utilized can be factors that contribute to a successful integration event.
  • a vector containing DNA encoding a protein of interest is stably integrated into the genome of eukaryotic cells (for example mammalian cells), resulting in the stable expression of transfected genes.
  • a gene that encodes a selectable marker can be introduced into host cells along with the gene of interest in order to identify and select clones that stably express a gene encoding a protein of interest.
  • Cells containing the gene of interest can be identified by drug selection wherein cells that have incorporated the selectable marker gene will survive in the presence of the drug. Cells that have not incorporated the gene for the selectable marker die. Surviving cells can then be screened for the production of the desired antibody molecule.
  • the bispecific antibodies disclosed herein are encoded in a vector for expression in a cell line.
  • a first vector comprises a polynucleotide sequence that encodes an anti-CD45 portion of a bispecific antibody
  • a second vector comprises a polynucleotide sequence that encodes an anti-PDl portion of a bispecific antibody
  • each vector is transfected into one or more cell lines for expression.
  • a single vector comprises a polynucleotide sequence that encodes an anti-CD45 portion of a bispecific antibody and an anti-PDl portion of the bispecific antibody and the vector is transfected into one or more cell lines for expression.
  • one or more vectors comprise polynucleotide sequences encoding a light chain or a fragment thereof and a heavy chain or a fragment thereof the bispecific antibody.
  • a first vector may comprise a polynucleotide sequence encoding a light chain, or fragment thereof, of an anti-CD45 portion of the bispecific antibody
  • a second vector may comprise a polynucleotide sequence encoding a light chain, or fragment thereof, of an anti-PDl portion of the bispecific antibody
  • a third vector may comprise a polynucleotide sequence encoding a heavy chain, or fragment thereof, of an anti-CD45 portion of the bispecific antibody
  • a fourth vector may comprise a polynucleotide sequence encoding a heavy chain, or fragment thereof, of an anti-PDl portion of the bispecific antibody.
  • all four vectors are transfected into one or more cell lines for expression.
  • the bispecific antibodies disclosed herein are encoded in a vector for expression in a cell line.
  • a first vector comprises a polynucleotide sequence that encodes an anti-CD43 portion of a bispecific antibody
  • a second vector comprises a polynucleotide sequence that encodes an anti-PDl portion of a bispecific antibody
  • each vector is transfected into one or more cell lines for expression.
  • a single vector comprises a polynucleotide sequence that encodes an anti-CD43 portion of a bispecific antibody and an anti-PDl portion of the bispecific antibody and the vector is transfected into one or more cell lines for expression.
  • one or more vectors comprise polynucleotide sequences encoding a light chain or a fragment thereof and a heavy chain or a fragment thereof the bispecific antibody.
  • a first vector may comprise a polynucleotide sequence encoding a light chain, or fragment thereof, of an anti-CD43 portion of the bispecific antibody
  • a second vector may comprise a polynucleotide sequence encoding a light chain, or fragment thereof, of an anti-PDl portion of the bispecific antibody
  • a third vector may comprise a polynucleotide sequence encoding a heavy chain, or fragment thereof, of an anti-CD43 portion of the bispecific antibody
  • a fourth vector may comprise a polynucleotide sequence encoding a heavy chain, or fragment thereof, of an anti-PDl portion of the bispecific antibody.
  • all four vectors are transfected into one or more cell lines for expression.
  • the anti-CD45 antibodies disclosed herein are encoded in a vector for expression in a cell line.
  • a vector comprises a polynucleotide sequence that encodes an anti-CD45 antibody, the vector is transfected into one or more cell lines for expression.
  • a first vector comprises a polynucleotide sequence that encodes an anti-CD45 heavy chain and a second vector comprises a polynucleotide sequence that encodes an anti-CD45 light chain, and each vector is transfected into one or more cell lines for expression.
  • one or more vectors comprise polynucleotide sequences encoding a light chain or a fragment thereof and a heavy chain or a fragment thereof the antibody.
  • a first vector may comprise a polynucleotide sequence encoding a light chain, or fragment thereof, of an anti-CD45 antibody
  • a second vector may comprise a polynucleotide sequence encoding a heavy chain, or fragment thereof, of an anti-CD45 portion of the antibody.
  • all vectors are transfected into one or more cell lines for expression.
  • a host cell strain which modulates the expression of the inserted sequences, or modifies and processes the nucleic acid in a specific fashion desired also may be chosen. Such modifications (for example, glycosylation and other post-translational modifications) and processing (for example, cleavage) of protein products may be important for the function of the antibody.
  • Different host cell strains have characteristic and specific mechanisms for the post- translational processing and modification of proteins and gene products. As such, appropriate host systems or cell lines can be chosen to ensure the correct modification and processing of the foreign antibody expressed.
  • eukaryotic host cells possessing the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product may be used.
  • Various culturing parameters can be used with respect to the host cell being cultured.
  • Appropriate culture conditions for mammalian cells are well known in the art (Cleveland WL, et al., J Immunol Methods, 1983, 56(2): 221-234) or can be determined by the skilled artisan (see, for example, Animal Cell Culture: A Practical Approach 2nd Ed., Rickwood, D. and Hames, B. D., eds. (Oxford University Press: New York, 1992)).
  • Cell culturing conditions can vary according to the type of host cell selected. Commercially available media can be utilized.
  • Monoclonal antibodies, antigen binding fragments thereof, and bispecific antibodies disclosed herein can be purified from any human or non-human cell which expresses the antibody, including those which have been transfected with expression constructs that express the antibody or fragments thereof.
  • the cell culture medium or cell lysate is centrifuged to remove particulate cells and cell debris.
  • the desired antibody molecule is isolated or purified away from contaminating soluble proteins and polypeptides by suitable purification techniques.
  • Non-limiting purification methods for proteins/antibodies include: size exclusion chromatography; affinity chromatography; ion exchange chromatography; ethanol precipitation; reverse phase HPLC; chromatography on a resin, such as silica, or cation exchange resin, e.g., DEAE; chromatofocusing; SDS-PAGE; ammonium sulfate precipitation; gel filtration using, e.g., Sephadex G-75, Sepharose; protein A sepharose chromatography for removal of immunoglobulin contaminants; and the like.
  • Other additives such as protease inhibitors (e.g., PMSF or proteinase K) can be used to inhibit proteolytic degradation during purification.
  • Purification procedures that can select for carbohydrates can also be used, e.g., ion-exchange soft gel chromatography, or HPLC using cation- or anion-exchange resins, in which the more acidic fraction(s) is/are collected.
  • the subject matter disclosed herein relates to a preventive medical treatment started after following diagnosis of a disease (e.g., cancer) in order to prevent the disease from worsening or curing the disease.
  • a disease e.g., cancer
  • the subject matter disclosed herein relates to prophylaxis of subjects who are believed to be at risk for moderate or severe disease associated with cancer or have previously been diagnosed with another disease, such as cancer.
  • the subjects can be administered the pharmaceutical composition described herein.
  • the invention contemplates using any of the antibodies produced by the systems and methods described herein.
  • the compositions described herein can be administered subcutaneously via syringe or any other suitable method know in the art.
  • the compound(s) or combination of compounds disclosed herein, or pharmaceutical compositions may be administered to a cell, mammal, or human by any suitable means.
  • methods of administration include, among others, (a) administration though oral pathways, which includes administration in capsule, tablet, granule, spray, syrup, or other such forms; (b) administration through non-oral pathways such as intraocular, intranasal, intraauricular, rectal, vaginal, intraurethral, transmucosal, buccal, or transdermal, which includes administration as an aqueous suspension, an oily preparation or the like or as a drip, spray, suppository, salve, ointment or the like; (c) administration via injection, including subcutaneously, intraperitoneally, intravenously, intramuscularly, intradermally, intraorbitally, intracapsularly, intraspinally, intrasternally, or the like, including infusion pump delivery;
  • administration locally such as by injection directly in the renal or cardiac area, e.g., by depot implantation; (e) administration topically; as deemed appropriate by those of skill in the art for bringing the compound or combination of compounds disclosed herein into contact with living tissue; (f) administration via inhalation, including through aerosolized, nebulized, and powdered formulations; (g) administration through implantation; and administration via electroporation.
  • one or more antibodies disclosed herein are prepared in a cocktail of DNA-encoding antibodies or mRNA-encoding antibodies and delivered by electroporation to a subject for in vivo expression of the encoded antibodies.
  • the effective in vivo dose to be administered and the particular mode of administration will vary depending upon the age, weight and species treated, and the specific use for which the compound or combination of compounds disclosed herein are employed.
  • the determination of effective dose levels can be accomplished by one skilled in the art using routine pharmacological methods. Typically, human clinical applications of products are commenced at lower dose levels, with dose level being increased until the desired effect is achieved. Alternatively, acceptable in vitro studies can be used to establish useful doses and routes of administration of the compositions identified by the present methods using established pharmacological methods.
  • Effective animal doses from in vivo studies can be converted to appropriate human doses using conversion methods known in the art (e.g., see Nair AB, Jacob S. A simple practice guide for dose conversion between animals and human. Journal of basic and clinical pharmacy. 2016 Mar;7(2):27.)
  • compositions prepared using methods of the invention can be used as a vaccine to promote an immune response against future disease (e.g., cancer) or infection (e.g., HIV).
  • future disease e.g., cancer
  • infection e.g., HIV
  • the antibodies are neutralizing antibodies.
  • the antibodies (or polynucleotides encoding antibodies) prepared using methods of the invention can be combined with additional pharmaceutical components.
  • a prophylactically effective or therapeutically effective amount is typically dependent on the weight of the subject being treated, the subject’s physical condition, the extensiveness of the condition to be treated, and the age of the subject being treated.
  • an anti-CD45xlPD-l or an anti-CD43xPD-l bispecific antibody, or an anti-CD45 antibody or polynucleotides encoding one or more antibodies, disclosed herein may be administered in an amount in the range of about 10 ng/kg body weight to about 100 mg/kg body weight per dose. In some embodiments, antibodies may be administered in an amount in the range of about 50 pg/kg body weight to about 5 mg/kg body weight per dose.
  • antibodies may be administered in an amount in the range of about 100 pg/kg body weight to about 10 mg/kg body weight per dose. In some embodiments, antibodies may be administered in an amount in the range of about 100 pg/kg body weight to about 20 mg/kg body weight per dose. In some embodiments, antibodies may be administered in an amount in the range of about 0.5 mg/kg body weight to about 20 mg/kg body weight per dose. In some embodiments, antibodies may be administered in an amount in the range of about 0.5 mg/kg body weight to about 10 mg/kg body weight per dose. In some embodiments, antibodies may be administered in an amount in the range of about 1 mg/kg body weight to about 5 mg/kg body weight per dose.
  • antibodies may be administered in an amount in the range of about 0.1 mg/kg body weight to about 0.5 mg/kg body weight per dose. In some embodiments, antibodies may be administered in a dose of at least about 100 pg/kg body weight, at least about 250 pg/kg body weight, at least about 500 pg/kg body weight, at least about 750 pg/kg body weight, at least about 3 mg/kg body weight, at least about 5 mg/kg body weight, or at least about 10 mg/kg body weight.
  • the dosage is adjusted to achieve a plasma antibody concentration of about 1-1000 pg/mL or about 25-300 pg/mL. In some embodiments, the dosage is adjusted to achieve a plasma antibody concentration of about 0.001 pg/mL to about 10 pg/mL. In some embodiments, the dosage is adjusted to achieve a plasma antibody concentration of about 1 pg/mL to about 10 pg/mL. In some embodiments, the dosage is adjusted to achieve a plasma antibody concentration of about 0.01 pg/mL to about 1 pg/mL. In some embodiments, the dosage is adjusted to achieve a plasma antibody concentration of about 0.01 pg/mL to about 0.1 pg/mL.
  • the constructs e.g., anti-CD45, anti-CD43, and anti-PD-1 sequences
  • pVaxl vector ThermoFisher, V26020, Kanamycin-resistant
  • Hindlll/Xbal enzyme sites Each construct was separately cloned into a vector and co-transfected into cells (see Example 4).
  • Plasmid DNA (2pg) and ddEEO was added.
  • The, 1 OX digestion buffer (ThermoFisher) was added followed by the addition of 0.5-1 pL of restriction enzymes. The total volume was 30pL. Incubation took place at 37°C for 2 hours. Then, a 1% agarose gel was run and the proper bands were cut out under a UV lamp. DNA fragments were extracted with a gel extraction kit.
  • Example 3 Ligation
  • PEI transfection 300 pg of PEI was added to 2.5 mL of OptiMEM to tube A, 100 pg of DNA plasmid was added to 2.5 mL of OptiMEM to tube B, and the solution was mixed well and incubated for 5 minutes at room temperature.
  • the solution in tube A was added to tube B, tube B was vortexed for 10 seconds, and incubated for 10-15 minutes at room temperature, then it was added to expi293 cells.
  • the cells were cultured in the 37°C CO2 shaking incubator. On day 5, the cells and the medium were centrifuged for 10 minutes at 3600 RPM. The supernatants were collected, 300pL of protein-A-agarose (Pierce Protein A Agarose beads, ThermoFisher Scientific, 20333) was added, and it was incubated on a rotator at room temperature for 2 days.
  • the OD280 of the eluted proteins was measured to roughly calculate the concentration of the antibody solution. Then, eluted antibodies were run in a PAGE gel with Ipg and 2 pg of BSA as an internal control. The correct antibody concentrations were estimated based on the intensity of the bands compared to the intensity of the BSA bands.
  • CD45 is more antigenic and more expressed in T cells compared to PD-1
  • a reduced affinity anti-CD45 arm will ensure that the bispecific species will bind in cis.
  • CD45 is expressed in cells other than T cells
  • a reduced affinity CD45 arm will follow the specificity binding of PD-1, which is limited to T cells. In this way, the antibody will be specific to T cell and not to other immune cells.
  • Three versions of anti-CD45xPD-l antibodies (Ml, M3, and M4) were created (i.e., anti-CD45MlPD-l, anti-CD45M3PD-l, and anti-CD45M4PD-l).
  • the CDR sequences to CD45 are novel as described herein and the CDR to PD-1 is adopted from pembrolizumab.
  • a version where CD45 was replaced with CD43 was also created using sequences described herein.
  • Jurkat and Raji cells were counted with trypan blue to establish viability and determine the number of cells. Then, 2xl0 5 Jurkat cells were added in 90pL of RPMI-10 to each well on a 96-well round bottom plate. Then, 2xl0 5 Raji cells were added in 90pL of RPMI-10 to each well. A final concentration of 50-100pg/ml of SEE was added in lOpL of RPMI-10 (1000-2000pg/ml working solution) to each well. Then, lOpL of antibody solution was added to each well with proper final concentrations (0.0001 to 10 ug/ml). Incubation for stimulation occurred for 24 hours and the supernatants were collected for human-IL-2 measurement (human IL-2 ELISA MAX Standard Set, Biolegend, 431801).
  • the first antibody was added in the blocking buffer and incubation occurred for 1 hour at room temperature.
  • the ELISA plate was then washed 6 times.
  • a second antibody was added (Sinobiological, goat anti-human IgG Fc secondary Ab, HRP, SSA001) in blocking buffer and incubation occurred for 1 hour at room temperature.
  • the ELISA plate was then washed 6 times again.
  • TMB substrate BiolLegend
  • a stop solution was then added and the ELISA plate was measured at OD450.
  • FIG. 2 shows that wild-type anti-CD45xPD-l bispecific Ab bind to PD-1 and CD45 (the anti-CD45 sequence for the anti-CD45 arm is the wild-type sequence, indicated by SEQ ID NO: 2 and the anti-PD-1 arm is indicated by SEQ ID NO: 26).
  • the Raji B cells were pretreated with SEE (50 ug/ml), and the Jurakt T cells were pretreated with anti-CD45xPD-l bispecific antibody, anti-PD-1 antibody, and control antibody (1 ug/ml), described herein, for 45 minutes.
  • SEE 50 ug/ml
  • Jurakt T cells were pretreated with anti-CD45xPD-l bispecific antibody, anti-PD-1 antibody, and control antibody (1 ug/ml), described herein, for 45 minutes.
  • live cocultured cells were imaged with confocal microscopy (Zeiss) for 30 minutes, and the number of the conjugates where PD-1 was enriched at the immune synapse was calculated using ImageJ.
  • FIG. 3 A shows representative images
  • the wild-type anti-CD45xPD-l bispecific antibody (the anti-CD45 sequence for the anti-CD45 arm is the wild-type sequence, indicated by SEQ ID NO: 2 and the anti-PD-1 arm is indicated by SEQ ID NO: 26) can activate T cells better than the generic anti- PD-1 antibody.
  • PBMC -based assay For the PBMC -based assay, after PBMC separation and wash, 4xl0 5 PBMCs/well were seeded to a 96-well round bottom plate, and SEE (50 pg/mL final) and antibodies were added in RPMI-10. The PBMCs were incubated at 37°C for 24 hours and the supernatants were harvested for human IL-2 measurement.
  • SEE 50 pg/mL final
  • IL-2 levels (pg/mL) in PBMC that were isolated from healthy volunteers and then pretreated with anti-CD43xPD-l (referred to as anti- CD43M1PD-1 herein) bispecific antibody, anti-CD45MlxPD-l bispecific antibody, anti- CD45M2xPD-l bispecific antibody, anti-PD-1 antibody, and control antibody (1 pg/mL) and SEE (50 pg/mL).
  • the anti-CD45MlxPD-l antibody produced the highest level of IL-2 secretion followed by the anti-CD45M2xPD-l antibody.
  • the experiments of Examples 8 and 9 show a correlation between the degree of PD-1 exclusion from the synapse and its lack of ability to prevent cell proliferation and cytokine secretion.
  • FIG. 6 shows ELISA binding curves for binding of four anti-PD-lxCD45 bispecific antibodies (wild-type anti-PD-lxCD45 and three mutant anti-PD-lxCD45) to human-PD-l-His- coated plates.
  • the three mutant antibodies (anti-PD-lxCD45-mutl, anti-PD-lxCD45-mut3, and anti-PD-lxCD45-mut4) had reduced affinity to CD45.
  • FIG. 7 shows IL-2 concentration in Jurkat-Raji co-culture in the presence of 10 pg/ml of 100 pg/ml SEE (Staphylococcal Enterotoxin E) and an anti-PD-1 antibody, wild-type anti-PD-lxCD45 antibody, or anti-PD-lxCD45-mut4 antibody compared to SEE alone with no antibody and no SEE no antibody controls.
  • Jurkat T cells stably overexpressing human-PD-1 and Raji B cells stably overexpressing human-PD-Ll were co-cultured for 24 hours in the presence of 10 ug/ml of each antibody and 100 pg/ml SEE (Staphylococcal Enterotoxin E).
  • IL-2 concentrations in the supernatants were determined by ELISA. These results show that anti-PD- lxCD45 antibodies (and especially anti-PD-lxCD45-mut4 antibody) lead to increased IL-2 concentrations in supernatants compared to controls or anti-PD-1 monospecific antibody.
  • FIG. 8A-B show binding curves to human-CD45RO-ECD-His-coated plates was quantified by ELISA.
  • FIG. 8 A shows binding curves for anti- CD45 antibody clones 023, 026, 027, and 028, CD45RO Monoclonal Antibody (UCHL1) (eBioscienceTM), anti-HEL-human IgGl isotype control, and blank.
  • FIG. 8B shows binding curves for anti-CD45 antibody clones 031, and 042, CD45RO Monoclonal Antibody (UCHL1) (eBioscienceTM), anti-HEL-human IgGl isotype control, and blank.
  • EC50 values were calculated with GraphPad Prism (vlO.2.1).
  • Table 1 shows EC50 values for or anti-CD45 antibody clones 023, 026, 027, and 028, CD45RO Monoclonal Antibody (UCHL1) (eBioscienceTM), and anti-HEL-human IgGl isotype control.
  • Table 2 shows EC50 values for or anti-CD45 antibody clones 031 and 042, CD45RO Monoclonal Antibody (UCHL1) (eBioscienceTM), and anti-HEL-human IgGl isotype control.
  • FIG. 9A-E shows SRP analysis of binding of anti-CD45 antibodies to captured human-CD45RO-ECD-His.
  • FIG. 9A shows binding of CD45RO Monoclonal Antibody (UCHL1) to captured human-CD45RO-ECD-His.
  • FIG. 9B shows binding of anti-CD45 antibody clone 23 to captured human-CD45RO-ECD-His.
  • FIG. 9C shows binding of anti-CD45 antibody clone 26 to captured human-CD45RO-ECD-His.
  • FIG. 9D shows binding of anti-CD45 antibody clone 27 to captured human-CD45RO-ECD-His.
  • FIG. 9E shows binding of anti-CD45 antibody clone 28 to captured human-CD45RO-ECD-His.
  • FIG. 9B shows binding of anti-CD45 antibody clone 31 to captured human-CD45RO-ECD-His.
  • Table 3 shows the binding ka (1/Ms), kd(l/s), and KD(M) for CD45RO Monoclonal Antibody (UCHL1), and anti-CD45 antibody clones 023, 026, 027, 028, and 031.
  • FIG. 10 shows binding of anti-CD45 antibodies to cell-expressed CD45 in Jurkat cells by flow cytometry.
  • FIG. 10 shows binding for anti-CD45 antibody clones 027, 042, 028, 031, 026, and 023 compared to unstained, sec-only, and non-relevant control. Wild type Jurkat T cells were incubated with either 10 pg/ml (left), 1 pg/ml (middle), or 0.1 pg/ml (right) of each anti-CD45 clone on ice for 30 minutes, washed twice, and then incubated for 30 minutes on ice with a fluorescent anti-human-Fc secondary antibody. Following two additional washes, cells were analyzed on a BD LSRFortessaTM flow cytometer. These results show that anti-CD45 antibodies bind to cell-expressed CD45 in Jurkat cells.
  • FIG. 11 shows binding of anti-CD45 antibodies to cell-expressed CD45 in Raji cells by flow cytometry.
  • FIG. 11 shows binding for anti-CD45 antibody clones 027, 042, 028, 031, 026, and 023 compared to unstained, sec-only, and non-relevant control.
  • Wild type Raji B cells were incubated with either 10 pg/ml (left), 1 pg/ml (middle), or 0.1 pg/ml (right) of each anti- CD45 clone on ice for 30 minutes, washed twice, and then incubated for 30 minutes on ice with a fluorescent anti-human-Fc secondary antibody. Following two additional washes, cells were analyzed on a BD LSRFortessaTM flow cytometer. These results show that anti-CD45 antibodies bind to cell-expressed CD45 in Raji cells.
  • FIG. 12A-B binding curves to human-PD-l-His-coated plates was quantified by ELISA.
  • FIG. 12A shows binding curves for anti-PDl antibody clones 01, 02, 03, and 07, anti -human PD-1 antibody Penbio (pembrolizumab), anti-HEL-human IgGl isotype control, and blank.
  • FIG. 12B shows binding curves for anti-PDl antibody clones 09, 51, 55, 79, and 80, anti -human PD-1 antibody Penbio (pembrolizumab), anti-HEL-human IgGl isotype control, and blank.
  • Table 4 shows EC50 values for anti-PDl antibody clones 01, 02, 03, and 07, antihuman PD-1 antibody Penbio (pembrolizumab), anti-HEL-human IgGl isotype control, and blank.
  • Table 5 shows EC50 values for anti-PDl antibody clones 09, 51, 55, 79, and 80, antihuman PD-1 antibody Penbio (pembrolizumab), anti-HEL-human IgGl isotype control, and blank.
  • EC50 values were calculated with GraphPad Prism (vl0.2.1).
  • FIG. 13 shows binding of anti-PD-1 clones 01, 02, 03, 07, 09, 51, 55, 79, and 80 to cell-expressed PD-1 by flow cytometry.
  • Jurkat T cells stably overexpressing human-PD-1 were incubated with 10 pg/ml (FIG. 13 left) or 1 pg/ml (FIG. 13 right) of each anti-PD-1 clone on ice for 30 minutes, washed twice, and then incubated for 30 minutes on ice with a fluorescent anti- human-Fc secondary antibody. Following two additional washes, cells were analyzed on a BD LSRFortessaTM flow cytometer. These results show that the anti-PD-1 clones bind to cell- expressed PD-1.
  • FIG. 14 shows anti-PD-1 clones 01, 02, 03, 07, 09, 51, 55, 79, and 80 blocking the binding of rhPD-L2 to cell-expressed PD-1.
  • Jurkat T cells stably overexpressing human-PD-1 were incubated with 10 pg/ml of each anti-PD-1 clone on ice for 30 minutes, washed twice, and then incubated for 1 hour on ice with 0.5 pg/ml (FIG. 14 left) or 5 pg/ml (FIG. 14 right) of recombinant human PD-L2 (with mouse-Fc).
  • FIG. 15 shows IL-2 concentrations following addition of anti -PD-1 clones 01, 02, 03, 07, 09, 51, 55, 79, or 80 and SEE (Staphylococcal Enterotoxin E) in Jurkat-Raji co-culture compared to no SEE and no antibody control and SEE and no antibody control.
  • Jurkat T cells stably overexpressing human-PD-1 and Raji B cells stably overexpressing human-PD-Ll were co-cultured for 24 hours in the presence of 10 pg/ml of each anti -PD-1 clone and 100 pg/ml SEE (Staphylococcal Enterotoxin E).
  • IL-2 concentrations in the supernatants were determined by ELISA.
  • FIG. 16 shows IL-2 concentrations determined by ELISA following addition of anti- PD-1 clones 01, 02, 03, 07, 09, 51, 55, 79, or 80 at different concentrations (10, 2, 0.5 or 0.1 pg/ml) and SEE (Staphylococcal Enterotoxin E) in Jurkat-Raji co-culture compared to no SEE and no antibody control and SEE and no antibody control.
  • SEE Staphylococcal Enterotoxin E
  • Jurkat T cells stably overexpressing human-PD-1 and Raji B cells stably overexpressing human-PD-Ll were co-cultured for 24 hours in the presence of different concentrations (10, 2, 0.5 or 0.1 pg/ml) of each anti-PD-1 clone and 100 pg/ml SEE (Staphylococcal Enterotoxin E).
  • SEE Staphylococcal Enterotoxin E
  • FIG. 17 shows concentrations of IL-2, IFNy, IL-6, IL-4 and IL-ip determined by ELISA in PBMCs in the presence of anti-PD-1 clones 01, 02, 03, 07, 09, 51, 55, 79, or 80 and SEE (Staphylococcal Enterotoxin E).
  • PBMCs from 3 healthy donors were incubated for 24 hours in the presence of 10 pg/ml of each anti-PD-1 clone and 100 pg/ml SEE (Staphylococcal Enterotoxin E).
  • the concentrations of IL-2, IFNy, IL-6, IL-4 and IL-ip in the supernatants were determined by ELISA.
  • experiments are designed to test these bispecific antibodies in vivo using syngeneic tumor model, in comparison to anti-PD-1 monoclonal antibodies.

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Abstract

L'invention concerne des méthodes de relocalisation de protéines de la synapse immunitaire pour moduler l'activation des lymphocytes T et l'utilisation de telles méthodes pour traiter une maladie chez un sujet en ayant besoin. L'invention concerne en outre des anticorps anti-CD45, dans certains modes de réalisation ayant une affinité réduite pour CD45, ainsi que des anticorps bispécifiques capables de moduler l'activité des lymphocytes T, les deux bras de l'anticorps bispécifique étant contre CD45 et PD-1 ou contre CD43 et PD-1. Dans certains modes de réalisation, l'anticorps bispécifique a une affinité réduite pour CD45 ou CD43.
PCT/US2024/047201 2023-09-18 2024-09-18 Anticorps bispécifiques anti-cd45 x pd-1 et anti-cd43 x pd-1 modulés par affinité pour traiter le cancer et l'auto-immunité Pending WO2025064498A1 (fr)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009055074A2 (fr) * 2007-10-25 2009-04-30 Wyeth Compositions et procédés thérapeutiques
US20190185566A1 (en) * 2016-05-13 2019-06-20 Hoffmann-La Roche Inc. Antigen Binding Molecules comprising a TNF family ligand trimer and PD1 binding moiety
US20210206848A1 (en) * 2018-05-17 2021-07-08 The Board Of Trustees Of The Leland Stanford Junior University Receptor inhibition by phosphatase recruitment
WO2021263169A2 (fr) * 2020-06-26 2021-12-30 Sorrento Therapeutics, Inc. Virus oncolytiques exprimant des protéines hybrides immunomodulatrices

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009055074A2 (fr) * 2007-10-25 2009-04-30 Wyeth Compositions et procédés thérapeutiques
US20190185566A1 (en) * 2016-05-13 2019-06-20 Hoffmann-La Roche Inc. Antigen Binding Molecules comprising a TNF family ligand trimer and PD1 binding moiety
US20210206848A1 (en) * 2018-05-17 2021-07-08 The Board Of Trustees Of The Leland Stanford Junior University Receptor inhibition by phosphatase recruitment
WO2021263169A2 (fr) * 2020-06-26 2021-12-30 Sorrento Therapeutics, Inc. Virus oncolytiques exprimant des protéines hybrides immunomodulatrices

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