Affinity-modulated Anti-CD45 x PD-1 and Anti-CD43 x PD-1 bispecific antibodies to treat cancer and autoimmunity

Abstract

The subject matter described here relates to 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, in some embodiments with reduced affinity for CD45, as well as bispecific antibodies capable of modulating T-cell activity, wherein the two arms of the bispecific antibody are against CD45 and PD-1 or against CD43 and PD-1. In certain embodiments the bispecific antibody has reduced affinity for CD45 or CD43.

Description

AFFINITY-MODULATED ANTI-CD45 X PD-1 AND ANTI-CD43 X PD-1 BISPECIFIC ANTIBODIES TO TREAT CANCER AND AUTOIMMUNITY

[0001] This application claims the benefit of and priority under 35 U.S.C. § 1 19(e) to US Provisional Serial No.: 63 / 583,555, filed September 18, 2023; and US Provisional Serial No.: 63 / 607,910, filed December 8, 2023, the contents of each of which are hereby incorporated by reference in their entireties.

[0002] This patent disclosure contains material that is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or the patent disclosure as it appears in the U.S. Patent and Trademark Office patent file or records but otherwise reserves any and all copyright rights.INCORPORATION BY REFERENCE

[0003] All documents cited herein are incorporated herein by reference in their entireties.GOVERNMENT SUPPORT

[0004] This invention was made with government support under AU25640, AI175498, CA231277, and AI150597 awarded by the National Institutes of Health. The government has certain rights in the invention.TECHNICAL FIELD

[0005] 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.BACKGROUND

[0006] 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 forprophylactic and / or therapeutic agents with improved specificity and efficacy, and reduced side effects.SUMMARY OF THE INVENTION

[0007] In certain aspects, described herein is a composition 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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.

[0008] In some embodiments, 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 (RANKL), CD62L, CD185 (CXCR5), CD19, CD15, CD14, CD194 (CCR4), CD57, KLRG1, IL17RB, NKp80, CD27, IL23R, VCAM1, CEACAM1, IL12R, IL23R, IL15R, IL6R, IL17RA (CD217), IL17RB, IL2R, TNFSF4 (CD252; OX40L), TNFRSF13C (CD268; BAFFR), TNFSF13B (CD257; BAFF), PTPRC (CD45), CD43, CD2, CD18, CDl la, CDl lb, CDl lc, CD49, CD29, CD22, CD43, ICAM1, CD134 (0X40), CD137 (4IBB), CD357 (GITR), CD256 (APRIL), CD267 (TACI), CD269 (BCMA), CD270 (HVEM), CD120a (TNFR1), CD120b (TNFR2), LTbR, CD70, 41BBL, Galectin 9, CD155, GITRL, TMIGD2, CD160, BTLA, CD270, CD96, CD226, CD112R, CD200R, A2aR, VISTA, B7-H7, CD270, LIGHT, FGL1, CD137L, CD155, CD112, CD277, KIR, SLAMF3, SLAMF7, or SIT1. In some embodiments,the protein for relocalization to the distal compartment of the immune synapse is a stimulatory receptor. In some embodiments, the protein for relocalization to the distal compartment is an inhibitory receptor.

[0009] In some embodiments, the bispecific antibody is any of the bi specific antibodies described herein.

[0010] In certain aspects, described herein is 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. In some embodiments, the molecule is an antibody or an antigen binding fragment thereof capable of localizing the protein to a distal compartment of the immune synapse. In some embodiments, 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.

[0011] In some embodiments, 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, CD5, CD69, CD183 (CXCR3), CD127 (IL17RA), CD254 (RANKL), CD62L, CD185 (CXCR5), CD19, CD15, CD14, CD194 (CCR4), CD57, KLRG1, IL17RB, NKp80, CD27, IL23R, CD352 (SLAMF6), VCAM1, CEACAM1, IL12R, IL23R, IL15R, IL6R, IL17RA (CD217), IL17RB, IL2R, TNFSF4 (CD252; OX40L), TNFRSF13C (CD268; BAFFR), TNFSF13B (CD257; BAFF), PTPRC (CD45), CD43, CD2, CD18, CDl la, CDl lb, CDl lc, CD49, CD29, CD22, CD43, ICAM1, CD134 (0X40), CD137 (4IBB), CD357 (GITR), CD256 (APRIL), CD267 (TACI), CD269 (BCMA), CD270 (HVEM), CD120a (TNFR1), CD120b (TNFR2), LTbR, CD70, 41BBL, Galectin 9, CD155, GITRL, TMIGD2, CD160, BTLA, CD270, CD96, CD226, CD112R, CD200R, A2aR, VISTA, B7-H7, CD270, LIGHT, FGL1, CD137L, CD155, CD112, CD277, KIR, SLAMF3, SLAMF7, or SIT1.

[0012] In certain aspects, described herein is 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 orantigen 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.

[0013] In certain aspects, described herein is 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.

[0014] In some embodiments, the first arm is encoded by a first polypeptide chain and the second arm is encoded by a second polypeptide chain that associate together. In some embodiments, the first arm comprises a linker between the first variable heavy domain and first variable light chain domain. In some embodiments, the second arm comprises a linker between the first variable heavy domain and first variable light chain domain. In some embodiments, the linker is a glycine-serine linker. In some embodiments, the first and second arms each further comprise a fragment, crystallizable (Fc) region. In some embodiments, the Fc region of the first arm comprises knob mutations and the Fc region of the second arm comprise hole mutations, or vice versa. In some embodiments, 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. In some embodiments, 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, and 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. In some embodiments, 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).

[0015] In certain aspects, described herein is 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 CD45protein 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.

[0016] In some embodiments, the first arm is encoded by a first polypeptide chain and the second arm is encoded by a second polypeptide chain that associate together. In some embodiments, the first arm comprises a linker between the first variable heavy domain and first variable light chain domain. In some embodiments, the second arm comprises a linker between the first variable heavy domain and first variable light chain domain. In some embodiments, the linker is a glycine-serine linker. In some embodiments, the first and second arms each further comprise a fragment, crystallizable (Fc) region. In some embodiments, the Fc region of the first arm comprises knob mutations and the Fc region of the second arm comprise hole mutations, or vice versa.

[0017] In some embodiments, 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. In some embodiments, 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, 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. In some embodiments, 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).

[0018] In some embodiments, 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. In some embodiments, the first arm comprises SEQ ID NO: 22, wherein the second arm comprises SEQ ID NO: 26. In some embodiments, 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).

[0019] In some embodiments, 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. In some embodiments, 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. In some embodiments, 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.

[0020] In some embodiments, 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.

[0021] In some embodiments, 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. In some embodiments, 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.

[0022] In certain aspects, described herein is a pharmaceutical composition comprising: the bispecific antibody described herein and a pharmaceutically acceptable carrier. In certain aspects, described herein is a method of preventing or treating cancer in a subject comprisingadministering to the subject an effective amount of the composition described herein. In some embodiments, 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. In certain aspects, 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. In some embodiments, the viral infection is HIV.

[0023] In certain aspects, described herein is a pharmaceutical composition comprising: the bispecific antibody described herein and a pharmaceutically acceptable carrier. In certain aspects, 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. In some embodiments, the diseases is selected from rheumatoid arthritis, systemic lupus erythematosus, psoriasis and psoriatic arthritis, spondyloarthropathy, type 1 diabetes, and multiple sclerosis.

[0024] In certain aspects, described herein is a kit for generating a bispecific antibody or fragment thereof, the kit comprising one or more vectors comprising a polynucleotide sequence encoding any of the bispecific antibodies described herein. In certain aspects, described herein is a kit for generating a bispecific antibody or fragment thereof, the kit 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. In some embodiments, 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.

[0025] In some embodiments, 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, wherein 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, wherein 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, and wherein 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 ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, and SEQ ID NO: 43. In some embodiments, 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. In some embodiments, the first arm comprises SEQ ID NO: 4. In some embodiments, the first arm comprises SEQ ID NO: 6. In some embodiments, the first arm comprises SEQ ID NO: 22.

[0026] In certain aspects, 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. In certain aspects, 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.

[0027] In some embodiments, 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. In some embodiments, the first arm comprises a first variable heavy chain domain and a first variable light chain domain, wherein the second arm comprises a second variable heavy chain domain and a second variable light chain domain, wherein 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. In some embodiments, 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 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. In someembodiments, the first arm comprises SEQ ID NO: 4. In some embodiments, the first arm comprises SEQ ID NO: 6. In some embodiments, the first arm comprises SEQ ID NO: 22.

[0028] In certain aspects, 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. In certain aspects, 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.

[0029] In certain aspects, described herein is a composition comprising: one or more vectors comprising a polynucleotide sequence encoding any of the bispecific antibodies described herein. In certain aspects, described herein is a composition 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.

[0030] In some embodiments, 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. In some embodiments, the first arm comprises a first variable heavy chain domain and a first variable light chain domain, wherein the second arm comprises a second variable heavy chain domain and a second variable light chain domain, wherein 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. In some embodiments, 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 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. In some embodiments, the first arm comprises SEQ ID NO: 4. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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, and wherein the portion of the first arm is capable of binding to the portion of the CD45 protein.

[0032] In some embodiments, 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, wherein 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, and wherein the portion of the first arm is capable of binding to the portion of the CD45 protein. In some embodiments, 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).

[0033] In some embodiments, 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. In some embodiments, 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).

[0034] In some embodiments, 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. In some embodiments, 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.

[0035] In some embodiments, 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. In some embodiments, 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.

[0036] In some embodiments, 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.DEFINITIONS

[0037] As used herein, 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. The term “immunoglobulin” (Ig) is used interchangeably with “antibody” herein. In some embodiments, the antibody binds to CD45. In some embodiments, the antibody binds to CD45 with reduced affinity.

[0038] 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 ordifferent targets. If the epitopes are on different targets, such targets may be on the same cell or different cells or cell types. In some embodiments, the bispecifc antibody comprises a first and second chain. In some embodiments, 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. In some embodiments, the first and second chains each further comprise a Fc domain. In some embodiments, the bispecific antibody comprises a first and a second heavy chain. In some embodiments, 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.BRIEF DESCRIPTION OF THE DRAWINGS

[0039] The patent or application file contains at least one drawing executed in color. To conform to the requirements for PCT patent applications, many of the figures presented herein are black and white representations of images originally created in color.

[0040] The following figures depict illustrative embodiments of the invention.

[0041] FIG. 1 shows pVaxl vector (ThermoFisher, V26020, Kanamycin-resistant) into which antibody constructs were cloned at HindHI / Xbal enzyme sites.

[0042] FIG. 2 shows ELISA binding for an anti-CD45xPD-l bispecific antibody to CD45 and PD-1. ELISA wells were coated with recombinant CD45 or recombinant ectodomain of PD-1 (2 pg / ml) overnight before incubation with different concentrations of the bispecific antibodies at room temperate. After 45 minutes, wells were washed, and the level of bound antibodies was detected with tagged secondary antibodies using a plate reader at an OD of 450. A representative experiment is shown (n=2).

[0043] 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-1was enriched at the immune synapse was calculated using ImageJ. FIG. 3 A shows representative images and FIG. 3B shows the quantitation of 4 independent experiments is included (n=4, 70-100 cells per experiment, SEM, *p<0.05).

[0044] 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). Cells were cultured at 37° overnight, and ELISA determined the levels of secreted IL-2 in the media (n=5, SEM, *p<0.001).

[0045] FIG. 5 shows the ELISA OD 450 values for binding curves for binding of wild-type anti-PD-lxCD45 bispecific antibody to human PD-l-His-coated plates. NC = no antibody control.

[0046] 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.

[0047] 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.

[0048] 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) (eBioscience™), 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) (eBioscience™), anti-HEL-human IgGl isotype control, and blank. EC50 values were calculated with GraphPad Prism (vl0.2.1).

[0049] 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-CD45antibody clone 28 to captured human-CD45RO-ECD-His. FIG. 9F shows binding of anti-CD45 antibody clone 31 to captured human-CD45RO-ECD-His.

[0050] 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.

[0051] 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.

[0052] 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).

[0053] 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.

[0054] 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.

[0055] 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.

[0056] 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.

[0057] 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).

[0058] 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.DETAILED DESCRIPTION

[0059] Disclosed herein are methods of relocalizing proteins of the immune synapse to modulate T cell functions and the use of such methods to treat or prevent disease (e.g., cancer, inflammation, autoimmunity) or infection (e.g., human immunodeficiency virus (HIV) in a subject in need thereof. In some embodiments the target protein of the immune synapse is relocalized to the distal compartment away from the immune synapse. In some embodiments, where the target protein is a stimulatory receptor (for example, CD2 or CD28), such relocalization will lead to T cell inhibition and is useful for treating diseases such as autoimmune disorders. In other embodiments, where 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.

[0060] Disclosed herein are 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)). Described herein are antibodies, including, but not limited to, bispecific antibodies that bind to CD45. In some embodiments, the antibodies or bispecific antibodies disclosed herein bind to CD45 with reduced affinity.

[0061] While monoclonal antibodies have been developed to inhibit the immune checkpoint PD-1 on T cells from binding to its ligands, PDL-1 and PDL-2 on cancer cells, these monospecific antibodies lack specificity and can cause adverse events in patients. Described herein are 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. In some embodiments, the novel anti-CD45xPD-l and anti-CD43xPD-l bispecific antibodies inhibit PD-1 signaling with enhanced specificity. In some embodiments, the bispecific antibodies disclosed herein bind to CD45 with reduced affinity.T Cells and The Immune Synapse

[0062] T cells have surface receptors that can act as immune checkpoint receptors, such as PD-1. See Speiser DE, Ho PC, Verdeil G. Regulatory Circuits of T Cell Function in Cancer. Nat Rev Immunol. 2016 Oct; 16(10): pp. 599-611, incorporated by reference in its entiretyherein. These receptors act as “checkpoints” to prevent excessive immune activation. However, cancer cells can exploit these checkpoint pathways to evade immune detection and attack. See Liu J, Chen Z, Li Y, Zhao W, Wu J and Zhang Z. PD- I / PD- / .7 Checkpoint Inhibitors in Tumor Immunotherapy. Front. Pharmacol. 2021 Sep; pp. 12: p. 731798, incorporated by reference in its entirety herein. 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. For example, with respect to proteins found on T cells, the T cell receptor (TCR) is localized to the center of the synapse, PD-1 and CD28 are located in the peripheral synapse, and LFA-1, CD43, and CD45 are found in the distal compartment of the synapse. See e.g., Delon J, Kaibuchi K, Germain RN. 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.PMID: 11728332. This organization is critical for the function of the synapse, where specific clusters of proteins are formed.

[0063] 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. He X, Xu C. Immune Checkpoint Signaling and Cancer Immunotherapy . Cell Res. 2020 Aug; 30(8): pp. 660-669, incorporated by reference in its entirety herein. For example, 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).

[0064] Without intending to be bound by any particular theory, the design of antibodies for the prevention or treatment of cancer (and autoimmunity) 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 (CD40L), CD278 (ICOS), CD25 (IL2R), CD152 (CTLA4), CXCR1, HLA- DR, CD16, CD5, CD69, CD183 (CXCR3), CD127 (IL17RA), CD254 (RANKL), CD62L, CD185 (CXCR5), CD19, CD15, CD14, CD194 (CCR4), CD57, KLRG1, IL17RB, NKp80, CD27, IL23R, VCAM1, CEACAM1, IL12R, IL23R, IL15R, IL6R, IL17RA (CD217), IL17RB, IL2R, TNFSF4 (CD252; OX40L), TNFRSF13C (CD268; BAFFR), TNFSF13B (CD257; BAFF), PTPRC (CD45), CD43, CD2, CD18, CDl la, CDl lb, CDl lc, CD49, CD29, CD22, CD43, ICAM1, CD134 (0X40), CD137 (4IBB), CD357 (GITR), CD256 (APRIL), CD267 (TACI), CD269 (BCMA), CD270 (HVEM), CD120a (TNFR1), CD120b (TNFR2), LTbR, CD70, 41BBL, Galectin 9, CD155, GITRL, TMIGD2, CD160, BTLA, CD270, CD96, CD226, CD112R, CD200R, A2aR, VISTA, B7-H7, CD270, LIGHT, FGL1, CD137L, CD155, CD112, CD277, KIR, SLAMF3, SLAMF7, SIT1) on the surface of the immune cells in the context of the immune synapse. Without intending to be bound by any particular theory, in addition to blocking ligand binding, it is hypothesized that changing the location 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), CD 196 (CCR6), CD56 (NCAM), CD366 (TIM3), CD45RA, CD 154 (CD40L), CD278 (ICOS), CD25 (IL2R), CD 152 (CTLA4), CXCR1, HLA-DR, CD 16, CD5, CD69, CD 183 (CXCR3), CD127 (IL17RA), CD254 (RANKL), CD62L, CD185 (CXCR5), CD19, CD15, CD14, CD194 (CCR4), CD57, KLRG1, IL17RB, NKp80, CD27, IL23R, VCAM1, CEACAM1, IL12R, IL23R, IL15R, IL6R, IL17RA (CD217), IL17RB, IL2R, TNFSF4 (CD252; OX40L), TNFRSF13C (CD268; BAFFR), TNFSF13B (CD257; BAFF), PTPRC (CD45), CD43, CD2, CD18, CDl la, CDl lb, CDl lc, CD49, CD29, CD22, CD43, ICAM1, CD134 (0X40), CD137 (4IBB), CD357 (GITR), CD256 (APRIL), CD267 (TACI), CD269 (BCMA), CD270 (HVEM), CD120a (TNFR1), CD120b (TNFR2), LTbR, CD70, 41BBL, Galectin 9, CD155, GITRL, TMIGD2, CD160, BTLA, CD270, CD96, CD226, CD112R, CD200R, A2aR, VISTA, B7-H7, CD270, LIGHT, FGL1, CD137L, CD155, CD112, CD277, KIR, SLAMF3, SLAMF7, SIT1) within the different compartments of the immune synapse could serve as an alternative, efficient, and safer approach to treating cancer patients.Compositions and Methods of Relocalizing Proteins of the Immune Synapse

[0065] Spatial proximity of receptors and proteins of the immune synapse and their downstream signaling molecules within the immune synapse is necessary for T-cell activation.For example, the clustering of SLAMF6 endodomain with CD3 is required for SLAMF6 mediated T-cell activation. This organization is critical for the function of the synapse, where specific clusters of proteins are formed. In another example, removal of PD-1 from the immune synapse prevents the inhibitory signals of T-cells, thereby preventing the tumor cells from evading the T cell immune response.

[0066] Described herein are compositions or methods for relocalizing proteins of the immune synapse to the different compartments of the immune synapse. Without intending to be bound by any particular theory, it is hypothesized that 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. Additionally, without intending to be bound by any particular theory, 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. In some embodiments, localizing proteins away from the immune synapse relocalizes proteins to the distal compartment of the immune synapse. Thus, 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.

[0067] Without intending to be bound by any particular theory, the rationale behind the design of the antibodies disclosed herein, including the bispecific antibodies, was that disruption of selected protein interactions through altered localization would serve as a strategy to affect T cell function better. In some embodiments, 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). Without intending to be bound by any particular theory, it is hypothesized that 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. In some embodiments, 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.

[0068] Described herein are inhibitors of proteins at the immune synapse capable of relocalizing the protein. In some embodiments 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. In some embodiments, 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. Without intending to be bound by any particular theory, 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), CXCR1, HLA-DR, CD16, CD5, CD69, CD183 (CXCR3), CD127 (IL17RA), CD254 (RANKL), CD62L, CD185 (CXCR5), CD19, CD15, CD14, CD194 (CCR4), CD57, KLRG1, IL17RB, NKp80, CD27, IL23R, CD352 (SLAMF6), VCAM1, CEACAM1, IL12R, IL23R, IL15R, IL6R, IL17RA (CD217), IL17RB, IL2R, TNFSF4 (CD252; OX40L), TNFRSF13C (CD268; BAFFR), TNFSF13B (CD257; BAFF), PTPRC (CD45), CD43, CD2, CD18, CDl la, CDl lb, CDl lc, CD49, CD29, CD22, CD43, ICAM1, CD134 (0X40), CD137 (4IBB), CD357 (GITR), CD256 (APRIL), CD267 (TACI), CD269 (BCMA), CD270 (HVEM), CD120a (TNFR1), CD120b (TNFR2), LTbR, CD70, 41BBL, Galectin 9, CD155, GITRL, TMIGD2, CD160, BTLA, CD270, CD96, CD226, CD112R, CD200R, A2aR, VISTA, B7-H7, CD270, LIGHT, FGL1, CD137L, CD155, CD112, CD277, KIR, SLAMF3, SLAMF7, or SIT1. In some embodiments, 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. In some embodiments, localizing proteins away from the immune synapse relocalizes proteins to the distal compartment of the immune synapse.

[0069] Without intending to be bound by any particular theory, it is hypothesized that bispecific antibodies that bind distal synaptic proteins and core synaptic proteins 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. Without intending to be bound by any particular theory, 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), CXCR1, HLA-DR, CD16, CD5, CD69, CD183 (CXCR3), CD127 (IL17RA), CD254 (RANKL), CD62L, CD185 (CXCR5), CD19, CD15, CD14, CD194 (CCR4), CD57, KLRG1, IL17RB, NKp80, CD27, IL23R, VCAM1, CEACAM1, IL12R, IL23R, IL15R, IL6R, IL17RA (CD217), IL17RB, IL2R, TNFSF4 (CD252; OX40L), TNFRSF13C (CD268; BAFFR), TNFSF13B (CD257; BAFF), PTPRC (CD45), CD43, CD2, CD18, CDl la, CDl lb, CDl lc, CD49, CD29, CD22, CD43, ICAM1, CD134 (0X40), CD137 (4IBB), CD357 (GITR), CD256 (APRIL), CD267 (TACI), CD269 (BCMA), CD270 (HVEM), CD120a (TNFR1), CD120b (TNFR2), LTbR, CD70, 41BBL, Galectin 9, CD155, GITRL, TMIGD2, CD160, BTLA, CD270, CD96, CD226, CD112R, CD200R, A2aR, VISTA, B7-H7, CD270, LIGHT, FGL1, CD137L, CD155, CD112, CD277, KIR, SLAMF3, SLAMF7, or SIT1 and the proteins at the distal compartment of the immune synapse are CD45, CD43, CD44, LFA-1, Talin, and CD2.

[0070] Described herein are improved versions of anti -PD-1 antibodies. A panel of novel anti-CD45xPD-l bispecific antibodies and anti-CD43xPD-l bispecific antibodies were designed and are described herein. In some embodiments, the novel bispecific antibodies disclosed herein bind to PD-1 and CD45 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 CD45 on another cell). In some embodiments, 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).

[0071] In some embodiments, the novel bispecific antibodies disclosed herein bind to a protein of the immune synapse for relocalization and CD45 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 ofthe immune synapse for relocalization on one cell, and the other arm of the antibody binds to CD45 on another cell). In some embodiments, 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).

[0072] Described herein are methods of treating cancer by causing proteins of immune synapse to be excluded from the immune synapse. Without intending to be bound by any particular theory, the design of the prevention or treatment of cancer is improved by taking into account the localization of proteins on the surface of the immune cells in the context of the immune synapse. Without intending to be bound by any particular theory, 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. In some embodiments, the composition comprises an antibody against the protein at the immune synapse. In some embodiments, 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), CD25 (IL2R), CD152 (CTLA4), CXCR1, HLA-DR, CD16, CD5, CD69, CD183 (CXCR3), CD127 (IL17RA), CD254 (RANKL), CD62L, CD185 (CXCR5), CD19, CD15, CD14, CD194 (CCR4), CD57, KLRG1, IL17RB, NKp80, CD27, IL23R, VCAM1, CEACAM1, IL12R, IL23R, IL15R, IL6R, IL17RA (CD217), IL17RB, IL2R, TNFSF4 (CD252; OX40L), TNFRSF13C (CD268; BAFFR), TNFSF13B (CD257; BAFF), PTPRC (CD45), CD43, CD2, CD18, CDl la, CDl lb, CDl lc, CD49, CD29, CD22, CD43, ICAM1, CD134 (0X40), CD137 (4IBB), CD357 (GITR), CD256 (APRIL), CD267 (TACI), CD269 (BCMA), CD270 (HVEM), CD120a (TNFR1), CD120b (TNFR2), LTbR, CD70, 41BBL, Galectin 9, CD155, GITRL, TMIGD2, CD160, BTLA, CD270, CD96, CD226, CD112R, CD200R, A2aR, VISTA, B7-H7, CD270, LIGHT, FGL1, CD137L, CD155, CD112, CD277, KIR, SLAMF3, SLAMF7, or SIT1. In some embodiments, 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, CD 154 (CD40L), CD278 (ICOS), CD25 (IL2R), CD152 (CTLA4), CXCR1, HLA-DR, CD16, CD5, CD69, CD183 (CXCR3), CD127 (IL17RA), CD254 (RANKL), CD62L, CD185 (CXCR5), CD19, CD15, CD14, CD194 (CCR4), CD57, KLRG1, IL17RB, NKp80, CD27, IL23R, CD352 (SLAMF6), VCAM1, CEACAM1, IL12R, IL23R, IL15R, IL6R, IL17RA (CD217), IL17RB, IL2R, TNFSF4 (CD252; OX40L), TNFRSF13C (CD268; BAFFR), TNFSF13B (CD257; BAFF), PTPRC (CD45), CD43, CD2, CD18, CDlla, CDllb, CDllc, CD49, CD29, CD22, CD43, ICAM1, CD134 (0X40), CD137 (4IBB), CD357 (GITR), CD256 (APRIL), CD267 (TACI), CD269 (BCMA), CD270 (HVEM), CD120a (TNFR1), CD120b (TNFR2), LTbR, CD70, 41BBL, Galectin 9, CD155, GITRL, TMIGD2, CD160, BTLA, CD270, CD96, CD226, CD112R, CD200R, A2aR, VISTA, B7-H7, CD270, LIGHT, FGL1, CD137L, CD155, CD112, CD277, KIR, SLAMF3, SLAMF7, or SIT1 are excluded from the immune synapse. In some embodiments any of the above proteins are relocalized to the distal compartment of the immune synapse. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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.

[0073] Without intending to be bound by any particular theory, it is hypothesized that relocalizing proteins at the immune synapse promotes T cell activation and disrupts inhibitory signals, and localizing proteins away from the immune synapse draws with it proteins necessary for inhibition of T cell activation. Additionally, without intending to be bound by any particular theory, 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. Thus, changing the location ofproteins within the different compartments of the immune synapse could serve as an alternative, efficient, and safer approach to treating cancer patients.

[0074] In some embodiments, 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. In some embodiments, 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. In some embodiments, 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 disease caused by any solid tumor that is not able to repair errors in its DNA that occur when the DNA is copied.

[0075] In some embodiments, 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 a viral infection. In some embodiments, the viral infection is caused by HIV in a subject.

[0076] Without intending to be bound by any particular theory, the rationale behind the design of the bispecific antibodies disclosed herein was that disruption of selected protein interactions through altered localization would serve as a strategy to affect T cell function better. The PD-1 pathway provides a clear example to demonstrate how changing the localization of a protein on the cell surface can impact T cell function. In some embodiments, 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. Without intending to be bound by any particular theory, it is hypothesized that a bispecific antibody to CD45 (or to CD43) and to PD-1 would bind to CD45 (or CD43), which would serve as an anchor, outside the synapse, and pull PD-1 away from the core of the synapse. In some embodiments, the novel bispecific antibodies disclosed herein bind to PD-1 and CD45 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 CD45 on another cell). In some embodiments, 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).

[0077] In some embodiments, described herein are anti-CD45xPD-l and anti-CD43xPD-l bispecific antibodies for addressing the limitation of PD-1 localization. In some embodiments, one or more of the anti-CD45xPD-l bispecific antibodies described herein function by binding to CD45 and pulling PD-1 away from the core of the synapse, disrupting downstream signaling and effector pathways, and / or enhancing T-cell function. In some embodiments, one or more of the anti-CD43xPD-l bispecific antibodies described herein function by binding to CD43 and pulling PD-1 away from the core of the synapse, disrupting downstream signaling and effector pathways, and / or enhancing T-cell function.

[0078] In some embodiments, the engineered bispecific antibodies disclosed herein (e.g., anti-CD45xPD-l and / or anti-CD43xPD-l bispecific antibodies) feature reduced affinity anti- CD45 or anti-CD43 arms. In some embodiments, the reduced affinity arms increase binding in cis and specificity for T cells. In some embodiments, the advantages of the disclosed anti- CD45xPD-l and / or anti-CD43xPD-l bispecific antibodies over current options include one or more of preventing PD-1 interaction with its ligands, specific targeting to T cells, and the facilitation of PD-1 dephosphorylation. In some embodiments, the anti-CD45xPD-l and / or anti- CD43xPD-l bispecific antibodies 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.

[0079] In some embodiments, disclosed herein are anti-CD45xPD-l bispecific antibodies that bind to CD45 and localize PD-1 away from the core of the synapse. In some embodiments, disclosed herein are anti-CD43xPD-l bispecific antibodies that bind to CD43 and localize PD-1 away from the core of the synapse. In some embodiments, altering the location of PD-1 disrupts downstream signaling and effector pathways, and enhances T-cell function.

[0080] In some embodiments, the anti-CD45xPD-l bispecific antibody has a modified affinity for CD45 relative to a preselected affinity threshold. For example, in some embodiments, the affinity of the anti-CD45xPD-l bispecific antibody to CD45 is lower than an anti-CD45 antibody known in the art. For example, in some embodiments, 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. In some embodiments, the affinity (e.g., measured as a dissociation constant (KD)) of the anti-CD45xPD-l bispecific antibody to CD45 is betweenabout 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).

[0081] In some embodiments, the anti-CD43xPD-l bispecific antibody has a modified affinity for CD43 relative to a preselected affinity threshold. For example, in some embodiments, the affinity of the anti-CD43xPD-l bispecific antibody to CD43 is lower than an anti-CD43 antibody known in the art. In some embodiments, 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). In some embodiments, 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).

[0082] In some embodiments, 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. In some embodiments, 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.

[0083] In some embodiments, an anti-CD45xPD-l and / or an anti-CD43xPD-l bispecific antibody, as disclosed herein, is used to prevent or treat cancer in a subject. In some embodiments, 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. In some embodiments, an anti-CD45xPD-l and / or an anti-CD43xPD-l bispecific antibody, 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.

[0084] In some embodiments, an anti-CD45xPD-l and / or an anti-CD43xPD-l bispecific antibody, as disclosed herein, is used to prevent or treat a viral infection. In some embodiments, the viral infection is caused by HIV in a subject.Antibodies

[0085] There are five classes of human antibodies (i.e., IgA, IgD, IgE, IgG, and IgM) and each have various isotypes (e.g., IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2). In some embodiments, the antibodies disclosed herein belong to the IgG class. IgG can be further divided into four subclasses: IgGl, IgG2, IgG3, and IgG4. Each subclass has a unique profile with respect to antigen binding, immune complex formation, complement activation, triggering of effector cells, half-life, and placental transport. E.g., see Gestur Vidarsson, et al., IgG Subclasses and Allotypes: From Structure to Effector Functions, 5 Frontiers in Immunology 520 (2014), incorporated by reference herein in its entirety.

[0086] 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. Similarly, 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. The part of the antibody formed by the lower hinge region and the CH2 / CH3 domains of the heavy chain is called “Fc” (“fragment crystalline”). See e.g., Basic and Clinical Immunology, 8th Edition, Daniel P. Sties, Abba I. Terr and Tristram G. Parsolw (eds), Appleton & Lange, Norwalk, CT, 1994, page 71 and Chapter 6, incorporated by reference herein in its entirety.

[0087] The variability in an antibody sequence is concentrated in three segments called complementarity determining regions (CDRs) (also called hypervariable regions (HVRs)) both in the light-chain and the heavy chain variable domains. The more highly conserved portions of variable domains are called the framework regions (FR). 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. See Kabat et al, Sequences of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, MD (1991), incorporated by reference in its entirety herein. The constant domains are not involved directly in the binding of antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibodydependent cellular toxicity.

[0088] By way of example, 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.

[0089] In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the monoclonal antibody comprises a first, second, third and fourth chain. In some embodiments, the first and third chains each comprise a VH domain and the second and fourth chains each comprise a VL domain. In some embodiments, the first and third chains each further comprises a CHI domain, a hinge domain, and a Fc domain. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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.

[0090] However, the antibodies disclosed herein (e.g., monoclonal antibodies, bispecific antibodies) 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. These antigen-binding fragments are contemplated herein and a person of skill in the art can readily synthesize such molecules using the sequences and identified domains of the heavy and light chains of the anti-CD45 antibodies disclosed here. For example, in some embodiments, the monoclonal antibody comprises a first and second chain that associate together. In some embodiments, 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. In some embodiments, the linker is a glycine-serine linker. In some embodiments, the Fc domain of the first chain comprises knob mutations and the Fc domain of the second chain comprise hole mutations, or vice versa. In some embodiments 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.

[0091] In some embodiments, the antibody is a bispecific antibody. In some embodiments, the bispecific antibody comprises a first and second chain. In some embodiments, 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. In some embodiments, the first and second chains each further comprise a Fc domain. In some embodiments, 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. In some embodiments, 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. If the epitopes are on different targets, such targets may be on the same cell or different cells or cell types. 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.

[0092] In some embodiments, 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. In some embodiments, 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. In some embodiments, 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 Acid Sequences of Anti-CD45 Portions of Anti-CD45xPD-l Bispecific Antibodies

[0093] In some embodiments, the anti-CD45 portion of the anti-CD45xPD-l bispecific antibody is 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 (VH) domain, a linker, a variable light chain (VL) domain, and an Fc portion. 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. 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. In some embodiments, instead of the “hole” mutations, 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.

[0094] In some embodiments, 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). In some embodiments, 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: 2.EVQLVESGGGLVQPGGSLRLSCAASGFTFNNY[W]MTWVRQAPGKGLEWVASISSSGG SIY[Y]PDSVKGRFTISRDNSKNTLYLOMNSLRAEDTAVYYCARDERrW]AGAMDAWG OGTTVTVSSGGGGSGGGGSGGGGSDIOMTOSPSSLSASVGDRVTITCKASQNINKNLD WYQOKPGKAPKLLIYETNNLOTGVPSRFSGSGSGTDFTLTISSLOPEDFATYYCYQHNS ^ YGGGY YLPXKSLEPKSCDKTHTCPPCPAPEFQGGPSVFLFPPKPKDTLYITREPEVTCV WDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCQV SNKALPAPIEKTISKAKGQPREPQVYVYPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFALVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 2). In some embodiments, 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. In some embodiments, the signal peptide is cleaved during post- translational modifications that occur in vitro or in vivo.

[0095] In some embodiments, 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. In some embodiments, the anti-CD45 portion (scFv-Fc) of the anti-CD45xPD-l bispecific antibody (without the signal peptide) 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. In some embodiments, 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). In some embodiments, 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.

[0096] Although the sequence above is depicted with a linker “GGGGSGGGGSGGGGS” (SEQ ID NO: 3) a person of skill in the art recognizes that numerous other flexible linkers known in the art can be used, including those enriched in small or hydrophilic amino acids. In some embodiments, the linker between the VH and VL domains comprises a glycine-serine linker. In some embodiments, any combination of glycine and serine residues can be used. In some embodiments, 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.

[0097] In some embodiments, 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. In some embodiments, 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.

[0098] In some embodiments, 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). In some embodiments, 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.EVQLVESGGGLVQPGGSLRLSCAASGFTFNNY[W]MTWVRQAPGKGLEWVASISSSGG SIY[Y]PDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDER[[A]]AGAMDAW GOGTTVTVSSGGGGSGGGGSGGGGSDIOMTOSPSSLSASVGDRVTITCKASQNINKNL DWYQOKPGKAPKLLIYETNNLOTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCYQHN ^ YGGGY YLP KSLEPKSCDKTHTCPPCPAPEFQGGPSVFLFPPKPKDTLYITREPEVTC VWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCQ VSNKALPAPIEKTISKAKGQPREPQVYVYPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFALVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (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. In some embodiments, one or more amino acids shown inside single square brackets can also be substituted relative to the sequence shown. Insome embodiments, 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. In some embodiments, the signal peptide is cleaved during post-translational modifications that occur in vitro or in vivo.

[0099] In some embodiments, 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.

[0100] In some embodiments, 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. In some embodiments, the anti-CD45 portion (scFv-Fc) of the anti-CD45xPD-l bispecific antibody (without the signal peptide) 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. In some embodiments, 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). In some embodiments, 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.

[0101] Although the sequence above is depicted with a linker “GGGGSGGGGSGGGGS” (SEQ ID NO: 3) a person of skill in the art recognizes that numerous other flexible linkers known in the art can be used, including those enriched in small or hydrophilic amino acids. In some embodiments, the linker between the VH and VL domains comprises a glycine-serine linker. In some embodiments, any combination of glycine and serine residues can be used. In some embodiments, 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.

[0102] In some embodiments, 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). In some embodiments, the amino acidsequence of the anti-CD45 portion (scFv-Fc) of an anti-CD45xPD-l bispecific antibody (without the signal peptide) comprises SEQ ID NO: 6.EVQLVESGGGLVQPGGSLRLSCAASGFTFNNY[[A]]MTWVRQAPGKGLEWVASISSSG GSIY[Y]PDSVKGRFTISRDNSKNTLYLOMNSLRAEDTAVYYCARDER[W]AGAMDAW GOGTTVTVSSGGGGSGGGGSGGGGSDIOMTOSPSSLSASVGDRVTITCKASQNINKNL DWYOQKPGKAPKLLIYETNNLOTGVPSRFSGSGSGTDFTLTISSLOPEDFATYYCYQHN ^ YGGGY YLP KSLEPKSCDKTHTCPPCPAPEFQGGPSVFLFPPKPKDTLYITREPEVTC VWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCQ VSNKALPAPIEKTISKAKGQPREPQVYVYPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFALVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (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. In some embodiments, one or more amino acids shown inside single square brackets can also be substituted relative to the sequence shown. In some embodiments, 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. In some embodiments, the signal peptide is cleaved during post-translational modifications that occur in vitro or in vivo.

[0103] In some embodiments, 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.

[0104] In some embodiments, 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. In some embodiments, the anti-CD45 portion (scFv-Fc) of the anti-CD45xPD-l bispecific antibody (without the signal peptide) 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. In some embodiments, 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). In some embodiments, 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 bispecificantibody 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.

[0105] Although the sequence above is depicted with a linker “GGGGSGGGGSGGGGS” (SEQ ID NO: 3) a person of skill in the art recognizes that numerous other flexible linkers known in the art can be used, including those enriched in small or hydrophilic amino acids. In some embodiments, the linker between the VH and VL domains comprises a glycine-serine linker. In some embodiments, any combination of glycine and serine residues can be used. In some embodiments, 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.

[0106] In some embodiments, 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). In some embodiments, 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: 8.EVQLVESGGGLVQPGGSLRLSCAASGFTFNNY[W]MTWVRQAPGKGLEWVASISSSGG SIY[[A]]PDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDER[W]AGAMDAW GOGTTVTVSSGGGGSGGGGSGGGGSDIOMTOSPSSLSASVGDRVTITCKASQNINKNL DWYQQKPGKAPKLLIYETNNLQTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCYQHN ^ YGGGY YLP KSLEPKSCDKTHTCPPCPAPEFQGGPSVFLFPPKPKDTLYITREPEVTC VWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCQ VSNKALPAPIEKTISKAKGQPREPQVYVYPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFALVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (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. In some embodiments, one or more amino acids shown inside single square brackets can also be substituted relative to the sequence shown. In some embodiments, 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. In some embodiments, the signal peptide is cleaved during post-translational modifications that occur in vitro or in vivo.

[0107] In some embodiments, 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.

[0108] In some embodiments, 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. In some embodiments, the anti-CD45 portion (scFv-Fc) of the anti-CD45xPD-l bispecific antibody (without the signal peptide) 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. In some embodiments, 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). In some embodiments, 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.

[0109] Although the sequence above is depicted with a linker “GGGGSGGGGSGGGGS” (SEQ ID NO: 3) a person of skill in the art recognizes that numerous other flexible linkers known in the art can be used, including those enriched in small or hydrophilic amino acids. In some embodiments, the linker between the VH and VL domains comprises a glycine-serine linker. In some embodiments, any combination of glycine and serine residues can be used. In some embodiments, 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.

[0110] In some embodiments, 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). In some embodiments, 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.EVQLVESGGGLVQPGGSLRLSCAASGFTFNNY[W]MTWVRQAPGKGLEWVASISSSGG SIY[[A]]PDS[[A]]KGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDER[W]AGAMDA WGOGTTVTVSSGGGGSGGGGSGGGGSDIOMTOSPSSLSASVGDRVTITCKASQNINKNLDWYQOKPGKAPKLLIYETNNLOTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCYQH ^^FYYGGGYYYEE KRLEPKSCDKTHTCPPCPAPEFOGGPSVFLFPPKPKDTLYITREPEVT CVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKC QVSNKALPAPIEKTISKAKGQPREPQVYVYPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFALVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP G (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. In some embodiments, one or more amino acids shown inside single square brackets can also be substituted relative to the sequence shown. In some embodiments, 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. In some embodiments, the signal peptide is cleaved during post-translational modifications that occur in vitro or in vivo.

[0111] In some embodiments, 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.

[0112] In some embodiments, 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. In some embodiments, the anti-CD45 portion (scFv-Fc) of the anti-CD45xPD-l bispecific antibody (without the signal peptide) 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. In some embodiments, 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). In some embodiments, 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.

[0113] Although the sequence above is depicted with a linker “GGGGSGGGGSGGGGS” (SEQ ID NO: 3) a person of skill in the art recognizes that numerous other flexible linkersknown in the art can be used, including those enriched in small or hydrophilic amino acids. In some embodiments, the linker between the VH and VL domains comprises a glycine-serine linker. In some embodiments, any combination of glycine and serine residues can be used. In some embodiments, 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.

[0114] In some embodiments, 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). In some embodiments, 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.EVOLOQSGPELEKPGASVKISCKASGYSFTGYNMNWVKOSNGKSLEWIGNIDPYYGGS DYSQKFLGKATLTVDKSSSTAYMHLKSLTSEDSAVYYCARSNMYDGEYYVMDYWGQ GTSVTVSSGGGGSGGGGSGGGGSDIVLTOSPASLAVSLGORATISCRASESVDNFGVSF MNWFOOKPGOPPKLLIYASSNRGSGVPARFSGSGSGTDFSLNVHPMEEDDAAMYFCQ QSKA V^GGG L X^EPKSCDKTHTCPPCPAPEFQGGPSVFLFPPKPKDTLYITREP EVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKE YKCQVSNKALPAPIEKTISKAKGQPREPQVYVYPPSRDELTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFALVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPG (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). In some embodiments, 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. In some embodiments, the signal peptide is cleaved during post-translational modifications that occur in vitro or in vivo.

[0115] In some embodiments, 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.

[0116] In some embodiments, 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. In some embodiments, the anti-CD45 portion (scFv-Fc) of the anti-CD45xPD-l bispecific antibody (without the signal peptide) 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. In some embodiments, 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). In some embodiments, 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.

[0117] Although the sequence above is depicted with a linker “GGGGSGGGGSGGGGS” (SEQ ID NO: 3) a person of skill in the art recognizes that numerous other flexible linkers known in the art can be used, including those enriched in small or hydrophilic amino acids. In some embodiments, the linker between the VH and VL domains comprises a glycine-serine linker. In some embodiments, any combination of glycine and serine residues can be used. In some embodiments, 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.

[0118] In some embodiments, 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). In some embodiments, 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.DVQLVESGGGLVQPGGSRKLSCAASGFTFSNFGMHWVRQAPEKGLEWVAYVSRGST TFYYADTVKGRFTISRDNPKNTLFLQMTSLRSEDTAMYYCARSATATWTMDYWGHG TSVTVSSGGGGSGGGGSGGGGSNIMMTOSPSSLAVSAGEKVTMSCKSSOSVFSSSNHM NYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYC Q ESSNVTFGGGSKLE KLEPKSCDKTHTCPPCPAPEFOGGPSVFLFPPKPKDTLYITREP EVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKE YKCQVSNKALPAPIEKTISKAKGQPREPQVYVYPPSRDELTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFALVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPG (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). In some embodiments, 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 IDNO: 30. In some embodiments, the signal peptide is cleaved during post-translational modifications that occur in vitro or in vivo.

[0119] In some embodiments, 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.

[0120] In some embodiments, 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. In some embodiments, the anti-CD45 portion (scFv-Fc) of the anti-CD45xPD-l bispecific antibody (without the signal peptide) 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. In some embodiments, 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). In some embodiments, 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.

[0121] Although the sequence above is depicted with a linker “GGGGSGGGGSGGGGS” (SEQ ID NO: 3) a person of skill in the art recognizes that numerous other flexible linkers known in the art can be used, including those enriched in small or hydrophilic amino acids. In some embodiments, the linker between the VH and VL domains comprises a glycine-serine linker. In some embodiments, any combination of glycine and serine residues can be used. In some embodiments, 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.

[0122] In some embodiments, 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). In some embodiments, 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.QVQLQQSAPELARPGASVRMSCKASGYTFTTYTMNWLTQRPGQGLEWIGYINPSSGY TEYNQKFKDKTSLTADTSSSTAYMQLSSLTSEHSAVYYCARASGYSSWFAYWGOGTL VTVSAGGGGSGGGGSGGGGSNIVMTOTPKFLLVSAGDRVTITCKASOSVNNDVGWYQ QKPGQSPKLLIYYASNRYTGVPDRFTGSGYGTDFTFTISTVQAEDLAVYFCQQDYNSPL YYGAGYYYEELYG£PKSCDKTHTCPPCPAPEFQGGPSVFLFPPKPKDTLYITREPEVTCVVVD VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCQVSNK ALPAPIEKnSKAKGQPREPQVYVYPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFALVSKLIVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (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). In some embodiments, 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. In some embodiments, the signal peptide is cleaved during post-translational modifications that occur in vitro or in vivo.

[0123] In some embodiments, 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.

[0124] In some embodiments, 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. In some embodiments, the anti-CD45 portion (scFv-Fc) of the anti-CD45xPD-l bispecific antibody (without the signal peptide) 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. In some embodiments, 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). In some embodiments, 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.

[0125] Although the sequence above is depicted with a linker “GGGGSGGGGSGGGGS” (SEQ ID NO: 3) a person of skill in the art recognizes that numerous other flexible linkersknown in the art can be used, including those enriched in small or hydrophilic amino acids. In some embodiments, the linker between the VH and VL domains comprises a glycine-serine linker. In some embodiments, any combination of glycine and serine residues can be used. In some embodiments, 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.

[0126] In some embodiments, 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). In some embodiments, 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.OVOLOQSGAELARPGASVKMSCKASGYTFTFYAILWVKOMPGOGLEWIGFINPSSGY TSYNQKFKDKATLTADKSSSTAYMQLSSLTSEDSAVYYCARDYGLDYWGQGTTLAVS SGGGGSGGGGSGGGGSDIVMTOAAPSVPVTPGESVSISCRSSKSLLHSNGNTYLYWFL ORPGOSPOLLIYRMSNLASGVPDRFSGSGSGTAFTLGISRVEAGDVGVYYCMQHLEYP UYG GY EYI LEPKSCDKTHTCPPCPAPEFQGGPSVFLFPPKPKDTLYITREPEVTCVW DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCQVSN KALPAPIEKTISKAKGQPREPQVYVYPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPE NNYKTTPPVLDSDGSFAL VSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (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). In some embodiments, 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. In some embodiments, the signal peptide is cleaved during post-translational modifications that occur in vitro or in vivo.

[0127] In some embodiments, 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.

[0128] In some embodiments, 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. In some embodiments, the anti-CD45 portion (scFv-Fc) of the anti-CD45xPD-l bispecific antibody (without the signal peptide) 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. In some embodiments, 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). In some embodiments, 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.

[0129] Although the sequence above is depicted with a linker “GGGGSGGGGSGGGGS” (SEQ ID NO: 3) a person of skill in the art recognizes that numerous other flexible linkers known in the art can be used, including those enriched in small or hydrophilic amino acids. In some embodiments, the linker between the VH and VL domains comprises a glycine-serine linker. In some embodiments, any combination of glycine and serine residues can be used. In some embodiments, 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.

[0130] In some embodiments, 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). In some embodiments, 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.DVQLVESGGGLVQPGGSRKLSCAASGFTFSSFGMHWVRQAPEKGLEWVAYINSGSTT FYYADTVKGRFTISRDNPKNTLFLQMTSLGSEDTAMYYCARSATATWTMDYWGQGT SVTVSSGGGGSGGGGSGGGGSNIMMTOSPSSLAVSAGEKVTMSCKSSOSVFVSSNOKN YLAWYQQKPGQSPKLLIYWASTRESAVPDRFTGSGSGTDFTLTIGSVQAEDLAVYYCH Q E ^ YGGGY EPX LEPKSCDKTHTCPPCPAPEFQGGPSVFLFPPKPKDTLYITREPE VTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEY KCQVSNKALPAPIEKTISKAKGQPREPQVYVYPPSRDELTKNQVSLTCLVKGFYPSDIAVEWE SNGQPENNYKTTPPVLDSDGSFALVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PG (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). In some embodiments, 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 IDNO: 33. In some embodiments, the signal peptide is cleaved during post-translational modifications that occur in vitro or in vivo.

[0131] In some embodiments, 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.

[0132] In some embodiments, 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. In some embodiments, the anti-CD45 portion (scFv-Fc) of the anti-CD45xPD-l bispecific antibody (without the signal peptide) 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. In some embodiments, 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). In some embodiments, 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.

[0133] Although the sequence above is depicted with a linker “GGGGSGGGGSGGGGS” (SEQ ID NO: 3) a person of skill in the art recognizes that numerous other flexible linkers known in the art can be used, including those enriched in small or hydrophilic amino acids. In some embodiments, the linker between the VH and VL domains comprises a glycine-serine linker. In some embodiments, any combination of glycine and serine residues can be used. In some embodiments, 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.

[0134] In some embodiments, 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). In some embodiments, 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.EVQLQQSGAELVKPGASVKLSCTASGFNIKDTFMHWVKLRPEQGLEWIGRIDPANGY TKYDPRFOGKATIIADTSSNTAYLOLSSLTSEDTAVYYCASGEYYALDYWGOGTSVTV SSGGGGSGGGGSGGGGSDIVLTOSPASLTVSLGORATISCRASKSVSTSGYSYMHWYQ QKPGOPPKLLIYLASNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCOHNRELPY JYGGGYYYLPXKLEPKSCDKTHTCPPCPAPEFQGGPSVFLFPPKPKDTLYITREPEVTCVWD VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCQVSNK ALPAPIEKnSKAKGQPREPQVYVYPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFALVSKLIVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (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). In some embodiments, 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. In some embodiments, the signal peptide is cleaved during post-translational modifications that occur in vitro or in vivo.

[0135] In some embodiments, 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.

[0136] In some embodiments, 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. In some embodiments, the anti-CD45 portion (scFv-Fc) of the anti-CD45xPD-l bispecific antibody (without the signal peptide) 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. In some embodiments, 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). In some embodiments, 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.

[0137] Although the sequence above is depicted with a linker “GGGGSGGGGSGGGGS” (SEQ ID NO: 3) a person of skill in the art recognizes that numerous other flexible linkersknown in the art can be used, including those enriched in small or hydrophilic amino acids. In some embodiments, the linker between the VH and VL domains comprises a glycine-serine linker. In some embodiments, any combination of glycine and serine residues can be used. In some embodiments, 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 of Anti-CD45 Portions of Anti-CD 45xPD-l Bispecific Antibodies

[0138] 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. In some embodiments, instead of the “hole” mutations, 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. In some embodiments, the nucleic acid sequences may be codon optimized.

[0139] In some embodiments, 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.ATGGGCTGGAGCTGCATTATCCTGTTCCTGGTGGCCACAGCCACCGGCGTGCACAG C (SEQ ID NO: 11). In some embodiments, the 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.GAGGTGCAGCTGGTTGAATCTGGCGGAGGACTGGTTCAGCCTGGCGGATCTCTGAG ACTGTCTTGTGCCGCCAGCGGCTTCACCTTCAACAACTACTGGATGACCTGGGTCCG ACAGGCCCCTGGCAAAGGACTTGAATGGGTCGCCAGCATCTCTAGCAGCGGCGGCA GCATCTACTACCCCGATTCTGTGAAGGGCAGATTCACCATCAGCCGGGACAACAGC AAGAACACCCTGTACCTGCAGATGAACAGCCTGAGAGCCGAGGACACCGCCGTGTA CTACTGTGCCAGAGATGAGAGATGGGCTGGCGCCATGGATGCTTGGGGACAGGGAA CAACCGTGACCGTTTCTTCTGGCGGCGGAGGAAGCGGAGGCGGAGGCTCCGGTGGT GGTGGATCTGACATCCAGATGACACAGAGCCCCAGCAGCCTGTCTGCCTCTGTGGG AGACAGAGTGACCATCACATGCAAGGCCAGCCAGAACATCAACAAGAACCTGGAT TGGTATCAGCAGAAGCCCGGCAAGGCCCCTAAGCTGCTGATCTACGAGACAAACAA CCTGCAGACCGGCGTGCCCAGCAGATTTTCTGGCTCTGGCAGCGGCACCGACTTCACCCTGACCATATCTAGCCTGCAGCCTGAGGACTTCGCCACCTACTACTGCTACCAGCA CAACAGCCGGTTCACCTTTGGCGGAGGCACCAAGCTGGAAATCAAGCGGCTCGAGC CCAAGAGCTGCGACAAGACCCACACCTGTCCTCCATGTCCTGCTCCAGAGTTTCAAG GCGGCCCTTCCGTGTTCCTGTTTCCTCCAAAGCCTAAGGACACCCTGTACATCACCC GCGAGCCTGAAGTGACCTGTGTGGTGGTGGATGTGTCCCACGAGGACCCCGAAGTG AAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAG AGAGGAACAGTACAACAGCACCTACAGAGTGGTGTCCGTGCTGACCGTGCTGCACC AGGATTGGCTGAACGGCAAAGAGTACAAGTGCCAGGTGTCCAACAAGGCCCTGCCT GCTCCTATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCTAGGGAACCTCAAGT GTACGTGTACCCTCCTAGCCGGGACGAGCTGACCAAGAATCAGGTGTCCCTGACCT GCCTCGTGAAGGGCTTCTACCCTTCCGACATCGCCGTGGAATGGGAGAGCAATGGC CAGCCTGAGAACAACTACAAGACAACCCCTCCTGTGCTGGACAGCGACGGCTCTTT TGCCCTGGTGTCCAAGCTGACAGTGGACAAGTCCAGATGGCAGCAGGGCAACGTGT TCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGAGCCTGTCTCCTGGATGA (SEQ ID NO: 12). In some embodiments, 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: 12. In some embodiments, 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.

[0140] In some embodiments, 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. In some embodiments, the nucleic acid sequence encoding a 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: 12. In some embodiments, 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). In some embodiments, 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-lbispecific 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.

[0141] Although the sequence above is depicted with a nucleic acid sequence encoding a linker “GGAGGCGGAGGTTCTGGCGGCGGAGGAAGTGGTGGCGGAGGCTCT” (SEQ ID NO: 13) a person of skill in the art recognizes that numerous other flexible linkers known in the art can be used, including those enriched in small or hydrophilic amino acids. In some embodiments, the linker between the VH and VL domains comprises a glycine-serine linker. In some embodiments, any combination of glycine and serine residues can be used. In some embodiments, the glycine-serine linker comprises a nucleic acid sequence encoding a linker that is between about 2 and 25 amino acids in length. In some embodiments, the nucleic acid sequence encodes a glycine-serine linker that is about 15 amino acids in length.

[0142] In some embodiments, 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. In some embodiments, 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.

[0143] In some embodiments, 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.ATGGGCTGGAGCTGCATTATCCTGTTCCTGGTGGCCACAGCCACCGGCGTGCACAG CG (SEQ ID NO: 11). In some embodiments, the nucleic acid sequence of the anti-CD45 portion (scFv-Fc) of an anti-CD45xPD-l bispecific antibody (without the signal peptide) comprises SEQ ID NO: 14.AGGTGCAGCTGGTTGAATCTGGCGGAGGACTGGTTCAGCCTGGCGGATCTCTGAGA CTGTCTTGTGCCGCCAGCGGCTTCACCTTCAACAACTACTGGATGACCTGGGTCCGA CAGGCCCCTGGCAAAGGACTTGAATGGGTCGCCAGCATCTCTAGCAGCGGCGGCAG CATC[[TAC]]TACCCCGATTCTGTGAAGGGCAGATTCACCATCAGCCGGGACAACAG CAAGAACACCCTGTACCTGCAGATGAACAGCCTGAGAGCCGAGGACACCGCCGTGT ACTACTGTGCCAGAGATGAGAGAGCCGCTGGCGCCATGGATGCTTGGGGACAGGGAACAACCGTGACCGTTTCTTCTGGCGGCGGAGGAAGCGGAGGCGGAGGCTCCGGTGG TGGTGGATCTGACATCCAGATGACACAGAGCCCCAGCAGCCTGTCTGCCTCTGTGG GAGACAGAGTGACCATCACATGCAAGGCCAGCCAGAACATCAACAAGAACCTGGA TTGGTATCAGCAGAAGCCCGGCAAGGCCCCTAAGCTGCTGATCTACGAGACAAACA ACCTGCAGACCGGCGTGCCCAGCAGATTTTCTGGCTCTGGCAGCGGCACCGACTTC ACCCTGACCATATCTAGCCTGCAGCCTGAGGACTTCGCCACCTACTACTGCTACCAG CACAACAGCCGGTTCACCTTTGGCGGAGGCACCAAGCTGGAAATCAAGCGGCTCGA GCCCAAGAGCTGCGACAAGACCCACACCTGTCCTCCATGTCCTGCTCCAGAGTTTCA AGGCGGCCCTTCCGTGTTCCTGTTTCCTCCAAAGCCTAAGGACACCCTGTACATCAC CCGCGAGCCTGAAGTGACCTGTGTGGTGGTGGATGTGTCCCACGAGGACCCCGAAG TGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCT AGAGAGGAACAGTACAACAGCACCTACAGAGTGGTGTCCGTGCTGACCGTGCTGCA CCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCCAGGTGTCCAACAAGGCCCTGC CTGCTCCTATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCTAGGGAACCTCAA GTGTACGTGTACCCTCCTAGCCGGGACGAGCTGACCAAGAATCAGGTGTCCCTGAC CTGCCTCGTGAAGGGCTTCTACCCTTCCGACATCGCCGTGGAATGGGAGAGCAATG GCCAGCCTGAGAACAACTACAAGACAACCCCTCCTGTGCTGGACAGCGACGGCTCT TTTGCCCTGGTGTCCAAGCTGACAGTGGACAAGTCCAGATGGCAGCAGGGCAACGT GTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCC TGAGCCTGTCTCCTGGATGA (SEQ ID NO: 14). In some embodiments, 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. In some embodiments, 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.

[0144] In some embodiments, 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. In some embodiments, the nucleic acid sequence encoding an anti- CD45 portion (scFv-Fc) of the anti-CD45xPD-l bispecific antibody (without the signal peptide) 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. In some embodiments, the nucleic acidsequence 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). In some embodiments, 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.

[0145] Although the sequence above is depicted with a nucleic acid sequence encoding a linker “GGAGGCGGAGGTTCTGGCGGCGGAGGAAGTGGTGGCGGAGGCTCT” (SEQ ID NO: 13) a person of skill in the art recognizes that numerous other flexible linkers known in the art can be used, including those enriched in small or hydrophilic amino acids. In some embodiments, the linker between the VH and VL domains comprises a glycine-serine linker. In some embodiments, any combination of glycine and serine residues can be used. In some embodiments, the glycine-serine linker comprises a nucleic acid sequence encoding a linker that is between about 2 and 25 amino acids in length. In some embodiments, the nucleic acid sequence encodes a glycine-serine linker that is about 15 amino acids in length. In some embodiments, 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.ATGGGCTGGAGCTGCATTATCCTGTTCCTGGTGGCCACAGCCACCGGCGTGCACAG CG (SEQ ID NO: 11). In some embodiments, the 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.ATGGGCTGGAGCTGCATTATCCTGTTCCTGGTGGCCACAGCCACCGGCGTGCACAG CGAGGTGCAGCTGGTTGAATCTGGCGGAGGACTGGTTCAGCCTGGCGGATCTCTGA GACTGTCTTGTGCCGCCAGCGGCTTCACCTTCAACAACTACGCCATGACCTGGGTCC GACAGGCCCCTGGCAAAGGACTTGAATGGGTCGCCAGCATCTCTAGCAGCGGCGGC AGCATCTACTACCCCGATTCTGTGAAGGGCAGATTCACCATCAGCCGGGACAACAG CAAGAACACCCTGTACCTGCAGATGAACAGCCTGAGAGCCGAGGACACCGCCGTGT ACTACTGTGCCAGAGATGAGAGATGGGCTGGCGCCATGGATGCTTGGGGACAGGGA ACAACCGTGACCGTTTCTTCTGGCGGCGGAGGAAGCGGAGGCGGAGGCTCCGGTGG TGGTGGATCTGACATCCAGATGACACAGAGCCCCAGCAGCCTGTCTGCCTCTGTGG GAGACAGAGTGACCATCACATGCAAGGCCAGCCAGAACATCAACAAGAACCTGGATTGGTATCAGCAGAAGCCCGGCAAGGCCCCTAAGCTGCTGATCTACGAGACAAACA ACCTGCAGACCGGCGTGCCCAGCAGATTTTCTGGCTCTGGCAGCGGCACCGACTTC ACCCTGACCATATCTAGCCTGCAGCCTGAGGACTTCGCCACCTACTACTGCTACCAG CACAACAGCCGGTTCACCTTTGGCGGAGGCACCAAGCTGGAAATCAAGCGGCTCGA GCCCAAGAGCTGCGACAAGACCCACACCTGTCCTCCATGTCCTGCTCCAGAGTTTCA AGGCGGCCCTTCCGTGTTCCTGTTTCCTCCAAAGCCTAAGGACACCCTGTACATCAC CCGCGAGCCTGAAGTGACCTGTGTGGTGGTGGATGTGTCCCACGAGGACCCCGAAG TGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCT AGAGAGGAACAGTACAACAGCACCTACAGAGTGGTGTCCGTGCTGACCGTGCTGCA CCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCCAGGTGTCCAACAAGGCCCTGC CTGCTCCTATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCTAGGGAACCTCAA GTGTACGTGTACCCTCCTAGCCGGGACGAGCTGACCAAGAATCAGGTGTCCCTGAC CTGCCTCGTGAAGGGCTTCTACCCTTCCGACATCGCCGTGGAATGGGAGAGCAATG GCCAGCCTGAGAACAACTACAAGACAACCCCTCCTGTGCTGGACAGCGACGGCTCT TTTGCCCTGGTGTCCAAGCTGACAGTGGACAAGTCCAGATGGCAGCAGGGCAACGT GTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGAGCCTGTCTCCTGGATGA (SEQ ID NO: 16). In some embodiments, 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: 16. In some embodiments, 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.

[0146] In some embodiments, 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. In some embodiments, the nucleci 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: 16. In some embodiments, 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). In someembodiments, 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.

[0147] Although the sequence above is depicted with a nucleic acid sequence encoding a linker “GGAGGCGGAGGTTCTGGCGGCGGAGGAAGTGGTGGCGGAGGCTCT” (SEQ ID NO: 13) a person of skill in the art recognizes that numerous other flexible linkers known in the art can be used, including those enriched in small or hydrophilic amino acids. In some embodiments, the linker between the VH and VL domains comprises a glycine-serine linker. In some embodiments, any combination of glycine and serine residues can be used. In some embodiments, the glycine-serine linker comprises a nucleic acid sequence encoding a linker that is between about 2 and 25 amino acids in length. In some embodiments, the nucleic acid sequence encodes a glycine-serine linker that is about 15 amino acids in length.

[0148] In some embodiments, 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.ATGGGCTGGAGCTGCATTATCCTGTTCCTGGTGGCCACAGCCACCGGCGTGCACAG CG (SEQ ID NO: 11) In some embodiments, the 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.ATGGGCTGGAGCTGCATTATCCTGTTCCTGGTGGCCACAGCCACCGGCGTGCACAG CGAGGTGCAGCTGGTTGAATCTGGCGGAGGACTGGTTCAGCCTGGCGGATCTCTGA GACTGTCTTGTGCCGCCAGCGGCTTCACCTTCAACAACTACTGGATGACCTGGGTCC GACAGGCCCCTGGCAAAGGACTTGAATGGGTCGCCAGCATCTCTAGCAGCGGCGGC AGCATCTACTACCCCGATTCTGTGAAGGGCAGATTCACCATCAGCCGGGACAACAG CAAGAACACCCTGTACCTGCAGATGAACAGCCTGAGAGCCGAGGACACCGCCGTGT ACTACTGTGCCAGAGATGAGAGATGGGCTGGCGCCATGGATGCTTGGGGACAGGGA ACAACCGTGACCGTTTCTTCTGGCGGCGGAGGAAGCGGAGGCGGAGGCTCCGGTGG TGGTGGATCTGACATCCAGATGACACAGAGCCCCAGCAGCCTGTCTGCCTCTGTGG GAGACAGAGTGACCATCACATGCAAGGCCAGCCAGAACATCAACAAGAACCTGGA TTGGTATCAGCAGAAGCCCGGCAAGGCCCCTAAGCTGCTGATCTACGAGACAAACA ACCTGCAGACCGGCGTGCCCAGCAGATTTTCTGGCTCTGGCAGCGGCACCGACTTC ACCCTGACCATATCTAGCCTGCAGCCTGAGGACTTCGCCACCTACTACTGCTACCAGCACAACAGCCGGTTCACCTTTGGCGGAGGCACCAAGCTGGAAATCAAGCGGCTCGA GCCCAAGAGCTGCGACAAGACCCACACCTGTCCTCCATGTCCTGCTCCAGAGTTTCA AGGCGGCCCTTCCGTGTTCCTGTTTCCTCCAAAGCCTAAGGACACCCTGTACATCAC CCGCGAGCCTGAAGTGACCTGTGTGGTGGTGGATGTGTCCCACGAGGACCCCGAAG TGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCT AGAGAGGAACAGTACAACAGCACCTACAGAGTGGTGTCCGTGCTGACCGTGCTGCA CCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCCAGGTGTCCAACAAGGCCCTGC CTGCTCCTATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCTAGGGAACCTCAA GTGTACGTGTACCCTCCTAGCCGGGACGAGCTGACCAAGAATCAGGTGTCCCTGAC CTGCCTCGTGAAGGGCTTCTACCCTTCCGACATCGCCGTGGAATGGGAGAGCAATG GCCAGCCTGAGAACAACTACAAGACAACCCCTCCTGTGCTGGACAGCGACGGCTCT TTTGCCCTGGTGTCCAAGCTGACAGTGGACAAGTCCAGATGGCAGCAGGGCAACGT GTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGAGCCTGTCTCCTGGATGA (SEQ ID NO: 18). In some embodiments, 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: 18. In some embodiments, 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.

[0149] In some embodiments, 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. In some embodiments, 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: 18. In some embodiments, 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). In some embodiments, 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 antibodycomprise 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.

[0150] Although the sequence above is depicted with a nucleic acid sequence encoding a linker “GGAGGCGGAGGTTCTGGCGGCGGAGGAAGTGGTGGCGGAGGCTCT” (SEQ ID NO: 13) a person of skill in the art recognizes that numerous other flexible linkers known in the art can be used, including those enriched in small or hydrophilic amino acids. In some embodiments, the linker between the VH and VL domains comprises a glycine-serine linker. In some embodiments, any combination of glycine and serine residues can be used. In some embodiments, the glycine-serine linker comprises a nucleic acid sequence encoding a linker that is between about 2 and 25 amino acids in length. In some embodiments, the nucleic acid sequence encodes a glycine-serine linker that is about 15 amino acids in length.

[0151] In some embodiments, 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.ATGGGCTGGAGCTGCATTATCCTGTTCCTGGTGGCCACAGCCACCGGCGTGCACAG CG (SEQ ID NO: 11). In some embodiments, the 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.ATGGGCTGGAGCTGCATTATCCTGTTCCTGGTGGCCACAGCCACCGGCGTGCACAG CGAGGTGCAGCTGGTTGAATCTGGCGGAGGACTGGTTCAGCCTGGCGGATCTCTGA GACTGTCTTGTGCCGCCAGCGGCTTCACCTTCAACAACTACTGGATGACCTGGGTCC GACAGGCCCCTGGCAAAGGACTTGAATGGGTCGCCAGCATCTCTAGCAGCGGCGGC AGCATCTACTACCCCGATTCTGCGAAGGGCAGATTCACCATCAGCCGGGACAACAG CAAGAACACCCTGTACCTGCAGATGAACAGCCTGAGAGCCGAGGACACCGCCGTGT ACTACTGTGCCAGAGATGAGAGATGGGCTGGCGCCATGGATGCTTGGGGACAGGGA ACAACCGTGACCGTTTCTTCTGGCGGCGGAGGAAGCGGAGGCGGAGGCTCCGGTGG TGGTGGATCTGACATCCAGATGACACAGAGCCCCAGCAGCCTGTCTGCCTCTGTGG GAGACAGAGTGACCATCACATGCAAGGCCAGCCAGAACATCAACAAGAACCTGGA TTGGTATCAGCAGAAGCCCGGCAAGGCCCCTAAGCTGCTGATCTACGAGACAAACA ACCTGCAGACCGGCGTGCCCAGCAGATTTTCTGGCTCTGGCAGCGGCACCGACTTC ACCCTGACCATATCTAGCCTGCAGCCTGAGGACTTCGCCACCTACTACTGCTACCAG CACAACAGCCGGTTCACCTTTGGCGGAGGCACCAAGCTGGAAATCAAGCGGCTCGA GCCCAAGAGCTGCGACAAGACCCACACCTGTCCTCCATGTCCTGCTCCAGAGTTTCAAGGCGGCCCTTCCGTGTTCCTGTTTCCTCCAAAGCCTAAGGACACCCTGTACATCAC CCGCGAGCCTGAAGTGACCTGTGTGGTGGTGGATGTGTCCCACGAGGACCCCGAAG TGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCT AGAGAGGAACAGTACAACAGCACCTACAGAGTGGTGTCCGTGCTGACCGTGCTGCA CCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCCAGGTGTCCAACAAGGCCCTGC CTGCTCCTATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCTAGGGAACCTCAA GTGTACGTGTACCCTCCTAGCCGGGACGAGCTGACCAAGAATCAGGTGTCCCTGAC CTGCCTCGTGAAGGGCTTCTACCCTTCCGACATCGCCGTGGAATGGGAGAGCAATG GCCAGCCTGAGAACAACTACAAGACAACCCCTCCTGTGCTGGACAGCGACGGCTCT TTTGCCCTGGTGTCCAAGCTGACAGTGGACAAGTCCAGATGGCAGCAGGGCAACGT GTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCC TGAGCCTGTCTCCTGGATGA (SEQ ID NO: 20). In some embodiments, 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. In some embodiments, 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.

[0152] In some embodiments, 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. In some embodiments, 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. In some embodiments, 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). In some embodiments, 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.

[0153] Although the sequence above is depicted with a nucleic acid sequence encoding a linker “GGAGGCGGAGGTTCTGGCGGCGGAGGAAGTGGTGGCGGAGGCTCT” (SEQ ID NO: 13) a person of skill in the art recognizes that numerous other flexible linkers known in the art can be used, including those enriched in small or hydrophilic amino acids. In some embodiments, the linker between the VH and VL domains comprises a glycine-serine linker. In some embodiments, any combination of glycine and serine residues can be used. In some embodiments, the glycine-serine linker comprises a nucleic acid sequence encoding a linker that is between about 2 and 25 amino acids in length. In some embodiments, the nucleic acid sequence encodes a glycine-serine linker that is about 15 amino acids in length.Amino Acid Sequences of Anti-CD43 Portion of Anti-CD43xPD-l Bispecific Antibodies

[0154] In some embodiments, 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. 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. In some embodiments, instead of the “hole” mutations, 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.

[0155] In some embodiments, 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.EVQLVESGGGLVQPGGSLRLSCVASGFTFSSFGMHWVRQAPGKGLEWVAYISSGSGN FYYVDTVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARSTYYHGSRGAMDYWG OGTLVTVSSGGGGSGGGGSGGGGSDVVMTOSPAFLSVTPGEKVTITCSASSSVSSMYW YQQKPDQAPKLLIYDTSKMASGVPSRF SGSGSGTDFTFTIS SLEAED AAT YYCQQWSSY YYVYYGGGY AS KSLEPKSCDKTHTCPPCPAPEFQGGPSVFLFPPKPKDTLYITREPEVTCV WDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCQVSNKALPAPIEKTISKAKGQPREPQVYVYPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFALVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 22). In some embodiments, 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. In some embodiments, the signal peptide is cleaved during post- translational modifications that occur in vitro or in vivo.

[0156] In some embodiments, 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. In some embodiments, the anti-CD43 portion (scFv-Fc) of the anti-CD43xPD-l bispecific antibody (without the signal peptide) 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. In some embodiments, 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). In some embodiments, 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.

[0157] Although the sequence above is depicted with a linker “GGGGSGGGGSGGGGS” (SEQ ID NO: 3) a person of skill in the art recognizes that numerous other flexible linkers known in the art can be used, including those enriched in small or hydrophilic amino acids. In some embodiments, the linker between the VH and VL domains comprises a glycine-serine linker. In some embodiments, any combination of glycine and serine residues can be used. In some embodiments, 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.

[0158] In some embodiments, 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. In some embodiments, the one or more amino acids of an amino acid sequence encoding one ormore 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 of Anti-CD43 Portion of Anti-CD43xPD-l Bispecific Antibodies

[0159] 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. In some embodiments, instead of the “hole” mutations, 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. In some embodiments, the nucleic acid sequences may be codon optimized.

[0160] In some embodiments, the nucleic acid sequence of the anti-CD43 portion (scFv-Fc) of the anti-CD43xPD-l bispecific antibody comprises a signal peptide comprising SEQ ID NO: 19.ATGGGCTGGAGCTGCATTATCCTGTTCCTGGTGGCCACAGCCACCGGCGTGCACAG CG (SEQ ID NO: 11). In some embodiments, the nucleic acid sequence of the anti-CD43 portion (scFv-Fc) of an anti-CD43xPD-l bispecific antibody (without the signal peptide) comprises SEQ ID NO: 24.AGGTGCAGCTGGTTGAATCTGGCGGAGGACTGGTTCAGCCTGGCGGATCTCTGAGA CTGAGCTGTGTGGCCAGCGGCTTCACCTTCAGCAGCTTTGGCATGCACTGGGTCCGA CAGGCCCCTGGCAAAGGACTTGAGTGGGTCGCCTACATCAGCAGCGGCAGCGGCAA CTTCTACTACGTGGACACCGTGAAGGGCAGATTCACCATCTCCAGAGACAACGCCA AGAACAGCCTGTACCTGCAGATGAACTCCCTGAGAGCCGAGGACACCGCCGTGTAC TACTGTGCCAGAAGCACCTACTACCACGGCAGCAGAGGCGCCATGGATTATTGGGG ACAGGGCACCCTGGTCACCGTTTCTAGCGGAGGCGGAGGTTCTGGCGGCGGAGGAA GTGGTGGCGGAGGCTCTGATGTGGTCATGACTCAGAGCCCTGCCTTCCTGTCTGTGA CCCCTGGCGAGAAAGTGACCATCACCTGTAGCGCCAGCAGCAGCGTGTCCAGCATG TACTGGTATCAGCAGAAGCCCGATCAGGCTCCCAAACTGCTGATCTACGACACCAG CAAGATGGCCTCTGGCGTGCCCAGCAGATTTTCTGGCTCTGGCAGCGGAACCGACTT CACCTTTACCATCAGCTCCCTGGAAGCCGAAGATGCCGCCACCTACTATTGCCAGCA GTGGTCTAGCTACCCTCCTATCACCTTTGGAGGCGGCACCAAGCTGGAAATCAAGCGGCTCGAGCCCAAGAGCTGCGACAAGACCCACACCTGTCCTCCATGTCCTGCTCCA GAGTTTCAAGGCGGCCCTTCCGTGTTCCTGTTTCCTCCAAAGCCTAAGGACACCCTG TACATCACCCGCGAGCCTGAAGTGACCTGTGTGGTGGTGGATGTGTCCCACGAGGA CCCCGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGA CCAAGCCTAGAGAGGAACAGTACAACAGCACCTACAGAGTGGTGTCCGTGCTGACC GTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCCAGGTGTCCAACAA GGCCCTGCCTGCTCCTATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCTAGGG AACCTCAAGTGTACGTGTACCCTCCTAGCCGGGACGAGCTGACCAAGAATCAGGTG TCCCTGACCTGCCTCGTGAAGGGCTTCTACCCTTCCGACATCGCCGTGGAATGGGAG AGCAATGGCCAGCCTGAGAACAACTACAAGACAACCCCTCCTGTGCTGGACAGCGA CGGCTCTTTTGCCCTGGTGTCCAAGCTGACAGTGGACAAGTCCAGATGGCAGCAGG GCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGAGCCTGTCTCCTGGATGA (SEQ ID NO: 24). In some embodiments, the nucleic acid sequence encoding the anti-CD43 portion (scFv-Fc) of an anti-CD43xPD-l bispecific antibody comprises SEQ ID NO: 11 immediately followed by SEQ ID NO: 24. In some embodiments, 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:24 can be determined from the amino acid sequence of SEQ ID NO: 22 as identified herein by a person of skill in the art.

[0161] In some embodiments, 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. In some embodiments, the nucleic acid sequence encoding an anti- CD43 portion (scFv-Fc) of the anti-CD43xPD-l bispecific antibody (without the signal peptide) 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. In some embodiments, 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). In some embodiments, 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-lbispecific 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.

[0162] Although the sequence above is depicted with a nucleic acid sequence encoding a linker “GGAGGCGGAGGTTCTGGCGGCGGAGGAAGTGGTGGCGGAGGCTCT” (SEQ ID NO: 13) a person of skill in the art recognizes that numerous other flexible linkers known in the art can be used, including those enriched in small or hydrophilic amino acids. In some embodiments, the linker between the VH and VL domains comprises a glycine-serine linker. In some embodiments, any combination of glycine and serine residues can be used. In some embodiments, the glycine-serine linker comprises a nucleic acid sequence encoding a linker that is between about 2 and 25 amino acids in length. In some embodiments, the nucleic acid sequence encodes a glycine-serine linker that is about 15 amino acids in length.

[0163] In some embodiments, 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. In some embodiments, 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.Amino Acid Sequences of Anti-PD-1 Portions of Anti-CD45xPD-l or Anti-CD43xPD-l Bispecific Antibodies

[0164] In some embodiments, 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. 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. In some embodiments, instead of the “knob” mutations, the amino acid sequence comprising the anti-PD-1 portion of an anti-CD45xPD-l bispecific antibody cancomprise 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.

[0165] In some embodiments, 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). In some embodiments, 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.OVOLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVROAPGOGLEWMGGINPSN GGTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWG OGTLVTVSSGGGGSGGGGSGGGGSDIVLTOSPATLSLSPGERATLSCRASKGVSTSGYS YLHWYOQKPGOAPRLLIYLASYLESGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCOH S^EPEYYGGGYKNP KSLEPKSCDKTHTCPPCPAPEFOGGPSVFLFPPKPKDTLYITREPE VTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEY KCQVSNKALPAPIEKTISKAKGQPREPQVYVLPPSRDELTKNQVSLLCLVKGFYPSDIAVEWE SNGQPENNYLTWPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPG (SEQ ID NO: 26). In some embodiments, 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. In some embodiments, the signal peptide is cleaved during post-translational modifications that occur in vitro or in vivo.

[0166] In some embodiments, 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. In some embodiments, the anti-PD-1 portion (scFv-Fc) of the anti-CD45xPD-l or the anti-CD43xPD-l bispecific antibody (without the signal peptide) 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. In some embodiments, 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). In some embodiments, the framework regions (FRs) of the variable heavy chain domain and the FRs of the variable light chain domainof 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.

[0167] Although the sequence above is depicted with a linker “GGGGSGGGGSGGGGS” (SEQ ID NO: 3) a person of skill in the art recognizes that numerous other flexible linkers known in the art can be used, including those enriched in small or hydrophilic amino acids. In some embodiments, the linker between the VH and VL domains comprises a glycine-serine linker. In some embodiments, any combination of glycine and serine residues can be used. In some embodiments, 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.

[0168] In some embodiments, 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). In some embodiments, 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.EVKLLESGGGLVQPGGSLKLSCAASGFDFSRYWMSWVRQAPGKGLEWIGEINPDSSTIK YTPSLKDKFIISRDNAKNTLCLQLSKVRSEDSALYYCARMGYRLFDSWGQGTTLTVSS GGGSGGGGSGGGGSNTVMTQ ATPS VP VTPGES VSTSCR SSK ST J HSDGDT YI Y WFEQR.P GQSPQLLIYRMSNLVSGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPYTFG GGYYYEE FiLEPKSCDKTHTCPPCPAPEFQGGPSVFLFPPKPKDTLYITREPEVTCVWDVSH EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCQVSNKALP APIEKTISKAKGQPREPQVYVLPPSRDELTKNQVSLLCLVKGFYPSDIAVEWESNGQPENNYL TWPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 35). In some embodiments, 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. In some embodiments, the signal peptide is cleaved during post-translational modifications that occur in vitro or in vivo.

[0169] In some embodiments, 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. In some embodiments, the anti-PD-1 portion (scFv-Fc) of the anti-CD45xPD-l or the anti-CD43xPD-l bispecific antibody (without the signal peptide) comprises an amino acidsequence 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. In some embodiments, 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). In some embodiments, 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.

[0170] Although the sequence above is depicted with a linker “GGGGSGGGGSGGGGS” (SEQ ID NO: 3) a person of skill in the art recognizes that numerous other flexible linkers known in the art can be used, including those enriched in small or hydrophilic amino acids. In some embodiments, the linker between the VH and VL domains comprises a glycine-serine linker. In some embodiments, any combination of glycine and serine residues can be used. In some embodiments, 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.

[0171] In some embodiments, 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). In some embodiments, 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.EVQLQQSGTVLARPGASVKMSCKASGNTFTSYWMHWVKQRPGQGLEWIGAIYPGYSD TRYNQKFKGKATLTAVTSTSTAYMELSSLTNEDSAVYYCTKFIATVGGYFDVWGAGTT VTVSSGGGGSGGGGSGGGGSOIVLTO SP AIMS S SLGERVTMTCT AS S S VS S S YLHW YQQ KPGSSPKLWIYSTSNLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCHQYHRSPPIFT YG GY EPAKEEPKSCDKTHTCPPCPAPEFQGGPSVFLFPPKPKDTLYITREPEVTCVWDVS HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCQVSNKAL PAPIEKTISKAKGQPREPQVYVLPPSRDELTKNQVSLLCLVKGFYPSDIAVEWESNGQPENNY LTWPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 36). In some embodiments, 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: 1immediately followed by SEQ ID NO: 36. In some embodiments, the signal peptide is cleaved during post-translational modifications that occur in vitro or in vivo.

[0172] In some embodiments, 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. In some embodiments, the anti-PD-1 portion (scFv-Fc) of the anti-CD45xPD-l or the anti-CD43xPD-l bispecific antibody (without the signal peptide) 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. In some embodiments, 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). In some embodiments, 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.

[0173] Although the sequence above is depicted with a linker “GGGGSGGGGSGGGGS” (SEQ ID NO: 3) a person of skill in the art recognizes that numerous other flexible linkers known in the art can be used, including those enriched in small or hydrophilic amino acids. In some embodiments, the linker between the VH and VL domains comprises a glycine-serine linker. In some embodiments, any combination of glycine and serine residues can be used. In some embodiments, 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.

[0174] In some embodiments, 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). In some embodiments, 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.QIQLVQSGPELKKPGETVKISCKASGYTFTDYSMHWVKQAPGKGLKWMGWIKIETGNP TYADDFKGRFAFSLETSASTAYLQINNLKNEDTATYFCARDYYGYYYYAMDYWGQGT S VT VS SGGGGSGGGGSGGGGSOIVLTOSPAIMSASLGERVTMTCTVS S SIS S S YLHW YQQKPGSSPKLWIYSTSNLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCHQYHRSPLT YGAGYYAFEKLEPKSCDKTHTCPPCPAPEFQGGPSVFLFPPKPKDTLYITREPEVTCVWDV SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCQVSNKA LPAPIEKTISKAKGQPREPQVYVLPPSRDELTKNQVSLLCLVKGFYPSDIAVEWESNGQPENN YLTWPPVLDSDGSFFL YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 37) In some embodiments, 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: 37. In some embodiments, the signal peptide is cleaved during post-translational modifications that occur in vitro or in vivo.

[0175] In some embodiments, 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. In some embodiments, the anti-PD-1 portion (scFv-Fc) of the anti-CD45xPD-l or the anti-CD43xPD-l bispecific antibody (without the signal peptide) 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. In some embodiments, 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). In some embodiments, 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.

[0176] Although the sequence above is depicted with a linker “GGGGSGGGGSGGGGS” (SEQ ID NO: 3) a person of skill in the art recognizes that numerous other flexible linkers known in the art can be used, including those enriched in small or hydrophilic amino acids. In some embodiments, the linker between the VH and VL domains comprises a glycine-serine linker. In some embodiments, any combination of glycine and serine residues can be used. In some embodiments, 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.

[0177] In some embodiments, 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 peptidecomprising SEQ ID NO: 1. MGWSCIILFLVATATGVHS (SEQ ID NO: 1). In some embodiments, 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.EVQLVESGGGLVQPGGSLRLSCAASGFTFSHYGMSWVRQTPDKRLELVATIDNNGGNT YYPDSVKGRFTISRDNAKNTLYLQMSSLKSEDTAMYYCARDAYYTYDYWYFDVWGA GTTVTVSSGGGGSGGGGSGGGGSDIOMTOTTSSLSASLGDRVTISCSASOGISNYLNWY QQKPDGTVKLLIYYTSSLHSGVPSRFSGSGSGTDYSLTISNLEPEDIATYYCQQYHKLPW YYGGGYKEPXKLEPKSCDKTHTCPPCPAPEFQGGPSVFLFPPKPKDTLYITREPEVTCVWD VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCQVSNK ALPAPIEKTISKAKGQPREPQVYVLPPSRDELTKNQVSLLCLVKGFYPSDIAVEWESNGQPEN NYLTWPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 38) In some embodiments, 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: 38. In some embodiments, the signal peptide is cleaved during post-translational modifications that occur in vitro or in vivo.

[0178] In some embodiments, 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. In some embodiments, the anti-PD-1 portion (scFv-Fc) of the anti-CD45xPD-l or the anti-CD43xPD-l bispecific antibody (without the signal peptide) 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. In some embodiments, 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). In some embodiments, 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.

[0179] Although the sequence above is depicted with a linker “GGGGSGGGGSGGGGS” (SEQ ID NO: 3) a person of skill in the art recognizes that numerous other flexible linkersknown in the art can be used, including those enriched in small or hydrophilic amino acids. In some embodiments, the linker between the VH and VL domains comprises a glycine-serine linker. In some embodiments, any combination of glycine and serine residues can be used. In some embodiments, 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.

[0180] In some embodiments, 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). In some embodiments, 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.EVKLVESGGGLVQPGGSLKLSCAASGFTFSSYTMSWVRQTPEKRLEWVAYISYGGGDT YYPDTVKGRFTISRDNAKNTLYLQMSSLKSEDTAMYYCARQKVDGYFVAMDYWSQG TSVTVSSGGGGSGGGGSGGGGSVIVLTOSPASLAVSLGORATISCRASERVDDYGISFM NWFQQKPGQPPKLLIYAASNQGSGVPARF SGSGSGTDF SLNIHPMEEDDTAMYFCQQN YYEVPN YYGGGYYYEE KLEPKSCDKTHTCPPCPAPEFQGGPSVFLFPPKPKDTLYITREPEVT CVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKC QVSNKALPAPIEKTISKAKGQPREPQVYVLPPSRDELTKNQVSLLCLVKGFYPSDIAVEWESN GQPENNYLTWPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP G (SEQ ID NO: 39). In some embodiments, 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. In some embodiments, the signal peptide is cleaved during post-translational modifications that occur in vitro or in vivo.

[0181] In some embodiments, 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. In some embodiments, the anti-PD-1 portion (scFv-Fc) of the anti-CD45xPD-l or the anti-CD43xPD-l bispecific antibody (without the signal peptide) 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. In some embodiments, 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 CDRsequences indicated above (in bold and underline). In some embodiments, 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.

[0182] Although the sequence above is depicted with a linker “GGGGSGGGGSGGGGS” (SEQ ID NO: 3) a person of skill in the art recognizes that numerous other flexible linkers known in the art can be used, including those enriched in small or hydrophilic amino acids. In some embodiments, the linker between the VH and VL domains comprises a glycine-serine linker. In some embodiments, any combination of glycine and serine residues can be used. In some embodiments, 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.

[0183] In some embodiments, 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). In some embodiments, 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.EVKLVESGGGLVKPGGSLKLSCAASGFTFSSYGMSWVRQTPEKRLEWVATISGGGRYT YYPDSVKGRFTISRDNAKNILYLQMSSLRSEDTALYYCASPYDGYYGAMDYWGQGTS VTVSSGGGGSGGGGSGGGGSDIVLTOSPASLAVSLGORATISCRASESVDNSGISFMNW FQQKPGQPPKLLIYAASNQGSGVPARFSGSGSGTDFSLNIHPMEEDDTAMYFCQQSKEV Y^YYGGGY EPX LEPKSCDKTHTCPPCPAPEFQGGPSVFLFPPKPKDTLYITREPEVTCW VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCQVS NKALPAPIEKTISKAKGQPREPQVYVLPPSRDELTKNQVSLLCLVKGFYPSDIAVEWESNGQP ENNYLTWPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 40). In some embodiments, 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. In some embodiments, the signal peptide is cleaved during post-translational modifications that occur in vitro or in vivo.

[0184] In some embodiments, 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 SEQID NO: 1. In some embodiments, the anti-PD-1 portion (scFv-Fc) of the anti-CD45xPD-l or the anti-CD43xPD-l bispecific antibody (without the signal peptide) 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. In some embodiments, 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). In some embodiments, 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.

[0185] Although the sequence above is depicted with a linker “GGGGSGGGGSGGGGS” (SEQ ID NO: 3) a person of skill in the art recognizes that numerous other flexible linkers known in the art can be used, including those enriched in small or hydrophilic amino acids. In some embodiments, the linker between the VH and VL domains comprises a glycine-serine linker. In some embodiments, any combination of glycine and serine residues can be used. In some embodiments, 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.

[0186] In some embodiments, 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). In some embodiments, 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.EVQLQQSGTVLARPGASVRMSCKASGYSFSTYWMHWVKQRPGQGLEWIGGIYPGNSD TNYNQKFKGKAKLTAVTSASTAYMDLSSLTNEDSAVYYCTGGYFFDVWGAGTTVTVS SGGGGSGGGGSGGGGSDIKMTOSPSSLYASLGERVTITCKASODINRYLTWFOQKPGKS PKTLIFRANRLVTGVPSRFIGSGSGQEYSLTISSLEYEDMGIYFCLQYDESPYTFGGGTKL PAKEEPKSCDKTHTCPPCPAPEFQGGPSVFLFPPKPKDTLYITREPEVTCVWDVSHEDPEVK FNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCQVSNKALPAPIEKTI SKAKGQPREPQVYVLPPSRDELTKNQVSLLCLVKGFYPSDIAVEWESNGQPENNYLTWPPVL DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 41).In some embodiments, 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. In some embodiments, the signal peptide is cleaved during post- translational modifications that occur in vitro or in vivo.

[0187] In some embodiments, 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. In some embodiments, the anti-PD-1 portion (scFv-Fc) of the anti-CD45xPD-l or the anti-CD43xPD-l bispecific antibody (without the signal peptide) 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. In some embodiments, 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). In some embodiments, 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.

[0188] Although the sequence above is depicted with a linker “GGGGSGGGGSGGGGS” (SEQ ID NO: 3) a person of skill in the art recognizes that numerous other flexible linkers known in the art can be used, including those enriched in small or hydrophilic amino acids. In some embodiments, the linker between the VH and VL domains comprises a glycine-serine linker. In some embodiments, any combination of glycine and serine residues can be used. In some embodiments, 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.

[0189] In some embodiments, 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). In some embodiments, 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.QIQLQQSGPEQVKPGASVKISCKASGYMFIDYYMNWVKQKPGQGLEWIGWIYPGSGSTKDNENFKGKATLTVDTS S STAYMQLS SLTSEDTAVYFC ARYGPRFFD VWGAGTT VTVS SGGGGSGGGGSGGGGSDIOMTOSPSSLSASLGERVNLTCRASOEISGYLSWLOQKPDGT IKRLIYVASTLDSGVPERFSGSRSGSDYSLTISSLESEDFADYYCLQYASYPYTFGGGTKL PAKEEPKSCDKTHTCPPCPAPEFQGGPSVFLFPPKPKDTLYITREPEVTCVWDVSHEDPEVK FNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCQVSNKALPAPIEKTI SKAKGQPREPQVYVLPPSRDELTKNQVSLLCLVKGFYPSDIAVEWESNGQPENNYLTWPPVL DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 42). In some embodiments, 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. In some embodiments, the signal peptide is cleaved during post- translational modifications that occur in vitro or in vivo.

[0190] In some embodiments, 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. In some embodiments, the anti-PD-1 portion (scFv-Fc) of the anti-CD45xPD-l or the anti-CD43xPD-l bispecific antibody (without the signal peptide) 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. In some embodiments, 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). In some embodiments, 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.

[0191] Although the sequence above is depicted with a linker “GGGGSGGGGSGGGGS” (SEQ ID NO: 3) a person of skill in the art recognizes that numerous other flexible linkers known in the art can be used, including those enriched in small or hydrophilic amino acids. In some embodiments, the linker between the VH and VL domains comprises a glycine-serine linker. In some embodiments, any combination of glycine and serine residues can be used. In some embodiments, 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.

[0192] In some embodiments, 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). In some embodiments, 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.EVKLLESGGGLVQPGGSLKLSCEVSGFDFSRDWMSWVRQAPGKGLEWIGQISPDSTSIN YKPSLKDKFIISRDNAKNTLYLQLSGVRSEDTALYHCARDTSGYPDYWGQGTSLTVSS GGGSGGGGSGGGGSDILLTOSPSSMSVSLGDTVSITCHASODISRNIGWLROKPGKSFKG LIYHGTNLEDGVPSRF SGSGSGAD YSLTIS SLESEDF AD YYC VQ YAQFPYTFGGGTKLEI KLEPKSCDKTHTCPPCPAPEFQGGPSVFLFPPKPKDTLYITREPEVTCVWDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCQVSNKALPAPIEKTIS KAKGQPREPQVYVLPPSRDELTKNQVSLLCLVKGFYPSDIAVEWESNGQPENNYLTWPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 43). In some embodiments, 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. In some embodiments, the signal peptide is cleaved during post- translational modifications that occur in vitro or in vivo.

[0193] In some embodiments, 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. In some embodiments, the anti-PD-1 portion (scFv-Fc) of the anti-CD45xPD-l or the anti-CD43xPD-l bispecific antibody (without the signal peptide) 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. In some embodiments, 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). In some embodiments, 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.

[0194] Although the sequence above is depicted with a linker “GGGGSGGGGSGGGGS” (SEQ ID NO: 3) a person of skill in the art recognizes that numerous other flexible linkers known in the art can be used, including those enriched in small or hydrophilic amino acids. In some embodiments, the linker between the VH and VL domains comprises a glycine-serine linker. In some embodiments, any combination of glycine and serine residues can be used. In some embodiments, 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 of Anti-PD-1 Portions of Anti-CD45xPD-l or Anti-CD43xPD-l Bispecific Antibodies

[0195] 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. In some embodiments, instead of the “knob” mutations, 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. In some embodiments, the nucleic acid sequences may be codon optimized.

[0196] In some embodiments, 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.ATGGGCTGGAGCTGCATTATCCTGTTCCTGGTGGCCACAGCCACCGGCGTGCACAG C (SEQ ID NO: 11). In some embodiments, the 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. CAGGTGCAGCTGGTGCAGTCCGGCGTGGAGGTGAAGAAGCCAGGCGCCTCCGTGAA GGTGTCTTGCAAGGCCAGCGGCTACACATTCACCAACTACTATATGTATTGGGTGCG GCAGGCACCTGGACAGGGCCTGGAGTGGATGGGAGGCATCAACCCATCCAATGGC GGCACAAACTTCAATGAGAAGTTTAAGAATCGGGTGACCCTGACCACAGATAGCTC CACCACAACCGCCTACATGGAGCTGAAGTCTCTGCAGTTTGACGATACAGCCGTGT ACTATTGCGCCCGGAGAGACTACAGATTCGATATGGGCTTTGACTATTGGGGCCAG GGCACACTGGTGACCGTGTCTAGCGGCGGCGGCGGCTCTGGAGGAGGAGGAAGCGGAGGAGGAGGATCCGACATCGTGCTGACACAGTCTCCAGCCACCCTGAGCCTGTCC CCAGGAGAGAGGGCCACACTGTCCTGTCGCGCCTCTAAGGGCGTGTCTACCAGCGG CTACAGCTATCTGCACTGGTACCAGCAGAAGCCAGGACAGGCACCTAGGCTGCTGA TCTACCTGGCAAGCTATCTGGAGTCCGGAGTGCCAGCAAGATTCTCCGGATCTGGA AGCGGAACCGACTTCACCCTGACCATCTCCTCTCTGGAGCCCGAGGATTTCGCCGTG TACTATTGTCAGCACAGCAGGGACCTGCCTCTGACATTTGGCGGCGGCACCAAGGT GGAGATCAAGCGCCTCGAGCCCAAGAGCTGCGACAAGACCCACACCTGTCCTCCAT GTCCTGCTCCAGAGTTTCAAGGCGGCCCTTCCGTGTTCCTGTTTCCTCCAAAGCCTA AGGACACCCTGTACATCACCCGCGAGCCTGAAGTGACCTGTGTGGTGGTGGATGTG TCCCACGAGGACCCCGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCA CAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAACAGCACCTACAGAGTGGTG TCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCCA GGTGTCCAACAAGGCCCTGCCTGCTCCTATCGAGAAAACCATCAGCAAGGCCAAGG GCCAGCCTAGGGAACCTCAGGTGTACGTGTTGCCTCCTAGCAGGGACGAGCTGACC AAGAATCAGGTGTCCCTGCTGTGCCTGGTCAAGGGCTTCTACCCTTCCGACATCGCC GTGGAATGGGAGAGCAATGGCCAGCCTGAGAACAACTACCTGACCTGGCCTCCTGT GCTGGATAGCGACGGCTCATTCTTCCTGTACAGCAAGCTGACAGTGGACAAGAGCA GATGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAAC CACTACACCCAGAAGTCCCTGAGCCTGTCTCCTGGATGA (SEQ ID NO: 28). In some embodiments, 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. In some embodiments, 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.

[0197] In some embodiments, 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. In some embodiments, the nucleic acid sequence encoding an anti-PD-1 portion (scFv-Fc) of the anti-CD45xPD-l or anti-CD43xPD-l bispecific antibody (without the signal peptide) 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.In some embodiments, 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). In some embodiments, 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.

[0198] Although the sequence above is depicted with a nucleic acid sequence encoding a linker “GGAGGCGGAGGTTCTGGCGGCGGAGGAAGTGGTGGCGGAGGCTCT” (SEQ ID NO: 13) a person of skill in the art recognizes that numerous other flexible linkers known in the art can be used, including those enriched in small or hydrophilic amino acids. In some embodiments, the linker between the VH and VL domains comprises a glycine-serine linker. In some embodiments, any combination of glycine and serine residues can be used. In some embodiments, the glycine-serine linker comprises a nucleic acid sequence encoding a linker that is between about 2 and 25 amino acids in length. In some embodiments, the nucleic acid sequence encodes a glycine-serine linker that is about 15 amino acids in length.Additional Amino Acid and Nucleic Acid Sequences of An Anti-PD-1 Portion of Anti- CD45xPD-l or Anti-CD43xPD-l Bispecific Antibodies

[0199] In some embodiments, 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. In some embodiments, 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. In some embodiments, 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 In some embodiments, 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, Camrelizumab, Sintilimab, Penpulimab, or any other anti-PD-1 antibody known in the art In some embodiments, the nucleic acid sequence 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 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. In some embodiments, 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. In some embodiments, 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 sequences of anti-PD-1 antibodies are described in the art and incorporated herein by reference as follows: Pembrolizumab (see USPN 8,168,757, USPN 8,354,509, USPN 8,900,587, the contents of each of which is hereby incorporated by reference in its entirety), Cemiplimab (see USPN 9,987,500 the contents of which is hereby incorporated by reference in its entirety), Retifanlimab (see US2019 / 0127467 the contents of which is hereby incorporated by reference in its entirety), Dostarlimab (see WO / 2021 / 058711 the contents of which is hereby incorporated by reference in its entirety), Zimberelimab (see CN106432494 the contents of which is hereby incorporated by reference in its entirety), Tiselelizumab (see USPN 8,735,553 the contents of which is hereby incorporated by reference in its entirety), Camrelizumab (see US2019 / 0309069 the contents of which is hereby incorporated by reference in its entirety), Sintilimab (see USPN 10,316,089 the contents of which is hereby incorporated by reference in its entirety), Penpulimab (see US2019 / 0321466 the contents of which is hereby incorporated by reference in its entirety). The CDRs of certain anti-PD-1 antibodies are described in Jeong TJ, Lee HT, Gu N, Jang YJ, Choi SB, Park UB, Lee SH, Heo YS, The High-Resolution Structure RevealsRemarkable Similarity in PD-1 Binding of Cemiplimab and ostarlimab, the FDA- Approved Antibodies for Cancer Immunotherapy. Biomedicines, 2022 Dec 6; 10(12):3154, the contents of which is hereby incorporated by reference in its entirety.Amino Acid Sequences of Anti-CD45xPD-l Bispecific Antibodies

[0200] 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.

[0201] 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.

[0202] 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.

[0203] 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.

[0204] 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.

[0205] 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. In some embodiments, a bispecific antibody comprises the anti-CD45 portion comprising SEQ ID NO: 29 and an anti-PD-1 portion comprising SEQ ID NO: 26.

[0206] 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. In some embodiments, a bispecific antibody comprises the anti-CD45 portion comprising SEQ ID NO: 30 and an anti-PD-1 portion comprising SEQ ID NO: 26.

[0207] 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. In some embodiments, a bispecific antibody comprises the anti-CD45 portion comprising SEQ ID NO: 31 and an anti-PD-1 portion comprising SEQ ID NO: 26.

[0208] 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. In some embodiments, a bispecific antibody comprises the anti-CD45 portion comprising SEQ ID NO: 32 and an anti-PD-1 portion comprising SEQ ID NO: 26.

[0209] 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. In some embodiments, a bispecific antibody comprises the anti-CD45 portion comprising SEQ ID NO: 33 and an anti-PD-1 portion comprising SEQ ID NO: 26.

[0210] 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. In some embodiments, a bispecific antibody comprises the anti-CD45 portion comprising SEQ ID NO: 34 and an anti-PD-1 portion comprising SEQ ID NO: 26.

[0211] In some embodiments, 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.Nucleic Acid Sequences of Anti-CD45xPD-l Bispecific Antibodies

[0212] 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.

[0213] 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.

[0214] 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.

[0215] 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.

[0216] 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.-n -Amino Acid Sequences of Anti-CD43xPD-l Bispecific Antibodies

[0217] 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.

[0218] In some embodiments, 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.Nucleic Acid Sequences of Anti-CD43xPD-l Bispecific Antibodies

[0219] 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.Structure of Anti-CD45xPD-l and Anti-CD43xPD-l Antibodies

[0220] In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, the anti-CD45xPD-l or the anti-CD43xPD-l bispecific antibody comprises a three-dimensional structure that is similar tothe 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.

[0221] In some embodiments, 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. In some embodiments, a metric of structural similarity between two antibodies can be obtained based on the output of a structure prediction algorithm known in the art. In some embodiments, the metric of structural similarity between two antibodies is based on a similarity distance.

[0222] In some embodiments, the structure of the anti-CD45xPD-l bispecific antibody allows the anti-CD45xPD-l bispecific antibody to bind to CD45 and PD-1. In some embodiments, 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. In some embodiments, the anti- CD45xPD-l bispecific antibody comprises means for binding CD45 and PD-1. In some embodiments, 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.

[0223] In some embodiments, the structure of the anti-CD43xPD-l bispecific antibody allows the anti-CD43xPD-l bispecific antibody to bind to CD43 and PD-1. In some embodiments, 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. In some embodiments, the anti- CD43xPD-l bispecific antibody comprises means for binding CD43 and PD-1. In some embodiments, means for binding CD43 and PD-1 comprises an anti-CD43xPD-l bispecificantibody 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.Monoclonal antibodies to CD45 and with Reduced Affinity for CD45

[0224] Described herein are monoclonal antibodies or antigen binding fragment thereof comprising an antigen binding site against CD45. In some embodiments, the antibodies have reduced affinity to CD45. In some embodiments, 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.

[0225] Described herein are amino acid sequences and nucleic acid sequences of anti-CD45 monoclonal antibodies or antigen binding fragment thereof. The monoclonal antibodies or antigen binding fragment thereof contemplated herein 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. In some embodiments, the amino acid sequences of the anti-CD45 portion is encoded by the nucleic acid sequences disclosed herein.

[0226] In some embodiments, 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. In some embodiments, 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.

[0227] In some embodiments, 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. In some embodiments, the Fc domain comprises the Fc domain sequence of SEQ ID NO: 2. In some embodiments, 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 knownin the art. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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: 2.

[0228] In some embodiments, 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. In some embodiments, the Fc domain comprises the VL domain sequence of SEQ ID NO: 4. In some embodiments, 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. In some embodiments, 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: 4. In some embodiments, 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. In some embodiments, 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.

[0229] In some embodiments, 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. In some embodiments, the Fc domain comprises the VL domain sequence of SEQ ID NO: 6. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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.

[0230] In some embodiments, 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. In some embodiments, the Fc domain comprises the VL domain sequence of SEQ ID NO: 8. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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.

[0231] In some embodiments, 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. In some embodiments, 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. In some embodiments, one or more amino acids shown inside single square brackets of SEQ ID NO: 10 can also be substituted relative to the sequence shown. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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.

[0232] In some embodiments, 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. In some embodiments, the Fc domain comprises the VL domain sequence of SEQ ID NO: 29. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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.

[0233] In some embodiments, 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. In some embodiments, the Fc domain comprises the VL domain sequence of SEQ ID NO: 30. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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.

[0234] In some embodiments, 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. In some embodiments, the Fc domain comprises the VL domain sequence of SEQ ID NO: 31. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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.

[0235] In some embodiments, 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. In some embodiments, the Fc domain comprises the VL domain sequence of SEQ ID NO: 32. In some embodiments, 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. In some embodiments, 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. In some embodiments, the anti-CD45 antibody comprises an amino acid sequence comprising the CDRs of the variable heavy chain domain and the CDRs of the variablelight chain domain of the anti-CD45 antibody, wherein the CDR sequences are indicated above (in bold and underline) in SEQ ID NO: 32. In some embodiments, 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.

[0236] In some embodiments, 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. In some embodiments, the Fc domain comprises the VL domain sequence of SEQ ID NO: 33. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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.

[0237] In some embodiments, 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. In some embodiments, the Fc domain comprises the VL domain sequence of SEQ ID NO: 34. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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.

[0238] In some embodiments, 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. In some embodiments, the signal peptide is cleaved during post-translational modifications that occur in vitro or in vivo.

[0239] In some embodiments 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. In some embodiments the monoclonal antibody is a scFv-Fc antibody comprising SEQ ID NO: 2.

[0240] In some embodiments 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. In some embodiments the monoclonal antibody is a scFv-Fc antibody comprising SEQ ID NO: 4.

[0241] In some embodiments 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. In some embodiments the monoclonal antibody is a scFv-Fc antibody comprising SEQ ID NO: 6.

[0242] In some embodiments 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. In some embodiments the monoclonal antibody is a scFv-Fc antibody comprising SEQ ID NO: 8.

[0243] In some embodiments 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. In some embodiments the monoclonal antibody is a scFv-Fc antibody comprising SEQ ID NO: 10.

[0244] In some embodiments 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. In some embodiments the monoclonal antibody is a scFv-Fc antibody comprising SEQ ID NO: 29.

[0245] In some embodiments 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. In some embodiments the monoclonal antibody is a scFv-Fc antibody comprising SEQ ID NO: 30.

[0246] In some embodiments 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. In some embodiments the monoclonal antibody is a scFv-Fc antibody comprising SEQ ID NO: 31.

[0247] In some embodiments 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. In some embodiments the monoclonal antibody is a scFv-Fc antibody comprising SEQ ID NO: 32.

[0248] In some embodiments 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. In some embodiments the monoclonal antibody is a scFv-Fc antibody comprising SEQ ID NO: 33.

[0249] In some embodiments 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. In some embodiments the monoclonal antibody is a scFv-Fc antibody comprising SEQ ID NO: 34.

[0250] In some embodiments, the amino acid sequence of the anti-CD45 (scFv-Fc) antibody comprises a signal peptide comprising SEQ ID NO: 1. MGWSCIILFLVATATGVHS (SEQ IDNO: 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.

[0251] 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, variable heavy chain and variable light chain domains, Fc portion, and any amino acids that are associated with reduced binding affinity) by translating the nucleic acid sequences into amino acid sequences. In some embodiments, 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.

[0252] In some embodiments, the monoclonal anti-CD45 antibody, or antigen binding fragment thereof, has a modified affinity for CD45 relative to a preselected affinity threshold. For example, in some embodiments, 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. For example, in some embodiments, 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. In some embodiments, 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).

[0253] In some embodiments, an anti-CD45 antibody, or antigen binding fragment thereof, as disclosed herein, is used to prevent or treat cancer in a subject. In some embodiments, 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. In some embodiments, 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.

[0254] In some embodiments, an anti-CD45 antibody, or antigen binding fragment thereof, as disclosed herein, is used to prevent or treat a viral infection. In some embodiments, the viral infection is caused by HIV in a subject.Non-limiting Embodiments of the Subject Matter

[0255] In certain aspects, described herein is a composition 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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.

[0256] In some embodiments, 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 (RANKL), CD62L, CD185 (CXCR5), CD19, CD15, CD14, CD194 (CCR4), CD57, KLRG1, IL17RB, NKp80, CD27, IL23R, VCAM1, CEACAM1, IL12R, IL23R, IL15R, IL6R, IL17RA (CD217), IL17RB, IL2R, TNFSF4 (CD252; OX40L), TNFRSF13C (CD268; BAFFR), TNFSF13B (CD257; BAFF), PTPRC (CD45), CD43, CD2, CD18, CDl la, CDl lb, CDl lc, CD49, CD29, CD22, CD43, ICAM1, CD134 (0X40), CD137 (4IBB), CD357 (GITR), CD256 (APRIL), CD267 (TACI), CD269 (BCMA), CD270 (HVEM), CD120a (TNFR1), CD120b (TNFR2), LTbR, CD70, 41BBL, Galectin 9, CD155, GITRL, TMIGD2, CD160, BTLA, CD270, CD96, CD226, CD112R, CD200R, A2aR, VISTA, B7-H7, CD270, LIGHT, FGL1,CD137L, CD155, CD112, CD277, KIR, SLAMF3, SLAMF7, or SIT1. In some embodiments, the protein for relocalization to the distal compartment of the immune synapse is a stimulatory receptor. In some embodiments, the protein for relocalization to the distal compartment is an inhibitory receptor.

[0257] In some embodiments, the bispecific antibody is any of the bi specific antibodies described herein.

[0258] In certain aspects, described herein is 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. In some embodiments, the molecule is an antibody or an antigen binding fragment thereof capable of localizing the protein to a distal compartment of the immune synapse. In some embodiments, 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.

[0259] In some embodiments, 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, CD5, CD69, CD183 (CXCR3), CD127 (IL17RA), CD254 (RANKL), CD62L, CD185 (CXCR5), CD19, CD15, CD14, CD194 (CCR4), CD57, KLRG1, IL17RB, NKp80, CD27, IL23R, CD352 (SLAMF6), VCAM1, CEACAM1, IL12R, IL23R, IL15R, IL6R, IL17RA (CD217), IL17RB, IL2R, TNFSF4 (CD252; OX40L), TNFRSF13C (CD268; BAFFR), TNFSF13B (CD257; BAFF), PTPRC (CD45), CD43, CD2, CD18, CDl la, CDl lb, CDl lc, CD49, CD29, CD22, CD43, ICAM1, CD134 (0X40), CD137 (4IBB), CD357 (GITR), CD256 (APRIL), CD267 (TACI), CD269 (BCMA), CD270 (HVEM), CD120a (TNFR1), CD120b (TNFR2), LTbR, CD70, 41BBL, Galectin 9, CD155, GITRL, TMIGD2, CD160, BTLA, CD270, CD96, CD226, CD112R, CD200R, A2aR, VISTA, B7-H7, CD270, LIGHT, FGL1, CD137L, CD155, CD112, CD277, KIR, SLAMF3, SLAMF7, or SIT1.

[0260] In certain aspects, described herein is an antibody or antigen binding fragment thereof which binds epitope on CD45 comprising three light chain CDRs and three heavy chainCDRs 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.

[0261] In certain aspects, described herein is 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.

[0262] In some embodiments, the first arm is encoded by a first polypeptide chain and the second arm is encoded by a second polypeptide chain that associate together. In some embodiments, the first arm comprises a linker between the first variable heavy domain and first variable light chain domain. In some embodiments, the second arm comprises a linker between the first variable heavy domain and first variable light chain domain. In some embodiments, the linker is a glycine-serine linker. In some embodiments, the first and second arms each further comprise a fragment, crystallizable (Fc) region. In some embodiments, the Fc region of the first arm comprises knob mutations and the Fc region of the second arm comprise hole mutations, or vice versa. In some embodiments, 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. In some embodiments, 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, and 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. In some embodiments, 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).

[0263] In certain aspects, described herein is a bispecific antibody or a fragment thereof, comprising: a first arm comprising a first variable heavy chain domain and a first variable lightchain 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.

[0264] In some embodiments, the first arm is encoded by a first polypeptide chain and the second arm is encoded by a second polypeptide chain that associate together. In some embodiments, the first arm comprises a linker between the first variable heavy domain and first variable light chain domain. In some embodiments, the second arm comprises a linker between the first variable heavy domain and first variable light chain domain. In some embodiments, the linker is a glycine-serine linker. In some embodiments, the first and second arms each further comprise a fragment, crystallizable (Fc) region. In some embodiments, the Fc region of the first arm comprises knob mutations and the Fc region of the second arm comprise hole mutations, or vice versa.

[0265] In some embodiments, 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. In some embodiments, 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, 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. In some embodiments, 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).

[0266] In some embodiments, 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. In some embodiments, the first arm comprises SEQ ID NO: 22, wherein the second arm comprises SEQ ID NO: 26. In some embodiments, 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).

[0267] In some embodiments, 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. In some embodiments, 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. In some embodiments, 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.

[0268] In some embodiments, 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.

[0269] In some embodiments, 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. In some embodiments, 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.

[0270] In certain aspects, described herein is a pharmaceutical composition comprising: the bispecific antibody described herein and a pharmaceutically acceptable carrier. In certain aspects, described herein is a method of preventing or treating cancer in a subject comprisingadministering to the subject an effective amount of the composition described herein. In some embodiments, 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. In certain aspects, 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. In some embodiments, the viral infection is HIV.

[0271] In certain aspects, described herein is a pharmaceutical composition comprising: the bispecific antibody described herein and a pharmaceutically acceptable carrier. In certain aspects, 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. In some embodiments, the diseases is selected from rheumatoid arthritis, systemic lupus erythematosus, psoriasis and psoriatic arthritis, spondyloarthropathy, type 1 diabetes, and multiple sclerosis.

[0272] In certain aspects, described herein is a kit for generating a bispecific antibody or fragment thereof, the kit comprising one or more vectors comprising a polynucleotide sequence encoding any of the bispecific antibodies described herein. In certain aspects, described herein is a kit for generating a bispecific antibody or fragment thereof, the kit 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. In some embodiments, 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.

[0273] In some embodiments, 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, wherein 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, wherein 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, and wherein 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 ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, or SEQ ID NO: 43. In some embodiments, 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. In some embodiments, the first arm comprises SEQ ID NO: 4. In some embodiments, the first arm comprises SEQ ID NO: 6. In some embodiments, the first arm comprises SEQ ID NO: 22.

[0274] In certain aspects, 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. In certain aspects, 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.

[0275] In some embodiments, 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. In some embodiments, the first arm comprises a first variable heavy chain domain and a first variable light chain domain, wherein the second arm comprises a second variable heavy chain domain and a second variable light chain domain, wherein 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. In some embodiments, 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 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. In someembodiments, the first arm comprises SEQ ID NO: 4. In some embodiments, the first arm comprises SEQ ID NO: 6. In some embodiments, the first arm comprises SEQ ID NO: 22.

[0276] In certain aspects, 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. In certain aspects, 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.

[0277] In certain aspects, described herein is a composition comprising: one or more vectors comprising a polynucleotide sequence encoding any of the bispecific antibodies described herein. In certain aspects, described herein is a composition 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.

[0278] In some embodiments, 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. In some embodiments, the first arm comprises a first variable heavy chain domain and a first variable light chain domain, wherein the second arm comprises a second variable heavy chain domain and a second variable light chain domain, wherein 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. In some embodiments, 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 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. In some embodiments, the first arm comprises SEQ ID NO: 4. In some embodiments, the first arm comprises SEQ ID NO: 6. In some embodiments, the first arm comprises SEQ ID NO: 22.

[0279] 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. In some embodiments, 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. In some embodiments, 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, and wherein the portion of the first arm is capable of binding to the portion of the CD45 protein.

[0280] In some embodiments, 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, wherein 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, and wherein the portion of the first arm is capable of binding to the portion of the CD45 protein. In some embodiments, 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).

[0281] In some embodiments, 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. In some embodiments, 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).

[0282] In some embodiments, 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. In some embodiments, 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.

[0283] In some embodiments, 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. In some embodiments, 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.

[0284] In some embodiments, 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.Compositions

[0285] In some embodiments, 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. In some embodiments, 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 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 SEQ ID NOs: 29 and any of 35-43, comprising SEQ ID NOs: 30 and any of 35-43, comprising SEQ ID NOs: 31 and any of 35-43, comprising SEQ ID NOs: 32 and any of 35- 43, comprising SEQ ID NOs: 33 and any of 35-43, or comprising SEQ ID NOs: 34 and any of 35-43. In some embodiments, 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. In some embodiments, the pharmaceutical composition is in the form of a spray, aerosol, gel, solution, emulsion, nanoparticle (e.g., lipid nanoparticle), or suspension.

[0286] The composition is preferably administered to a subject with 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.

[0287] In certain embodiments, the one or more antibodies (or one or more polynucleotides encoding 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 SEQ ID NOs: 29 and any of 35-43, comprising SEQ ID NOs: 30 and any of 35-43, comprising SEQ ID NOs: 31 and any of 35-43, comprising SEQ ID NOs: 32 and any of 35-43, comprising SEQ ID NOs: 33 and any of 35-43, or comprising SEQ ID NOs: 34 and any of 35-43, or antibody comprising the VH domain and VL domain of SEQ ID NOs: 4, 6, 8, 10, or 29-34) and a pharmaceutically acceptable carrier. In certain embodiments, the composition further comprises an adjuvant. In certain embodiments, the antibodies are conjugated with other molecules to increase their effectiveness as is known by those practiced in the art.

[0288] In some embodiments, the pharmaceutically acceptable carrier is selected from the group consisting of saline, Ringer's solution, dextrose solution, and a combination thereof. Other 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.

[0289] The phrase pharmaceutically acceptable carrier as used herein 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. Each carrier is acceptable in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of 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’s solution; ethyl alcohol; phosphate buffer solutions; and other non-toxic compatible substances employed in pharmaceutical formulations. The term 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.

[0290] 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. Typical carbohydrates useful in formulating compositions include but are not limited to sucrose, mannitol, lactose, trehalose, or glucose.

[0291] 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 notlimited to sorbitan trioleate, soya lecithin, and oleic acid. In certain cases, solution aerosols are preferred using solvents such as ethanol. Thus, 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. In some embodiments, 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.

[0292] In some embodiments, 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. So called stabilizers or preservatives may include, but are not limited to, amino acids, antioxidants, pH buffers, or salt buffers. Nonlimiting examples of antioxidants include butylated hydroxy anisole (BHA), ascorbic acid and derivatives thereof, tocopherol and derivatives thereof, butylated hydroxy anisole and cysteine. Nonlimiting examples of preservatives include parabens, such as methyl or propyl p- hydroxybenzoate and benzalkonium chloride. Additional nonlimiting examples of amino acids include glycine or proline.

[0293] 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.

[0294] In one embodiment, 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. Preferably, the vehicles, carriers, excipients, stabilizing agents and preservatives are pharmaceutically acceptable.

[0295] In some embodiments, 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. Accordingly, 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.

[0296] 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.

[0297] The invention also provides that a pharmaceutical composition can be packaged in a hermetically sealed container such as an ampoule or sachette indicating the quantity. In one embodiment, 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.

[0298] 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 ormore accessory ingredients. In general, 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.

[0299] 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.

[0300] 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 methods of spray freeze- drying proteins for pharmaceutical administration disclosed in Maa et cd.. Curr. Pharm. Biotechnol., 2001, 1, 283-302, are incorporated herein. In another embodiment, 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.

[0301] 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). 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).

[0302] 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.

[0303] 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. See, e.g., Remington's Pharmaceutical Sciences; Remington: The Science and Practice of Pharmacy supra; Pharmaceutical Dosage Forms and Drug Delivery Systems by Howard C., Ansel et al., Lippincott Williams & Wilkins; 7th edition (Oct. 1, 1999).

[0304] Generally, 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. In addition, the prophylactically and therapeutically effective dosage form may vary among different conditions. For example, 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. On the other hand, 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). These and other ways in which specific dosage forms encompassed by this invention will vary from one another and will be readily apparent to those skilled in the art. See, e.g., Remington's Pharmaceutical Sciences, 2005, Mack Publishing Co.; Remington: The Science and Practice of Pharmacy by Gennaro, Lippincott Williams & Wilkins; 20th edition (2003);Pharmaceutical Dosage Forms and Drug Delivery Systems by Howard C. Ansel et al., Lippincott Williams & Wilkins; 7th edition (Oct. 1, 1999); and Encyclopedia of Pharmaceutical Technology, edited by Swarbrick, J. & J.C. Boylan, Marcel Dekker, Inc., New York, 1988, which are incorporated herein by reference in their entirety.

[0305] 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. Similarly, 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. In this regard, 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.

[0306] Compositions can be formulated with appropriate carriers and adjuvants using techniques to yield compositions suitable for prophylaxis or treatment. The compositions caninclude 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.

[0307] In some embodiments, 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.Antibody Production

[0308] The antibodies (e.g., monoclonal antibodies, bispecific antibodies) disclosed herein can be produced by any method known in the art. In some embodiments, 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. E.g., See Examples 1-5. For example, 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.

[0309] In some embodiments, 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. For example, 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. Y., 1981)), recombinant DNA methods, phage-display technologies (see, e.g., Clackson et al, Nature, 352: 624-628 (1991); Marks et al, J. Mol Biol. 222: 581-597 (1992); Sidhu et al, J. Mol Biol. 338(2): 299-310 (2004); Lee et al, J. Mol Biol. 340(5): 1073-1093 (2004); Fellouse, Proc. Natl. Acad. ScL USA 101(34): 12467-12472 (2004); and Lee et al, J. Immunol. 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).

[0310] In some embodiments, expression of an antibody comprises expression vector(s) containing a polynucleotide that encodes an anti-CD45xlPD-l or an anti-CD43xPD-l bispecific antibody. In some embodiments, expression of an antibody comprises expression vector(s) containing a polynucleotide that encodes an anti-CD45 antibody. Methods that are well known to those skilled in the art can be used to construct expression vectors comprising antibody coding sequences and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Particular embodiments provide 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. In preferred embodiments, 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.

[0311] In some embodiments, 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. For example, 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. (1997) Protein Science 6:781-788, each of which are incorporated in its entirety herein. For example, the production of 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. In some embodiments, in one of the heavy chains referred as a “knob” variant, a small amino acid has been replaced with a larger one in the CH3 domain. Subsequently, in the other heavy chain, 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. For example, multispecific antibodies having KnH in their Fc regions can further comprise single variable domains linked to each Fcregion, or further comprise different heavy chain variable domains that pair with similar or different light chain variable domains. KnH technology can also be used to pair two different receptor extracellular domains together or any other polypeptide sequences that comprises different target recognition sequences (e.g., including affibodies, peptibodies and other Fc fusions). In some embodiments, the bispecific antibodies described herein are scFv-Fc antibodies. In some embodiments, 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.

[0312] 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. Typically, such 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. Alternatively, the relevant 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.

[0313] 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 nucleotidesequence 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. For stable amplification and expression of a desired protein, a vector containing DNA encoding a protein of interest (e.g., antibodies and fragments thereof) 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 (for example, resistance to antibiotics or drugs) 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.

[0314] In some embodiments, the bispecific antibodies disclosed herein are encoded in a vector for expression in a cell line. In some embodiments, 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, and each vector is transfected into one or more cell lines for expression. In some embodiments, 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. In some embodiments, 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. For example, in some embodiments, 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 ofthe 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, and / or a fourth vector may comprise a polynucleotide sequence encoding a heavy chain, or fragment thereof, of an anti-PDl portion of the bispecific antibody. In some embodiments, all four vectors are transfected into one or more cell lines for expression.

[0315] In some embodiments, the bispecific antibodies disclosed herein are encoded in a vector for expression in a cell line. In some embodiments, 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, and each vector is transfected into one or more cell lines for expression. In some embodiments, 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. In some embodiments, 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. For example, in some embodiments, 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, and / or a fourth vector may comprise a polynucleotide sequence encoding a heavy chain, or fragment thereof, of an anti-PDl portion of the bispecific antibody. In some embodiments, all four vectors are transfected into one or more cell lines for expression.

[0316] In some embodiments, the anti-CD45 antibodies disclosed herein are encoded in a vector for expression in a cell line. In some embodiments, a vector comprises a polynucleotide sequence that encodes an anti-CD45 antibody, the vector is transfected into one or more cell lines for expression. In some embodiments, 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. In some embodiments, 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. For example, in some embodiments, a first vector may comprise a polynucleotide sequence encoding a light chain, or fragment thereof, of an anti-CD45 antibody, a second vectormay comprise a polynucleotide sequence encoding a heavy chain, or fragment thereof, of an anti-CD45 portion of the antibody. In some embodiments, all vectors are transfected into one or more cell lines for expression.

[0317] 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. Thus, eukaryotic host cells possessing the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product may be used.

[0318] 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.

[0319] 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. For antibody recovery, isolation and / or purification, 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 forcarbohydrates 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.Methods of Treatment

[0320] In one embodiment, 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. In one embodiment, 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. In one embodiment, 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. In one embodiment, the compositions described herein can be administered subcutaneously via syringe or any other suitable method know in the art.

[0321] 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. Nonlimiting examples of 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;(d) 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.

[0322] In some embodiments, 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.- I l l -

[0323] As will be readily apparent to one skilled in the art, 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, that is the dose levels necessary to achieve the desired result, 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.)Methods of Prevention

[0324] In some embodiments, the 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). In some embodiments, the antibodies are neutralizing antibodies.

[0325] In some embodiments, the antibodies (or polynucleotides encoding antibodies) prepared using methods of the invention can be combined with additional pharmaceutical components.Dosage

[0326] A prophylactically effective or therapeutically effective amount is typically dependent on the weight of the subject being treated, the subj...

Claims

What is claimed is:

1. 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.

2. The bispecific antibody of claim 1, wherein the first arm is encoded by a first polypeptide chain and the second arm is encoded by a second polypeptide chain that associate together.

3. The bispecific antibody of claims 1-2, wherein the first arm comprises a linker between the first variable heavy domain and first variable light chain domain.

4. The bispecific antibody of claims 1-3, wherein the second arm comprises a linker between the first variable heavy domain and first variable light chain domain.

5. The bispecific antibody of claims 3-4, wherein the linker is a glycine-serine linker.

6. The bispecific antibody of claim 2, wherein the first and second arms each further comprise a fragment, crystallizable (Fc) region.

7. The bispecific antibody of claim 6, wherein the Fc region of the first arm comprises knob mutations and the Fc region of the second arm comprise hole mutations, or vice versa.

8. The bispecific antibody of claims 1-7, wherein 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.

9. The bispecific antibody of claims 1-7, wherein 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, 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.

10. The bispecific antibody of claims 8-9, wherein 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).

11. The bispecific antibody of claims 1-7, 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.

12. The bispecific antibody of claims 1-7, wherein the first arm comprises SEQ ID NO: 22, wherein the second arm comprises SEQ ID NO: 26.

13. The bispecific antibody of claims 11-12, wherein 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).

14. The bispecific antibody of claims 1-7, 8, or 11, wherein 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.

15. The bispecific antibody of claims 1-7, wherein 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.

16. The bispecific antibody of claims 1-7, wherein 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.

17. The bispecific antibody of claims 1-7, wherein the bispecific antibody is capable of disrupting downstream signaling of a PD-1 mediated response in a T cell.

18. The bispecific antibody of claims 1-7, wherein the bispecific antibody is capable of enhancing T cell function.

19. The bispecific antibody of claims 1-7, wherein 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.

20. The bispecific antibody of claims 1-7, wherein 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.

21. The bispecific antibody of claims 1-7, wherein the bispecific antibody is capable of disrupting a downstream signaling of a PD-1 mediated response in a T cell.

22. The bispecific antibody of claims 1-7, wherein the bispecific antibody is capable of enhancing T cell function.

23. The bispecific antibody of claims 1-7, wherein 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.

24. The bispecific antibody of claims 1-7, wherein 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.

25. The bispecific antibody of claim 24, wherein the bispecific antibody is capable of dephosphorylating a tail of the PD-1 protein.

26. The bispecific antibody of claim 24, wherein the bispecific antibody is capable of inducing a cytokine secretion in a T cell.

27. The bispecific antibody of claim 26, wherein the cytokine secretion is a secretion of IL-2.

28. A pharmaceutical composition comprising: the bispecific antibody of any of claims 1-27; and a pharmaceutically acceptable carrier.

29. A method of preventing or treating cancer in a subject comprising administering to the subject an effective amount of the composition of claim 28.

30. The method of claim 29, wherein 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.

31. A method of preventing or treating a viral infection in a subject comprising administering to the subject an effective amount of the composition of claim 28.

32. The method of claim 31, wherein the viral infection is HIV.

33. A kit for generating a bispecific antibody or fragment thereof, the kit comprising one or more vectors comprising a polynucleotide sequence encoding any of the bispecific antibodies of any of claims 1-27.

34. A kit for generating a bispecific antibody or fragment thereof, the kit 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.

35. The kit of claim 34, wherein the first vector and the second vector are the same vector.

36. The kit of claim 34, wherein the first vector and the second vector are two different vectors.

37. The kit of claims 34-36, wherein 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 wherein 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, wherein 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, 35, 36, 37, 38, 39, 40, 41, 42, or 43 and wherein a variable light chain domain of the second arm comprises an amino acid sequence of the variable light chain portion of SEQ ID NO: 26, 35, 36, 37, 38, 39, 40, 41, 42, or 43.

38. The kit of claims 34-36, wherein 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 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.

39. The kit of claim 38, wherein the first arm comprises SEQ ID NO: 4.

40. The kit of claim 38, wherein the first arm comprises SEQ ID NO: 6.

41. The kit of claim 38, wherein the first arm comprises SEQ ID NO: 22.

42. One or more host cells comprising: one or more vectors comprising a polynucleotide sequence encoding any of the bispecific antibodies of any of claims 1-27.

43. 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 iscapable 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.

44. The one or more host cells of claim 43, wherein the first vector and the second vector are the same vector.

45. The one or more host cells of claim 43, wherein the first vector and the second vector are two different vectors.

46. The one or more host cells of claim 43-45, wherein the first arm comprises a first variable heavy chain domain and a first variable light chain domain, wherein the second arm comprises a second variable heavy chain domain and a second variable light chain domain, wherein 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.

47. The one or more host cells of claim 43-45, wherein 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 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.

48. The one or more host cells of claim 47, wherein the first arm comprises SEQ ID NO: 4.

49. The one or more host cells of claim 47, wherein the first arm comprises SEQ ID50. The one or more host cells of claim 47, wherein the first arm comprises SEQ ID NO: 22.

51. A method of making a bispecific antibody or fragment thereof comprising: culturing the one or more host cells of claim 42 under conditions suitable for an expression of the one or more vectors; and recovering the bispecific antibody or fragment thereof.

52. A method of making a bispecific antibody or fragment thereof comprising: culturing the one or more host cells of any of claims 43-50 under conditions suitable for an expression of the first vector and the second vector; and recovering the bispecific antibody or fragment thereof.

53. A composition comprising: one or more vectors comprising a polynucleotide sequence encoding any of the bispecific antibodies of any of claims 1-27.

54. A composition 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.

55. The composition of claim 54, wherein the first vector and the second vector are the same vector.

56. The composition of claim 54, wherein the first vector and the second vector are two different vectors.

57. The composition of claim 54-56, wherein the first arm comprises a first variable heavy chain domain and a first variable light chain domain, wherein the second arm comprises a second variable heavy chain domain and a second variable lightchain domain, wherein 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.

58. The composition of claim 54-56, wherein 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 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.

59. The composition of claim 58, wherein the first arm comprises SEQ ID NO: 4.

60. The composition of claim 58, wherein the first arm comprises SEQ ID NO: 6.

61. The composition of claim 58, wherein the first arm comprises SEQ ID NO: 22.

62. 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.

63. The means of claim 62, wherein the means comprises a bispecific antibody or fragment thereof.

64. The means of claim 63, wherein 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; anda 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.

65. The means of claim 63, wherein 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, and wherein the portion of the first arm is capable of binding to the portion of the CD45 protein.

66. The means of claim 64, wherein 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, 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, and wherein the portion of the first arm is capable of binding to the portion of the CD45 protein.

67. The means of claim 66, wherein 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).

68. The means of claim 64, wherein 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.

69. The means of claim 68, wherein 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).

70. The means of claim 64, wherein 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.

71. The means of claim 70, wherein 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.

72. The means of claim 64, wherein the bispecific antibody is capable of disrupting downstream signaling of a PD-1 mediated response in a T cell.

73. The means of claim 64, wherein the bispecific antibody is capable of enhancing T cell function.

74. The means of claim 64, wherein 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.

75. The means of claim 64, wherein 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.

76. The means of claim 64, wherein the bispecific antibody is capable of disrupting a downstream signaling of a PD-1 mediated response in a T cell.

77. The means of claim 64, wherein the bispecific antibody is capable of enhancing T cell function.

78. The means of claim 64, wherein 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.

79. The means of claim 64, wherein 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.

80. The means of claim 79, wherein the bispecific antibody is capable of dephosphorylating a tail of the PD-1 protein.

81. The means of claim 79, wherein the bispecific antibody is capable of inducing a cytokine secretion in a T cell.

82. The means of claim 81, wherein the cytokine secretion is a secretion of IL-2.

83. A composition 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.

84. The composition of claim 83, wherein 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.

85. The composition of claim 84, wherein 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.

86. The composition of claim 83, wherein 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.

87. The composition of claim 86, wherein 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.

88. The composition of claims 83-87, wherein 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, CD154 (CD40L), CD278 (ICOS), CD25 (IL2R), CD 152 (CTLA4), CXCR1, HLA-DR, CD 16, CD5, CD69, CD183 (CXCR3), CD127 (IL17RA), CD254 (RANKL), CD62L,CD185 (CXCR5), CD19, CD15, CD14, CD194 (CCR4), CD57, KLRG1, IL17RB, NKp80, CD27, IL23R, VCAM1, CEACAM1, IL12R, IL23R, IL15R, IL6R, IL17RA (CD217), IL17RB, IL2R, TNFSF4 (CD252; OX40L), TNFRSF13C (CD268; BAFFR), TNFSF13B (CD257; BAFF), PTPRC (CD45), CD43, CD2, CD18, CDlla, CDllb, CDllc, CD49, CD29, CD22, CD43, ICAM1, CD134 (0X40), CD137 (4IBB), CD357 (GITR), CD256 (APRIL), CD267 (TACI), CD269 (BCMA), CD270 (HVEM), CD120a (TNFR1), CD120b (TNFR2), LTbR, CD70, 41BBL, Galectin 9, CD155, GITRL, TMIGD2, CD160, BTLA, CD270, CD96, CD226, CD112R, CD200R, A2aR, VISTA, B7-H7, CD270, LIGHT, FGL1, CD137L, CD155, CD112, CD277, KIR, SLAMF3, SLAMF7, or SITl.The composition of claims 83-88, wherein the bispecific antibody is any of the bispecific antibodies of claims 1-27.

89. 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.

90. The method of claim 89, wherein the molecule is an antibody or an antigen binding fragment thereof capable of localizing the protein to a distal compartment of the immune synapse.

91. The method of claim 90, wherein 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.

92. The method of claims 89-91, wherein 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, CD 154 (CD40L), CD278 (ICOS), CD25 (IL2R), CD 152 (CTLA4), CXCR1, HLA-DR, CD16, CD5, CD69, CD183 (CXCR3), CD127(IL17RA), CD254 (RANKL), CD62L, CD185 (CXCR5), CD19, CD15, CD14, CD 194 (CCR4), CD57, KLRG1, IL17RB, NKp80, CD27, IL23R, CD352 (SLAMF6), VCAM1, CEACAM1, IL12R, IL23R, IL15R, IL6R, IL17RA (CD217), IL17RB, IL2R, TNFSF4 (CD252; OX40L), TNFRSF13C (CD268; BAFFR), TNFSF13B (CD257; BAFF), PTPRC (CD45), CD43, CD2, CD18, CDlla, CDllb, CDllc, CD49, CD29, CD22, CD43, ICAM1, CD134 (0X40), CD137 (4IBB), CD357 (GITR), CD256 (APRIL), CD267 (TACI), CD269 (BCMA), CD270 (HVEM), CD120a (TNFR1), CD120b (TNFR2), LTbR, CD70, 41BBL, Galectin 9, CD155, GITRL, TMIGD2, CD160, BTLA, CD270, CD96, CD226, CD112R, CD200R, A2aR, VISTA, B7-H7, CD270, LIGHT, FGL1, CD137L, CD155, CD112, CD277, KIR, SLAMF3, SLAMF7, or SIT1.

93. An antibody or antigen binding fragment thereof which binds an 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.

94. The antibody or antigen binding fragment thereof of claim 93, 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.

95. An 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.

96. The antibody of claim 95, wherein the first arm is encoded by a first polypeptide chain and the second arm is encoded by a second polypeptide chain that associate together.

97. The antibody of claims 95-96, wherein the first arm comprises a linker between the first variable heavy domain and first variable light chain domain.

98. The antibody of claims 95-96, wherein the second arm comprises a linker between the first variable heavy domain and first variable light chain domain.

99. The antibody of claims 97-98, wherein the linker is a glycine-serine linker.

100. The antibody of claim 96, wherein the first and second arms each further comprise a fragment, crystallizable (Fc) region.

101. The antibody of claim 100, wherein the Fc region of the first arm comprises knob mutations and the Fc region of the second arm comprise hole mutations, or vice versa.

102. The antibody of claims 95-101, wherein 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.

103. The antibody of claims 95-101, wherein 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, and 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.

104. The antibody of claims 102-103, wherein 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).

Images