Bispecific antibodies targeting CD137 and their use for anti-cancer immunotherapy - Patent Application 20070123333

Abstract

Provided herein are antibodies or antigen-binding fragments thereof having binding specificity for p95HER2 or CD73, bispecific antibodies comprising a first antigen-binding region that binds to p95HER2 or CD73 and a second antigen-binding region that binds to an immune checkpoint molecule or an immune stimulatory molecule, and antibody-drug conjugates thereof. Also provided herein are pharmaceutical compositions comprising the antibodies, antigen-binding fragments thereof, bispecific antibodies or antibody-drug conjugates thereof, and methods of use thereof. Methods of use include methods of treating cancer. TIFF2024540604000013.tif101162

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority under 35 U.S.C. §119(e) of U.S. Provisional Application No. 63 / 281,347, filed November 19, 2021, and U.S. Provisional Application No. 63 / 392,474, filed July 26, 2022. The disclosures of the prior applications are considered part of the disclosure of this application and are hereby incorporated by reference in their entireties in the disclosure of this application.

[0002] Incorporating sequence tables The material in the attached Sequence Listing is incorporated by reference into this application. The attached Sequence Listing XML file, named AP1110_2WO.xml, was created on Nov. 18, 2022 and is 206kb.

[0003] FIELD OF THEINVENTION The present invention relates generally to antibodies and antigen-binding fragments thereof, and more specifically to antibodies and antigen-binding fragments thereof that have cytotoxicity against cancer cells and enhance T cell function. [Background technology]

[0004] Background information Cancer immunotherapy restores or enhances the natural defense against tumors by the human immune system. Such therapies usually target specific biomolecules on the surface of cancer cells, such as tumor-associated antigens (TAAs). Antitumor activity may be achieved by directing the host immune system to the TAAs, thereby establishing or inducing an adaptive immune response against cancer cells. Over the past decades, cancer therapy using monoclonal antibodies (mAbs) has been highly successful, and many of them have been approved for cancer treatment or clinical trials.

[0005] CD137 (4-1BB / TNFRSF9) was identified in 1989 as an inducible gene expressed in antigen-primed but not resting T cells. In addition, it is known to be expressed in dendritic cells (DCs), natural killer cells (NKs), activated CD4+ and CD8+ T lymphocytes, eosinophils, natural killer T cells (NKTs), and mast cells, but it has been investigated that myeloid-derived suppressor cells (MDSCs) do not express this molecule on their surface. Anti-CD137 antibodies show great potential in anticancer activity due to their ability to activate cytotoxic T cells and increase the production of interferon gamma (IFN-γ).

[0006] Antibody-based strategies for cancer therapy have progressed dramatically over the past two decades. Since rituximab was approved as the first monoclonal antibody (mAb) for the treatment of cancer in 1997, several mAbs have become the standard of care for the treatment of both solid tumors and hematological malignancies. Most of the approved mAbs (e.g., rituximab, trastuzumab, and cetuximab) target tumor-associated antigens on the surface of cancer cells and inhibit cell growth. Although several effective antibodies have emerged, long-term and sustained responses are still not possible, and resistance and recurrence remain major problems. Immunomodulatory antibodies have revolutionized cancer immunotherapy and helped to obtain breakthrough features.

[0007] In addition to checkpoint blockers such as ipilimumab and pembrolizumab, drugs targeting the tumor necrosis factor (TNF) superfamily of costimulatory receptors are in development. CD137 is one of the TNF receptor family targets moving into clinical trials. CD137 regulates many immune cells, including CD4+ and CD8+ T cells, regulatory T cells (Tregs), dendritic cells (DCs), and natural killer (NK) cells. Recent studies show that the addition of anti-CD137 mAbs can enhance the antitumor efficacy of immunomodulatory antibodies. However, there remains an unmet need for more specific and more effective antigen-binding molecules that target CD137. Summary of the Invention

[0008] The present invention is based on the discovery of anti-p95HER2 antibodies that exhibit superior activity in binding to p95HER2-expressing cancer cells and anti-CD73 antibodies that exhibit superior activity in neutralizing the enzymatic activity of CD73 in cell membrane-bound or soluble forms. The present invention is further based on the discovery that bispecific antibodies targeting p95HER2 and CD137 have dual functions of antibody-dependent cell-mediated cytotoxicity (ADCC) and p95HER2-dependent T cell activation against p95HER-positive cancer cells, and that bispecific antibodies targeting CD73 and CD137 have exceptional activity for enhancing T cell effector function and better inhibiting tumor growth in vivo than either antibody monotherapy or combination therapy.

[0009] In one embodiment, the present invention provides four antibodies or antigen-binding fragments thereof, including anti-p95HER2 antibody clone R4-3 (p95HER2 #R4-3), anti-p95HER2 antibody clone R4-5 (p95HER2 #R4-5), anti-p95HER2 antibody clone R4-11 (p95HER2 #R4-11), anti-p95HER2 antibody clone R4-15 (p95HER2 #R4-15), and anti-p95HER2 antibody clone R4-38 (p95HER2 #R4-38). In one aspect, the anti-p95HER2 antibody provided herein comprises a heavy chain variable (VH) region comprising an amino acid sequence having at least 80% sequence identity to a sequence selected from SEQ ID NO:1, SEQ ID NO:9, SEQ ID NO:17, SEQ ID NO:25, and SEQ ID NO:33, and a light chain variable (VL) region comprising an amino acid sequence having at least 80% sequence identity to a sequence selected from SEQ ID NO:2, SEQ ID NO:10, SEQ ID NO:18, SEQ ID NO:26, and SEQ ID NO:34.

[0010] In one aspect, the antibody or antigen-binding fragment has a VH region comprising an amino acid sequence having at least 80% identity to SEQ ID NO:1, and a VL region comprising an amino acid sequence having at least 80% identity to SEQ ID NO:2, and comprises (a) a VH CDR-H1, CDR-H2, and CDR-H3, wherein CDR-H1 comprises an amino acid sequence having at least 80% identity to SEQ ID NO:3, CDR-H2 comprises an amino acid sequence having at least 80% identity to SEQ ID NO:4, and CDR-H3 comprises an amino acid sequence having at least 80% identity to SEQ ID NO:5, and (b) a VL CDR-L1, CDR-L2, and CDR-L3, wherein CDR-L1 comprises an amino acid sequence having at least 80% identity to SEQ ID NO:6, CDR-L2 comprises an amino acid sequence having at least 80% identity to SEQ ID NO:7, and CDR-L3 comprises an amino acid sequence having at least 80% identity to SEQ ID NO:8.

[0011] In another embodiment, the antibody, or antigen-binding fragment thereof, comprises a VH region comprising an amino acid sequence having at least 80% identity to SEQ ID NO:9 and a VL region comprising an amino acid sequence having at least 80% identity to SEQ ID NO:10, and comprises (a) a VH CDR-H1, CDR-H2, and CDR-H3, wherein CDR-H1 comprises an amino acid sequence having at least 80% identity to SEQ ID NO:11, CDR-H2 comprises an amino acid sequence having at least 80% identity to SEQ ID NO:12, and CDR-H3 comprises an amino acid sequence having at least 80% identity to SEQ ID NO:13, and (b) a VL It comprises CDR-L1, CDR-L2, and CDR-L3 (wherein CDR-L1 comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 14, CDR-L2 comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 15, and CDR-L3 comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 16).

[0012] In one embodiment, the antibody, or antigen-binding fragment thereof, comprises a VH region comprising an amino acid sequence having at least 80% identity to SEQ ID NO: 17, and a VL region comprising an amino acid sequence having at least 80% identity to SEQ ID NO: 18, and comprises (a) a VH CDR-H1, CDR-H2, and CDR-H3, wherein CDR-H1 comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 19, CDR-H2 comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 20, and CDR-H3 comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 21, and (b) a VL It comprises CDR-L1, CDR-L2, and CDR-L3 (wherein CDR-L1 comprises an amino acid sequence having at least 80% identity to SEQ ID NO:22, CDR-L2 comprises an amino acid sequence having at least 80% identity to SEQ ID NO:23, and CDR-L3 comprises an amino acid sequence having at least 80% identity to SEQ ID NO:24).

[0013] In another embodiment, the antibody, or antigen-binding fragment thereof, comprises a VH region comprising an amino acid sequence having at least 80% identity to SEQ ID NO: 25 and a VL region comprising an amino acid sequence having at least 80% identity to SEQ ID NO: 26, and further comprises (a) a VH CDR-H1, CDR-H2, and CDR-H3, wherein CDR-H1 comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 27, CDR-H2 comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 28, and CDR-H3 comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 29, and (b) a VL It comprises CDR-L1, CDR-L2, and CDR-L3 (wherein CDR-L1 comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 30, CDR-L2 comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 31, and CDR-L3 comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 32).

[0014] In yet another aspect, the antibody, or antigen-binding fragment thereof, comprises a VH region comprising an amino acid sequence having at least 80% identity to SEQ ID NO: 33 and a VL region comprising an amino acid sequence having at least 80% identity to SEQ ID NO: 34, and wherein CDR-H1, CDR-H2, and CDR-H3 comprise an amino acid sequence having at least 80% identity to SEQ ID NO: 35, CDR-H2 comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 36, and CDR-H3 comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 37, and (b) a VL region comprising an amino acid sequence having at least 80% identity to SEQ ID NO: 38. It comprises CDR-L1, CDR-L2, and CDR-L3 (wherein CDR-L1 comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 38, CDR-L2 comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 39, and CDR-L3 comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 40).

[0015] In one embodiment, the antibody comprises an Fc domain. In some embodiments, the Fc domain is an IgG, IgE, IgM, IgD, IgA, or IgY domain. In various embodiments, the IgG domain is an IgG1, IgG2, IgG3, or IgG4 domain. In other embodiments, the IgG1 domain comprises the amino acid sequence of SEQ ID NO: 41. In various embodiments, the IgG1 domain comprises point mutations that alter or enhance antibody-dependent cellular cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC) compared to a wild-type IgG1 domain. In many embodiments, the point mutations comprise S241D / A332L / I334E mutations.

[0016] In another embodiment, the invention provides a bispecific antibody comprising a first antigen-binding region and a second antigen-binding region, wherein the first antigen-binding region binds to p95HER2.

[0017] In one embodiment, the bispecific antibody comprises a complementarity determining region (CDR)-H1 as set forth in SEQ ID NO: 19, a CDR-H2 as set forth in SEQ ID NO: 20, and a CDR-H3 as set forth in SEQ ID NO: 21, or a V sequence having 90% identity and antigen binding specificity to SEQ ID NO: 19, 20, or 21. H and a V region comprising CDR-L1, CDR-L2, and CDR-L3, wherein CDR-L1 is represented by SEQ ID NO: 22, CDR-L2 is represented by SEQ ID NO: 23, and CDR-L3 is represented by SEQ ID NO: 24, or a sequence having 90% identity and antigen-binding specificity to SEQ ID NO: 19, 20, or 21. L and a region. In another embodiment, the VH region has an amino acid sequence having at least 80% identity to SEQ ID NO: 17 and the antigen-binding specificity of SEQ ID NO: 19-21, and the VL region has an amino acid sequence having at least 80% identity to SEQ ID NO: 18 and the antigen-binding specificity of SEQ ID NO: 22-24. In one embodiment, the second antigen-binding region binds to an immune checkpoint molecule or an immunostimulatory molecule. In another embodiment, the antibody comprises a heavy chain sequence having the amino acid sequence of SEQ ID NO: 46 or 47, or a sequence having 90% identity thereto and the binding specificity thereof. In one embodiment, the bispecific antibody further comprises a light chain having the amino acid sequence of SEQ ID NO: 18. In another embodiment, the bispecific antibody comprises an Fc domain. In some embodiments, the Fc domain is an IgG domain, an IgE domain, an IgM domain, and an IgD domain, an IgA domain, or an IgY domain. In various embodiments, the Fc domain is an IgG domain. In many embodiments, the IgG domain is an IgG1 domain, an IgG2 domain, an IgG3 domain, or an IgG4 domain. In one embodiment, the scFv is linked to the C-terminus of the Fc domain. In another embodiment, the bispecific antibody comprises a linker between the Fab domain and the scFv domain. In some embodiments, the Fab fragment is linked to the N-terminus of the Fc domain. In various embodiments, the Fab comprises a p95HER2 binding site and the scFv comprises a CD137 binding site.

[0018] In one embodiment, the invention provides an antibody-drug conjugate comprising a therapeutic agent and any one of the antibodies or antigen-binding fragments thereof described herein, or any one of the bispecific antibodies described herein.

[0019] In one aspect, the therapeutic agent is covalently attached to the antibody, antigen-binding fragment, or bispecific antibody via a linker.

[0020] In another embodiment, the present invention provides a pharmaceutical composition comprising any one of the antibodies or antigen-binding fragments thereof described herein, any one of the bispecific antibodies described herein, or any one of the antibody-drug conjugates described herein, and at least one pharma- ceutically acceptable carrier.

[0021] In one aspect, the pharma- ceutically acceptable carrier is conjugated to the C-terminus of the amino acid sequence of the antibody, antigen-binding fragment, bispecific antibody, or antibody-drug conjugate.

[0022] In one embodiment, the present invention provides a method of treating cancer comprising administering to a subject in need thereof an effective amount of any one of the antibodies or antigen-binding fragments thereof described herein, any one of the bispecific antibodies described herein, or any one of the antibody-drug conjugates described herein, thereby treating the cancer. In one aspect, the cancer is selected from prostate cancer, lung cancer, non-small cell lung cancer (NSCLC), melanoma, lymphoma, breast cancer, head and neck cancer, renal cell carcinoma (RCC), ovarian cancer, kidney cancer, bladder cancer, uterine cancer, cervical cancer, ovarian cancer, liver cancer, gastric cancer, colon cancer, rectal cancer, oral cancer, pharyngeal cancer, pancreatic cancer, thyroid cancer, skin cancer, brain cancer, bone cancer, hematopoietic cancer, or leukemia.

[0023] In another embodiment, the present invention provides an isolated amino acid sequence set forth in SEQ ID NOs: 1-42 or 46-47.

[0024] In one embodiment, the invention provides an isolated nucleic acid sequence encoding any one of the antibodies or antigen-binding fragments thereof, or bispecific antibodies described herein. In another embodiment, the invention provides an isolated nucleic acid encoding any one of SEQ ID NOs: 1-42 or 46-47.

[0025] In further embodiments, the present invention provides four antibodies or antigen-binding fragments thereof, including anti-CD73 antibody clone 3-4 (CD73 #3-4), anti-CD73 antibody clone 3-16 (CD73 #3-16), anti-CD73 antibody clone 3-1-28 (CD73 #3-1-28), and anti-CD73 antibody clone 3-1-37 (CD73 #3-1-37), as well as three affinity matured antibodies derived from anti-CD73 clone 3-1-28, including anti-CD73 #3-1-28(S51A Q54E), anti-CD73 #3-1-28(S107D S109H), and anti-CD73 #3-1-28(S107D S109T A114S).

[0026] In one aspect, an anti-CD73 antibody provided herein comprises a heavy chain variable (VH) region comprising an amino acid sequence having at least 80% sequence identity to a sequence selected from SEQ ID NO:49, SEQ ID NO:57, SEQ ID NO:65, and SEQ ID NO:73, and a light chain variable (VL) region comprising an amino acid sequence having at least 80% sequence identity to a sequence selected from SEQ ID NO:50, SEQ ID NO:58, SEQ ID NO:66, and SEQ ID NO:74, SEQ ID NO:81, SEQ ID NO:83, and SEQ ID NO:85.

[0027] In one embodiment, the antibody or antigen-binding fragment comprises a VH region comprising an amino acid sequence having at least 80% identity to SEQ ID NO: 49, and a VL region comprising an amino acid sequence having at least 80% identity to SEQ ID NO: 50, and wherein: (a) VH CDR-H1, CDR-H2, and CDR-H3, wherein CDR-H1 comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 51, CDR-H2 comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 52, and CDR-H3 comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 53; and (b) VL It comprises CDR-L1, CDR-L2, and CDR-L3 (wherein CDR-L1 comprises an amino acid sequence having at least 80% identity to SEQ ID NO:54, CDR-L2 comprises an amino acid sequence having at least 80% identity to SEQ ID NO:55, and CDR-L3 comprises an amino acid sequence having at least 80% identity to SEQ ID NO:56).

[0028] In another embodiment, the antibody, or antigen-binding fragment thereof, comprises a VH region comprising an amino acid sequence having at least 80% identity to SEQ ID NO: 57 and a VL region comprising an amino acid sequence having at least 80% identity to SEQ ID NO: 58, and wherein: (a) VH CDR-H1, CDR-H2, and CDR-H3, wherein CDR-H1 comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 59, CDR-H2 comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 60, and CDR-H3 comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 61; and (b) a VL region comprising an amino acid sequence having at least 80% identity to SEQ ID NO: 62. It comprises CDR-L1, CDR-L2, and CDR-L3 (wherein CDR-L1 comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 62, CDR-L2 comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 63, and CDR-L3 comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 64).

[0029] In one embodiment, the antibody, or antigen-binding fragment thereof, comprises a VH region comprising an amino acid sequence having at least 80% identity to SEQ ID NO: 65, and a VL region comprising an amino acid sequence having at least 80% identity to SEQ ID NO: 66, and wherein: (a) VH CDR-H1, CDR-H2, and CDR-H3, wherein CDR-H1 comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 67, CDR-H2 comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 68, and CDR-H3 comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 69; and (b) VL It comprises CDR-L1, CDR-L2, and CDR-L3 (wherein CDR-L1 comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 70, CDR-L2 comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 71, and CDR-L3 comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 72).

[0030] In another embodiment, the antibody, or antigen-binding fragment thereof, comprises a VH region comprising an amino acid sequence having at least 80% identity to SEQ ID NO: 73 and a VL region comprising an amino acid sequence having at least 80% identity to SEQ ID NO: 74, and wherein CDR-H1, CDR-H2, and CDR-H3 comprise an amino acid sequence having at least 80% identity to SEQ ID NO: 75, CDR-H2, and CDR-H3 comprise an amino acid sequence having at least 80% identity to SEQ ID NO: 76, and CDR-H3 comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 77; and (b) a VL region comprising an amino acid sequence having at least 80% identity to SEQ ID NO: 78. It comprises CDR-L1, CDR-L2, and CDR-L3 (wherein CDR-L1 comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 78, CDR-L2 comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 79, and CDR-L3 comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 80).

[0031] In additional embodiments, the invention provides three affinity matured antibodies derived from anti-CD73 clone 3-1-28, including anti-CD73 #3-1-28(S51A Q54E), anti-CD73 #3-1-28(S107D S109H), and anti-CD73 #3-1-28(S107D S109T A114S).

[0032] In one embodiment, an anti-CD73 #3-1-28 (S51A Q54E) antibody or antigen-binding fragment thereof having a VL region comprising an amino acid sequence having at least 80% identity to SEQ ID NO: 81 comprises a VL CDR-L2 region comprising an amino acid sequence having at least 80% identity to SEQ ID NO: 82. In another embodiment, an anti-CD73 #3-1-28 (S107D S109H) antibody or antigen-binding fragment thereof having a VH region comprising an amino acid sequence having at least 80% identity to SEQ ID NO: 83 comprises a VH CDR-H3 region comprising an amino acid sequence having at least 80% identity to SEQ ID NO: 84. In yet another embodiment, an anti-CD73 #3-1-28 (S107D S109T A114S) antibody or antigen-binding fragment thereof having a VH region comprising an amino acid sequence having at least 80% identity to SEQ ID NO: 85 comprises a VH CDR-H3 region comprising an amino acid sequence having at least 80% identity to SEQ ID NO: 86.

[0033] In one embodiment, the antibody, or antigen-binding fragment thereof, comprises a VH region comprising an amino acid sequence having at least 80% identity to SEQ ID NO: 65, and a VL region comprising an amino acid sequence having at least 80% identity to SEQ ID NO: 81, and wherein: (a) VH CDR-H1, CDR-H2, and CDR-H3, wherein CDR-H1 comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 67, CDR-H2 comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 68, and CDR-H3 comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 69; and (b) VL It comprises CDR-L1, CDR-L2, and CDR-L3 (wherein CDR-L1 comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 70, CDR-L2 comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 82, and CDR-L3 comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 72).

[0034] In another embodiment, the antibody, or antigen-binding fragment thereof, comprises a VH region comprising an amino acid sequence having at least 80% identity to SEQ ID NO: 65 and a VL region comprising an amino acid sequence having at least 80% identity to SEQ ID NO: 83, and wherein: (a) VH CDR-H1, CDR-H2, and CDR-H3, wherein CDR-H1 comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 67, CDR-H2 comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 68, and CDR-H3 comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 69; and (b) VL It comprises CDR-L1, CDR-L2, and CDR-L3 (wherein CDR-L1 comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 70, CDR-L2 comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 84, and CDR-L3 comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 72).

[0035] In yet another aspect, the antibody, or antigen-binding fragment thereof, comprises a VH region comprising an amino acid sequence having at least 80% identity to SEQ ID NO: 65 and a VL region comprising an amino acid sequence having at least 80% identity to SEQ ID NO: 85, and further comprises (a) a VH CDR-H1, CDR-H2, and CDR-H3, wherein CDR-H1 comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 67, CDR-H2 comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 68, and CDR-H3 comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 69, and (b) a VL It comprises CDR-L1, CDR-L2, and CDR-L3 (wherein CDR-L1 comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 70, CDR-L2 comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 86, and CDR-L3 comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 72).

[0036] In one aspect, the antibodies or antigen-binding fragments thereof provided herein comprise an Fc domain. In some aspects, the Fc domain is an IgG, IgE, IgM, IgD, IgA, or IgY domain. In various aspects, the IgG domain is an IgG1, IgG2, IgG3, or IgG4 domain. In other aspects, the IgG1 domain comprises the amino acid sequence of SEQ ID NO: 87. In various aspects, the IgG1 comprises a point mutation that alters or reduces antibody-dependent cellular cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC) compared to wild-type IgG1. In many aspects, the point mutation comprises K297A and K322A mutations.

[0037] In another embodiment, the invention provides a bispecific antibody comprising a first antigen-binding region and a second antigen-binding region, wherein the first antigen-binding region binds to CD73.

[0038] In one embodiment the bispecific antibody comprises a VH region comprising CDR-H1, CDR-H2 and CDR-H3, wherein CDR-H1 is set forth in SEQ ID NO: 67, CDR-H2 is set forth in SEQ ID NO: 68 and CDR-H3 is set forth in SEQ ID NO: 69, or sequences with 90% identity and antigen binding specificity to SEQ ID NO: 67, 68 or 69, and a VL region comprising CDR-L1, CDR-L2 and CDR-L3, wherein CDR-L1 is set forth in SEQ ID NO: 70, CDR-L2 is set forth in SEQ ID NO: 71 and CDR-L3 is set forth in SEQ ID NO: 72, or sequences with 90% identity and antigen binding specificity to SEQ ID NO: 70, 71 or 72. In another embodiment, the bispecific antibody comprises a VH region comprising CDR-H1, CDR-H2 and CDR-H3, wherein CDR-H1 is set forth in SEQ ID NO: 67, CDR-H2 is set forth in SEQ ID NO: 68 and CDR-H3 is set forth in SEQ ID NO: 69, or sequences with 90% identity and antigen binding specificity to SEQ ID NO: 67, 68 or 69, and a VL region comprising CDR-L1, CDR-L2 and CDR-L3, wherein CDR-L1 is set forth in SEQ ID NO: 70, CDR-L2 is set forth in SEQ ID NO: 82 and CDR-L3 is set forth in SEQ ID NO: 72, or sequences with 90% identity and antigen binding specificity to SEQ ID NO: 70, 82 or 72. In one embodiment the bispecific antibody comprises a VH region comprising CDR-H1, CDR-H2 and CDR-H3, wherein CDR-H1 is set forth in SEQ ID NO: 67, CDR-H2 is set forth in SEQ ID NO: 68 and CDR-H3 is set forth in SEQ ID NO: 69, or sequences with 90% identity and antigen binding specificity to SEQ ID NO: 67, 68 or 69, and a VL region comprising CDR-L1, CDR-L2 and CDR-L3, wherein CDR-L1 is set forth in SEQ ID NO: 70, CDR-L2 is set forth in SEQ ID NO: 84 and CDR-L3 is set forth in SEQ ID NO: 72, or sequences with 90% identity and antigen binding specificity to SEQ ID NO: 70, 84 or 72.In another embodiment, the bispecific antibody comprises a VH region comprising CDR-H1, CDR-H2 and CDR-H3, wherein CDR-H1 is set forth in SEQ ID NO: 67, CDR-H2 is set forth in SEQ ID NO: 68 and CDR-H3 is set forth in SEQ ID NO: 69, or sequences with 90% identity and antigen binding specificity to SEQ ID NO: 67, 68 or 69, and a VL region comprising CDR-L1, CDR-L2 and CDR-L3, wherein CDR-L1 is set forth in SEQ ID NO: 70, CDR-L2 is set forth in SEQ ID NO: 86 and CDR-L3 is set forth in SEQ ID NO: 72, or sequences with 90% identity and antigen binding specificity to SEQ ID NO: 70, 86 or 72. In one aspect, the VH region has an amino acid sequence having at least 80% identity to SEQ ID NO: 65 and the antigen-binding specificity of SEQ ID NO: 67 to 69, and the VL region has an amino acid sequence having at least 80% identity to SEQ ID NO: 66 and the antigen-binding specificity of SEQ ID NO: 70 to 72. In another aspect, the VH region has an amino acid sequence having at least 80% identity to SEQ ID NO: 65 and the antigen-binding specificity of SEQ ID NO: 67 to 69, and the VL region has an amino acid sequence having at least 80% identity to SEQ ID NO: 81 and the antigen-binding specificity of SEQ ID NO: 70, 82, and 72. In one aspect, the VH region has an amino acid sequence having at least 80% identity to SEQ ID NO: 65 and the antigen-binding specificity of SEQ ID NO: 67 to 69, and the VL region has an amino acid sequence having at least 80% identity to SEQ ID NO: 82 and the antigen-binding specificity of SEQ ID NO: 70, 84, and 72. In another aspect, the VH region has an amino acid sequence having at least 80% identity to SEQ ID NO: 65 and the antigen-binding specificity of SEQ ID NOs: 67-69, and the VL region has an amino acid sequence having at least 80% identity to SEQ ID NO: 85 and the antigen-binding specificity of SEQ ID NOs: 70, 86 and 72. In one aspect, the second antigen-binding region binds to an immune checkpoint molecule or an immunostimulatory molecule. In one aspect, the antibody comprises a heavy chain sequence having the amino acid sequence of SEQ ID NO: 88, 89, 90 or 91, or a sequence having 90% identity thereto and the binding specificity thereof. In another aspect, the bispecific antibody further comprises a light chain having the amino acid sequence of SEQ ID NO: 66.

[0039] In one embodiment, the invention provides an antibody-drug conjugate comprising a therapeutic agent and any one of the antibodies or antigen-binding fragments thereof described herein, or any one of the bispecific antibodies described herein.

[0040] In another embodiment, the present invention provides a pharmaceutical composition comprising any one of the antibodies or antigen-binding fragments thereof described herein, any one of the bispecific antibodies described herein, or any one of the antibody-drug conjugates described herein, and at least one pharma- ceutically acceptable carrier.

[0041] In one embodiment, the invention provides a method of treating cancer comprising administering to a subject in need thereof an effective amount of any one of the antibodies or antigen-binding fragments thereof described herein, any one of the bispecific antibodies described herein, or any one of the antibody-drug conjugates described herein, thereby treating the cancer.

[0042] In another embodiment, the present invention provides an isolated amino acid sequence set forth in SEQ ID NOs: 49-86 or 88-91.

[0043] In one embodiment, the invention provides an isolated nucleic acid sequence encoding any one of the antibodies or antigen-binding fragments thereof, or bispecific antibodies described herein. In another embodiment, the invention provides an isolated nucleic acid encoding any one of SEQ ID NOs: 49-86 or 88-91. [Brief description of the drawings]

[0044] [Figure 1-1]Figures 1A-1C show the screening of phage clones targeted against p95HER2 by flow cytometer. Phage clones recognizing p95HER2 were enriched after four rounds of panning. Positive phage binders were selected by staining p95HER2-HEK293 cells with phage-containing supernatant. Figure 1A shows that phage clones with specific binding to p95HER2-HEK293 cells were determined by flow cytometer. Figure 1B shows that clones (numbered 1-40) that specifically bound to p95HER2-HEK293 cells with ΔMFI above 800 were selected for sequencing. Negative control (NC), stained with secondary antibody only. Positive control (PC), stained with both primary anti-p95HER2 ref antibody and secondary antibody. Figure 1C shows that phage clones with different sequences (marked in bold with grey background) were selected for conversion to monoclonal antibodies. The clones were designated R4-X, where X is the number in FIG. 1A. [Figure 1-2] See description of Figure 1-1. [Diagram 2] 1 shows the purity of Protein A purified anti-p95 HER2 antibody lead by SDS-PAGE. The purity and integrity of the one-step Protein A purified anti-p95 HER2 antibody lead was analyzed by SDS-PAGE under non-reducing and reducing conditions. [Diagram 3] Figures 3A-3B show the binding activity of purified anti-p95HER2 antibody leads by flow cytometer. Figure 3A shows that P95HER2-expressing HEK293 cells were surface stained with anti-p95HER2 antibodies (30, 3, 0.3 nM) for 1 hour on ice, and then incubated with A488-conjugated anti-human IgG secondary antibody for another hour on ice. Cells were analyzed by flow cytometer, and the fluorescence intensity of A488 was shown by numbers. Figure 3B shows that P95HER2-expressing NCI-H292 stable cells were surface stained with selected anti-p95HER2 antibodies and analyzed by flow cytometer. Binding curves and EC50 were shown. [Figure 4A]Figures 4A-4B show binding epitope mapping of anti-p95HER2 antibody leads. Figure 4A shows that anti-p95HER2 #R4-3, R4-5, R4-11, R4-15 and R4-38 antibodies showed broadly identical responses to peptides with the consensus motif KFPDE, with medium to high spot intensities and high signal-to-noise ratios. Antibody R4-3 showed a slightly weaker response, and antibody R4-38 showed the strongest response among these antibodies, even though the intensity levels did not vary significantly. Figure 4B is a heat map showing anti-p95HER2 antibodies binding to linear 5, 10 and 15 amino acid peptides with peptide-peptide overlap. Binding intensities are shown for each peptide and corrected for its own background value. [Figure 4B] See legend to Figure 4A. [Diagram 5] 1 shows a symmetric format of anti-p95HER2-CD137 bispecific antibody (bsAb). [Figure 6] Figures 6A-6B show protein aggregation and integrity of anti-p95HER2-CD137 bsAb by SEC-HPLC and μCE-SDS-PAGE, respectively. Figure 6A shows analysis of protein aggregation by SEC-HPLC. Figure 6B shows analysis of protein integrity by μCE-SDS-PAGE under non-reducing and reducing conditions, and the data are summarized in the table below. [Figure 7] Figures 7A-7B show the binding affinity of anti-p95HER2-CD137 bsAb to p95HER2 and CD137 antigens by biolayer interferometry. Figure 7A shows the binding kinetics to human p95HER2 antigen. Figure 7B shows the binding kinetics to human CD137 antigen. [Figure 8] Shows simultaneous binding to p95HER2 and CD137 antigens by anti-p95HER2-CD137 bsAb. [Figure 9]Figures 9A-9B show that anti-p95HER2-CD137 bsAb induces IFN-γ production in T cells when co-cultured with p95HER2-expressing NCI-H292 cells. Figure 9A shows the expression level of p95HER2 on p95HER2-expressing NCI-H292 cells. Figure 9B shows IFN-γ production in T cells when co-cultured with p95HER2-expressing NCI-H292 lung cancer cells. [Figure 10] Figures 10A-10B show antibody-dependent cell-mediated cytotoxicity of anti-p95HER2-CD137 bsAb p95HER2-expressing NCI-H292 cells. Figure 10A shows the expression levels of CD137 on CD137-expressing HEK293 cells (left). ADCC of anti-p95HER2-CD137 bsAb with normal or ADCC-enhanced IgG1 format (right). Figure 10B shows the expression levels of p95HER2 on p95HER2-expressing NCI-H292 cells (left). Anti-p95HER2-CD137 bsAb with normal IgG1 format induced dose-dependent ADCC similar to #R4-11 IgG1 monoclonal antibody (right). [Figure 11-1] Figures 11A-11C show the anti-tumor activity of antibody p95HER2-CD137 bsAb in an in vivo MDA-MB-231 breast cancer model. Figure 11A shows p95HER2 expression by MDA-MB-231 cells. Figure 11B shows tumor volume. Figure 11C shows tumor mass. [Figure 11-2] See description of Figure 11-1. [Figure 12] 1 shows the pharmacokinetic profile of anti-p95HER2-CD137 bsAb in vivo. [Figure 13-1] Figures 13A-13C show screening of phage clones targeted against CD73 by ELISA. Figure 13A shows the ELISA results of phage clones isolated from the third round of MDA-MB-231 cell-based panning. Figure 13B shows the ELISA results of phage clones isolated from the third round of CD73-overexpressing Expi293 cell-based panning in the third round. Figure 13C shows the list of clone names numbered in Figures 13A and 13B. [Figure 13-2] See description of Figure 13-1. [Figure 14] 1 shows the purity of Protein A purified anti-CD73 antibody lead by SDS-PAGE. [Figure 15] 1 shows binding of anti-CD73 antibodies to CD73-expressing MDA-MB-231 breast cancer cells. [Figure 16] Blockade of CD73 enzymatic activity by anti-CD73 antibodies. [Figure 17-1] Figures 17A-17C show the screening of affinity matured clones derived from anti-CD73 clone 3-1-28 antibody. Figure 17A shows the CD73 binding activity of the phage clones. Figure 17B shows the binding activity of the antibodies converted from the phage clones numbered in Figure 17A. Figure 17C shows the binding kinetics of the antibodies selected in Figure 17B. [Figure 17-2] See description of Figure 17-1. [Figure 18] Figures 18A-18B show blockade of membrane-bound and soluble CD73 enzymatic activity by affinity matured anti-CD73#3-1-28 antibody. Figure 18A shows membrane-bound CD73 expressed by MDA-MB-231 cells. Figure 18B shows soluble CD73 secreted by MDA-MB-231 cells. [Figure 19] 1 shows a symmetric format of anti-CD73-CD137 bispecific antibody (bsAb). [Figure 20A] Figures 20A-20B show protein aggregation and integrity of anti-CD73-CD137 bsAb by SEC-HPLC and μCE-SDS-PAGE, respectively. Figure 20A shows analysis of protein aggregation by SEC-HPLC. Figure 20B shows analysis of protein integrity by μCE-SDS-PAGE under non-reducing and reducing conditions. [Figure 20B-1] See legend to Figure 20A. [Figure 20B-2] See legend to Figure 20A. [Figure 21]Figures 21A-21B show the binding affinity of anti-CD73-CD137 bsAb to CD73 and CD137 antigens, respectively, measured by biolayer interferometry. Figure 21A shows the binding kinetics to human CD73 antigen. Figure 21B shows the binding kinetics to human CD137 antigen. [Figure 22] Shows simultaneous binding of CD73 and CD137 antigens by anti-CD73-CD137 bsAb. [Diagram 23] Blockade of CD73 enzymatic activity by anti-CD73-CD137 bsAb. Anti-CD73-CD137 bsAb showed comparable efficacy in inhibiting CD73 enzymatic activity to the parental anti-CD73 antibody. [Figure 24] Shows rescue of AMP-suppressed T cell activation by anti-CD73-CD137 bsAb. [Figure 25-1] Figures 25A-25C show that anti-CD73-CD137 bsAb induces IFN-γ production in T cells when co-cultured with CD73-expressing cancer cells. Figure 25A shows the expression levels of CD73 on MDA-MB-231 and NCI-H292 cells. Figure 25B shows IFN-γ production in T cells when co-cultured with CD73-expressing MDA-MB-231 (breast cancer cells). Figure 25C shows IFN-γ production in T cells when co-cultured with CD73-expressing NCI-H292 (lung cancer cells). [Figure 25-2] See description of Figure 25-1. [Figure 26-1] Figures 26A-26E show the anti-tumor activity of antibody CD73-CD137 bsAb in an in vivo MDA-MB-231 breast cancer model. Figure 26A shows tumor volume. Figure 26B shows tumor mass. Figure 26C shows the percentage of viable huCD45+ cells. Figure 26D shows the percentage of CD8+ T cells among CD3+ cells. Figure 26E shows the percentage of CD8+ T cells among CD4+ cells. [Figure 26-2] See description of Figure 26-1. [Figure 27] 1 shows the pharmacokinetic profile of anti-CD73-CD137 bsAb in vivo. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0045] Detailed Description of the Invention The present invention is based on the discovery of anti-p95HER2 antibodies that exhibit superior activity in binding to p95HER2-expressing cancer cells and anti-CD73 antibodies that exhibit superior activity in neutralizing the enzymatic activity of CD73 in cell membrane-bound or soluble forms. The present invention is further based on the discovery that bispecific antibodies targeting p95HER2 and CD137 have dual functions of antibody-dependent cell-mediated cytotoxicity (ADCC) and p95HER2-dependent T cell activation against p95HER-positive cancer cells, and that bispecific antibodies targeting CD73 and CD137 have exceptional activity for enhancing T cell effector function and better inhibiting tumor growth in vivo than either antibody monotherapy or combination therapy.

[0046] Before describing the compositions and methods of the present invention, it is to be understood that the invention is not limited to the particular compositions, methods, and experimental conditions described, as such compositions, methods, and conditions may vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments, and is not intended to be limiting, since the scope of the present invention is limited only by the appended claims.

[0047] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to "a method" includes one or more methods, and / or steps, of the type described herein that will be apparent to those skilled in the art upon reading this disclosure and so forth.

[0048] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0049] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs. Any methods and materials similar or equivalent to those described herein may be used in the practice or testing of the present invention, but it will be understood that modifications and variations are within the spirit and scope of the present disclosure. Preferred methods and materials are now described.

[0050] p95HER2 targeting peptide The receptor tyrosine kinase HER2 (ErbB2) is one of four members of the epidermal growth factor receptor family (HER1-4) and is overexpressed in different tumors, including 20-30% of breast and gastric cancers. HER1-4 share a similar structure, i.e., an extracellular ligand-binding domain, a short hydrophobic transmembrane region, and a cytoplasmic tyrosine kinase domain. Activation of the HER family is induced by ligand binding or receptor overexpression and results in cell proliferation, differentiation, adhesion, migration, and survival.

[0051] The recombinant humanized monoclonal antibody trastuzumab binds to the extracellular domain of HER2. Trastuzumab improves recurrence-free and overall survival in patients with HER2-positive early breast cancer. Furthermore, combination therapy of trastuzumab with chemotherapy improves survival and provides substantial clinical benefit in patients with HER2-positive advanced breast cancer. However, only 20-30% of patients with HER2-positive breast cancer respond to trastuzumab when administered as monotherapy. Patients who do not respond to trastuzumab are caused by several potential mechanisms leading to primary or acquired resistance, including activation of insulin-like growth factor receptor (IGF-1R) signaling, inactivation or loss of the gene for phosphatase and tensin homolog deleted on chromosome 10 (PTEN), and accumulation of p95HER2.

[0052] p95HER2 is a truncated form of the HER2 receptor that lacks the extracellular trastuzumab-binding domain, and approximately 30-40% of HER2-positive tumors express p95HER2. In some tumors, p95HER2 is the predominant HER2 form. p95HER2 is generated by selective initiation of translation from two methionine residues (611 and 687) or by proteolytic shedding of full-length HER2. The biological role of p95HER2 has not been fully evaluated, and the possible relationship between p95HER2 and full-length HER2 is unclear. p95HER2 has tyrosine kinase activity that is essential for xenograft tumor growth in nude mice. Due to its exclusive expression in cancerous tissues, p95HER2 would be a good target for cancer therapy. We developed a bispecific antibody that engages T cells and p95HER2-expressing cancer cells and showed effective antitumor activity in orthotopic MCF-7 breast cancer and PDX models. In addition, we developed p95HER2 CAR T cells that had significant activity against p95HER2-expressing primary tumors and metastases in the lung or brain in vivo.

[0053] Bispecific antibodies (bsAbs), blinatumomab (Blincyto®) and catumaxomab (Removab®), which engage TAA of tumor cells and CD3 of T cells, have shown anti-cancer efficacy and have been approved for the treatment of Philadelphia chromosome-negative B acute lymphoblastic leukemia (B-ALL) and malignant ascites associated with ovarian cancer. However, poor efficacy in solid tumors and systemic cytokine release of BiTEs limit their use. Compared to anti-CD3, anti-CD137 agonist antibodies may offer better therapeutic window and safety profile as proven efficacy and cross-linking-dependent agonist activity against solid tumors in clinical trials. Utilizing the cancer-specific p95HER2 expression and the target-dependent T cell activation platform of our invented cross-linking-dependent anti-CD137 single chain antibody, drugs with potent efficacy against p95HER2-positive solid tumors and satisfactory safety profile can be generated.

[0054] Provided herein are novel anti-p95HER2 monoclonal antibodies that were generated and screened for the strongest binding affinity. Also provided herein are novel bispecific antibodies that simultaneously target p95HER2 and CD137 to induce superior T cell activation in the presence of p95HER2.

[0055] Anti-p95HER2 antibody and its antigen-binding fragment In one embodiment, the present invention provides antibodies and antigen-binding fragments thereof that bind to p95HER2. In one aspect, the present invention provides four antibodies or antigen-binding fragments thereof, including anti-p95HER2 antibody clone R4-3 (p95HER2 #R4-3), anti-p95HER2 antibody clone R4-5 (p95HER2 #R4-5), anti-p95HER2 antibody clone R4-11 (p95HER2 #R4-11), anti-p95HER2 antibody clone R4-15 (p95HER2 #R4-15), and anti-p95HER2 antibody clone R4-38 (p95HER2 #R4-38). In one aspect, the anti-p95HER2 antibody provided herein comprises a heavy chain variable (VH) region comprising an amino acid sequence having at least 80% sequence identity to a sequence selected from SEQ ID NO:1, SEQ ID NO:9, SEQ ID NO:17, SEQ ID NO:25, and SEQ ID NO:33, and a light chain variable (VL) region comprising an amino acid sequence having at least 80% sequence identity to a sequence selected from SEQ ID NO:2, SEQ ID NO:10, SEQ ID NO:18, SEQ ID NO:26, and SEQ ID NO:34.

[0056] As used herein, the term "antibody" refers to an immunoglobulin molecule capable of specifically binding to an antigen. The term "antibody" includes, but is not limited to, monoclonal antibodies, polyclonal antibodies, human antibodies, humanized antibodies, chimeric antibodies, bispecific antibodies, and anti-idiotypic antibodies, unless the context clearly indicates otherwise. In one aspect, the antibodies provided herein include monoclonal antibodies. The antibodies provided herein include any isotype and class (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2). As used herein, "antigen-binding fragment" refers to a fragment or portion of an immunoglobulin molecule or antibody that has the ability to specifically bind to the same antigen as the immunoglobulin molecule or antibody. Exemplary antigen-binding fragments include scFv, Fab, or F(ab)2 fragments. As used herein, "antigen-binding region" refers to a portion of an antibody or immunoglobulin molecule that binds to an antigen or protein, e.g., by contacting the antigen or protein. An antigen-binding region generally includes a heavy chain variable (VH) region and a light chain variable (VL) region. An antigen-binding region generally includes one or more antigen-binding sites or paratopes.

[0057] The antibodies provided herein have a VH region comprising an amino acid sequence having at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range therebetween, to the sequence of SEQ ID NO:1, SEQ ID NO:9, SEQ ID NO:17, SEQ ID NO:25, or SEQ ID NO:33. The antibodies provided herein also include a VL region comprising an amino acid sequence having at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range therebetween, to the sequence of SEQ ID NO:2, SEQ ID NO:10, SEQ ID NO:18, SEQ ID NO:26, SEQ ID NO:34.

[0058] Generally, "sequence identity" or "sequence homology", which may be used interchangeably, refer to the exact nucleotide-by-nucleotide or amino acid-by-amino acid correspondence of two polynucleotide or polypeptide sequences, respectively. Typically, techniques for determining sequence identity include determining the nucleotide sequence of a polynucleotide and / or the amino acid sequence of the polypeptide encoded thereby, and comparing these sequences to a second nucleotide or amino acid sequence. As used herein, the term "percent sequence identity" or "percent identity", including "homology", refers to the percentage of amino acid residues or nucleotides in a sequence that are identical to the amino acid residues or nucleotides in a reference sequence, without considering any conservative substitutions as part of the sequence identity, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Thus, two or more sequences (polynucleotide or amino acid) can be compared by determining their "percent identity" (also referred to as "percent homology"). Percent identity to a reference sequence (e.g., a nucleic acid or amino acid sequence), which may be a sequence within a longer molecule (e.g., a polynucleotide or polypeptide), may be calculated as the number of exact matches between two optimally aligned sequences divided by the length of the reference sequence multiplied by 100. Percent identity may also be determined by comparing sequence information using, for example, advanced BLAST computer programs, including version 2.2.9 available from the National Institutes of Health.The BLAST program is based on the alignment method of Karlin and Altschul, Proc. Natl. Acad. Sci. USA 87:2264-2268 (1990), as discussed in Altschul, et al., J. Mol. Biol. 215:403-410 (1990), Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873-5877 (1993), and Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997). Briefly, the BLAST program defines identity as the number of identical aligned symbols (i.e., nucleotides or amino acids) divided by the total number of symbols in the shorter of the two sequences. The program may be used to determine the percent identity over the entire length of the sequences being compared. Default parameters are provided for optimizing searches using short query sequences, for example using the blastp program. This program also allows for masking off segments of the query sequence using SEG filters, as determined by the SEG program of Wootton and Federhen, Computers and Chemistry 17:149-163 (1993). The desired degree of sequence identity ranges from approximately 80% to 100%, and integer values ​​therebetween. The percent identity between the reference sequence and the claimed sequence can be at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, or at least 99.9%. In general, a perfect match indicates 100% identity over the entire length of the reference sequence.Additional programs and methods for comparing sequences and / or assessing sequence identity include the Needleman-Wunsch algorithm (see, e.g., EMBOSS Needle aligner, available at www.ebi.ac.uk / Tools / psa / emboss_needle / , optionally using default settings), the Smith-Waterman algorithm (see, e.g., EMBOSS Water aligner, available at www.ebi.ac.uk / Tools / psa / emboss_water / , optionally using default settings), the similarity search method of Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85, 2444, or computer programs that use these algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.). In some embodiments, references to percent sequence identity refer to sequence identity measured using BLAST (Basic Local Alignment Search Tool). In other embodiments, ClustalW is used for multiple sequence alignment. Optimal alignment can be assessed using any suitable parameters of the selected algorithm, including default parameters.

[0059] In one embodiment, the antibody or antigen-binding fragment thereof has an amino acid sequence having at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range therebetween, to SEQ ID NO:1. and a VL region comprising an amino acid sequence having at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range therebetween, to SEQ ID NO: 2. SEQ ID NO: 1 provides an amino acid sequence comprising the variable region of the anti-p95HER2 antibody clone #4-3 heavy chain. SEQ ID NO: 2 provides an amino acid sequence comprising the variable region of the anti-p95HER2 antibody clone #4-3 light chain.

[0060] The antigen-binding region of an antibody or an antigen-binding fragment thereof generally comprises a complementarity determining region (CDR). "Complementarity determining region (CDR)" refers to the hypervariable regions of VH and VL. CDRs comprise the target protein or antigen-binding site of an antibody that confers specificity to protein or antigen binding. VH and VL generally comprise three sequentially numbered CDRs. As used herein, CDR-H1, CDR-H2, and CDR-H3 refer to the three consecutively arranged CDRs of the heavy chain variable region (VH) as numbered from the N-terminus of the heavy chain polypeptide. As used herein, CDR-L1, CDR-L2, and CDR-L3 refer to the three consecutively arranged CDRs of the light chain variable region (VL) as numbered from the N-terminus of the light chain polypeptide.

[0061] In one embodiment, an antigen-binding region or antigen-binding fragment thereof of an antibody having a VH region comprising an amino acid sequence having at least about 80% identity to SEQ ID NO:1 and a VL region comprising an amino acid sequence having at least about 80% identity to SEQ ID NO:2 has at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 100% identity, at least about 101% identity, at least about 102% identity, at least about 103% identity, at least about 104% identity, at least about 105% identity, at least about 106% identity, at least about 107% identity, at least about 108% identity, at least about 109% identity, at least about 110% identity, at least about 111% identity, at least about 112% identity, at least about 113% identity, at least about 114% identity, at least about 115% identity, at least about 116% identity, at least about 117% identity, at least about 118% identity, at least about 119% identity, at least about 120% identity, at least about 121% identity, at least about 122% identity, at least about 123% identity, at least about 124% identity, at least about 125% identity, at least about 126% identity, at least about 127% identity, at least about 128% identity, at least about 129% identity, at least about 130% identity, at least about 131% identity, at least about 132% identity, at least CDR-H1 comprising an amino acid sequence having 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range therebetween, and CDR-H2 comprising an amino acid sequence having at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about and CDR-H2 comprising an amino acid sequence having at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range therebetween, and at least about 80% identity, at least about 85% identity to SEQ ID NO:5. and a CDR-H3 comprising an amino acid sequence having at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range therebetween.In another aspect, an antigen-binding region or antigen-binding fragment thereof of an antibody having a VH region comprising an amino acid sequence having at least about 80% identity to SEQ ID NO:1 and a VL region comprising an amino acid sequence having at least about 80% identity to SEQ ID NO:2 has at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 100% identity, at least about 101% identity, at least about 102% identity, at least about 103% identity, at least about 104% identity, at least about 105% identity, at least about 106% identity, at least about 107% identity, at least about 108% identity, at least about 109% identity, at least about 110% identity, at least about 111% identity, at least about 112% identity, at least about 113% identity, at least about 114% identity, at least about 115% identity, at least about 116% identity, at least about 117% identity, at least about 118% identity, at least about 119% identity, at least about 120% identity, at least about 121% identity, at least about 122% identity, at least about 123% identity, at least about 124% identity, at least about 125% identity, at least about 126% identity, at least about 127% identity, at least about 128% identity, at least about 129% identity, at least about 130% identity, at least about 131% identity, at least about 132% identity, at least CDR-L1 comprising an amino acid sequence having about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range therebetween, and CDR-L2 comprising an amino acid sequence having at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about and CDR-L2 comprising an amino acid sequence having at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range therebetween; and CDR-L2 comprising an amino acid sequence having at least about 80% identity, at least about 85% identity, or any number or range therebetween, to SEQ ID NO:8. and a CDR-L3 comprising an amino acid sequence having at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range therebetween.

[0062] In some embodiments, the antibody or antigen-binding fragment thereof has an amino acid sequence having at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range therebetween, to SEQ ID NO:9. and a VL region comprising an amino acid sequence having at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range therebetween, to SEQ ID NO: 10. SEQ ID NO: 9 provides an amino acid sequence comprising the variable region of the anti-p95HER2 antibody clone #4-5 heavy chain. SEQ ID NO: 10 provides an amino acid sequence comprising the variable region of the anti-p95HER2 antibody clone #4-5 light chain.

[0063] In one embodiment, an antigen-binding region of an antibody or antigen-binding fragment thereof comprising a VH region comprising an amino acid sequence having at least about 80% identity to SEQ ID NO:9 and a VL region comprising an amino acid sequence having at least about 80% identity to SEQ ID NO:10 has at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 100% identity, at least about 101% identity, at least about 102% identity, at least about 103% identity, at least about 104% identity, at least about 105% identity, at least about 106% identity, at least about 107% identity, at least about 108% identity, at least about 109% identity, at least about 1109% identity, at least about 11112 ... CDR-H1 comprising an amino acid sequence having 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range therebetween, and CDR-H2 comprising an amino acid sequence having at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about and CDR-H2 comprising an amino acid sequence having at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range therebetween; and CDR-H3 comprising an amino acid sequence having at least about 80% identity, at least about 85% identity, or any number or range therebetween, to SEQ ID NO: 13. and a CDR-H3 comprising an amino acid sequence having at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range therebetween.In another aspect, an antigen-binding region of an antibody or antigen-binding fragment thereof comprising a VH region comprising an amino acid sequence having at least about 80% identity to SEQ ID NO:9 and a VL region comprising an amino acid sequence having at least about 80% identity to SEQ ID NO:10 has at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 100% identity, at least about 101% identity, at least about 102% identity, at least about 103% identity, at least about 104% identity, at least about 105% identity, at least about 106% identity, at least about 107% identity, at least about 108% identity, at least about 109% identity, at least about 110% identity, at least about 111% identity, at least about 112% identity, at least about 113% identity, at least about 114% identity, at least about 115% identity, at least about 116% identity, at least about 117% identity, at least about 118% identity, at least about 119% identity, at least about 120% identity, at least about 121% identity, at least about 122% identity, at least about 123% identity, at least about 124% identity, at least about 125% identity, at least about 126% identity, at least about 127% identity, at least about 128% identity, at least about 129% identity, at least about 130% identity, at least about 131% identity, at least about 132% identity, at CDR-L1 comprising an amino acid sequence having about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range therebetween; and CDR-L2 comprising an amino acid sequence having at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range therebetween to SEQ ID NO:15. and CDR-L2 comprising an amino acid sequence having at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range therebetween, and a CDR-L3 having at least about 80% identity, at least about 85% identity, or any number or range therebetween, to SEQ ID NO: 16. and a CDR-L3 comprising an amino acid sequence having identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range therebetween.

[0064] In some embodiments, the antibody or antigen-binding fragment thereof has an amino acid sequence having at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range therebetween, to SEQ ID NO:17. and a VL region comprising an amino acid sequence having at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range therebetween, to SEQ ID NO: 18. SEQ ID NO: 17 provides an amino acid sequence comprising the variable region of the anti-p95HER2 antibody clone #4-11 heavy chain. SEQ ID NO: 18 provides an amino acid sequence comprising the variable region of the anti-p95HER2 antibody clone #4-11 light chain.

[0065] In one embodiment, an antigen-binding region of an antibody or antigen-binding fragment thereof comprising a VH region comprising an amino acid sequence having at least about 80% identity to SEQ ID NO: 17 and a VL region comprising an amino acid sequence having at least about 80% identity to SEQ ID NO: 18 has at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 100% identity, at least about 101% identity, at least about 102% identity, at least about 103% identity, at least about 104% identity, at least about 105% identity, at least about 106% identity, at least about 107% identity, at least about 108% identity, at least about 109% identity, at least about 110% identity, at least about 111% identity, at least about 112% identity, at least about 113% identity, at least about 114% identity, at least about 115% identity, at least about 116% identity, at least about 117% identity, at least about 118% identity, at least about 119 ...9% identity, at least about 119% identity, at least about 119% identity, at least about 119% identity, at least about 119% CDR-H1 comprising an amino acid sequence having about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range therebetween, and CDR-H2 comprising an amino acid sequence having at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range therebetween, to SEQ ID NO:20; and CDR-H2 comprising an amino acid sequence having at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range therebetween, and a CDR-H3 having at least about 80% identity, at least about 85% identity, or any number or range therebetween, to SEQ ID NO:21. and a CDR-H3 comprising an amino acid sequence having identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range therebetween.In another aspect, an antigen-binding region of an antibody or antigen-binding fragment thereof comprising a VH region comprising an amino acid sequence having at least about 80% identity to SEQ ID NO: 17 and a VL region comprising an amino acid sequence having at least about 80% identity to SEQ ID NO: 18 has at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 100% identity, at least about 101% identity, at least about 102% identity, at least about 103% identity, at least about 104% identity, at least about 105% identity, at least about 106% identity, at least about 107% identity, at least about 108% identity, at least about 109% identity, at least about 110% identity, at least about 111% identity, at least about 112% identity, at least about 113% identity, at least about 114% identity, at least about 115% identity, at least about 116% identity, at least about 117% identity, at least about 118% identity, at least about 119% identity, at least about 120% identity, at least about 121% identity, at least about 122% identity, at least about 125% identity, at least about 126% identity, at least about 127% identity, at least about 128% identity, at least about 129% identity, at least about 130% identity, at least about 131% identity, at least about 132% identity, at least about 133% identity, at least about 134% and CDR-L1 comprising an amino acid sequence having at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range therebetween to SEQ ID NO:23; and CDR-L2 comprising an amino acid sequence having at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range therebetween to SEQ ID NO:23. and CDR-L2 comprising an amino acid sequence having at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range therebetween, and a CDR-L3 having at least about 80% identity, at least about 85% identity, or any number or range therebetween, to SEQ ID NO:24. and a CDR-L3 comprising an amino acid sequence having identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range therebetween.

[0066] In some embodiments, the antibody or antigen-binding fragment thereof has an amino acid sequence having at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range therebetween, to SEQ ID NO:25. and a VL region comprising an amino acid sequence having at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range therebetween, to SEQ ID NO: 26. SEQ ID NO: 25 provides an amino acid sequence comprising the variable region of the anti-p95HER2 antibody clone #4-15 heavy chain. SEQ ID NO: 26 provides an amino acid sequence comprising the variable region of the anti-p95HER2 antibody clone #4-15 light chain.

[0067] In one embodiment, an antigen-binding region of an antibody or antigen-binding fragment thereof comprising a VH region comprising an amino acid sequence having at least about 80% identity to SEQ ID NO:25 and a VL region comprising an amino acid sequence having at least about 80% identity to SEQ ID NO:26 has at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 100% identity, at least about 101% identity, at least about 102% identity, at least about 103% identity, at least about 104% identity, at least about 105% identity, at least about 106% identity, at least about 107% identity, at least about 108% identity, at least about 109% identity, at least about 110% identity, at least about 111% identity, at least about 112% identity, at least about 113% identity, at least about 114% identity, at least about 115% identity, at least about 116% identity, at least about 117% identity, at least about 118% identity, at least about 119% identity, at least about 120% identity, at least about 121% identity, at least about 122% identity, at least about 123% identity, at least about 124% identity, at least about 125% identity, at least about 126% identity, at least about 127% identity, at least about 128% identity, at least about 129% identity, at least about 130% identity, at least about 131% identity, at least about 132% identity, CDR-H1 comprising an amino acid sequence having about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range therebetween, and CDR-H2 comprising an amino acid sequence having at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range therebetween, to SEQ ID NO:28. and CDR-H2 comprising an amino acid sequence having at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range therebetween, and a CDR-H3 having at least about 80% identity, at least about 85% identity, or any number or range therebetween, to SEQ ID NO:29. and a CDR-H3 comprising an amino acid sequence having identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range therebetween.In another aspect, an antigen-binding region of an antibody or antigen-binding fragment thereof comprising a VH region comprising an amino acid sequence having at least about 80% identity to SEQ ID NO:25 and a VL region comprising an amino acid sequence having at least about 80% identity to SEQ ID NO:26 has at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 100% identity, at least about 101% identity, at least about 102% identity, at least about 103% identity, at least about 104% identity, at least about 105% identity, at least about 106% identity, at least about 107% identity, at least about 108% identity, at least about 109% identity, at least about 110% identity, at least about 111% identity, at least about 112% identity, at least about 113% identity, at least about 114% identity, at least about 115% identity, at least about 116% identity, at least about 117% identity, at least about 118% identity, at least about 119% identity, at least about 120% identity, at least about 121% identity, at least about 122% identity, at least about 123% identity, at least about 124% identity, at least about 125% identity, at least about 126% identity, at least about 127% identity, at least about 128% identity, at least about 129% identity, at least about 130% identity, at least about 131% identity, at least about 132% identity, and CDR-L1 comprising an amino acid sequence having at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range therebetween to SEQ ID NO: 31; and CDR-L2 comprising an amino acid sequence having at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range therebetween to SEQ ID NO: 31. and CDR-L2 comprising an amino acid sequence having at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range therebetween, and a CDR-L3 having at least about 80% identity, at least about 85% identity, or any number or range therebetween, to SEQ ID NO:32. and a CDR-L3 comprising an amino acid sequence having identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range therebetween.

[0068] In some embodiments, the antibody or antigen-binding fragment thereof has an amino acid sequence having at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range therebetween, to SEQ ID NO:33. and a VL region comprising an amino acid sequence having at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range therebetween, to SEQ ID NO: 34. SEQ ID NO: 33 provides an amino acid sequence comprising the variable region of the anti-p95HER2 antibody clone #4-38 heavy chain. SEQ ID NO: 34 provides an amino acid sequence comprising the variable region of the anti-p95HER2 antibody clone #4-38 light chain.

[0069] In one embodiment, an antigen-binding region of an antibody or antigen-binding fragment thereof comprising a VH region comprising an amino acid sequence having at least about 80% identity to SEQ ID NO: 33 and a VL region comprising an amino acid sequence having at least about 80% identity to SEQ ID NO: 34 has at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 100% identity, at least about 101% identity, at least about 102% identity, at least about 103% identity, at least about 104% identity, at least about 105% identity, at least about 106% identity, at least about 107% identity, at least about 108% identity, at least about 109% identity, at least about 110% identity, at least about 111% identity, at least about 112% identity, at least about 113% identity, at least about 114% identity, at least about 115% identity, at least about 116% identity, at least about 117% identity, at least about 118% identity, at least about 119% identity, at least about 120% identity, at least about 121% identity, at least about 122% identity, at least about 123% identity, at least about 124% identity, at least about 125% identity, at least about 126% identity, at least about 127% identity, at least about 128% identity, at least about 129% identity, at least about 130% identity, at least about 131% identity, at least about 132% CDR-H1 comprising an amino acid sequence having about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range therebetween, and CDR-H2 comprising an amino acid sequence having at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range therebetween to SEQ ID NO:36; and CDR-H2 comprising an amino acid sequence having at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range therebetween, and a CDR-H3 having at least about 80% identity, at least about 85% identity, or any number or range therebetween, to SEQ ID NO:37. and a CDR-H3 comprising an amino acid sequence having identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range therebetween.In another aspect, an antigen-binding region of an antibody or antigen-binding fragment thereof comprising a VH region comprising an amino acid sequence having at least about 80% identity to SEQ ID NO: 33 and a VL region comprising an amino acid sequence having at least about 80% identity to SEQ ID NO: 34 has at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 100% identity, at least about 101% identity, at least about 102% identity, at least about 103% identity, at least about 104% identity, at least about 105% identity, at least about 106% identity, at least about 107% identity, at least about 108% identity, at least about 109% identity, at least about 110% identity, at least about 111% identity, at least about 112% identity, at least about 113% identity, at least about 114% identity, at least about 115% identity, at least about 116% identity, at least about 117% identity, at least about 118% identity, at least about 119% identity, at least about 120% identity, at least about 121% identity, at least about 122% identity, at least about 123% identity, at least about 124% identity, at least about 125% identity, at least about 126% identity, at least about 127% identity, at least about 128% identity, at least about 129% identity, at least about 130% identity, at least about 131% identity, at least about 132% and CDR-L1 comprising an amino acid sequence having at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range therebetween to SEQ ID NO: 39; and CDR-L2 comprising an amino acid sequence having at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range therebetween to SEQ ID NO: 39. and CDR-L2 comprising an amino acid sequence having at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range therebetween, and CDR-L3 comprising an amino acid sequence having at least about 80% identity, at least about 85% identity, or any number or range therebetween, to SEQ ID NO:40. and a CDR-L3 comprising an amino acid sequence having identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range therebetween.

[0070] The antibodies or antigen-binding fragments thereof provided herein further comprise an Fc domain. As used herein, the term Fc domain refers to an antibody region that includes at least the hinge region, the CH2 domain, and the CH3 domain, unless the context clearly indicates otherwise. The terms Fc domain and Fc region may be used interchangeably, unless the context clearly indicates otherwise. In certain embodiments, the Fc domain is an IgG domain, an IgE domain, an IgM domain, and an IgD domain, an IgA domain, or an IgY domain. Fc domains of any sequence and from any species can be used, including human, ape, monkey, mouse, rabbit, goat, sheep, guinea pig, horse, and others. In certain embodiments, the Fc domain is an engineered, i.e., non-naturally occurring, or recombinant Fc domain, generated, for example, using molecular biology techniques. In some embodiments, the IgG domain is an IgG1 domain, an IgG2 domain, an IgG3 domain, or an IgG4 domain.

[0071] Anti-p95HER2 bispecific antibody Bispecific molecules, such as bispecific antibodies (bsAbs), provide a means to simultaneously target multiple epitopes on the same or different molecular targets with a single therapeutic agent. Without being limited by theory, for example, compared to a mixture of two monoclonal antibodies (mAbs), bispecific molecules as cancer therapeutics have the potential to confer novel or more potent activities, reduce the cost of goods, and facilitate the development of new therapeutic regimens.

[0072] In some embodiments, provided herein is a bispecific antibody comprising a first antigen-binding region and a second antigen-binding region. Generally, the first antigen-binding region and the second antigen-binding region specifically bind to different antigens or targets. In some aspects, the first antigen-binding region and the second antigen-binding region bind to different epitopes within the same antigen or target.

[0073] In one embodiment, the invention provides a bispecific antibody comprising a first antigen-binding region and a second antigen-binding region, wherein the first antigen-binding region binds to p95HER2. The first antigen-binding region comprises a VH region comprising an amino acid sequence having at least 80% identity, at least 85% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, at least 99.5% identity, at least 99.9% identity, and any number or range therebetween, to a sequence selected from SEQ ID NO:18. and a VL region comprising an amino acid sequence having at least 80% identity, at least 85% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, at least 99.5% identity, at least 99.9% identity, and any number or range therebetween, to about 100-120 amino acids of the N-terminal sequence of a sequence selected from the group consisting of: In some embodiments, the second antigen-binding region of the bispecific antibodies provided herein binds to an immune checkpoint molecule, an immune stimulatory molecule, or a tumor antigen.

[0074] In one embodiment, the bispecific antibody comprises a complementarity determining region (CDR)-H1 as set forth in SEQ ID NO: 19, a CDR-H2 as set forth in SEQ ID NO: 20, and a CDR-H3 as set forth in SEQ ID NO: 21, or a V sequence having 90% identity and antigen binding specificity to SEQ ID NO: 19, 20, or 21. H and a V region comprising CDR-L1, CDR-L2, and CDR-L3, wherein CDR-L1 is represented by SEQ ID NO: 22, CDR-L2 is represented by SEQ ID NO: 23, and CDR-L3 is represented by SEQ ID NO: 24, or a sequence having 90% identity and antigen-binding specificity to SEQ ID NO: 19, 20, or 21.L In another aspect, the VH region has an amino acid sequence having at least 80% identity to SEQ ID NO: 17 and the antigen-binding specificity of SEQ ID NOs: 19-21, and the VL region has an amino acid sequence having at least 80% identity to SEQ ID NO: 18 and the antigen-binding specificity of SEQ ID NOs: 22-24.

[0075] Any combination of first and second antigen-binding regions can be included in the bispecific antibodies provided herein, including, for example, first and second antigen-binding regions that bind any immune checkpoint molecule, any immune stimulating molecule, or any tumor antigen. Thus, in some aspects, the second antigen-binding region of the bispecific antibodies provided herein binds any immune checkpoint molecule, any immune stimulating molecule, or any tumor antigen. In some aspects, the first and second antigen-binding regions bind to the same molecule. For example, the first and second antigen-binding regions can bind to the same epitope or different epitopes of the same molecule. In other aspects, the first and second antigen-binding regions bind to different molecules.

[0076] In one embodiment, a bispecific antibody having a first antigen-binding region that binds p95HER2 and a second antigen-binding region that binds CD137 binds p95HER2 and CD137 simultaneously (Figure 8). Without being limited by theory, designing a bispecific antibody that can bind both p95HER2 and CD137 may reduce the risk of liver toxicity and its associated lethality seen in clinical trials with anti-CD137 antibodies such as urelumab by restricting anti-p95HER2 binding activity to tumor sites that express p95HER2. In addition, it is believed that simultaneous binding to p95HER2 and CD137 may enhance T cell activation as a result of cross-linking (see also Example 8 below).

[0077] In some aspects, the first antigen-binding region and the second antigen-binding region comprise an scFv, F(ab)2, Fab, or any combination thereof. In one aspect, the first antigen-binding region comprises an scFv and the second antigen-binding region comprises a Fab. In another aspect, the scFv of the bispecific antibody provided herein binds to an immune checkpoint molecule, an immune stimulatory molecule, or a tumor antigen. In yet another aspect, the scFv of the bispecific antibody provided herein binds to CD137. In some aspects, the Fab of the bispecific antibody provided herein binds to an immune checkpoint molecule, an immune stimulatory molecule, or a tumor antigen. In one aspect, the Fab of the bispecific antibody provided herein binds to p95HER2. In another aspect, the scFv of the bispecific antibody provided herein comprises the amino acid sequence of SEQ ID NO: 48.

[0078] In some embodiments, the bispecific antibodies provided herein further comprise a linker between the VH and VL regions of the scFv. Any linker can be used. For example, the linker can comprise any amino acid sequence. The linker can be of any length, such as 1 amino acid, 2 amino acids, 3 amino acids, 4 amino acids, 5 amino acids, 6 amino acids, 7 amino acids, 8 amino acids, 9 amino acids, 10 amino acids, 11 amino acids, 12 amino acids, 13 amino acids, 14 amino acids, 15 amino acids, 16 amino acids, 17 amino acids, 18 amino acids, 19 amino acids, 20 amino acids, or more amino acids. The linker can also comprise multiple amino acid sequences. Any number of multiple amino acid sequences can be included in the linker. Exemplary linker sequences are provided in SEQ ID NO: 44, SEQ ID NO: 45, and SEQ ID NO: 160.

[0079] In some aspects, the bispecific antibodies provided herein comprise a heavy chain sequence of SEQ ID NO: 46. In another aspect, the bispecific antibodies provided herein further comprise a light chain sequence of SEQ ID NO: 18. A heavy chain sequence such as SEQ ID NO: 46 can comprise one or more G linkers (SEQ ID NO: 44), one or more G4S linkers (SEQ ID NO: 45 or SEQ ID NO: 160), or any multiple G or G4S linkers, although any other suitable linker can be used.

[0080] CD73 targeting peptides Immunomodulation of the adaptive immune system is an attractive area in cancer immunotherapy due to its minimal side effects and long-term protection from cancer recurrence. Despite the success of anti-PD-1 / PD-L1 therapy in advanced cancers, a significant proportion of patients remain unresponsive to these treatments, indicating that other immunosuppressive mechanisms coexist in the tumor microenvironment, with the adenosinergic pathway emerging as a major contributor.

[0081] Tumor hypoxia, chronic inflammation, and metabolic changes result in extracellular ATP accumulation that promotes antitumor activity through induction of inflammasome assembly and proinflammatory cytokine secretion. However, due to the presence of hypoxia, inflammatory cytokines, and TGF-β, higher levels of ectonucleotidases CD39 and CD73 are usually observed in solid tumors compared to corresponding nonmalignant tissues. Extracellularly accumulated ATP is sequentially hydrolyzed to adenosine by tumor-associated CD39 and CD73, which then mediates its regulatory function upon binding to adenosine receptors expressed by tumor cells, stromal cells, and infiltrating lymphocytes within the tumor.

[0082] In tumor cells, adenosine signaling through A2A and A2B receptors promotes proliferation, metastasis, and secretion of vascular endothelial growth factor (VEGF). In cancer-associated fibroblasts (CAFs), adenosine signaling through A2BR induces the expression of protumor fibroblast growth factor 2 (FGF2) and CXCL12, generating a protumor microenvironment. In tumor-infiltrating immune cells, A2A signaling inhibits effector T cell activation by suppressing proximal TCR signaling and simultaneously inducing the expression of co-inhibitory molecules PD-1, LAG3, and TIM3. In addition, A2A signaling promotes mouse Treg cell differentiation, proliferation, and CTLA-4 expression. In NK cells, A2A signaling blocks maturation and cytotoxicity. In professional antigen-presenting cells, macrophages and dendritic cells, A2A and A2B signaling downregulates the expression of antigen-presenting and costimulatory molecules while promoting M2 macrophage differentiation.

[0083] High levels of CD73 have been found in malignant tissues of various cancer types, including pancreatic, gastrointestinal, renal, and lung cancers, and corresponded to poor prognosis. High levels of soluble CD73 have also been observed in the circulation of patients with metastatic melanoma. Given the protumor function of adenosine and the relevance of CD73 in cancer prognosis, CD73 has become an attractive therapeutic target for cancer. In preclinical studies, the antitumor activity of small molecule inhibitors and antibodies targeting CD73 was attributed not only to inhibition of tumor cell growth and migration, but also to the involvement of adaptive immunity. In addition, combining anti-CD73 antibodies enhanced the antitumor activity of antibodies against other immune regulatory molecules, including CTLA-4, PD-1, and CD137. Synergistic antitumor activity in combination with anti-PD-1 antibodies was attributed to activation of A2A signaling, which enhanced PD-1 expression on tumor-specific CD8+T cells. Interestingly, tumor resistance to anti-CD137 antibody therapy was referred to upregulation of CD73 on tumor-infiltrating CD8+ T cells, such that combination therapy with anti-CD137 and CD73 antibodies elicited more effective antitumor activity.

[0084] In clinical trials, several anti-CD73 antibodies, including MEDI9447 (oleculab), CPI-006, BMS-986179, and NZV930, are undergoing phase I / II in monotherapy or combination therapy. Partial responses were observed in 4.8% (1 / 21) of patients with colorectal cancer and 10% (2 / 20) of patients with pancreatic cancer by treatment with oleculab and durvalumab (anti-PD-L1 antibody). Partial responses were observed in 13.5% (7 / 52) of patients with head and neck, pancreatic, prostate, anal, and renal cancer by combination of BMS-986179 and nivolumab (anti-PD-1 antibody).

[0085] Provided herein are novel anti-CD73 monoclonal antibodies that have been generated and screened for the strongest binding affinity and enzyme blocking activity. Also provided herein are novel bispecific antibodies that simultaneously target CD73 and CD137 or PD-L1 to induce superior T cell activation and in vivo anti-tumor activity upon co-culture with CD73+ tumor cells.

[0086] Anti-CD73 antibodies and antigen-binding fragments thereof In one embodiment, the present invention provides antibodies and antigen-binding fragments thereof that bind to CD73. In one aspect, the present invention provides four antibodies or antigen-binding fragments thereof, including anti-CD73 antibody clone 3-4 (CD73 #3-4), anti-CD73 antibody clone 3-16 (CD73 #3-16), anti-CD73 antibody clone 3-1-28 (CD73 #3-1-28), and anti-CD73 antibody clone 3-1-37 (CD73 #3-1-37). In another aspect, the present invention provides three affinity matured antibodies derived from anti-CD73 clone 3-1-28, including anti-CD73 #3-1-28(S51A Q54E), anti-CD73 #3-1-28(S107D S109H), and anti-CD73 #3-1-28(S107D S109T A114S).

[0087] In one aspect, an anti-CD73 antibody provided herein comprises a heavy chain variable (VH) region comprising an amino acid sequence having at least 80% sequence identity to a sequence selected from SEQ ID NO:49, SEQ ID NO:57, SEQ ID NO:65, and SEQ ID NO:73, and a light chain variable (VL) region comprising an amino acid sequence having at least 80% sequence identity to a sequence selected from SEQ ID NO:50, SEQ ID NO:58, SEQ ID NO:66, SEQ ID NO:74, SEQ ID NO:81, SEQ ID NO:83, and SEQ ID NO:85.

[0088] The antibodies provided herein have a VH region comprising an amino acid sequence having at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range therebetween, to the sequences of SEQ ID NO:49, SEQ ID NO:57, SEQ ID NO:65, and SEQ ID NO:73. The antibodies provided herein also include a VL region comprising an amino acid sequence having at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range therebetween, to the sequences of SEQ ID NO:50, SEQ ID NO:58, SEQ ID NO:66, and SEQ ID NO:74, SEQ ID NO:81, SEQ ID NO:83, and SEQ ID NO:85.

[0089] In one embodiment, the antibody or antigen-binding fragment thereof has an amino acid sequence having at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range therebetween, to SEQ ID NO:49. and a VL region comprising an amino acid sequence having at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range therebetween, to SEQ ID NO: 50. SEQ ID NO: 49 provides an amino acid sequence comprising the variable region of the anti-CD73 antibody clone #3-4 heavy chain. SEQ ID NO: 50 provides an amino acid sequence comprising the variable region of the anti-CD73 antibody clone #3-4 light chain.

[0090] In one embodiment, an antigen-binding region or antigen-binding fragment thereof of an antibody having a VH region comprising an amino acid sequence having at least about 80% identity to SEQ ID NO:49 and a VL region comprising an amino acid sequence having at least about 80% identity to SEQ ID NO:50 has an amino acid sequence having at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 100% identity, at least about 101% identity, at least about 102% identity, at least about 103% identity, at least about 104% identity, at least about 105% identity, at least about 106% identity, at least about 107% identity, at least about 108% identity, at least about 109% identity, at least about 110% identity, at least about 111% identity, at least about 112% identity, at least about 113% identity, at least about 114% identity, at least about 115% identity, at least about 116% identity, at least about 117% identity, at least about 118% identity, at least about 119% identity, at least about 120% identity, at least about 121% identity, at least about 122% identity, at least about 123% identity, at least about 124% identity, at least about 125% identity, at least about 126% identity, at least about 127% identity, at least about 128% identity, at least about 129% identity, at least about 130% identity, at least about 131% identity, at least about 1 and CDR-H1 comprising an amino acid sequence having at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range therebetween to SEQ ID NO:52; and CDR-H2 comprising an amino acid sequence having at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range therebetween to SEQ ID NO:52. and CDR-H2 comprising an amino acid sequence having at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range therebetween, and a CDR-H3 having at least about 80% identity, at least about 85% identity, or any number or range therebetween, to SEQ ID NO:53. and a CDR-H3 comprising an amino acid sequence having identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range therebetween.In another aspect, an antigen-binding region of an antibody or antigen-binding fragment thereof having a VH region comprising an amino acid sequence having at least about 80% identity to SEQ ID NO:49 and a VL region comprising an amino acid sequence having at least about 80% identity to SEQ ID NO:50 has at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 100% identity, at least about 101% identity, at least about 102% identity, at least about 103% identity, at least about 104% identity, at least about 105% identity, at least about 106% identity, at least about 107% identity, at least about 108% identity, at least about 109% identity, at least about 110% identity, at least about 111% identity, at least about 112% identity, at least about 113% identity, at least about 114% identity, at least about 115% identity, at least about 116% identity, at least about 117% identity, at least about 118% identity, at least about 119% identity, at least about 120% identity, at least about 121% identity, at least about 122% identity, at least about 123% identity, at least about 124% identity, at least about 125% identity, at least about 126% identity, at least about 127% identity, at least about 128% identity, at least about 129% identity, at least about 130% identity, at least about 131% identity, at least about 132% identity, CDR-L1 comprising an amino acid sequence having at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range therebetween, and CDR-L2 comprising an amino acid sequence having at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, CDR-L2 comprising an amino acid sequence having at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range therebetween; and CDR-L3 comprising an amino acid sequence having at least about 80% identity, at least about 85% identity, or any number or range therebetween, to SEQ ID NO:56. and a CDR-L3 comprising an amino acid sequence having identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range therebetween.

[0091] In some embodiments, the antibody or antigen-binding fragment thereof has an amino acid sequence having at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range therebetween, to SEQ ID NO:57. and a VL region comprising an amino acid sequence having at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range therebetween, to SEQ ID NO: 57. SEQ ID NO: 58 provides the amino acid sequence comprising the variable region of the anti-CD73 antibody clone #3-16 heavy chain.

[0092] In one embodiment, an antigen-binding region of an antibody or antigen-binding fragment thereof comprising a VH region comprising an amino acid sequence having at least about 80% identity to SEQ ID NO:57 and a VL region comprising an amino acid sequence having at least about 80% identity to SEQ ID NO:58 has at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 100% identity, at least about 101% identity, at least about 102% identity, at least about 103% identity, at least about 104% identity, at least about 105% identity, at least about 106% identity, at least about 107% identity, at least about 108% identity, at least about 109% identity, at least about 110% identity, at least about 111% identity, at least about 112% identity, at least about 113% identity, at least about 114% identity, at least about 115% identity, at least about 116% identity, at least about 117% identity, at least about 118% identity, at least about 119% identity, at least about 120% identity, at least about 121% identity, at least about 122% identity, at least about 123% identity, at least about 124% identity, at least about 125% identity, at least about 126% identity, at least about 127% identity, at least about 128% identity, at least about 129% identity, at least about 130% identity, at least about 131% identity, at least about 132% identity, CDR-H1 comprising an amino acid sequence having about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range therebetween, and CDR-H2 comprising an amino acid sequence having at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range therebetween, to SEQ ID NO:60; CDR-H2 comprising an amino acid sequence having at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range therebetween, and a CDR-H3 having at least about 80% identity, at least about 85% identity, or any number or range therebetween, to SEQ ID NO:61. and a CDR-H3 comprising an amino acid sequence having identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range therebetween.In another aspect, an antigen-binding region of an antibody or antigen-binding fragment thereof comprising a VH region comprising an amino acid sequence having at least about 80% identity to SEQ ID NO:57 and a VL region comprising an amino acid sequence having at least about 80% identity to SEQ ID NO:58 has at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 100% identity, at least about 101% identity, at least about 102% identity, at least about 103% identity, at least about 104% identity, at least about 105% identity, at least about 106% identity, at least about 107% identity, at least about 108% identity, at least about 109% identity, at least about 110% identity, at least about 111% identity, at least about 112% identity, at least about 113% identity, at least about 114% identity, at least about 115% identity, at least about 116% identity, at least about 117% identity, at least about 118% identity, at least about 119% identity, at least about 120% identity, at least about 121% identity, at least about 122% identity, at least about 123% identity, at least about 124% identity, at least about 125% identity, at least about 126% identity, at least about 127% identity, at least about 128% identity, at least about 129% identity, at least about 130% identity, at least about 131% identity, at least about 132% identity, and CDR-L1 comprising an amino acid sequence having at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range therebetween, to SEQ ID NO:63; and CDR-L2 comprising an amino acid sequence having at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range therebetween to SEQ ID NO:63. and CDR-L2 comprising an amino acid sequence having at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range therebetween, and a CDR-L3 having at least about 80% identity, at least about 85% identity, or any number or range therebetween, to SEQ ID NO:64. and a CDR-L3 comprising an amino acid sequence having identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range therebetween.

[0093] In some embodiments, the antibody or antigen-binding fragment thereof has an amino acid sequence having at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range therebetween, to SEQ ID NO:65. and a VL region comprising an amino acid sequence having at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range therebetween, to SEQ ID NO: 66. SEQ ID NO: 65 provides the amino acid sequence comprising the variable region of the anti-CD73 antibody clone #3-1-28 heavy chain. SEQ ID NO: 66 provides the amino acid sequence comprising the variable region of the anti-CD73 antibody clone #3-1-28 light chain.

[0094] In one embodiment, an antigen-binding region of an antibody or antigen-binding fragment thereof comprising a VH region comprising an amino acid sequence having at least about 80% identity to SEQ ID NO:65 and a VL region comprising an amino acid sequence having at least about 80% identity to SEQ ID NO:66 has at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 100% identity, at least about 101% identity, at least about 102% identity, at least about 103% identity, at least about 104% identity, at least about 105% identity, at least about 106% identity, at least about 107% identity, at least about 108% identity, at least about 109% identity, at least about 110% identity, at least about 111% identity, at least about 112% identity, at least about 113% identity, at least about 114% identity, at least about 115% identity, at least about 116% identity, at least about 117% identity, at least about 118% identity, at least about 119% identity, at least about 120% identity, at least about 121% identity, at least about 122% identity, at least about 123% identity, at least about 124% identity, at least about 125% identity, at least about 126% identity, at least about 127% identity, at least about 128% identity, at least about 129% identity, at least about 130% identity, at least about 131% identity, at least about 132% identity, CDR-H1 comprising an amino acid sequence having about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range therebetween, and CDR-H2 comprising an amino acid sequence having at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range therebetween to SEQ ID NO:68. and CDR-H2 comprising an amino acid sequence having at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range therebetween, and CDR-H3 comprising an amino acid sequence having at least about 80% identity, at least about 85% identity, or any number or range therebetween, to SEQ ID NO:69. and a CDR-H3 comprising an amino acid sequence having identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range therebetween.In another aspect, an antigen-binding region of an antibody or antigen-binding fragment thereof comprising a VH region comprising an amino acid sequence having at least about 80% identity to SEQ ID NO:65 and a VL region comprising an amino acid sequence having at least about 80% identity to SEQ ID NO:66 has at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 100% identity, at least about 101% identity, at least about 102% identity, at least about 103% identity, at least about 104% identity, at least about 105% identity, at least about 106% identity, at least about 107% identity, at least about 108% identity, at least about 109% identity, at least about 110% identity, at least about 111% identity, at least about 112% identity, at least about 113% identity, at least about 114% identity, at least about 115% identity, at least about 116% identity, at least about 117% identity, at least about 118% identity, at least about 119% identity, at least about 120% identity, at least about 121% identity, at least about 122% identity, at least about 123% identity, at least about 124% identity, at least about 125% identity, at least about 126% identity, at least about 127% identity, at least about 128% identity, at least about 129% identity, at least about 130% identity, at least about 131% identity, at least about 132% identity, and CDR-L1 comprising an amino acid sequence having at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range therebetween to SEQ ID NO: 71. and CDR-L2 comprising an amino acid sequence having at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range therebetween, and a CDR-L3 having at least about 80% identity, at least about 85% identity, or any number or range therebetween, to SEQ ID NO:72. and a CDR-L3 comprising an amino acid sequence having identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range therebetween.

[0095] In some embodiments, the antibody or antigen-binding fragment thereof has an amino acid sequence having at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range therebetween, to SEQ ID NO:73. and a VL region comprising an amino acid sequence having at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range therebetween, to SEQ ID NO: 74. SEQ ID NO: 73 provides the amino acid sequence comprising the variable region of the anti-CD73 antibody clone #3-1-37 heavy chain. SEQ ID NO: 74 provides the amino acid sequence comprising the variable region of the anti-CD73 antibody clone #3-1-37 light chain.

[0096] In one embodiment, an antigen-binding region of an antibody or antigen-binding fragment thereof comprising a VH region comprising an amino acid sequence having at least about 80% identity to SEQ ID NO: 73 and a VL region comprising an amino acid sequence having at least about 80% identity to SEQ ID NO: 74 has at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 100% identity, at least about 101% identity, at least about 102% identity, at least about 103% identity, at least about 104% identity, at least about 105% identity, at least about 106% identity, at least about 107% identity, at least about 108% identity, at least about 109% identity, at least about 110% identity, at least about 111% identity, at least about 112% identity, at least about 113% identity, at least about 114% identity, at least about 115% identity, at least about 116% identity, at least about 117% identity, at least about 118% identity, at least about 119% identity, at least about 120% identity, at least about 121% identity, at least about 122% identity, at least about 123% identity, at least about 124% identity, at least about 125% identity, at least about 126% identity, at least about 127% identity, at least about 128% identity, at least about 129% identity, at least about 130% identity, at least about 131% identity, at least about 132% CDR-H1 comprising an amino acid sequence having about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range therebetween, and CDR-H2 comprising an amino acid sequence having at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range therebetween, to SEQ ID NO:76. CDR-H2 comprising an amino acid sequence having at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range therebetween, and a CDR-H3 having at least about 80% identity, at least about 85% identity, or any number or range therebetween, to SEQ ID NO:77. and a CDR-H3 comprising an amino acid sequence having identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range therebetween.In another aspect, an antigen-binding region of an antibody or antigen-binding fragment thereof comprising a VH region comprising an amino acid sequence having at least about 80% identity to SEQ ID NO: 73 and a VL region comprising an amino acid sequence having at least about 80% identity to SEQ ID NO: 74 has at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 100% identity, at least about 101% identity, at least about 102% identity, at least about 103% identity, at least about 104% identity, at least about 105% identity, at least about 106% identity, at least about 107% identity, at least about 108% identity, at least about 109% identity, at least about 110% identity, at least about 111% identity, at least about 112% identity, at least about 113% identity, at least about 114% identity, at least about 115% identity, at least about 116% identity, at least about 117% identity, at least about 118% identity, at least about 119% identity, at least about 120% identity, at least about 121% identity, at least about 122% identity, at least about 123% identity, at least about 124% identity, at least about 125% identity, at least about 126% identity, at least about 127% identity, at least about 128% identity, at least about 129% identity, at least about 130% identity, at least about 131% identity, at least about 132% and CDR-L1 comprising an amino acid sequence having at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range therebetween to SEQ ID NO:79. and CDR-L2 comprising an amino acid sequence having at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range therebetween, and CDR-L3 comprising an amino acid sequence having at least about 80% identity, at least about 85% identity, or any number or range therebetween, to SEQ ID NO: 80. and a CDR-L3 comprising an amino acid sequence having identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range therebetween.

[0097] In some embodiments, the antibody or antigen-binding fragment thereof has an amino acid sequence having at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range therebetween, to SEQ ID NO:65. and a VL region comprising an amino acid sequence having at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range therebetween, to SEQ ID NO: 81. SEQ ID NO: 65 provides the amino acid sequence comprising the variable region of the anti-CD73 antibody clone #3-1-28 heavy chain. SEQ ID NO: 81 provides the amino acid sequence comprising the variable region of the anti-CD73 antibody clone #3-1-28(S51A Q54E) light chain.

[0098] In one embodiment, an antigen-binding region of an antibody or antigen-binding fragment thereof comprising a VH region comprising an amino acid sequence having at least about 80% identity to SEQ ID NO:65 and a VL region comprising an amino acid sequence having at least about 80% identity to SEQ ID NO:81 has at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 100% identity, at least about 101% identity, at least about 102% identity, at least about 103% identity, at least about 104% identity, at least about 105% identity, at least about 106% identity, at least about 107% identity, at least about 108% identity, at least about 109% identity, at least about 110% identity, at least about 111% identity, at least about 112% identity, at least about 113% identity, at least about 114% identity, at least about 115% identity, at least about 116% identity, at least about 117% identity, at least about 118% identity, at least about 119% identity, at least about 120% identity, at least about 121% identity, at least about 122% identity, at least about 123% identity, at least about 124% identity, at least about 125% identity, at least about 126% identity, at least about 127% identity, at least about 128% identity, at least about 129% identity, at least about 130% identity, at least about 131% identity, at least about 132% identity, CDR-H1 comprising an amino acid sequence having about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range therebetween, and CDR-H2 comprising an amino acid sequence having at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range therebetween to SEQ ID NO:68. and CDR-H2 comprising an amino acid sequence having at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range therebetween, and CDR-H3 comprising an amino acid sequence having at least about 80% identity, at least about 85% identity, or any number or range therebetween, to SEQ ID NO:69. and a CDR-H3 comprising an amino acid sequence having identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range therebetween.In another aspect, an antigen-binding region of an antibody or antigen-binding fragment thereof comprising a VH region comprising an amino acid sequence having at least about 80% identity to SEQ ID NO:65 and a VL region comprising an amino acid sequence having at least about 80% identity to SEQ ID NO:81 has at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 100% identity, at least about 101% identity, at least about 102% identity, at least about 103% identity, at least about 104% identity, at least about 105% identity, at least about 106% identity, at least about 107% identity, at least about 108% identity, at least about 109% identity, at least about 110% identity, at least about 111% identity, at least about 112% identity, at least about 113% identity, at least about 114% identity, at least about 115% identity, at least about 116% identity, at least about 117% identity, at least about 118% identity, at least about 119% identity, at least about 120% identity, at least about 121% identity, at least about 122% identity, at least about 123% identity, at least about 124% identity, at least about 125% identity, at least about 126% identity, at least about 127% identity, at least about 128% identity, at least about 129% identity, at least about 130% identity, at least about 131% identity, at least about 132% identity, and CDR-L1 comprising an amino acid sequence having at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range therebetween to SEQ ID NO: 82. and CDR-L2 comprising an amino acid sequence having at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range therebetween, and a CDR-L3 having at least about 80% identity, at least about 85% identity, or any number or range therebetween, to SEQ ID NO:72. and a CDR-L3 comprising an amino acid sequence having identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range therebetween.

[0099] In some embodiments, the antibody or antigen-binding fragment thereof has an amino acid sequence having at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range therebetween, to SEQ ID NO:65. and a VL region comprising an amino acid sequence having at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range therebetween, to SEQ ID NO: 83. SEQ ID NO: 65 provides the amino acid sequence comprising the variable region of the anti-CD73 antibody clone #3-1-28 heavy chain. SEQ ID NO: 83 provides the amino acid sequence comprising the variable region of the anti-CD73 antibody clone #3-1-28 (S107D S109H) light chain.

[0100] In one embodiment, an antigen-binding region of an antibody or antigen-binding fragment thereof comprising a VH region comprising an amino acid sequence having at least about 80% identity to SEQ ID NO:65 and a VL region comprising an amino acid sequence having at least about 80% identity to SEQ ID NO:83 has at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 100% identity, at least about 101% identity, at least about 102% identity, at least about 103% identity, at least about 104% identity, at least about 105% identity, at least about 106% identity, at least about 107% identity, at least about 108% identity, at least about 109% identity, at least about 110% identity, at least about 111% identity, at least about 112% identity, at least about 113% identity, at least about 114% identity, at least about 115% identity, at least about 116% identity, at least about 117% identity, at least about 118% identity, at least about 119% identity, at least about 120% identity, at least about 121% identity, at least about 122% identity, at least about 123% identity, at least about 124% identity, at least about 125% identity, at least about 126% identity, at least about 127% identity, at least about 128% identity, at least about 129% identity, at least about 130% identity, at least about 131% identity, at least about 132% identity, CDR-H1 comprising an amino acid sequence having about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range therebetween, and CDR-H2 comprising an amino acid sequence having at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range therebetween to SEQ ID NO:68. and CDR-H2 comprising an amino acid sequence having at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range therebetween, and CDR-H3 comprising an amino acid sequence having at least about 80% identity, at least about 85% identity, or any number or range therebetween, to SEQ ID NO:69. and a CDR-H3 comprising an amino acid sequence having identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range therebetween.In another aspect, an antigen-binding region of an antibody or antigen-binding fragment thereof comprising a VH region comprising an amino acid sequence having at least about 80% identity to SEQ ID NO:65 and a VL region comprising an amino acid sequence having at least about 80% identity to SEQ ID NO:83 has at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 100% identity, at least about 101% identity, at least about 102% identity, at least about 103% identity, at least about 104% identity, at least about 105% identity, at least about 106% identity, at least about 107% identity, at least about 108% identity, at least about 109% identity, at least about 110% identity, at least about 111% identity, at least about 112% identity, at least about 113% identity, at least about 114% identity, at least about 115% identity, at least about 116% identity, at least about 117% identity, at least about 118% identity, at least about 119% identity, at least about 120% identity, at least about 121% identity, at least about 122% identity, at least about 123% identity, at least about 124% identity, at least about 125% identity, at least about 126% identity, at least about 127% identity, at least about 128% identity, at least about 129% identity, at least about 130% identity, at least about 131% identity, at least about 132% identity, and CDR-L1 comprising an amino acid sequence having at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range therebetween to SEQ ID NO: 84; and CDR-L2 comprising an amino acid sequence having at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range therebetween to SEQ ID NO: 84. and CDR-L2 comprising an amino acid sequence having at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range therebetween, and a CDR-L3 having at least about 80% identity, at least about 85% identity, or any number or range therebetween, to SEQ ID NO:72. and a CDR-L3 comprising an amino acid sequence having identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range therebetween.

[0101] In some embodiments, the antibody or antigen-binding fragment thereof has an amino acid sequence having at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range therebetween, to SEQ ID NO:65. and a VL region comprising an amino acid sequence having at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range therebetween, to SEQ ID NO: 85. SEQ ID NO: 65 provides the amino acid sequence comprising the variable region of the anti-CD73 antibody clone #3-1-28 heavy chain. SEQ ID NO: 85 provides the amino acid sequence comprising the variable region of the anti-CD73 antibody clone #3-1-28 (S107D S109T A114S) light chain.

[0102] In one embodiment, an antigen-binding region of an antibody or antigen-binding fragment thereof comprising a VH region comprising an amino acid sequence having at least about 80% identity to SEQ ID NO:65 and a VL region comprising an amino acid sequence having at least about 80% identity to SEQ ID NO:85 has at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 100% identity, at least about 101% identity, at least about 102% identity, at least about 103% identity, at least about 104% identity, at least about 105% identity, at least about 106% identity, at least about CDR-H1 comprising an amino acid sequence having about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range therebetween, and CDR-H2 comprising an amino acid sequence having at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range therebetween to SEQ ID NO:68. and CDR-H2 comprising an amino acid sequence having at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range therebetween, and CDR-H3 comprising an amino acid sequence having at least about 80% identity, at least about 85% identity, or any number or range therebetween, to SEQ ID NO:69. and a CDR-H3 comprising an amino acid sequence having identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range therebetween.In another aspect, an antigen-binding region of an antibody or antigen-binding fragment thereof comprising a VH region comprising an amino acid sequence having at least about 80% identity to SEQ ID NO:65 and a VL region comprising an amino acid sequence having at least about 80% identity to SEQ ID NO:85 has at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 100% identity, at least about 101% identity, at least about 102% identity, at least about 103% identity, at least about 104% identity, at least about 105% identity, at least about 106% identity, at least about 107% identity, at least about 108% identity, at least about 109% identity, at least about 110% identity, at least about 111% identity, at least about 112% identity, at least about 113% identity, at least about 114% identity, at least about 115% identity, at least about 116% identity, at least about 117% identity, at least about 118% identity, at least about 119% identity, at least about 120% identity, at least about 121% identity, at least about 122% identity, at least about 123% identity, at least about 124% identity, at least about 125% identity, at least about 126% identity, at least about 127% identity, at least about 128% identity, at least about 129% identity, at least about 130% identity, at least about 131% identity, at least about 132% identity, and CDR-L1 comprising an amino acid sequence having at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range therebetween to SEQ ID NO: 86; and CDR-L2 comprising an amino acid sequence having at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range therebetween to SEQ ID NO: 86. and CDR-L2 comprising an amino acid sequence having at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range therebetween, and a CDR-L3 having at least about 80% identity, at least about 85% identity, or any number or range therebetween, to SEQ ID NO:72. and a CDR-L3 comprising an amino acid sequence having identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range therebetween.

[0103] The antibodies or antigen-binding fragments thereof provided herein further comprise an Fc domain. In certain embodiments, the Fc domain is an IgG domain, an IgE domain, an IgM domain, and an IgD domain, an IgA domain, or an IgY domain. In some embodiments, the IgG domain is an IgG1 domain, an IgG2 domain, an IgG3 domain, or an IgG4 domain.

[0104] Anti-CD73 bispecific antibody Provided herein are bifunctional proteins, including bispecific antibodies. As used herein, the term "bifunctional protein" refers to a protein having at least two functions. Non-limiting examples of bifunctional proteins include bispecific antibodies that can bind to two antigens. The bispecific antibodies provided herein can include, for example, an isolated functional scFv fragment that binds CD137 and is fused to the C-terminus of the Fc domain of an anti-CD73 antibody. In some embodiments, the C-terminally located scFv that binds CD137 in the fusion constructs provided herein is fused to the Fc domain of an antibody that binds to other immune-regulatory molecules, such as, for example, CD40 or CTLA-4. In other embodiments, the C-terminally located scFv can bind to immune-regulatory molecules, such as, for example, CD40 or CTLA-4.

[0105] In one embodiment, the invention provides a bispecific antibody comprising a first antigen-binding region and a second antigen-binding region, wherein the first antigen-binding region binds to CD73. The first antigen-binding region comprises a VH region comprising an amino acid sequence having at least 80% identity, at least 85% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, at least 99.5% identity, at least 99.9% identity, and any number or range therebetween, to a sequence selected from SEQ ID NO:66. and a VL region comprising an amino acid sequence having at least 80% identity, at least 85% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, at least 99.5% identity, at least 99.9% identity, and any number or range therebetween, to about 100-120 amino acids of the N-terminal sequence of a sequence selected from the group consisting of: In some embodiments, the second antigen-binding region of the bispecific antibodies provided herein binds to an immune checkpoint molecule, an immune stimulatory molecule, or a tumor antigen.

[0106] In one embodiment, the bispecific antibody comprises a complementarity determining region (CDR)-H1 as set forth in SEQ ID NO: 67, a CDR-H2 as set forth in SEQ ID NO: 68, and a CDR-H3 as set forth in SEQ ID NO: 69, or a V sequence having 90% identity and antigen binding specificity to SEQ ID NO: 67, 68, or 69. H and a V sequence having 90% identity and antigen-binding specificity to SEQ ID NO: 70, 71, or 72, wherein CDR-L1 is represented by SEQ ID NO: 70, CDR-L2 is represented by SEQ ID NO: 71, and CDR-L3 is represented by SEQ ID NO: 72,L In another aspect, the VH region has an amino acid sequence having at least 80% identity to SEQ ID NO: 65 and the antigen-binding specificity of SEQ ID NOs: 67-69, and the VL region has an amino acid sequence having at least 80% identity to SEQ ID NO: 66 and the antigen-binding specificity of SEQ ID NOs: 70-72.

[0107] The first antigen-binding region comprises a VH region comprising an amino acid sequence having at least 80% identity, at least 85% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, at least 99.5% identity, at least 99.9% identity, and any number or range therebetween, to a sequence selected from SEQ ID NO:65; and a VL region comprising an amino acid sequence having at least 80% identity, at least 85% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, at least 99.5% identity, at least 99.9% identity, and any number or range therebetween, to about 100-120 amino acids of the N-terminal sequence of a sequence selected from the group consisting of: In some embodiments, the second antigen-binding region of the bispecific antibodies provided herein binds to an immune checkpoint molecule, an immune stimulatory molecule, or a tumor antigen.

[0108] In one embodiment, the bispecific antibody comprises a complementarity determining region (CDR)-H1 as set forth in SEQ ID NO: 67, a CDR-H2 as set forth in SEQ ID NO: 68, and a CDR-H3 as set forth in SEQ ID NO: 69, or a V sequence having 90% identity and antigen binding specificity to SEQ ID NO: 67, 68, or 69. Hand a V region comprising CDR-L1, CDR-L2, and CDR-L3, wherein CDR-L1 is represented by SEQ ID NO: 70, CDR-L2 is represented by SEQ ID NO: 84, and CDR-L3 is represented by SEQ ID NO: 72, or a sequence having 90% identity and antigen-binding specificity to SEQ ID NO: 70, 84, or 72. L In another aspect, the VH region has an amino acid sequence having at least 80% identity to SEQ ID NO: 65 and the antigen-binding specificity of SEQ ID NOs: 67-69, and the VL region has an amino acid sequence having at least 80% identity to SEQ ID NO: 83 and the antigen-binding specificity of SEQ ID NOs: 70, 84, and 72.

[0109] The first antigen-binding region comprises a VH region comprising an amino acid sequence having at least 80% identity, at least 85% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, at least 99.5% identity, at least 99.9% identity, and any number or range therebetween, to a sequence selected from SEQ ID NO:65; and a VL region comprising an amino acid sequence having at least 80% identity, at least 85% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, at least 99.5% identity, at least 99.9% identity, and any number or range therebetween, to about 100-120 amino acids of the N-terminal sequence of a sequence selected from the group consisting of: In some embodiments, the second antigen-binding region of the bispecific antibodies provided herein binds to an immune checkpoint molecule, an immune stimulatory molecule, or a tumor antigen.

[0110] In one embodiment, the bispecific antibody comprises a complementarity determining region (CDR)-H1 as set forth in SEQ ID NO: 67, a CDR-H2 as set forth in SEQ ID NO: 68, and a CDR-H3 as set forth in SEQ ID NO: 69, or a V sequence having 90% identity and antigen binding specificity to SEQ ID NO: 67, 68, or 69. H and a V sequence having 90% identity and antigen-binding specificity to SEQ ID NO: 70, 86, or 72, wherein CDR-L1 is represented by SEQ ID NO: 70, CDR-L2 is represented by SEQ ID NO: 85, and CDR-L3 is represented by SEQ ID NO: 72, or a sequence having 90% identity and antigen-binding specificity to SEQ ID NO: 70, 86, or 72. L In another aspect, the VH region has an amino acid sequence having at least 80% identity to SEQ ID NO: 65 and the antigen-binding specificity of SEQ ID NOs: 67-69, and the VL region has an amino acid sequence having at least 80% identity to SEQ ID NO: 85 and the antigen-binding specificity of SEQ ID NOs: 70, 86, and 72.

[0111] In some aspects, the second antigen-binding region of the bispecific antibodies provided herein binds to an immune checkpoint molecule, an immune stimulatory molecule, or a tumor antigen.

[0112] Any combination of first and second antigen-binding regions can be included in the bispecific antibodies provided herein, including, for example, first and second antigen-binding regions that bind any immune checkpoint molecule, any immune stimulating molecule, or any tumor antigen. Thus, in some aspects, the second antigen-binding region of the bispecific antibodies provided herein binds any immune checkpoint molecule, any immune stimulating molecule, or any tumor antigen. In some aspects, the first and second antigen-binding regions bind to the same molecule. For example, the first and second antigen-binding regions can bind to the same epitope or different epitopes of the same molecule. In other aspects, the first and second antigen-binding regions bind to different molecules.

[0113] In one embodiment, a bispecific antibody having a first antigen-binding region that binds CD73 and a second antigen-binding region that binds CD137 binds CD73 and CD137 simultaneously (Figure 22). Without being limited by theory, designing a bispecific antibody that can bind both CD73 and CD137 may limit anti-CD73 binding activity to tumor sites that express CD73, thereby reducing the risk of liver toxicity and its associated lethality seen in clinical trials with anti-CD137 antibodies such as urelumab. In addition, it is believed that simultaneous binding to CD73 and CD137 may enhance T cell activation as a result of cross-linking (see also Example 21 below).

[0114] In some aspects, the first antigen-binding region and the second antigen-binding region comprise an scFv, F(ab)2, Fab, or any combination thereof. In one aspect, the first antigen-binding region comprises an scFv and the second antigen-binding region comprises a Fab. In another aspect, the scFv of the bispecific antibody provided herein binds to an immune checkpoint molecule, an immune stimulatory molecule, or a tumor antigen. In yet another aspect, the scFv of the bispecific antibody provided herein binds to CD137. In some aspects, the Fab of the bispecific antibody provided herein binds to an immune checkpoint molecule, an immune stimulatory molecule, or a tumor antigen. In one aspect, the Fab of the bispecific antibody provided herein binds to CD73. In one aspect, the antibody comprises a heavy chain sequence having an amino acid sequence of SEQ ID NO: 88, 89, 90, or 91, or a sequence having 90% identity thereto and the binding specificity thereof. In another aspect, the bispecific antibody further comprises a light chain having an amino acid sequence of SEQ ID NO: 66. In another embodiment, the scFv of the bispecific antibody provided herein comprises the amino acid sequence of SEQ ID NO:91.

[0115] In some embodiments, the bispecific antibodies provided herein further comprise a linker between the VH and VL regions of the scFv. Any linker can be used. For example, the linker can comprise any amino acid sequence. The linker can be of any length, such as 1 amino acid, 2 amino acids, 3 amino acids, 4 amino acids, 5 amino acids, 6 amino acids, 7 amino acids, 8 amino acids, 9 amino acids, 10 amino acids, 11 amino acids, 12 amino acids, 13 amino acids, 14 amino acids, 15 amino acids, 16 amino acids, 17 amino acids, 18 amino acids, 19 amino acids, 20 amino acids, or more amino acids. The linker can also comprise multiple amino acid sequences. Any number of multiple amino acid sequences can be included in the linker. Exemplary linker sequences are provided in SEQ ID NO: 44, SEQ ID NO: 45, and 160.

[0116] In some aspects, the bispecific antibodies provided herein comprise a heavy chain sequence of SEQ ID NO: 88, SEQ ID NO: 89, or SEQ ID NO: 91. In another aspect, the bispecific antibodies provided herein further comprise a light chain sequence of SEQ ID NO: 66 or SEQ ID NO: 81. The heavy chain sequences, such as SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 91, can include one or more G linkers (SEQ ID NO: 44), one or more G4S linkers (SEQ ID NO: 45 and SEQ ID NO: 160), or any multiple G or G4S linkers, although any other suitable linker can be used.

[0117] Antibody-drug conjugates In one embodiment, the invention provides an antibody-drug conjugate comprising a therapeutic agent and any one of the antibodies or antigen-binding fragments thereof described herein, or any one of the bispecific antibodies described herein. For example, the antibodies, antigen-binding fragments thereof, or bispecific antibodies include anti-p95HER2 antibodies, antigen-binding fragments thereof, bispecific antibodies targeting p95HER2 and a second target, anti-CD73 antibodies, antigen-binding fragments thereof, and bispecific antibodies targeting CD73 and a second target, such as those described herein.

[0118] The term "antibody drug conjugate", which may be used interchangeably with the term "immunoconjugate" or "conjugate" as used herein, refers to a compound or derivative thereof, e.g., a drug (i.e., an anti-cancer drug), linked to a cell-binding agent (i.e., an antibody or fragment thereof, or a bispecific antibody) and defined by the general formula: CLA, where C=cytotoxin, L=linker, A=cell-binding agent or antibody. Immunoconjugates can also be defined by the general formula in the reverse order: ALC.

[0119] In one aspect, the therapeutic agent is covalently attached to the antibody, antigen-binding fragment, or bispecific antibody via a linker.

[0120] "Therapeutic agent" refers to any drug, compound, or small molecule useful in the treatment of cancer that can be linked to a binding agent described herein. In various aspects, the therapeutic agent is a chemotherapeutic agent. Chemotherapeutic and anti-tumor agents are well-known cytotoxic agents, including, but not limited to: (i) anti-microtubule agents, including vinca alkaloids (vinblastine, vincristine, vinflunine, vindesine, and vinorelbine), taxanes (cabazitaxel, docetaxel, larotaxel, ortataxel, paclitaxel, and tesetaxel), epothilones (ixabepilone), and podophyllotoxins (etoposide and teniposide); (ii) antifolates (aminopterin, methotrexate, pemetrexate, cefotaxel ... antimetabolites, including deoxynucleoside analogues (azacytidine, capecitabine, carmofur, cladribine, clofarabine, cytarabine, decitabine, doxifluridine, floxuridine, fludarabine, fluorouracil, gemcitabine, hydroxycarbamide, mercaptopurine, nelarabine, pentostatin, tegafur, and thioguanine);(iii) topoisomerase I inhibitors (belotecan, camptothecin, cositecan, gimatecan, exatecan, irinotecan, lurtotecan, ciratecan, topotecan, and rubitecan) and topoisomerase II inhibitors (acrarubicin, amrubicin, daunorubicin, doxorubicin, epirubicin, etoposide, idarubicin, mervalone, mitoxantrone, novobiocin, pirarubicin, (iv) topoisomerase inhibitors, including nitrogen mustards (bendamustine, busulfan, chlorambucil, cyclophosphamide, estramustine phosphate, ifosamide, mechlorethamine, melphalan, prednimustine, trofosfamide, and uramustine), nitrosoureas (carmustine (BCNU), fotemustine), (v) anthracyclines including doxorubicin and daunorubicin; (vi) tetracyclines including tetracycline, tetrazolomide ... Derivatives of these compounds include epirubicin and idarubicin; pirarubicin, acurubicin, and mitoxantrone, bleomycin, mitomycin C, mitoxantrone, and actinomycin;(vi) FI inhibitors (tipifarnib), CDK inhibitors (abemaciclib, alvocidib, palbociclib, ribociclib, and seliclib), PrI inhibitors (bortezomib, carfilzomib, and ixazomib), PhI inhibitors (anagrelide), IMPDI inhibitors (tiazofurin), LI inhibitors (masoprocol), PARP inhibitors (niraparib, olaparib, and rucaparib), HDAC inhibitors (verinolide), (vii) receptor antagonists including ERA receptor antagonists (atrasentan), retinoid X receptor antagonists (bexarotene), sex steroid receptor antagonists (testosterone); (viii) ungrouped agents including amsacrine, trabectedin, retinoids (alitretinoin tretinoin) arsenic trioxide, asparagine depleting agents (asparaginase / pegaspargase), celecoxib, demecolcine elesclomol, elsamitrucin, etoglucide, lonidamine, lucantone, mitoguazone, mitotane, oblimersen, omacetaxine mepesuccinate, and eribulin.

[0121] Pharmaceutical Compositions In one embodiment, the invention provides a pharmaceutical composition comprising any one of the antibodies or antigen-binding fragments thereof described herein, any one of the bispecific antibodies described herein, or any one of the antibody-drug conjugates described herein, and at least one pharma- ceutically acceptable carrier.

[0122] For example, the pharmaceutical composition includes an anti-p95HER2 antibody, an antigen-binding fragment thereof, a bispecific antibody targeting p95HER2 and a second target, an antibody-drug conjugate thereof, an anti-CD73 antibody, an antigen-binding fragment thereof, a bispecific antibody targeting CD73 and a second target, or an antibody-drug conjugate thereof, such as those described herein.

[0123] As used herein, a "pharmaceutical composition" refers to a formulation that includes an active ingredient and, optionally, one or more pharma- ceutically acceptable carriers, diluents, or excipients. The term "active ingredient" can interchangeably refer to "active ingredient" and is meant to refer to any agent capable of inducing a desired effect upon administration. In one embodiment, the active ingredient comprises a biologically active molecule. As used herein, the phrase "biologically active molecule" refers to a molecule that has a biological effect intracellularly. In certain embodiments, the active molecule is an antibody, an antibody fragment, a bispecific antibody, or an antibody-drug conjugate, such as those described herein, or a morpholino.

[0124] By "pharmacologically acceptable" it is meant that the carrier, diluent or excipient is compatible with the other ingredients of the formulation and not deleterious to the recipient thereof or to the activity of the active ingredients of the formulation. Pharmaceutically acceptable carriers, excipients or stabilizers are well known in the art. See, e.g., Remington's Pharmaceutical Sciences, 16th edition, Osol, A. Ed. (1980). Pharmaceutically acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed and include, but are not limited to, buffers such as phosphate, citrate, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, erythritol, erythritol phosphates, and other suitable agents. The carrier may include, but is not limited to, liposomes, nanoparticles, ointments, micelles, gelatins, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as TWEEN™, PLURONICS™, or polyethylene glycol (PEG). Examples of carriers include, but are not limited to, liposomes, nanoparticles, ointments, micelles, microspheres, microparticles, creams, emulsions, and gels.Examples of excipients include, but are not limited to, antiadhesives such as magnesium stearate, binders such as sugars and their derivatives (sucrose, lactose, starch, cellulose, sugar alcohols, etc.), proteins such as gelatin and synthetic polymers, lubricants such as talc and silica, and preservatives such as antioxidants, vitamin A, vitamin E, vitamin C, retinyl palmitate, selenium, cysteine, methionine, citric acid, sodium sulfate, and parabens. Examples of diluents include, but are not limited to, water, alcohol, saline solution, glycols, mineral oil, and dimethyl sulfoxide (DMSO).

[0125] In some embodiments, the pharma- ceutically acceptable carrier is conjugated to the C-terminus of one or more polypeptides of the antibody or antigen-binding fragment. Any suitable means of conjugating the pharma- ceutically acceptable carrier can be used, including, for example, covalent conjugation and the use of a linker.

[0126] How to use In one embodiment, the invention provides a method of treating cancer comprising administering to a subject in need thereof an effective amount of any one of the antibodies or antigen-binding fragments thereof described herein, any one of the bispecific antibodies described herein, or any one of the antibody-drug conjugates described herein, thereby treating the cancer.

[0127] As used herein, the terms "treat", "treatment", "therapy", "therapeutic" and the like refer to obtaining a desired pharmacological and / or physiological effect, including, but not limited to, alleviating a disease or disorder, slowing or retarding its progression, reducing its effects or symptoms, preventing its onset, inhibiting, ameliorating its onset, obtaining a beneficial or desired result with respect to a disease, disorder, or medical condition, such as a therapeutic benefit and / or a prophylactic benefit. "Treatment", as used herein, includes any treatment of disease in a mammal, particularly a human, including (a) preventing the disease from occurring in a subject, including a subject predisposed to or at risk of acquiring the disease, but not yet diagnosed as having it, (b) inhibiting the disease, i.e., arresting its onset, and / or (c) relieving the disease, i.e., causing regression of the disease. Therapeutic benefit includes eradication or amelioration of the underlying disorder being treated. Therapeutic benefit is also achieved by eradication or amelioration of one or more of the physiological symptoms associated with the underlying disorder, such that improvement is observed in the subject, even though the subject may still suffer from the underlying disorder. In some embodiments, for prophylactic benefit, the treatment or composition for treatment is administered to a subject at risk of developing a particular disease, or to a subject who reports one or more of the physiological symptoms of the disease, even if the disease has not been diagnosed. The methods of the present disclosure may be used on any mammal or other animal. In some embodiments, the treatment results in a reduction or cessation of symptoms. A prophylactic effect includes delaying or eliminating the appearance of a disease or condition, delaying or eliminating the onset of symptoms of a disease or condition, slowing, halting, or reversing the progression of a disease or condition, or any combination thereof.

[0128] As used herein, the term "subject" refers to any individual or patient on whom the methods disclosed herein are performed. The term "subject" may be used interchangeably with the terms "individual" or "patient". A subject may be a human, or, as will be understood by those skilled in the art, an animal. Thus, other animals, including domestic animals, including rodents (including mice, rats, hamsters, and guinea pigs), cats, dogs, rabbits, cows, horses, goats, sheep, pigs, etc., as well as mammals, such as primates (including monkeys, chimpanzees, orangutans, and gorillas), are included in the definition of a subject.

[0129] As used herein, the term "effective amount" or "therapeutically effective amount" refers to an amount of an antibody, antigen-binding fragment thereof, or other composition described herein sufficient to induce an intended effect, including but not limited to disease treatment as defined herein. A therapeutically effective amount may vary depending on the intended therapeutic application (e.g., in vivo), or the patient and disease state to be treated, such as the patient's weight and age, the severity of the disease state, the mode of administration, etc., which can be readily determined by one of ordinary skill in the art. The term also applies to a dose that induces a specific response in target cells. A specific dose will vary depending on the particular antibody, antigen-binding fragment thereof, or other composition selected, the dosing regimen to be followed, whether it is administered in combination with other compounds, the timing of administration, the tissue to which it is administered, and the physical delivery system in which it is carried.

[0130] In some aspects, the antibodies or antigen-binding fragments thereof of the present invention are combined with other therapeutic agents such as radiation therapy, cytotoxic chemotherapy, immunotherapy including immune checkpoint inhibitors, and other immunomodulatory agents such as vaccines, interleukins, cytokines, chemokines, and biologics, either as monotherapy or as combination therapy. Exemplary interleukins for immunotherapy include IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-15, IL-18, IL-21, and IL-23. Exemplary cytokines for immunotherapy include interferons, TNF-α, TGF-β, G-CSF, and GM-CSF. Exemplary chemokines for immunotherapy include CCL3, CCL26, and CXCL7. Exemplary biologics include CAR T cell therapy, tumor infiltrating lymphocyte (TIL) therapy, and monoclonal antibodies, such as alemtuzumab, trastuzumab, ibritumomab tiuxetan, brentuximab vedotin, adotrastuzumab emtansine, blinatumomab, bevacizumab, and cetuximab. Antibodies also include checkpoint inhibitors, including PD-1 inhibitors, such as pembrolizumab, nivolumab, and cemiplimab, PD-L1 inhibitors, such as atezolizumab, avelumab, and durvalumab, CTLA-4 inhibitors, such as iprimumab, and other checkpoint inhibitors, such as anti-B7-H3 antibodies, anti-KIR antibodies, and anti-LAG3 antibodies.

[0131] In some embodiments, provided herein is a method of treating cancer in a subject comprising administering to the subject an amount of any of the bispecific antibodies, or antigen-binding fragments thereof, provided herein, effective to treat the cancer.

[0132] Cancer is a group of diseases involving abnormal cell growth that can invade or spread to other parts of the body. In 2015, approximately 90.5 million people were affected by cancer, with approximately 14.1 million new cases occurring annually, causing approximately 8.8 million deaths (15.7% of deaths). The most common types of cancer in men are lung, prostate, colorectal, and stomach cancer. In women, the most common types are breast, colorectal, lung, and cervical cancer. The term "cancer" refers to a group of diseases characterized by abnormal and uncontrolled cell growth that begins at one site (primary site) with the potential to invade and spread to other sites (secondary sites, metastases), which distinguishes cancer (malignant tumors) from benign tumors. Virtually all organs can be affected, resulting in over 100 types of cancer that can affect humans. Cancer can result from many causes, including genetic predisposition, viral infection, exposure to ionizing radiation, exposure to environmental pollutants, tobacco and / or alcohol use, obesity, poor diet, lack of physical activity, or any combination thereof. As used herein, "neoplasm" or "tumor," including grammatical variations thereof, refers to a new, abnormal growth of tissue, which may be benign or cancerous. In related aspects, neoplasm refers to a neoplastic disease or disorder, including, but not limited to, various cancers. For example, such cancers may include prostate cancer, pancreatic cancer, biliary tract cancer, colon cancer, rectal cancer, liver cancer, kidney cancer, lung cancer, testicular cancer, breast cancer, ovarian cancer, pancreatic cancer, brain cancer, head and neck cancer, melanoma, sarcoma, multiple myeloma, leukemia, lymphoma, and the like.

[0133] In some embodiments, the cancer is prostate cancer, lung cancer, non-small cell lung cancer (NSCLC), melanoma, lymphoma, breast cancer, head and neck cancer, renal cell carcinoma (RCC), ovarian cancer, kidney cancer, bladder cancer, uterine cancer, cervical cancer, ovarian cancer, liver cancer, gastric cancer, colon cancer, rectal cancer, oral cancer, pharyngeal cancer, pancreatic cancer, thyroid cancer, skin cancer, brain cancer, bone cancer, hematopoietic cancer, or leukemia.

[0134] Amino acid and nucleic acid sequences In some embodiments, provided herein is an isolated amino acid sequence encoding any of the antibodies, antigen-binding fragments thereof, or bispecific antibodies described herein.

[0135] The terms "peptide", "polypeptide" and "protein" are used interchangeably herein and refer to any chain of at least two amino acids linked by a covalently bonded chemical. As used herein, a polypeptide may refer to a complete amino acid sequence that encodes an entire protein or a portion thereof. A "protein coding sequence" or a sequence that "encodes" a particular polypeptide or peptide is a nucleic acid sequence that is transcribed (in the case of DNA) or translated in vitro or in vivo into a polypeptide (in the case of mRNA) when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxyl) terminus. Coding sequences include, but are not limited to, cDNA from prokaryotic or eukaryotic mRNA, genomic DNA sequences from prokaryotic or eukaryotic DNA, and even synthetic DNA sequences. A transcription termination sequence will usually be located 3' to the coding sequence.

[0136] In various embodiments, the amino acid sequences are as shown in SEQ ID NOs: 1-40 and 46-91.

[0137] Also provided herein are isolated nucleic acid sequences that encode the amino acid sequences provided herein.

[0138] As used herein, the term "nucleic acid" refers to a polynucleotide, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). Nucleic acids include, but are not limited to, genomic DNA, cDNA, mRNA, iRNA, miRNA, tRNA, ncRNA, rRNA, as well as recombinantly produced and chemically synthesized molecules, such as aptamers, plasmids, antisense DNA strands, shRNA, ribozymes, conjugated nucleic acids, and oligonucleotides. According to the present invention, nucleic acids can exist as single-stranded or double-stranded and linear or covalently circularly closed molecules. Nucleic acids can be isolated. The term "isolated nucleic acid" means that the nucleic acid has been (i) amplified in vitro, for example, via polymerase chain reaction (PCR), (ii) recombinantly produced by cloning, (iii) purified, for example, by cleavage and separation by gel electrophoresis, or (iv) synthesized, for example, by chemical synthesis. Nucleic acids can be used for introduction into cells, i.e., transfection, particularly in the form of RNA, which can be prepared by in vitro transcription from a DNA template. Additionally, the RNA can be modified prior to application by stabilizing sequences, capping, and polyadenylation.

[0139] In some embodiments, provided herein is an isolated nucleic acid sequence encoding any one of the amino acid sequences of SEQ ID NOs: 1-40 and 46-91.

[0140] In some embodiments, the present invention further provides for expression, purification, and characterization of anti-p95HER2 and anti-CD73 antibodies, as detailed in the Examples below. A signal sequence may be included in the expression constructs of the antibodies provided herein. Any suitable signal sequence may be used, for example, the sequence of SEQ ID NO: 43.

[0141] Below are presented examples illustrating anti-p95HER2 and anti-CD73 antibodies, antigen-binding fragments thereof, and bispecific antibodies thereof, contemplated for the described uses. The following examples are provided to further illustrate embodiments of the invention, but are not intended to limit the scope of the invention. They are typical of those that might be used, although alternatively, other procedures, methodologies, or techniques known to those of skill in the art may be used. EXAMPLES

[0142] Example 1 Generation of antibodies from OmniMab libraries To generate antibodies against p95HER2, panning was performed using an OmniMab phagemid library constructed by AP Biosciences Inc. against IgG variable region sequences collected from PBMCs of over 100 healthy donors. Phage display was performed using Hyperphage (M13K07ΔρIII, Progen, Heidelberg, Germany).

[0143] Panning was performed using p95HER2 overexpressing HEK293 cells in the first round, and then recombinant p95HER2 protein produced by Expi-293 cells in the following three rounds of selection. Briefly, for the first round of cell panning, the omniMab phage library was mixed with 1x 1% BSA in PBS. The phage / PBS mixture was added to a tube containing wild-type Expi293 cells for subtraction. Wild-type Expi293 cells were then collected by centrifugation, and the cells were washed three times with 0.5% BSA in PBS to collect phages that did not recognize Expi293 cells. The collected phages were also mixed with full-length p95HER2 transfected Expi293 under shaking. The transfected Expi293 cells were washed 10 times with 0.5% BSA in PBS, and the phages bound to the p95HER2 transfected Expi293 cells were eluted with TEA solution. The eluted phages were collected and infected into TG1 for phage rescue.

[0144] For the second to fourth rounds of solid-phase panning, secreted p95HER2 ECD (ERBB2_HUMAN amino acid positions 611-652) with signal peptide and FLAG-taq was produced on the Expi293 platform. The culture supernatant was collected by centrifugation as the target antigen for panning with. The immunotubes were pre-coated with anti-FLAG mouse antibody with PBS and loaded with various FLAG-Taq recombinant proteins. The immunotubes and phages from the previous panning rounds were mixed with 5% NFM or 1% BSA in PBS for blocking, and then the phage mixture was added to the pre-loaded immunotubes containing FLAG-tag recombinant proteins (except p95HER2 ECD-FLAG) for subtraction. The other immunotubes were added to the culture supernatant containing secreted p95HER2 ECD FLAG-tag. After incubation, the tubes were collected and washed three times with 1% tween-20 in PBS to collect the phages that did not recognize the immunotubes. The phage mixture is then transferred to an immunotube containing p95HER2 ECD with FLAG-taq for antigen binding. After incubation, the immunotube was washed 10 times with 1% tween-20 in PBS, and bound phages were eluted with TEA solution. The eluted phages were collected and infected with TG1 for phage rescue. Single clones specific for p95HER2 were then isolated, determined by FACS (Figures 1A-1C), and selected for sequencing to confirm the diversity of heavy and light chains. The reference antibodies used in this study were generated from SEQ ID NOs: 20 and 21 of US2018 / 118849A1 with a human IgG1 backbone and produced on the ExpiCHO platform.

[0145] The unique antibody sequences were then subcloned and expressed for further analysis.

[0146] Example 2 Subcloning, expression, and purification of p95HER2-specific binding protein in the form of IgG To rapidly screen candidates, the heavy and light chains of positive p95HER2 binders identified by FACS were amplified, digested, and subcloned into an in-house generated IgG expression vector with IgG1 constant region (SEQ ID NO: 41). After sequence verification, plasmids were prepared and transfected into ExpiCHO cells (Invitrogen) for antibody production. After 6 days of culture, antibodies were affinity purified from culture supernatants by Protein A chromatography. Purified antibodies were concentrated and dialyzed in PBS buffer. Antibody concentrations were determined using a NanoDrop2000 spectrophotometer. Antibody purity and integrity were determined by NuPAGE (ThermoFisher, Cat. NP0321BOX) under non-reducing or reducing conditions and visualized by Coomassie Brilliant Blue G-250 staining solution.

[0147] Figure 2 shows the PAGE results of the purified anti-p95HER2 antibody leads. The results showed that the protein had a molecular weight of about 145 kDa under non-reducing conditions, and the heavy and light chains had molecular weights of 55 kDa and 25 kDa, respectively, under reducing conditions. Most of the antibody leads showed a purity of over 90% from one-step Protein A chromatography.

[0148] 3A-3B show the binding activity of purified anti-p95HER2 antibody leads by flow cytometer.

[0149] Example 3 Binding epitope mapping of anti-p95HER2 monoclonal antibody Binding epitope mapping was performed by PEPperPRINT Inc. To determine the binding epitopes, the GS linker-elongated full-length p95HER2 peptide sequence was converted into linear 5, 10, and 15 amino acid peptides overlapping with 4, 9, and 14 amino acids, respectively.

[0150] Linear peptide microarrays were incubated with antibody leads at a concentration of 1 μg / ml, followed by staining with secondary antibodies (goat anti-human IgG (H+L) DyLight680) and control antibodies (mouse monoclonal anti-HA (12CA5) DyLight800), and read out with an Odyssey Imaging System (LI-COR, Inc). Quantification of spot intensity and peptide annotation was performed using PepSlide® Analyzer (SICASYS Software GmbH). The epitopes of the p95HER2 antibodies are shown and summarized in Figure 4A. The heat map of epitope mapping results showed that both anti-p95HER2# R4-3, R4-5, R4-11, R4-15 and R4-38 could strongly recognize the motif KFPDE (SEQ ID NO: 96). However, the signals from 10 and 15 amino acid peptides containing the KFPDE motif disclosed that the structure of p95HER2 is not a simple linear one. When the motif KFPDE was located at the end of the amino acid sequence, a relatively strong signal was generated, indicating that the amino acids in the vicinity of the motif KFPDE may affect the binding affinity of the anti-p95HER2 antibody (Figure 4B).

[0151] Example 4 Assessment of protein aggregation of anti-p95HER2-CD137 scFv bispecific antibody by SEC-HPLC Purified and concentrated antibodies (>1 mg / ml) were applied to determine variants and aggregation by SEC-HPLC analysis. SEC-HPLC was performed using a Waters ACQUITY Arc system with a Waters 2489 UV / Vis detector. Samples were loaded onto an XBridge Protein BEH SEC column (Waters, Cat. No. 186007640) using isocratic 25 mM sodium phosphate, 200 mM NaCl, pH 6.8 as mobile phase buffer for SEC separation. The flow rate was 0.4 mL / min and sample injection volume was 40 μg. Peaks were detected by absorbance at 280 nm. All samples were filtered through a 0.22 μm filter (Millipore, Cat. No. SLGP003RB) to remove precipitated protein material prior to injection onto the SEC column. Data was analyzed by Empower 3 software. In summary, the purity of the single-column Protein A-purified anti-p95HER2-CD137 antibody is greater than 95% (Figure 6A).

[0152] The general structure of a bispecific antibody is presented in FIG.

[0153] Example 5 Assessment of protein integrity of anti-p95HER2-CD137 bispecific antibody by MCE-SDS-PAGE All size variant analyses of purified antibodies were performed on a LabChip GXII instrument (Perkin Elmer, Inc.). The assays were performed using the "Protein Clear HR Assay" as manufacturer's protocol. Briefly, 2.5 μl of protein sample at 1 mg / mL was mixed with 18 μl of sample buffer. Sample buffer was prepared by mixing 700 μl of Protein Clear HR sample buffer with either 24.5 μl of 1 M DTT (for reduced assay) or 24.5 μl of 0.25 M NEM (for non-reduced assay). Samples were incubated at 70° C. for 10 min. After cooling to room temperature, 35 μl of water was added to each sample before loading onto the instrument. Samples were then analyzed using the Protein Clear HR Assay script. Meanwhile, chips were prepared according to the manufacturer's instructions and kept at 30° C. throughout the analysis. The excitation and detection wavelengths were 630 nm and 700 nm, respectively. In summary, the purity and integrity of one-column Protein A purified anti-p95HER2-CD137 antibody reaches about 95% (FIG. 6B).

[0154] Example 6 Binding affinity of anti-p95HER2-CD137 bispecific antibody by biolayer interferometry The affinity of anti-p95HER2-CD137 bsAb was determined by Biolayer Interferometry (ForteBIO, USA). To evaluate the binding affinity of anti-p95HER2-CD137 bsAb to CD137, the bsAb was loaded at 5 μg / mL onto AHC (Anti-Human IgG Fc Capture, ForteBIO, Cat. No. 18-5060) and allowed the signal to reach approximately 0.5 nm. The sensor was then exposed to a seven-series concentration analyte, which was CD137 (Cat. No. 41B-H5256) extracellular domain fused with mouse Fc tag recombinant protein. To determine the affinity of anti-p95HER2-CD137 bsAb to p95HER2, custom synthesized biotinylated Her2 p95 peptide (GenScript) was loaded at 5 ng / mL onto SA (Streptavidin, ForteBIO, Cat. No. 18-5019) biosensor for 1 min. The sensors were then exposed to 7 series of concentrated analytes that were bsAbs. Measurements were performed as follows: 1 min baseline in kinetic buffer, 1 min baseline in kinetic buffer, 5 min association in 7 different concentrations of analyte, and 5 min dissociation in kinetic buffer. Kinetic parameters were calculated and generated by Octet Data Acquisition and Analysis Software. In summary, the affinity (K D ) were 0.189 and 1.12 nM, respectively (see Figures 7A-7B).

[0155] Example 7 Simultaneous binding of p95HER2 and CD137 by anti-p95HER2#R4-11-CD137#54 bsAB The binding activity of the anti-p95HER2-CD137 bispecific antibody was determined by ForteBIO® (Menlo Park, Calif.) biosensor analysis. In this method, custom synthesized biotinylated Her2 p95 peptide (GenScript) was loaded onto a SA (streptavidin, ForteBIO, catalog number 18-5019) biosensor at 50 μg / mL for 5 minutes. The sensor was then exposed to the antibody sample at 100 nM for 5 minutes, followed by association with mouse Fc-tagged human CD137 protein (AcroBiosystems, catalog number 41B-H5256) at 100 nM for 5 minutes. Binding charts were generated by Octet Data Acquisition and Analysis Software. Taken together, these results show that the anti-p95HER2-CD137 bispecific antibody simultaneously recognizes p95HER2 and CD137 as determined by ForteBIO® biosensor analysis (Figure 8).

[0156] Example 8 p95HER2-dependent T cell activation induced by anti-p95HER2-CD137 bispecific antibody Human T cells were isolated using RosetteSep™ Human T Cell Enrichment Cocktail (STEMCELL Catalog No. 15061). Purified T cells (1 × 10 5 cells) were cultured in the presence of anti-CD3 (OKT3)-coated polybeads at a T cell:bead ratio of 1:1 with p95HER2-expressing NCI-H292 lung cancer cells (1 × 10 4 After 3 days, T cell activation was measured by IFN-γ production as measured by ELISA.

[0157] The expression level of p95HER2 on NCI-H292 lung cancer cells was confirmed by flow cytometry (Figure 9A). Compared with monotherapy or combination therapy with anti-p95HER2 and anti-CD137#54 antibodies, anti-p95HER2-CD137 bsAb induced stronger IFN-γ production (Figure 9B).

[0158] Example 9 Antibody-dependent cell-mediated cytotoxicity (ADCC) with anti-p95HER2-CD137 bispecific antibody For ADCC induction, CD16-NK92 effector cells were co-cultured with CD137-expressing HEK293 target cells (2 × 10 4 The cells were co-cultured with 1000 cells / mL of p95HER2-CD137 bsAbs. Two anti-p95HER2-CD137 bsAbs, #R4-11-#54 (with original IgG1 Fc) and #R4-11e5-#54 (with ADCC-enhancing IgG1 Fc, SEQ ID NO: 42), were applied in this study. After 6 hours, the cytotoxic activity was determined by CytoTox 96 (registered) Non-Radioactive Cytotoxicity Assay (Promega, G1780).

[0159] Compared to the isotype control, #R4-11e5-#54, but not #R4-11 and #R4-11-#54, induced ADCC in CD137-HEK293 cells, corresponding to the ADCC-enhancing mutations in the IgG1 Fc region of #R4-11e5-#54 (Figure 10A). #R4-11-#54 induced ADCC activity against p95HER2-NCI-H292 cells and its parent #R4-11 mAb (Figure 10B).

[0160] Taken together, these results indicated that #R4-11e5-#54 bsAb may be harmful to CD137-expressing cells, such as NK cells and T cells, which are important effector cells in cancer. Therefore, #R4-11-#54 bsAb with normal IgG1 Fc was selected for further development.

[0161] Example 10 Inhibition of tumor growth by anti-p95HER2-CD137 bispecific antibody in vivo. To verify the antitumor activity of the anti-p95HER2#4-11-CD137#54 bsAb, which does not cross-react with mouse CD137, human tumor cells (p95HER2-expressing MDA-MB-231) were premixed with human PBMCs and orthotopically implanted into the anterior mammary fat pad of female SCID beige mice to evaluate the antitumor activity in vivo. Ten days after tumor inoculation, equimolar amounts of the mAb (MW 150 kDa, 7.7 mg / kg) and the bsAb (MW 195 kDa, 10, 2, 0.4 mg / kg) were injected intraperitoneally twice a week. Tumor size (mm 3 The tumor volume (TGIv) or tumor weight (TGIw) was measured twice a week and calculated as (length x width x width) / 2. The tumor inhibition rate was calculated based on the tumor volume (TGIv) or tumor weight (TGIw) by the following equation: TGIv (%) = [1-(V of treatment group t -V 0 ) / (V of control group t -V 0 )]×100%, where V t : Mean tumor volume after treatment, V 0 : Mean tumor volume before treatment. TGIw(%)=(mean tumor weight in control group-mean tumor weight in treatment group) / mean tumor weight in control group×100%.

[0162] Taken together, these results show a significant inhibition of tumor growth upon treatment compared to treatment with PBS (Figures 11A-11C). w showed greater tumor inhibition of 95.6%, 91.5% and 93.7% at 10, 2 and 0.4 mg / kg, respectively, but the TGI v were 125.5%, 113% and 117%.

[0163] Example 11 Pharmacokinetic profile of anti-p95HER2-CD137 bispecific antibody in mice Bispecific anti-p95HER2-CD137 antibodies (5 mg / kg body weight) were administered to SCI-beige mice via intravenous bolus injection. Peripheral blood was collected at 0.5, 24, 72, 168, 264, 336, 504, 672 and 840 hours after injection. Plasma concentrations of antibodies were determined by ELISA. MaxiSorp plates (Invitrogen) were coated with CD137-Fc fusion protein (AP Biosciences, 0.5 μg / mL), after which plasma samples were serially diluted and anti-CD73-CD137#54 bsAb was applied as a standard curve. Bound antibodies were detected by CD73-Flag-His antigen (AP Biosciences) and HRP-conjugated anti-Flag antibody using TMB substrate. Plasma antibody concentrations were calculated by interpolation. PK parameters were calculated using PKSolver software. Anti-p95HER2-CD137 1 / 2 The PK profile showed a mean mean time of administration of 100 mg / mL for 336 hours and an AUC of 63,896 μg / ml*h (FIG. 12).

[0164] Example 12 Generation of antibodies from OmniMab libraries To generate antibodies against CD73, selection was performed using the OmniMab phagemid library, which was constructed by AP Biosciences Inc. (APBio Inc.) from a collection of peripheral blood mononuclear cells from over 100 healthy donors.

[0165] The first round of panning was performed using Hyperphage (M13K07ΔρIII, Progen, Heidelberg, Germany). Solution-phase panning was used for the selection and isolation of CD73-specific binders from the OmniMab library. Solution-phase panning was performed using recombinant CD73 protein with a flag tag and a polyhistidine tag at the C-terminus produced by the mammalian cell expression system used in the first round of selection. The second round of enrichment also used solution-phase panning. CD73-expressing Expi293 cells and breast cancer cell line MDA-MB-231 were separately used for the third round of enrichment. Specific CD73 binders were screened and isolated by direct ELISA and FACS after three rounds of panning (Figures 13A-13C). Phage clones specifically recognize human CD73 after three rounds of human CD73-specific enrichment process from the OmniMab phage library. Phage-containing supernatants were applied to CD73 ELISA and positive binders were numbered. MDA-MB-231 breast cancer cell lines were stained with CD73 phage supernatants (50 μl / well) isolated from MDA-MB-231 cell-based panning to examine CD73 binding activity. Figure 13B shows the ranking of binding activity of binders. Binders were isolated and sent for sequencing to confirm the sequence and diversity of the heavy chain.

[0166] Example 13 Subcloning, expression, and purification of CD73-specific binding proteins in the form of IgG To rapidly screen candidates, the heavy and light chains of positive CD73 binders identified by ELISA and FACS were amplified, digested, and subcloned into an IgG expression vector carrying an engineered IgG1 constant region (SEQ ID NO: 87) generated by APBio. After sequence verification, plasmids were prepared and transfected into ExpiCHO cells (Invitrogen) for antibody production. After 6 days of culture, antibodies secreted into serum-free medium were affinity purified from culture supernatants by Protein A chromatography. Purified antibodies were concentrated and then dialyzed in PBS buffer. The final concentration of dialyzed protein was determined using a NanoDrop2000 spectrophotometer, and purity and integrity were determined by SDS-PAGE with or without reducing reagent. Purified antibodies were analyzed by NuPAGE (ThermoFisher, Cat. No. NP0321BOX) under non-reducing or reducing conditions. When the dye front reaches the bottom of the gel, the voltage is turned off and the gel is removed from the glass plate. Place the gel in Coomassie Brilliant Blue G-250 staining solution in a plastic container and rock for several hours. The destaining solution can be poured into a designated waste container in a fume hood and replaced several times until the bands can be clearly visualized against the gel background.

[0167] Figure 14 shows a representative PAGE gel analysis of different batches of purified anti-CD137 antibody lead. The purity and integrity of the one-step Protein A purified anti-CD73 antibody lead was analyzed by SDS-PAGE under non-reducing and reducing conditions. The results showed that the protein had a molecular weight of about 145 kDa under non-reducing conditions, and the heavy and light chains had molecular weights of 55 kDa and 25 kDa, respectively, under reducing conditions. A purity of more than 90% could be obtained by one step of Protein A chromatography.

[0168] Example 14 Blockade of CD73 enzymatic activity by anti-CD73 antibodies MDA-MB-231 cells (2 x 10 4MDA-MB-231 cells (4.8×10 cells) were incubated with anti-CD73 antibodies in wells of a 96-well tissue culture plate for 30 min at 4° C. AMP (12.5 μM, Sigma, Cat. No. A1752) was added and incubated for another 1 h at 4° C. Supernatants were transferred to 96-well white plates and residual AMP was detected by AMP-Glo ​​assay according to the manufacturer's instructions (Promega, Cat. No. V5011). The percentage of enzyme activity inhibition was calculated as [(cells + AMP + antibody)-(cells + AMP)] / [AMP-(cells + AMP)]×100. To test the blocking activity of anti-CD73 antibodies against soluble CD73, MDA-MB-231 cells (4.8×10 cells) were incubated with anti-CD73 antibodies in wells of a 96-well tissue culture plate for 30 min at 4° C. AMP (12.5 μM, Sigma, Cat. No. A1752) was added and incubated for another 1 h at 4° C. Supernatants were transferred to 96-well white plates and residual AMP was detected by AMP-Glo ​​assay according to the manufacturer's instructions (Promega, Cat. No. V5011). The percentage of enzyme activity inhibition was calculated as [(cells + AMP + antibody)-(cells + AMP)] / [AMP-(cells + AMP)]×100. 6 Cells) were seeded into wells of a 24-well plate for 40 h, then the culture medium was collected and incubated with the antibodies for 1 h at 37° C. AMP (12.5 μM, Sigma, Cat. No. A1752) was added and incubated for another 2 h at 37° C., after which the same procedure as above was performed to detect the residual AMP and calculate the percentage of enzyme inhibition.

[0169] In summary, the binding affinity of anti-CD73 antibodies did not necessarily correspond to their blocking of enzymatic activity. Weaker blocking of CD73 enzymatic activity was observed in clones #3-6, #3-27, #3-34, #3-1-14, #3-1-21, and #3-1-31, although with better binding activity than clone #3-4. Interestingly, clone #3-1-28 showed the strongest blocking of CD73 enzymatic activity even with binding activity comparable to clone #3-4, and #3-1-28 was selected for affinity maturation (Figures 15 and 16). As shown in Figure 15, the binding activity of anti-CD73 antibody leads was evaluated by flow cytometer. MDA-MB-231 cells (1 × 10 5MDA-MB-231 cells (100 μg / ml) were surface stained with anti-CD73 antibody (4 μg / ml) for 30 min, followed by incubation with A488-conjugated anti-human IgG secondary antibody for 15 min on ice. Cells were analyzed by flow cytometer, and the A488 fluorescence intensity was shown by histogram (left) and numbers (table on the right). Blocking of CD73 enzymatic activity by anti-CD73 antibody lead was evaluated, as shown in Figure 16. Enzymatic activity of CD73 expressed by MDA-MB-231 on the cell surface was blocked by anti-CD73 antibody lead.

[0170] Corresponding to the improved binding affinity (Figures 17A-17C and Example 15), the CD73 enzyme blocking activity of anti-CD73 clones #3-1-28-v1, #3-1-28-v2, and #3-1-28-v3 was superior to their parent clone #3-1-28 (Figure 18). The IC of anti-CD73 #3-1-28-v1 (0.213 nM) and anti-CD73 #3-1-28-v2 (0.245 nM) in inhibiting the cell membrane-bound CD73 enzyme was 50 was approximately 2-fold lower than the IC50 of the parent #3-1-28 (0.49 nM) (Figure 18A). Interestingly, the IC50 of anti-CD73#3-1-28-v1 (0.187 nM), anti-CD73#3-1-28-v2 (0.273 nM), and anti-CD73#3-1-28-v3 (0.241 nM) in inhibiting the soluble CD73 enzyme was 0.018 nM. 50 is the IC of parent #3-1-28 (1.478 nM) 50 This was more than five-fold lower than in the control group (Figure 18B).

[0171] Furthermore, conversion of affinity matured anti-CD73 #3-1-28-v1, #3-1-28-v2, and #3-1-28-v3 into bispecific antibodies by linking CD137 scFv to the C-terminus of their Fc did not alter their enzyme-blocking activity (Figure 23).

[0172] Example 15 Affinity maturation of anti-CD73#3-1-28 Affinity matured antibodies can exhibit increased biological efficacy, regardless of whether the antibody is isolated from a hybridoma or human Fv phage library. Increased affinity can also allow for reduced dosage of therapeutic antibodies and reduce toxic side effects. Germline hotspot residues are most likely to have a large effect on affinity based on statistical analysis of independent monoclonal antibodies derived from the same germline gene that recognize the same epitope. Germline hotspots within antibody complementarity determining regions (CDRs) are naturally prone to hypermutation (Neuberger and Milstein, 1995). To improve the binding activity of #3-1-28, we introduced random mutations into LCDR2 / HCDR3 and generated phage display libraries with approximately 10^2 and 10^8 diversity in these two CDR regions, respectively. Panning of these hotspot libraries resulted in mutant antibodies with increased affinity. CD73-expressing MDA-MB-231 breast cancer cells were used for three rounds of cell-based panning enrichment. Binders with better binding activity than the parent phage #3-1-28 were screened by FACS (Figure 17A), and 27 binders were sequenced to confirm the diversity of the engineered CDR regions. The heavy and light chains of the positive CD73 binders identified by FACS were amplified, digested, and subcloned into an IgG expression vector carrying the engineered IgG1 constant region. The vector was transfected into a mammalian cell expression system for IgG production.

[0173] Based on the improved binding measured by ELISA and biolayer interferometry (Figures 17B and 17C), clones #1919-003, #1911-022, and #1911-030 were selected for further development of bispecific antibodies (with the general structure shown in Figure 19). To emphasize their origin from parent #3-1-28, clones #1919-003, #1911-022, and #1911-030 are hereafter renamed as #3-1-28-v1, #3-1-28-v2, and #3-1-28-v3, respectively. As shown in Figure 17A, the CD73 binding activity of phage clones derived from the small-scale affinity maturation library was determined by staining CD73-expressing MDA-MB-231 cells and analyzed by flow cytometer. Clones with better binding activity compared to parent phage clone #3-1-28 were numbered. As shown in Figure 17B, the binding activity of the antibodies converted from the phage clones numbered in Figure 17A was measured by CD73 ELISA. The bolded clones #1919-002, #1919-003, #1919-004, #1911-022, and #1911-030 showed better binding activity than the parent #3-1-28 antibody. As shown in Figure 17C, the binding kinetics of the antibodies selected in Figure 17B was measured by biolayer interferometry. To emphasize that they are derived from clone #3-1-28, clones #1919-003, #1911-022, and #1911-030 are hereafter named #3-1-28-v1, #3-1-28-v2, and #3-1-28-v3, respectively. The detected K D Based on the values, the affinity of #3-1-28-v1, #3-1-28-v2, and #3-1-28-v3 was improved by approximately 2-fold compared to the parent #3-1-28.

[0174] Example 16 Binding affinity of anti-CD73 monoclonal antibody and anti-CD73-CD137 bispecific antibody by biolayer interference analysis The affinity of anti-CD73 mAbs and bsAbs was determined by Biolayer Interferometry (ForteBIO, USA). In this method, anti-CD73 antibodies were loaded at 5 μg / mL on AHC (Anti-Human IgG Fc Capture, ForteBIO, Cat. No. 18-5060) and the signal was allowed to reach approximately 0.5 nm. The sensor was then exposed to a solution containing his-tagged human CD73 (AcroBiosystems, Cat. No. CD3-H52H7) with a total of 7 points in 2-fold serial dilutions from a top concentration of 30 nM. Measurements were performed as follows: 1 min baseline in kinetic buffer, 5 min association in hCD73-His containing solution, and 5 min dissociation into kinetic buffer. Kinetic parameters were calculated and generated by Octet Data Acquisition and Analysis Software.

[0175] In summary, the binding affinity of anti-CD73 #3-1-28-v1, #3-1-28-v2, and #3-1-28-v3 shows approximately 2-fold improvement after affinity maturation compared to the parent #3-1-28 (Figure 17C). The improved CD73 binding affinity of #3-1-28-v1, #3-1-28-v2, and #3-1-28-v3 remains after conversion to anti-CD73-CD137 bispecific antibodies (Figures 21A-21B).

[0176] Example 17 Assessment of protein aggregation of anti-CD73-CD137 scFv bispecific antibody by SEC-HPLC Purified and concentrated antibodies (>1 mg / ml) were applied to determine variants and aggregation by SEC-HPLC analysis. SEC-HPLC was performed using a Waters ACQUITY Arc system with a Waters 2489 UV / Vis detector. Samples were loaded onto an XBridge Protein BEH SEC column (Waters, Cat. No. 186007640) using isocratic 25 mM sodium phosphate, 200 mM NaCl, pH 6.8 as mobile phase buffer for SEC separation. The flow rate was 0.4 mL / min and sample injection volume was 40 μg. Peaks were detected by absorbance at 280 nm. All samples were filtered through a 0.22 μm filter (Millipore, Cat. No. SLGP003RB) to remove precipitated protein material prior to injection onto the SEC column. Data was analyzed by Empower 3 software. In summary, the purity of single-column Protein A purified anti-CD73-CD137 antibodies is greater than 90% (Figures 20A-20B).

[0177] Example 18 Assessment of protein integrity of anti-CD73-CD137 bispecific antibodies by MCE-SDS-PAGE All size variant analyses of purified antibodies were performed on a LabChip GXII instrument (Perkin Elmer, Inc.). The assays were performed using the "Protein Clear HR Assay" as manufacturer's protocol. Briefly, 2.5 μl of protein sample at 1 mg / mL was mixed with 18 μl of sample buffer. Sample buffer was prepared by mixing 700 μl of Protein Clear HR sample buffer with either 24.5 μl of 1 M DTT (for reduced assay) or 24.5 μl of 0.25 M NEM (for non-reduced assay). Samples were incubated at 70° C. for 10 min. After cooling to room temperature, 35 μl of water was added to each sample before loading onto the instrument. Samples were then analyzed using the Protein Clear HR Assay script. Meanwhile, chips were prepared according to the manufacturer's instructions and kept at 30° C. throughout the analysis. The excitation and detection wavelengths are 630 nm and 700 nm, respectively. In summary, the purity and integrity of one-column Protein A purified anti-CD73-CD137 antibody reaches about 95% (FIGS. 20A-20B).

[0178] Example 19 Antigen recognition by anti-CD73-CD137 bispecific antibodies The binding activity of AP601 was determined by ForteBIO® (Menlo Park, Calif.) biosensor analysis. In this method, His-tagged human CD73 protein (AcroBiosystems, Catalog No. CD3-H52H7) was loaded onto HIS1K (Anti-Penta-HIS) biosensor (Cat. No. 18-5120) at 5 μg / mL for 5 minutes. The sensor was then exposed to antibody samples at 100 nM for 5 minutes, followed by association with mouse Fc-tagged human CD137 protein (AcroBiosystems, Catalog No. 41B-H5256) at 100 nM for 5 minutes. Binding charts were generated by Octet Data Acquisition and Analysis Software.

[0179] Taken together, these results demonstrate that the anti-CD73-CD137 bispecific antibody simultaneously recognizes CD73 and CD137 as determined by ForteBIO® biosensor analysis (FIG. 22).

[0180] Example 20 Rescue of AMP-suppressed T cell proliferation by anti-CD73-CD137 bispecific antibody Human T cells were isolated using RosetteSep™ Human T Cell Enrichment Cocktail (STEMCELL Cat. No. 15061). Purified T cells were labeled with CFSE (ThermoFisher, Cat. No. C34554) and stimulated with human T activator CD3 / CD28 Dynabeads (ThermoFisher, Cat. No. 11131D) at a T cell:bead ratio of 1:0.5 in the presence of AMP (400 μM, Sigma, Cat. A1752). After 3 days, T cell activation was determined by IFN-γ production measured by ELISA and cell proliferation was analyzed by flow cytometry.

[0181] Taken together, corresponding to the binding affinity, anti-CD73-CD173 bispecific antibodies with improved affinity showed better rescue of AMP-suppressed T cell activation (Figure 24).

[0182] Example 21 CD73-dependent T cell activation induced by anti-CD73-CD137 bispecific antibody Human T cells were isolated using RosetteSep™ Human T Cell Enrichment Cocktail (STEMCELL Catalog No. 15061). Purified T cells (1 × 10 5 1 × 10 cells) were cultured at a T cell:bead ratio of 1:1 in the presence of anti-CD3 (OKT3)-coated polybeads with CD73-expressing MDA-MB-231 breast cancer cells and NCI-H292 lung cancer cells (1 × 10 4 After 3 days, T cell activation was measured by IFN-γ production as measured by ELISA.

[0183] CD73 expression levels on MDA-MB-231 breast cancer cells and NCI-H292 lung cancer cells were confirmed by flow cytometry (Figure 25A). Compared with monotherapy or combination therapy with anti-CD73 and anti-CD137#54 antibodies, anti-CD73-CD137 bsAb induced stronger IFN-γ production (Figures 25B-25C).

[0184] Example 22 Inhibition of tumor growth by anti-CD73#3-1-28-V2-CD137#54 bispecific antibody in vivo. To verify the antitumor activity of the anti-CD73#3-1-28-v2-CD137#54 bsAb, which does not cross-react with mouse CD73 and mouse CD137, human tumor cells (MDA-MB-231) were premixed with human PBMCs and orthotopically implanted into the anterior mammary fat pad of female SCID beige mice to evaluate the anticancer activity in vivo. Ten days after tumor inoculation, equimolar amounts of mAb (MW 150 kDa, 10 mg / kg) and bsAb (MW 195 kDa, 13 mg / kg) were injected intraperitoneally twice weekly. Tumor size (mm3) was measured twice weekly and calculated as (length x width x width) / 2. Tumor inhibition rate was calculated based on tumor volume (TGI v ) or tumor weight (TGI w ) was calculated according to the following equation: TGI v (%)=[1-(V of treatment group t -V 0 ) / (V of control group t -V 0 )]×100%, where Vt is the mean tumor volume after treatment, V 0 : Mean tumor volume before treatment. TGI w(%) = (mean tumor weight of control group - mean tumor weight of treatment group) / mean tumor weight of control group x 100%. For isolation of tumor infiltrating lymphocytes (TILs), dissected tumors were cut into small pieces and digested with RPMI-1640 medium containing collagenase IV (Worthington, Catalog No. LS004188), hyaluronidase (Sigma, Catalog No. H6254), and DNase I (Sigma, Catalog No. D5025) by gentleMACS (Miltenyi Biotec Inc.), followed by density gradient separation by Lymphoprep (STEMCELL, Catalog No. 07811). Isolated TILs were stained with cell type-specific markers and analyzed by flow cytometer.

[0185] Taken together, these results show significant inhibition of tumor growth upon treatment with 10 mg / kg of anti-CD73#3-1-28-v2-CD137#54 bsAb compared to treatment with anti-CD73 ref mAb (Figure 26). w showed greater tumor inhibition of 40.9% and 17.5% at 10 and 3 mg / kg, respectively, but the TGI v Consistent with the antitumor activity, anti-CD73#3-1-28-v2-CD137#54 bsAb specifically induced the expansion of CD8+, but reduced CD4+, T cells in the tumor (Figures 26A-26E).

[0186] Example 23 Anti-CD73-CD137 bispecific in mice Antibody Pharmacokinetic Profile Bispecific anti-CD73#3-1-28-v2-CD137#54 antibody (5 mg / kg body weight) was administered to SCI-beige mice via intravenous bolus injection. Peripheral blood was collected at 0.5, 24, 72, 168, 240, 336, 504, and 672 hours after injection. Plasma concentrations of antibodies were determined by ELISA. MaxiSorp plates (Invitrogen) were coated with CD137-Fc fusion protein (AP Biosciences, 0.5 μg / mL), after which plasma samples were serially diluted and anti-CD73-CD137#54 bsAb was applied as a standard curve. Bound antibodies were detected by CD73-Flag-His antigen (AP Biosciences) and HRP-conjugated anti-Flag antibody using TMB substrate. Plasma antibody concentrations were calculated by interpolation. PK parameters were calculated using PKSolver software. Anti-CD73#3-1-28-v3 is 1 / 2 showed a worse PK profile with t1 / 2 of 103 hours and AUC of 16438 μg / ml*h, whereas anti-CD73 #3-1-28, #3-1-28-v1, #3-1-28-v2-CD137#54 bsAbs showed comparable t1 / 2 and AUC (Figure 27).

[0187] array TIFF2024540604000002.tif42153TIFF2024540604000003.tif212153TIFF2024540604000004.tif214153TIFF20245406040 00005.tif212153TIFF2024540604000006.tif215153TIFF2024540604000007.tif216153TIFF2024540604000008.tif91153

[0188] References TIFF2024540604000009.tif109146TIFF2024540604000010.tif213147TIFF2024540604000011.tif216147TIFF2024540604000012.tif113146

[0189] Although the invention has been described with reference to the above examples, it will be understood that modifications and variations are encompassed within the spirit and scope of the invention. Accordingly, the invention is limited only by the scope of the following claims.

Claims

1. (i) a V comprising a complementarity determining region (CDR)-H1, CDR-H2, and CDR-H3, wherein CDR-H1 is represented by SEQ ID NO: 19, CDR-H2 is represented by SEQ ID NO: 20, and CDR-H3 is represented by SEQ ID NO: 21, or a sequence having 90% identity and antigen-binding specificity to SEQ ID NO: 19, 20, or 21; Ｈ and CDR-L1, CDR-L2, and CDR-L3, wherein CDR-L1 is represented by SEQ ID NO: 22, CDR-L2 is represented by SEQ ID NO: 23, and CDR-L3 is represented by SEQ ID NO: 24, or a sequence having 90% identity and antigen-binding specificity to SEQ ID NO: 22, 23, or 24. Ｌ region, (ii) V comprising CDR-H1, CDR-H2, and CDR-H3, wherein CDR-H1 is represented by SEQ ID NO: 11, CDR-H2 is represented by SEQ ID NO: 12, and CDR-H3 is represented by SEQ ID NO: 13, or a sequence having 90% identity and antigen-binding specificity to SEQ ID NO: 11, 12, or 13; Ｈ region, and CDR-L1, CDR-L2, and CDR-L3, wherein CDR-L1 is set forth in SEQ ID NO: 14, CDR-L2 is set forth in SEQ ID NO: 15, and CDR-L3 is set forth in SEQ ID NO: 16, or a sequence having 90% identity and antigen-binding specificity to SEQ ID NO: 14, 15, or 16. Ｌ region, (iii) V, comprising CDR-H1, CDR-H2, and CDR-H3, wherein CDR-H1 is represented by SEQ ID NO: 3, CDR-H2 is represented by SEQ ID NO: 4, and CDR-H3 is represented by SEQ ID NO: 5, or a sequence having 90% identity and antigen-binding specificity to SEQ ID NO: 3, 4, or 5. Ｈ region, and CDR-L1, CDR-L2, and CDR-L3, wherein CDR-L1 is set forth in SEQ ID NO: 6, CDR-L2 is set forth in SEQ ID NO: 7, and CDR-L3 is set forth in SEQ ID NO: 8, or a sequence having 90% identity and antigen-binding specificity to SEQ ID NO: 6, 7, or 8. Ｌ region, (iv) V, comprising CDR-H1, CDR-H2, and CDR-H3, wherein CDR-H1 is represented by SEQ ID NO: 27, CDR-H2 is represented by SEQ ID NO: 28, and CDR-H3 is represented by SEQ ID NO: 29, or a sequence having 90% identity and antigen-binding specificity to SEQ ID NO: 27, 28, or 29. Ｈ and CDR-L1, CDR-L2, and CDR-L3, wherein CDR-L1 is set forth in SEQ ID NO: 30, CDR-L2 is set forth in SEQ ID NO: 31, and CDR-L3 is set forth in SEQ ID NO: 32, or a sequence having 90% identity and antigen-binding specificity to SEQ ID NO: 30, 31, or 32. Ｌ Area, or (v) V comprising CDR-H1, CDR-H2, and CDR-H3, wherein CDR-H1 is represented by SEQ ID NO: 35, CDR-H2 is represented by SEQ ID NO: 36, and CDR-H3 is represented by SEQ ID NO: 37, or a sequence having 90% identity and antigen-binding specificity to SEQ ID NO: 35, 36, or 37. Ｈ and CDR-L1, CDR-L2, and CDR-L3, wherein CDR-L1 is set forth in SEQ ID NO: 38, CDR-L2 is set forth in SEQ ID NO: 39, and CDR-L3 is set forth in SEQ ID NO: 40, or a sequence having 90% identity and antigen-binding specificity to SEQ ID NO: 38, 39, or 40. Ｌ region Including, binds to p95HER2, An antibody or antigen-binding fragment thereof.

2. (i) the VH region has an amino acid sequence having at least 80% identity to SEQ ID NO: 17 and the antigen-binding specificity of SEQ ID NOs: 19-21, and the VL region has an amino acid sequence having at least 80% identity to SEQ ID NO: 18 and the antigen-binding specificity of SEQ ID NOs: 22-24; (ii) the VH region has an amino acid sequence having at least 80% identity to SEQ ID NO:9 and the antigen-binding specificity of SEQ ID NOs:11-13, and the VL region has an amino acid sequence having at least 80% identity to SEQ ID NO:10 and the antigen-binding specificity of SEQ ID NOs:14-16; (iii) the VH region has an amino acid sequence having at least 80% identity to SEQ ID NO: 1 and the antigen-binding specificity of SEQ ID NOs: 3-5, and the VL region has an amino acid sequence having at least 80% identity to SEQ ID NO: 2 and the antigen-binding specificity of SEQ ID NOs: 6-8; (iv) the VH region has an amino acid sequence having at least 80% identity to SEQ ID NO: 25 and the antigen-binding specificity of SEQ ID NOs: 27-29, and the VL region has an amino acid sequence having at least 80% identity to SEQ ID NO: 26 and the antigen-binding specificity of SEQ ID NOs: 30-32; or (v) the VH region has an amino acid sequence having at least 80% identity to SEQ ID NO: 33 and the antigen-binding specificity of SEQ ID NOs: 35-37, and the VL region has an amino acid sequence having at least 80% identity to SEQ ID NO: 34 and the antigen-binding specificity of SEQ ID NOs: 38-40; The antibody or antigen-binding fragment of claim 1.

3. (i) the VH region is represented by SEQ ID NO: 17, and the VL region is represented by SEQ ID NO: 18 or a sequence having 90% identity thereto and the binding specificity thereof; (ii) the VH region is represented by SEQ ID NO: 9, and the VL region is represented by SEQ ID NO: 10 or a sequence having 90% identity thereto and the binding specificity thereof; (iii) the VH region is represented by SEQ ID NO: 1, and the VL region is represented by SEQ ID NO: 2 or a sequence having 90% identity thereto and the binding specificity thereof; (iv) the VH region is set forth in SEQ ID NO: 25 and the VL region is set forth in SEQ ID NO: 26 or a sequence having 90% identity thereto and the binding specificity thereof; or (v) the VH region is represented by SEQ ID NO: 33, and the VL region is represented by SEQ ID NO: 34 or a sequence having 90% identity thereto and the binding specificity thereof; The antibody or antigen-binding fragment of claim 1.

4. A bispecific antibody comprising a first antigen-binding region and a second antigen-binding region, the first antigen-binding region binds to p95HER2; The antibody a VH region comprising CDR-H1, CDR-H2, and CDR-H3, wherein CDR-H1 is set forth in SEQ ID NO: 19, CDR-H2 is set forth in SEQ ID NO: 20, and CDR-H3 is set forth in SEQ ID NO: 21, or a sequence having 90% identity and antigen-binding specificity to SEQ ID NO: 19, 20, or 21; a V L region comprising CDR-L1, CDR-L2, and CDR-L3, wherein CDR-L1 is set forth in SEQ ID NO: 22, CDR-L2 is set forth in SEQ ID NO: 23, and CDR-L3 is set forth in SEQ ID NO: 24, or a sequence having 90% identity and antigen-binding specificity to SEQ ID NO: 22, 23, or 24; A bispecific antibody comprising:

5. 5. The bispecific antibody of claim 4, wherein the VH region has an amino acid sequence that is at least 80% identical to SEQ ID NO: 17 and has the antigen-binding specificity of SEQ ID NOs: 19-21, and the VL region has an amino acid sequence that is at least 80% identical to SEQ ID NO: 18 and has the antigen-binding specificity of SEQ ID NOs: 22-24.

6. 5. The bispecific antibody of claim 4, wherein the second antigen-binding region binds to an immune checkpoint molecule or an immunostimulatory molecule.

7. 5. The bispecific antibody of claim 4, comprising a heavy chain sequence having the amino acid sequence of SEQ ID NO: 46 or 47, or a sequence having 90% identity thereto and the binding specificity thereof.

8. 8. The bispecific antibody of claim 7, further comprising a light chain having the amino acid sequence of SEQ ID NO:

18.

9. (a) a therapeutic agent; (b) an antibody or antigen-binding fragment thereof, or a bispecific antibody, wherein the antibody or antigen-binding fragment thereof is: (i) a V H region comprising CDR-H1, CDR-H2, and CDR-H3, wherein CDR-H1 is set forth in SEQ ID NO: 19, CDR-H2 is set forth in SEQ ID NO: 20, and CDR-H3 is set forth in SEQ ID NO: 21, or a sequence having 90% identity to and antigen-binding specificity of SEQ ID NO: 19, 20, or 21; and a V L region comprising CDR-L1, CDR-L2, and CDR-L3, wherein CDR-L1 is set forth in SEQ ID NO: 22, CDR-L2 is set forth in SEQ ID NO: 23, and CDR-L3 is set forth in SEQ ID NO: 24, or a sequence having 90% identity to and antigen-binding specificity of SEQ ID NO: 22, 23, or 24; (ii) a V H region comprising CDR-H1, CDR-H2, and CDR-H3, wherein CDR-H1 is set forth in SEQ ID NO: 11, CDR-H2 is set forth in SEQ ID NO: 12, and CDR-H3 is set forth in SEQ ID NO: 13, or a sequence having 90% identity to and antigen-binding specificity of SEQ ID NO: 11, 12, or 13; and a V L region comprising CDR-L1, CDR-L2, and CDR-L3, wherein CDR-L1 is set forth in SEQ ID NO: 14, CDR-L2 is set forth in SEQ ID NO: 15, and CDR-L3 is set forth in SEQ ID NO: 16, or a sequence having 90% identity to and antigen-binding specificity of SEQ ID NO: 14, 15, or 16; (iii) a V H region comprising CDR-H1, CDR-H2, and CDR-H3, wherein CDR-H1 is set forth in SEQ ID NO: 3, CDR-H2 is set forth in SEQ ID NO: 4, and CDR-H3 is set forth in SEQ ID NO: 5, or a sequence having 90% identity to and antigen-binding specificity of SEQ ID NO: 3, 4, or 5; and a V L region comprising CDR-L1, CDR-L2, and CDR-L3, wherein CDR-L1 is set forth in SEQ ID NO: 6, CDR-L2 is set forth in SEQ ID NO: 7, and CDR-L3 is set forth in SEQ ID NO: 8, or a sequence having 90% identity to and antigen-binding specificity of SEQ ID NO: 6, 7, or 8; (iv) a V H region comprising CDR-H1, CDR-H2, and CDR-H3, wherein CDR-H1 is set forth in SEQ ID NO: 27, CDR-H2 is set forth in SEQ ID NO: 28, and CDR-H3 is set forth in SEQ ID NO: 29, or a sequence having 90% identity and antigen-binding specificity to SEQ ID NO: 27, 28, or 29, and a V L region comprising CDR-L1, CDR-L2, and CDR-L3, wherein CDR-L1 is set forth in SEQ ID NO: 30, CDR-L2 is set forth in SEQ ID NO: 31, and CDR-L3 is set forth in SEQ ID NO: 32, or a sequence having 90% identity and antigen-binding specificity to SEQ ID NO: 30, 31, or 32; or (v) a V H region comprising CDR-H1, CDR-H2, and CDR-H3, wherein CDR-H1 is set forth in SEQ ID NO: 35, CDR-H2 is set forth in SEQ ID NO: 36, and CDR-H3 is set forth in SEQ ID NO: 37, or a sequence having 90% identity to and antigen-binding specificity of SEQ ID NO: 35, 36, or 37; and a V L region comprising CDR-L1, CDR-L2, and CDR-L3, wherein CDR-L1 is set forth in SEQ ID NO: 38, CDR-L2 is set forth in SEQ ID NO: 39, and CDR-L3 is set forth in SEQ ID NO: 40, or a sequence having 90% identity to and antigen-binding specificity of SEQ ID NO: 38, 39, or 40. Including, the antibody or antigen-binding fragment thereof binds to p95HER2; the bispecific antibody comprises an antibody antigen-binding region that binds to p95HER2, and a second antigen-binding region. an antibody or antigen-binding fragment thereof, or a bispecific antibody; An antibody-drug conjugate comprising:

10. (i) a VH region comprising CDR-H1, CDR-H2, and CDR-H3, wherein CDR-H1 is set forth in SEQ ID NO: 67, CDR-H2 is set forth in SEQ ID NO: 68, and CDR-H3 is set forth in SEQ ID NO: 69, or a sequence having 90% identity and antigen-binding specificity to SEQ ID NO: 67, 68, or 69; and a VL region comprising CDR-L1, CDR-L2, and CDR-L3, wherein CDR-L1 is set forth in SEQ ID NO: 70, CDR-L2 is set forth in SEQ ID NO: 71, and CDR-L3 is set forth in SEQ ID NO: 72, or a sequence having 90% identity and antigen-binding specificity to SEQ ID NO: 70, 71, or 72; (ii) a VH region comprising CDR-H1, CDR-H2, and CDR-H3, wherein CDR-H1 is represented by SEQ ID NO: 59, CDR-H2 is represented by SEQ ID NO: 60, and CDR-H3 is represented by SEQ ID NO: 61, or a sequence having 90% identity and antigen-binding specificity to SEQ ID NO: 59, 60, or 61; and a VL region comprising CDR-L1, CDR-L2, and CDR-L3, wherein CDR-L1 is represented by SEQ ID NO: 62, CDR-L2 is represented by SEQ ID NO: 63, and CDR-L3 is represented by SEQ ID NO: 64, or a sequence having 90% identity and antigen-binding specificity to SEQ ID NO: 62, 63, or 64; (iii) a VH region comprising CDR-H1, CDR-H2, and CDR-H3, wherein CDR-H1 is represented by SEQ ID NO: 51, CDR-H2 is represented by SEQ ID NO: 52, and CDR-H3 is represented by SEQ ID NO: 53, or a sequence having 90% identity and antigen-binding specificity to SEQ ID NO: 51, 52, or 53; and a VL region comprising CDR-L1, CDR-L2, and CDR-L3, wherein CDR-L1 is represented by SEQ ID NO: 54, CDR-L2 is represented by SEQ ID NO: 55, and CDR-L3 is represented by SEQ ID NO: 56, or a sequence having 90% identity and antigen-binding specificity to SEQ ID NO: 54, 55, or 56; (iv) a VH region comprising CDR-H1, CDR-H2, and CDR-H3, wherein CDR-H1 is represented by SEQ ID NO: 75, CDR-H2 is represented by SEQ ID NO: 76, and CDR-H3 is represented by SEQ ID NO: 77, or a sequence having 90% identity and antigen-binding specificity to SEQ ID NO: 75, 76, or 77; and a VL region comprising CDR-L1, CDR-L2, and CDR-L3, wherein CDR-L1 is represented by SEQ ID NO: 78, CDR-L2 is represented by SEQ ID NO: 79, and CDR-L3 is represented by SEQ ID NO: 80, or a sequence having 90% identity and antigen-binding specificity to SEQ ID NO: 78, 79, or 80; (v) a VH region comprising CDR-H1, CDR-H2, and CDR-H3, wherein CDR-H1 is represented by SEQ ID NO: 67, CDR-H2 is represented by SEQ ID NO: 68, and CDR-H3 is represented by SEQ ID NO: 69, or a sequence having 90% identity and antigen-binding specificity to SEQ ID NO: 67, 68, or 69; and a VL region comprising CDR-L1, CDR-L2, and CDR-L3, wherein CDR-L1 is represented by SEQ ID NO: 70, CDR-L2 is represented by SEQ ID NO: 82, and CDR-L3 is represented by SEQ ID NO: 72, or a sequence having 90% identity and antigen-binding specificity to SEQ ID NO: 70, 82, or 72; (vi) a VH region comprising CDR-H1, CDR-H2, and CDR-H3, wherein CDR-H1 is represented by SEQ ID NO: 67, CDR-H2 is represented by SEQ ID NO: 68, and CDR-H3 is represented by SEQ ID NO: 69, or a sequence having 90% identity and antigen-binding specificity to SEQ ID NO: 67, 68, or 69; and a VL region comprising CDR-L1, CDR-L2, and CDR-L3, wherein CDR-L1 is represented by SEQ ID NO: 70, CDR-L2 is represented by SEQ ID NO: 84, and CDR-L3 is represented by SEQ ID NO: 72, or a sequence having 90% identity and antigen-binding specificity to SEQ ID NO: 70, 84, or 72; or (vii) a VH region comprising CDR-H1, CDR-H2, and CDR-H3, wherein CDR-H1 is represented by SEQ ID NO: 67, CDR-H2 is represented by SEQ ID NO: 68, and CDR-H3 is represented by SEQ ID NO: 69, or a sequence having 90% identity and antigen-binding specificity to SEQ ID NO: 67, 68, or 69; and a VL region comprising CDR-L1, CDR-L2, and CDR-L3, wherein CDR-L1 is represented by SEQ ID NO: 70, CDR-L2 is represented by SEQ ID NO: 86, and CDR-L3 is represented by SEQ ID NO: 72, or a sequence having 90% identity and antigen-binding specificity to SEQ ID NO: 70, 86, or 72. Including, binds to CD73, An antibody or antigen-binding fragment thereof.

11. (i) the VH region has an amino acid sequence having at least 80% identity to SEQ ID NO: 65 and the antigen-binding specificity of SEQ ID NOs: 67-69, and the VL region has an amino acid sequence having at least 80% identity to SEQ ID NO: 66 and the antigen-binding specificity of SEQ ID NOs: 70-72; (ii) the VH region has an amino acid sequence having at least 80% identity to SEQ ID NO: 57 and the antigen-binding specificity of SEQ ID NOs: 59-61, and the VL region has an amino acid sequence having at least 80% identity to SEQ ID NO: 58 and the antigen-binding specificity of SEQ ID NOs: 62-64; (iii) the VH region has an amino acid sequence having at least 80% identity to SEQ ID NO: 49 and the antigen-binding specificity of SEQ ID NOs: 51-53, and the VL region has an amino acid sequence having at least 80% identity to SEQ ID NO: 50 and the antigen-binding specificity of SEQ ID NOs: 54-56; (iv) the VH region has an amino acid sequence having at least 80% identity to SEQ ID NO: 73 and the antigen-binding specificity of SEQ ID NOs: 75-77, and the VL region has an amino acid sequence having at least 80% identity to SEQ ID NO: 74 and the antigen-binding specificity of SEQ ID NOs: 78-80; (v) the VH region has an amino acid sequence having at least 80% identity to SEQ ID NO: 65 and the antigen-binding specificity of SEQ ID NOs: 67-69, and the VL region has an amino acid sequence having at least 80% identity to SEQ ID NO: 81 and the antigen-binding specificity of SEQ ID NOs: 70, 82, and 72; (vi) the VH region has an amino acid sequence having at least 80% identity to SEQ ID NO: 65 and the antigen-binding specificity of SEQ ID NOs: 67-69, and the VL region has an amino acid sequence having at least 80% identity to SEQ ID NO: 83 and the antigen-binding specificity of SEQ ID NOs: 70, 84, and 72; or (vii) the VH region has an amino acid sequence having at least 80% identity to SEQ ID NO: 65 and the antigen-binding specificity of SEQ ID NOs: 67-69, and the VL region has an amino acid sequence having at least 80% identity to SEQ ID NO: 85 and the antigen-binding specificity of SEQ ID NOs: 70, 86, and 72; The antibody or antigen-binding fragment of claim 10.

12. (i) the VH region is represented by SEQ ID NO: 65, and the VL region is represented by SEQ ID NO: 66 or a sequence having 90% identity thereto and the binding specificity thereof; (ii) the VH region is represented by SEQ ID NO: 57, and the VL region is represented by SEQ ID NO: 58 or a sequence having 90% identity thereto and the binding specificity thereof; (iii) the VH region is represented by SEQ ID NO: 49, and the VL region is represented by SEQ ID NO: 50 or a sequence having 90% identity thereto and the binding specificity thereof; (iv) the VH region is represented by SEQ ID NO: 73, and the VL region is represented by SEQ ID NO: 74 or a sequence having 90% identity thereto and the binding specificity thereof; (v) the VH region is represented by SEQ ID NO: 65, and the VL region is represented by SEQ ID NO: 81 or a sequence having 90% identity and binding specificity thereto; (vi) the VH region is set forth in SEQ ID NO: 65 and the VL region is set forth in SEQ ID NO: 83 or a sequence having 90% identity thereto and the binding specificity thereof; or (vii) the VH region is represented by SEQ ID NO: 65, and the VL region is represented by SEQ ID NO: 85 or a sequence having 90% identity thereto and the binding specificity thereof; The antibody or antigen-binding fragment of claim 10.

13. 11. The antibody or antigen-binding fragment of claim 1 or 10, wherein the antibody comprises an Fc domain.

14. 14. The antibody or antigen-binding fragment of claim 13, wherein the Fc domain is an IgG domain, an IgE domain, an IgM domain, and an IgD domain, an IgA domain, or an IgY domain.

15. 15. The antibody or antigen-binding fragment of claim 14, wherein the IgG domain is an IgG1 domain, an IgG2 domain, an IgG3 domain, or an IgG4 domain.

16. 16. The antibody or antigen-binding fragment of claim 15, wherein the IgG1 domain comprises the amino acid sequence of SEQ ID NO: 41 or 87.

17. 16. The antibody or antigen-binding fragment of claim 15, wherein the IgG1 comprises point mutations that alter or enhance antibody-dependent cellular cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC) compared to wild-type IgG1.

18. 18. The antibody or antigen-binding fragment of claim 17, wherein the point mutation is K297A or K322A.

19. A bispecific antibody comprising a first antigen-binding region and a second antigen-binding region, the first antigen-binding region binds to CD73, and the antibody (i) a VH region comprising CDR-H1, CDR-H2, and CDR-H3, wherein CDR-H1 is set forth in SEQ ID NO: 67, CDR-H2 is set forth in SEQ ID NO: 68, and CDR-H3 is set forth in SEQ ID NO: 69, or a sequence having 90% identity and antigen-binding specificity to SEQ ID NO: 67, 68, or 69; and a VL region comprising CDR-L1, CDR-L2, and CDR-L3, wherein CDR-L1 is set forth in SEQ ID NO: 70, CDR-L2 is set forth in SEQ ID NO: 71, and CDR-L3 is set forth in SEQ ID NO: 72, or a sequence having 90% identity and antigen-binding specificity to SEQ ID NO: 70, 71, or 72; (ii) a VH region comprising CDR-H1, CDR-H2, and CDR-H3, wherein CDR-H1 is set forth in SEQ ID NO: 67, CDR-H2 is set forth in SEQ ID NO: 68, and CDR-H3 is set forth in SEQ ID NO: 69, or a sequence having 90% identity and antigen-binding specificity to SEQ ID NO: 67, 68, or 69; and a VL region comprising CDR-L1, CDR-L2, and CDR-L3, wherein CDR-L1 is set forth in SEQ ID NO: 70, CDR-L2 is set forth in SEQ ID NO: 82, and CDR-L3 is set forth in SEQ ID NO: 72, or a sequence having 90% identity and antigen-binding specificity to SEQ ID NO: 70, 82, or 72; (iii) a VH region comprising CDR-H1, CDR-H2, and CDR-H3, wherein CDR-H1 is set forth in SEQ ID NO: 67, CDR-H2 is set forth in SEQ ID NO: 68, and CDR-H3 is set forth in SEQ ID NO: 69, or a sequence having 90% identity and antigen-binding specificity to SEQ ID NO: 67, 68, or 69; and a VL region comprising CDR-L1, CDR-L2, and CDR-L3, wherein CDR-L1 is set forth in SEQ ID NO: 70, CDR-L2 is set forth in SEQ ID NO: 84, and CDR-L3 is set forth in SEQ ID NO: 72, or a sequence having 90% identity and antigen-binding specificity to SEQ ID NO: 70, 84, or 72; or (iv) a VH region comprising CDR-H1, CDR-H2, and CDR-H3, wherein CDR-H1 is represented by SEQ ID NO: 67, CDR-H2 is represented by SEQ ID NO: 68, and CDR-H3 is represented by SEQ ID NO: 69, or a sequence having 90% identity and antigen-binding specificity to SEQ ID NO: 67, 68, or 69; and a VL region comprising CDR-L1, CDR-L2, and CDR-L3, wherein CDR-L1 is represented by SEQ ID NO: 70, CDR-L2 is represented by SEQ ID NO: 86, and CDR-L3 is represented by SEQ ID NO: 72, or a sequence having 90% identity and antigen-binding specificity to SEQ ID NO: 70, 86, or 72. A bispecific antibody comprising:

20. (i) the VH region has an amino acid sequence having at least 80% identity to SEQ ID NO: 65 and the antigen-binding specificity of SEQ ID NOs: 67-69, and the VL region has an amino acid sequence having at least 80% identity to SEQ ID NO: 66 and the antigen-binding specificity of SEQ ID NOs: 70-72; (ii) the VH region has an amino acid sequence having at least 80% identity to SEQ ID NO: 65 and the antigen-binding specificity of SEQ ID NOs: 67-69, and the VL region has an amino acid sequence having at least 80% identity to SEQ ID NO: 81 and the antigen-binding specificity of SEQ ID NOs: 70, 82, and 72; (iii) the VH region has an amino acid sequence having at least 80% identity to SEQ ID NO: 65 and the antigen-binding specificity of SEQ ID NOs: 67-69, and the VL region has an amino acid sequence having at least 80% identity to SEQ ID NO: 82 and the antigen-binding specificity of SEQ ID NOs: 70, 84, and 72; or (iv) the VH region has an amino acid sequence having at least 80% identity to SEQ ID NO: 65 and the antigen-binding specificity of SEQ ID NOs: 67-69, and the VL region has an amino acid sequence having at least 80% identity to SEQ ID NO: 85 and the antigen-binding specificity of SEQ ID NOs: 70, 86, and 72; 20. The bispecific antibody of claim 19.

21. 20. The bispecific antibody of claim 19, wherein the second antigen-binding region binds to an immune checkpoint molecule or an immunostimulatory molecule.

22. 20. The bispecific antibody of claim 19, comprising a heavy chain sequence having the amino acid sequence of SEQ ID NO: 88, 89, 90 or 91, or a sequence with 90% identity thereto and binding specificity thereto.

23. 20. The bispecific antibody of claim 19, further comprising a light chain having the amino acid sequence of SEQ ID NO:

66.

24. 20. The bispecific antibody of claim 4 or 19, comprising an Fc domain.

25. 25. The bispecific antibody of claim 24, wherein the Fc domain is an IgG domain, an IgE domain, an IgM domain, and an IgD domain, an IgA domain, or an IgY domain.

26. 26. The bispecific antibody of claim 25, wherein the Fc domain is an IgG domain.

27. 27. The bispecific antibody of claim 26, wherein the IgG domain is an IgG1 domain, an IgG2 domain, an IgG3 domain, or an IgG4 domain.

28. The bispecific antibody of claim 24, wherein an scFv is linked to the C-terminus of the Fc domain.

29. 29. The bispecific antibody of claim 28, comprising a linker between the Fab domain and the scFv domain.

30. 30. The bispecific antibody of claim 29, wherein a Fab fragment is linked to the N-terminus of the Fc domain.

31. The bispecific antibody of claim 30, wherein the Fab comprises a p95HER2 binding site or a CD73 binding site and the scFv comprises a CD137 binding site. (a) a therapeutic agent; and (b) an antibody or antigen-binding fragment thereof, or a bispecific antibody, wherein the antibody or antigen-binding fragment thereof is: (i) a VH region comprising CDR-H1, CDR-H2, and CDR-H3, wherein CDR-H1 is set forth in SEQ ID NO: 67, CDR-H2 is set forth in SEQ ID NO: 68, and CDR-H3 is set forth in SEQ ID NO: 69, or a sequence having 90% identity and antigen-binding specificity to SEQ ID NO: 67, 68, or 69; and a VL region comprising CDR-L1, CDR-L2, and CDR-L3, wherein CDR-L1 is set forth in SEQ ID NO: 70, CDR-L2 is set forth in SEQ ID NO: 71, and CDR-L3 is set forth in SEQ ID NO: 72, or a sequence having 90% identity and antigen-binding specificity to SEQ ID NO: 70, 71, or 72; (ii) a VH region comprising CDR-H1, CDR-H2, and CDR-H3, wherein CDR-H1 is represented by SEQ ID NO: 59, CDR-H2 is represented by SEQ ID NO: 60, and CDR-H3 is represented by SEQ ID NO: 61, or a sequence having 90% identity and antigen-binding specificity to SEQ ID NO: 59, 60, or 61; and a VL region comprising CDR-L1, CDR-L2, and CDR-L3, wherein CDR-L1 is represented by SEQ ID NO: 62, CDR-L2 is represented by SEQ ID NO: 63, and CDR-L3 is represented by SEQ ID NO: 64, or a sequence having 90% identity and antigen-binding specificity to SEQ ID NO: 62, 63, or 64; (iii) a VH region comprising CDR-H1, CDR-H2, and CDR-H3, wherein CDR-H1 is represented by SEQ ID NO: 51, CDR-H2 is represented by SEQ ID NO: 52, and CDR-H3 is represented by SEQ ID NO: 53, or a sequence having 90% identity and antigen-binding specificity to SEQ ID NO: 51, 52, or 53; and a VL region comprising CDR-L1, CDR-L2, and CDR-L3, wherein CDR-L1 is represented by SEQ ID NO: 54, CDR-L2 is represented by SEQ ID NO: 55, and CDR-L3 is represented by SEQ ID NO: 56, or a sequence having 90% identity and antigen-binding specificity to SEQ ID NO: 54, 55, or 56; (iv) a VH region comprising CDR-H1, CDR-H2, and CDR-H3, wherein CDR-H1 is represented by SEQ ID NO: 75, CDR-H2 is represented by SEQ ID NO: 76, and CDR-H3 is represented by SEQ ID NO: 77, or a sequence having 90% identity and antigen-binding specificity to SEQ ID NO: 75, 76, or 77; and a VL region comprising CDR-L1, CDR-L2, and CDR-L3, wherein CDR-L1 is represented by SEQ ID NO: 78, CDR-L2 is represented by SEQ ID NO: 79, and CDR-L3 is represented by SEQ ID NO: 80, or a sequence having 90% identity and antigen-binding specificity to SEQ ID NO: 78, 79, or 80; (v) a VH region comprising CDR-H1, CDR-H2, and CDR-H3, wherein CDR-H1 is represented by SEQ ID NO: 67, CDR-H2 is represented by SEQ ID NO: 68, and CDR-H3 is represented by SEQ ID NO: 69, or a sequence having 90% identity and antigen-binding specificity to SEQ ID NO: 67, 68, or 69; and a VL region comprising CDR-L1, CDR-L2, and CDR-L3, wherein CDR-L1 is represented by SEQ ID NO: 70, CDR-L2 is represented by SEQ ID NO: 82, and CDR-L3 is represented by SEQ ID NO: 72, or a sequence having 90% identity and antigen-binding specificity to SEQ ID NO: 70, 82, or 72; (vi) a VH region comprising CDR-H1, CDR-H2, and CDR-H3, wherein CDR-H1 is represented by SEQ ID NO: 67, CDR-H2 is represented by SEQ ID NO: 68, and CDR-H3 is represented by SEQ ID NO: 69, or a sequence having 90% identity and antigen-binding specificity to SEQ ID NO: 67, 68, or 69; and a VL region comprising CDR-L1, CDR-L2, and CDR-L3, wherein CDR-L1 is represented by SEQ ID NO: 70, CDR-L2 is represented by SEQ ID NO: 84, and CDR-L3 is represented by SEQ ID NO: 72, or a sequence having 90% identity and antigen-binding specificity to SEQ ID NO: 70, 84, or 72; or (vii) a VH region comprising CDR-H1, CDR-H2, and CDR-H3, wherein CDR-H1 is represented by SEQ ID NO: 67, CDR-H2 is represented by SEQ ID NO: 68, and CDR-H3 is represented by SEQ ID NO: 69, or a sequence having 90% identity and antigen-binding specificity to SEQ ID NO: 67, 68, or 69; and a VL region comprising CDR-L1, CDR-L2, and CDR-L3, wherein CDR-L1 is represented by SEQ ID NO: 70, CDR-L2 is represented by SEQ ID NO: 86, and CDR-L3 is represented by SEQ ID NO: 72, or a sequence having 90% identity and antigen-binding specificity to SEQ ID NO: 70, 86, or 72. Including, the antibody or antigen-binding fragment thereof binds to CD73; the bispecific antibody comprises an antibody antigen-binding region that binds to CD73, and a second antigen-binding region. an antibody or antigen-binding fragment thereof, or a bispecific antibody; An antibody-drug conjugate comprising:

33. 33. The antibody-drug conjugate of claim 9 or 32, wherein the therapeutic agent is covalently attached to the antibody, antigen-binding fragment, or bispecific antibody via a linker.

34. A pharmaceutical composition comprising the antibody or antigen-binding fragment of any one of claims 1 to 3 or 10 to 13, the bispecific antibody of any one of claims 4 to 8 or 19 to 23, or the antibody-drug conjugate of claim 9 or 32, and at least one pharmaceutically acceptable carrier.

35. 35. The pharmaceutical composition of claim 34, wherein the pharmaceutically acceptable carrier is conjugated to the C-terminus of the amino acid sequence of the antibody, antigen-binding fragment, bispecific antibody, or antibody-drug conjugate.

36. 34. A composition for treating cancer in a subject, comprising a therapeutically effective amount of the antibody or antigen-binding fragment of any one of claims 1 to 3 or 10 to 13, the bispecific antibody of any one of claims 4 to 8 or 19 to 23, or the antibody-drug conjugate of claim 9 or 33.

37. 37. The composition of claim 36, wherein the cancer is selected from prostate cancer, lung cancer, non-small cell lung cancer (NSCLC), melanoma, lymphoma, breast cancer, head and neck cancer, renal cell carcinoma (RCC), ovarian cancer, kidney cancer, bladder cancer, uterine cancer, cervical cancer, ovarian cancer, liver cancer, stomach cancer, colon cancer, rectal cancer, oral cancer, pharyngeal cancer, pancreatic cancer, thyroid cancer, skin cancer, brain cancer, bone cancer, hematopoietic cancer, or leukemia.

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