Multispecific binding constructs against checkpoint molecules and their uses

Abstract

The present disclosure relates to compositions and methods for preventing tumor escape by reducing immune checkpoint inhibition. In some embodiments, compositions are provided herein that block the interaction between PD-1 and its ligands (e.g., PD-1 and / or PD-L2) while promoting the interaction between PD-1 and cells on which its ligands are expressed. The present disclosure also provides methods for using such compositions.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority to U.S. Provisional Application No. 62 / 760,801, filed on November 13, 2018, U.S. Provisional Application No. 62 / 855,580, filed on May 31, 2019, U.S. Application No. 62 / 898,991, filed on September 11, 2019, and U.S. Application No. 62 / 931,478, filed on November 6, 2019. The entireties of the above applications are incorporated herein by reference. Background Art

[0003] Cancer is one of the leading causes of death in the United States and worldwide. Although common treatments (such as surgery, radiation, chemotherapy, hormone therapy, targeted therapy, and immunotherapy) have reduced the rate of cancer-related deaths in the 20th century, as of 2012, 14.1 million new cancer cases were diagnosed worldwide, and 8.2 million cancer deaths occurred. Even though overall cancer survival rates have improved in the 20th century, cancer still causes one in seven deaths worldwide. See American Cancer Society, Global Cancer Facts & Figures, 3rd edition, Atlanta: American Cancer Society; 2015.

[0004] In recent years, increasing evidence has shown that the immune system plays an important role in the formation and progression of tumors. The fact that naturally occurring T cells with anti-tumor potential or activity exist in cancer patients has rationalized the development of oncology immunotherapy. Immune cells (such as T cells, macrophages, and natural killer cells) can exhibit anti-tumor activity and effectively control the emergence and growth of malignant tumors. Tumor-specific antigens or tumor-associated antigens can induce immune cells to recognize and eliminate malignant tumors (Chen & Mellman, (2013) Immunity 39(1):1-10). Despite the existence of tumor-specific immune responses, malignant tumors often escape or avoid immune attacks through various immune regulatory mechanisms, making it difficult to control the emergence and progression of tumors (Motz & Coukos, (2013) Immunity 39(1):61-730). Indeed, an emerging hallmark of cancer is the exploitation of such immunoregulatory mechanisms and the impairment of anti-tumor immune responses leading to tumor escape and evasion of immune killing (Hanahan and Weinberg (2011) Cell 144(5):646-674).

[0005] New approaches to cancer immunotherapy involve counteracting these immune evasion and escape mechanisms and inducing the endogenous immune system to reject the tumor. However, there remains a need for new therapeutic agents, particularly cancer therapeutics, that are effective against immune evasion. SUMMARY OF THE INVENTION

[0007] The present disclosure is based in part on new multispecific, multivalent constructs targeting both PD-1 and PD-L1, such as bispecific tetravalent constructs. As demonstrated herein, these multispecific constructs have improved in vitro and in vivo efficacy compared to combinations of individual antibodies, as well as compared to clinical checkpoint blockers. Also provided herein are novel monoclonal anti-PD-1 antibodies and antigen-binding fragments thereof, and novel monoclonal anti-PD-L1 antibodies and antigen-binding fragments thereof for such multispecific, multivalent constructs. Some of these novel monoclonal anti-PD-1 antibodies and novel monoclonal anti-PD-L1 antibodies have a common light chain, thereby allowing the generation of multispecific, multivalent constructs with surprisingly excellent drug-like properties (DLPs) and manufacturing affordability, as well as affinities similar to those of their parent antibodies. The present disclosure is also based in part on the following discovery: blocking the interaction between PD-1 expressed by an immune cell and its ligand (e.g., PD-L1 or PD-L2) expressed on a second cell, while bridging the immune cell with the second cell (e.g., another immune cell or a tumor cell expressing a PD-1 ligand) strongly enhances, for example, T cell proliferation, IFNγ production and secretion, and T cell cytolytic activity. Therefore, provided herein are compositions that block the interaction between PD-1 and its ligand while promoting the interaction (bridging) of cells on which PD-1 and its ligand (PD-L1 or PD-L2) are expressed. As exemplified herein, such compositions of the present disclosure having “blocking and bridging” capabilities provide superior anti-tumor efficacy (as measured by, for example, IFNγ production and secretion and in vivo activity) compared to, for example, a mixture of agents that bind to receptors and ligands in stoichiometric amounts alone, or a single agent that binds to a receptor or ligand. It was also found that the multispecific, multivalent constructs targeting both PD-1 and PD-L1 described herein cause the loss of PD-1 expression on the cell surface in a valence-dependent manner. This loss of PD-1 expression was not observed when a combination of parent antibodies was used in stoichiometrically equivalent amounts. Thus, the multispecific, multivalent constructs described herein that target both PD-1 and PD-L1 provide new immunotherapeutic agents with increased potency and efficacy for the treatment of cancer.

[0008] In some embodiments, any of the multispecific antigen-binding constructs disclosed herein binds to at least two different receptors or epitopes (e.g., PD-1 and PD-L1), wherein the two different receptors or epitopes bound by the multispecific antigen-binding construct are expressed on the surface of the same cell. For example, in some embodiments, the multispecific antigen-binding construct simultaneously binds to PD-1 and PD-L1, wherein PD-1 and PD-L1 are expressed on the surface of the same cell. In some embodiments, any of the multispecific antigen-binding constructs disclosed herein binds to at least two different receptors or epitopes (e.g., PD-1 and PD-L1), wherein the two different receptors or epitopes bound by the multispecific antigen-binding construct are expressed on the surface of two different cells. For example, in some embodiments, the multispecific antigen-binding construct simultaneously binds to PD-1 expressed on the surface of a first cell and a PD-1 ligand expressed on the surface of a second cell, e.g., PD-L1 or PD-L2.

[0009] In some embodiments, the present disclosure provides a multispecific antigen-binding construct comprising at least two antigen-binding arms, wherein the first arm binds to PD-1 expressed by an immune cell, and the second arm binds to a PD-1 ligand expressed by a second cell, wherein the multispecific antigen-binding construct blocks the interaction between PD-1 and the PD-1 ligand. In some embodiments, the PD-1 ligand is PD-L2. In some embodiments, the PD-1 ligand is PD-L1. In some embodiments, the immune cell is a T cell. In some embodiments, the T cell is a CD8+ T cell. In some embodiments, the immune cell is a natural killer (NK) cell. In some embodiments, the immune cell is a macrophage. In some embodiments, the second cell is a second immune cell. In some embodiments, the second immune cell is any one or more of a T cell, a B cell, a macrophage, a suppressor cell derived from the bone marrow, a dendritic cell, or a mesenchymal stromal cell. In some embodiments, the second immune cell is a regulatory T cell. In some embodiments, the second cell is a tumor cell. In some embodiments, the tumor cell line is selected from the group consisting of blood cancer, lymphoma, myeloma, leukemia, neurological cancer, melanoma, breast cancer, prostate cancer, colorectal cancer, lung cancer, head and neck cancer, gastrointestinal cancer, liver cancer, pancreatic cancer, genitourinary cancer, bone cancer, kidney cancer, and vascular cancer. In some embodiments, both arms have at least 1x10 -7 M, at least 1x10 -8 M, at least 1x10 -9 M or at least 1x10 -10 M's K DIn some embodiments, binding of one arm to its target does not block binding of the other arm to its target. In some embodiments, the first arm and the second arm bind to their respective targets and both arms remain bound simultaneously. In some embodiments, binding of the first arm and the second arm to their respective targets can bridge the immune cell to the second cell. In some embodiments, bridging of the immune cell to the second cell is determined by flow cytometry. In some embodiments, the first arm is an antagonist of PD-1.

[0010] In some embodiments of the multispecific antigen-binding construct, the first arm binds PD-1 and comprises: (a) a heavy chain variable region comprising (i) a CDRH1 comprising SEQ ID NO: 70 (FTFX1X2YAX3X4, wherein X1 = S, R, G, or N; X2 = D, S, N, A, R, or G; X3 = M or L; X4 = S, L, or N); (ii) a CDRH2 comprising SEQ ID NO: 71 (SAISNSGTYTYYA); and (iii) a CDRH3 comprising SEQ ID NO: 72 (ARGLDFIVGX5TGNDY, wherein X5 = A, Y, or R); and (b) a light chain variable region comprising: (i) a CDRL1 comprising SEQ ID NO: 9 (RASQSISSYLN); (ii) a CDRL2 comprising SEQ ID NO: 5 (AASSLQS); and (iii) a CDRL3 comprising SEQ ID NO: 10 (QQSYSTPLT).

[0011] In some embodiments, the CDRH1 of the first arm comprises SEQ ID NO: 73 (FTFSDYAMS), the CDRH2 of the first arm comprises SEQ ID NO: 71 (SAISNSGTYTYYA), and the CDRH3 of the first arm comprises SEQ ID NO: 74 (ARGLDFIVGATGNDY). In some embodiments, the CDRH1 of the first arm comprises SEQ ID NO: 73 (FTFSDYAMS), the CDRH2 of the first arm comprises SEQ ID NO: 71 (SAISNSGTYTYYA), and the CDRH3 of the first arm comprises SEQ ID NO: 75 (ARGLDFIVGYTGNDY). In some embodiments, the CDRH1 of the first arm comprises SEQ ID NO: 76 (FTFSSYAMS), the CDRH2 of the first arm comprises SEQ ID NO: 71 (SAISNSGTYTYYA), and the CDRH3 of the first arm comprises SEQ ID NO: 75 (ARGLDFIVGYTGNDY). In some embodiments, the CDRH1 of the first arm comprises SEQ ID NO: 77 (FTFSSYAML), the CDRH2 of the first arm comprises SEQ ID NO: 71 (SAISNSGTYTYYA), and the CDRH3 of the first arm comprises SEQ ID NO: 75 (ARGLDFIVGYTGNDY). In some embodiments, the CDRH1 of the first arm comprises SEQ ID NO: 78 (FTFSNYALS), the CDRH2 of the first arm comprises SEQ ID NO: 71 (SAISNSGTYTYYA), and the CDRH3 of the first arm comprises SEQ ID NO: 75 (ARGLDFIVGYTGNDY). In some embodiments, the CDRH1 of the first arm comprises SEQ ID NO: 79 (FTFSAYAMN), the CDRH2 of the first arm comprises SEQ ID NO: 71 (SAISNSGTYTYYA), and the CDRH3 of the first arm comprises SEQ ID NO: 75 (ARGLDFIVGYTGNDY). In some embodiments, the CDRH1 of the first arm comprises SEQ ID NO: 80 (FTFRSYAMS), the CDRH2 of the first arm comprises SEQ ID NO: 71 (SAISNSGTYTYYA), and the CDRH3 of the first arm comprises SEQ ID NO: 75 (ARGLDFIVGYTGNDY). In some embodiments, the CDRH1 of the first arm comprises SEQ ID NO: 81 (FTFGRYAMS), the CDRH2 of the first arm comprises SEQ ID NO: 71 (SAISNSGTYTYYA), and the CDRH3 of the first arm comprises SEQ ID NO: 75 (ARGLDFIVGYTGNDY).In some embodiments, the CDRH1 of the first arm comprises SEQ ID NO: 82 (FTFNSYAMS), the CDRH2 of the first arm comprises SEQ ID NO: 71 (SAISNSGTYTYYA), and the CDRH3 of the first arm comprises SEQ ID NO: 75 (ARGLDFIVGYTGNDY). In some embodiments, the CDRH1 of the first arm comprises SEQ ID NO: 83 (FTFSNYAMS), the CDRH2 of the first arm comprises SEQ ID NO: 71 (SAISNSGTYTYYA), and the CDRH3 of the first arm comprises SEQ ID NO: 74 (ARGLDFIVGATGNDY). In some embodiments, the CDRH1 of the first arm comprises SEQ ID NO: 84 (FTFSGYAMS), the CDRH2 of the first arm comprises SEQ ID NO: 71 (SAISNSGTYTYYA), and the CDRH3 of the first arm comprises SEQ ID NO: 85 (ARGLDFIVGRTGNDY). In some embodiments, the CDRH1 of the first arm comprises SEQ ID NO: 86 (FTFSSYAMN), the CDRH2 of the first arm comprises SEQ ID NO: 71 (SAISNSGTYTYYA), and the CDRH3 of the first arm comprises SEQ ID NO: 85 (ARGLDFIVGRTGNDY). In some embodiments, the CDRH1 of the first arm comprises SEQ ID NO: 80 (FTFRSYAMS), the CDRH2 of the first arm comprises SEQ ID NO: 71 (SAISNSGTYTYYA), and the CDRH3 of the first arm comprises SEQ ID NO: 85 (ARGLDFIVGRTGNDY).

[0012] In some embodiments, CDRL1 of the first arm comprises SEQ ID NO: 9 (RASQSISSYLN), CDRL2 of the first arm comprises SEQ ID NO: 5 (AASSLQS), and CDRL3 of the first arm comprises SEQ ID NO: 10 (QQSYSTPLT).

[0013] In some embodiments, the heavy chain variable region of the first arm comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 87. In some embodiments, the heavy chain variable region of the first arm comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 88. In some embodiments, the heavy chain variable region of the first arm comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 89. In some embodiments, the heavy chain variable region of the first arm comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 90. In some embodiments, the heavy chain variable region of the first arm comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 91. In some embodiments, the heavy chain variable region of the first arm comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 92. In some embodiments, the heavy chain variable region of the first arm comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 93. In some embodiments, the heavy chain variable region of the first arm comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 94. In some embodiments, the heavy chain variable region of the first arm comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 95. In some embodiments, the heavy chain variable region of the first arm comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 96. In some embodiments, the heavy chain variable region of the first arm comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 97. In some embodiments, the heavy chain variable region of the first arm comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 98. In some embodiments, the heavy chain variable region of the first arm comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 99. In some embodiments, the light chain variable region of the first arm comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 59.

[0014] In some embodiments, the second arm is an antagonist of PD-1 ligand. In some embodiments, the second arm is an antagonist of PD-L2. In some embodiments, the second arm is an antagonist of PD-L1. In some embodiments, the second arm binds PD-L1 and comprises: a. a heavy chain variable region comprising (i) a CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN); (ii) a CDRH2 comprising SEQ ID NO: 2 (GGIIPX1X2GX3ATYA, wherein X1 is V or I; X2 is F, L, or V; and X3 is T or A); and (iii) a CDRH3 comprising SEQ ID NO: 3 (ARLKX1ELKDAFDI, wherein X1 is G, F, or N); and b. a light chain variable region comprising: (i) a CDRL1 comprising SEQ ID NO: 4 (RASQX1ISSYLN, wherein X1 is S, W, or Q); (ii) a CDRL2 comprising SEQ ID NO: 5 (AASSLQS); and (iii) a CDRL3 comprising SEQ ID NO: 6 (X1QSYSTPLT, wherein X1 is Q or F).

[0015] In some embodiments, the CDRH1 of the second arm comprises SEQ ID NO: 1 (GTFSSYAIN), the CDRH2 of the second arm comprises SEQ ID NO: 7 (GGIIPILGAATYA), and the CDRH3 of the second arm comprises SEQ ID NO: 8 (ARLKGELKDAFDI). In some embodiments, the CDRH1 of the second arm comprises SEQ ID NO: 1 (GTFSSYAIN), the CDRH2 of the second arm comprises SEQ ID NO: 7 (GGIIPILGAATYA), and the CDRH3 of the second arm comprises SEQ ID NO: 8 (ARLKGELKDAFDI); the CDRL1 of the second arm comprises SEQ ID NO: 9 (RASQSISSYLN), the CDRL2 of the second arm comprises SEQ ID NO: 5 (AASSLQS), and the CDRL3 of the second arm comprises SEQ ID NO: 10 (QQSYSTPLT).

[0016] In some embodiments, the CDRH1 of the second arm comprises SEQ ID NO: 1 (GTFSSYAIN), the CDRH2 of the second arm comprises SEQ ID NO: 11 (GGIIPVFGTATYA), and the CDRH3 of the second arm comprises SEQ ID NO: 8 (ARLKGELKDAFDI); the CDRL1 of the second arm comprises SEQ ID NO: 9 (RASQSISSYLN), the CDRL2 of the second arm comprises SEQ ID NO: 5 (AASSLQS), and the CDRL3 of the second arm comprises SEQ ID NO: 10 (QQSYSTPLT). In some embodiments, the CDRH1 of the second arm comprises SEQ ID NO: 1 (GTFSSYAIN), the CDRH2 of the second arm comprises SEQ ID NO: 11 (GGIIPVFGTATYA), and the CDRH3 of the second arm comprises SEQ ID NO: 8 (ARLKGELKDAFDI). In some embodiments, CDRH1 of the second arm comprises SEQ ID NO: 1 (GTFSSYAIN), CDRH2 of the second arm comprises SEQ ID NO: 11 (GGIIPVFGTATYA), and CDRH3 of the second arm comprises SEQ ID NO: 8 (ARLKGELKDAFDI); CDRL1 of the second arm comprises SEQ ID NO: 12 (RASQWISSYLN), CDRL2 of the second arm comprises SEQ ID NO: 5 (AASSLQS), and CDRL3 of the second arm comprises SEQ ID NO: 10 (QQSYSTPLT). In some embodiments, CDRH1 of the second arm comprises SEQ ID NO: 1 (GTFSSYAIN), CDRH2 of the second arm comprises SEQ ID NO: 11 (GGIIPVFGTATYA), and CDRH3 of the second arm comprises SEQ ID NO: 8 (ARLKGELKDAFDI); CDRL1 of the second arm comprises SEQ ID NO: 13 (RASQQISSYLN), CDRL2 of the second arm comprises SEQ ID NO: 5 (AASSLQS), and CDRL3 of the second arm comprises SEQ ID NO: 10 (QQSYSTPLT).In some embodiments, CDRH1 of the second arm comprises SEQ ID NO: 1 (GTFSSYAIN), CDRH2 of the second arm comprises SEQ ID NO: 11 (GGIIPVFGTATYA), and CDRH3 of the second arm comprises SEQ ID NO: 8 (ARLKGELKDAFDI); CDRL1 of the second arm comprises SEQ ID NO: 9 (RASQSISSYLN), CDRL2 of the second arm comprises SEQ ID NO: 5 (AASSLQS), and CDRL3 of the second arm comprises SEQ ID NO: 10 (QQSYSTPLT).

[0017] In some embodiments, the CDRH1 of the second arm comprises SEQ ID NO: 1 (GTFSSYAIN), the CDRH2 of the second arm comprises SEQ ID NO: 15 (GGIIPIFGIANYA), and the CDRH3 of the second arm comprises SEQ ID NO: 8 (ARLKGELKDAFDI). In some embodiments, the CDRH1 of the second arm comprises SEQ ID NO: 1 (GTFSSYAIN), the CDRH2 of the second arm comprises SEQ ID NO: 15 (GGIIPIFGIANYA), and the CDRH3 of the second arm comprises SEQ ID NO: 8 (ARLKGELKDAFDI); the CDRL1 of the second arm comprises SEQ ID NO: 9 (RASQSISSYLN), the CDRL2 of the second arm comprises SEQ ID NO: 5 (AASSLQS), and the CDRL3 of the second arm comprises SEQ ID NO: 10 (QQSYSTPLT).

[0018] In some embodiments, the CDRH1 of the second arm comprises SEQ ID NO: 1 (GTFSSYAIN), the CDRH2 of the second arm comprises SEQ ID NO: 16 (GGIIPNFGTATYA), and the CDRH3 of the second arm comprises SEQ ID NO: 17 (ARLKGELKGAGDI). In some embodiments, the CDRH1 of the second arm comprises SEQ ID NO: 1 (GTFSSYAIN), the CDRH2 of the second arm comprises SEQ ID NO: 16 (GGIIPNFGTATYA), and the CDRH3 of the second arm comprises SEQ ID NO: 17 (ARLKGELKGAGDI); the CDRL1 of the second arm comprises SEQ ID NO: 9 (RASQSISSYLN), the CDRL2 of the second arm comprises SEQ ID NO: 5 (AASSLQS), and the CDRL3 of the second arm comprises SEQ ID NO: 10 (QQSYSTPLT).

[0019] In some embodiments, the CDRH1 of the second arm comprises SEQ ID NO: 1 (GTFSSYAIN), the CDRH2 of the second arm comprises SEQ ID NO: 11 (GGIIPVFGTATYA), and the CDRH3 of the second arm comprises SEQ ID NO: 18 (ARLKFELKDAFDI). In some embodiments, the CDRH1 of the second arm comprises SEQ ID NO: 1 (GTFSSYAIN), the CDRH2 of the second arm comprises SEQ ID NO: 11 (GGIIPVFGTATYA), the CDRH3 of the second arm comprises SEQ ID NO: 18 (ARLKFELKDAFDI), the CDRL1 of the second arm comprises SEQ ID NO: 9 (RASQSISSYLN), the CDRL2 of the second arm comprises SEQ ID NO: 5 (AASSLQS), and the CDRL3 of the second arm comprises SEQ ID NO: 10 (QQSYSTPLT).

[0020] In some embodiments, the CDRH1 of the second arm comprises SEQ ID NO: 1 (GTFSSYAIN), the CDRH2 of the second arm comprises SEQ ID NO: 11 (GGIIPVFGTATYA), and the CDRH3 of the second arm comprises SEQ ID NO: 19 (ARLKGELKDAFDE). In some embodiments, the CDRH1 of the second arm comprises SEQ ID NO: 1 (GTFSSYAIN), the CDRH2 of the second arm comprises SEQ ID NO: 11 (GGIIPVFGTATYA), the CDRH3 of the second arm comprises SEQ ID NO: 19 (ARLKGELKDAFDE), the CDRL1 of the second arm comprises SEQ ID NO: 9 (RASQSISSYLN), the CDRL2 of the second arm comprises SEQ ID NO: 5 (AASSLQS), and the CDRL3 of the second arm comprises SEQ ID NO: 10 (QQSYSTPLT).

[0021] In some embodiments, the CDRH1 of the second arm comprises SEQ ID NO: 1 (GTFSSYAIN), the CDRH2 of the second arm comprises SEQ ID NO: 11 (GGIIPVFGTATYA), and the CDRH3 of the second arm comprises SEQ ID NO: 20 (ARLKNELKDAFDI). In some embodiments, the CDRH1 of the second arm comprises SEQ ID NO: 1 (GTFSSYAIN), the CDRH2 of the second arm comprises SEQ ID NO: 11 (GGIIPVFGTATYA), the CDRH3 of the second arm comprises SEQ ID NO: 20 (ARLKNELKDAFDI), the CDRL1 of the second arm comprises SEQ ID NO: 9 (RASQSISSYLN), the CDRL2 of the second arm comprises SEQ ID NO: 5 (AASSLQS), and the CDRL3 of the second arm comprises SEQ ID NO: 10 (QQSYSTPLT).

[0022] In some embodiments, the CDRH1 of the second arm comprises SEQ ID NO: 1 (GTFSSYAIN), the CDRH2 of the second arm comprises SEQ ID NO: 21 (GGVIPFLGTANYA), and the CDRH3 of the second arm comprises SEQ ID NO: 22 (ARLKGILKDALDI). In some embodiments, the CDRH1 of the second arm comprises SEQ ID NO: 1 (GTFSSYAIN), the CDRH2 of the second arm comprises SEQ ID NO: 21 (GGVIPFLGTANYA), the CDRH3 of the second arm comprises SEQ ID NO: 22 (ARLKGILKDALDI), the CDRL1 of the second arm comprises SEQ ID NO: 9 (RASQSISSYLN), the CDRL2 of the second arm comprises SEQ ID NO: 5 (AASSLQS), and the CDRL3 of the second arm comprises SEQ ID NO: 10 (QQSYSTPLT).

[0023] In some embodiments, the CDRH1 of the second arm comprises SEQ ID NO: 1 (GTFSSYAIN), the CDRH2 of the second arm comprises SEQ ID NO: 29 (GRIIPIFGTADYA), and the CDRH3 of the second arm comprises SEQ ID NO: 8 (ARLKGELKDAFDI). In some embodiments, the CDRH1 of the second arm comprises SEQ ID NO: 1 (GTFSSYAIN), the CDRH2 of the second arm comprises SEQ ID NO: 29 (GRIIPIFGTADYA), the CDRH3 of the second arm comprises SEQ ID NO: 8 (ARLKGELKDAFDI), the CDRL1 of the second arm comprises SEQ ID NO: 9 (RASQSISSYLN), the CDRL2 of the second arm comprises SEQ ID NO: 5 (AASSLQS), and the CDRL3 of the second arm comprises SEQ ID NO: 10 (QQSYSTPLT).

[0024] In some embodiments, the CDRH1 of the second arm comprises SEQ ID NO: 1 (GTFSSYAIN), the CDRH2 of the second arm comprises SEQ ID NO: 31 (GGIIPILGTATYA), and the CDRH3 of the second arm comprises SEQ ID NO: 32 (ARRKGELKDAFDI). In some embodiments, the CDRH1 of the second arm comprises SEQ ID NO: 1 (GTFSSYAIN), the CDRH2 of the second arm comprises SEQ ID NO: 31 (GGIIPILGTATYA), the CDRH3 of the second arm comprises SEQ ID NO: 32 (ARRKGELKDAFDI), the CDRL1 of the second arm comprises SEQ ID NO: 9 (RASQSISSYLN), the CDRL2 of the second arm comprises SEQ ID NO: 5 (AASSLQS), and the CDRL3 of the second arm comprises SEQ ID NO: 10 (QQSYSTPLT).

[0025] In some embodiments, the CDRH1 of the second arm comprises SEQ ID NO: 1 (GTFSSYAIN), the CDRH2 of the second arm comprises SEQ ID NO: 33 (GGIIPIVATANYA), and the CDRH3 of the second arm comprises SEQ ID NO: 32 (ARRKGELKDAFDI). In some embodiments, the CDRH1 of the second arm comprises SEQ ID NO: 1 (GTFSSYAIN), the CDRH2 of the second arm comprises SEQ ID NO: 33 (GGIIPIVATANYA), the CDRH3 of the second arm comprises SEQ ID NO: 32 (ARRKGELKDAFDI), the CDRL1 of the second arm comprises SEQ ID NO: 9 (RASQSISSYLN), the CDRL2 of the second arm comprises SEQ ID NO: 5 (AASSLQS), and the CDRL3 of the second arm comprises SEQ ID NO: 10 (QQSYSTPLT).

[0026] In some embodiments, the CDRH1 of the second arm comprises SEQ ID NO: 1 (GTFSSYAIN), the CDRH2 of the second arm comprises SEQ ID NO: 34 (GGIIPIFGKATYA), and the CDRH3 of the second arm comprises SEQ ID NO: 32 (ARRKGELKDAFDI). In some embodiments, the CDRH1 of the second arm comprises SEQ ID NO: 1 (GTFSSYAIN), the CDRH2 of the second arm comprises SEQ ID NO: 34 (GGIIPIFGKATYA), the CDRH3 of the second arm comprises SEQ ID NO: 32 (ARRKGELKDAFDI), the CDRL1 of the second arm comprises SEQ ID NO: 9 (RASQSISSYLN), the CDRL2 of the second arm comprises SEQ ID NO: 5 (AASSLQS), and the CDRL3 of the second arm comprises SEQ ID NO: 10 (QQSYSTPLT).

[0027] In some embodiments, CDRH1 of the second arm comprises SEQ ID NO: 1 (GTFSSYAIN), CDRH2 of the second arm comprises SEQ ID NO: 11 (GGIIPVFGTATYA), and CDRH3 of the second arm comprises SEQ ID NO: 8 (ARLKGELKDAFDI); CDRL1 of the second arm comprises SEQ ID NO: 9 (RASQSISSYLN), CDRL2 of the second arm comprises SEQ ID NO: 5 (AASSLQS), and CDRL3 of the second arm comprises SEQ ID NO: 38 (FQSYSTPLT).

[0028] In some embodiments, the CDRH1 of the second arm comprises SEQ ID NO: 1 (GTFSSYAIN), the CDRH2 of the second arm comprises SEQ ID NO: 11 (GGIIPVFGTATYA), and the CDRH3 of the second arm comprises SEQ ID NO: 8 (ARLKGELKDAFDI); the CDRL1 of the second arm comprises SEQ ID NO: 9 (RASQSISSYLN), the CDRL2 of the second arm comprises SEQ ID NO: 5 (AASSLQS), and the CDRL3 of the second arm comprises SEQ ID NO: 39 (QQSYSTILT).

[0029] In some embodiments, the second arm comprises: a. a heavy chain variable region comprising (i) CDRH1 comprising SEQ ID NO: 14 (GTFSSYAFS), (ii) CDRH2 comprising SEQ ID NO: 11 (GGIIPVFGTATYA), and (iii) CDRH3 comprising SEQ ID NO: 8 (ARLKGELKDAFDI); and b. a light chain variable region comprising: (i) CDRL1 comprising SEQ ID NO: 9 (RASQSISSYLN); (ii) CDRL2 comprising SEQ ID NO: 5 (AASSLQS); and (iii) CDRL3 comprising SEQ ID NO: 10 (QQSYSTPLT).

[0030] In some embodiments, the second arm comprises a heavy chain variable region comprising (i) a CDRH1 comprising SEQ ID NO: 23 (GTFSSYAIS), (ii) a CDRH2 comprising SEQ ID NO: 24 (GGIIPIVGIANYA), and (iii) a CDRH3 comprising SEQ ID NO: 8 (ARLKGELKDAFDI). In some embodiments, the second arm comprises a light chain variable region comprising: (i) a CDRL1 comprising SEQ ID NO: 9 (RASQSISSYLN); (ii) a CDRL2 comprising SEQ ID NO: 5 (AASSLQS); and (iii) a CDRL3 comprising SEQ ID NO: 10 (QQSYSTPLT).

[0031] In some embodiments, the second arm comprises a heavy chain variable region comprising (i) a CDRH1 comprising SEQ ID NO: 23 (GTFSSYAIS), (ii) a CDRH2 comprising SEQ ID NO: 11 (GGIIPVFGTATYA), and (iii) a CDRH3 comprising SEQ ID NO: 25 (ARLKGEFKDAFDI). In some embodiments, the second arm comprises a light chain variable region comprising: (i) a CDRL1 comprising SEQ ID NO: 9 (RASQSISSYLN); (ii) a CDRL2 comprising SEQ ID NO: 5 (AASSLQS); and (iii) a CDRL3 comprising SEQ ID NO: 10 (QQSYSTPLT).

[0032] In some embodiments, the second arm comprises a heavy chain variable region comprising (i) a CDRH1 comprising SEQ ID NO: 23 (GTFSSYAIS), (ii) a CDRH2 comprising SEQ ID NO: 26 (GRIIPLFGTAHYA), and (iii) a CDRH3 comprising SEQ ID NO: 8 (ARLKGELKDAFDI). In some embodiments, the second arm comprises a light chain variable region comprising: (i) a CDRL1 comprising SEQ ID NO: 9 (RASQSISSYLN); (ii) a CDRL2 comprising SEQ ID NO: 5 (AASSLQS); and (iii) a CDRL3 comprising SEQ ID NO: 10 (QQSYSTPLT).

[0033] In some embodiments, the second arm comprises a heavy chain variable region comprising (i) a CDRH1 comprising SEQ ID NO: 23 (GTFSSYAIS), (ii) a CDRH2 comprising SEQ ID NO: 27 (GRINPILGTANYA), and (iii) a CDRH3 comprising SEQ ID NO: 28 (ARLKGELKDAFSI). In some embodiments, the second arm comprises a light chain variable region comprising: (i) a CDRL1 comprising SEQ ID NO: 9 (RASQSISSYLN); (ii) a CDRL2 comprising SEQ ID NO: 5 (AASSLQS); and (iii) a CDRL3 comprising SEQ ID NO: 10 (QQSYSTPLT).

[0034] In some embodiments, the second arm comprises a heavy chain variable region comprising (i) a CDRH1 comprising SEQ ID NO: 23 (GTFSSYAIS), (ii) a CDRH2 comprising SEQ ID NO: 11 (GGIIPVFGTATYA), and (iii) a CDRH3 comprising SEQ ID NO: 30 (ARLKGELKCAFDI). In some embodiments, the second arm comprises a light chain variable region comprising: (i) a CDRL1 comprising SEQ ID NO: 9 (RASQSISSYLN); (ii) a CDRL2 comprising SEQ ID NO: 5 (AASSLQS); and (iii) a CDRL3 comprising SEQ ID NO: 10 (QQSYSTPLT).

[0035] In some embodiments, the second arm comprises a heavy chain variable region comprising (i) a CDRH1 comprising SEQ ID NO: 122 (GTKSSYAIS), (ii) a CDRH2 comprising SEQ ID NO: 11 (GGIIPVFGTATYA), and (iii) a CDRH3 comprising SEQ ID NO: 30 (ARLKGELKCAFDI). In some embodiments, the second arm further comprises a light chain variable region comprising: (i) a CDRL1 comprising SEQ ID NO: 9 (RASQSISSYLN); (ii) a CDRL2 comprising SEQ ID NO: 5 (AASSLQS); and (iii) a CDRL3 comprising SEQ ID NO: 10 (QQSYSTPLT).

[0036] In some embodiments, the second arm comprises a heavy chain variable region comprising (i) a CDRH1 comprising SEQ ID NO: 36 (GPFRSHAVS), (ii) a CDRH2 comprising SEQ ID NO: 11 (GGIIPVFGTATYA), and (iii) a CDRH3 comprising SEQ ID NO: 37 (ARLKSELKDAFDI). In some embodiments, the second arm comprises a light chain variable region comprising: (i) a CDRL1 comprising SEQ ID NO: 9 (RASQSISSYLN); (ii) a CDRL2 comprising SEQ ID NO: 5 (AASSLQS); and (iii) a CDRL3 comprising SEQ ID NO: 10 (QQSYSTPLT).

[0037] In some embodiments, the second arm that binds PD-L1 comprises a heavy chain variable region comprising an amino acid sequence at least 90% identical to any one of SEQ ID NO: 35, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, or 58, and a light chain variable region comprising an amino acid sequence at least 90% identical to any one of SEQ ID NO: 59, 60, 61, 62, or 63.

[0038] In some embodiments, the heavy chain variable region of the second arm comprises an amino acid sequence at least 90% identical to SEQ ID NO: 35 (QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAINWVRQAPGQGLEWMGGIIPVFGTATYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARLKGELKDAFDIWGQGTMVTVSS). In some embodiments, the heavy chain variable region of the second arm comprises an amino acid sequence at least 90% identical to SEQ ID NO: 40 (QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAFSWVRQAPGQGLEWMGGIIPVFGTATYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARLKGELKDAFDIWGQGTLVTVSS). In some embodiments, the heavy chain variable region of the second arm comprises an amino acid sequence at least 90% identical to SEQ ID NO: 41 (QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAINWVRQAPGQGLEWMGGIIPIFGIANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARLKGELKDAFDIWGQGTLVTVSS). In some embodiments, the heavy chain variable region of the second arm comprises an amino acid sequence at least 90% identical to SEQ ID NO: 42 (QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAINWVRQAPGQGLEWMGGIIPNFGTATYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARLKGELKGAGDIWGQGTLVTVSS). In some embodiments, the heavy chain variable region of the second arm comprises an amino acid sequence at least 90% identical to SEQ ID NO:43 (QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAINWVRQAPGQGLEWMGGIIPVFGTATYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARLKFELKDAFDIWGQGTLVTVSS).In some embodiments, the heavy chain variable region of the second arm comprises an amino acid sequence at least 90% identical to SEQ ID NO: 44 (QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAINWVRQAPGQGLEWMGGIIPVFGTATYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARLKGELKDAFDEWGQGTLVTVSS). In some embodiments, the heavy chain variable region of the second arm comprises an amino acid sequence at least 90% identical to SEQ ID NO: 45 (QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAINWVRQAPGQGLEWMGGIIPVFGTATYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARLKGELKDAFDIWGQGTLVTAST). In some embodiments, the heavy chain variable region of the second arm comprises an amino acid sequence at least 90% identical to SEQ ID NO: 46 (QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAINWVRQAPGQGLEWMGGIIPVFGTATYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARLKNELKDAFDIWGQGTLVTVSS). In some embodiments, the heavy chain variable region of the second arm comprises an amino acid sequence at least 90% identical to SEQ ID NO: 47 (QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAINWVRQAPGQGLEWMGGVIPFLGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARLKGILKDALDIWGQGTLVTVSS). In some embodiments, the heavy chain variable region of the second arm comprises an amino acid sequence at least 90% identical to SEQ ID NO:48 (QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQDLEWMGGIIPIVGIANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARLKGELKDAFDIWGQGTLVTVSS).In some embodiments, the heavy chain variable region of the second arm comprises an amino acid sequence at least 90% identical to SEQ ID NO: 49 (QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPVFGTATYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARLKGEFKDAFDIWGQGTLVTVSS). In some embodiments, the heavy chain variable region of the second arm comprises an amino acid sequence at least 90% identical to SEQ ID NO: 50 (QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGRIIPLFGTAHYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARLKGELKDAFDIWGQGTLVTVSS). In some embodiments, the heavy chain variable region of the second arm comprises an amino acid sequence at least 90% identical to SEQ ID NO: 51 (QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGRINPILGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARLKGELKDAFSIWGQGTLVTVSS). In some embodiments, the heavy chain variable region of the second arm comprises an amino acid sequence at least 90% identical to SEQ ID NO: 52 (QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAINWVRQAPGQGLEWMGRIIPIFGTADYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARLKGELKDAFDIWGQGTLVTVSS). In some embodiments, the heavy chain variable region of the second arm comprises an amino acid sequence at least 90% identical to SEQ ID NO: 53 (QVQLVQSGAEVKKPGSSVKVSCKASGGKFSSYAISWVRQAPGQGLEWMGGIIPVFGTATYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARLKGELKCAFDIWGQGTLVTVSS).In some embodiments, the heavy chain variable region of the second arm comprises an amino acid sequence at least 90% identical to SEQ ID NO: 54 (QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAINWVRQAPGQGLEWMGGIIPILGTATYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARRKGELKDAFDIWGQGTLVTVSS). In some embodiments, the heavy chain variable region of the second arm comprises an amino acid sequence at least 90% identical to SEQ ID NO: 55 (QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAINWVRQAPGQGLEWMGGIIPILGAATYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARLKGELKDAFDIWGQGTLVTVSS). In some embodiments, the heavy chain variable region of the second arm comprises an amino acid sequence at least 90% identical to SEQ ID NO: 56 (QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAINWVRQAPGQGLEWMGGIIPIVATANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARRKGELKDAFDIWGQGTLVTVSS). In some embodiments, the heavy chain variable region of the second arm comprises an amino acid sequence at least 90% identical to SEQ ID NO: 57 (QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAINWVRQAPGQGLEWMGGIIPIFGKATYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARRKGELKDAFDIWGQGTLVTVSS). In some embodiments, the heavy chain variable region of the second arm comprises an amino acid sequence at least 90% identical to SEQ ID NO: 58 (QVQLVQSGAEVKKPGSSVKVSCKASGGPFRSHAVSWVRQAPGQGLEWMGGIIPVFGTATYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARLKSELKDAFDIWGQGTLVTVSS). In some embodiments, the light chain variable region of the second arm comprises an amino acid sequence at least 90% identical to SEQ ID NO: 59 (DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGGGTKVEIK).In some embodiments, the light chain variable region of the second arm comprises an amino acid sequence at least 90% identical to SEQ ID NO: 60 (DIQMTQSPSSLSASVGDRVTITCRASQWISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGGGTKVEIK). In some embodiments, the light chain variable region of the second arm comprises an amino acid sequence at least 90% identical to SEQ ID NO: 61 (DIQMTQSPSSLSASVGDRVTITCRASQQISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGGGTKVEIK). In some embodiments, the light chain variable region of the second arm comprises an amino acid sequence at least 90% identical to SEQ ID NO: 62 (DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCFQSYSTPLTFGGGTKVEIK). In some embodiments, the light chain variable region of the second arm comprises an amino acid sequence at least 90% identical to SEQ ID NO: 63 (DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTILTFGGGTKVEIK).

[0039] In some embodiments of any aspect described herein, the construct is a bispecific antibody. In some embodiments, the bispecific antibody is an antagonist of both PD-1 and PD-1 ligand. In some embodiments, the construct comprises a common light chain. In some embodiments, one or both of the arms are aptamers. In some embodiments, one or both of the arms are proteins other than antibodies. In some embodiments, the construct comprises at least two bispecific antibodies. In some embodiments, one of the at least two bispecific antibodies is monovalent for PD-1. In some embodiments, one of the at least two bispecific antibodies is monovalent for PD-1 ligand. In some embodiments, at least one of the arms is a bivalent antibody specific for PD-1. In some embodiments, at least one of the arms is a bivalent antibody specific for PD-L1. In some embodiments, at least one of the arms is a bivalent antibody specific for PD-1, and at least one of the arms is a bivalent antibody specific for PD-L1. In some embodiments, at least one of the arms is bivalent for PD-1. In some embodiments, at least one of the arms is bivalent for PD-L1. In some embodiments, at least one of the arms is bivalent for PD-1 and at least one of the arms is bivalent for PD-L1. In some embodiments, the bispecific antibody binds to two different epitopes on PD-1. In some embodiments, the bispecific antibody binds to two different epitopes on the PD-1 ligand.

[0040] In some embodiments, any of the multispecific antigen-binding constructs disclosed herein comprises at least two monospecific antibodies. In some embodiments, at least one of the monospecific antibodies is an anti-PD-1 antibody. In some embodiments, the anti-PD-1 antibody is a bivalent anti-PD-1 antibody. In some embodiments, at least one of the monospecific antibodies is an anti-PD-L1 antibody. In some embodiments, the anti-PD-1 antibody is a bivalent anti-PD-L1 antibody. In some embodiments, the construct comprises a bivalent anti-PD-1 antibody and a bivalent anti-PD-L1 antibody. In some embodiments, the construct is a fusion construct in which a polypeptide comprising the variable heavy chain of an anti-PD-1 antibody is fused to a polypeptide comprising the variable heavy chain of an anti-PD-L1 antibody. In some embodiments, the polypeptide comprising the variable heavy chain of an anti-PD-1 antibody is fused to a polypeptide comprising the variable heavy chain of an anti-PD-L1 antibody via a linker. In some embodiments, the fusion construct comprises a common light chain. In some embodiments, the N-terminal variable heavy chain of the fusion construct binds to PD-1 in the presence of a common light chain, and the C-terminal variable heavy chain of the fusion construct binds to PD-L1 in the presence of a common light chain. In some embodiments, the N-terminal variable heavy chain of the fusion construct binds to PD-L1 in the presence of a common light chain, and the C-terminal variable heavy chain of the fusion construct binds to PD-1 in the presence of a common light chain.

[0041] In some aspects and embodiments, the present disclosure provides a multispecific antigen-binding construct comprising at least two antigen-binding units, wherein the first antigen-binding unit binds to PD-1 and the second antigen-binding unit binds to a PD-1 ligand. In some embodiments, the first antigen-binding unit binds to PD-1 expressed by an immune cell. In some embodiments, the second antigen-binding unit binds to PD-1 expressed by a second cell. In some embodiments, the multispecific antigen-binding construct blocks the interaction between PD-1 and a PD-1 ligand (such as PD-L1 or PD-L2). In some embodiments, the multispecific antigen-binding construct blocks the interaction between PD-1 and a PD-1 ligand (such as PD-L1 or PD-L2). In some embodiments, the multispecific antigen-binding construct comprises at least two antigen-binding units that bind to PD-1. In some embodiments, the multispecific antigen-binding construct comprises two antigen-binding units that bind to PD-1. In some embodiments, the multispecific antigen-binding construct comprises at least two antigen-binding units that bind to a PD-1 ligand (such as PD-L1 or PD-L2). In some embodiments, the multispecific antigen-binding construct comprises two antigen-binding units that bind to a PD-1 ligand (such as PD-L1 or PD-L2). In some embodiments, the multispecific antigen-binding construct comprises at least four antigen-binding units, two of which bind to PD-1 and two bind to a PD-1 ligand (such as PD-L1 or PD-L2). In some embodiments, the multispecific antigen-binding construct comprises four antigen-binding units, two of which bind to PD-1 and two bind to a PD-1 ligand (such as PD-L1 or PD-L2). In some embodiments, each antigen-binding unit can independently bind to its cognate antigen, i.e., PD-1 or a PD-1 ligand, such as PD-L1 or PD-L2. In some embodiments, the multispecific antigen-binding construct promotes the loss of PD-1 expression from cells. In some embodiments, the loss of PD-1 expression is due to PD-1 shedding. In some embodiments, the multispecific antigen-binding construct blocks the interaction between PD-1 and a PD-1 ligand (such as PD-L1 or PD-L2). In some embodiments, the multispecific antigen-binding construct comprises a common light chain. For example, at least two antigen-binding units comprise a common light chain.

[0042] In some embodiments, the first antigen binding unit binds to PD-1 and comprises:

[0043] (a) a heavy chain variable region comprising (i) a CDRH1 comprising SEQ ID NO: 70 (FTFX1X2YAX3X4, wherein X1 = S, R, G, or N; X2 = D, S, N, A, R, or G; X3 = M or L; X4 = S, L, or N); (ii) a CDRH2 comprising SEQ ID NO: 71 (SAISNSGTYTYYA); and (iii) a CDRH3 comprising SEQ ID NO: 72 (ARGLDFIVGX5TGNDY, wherein X5 = A, Y, or R); and

[0044] (b) a light chain variable region comprising: (i) CDRL1 comprising SEQ ID NO: 9 (RASQSISSYLN); (ii) CDRL2 comprising SEQ ID NO: 5 (AASSLQS); and (iii) CDRL3 comprising SEQ ID NO: 10 (QQSYSTPLT).

[0045] In some such embodiments, the first antigen binding unit binds PD-1 and comprises:

[0046] (a) CDRH1 comprising SEQ ID NO:73 (FTFSDYAMS), CDRH2 comprising SEQ ID NO:71 (SAISNSGTYTYYA), and CDRH3 comprising SEQ ID NO:74 (ARGLDFIVGATGNDY);

[0047] (b) CDRH1 comprising SEQ ID NO:73 (FTFSDYAMS), CDRH2 comprising SEQ ID NO:71 (SAISNSGTYTYYA), and CDRH3 comprising SEQ ID NO:75 (ARGLDFIVGYTGNDY);

[0048] (c) CDRH1 comprising SEQ ID NO:76 (FTFSSYAMS), CDRH2 comprising SEQ ID NO:71 (SAISNSGTYTYYA), and CDRH3 comprising SEQ ID NO:75 (ARGLDFIVGYTGNDY);

[0049] (d) CDRH1 comprising SEQ ID NO:77 (FTFSSYAML), CDRH2 comprising SEQ ID NO:71 (SAISNSGTYTYYA), and CDRH3 comprising SEQ ID NO:75 (ARGLDFIVGYTGNDY);

[0050] (e) CDRH1 comprising SEQ ID NO:78 (FTFSNYALS), CDRH2 comprising SEQ ID NO:71 (SAISNSGTYTYYA), and CDRH3 comprising SEQ ID NO:75 (ARGLDFIVGYTGNDY);

[0051] (f) CDRH1 comprising SEQ ID NO:79 (FTFSAYAMN), CDRH2 comprising SEQ ID NO:71 (SAISNSGTYTYYA), and CDRH3 comprising SEQ ID NO:75 (ARGLDFIVGYTGNDY);

[0052] (g) CDRH1 comprising SEQ ID NO:80 (FTFRSYAMS), CDRH2 comprising SEQ ID NO:71 (SAISNSGTYTYYA), and CDRH3 comprising SEQ ID NO:75 (ARGLDFIVGYTGNDY);

[0053] (h) CDRH1 comprising SEQ ID NO:81 (FTFGRYAMS), CDRH2 comprising SEQ ID NO:71 (SAISNSGTYTYYA), and CDRH3 comprising SEQ ID NO:75 (ARGLDFIVGYTGNDY);

[0054] (i) a CDRH1 comprising SEQ ID NO: 82 (FTFNSYAMS), a CDRH2 comprising SEQ ID NO: 71 (SAISNSGTYTYYA), and a CDRH3 comprising SEQ ID NO: 75 (ARGLDFIVGYTGNDY);

[0055] (j) CDRH1 comprising SEQ ID NO:83 (FTFSNYAMS), CDRH2 comprising SEQ ID NO:71 (SAISNSGTYTYYA), and CDRH3 comprising SEQ ID NO:74 (ARGLDFIVGATGNDY);

[0056] (k) CDRH1 comprising SEQ ID NO:84 (FTFSGYAMS), CDRH2 comprising SEQ ID NO:71 (SAISNSGTYTYYA), and CDRH3 comprising SEQ ID NO:85 (ARGLDFIVGRTGNDY);

[0057] (1) a CDRH1 comprising SEQ ID NO: 86 (FTFSSYAMN), a CDRH2 comprising SEQ ID NO: 71 (SAISNSGTYTYYA), and a CDRH3 comprising SEQ ID NO: 85 (ARGLDFIVGRTGNDY); or

[0058] (m) CDRH1 comprising SEQ ID NO:80 (FTFRSYAMS), CDRH3 comprising SEQ ID NO:71 (SAISNSGTYTYYA), and CDRH3 comprising SEQ ID NO:85 (ARGLDFIVGRTGNDY).

[0059] In some embodiments, the first antigen binding unit binds to PD-1 and comprises:

[0060] (a) a heavy chain variable region comprising an amino acid sequence at least 90% identical to SEQ ID NO: 87;

[0061] (b) a heavy chain variable region comprising an amino acid sequence at least 90% identical to SEQ ID NO: 88;

[0062] (c) a heavy chain variable region comprising an amino acid sequence at least 90% identical to SEQ ID NO: 89;

[0063] (d) a heavy chain variable region comprising an amino acid sequence at least 90% identical to SEQ ID NO: 90;

[0064] (e) a heavy chain variable region comprising an amino acid sequence at least 90% identical to SEQ ID NO: 91;

[0065] (f) a heavy chain variable region comprising an amino acid sequence at least 90% identical to SEQ ID NO: 92;

[0066] (g) a heavy chain variable region comprising an amino acid sequence at least 90% identical to SEQ ID NO: 93;

[0067] (h) a heavy chain variable region comprising an amino acid sequence at least 90% identical to SEQ ID NO: 94;

[0068] (i) a heavy chain variable region comprising an amino acid sequence at least 90% identical to SEQ ID NO: 95;

[0069] (j) a heavy chain variable region comprising an amino acid sequence at least 90% identical to SEQ ID NO: 96;

[0070] (k) a heavy chain variable region comprising an amino acid sequence at least 90% identical to SEQ ID NO: 97;

[0071] (1) a heavy chain variable region comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 98; or

[0072] (m) a heavy chain variable region comprising an amino acid sequence at least 90% identical to SEQ ID NO:99.

[0073] In some embodiments, the first antigen binding unit binds to PD-1 and comprises a light chain variable region comprising an amino acid sequence at least 90% identical to SEQ ID NO:59.

[0074] In some embodiments, the second antigen binding unit binds to PD-L2. In some embodiments, the second antigen binding unit binds to PD-L1. In some embodiments, the second antigen binding unit binds to PD-L1 and comprises:

[0075] a. A heavy chain variable region comprising (i) a CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN); (ii) a CDRH2 comprising SEQ ID NO: 2 (GGIIPX1X2GX3ATYA, wherein X1 is V or I; X2 is F, L or V; and X3 is T or A); and (iii) a CDRH3 comprising SEQ ID NO: 3 (ARLKX1ELKDAFDI, wherein X1 is G, F or N); and

[0076] b. A light chain variable region comprising: (i) CDRL1 comprising SEQ ID NO: 4 (RASQX1ISSYLN, wherein X1 is S, W or Q); (ii) CDRL2 comprising SEQ ID NO: 5 (AASSLQS); and (iii) CDRL3 comprising SEQ ID NO: 6 (X1QSYSTPLT, wherein X1 is Q or F).

[0077] In some such embodiments, the second antigen binding unit binds PD-L1 and comprises:

[0078] (a) CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), CDRH2 comprising SEQ ID NO: 7 (GGIIPILGAATYA), and CDRH3 comprising SEQ ID NO: 8 (ARLKGELKDAFDI);

[0079] (b) a CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), a CDRH2 comprising SEQ ID NO: 7 (GGIIPILGAATYA), a CDRH3 comprising SEQ ID NO: 8 (ARLKGELKDAFDI); a CDRL1 comprising SEQ ID NO: 9 (RASQSISSYLN), a CDRL2 comprising SEQ ID NO: 5 (AASSLQS), and a CDRL3 comprising SEQ ID NO: 10 (QQSYSTPLT);

[0080] (c) a CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), a CDRH2 comprising SEQ ID NO: 11 (GGIIPVFGTATYA), a CDRH3 comprising SEQ ID NO: 8 (ARLKGELKDAFDI); a CDRL1 comprising SEQ ID NO: 9 (RASQSISSYLN), a CDRL2 comprising SEQ ID NO: 5 (AASSLQS), and a CDRL3 comprising SEQ ID NO: 10 (QQSYSTPLT);

[0081] (d) CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), CDRH2 comprising SEQ ID NO: 11 (GGIIPVFGTATYA), and CDRH3 comprising SEQ ID NO: 8 (ARLKGELKDAFDI);

[0082] (e) a CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), a CDRH2 comprising SEQ ID NO: 11 (GGIIPVFGTATYA), a CDRH3 comprising SEQ ID NO: 8 (ARLKGELKDAFDI); a CDRL1 comprising SEQ ID NO: 12 (RASQWISSYLN), a CDRL2 comprising SEQ ID NO: 5 (AASSLQS), and a CDRL3 comprising SEQ ID NO: 10 (QQSYSTPLT);

[0083] (f) CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), CDRH2 comprising SEQ ID NO: 11 (GGIIPVFGTATYA), CDRH3 comprising SEQ ID NO: 8 (ARLKGELKDAFDI); CDRL1 comprising SEQ ID NO: 13 (RASQQISSYLN), CDRL2 comprising SEQ ID NO: 5 (AASSLQS), and CDRL3 comprising SEQ ID NO: 10 (QQSYSTPLT);

[0084] (g) CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), CDRH2 comprising SEQ ID NO: 11 (GGIIPVFGTATYA), CDRH3 comprising SEQ ID NO: 8 (ARLKGELKDAFDI); CDRL1 comprising SEQ ID NO: 9 (RASQSISSYLN), CDRL2 comprising SEQ ID NO: 5 (AASSLQS), and CDRL3 comprising SEQ ID NO: 10 (QQSYSTPLT);

[0085] (h) CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), CDRH2 comprising SEQ ID NO: 15 (GGIIPIFGIANYA), and CDRH3 comprising SEQ ID NO: 8 (ARLKGELKDAFDI);

[0086] (i) a CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), a CDRH2 comprising SEQ ID NO: 15 (GGIIPIFGIANYA), a CDRH3 comprising SEQ ID NO: 8 (ARLKGELKDAFDI); a CDRL1 comprising SEQ ID NO: 9 (RASQSISSYLN), a CDRL2 comprising SEQ ID NO: 5 (AASSLQS), and a CDRL3 comprising SEQ ID NO: 10 (QQSYSTPLT);

[0087] (j) CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), CDRH2 comprising SEQ ID NO: 16 (GGIIPNFGTATYA), and CDRH3 comprising SEQ ID NO: 17 (ARLKGELKGAGDI);

[0088] (k) a CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), a CDRH2 comprising SEQ ID NO: 16 (GGIIPNFGTATYA), a CDRH3 comprising SEQ ID NO: 17 (ARLKGELKGAGDI); a CDRL1 comprising SEQ ID NO: 9 (RASQSISSYLN), a CDRL2 comprising SEQ ID NO: 5 (AASSLQS), and a CDRL3 comprising SEQ ID NO: 10 (QQSYSTPLT);

[0089] (1) CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), CDRH2 comprising SEQ ID NO: 11 (GGIIPVFGTATYA), and CDRH3 comprising SEQ ID NO: 18 (ARLKFELKDAFDI);

[0090] (m) a CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), a CDRH2 comprising SEQ ID NO: 11 (GGIIPVFGTATYA), a CDRH3 comprising SEQ ID NO: 18 (ARLKFELKDAFDI), a CDRL1 comprising SEQ ID NO: 9 (RASQSISSYLN), a CDRL2 comprising SEQ ID NO: 5 (AASSLQS), and a CDRL3 comprising SEQ ID NO: 10 (QQSYSTPLT);

[0091] (n) CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), CDRH2 comprising SEQ ID NO: 11 (GGIIPVFGTATYA), and CDRH3 comprising SEQ ID NO: 19 (ARLKGELKDAFDE);

[0092] (o) a CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), a CDRH2 comprising SEQ ID NO: 11 (GGIIPVFGTATYA), a CDRH3 comprising SEQ ID NO: 19 (ARLKGELKDAFDE), a CDRL1 comprising SEQ ID NO: 9 (RASQSISSYLN), a CDRL2 comprising SEQ ID NO: 5 (AASSLQS), and a CDRL3 comprising SEQ ID NO: 10 (QQSYSTPLT);

[0093] (p) CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), CDRH2 comprising SEQ ID NO: 11 (GGIIPVFGTATYA), and CDRH3 comprising SEQ ID NO: 20 (ARLKNELKDAFDI);

[0094] (q) a CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), a CDRH2 comprising SEQ ID NO: 11 (GGIIPVFGTATYA), a CDRH3 comprising SEQ ID NO: 20 (ARLKNELKDAFDI), a CDRL1 comprising SEQ ID NO: 9 (RASQSISSYLN), a CDRL2 comprising SEQ ID NO: 5 (AASSLQS), and a CDRL3 comprising SEQ ID NO: 10 (QQSYSTPLT);

[0095] (r) a CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), a CDRH2 comprising SEQ ID NO: 21 (GGVIPFLGTANYA), and a CDRH3 comprising SEQ ID NO: 22 (ARLKGILKDALDI);

[0096] (s) a CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), a CDRH2 comprising SEQ ID NO: 21 (GGVIPFLGTANYA), a CDRH3 comprising SEQ ID NO: 22 (ARLKGILKDALDI), a CDRL1 comprising SEQ ID NO: 9 (RASQSISSYLN), a CDRL2 comprising SEQ ID NO: 5 (AASSLQS), and a CDRL3 comprising SEQ ID NO: 10 (QQSYSTPLT);

[0097] (t) CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), CDRH2 comprising SEQ ID NO: 29 (GRIIPIFGTADYA), and CDRH3 comprising SEQ ID NO: 8 (ARLKGELKDAFDI);

[0098] (u) a CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), a CDRH2 comprising SEQ ID NO: 29 (GRIIPIFGTADYA), a CDRH3 comprising SEQ ID NO: 8 (ARLKGELKDAFDI), a CDRL1 comprising SEQ ID NO: 9 (RASQSISSYLN), a CDRL2 comprising SEQ ID NO: 5 (AASSLQS), and a CDRL3 comprising SEQ ID NO: 10 (QQSYSTPLT);

[0099] (v) a CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), a CDRH2 comprising SEQ ID NO: 31 (GGIIPILGTATYA), and a CDRH3 comprising SEQ ID NO: 32 (ARRKGELKDAFDI);

[0100] (w) a CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), a CDRH2 comprising SEQ ID NO: 31 (GGIIPILGTATYA), a CDRH3 comprising SEQ ID NO: 32 (ARRKGELKDAFDI), a CDRL1 comprising SEQ ID NO: 9 (RASQSISSYLN), a CDRL2 comprising SEQ ID NO: 5 (AASSLQS), and a CDRL3 comprising SEQ ID NO: 10 (QQSYSTPLT);

[0101] (x) a CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), a CDRH2 comprising SEQ ID NO: 33 (GGIIPIVATANYA), and a CDRH3 comprising SEQ ID NO: 32 (ARRKGELKDAFDI);

[0102] (y) a CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), a CDRH2 comprising SEQ ID NO: 33 (GGIIPIVATANYA), a CDRH3 comprising SEQ ID NO: 32 (ARRKGELKDAFDI), a CDRL1 comprising SEQ ID NO: 9 (RASQSISSYLN), a CDRL2 comprising SEQ ID NO: 5 (AASSLQS), and a CDRL3 comprising SEQ ID NO: 10 (QQSYSTPLT);

[0103] (z) CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), CDRH2 comprising SEQ ID NO: 34 (GGIIPIFGKATYA), and CDRH3 comprising SEQ ID NO: 32 (ARRKGELKDAFDI);

[0104] (aa) CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), CDRH2 comprising SEQ ID NO: 34 (GGIIPIFGKATYA), CDRH3 comprising SEQ ID NO: 32 (ARRKGELKDAFDI), CDRL1 comprising SEQ ID NO: 9 (RASQSISSYLN), CDRL2 comprising SEQ ID NO: 5 (AASSLQS), and CDRL3 comprising SEQ ID NO: 10 (QQSYSTPLT);

[0105] (bb) CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), CDRH2 comprising SEQ ID NO: 11 (GGIIPVFGTATYA), CDRH3 comprising SEQ ID NO: 8 (ARLKGELKDAFDI); CDRL1 comprising SEQ ID NO: 9 (RASQSISSYLN), CDRL2 comprising SEQ ID NO: 5 (AASSLQS), and CDRL3 comprising SEQ ID NO: 38 (FQSYSTPLT);

[0106] (cc) a CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), a CDRH2 comprising SEQ ID NO: 11 (GGIIPVFGTATYA), a CDRH3 comprising SEQ ID NO: 8 (ARLKGELKDAFDI); a CDRL1 comprising SEQ ID NO: 9 (RASQSISSYLN), a CDRL2 comprising SEQ ID NO: 5 (AASSLQS), and a CDRL3 comprising SEQ ID NO: 39 (QQSYSTILT);

[0107] (dd) CDRH1 comprising SEQ ID NO: 14 (GTFSSYAFS), CDRH2 comprising SEQ ID NO: 11 (GGIIPVFGTATYA), and CDRH3 comprising SEQ ID NO: 8 (ARLKGELKDAFDI); CDRL1 comprising SEQ ID NO: 9 (RASQSISSYLN), CDRL2 comprising SEQ ID NO: 5 (AASSLQS), and CDRL3 comprising SEQ ID NO: 10 (QQSYSTPLT);

[0108] (ee) CDRH1 comprising SEQ ID NO:23 (GTFSSYAIS), CDRH2 comprising SEQ ID NO:24 (GGIIPIVGIANYA), and CDRH3 comprising SEQ ID NO:8 (ARLKGELKDAFDI);

[0109] (ff) CDRH1 comprising SEQ ID NO:23 (GTFSSYAIS), CDRH2 comprising SEQ ID NO:24 (GGIIPIVGIANYA), and CDRH3 comprising SEQ ID NO:8 (ARLKGELKDAFDI); CDRL1 comprising SEQ ID NO:9 (RASQSISSYLN), CDRL2 comprising SEQ ID NO:5 (AASSLQS), and CDRL3 comprising SEQ ID NO:10 (QQSYSTPLT);

[0110] (gg) CDRH1 comprising SEQ ID NO:23 (GTFSSYAIS), CDRH2 comprising SEQ ID NO:11 (GGIIPVFGTATYA), and CDRH3 comprising SEQ ID NO:25 (ARLKGEFKDAFDI);

[0111] (hh) CDRH1 comprising SEQ ID NO:23 (GTFSSYAIS), CDRH2 comprising SEQ ID NO:11 (GGIIPVFGTATYA), and CDRH3 comprising SEQ ID NO:25 (ARLKGEFKDAFDI); CDRL1 comprising SEQ ID NO:9 (RASQSISSYLN), CDRL2 comprising SEQ ID NO:5 (AASSLQS), and CDRL3 comprising SEQ ID NO:10 (QQSYSTPLT);

[0112] (ii) a CDRH1 comprising SEQ ID NO: 23 (GTFSSYAIS), a CDRH2 comprising SEQ ID NO: 26 (GRIIPLFGTAHYA), and a CDRH3 comprising SEQ ID NO: 8 (ARLKGELKDAFDI);

[0113] (jj) CDRH1 comprising SEQ ID NO:23 (GTFSSYAIS), CDRH2 comprising SEQ ID NO:26 (GRIIPLFGTAHYA), and CDRH3 comprising SEQ ID NO:8 (ARLKGELKDAFDI); CDRL1 comprising SEQ ID NO:9 (RASQSISSYLN), CDRL2 comprising SEQ ID NO:5 (AASSLQS), and CDRL3 comprising SEQ ID NO:10 (QQSYSTPLT);

[0114] (kk) CDRH1 comprising SEQ ID NO:23 (GTFSSYAIS), CDRH2 comprising SEQ ID NO:27 (GRINPILGTANYA), and CDRH3 comprising SEQ ID NO:28 (ARLKGELKDAFSI);

[0115] (11) CDRH1 comprising SEQ ID NO:23 (GTFSSYAIS), CDRH2 comprising SEQ ID NO:27 (GRINPILGTANYA), and CDRH3 comprising SEQ ID NO:28 (ARLKGELKDAFSI); CDRL1 comprising SEQ ID NO:9 (RASQSISSYLN), CDRL2 comprising SEQ ID NO:5 (AASSLQS), and CDRL3 comprising SEQ ID NO:10 (QQSYSTPLT);

[0116] (mm) CDRH1 comprising SEQ ID NO:23 (GTFSSYAIS), CDRH2 comprising SEQ ID NO:11 (GGIIPVFGTATYA), and CDRH3 comprising SEQ ID NO:30 (ARLKGELKCAFDI);

[0117] (nn) CDRH1 comprising SEQ ID NO:23 (GTFSSYAIS), CDRH2 comprising SEQ ID NO:11 (GGIIPVFGTATYA), and comprising SEQ ID NO:30 (ARLKGELKCAFDI); CDRL1 comprising SEQ ID NO:9 (RASQSISSYLN), CDRL2 comprising SEQ ID NO:5 (AASSLQS), and CDRL3 comprising SEQ ID NO:10 (QQSYSTPLT);

[0118] (oo) a CDRH1 comprising SEQ ID NO: 36 (GPFRSHAVS), a CDRH2 comprising SEQ ID NO: 11 (GGIIPVFGTATYA), and a CDRH3 comprising SEQ ID NO: 37 (ARLKSELKDAFDI); or

[0119] (pp) CDRH1 comprising SEQ ID NO:36 (GPFRSHAVS), CDRH2 comprising SEQ ID NO:11 (GGIIPVFGTATYA), and CDRH3 comprising SEQ ID NO:37 (ARLKSELKDAFDI); CDRL1 comprising SEQ ID NO:9 (RASQSISSYLN), CDRL2 comprising SEQ ID NO:5 (AASSLQS), and CDRL3 comprising SEQ ID NO:10 (QQSYSTPLT).

[0120] In some such embodiments, the second antigen binding unit binds to PD-L1 and comprises a heavy chain variable region comprising an amino acid sequence at least 90% identical to any one of SEQ ID NO: 35, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57 or 58, and a light chain variable region comprising an amino acid sequence at least 90% identical to any one of SEQ ID NO: 59, 60, 61, 62 or 63.

[0121] In some embodiments, the second antigen binding unit binds to PD-L1 and comprises a heavy chain variable region comprising the following amino acid sequence:

[0122] (a) an amino acid sequence at least 90% identical to SEQ ID NO: 35;

[0123] (b) an amino acid sequence that is at least 90% identical to SEQ ID NO:40;

[0124] (c) an amino acid sequence at least 90% identical to SEQ ID NO: 41;

[0125] (d) an amino acid sequence at least 90% identical to SEQ ID NO:42;

[0126] (e) an amino acid sequence at least 90% identical to SEQ ID NO: 43;

[0127] (f) an amino acid sequence at least 90% identical to SEQ ID NO:44;

[0128] (g) an amino acid sequence at least 90% identical to SEQ ID NO:45;

[0129] (h) an amino acid sequence at least 90% identical to SEQ ID NO:46;

[0130] (i) an amino acid sequence at least 90% identical to SEQ ID NO: 47;

[0131] (j) an amino acid sequence at least 90% identical to SEQ ID NO: 48;

[0132] (k) an amino acid sequence at least 90% identical to SEQ ID NO:49;

[0133] (1) an amino acid sequence that is at least 90% identical to SEQ ID NO: 50;

[0134] (m) an amino acid sequence at least 90% identical to SEQ ID NO: 51;

[0135] (n) an amino acid sequence at least 90% identical to SEQ ID NO: 52;

[0136] (o) an amino acid sequence at least 90% identical to SEQ ID NO: 53;

[0137] (p) an amino acid sequence at least 90% identical to SEQ ID NO:54;

[0138] (q) an amino acid sequence at least 90% identical to SEQ ID NO: 55;

[0139] (r) an amino acid sequence at least 90% identical to SEQ ID NO:56;

[0140] (s) an amino acid sequence that is at least 90% identical to SEQ ID NO: 57; or

[0141] (t) an amino acid sequence at least 90% identical to SEQ ID NO:58.

[0142] In some embodiments, the second antigen binding unit binds to PD-L1 and comprises a light chain variable region comprising the following amino acid sequence:

[0143] (a) an amino acid sequence that is at least 90% identical to SEQ ID NO: 59;

[0144] (b) an amino acid sequence that is at least 90% identical to SEQ ID NO: 60;

[0145] (c) an amino acid sequence at least 90% identical to SEQ ID NO: 61;

[0146] (d) an amino acid sequence that is at least 90% identical to SEQ ID NO: 62; or

[0147] (e) an amino acid sequence that is at least 90% identical to SEQ ID NO: 63.

[0148] In some aspects and embodiments, provided herein are multispecific antigen-binding constructs comprising four antigen-binding units, wherein two antigen-binding units bind to PD-1 and two antigen-binding units bind to PD-L1, and wherein the construct comprises a heavy chain amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 100 or 102, and a light chain amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 101 or 103.

[0149] In some aspects and embodiments, provided herein are multispecific antigen-binding constructs comprising four antigen-binding units, wherein two antigen-binding units bind to PD-1 and two antigen-binding units bind to PD-L1, and wherein the construct comprises a heavy chain amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 100 and a light chain amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 101.

[0150] In some aspects and embodiments, provided herein are multispecific antigen-binding constructs comprising four antigen-binding units, wherein two antigen-binding units bind to PD-1 and two antigen-binding units bind to PD-L1, and wherein the construct comprises a heavy chain amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 102 and a light chain amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 103.

[0151] In some embodiments, the construct does not include an Fc domain. In some embodiments, the first arm or the second arm or both include a heavy chain comprising one or more immunoglobulin Fc modifications. In some embodiments, the immunoglobulin Fc domain of the heavy chain includes one or more amino acid mutations that promote heterodimerization of the first arm and the second arm. In some embodiments, the mutation is present in the CH3 domain of the heavy chain. In some embodiments, the multispecific antigen-binding construct is produced in a quadroma cell. In some embodiments, the construct includes one or more immunoglobulin constant region modifications. In some embodiments, the immunoglobulin constant region includes one or more amino acid mutations that promote heterodimerization of the antibody. In some embodiments, one or more mutations are present in the light chain constant region of one arm, and one or more mutations are present in the heavy chain constant region of the other arm. In some embodiments, the bispecific antibody is in the form of a group consisting of: bispecific IgG, bispecific antibody fragment, bispecific fusion protein, appended IgG, and bispecific antibody conjugate. In some embodiments, the Fc region has reduced effector function. In some embodiments, the Fc region enhances the half-life of the construct.

[0152] In some embodiments, the construct comprises a heavy chain amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 100 or 102. In some embodiments, the construct comprises a light chain amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 101 or 103. In some embodiments, the construct comprises a heavy chain amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 100, and wherein the construct comprises a light chain amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 101. In some embodiments, the construct comprises a heavy chain amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 102, and wherein the construct comprises a light chain amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 103.

[0153] In some embodiments, any of the multispecific antigen-binding constructs disclosed herein is aglycosylated. In some embodiments, the multispecific antigen-binding construct binds to human PD-1. In some embodiments, the multispecific antigen-binding construct binds to murine PD-1. In some embodiments, the multispecific antigen-binding construct binds to cynomolgus monkey PD-1. In some embodiments, the multispecific antigen-binding construct binds to human, murine, and cynomolgus monkey PD-1 with similar affinities.

[0154] In some embodiments, any of the multispecific antigen-binding constructs disclosed herein can reduce PD-1 levels on cells. In some embodiments, the multispecific antigen-binding construct can induce PD-1 degradation. In some embodiments, the multispecific antigen-binding construct can reduce PD-1 expression. In some embodiments, the multispecific antigen-binding construct can reduce PD-1 cell surface expression. In some embodiments, the multispecific antigen-binding construct can reduce PD-1 cell surface expression by inducing PD-1 to shed from the cell surface. In some embodiments, the multispecific antigen-binding construct can bind to both PD-1 and PD-L1 and reduce PD-1 levels on cells. In some embodiments, the multispecific antigen-binding construct can bind to both PD-1 and PD-L1 and induce PD-1 degradation. In some embodiments, the multispecific antigen-binding construct can bind to both PD-1 and PD-L1 and reduce PD-1 expression. In some embodiments, the multispecific antigen-binding construct can induce PD-1 to shed from immune cells. In some embodiments, the multispecific antigen-binding construct can bind to both PD-1 and PD-L1 and induce PD-1 to fall off from immune cells. In some embodiments, the multispecific antigen-binding construct can isolate PD-L1 so that PD-L1 cannot bind to CD80. In some embodiments, the multispecific antigen-binding construct can isolate PD-L1 so that PD-L1 cannot bind to CD80, and wherein CD80 can freely bind to CD28. In some embodiments, the cell is an immune cell, such as a T cell. In some embodiments, the immune cell (e.g., T cell) is a tumor infiltrating lymphocyte (TIL). In some embodiments, the engagement of the multispecific antigen-binding molecules described herein with PD-1 expressed by immune cells in the tumor microenvironment results in downregulation of PD-1 by immune cells. In some embodiments, the immune cell is a T cell. In some embodiments, the immune cell (e.g., T cell) is a tumor infiltrating lymphocyte (TIL).

[0155] In some embodiments, any of the multispecific antigen-binding constructs disclosed herein is capable of inducing interferon-γ levels that are at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% higher (e.g., as measured in a Staphylococcus aureus enterotoxin A ("SEA") assay) compared to a reference antigen-binding construct (e.g., pembrolizumab or atezolizumab) or compared to a reference combination of antigen-binding constructs (e.g., a composition comprising a PD-1 arm and a PD-L1 arm of a multispecific antigen-binding construct, wherein the PD-1 arm and the PD-L1 arm of the composition are not bound to each other). In some embodiments, the multispecific antigen-binding construct is capable of inducing interleukin-2 levels that are at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% higher (e.g., as measured in a SEA assay) compared to a reference antigen-binding construct (e.g., pembrolizumab or atezolizumab) or compared to a reference combination of antigen-binding constructs (e.g., a composition comprising a PD-1 arm and a PD-L1 arm of a multispecific antigen-binding construct, wherein the PD-1 arm and the PD-L1 arm of the composition are not bound to each other).

[0156] In some embodiments, the multispecific antigen-binding construct induces at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 200%, 300%, 400%, or 500% greater killing of tumor cells (e.g., leukemia cells, lymphoma cells, melanoma cells, or breast cancer cells) compared to a reference antigen-binding construct (e.g., pembrolizumab or atezolizumab) or compared to a reference combination of antigen-binding constructs (e.g., a composition comprising a PD-1 arm and a PD-L1 arm of the multispecific antigen-binding construct, wherein the PD-1 arm and the PD-L1 arm of the composition are not bound to each other).

[0157] In some embodiments, the multispecific antigen-binding construct is capable of prolonging survival of an individual having cancer (e.g., leukemia, lymphoma, melanoma, and / or breast cancer) by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 100%, 200%, 300%, 400%, or 500% longer than an individual administered a reference antigen-binding construct (e.g., pembrolizumab or atezolizumab) or a reference combination of antigen-binding constructs (e.g., a composition comprising a PD-1 arm and a PD-L1 arm of the multispecific antigen-binding construct, wherein the PD-1 arm and the PD-L1 arm of the composition do not bind to each other). In some embodiments, the multispecific antigen-binding construct is capable of inducing at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 100%, 200%, 300%, 400%, or 500% higher shedding of PD-1 from immune cells compared to untreated immune cells or compared to immune cells treated with a reference antigen-binding construct (e.g., pembrolizumab or atezolizumab) or a reference combination of antigen-binding constructs (e.g., a composition comprising a PD-1 arm and a PD-L1 arm of the multispecific antigen-binding construct, wherein the PD-1 arm and the PD-L1 arm of the composition are not bound to each other). In some embodiments, the multispecific antigen-binding construct reduces PD-1 levels by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 100%, 200%, 300%, 400%, or 500% compared to untreated immune cells or compared to immune cells treated with a reference antigen-binding construct (e.g., pembrolizumab or atezolizumab) or a reference combination of antigen-binding constructs (e.g., a composition comprising a PD-1 arm and a PD-L1 arm of the multispecific antigen-binding construct, wherein the PD-1 arm and the PD-L1 arm of the composition are not bound to each other).

[0158] In some embodiments, the engagement of the multispecific antigen-binding molecules described herein with PD-1 expressed by cells results in downregulation and / or loss of PD-1 cell surface expression of the cells. This downregulation or loss of cell surface expression may be due, for example, to shedding of extracellular PD-1 from the surface of immune cells. In some embodiments, the cells are immune cells, such as T cells. In some embodiments, the immune cells (e.g., T cells) are tumor infiltrating lymphocytes (TILs). In some embodiments, the engagement of the multispecific antigen-binding molecules described herein with PD-1 expressed by immune cells in the tumor microenvironment results in downregulation of the immune cell PD-1. In some embodiments, the immune cells are T cells. In some embodiments, the immune cells (e.g., T cells) are tumor infiltrating lymphocytes (TILs).

[0159] In some aspects, the present disclosure provides a method for treating a proliferative disorder in an individual in need thereof, comprising administering to the individual a therapeutically effective amount of any one of the multispecific antigen-binding constructs disclosed herein, thereby treating the proliferative disorder in the individual. In some embodiments, the proliferative disorder is cancer. In some embodiments, the cancer is selected from the group consisting of: blood cancer, neurological cancer, melanoma, breast cancer, lung cancer, head and neck cancer, gastrointestinal cancer, liver cancer, pancreatic cancer, genitourinary cancer, bone cancer, and vascular cancer. In some embodiments, the present disclosure provides a method for enhancing the immune response in an individual in need thereof, comprising administering to the individual a therapeutically effective amount of any one of the multispecific antigen-binding constructs disclosed herein, thereby enhancing the immune response in the individual. In some embodiments, the enhanced immune response includes any one or more of enhanced T cell function, enhanced NK cell function, or enhanced macrophage function. In some embodiments, the enhancement of T cell function is greater after administration of the multispecific antigen-binding construct compared to an agent that binds to PD-1 or a PD-1 ligand, or a mixture comprising an agent that binds to PD-1 and an agent that binds to a PD-1 ligand. In some embodiments, T cell function is any one or more of increased IFNγ production of T cells, enhanced T cell survival, increased T cell proliferation, or rescue of a self-exhausted T cell phenotype. In some embodiments, the enhanced T cell function is greater after administration of a multispecific antigen-binding construct compared to an agent that binds to PD-1 or a PD-1 ligand, or a mixture of an agent that binds to PD-1 and an agent that binds to a PD-1 ligand. In some embodiments, the multispecific antigen-binding construct is administered subcutaneously, intravenously, intradermally, intraperitoneally, orally, intramuscularly, or intracranially. In some embodiments, the multispecific antigen-binding construct binds to PD-1 and PD-L1 expressed on the surface of the same cell of an individual. In some embodiments, the multispecific antigen-binding construct binds to PD-1 expressed on the surface of a first cell of an individual, and wherein the multispecific antigen-binding construct binds to PD-L1 expressed on the surface of a second cell of an individual.

[0160] In some embodiments, the present disclosure provides an anti-PD-1 antibody or antigen-binding fragment thereof, wherein the anti-PD-1 antibody or antigen-binding fragment comprises: (a) a heavy chain variable region comprising (i) a CDRH1 comprising SEQ ID NO: 70 (FTFX1X2YAX3X4, wherein X1 = S, R, G, or N; X2 = D, S, N, A, R, or G; X3 = M or L; X4 = S, L, or N); (ii) a CDRH2 comprising SEQ ID NO: 71 (SAISNSGTYTYYA); and (iii) a CDRH3 comprising SEQ ID NO: 72 (ARGLDFIVGX5TGNDY, wherein X5 = A, Y, or R); and (b) a light chain variable region comprising: (i) a CDRL1 comprising SEQ ID NO: 9 (RASQSISSYLN); (ii) a CDRL2 comprising SEQ ID NO: 5 (AASSLQS); and (iii) a CDRH3 comprising SEQ ID NO: In some embodiments, the anti-PD-1 antibody or antigen-binding fragment comprises: (a) a heavy chain variable region comprising: (i) a CDRH1 comprising the amino acid sequence of any one of SEQ ID NOs: 73, 76, 77, 78, 79, 80, 81, 82, 83, 84, or 86; (ii) a CDRH2 comprising the amino acid sequence of SEQ ID NO: 71; and (iii) a CDRH3 comprising the amino acid sequence of any one of SEQ ID NOs: 74, 75, or 85; and (b) a light chain variable region comprising: (i) a CDRL1 comprising SEQ ID NO: 9 (RASQSISSYLN); (ii) a CDRL2 comprising SEQ ID NO: 5 (AASSLQS); and (iii) a CDRL3 comprising SEQ ID NO: 10 (QQSYSTPLT). In some embodiments, the heavy chain variable region comprises an amino acid sequence at least 85% identical to the amino acid sequence of any one of SEQ ID NO: 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99. In some embodiments, the light chain variable region comprises an amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO: 59.

[0161] In some embodiments, the present disclosure provides an anti-PD-L1 antibody or antigen-binding fragment thereof, wherein the anti-PD-L1 antibody or antigen-binding fragment comprises: a. a heavy chain variable region comprising (i) a CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN); (ii) a CDRH2 comprising SEQ ID NO: 2 (GGIIPX1X2GX3ATYA, wherein X1 is V or I; X2 is F, L, or V; and X3 is T or A); and (iii) a CDRH3 comprising SEQ ID NO: 3 (ARLKX1ELKDAFDI, wherein X1 is G, F, or N); and b. a light chain variable region comprising: (i) a CDRL1 comprising SEQ ID NO: 4 (RASQX1ISSYLN, wherein X1 is S, W, or Q); (ii) a CDRL2 comprising SEQ ID NO: 5 (AASSLQS); and (iii) a CDRH3 comprising SEQ ID NO: CDRL3 of NO:6(X1QSYSTPLT, where X1 is Q or F). In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment comprises: (a) a heavy chain variable region comprising (i) a CDRH1 comprising the amino acid sequence of any one of SEQ ID NO: 1, 14, 23, 36, or 122; (ii) a CDRH2 comprising the amino acid sequence of any one of SEQ ID NO: 11, 15, 16, 21, 24, 26, 27, 29, 31, 33, or 34; and (iii) a CDRH3 comprising the amino acid sequence of any one of SEQ ID NO: 8, 17, 18, 19, 20, 22, 25, 28, 30, 32, or 37; and (b) a light chain variable region comprising: (i) a CDRL1 comprising the amino acid sequence of any one of SEQ ID NO: 9, 12, or 13; (ii) a CDRL2 comprising the amino acid sequence of SEQ ID NO: 5; and (iii) a CDRL3 comprising the amino acid sequence of any one of SEQ ID NO: 10, 38, or 39. In some embodiments, the heavy chain variable region comprises an amino acid sequence at least 85% identical to the amino acid sequence of any one of SEQ ID NOs: 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, or 35. In some embodiments, the light chain variable region comprises an amino acid sequence at least 85% identical to the amino acid sequence of any one of SEQ ID NOs: 59, 60, 61, 62, or 63.

[0162] In some embodiments, the present disclosure provides a method of treating a proliferative disorder in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of any of the antibodies or antigen-binding constructs disclosed herein, thereby treating the proliferative disorder in the subject. In some embodiments, the proliferative disorder is cancer. In some embodiments, the cancer is selected from the group consisting of: blood cancer, neurological cancer, melanoma, breast cancer, lung cancer, head and neck cancer, gastrointestinal cancer, liver cancer, pancreatic cancer, genitourinary cancer, bone cancer, and vascular cancer.

[0163] In some embodiments, the present disclosure provides a method of enhancing an immune response in an individual in need thereof, comprising administering to the individual a therapeutically effective amount of any of the antibodies or antigen-binding constructs disclosed herein, thereby enhancing the immune response in the individual.

[0164] In some embodiments, any of the methods described herein may include detecting the presence or absence of PD-1 expression in one or more cells (or cell populations, such as TILs) before and / or after contact with a multispecific antigen-binding molecule described herein. For example, any of the methods described herein may include detecting the presence or absence of PD-1 expression in one or more cells (or cell populations, such as TILs) before and / or after administering a multispecific antigen-binding molecule described herein to an individual (e.g., a cancer patient). Such methods can be used, for example, to determine a therapeutically effective amount of a molecule for treating a given patient or patient population. Methods for detecting the presence, reduction, and / or absence of PD-1 expression are known to those skilled in the art, for example, using flow cytometry, Western blotting, ELISA, and the like.

[0165] In another aspect, the disclosure features a method comprising measuring the level of PD-1 expression by one or more cells (or cell populations, such as TILs) before and / or after contacting with a multispecific antigen-binding molecule described herein. In some embodiments, the method comprises measuring the level of PD-1 expression by one or more cells (or cell populations, such as TILs) before and / or after administering a multispecific antigen-binding molecule described herein to an individual (e.g., a cancer patient).

[0166] In another aspect, the disclosure features a method comprising measuring the level of PD-1 expression of one or more cells (or cell populations, such as TILs) before and / or after contact with a multispecific antigen-binding molecule described herein. For example, any of the methods described herein may comprise measuring the level of PD-1 expression of one or more cells (or cell populations, such as TILs) before and / or after administering a multispecific antigen-binding molecule described herein to an individual (e.g., a cancer patient). Such methods may be particularly useful for detecting or measuring the biological effects of the molecules described herein on an individual. In some embodiments, a decrease in the level of PD-1 expression of immune cells (e.g., TILs isolated from a patient) after treatment with a multispecific antigen-binding molecule described herein indicates that the molecule has a biological effect in the individual. In some embodiments, a decrease in the level of PD-1 expression of immune cells (e.g., TILs isolated from a patient) after treatment with a multispecific antigen-binding molecule described herein indicates that the patient should receive one or more doses of the molecule, or otherwise continue a therapy comprising the molecule.

[0167] In another aspect, the disclosure features a method for determining whether a biological effect occurs in a patient or patient population treated with a multispecific antigen binding molecule as described herein. The method includes detecting the presence or amount of PD-1 expression of one or more test immune cells (e.g., effector immune cells, such as effector immune cells in a tumor microenvironment) obtained from one or more patients to whom a multispecific antigen binding molecule as described herein has been applied, wherein PD-1 is expressed (e.g., cell surface expression) by one or more immune cells relative to a control expression level (e.g., PD-1 expression levels of immune cells of the same histological type as the test immune cells before the molecule is applied), indicating that a biological effect has been produced in the patient or patient population. In some embodiments, the method includes administering a multispecific antigen binding molecule before detection. In some embodiments, the method includes administering a multispecific antigen binding molecule to a patient or patient population having determined a biological effect. In some embodiments, the control PD-1 expression level is approximately the middle or average expression level of the PD-1 of immune cells of the same histological type in a population not yet diagnosed with cancer. In some embodiments, the control PD-1 expression level is approximately the median or average expression level of PD-1 in immune cells of the same histological type in a population of individuals who have not been administered a multispecific antigen-binding molecule and / or an agent that binds to and / or inhibits PD-1 as described herein.

[0168] In another aspect, the disclosure features a method for reducing the PD-1 expression of one or more immune cells in an individual (e.g., a cancer patient), the method comprising administering a multispecific antigen binding molecule as described herein to the individual, thereby reducing the PD-1 expression of one or more immune cells in the individual. In some embodiments, the method includes determining whether a reduction in the PD-1 expression of one or more immune cells in the patient occurs. In some embodiments, the method includes obtaining a biological sample (e.g., a tumor biopsy) comprising one or more immune cells from the individual (e.g., after administering the molecule to the individual). In some embodiments, the method includes measuring the level of PD-1 expression of one or more immune cells in the biological sample.

[0169] In yet another aspect, the disclosure features a method of inhibiting binding between PD-L1 and CD80 in a subject (e.g., a cancer patient), the method comprising administering to the subject a multispecific antigen-binding molecule described herein, thereby inhibiting binding between PD-L1 and CD80 in the subject. BRIEF DESCRIPTION OF THE DRAWINGS

[0171] This patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0172] Figure 1 Shown is the induction of interferon-gamma (IFNγ) in a mixed lymphocyte reaction (MLR) assay treated with various antibody mixtures or bispecific antibodies, including pembrolizumab x atezolizumab bispecific, nivolumab x atezolizumab bispecific, a mixture of KEYTRUDA and atezolizumab, and a mixture of nivolumab and atezolizumab, compared to KEYTRUDA alone. Results show the concentration of IFNγ in pg / mL at the final concentration of the test antibody as shown.

[0173] Figure 2 Shown is the induction of interferon-gamma (IFNγ) in a mixed lymphocyte reaction (MLR) assay treated with various monoclonal and bispecific antibodies, including pembrolizumab x nivolumab bispecific, atezolizumab x atezolizumab tetravalent fusion, nivolumab, and atezolizumab, compared to KEYTRUDA alone. Results show the concentration of IFNγ in pg / mL at the indicated final concentration of the test antibody.

[0174] Figure 3Schematic diagram and amino acid sequence of the pembrolizumab x atezolizumab bispecific are shown. Separate sequences are provided for the pembrolizumab_aglyco-IgG1-(G4S)4 heavy chain (H chain; SEQ ID NO: 104) and light chain (L chain; SEQ ID NO: 105), and for the atezolizumab_FabH-(G4S)4 heavy chain (H chain; SEQ ID NO: 106) and light chain (L chain; SEQ ID NO: 107).

[0175] Figure 4 Schematic diagram and amino acid sequences of the nivolumab x atezolizumab bispecific are shown. Separate sequences are provided for the nivolumab aglyco-IgG1-(G4S)4 heavy chain (H chain; SEQ ID NO: 108) and light chain (L chain; SEQ ID NO: 109), and for the atezolizumab FabH-(G4S)4 heavy chain (H chain; SEQ ID NO: 106) and light chain (L chain; SEQ ID NO: 107).

[0176] Figure 5 Schematic diagram and amino acid sequence of the 949aglyco-IgG1 x Atezolizumab bispecific are shown. Separate sequences are provided for the 949_aglyco-IgG1-(G4S)4 heavy chain (H chain; SEQ ID NO: 110) and light chain (L chain; SEQ ID NO: 111), and for the Atezolizumab_FabH-(G4S)4 heavy chain (H chain; SEQ ID NO: 106) and light chain (L chain; SEQ ID NO: 107).

[0177] Figure 6 Schematic diagram and amino acid sequence of the atezolizumab x nivolumab bispecific are shown. Sequences are provided for the atezolizumab-aglyco-IgG1-(G4S)4 heavy chain (H chain; SEQ ID NO: 112) and light chain (L chain; SEQ ID NO: 107), and the nivolumab-HC Fab-(G4S)4 heavy chain (H chain; SEQ ID NO: 113) and light chain (L chain; SEQ ID NO: 109).

[0178] Figure 7IFN-γ release in a mixed lymphocyte reaction (MLR) assay is shown as a function of the antibodies tested at various concentrations in pg / mL (right panel). These results indicate that the bispecific antibodies PD-1xPD-L1 (pembrolizumab x atezolizumab) or (nivolumab x atezolizumab) in a multispecific format induce greater IFN-γ responses at femtomolar concentrations compared to a mixture of pembrolizumab and atezolizumab or a mixture of nivolumab and atezolizumab. Size exclusion chromatography of the bispecific format for mAb precursors is shown (left panel).

[0179] Figure 8 An example of a workflow for identifying multispecific (e.g., bispecific) antibodies that demonstrate synergy is shown. The process involves unbiased screening of checkpoint blocker combinations in a mixed lymphocyte reaction (MLR) assay that measures IFN-γ release at various concentrations in pg / mL. In the second step of the illustrated workflow, a common light chain bispecific is generated to further test its efficacy; various bispecific formats are described here. The identified bispecific formats outperformed known PD-1 blockers in a T cell activation assay.

[0180] Figures 9A to 9B Bispecific 3 was shown to induce greater killing of K562-CD32-PDL1 target cells by CD3 / CD28-expanded T-cells compared to both an isotype control antibody and KEYTRUDA in an antigen-nonspecific T-cell assay ( Figure 9A ) and increased IFNγ production ( Figure 9B This increased killing by bispecific 3 was seen even at a low concentration of 0.01 nM.

[0181] Figures 10A to 10B The effect of bispecific 3 on tumor cell killing is shown. Figure 10A Bispecific 3 was shown to increase specific killing of K562-A2-CMV-PDL1 tumor antigen target cells by CMV-specific T-cells at low concentrations of 0.001 to 0.01 nM compared to both KEYTRUDA and the combination of mAb 1 and mAb 28, suggesting that bispecific 3 can be used to mediate antigen-specific killing of target cells at lower doses. Figure 10B Bispecific 3 was shown to be more effective than KEYTRUDA or the combination of mAb 1 and mAb 28 in specifically killing Raji-A2-CMV-PDL1 cell tumor antigen target cells by CMV-specific T-cells at a low concentration of 0.001 nM, again suggesting that bispecific 3 can be used to mediate antigen-specific killing of target cells at lower doses.

[0182] Figure 11In the SEA stimulation assay, Bispecific 3 was shown to induce more IL-2 than KEYTRUDA at all doses tested. Importantly, Bispecific 3 induced increased IL-2 production starting at lower antibody concentrations compared to both KEYTRUDA and mAb 1 and mAb 28.

[0183] Figures 12A to 12G Bispecific 3 was demonstrated to have the unique ability to cause internalization of PD-1 and subsequent degradation or loss of PD-1 expression, and this property was dependent on engagement of both the PD-1 and PD-L1 targeting arms of the molecule. Figures 12A to 12B It was demonstrated that only bispecific 3 resulted in internalization of PD-1 and subsequent degradation or loss of PD-1 expression when compared to an isotype control, KEYTRUDA, mAb 1 and mAb 28, atezolizumab, or atezolizumab and KEYTRUDA. Figure 12C As shown in , the ability of Bispecific 3 to drive PD-1 internalization was lost when the anti-PD-L1 antibody mAb1 was added to the wells at 50 nM 5 minutes before the addition of Bispecific 3. This suggests that the two arms of Bispecific 3 should be engaged to drive loss of PD-1 expression and / or internalization and / or degradation. Figure 12D The results showed that treatment with bispecific 3 increased the amount of PD-1 in the supernatant when both binding arms of the bispecific were engaged simultaneously. This effect was lost when the PD-L1 targeting arm was blocked by mAb 1. This suggests that bispecific 3 increases PD-1 shedding into the supernatant. Figure 12E It was demonstrated that the valency of the binding arms affects the extent of loss of PD-1 expression. Bispecific 5 was prepared, which has a first N-terminal Fab that binds PD-L1 based on the VH and VL sequences of mAb1 and a second N-terminal Fab that binds PD-1 based on the VH and VL sequences of mAb28. In other words, bispecific 5 has one monovalent arm that binds PD-L1 and one monovalent arm that binds PD-1, compared to bispecific 3 (which has a bivalent arm that binds PD-L1 and a bivalent arm that binds PD-1). As shown, loss of PD-1 expression begins to occur at higher doses of bispecific 5 (bivalent) compared to bispecific 3 (tetravalent), suggesting that the increased valency of bispecific 3 contributes to this difference. Figure 12F Pretreatment with Batimastat, a broad-spectrum inhibitor of multiple MMPs and ADAMs (shedding enzymes or proteases responsible for cleaving proteins from the cytoplasmic membrane), was demonstrated to greatly reduce the amount of cell-associated PD-1 loss, suggesting that PD-1 loss or shedding is due to cleavage by MMP or ADAM proteases. Figure 12GIt was shown that when bispecific 3 binds to PD-1 and PD-L1 in a trans configuration (ie, expressed by different cells), it primarily drives loss of cell surface PD-1 expression.

[0184] Figures 13A to 13B In vivo results using bispecific 3 are shown. Figure 13A Schematic diagram depicting the experimental protocol. Figure 13B Both the Bispecific 3 and the combination mAb1 and mAb28 groups demonstrated significant delays in tumor growth compared to both the isotype and KEYTRUDA groups. Furthermore, at day 24, there was a significant difference between the Bispecific 3 group and the group treated with the mAb1 and mAb28 combination, with Bispecific 3 causing a greater delay in tumor growth compared to the combination. The group without T cell transfer had more aggressively growing tumors than any of the groups containing T cells. In this model, KEYTRUDA had no advantage in delaying KACP tumor growth compared to the isotype control.

[0185] Figures 14A to 14B In vivo results using bispecific 3 in the K562-A2-CMV-PD-L1 tumor mouse model are shown. Figure 14A and 14B To illustrate the following graph: While each of the different treatment groups resulted in a delay in mean tumor growth compared to untreated mice, treatment with T cells and Bispecific 3 resulted in the greatest delay in mean tumor growth over time. The group without T cell transfer had more aggressively growing tumors than any of the groups containing T cells. In this model, KEYTRUDA had no advantage over the isotype control in delaying K562-A2-CMV-PD-L1 tumor growth.

[0186] Figures 15A to 15B In vivo results using bispecific 3 in several synergistic tumor models are shown. Figure 15A Graph showing that treatment with Bispecific 3 in the EMT-6 synergistic tumor model resulted in a greater delay in tumor growth compared to control treatment. Figure 15B Graph showing that treatment with Bispecific 3 in the MB49 synergy tumor model resulted in a greater delay in tumor growth compared to control treatment.

[0187] Figures 16A to 16B Shown are in vivo results using Bispecific 1 in the MC38-hPD-L1 model of humanized PD-1 / PD-L1 transgenic mice. Figure 16A Both KEYTRUDA and Bispecific 1 treatment were shown to effectively control tumor growth in mice bearing MC38-hPD-L1 tumors compared to control-treated mice. Figure 16Bis a graph of survival demonstrating that Bispecific 1 increased survival in mice bearing MC38-PD-L1 tumors compared to control-treated mice.

[0188] Figures 17A to 17F Depicting in vivo results using bispecific 3 in the B16F10-hPD-L1 model. Figure 17A Figure 2 is a series of graphs showing the effect of different treatments on tumor growth in B16F10-HuPD-L1 mice as measured at a 15-day cutoff. Different groups of mice (n=8) were treated with Bispecific 3, KEYTRUDA, Avelumab, the KEYTRUDA + Avelumab combination, or an isotype control antibody. Figure 17A Individual tumor volume traces are shown for each group. Metastases were identified in multiple mice that died before the tumor-sizing cutoff. Figure 17B Differences in mean tumor volume across treatment groups are shown, demonstrating that Bispecific 3 treatment delayed mean tumor growth in B16F10-HuPD-L1 mice significantly longer than any of the other treatments tested at day 15 post-tumor cell inoculation. ****, P < 0.0001; **, P < 0.01, *, P < 0.05, two-way ANOVA with Tukey's multiple comparison test. Figure 17B Figure 2 is a graph comparing the effects of different treatments on tumor volume in B16F10-HuPD-L1 mice measured at a 15-day cutoff. Metastases were identified in multiple mice that died before the tumor size cutoff. Figure 17B As shown in , Bispecific 3 treatment delayed mean tumor growth in B16F10-hPD-L1 mice significantly longer than any of the other treatments tested. Figure 17C is a graph of survival illustrating that treatment with Bispecific 3 increased survival of B16F10-hPD-L1 tumor-bearing mice compared to survival with any of the other treatments tested. Figure 17D Results showed that 21 days after tumor cell inoculation, Bispecific 3 treatment continued to delay mean tumor growth in B16F10-hPD-L1 mice significantly better than treatment with KEYTRUDA. Figure 17E is a graph of survival illustrating that treatment with Bispecific 3 increased survival of B16F10-HuPD-L1 tumor-bearing mice compared to survival with any of the other treatments tested. Figure 17F A table showing the number of tumor-free mice for each of the different treatment groups is provided. The group treated with Bispecific 3 had 3 tumor-free mice, while the combination of KEYTRUDA and avolumab had 1 tumor-free mouse.

[0189] Figures 18A to 18DBispecific 3 was demonstrated to have drug-like properties (DLPs) similar to well-behaved monoclonal antibodies and maintain parental PD-1 and PD-L1 binding. Figure 18A Bispecific 3 is shown to exhibit similar binding to human PD-1 expressing CHO cells as the parental clone mAb28 (upper panel) and similar binding to human PD-L1 expressing CHO cells as the parental clone mAbl (lower panel). Figure 18B Bispecific 3 is shown to show similar binding to CHO cells expressing cynomolgus PD-1 (upper panel) and similar binding to CHO cells expressing cynomolgus PD-L1 (lower panel) as the parental clone mAb28. Figure 18C Bispecific 3 is shown to exhibit similar binding to CHO cells expressing mouse PD-1 (upper panel) and to CHO cells expressing mouse PD-L1 (lower panel) as the parental clone mAb28. Figure 18D Shown are the size exclusion chromatography trace of bispecific 3 after protein A chromatography (top), which demonstrates a single peak with greater than 98% purity, and the differential scanning fluorimetry (DSF) trace of bispecific 3 (bottom), which demonstrates the high thermal stability of the molecule. Detailed Description of the Invention

[0191] Unless otherwise specified, all technical terms, annotations and other scientific terms used herein are intended to have the meanings commonly understood by those skilled in the art to which the present disclosure belongs. In some cases, for clarity and / or ease of reference, terms with commonly understood meanings are defined herein, and the inclusion of such definitions herein should not necessarily be interpreted as representing differences from what is commonly understood in the art. The techniques and processes described or referenced herein are generally understood by those skilled in the art and are generally adopted using conventional methods, such as, for example, the widely used molecular cloning methods described in Sambrook et al., Molecular Cloning: A Laboratory Manual 2nd edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Where appropriate, unless otherwise specified, the process involving the use of commercially available kits and reagents is generally implemented according to the protocol and / or parameters determined by the manufacturer.

[0192] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0193] Throughout this specification and the claims, the term "about" is used to modify, for example, the amounts, concentrations, volumes, process temperatures, process times, yields, flow rates, pressures, and similar values ​​and ranges thereof, of ingredients in compositions employed in the embodiments of the present disclosure. The term "about" refers to variations in the numerical amount that can occur, for example, by typical measurements and operating procedures used to prepare compounds, compositions, concentrates, or use formulations; by unintentional errors in these procedures; by differences in the manufacture, origin, or purity of the starting materials or ingredients used to perform the methods, and similar approximate considerations. The term "about" also encompasses amounts that vary due to aging of a formulation having a particular initial concentration or mixture, and amounts that vary due to mixing or processing of a formulation having a particular initial concentration or mixture. Where modified by the term "about," the appended claims include equivalents to these amounts. If the use of the term is not clear to one skilled in the art in the context in which it is used, "about" will mean up to plus or minus 10% of the specified value.

[0194] With respect to the binding of an antigen binding protein / region / arm to a target molecule, the terms "specifically binds," "specifically binds to," "specifically for," "selectively binds," and "selectively for" a specific antigen (e.g., a polypeptide target) or an epitope on a specific antigen mean that the binding can be measurably distinguished from non-specific or non-selective interactions. Specific binding can be measured, for example, by comparing the binding of the assay molecule to the binding of a control molecule. Specific binding can also be determined by competition with a control molecule similar to the target, such as an excess of unlabeled target. In this case, specific binding is indicated if the binding of the labeled target to the probe is competitively inhibited by excess unlabeled target.

[0195] The term "epitope" means a component of an antigen that can specifically bind to an antigen-binding protein. Epitopes are often composed of surface accessible amino acid residues and / or sugar side chains and may have specific three-dimensional structural characteristics, as well as specific charge characteristics. The difference between conformational and non-conformational epitopes is that binding to the former, but not the latter, is lost in the presence of denaturing solvents. An epitope may include amino acid residues that are directly involved in binding, and other amino acid residues that are not directly involved in binding. Known techniques for epitope determination can be used, such as, for example, testing antigen-binding proteins bound to antigen variants with different point mutations to determine the epitope to which the antigen-binding protein binds.

[0196] The percent "identity" between a polypeptide sequence and a reference sequence is defined as the percentage of amino acid residues in the polypeptide sequence that are identical to the amino acid residues in the reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignments for the purpose of determining percent amino acid sequence identity can be achieved in various ways that are within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, MEGALIGN (DNASTAR), CLUSTALW, or CLUSTAL OMEGA software. In some embodiments, alignment is performed using CLUSTAL OMEGA software. One skilled in the art can determine appropriate parameters for aligning sequences, including any algorithm required to achieve maximum alignment across the full length of the sequences being compared.

[0197] As used herein, the term "capable" means that an agent or method (e.g., any of the multispecific antigen-binding constructs or methods disclosed herein) has the ability to achieve a particular property under appropriate circumstances (as understood by those skilled in the art), but is not necessarily associated with that property at any particular time. For example, any of the multispecific antigen-binding constructs disclosed herein may be capable of binding to PD-1 and / or PD-L1 when administered to cells expressing PD-1 and / or PD-L1, but when the construct is in a composition lacking PD-1 or PD-L1 protein, the construct is not expected to bind to PD-1 and / or PD-L1.

[0198] "Conservative substitutions" or "conservative amino acid substitutions" refer to the replacement of one or more amino acids with one or more chemically or functionally similar amino acids. Conservative substitution tables providing similar amino acids are well known in the art. Polypeptide sequences having such substitutions are referred to as "conservatively modified variants" or "variants". Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles. Some examples of conservative substitutions can be found, for example, in Creighton, Proteins: Structures and Molecular Properties, 2nd Edition (1993) WH Freeman & Co., New York, NY.

[0199] Polypeptide disclosed herein can include non-naturally produced amino acid sequences.Such variants must have a sequence identity or similarity less than 100% with the starting molecule.In certain embodiments, variants will have an amino acid sequence of about 75% to less than 100% with the starting (e.g., naturally occurring or wild-type) polypeptide, more preferably about 80% to less than 100%, more preferably about 85% to less than 100%, more preferably about 90% to less than 100% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) and most preferably about 95% to less than 100% amino acid sequence identity or similarity (e.g., across the length of variant molecules).

[0200] As used herein, "antibody" can refer to both intact antibodies (e.g., intact immunoglobulins) and antibody portions, e.g., antigen binding portions. An antigen binding portion comprises at least one antigen binding domain. An example of an antigen binding domain is a H -V L The antigen binding domain formed by the dimer. Antibodies and antigen-binding portions can be described by the antigen to which they specifically bind. For example, a PD-L1 antibody or anti-PD-L1 antibody is an antibody that specifically binds to PD-L1.

[0201] V H and V L The V regions are further subdivided into regions of hypervariability (hypervariable regions (HVRs), also called complementarity determining regions (CDRs)) interspersed with more conserved regions. The more conserved regions are called framework regions (FRs). H and V L Typically, the antibody comprises three CDRs and four FRs arranged in the following order (from N-terminus to C-terminus): FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The CDRs are involved in antigen binding and confer antigen specificity and binding affinity to the antibody. (See Kabat et al. (1991) Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD.)

[0202] CDRs participate in antigen binding and confer antigen specificity and binding affinity to antibodies. Three CDRs are present in each of the heavy and light chain variable domains, which are designated CDR1, CDR2, and CDR3 for each variable domain. As used herein, the term "CDR set" refers to a group of three CDRs that appear in a single heavy or light chain variable domain that is capable of binding to a target antigen. The precise boundaries of these CDRs are defined differently in different systems. The three heavy chain CDRs may be referred to as CDRH1, CDRH2, and CDRH3, and the three light chain CDRs may be referred to as CDRL1, CDRL2, and CDRL3.

[0203] The system described by Kabat, also referred to as "numbering according to Kabat," "Kabat numbering," "Kabat definition," and "Kabat notation," provides an unambiguous residue numbering system applicable to any variable domain of an antibody and provides precise residue boundaries defining the three CDRs of each chain. (Kabat et al., Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md. (1987) and (1991), the contents of which are incorporated by reference in their entireties.) These CDRs are referred to as Kabat CDRs and include approximately residues 24-34 (CDR1), 50-56 (CDR2), and 89-97 (CDR3) in the light chain variable domain and 31-35 (CDR1), 50-65 (CDR2), and 95-102 (CDR3) in the heavy chain variable domain. When the CDRs are defined according to Kabat, the light chain FR residues are located at approximately residues 1-23 (LCFR1), 35-49 (LCFR2), 57-88 (LCFR3), and 98-107 (LCFR4), and the heavy chain FR residues are located at approximately residues 1-30 (HCFR1), 36-49 (HCFR2), 66-94 (HCFR3), and 103-113 (HCFR4) of the heavy chain residues. The "EU index as in Kabat" refers to the residue numbering of human IgG1 EU antibodies.

[0204] Other CDR numbering systems are also used in the art (see, for example, Table A). Chothia and colleagues found that certain sub-portions within the Kabat CDRs adopt nearly identical peptide backbone conformations, despite their great diversity at the amino acid sequence level. (Chothia et al. (1987) J. Mol. Biol. 196:901-917; and Chothia et al. (1989) Nature 342:877-883). These subportions may be designated L1, L2, and L3, or H1, H2, and H3, where "L" and "H" refer to the light and heavy chain regions, respectively. These CDRs may be referred to as "Chothia CDRs," "Chothia numbering," or "numbered according to Chothia," and comprise approximately residues 24-34 (CDR1), 50-56 (CDR2), and 89-97 (CDR3) in the light chain variable domain, and 26-32 (CDR1), 52-56 (CDR2), and 95-102 (CDR3) in the heavy chain variable domain. Mol. Biol. 196:901-917 (1987).

[0205] The system described by MacCallum, also referred to as "according to MacCallum numbering" or "MacCallum numbering," comprises approximately residues 30-36 (CDR1), 46-55 (CDR2), and 89-96 (CDR3) in the light chain variable domain, and 30-35 (CDR1), 47-58 (CDR2), and 93-101 (CDR3) in the heavy chain variable domain. MacCallum et al. ((1996) J. Mol. Biol. 262(5):732-745).

[0206] The system described by AbM, also referred to as "numbering according to AbM" or "AbM numbering" comprises approximately residues 24-34 (CDR1), 50-56 (CDR2), and 89-97 (CDR3) in the light chain variable domain, and 26-35 (CDR1), 50-58 (CDR2), and 95-102 (CDR3) in the heavy chain variable domain.

[0207] The IMGT (International Immunogenetics Information System) numbering of the variable region may also be used, which is the numbering of residues in the immunoglobulin variable heavy or light chain according to the method of IIMGT, as described in Lefranc, M.-P., "The IMGT unique numbering for immunoglobulins, T cell receptors and Ig-like domain", The Immunologist, 7, 132-136 (1999), and the entire text of which is expressly incorporated herein by reference. As used herein, "IMGT sequence numbering" or "numbering according to IMTG" refers to the numbering of the sequence encoding the variable region according to IMGT. For the heavy chain variable domain, when numbered according to IMGT, the hypervariable region ranges from amino acid positions 27 to 38 for CDR1, amino acid positions 56 to 65 for CDR2, and amino acid positions 105 to 117 for CDR3. For the light chain variable domain, the hypervariable region ranges from amino acid positions 27 to 38 for CDR1, from amino acid positions 56 to 65 for CDR2, and from amino acid positions 105 to 117 for CDR3, when numbered according to IMGT.

[0208] In some embodiments of the constructs and antigen-binding arms described herein, the CDRs described herein comprise approximately residues 24-34 (CDR1), 49-56 (CDR2), and 89-97 (CDR3) in the light chain variable domain, and 27-35 (CDR1), 49-60 (CDR2), and 93-102 (CDR3) in the heavy chain variable domain, when numbered according to Chothia numbering. In some embodiments, CDR2 in the light chain variable domain may comprise amino acids 49-56, when numbered according to Chothia numbering.

[0209] Table A: CDR Definition

[0210]

[0211] Although methods and materials similar or equivalent to those described herein can also be used to implement or test the methods and compositions disclosed herein, preferred methods and materials are described herein. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.

[0212] Various aspects of the disclosure are described in further detail below. Additional definitions are set forth throughout this specification.

[0213] The immune system has the ability to recognize and eliminate tumor cells; however, tumors can use a variety of strategies to escape immunity. Recent studies have shown that inhibition of immune checkpoint molecules promotes cancer progression through various anti-tumor inhibitory mechanisms. Blocking of immune checkpoints is one of the methods for activating or reactivating therapeutic anti-tumor immunity. Various ligands have been described for many homologous inhibitory immune checkpoint receptors. For example, Nair and Elkord, Immunology & Cell Biology (2018), 96: 21-33; and Jenkins et al., British J. of Cancer (2017), 118: 9-16.

[0214] The programmed death 1 (PD-1) protein is an inhibitory member of the extended CD28 / CTLA-4 family of T cell regulators (Okazaki et al. (2002) Curr Opin Immunol 14:391779-82; Bennett et al. (2003) J. Immunol. 170:711-8). Other members of the CD28 family include CD28, CTLA-4, ICOS, and BTLA. PD-1 is proposed to exist as a monomer that lacks the unpaired cysteine ​​residue characteristic of other CD28 family members. PD-1 is expressed on activated B cells, T cells, and monocytes.

[0215] The PD-1 gene encodes a 55kDa type I transmembrane protein (Agata et al. (1996) Int Immunol. 8:765-72). Although structurally similar to CTLA-4, PD-1 lacks the MYPPY motif that is important for B7-1 and B7-2 binding. Two ligands for PD-1 have been identified, PD-L1 (B7-H1) and PD-L2 (B7-DC), which have been shown to downregulate T cell activation after binding to PD-1 (Freeman et al. (2000) J. Exp. Med. 192:1027-34; Carter et al. (2002) Eur. J. Immunol. 32:634-43). Both PD-L1 and PD-L2 are B7 homologs that bind to PD-1 but not to other CD28 family members. PD-L1 is abundant in various human cancers (Dong et al. (2002) Nat. Med. 8:787-9).

[0216] PD-1 is known as an immunosuppressive protein that negatively regulates TCR signaling (Ishida, Y. et al. (1992) EMBO J. 11: 3887-3895; Blank, C. et al. (Epub 2006 Dec. 29) Immunol. Immunother. 56(5): 739-745). The interaction between PD-1 and PD-L1 can act as an immune checkpoint, which can lead to, for example, a decrease in tumor-infiltrating lymphocytes, a decrease in T cell receptor-mediated proliferation, and / or immune escape by cancer cells (Dong et al. (2003) J. Mol. Med. 81: 281-7; Blank et al. (2005) Cancer Immunol. Immunother. 54: 307-314; Konishi et al. (2004) Clin. Cancer Res. 10: 5094-100). Immunosuppression can be reversed by inhibiting the local interaction of PD-1 with PD-L1 or PD-L2; also, when the interaction of PD-1 and PD-L2 is blocked, the effects are additive (Iwai et al. (2002) Proc. Nat'l. Acad. Sci. USA 99:12293-7; Brown et al. (2003) J. Immunol. 170:1257-66).

[0217] PD-L1, also known as cluster of differentiation 274 (CD274) or B7 homolog 1 (B7-H1), is a 40kDa type 1 transmembrane protein that plays a role in suppressing the immune system during specific events such as pregnancy, tissue allografts, autoimmune diseases and other disease states, such as hepatitis. For example, human PD-L1 comprises the amino acid sequence of SEQ ID NO: 115 (UniProt Q9NZQ7). Normally, the immune system responds to foreign antigens associated with exogenous or endogenous danger signals, which triggers the proliferation of antigen-specific CD8+ T cells and / or CD4+ helper cells. In cancer, PD-L1 expressed on cancer cells binds to its ligand PD-1 on immune effector cells (e.g., T cells). The binding of PD-L1 to PD-1 transmits an inhibitory signal that reduces the proliferation of antigen-specific T cells in the lymph nodes while reducing the apoptosis of regulatory T cells (anti-inflammatory suppressor T cells). PD-1 / PD-L1 interaction also induces apoptosis of tumor-specific T cells and promotes CD4 + T cell differentiation into Foxp3 + Regulatory T cells (Tregs) promote tumor cell resistance to attack by cytotoxic T lymphocytes (CTLs), thereby allowing tumors to evade the host immune system.

[0218] The present disclosure relates to compositions and methods for suppressing tumor escape by reducing the immune checkpoint inhibition caused by the interaction between PD-1 and its ligand (e.g., PD-L1 and / or PD-L2). Specifically, provided herein are compositions comprising novel multispecific multivalent constructs (such as bispecific tetravalent constructs), which block the interaction between PD-1 and its ligand (e.g., PD-1 and / or PD-L2), while promoting the interaction (bridging) of cells expressing PD-1 and its ligand above. Compared to the combination of individual antibodies, and compared to clinical checkpoint blockers, such compositions of the present disclosure with "blocking and bridging" ability increase in vitro and in vivo efficacy and strongly enhance, for example, T cell proliferation, IFNγ production and / or secretion, T cell cytolytic activity, and / or self-functional exhaustion rescue T cells, to provide superior anti-tumor efficacy (biological effects that can be manifested by various methods, including but not limited to, for example, a reduction in tumor volume, a reduction in the number of tumor cells, a reduction in tumor cell proliferation, and / or a reduction in tumor cell survival). Also provided herein are novel monoclonal anti-PD-1 antibodies and antigen-binding fragments thereof, and novel monoclonal anti-PD-L1 antibodies and antigen-binding fragments thereof for use in such multispecific, multivalent constructs. Some of these novel monoclonal anti-PD-1 antibodies and novel monoclonal anti-PD-L1 antibodies share a common light chain, thereby allowing the generation of multispecific, multivalent constructs with excellent drug-like properties and manufacturability, as well as affinities similar to those of their parent antibodies.

[0219] Therefore, the present disclosure provides a multispecific antigen-binding construct comprising at least two antigen-binding arms or antigen-binding units, wherein the first antigen-binding arm or unit binds to PD-1 expressed by an immune cell, and the second antigen-binding arm or unit binds to one or more PD-1 ligands (e.g., PD-L1 and / or PD-L2) expressed by a second cell. In some embodiments, the multispecific antigen-binding construct blocks the interaction between PD-1 and its ligand. In some embodiments, the multispecific antigen-binding construct bridges cells on which PD-1 and its ligand are expressed to promote the interaction and / or efficacy of immune cells expressing PD-1. In some embodiments, at least one of the antigen-binding arms is bivalent for PD-1. In some embodiments, at least one of the antigen-binding arms is bivalent for PD-L1. In some embodiments, at least one of the antigen-binding arms is bivalent for PD-1, and at least one of the antigen-binding arms is bivalent for PD-L1. In some embodiments, the multispecific antigen-binding construct comprises at least two antigen-binding units that bind to PD-1. In some embodiments, the multispecific antigen-binding construct comprises two antigen-binding units that bind to PD-1. In some embodiments, the multispecific antigen-binding construct comprises at least two antigen-binding units that bind to PD-1 ligands (such as PD-L1 or PD-L2). In some embodiments, the multispecific antigen-binding construct comprises two antigen-binding units that bind to PD-1 ligands (such as PD-L1 or PD-L2). In some embodiments, the multispecific antigen-binding construct comprises at least four antigen-binding units, wherein two antigen-binding units bind to PD-1 and two antigen-binding units bind to PD-1 ligands (such as PD-L1 or PD-L2). In some embodiments, the multispecific antigen-binding construct comprises four antigen-binding units, wherein two antigen-binding units bind to PD-1 and two antigen-binding units bind to PD-1 ligands (such as PD-L1 or PD-L2). In some embodiments of any one of the aspects described herein, the construct is a bispecific antibody. In some embodiments, the bispecific antibody is an antagonist of both PD-1 and PD-1 ligands. In some embodiments, the construct comprises a common light chain. In some embodiments, one or both of the antigen-binding arms are aptamers. In some embodiments, one or both of the antigen-binding arms are proteins other than antibodies. In some embodiments, the construct comprises at least two antibodies. In some embodiments, at least one of the antigen-binding arms is a bivalent antibody specific for PD-1. In some embodiments, at least one of the antigen-binding arms is a bivalent antibody specific for PD-L1.In some embodiments, at least one of the antigen binding arms is a bivalent antibody specific for PD-1, and at least one of the antigen binding arms is a bivalent antibody specific for PD-L1, such that the construct is tetravalent. In some embodiments, the bispecific antibody binds to two different epitopes on PD-1. In some embodiments, the bispecific antibody binds to two different epitopes on the PD-1 ligand. In some aspects, novel isolated antibodies and antigen binding portions thereof that specifically bind to PD-L1 or PD-1 are also provided herein. In some embodiments, these novel isolated antibodies and antigen binding portions thereof that specifically bind to PD-L1 or PD-1 (such as CDRs, variable heavy chains, and / or variable light chains) can be used in one or more antigen binding arms or units of the multispecific antigen binding constructs described herein.

[0220] Thus, as used herein, the disclosed multispecific antigen-binding constructs comprise bispecific, trispecific, tetraspecific or multispecific antibodies or antigen-binding portions thereof. Preferably, the multispecific construct is bivalent for at least one, preferably two, antigen-binding arms, i.e., a trivalent bispecific, or a tetravalent bispecific molecule. In various aspects and embodiments, the multispecific constructs described herein comprise one or more antibodies and / or antigen-binding portions thereof. For example, the antigen-binding arm may comprise a variable heavy chain and / or a variable light chain or its complementary determining region (CDR) of a given antibody of PD-1 and / or a given antibody of PD-L1. Thus, in some embodiments of any one of the aspects described herein, the first antigen-binding arm, the second antigen-binding arm, the first antigen-binding unit, the second binding unit, or any combination thereof may comprise an antibody or an antigen-binding portion thereof. In some embodiments of any one of the aspects described herein, the first antigen-binding arm, the second antigen-binding arm, the first antigen-binding unit, the second binding unit, or any combination thereof is an antibody or an antigen-binding portion thereof.

[0221] A. PD-L1 antagonists

[0222] In some aspects and embodiments, the present disclosure provides anti-PD-L1 antagonists. In some embodiments, the anti-PD-L1 antagonist is any of the anti-PD-L1 antibodies or antigen-binding molecules disclosed herein. In some embodiments, the anti-PD-L1 antibody or antigen-binding molecule is not part of a multispecific antigen-binding construct, that is, the anti-PD-L1 antibody or antigen-binding molecule is not part of a protein construct that binds to multiple epitopes. In some embodiments, the anti-PD-L1 antibody or antigen-binding portion can be combined with different antibodies or antigen-binding portions to form a multispecific antigen-binding construct. In some embodiments, the multispecific antigen-binding construct can bind to an epitope on PD-L1 and an epitope on another protein. In some embodiments, the epitope on the other protein is on PD-1.

[0223] Thus, in some aspects, provided herein are antibodies, or antigen-binding portions thereof, that specifically bind to PD-L1. In some aspects, an antibody, or antigen-binding portion thereof, that specifically binds PD-L1 comprises (a) a heavy chain variable region comprising (i) a CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN); (ii) a CDRH2 comprising SEQ ID NO: 2 (GGIIPX1X2GX3ATYA, wherein X1 is V or I; X2 is F, L, or V; and X3 is T or A); and (iii) a CDRH3 comprising SEQ ID NO: 3 (ARLKX1ELKDAFDI, wherein X1 is G, F, or N); and (b) a light chain variable region comprising: (i) a CDRL1 comprising SEQ ID NO: 4 (RASQX1ISSYLN, wherein X1 is S, W, or Q); (ii) a CDRL2 comprising SEQ ID NO: 5 (AASSLQS); and (iii) a CDRL3 comprising SEQ ID NO: 6 (X1QSYSTPLT, wherein X1 is Q or F).

[0224] In some embodiments of these aspects and all such aspects described herein, CDRH1 comprises SEQ ID NO: 1 (GTFSSYAIN), CDRH2 comprises SEQ ID NO: 7 (GGIIPILGAATYA), and CDRH3 comprises SEQ ID NO: 8 (ARLKGELKDAFDI). In some embodiments of these aspects and all such aspects described herein, CDRH1 comprises SEQ ID NO: 1 (GTFSSYAIN), CDRH2 comprises SEQ ID NO: 7 (GGIIPILGAATYA), CDRH3 comprises SEQ ID NO: 8 (ARLKGELKDAFDI); CDRL1 comprises SEQ ID NO: 9 (RASQSISSYLN), CDRL2 comprises SEQ ID NO: 5 (AASSLQS), and CDRL3 comprises SEQ ID NO: 10 (QQSYSTPLT). A representative antibody having such heavy and light chain variable CDR regions is mAb1.

[0225] In some embodiments of these aspects and all such aspects described herein, CDRH1 comprises SEQ ID NO: 1 (GTFSSYAIN), CDRH2 comprises SEQ ID NO: 11 (GGIIPVFGTATYA), and CDRH3 comprises SEQ ID NO: 8 (ARLKGELKDAFDI). In some embodiments of these aspects and all such aspects described herein, CDRH1 comprises SEQ ID NO: 1 (GTFSSYAIN), CDRH2 comprises SEQ ID NO: 11 (GGIIPVFGTATYA), CDRH3 comprises SEQ ID NO: 8 (ARLKGELKDAFDI); CDRL1 comprises SEQ ID NO: 12 (RASQWISSYLN), CDRL2 comprises SEQ ID NO: 5 (AASSLQS), and CDRL3 comprises SEQ ID NO: 10 (QQSYSTPLT). A representative antibody having such heavy and light chain variable CDR regions is mAb2. In some embodiments of these aspects and all such aspects described herein, CDRH1 comprises SEQ ID NO: 1 (GTFSSYAIN), CDRH2 comprises SEQ ID NO: 11 (GGIIPVFGTATYA), CDRH3 comprises SEQ ID NO: 8 (ARLKGELKDAFDI); CDRL1 comprises SEQ ID NO: 13 (RASQQISSYLN), CDRL2 comprises SEQ ID NO: 5 (AASSLQS), and CDRL3 comprises SEQ ID NO: 10 (QQSYSTPLT). A representative antibody having such heavy and light chain variable CDR regions is mAb3.

[0226] In some embodiments of these aspects and all such aspects described herein, CDRH1 comprises SEQ ID NO: 1 (GTFSSYAIN), CDRH2 comprises SEQ ID NO: 11 (GGIIPVFGTATYA), CDRH3 comprises SEQ ID NO: 8 (ARLKGELKDAFDI); CDRL1 comprises SEQ ID NO: 9 (RASQSISSYLN), CDRL2 comprises SEQ ID NO: 5 (AASSLQS), and CDRL3 comprises SEQ ID NO: 10 (QQSYSTPLT). A representative antibody having such heavy and light chain variable CDR regions is mAb4. Another representative antibody having such heavy and light chain variable CDR regions is mAb24.

[0227] In some embodiments of these aspects and all such aspects described herein, CDRH1 comprises SEQ ID NO: 14 (GTFSSYAFS), CDRH2 comprises SEQ ID NO: 11 (GGIIPVFGTATYA), and CDRH3 comprises SEQ ID NO: 8 (ARLKGELKDAFDI). In some embodiments of these aspects and all such aspects described herein, CDRH1 comprises SEQ ID NO: 14 (GTFSSYAFS), CDRH2 comprises SEQ ID NO: 11 (GGIIPVFGTATYA), CDRH3 comprises SEQ ID NO: 8 (ARLKGELKDAFDI); CDRL1 comprises SEQ ID NO: 9 (RASQSISSYLN), CDRL2 comprises SEQ ID NO: 5 (AASSLQS), and CDRL3 comprises SEQ ID NO: 10 (QQSYSTPLT). A representative antibody having such heavy and light chain variable CDR regions is mAb5.

[0228] In some embodiments of these aspects and all such aspects described herein, CDRH1 comprises SEQ ID NO: 1 (GTFSSYAIN), CDRH2 comprises SEQ ID NO: 15 (GGIIPIFGIANYA), and CDRH3 comprises SEQ ID NO: 8 (ARLKGELKDAFDI). In some embodiments of these aspects and all such aspects described herein, CDRH1 comprises SEQ ID NO: 1 (GTFSSYAIN), CDRH2 comprises SEQ ID NO: 15 (GGIIPIFGIANYA), CDRH3 comprises SEQ ID NO: 8 (ARLKGELKDAFDI); CDRL1 comprises SEQ ID NO: 9 (RASQSISSYLN), CDRL2 comprises SEQ ID NO: 5 (AASSLQS), and CDRL3 comprises SEQ ID NO: 10 (QQSYSTPLT). A representative antibody having such heavy and light chain variable CDR regions is mAb6.

[0229] In some embodiments of these aspects and all such aspects described herein, CDRH1 comprises SEQ ID NO: 1 (GTFSSYAIN), CDRH2 comprises SEQ ID NO: 16 (GGIIPNFGTATYA), and CDRH3 comprises SEQ ID NO: 17 (ARLKGELKGAGDI). In some embodiments of these aspects and all such aspects described herein, CDRH1 comprises SEQ ID NO: 1 (GTFSSYAIN), CDRH2 comprises SEQ ID NO: 16 (GGIIPNFGTATYA), CDRH3 comprises SEQ ID NO: 17 (ARLKGELKGAGDI); CDRL1 comprises SEQ ID NO: 9 (RASQSISSYLN), CDRL2 comprises SEQ ID NO: 5 (AASSLQS), and CDRL3 comprises SEQ ID NO: 10 (QQSYSTPLT). A representative antibody having such heavy and light chain variable CDR regions is mAb7.

[0230] In some embodiments of these aspects and all such aspects described herein, CDRH1 comprises SEQ ID NO: 1 (GTFSSYAIN), CDRH2 comprises SEQ ID NO: 11 (GGIIPVFGTATYA), and CDRH3 comprises SEQ ID NO: 18 (ARLKFELKDAFDI). In some embodiments of these aspects and all such aspects described herein, CDRH1 comprises SEQ ID NO: 1 (GTFSSYAIN), CDRH2 comprises SEQ ID NO: 11 (GGIIPVFGTATYA), CDRH3 comprises SEQ ID NO: 18 (ARLKFELKDAFDI), CDRL1 comprises SEQ ID NO: 9 (RASQSISSYLN), CDRL2 comprises SEQ ID NO: 5 (AASSLQS), and CDRL3 comprises SEQ ID NO: 10 (QQSYSTPLT). A representative antibody having such heavy and light chain variable CDR regions is mAb8.

[0231] In some embodiments of these aspects and all such aspects described herein, CDRH1 comprises SEQ ID NO: 1 (GTFSSYAIN), CDRH2 comprises SEQ ID NO: 11 (GGIIPVFGTATYA), and CDRH3 comprises SEQ ID NO: 19 (ARLKGELKDAFDE). In some embodiments of these aspects and all such aspects described herein, CDRH1 comprises SEQ ID NO: 1 (GTFSSYAIN), CDRH2 comprises SEQ ID NO: 11 (GGIIPVFGTATYA), CDRH3 comprises SEQ ID NO: 19 (ARLKGELKDAFDE), CDRL1 comprises SEQ ID NO: 9 (RASQSISSYLN), CDRL2 comprises SEQ ID NO: 5 (AASSLQS), and CDRL3 comprises SEQ ID NO: 10 (QQSYSTPLT). A representative antibody having such heavy and light chain variable CDR regions is mAb9.

[0232] In some embodiments of these aspects and all such aspects described herein, CDRH1 comprises SEQ ID NO: 1 (GTFSSYAIN), CDRH2 comprises SEQ ID NO: 11 (GGIIPVFGTATYA), and CDRH3 comprises SEQ ID NO: 20 (ARLKNELKDAFDI). In some embodiments of these aspects and all such aspects described herein, CDRH1 comprises SEQ ID NO: 1 (GTFSSYAIN), CDRH2 comprises SEQ ID NO: 11 (GGIIPVFGTATYA), CDRH3 comprises SEQ ID NO: 20 (ARLKNELKDAFDI), CDRL1 comprises SEQ ID NO: 9 (RASQSISSYLN), CDRL2 comprises SEQ ID NO: 5 (AASSLQS), and CDRL3 comprises SEQ ID NO: 10 (QQSYSTPLT). A representative antibody having such heavy and light chain variable CDR regions is mAb10.

[0233] In some embodiments of these aspects and all such aspects described herein, CDRH1 comprises SEQ ID NO: 1 (GTFSSYAIN), CDRH2 comprises SEQ ID NO: 21 (GGVIPFLGTANYA), and CDRH3 comprises SEQ ID NO: 22 (ARLKGILKDALDI). In some embodiments of these aspects and all such aspects described herein, CDRH1 comprises SEQ ID NO: 1 (GTFSSYAIN), CDRH2 comprises SEQ ID NO: 21 (GGVIPFLGTANYA), CDRH3 comprises SEQ ID NO: 22 (ARLKGILKDALDI), CDRL1 comprises SEQ ID NO: 9 (RASQSISSYLN), CDRL2 comprises SEQ ID NO: 5 (AASSLQS), and CDRL3 comprises SEQ ID NO: 10 (QQSYSTPLT). A representative antibody having such heavy and light chain variable CDR regions is mAb 11.

[0234] In some embodiments of these aspects and all such aspects described herein, CDRH1 comprises SEQ ID NO: 23 (GTFSSYAIS), CDRH2 comprises SEQ ID NO: 24 (GGIIPIVGIANYA), and CDRH3 comprises SEQ ID NO: 8 (ARLKGELKDAFDI). In some embodiments of these aspects and all such aspects described herein, CDRH1 comprises SEQ ID NO: 23 (GTFSSYAIS), CDRH2 comprises SEQ ID NO: 24 (GGIIPIVGIANYA), CDRH3 comprises SEQ ID NO: 8 (ARLKGELKDAFDI), CDRL1 comprises SEQ ID NO: 9 (RASQSISSYLN), CDRL2 comprises SEQ ID NO: 5 (AASSLQS), and CDRL3 comprises SEQ ID NO: 10 (QQSYSTPLT). A representative antibody having such heavy and light chain variable CDR regions is mAb 12.

[0235] In some embodiments of these aspects and all such aspects described herein, CDRH1 comprises SEQ ID NO: 23 (GTFSSYAIS), CDRH2 comprises SEQ ID NO: 11 (GGIIPVFGTATYA), and CDRH3 comprises SEQ ID NO: 25 (ARLKGEFKDAFDI). In some embodiments of these aspects and all such aspects described herein, CDRH1 comprises SEQ ID NO: 23 (GTFSSYAIS), CDRH2 comprises SEQ ID NO: 11 (GGIIPVFGTATYA), CDRH3 comprises SEQ ID NO: 25 (ARLKGEFKDAFDI), CDRL1 comprises SEQ ID NO: 9 (RASQSISSYLN), CDRL2 comprises SEQ ID NO: 5 (AASSLQS), and CDRL3 comprises SEQ ID NO: 10 (QQSYSTPLT). A representative antibody having such heavy and light chain variable CDR regions is mAb 13.

[0236] In some embodiments of these aspects and all such aspects described herein, CDRH1 comprises SEQ ID NO: 23 (GTFSSYAIS), CDRH2 comprises SEQ ID NO: 26 (GRIIPLFGTAHYA), and CDRH3 comprises SEQ ID NO: 8 (ARLKGELKDAFDI). In some embodiments of these aspects and all such aspects described herein, CDRH1 comprises SEQ ID NO: 23 (GTFSSYAIS), CDRH2 comprises SEQ ID NO: 26 (GRIIPLFGTAHYA), CDRH3 comprises SEQ ID NO: 8 (ARLKGELKDAFDI), CDRL1 comprises SEQ ID NO: 9 (RASQSISSYLN), CDRL2 comprises SEQ ID NO: 5 (AASSLQS), and CDRL3 comprises SEQ ID NO: 10 (QQSYSTPLT). A representative antibody having such heavy and light chain variable CDR regions is mAb 14.

[0237] In some embodiments of these aspects and all such aspects described herein, CDRH1 comprises SEQ ID NO: 23 (GTFSSYAIS), CDRH2 comprises SEQ ID NO: 27 (GRINPILGTANYA), and CDRH3 comprises SEQ ID NO: 28 (ARLKGELKDAFSI). In some embodiments of these aspects and all such aspects described herein, CDRH1 comprises SEQ ID NO: 23 (GTFSSYAIS), CDRH2 comprises SEQ ID NO: 27 (GRINPILGTANYA), CDRH3 comprises SEQ ID NO: 28 (ARLKGELKDAFSI), CDRL1 comprises SEQ ID NO: 9 (RASQSISSYLN), CDRL2 comprises SEQ ID NO: 5 (AASSLQS), and CDRL3 comprises SEQ ID NO: 10 (QQSYSTPLT). A representative antibody having such heavy and light chain variable CDR regions is mAb 15.

[0238] In some embodiments of these aspects and all such aspects described herein, CDRH1 comprises SEQ ID NO: 1 (GTFSSYAIN), CDRH2 comprises SEQ ID NO: 29 (GRIIPIFGTADYA), and CDRH3 comprises SEQ ID NO: 8 (ARLKGELKDAFDI). In some embodiments of these aspects and all such aspects described herein, CDRH1 comprises SEQ ID NO: 1 (GTFSSYAIN), CDRH2 comprises SEQ ID NO: 29 (GRIIPIFGTADYA), CDRH3 comprises SEQ ID NO: 8 (ARLKGELKDAFDI), CDRL1 comprises SEQ ID NO: 9 (RASQSISSYLN), CDRL2 comprises SEQ ID NO: 5 (AASSLQS), and CDRL3 comprises SEQ ID NO: 10 (QQSYSTPLT). A representative antibody having such heavy and light chain variable CDR regions is mAb 16.

[0239] In some embodiments of these aspects and all such aspects described herein, CDRH1 comprises SEQ ID NO: 23 (GTFSSYAIS), CDRH2 comprises SEQ ID NO: 11 (GGIIPVFGTATYA), and CDRH3 comprises SEQ ID NO: 30 (ARLKGELKCAFDI). In some embodiments of these aspects and all such aspects described herein, CDRH1 comprises SEQ ID NO: 23 (GTFSSYAIS), CDRH2 comprises SEQ ID NO: 11 (GGIIPVFGTATYA), CDRH3 comprises SEQ ID NO: 30 (ARLKGELKCAFDI), CDRL1 comprises SEQ ID NO: 9 (RASQSISSYLN), CDRL2 comprises SEQ ID NO: 5 (AASSLQS), and CDRL3 comprises SEQ ID NO: 10 (QQSYSTPLT).

[0240] In some embodiments of these aspects and all such aspects described herein, CDRH1 comprises SEQ ID NO: 122 (GTKSSYAIS), CDRH2 comprises SEQ ID NO: 11 (GGIIPVFGTATYA), and CDRH3 comprises SEQ ID NO: 30 (ARLKGELKCAFDI). In some embodiments of these aspects and all such aspects described herein, CDRH1 comprises SEQ ID NO: 122 (GTKSSYAIS), CDRH2 comprises SEQ ID NO: 11 (GGIIPVFGTATYA), CDRH3 comprises SEQ ID NO: 30 (ARLKGELKCAFDI), CDRL1 comprises SEQ ID NO: 9 (RASQSISSYLN), CDRL2 comprises SEQ ID NO: 5 (AASSLQS), and CDRL3 comprises SEQ ID NO: 10 (QQSYSTPLT). A representative antibody having such heavy and light chain variable CDR regions is mAb 17.

[0241] In some embodiments of these aspects and all such aspects described herein, CDRH1 comprises SEQ ID NO: 1 (GTFSSYAIN), CDRH2 comprises SEQ ID NO: 31 (GGIIPILGTATYA), and CDRH3 comprises SEQ ID NO: 32 (ARRKGELKDAFDI). In some embodiments of these aspects and all such aspects described herein, CDRH1 comprises SEQ ID NO: 1 (GTFSSYAIN), CDRH2 comprises SEQ ID NO: 31 (GGIIPILGTATYA), CDRH3 comprises SEQ ID NO: 32 (ARRKGELKDAFDI), CDRL1 comprises SEQ ID NO: 9 (RASQSISSYLN), CDRL2 comprises SEQ ID NO: 5 (AASSLQS), and CDRL3 comprises SEQ ID NO: 10 (QQSYSTPLT). A representative antibody having such heavy and light chain variable CDR regions is mAb 18.

[0242] In some embodiments of these aspects and all such aspects described herein, CDRH1 comprises SEQ ID NO: 1 (GTFSSYAIN), CDRH2 comprises SEQ ID NO: 33 (GGIIPIVATANYA), and CDRH3 comprises SEQ ID NO: 32 (ARRKGELKDAFDI). In some embodiments of these aspects and all such aspects described herein, CDRH1 comprises SEQ ID NO: 1 (GTFSSYAIN), CDRH2 comprises SEQ ID NO: 33 (GGIIPIVATANYA), CDRH3 comprises SEQ ID NO: 32 (ARRKGELKDAFDI), CDRL1 comprises SEQ ID NO: 9 (RASQSISSYLN), CDRL2 comprises SEQ ID NO: 5 (AASSLQS), and CDRL3 comprises SEQ ID NO: 10 (QQSYSTPLT). A representative antibody having such heavy and light chain variable CDR regions is mAb 19.

[0243] In some embodiments of these aspects and all such aspects described herein, CDRH1 comprises SEQ ID NO: 1 (GTFSSYAIN), CDRH2 comprises SEQ ID NO: 34 (GGIIPIFGKATYA), and CDRH3 comprises SEQ ID NO: 32 (ARRKGELKDAFDI). In some embodiments of these aspects and all such aspects described herein, CDRH1 comprises SEQ ID NO: 1 (GTFSSYAIN), CDRH2 comprises SEQ ID NO: 34 (GGIIPIFGKATYA), CDRH3 comprises SEQ ID NO: 32 (ARRKGELKDAFDI), CDRL1 comprises SEQ ID NO: 9 (RASQSISSYLN), CDRL2 comprises SEQ ID NO: 5 (AASSLQS), and CDRL3 comprises SEQ ID NO: 10 (QQSYSTPLT). A representative antibody having such heavy and light chain variable CDR regions is mAb20.

[0244] In some embodiments of these aspects and all such aspects described herein, CDRH1 comprises SEQ ID NO: 36 (GPFRSHAVS), CDRH2 comprises SEQ ID NO: 11 (GGIIPVFGTATYA), and CDRH3 comprises SEQ ID NO: 37 (ARLKSELKDAFDI). In some embodiments of these aspects and all such aspects described herein, CDRH1 comprises SEQ ID NO: 36 (GPFRSHAVS), CDRH2 comprises SEQ ID NO: 11 (GGIIPVFGTATYA), CDRH3 comprises SEQ ID NO: 37 (ARLKSELKDAFDI), CDRL1 comprises SEQ ID NO: 9 (RASQSISSYLN), CDRL2 comprises SEQ ID NO: 5 (AASSLQS), and CDRL3 comprises SEQ ID NO: 10 (QQSYSTPLT). A representative antibody having such heavy and light chain variable CDR regions is mAb21.

[0245] In some embodiments of these aspects and all such aspects described herein, CDRH1 comprises SEQ ID NO: 1 (GTFSSYAIN), CDRH2 comprises SEQ ID NO: 11 (GGIIPVFGTATYA), CDRH3 comprises SEQ ID NO: 8 (ARLKGELKDAFDI); CDRL1 comprises SEQ ID NO: 9 (RASQSISSYLN), CDRL2 comprises SEQ ID NO: 5 (AASSLQS), and CDRL3 comprises SEQ ID NO: 38 (FQSYSTPLT). A representative antibody having such heavy and light chain variable CDR regions is mAb 22.

[0246] In some embodiments of these aspects and all such aspects described herein, CDRH1 comprises SEQ ID NO: 1 (GTFSSYAIN), CDRH2 comprises SEQ ID NO: 11 (GGIIPVFGTATYA), CDRH3 comprises SEQ ID NO: 8 (ARLKGELKDAFDI); CDRL1 comprises SEQ ID NO: 9 (RASQSISSYLN), CDRL2 comprises SEQ ID NO: 5 (AASSLQS), and CDRL3 comprises SEQ ID NO: 39 (QQSYSTILT). A representative antibody having such heavy and light chain variable CDR regions is mAb 23.

[0247] In some embodiments of these aspects and all such aspects described herein, CDRH1 comprises SEQ ID NO: 1 (GTFSSYAIN), CDRH2 comprises SEQ ID NO: 11 (GGIIPVFGTATYA), CDRH3 comprises SEQ ID NO: 8 (ARLKGELKDAFDI); CDRL1 comprises SEQ ID NO: 9 (RASQSISSYLN), CDRL2 comprises SEQ ID NO: 5 (AASSLQS), and CDRL3 comprises SEQ ID NO: 10 (QQSYSTPLT). A representative antibody having such heavy and light chain variable CDR regions is mAb 24.

[0248] In each case where a particular sequence is described, embodiments are also provided comprising a sequence at least 90% identical (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical) to the sequence (e.g., SEQ ID NOs: 1-34 and 36-39).

[0249] In some aspects, the present disclosure also provides an antibody, or antigen-binding portion thereof, that specifically binds PD-L1, wherein the antibody, or antigen-binding portion thereof, comprises: a heavy chain variable region comprising an amino acid sequence that is at least 90% identical (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical) to SEQ ID NO: 35, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, or 58, and a heavy chain variable region comprising an amino acid sequence that is at least 90% identical (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical) to SEQ ID NO: 35, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, NO:59, 60, 61, 62 or 63 has a light chain variable region of an amino acid sequence that is at least 90% identical (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical).

[0250] In some aspects, the disclosure also provides antibodies, or antigen-binding portions thereof, that specifically bind PD-L1, wherein the antibody, or antigen-binding portion thereof, comprises a heavy chain variable region comprising an amino acid sequence that is at least 90% identical (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical) to SEQ ID NO: 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, or 58 and a light chain variable region comprising an amino acid sequence that is at least 90% identical (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical) to SEQ ID NO: 59.

[0251] In some aspects, the present disclosure also provides antibodies, or antigen-binding portions thereof, that specifically bind PD-L1, wherein the antibody, or antigen-binding portion thereof, comprises a heavy chain variable region comprising an amino acid sequence that is at least 90% identical (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical) to SEQ ID NO: 35 and a light chain variable region comprising an amino acid sequence that is at least 90% identical (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical) to SEQ ID NO: 59, 60, 61, 62, or 63.

[0252] Antibodies mAb1 to mAb23 are affinity matured antibodies derived from the parent antibody mAb24, as described in the Examples. Affinity matured antibodies, or antigen binding portions thereof, are antibodies or antigen binding fragments having one or more alterations (e.g., in one or more CDRs or FRs) that result in an increase in the affinity of the antibody for its antigen compared to the parent antibody lacking the alterations. In some embodiments, the affinity matured antibodies have nanomolar or picomolar affinities for PD-L1. In some embodiments, the PD-L1 antibody, or antigen binding portion thereof, has an affinity of at least 1x10 -7 M, at least 1x10 -8 M, at least 1x10 -9 M, at least 1x10 -10 M, at least 1x10 -11 M, at least 1x10 -12 M, or at least 1x10 -13 M's K D .

[0253] Table 1 shows the binding affinities (K) of mAb1, mAb2, mAb3, mAb4, mAb5, mAb6, mAb7, mAb8, mAb9, mAb10, mAb11, mAb12, mAb13, mAb14, mAb15, mAb16, mAb17, mAb18, mAb19, mAb20, mAb21, mAb22, and mAb23 (i.e., an affinity matured variant of mAb24) to human PD-L1. D ). As used herein, the term K D Refers to the dissociation equilibrium constant of a specific antibody-antigen interaction. D =k d / k a As used herein, the term k d (sec -1 ) refers to the dissociation rate constant for a specific antibody-antigen interaction. This value is also called k off As used herein, the term k a (M -1 x sec -1 ) refers to the association rate constant for a specific antibody-antigen interaction. This value is also called k on value.

[0254] Table 1. Binding affinities of mAb1 to mAb23 to human PD-L1

[0255] mAbs <![CDATA[K D (nM)]]> Improvement multiple 1 4.16 1.1 2 2.2 2.0 3 0.9 4.8 4 4.9 0.9 5 27.1 0.2 6 35.9 0.1 7 -- -- 8 3.7 1.2 9 5.4 0.8 10 6.3 0.7 11 156 -- 12 358 -- 13 52.3 0.1 14 -- -- 15 -- -- 16 -- -- 17 -- -- 18 527 -- 19 817 -- 20 53.9 0.1 21 91.5 0.1 22 2.5 1.8 23 369 --

[0256] Table 2 provides cellular binding data for mAb1, mAb2, mAb3, mAb4, mAb5, mAb6, mAb7, mAb8, mAb9, mAb10, mAb11, mAb12, mAb13, mAb14, mAb15, mAb16, mAb17, mAb18, mAb19, mAb20, mAb21, mAb22, and mAb23 (i.e., an affinity matured variant of mAb24) to human PD-L1 ("huPDL1"), cynomolgus monkey PD-L1, or murine PD-L1 ("muPDL1"). Human or cynomolgus monkey PD-L1 was expressed on HEK cells; murine PD-L1 was expressed on A20 cells. Binding is expressed as EC 50 EC values ​​can be estimated by titrating different concentrations of mAb on cells exogenously expressing the antigen of interest. mAb binding can be detected and quantified using fluorescently labeled secondary antibodies. The data shown in Table 2 were fitted to a 1:1 binding model using built-in functions in GraphPad to generate EC values. 50 value.

[0257] Table 2. Cellular binding of mAb1 to mAb23 to human, cynomolgus monkey, and murine PD-L1 expressed on cells

[0258]

[0259]

[0260] *NB = no binding; ND = not determined; human and cynomolgus monkey PDL1 are expressed in HEK cells; murine PDL1 is expressed in A20 cells

[0261] The present disclosure also provides an antibody, or antigen-binding portion thereof, that specifically binds PD-L1, wherein the antibody, or antigen-binding portion thereof, comprises: a heavy chain variable region comprising an amino acid sequence that is at least 90% identical (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical) to any one of SEQ ID NO: 35, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, or 58, and ... NO:59, 60, 61, 62 or 63 has an amino acid sequence that is at least 90% identical (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical) to any one of NO:59, 60, 61, 62 or 63. In some embodiments, the heavy chain variable region comprises an amino acid sequence that differs from any one of SEQ ID NO: 35, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, or 58 by 15 amino acids or less, 14 amino acids or less, 13 amino acids or less, 12 amino acids or less, 11 amino acids or less, 10 amino acids or less, 9 amino acids or less, 8 amino acids or less, 7 amino acids or less, 6 amino acids or less, 5 amino acids or less, 4 amino acids or less, 3 amino acids or less, 2 amino acids or less, or 1 amino acid. In some embodiments, the light chain variable region comprises an amino acid sequence that differs from any one of SEQ ID NOs: 59, 60, 61, 62, or 63 by 15 amino acids or less, 14 amino acids or less, 13 amino acids or less, 12 amino acids or less, 11 amino acids or less, 10 amino acids or less, 9 amino acids or less, 8 amino acids or less, 7 amino acids or less, 6 amino acids or less, 5 amino acids or less, 4 amino acids or less, 3 amino acids or less, 2 amino acids or less, or 1 amino acid. Tables 3 and 4 provide the sequences of heavy chain variable sequences SEQ ID Nos: 35 and 40-58, and light chain variable sequences SEQ ID Nos: 59-63, respectively.

[0262] Table 3. Heavy chain anti-PD-L1 variable sequences

[0263]

[0264]

[0265]

[0266]

[0267] Table 4. Light chain variable sequences

[0268]

[0269] In some embodiments, the present disclosure also provides an antibody, or antigen binding portion thereof, that specifically binds to PD-L1, wherein the antibody, or antigen binding portion thereof, comprises the heavy chain CDRs of any one of the heavy chain variable regions of SEQ ID NO: 35, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, or 58, and the light chain CDRs of any one of the light chain variable regions of SEQ ID NO: 59, 60, 61, 62, or 63.

[0270] In some embodiments, the present disclosure also provides an antibody, or antigen binding portion thereof, that specifically binds PD-L1, wherein the antibody, or antigen binding portion thereof, comprises the heavy chain CDRs of any one of the heavy chain variable regions of SEQ ID NO: 35, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, or 58, and the light chain CDRs of any one of the light chain variable regions of SEQ ID NO: 59, 60, 61, 62, or 63, wherein the heavy and light chain CDR residues are numbered according to Kabat.

[0271] In some embodiments, the present disclosure also provides an antibody, or antigen binding portion thereof, that specifically binds PD-L1, wherein the antibody, or antigen binding portion thereof, comprises the heavy chain CDRs of any one of the heavy chain variable regions of SEQ ID NO: 35, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, or 58, and the light chain CDRs of any one of the light chain variable regions of SEQ ID NO: 59, 60, 61, 62, or 63, wherein the heavy and light chain CDR residues are numbered according to Chothia.

[0272] In some embodiments, the present disclosure also provides an antibody, or antigen binding portion thereof, that specifically binds PD-L1, wherein the antibody, or antigen binding portion thereof, comprises the heavy chain CDRs of any one of the heavy chain variable regions of SEQ ID NO: 35, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, or 58, and the light chain CDRs of any one of the light chain variable regions of SEQ ID NO: 59, 60, 61, 62, or 63, wherein the heavy and light chain CDR residues are numbered according to MacCallum.

[0273] In some embodiments, the present disclosure also provides an antibody, or antigen binding portion thereof, that specifically binds PD-L1, wherein the antibody, or antigen binding portion thereof, comprises the heavy chain CDRs of any one of the heavy chain variable regions of SEQ ID NO: 35, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, or 58, and the light chain CDRs of any one of the light chain variable regions of SEQ ID NO: 59, 60, 61, 62, or 63, wherein the heavy and light chain CDR residues are numbered according to AbM.

[0274] In some embodiments, the present disclosure also provides an antibody, or antigen binding portion thereof, that specifically binds PD-L1, wherein the antibody, or antigen binding portion thereof, comprises the heavy chain CDRs of any one of the heavy chain variable regions of SEQ ID NO: 35, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, or 58, and the light chain CDRs of any one of the light chain variable regions of SEQ ID NO: 59, 60, 61, 62, or 63, wherein the heavy and light chain CDR residues are numbered according to IMGT.

[0275] B. PD-1 antagonists

[0276] In some aspects and embodiments, the present disclosure provides anti-PD-1 antagonists. In some embodiments, the anti-PD-1 antagonist is any of the anti-PD-1 antibodies or antigen-binding molecules disclosed herein. In some embodiments, the anti-PD-1 antibody or antigen-binding molecule is not part of a multispecific antigen-binding construct, that is, the anti-PD-1 antibody or antigen-binding molecule is not part of a protein construct that binds to multiple epitopes. In some embodiments, the anti-PD-1 antibody or antigen-binding portion can be combined with different antibodies or antigen-binding portions to form a multispecific antigen-binding construct. In some embodiments, the multispecific antigen-binding construct can bind to an epitope on PD-1 and an epitope on another protein. In some embodiments, the epitope on the other protein is on PD-L1.

[0277] In some embodiments, any of the multispecific antigen-binding constructs disclosed herein comprises a PD-1 antagonist. In some embodiments, the PD-1 antagonist is an "inhibitory receptor." As used herein, "inhibitory receptor" generally refers to an immune checkpoint molecule that, when bound by its cognate ligand, causes suppression or inhibition of an immune response, such as those known to enhance tumor escape. However, in some examples as used herein, "inhibitory receptor" specifically refers to PD-1.

[0278] PD-1 is an immune checkpoint inhibitory receptor that contains an "immunoreceptor tyrosine-based inhibitory motif" or "ITIM" comprising a conserved sequence of amino acids (S / I / V / L)xYxx(I / V / L), where x is any amino acid. Methods for analyzing whether PD-1 activity has been inhibited are known in the art and can be readily designed by one skilled in the art. Such analyses include, for example, testing the effects of any downstream signaling pathway of PD-1 in vitro or in vivo. Following interaction of PD-1 with its ligand, the ITIM motif is phosphorylated by enzymes such as the Src kinase family, thereby allowing it to recruit other enzymes, such as the phosphotyrosine phosphatases SHP-1 and SHP-2, or the inositol-phosphatase known as SHIP. These phosphatases have been shown to reduce the activation of molecules involved in cell signaling. See, for example, Barrow and Trowsdale (2006) Eur J Immunol. 36(7):1646-53. Therefore, the phosphorylation state of the ITIM motif within PD-1 can be assessed using methods known in the art. Similarly, the presence of downstream factors (such as phosphotyrosine phosphatases) can also be examined. In addition, various cell-based assays and kits are known in the art for detecting the presence of downstream factors that are representative of the activity state of PD-1 (e.g., nuclear factor of activated T-cells (NFAT) as a measure of PD-1 inhibition). In other examples, simple binding assays can be used to determine whether the constructs of the present disclosure can block the binding of PD-1 to its ligand, as discussed above.

[0279] Thus, in some aspects, provided herein are antibodies, or antigen-binding portions thereof, that specifically bind to PD-1. In some aspects, an antibody, or antigen-binding portion thereof, that specifically binds to PD-1 comprises (a) a heavy chain variable region comprising (i) CDRH1 comprising SEQ ID NO: 70 (FTFX1X2YAX3X4, wherein X1 = S, R, G, or N; X2 = D, S, N, A, R, or G; X3 = M or L; X4 = S, L, or N); (ii) CDRH2 comprising SEQ ID NO: 71 (SAISNSGTYTYYA); and (iii) CDRH3 comprising SEQ ID NO: 72 (ARGLDFIVGX5TGNDY, wherein X5 = A, Y, or R); and (b) a light chain variable region comprising: (i) CDRL1 comprising SEQ ID NO: 9 (RASQSISSYLN); (ii) CDRL2 comprising SEQ ID NO: 5 (AASSLQS); and (iii) CDRL3 comprising SEQ ID NO: 10 (QQSYSTPLT).

[0280] In some embodiments of these aspects and all such aspects described herein, CDRH1 comprises SEQ ID NO: 73 (FTFSDYAMS), CDRH2 comprises SEQ ID NO: 71 (SAISNSGTYTYYA), and CDRH3 comprises SEQ ID NO: 74 (ARGLDFIVGATGNDY). In some embodiments of these aspects and all such aspects described herein, CDRH1 comprises SEQ ID NO: 73 (FTFSDYAMS); CDRH2 comprises SEQ ID NO: 71 (SAISNSGTYTYYA); CDRH3 comprises SEQ ID NO: 74 (ARGLDFIVGATGNDY); CDRL1 comprises SEQ ID NO: 9 (RASQSISSYLN); CDRL2 comprises SEQ ID NO: 5 (AASSLQS); and CDRL3 comprises SEQ ID NO: 10 (QQSYSTPLT). A representative antibody having such heavy and light chain variable CDR regions is mAb 25.

[0281] In some embodiments of these aspects and all such aspects described herein, CDRH1 comprises SEQ ID NO: 73 (FTFSDYAMS), CDRH2 comprises SEQ ID NO: 71 (SAISNSGTYTYYA), and CDRH3 comprises SEQ ID NO: 75 (ARGLDFIVGYTGNDY). In some embodiments of these aspects and all such aspects described herein, CDRH1 comprises SEQ ID NO: 73 (FTFSDYAMS); CDRH2 comprises SEQ ID NO: 71 (SAISNSGTYTYYA); CDRH3 comprises SEQ ID NO: 75 (ARGLDFIVGYTGNDY); CDRL1 comprises SEQ ID NO: 9 (RASQSISSYLN); CDRL2 comprises SEQ ID NO: 5 (AASSLQS); and CDRL3 comprises SEQ ID NO: 10 (QQSYSTPLT). A representative antibody having such heavy and light chain variable CDR regions is mAb 26.

[0282] In some embodiments of these aspects and all such aspects described herein, CDRH1 comprises SEQ ID NO: 76 (FTFSSYAMS), CDRH2 comprises SEQ ID NO: 71 (SAISNSGTYTYYA), and CDRH3 comprises SEQ ID NO: 75 (ARGLDFIVGYTGNDY). In some embodiments of these aspects and all such aspects described herein, CDRH1 comprises SEQ ID NO: 76 (FTFSSYAMS); CDRH2 comprises SEQ ID NO: 71 (SAISNSGTYTYYA); CDRH3 comprises SEQ ID NO: 75 (ARGLDFIVGYTGNDY); CDRL1 comprises SEQ ID NO: 9 (RASQSISSYLN); CDRL2 comprises SEQ ID NO: 5 (AASSLQS); and CDRL3 comprises SEQ ID NO: 10 (QQSYSTPLT). A representative antibody having such heavy and light chain variable CDR regions is mAb27.

[0283] In some embodiments of these aspects and all such aspects described herein, CDRH1 comprises SEQ ID NO: 77 (FTFSSYAML), CDRH2 comprises SEQ ID NO: 71 (SAISNSGTYTYYA), and CDRH3 comprises SEQ ID NO: 75 (ARGLDFIVGYTGNDY). In some embodiments of these aspects and all such aspects described herein, CDRH1 comprises SEQ ID NO: 77 (FTFSSYAML); CDRH2 comprises SEQ ID NO: 71 (SAISNSGTYTYYA); CDRH3 comprises SEQ ID NO: 75 (ARGLDFIVGYTGNDY); CDRL1 comprises SEQ ID NO: 9 (RASQSISSYLN); CDRL2 comprises SEQ ID NO: 5 (AASSLQS); and CDRL3 comprises SEQ ID NO: 10 (QQSYSTPLT). A representative antibody having such heavy and light chain variable CDR regions is mAb 28.

[0284] In some embodiments of these aspects and all such aspects described herein, CDRH1 comprises SEQ ID NO: 78 (FTFSNYALS), CDRH2 comprises SEQ ID NO: 71 (SAISNSGTYTYYA), and CDRH3 comprises SEQ ID NO: 75 (ARGLDFIVGYTGNDY). In some embodiments of these aspects and all such aspects described herein, CDRH1 comprises SEQ ID NO: 78 (FTFSNYALS); CDRH2 comprises SEQ ID NO: 71 (SAISNSGTYTYYA); CDRH3 comprises SEQ ID NO: 75 (ARGLDFIVGYTGNDY); CDRL1 comprises SEQ ID NO: 9 (RASQSISSYLN); CDRL2 comprises SEQ ID NO: 5 (AASSLQS); and CDRL3 comprises SEQ ID NO: 10 (QQSYSTPLT). A representative antibody having such heavy and light chain variable CDR regions is mAb 29.

[0285] In some embodiments of these aspects and all such aspects described herein, CDRH1 comprises SEQ ID NO: 79 (FTFSAYAMN), CDRH2 comprises SEQ ID NO: 71 (SAISNSGTYTYYA), and CDRH3 comprises SEQ ID NO: 75 (ARGLDFIVGYTGNDY). In some embodiments of these aspects and all such aspects described herein, CDRH1 comprises SEQ ID NO: 79 (FTFSAYAMN); CDRH2 comprises SEQ ID NO: 71 (SAISNSGTYTYYA); CDRH3 comprises SEQ ID NO: 75 (ARGLDFIVGYTGNDY); CDRL1 comprises SEQ ID NO: 9 (RASQSISSYLN); CDRL2 comprises SEQ ID NO: 5 (AASSLQS); and CDRL3 comprises SEQ ID NO: 10 (QQSYSTPLT). A representative antibody having such heavy and light chain variable CDR regions is mAb30.

[0286] In some embodiments of these aspects and all such aspects described herein, CDRH1 comprises SEQ ID NO: 80 (FTFRSYAMS), CDRH2 comprises SEQ ID NO: 71 (SAISNSGTYTYYA), and CDRH3 comprises SEQ ID NO: 75 (ARGLDFIVGYTGNDY). In some embodiments of these aspects and all such aspects described herein, CDRH1 comprises SEQ ID NO: 80 (FTFRSYAMS); CDRH2 comprises SEQ ID NO: 71 (SAISNSGTYTYYA); CDRH3 comprises SEQ ID NO: 75 (ARGLDFIVGYTGNDY); CDRL1 comprises SEQ ID NO: 9 (RASQSISSYLN); CDRL2 comprises SEQ ID NO: 5 (AASSLQS); and CDRL3 comprises SEQ ID NO: 10 (QQSYSTPLT). A representative antibody having such heavy and light chain variable CDR regions is mAb31.

[0287] In some embodiments of these aspects and all such aspects described herein, CDRH1 comprises SEQ ID NO: 81 (FTFGRYAMS), CDRH2 comprises SEQ ID NO: 71 (SAISNSGTYTYYA), and CDRH3 comprises SEQ ID NO: 75 (ARGLDFIVGYTGNDY). In some embodiments of these aspects and all such aspects described herein, CDRH1 comprises SEQ ID NO: 81 (FTFGRYAMS); CDRH2 comprises SEQ ID NO: 71 (SAISNSGTYTYYA); CDRH3 comprises SEQ ID NO: 75 (ARGLDFIVGYTGNDY); CDRL1 comprises SEQ ID NO: 9 (RASQSISSYLN); CDRL2 comprises SEQ ID NO: 5 (AASSLQS); and CDRL3 comprises SEQ ID NO: 10 (QQSYSTPLT). A representative antibody having such heavy and light chain variable CDR regions is mAb32.

[0288] In some embodiments of these aspects and all such aspects described herein, CDRH1 comprises SEQ ID NO: 82 (FTFNSYAMS), CDRH2 comprises SEQ ID NO: 71 (SAISNSGTYTYYA), and CDRH3 comprises SEQ ID NO: 75 (ARGLDFIVGYTGNDY). In some embodiments of these aspects and all such aspects described herein, CDRH1 comprises SEQ ID NO: 82 (FTFNSYAMS); CDRH2 comprises SEQ ID NO: 71 (SAISNSGTYTYYA); CDRH3 comprises SEQ ID NO: 75 (ARGLDFIVGYTGNDY); CDRL1 comprises SEQ ID NO: 9 (RASQSISSYLN); CDRL2 comprises SEQ ID NO: 5 (AASSLQS); and CDRL3 comprises SEQ ID NO: 10 (QQSYSTPLT). A representative antibody having such heavy and light chain variable CDR regions is mAb33.

[0289] In some embodiments of these aspects and all such aspects described herein, CDRH1 comprises SEQ ID NO: 83 (FTFSNYAMS), CDRH2 comprises SEQ ID NO: 71 (SAISNSGTYTYYA), and CDRH3 comprises SEQ ID NO: 74 (ARGLDFIVGATGNDY). In some embodiments of these aspects and all such aspects described herein, CDRH1 comprises SEQ ID NO: 83 (FTFSNYAMS); CDRH2 comprises SEQ ID NO: 71 (SAISNSGTYTYYA); CDRH3 comprises SEQ ID NO: 74 (ARGLDFIVGATGNDY); CDRL1 comprises SEQ ID NO: 9 (RASQSISSYLN); CDRL2 comprises SEQ ID NO: 5 (AASSLQS); and CDRL3 comprises SEQ ID NO: 10 (QQSYSTPLT). A representative antibody having such heavy and light chain variable CDR regions is mAb34.

[0290] In some embodiments of these aspects and all such aspects described herein, CDRH1 comprises SEQ ID NO: 84 (FTFSGYAMS), CDRH2 comprises SEQ ID NO: 71 (SAISNSGTYTYYA), and CDRH3 comprises SEQ ID NO: 85 (ARGLDFIVGRTGNDY). In some embodiments of these aspects and all such aspects described herein, CDRH1 comprises SEQ ID NO: 84 (FTFSGYAMS); CDRH2 comprises SEQ ID NO: 71 (SAISNSGTYTYYA); CDRH3 comprises SEQ ID NO: 85 (ARGLDFIVGRTGNDY); CDRL1 comprises SEQ ID NO: 9 (RASQSISSYLN); CDRL2 comprises SEQ ID NO: 5 (AASSLQS); and CDRL3 comprises SEQ ID NO: 10 (QQSYSTPLT). A representative antibody having such heavy and light chain variable CDR regions is mAb35.

[0291] In some embodiments of these aspects and all such aspects described herein, CDRH1 comprises SEQ ID NO: 86 (FTFSSYAMN), CDRH2 comprises SEQ ID NO: 71 (SAISNSGTYTYYA), and CDRH3 comprises SEQ ID NO: 85 (ARGLDFIVGRTGNDY). In some embodiments of these aspects and all such aspects described herein, CDRH1 comprises SEQ ID NO: 86 (FTFSSYAMN); CDRH2 comprises SEQ ID NO: 71 (SAISNSGTYTYYA); CDRH3 comprises SEQ ID NO: 85 (ARGLDFIVGRTGNDY); CDRL1 comprises SEQ ID NO: 9 (RASQSISSYLN); CDRL2 comprises SEQ ID NO: 5 (AASSLQS); and CDRL3 comprises SEQ ID NO: 10 (QQSYSTPLT). A representative antibody having such heavy and light chain variable CDR regions is mAb36.

[0292] In some embodiments of these aspects and all such aspects described herein, CDRH1 comprises SEQ ID NO: 80 (FTFRSYAMS), CDRH2 comprises SEQ ID NO: 71 (SAISNSGTYTYYA), and CDRH3 comprises SEQ ID NO: 85 (ARGLDFIVGRTGNDY). In some embodiments of these aspects and all such aspects described herein, CDRH1 comprises SEQ ID NO: 80 (FTFRSYAMS); CDRH2 comprises SEQ ID NO: 71 (SAISNSGTYTYYA); CDRH3 comprises SEQ ID NO: 85 (ARGLDFIVGRTGNDY); CDRL1 comprises SEQ ID NO: 9 (RASQSISSYLN); CDRL2 comprises SEQ ID NO: 5 (AASSLQS); and CDRL3 comprises SEQ ID NO: 10 (QQSYSTPLT). A representative antibody having such heavy and light chain variable CDR regions is mAb37.

[0293] In each case where a particular sequence is described, embodiments are also provided comprising a sequence having at least 85% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical) identity to the sequence (e.g., SEQ ID NOs: 5, 9, 10, or 71-86).

[0294] In some aspects, the present disclosure also provides antibodies, or antigen-binding portions thereof, that specifically bind to PD-1, wherein the antibodies, or antigen-binding portions thereof, comprise a heavy chain variable region comprising an amino acid sequence that is at least 85% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical) to any one of SEQ ID NOs: 87-99, and a light chain variable region comprising an amino acid sequence that is at least 85% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical) to SEQ ID NO: 59. In some embodiments, the heavy chain variable region comprises an amino acid sequence that is at least 85% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical) to SEQ ID NO: 90, and the light chain variable region comprises an amino acid sequence that is at least 85% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical) to SEQ ID NO: 59. Table 7 provides the sequences of the heavy chain variable sequences SEQ ID Nos: 87-99, and Table 4 provides the light chain variable sequence SEQ ID NO: 59. In some embodiments, the heavy chain variable region comprises an amino acid sequence that differs from any one of SEQ ID NOs: 87-99 by 15 amino acids or less, 14 amino acids or less, 13 amino acids or less, 12 amino acids or less, 11 amino acids or less, 10 amino acids or less, 9 amino acids or less, 8 amino acids or less, 7 amino acids or less, 6 amino acids or less, 5 amino acids or less, 4 amino acids or less, 3 amino acids or less, 2 amino acids or less, or 1 amino acid. In some embodiments, the light chain variable region comprises an amino acid sequence that differs from SEQ ID NO: 591 by 5 amino acids or less, 14 amino acids or less, 13 amino acids or less, 12 amino acids or less, 11 amino acids or less, 10 amino acids or less, 9 amino acids or less, 8 amino acids or less, 7 amino acids or less, 6 amino acids or less, 5 amino acids or less, 4 amino acids or less, 3 amino acids or less, 2 amino acids or less, or 1 amino acid.

[0295] Antibodies mAb26 to mAb37 are affinity matured antibodies derived from the parent antibody mAb25, as described in the Examples. Affinity matured antibodies, or antigen binding portions thereof, are antibodies or antigen binding fragments having one or more alterations (e.g., in one or more CDRs or FRs) that result in an increase in the affinity of the antibody for its antigen compared to the parent antibody lacking the alterations. In some embodiments, the affinity matured antibodies have nanomolar or picomolar affinities for PD-1. In some embodiments, the PD-1 antibody, or antigen binding portion thereof, has an affinity of at least 1x10 -7 M, at least 1x10 -8 M, at least 1x10 -9 M, at least 1x10 -10 M, at least 1x10 -11 M, at least 1x10 -12 M, or at least 1x10 -13 M's K D .

[0296] Tables 5 and 6 provide cellular binding data for mAb25, mAb26, mAb27, mAb28, mAb29, mAb30, mAb31, mAb32, mAb33, mAb34, mAb35, mAb36, and mAb37 (i.e., affinity matured variants of mAb25) to human PD-1 ("huPD-1"), cynomolgus monkey PD-1 ("cyPD-1"), or murine PD-1 ("muPD-1"). Human, cynomolgus monkey, or murine PD-1 was expressed on CHO cells. Binding is expressed as EC 50 EC values ​​can be estimated by titrating different concentrations of mAb on cells exogenously expressing the antigen of interest. mAb binding can be detected and quantified using fluorescently labeled secondary antibodies. The data shown in Table 5 were fitted to a 1:1 binding model using built-in functions in GraphPad, which yielded EC values ​​of 50 value.

[0297] Table 5: Binding affinity of mAb25 to mAb37 to human PD-1

[0298]

[0299]

[0300] Table 6. Cellular binding of mAb25 to mAb37 to human, cynomolgus monkey, and murine PD-1 expressed on cells

[0301]

[0302] *NB = no binding; PF = poor fit; human, cynomolgus monkey, and murine PD-1 expressed on CHO cells

[0303] Table 7. Heavy chain anti-PD-1 variable sequences

[0304]

[0305]

[0306]

[0307] In some embodiments, the present disclosure also provides an antibody, or antigen-binding portion thereof, that specifically binds to PD-L1, wherein the antibody, or antigen-binding portion thereof, comprises the heavy chain CDRs of any one of the heavy chain variable regions of SEQ ID NO: 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99, and the light chain CDRs of the light chain variable region of SEQ ID NO: 59.

[0308] In some embodiments, the present disclosure also provides an antibody, or antigen-binding portion thereof, that specifically binds to PD-L1, wherein the antibody, or antigen-binding portion thereof, comprises the heavy chain CDRs of any one of the heavy chain variable regions of SEQ ID NO: 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99, and the light chain CDRs of the light chain variable region of SEQ ID NO: 59, wherein the heavy and light chain CDR residues are numbered according to Kabat.

[0309] In some embodiments, the present disclosure also provides an antibody, or antigen-binding portion thereof, that specifically binds to PD-L1, wherein the antibody, or antigen-binding portion thereof, comprises the heavy chain CDRs of any one of the heavy chain variable regions of SEQ ID NO: 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99, and the light chain CDRs of the light chain variable region of SEQ ID NO: 59, wherein the heavy and light chain CDR residues are numbered according to Chothia.

[0310] In some embodiments, the present disclosure also provides an antibody, or antigen-binding portion thereof, that specifically binds PD-L1, wherein the antibody, or antigen-binding portion thereof, comprises the heavy chain CDRs of any one of the heavy chain variable regions of SEQ ID NO: 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99, and the light chain CDRs of the light chain variable region of SEQ ID NO: 59, wherein the heavy and light chain CDR residues are numbered according to MacCallum.

[0311] In some embodiments, the present disclosure also provides an antibody, or antigen-binding portion thereof, that specifically binds to PD-L1, wherein the antibody, or antigen-binding portion thereof, comprises the heavy chain CDRs of any one of the heavy chain variable regions of SEQ ID NO: 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99, and the light chain CDRs of the light chain variable region of SEQ ID NO: 59, wherein the heavy and light chain CDR residues are numbered according to AbM.

[0312] In some embodiments, the present disclosure also provides an antibody, or antigen-binding portion thereof, that specifically binds to PD-L1, wherein the antibody, or antigen-binding portion thereof, comprises the heavy chain CDRs of any one of the heavy chain variable regions of SEQ ID NO: 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99, and the light chain CDRs of the light chain variable region of SEQ ID NO: 59, wherein the heavy and light chain CDR residues are numbered according to IMGT.

[0313] C. Multispecific Antigen-Binding Constructs

[0314] In some aspects, the present disclosure provides compositions and methods for enhancing the immune response to tumor cells by inhibiting the interaction between PD-L1 and PD-1 (e.g., the interaction between PD-L1 expressed on tumor cells and PD-1 expressed on T cells). Antibodies or antigen-binding portions thereof that specifically or selectively bind to PD-L1 or PD-1 are provided. As used herein, the terms "specifically bind to," "specifically for," "selectively bind to," and "selectively for" PD-L1 or PD-1, or an epitope on PD-L1 or PD-1, mean that the binding can be measurably different from non-specific or non-selective interactions. In some embodiments, the antibody or antigen-binding portion thereof specifically binds to human PD-L1 or PD-1 and / or mouse PD-L1 or PD-1. Specific binding can be measured, for example, by comparing the binding of the assay molecule to the binding of a control molecule. Specific binding can also be measured by competition with a control molecule similar to the target, such as an excess of unlabeled target. In this case, specific binding is indicated if the binding of the labeled target to the probe is competitively inhibited by an excess of unlabeled target.

[0315] In some embodiments, any of the multispecific antigen-binding constructs disclosed herein binds to at least two different receptors or epitopes (e.g., PD-1 and PD-L1), wherein the two different receptors or epitopes bound by the multispecific antigen-binding construct are expressed on the surface of the same cell. For example, in some embodiments, the multispecific antigen-binding construct simultaneously binds to PD-1 and PD-L1, wherein PD-1 and PD-L1 are expressed on the surface of the same cell. In some embodiments, any of the multispecific antigen-binding constructs disclosed herein binds to at least two different receptors or epitopes (e.g., PD-1 and PD-L1), wherein the two different receptors or epitopes bound by the multispecific antigen-binding construct are expressed on the surface of two different cells. For example, in some embodiments, the multispecific antigen-binding construct simultaneously binds to PD-1 expressed on the surface of a first cell and PD-L1 expressed on the surface of a second cell.

[0316] In some embodiments, the multispecific antigen-binding construct binds to human PD-1. In some embodiments, the multispecific antigen-binding construct binds to murine PD-1. In some embodiments, the multispecific antigen-binding construct binds to cynomolgus monkey PD-1. In some embodiments, the multispecific antigen-binding construct binds to human, murine, and cynomolgus monkey PD-1 with similar affinities.

[0317] In some aspects and embodiments, the present disclosure provides a multispecific antigen-binding construct comprising at least two antigen-binding units, wherein the first antigen-binding unit binds to PD-1 and the second antigen-binding unit binds to a PD-1 ligand. In some embodiments, the first antigen-binding unit binds to PD-1 expressed by an immune cell. In some embodiments, the second antigen-binding unit binds to PD-1 expressed by a second cell. In some embodiments, the multispecific antigen-binding construct blocks the interaction between PD-1 and a PD-1 ligand (such as PD-L1 or PD-L2). In some embodiments, the multispecific antigen-binding construct blocks the interaction between PD-1 and a PD-1 ligand (such as PD-L1 or PD-L2). In some embodiments, the multispecific antigen-binding construct comprises at least two antigen-binding units that bind to PD-1. In some embodiments, the multispecific antigen-binding construct comprises two antigen-binding units that bind to PD-1. In some embodiments, the multispecific antigen-binding construct comprises at least two antigen-binding units that bind to a PD-1 ligand (such as PD-L1 or PD-L2). In some embodiments, the multispecific antigen-binding construct comprises two antigen-binding units that bind to a PD-1 ligand (such as PD-L1 or PD-L2). In some embodiments, the multispecific antigen-binding construct comprises at least four antigen-binding units, two of which bind to PD-1 and two bind to a PD-1 ligand (such as PD-L1 or PD-L2). In some embodiments, the multispecific antigen-binding construct comprises four antigen-binding units, two of which bind to PD-1 and two bind to a PD-1 ligand (such as PD-L1 or PD-L2). In some embodiments, each antigen-binding unit can independently bind to its cognate antigen, i.e., PD-1 or a PD-1 ligand (such as PD-L1 or PD-L2). In some embodiments, the multispecific antigen-binding construct promotes the loss of PD-1 expression from cells. In some embodiments, the loss of PD-1 expression is due to PD-1 shedding. In some embodiments, the multispecific antigen-binding construct blocks the interaction between PD-1 and a PD-1 ligand (such as PD-L1 or PD-L2). In some embodiments, the multispecific antigen-binding construct comprises a common light chain. For example, at least two antigen-binding units comprise a common light chain.

[0318] In some embodiments, the first antigen binding unit binds to PD-1 and comprises:

[0319] (a) a heavy chain variable region comprising (i) a CDRH1 comprising SEQ ID NO: 70 (FTFX1X2YAX3X4, wherein X1 = S, R, G, or N; X2 = D, S, N, A, R, or G; X3 = M or L; X4 = S, L, or N); (ii) a CDRH2 comprising SEQ ID NO: 71 (SAISNSGTYTYYA); and (iii) a CDRH3 comprising SEQ ID NO: 72 (ARGLDFIVGX5TGNDY, wherein X5 = A, Y, or R); and

[0320] (b) a light chain variable region comprising: (i) CDRL1 comprising SEQ ID NO: 9 (RASQSISSYLN); (ii) CDRL2 comprising SEQ ID NO: 5 (AASSLQS); and (iii) CDRL3 comprising SEQ ID NO: 10 (QQSYSTPLT).

[0321] In some such embodiments, the first antigen binding unit binds PD-1 and comprises:

[0322] (a) CDRH1 comprising SEQ ID NO:73 (FTFSDYAMS), CDRH2 comprising SEQ ID NO:71 (SAISNSGTYTYYA), and CDRH3 comprising SEQ ID NO:74 (ARGLDFIVGATGNDY);

[0323] (b) CDRH1 comprising SEQ ID NO:73 (FTFSDYAMS), CDRH2 comprising SEQ ID NO:71 (SAISNSGTYTYYA), and CDRH3 comprising SEQ ID NO:75 (ARGLDFIVGYTGNDY);

[0324] (c) CDRH1 comprising SEQ ID NO:76 (FTFSSYAMS), CDRH2 comprising SEQ ID NO:71 (SAISNSGTYTYYA), and CDRH3 comprising SEQ ID NO:75 (ARGLDFIVGYTGNDY);

[0325] (d) CDRH1 comprising SEQ ID NO:77 (FTFSSYAML), CDRH2 comprising SEQ ID NO:71 (SAISNSGTYTYYA), and CDRH3 comprising SEQ ID NO:75 (ARGLDFIVGYTGNDY);

[0326] (e) CDRH1 comprising SEQ ID NO:78 (FTFSNYALS), CDRH2 comprising SEQ ID NO:71 (SAISNSGTYTYYA), and CDRH3 comprising SEQ ID NO:75 (ARGLDFIVGYTGNDY);

[0327] (f) CDRH1 comprising SEQ ID NO:79 (FTFSAYAMN), CDRH2 comprising SEQ ID NO:71 (SAISNSGTYTYYA), and CDRH3 comprising SEQ ID NO:75 (ARGLDFIVGYTGNDY);

[0328] (g) CDRH1 comprising SEQ ID NO:80 (FTFRSYAMS), CDRH2 comprising SEQ ID NO:71 (SAISNSGTYTYYA), and CDRH3 comprising SEQ ID NO:75 (ARGLDFIVGYTGNDY);

[0329] (h) CDRH1 comprising SEQ ID NO:81 (FTFGRYAMS), CDRH2 comprising SEQ ID NO:71 (SAISNSGTYTYYA), and CDRH3 comprising SEQ ID NO:75 (ARGLDFIVGYTGNDY);

[0330] (i) a CDRH1 comprising SEQ ID NO: 82 (FTFNSYAMS), a CDRH2 comprising SEQ ID NO: 71 (SAISNSGTYTYYA), and a CDRH3 comprising SEQ ID NO: 75 (ARGLDFIVGYTGNDY);

[0331] (j) CDRH1 comprising SEQ ID NO:83 (FTFSNYAMS), CDRH2 comprising SEQ ID NO:71 (SAISNSGTYTYYA), and CDRH3 comprising SEQ ID NO:74 (ARGLDFIVGATGNDY);

[0332] (k) CDRH1 comprising SEQ ID NO:84 (FTFSGYAMS), CDRH2 comprising SEQ ID NO:71 (SAISNSGTYTYYA), and CDRH3 comprising SEQ ID NO:85 (ARGLDFIVGRTGNDY);

[0333] (1) a CDRH1 comprising SEQ ID NO: 86 (FTFSSYAMN), a CDRH2 comprising SEQ ID NO: 71 (SAISNSGTYTYYA), and a CDRH3 comprising SEQ ID NO: 85 (ARGLDFIVGRTGNDY); or

[0334] (m) CDRH1 comprising SEQ ID NO:80 (FTFRSYAMS), CDRH3 comprising SEQ ID NO:71 (SAISNSGTYTYYA), and CDRH3 comprising SEQ ID NO:85 (ARGLDFIVGRTGNDY).

[0335] In some embodiments, the first antigen binding unit binds to PD-1 and comprises:

[0336] (a) a heavy chain variable region comprising an amino acid sequence at least 90% identical to SEQ ID NO: 87;

[0337] (b) a heavy chain variable region comprising an amino acid sequence at least 90% identical to SEQ ID NO: 88;

[0338] (c) a heavy chain variable region comprising an amino acid sequence at least 90% identical to SEQ ID NO: 89;

[0339] (d) a heavy chain variable region comprising an amino acid sequence at least 90% identical to SEQ ID NO: 90;

[0340] (e) a heavy chain variable region comprising an amino acid sequence at least 90% identical to SEQ ID NO: 91;

[0341] (f) a heavy chain variable region comprising an amino acid sequence at least 90% identical to SEQ ID NO: 92;

[0342] (g) a heavy chain variable region comprising an amino acid sequence at least 90% identical to SEQ ID NO: 93;

[0343] (h) a heavy chain variable region comprising an amino acid sequence at least 90% identical to SEQ ID NO: 94;

[0344] (i) a heavy chain variable region comprising an amino acid sequence at least 90% identical to SEQ ID NO: 95;

[0345] (j) a heavy chain variable region comprising an amino acid sequence at least 90% identical to SEQ ID NO: 96;

[0346] (k) a heavy chain variable region comprising an amino acid sequence at least 90% identical to SEQ ID NO: 97;

[0347] (1) a heavy chain variable region comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 98; or

[0348] (m) a heavy chain variable region comprising an amino acid sequence at least 90% identical to SEQ ID NO:99.

[0349] In some embodiments, the first antigen binding unit binds to PD-1 and comprises a light chain variable region comprising an amino acid sequence at least 90% identical to SEQ ID NO:59.

[0350] In some embodiments, the second antigen binding unit binds to PD-L2. In some embodiments, the second antigen binding unit binds to PD-L1. In some embodiments, the second antigen binding unit binds to PD-L1 and comprises:

[0351] a. A heavy chain variable region comprising (i) a CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN); (ii) a CDRH2 comprising SEQ ID NO: 2 (GGIIPX1X2GX3ATYA, wherein X1 is V or I; X2 is F, L or V; and X3 is T or A); and (iii) a CDRH3 comprising SEQ ID NO: 3 (ARLKX1ELKDAFDI, wherein X1 is G, F or N); and

[0352] b. A light chain variable region comprising: (i) CDRL1 comprising SEQ ID NO: 4 (RASQX1ISSYLN, wherein X1 is S, W or Q); (ii) CDRL2 comprising SEQ ID NO: 5 (AASSLQS); and (iii) CDRL3 comprising SEQ ID NO: 6 (X1QSYSTPLT, wherein X1 is Q or F).

[0353] In some such embodiments, the second antigen binding unit binds PD-L1 and comprises:

[0354] (a) CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), CDRH2 comprising SEQ ID NO: 7 (GGIIPILGAATYA), and CDRH3 comprising SEQ ID NO: 8 (ARLKGELKDAFDI);

[0355] (b) a CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), a CDRH2 comprising SEQ ID NO: 7 (GGIIPILGAATYA), a CDRH3 comprising SEQ ID NO: 8 (ARLKGELKDAFDI); a CDRL1 comprising SEQ ID NO: 9 (RASQSISSYLN), a CDRL2 comprising SEQ ID NO: 5 (AASSLQS), and a CDRL3 comprising SEQ ID NO: 10 (QQSYSTPLT);

[0356] (c) a CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), a CDRH2 comprising SEQ ID NO: 11 (GGIIPVFGTATYA), a CDRH3 comprising SEQ ID NO: 8 (ARLKGELKDAFDI); a CDRL1 comprising SEQ ID NO: 9 (RASQSISSYLN), a CDRL2 comprising SEQ ID NO: 5 (AASSLQS), and a CDRL3 comprising SEQ ID NO: 10 (QQSYSTPLT);

[0357] (d) CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), CDRH2 comprising SEQ ID NO: 11 (GGIIPVFGTATYA), and CDRH3 comprising SEQ ID NO: 8 (ARLKGELKDAFDI);

[0358] (e) a CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), a CDRH2 comprising SEQ ID NO: 11 (GGIIPVFGTATYA), a CDRH3 comprising SEQ ID NO: 8 (ARLKGELKDAFDI); a CDRL1 comprising SEQ ID NO: 12 (RASQWISSYLN), a CDRL2 comprising SEQ ID NO: 5 (AASSLQS), and a CDRL3 comprising SEQ ID NO: 10 (QQSYSTPLT);

[0359] (f) CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), CDRH2 comprising SEQ ID NO: 11 (GGIIPVFGTATYA), CDRH3 comprising SEQ ID NO: 8 (ARLKGELKDAFDI); CDRL1 comprising SEQ ID NO: 13 (RASQQISSYLN), CDRL2 comprising SEQ ID NO: 5 (AASSLQS), and CDRL3 comprising SEQ ID NO: 10 (QQSYSTPLT);

[0360] (g) CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), CDRH2 comprising SEQ ID NO: 11 (GGIIPVFGTATYA), CDRH3 comprising SEQ ID NO: 8 (ARLKGELKDAFDI); CDRL1 comprising SEQ ID NO: 9 (RASQSISSYLN), CDRL2 comprising SEQ ID NO: 5 (AASSLQS), and CDRL3 comprising SEQ ID NO: 10 (QQSYSTPLT);

[0361] (h) CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), CDRH2 comprising SEQ ID NO: 15 (GGIIPIFGIANYA), and CDRH3 comprising SEQ ID NO: 8 (ARLKGELKDAFDI);

[0362] (i) a CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), a CDRH2 comprising SEQ ID NO: 15 (GGIIPIFGIANYA), a CDRH3 comprising SEQ ID NO: 8 (ARLKGELKDAFDI); a CDRL1 comprising SEQ ID NO: 9 (RASQSISSYLN), a CDRL2 comprising SEQ ID NO: 5 (AASSLQS), and a CDRL3 comprising SEQ ID NO: 10 (QQSYSTPLT);

[0363] (j) CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), CDRH2 comprising SEQ ID NO: 16 (GGIIPNFGTATYA), and CDRH3 comprising SEQ ID NO: 17 (ARLKGELKGAGDI);

[0364] (k) a CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), a CDRH2 comprising SEQ ID NO: 16 (GGIIPNFGTATYA), a CDRH3 comprising SEQ ID NO: 17 (ARLKGELKGAGDI); a CDRL1 comprising SEQ ID NO: 9 (RASQSISSYLN), a CDRL2 comprising SEQ ID NO: 5 (AASSLQS), and a CDRL3 comprising SEQ ID NO: 10 (QQSYSTPLT);

[0365] (1) CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), CDRH2 comprising SEQ ID NO: 11 (GGIIPVFGTATYA), and CDRH3 comprising SEQ ID NO: 18 (ARLKFELKDAFDI);

[0366] (m) a CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), a CDRH2 comprising SEQ ID NO: 11 (GGIIPVFGTATYA), a CDRH3 comprising SEQ ID NO: 18 (ARLKFELKDAFDI), a CDRL1 comprising SEQ ID NO: 9 (RASQSISSYLN), a CDRL2 comprising SEQ ID NO: 5 (AASSLQS), and a CDRL3 comprising SEQ ID NO: 10 (QQSYSTPLT);

[0367] (n) CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), CDRH2 comprising SEQ ID NO: 11 (GGIIPVFGTATYA), and CDRH3 comprising SEQ ID NO: 19 (ARLKGELKDAFDE);

[0368] (o) a CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), a CDRH2 comprising SEQ ID NO: 11 (GGIIPVFGTATYA), a CDRH3 comprising SEQ ID NO: 19 (ARLKGELKDAFDE), a CDRL1 comprising SEQ ID NO: 9 (RASQSISSYLN), a CDRL2 comprising SEQ ID NO: 5 (AASSLQS), and a CDRL3 comprising SEQ ID NO: 10 (QQSYSTPLT);

[0369] (p) CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), CDRH2 comprising SEQ ID NO: 11 (GGIIPVFGTATYA), and CDRH3 comprising SEQ ID NO: 20 (ARLKNELKDAFDI);

[0370] (q) a CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), a CDRH2 comprising SEQ ID NO: 11 (GGIIPVFGTATYA), a CDRH3 comprising SEQ ID NO: 20 (ARLKNELKDAFDI), a CDRL1 comprising SEQ ID NO: 9 (RASQSISSYLN), a CDRL2 comprising SEQ ID NO: 5 (AASSLQS), and a CDRL3 comprising SEQ ID NO: 10 (QQSYSTPLT);

[0371] (r) a CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), a CDRH2 comprising SEQ ID NO: 21 (GGVIPFLGTANYA), and a CDRH3 comprising SEQ ID NO: 22 (ARLKGILKDALDI);

[0372] (s) a CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), a CDRH2 comprising SEQ ID NO: 21 (GGVIPFLGTANYA), a CDRH3 comprising SEQ ID NO: 22 (ARLKGILKDALDI), a CDRL1 comprising SEQ ID NO: 9 (RASQSISSYLN), a CDRL2 comprising SEQ ID NO: 5 (AASSLQS), and a CDRL3 comprising SEQ ID NO: 10 (QQSYSTPLT);

[0373] (t) CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), CDRH2 comprising SEQ ID NO: 29 (GRIIPIFGTADYA), and CDRH3 comprising SEQ ID NO: 8 (ARLKGELKDAFDI);

[0374] (u) a CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), a CDRH2 comprising SEQ ID NO: 29 (GRIIPIFGTADYA), a CDRH3 comprising SEQ ID NO: 8 (ARLKGELKDAFDI), a CDRL1 comprising SEQ ID NO: 9 (RASQSISSYLN), a CDRL2 comprising SEQ ID NO: 5 (AASSLQS), and a CDRL3 comprising SEQ ID NO: 10 (QQSYSTPLT);

[0375] (v) a CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), a CDRH2 comprising SEQ ID NO: 31 (GGIIPILGTATYA), and a CDRH3 comprising SEQ ID NO: 32 (ARRKGELKDAFDI);

[0376] (w) a CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), a CDRH2 comprising SEQ ID NO: 31 (GGIIPILGTATYA), a CDRH3 comprising SEQ ID NO: 32 (ARRKGELKDAFDI), a CDRL1 comprising SEQ ID NO: 9 (RASQSISSYLN), a CDRL2 comprising SEQ ID NO: 5 (AASSLQS), and a CDRL3 comprising SEQ ID NO: 10 (QQSYSTPLT);

[0377] (x) a CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), a CDRH2 comprising SEQ ID NO: 33 (GGIIPIVATANYA), and a CDRH3 comprising SEQ ID NO: 32 (ARRKGELKDAFDI);

[0378] (y) a CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), a CDRH2 comprising SEQ ID NO: 33 (GGIIPIVATANYA), a CDRH3 comprising SEQ ID NO: 32 (ARRKGELKDAFDI), a CDRL1 comprising SEQ ID NO: 9 (RASQSISSYLN), a CDRL2 comprising SEQ ID NO: 5 (AASSLQS), and a CDRL3 comprising SEQ ID NO: 10 (QQSYSTPLT);

[0379] (z) CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), CDRH2 comprising SEQ ID NO: 34 (GGIIPIFGKATYA), and CDRH3 comprising SEQ ID NO: 32 (ARRKGELKDAFDI);

[0380] (aa) CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), CDRH2 comprising SEQ ID NO: 34 (GGIIPIFGKATYA), CDRH3 comprising SEQ ID NO: 32 (ARRKGELKDAFDI), CDRL1 comprising SEQ ID NO: 9 (RASQSISSYLN), CDRL2 comprising SEQ ID NO: 5 (AASSLQS), and CDRL3 comprising SEQ ID NO: 10 (QQSYSTPLT);

[0381] (bb) CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), CDRH2 comprising SEQ ID NO: 11 (GGIIPVFGTATYA), CDRH3 comprising SEQ ID NO: 8 (ARLKGELKDAFDI); CDRL1 comprising SEQ ID NO: 9 (RASQSISSYLN), CDRL2 comprising SEQ ID NO: 5 (AASSLQS), and CDRL3 comprising SEQ ID NO: 38 (FQSYSTPLT);

[0382] (cc) a CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), a CDRH2 comprising SEQ ID NO: 11 (GGIIPVFGTATYA), a CDRH3 comprising SEQ ID NO: 8 (ARLKGELKDAFDI); a CDRL1 comprising SEQ ID NO: 9 (RASQSISSYLN), a CDRL2 comprising SEQ ID NO: 5 (AASSLQS), and a CDRL3 comprising SEQ ID NO: 39 (QQSYSTILT);

[0383] (dd) CDRH1 comprising SEQ ID NO: 14 (GTFSSYAFS), CDRH2 comprising SEQ ID NO: 11 (GGIIPVFGTATYA), and CDRH3 comprising SEQ ID NO: 8 (ARLKGELKDAFDI); CDRL1 comprising SEQ ID NO: 9 (RASQSISSYLN), CDRL2 comprising SEQ ID NO: 5 (AASSLQS), and CDRL3 comprising SEQ ID NO: 10 (QQSYSTPLT);

[0384] (ee) CDRH1 comprising SEQ ID NO:23 (GTFSSYAIS), CDRH2 comprising SEQ ID NO:24 (GGIIPIVGIANYA), and CDRH3 comprising SEQ ID NO:8 (ARLKGELKDAFDI);

[0385] (ff) CDRH1 comprising SEQ ID NO:23 (GTFSSYAIS), CDRH2 comprising SEQ ID NO:24 (GGIIPIVGIANYA), and CDRH3 comprising SEQ ID NO:8 (ARLKGELKDAFDI); CDRL1 comprising SEQ ID NO:9 (RASQSISSYLN), CDRL2 comprising SEQ ID NO:5 (AASSLQS), and CDRL3 comprising SEQ ID NO:10 (QQSYSTPLT);

[0386] (gg) CDRH1 comprising SEQ ID NO:23 (GTFSSYAIS), CDRH2 comprising SEQ ID NO:11 (GGIIPVFGTATYA), and CDRH3 comprising SEQ ID NO:25 (ARLKGEFKDAFDI);

[0387] (hh) CDRH1 comprising SEQ ID NO:23 (GTFSSYAIS), CDRH2 comprising SEQ ID NO:11 (GGIIPVFGTATYA), and CDRH3 comprising SEQ ID NO:25 (ARLKGEFKDAFDI); CDRL1 comprising SEQ ID NO:9 (RASQSISSYLN), CDRL2 comprising SEQ ID NO:5 (AASSLQS), and CDRL3 comprising SEQ ID NO:10 (QQSYSTPLT);

[0388] (ii) a CDRH1 comprising SEQ ID NO: 23 (GTFSSYAIS), a CDRH2 comprising SEQ ID NO: 26 (GRIIPLFGTAHYA), and a CDRH3 comprising SEQ ID NO: 8 (ARLKGELKDAFDI);

[0389] (jj) CDRH1 comprising SEQ ID NO:23 (GTFSSYAIS), CDRH2 comprising SEQ ID NO:26 (GRIIPLFGTAHYA), and CDRH3 comprising SEQ ID NO:8 (ARLKGELKDAFDI); CDRL1 comprising SEQ ID NO:9 (RASQSISSYLN), CDRL2 comprising SEQ ID NO:5 (AASSLQS), and CDRL3 comprising SEQ ID NO:10 (QQSYSTPLT);

[0390] (kk) CDRH1 comprising SEQ ID NO:23 (GTFSSYAIS), CDRH2 comprising SEQ ID NO:27 (GRINPILGTANYA), and CDRH3 comprising SEQ ID NO:28 (ARLKGELKDAFSI);

[0391] (11) CDRH1 comprising SEQ ID NO:23 (GTFSSYAIS), CDRH2 comprising SEQ ID NO:27 (GRINPILGTANYA), and CDRH3 comprising SEQ ID NO:28 (ARLKGELKDAFSI); CDRL1 comprising SEQ ID NO:9 (RASQSISSYLN), CDRL2 comprising SEQ ID NO:5 (AASSLQS), and CDRL3 comprising SEQ ID NO:10 (QQSYSTPLT);

[0392] (mm) CDRH1 comprising SEQ ID NO:23 (GTFSSYAIS), CDRH2 comprising SEQ ID NO:11 (GGIIPVFGTATYA), and CDRH3 comprising SEQ ID NO:30 (ARLKGELKCAFDI);

[0393] (nn) CDRH1 comprising SEQ ID NO:23 (GTFSSYAIS), CDRH2 comprising SEQ ID NO:11 (GGIIPVFGTATYA), and comprising SEQ ID NO:30 (ARLKGELKCAFDI); CDRL1 comprising SEQ ID NO:9 (RASQSISSYLN), CDRL2 comprising SEQ ID NO:5 (AASSLQS), and CDRL3 comprising SEQ ID NO:10 (QQSYSTPLT);

[0394] (oo) a CDRH1 comprising SEQ ID NO: 36 (GPFRSHAVS), a CDRH2 comprising SEQ ID NO: 11 (GGIIPVFGTATYA), and a CDRH3 comprising SEQ ID NO: 37 (ARLKSELKDAFDI); or

[0395] (pp) CDRH1 comprising SEQ ID NO:36 (GPFRSHAVS), CDRH2 comprising SEQ ID NO:11 (GGIIPVFGTATYA), and CDRH3 comprising SEQ ID NO:37 (ARLKSELKDAFDI); CDRL1 comprising SEQ ID NO:9 (RASQSISSYLN), CDRL2 comprising SEQ ID NO:5 (AASSLQS), and CDRL3 comprising SEQ ID NO:10 (QQSYSTPLT).

[0396] In some such embodiments, the second antigen binding unit binds to PD-L1 and comprises a heavy chain variable region comprising an amino acid sequence at least 90% identical to any one of SEQ ID NO: 35, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57 or 58, and a light chain variable region comprising an amino acid sequence at least 90% identical to any one of SEQ ID NO: 59, 60, 61, 62 or 63.

[0397] In some embodiments, the second antigen binding unit binds PD-L1 and comprises a heavy chain variable region comprising:

[0398] (a) an amino acid sequence at least 90% identical to SEQ ID NO: 35;

[0399] (b) an amino acid sequence that is at least 90% identical to SEQ ID NO:40;

[0400] (c) an amino acid sequence at least 90% identical to SEQ ID NO: 41;

[0401] (d) an amino acid sequence at least 90% identical to SEQ ID NO:42;

[0402] (e) an amino acid sequence at least 90% identical to SEQ ID NO: 43;

[0403] (f) an amino acid sequence at least 90% identical to SEQ ID NO:44;

[0404] (g) an amino acid sequence at least 90% identical to SEQ ID NO:45;

[0405] (h) an amino acid sequence at least 90% identical to SEQ ID NO:46;

[0406] (i) an amino acid sequence at least 90% identical to SEQ ID NO: 47;

[0407] (j) an amino acid sequence at least 90% identical to SEQ ID NO: 48;

[0408] (k) an amino acid sequence at least 90% identical to SEQ ID NO:49;

[0409] (1) an amino acid sequence that is at least 90% identical to SEQ ID NO: 50;

[0410] (m) an amino acid sequence at least 90% identical to SEQ ID NO: 51;

[0411] (n) an amino acid sequence at least 90% identical to SEQ ID NO: 52;

[0412] (o) an amino acid sequence at least 90% identical to SEQ ID NO: 53;

[0413] (p) an amino acid sequence at least 90% identical to SEQ ID NO:54;

[0414] (q) an amino acid sequence at least 90% identical to SEQ ID NO: 55;

[0415] (r) an amino acid sequence at least 90% identical to SEQ ID NO:56;

[0416] (s) an amino acid sequence that is at least 90% identical to SEQ ID NO: 57; or

[0417] (t) an amino acid sequence at least 90% identical to SEQ ID NO:58.

[0418] In some embodiments, the second antigen binding unit binds PD-L1 and comprises a light chain variable region comprising:

[0419] (a) an amino acid sequence that is at least 90% identical to SEQ ID NO: 59;

[0420] (b) an amino acid sequence that is at least 90% identical to SEQ ID NO: 60;

[0421] (c) an amino acid sequence at least 90% identical to SEQ ID NO: 61;

[0422] (d) an amino acid sequence that is at least 90% identical to SEQ ID NO: 62; or

[0423] (e) an amino acid sequence that is at least 90% identical to SEQ ID NO: 63.

[0424] In some embodiments, the present disclosure provides multispecific antigen-binding constructs comprising any of the PD-1 antagonists disclosed herein and any of the antagonists of PD-1 ligands (such as PD-L1) disclosed herein. For example, bispecific 3 is a multispecific tetravalent antigen-binding construct that specifically binds to human PD-1 and human PD-L1. The construct comprises an anti-PD-1 IgG1 antibody (mAb28), wherein the heavy chain of the antibody is a fusion protein further comprising the heavy chain variable region of an anti-PD-L1 antibody (mAb1) at its C-terminus, and the anti-PD-L1 antibody is connected to the Fc region of an anti-BCMA antibody via a poly GGGS (SEQ ID NO: 120) linker. The light chains of the anti-PD-1 portion and the anti-PD-L1 portion of the construct are identical (SEQ ID NO: 101). Bispecific 3 (whose structure is composed of Figure 13A The example in (shown in ) comprises the heavy chain sequence described in SEQ ID NO: 100 and the light chain sequence described in SEQ ID NO: 101.

[0425] In some embodiments, the present disclosure provides multispecific antibodies, or antigen-binding portions thereof, that specifically bind PD-1 and PD-L1, wherein the antibody, or antigen-binding portion thereof, comprises a heavy chain region comprising an amino acid sequence that is at least 85% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical) to SEQ ID NO: 100 or 102 and a light chain region comprising an amino acid sequence that is at least 85% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical) to SEQ ID NO: 101 or 103.

[0426] In some embodiments, the present disclosure provides multispecific antibodies, or antigen-binding portions thereof, that specifically bind PD-1 and PD-L1, wherein the antibody, or antigen-binding portion thereof, comprises a heavy chain region comprising an amino acid sequence that is at least 85% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical) to SEQ ID NO: 100, and a light chain region comprising an amino acid sequence that is at least 85% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical) to SEQ ID NO: 101. In some embodiments, the heavy chain region comprises an amino acid sequence that differs from SEQ ID NO: 1001 by 5 amino acids or less, 14 amino acids or less, 13 amino acids or less, 12 amino acids or less, 11 amino acids or less, 10 amino acids or less, 9 amino acids or less, 8 amino acids or less, 7 amino acids or less, 6 amino acids or less, 5 amino acids or less, 4 amino acids or less, 3 amino acids or less, 2 amino acids or less, or 1 amino acid. In some embodiments, the light chain region comprises an amino acid sequence that differs from SEQ ID NO: 1011 by 5 amino acids or less, 14 amino acids or less, 13 amino acids or less, 12 amino acids or less, 11 amino acids or less, 10 amino acids or less, 9 amino acids or less, 8 amino acids or less, 7 amino acids or less, 6 amino acids or less, 5 amino acids or less, 4 amino acids or less, 3 amino acids or less, 2 amino acids or less, or 1 amino acid.

[0427] In some embodiments, the present disclosure provides multispecific antibodies, or antigen-binding portions thereof, that specifically bind PD-1 and PD-L1, wherein the antibody, or antigen-binding portion thereof, comprises a heavy chain region comprising an amino acid sequence that is at least 85% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical) to SEQ ID NO: 102, and a light chain region comprising an amino acid sequence that is at least 85% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical) to SEQ ID NO: 103. In some embodiments, the heavy chain region comprises an amino acid sequence that differs from SEQ ID NO: 1021 by 5 amino acids or less, 14 amino acids or less, 13 amino acids or less, 12 amino acids or less, 11 amino acids or less, 10 amino acids or less, 9 amino acids or less, 8 amino acids or less, 7 amino acids or less, 6 amino acids or less, 5 amino acids or less, 4 amino acids or less, 3 amino acids or less, 2 amino acids or less, or 1 amino acid. In some embodiments, the light chain region comprises an amino acid sequence that differs from SEQ ID NO: 1031 by 5 amino acids or less, 14 amino acids or less, 13 amino acids or less, 12 amino acids or less, 11 amino acids or less, 10 amino acids or less, 9 amino acids or less, 8 amino acids or less, 7 amino acids or less, 6 amino acids or less, 5 amino acids or less, 4 amino acids or less, 3 amino acids or less, 2 amino acids or less, or 1 amino acid.

[0428] In some aspects and embodiments, also provided herein are multispecific antigen-binding constructs comprising four antigen-binding units, wherein two antigen-binding units bind to PD-1 and two antigen-binding units bind to PD-L1, and wherein the construct comprises a heavy chain amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 100 or 102, and a light chain amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 101 or 103.

[0429] In some aspects and embodiments, also provided herein are multispecific antigen-binding constructs comprising four antigen-binding units, wherein two antigen-binding units bind to PD-1 and two antigen-binding units bind to PD-L1, and wherein the construct comprises a heavy chain amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 100 and a light chain amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 101.

[0430] In some aspects and embodiments, also provided herein are multispecific antigen-binding constructs comprising four antigen-binding units, wherein two antigen-binding units bind to PD-1 and two antigen-binding units bind to PD-L1, and wherein the construct comprises a heavy chain amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 102 and a light chain amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 103.

[0431] Table 8. Multispecific heavy chain sequences

[0432]

[0433]

[0434]

[0435] Table 9. Multispecific light chain sequences

[0436]

[0437] As used herein, the term "multispecific antigen-binding construct" refers to a bispecific, trispecific or multispecific antigen-binding construct, and an antigen-binding portion or fragment thereof. A multispecific antigen-binding construct can be a single multifunctional polypeptide or it can be a multimeric complex of two or more molecules (e.g., polypeptides and / or aptamers) covalently or non-covalently bound to each other. The term "multispecific antigen-binding construct" includes antibodies (or antigen-binding fragments thereof) that can be connected or co-expressed with another functional molecule (e.g., another peptide, protein and / or aptamer). For example, an antibody or its fragment can be functionally linked (e.g., by chemical coupling, genetic fusion, non-covalent binding or other means) to one or more other molecular entities (such as proteins or their fragments) to produce a bispecific or multispecific antigen-binding molecule with a second binding specificity. As used herein, the term "multispecific antigen-binding construct" also includes bispecific, trispecific or multispecific antibodies or their antigen-binding fragments. In certain embodiments, an antibody is functionally linked to another antibody or its antigen-binding fragment to produce a bispecific antibody with a second binding specificity. Bispecific and multispecific antibodies of the present disclosure are also described elsewhere herein.

[0438] As used herein, an antigen-binding "arm" refers to a unit, domain, region, or the like of a construct that forms an area of ​​a multispecific antigen-binding construct that binds to an antigen. Thus, a "first arm" and a "second arm" of a multispecific antigen-binding construct form separate binding regions of the construct, with each arm forming an antigen-binding unit. Generally, one "arm" (the first arm) differs from the other "arm" (the second arm) in its antigen binding or antigen specificity. Thus, in the example of a bispecific bivalent antibody, one arm of the antibody binds to antigen A, while the other arm of the antibody binds to antigen B. In some embodiments, in the example of a bispecific bivalent antibody, one arm of the antibody binds to antigen A, while the other arm of the antibody binds to antigen B or C (due to, for example, structural similarity and cross-reactivity with two antigens (such as PD-L1 and PD-L2)). See, e.g., U.S. Patent No. 9,845,356. Similarly, in the example of a tetravalent bispecific antibody (e.g., formed by joining two different antibodies), one "arm" refers to the region of the antibody that binds to antigen A (even if both binding sites of the bivalent antibody bind to antigen A), and the "other arm" refers to the region of the antibody that binds to antigen B (even if both binding sites of the bivalent antibody bind to antigen B). In some embodiments, in the example of a tetravalent bispecific antibody (e.g., formed by joining two different antibodies), one "arm" refers to the region of the antibody that binds to antigen A (even if both binding sites of the bivalent antibody bind to antigen A), and the "other arm" refers to the region of the antibody that binds to antigen B or C (even if both binding sites of the bivalent antibody can bind to antigen B or C). See, for example, U.S. Patent 9,845,356. It will be apparent to those skilled in the art that "first" and "second" can be used interchangeably.

[0439] When used to describe an antigen-binding construct or protein or antigen-binding arm, the term "valence" refers to the number of recognition (binding) sites in the antigen-binding construct or protein, regardless of whether those different recognition or binding sites are bound to the same epitope. Each recognition site specifically recognizes and therefore can bind to an epitope (binding site) on the antigen. When an antigen-binding protein comprises more than one recognition site (for example, when an antigen-binding protein is an IgG, it has two recognition sites in its variable region), each recognition site can specifically recognize the same epitope on the same antigen, or different epitopes, regardless of whether they are on the same or different antigens. Multivalence can increase avidity, that is, the binding strength between an antigen-binding arm or construct and a related antigen or target receptor. Avidity is related to the affinity (affinity) between its binding site on an epitope or antigenic determinant and an antigen-binding unit, and the actual number of related binding sites present on the antigen-binding unit.

[0440] In some embodiments, any of the multispecific antigen-binding constructs disclosed herein comprises a multivalent (e.g., bivalent) antibody or antigen-binding fragment, wherein at least two of the valencies specifically bind to PD-1. In some embodiments, any of the multispecific antigen-binding constructs disclosed herein comprises a multivalent (e.g., bivalent) antibody or antigen-binding fragment, wherein at least two of the valencies specifically bind to a PD-1 ligand (e.g., PD-L1 or PD-L2). In some embodiments, any of the multispecific antigen-binding constructs disclosed herein comprises a multivalent (e.g., bivalent) antibody or antigen-binding fragment, wherein at least two of the valencies specifically bind to PD-L1. In some embodiments, any of the multispecific antigen-binding constructs disclosed herein comprises a first multivalent (e.g., bivalent) antibody or antigen-binding fragment and a second multivalent (e.g., bivalent) antibody or antigen-binding fragment, wherein at least two of the valencies of the first multivalent antibody or antigen-binding fragment specifically bind to PD-1, and wherein at least two of the valencies of the second multivalent antibody or antigen-binding fragment specifically bind to PD-L1. In some embodiments, any of the multispecific antigen-binding constructs disclosed herein is a tetravalent construct, wherein the tetravalent construct comprises a first bivalent antibody or antigen-binding fragment and a second bivalent antibody or antigen-binding fragment, wherein the valencies of the first bivalent antibody or antigen-binding fragment are all specific for the same epitope on PD-1, and wherein the valencies of the second bivalent antibody or antigen-binding fragment are all specific for the same epitope on PD-L1. In some embodiments of such tetravalent constructs, the first and second bivalent antibodies, or antigen-binding fragments or portions thereof, utilize light chains having the same amino acid sequence. In other words, the tetravalent construct comprises a common light chain. For example, a light chain having the sequence of SEQ ID NO: 101 or SEQ ID NO: 103.

[0441] In some embodiments, the first arm is an antagonist of PD-1. In some embodiments, the second arm is an antagonist of a PD-1 ligand (e.g., PD-L1 and / or PD-L2). In some embodiments, the first arm is an antagonist of PD-1 and the second arm is an antagonist of a cognate PD-1 ligand (e.g., PD-L1 and / or PD-L2).

[0442] When referring to the biological activity of the antigen binding arm, the terms "antagonist", "antagonism" and "inhibition" indicate that the antigen binding arm binds to its target (e.g., PD-1) on each cell and partially or completely blocks, inhibits and / or reduces the biological response by PD-1. In some embodiments, inhibition in the presence of an antagonist is observed in a dose-dependent manner. In some embodiments, the measured signal (e.g., biological activity) is at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% or at least about 100% lower than the signal measured using a negative control under comparable conditions. Methods for identifying antagonists suitable for the methods of the present disclosure are also disclosed herein. For example, these methods include, but are not limited to, enzyme-linked immunosorbent assays (ELISA), FORTE Binding assays such as immunofluorescence systems and radioimmunoassays (RIA). These assays measure the ability of an antagonist to bind to a polypeptide of interest (e.g., PD-1 or its ligand) and thus indicate the ability of the antagonist to inhibit, neutralize, or block the activity of the polypeptide of interest. Functional assays can also be used to measure the efficacy of an antagonist, such as the ability of an antagonist to inhibit the function of a polypeptide. For example, a functional assay can include contacting a polypeptide with a candidate antagonist molecule and measuring a detectable change in one or more biological activities normally associated with the polypeptide. The efficacy of an antagonist is typically measured by its IC 50 The IC value (the concentration required to inhibit the agonist response by 50%) is defined. 50 The lower the value, the more potent the antagonist and the lower the concentration required to inhibit the maximal biological response.

[0443] In some embodiments, at least one antigen binding arm has at least 1×10 -7 M, at least 1x10 -8 M, at least 1x10 -9 M, at least 1x10 -10 M, at least 1x10 -11 M, at least 1x10 -12 M or at least 1x10 -13 M's K D In some embodiments, both antigen binding arms have the same or similar K D As used herein, the term "K D "(M) refers to the dissociation equilibrium constant of a specific antigen binding arm / antigen interaction. K D =k d / k a As used herein, the term k d(sec -1 ) refers to the dissociation rate constant of a specific antigen binding arm / antigen interaction. This value is also called k off As used herein, the term k a (M -1 x sec -1 ) refers to the association rate constant for a specific antigen binding arm / antigen interaction. This value is also called k on value.

[0444] In some embodiments, the binding of one arm (e.g., the first arm) of a multispecific antigen-binding construct to its target does not block the binding of another arm (e.g., the second arm) to its target. In some embodiments, the binding of one arm does not sterically hinder the binding of the second arm to its target. For example, after the first arm binds to PD-1, the second arm is free to bind to the ligand of PD-1 (e.g., PD-L1 and / or PD-L2). Thus, in some embodiments, the first arm and the second arm bind to their respective targets and both arms remain bound simultaneously.

[0445] In some embodiments, the binding of the first and second arms to their respective targets bridges the immune cell and the second cell together, bringing the two cells into close proximity. As used herein, "bridging" refers to joining two cell types (e.g., an immune cell expressing PD-1, and a second cell expressing its ligand - PD-L1), or bringing two cells into close proximity; the two cells do not necessarily have to be in physical contact. Thus, the multispecific antigen-binding construct acts as a connector (e.g., a bridge) between two cells, each of which expresses PD-1 or its ligand.

[0446] Methods for determining whether two cells are bridged or linked together by a construct of the present disclosure are known in the art. For example, in some embodiments, the bridging of an immune cell and a second cell is determined by, for example, flow cytometry, FRET, immunoprecipitation, microscopy, or a fluorescent plate reader.

[0447] In some embodiments, the combination of the first arm and the second arm of the multispecific construct with its respective target results in the reduction and / or shedding and / or degradation of the extracellular domain of the target (e.g., PD-1). As used herein, "reduction" refers to a process by which cells reduce the number of cellular components (such as RNA or protein). In the case of cell surface protein receptors, reduction can be produced by receptor internalization due to binding to any one of the ligands or constructs described herein. Falling off or extracellular domain falling off refers to a process by which cell surface proteins are proteolytically cleaved, causing their extracellular domains to be released into the extracellular environment. Non-limiting examples of the shedding enzymes regulating the shedding of the extracellular domain include members of the disintegrin and metalloproteinase (ADAM) family, such as ADAM8, ADAM9, ADAM10, ADAM12, ADAM15, ADAM17, and ADAM28, and matrix metalloproteinases (MMPs), such as MMP2, MMP3, MMP7, MMP9, and MMP14. The distance from the plasma membrane and the structure of the cleavage site region are believed to be more important than the specific sequence in the shedding of the extracellular domain. Protein degradation or proteolysis refers to a group of processes that result in the hydrolysis of one or more of the peptide bonds in a protein by catalytic or non-enzymatic (e.g., at very low or very high pH) proteolytic enzymes called proteases. In eukaryotic cells, two major pathways—the ubiquitin proteasome pathway and lysosomal proteolysis—mediate protein degradation. Methods for determining whether a target receptor is downregulated and / or shed and / or degraded by the multispecific constructs disclosed herein are known in the art and are described in the Examples, see, e.g., Figures 12A to 12C ; for example, flow cytometry, Western blotting, immunoprecipitation, microscopy, or fluorescence plate reader.

[0448] As described herein, the constructs of the present disclosure can bridge an immune cell expressing PD-1 and a second cell expressing its ligand, such as a second immune cell and / or a cancer or tumor cell. It is understood by those skilled in the art that the type of immune cell depends on the context of the disease to be treated; the specific type of immune cell can be easily determined based on the condition under consideration. In some embodiments, the immune cell is a T cell, for example, a regulatory T cell (also known as a suppressor T cell), including CD8+ T cells and CD4+ T cells, and subtypes such as CD4+ T cells. + FOXP3 + T reg cells, CD4 + FOXP3 - T reg cells, Tr1 cells, Th3 cells and T reg17 cells. In some embodiments, the immune cells are natural killer (NK) cells. In some embodiments, the immune cells are B cells. In some embodiments, the immune cells are macrophages.

[0449] Similarly, the type of the second cell depends on the condition under consideration. In some embodiments, the second cell (a cell expressing PD-1 ligand) is a second immune cell, for example, a regulatory immune cell. In some embodiments, the regulatory immune cell is any one or more of a regulatory T cell, a B cell, a macrophage, a suppressor cell derived from bone marrow, a dendritic cell, or a mesenchymal stromal cell. In some embodiments, the regulatory immune cell is a regulatory T cell, for example, a CD8+ T cell or a CD4+ T cell.

[0450] In some embodiments, the second cell is a tumor cell. As used herein, "tumor cell" is not only sometimes used interchangeably with "cancer cell", but also includes non-malignant (non-cancerous) cells that show increased proliferation compared to normal cells. In some embodiments, tumor cells are cancers that can be treated by blocking the interaction between PD-1 expressed by immune cells and its ligand (e.g., PD-L1 or PD-L2) expressed on the second cell, while bridging immune cells and tumor cells. In some embodiments, the tumor cell line is selected from the group consisting of: blood cancer, lymphoma, myeloma, leukemia, neurocarcinoma, melanoma, breast cancer, prostate cancer, colorectal cancer, lung cancer, head and neck cancer, gastrointestinal cancer, liver cancer, pancreatic cancer, genitourinary cancer, bone cancer, kidney cancer and vascular cancer.In some embodiments, the tumor cell is selected from the group consisting of: Kaposi's sarcoma, leukemia, acute lymphocytic leukemia, acute myeloid leukemia, myeloblastic promyelocytic myelomonocytic monocytic erythroleukemia, chronic leukemia, chronic myeloid (granulocytic) leukemia, chronic lymphocytic leukemia, mantle cell lymphoma, primary central nervous system lymphoma, Burkitt's lymphoma and marginal zone B-cell lymphoma, polycythemia vera lymphoma, Hodgkin's disease disease), non-Hodgkin's disease, multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, solid tumors, sarcomas and carcinomas, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, osteosarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon sarcoma, colorectal cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, uterine cancer, testicular cancer, lung cancer, small cell lung cancer, non-small cell lung cancer, bladder cancer, epithelial cancer, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma, nasopharyngeal cancer, esophageal cancer, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, brain and central nervous system (CNS) cancer, cervical cancer, choriocarcinoma, colorectal cancer, connective tissue cancer, digestive system cancer, endometrial cancer, esophageal cancer cancer), eye cancer, head and neck cancer, stomach cancer, intraepithelial neoplasia, kidney cancer, laryngeal cancer, liver cancer, lung cancer (small cell and large cell), melanoma, neuroblastoma; oral cancer (e.g., lip, tongue, mouth, and pharynx), ovarian cancer, pancreatic cancer, retinoblastoma, rhabdomyosarcoma, rectal cancer; respiratory cancer, sarcoma, skin cancer, stomach cancer, testicular cancer, thyroid cancer, uterine cancer, and urinary tract cancer.

[0451] As described herein, the multispecific antigen-binding constructs of the invention comprise bispecific, trispecific, tetraspecific, or multispecific antibodies (immunoglobulins), or antigen-binding portions or fragments thereof.

[0452] The term "immunoglobulin" refers to a class of structurally related proteins that generally comprise two pairs of polypeptide chains: a pair of light (L) chains and a pair of heavy (H) chains. In an "intact immunoglobulin," all four of these chains are interconnected by disulfide bonds. The structure of immunoglobulins has been well characterized. See, for example, Paul, Fundamental Immunology, 7th ed., Chapter 5 (2013) Lippincott Williams & Wilkins, Philadelphia, PA. Briefly, each heavy chain generally comprises a heavy chain variable region (V H ) and heavy chain constant region (C H The heavy chain constant region usually contains three domains, C H1 、C H2 and C H3 Each light chain typically comprises a light chain variable region (V L ) and the light chain constant region. The light chain constant region usually comprises a domain, abbreviated as C L The term "immunoglobulin" (Ig) is sometimes used interchangeably with the term "antibody" herein.

[0453] The term "antibody" describes an immunoglobulin molecule and is used herein in its broadest sense. Antibodies specifically include intact antibodies (e.g., intact immunoglobulins), and antibody fragments (such as antigen-binding fragments of antibodies), as described herein. Thus, "antibody" may refer to intact antibodies as well as antigen-binding fragments thereof. An antibody comprises at least one antigen-binding domain. An example of an antigen-binding domain is a V-binding domain. H -V L The antigen binding domain formed by the dimer. Antibodies can be described by the antigen to which they specifically bind. For example, a PD-1 antibody (or anti-PD-1 antibody) is an antibody that specifically binds to the inhibitory receptor PD-1.

[0454] V H and V L The V regions are further subdivided into regions of hypervariability ("hypervariable regions (HVRs)"; also called "complementarity determining regions" (CDRs)) interspersed with more conserved regions. The more conserved regions are called framework regions (FRs). H and V LGenerally, the antibody comprises three CDRs and four FRs arranged in the following order (from N-terminus to C-terminus): FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The CDRs are involved in antigen binding and confer antigen specificity and binding affinity to the antibody. See Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. (1991), Public Health Service, National Institutes of Health, Bethesda, MD, which is incorporated by reference in its entirety.

[0455] Light chains from vertebrate species can be assigned to one of two types, called kappa and lambda, based on the sequence of the constant domain.

[0456] Heavy chains from vertebrate species can be assigned to one of five different classes (or isotypes): IgA, IgD, IgE, IgG, and IgM. These classes are also designated α, δ, ε, γ, and μ, respectively. The IgG and IgA classes are further divided into subclasses based on differences in sequence and function. Humans express the following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.

[0457] Methods for producing and screening antibodies against desired targets are well known in the art. As described herein, methods for further modifying antibodies to obtain enhanced properties (e.g., enhanced affinity, chimerization, humanization) and generating antigen-binding fragments are well known in the art.

[0458] The term "chimeric antibody" refers to an antibody in which components of the heavy and / or light chains are derived from a particular source or species, while the remainder of the heavy and / or light chains are derived from a different source or species.

[0459] The "humanized" form of a non-human antibody is a chimeric antibody containing minimal sequences derived from a non-human antibody. A humanized antibody is generally a human immunoglobulin (recipient antibody) in which residues from one or more CDRs are replaced by residues from one or more CDRs of a non-human antibody (donor antibody). The donor antibody may be any suitable non-human antibody with the desired specificity, affinity, or biological effect, such as a mouse, rat, rabbit, chicken, or non-human primate antibody. In some instances, the selected framework region residues of the recipient antibody are replaced by corresponding framework region residues from the donor antibody. Humanized antibodies may also include residues not found in the recipient antibody or the donor antibody. Such modifications may be made to further refine antibody function. For further details, see Jones et al., (1986) Nature, 321:522-525; Riechmann et al., (1988) Nature, 332:323-329; and Presta, (1992) Curr. Op. Struct. Biol., 2:593-596, each of which is incorporated by reference in its entirety.

[0460] A "human antibody" is an antibody having an amino acid sequence that corresponds to the amino acid sequence of an antibody produced by a human or human cell, or derived from a non-human source that utilizes human antibody repertoires or human antibody coding sequences (e.g., obtained from a human source or de novo). Human antibodies specifically exclude humanized antibodies.

[0461] In some embodiments, antibody molecules include diabodies and single-chain molecules, as well as antigen-binding fragments of antibodies (e.g., Fab, F(ab')2, and Fv). For example, an antibody molecule may comprise a heavy (H) chain variable domain sequence (abbreviated herein as VH), and a light (L) chain variable domain sequence (abbreviated herein as VL). In some embodiments, an antibody molecule comprises or consists of a heavy chain and a light chain (referred to as a half antibody). In another example, an antibody molecule comprises two heavy (H) chain variable domain sequences and two light (L) chain variable domain sequences, thereby forming two antigen-binding sites, such as Fab, Fab', F(ab')2, Fc, Fd, Fd', Fv, single-chain antibodies (e.g., scFv), single variable domain antibodies, diabodies (Dab) (bivalent bispecific) and chimeric (e.g., humanized) antibodies, which can be produced by modifying whole antibodies or those synthesized de novo using recombinant DNA technology. These functional antibody fragments retain the ability to selectively bind to their respective antigens. Antibodies and antibody fragments can be from any class of antibodies, including but not limited to IgG, IgA, IgM, IgD and IgE, and from any subclass of antibodies (e.g., IgA1, IgA2, IgG1, IgG2, IgG3 and IgG4). The preparation of antibody molecules can be monoclonal or polyclonal. Antibody molecules can also be human, humanized, CDR-grafted or in vitro produced antibodies. The antibody can have a heavy chain constant region selected from, for example, IgG1, IgG2, IgG3 or IgG4. The antibody can also have a light chain selected from, for example, κ or λ. In some embodiments, the antibody comprises a light chain having a constant region corresponding to SEQ ID NO: 1. NO:64(ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVD KKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWL 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to an IgG1 heavy chain constant region of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to (e.g., NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK).In some embodiments, the antibody comprises a polypeptide having the sequence of SEQ ID NO: 68 (ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLN GKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK). In some embodiments, the antibody comprises an IgG4 heavy chain constant region having an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: ... NO:69(ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKV DKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLN or a combination thereof. The present invention further comprises an IgG4 heavy chain constant region having an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to that of the IgG4 heavy chain constant region of FIG.

[0462] Antigen-binding portions or fragments of antibody molecules are well known in the art and include, for example: (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a diabody (dAb) fragment, which consists of a VH domain; (vi) camelid or camelized variable domains; (vii) single-chain Fv (scFv), see, e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883; (viii) single-domain antibodies. These antibody fragments can be obtained using conventional techniques known to those with skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies.

[0463] Antibody molecules may also be single domain antibodies. Single domain antibodies may include antibodies whose complementary determining regions are part of a single domain polypeptide. Examples include, but are not limited to, heavy chain antibodies, antibodies naturally lacking light chains, single domain antibodies derived from conventional 4-chain antibodies, engineered antibodies, and single domain scaffolds other than those derived from antibodies. Single domain antibodies may be any of those in the art, or any future single domain antibodies. Single domain antibodies may be derived from any species, including but not limited to mice, humans, camels, llamas, fish, sharks, goats, rabbits, and cattle. According to another aspect of the present disclosure, single domain antibodies are naturally occurring single domain antibodies referred to as heavy chain antibodies lacking light chains. Such single domain antibodies are disclosed in WO 9404678. For clarity reasons, this variable domain derived from a heavy chain antibody naturally lacking light chains is referred to herein as a VHH or nanobody to distinguish it from the conventional VH of a four-chain immunoglobulin. Such VHH molecules can be derived from antibodies raised in species of the Camelidae family (e.g., camels, llamas, dromedaries, alpacas, and guanacos). Species other than Camelidae may produce heavy chain antibodies that naturally lack light chains; such VHHs are within the scope of the present disclosure.

[0464] In some embodiments, the multispecific antigen-binding construct comprises a bispecific antibody. Bispecific antibodies have specificity for no more than two antigens, but may have more than two binding sites, as described herein. The bispecific antibody molecule is characterized in that it has a first immunoglobulin variable domain sequence with binding specificity to a first antigen (e.g., PD-1) and a second immunoglobulin variable domain sequence with binding specificity to a second antigen (e.g., PD-1 ligand, such as PD-L1 ligand). In some embodiments, the bispecific antibody molecule comprises an scFv or fragment thereof with binding specificity to a first antigen, and an scFv or fragment thereof with binding specificity to a second antigen. See, for example, Kontermann & Brinkmann, (2015), Drug Discovery Today, 20 (7): 838-47, which is incorporated by reference in its entirety.

[0465] Various formats and methods that can be used to generate the multivalent and / or multispecific constructs described herein, such as multivalent and / or multispecific antibody formats of both asymmetric and symmetric architectures, are known in the art. Non-limiting examples of such formats include (i) Fc-less bispecific antibody formats, such as tandem single chain variable fragments (scFv2, taFv) and triabodies, including bispecific T cell engagers (BiTE) and bispecific killer cell engager (BiKE) molecules; bispecific single domain antibody fusion proteins comprising single domain antibodies, such as VH or VL domains, VHH, VNARs and nanobodies; diabodies and diabody derivatives, including tandem diabodies and dual affinity retargeting (DART) proteins; Fab fusion proteins; and other Fc-less fusion proteins by using heterodimeric peptides or miniantibodies from various proteins, for example, leucine zippers with a coiled coil structure; (ii) bispecific IgGs with asymmetric architectures, such as having heavy chains from two different antibodies.

[0014] The present invention relates to asymmetric IgGs comprising asymmetric Fc and light chains; bispecific IgGs with asymmetric Fc regions using a knob-in-hole approach, electrostatic interactions (steering) to avoid homodimerization of the CH3 domains, preferred heavy chain heterodimerization by introducing charge pairs into the hinge regions of IgG1 and IgG2, chain exchange engineered domain (SEED) heterodimers, and bispecific engagement of antibodies based on T cell receptor (BEAT) technology; asymmetric Fc and CH3 fusion proteins; (iii) bispecific antibodies with symmetric architectures, such as by fusing scFv, fusion domain antibodies and scaffold proteins, fusion Fab arms, and additional IgGs fusing additional variable heavy and light chain domains; modified IgG molecules; symmetric Fc- and CH3-based bispecific antibodies; and bispecific antibodies using immunoglobulin-derived homodimerization domains. See, for example, "The making of bispecific antibodies," Brinkmann and Kontermann, MABS 2017, Vol. 9:2, pp. 182-212, the contents of which are incorporated herein by reference in their entirety. See also, e.g., the "knob-in-hole" approach described in U.S. Pat. No. 5,731,168; electrostatically directed Fc pairing as described in, e.g., WO 09 / 089004, WO 06 / 106905, and WO 2010 / 129304; strand exchange engineered domain (SEED) heterodimer formation as described in, e.g., WO 07 / 110205; Fab arm exchange as described in, e.g., WO 08 / 119353, WO 2011 / 131746, and WO 2013 / 060867; diabody conjugates, e.g., cross-linking of antibodies using heterobifunctional reagents having amine-reactive groups and thiol-reactive groups to generate bispecific structures, as described in, e.g., U.S. Pat. Nos. 4,433,059; bispecific antibody determinants generated by reconstitution of half antibodies (heavy-light chain pairs or Fabs) from different antibodies by cycles of reduction and oxidation of disulfide bonds between the two heavy chains, as described, for example, in U.S. Pat. No. 4,444,878; trifunctional antibodies, for example, three Fab′ fragments cross-linked by sulfhydryl-reactive groups, as described, for example, in U.S. Pat. No. 5,273,743; biosynthetic binding proteins, for example, scFv pairs cross-linked by C-terminal tails, preferably by disulfide bonds or amine-reactive chemical cross-linking, as described, for example, in U.S. Pat. No. 5,534,254; bifunctional antibodies, for example , Fab fragments with different binding specificities dimerized by leucine zippers that have replaced constant domains (e.g., c-fos and c-jun), as described, for example, in U.S. Pat. No. 5,582,996; bispecific and oligospecific monovalent and oligovalent receptors, for example, the VH-CH1 regions of two antibodies (two Fab fragments) linked by a polypeptide spacer between the CH1 region of one antibody and the VH region of another antibody, typically with associated light chains, as described, for example, in U.S. Pat. No. 5,591,828; bispecific DNA-antibody conjugates, for example, cross-linking of antibodies or Fab fragments by double-stranded fragments of DNA, as described, for example, in U.S. Pat. 5,635,602; bispecific fusion proteins, e.g., expression constructs containing two scFvs with a hydrophilic helical peptide linker therebetween and intact constant regions, as described, e.g., in U.S. Pat. No. 5,637,481; multivalent and multispecific binding proteins, e.g., a dimer of polypeptides having a first domain binding region of an Ig heavy chain variable region and a second domain binding region of an Ig light chain variable region, generally referred to as diabodies (also including higher order structures created for bispecific, trispecific, or tetraspecific molecules), as described, e.g., in U.S. Pat. No. 5,837,242; polypeptides having a scFv further linked to the hinge and CH3 regions of an antibody with a peptide spacer. Minibody constructs of linked VL and VH chains, which can be dimerized to form bispecific / multivalent molecules, as described, for example, in U.S. Pat. No. 5,837,821; VH and VL domains linked in any orientation with a short peptide linker (e.g., 5 or 10 amino acids) or no linker at all, which can form dimers, trimers, and tetramers for forming bispecific diabodies, as described, for example, in U.S. Pat. No. 5,844,094; strings of VH domains (or VL domains in family members) linked at the C-terminus by peptide chains with cross-linkable groups, further associated with VL domains to form a series of FVs (or scFvs), as described, for example, in U.S. Pat. No. 5,864,019; and single-chain binding polypeptides having both VH and VL domains connected by a peptide linker are combined into multivalent structures by non-covalent or chemical cross-linking to form, for example, homologous bivalent, heterologous bivalent, trivalent and tetravalent structures using both scFV or diabody-type formats, as described in, for example, U.S. Patent No. 5,869,620. Additional exemplary multispecific and bispecific molecules and methods of making the same are found in, for example, U.S. Patent No. 5,910,573, U.S. Patent No. 5,932,448, U.S. Patent No. 5,959,083, U.S. Patent No. 5,989,830, U.S. Patent No. 6,005,079, U.S. Patent No. 6,239,259, U.S. Patent No. 6,294,353, U.S. Patent No. 6,333,396, U.S. Patent No. 6,476,198, U.S. Patent No. 6,511,663, U.S. Patent No. 6,670,453, U.S. Patent No. 6,743,896, U.S. Patent No. 6,809,185, U.S. Patent No. 6,833,441, U.S. Patent No. 7,129,330, U.S. Patent No. 7,183,076, U.S. Patent No. 7,521,056, U.S. Patent No. 7,527,787, U.S. Patent No. 7,534,866, U.S. Patent No. 7,612,181. US2002004587A1, US2002076406A1, US2002103345A1, US2003207346A1, US2003211078A1, US2004219643A1, US2004220388A1, US200 4242847A1, US2005003403A1, US2005004352A1, US2005069552A1, US2005079170A1, US2005100543A1, US2005136049A1, US2005136051A1, US2005163782A1, US2005266425A1, US2006083747A1, US2006120960A1, US2006204493A1, US2006263367A1, US2007004909A1, US2007087 381A1, US2007128150A1, US2007141049A1, US2007154901A1, US2007274985A1, US2008050370A1, US2008069820A1, US2008152645A1, US20 08171855A1, US2008241884A1, US2008254512A1, US2008260738A1, US2009130106A1, US2009148905A1, US2009155275A1, US2009162359A 1. US2009162360A1, US2009175851A1, US2009175867A1, US2009232811A1, US2009234105A1, US2009263392A1, US2009274649A1, EP346087 A2, WO0006605A2, WO02072635A2, WO04081051A1, WO06020258A2, WO2007044887A2, WO2007095338A2, WO2007137760A2, WO2008119353A1, WO2009021754A2, WO2009068630A1, WO9103493A1, WO9323537A1, WO9409131A1, WO9412625A2, WO9509917A1, WO9637621A2, WO9964460A1. The contents of the above applications are incorporated herein by reference in their entirety.

[0466] In some embodiments, the multispecific antigen-binding constructs of the present disclosure are bispecific antibodies. The bispecific antibodies of the present disclosure can be produced against PD-1 and PD-L1 or against PD-1 and PD-L2. The antibody arms of the bispecific antibodies can be produced by standard techniques, as disclosed herein. In some embodiments, any known antibody against PD-1 and its ligands can be used to produce the bispecific antibodies of the present disclosure. For example, such bispecific constructs have been exemplified herein (see, e.g., Figure 3 In this study, pembrolizumab (anti-PD-1 antibody) was combined with atezolizumab (anti-PD-L1 antibody); Figure 4 As exemplified herein, multispecific antigen-binding constructs (e.g., bispecific antibodies) described herein can be generated using antibodies known and / or available in the art.

[0467] In some embodiments, the bispecific antibodies are bivalent, e.g., one arm is monovalent against PD-1, and the other arm is monovalent against PD-L1 or PD-L2 or both—e.g., cross-reacting with two ligands). In some embodiments, the bispecific antibodies are tetravalent, such as the novel Bispecific 3 and Bispecific 4 antibodies described herein. For example, Figure 3 As described in , the pembrolizumab binding arm is bivalent against PD-1, each binding to the same epitope on PD-1, while the atezolizumab binding arm is bivalent against PD-L1, each binding to the same epitope on PD-L1. This can also be seen in, for example Figure 8 Bispecific formats in Figure 8 An exemplary common light chain bispecific is illustrated in the left panel of step 2 of the workflow presented in ). Figure 8 The exemplary common light chain bispecific format in (the format illustrated in the right figure of step 2 of the workflow) represents another example of a tetravalent bispecific format. Compared to a tetravalent bispecific format (in which the first antigen binding arm is attached to the second antigen binding arm on the opposite end of the Fc region), here each Fab of the first antigen binding arm is attached to each Fab of the second antigen binding arm. For example, a linker is used to connect one Fab of the first antigen binding arm to the Fab of the second antigen binding arm, wherein each antigen binding arm shares a common light chain. See, Figure 8 , the form illustrated in the right figure of step 2 of the workflow.

[0468] In some embodiments, the bispecific antibody is tetravalent, wherein one arm is divalent for PD-1, each binding to two different epitopes on PD-1. In some embodiments, the bispecific antibody is tetravalent, wherein one arm is divalent for the ligand of PD-1 (PD-L1 and / or PD-L2), each binding to two different epitopes on the ligand of PD-1. In some embodiments, the bispecific antibody is tetravalent, wherein one arm is divalent for PD-1, each binding to two different but overlapping epitopes on PD-1. In some embodiments, the bispecific antibody is tetravalent, wherein one arm is divalent for PD-1, each binding to two different but overlapping epitopes on PD-1 ligand (PD-L1 and / or PD-L2). In some embodiments, the bispecific antibody is tetravalent, wherein one arm is divalent for PD-1 and each binds to the same epitope on PD-1. In some embodiments, the bispecific antibody is tetravalent, wherein one arm is bivalent against the PD-1 ligand (PD-L1 and / or PD-L2) and each binds to the same epitope on the PD-1 ligand (PD-L1 and / or PD-L2). In some embodiments, the bispecific antibody is tetravalent, wherein one arm is bivalent against the same epitope on PD-1; and the other arm is bivalent against the same epitope on the PD-1 ligand (PD-L1 and / or PD-L2).

[0469] In some embodiments, the bispecific antibody is an antagonist of both PD-1 and PD-L1. In some embodiments, the bispecific antibody is an antagonist of both PD-1 and PD-L2. In some embodiments, the bispecific antibody is an antagonist of PD-1 and both the ligands PD / L1 and PD-L2 (e.g., cross-reacts with both ligands).

[0470] In certain embodiments, the first antigen binding arm and the second antigen binding arm are connected by a linker amino acid sequence having at least one amino group. Optionally, the linker amino acid sequence comprises GGGGS x (SEQ ID NO: 121), wherein x is an integer between 1 and 6, inclusive.

[0471] In some embodiments, the multispecific antigen-binding construct does not comprise an immunoglobulin Fc domain.

[0472] In some embodiments, the construct comprises an immunoglobulin Fc domain. In some embodiments, the first arm or the second arm or both of the constructs comprise a heavy chain comprising one or more immunoglobulin Fc modifications. In some embodiments, the immunoglobulin Fc domain of the heavy chain comprises one or more amino acid mutations that, for example, promote heterodimerization of the first and second arms, promote serum half-life, and / or modify effector function. In some embodiments, the mutation is present in the CH3 domain of the heavy chain. See, for example, Xu et al., mAbs 7(1):231-42, 2015.

[0473] While traditional Fc fusion proteins and antibodies are examples of non-directed interaction pairing, various engineered Fc domains have been designed to exhibit asymmetric interaction pairing (Spiess et al. (2015) Molecular Immunology 67(2A):95-106) to promote, for example, heterodimerization of a first antigen-binding arm and a second antigen-binding arm. Various methods are known in the art to increase the desired pairing of Fc-containing polypeptide chains in a single cell line to produce preferred asymmetric fusion proteins in acceptable yields [see, e.g., Klein et al. (2012) mAbs 4:653-663; and Spiess et al. (2015) Molecular Immunology 67(2 Part A):95-106]. Methods for achieving the desired pairing of Fc-containing polypeptides include, but are not limited to, charge-based pairing (electrostatic steering), "knob-in-hole" stereo pairing, SEEDbody pairing, and leucine zipper-based pairing. See, e.g., Ridgway et al. (1996) Protein Eng 9:617-621; Merchant et al. (1998) Nat Biotech 16:677-681; Davis et al. (2010) Protein Eng Des Sel 23:195-202; Gunasekaran et al. (2010); 285:19637-19646; Wranik et al. (2012) J Biol Chem 287:43331-43339; US5932448; WO 1993 / 011162; WO 2009 / 089004, and WO 2011 / 034605.

[0474] For example, one approach that can promote interactions between specific polypeptides is to engineer a protuberance into a cavity (knob-to-hole) complementary region, such as described in Arathoon et al., US7,183,076; Carter et al., US5,731,168; and Kumar et al., WO 2016 / 164089, the entireties of which are incorporated herein by reference. A "protuberance" is constructed by replacing a small amino acid side chain from the junction of a first polypeptide (e.g., a first interaction pair) with a larger side chain (e.g., tyrosine or tryptophan). A complementary "cavity" of the same or similar size as the protuberance is optionally created on the junction of a second polypeptide (e.g., a second interaction pair) by replacing a large amino acid side chain with a smaller one (e.g., alanine or threonine). In the event that a protuberance or cavity of appropriate location and size is present at the junction of the first or second polypeptide, it is only necessary to engineer the corresponding cavity or protuberance at the adjacent junction, respectively.

[0475] At neutral pH (7.0), aspartic acid and glutamic acid are negatively charged, and lysine, arginine, and histidine are positively charged. These charged residues can be used to promote the formation of heterodimers and simultaneously prevent the formation of homodimers. Attractive interactions occur between opposite charges, and repulsive interactions occur between similar charges. In part, by implementing site-directed mutagenesis of charged junction residues, the protein complexes disclosed herein utilize attractive interactions to promote heteromultimer formation (e.g., heterodimer formation), and optionally, utilize repulsive interactions to prevent homodimer formation (e.g., homodimer formation).

[0476] For example, the IgG1 CH3 domain junction contains four unique pairs of charged residues that participate in domain-domain interactions: Asp356-Lys439', Glu357-Lys370', Lys392-Asp399', and Asp399-Lys409' [residue numbering in the second chain is indicated by (')]. It should be noted that the numbering scheme used to indicate residues in the IgG1 CH3 domain herein is consistent with the EU numbering scheme of Kabat. Due to the 2-fold symmetry present in the CH3-CH3 domain interactions, each unique interaction will be represented twice in the structure (e.g., Asp-399-Lys409' and Lys409-Asp399'). In the wild-type sequence, K409-D399' supports the formation of both heterodimers and homodimers. A single mutation that switches charge polarity in the first chain (e.g., K409E; positive to negative) results in interactions that are unfavorable for the formation of first chain homodimers. Due to the repulsive interaction that occurs between the same charges, an unfavorable interaction (negative-negative; K409E-D399' and D399-K409E') is produced. Similar mutations (D399K'; negative to positive) that convert charge polarity in the second chain result in unfavorable interactions (K409'-D399K' and D399K-K409') for the formation of the second chain homodimer. However, at the same time, these two mutations (K409E and D399K') result in favorable interactions (K409E-D399K' and D399-K409') for the formation of heterodimers. The effect of electrostatic steering on heterodimer formation and homodimer prevention can be further enhanced by the mutation of other charged residues, which may or may not be paired with oppositely charged residues (including, for example, Arg355 and Lys360) in the second chain. See, for example, WO 2016 / 164089.

[0477] Therefore, in some embodiments, the multispecific antigen-binding constructs described herein (e.g., bispecific constructs) may comprise constant domains of immunoglobulins, including, for example, the Fc portion of an immunoglobulin. For example, the first arm may comprise an amino acid sequence derived from the Fc domain of an IgG (IgG1, IgG2, IgG3, or IgG4), IgA (IgA1 or IgA2), IgE, or IgM immunoglobulin. Optionally, the second arm may comprise an amino acid sequence derived from the Fc domain of an IgG (IgG1, IgG2, IgG3, or IgG4), IgA (IgA1 or IgA2), IgE, or IgM. Such immunoglobulin domains may comprise one or more amino acid modifications (e.g., deletions, additions, and / or substitutions) that promote heterodimer formation. In some embodiments, the multispecific antigen-binding construct is an IgG1 isotype. In some embodiments, the multispecific antigen-binding construct is an IgG1 isotype and comprises substitutions. In some embodiments, the multispecific antigen-binding construct is an IgG2 isotype. In some embodiments, the multispecific antigen-binding construct is an IgG3 isotype. In some embodiments, the multispecific antigen-binding construct is an IgG4 isotype. In some embodiments, the multispecific antigen-binding construct is an IgG4 isotype and comprises a substitution. In some embodiments, when numbered according to EU numbering, the substitution is at Ser228. In some embodiments, the substitution at Ser228 is S228P. In some embodiments, the first arm and the second arm comprise Fc domains derived from the same immunoglobulin class and subtype. In some embodiments, the first arm and the second arm comprise Fc domains derived from different immunoglobulin classes or subtypes. Similarly, the first arm and / or the second arm (e.g., an asymmetric pair or a non-guided interaction pair) comprise a modified constant domain of an immunoglobulin, including, for example, one or more amino acid modifications (e.g., deletions, additions, and / or substitutions) that promote heterodimer formation. Methods for producing Fc modifications with desired heterodimer formation are known in the art.

[0478] In some embodiments, the Fc domain can be modified to increase the serum half-life of the multispecific antigen-binding constructs disclosed herein. Fc domains comprising one or more mutations that enhance or reduce antibody binding to Fc receptors at acidic pH compared to neutral pH are known in the art. For example, the constructs disclosed herein may comprise a C-terminal portion of the Fc domain. H 2 or C H Mutations in region 3, wherein the mutations increase the affinity of the Fc domain for FcRn in an acidic environment (e.g., in endosomes at a pH range of about 5.5 to about 6.0). When administered to an animal, such mutations can result in an increase in the serum half-life of the construct. Methods for modifying Fc domains for desired characteristics (such as increased serum half-life) are known in the art.

[0479] In some embodiments, the constructs described herein comprise altered heavy chain constant regions that have reduced (or no) effector functions relative to their corresponding unaltered constant regions. The effector functions of the constant regions of the constructs described herein can be regulated by changing the properties of the constant regions or Fc regions. The altered effector functions include, for example, regulation of one or more of the following activities: antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), apoptosis, binding to one or more Fc-receptors, and proinflammatory responses. Regulation refers to an increase, reduction, or elimination of the effector function activity displayed by an individual antibody containing an altered constant region compared to the activity of the unaltered form of the constant region. In specific embodiments, regulation includes a situation where activity is abolished or completely absent.

[0480] An altered constant region having altered FcR binding affinity and / or ADCC activity and / or altered CDC activity is a polypeptide having enhanced or decreased FcR binding activity and / or ADCC activity and / or CDC activity compared to the unaltered form of the constant region. An altered constant region that exhibits increased binding to an FcR binds to at least one FcR with greater affinity than the unaltered polypeptide. An altered constant region that exhibits decreased binding to an FcR binds to at least one FcR with lower affinity than the unaltered form of the constant region. Such variants that exhibit reduced binding to an FcR can have little or no appreciable binding to an FcR as compared to the level of binding of a native sequence immunoglobulin constant or Fc region to an FcR, e.g., 0 to 50% (e.g., less than 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1%) of FcR binding. Similarly, the altered constant region showing regulated ADCC and / or CDC activity can display ADCC and / or CDC activity that is increased or decreased compared to the unaltered constant region. For example, in some embodiments, any one or more of the antibodies described herein comprising an altered constant region can display about 0 to 50% (e.g., less than 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1%) of the ADCC and / or CDC activity of the unaltered form of the constant region. The multispecific antigen-binding constructs described herein comprising altered constant regions that exhibit reduced ADCC and / or CDC can display reduced or no ADCC and / or CDC activity.

[0481] In some embodiments, the multispecific antigen-binding constructs described herein exhibit reduced or no effector function. In some embodiments, the multispecific antigen-binding constructs comprise a hybrid constant region or portion thereof, such as a G2 / G4 hybrid constant region (see, e.g., Burton et al. (1992) Adv Immun 51: 1-18; Canfield et al. (1991) J Exp Med 173: 1483-1491; and Mueller et al. (1997) Mol Immunol 34 (6): 441-452).

[0482] In some embodiments, the multispecific antigen-binding constructs described herein may contain altered constant regions that exhibit enhanced or reduced complement-dependent cytotoxicity (CDC). Modulated CDC activity can be achieved by introducing one or more amino acid substitutions, insertions, or deletions into the Fc region of an antibody. See, e.g., U.S. Patent No. 6,194,551.

[0483] The constructs and antigen binding arms described herein may partially comprise scaffold domains, proteins or portions, for example, without providing target receptor binding activity, but may provide molecules of portions or domains of the construct, which provide spatial organization, structural support, tools for connecting multiple receptor binding units, or other desired properties (e.g., improved half-life). Various scaffold technologies and compositions are known in the art and can be easily connected or incorporated into the antigen binding units described herein. Scaffold domains, proteins or portions may be derived from antibodies or not. Such scaffold proteins and their domains are generally obtained by combinatorial chemistry-based transformation of pre-existing antigen binding proteins.

[0484] Non-antibody protein scaffolds can be considered to fall into two structural categories: domain-sized constructs (in the range of 6 to 20 kDa) and constrained peptides (in the range of 2 to 4 kDa). Domain-sized non-antibody scaffolds include, but are not limited to, affibodies, affilins, anticalins, atrimers, DARPins, FN3 scaffolds (such as adnectins and centyrins), fynomers, Kunitz domains, probents, and OBodys. Peptide-sized non-antibody scaffolds include, for example, avimers, bicyclic peptides, and cysteine ​​knots. These non-antibody scaffolds and the basic proteins or peptides based thereon or derived therefrom are reviewed by, for example, Simeon and Chen, Protein Cell 9(1):3-14(2018); Vazquez-Lombardi et al., Drug Discovery Today 20:1271-1283(2015), and by Binz et al., Nature Biotechnol. 23:1257-1268(2005) (the contents of each of which are incorporated herein by reference in their entirety). Advantages of using non-antibody scaffolds include increased affinity, target neutralization, and stability. Various non-antibody scaffolds can also overcome some of the limitations of antibody scaffolds, such as tissue penetration, smaller size, and thermal stability. When bispecific constructs are required, some non-antibody scaffolds can also allow for easier construction, not being blocked by, for example, light chain-related issues. Methods for constructing constructs on non-antibody scaffolds are known to those skilled in the art. While not formally on an antibody scaffold, such constructs often comprise an antibody binding domain, whether in the form of a single domain antibody, scFv, or other antibody binding domain variant that provides specific target binding capability.

[0485] Thus, in some embodiments of any one of the aspects described herein, the construct may comprise a non-antibody scaffold protein. In some embodiments of any one of the aspects described herein, at least one of the receptor binding units may comprise a non-antibody scaffold protein. It will be appreciated by those skilled in the art that in some embodiments, the scaffold portion of the non-antibody scaffold protein may comprise, for example, an adhesin scaffold or portion derived from the tenth human fibronectin type III domain (10Fn3); an anticalin scaffold derived from human lipocalin (e.g., such as those described in, for example, WO2015 / 104406); an Eiffel affimer scaffold or protein fragment derived from the A domain of low-density-related protein (LRP) and / or very low-density lipoprotein receptor (VLDLR); a fynomer scaffold or portion of the SH3 domain of FYN tyrosine kinase; a Kunitz-type protease inhibitor (such as human trypsin inhibitor, aprotinin (bovine pancreatic trypsin inhibitor)); In some embodiments, the invention provides a scaffold or portion of a kunitz domain of a protein derived from a cytokine inhibitor (a kinase inhibitor), an Alzheimer's amyloid precursor protein, and a tissue factor pathway inhibitor; a knottin scaffold (cysteine ​​knot microprotein), such as a knottin scaffold based on a trypsin inhibitor from E. elaterium; an affibody scaffold or all or part of the Z domain of Staphylococcus aureus (S. aureus) protein A; a β-hairpin mimetic scaffold; a designed ankyrin repeat protein (DARPin) scaffold or an artificial protein scaffold based on ankyrin repeat (AR) protein; or any scaffold derived from or based on human transferrin, human CTLA-4, human crystallin, and human ubiquitin. For example, the binding sites of human transferrin for the human transferrin receptor can be diversified to create a different library of transferrin variants, some of which have acquired affinities for different antigens. See, for example, Ali et al. (1999) J. Biol. Chem. 274: 24066-24073. The portion of human transferrin that is not involved in binding the receptor remains uncharged and serves as a scaffold, similar to the framework region of an antibody, to present different binding sites. The library is then screened because, according to the methods described herein, the antibody library is directed against the target antigen of interest to identify those variants with the best selectivity and affinity for the target antigen. See, for example, Hey et al. (2005) TRENDSBiotechnol. 23 (10): 514-522.

[0486] D. Methods for Generating Multispecific Antigen-Binding Constructs

[0487] The present disclosure also features methods for producing any of the multispecific antigen-binding constructs described herein. In some embodiments, methods for producing constructs of the present disclosure include methods for preparing antibodies and / or fragments thereof as described herein. Such methods are well known in the art and may include, for example, immunizing an individual (e.g., a non-human mammal) with a suitable immunogen. For example, to produce antibodies that bind to PD-1, one skilled in the art may immunize a suitable individual (e.g., a non-human mammal, such as a rat, mouse, gerbil, hamster, dog, cat, pig, goat, horse, or non-human primate) with a full-length PD-1 polypeptide, such as a full-length human PD-1 polypeptide comprising the amino acid sequence set forth in SEQ ID NO.: 114 (GenBank Accession No. NP_005009.2; UniProtQ15116), an antigenic fragment thereof, and / or a variant thereof. Similarly, to generate antibodies that bind to a PD-1 ligand (e.g., PD-L1), one skilled in the art can immunize a suitable individual with a full-length PD-L1 polypeptide, such as a full-length human PD-L1 polypeptide comprising the amino acid sequence set forth in SEQ ID NO.: 115 (GenBank Accession No. NP_054862.1, UniProt Q9NZQ7), an antigenic fragment thereof, and / or a variant thereof. Similarly, to generate antibodies that bind to PD-L2, one skilled in the art can immunize a suitable individual with a full-length PD-L2 polypeptide, such as a full-length human PD-L2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO.: 116 (GenBank Accession No. NP_079515.2, UniProt Q9BQ51), an antigenic fragment thereof, and / or a variant thereof.

[0488] Those skilled in the art will appreciate that full-length polypeptides (PD-1, PD-L1, or PD-L2) can be used as antigens and that antibodies can be screened for desired binding properties (e.g., blocking PD-1 / ligand interactions; the ability to bridge cells expressing PD-1 and its ligand). Those skilled in the art will also appreciate that antigenic fragments of polypeptides (PD-1, PD-L1, or PD-L2) can be selected based on known structural features of the polypeptides. For example, the PD-1 / PD-L1 and PD-1 / PD-L2 interactions have been structurally well characterized (see, e.g., Zak, K. et al. (2015) Structure 23(12):2341-48; Ghiotto, M. et al. (2010) Int'l Immuno. 22(8):651-60; Freeman, G. (2008) PNAS 105(30):10275-76; Lazar-Molnar, E. et al. (2008) PNAS 105:10483-88, which are incorporated by reference in their entireties). Thus, for example, regions within PD-1, PD-L1, and / or PD-L2 based on receptor / ligand interface information available in the art can be used to design suitable antigen fragments with desired binding properties. For example, the PD-1 extracellular domain contains a single IgV domain typical of the CD28 family, whereas PD-L1 and PD-L2 are composed of IgV and IgC domains typical of the B7 family. The structures of PD-1, PD-L1, and / or PD-L2 exhibit a 1:1 stoichiometry, with interactions primarily between the faces of the IgV domain. The IgV domain comprises approximately 120 amino acids organized into nine parallel beta strands (ABCC'C"DEFG) connected by loops. PD-1 has been shown to bind to the beta face of PD-L1 (GFCC') or the beta face of PD-L2 (AGFC strand and FG loop) using the anterior beta face (GFCC' strand and CC', CC" and FG loops). In addition, six amino acids from the C, F, and G strands of PD-1 form a concave hydrophobic core that interacts with the F and G strands and the FG loop of PD-L2. Eight of the 14 residues involved in binding to PD-1 are identical or highly conserved between PD-L1 and PD-L2. Using this information, one skilled in the art can determine the appropriate antigenic region to generate an antibody with the desired properties. For example, one skilled in the art can generate antibodies that cross-react with both the ligands PD-L1 and PD-L2 (see, e.g., U.S. Patent 9,845,356).

[0489] A suitable individual (e.g., a non-human mammal) can be immunized with a suitable antigen and subsequently boosted multiple times sufficient to induce the production of antibodies in the mammal. An immunogen and an adjuvant can be administered to an individual (e.g., a non-human mammal). Adjuvants that can be used to produce antibodies in an individual include, but are not limited to, protein adjuvants; bacterial adjuvants, for example, whole bacteria (BCG, Corynebacterium parvum, or Salmonella minnesota) and bacterial components thereof, including cell wall skeleton, trehalose dimycolate, monophosphoryl lipid A, methanol extractable residue (MER) of tubercle bacillus, complete or incomplete Freund's adjuvant; viral adjuvants; chemical adjuvants, for example, aluminum hydroxide and iodoacetic acid and cholesterol hemisuccinate. Other adjuvants that can be used in methods of inducing an immune response include, for example, cholera toxin and parapoxvirus proteins. See also Bieg et al. (1999) Autoimmunity 31(1):15-24. See also, e.g., Lodmell et al. (2000) Vaccine 18: 1059-1066; Johnson et al. (1999) J Med Chem 42: 4640-4649; Baldridge et al. (1999) Methods 19: 103-107; and Gupta et al. (1995) Vaccine 13(14): 1263-1276.

[0490] In some embodiments, the method includes preparing a hybridoma cell line that secretes a monoclonal antibody that binds to an immunogen. For example, a suitable mammal (such as a laboratory mouse) is immunized with a polypeptide (e.g., PD-1, PD-L1, PD-L2) or antigen fragment as described above. Antibody-producing cells (e.g., B cells of the spleen) of the immunized mammal can be isolated 2 to 4 days after at least one booster immunization with the immunogen and then grown briefly in culture before being fused with cells of a suitable myeloma cell line. The cells can be fused in the presence of a fusion promoter (such as, for example, vaccinia virus or polyethylene glycol). The hybrid cells obtained in the fusion are cloned, and cell clones that secrete the desired antibodies are selected. For example, spleen cells of Balb / c mice immunized with a suitable immunogen can be fused with cells of the myeloma cell line PAI or the myeloma cell line Sp2 / 0-Ag 14. After fusion, the cells are expanded at regular intervals in a suitable culture medium supplemented with a selection medium (e.g., HAT medium) to prevent normal myeloma cells from growing beyond the desired hybridoma cells. The resulting hybridoma cells are then screened for secretion of the desired antibody (eg, an antibody that binds to PD-1).

[0491] In some embodiments, one skilled in the art can identify anti-PD-1 antibodies from a non-immune biased library, as described in, for example, U.S. Patent No. 6,300,064 (issued to Knappik et al.; Morphosys AG) and Schoonbroodt et al. (2005) Nucleic Acids Res 33(9):e81.

[0492] In some embodiments, the methods described herein can include or be combined with the use of, for example, phage display technology, bacterial display, yeast surface display, eukaryotic virus display, mammalian cell display, and cell-free (e.g., ribosome display) antibody screening technology (see, e.g., Etz et al. (2001) J Bacteriol 183:6924-6935; Cornelis (2000) Curr Opin Biotechnol 11:450-454; Klemm et al. (2000) Microbiology 146:3025-3032; Kieke et al. (1997) Protein Eng 10:1303-1310; Yeung et al. (2002) Biotechnol Prog 18:212-220; Boder et al. (2000) Methods Enzymology 328:430-444; Grabherr et al. (2001) Comb Chem High Throughput Screen 4:185-192; Michael et al. (1995) Gene Ther 2:660-668; Pereboev et al. (2001) J Virol 75:7107-7113; Schaffitzel et al. (1999) J Immunol Methods 231:119-135; and Hanes et al. (2000) Nat Biotechnol 18:1287-1292).

[0493] Methods for identifying antibodies using various phage display methods are known in the art. In phage display methods, functional antibody domains are displayed on the surface of phage particles carrying the polynucleotide sequence encoding them. The antigen binding domains of this phage display antibody can be utilized, such as Fab, Fv expressed from a library or a combinatorial antibody library (e.g., humans or murines) or Fv antibody fragments stabilized by disulfide bonds. The phage used in these methods is typically a filamentous phage, such as fd and M13. The antigen binding domain is expressed as a protein recombinantly fused to any one of the phage coat proteins pIII, pVIII or pIX. See, for example, Shi et al. (2010) JMB 397:385-396. Examples of phage display methods that can be used to prepare the immunoglobulins or fragments thereof described herein include those disclosed in Brinkman et al. (1995) J Immunol Methods 182:41-50; Ames et al. (1995) J Immunol Methods 184:177-186; Kettleborough et al. (1994) Eur J Immunol 24:952-958; Persic et al. (1997) Gene 187:9-18; Burton et al. (1994) Advances in Immunology 57:191-280; and PCT Publication Nos. WO 90 / 02809, WO 91 / 10737, WO 92 / 01047, WO 92 / 18619, WO 93 / 11236, WO 95 / 15982, and WO 95 / 20401. Suitable methods are also described, for example, in U.S. Pat. Nos. 5,698,426, 5,223,409, 5,403,484, 5,580,717, 5,427,908, 5,750,753, 5,821,047, 5,571,698, 5,427,908, 5,516,637, 5,780,225, 5,658,727, 5,733,743, and 5,969,108.

[0494] In some embodiments, mRNA collected from B cells of immunized mammals can be used to generate phage display antibody libraries. For example, a spleen cell sample containing B cells can be isolated from a mouse immunized with a PD-1 polypeptide as described above. mRNA can be isolated from the cells and converted into cDNA using standard molecular biology techniques. See, for example, Sambrook et al. (1989) "Molecular Cloning: A Laboratory Manual, 2nd Edition", Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Harlow and Lane (1988), supra; Benny KCLo (2004), supra; and Borrebaek (1995), supra. cDNAs encoding the variable regions of the heavy and light chain polypeptides of immunoglobulins are used to construct phage display libraries. Methods for generating such libraries are described, for example, in Merz et al. (1995) J Neurosci Methods 62(1-2):213-9; Di Niro et al. (2005) Biochem J 388(Pt 3):889-894; and Engberg et al. (1995) Methods Mol Biol 51:355-376.

[0495] In some embodiments, a combination of selection and screening can be used to identify antibodies of interest from, for example, a population of antibodies derived from hybridomas or a phage-displayed antibody library. Suitable methods are known in the art and are described, for example, in Hoogenboom (1997) Trends in Biotechnology 15: 62-70; Brinkman et al. (1995), supra; Ames et al. (1995), supra; Kettleborough et al. (1994), supra; Persic et al. (1997), supra; and Burton et al. (1994), supra. For example, a plurality of phagemid vectors encoding fusion proteins of a phage coat protein (e.g., pIII, pVIII, or pIX of M13 phage) and different antigen combination regions are generated using standard molecular biology techniques and then introduced into a bacterial (e.g., Escherichia coli (E. coli)) population. In some embodiments, expression of phage in bacteria may require the use of a helper phage. In some embodiments, a helper phage is not required (see, e.g., Chasteen et al. (2006) Nucleic Acids Res 34(21):e145). Phage produced from bacteria are recovered and then contacted with, for example, a target antigen bound to a solid support (immobilized). Phage and antigen can also be contacted in solution, followed by binding of the complex to a solid support.

[0496] Any immunological or biochemical method known in the art can be used to characterize the specificity and binding affinity of the subgroup of antibodies screened using the above method to a specific antigen (e.g., human PD-1). For example, the specific binding of the antibody to PD-1 can be determined, for example, using an immunological or biochemical method (such as, but not limited to, ELISA assay, SPR assay, immunoprecipitation assay, affinity chromatography, and equilibrium dialysis as described above). Immunoassays that can be used to analyze the immunospecific binding and cross-reactivity of antibodies include, but are not limited to, competitive and non-competitive assay systems using techniques such as Western blot, RIA, ELISA (enzyme-linked immunosorbent assay), "sandwich" immunoassay, immunoprecipitation assay, immunodiffusion assay, agglutination assay, complement fixation assay, immunoradiometric assay, fluorescent immunoassay, and protein A immunoassay. Such assays are conventional and well known in the art.

[0497] It will be appreciated that the above methods can also be used to determine, for example, whether an anti-PD-1 antibody does not bind to full-length human PD-1 and / or PD-1 protein.

[0498] In embodiments where the selected CDR amino acid sequence is a short sequence (e.g., less than 10 to 15 amino acids in length), nucleic acids encoding the CDRs can be chemically synthesized as described in, for example, Shiraishi et al. (2007) Nucleic Acids Symposium Series 51(1):129-130 and U.S. Patent No. 6,995,259. For a given nucleic acid sequence encoding an acceptor antibody, regions of the nucleic acid sequence encoding the CDRs can be replaced with chemically synthesized nucleic acids using standard molecular biology techniques. The 5' and 3' ends of the chemically synthesized nucleic acid can be synthesized to include sticky end restriction enzyme sites for cloning the nucleic acid into the nucleic acid encoding the variable region of the donor antibody. Alternatively, fragments of chemically synthesized nucleic acids that together encode the antibody can be joined together using DNA assembly techniques known in the art (e.g., Gibson assembly).

[0499] Any antibody selected can be further modified to produce an antigen-binding fragment as described herein, and / or manipulated using techniques known in the art to produce a multispecific antigen-binding construct as described herein. For example, a cross-linking method can be used to produce a bispecific structure using a heterobifunctional reagent having an amine-reactive group and a sulfhydryl-reactive group, as described in, for example, U.S. Patent No. 4,433,059; bispecific antibody determinants can be produced by recombining half antibodies (heavy chain-light chain pairs or Fabs) from different antibodies through a reduction and oxidation cycle of a disulfide bond between two heavy chains, as described in, for example, U.S. Patent No. 4,444,878; trifunctional antibodies, for example, three Fab' fragments can be cross-linked by a sulfhydryl-reactive group, as described in, for example, U.S. Patent No. 5,273,743. Other methods for producing bispecific constructs are described herein, for example, methods for producing bispecific constructs with a common light chain. Non-limiting examples of the amino acid sequence of the common light chain used in the constructs described herein include SEQ ID NOs: 59-63.

[0500] E. Expression and Purification of Multispecific Antigen-Binding Constructs

[0501] Various techniques known in the art of molecular biology and protein chemistry can be used to produce the multispecific antigen-binding constructs described herein. For example, a nucleic acid encoding a multispecific antigen-binding construct (as a single multifunctional polypeptide or as a separate molecule of a multimeric complex - for example, one antigen-binding arm is independent of another antigen-binding arm) can be inserted into an expression vector containing transcriptional and translational regulatory sequences, including, for example, promoter sequences, ribosome binding sites, transcriptional start and stop sequences, translational start and stop sequences, transcriptional termination signals, polyadenylation signals, and enhancer or activator sequences. The regulatory sequences include promoters and transcriptional start and stop sequences. In addition, the expression vector may include more than one replication system so that it can be maintained in two different organisms, for example, in mammalian or insect cells for expression and in prokaryotic hosts for cloning and amplification.

[0502] Several possible vector systems can be used to express cloned heavy and light chain polypeptides according to nucleic acid in mammalian cells. One type of vector relies on the integration of the desired gene sequence into the host cell genome. Cells that have stably integrated DNA can be selected by simultaneously introducing drug resistance genes, such as Escherichia coli gpt (Mulligan and Berg (1981) Proc Natl Acad Sci USA 78: 2072) or Tn5 neo (Southern and Berg (1982) Mol Appl Genet 1: 327). Selectable marker genes can be connected to the DNA gene sequence to be expressed, or introduced into the same cell by co-transfection (Wigler et al. (1979) Cell 16: 77). The second type of vector utilizes DNA elements that confer autonomous replication ability to extrachromosomal plasmids. These vectors may be derived from animal viruses such as bovine papillomavirus (Sarver et al. (1982) Proc Natl Acad Sci USA, 79:7147), cytomegalovirus, polyoma virus (Deans et al. (1984) Proc Natl Acad Sci USA 81:1292), or SV40 virus (Lusky and Botchan (1981) Nature 293:79).

[0503] The expression vector can be introduced into the cell in a manner suitable for the subsequent expression of the nucleic acid. The method of introduction is primarily indicated by the target cell type discussed below. Exemplary methods include CaPO precipitation, liposome fusion, cationic liposomes, electroporation, viral transfection, dextran-mediated transfection, polybrene-mediated transfection, protoplast fusion, and direct microinjection.

[0504] Suitable host cells for expression of antibodies or antigen-binding fragments thereof include yeast, bacteria, insects, plants, and mammalian cells. Of particular interest are bacteria (such as E. coli), fungi (such as Saccharomyces cerevisiae and Pichia pastoris), insect cells (such as SF9), mammalian cell lines (e.g., human cell lines), and primary cell lines.

[0505] In some embodiments, the antibodies or fragments thereof can be expressed and purified in transgenic animals (e.g., transgenic mammals). For example, antibodies can be produced in transgenic non-human mammals (e.g., rodents) and isolated from milk as described in, for example, Houdebine (2002) Curr Opin Biotechnol 13(6):625-629; van Kuik-Romeijn et al. (2000) Transgenic Res 9(2):155-159; and Pollock et al. (1999) J Immunol Methods 231(1-2):147-157.

[0506] The antibody and its fragment can be produced from cells by culturing host cells transformed with expression vectors containing nucleic acids encoding antibodies or their fragments under culture conditions and time sufficient to allow protein expression. Such conditions for protein expression will vary with the selection of expression vectors and host cells, and those skilled in the art can easily determine by routine experiments. For example, antibodies expressed in Escherichia coli can be refolded from inclusion bodies (see, for example, Hou et al. (1998) Cytokine 10:319-30). Bacterial expression systems and methods of using them are well known in the art (see Current Protocols in Molecular Biology, Wiley & Sons, and Molecular Cloning--A Laboratory Manual--3rd Edition, Cold Spring Harbor Laboratory Press, New York (2001)). The selection of codons, suitable expression vectors and suitable host cells will vary according to many factors and can be easily optimized as needed. The antibodies (or fragments thereof) described herein can be expressed in mammalian cells or in other expression systems, including but not limited to yeast, baculovirus, and or in vitro expression systems (see, e.g., Kaszubska et al. (2000) Protein Expression and Purification 18:213-220).

[0507] After expression, antibodies and fragments thereof can be isolated. Antibodies or fragments thereof can be isolated or purified by various methods known to those skilled in the art, depending on the other components present in the sample. Standard purification methods include electrophoresis, molecular techniques, immunological techniques, and chromatography techniques (including ion exchange, hydrophobic, affinity, and reversed-phase HPLC chromatography). For example, antibodies can be purified using standard anti-antibody columns (e.g., protein-A or protein-G columns). Ultrafiltration and diafiltration techniques can also be used in conjunction with protein concentration. See, for example, Scopes (1994) "Protein Purification, 3rd Edition", Springer-Verlag, New York City, New York. The necessary degree of purification will vary depending on the desired use. In some instances, it will be necessary not to purify the expressed antibody or fragment thereof.

[0508] Methods for determining the yield or purity of a purified antibody or fragment thereof are known in the art and include, for example, the Bradford assay, UV spectroscopy, biuret protein assay, Lowry protein assay, amidoblack protein assay, high pressure liquid chromatography (HPLC), mass spectrometry (MS), and gel electrophoresis (e.g., using a protein stain such as Coomassie blue or colloidal silver stain).

[0509] F. Modification of Multispecific Antigen-Binding Constructs

[0510] The multispecific antigen-binding construct can be modified into a single multifunctional polypeptide or into separate molecules of a multimeric complex, e.g., one antigen-binding arm is separated from another antigen-binding arm, according to its expression and purification. The modification can be covalent or non-covalent. Such modifications can be introduced into the antibody or antigen-binding fragment by, for example, reacting the targeted amino acid residues of the polypeptide with an organic derivatizing agent that reacts with selected side chains or terminal residues. Suitable sites for modification can be selected using any of a variety of criteria, including, for example, structural analysis or amino acid sequence analysis of the antibody or fragment.

[0511] The amino acid sequences provided herein are shown in single-letter amino acid codes, which are interchangeable with the three-letter amino acid codes. An amino acid refers to any monomeric unit that can be incorporated into a peptide, polypeptide, or protein. The 20 naturally occurring or genetically encoded α-amino acids are as follows: alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartic acid (Asp or D), cysteine ​​(Cys or C), glutamine (Gln or Q), glutamic acid (Glu or E), glycine (Gly or G), histidine (His or H), isoleucine (Ile or I), leucine (Leu or L), lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y), and valine (Val or V). The structures of the 20 natural amino acids are shown, for example, in Stryer et al., Biochemistry, 5th ed., Freeman and Company (2002). The term amino acid also includes unnatural amino acids, modified amino acids (e.g., with modified side chains and / or backbones), and amino acid analogs.

[0512] In the context of two or more nucleic acid or polypeptide sequences, the terms identical or percent identity refer to two or more sequences or subsequences that are identical or have a specified percentage (e.g., 90% or 95% or greater identity over a specified region) of nucleotide or amino acid residues that are the same, when compared and aligned for maximum correspondence over a comparison window or designated region, as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection.

[0513] Sequence identity or similarity is defined as the percentage of amino acid residues in the candidate sequence that are identical to the starting amino acid residue (i.e., identical residues), after aligning the sequences and introducing gaps (if necessary) to achieve the maximum percentage of sequence identity. Methods of sequence alignment for comparison are well known in the art. Optimal alignment of sequences for comparison can be performed, for example, by the local homology algorithm of Smith and Waterman (Adv. Appl. Math. 2:482, 1970), by the homology alignment algorithm of Needleman and Wunsch (J. Mol. Biol. 48:443, 1970), by the search similarity method of Pearson and Lipman (Proc. Natl. Acad. Sci. USA 85:2444, 1988), by computer implementations of these algorithms (e.g., GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by manual alignment and visual inspection (see, e.g., Ausubel et al., Current Protocols in Molecular Biology (1995 suppl)).

[0514] It will be appreciated that, as with all peptides, polypeptides, and proteins (including fragments thereof), additional modifications in the amino acid sequences of the constructs, antibodies, or antigen-binding portions thereof described herein (e.g., in the heavy chain variable region and / or light chain variable region) may occur without altering the properties or function of the antibody or antigen-binding fragment thereof. Such modifications include conservative amino acid substitutions, such that each of the described sequences optionally contains one or more conservative amino acid substitutions. The following groups each contain amino acids that are conservative substitutions for one another. These groups are examples and other conservative substitutions are known to those skilled in the art.

[0515] 1) Alanine (A), glycine (G);

[0516] 2) Aspartic acid (D), glutamic acid (E);

[0517] 3) Asparagine (N), glutamine (Q);

[0518] 4) Arginine (R), Lysine (K);

[0519] 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);

[0520] 6) Phenylalanine (F), tyrosine (Y), tryptophan (W);

[0521] 7) Serine (S), Threonine (T); and

[0522] 8) Cysteine ​​(C), Methionine (M)

[0523] For example, when reference is made to aspartic acid at a particular residue, conservative substitutions at that residue, e.g., glutamic acid, are also contemplated. Non-conservative substitutions, e.g., substitution of proline with glycine, are also contemplated.

[0524] In some embodiments, the construct, antibody, or antigen-binding portion thereof can be conjugated to a heterologous moiety. The heterologous moiety can be, for example, a heterologous polypeptide, a therapeutic agent (e.g., a toxin or a drug), or a detectable label, such as, but not limited to, a radiolabel, an enzymatic label, a fluorescent label, a heavy metal label, a luminescent label, or an affinity tag (such as biotin or streptavidin). Suitable heterologous polypeptides include, for example, antigenic tags for purification of antibodies or fragments (e.g., FLAG (DYKDDDDK) (SEQ ID NO: 117), polyhistidine (6-His; HHHHHH (SEQ ID NO: 118)), hemagglutinin (HA; YPYDVPDYA (SEQ ID NO: 119)), glutathione-S-transferase (GST), or maltose binding protein (MBP)). Heterologous polypeptides also include polypeptides (e.g., enzymes) that can be used as diagnostic or detectable markers, for example, luciferase, fluorescent proteins (e.g., green fluorescent protein (GFP)), or chloramphenicol acetyltransferase (CAT). Suitable radiolabels include, for example, 32 P. 33 P. 14 C. 125 I. 131 I. 35 S and 3 H. Suitable fluorescent labels include, but are not limited to, fluorescein, fluorescein isothiocyanate (FITC), green fluorescent protein (GFP), DYLIGHT TM 488, Phycoerythrin (PE), Propidium Iodide (PI), PerCP, PE-ALEXA 700, Cy5, allophycocyanin, and Cy7. Luminescent labels include, for example, any of various luminescent lanthanide (e.g., europium or terbium) chelates. For example, suitable europium chelates include europium chelates of diethylenetriaminepentaacetic acid (DTPA) or tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA). Enzymatic labels include, for example, alkaline phosphatase, CAT, luciferase, and horseradish peroxidase.

[0525] Two proteins (e.g., an antibody and a heterologous moiety) can be cross-linked using any of a number of known chemical cross-linking agents. Examples of such cross-linking agents are those that connect two amino acid residues via a linkage, including a "hindered" disulfide bond. In these linkages, the disulfide bond within the cross-linking unit is protected (by hindering groups on either side of the disulfide bond) from reduction by, for example, reduced glutathione or disulfide reductase. One suitable reagent, 4-succinyloxycarbonyl-α-methyl-α(2-pyridyldisulfide)toluene (SMPT), utilizes a terminal lysine on one of the proteins and a terminal cysteine ​​on the other to form this linkage between the two proteins. Heterobifunctional reagents that cross-link through different coupling moieties on each protein can also be used. Other useful cross-linking agents include, but are not limited to, reagents that link two amino groups (e.g., N-5-azido-2-nitrobenzoyloxysuccinimide), reagents that link two sulfhydryl groups (e.g., 1,4-bis-maleimidobutane), reagents that link an amino group and a sulfhydryl group (e.g., m-maleimidobenzoyl-N-hydroxysuccinimide ester), reagents that link an amino group and a carboxyl group (e.g., 4-[p-azidosalicylamido]butylamine), and reagents that link an amino group and the guanidinium group present in the arginine side chain (e.g., p-azidophenylglyoxal monohydrate).

[0526] In some embodiments, the radiolabel can be directly bound to the amino acid backbone of the antibody. Alternatively, the radiolabel can be included as part of a larger molecule (e.g., a meta-iodophenyl (mIP) derivative (see, e.g., Rogers et al. (1997) J Nucl Med 38: 1221-1229) or a chelate (e.g., to DOTA or DTPA)) that is bound to a free amino acid to form a protein of interest. 125 I] Iodophenyl-N-hydroxysuccinimide ([ 125 I]mIPNHS) 125 I), which in turn binds to the protein backbone. Methods for conjugating radiolabels or larger molecules / chelates containing them to the antibodies or antigen-binding fragments described herein are known in the art. Such methods include incubating the protein with the radiolabel under conditions (e.g., pH, salt concentration, and / or temperature) that promote binding of the radiolabel or chelate to the protein (see, e.g., U.S. Patent No. 6,001,329).

[0527] Methods for conjugating fluorescent tags (sometimes referred to as "fluorophores") to proteins (e.g., antibodies) are known in the art of protein chemistry. For example, a fluorophore can be conjugated to a free amine group (e.g., of a lysine) or sulfhydryl group (e.g., cysteine) of a protein using a succinyl (NHS) ester or tetrafluorophenyl (TFP) ester moiety attached to the fluorophore. In some embodiments, the fluorophore can be conjugated to a heterobifunctional cross-linker moiety, such as sulfo-SMCC. Suitable conjugation methods include incubating the antibody protein or a fragment thereof with the fluorophore under conditions that promote binding of the fluorophore to the protein. See, for example, Welch and Redvanly (2003) "Handbook of Radiopharmaceuticals: Radiochemistry and Applications", John Wiley and Sons (ISBN 0471495603).

[0528] In some embodiments, the antibody or fragment can be modified, for example, with a moiety that improves the stabilization and / or retention of the antibody in the circulation of, for example, blood, serum, or other tissues. For example, the antibody or fragment can be PEGylated or HESylated as described, for example, in Lee et al. (1999) Bioconjug Chem 10(6):973-8; Kinstler et al. (2002) Advanced Drug Deliveries Reviews 54:477-485; and Roberts et al. (2002) Advanced Drug Delivery Reviews 54:459-476 (Fresenius Kabi, Germany; see, e.g., et al. (2010) Int J Pharm 387(1-2):110-119). The stabilizing moiety can improve the stability or retention of the antibody (or fragment) by at least 1.5 (e.g., at least 2, 5, 10, 15, 20, 25, 30, 40, or 50 or more) fold.

[0529] In some embodiments, the antibodies or antigen-binding fragments thereof described herein may be glycosylated. In some embodiments, the antibodies or antigen-binding fragments thereof described herein may be enzymatically or chemically treated, or produced from cells, such that the antibodies or fragments have reduced or absent glycosylation. Methods for producing antibodies with reduced glycosylation are known in the art and are described, for example, in U.S. Patent No. 6,933,368; Wright et al. (1991) EMBO J 10(10):2717-2723; and Co et al. (1993) Mol Immunol 30:1361. In some embodiments, the antibodies or antigen-binding fragments thereof are aglycosylated.

[0530] G. Pharmaceutical Compositions and Formulations

[0531] The present disclosure also provides pharmaceutical compositions comprising the multispecific antigen-binding constructs of the present disclosure and a pharmaceutically acceptable diluent, carrier, solubilizer, emulsifier, preservative, and / or adjuvant for use with the methods disclosed herein. Such pharmaceutical compositions can be used in individuals suffering from, for example, a cancer as disclosed herein.

[0532] In certain embodiments, acceptable formulations are preferably non-toxic to the recipient at the dosages and concentrations employed. In certain embodiments, the formulations are for subcutaneous and / or intravenous administration. In certain embodiments, pharmaceutical compositions may include formulations for modifying, maintaining, or preserving, for example, the pH, osmotic pressure, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, absorption, or penetration of the composition. In certain embodiments, suitable formulators include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine, or lysine); antimicrobial agents; antioxidants (such as ascorbic acid, sodium sulfite, or sodium bisulfite); buffers (such as borates, bicarbonates, Tris-HCl, citrates, phosphates, or other organic acids); bulking agents (such as mannitol or glycine); chelating agents (such as ethylenediaminetetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, β-cyclodextrin, or hydroxypropyl-β-cyclodextrin); fillers; monosaccharides; disaccharides; and other carbohydrates (such as glucose, mannose, or dextrin); proteins (such as serum albumin, gelatin, or immunoglobulins); colorants, flavorings, and diluents; emulsifiers; hydrophilic polymers (such as polyvinylpyrrolidone); low molecular weight the polypeptide; a salt-forming counterion (such as sodium); a preservative (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenylethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid or hydrogen peroxide); a solvent (such as glycerol, propylene glycol or polyethylene glycol); a sugar alcohol (such as mannitol or sorbitol); a suspending agent; a surfactant or wetting agent (such as pluronic, PEG, sorbitan esters, polysorbates (such as polysorbate 20, polysorbate 80), triton, tromethamine, lecithin, cholesterol, tyloxapal); a stability enhancer (such as sucrose or sorbitol); a tonicity enhancer (such as an alkali metal halide (preferably sodium chloride or potassium chloride), mannitol, sorbitol); a delivery vehicle; a diluent; an excipient and / or a pharmaceutical adjuvant. (Remington's Pharmaceutical Sciences, 18th ed., A.R. Gennaro, ed., Mack Publishing Company (1995)). In certain embodiments, the formulation comprises PBS; 20 mM NaOAC, pH 5.2, 50 mM NaCl; and / or 10 mM NAOAC, pH 5.2, 9% sucrose. In certain embodiments, the optimal pharmaceutical composition will be determined by one skilled in the art based on, for example, the intended route of administration, the delivery form, and the desired dosage. See, for example, Remington's Pharmaceutical Sciences, supra.In certain embodiments, such compositions may affect the physical state, stability, rate of in vivo release, and / or in vivo clearance of the multispecific antigen-binding construct.

[0533] In certain embodiments, the main medium or carrier in the pharmaceutical composition may be naturally aqueous or non-aqueous. For example, in certain embodiments, a suitable medium or carrier may be water for injection, physiological saline solution or artificial cerebrospinal fluid, which may be supplemented with other substances commonly found in compositions for parenteral administration. In certain embodiments, saline comprises isotonic phosphate buffered saline. In certain embodiments, neutral buffered saline or saline mixed with serum albumin is another exemplary medium. Therefore, in certain embodiments, the pharmaceutical composition comprises a Tris buffer of about pH 7.0 to 8.5 or an acetate buffer of about pH 4.0 to 5.5, which may further comprise sorbitol or a suitable substitute. In certain embodiments, a composition comprising a multispecific antigen-binding construct disclosed herein can be prepared in the form of a lyophilized cake or aqueous solution by mixing the selected composition with the desired purity with an optional formulation (Remington's Pharmaceutical Sciences, see above) for storage. In addition, in certain embodiments, a suitable excipient (such as sucrose) can be used to formulate a composition comprising a multispecific antigen-binding construct disclosed herein into a lyophilized product.

[0534] In certain embodiments, the pharmaceutical composition can be selected for parenteral delivery. In certain embodiments, the composition can be selected for inhalation or for delivery through the digestive tract, such as orally. The preparation of such pharmaceutically acceptable compositions is within the capabilities of those skilled in the art.

[0535] In certain embodiments, the formulation components are present in concentrations acceptable to the site of administration.In certain embodiments, a buffer is used to maintain the composition at physiological pH or at slightly lower pH, typically within the pH range of about 5 to about 8.

[0536] In certain embodiments, when considering parenteral administration, the therapeutic composition can be in the form of a pyrogen-free parenteral acceptable aqueous solution, which includes a multispecific antigen binding construct in a pharmaceutically acceptable vehicle. In certain emb...

Claims

1. A multispecific antigen-binding construct comprising at least two antigen-binding arms, wherein: The first arm binds to PD-1 and comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region of the first arm comprises CDRH1 of SEQ ID NO: 77, CDRH2 of SEQ ID NO: 71, and CDRH3 of SEQ ID NO: 75, The second arm binds to the PD-1 ligand and comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region of the second arm comprising CDRH1 of SEQ ID NO: 1, CDRH2 of SEQ ID NO: 7, and CDRH3 of SEQ ID NO: 8; in, The light chain variable region of the first arm and the light chain variable region of the second arm both comprise CDRL1 of SEQ ID NO: 9, CDRL2 of SEQ ID NO: 5, and CDRL3 of SEQ ID NO: 10; and Wherein, the multispecific antigen-binding construct blocks the interaction between PD-1 and PD-1 ligand.

2. The multispecific antigen-binding construct of claim 1, wherein the heavy chain variable region of the first arm comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:

90.

3. The multispecific antigen-binding construct of claim 1, wherein the heavy chain variable region of the first arm comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:

90.

4. The multispecific antigen-binding construct of claim 1, wherein the heavy chain variable region of the second arm comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:

55.

5. The multispecific antigen-binding construct of claim 1, wherein the heavy chain variable region of the second arm comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:

55.

6. The multispecific antigen-binding construct of claim 1, wherein the first and second antigen-binding arms both comprise a common light chain variable region.

7. The multispecific antigen-binding construct of claim 1, wherein the construct comprises at least two arms that bind to PD-1 and at least two arms that bind to PD-L1.

8. The multispecific antigen-binding construct of claim 1, wherein the construct comprises a bivalent antibody specific for PD-1 and a bivalent antibody specific for PD-L1, wherein the bivalent antibody specific for PD-1 and the bivalent antibody specific for PD-L1 are linked.

9. The multispecific antigen-binding construct of claim 1 , wherein the heavy chain of the first arm has an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 100, and wherein the light chains of the first arm and the second arm have an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:

101.

10. The multispecific antigen-binding construct of claim 1, wherein the heavy chain of the first arm has an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 100, and wherein the light chains of the first arm and the second arm have an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:

101.

11. The multispecific antigen-binding construct of claim 1, wherein the heavy chain of the first arm has the amino acid sequence of SEQ ID NO: 100, and wherein the light chains of the first arm and the second arm have the amino acid sequence of SEQ ID NO:

101.

12. The multispecific antigen-binding construct of claim 1, wherein the heavy chain variable region of the first arm comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:

90.

13. The multispecific antigen-binding construct of claim 1, wherein the heavy chain variable region of the first arm comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO:

90.

14. The multispecific antigen-binding construct of claim 1, wherein the heavy chain variable region of the first arm comprises the amino acid sequence of SEQ ID NO:

90.

15. The multispecific antigen-binding construct of claim 1, wherein the heavy chain variable region of the second arm comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:

55.

16. The multispecific antigen-binding construct of claim 1, wherein the heavy chain variable region of the second arm comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO:

55.

17. The multispecific antigen-binding construct of claim 1, wherein the heavy chain variable region of the second arm comprises the amino acid sequence of SEQ ID NO:

55.

18. The multispecific antigen-binding construct of claim 1, wherein the heavy chain of the first arm has an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 100, and wherein the light chains of the first arm and the second arm have an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:

101.

19. The multispecific antigen-binding construct of claim 1, wherein the heavy chain of the first arm has an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 100, and wherein the light chains of the first arm and the second arm have an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO:

101.

20. The multispecific antigen-binding construct of claim 1, wherein the first arm binds to PD-1 expressed by immune cells.

21. The multispecific antigen-binding construct of claim 1, wherein the second arm binds a PD-1 ligand expressed by a second cell.

22. The multispecific antigen-binding construct of claim 21, wherein the second cell is a tumor cell and the PD-1 ligand is PD-L1.

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