Multispecific binding constructs for checkpoint molecules and uses thereof

Abstract

To provide novel therapeutics that effectively counteract immune evasion.SOLUTION: The present disclosure relates to compositions and methods for inhibiting tumor evasion by reducing immune checkpoint suppression. In some embodiments, provided herein are compositions that block the interaction between PD-1 and its ligand (e.g., PD-1 and / or PD-L2) while promoting the interaction of cells on which PD-1 and its ligand are expressed. Also provided are methods of using such compositions.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present disclosure relates to multispecific binding constructs to checkpoint molecules and uses thereof. [Background technology]

[0002] Cancer is one of the leading causes of death both in the United States and worldwide. Common treatments such as surgery, radiation, chemotherapy, hormone therapy, targeted therapy, and immunotherapy reduced the rate of cancer-related deaths during the 20th century, but as of 2012, there were 14.1 million newly diagnosed cancer cases and 8.2 million cancer deaths worldwide. Although there have been improvements in overall cancer survival rates during the 20th century, cancer still causes 1 in 7 deaths worldwide. See Non-Patent Document 1.

[0003] In recent years, increasing evidence suggests that the immune system acts as a critical barrier against tumor formation and progression. The principle that naturally occurring T cells with anti-tumor potential or activity exist in patients with cancer has rationalized the development of immunotherapeutic approaches in oncology. Immune cells such as T cells, macrophages, and natural killer cells exhibit anti-tumor activity and can effectively control the emergence and growth of malignant tumors. Tumor-specific or associated antigens can induce immune cells to recognize and eliminate malignant tumors (Non-Patent Document 2). Despite the existence of tumor-specific immune responses, malignant tumors often escape or evade immune attack through various immunoregulatory mechanisms, resulting in a failure to control tumor emergence and progression (Non-Patent Document 3). Indeed, an emerging hallmark of cancer is the inability to utilize these immunoregulatory mechanisms and mount an anti-tumor immune response, resulting in tumor escape and evasion from immunological killing (Non-Patent Document 4). [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] American Cancer Society, Global Cancer Facts & Figures 3rd Edition, American Cancer Society [Atlanta, USA], 2015 [Non-patent document 2] Chen and Mellman, (2013) Immunity 39(1):1-10 [Non-patent document 3] Motz and Coukos, (2013) Immunity 39(1):61-730 [Non-patent document 4] Hanahan and Weinberg (2011) Cell 144(5):646-674 Summary of the Invention [Problem to be solved by the invention]

[0005] Novel approaches in cancer immunotherapy involve countering these immune evasion and escape mechanisms and inducing the endogenous immune system to reject tumors. However, there remains a need for novel therapies that effectively counter immune evasion, particularly in cancer therapy. [Means for solving the problem]

[0006] The present disclosure is based in part on novel multispecific and multivalent constructs, e.g., bispecific and tetravalent constructs, that target both PD-1 and PD-L1. As demonstrated herein, these multispecific constructs have improved in vitro and in vivo efficacy compared to clinical checkpoint blockade agents, as well as to combinations of individual antibodies. 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 and multivalent constructs. Some of these novel monoclonal anti-PD-1 antibodies and novel monoclonal anti-PD-L1 antibodies share a common light chain, thereby enabling the generation of multispecific and multivalent constructs with affinities similar to their parent antibodies, as well as surprisingly excellent drug-like properties (DLPs) and ease of production. The present disclosure is also based, in part, on the discovery that 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 and 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. Thus, provided herein are compositions that block the interaction between PD-1 and its ligand (PD-L1 or PD-L2) while promoting the interaction (bridging) between cells expressing these ligands. As exemplified herein, such compositions of the present disclosure capable of "blocking and bridging" provide superior anti-tumor efficacy (e.g., as measured by IFNγ production and secretion and in vivo activity) compared to, for example, cocktails having stoichiometric amounts of agents that bind to the receptor and ligand separately, or single agents that bind to either the receptor or the ligand. It was also found that the multispecific and multivalent constructs targeting both PD-1 and PD-L1 described herein caused loss of expression of PD-1 on the cell surface in a valency-dependent manner.This loss of PD-1 expression was not observed when the parent antibody combination was used in stoichiometrically equivalent amounts. Thus, the multispecific and multivalent constructs targeting both PD-1 and PD-L1 described herein provide novel immunotherapeutic agents with increased potency and efficacy for use in the treatment of cancer.

[0007] In some embodiments, any multispecific antigen-binding construct disclosed herein binds to at least two different receptors or epitopes (e.g., PD-1 and PD-L1), and 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, a multispecific antigen-binding construct simultaneously binds to PD-1 and PD-L1, and PD-1 and PD-L1 are expressed on the surface of the same cell. In some embodiments, any multispecific antigen-binding construct disclosed herein binds to at least two different receptors or epitopes (e.g., PD-1 and PD-L1), and 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, a multispecific antigen-binding construct simultaneously binds to PD-1 expressed on the surface of a first cell and a PD-1 ligand, e.g., PD-L1 or PD-L2, expressed on the surface of a second cell.

[0008] In some embodiments, the present disclosure provides a multispecific antigen-binding construct comprising at least two antigen-binding arms, wherein a first arm binds to PD-1 expressed by an immune cell and a second arm binds to a PD-1 ligand expressed by a second cell, such that the multispecific antigen-binding construct blocks the interaction of 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 myeloid-derived suppressor cell, 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 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, renal cancer, and vascular cancer. In some embodiments, both arms contain at least 1 x 10 -7 M, at least 1 × 10 -8 M, at least 1 × 10 -9 M, or at least 1 × 10 -10 K of M DIn some embodiments, the binding of one arm to its target does not block the 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, the binding of the first arm and the second arm to their respective targets can bridge the immune cell and the second cell together. In some embodiments, the bridging of the immune cell and the second cell is determined by flow cytometry. In some embodiments, the first arm is a PD-1 antagonist.

[0009] In some embodiments of the multispecific antigen-binding construct, the first arm binds to PD-1 and comprises: (a) a heavy chain variable region comprising: (i) a CDRH1 comprising SEQ ID NO: 70 (FTFX1X2YAX3X4, where X1 = S, R, G, or N, X2 = D, S, N, A, R, or G, X3 = M or L, and X4 = S, L, or N), (ii) a CDRH2 comprising SEQ ID NO: 71 (SAISNSGTYTYYA), and (iii) a CDRH3 comprising SEQ ID NO: 72 (ARGLDFIVGX5TGNDY, where 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).

[0010] In some embodiments, CDRH1 of the first arm comprises SEQ ID NO: 73 (FTFSDYAMS), CDRH2 of the first arm comprises SEQ ID NO: 71 (SAISNSGTYTYYA), and CDRH3 of the first arm comprises SEQ ID NO: 74 (ARGLDFIVGATGNDY). In some embodiments, CDRH1 of the first arm comprises SEQ ID NO: 73 (FTFSDYAMS), CDRH2 of the first arm comprises SEQ ID NO: 71 (SAISNSGTYTYYA), and CDRH3 of the first arm comprises SEQ ID NO: 75 (ARGLDFIVGYTGNDY). In some embodiments, CDRH1 of the first arm comprises SEQ ID NO: 76 (FTFSSYAMS), CDRH2 of the first arm comprises SEQ ID NO: 71 (SAISNSGTYTYYA), and CDRH3 of the first arm comprises SEQ ID NO: 75 (ARGLDFIVGYTGNDY). In some embodiments, CDRH1 of the first arm comprises SEQ ID NO:77 (FTFSSYAML), CDRH2 of the first arm comprises SEQ ID NO:71 (SAISNSGTYTYYA), and CDRH3 of the first arm comprises SEQ ID NO:75 (ARGLDFIVGYTGNDY). In some embodiments, CDRH1 of the first arm comprises SEQ ID NO:78 (FTFSNYALS), CDRH2 of the first arm comprises SEQ ID NO:71 (SAISNSGTYTYYA), and CDRH3 of the first arm comprises SEQ ID NO:75 (ARGLDFIVGYTGNDY). In some embodiments, CDRH1 of the first arm comprises SEQ ID NO:79 (FTFSAYAMN), CDRH2 of the first arm comprises SEQ ID NO:71 (SAISNSGTYTYYA), and CDRH3 of the first arm comprises SEQ ID NO:75 (ARGLDFIVGYTGNDY). In some embodiments, CDRH1 of the first arm comprises SEQ ID NO: 80 (FTFRSYAMS), CDRH2 of the first arm comprises SEQ ID NO: 71 (SAISNSGTYTYYA), and CDRH3 of the first arm comprises SEQ ID NO: 75 (ARGLDFIVGYTGNDY). In some embodiments, CDRH1 of the first arm comprises SEQ ID NO: 81 (FTFGRYAMS), CDRH2 of the first arm comprises SEQ ID NO: 71 (SAISNSGTYTYYA), and CDRH3 of the first arm comprises SEQ ID NO: 75 (ARGLDFIVGYTGNDY).In some embodiments, CDRH1 of the first arm comprises SEQ ID NO: 82 (FTFNSYAMS), CDRH2 of the first arm comprises SEQ ID NO: 71 (SAISNSGTYTYYA), and CDRH3 of the first arm comprises SEQ ID NO: 75 (ARGLDFIVGYTGNDY). In some embodiments, CDRH1 of the first arm comprises SEQ ID NO: 83 (FTFSNYAMS), CDRH2 of the first arm comprises SEQ ID NO: 71 (SAISNSGTYTYYA), and CDRH3 of the first arm comprises SEQ ID NO: 74 (ARGLDFIVGATGNDY). In some embodiments, CDRH1 of the first arm comprises SEQ ID NO: 84 (FTFSGYAMS), CDRH2 of the first arm comprises SEQ ID NO: 71 (SAISNSGTYTYYA), and CDRH3 of the first arm comprises SEQ ID NO: 85 (ARGLDFIVGRTGNDY). In some embodiments, CDRH1 of the first arm comprises SEQ ID NO: 86 (FTFSSYAMN), CDRH2 of the first arm comprises SEQ ID NO: 71 (SAISNSGTYTYYA), and CDRH3 of the first arm comprises SEQ ID NO: 85 (ARGLDFIVGRTGNDY). In some embodiments, CDRH1 of the first arm comprises SEQ ID NO: 80 (FTFRSYAMS), CDRH2 of the first arm comprises SEQ ID NO: 71 (SAISNSGTYTYYA), and CDRH3 of the first arm comprises SEQ ID NO: 85 (ARGLDFIVGRTGNDY).

[0011] 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).

[0012] In some embodiments, the heavy chain variable region of the first arm comprises an amino acid sequence 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 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 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 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 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 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 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 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 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 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 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 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 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 at least 90% identical to SEQ ID NO: 59.

[0013] In some embodiments, the second arm is a PD-1 ligand antagonist. In some embodiments, the second arm is a PD-L2 antagonist. In some embodiments, the second arm is a PD-L1 antagonist. In some embodiments, the second arm binds to PD-L1 and comprises a heavy chain variable region comprising: (i) a CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN); (ii) a CDRH2 comprising SEQ ID NO: 2 (GGIIPX1X2GX3ATYA, where 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, where X1 is G, F, or N); and b. a light chain variable region comprising: (i) a CDRL1 comprising SEQ ID NO: 4 (RASQX1ISSYLN, where 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, where X1 is Q or F).

[0014] In some embodiments, CDRH1 of the second arm comprises SEQ ID NO: 1 (GTFSSYAIN), CDRH2 of the second arm comprises SEQ ID NO: 7 (GGIIPILGAATYA), and 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: 7 (GGIIPILGAATYA), 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).

[0015] 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), 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). 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). 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), 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), 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), 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).

[0016] In some embodiments, CDRH1 of the second arm comprises SEQ ID NO: 1 (GTFSSYAIN), CDRH2 of the second arm comprises SEQ ID NO: 15 (GGIIPIFGIANYA), and 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: 15 (GGIIPIFGIANYA), 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, CDRH1 of the second arm comprises SEQ ID NO: 1 (GTFSSYAIN), CDRH2 of the second arm comprises SEQ ID NO: 16 (GGIIPNFGTATYA), and CDRH3 of the second arm comprises SEQ ID NO: 17 (ARLKGELKGAGDI). In some embodiments, CDRH1 of the second arm comprises SEQ ID NO: 1 (GTFSSYAIN), CDRH2 of the second arm comprises SEQ ID NO: 16 (GGIIPNFGTATYA), CDRH3 of the second arm comprises SEQ ID NO: 17 (ARLKGELKGAGDI), 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).

[0018] 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: 18 (ARLKFELKDAFDI). 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), CDRH3 of the second arm comprises SEQ ID NO: 18 (ARLKFELKDAFDI), 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).

[0019] 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: 19 (ARLKGELKDAFDE). 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), CDRH3 of the second arm comprises SEQ ID NO: 19 (ARLKGELKDAFDE), 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).

[0020] 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: 20 (ARLKNELKDAFDI). 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), CDRH3 of the second arm comprises SEQ ID NO: 20 (ARLKNELKDAFDI), 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).

[0021] In some embodiments, CDRH1 of the second arm comprises SEQ ID NO: 1 (GTFSSYAIN), CDRH2 of the second arm comprises SEQ ID NO: 21 (GGVIPFLGTANYA), and CDRH3 of the second arm comprises SEQ ID NO: 22 (ARLKGILKDALDI). In some embodiments, CDRH1 of the second arm comprises SEQ ID NO: 1 (GTFSSYAIN), CDRH2 of the second arm comprises SEQ ID NO: 21 (GGVIPFLGTANYA), CDRH3 of the second arm comprises SEQ ID NO: 22 (ARLKGILKDALDI), 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).

[0022] In some embodiments, CDRH1 of the second arm comprises SEQ ID NO: 1 (GTFSSYAIN), CDRH2 of the second arm comprises SEQ ID NO: 29 (GRIIPIFGTADYA), and 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: 29 (GRIIPIFGTADYA), 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).

[0023] In some embodiments, CDRH1 of the second arm comprises SEQ ID NO: 1 (GTFSSYAIN), CDRH2 of the second arm comprises SEQ ID NO: 31 (GGIIPILGTATYA), and CDRH3 of the second arm comprises SEQ ID NO: 32 (ARRKGELKDAFDI). In some embodiments, CDRH1 of the second arm comprises SEQ ID NO: 1 (GTFSSYAIN), CDRH2 of the second arm comprises SEQ ID NO: 31 (GGIIPILGTATYA), CDRH3 of the second arm comprises SEQ ID NO: 32 (ARRKGELKDAFDI), 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).

[0024] In some embodiments, CDRH1 of the second arm comprises SEQ ID NO: 1 (GTFSSYAIN), CDRH2 of the second arm comprises SEQ ID NO: 33 (GGIIPIVATANYA), and CDRH3 of the second arm comprises SEQ ID NO: 32 (ARRKGELKDAFDI). In some embodiments, CDRH1 of the second arm comprises SEQ ID NO: 1 (GTFSSYAIN), CDRH2 of the second arm comprises SEQ ID NO: 33 (GGIIPIVATANYA), CDRH3 of the second arm comprises SEQ ID NO: 32 (ARRKGELKDAFDI), 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).

[0025] In some embodiments, CDRH1 of the second arm comprises SEQ ID NO: 1 (GTFSSYAIN), CDRH2 of the second arm comprises SEQ ID NO: 34 (GGIIPIFGKATYA), and CDRH3 of the second arm comprises SEQ ID NO: 32 (ARRKGELKDAFDI). In some embodiments, CDRH1 of the second arm comprises SEQ ID NO: 1 (GTFSSYAIN), CDRH2 of the second arm comprises SEQ ID NO: 34 (GGIIPIFGKATYA), CDRH3 of the second arm comprises SEQ ID NO: 32 (ARRKGELKDAFDI), 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).

[0026] 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), 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).

[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), 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: 39 (QQSYSTILT).

[0028] In some embodiments, the second arm comprises a. a heavy chain variable region comprising: (i) a CDRH1 comprising SEQ ID NO: 14 (GTFSSYAFS), (ii) a CDRH2 comprising SEQ ID NO: 11 (GGIIPVFGTATYA), and (iii) a CDRH3 comprising SEQ ID NO: 8 (ARLKGELKDAFDI), 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).

[0029] 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).

[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: 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).

[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: 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).

[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: 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).

[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: 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).

[0034] 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).

[0035] 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).

[0036] 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 NOs: 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 NOs: 59, 60, 61, 62, or 63.

[0037] 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).

[0038] 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 a PD-1 ligand. In some embodiments, the construct comprises a common light chain. In some embodiments, one or both of the arms is an aptamer. In some embodiments, one or both of the arms is a protein other than an antibody. 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 a 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.

[0039] In some embodiments, any multispecific antigen-binding construct 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 by 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 PD-1 in the presence of a common light chain, and the C-terminal variable heavy chain of the fusion construct binds PD-L1 in the presence of a common light chain. In some embodiments, the N-terminal variable heavy chain of the fusion construct binds PD-L1 in the presence of a common light chain, and the C-terminal variable heavy chain of the fusion construct binds PD-1 in the presence of a common light chain.

[0040] In some aspects and embodiments, the present disclosure provides a multispecific antigen-binding construct comprising at least two antigen-binding units, wherein a first antigen-binding unit binds to PD-1 and a 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 of PD-1 and a PD-1 ligand, e.g., PD-L1 or PD-L2. In some embodiments, the multispecific antigen-binding construct blocks the interaction of PD-1 and a PD-1 ligand, e.g., 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, e.g., 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, e.g., 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 of which bind to a PD-1 ligand, e.g., 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 of which bind to a PD-1 ligand, e.g., 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, e.g., 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 of PD-1 and a PD-1 ligand, e.g., PD-L1 or PD-L2. In some embodiments, the multispecific antigen-binding construct comprises a common light chain. For example, at least two of the antigen-binding units comprise a common light chain.

[0041] In some embodiments, the antigen-binding first unit binds to PD-1 and (a) a heavy chain variable region comprising: (i) a CDRH1 comprising SEQ ID NO: 70 (FTFX1X2YAX3X4, where X1 = S, R, G, or N, X2 = D, S, N, A, R, or G, X3 = M or L, and X4 = S, L, or N); (ii) a CDRH2 comprising SEQ ID NO: 71 (SAISNSGTYTYYA); and (iii) a CDRH3 comprising SEQ ID NO: 72 (ARGLDFIVGX5TGNDY, where 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). Includes:

[0042] In some such embodiments, the antigen-binding first unit binds to PD-1 and (a) CDRH1 comprising SEQ ID NO: 73 (FTFSDYAMS), CDRH2 comprising SEQ ID NO: 71 (SAISNSGTYTYYA), and CDRH3 comprising SEQ ID NO: 74 (ARGLDFIVGATGNDY); (b) CDRH1 comprising SEQ ID NO: 73 (FTFSDYAMS), CDRH2 comprising SEQ ID NO: 71 (SAISNSGTYTYYA), and CDRH3 comprising SEQ ID NO: 75 (ARGLDFIVGYTGNDY); (c) CDRH1 comprising SEQ ID NO: 76 (FTFSSYAMS), CDRH2 comprising SEQ ID NO: 71 (SAISNSGTYTYYA), and CDRH3 comprising SEQ ID NO: 75 (ARGLDFIVGYTGNDY); (d) CDRH1 comprising SEQ ID NO: 77 (FTFSSYAML), CDRH2 comprising SEQ ID NO: 71 (SAISNSGTYTYYA), and CDRH3 comprising SEQ ID NO: 75 (ARGLDFIVGYTGNDY); (e) CDRH1 comprising SEQ ID NO: 78 (FTFSNYALS), CDRH2 comprising SEQ ID NO: 71 (SAISNSGTYTYYA), and CDRH3 comprising SEQ ID NO: 75 (ARGLDFIVGYTGNDY); (f) CDRH1 comprising SEQ ID NO: 79 (FTFSAYAMN), CDRH2 comprising SEQ ID NO: 71 (SAISNSGTYTYYA), and CDRH3 comprising SEQ ID NO: 75 (ARGLDFIVGYTGNDY); (g) CDRH1 comprising SEQ ID NO: 80 (FTFRSYAMS), CDRH2 comprising SEQ ID NO: 71 (SAISNSGTYTYYA), and CDRH3 comprising SEQ ID NO: 75 (ARGLDFIVGYTGNDY); (h) CDRH1 comprising SEQ ID NO: 81 (FTFGRYAMS), CDRH2 comprising SEQ ID NO: 71 (SAISNSGTYTYYA), and CDRH3 comprising SEQ ID NO: 75 (ARGLDFIVGYTGNDY); (i) CDRH1 comprising SEQ ID NO: 82 (FTFNSYAMS), CDRH2 comprising SEQ ID NO: 71 (SAISNSGTYTYYA), and CDRH3 comprising SEQ ID NO: 75 (ARGLDFIVGYTGNDY); (j) CDRH1 comprising SEQ ID NO: 83 (FTFSNYAMS), CDRH2 comprising SEQ ID NO: 71 (SAISNSGTYTYYA), and CDRH3 comprising SEQ ID NO: 74 (ARGLDFIVGATGNDY); (k) CDRH1 comprising SEQ ID NO: 84 (FTFSGYAMS), CDRH2 comprising SEQ ID NO: 71 (SAISNSGTYTYYA), and CDRH3 comprising SEQ ID NO: 85 (ARGLDFIVGRTGNDY); (l) CDRH1 comprising SEQ ID NO: 86 (FTFSSYAMN), CDRH2 comprising SEQ ID NO: 71 (SAISNSGTYTYYA), and CDRH3 comprising SEQ ID NO: 85 (ARGLDFIVGRTGNDY), or (m) CDRH1 comprising SEQ ID NO: 80 (FTFRSYAMS), CDRH3 comprising SEQ ID NO: 71 (SAISNSGTYTYYA), and CDRH4 comprising SEQ ID NO: 85 (ARGLDFIVGRTGNDY) Includes:

[0043] In some embodiments, the antigen-binding first unit binds to PD-1 and (a) a heavy chain variable region comprising an amino acid sequence at least 90% identical to SEQ ID NO: 87; (b) a heavy chain variable region comprising an amino acid sequence at least 90% identical to SEQ ID NO: 88; (c) a heavy chain variable region comprising an amino acid sequence at least 90% identical to SEQ ID NO: 89; (d) a heavy chain variable region comprising an amino acid sequence at least 90% identical to SEQ ID NO: 90; (e) a heavy chain variable region comprising an amino acid sequence at least 90% identical to SEQ ID NO: 91; (f) a heavy chain variable region comprising an amino acid sequence at least 90% identical to SEQ ID NO: 92; (g) a heavy chain variable region comprising an amino acid sequence at least 90% identical to SEQ ID NO: 93; (h) a heavy chain variable region comprising an amino acid sequence at least 90% identical to SEQ ID NO: 94; (i) a heavy chain variable region comprising an amino acid sequence at least 90% identical to SEQ ID NO: 95; (j) a heavy chain variable region comprising an amino acid sequence at least 90% identical to SEQ ID NO: 96; (k) a heavy chain variable region comprising an amino acid sequence at least 90% identical to SEQ ID NO: 97; (l) a heavy chain variable region comprising an amino acid sequence at least 90% identical to SEQ ID NO: 98; or (m) comprises a heavy chain variable region comprising an amino acid sequence at least 90% identical to SEQ ID NO: 99.

[0044] In some embodiments, the antigen-binding first 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. In some embodiments, the antigen-binding second unit binds to PD-L2. In some embodiments, the antigen-binding second unit binds to PD-L1. In some embodiments, the antigen-binding second unit binds to PD-L1. 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, where 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, where X1 is G, F, or N), and b. A light chain variable region comprising: (i) a CDRL1 comprising SEQ ID NO: 4 (RASQX1ISSYLN, where 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, where X1 is Q or F). Includes:

[0045] In some such embodiments, the antigen-binding second unit binds to PD-L1 and (a) CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), CDRH2 comprising SEQ ID NO: 7 (GGIIPILGAATYA), and CDRH3 comprising SEQ ID NO: 8 (ARLKGELKDAFDI); (b) CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), CDRH2 comprising SEQ ID NO: 7 (GGIIPILGAATYA), 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); (c) 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); (d) CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), CDRH2 comprising SEQ ID NO: 11 (GGIIPVFGTATYA), and CDRH3 comprising SEQ ID NO: 8 (ARLKGELKDAFDI); (e) 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: 12 (RASQWISSYLN), CDRL2 comprising SEQ ID NO: 5 (AASSLQS), and CDRL3 comprising SEQ ID NO: 10 (QQSYSTPLT); (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); (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); (h) CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), CDRH2 comprising SEQ ID NO: 15 (GGIIPIFGIANYA), and CDRH3 comprising SEQ ID NO: 8 (ARLKGELKDAFDI); (i) CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), CDRH2 comprising SEQ ID NO: 15 (GGIIPIFGIANYA), 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 (CQQSYSTPLTF); (j) CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), CDRH2 comprising SEQ ID NO: 16 (GGIIPNFGTATYA), and CDRH3 comprising SEQ ID NO: 17 (ARLKGELKGAGDI); (k) CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), CDRH2 comprising SEQ ID NO: 16 (GGIIPNFGTATYA), CDRH3 comprising SEQ ID NO: 17 (ARLKGELKGAGDI), CDRL1 comprising SEQ ID NO: 9 (RASQSISSYLN), CDRL2 comprising SEQ ID NO: 5 (AASSLQS), and CDRL3 comprising SEQ ID NO: 10 (QQSYSTPLT); (l) CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), CDRH2 comprising SEQ ID NO: 11 (GGIIPVFGTATYA), and CDRH3 comprising SEQ ID NO: 18 (ARLKFELKDAFDI); (m) CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), CDRH2 comprising SEQ ID NO: 11 (GGIIPVFGTATYA), CDRH3 comprising SEQ ID NO: 18 (ARLKFELKDAFDI), CDRL1 comprising SEQ ID NO: 9 (RASQSISSYLN), CDRL2 comprising SEQ ID NO: 5 (AASSLQS), and CDRL3 comprising SEQ ID NO: 10 (QQSYSTPLT); (n) CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), CDRH2 comprising SEQ ID NO: 11 (GGIIPVFGTATYA), and CDRH3 comprising SEQ ID NO: 19 (ARLKGELKDAFDE); (o) CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), CDRH2 comprising SEQ ID NO: 11 (GGIIPVFGTATYA), CDRH3 comprising SEQ ID NO: 19 (ARLKGELKDAFDE), CDRL1 comprising SEQ ID NO: 9 (RASQSISSYLN), CDRL2 comprising SEQ ID NO: 5 (AASSLQS), and CDRL3 comprising SEQ ID NO: 10 (QQSYSTPLT); (p) CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), CDRH2 comprising SEQ ID NO: 11 (GGIIPVFGTATYA), and CDRH3 comprising SEQ ID NO: 20 (ARLKNELKDAFDI); (q) CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), CDRH2 comprising SEQ ID NO: 11 (GGIIPVFGTATYA), CDRH3 comprising SEQ ID NO: 20 (ARLKNELKDAFDI), CDRL1 comprising SEQ ID NO: 9 (RASQSISSYLN), CDRL2 comprising SEQ ID NO: 5 (AASSLQS), and CDRL3 comprising SEQ ID NO: 10 (QQSYSTPLT); (r) CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), CDRH2 comprising SEQ ID NO: 21 (GGVIPFLGTANYA), and CDRH3 comprising SEQ ID NO: 22 (ARLKGILKDALDI); (s) CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), CDRH2 comprising SEQ ID NO: 21 (GGVIPFLGTANYA), CDRH3 comprising SEQ ID NO: 22 (ARLKGILKDALDI), CDRL1 comprising SEQ ID NO: 9 (RASQSISSYLN), CDRL2 comprising SEQ ID NO: 5 (AASSLQS), and CDRL3 comprising SEQ ID NO: 10 (QQSYSTPLT); (t) CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), CDRH2 comprising SEQ ID NO: 29 (GRIIPIFGTADYA), and CDRH3 comprising SEQ ID NO: 8 (ARLKGELKDAFDI); (u) CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), CDRH2 comprising SEQ ID NO: 29 (GRIIPIFGTADYA), 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); (v) CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), CDRH2 comprising SEQ ID NO: 31 (GGIIPILGTATYA), and CDRH3 comprising SEQ ID NO: 32 (ARRKGELKDAFDI); (w) CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), CDRH2 comprising SEQ ID NO: 31 (GGIIPILGTATYA), 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); (x) CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), CDRH2 comprising SEQ ID NO: 33 (GGIIPIVATANYA), and CDRH3 comprising SEQ ID NO: 32 (ARRKGELKDAFDI); (y) CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), CDRH2 comprising SEQ ID NO: 33 (GGIIPIVATANYA), 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); (z) CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), CDRH2 comprising SEQ ID NO: 34 (GGIIPIFGKATYA), and CDRH3 comprising SEQ ID NO: 32 (ARRKGELKDAFDI); (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); (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); (cc) 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: 39 (QQSYSTILT); (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); (ee) CDRH1 comprising SEQ ID NO: 23 (GTFSSYAIS), CDRH2 comprising SEQ ID NO: 24 (GGIIPIVGIANYA), and CDRH3 comprising SEQ ID NO: 8 (ARLKGELKDAFDI); (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); (gg) CDRH1 comprising SEQ ID NO: 23 (GTFSSYAIS), CDRH2 comprising SEQ ID NO: 11 (GGIIPVFGTATYA), and CDRH3 comprising SEQ ID NO: 25 (ARLKGEFKDAFDI); (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); (ii) CDRH1 comprising SEQ ID NO: 23 (GTFSSYAIS), CDRH2 comprising SEQ ID NO: 26 (GRIIPLFGTAHYA), and CDRH3 comprising SEQ ID NO: 8 (ARLKGELKDAFDI); (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); (kk) CDRH1 comprising SEQ ID NO: 23 (GTFSSYAIS), CDRH2 comprising SEQ ID NO: 27 (GRINPILGTANYA), and CDRH3 comprising SEQ ID NO: 28 (ARLKGELKDAFSI); (ll) 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); (mm) CDRH1 comprising SEQ ID NO: 23 (GTFSSYAIS), CDRH2 comprising SEQ ID NO: 11 (GGIIPVFGTATYA), and CDRH3 comprising SEQ ID NO: 30 (ARLKGELKCAFDI); (nn) CDRH1 comprising SEQ ID NO: 23 (GTFSSYAIS), CDRH2 comprising SEQ ID NO: 11 (GGIIPVFGTATYA), and CDRL1 comprising SEQ ID NO: 9 (RASQSISSYLN), CDRL2 comprising SEQ ID NO: 5 (AASSLQS), and CDRL3 comprising SEQ ID NO: 10 (QQSYSTPLT), comprising SEQ ID NO: 30 (ARLKGELKCAFDI); (oo) CDRH1 comprising SEQ ID NO: 36 (GPFRSHAVS), CDRH2 comprising SEQ ID NO: 11 (GGIIPVFGTATYA), and CDRH3 comprising SEQ ID NO: 37 (ARLKSELKDAFDI), or (pp) CDRH1 containing SEQ ID NO:36 (GPFRSHAVS), CDRH2 containing SEQ ID NO:11 (GGIIPVFGTATYA), and CDRH3 containing SEQ ID NO:37 (ARLKSELKDAFDI), CDRL1 containing SEQ ID NO:9 (RASQSISSYLN), CDRL2 containing SEQ ID NO:5 (AASSLQS), and CDRL3 containing SEQ ID NO:10 (QQSYSTPLT) Includes:

[0046] In some such embodiments, the antigen-binding second 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 NOs: 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 NOs: 59, 60, 61, 62, or 63.

[0047] In some embodiments, the antigen-binding second unit binds to PD-L1 and (a) an amino acid sequence at least 90% identical to SEQ ID NO: 35; (b) an amino acid sequence at least 90% identical to SEQ ID NO: 40; (c) an amino acid sequence at least 90% identical to SEQ ID NO: 41; (d) an amino acid sequence at least 90% identical to SEQ ID NO: 42; (e) an amino acid sequence at least 90% identical to SEQ ID NO: 43; (f) an amino acid sequence at least 90% identical to SEQ ID NO: 44; (g) an amino acid sequence at least 90% identical to SEQ ID NO: 45; (h) an amino acid sequence at least 90% identical to SEQ ID NO: 46; (i) an amino acid sequence at least 90% identical to SEQ ID NO: 47; (j) an amino acid sequence at least 90% identical to SEQ ID NO: 48; (k) an amino acid sequence at least 90% identical to SEQ ID NO: 49; (l) an amino acid sequence at least 90% identical to SEQ ID NO: 50; (m) an amino acid sequence at least 90% identical to SEQ ID NO: 51; (n) an amino acid sequence at least 90% identical to SEQ ID NO: 52; (o) an amino acid sequence at least 90% identical to SEQ ID NO: 53; (p) an amino acid sequence at least 90% identical to SEQ ID NO: 54; (q) an amino acid sequence at least 90% identical to SEQ ID NO: 55; (r) an amino acid sequence at least 90% identical to SEQ ID NO: 56; (s) an amino acid sequence at least 90% identical to SEQ ID NO: 57; or (t) an amino acid sequence at least 90% identical to SEQ ID NO: 58 The heavy chain variable region comprises:

[0048] In some embodiments, the antigen-binding second unit binds to PD-L1 and (a) an amino acid sequence at least 90% identical to SEQ ID NO: 59; (b) an amino acid sequence at least 90% identical to SEQ ID NO: 60; (c) an amino acid sequence at least 90% identical to SEQ ID NO: 61; (d) an amino acid sequence at least 90% identical to SEQ ID NO: 62; or (e) an amino acid sequence at least 90% identical to SEQ ID NO: 63 The light chain variable region comprises:

[0049] Also provided herein in some aspects and embodiments is a multispecific antigen-binding construct comprising four antigen-binding units, wherein two of the antigen-binding units bind to PD-1 and two of the antigen-binding units bind to PD-L1, and wherein the construct comprises a heavy chain amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO: 100 or 102, and a light chain amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO: 101 or 103.

[0050] Also provided herein in some aspects and embodiments is a multispecific antigen-binding construct comprising four antigen-binding units, wherein two of the antigen-binding units bind to PD-1 and two of the antigen-binding units bind to PD-L1, and wherein the construct comprises a heavy chain amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO: 100 and a light chain amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO: 101.

[0051] Also provided herein in some aspects and embodiments is a multispecific antigen-binding construct comprising four antigen-binding units, wherein two of the antigen-binding units bind to PD-1 and two of the antigen-binding units bind to PD-L1, and wherein the construct comprises a heavy chain amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO: 102 and a light chain amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO: 103.

[0052] In some embodiments, the construct does not comprise an Fc domain. In some embodiments, the first arm, the second arm, or both 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 promote heterodimerization of the first and second arms. In some embodiments, the mutations are present in the CH3 domain of the heavy chain. In some embodiments, the multispecific antigen-binding construct is produced in quadroma cells. In some embodiments, the construct comprises one or more immunoglobulin constant region modifications. In some embodiments, the immunoglobulin constant region comprises 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 a format selected from the group consisting of a bispecific IgG, a bispecific antibody fragment, a bispecific fusion protein, an appended IgG, and a 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.

[0053] 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 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 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.

[0054] In some embodiments, any of the multispecific antigen-binding constructs disclosed herein is aglycosylated. In some embodiments, the multispecific antigen-binding construct is capable of binding to human PD-1. In some embodiments, the multispecific antigen-binding construct is capable of binding to mouse PD-1. In some embodiments, the multispecific antigen-binding construct is capable of binding to cynomolgus PD-1. In some embodiments, the multispecific antigen-binding construct is capable of binding to human, mouse, and cynomolgus PD-1 with similar affinity.

[0055] In some embodiments, any of the multispecific antigen-binding constructs disclosed herein are capable of reducing PD-1 levels on cells. In some embodiments, the multispecific antigen-binding constructs are capable of inducing PD-1 degradation. In some embodiments, the multispecific antigen-binding constructs are capable of reducing PD-1 expression. In some embodiments, the multispecific antigen-binding constructs are capable of reducing PD-1 cell surface expression. In some embodiments, the multispecific antigen-binding constructs are capable of reducing PD-1 cell surface expression by inducing PD-1 shedding from the cell surface. In some embodiments, the multispecific antigen-binding constructs bind to both PD-1 and PD-L1 and reduce PD-1 levels on cells. In some embodiments, the multispecific antigen-binding constructs bind to both PD-1 and PD-L1 and induce PD-1 degradation. In some embodiments, the multispecific antigen-binding constructs bind to both PD-1 and PD-L1 and reduce PD-1 expression. In some embodiments, the multispecific antigen-binding constructs are capable of inducing PD-1 shedding from immune cells. In some embodiments, the multispecific antigen-binding construct can bind to both PD-1 and PD-L1 and induce PD-1 shedding from immune cells. In some embodiments, the multispecific antigen-binding construct can sequester PD-L1 so that it cannot bind to CD80. In some embodiments, the multispecific antigen-binding construct can sequester PD-L1 so that it cannot bind to CD80, which is free to bind to CD28. In some embodiments, the cell is an immune cell, e.g., a T cell. In some embodiments, the immune cell (e.g., a T cell) is a tumor-infiltrating lymphocyte (TIL). In some embodiments, engagement of a multispecific antigen-binding molecule described herein with PD-1 expressed by immune cells in the tumor microenvironment results in downregulation of PD-1 by the immune cell. In some embodiments, the immune cell is a T cell. In some embodiments, the immune cell (e.g., a T cell) is a tumor-infiltrating lymphocyte (TIL).

[0056] In some embodiments, any multispecific antigen-binding construct disclosed herein may induce at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% higher interferon-gamma levels (e.g., interferon-gamma levels as measured in a Staphylococcus aureus enterotoxin A ("SEA") assay) compared to a reference antigen-binding construct (e.g., pembrolizumab or atezolizumab) or reference combination of antigen-binding constructs (e.g., a composition comprising the PD-1 and PD-L1 arms of a multispecific antigen-binding construct, wherein the PD-1 and PD-L1 arms in the composition are not conjugated 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%, or 99% higher interleukin-2 levels (e.g., interleukin-2 levels as measured in an SEA assay) compared to a reference antigen-binding construct (e.g., pembrolizumab or atezolizumab) or reference combination of antigen-binding constructs (e.g., a composition comprising the PD-1 and PD-L1 arms of a multispecific antigen-binding construct, wherein the PD-1 and PD-L1 arms in the composition are not conjugated to each other).

[0057] 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, or breast cancer cells) compared to a reference antigen-binding construct (e.g., pembrolizumab or atezolizumab) or reference combination of antigen-binding constructs (e.g., a composition comprising the PD-1 and PD-L1 arms of a multispecific antigen-binding construct, wherein the PD-1 and PD-L1 arms in the composition are not conjugated to each other).

[0058] In some embodiments, the multispecific antigen-binding construct may prolong survival of a subject with 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 a subject administered a reference antigen-binding construct (e.g., pembrolizumab or atezolizumab) or a reference combination of antigen-binding constructs (e.g., a composition comprising the PD-1 and PD-L1 arms of a multispecific antigen-binding construct, wherein the PD-1 and PD-L1 arms in the composition are not conjugated to each other). In some embodiments, the multispecific antigen-binding construct may induce 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% greater 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 the PD-1 and PD-L1 arms of a multispecific antigen-binding construct, wherein the PD-1 and PD-L1 arms in the composition are not conjugated to each other).In some embodiments, the multispecific antigen-binding construct exhibits a higher affinity for PD-1 than untreated immune cells or 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 the PD-1 and PD-L1 arms of a multispecific antigen-binding construct, wherein the PD-1 and PD-L1 arms in the composition are not conjugated to each other). The PD-1 levels can be reduced 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% (e.g., by causing PD-1 shedding from the cell surface and / or inducing PD-1 degradation and / or reducing PD-1 expression).

[0059] In some embodiments, engagement of a multispecific antigen-binding molecule described herein with PD-1 expressed by a cell results in downregulation and / or loss of cell surface expression of PD-1 by the cell. Such downregulation or loss of cell surface expression can be due, for example, in part, to shedding of extracellular PD-1 from the surface of the immune cell. In some embodiments, the cell is an immune cell, e.g., a T cell. In some embodiments, the immune cell (e.g., a T cell) is a tumor-infiltrating lymphocyte (TIL). In some embodiments, engagement of a multispecific antigen-binding molecule described herein with PD-1 expressed by an immune cell in the tumor microenvironment results in downregulation of PD-1 by the immune cell. In some embodiments, the immune cell is a T cell. In some embodiments, the immune cell (e.g., a T cell) is a tumor-infiltrating lymphocyte (TIL).

[0060] In some aspects, 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 multispecific 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 hematological 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 of enhancing an immune response in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of any of the multispecific antigen-binding constructs disclosed herein, thereby enhancing the immune response in the subject. In some embodiments, the enhanced immune response comprises 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 with administration of the multispecific antigen-binding construct compared to an agent that binds to either PD-1 or a PD-1 ligand, or a cocktail comprising an agent that binds to PD-1 and an agent that binds to a PD-1 ligand. In some embodiments, the T cell function is one or more of increased IFNγ production from T cells, enhanced T cell survival, increased T cell proliferation, or rescue from an exhausted T cell phenotype. In some embodiments, the enhanced T cell function is greater with administration of the multispecific antigen-binding construct compared to an agent that binds to either PD-1 or a PD-1 ligand, or a cocktail comprising 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 cells in a subject. In some embodiments, the multispecific antigen-binding construct binds to PD-1 expressed on the surface of a first cell in the subject, and the multispecific antigen-binding construct binds to PD-L1 expressed on the surface of a second cell in the subject.

[0061] In some embodiments, the disclosure provides an anti-PD1 antibody or antigen-binding fragment thereof comprising: (a) a heavy chain variable region comprising: (i) a CDRH1 comprising SEQ ID NO: 70 (FTFX1X2YAX3X4, where X1 = S, R, G, or N, X2 = D, S, N, A, R, or G, X3 = M or L, and X4 = S, L, or N), (ii) a CDRH2 comprising SEQ ID NO: 71 (SAISNSGTYTYYA), and (iii) a CDRH3 comprising SEQ ID NO: 72 (ARGLDFIVGX5TGNDY, where 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). 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 NOs: 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 that is at least 85% identical to the amino acid sequence of SEQ ID NO:59.

[0062] In some embodiments, the disclosure provides an anti-PD-L1 antibody or antigen-binding fragment thereof, comprising a heavy chain variable region comprising: (i) a CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN); (ii) a CDRH2 comprising SEQ ID NO: 2 (GGIIPX1X2GX3ATYA, where 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, where X1 is G, F, or N); and b. a light chain variable region comprising: (i) a CDRL1 comprising SEQ ID NO: 4 (RASQX1ISSYLN, where 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, 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 NOs: 1, 14, 23, 36, or 122; (ii) a CDRH2 comprising the amino acid sequence of any one of SEQ ID NOs: 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 NOs: 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 NOs: 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 NOs: 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.

[0063] 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 hematological 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.

[0064] In some embodiments, the present disclosure provides a method of enhancing an immune response 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 enhancing the immune response in the subject.

[0065] In some embodiments, any of the methods described herein can involve detecting the presence or absence of PD-1 expression by one or more cells (or populations of cells, such as TILs) before and / or after contact with a multispecific antigen-binding molecule described herein. For example, any of the methods described herein can involve detecting the presence or absence of PD-1 expression by one or more cells (or populations of cells, such as TILs) before and / or after administration of a multispecific antigen-binding molecule described herein to a subject (e.g., a cancer patient). Such methods are useful, for example, in determining a therapeutically effective amount of a molecule for use in treating a given patient or patient population. Methods for detecting the presence, reduction, and / or absence of PD-1 expression are known to those of skill in the art and use, for example, flow cytometry, Western blotting, ELISA, etc.

[0066] In another aspect, the disclosure features a method that includes measuring the level of PD-1 expression by one or more cells (or populations of cells, such as TILs) before and / or after contact with a multispecific antigen-binding molecule described herein. In some embodiments, the method includes measuring the level of PD-1 expression by one or more cells (or populations of cells, such as TILs) before and / or after administration of a multispecific antigen-binding molecule described herein to a subject (e.g., a cancer patient).

[0067] In yet another aspect, the disclosure features a method including measuring the level of PD-1 expression by one or more cells (or populations of cells, such as TILs) before and / or after contact with a multispecific antigen-binding molecule described herein. For example, any of the methods described herein can involve measuring the level of PD-1 expression by one or more cells (or populations of cells, such as TILs) before and / or after administration of a multispecific antigen-binding molecule described herein to a subject (e.g., a cancer patient). Such methods are useful, among other things, for detecting or measuring the biological effect of a molecule described herein on a subject. In some embodiments, a reduction in the level of PD-1 expression by immune cells (e.g., TILs isolated from a patient) after treatment with a multispecific antigen-binding molecule described herein indicates that the molecule had a biological effect in the subject. In some embodiments, a reduction in the level of PD-1 expression by 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 therapy including the molecule.

[0068] In yet another aspect, the disclosure features a method for determining whether a biological effect has occurred in a patient or population of patients treated with a multispecific antigen-binding molecule described herein. The method includes detecting the presence or amount of PD-1 expression by one or more test immune cells (e.g., effector immune cells, e.g., effector immune cells in a tumor microenvironment) obtained from one or more patients administered a multispecific antigen-binding molecule described herein, wherein a reduced level of PD-1 expression (e.g., cell surface expression) of PD-1 by the one or more immune cells compared to a control expression level (e.g., the expression level of PD-1 by immune cells of the same histological type as the test immune cells prior to administration of the molecule) indicates that a biological effect has occurred in the patient or population of patients. In some embodiments, the method includes administering the multispecific antigen-binding molecule prior to detection. In some embodiments, the method includes administering the multispecific antigen-binding molecule to a patient or population of patients for whom the occurrence of a biological effect has been determined. In some embodiments, the control PD-1 expression level is about the median or mean expression level of PD-1 by immune cells of the same histological type in a population of subjects not diagnosed with cancer. In some embodiments, the control PD-1 expression level is about the median or mean expression level of PD-1 by immune cells of the same histological type in a population of subjects not administered a multispecific antigen-binding molecule and / or an agent that binds to and / or inhibits PD-1 as described herein.

[0069] In yet another aspect, the disclosure features a method of reducing expression of PD-1 by one or more immune cells in a subject (e.g., a cancer patient), the method including administering to the subject a multispecific antigen-binding molecule described herein, thereby reducing expression of PD-1 by one or more immune cells in the subject. In some embodiments, the method includes determining whether a reduction in PD-1 expression by one or more immune cells has occurred in the patient. In some embodiments, the method includes obtaining from the subject a biological sample (e.g., a tumor biopsy) containing one or more immune cells (e.g., after administration of the molecule to the subject). In some embodiments, the method includes measuring the level of PD-1 expression by the one or more immune cells in the biological sample.

[0070] 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 including administering to the subject a multispecific antigen-binding molecule described herein, thereby inhibiting binding between PD-L1 and CD80 in the subject.

[0071] The patent or application file contains at least one color drawing. 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. [Brief explanation of the drawings]

[0072] [Figure 1] Figure 1 shows the induction of interferon-gamma (IFNγ) in mixed lymphocyte reaction (MLR) assays treated with various antibody cocktails or bispecific antibodies, including pembrolizumab x atezolizumab bispecific, nivolumab x atezolizumab bispecific, a cocktail of KEYTRUDA and atezolizumab, and a cocktail of nivolumab and atezolizumab, compared to KEYTRUDA alone. Results show the concentration of IFNγ in pg / mL at the final concentrations of antibodies tested as indicated. [Figure 2]Figure 1 shows the induction of interferon-gamma (IFNγ) in mixed lymphocyte reaction (MLR) assays 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 final concentration of antibody tested as indicated. [Figure 3] Schematic and amino acid sequences for 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 the atezolizumab_FabH-(G4S)4 heavy chain (H chain; SEQ ID NO: 106) and light chain (L chain; SEQ ID NO: 107). [Figure 4] Schematic and amino acid sequences for 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 the atezolizumab FabH-(G4S)4 heavy chain (H chain; SEQ ID NO: 106) and light chain (L chain; SEQ ID NO: 107). [Figure 5] Schematic and amino acid sequences for the 949 aglyco-IgG1 x atezolizumab bispecific. 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 the atezolizumab_FabH-(G4S)4 heavy chain (H chain; SEQ ID NO: 106) and light chain (L chain; SEQ ID NO: 107). [Figure 6] Schematic and amino acid sequences for the atezolizumab x nivolumab bispecific are shown. Separate 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 nivolumab_HC Fab-(G4S)4 heavy chain (H chain; SEQ ID NO: 113) and light chain (L chain; SEQ ID NO: 109). [Figure 7]The right panel shows IFN-γ release in pg / mL in a mixed lymphocyte reaction (MLR) assay as a function of antibody tested at various concentrations. These results indicate that the bispecific antibody PD-1 × PD-L1 (pembrolizumab × atezolizumab) or (nivolumab × atezolizumab) in a multispecific format induces a greater IFN-γ response at femtomolar concentrations compared to the cocktails of pembrolizumab and atezolizumab or nivolumab and atezolizumab. Size-exclusion chromatography of the bispecific format on the mAb precursor is shown (left panel). [Figure 8] An example workflow for identifying multispecific (e.g., bispecific) antibodies that demonstrate synergy is shown. The process involves unbiased screening of combinations of checkpoint blockers in a mixed lymphocyte reaction (MLR) assay, measuring IFN-γ release in pg / mL at various concentrations. In the second step of the illustrated workflow, common light chain bispecifics were generated to further test their efficacy, where various bispecific formats are depicted. The identified bispecific formats outperformed known PD-1 blockers in a T cell activation assay. [Figure 9A] Figure 9A shows that bispecific 3 induced higher killing of K562-CD32-PDL1 target cells by CD3 / CD28-expanded T cells in an antigen-nonspecific T cell assay compared to both an isotype control antibody and Keytruda. This increased killing by bispecific 3 was seen even at concentrations as low as 0.01 nM. [Figure 9B] Figure 9A shows that bispecific 3 induced higher killing of K562-CD32-PDL1 target cells by CD3 / CD28-expanded T cells in an antigen-nonspecific T cell assay compared to both an isotype control antibody and Keytruda. This increased killing by bispecific 3 was seen even at concentrations as low as 0.01 nM. [Figure 10A]Figure 10A shows the effect of bispecific 3 on tumor cell killing. Compared to both Keytruda and the combination of mAb1 and mAb28, bispecific 3 increased specific killing of K562-A2-CMV-PDL1 tumor antigen target cells by CMV-specific T cells at concentrations as low as 0.001-0.01 nM, indicating that bispecific 3 can be used to mediate antigen-specific killing of target cells at lower doses. [Figure 10B] Figure 10B shows the effect of bispecific 3 on tumor cell killing. Figure 10B shows that bispecific 3 was more effective than either Keytruda or the combination of mAb1 and mAb28 in specific killing of Raji-A2-CMV-PDL1 cell tumor antigen target cells by CMV-specific T cells at concentrations as low as 0.001 nM, again indicating that bispecific 3 can be used to mediate antigen-specific killing of target cells at lower doses. [Figure 11] Bispecific 3 induces more IL-2 than Keytruda at all doses tested in the SEA stimulation assay. Importantly, bispecific 3 induces increased IL-2 production starting at lower concentrations of antibody compared to Keytruda and both mAb1 and mAb28. [Figure 12A] Bispecific 3 demonstrates that it has the unique ability to cause PD-1 internalization and subsequent degradation or loss of expression, and that this property is dependent on engagement of both PD-1 and PD-L1 targeting arms of the molecule. Figures 12A-12B demonstrate that only bispecific 3 results in PD-1 internalization and subsequent degradation or loss of expression when compared to isotype control, Keytruda, mAb1 and mAb28, atezolizumab, or atezolizumab and Keytruda. [Figure 12B]Bispecific 3 demonstrates that it has the unique ability to cause PD-1 internalization and subsequent degradation or loss of expression, and that this property is dependent on engagement of both PD-1 and PD-L1 targeting arms of the molecule. Figures 12A-12B demonstrate that only bispecific 3 results in PD-1 internalization and subsequent degradation or loss of expression when compared to isotype control, Keytruda, mAb1 and mAb28, atezolizumab, or atezolizumab and Keytruda. [Figure 12C] These results demonstrate that bispecific 3 has the unique ability to cause PD-1 internalization and subsequent degradation or loss of expression, and that this property is dependent on engagement of both PD-1 and PD-L1 targeting arms of the molecule. Additionally, as shown in Figure 12C, when the anti-PD-L1 antibody, mAb1, was added to the wells at 50 nM 5 minutes before adding bispecific 3, the ability of bispecific 3 to promote PD-1 internalization was abolished. This suggests that both arms of bispecific 3 must be engaged to promote loss of expression and / or internalization and / or degradation of PD-1. [Figure 12D] We demonstrate that bispecific 3 has the unique ability to cause PD-1 internalization and subsequent degradation or loss of expression, and that this property is dependent on engagement of both the PD-1 and PD-L1 targeting arms of the molecule. Figure 12D shows that treatment with bispecific 3 increases the amount of PD-1 in the supernatant when both binding arms of the bispecific are simultaneously engaged. This effect is abolished when the PD-L1 targeting arm is blocked with mAb1, suggesting that bispecific 3 increases PD-1 shedding into the supernatant. [Figure 12E]Bispecific 3 demonstrates that it has the unique ability to cause PD-1 internalization and subsequent degradation or loss of expression, and that this property is dependent on engagement of both PD-1 and PD-L1 targeting arms of the molecule. Figure 12E demonstrates that the valency of the binding arms affects the extent of loss of PD-1 expression. Bispecific 5 was generated with a first N-terminal Fab that binds to PD-L1 based on the VH and VL sequences of mAb1 and a second N-terminal Fab that binds to PD-1 based on the VH and VL sequences of mAb28. In other words, compared to bispecific 3, which has a bivalent arm that binds to PD-L1 and a bivalent arm that binds to PD-1, bispecific 5 has one monovalent arm that binds to PD-L1 and one monovalent arm that binds to 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 accounts for this difference. [Figure 12F] We demonstrate that bispecific 3 has the unique ability to cause PD-1 internalization and subsequent degradation or loss of expression, and that this property is dependent on engagement of both PD-1 and PD-L1 targeting arms of the molecule. Figure 12F demonstrates that pretreatment with batimastat, a broad-spectrum inhibitor of multiple MMPs and ADAMs, sheddases or proteases responsible for cleaving proteins from the plasma membrane of cells, greatly reduced 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 12G] Bispecific 3 demonstrates that it has the unique ability to cause PD-1 internalization and subsequent degradation or loss of expression, and that this property is dependent on engagement of both PD-1 and PD-L1 targeting arms of the molecule. Figure 12G suggests that bispecific 3 primarily promotes loss of cell surface PD-1 expression when it binds to PD-1 and PD-L1 that are in the trans configuration, i.e., expressed by different cells. [Figure 13A]In vivo results are shown using bispecific 3. Figure 13A depicts a schematic of the experimental protocol. [Figure 13B] Figure 13B shows in vivo results using bispecific 3. Figure 13B demonstrates that both the bispecific 3 group and the combination mAb1 and mAb28 group had a significant delay in tumor growth compared to both the isotype and Keytruda groups. Additionally, at day 24, there was a significant difference between the bispecific 3 group and the group treated with the combination of mAb1 and mAb28, with bispecific 3 causing a greater delay in tumor growth compared to the combination. The no T cell transfer group had tumors that grew more aggressively than any group containing T cells. In this model, Keytruda conferred no benefit in delaying KACP tumor growth compared to the isotype control. [Figure 14A] Figures 14A and 14B show in vivo results using bispecific 3 in the K562-A2-CMV-PD-L1 tumor mouse model. Figures 14A and 14B are graphs illustrating that 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 T cell-free group had tumors that grew more aggressively than any group containing T cells. In this model, Keytruda conferred no benefit in delaying K562-A2-CMV-PD-L1 tumor growth compared to the isotype control. [Figure 14B]Figures 14A and 14B show in vivo results using bispecific 3 in the K562-A2-CMV-PD-L1 tumor mouse model. Figures 14A and 14B are graphs illustrating that 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 T cell-free group had tumors that grew more aggressively than any group containing T cells. In this model, Keytruda conferred no benefit in delaying K562-A2-CMV-PD-L1 tumor growth compared to the isotype control. [Figure 15A]

[00137] Figure 15 illustrates in vivo results using bispecific 3 in several syngeneic tumor models. Figure 15A is a graph showing that treatment of an EMT-6 syngeneic tumor model with bispecific 3 resulted in a greater delay in tumor growth compared to control treatment. [Figure 15B]

[00147] Figure 15B illustrates in vivo results using bispecific 3 in several syngeneic tumor models. Figure 15B is a graph showing that treatment of an MB49 syngeneic tumor model with bispecific 3 resulted in a greater delay in tumor growth compared to control treatment. [Figure 16A] Figure 16 shows in vivo results using bispecific 1 in the MC38-hPD-L1 model in humanized PD-1 / PD-L1 transgenic mice. Figure 16A shows that both KEYTRUDA and bispecific 1 treatment effectively controlled tumor growth in MC38-hPD-L1 tumor mice compared to control-treated mice. [Figure 16B] Figure 16B shows in vivo results using bispecific 1 in the MC38-hPD-L1 model in humanized PD-1 / PD-L1 transgenic mice. Figure 16B is a survival graph illustrating that bispecific 1 increased survival of MC38-PD-L1 tumor-bearing mice compared to control-treated mice. [Figure 17A]Figure 17A illustrates in vivo results using bispecific 3 in the B16F10-hPD-L1 model. Figure 17A is a series of graphs showing the effect of different treatments, respectively, on tumor growth measured at a 15-day cutoff in B16F10-HuPD-L1 mice. Different groups of mice (n=8) were treated with bispecific 3, Keytruda, avelumab, the combination of Keytruda and avelumab, or an isotype control antibody. Figure 17A shows individual tumor volume traces for each group. Metastases were identified in multiple mice that died before the tumor sizing cutoff. [Figure 17B] Figure 17B illustrates in vivo results using bispecific 3 in the B16F10-hPD-L1 model. Figure 17B shows the difference in mean tumor volume between treatment groups, demonstrating that by 15 days post-tumor cell inoculation, bispecific 3 treatment delayed mean tumor growth significantly longer than any other treatment tested in B16F10-HuPD-L1 mice. ****, P<0.0001; **, P<0.01; *, P<0.05, two-way ANOVA and Tukey's multiple comparisons test. Figure 17B is a graph comparing the effect of different treatments on tumor volume measured at a 15-day cutoff in B16F10-HuPD-L1 mice. Metastases were identified in multiple mice that died before the tumor sizing cutoff. As shown in Figure 17B, bispecific 3 treatment delayed mean tumor growth significantly longer than any other treatment tested in B16F10-hPD-L1 mice. [Figure 17C] Figure 17C illustrates in vivo results using bispecific 3 in the B16F10-hPD-L1 model. Figure 17C is a survival graph illustrating that treatment with bispecific 3 increased survival of mice with B16F10-hPD-L1 tumors compared to survival with any of the other treatments tested. [Figure 17D]Figure 17D illustrates in vivo results using bispecific 3 in the B16F10-hPD-L1 model. Figure 17D shows that by day 21 post tumor cell inoculation, bispecific 3 treatment continued to delay mean tumor growth significantly better than treatment with Keytruda in B16F10-hPD-L1 mice. [Figure 17E] Figure 17E illustrates in vivo results using bispecific 3 in the B16F10-hPD-L1 model. Figure 17E is a survival graph illustrating that treatment with bispecific 3 increased survival of mice with B16F10-HuPD-L1 tumors compared to survival with any of the other treatments tested. [Figure 17F] Figure 17F illustrates in vivo results using bispecific 3 in the B16F10-hPD-L1 model. Figure 17F provides a table showing the number of tumor-free mice for each of the different treatment groups. The group treated with bispecific 3 had 3 mice that were tumor-free, while the combination of Keytruda and avelumab had 1 mouse that was tumor-free. [Figure 18A] Bispecific 3 demonstrates that it has drug-like properties (DLP) similar to well-behaved monoclonal antibodies and maintains parental PD-1 and PD-L1 binding. Figure 18A shows that bispecific 3 exhibits similar binding to parental clone mAb28 to CHO cells expressing human PD-1 (top) and similar binding to parental clone mAb1 to CHO cells expressing human PD-L1 (bottom). [Figure 18B] Bispecific 3 demonstrates that it has drug-like properties (DLP) similar to well-behaved monoclonal antibodies and maintains parental PD-1 and PD-L1 binding. Figure 18B shows that bispecific 3 exhibits similar binding to parental clone mAb28 on CHO cells expressing cynomolgus PD-1 (top) and similar binding to parental clone mAb1 on CHO cells expressing cynomolgus PD-L1 (bottom). [Figure 18C]Bispecific 3 demonstrates that it has drug-like properties (DLP) similar to well-behaved monoclonal antibodies and maintains parental PD-1 and PD-L1 binding. Figure 18C shows that bispecific 3 exhibits similar binding to parental clone mAb28 to CHO cells expressing mouse PD-1 (top) and similar binding to parental clone mAb1 to CHO cells expressing mouse PD-L1 (bottom). [Figure 18D] Bispecific 3 demonstrates drug-like properties (DLP) similar to well-behaved monoclonal antibodies and maintains parental PD-1 and PD-L1 binding. Figure 18D shows a size-exclusion chromatography trace of bispecific 3 after protein A chromatography (top), demonstrating a single peak with greater than 98% purity, and a differential scanning fluorimetry (DSF) trace of bispecific 3 (bottom), demonstrating that the molecule has high thermal stability. DETAILED DESCRIPTION OF THE INVENTION

[0073] Unless otherwise defined, all technical terms, notations, and other scientific terms used herein are intended to have the meaning commonly understood by one of ordinary skill in the art to which this disclosure pertains. In some cases, terms having a commonly understood meaning are defined herein for clarity and / or ready reference, and the inclusion of such definitions herein should not necessarily be construed as representing a difference to that commonly understood in the art. The techniques and procedures described or referenced herein are generally well understood and may be readily understood by those of ordinary skill in the art using conventional methodology, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual 2nd ed. (1989) Cold Spring Harbor Laboratory Press. These methods are generally used using widely available molecular cloning methodologies such as those described in the Molecular Cloning Methods of the 1990s and 2000s (Biochem. J. Clin. Microbiol. 1990, 1991, 1992, 1993, 1994, 1995, 1996, 1997, 1998, 1999, 2000, 2001, 2002, 2003, 2004, 2005, 2006, 2007, 2008, 2009, 2010, 2011, 2012, 2013, 2014, 2015, 2016, 2017, 2018, 2019, 2020, 2021, 2022,

[0074] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. In the specification and claims, the term "about" is used to modify, for example, the amount, concentration, volume, process temperature, process time, yield, flow rate, pressure, and similar values ​​and ranges of raw ingredients in a composition used in describing embodiments of the present disclosure. The term "about" refers to variations in numerical values ​​that may occur, for example, through typical measuring and handling procedures used to prepare a compound, composition, concentrate, or formulation; through unintentional errors in these procedures; differences in the production, source, or purity of starting materials or raw ingredients used to carry out the method, and similar considerations of approximation. The term "about" also encompasses amounts that differ due to aging of a formulation having a specific initial concentration or mixture, and amounts that differ due to mixing or processing of a formulation having a specific initial concentration or mixture. When modified by the term "about," the claims appended hereto include equivalents of these amounts. If there are uses of the term that are not clear to a person skilled in the art given the context in which it is used, "about" means up to plus or minus 10% of the specific value.

[0075] With respect to the binding of an antigen-binding protein / region / arm to a target molecule, terms such as "specific binding," "specifically binds," "specific," "selectively binds," and "selective," when referring to a specific antigen (e.g., a polypeptide target) or an epitope on a specific antigen, refer to binding that is measurably different from nonspecific or nonselective interactions. Specific binding can be measured, for example, by determining the binding of a molecule compared 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 when the binding of the labeled target to the probe is competitively inhibited by an excess of unlabeled target.

[0076] The term "epitope" refers to a component of an antigen capable of specific binding to an antigen-binding protein. Epitopes frequently consist 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. Conformational and nonconformational epitopes are distinguished in that the binding to the former is lost in the presence of denaturing solvents, while the binding to the latter is not. An epitope can include amino acid residues directly involved in binding and other amino acid residues not directly involved in binding. The epitope bound by an antigen-binding protein can be determined using known techniques for determining epitopes, such as testing the binding of the antigen-binding protein to antigen variants with different point mutations.

[0077] The "identity" percentage 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, if necessary, introducing gaps to achieve the maximum sequence identity percentage.Alignment for determining the percent amino acid sequence identity can be achieved in various ways within the skill of those skilled in 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.Those skilled in the art can determine the appropriate parameters for aligning sequences, including any algorithms required to achieve maximum alignment over the entire length of the sequences being compared.

[0078] The term "capable of," as used herein, means that an agent or method (e.g., any multispecific antigen-binding construct or method disclosed herein) has the ability to achieve a specified property in the appropriate context (as would be understood by one of skill in the art), but does not require being associated with that property at any particular time. For example, any multispecific antigen-binding construct disclosed herein may be capable of binding to PD-1 and / or PD-L1 when administered to cells that express PD-1 and / or PD-L1, but the construct would not be expected to bind to PD-1 and / or PD-L1 when in a composition lacking PD-1 or PD-L1 protein.

[0079] "Conservative substitution" or "conservative amino acid substitution" refers to the substitution 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 with such substitutions are known as "conservatively modified variants" or "variants." Such conservatively modified variants are additionally, but without excluding, polymorphic variants, interspecies homologs, and alleles. Some examples of conservative substitutions can be found, for example, in Creighton, Proteins: Structures and Molecular Properties 2nd ed. (1993) W.H. Freeman & Co. (New York, NY, USA).

[0080] The polypeptides disclosed herein can include non-naturally occurring amino acid sequences. Such variants necessarily have less than 100% sequence identity or similarity with the starting molecule. In certain embodiments, the variants have an amino acid sequence identity or similarity of about 75% to less than 100%, 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% with the amino acid sequence of the starting (e.g., naturally occurring or wild-type) polypeptide, for example, over the length of the variant molecule.

[0081] "Antibody," as used herein, can refer to an intact antibody (e.g., an intact immunoglobulin) and an antibody portion, e.g., an antigen-binding portion. An antigen-binding portion comprises at least one antigen-binding domain. One example of an antigen-binding domain is V H -V LThe antigen-binding domain is formed by a 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.

[0082] V H and V L The region can be further divided 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). Each V H and V L generally contain 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., National Institutes of Health, Public Health Service, Bethesda, Maryland, USA.) CDRs are involved in antigen binding and confer antigen specificity and binding affinity to antibodies.There are three CDRs in each heavy and light chain variable domain, and these are called CDR1, CDR2 and CDR3 for each variable domain.The term "CDR set" as used herein refers to a group of three CDRs occurring in a single heavy or light chain variable domain that can bind to target antigen.The exact boundaries of these CDRs are defined differently according to different systems.The three heavy chain CDRs can be called CDRH1, CDRH2 and CDRH3, and the three light chain CDRs can be called CDRL1, CDRL2 and CDRL3.

[0083] The system described by Kabat, also referred to as "numbered according to Kabat," "Kabat numbering," "Kabat definition," and "Kabat labeling," provides an unambiguous residue numbering system applicable to any antibody variable domain 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, USA (1987) and (1991); the entire contents of which are incorporated by reference.) 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 residues 31-35 (CDR1), 50-65 (CDR2), and 95-102 (CDR3) in the heavy chain variable domain. When CDRs are defined according to Kabat, light chain FR residues are located at approximately residues 1-23 (LCFR1), 35-49 (LCFR2), 57-88 (LCFR3), and 98-107 (LCFR4), and heavy chain FR residues are located at approximately residues 1-30 (HCFR1), 36-49 (HCFR2), 66-94 (HCFR3), and 103-113 (HCFR4) in the heavy chain. "EU index as in Kabat" refers to the residue numbering of a human IgG1 EU antibody.

[0084] Other CDR numbering systems are also used in the art (see, e.g., Table A). Chothia and coworkers found that certain subportions within Kabat CDRs adopt nearly identical peptide backbone conformations despite 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 are designated as L1, L2, and L3 or H1, H2, and H3, with "L" and "H" designating the light and heavy chain regions, respectively. These CDRs, sometimes referred to as "Chotia CDRs," "Chotia numbering," or "numbered according to Chothia," include approximately residues 24-34 (CDR1), 50-56 (CDR2), and 89-97 (CDR3) in the light chain variable domain, and residues 26-32 (CDR1), 50-56 or 52-56 (CDR2), and 95-102 (CDR3) in the heavy chain variable domain. Mol. Biol. 196:901-917 (1987).

[0085] The system described by MacCallum, also referred to as "numbered according to MacCallum" or "MacCallum numbering," includes 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. al.) ((1996) J. Mol. Biol. 262(5):732-745).

[0086] The system described by AbM, also referred to as "numbering according to AbM" or "AbM numbering," includes 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.

[0087] The IMGT (INTERNATIONAL IMMUNOGENETICS INFORMATION SYSTEM) numbering of variable regions can also be used, which is based on Lefranc, M.-P., "The IMGT unique numbering for immunoglobulin, T-cell receptor and Ig-like domains." "IMGT sequence numbering" refers to the numbering of residues in an immunoglobulin variable heavy or light chain according to the IMGT method as described in "Numbering for Immunoglobulins, T Cell Receptors and Ig-like Domains," Immunologist, 7, 132-136 (1999), which is expressly incorporated herein by reference in its entirety. As used herein, "IMGT sequence numbering" or "numbered according to IMGT" refers to the numbering of sequences encoding variable regions according to IMGT. For heavy chain variable domains, when numbered according to IMGT, the hypervariable region spans amino acid positions 27-38 for CDR1, amino acid positions 56-65 for CDR2, and amino acid positions 105-117 for CDR3. For light chain variable domains, when numbered according to IMGT, the hypervariable region spans amino acid positions 27-38 for CDR1, amino acid positions 56-65 for CDR2, and amino acid positions 105-117 for CDR3.

[0088] In some embodiments of the constructs and antigen-binding arms described herein, the CDRs described herein, when numbered according to Chothia numbering, 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. In some embodiments, CDR2 in the light chain variable domain can comprise amino acids 49-56 when numbered according to Chothia numbering.

[0089] [Table 1]

[0090] Preferred methods and materials are described herein, although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the methods and compositions of the disclosure. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference.

[0091] Various aspects of the invention are described in further detail below. Additional definitions are set forth throughout the specification. Although the immune system has the ability to recognize and eliminate tumor cells, tumors can employ multiple strategies to evade immunity. Recent studies have shown that inhibitory immune checkpoint molecules promote cancer progression through various antitumor inhibitory mechanisms. Blockade of immune checkpoints is one approach to therapeutically activate or reactivate antitumor immunity. Various ligands have been described for numerous cognate inhibitory immune checkpoint receptors. For example, these are reviewed in Nair & Elkord, Immunology & Cell Biology (2018), 96:21-33; and Jenkins et al., British Journal of Cancer (2017), 118:9-16.

[0092] 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 exists as a monomer and has been suggested to lack the unpaired cysteine ​​residue characteristic of other CD28 family members. PD-1 is expressed on activated B cells, T cells, and monocytes.

[0093] The PD-1 gene encodes a 55-kDa 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, PD-L1 (B7-H1) and PD-L2 (B7-DC), have been identified and shown to downregulate T cell activation upon 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 other CD28 family members. PD-L1 is abundant in a variety of human cancers (Dong et al. (2002) Nat. Med. 8:787-9).

[0094] PD-1 is known as an immunoinhibitory 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 reduction in tumor-infiltrating lymphocytes, a reduction in T cell receptor-mediated proliferation, and / or immune escape by cancerous 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). Immune suppression can be reversed by inhibiting the local interaction of PD-1 with PD-L1 or PD-L2, and the effect is additive when PD-1 interaction with PD-L2 is also blocked (Iwai et al. (2002) Proc. Nat'l. Acad. Sci. USA 99:12293-7; Brown et al. (2003) J. Immunol. 170:1257-66).

[0095] PD-L1, also known as cluster of differentiation 274 (CD274) or B7 homolog 1 (B7-H1), is a 40-kDa 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 contains 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, triggering 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, such as T cells. Binding of PD-L1 to PD-1 transmits an inhibitory signal that reduces the proliferation of antigen-specific T cells in lymph nodes, while simultaneously reducing apoptosis in regulatory T cells (anti-inflammatory, suppressive T cells). PD-1 / PD-L1 interaction also induces apoptosis of tumor-specific T cells and Foxp3 + CD4 on regulatory T cells + It promotes T cell differentiation and promotes tumor cell resistance to cytotoxic T lymphocyte (CTL) attack, thus enabling tumors to evade the host immune system.

[0096] The present disclosure relates to compositions and methods for inhibiting tumor escape by reducing immune checkpoint suppression resulting from the interaction between PD-1 and its ligands (e.g., PD-L1 and / or PD-L2). In particular, provided herein are compositions comprising novel multispecific and multivalent constructs, such as bispecific and tetravalent constructs, that block the interaction between PD-1 and its ligands (e.g., PD-1 and / or PD-L2) while promoting the interaction (bridging) of cells expressing PD-1 and its ligands. Such compositions of the present disclosure, capable of "blocking and bridging," have increased potency in vitro and in vivo, for example, by strongly enhancing T cell proliferation, IFNγ production and / or secretion, T cell cytolytic activity, and / or rescue of T cells from functional exhaustion, providing superior anti-tumor efficacy (e.g., biological effects that may be manifested by various means, including, but not limited to, reduced tumor volume, reduced tumor cell number, reduced tumor cell proliferation, and / or reduced tumor cell survival) compared to combinations of individual antibodies as well as clinical checkpoint blockade agents. 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 and multivalent constructs. Some of these novel monoclonal anti-PD-1 antibodies and novel monoclonal anti-PD-L1 antibodies share a common light chain, thereby enabling the generation of multispecific and multivalent constructs with similar affinities to their parent antibodies, as well as superior drug-like properties and manufacturability.

[0097] Thus, the present disclosure provides multispecific antigen-binding constructs comprising at least two antigen-binding arms or units, wherein a first antigen-binding arm or unit binds to PD-1 expressed by an immune cell and a 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 of PD-1 and its ligand. In some embodiments, the multispecific antigen-binding construct bridges cells expressing PD-1 and its ligand, promoting 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 a PD-1 ligand, e.g., 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, e.g., 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 of which bind to a PD-1 ligand, e.g., 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 of which bind to a PD-1 ligand, e.g., PD-L1 or PD-L2. 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 a PD-1 ligand. In some embodiments, the construct comprises a common light chain. In some embodiments, one or both of the antigen-binding arms is an aptamer. In some embodiments, one or both of the antigen-binding arms is a protein other than an antibody. 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, thereby making the construct 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 a PD-1 ligand. Also provided herein in some aspects are novel isolated antibodies, and antigen-binding portions thereof, that specifically bind to PD-L1 or PD-1. In some embodiments, these novel isolated antibodies that specifically bind to PD-L1 or PD-1, and antigen-binding portions thereof, e.g., CDRs, variable heavy chains, and / or variable light chains, may be used in one or more antigen-binding arms or units of the multispecific antigen-binding constructs described herein.

[0098] Thus, as described herein, the disclosed multispecific antigen-binding constructs include bispecific, trispecific, tetraspecific, or multispecific antibodies or antigen-binding portions thereof. The described multispecific constructs are preferably bivalent for at least one, and preferably both, antigen-binding arms, i.e., bispecific and trivalent, or bispecific and tetravalent molecules. The multispecific constructs described herein, in various aspects and embodiments, can comprise one or more antibodies and / or antigen-binding portions thereof. For example, an antigen-binding arm can comprise the variable heavy chain and / or variable light chain, or complementarity-determining regions (CDRs) thereof, of a given antibody against PD-1 and / or a given antibody against PD-L1. Thus, in some embodiments of any aspect described herein, the first antigen-binding arm, the second antigen-binding arm, the first unit of antigen-binding, the second unit of antigen-binding, or any combination thereof, can comprise an antibody or antigen-binding portion thereof. In some embodiments of any aspect described herein, the first antigen-binding arm, the second antigen-binding arm, the first unit of antigen binding, the second unit of binding, or any combination thereof is an antibody or antigen-binding portion thereof.

[0099] A. PD-L1 antagonist In some aspects and embodiments, the present disclosure provides an anti-PD-L1 antagonist. In some embodiments, the anti-PD-L1 antagonist is any anti-PD-L1 antibody or antigen-binding molecule disclosed herein. In some embodiments, the anti-PD-L1 antibody or antigen-binding molecule is not part of a multispecific antigen-binding construct, i.e., the anti-PD-L1 antibody or antigen-binding molecule is not part of a protein construct that binds to multiple epitopes. In some embodiments, an anti-PD-L1 antibody or antigen-binding moiety can be combined with a different antibody or antigen-binding moiety to form a multispecific antigen-binding construct. In some embodiments, the multispecific antigen-binding construct is capable of binding 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.

[0100] Thus, in some embodiments, provided herein are antibodies, or antigen-binding portions thereof, that specifically bind to PD-L1. In some embodiments, the antibodies, or antigen-binding portions thereof, that specifically bind to PD-L1 comprise: (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, where 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, where X1 is G, F, or N), and (b) a light chain variable region comprising: (i) a CDRL1 comprising SEQ ID NO: 4 (RASQX1ISSYLN, where 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, where X1 is Q or F).

[0101] 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). An exemplary antibody having such heavy and light chain variable CDR regions is mAb1.

[0102] 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). An exemplary 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). An exemplary antibody having such heavy and light chain variable CDR regions is mAb3.

[0103] 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). An exemplary antibody having such heavy and light chain variable CDR regions is mAb4. Another exemplary antibody having such heavy and light chain variable CDR regions is mAb24.

[0104] 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). An exemplary antibody having such heavy and light chain variable CDR regions is mAb5.

[0105] 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). An exemplary antibody having such heavy and light chain variable CDR regions is mAb6.

[0106] 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, 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). An exemplary antibody having such heavy and light chain variable CDR regions is mAb7.

[0107] 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). An exemplary antibody having such heavy and light chain variable CDR regions is mAb8.

[0108] 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). An exemplary antibody having such heavy and light chain variable CDR regions is mAb9.

[0109] 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). An exemplary antibody having such heavy and light chain variable CDR regions is mAb10.

[0110] 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). An exemplary antibody having such heavy and light chain variable CDR regions is mAb11.

[0111] 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). An exemplary antibody having such heavy and light chain variable CDR regions is mAb12.

[0112] 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). An exemplary antibody having such heavy and light chain variable CDR regions is mAb13.

[0113] 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). An exemplary antibody having such heavy and light chain variable CDR regions is mAb14.

[0114] 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). An exemplary antibody having such heavy and light chain variable CDR regions is mAb15.

[0115] 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). An exemplary antibody having such heavy and light chain variable CDR regions is mAb16.

[0116] 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).

[0117] 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). An exemplary antibody having such heavy and light chain variable CDR regions is mAb17.

[0118] 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). An exemplary antibody having such heavy and light chain variable CDR regions is mAb18.

[0119] 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). An exemplary antibody having such heavy and light chain variable CDR regions is mAb19.

[0120] 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). An exemplary antibody having such heavy and light chain variable CDR regions is mAb20.

[0121] 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). An exemplary antibody having such heavy and light chain variable CDR regions is mAb21.

[0122] 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). An exemplary antibody having such heavy and light chain variable CDR regions is mAb22.

[0123] 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). An exemplary antibody having such heavy and light chain variable CDR regions is mAb23.

[0124] 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). An exemplary antibody having such heavy and light chain variable CDR regions is mAb24.

[0125] In each case, where a specific sequence is referenced, embodiments are also provided that include sequences having at least 90% identity (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% identity) to the referenced sequence (e.g., SEQ ID NOs: 1-34 and 36-39).

[0126] The disclosure also provides, in some embodiments, an antibody or antigen-binding portion thereof that specifically binds to PD-L1, and 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%, at least 99%, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least 107%, at least 108%, at least 109%, at least 110%, at least 111%, at least 112%, at least 113%, at least 114%, at least 115%, at least 116%, at least 117%, at least 118%, at least 119%, at least 120%, at least 121%, at least 122%, at least 123%, at least 124%, at least 125%, at least 126%, at least 127%, at least 128%, at least 129%, at least 130%, at least 131%, at least 132%, at least 133%, at least 134%, at least 135%, at least 136%, at least 137%, at least 138%, at least 139%, at least 140%, at least 141%, at least 142%, at least 143%, at least 144%, at least 145%, at least 146%, at least 147%, at least 148%, at least 149%, at least 150%, at least 151%, at least 152%, at least 153%, at least 154%, at least 155%, at least 156%, at least 157%, at and a light chain variable region comprising an amino acid 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 SEQ ID NO: 59, 60, 61, 62, or 63.

[0127] The present disclosure also provides, in some embodiments, an antibody or antigen-binding portion thereof that specifically binds to PD-L1, wherein the antibody or antigen-binding portion thereof comprises a heavy chain variable region comprising an amino acid 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 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 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.

[0128] The present disclosure also provides, in some embodiments, an antibody or antigen-binding portion thereof that specifically binds to PD-L1, wherein the antibody or antigen-binding portion thereof comprises a heavy chain variable region comprising an amino acid 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 SEQ ID NO: 35 and a light chain variable region comprising an amino acid 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 SEQ ID NO: 59, 60, 61, 62, or 63.

[0129] Antibodies mAb1-mAb23 are affinity matured antibodies derived from parent antibody mAb24, as described in the Examples. Affinity matured antibodies or antigen-binding fragments 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 improvement in the affinity of the antibody for its antigen, compared to a parent antibody lacking the alterations. In some embodiments, affinity matured antibodies have nanomolar or picomolar affinity for PD-L1. In some embodiments, PD-L1 antibodies or antigen-binding portions thereof have an affinity of at least 1 x 10 -7 M, at least 1 × 10 -8 M, at least 1 × 10 -9 M, at least 1 × 10 -10 M, at least 1 × 10 -11 M, at least 1 × 10 -12 M, or at least 1 × 10 -13 K of M D It has.

[0130] Table 1 shows the binding affinity (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., affinity-matured variant of mAb24) to human PD-L1. D ) indicates the term K D As used herein, K refers to the dissociation equilibrium constant of a particular antibody-antigen interaction. D =k d / k a . term k d (seconds -1 ) as used herein refers to the dissociation rate constant of a specific antibody-antigen interaction. The value k off Also called value. The term k a (M -1 × seconds -1 ) as used herein refers to the association rate constant of a specific antibody-antigen interaction. The value k on Also called the value.

[0131] [Table 2]

[0132] Table 2 provides cell 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., affinity matured variant of mAb24) against human PD-L1 ("huPDL1"), cynomolgus PD-L1, or mouse PD-L1 ("muPDL1"). Human or cynomolgus PD-L1 was expressed on HEK cells, and mouse PD-L1 was expressed on A20 cells. Binding was measured using EC 50 The EC values ​​are expressed as mAb binding values, which can be estimated from titrating different concentrations of mAb on cells exogenously expressing the antigen of interest. Fluorescently tagged secondaries can be used to detect and quantify mAb binding. The data shown in Table 2 were fitted to a 1:1 binding model using built-in functions in GRAPHPAD to obtain the EC values. 50 got the value.

[0133] [Table 3]

[0134] The disclosure also provides an antibody or antigen-binding portion thereof that specifically binds to PD-L1, wherein the antibody or antigen-binding portion thereof 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 more) to any one of SEQ ID NOs: 35, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, or 58. or at least 99% identical) to any one of SEQ ID NOs: 59, 60, 61, 62, or 63, and a light chain variable region comprising an amino acid 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 any one of SEQ ID NOs: 59, 60, 61, 62, or 63. In some embodiments, the heavy chain variable region comprises an amino acid sequence that differs by 15 or fewer amino acids, 14 or fewer amino acids, 13 or fewer amino acids, 12 or fewer amino acids, 11 or fewer amino acids, 10 or fewer amino acids, 9 or fewer amino acids, 8 or fewer amino acids, 7 or fewer amino acids, 6 or fewer amino acids, 5 or fewer amino acids, 4 or fewer amino acids, 3 or fewer amino acids, 2 or fewer amino acids, or 1 amino acid from any one of SEQ ID NOs: 35, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, or 58.In some embodiments, the light chain variable region comprises an amino acid sequence that differs by 15 or fewer amino acids, 14 or fewer amino acids, 13 or fewer amino acids, 12 or fewer amino acids, 11 or fewer amino acids, 10 or fewer amino acids, 9 or fewer amino acids, 8 or fewer amino acids, 7 or fewer amino acids, 6 or fewer amino acids, 5 or fewer amino acids, 4 or fewer amino acids, 3 or fewer amino acids, 2 or fewer amino acids, or 1 amino acid from any one of SEQ ID NOs: 59, 60, 61, 62, or 63. Tables 3 and 4 provide the sequences of the heavy chain variable sequences of SEQ ID NOs: 35 and 40-58, and the light chain variable sequences of SEQ ID NOs: 59-63, respectively.

[0135] [Table 4]

[0136] [Table 5]

[0137] The present disclosure also provides, in some embodiments, an antibody or antigen-binding portion thereof that specifically binds to PD-L1, wherein the antibody or antigen-binding portion thereof comprises any of the heavy chain CDRs of the heavy chain variable region 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 any of the light chain CDRs of the light chain variable region of SEQ ID NO: 59, 60, 61, 62, or 63.

[0138] The present disclosure also provides, in some embodiments, 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 of the heavy chain variable regions of SEQ ID NOs: 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 of the light chain variable regions of SEQ ID NOs: 59, 60, 61, 62, or 63, wherein the heavy and light chain CDR residues are numbered according to Kabat.

[0139] The present disclosure also provides, in some embodiments, 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 of the heavy chain variable regions of SEQ ID NOs: 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 of the light chain variable regions of SEQ ID NOs: 59, 60, 61, 62, or 63, wherein the heavy and light chain CDR residues are numbered according to Chothia.

[0140] The present disclosure also provides, in some embodiments, an antibody or antigen-binding portion thereof that specifically binds to PD-L1, the antibody or antigen-binding portion thereof comprising the heavy chain CDRs of any of the heavy chain variable regions of SEQ ID NOs: 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 of the light chain variable regions of SEQ ID NOs: 59, 60, 61, 62, or 63, wherein the heavy and light chain CDR residues are numbered according to MacCallum.

[0141] The present disclosure also provides, in some embodiments, an antibody or antigen-binding portion thereof that specifically binds to PD-L1, the antibody or antigen-binding portion thereof comprising any of the heavy chain CDRs of the heavy chain variable region 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 any of the light chain CDRs of the light chain variable region of SEQ ID NO: 59, 60, 61, 62, or 63, wherein the heavy and light chain CDR residues are numbered according to AbM.

[0142] The present disclosure also provides, in some embodiments, an antibody or antigen-binding portion thereof that specifically binds to PD-L1, the antibody or antigen-binding portion thereof comprising any of the heavy chain CDRs of the heavy chain variable region 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 any of the light chain CDRs of the light chain variable region of SEQ ID NO: 59, 60, 61, 62, or 63, wherein the heavy and light chain CDR residues are numbered according to IMGT.

[0143] B. PD-1 antagonist In some aspects and embodiments, the present disclosure provides an anti-PD-1 antagonist. In some embodiments, the anti-PD-1 antagonist is any anti-PD-1 antibody or antigen-binding molecule disclosed herein. In some embodiments, the anti-PD-1 antibody or antigen-binding molecule is not part of a multispecific antigen-binding construct, i.e., the anti-PD-1 antibody or antigen-binding molecule is not part of a protein construct that binds to multiple epitopes. In some embodiments, an anti-PD-1 antibody or antigen-binding moiety can be combined with a different antibody or antigen-binding moiety 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.

[0144] 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, e.g., one known to promote tumor escape. However, in some cases, as used herein, "inhibitory receptor" specifically refers to PD-1.

[0145] PD-1 is an immune checkpoint inhibitory receptor containing an "immunoreceptor tyrosine inhibitory motif" or "ITIM," which comprises a conserved sequence of amino acids (S / I / V / L)xYxx(I / V / L) (where x is any amino acid). Methods for assaying whether PD-1 activity is inhibited are known in the art and can be easily designed by those skilled in the art. Such assays include, for example, testing the effects of any downstream signaling pathway of PD-1 in vitro or in vivo. After PD-1 interacts with its ligand, the ITIM motif is phosphorylated by enzymes, such as those of the Src kinase family, allowing the recruitment of other enzymes, such as the phosphotyrosine phosphatases SHP-1 and SHP-2, or an inositol phosphatase called SHIP. These phosphatases have been shown to reduce the activation of molecules involved in cell signaling. See, e.g., Barrow and Trowsdale (2006) Eur J Immunol. 36(7): 1646-53. Therefore, the phosphorylation status of ITIM motifs in PD-1 can be assessed using methods known in the art. The presence of downstream factors, such as phosphotyrosine phosphatases, can also be examined. Furthermore, various cell-based assays and kits are known in the art that detect the presence of downstream factors (e.g., nuclear factor of activated T cells—NFAT as a measure of PD-1 inhibition) as a surrogate for PD-1 activity status. In another example, a simple binding assay can be used to determine whether a construct of the present disclosure can block binding of PD-1 and its ligand, as discussed above.

[0146] Thus, in some embodiments, provided herein are antibodies, or antigen-binding portions thereof, that specifically bind to PD-1. In some embodiments, the antibodies, or antigen-binding portions thereof, that specifically bind to PD-1 comprise: (a) a heavy chain variable region comprising: (i) a CDRH1 comprising SEQ ID NO: 70 (FTFX1X2YAX3X4, where X1 = S, R, G, or N, X2 = D, S, N, A, R, or G, X3 = M or L, and X4 = S, L, or N), (ii) a CDRH2 comprising SEQ ID NO: 71 (SAISNSGTYTYYA), and (iii) a CDRH3 comprising SEQ ID NO: 72 (ARGLDFIVGX5TGNDY, where 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).

[0147] 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). An exemplary antibody having such heavy and light chain variable CDR regions is mAb25.

[0148] 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). An exemplary antibody having such heavy and light chain variable CDR regions is mAb26.

[0149] 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). An exemplary antibody having such heavy and light chain variable CDR regions is mAb27.

[0150] 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). An exemplary antibody having such heavy and light chain variable CDR regions is mAb28.

[0151] 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). An exemplary antibody having such heavy and light chain variable CDR regions is mAb29.

[0152] 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). An exemplary antibody having such heavy and light chain variable CDR regions is mAb30.

[0153] 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). An exemplary antibody having such heavy and light chain variable CDR regions is mAb31.

[0154] 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). An exemplary antibody having such heavy and light chain variable CDR regions is mAb32.

[0155] 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). An exemplary antibody having such heavy and light chain variable CDR regions is mAb33.

[0156] 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). An exemplary antibody having such heavy and light chain variable CDR regions is mAb34.

[0157] 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). An exemplary antibody having such heavy and light chain variable CDR regions is mAb35.

[0158] 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). An exemplary antibody having such heavy and light chain variable CDR regions is mAb36.

[0159] 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). An exemplary antibody having such heavy and light chain variable CDR regions is mAb37.

[0160] In each case, where a specific sequence is referenced, embodiments are also provided that include a sequence having at least 85% identity (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% identity) to the referenced sequence (e.g., SEQ ID NOs: 5, 9, 10, or 71-86).

[0161] In some embodiments, the present disclosure also provides an antibody or antigen-binding portion thereof that specifically binds to PD-1, the antibody or antigen-binding portion thereof comprising a heavy chain variable region comprising an amino acid sequence 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 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 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 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 heavy chain variable sequences of SEQ ID NOs: 87-99, and Table 4 provides the light chain variable sequence of SEQ ID NO: 59, respectively. In some embodiments, the heavy chain variable region comprises an amino acid sequence that differs by 15 or fewer amino acids, 14 or fewer amino acids, 13 or fewer amino acids, 12 or fewer amino acids, 11 or fewer amino acids, 10 or fewer amino acids, 9 or fewer amino acids, 8 or fewer amino acids, 7 or fewer amino acids, 6 or fewer amino acids, 5 or fewer amino acids, 4 or fewer amino acids, 3 or fewer amino acids, 2 or fewer amino acids, or 1 amino acid from any one of SEQ ID NOs: 87-99.In some embodiments, the light chain variable region comprises an amino acid sequence that differs from SEQ ID NO:59 by 15 or fewer amino acids, 14 or fewer amino acids, 13 or fewer amino acids, 12 or fewer amino acids, 11 or fewer amino acids, 10 or fewer amino acids, 9 or fewer amino acids, 8 or fewer amino acids, 7 or fewer amino acids, 6 or fewer amino acids, 5 or fewer amino acids, 4 or fewer amino acids, 3 or fewer amino acids, 2 or fewer amino acids, or 1 amino acid.

[0162] Antibodies mAb26-mAb37 are affinity matured antibodies derived from 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 improvement in the affinity of the antibody for its antigen, compared to a parent antibody lacking the alterations. In some embodiments, affinity matured antibodies have nanomolar or picomolar affinity for PD-1. In some embodiments, PD-1 antibodies or antigen-binding portions thereof have an affinity of at least 1×10 -7 M, at least 1 × 10 -8 M, at least 1 × 10 -9 M, at least 1 × 10 -10 M, at least 1 × 10 -11 M, at least 1 × 10 -12 M, or at least 1 × 10 -13 K of M D It has.

[0163] Tables 5 and 6 provide cell binding data for mAb25, mAb26, mAb27, mAb28, mAb29, mAb30, mAb31, mAb32, mAb33, mAb34, mAb35, mAb36, and mAb37 (i.e., affinity matured variants of mAb25) against human PD-1 ("huPD-1"), cynomolgus monkey PD-1 ("cyPD-1"), or mouse PD-1 ("muPD-1"). Human, cynomolgus monkey, or mouse PD-1 was expressed on CHO cells. Binding was measured using EC 50 The EC values ​​are expressed as mAb binding values, which can be estimated from titrating different concentrations of mAb on cells exogenously expressing the antigen of interest. Fluorescently tagged secondary antibodies can be used to detect and quantitate mAb binding. The data shown in Table 5 were fitted to a 1:1 binding model using built-in functions in GRAPHPAD to obtain the EC values. 50 got the value.

[0164] [Table 6]

[0165] [Table 7]

[0166] [Table 8]

[0167] The present disclosure also provides, in some embodiments, 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 of the heavy chain variable regions of SEQ ID NOs: 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.

[0168] The present disclosure also provides, in some embodiments, an antibody or antigen-binding portion thereof that specifically binds to PD-L1, the antibody or antigen-binding portion thereof comprising the heavy chain CDRs of any of the heavy chain variable regions of SEQ ID NOs: 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.

[0169] The present disclosure also provides, in some embodiments, an antibody or antigen-binding portion thereof that specifically binds to PD-L1, the antibody or antigen-binding portion thereof comprising the heavy chain CDRs of any of the heavy chain variable regions of SEQ ID NOs: 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.

[0170] The present disclosure also provides, in some embodiments, 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 of the heavy chain variable regions of SEQ ID NOs: 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 McCallum.

[0171] The present disclosure also provides, in some embodiments, an antibody or antigen-binding portion thereof that specifically binds to PD-L1, the antibody or antigen-binding portion thereof comprising the heavy chain CDRs of any of the heavy chain variable regions of SEQ ID NOs: 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.

[0172] The present disclosure also provides, in some embodiments, an antibody or antigen-binding portion thereof that specifically binds to PD-L1, the antibody or antigen-binding portion thereof comprising the heavy chain CDRs of any of the heavy chain variable regions of SEQ ID NOs: 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.

[0173] C. Multispecific antigen-binding constructs In some aspects, the present disclosure provides compositions and methods for enhancing immune responses against 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," "specific," "selectively bind," and "selective" with respect to PD-L1 or PD-1, or an epitope on PD-L1 or PD-1, refer to binding that is measurably different from nonspecific or nonselective 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 determining binding of a molecule compared to 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. Specific binding is then indicated when the binding of the labeled target to the probe is competitively inhibited by excess unlabeled target.

[0174] In some embodiments, any multispecific antigen-binding construct disclosed herein binds to at least two different receptors or epitopes (e.g., PD-1 and PD-L1), and 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, a multispecific antigen-binding construct simultaneously binds to PD-1 and PD-L1, and PD-1 and PD-L1 are expressed on the surface of the same cell. In some embodiments, any multispecific antigen-binding construct disclosed herein binds to at least two different receptors or epitopes (e.g., PD-1 and PD-L1), and 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, a 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.

[0175] In some embodiments, the multispecific antigen-binding construct is capable of binding to human PD-1. In some embodiments, the multispecific antigen-binding construct is capable of binding to mouse PD-1. In some embodiments, the multispecific antigen-binding construct is capable of binding to cynomolgus PD-1. In some embodiments, the multispecific antigen-binding construct is capable of binding to human, mouse, and cynomolgus PD-1 with similar affinity.

[0176] In some aspects and embodiments, the present disclosure provides a multispecific antigen-binding construct comprising at least two antigen-binding units, wherein a first antigen-binding unit binds to PD-1 and a 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 of PD-1 and a PD-1 ligand, e.g., PD-L1 or PD-L2. In some embodiments, the multispecific antigen-binding construct blocks the interaction of PD-1 and a PD-1 ligand, e.g., 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, e.g., 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, e.g., 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 of which bind to a PD-1 ligand, e.g., 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 of which bind to a PD-1 ligand, e.g., 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, e.g., 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 of PD-1 and a PD-1 ligand, e.g., PD-L1 or PD-L2. In some embodiments, the multispecific antigen-binding construct comprises a common light chain. For example, at least two of the antigen-binding units comprise a common light chain.

[0177] In some embodiments, the antigen-binding first unit binds to PD-1 and (a) a heavy chain variable region comprising: (i) a CDRH1 comprising SEQ ID NO: 70 (FTFX1X2YAX3X4, where X1 = S, R, G, or N, X2 = D, S, N, A, R, or G, X3 = M or L, and X4 = S, L, or N); (ii) a CDRH2 comprising SEQ ID NO: 71 (SAISNSGTYTYYA); and (iii) a CDRH3 comprising SEQ ID NO: 72 (ARGLDFIVGX5TGNDY, where 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). Includes:

[0178] In some such embodiments, the antigen-binding first unit binds to PD-1 and (a) CDRH1 comprising SEQ ID NO: 73 (FTFSDYAMS), CDRH2 comprising SEQ ID NO: 71 (SAISNSGTYTYYA), and CDRH3 comprising SEQ ID NO: 74 (ARGLDFIVGATGNDY); (b) CDRH1 comprising SEQ ID NO: 73 (FTFSDYAMS), CDRH2 comprising SEQ ID NO: 71 (SAISNSGTYTYYA), and CDRH3 comprising SEQ ID NO: 75 (ARGLDFIVGYTGNDY); (c) CDRH1 comprising SEQ ID NO: 76 (FTFSSYAMS), CDRH2 comprising SEQ ID NO: 71 (SAISNSGTYTYYA), and CDRH3 comprising SEQ ID NO: 75 (ARGLDFIVGYTGNDY); (d) CDRH1 comprising SEQ ID NO: 77 (FTFSSYAML), CDRH2 comprising SEQ ID NO: 71 (SAISNSGTYTYYA), and CDRH3 comprising SEQ ID NO: 75 (ARGLDFIVGYTGNDY); (e) CDRH1 comprising SEQ ID NO: 78 (FTFSNYALS), CDRH2 comprising SEQ ID NO: 71 (SAISNSGTYTYYA), and CDRH3 comprising SEQ ID NO: 75 (ARGLDFIVGYTGNDY); (f) CDRH1 comprising SEQ ID NO: 79 (FTFSAYAMN), CDRH2 comprising SEQ ID NO: 71 (SAISNSGTYTYYA), and CDRH3 comprising SEQ ID NO: 75 (ARGLDFIVGYTGNDY); (g) CDRH1 comprising SEQ ID NO: 80 (FTFRSYAMS), CDRH2 comprising SEQ ID NO: 71 (SAISNSGTYTYYA), and CDRH3 comprising SEQ ID NO: 75 (ARGLDFIVGYTGNDY); (h) CDRH1 comprising SEQ ID NO: 81 (FTFGRYAMS), CDRH2 comprising SEQ ID NO: 71 (SAISNSGTYTYYA), and CDRH3 comprising SEQ ID NO: 75 (ARGLDFIVGYTGNDY); (i) CDRH1 comprising SEQ ID NO: 82 (FTFNSYAMS), CDRH2 comprising SEQ ID NO: 71 (SAISNSGTYTYYA), and CDRH3 comprising SEQ ID NO: 75 (ARGLDFIVGYTGNDY); (j) CDRH1 comprising SEQ ID NO: 83 (FTFSNYAMS), CDRH2 comprising SEQ ID NO: 71 (SAISNSGTYTYYA), and CDRH3 comprising SEQ ID NO: 74 (ARGLDFIVGATGNDY); (k) CDRH1 comprising SEQ ID NO: 84 (FTFSGYAMS), CDRH2 comprising SEQ ID NO: 71 (SAISNSGTYTYYA), and CDRH3 comprising SEQ ID NO: 85 (ARGLDFIVGRTGNDY); (l) CDRH1 comprising SEQ ID NO: 86 (FTFSSYAMN), CDRH2 comprising SEQ ID NO: 71 (SAISNSGTYTYYA), and CDRH3 comprising SEQ ID NO: 85 (ARGLDFIVGRTGNDY), or (m) CDRH1 comprising SEQ ID NO: 80 (FTFRSYAMS), CDRH3 comprising SEQ ID NO: 71 (SAISNSGTYTYYA), and CDRH4 comprising SEQ ID NO: 85 (ARGLDFIVGRTGNDY) Includes:

[0179] In some embodiments, the antigen-binding first unit binds to PD-1 and (a) a heavy chain variable region comprising an amino acid sequence at least 90% identical to SEQ ID NO: 87; (b) a heavy chain variable region comprising an amino acid sequence at least 90% identical to SEQ ID NO: 88; (c) a heavy chain variable region comprising an amino acid sequence at least 90% identical to SEQ ID NO: 89; (d) a heavy chain variable region comprising an amino acid sequence at least 90% identical to SEQ ID NO: 90; (e) a heavy chain variable region comprising an amino acid sequence at least 90% identical to SEQ ID NO: 91; (f) a heavy chain variable region comprising an amino acid sequence at least 90% identical to SEQ ID NO: 92; (g) a heavy chain variable region comprising an amino acid sequence at least 90% identical to SEQ ID NO: 93; (h) a heavy chain variable region comprising an amino acid sequence at least 90% identical to SEQ ID NO: 94; (i) a heavy chain variable region comprising an amino acid sequence at least 90% identical to SEQ ID NO: 95; (j) a heavy chain variable region comprising an amino acid sequence at least 90% identical to SEQ ID NO: 96; (k) a heavy chain variable region comprising an amino acid sequence at least 90% identical to SEQ ID NO: 97; (l) a heavy chain variable region comprising an amino acid sequence at least 90% identical to SEQ ID NO: 98; or (m) comprises a heavy chain variable region comprising an amino acid sequence at least 90% identical to SEQ ID NO: 99.

[0180] In some embodiments, the antigen-binding first 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. In some embodiments, the antigen-binding second unit binds to PD-L2. In some embodiments, the antigen-binding second unit binds to PD-L1. In some embodiments, the antigen-binding second unit binds to PD-L1. 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, where 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, where X1 is G, F, or N), and b. A light chain variable region comprising: (i) a CDRL1 comprising SEQ ID NO: 4 (RASQX1ISSYLN, where 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, where X1 is Q or F). Includes:

[0181] In some such embodiments, the antigen-binding second unit binds to PD-L1 and (a) CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), CDRH2 comprising SEQ ID NO: 7 (GGIIPILGAATYA), and CDRH3 comprising SEQ ID NO: 8 (ARLKGELKDAFDI); (b) CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), CDRH2 comprising SEQ ID NO: 7 (GGIIPILGAATYA), 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); (c) 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); (d) CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), CDRH2 comprising SEQ ID NO: 11 (GGIIPVFGTATYA), and CDRH3 comprising SEQ ID NO: 8 (ARLKGELKDAFDI); (e) 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: 12 (RASQWISSYLN), CDRL2 comprising SEQ ID NO: 5 (AASSLQS), and CDRL3 comprising SEQ ID NO: 10 (QQSYSTPLT); (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); (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); (h) CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), CDRH2 comprising SEQ ID NO: 15 (GGIIPIFGIANYA), and CDRH3 comprising SEQ ID NO: 8 (ARLKGELKDAFDI); (i) CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), CDRH2 comprising SEQ ID NO: 15 (GGIIPIFGIANYA), 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); (j) CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), CDRH2 comprising SEQ ID NO: 16 (GGIIPNFGTATYA), and CDRH3 comprising SEQ ID NO: 17 (ARLKGELKGAGDI); (k) CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), CDRH2 comprising SEQ ID NO: 16 (GGIIPNFGTATYA), CDRH3 comprising SEQ ID NO: 17 (ARLKGELKGAGDI), CDRL1 comprising SEQ ID NO: 9 (RASQSISSYLN), CDRL2 comprising SEQ ID NO: 5 (AASSLQS), and CDRL3 comprising SEQ ID NO: 10 (QQSYSTPLT); (l) CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), CDRH2 comprising SEQ ID NO: 11 (GGIIPVFGTATYA), and CDRH3 comprising SEQ ID NO: 18 (ARLKFELKDAFDI); (m) CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), CDRH2 comprising SEQ ID NO: 11 (GGIIPVFGTATYA), CDRH3 comprising SEQ ID NO: 18 (ARLKFELKDAFDI), CDRL1 comprising SEQ ID NO: 9 (RASQSISSYLN), CDRL2 comprising SEQ ID NO: 5 (AASSLQS), and CDRL3 comprising SEQ ID NO: 10 (QQSYSTPLT); (n) CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), CDRH2 comprising SEQ ID NO: 11 (GGIIPVFGTATYA), and CDRH3 comprising SEQ ID NO: 19 (ARLKGELKDAFDE); (o) CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), CDRH2 comprising SEQ ID NO: 11 (GGIIPVFGTATYA), CDRH3 comprising SEQ ID NO: 19 (ARLKGELKDAFDE), CDRL1 comprising SEQ ID NO: 9 (RASQSISSYLN), CDRL2 comprising SEQ ID NO: 5 (AASSLQS), and CDRL3 comprising SEQ ID NO: 10 (QQSYSTPLT); (p) CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), CDRH2 comprising SEQ ID NO: 11 (GGIIPVFGTATYA), and CDRH3 comprising SEQ ID NO: 20 (ARLKNELKDAFDI); (q) CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), CDRH2 comprising SEQ ID NO: 11 (GGIIPVFGTATYA), CDRH3 comprising SEQ ID NO: 20 (ARLKNELKDAFDI), CDRL1 comprising SEQ ID NO: 9 (RASQSISSYLN), CDRL2 comprising SEQ ID NO: 5 (AASSLQS), and CDRL3 comprising SEQ ID NO: 10 (QQSYSTPLT); (r) CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), CDRH2 comprising SEQ ID NO: 21 (GGVIPFLGTANYA), and CDRH3 comprising SEQ ID NO: 22 (ARLKGILKDALDI); (s) CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), CDRH2 comprising SEQ ID NO: 21 (GGVIPFLGTANYA), CDRH3 comprising SEQ ID NO: 22 (ARLKGILKDALDI), CDRL1 comprising SEQ ID NO: 9 (RASQSISSYLN), CDRL2 comprising SEQ ID NO: 5 (AASSLQS), and CDRL3 comprising SEQ ID NO: 10 (QQSYSTPLT); (t) CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), CDRH2 comprising SEQ ID NO: 29 (GRIIPIFGTADYA), and CDRH3 comprising SEQ ID NO: 8 (ARLKGELKDAFDI); (u) CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), CDRH2 comprising SEQ ID NO: 29 (GRIIPIFGTADYA), 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); (v) CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), CDRH2 comprising SEQ ID NO: 31 (GGIIPILGTATYA), and CDRH3 comprising SEQ ID NO: 32 (ARRKGELKDAFDI); (w) CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), CDRH2 comprising SEQ ID NO: 31 (GGIIPILGTATYA), 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); (x) CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), CDRH2 comprising SEQ ID NO: 33 (GGIIPIVATANYA), and CDRH3 comprising SEQ ID NO: 32 (ARRKGELKDAFDI); (y) CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), CDRH2 comprising SEQ ID NO: 33 (GGIIPIVATANYA), 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); (z) CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), CDRH2 comprising SEQ ID NO: 34 (GGIIPIFGKATYA), and CDRH3 comprising SEQ ID NO: 32 (ARRKGELKDAFDI); (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); (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); (cc) 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: 39 (QQSYSTILT); (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); (ee) CDRH1 comprising SEQ ID NO: 23 (GTFSSYAIS), CDRH2 comprising SEQ ID NO: 24 (GGIIPIVGIANYA), and CDRH3 comprising SEQ ID NO: 8 (ARLKGELKDAFDI); (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); (gg) CDRH1 comprising SEQ ID NO: 23 (GTFSSYAIS), CDRH2 comprising SEQ ID NO: 11 (GGIIPVFGTATYA), and CDRH3 comprising SEQ ID NO: 25 (ARLKGEFKDAFDI); (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); (ii) CDRH1 comprising SEQ ID NO: 23 (GTFSSYAIS), CDRH2 comprising SEQ ID NO: 26 (GRIIPLFGTAHYA), and CDRH3 comprising SEQ ID NO: 8 (ARLKGELKDAFDI); (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); (kk) CDRH1 comprising SEQ ID NO: 23 (GTFSSYAIS), CDRH2 comprising SEQ ID NO: 27 (GRINPILGTANYA), and CDRH3 comprising SEQ ID NO: 28 (ARLKGELKDAFSI); (ll) 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); (mm) CDRH1 comprising SEQ ID NO: 23 (GTFSSYAIS), CDRH2 comprising SEQ ID NO: 11 (GGIIPVFGTATYA), and CDRH3 comprising SEQ ID NO: 30 (ARLKGELKCAFDI); (nn) CDRH1 comprising SEQ ID NO: 23 (GTFSSYAIS), CDRH2 comprising SEQ ID NO: 11 (GGIIPVFGTATYA), and CDRL1 comprising SEQ ID NO: 9 (RASQSISSYLN), CDRL2 comprising SEQ ID NO: 5 (AASSLQS), and CDRL3 comprising SEQ ID NO: 10 (QQSYSTPLT), comprising SEQ ID NO: 30 (ARLKGELKCAFDI); (oo) CDRH1 comprising SEQ ID NO: 36 (GPFRSHAVS), CDRH2 comprising SEQ ID NO: 11 (GGIIPVFGTATYA), and CDRH3 comprising SEQ ID NO: 37 (ARLKSELKDAFDI), or (pp) CDRH1 containing SEQ ID NO:36 (GPFRSHAVS), CDRH2 containing SEQ ID NO:11 (GGIIPVFGTATYA), and CDRH3 containing SEQ ID NO:37 (ARLKSELKDAFDI), CDRL1 containing SEQ ID NO:9 (RASQSISSYLN), CDRL2 containing SEQ ID NO:5 (AASSLQS), and CDRL3 containing SEQ ID NO:10 (QQSYSTPLT) Includes:

[0182] In some such embodiments, the antigen-binding second 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 NOs: 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 NOs: 59, 60, 61, 62, or 63.

[0183] In some embodiments, the antigen-binding second unit binds to PD-L1 and (a) an amino acid sequence at least 90% identical to SEQ ID NO: 35; (b) an amino acid sequence at least 90% identical to SEQ ID NO: 40; (c) an amino acid sequence at least 90% identical to SEQ ID NO: 41; (d) an amino acid sequence at least 90% identical to SEQ ID NO: 42; (e) an amino acid sequence at least 90% identical to SEQ ID NO: 43; (f) an amino acid sequence at least 90% identical to SEQ ID NO: 44; (g) an amino acid sequence at least 90% identical to SEQ ID NO: 45; (h) an amino acid sequence at least 90% identical to SEQ ID NO: 46; (i) an amino acid sequence at least 90% identical to SEQ ID NO: 47; (j) an amino acid sequence at least 90% identical to SEQ ID NO: 48; (k) an amino acid sequence at least 90% identical to SEQ ID NO: 49; (l) an amino acid sequence at least 90% identical to SEQ ID NO: 50; (m) an amino acid sequence at least 90% identical to SEQ ID NO: 51; (n) an amino acid sequence at least 90% identical to SEQ ID NO: 52; (o) an amino acid sequence at least 90% identical to SEQ ID NO: 53; (p) an amino acid sequence at least 90% identical to SEQ ID NO: 54; (q) an amino acid sequence at least 90% identical to SEQ ID NO: 55; (r) an amino acid sequence at least 90% identical to SEQ ID NO: 56; (s) an amino acid sequence at least 90% identical to SEQ ID NO: 57; or (t) an amino acid sequence at least 90% identical to SEQ ID NO: 58 The heavy chain variable region comprises:

[0184] In some embodiments, the antigen-binding second unit binds to PD-L1 and (a) an amino acid sequence at least 90% identical to SEQ ID NO: 59; (b) an amino acid sequence at least 90% identical to SEQ ID NO: 60; (c) an amino acid sequence at least 90% identical to SEQ ID NO: 61; (d) an amino acid sequence at least 90% identical to SEQ ID NO: 62; or (e) an amino acid sequence at least 90% identical to SEQ ID NO: 63 The light chain variable region comprises:

[0185] 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 a PD-1 ligand, 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), a fusion protein in which the heavy chain of the antibody further comprises the heavy chain variable region of an anti-PD-L1 antibody (mAb1) at its C-terminus, connected to the Fc region of an anti-BCMA antibody by a poly-GGGS (SEQ ID NO: 120) linker. The light chains of the anti-PD-1 and anti-PD-L1 portions of the construct are identical (SEQ ID NO: 101). Bispecific 3, the construct depicted in Figure 13A, comprises the heavy chain sequence set forth in SEQ ID NO: 100 and the light chain sequence set forth in SEQ ID NO: 101.

[0186] In some embodiments, the present disclosure provides a multispecific antibody or antigen-binding portion thereof that specifically binds to PD-1 and PD-L1, wherein the multispecific antibody or antigen-binding portion thereof comprises a heavy chain region comprising an amino acid sequence 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 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.

[0187] In some embodiments, the present disclosure provides a multispecific antibody or antigen-binding portion thereof that specifically binds to PD-1 and PD-L1, wherein the multispecific antibody or antigen-binding portion thereof comprises a heavy chain region comprising an amino acid sequence 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 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 by 15 or fewer amino acids, 14 or fewer amino acids, 13 or fewer amino acids, 12 or fewer amino acids, 11 or fewer amino acids, 10 or fewer amino acids, 9 or fewer amino acids, 8 or fewer amino acids, 7 or fewer amino acids, 6 or fewer amino acids, 5 or fewer amino acids, 4 or fewer amino acids, 3 or fewer amino acids, 2 or fewer amino acids, or 1 amino acid from SEQ ID NO: 100. In some embodiments, the light chain region comprises an amino acid sequence that differs by 15 or fewer amino acids, 14 or fewer amino acids, 13 or fewer amino acids, 12 or fewer amino acids, 11 or fewer amino acids, 10 or fewer amino acids, 9 or fewer amino acids, 8 or fewer amino acids, 7 or fewer amino acids, 6 or fewer amino acids, 5 or fewer amino acids, 4 or fewer, 3 or fewer amino acids, 2 or fewer, or 1 amino acid from SEQ ID NO: 101.

[0188] In some embodiments, the present disclosure provides a multispecific antibody or antigen-binding portion thereof that specifically binds to PD-1 and PD-L1, wherein the multispecific antibody or antigen-binding portion thereof comprises a heavy chain region comprising an amino acid sequence 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 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 by 15 or fewer amino acids, 14 or fewer amino acids, 13 or fewer amino acids, 12 or fewer amino acids, 11 or fewer amino acids, 10 or fewer amino acids, 9 or fewer amino acids, 8 or fewer amino acids, 7 or fewer amino acids, 6 or fewer amino acids, 5 or fewer amino acids, 4 or fewer amino acids, 3 or fewer amino acids, 2 or fewer amino acids, or 1 amino acid from SEQ ID NO: 102. In some embodiments, the light chain region comprises an amino acid sequence that differs by 15 or fewer amino acids, 14 or fewer amino acids, 13 or fewer amino acids, 12 or fewer amino acids, 11 or fewer amino acids, 10 or fewer amino acids, 9 or fewer amino acids, 8 or fewer amino acids, 7 or fewer amino acids, 6 or fewer amino acids, 5 or fewer amino acids, 4 or fewer, 3 or fewer amino acids, 2 or fewer, or 1 amino acid from SEQ ID NO: 103.

[0189] Also provided herein in some aspects and embodiments is a multispecific antigen-binding construct comprising four antigen-binding units, wherein two of the antigen-binding units bind to PD-1 and two of the antigen-binding units bind to PD-L1, and wherein the construct comprises a heavy chain amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO: 100 or 102, and a light chain amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO: 101 or 103.

[0190] Also provided herein in some aspects and embodiments is a multispecific antigen-binding construct comprising four antigen-binding units, wherein two of the antigen-binding units bind to PD-1 and two of the antigen-binding units bind to PD-L1, and wherein the construct comprises a heavy chain amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO: 100 and a light chain amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO: 101.

[0191] Also provided herein in some aspects and embodiments is a multispecific antigen-binding construct comprising four antigen-binding units, wherein two of the antigen-binding units bind to PD-1 and two of the antigen-binding units bind to PD-L1, and wherein the construct comprises a heavy chain amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO: 102 and a light chain amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO: 103.

[0192] [Table 9]

[0193] [Table 10]

[0194] The term "multispecific antigen-binding construct" as used herein refers to bispecific, trispecific, or multispecific antigen-binding constructs, and antigen-binding portions or fragments thereof. A multispecific antigen-binding construct can be a single multifunctional polypeptide or a multimeric complex of two or more molecules (e.g., polypeptides and / or aptamers) covalently or noncovalently associated with each other. The term "multispecific antigen-binding construct" includes an antibody (or antigen-binding fragment thereof) that can be linked to or coexpressed with another functional molecule, e.g., another peptide, protein, and / or aptamer. For example, an antibody or fragment thereof can be functionally linked (e.g., by chemical coupling, genetic fusion, noncovalent association, or otherwise) to one or more other molecular entities, such as a protein or fragment thereof, 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 antigen-binding fragments thereof. In certain embodiments, an antibody is operably linked to another antibody or antigen-binding fragment thereof to produce a bispecific antibody having a second binding specificity. Bispecific and multispecific antibodies of the invention are described elsewhere herein.

[0195] As used herein, an antigen-binding "arm" refers to a unit, domain, region, or the like of a multispecific antigen-binding construct that forms the antigen-binding compartment of the construct. Thus, the "first arm" forms a binding compartment of the multispecific antigen-binding construct that is separate from the "second arm" of the construct, and each arm forms an antigen-binding unit. Generally, one "arm" (first arm) is distinct from the other "arm" (second arm) in its antigen-binding or antigen-specificity. Thus, in an example of a bispecific, bivalent antibody, one arm of the antibody binds to antigen A, and the other arm of the antibody binds to antigen B. In some embodiments, in an example of a bispecific, bivalent antibody, one arm of the antibody binds to antigen A, and the other arm of the antibody binds to antigen B or C (e.g., cross-reacts with two antigens, such as PD-L1 and PD-L2, due to structural similarities). 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 section of the antibody that binds to antigen A (even if two binding sites of the bivalent antibody bind to antigen A), and the "other arm" refers to the section of the antibody that binds to antigen B (even if two 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 section of the antibody that binds to antigen A (even if two binding sites of the bivalent antibody bind to antigen A), and the "other arm" refers to the section of the antibody that binds to antigen B or C (even if two binding sites of the bivalent antibody can bind to antigen B or C). See, e.g., U.S. Pat. No. 9,845,356. As will be apparent to one of skill in the art, "first" and "second" may be used interchangeably.

[0196] The term "valency," when used to describe an antigen-binding construct or protein or antigen-binding arm, refers to the number of recognition (binding) sites in the antigen-binding construct or protein, regardless of whether different recognition or binding sites bind to the same epitope. Each recognition site can specifically recognize and therefore bind to one epitope (binding site) on an antigen. If an antigen-binding protein contains more than one recognition site (e.g., if the antigen-binding protein is an IgG with two recognition sites in its variable region), each recognition site can specifically recognize the same epitope on the same antigen, or different epitopes, whether on the same antigen or different antigens. Multivalency can increase the avidity, i.e., the strength of binding, between the antigen-binding arm or construct and the associated antigen or target receptor. Avidity relates to both the affinity between an epitope or antigenic determinant and its binding site on the antigen-binding unit and the actual number of associated binding sites present on the antigen-binding unit.

[0197] In some embodiments, any multispecific antigen-binding construct disclosed herein comprises a multivalent (e.g., bivalent) antibody or antigen-binding fragment, at least two of whose valencies specifically bind to PD-1. In some embodiments, any multispecific antigen-binding construct disclosed herein comprises a multivalent (e.g., bivalent) antibody or antigen-binding fragment, at least two of whose valencies specifically bind to a PD-1 ligand (e.g., PD-L1 or PD-L2). In some embodiments, any multispecific antigen-binding construct disclosed herein comprises a multivalent (e.g., bivalent) antibody or antigen-binding fragment, at least two of whose valencies specifically bind to PD-L1. In some embodiments, any multispecific antigen-binding construct 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 valencies of the first multivalent antibody or antigen-binding fragment specifically bind to PD-1 and at least two valencies of the second multivalent antibody or antigen-binding fragment specifically bind to PD-L1. In some embodiments, any multispecific antigen-binding construct 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 both valencies of the first bivalent antibody or antigen-binding fragment are specific for the same epitope on PD-1 and both valencies of the second bivalent antibody or antigen-binding fragment are 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 use light chains with 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.

[0198] In some embodiments, the first arm is a PD-1 antagonist. In some embodiments, the second arm is a PD-1 ligand antagonist, e.g., PD-L1 and / or PD-L2. In some embodiments, the first arm is a PD-1 antagonist and the second arm is a cognate PD-1 ligand antagonist, e.g., PD-L1 and / or PD-L2.

[0199] The terms "antagonist," "antagonize," and "inhibit," when used to refer to the biological activity of an antigen-binding arm, indicate that the antigen-binding arm binds to its target (e.g., PD-1) on the respective cell and partially or fully blocks, inhibits, and / or reduces a PD-1-mediated biological response. 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 use in the disclosed methods are also disclosed herein. For example, these methods include, but are not limited to, binding assays such as enzyme-linked immunosorbent assay (ELISA), FORTE BIO (Copyright) system, and radioimmunoassay (RIA). These assays determine 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. The efficacy of an antagonist, such as the ability of an antagonist to inhibit the function of a polypeptide, can also be determined using a functional assay. For example, a functional assay may 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 potency of an antagonist is typically determined by its IC 50 It is defined by the IC value (the concentration required to inhibit 50% of the agonist response). 50The lower the value, the more potent the antagonist and the lower the concentration required to inhibit the maximal biological response.

[0200] In some embodiments, at least one antigen-binding arm comprises at least 1 x 10 -7 M, at least 1 × 10 -8 M, at least 1 × 10 -9 M, at least 1 × 10 -10 M, at least 1 × 10 -11 M, at least 1 × 10 -12 M, or at least 1 × 10 -13 K of M D In some embodiments, both antigen-binding arms have the same or similar K D "K D The term "(M)" as used herein refers to the dissociation equilibrium constant of a particular antigen-binding arm / antigen interaction. D =k d / k a . "k d ”(seconds -1 The term k ) as used herein refers to the dissociation rate constant of a specific antigen-binding arm / antigen interaction. This value is k off Also called the k value. a " (M -1 × seconds -1 The term k ) as used herein refers to the association rate constant of a specific antigen-binding arm / antigen interaction. This value is known as k on Also called the value.

[0201] In some embodiments, binding of one arm (e.g., a first arm) of a multispecific antigen-binding construct to its target does not block binding of the other arm (e.g., a second arm) to its target. In some embodiments, binding of one arm does not sterically hinder binding of the second arm to its target. For example, when the first arm binds to PD-1, the second arm is free to bind to a 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.

[0202] In some embodiments, binding of the first arm and the second arm 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., one immune cell expressing PD-1 and a second cell expressing its ligand, PD-L1) or bringing the two cells into close proximity; the two cells do not need to be in physical contact. Thus, the multispecific antigen-binding construct acts as a connector (e.g., a bridge) to two cells, each of which expresses either PD-1 or its ligand.

[0203] Methods for determining whether two cells are bridged or connected together by a construct of the present invention are known in the art. For example, in some embodiments, bridging of an immune cell and a second cell is determined by, for example, flow cytometry, FRET, immunoprecipitation, microscopy, or a fluorescent plate reader.

[0204] In some embodiments, binding of the first and second arms of the multispecific construct to their respective targets results in downregulation of the target, e.g., PD-1, and / or ectodomain shedding and / or degradation. As used herein, "downregulation" refers to the process by which a cell reduces the amount of a cellular component, such as RNA or protein. In the case of a cell surface protein receptor, downregulation can occur through receptor internalization as a result of binding to a ligand or any of the constructs described herein. Shedding or ectodomain shedding refers to the process by which cell surface proteins are proteolytically cleaved, resulting in the release of their ectodomains into the extracellular environment. Non-limiting examples of sheddases that regulate ectodomain shedding 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 and structure of the cleavage site region from the plasma membrane are thought to be more important than the specific sequence in ectodomain shedding. Proteolysis or protein hydrolysis refers to a set of processes that result in the hydrolysis of one or more peptide bonds in proteins, either through catalysis by protein-degrading enzymes called proteases or non-enzymatically, for example, at very low or very high pH. In eukaryotic cells, two major pathways mediate protein degradation: the ubiquitin-proteasome pathway and lysosomal proteolysis. Methods for determining whether a target receptor is downregulated and / or shedding and / or degraded by the multispecific constructs disclosed herein are known in the art and described in the Examples (see, e.g., Figures 12A-12C), and include, for example, flow cytometry, Western blotting, immunoprecipitation, microscopy, or a fluorescent plate reader.

[0205] As described herein, the constructs of the present invention can bridge immune cells that express PD-1 and a second cell, e.g., a second immune cell, and / or a cancer or tumor cell, that expresses its ligand. As one of skill in the art will recognize, the type of immune cell will depend on the context of the disease being treated, and the specific type of immune cell can be readily determined depending on the disorder under consideration. In some embodiments, the immune cell is a T cell, e.g., a regulatory T cell (also known as a suppressor T cell), including CD8+ T cells and CD4+ T cells, and subtypes, e.g., CD4 + FOXP3 + T reg cells, CD4 + FOXP3 - T reg cells, Tr1 cells, Th3 cells, and T reg 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.

[0206] Similarly, the type of second cell depends on the disorder being considered. In some embodiments, the second cell (a cell expressing a PD-1 ligand) is a second immune cell, e.g., 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 myeloid-derived suppressor cell, a dendritic cell, or a mesenchymal stromal cell. In some embodiments, the regulatory immune cell is a regulatory T cell, e.g., a CD8+ T cell or a CD4+ T cell.

[0207] In some embodiments, the second cell is a tumor cell. As used herein, "tumor cell" may be used interchangeably with "cancer cell," but also encompasses non-malignant (non-cancerous) cells that exhibit increased proliferation compared to normal cells. In some embodiments, the tumor cell is a cancer that can be treated by 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 and bridging the immune cell and the tumor cell. In some embodiments, the tumor cell is selected from the group consisting of hematological 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, renal cancer, and vascular cancer.In some embodiments, the tumor cells are selected from the group consisting of Kaposi's sarcoma, leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, myeloblastic, promyelocytic, myelomonocytic, monocytic erythroleukemia, chronic leukemia, chronic myelocytic (granulocytic) leukemia, chronic lymphocytic leukemia, mantle cell lymphoma, primary central nervous system lymphoma, Burkitt's lymphoma and marginal zone B-cell lymphoma, polycythemia vera, and leukemia. Lymphoma), Hodgkin's disease, non-Hodgkin's disease, multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, solid tumor, sarcoma, carcinoma, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, osteosarcoma, chordoma, angiosarcoma, endothelial sarcoma, lymphangiosarcoma, lymphoendothelial sarcoma, synovium, 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, cholangiocarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, uterine cancer, testicular tumor, lung cancer, small cell lung cancer, non-small cell lung cancer, bladder cancer, epithelial carcinoma, neuro Selected from the group consisting of glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma, nasopharyngeal carcinoma, esophageal cancer, basal cell carcinoma, biliary tract 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, eye cancer, head and neck cancer, gastric cancer, intraepithelial neoplasia, kidney cancer, laryngeal cancer, liver cancer, lung cancer (small cell, large cell), melanoma, neuroblastoma; oral cancer (e.g., lip, tongue, mouth, and pharynx), ovarian cancer, pancreatic cancer, retinoblastoma, rhabdomyosarcoma, rectal cancer; respiratory system cancer, sarcoma, skin cancer, stomach cancer, testicular cancer, thyroid cancer, uterine cancer, and urinary system cancer.

[0208] As described herein, the multispecific antigen-binding constructs of the present invention include bispecific, trispecific, tetraspecific, or multispecific antibodies (immunoglobulins) or antigen-binding portions or fragments thereof.

[0209] The term "immunoglobulin" refers to a class of structurally related proteins that generally contain two pairs of polypeptide chains: one pair of light (L) chains and one pair of heavy (H) chains. In an "intact immunoglobulin," all four of these chains are interconnected by disulfide bonds. The structure of immunoglobulins is well characterized. See, for example, Paul, Fundamental Immunology 7th ed., Ch. 5 (2013), Lippincott Williams & Wilkins (Philadelphia, PA, USA). Briefly, each heavy chain typically contains a heavy chain variable region (V H ) and the heavy chain constant region (C H The heavy chain constant region typically comprises three domains: C H1 , C H2 , and C H3 Each light chain typically comprises a light chain variable region (V L ) and a light chain constant region. The light chain constant region typically comprises C L The term "immunoglobulin" (Ig) is sometimes used interchangeably with the term "antibody" herein.

[0210] The term "antibody" describes a type of immunoglobulin molecule and is used herein in its broadest sense. Antibodies specifically include intact antibodies (e.g., intact immunoglobulins) and antibody fragments, e.g., antigen-binding fragments of antibodies as described herein. Thus, "antibody" can refer to intact antibodies as well as antigen-binding fragments thereof. An antibody comprises at least one antigen-binding domain. One example of an antigen-binding domain is the V H -V L The antigen-binding domain is formed by a dimer. Antibodies can be described by the antigen to which they specifically bind. For example, PD-1 antibodies, alternatively referred to as anti-PD-1 antibodies, are antibodies that specifically bind to the inhibitory receptor PD-1.

[0211] V H and V L The region can be further divided 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). Each V H and V L generally contain 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, Maryland, USA (incorporated herein by reference).

[0212] Light chains from any vertebrate species can be assigned to one of two types, called kappa and lambda, based on the sequence of their constant domain. 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 called 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.

[0213] Methods for generating and screening antibodies against desired targets are well known in the art. Methods for further modifying antibodies for enhanced properties (e.g., enhanced affinity, chimerization, humanization), as well as generating antigen-binding fragments as described herein, are also well known in the art.

[0214] The term "chimeric antibody" refers to an antibody in which components of the heavy and / or light chain are derived from a particular source or species, while the remainder of the heavy and / or light chain are derived from a different source or species.

[0215] "Humanized" forms of non-human antibodies are chimeric antibodies that contain minimal sequence derived from the non-human antibody. Humanized antibodies are generally human immunoglobulins (recipient antibodies) 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 can be any suitable non-human antibody, such as a mouse, rat, rabbit, chicken, or non-human primate antibody, having the desired specificity, affinity, or biological effect. In some cases, selected framework region residues of the recipient antibody are replaced by the corresponding framework region residues from the donor antibody. Humanized antibodies can also contain residues that are found in neither the recipient antibody nor the donor antibody. Such modifications can be made to further refine antibody function. For further details, see Jones et al. al.), (1986) Nature, 321:522-525; Riechmann et al., (1988) Nature, 332:323-329; and Presta, (1992) Curr. Op. See Struct. Biol., 2:593-596, each of which is incorporated herein by reference.

[0216] A "human antibody" is one that has an amino acid sequence that corresponds to that of an antibody produced by a human or human cell, or is derived from a non-human source that utilizes a human antibody repertoire or human antibody coding sequences (e.g., obtained from a human source or designed de novo). Human antibodies specifically exclude humanized antibodies.

[0217] In some embodiments, antibody molecules include antigen-binding fragments of antibodies (e.g., Fab, F(ab')2, and Fv), in addition to diabodies and single-chain molecules. For example, an antibody molecule can include 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 one heavy chain and one 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 (DAbs) (bivalent and bispecific), and chimeric (e.g., humanized) antibodies, which can be produced by modification of whole antibodies or 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 antibody, including, but not limited to, IgG, IgA, IgM, IgD, and IgE, and from any subclass of antibody (e.g., IgA1, IgA2, IgG1, IgG2, IgG3, and IgG4). Preparations of antibody molecules can be monoclonal or polyclonal. Antibody molecules can also be human, humanized, CDR-grafted, or in vitro generated. The antibody can have a heavy chain constant region chosen from, for example, IgG1, IgG2, IgG3, or IgG4. The antibody can also have a light chain chosen from, for example, kappa or lambda.In some embodiments, the antibody comprises the sequence of SEQ ID NO: 64 (ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLN 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of the IgG1 heavy chain constant region of the present invention (GKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK). In some embodiments, the antibody comprises the sequence of SEQ ID NO: 68 (ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGK EYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK).In some embodiments, the antibody comprises the sequence of SEQ ID NO: 69 (ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNG

[0039] The present invention relates to an IgG4 heavy chain constant region having an amino acid sequence at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of the IgG4 heavy chain constant region of the present invention (e.g., KEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG).

[0218] Antigen-binding portions or fragments of antibody molecules are well known in the art and include, for example, (i) a Fab fragment, which is a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) a F(ab')2 fragment, which is 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) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (v) a diabody (dAb) fragment consisting of a VH domain; (vi) a camelid or camelized variable domain; and (vii) a single-chain Fv (scFv) (see, e.g., Bird et al. (viii) single-domain antibodies. These antibody fragments are obtained using conventional techniques known to those of skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies.

[0219] The antibody molecule can also be a single-domain antibody. Single-domain antibodies can include antibodies whose complementary determining regions are portions of a single-domain polypeptide. Examples include, but are not limited to, heavy-chain antibodies, antibodies naturally devoid of light chains, single-domain antibodies derived from conventional four-chain antibodies, engineered antibodies, and single-domain scaffolds other than those derived from antibodies. The single-domain antibody can be any known in the art or any future single-domain antibody. The single-domain antibody can be derived from any species, including, but not limited to, mouse, human, camel, llama, fish, shark, goat, rabbit, and cow. According to another aspect of the present invention, the single-domain antibody is a naturally occurring single-domain antibody known as a heavy-chain antibody devoid of light chains. Such single-domain antibodies are disclosed, for example, in WO 9404678. For clarity, this variable domain derived from a heavy-chain antibody naturally devoid of light chains is known herein as a VHH or nanobody to distinguish it from the conventional VH of four-chain immunoglobulins. Such VHH molecules can be derived from antibodies raised in Camelidae species, such as camel, llama, dromedary, alpaca and guanaco. Other species besides Camelidae may produce heavy chain antibodies naturally devoid of light chains, and such VHHs are within the scope of the present invention.

[0220] In some embodiments, the multispecific antigen-binding construct comprises a bispecific antibody. A bispecific antibody has specificity for no more than two antigens, but can have more than two binding sites, as described herein. A bispecific antibody molecule is characterized by a first immunoglobulin variable domain sequence having binding specificity for a first antigen (e.g., PD-1) and a second immunoglobulin variable domain sequence having binding specificity for a second antigen (e.g., a PD-1 ligand, such as a PD-L1 ligand). In some embodiments, the bispecific antibody molecule comprises an scFv or fragment thereof having binding specificity for a first antigen and an scFv or fragment thereof having binding specificity for a second antigen. See, e.g., Kontermann and Brinkmann, (2015), Drug Discovery Today, 20(7):838-47 (incorporated herein by reference).

[0221] A variety of formats and methods are known in the art that can be used to generate the multivalent and / or multispecific constructs described herein, including multivalent and / or multispecific antibody formats of both asymmetric and symmetric architectures. Non-limiting examples of such formats include: (i) Fc-less bispecific antibody formats, e.g., tandem single-chain variable fragments (scFv2, taFv) and triplebodies, e.g., bispecific T cell engager (BiTE) and bispecific killer cell engager (BiKE) molecules; bispecific single domain antibody fusion proteins, including single domain antibodies, e.g., VH or VL domains, VHH, VNAR, and nanobodies; diabodies and diabody derivatives, e.g., tandem diabodies and dual-affinity retargeting (DAT) molecules; (ii) bispecific IgGs with asymmetric architectures, e.g., asymmetric IgGs with heavy and light chains from two different antibodies; knob-into-hole approaches, electrostatic interactions (steering) to avoid homodimerization of CH3 domains, preferential heavy chain heterodimerization by introducing charge pairs into the hinge region of IgG1 and IgG2, strand-exchange engineered domain (SEED) heterodimers, and bispecific engagement by antibodies based on the T cell receptor. Bispecific IgGs with asymmetric Fc regions using BEAT (Biased Antibody-T Cell Receptor) technology; asymmetric Fc and CH3 fusion proteins; (iii) bispecific antibodies with symmetric architectures, such as IgGs augmented by fusion of scFvs, fusions of domain antibodies and scaffold proteins, fusions of Fab arms, and fusions of additional variable heavy and light chain domains; engineered 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 entire contents of which are incorporated herein by reference). For example, the "knob in a hole" technique described in U.S. Pat. No. 5,731,168 is also well known. electrostatic steering Fc pairing, e.g., as described in WO 09 / 089004, WO 06 / 106905, and WO 2010 / 129304; strand exchange engineering domain (SEED) heterodimerization, e.g., as described in WO 07 / 110205; Fab arm exchange, e.g., as described in WO 08 / 119353, WO 2011 / 131746, and WO 2013 / 060867; amine-reactive groups and strand exchange heterodimerization, e.g., as described in U.S. Pat. No. 4,433,059; Bispecific antibody conjugates, by antibody cross-linking to generate bispecific structures using heterobifunctional reagents with sulfhydryl-reactive groups; bispecific antibody determinants generated by recombination of half antibodies (heavy-light chain pairs or Fab) from different antibodies through cycles of reduction and oxidation of the disulfide bond between the two heavy chains, as described, for example, in U.S. Pat. No. 4,444,878; trifunctional antibodies, e.g., three Fab' fragments cross-linked through sulfhdryl-reactive groups, as described, for example, in U.S. Pat. No. 5,273,743;Biosynthetic binding proteins, such as pairs of scFvs cross-linked through their C-terminal tails, preferably via disulfide or amine reactive chemical bridges, as described in U.S. Pat. No. 5,534,254; bifunctional antibodies, such as Fab fragments with different binding specificities dimerized through leucine zippers (e.g., c-fos and c-jun) replacing the constant domains, as described in U.S. Pat. No. 5,582,996; bispecific and oligomeric antibodies, such as those described in U.S. Pat. No. 5,591,828; Monovalent and oligovalent receptors of specificity, e.g., the VH-CH1 regions of two antibodies (two Fab fragments) linked through a polypeptide spacer between the CH1 region of one antibody and the VH region of the other antibody, typically with the associated light chain; bispecific DNA-antibody conjugates, e.g., cross-linking of antibodies or Fab fragments through a double-stranded piece of DNA, as described, e.g., in U.S. Pat. No. 5,635,602; bispecific fusion proteins, e.g., those combining antibodies with full-length constant regions, as described, e.g., in U.S. Pat. No. 5,637,481; expression constructs containing two scFvs with a hydrophilic helical peptide linker between them; multivalent and multispecific binding proteins, such as dimers of polypeptides having a first domain with a binding region of an Ig heavy chain variable region and a second domain with a binding region of an Ig light chain variable region, commonly referred to as diabodies, as described, for example, in U.S. Pat. No. 5,837,242 (higher order structures creating bispecific, trispecific, or tetraspecific molecules are also encompassed); Minibody constructs with linked VL and VH chains further connected to the antibody hinge and CH3 regions using peptide spacers, which can dimerize to form bispecific / multivalent molecules, such as those described in U.S. Pat. No. 5,844,094; VH and VL domains linked in either orientation using short peptide linkers (e.g., 5 or 10 amino acids) or no linker at all, which can dimerize to form bispecific diabodies; trimers and tetramers, such as those described in U.S. Pat. No. 5,844,094;See also strings of VH domains (or VL domains in family members) connected by peptide linkages with crosslinkable groups at the C-terminus further associated with the VL domain to form strings of FVs (or scFvs), as described in U.S. Pat. No. 5,864,019; and single-chain binding polypeptides having both VH and VL domains linked through peptide linkers combined into multivalent structures through non-covalent or chemical crosslinking to form, for example, homobivalent, heterobivalent, trivalent, and tetravalent structures using both scFV-type or diabody-type formats, as described, for example, in U.S. Pat. No. 5,869,620. 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The contents of the above-referenced applications are incorporated herein by reference.

[0222] In some embodiments, the multispecific antigen-binding constructs of the present invention are bispecific antibodies. Bispecific antibodies according to the present disclosure can be generated against PD-1 and PD-L1, or against PD-1 and PD-L2. The antibody arms of the bispecific antibody can be generated by standard techniques, as disclosed herein. In some embodiments, any known antibody against PD-1 and its ligand can be used to generate bispecific antibodies according to the present disclosure. For example, such bispecific constructs are exemplified herein (see, e.g., pembrolizumab (PD-1 antibody) conjugated with atezolizumab (PD-L1 antibody) in Figure 3; nivolumab (PD-1 antibody) conjugated with atezolizumab (PD-L1 antibody) in Figure 4). As exemplified herein, the multispecific antigen-binding constructs (e.g., bispecific antibodies) described herein can be generated using antibodies known and / or available in the art.

[0223] In some embodiments, the bispecific antibody is bivalent, e.g., one arm is monovalent for PD-1, and the other arm is monovalent for either PD-L1 or PD-L2, or both (e.g., cross-reacts with both ligands). In some embodiments, the bispecific antibody is tetravalent, such as the novel Bispecific 3 and Bispecific 4 antibodies described herein. For example, as illustrated in Figure 3, the pembrolizumab-binding arms are bivalent for PD-1, each binding to the same epitope on PD-1, and the atezolizumab-binding arms are bivalent for PD-L1, each binding to the same epitope on PD-L1. This can also be seen, for example, in the bispecific format in Figure 8 (the exemplary common light chain bispecific illustrated in the left panel of step 2 of the workflow presented in Figure 8). The exemplary common light chain bispecific format in Figure 8 (the format illustrated in the right panel of step 2 of the workflow) represents another example of a tetravalent bispecific format. In contrast to the tetravalent bispecific format in which a first antigen-binding arm is conjugated to a second antigen-binding arm on the opposite end of the Fc region, each Fab of the first antigen-binding arm here is conjugated to each Fab of the second antigen-binding arm. For example, one Fab of the first antigen-binding arm is linked to a Fab of the second antigen-binding arm using a linker, and each antigen-binding arm shares a common light chain. See the format illustrated in Figure 8, right panel of step 2 of the workflow.

[0224] In some embodiments, the bispecific antibody is tetravalent, with one arm bivalent for PD-1, each binding to two different epitopes on PD-1. In some embodiments, the bispecific antibody is tetravalent, with one arm bivalent for PD-1 ligand (PD-L1 and / or PD-L2), each binding to two different epitopes on PD-1 ligand. In some embodiments, the bispecific antibody is tetravalent, with one arm bivalent for PD-1, each binding to two different but overlapping epitopes on PD-1. In some embodiments, the bispecific antibody is tetravalent, with one arm bivalent for PD-1 ligand (PD-L1 and / or PD-L2), 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, with one arm bivalent for PD-1, each binding to the same epitope on PD-1. In some embodiments, the bispecific antibody is tetravalent, with one arm bivalent for the PD-1 ligand (PD-L1 and / or PD-L2), each binding to the same epitope on the PD-1 ligand (PD-L1 and / or PD-L2). In some embodiments, the bispecific antibody is tetravalent, with one arm bivalent for the same epitope on PD-1, and the other arm bivalent for the same epitope on the PD-1 ligand (PD-L1 and / or PD-L2).

[0225] 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 ligands PD / L1 and PD-L2 (e.g., cross-reacts with both ligands).

[0226] In certain embodiments, the first antigen-binding arm and the second antigen-binding arm are linked by at least one amino acid linker amino acid sequence. Optionally, the linker amino acid sequence is GGGGS x (SEQ ID NO: 121) (where x is an integer from 1 to 6, inclusive).

[0227] In some embodiments, the multispecific antigen-binding construct does not comprise an immunoglobulin Fc domain. In some embodiments, the construct comprises an immunoglobulin Fc domain. In some embodiments, the first arm or the second arm, or both, of the construct 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 mutations are present in the CH3 domain of the heavy chain (see, e.g., Xu et al., mAbs 7(1):231-42, 2015).

[0228] While traditional Fc fusion proteins and antibodies are examples of unguided interaction pairs, various engineered Fc domains have been designed as asymmetric interaction pairs, for example, to promote heterodimerization of a first antigen-binding arm and a second antigen-binding arm (Spiess et al. (2015) Molecular Immunology 67(2A):95-106). Various methods are known in the art for increasing the desired pairing of Fc-containing polypeptide chains in a single cell line to produce a preferred asymmetric fusion protein in acceptable yields (see, for example, Klein et al. (2012) mAbs 4:653-663; and Spiess et al. (2015) Molecular Immunology 67(2PartA):95-106). Methods for achieving desired pairing of Fc-containing polypeptides include, but are not limited to, charge-based pairing (electrostatic steering), "knob-into-hole" steric pairing, SEED body pairing, and leucine zipper-based pairing. For example, see 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; U.S. Patent No. 5,932,448; and WO 1993 / 011162, WO 2009 / 089004, and WO 2011 / 034605.

[0229] For example, one means by which interactions between specific polypeptides can be promoted is by engineering protuberance-into-cavity (knob-into-hole) complementary regions, as described in Arathoon et al., U.S. Pat. No. 7,183,076; Carter et al., U.S. Pat. No. 5,731,168; and Kumar et al., WO 2016 / 164089 (incorporated herein by reference). A "protuberance" (protuberance) is constructed by replacing a small amino acid side chain from the interface of a first polypeptide (e.g., a first interacting pair) with a larger side chain (e.g., tyrosine or tryptophan). A complementary "cavity" of identical or similar size to the protuberance is optionally created on the interface of the second polypeptide (e.g., the second interacting pair) by replacing large amino acid side chains with smaller ones (e.g., alanine or threonine). Once a protuberance or cavity of suitable location and dimensions is present at the interface of either the first or second polypeptide, it is only necessary to engineer a corresponding cavity or protuberance, respectively, at the adjacent interface.

[0230] At neutral pH (7.0), aspartic acid and glutamic acid are negatively charged, while lysine, arginine, and histidine are positively charged. These charged residues can be used to promote heterodimer formation while simultaneously discouraging homodimer formation. Attractive interactions occur between opposite charges, and repulsive interactions occur between like charges. In part, the protein complexes disclosed herein use attractive interactions to promote heteromultimer formation (e.g., heterodimer formation) and optionally repulsive interactions to discourage homodimer formation (e.g., homodimer formation) by performing site-directed mutagenesis of charged interface residues.

[0231] For example, the IgG1 CH3 domain interface contains four unique pairs of charged residues involved 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 here to designate residues in the IgG1 CH3 domain follows Kabat's EU numbering scheme. Due to the two-fold symmetry present in CH3-CH3 domain interactions, each unique interaction appears twice in the structure (e.g., Asp-399-Lys409' and Lys409-Asp399'). In the wild-type sequence, K409-D399' favors the formation of both heterodimers and homodimers. A single mutation that switches charge polarity in the first chain (e.g., K409E; from positive to negative) leads to interactions that are unfavorable for the formation of homodimers of the first chain. The unfavorable interactions arise due to repulsive interactions between like-charged chains (negative-negative; K409E-D399' and D399-K409E'). A similar mutation that switches charge polarity in the second chain (D399K'; from negative to positive) leads to interactions that are unfavorable for the formation of homodimers of the second chain (K409'-D399K' and D399K-K409'). However, these two mutations (K409E and D399K') simultaneously lead to interactions that are favorable for the formation of heterodimers (K409E-D399K' and D399-K409'). The electrostatic steering effect on heterodimer formation and homodimer inhibition can be further enhanced by mutation of additional charged residues, which may or may not pair with oppositely charged residues in the second chain, including, for example, Arg355 and Lys360 (see, e.g., WO 2016 / 164089).

[0232] Thus, in some embodiments, the multispecific antigen-binding constructs (e.g., bispecific constructs) described herein can comprise an immunoglobulin constant domain, including, for example, the Fc portion of an immunoglobulin. For example, the first arm can comprise an amino acid sequence derived from the Fc domain of an IgG (IgG1, IgG2, IgG3, or IgG4), IgA (IgAl or IgA2), IgE, or IgM immunoglobulin. Optionally, the second arm can comprise an amino acid sequence derived from the Fc domain of an IgG (IgG1, IgG2, IgG3, or IgG4), IgA (IgAl or IgA2), IgE, or IgM immunoglobulin. Such immunoglobulin domains can 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, the substitution is at Ser228 when numbered according to EU numbering. In some embodiments, the substitution at Ser228 is S228P. In some embodiments, the first arm and the second arm comprise Fc domains from the same immunoglobulin class and subtype. In some embodiments, the first arm and the second arm comprise Fc domains 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 immunoglobulin constant domain, e.g., containing one or more amino acid modifications (e.g., deletions, additions, and / or substitutions) that promote heterodimer formation. Methods for generating Fc modifications with desired heterodimer formation are known in the art.

[0233] In some embodiments, the Fc domain can be modified to enhance the serum half-life of the multispecific antigen-binding constructs disclosed herein. For example, Fc domains containing 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 can be modified to enhance or reduce the serum half-life of the Fc domain. H 2 or C H The Fc domain can contain mutations in the three regions that increase the affinity of the Fc domain for FcRn in acidic environments (e.g., in endosomes, where the pH ranges from about 5.5 to about 6.0). Such mutations can result in increased serum half-life of the construct when administered to an animal. Methods for modifying Fc domains for desired characteristics, such as increased serum half-life, are known in the art.

[0234] In some embodiments, the constructs described herein comprise an altered heavy chain constant region that has reduced effector function (or no effector function) relative to its corresponding unaltered constant region. Effector function associated with the constant region of the constructs described herein can be modulated by altering the properties of the constant or Fc region. Altered effector function includes, for example, modulation in 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 pro-inflammatory responses. Modulation refers to an increase, decrease, or elimination of effector function activity exhibited by a subject antibody containing an altered constant region compared to the activity of the unaltered form of the constant region. In specific embodiments, modulation includes a situation in which activity is abolished or completely absent.

[0235] An altered constant region with altered FcR binding affinity and / or ADCC activity and / or altered CDC activity is a polypeptide that has either increased 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 a higher affinity than the unaltered form of the polypeptide. An altered constant region that exhibits decreased binding to an FcR binds to at least one FcR with a lower affinity than the unaltered form of the constant region. Such variants that exhibit reduced binding to FcR may have little or no appreciable binding to FcR, for example, 0-50% (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%) binding to FcR compared to the level of binding of a native sequence immunoglobulin constant or Fc region to FcR. Similarly, an altered constant region that exhibits modulated ADCC and / or CDC activity can exhibit either increased or decreased ADCC and / or CDC activity 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 exhibit about 0-50% (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 an altered constant region that exhibits reduced ADCC and / or CDC may exhibit reduced ADCC and / or CDC activity, or no ADCC and / or CDC activity at all.

[0236] 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, such as a G2 / G4 hybrid constant region, or a portion thereof (e.g., Burton et al. (1992) Adv. Immun. 51:1-18; Canfield et al. (1993) Adv. Immun. 51:1-18). (see Mueller et al.) (1991) J. Exp. Med. 173:1483-1491; and Mueller et al. (1997) Mol. Immunol. 34(6):441-452).

[0237] In some embodiments, the multispecific antigen-binding construct may contain an altered constant region that exhibits 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 the antibody. See, e.g., U.S. Patent No. 6,194,551.

[0238] The constructs and antigen-binding arms described herein may comprise, in part, scaffold domains, proteins, or moieties, such as molecules that do not provide target receptor binding activity but can provide spatial organization, structural support, a means for linking multiple receptor-binding units, or other desirable features, such as improved half-life, of the construct or domain. Various scaffold technologies and compositions are known in the art and can be easily linked or conjugated to the antigen-binding units described herein. The scaffold domains, proteins, or moieties may be derived from antibodies or may not be derived from antibodies. Such scaffold proteins and their domains are generally obtained through combinatorial chemistry-based adaptation of existing antigen-binding proteins.

[0239] Non-antibody protein scaffolds can be considered to fall into two structural categories: domain-sized constructs (in the 6-20 kDa range) and constrained peptides (in the 2-4 kDa range). Domain-sized non-antibody scaffolds include, but are not limited to, affibodies, affilins, anticalins, atrimers, DARPins, FN3 scaffolds (e.g., adnectins and centrins), finomers, Kunitz domains, pronectins, 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 on which they are based or derived have been reviewed, for example, by Simeon and Chen, Protein Cell 9(1):3-14 (2018); Vazquez-Lombardi et al., Drug Discovery Today 20:1271-1283 (2015), and Binz et al., Nature Biotechnol. 23:1257-1268 (2005), the entire contents of each of which are incorporated herein by reference. 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, for example, in terms of tissue penetration, smaller size, and thermal stability. Some non-antibody scaffolds can also allow for easier construction, for example, without being hindered by light chain association issues when bispecific constructs are desired. 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 include 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.

[0240] Thus, in some embodiments of any aspect described herein, the construct can comprise a non-antibody scaffold protein, hi some embodiments of any aspect described herein, at least one of the receptor binding units can comprise a non-antibody scaffold protein. The scaffold portion of the non-antibody scaffold protein may, in some embodiments, be, for example, an adnectin scaffold or portion derived from human tenth fibronectin type III domain (10Fn3); an anticalin scaffold derived from human lipocalin (e.g., such as those described in WO 2015 / 104406); an avimer scaffold or protein fragment derived from the A domain of low density related protein (LRP) and / or very low density lipoprotein receptor (VLDLR); a finomer scaffold or portion of the SH3 domain of FYN tyrosine kinase; a Kunitz domain scaffold or portion of a Kunitz-type protease inhibitor, such as human trypsin inhibitor, aprotinin (bovine pancreatic trypsin inhibitor), Alzheimer's amyloid precursor protein, and tissue factor pathway inhibitor; E. elaeterium (E. Those skilled in the art will understand that the scaffolds may include knottin scaffolds (cysteine ​​knot miniproteins) such as those based on the trypsin inhibitor from S. elaterium; affibody scaffolds or all or part of the Z domain of S. aureus protein A; β-hairpin mimetic scaffolds; designed ankyrin repeat protein (DARPin) scaffolds or artificial protein scaffolds based on ankyrin repeat (AR) proteins; or any scaffold derived from or based on human transferrin, human CTLA-4, human crystallin, and human ubiquitin. For example, the binding site of human transferrin for the human transferrin receptor can be diversified to generate a diverse library of transferrin variants, some of which have acquired affinity for different antigens.See, e.g., Ali et al. (1999) J. Biol. Chem. 274:24066-24073. Portions of human transferrin that are not involved in receptor binding remain unchanged and serve as scaffolds, like the framework regions of antibodies, to display variant binding sites. The library is then screened against the target antigen of interest as an antibody library and according to the methods described herein to identify variants with optimal selectivity and affinity for the target antigen. See, e.g., Hey et al. (2005) TRENDS Biotechnol. 23(10):514-522.

[0241] D. Methods for Producing Multispecific Antigen-Binding Constructs The present disclosure also features methods of producing any of the multispecific antigen-binding constructs described herein. In some embodiments, methods of producing the constructs of the invention include methods of preparing antibodies and / or fragments thereof as described herein. Such methods are well known in the art and can include, for example, immunizing a subject (e.g., a non-human mammal) with an appropriate immunogen. For example, to generate antibodies that bind to PD-1, one skilled in the art can immunize a suitable subject (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 depicted in SEQ ID NO: 114 (GenBank Accession No. NP_005009.2; UniProt Q15116), an antigenic fragment thereof, and / or a variant thereof. Similarly, to generate antibodies that bind to a ligand of PD-1 (e.g., PD-L1), one skilled in the art could immunize a suitable subject with a full-length PD-L1 polypeptide, such as the full-length human PD-L1 polypeptide comprising the amino acid sequence depicted in SEQ ID NO: 115 (GenBank Accession No. NP 054862.1, UniProt Q9NZQ7), antigenic fragments thereof, and / or variants thereof. Similarly, to generate antibodies that bind to PD-L2, one skilled in the art could immunize a suitable subject with a full-length PD-L2 polypeptide, such as the full-length human PD-L2 polypeptide comprising the amino acid sequence depicted in SEQ ID NO: 116 (GenBank Accession No. NP_079515.2, UniProt Q9BQ51), antigenic fragments thereof, and / or variants thereof.

[0242] As one of skill in the art will recognize, the full-length polypeptide (PD-1, PD-L1, or PD-L2) can be used as the antigen, and antibodies can be screened for the desired binding properties (e.g., blocking of the PD-1 / ligand interaction; ability to bridge cells on which PD-1 and its ligand are expressed). As one of skill in the art will also recognize, antigenic fragments of the polypeptide (PD-1, PD-L1, or PD-L2) can be selected based on known structural features of the polypeptide. 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; incorporated herein by reference). Therefore, regions within PD-1, PD-L1, and / or PD-L2, for example, based on receptor / ligand interface information available in the art, can be used to design suitable antigenic fragments with desired binding properties. For example, the PD-1 ectodomain contains a single IgV domain, typical of the CD28 family, while 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 faces of the IgV domain. The IgV domain contains approximately 120 amino acids organized into nine parallel beta strands (ABCC'C''DEFG), with loops connecting the strands. PD-1 has been shown to bind to the beta face of PD-L1 (GFCC') or PD-L2 (AGFC strand and FG loop) using its front beta face (GFCC' strand and CC', CC'' and FG loop).Additionally, 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 as well as 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 such information, one skilled in the art can determine suitable antigenic regions for generating antibodies with desired properties. For example, one skilled in the art can generate antibodies that cross-react with both ligands, PD-L1 and PD-L2 (see, e.g., U.S. Patent No. 9,845,356).

[0243] A suitable subject (e.g., a non-human mammal) can be immunized with an appropriate antigen, with subsequent booster immunizations sufficient to induce antibody production by the mammal. The immunogen can be administered to the subject (e.g., a non-human mammal) along with an adjuvant. Adjuvants useful in producing antibodies in a subject include, but are not limited to, protein adjuvants; bacterial adjuvants, such as whole bacteria (BCG, Corynebacterium parvum, or Salmonella minnesota) and bacterial components including cell wall skeleton, trehalose dimycolate, monophosphoryl lipid A, methanol-extractable residue of Mycobacterium tuberculosis (M. tuberculosis) (MER), complete or incomplete Freund's adjuvant; viral adjuvants; and chemical adjuvants, such as aluminum hydroxide, and iodoacetate and cholesteryl hemisuccinate. Other adjuvants that can be used in methods for inducing immune responses include, for example, cholera toxin and parapoxvirus proteins. See also Bieg et al. (1999) Autoimmunity 31(1):15-24. See also, for example, 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.

[0244] In some embodiments, the method involves preparing a hybridoma cell line secreting a monoclonal antibody that binds to the 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 antigenic fragment as described above. Antibody-producing cells (e.g., splenic B cells) from the immunized mammal can be isolated 2-4 days after at least one booster immunization with the immunogen and then grown in culture for a short period before fusion with cells of a suitable myeloma cell line. The cells can be fused in the presence of a fusion promoter, such as vaccinia virus or polyethylene glycol. The hybrid cells resulting from the fusion are cloned, and cell clones secreting the desired antibody are selected. For example, spleen cells from a Balb / c mouse immunized with a suitable immunogen can be fused with cells of the myeloma cell line PAI or the myeloma cell line Sp2 / 0-Ag14. After fusion, the cells are expanded in a suitable culture medium supplemented with a selective medium, e.g., HAT medium, at periodic intervals to prevent normal myeloma cells from overgrowing the desired hybridoma cells. The resulting hybridoma cells are then screened for secretion of the desired antibody, e.g., an antibody that binds to PD-1.

[0245] In some embodiments, one of skill in the art can identify anti-PD-1 antibodies from non-immune biased libraries, such as those described in U.S. Pat. No. 6,300,064 (to Knappik et al.; Morphosys AG) and Schoonbroodt et al. (2005) Nucleic Acids Res 33(9):e81.

[0246] In some embodiments, the methods described herein can involve or be used in combination with, for example, phage display techniques, bacterial display, yeast surface display, eukaryotic viral display, mammalian cell display, and cell-free (e.g., ribosome display) antibody screening techniques (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).

[0247] 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 that carry the polynucleotide sequences encoding them. Such phage can be used to display antigen-binding domains of antibodies, such as Fab, Fv, or disulfide-stabilized Fv antibody fragments, expressed from repertoire or combinatorial antibody libraries (e.g., human or murine). The phages used in these methods are typically filamentous phages, such as fd and M13. The antigen-binding domains are expressed as recombinant fusion proteins to either the phage coat protein pIII, pVIII, or pIX. See, for example, Shi et al. See, e.g., Brinkman et al. (1995) J. Immunol. Methods 182:41-50; Ames et al. (2010) JMB 397:385-396. Examples of phage display methods that can be used to generate the immunoglobulins, or fragments thereof, described herein include Brinkman et al. (1995) J. Immunol. Methods 182:41-50; Ames et al. (2010) JMB 397:385-396. 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 those disclosed in 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.

[0248] In some embodiments, phage display antibody libraries can be generated using mRNA collected from B cells from an immunized mammal. For example, a spleen cell sample containing B cells can be isolated from a mouse immunized with a PD-1 polypeptide as described above. The mRNA can be isolated from the cells and converted to cDNA using standard molecular biology techniques. See, for example, Sambrook et al. (1989) "Molecular Cloning: A Laboratory Manual, 2 ndEdition,” Cold Spring Harbor Laboratory Press [New York]; Harlow and Lane (1988), supra; Benny K.C. See, e.g., Merz et al. (1995) J. Immunol. 1999, 143:131-134, 1999. See, e.g., Lo (2004), supra; and Borrebaek (1995), supra. Phage display libraries are constructed using cDNAs encoding the variable regions of immunoglobulin heavy and light chain polypeptides. Methods for generating such libraries are described, for example, in Merz et al. (1995) J. Immunol. 1999, 143:131-134, 1999. 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.

[0249] In some embodiments, a combination of selection and screening can be used to identify antibodies of interest, for example, from a population of hybridoma-derived antibodies or a phage-display 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, multiple phagemid vectors, each encoding a fusion protein of a bacteriophage coat protein (e.g., pIII, pVIII, or pIX of M13 phage) and a different antigen combination region, are produced using standard molecular biology techniques and then introduced into a population of bacteria (e.g., E. coli). Expression of bacteriophage in bacteria may, in some embodiments, 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). The phage produced from the bacteria is recovered and then contacted with a target antigen, for example, bound to a (immobilized) solid support. The phage can also be contacted with an antigen in solution, and the complex is then bound to a solid support.

[0250] The antibody subpopulations screened using the above methods can be characterized for their specificity and binding affinity to a particular antigen (e.g., human PD-1) using any immunological or biochemical-based method known in the art. For example, the specific binding of an antibody to PD-1 can be determined using immunological or biochemical-based methods, such as, but not limited to, ELISA assays, SPR assays, immunoprecipitation assays, 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 blots, RIAs, ELISAs (enzyme-linked immunosorbent assays), "sandwich" immunoassays, immunoprecipitation assays, immunodiffusion assays, agglutination assays, complement fixation assays, immunoradiometric assays, fluorescent immunoassays, and protein A immunoassays. Such assays are conventional and well known in the art.

[0251] 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. In embodiments where the selected CDR amino acid sequence is a short sequence (e.g., less than 10-15 amino acids in length), the nucleic acid encoding the CDR can be prepared using a method such as that 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, the region of the nucleic acid sequence encoding the CDR can be replaced with a chemically synthesized nucleic acid using standard molecular biology techniques. The 5' and 3' ends of the chemically synthesized nucleic acid can be synthesized to contain cohesive end restriction enzyme sites for use in cloning the nucleic acid into the nucleic acid encoding the variable region of the donor antibody. Alternatively, chemically synthesized nucleic acid fragments that can together encode an antibody can be joined using DNA assembly techniques known in the art (e.g., Gibson assembly).

[0252] Any antibody of choice can be further modified to generate antigen-binding fragments as described herein and / or manipulated using techniques known in the art to generate multispecific antigen-binding constructs as described herein. For example, cross-linking methods can be used to generate bispecific structures using heterobifunctional reagents with amine-reactive and sulfhydryl-reactive groups, as described, for example, in U.S. Pat. No. 4,433,059; bispecific antibody determinants can be generated by recombining half antibodies (heavy chain-light chain pairs or Fab) from different antibodies through cycles of reduction and oxidation of the disulfide bond between the two heavy chains, as described, for example, in U.S. Pat. No. 4,444,878; trifunctional antibodies, e.g., three Fab' fragments, can be cross-linked through sulfhydryl-reactive groups, as described, for example, in U.S. Pat. No. 5,273,743. Other methods for generating bispecific constructs, such as those for generating bispecific constructs with a common light chain, are described herein. Non-limiting examples of consensus light chain amino acid sequences used in the constructs described herein include SEQ ID NOs: 59-63.

[0253] E. Expression and Purification of Multispecific Antigen-Binding Constructs The multispecific antigen-binding constructs described herein can be produced using various techniques known in the art of molecular biology and protein chemistry.For example, the nucleic acid encoding the multispecific antigen-binding construct (as a single multifunctional polypeptide or as separate molecules of a multimeric complex, for example, as one antigen-binding arm separate from other antigen-binding arms) can be inserted into an expression vector containing transcriptional and translational regulatory sequences, such as promoter sequences, ribosomal binding sites, transcriptional start and stop sequences, translational start and stop sequences, transcription terminator signals, polyadenylation signals, and enhancer or activator sequences.Regulatory sequences include promoters and transcriptional start and stop sequences.In addition, the expression vector can contain 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.

[0254] Several possible vector systems are available for the expression of cloned heavy and light chain polypeptides from nucleic acids in mammalian cells. One class of vectors relies on integration of the desired gene sequences into the host cell genome. Cells with stably integrated DNA can be selected by co-introducing a drug resistance gene such as E. 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). The selectable marker gene can either be linked to the DNA gene sequences to be expressed or introduced into the same cell by co-transfection (Wigler et al. (1979) Cell 16:77). The second class of vectors utilizes DNA elements that confer autonomous replication capability to extrachromosomal plasmids. These vectors can be derived from animal viruses such as bovine papillomavirus (Sarver et al. (1982) Proc. Natl. Acad. Sci. USA, 79:7147), cytomegalovirus, polyomavirus (Deans et al. (1984) Proc. Natl. Acad. Sci. USA 81:1292), or SV40 virus (Lusky and Botchan (1981) Nature 293:79).

[0255] The expression vector can be introduced into cells in a manner suitable for subsequent expression of the nucleic acid. The method of introduction is generally determined by the targeted cell type, as discussed below. Exemplary methods include CaPO precipitation, liposome fusion, cationic liposomes, electroporation, viral infection, dextran-mediated transfection, polybrene-mediated transfection, protoplast fusion, and direct microinjection.

[0256] Suitable host cells for expression of antibodies or antigen-binding fragments thereof include yeast, bacteria, insect, plant, 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), as well as primary cell lines.

[0257] In some embodiments, antibodies or fragments thereof can be expressed in and purified from transgenic animals (e.g., transgenic mammals). For example, antibodies can be purified from transgenic mammals, e.g., antibodies described in, e.g., 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. It can be produced in transgenic non-human mammals (e.g., rodents) and isolated from milk, as described in (Illegible text - likely OCR error) et al. (1999) J. Immunol. Methods 231(1-2):147-157.

[0258] Antibodies and fragments thereof can be produced from cells transformed with an expression vector containing nucleic acid encoding the antibody or fragment by culturing the host cells under conditions and for a period of time sufficient to allow expression of the protein. Such conditions for protein expression vary with the choice of expression vector and host cell and are readily ascertained by one of ordinary skill in the art through routine experimentation. For example, antibodies expressed in E. coli can be refolded from inclusion bodies (see, e.g., Hou et al. (1998) Cytokine 10:319-30). Bacterial expression systems and methods for their use are known in the art (see, Current Protocols in Molecular Biology, Wiley & Sons; and Molecular Cloning—A Laboratory Manual, 3rd Ed., Cold Spring Harbor Laboratory Press, New York (2001)). The selection of codons, suitable expression vectors, and suitable host cells will vary depending on numerous factors and can be easily optimized as needed. The antibodies (or fragments thereof) described herein can be expressed in mammalian cells or other expression systems, including but not limited to yeast, baculovirus, and in vitro expression systems (see, e.g., Kaszubska et al. (2000) Protein Expression and Purification 18:213-220).

[0259] After expression, antibodies and their fragments can be isolated. Antibodies or their fragments can be isolated or purified in various ways known in the art, depending on what other components are present in the sample. Standard purification methods include electrophoretic, molecular, immunological, and chromatographic techniques, including ion exchange, hydrophobic, affinity, and reverse-phase HPLC chromatography. For example, antibodies can be purified using a standard anti-antibody column (e.g., Protein A or Protein G column). Ultrafiltration and diafiltration techniques are also useful in combination with protein concentration. See, for example, Scopes (1994) Protein Purification, 3 rd See, e.g., 1999 American Institute of Biological Chemistry, vol. 10, no. 10, pp. 111-114, Springer-Verlag, New York City, NY, USA. The degree of purification required will vary depending on the desired use. In some cases, purification of the expressed antibody or fragment thereof is not necessary.

[0260] Methods for determining the yield or purity of a purified antibody or fragment thereof are known in the art and include, for example, Bradford assay, UV spectroscopy, Biuret protein assay, Lowry protein assay, Amido Black protein assay, high pressure liquid chromatography (HPLC), mass spectrometry (MS), and gel electrophoresis methods (e.g., using protein stains such as Coomassie blue or colloidal silver stains).

[0261] F. Modification of Multispecific Antigen-Binding Constructs Multispecific antigen-binding constructs can be modified after their expression and purification, either as a single multifunctional polypeptide or as separate molecules of a multimeric complex, e.g., one antigen-binding arm separated from the other antigen-binding arms. Modifications can be covalent or non-covalent. Such modifications can be introduced into antibodies or antigen-binding fragments, for example, by reacting targeted amino acid residues of the polypeptide with organic derivatizing agents capable of reacting 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.

[0262] The amino acid sequences provided herein are shown in one-letter amino acid code, which may be used interchangeably with the three-letter amino acid code. 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 alpha-amino acids are: 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 these 20 naturally occurring amino acids are described, for example, in Stryer et al., Biochemistry, 5 th ed., Freeman and Company (2002). The term amino acid also includes unnatural amino acids, modified amino acids (e.g., having modified side chains and / or backbones), and amino acid analogs.

[0263] The terms identical or percent identity in the context of two or more nucleic acid or polypeptide sequences refer to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides or amino acid residues that are the same (e.g., 90%, or 95% or higher identity over a specified region) 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.

[0264] Identity or similarity with respect to sequences is defined as the percentage of amino acid residues in a candidate sequence that are identical (i.e., the same residues) with the starting amino acid residues after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Methods for aligning sequences for comparison are well known in the art. Optimal alignment of comparison sequences can be achieved, 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 similarity search method of Pearson and Lipman (Proc. Natl. Acad. Sci. USA 85:2444, 1988), or by computerized implementations of these algorithms (e.g., GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wisconsin, USA). This can be performed by automated alignment (see, for example, Ausubel et al., Current Protocols in Molecular Biology (1995 supplement)) or by manual alignment and visual inspection (see, for example, Ausubel et al., Current Protocols in Molecular Biology (1995 supplement)).

[0265] As with all peptides, polypeptides, and proteins, including fragments, it is understood that additional modifications may be made in the amino acid sequences of the constructs, antibodies, or antigen-binding portions thereof, described herein, for example, in the heavy chain variable region and / or light chain variable region, that do not alter the properties or function of the antibody or antigen-binding fragment thereof. Such modifications include conservative amino acid substitutions, such that each referenced sequence optionally contains one or more conservative amino acid substitutions. The following groups each contain amino acids that are conservative substitutions for each other. These groups are exemplary, and other conservative substitutions are known to those skilled in the art.

[0266] 1) Alanine (A), Glycine (G); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); 6) phenylalanine (F), tyrosine (Y), tryptophan (W); 7) serine (S), threonine (T), and 8) Cysteine ​​(C), Methionine (M) For example, if an aspartic acid at a particular residue is mentioned, a conservative substitution at that residue is also envisioned, e.g., glutamic acid. Non-conservative substitutions are also envisioned, e.g., substitution of a proline with a glycine.

[0267] In some embodiments, the construct, antibody, or antigen-binding portion thereof may be conjugated to a heterologous moiety. The heterologous moiety can be, for example, a heterologous polypeptide, a therapeutic agent (e.g., a toxin or drug), or a detectable label, including, but not limited to, a radioactive label, an enzyme 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 use in purifying 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 useful as diagnostic or detectable markers (e.g., enzymes), such as 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 3H. Suitable fluorescent labels include, but are not limited to, fluorescein, fluorescein isothiocyanate (FITC), green fluorescent protein (GFP), DYLIGHT™ 488, phycoerythrin (PE), propidium iodide (PI), PerCP, PE-ALEXA FLUOR® 700, Cy5, allophycocyanin, and Cy7. Luminescent labels include, for example, any of a variety of luminescent lanthanide (e.g., europium or terbium) chelates. For example, suitable europium chelates include the 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.

[0268] Two proteins (e.g., an antibody and a heterologous moiety) can be crosslinked using any of a number of known chemical crosslinkers. Examples of such crosslinkers are those that link two amino acid residues via a linkage containing a "hindered" disulfide bond. In these linkages, the disulfide bond within the bridging unit is protected from reduction (by blocking groups on either side of the disulfide bond), e.g., by the action of reduced glutathione or the enzyme disulfide reductase. One suitable reagent, 4-succinimidyloxycarbonyl-α-methyl-α(2-pyridyldithio)toluene (SMPT), forms such a linkage between two proteins by utilizing a terminal lysine on one of the proteins and a terminal cysteine ​​on the other. Heterobifunctional reagents that crosslink via different coupling moieties on each protein can also be used. Other useful crosslinkers include, but are not limited to, reagents that link two amino groups (e.g., N-5-azido-2-nitrobenzoyloxysuccinimide), two sulfhydryl groups (e.g., 1,4-bis-maleimidobutane), an amino group and a sulfhydryl group (e.g., m-maleimidobenzoyl-N-hydroxysuccinimide ester), an amino group and a carboxyl group (e.g., 4-[p-azidosalicylamido]butylamine), and an amino group and a guanidinium group present in the side chain of arginine (e.g., p-azidophenylglyoxal monohydrate).

[0269] In some embodiments, the radiolabel can be directly conjugated to the amino acid backbone of the antibody. Alternatively, the radiolabel can be attached to a larger molecule attached to the protein backbone (e.g., a meta-[ 125 Iodophenyl-N-hydroxysuccinimide ([ 125 I]mIPNHS 125The radiolabel can be included as part of a radiolabel (e.g., see Rogers et al. (1997) J. Nucl. Med. 38:1221-1229) or a chelate (e.g., DOTA or DTPA). Methods for conjugating radiolabels or larger molecules / chelates containing same to the antibodies or antigen-binding fragments described herein are known in the art. Such methods involve 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. Pat. No. 6,001,329).

[0270] Methods for conjugating fluorescent labels (sometimes referred to as "fluorophores") to proteins (e.g., antibodies) are known in the art of protein chemistry. For example, fluorophores can be conjugated to free amino groups (e.g., of lysine) or sulfhydryl groups (e.g., of cysteine) of proteins using succinimidyl (NHS) ester or tetrafluorophenyl (TFP) ester moieties attached to the fluorophore. In some embodiments, fluorophores can be conjugated to heterobifunctional cross-linker moieties such as sulfo-SMCC. A suitable conjugation method involves incubating an antibody protein or a fragment thereof with the fluorophore under conditions conducive to 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).

[0271] In some embodiments, the antibody or fragment can be modified with a moiety that improves stabilization and / or retention of the antibody in circulation, e.g., in blood, serum, or other tissues. For example ... as described in, e.g., 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 Deliveries Reviews 54:459-476, or HESylated (Fresenius Kabi, Germany) (see, e.g., Pavisic 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 a factor of at least, e.g., 1.5 (e.g., at least 2, 5, 10, 15, 20, 25, 30, 40, or 50 or more).

[0272] In some embodiments, the antibodies or antigen-binding fragments thereof described herein can be glycosylated. In some embodiments, the antibodies or antigen-binding fragments thereof described herein can be subjected to enzymatic or chemical treatment or produced from cells so that the antibodies or fragments have reduced or no glycosylation. Methods for producing antibodies with reduced glycosylation are known in the art, and are described, for example, in U.S. Pat. No. 6,933,368; Wright et al. (1991) EMBO J. 10(10):2717-2723; and Co et al. et al. (1993) Mol. Immunol. 30: 1361. In some embodiments, the antibody or antigen-binding fragment thereof is aglycosylated.

[0273] G. Pharmaceutical Compositions and Formulations The present disclosure also provides pharmaceutical compositions comprising the multispecific antigen-binding constructs of the present disclosure together with a pharmaceutically acceptable diluent, carrier, solubilizer, emulsifier, preservative, and / or adjuvant for use with the methods disclosed herein. Such pharmaceutical compositions can be used, for example, in subjects with cancer, as disclosed herein.

[0274] In certain embodiments, acceptable formulation materials are preferably non-toxic to recipients at the dosages and concentrations used. In certain embodiments, the formulation materials are for sc and / or IV administration. In certain embodiments, pharmaceutical compositions can contain formulation materials to modify, maintain, or preserve, for example, pH, osmolality, viscosity, transparency, color, isotonicity, odor, sterility, stability, dissolution or release rate, adsorption, or permeation of the composition.In certain embodiments, suitable formulation materials include amino acids (e.g., glycine, glutamine, asparagine, arginine, or lysine); antimicrobial agents; antioxidants (e.g., ascorbic acid, sodium sulfite, or sodium bisulfite); buffers (e.g., borate, bicarbonate, Tris-HCl, citrate, phosphate, or other organic acids); bulking agents (e.g., mannitol or glycine); chelating agents (e.g., ethylenediaminetetraacetic acid (EDTA)); complexing agents (e.g., caffeine, polyvinylpyrrolidone, beta-cyclodextrin, or hydroxypropyl-beta-cyclodextrin); bulking agents; monosaccharides, disaccharides, and other carbohydrates (e.g., glucose, mannose, or dextrin); proteins (e.g., serum albumin, gelatin, or immunoglobulins); colors, flavors, and diluents; emulsifiers; hydrophilic polymers (e.g., polyvinylpyrrolidone); low molecular weight polypeptides; salt forms preservatives (e.g., benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide); solvents (e.g., glycerin, propylene glycol, or polyethylene glycol); sugar alcohols (e.g., mannitol or sorbitol); suspending agents; surfactants or wetting agents (e.g., pluronic, PEG, sorbitan esters, polysorbates such as polysorbate 20, polysorbate 80, triton, tromethamine, lecithin, cholesterol, tyloxapal); stability enhancers (e.g., sucrose or sorbitol); tonicity enhancers (e.g., alkali metal halides, preferably sodium or potassium chloride, mannitol, sorbitol); delivery solvents; diluents; excipients and / or pharmaceutical adjuvants. (Remington's Pharmaceutical Sciences, 18th Edition, AR 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 of skill in the art depending, for example, on the intended route of administration, delivery format, and desired dosage. See, e.g., Remington's Pharmaceutical Sciences, supra. In certain embodiments, such compositions may affect the physical state, stability, rate of in vivo release, and / or rate of in vivo clearance of the multispecific antigen-binding construct.

[0275] In certain embodiments, the primary solvent or carrier in a pharmaceutical composition can be either aqueous or non-aqueous in nature. For example, in certain embodiments, a suitable solvent or carrier can be water for injection, saline solution, or artificial cerebrospinal fluid, optionally supplemented with other materials common 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 a further exemplary solvent. In certain embodiments, a pharmaceutical composition comprises a Tris buffer of about pH 7.0-8.5 or an acetate buffer of about pH 4.0-5.5, which may further contain sorbitol or a suitable substitute therefor. In certain embodiments, compositions comprising the multispecific antigen-binding constructs disclosed herein can be prepared for storage by mixing a selected composition having the desired purity in the form of a lyophilized cake or aqueous solution with optional formulation substances (Remington's Pharmaceutical Sciences, supra). Furthermore, in certain embodiments, compositions comprising the multispecific antigen-binding constructs disclosed herein may be formulated as a lyophilizate using appropriate excipients such as sucrose.

[0276] In certain embodiments, pharmaceutical compositions can be selected for parenteral delivery. In certain embodiments, compositions can be selected for delivery through the digestive...

Claims

1. A pharmaceutical composition for the treatment of a proliferative disorder and / or enhancing an immune response, comprising a multispecific antigen-binding construct comprising at least two antigen-binding arms, a first arm binds to PD-1 and comprises a light chain variable region and a heavy chain variable region comprising a CDRH1 of SEQ ID NO: 77, a CDRH2 of SEQ ID NO: 71, and a CDRH3 of SEQ ID NO: 75; the second arm binds to a PD-1 ligand and comprises a light chain variable region and a heavy chain variable region comprising a CDRH1 of SEQ ID NO: 1, a CDRH2 of SEQ ID NO: 7, and a CDRH3 of SEQ ID NO: 8; the light chain variable region of the first arm and the light chain variable region of the second arm each comprise a CDRL1 of SEQ ID NO:9, a CDRL2 of SEQ ID NO:5, and a CDRL3 of SEQ ID NO:10; A pharmaceutical composition wherein the multispecific antigen-binding construct blocks the interaction of PD-1 and the PD-1 ligand.

2. 2. The pharmaceutical composition of claim 1, wherein the proliferative disorder is a cancer selected from the group consisting of hematological 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.

3. 2. The pharmaceutical composition of claim 1, wherein the heavy chain variable region of the first arm comprises an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO:

90.

4. 2. The pharmaceutical composition 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.

5. The pharmaceutical composition of claim 1 , wherein the first and second antigen-binding arms each comprise a common light chain variable region.

6. 2. The pharmaceutical composition 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.

7. the construct comprises a bivalent antibody specific for PD-1 and a bivalent antibody specific for PD-L1; The pharmaceutical composition of claim 1, wherein the bivalent antibody specific for PD-1 and the bivalent antibody specific for PD-L1 are linked together.

8. the heavy chain of the first arm has an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 100; 2. The pharmaceutical composition of claim 1, 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.

9. the heavy chain of the first arm has the amino acid sequence of SEQ ID NO: 100; 2. The pharmaceutical composition of claim 1, wherein the light chains of the first arm and the second arm have the amino acid sequence of SEQ ID NO:

101.

10. 2. The pharmaceutical composition of claim 1, wherein the heavy chain variable region of the first arm comprises an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:

90.

11. 2. The pharmaceutical composition 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.

12. 2. The pharmaceutical composition of claim 1, wherein the heavy chain variable region of the first arm comprises the amino acid sequence of SEQ ID NO:

90.

13. 2. The pharmaceutical composition 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.

14. 2. The pharmaceutical composition 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.

15. 2. The pharmaceutical composition of claim 1, wherein the heavy chain variable region of the second arm comprises the amino acid sequence of SEQ ID NO:

55.

16. the heavy chain of the first arm has an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 100; 2. The pharmaceutical composition of claim 1, 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.

17. the heavy chain of the first arm has an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO: 100; 2. The pharmaceutical composition of claim 1, 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.

18. The pharmaceutical composition of claim 1, wherein the first arm binds to PD-1 expressed by an immune cell.

19. 2. The pharmaceutical composition of claim 1, wherein the second arm binds to a PD-1 ligand expressed by a second cell.

20. 20. The pharmaceutical composition of claim 19, wherein the second cell is a tumor cell and the PD-1 ligand is PD-L1.

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