Multiple specific binding constructs for checkpoint molecules and their use

Abstract

This provides a novel treatment that effectively counteracts immune evasion. [Solution] This disclosure relates to compositions and methods for inhibiting tumor escape by reducing immune checkpoint suppression. In some embodiments, compositions are provided herein that block the interaction between PD-1 and its ligand (e.g., PD-1 and / or PD-L2) while promoting the interaction between cells expressing PD-1 and its ligand. Methods for using such compositions are also provided.

Description

[Technical Field]

[0001] This disclosure relates to multispecific binding constructs for checkpoint molecules and their use. [Background technology]

[0002] Cancer is one of the leading causes of death in both the United States and worldwide. While common treatments such as surgery, radiation, chemotherapy, hormone therapy, targeted therapy, and immunotherapy reduced cancer-related death rates throughout the 20th century, as of 2012, there were 14.1 million newly diagnosed cancer cases and 8.2 million cancer deaths worldwide. Despite improvements in overall cancer survival rates during the 20th century, cancer still accounts for one in seven deaths worldwide. See Non-Patent Literature 1.

[0003] In recent years, increasing evidence suggests that the immune system acts as a crucial barrier against tumor formation and progression. The principle that naturally occurring T cells with antitumor potential or activity are present in patients with cancer has streamlined the development of immunotherapeutic approaches in oncology. Immune cells such as T cells, macrophages, and natural killer cells can exhibit antitumor activity and effectively control the emergence and growth of malignant tumors. Tumor-specific or related antigens can induce immune cells to recognize and eliminate malignant tumors (Non-Patent Literature 2). Despite the presence of tumor-specific immune responses, malignant tumors often evade or avoid immune attack through various immunomodulatory mechanisms, resulting in a failure to control tumor emergence and progression (Non-Patent Literature 3). Indeed, an emerging characteristic of cancer is the utilization of these immunomodulatory mechanisms and the inability of antitumor immune responses, resulting in tumor evasion and escape from immunological killing (Non-Patent Literature 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 [Overview of the project] [Problems that the invention aims to solve]

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

[0006] This 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 exhibit enhanced in vitro and in vivo efficacy compared to clinical checkpoint blockers, as well as compared to combinations of individual antibodies. Novel monoclonal anti-PD-1 antibodies and their antigen-binding fragments, as well as novel monoclonal anti-PD-L1 antibodies and their antigen-binding fragments, for use in such multispecific and multivalent constructs are also provided herein. Some of these novel monoclonal anti-PD-1 antibodies and novel monoclonal anti-PD-L1 antibodies share a common light chain, thereby enabling the creation of multispecific and multivalent constructs that have similar affinity to their parent antibodies, in addition to remarkably superior drug-like properties (DLP) and ease of production. This disclosure is also partly based on the discovery that blocking the interaction between PD-1 expressed by immune cells 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 the PD-1 ligand), strongly enhances, for example, T cell proliferation, IFNγ production and secretion, and T cell cytolytic activity. Therefore, compositions are provided herein that block the interaction between PD-1 and its ligand (PD-L1 or PD-L2) while promoting the interaction (bridging) between cells expressing PD-1 and its ligand (PD-L1 or PD-L2). Such compositions of this disclosure having the ability to “block and bridge” as exemplified herein offer superior antitumor efficacy (e.g., as measured by IFNγ production and secretion and in vivo activity) compared to, for example, a cocktail having stoichiometric amounts of agents separately bound to the receptor and ligand, or a single agent bound to either the receptor or the ligand. The multispecific and multivalent constructs that target both PD-1 and PD-L1 described herein have also been found to induce a loss of PD-1 expression on the cell surface in a valency-dependent manner.This loss of PD-1 expression was not observed when the parental antibody combination was used in stoichiometrically equivalent amounts. Therefore, 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, the multispecific antigen-binding construct binds simultaneously 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, the multispecific antigen-binding construct binds simultaneously to PD-1 expressed on the surface of a first cell and to a PD-1 ligand, e.g., PD-L1 or PD-L2, expressed on the surface of a second cell.

[0008] In some embodiments, the disclosure provides a multispecific antigen-binding construct comprising at least two antigen-binding arms, wherein the first arm binds to PD-1 expressed by an immune cell, and the second arm binds to a PD-1 ligand expressed by a second cell, thereby blocking the interaction between PD-1 and the PD-1 ligand. In some embodiments, the PD-1 ligand is PD-L2. In some embodiments, the PD-1 ligand is PD-L1. In some embodiments, the immune cell is a T cell. In some embodiments, the T cell is a CD8+ T cell. In some embodiments, the immune cell is a natural killer (NK) cell. In some embodiments, the immune cell is a macrophage. In some embodiments, the second cell is a second immune cell. In some embodiments, the second immune cell is one or more of the following: 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 hematological cancers, lymphomas, myelomas, leukemias, neurological cancers, melanomas, breast cancers, prostate cancers, colorectal cancers, lung cancers, head and neck cancers, gastrointestinal cancers, liver cancers, pancreatic cancers, genitourinary cancers, bone cancers, kidney cancers, and vascular cancers. In some embodiments, both arms are at least 1 × 10⁻⁶ -7 M, at least 1 × 10 -8 M, at least 1 × 10 -9 M, or at least 1 × 10 -10 M's K DIt has the following characteristics. In some embodiments, binding of one arm to its target does not block binding of the other arm to its target. In some embodiments, the first and second arms bind to their respective targets, and both arms remain bound simultaneously. In some embodiments, the binding of the first and second arms to their respective targets can cause bridging to bring the immune cell and the second cell together. In some embodiments, 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 includes a heavy chain variable region comprising (a)(i) CDRH1 containing 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) CDRH2 containing SEQ ID NO: 71 (SAISNSGTYTYYA), and (iii) CDRH3 containing SEQ ID NO: 72 (ARGLDFIVGX5TGNDY, where X5=A, Y, or R), and (b) a light chain variable region comprising (i) CDRL1 containing SEQ ID NO: 9 (RASQSISSYLN), (ii) CDRL2 containing SEQ ID NO: 5 (AASSLQS), and (iii) CDRL3 containing SEQ ID NO: 10 (QQSYSTPLT).

[0010] In some embodiments, CDRH1 of the first arm includes sequence number 73 (FTFSDYAMS), CDRH2 of the first arm includes sequence number 71 (SAISNSGTYTYYA), and CDRH3 of the first arm includes sequence number 74 (ARGLDFIVGATGNDY). In some embodiments, CDRH1 of the first arm includes sequence number 73 (FTFSDYAMS), CDRH2 of the first arm includes sequence number 71 (SAISNSGTYTYYA), and CDRH3 of the first arm includes sequence number 75 (ARGLDFIVGYTGNDY). In some embodiments, CDRH1 of the first arm includes sequence number 76 (FTFSSYAMS), CDRH2 of the first arm includes sequence number 71 (SAISNSGTYTYYA), and CDRH3 of the first arm includes sequence number 75 (ARGLDFIVGYTGNDY). In some embodiments, CDRH1 of the first arm includes sequence number 77 (FTFSSYAML), CDRH2 of the first arm includes sequence number 71 (SAISNSGTYTYYA), and CDRH3 of the first arm includes sequence number 75 (ARGLDFIVGYTGNDY). In some embodiments, CDRH1 of the first arm includes sequence number 78 (FTFSNYALS), CDRH2 of the first arm includes sequence number 71 (SAISNSGTYTYYA), and CDRH3 of the first arm includes sequence number 75 (ARGLDFIVGYTGNDY). In some embodiments, CDRH1 of the first arm includes sequence number 79 (FTFSAYAMN), CDRH2 of the first arm includes sequence number 71 (SAISNSGTYTYYA), and CDRH3 of the first arm includes sequence number 75 (ARGLDFIVGYTGNDY). In some embodiments, CDRH1 of the first arm includes sequence number 80 (FTFRSYAMS), CDRH2 of the first arm includes sequence number 71 (SAISNSGTYTYYA), and CDRH3 of the first arm includes sequence number 75 (ARGLDFIVGYTGNDY).In some embodiments, CDRH1 of the first arm includes sequence number 82 (FTFNSYAMS), CDRH2 of the first arm includes sequence number 71 (SAISNSGTYTYYA), and CDRH3 of the first arm includes sequence number 75 (ARGLDFIVGYTGNDY). In some embodiments, CDRH1 of the first arm includes sequence number 83 (FTFSNYAMS), CDRH2 of the first arm includes sequence number 71 (SAISNSGTYTYYA), and CDRH3 of the first arm includes sequence number 74 (ARGLDFIVGATGNDY). In some embodiments, CDRH1 of the first arm includes sequence number 84 (FTFSGYAMS), CDRH2 of the first arm includes sequence number 71 (SAISNSGTYTYYA), and CDRH3 of the first arm includes sequence number 85 (ARGLDFIVGRTGNDY). In some embodiments, CDRH1 of the first arm includes sequence number 86 (FTFSSYAMN), CDRH2 of the first arm includes sequence number 71 (SAISNSGTYTYYA), and CDRH3 of the first arm includes sequence number 85 (ARGLDFIVGRTGNDY). In some embodiments, CDRH1 of the first arm includes sequence number 80 (FTFRSYAMS), CDRH2 of the first arm includes sequence number 71 (SAISNSGTYTYYA), and CDRH3 of the first arm includes sequence number 85 (ARGLDFIVGRTGNDY).

[0011] In some embodiments, CDRL1 of the first arm includes sequence number 9 (RASQSISSYLN), CDRL2 of the first arm includes sequence number 5 (AASSLQS), and CDRL3 of the first arm includes sequence number 10 (QQSYSTPLT).

[0012] In some embodiments, the heavy chain variable region of the first arm contains at least 90% identical amino acid sequences to SEQ ID NO: 87. In some embodiments, the heavy chain variable region of the first arm contains at least 90% identical amino acid sequences to SEQ ID NO: 88. In some embodiments, the heavy chain variable region of the first arm contains at least 90% identical amino acid sequences to SEQ ID NO: 89. In some embodiments, the heavy chain variable region of the first arm contains at least 90% identical amino acid sequences to SEQ ID NO: 90. In some embodiments, the heavy chain variable region of the first arm contains at least 90% identical amino acid sequences to SEQ ID NO: 91. In some embodiments, the heavy chain variable region of the first arm contains at least 90% identical amino acid sequences to SEQ ID NO: 92. In some embodiments, the heavy chain variable region of the first arm contains at least 90% identical amino acid sequences to SEQ ID NO: 93. In some embodiments, the heavy chain variable region of the first arm contains at least 90% identical amino acid sequences to SEQ ID NO: 94. In some embodiments, the heavy chain variable region of the first arm contains at least 90% identical amino acid sequences to SEQ ID NO: 95. In some embodiments, the heavy chain variable region of the first arm contains at least 90% identical amino acid sequences to SEQ ID NO: 96. In some embodiments, the heavy chain variable region of the first arm contains at least 90% identical amino acid sequences to SEQ ID NO: 97. In some embodiments, the heavy chain variable region of the first arm contains at least 90% identical amino acid sequences to SEQ ID NO: 98. In some embodiments, the heavy chain variable region of the first arm contains at least 90% identical amino acid sequences to SEQ ID NO: 99. In some embodiments, the light chain variable region of the first arm contains at least 90% identical amino acid sequences 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 is coupled to PD-L1 and includes a heavy chain variable region including a. (i) CDRH1 containing sequence number 1 (GTFSSYAIN), (ii) CDRH2 containing sequence number 2 (GGIIPX1X2GX3ATYA, where X1 is V or I, X2 is F, L, or V, and X3 is T or A), and (iii) CDRH3 containing sequence number 3 (ARLKX1ELKDAFDI, where X1 is G, F, or N), and b. a light chain variable region including (i) CDRL1 containing sequence number 4 (RASQX1ISSYLN, where X1 is S, W, or Q), (ii) CDRL2 containing sequence number 5 (AASSLQS), and (iii) CDRL3 containing sequence number 6 (X1QSYSTPLT, where X1 is Q or F).

[0014] In some embodiments, CDRH1 of the second arm includes sequence number 1 (GTFSSYAIN), CDRH2 of the second arm includes sequence number 7 (GGIIPILGAATYA), and CDRH3 of the second arm includes sequence number 8 (ARLKGELKDAFDI). In some embodiments, CDRH1 of the second arm includes sequence number 1 (GTFSSYAIN), CDRH2 of the second arm includes sequence number 7 (GGIIPILGAATYA), CDRH3 of the second arm includes sequence number 8 (ARLKGELKDAFDI), CDRL1 of the second arm includes sequence number 9 (RASQSISSYLN), CDRL2 of the second arm includes sequence number 5 (AASSLQS), and CDRL3 of the second arm includes sequence number 10 (QQSYSTPLT).

[0015] In some embodiments, CDRH1 of the second arm includes sequence number 1 (GTFSSYAIN), CDRH2 of the second arm includes sequence number 11 (GGIIPVFGTATYA), CDRH3 of the second arm includes sequence number 8 (ARLKGELKDAFDI), CDRL1 of the second arm includes sequence number 9 (RASQSISSYLN), CDRL2 of the second arm includes sequence number 5 (AASSLQS), and CDRL3 of the second arm includes sequence number 10 (QQSYSTPLT). In some embodiments, CDRH1 of the second arm includes sequence number 1 (GTFSSYAIN), CDRH2 of the second arm includes sequence number 11 (GGIIPVFGTATYA), and CDRH3 of the second arm includes sequence number 8 (ARLKGELKDAFDI). In some embodiments, CDRH1 of the second arm includes sequence number 1 (GTFSSYAIN), CDRH2 of the second arm includes sequence number 11 (GGIIPVFGTATYA), CDRH3 of the second arm includes sequence number 8 (ARLKGELKDAFDI), CDRL1 of the second arm includes sequence number 12 (RASQWISSYLN), CDRL2 of the second arm includes sequence number 5 (AASSLQS), and CDRL3 of the second arm includes sequence number 10 (QQSYSTPLT). In some embodiments, CDRH1 of the second arm includes sequence number 1 (GTFSSYAIN), CDRH2 of the second arm includes sequence number 11 (GGIIPVFGTATYA), CDRH3 of the second arm includes sequence number 8 (ARLKGELKDAFDI), CDRL1 of the second arm includes sequence number 13 (RASQQISSYLN), CDRL2 of the second arm includes sequence number 5 (AASSLQS), and CDRL3 of the second arm includes sequence number 10 (QQSYSTPLT). In some embodiments, CDRH1 of the second arm includes sequence number 1 (GTFSSYAIN), CDRH2 of the second arm includes sequence number 11 (GGIIPVFGTATYA), CDRH3 of the second arm includes sequence number 8 (ARLKGELKDAFDI), CDRL1 of the second arm includes sequence number 9 (RASQSISSYLN), CDRL2 of the second arm includes sequence number 5 (AASSLQS), and CDRL3 of the second arm includes sequence number 10 (QQSYSTPLT).

[0016] In some embodiments, CDRH1 of the second arm includes sequence number 1 (GTFSSYAIN), CDRH2 of the second arm includes sequence number 15 (GGIIPIFGIANYA), and CDRH3 of the second arm includes sequence number 8 (ARLKGELKDAFDI). In some embodiments, CDRH1 of the second arm includes sequence number 1 (GTFSSYAIN), CDRH2 of the second arm includes sequence number 15 (GGIIPIFGIANYA), CDRH3 of the second arm includes sequence number 8 (ARLKGELKDAFDI), CDRL1 of the second arm includes sequence number 9 (RASQSISSYLN), CDRL2 of the second arm includes sequence number 5 (AASSLQS), and CDRL3 of the second arm includes sequence number 10 (QQSYSTPLT).

[0017] In some embodiments, CDRH1 of the second arm includes sequence number 1 (GTFSSYAIN), CDRH2 of the second arm includes sequence number 16 (GGIIPNFGTATYA), and CDRH3 of the second arm includes sequence number 17 (ARLKGELKGAGDI). In some embodiments, CDRH1 of the second arm includes sequence number 1 (GTFSSYAIN), CDRH2 of the second arm includes sequence number 16 (GGIIPNFGTATYA), CDRH3 of the second arm includes sequence number 17 (ARLKGELKGAGDI), CDRL1 of the second arm includes sequence number 9 (RASQSISSYLN), CDRL2 of the second arm includes sequence number 5 (AASSLQS), and CDRL3 of the second arm includes sequence number 10 (QQSYSTPLT).

[0018] In some embodiments, CDRH1 of the second arm includes sequence number 1 (GTFSSYAIN), CDRH2 of the second arm includes sequence number 11 (GGIIPVFGTATYA), and CDRH3 of the second arm includes sequence number 18 (ARLKFELKDAFDI). In some embodiments, CDRH1 of the second arm includes sequence number 1 (GTFSSYAIN), CDRH2 of the second arm includes sequence number 11 (GGIIPVFGTATYA), CDRH3 of the second arm includes sequence number 18 (ARLKFELKDAFDI), CDRL1 of the second arm includes sequence number 9 (RASQSISSYLN), CDRL2 of the second arm includes sequence number 5 (AASSLQS), and CDRL3 of the second arm includes sequence number 10 (QQSYSTPLT).

[0019] In some embodiments, CDRH1 of the second arm includes sequence number 1 (GTFSSYAIN), CDRH2 of the second arm includes sequence number 11 (GGIIPVFGTATYA), and CDRH3 of the second arm includes sequence number 19 (ARLKGELKDAFDE). In some embodiments, CDRH1 of the second arm includes sequence number 1 (GTFSSYAIN), CDRH2 of the second arm includes sequence number 11 (GGIIPVFGTATYA), CDRH3 of the second arm includes sequence number 19 (ARLKGELKDAFDE), CDRL1 of the second arm includes sequence number 9 (RASQSISSYLN), CDRL2 of the second arm includes sequence number 5 (AASSLQS), and CDRL3 of the second arm includes sequence number 10 (QQSYSTPLT).

[0020] In some embodiments, CDRH1 of the second arm includes sequence number 1 (GTFSSYAIN), CDRH2 of the second arm includes sequence number 11 (GGIIPVFGTATYA), and CDRH3 of the second arm includes sequence number 20 (ARLKNELKDAFDI). In some embodiments, CDRH1 of the second arm includes sequence number 1 (GTFSSYAIN), CDRH2 of the second arm includes sequence number 11 (GGIIPVFGTATYA), CDRH3 of the second arm includes sequence number 20 (ARLKNELKDAFDI), CDRL1 of the second arm includes sequence number 9 (RASQSISSYLN), CDRL2 of the second arm includes sequence number 5 (AASSLQS), and CDRL3 of the second arm includes sequence number 10 (QQSYSTPLT).

[0021] In some embodiments, CDRH1 of the second arm includes sequence number 1 (GTFSSYAIN), CDRH2 of the second arm includes sequence number 21 (GGVIPFLGTANYA), and CDRH3 of the second arm includes sequence number 22 (ARLKGILKDALDI). In some embodiments, CDRH1 of the second arm includes sequence number 1 (GTFSSYAIN), CDRH2 of the second arm includes sequence number 21 (GGVIPFLGTANYA), CDRH3 of the second arm includes sequence number 22 (ARLKGILKDALDI), CDRL1 of the second arm includes sequence number 9 (RASQSISSYLN), CDRL2 of the second arm includes sequence number 5 (AASSLQS), and CDRL3 of the second arm includes sequence number 10 (QQSYSTPLT).

[0022] In some embodiments, CDRH1 of the second arm includes sequence number 1 (GTFSSYAIN), CDRH2 of the second arm includes sequence number 29 (GRIIPIFGTADYA), and CDRH3 of the second arm includes sequence number 8 (ARLKGELKDAFDI). In some embodiments, CDRH1 of the second arm includes sequence number 1 (GTFSSYAIN), CDRH2 of the second arm includes sequence number 29 (GRIIPIFGTADYA), CDRH3 of the second arm includes sequence number 8 (ARLKGELKDAFDI), CDRL1 of the second arm includes sequence number 9 (RASQSISSYLN), CDRL2 of the second arm includes sequence number 5 (AASSLQS), and CDRL3 of the second arm includes sequence number 10 (QQSYSTPLT).

[0023] In some embodiments, CDRH1 of the second arm includes sequence number 1 (GTFSSYAIN), CDRH2 of the second arm includes sequence number 31 (GGIIPILGTATYA), and CDRH3 of the second arm includes sequence number 32 (ARRKGELKDAFDI). In some embodiments, CDRH1 of the second arm includes sequence number 1 (GTFSSYAIN), CDRH2 of the second arm includes sequence number 31 (GGIIPILGTATYA), CDRH3 of the second arm includes sequence number 32 (ARRKGELKDAFDI), CDRL1 of the second arm includes sequence number 9 (RASQSISSYLN), CDRL2 of the second arm includes sequence number 5 (AASSLQS), and CDRL3 of the second arm includes sequence number 10 (QQSYSTPLT).

[0024] In some embodiments, CDRH1 of the second arm includes sequence number 1 (GTFSSYAIN), CDRH2 of the second arm includes sequence number 33 (GGIIPIVATANYA), and CDRH3 of the second arm includes sequence number 32 (ARRKGELKDAFDI). In some embodiments, CDRH1 of the second arm includes sequence number 1 (GTFSSYAIN), CDRH2 of the second arm includes sequence number 33 (GGIIPIVATANYA), CDRH3 of the second arm includes sequence number 32 (ARRKGELKDAFDI), CDRL1 of the second arm includes sequence number 9 (RASQSISSYLN), CDRL2 of the second arm includes sequence number 5 (AASSLQS), and CDRL3 of the second arm includes sequence number 10 (QQSYSTPLT).

[0025] In some embodiments, CDRH1 of the second arm includes sequence number 1 (GTFSSYAIN), CDRH2 of the second arm includes sequence number 34 (GGIIPIFGKATYA), and CDRH3 of the second arm includes sequence number 32 (ARRKGELKDAFDI). In some embodiments, CDRH1 of the second arm includes sequence number 1 (GTFSSYAIN), CDRH2 of the second arm includes sequence number 34 (GGIIPIFGKATYA), CDRH3 of the second arm includes sequence number 32 (ARRKGELKDAFDI), CDRL1 of the second arm includes sequence number 9 (RASQSISSYLN), CDRL2 of the second arm includes sequence number 5 (AASSLQS), and CDRL3 of the second arm includes sequence number 10 (QQSYSTPLT).

[0026] In some embodiments, CDRH1 of the second arm includes sequence number 1 (GTFSSYAIN), CDRH2 of the second arm includes sequence number 11 (GGIIPVFGTATYA), CDRH3 of the second arm includes sequence number 8 (ARLKGELKDAFDI), CDRL1 of the second arm includes sequence number 9 (RASQSISSYLN), CDRL2 of the second arm includes sequence number 5 (AASSLQS), and CDRL3 of the second arm includes sequence number 38 (FQSYSTPLT).

[0027] In some embodiments, CDRH1 of the second arm includes sequence number 1 (GTFSSYAIN), CDRH2 of the second arm includes sequence number 11 (GGIIPVFGTATYA), CDRH3 of the second arm includes sequence number 8 (ARLKGELKDAFDI), CDRL1 of the second arm includes sequence number 9 (RASQSISSYLN), CDRL2 of the second arm includes sequence number 5 (AASSLQS), and CDRL3 of the second arm includes sequence number 39 (QQSYSTILT).

[0028] In some embodiments, the second arm includes a heavy chain variable region comprising a. (i) CDRH1 containing sequence number 14 (GTFSSYAFS), (ii) CDRH2 containing sequence number 11 (GGIIPVFGTATYA), and (iii) CDRH3 containing sequence number 8 (ARLKGELKDAFDI), and b. a light chain variable region comprising a CDRL1 containing sequence number 9 (RASQSISSYLN), (ii) CDRL2 containing sequence number 5 (AASSLQS), and (iii) CDRL3 containing sequence number 10 (QQSYSTPLT).

[0029] In some embodiments, the second arm includes a heavy chain variable region including (i) CDRH1 containing sequence number 23 (GTFSSYAIS), (ii) CDRH2 containing sequence number 24 (GGIIPIVGIANYA), and (iii) CDRH3 containing sequence number 8 (ARLKGELKDAFDI). In some embodiments, the second arm includes a light chain variable region including (i) CDRL1 containing sequence number 9 (RASQSISSYLN), (ii) CDRL2 containing sequence number 5 (AASSLQS), and (iii) CDRL3 containing sequence number 10 (QQSYSTPLT).

[0030] In some embodiments, the second arm includes a heavy chain variable region including (i) CDRH1 containing sequence number 23 (GTFSSYAIS), (ii) CDRH2 containing sequence number 11 (GGIIPVFGTATYA), and (iii) CDRH3 containing sequence number 25 (ARLKGEFKDAFDI). In some embodiments, the second arm includes a light chain variable region including (i) CDRL1 containing sequence number 9 (RASQSISSYLN), (ii) CDRL2 containing sequence number 5 (AASSLQS), and (iii) CDRL3 containing sequence number 10 (QQSYSTPLT).

[0031] In some embodiments, the second arm includes a heavy chain variable region including (i) CDRH1 containing sequence number 23 (GTFSSYAIS), (ii) CDRH2 containing sequence number 26 (GRIIPLFGTAHYA), and (iii) CDRH3 containing sequence number 8 (ARLKGELKDAFDI). In some embodiments, the second arm includes a light chain variable region including (i) CDRL1 containing sequence number 9 (RASQSISSYLN), (ii) CDRL2 containing sequence number 5 (AASSLQS), and (iii) CDRL3 containing sequence number 10 (QQSYSTPLT).

[0032] In some embodiments, the second arm includes a heavy chain variable region including (i) CDRH1 containing sequence number 23 (GTFSSYAIS), (ii) CDRH2 containing sequence number 27 (GRINPILGTANYA), and (iii) CDRH3 containing sequence number 28 (ARLKGELKDAFSI). In some embodiments, the second arm includes a light chain variable region including (i) CDRL1 containing sequence number 9 (RASQSISSYLN), (ii) CDRL2 containing sequence number 5 (AASSLQS), and (iii) CDRL3 containing sequence number 10 (QQSYSTPLT).

[0033] In some embodiments, the second arm includes a heavy chain variable region including (i) CDRH1 containing sequence number 23 (GTFSSYAIS), (ii) CDRH2 containing sequence number 11 (GGIIPVFGTATYA), and (iii) CDRH3 containing sequence number 30 (ARLKGELKCAFDI). In some embodiments, the second arm includes a light chain variable region including (i) CDRL1 containing sequence number 9 (RASQSISSYLN), (ii) CDRL2 containing sequence number 5 (AASSLQS), and (iii) CDRL3 containing sequence number 10 (QQSYSTPLT).

[0034] In some embodiments, the second arm includes a heavy chain variable region including (i) CDRH1 containing sequence number 122 (GTKSSYAIS), (ii) CDRH2 containing sequence number 11 (GGIIPVFGTATYA), and (iii) CDRH3 containing sequence number 30 (ARLKGELKCAFDI). In some embodiments, the second arm further includes a light chain variable region including (i) CDRL1 containing sequence number 9 (RASQSISSYLN), (ii) CDRL2 containing sequence number 5 (AASSLQS), and (iii) CDRL3 containing sequence number 10 (QQSYSTPLT).

[0035] In some embodiments, the second arm includes a heavy chain variable region including (i) CDRH1 containing sequence number 36 (GPFRSHAVS), (ii) CDRH2 containing sequence number 11 (GGIIPVFGTATYA), and (iii) CDRH3 containing sequence number 37 (ARLKSELKDAFDI). In some embodiments, the second arm includes a light chain variable region including (i) CDRL1 containing sequence number 9 (RASQSISSYLN), (ii) CDRL2 containing sequence number 5 (AASSLQS), and (iii) CDRL3 containing sequence number 10 (QQSYSTPLT).

[0036] In some embodiments, the second arm that binds to PD-L1 includes a heavy chain variable region containing an amino acid sequence that is 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 containing an amino acid sequence that is 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 contains at least 90% identical amino acid sequences to SEQ ID NO: 35 (QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAINWVRQAPGQGLEWMGGIIPVFGTATYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARLKGELKDAFDIWGQGTMVTVSS). In some embodiments, the heavy chain variable region of the second arm contains at least 90% identical amino acid sequences to SEQ ID NO: 40 (QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAFSWVRQAPGQGLEWMGGIIPVFGTATYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARLKGELKDAFDIWGQGTLVTVSS). In some embodiments, the heavy chain variable region of the second arm contains at least 90% identical amino acid sequences to SEQ ID NO: 41 (QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAINWVRQAPGQGLEWMGGIIPIFGIANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARLKGELKDAFDIWGQGTLVTVSS). In some embodiments, the heavy chain variable region of the second arm contains at least 90% identical amino acid sequences to SEQ ID NO: 42 (QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAINWVRQAPGQGLEWMGGIIPNFGTATYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARLKGELKGAGDIWGQGTLVTVSS). In some embodiments, the heavy chain variable region of the second arm contains an amino acid sequence that is at least 90% identical to SEQ ID NO: 43 (QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAINWVRQAPGQGLEWMGGIIPVFGTATYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARLKFELKDAFDIWGQGTLVTVSS).In some embodiments, the heavy chain variable region of the second arm contains at least 90% identical amino acid sequences to SEQ ID NO: 44 (QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAINWVRQAPGQGLEWMGGIIPVFGTATYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARLKGELKDAFDEWGQGTLVTVSS). In some embodiments, the heavy chain variable region of the second arm contains at least 90% identical amino acid sequences to SEQ ID NO: 45 (QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAINWVRQAPGQGLEWMGGIIPVFGTATYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARLKGELKDAFDIWGQGTLVTAST). In some embodiments, the heavy chain variable region of the second arm contains at least 90% identical amino acid sequences to SEQ ID NO: 46 (QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAINWVRQAPGQGLEWMGGIIPVFGTATYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARLKNELKDAFDIWGQGTLVTVSS). In some embodiments, the heavy chain variable region of the second arm contains at least 90% identical amino acid sequences to SEQ ID NO: 47 (QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAINWVRQAPGQGLEWMGGVIPFLGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARLKGILKDALDIWGQGTLVTVSS). In some embodiments, the heavy chain variable region of the second arm contains an amino acid sequence that is at least 90% identical to SEQ ID NO: 48 (QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQDLEWMGGIIPIVGIANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARLKGELKDAFDIWGQGTLVTVSS).In some embodiments, the heavy chain variable region of the second arm contains at least 90% identical amino acid sequences to SEQ ID NO: 49 (QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPVFGTATYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARLKGEFKDAFDIWGQGTLVTVSS). In some embodiments, the heavy chain variable region of the second arm contains at least 90% identical amino acid sequences to SEQ ID NO: 50 (QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGRIIPLFGTAHYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARLKGELKDAFDIWGQGTLVTVSS). In some embodiments, the heavy chain variable region of the second arm contains at least 90% identical amino acid sequences to SEQ ID NO: 51 (QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGRINPILGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARLKGELKDAFSIWGQGTLVTVSS). In some embodiments, the heavy chain variable region of the second arm contains at least 90% identical amino acid sequences to SEQ ID NO: 52 (QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAINWVRQAPGQGLEWMGRIIPIFGTADYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARLKGELKDAFDIWGQGTLVTVSS). In some embodiments, the heavy chain variable region of the second arm contains at least 90% identical amino acid sequences to SEQ ID NO: 53 (QVQLVQSGAEVKKPGSSVKVSCKASGGKFSSYAISWVRQAPGQGLEWMGGIIPVFGTATYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARLKGELKCAFDIWGQGTLVTVSS).In some embodiments, the heavy chain variable region of the second arm contains at least 90% identical amino acid sequences to SEQ ID NO: 54 (QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAINWVRQAPGQGLEWMGGIIPILGTATYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARRKGELKDAFDIWGQGTLVTVSS). In some embodiments, the heavy chain variable region of the second arm contains at least 90% identical amino acid sequences to SEQ ID NO: 55 (QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAINWVRQAPGQGLEWMGGIIPILGAATYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARLKGELKDAFDIWGQGTLVTVSS). In some embodiments, the heavy chain variable region of the second arm contains at least 90% identical amino acid sequences to SEQ ID NO: 56 (QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAINWVRQAPGQGLEWMGGIIPIVATANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARRKGELKDAFDIWGQGTLVTVSS). In some embodiments, the heavy chain variable region of the second arm contains at least 90% identical amino acid sequences to SEQ ID NO: 57 (QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAINWVRQAPGQGLEWMGGIIPIFGKATYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARRKGELKDAFDIWGQGTLVTVSS). In some embodiments, the heavy chain variable region of the second arm contains an amino acid sequence that is at least 90% identical to SEQ ID NO: 58 (QVQLVQSGAEVKKPGSSVKVSCKASGGPFRSHAVSWVRQAPGQGLEWMGGIIPVFGTATYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARLKSELKDAFDIWGQGTLVTVSS).In some embodiments, the light chain variable region of the second arm contains at least 90% identical amino acid sequences to SEQ ID NO: 59 (DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGGGTKVEIK). In some embodiments, the light chain variable region of the second arm contains at least 90% identical amino acid sequences to SEQ ID NO: 60 (DIQMTQSPSSLSASVGDRVTITCRASQWISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGGGTKVEIK). In some embodiments, the light chain variable region of the second arm contains at least 90% identical amino acid sequences to SEQ ID NO: 61 (DIQMTQSPSSLSASVGDRVTITCRASQQISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGGGTKVEIK). In some embodiments, the light chain variable region of the second arm contains at least 90% identical amino acid sequences to SEQ ID NO: 62 (DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCFQSYSTPLTFGGGTKVEIK). In some embodiments, the light chain variable region of the second arm contains an amino acid sequence that is at least 90% identical to SEQ ID NO: 63 (DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTILTFGGGTKVEIK).

[0038] In some embodiments of any part of the embodiments described herein, the construct is a bispecific antibody. In some embodiments, the bispecific antibody is an antagonist of both PD-1 and the PD-1 ligand. In some embodiments, the construct contains a common light chain. In some embodiments, one or both arms are aptamers. In some embodiments, one or both arms are proteins other than antibodies. In some embodiments, the construct contains 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 the PD-1 ligand. In some embodiments, at least one arm is a bivalent antibody specific to PD-1. In some embodiments, at least one arm is a bivalent antibody specific to PD-L1. In some embodiments, at least one arm is a bivalent antibody specific to PD-1 and at least one arm is a bivalent antibody specific to PD-L1. In some embodiments, at least one arm is bivalent for PD-1. In some embodiments, at least one arm is bivalent for PD-L1. In some embodiments, at least one arm is bivalent for PD-1 and at least one arm 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 containing the variable heavy chain of an anti-PD-1 antibody is fused to a polypeptide containing the variable heavy chain of an anti-PD-L1 antibody. In some embodiments, the polypeptide containing the variable heavy chain of an anti-PD-1 antibody is fused to a polypeptide containing 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 to PD-1 in the presence of a common light chain, and the C-terminal variable heavy chain of the fusion construct binds to PD-L1 in the presence of a common light chain. In some embodiments, the N-terminal variable heavy chain of the fusion construct binds to PD-L1 in the presence of a common light chain, and the C-terminal variable heavy chain of the fusion construct binds to PD-1 in the presence of a common light chain.

[0040] In some embodiments and aspects, the disclosure provides a multispecific antigen-binding construct comprising at least two units of antigen binding, wherein a first unit of antigen binding binds to PD-1 and a second unit of antigen binding binds to a PD-1 ligand. In some embodiments, the first unit of antigen binding binds to PD-1 expressed by an immune cell. In some embodiments, the second unit of antigen binding binds to PD-1 expressed by a second cell. In some embodiments, the multispecific antigen-binding construct blocks the interaction between PD-1 and a PD-1 ligand, e.g., PD-L1 or PD-L2. In some embodiments, the multispecific antigen-binding construct blocks the interaction between 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 units of antigen binding that bind to PD-1. In some embodiments, the multispecific antigen-binding construct comprises two units of antigen binding that bind to PD-1. In some embodiments, the multispecific antigen-binding construct includes at least two units of antigen binding that bind to a PD-1 ligand, e.g., PD-L1 or PD-L2. In some embodiments, the multispecific antigen-binding construct includes two units of antigen binding that bind to a PD-1 ligand, e.g., PD-L1 or PD-L2. In some embodiments, the multispecific antigen-binding construct includes at least four units of antigen binding, 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 includes four units of antigen binding, 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 unit of antigen binding can independently bind to its cognitive 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 between PD-1 and a PD-1 ligand, such as PD-L1 or PD-L2. In some embodiments, the multispecific antigen-binding construct includes a common light chain. For example, at least two units of antigen binding include a common light chain.

[0041] In some embodiments, the first antigen-binding unit is bound to PD-1. (a)(i) CDRH1 containing sequence number 70 (FTFX1X2YAX3X4, where X1=S, R, G, or N, X2=D, S, N, A, R, or G, X3=M or L, X4=S, L, or N), (ii) CDRH2 containing sequence number 71 (SAISNSGTYTYYA), and (iii) CDRH3 containing sequence number 72 (ARGLDFIVGX5TGNDY, where X5=A, Y, or R), and (b) Light chain variable region including (i) CDRL1 containing sequence number 9 (RASQSISSYLN), (ii) CDRL2 containing sequence number 5 (AASSLQS), and (iii) CDRL3 containing sequence number 10 (QQSYSTPLT). Includes.

[0042] In some such embodiments, the first unit of antigen binding is bound to PD-1, (a) CDRH1 containing sequence number 73 (FTFSDYAMS), CDRH2 containing sequence number 71 (SAISNSGTYTYYA), and CDRH3 containing sequence number 74 (ARGLDFIVGATGNDY), (b) CDRH1 containing sequence number 73 (FTFSDYAMS), CDRH2 containing sequence number 71 (SAISNSGTYTYYA), and CDRH3 containing sequence number 75 (ARGLDFIVGYTGNDY), (c) CDRH1 containing sequence number 76 (FTFSSYAMS), CDRH2 containing sequence number 71 (SAISNSGTYTYYA), and CDRH3 containing sequence number 75 (ARGLDFIVGYTGNDY), (d) CDRH1 containing sequence number 77 (FTFSSYAML), CDRH2 containing sequence number 71 (SAISNSGTYTYYA), and CDRH3 containing sequence number 75 (ARGLDFIVGYTGNDY), (e) CDRH1 containing sequence number 78 (FTFSNYALS), CDRH2 containing sequence number 71 (SAISNSGTYTYYA), and CDRH3 containing sequence number 75 (ARGLDFIVGYTGNDY), (f) CDRH1 containing sequence number 79 (FTFSAYAMN), CDRH2 containing sequence number 71 (SAISNSGTYTYYA), and CDRH3 containing sequence number 75 (ARGLDFIVGYTGNDY), (g) CDRH1 containing sequence number 80 (FTFRSYAMS), CDRH2 containing sequence number 71 (SAISNSGTYTYYA), and CDRH3 containing sequence number 75 (ARGLDFIVGYTGNDY), (h) CDRH1 containing sequence number 81 (FTFGRYAMS), CDRH2 containing sequence number 71 (SAISNSGTYTYYA), and CDRH3 containing sequence number 75 (ARGLDFIVGYTGNDY), (i) CDRH1 containing sequence number 82 (FTFNSYAMS), CDRH2 containing sequence number 71 (SAISNSGTYTYYA), and CDRH3 containing sequence number 75 (ARGLDFIVGYTGNDY), (j) CDRH1 containing sequence number 83 (FTFSNYAMS), CDRH2 containing sequence number 71 (SAISNSGTYTYYA), and CDRH3 containing sequence number 74 (ARGLDFIVGATGNDY), (k) CDRH1 containing sequence number 84 (FTFSGYAMS), CDRH2 containing sequence number 71 (SAISNSGTYTYYA), and CDRH3 containing sequence number 85 (ARGLDFIVGRTGNDY), (l) CDRH1 containing sequence number 86 (FTFSSYAMN), CDRH2 containing sequence number 71 (SAISNSGTYTYYA), and CDRH3 containing sequence number 85 (ARGLDFIVGRTGNDY), or (m) CDRH1 containing sequence number 80 (FTFRSYAMS), CDRH3 containing sequence number 71 (SAISNSGTYTYYA), and CDRH3 containing sequence number 85 (ARGLDFIVGRTGNDY). Includes.

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

[0044] In some embodiments, the first antigen-binding unit is bound to PD-1 and includes a light chain variable region having at least 90% identical amino acid sequence to SEQ ID NO: 59. In some embodiments, the second antigen-binding unit binds to PD-L2. In some embodiments, the second antigen-binding unit binds to PD-L1. In some embodiments, the second antigen-binding unit binds to PD-L1, a. A heavy chain variable region including (i) CDRH1 containing sequence number 1 (GTFSSYAIN), (ii) CDRH2 containing sequence number 2 (GGIIPX1X2GX3ATYA, where X1 is V or I, X2 is F, L, or V, and X3 is T or A), and (iii) CDRH3 containing sequence number 3 (ARLKX1ELKDAFDI, where X1 is G, F, or N), and b. Light chain variable region containing (i) CDRL1 containing sequence number 4 (RASQX1ISSYLN, where X1 is S, W, or Q), (ii) CDRL2 containing sequence number 5 (AASSLQS), and (iii) CDRL3 containing sequence number 6 (X1QSYSTPLT, where X1 is Q or F). Includes.

[0045] In some such embodiments, the second unit of antigen binding is bound to PD-L1, (a) CDRH1 containing sequence number 1 (GTFSSYAIN), CDRH2 containing sequence number 7 (GGIIPILGAATYA), and CDRH3 containing sequence number 8 (ARLKGELKDAFDI), (b) CDRH1 containing sequence number 1 (GTFSSYAIN), CDRH2 containing sequence number 7 (GGIIPILGAATYA), CDRH3 containing sequence number 8 (ARLKGELKDAFDI), CDRL1 containing sequence number 9 (RASQSISSYLN), CDRL2 containing sequence number 5 (AASSLQS), and CDRL3 containing sequence number 10 (QQSYSTPLT), (c) CDRH1 containing sequence number 1 (GTFSSYAIN), CDRH2 containing sequence number 11 (GGIIPVFGTATYA), CDRH3 containing sequence number 8 (ARLKGELKDAFDI), CDRL1 containing sequence number 9 (RASQSISSYLN), CDRL2 containing sequence number 5 (AASSLQS), and CDRL3 containing sequence number 10 (QQSYSTPLT), (d) CDRH1 containing sequence number 1 (GTFSSYAIN), CDRH2 containing sequence number 11 (GGIIPVFGTATYA), and CDRH3 containing sequence number 8 (ARLKGELKDAFDI), (e) CDRH1 containing sequence number 1 (GTFSSYAIN), CDRH2 containing sequence number 11 (GGIIPVFGTATYA), CDRH3 containing sequence number 8 (ARLKGELKDAFDI), CDRL1 containing sequence number 12 (RASQWISSYLN), CDRL2 containing sequence number 5 (AASSLQS), and CDRL3 containing sequence number 10 (QQSYSTPLT), (f) CDRH1 containing sequence number 1 (GTFSSYAIN), CDRH2 containing sequence number 11 (GGIIPVFGTATYA), CDRH3 containing sequence number 8 (ARLKGELKDAFDI), CDRL1 containing sequence number 13 (RASQQISSYLN), CDRL2 containing sequence number 5 (AASSLQS), and CDRL3 containing sequence number 10 (QQSYSTPLT), (g) CDRH1 containing sequence number 1 (GTFSSYAIN), CDRH2 containing sequence number 11 (GGIIPVFGTATYA), CDRH3 containing sequence number 8 (ARLKGELKDAFDI), CDRL1 containing sequence number 9 (RASQSISSYLN), CDRL2 containing sequence number 5 (AASSLQS), and CDRL3 containing sequence number 10 (QQSYSTPLT), (h) CDRH1 containing sequence number 1 (GTFSSYAIN), CDRH2 containing sequence number 15 (GGIIPIFGIANYA), and CDRH3 containing sequence number 8 (ARLKGELKDAFDI), (i) CDRH1 containing sequence number 1 (GTFSSYAIN), CDRH2 containing sequence number 15 (GGIIPIFGIANYA), CDRH3 containing sequence number 8 (ARLKGELKDAFDI), CDRL1 containing sequence number 9 (RASQSISSYLN), CDRL2 containing sequence number 5 (AASSLQS), and CDRL3 containing sequence number 10 (CQQSYSTPLTF), (j) CDRH1 containing sequence number 1 (GTFSSYAIN), CDRH2 containing sequence number 16 (GGIIPNFGTATYA), and CDRH3 containing sequence number 17 (ARLKGELKGAGDI), (k) CDRH1 containing sequence number 1 (GTFSSYAIN), CDRH2 containing sequence number 16 (GGIIPNFGTATYA), CDRH3 containing sequence number 17 (ARLKGELKGAGDI), CDRL1 containing sequence number 9 (RASQSISSYLN), CDRL2 containing sequence number 5 (AASSLQS), and CDRL3 containing sequence number 10 (QQSYSTPLT), (l) CDRH1 containing sequence number 1 (GTFSSYAIN), CDRH2 containing sequence number 11 (GGIIPVFGTATYA), and CDRH3 containing sequence number 18 (ARLKFELKDAFDI), (m) CDRH1 containing sequence number 1 (GTFSSYAIN), CDRH2 containing sequence number 11 (GGIIPVFGTATYA), CDRH3 containing sequence number 18 (ARLKFELKDAFDI), CDRL1 containing sequence number 9 (RASQSISSYLN), CDRL2 containing sequence number 5 (AASSLQS), and CDRL3 containing sequence number 10 (QQSYSTPLT), (n) CDRH1 containing sequence number 1 (GTFSSYAIN), CDRH2 containing sequence number 11 (GGIIPVFGTATYA), and CDRH3 containing sequence number 19 (ARLKGELKDAFDE), (o) CDRH1 containing sequence number 1 (GTFSSYAIN), CDRH2 containing sequence number 11 (GGIIPVFGTATYA), CDRH3 containing sequence number 19 (ARLKGELKDAFDE), CDRL1 containing sequence number 9 (RASQSISSYLN), CDRL2 containing sequence number 5 (AASSLQS), and CDRL3 containing sequence number 10 (QQSYSTPLT), (p) CDRH1 containing sequence number 1 (GTFSSYAIN), CDRH2 containing sequence number 11 (GGIIPVFGTATYA), and CDRH3 containing sequence number 20 (ARLKNELKDAFDI), (q) CDRH1 containing sequence number 1 (GTFSSYAIN), CDRH2 containing sequence number 11 (GGIIPVFGTATYA), CDRH3 containing sequence number 20 (ARLKNELKDAFDI), CDRL1 containing sequence number 9 (RASQSISSYLN), CDRL2 containing sequence number 5 (AASSLQS), and CDRL3 containing sequence number 10 (QQSYSTPLT), (r) CDRH1 containing sequence number 1 (GTFSSYAIN), CDRH2 containing sequence number 21 (GGVIPFLGTANYA), and CDRH3 containing sequence number 22 (ARLKGILKDALDI), (s) CDRH1 containing sequence number 1 (GTFSSYAIN), CDRH2 containing sequence number 21 (GGVIPFLGTANYA), CDRH3 containing sequence number 22 (ARLKGILKDALDI), CDRL1 containing sequence number 9 (RASQSISSYLN), CDRL2 containing sequence number 5 (AASSLQS), and CDRL3 containing sequence number 10 (QQSYSTPLT), (t) CDRH1 containing sequence number 1 (GTFSSYAIN), CDRH2 containing sequence number 29 (GRIIPIFGTADYA), and CDRH3 containing sequence number 8 (ARLKGELKDAFDI), (u) CDRH1 containing sequence number 1 (GTFSSYAIN), CDRH2 containing sequence number 29 (GRIIPIFGTADYA), CDRH3 containing sequence number 8 (ARLKGELKDAFDI), CDRL1 containing sequence number 9 (RASQSISSYLN), CDRL2 containing sequence number 5 (AASSLQS), and CDRL3 containing sequence number 10 (QQSYSTPLT), (v) CDRH1 containing sequence number 1 (GTFSSYAIN), CDRH2 containing sequence number 31 (GGIIPILGTATYA), and CDRH3 containing sequence number 32 (ARRKGELKDAFDI), (w) CDRH1 containing sequence number 1 (GTFSSYAIN), CDRH2 containing sequence number 31 (GGIIPILGTATYA), CDRH3 containing sequence number 32 (ARRKGELKDAFDI), CDRL1 containing sequence number 9 (RASQSISSYLN), CDRL2 containing sequence number 5 (AASSLQS), and CDRL3 containing sequence number 10 (QQSYSTPLT), (x) CDRH1 containing sequence number 1 (GTFSSYAIN), CDRH2 containing sequence number 33 (GGIIPIVATANYA), and CDRH3 containing sequence number 32 (ARRKGELKDAFDI), (y) CDRH1 containing sequence number 1 (GTFSSYAIN), CDRH2 containing sequence number 33 (GGIIPIVATANYA), CDRH3 containing sequence number 32 (ARRKGELKDAFDI), CDRL1 containing sequence number 9 (RASQSISSYLN), CDRL2 containing sequence number 5 (AASSLQS), and CDRL3 containing sequence number 10 (QQSYSTPLT), (z) CDRH1 containing sequence number 1 (GTFSSYAIN), CDRH2 containing sequence number 34 (GGIIPIFGKATYA), and CDRH3 containing sequence number 32 (ARRKGELKDAFDI), (aa) CDRH1 containing sequence number 1 (GTFSSYAIN), CDRH2 containing sequence number 34 (GGIIPIFGKATYA), CDRH3 containing sequence number 32 (ARRKGELKDAFDI), CDRL1 containing sequence number 9 (RASQSISSYLN), CDRL2 containing sequence number 5 (AASSLQS), and CDRL3 containing sequence number 10 (QQSYSTPLT), (bb) CDRH1 containing sequence number 1 (GTFSSYAIN), CDRH2 containing sequence number 11 (GGIIPVFGTATYA), CDRH3 containing sequence number 8 (ARLKGELKDAFDI), CDRL1 containing sequence number 9 (RASQSISSYLN), CDRL2 containing sequence number 5 (AASSLQS), and CDRL3 containing sequence number 38 (FQSYSTPLT), (cc) CDRH1 containing sequence number 1 (GTFSSYAIN), CDRH2 containing sequence number 11 (GGIIPVFGTATYA), CDRH3 containing sequence number 8 (ARLKGELKDAFDI), CDRL1 containing sequence number 9 (RASQSISSYLN), CDRL2 containing sequence number 5 (AASSLQS), and CDRL3 containing sequence number 39 (QQSYSTILT), (dd) CDRH1 containing sequence number 14 (GTFSSYAFS), CDRH2 containing sequence number 11 (GGIIPVFGTATYA), CDRH3 containing sequence number 8 (ARLKGELKDAFDI), CDRL1 containing sequence number 9 (RASQSISSYLN), CDRL2 containing sequence number 5 (AASSLQS), and CDRL3 containing sequence number 10 (QQSYSTPLT), (ee) CDRH1 containing sequence number 23 (GTFSSYAIS), CDRH2 containing sequence number 24 (GGIIPIVGIANYA), and CDRH3 containing sequence number 8 (ARLKGELKDAFDI), (ff) CDRH1 containing sequence number 23 (GTFSSYAIS), CDRH2 containing sequence number 24 (GGIIPIVGIANYA), CDRH3 containing sequence number 8 (ARLKGELKDAFDI), CDRL1 containing sequence number 9 (RASQSISSYLN), CDRL2 containing sequence number 5 (AASSLQS), and CDRL3 containing sequence number 10 (QQSYSTPLT), CDRH1 containing (gg) Sequence ID 23 (GTFSSYAIS), CDRH2 containing Sequence ID 11 (GGIIPVFGTATYA), and CDRH3 containing Sequence ID 25 (ARLKGEFKDAFDI), (hh) CDRH1 containing sequence number 23 (GTFSSYAIS), CDRH2 containing sequence number 11 (GGIIPVFGTATYA), CDRH3 containing sequence number 25 (ARLKGEFKDAFDI), CDRL1 containing sequence number 9 (RASQSISSYLN), CDRL2 containing sequence number 5 (AASSLQS), and CDRL3 containing sequence number 10 (QQSYSTPLT), (ii) CDRH1 containing sequence number 23 (GTFSSYAIS), CDRH2 containing sequence number 26 (GRIIPLFGTAHYA), and CDRH3 containing sequence number 8 (ARLKGELKDAFDI), (jj) CDRH1 containing sequence number 23 (GTFSSYAIS), CDRH2 containing sequence number 26 (GRIIPLFGTAHYA), CDRH3 containing sequence number 8 (ARLKGELKDAFDI), CDRL1 containing sequence number 9 (RASQSISSYLN), CDRL2 containing sequence number 5 (AASSLQS), and CDRL3 containing sequence number 10 (QQSYSTPLT), (kk) CDRH1 containing sequence number 23 (GTFSSYAIS), CDRH2 containing sequence number 27 (GRINPILGTANYA), and CDRH3 containing sequence number 28 (ARLKGELKDAFSI), (ll) CDRH1 containing sequence number 23 (GTFSSYAIS), CDRH2 containing sequence number 27 (GRINPILGTANYA), CDRH3 containing sequence number 28 (ARLKGELKDAFSI), CDRL1 containing sequence number 9 (RASQSISSYLN), CDRL2 containing sequence number 5 (AASSLQS), and CDRL3 containing sequence number 10 (QQSYSTPLT), CDRH1 containing (mm) Sequence ID 23 (GTFSSYAIS), CDRH2 containing Sequence ID 11 (GGIIPVFGTATYA), and CDRH3 containing Sequence ID 30 (ARLKGELKCAFDI), (nn) CDRH1 containing sequence number 23 (GTFSSYAIS), CDRH2 containing sequence number 11 (GGIIPVFGTATYA), CDRL1 containing sequence number 9 (RASQSISSYLN) and sequence number 30 (ARLKGELKCAFDI), CDRL2 containing sequence number 5 (AASSLQS), and CDRL3 containing sequence number 10 (QQSYSTPLT), (oo) CDRH1 containing sequence number 36 (GPFRSHAVS), CDRH2 containing sequence number 11 (GGIIPVFGTATYA), and CDRH3 containing sequence number 37 (ARLKSELKDAFDI), or (pp) CDRH1 containing sequence number 36 (GPFRSHAVS), CDRH2 containing sequence number 11 (GGIIPVFGTATYA), CDRH3 containing sequence number 37 (ARLKSELKDAFDI), CDRL1 containing sequence number 9 (RASQSISSYLN), CDRL2 containing sequence number 5 (AASSLQS), and CDRL3 containing sequence number 10 (QQSYSTPLT) Includes.

[0046] In some such embodiments, the second antigen-binding unit is bound to PD-L1 and includes a heavy chain variable region having an amino acid sequence that is 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 having an amino acid sequence that is at least 90% identical to any one of SEQ ID NOs. 59, 60, 61, 62, or 63.

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

[0048] In some embodiments, the second antigen-binding unit is bound to PD-L1. (a) At least 90% identical amino acid sequence to SEQ ID NO: 59, (b) At least 90% identical amino acid sequence to SEQ ID NO: 60, (c) At least 90% identical amino acid sequence to SEQ ID NO: 61, (d) an amino acid sequence that is at least 90% identical to SEQ ID NO: 62, or (e) Amino acid sequence identical to at least 90% of SEQ ID NO: 63 Includes a light chain variable region.

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

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

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

[0052] In some embodiments, the construct does not contain an Fc domain. In some embodiments, the first arm or the second arm, or both, contains a heavy chain containing one or more immunoglobulin Fc modifications. In some embodiments, the immunoglobulin Fc domain of the heavy chain contains one or more amino acid mutations that promote heterodimerization of the first and second arms. In some embodiments, the mutations are located 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 contains one or more immunoglobulin constant region modifications. In some embodiments, the immunoglobulin constant region contains one or more amino acid mutations that promote heterodimerization of the antibody. In some embodiments, one or more mutations are located in the light chain constant region of one arm, and one or more mutations are located in the heavy chain constant region of another arm. In some embodiments, the bispecific antibody is in a format selected from the group consisting of bispecific IgG, bispecific antibody fragments, bispecific fusion proteins, appended IgG, and bispecific antibody conjugates. In some embodiments, the Fc region has reduced effector function. In some embodiments, the Fc region extends the half-life of the structure.

[0053] In some embodiments, the construct includes 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 includes 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 includes 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 includes 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 includes 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 includes 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 multispecific antigen-binding construct disclosed herein is nonglycosylated. In some embodiments, the multispecific antigen-binding construct can bind to human PD-1. In some embodiments, the multispecific antigen-binding construct can bind to mouse PD-1. In some embodiments, the multispecific antigen-binding construct can bind to cynomolgus monkey PD-1. In some embodiments, the multispecific antigen-binding construct can bind to human, mouse, and cynomolgus monkey PD-1 with similar affinity.

[0055] In some embodiments, any multispecific antigen-binding construct disclosed herein can reduce PD-1 levels on cells. In some embodiments, multispecific antigen-binding constructs can induce PD-1 degradation. In some embodiments, multispecific antigen-binding constructs can reduce PD-1 expression. In some embodiments, multispecific antigen-binding constructs can reduce PD-1 cell surface expression. In some embodiments, multispecific antigen-binding constructs can reduce PD-1 cell surface expression by inducing shedding of PD-1 from the cell surface. In some embodiments, multispecific antigen-binding constructs can bind to both PD-1 and PD-L1, reducing PD-1 levels on cells. In some embodiments, multispecific antigen-binding constructs can bind to both PD-1 and PD-L1, inducing PD-1 degradation. In some embodiments, multispecific antigen-binding constructs can bind to both PD-1 and PD-L1, reducing PD-1 expression. In some embodiments, multispecific antigen-binding constructs can induce shedding of PD-1 from immune cells. In some embodiments, the multispecific antigen-binding construct can bind to both PD-1 and PD-L1, inducing PD-1 shedding from immune cells. In some embodiments, the multispecific antigen-binding construct can sequester PD-L1 so that PD-L1 cannot bind to CD80. In some embodiments, the multispecific antigen-binding construct can sequester PD-L1 so that PD-L1 cannot bind to CD80, allowing CD80 to freely bind to CD28. In some embodiments, the cells are immune cells, e.g., T cells. In some embodiments, the immune cells (e.g., T cells) are tumor-infiltrating lymphocytes (TILs). In some embodiments, engagement of the multispecific antigen-binding molecules described herein to PD-1 expressed by immune cells in the tumor microenvironment results in downregulation of PD-1 by the immune cells. In some embodiments, the immune cells are T cells. In some embodiments, the immune cells (e.g., T cells) are tumor-infiltrating lymphocytes (TILs).

[0056] In some embodiments, any multispecific antigen-binding construct disclosed herein can induce interferon-gamma levels (e.g., interferon-gamma levels as measured in a Staphylococcus aureus enterotoxin A ("SEA") assay) that are at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% higher compared to 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, a multispecific antigen-binding construct can induce interleukin-2 levels (e.g., interleukin-2 levels as measured in a SEA assay) that are at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% higher compared to 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).

[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% higher killing of tumor cells (e.g., leukemia cells, lymphoma cells, melanoma cells, or breast cancer cells) compared to a reference antigen-binding construct (e.g., pembrolizumab or atezolizumab) or a reference combination of antigen-binding constructs (e.g., a composition comprising the PD-1 and PD-L1 arms of the 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, multispecific antigen-binding constructs can extend the survival of subjects 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 subjects 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, multispecific antigen-binding constructs can induce PD-1 shedding from immune cells at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 100%, 200%, 300%, 400%, or 500% higher 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).In some embodiments, the multispecific antigen-binding construct is compared to 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 the multispecific antigen-binding construct, wherein the PD-1 and PD-L1 arms in the composition are not conjugated to each other), (for example) For example, by causing PD-1 shedding from the cell surface and / or inducing PD-1 degradation and / or reducing PD-1 expression, 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%.

[0059] In some embodiments, engagement of the multispecific antigen-binding molecules described herein to PD-1 expressed by cells results in downregulation and / or loss of cell surface expression of PD-1 by the cells. Such downregulation or loss of cell surface expression can be partly attributable to, for example, shedding of extracellular PD-1 from the surface of immune cells. In some embodiments, the cells are immune cells, e.g., T cells. In some embodiments, the immune cells (e.g., T cells) are tumor-infiltrating lymphocytes (TILs). In some embodiments, engagement of the multispecific antigen-binding molecules described herein to PD-1 expressed by immune cells in the tumor microenvironment results in downregulation of PD-1 by the immune cells. In some embodiments, the immune cells are T cells. In some embodiments, the immune cells (e.g., T cells) are tumor-infiltrating lymphocytes (TILs).

[0060] In some embodiments, the Disclosure provides a method for treating a proliferative disorder in a subject requiring such treatment, comprising the step of administering to the subject a therapeutically effective amount of any multispecific antigen-binding construct 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 cancers, neurological cancers, melanoma, breast cancer, lung cancer, head and neck cancers, gastrointestinal cancers, liver cancers, pancreatic cancers, genitourinary cancers, bone cancers, and vascular cancers. In some embodiments, the Disclosure provides a method for enhancing an immune response in a subject requiring such treatment, comprising the step of administering to the subject a therapeutically effective amount of any multispecific antigen-binding construct 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 the administration of a multispecific antigen-binding construct compared to a cocktail containing an agent that binds to either PD-1 or a PD-1 ligand, or an agent that binds to both PD-1 and a PD-1 ligand. In some embodiments, T cell function is one or more of the following: increased IFNγ production from T cells, enhanced T cell survival, increased T cell proliferation, or rescue from exhausted T cell phenotype. In some embodiments, the enhanced T cell function is greater with the administration of a multispecific antigen-binding construct compared to a cocktail containing an agent that binds to either PD-1 or a PD-1 ligand, or an agent that binds to both PD-1 and a PD-1 ligand. In some embodiments, the multispecific antigen-binding construct is administered subcutaneously, intravenously, intradermally, intraperitoneally, or 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 the 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 present disclosure provides an anti-PD1 antibody or an antigen-binding fragment thereof comprising a heavy chain variable region including (a)(i) 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) CDRH2 comprising SEQ ID NO: 71 (SAISNSGTYTYYA), and (iii) CDRH3 comprising SEQ ID NO: 72 (ARGLDFIVGX5TGNDY, where X5=A, Y, or R), and (b)(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). In some embodiments, the anti-PD-1 antibody or antigen-binding fragment includes a heavy chain variable region comprising (a) a CDRH1 comprising any one amino acid sequence 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 NOs. 71, and (iii) a CDRH3 comprising any one amino acid sequence of SEQ ID NOs. 74, 75, or 85, and (b) a light chain variable region comprising (i) a CDRL1 comprising SEQ ID NOs. 9 (RASQSISSYLN), (ii) a CDRL2 comprising SEQ ID NOs. 5 (AASSLQS), and (iii) a CDRL3 comprising SEQ ID NOs. 10 (QQSYSTPLT). In some embodiments, the heavy chain variable region comprises an amino acid sequence that is at least 85% identical to any one amino acid sequence 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 contains 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 present disclosure provides an anti-PD-L1 antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region comprising: a. a heavy chain variable region comprising: (i) CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN); (ii) 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) CDRH3 comprising SEQ ID NO: 3 (ARLKX1ELKDAFDI, where X1 is G, F, or N); and b. a light chain variable region comprising: (i) CDRL1 comprising SEQ ID NO: 4 (RASQX1ISSYLN, where X1 is S, W, or Q); (ii) CDRL2 comprising SEQ ID NO: 5 (AASSLQS); and (iii) 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 heavy chain variable region including (a) a CDRH1 containing any one amino acid sequence of SEQ ID NO: 1, 14, 23, 36, or 122, (ii) a CDRH2 containing any one amino acid sequence of SEQ ID NO: 11, 15, 16, 21, 24, 26, 27, 29, 31, 33, or 34, and (iii) a CDRH3 containing any one amino acid sequence of SEQ ID NO: 8, 17, 18, 19, 20, 22, 25, 28, 30, 32, or 37, and (b) a light chain variable region including a CDRL1 containing any one amino acid sequence of SEQ ID NO: 9, 12, or 13, (ii) a CDRL2 containing the amino acid sequence of SEQ ID NO: 5, and (iii) a CDRL3 containing any one amino acid sequence of SEQ ID NO: 10, 38, or 39. In some embodiments, the heavy chain variable region contains at least 85% identical amino acid sequences to any one of the amino acid sequences 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 contains at least 85% identical amino acid sequences to any one of the amino acid sequences of SEQ ID NOs: 59, 60, 61, 62, or 63.

[0063] In some embodiments, the Disclosure provides a method for treating a proliferative disorder in a subject requiring such treatment, comprising the step of administering to the subject a therapeutically effective amount of any antibody or antigen-binding construct 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 cancers, neurological cancers, melanoma, breast cancer, lung cancer, head and neck cancers, gastrointestinal cancers, liver cancers, pancreatic cancers, genitourinary cancers, bone cancers, and vascular cancers.

[0064] In some embodiments, the Disclosure provides a method for enhancing an immune response in a subject in need thereof, comprising the step of administering to the subject a therapeutically effective amount of any antibody or antigen-binding construct disclosed herein, thereby enhancing an immune response in the subject.

[0065] In some embodiments, any method described herein may involve a step of detecting the presence or absence of PD-1 expression in one or more cells (or a population of cells such as TILs) before and / or after contact with the multispecific antigen-binding molecule described herein. For example, any method described herein may involve a step of detecting the presence or absence of PD-1 expression in one or more cells (or a population of cells such as TILs) before and / or after administration of the 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 dose of a molecule for use in the treatment of a given patient or patient population. Methods for detecting the presence, reduction, and / or absence of PD-1 expression are known to those skilled in the art, and examples include flow cytometry, Western blotting, ELISA, etc.

[0066] In another embodiment, the present disclosure features a method comprising the step of measuring the level of PD-1 expression in one or more cells (or a population of cells such as TILs) before and / or after contact with a multispecific antigen-binding molecule described herein. In some embodiments, the method comprises the step of measuring the level of PD-1 expression in one or more cells (or a population 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 embodiment, the Disclosure features a method comprising the step of measuring the level of PD-1 expression in one or more cells (or a population of cells such as TILs) before and / or after contact with the multispecific antigen-binding molecules described herein. For example, any method described herein may involve the step of measuring the level of PD-1 expression in one or more cells (or a population of cells such as TILs) before and / or after administration of the multispecific antigen-binding molecules described herein to a subject (e.g., a cancer patient). Such methods are useful, among others, for detecting or measuring the biological effect of the molecules described herein on a subject. In some embodiments, a reduction in the level of PD-1 expression in immune cells (e.g., TILs isolated from a patient) after treatment with the multispecific antigen-binding molecules described herein indicates that the molecule had a biological effect on the subject. In some embodiments, a reduction in the level of PD-1 expression in immune cells (e.g., TILs isolated from a patient) after treatment with the multispecific antigen-binding molecules described herein indicates that the patient should be given one or more doses of the molecule or otherwise continue therapy containing the molecule.

[0068] In yet another embodiment, the Disclosure features a method for determining whether a biological effect has occurred in a patient or population of patients treated with the multispecific antigen-binding molecules described herein. The method includes detecting the presence or amount of PD-1 expression in one or more test immune cells (e.g., effector immune cells, e.g., effector immune cells in the tumor microenvironment) obtained from one or more patients administered the multispecific antigen-binding molecules described herein, wherein a reduced level of PD-1 expression (e.g., cell surface expression) in one or more immune cells compared to a control expression level (e.g., the level of PD-1 expression in immune cells of the same histological type as the test immune cells before 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 before detection. In some embodiments, the method includes administering the multispecific antigen-binding molecule to the patient or population of patients in which the appearance of a biological effect has been determined. In some embodiments, the control PD-1 expression level is approximately the median or mean expression level of PD-1 in 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 approximately the median or mean expression level of PD-1 in immune cells of the same histological type in a population of subjects not administered with the multispecific antigen-binding molecules and / or agents that bind to and / or inhibit PD-1 as described herein.

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

[0070] In yet another aspect, the present disclosure is characterized by a method for inhibiting the binding between PD-L1 and CD80 in a subject (e.g., a cancer patient), comprising the step of administering a multispecific antigen-binding molecule described herein to the subject, thereby inhibiting the binding between PD-L1 and CD80 in the subject.

[0071] The patent or application documents shall contain at least one color drawing. A copy of the published patent or patent application containing the color drawing shall be provided by the Office upon request, with payment of the required fees. [Brief explanation of the drawing]

[0072] [Figure 1] This 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. The results show the concentration of IFNγ as pg / mL at the final concentration of the antibody tested as indicated. [Figure 2]This 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 IFNγ concentration as pg / mL at the final concentrations of the tested antibody as indicated. [Figure 3] The diagram and amino acid sequences for the pembrolizumab × atezolizumab dual-specific product are shown. Separate sequences are given for pembrolizumab_aglyco-IgG1-(G4S) quadruplex (H chain; SEQ ID NO: 104) and light chain (L chain; SEQ ID NO: 105), and for atezolizumab_FabH-(G4S) quadruplex (H chain; SEQ ID NO: 106) and light chain (L chain; SEQ ID NO: 107). [Figure 4] The diagram and amino acid sequences for the nivolumab × atezolizumab dual specific product are shown. The sequences for nivolumab aglyco-IgG1-(G4S) quadruplex (H chain; SEQ ID NO: 108) and light chain (L chain; SEQ ID NO: 109), and atezolizumab FabH-(G4S) quadruplex (H chain; SEQ ID NO: 106) and light chain (L chain; SEQ ID NO: 107) are given separately. [Figure 5] The diagram and amino acid sequence of the 949 Agricole-IgG1 × Atezolizumab dual specific product are shown. Separate sequences are given for 949 Agricole-IgG1-(G4S) quadruplex (H chain; SEQ ID NO: 110) and light chain (L chain; SEQ ID NO: 111), and for atezolizumab_FabH-(G4S) quadruplex (H chain; SEQ ID NO: 106) and light chain (L chain; SEQ ID NO: 107). [Figure 6] The diagram and amino acid sequences for the atezolizumab × nivolumab dual specific product are shown. Separate sequences are given for atezolizumab_aglyco-IgG1-(G4S) quadruplex (H chain; SEQ ID NO: 112) and light chain (L chain; SEQ ID NO: 107), and for nivolumab_HC Fab-(G4S) quadruplex (H chain; SEQ ID NO: 113) and light chain (L chain; SEQ ID NO: 109). [Figure 7]The right panel shows IFN-γ release at pg / mL as a function of antibodies tested at various concentrations in a mixed lymphocyte reaction (MLR) assay. These results indicate that the bispecific antibody PD-1×PD-L1 (pembrolizumab×atezolizumab) or (nivolumab×atezolizumab) in multispecific format induces a greater IFN-γ response at femtomole concentrations compared to cocktails of pembrolizumab and atezolizumab or nivolumab and atezolizumab. The left panel shows size exclusion chromatography of the bispecific format against mAb precursors. [Figure 8] An example workflow for identifying multispecific (e.g., bispecific) antibodies demonstrating synergistic effects is presented. The process involves unbiased screening of checkpoint blocker combinations in a mixed lymphocyte reaction (MLR) assay measuring IFN-γ release at various concentrations of pg / mL. In the second step of the illustrated workflow, common light chain bispecifics are generated and their efficacy is further tested, depicting various bispecificity formats. The identified bispecificity formats outperformed known PD-1 blockers in a T-cell activation assay. [Figure 9A] The dual-specific agent 3 induced higher killing of K562-CD32-PDL1 target cells by CD3 / CD28 expanding T cells (Figure 9A) and increased IFNγ production (Figure 9B) in antigen-nonspecific T cell assays compared to both isotype control antibodies and Keytruda. This increased killing by the dual-specific agent 3 was observed even at a low concentration of 0.01 nM. [Figure 9B] The dual-specific agent 3 induced higher killing of K562-CD32-PDL1 target cells by CD3 / CD28 expanding T cells (Figure 9A) and increased IFNγ production (Figure 9B) in antigen-nonspecific T cell assays compared to both isotype control antibodies and Keytruda. This increased killing by the dual-specific agent 3 was observed even at a low concentration of 0.01 nM. [Figure 10A]The effect of dual-specific compound 3 on tumor cell killing is demonstrated. Figure 10A shows that, compared to both Keytruda and the combination of mAb1 and mAb28, dual-specific compound 3 increased the specific killing of K562-A2-CMV-PDL1 tumor antigen target cells by CMV-specific T cells at low concentrations of 0.001–0.01 nM, indicating that dual-specific compound 3 can mediate antigen-specific killing of target cells at lower doses. [Figure 10B] The effect of dual-specific agent 3 on tumor cell killing is demonstrated. Figure 10B shows that dual-specific agent 3 was more effective than either Keytruda or the combination of mAb1 and mAb28 in the specific killing of Raji-A2-CMV-PDL1 tumor antigen target cells by CMV-specific T cells at a low concentration of 0.001 nM, again indicating that dual-specific agent 3 can mediate antigen-specific killing of target cells at lower doses. [Figure 11] Dual-specific compound 3 induced more IL-2 than Keytruda at all tested doses in the SEA stimulation assay. Importantly, dual-specific compound 3 induced increased IL-2 production at lower antibody concentrations compared to Keytruda and both mAb1 and mAb28. [Figure 12A] We demonstrate that dual-specific compound 3 possesses a unique ability to induce internal translocation of PD-1 and subsequent degradation or loss of expression, and that this property depends on the engagement of both PD-1 and PD-L1 targeting the molecular arms. Figures 12A–12B demonstrate that only dual-specific compound 3 results in internal translocation of PD-1 and subsequent degradation or loss of expression when compared to isotype controls, Keytruda, mAb1 and mAb28, atezolizumab, or atezolizumab and Keytruda. [Figure 12B]We demonstrate that dual-specific compound 3 possesses a unique ability to induce internal translocation of PD-1 and subsequent degradation or loss of expression, and that this property depends on the engagement of both PD-1 and PD-L1 targeting the molecular arms. Figures 12A–12B demonstrate that only dual-specific compound 3 results in internal translocation of PD-1 and subsequent degradation or loss of expression when compared to isotype controls, Keytruda, mAb1 and mAb28, atezolizumab, or atezolizumab and Keytruda. [Figure 12C] We demonstrate that dual-specific compound 3 possesses a unique ability to induce internal translocation of PD-1 and subsequent degradation or loss of expression, and that this property depends on the engagement of both PD-1 and PD-L1 targeting the molecular arms. Additionally, as shown in Figure 12C, when anti-PD-L1 antibody, mAb1, was added to the well at 50 nM five minutes prior to adding dual-specific compound 3, the ability of dual-specific compound 3 to promote internal translocation of PD-1 was lost. This suggests that both arms of dual-specific compound 3 should be engaged to promote loss of expression and / or internal translocation and / or degradation of PD-1. [Figure 12D] We demonstrate that dual-specific compound 3 possesses a unique ability to induce internal translocation of PD-1 and subsequent degradation or loss of expression, and that this property depends on the engagement of both PD-1 and PD-L1 targeting the molecular arms. Figure 12D shows that treatment with dual-specific compound 3 increases the amount of PD-1 in the supernatant when both binding arms of the dual-specific compound are simultaneously engaged. This effect is lost when the PD-L1 targeting arm is blocked by mAb1. This suggests that dual-specific compound 3 increases the shedding of PD-1 into the supernatant. [Figure 12E]We demonstrate that dual-specific compound 3 possesses a unique ability to induce internal translocation of PD-1 and subsequent degradation or loss of expression, and that this property depends on the engagement of both PD-1 and PD-L1 targeting the molecular arms. Figure 12E demonstrates that the valence of the binding arm affects the degree of PD-1 expression loss. We constructed dual-specific compound 5 having 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 dual-specific compound 3, which has a divalent arm that binds to PD-L1 and a divalent arm that binds to PD-1, dual-specific compound 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 at higher doses of bivalent 5 (divalent) compared to bivalent 3 (tetravalent), suggesting that the increased valency of bivalent 3 is responsible for this difference. [Figure 12F] The dual specific substance 3 possesses a unique ability to induce internal translocation of PD-1 and subsequent degradation or loss of expression, and demonstrates that this property depends on the engagement of both PD-1 and PD-L1 targeting the molecular arms. Figure 12F demonstrates that pretreatment with bacimast, a broad-spectrum inhibitor of multiple MMPs and ADAMs, which are schidases or proteases responsible for cleaving proteins from the cell plasma membrane, significantly reduces 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] We demonstrate that the bispecific compound 3 possesses a unique ability to induce internal translocation of PD-1 and subsequent degradation or loss of expression, and that this property depends on the engagement of both PD-1 and PD-L1 targeting the molecular arms. Figure 12G suggests that the bispecific compound 3 promotes the loss of cell surface PD-1 expression primarily when it binds to PD-1 and PD-L1 in trans configuration, i.e., expressed by different cells. [Figure 13A]The in vivo results using dual-specific material 3 are shown. Figure 13A illustrates the experimental protocol. [Figure 13B] The in vivo results using dual-specific agent 3 are shown. Figure 13B demonstrates that both the dual-specific agent 3 group and the combined mAb1 and mAb28 group had a significant delay in tumor growth compared to both the isotype group and the Keytruda group. Additionally, at day 24, there was a significant difference between the dual-specific agent 3 group and the group treated with the combination of mAb1 and mAb28, with dual-specific agent 3 causing a greater delay in tumor growth compared to the combination. The T-cell-free group had more aggressively growing tumors than any group containing T cells. In this model, Keytruda did not provide any benefit in delaying KACP tumor growth compared to the isotype control. [Figure 14A] The in vivo results using dual-specific material 3 in a K562-A2-CMV-PD-L1 tumor mouse model are shown. Figures 14A and 14B are graphs illustrating that each of the different treatment groups resulted in a delay in mean tumor growth compared to untreated mice, but treatment with T cells and dual-specific material 3 resulted in the greatest delay in mean tumor growth over time. The T-cell-free groups had tumors that grew more rapidly than any group containing T cells. In this model, Keytruda did not provide any benefit in delaying K562-A2-CMV-PD-L1 tumor growth compared to isotype controls. [Figure 14B]The in vivo results using dual-specific material 3 in a K562-A2-CMV-PD-L1 tumor mouse model are shown. Figures 14A and 14B are graphs illustrating that each of the different treatment groups resulted in a delay in mean tumor growth compared to untreated mice, but treatment with T cells and dual-specific material 3 resulted in the greatest delay in mean tumor growth over time. The T-cell-free groups had tumors that grew more rapidly than any group containing T cells. In this model, Keytruda did not provide any benefit in delaying K562-A2-CMV-PD-L1 tumor growth compared to isotype controls. [Figure 15A] The in vivo results using dual-specific material 3 in several syngeneic tumor models are illustrated. Figure 15A is a graph showing that treatment of the EMT-6 syngeneic tumor model with dual-specific material 3 resulted in a greater delay in tumor growth compared to the control treatment. [Figure 15B] The in vivo results using dual-specific material 3 in several syngeneic tumor models are illustrated. Figure 15B is a graph showing that treatment of the MB49 syngeneic tumor model with dual-specific material 3 resulted in a greater delay in tumor growth compared to the control treatment. [Figure 16A] Figure 16A shows in vivo results using dual-specific material 1 in the MC38-hPD-L1 model in humanized PD-1 / PD-L1 transgenic mice. Both treatment with Keytruda and dual-specific material 1 effectively controlled tumor growth in MC38-hPD-L1 tumor mice compared to control-treated mice. [Figure 16B] Figure 16B shows in vivo results using dual-specific material 1 in the MC38-hPD-L1 model in humanized PD-1 / PD-L1 transgenic mice. The survival graph illustrates that dual-specific material 1 increased the survival of MC38-PD-L1 tumor mice compared to control-treated mice. [Figure 17A]The in vivo results using dual-specific agent 3 in the B16F10-hPD-L1 model are illustrated. Figure 17A is a series of graphs showing the effects of different treatments on tumor growth measured at the 15-day cutoff in B16F10-HuPD-L1 mice. Different groups of mice (n=8) were treated with dual-specific agent 3, Keytruda, avelumab, a combination of Keytruda and avelumab, or an isotype control antibody. Figure 17A shows individual tumor volume traces for each group. Metastasis was identified in several mice that died before the tumor sizing cutoff. [Figure 17B] The in vivo results using dual-specific material 3 in the B16F10-hPD-L1 model are illustrated. Figure 17B shows the difference in mean tumor volume between treatment groups and demonstrates that, up to 15 days after tumor cell inoculation, dual-specific material 3 treatment significantly delayed mean tumor growth for a longer period than any other treatment tested in B16F10-hPD-L1 mice. ****, P<0.0001;**, P<0.01, *, P<0.05, two-way ANOVA and Tukey's multiple comparison test. Figure 17B is a graph comparing the effects of different treatments on tumor volume measured at the 15-day cutoff in B16F10-HuPD-L1 mice. Metastasis was identified in several mice that died before the tumor sizing cutoff. As shown in Figure 17B, dual-specific material 3 treatment significantly delayed mean tumor growth for a longer period than any other treatment tested in B16F10-hPD-L1 mice. [Figure 17C] Figure 17C shows the in vivo results using dual specific agent 3 in the B16F10-hPD-L1 model. The survival graph illustrates that treatment with dual specific agent 3 increased the survival of B16F10-hPD-L1 tumor mice compared to survival with any of the other treatments tested. [Figure 17D]Figure 17D illustrates the in vivo results using dual-specific compound 3 in the B16F10-hPD-L1 model. Up to day 21 after tumor cell inoculation, treatment with dual-specific compound 3 significantly and effectively delayed mean tumor growth in B16F10-hPD-L1 mice compared to treatment with Keytruda. [Figure 17E] Figure 17E illustrates the in vivo results using dual-specific material 3 in the B16F10-hPD-L1 model. The survival graph shows that treatment with dual-specific material 3 increased the survival of B16F10-HuPD-L1 tumor mice compared to survival with any of the other treatments tested. [Figure 17F] The in vivo results using dual specific agent 3 in the B16F10-hPD-L1 model are illustrated. Figure 17F provides a table showing the number of tumor-free mice for each of the different treatment groups. The group treated with dual specific agent 3 had 3 tumor-free mice, while the combination of keytruda and avelumab had 1 tumor-free mouse. [Figure 18A] Bi-specific substance 3 exhibits drug-like properties (DLP) similar to those of a well-behaving monoclonal antibody, demonstrating its ability to maintain parental PD-1 and PD-L1 binding. Figure 18A shows that bi-specific substance 3 exhibits binding similar to that of parental clone mAb28 to human PD-1-expressing CHO cells (top) and similar to that of parental clone mAb1 to human PD-L1-expressing CHO cells (bottom). [Figure 18B] Bi-specific substance 3 exhibits drug-like properties (DLP) similar to those of a well-behaving monoclonal antibody, demonstrating its ability to maintain parental PD-1 and PD-L1 binding. Figure 18B shows that bi-specific substance 3 exhibits binding similar to that of parental clone mAb28 to cynomolgus monkey PD-1-expressing CHO cells (top) and similar to that of parental clone mAb1 to cynomolgus monkey PD-L1-expressing CHO cells (bottom). [Figure 18C]Bi-specific substance 3 exhibits drug-like properties (DLP) similar to those of a well-behaving monoclonal antibody, demonstrating its ability to maintain parental PD-1 and PD-L1 binding. Figure 18C shows that bi-specific substance 3 exhibits binding similar to that of parental clone mAb28 to mouse PD-1-expressing CHO cells (top) and similar to that of parental clone mAb1 to mouse PD-L1-expressing CHO cells (bottom). [Figure 18D] Bi-specific product 3 exhibits drug-like properties (DLP) similar to those of a well-behaving monoclonal antibody, demonstrating its maintenance of parental PD-1 and PD-L1 binding. Figure 18D shows the size exclusion chromatography trace of bi-specific product 3 after protein A chromatography (top), demonstrating a single peak with a purity of over 98%, and the differential scanning fluorescence (DSF) trace of bi-specific product 3 (bottom), demonstrating the high thermal stability of the molecule. [Modes for carrying out the invention]

[0073] Unless otherwise defined, all technical terms, notations, and other scientific terms used herein are intended to have meanings that are generally understood by those skilled in the art to which this disclosure relates. In some cases, terms that have generally understood meanings are defined herein for clarity and / or immediate reference, and the inclusion of such definitions herein should not necessarily be construed as representing a difference from the generally understood meaning in the art. The techniques and procedures described or referenced herein are generally well understood and, by those skilled in the art, refer to prior methodologies, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual 2nd ed. (1989), Cold Spring Harbor Laboratory Press. It is commonly performed using widely used molecular cloning methodologies, such as those described in Laboratory Press (located in New York, USA). Where appropriate, procedures involving the use of commercially available kits and reagents are generally carried out according to the manufacturer's defined protocols and / or parameters unless otherwise noted.

[0074] As used herein, the singular forms "a," "an," and "the" refer to multiple objects unless the context explicitly indicates otherwise. In the specification and claims, the term “about” is used, for example, in the description of embodiments of this disclosure, to modify the amount, concentration, volume, process temperature, process time, yield, flow rate, pressure, and similar values ​​of the raw material components in a composition, as well as the ranges thereof. The term “about” refers to variations in quantity that may occur, for example, through typical measurement and handling procedures used for the preparation of a compound, composition, concentrate, or formulation; through unintended errors in these procedures; through differences in the production, source, or purity of the starting materials or raw material components used to carry out the method, and through consideration of similar approximations. The term “about” also includes different amounts resulting from aging of a formulation having a specific initial concentration or mixture, and different amounts resulting from mixing or processing of a formulation having a specific initial concentration or mixture. Where modified by the term “about,” the claims attached herein include equal amounts of these quantities. Where there is use of the term that is not clear to a person skilled in the art considering the context in which it is used, “about” means plus or minus 10% of the specific value.

[0075] Regarding the binding of antigen-binding proteins / regions / arms to target molecules, terms such as "specific binding," "specific," "selective binding," and "selective" mean binding that is measurably different from nonspecific or nonselective interactions when relating to a specific antigen (e.g., a polypeptide target) or an epitope on a specific antigen. Specific binding can be measured, for example, by determining the binding of a molecule by comparing it to the binding of a control molecule. Specific binding can also be determined by competition with a target-like control molecule, such as an excess of unlabeled targets. In this case, specific binding is indicated when the binding of a labeled target to a probe is competitively inhibited by an excess of unlabeled targets.

[0076] The term "epitope" refers to an antigenic component capable of specific binding to an antigen-binding protein. Epitopes frequently consist of amino acid residues and / or sugar side chains accessible to the surface and may possess specific electrical characteristics in addition to specific three-dimensional structural features. Conformational and non-conformational epitopes are distinguished in that binding to the latter is not, while binding to the former is lost in the presence of denaturing solvents. Epitopes can include amino acid residues directly involved in binding and other amino acid residues not directly involved in binding. The epitope to which an antigen-binding protein binds can be determined using known techniques for epitope determination, such as testing the binding of the antigen-binding protein to antigenic 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 those in the reference sequence, after the sequences have been aligned and gaps introduced where necessary to achieve the maximum possible sequence identity percentage. Alignment for the purpose of determining the amino acid sequence identity percentage can be achieved in various ways within the technical scope of a person skilled in the art, 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. A person skilled in the art can determine appropriate parameters for aligning the sequences, including any algorithm required to achieve the maximum possible alignment over the entire length of the sequences being compared.

[0078] The term "may" 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 a suitable context (as understood by those skilled in the art), but it is not required that it be associated with that property at any specific point in time. For example, any multispecific antigen-binding construct disclosed herein may bind to PD-1 and / or PD-L1 when administered to cells expressing PD-1 and / or PD-L1, but the construct is not expected to bind to PD-1 and / or PD-L1 when in a composition lacking the PD-1 or PD-L1 protein.

[0079] A “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. Tables of conservative substitutions that provide similar amino acids are well known in the art. Polypeptide sequences having such substitutions are known as “conservatively modified variants” or “variants.” Such conservatively modified variants are, additionally, polymorphic variants, interspecific homologs, and alleles. Some examples of conservative substitutions can be found, for example, in Creighton, Proteins: Structures and Molecular Properties 2nd ed. (1993) by WH Freeman & Co. [New York, New York, USA].

[0080] Polypeptides disclosed herein may include amino acid sequences that do not exist in nature. Such variants will necessarily have less than 100% sequence identity or similarity to the starting molecule. In certain embodiments, the variant has, for example, an amino acid sequence with 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% amino acid sequence identity or similarity to the amino acid sequence of the starting (e.g., naturally occurring or wild-type) polypeptide over the length of the variant molecule.

[0081] As used herein, "antibody" may refer to an intact antibody (e.g., intact immunoglobulin) and an antibody moiety, such as an antigen-binding moiety. The antigen-binding moiety includes at least one antigen-binding domain. One example of an antigen-binding domain is V H -V LIt is an antigen-binding domain formed by a dimer. Antibodies and antigen-binding portions can be described by the antigens to which they specifically bind. For example, a PD-L1 antibody, or an anti-PD-L1 antibody, is an antibody that specifically binds to PD-L1.

[0082] V H and V L regions can be further divided into regions of hypervariability (also called hypervariable regions (HVRs) or complementarity-determining regions (CDRs)) with more conserved regions interspersed. The more conserved regions are called framework regions (FRs). Each V H and V L generally contains three CDRs and four FRs arranged in the following order (from the N-terminus to the C-terminus): FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The CDRs are involved in antigen binding and confer antigen specificity and binding affinity to the antibody. (See Kabat et al. (1991) Sequences of Proteins of Immunological Interest 5th ed., Public Health Service, National Institutes of Health, Bethesda, Maryland, USA.) The CDRs are involved in antigen binding and confer antigen specificity and binding affinity to the antibody. There are three CDRs in each of the variable domains of the heavy and light chains, and these are designated CDR1, CDR2, and CDR3 for each variable domain. The term "CDR set" as used herein refers to the group of three CDRs occurring in a single heavy or light chain variable domain that can bind to a target antigen. The exact boundaries of these CDRs are defined differently according to different systems. The three heavy chain CDRs can be designated CDRH1, CDRH2, and CDRH3, and the three light chain CDRs can be designated CDRL1, CDRL2, and CDRL3.

[0083] The system described by Kabat, also known as "numbered according to Kabat," "Kabat numbering," "Kabat definition," and "Kabat labeling," provides a clear residue numbering system applicable to any variable domain of an antibody, and provides precise residue boundaries defining three CDRs in each chain. (Kabat et al., Sequences of Proteins of Immunological Interest, National Institutes of Health [Bethesda, Maryland, USA] (1987) and (1991); the entire content is referenced by reference). These CDRs are called Kabat CDRs and include 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. If CDR is defined according to Kabat, the light chain FR residues are located approximately at residues 1-23 (LCFR1), 35-49 (LCFR2), 57-88 (LCFR3), and 98-107 (LCFR4), and the heavy chain FR residues are located approximately at residues 1-30 (HCFR1), 36-49 (HCFR2), 66-94 (HCFR3), and 103-113 (HCFR4) within the heavy chain. "EU index as in Kabat" refers to the residue numbering of human IgG1 EU antibodies.

[0084] Other CDR numbering systems are also used in the art (see, for example, Table A). Chothia and collaborators found that certain sub-regions within Kabat CDRs adopt nearly identical peptide skeletal conformations despite significant 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 sub-regions are designated as L1, L2, and L3 or H1, H2, and H3, where "L" and "H" designate the light chain and heavy chain regions, respectively. These CDRs are sometimes referred to as "Cotia CDRs," "Cotia numbering," or "numbered according to Cotia," and include 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 residues 30-36 (CDR1), 46-55 (CDR2), and 89-96 (CDR3) in the light chain variable domain, and residues 30-35 (CDR1), 47-58 (CDR2), and 93-101 (CDR3) in the heavy chain variable domain. MacCallum et al. al.) ((1996) J. Mol. Biol. 262(5):732-745).

[0086] The AbM-described system, also known as "AbM-based numbering" or "AbM numbering," includes residues 24-34 (CDR1), 50-56 (CDR2), and 89-97 (CDR3) in the light chain variable domain, and residues 26-35 (CDR1), 50-58 (CDR2), and 95-102 (CDR3) in the heavy chain variable domain.

[0087] The IMGT (International Immunogenetics Information System) numbering for variable regions can also be used, which is the IMGT-specific numbering for immunoglobulins, T cell receptors, and Ig-like domains, as developed by Lefranc, M.-P. This refers to the numbering of residues in immunoglobulin variable heavy or light chains according to the IMGT method, as described in “Numbering for Immunoglobulins, T Cell Receptors and Ig-like Domains,” Immunologist, 7, 132-136 (1999) (the entire text expressly incorporated herein by reference). 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 extends from 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 extends from 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 CDR described herein, when numbered according to Cotia numbering, comprises approximately residues 24-34 (CDR1), 49-56 (CDR2), and 89-97 (CDR3) in the light chain variable domain, and residues 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 may contain amino acids 49-56, when numbered according to Cotia numbering.

[0089] [Table 1]

[0090] Preferred methods and materials are described herein, but similar or equivalent methods and materials may also be used in the implementation or testing of the methods and compositions of this disclosure. All publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference.

[0091] Various aspects of the present invention are described in further detail below. Additional definitions are stated throughout this specification. While the immune system has the ability to recognize and eliminate tumor cells, tumors can use multiple strategies to evade immunity. Recent studies have shown that inhibitory immune checkpoint molecules promote cancer progression through various antitumor inhibitory mechanisms. Blocking immune checkpoints is one approach to activating or reactivating therapeutic antitumor immunity. Various ligands have been described for numerous cognitive inhibitory immune checkpoint receptors. These are reviewed, for example, in Nair & Elkord, Immunology & Cell Biology (2018), 96:21-33; and Jenkins et al., British J. of Cancer (2017), 118:9-16.

[0092] Programmed cell 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 is suggested to lack the unpaired cysteine ​​residues 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). Structurally similar to CTLA-4, PD-1 lacks the MYPPY motif crucial 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 to other CD28 family members. PD-L1 is abundant in various human cancers (Dong et al. (2002) Nat. Med. 8:787-9).

[0094] PD-1 is known as an immunosuppressive protein that negatively modulates 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 decrease in T-cell receptor-mediated proliferation, and / or immune evasion by cancer cells (Dong et al. (2003) J. Mol. Med. 81:281-7; Blank et al. (2005) Cancer Immunol. Immunother. 54:307-314; Konishi et al. (2004) Clin. Cancer Res. (10:5094-100). Immunosuppression can be reversed by inhibiting the local interaction of PD-1 with PD-L1 or PD-L2, and the effect is additive when the interaction of PD-1 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 differentiation cluster 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 allogeneic grafting, 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 that trigger 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. The 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 interactions also induce apoptosis in tumor-specific T cells, and Foxp3 + CD4 to regulatory T cells + This promotes T cell differentiation and enhances the resistance of tumor cells to cytotoxic T lymphocyte (CTL) attack, thereby allowing tumors to evade the host immune system.

[0096] This disclosure relates to compositions and methods for inhibiting tumor escape by reducing immune checkpoint suppression resulting from interactions between PD-1 and its ligands (e.g., PD-L1 and / or PD-L2). In particular, compositions comprising novel multispecific and multivalent constructs, such as bispecific and tetravalent constructs, that block the interaction between PD-1 and its ligands while promoting the interaction (bridging) of cells expressing PD-1 and its ligands (e.g., PD-1 and / or PD-L2). Such compositions of this disclosure having the ability to "block and bridge" have increased potency in vitro and in vivo, and provide superior antitumor efficacy (biological effects that may manifest by various means, including but not limited to reductions in tumor volume, reductions in the number of tumor cells, reductions in tumor cell proliferation, and / or reductions in tumor cell survival) compared to clinical checkpoint blockers, as well as compared to combinations of individual antibodies, by strongly enhancing, for example, T cell proliferation, IFNγ production and / or secretion, T cell cytolytic activity, and / or T cell rescue from functional exhaustion. Novel monoclonal anti-PD-1 antibodies and their antigen-binding fragments, as well as novel monoclonal anti-PD-L1 antibodies and their antigen-binding fragments, for use in such multispecific and multivalent constructs are also provided herein. Some of these novel monoclonal anti-PD-1 antibodies and novel monoclonal anti-PD-L1 antibodies share a common light chain, thereby enabling the creation of multispecific and multivalent constructs with similar affinity to their parent antibodies, in addition to excellent drug-like properties and producibility.

[0097] Accordingly, the Disclosure provides a multispecific antigen-binding construct comprising at least two antigen-binding arms or units, wherein the first antigen-binding arm or unit binds to PD-1 expressed by an immune cell, and the second antigen-binding arm or unit binds to one or more PD-1 ligands (e.g., PD-L1 and / or PD-L2) expressed by a second cell. In some embodiments, the multispecific antigen-binding construct blocks the interaction of PD-1 and its ligands. In some embodiments, the multispecific antigen-binding construct bridges cells expressing PD-1 and its ligands to promote the interaction and / or efficacy of immune cells expressing PD-1. In some embodiments, at least one of the antigen-binding arms is bivalent with respect to PD-1. In some embodiments, at least one of the antigen-binding arms is bivalent with respect to PD-L1. In some embodiments, at least one of the antigen-binding arms is bivalent with respect to PD-1 and at least one of the antigen-binding arms is bivalent with respect to PD-L1. In some embodiments, the multispecific antigen-binding construct includes at least two units of antigen binding that bind to PD-1. In some embodiments, the multispecific antigen-binding construct includes two units of antigen binding that bind to PD-1. In some embodiments, the multispecific antigen-binding construct includes at least two units of antigen binding that bind to a PD-1 ligand, e.g., PD-L1 or PD-L2. In some embodiments, the multispecific antigen-binding construct includes two units of antigen binding that bind to a PD-1 ligand, e.g., PD-L1 or PD-L2. In some embodiments, the multispecific antigen-binding construct includes at least four units of antigen binding, 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 includes four units of antigen binding, 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 part of the herein, the construct is a bispecific antibody.In some embodiments, the bispecific antibody is an antagonist of both PD-1 and the PD-1 ligand. In some embodiments, the construct contains a common light chain. In some embodiments, one or both of the antigen-binding arms are aptamers. In some embodiments, one or both of the antigen-binding arms are proteins other than antibodies. In some embodiments, the construct contains at least two antibodies. In some embodiments, at least one of the antigen-binding arms is a bivalent antibody specific to PD-1. In some embodiments, at least one of the antigen-binding arms is a bivalent antibody specific to PD-L1. In some embodiments, at least one of the antigen-binding arms is a bivalent antibody specific to PD-1, and at least one of the antigen-binding arms is a bivalent antibody specific to 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 the PD-1 ligand. In some embodiments, novel isolated antibodies that specifically bind to PD-L1 or PD-1 and their antigen-binding moieties are also provided herein. In some embodiments, these novel isolated antibodies that specifically bind to PD-L1 or PD-1, as well as their antigen-binding moieties, such as CDRs, variable heavy chains, and / or variable light chains, may be used in one or more arms or units of antigen binding in the multispecific antigen-binding constructs described herein.

[0098] Therefore, as described herein, the disclosed multispecific antigen-binding constructs include a bispecific, triplicate, quadruplicate, or multispecific antibody or its antigen-binding moiety. The described multispecific constructs are preferably bivalent with respect to at least one, preferably both, antigen-binding arms, i.e., a bispecific and trivalent, or bispecific and tetravalent molecule. The multispecific constructs described herein may, in various embodiments and models, include one or more antibodies and / or their antigen-binding moieties. For example, the antigen-binding arm may include a variable heavy chain and / or a variable light chain, or its complementarity-determining region (CDR), of a given antibody against PD-1 and / or a given antibody against PD-L1. Therefore, in some embodiments of any of the embodiments described herein, a first antigen-binding arm, a second antigen-binding arm, a first unit of antigen binding, a second unit of binding, or any combination thereof may include an antibody or its antigen-binding moiety. In some embodiments of any part of the herein, the first antigen-binding arm, the second antigen-binding arm, the first antigen-binding unit, the second binding unit, or any combination thereof, is an antibody or its antigen-binding portion.

[0099] A. PD-L1 antagonist In some embodiments and aspects, the Disclosure provides anti-PD-L1 antagonists. 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; that is, the anti-PD-L1 antibody or antigen-binding molecule is not part of a protein construct that binds to multiple epitopes. In some embodiments, the anti-PD-L1 antibody or antigen-binding 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-L1 and an epitope on another protein. In some embodiments, the epitope on the other protein is on PD-1.

[0100] Therefore, in some embodiments, an antibody or its antigen-binding portion that specifically binds to PD-L1 is provided herein. In some embodiments, the antibody or its antigen-binding portion that specifically binds to PD-L1 includes a heavy chain variable region comprising (a) CDRH1 comprising (i) SEQ ID NO: 1 (GTFSSYAIN), (ii) 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) CDRH3 comprising SEQ ID NO: 3 (ARLKX1ELKDAFDI, where X1 is G, F, or N), and (b) a light chain variable region comprising (i) CDRL1 comprising SEQ ID NO: 4 (RASQX1ISSYLN, where X1 is S, W, or Q), (ii) CDRL2 comprising SEQ ID NO: 5 (AASSLQS), and (iii) CDRL3 comprising SEQ ID NO: 6 (X1QSYSTPLT, where X1 is Q or F).

[0101] In these embodiments and some embodiments of all such embodiments 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 these embodiments and some embodiments of all such embodiments described herein, CDRH1 comprises SEQ ID NO: 1 (GTFSSYAIN), CDRH2 comprises SEQ ID NO: 7 (GGIIPILGAATYA), CDRH3 comprises SEQ ID NO: 8 (ARLKGELKDAFDI), CDRL1 comprises SEQ ID NO: 9 (RASQSISSYLN), CDRL2 comprises SEQ ID NO: 5 (AASSLQS), and CDRL3 comprises SEQ ID NO: 10 (QQSYSTPLT). A representative antibody having such heavy and light chain variable CDR regions is mAb1.

[0102] In these embodiments and some embodiments of all such embodiments 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 these embodiments and some embodiments of all such embodiments described herein, CDRH1 comprises SEQ ID NO: 1 (GTFSSYAIN), CDRH2 comprises SEQ ID NO: 11 (GGIIPVFGTATYA), CDRH3 comprises SEQ ID NO: 8 (ARLKGELKDAFDI), CDRL1 comprises SEQ ID NO: 12 (RASQWISSYLN), CDRL2 comprises SEQ ID NO: 5 (AASSLQS), and CDRL3 comprises SEQ ID NO: 10 (QQSYSTPLT). A typical antibody having such heavy and light chain variable CDR regions is mAb2. In these embodiments and some embodiments of all such embodiments described herein, CDRH1 comprises SEQ ID NO: 1 (GTFSSYAIN), CDRH2 comprises SEQ ID NO: 11 (GGIIPVFGTATYA), CDRH3 comprises SEQ ID NO: 8 (ARLKGELKDAFDI), CDRL1 comprises SEQ ID NO: 13 (RASQQISSYLN), CDRL2 comprises SEQ ID NO: 5 (AASSLQS), and CDRL3 comprises SEQ ID NO: 10 (QQSYSTPLT). A representative antibody having such heavy and light chain variable CDR regions is mAb3.

[0103] In these embodiments and in some embodiments of all such embodiments described herein, CDRH1 comprises SEQ ID NO: 1 (GTFSSYAIN), CDRH2 comprises SEQ ID NO: 11 (GGIIPVFGTATYA), CDRH3 comprises SEQ ID NO: 8 (ARLKGELKDAFDI), CDRL1 comprises SEQ ID NO: 9 (RASQSISSYLN), CDRL2 comprises SEQ ID NO: 5 (AASSLQS), and CDRL3 comprises SEQ ID NO: 10 (QQSYSTPLT). A representative antibody having such heavy and light chain variable CDR regions is mAb4. Another representative antibody having such heavy and light chain variable CDR regions is mAb24.

[0104] In these embodiments and some embodiments of all such embodiments 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 these embodiments and some embodiments of all such embodiments described herein, CDRH1 comprises SEQ ID NO: 14 (GTFSSYAFS), CDRH2 comprises SEQ ID NO: 11 (GGIIPVFGTATYA), CDRH3 comprises SEQ ID NO: 8 (ARLKGELKDAFDI), CDRL1 comprises SEQ ID NO: 9 (RASQSISSYLN), CDRL2 comprises SEQ ID NO: 5 (AASSLQS), and CDRL3 comprises SEQ ID NO: 10 (QQSYSTPLT). A representative antibody having such heavy and light chain variable CDR regions is mAb5.

[0105] In these embodiments and some embodiments of all such embodiments 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 these embodiments and some embodiments of all such embodiments described herein, CDRH1 comprises SEQ ID NO: 1 (GTFSSYAIN), CDRH2 comprises SEQ ID NO: 15 (GGIIPIFGIANYA), CDRH3 comprises SEQ ID NO: 8 (ARLKGELKDAFDI), CDRL1 comprises SEQ ID NO: 9 (RASQSISSYLN), CDRL2 comprises SEQ ID NO: 5 (AASSLQS), and CDRL3 comprises SEQ ID NO: 10 (QQSYSTPLT). A representative antibody having such heavy and light chain variable CDR regions is mAb6.

[0106] In these embodiments and some embodiments of all such embodiments 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 these embodiments and some embodiments of all such embodiments 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). A representative antibody having such heavy and light chain variable CDR regions is mAb7.

[0107] In these embodiments and some embodiments of all such embodiments 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 these embodiments and some embodiments of all such embodiments described herein, CDRH1 comprises SEQ ID NO: 1 (GTFSSYAIN), CDRH2 comprises SEQ ID NO: 11 (GGIIPVFGTATYA), CDRH3 comprises SEQ ID NO: 18 (ARLKFELKDAFDI), CDRL1 comprises SEQ ID NO: 9 (RASQSISSYLN), CDRL2 comprises SEQ ID NO: 5 (AASSLQS), and CDRL3 comprises SEQ ID NO: 10 (QQSYSTPLT). A representative antibody having such heavy and light chain variable CDR regions is mAb8.

[0108] In these embodiments and some embodiments of all such embodiments 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 these embodiments and some embodiments of all such embodiments described herein, CDRH1 comprises SEQ ID NO: 1 (GTFSSYAIN), CDRH2 comprises SEQ ID NO: 11 (GGIIPVFGTATYA), CDRH3 comprises SEQ ID NO: 19 (ARLKGELKDAFDE), CDRL1 comprises SEQ ID NO: 9 (RASQSISSYLN), CDRL2 comprises SEQ ID NO: 5 (AASSLQS), and CDRL3 comprises SEQ ID NO: 10 (QQSYSTPLT). A representative antibody having such heavy and light chain variable CDR regions is mAb9.

[0109] In these embodiments and some embodiments of all such embodiments 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 these embodiments and some embodiments of all such embodiments described herein, CDRH1 comprises SEQ ID NO: 1 (GTFSSYAIN), CDRH2 comprises SEQ ID NO: 11 (GGIIPVFGTATYA), CDRH3 comprises SEQ ID NO: 20 (ARLKNELKDAFDI), CDRL1 comprises SEQ ID NO: 9 (RASQSISSYLN), CDRL2 comprises SEQ ID NO: 5 (AASSLQS), and CDRL3 comprises SEQ ID NO: 10 (QQSYSTPLT). A representative antibody having such heavy and light chain variable CDR regions is mAb10.

[0110] In these embodiments and some embodiments of all such embodiments 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 these embodiments and some embodiments of all such embodiments described herein, CDRH1 comprises SEQ ID NO: 1 (GTFSSYAIN), CDRH2 comprises SEQ ID NO: 21 (GGVIPFLGTANYA), CDRH3 comprises SEQ ID NO: 22 (ARLKGILKDALDI), CDRL1 comprises SEQ ID NO: 9 (RASQSISSYLN), CDRL2 comprises SEQ ID NO: 5 (AASSLQS), and CDRL3 comprises SEQ ID NO: 10 (QQSYSTPLT). A representative antibody having such heavy and light chain variable CDR regions is mAb11.

[0111] In these embodiments and some embodiments of all such embodiments 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 these embodiments and some embodiments of all such embodiments described herein, CDRH1 comprises SEQ ID NO: 23 (GTFSSYAIS), CDRH2 comprises SEQ ID NO: 24 (GGIIPIVGIANYA), CDRH3 comprises SEQ ID NO: 8 (ARLKGELKDAFDI), CDRL1 comprises SEQ ID NO: 9 (RASQSISSYLN), CDRL2 comprises SEQ ID NO: 5 (AASSLQS), and CDRL3 comprises SEQ ID NO: 10 (QQSYSTPLT). A representative antibody having such heavy-chain and light-chain variable CDR regions is mAb12.

[0112] In these embodiments and some embodiments of all such embodiments 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 these embodiments and some embodiments of all such embodiments described herein, CDRH1 comprises SEQ ID NO: 23 (GTFSSYAIS), CDRH2 comprises SEQ ID NO: 11 (GGIIPVFGTATYA), CDRH3 comprises SEQ ID NO: 25 (ARLKGEFKDAFDI), CDRL1 comprises SEQ ID NO: 9 (RASQSISSYLN), CDRL2 comprises SEQ ID NO: 5 (AASSLQS), and CDRL3 comprises SEQ ID NO: 10 (QQSYSTPLT). A representative antibody having such heavy and light chain variable CDR regions is mAb13.

[0113] In these embodiments and some embodiments of all such embodiments 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 these embodiments and some embodiments of all such embodiments described herein, CDRH1 comprises SEQ ID NO: 23 (GTFSSYAIS), CDRH2 comprises SEQ ID NO: 26 (GRIIPLFGTAHYA), CDRH3 comprises SEQ ID NO: 8 (ARLKGELKDAFDI), CDRL1 comprises SEQ ID NO: 9 (RASQSISSYLN), CDRL2 comprises SEQ ID NO: 5 (AASSLQS), and CDRL3 comprises SEQ ID NO: 10 (QQSYSTPLT). A representative antibody having such heavy and light chain variable CDR regions is mAb14.

[0114] In these embodiments and some embodiments of all such embodiments 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 these embodiments and some embodiments of all such embodiments described herein, CDRH1 comprises SEQ ID NO: 23 (GTFSSYAIS), CDRH2 comprises SEQ ID NO: 27 (GRINPILGTANYA), CDRH3 comprises SEQ ID NO: 28 (ARLKGELKDAFSI), CDRL1 comprises SEQ ID NO: 9 (RASQSISSYLN), CDRL2 comprises SEQ ID NO: 5 (AASSLQS), and CDRL3 comprises SEQ ID NO: 10 (QQSYSTPLT). A representative antibody having such heavy and light chain variable CDR regions is mAb15.

[0115] In these embodiments and some embodiments of all such embodiments 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 these embodiments and some embodiments of all such embodiments described herein, CDRH1 comprises SEQ ID NO: 1 (GTFSSYAIN), CDRH2 comprises SEQ ID NO: 29 (GRIIPIFGTADYA), CDRH3 comprises SEQ ID NO: 8 (ARLKGELKDAFDI), CDRL1 comprises SEQ ID NO: 9 (RASQSISSYLN), CDRL2 comprises SEQ ID NO: 5 (AASSLQS), and CDRL3 comprises SEQ ID NO: 10 (QQSYSTPLT). A representative antibody having such heavy and light chain variable CDR regions is mAb16.

[0116] In these embodiments and some embodiments of all such embodiments described herein, CDRH1 includes sequence number 23 (GTFSSYAIS), CDRH2 includes sequence number 11 (GGIIPVFGTATYA), and CDRH3 includes sequence number 30 (ARLKGELKCAFDI). In these embodiments and some embodiments of all such embodiments described herein, CDRH1 includes sequence number 23 (GTFSSYAIS), CDRH2 includes sequence number 11 (GGIIPVFGTATYA), CDRH3 includes sequence number 30 (ARLKGELKCAFDI), CDRL1 includes sequence number 9 (RASQSISSYLN), CDRL2 includes sequence number 5 (AASSLQS), and CDRL3 includes sequence number 10 (QQSYSTPLT).

[0117] In these embodiments and some embodiments of all such embodiments 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 these embodiments and some embodiments of all such embodiments described herein, CDRH1 comprises SEQ ID NO: 122 (GTKSSYAIS), CDRH2 comprises SEQ ID NO: 11 (GGIIPVFGTATYA), CDRH3 comprises SEQ ID NO: 30 (ARLKGELKCAFDI), CDRL1 comprises SEQ ID NO: 9 (RASQSISSYLN), CDRL2 comprises SEQ ID NO: 5 (AASSLQS), and CDRL3 comprises SEQ ID NO: 10 (QQSYSTPLT). A representative antibody having such heavy and light chain variable CDR regions is mAb17.

[0118] In these embodiments and some embodiments of all such embodiments 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 these embodiments and some embodiments of all such embodiments described herein, CDRH1 comprises SEQ ID NO: 1 (GTFSSYAIN), CDRH2 comprises SEQ ID NO: 31 (GGIIPILGTATYA), CDRH3 comprises SEQ ID NO: 32 (ARRKGELKDAFDI), CDRL1 comprises SEQ ID NO: 9 (RASQSISSYLN), CDRL2 comprises SEQ ID NO: 5 (AASSLQS), and CDRL3 comprises SEQ ID NO: 10 (QQSYSTPLT). A representative antibody having such heavy and light chain variable CDR regions is mAb18.

[0119] In these embodiments and some embodiments of all such embodiments 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 these embodiments and some embodiments of all such embodiments described herein, CDRH1 comprises SEQ ID NO: 1 (GTFSSYAIN), CDRH2 comprises SEQ ID NO: 33 (GGIIPIVATANYA), CDRH3 comprises SEQ ID NO: 32 (ARRKGELKDAFDI), CDRL1 comprises SEQ ID NO: 9 (RASQSISSYLN), CDRL2 comprises SEQ ID NO: 5 (AASSLQS), and CDRL3 comprises SEQ ID NO: 10 (QQSYSTPLT). A representative antibody having such heavy and light chain variable CDR regions is mAb19.

[0120] In these embodiments and some embodiments of all such embodiments 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 these embodiments and some embodiments of all such embodiments described herein, CDRH1 comprises SEQ ID NO: 1 (GTFSSYAIN), CDRH2 comprises SEQ ID NO: 34 (GGIIPIFGKATYA), CDRH3 comprises SEQ ID NO: 32 (ARRKGELKDAFDI), CDRL1 comprises SEQ ID NO: 9 (RASQSISSYLN), CDRL2 comprises SEQ ID NO: 5 (AASSLQS), and CDRL3 comprises SEQ ID NO: 10 (QQSYSTPLT). A representative antibody having such heavy and light chain variable CDR regions is mAb20.

[0121] In these embodiments and some embodiments of all such embodiments 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 these embodiments and some embodiments of all such embodiments described herein, CDRH1 comprises SEQ ID NO: 36 (GPFRSHAVS), CDRH2 comprises SEQ ID NO: 11 (GGIIPVFGTATYA), CDRH3 comprises SEQ ID NO: 37 (ARLKSELKDAFDI), CDRL1 comprises SEQ ID NO: 9 (RASQSISSYLN), CDRL2 comprises SEQ ID NO: 5 (AASSLQS), and CDRL3 comprises SEQ ID NO: 10 (QQSYSTPLT). A representative antibody having such heavy and light chain variable CDR regions is mAb21.

[0122] In these embodiments and some embodiments of all such embodiments described herein, CDRH1 comprises SEQ ID NO: 1 (GTFSSYAIN), CDRH2 comprises SEQ ID NO: 11 (GGIIPVFGTATYA), CDRH3 comprises SEQ ID NO: 8 (ARLKGELKDAFDI), CDRL1 comprises SEQ ID NO: 9 (RASQSISSYLN), CDRL2 comprises SEQ ID NO: 5 (AASSLQS), and CDRL3 comprises SEQ ID NO: 38 (FQSYSTPLT). A representative antibody having such heavy-chain and light-chain variable CDR regions is mAb22.

[0123] In these embodiments and some embodiments of all such embodiments described herein, CDRH1 comprises SEQ ID NO: 1 (GTFSSYAIN), CDRH2 comprises SEQ ID NO: 11 (GGIIPVFGTATYA), CDRH3 comprises SEQ ID NO: 8 (ARLKGELKDAFDI), CDRL1 comprises SEQ ID NO: 9 (RASQSISSYLN), CDRL2 comprises SEQ ID NO: 5 (AASSLQS), and CDRL3 comprises SEQ ID NO: 39 (QQSYSTILT). A representative antibody having such heavy and light chain variable CDR regions is mAb23.

[0124] In these embodiments and some embodiments of all such embodiments described herein, CDRH1 comprises SEQ ID NO: 1 (GTFSSYAIN), CDRH2 comprises SEQ ID NO: 11 (GGIIPVFGTATYA), CDRH3 comprises SEQ ID NO: 8 (ARLKGELKDAFDI), CDRL1 comprises SEQ ID NO: 9 (RASQSISSYLN), CDRL2 comprises SEQ ID NO: 5 (AASSLQS), and CDRL3 comprises SEQ ID NO: 10 (QQSYSTPLT). A representative antibody having such heavy and light chain variable CDR regions is mAb24.

[0125] Embodiments are also provided that, in each case where a particular sequence is mentioned, 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) with respect to the mentioned sequence (e.g., sequence numbers 1-34 and 36-39).

[0126] In some embodiments, this disclosure also relates to an antibody or its antigen-binding moiety that specifically binds to PD-L1 and is at least 90% identical to SEQ ID NOs. 35, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, or 58 (for example, 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 9 The present invention provides an antibody or its antigen-binding moiety comprising a heavy chain variable region containing an amino acid sequence that is 8% or at least 99% identical, and a light chain variable region containing an amino acid sequence that is at least 90% identical (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical) to SEQ ID NOs. 59, 60, 61, 62, or 63.

[0127] The disclosure also provides, in some embodiments, an antibody or an antigen-binding moiety thereof that specifically binds to PD-L1, comprising a heavy chain variable region containing an amino acid sequence that is at least 90% identical (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) to SEQ ID NOs. 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 containing an amino acid sequence that is at least 90% identical (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) to SEQ ID NOs.

[0128] The disclosure also provides, in some embodiments, an antibody or an antigen-binding moiety thereof that specifically binds to PD-L1, comprising a heavy chain variable region containing an amino acid sequence that is at least 90% identical to SEQ ID NO: 35 (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) and a light chain variable region containing an amino acid sequence that is at least 90% identical to SEQ ID NOs: 59, 60, 61, 62, or 63 (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).

[0129] Antibodies mAb1 to mAb23 are affinity-mature antibodies derived from the parent antibody mAb24, as described in the examples. The affinity-mature antibody or its antigen-binding moiety is an antibody or antigen-binding fragment having one or more modifications (e.g., in one or more CDRs or FRs) that result in an improved affinity of the antibody to the antigen compared to the parent antibody lacking such modifications. In some embodiments, the affinity-mature antibody has an affinity of nanomolar or picomolar concentration for PD-L1. In some embodiments, the PD-L1 antibody or its antigen-binding moiety has at least 1 × 10⁻⁶ molecules. -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 M's K D It holds.

[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. Term K D When used herein, K refers to the dissociation equilibrium constant of a specific antibody-antigen interaction. D =k d / k a . term k d (seconds -1 ) refers to the specific antibody-antigen interaction dissociation rate constant, as used herein. The value is k off Also called value. Term k a (M -1 × seconds -1 ) refers to the specific antibody-antigen interaction association rate constant, as used herein. The value is k on It is also called a 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 variants of mAb24) against human PD-L1 ("huPDL1"), cynomolgus monkey PD-L1, or mouse PD-L1 ("muPDL1"). Human or cynomolgus monkey PD-L1 was expressed on HEK cells, and mouse PD-L1 was expressed on A20 cells. Binding was observed in EC cells. 50 This is expressed as a value, which can be estimated by titrating different concentrations of mAbs on cells exogenously expressing the antigen of interest. mAb binding can be detected and quantified using fluorescently tagged secondary products (secondaries). The data shown in Table 2 can be fitted to a 1:1 binding model using the built-in function in GRAPHPAD for EC 50 The value was obtained.

[0133] [Table 3]

[0134] This disclosure also relates to an antibody or its antigen-binding moiety that specifically binds to PD-L1 and is at least 90% identical (for example, 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%) 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. The present invention provides an antibody or its antigen-binding moiety, comprising a heavy chain variable region containing an amino acid sequence that is at least 99% identical to (or at least 99% identical to) any one of SEQ ID NOs. 59, 60, 61, 62, or 63, and a light chain variable region containing an amino acid sequence that is at least 90% identical (for example, 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). In some embodiments, the heavy chain variable region includes an amino acid sequence that differs by 15 amino acids or less, 14 amino acids or less, 13 amino acids or less, 12 amino acids or less, 11 amino acids or less, 10 amino acids or less, 9 amino acids or less, 8 amino acids or less, 7 amino acids or less, 6 amino acids or less, 5 amino acids or less, 4 amino acids or less, 3 amino acids or less, 2 amino acids or less, or 1 amino acid 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 contains an amino acid sequence that differs from any one of SEQ ID NOs: 59, 60, 61, 62, or 63 by 15 amino acids or less, 14 amino acids or less, 13 amino acids or less, 12 amino acids or less, 11 amino acids or less, 10 amino acids or less, 9 amino acids or less, 8 amino acids or less, 7 amino acids or less, 6 amino acids or less, 5 amino acids or less, 4 amino acids or less, 3 amino acids or less, 2 amino acids or less, or 1 amino acid. Tables 3 and 4 provide the sequences of 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 disclosure also provides, in some embodiments, an antibody or an antigen-binding moiety thereof that specifically binds to PD-L1, comprising a heavy chain CDR from 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 a light chain CDR from any of the light chain variable regions of SEQ ID NOs. 59, 60, 61, 62, or 63.

[0138] The disclosure also provides, in some embodiments, an antibody or an antigen-binding moiety thereof that specifically binds to PD-L1, comprising a heavy chain CDR from 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 a light chain CDR from any of the light chain variable regions of SEQ ID NOs. 59, 60, 61, 62, or 63, wherein the heavy chain and light chain CDR residues are numbered according to Kabat.

[0139] The disclosure also provides, in some embodiments, an antibody or its antigen-binding moiety that specifically binds to PD-L1, comprising a heavy chain CDR from 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 a light chain CDR from any of the light chain variable regions of SEQ ID NOs. 59, 60, 61, 62, or 63, wherein the heavy chain and light chain CDR residues are numbered according to Chothia.

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

[0141] The disclosure also provides, in some embodiments, an antibody or its antigen-binding moiety that specifically binds to PD-L1, comprising a heavy chain CDR from 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 a light chain CDR from any of the light chain variable regions of SEQ ID NOs. 59, 60, 61, 62, or 63, wherein the heavy chain and light chain CDR residues are numbered according to AbM.

[0142] The disclosure also provides, in some embodiments, an antibody or an antigen-binding moiety thereof that specifically binds to PD-L1, comprising a heavy chain CDR from 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 a light chain CDR from any of the light chain variable regions of SEQ ID NOs. 59, 60, 61, 62, or 63, wherein the heavy chain and light chain CDR residues are numbered according to IMGT.

[0143] B.PD-1 Antagonist In some embodiments and aspects, the Disclosure provides anti-PD-1 antagonists. 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; that is, the anti-PD-1 antibody or antigen-binding molecule is not part of a protein construct that binds to multiple epitopes. In some embodiments, the anti-PD-1 antibody or antigen-binding 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 multispecific antigen-binding construct 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 a cognitive ligand, causes suppression or inhibition of the immune response, such as those known to enhance tumor evasion. However, in some cases, as used herein, “inhibitory receptor” specifically refers to PD-1.

[0145] PD-1 is an immune checkpoint inhibitor receptor containing an "immune receptor tyrosine inhibitory motif" or "ITIM" which includes 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 readily designed by those skilled in the art. Such assays include, for example, the step of testing the effect 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 enzymes of the Src kinase family, which then allows it to recruit other enzymes, such as phosphotyrosine phosphatases SHP-1 and SHP-2, or inositol phosphatases called SHIP. These phosphatases have been shown to reduce the activation of molecules involved in cellular signaling. See, for example, Barrow and Trowsdale (2006) Eur J Immunol. 36(7): 1646-53. Therefore, the phosphorylation status of the ITIM motif in PD-1 can be assessed using methods known in the art. The presence of downstream factors, such as phosphotyrosine phosphatases, can also be investigated. Furthermore, various cell-based assays and kits are known in the art to detect the presence of downstream factors (e.g., activated T cell nuclear factor-NFAT as a measure of PD-1 inhibition) as a surrogate for the PD-1 activity status. In other examples, simple binding assays can be used to determine whether the constructs of the present disclosure can block binding PD-1 and its ligands, as discussed above.

[0146] Therefore, in some embodiments, an antibody or its antigen-binding portion that specifically binds to PD-1 is provided herein. In some embodiments, the antibody or its antigen-binding portion that specifically binds to PD-1 includes a heavy chain variable region comprising (a) a CDRH1 comprising (i) 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 these embodiments and some embodiments of all such embodiments 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 these embodiments and some embodiments of all such embodiments described herein, CDRH1 comprises SEQ ID NO: 73 (FTFSDYAMS), CDRH2 comprises SEQ ID NO: 71 (SAISNSGTYTYYA), CDRH3 comprises SEQ ID NO: 74 (ARGLDFIVGATGNDY), CDRL1 comprises SEQ ID NO: 9 (RASQSISSYLN), CDRL2 comprises SEQ ID NO: 5 (AASSLQS), and CDRL3 comprises SEQ ID NO: 10 (QQSYSTPLT). A representative antibody having such heavy-chain and light-chain variable CDR regions is mAb25.

[0148] In these embodiments and some embodiments of all such embodiments 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 these embodiments and some embodiments of all such embodiments described herein, CDRH1 comprises SEQ ID NO: 73 (FTFSDYAMS), CDRH2 comprises SEQ ID NO: 71 (SAISNSGTYTYYA), CDRH3 comprises SEQ ID NO: 75 (ARGLDFIVGYTGNDY), CDRL1 comprises SEQ ID NO: 9 (RASQSISSYLN), CDRL2 comprises SEQ ID NO: 5 (AASSLQS), and CDRL3 comprises SEQ ID NO: 10 (QQSYSTPLT). A representative antibody having such heavy-chain and light-chain variable CDR regions is mAb26.

[0149] In these embodiments and some embodiments of all such embodiments 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 these embodiments and some embodiments of all such embodiments described herein, CDRH1 comprises SEQ ID NO: 76 (FTFSSYAMS), CDRH2 comprises SEQ ID NO: 71 (SAISNSGTYTYYA), CDRH3 comprises SEQ ID NO: 75 (ARGLDFIVGYTGNDY), CDRL1 comprises SEQ ID NO: 9 (RASQSISSYLN), CDRL2 comprises SEQ ID NO: 5 (AASSLQS), and CDRL3 comprises SEQ ID NO: 10 (QQSYSTPLT). A representative antibody having such heavy-chain and light-chain variable CDR regions is mAb27.

[0150] In these embodiments and some embodiments of all such embodiments 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 these embodiments and some embodiments of all such embodiments described herein, CDRH1 comprises SEQ ID NO: 77 (FTFSSYAML), CDRH2 comprises SEQ ID NO: 71 (SAISNSGTYTYYA), CDRH3 comprises SEQ ID NO: 75 (ARGLDFIVGYTGNDY), CDRL1 comprises SEQ ID NO: 9 (RASQSISSYLN), CDRL2 comprises SEQ ID NO: 5 (AASSLQS), and CDRL3 comprises SEQ ID NO: 10 (QQSYSTPLT). A representative antibody having such heavy-chain and light-chain variable CDR regions is mAb28.

[0151] In these embodiments and some embodiments of all such embodiments 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 these embodiments and some embodiments of all such embodiments described herein, CDRH1 comprises SEQ ID NO: 78 (FTFSNYALS), CDRH2 comprises SEQ ID NO: 71 (SAISNSGTYTYYA), CDRH3 comprises SEQ ID NO: 75 (ARGLDFIVGYTGNDY), CDRL1 comprises SEQ ID NO: 9 (RASQSISSYLN), CDRL2 comprises SEQ ID NO: 5 (AASSLQS), and CDRL3 comprises SEQ ID NO: 10 (QQSYSTPLT). A representative antibody having such heavy-chain and light-chain variable CDR regions is mAb29.

[0152] In these embodiments and some embodiments of all such embodiments 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 these embodiments and some embodiments of all such embodiments described herein, CDRH1 comprises SEQ ID NO: 79 (FTFSAYAMN), CDRH2 comprises SEQ ID NO: 71 (SAISNSGTYTYYA), CDRH3 comprises SEQ ID NO: 75 (ARGLDFIVGYTGNDY), CDRL1 comprises SEQ ID NO: 9 (RASQSISSYLN), CDRL2 comprises SEQ ID NO: 5 (AASSLQS), and CDRL3 comprises SEQ ID NO: 10 (QQSYSTPLT). A representative antibody having such heavy and light chain variable CDR regions is mAb30.

[0153] In these embodiments and some embodiments of all such embodiments 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 these embodiments and some embodiments of all such embodiments described herein, CDRH1 comprises SEQ ID NO: 80 (FTFRSYAMS), CDRH2 comprises SEQ ID NO: 71 (SAISNSGTYTYYA), CDRH3 comprises SEQ ID NO: 75 (ARGLDFIVGYTGNDY), CDRL1 comprises SEQ ID NO: 9 (RASQSISSYLN), CDRL2 comprises SEQ ID NO: 5 (AASSLQS), and CDRL3 comprises SEQ ID NO: 10 (QQSYSTPLT). A representative antibody having such heavy-chain and light-chain variable CDR regions is mAb31.

[0154] In these embodiments and some embodiments of all such embodiments 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 these embodiments and some embodiments of all such embodiments described herein, CDRH1 comprises SEQ ID NO: 81 (FTFGRYAMS), CDRH2 comprises SEQ ID NO: 71 (SAISNSGTYTYYA), CDRH3 comprises SEQ ID NO: 75 (ARGLDFIVGYTGNDY), CDRL1 comprises SEQ ID NO: 9 (RASQSISSYLN), CDRL2 comprises SEQ ID NO: 5 (AASSLQS), and CDRL3 comprises SEQ ID NO: 10 (QQSYSTPLT). A representative antibody having such heavy-chain and light-chain variable CDR regions is mAb32.

[0155] In these embodiments and some embodiments of all such embodiments described herein, CDRH1 includes SEQ ID NO: 82 (FTFNSYAMS), CDRH2 includes SEQ ID NO: 71 (SAISNSGTYTYYA), and CDRH3 includes SEQ ID NO: 75 (ARGLDFIVGYTGNDY). In these embodiments and some embodiments of all such embodiments described herein, CDRH1 includes SEQ ID NO: 82 (FTFNSYAMS), CDRH2 includes SEQ ID NO: 71 (SAISNSGTYTYYA), CDRH3 includes SEQ ID NO: 75 (ARGLDFIVGYTGNDY), CDRL1 includes SEQ ID NO: 9 (RASQSISSYLN), CDRL2 includes SEQ ID NO: 5 (AASSLQS), and CDRL3 includes SEQ ID NO: 10 (QQSYSTPLT). A representative antibody having such heavy-chain and light-chain variable CDR regions is mAb33.

[0156] In these embodiments and some embodiments of all such embodiments 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 these embodiments and some embodiments of all such embodiments described herein, CDRH1 comprises SEQ ID NO: 83 (FTFSNYAMS), CDRH2 comprises SEQ ID NO: 71 (SAISNSGTYTYYA), CDRH3 comprises SEQ ID NO: 74 (ARGLDFIVGATGNDY), CDRL1 comprises SEQ ID NO: 9 (RASQSISSYLN), CDRL2 comprises SEQ ID NO: 5 (AASSLQS), and CDRL3 comprises SEQ ID NO: 10 (QQSYSTPLT). A representative antibody having such heavy-chain and light-chain variable CDR regions is mAb34.

[0157] In these embodiments and some embodiments of all such embodiments 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 these embodiments and some embodiments of all such embodiments described herein, CDRH1 comprises SEQ ID NO: 84 (FTFSGYAMS), CDRH2 comprises SEQ ID NO: 71 (SAISNSGTYTYYA), CDRH3 comprises SEQ ID NO: 85 (ARGLDFIVGRTGNDY), CDRL1 comprises SEQ ID NO: 9 (RASQSISSYLN), CDRL2 comprises SEQ ID NO: 5 (AASSLQS), and CDRL3 comprises SEQ ID NO: 10 (QQSYSTPLT). A representative antibody having such heavy-chain and light-chain variable CDR regions is mAb35.

[0158] In these embodiments and some embodiments of all such embodiments 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 these embodiments and some embodiments of all such embodiments described herein, CDRH1 comprises SEQ ID NO: 86 (FTFSSYAMN), CDRH2 comprises SEQ ID NO: 71 (SAISNSGTYTYYA), CDRH3 comprises SEQ ID NO: 85 (ARGLDFIVGRTGNDY), CDRL1 comprises SEQ ID NO: 9 (RASQSISSYLN), CDRL2 comprises SEQ ID NO: 5 (AASSLQS), and CDRL3 comprises SEQ ID NO: 10 (QQSYSTPLT). A representative antibody having such heavy and light chain variable CDR regions is mAb36.

[0159] In these embodiments and some embodiments of all such embodiments 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 these embodiments and some embodiments of all such embodiments described herein, CDRH1 comprises SEQ ID NO: 80 (FTFRSYAMS), CDRH2 comprises SEQ ID NO: 71 (SAISNSGTYTYYA), CDRH3 comprises SEQ ID NO: 85 (ARGLDFIVGRTGNDY), CDRL1 comprises SEQ ID NO: 9 (RASQSISSYLN), CDRL2 comprises SEQ ID NO: 5 (AASSLQS), and CDRL3 comprises SEQ ID NO: 10 (QQSYSTPLT). A representative antibody having such heavy and light chain variable CDR regions is mAb37.

[0160] Embodiments are also provided that, in each case where a particular sequence is mentioned, include sequences 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 sequence mentioned (e.g., sequence numbers 5, 9, 10, or 71-86).

[0161] The disclosure also provides, in some embodiments, an antibody or an antigen-binding moiety thereof that specifically binds to PD-1, comprising a heavy chain variable region containing 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) of amino acid sequences to any one of SEQ ID NOs. 87 to 99, and a light chain variable region containing 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) of amino acid sequences to SEQ ID NO. 59. In some embodiments, the heavy chain variable region includes 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) of the amino acid sequence to SEQ ID NO: 90, and the light chain variable region includes 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) of the amino acid sequence to SEQ ID NO: 59. Table 7 provides the heavy chain variable sequences for SEQ ID NOs. 87 to 99, and Table 4 provides the light chain variable sequences for SEQ ID NO: 59. In some embodiments, the heavy chain variable region includes an amino acid sequence that differs from any one of sequence numbers 87 to 99 by 15 amino acids or less, 14 amino acids or less, 13 amino acids or less, 12 amino acids or less, 11 amino acids or less, 10 amino acids or less, 9 amino acids or less, 8 amino acids or less, 7 amino acids or less, 6 amino acids or less, 5 amino acids or less, 4 amino acids or less, 3 amino acids or less, 2 amino acids or less, or 1 amino acid.In some embodiments, the light chain variable region comprises an amino acid sequence that differs by 15 amino acids or less, 14 amino acids or less, 13 amino acids or less, 12 amino acids or less, 11 amino acids or less, 10 amino acids or less, 9 amino acids or less, 8 amino acids or less, 7 amino acids or less, 6 amino acids or less, 5 amino acids or less, 4 amino acids or less, 3 amino acids or less, 2 amino acids or less, or 1 amino acid from SEQ ID NO: 59.

[0162] Antibodies mAb26-mAb37 are affinity matured antibodies derived from the parental antibody mAb25 as described in the Examples. An affinity matured antibody or antigen-binding portion thereof is an antibody or antigen-binding fragment having a change that results in an improvement in the affinity of the antibody for its antigen as compared to the parental antibody lacking the one or more changes (e.g., in one or more CDRs or FRs). In some embodiments, the affinity matured antibody has a nanomolar or picomolar affinity for PD-1. In some embodiments, the PD-1 antibody or antigen-binding portion thereof has a K -7 of 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 M. D

[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 expressed as EC 50 values, which can be estimated by titrating different concentrations of mAb on cells that externally express the antigen of interest. Fluorescently tagged secondary reagents can be used to detect and quantify mAb binding. The data shown in Table 5 was fit to a 1:1 binding model using the built-in function in GRAPHPAD to obtain EC 50 values.

[0164] [Table 6]

[0165] [Table 7]

[0166] [Table 8]

[0167] The present disclosure also provides, in some embodiments, an antibody or an antigen-binding portion thereof that specifically binds to PD-L1 and comprises any heavy chain CDR of the heavy chain variable region of SEQ ID NO: 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99, and the light chain CDR of the light chain variable region of SEQ ID NO: 59.

[0168] The disclosure also provides, in some embodiments, an antibody or its antigen-binding moiety that specifically binds to PD-L1, comprising a heavy chain CDR from 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 a light chain CDR from the light chain variable region of SEQ ID NO. 59, wherein the heavy chain and light chain CDR residues are numbered according to Kabat.

[0169] The disclosure also provides, in some embodiments, an antibody or its antigen-binding moiety that specifically binds to PD-L1, comprising a heavy chain CDR from 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 a light chain CDR from the light chain variable region of SEQ ID NOs. 59, wherein the heavy chain and light chain CDR residues are numbered according to Cotia.

[0170] The disclosure also provides, in some embodiments, an antibody or its antigen-binding moiety that specifically binds to PD-L1, comprising a heavy chain CDR from 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 a light chain CDR from the light chain variable region of SEQ ID NO. 59, wherein the heavy chain and light chain CDR residues are numbered according to the McCollum.

[0171] The disclosure also provides, in some embodiments, an antibody or its antigen-binding moiety that specifically binds to PD-L1, comprising a heavy chain CDR from 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 a light chain CDR from the light chain variable region of SEQ ID NOs. 59, wherein the heavy chain and light chain CDR residues are numbered according to AbM.

[0172] The disclosure also provides, in some embodiments, an antibody or its antigen-binding moiety that specifically binds to PD-L1, comprising a heavy chain CDR from 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 a light chain CDR from the light chain variable region of SEQ ID NOs. 59, wherein the heavy chain and light chain CDR residues are numbered according to IMGT.

[0173] C. Multispecific antigen binding constructs This disclosure provides compositions and methods for enhancing an immune response against tumor cells by inhibiting interactions between PD-L1 and PD-1, for example, the interaction between PD-L1 expressed on tumor cells and PD-1 expressed on T cells, in some embodiments. Antibodies or their antigen-binding moieties 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” to PD-L1 or PD-1, or to an epitope on PD-L1 or PD-1, mean binding that is measurably different from nonspecific or nonselective interactions. In some embodiments, the antibody or its antigen-binding moiety 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 the binding of the molecule by comparing it to the binding of a control molecule. Specific binding can also be determined by competition with a target-like control molecule, such as an excessive amount of unlabeled target. In this case, specific binding is indicated when the binding of the labeled target to the probe is competitively inhibited by excessive unlabeled target activity.

[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, the multispecific antigen-binding construct binds simultaneously 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, the multispecific antigen-binding construct binds simultaneously 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 can bind to human PD-1. In some embodiments, the multispecific antigen-binding construct can bind to mouse PD-1. In some embodiments, the multispecific antigen-binding construct can bind to cynomolgus monkey PD-1. In some embodiments, the multispecific antigen-binding construct can bind to human, mouse, and cynomolgus monkey PD-1 with similar affinity.

[0176] In some embodiments and aspects, the disclosure provides a multispecific antigen-binding construct comprising at least two units of antigen binding, wherein a first unit of antigen binding binds to PD-1 and a second unit of antigen binding binds to a PD-1 ligand. In some embodiments, the first unit of antigen binding binds to PD-1 expressed by an immune cell. In some embodiments, the second unit of antigen binding binds to PD-1 expressed by a second cell. In some embodiments, the multispecific antigen-binding construct blocks the interaction between PD-1 and a PD-1 ligand, e.g., PD-L1 or PD-L2. In some embodiments, the multispecific antigen-binding construct blocks the interaction between 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 units of antigen binding that bind to PD-1. In some embodiments, the multispecific antigen-binding construct comprises two units of antigen binding that bind to PD-1. In some embodiments, the multispecific antigen-binding construct includes at least two units of antigen binding that bind to a PD-1 ligand, e.g., PD-L1 or PD-L2. In some embodiments, the multispecific antigen-binding construct includes two units of antigen binding that bind to a PD-1 ligand, e.g., PD-L1 or PD-L2. In some embodiments, the multispecific antigen-binding construct includes at least four units of antigen binding, 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 includes four units of antigen binding, 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 unit of antigen binding can independently bind to its cognitive 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 between PD-1 and a PD-1 ligand, such as PD-L1 or PD-L2. In some embodiments, the multispecific antigen-binding construct includes a common light chain. For example, at least two units of antigen binding include a common light chain.

[0177] In some embodiments, the first antigen-binding unit is bound to PD-1. (a)(i) CDRH1 containing sequence number 70 (FTFX1X2YAX3X4, where X1=S, R, G, or N, X2=D, S, N, A, R, or G, X3=M or L, X4=S, L, or N), (ii) CDRH2 containing sequence number 71 (SAISNSGTYTYYA), and (iii) CDRH3 containing sequence number 72 (ARGLDFIVGX5TGNDY, where X5=A, Y, or R), and (b) Light chain variable region including (i) CDRL1 containing sequence number 9 (RASQSISSYLN), (ii) CDRL2 containing sequence number 5 (AASSLQS), and (iii) CDRL3 containing sequence number 10 (QQSYSTPLT). Includes.

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

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

[0180] In some embodiments, the first antigen-binding unit is bound to PD-1 and includes a light chain variable region having at least 90% identical amino acid sequence to SEQ ID NO: 59. In some embodiments, the second antigen-binding unit binds to PD-L2. In some embodiments, the second antigen-binding unit binds to PD-L1. In some embodiments, the second antigen-binding unit binds to PD-L1, a. A heavy chain variable region including (i) CDRH1 containing sequence number 1 (GTFSSYAIN), (ii) CDRH2 containing sequence number 2 (GGIIPX1X2GX3ATYA, where X1 is V or I, X2 is F, L, or V, and X3 is T or A), and (iii) CDRH3 containing sequence number 3 (ARLKX1ELKDAFDI, where X1 is G, F, or N), and b. Light chain variable region containing (i) CDRL1 containing sequence number 4 (RASQX1ISSYLN, where X1 is S, W, or Q), (ii) CDRL2 containing sequence number 5 (AASSLQS), and (iii) CDRL3 containing sequence number 6 (X1QSYSTPLT, where X1 is Q or F). Includes.

[0181] In some such embodiments, the second unit of antigen binding is bound to PD-L1, (a) CDRH1 containing sequence number 1 (GTFSSYAIN), CDRH2 containing sequence number 7 (GGIIPILGAATYA), and CDRH3 containing sequence number 8 (ARLKGELKDAFDI), (b) CDRH1 containing sequence number 1 (GTFSSYAIN), CDRH2 containing sequence number 7 (GGIIPILGAATYA), CDRH3 containing sequence number 8 (ARLKGELKDAFDI), CDRL1 containing sequence number 9 (RASQSISSYLN), CDRL2 containing sequence number 5 (AASSLQS), and CDRL3 containing sequence number 10 (QQSYSTPLT), (c) CDRH1 containing sequence number 1 (GTFSSYAIN), CDRH2 containing sequence number 11 (GGIIPVFGTATYA), CDRH3 containing sequence number 8 (ARLKGELKDAFDI), CDRL1 containing sequence number 9 (RASQSISSYLN), CDRL2 containing sequence number 5 (AASSLQS), and CDRL3 containing sequence number 10 (QQSYSTPLT), (d) CDRH1 containing sequence number 1 (GTFSSYAIN), CDRH2 containing sequence number 11 (GGIIPVFGTATYA), and CDRH3 containing sequence number 8 (ARLKGELKDAFDI), (e) CDRH1 containing sequence number 1 (GTFSSYAIN), CDRH2 containing sequence number 11 (GGIIPVFGTATYA), CDRH3 containing sequence number 8 (ARLKGELKDAFDI), CDRL1 containing sequence number 12 (RASQWISSYLN), CDRL2 containing sequence number 5 (AASSLQS), and CDRL3 containing sequence number 10 (QQSYSTPLT), (f) CDRH1 containing sequence number 1 (GTFSSYAIN), CDRH2 containing sequence number 11 (GGIIPVFGTATYA), CDRH3 containing sequence number 8 (ARLKGELKDAFDI), CDRL1 containing sequence number 13 (RASQQISSYLN), CDRL2 containing sequence number 5 (AASSLQS), and CDRL3 containing sequence number 10 (QQSYSTPLT), (g) CDRH1 containing sequence number 1 (GTFSSYAIN), CDRH2 containing sequence number 11 (GGIIPVFGTATYA), CDRH3 containing sequence number 8 (ARLKGELKDAFDI), CDRL1 containing sequence number 9 (RASQSISSYLN), CDRL2 containing sequence number 5 (AASSLQS), and CDRL3 containing sequence number 10 (QQSYSTPLT), (h) CDRH1 containing sequence number 1 (GTFSSYAIN), CDRH2 containing sequence number 15 (GGIIPIFGIANYA), and CDRH3 containing sequence number 8 (ARLKGELKDAFDI), (i) CDRH1 containing sequence number 1 (GTFSSYAIN), CDRH2 containing sequence number 15 (GGIIPIFGIANYA), CDRH3 containing sequence number 8 (ARLKGELKDAFDI), CDRL1 containing sequence number 9 (RASQSISSYLN), CDRL2 containing sequence number 5 (AASSLQS), and CDRL3 containing sequence number 10 (QQSYSTPLT), (j) CDRH1 containing sequence number 1 (GTFSSYAIN), CDRH2 containing sequence number 16 (GGIIPNFGTATYA), and CDRH3 containing sequence number 17 (ARLKGELKGAGDI), (k) CDRH1 containing sequence number 1 (GTFSSYAIN), CDRH2 containing sequence number 16 (GGIIPNFGTATYA), CDRH3 containing sequence number 17 (ARLKGELKGAGDI), CDRL1 containing sequence number 9 (RASQSISSYLN), CDRL2 containing sequence number 5 (AASSLQS), and CDRL3 containing sequence number 10 (QQSYSTPLT), (l) CDRH1 containing sequence number 1 (GTFSSYAIN), CDRH2 containing sequence number 11 (GGIIPVFGTATYA), and CDRH3 containing sequence number 18 (ARLKFELKDAFDI), (m) CDRH1 containing sequence number 1 (GTFSSYAIN), CDRH2 containing sequence number 11 (GGIIPVFGTATYA), CDRH3 containing sequence number 18 (ARLKFELKDAFDI), CDRL1 containing sequence number 9 (RASQSISSYLN), CDRL2 containing sequence number 5 (AASSLQS), and CDRL3 containing sequence number 10 (QQSYSTPLT), (n) CDRH1 containing sequence number 1 (GTFSSYAIN), CDRH2 containing sequence number 11 (GGIIPVFGTATYA), and CDRH3 containing sequence number 19 (ARLKGELKDAFDE), (o) CDRH1 containing sequence number 1 (GTFSSYAIN), CDRH2 containing sequence number 11 (GGIIPVFGTATYA), CDRH3 containing sequence number 19 (ARLKGELKDAFDE), CDRL1 containing sequence number 9 (RASQSISSYLN), CDRL2 containing sequence number 5 (AASSLQS), and CDRL3 containing sequence number 10 (QQSYSTPLT), (p) CDRH1 containing sequence number 1 (GTFSSYAIN), CDRH2 containing sequence number 11 (GGIIPVFGTATYA), and CDRH3 containing sequence number 20 (ARLKNELKDAFDI), (q) CDRH1 containing sequence number 1 (GTFSSYAIN), CDRH2 containing sequence number 11 (GGIIPVFGTATYA), CDRH3 containing sequence number 20 (ARLKNELKDAFDI), CDRL1 containing sequence number 9 (RASQSISSYLN), CDRL2 containing sequence number 5 (AASSLQS), and CDRL3 containing sequence number 10 (QQSYSTPLT), (r) CDRH1 containing sequence number 1 (GTFSSYAIN), CDRH2 containing sequence number 21 (GGVIPFLGTANYA), and CDRH3 containing sequence number 22 (ARLKGILKDALDI), (s) CDRH1 containing sequence number 1 (GTFSSYAIN), CDRH2 containing sequence number 21 (GGVIPFLGTANYA), CDRH3 containing sequence number 22 (ARLKGILKDALDI), CDRL1 containing sequence number 9 (RASQSISSYLN), CDRL2 containing sequence number 5 (AASSLQS), and CDRL3 containing sequence number 10 (QQSYSTPLT), (t) CDRH1 containing sequence number 1 (GTFSSYAIN), CDRH2 containing sequence number 29 (GRIIPIFGTADYA), and CDRH3 containing sequence number 8 (ARLKGELKDAFDI), (u) CDRH1 containing sequence number 1 (GTFSSYAIN), CDRH2 containing sequence number 29 (GRIIPIFGTADYA), CDRH3 containing sequence number 8 (ARLKGELKDAFDI), CDRL1 containing sequence number 9 (RASQSISSYLN), CDRL2 containing sequence number 5 (AASSLQS), and CDRL3 containing sequence number 10 (QQSYSTPLT), (v) CDRH1 containing sequence number 1 (GTFSSYAIN), CDRH2 containing sequence number 31 (GGIIPILGTATYA), and CDRH3 containing sequence number 32 (ARRKGELKDAFDI), (w) CDRH1 containing sequence number 1 (GTFSSYAIN), CDRH2 containing sequence number 31 (GGIIPILGTATYA), CDRH3 containing sequence number 32 (ARRKGELKDAFDI), CDRL1 containing sequence number 9 (RASQSISSYLN), CDRL2 containing sequence number 5 (AASSLQS), and CDRL3 containing sequence number 10 (QQSYSTPLT), (x) CDRH1 containing sequence number 1 (GTFSSYAIN), CDRH2 containing sequence number 33 (GGIIPIVATANYA), and CDRH3 containing sequence number 32 (ARRKGELKDAFDI), (y) CDRH1 containing sequence number 1 (GTFSSYAIN), CDRH2 containing sequence number 33 (GGIIPIVATANYA), CDRH3 containing sequence number 32 (ARRKGELKDAFDI), CDRL1 containing sequence number 9 (RASQSISSYLN), CDRL2 containing sequence number 5 (AASSLQS), and CDRL3 containing sequence number 10 (QQSYSTPLT), (z) CDRH1 containing sequence number 1 (GTFSSYAIN), CDRH2 containing sequence number 34 (GGIIPIFGKATYA), and CDRH3 containing sequence number 32 (ARRKGELKDAFDI), (aa) CDRH1 containing sequence number 1 (GTFSSYAIN), CDRH2 containing sequence number 34 (GGIIPIFGKATYA), CDRH3 containing sequence number 32 (ARRKGELKDAFDI), CDRL1 containing sequence number 9 (RASQSISSYLN), CDRL2 containing sequence number 5 (AASSLQS), and CDRL3 containing sequence number 10 (QQSYSTPLT), (bb) CDRH1 containing sequence number 1 (GTFSSYAIN), CDRH2 containing sequence number 11 (GGIIPVFGTATYA), CDRH3 containing sequence number 8 (ARLKGELKDAFDI), CDRL1 containing sequence number 9 (RASQSISSYLN), CDRL2 containing sequence number 5 (AASSLQS), and CDRL3 containing sequence number 38 (FQSYSTPLT), (cc) CDRH1 containing sequence number 1 (GTFSSYAIN), CDRH2 containing sequence number 11 (GGIIPVFGTATYA), CDRH3 containing sequence number 8 (ARLKGELKDAFDI), CDRL1 containing sequence number 9 (RASQSISSYLN), CDRL2 containing sequence number 5 (AASSLQS), and CDRL3 containing sequence number 39 (QQSYSTILT), (dd) CDRH1 containing sequence number 14 (GTFSSYAFS), CDRH2 containing sequence number 11 (GGIIPVFGTATYA), CDRH3 containing sequence number 8 (ARLKGELKDAFDI), CDRL1 containing sequence number 9 (RASQSISSYLN), CDRL2 containing sequence number 5 (AASSLQS), and CDRL3 containing sequence number 10 (QQSYSTPLT), (ee) CDRH1 containing sequence number 23 (GTFSSYAIS), CDRH2 containing sequence number 24 (GGIIPIVGIANYA), and CDRH3 containing sequence number 8 (ARLKGELKDAFDI), (ff) CDRH1 containing sequence number 23 (GTFSSYAIS), CDRH2 containing sequence number 24 (GGIIPIVGIANYA), CDRH3 containing sequence number 8 (ARLKGELKDAFDI), CDRL1 containing sequence number 9 (RASQSISSYLN), CDRL2 containing sequence number 5 (AASSLQS), and CDRL3 containing sequence number 10 (QQSYSTPLT), CDRH1 containing (gg) Sequence ID 23 (GTFSSYAIS), CDRH2 containing Sequence ID 11 (GGIIPVFGTATYA), and CDRH3 containing Sequence ID 25 (ARLKGEFKDAFDI), (hh) CDRH1 containing sequence number 23 (GTFSSYAIS), CDRH2 containing sequence number 11 (GGIIPVFGTATYA), CDRH3 containing sequence number 25 (ARLKGEFKDAFDI), CDRL1 containing sequence number 9 (RASQSISSYLN), CDRL2 containing sequence number 5 (AASSLQS), and CDRL3 containing sequence number 10 (QQSYSTPLT), (ii) CDRH1 containing sequence number 23 (GTFSSYAIS), CDRH2 containing sequence number 26 (GRIIPLFGTAHYA), and CDRH3 containing sequence number 8 (ARLKGELKDAFDI), (jj) CDRH1 containing sequence number 23 (GTFSSYAIS), CDRH2 containing sequence number 26 (GRIIPLFGTAHYA), CDRH3 containing sequence number 8 (ARLKGELKDAFDI), CDRL1 containing sequence number 9 (RASQSISSYLN), CDRL2 containing sequence number 5 (AASSLQS), and CDRL3 containing sequence number 10 (QQSYSTPLT), (kk) CDRH1 containing sequence number 23 (GTFSSYAIS), CDRH2 containing sequence number 27 (GRINPILGTANYA), and CDRH3 containing sequence number 28 (ARLKGELKDAFSI), (ll) CDRH1 containing sequence number 23 (GTFSSYAIS), CDRH2 containing sequence number 27 (GRINPILGTANYA), CDRH3 containing sequence number 28 (ARLKGELKDAFSI), CDRL1 containing sequence number 9 (RASQSISSYLN), CDRL2 containing sequence number 5 (AASSLQS), and CDRL3 containing sequence number 10 (QQSYSTPLT), CDRH1 containing (mm) Sequence ID 23 (GTFSSYAIS), CDRH2 containing Sequence ID 11 (GGIIPVFGTATYA), and CDRH3 containing Sequence ID 30 (ARLKGELKCAFDI), (nn) CDRH1 containing sequence number 23 (GTFSSYAIS), CDRH2 containing sequence number 11 (GGIIPVFGTATYA), CDRL1 containing sequence number 9 (RASQSISSYLN) and sequence number 30 (ARLKGELKCAFDI), CDRL2 containing sequence number 5 (AASSLQS), and CDRL3 containing sequence number 10 (QQSYSTPLT), (oo) CDRH1 containing sequence number 36 (GPFRSHAVS), CDRH2 containing sequence number 11 (GGIIPVFGTATYA), and CDRH3 containing sequence number 37 (ARLKSELKDAFDI), or (pp) CDRH1 containing sequence number 36 (GPFRSHAVS), CDRH2 containing sequence number 11 (GGIIPVFGTATYA), CDRH3 containing sequence number 37 (ARLKSELKDAFDI), CDRL1 containing sequence number 9 (RASQSISSYLN), CDRL2 containing sequence number 5 (AASSLQS), and CDRL3 containing sequence number 10 (QQSYSTPLT) Includes.

[0182] In some such embodiments, the second antigen-binding unit is bound to PD-L1 and includes a heavy chain variable region containing at least 90% identical amino acid sequences 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 containing at least 90% identical amino acid sequences to any one of SEQ ID NOs. 59, 60, 61, 62, or 63.

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

[0184] In some embodiments, the second antigen-binding unit is bound to PD-L1. (a) At least 90% identical amino acid sequence to SEQ ID NO: 59, (b) At least 90% identical amino acid sequence to SEQ ID NO: 60, (c) At least 90% identical amino acid sequence to SEQ ID NO: 61, (d) an amino acid sequence that is at least 90% identical to SEQ ID NO: 62, or (e) Amino acid sequence identical to at least 90% of SEQ ID NO: 63 Includes a light chain variable region.

[0185] In some embodiments, the disclosure provides multispecific antigen-binding constructs comprising any PD-1 antagonist disclosed herein and any antagonist of a PD-1 ligand such as PD-L1 disclosed herein. For example, dual-specific construct 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), which is a fusion protein in which the antibody heavy chain further comprises a heavy chain variable region of an anti-PD-L1 antibody (mAb1) linked to the Fc region of an anti-BCMA antibody by a poly-GGGS (SEQ ID NO: 120) linker at its C-terminus. The light chains of the anti-PD-1 and anti-PD-L1 portions of the construct are identical (SEQ ID NO: 101). Dual-specific construct 3, the construct illustrated in Figure 13A, comprises the heavy chain sequence referred to in SEQ ID NO: 100 and the light chain sequence referred to in SEQ ID NO: 101.

[0186] In some embodiments, the disclosure provides a multispecific antibody or its antigen-binding moiety that specifically binds to PD-1 and PD-L1, comprising a heavy chain region containing 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%) of the amino acid sequence to SEQ ID NO: 100 or 102, and a light chain region containing 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%) of the amino acid sequence to SEQ ID NO: 101 or 103.

[0187] In some embodiments, the disclosure provides a multispecific antibody or its antigen-binding moiety that specifically binds to PD-1 and PD-L1, comprising a heavy chain region containing 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) of amino acid sequences to SEQ ID NO: 100, and a light chain region containing 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) of amino acid sequences to SEQ ID NO: 101. In some embodiments, the heavy chain region includes an amino acid sequence that differs by only 15 amino acids or less, 14 amino acids or less, 13 amino acids or less, 12 amino acids or less, 11 amino acids or less, 10 amino acids or less, 9 amino acids or less, 8 amino acids or less, 7 amino acids or less, 6 amino acids or less, 5 amino acids or less, 4 amino acids or less, 3 amino acids or less, 2 amino acids or less, or 1 amino acid from SEQ ID NO: 100. In some embodiments, the light chain region includes an amino acid sequence that differs by only 15 amino acids or less, 14 amino acids or less, 13 amino acids or less, 12 amino acids or less, 11 amino acids or less, 10 amino acids or less, 9 amino acids or less, 8 amino acids or less, 7 amino acids or less, 6 amino acids or less, 5 amino acids or less, 4 amino acids or less, 3 amino acids or less, 2 amino acids or less, or 1 amino acid from SEQ ID NO: 101.

[0188] In some embodiments, the disclosure provides a multispecific antibody or its antigen-binding moiety that specifically binds to PD-1 and PD-L1, comprising a heavy chain region containing 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) of amino acid sequences to SEQ ID NO: 102, and a light chain region containing 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) of amino acid sequences to SEQ ID NO: 103. In some embodiments, the heavy chain region includes an amino acid sequence that differs by only 15 amino acids or less, 14 amino acids or less, 13 amino acids or less, 12 amino acids or less, 11 amino acids or less, 10 amino acids or less, 9 amino acids or less, 8 amino acids or less, 7 amino acids or less, 6 amino acids or less, 5 amino acids or less, 4 amino acids or less, 3 amino acids or less, 2 amino acids or less, or 1 amino acid from SEQ ID NO: 102. In some embodiments, the light chain region includes an amino acid sequence that differs by only 15 amino acids or less, 14 amino acids or less, 13 amino acids or less, 12 amino acids or less, 11 amino acids or less, 10 amino acids or less, 9 amino acids or less, 8 amino acids or less, 7 amino acids or less, 6 amino acids or less, 5 amino acids or less, 4 amino acids or less, 3 amino acids or less, 2 amino acids or less, or 1 amino acid from SEQ ID NO: 103.

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

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

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

[0192] [Table 9]

[0193] [Table 10]

[0194] The term “multispecific antigen-binding construct” as used herein refers to a bispecific, triplicate, or multispecific antigen-binding construct, and its antigen-binding portion or fragment. A multispecific antigen-binding construct may be a single multifunctional polypeptide or a multimer 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 its antigen-binding fragment) that can be linked to or co-expressed with another functional molecule, e.g., another peptide, protein, and / or aptamer. For example, an antibody or its fragment can be functionally linked (e.g., by chemical coupling, gene fusion, noncovalent association, or otherwise) to one or more other molecular entities, such as a protein or its fragment, to produce a bispecific or multispecific antigen-binding molecule having a second binding specificity. As used herein, the term “multispecific antigen-binding construct” also includes a bispecific, triplicate, or multispecific antibody or its antigen-binding fragment. In certain embodiments, an antibody is functionally ligated to another antibody or its antigen-binding fragment to produce a bispecific antibody having a second binding specificity. The bispecific and multispecific antibodies of the present invention are described elsewhere in this specification.

[0195] As used herein, the antigen-binding “arm” refers to a unit, domain, or region of a multispecific antigen-binding construct that forms a compartment of the construct that binds to an antigen. 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 a unit of antigen binding. Generally, one “arm” (the first arm) is distinct from the other “arm” (the second arm) in its antigen-binding or antigen-specificity. Thus, in the example of a bispecific bivalent antibody, one arm of the antibody binds to antigen A and the other arm of the antibody binds to antigen B. In some embodiments, in the example of a bispecific bivalent antibody, one arm of the antibody binds to antigen A and the other arm of the antibody binds to antigen B or C (for example, cross-reacting with two antigens such as PD-L1 and PD-L2 due to structural similarity). See, for example, U.S. Patent No. 9,845,356. Similarly, in the case of a tetravalent bispecific antibody (for example, formed by conjugating two different antibodies), one “arm” refers to the antibody compartment 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 antibody compartment that binds to antigen B (even if two binding sites of the bivalent antibody bind to antigen B). In some embodiments, in the case of a tetravalent bispecific antibody (for example, formed by conjugating two different antibodies), one “arm” refers to the antibody compartment 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 antibody compartment that binds to antigen B or C (even if two binding sites of the bivalent antibody can bind to antigen B or C). See, for example, U.S. Patent No. 9,845,356. As will be apparent to those skilled in the art, “first” or “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 (for example, 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 a different antigen. Polyvalency can increase the avidity, i.e., the strength of binding, between the antigen-binding arm or construct and the antigen or target receptor involved. Avidity relates to both the affinity between the epitope or antigenic determinant and its binding site on the antigen-binding unit, and the actual number of 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 in which at least two 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 in which at least two 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 in which at least two 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 comprising 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 to the same epitope on PD-1, and both valencies of the second bivalent antibody or antigen-binding fragment are specific to the same epitope on PD-L1. In some embodiments of such a tetravalent construct, the first and second bivalent antibodies or their antigen-binding fragments or portions use light chains having the same amino acid sequence. In other words, the tetravalent construct contains 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, e.g., an antagonist of PD-L1 and / or PD-L2. In some embodiments, the first arm is a PD-1 antagonist, and the second arm is a cognitive PD-1 ligand, e.g., an antagonist of PD-L1 and / or PD-L2.

[0199] When the terms “antagonist,” “antagonize,” and “inhibit” are used to refer to the biological activity of an antigen-binding arm, it means that the antigen-binding arm binds to its target (e.g., PD-1) on each cell to partially or completely block, inhibit, and / or reduce the biological response mediated by PD-1. In some embodiments, inhibition in the presence of an antagonist is observed in a dose-dependent manner. In some embodiments, the measured signal (e.g., biological activity) is at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% lower than the signal measured using a negative control under equivalent conditions. Methods for identifying antagonists suitable for use in the methods of this disclosure are also disclosed herein. Examples of such methods include, but are not limited to, binding assays such as enzyme-linked immunosorbent assays (ELISA), FORTE BIO® systems, and radioimmunoassays (RIA). These assays determine the ability of an antagonist to bind to a target polypeptide (e.g., PD-1 or its ligand) and thus indicate the antagonist's ability to inhibit, neutralize, or block the activity of the target polypeptide. The efficacy of an antagonist, such as its ability to inhibit the function of a polypeptide, can also be determined using functional assays. For example, a functional assay may include the steps of contacting a polypeptide with a candidate antagonist molecule and measuring a detectable change in one or more biological activities typically associated with the polypeptide. The potency of an antagonist is usually determined by its IC (Impulse Control Value). 50 It is defined by the value (the concentration required to inhibit 50% of the agonist response). IC 50The lower the value, the greater the antagonist's potency, and the lower the concentration required to inhibit the maximum biological response.

[0200] In some embodiments, at least one antigen-binding arm is 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 M's K D In some embodiments, both antigen-binding arms have the same or similar K D It has "K D The term (M), as used herein, refers to the specific antigen-binding arm / antigen interaction dissociation equilibrium constant. D =k d / k a . "k d ”(seconds -1 The term ) as used herein refers to the specific antigen-binding arm / antigen interaction dissociation rate constant. This value is k off Also called value. a (M -1 × seconds -1 The term ) as used herein refers to the specific antigen-binding arm / antigen interaction association rate constant. This value is k on It is also called a value.

[0201] In some embodiments, the binding of one arm of a multispecific antigen-binding construct (e.g., the first arm) to its target does not block the binding of the other arm (e.g., the second arm) to its target. In some embodiments, the binding of one arm does not sterically interfere with the 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 the ligand of PD-1 (e.g., PD-L1 and / or PD-L2). Therefore, in some embodiments, the first and second arms bind to their respective targets, and both arms remain bound simultaneously.

[0202] In some embodiments, the binding of the first and second arms to their respective targets causes bridging to bring an immune cell and a 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, and 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 expressing either PD-1 or its ligand.

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

[0204] In some embodiments, the binding of the first and second arms of a multispecific construct to their respective targets results in downregulation and / or shedding and / or degradation of the target, e.g., PD-1. As used herein, “downregulation” refers to the process by which a cell reduces the amount of cellular components such as RNA or protein. In the case of cell surface protein receptors, downregulation may occur through the internal translocation of the receptor as a consequence of binding to a ligand or any construct described herein. Shedding or shedding of the ectodomain refers to the process by which cell surface proteins are cleaved by proteolysis, resulting in the release of their ectodomains into the extracellular environment. Non-limiting examples of schidases that regulate ectodomain shedding include members of the disintegrin and metalloproteinase (ADAM) families, e.g., ADAM8, ADAM9, ADAM10, ADAM12, ADAM15, ADAM17, and ADAM28, as well as matrix metalloproteinases (MMPs), e.g., MMP2, MMP3, MMP7, MMP9, and MMP14. The distance and structure of the cleavage site region from the plasma membrane are considered more important than specific sequences in ectodomain shedding. Proteolysis or proteolysis refers to a set of processes that result in the hydrolysis of one or more peptide bonds in a protein, either through the catalytic action of proteolytic enzymes called proteases, or non-enzymatically, for example, at very low or very high pH. In eukaryotic cells, two major pathways, the ubiquitin-proteasome pathway and lysosomal protein hydrolysis, mediate 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 have been described in examples (see, for example, Figures 12A–12C), and include, for example, flow cytometry, Western blotting, immunoprecipitation, microscopy, or fluorescence plate reading.

[0205] As described herein, constructs of the present invention can bridge immune cells expressing PD-1, and second cells expressing its ligand, such as second immune cells, and / or cancer or tumor cells. As those skilled in the art will recognize, the type of immune cell depends on the context of the disease being treated, and the specific type of immune cell can be readily determined depending on the disorder being considered. In some embodiments, the immune cells are T cells, such as regulatory T cells (also known as suppressor T cells), which include CD8+ T cells and CD4+ T cells, as well as subtypes, such as CD4 + FOXP3 + T reg cells, CD4 + FOXP3 - T reg Cells, Tr1 cells, Th3 cells, and T reg 17 cells are included. 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 (the cell expressing the PD-1 ligand) is a second immune cell, e.g., a regulatory immune cell. In some embodiments, the regulatory immune cell is one or more of the following: regulatory T cells, B cells, macrophages, myeloid suppressor cells, dendritic cells, or mesenchymal stromal cells. 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 it also includes non-malignant (non-cancerous) cells that exhibit increased proliferation compared to normal cells. In some embodiments, tumor cells are cancers that can be treated by blocking the interaction between PD-1 expressed by immune cells and its ligand (e.g., PD-L1 or PD-L2) expressed on the second cell, as well as by bridging the immune cells and tumor cells. In some embodiments, tumor cells are selected from the group consisting of hematological cancers, lymphomas, myelomas, leukemias, neurological cancers, melanomas, breast cancers, prostate cancers, colorectal cancers, lung cancers, head and neck cancers, gastrointestinal cancers, liver cancers, pancreatic cancers, genitourinary cancers, bone cancers, kidney cancers, and vascular cancers.In some embodiments, tumor cells include Kaposi's sarcoma, leukemia, acute lymphoblastic leukemia, acute myeloid leukemia, myeloblastic, promyelocytic, myelomonocytic, monocytic erythroleukemia, chronic leukemia, chronic myeloid (granulocytic) leukemia, chronic lymphocytic leukemia, mantle cell lymphoma, primary central nervous system lymphoma, Burkitt lymphoma, and marginal zone B-cell lymphoma, and polycythemia vera. Lymphoma, Hodgkin's disease, non-Hodgkin's disease, multiple myeloma, Waldenström macroglobulinemia, heavy chain disease, solid tumors, sarcomas, carcinomas, fibrosarcomas, myxosarcomas, liposarcomas, chondrosarcomas, osteogenic sarcomas, osteosarcomas, chordomas, angiosarcomas, endosarcomas, lymphangiosarcomas, lymphoendosarcomas, synoviomas, mesotheliomas, Ewing's tumors, leiomyosarcomas Rhabdomyosarcoma, colonic 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, seminomastoma, fetal carcinoma, Wilms' tumor, cervical cancer, uterine cancer, testicular cancer, lung cancer, small cell lung cancer, non-small cell lung cancer, bladder cancer, epithelial carcinoma, nerve cancer The following are selected from the group consisting of gliomas, astrocytomas, medulloblastomas, craniopharyngiomas, ependymomas, pineal glands, hemangioblastomas, acoustic neuromas, oligodendrogliomas, meningiomas, melanomas, neuroblastomas, retinoblastomas, nasopharyngeal cancers, esophageal cancers, basal cell carcinomas, biliary tract cancers, bladder cancers, bone cancers, brain and central nervous system (CNS) cancers, cervical cancers, choriocarcinomas, colorectal cancers, connective tissue cancers, digestive system cancers, endometrial cancers, esophageal cancers, eye cancers, head and neck cancers, gastric cancers, carcinomas in situ, kidney cancers, laryngeal cancers, liver cancers, lung cancers (small cell and large cell), melanomas, neuroblastomas; oral cancers (e.g., lips, tongue, mouth and pharynx), ovarian cancers, pancreatic cancers, retinoblastomas, rhabdomyosarcomas, rectal cancers; respiratory system cancers, sarcomas, skin cancers, gastric cancers, testicular cancers, thyroid cancers, uterine cancers, and urinary system cancers.

[0208] As described herein, the multispecific antigen-binding constructs of the present invention include a bispecific, triplicate, quadruplicate, or multispecific antibody (immunoglobulin) or its antigen-binding moiety or fragment.

[0209] The term "immunoglobulin" refers to a structurally related class of proteins that generally include two pairs of polypeptide chains: one pair of light (L) chains and one pair of heavy (H) chains. In "intact immunoglobulins," 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, Pennsylvania, USA]. Briefly speaking, each heavy chain typically contains a heavy chain variable region (V H ) and heavy chain constant region (C H ) includes. The heavy chain constant region typically consists of three domains, C H1 , C H2 , and C H3 Each light chain typically has a light chain variable region (V L ) and the light chain steady region. The light chain steady region is typically C L It contains one domain, abbreviated as . The term "immunoglobulin" (Ig) may be used interchangeably with the term "antibody" in this specification.

[0210] The term "antibody" is used herein in its broadest sense to describe a type of immunoglobulin molecule. Antibodies include, in particular, intact antibodies (e.g., intact immunoglobulins) and antibody fragments, such as antigen-binding fragments of antibodies as described herein. Therefore, "antibody" can refer to both intact antibodies and their antigen-binding fragments. An antibody contains at least one antigen-binding domain. One example of an antigen-binding domain is V H -V L This is an antigen-binding domain formed by a dimer. Antibodies can be described by the antigen to which they specifically bind. For example, a PD-1 antibody, substituted for an anti-PD-1 antibody, is an antibody that specifically binds to the inhibitory receptor PD-1.

[0211] V H and V L The region can be further divided into hypervariable regions (also called complementary determination regions (CDRs) or "hypervariable regions" (HVRs)) where more conserved regions are scattered. The more conserved regions are called framework regions (FRs). Each V H and V L Generally, it contains three CDRs and four FRs arranged in the following order (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] (referenced herein).

[0212] Light chains from vertebrate species can be assigned to one of two types, called kappa and lambda, based on the sequence of their constant domains. Heavy chains from vertebrate species can be assigned to one of five distinct classes (or isotypes): IgA, IgD, IgE, IgG, and IgM. These classes are also designated α, δ, ε, γ, and μ, respectively. The IgG and IgA classes are further divided into subclasses based on differences in sequence and function. Humans express the following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.

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

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

[0215] The "humanized" form of a non-human antibody is a chimeric antibody containing the smallest sequence derived from the non-human antibody. A humanized antibody is generally a human immunoglobulin (recipient antibody) in which one or more residues from a CDR are replaced by one or more residues from a non-human antibody (donor antibody) CDR. The donor antibody can be any suitable non-human antibody, such as a mouse, rat, rabbit, chicken, or non-human primate antibody, possessing the desired specificity, affinity, or biological effect. In some cases, selected framework region residues of the recipient antibody are replaced by corresponding framework region residues from the donor antibody. Humanized antibodies can also contain residues not found in either the recipient or 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] "Human antibodies" are those that have an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human or human cell, or that originate from a non-human source that utilizes the human antibody repertoire or a human antibody coding sequence (e.g., obtained from a human source or designed de novo). Human antibodies specifically exclude humanized antibodies.

[0217] In some embodiments, the antibody molecule comprises an antigen-binding fragment of the antibody (e.g., Fab, F(ab')2, and Fv) in addition to the diabody and single-chain molecule. For example, the antibody molecule may 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, the antibody molecule comprises or consists of one heavy chain and one light chain (referred to as a half-antibody). In another example, the 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, diabody (Dab) (bivalent and bispecific), and chimeric (e.g., humanized) antibodies, which can be produced by modification of a whole antibody or may be synthesized de novo using recombinant DNA technology. These functional antibody fragments retain the ability to selectively bind to their respective antigens. Antibodies and antibody fragments can be from any class of antibodies, including but not limited to IgG, IgA, IgM, IgD, and IgE, as well as from any subclass of antibodies (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 graft, or in vitro-generated antibodies. Antibodies can have a heavy chain constant region selected from, for example, IgG1, IgG2, IgG3, or IgG4. Antibodies can also have a light chain selected from, for example, kappa or lambda.In some embodiments, the antibody is sequence number 64 (ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLN It contains an IgG1 heavy chain constant region having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical amino acid sequence to GKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK). In some embodiments, the antibody is sequence number 68 (ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGK It contains an IgG 4 heavy chain constant region having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical amino acid sequence to EYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK).In some embodiments, the antibody is sequence number 69 (ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNG It contains an IgG 4 heavy chain constant region having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical amino acid sequence to KEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG).

[0218] Antigen-binding portions or fragments of antibody molecules are well known in the art, for example, (i) Fab fragments, which are monovalent fragments consisting of VL, VH, CL, and CH1 domains; (ii) F(ab')2 fragments, which are bivalent fragments containing two Fab fragments linked by a disulfide bridge in the hinge region; (iii) Fd fragments consisting of VH and CH1 domains; (iv) Fv fragments consisting of VL and VH domains of a single arm of the antibody; (v) diabody (dAb) fragments consisting of a VH domain; (vi) camelid or camelidization variable domains; (vii) single-chain Fv (scFv) (e.g., Bird et al.) See (al.) (1988) Science 242:423-426; Houston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883); (viii) Includes single-domain antibodies. These antibody fragments are obtained using prior art known to those skilled in the art, and the fragments are screened for utility in the same manner as intact antibodies.

[0219] Antibody molecules can also be single-domain antibodies. Single-domain antibodies may include antibodies whose complementary determining regions are a portion of a single-domain polypeptide. Examples include, but are not limited to, heavy-chain antibodies, naturally deficient light-chain antibodies, single-domain antibodies derived from conventional four-chain antibodies, engineered antibodies, and single-domain scaffolds other than those derived from antibodies. A single-domain antibody can be any single-domain antibody in the art or any future single-domain antibody. A single-domain antibody can be derived from any species, including but not limited to mice, humans, camels, llamas, fish, sharks, goats, rabbits, and cattle. According to another aspect of the invention, a single-domain antibody is a naturally occurring single-domain antibody known as a light-chain deficient heavy-chain antibody. Such a single-domain antibody is disclosed, for example, in International Publication No. 9404678. For clarity, this variable domain derived from a naturally deficient light-chain heavy-chain antibody is referred to herein as VHH or nanobody to distinguish it from the conventional VH of four-chain immunoglobulins. Such VHH molecules can be derived from antibodies produced in camelid species, such as camels, llamas, dromedaries, alpacas, and guanacos. Other species besides camelids may naturally produce heavy-chain antibodies lacking light chains, and such VHHs are within the scope of this invention.

[0220] In some embodiments, the multispecific antigen-binding construct includes a bispecific antibody. A bispecific antibody has specificity for two or fewer antigens, but may 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 PD-L1 ligand). In some embodiments, the bispecific antibody molecule includes 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, for example, Kontermann and Brinkmann, (2015), Drug Discovery Today, 20(7):838-47 (as incorporated herein).

[0221] Various formats and methods are known in the art that can be used to generate the multivalent and / or multispecific constructs described herein, such as multivalent and / or multispecific antibody formats of both asymmetric and symmetric architectures. Non-limiting examples of such formats include: (i) Fc-less bispecific antibody formats, e.g., tandem single-chain variable fragments (scFv2, taFv) and triple bodies, e.g., bispecific T cell engager (BiTE) and bispecific killer cell engager (BiKE) molecules; bispecific single-domain antibody fusion proteins, e.g., VH or VL domains, VHH, VNAR and nanobodies; diabodies and diabody derivatives, e.g., tandem diabodies and dual-affinity retargeting. (ii) retargeting) (DART) proteins; Fab fusion proteins; and heterodimerized peptides or mini-antibodies from various proteins, e.g., other Fc-less fusion proteins through the use of leucine zippers having a coiled-coil structure; (ii) bispecific IgG with an asymmetric architecture, e.g., asymmetric IgG with heavy and light chains from two different antibodies; knob-into-hole approach, electrostatic interaction (steering) to avoid homodimerization of the CH3 domain, preferential heavy chain heterodimerization by introducing charge pairs into the hinge regions of IgG1 and IgG2, strand-exchange engineered domain (SEED) heterodimers, and bispecific engagement by antibodies based on the T cell receptor (iii) Bispecific IgG having an asymmetric Fc region using T cell receptor (BEAT) technology; asymmetric Fc and CH3 fusion proteins; (iii) Bispecific antibodies having a symmetric architecture, e.g., additional IgG by scFv fusion, fusions of domain antibodies and scaffold proteins, Fab arm fusions, and fusions of additional variable heavy and light chain domains; modified IgG molecules; symmetric Fc and CH3-based bispecific antibodies; and bispecific antibodies using immunoglobulin-derived homodimerized domains. See, for example, “The making of bispecific antibodies,” Brinkmann and Kontermann, MABS 2017, Vol. 9:2, pp. 182-212 (the entire content of which is incorporated herein by reference). See, for example, “knob in a hole” as described in U.S. Patent No. 5,731,168. hole) approach; e.g., electrostatic steering Fc pairing as described in International Publication Nos. 09 / 089004, 06 / 106905 and 2010 / 129304; e.g., chain exchange domain (SEED) heterodimer formation as described in International Publication No. 07 / 110205; e.g., Fab arm exchange as described in International Publication Nos. 08 / 119353, 2011 / 131746 and 2013 / 060867; e.g., amine-reactive group and s as described in U.S. Patent No. 4,433,059 Bi-antibody conjugates by antibody crosslinking to generate bispecific structures using heterobifunctional reagents having sulfhydryl-reactive groups; for example, bispecific antibody determinants produced by the recombination of half-antibodies (heavy-light chain pairs or Fabs) from different antibodies through a reduction and oxidation cycle of the disulfide bond between two heavy chains, as described in U.S. Patent No. 4,444,878; for example, trifunctional antibodies, e.g., three Fab' fragments crosslinked via sulfhydryl-reactive groups, as described in U.S. Patent No. 5,273,743; for example,Biosynthetic binding proteins, such as those described in U.S. Patent No. 5,534,254, e.g., pairs of scFv crosslinked through the C-terminal tail, preferably via disulfide or amine-reactive chemical crosslinks; bifunctional antibodies, such as those described in U.S. Patent No. 5,582,996, e.g., Fab fragments with different binding specificities dimerized via leucine zippers (e.g., c-fos and c-jun) with a constant domain replaced; bispecific and oligo- Specific monovalent and oligovalent receptors, e.g., the VH-CH1 regions of two antibodies (two Fab fragments) linked via a polypeptide spacer between the CH1 region of one antibody and the VH region of another antibody, typically having associated light chains; bispecific DNA-antibody conjugates, e.g., as described in U.S. Patent No. 5,635,602, e.g., crosslinking of antibodies or Fab fragments through double-stranded DNA fragments; bispecific fusion proteins, e.g., as described in U.S. Patent No. 5,637,481, e.g., those with full-length constant regions Expression constructs containing two scFv having a hydrophilic helical peptide linker between them; for example, polyvalent and multispecific binding proteins, such as those described in U.S. Patent No. 5,837,242, e.g., dimers of polypeptides having a first domain with a binding region of the Ig heavy chain variable region and a second domain with a binding region of the Ig light chain variable region, commonly referred to as a diabody (higher-order structures that produce bispecific, tripspecific, or quadrispecific molecules are also included); for example, as described in U.S. Patent No. 5,837,821 Mini-body constructs having linked VL and VH chains further connected to the antibody hinge region and CH3 region using peptide spacers, which can be dimerized to form bispecific / polyvalent molecules; VH and VL domains linked in either direction using short peptide linkers (e.g., 5 or 10 amino acids) or without linkers at all, which can be dimerized to form bispecific diaboves; trimers and tetramers, for example, as described in U.S. Patent No. 5,844,094; for example,See also, for example, U.S. Patent No. 5,864,019, a chain of VH domains (or VL domains in family members) linked by peptide linkage using a crosslinkable group at the C-terminus that further associates with a VL domain to form a series of FVs (or scFvs); and also, for example, U.S. Patent No. 5,869,620, in which single-chain binding polypeptides having both VH domains and VL domains linked via a peptide linker are combined into a polyvalent structure through non-covalent or chemical crosslinking to form homodivalent, heterodivalent, trivalent, and tetravalent structures using both scFV and diabody formats. Additional exemplary multispecific and bispecific molecules and methods for producing them are, for example, U.S. Patent Nos. 5,910,573, 5,932,448, 5,959,083, 5,989,830, 6,005,079, 6,239,259, 6,294,353, 6,333,396, 6,476,198, 6,511,663, 6,670,453, 6,743,896, 6,809,185, 6,833,441, 7,129,330, and 7,183,076. U.S. Patent No. 7,521,056, U.S. Patent No. 7,527,787, U.S. Patent No. 7,534,866, U.S. Patent No. 7,612,181, U.S. Patent Publication No. US2002004587, U.S. Patent Publication No. 2002076406, U.S. Patent Publication No. 2002103345, U.S. Patent Publication No. 2003207346 Specification, U.S. Patent Application Publication No. 2003211078, U.S. Patent Application Publication No. 2004219643, U.S. Patent Application Publication No. 2004220388, U.S. Patent Application Publication No. 2004242847, U.S. Patent Application Publication No. 2005003403, U.S. Patent Application Publication No. 2005004352, U.S. Patent Application Publication No. 2005069552,U.S. Patent Application Publication No. 2005079170, U.S. Patent Application Publication No. 2005100543, U.S. Patent Application Publication No. 2005136049, U.S. Patent Application Publication No. 2005136051, U.S. Patent Application Publication No. 2005163782, U.S. Patent Application Publication No. 2005266425, U.S. Patent Application Publication No. 2006083747, U.S. Patent Application Publication No. 2006120960, U.S. Patent Application Publication No. 2006204493, U.S. Patent Application Publication No. 263367, U.S. Patent Application Publication No. 2 Specification No. 007004909, U.S. Patent Application Publication No. 2007087381, U.S. Patent Application Publication No. 2007128150, U.S. Patent Application Publication No. 2007141049, U.S. Patent Application Publication No. 2007154901, U.S. Patent Application Publication No. 2007274985, U.S. Patent Application Publication No. 2008050370, U.S. Patent Application Publication No. 2008069820, U.S. Patent Application Publication No. 2008152645, U.S. Patent Application Publication No. 2008171855, U.S. Patent Application Publication No. 2008241884 Specification, U.S. Patent Application Publication No. 2008254512, U.S. Patent Application Publication No. 2008260738, U.S. Patent Application Publication No. 2009130106, U.S. Patent Application Publication No. 2009148905, U.S. Patent Application Publication No. 2009155275, U.S. Patent Application Publication No. 2009162359, U.S. Patent Application Publication No. 2009162360, U.S. Patent Application Publication No. 2009175851, U.S. Patent Application Publication No. 2009175867, U.S. Patent Application Publication No. 2009232811, U.S. Patent Application Publication No. 2009234105, U.S. Patent Application Publication No. 2009263392, U.S. Patent Application Publication No. 2009274649, European Patent Application Publication No. 346087, and International Publication No. 0006605, International Publication No. 02072635, International Publication No. 04081051, International Publication No. 06020258, International Publication No. 2007044887, International Publication No. 2007095338, International Publication No. 2007137760, International Publication No. 2008119353, International Publication No. 2009021754, International Publication No. 2009068630,This information is found in International Publications 9103493, 9323537, 9409131, 9412625, 9509917, 9637621, and 9964460. The contents of the applications referenced above 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 this disclosure may be generated against PD-1 and PD-L1, or against PD-1 and PD-L2. The antibody arms of the bispecific antibodies may be generated by standard techniques as disclosed herein. In some embodiments, bispecific antibodies according to this disclosure can be generated using any known antibody against PD-1 and its ligand. For example, such bispecific constructs are illustrated herein (see, for example, pembrolizumab (PD-1 antibody) conjugated with atezolizumab (PD-L1 antibody) in Figure 3; and nivolumab (PD-1 antibody) conjugated with atezolizumab (PD-L1 antibody) in Figure 4). As illustrated herein, the multispecific antigen-binding constructs (e.g., bispecific antibodies) described herein may be generated using antibodies known and / or available in the art.

[0223] In some embodiments, the bispecific antibody is bivalent, for example, 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-reacting with both ligands). In some embodiments, the bispecific antibody is tetravalent, such as the novel bispecificity 3 and bispecificity 4 antibodies described herein. For example, as illustrated in Figure 3, the pembrolizumab-binding arm is bivalent for PD-1, each binding to the same epitope on PD-1, and the atezolizumab-binding arm is bivalent for PD-L1, each binding to the same epitope on PD-L1. This can also be seen, for example, in the bispecificity format in Figure 8 (an exemplary common light chain bispecificity illustrated in the left panel of step 2 of the workflow presented in Figure 8). The exemplary common light chain bispecificity format in Figure 8 (the format illustrated in the right panel of step 2 of the workflow) represents another example of a tetravalent bispecificity format. In contrast to the tetravalent bispecificity format in which the first antigen-binding arm is joined to the second antigen-binding arm on the opposite end of the Fc region, here each Fab of the first antigen-binding arm is joined 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 Figure 8, the format illustrated in the right panel of step 2 of the workflow.

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

[0226] In a particular embodiment, the first antigen-binding arm and the second antigen-binding arm are linked by at least one aminolinker amino acid sequence. Optionally, the linker amino acid sequence is GGGGS x (Sequence code 121) (x is an integer from 1 to 6, including 1 and 6).

[0227] In some embodiments, the multispecific antigen-binding construct does not contain an immunoglobulin Fc domain. In some embodiments, the construct includes an immunoglobulin Fc domain. In some embodiments, the first arm or the second arm of the construct, or both, include a heavy chain containing one or more immunoglobulin Fc modifications. In some embodiments, the immunoglobulin Fc domain of the heavy chain includes one or more amino acid mutations that, for example, promote heterodimerization of the first and second arms, extend the serum half-life, and / or modify effector function. In some embodiments, the mutations reside in the CH3 domain of the heavy chain (see, e.g., Xu et al., mAbs 7(1):231-42, 2015).

[0228] Traditional Fc fusion proteins and antibodies are examples of unguided interaction pairs; however, various engineered Fc domains have been designed as asymmetric interaction pairs to promote heterodimerization of the first and second antigen-binding arms, for example (Spiess et al. (2015) Molecular Immunology 67(2A):95-106). Various methods are known in the art to increase the formation of desired pairs of Fc-containing polypeptide chains in single cell lines to produce preferred asymmetric fusion proteins in acceptable yields [see, e.g., Klein et al. (2012) mAbs 4:653-663; and Spiess et al. (2015) Molecular Immunology 67(2PartA):95-106]. Methods for obtaining desired pair formation of Fc-containing polypeptides include, but are not limited to, charge-based pair formation (electrostatic steering), "knob-into-hole" stereopair formation, seed body pair formation, and leucine zipper-based pair formation. For example, 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. See 287:43331-43339; U.S. Patent No. 5,932,448; and International Publication Nos. 1993 / 011162, 2009 / 089004, and 2011 / 034605.

[0229] For example, one means of promoting interactions between specific polypeptides is by manipulating protuberance-into-cavity (knob-into-hole) complementary regions, as described by Arathoon et al., U.S. Patent No. 7,183,076; Carter et al., U.S. Patent No. 5,731,168; and Kumar et al., International Publication No. 2016 / 164089 (as incorporated herein). The “protuberance” (protrusion) is constructed by replacing a smaller amino acid side chain from the interface of the first polypeptide (e.g., the first interaction pair) with a larger side chain (e.g., tyrosine or tryptophan). A complementary “cavity” of the same or similar size as the protuvément is optionally created on the interface of the second polypeptide (e.g., the second interaction pair) by replacing the larger amino acid side chain with a smaller one (e.g., alanine or threonine). If a protuvément or cavity of suitable location and size exists at the interface of either the first or second polypeptide, it is only necessary to manipulate the corresponding cavity or protuvément at the adjacent interface.

[0230] At a 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 heterodimerization and simultaneously inhibit homodimerization. Attractive interactions occur between opposite charges, and repulsive interactions occur between similar charges. Partially, the protein complexes disclosed herein use attractive interactions to promote heteromultimerization (e.g., heterodimerization) by performing site-directed mutagenesis of charged interface residues, and optionally use repulsive interactions to inhibit homodimerization (e.g., homodimerization).

[0231] For example, the IgG1 CH3 domain interface contains four unique charge residue pairs 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 specify residues in the IgG1 CH3 domain follows Kabat's EU numbering scheme. Due to the double symmetry present in the 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' prefers the formation of both heterodimers and homodimers. A single mutation in the first chain that reverses charge polarity (e.g., K409E; positive to negative charge) leads to an interaction unfavorable for homodimer formation of the first chain. This unfavorable interaction is due to repulsive interactions between identical charges (negative-negative; K409E-D399' and D399-K409E'). A similar mutation in the second chain that reverses charge polarity (D399K'; negative to positive) leads to an interaction unfavorable for homodimer formation of the second chain (K409'-D399K' and D399K-K409'). However, these two mutations (K409E and D399K') simultaneously lead to an interaction favorable for heterodimer formation (K409E-D399K' and D399-K409'). The electrostatic steering effect on heterodimer formation and homodimerization can be further enhanced by mutations in additional charged residues, which may or may not be paired with oppositely charged residues in the second chain, such as Arg355 and Lys360 (see, for example, International Publication 2016 / 164089).

[0232] Therefore, in some embodiments, the multispecific antigen-binding constructs described herein (e.g., bispecific constructs) may include a constant domain of immunoglobulin, for example, containing the Fc portion of the immunoglobulin. For example, the first arm may include an amino acid sequence derived from the Fc domain of IgG (IgG1, IgG2, IgG3, or IgG4), IgA (IgAl or IgA2), IgE, or IgM immunoglobulin. Optionally, the second arm may include an amino acid sequence derived from the Fc domain of IgG (IgG1, IgG2, IgG3, or IgG4), IgA (IgAl or IgA2), IgE, or IgM. Such immunoglobulin domains may include one or more amino acid modifications (e.g., deletions, additions, and / or substitutions) that promote heterodimerization. In some embodiments, the multispecific antigen-binding construct is the IgG1 isotype. In some embodiments, the multispecific antigen-binding construct is the IgG1 isotype and includes substitutions. In some embodiments, the multispecific antigen-binding construct is the IgG2 isotype. In some embodiments, the multispecific antigen-binding construct is the IgG3 isotype. In some embodiments, the multispecific antigen-binding construct is the IgG4 isotype. In some embodiments, the multispecific antigen-binding construct is the IgG4 isotype and includes substitutions. In some embodiments, the substitution is at Ser228, as numbered according to EU numbering. In some embodiments, the substitution at Ser228 is S228P. In some embodiments, the first and second arms include Fc domains derived from the same immunoglobulin class and subtype. In some embodiments, the first and second arms include Fc domains derived from different immunoglobulin classes or subtypes. Similarly, the first and / or second arms (e.g., an asymmetric pair or a non-guiding interaction pair) include a constant domain of the modified immunoglobulin, for example, one or more amino acid modifications (e.g., deletions, additions, and / or substitutions) that promote heterodimerization. Methods for generating Fc modifications having desired heterodimerization 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 decrease the binding of an antibody to an Fc receptor at acidic pH compared to neutral pH are known in the art. For example, the constructs disclosed herein can contain mutations in the C H 2 or C H 3 region, and the mutations increase the affinity of the Fc domain for FcRn in an acidic environment (e.g., in an endosome where the pH ranges from about 5.5 to about 6.0). Such mutations can result in an increase in the serum half-life of the construct when administered to an animal. Methods for modifying the Fc domain for desired characteristics such as an enhanced serum half-life are known in the art.

[0234] In some embodiments, the constructs described herein include a modified heavy-chain constant region having reduced effector function (or no effector function) compared to its corresponding unmodified constant region. The effector function associated with the constant region of the constructs described herein can be modulated by changing the properties of the constant or Fc region. Modified effector functions include, for example, the following activities: antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), apoptosis, binding to one or more Fc receptors, and modulation in one or more of the pro-inflammatory responses. Modulation refers to an increase, decrease, or elimination of the effector function activity presented by the subject antibody containing the modified constant region compared to the activity of the unmodified form of the constant region. In specific embodiments, modulation includes situations where the activity is lost or completely absent.

[0235] A modified constant region having altered FcR binding affinity and / or ADCC activity and / or altered CDC activity is a polypeptide having either increased or decreased FcR binding activity and / or ADCC activity and / or CDC activity compared to the unmodified constant region. A modified constant region showing increased binding to FcR binds to at least one FcR with higher affinity than the unmodified polypeptide. A modified constant region showing decreased binding to FcR binds to at least one FcR with lower affinity than the unmodified constant region. Such variants exhibiting reduced binding to FcR may have little to no recognizable binding to FcR, for example, 0–50% (e.g., 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 less than 1%) compared to the level of binding to FcR of the native sequence immunoglobulin constant or Fc region. Similarly, a modified constant region exhibiting modulated ADCC and / or CDC activity may exhibit either increased or decreased ADCC and / or CDC activity compared to an unmodified constant region. For example, in some embodiments, one or more of the antibodies described herein, including a modified constant region, may exhibit approximately 0–50% of the ADCC and / or CDC activity of the unmodified form of the constant region (e.g., 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 less than 1%).The multispecific antigen-binding constructs described herein, which include an altered constant region exhibiting reduced ADCC and / or CDC activity, 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 construct includes 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. See 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 a modified constant region exhibiting 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, for example, U.S. Patent No. 6,194,551.

[0238] The constructs and antigen-binding arms described herein may, in part, include scaffold domains, proteins, or moieties that do not provide target receptor-binding activity but can provide a portion or domain of the construct that provides spatial organization, structural support, a means of linking multiple receptor-binding units, or other desired features, such as an improved half-life. Various scaffolding techniques and compositions are known in the art and can be readily linked or conjugated to the antigen-binding units described herein. The scaffold domains, proteins, or moieties may be antibody-derived or non-antibody-derived. 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 (ranging from 6 to 20 kDa) and constrained peptides (ranging from 2 to 4 kDa). Examples of domain-sized non-antibody scaffolds include, but are not limited to, afibodies, affilins, antikalins, atrimers, DARPin, FN3 scaffolds (e.g., adonectin and centinlin), finomers, Knitz domains, pronectin, and OBody. Examples of peptide-sized non-antibody scaffolds include avimers, bicyclic peptides, and cysteine ​​knots. These non-antibody scaffolds and their base or derived basal proteins or peptides 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 are incorporated into this specification 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 permeability, smaller size, and thermal stability. Some non-antibody scaffolds can also allow for easier construction without being hindered by issues such as light chain association, for example, when bispecific constructs are desired. Methods for constructing constructs on non-antibody scaffolds are known to those skilled in the art. Although not formally on antibody scaffolds, such constructs often contain an antibody-binding domain, whether in the form of a single-domain antibody, scFv, or other antibody-binding domain variant, providing specific target-binding capability.

[0240] Therefore, in some embodiments of any aspect described herein, the construct may include a non-antibody scaffold protein. In some embodiments of any aspect described herein, at least one of the receptor-binding units may include a non-antibody scaffold protein. The scaffold portion of non-antibody scaffold proteins is, in some embodiments, for example, an adnectin scaffold or portion derived from the human fibronectin type 10 domain (10Fn3); an anticarin scaffold derived from human lipocalin (e.g., as described in International Publication No. 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 Knitz domain scaffold or portion of Knitz-type protease inhibitors such as human trypsin inhibitors, aprotinin (bovine pancreatic trypsin inhibitor), Alzheimer's amyloid precursor protein, and tissue factor pathway inhibitors; E. elaterium (E. Those skilled in the art will understand that these may include nottin scaffolds (cysteine ​​knot miniprotein) based on trypsin inhibitors from elaterium; affibodi scaffolds or the whole 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) protein; or any scaffolds derived from or based on human transferrin, human CTLA-4, human crystallin, and human ubiquitin. For example, the binding sites of human transferrin for human transferrin receptors can be diversified to create diverse libraries of transferrin variants in which parts acquire affinity for different antigens.See, for example, Ali et al. (1999) J. Biol. Chem. 274:24066-24073. The portion of human transferrin not involved in receptor binding remains unchanged and acts as a scaffold, like a framework region of the antibody, for presenting the variant binding site. The library is then screened against the target antigen of interest as an antibody library and according to the method described herein, to identify variants with optimal selectivity and affinity for the target antigen. See, for example, Hey et al. (2005) TRENDS Biotechnol. 23(10):514-522.

[0241] D. Method for producing multispecific antigen-binding constructs This disclosure also features methods for producing any multispecific antigen-binding constructs described herein. In some embodiments, the methods for producing the constructs of the present invention include methods for preparing antibodies and / or fragments thereof, as described herein. Such methods are well known in the art and may include, for example, the step of immunizing a subject (e.g., a non-human mammal) with a suitable immunogen. For example, to produce an antibody that binds to PD-1, a person 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 human PD-1 polypeptide, such as a full-length human PD-1 polypeptide containing the amino acid sequence described in SEQ ID NO. 114 (GenBank accession number NP_005009.2; UniProt Q15116), its antigenic fragment, and / or variant. Similarly, to produce antibodies that bind to a ligand of PD-1 (e.g., PD-L1), a person skilled in the art can immunize a suitable target using a full-length human PD-L1 polypeptide, such as the full-length human PD-L1 polypeptide containing the amino acid sequence described in SEQ ID NO: 115 (GenBank accession number NP 054862.1, UniProt Q9NZQ7), its antigenic fragment, and / or variants. Similarly, to produce antibodies that bind to PD-L2, a person skilled in the art can immunize a suitable target using a full-length human PD-L2 polypeptide, such as the full-length human PD-L2 polypeptide containing the amino acid sequence described in SEQ ID NO: 116 (GenBank accession number NP_079515.2, UniProt Q9BQ51), its antigenic fragment, and / or variants.

[0242] As those skilled in the art will recognize, full-length polypeptides (PD-1, PD-L1, or PD-L2) can be used as antigens, and antibodies can be screened for desired binding properties (e.g., blockade of PD-1 / ligand interactions; the ability to bridge cells expressing PD-1 and its ligand). As those skilled in the art will also recognize, antigenic fragments of polypeptides (PD-1, PD-L1, or PD-L2) can be selected based on known structural features of the polypeptide. For example, PD-1 / PD-L1 and PD-1 / PD-L2 interactions are well-characterized structurally (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, based on information on receptor / ligand interfaces available in the art, suitable antigenic fragments with desired binding properties can be designed using regions, for example, within PD-1, PD-L1, and / or PD-L2. For example, the PD-1 ectodomain contains a single IgV domain typical of the CD28 family, while PD-L1 and PD-L2 consist 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 and primarily involve inter-face interactions 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).Furthermore, the six amino acids of the C, F, and G strands of PD-1 form a recessed hydrophobic core that interacts with the F and G strands of PD-L2, as well as the FG loop. 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, a person skilled in the art can determine a suitable antigenic region for producing an antibody with desirable properties. For example, a person skilled in the art can produce an antibody that cross-reacts with both ligands PD-L1 and PD-L2 (see, for example, U.S. Patent No. 9,845,356).

[0243] Suitable subjects (e.g., non-human mammals) can be immunized with appropriate antigens along with a sufficient number of subsequent booster immunizations to induce antibody production by the mammal. The immunogen can be administered to the subject (e.g., non-human mammals) together with an adjuvant. Adjuvants useful in antibody production in the subject include, but are not limited to, protein adjuvants; bacterial adjuvants, e.g., whole bacteria (BCG, Corynebacterium parvum, or Salmonella minnesota), and bacterial components including cell wall skeletons, trehalose dimycolic acid, monophosphoryl lipid A, methanol extractable residue (MER) of Mycobacterium tuberculosis, and complete or incomplete Freund's adjuvants; viral adjuvants; and chemical adjuvants, e.g., aluminum hydroxide, and iodoacetate and cholesteryl hemisuccinate. Other adjuvants that can be used in methods to induce an immune response include, for example, cholera toxin and parapoxvirus protein. 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 that secretes monoclonal antibodies bound to an immunogen. For example, a suitable mammal, such as a laboratory mouse, is immunized with a polypeptide (e.g., PD-1, PD-L1, PD-L2) or antigenic fragment as described above. Antibody-producing cells from the immunized mammal (e.g., spleen B cells) can be isolated 2-4 days after at least one booster immunization with the immunogen and then briefly grown in culture 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 obtained in the fusion are cloned, and cell clones that secrete 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 and grown at regular intervals in a suitable culture medium supplemented with a selective medium, such as HAT medium, to prevent normal myeloma cells from overgrowing beyond the desired hybridoma cells. The resulting hybridoma cells are then screened for the secretion of a desired antibody, such as an antibody that binds to PD-1.

[0245] In some embodiments, those skilled in the art can identify anti-PD-1 antibodies from non-immuno-biased libraries, such as those described in U.S. Patent No. 6,300,064 (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 be used with or in combination with, for example, phage display technology, bacterial display, yeast surface display, eukaryotic virus display, mammalian cell display, and cell-free (e.g., ribosome display) antibody screening technology (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. (See 18:1287-1292).

[0247] Methods for identifying antibodies using various phage display techniques are known in the art. In phage display techniques, the functional antibody domain is presented on the surface of a phage particle having the polynucleotide sequence encoding it. Such phages can be used to present the antigen-binding domain of antibodies such as Fab, Fv, or disulfide-bonded Fv antibody fragments expressed from a repertoire or combinatorial antibody library (e.g., human or mouse). The phages used in these methods are typically filamentous phages such as fd and M13. The antigen-binding domain is expressed as a recombinant fusion protein to one of the phage coat proteins pIII, pVIII, or pIX. For example, Shi et al. See al. (2010) JMB 397:385-396. Examples of phage display methods that can be used to make immunoglobulins or fragments thereof as described herein include Brinkman et al. (1995) J. Immunol. Methods 182:41-50; Ames et al. Examples include those disclosed in PCT International Publications 90 / 02809, 91 / 10737, 92 / 01047, 92 / 18619, 93 / 11236, 95 / 15982, and 95 / 20401. Preferred methods are also described, for example, in U.S. Patent 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 immunized mammals. For example, a spleen cell sample containing B cells can be isolated from a mouse immunized with the PD-1 polypeptide as described above. The mRNA can be isolated from the cells and converted to cDNA using standard molecular biology techniques. For example, Sambrook et al. (1989) “Molecular Cloning: A Laboratory Manual, 2 ndEdition, Cold Spring Harbor Laboratory Press [located in New York]; Harlow and Lane (1988), cited above; Benny KC See Lo) (2004), cited above; and Borrebaek (1995), cited above. Phage display libraries are constructed using cDNA encoding the variable regions of the heavy and light chain polypeptides of immunoglobulins. Methods for generating such libraries are, for example, seen Merz et al. (1995) J This method is described in 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 the antibody of interest from, for example, a population of hybridoma-derived antibodies or a phage display antibody library. Preferred methods are known in the art and are described, for example, Hoogenboom (1997) Trends in Biotechnology 15:62-70; Brinkman et al. (1995), cited above; Ames et al. (1995), cited above; Kettleborough et al. (1994), cited above; Persic et al. (1997), cited above; and Burton et al. (1994), cited above. For example, multiple phagemid vectors encoding fusion proteins of bacteriophage coat proteins (e.g., pIII, pVIII, or pIX of the M13 phage) and different antigen combination regions are fabricated using standard molecular biology techniques and then introduced into a population of bacteria (e.g., Escherichia coli). Bacteriophage expression in bacteria may require the use of helper phages in some embodiments. In some embodiments, helper phages are not required (see, e.g., Chasteen et al. (2006) Nucleic Acids Res. 34(21):e145). The phages produced from the bacteria are then collected and contacted with a target antigen, for example, bound to a (immobilized) solid support. The phages can also be contacted with an antigen in solution, and the complex is subsequently bound to a solid support.

[0250] Subpopulations of antibodies screened using the above method can be characterized for 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 blotting, RIA, ELISA (enzyme-coupled immunosorbent assay), "sandwich" immunoassays, immunoprecipitation assays, immunodiffusion assays, agglutination assays, complement-immobilized assays, immunoradiometric assays, fluorescence immunoassays, and protein A immunoassays. Such assays are conventional and well known in the art.

[0251] It is understood that the above method can also be used to determine, for example, whether an anti-PD-1 antibody does not bind to the 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 is, for example, Shiraishi et al. (2007) Nucleic Acids Symposium It can be chemically synthesized as described in 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 include adherent-end restriction enzyme sites for use in cloning the nucleic acid to 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 selected antibody 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, a crosslinking method can be used to generate a bispecific structure using a heterobifunctional reagent having an amine-reactive group and a sulfhydryl-reactive group, as described in U.S. Patent No. 4,433,059; a bispecific antibody determinant can be generated by recombining half-antibodies (heavy-light chain pairs or Fabs) from different antibodies through a reduction-oxidation cycle of the disulfide bond between two heavy chains, as described in U.S. Patent No. 4,444,878; a trifunctional antibody, for example, three Fab' fragments, can be crosslinked via a sulfhydryl-reactive group, as described in U.S. Patent No. 5,273,743. Other methods for generating bispecific constructs, for example, methods for generating bispecific constructs having a common light chain, are described herein. Non-limiting examples of common 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 prepared using various techniques known in the fields of molecular biology and protein chemistry. For example, nucleic acids encoding a multispecific antigen-binding construct (as a single multifunctional polypeptide or as separate molecules of a multimer complex, e.g., as one antigen-binding arm separately from other antigen-binding arms) can be inserted into an expression vector containing transcriptional and translational regulatory sequences, such as promoter sequences, ribosome binding sites, transcription start and stop sequences, translation start and stop sequences, transcriptional terminator signals, polyadenylation signals, and enhancer or activator sequences. Regulatory sequences include promoters and transcription start and stop sequences. Additionally, the expression vector may contain one or more replication systems so that it can be maintained in two different organisms, e.g., in mammalian or insect cells for expression and in a prokaryotic host for cloning and amplification.

[0254] Several possible vector systems are available for the expression of heavy and light chain polypeptides cloned from nucleic acids in mammalian cells. One class of vectors relies on the integration of the desired gene sequence into the host cell genome. Cells with stably integrated DNA can be selected by simultaneously introducing drug resistance genes such as E. coli (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 selection marker gene can be either ligated to the DNA gene sequence to be expressed or introduced into the same cell by co-transfection (Wigler et al. (1979) Cell 16:77). A second class of vectors utilizes DNA elements that confer autonomous replication ability 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] Expression vectors can be introduced into cells in a manner suitable for subsequent nucleic acid expression. The method of introduction is generally determined by the targeted cell type, which is discussed below. Exemplary methods include CaPO4 precipitation, liposome fusion, cationic liposomes, electroporation, viral infection, dextran-mediated transfection, polybren-mediated transfection, protoplast fusion, and direct microinjection.

[0256] Suitable host cells for the expression of antibodies or their antigen-binding fragments include yeast, bacteria, insects, plants, and mammalian cells. Of particular interest are bacteria such as Escherichia coli (E. coli), fungi such as Saccharomyces cerevisiae and Pichia pastoris, insect cells such as SF9, mammalian cell lines (e.g., human cell lines), and primary cell lines.

[0257] In some embodiments, antibodies or fragments thereof can be expressed in transgenic animals (e.g., transgenic mammals) and purified therefrom. For example, antibodies can be expressed in 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. As described in al.)(1999)J. Immunol. Methods 231(1-2):147-157, it is produced in transgenic non-human mammals (e.g., rodents) and can be isolated from milk.

[0258] Antibodies and their fragments can be produced from cells by culturing transformed host cells with an expression vector containing nucleic acids encoding the antibody or fragment for a period of time and under conditions sufficient to enable protein expression. Such conditions for protein expression vary with the selection of the expression vector and host cell and are readily apparent to those skilled in the art through conventional experiments. For example, antibodies expressed in Escherichia coli (E. coli) can be refolded from inclusion bodies (see, e.g., Hou et al. (1998) Cytokine 10:319-30). Bacterial expression systems and methods of use are well 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 varies depending on numerous factors and can be readily optimized as needed. The antibodies (or fragments thereof) described herein can be expressed in mammalian cells or in other expression systems, including but not limited to yeast, baculovirus, and in vitro expression systems (see, for example, 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 by various methods known in the art, depending on the other components present in the sample. Standard purification methods include electrophoresis, molecular, immunological, and chromatographic techniques, with chromatographic techniques including ion exchange, hydrophobic, affinity, and reverse-phase HPLC chromatography. For example, antibodies can be purified using standard anti-antibody columns (e.g., Protein A or Protein G columns). Ultrafiltration and diafiltration techniques are also useful in combination with protein concentration. See, for example, Scopes (1994) Protein Purification, 3 rd See edition, Springer-Verlag [New York City, New York, USA]. The required degree of purification varies depending on the desired use. In some cases, purification of the expressed antibody or its fragments is not necessary.

[0260] Methods for determining the yield or purity of purified antibodies or fragments thereof are known in the art, and include, for example, the 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 (using, for example, protein stains such as Coomassie blue or colloidal silver stain).

[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 in a multimer complex, for example, as a single antigen-binding arm separated from other antigen-binding arms. The modifications can be covalent or non-covalent. Such modifications can be introduced into an antibody or antigen-binding fragment, for example, by reacting targeted amino acid residues of the polypeptide with an organic derivatizing agent that can react with selected side-chain 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 represented by single-letter amino acid codes that can be used interchangeably with three-letter amino acid codes. An amino acid refers to any monomeric unit that can be incorporated into a peptide, polypeptide, or protein. The 20 naturally occurring or genetically encoded alpha-amino acids are as follows: alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartic acid (Asp or D), cysteine ​​(Cys or C), glutamine (Gln or Q), glutamic acid (Glu or E), glycine (Gly or G), histidine (His or H), isoleucine (Ile or I), leucine (Leu or L), lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y), and valine (Val or V). The structures of these 20 naturally occurring amino acids can be found, for example, in Stryer et al., Biochemistry, 5. th ed., As shown in Freeman and Company (2002), the term amino acid also includes unnatural amino acids, modified amino acids (e.g., those with modified side chains and / or skeletons), and amino acid analogs.

[0263] In the context of two or more nucleic acid or polypeptide sequences, the terms identical or percentage of identity refer to two or more sequences or subsequences that are identical or have a specified percentage of identical nucleotide or amino acid residues (e.g., 90%, 95%, or higher identity over a specified region) when compared and aligned for maximum match over a comparison window, such as when measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection, or when compared over a specified region.

[0264] Sequence identity or similarity is defined as the percentage of amino acid residues in a candidate sequence that are identical (i.e., the same residue) to the starting amino acid residue, after the sequences have been aligned and gaps introduced where necessary to achieve the maximum possible sequence identity percentage. Methods for aligning sequences for comparison are well known in the art. The optimal alignment of sequences for comparison can be determined, 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 embodiments of these algorithms (e.g., GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science, Wisconsin, USA, Dr. Madison, This can be done by [Madison] location, 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 occur to the amino acid sequences of the constructs described herein, antibodies, or their antigen-binding portions, for example, in the heavy chain variable region and / or light chain variable region, without altering the properties or function of the antibody or its antigen-binding fragment. Such modifications include conservative amino acid substitutions, where each mentioned sequence optionally contains one or more conservative amino acid substitutions. The following groups each contain amino acids that are conserved substitutions with respect to each other. These groups are illustrative, 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, when aspartic acid is mentioned at a particular residue, a conserved substitution at that residue, such as glutamic acid, is also assumed. A non-conservative substitution, such as proline substitution at glycine, is also assumed.

[0267] In some embodiments, a construct, antibody or antigen-binding portion thereof can be conjugated to a heterologous moiety. The heterologous moiety can be, for example, a heterologous polypeptide, a therapeutic agent (e.g., a toxin or a drug), or a detectable label, and the detectable label can be, but is 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 the purification of an antibody or fragment (e.g., FLAG (DYKDDDDK) (SEQ ID NO: 117), polyhistidine (6-His; HHHHHH) (SEQ ID NO: 118), hemagglutinin (HA; YPYDVPDYA) (SEQ ID NO: 119), glutathione-S-transferase (GST), or maltose binding protein (MBP)). Heterologous polypeptides also include polypeptides (e.g., enzymes) useful as diagnostic or detectable markers, such as luciferase, fluorescent proteins (e.g., green fluorescent protein (GFP)), or chloramphenicol acetyltransferase (CAT). Suitable radioactive labels include, for example, 32 P, 33 P, 14 C, 125 I, 131 I, 35 S, and 3H is one example. 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. Examples of luminescence labels include any of the various luminescent lanthanides (e.g., europium or terbium) chelates. For example, suitable europium chelates include diethylenetriaminepentaacetic acid (DTPA) or tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) europium chelates. Examples of enzymatic labels include alkaline phosphatase, CAT, luciferase, and horseradish peroxidase.

[0268] Two proteins (e.g., an antibody and a heterologous portion) can be crosslinked using any of a number of known chemical crosslinking agents. An example of such a crosslinking agent is one that links two amino acid residues via a linkage containing a "hindered" disulfide bond. In these linkages, the disulfide bond within the crosslinking unit is protected from reduction (by interfering with the group on either side of the disulfide bond) by, for example, reduced glutathione or the enzyme disulfide reductase. One suitable reagent, 4-succinimidyloxycarbonyl-α-methyl-α(2-pyridyldithio)toluene (SMPT), utilizes the terminal lysine in one protein and the terminal cysteine ​​in the other to form such a linkage between the two proteins. Heterobifunctional reagents that crosslink by 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-azidosalicylamide]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 a larger molecule bound to the protein backbone (e.g., meta-iodophenyl (mIP) derivative of the relevant protein that binds to a free amino group). 125 I] Iodophenyl-N-hydroxysuccinimide ([ 125 I] During the miIPNHS 125I (see, for example, Rogers et al. (1997) J. Nucl. Med. 38:1221-1229) or chelate (e.g., DOTA or DTPA) may be included as part of the radiolabeled material. Methods for conjugating radiolabeled materials or larger molecules / chelates containing them to antibody or antigen-binding fragments as described herein are known in the art. Such methods involve incubating the protein with the radiolabeled material under conditions that promote the binding of the radiolabeled material or chelate to the protein (e.g., pH, salt concentration, and / or temperature) (see, for example, U.S. Patent No. 6,001,329).

[0270] Methods for conjugating fluorescent labels (sometimes referred to as "fluorophores") to proteins (e.g., antibodies) are well known in the field of protein chemistry. For example, fluorophores can be conjugated to the free amino group (e.g., lysine) or sulfhydryl group (e.g., cysteine) of a protein using succinimidyl (NHS) or tetrafluorophenyl (TFP) ester moieties attached to the fluorophore. In some embodiments, fluorophores can be conjugated to heterobifunctional crosslinker moieties such as sulfo-SMCC. A preferred conjugation method involves incubating an antibody protein or fragment thereof with the fluorophore under conditions that promote the 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 may be modified with a moiety that improves the stabilization and / or retention of the antibody in circulation, for example, in blood, serum, or other tissues. For example, the antibody or fragment may be modified with a moiety that improves the stabilization and / or retention of the antibody in circulation, for example, in blood, serum, or other tissues. For example, 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 The antibody (or fragment) can be PEGylated or HES-modified (Fresenius Kabi, Germany) as described in Reviews 54:459-476 (see, for example, Pavisic et al. (2010) Int. J. Pharm. 387(1-2):110-119). The stabilizing portion can improve the stability or retention of the antibody (or fragment) by at least, for example, 1.5 times (e.g., at least 2, 5, 10, 15, 20, 25, 30, 40, or 50 or greater).

[0272] In some embodiments, the antibodies or antigen-binding fragments described herein can be glycosylated. In some embodiments, the antibodies or antigen-binding fragments described herein can be subjected to enzymatic or chemical treatment, or produced from cells, so that the antibody or fragment has reduced glycosylation or no glycosylation. Methods for producing antibodies with reduced glycosylation are known in the art, for example, U.S. Patent 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 its antigen-binding fragment is nonglycosylated.

[0273] G. Pharmaceutical compositions and formulations This disclosure also provides pharmaceutical compositions comprising the multispecific antigen-binding constructs of this disclosure together with pharmaceutically acceptable diluents, carriers, solubilizers, emulsifiers, preservatives and / or adjuvants used in conjunction with the methods disclosed herein. Such pharmaceutical compositions may be used, for example, in subjects with cancer, as disclosed herein.

[0274] In certain embodiments, the acceptable formulation materials are preferably non-toxic to the recipient at the dosage and concentration used. In certain embodiments, the formulation materials are for sc and / or IV administration. In certain embodiments, the pharmaceutical composition may contain formulation materials for modifying, maintaining, or preserving the composition's properties, such as pH, osmolality by weight, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption or permeation.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); fillers (e.g., mannitol or glycine); chelating agents (e.g., ethylenediaminetetraacetic acid (EDTA)); complexing agents (e.g., caffeine, polyvinylpyrrolidone, beta-cyclodextrin, or hydroxypropyl-beta-cyclodextrin); fillers; monosaccharides, disaccharides, and other carbohydrates (e.g., glucose, mannose, or dextrin); proteins (e.g., serum albumin, gelatin, or imm...

Claims

[Claim 1] The invention described herein.

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