Anti-Programmed Cell Death Ligand 1 (PD-L1) Antibodies

Abstract

The present invention provides anti-PD-L1 antigen-binding molecules, including antibodies and antigen-binding fragments thereof, and uses of the anti-PD-L1 antigen-binding molecules to treat various diseases, including cancer.

Description

[Technical field]

[0001] [Cross reference] This application claims priority to U.S. Provisional Application No. 63 / 314,925, filed February 28, 2022, the entire contents of which are incorporated herein by reference. The present invention relates to antigen-binding molecules, such as antibodies or antigen-binding fragments thereof, for cancer treatment, in particular anti-programmed cell death-ligand 1 (PD-L1) antibodies for use in cancer immunotherapy. [Background technology]

[0002] The adaptive immune response is mediated by two major immune cell subsets: T lymphocytes (CD4+ and CD8+ T cells) and B lymphocytes. While B cells play a major role in secreting antibodies and acting as antigen-presenting cells, T cells have been extensively examined for their special effector functions in various physiological responses, including acute and chronic infections, cancer and autoimmunity, and immune homeostasis. Two distinct signals are required for T cell activation. One is ligation via binding of the T cell receptor (TCR) to peptide-MHC, and the other is a positive co-stimulatory signal, such as the interaction between CD28 on T cells and CD80 (also called B7.1) and / or CD86 (also called B7.2) on antigen-presenting cells. Early in the activation process, negative regulators, including cytotoxic T lymphocyte antigen 4 (CTLA4; also called CD152), are induced to oppose the activation program and compete with CD28 for the ligands CD80 and CD86. Programmed cell death protein 1 (PD1; also known as PDCD1 and CD279) is also expressed during T cell activation and counters positive signals via the TCR and CD28 by binding to its ligands, programmed cell death 1 ligand 1 (PD-L1; also known as CD274 and B7-H1) and / or PD-L2 (also known as CD273 and B7-DC).

[0003] PD-L1 is a 53 kDa type I transmembrane protein that is expressed on many immune cell subsets, including activated exhausted / anergy T cells, B cells, myeloid cells (including monocytes, mast cells, and dendritic cells), and some cancer cells. PD-L1 is one of the “co-inhibitory” receptors that act as a resting point for adaptive immune responses, acting as an immune checkpoint that effector T cells must pass to exert their full function. Under pathological conditions, PD-L1 expression is found to be significantly upregulated in many tumors, including non-small cell lung cancer (NSCLC), bladder cancer, hepatocellular carcinoma, breast cancer, and pancreatic cancer. Increased expression of PD-L1 on tumor cells may modulate the tumor microenvironment (TME) and promote immune evasion of tumor cells by 1) stimulating regulatory T cells to enhance immune suppression, and 2) promoting T cell exhaustion / anergy to reduce inflammation.

[0004] Since treatment with PD-1 pathway inhibitors (also known as "checkpoint blockade") has proven capable of promoting sustained antitumor immune responses, the monoclonal antibodies pembrolizumab (anti-PD-1; Merck & Co), nivolumab (anti-PD-1; Bristol Myers Squibb), atezolizumab (anti-PD-L1; Roche / Genentech), and durvalumab (anti-PD-L1; AstraZeneca) and avelumab (anti-PD-L1; EMD Millipore) have been used to treat PD-1-associated leukemia (CI) and leukemia (CI-ALT). Serono), cemiplimab (anti-PD-1; Regeneron), and dostallimab (anti-PD-1; GlaxoSmithKline) have been approved by the U.S. Food and Drug Administration to treat more than 20 types of cancer, including melanoma, bladder cancer, non-small cell lung cancer (NSCLC), hepatocellular carcinoma (HCC), renal cell carcinoma (RCC), head and neck squamous cell carcinoma (HNSC), Hodgkin lymphoma, Merkel cell carcinoma, and adult and pediatric solid tumors with high microsatellite instability or mismatch repair deficiency (see Cancer Institute: https: / / www.cancerresearch.org / fda-approval-timeline-of-active-immunotherapies).

[0005] Despite the revolutionary cure-like survival benefits observed in clinical trials, it is estimated that only a small number of cancer patients would respond positively to PD-1 / PD-L1 blockade therapy, and primary or acquired resistance may ultimately lead to cancer progression in patients with clinical responses. To overcome the limitations of therapeutic resistance, significant efforts are being made to improve or develop anti-PD-1 / PD-L1-based immunotherapeutic strategies with better clinical responses and reduced immune-mediated toxicity. There is a need in the art for new therapeutic approaches based on new antigen-binding molecules (e.g., antibodies or antigen-binding fragments thereof that specifically bind to PD-L1) for use in the treatment of diseases (e.g., cancer). Summary of the Invention

[0006] The present invention provides antigen binding molecules, such as anti-PD-L1 antibodies or antigen-binding fragments thereof, for binding (e.g., specifically binding) to PD-L1. PD-L1 may be present on the surface of a cell (e.g., a mammalian cell, such as a mammalian tumor cell, such as a mouse tumor cell, a cynomolgus monkey tumor cell, or a human tumor cell). The present invention also provides methods of using antigen binding molecules of the invention (e.g., anti-PD-L1 antibodies or antigen-binding fragments thereof) for specifically binding to PD-L1. Binding of an antigen binding molecule of the invention (e.g., an anti-PD-L1 antibody or antigen-binding fragment thereof) can block PD-1 / PD-L1 interaction or induce internalization of a complex of PD-L1 and the antigen-binding molecule. Binding of an antigen binding molecule according to the invention (e.g. an anti-PD-L1 antibody or antigen-binding fragment thereof) can be used to treat a subject who may benefit from modulation (e.g. inhibition) of the PD-1 / PD-L1 interaction, such as a subject suffering from or susceptible to a PD-L1-associated condition (e.g. a disease or disorder characterized by aberrant expression of PD-L1).

[0007] Thus, in one aspect, the invention provides an isolated antigen-binding molecule, such as an antibody or antigen-binding fragment thereof, that binds to human PD-L1, the antibody comprising a heavy chain variable (VH) domain comprising, from N-terminus to C-terminus, three heavy chain complementarity determining regions (CDRs): HCDR1, HCDR2, and HCDR3, and a light chain variable (VL) domain comprising, from N-terminus to C-terminus, three light chain complementarity determining regions (CDRs): LCDR1, LCDR2, and LCDR3, wherein: (a) HCDR1 is selected from the group consisting of X1-NHYM-X2 (SEQ ID NO:), X3-X4-YYMC (SEQ ID NO:), NNYYMS (SEQ ID NO:8), RYFYMS (SEQ ID NO:11), and SA; X35-I-X36-DYA-X37-WAKG (SEQ ID NO: 12), where X1 is N or S, X2 is C or S, X3 is A, N or S, and X4 is A, N or S; and (b) HCDR2 comprises an amino acid sequence selected from the group consisting of X32-I-X33-X34-GS-X35-I-X36-DYA-X37-WAKG (SEQ ID NO: 12), where X1 is N or S, X2 is C or S, X3 is A, N or S, and X4 is A, N or S; and (c) HCDR3 comprises the amino acid sequence shown in WTSGGGGFGL (SEQ ID NO: 42); (d) LCDR1 comprises the amino acid sequence shown in QSSQ-X65-X66-Y-X67-X68-YL-X69 (SEQ ID NO:), QSSQ-X70-IYSDYL-X71 (SEQ ID NO:) and QSSQS-X72-Y-X73-NYL-X74 (SEQ ID NO:), where X65 is N, S or T, X66 is I or V, X67 is N or S, and X68 is D or N; X69 is A, C, F or S, X70 is N or T, X71 is F or S, X72 is I or V, X73 is N or S, and X74 is A, C or S; (e) LCDR2 comprises an amino acid sequence selected from the group consisting of X98-X99-X100-TLAS (SEQ ID NO:), X101-X102-STLAS (SEQ ID NO:) and STATLAS (SEQ ID NO:72), where X98 is D, G, Q, S or Y, X99 is A or T, and X100 is A or S;X101 is D, G, Q, or Y, and X102 is A or T; and (f) LCDR3 comprises the amino acid sequence set forth in QGYYSGYIWT (SEQ ID NO: 83).

[0008] In each aspect of the invention and embodiments thereof, the antibody is an antigen-binding fragment of the antibody. In each aspect of the invention and embodiments thereof, the human PD-L1 comprises the sequence set forth in SEQ ID NO:

[0009] In one embodiment, (a) HCDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 12, (b) HCDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 22 to 31, (c) HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 42, (d) LCDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 53 to 59, (e) LCDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 69 to 73, and (f) LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 83.

[0010] In another embodiment, the isolated antigen-binding molecule, e.g., an antibody or antigen-binding fragment thereof, comprises: (a) an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 22, an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 42, an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 53, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 69, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 83; (b) an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 2, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 23, an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 42, an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 53, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 69, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 83; (c) an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 3, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 23, an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 42, an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 54, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 70. and LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 83; (d) HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 4, HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 24, HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 42, LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 55, LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 71, and LCDR3 comprising the amino acid sequence shown in SEQ ID NO: 83; (e) HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 5, HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 25, HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 42, LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 56, LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 69, and LCDR3 comprising the amino acid sequence shown in SEQ ID NO: 83; (f) HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 6, HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 26, HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 42, LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 57, and LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 71;and LCDR3 having the amino acid sequence shown in SEQ ID NO: 83; (g) HCDR1 having the amino acid sequence shown in SEQ ID NO: 7, HCDR2 having the amino acid sequence shown in SEQ ID NO: 27, HCDR3 having the amino acid sequence shown in SEQ ID NO: 42, LCDR1 having the amino acid sequence shown in SEQ ID NO: 56, LCDR2 having the amino acid sequence shown in SEQ ID NO: 69, and LCDR3 having the amino acid sequence shown in SEQ ID NO: 83; (h) HCDR1 having the amino acid sequence shown in SEQ ID NO: 8, HCDR2 having the amino acid sequence shown in SEQ ID NO: 28, HCDR3 having the amino acid sequence shown in SEQ ID NO: 42, LCDR1 having the amino acid sequence shown in SEQ ID NO: 53, LCDR2 having the amino acid sequence shown in SEQ ID NO: 69, and LCDR3 having the amino acid sequence shown in SEQ ID NO: 83; (i) HCDR1 having the amino acid sequence shown in SEQ ID NO: 9, HCDR2 having the amino acid sequence shown in SEQ ID NO: 27, HCDR3 having the amino acid sequence shown in SEQ ID NO: 42, LCDR1 having the amino acid sequence shown in SEQ ID NO: 53, LCDR2 having the amino acid sequence shown in SEQ ID NO: 69, and LCDR3 having the amino acid sequence shown in SEQ ID NO: 83. (j) an HCDR1 comprising the amino acid sequence shown in SEQ ID NO:5, an HCDR2 comprising the amino acid sequence shown in SEQ ID NO:29, an HCDR3 comprising the amino acid sequence shown in SEQ ID NO:42, an LCDR1 comprising the amino acid sequence shown in SEQ ID NO:53, an LCDR2 comprising the amino acid sequence shown in SEQ ID NO:69, and an LCDR3 comprising the amino acid sequence shown in SEQ ID NO:83, (k) an HCDR1 comprising the amino acid sequence shown in SEQ ID NO:11, an HCDR2 comprising the amino acid sequence shown in SEQ ID NO:30, an HCDR3 comprising the amino acid sequence shown in SEQ ID NO:42, an LCDR1 comprising the amino acid sequence shown in SEQ ID NO:58, an LCDR2 comprising the amino acid sequence shown in SEQ ID NO:72, and an LCDR3 comprising the amino acid sequence shown in SEQ ID NO:83; or (l) an HCDR1 comprising the amino acid sequence shown in SEQ ID NO:12, an HCDR2 comprising the amino acid sequence shown in SEQ ID NO:31, an HCDR3 comprising the amino acid sequence shown in SEQ ID NO:42, an LCDR1 comprising the amino acid sequence shown in SEQ ID NO:59, an LCDR2 comprising the amino acid sequence shown in SEQ ID NO:73, and an LCDR3 comprising the amino acid sequence shown in SEQ ID NO:83.

[0011] In yet another embodiment, the isolated antigen-binding molecule, for example, an antibody or antigen-binding fragment thereof, comprises (a) a heavy chain variable region (HCVR) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 92 to 103, and (b) a light chain variable region (LCVR) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 118 to 129.

[0012] In yet another embodiment, the isolated antigen-binding molecule, e.g., an antibody or antigen-binding fragment thereof, comprises: (a) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 92, and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 118; (b) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 93, and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 119; (c) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 94, and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 120; (d) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 95, and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 121; (e) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 96, and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 122; (f) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 97, and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 123. (g) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 98 and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 124, (h) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 99 and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 125, (i) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 100 and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 126, (j) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 101 and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 127, (k) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 102 and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 128; or (l) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 103 and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 129.

[0013] In one aspect, the invention provides an isolated antigen binding molecule, such as an antibody or antigen-binding fragment thereof, that binds to human PD-L1. The isolated antigen-binding molecule, for example, an antibody or an antigen-binding fragment thereof, comprises a heavy-chain variable (VH) domain comprising, from the N-terminus to the C-terminus, three heavy-chain complementarity determining regions (CDRs), HCDR1, HCDR2, and HCDR3; and a light-chain variable (VL) domain comprising, from the N-terminus to the C-terminus, three light-chain complementarity determining regions (CDRs), LCDR1, LCDR2, and LCDR3, wherein (a) HCDR1 comprises an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 12, (b) HCDR2 comprises an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 22 to 31, and (c) HCDR3 comprises an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 22 to 31, and (c) HCDR3 comprises an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identity with the amino acid sequence shown in SEQ ID NO: 42; (d) LCDR1 comprises an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 53 to 59; (e) LCDR2 comprises an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 69 to 73, and (f) LCDR3 comprises an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identity to the amino acid sequence shown in SEQ ID NO: 83.

[0014] In another aspect, the present invention provides an isolated antigen-binding molecule, e.g., an antibody or antigen-binding fragment thereof, that binds to human PD-L1. The isolated antigen-binding molecule, e.g., an antibody or antigen-binding fragment thereof, comprises (a) a heavy chain variable region (HCVR) comprising an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 92 to 103, and (b) a light chain variable region (LCVR) comprising an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 118 to 129.

[0015] In yet another aspect, the present invention provides an isolated antigen-binding molecule, e.g., an antibody or antigen-binding fragment thereof, that binds to human PD-L1. The isolated antigen-binding molecule, e.g., an antibody or antigen-binding fragment thereof, comprises a heavy chain variable (VV) domain that comprises, from the N-terminus to the C-terminus, three heavy chain complementarity determining regions (CDRs): HCDR1, HCDR2, and HCDR3. H ) domain and three light chain complementarity determining regions (CDRs), LCDR1, LCDR2, and LCDR3, from the N-terminus to the C-terminus. L) domain, wherein (a) HCDR1 comprises the amino acid sequence shown in X8-D-X9-YMS (SEQ ID NO: 1), where X8 is D or G, and X9 is W or Y; (b) HCDR2 comprises the amino acid sequence shown in SIY-X42-GSLN-X43-YYATWAKG (SEQ ID NO: 1), where X42 is S or T, and X43 is I, S, or T; (c) HCDR3 comprises the amino acid sequence shown in R-X57-KN-X58-D-X59-G-X60-FDL (SEQ ID NO: 1), where X57 is H, N, or T, X58 is A or G, X59 is W or Y, and X60 is H or Y; and (d) LCDR1 comprises the amino acid sequence shown in QAS-X76-SI-X77-X78-X79-LA (SEQ ID NO: 1), QAS-X80-SI NDRLA (SEQ ID NO: 67), QASQSI-X81-X82-ALA (SEQ ID NO: 68), QASQSISTALA (SEQ ID NO: 67), and QASQSIGNALA (SEQ ID NO: 68) (wherein X76 is E or Q, X77 is G, N or S, X78 is D, N or T, X79 is A or R, X80 is E or Q, X81 is G or S, and X82 is N or T); (e) LCDR2 comprises the amino acid sequence set forth in X106-X107-STLAS (SEQ ID NO: 67), (wherein X106 is A or G and X107 is A or T); and (f) LCDR3 comprises the amino acid sequence set forth in QQGWTVSSL-X111-NA (SEQ ID NO: 68), (wherein X111 is D or E).

[0016] In one embodiment, (a) HCDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 19, 20, and 21; (b) HCDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 38, 39, 40, and 41; (c) HCDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 49, 50, 51, and 52; (d) LCDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 65, 66, 67, and 68; (e) LCDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 80, 71, and 82; and (f) LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 90 or 91.

[0017] In another embodiment, the isolated antigen-binding molecule, e.g., an antibody or antigen-binding fragment thereof, comprises: (a) an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 19, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 38, an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 49, an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 65, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 80, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 90; (b) an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 19, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 39, an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 50, an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 66, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 71, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 90; (c) an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 20, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 39, an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 51, an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 67, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 80. and LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 90; (d) HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 19, HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 39, HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 52, LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 66, LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 80, and LCDR3 comprising the amino acid sequence shown in SEQ ID NO: 91; (e) HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 19, HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 40, HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 49, LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 66, LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 71, and LCDR3 comprising the amino acid sequence shown in SEQ ID NO: 90; (f) HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 21, HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 41, HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 49, LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 68, and LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 82;and LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 90; (g) HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 19, HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 39, HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 49, LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 66, LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 80, and LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 90; or (h) HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 19, HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 39, HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 49, LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 66, LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 71, and LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 90.

[0018] In another embodiment, the isolated antigen-binding molecule, for example, an antibody or antigen-binding fragment thereof, comprises (a) a heavy chain variable region (HCVR) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 110 to 117, and (b) a light chain variable region (LCVR) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 136 to 143.

[0019] In yet another embodiment, the isolated antigen-binding molecule, e.g., an antibody or antigen-binding fragment thereof, comprises: (a) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 110, and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 136; (b) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 111, and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 137; (c) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 112, and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 138; (d) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 113, and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 139; 9, (e) an HCVR comprising an amino acid sequence set forth in SEQ ID NO: 114 and an LCVR comprising an amino acid sequence set forth in SEQ ID NO: 140, (f) an HCVR comprising an amino acid sequence set forth in SEQ ID NO: 115 and an LCVR comprising an amino acid sequence set forth in SEQ ID NO: 141, (g) an HCVR comprising an amino acid sequence set forth in SEQ ID NO: 116 and an LCVR comprising an amino acid sequence set forth in SEQ ID NO: 142; or (h) an HCVR comprising an amino acid sequence set forth in SEQ ID NO: 117 and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 143.

[0020] In one aspect, the invention provides an isolated antigen-binding molecule, e.g., an antibody or antigen-binding fragment thereof, that binds to human PD-L1. The isolated antigen-binding molecule, e.g., an antibody or antigen-binding fragment thereof, comprises a heavy chain variable (VH) domain comprising, from N-terminus to C-terminus, three heavy chain complementarity determining regions (CDRs): HCDR1, HCDR2, and HCDR3, and a light chain variable (VL) domain comprising, from N-terminus to C-terminus, three light chain complementarity determining regions (CDRs): LCDR1, LCDR2, and LCDR3, wherein: (a) HCDR1 is selected from the sequences of SEQ ID NOs: 19, 20, and 21; HCDR2 comprises an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 38, 39, 40, and 41; (b) HCDR3 comprises an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 38, 39, 40, and 41; and (c) HCDR4 comprises an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 38, 39, 40, and 41. (d) LCDR1 comprises an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 49, 50, 51, and 52; and (d) LCDR2 comprises an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 65, 66, 67, and 68. (e) LCDR2 comprises an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 80, 71, and 82; and (f) LCDR3 comprises an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identity to an amino acid sequence set forth in SEQ ID NO: 90 or 91.

[0021] In another aspect, the present invention provides an isolated antigen-binding molecule, e.g., an antibody or antigen-binding fragment thereof, that binds to human PD-L1. The isolated antigen-binding molecule, e.g., an antibody or antigen-binding fragment thereof, comprises (a) a heavy chain variable region (HCVR) comprising an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 110 to 117, and (b) a light chain variable region (LCVR) comprising an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 136 to 143.

[0022] In yet another aspect, the present invention provides an isolated antigen-binding molecule, e.g., an antibody or antigen-binding fragment thereof, that binds to human PD-L1. The isolated antigen-binding molecule, e.g., an antibody or antigen-binding fragment thereof, comprises a heavy chain variable (VV) domain that comprises, from the N-terminus to the C-terminus, three heavy chain complementarity determining regions (CDRs): HCDR1, HCDR2, and HCDR3. H ) domain and three light chain complementarity determining regions (CDRs), LCDR1, LCDR2, and LCDR3, from the N-terminus to the C-terminus. L) domain, wherein (a) HCDR1 comprises an amino acid sequence selected from the group consisting of SGYDMS (SEQ ID NO: 15) and SGYDMC (SEQ ID NO: 16); (b) HCDR2 comprises an amino acid sequence set forth in X38-IFT-X39-SGSTWYANWAKG (SEQ ID NO: 17), where X38 is C or S and X39 is G or T; and (c) HCDR3 comprises an amino acid sequence set forth in T-X51-DG-X52-GSFYMNL (SEQ ID NO: 18), where X51 is K or R and X (d) LCDR1 comprises the amino acid sequence shown in QASGTIGSNLA (SEQ ID NO: 63) or QASQTIGSNLA (SEQ ID NO: 62), (e) LCDR2 comprises the amino acid sequence shown in KAFTLAS (SEQ ID NO: 76) or KTFTLAS (SEQ ID NO: 77), and (f) LCDR3 comprises the amino acid sequence shown in QQGATRINIDNA (SEQ ID NO: 86) or QQGASRINIDNA (SEQ ID NO: 87).

[0023] In one embodiment, (a) HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 34 or 35, and (b) HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 45 or 46.

[0024] In another embodiment of the isolated antigen-binding molecule, e.g., an antibody or antigen-binding fragment thereof, (a) HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 15, HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 34, HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 45, LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 62, LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 76, and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 86, or (b) HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 16, HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 35, HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 46, LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 63, LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 77, and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 87.

[0025] In yet another embodiment, the isolated antigen-binding molecule, e.g., an antibody or antigen-binding fragment thereof, comprises (a) a heavy chain variable region (HCVR) comprising the amino acid sequence set forth in SEQ ID NO: 106 or 107, and (b) a light chain variable region (LCVR) comprising the amino acid sequence set forth in SEQ ID NO: 132 or 133.

[0026] In yet another embodiment, the isolated antigen-binding molecule, e.g., an antibody or antigen-binding fragment thereof, comprises (a) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 106, and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 132, or (b) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 107, and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 133.

[0027] In one aspect, the invention provides an isolated antigen binding molecule, such as an antibody or antigen-binding fragment thereof, that binds to human PD-L1. The isolated antigen-binding molecule, e.g., an antibody or antigen-binding fragment thereof, comprises a heavy chain variable (VH) domain comprising, from N-terminus to C-terminus, three heavy chain complementarity determining regions (CDRs), HCDR1, HCDR2, and HCDR3; and a light chain variable (VL) domain comprising, from N-terminus to C-terminus, three light chain complementarity determining regions (CDRs), LCDR1, LCDR2, and LCDR3, wherein (a) HCDR1 comprises an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identity with the amino acid sequence set forth in SEQ ID NO: 15 or 16; (b) HCDR2 comprises an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identity with the amino acid sequence set forth in SEQ ID NO: 34 or 35; and (c) HC DR3 comprises an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identity to the amino acid sequence shown in SEQ ID NO: 45 or 46; (d) LCDR1 comprises an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identity to the amino acid sequence shown in SEQ ID NO: 62 or 63; (e) ) LCDR2 comprises an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identity to the amino acid sequence set forth in SEQ ID NO: 76 or 77, and (f) LCDR3 comprises an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identity to the amino acid sequence set forth in SEQ ID NO: 86 or 87.

[0028] In another aspect, the present invention provides an isolated antigen-binding molecule, e.g., an antibody or antigen-binding fragment thereof, that binds to human PD-L1. The isolated antigen-binding molecule, e.g., an antibody or antigen-binding fragment thereof, comprises (a) a heavy chain variable region (HCVR) comprising an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or about 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 106 and 107, and (b) a light chain variable region (LCVR) comprising an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or about 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 132 and 133.

[0029] In one aspect, the present invention provides an isolated antigen-binding molecule, e.g., an antibody or antigen-binding fragment thereof, that binds to human PD-L1. The isolated antigen-binding molecule, e.g., an antibody or antigen-binding fragment thereof, comprises a heavy chain variable (VV) domain that comprises, from the N-terminus to the C-terminus, three heavy chain complementarity determining regions (CDRs), HCDR1, HCDR2, and HCDR3. H ) domain and a light chain variable (V) domain, which includes, from the N-terminus to the C-terminus, three light chain complementarity determining regions (CDRs), LCDR1, LCDR2, and LCDR3. L) domain, wherein (a) HCDR1 comprises an amino acid sequence selected from the group consisting of STYALG (SEQ ID NO: 17) and STYAMG (SEQ ID NO: 18); (b) HCDR2 comprises an amino acid sequence set forth in SIGGATY-X40-A-X41-WAKG (SEQ ID NO: 19), where X40 is F or Y and X41 is S or T; and (c) HCDR3 comprises an amino acid sequence set forth in ARNVD-X53-IYL-X54-AF-X55-X56 (SEQ ID NO: 20), where X53 is I or S, X54 is D or N, and X55 is X56 is I or T), (d) LCDR1 comprises the amino acid sequence shown in QASQNIYNNLA (SEQ ID NO: 64), (e) LCDR2 comprises the amino acid sequence shown in X104-X105-STLAS (SEQ ID NO: ) (wherein X104 is R or S and X105 is A or S), and (f) LCDR3 comprises the amino acid sequence shown in QTYYLT-X109-T-X110-NA (SEQ ID NO: ) (wherein X109 is S or T and X110 is I or T).

[0030] In one embodiment, (a) HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 36 or 37, (b) HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 47 or 48, (c) LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 78 or 79, and (d) LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 88 or 89.

[0031] In another embodiment, (a) HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 17, HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 36, HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 47, LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 64, LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 78, and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 88, or (b) HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 18, HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 37, HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 48, LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 64, LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 79, and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 89.

[0032] In yet another embodiment, the isolated antigen-binding molecule, e.g., an antibody or antigen-binding fragment thereof, comprises (a) a heavy chain variable region (HCVR) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 108 and 109, and (b) a light chain variable region (LCVR) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 134 and 135.

[0033] In yet another embodiment, the isolated antigen-binding molecule, e.g., an antibody or antigen-binding fragment thereof, comprises: (a) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 108, and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 134; or (b) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 109, and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 135.

[0034] In one aspect, the invention provides an isolated antigen binding molecule, such as an antibody or antigen-binding fragment thereof, that binds to human PD-L1. The isolated antigen-binding molecule, e.g., an antibody or antigen-binding fragment thereof, comprises a heavy chain variable (VH) domain comprising, from N-terminus to C-terminus, three heavy chain complementarity determining regions (CDRs), HCDR1, HCDR2, and HCDR3; and a light chain variable (VL) domain comprising, from N-terminus to C-terminus, three light chain complementarity determining regions (CDRs), LCDR1, LCDR2, and LCDR3, wherein (a) HCDR1 comprises an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identity to the amino acid sequence set forth in SEQ ID NO: 17 or 18; (b) HCDR2 comprises an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identity to the amino acid sequence set forth in SEQ ID NO: 36 or 37; and (c) HCDR3 comprises an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identity with the amino acid sequence shown in SEQ ID NO: 47 or 48; (d) LCDR1 comprises an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identity with the amino acid sequence shown in SEQ ID NO: 64; and (e) (f) LCDR2 comprises an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identity to the amino acid sequence set forth in SEQ ID NO: 78 or 79, and (f) LCDR3 comprises an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identity to the amino acid sequence set forth in SEQ ID NO: 88 or 89.

[0035] In another aspect, the present invention provides an isolated antigen-binding molecule, e.g., an antibody or antigen-binding fragment thereof, that binds to human PD-L1. The isolated antigen-binding molecule, e.g., an antibody or antigen-binding fragment thereof, comprises (a) a heavy chain variable region (HCVR) comprising an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or about 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 108 and 109, and (b) a light chain variable region (LCVR) comprising an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or about 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 134 and 135.

[0036] In yet another aspect, the present invention provides an isolated antigen-binding molecule, e.g., an antibody or antigen-binding fragment thereof, that binds to human PD-L1. The isolated antigen-binding molecule, e.g., an antibody or antigen-binding fragment thereof, comprises a heavy chain variable (VV) domain that comprises, from the N-terminus to the C-terminus, three heavy chain complementarity determining regions (CDRs): HCDR1, HCDR2, and HCDR3. H ) domain and three light chain complementarity determining regions (CDRs), LCDR1, LCDR2, and LCDR3, from the N-terminus to the C-terminus. L ) domain, wherein (a) HCDR1 comprises the amino acid sequence set forth in SSYYMS (SEQ ID NO: 13), (b) HCDR2 comprises the amino acid sequence set forth in CISGGVTDNAYYASWAKG (SEQ ID NO: 32), (c) HCDR3 comprises the amino acid sequence set forth in DSSSGYFFLL (SEQ ID NO: 43), (d) LCDR1 comprises the amino acid sequence set forth in QASQNIYSNLA (SEQ ID NO: 60), (e) LCDR2 comprises the amino acid sequence set forth in GASNLRS (SEQ ID NO: 74), and (f) LCDR3 comprises the amino acid sequence set forth in QEGYSIGNVDNP (SEQ ID NO: 84).

[0037] In one embodiment, the isolated antigen-binding molecule, such as an antibody or antigen-binding fragment thereof, comprises a HCVR comprising the amino acid sequence set forth in SEQ ID NO: 104 and a LCVR comprising the amino acid sequence set forth in SEQ ID NO:130.

[0038] In one aspect, the invention provides an isolated antigen-binding molecule, such as an antibody or antigen-binding fragment thereof, that binds to human PD-L1. The isolated antigen-binding molecule, e.g., an antibody or antigen-binding fragment thereof, comprises a heavy chain variable (VH) domain comprising, from N-terminus to C-terminus, three heavy chain complementarity determining regions (CDRs), HCDR1, HCDR2, and HCDR3; and a light chain variable (VL) domain comprising, from N-terminus to C-terminus, three light chain complementarity determining regions (CDRs), LCDR1, LCDR2, and LCDR3, wherein (a) HCDR1 comprises an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identity with the amino acid sequence set forth in SEQ ID NO: 13; (b) HCDR2 comprises an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identity with the amino acid sequence set forth in SEQ ID NO: 32; c) HCDR3 comprises an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identity with the amino acid sequence set forth in SEQ ID NO: 43; (d) LCDR1 comprises an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identity with the amino acid sequence set forth in SEQ ID NO: 60; (e) LCDR2 comprises an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identity to the amino acid sequence set forth in SEQ ID NO:74, and (f) LCDR3 comprises an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identity to the amino acid sequence set forth in SEQ ID NO:84.

[0039] In another aspect, the present invention provides an isolated antigen-binding molecule, e.g., an antibody or antigen-binding fragment thereof, that binds to human PD-L1. The isolated antigen-binding molecule, e.g., an antibody or antigen-binding fragment thereof, comprises (a) a heavy chain variable region (HCVR) comprising an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or about 100% identity to the amino acid sequence set forth in SEQ ID NO: 104, and (b) a light chain variable region (LCVR) comprising an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or about 100% identity to the amino acid sequence set forth in SEQ ID NO: 130.

[0040] In one aspect, the present invention provides an isolated antigen-binding molecule, such as an antibody or antigen-binding fragment thereof, that binds to human PD-L1. The isolated antigen-binding molecule, such as an antibody or antigen-binding fragment thereof, comprises a heavy chain variable (VV) domain that comprises, from the N-terminus to the C-terminus, three heavy chain complementarity determining regions (CDRs): HCDR1, HCDR2, and HCDR3. H ) domain and three light chain complementarity determining regions (CDRs), LCDR1, LCDR2, and LCDR3, from the N-terminus to the C-terminus. L ) domain, wherein (a) HCDR1 comprises the amino acid sequence set forth in SSYYMI (SEQ ID NO: 14), (b) HCDR2 comprises the amino acid sequence set forth in YVYTGSGNTWYASWAKG (SEQ ID NO: 33), (c) HCDR3 comprises the amino acid sequence set forth in ASGADGVYDWGWDI (SEQ ID NO: 44), (d) LCDR1 comprises the amino acid sequence set forth in QASQSIYSLLA (SEQ ID NO: 61), (e) LCDR2 comprises the amino acid sequence set forth in GASNLES (SEQ ID NO: 75), and (f) LCDR3 comprises the amino acid sequence set forth in QNNYDSGRIYGLA (SEQ ID NO: 85).

[0041] In one embodiment, the isolated antigen-binding molecule, such as an antibody or antigen-binding fragment thereof, comprises a HCVR comprising the amino acid sequence set forth in SEQ ID NO: 105 and a LCVR comprising the amino acid sequence set forth in SEQ ID NO:131.

[0042] In another aspect, the present invention provides an isolated antigen binding molecule, such as an antibody or antigen-binding fragment thereof, that binds to human PD-L1. The isolated antigen-binding molecule, e.g., an antibody or antigen-binding fragment thereof, comprises a heavy chain variable (VH) domain comprising, from the N-terminus to the C-terminus, three heavy chain complementarity determining regions (CDRs), HCDR1, HCDR2, and HCDR3; and a light chain variable (VL) domain comprising, from the N-terminus to the C-terminus, three light chain complementarity determining regions (CDRs), LCDR1, LCDR2, and LCDR3, in which (a) HCDR1 comprises an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identity with the amino acid sequence set forth in SEQ ID NO: 14; (b) HCDR2 comprises an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identity with the amino acid sequence set forth in SEQ ID NO: 33; and (c) an H CDR3 comprises an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identity with the amino acid sequence shown in SEQ ID NO: 44; (d) LCDR1 comprises an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identity with the amino acid sequence shown in SEQ ID NO: 61; (e) LCDR2 comprises an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identity with the amino acid sequence shown in SEQ ID NO: 75; and (f) LCDR3 comprises an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identity with the amino acid sequence shown in SEQ ID NO: 85.

[0043] In yet another aspect, the present invention provides an isolated antigen-binding molecule, e.g., an antibody or antigen-binding fragment thereof, that binds to human PD-L1, The isolated antigen-binding molecule, e.g., an antibody or antigen-binding fragment thereof, comprises (a) a heavy chain variable region (HCVR) comprising an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or about 100% identity to the amino acid sequence set forth in SEQ ID NO: 105, and (b) a light chain variable region (LCVR) comprising an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or about 100% identity to the amino acid sequence set forth in SEQ ID NO: 131.

[0044] In one embodiment, binding of the antibody to PD-L1 or cell surface PD-L1 is measured using, for example, a flow cytometric assay or an ELISA assay as described in Examples 3, 4, and 5, or a substantially similar assay. In another embodiment, competitive binding of the antibody to PD-L1 or cell surface PD-L1 is measured using an assay known in the art, such as the assay described in Harms, et al., "Microtiter plate-based antibody-competition assay to determine binding affinities and plasma / blood stability of CXCR4 ligands, Scientific Reports, 2020:10:16036, doi.org / 10.1038 / s41598-020-73012-4," or a substantially similar assay. In yet another embodiment, blockade of the interaction of PD-1 and PD-L1 is measured using an assay as described in Example 5, or a substantially similar assay. In another embodiment, the enhanced immune response is measured using an assay as described in Example 5, or a substantially similar assay. + This is measured using methods known in the art, such as an increase in the number of T cells.

[0045] In one embodiment, the antibody blocks PD-1 / PD-L1 interaction by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%. In another embodiment, the antibody enhances the immune response by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, 1.5-fold, 2-fold, 4-fold, or more.

[0046] In yet another embodiment, the antibody specifically binds to human PD-L1 and / or cynomolgus PD-L1. In yet another embodiment, the antibody specifically binds to human PD-L1 and / or cynomolgus PD-L1 with similar affinity. In one embodiment, the antibody does not bind to non-primate PD-L1, or binds to non-primate PD-L1 with significantly lower affinity than human PD-L1 and / or cynomolgus PD-L1. In each aspect of the invention and embodiments thereof, the cynomolgus PD-L1 comprises the sequence shown in

[0047] In one embodiment, the invention provides an isolated antigen binding molecule, e.g., an antibody, that binds to human PD-L1 competitively with an antibody of any of the embodiments.

[0048] In one embodiment, the antigen-binding molecule, e.g., antibody, is a humanized antibody or a chimeric antibody. In another embodiment, the antibody comprises a heavy chain constant region of a class selected from the group consisting of IgA, IgD, IgE, IgG, and IgM. In yet another embodiment, the antibody comprises a heavy chain constant region of an IgG class, where the IgG is selected from the group consisting of IgG1, IgG2, IgG3, and IgG4.

[0049] In another aspect, the invention provides an isolated polynucleotide encoding the antigen-binding molecule (e.g., an antibody of any aspect and embodiment), its HCVR, its LCVR, its light chain, its heavy chain, or an antigen-binding fragment thereof.

[0050] In yet another aspect, the present invention provides an expression vector comprising the polynucleotide.

[0051] In yet another aspect, the present invention provides a recombinant cell comprising the polynucleotide or expression vector.

[0052] In one aspect, the invention provides a method for making an antigen binding molecule, e.g., an antibody in any of the aspects and embodiments, comprising expressing the antibody in a recombinant cell and isolating the expressed antibody.

[0053] In one aspect, the invention provides a pharmaceutical composition comprising an antigen-binding molecule (e.g., an antibody) of any of the aspects and embodiments and a pharma- ceutically acceptable carrier or diluent.

[0054] In one embodiment, the antibody is present in the pharmaceutical composition in an amount effective to (a) specifically bind to human or cynomolgus PD-L1 on the cell surface, (b) block the interaction of PD-1 with PD-L1, (c) induce ADCC, (d) induce internalization of PD-L1, (e) provide an immune response in an immune cell, or (f) any combination of (a)-(e).

[0055] In one aspect, the invention provides a method for blocking binding of PD-1 to PD-L1 expressed on a cell surface, comprising contacting a cell with an isolated antibody of any aspect or a pharmaceutical composition of any aspect, thereby blocking binding of PD-1 to PD-L1. In one embodiment, binding of PD-1 to PD-L1 is reduced by at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or about 100%. In another embodiment, the method is used to treat cancer.

[0056] In another aspect, the invention provides a method for enhancing an immune response in a subject, comprising administering to the subject an isolated antibody of any aspect or a pharmaceutical composition of any aspect, thereby enhancing an immune response in the subject. In one embodiment, the immune response includes, but is not limited to, a) reversing T cell inactivation and function, b) promoting T cell proliferation, c) enhancing NK cell function, d) enhancing T cell-mediated anti-tumor immune response, or e) reducing immunosuppression in the tumor microenvironment. In some embodiments, the method of the invention increases the immune response by at least about 10%, about 20%, about 50%, about 60%, about 70%, about 80%, about 90%, about 1-fold, about 2-fold, about 4-fold, or more compared to baseline levels.

[0057] In yet another aspect, the invention provides a method for inhibiting tumor growth in a subject comprising administering to the subject an isolated antibody of any of the aspects, or a pharmaceutical composition of any of the aspects, thereby inhibiting tumor growth.

[0058] In yet another aspect, a method for treating cancer in a subject is provided, comprising administering to the subject an isolated antibody according to any of the aspects or a pharmaceutical composition according to any of the aspects, thereby treating the cancer. In one embodiment, the cancer is any of the cancers described herein. In one particular embodiment, the cancer is selected from the group consisting of non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), head and neck squamous cell carcinoma (HNSCC), bladder cancer (BC), esophageal squamous cell carcinoma (ESCC), triple-negative breast cancer (TNBC), renal cell carcinoma (RCC), colorectal cancer (CRC), hepatocellular carcinoma (HCC), large B-cell lymphoma (BCL), Merkel cell carcinoma (MCC), cervical cancer (CC), classical Hodgkin lymphoma (cHL) microsatellite instability-high (MSI-H), and mismatch repair deficient (dMMR).

[0059] In one embodiment, the method of any of the above aspects results in activating T cells and inducing said T cells to kill tumor target cells.

[0060] In another embodiment, the method of any of the above aspects further comprises administering an additional therapeutic agent. In one embodiment, the additional therapeutic agent comprises any of the therapeutic agents described herein. In another embodiment, the additional therapeutic agent comprises an anti-tumor agent, radiation therapy, a chemotherapeutic agent, surgery, a cancer vaccine, an agonist for a stimulatory receptor on an immune cell, a cytokine, cell therapy, or a checkpoint inhibitor. In one embodiment, the additional therapeutic agent is an antibody, such as a multispecific antibody (e.g., a bispecific antibody).

[0061] In yet another embodiment, the checkpoint inhibitor is selected from the group consisting of TIGIT, CTLA-4, LAG-3, TIM-3, neuropeptides, BTLA, CECAM-1, CECAM-5, IL-1R8, VISTA, LAIR1, LILRB1, LILRB2, LILRB3, LILRB4, LILRB5, CD96, CD112R, CD160, 2B4, TGFβ-R, KIR, NKG2A, MICA, MICB, A2aR, A2bR, and any combination thereof. In one embodiment, the CTLA inhibitor is selected from the group consisting of ipilimumab, kadonilimab, and YH001 (Yuwa Pharmaceutical).

[0062] In yet another embodiment, the additional therapeutic agent is an agonist for a stimulatory receptor on an immune cell, and the agonist is selected from the group consisting of OX40, CD2, CD16, CD27, CDS, ICAM-1, LFA-1, ICOS (CD278), 4-1BB (CD137), GITR, CD28, CD30, CD40, BAFFR, HVEM, CD7, LIGHT, NKG2C, NKG2D, SLAMF7, NKp46, NKp80, CD160, B7-H3, CD83 ligand, and any combination thereof.

[0063] In one embodiment, the additional therapeutic agent is formulated in the same pharmaceutical composition as the antibody, hi another embodiment, the additional therapeutic agent is formulated in a different pharmaceutical composition than the antibody.

[0064] In yet another embodiment, the additional therapeutic agent is administered before and / or after administration of the antigen binding molecule (e.g., an antibody). In yet another embodiment, the additional therapeutic agent is administered after administration of the antigen binding molecule (e.g., an antibody). In yet another embodiment, the additional therapeutic agent is administered simultaneously with the antigen binding molecule (e.g., an antibody).

[0065] In one aspect, the invention provides a method for inducing antibody-dependent cellular cytotoxicity (ADCC) of PD-L1 expressing cells comprising inducing ADCC of PD-L1 expressing cells by contacting the cells with an isolated antibody of any of the aspects, or a pharmaceutical composition of any of the aspects.

[0066] In another aspect, the invention provides a method for killing a PD-L1 expressing cell comprising contacting the cell with a pharmaceutical composition of any of the aspects, or an isolated antibody of any of the aspects conjugated to a cytotoxic agent, thereby killing the PD-L1 expressing cell.

[0067] In one embodiment, the invention provides a kit comprising the pharmaceutical composition of any aspect, hi one embodiment, the pharmaceutical composition further comprises one or more additional therapeutic agents as described herein. [Brief description of the drawings]

[0068] [Figure 1] FIG. 1 shows that exemplary antibodies of the disclosure effectively block the interaction of PD-1 with PD-L1. [Diagram 2] Figures 2A, 2B, and 2C show that exemplary antibodies of the disclosure induce internalization upon binding to cell surface PD-L1. [Diagram 3] FIG. 3 shows that exemplary antibodies of the disclosure conjugated to a cytotoxic agent elicit potent killing of PD-L1 expressing cells. [Figure 4] Figures 4A and 4B show epitope binning of exemplary antibodies of the disclosure with Avelumab, as well as certain other anti-PD-L1 antibodies with Avelumab. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0069] The present invention and the accompanying drawings will now be described in a manner to enable one skilled in the art to practice the invention. However, those skilled in the art will understand that the invention described below may be practiced without these specific details or may be used for purposes other than those described herein. Indeed, in light of the present disclosure, the invention can be modified and used in conjunction with products and techniques known to those skilled in the art. The drawings and descriptions are intended to be illustrative of various aspects of the invention and are not intended to narrow the scope of the appended claims. Further, it will be understood that the drawings may illustrate various aspects of the invention alone and that elements of one drawing may be used in conjunction with elements shown in other drawings.

[0070] It will be understood that references to aspects, features, advantages, or similar terms throughout this specification do not imply that all aspects and advantages should or have been realized by the present invention in any single embodiment of the present invention. Rather, the language referring to aspects and advantages is understood to mean that the particular aspect, feature, advantage, or characteristic described in connection with an embodiment is included in at least one embodiment of the present invention. Thus, references to aspects and advantages throughout this specification, and similar terms, do not necessarily refer to the same embodiment.

[0071] The aspects, features, advantages, and characteristics described herein may be combined in any suitable manner in one or more additional embodiments. Moreover, those skilled in the art will recognize that the invention may be practiced without one or more of the specific aspects or advantages in a particular embodiment. In other cases, other aspects, features, and advantages may be recognized and claimed for a particular embodiment that may not be present in all embodiments of the invention.

[0072] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Those skilled in the art will recognize that many techniques and materials similar or equivalent to those described herein can be used in the practice of each aspect and embodiment of the present invention. The aspects and embodiments described in this application are not limited to the methods and materials described.

[0073] Additionally, any references, issued patents, or published patent applications cited herein are expressly incorporated by reference in their entirety for the teachings herein.

[0074] 1. Definition To facilitate understanding of this disclosure, several terms are first defined. It should be noted that when a value or range of values ​​for a parameter is listed, the intermediate values ​​and ranges of the listed values ​​are also intended to be part of this disclosure.

[0075] Use of the terms "a," "an," and "the above," and similar referents in the context of describing the present invention (particularly in the context of the claims), should be construed to include both the singular and the plural (i.e., one or more), unless otherwise stated herein or clearly contradicted by context. The terms "including," "having," "including," and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to"), unless otherwise indicated. The recitation of ranges of values ​​herein is intended to serve as a shorthand method of individually referring to each individual value recited or falling within the range, unless otherwise indicated herein, and each individual value is incorporated into the specification as if it were individually listed.

[0076] When "in one embodiment," "in another embodiment," "in other embodiments," "in some embodiments," or "in a particular embodiment" is used, the disclosure should be interpreted as encompassing any combination of features defining different embodiments described therein, unless the features are incombinable with each other, mutually exclusive, or otherwise expressly disclaimed herein.

[0077] The terms "about" or "approximately" generally mean within 10%, preferably within 5%, and more preferably within 1% of a given value or range.

[0078] Ranges may be expressed herein as from "about" one particular value, and / or to "about" another particular value. When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when values ​​are expressed as approximations, by use of the antecedent "about," it will be understood that the particular value constitutes another embodiment. It will be further understood that the endpoints of each range are not only distinct but independent of the other endpoint. Additionally, there are a number of values ​​disclosed herein, and each value will be understood to include "about" that particular value in addition to the value itself. For example, if the value "10" is disclosed, then "about 10" is also disclosed.

[0079] The term "reagent" as used herein refers to any substance, compound (e.g., molecule), supramolecular complex, material, or combination or mixture thereof. A compound may be any substance represented by a chemical formula, chemical structure, or sequence. Examples of reagents include, for example, small molecules, polypeptides, nucleic acids (e.g., RNAi agents, antisense oligonucleotides, aptamers), lipids, polysaccharides, and the like. In general, the reagents can be obtained using any suitable method known in the art. Typically, one of skill in the art will select an appropriate method based, for example, on the nature of the reagent. The reagents can be at least partially purified. In some embodiments, the reagents can be provided as part of a composition. The composition can also include, in various embodiments, in addition to the reagent, for example, counterions, aqueous or non-aqueous diluents or carriers, buffers, preservatives, or other components. In some embodiments, the reagents can be provided as salts, esters, hydrates, or solvates. In some embodiments, the reagents are cell-permeable, e.g., within the scope of typical reagents that are taken up by cells and act in cells (e.g., mammalian cells) to produce a biological effect. Certain compounds may exist in particular geometric or stereoisomeric forms. Such compounds include cis- and trans-isomers, E- and Z-isomers, R- and S-enantiomers, diastereomers, (D)-isomers, (L)-isomers, (-)- and (+)-isomers, racemic mixtures thereof, and other mixtures thereof, and are included in various embodiments of the present disclosure, unless otherwise indicated. Certain compounds may exist in various protonation states, have various configurations, exist as solvates (e.g., in water (i.e., hydrates) or common solvents), and / or have different crystal forms (e.g., polymorphs) or different tautomeric forms. Embodiments exhibiting such alternative protonation states, configurations, solvates, and forms are encompassed by the present disclosure, where applicable.

[0080] In certain embodiments, depending on the context, "agent" also encompasses therapeutic methods such as radiation therapy, chemotherapy, or surgery.

[0081] The term "agonist" refers to a substance that promotes (i.e., induces, causes, enhances, or increases) the biological activity or effect of another molecule. The term "agonist" includes substances that bind to receptors (e.g., antibodies) and substances that promote receptor function without binding to the receptor (e.g., promote receptor function by activating associated proteins).

[0082] The term "amino acid" refers to the 20 common naturally occurring amino acids, including alanine (Ala; A), arginine (Arg; R), asparagine (Asn; N), aspartic acid (ASP; D), cysteine ​​(Cys; C), glutamic acid (Glu; E), glutamine (Gin; Q), glycine (Gly; G); histidine (His; H), isoleucine (He; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V).

[0083] The terms "antagonist" or "inhibitor" refer to a substance that prevents, blocks, suppresses, neutralizes, or decreases the biological activity or effect of another molecule (eg, a receptor).

[0084] The term "antibody" as used herein refers to any antigen-binding molecule or molecular complex that contains at least one complementarity determining region (CDR) that specifically binds to or interacts with a particular antigen (e.g., PD-L1). The term "antibody" includes immunoglobulin molecules that contain four polypeptide chains (i.e., two heavy (H) and two light (L) chains inter-connected by disulfide bonds), as well as multimers thereof (e.g., IgM). Each heavy chain contains a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region is a CDR that is a nucleotide sequence that is linked to the CDR by the CDR domain. H 1. C H 2 and C HEach light chain contains three domains, designated CVR and VL. Each light chain contains a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region contains one domain (CVR). L 1) is included. H and V L The regions can be further subdivided into regions of hypervariability called complementarity determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). H and V L is composed of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In different embodiments of the invention, the FRs of an anti-PD-L1 antibody (or antigen-binding fragment thereof) may be identical to mouse or human germline sequences, or may be naturally or artificially modified. An amino acid consensus sequence can be determined based on a side-by-side analysis of two or more CDRs.

[0085] The term "antibody" as used herein also encompasses antigen-binding fragments of intact antibody molecules. The terms "antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody as used herein encompass any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. Antigen-binding fragments of antibodies can be obtained from intact antibody molecules by using any suitable standard technique, such as, for example, proteolysis or recombinant genetic engineering techniques (including the manipulation and expression of DNA encoding antibody variable and optionally constant regions).

[0086] Non-limiting examples of antigen-binding fragments include (i) Fab fragments, (ii) F(ab')2 fragments, (iii) Fd fragments, (iv) Fv fragments, (v) single chain Fv (scFv) molecules, (vi) dAb fragments, and (vii) minimal recognition units consisting of amino acid residues that mimic a hypervariable region of an antibody (e.g., an isolated complementarity determining region (CDR) such as a CDR3 peptide) or a constrained FR3-CDR3-FR4 peptide. Domain-specific antibodies, single domain antibodies, domain deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and other modified molecules such as the variable IgNAR domains of sharks are also encompassed by the term "antigen-binding fragment" as used herein.

[0087] When describing a sequence of polypeptide domains using hyphens between individual domains (eg, CH2-CH3), it will be understood that the order of the described domains is from N-terminus to C-terminus.

[0088] An antigen-binding fragment of an antibody typically comprises at least one variable domain, which may be of any size or consist of amino acids and generally comprises at least one CDR adjacent to or in frame with one or more framework sequences. H Domain and V L In the antigen-binding fragment in which the V domain is linked H and V L The domains may be positioned relative to each other in any suitable arrangement. For example, the variable region may be a dimer, with the V H -V H , V H -V L Or V L -V L Alternatively, the antigen-binding fragment of the antibody may comprise a dimer of monomer V H Or V L It may also include a domain.

[0089] In certain embodiments, an antigen-binding fragment of an antibody may comprise at least one variable domain covalently linked to at least one constant domain. Non-limiting exemplary structural arrangements of variable and constant domains contained in an antigen-binding fragment of an antibody of the invention include: (i) a V H -C H 1, (ii) V H -C H 2. (iii) V H -C H 3. (iv) V H -C H 1-C H 2. (v) V H -C H 1-C H 2-C H 3. (vi) V H -C H 2-C H 3. (vii) V H -C L , (viii) V L -C H 1, (ix) V L -C H 2. (x)V L -C H 3. (xi) V L -C H 1-C H 2. (xii) V L -C H 1-C H 2-C H 3. (xiii) V L -C H 2-C H 3, and (xiv) V L -C LIn any structural arrangement of the variable and constant domains (including any of the exemplary arrangements described above), the variable and constant domains may be directly linked to each other or may be linked by a complete or partial hinge or linker region. The hinge region may consist of at least two (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids to form a flexible or semi-flexible linkage between adjacent variable and / or constant domains within a single polypeptide molecule. Furthermore, antigen-binding fragments may be non-covalently linked to each other and / or to one or more monomeric V H Or V L The variable domains may comprise homodimers or heterodimers (or other multimers) of any of the variable and constant domain arrangements described above, in which the domains are non-covalently associated (e.g., by disulfide bonds).

[0090] As with intact antibody molecules, antigen-binding fragments may be monospecific or multispecific (e.g., bispecific). Multispecific antigen-binding fragments of antibodies typically contain at least two different variable domains. Each variable domain can specifically bind to a single antigen or to a different epitope on the same antigen. Any multispecific antibody format (including the exemplary bispecific antibody formats disclosed herein) may be adapted for use in conjunction with the antigen-binding fragments of antibodies of the invention, using techniques commonly available in the art.

[0091] The antibody according to the present invention may be an isolated antibody. As used herein, an "isolated" molecule (e.g., an isolated antibody or an isolated polypeptide) refers to a molecule (e.g., an antibody) that has been identified and separated and / or recovered from at least one component of its natural environment. For example, a molecule (e.g., an antibody) that has been separated or removed from at least one component of an organism, or from a tissue or cell in which the antibody naturally occurs or is naturally produced, is an "isolated" molecule (e.g., an antibody) for purposes of the present invention. An isolated molecule (e.g., an antibody) also includes an in situ molecule (e.g., an antibody) in a recombinant cell. In certain embodiments, an isolated molecule (e.g., an antibody) is a molecule (e.g., an antibody) that has been subjected to at least one purification or isolation step. According to certain embodiments, an isolated molecule (e.g., an antibody) may be substantially free of other cellular material and / or chemicals.

[0092] The present invention also includes one-arm antibodies that bind to PD-L1. As used herein, "one-arm antibody" refers to an antigen-binding molecule that comprises a single antibody heavy chain and a single antibody light chain. The one-arm antibodies described in the present invention may comprise any of the HCVR / LCVR or CDR amino acid sequences shown in Tables 1-20.

[0093] The anti-PD-L1 antibodies or antigen-binding fragments thereof described herein may contain one or more amino acid substitutions, insertions and / or deletions in the framework and / or CDR regions of the heavy and light chain variable regions compared to the corresponding germline sequences from which the antigen-binding molecule (e.g., an antibody or antigen-binding fragment thereof) is derived. Such mutations can be readily ascertained by comparing the amino acid sequences disclosed herein to germline sequences available, for example, from public antibody sequence databases. The invention includes antibodies and antigen-binding fragments thereof derived from any of the amino acid sequences disclosed herein, where one or more amino acids in one or more framework and / or CDR regions have been mutated to the corresponding residue in the germline sequence from which the antibody is derived, or to the corresponding residue in another human germline sequence, or to a conservative amino acid substitution of the corresponding germline residue (such sequence changes are collectively referred to herein as "germline mutations"). Starting from the heavy and light chain variable region sequences disclosed herein, one of skill in the art can generate numerous antibodies and antigen-binding fragments containing one or more individual germline mutations or combinations thereof. In certain embodiments, the V H and / or V LAll framework and / or CDR residues within the domains are mutated back to the residues of the original germline sequence from which the antibody is derived. In other embodiments, only certain residues are mutated back to the original germline sequence, for example only the mutated residues found within the first 8 amino acids of FR1 or the last 8 amino acids of FR4, or only the mutated residues found within CDR1, CDR2 or CDR3. In other embodiments, one or more of the framework and / or CDR residues are mutated to the corresponding residue in a different germline sequence (i.e., a germline sequence different from the germline sequence from which the antibody is derived). Furthermore, the antibodies or antigen-binding domains thereof of the invention can comprise any combination of two or more germline mutations in the framework and / or CDR regions, for example, certain individual residues are mutated to the corresponding residue in a particular germline sequence and certain other residues that differ from the original germline sequence are maintained or are mutated to the corresponding residue in a different germline sequence. Once obtained, antibodies or antigen-binding fragments thereof that contain one or more germline mutations can be readily measured for one or more desired properties, such as improved binding specificity, increased binding affinity, improved or enhanced antagonistic or agonistic biological properties (as the case may be), reduced immunogenicity, etc. Antibodies or antigen-binding fragments thereof obtained in such general manner are encompassed by the present invention.

[0094] The invention also includes anti-PD-L1 antibodies that comprise variants of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein. Exemplary variants included in aspects of the invention include variants of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein that have one or more conservative substitutions. For example, the invention includes anti-PD-L1 antibodies and antigen binding proteins that have HCVR, LCVR, and / or CDR amino acid sequences that include no more than 10, no more than 8, no more than 6, no more than 4, etc., conservative amino acid substitutions compared to any of the HCVR, LCVR, and / or CDR amino acid sequences shown in the tables herein.

[0095] Light chains are classified as kappa or lambda (K, λ). Each heavy chain class can be combined with either kappa or lambda light chains. Generally, when immunoglobulins are produced by hybridomas, B cells or genetically engineered host cells, the light and heavy chains are covalently linked to each other, and the "tail" portions of the two heavy chains are linked to each other by covalent disulfide bonds or non-covalent bonds. In the heavy chains, the amino acid sequences run from the N-terminus at the forked ends of the Y configuration to the C-terminus at the bottom of each chain.

[0096] As used herein, the term "light chain constant region" or "C L " are used interchangeably herein and refer to amino acid sequences derived from an antibody light chain. Preferably, the light chain constant region comprises at least one of a constant kappa domain or a constant lambda domain.

[0097] As used herein, the term "heavy chain constant region" includes an amino acid sequence derived from an immunoglobulin heavy chain. A polypeptide containing a heavy chain constant region is H 1 domain, a hinge (e.g., upper, middle and / or lower hinge region) domain, C H 2 domain, C H For example, the antigen-binding polypeptides used in the present disclosure include at least one of the C3 domains, or variants or fragments thereof. H A polypeptide chain containing one domain; C H 1 domain and at least a portion of the hinge domain and C H A polypeptide chain containing two domains; C H 1 Domain and C H A polypeptide chain containing three domains; C H 1 domain and at least a portion of the hinge domain and C H A polypeptide chain comprising three domains, or C H 1 domain and at least a portion of the hinge domain and C H 2 Domain and C H In some embodiments, the polypeptide of the present disclosure may comprise a polypeptide chain comprising a C HIn addition, the antibodies used in the present disclosure comprise a polypeptide chain comprising three domains. H It may be missing at least a portion of the C2 domain (e.g., H It should be understood that the heavy chain constant region may be modified so that its amino acid sequence does not differ from that of a naturally occurring immunoglobulin molecule.

[0098] The heavy chain constant regions of the antibodies disclosed herein may be derived from different immunoglobulin molecules. For example, the heavy chain constant region of the polypeptide may be derived from a C1 molecule. H In another example, the heavy chain constant region can comprise a hinge region derived, in part, from an IgG1 molecule and, in part, from an IgG3 molecule. In another example, the heavy chain portion can comprise a chimeric hinge derived, in part, from an IgG1 molecule and, in part, from an IgG4 molecule.

[0099] The subunit structures and three-dimensional configurations of the constant regions of the various immunoglobulin classes are well known. As used herein, the term "VH domain" comprises the N-terminal variable domain of an immunoglobulin heavy chain. H The "C1 domain" comprises the first (most N-terminal) constant region domain of an immunoglobulin heavy chain. H One domain is adjacent to the VH domain and N-terminal to the hinge region of the immunoglobulin heavy chain molecule.

[0100] As used herein, the term "C H The "C2 domain" includes, for example, the portion of an antibody heavy chain molecule extending from about residue 244 to residue 360, based on common numbering schemes (residues 244-360, Kabat numbering system; or residues 231-340, EU numbering system). H The 2 domain is unique in that it is not closely matched to another domain. Rather, two N-linked branched glycans match the two C domains of an intact native IgG molecule. H It is inserted between the two domains. HThe three domains are H It extends from domain 2 to the C-terminus of the IgG molecule and contains approximately 108 residues.

[0101] As used herein, the term "hinge region" refers to a region H 1 domain C H The hinge region comprises the portion of the heavy chain molecule that connects the two N-terminal antigen-binding domains. This hinge region contains approximately 25 residues and is flexible, allowing the two N-terminal antigen-binding domains to move independently. The hinge region can be subdivided into three distinct domains: the upper, middle and lower hinge regions.

[0102] As used herein, the term "disulfide bond" includes a covalent bond formed between two sulfur atoms. Cysteine ​​contains a thiol group that can form a disulfide bond or crosslink with a second thiol group. In most naturally occurring IgG molecules, C H 1 and C L The regions are linked by disulfide bonds, and the two heavy chains are linked by two disulfide bonds at positions corresponding to 239 and 242 according to the Kabat numbering system (and positions 226 and 229 according to the EU numbering system).

[0103] The term "antibody" encompasses various broad classes of polypeptides that can be distinguished biochemically. Those skilled in the art will appreciate that heavy chains are classified as alpha, delta, epsilon, gamma, and mu (or α, δ, ε, γ, and μ), as well as several subclasses (e.g., γ1-γ4). The nature of the chain determines the "class" of the antibody as IgG, IgM, IgA, IgD, or IgE, respectively. Immunoglobulin subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgG5, etc., are well characterized and known to confer functional specialization. In accordance with the present disclosure, modified forms of each of these classes and isotypes are readily discernible to one of skill in the art and are therefore within the scope of the present disclosure. All immunoglobulin classes are within the scope of the present disclosure, and for the following discussion, the discussion will generally be directed to the IgG class of immunoglobulin molecules.

[0104] The antibodies of the present disclosure include, but are not limited to, polyclonal antibodies, monoclonal antibodies, multispecific antibodies, bispecific antibodies, trispecific antibodies, human antibodies, humanized antibodies, primate antibodies, chimeric antibodies, and single chain antibodies. The antibodies disclosed herein may be from any animal origin, including birds and mammals. Preferably, the antibodies are from human, mouse, donkey, rabbit, goat, guinea pig, camel, llama, horse, or chicken. In some embodiments, the variable region may be from a chondrictoid (e.g., from a shark).

[0105] As used herein, the term "chimeric antibody" refers to an antibody in which the immunoreactive region or site is obtained or derived from a first species and the constant region (complete, partial or modified according to the present disclosure) is obtained from a second species. In certain embodiments, the target binding region or site is derived from a non-human source (e.g., mouse or primate) and the constant region is derived from a human.

[0106] The term "humanized antibody" as used herein refers to a genetically engineered non-human antibody that contains a human antibody constant region and a non-human variable region that has been modified to have a high degree of sequence identity to the human variable region. This can be achieved by grafting the six non-human antibody complementarity determining regions (CDRs) that form the antigen binding site onto the homologous human acceptor framework regions (FRs). Substitution of framework residues from the parent antibody (i.e., non-human antibody) into the human framework regions may be required to reconstitute the binding affinity and specificity of the parent antibody (back mutations). Homology modeling may help to identify amino acid residues in the framework regions that are important for the binding properties of the antibody. Thus, a humanized antibody may contain non-human CDR sequences, primarily human framework regions, optionally including one or more amino acid back mutations to the non-human amino acid sequences, and a fully human constant region. Optionally, other amino acid modifications (not necessarily back mutations) may be applied to obtain a humanized antibody with favorable properties such as affinity and biochemical properties.

[0107] "Single variable chain fragment" or "scFv" refers to a fusion protein of the variable heavy (VH) and variable light (VL) regions of an immunoglobulin. In some embodiments, these regions are linked by a short linker peptide of about 10-25 amino acids. The linker is rich in glycines for increased flexibility and serines or threonines for increased solubility, and can link the N-terminus of VH to the C-terminus of VL, or vice versa. The protein retains the specificity of the original immunoglobulin despite the removal of the constant regions and the introduction of the linker.

[0108] In some embodiments, the antibody of the present invention may be a recombinant antibody. The term "recombinant antibody" as used herein is intended to include all antibodies that are made, expressed, produced or isolated by recombinant means, including, for example, antibodies expressed using a recombinant expression vector transfected into a host cell, antibodies isolated from a recombinant combinatorial antibody library, or antibodies made, expressed, produced or isolated by any other means including splicing of immunoglobulin gene sequences to other DNA sequences. Such recombinant antibodies have variable and constant regions derived from germline immunoglobulin sequences. However, in certain embodiments, such recombinant antibodies are sequences that have been modified by in vitro mutagenesis to produce an amino acid sequence of the VH and VL regions of the recombinant antibody that is derived from and related to germline VH and VL sequences, but may not naturally exist in an antibody germline library in vivo.

[0109] The term "conjugate" or "antibody conjugate" refers to an antibody bound to one or more reagents. The antibody may be covalently bound to the reagent via a covalent bond or a linker. In certain embodiments, the linker is covalently bound to the antibody and covalently bound to the reagent. In certain embodiments, the linker is bound to the antibody and / or the reagent via non-covalent means. In certain embodiments, the linker is bound to the reagent via a covalent bond and to the antibody via a specific bond. In certain embodiments, the linker is a moiety that can specifically bind to the antibody, for example, an antibody that binds to the Fc region of the antibody.

[0110] The term "epitope" refers to an antigenic determinant that interacts with a specific antigen-binding site in the variable region of an antibody molecule, called the paratope. A single antigen can have multiple epitopes. Thus, different antibodies can bind to different regions on the antigen and have different biological effects. Epitopes can be either conformational or linear. Conformational epitopes are generated by spatially juxtaposed amino acids from different segments of a linear polypeptide chain. Linear epitopes are generated by adjacent amino acid residues within a polypeptide chain. In certain circumstances, epitopes may include carbohydrate, phosphoryl, or sulfonyl moieties on an antigen.

[0111] The term "immunoconjugate" refers to an antibody covalently fused to a peptide or small molecule drug, which can be attached to the C-terminus of the constant heavy chain or the N-terminus of the variable light and / or variable heavy chain.

[0112] The terms "improvement," "increase," or "decrease" as used in this context refer to a value or parameter relative to a baseline / control / reference measurement, e.g., a measurement in a cell or tissue prior to initiation of a treatment described herein, a measurement in a cell or tissue in the absence of a treatment described herein, a measurement in the same individual prior to initiation of a treatment described herein, or a measurement in a control individual in the absence of a treatment described herein (or a standard measurement in multiple control individuals, e.g., an average of multiple control individuals). A "control individual" refers to an individual with a similar condition, e.g., an individual afflicted with the same cell proliferative disorder as the treated individual, an individual of approximately the same age as the treated individual (to ensure that the stage of disease in the treated and control individuals is comparable). The treated individual (also referred to as a "patient" or "subject") may be a fetus, infant, child, adolescent, or adult afflicted with a cell proliferative disorder.

[0113] The term "recombinant" as used herein refers to a polypeptide or polynucleotide that does not occur in nature and can be made by combining polynucleotides or polypeptides in an arrangement in which they are not normally together. The term "recombinant cell" is defined herein as a non-naturally occurring host cell that contains one or more (e.g., two, several) heterologous polynucleotides.

[0114] The term "prevention" refers to a reduction in the occurrence of disease symptoms (e.g., associated with PD-L1 activity or function) in a patient. As noted above, prevention may be complete (no detectable symptoms) or partial, meaning that there are fewer symptoms observed than in the absence of treatment.

[0115] The phrase "small molecule drug" refers to a molecular entity, typically organic or organometallic rather than polymeric, that has pharmaceutical activity and has a molecular weight of less than about 2 kDa, less than about 1 kDa, less than about 900 Da, less than about 800 Da, or less than about 700 Da. The term encompasses most pharmaceutical compounds that are referred to as "drugs" other than proteins or nucleic acids, although small peptides or nucleic acid analogs can be considered small molecule drugs. Examples include chemotherapeutic anticancer drugs and enzyme inhibitors. Small molecule drugs can be synthetic, semi-synthetic (i.e., from naturally occurring precursors), or obtained by biological means.

[0116] The term "specific binding" or "specifically binds" as used herein refers to the ability to discriminate between possible binding partners in the environment in which the binding occurs. In some embodiments, an antibody that interacts (e.g., preferentially interacts) with one particular antigen in the presence of other possible antibodies is said to "specifically bind" to the antigen with which it interacts. In some embodiments, specific binding is assessed by detecting or determining the degree of binding between an antibody and its targeted antigen. In some embodiments, specific binding is assessed by detecting or determining the degree of dissociation of an antibody-antigen complex. In some embodiments, specific binding is assessed by detecting or determining the ability of an antibody to compete with an alternative interaction between its target and another antibody. In some embodiments, specific binding is assessed by performing such detection or determination over a range of concentrations. In general, an antibody binds to an epitope through its antigen-binding domain, and such binding necessarily involves some complementarity between the antigen-binding domain and the epitope. Thus, an antibody is said to "specifically bind" to an epitope when it is easier to bind to the epitope through the antigen-binding domain than to bind to a random, unrelated epitope. The term "specificity" is used herein to qualify the relative affinity with which a particular antibody binds to a particular epitope. For example, antibody "A" can be considered to have a higher specificity for a given epitope than antibody "B," or antibody "A" can be considered to have a higher specificity for epitope "C" than for related epitope "D." In some embodiments, the complex formed by an antibody or antigen-binding fragment thereof and an antigen has a dissociation constant (K d ) is 10 -6 M or less, 10 -7 M or less, 10 -8 M or less, 10 -9 M or less, or 10 -10The antibody or antigen-binding fragment thereof "has specificity" for an antigen if M or less. In some embodiments, specific binding of an antigen-binding molecule (e.g. an anti-human PD-L1 antibody or antigen-binding fragment thereof) can be demonstrated by preferential binding of the antigen-binding molecule to human PD-L1 expressed on the cell surface, using the assays described in Examples 4-7, or substantially similar methods.

[0117] When referring to a nucleic acid or a fragment thereof, the term "substantial identity" or "substantially identical" refers to having at least about 95%, more preferably at least about 96%, 97%, 98% or 99% nucleotide base identity with a nucleotide sequence when optimally aligned to another nucleic acid (or its complementary strand) with appropriate nucleotide insertions or deletions, as measured by any well-known sequence identity algorithm, such as FASTA, BLAST or Gap, as discussed below. In some cases, a nucleic acid molecule having substantial identity to a reference nucleic acid molecule can encode a polypeptide having an amino acid sequence identical or substantially similar to the polypeptide encoded by the reference nucleic acid molecule.

[0118] The term "substantial similarity" or "substantial similarity" as applied to polypeptides means that two peptide sequences share at least 95% sequence identity, and even more preferably at least 98% or 99% sequence identity, when optimally aligned, such as by a program (e.g., GAP or BESTFIT) using default gap weights. Preferably, non-identical residue positions differ by conservative amino acid substitutions. A "conservative amino acid substitution" means that an amino acid residue is replaced by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, conservative amino acid substitutions do not substantially change the functional properties of a protein. In cases where two or more amino acid sequences differ from each other by conservative substitutions, the percent sequence identity or similarity can be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art. See, e.g., Pearson (1994) Methods Mol. Biol. 24:307-331. Examples of amino acid groups with side chains of similar chemical properties include: (1) aliphatic side chains: glycine, alanine, valine, leucine, isoleucine, (2) aliphatic hydroxyl group side chains: serine, threonine, (3) amide-containing side chains: asparagine, glutamine, (4) aromatic side chains: phenylalanine, tyrosine, tryptophan, (5) basic side chains: lysine, arginine, and histidine, (6) acidic side chains: aspartic acid and glutamic acid, and (7) sulfur-containing side chains: cysteine ​​and methionine. Preferred conservative amino acid substitution groups are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine. Alternatively, a conservative substitution means any change that has a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256: 1443-1445. A "moderate conservative substitution" means any change that has a non-negative value in the PAM250 log-likelihood matrix.

[0119] The sequence similarity of polypeptides, also called sequence identity, is typically measured using sequence analysis software. Protein analysis software matches similar sequences using measures of similarity assigned to various substitutions, deletions, and other modifications (including conservative amino acid substitutions). For example, GCG software includes programs such as Gap and Bestfit, which can determine sequence homology or sequence identity between closely related polypeptides (homologous polypeptides from different species, or homologous polypeptides of wild-type proteins and their variants) using default arguments. See, for example, GCG version 6.1. Polypeptide sequences can also be compared using FASTA, a program in GCG version 6.1, using default or recommended arguments. FASTA (e.g., FASTA2 and FASTA3) provides alignment and percent sequence identity of the best overlapping regions between the query and search sequences (Pearson (2000), supra). Another preferred algorithm for comparing the sequences of the present invention to a database containing a large number of sequences from different organisms is the computer program BLAST, particularly BLASTP or TBLASTN, using default arguments. See, e.g., Altschul et al. (1990) J. Mol. Biol. 215:403-410 and Altschul et al. (1997) Nucleic Acids Res. 25:3389-402.

[0120] The terms "therapeutically effective amount", "pharmacologically effective amount", and "physiologically effective amount" are used interchangeably and refer to an amount of an active agent sufficient to ameliorate at least one symptom of a disease or disorder. For example, for a given parameter, a therapeutically effective amount indicates an increase or decrease of at least 5%, 10%, 15%, 20%, 25%, 40%, 50%, 60%, 75%, 80%, 90%, 95%, 99%, or at least 100%. A therapeutically effective amount can also be expressed as a "-fold" increase or decrease. For example, a therapeutically effective amount can have at least a 1.2-fold, 1.5-fold, 2-fold, 5-fold, or more effect over a control. The exact amount depends on many factors, such as the particular active agent, the components and physical characteristics of the composition, the target patient population, and patient considerations including weight, sex, etc., and can be readily determined by one of skill in the art based on the information provided herein or available in the relevant literature.

[0121] The term "treatment" refers to the improvement of one or more symptoms associated with a disease or disorder. Thus, these terms refer to any indicator of success in treating or improving an injury, disease, pathology or disorder, including prevention or delay of onset of one or more symptoms of a disease or disorder; reduction in the severity or frequency of one or more symptoms of a disease or disorder; reduction of any objective or subjective parameters; remission; reduction of symptoms or making an injury, pathology or disorder more tolerable to the patient; slowing down the rate of degeneration or decline; reduction in the debilitating end point of degeneration; and / or improvement of the patient's physical or mental health. "Treatment" may also include prophylactic treatment.

[0122] The phrases "patient in need thereof," "patient in need of treatment," "subject in need thereof," or "subject in need of treatment" include subjects (e.g., mammalian subjects) who may benefit from the administration of an antibody of the present disclosure in the treatment of a cell proliferative disorder.

[0123] The term "variant" as used herein refers to a polypeptide or polynucleotide (e.g., an antibody or polynucleotide) resulting from the incorporation of one or more amino acid or nucleotide insertions, substitutions, or deletions in a precursor polypeptide or polynucleotide (e.g., a "parent" polypeptide or polynucleotide). In certain embodiments, a variant polypeptide or polynucleotide has at least about 85% amino acid or nucleotide sequence identity, e.g., about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% amino acid or nucleotide sequence identity, with the entire amino acid or nucleotide sequence of the parent polypeptide or polynucleotide. A protein or peptide variant substantially maintains the structure, function, or activity of the protein. For example, an antibody variant maintains and / or modulates the function or activity of specifically binding to its antigen (e.g., inhibits the activity of the antigen). In the case of a polynucleotide, the variant maintains the function or activity of the parent polynucleotide. For example, a variant polynucleotide can encode a protein or peptide that has a similar function or activity as the polypeptide encoded by the parent polynucleotide.

[0124] The term "PD-L1" as used herein refers to programmed cell death ligand 1. The term "PD-L1" includes native PD-L1 from all mammalian species, e.g., human, primate, rodent, canine, feline, equine, and bovine, unless otherwise indicated (e.g., specific reference to human PD-L1). The nucleotide and amino acid sequences of PD-L1 are known and can be seen, for example, in GenBank Accession Nos. NP_054862.1, XP_015292694.1, NP_068693.1, XP_014973151.1, NP_068693.1, NP_001178883.1, XP_005615993.1, and G1SUI3-1, the entire contents of each of which are incorporated herein by reference. An exemplary human PD-L1 amino acid sequence is shown below: (Sequence Number) MRIFAVFIFMTYWHLLNAFTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHG EEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKINQR ILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCT FRRLDPEENHTAELVIPELPLAHPPNERTHLVILGAILLCLGVALTFIFRLRKGRMMDVKKCGIQDTNSK KQSDTHLEET An exemplary cynomolgus monkey PD-L1 amino acid sequence is shown below: (Sequence Number) MRIFAVFIFTIYWHLLNAFTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLTSLIVYWEMEDKNIIQFVHG EEDLKVQHSNYRQRAQLLKDQLSLGNAALRITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKINQR ILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLLNVTSTLRINTTANEIFYCI FRRLDPEENHTAELVIPELPLALPPNERTHLVILGAIFLLLGVALTFIFYLRKGRMMDMKKCGIRVTTNSK KQRGKNIRRNWEVEGKGNKKLKQ

[0125] The term "anti-PD-L1 antibody" or "PD-L1 antibody" refers to an antibody or polypeptide that specifically binds to PD-L1. In certain embodiments, the anti-PD-L1 antibody can inhibit the biological activity of PD-L1 and / or downstream signaling pathways mediated by PD-L1. The anti-PD-L1 antibody includes an antibody or polypeptide that includes one or more antigen-binding domains in the form of CDRs or variable regions. In certain embodiments, the anti-PD-L1 antibodies of the invention inhibit, antagonize, suppress or reduce (to any extent, including significantly) the biological activity of PD-L1, including downstream events mediated by PD-L1, such as PD-L1 binding, PD1 / PD-L1 interaction and downstream signaling, inhibition of anti-tumor immune responses, and immune suppression in immune deficiency disease states. In certain embodiments, the anti-PD-L1 antibodies of the invention can be internalized into tumor cells expressing PD-L1 and induce tumor cell killing by induction of antibody drug conjugates.

[0126] II. PD-L1 antibodies and antigen-binding proteins The present invention provides PD-L1 antigen binding molecules that specifically bind to PD-L1. As used herein, the term "antigen binding molecule" refers to a protein, polypeptide, or molecular complex comprising or consisting of at least one complementarity determining region (CDR) alone or in combination with one or more other CDRs and / or framework regions (FRs), that specifically binds to a particular antigen. As defined elsewhere herein, in some embodiments, the antigen binding molecule is an antibody or an antigen-binding fragment thereof. In some embodiments, the antigen binding molecule according to the present invention inhibits one or more of the biological functions of PD-L1.

[0127] In some embodiments, the PD-L1 is human PD-L1. An exemplary human PD-L1 includes the amino acid sequence set forth in SEQ ID NO:. In some embodiments, the PD-L1 is cynomolgus PD-L1. An exemplary cynomolgus PD-L1 includes the amino acid sequence set forth in SEQ ID NO:.

[0128] 1. Exemplary antigen-binding molecule sequences A PD-L1 antigen binding molecule may be in the form of a monoclonal antibody, one or more polypeptide fragments that contain one or more PD-L1 antigen binding domains, or one or more nucleic acids encoding one or more PD-L1 binding domains.

[0129] In various exemplary embodiments of the present invention, an antigen-binding molecule, such as an anti-PD-L1 antibody or antigen-binding fragment thereof, comprises (1) a heavy chain variable region comprising three complementarity determining regions (HCDRs), HCDR1, HCDR2, and HCDR3, and (2) a light chain variable region comprising three complementarity determining regions (LCDRs), LCDR1, LCDR2, and LCDR3, wherein the antigen-binding molecule specifically binds to human PD-L1. The amino acid sequences of exemplary HCDRs and LCDRs corresponding to exemplary anti-human PD-L1 monoclonal antibodies disclosed in the present invention are shown in Tables 1-5.

[0130] The amino acid sequence boundaries of the CDRs can be determined by one of skill in the art using any of a number of known numbering schemes, such as those described in Kabat et al., supra (the "Kabat" numbering scheme); Al-Lazikani et al., 1997, J. Mol. Biol., 273:927-948 (the "Chothia" numbering scheme); MacCallum et al., 1996, J. Mol. Biol. 262:732-745 (the "Contact" numbering scheme); Lefranc et al., Dev. Comp. Immunol., 2003, 27:55-77 (the "IMGT" numbering scheme); and Honegge and Pluckthun, J. Mol. Biol, 2001, 309:657-70 (the "AHo" numbering scheme), each of which is incorporated by reference in its entirety. Tables 1 and 2 show the sequences of the heavy chain CDRs of exemplary antibodies of the invention according to the Kabat numbering scheme and the IMGT numbering scheme, respectively. Table 3 shows the sequences of the heavy chain CDRs of exemplary antibodies of the invention, where the CDR sequences are defined by the CDRs combinations based on the Kabat and IMGT numbering schemes. Tables 4 and 5 show the sequences of the light chain CDRs of exemplary antibodies of the invention according to the Kabat numbering scheme and the IMGT numbering scheme.

[0131] In certain embodiments, the invention includes antigen-binding molecules, such as anti-PD-L1 antibodies, or antigen-binding fragments thereof, that comprise CDRs defined according to the Kabat and IMGT numbering schemes, or a combination thereof. Thus, in a particular embodiment, the present invention provides an HCDR1 having an amino acid sequence that is about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identical to an amino acid sequence selected from the HCDR1 sequences listed in Tables 1 and 6; (2) an HCDR2 having an amino acid sequence that is about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identical to an amino acid sequence selected from the HCDR2 sequences listed in Tables 1 and 9; (3) an HCDR3 having an amino acid sequence that is about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identical to an amino acid sequence selected from the HCDR3 sequences listed in Tables 1 and 11; (4) an HCDR1 having an amino acid sequence that is about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identical to an amino acid sequence selected from the HCDR3 sequences listed in Tables 4 and 5; (5) an LCDR2 having an amino acid sequence that has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or about 100% identity to an amino acid sequence selected from an LCDR1 sequence listed in Tables 4 and 13; (6) an LCDR3 having an amino acid sequence that has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or about 100% identity to an amino acid sequence selected from an LCDR2 sequence listed in Tables 4 and 15; and (7) an LCDR3 having an amino acid sequence that has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or about 100% identity to an amino acid sequence selected from an LCDR3 sequence listed in Tables 4 and 16.

[0132] In a particular embodiment, the present invention provides an HCDR1 having an amino acid sequence that is about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identical to an amino acid sequence selected from the HCDR1 sequences listed in Tables 2 and 7; (2) an HCDR2 having an amino acid sequence that is about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identical to an amino acid sequence selected from the HCDR2 sequences listed in Tables 2 and 10; (3) an HCDR3 having an amino acid sequence that is about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identical to an amino acid sequence selected from the HCDR3 sequences listed in Tables 2 and 12; (4) an HCDR1 having an amino acid sequence that is about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identical to an amino acid sequence selected from the HCDR3 sequences listed in Tables 5 and 13; (5) an LCDR2 having an amino acid sequence that has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or about 100% identity to an amino acid sequence selected from an LCDR2 sequence listed in Table 5; and (6) an LCDR3 having an amino acid sequence that has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or about 100% identity to an amino acid sequence selected from an LCDR3 sequence listed in Tables 5 and 16.

[0133] In a particular embodiment, the present invention provides an HCDR1 having an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identity with an amino acid sequence selected from the HCDR1 sequences listed in Tables 3 and 8; (2) an HCDR2 having an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identity with an amino acid sequence selected from the HCDR2 sequences listed in Tables 3 and 9; (3) an HCDR3 having an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identity with an amino acid sequence selected from the HCDR3 sequences listed in Tables 3 and 11; (5) an LCDR2 having an amino acid sequence that has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or about 100% identity to an amino acid sequence selected from an LCDR1 sequence listed in Tables 4 and 15; and (6) an LCDR3 having an amino acid sequence that has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or about 100% identity to an amino acid sequence selected from an LCDR3 sequence listed in Tables 4 and 16.

[0134] As used herein, a "position" in a CDR refers to an amino acid counted from the N-terminus of the CDR. For example, position numbered 1 or the first position in HCDR1 refers to the first amino acid in HCDR1. Thus, in clone 27A2, position numbered 1 in HCDR1 based on the Kabat numbering scheme is a serine (S).

[0135] [Table 1]

[0136] [Table 2]

[0137] [Table 3]

[0138] [Table 4]

[0139] [Table 5]

[0140] Tables 6-16 show consensus sequences for some of the CDRs in some exemplary antibodies of the disclosure.

[0141] [Table 6]

[0142] As used herein, X1 is N or S, X2 is C or S, X3 is A, N or S, X4 is A, N or S, X5 is C or S, X6 is C or S, X7 is L or M, X8 is G or D, and X9 is W or Y.

[0143] [Table 7]

[0144] As used herein, X10 is A or S, X11 is A, N or S, X12 is H or Y, X13 is G or S, X14 is A, N or S, X15 is F or I, X16 is D or S, X17 is D or G, and X18 is W or Y.

[0145] [Table 8]

[0146] As used herein, X19 is A or S, X20 is A, N or S, X21 is H or Y, X22 is C or S, X23 is G or S, X24 is A, N or S, X25 is C or S, X26 is C or S, X27 is F or I, X28 is D or S, X29 is L or M, X30 is D or G, and X31 is W or Y.

[0147] [Table 9]

[0148] As used herein, X32 is C or S, X33 is G or S, X34 is I, T or V, X35 is A, D, G or Y, X36 is S or T, X37 is N or S, X38 is C or S, X39 is G or T, X40 is F or Y, X41 is S or T, X42 is S or T, and X43 is I, S or T.

[0149] [Table 10]

[0150] As used herein, X44 is G or S, X45 is I, T or V, X46 is A, D or G, X47 is I or V, X48 is A or D, X49 is G or T and X50 is S or T.

[0151] [Table 11]

[0152] As used herein, X51 is K or R, X52 is A or V, X53 is I or S, X54 is D or N, X55 is D or H, X56 is I or T, X57 is H, N or T, X58 is A or G, X59 is W or Y, and X60 is H or Y.

[0153] [Table 12]

[0154] As used herein, X61 is H, N or T; X62 is A or G; X63 is W or Y; and X64 is H or Y.

[0155] [Table 13]

[0156] As used herein, X65 is N, S or T, X66 is I or V, X67 is N or S, X68 is D or N, X69 is A, C, F or S, X70 is N or T, X71 is F or S, X72 is I or V, X73 is N or S, X74 is A, C or S, X75 is G or Q, X76 is E or Q, X77 is G, N or S, X78 is D, N or T, X79 is A or R, X80 is E or Q, X81 is G or S, and X82 is N or T.

[0157] [Table 14]

[0158] As used herein, X83 is N, S or T, X84 is I or V, X85 is N or S, X86 is D or N, X87 is N or T, X88 is I or V, X89 is N or S, X90 is G or Q, X91 is E or Q, X92 is G, N or S, X93 is D, N or T, X94 is A or R, X95 is E or Q, X96 is G or S and X97 is N or T.

[0159] [Table 15]

[0160] As used herein, X98 is D, G, Q, S or Y, X99 is A or T, X100 is A or S, X101 is D, G, Q or Y, X102 is A or T, X103 is A or T, X104 is R or S, X105 is A or S, X106 is A or G, and X107 is A or T.

[0161] [Table 16]

[0162] As used herein, X108 is S or T, X119 is S or T, X110 is I or T, and X111 is D or E.

[0163] In some embodiments, the antibody or antigen-binding fragment thereof comprises (1) a heavy chain variable region (HCVR) having an amino acid sequence that is about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identical to an amino acid sequence selected from the HCVR sequences listed in Tables 17 and 18, and (2) a light chain variable region (LCVR) having an amino acid sequence that is about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identical to an amino acid sequence selected from the LCVR sequences listed in Tables 19 and 20, wherein the antibody or antigen-binding fragment thereof specifically binds to human PD-L1. Exemplary HCVR and LCVR amino acid sequences corresponding to exemplary anti-human PD-L1 monoclonal antibodies disclosed herein are set forth in Tables 17-20.

[0164] [Table 17-1] [Table 17-2]

[0165] [Table 18-1] [Table 18-2]

[0166] [Table 19-1] [Table 19-2]

[0167] [Table 20]

[0168] In certain embodiments, the antigen-binding molecule of the present invention, e.g., an antibody or an antigen-binding fragment thereof, is post-translationally modified. Examples of post-translational modifications include cleavage of lysine at the C-terminus of the heavy chain by carboxypeptidase, modification of glutamine or glutamic acid to pyroglutamic acid at the N-terminus of the heavy and light chains by pyroglutamylation, glycosylation, oxidation, deamidation, and glycation. Such post-translational modifications are known to occur in various antibodies (see journal of Pharmaceutical Sciences, 2008, Vol. 97, p. 2426-2447, which is incorporated herein by reference in its entirety). Examples of post-translationally modified antigen-binding molecules, e.g., antibodies or antigen-binding fragments thereof, include antigen-binding molecules that have undergone pyroglutamylation at the N-terminus of the heavy chain variable region and / or deletion of lysine at the C-terminus of the heavy chain, e.g., antibodies or antigen-binding fragments thereof. The sequences of exemplary antigen-binding molecules with pyroglutamylated N-terminus are shown in Table 7. As used herein, "pE," when used to describe an amino acid in a polypeptide, refers to pyroglutamic acid.

[0169] 2. Variants of antigen-binding molecules In certain embodiments, the PD-L1 antigen binding molecule described herein, such as the anti-PD-L1 antibody, is a monoclonal antibody, a chimeric antibody, a humanized antibody, a Fab, (Fab) 2、 It may also be a multispecific antibody comprising an scFv or other binding specificities as described herein.

[0170] In certain embodiments, the anti-PD-L1 antibodies described in the invention may be linked to an Fc that contains one or more modifications to alter one or more functional properties of the antibody, such as serum half-life, complement binding, Fc receptor binding, and / or antigen-dependent cellular cytotoxicity. Additionally, the antibodies described in the invention may be chemically modified (e.g., one or more chemical moieties are linked to the antibody) or modified to alter its glycosylation to alter one or more functional properties of the antibody. More specifically, in certain embodiments, the antibodies described in the invention may contain modifications in the Fc region to generate Fc variants that have (a) increased or decreased antibody-dependent cell-mediated cytotoxicity (ADCC), (b) increased or decreased complement-mediated cytotoxicity (CDC), (c) increased or decreased affinity for C1q, and / or (d) greater or lesser affinity for Fc receptors than the parent Fc. Such Fc region variants generally contain at least one amino acid modification in the Fc region. Combining amino acid modifications may be particularly desirable. For example, the Fc region variant can include substitutions of two, three, four, five, etc. at specific Fc region positions, eg, as described herein.

[0171] For applications where effector functions should be avoided entirely, e.g. where antigen binding alone is sufficient to produce the desired therapeutic effect and effector functions only result in undesirable side effects (or increase the risk of same), one may use IgG4 antibodies or IgG1 L234F, L235E and P331S variants which have no ADCC effect, or one may design antibodies or fragments which lack the Fc region or a large part of it, or one may mutate the Fc to completely eliminate glycosylation (e.g. N297A). Alternatively, one may use human IgG2 (C1H1) antibodies which have no effector functions and lack the ability to bind FcγRs (like IgG2) and activate complement (like IgG4). H 1 domain and hinge region) and human IgG4 (C H 2 and C HHybrid constructs of the IgG4 constant domain (3 domains) may be made. When an IgG4 constant domain is used, it is usually preferred to include the substitution S228P, which mimics the hinge sequence of IgG1, and the R409K mutation, which stabilizes the IgG4 molecule by blocking Fab arm exchange, thereby reducing Fab arm exchange between the therapeutic antibody and endogenous IgG4 in the patient being treated.

[0172] In certain embodiments, the anti-PD-L1 antibodies or fragments thereof can be modified to increase their biological half-life. Various methods can be used, such as, for example, increasing the binding affinity of the Fc region to FcRn. In one embodiment, the antibody is H 1 or CL region of the IgG Fc region. H It contains a salvage receptor binding epitope from two loops of two domains. The numbering of residues in the Fc region is that of the EU index of Kabat. The sequence variants disclosed herein are provided by reference to the residue number, followed by the amino acid substituted for the natural amino acid, optionally preceded by the natural residue at that position. When multiple amino acids can be present at a given position, for example when the sequence differs between natural isotypes, or when multiple mutations are substituted at that position, they are separated by slashes (e.g., "X / Y / Z").

[0173] Exemplary Fc variants that increase binding to FcRn and / or improve pharmacokinetic properties include substitutions at positions 259, 308, and 434, such as, for example, 259I, 308F, 428L, 428M, 434S, 434H, 434F, 434Y, and 434M. Other variants that increase binding of Fc to FcRn include 250E, 250Q, 428L, 428F, 250Q / 428L (Hinton et al., 2004, J. Biol. Chem. 279(8):6213-6216; Hinton et al. 2006 Journal of Immunology 176:346-356), 256A, 272A, 305A, 307A, 31IA, 312A, 378Q, 380A, 382A, 434A (Shields et al. al. (2001) J. Biol. Chem., 276(9):6591-6604), 252F, 252Y, 252W, 254T, 256Q, 256E, 256D, 433R, 434F, 434Y, 252Y / 254T / 256E, 433K / 434F / 436H (Dall'Acqua et al. (2002) J. Immunol., 169:5171-5180, Dall'Acqua et al. (2006) J. Biol. Chem., 281:23514-23524, and U.S. Patent No. 8,367,805).

[0174] Modifications of certain conserved residues (I253, H310, Q311, H433, N434) in IgG Fc, such as the N434A variant (Yeung et al. (2009) J. Immunol. 182:7663), have been proposed to increase affinity for FcRn and extend antibody half-life in circulation (WO98 / 023289). An Fc variant containing the combination of M428L and N434S has been shown to improve binding to FcRn and extend serum half-life by up to five-fold (Zalevsky et al. (2010) Nat. Biotechnol. 28:157). Fc variants containing the modified combination of T307A, E380A and N434A also extended the half-life of IgG1 antibodies (Petkova et al. (2006) Int. Immunol. 18:1759). Furthermore, Fc variants containing the combination of M252Y-M428L, M428L-N434H, M428L-N434F, M428L-N434Y, M428L-N434A, M428L-N434M and M428L-N434S have also been shown to extend the half-life (US 2006 / 173170). Furthermore, it has been reported that Fc variants containing the combination of M252Y, S254T and T256E increase the half-life by almost 4-fold. (Dall'Acqua et al. (2006) J. Biol. Chem. 281:23514).

[0175] In certain embodiments, the PD-L1 antigen-binding molecule described in the present invention is a bispecific antibody comprising a first targeting domain that specifically binds to PD-L1 and a second targeting domain that specifically binds to another epitope on PD-L1 or another protein. In some embodiments, the first targeting domain comprises an antigen-binding fragment from any of the PD-L1 antibodies described in the present invention. In certain embodiments, the first targeting domain of the bispecific antibody specifically binds to PD-L1 and the second targeting domain specifically binds to a protein expressed on the surface of an immune cell, such as a T cell, a NK cell, a NK T cell, a macrophage, a dendritic cell, or a myeloid-derived suppressor cell (MDSC). Without wishing to be bound by any theory, it is hypothesized that the bispecific antibody may bind to PD-L1 on the surface of tumor cells and to a protein expressed on the surface of immune cells. Thus, the bispecific antibody promotes the killing of tumor cells by immune cells.

[0176] In certain embodiments, the antigen-binding molecule described in the present invention, such as an anti-PD-L1 antibody or antigen-binding fragment thereof, is chemically conjugated to one or more therapeutically active peptides and / or small molecule drugs to form an antibody drug conjugate (ADC). The peptide or small molecule drug is, for example, bound to a reduced SH group and / or a carbohydrate side chain. Methods for making covalent or non-covalent conjugates of peptides or small molecule drugs with antibodies are known in the art, and any such known methods may be utilized. Without wishing to be bound by any theory, it is hypothesized that the ADC of the present invention may bind to PD-L1 expressed on the surface of tumor cells. Thus, by internalization of the antibody-antigen complex, the drug can be introduced into the tumor cells, allowing the tumor cells to be killed by the PD-L1-bound drug of the present invention.

[0177] In some embodiments, the peptide or small molecule drug is linked to the hinge region of the reduced antibody component via a disulfide bond. Alternatively, such drugs are linked using a heterobifunctional crosslinker (N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP)). General techniques for such linkages are well known in the art. In some aspects, the peptide or small molecule drug is linked via a carbohydrate moiety in the Fc region of the antibody. The carbohydrate group can be used to increase the loading of the same drug linked to a thiol group, or the carbohydrate moiety can be used to link a different therapeutic or diagnostic agent. Methods for linking peptide inhibitors or small molecule drugs to antibodies via the carbohydrate moiety of the antibody are well known to those skilled in the art. For example, in one embodiment, the method involves reacting an antibody component having an oxidized carbohydrate moiety with a carrier polymer having at least one free amine functional group. This reaction results in the linkage of an initial Schiff base (imine), which is reduced to a secondary amine to stably form the final conjugate. Exemplary methods for conjugating small molecule drugs and peptides to antibodies are described in US Patent Application Publication No. 2014 / 0356385.

[0178] 3. Biological properties of antibodies and antigen-binding molecules The present invention includes antibodies and antigen-binding fragments thereof that bind to human and cynomolgus PD-L1. The present invention includes PD-L1 antigen-binding molecules, such as PD-L1 antibodies or antigen-binding fragments thereof, that can specifically bind to cell-surface-expressed human and cynomolgus monkey PD-L1 and inhibit the activity or function of PD-L1. In certain embodiments, the antigen-binding molecules block the interaction between cell-surface-expressed human PD-L1 and a PD-L1 agonist, such as PD-1. The extent to which a PD-L1 antigen-binding protein, such as a PD-L1 antibody or antigen-binding fragment thereof, blocks the interaction between PD-1 and cell-surface-expressed PD-L1, can be assessed by the assay described in Example 5 or a substantially similar assay. The present invention includes antigen-binding molecules, such as antibodies or antigen-binding fragments thereof, that block the interaction between cell-surface-expressed human PD-L1 and PD-1, and can be assessed by the IC measured using the assay described in Example 5 or a substantially similar assay. 50 The value is about 1 nM, about 0.75 nM, about 0.5 nM or less, or about 0.2 to 1.0 nM.

[0179] The present invention includes PD-L1 antigen-binding molecules, such as PD-L1 antibodies or antigen-binding fragments thereof, that can bind to PD-L1 in competition with PD-L1 to block the interaction between PD-L1 and PD-1 protein. In certain embodiments, the antigen-binding molecules described in the present invention, such as PD-L1 antibodies or antigen-binding fragments thereof, compete with PD-1 to bind to PD-L1 to block the interaction between PD-L1 and PD-1, as measured using a competitive ELISA assay described in Example 5. In certain embodiments, the present invention includes antigen-binding molecules that have an IC of about 10 nM, about 7.5 nM, about 5.0 nM, about 4.0 nM, about 3.0 nM, about 2.5 nM, about 2.0 nM, about 1.5 nM, about 1.0 nM or less, or about 1.0-10 nM, as measured using a competitive ELISA assay described in Example 5, or a substantially similar assay. 50 The values ​​include antigen-binding molecules, such as antibodies or antigen-binding fragments thereof, that compete with PD-L1 to bind to PD-L1 and block the interaction between PD-1 and PD-L1.

[0180] The present invention includes PD-L1 antigen binding molecules, such as PD-L1 antibodies or antigen-binding fragments thereof, that specifically bind to human PD-L1 or non-human primate PD-L1. In certain embodiments, the binding of the antigen binding molecules of the invention to PD-L1 family members other than PD-L1, or to PD-L1 from certain non-human mammals (e.g. mouse PD-L1), is undetectable or weak, as measured using the assay described in Example 3 or a substantially similar assay. In certain embodiments, the present invention provides a compound having an EC50 of about 0.2 nM, 0.1 nM, 0.09 nM, 0.08 nM, 0.07 nM, 0.06 nM, 0.05 nM, 0.04 nM, 0.03 nM, about 0.02 nM, about 0.01 nM, about 0.009 nM, about 0.008 nM, about 0.007 nM or less, or between about 0.007 nM and 0.2 nM, as measured using the assay described in Example 3 or a substantially similar assay. 50 Values ​​include antigen-binding molecules, such as antibodies or antigen-binding fragments thereof, that bind to human PD-L1 or cynomolgus PD-L1, and that do not bind to PD-L1 family members other than PD-L1 or to PD-L1 from certain non-human mammals (e.g., mouse PD-L1).

[0181] The present invention includes PD-L1 antigen binding molecules, e.g., PD-L1 antibodies, that bind with high affinity to human or cynomolgus PD-L1. In certain embodiments, the present invention includes PD-L1 antigen binding molecules, e.g., PD-L1 antibodies, that bind with high affinity to human or cynomolgus PD-L1. In certain embodiments, the present invention includes PD-L1 antibodies that have a K of about 10 nM, about 5 nM, about 2 nM, about 1 nM, about 0.9 nM, about 0.8 nM, about 0.7 nM, about 0.6 nM, about 0.5 nM, about 0.4 nM, about 0.3 nM, about 0.2 nM, about 0.1 nM, about 0.09 nM, about 0.08 nM, about 0.05 nM, about 0.04 nM, about 0.03 nM, about 0.02 nM, about 0.01 nM or less, or between about 0.01 nM and 10 nM, as measured using the assay described in Example 3, or a substantially similar assay. D Values ​​include antigen-binding molecules, such as antibodies or antigen-binding fragments thereof, that bind to human or cynomolgus PD-L1.

[0182] The present invention includes anti-PD-L1 antigen binding molecules, such as PD-L1 antibodies or antigen-binding fragments thereof, that specifically bind to PD-L1 expressing cells, such as cells that express endogenous human and / or non-human primate PD-L1 (e.g. cynomolgus PD-L1) expressed on the surface of immune cells of a human or non-human primate, as measured using the assay described in Example 4, or a substantially similar assay. In certain embodiments, the present invention provides a compound having an EC50 of about 20 nM, about 18 nM, about 15 nM, about 10 nM, about 10 nM, about 5 nM, about 4 nM, about 3 nM, about 2 nM, about 1 nM, about 0.9 nM, about 0.8 nM, about 0.7 nM, about 0.6 nM, about 0.5 nM, about 0.4 nM, about 0.3 nM, about 0.2 nM, about 0.1 nM or less, or between about 0.1 nM and 20 nM, as measured using the assay described in Example 4 or a substantially similar assay. 50 Values ​​include anti-PD-L1 antigen binding molecules, such as anti-PD-L1 antibodies or antigen-binding fragments thereof, that bind to human or non-human primate PD-L1 cells.

[0183] The present invention includes PD-L1 antigen-binding molecules, such as PD-L1 antibodies or antigen-binding fragments thereof, that induce antibody-dependent cellular cytotoxicity (ADCC) as measured using the assay described in Example 6, or a substantially similar assay. In certain embodiments, the present invention includes antibodies with an EC of about 10 nM, about 5 nM, about 4 nM, about 3 nM, about 2 nM, about 1 nM, about 0.9 nM, about 0.8 nM, about 0.7 nM, about 0.6 nM, about 0.5 nM, about 0.4 nM, about 0.3 nM, about 0.2 nM, about 0.1 nM or less, or about 0.1 nM to about 10 nM, about 0.1 nM to 2.5 nM, or about 1.0 nM to 5.0 nM, as measured using the assay described in Example 6, or a substantially similar assay. 50 and the like. Examples of such antibodies include anti-PD-L1 antigen-binding molecules that induce ADCC, such as anti-PD-L1 antibodies or antigen-binding fragments thereof.

[0184] The present invention includes PD-L1 antigen binding molecules, such as PD-L1 antibodies or antigen-binding fragments thereof, that induce internalization upon binding to cell surface-expressed PD-L1 (e.g., human or cynomolgus PD-L1) as measured using the assay described in Example 7, or a substantially similar assay. In certain embodiments, the present invention includes PD-L1 antibodies or antigen-binding fragments thereof that induce internalization upon binding to cell surface-expressed PD-L1 (e.g., human or cynomolgus PD-L1) as measured using the assay described in Example 7, or a substantially similar assay. In certain embodiments, the present invention includes PD-L1 antibodies or antigen-binding fragments thereof that induce internalization upon binding to cell surface-expressed PD-L1 (e.g., human or cynomolgus PD-L1) as measured using the assay described in Example 7, or a substantially similar assay. , about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40% or more, or about 4% to about 40% of the anti-PD-L1 antigen-binding molecule, such as an anti-PD-L1 antibody or antigen-binding fragment thereof, that induces internalization of a cell surface complex upon binding of the PD-L1 antigen-binding molecule (e.g., an anti-PD-L1 antibody or antigen-binding fragment thereof) to cell surface PD-L1 (e.g., human or cynomolgus PD-L1).

[0185] The present invention includes PD-L1 antigen binding molecules, such as PD-L1 antibodies or antigen-binding fragments thereof, that induce a cytotoxic response of PD-L1-expressing cells when the PD-L1 antigen binding molecule (e.g. PD-L1 antibodies or antigen-binding fragments thereof) is conjugated to a cytotoxic agent, as measured using the assay described in Example 8, or a substantially similar assay.

[0186] The present invention includes PD-L1 antigen binding molecules, such as PD-L1 antibodies or antigen-binding fragments thereof, that belong to the same epitopic region as avelumab or a different epitopic region, as measured using the assay described in Example 9, or a substantially similar assay.

[0187] 4. Species selectivity and species cross-reactivity According to certain embodiments, the present invention provides antigen binding molecules that bind to human PD-L1 but do not bind to PD-L1 of other species.The present invention also includes antigen binding molecules that bind to human PD-L1 and PD-L1 of one or more non-human species (e.g., non-human primates).

[0188] According to certain exemplary embodiments of the invention, there is provided an antigen binding molecule that binds to human PD-L1 and may or may not further bind to one or more of mouse, rat, guinea pig, hamster, gerbil, pig, cat, dog, rabbit, goat, sheep, cow, horse, camel, cynomolgus monkey, marmoset, rhesus monkey, or chimpanzee PD-L1. For example, in certain exemplary embodiments of the invention, there is provided an antigen binding molecule that comprises an antigen binding domain that binds to human PD-L1 and non-human primate (e.g., cynomolgus monkey) PD-L1.

[0189] 3. Therapeutic Uses of Anti-PD-L1 Antigen Binding Molecules The anti-PD-L1 antigen-binding molecules (including antibodies, antigen-binding fragments thereof, and multispecific antibodies thereof) described in the present invention have numerous in vitro, in vivo, and ex vivo applications related to enhancing immune responses by blocking adenosine and other signaling in the treatment of cancer. Without wishing to be bound by any theory, it is hypothesized that the antigen-binding molecules (e.g., anti-PD-L1 antibodies or antigen-binding fragments thereof) described in the present invention bind to PD-L1 expressed on the cell surface and block the interaction with PD-1, and such binding results in various effects, such as enhancing immune activity by blocking the interaction between PD-1 and PD-L1. Thus, the antigen-binding molecules (and therapeutic compositions comprising same) described in the present invention are useful, inter alia, for treating any disease or disorder that would benefit from blocking the PD-1 / PD-L1 interaction (e.g., stimulating and / or activating an immune response). Given the expression of PD-L1 and the diverse effects mediated by the interaction of PD-1 with PD-L1, the anti-PD-L1 antigen-binding molecules (e.g. antibodies or antigen-binding fragments thereof) described in the invention can be used alone, or in combination with a variety of active agents, to treat a wide range of diseases or disorders, including various cancers.

[0190] Thus, the invention provides a method of blocking the interaction between PD-1 and PD-L1, comprising contacting a cell with an antigen-binding molecule (e.g., an anti-PD-L1 antibody or antigen-binding fragment thereof) according to the invention. Blockade of the interaction between PD-1 and PD-L1 can be measured by a method described in Example 5 or a substantially similar method. In certain embodiments, the methods of the invention result in blocking of the interaction between PD-1 and PD-L1 by at least about 10%, about 20%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95% or more compared to baseline levels.

[0191] In some embodiments, antigen binding molecules of the invention (e.g., anti-PD-L1 antibodies or antigen-binding fragments thereof) are administered to cultured cells (in vitro) or to human subjects (in vivo or ex vivo) to enhance immunity to various diseases. Thus, in one embodiment, a method for stimulating an immune response in a subject in need thereof comprises administering to the subject an anti-PD-L1 antibody, antigen-binding fragment thereof (e.g., anti-PD-L1 HCVR and LCVR), or multispecific anti-PD-L1 antibody described herein to enhance, stimulate, upregulate an immune response in the subject, e.g., to inhibit tumor growth, stimulate anti-tumor T cell immunity, and / or stimulate anti-bacterial immunity.

[0192] In one aspect, a method for enhancing an immune response (e.g., a T cell response) in a subject comprises administering to the subject an anti-PD-L1 antibody described herein, thereby enhancing an immune response (e.g., a T cell response) in the subject. In some embodiments, the subject is a subject suffering from a tumor, and an immune response against the tumor is enhanced. The tumor may be a solid tumor or a liquid tumor, e.g., a hematological malignancy. In certain embodiments, the tumor is an immunogenic tumor. In other embodiments, the tumor is a non-immunogenic tumor. In other embodiments, the subject is a subject bearing a pathogen, and an immune response against the pathogen is enhanced by administration of an anti-PD-L1 antibody described herein. The immune response includes, but is not limited to, a) reversing T cell inactivation and function, b) promoting T cell proliferation, c) enhancing NK cell function, or d) enhancing a T cell-mediated anti-tumor immune response.

[0193] In certain embodiments, the methods described herein increase the immune response by at least about 10%, about 20%, about 50%, about 60%, about 70%, about 80%, about 90%, about 1-fold, about 2-fold, about 4-fold, or more compared to baseline levels.

[0194] Preferred subjects include human patients in whom an enhanced immune response is desired. The method is particularly suitable for treating human patients suffering from disorders that can be treated by enhancing the immune response (e.g., T cell-mediated immune response). The method is particularly suitable for treating cancer, chronic infectious diseases, and chronic inflammatory diseases. The antibody used in the disclosed method described in the present invention is preferably a human antibody or a humanized antibody.

[0195] In one embodiment, a method for inhibiting tumor cell growth in a subject comprises administering to the subject an anti-PD-L1 antibody as described herein, thereby inhibiting tumor growth in the subject. Inhibition of tumor growth can be measured by a variety of methods. Tumor growth can be measured, for example, using the methods described in Talkington, A and Durrett, R, Estimating Tumor Growth Rates in vivo, Bull Math Biol., 2015 Oct.:77(10):1934-54, available at https: / / www.ncbi.nlm.nih.gov / pmc / articles / PMC4764475 / , the entire contents of which are incorporated herein by reference. Inhibition of tumor growth can also be measured by a reduction in tumor size. In certain embodiments, the methods of the invention result in tumor growth being inhibited by at least about 10%, about 20%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or more, compared to baseline levels.

[0196] In a specific embodiment, the antigen binding molecules of the invention (e.g., anti-PD-L1 antibodies or antigen-binding fragments thereof) are used to reduce immunosuppression in the tumor microenvironment. Such reduction can be measured by a variety of methods. The level of immunosuppression in the tumor microenvironment can be measured by the presence and / or abundance of certain biomarkers in the tumor (e.g., PD-L1, CD39, CD73, LAG-3, IL-10, or TGF-β). The level of immunosuppression ... Tregs (T reg)It can also be measured by the ratio of CD8+ cytotoxic T cells to T cells. reg In certain embodiments, the antigen binding molecules described in the invention (e.g., anti-PD-L1 antibodies or antigen-binding fragments thereof) reduce the level of immune suppression in the tumor microenvironment by at least about 10%, about 20%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or more compared to baseline levels.

[0197] T from the tumor microenvironment of a subject having a tumor (e.g., a cancerous tumor). reg Also encompassed herein are methods for depleting T cells in the tumor microenvironment. reg This involves administering to a subject a therapeutically effective amount of an anti-PD-L1 antibody described herein that comprises an Fc that stimulates cell depletion, for example an Fc with appropriate effector function or an Fc with enhanced effector function via one or more activating Fc receptors.

[0198] In certain preferred embodiments, the subject suffers from a cell proliferative disease or cancer. The antigen-binding molecules described herein (e.g., anti-PD-L1 antibodies or antigen-binding fragments thereof) can enhance the patient's immune response against cancerous cells by blocking PD-1 / PD-L1 interaction. Thus, the present invention provides a method of treating a subject with cancer, comprising administering to the subject an anti-PD-L1 antigen-binding molecule described herein (e.g., an antibody or antigen-binding fragment thereof) to treat the subject (e.g., inhibit or reduce cancerous tumor growth and / or cause tumor regression). The anti-PD-L1 antibodies can be used alone to inhibit cancerous tumor growth. Alternatively, the anti-PD-L1 antibodies can be used in conjunction with one or more other targeted active agents, such as other anti-cancer targeted drugs, immunogenic agents, general cancer therapeutic agents, or other antibodies. The antigen-binding molecules described herein can be used, for example, to treat primary and / or metastatic tumors. The present invention also includes a method for treating residual cancer in a subject. As used herein, the term "residual cancer" refers to the presence or persistence of one or more cancer cells in a subject following treatment with an anti-cancer therapy.

[0199] Thus, in one aspect, a method for treating cancer comprises administering to a subject in need thereof a therapeutically effective amount of an anti-PD-L1 antibody according to the invention. Preferably, the antibody inhibits the activity of human anti-PD-L1 and comprises one or more HCVRs and LCVRs according to the invention. Furthermore, the anti-PD-L1 antigen binding molecule (e.g., antibody) used in the method can comprise a non-human anti-PD-L1 antibody that has been chimeric or humanized therefrom. Efficacy for treating cancer can be measured by various methods. For example, efficacy for treating cancer can be measured by improved survival rate or reduction in tumor size. In certain embodiments, the method according to the invention increases efficacy for treating cancer by at least about 10%, about 20%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 1-fold, about 2-fold, about 4-fold, or more compared to baseline levels. In the context of cancer treatment, when the PD-L1 antigen binding molecule according to the invention is the only therapeutic agent, baseline levels refer to efficacy with a placebo. When the PD-L1 antigen binding molecules of the invention are used in combination with other therapeutic agents, the baseline level refers to efficacy with a placebo or the other therapeutic agent.

[0200] Cancers whose proliferation can be inhibited using the antibodies of the present invention include a wide variety of cancers, particularly those that are unresponsive or prone to become unresponsive to monotherapy with other antibodies or chemotherapeutic drugs. Non-limiting examples of cancers for treatment include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, squamous non-small cell lung cancer (NSCLC), non-NSCLC, glioma, gastrointestinal cancer, renal cancer (e.g., clear cell carcinoma), ovarian cancer, liver cancer, colorectal cancer, endometrial cancer, renal cancer (e.g., renal cell carcinoma (RCC)), prostate cancer (e.g., hormone-refractory prostate cancer), thyroid cancer, neuroblastoma, pancreatic cancer, glioblastoma (e.g., glioblastoma multiforme), cervical cancer, gastric cancer, bladder cancer, liver cancer, breast cancer, colon cancer and head and neck cancer, gastric cancer, germ cell tumors. , pediatric sarcoma, sinonasal natural killer cell, melanoma (e.g. metastatic melanoma such as cutaneous or intraocular melanoma), bone cancer, skin cancer, uterine cancer, anal cancer, testicular cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, esophageal cancer, small intestine cancer, endocrine system cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, pediatric solid tumors, ureteral cancer, renal pelvis cancer, central nervous system tumors (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma sarcoma), epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, cancers induced by environmental factors (including asbestos-induced cancer, virus-associated cancers, e.g., human papillomavirus (HPV)-associated tumors), and hematological malignancies derived from either of the two major blood cell lineages: myeloid cells (which produce granulocytes, erythrocytes, platelets, macrophages, and mast cells) or lymphoid cells (which produce B, T, NK, and plasma cells), e.g., all types of leukemia, lymphoma, myeloma (e.g., acute leukemia, lymphoma, myeloma, etc.). acute, chronic, lymphocytic and / or myeloid leukemias, such as ALL, acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CIVIL), AML with anaplastic progression (MO), myeloblastic leukemia (M1), myeloblastic leukemia (M2; cell maturation), promyelocytic leukemia (M3 or M3 subtype [M3V]), myelomonocytic leukemia (M4 or M4 subtype with eosinophilia [M4E]), monocytic leukemia (M5), erythroleukemia (M6), megakaryoblastic leukemia (M7), isolated granulocytic sarcoma, chloroma;Hodgkin's lymphoma (HL), non-Hodgkin's lymphoma (NEIL), B-cell lymphoma, T-cell lymphoma, lymphoplasmacytoid lymphoma, monocytoid B-cell lymphoma, mucosa-associated lymphoid tissue (MALT) lymphoma, anaplastic (e.g., Ki 1+)Large cell lymphoma, adult T-cell lymphoma / leukemia, mantle cell lymphoma, angioimmunoblastic T-cell lymphoma, angiocentric lymphoma, intestinal T-cell lymphoma, primary mediastinal B-cell lymphoma, precursor T-lymphoblastic lymphoma, T-lymphoblastic lymphoma, and lymphoma / leukemia (T-Lbly / T-ALL), peripheral T-cell lymphoma, lymphoblastic lymphoma, post-transplant lymphoproliferative disorder, true histiocytic lymphoma, primary central nervous system lymphoma, primary effusion lymphoma, lymphoblastic lymphoma (LBL), lymphoid hematopoietic neoplasms, acute lymphoblastic leukemia, diffuse large B-cell lymphoma, Burkitt's lymphoma lymphoma, such as follicular lymphoma, diffuse histiocytic lymphoma (DHL), immunoblastic large cell lymphoma, precursor B-cell lymphoblastic lymphoma, cutaneous T-cell lymphoma (CTLC) (also called mycosis fungoides or Sézary syndrome), lymphoplasmacytoid lymphoma with Waldenstrom's macroglobulinemia (LPL); myeloma, such as IgG myeloma, light chain myeloma, non-secretory myeloma, smoldering myeloma (also called asymptomatic myeloma), solitary plasmacytoma, and multiple myeloma; chronic lymphocytic leukemia (CLL), hairy cell lymphoma; hematopoietic tumors of the bone marrow system, tumors of mesenchymal origin (fibrosarcoma, rhabdomyosarcoma, etc.); seminoma , teratomas, tumors of the central and peripheral nervous system (astrocytoma, schwannoma, etc.); tumors of mesenchymal origin (fibrosarcoma, rhabdomyosarcoma, osteosarcoma, etc.); other tumors (melanoma, xeroderma pigmentosum, keratoacanthoma, seminoma, follicular thyroid carcinoma, teratocarcinoma, etc.); hematopoietic tumors of the lymphatic system (e.g. T-cell and B-cell tumors including but not limited to T-cell disorders such as T-cell prolymphocytic leukemia (T-PLL) including small cell and cerebral types, large granular lymphocytic leukemia (LGL) of the preferred T-cell type, a / d T-NHL hepatosplenic lymphoma, peripheral / postthymic T-cell lymphoma (pleomorphic and immunoblastic subtypes), angiocentric (nasal) T-cell lymphoma); head and neck cancer, renal cancer, rectal cancer, thyroid cancer;acute myeloid lymphoma, and any combination of the above cancers. The methods described herein can also be used to treat metastatic cancers, refractory cancers (e.g., cancers that are difficult to treat with conventional immunotherapy (e.g., using blocking antibodies against CTLA-4 or PD-1)), and recurrent cancers;

[0201] In some embodiments, treatment of cancer patients with anti-PD-L1 antibodies and / or other active agents described in the invention may result in long-term durable responses, including long-term survival of at least 1, 2, 3, 4, 5, 10 or more years and / or recurrence-free survival of at least 1, 2, 3, 4, 5, or 10 or more years, compared to conventional standard of care. In certain embodiments, treatment of cancer patients with anti-PD-L1 antibodies and / or other active agents described in the invention prevents cancer recurrence or delays cancer recurrence for, for example, 1, 2, 3, 4, 5, or 10 or more years. Anti-PD-L1 therapy may be used as a first-line or second-line therapy.

[0202] In some embodiments, ex vivo activation and expansion of antigen-specific T cells in the presence of anti-PD-L1 antibodies and adoptive transfer of these cells into a receptor is used to prime antigen-specific T cells against cancer or viral infection by increasing the frequency and activity of the adoptively transferred T cells.

[0203] Suitable routes for administering the antigen-binding molecules of the invention described herein, such as anti-PD-L1 antibodies or antigen-binding fragments thereof (e.g., humanized monoclonal antibodies, multispecific antibodies, and immunoconjugates), in vivo, ex vivo, or in vitro, will be well known to those of skill in the art and may be selected as desired. For example, the antibody compositions may be administered by parenteral injection (e.g., intravenous or subcutaneous). The appropriate dosage will depend on the age and weight of the subject, as well as the concentration and / or formulation of the antibody composition, as described below.

[0204] The term "cell proliferative disorder" refers to a disorder characterized by abnormal proliferation of cells. A proliferative disorder does not imply any limitation with respect to the rate of cell proliferation, but simply indicates a loss of normal controls that affect proliferation and cell division. Thus, in some embodiments, cells of a proliferative disorder may have the same cell division rate as normal cells, but do not respond to signals that limit such proliferation. Within the scope of "cell proliferative disorder" is a neoplasm, cancer, or tumor.

[0205] The term "cancer" refers to any one of a variety of malignant tumors characterized by the proliferation of cells capable of invading surrounding tissues and / or metastasizing to new sites of attachment, including solid and lymphatic cancers, including carcinomas, sarcomas, adenocarcinomas, melanomas, leukemias, lymphomas, germ cell tumors, and blastomas. Exemplary cancers that can be treated by the compositions and methods of the present invention include brain cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, head and neck cancer, kidney cancer, lung cancer, non-small cell lung cancer, mesothelioma, ovarian cancer, prostate cancer, gastric cancer, and uterine cancer, leukemia, and medulloblastoma.

[0206] The term "carcinoma" refers to a malignant growth of epithelial cells tending to infiltrate surrounding tissues and give rise to metastases. Exemplary carcinomas include, for example, acinar carcinoma, acinous carcinoma, adenoid cystic carcinoma, carcinoma adenomatosum, carcinoma of adrenal cortex, alveolar carcinoma, alveolar cell carcinoma, basal cell carcinoma, carcinoma basocellulare, basaloid carcinoma, basosquamous cell carcinoma, bronchioalveolar carcinoma, bronchiolar carcinoma, bronchogenic carcinoma, cerebriform carcinoma, and pulmonary carcinoma. cholangiocellular carcinoma, chorionic carcinoma, colloid carcinoma, comedo carcinoma, corpus carcinoma, cribriform carcinoma, carcinoma en cuirasse, carcinoma cutaneum, cylindrical carcinoma, cylindrical cell carcinoma duct carcinoma, carcinoma durum, embryonal carcinoma, encephaloid carcinoma, epiennoid carcinoma, carcinoma epitheliale adenoides, exophytic carcinomacarcinoma, carcinoma ex ulcere, carcinoma fibrosum, gelatiniform carcinoma, gelatinous carcinoma, giant cell carcinoma, carcinoma gigantocellulare, glandular carcinoma, granulosa cell carcinoma, hair-matrix carcinoma, hematoid carcinoma, hepatocellular carcinoma, hurthle cell carcinoma, hyaline carcinoma, hypemephroid carcinoma, infantile embryonal carcinoma, carcinoma in situ, intraepidermal carcinoma, intraepithelial carcinoma, Krompecher's carcinoma, Kulchitzky-cell carcinoma, large-cell carcinoma, lenticular carcinoma, carcinoma lenticulare, lipomatous carcinoma, lymphoepithelial carcinoma, carcinoma medullare, medullary carcinoma, melanotic carcinoma, carcinoma molle, mucinous carcinoma, carcinoma lenticulare muciparum, carcinoma mucocellulare, mucoepidermoid carcinoma, carcinoma mucosum, mucouscarcinoma, carcinoma myxomatodes, naspharyngeal carcinoma, oat cell carcinoma, carcinoma ossificans, osteoid carcinoma, pancreatic ductal adenocarcinoma, papillary carcinoma, periportal carcinoma, preinvasive carcinoma, prickle cell carcinoma, pultaceous carcinoma, renal cell carcinoma of kidney, reserve cell carcinoma, carcinoma sarcomatodes, schneiderian carcinoma, scirrhous carcinoma carcinoma, carcinoma scroti, signet-ring cell carcinoma, carcinoma simplex, small-cell carcinoma, solanoid carcinoma, spheroidal cell carcinoma, spindle cell carcinoma, carcinoma spongiosum, squamous carcinoma, squamous cell carcinoma, string carcinoma, carcinoma telangiectaticum, carcinoma telangiectodes, transitional cell carcinoma, carcinoma tuberosum, tuberous carcinoma, verrucous carcinoma carcinoma) and choriocarcinoma (carcinoma villosum).

[0207] The term "sarcoma" refers to a tumor made up of a substance like embryonic connective tissue and is generally composed of closely packed cells embedded in a fibrillar or homogeneous substance. Exemplary sarcomas include, for example, chondrosarcoma, fibrosarcoma, lymphosarcoma, melanoma, myxosarcoma, osteosarcoma, Abemethy's sarcoma, liposarcoma, and sarcoma of the ovarian and ventral sarcomas. sarcoma, liposarcoma, alveolar soft part sarcoma, ameloblastoma, botryoid sarcoma, chloroma sarcoma, choriocarcinoma, embryonal sarcoma, Wilms' tumor, endometrial sarcoma, stromal sarcoma, Ewing's sarcoma, fascial sarcoma, fibroblastic sarcoma, giant cell tumor, granulocytic sarcoma sarcoma, Hodgkin's sarcoma, idiopathic multiple pigmented hemorrhagic sarcoma, immunoblastic sarcoma of B cells, lymphoma (non-Hodgkin's lymphoma, etc.), immunoblastic sarcoma of T cells, Jensen's sarcoma, Kaposi's sarcoma, Kupffer cell sarcoma, angiosarcoma, leukocytic sarcoma sarcoma, malignant mesenchymomasarcoma, parosteal sarcoma, reticulocytic sarcoma, Rous sarcoma, serous sarcoma, synovial sarcoma, and telangiectatic sarcoma.

[0208] The term "melanoma" refers to tumors arising from the melanocytic system of the skin and other organs. Melanomas include, for example, acryl-lentiginous melanoma, amelanotic melanoma, benign juvenile melanoma, Cloudman's melanoma, S91 melanoma, Harding-Passey melanoma, juvenile melanoma, lentigo maligna melanoma, malignant melanoma, nodular melanoma, subungal melanoma, and superficial spreading melanoma.

[0209] The term "lymphoma" refers to cancers that affect hematopoietic and lymphatic tissues and arise from lymphocytes (blood cells found primarily in lymph nodes, spleen, thymus, and bone marrow). There are two main types of lymphoma: non-Hodgkin's lymphoma and Hodgkin's disease. Hodgkin's disease accounts for approximately 15% of all diagnosed lymphomas and is a Reed-Sternberg malignant B-lymphocyte-associated cancer. Non-Hodgkin's lymphoma (NHL) is classified based on the rate at which the cancer grows and the type of infiltrating cells. NHL can be aggressive (high-grade) or indolent (low-grade). Based on the type of infiltrating cells, there are B-cell NHL and T-cell NHL. Exemplary B cell lymphomas include, but are not limited to, small lymphocytic lymphoma, mantle cell lymphoma, follicular lymphoma, marginal zone lymphoma, extranodal MALT lymphoma, lymphonodemonocytoid B cell lymphoma, splenic lymphoma, diffuse large B cell lymphoma, Burkitt's lymphoma, lymphoblastic lymphoma, immunoblastic large cell lymphoma, or precursor B cell lymphoblastic lymphoma. Exemplary T cell lymphomas include, but are not limited to, cutaneous T cell lymphoma, peripheral T cell lymphoma, anaplastic large cell lymphoma, mycosis fungoides, and precursor T lymphoblastic lymphoma.

[0210] The term "leukemia" refers to a progressive, malignant disease of the hematopoietic system, generally characterized by the abnormal proliferation and development of white blood cells and their precursors in the blood and bone marrow. Exemplary leukemias include, for example, acute nonlymphocytic leukemia, chronic lymphocytic leukemia, acute granulocytic leukemia, chronic granulocytic leukemia, acute promyelocytic leukemia, adult T-cell leukemia, nonleukemic leukemia, leukemic leukemia, basophilic leukemia, blastic leukemia, bovine leukemia, chronic myelocytic leukemia, leukemia cutis, fetal leukemia, eosinophilic leukemia, Gross' leukemia, hairy cell leukemia, hemoblastic leukemia, hemocytoblastic leukemia, hemo ... leukemia, histiocytic leukemia, stem cell leukemia, acute monocytic leukemia, leukopenic leukemia, lymphatic leukemia, lymphoblastic leukemia, lymphocytic leukemia, lymphogenous leukemia, lymphoid leukemia, lymphosarcoma cell leukemia, mast cell leukemia, megakaryocytic leukemia, micromyeloblastic leukemia, monocytic leukemia, myeloblastic leukemia, myelocytic leukemia, myeloid granulocytic leukemia, myelomonocytic leukemia, Naegli leukemia, plasma cell leukemia, plasmacytic leukemia, promyelocytic leukemia, Rieder cell leukemia, Schilling's leukemia, stem cell leukemia, subleukemic leukemia, and anaplastic cell leukemia.

[0211] Other cancers include, for example, multiple myeloma, neuroblastoma, breast cancer, ovarian cancer, lung cancer, rhabdomyosarcoma, primary thrombocythemia, primary macroglobulinemia, small cell lung cancer, primary brain tumors, gastric cancer, colon cancer, malignant insulinoma, malignant carcinoid, precancerous skin lesions, testicular cancer, thyroid cancer, neuroblastoma, esophageal cancer, genitourinary cancer, malignant hypercalcemia, cervical cancer, endometrial cancer, and adrenal cortical carcinoma.

[0212] 4. Combination therapy In another aspect, the present invention provides therapeutic compositions and combination therapies for enhancing antigen-specific T cell responses, reducing immunosuppression, and / or reducing tumor growth in a subject. The present invention encompasses compositions and therapeutic formulations comprising a combination of any of the exemplary antigen binding molecules (e.g., herein) with one or more additional therapeutic agents, as well as methods of treatment comprising administering such compositions to a subject in need thereof. As used herein, the term "additional therapeutic agent" refers to any agent used to treat a disease or disorder, and any method of treatment for a particular disease or disorder. For example, radiation therapy and surgery are considered "additional therapeutic agents" when used in combination with an antigen binding molecule (e.g., an anti-PD-L1 antibody or antigen-binding fragment thereof) of the present invention.

[0213] In certain embodiments, the additional therapeutic agent may be an agent that blocks the interaction between PD-1 and PD-L1 other than the antigen binding molecule of the present invention (e.g., an anti-PD-L1 antibody or antigen-binding fragment thereof). Exemplary inhibitors for inhibition of PD1 / PD-L1 interaction include, but are not limited to, pembrolizumab, nivolumab, atezolizumab, avelumab, durvalumab, BMS-936559, sulfamonomethoxine 1, and sulfamethizole 2.

[0214] In some embodiments, an anti-PD-L1 antigen binding molecule (e.g. an anti-PD-L1 antibody or antigen-binding fragment thereof) described in the invention is co-administered with one or more additional therapeutic agents in an amount effective to stimulate an immune response and / or apoptosis, to further enhance, stimulate or upregulate an immune response and / or apoptosis in a subject. Furthermore, the one or more additional therapeutically active agents are administered before or after treatment with the anti-PD-L1 antibody.

[0215] In certain embodiments, the anti-PD-L1 antibodies described in the invention are used in combination or concomitantly with one or more additional active agents (such as anti-cancer antibodies or polypeptides, chemotherapeutic agents and radiotoxicants). In other embodiments, the anti-PD-L1 antibodies described in the invention are used in combination or concomitantly with standard cancer treatments (such as surgery or radiation).

[0216] Combining anti-PD-L1 antibodies with these active agents or therapeutic approaches can address clinical deficiencies related to drug resistance, changes in the antigenicity of tumor cells that become unresponsive to the antibody, and toxicity (by administering lower doses of one or more agents). PD-L1 blockade is particularly suitable for use in combination with otherwise refractory chemotherapy regimens. In such cases, enhanced efficacy can be achieved, but the dose of chemotherapy administered can be reduced (Mokyr et al. (1998) Cancer Research 58:5301-5304). PD-L1 blockade by radiotherapy or chemotherapy is based on the rationale that cell death is promoted as a result of the cytotoxic effects of radiation and most chemotherapy compounds, which can further result in increased levels of tumor antigens in the antigen presentation pathway. Other combination therapies that may act additively or synergistically with PD-L1 inhibition through cell death are surgery and hormone withdrawal or inhibition. Either regimen generates additional sources of tumor antigens in the host.

[0217] In some embodiments, the anti-PD-L1 antibodies described herein are linked to another active agent in the form of an immunoconjugate, immunoconjugate, or fusion protein. Alternatively, the anti-PD-L1 antibodies can be administered separately from the additional active agent. In this case, the anti-PD-L1 antibodies and additional antagonists can be administered before, after, or simultaneously with the additional active agent, or can be used in combination with other known therapies (e.g., other anti-cancer agents, radiation, etc.). Thus, the present invention provides compositions and methods for providing two or more anti-cancer agents that act additively or synergistically via different mechanisms to beneficially provide cytotoxicity and immunoprotection in human cancer cells.

[0218] For example, in some embodiments, the anti-PD-L1 antibodies described herein may be used in combination with other agents, such as alkylating agents, anthracycline antibiotics, antimetabolites, antidotes, interferons, polyclonal or monoclonal antibodies, EGFR inhibitors, HER2 inhibitors, histone deacetylase inhibitors, hormones, mitotic inhibitors, phosphatidylinositol-3-kinase (PI3K) inhibitors, Akt inhibitors, mammalian target of rapamycin (mTOR) inhibitors, proteasome inhibitors, poly(ADP-ribose) inhibitors, ribozymes ... ) polymerase (PARP) inhibitors, Ras / MAPK pathway inhibitors, centrosome declustering agents, multikinase inhibitors, serine / threonine kinase inhibitors, tyrosine kinase inhibitors, VEGF / VEGFR inhibitors, taxanes or taxane derivatives, aromatase inhibitors, anthracyclines, microtubule targeting agents, topoisomerase poisons, inhibitors of molecular targets or enzymes (e.g., kinases or protein methyltransferases), cytidine analogs, or combinations thereof.

[0219] Exemplary alkylating agents include, but are not limited to, cyclophosphamide (Cytoxan, Neosar), chlorambucil (Leukeran), melphalan (Alkeran), carmustine (BiCNU), busulfan (Busulfex), lomustine (CeeNU), dacarbazine (DTIC-Dome), oxaliplatin (Eloxatin), carmustine (Gliadel), ifosfamide (Ifex), mechlorethamine (Mustargen), busulfan (Myleran), carboplatin (Paraplatin), cisplatin (CDDP, Platinol), temozolomide (Temodar), thiotepa (Thioplex), bendamustine (Treanda), or streptozocin (Zanosar).

[0220] Exemplary anthracycline antibiotics include, but are not limited to, doxorubicin (Adriamycin), liposomal doxorubicin (Doxil), mitoxantrone (Novantron), bleomycin (Blenoxane), daunorubicin (Cerubidine), liposomal daunorubicin (DaunoXome), dactinomycin (Cosmegen), epirubicin (Ellence), idarubicin (Idamycin), plicamycin (Mithracin), mitomycin (Mutamycin), pentostatin (Nipent), or valrubicin (Valstar).

[0221] Exemplary antimetabolites include fluorouracil (Adrucil), capecitabine (Xeloda), hydroxyurea (Hydrea), mercaptopurine (Purinethol), pemetrexed (Alimta), fludarabine (Fludara), nelarabine (Arranon), cladribine (Cladribine Novaplus), clofarabine (Clolar), cytarabine (Cytosar-U), decitabine (Dacogen), cytarabine liposomal (DepoCyt), hydroxyurea (Droxia), pralatrexate (Folotyn), floxuridine (FUDR), gemcitabine (Gemzar), cladribine (Leustatin), fludarabine (Oforta), methotrexate (MTX, Rheumatrex), methotrexate (Trexall), thioguanine (Tabloid), TS-1 or cytarabine (Tarabine PFS).

[0222] Exemplary antidotes include, but are not limited to, amifostine (Ethyol) or mesna (Mesnex).

[0223] Exemplary interferons include, but are not limited to, interferon alpha-2b (Intron A) or interferon alpha-2a (Roferon-A).

[0224] Exemplary polyclonal or monoclonal antibodies include, but are not limited to, trastuzumab (Herceptin), ofatumumab (Arzerra), bevacizumab (Avastin), rituximab (Rituxan), cetuximab (Erbitux), panitumumab (Vectibix), tositumomab / iodine-131 tositumomab (Bexxar), alemtuzumab (Campath), ibritumomab (Zevalin, In-111, Y-90 Zevalin), gemtuzumab (Mylotarg), eculizumab (Soliris), or denosumab.

[0225] Exemplary EGFR inhibitors include, but are not limited to, gefitinib (Iressa), lapatinib (Tykerb), cetuximab (Erbitux), erlotinib (Tarceva), panitumumab (Vectibix), PKI-166, canertinib (CI-1033), matuzumab (Emd7200) or EKB-569.

[0226] Exemplary HER2 inhibitors include, but are not limited to, trastuzumab (Herceptin), lapatinib (Tykerb) or AC-480.

[0227] Exemplary histone deacetylase inhibitors include, but are not limited to, vorinostat (Zolinza), valproic acid, romidepsin, entinostat, abexinostat, gibinostat, and mocetinostat.

[0228] Exemplary hormones include tamoxifen (Soltamox, Nolvadex), raloxifene (Evista), megestrol (Megace), leuprolide (Lupron, Lupron Depot, Eligard, Viadur), fulvestrant (Faslodex), letrozole (Femara), triptorelin (Trelstar LA, Trelstar These include, but are not limited to, fluoxymesterone (Androxy, Halotestin), medroxyprogesterone (Provera, Depo-Provera), estramustine (Emcyt), flutamide (Eulexin), toremifene (Fareston), degarelix (Firmagon), nilutamide (Nilandron), abarelix (Plenaxis), or testolactone (Teslac).

[0229] Exemplary mitotic inhibitors include, but are not limited to, paclitaxel (Taxol, Onxol, Abraxane), docetaxel (Taxotere), vincristine (Oncovin, Vincasar PFS), vinblastine (Velban), etoposide (Toposar, Etopophos, VePesid), teniposide (Vumon), ixabepilone (Ixempra), nocodazole, epothilones, vinorelbine (Navelbine), camptothecin (CPT), irinotecan (Camptosar), topotecan (Hycamtin), amsacrine, or lamellarin D (LAM-D).

[0230] Exemplary phosphatidylinositol-3-kinase (PI3K) inhibitors include wortmannin (an irreversible inhibitor of PI3K), demethoxyviridin (a derivative of wortmannin), LY294002 (a reversible inhibitor of PI3K), BKM120 (Buparlisib), idelalisib (a PI3K-delta inhibitor), duvelisib (IPI-145, a PI3K-delta / gamma inhibitor), alpelisib (BYL719, an alpha-specific PI3K inhibitor), TGR 1202 (formerly known as RP5264), an oral PI3K-delta inhibitor, copanlisib (BAY 80-6946, which is primarily an inhibitor of the PI3K-alpha / delta isoforms).

[0231] Exemplary Akt inhibitors include, but are not limited to, miltefosine, AZD5363, GDC-0068, MK2206, perifosine, RX-0201, PBI-05204, GSK2141795, and SR13668.

[0232] Exemplary MTOR inhibitors include, but are not limited to, everolimus (Afinitor) or temsirolimus (Torisel), rapamune, ridaforolimus, deforolimus (AP23573), AZD8055 (AstraZeneca), OSI-027 (OSI), INK-128, BEZ235, PI-103, Torin 1, PP242, PP30, Ku-0063794, WAY-600, WYE-687, WYE-354, and CC-223.

[0233] Exemplary proteasome inhibitors include, but are not limited to, bortezomib (PS-341), ixazomib (MLN 2238), MLN 9708, delanzomib (CEP-18770), carfilzomib (PR-171), YU101, oprozomib (ONX-0912), marizomib (NPI-0052), and disulfiram.

[0234] Exemplary PARP inhibitors include, but are not limited to, olaparib, iniparib, velaparib, BMN-673, BSI-201, AG014699, ABT-888, GPI21016, MK4827, INO-1001, CEP-9722, PJ-34, Tiq-A, Phen, PF-01367338, and combinations thereof.

[0235] Exemplary Ras / MAPK pathway inhibitors include, but are not limited to, trametinib, selumetinib, cobimetinib, CI-1040, PD0325901, AS703026, R04987655, R05068760, AZD6244, GSK1120212, TAK-733, U0126, MEK162, and GDC-0973.

[0236] Exemplary centrosome de-clustering agents include, but are not limited to, griseofulvin; noscapine, brominated noscapine (e.g., 9-bromonoscapine), reduced bromonoscapine (RBN), N-(3-bromobenzyl)noscapine, aminoscapine, and their water-soluble derivatives; CW069; phenanthridine-derived poly(ADP-ribose) polymerase inhibitors; PJ-34; N2-(3-pyridylmethyl)-5-nitro-2-furamide, N2-(2-thienylmethyl)-5-nitro-2-furamide, and N2-benzyl-5-nitro-2-furamide.

[0237] Exemplary multikinase inhibitors include, but are not limited to, regorafenib, sorafenib (Nexavar), sunitinib (Sutent), BIBW 2992, E7080, Zd6474, PKC-412, motesanib, or AP24534.

[0238] Exemplary serine / threonine kinase inhibitors include, but are not limited to, ruboxistaurin; eril / fasudil hydrochloride; flavopiridol; seliciclib (CYC202, Roscovitrine), SNS-032 (BMS-387032), Pkc412, bryostatin, KAI-9803, SF1126, VX-680, Azd1152, Arry-142886 (AZD-6244), SCIO-469, GW681323, CC-401, CEP-1347, or PD 332991.

[0239] Exemplary tyrosine kinase inhibitors include erlotinib (Tarceva), gefitinib (Iressa), imatinib (Gleevec), sorafenib (Nexavar), sunitinib (Sutent), trastuzumab (Herceptin), bevacizumab (Avastin), rituximab (Rituxan), lapatinib (Tykerb), cetuximab (Erbitux), panitumumab (Vectibix), evetinib (Evetin), erythropoie ... These include, but are not limited to, rolimus (Afinitor), alemtuzumab (Campath), gemtuzumab (Mylotarg), temsirolimus (Torisel), pazopanib (Votrient), dasatinib (Sprycel), nilotinib (Tasigna), vatalanib (Ptk787, ZK222584), CEP-701, SU5614, MLN518, XL999, VX-322, Azd0530, BMS-354825, SKI-606 CP-690, AG-490, WHI-P154, WHI-P131, AC-220, or AMG888.

[0240] Exemplary VEGF / VEGFR inhibitors include, but are not limited to, bevacizumab (Avastin), sorafenib (Nexavar), sunitinib (Sutent), ranibizumab, pegaptanib, or vandetanib.

[0241] Exemplary microtubule targeting agents include, but are not limited to, paclitaxel, docetaxel, vincristine, vinblastine, nocodazole, epothilones, and navelbine.

[0242] Exemplary topoisomerase poisons include, but are not limited to, teniposide, etoposide, adriamycin, camptothecin, daunorubicin, dactinomycin, mitoxantrone, amsacrine, epirubicin, and idarubicin.

[0243] Exemplary taxanes or taxane derivatives include, but are not limited to, paclitaxel and docetaxol.

[0244] Exemplary general chemotherapeutic, antitumor, and antiproliferative agents include altretamine (Hexalen), isotretinoin (Accutane, Amnesteem, Claravis, Sotret), tretinoin (Vesanoid), azacytidine (Vidaza), bortezomib (Velcade), asparaginase (Elspar), levamisole (Ergamisol), mitotane (Lysodren), procarbazine (Matulane), pegaspargase (Oncaspar), denileukin diftitox (ODT), and rituximab (Tregs). ntak), porfimer (Photofrin), aldesleukin (Proleukin), lenalidomide (Revlimid), bexarotene (Targretin), thalidomide (Thalomid), temsirolimus (Torisel), arsenic trioxide (Trisenox), verteporfin (Visudyne), mimosine (Leucenol), (1M tegafur-0.4M 5-chloro-2,4-dihydroxypyrimidine-1M potassium oxonate) or lovastatin.

[0245] In certain embodiments, PD-L1 blockade is performed in combination with CD3 stimulation (e.g., by co-incubation with cells expressing membrane CD3) before, simultaneously with, or after treatment with an anti-PD-L1 antibody. For example, in one embodiment, a method for enhancing an antigen-specific T cell response comprises contacting T cells with an anti-PD-L1 antibody described herein and a CD3-expressing cell, whereby the antigen-specific T cell response is enhanced and PD-L1-mediated immune suppression is reduced. Suitable indicators of antigen-specific T cell response can be used to measure the antigen-specific T cell response. Non-limiting examples of such suitable indicators include increased T cell proliferation in the presence of the antibody and / or increased cytokine production in the presence of the antibody. In a preferred embodiment, production of interleukin-2 and / or interferon-γ by antigen-specific T cells is enhanced.

[0246] In some embodiments, the anti-PD-L1 antibodies described in the present invention can also be used in combination with bispecific antibodies that target Fcα or Fcγ receptor expressing effector cells to tumor cells (see, e.g., U.S. Patent Nos. 5,922,845 and 5,837,243). Such bispecific antibodies are used to target two different antigens. For example, anti-Fc receptor / anti-tumor antigen (e.g., Her-2 / neu) bispecific antibodies have been used to target macrophages to tumor sites. This targeting can more effectively activate tumor-specific responses. The T cell arm of these responses becomes enhanced by the inhibition of PD-L1. Alternatively, antigens can be delivered directly to DCs using bispecific antibodies that bind tumor antigens and dendritic cell-specific cell surface markers.

[0247] In all of the above methods, PD-L1 blockade can be combined with other means of immunotherapy, such as cytokine therapy (e.g., interferon, GM-CSF, G-CSF, IL-2) or bispecific antibody therapy with two different binding specificities, to provide enhanced tumor antigen presentation.

[0248] In some embodiments, the additional therapeutic agent for any of the above-mentioned methods of treatment, antigen-binding molecules, or pharmaceutical compositions is an immunostimulatory agent selected from the group consisting of: (a) an agent that inhibits signal transduction of an inhibitory receptor (immune checkpoint) or its ligand (immune checkpoint inhibitor) of an immune cell, or a nucleic acid encoding such an agent; (b) an agonist for a stimulatory receptor of an immune cell, or a nucleic acid encoding such an agonist; (c) a cytokine or a nucleic acid encoding a cytokine; (d) an oncolytic virus or a nucleic acid encoding an oncolytic virus; (e) a T cell expressing a chimeric antigen receptor; (f) a bispecific or multispecific T cell targeting antibody or a nucleic acid encoding such an antibody; (g) an anti-TGF-β antibody or a nucleic acid encoding such an antibody; (h) a TGF-β trap or a nucleic acid encoding such a trap; (i) a vaccine against a cancer-associated antigen (including such an antigen or a nucleic acid encoding such an antigen); (j) a cell therapy, and (k) a combination thereof. In some embodiments, the additional therapeutic agent is an agent that inhibits signal transduction of an inhibitory receptor or its ligand of an immune cell, or a nucleic acid encoding such an agent. The inhibitory receptor or its ligand is selected from the group consisting of TIGIT, CTLA-4, PD-1, PD-Ll, PD-L2, LAG-3, TIM-3, neuritin, BTLA, CECAM-1, CECAM-5, IL-1R8, VISTA, LAIR1, LILRB1, LILRB2, LILRB3, LILRB4, LILRB5, CD96, CD112R, CD 160, 2B4, TGFβ-R, KIR, NKG2A, MICA, MICB, A2aR, A2bR, and combinations thereof. In some embodiments, the additional therapeutic agent is an agonist for a stimulatory receptor on an immune cell, or a nucleic acid encoding such an agonist.The agonist is selected from the group consisting of OX40, CD2, CD16, CD27, CDS, ICAM-1, LFA-1 (CDlla / CD18), ICOS (CD278), 4-1BB (CD 137), GITR, CD28, CD30, CD40, BAFFR, HVEM, CD7, LIGHT, KG2C, SLAMF7, NKG2C, NKG2D, NKp46, NKp80, CD160, B7-H3, CD83 ligand, and combinations thereof. In some embodiments, the additional therapeutic agent is a cytokine or a nucleic acid encoding a cytokine selected from the group consisting of IL-2, IL-5, IL-7, IL-12, IL-15, IL-2I, and combinations thereof. In some embodiments, the additional therapeutic agent is an oncolytic virus or a nucleic acid encoding an oncolytic virus selected from herpes simplex virus, vesicular stomatitis virus, adenovirus, Newcastle disease virus, vaccinia virus, Maraba virus, and combinations thereof. In some embodiments, the additional therapeutic agent is a cell therapy. The cell therapy includes T cells, NK cells, or macrophages bearing a chimeric antigen receptor (CAR). In some embodiments, the cell therapy includes a bi- or multispecific T cell targeting antibody.

[0249] In certain embodiments, the invention provides a method for treating a disease or disorder (e.g., cancer) in a subject. The method comprises administering to the subject an antigen-binding molecule of the invention (e.g., an anti-PD-L1 antibody or antigen-binding fragment thereof), alone or in combination with a second or more additional therapeutic agents, wherein the subject has previously received treatment with a first or more additional therapeutic agents. In certain embodiments, the first or more additional therapeutic agent treatment may exhibit low efficacy in treating the disease in the subject. For example, the first or more therapeutic agent treatment may be a treatment with an anti-PD-1 antibody to which the subject may be resistant. In some embodiments, the second or more additional therapeutic agents are the same as the first or more additional therapeutic agents. In some embodiments, the second or more additional therapeutic agents are different from the first or more additional therapeutic agents.

[0250] In certain embodiments, the immune checkpoint inhibitor is an antibody that specifically interacts with an immune checkpoint. In some embodiments, the additional therapeutic agent comprises an immune stimulant. In some embodiments, the immune checkpoint inhibitor is an anti-CTLA-4 antibody (e.g., ipilimumab) and combinations thereof. In some embodiments, the immune checkpoint inhibitor is pembrolizumab. In some embodiments, the immune checkpoint inhibitor is nivolumab. In some embodiments, the immune checkpoint inhibitor is atezolizumab.

[0251] In some embodiments, the anti-PD-L1 antibodies are administered in combination or simultaneously with an immunogenic agent, non-limiting examples of which include cancer cells, tumor vaccines, and cells transfected with genes encoding purified tumor antigens (including recombinant proteins, peptides, carbohydrate molecules), oncolytic viruses, immune stimulating cytokines, etc.

[0252] In certain embodiments, the anti-PD-L1 antibodies are administered in conjunction with an antigen of interest or an antigen known to be present in the subject being treated (e.g., a subject with a tumor or a virus) to enhance antigen-specific immunity. When the anti-PD-L1 antibody is administered in conjunction with another agent, the two can be administered separately or simultaneously.

[0253] In certain embodiments, any one or more anti-PD-L1 antibodies described herein may be used in combination with an antigen of interest (e.g., a vaccine) to enhance an antigen-specific immune response. Thus, in one embodiment, a method for enhancing an immune response to an antigen in a subject comprises administering to the subject (i) the antigen and (ii) a PD-L1-based antibody, thereby enhancing the immune response to the antigen. The antigen may be, for example, a tumor antigen, a viral antigen, a bacterial antigen, or an antigen derived from a pathogen. Non-limiting examples of such antigens include those described in the sections above, such as the tumor antigens (or tumor vaccines) described above, or antigens derived from viruses, bacteria, or other pathogens described above.

[0254] In view of the synergistic benefits of the active agent composition, in certain embodiments, the anti-PD-L1 antibody and the additional active agent are each administered to a subject in need thereof at sub-therapeutic doses compared to the doses used in monotherapy.

[0255] In certain embodiments, PD-L1 inhibition is combined with standard cancer treatments (e.g., surgery, radiation, and chemotherapy). In such cases, the dose of chemotherapy administered may be reduced. The combination of PD-L1 inhibition and chemotherapy is believed to promote apoptosis and increase the presentation of tumor antigens for cytotoxic immunity. Other synergistic combination therapies include the combination of PD-L1 inhibition with radiation, surgery, or hormone ablation or inhibition. Each regimen generates a source of tumor antigens in the host, respectively.

[0256] The additional therapeutic agent may be administered prior to, simultaneously with, or after administration of the antigen-binding molecule of the present invention (for the purposes of this disclosure, such an administration regimen is considered a "combination" of the antigen-binding molecule with the additional therapeutically active ingredient).

[0257] The present invention includes pharmaceutical compositions comprising an antigen-binding molecule according to the invention in combination with one or more additional therapeutic agents as described herein.

[0258] V. Nucleic Acids and Host Cells Expressing Anti-PD-L1 Antibodies In another aspect, the invention provides nucleic acids encoding an antigen-binding molecule (e.g. an anti-PD-L1 antibody or antigen-binding fragment thereof) according to the invention, and expression vectors comprising such nucleic acids. In some embodiments, the nucleic acid encodes the HCVR and / or LCVR fragment of an antibody or fragment according to the embodiments described herein, or any of the other antibodies and antibody fragments described herein.

[0259] DNA encoding the antigen-binding site in a monoclonal antibody can be isolated and sequenced from hybridoma cells by conventional methods (e.g., using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of a monoclonal antibody). Alternatively, the amino acid sequence of the immunoglobulin of interest can be determined by direct protein sequencing, and an appropriate encoding nucleotide sequence can be designed according to a universal codon table. In other cases, the nucleotide and amino acid sequences of the antigen-binding site or other immunoglobulin sequences, including constant regions, hinge regions, etc., can be obtained from published sources well known in the art.

[0260] Expression vectors may be used to synthesize antibodies of this disclosure in in vitro cultured cells or may be administered directly to a patient to express antibodies of this disclosure in vivo or ex vivo. As used herein, "expression vector" refers to a viral or non-viral vector that contains a polynucleotide encoding one or more of the antibodies of this disclosure in a form suitable for expressing the polynucleotide in a host cell for antibody production or direct administration as a therapeutic agent.

[0261] A nucleic acid sequence is said to be "operably linked" to another nucleic acid sequence when the former is in a functional relationship with the latter. For example, DNA of a presequence or signal peptide is operably linked to DNA of a polypeptide if it is expressed as a preprotein involved in the secretion of the polypeptide. A promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence. Or, a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, "operably linked" means that the DNA sequences being linked are contiguous, and in the case of a signal peptide, contiguous and in reading phase. Enhancers, however, need not be contiguous. Linkage is accomplished by ligation at convenient restriction sites. If no such sites exist, synthetic oligonucleotide adaptors or linkers can be used in accordance with conventional methods.

[0262] The nucleic acid sequence for expressing the antibody of the present disclosure generally comprises an N-terminal signal peptide sequence that is removed from the mature protein.Since the signal peptide sequence can affect the expression level, the polynucleotide can encode any one of a variety of different N-terminal signal peptide sequences.It should be understood by those skilled in the art that the design of the expression vector may depend on factors such as the selection of the host cell to be transformed, the expression level of the protein of interest, etc.

[0263] The above-mentioned "regulatory sequence" refers to a DNA sequence necessary for the expression of an operably linked coding sequence in one or more host organisms. The term "regulatory sequence" includes promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cells, or those that direct expression of a nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). Expression vectors generally include sequences for transcription termination, and may further include one or more factors that positively affect mRNA stability.

[0264] The expression vector contains one or more transcription control elements, such as a promoter and / or enhancer, to direct the expression of the antibody of the present disclosure. A promoter comprises a DNA sequence that functions to initiate transcription from a relatively fixed position relative to the transcription start site. A promoter contains core elements required for basic interaction of RNA polymerase with transcription factors and can function in cooperation with other upstream elements and response elements.

[0265] The term "promoter" as used herein should be broadly construed to include, for example, transcriptional regulators (TREs) from genomic genes or chimeric TREs thereof (including TATA boxes or initiator elements for accurate transcription initiation), and further to include or not include other TREs (i.e., upstream activating sequences, transcription factor binding sites, enhancers, and silencers) that regulate the activation or repression of operably linked genes in response to developmental and / or external stimuli, as well as trans-acting regulatory proteins or nucleic acids. A promoter may contain genomic fragments or may contain chimeras combining one or more TREs.

[0266] Preferred promoters are those that can direct high levels of expression in the target cells of interest. Promoters include constitutive promoters (e.g., HCMV, SV40, elongation factor-1α (EF-1α) promoters) or promoters that are preferentially expressed in a particular cell type of interest. Enhancers generally refer to DNA sequences that function away from the transcription start site and can be located at the 5' or 3' end of the transcription unit. Additionally, enhancers can be present within introns and within coding sequences. Enhancers typically have a length of 10-300 bp and function in cis. Enhancers function to increase and / or regulate the transcription of nearby promoters. Preferred enhancers are those that direct high levels of expression in antibody-producing cells. Cell- or tissue-specific transcriptional regulators (TREs) can be incorporated into the expression vector to restrict expression to the desired cell type. Expression vectors can be designed to promote expression of the antibodies of the present disclosure in one or more cell types.

[0267] To co-express the individual chains of the antibody of the present disclosure, appropriate splice donor and splice acceptor sequences can be incorporated to express two products. Alternatively, an internal ribosome binding sequence (IRES) or 2A peptide sequence can be used to express multiple products from one promoter. An IRES provides a structure to which ribosomes can bind that does not necessarily have to be at the 5' end of the mRNA. Thus, an IRES can direct ribosomes to begin translation at a second start codon in the mRNA, allowing multiple polypeptides to be generated from a single mRNA. 2A peptides contain short sequences that mediate co-translational self-cleavage of peptides upstream and downstream of the 2A site, thus allowing the generation of equimolar amounts of two different proteins from a single transcript. CHYSEL is a non-limiting example of a 2A peptide that causes translating eukaryotic ribosomes to release the growing polypeptide chain they are synthesizing without dissociating from the mRNA. As the ribosomes continue translation, they generate a second polypeptide.

[0268] Expression vectors may include viral or non-viral vectors. Viral vectors can be derived from adeno-associated viruses (AAV), adenoviruses, herpes viruses, vaccinia viruses, polio viruses, pox viruses, retroviruses (including lentiviruses such as HIV-1 and HIV-2), Sindbis virus and other RNA viruses, alphaviruses, astroviruses, coronaviruses, orthomyxoviruses, papovaviruses, paramyxoviruses, parvoviruses, picornaviruses, togaviruses, etc. Non-viral vectors are simply "naked" expression vectors that are not packaged with viral derived components (e.g., capsid or envelope).

[0269] In some cases, these vectors may be engineered to target specific diseases or cell populations using targeting properties inherent or engineered into the viral vector. Specific cells are "targeted" for delivery and expression of polynucleotides. Thus, in this case, the term "targeting" may be based on the use of endogenous or heterologous binding agents in the form of capsids, envelope proteins, antibodies for delivery to specific cells, the use of tissue-specific regulatory elements to restrict expression to specific cell subsets, or both.

[0270] In some embodiments, expression of the antibody chains is under the control of regulatory sequences, such as tissue-specific or ubiquitous promoters. In some embodiments, a ubiquitous promoter (e.g., a CMV promoter, a CMV-chicken β-actin hybrid (CAG) promoter, a tissue-specific or tumor-specific promoter) controls expression of the heavy or light chain or single chain derivatives of a particular antibody.

[0271] Non-viral expression vectors can be utilized for non-viral gene transfer by direct injection of naked DNA or encapsulation of antibody-encoding polynucleotides in liposomes, microparticles, microcapsules, virus-like particles, or erythrocyte ghosts. Such compositions can be further linked to targeting domains by chemical conjugation to facilitate targeted delivery and / or introduction of the nucleic acid into desired cells of interest. Additionally, plasmid vectors can be incubated with synthetic gene transfer molecules (e.g., polymeric DNA-binding cations such as polylysine, protamine, and albumin) and can be linked to cell targeting ligands (e.g., asialoorosomucoid, insulin, galactose, lactose, or transferrin).

[0272] Alternatively, naked DNA can be used. The uptake efficiency of naked DNA can be improved by compression or the use of biodegradable latex beads. Such delivery can be further improved by treating the beads to increase their hydrophobicity, facilitating the disruption of endosomes and release of the DNA into the cytoplasm.

[0273] 6. Method for producing anti-PD-L1 antibodies In another aspect, the invention provides a host cell transformed with a nucleic acid or expression vector encoding an anti-PD-L1 HCVR and / or LCVR, which may be a bacterial or eukaryotic cell capable of expressing the nucleic acid or expression vector encoding the anti-PD-L1 HCVR and / or LCVR, or any of the other combination antibodies or antagonists described in the invention.

[0274] In another aspect, the methods for making the antibodies of the disclosure comprise culturing a host cell transformed with a nucleic acid or expression vector encoding an anti-PD-L1 HCVR and / or LCVR under conditions that allow for the production of the antibody or fragment, and purifying the antibody from the cells.

[0275] In an additional aspect, the invention provides a method for producing an antibody, comprising culturing cells that transiently or stably express one or more constructs encoding one or more polypeptide chains in the antibody, and purifying the antibody from the cultured cells. Any cell capable of producing a functional antibody can be used. In a preferred embodiment, the cell expressing the antibody is a eukaryotic or mammalian cell, preferably a human cell. Cells of various tissue cell types can be used to express the antibody. In other embodiments, the cell is a yeast cell, an insect cell, or a bacterial cell. Preferably, the antibody-producing cell is stably transformed with a vector expressing the antibody.

[0276] One or more expression vectors encoding the heavy or light chains of the antibody can be introduced into the cells by any conventional method, such as, for example, naked DNA techniques, cationic lipid-mediated transfection, polymer-mediated transfection, peptide-mediated transfection, viral-mediated infection, physical or chemical agents or treatments, electroporation, etc. Furthermore, the cells can be transfected with one or more expression vectors for expressing the antibody together with a selection marker that facilitates the selection of stably transformed clones expressing the antibody. The antibodies produced by such cells can be collected and / or purified according to techniques known in the art, such as centrifugation, chromatography, etc.

[0277] Examples of suitable selection markers for mammalian cells include dihydrofolate reductase (DHFR), thymidine kinase, neomycin, neomycin analog G418, hygromycin, and puromycin. When such selection markers are successfully introduced into a mammalian host cell, the transformed mammalian host cell can survive when placed under selective pressure. There are two widely used selection schemes. The first scheme is based on the metabolism of the cell and the use of mutant cell lines that are deficient in the ability to grow independent of supplemented media. Examples include CHO DHFR, thymidine kinase, neomycin, neomycin analog G418, hygromycin, and puromycin. -These include mouse LTV cells and mouse LTV cells. These cells cannot grow without the addition of nutrients such as thymidine and hypoxanthine. These cells lack certain genes required for a complete nucleotide synthesis pathway and therefore cannot survive unless the missing nucleotides are provided in a supplemented medium. As an alternative to supplementing the medium, an intact DHFR or TK gene can be introduced into cells lacking the corresponding gene, thus altering their growth requirements. Individual cells that have not been transformed with the DHFR or TK genes will not be able to survive in unsupplemented medium.

[0278] The second scheme, dominant selection, refers to a selection scheme used with any cell type and does not require the use of mutant cell lines. In these schemes, a drug is usually used to stop the growth of the host cells. The cells with the new gene express a protein that conveys drug resistance and survive the selection. Examples of such dominant selection include the use of drugs such as neomycin, mycophenolic acid, or hygromycin. Three examples use bacterial genes under eukaryotic control to convey resistance to the appropriate drug G418 or neomycin (geneticin), xgpt (mycophenolic acid), or hygromycin, respectively. Other drugs include the neomycin analog G418 and puromycin.

[0279] Exemplary antibody-expressing cells include human T-cell leukemia cells, human embryonic kidney (HEK) 293, Chinese hamster ovary (CHO) cells, mouse WEHI fibrosarcoma cells, as well as unicellular protozoan species (e.g., Leishmania tarentolae). Additionally, stably transformed antibody-producing cell lines can be generated using primary cells immortalized with c-myc or other immortalizing agents.

[0280] In one embodiment, the cell line comprises a stably transformed Leishmania cell line (e.g., Leishmania tarentolae). Leishmania can provide a robust, fast-growing, single-cell host for high-level expression of eukaryotic proteins that exhibit mammalian-type glycosylation patterns. Commercially available Leishmania eukaryotic expression kits are available (Jena Bioscience GmbH, Jena, Germany).

[0281] In some embodiments, the cell lines express at least 1 mg, at least 2 mg, at least 5 mg, at least 10 mg, at least 20 mg, at least 50 mg, or at least 100 mg of antibody per liter of culture medium.

[0282] Antibodies according to the invention can be cultured and maintained in any suitable medium (e.g., RPMI, DMEM, and AIM V®) and isolated from antibody-expressing cells. Antibodies can be purified using conventional protein purification methods (e.g., affinity purification, chromatography, etc.), including the use of Protein A or Protein G immunoaffinity purification. In some embodiments, the antibodies are designed to be secreted into the culture supernatant and isolated from the supernatant.

[0283] 7. Pharmaceutical Compositions and Methods of Administration In one aspect, a pharmaceutical composition described in the invention comprises an antigen-binding molecule described in the invention (e.g. an anti-PD-L1 antibody or antigen-binding fragment thereof) and a pharma- ceutically acceptable carrier. In other embodiments, the PD-L1 antibody or antigen-binding fragment thereof is in combination with a pharma- ceutically acceptable carrier. As discussed herein, an anti-PD-L1 composition may comprise one or more distinct antibodies, one or more multispecific antibodies, one or more fusion proteins, one or more immunoconjugates, or a combination thereof.

[0284] The present invention provides pharmaceutical compositions comprising the antigen-binding molecules according to the present invention. The pharmaceutical compositions according to the present invention are formulated with suitable carriers, excipients, and other agents capable of improving transport, delivery, tolerance, etc. A large number of suitable formulations are described in formularies known to all pharmaceutical chemists (Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA). These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid-containing vesicles (cationic or anionic) (e.g., LIPOFECTIN, 1997, 19 ... TM Excipients for parenteral formulations include, for example, glycerol-based formulations (PDA, Life Technologies, Carlsbad, Calif.), DNA conjugates, anhydrous absorbent pastes, oil-in-water and water-in-oil emulsions, carbowax emulsions (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. See also, Powell et al. "Compendium of excipients for parenteral formulations" PDA (1998) J Pharm Sci Technol 52:238-311.

[0285] The dose of the antigen-binding molecule administered to a patient may vary depending on the age and physique of the patient, the target disease, disorder, the route of administration, and the like. A preferred dose is generally calculated according to body weight or body surface area. When the bispecific antigen-binding molecule of the present invention is used to treat an adult patient, it is preferable to intravenously administer the bispecific antigen-binding molecule of the present invention at a single dose of about 0.01 to about 20 mg / kg body weight, more preferably about 0.02 to about 7 mg / kg body weight, about 0.03 to about 5 mg / kg body weight, or about 0.05 to about 3 mg / kg body weight. Depending on the severity of the condition, the frequency and duration of treatment can be adjusted. The effective dose and schedule for administering the bispecific antigen-binding molecule can be determined empirically. For example, the progress of the patient can be monitored by periodic evaluation, and the dose adjusted accordingly. Furthermore, interspecies scaling of doses can be performed using methods well known in the art (e.g., Mordenti et al., 1991, Pharmaceut. Res. 8:1351).

[0286] Various delivery systems are known and can be used to administer the pharmaceutical compositions of the present invention, including, for example, encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing mutant viruses, and receptor-mediated endocytosis (see, for example, Wu et al., 1987, J. BioI. Chem. 262:4429-4432). Methods of introduction include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The compositions may be administered by any convenient route, such as, for example, infusion or bolus administration, absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal mucosa, intestinal mucosa, etc.), and may be administered together with other bioactive agents. Administration may be systemic or local.

[0287] The pharmaceutical composition of the present invention can be delivered subcutaneously or intravenously using a standard needle and syringe. Moreover, for subcutaneous delivery, a pen delivery device can be easily applied to deliver the pharmaceutical composition of the present invention. Such a pen delivery device can be reusable or disposable. Reusable pen delivery devices generally utilize a replaceable cartridge containing the pharmaceutical composition. Once the pharmaceutical composition in the cartridge has been administered and the cartridge is empty, the empty cartridge can be easily discarded and replaced with a new cartridge containing the pharmaceutical composition. The pen delivery device can then be reused. Disposable pen delivery devices do not have a replaceable cartridge. Rather, disposable pen delivery devices are pre-filled with the pharmaceutical composition held in a reservoir within the device. Once the reservoir is empty of the pharmaceutical composition, the entire device is discarded.

[0288] In certain cases, the pharmaceutical composition can be delivered in a controlled release system. In one embodiment, a pump can be used (see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201). In another embodiment, a polymeric material can be used (see Medical Applications of Controlled Release, Langer and Wise (eds.), 1974, CRC Pres., Boca Raton, Florida). In yet another embodiment, the controlled release system can be placed close to the target of the composition, thus requiring only a fraction of the systemic dose (see, for example, Goodson, 1984, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138). Other controlled release systems are described in the review by Langer, 1990, Science 249:1527-1533.

[0289] The injections include dosage forms such as intravenous, subcutaneous, intradermal and intramuscular injections, drip infusions, etc. These injections can be prepared by known methods. For example, the injections can be prepared by dissolving, suspending or emulsifying the above-mentioned antibody or its salt in a sterile aqueous or oily medium for general injection. The aqueous medium for injection includes, for example, physiological saline, isotonic solutions containing glucose and other adjuvants, and may be used in combination with a suitable solubilizing agent such as alcohols (e.g., ethanol), polyalcohols (e.g., propylene glycol, polyethylene glycol), nonionic surfactants (e.g., polysorbate 80, HCO-(polyoxyethylene (50 mol)-hydrogenated castor oil adduct)), etc. As the oily medium, for example, sesame oil, soybean oil, etc. are used, and may be used in combination with a solubilizing agent such as benzyl benzoate, benzyl alcohol, etc. The prepared injection is preferably filled into a suitable ampoule.

[0290] It is particularly preferred that the above-mentioned pharmaceutical composition for oral or parenteral use is prepared in a dosage form of a unit dose suitable for the dose of the active ingredient. Such dosage forms of unit doses include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc. The content of the antibody is generally about 5 to about 500 mg per dosage form in a unit dose. In particular, it is preferred that the antibody is contained in the form of an injection at about 5 to about 100 mg, and in the case of other dosage forms, the antibody is contained at about 10 to about 250 mg.

[0291] In another embodiment, a method for treating a cell proliferative disorder, such as cancer, a chronic infection, or an immunodeficiency state, comprises administering to a subject in need thereof a pharmaceutical composition containing an anti-PD-L1 antibody, or antigen-binding fragment thereof, as described herein, in combination with a pharma- ceutical acceptable carrier. In some embodiments, the method restores, enhances, or potentiates lymphocyte activity in a subject in need thereof. In certain preferred embodiments, the antibody or fragment is a human or humanized anti-PD-L1 antibody that reduces or abolishes signaling through PD-L1.

[0292] In some embodiments, administration of the pharmaceutical composition increases lymphocyte (e.g., T cell) activity in patients who would benefit from increased lymphocyte activity or have a disease (e.g., T cell) caused or characterized by immune suppression, immune suppressive cells, or PD-L1 overexpression generated, for example, in CD4 T cells, CD8 T cells. The methods described herein are particularly useful in patients with solid tumors, for example, where the tumor microenvironment is suspected to contribute to reduced recognition by the immune system (immune escape). The tumor may be characterized, for example, by PD-L1 expressing (or overexpressing) immune cells (e.g., CD4 T cells, CD8 T cells, T-regs, B cells).

[0293] In certain embodiments, the methods and compositions are utilized in the treatment of various cancers and other proliferative disorders. These methods serve to reduce the levels of PD-L1, which can inhibit the anti-tumor activity of lymphocytes, and are applicable to a wide range of cancers, particularly solid cancers, where PD-L1 in the tumor microenvironment is known to suppress anti-tumor immune responses.

[0294] Non-limiting examples of cancers that may be treated using an antigen-binding molecule (e.g., an anti-PD-L1 antibody or an antigen-binding fragment thereof) according to the present invention include liver cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, breast cancer, lung cancer, non-small cell lung cancer (NSCLC), castration-resistant prostate cancer (CRPC), melanoma, uterine cancer, colon cancer, rectal cancer, anal cancer, gastric cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, non-Hodgkin's lymphoma, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, childhood solid tumors, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, renal pelvis cancer, central nervous system (CNS) neoplasms, primary CNS lymphoma, tumor vascular neoplasms, Spinal axis tumors, brain stem gliomas, pituitary adenomas, Kaposi's sarcoma, epidermoid carcinomas, squamous cell carcinomas, environmentally induced cancers (asbestos induced cancers), hematopoietic malignancies (multiple myeloma, B cell lymphoma, Hodgkin's lymphoma / primary mediastinal B cell lymphoma, non-Hodgkin's lymphoma, acute myeloid lymphoma, chronic myeloid leukemia, chronic lymphocytic leukemia, follicular lymphoma, diffuse large B cell lymphoma, Burkitt's lymphoma, immunoblastic large cell lymphoma, precursor B lymphoblastic lymphoma, mantle cell lymphoma, acute lymphoblastic leukemia, mycosis fungoides, anaplastic large cell lymphoma, T cell lymphoma, precursor T lymphoblastic lymphoma, etc.), or combinations of these cancers. The present disclosure is also applicable to the treatment of metastatic cancers. Patients can be tested or selected for one or more of the above clinical features before, during, or after treatment.

[0295] In one embodiment, the anti-PD-L1 antibody has at least an EC 50 , optional EC 70 , optionally substantive EC 100 In one embodiment, the anti-PD-L1 antibody is administered in an amount effective to achieve and / or maintain (e.g., for 1, 2, 3, 4 weeks, and / or until thereafter administration of the antigen-binding compound) a blood concentration of at least the EC 50 , optional EC 70 , optionally substantive EC100 In one embodiment, an activity amount of an anti-PD-L1 antibody is an amount effective to achieve an EC 50 , optional EC 70 , optionally substantive EC 100 is an amount effective to achieve (or maintain).

[0296] Optionally, in one embodiment, for antibodies that deplete PD-L1-expressing tumor cells by ADCC (e.g., may be fully effective at concentrations similar to or significantly lower than those that result in receptor saturation), the anti-PD-L1 antibody is primarily an inhibitor (having no substantial Fcγ receptor-mediated activity) and is administered in an amount effective to neutralize the enzymatic activity of PD-L1 for the desired period of time (e.g., one week, two weeks, one month) between successive administrations of the anti-PD-L1 antibody.

[0297] In one embodiment, the anti-PD-L1 antibody inhibits PD-L1-mediated immune suppression in T cells by at least EC 50 , optional EC 70 , optionally substantive EC 100 In one embodiment, the amount of anti-PD-L1 antibody is administered in an amount effective to achieve and / or maintain (for 1, 2, 3, 4 weeks, and / or until a subsequent administration of the anti-PD-L1 antibody) a blood concentration of EC 50 , optional EC 70 , optionally substantive EC 100 is an amount effective to achieve (or maintain).

[0298] In one embodiment, the invention provides a method of treating or preventing cancer in an individual. The method comprises administering to an individual having the disease an amount of an anti-PD-L1 antibody that achieves or maintains a circulating concentration within a predetermined period of time. Optionally, this concentration is higher than the concentration required for 50%, 70% or complete (e.g., 90%) receptor saturation of circulating PD-L1 expressing cells in an extravascular tissue of the subject (e.g., a tumor or tumor environment) (e.g., as assessed in PBMCs). Optionally, the concentration achieved is at least 20%, 50% or 100% higher than the concentration required for specific receptor saturation.

[0299] In one embodiment, the invention provides a method of treating or preventing cancer in an individual, the method comprising achieving or maintaining a predetermined concentration in the circulation, and optionally in an extravascular tissue of a subject (e.g., a tumor or tumor environment), for a predetermined period of time, and detecting an EC 50 , optional EC 70 or optional EC 100 Optionally, the concentration achieved is greater than the EC50 for binding to PD-L1-expressing cells. 50 , optional EC 70 or optional EC 100 at least 20%, 50% or 100% higher than

[0300] In any embodiment, the antibody has an EC 50 , optional EC 70 or optional EC 100 is 0.5 to 100 ng / ml, optionally 1 to 100 ng / ml, optionally 30 to 100 ng / ml, for example, about 30 to 90 ng / ml. For example, EC 50 may be about 30, 37, 39, 43, 57, 58, 61, 62, 90, 95, 143 ng / ml.

[0301] EC to neutralize PD-L1 enzymatic activity using anti-PD-L1 antibodies 50 For example, EC may be about 0.01 μg / ml to 1 μg / ml, optionally 0.1 μg / ml to 10 μg / ml, optionally 0.1 μg / ml to 1 μg / ml. 50 may be about 0.1 μg / ml, about 0.2 μg / ml, or about 0.3 μg / ml. Thus, the anti-PD-L1 antibody is administered in an amount to achieve and / or maintain a blood concentration of, for example, at least 0.1 μg / ml, optionally at least 0.2 μg / ml, optionally at least 1 μg / ml, or optionally at least 2 μg / ml.

[0302] When targeting tissues outside the vasculature (e.g., the tumor environment in the treatment of solid tumors), doses that are generally about 10-fold higher will be necessary compared to doses that provide corresponding concentrations in the circulation. Doses of anti-PD-L1 antibodies administered to achieve (and / or maintain) circulating (blood) concentrations of about 1 μg / ml, 2 μg / ml, 10 μg / ml, or 20 μg / ml are expected to achieve (and / or maintain) extravascular tissue (e.g., tumor tissue) concentrations of about 0.1 μg / ml, 0.2 μg / ml, 1 μg / ml, 2 μg / ml, respectively.

[0303] In one embodiment, the anti-PD-L1 antibody is administered in an amount to achieve and / or maintain a tissue (e.g., tumor milieu) concentration of, for example, at least 0.1 μg / ml, optionally at least 0.2 μg / ml, optionally at least 1 μg / ml, or optionally at least 2 μg / ml. The antibody is administered in an amount to achieve and / or maintain a blood concentration of, for example, at least about 1 μg / ml, 2 μg / ml, 10 μg / ml, or 20 μg / ml (e.g., 1-100 μg / ml, 10-100 μg / ml, 1-50 μg / ml, 1-20 μg / ml, or 1-10 μg / ml). Dosage can be adjusted to maintain a desired concentration within a predetermined period of time following administration (e.g., 1, 2, 3, 4 weeks, etc.).

[0304] In some embodiments, the amount of anti-PD-L1 antibody is at least the EC 70 or EC 100 The antibody is administered in an amount to achieve and / or maintain a blood or extravascular tissue (e.g., tumor environment) concentration of at least about 1 μg / ml, 2 μg / ml, 10 μg / ml, or 20 μg / ml, for example.

[0305] EC of selected PD-L1 antibodies 50、 EC 70 and E.C. 100 The EC value can be assessed, for example, in a cellular assay that blocks the PD-1 / PD-L1 interaction. 50 " refers to the concentration of anti-PD-L1 antibody that produces 50% of the maximum response or effect with respect to neutralizing enzymatic activity. 70 " refers to the concentration of anti-PD-L1 antibody that produces 70% of the maximum response or effect. "EC 100 " refers to the effective concentration of an anti-PD-L1 antibody that produces a maximal response or effect in terms of neutralizing such enzymatic activity. In certain embodiments, depending on the context, the EC 50 , E.C. 70 , or EC 100 and IC50, respectively, to reflect that an antigen-binding molecule (e.g., an anti-PD-L1 antibody or antigen-binding fragment thereof) inhibits the activity of PD-L1. 50 ,I C 70 , or IC 100 It is called IC xx refers to the concentration of a drug required to inhibit a biological process by xx%.

[0306] In some embodiments, particularly for the treatment of solid tumors, the concentration achieved is at least the EC 50、 Optionally about EC100 or at least about EC 100 The concentration is designed to be in tissues (outside the vasculature, e.g., within a tumor or tumor environment) corresponding to:

[0307] In one embodiment, the amount of anti-PD-L1 antibody is 1-20 mg / kg body weight, hi one embodiment, the amount is administered to the individual weekly, biweekly, monthly or bimonthly.

[0308] In one embodiment, a method for treating cancer in a subject in need thereof comprises administering to the individual an effective amount of an anti-PD-L1 antibody of the present disclosure for at least one administration cycle (optionally at least two, three, four or more administration cycles), wherein said cycle is eight weeks or less in duration, and wherein in each of said at least one cycle, the anti-PD-L1 antibody is administered once, twice, three or four times at a dose of 1-20 mg / kg body weight. In one embodiment, the anti-PD-L1 antibody is administered by intravenous infusion.

[0309] Suitable treatment protocols for treating a subject (e.g., a human) include, for example, administering to the patient an amount of an anti-PD-L1 antibody disclosed herein, where the method includes at least one dosing cycle comprising administering the anti-PD-L1 antibody in at least one dose. Optionally, the anti-PD-L1 antibody is administered at least 2, 3, 4, 5, 6, 7, or 8 times. In one embodiment, the dosing cycle is 2 to 8 weeks long.

[0310] In one embodiment, a method for treating or preventing a disease (e.g., cancer, solid tumor, hematological tumor) in an individual comprises administering to the individual at least one dosing cycle of an anti-PD-L1 antibody for neutralizing enzymatic activity of PD-L1, the dosing cycle comprising at least a first and a second (and optionally a third, fourth, fifth, sixth, seventh, and / or eighth or more) administrations of the anti-PD-L1 antibody, wherein the anti-PD-L1 antibody is administered in an amount effective to achieve or maintain, between two successive administrations, a blood (serum) concentration of the anti-PD-L1 antibody within a range of at least 0.1 μg / ml, at least 0.2 μg / ml, at least 1 μg / ml, at least 2 μg / ml, at least 10 μg / ml, at least 20 μg / ml, 1-100 μg / ml, 1-50 μg / ml, 1-20 μg / ml, 1-10 μg / ml, or between any of the aforementioned concentrations.

[0311] In one embodiment, a particular continuous blood level is maintained, where the blood level does not substantially fall below a particular blood level for a particular period of time (e.g., between two administrations of the antibody, for a number of weeks, 1 week, 2 weeks, 3 weeks, 4 weeks). In other words, although the blood level may vary during a specified period of time, the particular blood level that is maintained represents a minimum or "trough" level.

[0312] In one embodiment, a therapeutically active amount of an anti-PD-L1 antibody will produce (at least) EC50 / 200 in the blood and / or tissues to neutralize the enzymatic activity of PD-L1 for at least about one week, about two weeks, or about one month following administration of the antibody. 50 Concentration, optional EC 70 Concentration, optional EC 100 The amount of antibody is sufficient to provide a concentration.

[0313] Prior to or during treatment with an anti-PD-L1 antibody of the disclosure, the expression levels of PD-L1 and / or PD-1 in cells, the percentage of PD-L1-expressing cells and / or PD-1-expressing cells can be assessed in and / or near the patient's tumor to determine whether the patient is suitable for and likely to respond to treatment. Increased levels or expression indicates that the individual is suitable for (e.g., likely to benefit from) treatment with an anti-PD-L1 antibody of the disclosure.

[0314] In some embodiments, assessing the expression levels of PD-L1 and / or PD-1 in and / or near a patient's tumor includes obtaining from the subject a biological sample of human tissue selected from the group consisting of tissues from a cancer patient, such as cancer tissue, tissue near or surrounding the cancer, cancer adjacent tissue, adjacent non-tumor tissue, or normal adjacent tissue, and the expression levels of PD-L1 and / or PD-1 in the tissue. The expression levels or nucleotide concentrations obtained from the patient can be compared to reference levels (e.g., levels in healthy individuals).

[0315] In view of the above, in certain embodiments, the methods include the steps of: (a) measuring expression levels of PD-L1 and / or PD-1 in the tumor environment, optionally within the tumor and / or nearby tissues, and determining that the tumor environment exhibits increased PD-L1 levels and / or PD-1 levels relative to their corresponding reference levels; and (b) administering to the individual an anti-PD-L1 antibody.

[0316] In certain embodiments, determining the level of PD-L1 and / or PD-L1 in the tumor environment includes obtaining a biological sample from the subject containing cancer tissue and / or tissue near or surrounding the cancer (e.g., cancer adjacent tissue, adjacent non-tumor tissue, or normal adjacent tissue) and detecting the level and / or relative percentage of PD-L1 and / or PD-1. PD-L1 and / or PD-1 expressing cells include, for example, tumor cells, CD4 T cells, CD8 T cells, B cells, and combinations thereof. PD-L1 and / or PD-1 expression levels can be determined by assessing PD-L1 and / or PD-1 mRNA expression (e.g., by RT-PCR) or polypeptide expression (e.g., Western blot, immunofluorescence staining) using techniques well known to those of skill in the art, compared to a reference level in a healthy subject or a reference level before treatment.

[0317] A subject with cancer can be treated with an anti-PD-L1 antibody with or without assessing the levels of PD-L1 and PD-1 in the tumor microenvironment (e.g., tumor cells, CD4 T cells, CD8 T cells, B cells).

[0318] A determination that the biological sample contains cells that overexpress PD-L1 and / or PD-1 relative to the reference indicates that the subject has a cancer that may benefit from treatment with an agent that blocks the PD-1 / PD-L1 interaction. In some embodiments, the term "overexpression" is used in reference to PD-L1 and / or PD-1 polypeptide being expressed in at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or more of the cells taken from a particular patient, e.g., tumor cells or lymphocytes taken from a subject.

[0319] In one embodiment, a method for treating or preventing cancer in a subject in need thereof comprises: (a) detecting the percentage and / or degree of expression of cells corresponding to PD-L1 and / or PD-1 within the tumor environment, optionally within the tumor and / or adjacent tissues, and determining that the tumor environment contains cells that overexpress PD-L1 and / or PD-1 at an increased level, optionally compared to a suitable reference level; and (b) administering to the subject an anti-PD-L1 antibody. In one embodiment, the cells are tumor cells. In another embodiment, the cells within the tumor environment, tumor and / or adjacent tissues are non-malignant immune cells (e.g. T cells).

[0320] In some embodiments, measuring the level of expression of PD-L1 and / or PD-1 within the tumor environment comprises obtaining a biological sample from an individual that contains cancerous tissue and / or tissue near or surrounding the cancer (e.g., cancer-adjacent tissue, adjacent non-tumor tissue, or normal adjacent tissue), contacting the cells with an antibody bound to a PD-L1 and / or PD-1 polypeptide, and detecting the percentage of cells and / or the level of expression that corresponds to PD-L1 and / or PD-1. In certain embodiments, expression of PD-L1 and / or PD-1 is assessed by their cell surface expression using immunohistochemistry assays.

[0321] The antibody composition can be used as a monotherapy or as a combination therapy with one or more other therapeutic agents, including agents that are typically used for the particular therapeutic purpose for which the antibody is administered. See "Combination Therapy" above. The additional therapeutic agent is typically administered in the amount and treatment regimen that is typically used in the monotherapy used for the particular disease or disorder being treated. Such therapeutic agents include, but are not limited to, anti-cancer agents and chemotherapeutic agents.

[0322] As noted above, a method of using a pharmaceutical composition according to the present invention comprises the step of administering to a subject in need thereof an effective amount of a pharmaceutical composition according to the present disclosure.

[0323] Any suitable route or mode of administration can be used to provide a patient with a therapeutically or prophylactically effective dose of the antibody. Exemplary routes or modes of administration include parenteral (e.g., intravenous, intraarterial, intramuscular, subcutaneous, intratumor), oral, topical (intranasal, transdermal, intradermal or intraocular), mucosal (e.g., nasal, sublingual, buccal, rectal, vaginal), inhalation, intralymphatic, intraspinal, intracranial, intraperitoneal, intratracheal, intravesical, intrathecal, intraintestinal, intrapulmonary, intralymphatic, intracavity, intraorbital, intracapsular and transurethral, ​​as well as local delivery via a catheter or stent.

[0324] Pharmaceutical compositions comprising anti-PD-L1 antibodies according to the present disclosure may be formulated in any pharma- ceutically acceptable carrier or excipient. As used herein, the term "pharma-ceutically acceptable carrier" includes any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The pharmaceutical composition may include a suitable solid or gel phase carrier or excipient. Exemplary carriers or excipients include, but are not limited to, calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols. Exemplary pharma-ceutically acceptable carriers include water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, and combinations of one or more thereof. In many cases, it is preferred to include an isotonic agent in the composition, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride. Pharmaceutically acceptable carriers may further include minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives, or buffers, which enhance the shelf life or effectiveness of the therapeutic agent.

[0325] In certain preferred embodiments, the therapeutically active agent may be formulated in a pharmaceutical composition suitable for parenteral administration. Pharmaceutical compositions for parenteral administration may be formulated by injection, e.g., by bolus injection or continuous infusion.

[0326] Suitable buffering agents include, but are not limited to, sodium succinate, sodium citrate, sodium phosphate, or potassium phosphate. Sodium chloride at a concentration of 0-300 mM (for liquid dosage forms, the optimal concentration is 150 mM) can be used to modify the toxicity of the solution. Lyophilized dosage forms may include a cryoprotectant, primarily 0-10% sucrose (optimally 0.5-1.0%). Other suitable cryoprotectants include trehalose and lactose. Bulking agents, which can be included for lyophilized dosage forms, are primarily 1-10% mannitol (optimally 2-4%). ​​Stabilizers, which can be used in both liquid and lyophilized dosage forms, are primarily L-methionine at 1-50 mM (optimally 5-10 mM). Other suitable bulking agents include glycine, arginine, and 0-0.05% polysorbate-80 (optimally 0.005-0.01%). Other surfactants include, but are not limited to, polysorbate 20 and BRIJ surfactants.

[0327] Therapeutic formulations can be lyophilized and stored as sterile powders, preferably under vacuum, and then reconstituted with bacteriostatic water (e.g., containing a benzyl alcohol preservative) or sterile water prior to injection. The therapeutic agent in the pharmaceutical composition can be formulated in a "therapeutically effective amount" or a "prophylactically effective amount." A "therapeutically effective amount" refers to an amount effective to achieve the desired therapeutic result at the dosage and time period required. The therapeutically effective amount of an antibody or active agent can vary depending on the disorder being treated, the severity and course of the disorder, the method of administration, whether the antibody or active agent is administered for prophylactic or therapeutic purposes, the bioavailability of the particular agent, the ability of the antibody to elicit a desired response in the individual, previous treatments, the age, weight, and sex of the patient, the patient's clinical history and response to the antibody, the type of antibody used, the discretion of the attending physician, and the like. A therapeutically effective amount is also an amount in which the therapeutically beneficial effects outweigh any toxic or deleterious effects of the recombinant vector. A "prophylactically effective amount" refers to an amount effective to achieve the desired prophylactic result at the dosage and time period required.

[0328] Preferably, the polypeptide domains used in the antibodies or additional active agents described herein are derived from the same host to which they are administered, in order to reduce the inflammatory response to the administered therapeutic agent. As alluded to above, the therapeutic agent may be suitably administered to the subject once or over a series of treatments, or may be administered to the patient at any time following diagnosis. The PD-L1 antibodies may be administered as monotherapy or in combination with additional active agents or therapies useful in treating the disorder in question.

[0329] As a general proposition, a therapeutically or prophylactically effective amount of a PD-L1 antibody (or additional active agent) will be administered in the range of about 1 ng / kg body weight / day to about 100 mg / kg body weight / day, whether administered in single or multiple doses.In a specific embodiment, each PD-L1 antibody or active agent is administered at a dose of about 1 ng / kg body weight / day to about 10 mg / kg body weight / day, about 1 ng / kg body weight / day to about 1 mg / kg body weight / day, about 1 ng / kg body weight / day to about 100 μg / kg body weight / day, about 1 ng / kg body weight / day to about 10 μg / kg body weight / day, about 1 ng / kg body weight / day to about 1 μg / kg body weight / day, about 1 ng / kg body weight / day to about 100 ng / kg body weight / day, about 1 ng / kg body weight / day to about 10 ng / kg body weight / day, about 10 ng / kg body weight / day to about 100 mg / kg body weight / day, or about 10 ng / kg body weight / day. weight / day ~ approx. 10 mg / kg body weight / day, approx. 10 ng / kg body weight / day ~ approx. 1 mg / kg body weight / day, approx. 10 ng / kg body weight / day ~ approx. 100 μg / kg body weight / day, approx. 10 ng / kg body weight / day ~ approx. 10 μg / kg body weight / day, approximately 10 ng / kg body weight / day ~ approx. 1 μg / kg body weight / day, 10ng / kg body weight / day ~ approx. 100ng / kg body weight / day, approx. 100ng / kg body weight / day ~ approx. 100 mg / kg body weight / day, approx. 100ng / kg body weight / day ~ approx. 10 mg / kg body weight / day, approx. 100ng / kg body weight / day ~ approx. 1 mg / kg body weight / day, approx. weight / day ~ approx. 100 μg / kg body weight / day, approx. 100 ng / kg body weight / day ~ approx. 10 μg / kg body weight / day, approx. 100 ng / kg body weight / day ~ approx. 1 μg / kg body weight / day, approx. 1 μg / kg body weight / day ~ approx. 100 mg / kg body weight / day, approx. 1 μg / kg body weight / day ~ approx. 10 mg / k g body weight / day, approximately 1 μg / kg body weight / day to approximately 1 mg / kg body weight / day, approximately 1 μg / kg body weight / day to approximately 100 μg / kg body weight / day, approximately 1 μg / kg body weight / day to approximately 10 μg / kg body weight / day, approximately 10 μg / kg body weight / day to approximately 100 mg / kg body weight / day, approximately 10 μg / kg body weight It is administered in the range of about 10 mg / kg body weight / day, about 10 μg / kg body weight / day to about 1 mg / kg body weight / day, about 10 μg / kg body weight / day to about 100 μg / kg body weight / day to about 100 mg / kg body weight / day, about 100 μg / kg body weight / day to about 100 mg / kg body weight / day, about 100 μg / kg body weight / day to about 10 mg / kg body weight / day, about 100 μg / kg body weight / day to about 1 mg / kg body weight / day, about 1 mg / kg body weight / day to about 100 mg / kg body weight / day, about 1 mg / kg body weight / day to about 10 mg / kg body weight / day, about 10 mg / kg body weight / day to about 100 mg / kg body weight / day.

[0330] In other embodiments, the PD-L1 antibody and / or active agent is administered at a dose of 500 μg to 20 g every three days, or 25 mg / kg body weight every three days.

[0331] In other embodiments, the dose of each PD-L1 antibody and / or active agent may be, for a single administration, about 10 ng to about 100 ng, about 10 ng to about 1 μg, about 10 ng to about 10 μg, about 10 ng to about 100 μg, about 10 ng to about 1 mg, about 10 ng to about 10 mg, about 10 ng to about 100 mg, about 10 ng to about 1000 mg, about 10 ng to about 10,000 mg, about 10 0ng to about 1μg, about 100ng to about 10μg, about 100ng to about 100μg, about 100ng to about 1mg, about 100ng to about 10mg, about 100ng to about 100mg, about 100ng ~about 1000mg, about 100ng to about 10,000mg, about 1μg to about 10μg, about 1μg to about 100μg, about 1μg to about 1mg, about 1μg to about 10mg, about 1μg to about 100mg, Approximately 1μg to approximately 1000mg, approximately 1μg to approximately 10,000mg, approximately 10μg to approximately 100μg, approximately 10μg to approximately 1mg, approximately 10μg to approximately 10mg, approximately 10μg to approximately 100mg, approximately 10μ g ~ about 1000mg, about 10μg - about 10,000mg, about 100μg - about 1mg, about 100μg - about 10mg, about 100μg - about 100mg, about 100μg - about 1000mg, about 1 The antibody of the present disclosure may be administered daily, every 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, or 4 weeks.

[0332] In other specific embodiments, the dosage of each PD-L1 antibody or active agent may be about 0.0006 mg / day, 0.001 mg / day, 0.003 mg / day, 0.006 mg / day, 0.01 mg / day, 0.03 mg / day, 0.06 mg / day, 0.1 mg / day, 0.3 mg / day, 0.6 mg / day, 1 mg / day, 3 mg / day, 6 mg / day, 10 mg / day, 30 mg / day, 60 mg / day, 100 mg / day, 300 mg / day, 600 mg / day, 1000 mg / day, 2000 mg / day, 5000 mg / day, or 10,000 mg / day.

[0333] In certain embodiments, the coding sequence for the PD-L1 antibody and / or additional active agent is incorporated into an expression vector (e.g., a viral or non-viral vector) suitable for expressing an effective amount of the PD-L1 antibody or additional active agent in a subject in need of treatment by the above-mentioned methods. For example, in certain embodiments involving administration of one or more recombinant AAV (rAAV) viruses, the pharmaceutical composition comprises at least 10 10 Pieces, at least 10 11 Pieces, at least 10 12 Pieces, at least 10 13 pcs. or at least 10 14 In certain embodiments, the pharmaceutical composition may comprise at least 10 genome copies (GC) or recombinant viral particles per subject, or any range thereof. 10 Pieces, at least 10 11 Pieces, at least 10 12 Pieces, at least 10 13 Pieces, at least 10 14 Pieces, at least 10 15 The recombinant virus, such as an rAAV, may comprise up to 100 genomic copies or up to 100 genomic copies of the recombinant virus particle, or any range thereof.

[0334] Dosages can be tested in one or more art-recognized animal models appropriate for any particular cell proliferative or immunodeficiency disease state.

[0335] Delivery methods also include polycation-condensed DNA with or without linkage to killed viruses, ligand-linked DNA, liposomes, eukaryotic delivery vehicle cells, deposition of photopolymerized hydrogel materials, use of handheld gene transfer particle guns, ionizing radiation, neutralization of nuclear charges or fusion with cell membranes, particle-mediated gene transfer, and the like.

[0336] 8. Diagnostic Uses of Antibodies Antigen binding molecules (e.g., antibodies or antigen-binding fragments thereof) of the invention can also be used to detect and / or measure human or cynomolgus PD-L1, or cells expressing human or cynomolgus PD-L1, in a sample, e.g., for diagnostic purposes. For example, anti-PD-L1 antibodies or antigen-binding fragments thereof can be used to diagnose disorders or diseases characterized by aberrant expression (e.g., overexpression, underexpression, lack of expression, etc.) of PD-L1. An exemplary diagnostic assay for PD-L1 comprises, e.g., contacting a sample obtained from a patient with an antibody of the invention that has been labeled with a detectable label or reporter molecule. Alternatively, unlabeled antibodies can be used for diagnostic purposes in combination with a secondary antibody that is itself detectably labeled. The detectable label or reporter molecule can be, for example, 3 H, 14 C. 18 F, 32 p, 35 S or 125The PD-L1 may be a radioisotope such as I; a fluorescent or chemiluminescent moiety such as fluorescein isothiocyanate or rhodamine; or an enzyme such as alkaline phosphatase, β-galactosidase, horseradish peroxidase, or luciferase. Specific exemplary assays that can be used to detect or measure PD-L1 in a sample include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence-activated cell sorting (FACS). Samples that can be used in the PD-L1 diagnostic assays described in the present invention include any tissue or body fluid sample taken from a patient that contains a detectable amount of PD-L1 protein or a fragment thereof under normal or pathological conditions. In general, the level of PD-L1 in a particular sample taken from a healthy patient (e.g., a patient not suffering from a disease or disorder associated with abnormal PD-L1 levels or activity) is measured to initially establish a baseline or standard level of PD-L1. This baseline level of PD-L1 can then be compared to the level of PD-L1 measured in samples taken from individuals suspected of having a PD-L1-associated disease or disorder.

[0337] Moreover, the anti-PD-L1 antibodies described herein can be used to purify human PD-L1 through immunoaffinity purification.

[0338] 9. Kit Any of the compositions described herein, such as the anti-PD-L1 antigen binding molecules of the invention, and / or an additional therapeutic agent, may be comprised in a kit. In a non-limiting example, the kit comprises an antigen binding molecule, such as an antibody or antigen-binding fragment thereof. In certain embodiments, the kit further comprises an additional therapeutic agent described herein.

[0339] The kit may further comprise reagents or instructions for treating a disease or disorder, and may also comprise one or more buffers.

[0340] The above components of the kits may be packaged either in aqueous media or in lyophilized form. The container means of the kits will generally include at least one vial, test tube, flask, bottle, syringe or other container means into which a component may be placed, and preferably suitably aliquoted. Where there is more than one component in the kit (such as labeling reagents and labels which may be packaged together), the kits will generally include second, third or other container means for separately placing the other components. The kits may also include a second container means for containing a sterile pharma- ceutically acceptable buffer and / or other diluent. Additionally, vials may contain various combinations of components. Kits of the present invention will also typically include a means for containing the compositions of the present invention (e.g., the anti-PD-L1 antigen binding molecules and / or additional therapeutic agents) and any other reagent containers in close confinement for commercial sale.

[0341] When the above components of the kit are provided as one and / or more liquid solutions, the liquid solution is an aqueous solution, with a sterile aqueous solution being particularly preferred.Furthermore, the components of the kit can be provided as dry powder.When reagents and / or components are provided as dry powder, the powder can be reconstituted by adding a suitable solvent.It is also envisioned that the solvent can be provided in a separate container means.

[0342] The present invention is further illustrated, but not limited, by the following examples. All references, patents, published patent application contents, and figures and tables cited throughout this application are hereby incorporated by reference. EXAMPLES

[0343] Example 1: Preparation of rabbit anti-human PD-L1 monoclonal antibody Immunized rabbits Two New Zealand White rabbits (4-6 weeks old) were immunized by injecting the rabbits with human PD-L1 protein according to an IACUC-approved regimen by Capralogics. Briefly, rabbits were immunized with recombinant proteins containing the human PD-1 extracellular domain (ECD) and rabbit Fc (BonOpus) or the D1 domain and rabbit Fc (Yurogen). Serum samples were collected before the first immunization and 14 days after the second immunization.

[0344] Serum titration was performed using an antigen-specific enzyme-linked immunosorbent assay (ELISA). Briefly, microplates were precoated with 25 μl of 1 μg / ml human PD-L1-His (Sino Biological, Cat. No. 10084-H08H) or cynomolgus monkey PD-L1-His protein (Sino Biological, Cat. 90251-C08H). Rabbit sera were diluted 1:1000, followed by a 7-point 3-fold serial dilution. The serially diluted sera were added to the assay buffer. HRP-labeled goat anti-rabbit IgG (H+L) antibody (Jackson Immuno Research, Cat. No. 111-036-045) was used for detection at a dilution of 1:5000. Color was developed with 100 μl / well of TMB substrate. Stop solution (1N H2SO 4) was added at 50 μl / well.

[0345] Rabbits with high titers of specific antibodies were then euthanized and the spleens were aseptically isolated. Sera from immune rabbits were also subjected to PD-1:PD-L1 binding competitive ELISA (cELISA).Briefly, microplates were precoated with 1 μg / μl human PD-L1-mFc fusion protein (Sino Biological, Cat #10084-H05H) overnight at 4°C. Plates were then blocked and 100 μl / well of the following was added: (1) 0.5 μg / ml human PD-1-His-hFc-biotin fusion protein (Sino Biological, Catalog No. 10377-H03H-B) alone; (2) 0.5 μg / ml human PD-1-His-hFc-biotin fusion protein mixed with diluted Imfinzi (Durvalumab) (starting at 6000 ng / ml, followed by an eight-point serial dilution); or (3) 0.5 μg / ml human PD-1-His-hFc-biotin fusion protein mixed with diluted rabbit serum (starting at a 1:10 dilution, followed by an eight-point serial dilution). HRP-conjugated neutravidin (ThermoFisher, Catalog No. 31001) was used for detection at a dilution of 1:20000. Color was developed with 100 μl / well of TMB substrate. Stop solution (1N HSO 4) was added at 50 μl / well.

[0346] The results of cELISA (data not shown) indicate that the sera from immune rabbits were able to inhibit the PD-1:PD-L1 interaction. A commercially available anti-PD-L1 antibody (durvalumab) was used as a positive control.

[0347] B cell sorting Spleen tissue was harvested from immunized rabbits and homogenized. Spleen cells were either used immediately for sorting or flash frozen and stored at -196°C for a period before thawing for further sorting. Rabbit B cells were stained with biotinylated human PD-L1 D1-His-Avi (Yurogen), human PD-L1 ECD-His (Sino Biological, Cat. No. 10084-H08H), human PD-L1 mouse Fc fusion protein (Sino Biological, Cat #10084-H05H), or cynomolgus PD-L1 ECD-His (Sino Biological, Cat. 90251-C08H). If the selection antigen was not biotinylated, biotinylation was performed. Briefly, a 10 mM stock solution of Sulfo-NHS-LC-Biotin (ThermoFisher Cat. No. A39257) was prepared by dissolving 1 mg of biotin in 180 μL of deionized water on ice. Based on the amount of protein to be biotinylated, a 20-fold molar ratio of biotin was added to the protein solution and the mixture was incubated on ice for 2 h. Slide-A-Lyzer TMFree biotin was removed by dialyzing the biotinylated protein against 1x PBS in a mini dialysis kit (3.5K MWCO 0.1 mL) with at least two buffer exchanges at a minimum of 3 h intervals (ThermoFisher, catalog no. 69550). For each sorting, multiple vials of selected rabbit spleen cells were thawed and cultured overnight in B cell medium (RPMI-1640, 15% FBS, 1x HEPES, 1x 2-ME (1-mercaptoethanol), 1% penicillin-streptomycin) before sorting. Also, 96-well B cell culture plates were prepared the day before. Briefly, a mixture of irradiated YC1 and a proprietary growth factor cocktail in B cell medium was dispensed into 96-well culture plates (120 μL / well). On the day of sorting, floating and loosely attached splenocytes were harvested by gently pipetting the medium onto the culture surface of the flask. The cells were transferred to a conical tube and spun at 400×g for 3 minutes. The cell pellet was washed twice with chilled FACS buffer (1×PBS+0.5%BSA). Biotinylated PD-L1 protein was added at 5μg / ml (final concentration). The mixture was incubated at room temperature for 20 minutes. The labeled mixture was then spun at 400×g for 3 minutes, the cells were resuspended in FACS buffer, and the cells were transferred to a 1.5ml amber Eppendorf tube. NeutrAvidin-Dy (diluted 1:300, Invitrogen Cat. No. 22842) and FITC-conjugated anti-rabbit IgM antibody (diluted 1:500, Novus, Cat. No. MB7173) were then added, and the mixture was incubated at 4°C for 15-30 minutes. The labeled mixture was centrifuged at 400×g for 3 minutes. The cells were washed twice with cold FACS buffer. The washed cell pellet was diluted with chilled 1x PBS + 1% FBS for ~10 7 Cells were resuspended at 1000x cells / ml. At least 10 min before sorting, 7-AAD (1 μg / mL, final concentration) was added to discriminate between live and dead cells. Single 7-AAD- / IgM- / Dy594+ cells were sorted into each well of a seeded 96-well plate. Sorted B cells were placed in 96-well rabbit B cell complete medium (Yurogen) and cultured at 37°C, 5% CO2 for 9–12 days.

[0348] Screening of single B cell cultures On days 9-12 after sorting, 15 μL of B cell culture supernatant was collected for antigen-specific ELISA assay as described above. On days 10-13 after sorting, the B cell culture plates were centrifuged at 400×g for 3 min. Supernatants from positive clones (OD greater than cutoff 0.3 or cutoff 0.9 from antigen-specific ELISA) were selected and subjected to PD-1:PD-L1 cELISA (above). Supernatants from positive clones were also subjected to cell binding assay. Briefly, first, MDA-MB-231 cells (known to express human PD-L1) were plated in 96-well plates (1×10 5 Cells were added to 100 μl of B cell culture (100 μl / well) of undiluted B cell culture supernatant sample and incubated with the cells for 30 min on ice. After washing with FACS buffer, 100 μl of 1 μg / ml Alex Fluor 647-conjugated goat anti-rabbit IgG was added for B cell culture detection and incubated for 30 min on ice. Cells were washed twice and then resuspended in 100 μl of FACS buffer. 100 μl of 1:50 diluted 7-AAD was added to the cell suspension for live / dead cell staining and ready for FACS analysis (BD Accuri C6 plus or LSR II flow cytometer, HTS). Purified rabbit anti-hPD-L1 clone 8C6 (Yurogen) was used as a positive control. Anti-HEL.hIgG1.FES or rabbit IgG format was used as an isotype control.

[0349] Ten to thirteen days after sorting, cells of cELISA+FACS+ clones were collected, stored in 100 μL DNA / RNA conservation tubes (Zymo, catalog number R1100-250), and transferred to 250 μL PCR tubes.

[0350] V H and V L Cloning (or sequencing) of the gene encoding Total RNA from selected clones was purified from cell pellets stored in DNA / RNA protection tubes using the RNeasy Mini Kit (Zymo, Cat. No. R1051) according to the manufacturer's protocol. 36 μl of nuclease-free water (Ambion, Cat. No. AM9937) was used to elute the total RNA. 11 μl of total RNA from each clone was mixed with 1 μl of oligo(dT)12-18 primer (Invitrogen, Cat. No. 58862) and 1 μl of dNTPs (10 mM, ThermoScientific, Cat. No. R0182) and then heated at 65°C for 5 min. To each clone mixture, 4 μl of 5× FirstStrand buffer, 1 μl of 100 mM DTT, 1 μl of RNAaseOUT (Invitrogen, Cat. No. 10777-019) and 1 μl of SuperScript III reverse transcriptase (Invitrogen, Cat. No. 18080-044) were then added. Reverse transcription reactions were carried out at 50° C. for 1 hour and 75° C. for 15 minutes to inactivate the SuperScript III enzyme. After cDNA synthesis, the VH and VL genes were amplified separately with the VH and VL variable region primer pairs using Go-Taq enzyme mix or Kapa HIFI HotStart enzyme. The VH PCR reaction contained 5 μl of cDNA template, 0.5 μl of VH 5' nested primer (10 μm), 0.5 μl of VH 3' nested primer (10 μm), 2.5 μl of 10× PCR buffer, 1 μl of 50 mM MgCl2, 0.2 μl of Taq DNA polymerase (ThermoFisher, Cat. No. 10342053) and 15.5 μl of nuclease-free water, and the VH PCR conditions were 94°C for 3 min, followed by 40 cycles (94°C for 30 s, 55°C for 30 s, 72°C for 45 s) and a final extension step at 72°C for 5 min. The VL PCR reaction contained 5 μl of cDNA template, 0.75 μl of VL 5' nested primer (10 μm), 0.75 μl of VL 3' nested primer (10 μm), 2.5 μl of 10x PCR buffer, 1 μl of 50 mM MgCl2, 0.2 μl of Platinum TMThe incubation conditions included Taq DNA polymerase (ThermoFisher, Cat. No. 10966018) and 15 μl of nuclease-free water. The VL PCR conditions were 98°C for 3 min, followed by 40 cycles (95°C for 15 s, 64°C for 15 s, 72°C for 30 s) and a final extension step at 72°C for 5 min. The VH and VL PCR products were separated on a 1% agarose gel electrophoresis system. The expected size of the VH and VL amplicons is ~500 bp. The corresponding bands of VH and VL for each clone were excised from the gel, and VH and VL were extracted from the gel using NucleoSpin® Gel and PCR Clean-Up Kit (Macherey-Nagel, Cat. No. 740609.250) according to the manufacturer's protocol. VH and VL PCR products were eluted using 12-30 μl of elution buffer depending on the amount of PCR product. Linear expression module cassette (LEM) PCR products were constructed by overlap PCR of a C fragment containing the CMV promoter, VH or VL, and a rabbit IgG heavy chain CH1 fragment, human IgG1 CH2 and CH3 fragments, or an H fragment containing the light chain constant region followed by the SV40 transcription terminator and polyA signal sequence. The LEM heavy chain PCR reaction contained 20 ng (1-5 μl) VH fragment, 1 μl CH_RK fragment (10 ng / μl), 1 μl HH_RK_hIgG1Fc fragment (10 ng / μl), 1 μl 5' primer C fragment 3 (10 μM), 1 μl 3' primer H fragment 4 (10 μM), 10 μl 5× LongAmpTaq reaction buffer, 1.5 μl dNTPs (10 mM), 2 μl LongAmp Taq DNA polymerase (New England BioLabs, catalogue no. E5200S) and 31.5 μl nuclease-free water.The LEM light chain PCR reaction contained 20 ng (1-5 μl) of VL fragment, 1 μl of CL_RK fragment (10 ng / μl), 1 μl of HL_RK fragment (10 ng / μl), 1 μl of 5' primer C fragment 3 (10 μM), 1 μl of 3' primer H fragment 4 (10 μM), 10 μl of 5× LongAmp Taq reaction buffer, 1.5 μl of dNTPs (10 mM), 2 μl of LongAmp Taq DNA polymerase, and 31.5 μl of nuclease-free water. The PCR cycle conditions were 94°C for 3 min, repeated for 35 cycles (94°C for 30 s, 55°C for 40 s, 68°C for 3 min and 30 s), and a final cycle of 68°C for 5 min. 5 μl of PCR product was used to detect the size and yield of the amplified fragment by 0.8% agarose gel electrophoresis (shown below). The remaining PCR product was purified using NucleoSpin® Gel and PCR Clean-Up Kit according to the manufacturer's protocol. LEM PCR product was eluted using 32 μl of elution buffer. Sequencing of LEM constructs was performed by Quintara Bioscience (Quintara, Cambridge, MA).

[0351] Screening for LEM transfection Cell binding assays (as described above) were performed on the supernatant samples obtained after LEM transfection. Briefly, 50 μl of LEM transfection supernatant samples (undiluted or diluted 1:5, 1:10 or 1:50) were incubated with cells for 30 min on ice. After washing with FACS buffer, 100 μl of 1 μg / ml Alex Fluor 647-conjugated anti-human IgG was added and incubated for 30 min on ice. Cells were resuspended in 100 μl of FACS buffer after two washes. 100 μl of 1:50 diluted 7-AAD was added to the cell suspension for live / dead cell staining and ready for FACS analysis (BD Accuri C6 plus or LSR II flow cytometer, HTS). Durvalumab was used as a positive control for LEM transfection supernatant samples. Anti-HEL.hIgG1.FES or rabbit IgG format (BioIntron) were used as isotype controls.

[0352] Example 2: Cloning and expression of genes encoding antibody variable regions of anti-PD-L1 antibodies The VH and VL coding genes of each clone were cloned into Yurogen's expression vector consisting of human IgG1 Fc and rabbit CH1. After sequence confirmation, the IgG heavy and light chain plasmids were used for small-scale antibody production in HEK293T cells. Briefly, ∼5 × 10 6HEK293T cells were seeded in a 12-well plate. For each clone, 500ng of heavy chain LEM PCR product and 500ng of light chain LEM PCR product were diluted in 45μL of Opti-MEM medium (Gibco, Cat. No. 31985-070). 3μL of Lipofectamine 2000 reagent (Invitrogen, Cat. No. 11668019) was diluted in 47μL of Opti-MEM medium. The diluted LEM PCR product and Lipofectamine were mixed and incubated at room temperature for 10 minutes. The HEK293T medium was replaced with 1mL of Opti-MEM medium. The mixed solution was added dropwise to each well. The plate was swirled gently to thoroughly mix the transfection reagent and medium. The transfected cells were incubated at 37°C, 5% CO2 for 48-72 hours, and then the supernatant was collected for further screening.

[0353] Example 3: Specific binding of anti-PD-L1 antibodies to PD-L1 Binding specificity The purified antibodies were tested by FACS to confirm the binding and affinity to human and cynomolgus PD-L1-His recombinant proteins. Briefly, Maxisorp plates (Nunc) were coated with 0.5μg / ml human PD-L1-His (Sino Biological, Catalog No. 10084-H08H) or cynomolgus PD-L1-His (Sino Biological, Catalog No. 90251-C08H) protein diluted in 1xPBS (GIBCO) at 100ul / well overnight at 4°C. The plates were washed three times with 300μl / well washing buffer (1xPBST, 20xPBST (Thermo Scientific) diluted with distilled water) and then blocked with 300μl / well blocking buffer (1xPBST / 1%BSA) for 2 hours at room temperature on a shaker (100rpm). Plates were washed and test antibodies serially diluted in assay buffer (PBST / 1% BSA) were added at 100 μl / well. Antibodies were incubated for 2 hours at room temperature on a shaker at 100 rpm. Plates were washed and 100 μL of horseradish peroxidase (HRP)-conjugated detection antibody was added to each well and incubated for 1 hour at room temperature on a shaker at 100 rpm. Plates were washed and TMB substrate was added at 100 μl / well and developed for 10 minutes at room temperature in the dark. Stop solution (1N HSO 4000) was added at 50 μl / well. 4) After addition, data values ​​were read at 450 nm on a plate reader (SpectraMax M5).

[0354] Table 21 shows that the exemplary antibodies of the invention specifically bound to both human and cynomolgus PD-L1. Durvalumab (AstraZeneca) was used as a positive control. Anti-HEL isotype control (BioIntron) was used as a negative control.

[0355] [Table 21]

[0356] Similar assays were performed to test whether exemplary antibodies of the disclosure bind to mouse PD-L1 (Sino Biological, Catalog No. 50010-M08H), rat PD-L1 (Sino Biological, Catalog No. 80450-R08H), dog PD-L1 (Acro Biosystems, Catalog No. PDL-C52H4) or rabbit PD-L1 (Acro Biosystems, Catalog No. PDL-R52H6). No binding of exemplary antibodies of the disclosure to mouse, rat, dog or rabbit PD-L1 was detected.

[0357] Similar assays were performed to test whether the exemplary antibodies of the disclosure bind to other proteins in the same family as PD-L1. No binding of the exemplary antibodies of the disclosure to CD80 (Sino Biological, Catalog No. 10698-H08H), CD86 (Sino Biological, Catalog No. 10699-H08H), BH-H2 (Sino Biological, Catalog No. 11559-H08H), B7-H3 (Sino Biological, Catalog No. 11188-H08H) and PD-L2 (Sino Biological, Catalog No. 10292-H08H) was detected.

[0358] binding affinity The binding affinity of exemplary anti-PD-L1 antibodies of the present disclosure was measured. The assay was performed according to the protocol recommended by Gator or Gator Plus (Gator Bio Inc). Briefly, human PD-L1-His (Sino Biological, Catalog No. 10084-H08H) was serially diluted 4-fold starting at 4μg / ml. After regenerating three times with Refinement Buffer (Gator Bio, Catalog No. 120012) and neutralizing the HFC probe (Gator Bio, Catalog No. 160003), 1μg / ml of the antibody to be tested was immobilized on the HFC probe in Q Buffer (Gator Bio, Catalog No. 120010). After a baseline step, the antibody-immobilized probe was immersed in the antigen solution. Antigen-antibody binding and dissociation were measured with a Gator BLI machine. Affinities were calculated using Gator Bio analysis software.

[0359] Table 22 summarizes the binding affinity to human PD-L1 using Gator. Durvalumab (AstraZeneca) was used as a positive control. Anti-HEL isotype control (BioIntron) was used as a negative control.

[0360] [Table 22]

[0361] [Table 23]

[0362] Example 4: Specific binding of anti-PD-L1 antibodies to PD-L1-expressing cells The assay described in this Example is designed to determine whether the anti-PD-L1 antibodies of the disclosure specifically bind to PD-L1-expressing cells.

[0363] In this assay, MDA-MB-231-GFP cells (known to express human PD-L1) were harvested from fresh culture. The cells were then cultured at 10 6 The cells were resuspended in FACS buffer (1xDPBS / 1%FBS) at a concentration of 1:100 / ml. 100μl of cells were dispensed per well of a 96-well plate. Antibodies to be tested were serially diluted 1:3 starting at 15ug / mL and then added to each well. The antibodies were mixed with the cells and the mixture was incubated on ice for 1 hour. After washing with FACS buffer, 100μl of diluted PE-conjugated anti-human Fc secondary antibody (Jackson Immuno Research, Cat. No. 109-116-098) was added to each well and incubated on ice for 30 minutes. After washing with FACS buffer, the stained cells were resuspended in diluted 7-ADD (Biolegend, Cat. No. 420404, diluted 1:100) and analyzed on a Guava 11HT (Luminex). Data was analyzed using Flowjo software.

[0364] Table 24 shows that exemplary antibodies of the disclosure specifically bound to PD-L1 expressed on the surface of MDA-MD-231-GFP cells. Durvalumab (AstraZeneca) was used as a positive control. Anti-HEL isotype control (BioIntron) was used as a negative control.

[0365] [Table 24]

[0366] Example 5: Inhibition of PD-1 / PD-L1 interaction by anti-PD-L1 antibodies The assay in this example determines whether an anti-PD-L1 antibody of the disclosure can inhibit PD-1 / PD-L1 interaction.

[0367] cELISA assay A competitive ELISA (cELISA) assay was used to determine the blockade of PD-1 / PD-L1 interaction by the exemplary anti-PD-L1 antibodies of the present disclosure. Briefly, microplates were pre-coated with 1 μg / μl human PD-L1-mFc fusion protein (Sino Biological, Cat. No. 10084-H05H) diluted in 1 x PBS (GIBCO) and incubated overnight at 4°C. The plates were washed four times with 300 μl / well washing buffer (1xPBST, 20xPBST (Thermo Scientific) diluted with distilled water), then 300 μl / well blocking buffer (1xPBST / 3%BSA) was added and blocked for 2 hours at room temperature on a shaker (100 rpm). Plates were washed and 100 μl / well of the following was added: (1) 1 μg / ml human PD-1-His-hFc-biotin fusion protein (Sino Biological, cat. no. 10377-H03H-B) alone; (2) human PD-1-His-hFc-biotin fusion protein mixed with BAVENCIO (avelumab) or Imfinzi (durvalumab) or Anti-HEL isotype control antibody (BioIntron) (12-point 1:2-fold serial dilutions starting at 50 nM); or (3) human PD-1-His-hFc-biotin fusion protein mixed with test anti-PD-L1 antibodies (12-point 1:2-fold serial dilutions starting at 50 nM). HRP-conjugated avidin (Invitrogen, Cat. No. A2664) diluted 1:20,000 was added to each well at 100 μl / well and incubated for 1 h at room temperature on a shaker at 100 rpm. Plates were washed and TMB substrate was added at 100 μl / well and developed for 10 min at room temperature in the dark. Stop solution (1N HSO) was added at 50 μl / well. 4) After addition, the plate was read at 450 nm in a plate reader (SpectraMax M5).

[0368] Table 25 shows the IC of exemplary antibodies of the disclosure that block the interaction of PD-1 with PD-L1. 50The values ​​are summarized below. As positive controls, commercially available anti-PD-L1 antibodies Imfinzi (durvalumab) and Bavencio (avelumab) were used. As shown in Table 25 and Figure 1, the exemplary anti-PD-L1 antibodies of the present disclosure could effectively block the interaction between PD-1 and PD-L1.

[0369] [Table 25]

[0370] Promega Assay The assay uses a PD1 / PD-L1 Blocking Assay Kit (Promega, Cat. No. J1255) to measure whether an exemplary antibody of the invention can block the interaction of PD1 with cell surface PD-L1. This kit can also be used to measure the potency of an antibody to block PD1 / PD-L1 interaction. The assay was performed according to the manufacturer's manual. Briefly, 100μl of CHO-K1-hPD-L1 cell suspension in F-12 medium containing 10% FBS was added to each well of a cell culture treated flat bottom white 96-plate and cultured at 37°C in a CO2 incubator for 16 hours. Antibodies to be tested or the positive control commercial anti-PD-L1 antibodies Imfinzi (Durvalumab), Bavencio (Avelumab), Atezolizumab analog (BioIntron), or SGN-PD-L1 analog anti-PD-L1 antibody (BioIntron) (or Anti-HEL isotype control antibody) were serially diluted in 1xRPMI / 1%FBS. After removing the overnight culture medium, 40μl of each diluted antibody was added to each well, followed by 40μl of effector PD-L1 cell suspension to each well. The plates were incubated at 37°C in a CO2 incubator for 6 hours. Then, 80μl of BIO-Glo reagent was added to each well, and the plates were immediately processed to measure the luminescence signal on a plate reader (SpectraMax M5).

[0371] Table 26 shows the IC of exemplary antibodies of the disclosure that block the interaction of PD-1 with cell surface PD-L1. 50 The values ​​are summarized in Table 26. Durvalumab was used as a positive control. As shown in Table 26, the exemplary anti-PD-L1 antibodies of the present disclosure could effectively block the interaction between PD-1 and cell surface PD-L1.

[0372] [Table 26]

[0373] Table 27 shows the IC of exemplary antibodies of the disclosure for blocking the interaction of PD-1 with cell surface PD-L1 in a separate assay. 50 The values ​​are summarized in Table 27. Durvalumab, avelumab, atezolizumab analog, and SGN-PD-L1 analog were used as positive controls. All antibodies were tested in duplicate wells, except for avelumab, which was tested in quintuplicate wells. As shown in Table 27, the exemplary anti-PD-L1 antibodies of the present disclosure were able to effectively block the interaction between PD-1 and cell surface PD-L1.

[0374] [Table 27]

[0375] Example 6: ADCC of anti-PD-L1 antibodies The antibody-dependent cellular cytotoxicity (ADCC) of exemplary anti-PD-L1 antibodies of the disclosure was measured in this experiment.

[0376] InvivoGen assay using PD-L1 expressing Raji cells NFAT-CD16 Lucia luciferase reporter Jurkat cells and PD-L1 expressing Raji cells were purchased from InvivoGen and expanded for the assay. First, 20μl of the antibody to be tested was added to each well of a flat-bottom 96-well cell culture plate. Then, 90μl of Raji-PD-L1 cells (~100,000 cells) were added to each well. The plate was incubated at 37°C in a CO2 incubator for 1 hour, after which 90μl of Jurkat-Lucia TM -NFAT-CD16 cells (-200,000 cells) were added to each well and the plate was incubated at 37°C in a CO2 incubator for 6 hours. TM 20 μl of NFAT CD16 cell culture supernatant was transferred to each well of a 96-well white (opaque, Corning) plate, followed by 50 μl of QUANTI-Luc TM Substrate (InvivoGen: prepare in advance according to the vendor's instructions) was added to each well. The plate was immediately processed to measure the luminescence signal in a plate reader (SpectraMax M5). Anti-HEL isotype control antibody was used as a negative control.

[0377] Table 28 shows the ADCC EC values ​​of exemplary anti-PD-L1 antibodies of the disclosure. 50 This is a summary of the above.

[0378] [Table 28]

[0379] InvivoGen Assay with MDA-MB-231 GFP Cells This assay is similar to the InvivoGen assay above, except that Raji-PD-L1 cells were replaced with MDA-MB-231 GFP cells. Avelumab and durvalumab were used as positive controls. Anti-HEL isotype control antibody was used as negative control. Table 29 shows the EC of ADCC of exemplary anti-PD-L1 antibodies of the disclosure. 50 It is a collection of values.

[0380] [Table 29]

[0381] Example 7: Anti-PD-L1 antibodies and internalization of PD-L1 This example measures the internalization of a complex formed by an exemplary anti-PD-L1 antibody of the disclosure and PD-L1 expressed on the cell surface.

[0382] FACS-based internalization assay Approximately 100,000 (~10 5) MDB-MA-231 cells were incubated on ice with the antibodies to be tested at the indicated concentrations (2, 1, 0.5 and 0.25μg / mL). As a positive control, the commercially available ADC drug Trodelvy (sacituzumab govitecan-hziy, Immunomedics) was used. The internalization of the commercially available anti-PD-L1 antibodies durvalumab and avelumab was also tested. After 1 hour of incubation, two aliquots were removed from the mixture and transferred to a 37°C incubator for 2 or 4 hours of incubation, respectively. Samples taken at different time points were washed twice with cold FACS buffer and then stained with PE-conjugated anti-human Fc secondary antibody (Jackson Immuno Research) on ice for 1 hour. After washing twice with cold FACS buffer, cells were stained with 7-AAD (Biolegend) and then fixed with 2% paraformaldehyde (diluted from 10% paraformaldehyde (MilliporeSigma, Cat. No. R04586)). MDB-MA-231 cells are known to express both PD-L1 and Trop2. Sacituzumab is known to be an anti-Trop2 antibody that induces efficient internalization upon binding to cell surface Trop2.

[0383] The samples were then analyzed on a Guava 11HT (Luminex) and data analysis was performed with Flowjo software. The internalization rate was calculated as follows:

[0384] % Internalization = {[(MFI of sample on ice)-(MFI of sample at specified incubation time at 37 °C)] / (MFI of sample on ice)} x 100

[0385] Table 30 summarizes the internalization of complexes formed by different anti-PD-L1 antibodies and PD-L1 expressed on the surface of MDA-MB-231 cells. Some results show that the exemplary anti-PD-L1 antibodies induced internalization upon binding to cell surface PD-L1.

[0386] [Table 30]

[0387] Figure 2A shows that incubation of MDA-MB-231 cells with 1 μg / ml 33E8 for 4 hours at 37°C induced the highest level of internalization (shown as percentage of internalization) compared to all other anti-PD-L1 antibodies, including durvalumab. 32E2 or avelumab induced the lowest level of internalization. Figure 2B shows that incubation of MDA-MB-231-GFP cells with 1 μg / ml 33E8 for 1 hour, 2 hours, and 4 hours induced higher levels of internalization (shown as percentage of internalization) compared to all other anti-PD-L1 antibodies, including avelumab, durvalumab, atezolizumab analog (BioIntron), and SGN-PDL1 analog (BioIntron).

[0388] Dye-based internalization assay Invitrogen TM Zenon TM pHrodo TM The internalization properties of exemplary anti-PD-L1 antibodies were determined using the iFL RED human IgG labeling reagent (Cat. No. Z25612) according to the manufacturer's protocol. TM pHrodo TM Internalization of antibodies conjugated with the iFL RED human IgG labeling reagent increases the fluorescent signal in low pH intracellular compartments within the cell, providing a way by which internalization can be visualized.

[0389] Briefly, 4x standard solutions (24μg / ml) of each antibody were prepared in complete DMEM medium (10% FBS, 1xPen / Strep). 25μL of the 4x antibody standard solution was dispensed into each well of a clear-bottom 96-well plate. 25μL of the 4x Zenon standard solution was then added to the wells containing the antibody. The mixture of antibody and Zenon solution was incubated at room temperature for 5 minutes to allow the formation of the labeling complex, and then 2x10 6 50 μL of human PD-L1-expressing Raji cells at 1000 μg / mL were added to the wells. Cells were incubated with the labeled complex under standard cell culture conditions. After 4 to 24 h of incubation, cells were analyzed under an immunofluorescence microscope (Invitrogen EVOS Cell Imaging System M5000) to visualize the internalization of the antibody by the cells. As shown in Figure 2C, the induction of internalization can be visualized in the representative photographs, which show that internalization is induced by 33E8 and SGN-PDL1 analog antibodies, but not by Anti-HEL isotype control antibody. Hoechst 33342 (Invitrogen, Cat. No. H3570), a common cell-permeable nuclear counterstain that fluoresces blue, was used to distinguish condensed nuclei within the cells.

[0390] Example 8: Cytotoxicity of anti-PD-L1 antibodies conjugated to cytotoxic agents This study investigated whether exemplary antibodies of the disclosure conjugated to a cytotoxic agent could induce the killing of PD-L1 expressing cells. In this study, the cytotoxic agent deruxtecan was conjugated to an exemplary antibody of the disclosure via an anti-hFc cross-linking antibody that specifically binds to the Fc region of the antibody.

[0391] Briefly, Raji cells (InvivoGen) expressing PD-L1 were dispensed in 160 μL of culture medium into opaque-walled 96-well plates. Antibody conjugates were prepared by mixing 40 μL of anti-hFc antibody (Biolegend, Cat. No. 410701) conjugated with deruxtecan (MedKoo Bioscience, Cat. No. 2071060) via a stable thiol ether bond (made by CellMosaic Inc.) with: (1) an anti-PD-L1 antibody according to the disclosure, wherein the tested Fc region is human Fc (hFc), at 500 nM; (2) 500 nM of the commercially available anti-PD-L1 antibody Imfinzi (durvalumab) (3) 500 nM of the anti-PD-L1 antibody atezolizumab or an analog of SGN-PDL1 (in Figure 3, data for analog antibodies are indicated with *); (4) 500 nM of anti-HEL isotype control antibody, or (5) Medium only.

[0392] The antibody conjugate was divided into two aliquots and added to the wells containing Raji cells. The cells mixed with the antibody conjugate were incubated at 37°C, 5% CO2 for 3 days. The microtubule polymerization inhibitor colchicine, 5 μM, was used as a positive cytotoxicity control. After 3 days of incubation, 90 ul of the supernatant was removed from each well, and 100 μl of fresh CellTiter-Glo (Promega, Cat. No. G7571) was added to each well of the 96-well plate. The 96-well plate was then placed on an orbital shaker and shaken at 120 rpm for 2 minutes to induce cell lysis. The cell lysate was incubated at room temperature for 10 minutes to stabilize the luminescence signal, after which the luminescence signal was measured with a plate reader (SpectraMax5) to examine cell viability. There was a direct relationship between the intensity of the luminescence signal and the number of viable cells in each well. The results are shown in Figure 3.

[0393] Example 9: Epitope binning of anti-PD-L1 antibodies This study investigated whether certain exemplary antibodies of the present invention belong to the same epitope bin as commercially available avelumab, i.e., whether certain exemplary antibodies of the present disclosure bind to the same or similar epitopes on the PD-L1 protein as avelumab.

[0394] A competitive ELISA (cELISA) assay was used to determine whether the exemplary anti-PD-L1 antibodies of the present disclosure belong to the same epitope bin as avelumab. Briefly, NUNC Maxisorp flat-bottom 96-well microplates were pre-coated with 0.5 μg / μl recombinant human PD-L1-His protein (Sino Biologicals, Cat. No. 10084-H08H) and 1xPBS (GIBCO) and incubated overnight at 4°C. The plates were washed 4 times with 300 μl / well of washing buffer (1xPBST, 20xPBST (Thermo Scientific) diluted with distilled water) and then blocked for 2 hours at room temperature on a shaker (100 rpm) by adding 300 μl / well of blocking buffer (1xPBST / 3%BSA). The plates were washed and 50 μl / well of diluted anti-PD-L1 antibodies to be tested (starting at 20 μg / ml, followed by 11-point 1:2-fold serial dilutions) were added. Plates containing the anti-PD-L1 antibody solution were incubated at 37°C for 1 hour, then 50 μl / well of 0.4 μg / ml biotinylated avelumab (Thermo Scientific TM EZ-Link TM Micro Sulfo-NHS-LC-Biotinylation Kit, Cat. No. 0021935; prepared according to the manufacturer's manual). The plate containing the mixture was incubated at 37°C for 1 h. HRP-conjugated avidin (Invitrogen, Cat. No. A2664) was diluted 1:2000 for detection. Avelumab was used as a positive control.

[0395] Figures 4A and 4B show that exemplary antibodies 27A2, 56H3, 33H7, 28H3, 74A9, durvalumab, avelumab and avelumab analogs, atezolizumab, and SGN-PDL1 antibody were able to effectively block the binding of biotinylated avelumab to human PD-L1-His protein, indicating that these antibodies belong to the same epitope. Antibodies 32E2 and 33E8 only effectively blocked the binding of avelumab to PD-L1 at concentrations of 5 μg / ml or higher, indicating that these antibodies do not belong to the same epitope as avelumab, but that their epitopes may be related. Antibodies 55H3 / 76D6, 56E5, 57B10, 57F11, 62E6, 69D10, 73C8, and 74F4 of the present disclosure did not demonstrate blocking of the binding of avelumab to the PD-L1 protein under experimental conditions (data not shown), indicating that these antibodies belong to a different epitope than avelumab.

[0396] The above description is intended to teach those skilled in the art how to practice the invention and is not intended to detail all obvious modifications and variations of the invention that would become apparent to those skilled in the art upon reading the description. However, all such obvious modifications and variations are intended to be included within the scope of the invention as defined by the following claims. The claims are intended to cover the recited elements and steps in any order that is effective to achieve the intended purpose, unless the context specifically indicates to the contrary.

Claims

1. From the N-terminus to the C-terminus, the heavy chain variable (V) domain contains three heavy chain complementarity determining regions (CDRs): HCDR1, HCDR2, and HCDR3. Ｈ ) domain, From the N-terminus to the C-terminus, the light chain variable (V) domain contains three light chain complementarity determining regions (CDRs): LCDR1, LCDR2, and LCDR3. Ｌ ) domain, 1. An isolated antibody or antigen-binding fragment thereof that binds to human programmed cell death ligand 1 (PD-L1), (a) HCDR1 comprises an amino acid sequence selected from the group consisting of: X1-N-H-Y-M-X2 (SEQ ID NO:), X3-X4-Y-Y-M-C (SEQ ID NO:), N-N-Y-Y-M-S (SEQ ID NO:8), R-Y-F-Y-M-S (SEQ ID NO:11), and S-A-Y-W-I-C (SEQ ID NO:12), where X1 is N or S, X2 is C or S, X3 is A, N or S, and X4 is A, N or S; (b) HCDR2 comprises the amino acid sequence set forth in: X32-I-X33-X34-GS-X35-I-X36-D-Y-A-X37-W-A-K-G (SEQ ID NO:), wherein X32 is C or S, X33 is G or S, X34 is I, T or V, X35 is A, D, G or Y, X36 is S or T, and X37 is N or S; (c) HCDR3 comprises the amino acid sequence set forth in WTSGGGGFGL (SEQ ID NO: 42); (d) LCDR1 comprises an amino acid sequence selected from the group consisting of Q-S-S-Q-X65-X66-Y-X67-X68-Y-L-X69 (SEQ ID NO:), Q-S-S-Q-X70-I-Y-S-D-Y-L-X71 (SEQ ID NO:), and Q-S-S-Q-S-X72-Y-X73-N-Y-L-X74 (SEQ ID NO:), wherein X65 is N, S or T, X66 is I or V, X67 is N or S, X68 is D or N, X69 is A, C, F or S, X70 is N or T, X71 is F or S, X72 is I or V, X73 is N or S, and X74 is A, C or S; (e) LCDR2 comprises an amino acid sequence selected from the group consisting of: X98-X99-X100-T-L-A-S (SEQ ID NO:), X101-X102-S-T-L-A-S (SEQ ID NO:), and S-T-A-T-L-A-S (SEQ ID NO:72), where X98 is D, G, Q, S or Y, X99 is A or T, X100 is A or S, X101 is D, G, Q or Y, and X102 is A or T; (f) An isolated antibody or antigen-binding fragment thereof, wherein LCDR3 comprises the amino acid sequence shown in QGYY-SGYI-WT (sequence number 83).

2. (a) HCDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-12; (b) HCDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 22-31; (c) HCDR3 comprises the amino acid sequence set forth in SEQ ID NO:42; (d) LCDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 53-59; (e) LCDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 69-73; (f) The isolated antibody or antigen-binding fragment thereof of claim 1, wherein the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO:

83.

3. The antibody, (a) an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO:1, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO:22, an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO:42, an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO:53, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO:69, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO:83; (b) HCDR1 comprising the amino acid sequence set forth in SEQ ID NO:2, HCDR2 comprising the amino acid sequence set forth in SEQ ID NO:23, HCDR3 comprising the amino acid sequence set forth in SEQ ID NO:42, LCDR1 comprising the amino acid sequence set forth in SEQ ID NO:53, LCDR2 comprising the amino acid sequence set forth in SEQ ID NO:69, and LCDR3 comprising the amino acid sequence set forth in SEQ ID NO:83; (c) an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO:3, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO:23, an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO:42, an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO:54, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO:70, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO:83; (d) an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO:4, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO:24, an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO:42, an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO:55, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO:71, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO:83; (e) HCDR1 comprising the amino acid sequence set forth in SEQ ID NO:5, HCDR2 comprising the amino acid sequence set forth in SEQ ID NO:25, HCDR3 comprising the amino acid sequence set forth in SEQ ID NO:42, LCDR1 comprising the amino acid sequence set forth in SEQ ID NO:56, LCDR2 comprising the amino acid sequence set forth in SEQ ID NO:69, and LCDR3 comprising the amino acid sequence set forth in SEQ ID NO:83; (f) an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO:6, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO:26, an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO:42, an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO:57, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO:71, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO:83; (g) HCDR1 comprising the amino acid sequence set forth in SEQ ID NO:7, HCDR2 comprising the amino acid sequence set forth in SEQ ID NO:27, HCDR3 comprising the amino acid sequence set forth in SEQ ID NO:42, LCDR1 comprising the amino acid sequence set forth in SEQ ID NO:56, LCDR2 comprising the amino acid sequence set forth in SEQ ID NO:69, and LCDR3 comprising the amino acid sequence set forth in SEQ ID NO:83; (h) HCDR1 comprising the amino acid sequence shown in SEQ ID NO:8, HCDR2 comprising the amino acid sequence shown in SEQ ID NO:28, HCDR3 comprising the amino acid sequence shown in SEQ ID NO:42, LCDR1 comprising the amino acid sequence shown in SEQ ID NO:53, LCDR2 comprising the amino acid sequence shown in SEQ ID NO:69, and LCDR3 comprising the amino acid sequence shown in SEQ ID NO:83; (i) HCDR1 comprising the amino acid sequence set forth in SEQ ID NO:9, HCDR2 comprising the amino acid sequence set forth in SEQ ID NO:27, HCDR3 comprising the amino acid sequence set forth in SEQ ID NO:42, LCDR1 comprising the amino acid sequence set forth in SEQ ID NO:53, LCDR2 comprising the amino acid sequence set forth in SEQ ID NO:69, and LCDR3 comprising the amino acid sequence set forth in SEQ ID NO:83; (j) HCDR1 comprising the amino acid sequence shown in SEQ ID NO:5, HCDR2 comprising the amino acid sequence shown in SEQ ID NO:29, HCDR3 comprising the amino acid sequence shown in SEQ ID NO:42, LCDR1 comprising the amino acid sequence shown in SEQ ID NO:53, LCDR2 comprising the amino acid sequence shown in SEQ ID NO:69, and LCDR3 comprising the amino acid sequence shown in SEQ ID NO:83; (k) an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 30, an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 42, an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 58, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 72, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 83, or (l) The isolated antibody or antigen-binding fragment thereof of claim 2, comprising an HCDR1 having an amino acid sequence set forth in SEQ ID NO: 12, an HCDR2 having an amino acid sequence set forth in SEQ ID NO: 31, an HCDR3 having an amino acid sequence set forth in SEQ ID NO: 42, an LCDR1 having an amino acid sequence set forth in SEQ ID NO: 59, an LCDR2 having an amino acid sequence set forth in SEQ ID NO: 73, and an LCDR3 having an amino acid sequence set forth in SEQ ID NO:

83.

4. The antibody, (a) a heavy chain variable region (HCVR) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 92-103; (b) a light chain variable region (LCVR) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 118-129.

5. The antibody, (a) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 92, and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 118; (b) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 93, and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 119; (c) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 94, and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 120; (d) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 95, and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 121; (e) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 96, and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 122; (f) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 97, and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 123; (g) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 98, and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 124; (h) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 99, and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 125; (i) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 100, and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 126; (j) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 101, and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 127; (k) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 102, and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 128; or (l) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 103, and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 129, the isolated antibody or antigen-binding fragment thereof of claim 4.

6. a heavy chain variable (VH) domain comprising, from N-terminus to C-terminus, three heavy chain complementarity determining regions (CDRs): HCDR1, HCDR2, and HCDR3; a light chain variable (VL) domain comprising, from N-terminus to C-terminus, three light chain complementarity determining regions (CDRs): LCDR1, LCDR2, and LCDR3; An isolated antibody or antigen-binding fragment thereof that binds to human PD-L1, (a) HCDR1 comprises an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-12; (b) HCDR2 comprises an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 22-31; (c) HCDR3 comprises an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identity to the amino acid sequence set forth in SEQ ID NO:42; (d) LCDR1 comprises an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 53-59; (e) LCDR2 comprises an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 69-73; (f) An isolated antibody or antigen-binding fragment thereof, wherein LCDR3 comprises an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identity to the amino acid sequence set forth in SEQ ID NO:

83.

7. An isolated antibody or antigen-binding fragment thereof that binds to human PD-L1, (a) a heavy chain variable region (HCVR) comprising an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 92-103; (b) an isolated antibody or antigen-binding fragment thereof, comprising a light chain variable region (LCVR) comprising an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 118-129.

8. From the N-terminus to the C-terminus, the heavy chain variable (V) domain contains three heavy chain complementarity determining regions (CDRs): HCDR1, HCDR2, and HCDR3. Ｈ ) domain, From the N-terminus to the C-terminus, the light chain variable (V) domain contains three light chain complementarity determining regions (CDRs): LCDR1, LCDR2, and LCDR3. Ｌ ) domain, 1. An isolated antibody or antigen-binding fragment thereof that binds to human programmed cell death ligand 1 (PD-L1), (a) HCDR1 comprises the amino acid sequence shown in X8-D-X9-Y-M-S (SEQ ID NO:), where X8 is D or G and X9 is W or Y; (b) HCDR2 comprises the amino acid sequence set forth in S-I-Y-X42-G-S-L-N-X43-Y-Y-A-T-W-A-K-G (SEQ ID NO:), wherein X42 is S or T and X43 is I, S or T; (c) HCDR3 comprises the amino acid sequence shown in R-X57-K-N-X58-D-X59-G-X60-F-D-L (SEQ ID NO:), where X57 is H, N or T, X58 is A or G, X59 is W or Y, and X60 is H or Y; (d) LCDR1 comprises an amino acid sequence selected from the group consisting of Q-A-S-X76-S-I-X77-X78-X79-L-A (SEQ ID NO: 67), Q-A-S-Q-S-I-X81-X82-A-L-A (SEQ ID NO: 68), where X76 is E or Q, X77 is G, N or S, X78 is D, N or T, X79 is A or R, X80 is E or Q, X81 is G or S, and X82 is N or T; (e) LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: X106-X107-S-T-L-A-S, wherein X106 is A or G, and X107 is A or T; (f) An isolated antibody or antigen-binding fragment thereof, wherein LCDR3 comprises the amino acid sequence set forth in QQ-G-WT-V-S-S-L-X111-N-A (sequence number) (wherein X111 is D or E).

9. (a) HCDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 19, 20, and 21; (b) HCDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 38, 39, 40, and 41; (c) HCDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 49, 50, 51, and 52; (d) LCDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 65, 66, 67, and 68; (e) LCDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 80, 71, and 82; (f) The isolated antibody or antigen-binding fragment thereof of claim 8, wherein LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 90 or 91.

10. The antibody, (a) an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 19, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 38, an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 49, an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 65, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 80, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 90; (b) HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 19, HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 39, HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 50, LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 66, LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 71, and LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 90; (c) HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 20, HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 39, HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 51, LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 67, LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 80, and LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 90; (d) an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 19, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 39, an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 52, an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 66, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 80, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 91; (e) HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 19, HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 40, HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 49, LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 66, LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 71, and LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 90; (f) an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO:21, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO:41, an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO:49, an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO:68, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO:82, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO:90; (g) an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 19, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 39, an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 49, an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 66, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 80, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 90, or (h) An isolated antibody or antigen-binding fragment thereof according to claim 9, comprising: an HCDR1 having an amino acid sequence set forth in SEQ ID NO: 19; an HCDR2 having an amino acid sequence set forth in SEQ ID NO: 39; an HCDR3 having an amino acid sequence set forth in SEQ ID NO: 49; an LCDR1 having an amino acid sequence set forth in SEQ ID NO: 66; an LCDR2 having an amino acid sequence set forth in SEQ ID NO: 71; and an LCDR3 having an amino acid sequence set forth in SEQ ID NO:

90.

11. The antibody, (a) a heavy chain variable region (HCVR) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 110-117; (b) a light chain variable region (LCVR) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 136-143.

12. The antibody, (a) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 110, and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 136; (b) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 111, and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 137; (c) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 112, and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 138; (d) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 113, and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 139; (e) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 114, and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 140; (f) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 115, and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 141; (g) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 116, and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 142; or (h) The isolated antibody or antigen-binding fragment thereof of claim 11, comprising an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 117, and an LCVR comprising the amino acid sequence set forth in SEQ ID NO:

143.

13. a heavy chain variable (VH) domain comprising, from N-terminus to C-terminus, three heavy chain complementarity determining regions (CDRs): HCDR1, HCDR2, and HCDR3; a light chain variable (VL) domain comprising, from N-terminus to C-terminus, three light chain complementarity determining regions (CDRs): LCDR1, LCDR2, and LCDR3; An isolated antibody or antigen-binding fragment thereof that binds to human PD-L1, (a) HCDR1 comprises an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 19, 20, and 21; (b) HCDR2 comprises an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 38, 39, 40, and 41; (c) HCDR3 comprises an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 49, 50, 51, and 52; (d) LCDR1 comprises an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 65, 66, 67, and 68; (e) LCDR2 comprises an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 80, 71, and 82; (f) An isolated antibody or antigen-binding fragment thereof, wherein LCDR3 comprises an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identity to the amino acid sequence set forth in SEQ ID NO: 90 or 91.

14. An isolated antibody or antigen-binding fragment thereof that binds to human PD-L1, (a) a heavy chain variable region (HCVR) comprising an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 110-117; (b) an isolated antibody or antigen-binding fragment thereof, comprising a light chain variable region (LCVR) comprising an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 136-143.

15. From the N-terminus to the C-terminus, the heavy chain variable (V) domain contains three heavy chain complementarity determining regions (CDRs): HCDR1, HCDR2, and HCDR3. Ｈ ) domain, From the N-terminus to the C-terminus, the light chain variable (V) domain contains three light chain complementarity determining regions (CDRs): LCDR1, LCDR2, and LCDR3. Ｌ ) domain, An isolated antibody or antigen-binding fragment thereof that binds to human PD-L1, (a) HCDR1 comprises an amino acid sequence selected from the group consisting of S-G-Y-D-M-S (SEQ ID NO: 15) and S-G-Y-D-M-C (SEQ ID NO: 16); (b) HCDR2 comprises the amino acid sequence set forth in: X38-I-F-T-X39-S-G-S-T-W-Y-A-N-W-A-K-G (SEQ ID NO:), wherein X38 is C or S and X39 is G or T; (c) HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: T-X51-D-G-X52-G-S-F-Y-M-N-L (wherein X51 is K or R and X52 is A or V); (d) LCDR1 comprises the amino acid sequence shown in Q-A-S-G-T-I-G-S-N-L-A (SEQ ID NO: 63) or Q-A-S-Q-T-I-G-S-N-L-A (SEQ ID NO: 62); (e) LCDR2 comprises the amino acid sequence set forth in K-A-F-T-L-A-S (SEQ ID NO: 76) or K-T-F-T-L-A-S (SEQ ID NO: 77); (f) An isolated antibody or antigen-binding fragment thereof, wherein LCDR3 comprises the amino acid sequence shown in QQ-G-A-T-R-I-N-I-D-N-A (sequence number 86) or QQ-G-A-S-R-I-N-I-D-N-A (sequence number 87).

16. (a) HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 34 or 35; (b) the isolated antibody or antigen-binding fragment thereof of claim 15, wherein HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 45 or 46.

17. (a) HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 15, HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 34, HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 45, LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 62, LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 76, and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 86, or (b) The isolated antibody or antigen-binding fragment thereof of claim 16, wherein HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 16, HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 35, HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 46, LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 63, LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 77, and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO:

87.

18. The antibody, (a) a heavy chain variable region (HCVR) comprising the amino acid sequence set forth in SEQ ID NO: 106 or 107; (b) a light chain variable region (LCVR) comprising the amino acid sequence set forth in SEQ ID NO: 132 or 133.

19. The antibody, (a) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 106, and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 132; or (b) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 107, and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 133;

20. a heavy chain variable (VH) domain comprising, from N-terminus to C-terminus, three heavy chain complementarity determining regions (CDRs): HCDR1, HCDR2, and HCDR3; a light chain variable (VL) domain comprising, from N-terminus to C-terminus, three light chain complementarity determining regions (CDRs): LCDR1, LCDR2, and LCDR3; An isolated antibody or antigen-binding fragment thereof that binds to human PD-L1, (a) HCDR1 comprises an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identity to the amino acid sequence set forth in SEQ ID NO: 15 or 16; (b) HCDR2 comprises an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identity to the amino acid sequence set forth in SEQ ID NO: 34 or 35; (c) HCDR3 comprises an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identity to the amino acid sequence set forth in SEQ ID NO: 45 or 46; (d) LCDR1 comprises an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identity to the amino acid sequence set forth in SEQ ID NO: 62 or 63; (e) LCDR2 comprises an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identity to the amino acid sequence set forth in SEQ ID NO: 76 or 77; (f) An isolated antibody or antigen-binding fragment thereof, wherein LCDR3 comprises an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identity to the amino acid sequence set forth in SEQ ID NO: 86 or 87.

21. An isolated antibody or antigen-binding fragment thereof that binds to human PD-L1, (a) a heavy chain variable region (HCVR) comprising an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 106 and 107; (b) an isolated antibody or antigen-binding fragment thereof, comprising a light chain variable region (LCVR) comprising an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 132 and 133.

22. From the N-terminus to the C-terminus, there is a heavy chain variable (V) domain that contains three heavy chain complementarity determining regions (CDRs), HCDR1, HCDR2, and HCDR3. Ｈ ) domain, From the N-terminus to the C-terminus, there is a light chain variable (VV) region that contains three light chain complementarity determining regions (CDRs), LCDR1, LCDR2, and LCDR3. Ｌ ) domain, An isolated antibody or antigen-binding fragment thereof that binds to human PD-L1, (a) HCDR1 comprises an amino acid sequence selected from the group consisting of ST-Y-A-L-G (SEQ ID NO:17) and ST-Y-A-M-G (SEQ ID NO:18); (b) HCDR2 comprises the amino acid sequence shown in S-I-S-I-G-G-A-T-Y-X40-A-X41-W-A-K-G (SEQ ID NO:), where X40 is F or Y and X41 is S or T; (c) HCDR3 comprises the amino acid sequence set forth in A-R-N-V-D-X53-I-Y-L-X54-A-F-X55-X56 (SEQ ID NO:), wherein X53 is I or S, X54 is D or N, X55 is D or H, and X56 is I or T; (d) LCDR1 comprises the amino acid sequence set forth in Q-A-S-Q-N-I-Y-N-N-L-A (SEQ ID NO:64); (e) LCDR2 comprises the amino acid sequence set forth in X104-X105-S-T-L-A-S (SEQ ID NO:), where X104 is R or S, and X105 is A or S; (f) An isolated antibody or antigen-binding fragment thereof, wherein LCDR3 comprises the amino acid sequence set forth in Q-T-Y-Y-L-T-X109-T-X110-N-A (sequence number) (wherein X109 is S or T and X110 is I or T).

23. (a) HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 36 or 37; (b) HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 47 or 48; (c) LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 78 or 79; (d) the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 88 or 89;

24. (a) HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 17, HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 36, HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 47, LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 64, LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 78, and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 88, or (b) The isolated antibody or antigen-binding fragment thereof of claim 23, wherein HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 18, HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 37, HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 48, LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 64, LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 79, and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO:

89.

25. (a) a heavy chain variable region (HCVR) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 108 and 109; (b) a light chain variable region (LCVR) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 134 and 135.

26. The antibody, (a) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 108, and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 134; or (b) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 109, and an LCVR comprising the amino acid sequence set forth in SEQ ID NO:

135. The isolated antibody or antigen-binding fragment thereof of claim 25,

27. a heavy chain variable (VH) domain comprising, from N-terminus to C-terminus, three heavy chain complementarity determining regions (CDRs): HCDR1, HCDR2, and HCDR3; a light chain variable (VL) domain comprising, from N-terminus to C-terminus, three light chain complementarity determining regions (CDRs): LCDR1, LCDR2, and LCDR3; An isolated antibody or antigen-binding fragment thereof that binds to human PD-L1, (a) HCDR1 comprises an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identity to the amino acid sequence set forth in SEQ ID NO: 17 or 18; (b) HCDR2 comprises an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identity to the amino acid sequence set forth in SEQ ID NO: 36 or 37; (c) HCDR3 comprises an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identity to the amino acid sequence set forth in SEQ ID NO: 47 or 48; (d) LCDR1 comprises an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identity to the amino acid sequence set forth in SEQ ID NO:64; (e) LCDR2 comprises an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identity to the amino acid sequence set forth in SEQ ID NO: 78 or 79; (f) An isolated antibody or antigen-binding fragment thereof, wherein LCDR3 comprises an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identity to the amino acid sequence set forth in SEQ ID NO: 88 or 89.

28. An isolated antibody or antigen-binding fragment thereof that binds to human PD-L1, (a) a heavy chain variable region (HCVR) comprising an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 108 and 109; (b) an isolated antibody or antigen-binding fragment thereof, comprising a light chain variable region (LCVR) comprising an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 134 and 135.

29. From the N-terminus to the C-terminus, the heavy chain variable (V) domain contains three heavy chain complementarity determining regions (CDRs): HCDR1, HCDR2, and HCDR3. Ｈ ) domain, From the N-terminus to the C-terminus, the light chain variable (V) domain contains three light chain complementarity determining regions (CDRs): LCDR1, LCDR2, and LCDR3. Ｌ ) domain, An isolated antibody or antigen-binding fragment thereof that binds to human PD-L1, (a) HCDR1 comprises the amino acid sequence shown in S-S-Y-Y-M-S (SEQ ID NO: 13); (b) HCDR2 comprises the amino acid sequence set forth in SEQ ID NO:32; (c) HCDR3 comprises the amino acid sequence set forth in D-SS-S-G-Y-F-F-L-L (SEQ ID NO: 43); (d) LCDR1 comprises the amino acid sequence set forth in QA-S-Q-N-I-Y-S-N-L-A (SEQ ID NO:60); (e) LCDR2 comprises the amino acid sequence set forth in G-A-S-N-L-R-S (SEQ ID NO:74); (f) An isolated antibody or antigen-binding fragment thereof, wherein LCDR3 comprises the amino acid sequence set forth in QE-G-Y-SI-G-N-V-D-N-P (sequence number 84).

30. 26. The isolated antibody or antigen-binding fragment thereof of claim 25, wherein the antibody comprises an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 104, and an LCVR comprising the amino acid sequence set forth in SEQ ID NO:

130.

31. a heavy chain variable (VH) domain comprising, from N-terminus to C-terminus, three heavy chain complementarity determining regions (CDRs): HCDR1, HCDR2, and HCDR3; a light chain variable (VL) domain comprising, from N-terminus to C-terminus, three light chain complementarity determining regions (CDRs): LCDR1, LCDR2, and LCDR3; An isolated antibody or antigen-binding fragment thereof that binds to human PD-L1, (a) HCDR1 comprises an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identity to the amino acid sequence set forth in SEQ ID NO:13; (b) HCDR2 comprises an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identity to the amino acid sequence set forth in SEQ ID NO: 32; (c) HCDR3 comprises an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identity to the amino acid sequence set forth in SEQ ID NO: 43; (d) LCDR1 comprises an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identity to the amino acid sequence set forth in SEQ ID NO:60; (e) LCDR2 comprises an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identity to the amino acid sequence set forth in SEQ ID NO:74; (f) An isolated antibody or antigen-binding fragment thereof, wherein LCDR3 comprises an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identity to the amino acid sequence set forth in SEQ ID NO:

84.

32. An isolated antibody or antigen-binding fragment thereof that binds to human PD-L1, (a) a heavy chain variable region (HCVR) comprising an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identity to the amino acid sequence set forth in SEQ ID NO: 104; (b) an isolated antibody or antigen-binding fragment thereof, comprising a light chain variable region (LCVR) comprising an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identity to the amino acid sequence set forth in SEQ ID NO:

130.

33. From the N-terminus to the C-terminus, the heavy chain variable (V) domain contains three heavy chain complementarity determining regions (CDRs): HCDR1, HCDR2, and HCDR3. Ｈ ) domain, From the N-terminus to the C-terminus, the light chain variable (V) domain contains three light chain complementarity determining regions (CDRs): LCDR1, LCDR2, and LCDR3. Ｌ ) domain, An isolated antibody or antigen-binding fragment thereof that binds to human PD-L1, (a) HCDR1 comprises the amino acid sequence shown in S-S-Y-Y-M-I (SEQ ID NO: 14); (b) HCDR2 comprises the amino acid sequence set forth in Y-V-Y-T-G-S-G-N-T-W-Y-A-S-W-A-K-G (SEQ ID NO: 33); (c) HCDR3 comprises the amino acid sequence set forth in A-S-G-A-D-G-V-Y-D-W-G-W-D-I (SEQ ID NO: 44); (d) LCDR1 comprises the amino acid sequence set forth in Q-A-S-Q-S-I-Y-S-L-L-A (SEQ ID NO:61); (e) LCDR2 comprises the amino acid sequence set forth in G-A-S-N-L-E-S (SEQ ID NO:75); (f) An isolated antibody or antigen-binding fragment thereof, wherein LCDR3 comprises the amino acid sequence set forth in QNNY-DSG-RI-Y-GL-A (sequence number 85).

34. 30. The isolated antibody or antigen-binding fragment thereof of claim 29, wherein the antibody comprises an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 105, and an LCVR comprising the amino acid sequence set forth in SEQ ID NO:

131.

35. a heavy chain variable (VH) domain comprising, from N-terminus to C-terminus, three heavy chain complementarity determining regions (CDRs): HCDR1, HCDR2, and HCDR3; a light chain variable (VL) domain comprising, from N-terminus to C-terminus, three light chain complementarity determining regions (CDRs): LCDR1, LCDR2, and LCDR3; An isolated antibody or antigen-binding fragment thereof that binds to human PD-L1, (a) HCDR1 comprises an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identity to the amino acid sequence set forth in SEQ ID NO:14; (b) HCDR2 comprises an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identity to the amino acid sequence set forth in SEQ ID NO: 33; (c) HCDR3 comprises an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identity to the amino acid sequence set forth in SEQ ID NO:44; (d) LCDR1 comprises an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identity to the amino acid sequence set forth in SEQ ID NO:61; (e) LCDR2 comprises an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identity to the amino acid sequence set forth in SEQ ID NO:75; (f) An isolated antibody or antigen-binding fragment thereof, wherein LCDR3 comprises an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identity to the amino acid sequence set forth in SEQ ID NO:

85.

36. An isolated antibody or antigen-binding fragment thereof that binds to human PD-L1, (a) a heavy chain variable region (HCVR) comprising an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identity to the amino acid sequence set forth in SEQ ID NO: 105; (b) an isolated antibody or antigen-binding fragment thereof, comprising a light chain variable region (LCVR) comprising an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identity to the amino acid sequence set forth in SEQ ID NO:

131.

37. 37. The isolated antibody or antigen-binding fragment thereof of any one of claims 1 to 36, wherein the N-terminus of the heavy and / or light chain is a pyroglutamic acid (pE) residue.

38. (i) the antibody binds to human PD-L1 competitively with a monoclonal antibody selected from the group consisting of 27A2, 26G2, 28A12, 72G5, 67B6, 35C9, 65B6, 56H3, 39C10, 27C10, 33H7, 30A6, 32E2, 28B3, 33E8, 53G7, 74A9, 38D10, 55H3 / 76D6, 56E5, 57B10, 57F11, 62E6, 69D10, 73C8, and 74F4; (ii) the antibody specifically binds to human and / or cynomolgus PD-L1; (iii) the antibody specifically binds to human PD-L1 on the cell surface; (iv) the antibody inhibits the interaction of PD-1 and PD-L1; (v) the antibody enhances an immune response; (vi) the antibody induces ADCC; (vii) the antibody induces internalization of PD-L1.

38. An isolated antibody or antigen-binding fragment thereof according to any one of claims 1 to 37.

39. An isolated antibody or antigen-binding fragment thereof that binds to human PD-L1 competitively with the antibody of any one of claims 1 to 38.

40. 40. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 39, which is a humanized or chimeric antibody.

41. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 40, wherein the antibody comprises a heavy chain constant region of a class selected from the group consisting of IgA, IgD, IgE, IgG and IgM.

42. 42. The antibody or antigen-binding fragment thereof of claim 41, wherein the antibody comprises a heavy chain constant region of the IgG class, the IgG being selected from the group consisting of IgG1, IgG2, IgG3 and IgG4.

43. 43. An isolated polynucleotide encoding the antibody or antigen-binding fragment thereof, its HCVR, its LCVR, its light chain, its heavy chain, or its antigen-binding fragment thereof according to any one of claims 1 to 42.

44. 44. An expression vector comprising the polynucleotide of claim 43.

45. 45. A recombinant cell comprising the polynucleotide of claim 43 or the expression vector of claim 44.

46. 46. ​​A method for producing the antibody or antigen-binding fragment thereof of any one of claims 1 to 42, comprising expressing the antibody in a recombinant cell of claim 45 and isolating the expressed antibody.

47. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 42 and a pharma- ceutically acceptable carrier or diluent.

48. 48. The pharmaceutical composition of claim 47, wherein the pharmaceutical composition comprises the antibody or antigen-binding fragment thereof in an amount effective to (a) specifically bind to cell surface human or cynomolgus monkey PD-L1, (b) block the interaction between PD-1 and PD-L1, (c) induce ADCC, (d) induce internalization of PD-L1, (e) provide an immune response in immune cells, or (f) any combination of (a)-(e).

49. A method for blocking binding between PD-1 and PD-L1 expressed on a cell surface, comprising contacting the cell with an isolated antibody or antigen-binding fragment thereof of any one of claims 1 to 42, or a pharmaceutical composition of claim 47 or 48, thereby blocking binding between PD-1 and PD-L1 expressed on the cell surface.

50. A method for enhancing an immune response in a subject, comprising administering to the subject an isolated antibody or antigen-binding fragment thereof described in any one of claims 1 to 42, or a pharmaceutical composition described in claim 47 or 48, thereby enhancing the immune response in the subject.

51. 47. A method for inhibiting the growth of a tumor in a subject, comprising administering to the subject an isolated antibody or antigen-binding fragment thereof of any one of claims 1 to 42, or a pharmaceutical composition of claim 47 or 48, thereby inhibiting the growth of the tumor.

52. 50. A method for treating cancer in a subject, comprising administering to the subject an isolated antibody or antigen-binding fragment thereof of any one of claims 1 to 42, or a pharmaceutical composition of claim 47 or 48, thereby treating the cancer.

53. The method of any one of claims 49 to 52, wherein said method activates T cells and induces said T cells to kill tumor target cells.

54. 54. The method of any one of claims 49 to 53, further comprising administering an additional therapeutic agent.

55. 55. The method of claim 54, wherein the additional therapeutic agent comprises an anti-tumor agent, radiation therapy, a chemotherapeutic agent, surgery, a cancer vaccine, an agonist for a stimulatory receptor on an immune cell, a cytokine, cell therapy, or a checkpoint inhibitor.

56. 56. The method of claim 55, wherein the checkpoint inhibitor is selected from the group consisting of PD-1, PD-L1, TIGIT, CTLA-4, LAG-3, TIM-3, neuropeptide, BTLA, CECAM-1, CECAM-5, IL-1R8, VISTA, LAIR1, LILRB1, LILRB2, LILRB3, LILRB4, LILRB5, CD96, CD112R, CD160, 2B4, TGFβ-R, KIR, NKG2A, MICA, MICB, A2aR, A2bR, and any combination thereof.

57. 57. The method of claim 56, wherein the CTLA4 inhibitor is selected from the group consisting of ipilimumab, kadonilimab, and YH001 (Yuwa Pharmaceutical).

58. 57. The method of claim 56, wherein the additional therapeutic agent is an agonist for a stimulatory receptor on immune cells selected from the group consisting of OX40, CD2, CD16, CD27, CDS, ICAM-1, LFA-1, ICOS (CD278), 4-1BB (CD137), GITR, CD28, CD30, CD40, BAFFR, HVEM, CD7, LIGHT, NKG2C, NKG2D, SLAMF7, NKp46, NKp80, CD160, B7-H3, CD83 ligand, and any combination thereof.

59. The method of any one of claims 54 to 58, wherein the additional therapeutic agent is formulated in the same pharmaceutical composition as the antibody.

60. The method of any one of claims 54 to 58, wherein the additional therapeutic agent is formulated in a different pharmaceutical composition than the antibody.

61. The method of any one of claims 54-58 and 60, wherein the additional therapeutic agent is administered before and / or after administration of the antibody.

62. The method of any one of claims 54 to 60, wherein the additional therapeutic agent is administered simultaneously with the antibody or antigen-binding fragment thereof.

63. A method for inducing antibody-dependent cellular cytotoxicity (ADCC) of PD-L1 expressing cells, comprising contacting the cells with an isolated antibody or antigen-binding fragment thereof according to any one of claims 1 to 42, or a pharmaceutical composition according to claim 47 or 48, thereby inducing antibody-dependent cellular cytotoxicity (ADCC) of PD-L1 expressing cells.

64. 50. A method for killing a PD-L1 expressing cell, comprising contacting the cell with the pharmaceutical composition of claim 47 or 48, or the isolated antibody or antigen-binding fragment thereof of any one of claims 1-42 conjugated to a cytotoxic agent, thereby killing the PD-L1 expressing cell.

65. 49. A kit comprising the pharmaceutical composition of claim 47 or 48.

66. 66. The kit of claim 65, further comprising an additional therapeutic agent.

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