Anti-programmed death ligand 1 (PD-L1) antibodies

Abstract

The invention provides anti-PD-L1 antigen binding molecules, including antibodies and antigen binding fragments thereof, and methods of using the same to treat various diseases, including cancer. The antibodies may be delivered to a subject in the form of a pharmaceutical composition for the treatment of various diseases, including various types of cancer. Administration can activate the immune system and then target tumor cells in a subject. Other therapeutic agents, including other drugs, may be administered in combination with the molecules of the invention to treat subjects.

Description

This application claims priority to U.S. Provisional Application No. 63 / 314,925, filed on February 28, 2022, the entire contents of which are incorporated herein by reference. Technical Field

[0001] The present invention relates to antigen-binding molecules, such as antibodies or antigen-binding fragments thereof, for cancer treatment, in particular anti-programmed death ligand 1 (PD-L1) antibodies for cancer immunotherapy. Background Art

[0002] The adaptive immune response is mediated by two major subsets of immune cells: T lymphocytes (CD4 + T cells and CD8 + T cells) and B lymphocytes. While B cells play an important role in secreting antibodies and acting as antigen-presenting cells, T cells have been extensively explored for their outstanding effector functions in a variety of physiological responses, including acute and chronic infections, cancer and autoimmunity and immune homeostasis. T cell activation requires two distinct signals, ligation via peptide-MHC binding to the T cell receptor (TCR) and positive co-stimulatory signals, such as the interaction between CD28 on T cells and CD80 (also known as B7.1) and / or CD86 (also known as B7.2) on antigen-presenting cells. Early in the activation process, negative regulators, including cytotoxic T lymphocyte antigen 4 (CTLA4; also known as CD152), are induced to counteract 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 binds via 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), counteracting the positive signaling caused by TCR and CD28.

[0003] PD-L1 is a 53kDa type 1 transmembrane protein expressed by many immune cell subsets, including activated and exhausted / disabled T cells, B cells, and myeloid cells (including monocytes, mast cells, and dendritic cells), as well as some cancer cells. PD-L1 is one of the “co-inhibitory” receptors that serves as a resting point for the adaptive immune response, as an immune checkpoint that effector T cells must pass to fully function. Under pathological conditions, PD-L1 expression has been found to be significantly upregulated in many tumors, including but not limited to non-small cell lung cancer (NSCLC), bladder cancer, hepatocellular carcinoma, breast cancer, and pancreatic cancer. Increased PD-L1 expression on tumor cells can modulate the tumor microenvironment (TME) and promote immune evasion of tumor cells in two ways: 1) by stimulating regulatory T cells to enhance immunosuppression, and 2) by driving T cell exhaustion / disability to reduce inflammation.

[0004] Treatment with PD-1 pathway inhibitors (also known as “checkpoint blockade”) has been successful in promoting durable antitumor immune responses, and this success has led to the FDA approval of the monoclonal antibodies pembrolizumab (anti-PD-1; Merck), nivolumab (anti-PD-1; Bristol-Myers Squibb), atezolizumab (anti-PD-L1; Roche / Genentech), and durvalumab (anti-PD-L1; AstraZeneca), avelumab (anti-PD-L1; EMD Serono), Cemiplimab (anti-PD-1; Regeneron), and Dostarlimab (anti-PD-1, GlaxoSmithKline) are used to treat more than 20 cancers, 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 (refer to Cancer Research Institute, https: / / www.cancerresearch.org / fda-approval-timeline-of-active-immunoth erapies).

[0005] Despite the revolutionary cure-like survival benefits observed in trials, it is estimated that only a minority of cancer patients 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 treatment resistance, tremendous efforts have been made to improve or develop new anti-PD-1 / PD-L1 based immunotherapy strategies to achieve better clinical responses and reduced immune-mediated toxicity. There is a need in the art for novel therapies based on novel antigen binding molecules (e.g., antibodies or antigen binding fragments thereof that specifically bind to PD-L1) for 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) PD-L1. PD-L1 can be located 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 a method for specifically binding PD-L1 using an antigen binding molecule (e.g., an anti-PD-L1 antibody or an antigen binding fragment thereof) of the present invention. The binding of the antigen binding molecule (e.g., an anti-PD-L1 antibody or an antigen binding fragment thereof) of the present invention can inhibit the PD-1 / PD-L1 interaction or induce endocytosis of a complex of PD-L1 and the antigen binding molecule. The binding of the antigen binding molecule (e.g., an anti-PD-L1 antibody or an antigen binding fragment thereof) of the present invention can be used to treat a subject who can benefit from regulating (e.g., inhibiting) the PD-1 / PD-L1 interaction, such as a subject suffering from or susceptible to a PD-L1-related disease (e.g., a disease or condition characterized by abnormal expression of PD-L1).

[0007] Therefore, in one aspect, the present invention provides an isolated antigen binding molecule, e.g., an antibody or an antigen binding fragment thereof, that binds to human PD-L1. The antibody comprises a heavy chain variable (VH) domain comprising three heavy chain complementary determining regions (CDRs) from N-terminus to C-terminus: HCDR1, HCDR2, and HCDR3; and a light chain variable (VL) domain comprising three light chain complementary determining regions (CDRs) from N-terminus to C-terminus: LCDR1, LCDR2, and LCDR3; wherein (a) HCDR1 comprises an amino acid sequence selected from the following: X1-NHYM-X2 (SEQ ID NO:), X3-X4-YYMC (SEQ ID NO:), NNYYMS (SEQ ID NO: 8), RYFYMS (SEQ ID NO: 11), and SAY-WIC (SEQ ID NO: 12). NO:12); wherein 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 X32-I-X33-X34-GS-X35-I-X36-DYA-X37-WAKG (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 WTSGGGGFGL (SEQ ID NO:42); (d) LCDR1 comprises an amino acid sequence selected from the group consisting of QSSQ-X65-X66-Y-X67-X68-YL-X69 (SEQ ID NO:), QSSQ-X70-IYSDYL-X71 (SEQ ID NO:), NO:) and QSSQS-X72-Y-X73-NYL-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, and 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:). NO:72); wherein 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; and (f) LCDR3 comprises the amino acid sequence QGYYSGYIWT (SEQ ID NO:83).

[0008] In various aspects and embodiments of the present invention, the antibody is an antigen-binding fragment of the antibody. In various aspects and embodiments of the present invention, the human PD-L1 comprises the sequence shown in SEQ ID NO:.

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

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

[0011] In another embodiment, the isolated antigen-binding molecule (e.g., 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: 92-103; and (b) a light chain variable region (LCVR) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 118-129.

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

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

[0014] In another aspect, the present invention provides an isolated antigen binding molecule, such as an antibody or an antigen binding fragment thereof, that binds to human PD-L1. The isolated antigen binding molecule, such as an antibody or an antigen binding fragment thereof, comprises (a) a heavy chain variable region (HCVR), the heavy chain variable region comprising an amino acid sequence selected from SEQ ID NO: 92-103 having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identical amino acid sequence; and (b) a light chain variable region (LCVR), the light chain variable region comprising an amino acid sequence selected from SEQ ID NO: 118-129 having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identical amino acid sequence.

[0015] In another aspect, the present invention provides an isolated antigen-binding molecule, such as an antibody or an antigen-binding fragment thereof, that binds to human PD-L1. The isolated antigen-binding molecule, such as an antibody or an antigen-binding fragment thereof, comprises a heavy chain variable (V H ) domain, the heavy chain variable domain comprising three heavy chain complementarity determining regions (CDRs) from N-terminus to C-terminus: HCDR1, HCDR2 and HCDR3; and a light chain variable (V L ) domain, comprising three light chain complementary determining regions (CDRs) from N-terminus to C-terminus: LCDR1, LCDR2 and LCDR3; wherein (a) HCDR1 comprises the amino acid sequence X8-D-X9-YMS (SEQ ID NO:); wherein X8 is D or G, and X9 is W or Y; (b) HCDR2 comprises the amino acid sequence SIY-X42-GSLN-X43-YYATWAKG (SEQ ID NO:), wherein X42 is S or T, and X43 is I, S or T; (c) HCDR3 comprises the amino acid sequence R-X57-KN-X58-D-X59-G-X60-FDL (SEQ ID NO:). NO:), wherein 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 QAS-X76-SI-X77-X78-X79-LA (SEQ ID NO:), QAS-X80-SINDRLA (SEQ ID NO:), QASQSI-X81-X82-ALA (SEQ ID NO:), 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 X106-X107-STLAS (SEQ ID NO: NO:); wherein X106 is A or G, and X107 is A or T; and (f) LCDR3 comprises the amino acid sequence QQGWTVSSL-X111-NA (SEQ ID NO:), 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 an amino acid sequence shown in SEQ ID NOs: 90 or 91.

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

[0018] In another embodiment, the isolated antigen-binding molecule, such as 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: 110-117; and (b) a light chain variable region (LCVR) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 136-143.

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

[0020] In one aspect, the present invention provides an isolated antigen binding molecule, such as an antibody or an antigen binding fragment thereof, that binds to human PD-L1. The isolated antigen binding molecule, such as an antibody or an antigen binding fragment thereof, comprises a heavy chain variable (VH) domain, the heavy chain variable domain comprising three heavy chain complementary determining regions (CDRs) from N-terminus to C-terminus: HCDR1, HCDR2, and HCDR3; and a light chain variable (VL) domain, the light chain variable domain comprising three light chain complementary determining regions (CDRs) from N-terminus to C-terminus: 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% identical to an amino acid sequence selected from SEQ ID NO: 19, 20, and 21; (b) HCDR2 comprises an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identical to an amino acid sequence selected from SEQ ID NO: 19, 20, and 21; (c) HCDR2 comprises an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identical to an amino acid sequence selected from SEQ ID NO: 19, 20, and 21; (d) HCDR2 comprises an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identical to an amino acid sequence selected from SEQ ID NO: 19, 20, and 21; (e) HCDR2 comprises an amino acid sequence having about 80%, 85%, 90%, 9 NO:38, 39, 40 and 41 have an amino acid sequence that is about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identical; (c) HCDR3 comprises 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 SEQ ID NO:49, 50, 51 and 52; (d) LCDR1 comprises 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 SEQ ID NO:65, 66, 67 and 68; (e) LCDR2 comprises 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 SEQ ID NO: NO:80, 71 and 82 have amino acid sequences that are about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identical; and (f) LCDR3 comprises an amino acid sequence that is about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identical to the amino acid sequence shown in SEQ ID NO:90 or 91.

[0021] In another aspect, the present invention provides an isolated antigen binding molecule, such as an antibody or an antigen binding fragment thereof, that binds to human PD-L1. The isolated antigen binding molecule, such as an antibody or an antigen binding fragment thereof, comprises (a) a heavy chain variable region (HCVR), the heavy chain variable region comprising an amino acid sequence selected from SEQ ID NO: 110-117 having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identical amino acid sequence; and (b) a light chain variable region (LCVR), the light chain variable region comprising an amino acid sequence selected from SEQ ID NO: 136-143 having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identical amino acid sequence.

[0022] In another aspect, the present invention provides an isolated antigen-binding molecule, such as an antibody or an antigen-binding fragment thereof, that binds to human PD-L1. The isolated antigen-binding molecule, such as an antibody or an antigen-binding fragment thereof, comprises a heavy chain variable (V H ) domain, the heavy chain variable domain comprising three heavy chain complementarity determining regions (CDRs) from N-terminus to C-terminus: HCDR1, HCDR2 and HCDR3; and a light chain variable (V L ) domain, the light chain variable domain comprises three light chain complementary determining regions (CDRs) from N-terminus to C-terminus: LCDR1, LCDR2 and LCDR3; wherein (a) HCDR1 comprises an amino acid sequence selected from SGYDMS (SEQ ID NO: 15) and SGYDMC (SEQ ID NO: 16); (b) HCDR2 comprises an amino acid sequence X38-IFT-X39-SGSTWYANWAKG (SEQ ID NO:), wherein X38 is C or S, and X39 is G or T; (c) HCDR3 comprises an amino acid sequence T-X51-DG-X52-GSFYMNL (SEQ ID NO:); wherein X51 is K or R, and X52 is A or V; (d) LCDR1 comprises an amino acid sequence QASGTIGSNLA (SEQ ID NO: 63) or QASQTIGSNLA (SEQ ID NO: 62); (e) LCDR2 comprises an amino acid sequence KAFTLAS (SEQ ID NO: 64). NO:76) or KTFTLAS (SEQ ID NO:77); and (f) LCDR3 comprises the amino acid sequence QQGATRINIDNA (SEQ ID NO:86) or QQGASRNIDNA (SEQ ID NO:87).

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

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

[0025] In 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 of SEQ ID NO: 106 or 107; and (b) a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 132 or 133.

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

[0027] In one aspect, the present invention provides an isolated antigen-binding molecule, such as an antibody or an antigen-binding fragment thereof, that binds to human PD-L1. The isolated antigen-binding molecule, such as an antibody or an antigen-binding fragment thereof, comprises a heavy chain variable (VH) domain, the heavy chain variable domain comprising three heavy chain complementary determining regions (CDRs) from N-terminus to C-terminus: HCDR1, HCDR2 and HCDR3; and a light chain variable (VL) domain, the light chain variable domain comprising three light chain complementary determining regions (CDRs) from N-terminus to C-terminus: 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 shown 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 shown 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 with the amino acid sequence shown in SEQ ID NO: NO:45 or 46 has an amino acid sequence that is about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identical; (d) LCDR1 comprises an amino acid sequence that is about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identical to the amino acid sequence set forth in SEQ ID NO:62 or 63; (e) LCDR2 comprises an amino acid sequence that is about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identical to the amino acid sequence set forth in SEQ ID NO:76 or 77; and (f) LCDR3 comprises an amino acid sequence that is about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identical 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, such as an antibody or an antigen binding fragment thereof, that binds to human PD-L1. The isolated antigen binding molecule, such as an antibody or an antigen binding fragment thereof, comprises (a) a heavy chain variable region (HCVR) comprising an amino acid sequence selected from SEQ ID NOs: 106 and 107 having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identical amino acid sequence; and (b) a light chain variable region (LCVR) comprising an amino acid sequence selected from SEQ ID NOs: 132 and 133 having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identical amino acid sequence.

[0029] In one aspect, the present invention provides an isolated antigen-binding molecule, such as an antibody or an antigen-binding fragment thereof, that binds to human PD-L1. The isolated antigen-binding molecule, such as an antibody or an antigen-binding fragment thereof, comprises a heavy chain variable (V H ) domain, the heavy chain variable domain comprising three heavy chain complementarity determining regions (CDRs) from N-terminus to C-terminus: HCDR1, HCDR2 and HCDR3; and a light chain variable (V L ) domain, the light chain variable domain comprises three light chain complementary determining regions (CDRs) from N-terminus to C-terminus: LCDR1, LCDR2 and LCDR3; wherein (a) HCDR1 comprises an amino acid sequence selected from STYALG (SEQ ID NO: 17) and STYAMG (SEQ ID NO: 18); (b) HCDR2 comprises an amino acid sequence SISIGGATY-X40-A-X41-WAKG (SEQ ID NO:), wherein X40 is F or Y, and X41 is S or T; (c) HCDR3 comprises an amino acid sequence ARNVD-X53-IYL-X54-AF-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 an amino acid sequence QASQNIYNNLA (SEQ ID NO: IDNO:64); (e) LCDR2 comprises the amino acid sequence 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 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 shown in SEQ ID NO:36 or 37; (b) HCDR3 comprises the amino acid sequence shown in SEQ ID NO:47 or 48; (c) LCDR2 comprises the amino acid sequence shown in SEQ ID NO:78 or 79; and (d) LCDR3 comprises the amino acid sequence shown in SEQ ID NO:88 or 89.

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

[0032] In 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 another embodiment, the isolated antigen-binding molecule (e.g., antibody or antigen-binding fragment thereof) comprises (a) a HCVR comprising the amino acid sequence shown in SEQ ID NO:108, and a LCVR comprising the amino acid sequence shown in SEQ ID NO:134; or (b) a HCVR comprising the amino acid sequence shown in SEQ ID NO:109, and a LCVR comprising the amino acid sequence shown in SEQ ID NO:135.

[0034] In one aspect, the present invention provides an isolated antigen-binding molecule, such as an antibody or an antigen-binding fragment thereof, that binds to human PD-L1. The isolated antigen-binding molecule, such as an antibody or an antigen-binding fragment thereof, comprises a heavy chain variable (VH) domain, the heavy chain variable domain comprising three heavy chain complementary determining regions (CDRs) from N-terminus to C-terminus: HCDR1, HCDR2 and HCDR3; and a light chain variable (VL) domain, the light chain variable domain comprising three light chain complementary determining regions (CDRs) from N-terminus to C-terminus: 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 shown 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 with the amino acid sequence shown 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 with the amino acid sequence shown in SEQ ID NO: 36 or 37; NO:47 or 48 has an amino acid sequence that is about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identical; (d) LCDR1 comprises an amino acid sequence that is about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identical to the amino acid sequence set forth in SEQ ID NO:64; (e) LCDR2 comprises an amino acid sequence that is about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identical to the amino acid sequence set forth in SEQ ID NO:78 or 79; and (f) LCDR3 comprises an amino acid sequence that is about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identical 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, such as an antibody or an antigen binding fragment thereof, that binds to human PD-L1. The isolated antigen binding molecule, such as an antibody or an antigen binding fragment thereof, comprises (a) a heavy chain variable region (HCVR) comprising an amino acid sequence selected from SEQ ID NOs: 108 and 109 having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identical amino acid sequence; and (b) a light chain variable region (LCVR) comprising an amino acid sequence selected from SEQ ID NOs: 134 and 135 having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identical amino acid sequence.

[0036] In another aspect, the present invention provides an isolated antigen-binding molecule, such as an antibody or an antigen-binding fragment thereof, that binds to human PD-L1. The isolated antigen-binding molecule, such as an antibody or an antigen-binding fragment thereof, comprises a heavy chain variable (V H ) domain, the heavy chain variable domain comprising three heavy chain complementarity determining regions (CDRs) from N-terminus to C-terminus: HCDR1, HCDR2 and HCDR3; and a light chain variable (V L ) domain, the light chain variable domain comprising three light chain complementary determining regions (CDRs) from N-terminus to C-terminus: LCDR1, LCDR2 and LCDR3; wherein (a) HCDR1 comprises the amino acid sequence SSYYMS (SEQ ID NO: 13); (b) HCDR2 comprises the amino acid sequence CISGGVTDNAYYASWAKG (SEQ ID NO: 32); (c) HCDR3 comprises the amino acid sequence DSSSGYFFLL (SEQ ID NO: 43); (d) LCDR1 comprises the amino acid sequence QASQNIYSNLA (SEQ ID NO: 60); (e) LCDR2 comprises the amino acid sequence GASNLRS (SEQ ID NO: 74); and (f) LCDR3 comprises the amino acid sequence 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 shown in SEQ ID NO:104, and a LCVR comprising the amino acid sequence shown in SEQ ID NO:130.

[0038] In one aspect, the present invention provides an isolated antigen-binding molecule, such as an antibody or an antigen-binding fragment thereof, that binds to human PD-L1. The isolated antigen-binding molecule, such as an antibody or an antigen-binding fragment thereof, comprises a heavy chain variable (VH) domain, the heavy chain variable domain comprising three heavy chain complementary determining regions (CDRs) from N-terminus to C-terminus: HCDR1, HCDR2 and HCDR3; and a light chain variable (VL) domain, the light chain variable domain comprising three light chain complementary determining regions (CDRs) from N-terminus to C-terminus: 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 shown 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 shown 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 shown in SEQ ID NO: NO:43 has an amino acid sequence that is about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identical; (d) LCDR1 comprises an amino acid sequence that is about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identical to the amino acid sequence set forth in SEQ ID NO:60; (e) LCDR2 comprises an amino acid sequence that is about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identical to the amino acid sequence set forth in SEQ ID NO:74; and (f) LCDR3 comprises an amino acid sequence that is about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identical 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, such as an antibody or an antigen binding fragment thereof, that binds to human PD-L1. The isolated antigen binding molecule, such as an antibody or an 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% identical to the amino acid sequence shown 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% to about 100% identical to the amino acid sequence shown in SEQ ID NO: 130.

[0040] In one aspect, the present invention provides an isolated antigen-binding molecule, such as an antibody or an antigen-binding fragment thereof, that binds to human PD-L1. The isolated antigen-binding molecule, such as an antibody or an antigen-binding fragment thereof, comprises a heavy chain variable (V H ) domain, the heavy chain variable domain comprising three heavy chain complementarity determining regions (CDRs) from N-terminus to C-terminus: HCDR1, HCDR2 and HCDR3; and a light chain variable (V L ) domain, the light chain variable domain comprising three light chain complementary determining regions (CDRs) from N-terminus to C-terminus: LCDR1, LCDR2 and LCDR3; wherein (a) HCDR1 comprises the amino acid sequence SSYYMI (SEQ ID NO: 14); (b) HCDR2 comprises the amino acid sequence YVYTGSGNTWYASWAKG (SEQ ID NO: 33); (c) HCDR3 comprises the amino acid sequence ASGADGVYDWGWDI (SEQ ID NO: 44); (d) LCDR1 comprises the amino acid sequence QASQSIYSLLA (SEQ ID NO: 61); (e) LCDR2 comprises the amino acid sequence GASNLES (SEQ ID NO: 75); and (f) LCDR3 comprises the amino acid sequence 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 shown in SEQ ID NO:105, and a LCVR comprising the amino acid sequence shown in SEQ ID NO:131.

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

[0043] In another aspect, the present invention provides an isolated antigen binding molecule, such as an antibody or an antigen binding fragment thereof, that binds to human PD-L1. The isolated antigen binding molecule, such as an antibody or an 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% identical to the amino acid sequence shown 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% to about 100% identical to the amino acid sequence shown in SEQ ID NO: 131.

[0044] In one embodiment, a flow cytometry-based test or an ELISA-based test (as described in Examples 3, 4, and 5) or a substantially similar test thereof is used to determine the binding of the antibody to PD-L1 or cell surface PD-L1. In another embodiment, a test known in the art is used to determine the competitive binding of the antibody to PD-L1 or cell surface PD-L1, such as the test described in "Microtiter plate-based antibody competition test to determine the binding affinity and plasma / blood stability of CXCR4 ligands, Scientific Reports, 2020:10:16036, doi.org / 10.1038 / s41598-020-73012-4" by Harms et al., or a substantially similar test thereof. In yet another embodiment, the blocking of the interaction between PD-1 and PD-L1 is determined using the test described in Example 5 or a test substantially similar thereto. In another embodiment, the enhancement of the immune response, such as an increase in the concentration of inflammatory cytokines in tissues, an increase in the number of cytotoxic CD8+T cells, is determined using methods well known in the art.

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

[0046] In yet another embodiment, the antibody specifically binds to human PD-L1 and / or cynomolgus monkey PD-L1. In yet another embodiment, the antibody specifically binds to human PD-L1 and / or cynomolgus monkey 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 monkey PD-L1. In various aspects of the invention and embodiments thereof, cynomolgus monkey PD-L1 comprises a sequence such as.

[0047] In one aspect, the invention provides an isolated antigen binding molecule, such as an antibody, which competes with the antibody of any aspect for binding to human PD-L1.

[0048] In one embodiment, the antigen binding molecule, such as an antibody, is a humanized antibody or a chimeric antibody. In another embodiment, the antibody comprises a heavy chain constant region selected from IgA, IgD, IgE, IgG or IgM. In another embodiment, the antibody comprises a heavy chain constant region of IgG, wherein IgG is selected from IgG1, IgG2, IgG3 and IgG4.

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

[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 present invention provides a method for preparing the antigen-binding molecule, such as the antibody of any aspect and various embodiments thereof, the method comprising expressing the antibody in a recombinant cell and isolating the expressed antibody.

[0053] In one aspect, the present invention provides a pharmaceutical composition comprising an antigen binding molecule, such as an antibody, according to any aspect and various embodiments, and a pharmaceutically acceptable carrier or diluent.

[0054] In one embodiment, the pharmaceutical composition comprises an effective amount of the antibody or its antigen-binding fragment so that it (a) specifically binds to human or cynomolgus monkey PD-L1 on the cell surface; (b) blocks the interaction between PD-1 and PD-L1; (c) induces ADCC; (d) induces endocytosis of PD-L1; (e) provides an immune response of immune cells; or (f) achieves any combination of (a)-(e).

[0055] In one aspect, the present invention provides a method for blocking the binding of PD-1 to PD-L1 expressed on the cell surface, comprising contacting the cell with an isolated antibody of any aspect or a pharmaceutical composition of any aspect, thereby blocking the binding of PD-1 to PD-L1. In one embodiment, the 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] On the other hand, the present invention provides a method for enhancing the immune response of a subject. The method comprises administering to a subject an isolated antibody of any aspect or a pharmaceutical composition of any aspect, thereby enhancing the immune response of 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 present invention increases the immune response by at least about 10%, about 20%, about 50%, about 60%, about 70%, about 80%, about 90%, about 1 times, about 2 times, about 4 times or more compared to baseline levels.

[0057] The present invention also provides a method for inhibiting tumor growth in a subject, the method comprising administering the isolated antibody of any aspect or the pharmaceutical composition of any aspect to the subject, thereby inhibiting tumor growth.

[0058] On the other hand, the present invention provides a method for treating a subject's cancer, comprising administering an antibody of any aspect of the separation or a pharmaceutical composition of any aspect, thereby treating cancer. In one embodiment, the cancer is any cancer described herein. In a specific embodiment, the cancer is selected from 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 deficiency (dMMR).

[0059] In one embodiment, the method of any of the above aspects activates T cells and directs the T cells to kill tumor target cells.

[0060] In another embodiment, the method of any of the above aspects further comprises administering other therapeutic agents. In one embodiment, the other therapeutic agents include any therapeutic agent described herein. In another embodiment, the other therapeutic agents include anti-tumor agents, radiotherapy, chemotherapeutic agents, surgery, cancer vaccines, agonists of immune cell stimulating receptors, cytokines, cell therapy or checkpoint inhibitors. In one embodiment, the other therapeutic agents are antibodies, including multispecific antibodies, such as bispecific antibodies.

[0061] In another embodiment, the checkpoint inhibitor is selected from 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 inhibitors and any combination thereof. In one embodiment, the CTLA-4 inhibitor is selected from ipilimumab, cardonilimumab and YH001 (Yuhe Pharmaceutical).

[0062] In another embodiment, the additional therapeutic agent is an agonist of an immune cell stimulating receptor selected from 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 other therapeutic agent is formulated in the same pharmaceutical composition as the antibody. In another embodiment, the other therapeutic agent is formulated in a different pharmaceutical composition than the antibody.

[0064] In yet another embodiment, the other therapeutic agent is administered before the antigen binding molecule (e.g., antibody). In yet another embodiment, the other therapeutic agent is administered after the antigen binding molecule (e.g., antibody). In yet another embodiment, the other therapeutic agent is administered simultaneously with the antigen binding molecule (e.g., antibody).

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

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

[0067] In one aspect, the present invention provides a kit, the kit comprising the pharmaceutical composition of any aspect. In one embodiment, the pharmaceutical composition further comprises one or more other therapeutic agents as described herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0068] Figure 1 The results show that the exemplary antibodies of the present disclosure effectively block the interaction between PD-1 and PD-L1.

[0069] Figure 2A , 2B 2 and 2C show that exemplary antibodies of the present disclosure induce endocytosis upon binding to cell surface PD-L1.

[0070] Figure 3 The exemplary antibodies of the present disclosure conjugated to cytotoxic agents are shown to elicit potent killing of PD-L1 expressing cells.

[0071] Figure 4A and 4B Schematic diagram of the epitope classification of exemplary antibodies of the present disclosure and Avelumab, and the epitope classification of certain other anti-PD-L1 antibodies and Avelumab. Specific implementation plan

[0072] The present invention and the accompanying drawings are now described to enable those skilled in the art to practice the invention. However, it should be understood by those skilled in the art that the invention described below may be practiced without the use of these specific details, or that it may be used for purposes other than those described herein. Indeed, in view of this disclosure, it may be modified and used in conjunction with products and techniques known to those skilled in the art. The accompanying drawings and descriptions are intended to illustrate various aspects of the present invention and are not intended to narrow the scope of the claims. In addition, it should be understood that the accompanying drawings may show various aspects of the present invention individually, and that elements in one figure may be used in conjunction with elements shown in other figures.

[0073] It should be understood that references to aspects, features, advantages, or similar language throughout this specification do not mean that all aspects and advantages can be achieved by the present invention, nor do they mean that they should or have been achieved in any single embodiment of the present invention. Instead, language referencing aspects and advantages should be understood as meaning that the specific aspects, features, advantages, or properties described in connection with an embodiment should be included in at least one embodiment of the present invention. Therefore, discussion of aspects and advantages and similar language throughout this specification may, but do not necessarily, refer to the same embodiment.

[0074] The aspects, features, advantages and properties described herein may be combined in any suitable manner in one or more further embodiments. In addition, those skilled in the relevant art will recognize that the present invention may be practiced without one or more specific aspects or advantages of a particular embodiment. In other cases, other aspects, features and advantages may be recognized and claimed in certain embodiments, and these aspects, features and advantages may not be present in all embodiments of the present invention.

[0075] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those 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 the techniques and materials described herein can be used to implement various aspects and embodiments of the present invention. The aspects and embodiments described in this application are not limited to the methods and materials described.

[0076] Furthermore, any cited reference, any issued patent, or patent application publication described in this application is expressly incorporated herein by reference for its teachings of the present invention. I. Definition

[0077] In order that the present invention may be more readily understood, certain terms are first defined. In addition, it should be noted that whenever a parameter value or a range of values ​​is listed, it is intended to indicate that the values ​​and ranges intermediate to the listed values ​​are also part of the present invention.

[0078] Unless otherwise indicated herein or clearly contradicted by context, the terms "a" and "an" and "the" and similar designators used in the context of describing the invention (especially in the context of the claims) should be interpreted to cover both the singular and the plural (i.e., one or more). Unless otherwise indicated herein or clearly contradicted by context, the terms "a" and "an" and "the" and similar designators used in the context of describing the invention (especially in the context of the claims) should be interpreted to cover both the singular and the plural (i.e., one or more). Unless otherwise indicated herein, the terms "comprising", "having", "including" and "containing" should be interpreted as open-ended terms (i.e., meaning "including but not limited to"). Unless otherwise indicated herein, the recitation of numerical ranges herein is intended merely to serve as a shorthand method of referring individually to each individual numerical value cited or to each individual numerical value falling within the range, and each individual numerical value is incorporated into the specification as if it were individually recited.

[0079] When the phrases "in one embodiment," "in another embodiment," "in other embodiments," "in some embodiments," or "in certain embodiments" are used, the disclosure should be understood to include any combination of features that define the different embodiments described herein, unless these features cannot be combined with each other, or these features are mutually exclusive or are explicitly disclaimed herein.

[0080] The term "about" or "approximately" generally means within 10%, preferably within 5%, or more preferably within 1% of a given value or range.

[0081] Ranges can 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 one particular value and / or to another particular value. Similarly, when a value is expressed as an approximation by using the antecedent "about", it is understood that the particular value constitutes another embodiment. It should also be further understood that the endpoints of each range are both significant and independent relative to the other endpoint. It should also be understood that a large number of values ​​are disclosed herein, and each value also includes the "about" value of the particular value in addition to the value itself. For example, if the value "10" is disclosed, "about 10" is also disclosed.

[0082] The term "agent" as used herein refers to any substance, compound (e.g., molecule), supramolecular complex, material, or combination or mixture thereof. A compound can be any agent represented by a chemical formula, chemical structure, or sequence. Examples of agents include, for example, small molecules, polypeptides, nucleic acids (e.g., RNAi agents, antisense oligonucleotides, nucleic acid aptamers), lipids, polysaccharides, etc. Generally, any suitable method known in the art can be used to obtain the agent. A person of ordinary skill will select a suitable method based on, for example, the properties of the agent. The agent can be at least partially purified. In some embodiments, the agent can be provided as part of a composition, and in various embodiments, in addition to the agent, the composition can include, for example, counterions, aqueous or non-aqueous diluents or carriers, buffers, preservatives, or other ingredients. In some embodiments, the agent can be provided in the form of salts, esters, hydrates, or solvates. In some embodiments, the agent is permeable to cells, and is within the range of typical agents that are, for example, absorbed by cells and act intracellularly (e.g., act intracellularly to produce biological effects). Certain compounds can exist in specific 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 unless otherwise indicated, such compounds are included in various embodiments of the present disclosure. Certain compounds may exist in multiple or protonated states, may have multiple configurations, may exist as solvates (e.g., with water (i.e., hydrates) or common solvents) and / or may have different crystalline forms (e.g., polymorphs) or different tautomeric forms. The present disclosure includes embodiments exhibiting such alternative protonation states, configurations, solvates, and forms, where applicable.

[0083] In certain embodiments, "agent" also includes treatment methods, such as radiation therapy, chemotherapy, or surgery, depending on the context.

[0084] The term "agonist" refers to a substance that promotes (i.e., induces, causes, enhances, or increases) the biological activity or action of another molecule. The term agonist includes substances that bind to receptors (e.g., antibodies) and substances that promote receptor function but do not bind to it (e.g., promote receptor function by activating related proteins).

[0085] The term "amino acid" refers to the twenty common natural amino acids. Natural amino acids include 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).

[0086] The term "antagonist" or "inhibitor" refers to a substance that prevents, blocks, inhibits, neutralizes or reduces the biological activity or effect of another molecule (eg, a receptor).

[0087] The term "antibody" as used herein refers to any antigen binding molecule or molecular complex comprising at least one complementary determining region (CDR) that specifically binds to or interacts with a specific antigen (e.g., PD-L1). The term "antibody" includes immunoglobulin molecules comprising four polypeptide chains (i.e., two heavy (H) chains and two light (L) chains interconnected by disulfide bonds), and multimers thereof (e.g., IgM). Each heavy chain comprises a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region comprises three domains, namely, C H 1. C H 2 and C H 3. 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 a domain (C L 1). V H and V L The V region can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FRs). H and V LIt consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In different embodiments of the present invention, the FRs of the anti-PD-L1 antibody (or its antigen-binding fragment) may be identical to the mouse or human germline sequence, or may be naturally or artificially modified. The amino acid consensus sequence may be defined based on a side-by-side analysis of two or more CDRs.

[0088] The term "antibody" as used herein also includes antigen-binding fragments of intact antibody molecules. The terms "antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody, etc. as used herein include 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 using any suitable standard techniques, such as proteolysis or recombinant genetic engineering techniques (including manipulation and expression of DNA encoding antibody variable regions and optional constant regions).

[0089] 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, which consist of amino acid residues that mimic antibody hypervariable regions (e.g., isolated complementary determining regions (CDRs), such as CDR3 peptides) or restricted FR3-CDR3-FR4 peptides. Other engineered molecules, such as domain-specific antibodies, single domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, double antibodies, three antibodies, four antibodies, mini antibodies, nano antibodies (e.g., monovalent nano antibodies, bivalent nano antibodies, etc.), small modular immunopharmaceuticals (SMIPs) and shark variable IgNAR domains are also included in the expression "antigen binding fragment" used herein.

[0090] When a polypeptide domain arrangement is described with hyphens between the various domains (eg, CH2-CH3), it is understood that the order in which the domains are listed is from N-terminus to C-terminus.

[0091] The antigen-binding fragment of an antibody generally comprises at least one variable domain. The variable domain may be of any size or amino acid composition 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 with connected domains, V H and V LThe domains may be positioned relative to each other in any suitable arrangement. For example, the variable region may be a dimer and comprise V H -V H 、V H -V L or V L -V L Alternatively, the antigen-binding fragment of an antibody may contain a monomer V H or V L Structural domain.

[0092] In certain embodiments, the antigen-binding fragment of an antibody may contain at least one variable domain covalently linked to at least one constant domain. Non-limiting exemplary configurations of variable and constant domains comprised by the antigen-binding fragment of an antibody of the invention include: (i) 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 L. In any configuration of variable domains and constant domains (including any of the exemplary configurations listed above), the variable domains and constant domains may be directly connected to each other or may be connected by a complete or partial hinge or linker region. The hinge region may be composed of at least 2 (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids, thereby forming a flexible or semi-flexible connection between adjacent variable and / or constant domains in a single polypeptide molecule. In addition, the antigen-binding fragment may comprise homodimers or heterodimers (or other multimers) of any of the variable domain and constant domain configurations listed above, which are non-covalently linked to each other and / or to one or more monomer V H or V L The domains are non-covalently linked (eg, via disulfide bonds).

[0093] As with complete antibody molecules, antigen-binding fragments can be monospecific or multispecific (e.g., bispecific). Multispecific antigen-binding fragments of antibodies typically contain at least two different variable domains, each of which is capable of specifically binding to a separate antigen or a different epitope on the same antigen. By using conventional techniques available in the art, any multispecific antibody format (including the exemplary bispecific antibody formats disclosed herein) can be applied to the case of antigen-binding fragments of the antibodies of the present invention.

[0094] Antibodies of the present invention can be separated antibodies. "Separated" molecules used herein, such as separated antibodies or separated polypeptides, refer to molecules, such as antibodies, that have been identified and separated and / or recovered from at least one component of their natural environment. For example, molecules (such as antibodies) separated or removed from at least one component of an organism, or from tissues or cells that naturally exist or naturally produce antibodies are "separated" molecules (such as antibodies) for the purposes of the present invention. Separated molecules (such as antibodies) also include in situ molecules (such as antibodies) in recombinant cells. In certain embodiments, separated molecules (such as antibodies) are molecules (such as antibodies) that have been through at least one purification or separation step. According to certain embodiments, separated molecules (such as antibodies) can be substantially free of other cell materials and / or chemicals.

[0095] The present invention also includes a single-armed antibody that binds to PD-L1. As used herein, a "single-armed antibody" refers to an antigen binding molecule comprising a single antibody heavy chain and a single antibody light chain. The single-armed antibody of the present invention may comprise any HCVR / LCVR or CDR amino acid sequence listed in Tables 1-20.

[0096] Compared to the corresponding germline sequence from which an antigen binding molecule, such as an antibody or antigen binding fragment is derived, the anti-PD-L1 antibody or its antigen binding fragment described herein may include one or more amino acid substitutions, insertions and / or deletions in the framework region and / or CDR region of its heavy chain and light chain variable domains. Such mutations can be easily determined by comparing the amino acid sequences disclosed herein with germline sequences that can be obtained from, for example, a public antibody sequence database. The present invention includes antibodies and antigen binding fragments thereof obtained from any amino acid sequence disclosed herein, wherein one or more amino acids in one or more framework regions and / or CDR regions are mutated to the corresponding residues in the germline sequence from which the antibody is derived, or the corresponding residues in another human germline sequence, or conservative amino acid substitutions of the corresponding germline residues (such sequence changes are collectively referred to herein as "germline mutations"). A person of ordinary skill in the art can prepare a large number of antibodies and antigen binding fragments starting with the heavy chain and light chain variable region sequences disclosed herein, which contain one or more individual germline mutations or combinations thereof. In certain embodiments, V H and / or V L All framework regions and / or CDR residues in the domain are mutated back to the residues in the original germline sequence of the antibody source. In other embodiments, only some residues are mutated back to the original germline sequence, for example, only in the first 8 amino acids of FR1 or in the last 8 amino acids of FR4, or only in CDR1, CDR2 or CDR3. In other embodiments, one or more framework regions and / or CDR residues are mutated to the corresponding residues of different germline sequences (i.e., germline sequences different from the germline sequences of the antibody source). In addition, the antibody of the present invention or its antigen-binding domain can comprise any combination of two or more germline mutations in the framework region and / or CDR region, for example, wherein some individual residues are mutated to the corresponding residues of a specific germline sequence, and some other residues different from the original germline sequence are retained or mutated to the corresponding residues of different germline sequences. Once an antibody or its antigen-binding fragment containing one or more germline mutations is obtained, it can be easily tested for one or more desired properties, such as improved binding specificity, increased binding affinity, improved or enhanced antagonistic or exciting biological properties (as the case may be), reduced immunogenicity, etc. Antibodies or antigen-binding fragments thereof obtained in this general manner are encompassed by the present invention.

[0097] The present invention also includes anti-PD-L1 antibodies comprising variants of any HCVR, LCVR and / or CDR amino acid sequences disclosed herein. Exemplary variants included in this aspect of the present invention include variants of any HCVR, LCVR and / or CDR amino acid sequences disclosed herein having one or more conservative substitutions. For example, the present invention includes an anti-PD-L1 antibody and antigen binding protein having a HCVR, LCVR and / or CDR amino acid sequence comprising 10 or less, 8 or less, 6 or less, 4 or less, etc. conservative amino acid substitutions relative to any HCVR, LCVR and / or CDR amino acid sequence listed in the table herein.

[0098] Light chains are classified as either kappa or lambda (κ, λ). Each heavy chain class can be combined with a kappa or lambda light chain. Generally, light and heavy chains are covalently bonded to each other, and when immunoglobulins are produced by hybridomas, B cells, or genetically engineered host cells, the "tails" of the two heavy chains are bonded to each other by covalent disulfide bonds or non-covalent bonds. In the heavy chain, its amino acid sequence is from the N-terminus at the end of the Y-configuration bifurcation to the C-terminus at the bottom of each chain.

[0099] As used herein, the term "light chain constant region" or "CL" is used interchangeably herein to refer to an amino acid sequence 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.

[0100] The term "heavy chain constant region" as used herein includes an amino acid sequence derived from an immunoglobulin heavy chain. The polypeptide comprising the heavy chain constant region includes at least one of the following groups: a CH1 domain, a hinge (e.g., the upper, middle and / or lower part of the hinge region) domain, a CH2 domain, a CH3 domain, or a variant or fragment thereof. For example, the antigen-binding polypeptide used in the present disclosure may include a polypeptide chain comprising a CH1 domain; a polypeptide chain comprising a CH1 domain, at least a portion of a hinge domain and a CH2 domain; a polypeptide chain comprising a CH1 domain and a CH3 domain; a polypeptide chain comprising a CH1 domain, at least a portion of a hinge domain and a CH3 domain, or a polypeptide chain comprising a CH1 domain, at least a portion of a hinge domain, a CH2 domain and a CH3 domain. In some embodiments, the polypeptide of the present invention includes a polypeptide chain comprising a CH3 domain. In addition, the antibodies used in the present disclosure may lack at least a portion of a CH2 domain (e.g., all or part of a CH2 domain). It should be understood that the heavy chain constant region may be modified so that it is different from a natural immunoglobulin molecule in terms of amino acid sequence.

[0101] The heavy chain constant region of the antibodies disclosed herein may be derived from different immunoglobulin molecules. For example, the heavy chain constant region of the polypeptide may comprise a polypeptide derived from IgG 1The CH1 domain of the molecule and the IgG 3 In another example, the heavy chain constant region may comprise a portion derived from IgG 1 Molecules and partly derived from IgG 3 In another example, the heavy chain portion may include a portion derived from IgG 1 Molecules and partly derived from IgG 4 The chimeric hinge of the molecule.

[0102] The subunit structures and three-dimensional configurations of the constant regions of various immunoglobulin classes are well known. The term "VH domain" as used herein includes the N-terminal variable domain of an immunoglobulin heavy chain, and the term "CH1 domain" includes the first (most N-terminal) constant region domain of an immunoglobulin heavy chain. The CH1 domain is adjacent to the VH domain and is located at the N-terminus of the hinge region of an immunoglobulin heavy chain molecule.

[0103] The term "CH2 domain" as used herein includes the portion of the heavy chain molecule extending from, for example, about residue 244 to residue 360 ​​of an antibody, wherein the numbering uses the conventional numbering scheme (residues 244 to 360, Kabat numbering system; or residues 231-340, EU numbering system). The CH2 domain is unique in that it is not closely paired with another domain. In contrast, there are two N-linked branched sugar chains inserted between the two CH2 domains of a complete native IgG molecule. The CH3 domain extends from the CH2 domain to the C-terminus of the IgG molecule and comprises approximately 108 residues.

[0104] The term "hinge region" as used herein includes the portion of the heavy chain molecule that connects the CH1 domain to the CH2 domain. The hinge region contains approximately 25 residues and is flexible, thus allowing the two N-terminal antigen binding regions to move independently. The hinge region can be subdivided into three different domains: the upper hinge domain, the middle hinge domain, and the lower hinge domain.

[0105] The term "disulfide bond" as used herein includes a covalent bond formed between two sulfur atoms. The amino acid cysteine ​​contains a thiol group that can form a disulfide bond or bridge with a second thiol group. In most naturally occurring IgG molecules, the CH1 and CL regions are linked by disulfide bonds, and the two heavy chains are linked by two disulfide bonds corresponding to positions 239 and 242 (Kabat numbering system), which are positions 226 or 229 in the EU numbering system.

[0106] The term "antibody" includes various broad classes of polypeptides that can be distinguished biochemically. It will be appreciated by those skilled in the art that heavy chains are classified as alpha, delta, epsilon, gamma and mu (or α, δ, ε, γ and μ) and some subclasses therein (e.g., γ1-γ4). The nature of the chain determines the "class" of the antibody, which is IgG, IgM, IgA, IgD or IgE, respectively. Immunoglobulin subclasses (isotypes) such as IgG1, IgG2, IgG3, IgG4, IgG5, etc. have been fully characterized and are known to confer functional specialization. In view of the present disclosure, modified forms of each of these classes and isotypes are easily discernible to those skilled in the art and are therefore included within the scope of the present disclosure. All immunoglobulin classes are within the scope of the present disclosure, and the following discussion is generally directed to immunoglobulin molecules of the IgG class.

[0107] Antibodies disclosed herein 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. Antibodies disclosed herein may be from any animal source, including birds and mammals. Preferably, the antibody is an antibody of a human, mouse, donkey, rabbit, goat, guinea pig, camel, llama, horse, or chicken. In some embodiments, the variable region may be derived from an aquatic species (e.g., from a shark).

[0108] The phrase "chimeric antibody" as used herein refers to an antibody in which the immunoreactive region or site is obtained or derived from a first species and the constant region (which may be 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 of non-human origin (e.g., mouse or primate) and the constant region is of human origin.

[0109] The term "humanized antibody" as used herein refers to a genetically engineered non-human antibody comprising a human antibody constant domain and a modified non-human variable domain with a high degree of sequence homology to a human variable domain. This can be achieved by transplanting the six non-human antibody complementary determining regions (CDRs) that form an antigen binding site onto a homologous human receptor framework region (FR). In order to rebuild the binding affinity and specificity of the parental antibody, the framework region residues of the parental antibody (i.e., non-human antibody) need to be replaced with human framework region residues (reverse mutations). Structural homology modeling can help identify amino acid residues that are important to antibody binding properties in the framework region. Therefore, a humanized antibody can include a non-human CDR sequence, mainly a human framework region that optionally includes one or more amino acid reverse mutations to a non-human amino acid sequence, and a fully human constant region. Alternatively, other amino acid modifications (which are not necessarily reverse mutations) can be applied to obtain humanized antibodies with preferred features such as affinity and biochemical properties.

[0110] "Variable single-chain fragment" or "scFv" refers to a fusion protein of the variable region of the heavy chain (VH) and the variable region of the light chain (VL) of an immunoglobulin. In some aspects, these regions are connected by a short connecting peptide of 10 to about 25 amino acids. The connecting peptide can be rich in glycine to improve flexibility, or rich in serine or threonine to improve solubility, and the N-terminus of VH can be connected to the C-terminus of VL, and vice versa. Although the constant region is removed and the connecting peptide is introduced in the protein, it still retains the specificity of the original immunoglobulin.

[0111] In some embodiments, the antibodies of the present invention can be recombinant antibodies. The term "recombinant antibody" as used herein is intended to include all antibodies prepared, expressed, produced or separated by a recombinant method, such as antibodies expressed by a recombinant expression vector transfected into a host cell, antibodies separated from a recombinant combinatorial antibody library, or antibodies prepared, expressed, produced or separated by any other method involving splicing immunoglobulin gene sequences to other DNA sequences. Such recombinant antibodies have variable regions and constant regions derived from germline immunoglobulin sequences. However, in certain embodiments, such recombinant antibodies are subject to in vitro mutagenesis, so that the amino acid sequences of the VH and VL regions of the recombinant antibodies are derived from and related to germline VH and VL sequences, but may not naturally exist in sequences in the antibody germline library in vivo.

[0112] The term "conjugate" or "antibody conjugate" refers to an antibody linked to one or more agents. The antibody can be covalently linked to the agent via a covalent bond or a linker. In certain embodiments, the linker is covalently bound to the antibody and also covalently bound to the agent. In certain embodiments, the linker is non-covalently linked to the antibody and / or the agent. In certain embodiments, the linker is covalently linked to the agent and is linked to the antibody via specific binding. In certain embodiments, the linker is a portion that can specifically bind to the antibody, for example, an antibody that binds to the Fc region of an antibody.

[0113] 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 a paratope. A single antigen may have multiple epitopes. Thus, different antibodies may bind to different regions of the antigen and may have different biological effects. Epitopes may be conformational or linear. Conformational epitopes are produced by the juxtaposition of amino acids in space in different segments of a linear polypeptide chain. Linear epitopes are produced by adjacent amino acid residues in a polypeptide chain. In some cases, an epitope may include a sugar, phosphoryl, or sulfonyl moiety on the antigen.

[0114] The term "immunoconjugate" refers to an antibody fused to a peptide or small molecule drug by a covalent bond. The peptide or small molecule drug can be linked to the C-terminus of the constant heavy chain or the N-terminus of the variable light chain and / or heavy chain.

[0115] As used herein, the terms "improve", "increase" or "decrease" refer to a value or parameter measured relative to a baseline / control / reference, such as a measurement in a cell or tissue before the start of a treatment described herein, or a measurement in a cell or tissue without a treatment described herein, a measurement in the same individual before starting a treatment described herein, or a measurement in a control individual without a treatment described herein (or a standard measurement in multiple control individuals, such as an average of multiple control individuals). A "control individual" is an individual with a similar condition, for example, an individual with the same cell proliferative disease as the individual being treated, and whose age is approximately the same as the individual being treated (to ensure that the stage of the disease is comparable between the treated individual and the control individual). The individual being treated (also referred to as a "patient" or "subject") can be a fetus, infant, child, adolescent or adult with a cell proliferative disease.

[0116] The term "recombinant" as used herein refers to a polypeptide or polynucleotide that does not occur in nature, which can be produced by combining polynucleotides or polypeptides in an arrangement that does not normally occur 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.

[0117] The term "prevention" refers to reducing the occurrence of disease symptoms (e.g., related to PD-L1 activity or its function) in a patient. As described above, the prevention can be complete (no detectable symptoms) or partial, such that fewer symptoms are observed than would be possible without treatment.

[0118] The phrase "small molecule drug" refers to a molecular entity, usually organic or organometallic, not a polymer, that is pharmaceutically active 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 includes most pharmaceutical compounds referred to as "drugs" other than proteins or nucleic acids, but small peptides or nucleic acid analogs may be considered small molecule drugs. Examples include chemotherapeutic anticancer drugs and enzyme inhibitors. Small molecule drugs can be obtained by synthesis, semi-synthesis (i.e., from naturally occurring precursors) or biological methods.

[0119] The term "specific binding" or "specifically binding" as used herein refers to the ability to distinguish possible binding partners in an environment where binding occurs. In some embodiments, when other potential antibodies are present, an antibody that interacts with a specific antigen, such as a preferential interaction, is referred to as "specifically binding" to the antigen that interacts with it. 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 another antibody for an alternative interaction with its target. In some embodiments, specific binding is assessed by performing such detection or determination over a range of concentrations. Typically, an antibody binds to an epitope through its antigen binding domain, and the binding requires a certain complementarity between the antigen binding domain and the epitope. Therefore, when an antibody binds to an epitope through its antigen binding domain more easily than it binds to a random, unrelated epitope, the antibody is referred to as "specifically binding" the epitope. The term "specificity" is used herein to define the relative affinity of a specific antibody to bind to a specific epitope. For example, antibody "A" may be considered to have a higher specificity for a given epitope than antibody "B," or antibody "A" may be considered to have a higher specificity for binding epitope "C" than for the related epitope "D." In some embodiments, if the dissociation constant (K) of the complex formed by the antibody or antigen-binding fragment thereof and the antigen is d ) is 10 -6 M or smaller, 10 -7 M or smaller, 10 -8 M or smaller, 10 -9 M or smaller, or 10 -10 M or less, then the antibody or antibody fragment is "specific" for the antigen. In certain embodiments, the specific binding of the antigen-binding molecule (e.g., anti-human PD-L1 antibody or antigen-binding fragment thereof) is shown by the preferential binding of the antigen-binding molecule to human PD-L1 expressed on the cell surface using the detection methods described in Examples 4-7, or substantially similar methods.

[0120] The term "substantially identical" or "substantially identical" when referring to a nucleic acid or a fragment thereof means that when optimally aligned with another nucleic acid (or its complementary strand) with appropriate nucleotide insertions or deletions, there is at least about 95% identity, more preferably at least about 96%, 97%, 98% or 99% identity of the nucleotide bases to that nucleotide sequence as determined by any well-known sequence identity algorithm, such as FASTA, BLAST or the Gap method, 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 that is identical or substantially similar to that of the polypeptide encoded by the reference nucleic acid molecule.

[0121] When applied to polypeptides, the term "substantial similarity" or "substantially similar" means that two peptide sequences share at least 95% identical sequence, even more preferably at least 98% or 99% sequence identity when optimally aligned by programs such as GAP or BESTFIT using default gap weights. Preferably, residue positions that are not identical differ by conservative amino acid substitutions. "Conservative amino acid substitutions" refer to substitutions of one amino acid residue by another amino acid residue having a similar chemical property (e.g., charge or hydrophobicity) side chain (R group). In general, conservative amino acid substitutions do not substantially change the functional properties of proteins. In the case where two or more amino acid sequences differ from each other by conservative substitutions, the sequence identity percentage or similarity can be adjusted upward to correct for the conservatism of the substitution. Methods for making such adjustments are well known to those skilled in the art. See, for example, 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, and isoleucine; (2) aliphatic hydroxyl side chains: serine and threonine; (3) amide-containing side chains: asparagine and glutamine; (4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; (5) basic side chains: lysine, arginine, and histidine; (6) acidic side chains: aspartic acid and glutamic acid, (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 is any change that has a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256:1443-1445. A "moderately conservative" replacement is any change that has a non-negative value in the PAM250 log-likelihood matrix.

[0122] The sequence similarity of polypeptides, also referred to as sequence identity, is usually determined using sequence analysis software. Protein analysis software uses similarity metrics assigned to various substitutions, deletions and other modifications (including conservative amino acid substitutions) to match similar sequences. For example, GCG software includes programs such as Gap and Bestfit, which can use default parameters to determine the sequence homology or sequence identity between closely related polypeptides (e.g., homologous polypeptides from different biological species, or homologous polypeptides of wild-type proteins and their mutant proteins). See, for example, GCG version 6.1. Polypeptide sequences can also be compared using default or recommended parameters by FASTA, which is a program in GCG version 6.1. FASTA (e.g., FASTA2 and FASTA3) provides comparison and sequence identity percentages of the best overlapping regions between query and search sequences (Pearson (2000) supra). When comparing the sequence of the present invention with a database containing a large number of sequences from different organisms, another preferred algorithm is a computer program BLAST using default parameters, especially BLASTP or TBLASTN. See, e.g., Altschul et al., (1990) J. Mol. Biol. 215:403-410 and Altschul et al., (1997) Nucleic Acids Res. 25:3389-402.

[0123] The terms "therapeutically effective amount", "pharmacologically effective amount" and "physiologically effective amount" are used interchangeably and refer to the amount of an active agent sufficient to improve at least one symptom of a disease or condition. For example, for a given parameter, a therapeutically effective amount will show an increase or decrease of at least 5%, 10%, 15%, 20%, 25%, 40%, 50%, 60%, 75%, 80%, 90%, 95%, 99% or at least 100%. The therapeutic effect can also be expressed as an increase or decrease in "folds". For example, a therapeutically effective amount can have an effect of at least 1.2 times, 1.5 times, 2 times, 5 times or more than a control. The exact amount will depend on many factors, such as the specific active agent, the components and physical properties of the composition, the expected patient population, patient considerations, including weight, gender, etc., and can be easily determined by those skilled in the art based on the information provided herein or other information provided in the relevant literature.

[0124] The terms "treat" and "treat" refer to ameliorating one or more symptoms associated with a disease or condition. Thus, these terms refer to any indication of success in treating or ameliorating an injury, disease, pathology, or condition, including preventing or delaying the onset of one or more symptoms of a disease or condition; reducing the severity or frequency of one or more symptoms of a disease or condition; any objective or subjective parameter, such as relief, remission; symptom reduction or making the injury, pathology, or condition more tolerable to the patient; slowing the rate of degeneration or decline; reducing frailty at the degenerative endpoint; and / or improving the patient's physical or mental well-being. "Treatment" may also include prophylactic treatment.

[0125] The phrases "a patient in need thereof," "a patient in need of treatment," or "a subject in need of treatment" include subjects (eg, mammalian subjects) who would benefit from administration of an antibody of the disclosure in the treatment of a cell proliferative disorder.

[0126] The term "variant" as used herein refers to a polypeptide or polynucleotide, such as an antibody or polynucleotide, obtained by inserting, replacing or deleting one or more amino acids or nucleotides 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% of the amino acid or nucleotide sequence of the full amino acid or nucleotide sequence of the parent polypeptide or polynucleotide, such as about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% of the amino acid or nucleotide sequence is identical. The variant of a protein or peptide substantially maintains the structure, function or activity of the protein. For example, a variant of an antibody maintains the function or activity and / or regulation of its antigen specifically bound, such as suppressing antigen activity. In the case of a polynucleotide, its variant maintains the function or activity of its parent polynucleotide. For example, a variant polynucleotide can encode a protein or peptide having a similar function or activity to a polypeptide encoded by a parent polynucleotide.

[0127] The term "PD-L1" as used herein refers to programmed death ligand 1. Unless otherwise specified (e.g., specific reference to human PD-L1), the term "PD-L1" includes natural PD-L1 from all mammalian species, such as humans, primates, rodents, canines, felines, equines, and bovines. The nucleotide and amino acid sequences of PD-L1 are known and can be found in, for example, 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. The following is an exemplary human PD-L1 amino acid sequence: (SEQ ID NO:) MRIFAVFIFMTYWHLLNAFTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQ FVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVN APYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRI NTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNERTHLVILGAILLCLGVALTFIFRLRKGRMMD VKKCGIQDTNSKKQSDTHLEET

[0128] The exemplary amino acid sequence of cynomolgus macaque PD-L1 is shown below: (SEQ ID NO:) MRIFAVFIFTIYWHLLNAFTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLTSLIVYWEMEDKNIIQFVHG EEDLKVQHSNYRQRAQLLKDQLSLGNAALRITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKIN QRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLLNVTSTLRINTTANEIFYC IFRRLDPEENHTAELVIPELPLALPPNERTHLVILGAIFLLLGVALTFIFYLRKGRMMDMKKCGIRVTNSK KQRGKNIRRNWEVEGKGNKKLKQ

[0129] 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 is capable of inhibiting PD-L1 biological activity and / or inhibiting downstream signaling pathways mediated by PD-L1. Anti-PD-L1 antibodies include antibodies or polypeptides containing one or more CDR or variable region-formed antigen binding domains. In certain embodiments, the anti-PD-L1 antibodies of the present invention (to any extent, including significantly) block, antagonize, inhibit or reduce PD-L1 biological activity, including downstream events mediated by PD-L1, such as PD-L1 binding, PD1 / PD-L1 interactions and downstream signaling, inhibition of anti-tumor immune responses, and immunosuppression in immune-damaged disease states. In certain embodiments, the anti-PD-L1 antibodies of the present invention are capable of endocytosis into tumor cells expressing PD-L1 and triggering antibody-drug conjugate-induced tumor cell killing. 2. PD-L1 Antibodies and Antigen Binding Proteins

[0130] The present invention provides a PD-L1 antigen binding molecule that specifically binds to PD-L1. The term "antigen binding molecule" used herein refers to a protein, polypeptide or molecular complex that specifically binds to a specific antigen, which contains or consists of at least one complementary determining region (CDR), which exists alone or in combination with one or more other CDRs and / or framework regions (FRs). As the term is defined elsewhere herein, in certain embodiments, the antigen binding molecule is an antibody or an antigen binding fragment thereof. In certain embodiments, the antigen binding molecule described in the present invention inhibits one or more biological functions of PD-L1.

[0131] In certain embodiments, PD-L1 is human PD-L1. An exemplary human PD-L1 comprises the amino acid sequence shown in SEQ ID NO:. In some embodiments, the PD-L1 is cynomolgus monkey PD-L1. An exemplary cynomolgus monkey PD-L1 comprises the amino acid sequence shown in SEQ ID NO:.

[0132] 1. Sequences of Exemplary Antigen Binding Molecules

[0133] The PD-L1 antigen binding molecule can be in the form of a monoclonal antibody; in the form of one or more polypeptide fragments containing one or more PD-L1 antigen binding domains; or in the form of one or more nucleic acids encoding one or more PD-L1 binding domains.

[0134] In various exemplary embodiments of the present invention, an antigen binding molecule, such as an anti-PD-L1 antibody or an 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 the exemplary anti-human PD-L1 monoclonal antibodies disclosed herein are shown in Tables 1-5.

[0135] The amino acid sequence boundaries of a CDR can be determined by one of skill in the art using any of a number of known numbering schemes, including the scheme described by Kabat et al., supra (the "Kabat" numbering scheme); the scheme described by Al-Lazikani et al., 1997, J. Mol. Biol., 273:927-948 (the "Chothia" numbering scheme); the scheme described by MacCallum et al., 1996, J. Mol. Biol. 262:732-745 (the "Contact" numbering scheme); the scheme described by Lefranc et al., Dev. Comp. Immunol., 2003, 27:55-77 (the "IMGT" numbering scheme); and the scheme described by Honegge and Pluckthun, J. Mol. Biol, 2001, 309:657-70 (the "AHo" numbering scheme); each of which is incorporated herein by reference in its entirety. Tables 1 and 2 show the heavy chain CDRs sequences of exemplary antibodies of the present invention obtained according to the Kabat numbering scheme and the IMGT numbering scheme, respectively. Table 3 shows the sequences of heavy chain CDRs of exemplary antibodies of the present invention, wherein the CDR sequences are defined according to the combination of CDRs based on the Kabat and IMGT numbering schemes. Tables 4 and 5 show the light chain CDR sequences of exemplary antibodies of the present invention obtained according to the Kabat numbering scheme and the IMGT numbering scheme.

[0136] In certain embodiments, the present invention includes antigen binding molecules, such as anti-PD-L1 antibodies or antigen binding fragments thereof, comprising CDRs defined based on the Kabat and IMGT numbering schemes or a combination thereof. Thus, in certain embodiments, the present invention includes an antigen-binding molecule, such as an anti-PD-L1 antibody or an antigen-binding fragment thereof, comprising (1) a 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 group consisting of the HCDR1 sequences listed in Tables 1 and 6; (2) a 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 group consisting of the HCDR2 sequences listed in Tables 1 and 9; (3) a 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 group consisting of the HCDR4 sequences listed in Tables 1 and 11. (4) a LCDR1 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 group consisting of the LCDR1 sequences listed in Tables 4 and 13; (5) a LCDR2 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 group consisting of the LCDR2 sequences listed in Tables 4 and 15; and (6) a LCDR3 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 group consisting of the LCDR3 sequences listed in Tables 4 and 16.

[0137] In certain embodiments, the present invention relates to an antigen-binding molecule, such as an anti-PD-L1 antibody or an antigen-binding fragment thereof, comprising (1) a HCDR1 having an amino acid sequence selected from the group consisting of the HCDR1 sequences listed in Tables 2 and 7 that is about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identical; (2) a HCDR2 having an amino acid sequence selected from the group consisting of the HCDR2 sequences listed in Tables 2 and 10 that is about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identical; (3) a HCDR3 having an amino acid sequence selected from the group consisting of the HCDR4 sequences listed in Tables 2 and 12 that is about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identical. (4) a LCDR1 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 LCDR1 sequences listed in Tables 5 and 14; (5) a LCDR2 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 LCDR2 sequences listed in Table 5; and (6) a LCDR3 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 LCDR3 sequences listed in Tables 5 and 16.

[0138] In certain embodiments, the present invention relates to an antigen-binding molecule, such as an anti-PD-L1 antibody or an antigen-binding fragment thereof, comprising (1) a 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 group consisting of the HCDR1 sequences listed in Tables 3 and 8; (2) a 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 group consisting of the HCDR2 sequences listed in Tables 3 and 9; (3) a 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 group consisting of the HCDR4 sequences listed in Tables 3 and 11. (4) a LCDR1 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 group consisting of the LCDR1 sequences listed in Tables 4 and 13; (5) a LCDR2 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 group consisting of the LCDR2 sequences listed in Tables 4 and 15; and (6) a LCDR3 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 group consisting of the LCDR3 sequences listed in Tables 4 and 16.

[0139] As used herein, a "position" in a CDR refers to an amino acid counted from the N-terminus of the CDR. For example, position 1 or position 1 in HCDR1 refers to the first amino acid in HCDR1. Thus, in clone 27A2, position 1 of HCDR1 based on the Kabat numbering scheme is serine (S).

[0140] Table 1: Amino acid sequences of heavy chain CDRs of exemplary antibodies (Kabat numbering scheme)

[0141] Table 2: Amino acid sequences of heavy chain CDRs of exemplary antibodies (IMGT numbering scheme)

[0142] Table 3: Amino acid sequences of heavy chain CDRs of exemplary antibodies (combination of Kabat and IMGT numbering schemes)

[0143] Table 4: Amino acid sequences of light chain CDRs of exemplary antigen binding molecules (Kabat numbering scheme)

[0144] Table 5: Amino acid sequences of light chain CDRs of exemplary antigen binding molecules (IMGT numbering scheme)

[0145] Tables 6-16 show the consensus sequences of several CDRs in several exemplary antibodies of the present disclosure.

[0146] Table 6: Consensus sequences of heavy chain CDR1 of exemplary antigen binding molecules (Kabat numbering scheme) 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.

[0147] Table 7: Consensus sequences of heavy chain CDR1 of exemplary antigen binding molecules (IMGT numbering scheme) Clone HCDR1 SEQ ID NO: 27A2 GFSFSSNHY 26G2 GFSFSSNHY 28A12 GFSFSSAYY 72G5 GFSFSASYY N / A G-F-S-F-S-X10-X11-X12-Y 67B6 EFSFSSNYY 65B6 EFSFGANYY 56H3 EFSFSNNYY 39C10 EFSFSNNYY 27C10 EFSFSSNYY N / A E-F-S-F-X13-X14-N-Y-Y 74A9 GFSLSTYA 38D10 GIDLSTYA N / A G-X15-X16-L-S-T-Y-A 55H3 / 76D6 GFSFSGDYY 56E5 GFSFSGDYY 57B10 GFSFSDDYY 57F11 GFSFSGDYY 62E6 GFSFSGDYY 69D10 GFSFSGDWY 73C8 GFSFSGDYY 74F4 GFSFSGDYY N / A G-F-S-F-S-X17-D-X18-Y 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.

[0148] Table 8: Consensus sequences of heavy chain CDR1 of exemplary antigen binding molecules (combination of Kabat and IMGT numbering schemes) Clone HCDR1 SEQ ID NO: 27A2 GFSFSSNHYMS 26G2 GFSFSSNHYMC 28A12 GFSFSSAYYMC 72G5 GFSFSASYYMC N / A G-F-S-F-S-X19-X20-X21-Y-M-X22 67B6 EFSFSSNYYMC 65B6 EFSFGANYYMC 56H3 EFSFSNNYYMS 39C10 EFSFSNNYYMC 27C10 EFSFSSNYYMC N / A E-F-S-F-X23-X24-N-Y-Y-M-X25 33E8 GFYFSSGYDMS 53G7 GFYFSSGYDMC N / A G-F-Y-F-S-S-G-Y-D-M-X26 74A9 GFSLSTYALG 38D10 GIDLSTYAMG N / A G-X27-X28-L-S-T-Y-A-X29-G 55H3 / 76D6 GFSFSGDYYMS 56E5 GFSFSGDYYMS 57B10 GFSFSDDYYMS 57F11 GFSFSGDYYMS 62E6 GFSFSGDYYMS 69D10 GFSFSGDWYMS 73C8 GFSFSGDYYMS 74F4 GFSFSGDYYMS N / A G-F-S-F-S-X30-D-X31-Y-M-S 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.

[0149] Table 9: Consensus sequences of heavy chain CDR2 of exemplary antigen binding molecules (Kabat numbering scheme) 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.

[0150] Table 10: Consensus sequences of heavy chain CDR2 of exemplary antigen binding molecules (IMGT numbering scheme) 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: Consensus sequences of heavy chain CDR3 of exemplary antigen binding molecules (Kabat numbering scheme) Clone HCDR3 SEQ ID NO: 33E8 TKDGVGSFYMNL 53G7 TRDGAGSFYMNL N / A T-X51-D-G-X52-G-S-F-Y-M-N-L 74A9 ARNVDSIYLDAFHT 38D10 ARNVDIIYLNAFDI N / A A-R-N-V-D-X53-I-Y-L-X54-A-F-X55-X56 55H3 / 76D6 RNKNADYGHFDL 56E5 RHKNADYGHFDL 57B10 RNKNADWGHFDL 57F11 RTKNGDYGYFDL 62E6 RNKNADYGHFDL 69D10 RNKNADYGHFDL 73C8 RNKNADYGHFDL 74F4 RNKNADYGHFDL N / A R-X57-K-N-X58-D-X59-G-X60-F-D-L 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.

[0152] Table 12: Consensus sequences of heavy chain CDR3s of exemplary antigen binding molecules (IMGT numbering scheme) Clone HCDR3 SEQ ID NO: 33E8 TKDGVGSFYMNL 53G7 TRDGAGSFYMNL N / A T-X51-D-G-X52-G-S-F-Y-M-N-L 74A9 ARNVDSIYLDAFHT 38D10 ARNVDIIYLNAFDI N / A A-R-N-V-D-X53-I-Y-L-X54-A-F-X55-X56 55H3 / 76D6 ARRNKNADYGHFDL 56E5 ARRHKNADYGHFDL 57B10 ARRNKNADWGHFDL 57F11 ARRTKNGDYGYFDL 62E6 ARRNKNADYGHFDL 69D10 ARRNKNADYGHFDL 73C8 ARRNKNADYGHFDL 74F4 ARRNKNADYGHFDL N / A A-R-R-X61-K-N-X62-D-X63-G-X64-F-D-L 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.

[0153] Table 13: Consensus sequences of light chain CDR1 of exemplary antigen binding molecules (Kabat numbering scheme) 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.

[0154] Table 14: Consensus sequences of light chain CDR1 of exemplary antigen binding molecules (IMGT numbering scheme) 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.

[0155] Table 15: Consensus sequences of light chain CDR2 of exemplary antigen binding molecules (Kabat numbering scheme) 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.

[0156] Table 16: Consensus sequences of light chain CDR3s of exemplary antigen binding molecules (combination of Kabat and IMGT numbering schemes) Clone LCDR3 SEQ ID NO: 33E8 QQGATRINIDNA 53G7 QQGASRINIDNA N / A Q-Q-G-A-X108-R-I-N-I-D-N-A 74A9 QTYYLTTTTNA 38D10 QTYYLTSTINA N / A Q-T-Y-Y-L-T-X109-T-X110-N-A 55H3 / 76D6 QQGWTVSSLDNA 56E5 QQGWTVSSLDNA 57B10 QQGWTVSSLDNA 57F11 QQGWTVSSLENA 62E6 QQGWTVSSLDNA 69D10 QQGWTVSSLDNA 73C8 QQGWTVSSLDNA 74F4 QQGWTVSSLDNA N / A Q-Q-G-W-T-V-S-S-L-X111-N-A As used herein, X108 is S or T, X119 is S or T, X110 is I or T, and X111 is D or E.

[0157] 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 group consisting of 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 group consisting of 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 shown in Tables 17-20.

[0158] Table 17: Amino acid sequences of HCVRs of exemplary antigen binding molecules

[0159] Table 18: Amino acid sequences of pyroglutamylated HCVRs of exemplary antigen binding molecules

[0160] Table 19: Amino Acid Sequences of LCVRs of Exemplary Antigen Binding Molecules

[0161] Table 20: Amino acid sequences of pyroglutamylated LCVRs of exemplary antigen binding molecules

[0162] In certain embodiments, the antigen binding molecules of the present invention, such as antibodies or their antigen binding fragments, are modified after translation. 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 at the N-terminus of the heavy chain and light chain to pyroglutamic acid by pyroglutamylation; glycosylation; oxidation; deamidation; and saccharification, which are known to occur in various antibodies (see journal of Pharmaceutical Sciences, 2008, Vol. 97, pp. 2426-2447, which are incorporated herein by reference in their entirety). Examples of post-translationally modified antigen binding molecules such as antibodies or their antigen binding fragments include antigen binding molecules such as antibodies or their antigen binding fragments that have been pyroglutamylated at the N-terminus of the heavy chain variable region and / or antigen binding molecules such as antibodies or their antigen binding fragments that have deleted lysine at the C-terminus of the heavy chain. The sequences of exemplary antigen binding molecules of pyroglutamylation at the N-terminus are listed in Table 7. When used to represent amino acids in polypeptides, "pE" as used herein refers to pyroglutamic acid.

[0163] 2. Variants of Antigen Binding Molecules

[0164] In certain embodiments, the PD-L1 antigen binding molecule of the present invention, such as the anti-PD-L1 antibody, can be a monoclonal antibody, a chimeric antibody, a humanized antibody, a Fab, a (Fab) 2 , scFv, or multispecific antibodies comprising other binding specificities described herein.

[0165] In certain embodiments, the anti-PD-L1 antibodies described herein may be linked to an Fc comprising one or more modifications to change one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and / or antigen-dependent cellular cytotoxicity. In addition, the antibodies described herein may be chemically modified (e.g., one or more chemical moieties are attached to the antibody) or modified to change glycosylation, thereby changing one or more functional properties of the antibody. More specifically, in certain embodiments, the antibodies described herein may include modifications in the Fc region to produce Fc variants having the following characteristics: (a) increased or decreased antibody-dependent cell-mediated cytotoxicity (ADCC), (b) increased or decreased complement-mediated cytotoxicity (CDC), (c) increased or decreased affinity for Clq, and / or (d) increased or decreased affinity for Fc receptors than the parent Fc. Such Fc region variants typically include at least one amino acid modification in the Fc region. It is highly desirable to combine amino acid modifications. For example, an Fc region variant may include two, three, four, five, etc. substitutions at specific Fc region sites, e.g., as described herein.

[0166] For purposes where effector functions need to be avoided completely, for example, when antigen binding alone is sufficient to produce the desired therapeutic effect and effector functions only result in undesirable side effects (or increase their risk), IgG4 antibodies or IgG1 L234F, L235E and P331S variants without ADCC effects can be used, or antibodies or fragments lacking the Fc region or most of it can be designed, or Fc can be mutated to completely eliminate glycosylation (e.g., N297A). Alternatively, a hybrid construct of human IgG2 (CH1 domain and hinge region) and human IgG4 (CH2 and CH3 domains) can be generated that has no effector function and lacks the ability to bind FcγRs (like IgG2) and activate complement (like IgG4). When using an IgG4 constant domain, it is generally preferred to include an S228P substitution that mimics the IgG1 hinge sequence and an R409K mutation that prevents Fab arm exchange and thus stabilizes the IgG4 molecule, thereby reducing the Fab arm exchange between therapeutic antibodies and endogenous IgG4 in patients being treated.

[0167] In certain embodiments, the anti-PD-L1 antibody or fragment thereof may be modified to provide an extended biological half-life. Various methods may be used, including, for example, methods for increasing the binding affinity of the Fc region to FcRn. In one embodiment, the CH1 or CL region of the antibody is changed to include a salvage receptor binding epitope from two loops of the CH2 domain of the Fc region of IgG, as described in U.S. Patents 5,278,288 and 6,121,022. The residue numbering in the Fc region is the residue numbering of the EU index of Kabat. The sequence variants disclosed herein are provided with reference to the residue numbering, and the residue numbering is followed by an amino acid that replaces a naturally occurring amino acid, optionally preceded by a natural residue at the site. In the case where multiple amino acids may be present at a given site, for example, if the sequences between naturally occurring isoforms are different, or if there are multiple mutations that can be substituted at the site, they are separated by slashes (e.g., "X / Y / Z").

[0168] Exemplary Fc variants that increase binding to FcRn and / or improve pharmacokinetic properties include substitutions at positions 259, 308, and 434, including, e.g., 259I, 308F, 428L, 428M, 434S, 434H, 434F, 434Y, and 434M. Other variants that increase Fc binding 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, 311A, 312A, 378Q, 380A, 382A, 434A (Shields et al., (2001) J. Biol. Chem. Hem., 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 8,367,805).

[0169] Modification of certain conserved residues in IgG Fc (I253, H310, Q311, H433, N434), such as the N434A variant (Yeung et al., (2009) J. Immunol. 182: 7663), can increase affinity with FcRn, thereby extending the half-life of the antibody in circulation (WO98 / 023289). An Fc variant comprising a combination of M428L and N434S has been shown to increase binding to FcRn and extend serum half-life by five times (Zalevsky et al., (2010) Nat. Biotechnol. 28: 157). An Fc variant comprising a combination of T307A, E380A and N434A modifications also extends the half-life of IgG1 antibodies (Petkova et al., (2006) Int. Immunol. 18: 1759). In addition, the Fc variants comprising the combination of M252Y-M428L, M428L-N434H, M428L-N434F, M428L-N434Y, M428L-N434A, M428L-N434M and M428L-N434S can also extend the half-life (US2006 / 173170). In addition, it is reported that the combined Fc variants comprising M252Y, S254T and T256E can extend the half-life by nearly 4 times (Dall'Acqua et al., (2006) J.Biol.Chem.281:23514).

[0170] In certain embodiments, the PD-L1 antigen binding molecule of 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 or another protein in PD-L1. In some embodiments, the first targeting domain comprises an antigen binding fragment of any PD-L1 antibody 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 proteins expressed on the surface of immune cells, such as T cells, NK cells, NKT cells, macrophages, dendritic cells, or myeloid suppressor cells (MDSC). Without wishing to be bound by any theory, it is assumed that bispecific antibodies can bind to PD-L1 on the surface of tumor cells and proteins expressed on the surface of immune cells. Therefore, bispecific antibodies help immune cells kill tumor cells.

[0171] In certain embodiments, the antigen binding molecules (e.g., anti-PD-L1 antibodies or antigen binding fragments thereof) of the present invention are chemically coupled to one or more therapeutically active peptides and / or small molecule drugs to form antibody drug conjugates (ADCs). The peptide or small molecule drug can be attached to, for example, a reduced SH group and / or a carbohydrate side chain. Methods for preparing covalent or non-covalent conjugates of peptides or small molecule drugs with antibodies are known in the art and any such known methods can be used. Without being bound by any theory, we assume that the ADC of the present invention can bind to PD-L1 expressed on the surface of tumor cells, introduce the drug into tumor cells through endocytosis of the antibody-antigen complex, and thereby kill the tumor cells through the drug coupled to the PD-L1 of the present invention.

[0172] In some embodiments, the peptide or small molecule drug is connected to the hinge region of the reduced antibody component via a disulfide bond. Alternatively, such agents can be connected using a heterobifunctional cross-linking agent (e.g., N-succinyl 3-(2-pyridyldithio) propionate (SPDP)). The general techniques for this coupling are well known in the art. In some embodiments, the peptide or small molecule drug is coupled via a carbohydrate moiety in the Fc region of the antibody. The carbohydrate group can be used to increase the load of the same reagent bound to the thiol group, or the carbohydrate moiety can be used to bind different therapeutic agents or diagnostic agents. Methods for coupling peptide inhibitors or small molecule drugs to antibodies via antibody carbohydrate moieties 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. The reaction causes an initial Schiff base (imine) connection, which can be stabilized by reduction to a secondary amine to form a final conjugate. Exemplary methods for coupling small molecule drugs and peptides to antibodies are described in U.S. Patent Publication 2014 / 0356385.

[0173] 3. Biological properties of antibodies and antigen-binding molecules

[0174] The present invention includes an antibody and antigen-binding fragment thereof that binds to human and cynomolgus monkey PD-L1.

[0175] The present invention includes a PD-L1 antigen binding molecule, such as a PD-L1 antibody or an antigen binding fragment thereof, which can specifically bind to human and cynomolgus monkey PD-L1 expressed on the cell surface and inhibit PD-L1 activity or function. According to certain embodiments, the antigen binding molecule blocks the interaction between human PD-L1 expressed on the cell surface and a PD-L1 agonist (e.g., PD-1). The degree to which the PD-L1 antigen binding protein (e.g., a PD-L1 antibody or an antigen binding fragment thereof) blocks the interaction between PD-1 and PD-L1 expressed on the cell surface can be assessed by the detection method described in Example 5 or a substantially similar method. The present invention includes an antigen binding molecule, such as an antibody or an antigen binding fragment thereof, which blocks the interaction between human PD-L1 expressed on the cell surface and PD-1, and its IC is measured by 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 lower, or between about 0.2 and 1.0 nM.

[0176] The present invention relates to a PD-L1 antigen binding molecule, such as a PD-L1 antibody or an antigen binding fragment thereof, which is capable of competing with PD-1 for binding to PD-L1 and blocking the interaction between PD-L1 and PD-1 protein. In certain embodiments, the antigen binding molecule described in the present invention, such as a PD-L1 antibody or an antigen binding fragment thereof, competes with PD-1 for binding to PD-L1 and blocks the interaction between PD-L1 and PD1, as determined using the competitive ELISA test described in Example 5. In certain embodiments, the present invention relates to an antigen binding molecule, such as an antibody or an antigen binding fragment thereof, which competes with PD-1 for binding to PD-L1 and blocks the interaction between PD-1 and PD-L1, and its IC 50 The value is 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 between about 1.0 and 10 nM, as determined using the competitive ELISA assay described in Example 5 or a substantially similar assay.

[0177] The present invention includes PD-L1 antigen binding molecules, such as PD-L1 antibodies or antigen binding fragments thereof, which specifically bind to human PD-L1 or non-human primate PD-L1. In certain embodiments, the antigen binding molecules of the present invention bind to PD-L1 family members other than PD-L1 or PD-L1 from certain non-human mammals (e.g., mouse PD-L1) undetectable or weakly, as determined using the test described in Example 3 or a substantially similar test. In certain embodiments, the present invention relates to antigen binding molecules, such as antibodies or antigen binding fragments thereof, which bind to human PD-L1 or cynomolgus monkey PD-L1 EC 50 The value is about 0.2nM, 0.1nM, 0.09nM, 0.08nM, 0.07nM, 0.06nM, 0.05nM, 0.04nM, 0.03nM, about 0.02nM, about 0.01nM, about 0.009nM, about 0.008nM, about 0.007nM or lower, or between about 0.007nM and 0.2nM, as determined using the assay described in Example 3 or a substantially similar assay. And the antigen binding molecule, e.g., PD-L1 antibody or antigen binding fragment thereof, does not bind to PD-L1 family members other than PD-L1 or PD-L1 from certain non-human mammals (e.g., mouse PD-L1).

[0178] The present invention relates to a PD-L1 antigen binding molecule, such as a PD-L1 antibody, which binds to human or cynomolgus monkey PD-L1 with high affinity. In certain embodiments, the present invention relates to an antigen binding molecule, such as an antibody or an antigen binding fragment thereof, which binds to human or cynomolgus monkey PD-L1, and its K D The value is about 10nM, about 5nM, about 2nM, about 1nM, about 0.9nM, about 0.8nM, about 0.7nM, about 0.6nM, about 0.5nM, about 0.4nM, about 0.3nM, about 0.2nM, about 0.1nM, about 0.09nM, about 0.08nM, about 0.05nM, about 0.04nM, about 0.03nM, about 0.02nM, about 0.01nM or less, or between about 0.01nM and 10nM, as determined using the assay described in Example 3 or a substantially similar assay.

[0179] The present invention relates to PD-L1 antigen binding molecules, such as PD-L1 antibodies or antigen binding fragments thereof, which specifically bind to cells expressing PD-L1, such as cells expressing endogenous human and / or non-human primate PD-L1 (e.g., cynomolgus monkey PD-L1) on the surface of human or non-human primate immune cells, as determined using the assay described in Example 4 or a substantially similar assay. In certain embodiments, the present invention relates to anti-PD-L1 antigen binding molecules, such as anti-PD-L1 antibodies or antigen binding fragments thereof, which bind to cells expressing human or non-human primate PD-L1, and whose EC 50 The value is about 20nM, about 18nM, about 15nM, about 10nM, about 10nM, about 5nM, about 4nM, about 3nM, about 2nM, about 1nM, about 0.9nM, about 0.8nM, about 0.7nM, about 0.6nM, about 0.5nM, about 0.4nM, about 0.3nM, about 0.2nM, about 0.1nM or less, or between about 0.1nM and 20nM, as determined using the assay described in Example 4 or a substantially similar assay.

[0180] The present invention relates to PD-L1 antigen binding molecules, such as PD-L1 antibodies or antigen binding fragments thereof, which induce antibody-dependent cellular cytotoxicity (ADCC) as determined using the assay described in Example 6 or a substantially similar assay. In certain embodiments, the present invention relates to anti-PD-L1 antigen binding molecules, such as anti-PD-L1 antibodies or antigen binding fragments thereof, which induce ECs for ADCC. 50 The value is about 10nM, about 5nM, about 4nM, about 3nM, about 2nM, about 1nM, about 0.9nM, about 0.8nM, about 0.7nM, about 0.6nM, about 0.5nM, about 0.4nM, about 0.3nM, about 0.2nM, about 0.1nM or less, or between about 0.1nM to about 10nM, between about 0.1nM to 2.5nM, or between about 1.0nM to 5.0nM, as determined using the assay described in Example 6 or a substantially similar assay.

[0181] The present invention relates to PD-L1 antigen binding molecules, such as PD-L1 antibodies or antigen-binding fragments thereof, which induce endocytosis when bound to PD-L1 (e.g., human or cynomolgus monkey PD-L1) expressed on the surface of a cell, as determined using the assay described in Example 7 or a substantially similar assay. In certain embodiments, the present invention relates to anti-PD-L1 antigen binding molecules, such as anti-PD-L1 antibodies or antigen-binding fragments thereof, which induce about 4%, about 5%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40% or more, or between about 4% and about 40% of the endocytosis of the cell surface complex formed by the binding of the PD-L1 antigen binding molecule (e.g., anti-PD-L1 antibody or antigen binding fragment thereof) to the cell surface PD-L1 (e.g., human or cynomolgus monkey PD-L1) as determined using the assay described in Example 7 or a substantially similar assay.

[0182] The present invention relates to a PD-L1 antigen binding molecule, such as a PD-L1 antibody or an antigen-binding fragment thereof, which, when coupled to a cytotoxic agent, induces a cytotoxic response in cells expressing PD-L1, as determined using the assay described in Example 8 or a substantially similar assay.

[0183] The present invention relates to PD-L1 antigen binding molecules, such as PD-L1 antibodies or antigen binding fragments thereof, which belong to the same epitope region as Avelumab or a different epitope region, as determined using the test described in Example 9 or a substantially similar test.

[0184] 4. Species selectivity and species cross-reactivity

[0185] According to certain embodiments, the present invention provides an antigen binding molecule that binds to human PD-L1 but not to PD-L1 of other species. The present invention also includes an antigen binding molecule that binds to human PD-L1 and PD-L1 of one or more non-human species (e.g., non-human primates).

[0186] According to certain exemplary embodiments of the present invention, the present invention provides an antigen binding molecule that binds to human PD-L1 and may or may not bind (as the case may be) to mouse, rat, guinea pig, hamster, gerbil, pig, cat, dog, rabbit, goat, sheep, cattle, horse, camel, cynomolgus monkey, marmoset, rhesus monkey or chimpanzee PD-L1. For example, in a specific exemplary embodiment of the present invention, the present invention provides an antigen binding molecule comprising an antigen binding domain that binds to human PD-L1 and non-human primate (e.g., cynomolgus monkey) PD-L1. III. Therapeutic Uses of Anti-PD-L1 Antigen Binding Molecules

[0187] The anti-PD-L1 antigen binding molecules (including antibodies, antigen-binding fragments thereof and multispecific antibodies thereof) of the present invention have a variety of in vitro, in vivo and ex vivo uses, which are related to enhancing immune responses by blocking adenosine signaling and other signaling in cancer treatment. Without wishing to be bound by any theory, it is assumed that the antigen binding molecules (e.g., anti-PD-L1 antibodies or antigen-binding fragments thereof) of the present invention bind to PD-L1 expressed on the cell surface and inhibit its interaction with PD-1, and this binding can produce a variety of effects, such as blocking the interaction between PD-1 and PD-L1 can enhance immune activity. Therefore, the antigen binding molecules (and therapeutic compositions comprising the same) of the present invention, among others, can be used to treat any disease or condition that can benefit from blocking the PD-1 / PD-L1 interaction (e.g., stimulating and / or activating an immune response). In view of the diverse effects mediated by the expression of PD-L1 and the interaction between PD-1 and PD-L1, the anti-PD-L1 antigen binding molecules (e.g., antibodies or antigen-binding fragments thereof) of the present invention can be used alone or in combination with a variety of active agents to treat a wide range of diseases or conditions, including a variety of cancers.

[0188] Therefore, the present invention provides a method for blocking the interaction between PD-1 and PD-L1, comprising contacting a cell with an antigen binding molecule described in the present invention (e.g., an anti-PD-L1 antibody or an antigen binding fragment thereof). The blocking of the interaction between PD-1 and PD-L1 can be determined by the method described in Example 5 or a substantially similar method. In certain embodiments, the methods described herein block 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.

[0189] In some embodiments, the antigen binding molecules described in the present invention (e.g., anti-PD-L1 antibodies or antigen binding fragments thereof) are administered to (in vitro) cultured cells or to human subjects (in vivo or ex vivo) to enhance immunity against a variety of diseases. In one embodiment, a method of stimulating an immune response in a subject in need thereof comprises administering to the subject an anti-PD-L1 antibody, an antigen binding fragment thereof (e.g., HCVR and LCVR of anti-PD-L1), or a multispecific anti-PD-L1 antibody described herein to enhance, stimulate, or upregulate an immune response in the subject, thereby, for example, inhibiting tumor growth, stimulating anti-tumor T cell immunity, and / or stimulating anti-microbial immunity.

[0190] On the one hand, the method of enhancing the immune response (e.g., T cell response) of a subject comprises administering an anti-PD-L1 antibody as described herein to a subject to enhance the immune response (e.g., T cell response) in the subject. In some embodiments, the subject is a subject with a tumor, and the immune response to the tumor is enhanced. The tumor may be a solid tumor or a liquid tumor, such as a hematological malignancy. In certain embodiments, the tumor is an immunogenic tumor. In other embodiments, the tumor is non-immunogenic. In other embodiments, the subject is a subject carrying a pathogen, and its immune response to the pathogen is enhanced due to the administration of an anti-PD-L1 antibody as 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 T cell-mediated anti-tumor immune response.

[0191] 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.

[0192] Preferred subjects include human patients who need to enhance immune response. The method is particularly suitable for treating human patients with conditions that can be treated by enhancing immune response (e.g., T cell-mediated immune response). The method is particularly suitable for treating cancer, chronic infection and chronic inflammatory diseases. Preferably, the antibodies used in the methods of the present disclosure described herein are human antibodies or humanized antibodies.

[0193] In one embodiment, the method of inhibiting the growth of tumor cells in a subject comprises administering to the subject an anti-PD-L1 antibody as described herein, thereby inhibiting the growth of a tumor in the subject. The inhibition of tumor growth can be determined by a variety of methods. The growth of a tumor can be determined using the following methods: for example, the method described in Talkington, A and Durrett, R, Estimating Tumor Growth Rates in vivo, Bull Math Biol., 2015 Oct.: 77 (10): 1934-54 (available in https: / / www.ncbi.nlm.nih.gov / pmc / articles / PMC4764475 / Inhibition of tumor growth can also be measured by a reduction in tumor size. In certain embodiments, the methods described herein inhibit tumor growth 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.

[0194] In certain embodiments, the antigen binding molecules described herein, such as anti-PD-L1 antibodies or antigen binding fragments thereof, can be used to reduce immunosuppression in the tumor microenvironment. This reduction can be measured by various methods. For example, the level of immunosuppression in the tumor microenvironment can be measured by the presence and / or abundance of certain biomarkers (e.g., PD-L1, CD39, CD73, LAG-3, IL-10, or TGF-β) in the tumor. It can also be measured by comparing the level of CD8+ cytotoxic T cells with regulatory T (Treg) cells in the tumor. reg The level of immunosuppression is determined by the ratio of CD8+ cytotoxic T cells to T cells. reg In certain embodiments, compared to the baseline level, the antigen binding molecules described in the present invention, such as anti-PD-L1 antibodies or antigen binding fragments thereof, reduce the level of immunosuppression 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.

[0195] Also included herein is the elimination of T cells from the tumor microenvironment of a subject having a tumor (e.g., a cancerous tumor). reg A method for stimulating the elimination of T cells in a tumor microenvironment comprises administering to a subject a therapeutically effective amount of an anti-PD-L1 antibody described herein. reg The cellular Fc may be, for example, an Fc with suitable effector function or enhanced effector function (caused by one or more activated Fc receptors).

[0196] In certain preferred embodiments, the subject suffers from a cell proliferative disease or cancer. Blocking the PD-1 / PD-L1 interaction with an antigen binding molecule described in the present invention (e.g., an anti-PD-L1 antibody or an antigen binding fragment thereof) can enhance the patient's immune response to cancer cells. Therefore, the present invention provides a method for treating a subject with cancer, comprising administering an anti-PD-L1 antigen binding molecule described herein, such as an antibody or an antigen binding fragment thereof, to the subject, so that the subject is treated, for example, to inhibit or reduce the growth of cancerous tumors and / or to cause tumor regression. The anti-PD-L1 antibody can be used alone to inhibit the growth of cancerous tumors. Alternatively, the anti-PD-L1 antibody can be used in combination with one or more other targeted active agents, such as other anti-cancer targets, immunogenic agents, standard cancer treatments or other antibodies, as described below. The antigen binding molecules described in the present invention can be used to treat, for example, primary and / or metastatic tumors. The present invention also includes a method for treating residual cancer in a subject. The term "residual cancer" as used herein refers to one or more cancer cells present or persisting in a subject after treatment with an anti-cancer therapy.

[0197] In one aspect, a method for treating cancer comprises administering a therapeutically effective amount of an anti-PD-L1 antibody described herein to a subject in need thereof. Preferably, the antibody inhibits the activity of human PD-L1 and comprises one or more HCVRs and LCVRs described herein. In addition, the anti-PD-L1 antigen binding molecule (eg, antibody) used in the method may include a chimeric or humanized non-human anti-PD-L1 antibody thereof. The efficacy of treating cancer can be determined by a variety of methods. For example, the efficacy of treating cancer can be measured by an increase in survival rate or a reduction in tumor size. In certain embodiments, the method described herein increases the efficacy of treating cancer by at least about 10%, about 20%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 1 times, about 2 times, about 4 times or more, compared to baseline levels. In the context of cancer treatment, if the PD-L1 antigen binding molecule described in the present invention is the only therapeutic agent, the baseline level refers to the therapeutic effect of using a placebo; if the PD-L1 antigen binding molecule described in the present invention is used in combination with other therapeutic agents, the baseline level refers to the therapeutic effect of using a placebo or other therapeutic agents.

[0198] Cancers whose growth can be inhibited by the antibodies of the present invention include a variety of cancers, especially those that do not respond or tend to not respond to monotherapy with other antibodies or chemotherapeutic agents. 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 (glioblastoma multiforme), cervical cancer, gastric cancer, bladder cancer, hepatoma, breast cancer, colon cancer and head and neck cancer, stomach cancer, germ cell carcinoma, tumors, pediatric sarcomas, sinonasal natural killer cells, melanoma (e.g., metastatic malignant melanoma (e.g., skin or intraocular malignant melanoma)), bone cancer, skin cancer, uterine cancer, cancer of the anal region, 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, solid tumors in children, ureteral cancer, renal pelvis cancer, central nervous system (CNS) tumors, primary CNS lymphoma, tumor angiogenesis, spinal cord axis tumors, brain stem gliomas, pituitary adenomas, Kaposi Sarcoma (Kaposi'ssarcoma), epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers (including asbestos-induced cancers, virus-related cancers (such as human papillomavirus (HPV)-related tumors) and hematological malignancies (derived from either of the two major blood cell lineages, the myeloid cell line (producing granulocytes, erythrocytes, platelets, macrophages and mast cells) or the lymphoid cell lineage (producing B, T, NK and plasma cells), such as all types of leukemias, lymphomas and myelomas (such as acute, chronic, lymphocytic and / or myeloid leukemias diseases, such as acute leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL) chronic myeloid leukemia (CIVIL), undifferentiated AML (MO), myeloblastic leukemia (M1), myeloblastic leukemia (M2; cell maturity), promyelocytic leukemia (M3 or M3 variant [M3V]), myelomonocytic leukemia (M4 or M4 variant with eosinophilia [M4E]), monocytic leukemia (M5), erythroleukemia (M6), megakaryocytic leukemia (M7), solitary myeloid leukemia sarcoma, and chloroma;Lymphoma (e.g., 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., Ki1+) 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; and lymphoma / leukemia (T-Lbly / T-ALL), peripheral T-cell lymphoma, lymphoma B cell 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, follicular lymphoma, diffuse histiocytic lymphoma (DHL), immunoblastic large cell lymphoma, precursor B lymphoblastic lymphoma, cutaneous T-cell lymphoma (CTLC) (also known as mycosis fungoides or Sezary syndrome), and Waldenstrom's macroglobulinemia (Waldenstrom's macroglobulinemia). enstrom'smacroglobulinemia); myeloma (e.g., IgG myeloma, light chain myeloma, nonsecretory myeloma, smoldering myeloma (also called indolent myeloma), solitary plasmacytoma, and multiple myeloma), chronic lymphocytic leukemia (CLL), hairy cell lymphoma; myeloid hematopoietic tumors, tumors of mesenchymal origin (including fibrosarcoma and rhabdomyosarcoma); seminoma, teratoma, central and peripheral nerve tumors (including astrocytoma, schwannoma); tumors of mesenchymal origin (including fibrosarcoma, rhabdomyosarcoma, and osteosarcoma); and other tumors (including melanoma, xeroderma pigmentosum, keratoacanthoma, seminoma, follicular thyroid carcinoma and teratoma), hematopoietic tumors of the lymphoid lineage (e.g., T-cell and B-cell tumors, including but not limited to T-cell diseases, such as T-prolymphocytic leukemia (T-PLL), including small cell and brain-like cell types; large granular lymphocytic leukemia (LGL), preferably of T-cell type; a / dT-NHL hepatosplenic lymphoma; peripheral / post-thymic T-cell lymphoma (polymorphic 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 refractory to previous immunotherapy (e.g., using antibodies that block CTLA-4 or PD-1)) and relapsed cancers. ;

[0199] In some embodiments, treatment of cancer patients with the anti-PD-L1 antibodies and / or other active agents described herein can produce long-term durable responses, including long-term survival of at least 1, 2, 3, 4, 5, 10 years or more and / or recurrence-free survival of at least 1, 2, 3, 4, 5, 10 years or more, relative to the current standard of care. In certain embodiments, treatment of cancer patients with the anti-PD-L1 antibodies and / or other active agents described herein can prevent cancer recurrence or delay cancer recurrence by, for example, 1, 2, 3, 4, 5, 10 years or more. The anti-PD-L1 treatment can be used as primary or secondary treatment.

[0200] 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 to a recipient can increase the frequency and activity of the adoptively transferred T cells to stimulate antigen-specific T cells to fight cancer or viral infection.

[0201] The routes described herein suitable for in vivo, ex vivo or in vitro administration of the antigen-binding molecules (e.g., anti-PD-L1 antibodies and antigen-binding fragments thereof) of the present invention are well known in the art and can be selected by a person of ordinary skill. For example, the antibody composition can be administered by parenteral injection (e.g., intravenous or subcutaneous). As further described below, the appropriate dose depends on the age and weight of the subject and the concentration and / or formulation of the antibody composition.

[0202] The term "cell proliferative disorder" refers to a disorder characterized by abnormal proliferation of cells. A proliferative disease does not imply any restriction on the rate of cell growth, but only a loss of normal control over growth and cell division. Thus, in some embodiments, cells of a proliferative disorder may have the same rate of cell division as normal cells but do not respond to signals that restrict such growth. The scope of "cell proliferative disorder" includes neoplasms, cancers, or tumors.

[0203] The term "cancer" refers to any of a variety of malignancies characterized by cell proliferation with the ability to invade surrounding tissues and / or metastasize to new colonization sites, including carcinomas, sarcomas, adenocarcinomas, melanomas, leukemias, lymphomas, germ cell tumors, and blastomas, including solid cancers and lymphoid cancers. Exemplary cancers that can be treated according to 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 and uterine cancer, leukemia, and medulloblastoma.

[0204] The term "carcinoma" refers to a malignant growth of epithelial cells that tends to infiltrate surrounding tissues and give rise to metastases. Exemplary carcinomas include, e.g., acinar carcinoma, acinous carcinoma, adenocystic carcinoma, adenoid cystic carcinoma, carcinoma adenomatosum, adrenocortical carcinoma, alveolar carcinoma, alveolar cell carcinoma, basal cell carcinoma, carcinoma basocellulare, basaloid carcinoma, basosquamous cell carcinoma, bronchioalveolar carcinoma, bronchiolar carcinoma, bronchogenic carcinoma, cerebriform carcinoma, cholangiocellular carcinoma, chorionic carcinoma, colloid carcinoma ), comedo carcinoma, corpus carcinoma, cribriform carcinoma, carcinoma encuirasse, carcinoma cutaneum, cylindrical carcinoma, cylindrical cell carcinoma, ductal carcinoma, carcinoma durum, embryonal carcinoma, encephaloid carcinoma, epiennoid carcinoma, carcinoma epithelia leadenoides, exophytic carcinoma, carcinoma exulcere, carcinoma fibrosum, gelatinous carcinoma, gelatinous carcinoma, giant cell carcinoma,giant cell carcinoma, glandular carcinoma, granular cell carcinoma, hair-matrix carcinoma, hematoid carcinoma, hepatocellular carcinoma, hurthlecell 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 inoma、lymphoepithelial carcinoma、medullary carcinoma、melanotic carcinoma、carcinoma molle、mucinous carcinoma、carcinoma muciparum、carcinoma mucocellulare、mucoepidermoid carcinoma、carcinoma mucosum、mucous carcinoma、carcinoma myxomatodes、naspharyngeal carcinoma、oat cell carcinoma、carcinoma ossificans、osteoid carcinoma、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 scroti, signet-ring cell carcinoma, carcinoma simplex, small-cell carcinoma, solanoid carcinoma, spheroidal cell carcinoma, spindle cell carcinoma, medullary carcinoma (carcinoma spongiosum), squamous cell carcinoma (squamous carcinoma), squamous cell carcinoma (squamous cell carcinoma), string carcinoma, angiectatic carcinoma (carcinomate langiectaticum), angiectodes carcinoma (carcinomate langiectodes), transitional cell carcinoma (transitional cell carcinoma), tuberous carcinoma (carcinoma tuberosum), tuberous carcinoma, verrucous carcinoma and villous carcinoma (carcinoma villosum).

[0205] The term "sarcoma" refers to a tumor composed of a substance such as embryonic connective tissue, usually composed of tightly packed cells embedded in a fibrous or homogeneous substance. Exemplary sarcomas include, for example, chondrosarcoma, fibrosarcoma, lymphosarcoma, melanosarcoma, myxosarcoma, osteosarcoma, Abernethy's sarcoma, adiposesarcoma, liposarcoma, alveolar soft part sarcoma, ameloblastic sarcoma, botryoid sarcoma, chloroma sarcoma, choriocarcinoma, embryonal sarcoma, Wilms tumor sarcoma, sarcoma of the umbilical cord ... ′tumorsarcoma), endometrial sarcoma, stromal sarcoma, Ewing's sarcoma, fascial sarcoma, fibroblastic sarcoma, giant cell sarcoma, granulocytic sarcoma, Hodgkin's sarcoma, idiopathic multiple pigmentation Hemorrhagic sarcoma (idiopathic multiple pigmented hemorrhagic sarcoma), B-cell immunoblastic sarcoma (immunoblastic sarcoma of B cells), lymphoma (e.g., non-Hodgkin's lymphoma), T-cell immunoblastic sarcoma (immunoblastic sarcoma of T-cells), Jensen's sarcoma, Kaposi's sarcoma, hepatic macrophage sarcoma (Kupffer cells sarcoma), angiosarcoma (angiosarcoma), leukemic sarcoma (leukosarcoma), malignant mesenchymomas sarcoma, parosteal sarcoma, reticulocytic sarcoma, Rous sarcoma, serocystic sarcoma, synovial sarcoma and telangiectatic sarcoma.

[0206] The term "melanoma" refers to a tumor arising from the melanocyte system of the skin and other organs. Melanomas include, for example, acral-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.

[0207] The term "lymphoma" refers to cancers that affect the hematopoietic and lymphoid tissues and arise from lymphocytes (blood cells found primarily in the lymph nodes, spleen, thymus, and bone marrow). The two main types of lymphoma are non-Hodgkin lymphoma and Hodgkin's disease. Hodgkin's disease accounts for about 15% of all diagnosed lymphomas. It is a cancer associated with Reed-Sternberg malignant B lymphocytes. Non-Hodgkin lymphoma (NHL) can be classified based on how fast the cancer grows and the types of cells involved. There are aggressive (high-grade) and indolent (low-grade) types of NHL. Based on the types of cells involved, there are B-cell 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 of mucosa-associated lymphoid tissue, nodal (monocytoid B-cell) lymphoma, splenic lymphoma, diffuse large B cell lymphoma, Burkitt lymphoma, lymphoblastic lymphoma, immunoblastic large cell lymphoma, or precursor B-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.

[0208] The term "leukemia" refers to a progressive malignant disease of the hematopoietic organs, usually characterized by abnormal proliferation and development of leukocytes and their precursors in the blood and bone marrow. Exemplary leukemias include, for example, acute non-lymphocytic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, chronic myeloid leukemia, acute promyelocytic leukemia, adult T-cell leukemia, aleukemicleukemia, aleukocythemicleukemia, basophylicleukemia, blast cell leukemia, bovine leukemia, chronic myeloid leukemia, cutaneous leukemia, embryonic leukemia, and leukemia. nalleukemia), eosinophilicleukemia, Gross'leukemia, hairy cell leukemia, hemoblasticleukemia, hemocytoblasticleukemia, histiocytic leukemia, stem cell leukemia, acute monocytic leukemia, leukopenic leukemia, lymphatic leukemia, lymphoblastic leukemia lymphoblastic leukemia, lymphocytic leukemia, lymphoblastic leukemia, lymphoid leukemia, lymphosarcoma cell leukemia, mast cell leukemia, megakaryocytic leukemia, micromyeloblastic leukemia, monocytic leukemia, myeloblastic leukemia, myeloid leukemia, myeloid granulocytic leukemia mia), myelomonocytic leukemia, Naegeli leukemia, plasma cell leukemia, plasmacyticleukemia, promyelocytic leukemia, Rieder cell leukemia, Schilling's leukemia, stem cell leukemia, subleukemic leukemia, and undifferentiated cell leukemia.

[0209] Other cancers include, for example, multiple myeloma, neuroblastoma, breast cancer, ovarian cancer, lung cancer, rhabdomyosarcoma, essential thrombocythemia, essential macroglobulinemia, small cell lung cancer, primary brain tumors, gastric cancer, colon cancer, malignant pancreatic insular tumors, malignant carcinoids, precancerous skin lesions, testicular cancer, thyroid cancer, neuroblastoma, esophageal cancer, genitourinary tract cancer, hypercalcemia of malignancy, cervical cancer, endometrial cancer, and adrenocortical carcinoma. 4. Combination Therapy

[0210] On the other hand, the present invention provides therapeutic compositions and combination therapies for enhancing antigen-specific T cell responses, reducing immunosuppression and / or slowing tumor growth in subjects. The present invention includes compositions and therapeutic preparations comprising any (e.g., herein) exemplary antigen binding molecules in combination with one or more other therapeutic agents, and treatment methods including administering such compositions to subjects in need. The term "other therapeutic agent" as used herein refers to any agent that can be used to treat a disease or condition, and any method for treating certain diseases or conditions. For example, when radiotherapy and surgery are used in combination with antigen binding molecules such as anti-PD-L1 antibodies or antigen binding fragments thereof described in the present invention, they are considered "other therapeutic agents".

[0211] In certain embodiments, the other therapeutic agent may be an agent that blocks the interaction between PD-1 and PD-L1 that is different from the antigen binding molecule described in the present invention (e.g., an anti-PD-L1 antibody or an antigen binding fragment thereof). Exemplary blockers of the interaction between PD-1 and PD-L1 include, but are not limited to, pembrolizumab, nivolumab, atezolizumab, avelumab, durvalumab, BMS-9365599, sulfamethoxazole 1, and sulfamethoxazole 2.

[0212] In some embodiments, the anti-PD-L1 antigen binding molecules described in the present invention, such as anti-PD-L1 antibodies or antigen binding fragments thereof, are co-administered with one or more other therapeutic agents, wherein the other therapeutic agents have an amount effective to stimulate immune response and / or apoptosis, to further enhance, stimulate or upregulate immune response and / or apoptosis in the subject. In addition, the one or more other therapeutic agents are administered before or after treatment with the anti-PD-L1 antibody.

[0213] In certain embodiments, the anti-PD-L1 antibodies described herein are co-administered or co-administered with one or more other active agents (e.g., anti-cancer antibodies or polypeptides, chemotherapeutic agents, and radiotoxic agents). In other embodiments, the anti-PD-L1 antibodies described herein are co-administered or co-administered with standard cancer treatments (e.g., surgery or radiotherapy).

[0214] The co-administration of the anti-PD-L1 antibody with these active agents or treatments can address the following clinical deficiencies: drug resistance, changes in antigenicity that cause tumor cells to not react with antibodies, and toxicity (by administering lower doses of one or more agents). PD-L1 blockade is particularly suitable for use in combination with chemotherapy regimens that are difficult to work in other cases. In these cases, it is possible to achieve enhanced efficacy while reducing the dose of the chemotherapeutic agent administered (Mokyr et al., (1998) Cancer Research 58: 5301-5304). The basic principle of blocking PD-L1 with radiotherapy or chemotherapy is based on the fact that the cytotoxic effects of radiotherapy and most chemotherapeutic compounds promote cell death, which can further lead to an increase in tumor antigen levels in the antigen presentation pathway. Other combination therapies that act additively or synergistically with PD-L1 inhibition through cell death include surgery and hormone deprivation or inhibition. Each of these regimens further produces a source of tumor antigens in the host.

[0215] In some embodiments, the anti-PD-L1 antibody described herein is linked to another active agent in the form of an immune complex, an immunoconjugate, or a fusion protein. Alternatively, the anti-PD-L1 antibody can be administered separately from the other active agent. In this case, the anti-PD-L1 antibody and other antagonists can be administered before, after, or simultaneously with the other active agents, or can be co-administered with other known therapies (e.g., other anticancer agents, radiation, etc.). Therefore, the present invention provides a composition and method for providing two or more anticancer agents that act additively or synergistically through different mechanisms to beneficially exert cytotoxic and immunoprotective effects in human cancer cells.

[0216] For example, in some embodiments, the anti-PD-L1 antibodies described herein can be combined with the following anti-cancer agents: for example, 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) 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-targeted drugs, topoisomerase-toxic drugs, inhibitors of molecular targets or enzymes (e.g., kinases or protein methyltransferases), cytidine analogs, or a combination thereof.

[0217] 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); bischloromethyldiethylamine (Mustargen); busulfan (Myleran); carboplatin (Paraplatin); cisplatin (CDDP; Platinol); temozolomide (Temodar); thiotepa (Thioplex); bendamustine (Treanda); or streptozotocin (Zanosar).

[0218] Exemplary anthracyclines include, but are not limited to, Adriamycin; Doxil; Novantrone; Blenoxane; Cerubidine; Daunorubicin; Cosmegen; Ellence; Idamycin; Mithracin; Mutamycin; Nipent; or Valstar.

[0219] Exemplary antimetabolites include, but are not limited to, 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).

[0220] Exemplary antidotes include, but are not limited to, amifostine (Ethyol) or mesna (Mesnex).

[0221] Exemplary interferons include, but are not limited to, interferon alpha-2b (IntronA) or interferon alpha-2a (Roferon-A).

[0222] 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 tiuxetan (Zevalin; In-111; Y-90Zevalin); gemtuzumab (Mylotarg); eculizumab (Soliris) or denosumab.

[0223] 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.

[0224] Exemplary HER2 inhibitors include, but are not limited to, trastuzumab (Herceptin); lapatinib (Tykerb) or AC-480.

[0225] Exemplary histone deacetylase inhibitors include, but are not limited to, vorinostat (Zolinza), valproic acid, romidepsin, entinostat, abexinostat, givinostat, and mocetinostat.

[0226] Exemplary hormones include, but are not limited to, tamoxifen (Soltamox; Nolvadex); raloxifene (Evista); megestrol acetate (Megace); leuprolide (Lupron; Lupron Depot; Eligard; Viadur); fulvestrant (Faslodex); letrozole (Femara); triptorelin (Trelstar LA; Trelstar Depot); exemestane (Aromasin); goserelin (Zoladex); bicalutamide (Casodex); anastrozole (Arimidex); fluoxymesterone (Androxy; Halotestin); medroxyprogesterone acetate (Provera; Depo-Provera); estramustine (Emcyt); flutamide (Eulexin); toremifene (Fareston); degarelix (Firmagon); nilutamide (Nilandron); abarelix (Plenaxis); or testolactone (Teslac).

[0227] 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; epothilone; vinorelbine (Navelbine); camptothecin (CPT); irinotecan (Camptosar); topotecan (Hycamtin); amsacrine or lamellarin D (LAM-D).

[0228] 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 PI3KDelta inhibitor); duvelisib (IPI-145, a PI3Kdelta and gamma inhibitor); apelisib (BYL719, an alpha-specific PI3K inhibitor); TGR1202 (formerly known as RP5264, an oral PI3Kδ inhibitor); and cupanisib (BAY80-6946, an inhibitor of predominantly PI3Kα,δ isoforms).

[0229] Exemplary Akt inhibitors include, but are not limited to, miltefosine, AZD5363, GDC-0068, MK2206, perifosine, RX-0201, PBI-05204, GSK2141795, and SR13668.

[0230] 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, Torin1, PP242, PP30, Ku-0063794, WAY-600, WYE-687, WYE-354, and CC-223.

[0231] Exemplary proteasome inhibitors include, but are not limited to, bortezomib (PS-341), ixazomib (MLN2238), MLN9708, delanzomib (CEP-18770), carfilzomib (PR-171), YU101, oprozomib (ONX-0912), marizomib (NPI-0052), and disulfiram.

[0232] Exemplary PARP inhibitors include, but are not limited to, olaparib, iniparib, veliparib, BMN-673, BSI-201, AG014699, ABT-888, GPI21016, MK4827, INO-1001, CEP-9722, PJ-34, Tiq-A, Phen, PF-01367338, and combinations thereof.

[0233] 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, MEK1627, and GG.

[0234] Exemplary centrosome declustering agents include, but are not limited to, griseofulvin; narcotine, narcotine derivatives, such as brominated narcotine (e.g., 9-bromonacotine), reduced bromonacotine (RBN), N-(3-bromobenzyl)narcotine, aminonarcotine, and water-soluble derivatives thereof; CW069; phenanthrene-derived poly (ADP-ribose) polymerase inhibitor, PJ-34; N2-(3-pyridylmethyl)-5-nitro-2-furanamide, N2-(2-thienylmethyl)-5-nitro-2-furanamide, and N2-benzyl-5-nitro-2-furanamide.

[0235] Exemplary multi-kinase inhibitors include, but are not limited to, regorafenib; sorafenib (Nexavar); sunitinib (Sutent); BIBW2992; E7080; Zd6474; PKC-412; motesanib; or AP24534.

[0236] Exemplary serine / threonine kinase inhibitors include, but are not limited to, rubustard; eril / easudil hydrochloride; flavopiridol; cylicidin (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 PD332991.

[0237] Exemplary tyrosine kinase inhibitors include, but are not limited to, erlotinib (Tarceva); gefitinib (Iressa); imatinib (Gleevec); sorafenib (Nexavar); sunitinib (Sutent); trastuzumab (Herceptin); bevacizumab (Avastin); rituximab (Rituxan); lapatinib (Tykerb); cetuximab (Erbitux); panitumumab (Vectibix); everolimus (Afinitor); alemtuzumab (Campath) ; gemtuzumab uzumab (Mylotarg); temsirolimus (Torisel); pazopanib (Votrient); dasatinib (Sprycel); nilotinib (Tasigna); vatalanib (Ptk787; ZK222584); CEP-701; SU5614; MLN518; XL999; VX-322; azd0530; BMS-354825; SKI-606CP-690; AG-490; WHI-P154; WHI-P131; AC-220; or AMG888.

[0238] Exemplary VEGF / VEGFR inhibitors include, but are not limited to, bevacizumab (Avastin); sorafenib (Nexavar); sunitinib (Sutent); ranibizumab; pegaptanib; or vandetinib.

[0239] Exemplary microtubule-targeting drugs include, but are not limited to, paclitaxel, docetaxel, vincristine, vinblastine, nocodazole, epothilones, and vinorelbine.

[0240] Exemplary topoisomerase toxic drugs include, but are not limited to, teniposide, etoposide, doxorubicin, camptothecin, daunorubicin, dactinomycin, mitoxantrone, amsacrine, epirubicin, and idarubicin.

[0241] Exemplary taxanes or taxane derivatives include, but are not limited to, paclitaxel and docetaxel.

[0242] Exemplary general chemotherapeutic agents, anti-tumor agents, anti-proliferative agents include, but are not limited to, hexamethylmelamine (Hexalen); isotretinoin (Accutane; Amnesteem; Claravis; Sotret); tretinoin (Vesanoid); azacitidine (Vidaza); bortezomib (Velcade); asparaginase (Elspar); levamisole (Ergamisol); mitotane (Lysodren); procarbazine (Matulane); pegaspargase (Oncaspar); denileukin diftitox (Ontak); porfimer (Photofrin); aldesleukin (Proleukin); lenalidomide (Revlimid); bexarotene (Targretin); thalidomide (Thalomid); temsirolimus (Torisel); arsenic trioxide (Trisenox); verteporfin (Visudyne); mimosapinol (Leucenol); (1M tegafur-0.4M 5-chloro-2,4-dihydroxypyrimidine-1M potassium oxide) or lovastatin.

[0243] In certain embodiments, PD-L1 inhibition is combined with CD3 stimulation, and CD3 stimulation is performed before, simultaneously with, or after anti-PD-L1 antibody treatment. For example, in one embodiment, the method of enhancing antigen-specific T cell responses comprises contacting T cells with anti-PD-L1 antibodies and cells expressing CD3 as described herein, so that antigen-specific T cell responses are enhanced and PD-L1-mediated immunosuppression is reduced. Any suitable indicator of antigen-specific T cell responses can be used to measure antigen-specific T cell responses. Non-limiting examples of such suitable indicators include increased T cell proliferation in the presence of antibodies and / or increased cytokine production in the presence of antibodies. In a preferred embodiment, the production of interleukin-2 and / or interferon-γ by the antigen-specific T cells is enhanced.

[0244] In some embodiments, the anti-PD-L1 antibodies described herein can also be used in combination with bispecific antibodies that target effector cells expressing Fcα or Fcγ receptors to tumor cells (see, e.g., U.S. Patents 5,922,845 and 5,837,243). Such bispecific antibodies can be used to target two different antigens. For example, bispecific antibodies against Fc receptors / anti-tumor antigens (e.g., Her-2 / neu) have been used to target macrophages to tumor sites. This targeting may more effectively activate tumor-specific responses. The T cell arm of these responses will be enhanced by the inhibition of PD-L1. Alternatively, bispecific antibodies that bind tumor antigens and dendritic cell-specific cell surface markers can be used to deliver antigens directly to DCs.

[0245] In all of the above approaches, PD-L1 inhibition can be combined with other forms of immunotherapy, such as cytokine therapy (e.g., interferon, GM-CSF, G-CSF, IL-2), or bispecific antibody therapy using two different binding specificities to provide enhanced tumor antigen presentation.

[0246] In some embodiments, the other therapeutic agent used in any of the aforementioned treatment methods, antigen binding molecules or pharmaceutical compositions is an immunostimulatory agent selected from the following: (a) an agent that blocks signaling of an immune cell inhibitory receptor (immune checkpoint) or its ligand (immune checkpoint inhibitor) or a nucleic acid encoding such an agent; (b) an agonist of an immune cell stimulating receptor 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 for a cancer-associated antigen, including such an antigen or a nucleic acid encoding such an antigen, (j) cell therapy, and (k) a combination of the above. In some embodiments, the additional therapeutic agent is an agent that blocks signaling of an inhibitory receptor or its ligand of an immune cell, or a nucleic acid encoding such an agent, wherein the inhibitory receptor or its ligand is selected from TIGIT, CTLA-4, PD-1, PD-L1, PD-L2, LAG-3, TIM-3, neurite protein, 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 combinations thereof. In some embodiments, the other therapeutic agents are agonists of immune cell stimulating receptors or nucleic acids encoding such agonists, and the agonists are selected from OX40, CD2, CD16, CD27, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD28, CD30, CD40, BAFFR, HVEM, CD7, LIGHT, KG2C, SLAMF7, NKG2C, NKG2D, NKp46, NKp80, CD160, B7-H3, CD83 ligands and combinations thereof. In some embodiments, the other therapeutic agents are cytokines or nucleic acids encoding cytokines selected from IL-2, IL-5, IL-7, IL-12, IL-15, IL-2I and combinations thereof. In some embodiments, the other therapeutic agents are oncolytic viruses or nucleic acids encoding oncolytic viruses selected from herpes simplex virus, vesicular stomatitis virus, adenovirus, Newcastle disease virus, vaccinia virus, Maraba virus and combinations thereof. In some embodiments, the other therapeutic agents are cell therapy. Cell therapy may include T cells, NK cells or macrophages with chimeric antigen receptors (CAR).In some embodiments, the cell therapy comprises a bispecific or multispecific T cell targeting antibody.

[0247] In certain embodiments, the present invention provides a method for treating a disease or condition such as cancer in a subject. The method includes administering an antigen binding molecule of the present invention, such as an anti-PD-L1 antibody or an antigen binding fragment thereof, alone or in combination with a second or more other therapeutic agents to a subject, wherein the subject has previously been treated with a first or more other therapeutic agents. In certain embodiments, low efficacy may be exhibited when treating a subject's disease with a first or more other therapeutic agents. For example, treatment with the first or more therapeutic agents may be treatment with an anti-PD1 antibody, to which the subject may show resistance. In some embodiments, the second or more other therapeutic agents are the same as the first or more other therapeutic agents. In some embodiments, the second or more other therapeutic agents are different from the first or more other therapeutic agents.

[0248] In certain embodiments, the immune checkpoint inhibitor is an antibody that specifically interacts with an immune checkpoint. In some embodiments, the other therapeutic agents include immunostimulants. In some aspects, the immune checkpoint inhibitor is an anti-CTLA-4 antibody (e.g., ipilimumab), and combinations thereof. In some aspects, the immune checkpoint inhibitor is pembrolizumab. In some aspects, the immune checkpoint inhibitor is nivolumab. In some aspects, the immune checkpoint inhibitor is atezolizumab.

[0249] In some embodiments, the anti-PD-L1 antibody is combined with or administered simultaneously with an immunogenic agent. Non-limiting examples of immunogenic agents include cancer cells, tumor vaccines, and purified tumor antigens (including recombinant proteins, peptides, and carbohydrate molecules); oncolytic viruses; cells transfected with genes encoding immunostimulatory cytokines, etc.

[0250] In certain embodiments, the anti-PD-L1 antibody is administered together with a target antigen or an antigen known to be present in the subject to be treated (e.g., a tumor-bearing or virus-bearing subject) to enhance antigen-specific immunity. When the anti-PD-L1 antibody is administered together with another agent, the two can be administered separately or simultaneously.

[0251] In certain embodiments, the antigen-specific immune response can be enhanced by co-administering one or more of any of the anti-PD-L1 antibodies described herein with an antigen of interest (e.g., a vaccine). Therefore, in one embodiment, a method for enhancing an immune response to an antigen in a subject comprises administering to the subject: (i) an antigen; (ii) an antibody based on PD-L1, thereby enhancing the subject's immune response to the antigen. The antigen may be, for example, a tumor antigen, a viral antigen, a bacterial antigen, or an antigen from a pathogen. Non-limiting examples of such antigens include the antigens described in the above section, such as the tumor antigens (or tumor vaccines) discussed above, or antigens from viruses, bacteria, or other pathogens described above.

[0252] In view of the synergistic benefit of the active agent combination, in certain embodiments, the anti-PD-L1 antibody and the other active agent are each administered to a subject in need thereof at a subtherapeutic dose relative to the dose used in their monotherapy.

[0253] In certain embodiments, PD-L1 inhibition is combined with standard cancer treatments (e.g., surgery, radiotherapy, and chemotherapy). In these cases, it is possible to reduce the dosage of chemotherapeutic agents. The combined use of PD-L1 inhibition and chemotherapy can exacerbate apoptosis and promote tumor antigen presentation to achieve killer cell immunity. Other synergistic combination therapies include PD-L1 inhibition combined with radiotherapy, surgery, or hormone deprivation or suppression. Each of these regimens produces a source of tumor antigens in the host.

[0254] The other therapeutic agent may be administered before, simultaneously with, or after administration of the antigen binding molecules described herein; (for the purposes of this disclosure, such administration regimens are considered to be administered "in combination" with the antigen binding molecules and other therapeutically active ingredients).

[0255] The present invention includes a pharmaceutical composition in which the antigen binding molecules described herein are co-formulated with one or more other therapeutic agents described herein. 5. Nucleic Acids and Host Cells Expressing Anti-PD-L1 Antibodies

[0256] On the other hand, the present invention provides a nucleic acid encoding an antigen binding molecule (e.g., an anti-PD-L1 antibody or an antigen binding fragment thereof) described in the present invention, and an expression vector comprising such a nucleic acid. In some embodiments, the nucleic acid encodes the HCVR and / or LCVR fragment of the antibody or fragment in the embodiments described herein, or encodes any other antibody and antibody fragment described herein.

[0257] DNA encoding the antigen binding site in the monoclonal antibody can be isolated and sequenced from hybridoma cells using conventional methods (e.g., using oligonucleotide probes that can specifically bind to genes encoding the heavy and light chains of the monoclonal antibody). Alternatively, the amino acid sequence of the target immunoglobulin can be determined by direct protein sequencing, and a suitable encoding nucleotide sequence can be designed based on 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 public sources well known in the art.

[0258] Expression vectors can be used to synthesize the antibodies described in this disclosure in cells cultured in vitro, or they can be directly administered to a patient to express the antibodies described in 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 antibodies described in this disclosure in a form suitable for expressing the polynucleotide in a host cell for the purpose of antibody preparation or direct administration as a therapeutic agent.

[0259] When one nucleic acid sequence is placed in a functional relationship with another nucleic acid sequence, it is said that one nucleic acid sequence is "operably linked" to another nucleic acid sequence. For example, if the DNA of the presequence or signal peptide expresses a preprotein that participates in the secretion of the polypeptide, the DNA of the presequence or signal peptide is operably linked to the DNA of the polypeptide; if a promoter or enhancer affects the transcription of the sequence, the promoter or enhancer is operably linked to the coding sequence; or if a ribosome binding site is positioned to facilitate translation, the ribosome binding site is operably linked to the coding sequence. Generally, "operably linked" means that the DNA sequence being linked is continuous, and in the presence of a signal peptide, it is continuous and in the reading phase. However, enhancers do not have to be continuous. Connection is accomplished by connecting at convenient restriction sites. If such sites do not exist, synthetic oligonucleotide adapters or joints can be used according to conventional practices.

[0260] The nucleic acid sequence used to express the antibodies described in the present disclosure generally includes an N-terminal signal peptide sequence 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 will be appreciated by those skilled in the art that the design of the expression vector may depend on the following factors: the choice of the host cell to be transformed, the expression level of the target protein, etc.

[0261] The above-mentioned "regulatory sequence" refers to a DNA sequence necessary for expressing 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 regulatory sequences that direct constitutive expression of nucleotide sequences in many types of host cells, or regulatory sequences that direct expression of nucleotide sequences only in certain host cells (e.g., tissue-specific regulatory sequences). Expression vectors typically contain sequences for transcription termination and may additionally contain one or more elements that have a positive effect on mRNA stability.

[0262] The expression vector comprises one or more transcriptional regulatory elements, including a promoter and / or an enhancer, for directing the expression of the antibodies described in the present disclosure. The promoter comprises a DNA sequence, the function of which is to initiate transcription from a relatively fixed position relative to the transcription start site. The promoter comprises the core elements required for the basic interaction of RNA polymerase and transcription factors, and can operate with other upstream elements and response elements.

[0263] The term "promoter" as used herein should be broadly interpreted to include, for example, transcriptional regulatory elements (TREs) from genomic genes or chimeric TREs thereof, including TATA boxes or initiation elements for precise transcription initiation, with or without other TREs (i.e., upstream activation sequences, transcription factor binding sites, enhancers and silencers), which regulate the activation or repression of operably linked genes in response to development and / or external stimuli, and trans-acting regulatory proteins or nucleic acids. A promoter may comprise a genomic fragment or it may comprise a chimera of one or more TREs combined together.

[0264] Preferred promoters are promoters that can guide high-level expression in target target cells. The promoter may include constitutive promoters (e.g., promoters of HCMV, SV40, elongation factor-1α (EF-1α)), or promoters that show preferential expression in specific target cell types. An enhancer generally refers to a DNA sequence that acts away from the transcription start site, which may be located at the 5' or 3' end of the transcription unit. In addition, an enhancer may be within an intron and within a coding sequence. Its length is generally between 10 and 300 bp, and it acts in a cis manner. The function of an enhancer is to increase and / or regulate the transcription of nearby promoters. Preferred enhancers include enhancers that guide high-level expression in cells that produce antibodies. Cell or tissue-specific transcriptional regulatory elements (TREs) may be incorporated into expression vectors to limit expression to desired cell types. Expression vectors may be designed to promote expression of antibodies described in the present disclosure in one or more cell types.

[0265] In order to co-express the independent chains of the antibodies of the present disclosure, suitable splice donor and splice acceptor sequences can be incorporated to express two products. Alternatively, an internal ribosome binding sequence (IRES) or a 2A peptide sequence can be used to express multiple products from one promoter. IRES provides a structure that a ribosome can bind to, which does not need to be located at the 5' end of the mRNA. Therefore, it can guide the ribosome to start translation at the second start codon in the mRNA, so that a single mRNA produces more than one polypeptide. The 2A peptide comprises a short sequence that mediates the co-translational self-cleavage of the upstream and downstream peptides of the 2A site, so that a single transcript of equimolar amounts produces two different proteins. CHYSEL is a non-limiting example of a 2A peptide that allows the translating eukaryotic ribosome to release the growing polypeptide chain it is synthesizing without dissociating from the mRNA. The ribosome continues to translate, thereby producing a second polypeptide.

[0266] Expression vectors may include viral vectors or non-viral vectors. Viral vectors may be derived from adeno-associated virus (AAV), adenovirus, herpes virus, vaccinia virus, polio virus, poxvirus, retrovirus (including lentivirus, such as HIV-1 and HIV-2), Sindbis virus and other RNA viruses, alphavirus, astrovirus, coronavirus, orthomyxovirus, papovavirus, paramyxovirus, parvovirus, picornavirus, togavirus, etc. Non-viral vectors are simply "naked" expression vectors that are not packaged with virus-derived components (e.g., capsid and / or envelope).

[0267] In some cases, these vectors can be engineered to target certain diseases or cell populations by using targeting properties inherent to or engineered into the viral vectors. Specific cells can be "targeted" for delivery and expression of polynucleotides. In this context, the term "targeting" refers to delivery to specific cells using endogenous or heterologous binding agents in the form of capsids, envelope proteins, antibodies, or to the use of tissue-specific regulatory elements to limit expression to specific cell subsets, or both.

[0268] In some embodiments, the expression of the antibody chain is under the control of a regulatory element (e.g., a tissue-specific or ubiquitous promoter). 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 the expression of a particular antibody heavy or light chain or single chain derivative.

[0269] Non-viral expression vectors can be used for non-viral gene transfer by direct injection of naked DNA or by encapsulating the polynucleotide encoding the antibody in liposomes, microparticles, microcapsules, virus-like particles or erythrocyte ghosts. Such compositions can be further connected to a targeting domain by chemical coupling to facilitate targeted delivery of nucleic acids and / or entry into desired target cells. In addition, plasmid vectors can be incubated with synthetic gene transfer molecules (e.g., polymeric DNA binding cations such as polylysine, protamine, and albumin) and connected to cell targeting ligands (e.g., asialomucoids, insulin, galactose, lactose, or transferrin).

[0270] Alternatively, naked DNA can be used. The uptake efficiency of naked DNA can be improved by compression or the use of biodegradable latex beads. This delivery can be further improved by treating the beads to increase hydrophobicity and promote disruption of endosomes and release of DNA into the cytoplasm. VI. Methods for producing anti-PD-L1 antibodies

[0271] In another aspect, the present invention provides a host cell transformed with a nucleic acid or expression vector encoding an anti-PD-L1 HCVR and / or LCVR. The host cell may be a bacterial or eukaryotic cell capable of expressing a nucleic acid or expression vector encoding an anti-PD-L1 HCVR and / or LCVR or any other co-administered antibody or antagonist described herein.

[0272] In another aspect, a method of making an antibody of the present disclosure comprises culturing a host cell transformed with a nucleic acid or expression vector encoding an anti-PD-L1 HCVR and / or LCVR under conditions that permit production of the antibody or fragment thereof, and purifying the antibody from the cell.

[0273] On the other hand, the present invention provides a method for preparing an antibody, comprising culturing cells that can transiently or stably express one or more constructs encoding one or more polypeptide chains in an 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 cell producing the antibody is stably transformed with a vector expressing the antibody.

[0274] One or more expression vectors encoding antibody heavy chains or light chains can be introduced into cells by any conventional method, including, for example, naked DNA technology, cationic lipid-mediated transfection, polymer-mediated transfection, peptide-mediated transfection, virus-mediated infection, physical or chemical reagents or treatments, electroporation, etc. In addition, cells can be transfected with one or more expression vectors expressing antibodies together with a selection marker that helps select stable transformed clones expressing the antibody. Antibodies produced by such cells can be collected and / or purified according to techniques known in the art, such as centrifugation, chromatography, etc.

[0275] Examples of selectable markers suitable for mammalian cells include dihydrofolate reductase (DHFR), thymidine kinase, neomycin, neomycin analog G418, hydromycin and puromycin. When such selectable markers are successfully transferred into mammalian host cells, the transformed mammalian host cells can survive under the selection pressure. There are two widely used different selection schemes. The first type is based on the metabolism of the cell and uses mutant cell lines that lack the ability to grow independently of supplemented medium. Two examples are CHODHFR - cells and mouse LTV cells. These cells cannot grow without the addition of nutrients such as thymidine or hypoxanthine. Because these cells lack certain genes necessary for the complete nucleotide synthesis pathway, they cannot survive unless the missing nucleotides are provided in the supplemented medium. Another way to supplement the medium is to introduce the complete DHFR or TK gene into cells lacking the corresponding gene, thereby changing their growth requirements. Individual cells that have not been transformed with the DHFR or TK gene will not survive in unsupplemented medium.

[0276] The second category is dominant selection, which refers to selection schemes that can be used in any cell type and do not require the use of mutant cell lines. In these schemes, drugs are usually used to prevent the growth of host cells. Cells with new genes will express proteins that convey drug resistance and will survive the selection. Examples of this dominant selection include the use of drugs neomycin, mycophenolic acid or hygromycin. In these three examples, bacterial genes under the control of eukaryotic organisms are resistant to the appropriate drugs G418 or neomycin (Geneticin), xgpt (mycophenolic acid) or hygromycin, respectively. Other drugs include neomycin analogs G418 and puromycin.

[0277] Exemplary cells expressing antibodies include human T lymphocyte leukemia cells, human embryonic kidney (HEK) 293 cells, Chinese hamster ovary (CHO) cells, mouse WEHI fibrosarcoma cells, and unicellular protozoan species, such as Leishmania (Leishmania tarentolae). In addition, primary cells immortalized with c-myc or other immortalizing agents can be used to generate stable transformed antibody-producing cell lines.

[0278] In one embodiment, the cell line includes a stably transformed Leishmania cell line, such as Leishmania tarentolae. Leishmania can provide a powerful, fast-growing unicellular host that can be used for high-level expression of eukaryotic proteins that exhibit mammalian-type glycosylation patterns. Leishmania eukaryotic expression kits (Jena Bioscience GmbH, Jena, Germany) can be purchased commercially.

[0279] In some embodiments, the cell line expresses 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 the antibody per liter of culture.

[0280] The antibodies of the present invention can be cultured in any suitable culture medium (e.g., RPMI, DMEM and ) and then separated from the antibody expressing cells. Conventional protein purification methods (e.g., affinity purification, chromatography, etc.) can be used to purify antibodies, including the use of protein-A or protein-G immunoaffinity purification. In some embodiments, the antibody is designed to be secreted into the culture supernatant to be separated therefrom. VII. Pharmaceutical Composition and Method of Administration

[0281] In one aspect, the pharmaceutical composition of the present invention includes an antigen binding molecule described herein (e.g., a PD-L1 antibody or an antigen binding fragment thereof), and a pharmaceutically acceptable carrier. In other embodiments, the PD-L1 antibody or antigen binding fragment thereof is administered in combination with a pharmaceutically acceptable carrier. As described herein, the anti-PD-L1 composition may include one or more different antibodies, one or more multispecific antibodies, one or more fusion proteins, one or more immunoconjugates, or a combination thereof.

[0282] The present invention provides a pharmaceutical composition comprising the antigen binding molecules of the present invention. The pharmaceutical composition of the present invention is formulated with suitable carriers, excipients and other agents that can improve transfer, delivery, tolerance, etc. Many suitable formulations are included in the formulas 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, lipids (cationic or anionic) containing vesicles (e.g., LIPOFECTIN TM, Life Technologies, Carlsbad, CA), DNA conjugates, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, carbowax emulsions (polyethylene glycols of various molecular weights), semisolid gels, and semisolid mixtures containing carbowaxes. See also Powell et al., "Compendium of excipients for parenteral formulations" PDA (1998) J Pharm Sci Technol 52: 238-311.

[0283] The dosage of the antigen binding molecules applied to the patient may vary according to the patient's age and body shape, target disease, illness, route of administration, etc. The preferred dosage is usually calculated according to body weight or body surface area. When the bispecific antigen binding molecules of the present invention are used to treat adult patients, a favorable scheme is to administer the bispecific antigen binding molecules of the present invention intravenously in a single dose of about 0.01 to about 20 mg / kg body weight, and a more preferred dosage is about 0.02 to about 7, about 0.03 to about 5, or about 0.05 to about 3 mg / kg body weight. Depending on the severity of the disease, the frequency and duration of treatment can be adjusted. The effective dose and time schedule for administering the bispecific antigen binding molecules can be determined empirically; for example, the patient's disease progression can be monitored by regular assessment, and the dosage can be adjusted accordingly. In addition, interspecies scaling of dosage can be performed using methods well known in the art (e.g., Mordenti et al., 1991, Pharmaceut. Res. 8: 1351).

[0284] A variety of delivery systems are known and can be used to administer the pharmaceutical compositions of the invention, e.g., liposome encapsulation, microparticles, microcapsules, recombinant cells expressing mutant viruses, receptor-mediated endocytosis (see, e.g., Wu et al., 1987, J. Biol. Chem. 262:4429-4432). Methods of introduction include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The composition can be administered by any convenient route, e.g., by infusion or bolus, absorbed through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.), and the composition can be administered with other bioactive agents. It can be systemic or topical.

[0285] The pharmaceutical composition of the present invention can be delivered subcutaneously or intravenously with a standard needle and syringe. In addition, with respect to subcutaneous delivery, pen delivery devices are easily used to deliver the pharmaceutical composition of the present invention. Such pen delivery devices can be reusable or disposable. Reusable pen delivery devices typically use a replaceable cartridge containing the pharmaceutical composition. Once all of the pharmaceutical composition in the cartridge has been administered and the cartridge is empty, the empty cartridge can be discarded and replaced with a new cartridge containing the pharmaceutical composition. The pen delivery device can then be reused. In a disposable pen delivery device, there is no replaceable cartridge. In contrast, the disposable pen delivery device is pre-filled with the pharmaceutical composition, which is stored in a reservoir within the device. Once the pharmaceutical composition in the reservoir is emptied, the entire device is discarded.

[0286] In some 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 polymer material can be used; see, Medical Applications of Controlled Release, Langer and Wise (eds.), 1974, CRC Pres., Boca Raton, Florida. In yet another embodiment, a controlled release system can be placed near the target of the composition, so that only a portion of the systemic dose is required (see, e.g., Goodson, 1984, in Medical Applications of Controlled Release, supra, Vol. 2, pp. 115-138). Other controlled release systems are discussed in the review by Langer, 1990, Science 249: 1527-1533.

[0287] The injection can include dosage forms for intravenous, subcutaneous, intradermal and intramuscular injection, infusion, etc. These injections can be prepared by known methods. For example, the above-mentioned antibody or its salt can be dissolved, suspended or emulsified in a sterile aqueous medium or oily medium conventionally used for injection to prepare an injectable preparation. The aqueous medium for injection includes, for example, physiological saline, isotonic solution containing adjuvants such as glucose, etc., which can be used in combination with the following appropriate solubilizers: for example, alcohols (such as ethanol), polyols (such as propylene glycol, polyethylene glycol), nonionic surfactants (such as polysorbate 80, HCO-50 (adduct of polyoxyethylene (50 mol) hydrogenated castor oil)), etc. The oily medium includes, for example, sesame oil, soybean oil, etc., which can be used in combination with solubilizers such as benzyl benzoate and benzyl alcohol. The prepared injection is preferably contained in a suitable ampoule.

[0288] It is very beneficial to prepare the above-mentioned pharmaceutical composition for oral or parenteral use into a unit dosage form suitable for the dosage of the active ingredient. Such unit dosage forms include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc. The content of the above-mentioned antibody is usually about 5 to about 500 mg per dosage form in a unit dose; in particular, in the form of an injection, it is preferably about 5 to about 100 mg of the above-mentioned antibody, and for other dosage forms, it is preferably about 10 to about 250 mg.

[0289] In another aspect, a method for treating a cell proliferative disorder such as cancer, a chronic infection, or an immunocompromised state comprises administering to a subject in need thereof a pharmaceutical composition comprising the anti-PD-L1 antibody or antigen-binding fragment thereof in combination with a pharmaceutically acceptable carrier. In some embodiments, the method restores, enhances, or enhances 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 eliminates PD-L1 signaling.

[0290] In some embodiments, the pharmaceutical composition is administered to increase the activity of lymphocytes (e.g., T cells) in patients with a disease, wherein the increase in lymphocyte activity is beneficial to the disease, or the disease is caused or characterized by immunosuppression, immunosuppressive cells, or PD-L1 overexpression produced by, for example, CD4T cells and CD8T cells. The methods described herein are particularly useful, for example, in patients with solid tumors, the tumor microenvironment may contribute to reducing the recognition of the immune system (immune escape). For example, a tumor may be characterized by immune cells expressing (or overexpressing) PD-L1, such as CD4T cells, CD8T cells, T-regs, and B cells.

[0291] In certain embodiments, the methods and compositions are used to treat a variety of cancers and other proliferative diseases. Since these methods can reduce PD-L1 levels, thereby inhibiting the anti-tumor activity of lymphocytes, they are applicable to a very wide range of cancers, especially solid tumors, where PD-L1 in the tumor microenvironment of solid tumors inhibits anti-tumor immune responses.

[0292] Non-limiting cancers treated using the antigen binding molecules described in the present invention (e.g., anti-PD-L1 antibodies or antigen binding fragments thereof) include, for example, 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, solid tumors in children, lymphocytic lymphoma, bladder cancer, kidney or ureter, renal pelvis cancer, central nervous system (CNS) tumors, primary C NS lymphoma, tumor angiogenesis, spinal axis tumors, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, environmentally induced cancers (including cancers induced by asbestos), hematological malignancies, including, for example, multiple myeloma, B cell lymphoma, Hodgkin's lymphoma / primary mediastinal B cell lymphoma, non-Hodgkin's lymphoma, acute myeloid lymphoma, chronic myeloid leukemia, chronic lymphatic leukemia, follicular lymphoma, diffuse large B cell lymphoma, Burkitt's lymphoma, immunoblastic large cell lymphoma, precursor B lymphoblastic lymphoma, mantle cell lymphoma, acute lymphocytic leukemia, mycosis fungoides, anaplastic large cell lymphoma, T cell lymphoma and precursor T lymphoblastic lymphoma, or any combination of these cancers. The present disclosure is also applicable to the treatment of metastatic cancer. Patients may be tested or selected for one or more of the above clinical attributes before, during or after treatment.

[0293] In one embodiment, an effective amount of the anti-PD-L1 antibody is administered to achieve and / or maintain (e.g., for 1, 2, 3, 4 weeks, and / or until the antigen binding compound is subsequently administered) a concentration in the blood of the individual of at least EC 50 , optionally EC 70 , optionally substantially EC 100 In one embodiment, the active amount of the anti-PD-L1 antibody is to achieve at least an EC of neutralizing the enzymatic activity of PD-L1 in the extravascular tissue of the individual. 50 , optionally EC 70 , optionally substantially EC 100 In one embodiment, the active amount of the anti-PD-L1 antibody is to achieve (or maintain) an EC of inhibiting the enzymatic activity of neutralizing PD-L1 in an individual. 50 , optionally EC 70 , optionally substantially EC 100 An effective amount.

[0294] Optionally, in one embodiment, in contrast to some antibodies that eliminate PD-L1-expressing tumor cells by ADCC (which, for example, may exert full efficacy at concentrations equal to or significantly below those that provide receptor saturation), the anti-PD-L1 antibody is primarily a blocker (without substantial Fcγ receptor-mediated activity) and is administered in an effective amount to neutralize the enzymatic activity of PD-L1 for a desired period of time (e.g., 1 week, 2 weeks, a month) until the next continuous administration of the anti-PD-L1 antibody.

[0295] In one embodiment, an effective amount of the anti-PD-L1 antibody is administered to achieve and / or maintain (e.g., for 1, 2, 3, 4 weeks, and / or until subsequent administration of the anti-PD-L1 antibody) at least an EC of 100% for inhibiting PD-L1-mediated T cell immunosuppression in an individual. 50 , optionally EC 70 , optionally substantially EC 100 In one embodiment, the amount of the anti-PD-L1 antibody is an amount that achieves (or maintains) its EC to inhibit PD-L1-mediated immunosuppression in the extravascular tissue of the individual. 50 , optionally EC 70 , optionally substantially EC 100 An effective amount.

[0296] In one embodiment, the present invention provides a method of treating or preventing cancer in an individual, the method comprising administering to an individual with the disease an amount of an anti-PD-L1 antibody that achieves or maintains a circulating concentration for a specified period of time, optionally in a target extravascular tissue (e.g., a tumor or tumor environment) that is higher than the concentration required to achieve 50%, 70%, or complete (e.g., 90%) receptor saturation of circulating cells expressing PD-L1 (e.g., assessed in PBMCs). Optionally, the concentration achieved is at least 20%, 50%, or 100% higher than the concentration required for saturation of a specific receptor.

[0297] In one embodiment, the invention provides a method of treating or preventing cancer in an individual, the method comprising administering to the individual an amount of an anti-PD-L1 antibody that achieves or maintains a concentration in the circulation, optionally in a target extravascular tissue (e.g., a tumor or tumor environment) for a specific period of time, that is above the EC for binding to cells expressing PD-L1. 50 , optionally EC 70 , or optionally EC 100 Optionally, the concentration achieved is less than the EC for binding to cells expressing PD-L1. 50 , optionally EC 70 , or optionally EC 100 At least 20%, 50% or 100% higher.

[0298] In any embodiment, for example, the antibody binds to ECs of PD-L1 expressing cells in human PBMCs. 50 , optionally EC 70 , or optionally EC 100 Between 0.5-100 ng / ml, optionally 1-100 ng / ml, optionally 30-100 ng / ml, for example, about 30-90 ng / ml. For example, the EC 50 It can be about 30, 37, 39, 43, 57, 58, 61, 62, 90, 95, 143 ng / ml.

[0299] Neutralization of PD-L1 enzymatic activity by EC with anti-PD-L1 antibody 50 It can be, for example, between about 0.01 μg / ml and 1 μg / ml, optionally between 0.1 μg / ml and 10 μg / ml, optionally between 0.1 μg / ml and 1 μg / ml. For example, the EC 50 It may be about 0.1 μg / ml, about 0.2 μg / ml or about 0.3 μg / ml. Thus, for example, an amount of the anti-PD-L1 antibody is administered to achieve and / or maintain a blood concentration of 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.

[0300] When tissues outside the vasculature are targeted (tumor environment, for example, in the treatment of solid tumors), approximately 10-fold higher doses are generally required compared to doses that provide corresponding concentrations in the circulation. An administration amount of anti-PD-L1 antibody that achieves (and / or maintains) a concentration of about 1 μg / ml, 2 μg / ml, 10 μg / ml, or 20 μg / ml in the circulation (blood) is expected to achieve (and / or maintain) an extravascular tissue (e.g., tumor tissue) concentration of about 0.1 μg / ml, 0.2 μg / ml, 1 μg / ml, 2 μg / ml, respectively.

[0301] In one embodiment, for example, a certain amount of anti-PD-L1 antibody is administered to reach and / or maintain a tissue (eg, tumor environment) concentration of 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 can be administered, for example, in a certain amount, and the amount of the antibody can achieve and / or maintain a blood concentration of at least about 1 μg / ml, 2 μg / ml, 10 μg / ml, or 20 μg / ml, for example, 1-100 μg / ml, 10-100 μg / ml, 1-50 μg / ml, 1-20 μg / ml, or 1-10 μg / ml. The dosage can be adjusted to maintain the desired concentration within a specified time period (eg, 1, 2, 3, 4 weeks, etc.) after administration.

[0302] In some embodiments, an amount of anti-PD-L1 antibody is administered to obtain at least an EC for neutralizing the enzymatic activity of PD-L1 in blood (serum) or extravascular tissue (e.g., tumor environment). 70 or EC 100 The antibody can be administered, for example, in an amount that can 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.

[0303] EC of a given PD-L1 antibody 50 ,EC 70 and EC 100 The value of can be evaluated, for example, in a cell-based assay to block the PD-1 / PD-L1 interaction. The "EC 50 " refers to the concentration of anti-PD-L1 antibody that can produce 50% of the maximum reaction or effect of neutralizing enzyme activity. The "EC 70 ” refers to the concentration of anti-PD-L1 antibody that produces 70% of the maximal response or effect. The “EC 100 " refers to the effective concentration of anti-PD-L1 antibody that can produce the maximum response or effect of neutralizing enzyme activity. In certain embodiments, depending on the context, EC 50 ,EC 70 or EC 100 Can be referred to as IC 50 、IC 70 or IC 100 This reflects the activity of the antigen-binding molecule (eg, anti-PD-L1 antibody or antigen-binding fragment thereof) in inhibiting PD-L1. xx It is the concentration of drug required to inhibit a biological process by xx%.

[0304] In some embodiments, particularly for the treatment of solid tumors, the concentrations achieved are designed to achieve concentrations in tissues (outside the vasculature, e.g., in a tumor or tumor environment) that correspond to at least an EC for neutralization of enzyme activity. 50 , optionally about or at least about EC 100 .

[0305] In one embodiment, the amount of anti-PD-L1 antibody is between 1 and 20 mg / kg body weight. In one embodiment, the amount is administered to the individual weekly, biweekly, monthly, or bimonthly.

[0306] In one embodiment, a method of 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 2, 3, 4 or more administration cycles), wherein the cycle is eight weeks or less, wherein for each of at least one cycle, one, two, three or four doses of the anti-PD-L1 antibody are administered at a dose of 1-20 mg / kg body weight. In one embodiment, the anti-PD-L1 antibody is administered by intravenous infusion.

[0307] A treatment regimen suitable for treating, for example, a human subject comprises, for example, administering an anti-PD-L1 antibody to a patient in an amount disclosed herein, wherein the method comprises at least one administration cycle, wherein at least one dose of the anti-PD-L1 antibody is administered in one administration cycle. Optionally, at least 2, 3, 4, 5, 6, 7 or 8 doses of the anti-PD-L1 antibody are administered. In one embodiment, the administration cycle is 2 to 8 weeks.

[0308] In one embodiment, a method of treating or preventing a disease (e.g., cancer, a solid tumor, a hematological tumor) in a subject comprises administering to the subject an anti-PD-L1 antibody that neutralizes the enzymatic activity of PD-L1 for at least one administration cycle, the administration cycle comprising at least a first and a second (and optionally a 3rd, 4th, 5th, 6th, 7th and / or 8th or more) administration of the anti-PD-L1 antibody, wherein the anti-PD-L1 antibody is administered in an effective amount to achieve or maintain an anti-PD-L1 antibody blood (serum) concentration between two consecutive administrations in an amount 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 a range between any of the foregoing concentrations.

[0309] In one embodiment, a specified continuous blood concentration is maintained, wherein the blood concentration does not significantly fall below a specified blood concentration within a specified time period (e.g., between two antibody administrations, weeks, 1 week, 2 weeks, 3 weeks, 4 weeks). In other words, although the blood concentration may change within a specified time period, the maintained specified blood concentration represents a minimum or "trough" concentration.

[0310] In one embodiment, a therapeutically active amount of an anti-PD-L1 antibody is one that is able to provide (at least) EC 50 concentration, optionally EC 70 concentration, optionally EC 100The concentration is an amount of such antibody that neutralizes the enzymatic activity of PD-L1 for a period of time after administration of the antibody, the period of time being at least about 1 week, about 2 weeks, or about 1 month.

[0311] Before or during treatment with the anti-PD-L1 antibodies described in the present disclosure, the expression level of PD-L1 and / or PD-1 in cells can be assessed in and / or near the patient's tumor; the percentage of PD-L1 expressing cells and / or PD-1 expressing cells to assess whether the patient is suitable for treatment and whether the patient may respond to treatment. An increase in the aforementioned level or expression may indicate that the individual is suitable for (e.g., may benefit from) treatment with the anti-PD-L1 antibodies described in the present disclosure.

[0312] In some embodiments, the step of evaluating the expression level and of PD-L1 and / or PD-1 in and / or near a patient's tumor tissue sample comprises obtaining a biological sample of human tissue from a subject, wherein the human tissue is selected from a tissue of a cancer patient, such as cancer tissue, tissue near or surrounding cancer, cancer adjacent tissue, adjacent non-tumor tissue, or normal adjacent tissue, and the expression level and of PD-L1 and / or PD-1 in the tissue. The expression level or nucleotide concentration in the patient can be compared with a reference level (e.g., the level of a healthy individual).

[0313] In view of the foregoing, in certain embodiments, the method comprises the steps of: (a) determining the expression level of PD-L1 and / or PD-1 in a tumor environment, optionally within a tumor and / or adjacent tissue, and upon determining that the level of PD-L1 and / or PD-1 in the tumor environment is increased compared to its corresponding reference level, (b) administering an anti-PD-L1 antibody to the individual.

[0314] In certain embodiments, determining the level of PD-L1 and / or PD-1 in the tumor environment includes obtaining a biological sample containing cancerous tissue and / or tissue close to or located around the tumor (e.g., cancer adjacent tissue, adjacent non-tumor tissue, or normal adjacent tissue) from a subject, and detecting the level and / or relative percentage of PD-L1 expressing cells and / or PD-1 expressing cells. PD-L1 and / or PD-1 expressing cells may include, for example, tumor cells, CD4 T cells, CD8 T cells, B cells, and combinations thereof. Techniques well known to those of ordinary skill in the art can be used to assess the mRNA expression of PD-L1 and / or PD-1 (by, for example, RT-PCR) or polypeptide expression (by, for example, Western blotting, immunofluorescence staining) by comparing with reference levels of healthy subjects or with reference levels before treatment to determine the expression level of PD-L1 and / or PD-1.

[0315] Anti-PD-L1 antibodies can be used to treat subjects with cancer with or without assessing PD-L1 and / or PD-1 levels in the tumor microenvironment (e.g., tumor cells, CD4 T cells, CD8 T cells, B cells).

[0316] The result of the determination that the biological sample includes cells that overexpress PD-L1 and / or PD-1 as compared to a reference indicates that the subject has cancer that may benefit from treatment with a PD-1 / PD-L1 interaction blocker. In some embodiments, the term "overexpression" refers to expression of PD-L1 and / or PD-1 polypeptides in a large number of cells taken from a given patient, such as 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 tumor cells or lymphocytes taken from a subject.

[0317] In one embodiment, a method for treating or preventing cancer in a subject in need thereof comprises the following steps: (a) detecting the percentage and / or degree of expression of PD-L1 and / or PD-1 in the tumor environment, optionally in the tumor and / or in adjacent tissues, and after determining that the tumor environment includes cells that overexpress PD-L1 and / or PD-1, optionally with increased levels of PD-L1 and / or PD-1 compared to a suitable reference level, (b) administering an anti-PD-L1 antibody to the subject. In one embodiment, the cell is a tumor cell. In another embodiment, the cell in the tumor environment, tumor and / or adjacent tissue is a non-malignant immune cell, such as a T cell.

[0318] In some embodiments, determining the expression level of PD-L1 and / or PD-1 in a tumor environment comprises obtaining a biological sample from an individual, the biological sample comprising cancer tissue and / or tissue close to or surrounding cancer (e.g., cancer adjacent tissue, adjacent non-tumor tissue, or normal adjacent tissue), contacting cells with antibodies that bind to PD-L1 and / or PD-1 polypeptides, and detecting the cell percentage and / or expression level of PD-L1 and / or PD-1. In certain embodiments, the expression of PD-L1 and / or PD-1 is assessed by cell surface expression of PD-L1 and / or PD-1 using immunohistochemistry.

[0319] The antibody compositions can be used as a monotherapy or in combination therapy with one or more other therapeutic agents, including agents that are commonly used for the specific therapeutic purpose for which the antibody is administered. See "combination therapy" above. The agent is typically administered in an amount and treatment regimen commonly used in monotherapy with the other therapeutic agent to treat the specific disease or condition. Such therapeutic agents include, but are not limited to, anticancer agents and chemotherapeutic agents.

[0320] As described above, the methods of using the pharmaceutical compositions described herein include administering to a subject in need thereof an effective amount of the pharmaceutical composition described in the present disclosure.

[0321] Any suitable route or mode of administration can be used to provide a therapeutically or prophylactically effective dose of the antibody to the patient. Exemplary routes or modes of administration include parenteral (e.g., intravenous, intraarterial, intramuscular, subcutaneous, intratumoral), oral, topical (nasal, transdermal, intradermal or intraocular), mucosal (e.g., nasal, sublingual, oral, rectal, vaginal), inhalation, intralymphatic, intraspinal, intracranial, intraperitoneal, intratracheal, intravesical, intrathecal, enteral, intrapulmonary, intralymphatic, intracavitary, intraorbital, intracapsular and transurethral administration, and local delivery via catheter or stent.

[0322] The pharmaceutical composition comprising the anti-PD-L1 antibody described in the present disclosure can be formulated in any pharmaceutically acceptable carrier or excipient. The term "pharmaceutically acceptable carrier" as used herein includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents and absorption delaying agents that are physiologically compatible. 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 glycol. Exemplary pharmaceutically acceptable carriers include one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, and the like, and combinations thereof. In many cases, it is preferred to include an isotonic agent, such as a sugar, a polyol such as mannitol, sorbitol, or sodium chloride in the composition. A pharmaceutically acceptable carrier may also include a small amount of auxiliary substances, such as a wetting agent or emulsifier, a preservative, or a buffer, which may increase the shelf life or effectiveness of the therapeutic agent.

[0323] In certain preferred embodiments, the therapeutically active agent may be incorporated into a pharmaceutical composition suitable for parenteral administration.The pharmaceutical composition for parenteral administration may be formulated by injection, for example, by bolus injection or continuous infusion.

[0324] Suitable buffers include, but are not limited to, sodium succinate, sodium citrate, sodium phosphate or potassium phosphate. Sodium chloride at a concentration of 0-300 mM (optimal concentration of 150 mM for liquid dosage forms) can be used to change the toxicity of the solution. Lyophilized dosage forms can include cryoprotectants, mainly 0-10% sucrose (optimal 0.5-1.0%). Other suitable cryoprotectants include trehalose and lactose. Lyophilized dosage forms can include fillers, mainly 1-10% mannitol (optimal 2-4%). ​​Stabilizers can be used for liquid dosage forms and lyophilized dosage forms, mainly 1-50 mM L-methionine (optimal 5-10 mM). Other suitable fillers include glycine, arginine, and 0-0.05% polysorbate-80 (optimal 0.005-0.01%). Other surfactants include, but are not limited to polysorbate 20 and BRIJ surfactants.

[0325] The therapeutic agent preparation can be lyophilized and stored as a sterile powder, preferably stored under vacuum, and then reconstituted in bacteriostatic water (including, for example, benzyl alcohol preservative) or sterile water before injection. The therapeutic agent in the pharmaceutical composition can be formulated as a "therapeutically effective amount" or a "preventive effective amount". "Therapeutically effective amount" refers to the amount of the desired therapeutic result effectively achieved within the necessary dosage and time period. The therapeutic effective amount of an antibody or an active agent can vary according to the following circumstances: the disease to be treated, the severity and course of the disease, the mode of administration, whether the antibody or active agent is administered for preventive purposes or therapeutic purposes, the bioavailability of a specific agent, the ability of the antibody to cause a desired response in an individual, previous treatment, the age, weight and sex of the patient, the clinical history of the patient and the reaction to the antibody, the type of antibody used, the discretion of the attending physician, etc. The therapeutic effective amount is also the amount of any toxic or harmful effect of the therapeutic beneficial effect greater than the recombinant vector. "Preventive effective amount" refers to the amount of the desired preventive result effectively achieved within the necessary dosage and time period.

[0326] Preferably, the polypeptide domains used in the antibodies or other active agents described herein are derived from the same host in which the polypeptide domains are administered to reduce the inflammatory response caused by administration of the therapeutic agent. As suggested above, the therapeutic agent is suitably administered to the subject at one time or over a series of treatments, and may be administered to the patient at any time from diagnosis. The PD-L1 antibody may be administered as a sole therapy or in combination with other active agents or therapies useful for treating the disorder in question.

[0327] Whether administered once or multiple times, it is generally recommended that a therapeutically effective amount or a prophylactically effective amount of a PD-L1 antibody (or other active agent) be administered in the range of about 1 ng / kg body weight / day to about 100 mg / kg body weight / day.In a specific embodiment, each PD-L1 antibody or active agent is administered in a range 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, about 10 ng / kg body weight / day kg body weight / day to about 10 mg / kg body weight / day, about 10 ng / kg body weight / day to about 1 mg / kg body weight / day, about 10 ng / kg body weight / day to about 100 μg / kg body weight / day, about 10 ng / kg body weight / day to about 10 μg / kg body weight / day, about 10 ng / kg body weight / day to about 1 μg / kg body weight / day, 10 ng / kg body weight / day to about 100 ng / kg body weight / day, about 100 ng / kg body weight / day to about 100 mg / kg body weight / day, about 100 ng / kg body weight / day to about 10 mg / kg body weight / day, about 100 n g / kg body weight / day to about 100 μg / kg body weight / day, about 100 ng / kg body weight / day to about 10 μg / kg body weight / day, about 100 ng / kg body weight / day to about 1 μg / kg body weight / day, about 1 μg / kg body weight / day to about 100 mg / kg body weight / day, about 1 μg / kg body weight / day to about 100 mg / kg body weight / day, about 1 μg / kg body weight / day to about 10 mg / kg body weight / day, about 1 μg / kg body weight / day to about 1 mg / kg body weight / day, about 1 μg / kg body weight / day to about 100 μg / kg body weight / day, about 10 μg / kg body weight / day to about 100 mg / kg body weight / day, about 10 μg / kg body weight / day to 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, 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 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 10 mg / kg body weight / day to about 100 mg / kg body weight / day.

[0328] 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.

[0329] In other embodiments, each PD-L1 antibody and / or active agent is administered in a range of 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 ... about 1 μg, about 100 ng to about 10 μg, about 100 ng to about 100 μg, about 100 ng to about 1 mg, about 100 ng to about 10 mg, about 100 ng to about 100 mg, about 100 ng to about 1000 mg, about 100 ng to about 10,000 mg, about 1 μg to about 10 μg, about 1 μg to about 100 μg, about 1 μg to about 1 mg, about 1 μg to about 10 mg, about 1 μg to about 100 mg, about 1 μg to about 1000 mg, about 1 μg to about 10,000 mg, about 10 μg to about 100 μg, about 10 μg to about 1 mg, about 10 μg to about 10 mg, about 10 μg to about 100 mg, about 10 μg to about 1000 mg, about 10 μg to about 10,000 mg, about 100 μg to about 1 mg, about 100 μg to about 10 mg, about 100 μg to about 100 mg, about 100 μg to about 1000 mg, About 100 μg to about 10,000 mg, about 1 mg to about 10 mg, about 1 mg to about 100 mg, about 1 mg to about 1000 mg, about 1 mg to about 10,000 mg, about 10 mg to about 100 mg, about 10 mg to about 1000 mg, about 10 mg to about 10,000 mg, about 100 mg to about 1000 mg, about 100 mg to about 10,000 mg, or about 1000 mg to about 10,000 mg. The antibodies described in the present disclosure can be administered every day, every 2, 3, 4, 5, 6 or 7 days, or every 1, 2, 3 or 4 weeks.

[0330] In other specific embodiments, the amount of each PD-L1 antibody or active agent administered can be the following dosage: 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.

[0331] In certain embodiments, the coding sequence of the PD-L1 antibody and / or other active agent is incorporated into a suitable expression vector (e.g., a viral or non-viral vector) to express an effective amount of the PD-L1 antibody or other active agent in a subject in need of treatment according to the above methods. In certain embodiments comprising administering, for example, one or more recombinant AAV (rAAV) viruses, the pharmaceutical composition may contain rAAVs in an amount of at least 10 10 , at least 10 11 , at least 10 12 , at least 10 13 , or at least 10 14 genome copies (GC) or recombinant virus particles, or any range thereof. In certain embodiments, the pharmaceutical composition comprises an effective amount of the recombinant virus, such as rAAV, in an amount of at least 10 per subject. 10 , at least 10 11 , at least 10 12 , at least 10 13 , at least 10 14 , at least 10 15 genome copies or recombinant viral particle genome copies, or any range thereof.

[0332] Doses can be tested in one or several art-accepted animal models appropriate for any particular cell proliferative disorder or immunocompromised disease state.

[0333] Delivery methods can also include the use of polycation-concentrated DNA linked or unlinked to killed viruses, ligand-linked DNA, liposomes, eukaryotic cell delivery vector cells, deposition of photopolymerized hydrogel materials, use of handheld gene transfer particle guns, ionizing radiation, neutralization or fusion of nuclear charges with cell membranes, particle-mediated gene transfer, and the like. 8. Diagnostic Uses of Antibodies

[0334] The antigen binding molecules (e.g., antibodies or antigen binding fragments thereof) described in the present invention can also be used to detect and / or measure human or cynomolgus monkey PD-L1, or human or cynomolgus monkey PD-L1 expressing cells in a sample, for example, for diagnostic purposes. For example, anti-PD-L1 antibodies or antigen binding fragments thereof can be used to diagnose a condition or disease characterized by abnormal expression of PD-L1 (e.g., overexpression, underexpression, lack of expression, etc.). Exemplary diagnostic assays for PD-L1 include, for example, contacting a sample obtained from a patient with an antibody described in the present invention, wherein the antibody is labeled with a detectable marker or reporter molecule. Alternatively, an unlabeled antibody can be combined with a secondary antibody that is itself detectably labeled for diagnostic applications. The detectable marker or reporter molecule can be a radioactive isotope, such as 3 H. 14 C. 18 F. 32 p. 35 S or 125 I; fluorescent or chemiluminescent moieties, such as fluorescein isothiocyanate or rhodamine; or enzymes, 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 for the PD-L1 diagnostic assay described in the present invention include any tissue or fluid sample that can be obtained from a patient, which contains a detectable amount of PD-L1 protein or fragments thereof under normal or pathological conditions. Typically, the level of PD-L1 in a specific sample obtained from a healthy patient (e.g., a patient who does not suffer from a disease or condition associated with abnormal PD-L1 levels or activity) is measured to preliminarily establish a baseline or standard level of PD-L1. The baseline level of PD-L1 can then be compared with the level of PD-L1 measured in a sample obtained from an individual suspected of having a PD-L1-related disease or condition.

[0335] In addition, human PD-L1 can be purified by immunoaffinity purification using the anti-PD-L1 antibodies described herein. IX. Test kit

[0336] Any composition described herein, such as the anti-PD-L1 antigen binding molecules described herein, and / or other therapeutic agents, can be included in a kit. In a non-limiting example, the kit comprises an antigen binding molecule, such as an antibody or an antigen binding fragment thereof. In certain embodiments, the kit further comprises other therapeutic agents described herein.

[0337] The kit may also include reagents or instructions for treating a disease or condition. It may also include one or more buffers.

[0338] The components of the kit can be packaged in aqueous medium or lyophilized form. The container device of the kit generally includes at least one vial, test tube, flask, bottle, syringe or other container device, and the components can be placed, preferably, appropriately sub-packed in the container device. If there is more than one component in the kit (the labeling reagent and the label can be packaged together), the kit generally also includes a second, third or other container, and the other components can be placed separately in the other containers. The kit may also include a second container device for accommodating sterile pharmaceutically acceptable buffer and / or other diluents. In addition, various combinations of components may be included in the vial. The kit of the present invention generally also includes a device for accommodating the composition of the present invention (e.g., anti-PD-L1 antigen binding molecules and / or other therapeutic agents), and any other reagent container for commercial sale that is sealed.

[0339] When the components of the kit are provided in the form of one and / or more liquid solutions, the liquid solution is an aqueous solution, particularly preferably a sterile aqueous solution. In addition, the components of the kit may be provided in the form of a dry powder. When the reagents and / or components are provided in the form of a dry powder, the dry powder may be reconstituted by adding a suitable solvent. It is contemplated that the solvent may also be provided in another container means.

[0340] The present invention is further illustrated by the following examples, which should not be construed as limiting the present invention.The contents of all references, patents and published patent applications cited throughout this application and the figures and tables are incorporated herein by reference. Example Example 1: Generation of rabbit anti-human PD-L1 monoclonal antibody

[0341] Rabbit immunization

[0342] Two New Zealand white rabbits (4-6 weeks old) were immunized with human PD-L1 protein by Capralogics according to an IACUC-approved protocol. Briefly, rabbits were immunized with recombinant proteins containing either the human PD-L1 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.

[0343] Serum titers were determined using an antigen-specific enzyme-linked immunosorbent assay (ELISA). Briefly, microplates were pre-coated with 25 μl of 1 μg / ml human PD-L1-His (Sino Biological, Cat#10084-H08H) or cynomolgus monkey PD-L1-His protein (Sino Biological, Cat.90251-C08H). Rabbit serum was diluted 1:1000 and then 7-point 3-fold serial dilutions were performed. The serially diluted serum was added to the assay buffer. HRP-labeled goat anti-rabbit IgG (H+L) antibody (Jackson Immuno Research, Cat#111-036-045) was diluted 1:5000 for detection, 100 μl / well TMB substrate was used for color development, and 50 μl / well of stop solution (1N H 2 SO 4 ).

[0344] Rabbits with high titers of specific antibodies were then selected for euthanasia and their spleens were isolated in a sterile manner.

[0345] PD-1:PD-L1 binding competition ELISA (cELISA) was performed on immune rabbit sera. Briefly, the microplate was pre-coated with 1 μg / μl human PD-L1-mFc fusion protein (Sino Biological, Cat#10084-H05H) at 4°C overnight; the microplate was then blocked and the following substances were added at 100 μl / well: (1) 0.5 μg / ml human PD-1-His-hFc-Biotin fusion protein (Sino Biological, Cat#10377-H03H-B) alone, (2) 0.5 μg / ml human PD-1-His-hFc-Biotin fusion protein mixed with diluted Imfinzi (Durvalumab) (starting from 6000 ng / ml, followed by 8-point serial dilutions); or (3) 0.5 μg / ml human PD-1-His-hFc-Biotin fusion protein mixed with diluted rabbit serum (starting from a 1:10 dilution, followed by 8-point serial dilutions). HRP-conjugated Neutratavidin (ThermoFisher, Cat#31001) was diluted 1:20000 and used for detection. 100 μl / well TMB substrate was used for color development. 50 μl / well stop solution (1N H 2 SO 4 ).

[0346] cELISA results (data not shown) showed that immune rabbit serum could block PD-1:PD-L1 interaction, and a commercially available anti-PD-L1 antibody (Durvalumab) was used as a positive control.

[0347] B cell sorting

[0348] Spleen tissues of immunized rabbits were collected and homogenized. Splenocytes were either used immediately for sorting or snap frozen, stored at -196°C, and thawed later for further sorting. Biotinylated human PD-L1 D1-His-Avi (Yurogen), human PD-L1ECD-His (Sino Biological, Cat#10084-H08H), human PD-L1 mouse Fc fusion protein (Sino Biological, Cat#10084-H05H), or cynomolgus monkey PD-L1 ECD-His (Sino Biological, Cat.90251-C08H) were used to stain rabbit B cells. If the sorting antigen was not biotinylated, it was biotinylated. Briefly, 1 mg of biotin was dissolved in 180 μL of deionized water and a 10 mM Sulfo-NHS-LC-biotin (ThermoFisher Cat#A39257) stock solution was prepared on ice. Depending on the amount of protein to be biotinylated, add 20-fold molar ratio of biotin to the protein solution and incubate the mixture on ice for 2 h. TMBiotinylated proteins were dialyzed against 1×PBS in a microdialysis cup (3.5K MWCO, 0.1 mL) (ThermoFisher, Cat#69550) to remove free biotin, changing the buffer at least twice, 3 hours apart. For each sorting, multiple tubes of splenocytes from selected rabbits were thawed and cultured overnight in B cell culture medium (RPMI-1640, 15% FBS, 1×HEPES, 1×2-ME (β-mercaptoethanol), 1% penicillin / streptomycin) before sorting. 96-well B cell culture plates were also prepared one day in advance. Briefly, YC1 and a proprietary growth factor cocktail in irradiated B cell culture medium were added to 96-well culture plates (120 μL / well). On the day of sorting, suspended and loosely attached splenocytes were collected by gently pipetting the culture medium onto the culture surface of the flask. The cells were transferred to a conical tube and spun at 400×g for 3 minutes. Wash the cell pellet twice with pre-cooled FACS buffer (1×PBS+0.5% BSA). Add 5μg / ml (final concentration) of biotinylated PD-L1 protein. Incubate the mixture at room temperature for 20 minutes. Spin the labeled mixture at 400×g for 3 minutes, resuspend the cells in FACS buffer, and transfer the cells to a 1.5ml brown Eppendorf tube. Add NeutrAvidin-Dy594 (diluted 1:300, Invitrogen Cat#22842) and FITC-conjugated anti-rabbit IgM antibody (diluted 1:500, Novus, Cat#MB7173), and incubate the mixture at 4°C for 15-30 minutes. Centrifuge the labeling mixture at 400×g for 3 minutes. Wash the cells twice with ice-cold FACS buffer. Centrifuge the washed cell pellet at ~10 7 Resuspend in pre-chilled 1× PBS + 1% FBS at a concentration of 10 cells / ml. At least 10 minutes before sorting, add 7-AAD (1 μg / ml, final concentration) to distinguish live / dead cells. Sorted a single 7-AAD- / IgM- / Dy594+ cell into each well of a 96-well plate seeded with YC1. Sorted B cells were incubated in rabbit B cell complete medium (Yurogen) at 37°C and 5% CO 2 The culture conditions were maintained for 9-12 days.

[0349] Screening of single B cell cultures

[0350] On days 9 to 12 after sorting, 15 μL of B cell culture supernatant was collected for antigen-specific ELISA assay as described above. On days 10 to 13 after sorting, the B cell culture plate was centrifuged at 400 × g for 3 minutes. The supernatant of positive clones (OD greater than the cutoff value of 0.3 or the cutoff value of 0.9 for antigen-specific ELISA) was selected and subjected to PD-1:PD-L1c ELISA (as described above). The supernatant of positive clones was also subjected to cell binding assay. Briefly, MDA-MB-231 cells (known to express human PD-L1) were first added to a 96-well plate (1x10 5 cells). Incubate 50 μl of B cell culture (undiluted) supernatant sample with cells on ice for 30 minutes. After washing with FACS buffer, add 100 μl of 1 μg / ml Alex Fluor647-conjugated goat anti-rabbit IgG for B cell culture detection and incubate on ice for 30 minutes. After two washes, resuspend the cells with 100 μl of FACS buffer. Add 100 μl of 7-AAD diluted 1:50 to the cell suspension for live / dead cell staining, and then perform 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.

[0351] cELISA was collected on days 10 to 13 after sorting + FACS + The cloned cells were stored in 100 μL of DNA / RNA protection reagent (Zymo, Cat# R1100-250) and then transferred to a 250 μL PCR tube.

[0352] V H and V L Cloning (or sequencing) of the coding gene

[0353] Total RNA of selected clones was purified from cell pellets stored in DNA / RNA protection reagent using RNeasy Mini kit (Zymo, Cat#R1051) according to the manufacturer's operating manual. Total RNA was eluted using 36 μl of nuclease-free water (Ambion, cat#AM9937). For each clone, 11 μl of total RNA was mixed with 1 μl of oligo (dT) 12-18 primer (Invitrogen, Cat#58862) and 1 μl of dNTPs (10 mM, Thermo Scientific, Cat#R0182), and then heated at 65°C for 5 minutes. Then, for each clone mixture, 4 μl of 5×FirstStrand buffer, 1 μl of 100 mM DTT, 1 μl of RNAaseOUT (Invitrogen, Cat#10777-019) and 1 μl of SuperScript III reverse transcriptase (Invitrogen, Cat#18080-044) were added. Reverse transcription was performed at 50°C for 1 hour and at 75°C for 15 minutes to inactivate the SuperScript III enzyme. After cDNA synthesis, VH and VL genes were amplified using VH and VL variable region primer pairs, respectively, with 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 MgCl 2 , 0.2 μl of Taq DNA polymerase (ThermoFisher, Cat#10342053) and 15.5 μl of nuclease-free water. The conditions for VH PCR were 94°C for 3 min, followed by 40 cycles of 94°C for 30 sec, 55°C for 30 sec, 72°C for 45 sec, and finally extension at 72°C for 5 min. The reaction for VL PCR 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 10× PCR buffer, 1 μl of 50 mM MgCl 2 , 0.2 μl of Platinum TMTaq DNA polymerase (ThermoFisher, Cat#10966018) and 15 μl of nuclease-free water. The conditions for VL PCR were 3 min at 98°C, followed by 40 cycles (95°C for 15 sec, 64°C for 15 sec, 72°C for 30 sec) and a final extension at 72°C for 5 min. The PCR products of VH and VL were separated on a 1% agarose gel electrophoresis system. The expected size of VH and VL amplicons was ~500 bp. The corresponding bands of VH and VL of each clone were cut out from the gel and used according to the manufacturer's operating instructions. Gel and PCR Clea-Up kit (Macherey-Nagel, Cat#740609.250) was used to extract VH and VL from the gel. Depending on the amount of PCR product, 12-30 μl elution buffer was used to elute the PCR products of VH and VL. Linear expression module cassette (LEM) PCR products were constructed by overlapping PCR, wherein the C fragment contained a CMV promoter, VH or VL, and the H fragment contained a rabbit IgG heavy chain CH1 fragment, a human IgG1 CH2 and CH3 fragment or a light chain constant region, followed by an SV40 transcription terminator and a poly A 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-frag 3 (10 μM), 1 μl 3' primer H-frag 4 (10 μM), 10 μl 5× LongAmp Taq reaction buffer, 1.5 μl dNTPs (10 mM), 2 μl LongAmp Taq DNA polymerase (New England BioLabs, Cat# 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-frag 3 (10 μM), 1 μl of 3' primer H-frag 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 minutes; 35 cycles: 94°C for 30 seconds, 55°C for 40 seconds, 68°C for 3 minutes and 30 seconds; the last cycle: 68°C for 5 minutes. 5 μl of PCR product was used to check the amplified fragment size and yield by 0.8% agarose gel electrophoresis (as shown below). The remaining PCR products were used according to the manufacturer’s instructions. Gel and PCR Clean-Up Kit purification. The LEM PCR product was eluted using 32 μl of elution buffer. The LEM construct was sequenced by Quintara Bioscience (Quintara, Cambridge, MA).

[0354] Screening of LEM transfection

[0355] Cell binding assays were performed on supernatant samples obtained after LEM transfection (as described above). Briefly, 50 μl of LEM transfection supernatant samples (undiluted or diluted 1:5, 1:10 or 1:50) were incubated with cells on ice for 30 minutes. After washing with FACS buffer, 100 μl of 1 μg / ml Alex Fluor 647-coupled anti-human IgG was added and incubated on ice for 30 minutes. After two washes, the cells were resuspended with 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 then prepared 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 formats (BioIntron) were used as isotype controls. Example 2: Cloning and expression of antibody variable region genes of anti-PD-L1 antibodies

[0356] The genes encoding the VH and VL of each clone were cloned into a Yurogen expression vector consisting of human IgG1 Fc and rabbit CH1. After sequence confirmation, the IgG heavy and light chain plasmids were used to produce antibodies on a small scale in HEK293T cells. Briefly, ∼5 × 10 6 HEK293T cells were seeded into 12-well plates. For each clone, 500 ng of heavy chain LEM PCR product and 500 ng of light chain LEM PCR product were diluted with 45 μL of Opti-MEM medium (Gibco, Cat#31985-070). 3 μL of Lipofectamine 2000 reagent (Invitrogen, Cat#11668019) was diluted with 47 μL 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 1 mL of Opti-MEM medium. The mixed solution was added dropwise to each well. Gently rotate the culture plate to thoroughly mix the transfection reagent and medium. The transfected cells were incubated at 37°C, 5% CO 2 The cells were incubated for 48-72 hours under the same conditions and the supernatant was then harvested for further screening. Example 3: Specific binding of anti-PD-L1 antibodies to PD-L1

[0357] Binding specificity

[0358] Purified antibodies were tested by FACS to confirm their binding and affinity to human and cynomolgus PD-L1-His recombinant proteins. Briefly, 0.5 μg / ml of human PD-L1-His (Sino Biological, Cat#10084-H08H) or cynomolgus PD-L1-His (Sino Biological, Cat.90251-C08H) protein diluted in 1xPBS (GIBCO) was coated on Maxisorp plates (Nunc) at 100 μl / well overnight at 4°C. The wash buffer (1xPBST, diluted from 20xPBST (Thermo Scientific) with distilled water) was washed three times with 300 μl / well, and then 300 μl / well of blocking buffer (1xPBST / 1% BSA) was added and blocked at room temperature for 2 hours on a shaker (100 rpm). After washing the plate, add 100 μl / well of detection antibody diluted in detection buffer (PBST / 1% BSA) and incubate at room temperature for 2 hours on a shaker at 100 rpm. After washing the plate, add 100 μl of horseradish peroxidase (HRP)-conjugated detection antibody to each well and incubate at room temperature for 1 hour on a shaker at 100 rpm. After washing the plate, add 100 μl / well of TMB substrate and color develop at room temperature for 10 minutes in the dark. Add 50 μl / well of stop solution (1NH 2 SO 4 ) and read the values ​​at 450 nm on a microplate reader (SpectraMax M5).

[0359] Table 21 shows that exemplary antibodies of the invention specifically bind to human and cynomolgus monkey PD-L1. Durvalumab (AstraZeneca) was used as a positive control. Anti-HEL isotype control (BioIntron) was used as a negative control.

[0360] Table 21. EC50 (nM) of Anti-PD-L1 Antibodies Binding to Human and Cynomolgus Monkey PD-L1 Protein Antibody Human PD-L1 Cynomolgus monkey PD-L1 27A2 0.0146 0.0113 28B3 0.0159 0.0177 32E2 0.1062 0.1787 33E8 0.0399 0.0414 33H7 0.0171 0.0130 55H3 / 76D6 0.0199 0.0126 56E5 0.0184 0.0078 56H3 0.0172 0.0076 57B10 0.0186 0.0071 57F11 0.0216 0.0138 62E6 0.0240 0.0188 69D10 0.0215 0.0177 73C8 0.0160 0.0121 74A9 0.0229 0.0198 74F4 0.0157 0.0125 Durvalumab 0.0158 0.0081 Anti-HEL n.a. n.a.

[0361] Similar tests were performed to detect whether the exemplary antibodies of the present disclosure bind to mouse PD-L1 (SinoBiological, Cat#50010-M08H), rat PD-L1 (Sino Biological, Cat#80450-R08H), canine PD-L1 (Acro Biosystems, Cat#PDL-C52H4), or rabbit PD-L1 (Acro Biosystems, Cat#PDL-R52H6). No binding of the exemplary antibodies of the present disclosure to mouse, rat, canine, or rabbit PD-L1 was detected.

[0362] Similar tests were performed to detect whether the exemplary antibodies of the present disclosure bind to other proteins belonging to the same PD-L1 family. The exemplary antibodies of the present disclosure did not show binding to CD80 (Sino Biological, Cat#10698-H08H), CD86 (Sino Biological, Cat#10699-H08H), BH-H2 (Sino Biological, Cat#11559-H08H), B7-H3 (Sino Biological, Cat#11188-H08H) and PD-L2 (Sino Biological, Cat#10292-H08H).

[0363] Binding affinity

[0364] The binding affinity of the exemplary anti-PD-L1 antibodies of the present disclosure was determined. The assay was performed according to the protocol recommended by Gator or Gator Plus (Gator Bio Inc.). In short, human PD-L1-His (Sino Biological, Cat#10084-H08H) was serially diluted 4 times from 4 μg / ml. After three regenerations in the generation buffer (Gator Bio, Cat#120012) and neutralization of the HFC probe (Gator Bio, Cat#160003), 1 μg / ml of the antibody to be tested was fixed to the HFC probe in Q buffer (GatorBio, Cat#120010). After the baseline step, the probe with the antibody fixed was immersed in the antigen solution. The binding and dissociation of antigen and antibody were measured on the BLI instrument Gator. Affinity was calculated using Gator Bio analysis software.

[0365] Table 22 below summarizes the binding affinity to human PD-L1 using Gator analysis. Durvalumab (AstraZeneca) was used as a positive control. Anti-HEL isotype control (BioIntron) was used as a negative control.

[0366] Table 22. Binding affinity of anti-PD-L1 antibodies to human PD-L1 Antibody <![CDATA[Kon(x10 5 ,1 / Ms)]]> <![CDATA[Koff(x 10 -4 ,1 / s)]]> <![CDATA[K D (nM)]]> 27A2 6.43 0.36 0.06 28B3 8.10 0.76 0.09 32E2 3.71 1.13 0.31 33E8 3.61 4.18 1.16 33H7 5.36 3.29 0.61 55H3 / 76D6 4.98 3.17 0.64 56E5 7.48 5.60 0.75 56H3 6.86 3.99 0.58 57B10 8.10 0.77 0.09 57F11 3.71 1.13 0.31 62E6 7.88 3.42 0.43 69D10 7.18 65.3 9.09 73C8 8.04 0.939 0.12 74A9 6.43 2.34 0.36 74F4 6.48 2.38 0.36 Durvalumab 7.27 2.56 0.35

[0367] Table 23. Binding affinity of anti-PD-L1 antibodies to cynomolgus monkey PD-L1 *2 parallel tests Example 4: Specific binding of anti-PD-L1 antibodies to cells expressing PD-L1

[0368] The assay described in this example is to determine whether the anti-PD-L1 antibodies of the present disclosure specifically bind to cells expressing PD-L1.

[0369] In this test, MDA-MB-231-GFP cells (known to express human PD-L1) were collected from fresh culture. The cells were then suspended in FACS buffer (1xDPBS / 1% FBS) at a concentration of 10 6 / ml. 100μl of cells were added to each of the 96-well plates. The antibody to be tested was diluted from 15μg / mL in a 1:3 gradient and then added to each well. The antibody was mixed with the cells and the mixture was incubated on ice for 1 hour. After washing with FACS buffer, 100μl of diluted PE-coupled anti-human Fc secondary antibody (Jackson Immuno Research, Cat#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#420404, used at a 1:100 dilution) and analyzed on Guava 11HT (Luminex). Data were analyzed using Flowjo software.

[0370] Table 24 shows that exemplary antibodies of the present invention specifically bind 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.

[0371] Table 24. Binding of anti-PD-L1 antibodies to MDA-MB-231-GFP cells Example 5: Anti-PD-L1 antibodies block PD-1 / PD-L1 interaction

[0372] The test in this example is to determine whether the anti-PD-L1 antibodies of the present disclosure can block the PD-1 / PD-L1 interaction.

[0373] cELISA test

[0374] Competitive ELISA (cELISA) test was used to determine the blocking of PD-1 / PD-L1 interaction by exemplary anti-PD-L1 antibodies disclosed herein. Briefly, microplates were pre-coated with 1 μg / μl human PD-L1-mFc fusion protein (Sino Biological, Cat#10084-H05H) diluted with 1xPBS (GIBCO) and incubated overnight at 4°C; the plates were washed four times with 300 μl / well of wash buffer (1xPBST, diluted with distilled water from 20xPBST (Thermo Scientific)), followed by addition of 300 μl / well blocking buffer (1xPBST / 3% BSA) at room temperature on a shaker (100 rpm) for 2 hours. The plates were washed and the following reagents were added at 100 μl / well: (1) 1 μg / ml human PD-1-His-hFc-Biotin fusion protein alone (Sino Biological, Cat#10377-H03H-B), (2) mixed human PD-1-His-hFc-Biotin fusion protein and diluted Bavencio (Avelumab) or Imfinzi (Durvalumab) or anti-HEL isotype control antibody (BioIntron), starting at a concentration of 50 nM, followed by a 12-point 1:2-fold serial dilution; or (3) mixed human PD-1-His-hFc-Biotin fusion protein and diluted test anti-PD-L1 antibody, starting at a concentration of 50 nM, followed by a 12-point 1:2-fold serial dilution. 100 μl / well of 1:20,000 diluted HRP-coupled avidin (HRP-Avidin, Invitrogen, Cat#A2664) was added to each well and incubated at room temperature for 1 hour on a shaker at 100 rpm. After washing the plate, 100 μl / well of TMB substrate was added and color was developed at room temperature in the dark for 10 minutes. 50 μl / well stop solution (1N H 2 SO 4 ) and read the values ​​at 450 nm on a plate reader (SpectraMax M5).

[0375] Table 25 summarizes the IC values ​​of exemplary antibodies of the present disclosure blocking the interaction between PD-1 and PD-L1. 50 Value. Commercially available anti-PD-L1 antibodies Imfinzi (Durvalumab) and Bavencio (Avelumab) were used as positive controls. As shown in Table 25 andFigure 1 As shown, the exemplary anti-PD-L1 antibodies of the present disclosure can effectively block the interaction between PD-1 and PD-L1.

[0376] Table 25. IC of anti-PD-L1 antibodies blocking PD-1 / PD-L1 interaction 50 value Antibody <![CDATA[IC 50 (nM)]]> 27A2 1.9 28B3 1.5 32E2 7.5 33E8 3.1 33H7 2.1 56H3 2.1 74A9 2.3 Durvalumab 1.4* Avelumab 11.2* *: Average value of three parallel tests

[0377] Promega Testing

[0378] This test uses a PD1 / PD-L1 blocking test kit (Promega, Cat. J1255) to determine whether the exemplary antibodies of the present invention can block the interaction between PD1 and cell surface PD-L1. The kit can also be used to determine the activity of the antibody in blocking the PD1 / PD-L1 interaction. The test was performed according to the manufacturer's operating manual. Briefly, 100 μl of CHO-K1-hPD-L1 cell suspension (in F-12 medium containing 10% FBS) was added to a single well of a flat-bottom white 96 plate treated with cell culture and incubated at 37°C in CO 2 Incubate in an incubator for 16 hours. The antibody to be tested or the positive control commercially available anti-PD-L1 antibody Imfinzi (Durvalumab), Bavencio (Avelumab), Atezolizumab analog (BioIntron) or SGN-PD-L1 analog anti-PD-L1 antibody (BioIntron) (or anti-HEL isotype control antibody) was serially diluted with 1xRPMI / 1% FBS. The overnight culture medium was removed, and 40 μl of each diluted antibody was added to each well, followed by 40 μl of effector PD-1 cell suspension in each well. The plate was incubated at 37°C with CO 2 Then, 80 μl of BIO-Glo reagent was added to each well and the plate was immediately operated to measure the luminescent signal on a plate reader (SpectraMax M5).

[0379] Table 26 summarizes the IC values ​​of exemplary antibodies of the present disclosure blocking the interaction of PD-1 with cell surface PD-L1. 50 Durvalumab was used as a positive control. As shown in Table 27, the exemplary anti-PD-L1 antibodies of the present disclosure can effectively block the interaction between PD-1 and cell surface PD-L1.

[0380] Table 26. IC of anti-PD-L1 antibodies blocking PD-1 / PD-L1 interaction 50 value Antibody <![CDATA[IC 50 (nM)]]> 27A2 0.23 28B3 0.24 33H7 0.52 55H3 / 76D6 0.97 56E5 0.41 56H3 0.36 57B10 0.47 62E6 0.41 74A9 0.29 Durvalumab 0.14* *: Average value of three parallel tests

[0381] Table 27 summarizes the IC values ​​of exemplary antibodies of the present disclosure for blocking the interaction of PD-1 with cell surface PD-L1 in another assay. 50 Value. Durvalumab, Avelumab, Atezolizumab analogs, and SGN-PD-L1 analogs were used as positive controls. Except for Avelumab, which was tested in five parallel wells, the remaining antibodies were tested in two parallel wells. As shown in Table 27, the exemplary anti-PD-L1 antibodies of the present disclosure can effectively block the interaction between PD-1 and cell surface PD-L1.

[0382] Table 27. IC of anti-PD-L1 antibodies blocking PD-1 / PD-L1 interaction 50 value Antibody <![CDATA[IC 50 (nM)]]> 27A2 0.35 28B3 0.33 33E8 0.38 74A9 0.46 SGN-PDL1* 0.68 Atezolizumab* 0.33 Durvalumab 0.43 Avelumab 0.49** *: Similar to antibodies **: Average value of five parallel tests Example 6: ADCC of anti-PD-L1 antibodies

[0383] In this experiment, antibody-dependent cellular cytotoxicity (ADCC) of exemplary anti-PD-L1 antibodies of the present disclosure was determined.

[0384] InvivoGen testing using Raji cells expressing PD-L1

[0385] NFAT-CD16 Lucia luciferase reporter Jurkat cells and Raji cells expressing PD-L1 were purchased from InvivoGen and expanded for testing. First, 20 μl of the antibody to be tested was added to each well of a flat-bottom 96-well cell culture plate. Subsequently, 90 μl of Raji-PD-L1 cells (~100,000 cells) was added to each well. The plates were incubated at 37°C with CO 2 After incubation in the incubator for 1 hour, 90 μl of Jurkat-Lucia TM -NFAT-CD16 cells (~200,000 cells) were added to each well and the plates were incubated at 37°C in a CO atmosphere. 2 Incubate in the incubator for 6 hours. Take 20 μl of the co-incubated Raji-PD-L1 and Jurkat-Lucia TM NFAT CD16 cell culture supernatant was transferred to a single well of a 96-well white (opaque, Corning) plate, and 50 μl of QUANTI-Luc was added to each well. TMSubstrate (InvivoGen, prepared in advance according to the supplier's instructions). The plate was immediately processed to measure the luminescent signal on a plate reader (SpectraMax M5). An anti-HEL isotype control antibody was used as a negative control.

[0386] Table 28. EC of ADCC of anti-PD-L1 antibodies 50 value Antibody <![CDATA[EC 50 (nM)]]> 28B3 4.2 32E2 4.0 33E8 3.6 55H3 / 76D6 3.1 56E5 1.9 57B10 3.7 69D10 2.2 73C8 1.0 74A9 2.3 74F4 1.0 Anti-HEL n.a.

[0387] InvivoGen testing using MDA-MB-231GFP cells

[0388] This test is similar to the InvivoGen test described above, except that Raji-PD-L1 cells were replaced with MDA-MB-231GFP cells. Avelumab and Durvalumab were used as positive controls. An anti-HEL isotype control antibody was used as a negative control. Table 29 summarizes the EC values ​​for ADCC of exemplary anti-PD-L1 antibodies of the present disclosure. 50 value.

[0389] Table 29. EC of ADCC of anti-PD-L1 antibodies 50 value Antibody <![CDATA[EC 50 (nM)]]> 27A2 2.3 28B3 0.8 32E2 1.8 33E8 0.5 33H7 0.1 55H3 / 76D6 0.5 56E5 0.2 56H3 0.2 57B10 0.5 57F11 0.2 62E6 0.1 69D10 1.0 73C8 0.2 74A9 0.1 Avelumab* 0.4 Anti-HEL* n.a. *: Average value of 2 parallel tests Example 7: Anti-PD-L1 Antibodies and Internalization of PD-L1

[0390] This example is used to measure the endocytosis of the complex formed by the exemplary anti-PD-L1 antibody of the present invention and PD-L1 expressed on the cell surface.

[0391] FACS-based endocytosis analysis

[0392] About 100,000 (~10 5) MDB-MA-231 cells were incubated on ice with the test antibodies at the specified concentrations (2, 1, 0.5 and 0.25 μg / ml). The commercially available ADC drug Trodelvy (Sacituzumab govitecan-hziy, Immunomedics) was used as a positive control. The internalization of the commercially available anti-PD-L1 antibodies Durvalumab and Avelumab was also tested. After one hour of incubation, two portions were separated from the mixture and transferred to a 37°C incubator for incubation for 2 or 4 hours, respectively. Samples collected at different time points were washed twice with cold FACS buffer and then stained with PE-conjugated anti-human Fc secondary antibody (Jackson ImmunoResearch) on ice for 1 hour. After washing twice with cold FACS buffer, the cells were stained with 7-AAD (Biolegend) and then fixed with 2% paraformaldehyde (diluted from 10% paraformaldehyde (MilliporeSigma, Cat#R04586)). MDB-MA-231 cells are known to express PD-L1 and Trop2. Sacituzumab is an anti-Trop2 antibody that is known to induce efficient endocytosis after binding to Trop2 on the cell surface.

[0393] Samples were analyzed using Guava 11HT (Luminex) and data were analyzed using FlowJo software. The endocytosis rate was calculated as follows:

[0394] Internalization percentage % = {[(MFI of sample on ice) - (MFI of sample at 37°C, specified incubation time)] / (MFI of sample on ice)} × 100

[0395] Table 30 summarizes the endocytosis of the complexes formed by different anti-PD-L1 antibodies and PD-L1 expressed on the surface of MDA-MB-231 cells. The results show that the exemplary anti-PD-L1 antibodies induce endocytosis after binding to cell surface PD-L1.

[0396] Table 30: Anti-PD-L1 antibody-induced endocytosis

[0397] Figure 2A The results showed that incubation of MDA-MB-231 cells with 1 μg / ml of 33E8 for 4 hours at 37°C induced the highest level of endocytosis (shown as percentage of endocytosis) compared with all other anti-PD-L1 antibodies including Durvalumab. The lowest level of endocytosis was induced by 32E2 or Avelumab. Figure 2BIt was shown that incubation of MDA-MB-231-GFP cells with 1 μg / ml of 33E8 for 1, 2, and 4 hours induced higher levels of endocytosis (shown as percentage of endocytosis) compared to all other anti-PD-L1 antibodies including Avelumab, Durvelumab, Atezolizuab analogs (BioIntron), and SGN-PDL1 analogs (BioIntron).

[0398] Dye-based endocytosis assays

[0399] According to the manufacturer's instructions, Invitrogen TM Zenon TM pHrodo TM iFL RED Human IgG Labeling Reagent (Cat# Z25612) was used to determine the endocytic properties of an exemplary anti-PD-L1 antibody. TM pHrodo TM Internalized antibodies bound by the iFL RED Human IgG Labeling Reagent will increase the fluorescent signal in low pH intracellular compartments within the cell, thus providing a method to visualize endocytosis.

[0400] Briefly, a 4x working solution (24 μg / ml) of each antibody was prepared in complete DMEM medium (10% FBS, 1x Pen / Strep). 25 μL of the 4x antibody working solution was aliquoted into each well of a clear bottom 96-well plate. 25 μL of the 4x Zenon working 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 form the labeled complex, and then 50 μL of 2x10 6 / mL Raji cells expressing human PD-L1. Cells were incubated with the labeled complexes under standard cell culture conditions. After 4 to 24 hours of incubation, cells were analyzed under an immunofluorescence microscope (Invitrogen EVOS Cell Imaging System M5000) to observe the cellular internalization of antibodies. Figure 2C As shown, induction of endocytosis can be seen in representative images, indicating that 33E8 and SGN-PDL1 analog antibodies can induce endocytosis, but anti-HEL isotype control antibodies cannot. Hoechst 33342 (Invitrogen, Cat. H3570) is a commonly used cell-permeable nuclear counterstain that emits blue fluorescence and is used to distinguish condensed nuclei within cells. Example 8: Cytotoxicity of anti-PD-L1 antibodies conjugated to cytotoxic agents

[0401] This study determined whether exemplary antibodies of the disclosure coupled to cytotoxic agents could induce killing of cells expressing PD-L1.

[0402] In this study, the cytotoxic agent Deruxtecan was conjugated to an exemplary antibody of the present disclosure via an anti-hFc bridging antibody that specifically binds to the Fc region of the antibody.

[0403] Briefly, Raji cells expressing PD-L1 (InvivoGen) were plated at a density of 2500 cells / well in an opaque wall 96-well plate with 160 μl of medium per well. 40 μl of 200 nM anti-hFc antibody (Biolegend, Cat#410701) coupled via a stable thiol-ether bond (prepared by CellMosaic Inc.) and Deruxtecan (MedKoo Bioscience, Cat#2071060) were mixed with the following substances to prepare the antibody conjugate:

[0404] (1) 500 nM of the anti-PD-L1 antibody of the present invention to be tested, whose Fc region is human Fc (hFc),

[0405] (2) 500 nM commercially available anti-PD-L1 antibody Imfinzi (Durvalumab),

[0406] (3) 500 nM anti-PD-L1 antibody Atezolizumab or SGN-PDL1 analogs ( Figure 3 Similar antibody data are marked with *).

[0407] (4) 500 nM anti-HEL isotype control antibody, or

[0408] (5) Culture medium only.

[0409] The antibody conjugates were added in duplicate to the wells containing Raji cells. The cells mixed with the antibody conjugates were incubated at 37°C, 5% CO 2 Incubate for 3 days at 4 °C. 5 μM of the microtubule polymerization inhibitor colchicine was used as a positive cytotoxicity control. After three days of incubation, 90 μl of supernatant was removed from each well, and 100 μl of freshly prepared CellTiter-Glo (Promega, Cat# 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 luminescent signal, and then the luminescent signal was detected on a plate reader (SpectraMax5) to determine cell viability. There is a direct relationship between the intensity of the luminescent signal and the number of live cells in each well. The results are shown in Figure 2. Figure 3 shown. Example 9: Epitope classification of anti-PD-L1 antibodies

[0410] This study determined whether certain exemplary antibodies of the present invention belong to the same epitope group as the commercially available Avelumab, that is, whether certain exemplary antibodies of the present disclosure bind to the same or similar epitope on the PD-L1 protein as Avelumab.

[0411] 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 group 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#10084-H08H) and 1xPBS (GIBCO) and incubated overnight at 4°C. The plates were washed four times with 300 μl / well wash buffer (1xPBST, diluted from 20xPBST (Thermo Scientific) with distilled water), followed by addition of 300 μl / well blocking buffer (1xPBST / 3% BSA) for 2 hours at room temperature on a shaker (100 rpm). The plates were washed and the diluted anti-PD-L1 antibodies to be tested were added at 50 μl / well, starting at 20 μg / ml, followed by 11 point 1:2 serial dilutions. The plate containing the anti-PD-L1 antibody solution was incubated at 37°C for one hour, and then 0.4 μg / ml biotinylated Avelumab (Thermo Scientific TM EZ-Link TM Micro Sulfo-NHS-LC-Biotinylation Kit, Cat#0021935, prepared according to the manufacturer's manual). The plate containing the mixture was incubated at 37°C for one hour. HRP-conjugated avidin (Invitrogen, Cat#A2664) was used for detection at a dilution of 1:2000. Avelumab was used as a positive control.

[0412] Figure 4A and 4BIt is shown that exemplary antibodies 27A2, 56H3, 33H7, 28H3, 74A9, Durvalumab, Avelumab and analogs Avelumab, Atezolizumab, SGN-PDL1 antibodies can effectively block the binding of biotinylated Avelumab to human PD-L1-His protein, indicating that these antibodies belong to the same epitope group. Antibodies 32E2 and 33E8 only show effective blocking of 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 group as Avelumab, but their epitope groups may be related. Antibodies 55H3 / 76D6, 56E5, 57B10, 57F11, 62E6, 69D10, 73C8, and 74F4 of the present invention did not show blocking of the binding of Avelumab to PD-L1 protein under experimental conditions (data not shown), indicating that these antibodies belong to different epitope groups from Avelumab.

[0413] The above description is intended to guide those of ordinary skill in the art in practicing the present invention, rather than to describe in detail all obvious modifications and variations, which will become apparent to those skilled in the art after reading this specification. However, all such obvious modifications and variations should be included within the scope of the present invention, which is defined by the appended claims. Unless the context specifically indicates otherwise, the claims are intended to cover the claimed components and steps in any order that is effective to meet their intended objectives.

Claims

1. An isolated antibody or antigen-binding fragment thereof that binds to human programmed death ligand 1 (PD-L1), comprising Heavy chain variable (V H ) domain, the heavy chain variable domain comprising three heavy chain complementarity determining regions (CDRs) from N-terminus to C-terminus: HCDR1, HCDR2 and HCDR3; and Light chain variable (V L ) domain, the light chain variable domain comprises three light chain complementary determining regions (CDRs) from N-terminus to C-terminus: LCDR1, LCDR2 and LCDR3; wherein (a) HCDR1 comprises an amino acid sequence 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 SAYWIC (SEQ ID NO:12); wherein 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 X32-I-X33-X34-GS-X35-I-X36-DYA-X37-WAKG (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 WTSGGGGFGL (SEQ ID NO: 42); (d) LCDR1 comprises an amino acid sequence selected from the group consisting of: 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:); wherein X65 is N, S or T, X66 is I or V, X67 is N or S, X68 is D or N, and 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); wherein 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; and (f) LCDR3 comprises the amino acid sequence QGYYSGYIWT (SEQ ID NO: 83).

2. The isolated antibody or antigen-binding fragment thereof according to claim 1, wherein: (a) HCDR1 comprises an amino acid sequence selected from 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 shown 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; and (f) LCDR3 comprises the amino acid sequence shown in SEQ ID NO:

83.

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

83.

4. The isolated antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein the antibody comprises: (a) a heavy chain variable region (HCVR) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 92-103; and (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 isolated antibody or antigen-binding fragment thereof according to claim 4, wherein the antibody comprises: (a) a HCVR comprising the amino acid sequence set forth in SEQ ID NO:92, and a LCVR comprising the amino acid sequence set forth in SEQ ID NO:118; (b) a HCVR comprising the amino acid sequence set forth in SEQ ID NO:93, and a LCVR comprising the amino acid sequence set forth in SEQ ID NO:119; (c) a HCVR comprising the amino acid sequence set forth in SEQ ID NO:94, and a LCVR comprising the amino acid sequence set forth in SEQ ID NO:120; (d) a HCVR comprising the amino acid sequence set forth in SEQ ID NO:95, and a LCVR comprising the amino acid sequence set forth in SEQ ID NO:121; (e) a HCVR comprising the amino acid sequence set forth in SEQ ID NO:96, and a LCVR comprising the amino acid sequence set forth in SEQ ID NO:122; (f) a HCVR comprising the amino acid sequence set forth in SEQ ID NO:97, and a LCVR comprising the amino acid sequence set forth in SEQ ID NO:123; (g) a HCVR comprising the amino acid sequence set forth in SEQ ID NO:98, and a LCVR comprising the amino acid sequence set forth in SEQ ID NO:124; (h) a HCVR comprising the amino acid sequence set forth in SEQ ID NO:99, and a LCVR comprising the amino acid sequence set forth in SEQ ID NO:125; (i) a HCVR comprising the amino acid sequence set forth in SEQ ID NO: 100, and a LCVR comprising the amino acid sequence set forth in SEQ ID NO: 126; (j) a HCVR comprising the amino acid sequence set forth in SEQ ID NO:101, and a LCVR comprising the amino acid sequence set forth in SEQ ID NO:127; (k) a HCVR comprising the amino acid sequence set forth in SEQ ID NO:102, and a LCVR comprising the amino acid sequence set forth in SEQ ID NO:128; or (l) a HCVR comprising the amino acid sequence set forth in SEQ ID NO:103, and a LCVR comprising the amino acid sequence set forth in SEQ ID NO:

129.

6. An isolated antibody or antigen-binding fragment thereof that binds to human PD-L1, 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 three light chain complementarity determining regions (CDRs) from N-terminus to C-terminus: LCDR1, LCDR2 and LCDR3; wherein (a) HCDR1 comprises 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 SEQ ID NOs: 1-12; (b) HCDR2 comprises 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 SEQ ID NOs: 22-31; (c) HCDR3 comprises an amino acid sequence that is about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identical to the amino acid sequence set forth in SEQ ID NO:42; (d) LCDR1 comprises 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 SEQ ID NOs: 53-59; (e) LCDR2 comprises 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 SEQ ID NOs: 69-73; and (f) LCDR3 comprises an amino acid sequence that is about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identical 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, comprising: (a) a heavy chain variable region (HCVR) comprising 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 group consisting of SEQ ID NOs: 92-103; and (b) a light chain variable region (LCVR) comprising 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 group consisting of SEQ ID NOs: 118-129.

8. An isolated antibody or antigen-binding fragment thereof that binds to human programmed death ligand 1 (PD-L1), comprising Heavy chain variable (V H ) domain, the heavy chain variable domain comprising three heavy chain complementarity determining regions (CDRs) from N-terminus to C-terminus: HCDR1, HCDR2 and HCDR3; and Light chain variable (V L ) domain, comprising three light chain complementary determining regions (CDRs) from the N-terminus to the C-terminus: LCDR1, LCDR2 and LCDR3; (a) HCDR1 comprises the amino acid sequence X8-D-X9-YMS (SEQ ID NO: ); wherein X8 is D or G, and X9 is W or Y; (b) HCDR2 comprises the amino acid sequence SIY-X42-GSLN-X43-YYATWAKG (SEQ ID NO:), wherein X42 is S or T, and X43 is I, S or T; (c) HCDR3 comprises the amino acid sequence R-X57-KN-X58-D-X59-G-X60-FDL (SEQ ID NO:), wherein 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 a member selected from the group consisting of QAS-X76-SI-X77-X78-X79-LA (SEQ ID NO:), QAS-X80-SINDRLA (SEQ ID NO:), The amino acid sequences of QASQSI-X81-X82-ALA (SEQ ID NO:), 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 X106-X107-STLAS (SEQ ID NO: ); wherein X106 is A or G, and X107 is A or T; and (f) LCDR3 comprises the amino acid sequence QQGWTVSSL-X111-NA (SEQ ID NO:), wherein X111 is D or E.

9. The isolated antibody or antigen-binding fragment thereof according to claim 8, wherein: (a) HCDR1 comprises an amino acid sequence selected from 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 shown in SEQ ID NO: 90 or 91.

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

11. The isolated antibody or antigen-binding fragment thereof according to any one of claims 8 to 10, wherein the antibody comprises: (a) a heavy chain variable region (HCVR) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 110-117; and (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 isolated antibody or antigen-binding fragment thereof according to claim 11, wherein the antibody comprises: (a) a HCVR comprising the amino acid sequence set forth in SEQ ID NO:110, and a LCVR comprising the amino acid sequence set forth in SEQ ID NO:136; (b) a HCVR comprising the amino acid sequence set forth in SEQ ID NO:111, and a LCVR comprising the amino acid sequence set forth in SEQ ID NO:137; (c) a HCVR comprising the amino acid sequence set forth in SEQ ID NO:112, and a LCVR comprising the amino acid sequence set forth in SEQ ID NO:138; (d) a HCVR comprising the amino acid sequence set forth in SEQ ID NO:113, and a LCVR comprising the amino acid sequence set forth in SEQ ID NO:139; (e) a HCVR comprising the amino acid sequence set forth in SEQ ID NO:114, and a LCVR comprising the amino acid sequence set forth in SEQ ID NO:140; (f) a HCVR comprising the amino acid sequence set forth in SEQ ID NO:115, and a LCVR comprising the amino acid sequence set forth in SEQ ID NO:141; (g) a HCVR comprising the amino acid sequence set forth in SEQ ID NO:116, and a LCVR comprising the amino acid sequence set forth in SEQ ID NO:142; or (h) a HCVR comprising the amino acid sequence shown in SEQ ID NO:117, and a LCVR comprising the amino acid sequence shown in SEQ ID NO:

143.

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

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

15. An isolated antibody or antigen-binding fragment thereof that binds to human PD-L1, comprising: Heavy chain variable (V H ) domain, the heavy chain variable domain comprising three heavy chain complementarity determining regions (CDRs) from N-terminus to C-terminus: HCDR1, HCDR2 and HCDR3; and Light chain variable (V L ) domain, the light chain variable domain comprising three light chain complementary determining regions (CDRs) from N-terminus to C-terminus: LCDR1, LCDR2 and LCDR3; wherein (a) HCDR1 comprises an amino acid sequence selected from SGYDMS (SEQ ID NO: 15) and SGYDMC (SEQ ID NO: 16); (b) HCDR2 comprises the amino acid sequence X38-IFT-X39-SGSTWYANWAKG (SEQ ID NO: ), wherein X38 is C or S, and X39 is G or T; (c) HCDR3 comprises the amino acid sequence T-X51-DG-X52-GSFYMNL (SEQ ID NO: ); wherein X51 is K or R, and X52 is A or V; (d) LCDR1 comprises the amino acid sequence QASGTIGSNLA (SEQ ID NO: 63) or QASQTIGSNLA (SEQ ID NO: 62); (e) LCDR2 comprises the amino acid sequence KAFTLAS (SEQ ID NO:76) or KTFTLAS (SEQ ID NO:77); and (f) LCDR3 comprises the amino acid sequence QQGATRINIDNA (SEQ ID NO: 86) or QQGASRINIDNA (SEQ ID NO: 87).

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

17. The isolated antibody or antigen-binding fragment thereof according to claim 16, wherein (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 shown in SEQ ID NO:16, HCDR2 comprises the amino acid sequence shown in SEQ ID NO:35, HCDR3 comprises the amino acid sequence shown in SEQ ID NO:46, LCDR1 comprises the amino acid sequence shown in SEQ ID NO:63, LCDR2 comprises the amino acid sequence shown in SEQ ID NO:77, and LCDR3 comprises the amino acid sequence shown in SEQ ID NO:

87.

18. The isolated antibody or antigen-binding fragment thereof according to any one of claims 15 to 17, wherein the antibody comprises: (a) a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 106 or 107; and (b) a light chain variable region (LCVR), said light chain variable region comprising the amino acid sequence of SEQ ID NO: 132 or 133.

19. The isolated antibody or antigen-binding fragment thereof according to claim 18, wherein the antibody comprises: (a) a HCVR comprising the amino acid sequence set forth in SEQ ID NO:106, and a LCVR comprising the amino acid sequence set forth in SEQ ID NO:132; or (b) a HCVR comprising the amino acid sequence set forth in SEQ ID NO:107, and a LCVR comprising the amino acid sequence set forth in SEQ ID NO:

133.

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

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

22. An isolated antibody or antigen-binding fragment thereof that binds to human PD-L1, comprising: Heavy chain variable (V H ) domain, the heavy chain variable domain comprising three heavy chain complementarity determining regions (CDRs) from N-terminus to C-terminus: HCDR1, HCDR2 and HCDR3; and Light chain variable (V L ) domain, the light chain variable domain comprising three light chain complementary determining regions (CDRs) from N-terminus to C-terminus: LCDR1, LCDR2 and LCDR3; 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 the amino acid sequence SISIGGATY-X40-A-X41-WAKG (SEQ ID NO:), wherein X40 is F or Y, and X41 is S or T; (c) HCDR3 comprises the amino acid sequence ARNVD-X53-IYL-X54-AF-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 QASQNIYNNLA (SEQ ID NO: 64); (e) LCDR2 comprises the amino acid sequence 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 QTYYLT-X109-T-X110-NA (SEQ ID NO:), wherein X109 is S or T, and X110 is I or T.

23. The isolated antibody or antigen-binding fragment thereof according to claim 22, wherein (a) HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 36 or 37; (b) HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 47 or 48; (c) LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 78 or 79; and (d) LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 88 or 89.

24. The isolated antibody or antigen-binding fragment thereof according to claim 23, wherein (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 shown in SEQ ID NO:18, HCDR2 comprises the amino acid sequence shown in SEQ ID NO:37, HCDR3 comprises the amino acid sequence shown in SEQ ID NO:48, LCDR1 comprises the amino acid sequence shown in SEQ ID NO:64, LCDR2 comprises the amino acid sequence shown in SEQ ID NO:79, and LCDR3 comprises the amino acid sequence shown in SEQ ID NO:

89.

25. The isolated antibody or antigen-binding fragment thereof according to any one of claims 22 to 24, wherein the antibody 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.

26. The isolated antibody or antigen-binding fragment thereof of claim 25, wherein the antibody comprises: (a) a HCVR comprising the amino acid sequence set forth in SEQ ID NO:108, and a LCVR comprising the amino acid sequence set forth in SEQ ID NO:134; or (b) a HCVR comprising the amino acid sequence set forth in SEQ ID NO:109, and a LCVR comprising the amino acid sequence set forth in SEQ ID NO:

135.

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

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

29. An isolated antibody or antigen-binding fragment thereof that binds to human PD-L1, comprising: Heavy chain variable (V H ) domain, the heavy chain variable domain comprising three heavy chain complementarity determining regions (CDRs) from N-terminus to C-terminus: HCDR1, HCDR2 and HCDR3; and Light chain variable (V L ) domain, the light chain variable domain comprising three light chain complementary determining regions (CDRs) from N-terminus to C-terminus: LCDR1, LCDR2 and LCDR3; wherein (a) HCDR1 comprises the amino acid sequence SSYYMS (SEQ ID NO: 13); (b) HCDR2 comprises the amino acid sequence CISGGVTDNAYYASWAKG(SEQ ID NO:32); (c) HCDR3 comprises the amino acid sequence DSSSGYFFLL (SEQ ID NO: 43); (d) LCDR1 comprises the amino acid sequence QASQNIYSNLA (SEQ ID NO: 60); (e) LCDR2 comprises the amino acid sequence GASNLRS (SEQ ID NO:74); and (f) LCDR3 comprises the amino acid sequence QEGYSIGNVDNP (SEQ ID NO: 84).

30. The isolated antibody or antigen-binding fragment thereof of claim 25, wherein the antibody 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.

31. An isolated antibody or antigen-binding fragment thereof that binds to human PD-L1, 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 three light chain complementarity determining regions (CDRs) from N-terminus to C-terminus: LCDR1, LCDR2 and LCDR3; wherein (a) HCDR1 comprises an amino acid sequence that is about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identical to the amino acid sequence set forth in SEQ ID NO:13; (b) HCDR2 comprises an amino acid sequence that is about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identical to the amino acid sequence set forth in SEQ ID NO:32; (c) HCDR3 comprises an amino acid sequence that is about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identical to the amino acid sequence set forth in SEQ ID NO:43; (d) LCDR1 comprises an amino acid sequence that is about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identical to the amino acid sequence set forth in SEQ ID NO:60; (e) LCDR2 comprises an amino acid sequence that is about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identical to the amino acid sequence set forth in SEQ ID NO:74; and (f) LCDR3 comprises an amino acid sequence that is about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identical 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, comprising: (a) a heavy chain variable region (HCVR) comprising an amino acid sequence that is about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identical to the amino acid sequence of SEQ ID NO: 104; and (b) a light chain variable region (LCVR) comprising an amino acid sequence that is about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identical to the amino acid sequence of SEQ ID NO:

130.

33. An isolated antibody or antigen-binding fragment thereof that binds to human PD-L1, comprising: Heavy chain variable (V H ) domain, the heavy chain variable domain comprising three heavy chain complementarity determining regions (CDRs) from N-terminus to C-terminus: HCDR1, HCDR2 and HCDR3; and Light chain variable (V L ) domain, the light chain variable domain comprises three light chain complementary determining regions (CDRs) from N-terminus to C-terminus: LCDR1, LCDR2 and LCDR3; wherein (a) HCDR1 comprises the amino acid sequence SSYYMI (SEQ ID NO: 14); (b) HCDR2 comprises the amino acid sequence YVYTGSGNTWYASWAKG(SEQ ID NO:33); (c) HCDR3 comprises the amino acid sequence ASGADGVYDWGWDI (SEQ ID NO: 44); (d) LCDR1 comprises the amino acid sequence QASQSIYSLLA (SEQ ID NO: 61); (e) LCDR2 comprises the amino acid sequence GASNLES (SEQ ID NO:75); and (f) LCDR3 comprises the amino acid sequence QNNYDSGRIYGLA (SEQ ID NO: 85).

34. The isolated antibody or antigen-binding fragment thereof of claim 29, wherein the antibody 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.

35. An isolated antibody or antigen-binding fragment thereof that binds to human PD-L1, 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 three light chain complementarity determining regions (CDRs) from N-terminus to C-terminus: LCDR1, LCDR2 and LCDR3; wherein (a) HCDR1 comprises an amino acid sequence that is about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identical to the amino acid sequence set forth in SEQ ID NO:14; (b) HCDR2 comprises an amino acid sequence that is about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identical to the amino acid sequence set forth in SEQ ID NO:33; (c) HCDR3 comprises an amino acid sequence that is about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identical to the amino acid sequence set forth in SEQ ID NO:44; (d) LCDR1 comprises an amino acid sequence that is about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identical to the amino acid sequence set forth in SEQ ID NO:61; (e) LCDR2 comprises an amino acid sequence that is about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identical to the amino acid sequence set forth in SEQ ID NO:75; and (f) LCDR3 comprises an amino acid sequence that is about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identical 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, comprising: (a) a heavy chain variable region (HCVR) comprising an amino acid sequence that is about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identical to the amino acid sequence of SEQ ID NO: 105; and (b) a light chain variable region (LCVR) comprising an amino acid sequence that is about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identical to the amino acid sequence of SEQ ID NO:

131.

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

38. The isolated antibody or antigen-binding fragment thereof of any one of claims 1 to 37, wherein (i) the antibody competes for binding to human PD-L1 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 monkey PD-L1; (iii) the antibody specifically binds to human PD-L1 on the cell surface; (iv) the antibody blocks the interaction between PD-1 and PD-L1; (v) the antibodies enhance immune responses; (vi) the antibody induces ADCC; and (vii) The antibody induces internalization of PD-L1.

39. An isolated antibody or antigen-binding fragment thereof, which competes with the antibody of any one of claims 1-38 for binding to human PD-L1.

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

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

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

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 of any one of claims 1-42. An expression vector comprising the polynucleotide of claim 43 .

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

46. ​​A method for preparing the antibody or antigen-binding fragment thereof of any one of claims 1-42, comprising expressing the antibody in the 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 pharmaceutically acceptable carrier or diluent.

48. The pharmaceutical composition according to claim 47, wherein the pharmaceutical composition comprises an effective amount of the antibody or antigen-binding fragment thereof so that it (a) specifically binds to human or cynomolgus monkey PD-L1 on the cell surface; (b) blocks the interaction between PD-1 and PD-L1; (c) induces ADCC; (d) induces endocytosis of PD-L1; (e) provides an immune response of immune cells; or (f) achieves any combination of (a)-(e).

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

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

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

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

53. The method of any one of claims 49-52, wherein the method activates T cells and directs them to kill tumor target cells.

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

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

56. The method of claim 55, wherein the checkpoint inhibitor is selected from inhibitors of PD-1, PD-L1, 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.

57. The method of claim 56, wherein the CTLA-4 inhibitor is selected from ipilimumab, cadunimumab, and YH001 (Euhe Pharmaceutical).

58. The method of claim 56, wherein the other therapeutic agent is an agonist of an immune cell stimulating receptor 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 ligands, and any combination thereof.

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

60. The method of any one of claims 54-58, wherein the other therapeutic agent is formulated in a different pharmaceutical composition from the antibody.

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

62. The method of any one of claims 54-60, wherein the other 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 the isolated antibody or antigen-binding fragment thereof according to any one of claims 1 to 42 or the pharmaceutical composition according to claim 47 or 48, thereby inducing antibody-dependent cellular cytotoxicity (ADCC) of PD-L1-expressing cells.

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

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

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

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