Anti-adenosine receptor (A2RA) antibodies and their uses

Abstract

The present invention provides anti-A2aR antigen binding molecules, e.g., antibodies and antigen-binding fragments thereof, and further provides methods for using the anti-A2aR antigen binding molecules to treat multiple diseases associated with abnormalities in adenosine receptor signaling, such as cancer, chronic diseases, chronic infections, autoimmune diseases, inflammatory diseases, neurodegenerative diseases, and fibrosis.

Description

cross reference

[0001] This application claims priority to U.S. Provisional Application No. 63 / 325,578, filed March 30, 2022, the entire contents of which are incorporated herein by reference.

[0002] This application is related to International Patent Application PCT / US21 / 52819, filed September 30, 2021. The entire contents of the above filed application are incorporated herein by reference. [Technical field]

[0003] The present invention relates to antibodies, in particular adenosine receptor A2aR antibodies, that are useful in the treatment of several types of diseases, including cancer. [Background technology]

[0004] The immune system plays an important role in the identification and elimination of tumor cells. Tumor cells use various mechanisms to evade immune-mediated tumor cell destruction. In these pathways, tumor cells increase their levels of and responsiveness to adenosine via the adenosine receptor signaling pathway, which is a highly effective inhibitor of effector T cell function, to evade immune defenses.

[0005] Adenosine is a purine nucleoside generated by the decomposition of adenosine triphosphate (ATP). Under adverse conditions such as hypoxia, local ischemia, inflammation, or cancer, the extracellular level of adenosine increases significantly. Once adenosine is released, cell signaling pathways are activated by interaction with four known G protein-coupled receptors: adenosine A1 receptor subtype (A1R), adenosine A2A receptor subtype (A2aR), adenosine A2B receptor subtype (A2bR), and adenosine A3 receptor subtype (A3R).

[0006] Adenosine levels are primarily controlled by the activity of CD39 and CD73, two extracellular enzymes that act together in a two-step reaction to convert inflammatory ATP to immunosuppressive adenosine. CD39 hydrolyzes ATP to AMP, which is further hydrolyzed by CD73 to adenosine, which is readily accessible to most cells. In addition, tumor cells release intracellularly accumulated ATP (which is normally cell-impermeable) into the extracellular space upon cell death as a result of metabolic or oxygen deprivation stress.

[0007] In the tumor microenvironment, adenosine produced by CD73 promotes tumor cell growth and survival and inhibits antitumor immune responses. High levels of CD73 expression by cancer cells and their accumulation in tumor tissues are associated with poor overall survival and recurrence-free survival in patients with breast and ovarian cancer. CD73 and adenosine support the formation of new blood vessels in cancer cells, and the metastasis and survival of cancer cells. Adenosine mediates the expression of A2A (or A2A3) on T cells. 2A ) receptor (A2aR) and activates an intracellular signaling cascade that leads to inhibition of T cell activation and function. A2aR is a member of the G protein-coupled adenosine receptor family, which also includes A1R, A2bR, and A3R, and is an anti-inflammatory effector of extracellular adenosine through its predominant expression in cells of the brain and lymphoid tissues.

[0008] Abnormally high concentrations of adenosine in the tumor microenvironment trigger activation of A2aR, initiating a negative feedback loop that allows tumors to evade immune recognition. Specifically, adenosine-mediated activation of A2aR inhibits IFNγ production and upregulates CD8 + It inhibits the activity of multiple types of antitumor immune cells, including T cells, dendritic cells, natural killer cells, and M1 macrophages, and also suppresses the activity of bone marrow-derived suppressor cells (MDSCs) and regulatory T (T reg It has also been suggested that activation of A2aRs on tumor cells promotes tumor cell metastasis.

[0009] Although several small molecule antagonists of A2A receptors have entered the clinical testing stage to treat Parkinson's disease and cancer, blockade of A2aR with biopharmaceutical candidates in the treatment of cancer is still lacking. Mice treated with A2aR antagonists (e.g., ZM241385) show significant delay in tumor growth due to reduced immune suppression against effector T cells. This was further highlighted by A2aR knockout mice that showed increased tumor rejection. Also, the combination of blockade of A2aR with small molecule antagonists and inhibition of PD-1 / PD-L1 or CTLA-4 with monoclonal antibodies has synergistic effects in enhancing immune responses compared to blockade of single PD-1 / PD-L1 or CTLA-4 pathways.

[0010] Thus, modulation of A2aR activity, adenosine concentration and / or CD39 / CD73 expression, and effector immune cell activation in the tumor microenvironment represents an attractive therapeutic strategy to limit tumor progression, improve antitumor immune responses, avoid treatment-induced immune deviation, and potentially limit toxicity to normal tissues. There is a need in the art for compositions and methods for treating cancer by modulating (e.g., inhibiting) A2aR activity in immune cells. Summary of the Invention

[0011] The present invention provides antigen-binding molecules (e.g., anti-A2aR antibodies or antigen-binding fragments thereof) for regulating (e.g., enhancing or suppressing) the activity of A2aR by specifically binding to A2aR. The A2aR may be present on the surface of a cell, such as a mammalian cell (e.g., a mammalian immune cell, such as a mouse immune cell, a cynomolgus monkey immune cell, or a human immune cell). The present invention further provides methods in which the antigen-binding molecules (e.g., anti-A2aR antibodies or antigen-binding fragments thereof) of the present invention are used to regulate (e.g., suppress) the activity of A2aR or to treat a subject (e.g., a subject suffering from or susceptible to an A2aR-related disease) that would benefit from regulating (e.g., suppressing) the activity of A2aR. In some embodiments, the antigen-binding molecules (e.g., anti-A2aR antibodies or antigen-binding fragments thereof) of the present invention are humanized.

[0012] Thus, in one aspect, the present invention provides an isolated antigen-binding molecule, e.g., an antibody or antigen-binding fragment thereof, that binds to the human adenosine A2A receptor (A2aR). The antibody comprises a heavy chain variable region (VH) comprising, from N-terminus to C-terminus, three heavy chain complementarity-determining regions (CDRs), HCDR1, HCDR2, and HCDR3, and a light chain variable region (VL) comprising, from N-terminus to C-terminus, three light chain complementarity-determining regions (CDRs), LCDR1, LCDR2, and LCDR3; wherein (a) the HCDR1 comprises the amino acid sequence shown in GFX1FTX2X3WMN (SEQ ID NO: 1), where X1 is A or T, X2 is R or S, and X3 is F or Y; and (b) the HCDR2 comprises the amino acid sequence shown in RI DPX4DSEX5X6YX7X8KFWX9 (SEQ ID NO: 1) (wherein X4 is S or Y, X5 is A or T, X6 is H or Q, X7 is A, H, or N, X8 is A or H, and X9 is D or G), (c) the HCDR3 comprises the amino acid sequence represented by GRSLYGKGDY (SEQ ID NO: 10) or LRSLYGKGDY (SEQ ID NO: 11), (d) the LCDR1 comprises the amino acid sequence represented by RSSQSX 11 VHX 12NGNTYLE (sequence number) (where X 11 is L or I, and X 12 is R or S; (e) the LCDR2 comprises the amino acid sequence set forth in KVSNRFS (SEQ ID NO: 14); and (f) the LCDR3 comprises the amino acid sequence set forth in FQGSHVPLT (SEQ ID NO: 15) or YQGSHVPLT (SEQ ID NO: 16); further wherein the antibody or antigen-binding fragment thereof comprises a light chain framework region 4 (LFR4) comprising the amino acid sequence set forth in FGGGTKVEIK (SEQ ID NO: 50). In one embodiment, (a) the HCDR1 comprises an amino acid sequence selected from the group consisting of the amino acid sequences shown in SEQ ID NOs: 1, 2, and 3, (b) the HCDR2 comprises an amino acid sequence selected from the group consisting of the amino acid sequences shown in SEQ ID NOs: 4 to 9, and 89, (c) the HCDR3 comprises an amino acid sequence shown in SEQ ID NO: 10 or 11, (d) the LCDR1 comprises an amino acid sequence shown in SEQ ID NO: 12 or 13, (e) the LCDR2 comprises an amino acid sequence shown in SEQ ID NO: 14, and (f) the LCDR3 comprises an amino acid sequence shown in SEQ ID NO: 15 or 16.

[0013] In other embodiments, the isolated antibody or antigen-binding fragment thereof comprises: (a) an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 4, an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 10, an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 12, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 14, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 15; (b) an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5, an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 10, an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 12, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 14, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 15; (c) an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 2, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 6, an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 11, an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 13, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 14, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 16. (d) an HCDR1 comprising the amino acid sequence shown in SEQ ID NO:2, an HCDR2 comprising the amino acid sequence shown in SEQ ID NO:7, an HCDR3 comprising the amino acid sequence shown in SEQ ID NO:11, an LCDR1 comprising the amino acid sequence shown in SEQ ID NO:13, an LCDR2 comprising the amino acid sequence shown in SEQ ID NO:14, and an LCDR3 comprising the amino acid sequence shown in SEQ ID NO:16; (e) an HCDR1 comprising the amino acid sequence shown in SEQ ID NO:3, an HCDR2 comprising the amino acid sequence shown in SEQ ID NO:8, an HCDR3 comprising the amino acid sequence shown in SEQ ID NO:11, an LCDR1 comprising the amino acid sequence shown in SEQ ID NO:12, an LCDR2 comprising the amino acid sequence shown in SEQ ID NO:14, and an LCDR3 comprising the amino acid sequence shown in SEQ ID NO:16; (f) an HCDR1 comprising the amino acid sequence shown in SEQ ID NO:3, an HCDR2 comprising the amino acid sequence shown in SEQ ID NO:9, an HCDR3 comprising the amino acid sequence shown in SEQ ID NO:11, an LCDR1 comprising the amino acid sequence shown in SEQ ID NO:12, an LCDR2 comprising the amino acid sequence shown in SEQ ID NO:14, and an LCDR3 comprising the amino acid sequence shown in SEQ ID NO:16;or (g) an HCDR1 comprising the amino acid sequence shown in SEQ ID NO:3, an HCDR2 comprising the amino acid sequence shown in SEQ ID NO:89, an HCDR3 comprising the amino acid sequence shown in SEQ ID NO:11, an LCDR1 comprising the amino acid sequence shown in SEQ ID NO:12, an LCDR2 comprising the amino acid sequence shown in SEQ ID NO:14, and an LCDR3 comprising the amino acid sequence shown in SEQ ID NO:16.

[0014] In another aspect, the present invention provides an antigen-binding molecule that binds to the human adenosine A2A receptor (A2aR), e.g., an isolated antibody or antigen-binding fragment thereof. The antibody or antigen-binding fragment thereof comprises a heavy chain variable (VH) domain comprising three heavy chain complementarity determining regions (CDRs), HCDR1, HCDR2, and HCDR3, from N-terminus to C-terminus, and a light chain variable (VL) domain comprising three light chain complementarity determining regions (CDRs), LCDR1, LCDR2, and LCDR3, from N-terminus to C-terminus; wherein (a) the HCDR1 is as set forth in GFX1FTX2X3WMN (SEQ ID NO:), where X1 is A or T, X2 is R or S, and X3 is F or Y. (b) the HCDR2 comprises an amino acid sequence selected from the group consisting of the amino acid sequences shown in RIDPYDSETQYAHKFWD (SEQ ID NO: 5), RIDPSDSEAHYAHKFWG (SEQ ID NO: 7), RIDPSDSETHYNAKFWG (SEQ ID NO: 9), and RIDPSDSETHYAHKFWG (SEQ ID NO: 89); (c) the HCDR3 comprises the amino acid sequence shown in GRSLYGKGDY (SEQ ID NO: 10) or LRSLYGKGDY (SEQ ID NO: 11); and (d) the LCDR1 comprises the amino acid sequence shown in RSSQSX 11 VHX 12 NGNTYLE (sequence number) (where X 11 is L or I, and X 12is R or S.), (e) the LCDR2 comprises the amino acid sequence of KVSNRFS (SEQ ID NO: 14), and (f) the LCDR3 comprises the amino acid sequence of FQGSHVPLT (SEQ ID NO: 15) or YQGSHVPLT (SEQ ID NO: 16). In one embodiment, (a) the HCDR1 comprises an amino acid sequence selected from the group consisting of the amino acid sequences shown in SEQ ID NOs: 1, 2 and 3, (b) the HCDR2 comprises an amino acid sequence selected from the group consisting of the amino acid sequences shown in SEQ ID NOs: 5, 7, 9 and 89, (c) the HCDR3 comprises an amino acid sequence shown in SEQ ID NO: 10 or 11, (d) the LCDR1 comprises an amino acid sequence shown in SEQ ID NO: 12 or 13, (e) the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 14, and (f) the LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 15 or 16.

[0015] In other embodiments, the isolated antibody or antigen-binding fragment thereof comprises (a) an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO:1, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO:5, an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO:10, an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO:12, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO:14, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO:15; (b) an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO:2, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO:7, an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO:11, an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO:13, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO:14, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO:16. (c) an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 3, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 9, an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 11, an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 12, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 14, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 16, or (d) an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 3, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 89, an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 11, an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 12, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 14, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 16. In yet other embodiments, the isolated antibody or antigen-binding fragment thereof further comprises a light chain framework region (FR4) comprising the amino acid sequence set forth in FGGGTKVEIK (SEQ ID NO: 50).

[0016] In one embodiment of each aspect of the present invention and any one of the embodiments thereof, the isolated antibody or antigen-binding fragment thereof according to any one of claims 1 to 6 further comprises: (a) a heavy chain framework region 1 (HFR1) comprising the amino acid sequence represented by X14-VQLVQSGAEVKKPGASVKVSC-K-X15-S (SEQ ID NO:) or EVQLVQSGAEVKKPGESLRIS-CK-X16-S (SEQ ID NO:) (wherein X14 is E or Q, X15 is A or T, and X16 is A or T); (b) a heavy chain framework region 1 (HFR2) comprising the amino acid sequence represented by WVRQ-X17-PG-X18-GLEW-X19-G (SEQ ID NO:) (wherein X17 is A or M, X18 is K or Q, and X19 is I or M). (c) a heavy chain framework region 2 (HFR2) comprising the amino acid sequence shown in R-X20-T-X21-T-X22-D-X23-S-X24-STVYMELSSLRSEDTAVYYC (SEQ ID NO:) or HATLSVDKSSSTVYLQWSSLKASDTAMYYC (SEQ ID NO:29) (wherein X20 is A or V, X21 is L or M, X22 is R or V, X23 is K or T, and X24 is S); and (d) a heavy chain framework region 4 (HFR4) comprising the amino acid sequence shown in WGQGTTVTVSS (SEQ ID NO:32) or WGHGTTVTVSS (SEQ ID NO:33). In other embodiments, (a) the HFR1 comprises an amino acid sequence selected from the group consisting of the amino acid sequences shown in SEQ ID NOs: 17 to 21, (b) the HFR2 comprises an amino acid sequence selected from the group consisting of the amino acid sequences shown in SEQ ID NOs: 22, 23, and 24, (c) the HFR3 comprises an amino acid sequence selected from the group consisting of the amino acid sequences shown in SEQ ID NOs: 26 to 31, and (d) the HFR4 comprises the amino acid sequence shown in SEQ ID NO: 32 or 33.

[0017] In other embodiments, the isolated antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising, from N-terminus to C-terminus, the structure: HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4; wherein HFR1, HFR2, HFR3, and HFR4 have the following structures: (a) the sequences set forth in SEQ ID NO:17, SEQ ID NO:22, SEQ ID NO:25, and SEQ ID NO:32, respectively; (b) the sequences set forth in SEQ ID NO:18, SEQ ID NO:22, SEQ ID NO:26, and SEQ ID NO:32, respectively; (c) the sequences set forth in SEQ ID NO:18, SEQ ID NO:23, SEQ ID NO:26, and SEQ ID NO:32, respectively; (d) the sequences set forth in SEQ ID NO:18, SEQ ID NO:23, SEQ ID NO:27, and SEQ ID NO:32, respectively; (e) the sequences set forth in SEQ ID NO:18, SEQ ID NO:23, SEQ ID NO:28, and SEQ ID NO:32, respectively; (f) the sequences set forth in SEQ ID NO:19, SEQ ID NO:24, SEQ ID NO:29, and SEQ ID NO:32, respectively; (g) the sequences set forth in SEQ ID NO:19, SEQ ID NO:24, SEQ ID NO:29, and SEQ ID NO:32, respectively; (h) the sequences shown in SEQ ID NO:18, SEQ ID NO:22, SEQ ID NO:26, and SEQ ID NO:33, respectively; (i) the sequences shown in SEQ ID NO:18, SEQ ID NO:23, SEQ ID NO:27, and SEQ ID NO:33, respectively; (j) the sequences shown in SEQ ID NO:18, SEQ ID NO:23, SEQ ID NO:28, and SEQ ID NO:33, respectively; (k) the sequences shown in SEQ ID NO:20, SEQ ID NO:24, SEQ ID NO:29, and SEQ ID NO:33, respectively; (l) the sequences shown in SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:26, and SEQ ID NO:32, respectively; (m) the sequences shown in SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:30, and SEQ ID NO:32, respectively; (n) the sequences shown in SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:31, and SEQ ID NO:32, respectively; or (o) the sequences shown in SEQ ID NO:20, SEQ ID NO:24, SEQ ID NO:29, and SEQ ID NO:32, respectively.

[0018] In one embodiment of each aspect of the invention, and any one of its embodiments, the isolated antibody or antigen-binding fragment thereof comprises: (a) a light chain framework region 1 (LFR1) comprising the amino acid sequence set forth in D-X26-VMTQ-X27-P-X28-SL-X29-V-X30-X31-G-X32-PASISC (SEQ ID NO: 36), or DIVMTQSPDSLAVSLGERATINC (SEQ ID NO: 36), wherein X26 is I or V, X27 is S or T, X28 is D, L, or P, X29 is A, P, or S, X30 is N, S, or T, X31 is L or P, and X32 is E or Q; -X38-LIY (sequence number) (wherein X33 is F or Y, X34 is L or Q, X35 is K or R, X36 is P or S, X37 is K, Q or R, and X38 is L or R), and (c) a light chain framework region 3 (LFR3) comprising the amino acid sequence represented by GVPDRFSGSGSG-X39-DFTL-X40-IS-X41-X42-X43-AEDV-X44-VYYC (sequence number) (wherein X39 is S or T, X40 is K or T, X41 is R, S, or W, X42 is L or V, X43 is E or Q, and X44 is A or G). In one embodiment, (a) the LFR1 includes an amino acid sequence selected from the group consisting of the amino acid sequences shown in SEQ ID NOs: 34 to 41, (b) the LFR2 includes an amino acid sequence selected from the group consisting of the amino acid sequences shown in SEQ ID NOs: 42 to 46, and (c) the LFR3 includes an amino acid sequence selected from the group consisting of the amino acid sequences shown in SEQ ID NOs: 47, 48, and 49.

[0019] In another embodiment, the isolated antibody or antigen-binding fragment thereof comprises a light chain variable region comprising, from N-terminus to C-terminus, the structure LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4, wherein LFR1, LFR2, LFR3, and LFR4 have the sequences set forth in SEQ ID NOs: 34, 42, 47, and 50, respectively; (b) SEQ ID NOs: 34, 43, 47, and 50, respectively; (c) SEQ ID NOs: 34, 44, 47, and 50, respectively; (d) SEQ ID NOs: 35, 45, 47, and 50, respectively. and SEQ ID NO:50, (e) the sequences as set forth in SEQ ID NO:36, SEQ ID NO:46, SEQ ID NO:48 and SEQ ID NO:50, respectively, (f) the sequences as set forth in SEQ ID NO:37, SEQ ID NO:45, SEQ ID NO:47 and SEQ ID NO:50, respectively, (g) the sequences as set forth in SEQ ID NO:38, SEQ ID NO:45, SEQ ID NO:47 and SEQ ID NO:50, respectively, (h) the sequences as set forth in SEQ ID NO:39, SEQ ID NO:45, SEQ ID NO:49 and SEQ ID NO:50, respectively, (i) the sequences as set forth in SEQ ID NO:40, SEQ ID NO:45, SEQ ID NO:49 and SEQ ID NO:50, respectively, or (j) the sequences as set forth in SEQ ID NO:41, SEQ ID NO:45, SEQ ID NO:47 and SEQ ID NO:50, respectively.

[0020] In one embodiment, the isolated 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: 51 to 70, and (b) a light chain variable region (LCVR) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 71 to 88.

[0021] In one aspect, the present invention provides an antigen-binding molecule, e.g., an isolated antibody or antigen-binding fragment thereof, that binds to the human adenosine A2A receptor (A2aR), the antibody or antigen-binding fragment thereof comprising a heavy chain variable (VH) domain comprising, from N-terminus to C-terminus, three heavy chain complementarity determining regions (CDRs), HCDR1, HCDR2, and HCDR3, and a light chain variable (VL) domain comprising, from N-terminus to C-terminus, three light chain complementarity determining regions (CDRs), LCDR1, LCDR2, and LCDR3; wherein (a) the HCDR1 has a homology of about 80%, 85%, 90%, 100%, 120%, 140%, 160%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 310%, 320%, 330%, 340%, 350%, 360%, 370%, 380%, 390%, 400%, 410%, 420%, 430%, 440%, 450%, 460%, 470%, 480%, 490%, 500%, 510%, 520%, 530%, 540%, 550%, 560%, 570%, 580%, 590%, 600%, 610%, 620%, 630%, 640%, 650%, 660%, 670%, 680%, 690%, 700%, 710%, 720%, 730%, 740%, 750%, 760%, 770%, 780 (b) the HCDR2 comprises an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identity to an amino acid sequence selected from the group consisting of the amino acid sequences shown in SEQ ID NOs: 4 to 9 and 89; (c) the HCDR3 comprises an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identity to the amino acid sequence shown in SEQ ID NO: 10 or 11. (d) the LCDR1 comprises an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identity to the amino acid sequence shown in SEQ ID NO:12 and 13; (e) the LCDR2 comprises an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identity to the amino acid sequence shown in SEQ ID NO:14; and (f) the LCDR3 comprises an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identity to the amino acid sequence shown in SEQ ID NO:15. and wherein the antibody or antigen-binding fragment thereof comprises an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or about 100% identity to an amino acid sequence selected from the group consisting of the amino acid sequences set forth in SEQ ID NO:15 and 16; further wherein the antibody or antigen-binding fragment thereof comprises a light chain framework region 4 (FR4) comprising an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or about 100% identity to the sequence set forth in FGGGTKVEIK (SEQ ID NO:50).In one embodiment, the heavy chain variable region (VH) domain comprises, from the N-terminus to the C-terminus, four framework regions (FR) HFR1, HFR2, HFR3, and HFR4, and the light chain variable region (VL) domain comprises, from the N-terminus to the C-terminus, four framework regions (FR) LFR1, LFR2, LFR3, and LFR4; wherein (a) the HFR1 has a similar affinity to the amino acid sequences selected from the group consisting of the amino acid sequences shown in SEQ ID NOs: 17 to 21, and is about 80%, 85%, 90%, 95%, 96%, 97%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 310%, 320%, 330%, 340%, 350%, 360%, 370%, 380%, 390%, 400%, 410%, 420%, 430%, 440%, 450%, 460%, 470%, 480%, 490%, 500%, 510%, 520%, 530%, 540%, 550%, 560%, 570%, 580%, 590%, 600%, 610%, 620%, 630%, 640%, 650%, 660%, 670%, 680%, 690%, 700%, 710%, 720%, 730%, 740%, 750%, 760%, %, 98%, 99% or about 100% identity to an amino acid sequence selected from the group consisting of the amino acid sequences shown in SEQ ID NOs: 22, 23, and 24; and (c) the HFR3 comprises an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or less identity to an amino acid sequence selected from the group consisting of the amino acid sequences shown in SEQ ID NOs: 25 to 31. (d) the HFR4 comprises an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identity to an amino acid sequence selected from the group consisting of the amino acid sequences shown in SEQ ID NOs: 32 and 33; and (e) the LFR1 comprises an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identity to an amino acid sequence selected from the group consisting of the amino acid sequences shown in SEQ ID NOs: 34 to 41. (f) the LFR2 comprises an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identity to an amino acid sequence selected from the group consisting of the amino acid sequences shown in SEQ ID NOs: 42 to 46; and (g) the LFR3 comprises an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identity to an amino acid sequence selected from the group consisting of the amino acid sequences shown in SEQ ID NOs: 47, 48, and 49.

[0022] In one aspect, the present invention provides an antigen-binding molecule, for example, an isolated antibody or antigen-binding fragment thereof that binds to the human adenosine A2A receptor (A2aR). The antibody or antigen-binding fragment thereof comprises (a) a heavy chain variable region (HCVR) comprising an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identity to an amino acid sequence selected from SEQ ID NOs: 51 to 70, and (b) a light chain variable region (LCVR) comprising an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identity to an amino acid sequence selected from SEQ ID NOs: 71 to 88.

[0023] In one aspect, the invention provides an antigen-binding molecule, e.g., an isolated antibody or antigen-binding fragment thereof, that binds to the human adenosine A2A receptor (A2aR), the antibody or antigen-binding fragment thereof comprising (a) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:60 and a light chain comprising the amino acid sequence set forth in SEQ ID NO:80, (b) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:62 and a light chain comprising the amino acid sequence set forth in SEQ ID NO:78, (c) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:62 and a light chain comprising the amino acid sequence set forth in SEQ ID NO:80, (d) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:62 and a light chain comprising the amino acid sequence set forth in SEQ ID NO:83, (e) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:67 and a light chain comprising the amino acid sequence set forth in SEQ ID NO:86, or (f) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:67 and a light chain comprising the amino acid sequence set forth in SEQ ID NO:87.

[0024] In one embodiment of each aspect of the invention, and in any one of its embodiments, the N-terminus of the heavy and / or light chain of the isolated antibody or antigen-binding fragment thereof is a pyroglutamic acid (pE) residue. In another embodiment, the A2aR is human A2aR (SEQ ID NO:).

[0025] In other embodiments, the isolated antibody or antigen-binding fragment thereof (i) binds to human A2aR in competition with a monoclonal antibody selected from the group consisting of 1B5-3D7, 3F6-9G5, and 3F8-12E9; (ii) inhibits activity of A2aR; (iii) improves immune response; (iv) specifically binds to human A2aR on the cell surface; (v) reduces cAMP concentration in tissues; (vi) reduces activity of protein kinase A; (vii) reduces phosphorylation of cAMP response factors in the A2aR signaling pathway; (viii) specifically binds to human and / or cynomolgus A2aR; (ix) induces internalization of A2aR; or (x) achieves any combination of (i)-(ix).

[0026] In one embodiment, binding of the antibody to A2aR or cell surface A2aR is measured by flow cytometry assays described in Examples 2 and 5 or substantially similar thereto. In another embodiment, competition of the antibody for binding to A2aR or cell surface A2aR is determined by assays known in the art, including, for example, the assay described in Harms, et al., "Microtiter plate-based antibody-competition assay to determine binding affinities and plasma / blood stability of CXCR4 ligands, Scientific Reports, 2020:10:16036, doi.org / 10.1038 / s41598-020-73012-4," or substantially similar thereto. In yet another embodiment, reduction in cAMP concentration is determined by the assay described in Example 3 or substantially similar thereto. In yet another embodiment, inhibition of A2aR activity is determined by the assay described in Example 4 or substantially similar thereto. In one embodiment, the decreased protein kinase A activity and / or decreased phosphorylation of cAMP response elements in the A2aR signaling pathway is determined by the method described in Karege, et al., "A non-radioactive assay for the cAMP-dependent protein kinase activity in rat brain homogenates and age-related changes in hippocampus and cortex, Brain Res., 2001 Jun 8;903(1-2):86-93, doi:10.1016 / s0006-8993(01)02409-x" or a method substantially similar thereto. In another embodiment, the enhanced immune response is determined by an increase in inflammatory cytokines produced by activated immune cells, increased levels of inflammatory cytokines in tissues, and increased levels of activated CD25 + and / or CD69 + CD4 + T cells and cytotoxic CD8+ This is determined by methods well known in the art, such as an increase in the number of T cells, or by the assay described in Example 6 or an assay substantially similar thereto.

[0027] In one aspect, the invention provides an antigen binding molecule, e.g., an isolated antibody or antigen binding fragment thereof, that binds to human A2aR in competition with an antibody or antigen binding fragment thereof described in any one aspect and any one embodiment thereof described in the invention.

[0028] In one embodiment, the antibody or antigen-binding fragment thereof is a humanized antibody or a chimeric antibody. In another embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region selected from the group consisting of IgA, IgD, IgE, IgG, or IgM. In yet another embodiment, the heavy chain constant region is an IgG class constant region selected from the group consisting of IgG1, IgG2, IgG3, and IgG4. In yet another embodiment, the heavy chain constant region is a human heavy chain constant region.

[0029] In another aspect, the invention provides an isolated polynucleotide encoding an antibody or antigen-binding fragment thereof, its HCVR, its LCVR, its light chain, its heavy chain, or its antigen-binding fragment thereof according to any aspect and embodiment.

[0030] In yet another aspect, the invention provides an expression vector comprising any of the polynucleotides of the invention.

[0031] In yet another aspect, the present invention provides a recombinant cell comprising any of the polynucleotides or expression vectors of the invention.

[0032] In one aspect, the invention provides a method for making a polypeptide selected from the group consisting of an antibody and antigen-binding fragment thereof, its HCVR, its LCVR, its light chain, its heavy chain, and its antigen-binding fragment thereof according to any aspect and embodiment, the method comprising expressing the polypeptide in a recombinant cell of the invention, and optionally further comprising isolating the expressed polypeptide.

[0033] In another embodiment, the invention provides a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof according to any aspect and any embodiment of the invention and a pharma- ceutically acceptable carrier or diluent. In one embodiment, the pharmaceutical composition contains an amount of the antibody or antigen-binding fragment thereof effective to (a) specifically bind to human or cynomolgus A2aR on the cell surface, (b) reduce cAMP concentration in tissue, (c) inhibit activity of human A2aR, (d) reduce phosphorylation of cAMP response factors in the A2aR signaling pathway, (e) improve immune response of immune cells, (f) reduce activity of protein kinase A, or (g) any combination of (a)-(f).

[0034] In one aspect, the invention provides a method for inhibiting activity of A2aR expressed on a cell surface, the method comprising contacting the cell with an isolated antibody or antigen-binding fragment thereof according to any aspect and any embodiment of the invention, or a pharmaceutical composition according to any aspect and any embodiment of the invention, thereby inhibiting activity of A2aR in the cell. In one embodiment, the cell is endogenous to a subject. In another embodiment, the subject is human. In yet another embodiment, the subject is suffering from cancer. In one embodiment, the method is used to treat cancer in a subject.

[0035] In another aspect, the invention provides a method for enhancing an immune response in a subject, the method comprising administering to the subject an isolated antibody or antigen-binding fragment thereof according to any aspect and any embodiment of the invention, or a pharmaceutical composition according to any aspect and any embodiment of the invention, thereby enhancing an immune response in the subject. In one embodiment, the subject is a human. In another embodiment, the subject is suffering from cancer. In one embodiment, the method is used to treat cancer in a subject.

[0036] In yet another aspect, the invention provides a method for inhibiting tumor growth in a subject, the method comprising administering to the subject an isolated antibody or antigen-binding fragment thereof according to any aspect and any embodiment of the invention, or a pharmaceutical composition according to any aspect and any embodiment of the invention, thereby inhibiting tumor growth. In one embodiment, the subject is a human. In another embodiment, the method is used to treat cancer in a subject.

[0037] In yet another aspect, the invention provides a method of treating cancer in a subject, comprising administering to the subject an isolated antibody or antigen-binding fragment thereof according to any aspect and any embodiment of the invention, or a pharmaceutical composition according to any aspect and any embodiment of the invention, thereby treating the cancer.

[0038] In one embodiment of some aspects of the invention, the method activates and induces T cells to kill tumor target cells.

[0039] In one embodiment of some aspects of the invention, the method further comprises administering to the subject an additional therapeutic agent.

[0040] In one embodiment, the additional therapeutic agent comprises an anti-tumor agent, radiation therapy, a chemotherapeutic agent, surgery, a cancer vaccine, an agonist for a stimulatory receptor on an immune cell, a cytokine, a cell therapy, or a checkpoint inhibitor.

[0041] In other embodiments, the checkpoint inhibitor is an agent that inhibits CTLA-4, PD-1, PD-Ll, PD-L2, TIGIT, LAG-3, TIM-3, B7-H3, B7-H4, CD73, PVRIG / PVRL2, Neuritin, BTLA, CECAM-1, CECAM-5, CECAM6, IL-1R8, VISTA, LAIR1, LILRB1, LILRB2, LILRB3, LILRB4, LILRB5, CD47, SIRPa, CD200R, CD96, CD112R, 2B4, TGFβ-R, KIR, NKG2A, SEMA4D, Axl, MerTK, GAS6, TNFR2, GARP, CCR8, IDO, NOX2, SIGLEC7, SIGLEC15, and / or combinations thereof.

[0042] In yet other embodiments, the agent inhibits the interaction between PD-1 and PD-L1 and is selected from the group consisting of pembrolizumab, nivolumab, atezolizumab, avelumab, durvalumab, BMS-936559, sintilimab, toripalimab, ticilelitumab, camrelizumab, embafolimab, sugemalimab, penprimab, cadnilimab, sulfamonomethoxine, and sulfamethizole.

[0043] In yet other embodiments, the CTLA-4 inhibitor is ipilimumab, kadonilimab, YH001 (Eucure Biopharma), ADG116, or ADG126 (Tianyan Pharmaceutical).

[0044] In one embodiment, the additional therapeutic agent is an agonist for an immune cell stimulatory receptor selected from the group consisting of OX40, CD2, CD27, CD3, ICAM-1, LFA-1, ICOS (CD278), 4-1BB (CD137), GITR, CD28, CD30, CD40, BAFFR, HVEM, CD7, LIGHT, NKG2C, NKG2D, SLAMF7, NKp46, NKp80, CD160, and combinations thereof.

[0045] In other embodiments, the additional therapeutic agent is formulated together with the antibody or antigen-binding fragment thereof in the same pharmaceutical composition.

[0046] In yet other embodiments, the additional therapeutic agent is formulated in a separate pharmaceutical composition from the antibody or antigen-binding fragment thereof.

[0047] In one embodiment, the additional therapeutic agent is administered before and / or after administration of the antibody or antigen-binding fragment thereof, hi another embodiment, the additional therapeutic agent is administered simultaneously with the antibody or antigen-binding fragment thereof.

[0048] In one aspect, the invention provides a kit comprising an isolated antibody or antigen-binding fragment thereof according to any aspect and any embodiment of the invention, or a pharmaceutical composition according to any aspect and any embodiment of the invention, hi one embodiment, the kit comprises an additional therapeutic agent. [Brief description of the drawings]

[0049] [Figure 1] 1A and 1B are graphs showing blockade of activity of cell surface expressed human A2aR by exemplary humanized anti-A2aR antibodies of the invention and the 3F6-9G5 antibody (disclosed in '601). RLU (Relative Light Unit): Relative Light Unit. Hu3F6-VH3 / VL5: Hu3F6-H3 / Hu3F6-L5, Hu3F6-VH5 / VL5: Hu3F6-H5 / Hu3F6-L5.

[0050] [Diagram 2] Figure 2 shows that exemplary humanized anti-A2aR antibodies of the invention and the 3F6-9G5 antibody (disclosed in '601) induce internalization upon binding to A2aR on the cell surface. MFI (Mean Fluorescence Intensity): Mean Fluorescence Intensity. Hu3F6-VH3 / VL5: Hu3F6-H3 / Hu3F6-L5, Hu3F6-VH5 / VL5: Hu3F6-H5 / Hu3F6-L5.

[0051] [Diagram 3] Figure 3 shows the intracellular localization of exemplary humanized anti-A2aR antibodies of the invention and the 3F6-9G5 antibody (disclosed in '601) upon incubation with cells expressing human A2aR. Hu3F6-VH3 / VL5: Hu3F6-H3 / Hu3F6-L5; Hu3F6-VH5 / VL5: Hu3F6-H5 / Hu3F6-L5.

[0052] [Figure 4] Figures 4A and 4B show that exemplary humanized anti-A2aR antibodies of the invention and the 3F6-9G5 antibody (disclosed in '601) bind to several human and cynomolgus monkey CD4+ and CD8+ T cells. Hu3F6-VH3 / VL5: Hu3F6-H3 / Hu3F6-L5; Hu3F6-VH5 / VL5: Hu3F6-H5 / Hu3F6-L5.

[0053] [Diagram 5] 5A and 5B are images showing that an exemplary humanized anti-A2aR antibody of the present invention and the 3F6-9G5 antibody (disclosed in '601) were able to restore intracellular IFNγ and IL2 production by Dynabeads-activated CD4+ T cells in the presence of 0.1 μM NECA. NECA is an A2aR agonist that can induce immune suppression, e.g., suppression of T cell function. Hu3F6-VH3 / VL5: Hu3F6-H3 / Hu3F6-L5; Hu3F6-VH5 / VL5: Hu3F6-H5 / Hu3F6-L5. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0054] The present invention and the accompanying drawings are described herein to enable those skilled in the art to practice the invention. However, those skilled in the art will understand that the invention described below may be practiced without these specific details or may be used for purposes other than those described herein. Indeed, in light of the present disclosure, the present invention may be modified or used in conjunction with products and techniques known to those skilled in the art. The drawings and descriptions are intended as illustrations of various aspects of the invention and are not intended as narrowing the scope of the appended claims. Furthermore, it is understood that while the drawings may depict aspects of the invention independently, elements in one drawing may be used in conjunction with elements shown in other drawings.

[0055] References throughout this specification to aspects, features, advantages, or similar language should not be understood to mean that all aspects and advantages that may be realized using the present invention should or are in any single embodiment of the present invention. Rather, language referring to each aspect and advantage should be understood to mean that the specific aspect, feature, advantage, or property described in conjunction with the embodiment is included in at least one embodiment of the present invention. Thus, discussions of aspects and advantages throughout this specification and similar language may, but do not necessarily, refer to the same embodiment.

[0056] The described aspects, features, advantages and characteristics of the present invention may be combined in any suitable manner in one or more further embodiments.Furthermore, those skilled in the art will recognize that the present invention may be practiced without one or more of the specific aspects or advantages of a particular embodiment.In other cases, other aspects, features and advantages may be recognized and claimed in an embodiment, but those aspects, features and advantages may not be present in all embodiments of the present invention.

[0057] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Those skilled in the art will recognize many techniques and materials similar or equivalent to those described herein that can be used in the practice of each aspect and embodiment of the present invention. The described aspects and embodiments of this application are not limited to the methods and materials described.

[0058] Additionally, any cited references, any issued patents or published patent applications mentioned in this application for their teachings herein are expressly incorporated herein by reference.

[0059] 1. Definition

[0060] So that the present invention may be more readily understood, certain terms are first defined. Furthermore, it should be noted that when a parameter value or range of values ​​is recited, all intermediate values ​​and ranges of the recited values ​​are also intended to be part of the invention.

[0061] Unless otherwise indicated herein or clearly contradicted by context, the terms "a," "an," "said," and similar referential words used in the context of describing the present invention (particularly in the context of the claims) should be construed to include both the singular and the plural (i.e., one or more). The terms "comprise," "have," "include," and "contain" should be construed as open-ended terms (i.e., meaning "including, but not limited to") unless otherwise indicated. The recitation of ranges of values ​​herein is merely intended to serve as a shorthand method of referring individually to each separate value recited or falling within the range, unless otherwise indicated herein, and each separate value is incorporated herein as if it were individually recited.

[0062] When the phrases "in one embodiment," "in another embodiment," "in other embodiments," "in some embodiments," or "in an embodiment" are used, the disclosure should be construed to encompass any combination of features defining different embodiments described therein, unless the features are mutually incombinable, mutually exclusive, or otherwise expressly disclaimed herein.

[0063] The term "about" or "approximately", for example, when used with respect to one or more of the values ​​described herein, refers to a value similar to said reference value. In some embodiments, unless otherwise stated or clear from the context, the term "about" or "approximately" refers to a range of values ​​of 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater or less) of said reference value. In some embodiments, "about" or "about" should be understood to mean a difference of about two standard deviations from the mean. In some embodiments, "about" or "about" means to include up to ±10% (e.g., ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, ±1% or less). In some embodiments, "about" or "approximately" means ±5%. When "approximately" or "about" appears before a series of numerical values ​​or ranges, it should be understood that each numerical value in the series or range can be modified by "approximately / about."

[0064] Ranges may be expressed herein as from "about" one particular value and / or to "about" another particular value. When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when values ​​are expressed as approximations, by use of the antecedent "about," it is to be understood that the particular value constitutes another embodiment. Moreover, it is to be understood that the endpoints of each range are not only distinct but independent of the other endpoint. It is also to be understood that a number of values ​​are disclosed herein, and each value includes "about" that particular value in addition to the value itself. For example, when the value "10" is disclosed, "about 10" is also disclosed. When a value is disclosed, it is also understood that "less than or equal to" (i.e., that value), "greater than or equal to" (i.e., that value), and possible ranges between values ​​are also disclosed, as appropriately understood by one of ordinary skill in the art. For example, when the value "10" is disclosed, "less than or equal to 10" as well as "greater than or equal to 10" are also disclosed.

[0065] As used herein, the terms "administer" and "administration" refer to giving an agent, e.g., an antibody, to a subject. In some embodiments, "administering" refers to administering an antibody or antibody conjugate to a subject in a physiologically and / or (e.g., and) pharmacologically useful manner (e.g., treating a disease in a subject). Non-limiting examples of routes of administration include intravenous, intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intraarticular, intrasynovial, or intrathecal routes. In some embodiments, the route of administration is subcutaneous administration.

[0066] The term "agent" as used herein means any substance, compound (e.g., molecule), supramolecular complex, material, or combination or mixture thereof. A compound can be any agent that can be 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, aptamers), lipids, polysaccharides, and the like. Generally, agents are obtained using any suitable method known in the art. The skilled artisan will select the appropriate method based, for example, on the nature of the agent. An agent can be at least partially purified. In some embodiments, an agent can be provided as part of a composition that, in some embodiments, may contain, in addition to the agent, for example, counterions, aqueous or non-aqueous diluents or carriers, buffers, preservatives, or other components. In some embodiments, an agent can be provided as a salt, ester, hydrate, or solvate. In some embodiments, an agent is cell-permeable, for example, within the range of typical agents that are taken up by cells and act intracellularly (e.g., act in mammalian cells to produce a biological effect). Certain compounds can exist in specific geometric or stereoisomeric forms. 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 are included and are included in the various embodiments of the disclosure unless otherwise indicated. Certain compounds may exist in various protonation states, may have various configurations, may exist as solvates (e.g., with water (i.e., hydrates) or with common solvents), and / or may have different crystal forms (e.g., polymorphs) or different tautomeric forms. Embodiments exhibiting such alternative protonation states, configurations, solvates, and forms are encompassed by the disclosure, where applicable.

[0067] In certain embodiments, depending on the context, "medicament" also encompasses therapeutic methods such as radiation therapy, chemotherapy, or surgery.

[0068] The term "amino acid" refers to the 20 common naturally occurring amino acids, including alanine (Ala; A), arginine (Arg; R), asparagine (Asn; N), aspartic acid (Asp; D), cysteine ​​(Cys; C); glutamic acid (Glu; E), glutamine (Gin; Q), glycine (Gly; G), histidine (His; H), isoleucine (He; I), leucine (Leu; L), lysine (Lys; K), histidine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V).

[0069] The term "antagonist" or "inhibitor" refers to a substance that prevents, blocks, suppresses, neutralizes, or decreases the biological activity or effect of another molecule (eg, a receptor).

[0070] The term "agonist" refers to a substance that promotes (i.e., induces, causes, enhances, or increases) the biological activity or effect of another molecule. The term agonist encompasses substances that bind to receptors (e.g., antibodies) and substances that promote receptor function without binding to the receptor (e.g., by activating an associated protein).

[0071] As used herein, "antibody" refers to any antigen-binding molecule or molecular complex that contains at least one complementarity determining region (CDR) that specifically binds to or interacts with a particular antigen (e.g., A2aR). The term "antibody" includes immunoglobulin molecules that contain 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 contains a heavy chain variable region (referred to herein as HCVR, VH or VH). H The heavy chain constant region is abbreviated as C H 1 or CH1, C H 2 or CH2, and C HEach light chain comprises three domains, such as a light chain variable region (herein referred to as LCVR, VL or VL). L The light chain constant region comprises one domain (CL or C). L ) is included. V H and V L The regions can be further subdivided into regions of hypervariability called complementarity determining regions (CDRs) interspersed with more conserved regions called framework regions (FRs). The heavy chain framework regions are abbreviated herein as HFRs and the light chain framework regions are abbreviated herein as LFRs. Each V H and V L consists of three CDRs and four FRs in the following order from amino to carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In different embodiments of the invention, the FRs of the anti-A2aR antibody (or antigen-binding fragment thereof) can be identical to mouse or human germline sequences, or can be naturally or artificially modified. An amino acid consensus sequence can be defined based on a parallel analysis of two or more CDRs.

[0072] The term "antibody" as used herein also includes antigen-binding fragments of a complete antibody molecule. As used herein, the terms "antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody, and the like 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 derived from complete antibody molecules by any suitable standard technique, such as proteolytic digestion or recombinant gene technology involving the manipulation and expression of DNA encoding the variable and optionally selected constant regions of the antibody. Such DNA is known and / or readily available, for example, from commercial sources, DNA libraries (including, for example, phage-antibody libraries), or can be synthesized. The DNA can be sequenced and engineered, for example, chemically or using molecular biology techniques, to align one or more variable and / or constant regions in the appropriate configuration, to introduce codons, to generate cysteine ​​residues, to modify, add, or delete amino acids.

[0073] Non-limiting examples of antigen-binding fragments include (i) Fab fragments, (ii) F(ab')2 fragments, (iii) Fd fragments, (iv) Fv fragments, (v) single chain Fv (scFv) molecules, (vi) dAb fragments, and (vii) minimal recognition units consisting of amino acid residues that mimic the hypervariable regions (e.g., isolated complementarity determining regions (CDRs) such as CDR3 peptides) of an antibody or constrained FR3-CDR3-FR4 peptides. Domain-specific antibodies and other engineered molecules such as single domain antibodies, domain deleted antibodies, chimeric antibodies, CDR grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains are encompassed by the term "antigen-binding fragment" as used herein.

[0074] Antigen-binding fragments of antibodies typically contain at least one variable domain, which may be of any size or amino acid composition and generally contains at least one CDR adjacent to or in frame with one or more framework sequences. L V concatenated with domain H The antigen-binding fragment containing the V domain includes the H and V L The domains can be arranged relative to each other in any suitable arrangement. For example, the variable region may be a dimer and may have a V H -V H , V H -V L Or V L -V L An antigen-binding fragment of an antibody may comprise a dimer, or may comprise a monomeric VH or VL domain.

[0075] In certain embodiments, an antigen-binding fragment of an antibody can comprise at least one variable domain covalently linked to at least one constant domain. Non-limiting exemplary arrangements of variable and constant domains in an antigen-binding fragment of an antibody of the invention include: (i) a V H -C H 1, (ii) V H -C H 2. (iii) V H -C H 3. (iv) V H -C H 1-C H 2. (v) V H -C H 1-C H 2-C H 3. (vi) V H -C H 2-C H 3. (vii) V H -C L , (viii) V L -C H 1, (ix) V L -C H 2. (x)V L -C H 3. (xi) V L-C H 1-C H 2. (xii) V L -C H 1-C H 2-C H 3. (xiii) V L -C H 2-C H 3, and (xiv) V L -C L In any arrangement of variable and constant domains (including any of the exemplary arrangements listed above), the variable and constant domains may be directly linked to each other or may be linked by a complete or partial hinge or linker region. The hinge region comprises at least two (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids, thereby providing a flexible or semi-flexible linkage between adjacent variable and / or constant domains in a single polypeptide molecule. Furthermore, antigen-binding fragments may be linked to each other and / or to one or more monomeric V H Or V L The domains may include homo- or hetero-dimers (or other multimers) of any of the above listed variable and constant domain configurations non-covalently linked (e.g., linked by disulfide bonds). When describing sequences of polypeptide domains with hyphens (e.g., CH2-CH3) between individual domains, it is understood that the order of the described domains is from N-terminus to C-terminus.

[0076] As with intact 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 can specifically bind to a single antigen or to a different epitope on the same antigen. Any of the multispecific antibody formats, including the exemplary bispecific antibody formats disclosed herein, can be adapted for use in conjunction with the antigen-binding fragments of antibodies of the invention using conventional techniques available in the art.

[0077] The antibody according to the present invention may be an isolated antibody. As used herein, an "isolated" molecule (e.g., an isolated antibody or an isolated polypeptide) refers to a molecule (e.g., an antibody) that has been identified and separated and / or recovered from at least one component of its natural environment. For example, a molecule (e.g., an antibody) that has been separated or recovered from at least one component of an organism, or a tissue or cell in which the antibody naturally occurs or is naturally produced, is an "isolated" molecule (e.g., an antibody) for purposes of the present invention. An isolated molecule (e.g., an antibody) also includes an in situ molecule (e.g., an antibody) in a recombinant cell. In some embodiments, an isolated molecule (e.g., an antibody) is a molecule (e.g., an antibody) that has been subjected to at least one purification or isolation step. According to some embodiments, an isolated molecule (e.g., an antibody) may be substantially free of other cellular material and / or chemicals.

[0078] The present invention further includes a one-arm antibody that binds to A2aR. As used herein, "one-arm antibody" refers to an antigen-binding molecule that comprises a single antibody heavy chain and a single antibody light chain. The one-arm antibody described in the present invention may comprise any of the amino acid sequences of HCVR / LCVR or CDR shown in Tables 1 to 19.

[0079] The anti-A2aR antibodies or antigen-binding fragments thereof described herein may contain one or more amino acid substitutions, insertions and / or deletions in the framework and / or CDR regions of the variable regions of the heavy and light chains compared to the corresponding germline sequences from the antigen-binding molecule or antigen-binding domain. Such mutations can be readily ascertained by comparing the amino acid sequences disclosed herein to germline sequences available, for example, from public antibody sequence databases. The present invention encompasses antibodies and antigen-binding fragments thereof derived from any amino acid sequence disclosed herein, in which one or more amino acids in one or more framework and / or CDR regions are mutated to the corresponding residue in the germline sequence from which the antibody is derived, or to the corresponding residue in another human germline sequence, or to a conservative amino acid substitution of the corresponding germline residue (such sequence changes are collectively referred to herein as "germline mutations"). Starting from the heavy and light chain variable region sequences disclosed herein, one skilled in the art can generate numerous antibodies and antigen-binding fragments containing one or more individual germline mutations or combinations thereof. In one embodiment, the V H and / or V LAll framework region and / or CDR residues within the domains are mutated back to the residues of the original germline sequence from which the antibody is derived. In other embodiments, only certain residues are mutated back to the original germline sequence, for example only the mutated residues found within the first 8 amino acids of FR1 or the last 8 amino acids of FR4, or only the mutated residues found in CDR1, CDR2 or CDR3. In other embodiments, one or more of the framework and / or CDR residues are mutated to the corresponding residue in a different germline sequence (i.e., a germline sequence that differs from the germline sequence from which the antibody is derived). Furthermore, the antibodies or antigen-binding fragments thereof of the invention can include any combination of two or more germline mutations in the framework and / or CDR regions, for example, certain individual residues are mutated to the corresponding residue in a particular germline sequence and certain other residues that differ from the original germline sequence are maintained or are mutated to the corresponding residue in a different germline sequence. Once obtained, antibodies or antigen-binding fragments thereof that contain one or more germline mutations can be readily measured for one or more desired properties, such as improved binding specificity, increased binding affinity, improved or enhanced antagonistic or agonistic biological properties (as the case may be), reduced immunogenicity, etc. Antibodies or antigen-binding fragments thereof obtained in such general manner are encompassed by the present invention.

[0080] The invention further includes anti-A2aR antibodies that comprise variants of any of the HCVR, LCVR and / or CDR amino acid sequences disclosed herein. Exemplary variants included in this aspect of the invention include variants of the HCVR, LCVR and / or CDR amino acid sequences disclosed herein that have one or more conservative substitutions. For example, the invention includes anti-A2aR antibodies and antigen binding proteins that have HCVR, LCVR and / or CDR amino acid sequences that include no more than 10, no more than 8, no more than 6, no more than 4, etc., conservative amino acid substitutions compared to any of the HCVR, LCVR and / or CDR amino acid sequences shown in the tables herein.

[0081] Light chains are classified as kappa or lambda (K, λ). Each heavy chain class can be combined with either kappa or lambda light chains. Generally, when immunoglobulins are produced by hybridomas, B cells or genetically engineered host cells, the light and heavy chains are covalently linked to each other, and the "tail" portions of the two heavy chains are linked to each other by covalent disulfide bonds or non-covalent bonds. In the heavy chains, the amino acid sequences run from the N-terminus at the forked ends of the Y configuration to the C-terminus at the bottom of each chain.

[0082] As used herein, the terms "light chain constant region" and "CL" are used interchangeably herein and 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.

[0083] The term "heavy chain constant region" as used herein includes amino acid sequences derived from an immunoglobulin heavy chain. A polypeptide comprising a heavy chain constant region comprises at least one of a CH1 domain, a hinge (e.g., upper, middle and / or lower part of the hinge region), a CH2 domain, a CH3 domain, or variants or fragments thereof. For example, an antigen-binding polypeptide used in the present disclosure can comprise 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, a polypeptide of the invention comprises a polypeptide chain comprising a CH3 domain. Additionally, an antibody used in the present disclosure may lack at least a portion of a CH2 domain (e.g., all or a portion of a CH2 domain). It should be understood that a heavy chain constant region may be modified such that its amino acid sequence is no longer different from a naturally occurring immunoglobulin molecule.

[0084] The heavy chain constant regions of the antibodies disclosed herein can be derived from different immunoglobulin molecules. For example, the heavy chain constant region of the polypeptide can comprise a CH1 domain derived from an IgG1 molecule and a hinge region derived from an IgG3 molecule. In another example, the heavy chain constant region can comprise a hinge region derived, in part, from an IgG1 molecule and in part from an IgG3 molecule. In another example, the heavy chain portion can comprise a chimeric hinge derived, in part, from an IgG1 molecule and in part from an IgG4 molecule.

[0085] A "light chain-heavy chain pair" refers to an assembly of a light chain and a heavy chain that can form a dimer via disulfide bonds between the CL domain of the light chain and the CH1 domain of the heavy chain.

[0086] The subunit structures and three-dimensional configurations of the constant regions of various immunoglobulin classes are well known. As used herein, the term "VH domain" 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 N-terminal to the hinge region of the immunoglobulin heavy chain molecule.

[0087] The term "CH2 domain" as used herein includes, for example, that portion of a heavy chain molecule extending from about residue 244 to residue 360 ​​of an antibody based on common numbering schemes (residues 244-360 according to the Kabat numbering system, or residues 231-340 according to the EU numbering system). Rather, the CH2 domain is unique in that it is not closely matched to another domain. Two N-linked branched glycans are inserted between the two CH2 domains in an intact native IgG molecule. The CH3 domain extends from the CH2 domain to the C-terminus of the IgG molecule and comprises about 108 residues.

[0088] The term "hinge region" as used herein includes the portion of a heavy chain molecule that connects the CH1 domain to the CH2 domain. The hinge region comprises approximately 25 residues and is flexible, allowing the two N-terminal antigen-binding regions to move independently. The hinge region is subdivided into three distinct domains: the upper, middle and lower hinge regions.

[0089] 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 according to the Kabat numbering system (positions 226 or 229 according to the EU numbering system).

[0090] The term "antibody" encompasses a wide variety of classes of biochemically distinct polypeptides. Those skilled in the art will appreciate that heavy chains are classified as alpha, delta, epsilon, gamma, and mu (or α, δ, ε, γ, and μ), as well as several subclasses (e.g., γ1-γ4). The nature of the chain determines the "class" of the antibody, as IgG, IgM, IgA, IgD, or IgE, respectively. Immunoglobulin subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, etc., are well characterized and are known to confer functional specialization. In accordance with the present disclosure, modified forms of each of these classes and isotypes are readily discernible to one of skill in the art and are therefore within the scope of the present disclosure. All immunoglobulin classes are within the scope of the present disclosure. The following discussion generally relates to the IgG class of immunoglobulin molecules.

[0091] The antibodies of the present disclosure include, but are not limited to, polyclonal antibodies, monoclonal antibodies, multispecific antibodies, bispecific antibodies, trispecific antibodies, human antibodies, humanized antibodies, primate antibodies, chimeric antibodies, and single chain antibodies. The antibodies disclosed herein may be from any animal origin, including birds and mammals. Preferably, the antibodies are from human, mouse, donkey, rabbit, goat, guinea pig, camel, llama, horse, or chicken. In some embodiments, the variable region may be from a chondrictoid (e.g., from a shark).

[0092] The term "humanized antibody" as used herein refers to a genetically engineered non-human antibody comprising a human antibody constant region and a non-human variable region modified to have a high degree of sequence identity to the human variable region. This can be achieved by grafting the six non-human antibody complementarity determining regions (CDRs) that form the antigen binding site onto the homologous human acceptor framework regions (FRs). Substitution of framework residues from the parent antibody (i.e., non-human antibody) into the human framework regions may be required to reconstitute the binding affinity and specificity of the parent antibody (back mutations). Homology modeling may help to identify amino acid residues in the framework regions that are important for the binding properties of the antibody. Thus, a humanized antibody may comprise non-human CDR sequences, primarily human framework regions, optionally including one or more amino acid back mutations to the non-human amino acid sequences, and a fully human constant region. Optionally, additional amino acid modifications (not necessarily back mutations) may be applied to obtain a humanized antibody with favorable properties such as affinity and biochemical properties.

[0093] The term "chimeric antibody" as used herein refers to antibodies in which a portion of the heavy and / or light chain is substantially identical, identical or corresponding to a sequence corresponding to an antibody from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is substantially identical, identical or corresponding to a sequence corresponding to an antibody from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies so long as they exhibit the desired biological activity. In certain embodiments, a chimeric antibody comprises an immunoreactive region or site, such as a heavy and / or light chain variable region, obtained or derived from a first species, and a constant region (which may be complete, partial or modified according to the present disclosure) obtained from a second species. In certain embodiments, the target binding region or site is derived from a non-human source (e.g., mouse or primate) and the constant region is derived from a human. Humanized antibodies are a special type of chimeric antibody.

[0094] "Single variable chain fragment" or "scFv" refers to a fusion protein of the variable heavy (VH) and variable light (VL) chains of an immunoglobulin. In some embodiments, these regions are linked by a short linker peptide of about 10-25 amino acids. The linker is rich in glycine for improved flexibility or serine or threonine for improved solubility and can link the N-terminus of VH to the C-terminus of VL, or vice versa. The protein retains the specificity of the original immunoglobulin despite the removal of the constant regions and the introduction of the linker.

[0095] IgG is a standard immunoglobulin molecule that contains two identical light chain polypeptides of molecular weight approximately 23,000 daltons, and two identical heavy chain polypeptides of molecular weight 53,000 to 70,000. The four chains are typically linked by disulfide bonds in a "Y" configuration, where the light chains sandwich the heavy chains starting at the mouth of the "Y" and continuing through the variable region.

[0096] The term "biological activity" refers to any biological property of a molecule, whether naturally occurring in the body or provided or achieved by recombinant means. Biological activities include, but are not limited to, binding to a receptor, inducing cell proliferation, inhibiting cell growth, inducing other cytokines, inducing apoptosis, and enzymatic activity.

[0097] "Conjugate" or "drug 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 some embodiments, the linker is covalently linked to the antibody and also to the agent. In some embodiments, the linker is non-covalently linked to the antibody and / or the agent. In some embodiments, the linker is covalently linked to the agent and is linked to the antibody via a specific bond. In some embodiments, the linker is a moiety that can specifically bind to the antibody, for example, an antibody that binds to the Fc region of the antibody. In some embodiments, the complex comprises an antibody linked to one or more agents via a non-covalent bond.

[0098] The term "control" or "reference" when referring to a substance refers to a composition used as a standard or comparison point against which other test results are measured. In some embodiments, a "control" or "reference" is a composition that is known to contain no analyte ("negative control") or to contain the analyte ("positive control"). A positive control can contain a known concentration of the analyte. "Control" and "positive control" can refer to a composition that contains a known concentration of the analyte. A "positive control" can be used to establish a measurement performance characteristic that is a useful indicator for the integrity of an agent (e.g., an analyte). In some embodiments, a suitable "control" or "reference" is to change only one element to determine the role of the element. In some embodiments, a control refers to, for example, the target gene (e.g., in a cell or subject) level before being treated with an RNAi agent described herein.

[0099] The term "control" or "reference" also refers to the baseline level of a measurement, depending on the context in which it is used. The measured baseline level is a standard or comparison point against which the measured results are compared. In some embodiments, "control" or "reference" refers to the detected level of a biological activity or substance in a cell, tissue, organ or subject when the cell, tissue, organ or subject is not treated with an agent (e.g., an RNAi formulation), such as the expression level of a gene, the mRNA copy number of the gene, or the level of the protein encoded by the gene. In some embodiments, "control" or "reference" refers to the average measured level of some biological activity or substance (e.g., a certain enzyme activity in the liver) in a cell, tissue, organ or subject measured in a healthy group (e.g., a general population of people not suffering from a disease or disorder, such as liver disease, within a specific geographic or demographic limit, or within possible limits suitable for the study of any disease or disorder).

[0100] The term "control" may be applied to a "control individual." A "control individual" is an individual with a similar disease, e.g., the same cell proliferative disorder as the treated individual, and of approximately the same age as the treated individual (to ensure that the stage of the disease is comparable in treated and control individuals). The treated individual (also referred to as a "patient" or "subject") may be a fetus, infant, child, adolescent, or adult suffering from a cell proliferative disorder.

[0101] The term "reference" can also be used for "reference sequence." The term "reference sequence" refers to a sequence, e.g., a nucleic acid sequence or an amino acid sequence, that serves as the basis for sequence comparison.

[0102] The term "epitope" refers to an antigenic determinant that interacts with a specific antigen-binding site in the variable region of an antibody molecule, called the paratope. A single antigen can have multiple epitopes. Thus, different antibodies can bind to different regions on the antigen and have different biological effects. Epitopes can be either conformational or linear. Conformational epitopes are generated by spatially juxtaposed amino acids from different segments of a linear polypeptide chain. Linear epitopes are generated by adjacent amino acid residues within a polypeptide chain. In some cases, epitopes may include portions of sugars, phosphoryl groups, or sulfonyl groups on the antigen.

[0103] The term "immunoconjugate" refers to an antibody to which an agent is linked. In some embodiments, the agent in the immunoconjugate is a therapeutic or diagnostic agent. In some embodiments, the agent is a peptide or small molecule drug. The peptide or small molecule drug can be attached to the C-terminus of the constant heavy chain or the N-terminus of the variable light chain and / or variable heavy chain.

[0104] The term "immunoconjugate" refers to an antibody covalently fused to a peptide or small molecule drug, which can be attached to the C-terminus of the constant heavy chain or the N-terminus of the variable light and / or variable heavy chain.

[0105] As used herein, the terms "improvement," "enhancement," or "reduction" refer to a value or parameter relative to a baseline / control / reference measurement, e.g., a measurement in a cell or tissue prior to initiation of a treatment described herein, a measurement in a cell or tissue in the absence of a treatment described herein, a measurement in the same individual prior to initiation of a treatment described herein, or a measurement in a control individual in the absence of a treatment described herein (or a standard measurement in multiple control individuals, e.g., an average value of multiple control individuals).

[0106] In the context of describing levels of drug activity, the terms "inhibition," "reduction," and "decrease" refer to a statistically significant decrease in such levels, e.g., at least 10%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% reduction, or may be below the level of detection of the detection method.

[0107] The term "prevention" refers to a reduction in the occurrence of disease symptoms in a subject. As noted above, the prevention may be complete (no detectable symptoms) or partial, such that symptoms are seen to be less prevalent than in the absence of treatment.

[0108] The term "recombinant" as used herein, when referring to a polypeptide or polynucleotide, refers to a polypeptide or polynucleotide that does not occur in nature and that can be created by combining polynucleotides or polypeptides in an arrangement that does not normally occur together. The term "recombinant cell" as used herein refers to a non-naturally occurring host cell that contains one or more (e.g., two, several) heterologous polynucleotides. The term "host cell" refers to any cell type that is susceptible to transformation, transfection, conductance, etc., of a nucleic acid construct or expression vector.

[0109] The phrase "small molecule drug" refers to a molecular entity that is an organic or organometallic that has pharmaceutical activity and has a molecular weight of less than about 2 kDa, less than about 1 kDa, less than about 900 Da, less than about 800 Da, or less than about 700 Da, but is not polymeric. The term encompasses most pharmaceutical compounds that are called "drugs" other than proteins or nucleic acids. However, small peptides or nucleic acid analogs are considered small molecule drugs. Examples include chemotherapy anticancer drugs and enzyme inhibitors. Small molecule drugs can be synthetic, semi-synthetic (i.e., from naturally occurring precursors), or biologically derived.

[0110] The term "specifically binds" or "specifically binds" as used herein refers to the ability to discriminate between possible binding partners in the environment in which the binding occurs. In some embodiments, an antibody that interacts (e.g., preferentially interacts) with one particular antigen in the presence of other possible antibodies is said to "specifically bind" to the antigen with which it interacts. In some embodiments, specific binding is assessed by detecting or determining the degree of binding between the antibody and its targeted antigen. In some embodiments, specific binding is assessed by detecting or determining the level of dissociation of the antibody-antigen complex. In some embodiments, specific binding is assessed by detecting or determining the ability of the antibody to compete with alternative interactions between its target and another antibody. In some embodiments, specific binding is assessed by performing such detection or determination over a range of concentrations. Generally, an antibody binds to an epitope through its antigen-binding domain, and such binding entails some complementarity between the antigen-binding domain and the epitope. Thus, an antibody is said to "specifically bind" to an epitope when it is easier to bind to the epitope through the antigen-binding domain than to bind to a random, unrelated epitope. The term "specificity" is used herein to qualify the relative affinity with which a particular antibody binds to a particular epitope. For example, antibody "A" can be considered to have a higher specificity for a given epitope than antibody "B," or antibody "A" can be considered to have a higher specificity for epitope "C" than for related epitope "D." In some embodiments, the complex formed by the antibody or antigen-binding fragment thereof and the antigen has a dissociation constant (Kd) of 10 or greater. -6 M or less, 10 -7 M or less, 10 -8 M or less, 10 -9 M or less, or 10 -10If the antibody or antigen-binding fragment thereof has a specificity for the antigen, then the antibody or antigen-binding fragment thereof "has specificity" for the antigen. In one embodiment, specific binding of an antigen-binding molecule (e.g., an anti-human A2aR antibody or antigen-binding fragment thereof) can be demonstrated by preferential binding of the antigen-binding molecule to human A2aR expressed on a cell surface using the assay methods described in Examples 4 to 7 or substantially similar methods.

[0111] The term "subject" as used herein refers to a mammal. In some embodiments, the subject is a non-human primate or rodent. In some embodiments, the subject is a human. In some embodiments, the subject is a patient, e.g., a human patient suffering from or suspected of a disease. In some embodiments, the subject is a human patient suffering from or suspected of a TAZ disease or a TAZ-related disease.

[0112] When referring to a nucleic acid or a fragment thereof, the term "substantial identity" or "substantially identical" refers to a nucleotide sequence that, when optimally aligned to another nucleic acid (or its complementary strand), with appropriate nucleotide insertions or deletions, has at least about 95%, more preferably at least about 96%, 97%, 98% or 99% nucleotide base identity with a nucleotide sequence as measured by any well-known sequence identity algorithm, such as FASTA, BLAST or Gap, as discussed below. In some cases, a nucleic acid molecule that has substantial identity to a reference nucleic acid molecule can encode a polypeptide having an amino acid sequence identical or substantially similar to the polypeptide encoded by the reference nucleic acid molecule.

[0113] The term "substantial similarity" or "substantial similarity" as applied to polypeptides means that two peptide sequences share at least 95% sequence identity, and even more preferably at least 98% or 99% sequence identity, when optimally aligned, such as by a program (e.g., GAP or BESTFIT) using default gap weights. Preferably, non-identical residue positions differ by conservative amino acid substitutions. A "conservative amino acid substitution" means that an amino acid residue is replaced by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, conservative amino acid substitutions do not substantially change the functional properties of a protein. In cases where two or more amino acid sequences differ from each other by conservative substitutions, the percent sequence identity or similarity can be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art. See, e.g., Pearson (1994) Methods Mol. Biol. 24:307-331. Examples of amino acid groups with side chains of similar chemical properties include: (1) aliphatic side chains: glycine, alanine, valine, leucine, isoleucine, (2) aliphatic hydroxyl group side chains: serine, threonine, (3) amide-containing side chains: asparagine, glutamine, (4) aromatic side chains: phenylalanine, tyrosine, tryptophan, (5) basic side chains: lysine, arginine, and histidine, (6) acidic side chains: aspartic acid and glutamic acid, and (7) sulfur-containing side chains: cysteine ​​and methionine. Preferred conservative amino acid substitution groups are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine. Alternatively, a conservative substitution means any change that has a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256:1443-1445. A "moderate conservative substitution" means any change that has a non-negative value in the PAM250 log-likelihood matrix.

[0114] The sequence similarity of polypeptides, also called sequence identity, is typically measured using sequence analysis software. Protein analysis software matches similar sequences using measures of similarity assigned to various substitutions, deletions, and other modifications (including conservative amino acid substitutions). For example, GCG software includes programs such as Gap and Bestfit, which can determine sequence homology or sequence identity between closely related polypeptides (homologous polypeptides from different species, or homologous polypeptides of wild-type proteins and their variants) using default arguments. See, for example, GCG version 6.1. Polypeptide sequences can also be compared using FASTA, a program in GCG version 6.1, using default or recommended arguments. FASTA (e.g., FASTA2 and FASTA3) provides alignment and percent sequence identity of the best overlapping regions between the query sequence and the search sequence (Pearson (2000), supra). Another preferred algorithm for comparing the sequences of the present invention to a database containing a large number of sequences from different organisms is the computer program BLAST, particularly BLASTP or TBLASTN, using default arguments. See, e.g., Altschul et al. (1990) J. Mol. Biol. 215:403-410 and Altschul et al. (1997) Nucleic Acids Res. 25:3389-402. The term "sequence identity" also refers to a comparison between a pair of nucleic acid or polypeptide molecules, i.e., the relatedness between two nucleotide sequences. Generally, sequence alignment is to obtain the closest match.

[0115] The term "treatment" refers to the improvement of one or more symptoms associated with a disease or disorder. Thus, these terms refer to any indication of successful treatment or improvement of an injury, disease, pathology, or disorder, including prevention or delay of onset of one or more symptoms of a disease or disorder; reduction in the severity or frequency of one or more symptoms of a disease or disorder; reduction of any objective or subjective parameters; remission; reduction of symptoms or making an injury, pathology, or disorder more tolerable to a patient; slowing down the rate of degeneration or decline; reduction in the debilitating end point of degeneration; and / or improvement of the patient's physical or mental health. "Treatment" may also include prophylactic treatment.

[0116] The phrase "patient in need thereof," "patient in need of treatment," or "subject in need of treatment" includes subjects (e.g., mammalian subjects) who may benefit from the administration of an antibody of the present disclosure in the treatment of a cell proliferative disorder.

[0117] The terms "therapeutically effective amount", "pharmacologically effective amount", "physiologically effective amount" or "effective amount" are used interchangeably and refer to an amount of an active agent sufficient to ameliorate at least one symptom of a disease or disorder. For example, for a given parameter, a therapeutically effective amount indicates an increase or decrease of at least 5%, 10%, 15%, 20%, 25%, 40%, 50%, 60%, 75%, 80%, 90%, 95%, 99%, or at least 100%. The therapeutic effect can also be expressed as a "-fold" increase or decrease. For example, a therapeutically effective amount can have at least a 1.2-fold, 1.5-fold, 2-fold, 5-fold, or more effect over a control. The exact amount depends on many factors, such as the particular active agent, the components and physical characteristics of the composition, the target patient population, and patient considerations including weight, sex, etc., and can be readily determined by one of skill in the art based on the information provided herein or available in the relevant literature.

[0118] The term "variant" as used herein refers to a polypeptide or polynucleotide (e.g., an antibody or polynucleotide) resulting from the incorporation of one or more amino acid or nucleotide insertions, substitutions, or deletions in a precursor polypeptide or polynucleotide (e.g., a "parent" polypeptide or polynucleotide). In certain embodiments, the variant polypeptide or polynucleotide has at least about 85% amino acid or nucleotide sequence identity, e.g., about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% amino acid or nucleotide sequence identity, with the entire amino acid or nucleotide sequence of the parent polypeptide or polynucleotide. A protein or peptide variant substantially maintains the structure, function, or activity of the protein. For example, an antibody variant maintains and / or modulates its antigen-specific binding function or activity (e.g., inhibits antigen activity). In the case of a polynucleotide, the variant maintains the function or activity of the parent polynucleotide. For example, a variant polynucleotide can encode a protein or peptide that has a similar function or activity as the polypeptide encoded by the parent polynucleotide.

[0119] The term "A2aR" as used herein refers to adenosine A2A receptor.Unless otherwise indicated, for example, by specific reference to human A2aR, the term "A2aR" encompasses the native A2aR of all mammalian species, for example, from humans, primates, rodents, canines, felines, equines, and bovines.The nucleotide and amino acid sequences of A2aR are known and are described, for example, in GenBank Accession Nos. NP_000666.2, NP_033760.2, XP_038954384.1, EHH65694.1, EAW59658.1, XP_015313061.1, NP_445746.3, and XP_001095531.1, the entire contents of each of which are incorporated herein by reference.

[0120] An exemplary human A2aR amino acid sequence is shown below. MPIMGSSVYITVELAIAVLAILGNVLVCWAVWLNSNLQNVTNYFVVSLAAADIAVGVLAIPFAITISTGFCAACHGCLFIACFVLVLTQSSIFSLLAIAIDRYIAIRIPLRYNGLVTGTRAKGIIAICWVLSFAIGLTPMLGWNNCGQPKEGKNHSQGCGEGQVACLFEDVVPMNYMVYFNFFACVLVPLLLMLGVYLRIFLAARRQLKQMESQPLPGERARSTLQKEVHAAKSLAIIVGLFALCWLPLHIINCFTFFCPDCSHAPLWLMYLAIVLSHTNSVVNPFIYAYRIREFRQTFRKIIRSHVLRQQEPFKAAGTSARVLAAHGSDGEQVSLRLNGHPPGVWANGSAPHPERRPNGYALGLVSGGSAQESQGNTGLPDVELLSHELKGVCPEPPGLDDPLAQDGAGVS (sequence number)

[0121] An exemplary human A2aR amino acid sequence is shown below. VPIMGSSVYITVELAIAVLAILGNVLVCWAVWLNSNLQNVTNYFVVSLAAADIAVGVLAIPFAITISTGFCAACHGCLFIACFVLVLTQSSIFSLLAIAIDRY IAIRIPLRYNGLVTGTRAKGIIAICWVLSFAIGLTPMLGWNNCGQPKEGKNHSQGCGEGQVACLFEDVVPMNYMVYFNFFACVLVPLLLMLGVYLRIFLAARR QLKQMESQPLPGERARSTLQKEVHAAKSLAIIVGLFALCWLPLHIINCFTFFCPDCNHAPLWLMYLAIVLSHTNSVVNPFIYAYRIREFRQTFRKIIRSHVLRQQEPFKAAGTSARVLAAHGSDGEQVSLRLNGHPPGVWANGSAHPPERRPNGYALGLVSGGSTQESQGNTSLPDVELLSHELKGVCPEPPPGLDDPLAQGGAGVS (Sequence Number)

[0122] The term "anti-A2aR antibody" or "A2aR antibody" refers to an antibody or polypeptide that specifically binds to A2aR. In certain embodiments, an anti-A2aR antibody can inhibit A2aR biological activity and / or downstream signaling pathways mediated by A2aR. Anti-A2aR antibodies encompass antibodies or polypeptides that contain one or more antigen-binding domains in the form of CDRs or variable regions. In certain embodiments, the anti-A2aR antibodies of the present invention block, antagonize, inhibit or reduce (to any extent, including significantly) A2aR biological activity, including A2aR binding and downstream signaling and downstream events mediated by A2aR, such as stimulation of tumor growth, inhibition of anti-tumor immune responses, and immunosuppression in immune-compromised disease states.

[0123] As used herein, the term "A2aR disease" or "A2aR-related disease" refers to a disease or disorder caused by or associated with A2aR expressed and / or activated. The term "A2aR disease" or "A2aR-related disease" includes diseases, disorders, or conditions that can benefit from modulation (e.g., inhibition) of A2aR gene expression, replication, or protein activity. In certain embodiments, the A2aR disease or A2aR-related disease is cancer.

[0124] II. A2aR antibodies and antigen-binding molecules

[0125] The present invention provides an A2aR antigen binding molecule that specifically binds to A2aR. The term "antigen binding molecule" as used herein refers to a protein, polypeptide, or molecular complex that consists of at least one complementarity determining region (CDR), either alone or in combination with one or more other CDRs and / or framework regions (FRs), that specifically binds to a particular antigen. As that term is defined elsewhere herein, in one embodiment, the antigen binding molecule is an antibody or an antigen-binding fragment thereof. In one embodiment, the antigen binding molecule according to the present invention inhibits one or more biological functions of A2aR.

[0126] 1. Humanized chimeric antigen-binding molecules

[0127] The present invention provides chimeric antigen binding molecules, such as chimeric antibodies or antigen-binding fragments thereof, capable of specifically binding to A2aR. In one embodiment, the present invention provides humanized A2aR antigen binding molecules, such as humanized antibodies or antigen-binding fragments thereof.

[0128] The present invention encompasses humanized antibodies. Several methods for humanizing non-human antibodies are known in the art. For example, one or more amino acids of non-human origin can be introduced into a humanized antibody. These non-human amino acid residues are generally referred to as "imported" residues and usually come from the "imported" variable domain. Humanization is essentially performed according to the method of Winter and coworkers (Jones et al. (1986), Nature 321:522-525; Riechmann et al. (1988), Nature 332:323-327; Verhoeyen et al. (1988), Science 239:1534-1536), i.e., by replacement of the hypervariable region sequences with the corresponding sequences of a human antibody. Such "humanized" antibodies are thus chimeric antibodies (U.S. Pat. No. 4,816,567) in which less than a complete human variable domain is replaced by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some hypervariable region residues and possibly some FR residues are substituted by residues at analogous sites in rodent antibodies.

[0129] The selection of human variable domains (light and heavy chains) for producing humanized antibodies is important to reduce antigenicity. According to the so-called "best-fit" method, the sequence of the variable domain of a rodent antibody is screened against the entire library of known human variable domain sequences, and the human sequence that is closest to the rodent sequence is accepted as the human framework (FR) of the humanized antibody (see, for example, Sims et al. (1993) J Immunol. 151:2296; Chothia et al. (1987) J Mal. Biol. 196:901). Another method uses a specific framework derived from the consensus sequence of all human antibodies of a certain subgroup of light or heavy chains. The same framework can be used for several types of humanized antibodies (see, for example, Carter et al. (1992) Proc. Natl. Acad. Sci. USA, 89:4285; Presta et al. (1993) J Immunol. 151:2623).

[0130] Moreover, it is generally desired to humanize antibodies while retaining high affinity for the antigen and other favorable biological properties. To achieve this goal, humanized antibodies are generated by a method of analysis of the parental sequences and various conceptual humanized products with three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are well known to those skilled in the art and are publicly available. Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the possible role of the residues in the functioning of the candidate immunoglobulin sequence (i.e., analysis of residues which influence the ability of the candidate immunoglobulin to bind its antigen). In this manner, FR residues can be selected and combined from the consensus and import sequences such that the desired antibody characteristic (such as increased affinity for the target antigen) is achieved. In general, the hypervariable region residues are directly and most essentially involved in altering antigen binding.

[0131] The present invention encompasses chimeric antibodies with different "levels" or "degrees". In some embodiments, the chimeric antibodies of the present invention are "fully" humanized, i.e., contain sequences substantially identical or identical to the corresponding human FR sequences in both the heavy and light chain variable regions. In some embodiments, the chimeric antibodies of the present invention are "partially" humanized, e.g., contain sequences substantially identical or identical to the corresponding human FR sequences in one, two, three, four, five, six, or seven FR regions in the heavy and light chain variable regions, but contain sequences substantially identical or identical to the corresponding FR sequences in other species, e.g., mouse, rat, rabbit, chicken, and Chinese hamster FR sequences, in the remaining FR regions.

[0132] In certain embodiments, the A2aR is a human A2aR. An exemplary human A2aR has the amino acid sequence set forth in SEQ ID NO:[[xx]]. In some embodiments, the A2aR is a cynomolgus monkey A2aR. An exemplary cynomolgus monkey A2aR has the amino acid sequence set forth in SEQ ID NO:[[xx]].

[0133] 2. Exemplary antigen-binding molecule sequences

[0134] The A2aR antigen-binding molecule may be in the form of a monoclonal antibody, one or more polypeptide fragments containing one or more A2aR antigen-binding domains, or one or more nucleic acids encoding one or more A2aR binding domains. In one embodiment, the present invention provides a humanized A2aR antibody or an antigen-binding fragment thereof. The humanized antibody is a humanized version of the antibody disclosed in International Patent Publication No. WO2022 / 072601 (601 disclosure of International Patent Application PCT / US21 / 52819, filed September 30, 2021). The heavy and light chain variable regions ("variable regions") of the humanized antibody are named as follows: The variable regions derived from clone 1B5-3D7 in the 601 disclosure are referred to as "Hu1B5-Hn" and "Hu1B5-Ln" (representing the heavy and light chain variable regions, respectively). The variable regions derived from clone 3F6-9G5 in the 601 disclosure are referred to as "Hu3F6-Hn" and "Hu3F6-Ln" (representing the heavy chain variable region and the light chain variable region, respectively). The variable regions derived from clone 3F8-12E9 in the 601 disclosure are referred to as "Hu3F8-Hn" and "Hu3F8-Ln" (representing the heavy chain variable region and the light chain variable region, respectively). In the above nomenclature, "n" in "Hn" and "Ln" refers to a number. The number "n" at the named end of each heavy and light chain is used for identification purposes only. Thus, when one heavy chain and one light chain have the same other nomenclature except "H" and "L" (representing the heavy chain and the light chain, respectively), the nomenclature bears no significance to any function or property of the antibody composed of both those particular heavy and light chains. For example, the nomenclature of Hu1B5-H1 and Hu1B5-L1 is exactly the same except for "H" and "L", but the number "1" at the end of these nomenclature is used only for identification. An antibody consisting of Hu1B5-H1 and Hu1B5-L1 is equivalent to an antibody consisting of Hu1B5-H1 and another light chain (e.g., Hu1B5-L2). The antibody can be represented by its heavy and light chains in the form of "clone-Hm / clone-Ln", where "m" may be the same as or different from "n".For example, an antibody designated Hu3F6-H3 / Hu3F6-L5 refers to an antibody consisting of the heavy chain Hu3F6-H3 and the light chain Hu3F6-L5, both of which are derived from clone 3F6-9G5 in the 601 disclosure.

[0135] In exemplary embodiments of the present invention, an antigen-binding molecule, e.g., an anti-A2aR antibody or antigen-binding fragment thereof, comprises: (1) a heavy chain variable region comprising three complementarity determining regions (HCDRs), HCDR1, HCDR2, and HCDR3, wherein HCDR1 has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, and 3; HCDR2 has an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 4 to 9 and 89; HCDR3 has an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identity to an amino acid sequence shown in SEQ ID NO: 10 or 11. and (2) a light chain variable region comprising three complementarity determining regions (LCDRs) designated LCDR1, LCDR2, and LCDR3, wherein LCDR1 has an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 12 and 13; LCDR2 has an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identity to an amino acid sequence shown in SEQ ID NO: 14. and LCDR3 has an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 15 or 16.), wherein the antigen binding molecule specifically binds to human A2aR. Amino acid sequences corresponding to exemplary HCDRs and LCDRs of exemplary anti-human A2aR monoclonal antibodies disclosed in the present invention are shown in Tables 1 and 2.

[0136] The amino acid sequence boundaries of the CDRs can be determined by one of skill in the art using any of a number of known numbering schemes, such as those described in Kabat et al., supra (the "Kabat" numbering scheme); Al-Lazikani et al., 1997, J. Mol. Biol., 273:927-948 (the "Chothia" numbering scheme); MacCallum et al., 1996, J. Mol. Biol. 262:732-745 (the "Contact" numbering scheme); Lefranc et al., Dev. Comp. Immunol., 2003, 27:55-77 (the "IMGT" numbering scheme); and Honegge and Pluckthun, J. Mol. Biol, 2001, 309:657-70 (the "AHo" numbering scheme), each of which is incorporated by reference in its entirety. The amino acid sequence boundaries of the CDRs can also be determined by a combination of two numbering schemes (e.g., the Kabat and IMGT numbering schemes). Table 1 shows the heavy chain CDR sequences of exemplary antibodies of the invention, where the CDR sequences are defined by combining the CDRs based on the Kabat and IMGT numbering schemes. Table 2 shows the light chain CDR sequences of exemplary antibodies of the invention, where the CDR sequences are defined by combining the CDRs based on the Kabat and IMGT numbering schemes. In Tables 1 and 2, 1B5 and 1B5-2 are the CDRs from clone 1B5-3D7 disclosed in '601. 3F6 and 3F6-2 are the CDRs from clone 3F6-9G5 disclosed in '601. 3F8 and 3F8-2 are the CDRs from clone 3F8-12E9 disclosed in '601.

[0137] In one embodiment, the present invention relates to an antigen-binding molecule, e.g., an anti-A2aR antibody or antigen-binding fragment thereof, that comprises CDRs defined based on a combination of the Kabat and IMGT numbering schemes. Thus, in one embodiment, the present invention relates to an antigen-binding molecule, e.g., an anti-A2aR antibody or antigen-binding fragment thereof, that comprises CDRs defined based on a combination of the Kabat and IMGT numbering schemes. Accordingly ... that comprises CDRs defined based on a combination of the Kabat and IMGT numbering schemes. Accordingly, in one embodiment, the present invention relates to an antigen-binding molecule, e.g., an anti-A2aR antibody or antigen-binding fragment thereof that comprises CDRs defined based on a combination of the Kabat and IMGT numbering schemes. Accordingly, in one embodiment, the present invention relates to an antigen-binding molecule, e.g., an anti-A2aR antibody or antigen-binding fragment thereof that comprises CDRs defined based on a combination of the Kabat and IMGT numbering schemes. %, 96%, 97%, 98%, 99% or about 100% identity to an amino acid sequence selected from the group consisting of the HCDR3 sequences listed in Table 1, or containing one, two, three, four, five or six mutations; (3) an HCDR3 having an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or about 100% identity to an amino acid sequence selected from the group consisting of the HCDR3 sequences listed in Table 1, or containing one or two mutations; (4) an HCDR3 having an amino acid sequence selected from the group consisting of the HCDR3 sequences listed in Table 1, or containing one or two mutations; (5) an 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 Table 2, or that contains one or two mutations; and (6) an 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 LCDR3 sequences listed in Table 2, or that contains one or two mutations. The term "mutation" as used herein refers to a change or difference (substitution, insertion or deletion) in an amino acid sequence compared to a reference. In some embodiments, the mutation is a conservative substitution.

[0138] 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 in HCDR1 refers to the first amino acid in HCDR1. Thus, in mAB 1B5, position 1 in HCDR1 according to a numbering scheme based on a combination of the Kabat and IMGT numbering schemes is glycine (G).

[0139] Certain positions of the CDRs as shown in Tables 1 and 2 are conserved. For example, the first position of HCDR1 in Table 1 is highly conserved and contains "G". On the other hand, certain positions in the CDRs are less conserved. For example, the third position of HCDR1 in Table 1 is less conserved and may be "A" or "T". In some embodiments, mutations occur at one or more of the less conserved positions of the CDRs.

[0140] [Table 1]

[0141] As used herein, X1 is A or T, X2 is R or S, X3 is F or Y, X4 is S or Y, X5 is A or T, X6 is H or Q, X7 is A, H, or N, X8 is A or H, X9 is D or G, and X 10 is G or L.

[0142] [Table 2]

[0143] As used herein, X 11 is L or I, and X 12 is R or S, and X 13 is F or Y.

[0144] In one embodiment, the present invention provides a humanized antibody. Tables 3 to 8 show the CDR and FR sequences of exemplary humanized antibodies or antigen-binding fragments thereof of the present invention. [Table 3] [Table 4] [Table 5] [Table 6] [Table 7] [Table 8]

[0145] Tables 9-16 show the FR sequences of some exemplary antibodies of the present invention. [Table 9]

[0146] 1 represents Hu3F6-H1 and Hu3F8-H1.

[0147] 2 represents Hu1B5-H2.1, Hu1B5-H3, Hu1B5-H4, Hu1B5-H5, Hu3F6-H2, Hu3F6-H3, Hu3F6-H4, and Hu3F6-H5.

[0148] 3 represents Hu3F8-H6.

[0149] 4 represents Hu3F8-H2, Hu3F8-H3, Hu3F8-H4, Hu3F8-H5, and Hu3F8-5.1.

[0150] As used herein, X14 is E or Q, X15 is A or T, and X16 is A or T. [Table 10]

[0151] 1 represents Hu1B5-H2, Hu1B5-H2, Hu3F6-H1, Hu3F6-H2, Hu3F8-H1, and Hu3F8-H2.

[0152] 2 represents Hu1B5-3, Hu1B5-H4, Hu1B5-H5, Hu3F6-H3, Hu3F6-H4, Hu3F6-H5, Hu3F8-H3, Hu3F8-H4, Hu3F8-H5, and Hu3F8-H5.1.

[0153] 3 represents Hu3F6-H6 and Hu3F8-H6.

[0154] As used herein, X17 is A or M, X18 is K or Q, and X19 is I or M. [Table 11]

[0155] 1 represents Hu3F6-H1 and Hu3F8-H1.

[0156] 2 represents Hu1B5-H2.1, Hu3F6-H2, and Hu3F8-H2.

[0157] 3 represents Hu3F6-H3.

[0158] 4 represents Hu1B5-H5, Hu3F6-H4, and Hu3F6-H5.

[0159] 5represents Hu3F6-H6 and Hu3F8-H6.

[0160] 6 represents Hu3F8-H5 and Hu3F8-H5.1.

[0161] As used herein, X20 is A or V, X21 is L or M, X22 is R or V, X23 is K or T, and X24 is S or T. [Table 12]

[0162] 1 represents the heavy chains derived from 1B5-3D7 and 3F8-12E9.

[0163] 2 represents the heavy chain derived from 3F6-9G5.

[0164] As used herein, X25 is H or Q. [Table 13]

[0165] 1 represents Hu1B5-L2, Hu1B5-L3, Hu3F6-L1, Hu3F6-L2, Hu3F6-L3, Hu3F8-L1, Hu3F8-L2, and Hu3F8-L3.

[0166] 2 represents Hu3F6-L8 and Hu3F8-L5.

[0167] As used herein, X26 is I or V, X27 is S or T, X28 is D, L or P, X29 is A, P or S, X30 is N, S or T, X31 is L or P, and X32 is E or Q. [Table 14]

[0168] 1 represents Hu3F6-L1 and Hu3F8-L1.

[0169] 2 represents Hu3F6-L2 and Hu3F8-L2.

[0170] 3 represents Hu3F6-L3 and Hu3F8-L3.

[0171] 4 represents Hu3F6-L4, Hu3F6-L5, Hu3F6-L6, Hu3F6-L7, Hu3F6-L8, Hu3F8-L4, and Hu3F8-L5.

[0172] As used herein, X33 is F or Y, X34 is L or Q, X35 is K or R, X36 is P or S, X37 is K, Q or R, and X38 is L or R. [Table 15]

[0173] 1 represents Hu1B5-L2, Hu1B5-L3, Hu1B5-L4, Hu3F6-L1, Hu3F6-L2, Hu3F6-L3, Hu3F6-L4, Hu3F6-L5, Hu3F6-L8, Hu3F8-L1, Hu3F8-L2, Hu3F8-L3, Hu3F8-L4, and Hu3F8-L5.

[0174] 2 represents Hu3F6-L7.

[0175] As used herein, X39 is S or T, X40 is K or T, X41 is R, S or W, X42 is L or V, X43 is E or Q, and X44 is A or G. [Table 16]

[0176] 1 represents all light chains.

[0177] In some embodiments, the antibody or antigen-binding fragment thereof comprises: (1) a heavy chain variable region (HCVR) comprising an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 51-70; and (2) a light chain variable region (LCVR) comprising an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 71-88, wherein the antibody or antigen-binding fragment thereof specifically binds to human A2aR. Tables 18 and 19 show the amino acid sequences of exemplary HCVRs and LCVRs of exemplary anti-human A2aR monoclonal antibodies disclosed in the present invention. Tables 19 and 20 show exemplary nucleotide sequences of DNA encoding the HCVRs and LCVRs of exemplary anti-human A2aR antibodies of the present invention, respectively. [Table 17-1] [Table 17-2] [Table 18-1] [Table 18-2] [Table 19-1] [Table 19-2] [Table 20-1] [Table 20-2] [Table 20-3] [Table 21-1] [Table 21-2] [Table 21-3]

[0178] In one embodiment, the antigen-binding molecule of the present invention, e.g., an antibody or an antigen-binding fragment thereof, is post-translationally modified. Examples of post-translational modifications include cleavage of lysine at the C-terminus of the heavy chain by carboxypeptidase, modification of glutamine or glutamic acid to pyroglutamic acid at the N-terminus of the heavy and light chains by pyroglutamylation, glycosylation, oxidation, deamidation, and glycation. Such post-translational modifications are known to occur in various antibodies (see Journal of Pharmaceutical Sciences, 2008, Vol. 97, p. 2426-2447, the entire contents of which are incorporated herein by reference). Examples of post-translationally modified antigen-binding molecules, e.g., antibodies or antigen-binding fragments thereof, include antigen-binding molecules, e.g., antibodies or antigen-binding fragments thereof, that have undergone pyroglutamylation at the N-terminus of the heavy chain variable region and / or deletion of lysine at the C-terminus of the heavy chain. The sequences of exemplary antigen-binding molecules with pyroglutamylated N-terminus are shown in Table 7. As used herein, "pE," when used to describe an amino acid in a polypeptide, refers to pyroglutamic acid.

[0179] 2. Variants of antigen-binding molecules

[0180] In one embodiment, the A2aR antigen binding molecule according to the present invention, e.g., the anti-A2aR antibody, may be a monoclonal antibody, a chimeric antibody, a humanized antibody, a Fab, a F(ab')2, a scFv, or a multispecific antibody comprising other binding specificities as described herein.

[0181] Thus, in certain embodiments, the anti-A2aR antibodies described herein may be linked to an Fc that includes one or more modifications to modify one or more functional properties of the antibody, such as serum half-life, complement binding, Fc receptor binding, and / or antigen-dependent cellular cytotoxicity. Additionally, the antibodies described herein may be chemically modified (e.g., one or more chemical moieties are linked to the antibody) or modified to modify its glycosylation to modify 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 generate Fc variants that have (a) increased or decreased antibody-dependent cell-mediated cytotoxicity (ADCC), (b) increased or decreased complement-mediated cytotoxicity (CDC), (c) increased or decreased affinity for C1q, and / or (d) higher or lower affinity for Fc receptors than the parent Fc. Such Fc region variants generally include at least one amino acid modification in the Fc region. Combining amino acid modifications may be particularly desirable. For example, the Fc region variant can include substitutions of two, three, four, five, etc. at specific Fc region positions, eg, as described herein.

[0182] For applications requiring effector function, some effector function-enhanced Fc can be used. In some embodiments, when the antibody is intended to induce ADCC, ADCC-enhanced IgG1 S239D, A330L, I332E or S298A, E333A, K334A or F243L, R292P, Y300L, V305I, and P396L can be used. In some embodiments, to enhance the ADCC of a "Y"-shaped bivalent antibody, substitutions such as L234Y, L235Q, G236W, S239M, H268D, D270E, S298A are introduced in one heavy chain and substitutions such as D270E, K326D, A330M, K334E are introduced in the other heavy chain.

[0183] In some embodiments, when the antibody is intended to induce ADCP, the ADCP-enhanced versions G236A, S239D, and I332E can be used. In certain embodiments, when the antibody is intended to induce CDC, the CDC-enhanced versions K326W, E33S or S267E, H268F, S324T or E345R, E430G, and S440Y can be used.

[0184] For applications where effector functions should be completely avoided, for example where antigen binding alone is sufficient to produce the desired therapeutic effect and effector functions do not lead to (or increase the risk of) undesirable side effects, IgG4 antibodies or IgG1 L234F, L235E, P331S or L234A, L235A, and P239G with no ADCC effect may be used, antibodies or fragments lacking the Fc region or a large portion thereof may be designed, or the Fc may be mutated to completely eliminate glycosylation (e.g., N297A, N297Q, or N297G). Alternatively, hybrid constructs of human IgG2 (CH1 domain and hinge region) and human IgG4 (CH2 and CH3 domains) may be made that lack effector functions and the ability to bind FcγRs (e.g., IgG2) and activate complement (e.g., IgG4). When using an IgG4 constant domain, it is usually preferred to include the substitution S228P, which mimics the hinge sequence of IgG1, and the R409K mutation, which stabilizes the IgG4 molecule by blocking Fab arm exchange, thereby reducing Fab arm exchange between the therapeutic antibody and endogenous IgG4 in the patient being treated.

[0185] In some embodiments, the anti-A2aR antibody or fragment thereof can be modified to increase biological half-life. Various methods can be used, such as increasing the binding affinity of the Fc region to FcRn. In one embodiment, the antibody contains a salvage receptor binding epitope in two loops of the CH2 domain of the Fc region of IgG by modifying the CH1 or CL region as described in US Patents 5,869,046 and 6,121,022. The numbering of residues in the Fc region is that of the EU index of Kabat. The sequence variants disclosed herein are provided by reference to the residue number followed by the amino acid substituted at the naturally occurring amino acid position and optionally preceding the naturally occurring residue at that position. When multiple amino acids can be present at a given position, e.g., when sequences differ between naturally occurring isotypes, or when multiple mutations are substituted at that position, they are separated by slashes (e.g., "X / Y / Z").

[0186] Exemplary Fc variants that improve binding to FcRn and / or improve pharmacokinetic properties include substitutions at positions 259, 308, and 434, including, for example, 259I, 308F, 428L, 428M, 434S, 434H, 434F, 434Y, and 434M. Other variants that improve 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, and 434A (Shields et al., 2006). al, (2001) J. Biol. Chem., 276(9):6591-6604), 252F, 252Y, 252W, 254T, 256Q, 256E, 256D, 433R, 434F, 434Y, 252Y / 254T / 256E, 433K / 434F / 436H (Dall'Acqua et al, (2002) J. Immunol., 169:5171-5180; Dall'Acqua et al, (2006) J. Biol. Chem., 281:23514-23524; and U.S. Patent No. 8,367,805).

[0187] Modifications of certain conserved residues (I253, H310, Q311, H433, N434) in IgG Fc, such as the N434A variant (Yeung et al., (2009) J. Immunol. 182:7663), have been proposed to increase affinity for FcRn and extend antibody half-life in circulation (WO98 / 023289). A combined Fc variant containing M428L and N434S was shown to improve binding to FcRn and extend serum half-life by up to five-fold (Zalevsky et al., (2010) Nat. Biotechnol. 28:157). A combined Fc variant containing the T307A, E380A and N434A modifications also extended the half-life of an IgG1 antibody (Petkova et al, (2006) Int. Immunol. 18:1759). Additionally, it has been shown that combination Fc variants including M252Y-M428L, M428L-N434H, M428L-N434F, M428L-N434Y, M428L-N434A, M428L-N434M, and M428L-N434S extend half-life ((US 2006 / 173170)). Additionally, it has been reported that combination Fc variants including M252Y, S254T, and T256E increase half-life by nearly four-fold (Dall'Acqua et al, (2006) J. Biol. Chem. 281:23514).

[0188] In some embodiments, the A2aR antigen binding molecule described herein is a bispecific antibody comprising a first targeting domain that specifically binds to A2aR and a second targeting domain that specifically binds to another epitope on A2aR or another protein. In some embodiments, the first targeting domain comprises an antigen-binding fragment of any of the A2aR antibodies described herein.

[0189] In one embodiment, the antigen-binding molecule (e.g., anti-A2aR antibody or antigen-binding fragment thereof) according to the present invention is chemically conjugated to one or more therapeutically active peptides and / or small molecule pharmaceuticals. The peptides or small molecule pharmaceuticals can be linked, for example, to reduced SH groups and / or carbohydrate side chains. Methods for making covalent or non-covalent conjugates of peptides or small molecule pharmaceuticals with antibodies are known in the art, and any such known methods can be utilized.

[0190] In some embodiments, the peptide or small molecule drug is linked to the hinge region of the reduced antibody component via a disulfide bond. Alternatively, such drugs are linked using a heterobifunctional crosslinker (N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP)). General techniques for such conjugation are well known in the art. In some embodiments, the peptide or small molecule drug is conjugated via a carbohydrate moiety in the Fc region of the antibody. The carbohydrate group can be used to increase the loading of the same drug bound to a thiol group, or the carbohydrate moiety can be used to attach a different therapeutic or diagnostic agent. Methods for conjugating peptide inhibitors or small molecule drugs to antibodies via the carbohydrate moiety of the antibody are well known to those skilled in the art. For example, in one embodiment, the method involves reacting an antibody component bearing an oxidized carbohydrate moiety with a carrier polymer bearing at least one free amine functional group. This reaction results in an initial Schiff base (imine) linkage, which is stabilized to form the final conjugate by reduction to a secondary amine. Exemplary methods for conjugating small molecule drugs to peptides and antibodies are described in US Patent Application Publication No. 2014 / 0356385.

[0191] The size ranges of the A2aR antibodies (including fragments and multispecific forms thereof) are 50kD-300kD, 50kD-250kD, 60kD-250kD, 80kD-250kD, 100kD-250kD, 125kD-250kD, 150kD-250kD, 60kD-225kD, 75kD-225kD, 100kD-225kD, 125kD-225kD, 150kD-225kD, 60kD-200kD, 75 In one embodiment, the inventive subject matter may be any range contained within any combination of integers recited in the above ranges, or any range determined by any combination of integers between the above ranges.

[0192] 3. Biological properties of antibodies and antigen-binding molecules

[0193] The present invention includes antibodies and antigen-binding fragments thereof that bind to human and cynomolgus A2aR.

[0194] The present invention includes A2aR antigen binding molecules, e.g., A2aR antibodies or antigen-binding fragments thereof, that can specifically bind to human and cynomolgus monkey A2aR expressed on a cell surface and inhibit the activity or function of A2aR. In one embodiment, the antigen binding molecules block the interaction of human A2aR expressed on a cell surface with an A2aR agonist. The extent to which an A2aR antigen binding protein (e.g., an A2aR antibody or antigen-binding fragment thereof) blocks A2aR activity can be assessed by the assay described in Example 3 or a substantially similar assay. The present invention includes A2aR antigen binding molecules that block the interaction of human A2aR expressed on a cell surface with an A2aR agonist and have an IC determined by the assay described in Example 3 or a substantially similar assay. 50 The present invention also includes antigen-binding molecules, for example antibodies, having a value of 0.7 nM to about 20 nM or less.

[0195] 4. Species selectivity and species cross-reactivity

[0196] In some embodiments, the present invention provides antigen-binding molecules that bind to human A2aR but not to A2aR of other species. The present invention further includes antigen-binding molecules that bind to human A2aR and A2aR from one or more non-human species (e.g., non-human primates).

[0197] According to certain exemplary embodiments of the present invention, antigen-binding molecules are provided that bind to human A2aR and may or may not bind to A2aR from one or more of mouse, rat, guinea pig, hamster, gerbil, pig, cat, dog, rabbit, goat, sheep, cow, horse, camel, cynomolgus monkey, marmoset, rhesus monkey, and chimpanzee. For example, in certain exemplary embodiments of the present invention, the present invention provides antigen-binding molecules comprising an antigen-binding domain that binds to human A2aR and non-human primate (e.g., cynomolgus monkey) A2aR.

[0198] III. Therapeutic uses of anti-A2aR antigen binding molecules

[0199] The anti-A2aR antigen-binding molecules (antibodies, antigen-binding fragments thereof, and multispecific antibodies thereof) described in the present invention have numerous in vitro, in vivo, and ex vivo applications related to enhancing immune responses by blocking adenosine signaling and other signaling in the treatment of cancer. Without wishing to be bound by any theory, it is hypothesized that the antigen-binding molecules described in the present invention (e.g., anti-A2aR antibodies or antigen-binding fragments thereof) bind to and inhibit the activity of A2aR expressed on the cell surface, and inhibition of A2aR activity results in, for example, a reduction in the concentration of cAMP within the cell. Thus, the antigen-binding molecules described in the present invention (and therapeutic compositions comprising them) are particularly useful for treating any disease or disorder that would benefit from inhibiting the activity of A2aR (e.g., stimulating and / or activating an immune response). Given the widespread expression of A2aR and the diverse effects mediated by adenosine and A2aR, the anti-A2aR antigen binding molecules (e.g., antibodies or antigen-binding fragments thereof) described in the present invention can be used alone or in combination with a wide variety of active agents to treat a wide range of diseases or disorders, including various cancers.

[0200] Thus, the present invention provides a method for reducing the intracellular cAMP concentration, comprising contacting a cell with an antigen-binding molecule (e.g., an anti-A2aR antibody or an antigen-binding fragment thereof) according to the present invention. The reduction in the intracellular cAMP concentration can be measured by the method described in Example 4 or a substantially similar method. In one embodiment, the method according to the present invention reduces the intracellular cAMP concentration 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.

[0201] In some embodiments, antigen-binding molecules described herein (e.g., anti-A2aR antibodies or antigen-binding fragments thereof) are administered to cultured cells (in vitro) or human subjects (in vivo or ex vivo) to enhance immunity against various diseases. Thus, in one embodiment, a method for stimulating an immune response in a subject in need thereof comprises administering to the subject an anti-A2aR antibody, antigen-binding fragment thereof (e.g., anti-A2aR HCVR and LCVR), or multispecific anti-A2aR antibody described herein, such that an immune response is enhanced, stimulated, upregulated in the subject, e.g., for suppression of tumor growth, stimulation of anti-tumor T cell immunity, and / or stimulation of anti-microbial immunity.

[0202] In one aspect, a method for enhancing an immune response (e.g., a T cell response) in a subject comprises administering to the subject an anti-A2aR antibody described herein to enhance an immune response (e.g., a T cell response) in the subject. In some embodiments, the subject is a tumor-bearing subject, and an immune response against the tumor is enhanced. The tumor may be a solid tumor or a liquid tumor, e.g., a hematological malignancy. In some embodiments, the tumor is an immunogenic tumor. In some other embodiments, the tumor is a non-immunogenic tumor. In some other embodiments, the subject is a pathogen-bearing subject, and an immune response against the pathogen is enhanced by administration of an anti-A2aR antibody described herein. The immune response includes, but is not limited to, a) promoting effector T cell function, b) reducing Treg activity, c) preventing Treg expansion, d) promoting enhanced NK cell function, or e) promoting type 1 activation of antigen-presenting cells.

[0203] 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.

[0204] Preferred subjects include human patients in whom an enhanced immune response is desired. The method is particularly suitable for treating human patients suffering from disorders that can be treated by enhancing the immune response (e.g., T cell-mediated immune response). The method is particularly suitable for treating cancer, chronic infectious diseases, and chronic inflammatory diseases. The antibody used in the disclosed method described in the present invention is preferably a human antibody or a humanized antibody.

[0205] In one embodiment, a method for inhibiting tumor cell growth in a subject comprises administering to the subject an anti-A2aR antibody as described herein to inhibit tumor growth in the subject. Inhibition of tumor growth can be measured by a variety of methods. Tumor growth can be measured, for example, using the methods described in Talkington, A and Durrett, R, Estimating Tumor Growth Rates in vivo, Bull Math Biol., 2015 Oct.:77(10):1934-54 (available at https: / / www.ncbi.nlm.nih.gov / pmc / articles / PMC4764475 / , the entire contents of which are incorporated herein by reference). Inhibition of tumor growth can also be measured by a reduction in tumor size. In one embodiment, 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.

[0206] In one embodiment, the antigen-binding molecules of the present invention (e.g., anti-A2aR antibodies or antigen-binding fragments thereof) are used to reduce immunosuppression in the tumor microenvironment. Such reduction can be measured by various methods. The level of immunosuppression in the tumor microenvironment can be measured by the presence and / or abundance of certain biomarkers in the tumor (e.g., PD-L1, CD73, IL-10, or TGF-β). The level of immunosuppression can be measured by the expression of regulatory T (T regIn general, immunosuppression can be measured by the ratio of CD8+ cytotoxic T cells to T reg In one embodiment, the antigen-binding molecule (e.g., an anti-A2aR antibody or antigen-binding fragment thereof) described in the present invention reduces the level of immune suppression in the tumor microenvironment by at least about 10%, about 20%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95% or more compared to baseline levels.

[0207] T in the tumor microenvironment reg and administering to the subject a therapeutically effective amount of an anti-A2aR antibody described herein that comprises an Fc that stimulates depletion of A2aR cells from the tumor microenvironment of a subject having a tumor (e.g., a cancerous tumor). reg Methods for depleting cells are also encompassed herein. The Fc may be, for example, an Fc with appropriate effector function or with enhanced effector function (caused by one or more activating Fc receptors).

[0208] In one preferred embodiment, T reg Depletion of T in the tumor microenvironment eff without significant depletion or inhibition of T outside the tumor microenvironment eff Cells and T reg In one embodiment, the subject has a tumor microenvironment that is associated with a significant depletion or inhibition of T cells. eff T than on cells reg In one embodiment, the anti-A2aR antibody detects T cells in tumors that have higher levels of A2aR. reg and / or T in tumor-infiltrating lymphocytes (TILs) regs can be depleted.

[0209] In a preferred embodiment, the subject has a cell proliferative disease or cancer. Blockade of adenosine signaling via A2aR by an antigen binding molecule (e.g., an anti-A2aR antibody or antigen-binding fragment thereof) described in the present invention can enhance the immune response against cancerous cells in the patient. Thus, the present invention provides a method for treating a subject with cancer, comprising administering to the subject an anti-A2aR antigen binding molecule (e.g., an antibody or antigen-binding fragment thereof) described herein such that the subject is treated, e.g., cancerous tumor growth is inhibited or reduced and / or tumor regression occurs. The anti-A2aR antibody may be used alone to inhibit cancerous tumor growth. Alternatively, the anti-A2aR antibody may be combined with one or more other targeted active agents, such as, for example, 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 primary and / or metastatic tumors. The present invention also includes a method for treating residual cancer in a subject. As used herein, the term "residual cancer" refers to the presence or persistence of one or more cancerous cells in a subject following treatment with an anti-cancer therapy.

[0210] Thus, in one aspect, a method of treating cancer comprises administering to a subject in need thereof a therapeutically effective amount of an anti-A2aR antibody as described herein. Preferably, the antibody inhibits the activity of human A2aR and comprises one or more HCVRs and LCVRs as described herein. Furthermore, the anti-A2aR antigen binding molecule (e.g., an antibody) used in the method can comprise a chimeric or humanized non-human anti-A2aR antibody thereof. The efficacy of treating cancer can be measured by various methods. For example, it can be measured by improved survival rate or reduced tumor size. In one embodiment, according to the method of the present invention, the efficacy of treating cancer is improved by at least about 10%, about 20%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 1-fold, about 2-fold, about 4-fold or more compared to baseline levels. When used in relation to cancer treatment, the baseline level refers to the therapeutic effect of a placebo when the A2aR antigen binding molecule described in the present invention is the only therapeutic agent, and refers to the therapeutic effect of a placebo or an additional therapeutic agent when the A2aR antigen binding molecule described in the present invention is used in combination with an additional therapeutic agent.

[0211] Potential cancers whose growth may be inhibited by the antibodies according to the invention include a wide variety of cancers, particularly those that are unresponsive or have a tendency to become unresponsive to monotherapy with other antibodies or chemotherapeutic agents. Non-limiting examples of cancers that may be treated 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, kidney 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, stomach cancer, bladder cancer, hepatoma, breast cancer, colon cancer, and head and neck cancer, gastric cancer, and other cancers. ) cancer, germ cell tumors, childhood sarcomas, sinus natural killer, melanoma (e.g., metastatic malignant melanoma (e.g., cutaneous or intraocular malignant melanoma)), bone cancer, skin cancer, uterine cancer, anal region cancer, testicular cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, esophageal cancer, small intestine cancer, endocrine system cancer, parathyroid cancer, adrenal gland cancer, soft tissue sarcoma, urethral cancer, penile cancer, childhood solid tumors, ureteral cancer, renal pelvis cancer, central nervous system (CNS) neoplasms, primary CNS lymphoma, tumor angiogenesis, spinal axis tumors, brain stem gliomas, pituitary adenomas, Kaposi's sarcoma sarcoma), epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers including asbestos-induced cancer, virus-associated cancers (e.g., human papillomavirus (HPV)-associated tumors), and hematological malignancies derived from either of the two major blood cell lineages, the myeloid cell lineage (which produces granulocytes, erythrocytes, platelets, macrophages and mast cells) and the lymphoid cell lineage (which produces B, T, NK and plasma cells), e.g., all types of leukemia, lymphoma and myeloma, e.g., acute, chronic, lymphocytic and / or myeloid leukemia, e.g., acute leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), and chronic myeloid leukemia (CIVIL), anaplastic AML (MO), myeloblastic leukemia (M1), myeloblastic leukemia (M2;with eosinophilic maturation), promyelocytic leukemia (M3 or M3 variant [M3V]), myelomonocytic leukemia (M4, or M4 variant [M4E] with eosinophilia), monocytic leukemia (M5), erythroleukemia (M6), megakaryoblastic leukemia (M7), solitary granulocytic sarcoma, and chloroma; lymphomas, e.g., Hodgkin lymphoma (HL), non-Hodgkin lymphoma (NEIL), B-cell lymphoma, T-cell lymphoma, lymphoplasmacytic 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, lymphoblastic lymphoma, post-transplant lymphoproliferative disorder, true histiocytic lymphoma, primary central nervous system lymphoma, primary effusion lymphoma, lymphoblastic lymphoma (LBL), hematopoietic neoplasms of lymphoid lineage, acute lymphoblastic leukemia, diffuse large B-cell lymphoma, Burkitt's lymphoma lymphoma, follicular lymphoma, diffuse histiocytic lymphoma (DHL), immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma, cutaneous T-cell lymphoma (CTLC) (also called mycosis fungoides or Sézary syndrome), and lymphoplasmacytic lymphoma with Waldenström's macroglobulinemia (LPL); myelomas, e.g., IgG myeloma, light chain myeloma, non-secretory myeloma, smoldering myeloma (also called indolent myeloma), solitary plasmacytoma plasmocytoma), and multiple myeloma, chronic lymphocytic leukemia (CLL), hairy cell lymphoma; hematopoietic tumors of myeloid lineage, tumors of mesenchymal origin (including fibrosarcoma and rhabdomyoscarcoma); seminoma, teratoma, tumors of the central and peripheral nerves (including astrocytoma, schwannoma); tumors of mesenchymal origin (including fibrosarcoma, rhabdomyosarcoma, and osteosarcoma); and other tumors such as melanoma, xeroderma pigmentosum, keratoacanthoma, seminoma, thyroid follicular carcinoma and teratocarcinoma, hematopoietic tumors of the lymphatic system, e.g., T-cell tumors and B-cell tumors including, but not limited to, T-prolymphocytic leukemia (T-PLL), including those of the small cell and cerebrospinal cell types; preferred T-cell type large granular lymphocytic leukemia (LGL); a / d T-NHL hepatosplenic lymphoma; peripheral / retrothymic T-cell lymphoma (pleomorphic and immunoblastic subtypes); angiocentric (nasal) T-cell lymphoma; head and neck cancer, renal cancer, rectal cancer, thyroid cancer; acute myeloid lymphoma, and any combination of the above cancers.The methods described herein can also be used to treat metastatic cancers, refractory cancers (e.g., cancers that are refractory to previous immunotherapy, such as with CTLA-4 or PD-1 blocking antibodies), and recurrent cancers.

[0212] In some embodiments, treatment of cancer patients with anti-A2aR antibodies and / or other active agents described herein can result in long-term, sustainable responses compared to current standard of care, such as long-term survival, for example, at least 1, 2, 3, 4, 5, 10 years or longer, and / or recurrence-free survival, for example, at least 1, 2, 3, 4, 5, 10 years or longer. In some embodiments, treatment of cancer patients with anti-A2aR antibodies and / or other active agents described herein can prevent or delay cancer recurrence, for example, for 1, 2, 3, 4, 5, 10 years or longer. The anti-A2aR treatment can be used as a first-line or second-line treatment.

[0213] Bone marrow transplantation is currently used to treat various tumors of hematopoietic origin. However, this treatment results in graft-versus-host disease. In contrast, A2aR inhibition can be used to increase the effectiveness of donor engrafted tumor-specific T cells by reducing graft-versus-tumor responses.

[0214] In some embodiments, ex vivo activation and expansion of antigen-specific T cells in the presence of anti-A2aR antibodies and adoptive transfer of these cells into a recipient can be used to stimulate antigen-specific T cells against cancer or viral infection by increasing the frequency and activity of the adoptively transferred T cells.

[0215] Suitable routes for administering the antigen-binding molecules of the present invention described herein, such as anti-A2aR antibodies or antigen-binding fragments thereof (e.g., humanized monoclonal antibodies, multispecific antibodies, and immunoconjugates), in vivo, ex vivo, or in vitro, are well known in the art and can be selected by those skilled in the art. For example, the antibody compositions can be administered by parenteral injection (e.g., intravenous or subcutaneous). The appropriate dosage depends on the age and weight of the subject and the concentration and / or formulation of the antibody composition, as further described below.

[0216] Increased A2aR activity is involved in neurodegenerative diseases. Thus, in one embodiment, the present invention provides a method for treating a neurodegenerative disease, comprising administering an antigen-binding molecule (e.g., an anti-A2aR antibody or an antigen-binding fragment thereof) according to the present invention to a subject in need thereof to treat the neurodegenerative disease.

[0217] The term "cell proliferative disorder" refers to a disorder characterized by abnormal proliferation of cells. A proliferative disorder does not imply any limitation with respect to the rate of cell proliferation, but simply indicates a loss of normal controls that affect proliferation and cell division. Thus, in some embodiments, cells of a proliferative disorder may have the same rate of cell division as normal cells, but do not respond to signals that limit such proliferation. Within the scope of "cell proliferative disorder" is a neoplasm, cancer, or tumor.

[0218] The term "cancer" refers to any one of a variety of malignant neoplasms characterized by the proliferation of cells capable of invading surrounding tissues and / or metastasizing to new colonization sites. Cancer includes carcinomas, sarcomas, adenocarcinomas, melanomas, leukemias, lymphomas, germ cell tumors, and blastomas, including both solid and lymphatic cancers. Exemplary cancers that may be treated by the compositions and methods described herein include brain cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, head and neck cancer, renal cancer, lung cancer, non-small cell lung cancer, mesothelioma, ovarian cancer, prostate cancer, gastric and uterine cancer, leukemia, and medulloblastoma.

[0219] The term "cancer" refers to a malignant growth of epithelial cells tending to invade surrounding tissues and give rise to metastases. Exemplary cancers include, for example, acinar carcinoma, acinous carcinoma, adenocystic carcinoma, adenoid cystic carcinoma, carcinoma adenomatosum, carcinoma of the adrenal cortex, pulmonary foam carcinoma, pulmonary foam 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 en cuirasse, carcinoma cutaneum, cylindrical carcinoma, cylindrical cell carcinoma, ductal carcinoma, carcinoma durum, embryonal carcinoma carcinoma), encephaloid carcinoma, epiennoid carcinoma, carcinoma epitheliale adenoides, exophytic carcinoma, carcinoma ex ulcere, carcinoma fibrosum, gelatiniform carcinoma, gelatinous carcinomacarcinoma), giant cell carcinoma, carcinoma gigantocellulare, glandular carcinoma, granular cell carcinoma, hair-matrix carcinoma, hematoid carcinoma, hepatocellular carcinoma, Hurthle cell carcinoma, hyaline carcinoma, hypernephroid carcinoma, infantile embryonal carcinoma, carcinoma in situ, intraepidermal carcinoma, intraepithelial carcinoma, Krompecher’s carcinoma, Kulchitzky-cell carcinoma, large-cell carcinoma, lenticular carcinoma, carcinoma lenticulare, lipomatous carcinoma, lymphoepithelial carcinoma, carcinoma medullare, medullary carcinoma, melanotic carcinoma, carcinoma molle, mucinous carcinoma, carcinoma muciparum, carcinoma mucocellulare, mucoepidermoid carcinoma, carcinoma mucosum, mucous carcinoma, carcinoma myxomatodes, naspharyngeal carcinoma, oat cell carcinoma, carcinomaossificans), osteoid carcinoma, pancreatic ductal adenocarcinoma, papillary carcinoma, periportal carcinoma, preinvasive carcinoma, prickle cell carcinoma, pultaceous carcinoma, renal cell carcinoma of the 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, carcinoma spongiosum, squamous carcinoma, squamous cell carcinoma, string carcinoma, carcinoma telangiectaticum, carcinoma telangiectodes, transitional cell carcinoma, carcinoma tuberosum, tuberous carcinoma, verrucous carcinoma and carcinoma villosum.

[0220] The term "sarcoma" refers to a tumor made up of a substance similar to embryonic connective tissue and is generally made up of closely packed cells embedded in a fibrillar or homogeneous substance.Exemplary sarcomas include, for example, chondrosarcoma, fibrosarcoma, lymphosarcoma, melanosarcoma, myxosarcoma, osteosarcoma, Abemethy's sarcoma, adipose sarcoma, lipo sarcoma, alveolar soft part sarcoma, ameloblastic sarcoma, botryoid sarcoma, chloroma sarcoma, choriocarcinoma, embryonal sarcoma, Wilms' tumor sarcoma, endometrial sarcoma, stromal sarcoma, Ewing's sarcoma, fascial sarcoma, fibroblastic sarcoma, and fibrosarcoma. sarcoma, giant cell sarcoma, granulocytic sarcoma, Hodgkin's sarcoma, idiopathic multiple pigmented hemorrhagic sarcoma, immunoblastic sarcoma of B cells, lymphoma (e.g., non-Hodgkin's lymphoma), immunoblastic sarcoma of T-cells, Jensen's sarcoma, Kaposi's sarcoma, Kupffer cell sarcoma, angiosarcoma, leukosarcoma, malignant mesenchymoma Sarcoma of the ulcerative colitis includes sarcoma of the ulcerative colitis, parosteal sarcoma, reticulocytic sarcoma, Rous sarcoma, serocystic sarcoma, synovial sarcoma, and telangiectatic sarcoma.

[0221] The term "melanoma" refers to tumors arising from the melanocyte lineage of the skin and other organs. Melanoma includes, for example, acryl-lentiginous melanoma, amelanotic melanoma, benign juvenile melanoma, Cloudman's melanoma, S91 melanoma, Harding-Passey melanoma, juvenile melanoma, lentigo maligna melanoma, malignant melanoma, nodular melanoma, subungal melanoma, and superficial spreading melanoma.

[0222] The term "lymphoma" refers to a group of cancers that affect the hematopoietic and lymphatic tissues, beginning in lymphocytes (blood cells found primarily in lymph nodes, spleen, thymus, and bone marrow). The two main types of lymphoma are non-Hodgkin's lymphoma and Hodgkin's disease. Hodgkin's disease accounts for approximately 15% of all diagnosed lymphomas. It is a cancer associated with Reed-Sternberg malignant B lymphocytes. Non-Hodgkin's lymphoma (NHL) can be classified based on how fast the cancer grows and the type of cells involved. There are high-grade and low-grade NHL. Based on the type of cells involved, there are B-cell and T-cell NHL. Exemplary B-cell lymphomas include small lymphocytic lymphoma, mantle cell lymphoma, follicular lymphoma, marginal zone lymphoma, extranodal (MALT) lymphoma, nodal (monocytoid B-cell) lymphoma, splenic lymphoma, diffuse large B-cell lymphoma, Burkitt's 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.

[0223] The term "leukemia" refers to progressive, malignant diseases of the blood-forming organs and is generally characterized by a distorted proliferation and development of leukocytes and their precursors in the blood and bone marrow. Exemplary leukemias include, for example, acute nonlymphocytic leukemia, chronic lymphocytic leukemia, acute granulocytic leukemia, chronic granulocytic leukemia, acute promyelocytic leukemia, adult T-cell leukemia, aleukemic leukemia, aleukocythemic leukemia, basophylic leukemia. leukemia), blast cell leukemia, bovine leukemia, chronic myelocytic leukemia, cutaneous leukemia, embryonal leukemia leukemia), eosinophilicleukemia, gross'leukemia, hairy cell leukemia, hemoblastic leukemia, hemocytoblastic leukemia. leukemia, histiocytic leukemia, stem cell leukemia, acute monocytic leukemia, leukopenic leukemia, lymphatic leukemia, lymphoblastic leukemia, lymphocytic leukemia, lymphogenous leukemia, lymphoid leukemia, lymphosarcoma cell leukemia, mast cell leukemia, megakaryocytic leukemia, micromyeloblastic leukemia, monocytic leukemia, myeloblastic leukemia, myeloid granulocytic leukemia, myelomonocytic leukemia, Naegeli leukemia, plasma cell leukemia These include myelocytic leukemia, plasmacytic leukemia, promyelocytic leukemia, Rieder cell leukemia, Schilling's leukemia, stem cell leukemia, subleukemia, and anaplastic cell leukemia.

[0224] Other cancers include, for example, multiple myeloma, neuroblastoma, breast cancer, ovarian cancer, lung cancer, rhabdomyosarcoma, primary thrombocytosis, primary macroglobulinemia, small cell lung tumor, primary brain tumor, gastric cancer, colon cancer, malignant pancreatic insulinoma, malignant carcinoid, premalignant skin lesions, testicular cancer, thyroid cancer, neuroblastoma, esophageal cancer, genitourinary cancer, malignant hypercalcemia, cervical cancer, endometrial cancer, and adrenal cortical carcinoma.

[0225] 4. Combination therapy

[0226] In another aspect, the present invention provides therapeutic compositions and combination therapies for enhancing antigen-specific T cell responses, reducing immunosuppression, and / or reducing tumor growth in a subject. The present invention includes compositions and therapeutic formulations, for example, comprising any of the exemplified antigen-binding molecules in combination with one or more additional therapeutic agents, and therapeutic methods of administering such combinations to a subject in need thereof. As used herein, the term "additional therapeutic agent" refers to any agent that can be used to treat a disease or disorder, and any method that can be used to treat a disease or disorder. For example, radiation therapy and surgery are considered "additional therapeutic agents" when used in combination with an antigen-binding molecule (e.g., an anti-A2aR antibody or an antigen-binding fragment thereof) described in the present invention.

[0227] In some embodiments, the additional therapeutic agent may be an A2aR antagonist other than the antigen-binding molecule (e.g., an anti-A2aR antibody or antigen-binding fragment thereof) described herein. Exemplary A2aR antagonists include, but are not limited to, AZD4635 (AstraZeneca), NIR178 (Novartis), AB928 (Arcus), CPI-444 (Corvus), EOS850 (iTeos), and MK-3814 (Merck Sharp and Dolme).

[0228] In one embodiment, the additional therapeutic agent can be administered in the form of an antibody or antibody fragment thereof, directed against another adenosine signaling pathway member, such as an A1aR antagonist, an A2bR antagonist, an A3R antagonist, a CD39 antagonist, a CD73 antagonist, or a combination thereof. Exemplary CD39 antagonists include, but are not limited to, exemplary anti-CD39 antibodies and antigen-binding sites thereof (described in U.S. Patent Nos. 10,738,128, 10,662,253, and 10,556,959). Exemplary small molecule CD73 antagonists include, but are not limited to, AB421, MEDI9447, and BMS-986179. Exemplary anti-CD73 antibodies and their antigen binding sites are described in Patent Nos. 10,766,966, 10,584,169, 10,556,968, and 10,167,343.

[0229] In some embodiments, the anti-A2aR antigen binding molecules (e.g., anti-A2aR antibodies or antigen-binding fragments thereof) described herein are administered together with an effective amount of one or more additional agents to stimulate an immune response and / or apoptosis in order to further enhance, stimulate or upregulate the immune response and / or apoptosis in a subject. Furthermore, the one or more other therapeutically active agents are administered before or after treatment with the anti-A2aR antibody.

[0230] In certain embodiments, the anti-A2aR antibodies described herein are administered in combination with or concurrently with one or more other active agents (e.g., anti-cancer antibodies or polypeptides, chemotherapeutic agents, and radiotoxic agents). In some other embodiments, the anti-A2aR antibodies described herein are administered in combination with or concurrently with standard cancer therapies (e.g., surgery or radiation).

[0231] Co-administration of the anti-A2aR antibodies with these active agents or therapeutic procedures can address clinical defects related to drug resistance, changes in the antigenicity of tumor cells that cause them to become unresponsive to antibodies, and toxicity (due to administration of low doses of one or more agents). A2aR inhibition is particularly well suited for use in combination with other refractory chemotherapy regimens. In these cases, the dose of the chemotherapy agent administered can be reduced while achieving enhanced efficacy (Mokyr et al, (1998) Cancer Research 58:5301-5304). The basic principle of inhibiting A2aR by radiation or chemotherapy is based on the cytotoxic effect of radiation and most chemotherapy compounds promoting cell death, which further results in increased levels of tumor antigens in the antigen presentation pathway. Other combination therapies that act additively or synergistically with A2aR inhibition through cell death are surgery and hormone deprivation or inhibition. Each of these protocols creates an additional source of tumor antigens in the host.

[0232] In some embodiments, the anti-A2aR antibodies described herein are linked to another active agent in the form of an immunoconjugate, immune complex, or fusion protein. Alternatively, the anti-A2aR antibodies may be administered separately from the other active agent, in which case the anti-A2aR antibodies and other antagonists are administered before, after, or simultaneously with the other active agent, or with other known treatments (e.g., other anti-cancer agents, radiation, etc.). Thus, the present invention provides compositions and methods for providing two or more anti-cancer agents that act additively or synergistically through different mechanisms to beneficially provide both cytotoxic and immunoprotective effects in human cancer cells.

[0233] For example, in some embodiments, the anti-A2aR antibodies described herein may be used in combination with other agents, such as, 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) polysaccharide inhibitors, and the like. The present invention may be combined with anti-cancer agents such as PARP inhibitors, Ras / MAPK pathway inhibitors, centrosome declustering agents, multikinase inhibitors, serine / threonine kinase inhibitors, tyrosine kinase inhibitors, VEGF / VEGFR inhibitors, taxanes or taxane derivatives, aromatase inhibitors, anthracyclines, microtubule targeting agents, topoisomerase poisons, inhibitors of molecular targets or enzymes (e.g., kinases or protein methyltransferases), cytidine analogs, or combinations thereof.

[0234] Exemplary alkylating agents include, but are not limited to, cyclophosphamide (Cytoxan, Neosar), chlorambucil (Leukeran), melphalan (Alkeran), carmustine (BiCNU), busulfan (Busulfex), lomustine (CeeNU), dacarbazine (DTIC-Dome), oxaliplatin (Eloxatin), carmustine (Gliadel), ifosfamide (Ifex), mechlorethamine (Mustargen), busulfan (Myleran), carboplatin (Paraplatin), cisplatin (CDDP, Platinol), temozolomide (Temodar), thiotepa (Thioplex), bendamustine (Treanda), or streptozocin (Zanosar).

[0235] Exemplary anthracycline antibiotics include, but are not limited to, doxorubicin (Adriamycin), liposomal doxorubicin (Doxil), mitoxantrone (Novantron), bleomycin (Blenoxane), daunorubicin (Cerubidine), liposomal daunorubicin (DaunoXome), dactinomycin (Cosmegen), epirubicin (Ellence), idarubicin (Idamycin), plicamycin (Mithracin), mitomycin (Mutamycin), pentostatin (Nipent), or valrubicin (Valstar).

[0236] Exemplary antimetabolites include fluorouracil (Adrucil), capecitabine (Xeloda), hydroxyurea (Hydrea), mercaptopurine (Purinethol), pemetrexed (Alimta), fludarabine (Fludara), nelarabine (Arranon), cladribine (Cladribine), and the like. 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).

[0237] Exemplary antidotes include, but are not limited to, amifostine (Ethyol) or mesna (Mesnex).

[0238] Exemplary interferons include, but are not limited to, interferon alpha-2b (Intron A) or interferon alpha-2a (Roferon-A).

[0239] Exemplary polyclonal or monoclonal antibodies include, but are not limited to, trastuzumab (Herceptin), ofatumumab (Arzerra), bevacizumab (Avastin), rituximab (Rituxan), cetuximab (Erbitux), panitumumab (Vectibix), tositumomab / iodine-131 tositumomab (Bexxar), alemtuzumab (Campath), ibritumomab (Zevalin, In-111, Y-90 Zevalin), gemtuzumab (Mylotarg), eculizumab (Soliris), or denosumab.

[0240] 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.

[0241] Exemplary HER2 inhibitors include, but are not limited to, trastuzumab (Herceptin), lapatinib (Tykerb) or AC-480.

[0242] Exemplary histone deacetylase inhibitors include, but are not limited to, vorinostat (Zolinza), valproic acid, romidepsin, entinostat, abexinostat, givinostat, and mocetinostat.

[0243] Exemplary hormones include tamoxifen (Soltamox, Nolvadex), raloxifene (Evista), megestrol (Megace), leuprolide (Lupron, Lupron Depot, Eligard, Viadur), fulvestrant (Faslodex), letrozole (Femara), triptorelin (Trelstar LA, Trelstar These include, but are not limited to, fluoxymesterone (Androxy, Halotestin), medroxyprogesterone (Provera, Depo-Provera), estramustine (Emcyt), flutamide (Eulexin), toremifene (Fareston), degarelix (Firmagon), nilutamide (Nilandron), abarelix (Plenaxis), or testolactone (Teslac).

[0244] Exemplary mitotic inhibitors include, but are not limited to, paclitaxel (Taxol, Onxol, Abraxane), docetaxel (Taxotere), vincristine (Oncovin, Vincasar PFS), vinblastine (Velban), etoposide (Toposar, Etopophos, VePesid), teniposide (Vumon), ixabepilone (Ixempra), nocodazole, epothilones, vinorelbine (Navelbine), camptothecin (CPT), irinotecan (Camptosar), topotecan (Hycamtin), amsacrine, or lamellarin D (LAM-D).

[0245] Exemplary phosphatidylinositol-3-kinase (PI3K) inhibitors include wortmannin (an irreversible inhibitor of PI3K), demethoxyviridin (a derivative of wortmannin), LY294002 (a reversible inhibitor of PI3K), BKM120 (Buparlisib), idelalisib (a PI3K-delta inhibitor), duvelisib (IPI-145, a PI3K-delta / gamma inhibitor), alpelisib (BYL719, an alpha-specific PI3K inhibitor), TGR 1202 (formerly known as RP5264, an oral PI3K-delta inhibitor), and copanlisib (BAY 80-6946, an inhibitor primarily of the PI3K-alpha / delta isoforms).

[0246] Exemplary Akt inhibitors include, but are not limited to, miltefosine, AZD5363, GDC-0068, MK2206, perifosine, RX-0201, PBI-05204, GSK2141795, and SR13668.

[0247] 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.

[0248] Exemplary proteasome inhibitors include, but are not limited to, bortezomib (PS-341), ixazomib (MLN 2238), MLN 9708, delanzomib (CEP-18770), carfilzomib (PR-171), YU101, oprozomib (ONX-0912), marizomib (NPI-0052), and disulfiram.

[0249] Exemplary PARP inhibitors include, but are not limited to, olaparib, iniparib, velaparib, BMN-673, BSI-201, AG014699, ABT-888, GPI21016, MK4827, INO-1001, CEP-9722, PJ-34, Tiq-A, Phen, PF-01367338, and combinations thereof.

[0250] 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 GDC-0973.

[0251] Exemplary centrosomal cluster disrupting agents include, but are not limited to, griseofulvin; noscapine, noscapine derivatives, such as brominated noscapine (e.g., 9-bromonoscapine), reduced bromonoscapine (RBN), N-(3-bromobenzyl)noscapine, aminonoscapine and their water-soluble derivatives; CW069; the phenanthridine-derived poly(ADP-ribose) polymerase inhibitor, PJ-34; N2-(3-pyridylmethyl)-5-nitro-2-furamide, N2-(2-thienylmethyl)-5-nitro-2-furamide and N2-benzyl-5-nitro-2-furamide.

[0252] Exemplary multikinase inhibitors include, but are not limited to, regorafenib, sorafenib (Nexavar), sunitinib (Sutent), BIBW 2992, E7080, Zd6474, PKC-412, motesanib, or AP24534.

[0253] Exemplary target serine / threonine kinase inhibitors include, but are not limited to, ruboxistaurin, eril / fasudil hydrochloride, flavopiridol, seliciclib (CYC202; Roscovitrine), SNS-032 (BMS-387032), Pkc412, bryostatin, KAI-9803, SF1126, VX-680, Azd1152, Arry-142886 (AZD-6244), SCIO-469, GW681323, CC-401, CEP-1347, or PD 332991.

[0254] Exemplary tyrosine kinase inhibitors include erlotinib (Tarceva), gefitinib (Iressa), imatinib (Gleevec), sorafenib (Nexavar), sunitinib (Sutent), trastuzumab (Herceptin), bevacizumab (Avastin), rituximab (Rituxan), lapatinib (Tykerb), cetuximab (Erbitux), panitumumab (Vectibix), evetinib (Evetin), erythropoie ... These include, but are not limited to, rolimus (Afinitor), alemtuzumab (Campath), gemtuzumab (Mylotarg), temsirolimus (Torisel), pazopanib (Votrient), dasatinib (Sprycel), nilotinib (Tasigna), vatalanib (Ptk787, ZK222584), CEP-701, SU5614, MLN518, XL999, VX-322, Azd0530, BMS-354825, SKI-606 CP-690, AG-490, WHI-P154, WHI-P131, AC-220, or AMG888.

[0255] Exemplary VEGF / VEGFR inhibitors include, but are not limited to, bevacizumab (Avastin), sorafenib (Nexavar), sunitinib (Sutent), ranibizumab, pegaptanib, or vandetanib.

[0256] Exemplary microtubule targeting agents include, but are not limited to, paclitaxel, docetaxel, vincristine, vinblastine, nocodazole, epothilones, and navelbine.

[0257] Exemplary topoisomerase poisons include, but are not limited to, teniposide, etoposide, adriamycin, camptothecin, daunorubicin, dactinomycin, mitoxantrone, amsacrine, epirubicin, and idarubicin.

[0258] Exemplary taxanes or taxane derivatives include, but are not limited to, paclitaxel and docetaxol.

[0259] Exemplary general chemotherapy, antitumor, and antiproliferative agents include altretamine (Hexalen), isotretinoin (Accutane, Amnesteem, Claravis, Sotret), tretinoin (Vesanoid), azacytidine (Vidaza), bortezomib (Velcade), asparaginase (Elspar), levamisole (Ergamisol), mitotane (Lysodren), procarbazine (Matulane), pegaspargase (Oncaspar), denileukin diftitox (ODT), and rituximab (Tregs). ntak), porfimer (Photofrin), aldesleukin (Proleukin), lenalidomide (Revlimid), bexarotene (Targretin), thalidomide (Thalomid), temsirolimus (Torisel), arsenic trioxide (Trisenox), verteporfin (Visudyne), mimosine (Leucenol), (1M tegafur-0.4M 5-chloro-2,4-dihydroxypyrimidine-1M potassium oxonate) or lovastatin.

[0260] In some embodiments, A2aR inhibition is performed in combination with CD3 stimulation (e.g., by co-incubation with cells expressing membrane CD3) before, simultaneously with, or after treatment with anti-A2aR antibodies. For example, in one embodiment, a method for enhancing antigen-specific T cell responses includes contacting T cells with anti-A2aR antibodies described herein and CD3-expressing cells, such that antigen-specific T cell responses are enhanced and A2aR-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 the antibody and / or increased cytokine production in the presence of the antibody. In a preferred embodiment, interleukin-2 and / or interferon-γ production by the antigen-specific T cells is enhanced.

[0261] In some embodiments, the anti-A2aR antibodies described herein can also be used in combination with bispecific antibodies that direct Fcα or Fcγ receptor-expressing effector cells to tumor cells (see, e.g., U.S. Patent Nos. 5,922,845 and 5,837,243). Such bispecific antibodies can be used to target two separate antigens. For example, anti-Fc receptor / anti-tumor antigen (e.g., Her-2 / neu) bispecific antibodies have been used to direct macrophages to the site of tumors. Such targeting can more effectively activate tumor-specific responses. The T cell arm of these responses can be enhanced by inhibition of A2aR. Alternatively, antigens can be delivered directly to DCs using bispecific antibodies that bind tumor antigens and dendritic cell-specific cell surface markers.

[0262] In all of the above methods, A2aR 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 presentation of tumor antigens.

[0263] In some embodiments, the additional therapeutic agent used in any of the above-mentioned methods of treatment, in the antigen binding molecule, or in the pharmaceutical composition is an immunostimulatory agent selected from the group consisting of: (a) an agent that blocks signaling of an inhibitory receptor (immune checkpoint) or its ligand on an immune cell (immune checkpoint inhibitor), or a nucleic acid encoding such an agent; (b) an agonist for a stimulatory receptor on an immune cell, or a nucleic acid encoding such an agonist; (c) a cytokine or a nucleic acid encoding a cytokine; (d) an oncolytic virus or a nucleic acid encoding an oncolytic virus; (e) a T cell expressing a chimeric antigen receptor; (f) a bispecific or multispecific antibody against a T cell, or a nucleic acid encoding such an antibody; (g) an anti-TGF-β antibody or a nucleic acid encoding such an antibody; (h) a TGF-β trap or a nucleic acid encoding such a trap; (i) a vaccine against a cancer-associated antigen comprising such an antigen or a nucleic acid encoding such an antigen; (j) a cell therapy; and (k) a combination thereof. In some embodiments, the additional therapeutic agent is an agent that blocks signaling of an inhibitory receptor or its ligand on an immune cell, or a nucleic acid encoding such an agent, and the inhibitory receptor or its ligand is selected from the group consisting of PD-1, PD-L1, TIGIT, CTLA-4, PD-L2, LAG-3, TIM-3, B7-H3, B7-H4, CD73, PVRIG / PVRL2, neuritin, BTLA, CECAM-1, CECAM-5, CE CAM6, IL-1R8, VISTA, LAIR1, LILRB1, LILRB2, LILRB3, LILRB4, LILRB5, CD47, SIRPa, CD200R, CD96, CD112R, 2B4, TGFβ-R, KIR, NKG2A, SEMA4D, Axl, MerTK, GAS6, TNFR2, GARP, CCR8, IDO, NOX2, SIGLEC7, SIGLEC15, and combinations thereof. In some embodiments, the additional therapeutic agent is an agonist for a stimulatory receptor on an immune cell, or a nucleic acid encoding such an agonist.The agonist is selected from the group consisting of OX40, CD2, CD3, CD7, CD16, CD27, CD28, CD30, CD40, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), 4-1BB (CD 137), GITR, BAFFR, HVEM, LIGHT, KG2C, SLAMF7, NKG2C, NKG2D, NKp46, NKp80, CD160, and combinations thereof. In some embodiments, the additional therapeutic agent is a cytokine or a nucleic acid encoding a cytokine selected from the group consisting of IL-2, IL-5, IL-7, IL-12, IL-15, IL-2I, and combinations thereof. In some embodiments, the additional therapeutic agent is an oncolytic virus or a nucleic acid encoding an oncolytic virus selected from herpes simplex virus, vesicular stomatitis virus, adenovirus, Newcastle disease virus, vaccinia virus, Maraba virus, and combinations thereof. In some embodiments, the additional therapeutic agent is a cell therapy. The cell therapy can include T cells, NK cells, or macrophages with a chimeric antigen receptor (CAR). In some embodiments, the cell therapy includes a bispecific or multispecific T cell targeting antibody.

[0264] As used herein, the term "immune checkpoint protein" refers to a receptor and / or its ligand expressed on an immune cell (e.g., a T cell). The binding of the ligand to the receptor reduces or inhibits the immune response of the immune cell. An immune checkpoint inhibitor is an agent that reduces or inhibits the binding of the ligand to the receptor.

[0265] As used herein, the term "stimulatory receptor of an immune cell" refers to a receptor on an immune cell. When the stimulatory receptor of an immune cell binds to its ligand, the immune response of the immune cell is enhanced.

[0266] In one embodiment, the present invention provides a method for treating a disease or disorder (e.g., cancer) in a subject. The method includes administering an antigen-binding molecule of the present invention, such as an anti-A2aR antibody or an antigen-binding fragment thereof, alone or in combination with a second one or more additional therapeutic agents to a subject, where the subject has previously been treated with a first one or more additional therapeutic agents. In one embodiment, the treatment with the first one or more additional therapeutic agents may show low efficacy in treating a disease in the subject. For example, the treatment with the first one or more therapeutic agents may be treatment with an anti-PD1 antibody for a subject that may show resistance. In some embodiments, the second one or more additional therapeutic agents are the same as the first one or more additional therapeutic agents. In some embodiments, the second one or more additional therapeutic agents are different from the first one or more additional therapeutic agents.

[0267] In some embodiments, the immune checkpoint inhibitor is an antibody that specifically interacts with an immune checkpoint. In some embodiments, the additional therapeutic agent comprises an immune stimulant. In some aspects, the immune checkpoint inhibitor is selected from the group consisting of an anti-PD-1 antibody (e.g., pembrolizumab or nivolumab), an anti-PD-L antibody (e.g., atezolizumab), 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.

[0268] In some embodiments, the additional therapeutic agent is an agent that inhibits the interaction of PD-1 and PD-L1. In some aspects, the additional therapeutic agent that inhibits the interaction of PD-1 and PD-L1 is selected from the group consisting of an antibody, a peptidomimetic, and a small molecule. In some aspects, the additional therapeutic agent that inhibits the interaction of PD-1 and PD-L1 is selected from the group consisting of pembrolizumab, nivolumab, atezolizumab, avelumab, durvalumab, BMS-936559, sintilimab, toripalimab, ticilelitumab, camrelizumab, embafolimab, sugemalimab, pemplimab, cadnilimab, sulfamonomethoxine, and sulfamethizole.

[0269] In some embodiments, the anti-A2aR antibodies are combined or co-administered with an immunogenic agent, non-limiting examples of which include cancer cells, tumor vaccines, and purified tumor antigens (including recombinant proteins, peptides, and carbohydrate molecules), oncolytic viruses, cells transfected with genes encoding immune-stimulating cytokines, and the like.

[0270] In one embodiment, the anti-A2aR antibody is administered together with an antigen of interest or an antigen known to be present in the subject being treated (e.g., a tumor-bearing or virus-bearing subject) to enhance antigen-specific immunity. When the anti-A2aR antibody is administered together with another agent, both of them can be administered separately or simultaneously.

[0271] In certain embodiments, the anti-A2aR antibodies described herein can be used to enhance antigen-specific immune responses by co-administration of any one or more of these antibodies with an antigen of interest (e.g., a vaccine). Thus, in one embodiment, a method for enhancing an immune response to an antigen in a subject comprises administering to the subject (i) the antigen and (ii) an antibody based on A2aR, such that an immune response to the antigen is enhanced in the subject. The antigen can be, for example, a tumor antigen, a viral antigen, a bacterial antigen, or an antigen derived from a pathogen. Non-limiting examples of such antigens include the antigens described above, such as the tumor antigens (or tumor vaccines) discussed above, or antigens derived from viruses, bacteria, or other pathogens.

[0272] In view of the benefits associated with a synergistic active agent composition, in one embodiment, the anti-A2aR antibody and the other active agent are each administered to a subject in need thereof at a sub-therapeutic dose compared to the dose used in monotherapy therewith.

[0273] In some embodiments, A2aR inhibition is combined with standard cancer treatments (e.g., surgery, radiation, and chemotherapy). In these cases, the dose of chemotherapy reagents administered may be reduced. It is believed that the combination of A2aR inhibition and chemotherapy can enhance apoptosis and increase tumor antigen presentation for cytotoxic immunity. Other synergistic combination therapies include the combination of A2aR inhibition with radiation therapy, surgery, or hormone deprivation or inhibition. Each of these protocols creates a source of tumor antigens in the host.

[0274] The additional therapeutic agent may be administered prior to, simultaneously with, or after administration of an antigen-binding molecule according to the present invention (for the purposes of this disclosure, such an administration regimen is considered a "combination" of the antigen-binding molecule with an additional therapeutically active ingredient).

[0275] The present invention includes pharmaceutical compositions prepared together with an antigen-binding molecule according to the present invention and one or more additional therapeutic agents described herein.

[0276] V. Nucleic acids and host cells for expressing anti-A2aR antibodies

[0277] In another aspect, the invention provides nucleic acids encoding antigen-binding molecules (e.g., anti-A2aR antibodies or antigen-binding fragments thereof) according to the invention, and expression vectors comprising such nucleic acids. In some embodiments, the nucleic acid encodes the HCVR and / or LCVR fragment of an antibody or fragment according to embodiments described herein, or encodes any other antibody and antibody fragment described herein.

[0278] DNA encoding the antigen-binding sites in a monoclonal antibody can be isolated and sequenced from hybridoma cells by conventional methods (e.g., using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of a monoclonal antibody). Alternatively, the amino acid sequence from the immunoglobulin of interest can be determined by direct protein sequencing, and appropriate encoding nucleotide sequences can be designed according to a universal codon table. In other cases, the nucleotide and amino acid sequences of the antigen-binding site or other immunoglobulin sequences, including constant regions, hinge regions, etc., can be obtained from public sources well known in the art.

[0279] Expression vectors may be used to synthesize antibodies of this disclosure in in vitro cultured cells or may be administered directly to a patient to express the antibodies of this disclosure in vivo or ex vivo. As used herein, "expression vector" refers to a viral or non-viral vector that contains a polynucleotide encoding one or more of the antibodies of this disclosure in a form suitable for expressing the polynucleotide in a host cell for antibody production or direct administration as a therapeutic agent.

[0280] A nucleic acid sequence is said to be "operably linked" to another nucleic acid sequence when the former is in a functional relationship with the latter. For example, DNA of a presequence or signal peptide is operably linked to DNA of a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide. A promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence. Or, a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, "operably linked" means that the DNA sequences being linked are contiguous, and in the case of a signal peptide, contiguous and in reading phase. Enhancers, however, need not be contiguous. Linkage is accomplished by ligation at convenient restriction sites. If such sites do not exist, synthetic oligonucleotide adaptors or linkers can be used in accordance with conventional methods.

[0281] The nucleic acid sequence for expressing the antibody of the present disclosure generally comprises an N-terminal signal peptide sequence that is removed from the mature protein.Since the signal peptide sequence can affect the expression level, the polynucleotide can encode any one of a variety of different N-terminal signal peptide sequences.It should be understood by those skilled in the art that the design of the expression vector can depend on factors such as the selection of the host cell to be transformed, the expression level of the protein of interest, etc.

[0282] The above-mentioned "regulatory sequence" refers to a DNA sequence necessary for the expression of an operably linked coding sequence in one or more host organisms. The term "regulatory sequence" includes promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cells, or those that direct expression of a nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). Expression vectors generally include sequences for transcription termination, and may further include one or more factors that positively affect mRNA stability.

[0283] The expression vector contains one or more transcription control elements, such as a promoter and / or enhancer, to direct the expression of the antibody of the present disclosure. A promoter comprises a DNA sequence that functions to initiate transcription from a relatively fixed position relative to the transcription start site. A promoter contains core elements required for basic interaction between RNA polymerase and transcription factors, and can function in cooperation with other upstream elements and response elements.

[0284] The term "promoter" as used herein should be interpreted broadly to include, for example, transcriptional regulators (TREs) from genomic genes or chimeric TREs thereof (including a TATA box or initiator element for accurate transcription initiation), and to include or not include additional TREs (i.e., upstream activating sequences, transcription factor binding sites, enhancers, and silencers) that regulate the activation or repression of genes operably linked thereto in response to developmental and / or external stimuli, as well as trans-acting regulatory proteins or nucleic acids. A promoter may contain genomic fragments or may contain chimeras in which one or more TREs are combined.

[0285] Preferred promoters are those that can direct high levels of expression in the target cells of interest. Promoters include constitutive promoters (e.g., HCMV, SV40, elongation factor-1α (EF-1α) promoters) or promoters that are preferentially expressed in a particular cell type of interest. Enhancers generally refer to DNA sequences that function away from the transcription start site and may be located at the 5' or 3' end of the transcription unit. Additionally, enhancers can be found within introns and within coding sequences. Enhancers usually have a length of 10-300 bp and function in cis. Enhancers function to increase and / or regulate the transcription of nearby promoters. Preferred enhancers are those that direct high levels of expression in antibody-producing cells. Cell- or tissue-specific transcriptional regulators (TREs) can be incorporated into the expression vector to restrict expression to the desired cell type. Pol III promoters (H1 or U6) are particularly useful for expressing shRNAs in which siRNAs are expressed. Expression vectors can be designed to facilitate expression of the antibodies of the disclosure in one or more cell types.

[0286] siRNA is a double-stranded RNA that can be engineered to induce sequence-specific post-transcriptional gene silencing of mRNA.Synthetically produced siRNA structurally mimics the type of siRNA that is normally processed in cells by the enzyme Dicer.When expressed from an expression vector, the expression vector is engineered to transcribe short double-stranded hairpin-like RNA (shRNA) that is processed into targeting siRNA inside the cell.Synthetic siRNA and shRNA can be designed using well-known algorithms and synthesized using conventional DNA / RNA synthesizers.

[0287] To co-express the individual chains of the antibody of the present disclosure, appropriate splice donor and splice acceptor sequences can be incorporated to express two products. Alternatively, an internal ribosome binding sequence (IRES) or 2A peptide sequence can be used to express multiple products from one promoter. An IRES provides a structure to which ribosomes can bind that does not necessarily have to be at the 5' end of the mRNA. Thus, an IRES can direct ribosomes to begin translation at a second start codon in the mRNA, allowing multiple polypeptides to be produced from a single mRNA. 2A peptides contain short sequences that mediate co-translational self-cleavage of peptides upstream and downstream of the 2A site, allowing two different proteins to be produced in equimolar amounts from a single transcript. CHYSEL is a non-limiting example of a 2A peptide that causes translating eukaryotic ribosomes to release the growing polypeptide chain they are synthesizing without dissociating from the mRNA. As the ribosomes continue translation, they produce a second polypeptide.

[0288] Expression vectors may include viral or non-viral vectors. Viral vectors can be derived from adeno-associated viruses (AAV), adenoviruses, herpes viruses, vaccinia viruses, polio viruses, pox viruses, retroviruses (including lentiviruses such as HIV-1 and HIV-2), Sindbis virus and other RNA viruses, alphaviruses, astroviruses, coronaviruses, orthomyxoviruses, papovaviruses, paramyxoviruses, parvoviruses, picornaviruses, togaviruses, and the like. Non-viral vectors are simply "naked" expression vectors that are not packaged with viral derived components (e.g., capsid or envelope).

[0289] In some cases, these vectors may be engineered to target specific diseases or cell populations using targeting properties inherent or engineered into the viral vector. Specific cells are "targeted" for delivery and expression of polynucleotides. Thus, in this case, the term "targeting" may be based on the use of endogenous or heterologous binding agents in the form of capsids, envelope proteins, antibodies for delivery to specific cells, the use of tissue-specific regulators to restrict expression to specific cell subsets, or both.

[0290] In some embodiments, expression of the antibody chains is under the control of regulatory sequences, such as tissue-specific or ubiquitous promoters. In some embodiments, a ubiquitous promoter (e.g., a CMV promoter, a CMV-chicken β-actin hybrid (CAG) promoter, a tissue-specific or tumor-specific promoter) controls expression of the heavy or light chain or single chain derivatives of a particular antibody.

[0291] Non-viral expression vectors can be utilized for non-viral gene transfer by directly injecting naked DNA or encapsulating antibody-encoding polynucleotides in liposomes, microparticles, microcapsules, virus-like particles, or erythrocyte ghosts. Such compositions can be further linked to targeting domains by chemical conjugation to facilitate targeted delivery and / or introduction of the nucleic acid into desired target cells. Additionally, plasmid vectors can be incubated with synthetic gene transfer molecules (e.g., polymeric DNA-binding cations such as polylysine, protamine, and albumin) and can be linked to cell targeting ligands (e.g., asialoorosomucoid, insulin, galactose, lactose, or transferrin).

[0292] Alternatively, naked DNA can be used. The efficiency of uptake of naked DNA can be improved by compression or the use of biodegradable latex beads. Such delivery can be further improved by treating the beads to increase their hydrophobicity, facilitating the disruption of endosomes and release of the DNA into the cytoplasm.

[0293] 6. Method for producing anti-A2aR antibody

[0294] In another aspect, the invention provides a host cell transformed with a nucleic acid or expression vector encoding an anti-A2aR HCVR and / or LCVR, which may be a bacterial or eukaryotic cell capable of expressing the nucleic acid or expression vector encoding the anti-A2aR HCVR and / or LCVR, or any of the other co-administered antibodies or antagonists described herein.

[0295] In other aspects, methods of making the antibodies of the disclosure include culturing host cells transformed with a nucleic acid or expression vector encoding an anti-A2aR HCVR and / or LCVR under conditions that allow production of the antibody or fragment, and purifying the antibody from the cells.

[0296] In another aspect, the invention provides a method for producing an antibody, comprising culturing cells that transiently or stably express one or more constructs encoding one or more polypeptide chains in the antibody, and purifying the antibody from the cultured cells. Any cell capable of producing a functional antibody can be used. In a preferred embodiment, the cell expressing the antibody is a eukaryotic or mammalian cell, preferably a human cell. Cells of various tissue cell types can be used to express the antibody. In another embodiment, the cell is a yeast cell, an insect cell, or a bacterial cell. Preferably, the antibody-producing cell is stably transformed with a vector expressing the antibody.

[0297] One or more expression vectors encoding the heavy or light chains of the antibody can be introduced into the cells by any conventional method, such as, for example, naked DNA techniques, cationic lipid-mediated transfection, polymer-mediated transfection, peptide-mediated transfection, viral-mediated infection, physical or chemical agents or treatments, electroporation, etc. Furthermore, the cells can be transfected with one or more expression vectors for expressing the antibody together with a selection marker that facilitates the selection of stably transformed clones expressing the antibody. The antibodies produced by such cells can be collected and / or purified according to techniques known in the art, such as centrifugation, chromatography, etc.

[0298] Examples of suitable selection markers for mammalian cells include dihydrofolate reductase (DHFR), thymidine kinase, neomycin, neomycin analog G418, hygromycin and puromycin. When such selection markers are successfully introduced into a mammalian host cell, the transformed mammalian host cell can survive when placed under selective pressure. There are two widely used selection schemes. The first scheme is based on the use of mutant cell lines that lack the ability to grow independent of supplemented media and the metabolism of the cells. Two illustrative examples are CHO DHFR, thymidine kinase, neomycin analog G418, hygromycin and puromycin. - These include mouse LTV cells and mouse LTV cells. These cells cannot grow without the addition of nutrients such as thymidine and hypoxanthine. These cells lack certain genes required for a complete nucleotide synthesis pathway and therefore cannot survive unless the missing nucleotides are provided in a supplemented medium. As an alternative to supplementing the medium, an intact DHFR or TK gene can be introduced into cells lacking the corresponding gene, thus altering their growth requirements. Individual cells that have not been transformed with the DHFR or TK genes will not be able to survive in unsupplemented medium.

[0299] The second scheme, dominant selection, refers to a selection scheme used for any cell type and does not require the use of mutant cell lines. In these schemes, a drug is usually used to stop the growth of the host cells. The cells with the new gene express a protein that conveys drug resistance and survive the selection. Examples of such dominant selection include the use of drugs such as neomycin, mycophenolic acid, or hygromycin. Three examples use bacterial genes under eukaryotic control to convey resistance to the appropriate drug G418 or neomycin (geneticin), xgpt (mycophenolic acid), or hygromycin, respectively. Other drugs include the neomycin analog G418 and puromycin.

[0300] Exemplary antibody-expressing cells include human T-cell leukemia cells (Jurkat), human embryonic kidney cells (HEK)293, Chinese hamster ovary (CHO) cells, mouse WEHI fibrosarcoma cells, as well as unicellular protozoan species (e.g., Leishmania tarentolae). Additionally, stably transformed antibody-producing cell lines can be generated using primary cells immortalized with c-myc or other immortalizing agents.

[0301] In one embodiment, the cell line comprises a stably transformed Leishmania cell line (e.g., Leishmania tarentolae). Leishmania is known to provide a robust and fast-growing unicellular host for high-level expression of eukaryotic proteins that exhibit mammalian-type glycosylation patterns. Commercially available Leishmania eukaryotic expression kits are available (Jena Bioscience GmbH, Jena, Germany).

[0302] In some embodiments, the cell lines express at least 1 mg, at least 2 mg, at least 5 mg, at least 10 mg, at least 20 mg, at least 50 mg, or at least 100 mg of antibody per liter of culture medium.

[0303] Antibodies according to the invention can be cultured and maintained in any suitable medium (e.g., RPMI, DMEM, and AIM V®) and isolated from antibody-expressing cells. Antibodies can be purified using conventional protein purification methods (e.g., affinity purification, chromatography, etc.), including the use of Protein A or Protein G immunoaffinity purification. In some embodiments, the antibodies are designed to be secreted into the culture supernatant and isolated from the supernatant.

[0304] VII. Pharmaceutical Compositions and Administration Methods

[0305] In one aspect, the pharmaceutical composition of the present invention comprises an antigen-binding molecule described herein (e.g., an A2aR antibody or antigen-binding fragment thereof) and a pharma- ceutically acceptable carrier. In some other embodiments, the A2aR antibody or antigen-binding fragment thereof is administered in combination with a pharma- ceutically acceptable carrier. As described above, the anti-A2aR 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.

[0306] The present invention provides a pharmaceutical composition comprising an antigen-binding molecule according to the present invention. The pharmaceutical composition according to the present invention is formulated with suitable carriers, excipients, and other agents capable of improving transport, delivery, tolerance, etc. A large number of suitable formulations are described in formularies known to all pharmaceutical chemists (Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA). These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic)-containing vesicles (e.g., LIPOFECTIN, 1997, 19 ...0, 1990, 1991, 1992, 1993, 1994, 1 TMExcipients for parenteral formulations include, for example, glycerols, glycerols, glycerols, sorbitol ...

[0307] The dose of the antigen-binding molecule administered to a patient may vary depending on the age and physique of the patient, the disease or disorder of interest, the route of administration, and the like. A preferred dose is generally calculated according to body weight or body surface area. When used to treat an adult patient, the bispecific antigen-binding molecule according to the present invention is advantageously administered intravenously at a single dose of about 0.01 to about 20 mg / kg body weight, more preferably at a single dose of about 0.02 to about 7 mg / kg body weight, about 0.03 to about 5 mg / kg body weight, or about 0.05 to about 3 mg / kg body weight. Depending on the severity of the condition, the frequency and duration of treatment can be adjusted. The effective dose and schedule for administering the bispecific antigen-binding molecule can be determined empirically. For example, the progress of the patient can be monitored by periodic evaluation, and the dose adjusted accordingly. Furthermore, interspecies scaling of doses can be performed using methods well known in the art (e.g., Mordenti et al., 1991, Pharmaceut. Res. 8:1351).

[0308] A variety of delivery systems are known and may be used to administer the pharmaceutical compositions of the present invention, including, for example, encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing mutant viruses, and receptor-mediated endocytosis (see, for example, Wu et al., 1987, J. BioI. Chem. 262:4429-4432). Methods of introduction include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The compositions may be administered by any convenient route, such as, for example, infusion or bolus administration, absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal mucosa, intestinal mucosa, etc.), and may be administered together with other bioactive agents. Administration may be systemic or local.

[0309] The pharmaceutical composition of the present invention can be delivered subcutaneously or intravenously using a standard needle and syringe. Moreover, for subcutaneous delivery, a pen delivery device can be easily applied to deliver the pharmaceutical composition of the present invention. Such a pen delivery device can be reusable or disposable. Reusable pen delivery devices generally utilize a replaceable cartridge containing the pharmaceutical composition. Once the pharmaceutical composition in the cartridge is all administered and the cartridge is empty, the empty cartridge can be easily discarded and replaced with a new cartridge containing the pharmaceutical composition. The pen delivery device can then be reused. Disposable pen delivery devices do not have a replaceable cartridge. Conversely, disposable pen delivery devices are pre-filled with the pharmaceutical composition held in a reservoir within the device. Once the reservoir is empty of the pharmaceutical composition, the entire device is discarded.

[0310] 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 other embodiments, polymeric materials can be used (see Medical Applications of Controlled Release, Langer and Wise (eds.), 1974, CRC Pres., Boca Raton, Florida). In still other embodiments, the controlled release system can be placed close to 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 described in the review by Langer, 1990, Science 249:1527-1533.

[0311] The injections include dosage forms such as intravenous, subcutaneous, intradermal and intramuscular injections, drip infusions, etc. These injections can be prepared by known methods. For example, the injections can be prepared by dissolving, suspending or emulsifying the above-mentioned antibody or its salt in a sterile aqueous or oily medium for general injection. The aqueous medium for injection includes, for example, physiological saline, isotonic solutions containing glucose and other adjuvants, and may be used in combination with a suitable solubilizing agent such as alcohols (e.g., ethanol), polyalcohols (e.g., propylene glycol, polyethylene glycol), nonionic surfactants (e.g., polysorbate 80, HCO-(polyoxyethylene (50 mol)-hydrogenated castor oil adduct)), etc. As the oily medium, for example, sesame oil, soybean oil, etc. are used, and may be used in combination with a solubilizing agent such as benzyl benzoate, benzyl alcohol, etc. The prepared injection is preferably filled into a suitable ampoule.

[0312] It is particularly preferred that the above-mentioned pharmaceutical composition for oral or parenteral use is prepared in a dosage form of a unit dose suitable for the dose of the active ingredient. Such dosage forms of unit doses include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc. The content of the antibody is generally about 5 to about 500 mg per dosage form in a unit dose. In particular, in the case of injections, the antibody is preferably contained in an amount of about 5 to about 100 mg, and in the case of other dosage forms, the antibody is preferably contained in an amount of about 10 to about 250 mg.

[0313] In another aspect, a method for treating a cell proliferative disorder (e.g., cancer, a chronic infection, or an immunodeficiency disorder) comprises administering to a subject in need thereof a pharmaceutical composition comprising an anti-A2aR antibody or antigen-binding fragment thereof described herein in combination with a pharma- ceutically acceptable carrier. In some embodiments, the method restores, enhances, or potentiates lymphocyte activity in a subject in need thereof. In a preferred embodiment, the antibody or fragment is a human or humanized anti-A2aR antibody that reduces or abolishes signaling through A2aR.

[0314] In some embodiments, administration of the pharmaceutical composition increases lymphocyte (e.g., T cell) activity in patients with diseases that benefit from increased lymphocyte activity, or in patients with diseases caused or characterized by immunosuppression, immunosuppressive cells, or adenosine produced by CD4 T cells, CD8 T cells, B cells, etc. The methods described herein are particularly useful, for example, in patients with solid tumors in which the tumor microenvironment (and adenosine production therein) is suspected to contribute to lack of recognition by the immune system (immune escape). Tumors can be characterized, for example, by immune cells, such as CD4 T cells, CD8 T cells, T-regs, B cells, etc., that express (or overexpress) A2aR.

[0315] In some embodiments, the methods and compositions are utilized for the treatment of various cancers and other proliferative diseases. These methods function to reduce adenosine levels that can inhibit the anti-tumor activity of lymphocytes, and are therefore applicable to a wide range of cancers, particularly solid tumors, where adenosine in the tumor microenvironment is known to suppress anti-tumor immune responses.

[0316] Non-limiting examples of cancers that may be treated using an antigen-binding molecule (e.g., an anti-A2aR antibody or an antigen-binding fragment thereof) according to the present invention include, for example, liver cancer, bone cancer, pancreatic cancer, skin cancer, head and 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, pediatric solid tumors, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, renal pelvis cancer, central nervous system cancer, and / or pulmonary arterial cancer. and malignancies including, but not limited to, neoplasms of the central nervous system (CNS), primary CNS lymphomas, tumor angiogenesis, spinal axis tumors, brain stem gliomas, pituitary adenomas, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, cancers induced by environmental factors (including asbestos induced cancers), hematological malignancies including multiple myeloma, B-cell lymphoma, Hodgkin's lymphoma / primary mediastinal B-cell lymphoma, non-Hodgkin's lymphoma, acute myeloid lymphoma, chronic myeloid leukemia, chronic lymphoid leukemia, follicular lymphoma, diffuse large B-cell lymphoma, Burkitt's lymphoma, immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma, mantle cell lymphoma, acute lymphoblastic leukemia, mycosis fungoides, anaplastic large cell lymphoma, T-cell lymphoma, and precursor T-lymphoblastic lymphoma, or any combination of such cancers. The present disclosure is also applicable to the treatment of metastatic cancer. Patients can be tested or selected for one or more of the above clinical features before, during, or after treatment.

[0317] In one embodiment, the anti-A2aR antibody has at least an EC 50 , optional EC 70 , optionally substantive EC 100 In one embodiment, the anti-A2aR antibody is administered in an amount effective to achieve and / or maintain (e.g., for 1, 2, 3, 4 weeks and / or until a subsequent administration of the antigen-binding compound) a blood concentration of at least the EC 50 , optional EC 70 , optionally substantive EC 100 In one embodiment, an active amount of an anti-A2aR antibody is an amount effective to achieve an EC 50 , optional EC 70 , optionally substantive EC 100 is an amount effective to achieve (or maintain)

[0318] Optionally, in one embodiment, as opposed to an antibody that depletes A2aR-expressing tumor cells by ADCC (e.g., can exert its full efficacy at concentrations equivalent to or significantly lower than those that result in receptor saturation), the anti-A2aR antibody is primarily a blocker (having no substantial Fcγ receptor-mediated activity) and is administered in an amount effective to neutralize A2aR enzymatic activity for a desired period of time (e.g., one week, two weeks, one month) until the next consecutive administration of the anti-A2aR antibody.

[0319] In one embodiment, the anti-A2aR antibody is administered to an individual at least EC 50 , optional EC 70 , optionally substantive EC 100In one embodiment, the amount of anti-A2aR antibody is administered in an amount effective to achieve and / or maintain (for 1, 2, 3, 4 weeks and / or until subsequent administration of an anti-A2aR antibody) a blood concentration of EC 200 / mL or more for inhibiting A2aR-mediated catabolism of AMP to adenosine in the vascular tissue of the individual. 50 , optional EC 70 , optionally substantive EC 100 is an amount effective to achieve (or maintain)

[0320] In one embodiment, the invention provides a method for treating or preventing cancer in an individual, comprising administering to the diseased individual an amount of an anti-A2aR antibody that can achieve or maintain, over a predetermined period of time, a circulating concentration (e.g., as assessed in PBMCs) that is higher than the concentration required for 50%, 70% or complete (e.g., 90%) receptor saturation of circulating A2aR-expressing cells in an extravascular tissue of interest (e.g., a tumor or tumor environment). Optionally, the concentration achieved is at least 20%, 50% or 100% higher than the concentration required for specific receptor saturation.

[0321] In one embodiment, the present invention provides a concentration that can be achieved or maintained over a period of time in the circulation, and optionally in an extravascular tissue of interest (e.g., a tumor or tumor environment), i.e., the EC 50 , optional EC 70 or optional EC 100 The present invention provides a method for treating or preventing cancer in an individual, comprising administering to the individual an anti-A2aR antibody in an amount that is greater than the EC 50 , optional EC 70 or optional EC 100 at least 20%, 50% or 100% higher than

[0322] In any embodiment, for example, the antibody has an EC 50 , optional EC 70or optional EC 100 is 0.5 to 100 ng / ml, optionally 1 to 100 ng / ml, optionally 30 to 100 ng / ml, for example, about 30 to 90 ng / ml. For example, EC 50 may be about 30, 37, 39, 43, 57, 58, 61, 62, 90, 95, 143 ng / ml.

[0323] The EC50 for neutralizing the enzymatic activity of A2aR using an anti-A2aR antibody may be, for example, about 0.01 μg / ml to 1 μg / ml, optionally 0.1 μg / ml to 10 μg / ml, optionally 0.1 μg / ml to 1 μg / ml. For example, EC 50 may be about 0.1 μg / ml, about 0.2 μg / ml, or about 0.3 μg / ml. Thus, the anti-A2aR antibody is administered in an amount to achieve and / or maintain a blood concentration of, for example, at least 0.1 μg / ml, optionally at least 0.2 μg / ml, optionally at least 1 μg / ml, or optionally at least 2 μg / ml.

[0324] When targeting tissues outside the vasculature (e.g., the tumor environment in the treatment of solid tumors), doses generally about 10-fold higher will be required compared to doses that provide corresponding concentrations in the circulation. Doses of anti-A2aR antibodies administered to achieve (and / or maintain) circulating (blood) concentrations of about 1 μg / ml, 2 μg / ml, 10 μg / ml, or 20 μg / ml are expected to achieve (and / or maintain) extravascular tissue (e.g., tumor tissue) concentrations of about 0.1 μg / ml, 0.2 μg / ml, 1 μg / ml, 2 μg / ml, respectively.

[0325] In one embodiment, the anti-A2aR antibody is administered in an amount to achieve and / or maintain a tissue (e.g., tumor milieu) concentration of, for example, at least 0.1 μg / ml, optionally at least 0.2 μg / ml, optionally at least 1 μg / ml, or optionally at least 2 μg / ml. The antibody is administered in an amount to achieve and / or maintain a blood concentration of, for example, at least about 1 μg / ml, 2 μg / ml, 10 μg / ml, or 20 μg / ml (e.g., 1-100 μg / ml, 10-100 μg / ml, 1-50 μg / ml, 1-20 μg / ml, or 1-10 μg / ml). Dosage can be adjusted to maintain a desired concentration within a predetermined period of time following administration (e.g., 1, 2, 3, 4 weeks, etc.).

[0326] In some embodiments, the amount of anti-A2aR antibody is at least an EC50 concentration for neutralization of the enzymatic activity of A2aR in blood (serum) or extravascular tissue (e.g., the tumor environment). 70 or EC 100 The antibody is administered in an amount to achieve and / or maintain a blood or extravascular tissue (e.g., tumor environment) concentration of, for example, at least about 1 μg / ml, 2 μg / ml, 10 μg / ml, or 20 μg / ml.

[0327] EC of the specified A2aR antibody 50、 EC 70 and E.C. 100 The EC value can be assessed, for example, in a cellular assay for neutralizing A2aR. 50 " refers to the concentration of anti-A2aR antibody that produces 50% of the maximum response or effect with respect to neutralization of the enzyme activity of A2aR. 70 " refers to the concentration of anti-A2aR antibody that produces 70% of the maximum response or effect. "EC 100 " refers to the effective concentration of anti-A2aR antibody that produces a maximal response or effect with respect to neutralizing such enzyme activity. In certain embodiments, depending on the context, the EC 50 , E.C. 70 or EC 100and IC20 are used to reflect that an antigen-binding molecule (e.g., an anti-A2aR antibody or an antigen-binding fragment thereof) inhibits the activity of A2aR, respectively. 50 ,I C 70 , or IC 100 It is referred to as IC. xx refers to the concentration of a drug required to inhibit a biological process by xx%.

[0328] In some embodiments, particularly in the case of treatment of solid tumors, the concentration achieved is at least the EC 50、 Optionally about EC100 or at least about EC 100 The concentration is designed to be in tissues (outside the vasculature, e.g., within a tumor or tumor environment) corresponding to:

[0329] In one embodiment, the amount of anti-A2aR antibody is 1-20 mg / kg body weight, hi one embodiment, the amount is administered to an individual weekly, biweekly, monthly or bimonthly.

[0330] In one embodiment, a method for treating cancer in a subject in need thereof comprises administering to an individual an effective amount of an anti-A2aR antibody of the present disclosure for at least one administration cycle (optionally at least two, three, four or more administration cycles), wherein said cycle is eight weeks or less in duration, and wherein in each of said at least one cycle, said anti-A2aR antibody is administered once, twice, three or four times at a dose of 1-20 mg / kg body weight. In one embodiment, said anti-A2aR antibody is administered by intravenous infusion.

[0331] Suitable treatment protocols for treating a subject (e.g., a human) include, for example, administering to the patient an amount of an anti-A2aR antibody disclosed herein, where the method includes at least one dosing cycle comprising administering the anti-A2aR antibody in at least one dose. Optionally, the anti-A2aR antibody is administered at least 2, 3, 4, 5, 6, 7, or 8 times. In one embodiment, the dosing cycle is 2 to 8 weeks.

[0332] In one embodiment, a method for treating or preventing a disease (e.g., cancer, solid tumor, hematological tumor) in an individual comprises administering to the individual at least one dosing cycle of an anti-A2aR antibody for neutralizing enzymatic activity of A2aR, the dosing cycle comprising at least a first and a second (and optionally a third, fourth, fifth, sixth, seventh, and / or eighth or more) administrations of the anti-PD-L1 antibody, wherein the anti-A2aR antibody is administered in an amount effective to achieve or maintain, between two successive doses, a blood (serum) concentration of the anti-PD-L1 antibody within a range of at least 0.1 μg / ml, at least 0.2 μg / ml, at least 1 μg / ml, at least 2 μg / ml, at least 10 μg / ml, at least 20 μg / ml, 1-100 μg / ml, 1-50 μg / ml, 1-20 μg / ml, 1-10 μg / ml, or between any of the aforementioned concentrations.

[0333] In one embodiment, a particular continuous blood level is maintained, where the blood level does not substantially fall below a particular blood level for a particular period of time (e.g., between two administrations of the antibody, for a number of weeks, 1 week, 2 weeks, 3 weeks, 4 weeks). In other words, although the blood level may vary during a specified period of time, the particular blood level maintained represents a minimum or "trough" level.

[0334] In one embodiment, a therapeutically active amount of an anti-A2aR antibody is one that achieves (at least) EC50 / mL in blood and / or tissues to neutralize enzymatic activity of A2aR for at least about one week, about two weeks, or about one month after administration of the antibody. 50 Concentration, optional EC 70 Concentration, optional EC 100 The amount of antibody is sufficient to provide a concentration.

[0335] Prior to or during a course of treatment with an anti-A2aR antibody of the present disclosure, cellular expression levels of A2aR, CD39 and / or CD73, percentage of A2aR-expressing cells, CD39-expressing cells and / or CD73-expressing cells; and / or levels of adenosine, ADP and / or AMP can be assessed in and / or near the patient's tumor to determine whether the patient is suitable for and likely to respond to treatment. Increased levels or expression can indicate that the individual is suitable for (e.g., likely to benefit from) treatment with an anti-A2aR antibody of the present disclosure.

[0336] In some embodiments, assessing the expression levels of A2aR, CD39 and / or CD73 and the concentrations of adenosine, ADP and / or AMP in and / or near the tumor of a patient includes obtaining from the subject a biological sample of tissue from a cancer patient, e.g., human tissue selected from the group consisting of cancer tissue, tissue proximal to or in the periphery of the cancer, cancer adjacent tissue, adjacent non-neoplastic tissue or adjacent normal tissue, and the expression levels of A2aR, CD39 and / or CD73 and the concentrations of adenosine, ADP and / or AMP in the tissue. The expression levels or nucleotide concentrations obtained from the patient can be compared to a reference level (e.g., a level in a healthy individual).

[0337] A decrease in the levels of adenosine, ADP and / or AMP after administration (or administration of the antibody) compared to levels before treatment (or administration of the antibody) may indicate that the individual is benefiting from treatment with an anti-A2aR antibody of the present disclosure (including, but not limited to, an antibody that inhibits substrate-bound A2aR). Optionally, if the patient is benefiting from treatment with the anti-A2aR antibody, the method may further include administering to the patient an additional dose of an anti-A2aR antibody, alone or in combination with another active agent (e.g., continuing the treatment).

[0338] From the above, in one embodiment, the method comprises the steps of (a) determining the expression levels of A2aR, CD39 and / or CD73 and / or the concentrations of adenosine, ADP and / or AMP in the tumor environment, optionally within the tumor and / or adjacent tissue, and, upon determining that the tumor environment exhibits increased levels of A2aR, CD39, CD73, adenosine, ADP and / or AMP compared to their corresponding reference levels, (b) administering an anti-A2aR antibody to the individual.

[0339] In certain embodiments, determining the levels of A2aR, CD39, CD73, adenosine, ADP and / or AMP within the tumor environment includes obtaining a biological sample from a subject that contains cancer tissue and / or tissue proximal to or in the periphery of the cancer (e.g., cancer-adjacent tissue, adjacent non-neoplastic tissue or adjacent normal tissue) and detecting the level and / or relative percentage of A2aR-expressing cells, CD39-expressing cells and / or CD73-expressing cells, and / or the levels of adenosine, ADP and / or AMP. A2aR-expressing cells, CD39-expressing cells and / or CD73-expressing cells may include, for example, tumor cells, CD4 T cells, CD8 T cells, B cells, and combinations thereof. The expression levels of A2aR, CD39, CD73 can be determined by assessing their mRNA expression (e.g., by RT-PCR) or polypeptide expression (e.g., by Western blotting, immunofluorescence staining) compared to reference levels corresponding to healthy subjects or compared to reference levels before treatment, using techniques well known to those skilled in the art.

[0340] A subject suffering from cancer can be treated with an anti-A2aR antibody with or without assessment of A2aR, CD39, CD73, adenosine, ADP and / or AMP levels in the tumor microenvironment (e.g., in tumor cells, CD4 T cells, CD8 T cells, B cells).

[0341] An assay result that the biological sample contains cells that overexpress A2aR, CD39 and / or CD73 and / or contain elevated levels of adenosine, ADP and / or AMP compared to the reference indicates that the subject suffers from a cancer that may benefit from treatment with an agent that inhibits A2aR. In some embodiments, the term "overexpressed" is used in reference to A2aR, CD39 and / or CD73 polypeptides expressed in a significant number of cells taken from a given patient, e.g., at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or more of the tumor cells or lymphocytes taken from the subject.

[0342] In one embodiment, a method for treating or preventing cancer in a subject in need thereof comprises: (a) detecting the percentage and / or degree of expression of cells corresponding to A2aR, CD39 and / or CD73 in the tumor milieu, optionally in the tumor and / or adjacent tissues, and, upon assay results that the tumor milieu optionally contains cells that overexpress A2aR, CD39 and / or CD73 at an increased level compared to a suitable reference level, (b) administering an anti-A2aR antibody to the subject. In one embodiment, the cells are tumor cells. In other embodiments, the cells in the tumor milieu, tumor and / or adjacent tissues are non-malignant immune cells, e.g., T cells.

[0343] In some embodiments, determining the degree of A2aR, CD39 and / or CD73 expression within the tumor environment comprises obtaining a biological sample from an individual that includes cancer tissue and / or tissue proximal to or in the periphery of the cancer (e.g., cancer-adjacent tissue, adjacent non-neoplastic tissue or adjacent normal tissue), contacting cells with an antibody that binds to an A2aR polypeptide, a CD39 polypeptide and / or a CD73 polypeptide, and detecting the percentage of cells corresponding to and / or the degree of expression of A2aR, CD39 and / or CD73. In certain embodiments, expression of A2aR, CD39 and / or CD73 is assessed by their cell surface expression using immunohistochemistry assays.

[0344] The antibody composition can be used as a monotherapy or as a combination therapy with one or more other therapeutic agents, including agents that are typically used for the particular therapeutic purpose for which the antibody is administered. See "Combination Therapy" above. The additional therapeutic agent is typically administered in the amount and treatment regimen typically used for the monotherapy used for the particular disease or disorder being treated. Such therapeutic agents include, but are not limited to, anti-cancer agents and chemotherapeutic agents.

[0345] As described above, a method of using a pharmaceutical composition according to the present invention comprises administering to a subject in need thereof an effective amount of a pharmaceutical composition according to the present disclosure.

[0346] Any suitable route or mode of administration can be used to provide a patient with a therapeutically or prophylactically effective dose of the antibody. Exemplary routes or modes of administration include parenteral (e.g., intravenous, intraarterial, intramuscular, subcutaneous, intratumor), oral, topical (intranasal, transdermal, intradermal or intraocular), mucosal (e.g., nasal, sublingual, buccal, rectal, vaginal), inhalation, intralymphatic, intraspinal, intracranial, intraperitoneal, intratracheal, intravesical, intrathecal, intraintestinal, intrapulmonary, intralymphatic, intracavity, intraorbital, intracapsular and transurethral, ​​as well as local delivery via a catheter or stent.

[0347] Pharmaceutical compositions comprising anti-A2aR antibodies according to the present disclosure can be formulated in any pharma- ceutically acceptable carrier or excipient. The term "pharmaceutically acceptable carrier" as used herein includes any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. Pharmaceutical compositions may include suitable solid or gel phase carriers or excipients. Exemplary carriers or excipients include, but are not limited to, calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols. Exemplary pharma- ceutically acceptable carriers include water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, and combinations of one or more thereof. In many cases, it is preferred to include an isotonic agent in the composition, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride. Pharmaceutically acceptable carriers may further include minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives, or buffers that enhance the shelf life or effectiveness of the therapeutic agent.

[0348] In certain preferred embodiments, the pharmaceutical compositions can be prepared for use for parenteral administration by injection, eg, by bolus injection or continuous infusion.

[0349] Suitable buffering agents include, but are not limited to, sodium succinate, sodium citrate, sodium phosphate, or potassium phosphate. Sodium chloride can be used at a concentration of 0-300 mM (optimum concentration is 150 mM for liquid dosage forms) to adjust the toxicity of the solution. Cryoprotectants can be included for lyophilized dosage forms, primarily 0-10% sucrose (optimum 0.5-1.0%). Other suitable cryoprotectants include trehalose and lactose. Bulking agents can be included for lyophilized dosage forms, primarily 1-10% mannitol (optimum 2-4%). ​​Stabilizers can be used in both liquid and lyophilized dosage forms, primarily 1-50 mM L-methionine (optimum 5-10 mM). Other suitable bulking agents include glycine, arginine, and 0-0.05% polysorbate-80 (optimum 0.005-0.01%). Other surfactants include, but are not limited to, Polysorbate 20 and BRIJ® surfactants.

[0350] Therapeutic formulations can be lyophilized and stored as sterile powders, preferably under vacuum, and then reconstituted with bacteriostatic water (e.g., containing a benzyl alcohol preservative) or sterile water prior to injection. The therapeutic agent in the pharmaceutical composition can be formulated in a "therapeutically effective amount" or a "prophylactically effective amount." A "therapeutically effective amount" refers to an amount effective to achieve the desired therapeutic result at the dosage and for the period required. The therapeutically effective amount of an antibody or active agent can vary depending on the disorder being treated, the severity and course of the disorder, the method of administration, whether the antibody or active agent is administered for prophylactic or therapeutic purposes, the bioavailability of the particular agent, the ability of the antibody to elicit a desired response in the individual, previous treatments, the age, weight, and sex of the patient, the patient's clinical history and response to the antibody, the type of antibody used, the discretion of the attending physician, and the like. A therapeutically effective amount can also be an amount in which the therapeutically beneficial effects outweigh any toxic or deleterious effects of the recombinant vector. A "prophylactically effective amount" refers to an amount effective to achieve the desired prophylactic result at the dosage and for the period required.

[0351] Preferably, the polypeptide domains used in the antibodies or additional active agents described herein are derived from the same host to which they are administered, in order to reduce the inflammatory response to the administered therapeutic agent. As alluded to above, the therapeutic agent may be administered to the subject suitably once or over a series of treatments, or may be administered to the patient at any time following diagnosis. The A2aR antibody may be administered as a monotherapy or in combination with additional active agents or therapies useful in treating the disorder in question.

[0352] As a general proposition, a therapeutically or prophylactically effective amount of A2aR antibody (or additional active agent) will be administered in the range of about 1 ng / kg body weight / day to about 100 mg / kg body weight / day, whether administered in one or multiple doses.In a specific embodiment, each A2aR antibody or active agent is administered at a concentration of from about 1 ng / kg body weight / day to about 10 mg / kg body weight / day, from about 1 ng / kg body weight / day to about 1 mg / kg body weight / day, from about 1 ng / kg body weight / day to about 100 μg / kg body weight / day, from about 1 ng / kg body weight / day to about 10 μg / kg body weight / day, from about 1 ng / kg body weight / day to about 1 μg / kg body weight / day, from about 1 ng / kg body weight / day to about 100 ng / kg body weight / day, from about 1 ng / kg body weight / day to about 10 ng / kg body weight / day, from about 10 ng / kg body weight / day to about 100 mg / kg body weight / day, or from about 10 ng / kg body weight / day ~ approx. 10 mg / kg body weight / day, approx. 10 ng / kg body weight / day ~ approx. 1 mg / kg body weight / day, approx. 10 ng / kg body weight / day ~ approx. 100 μg / kg body weight / day, approx. 10 ng / kg body weight / day ~ approx. 10 μg / kg body weight / day, approx. 10 ng / kg body weight / day ~ approx. 1 μg / kg Body weight / day, 10ng / kg body weight / day ~ approx. 100ng / kg body weight / day, approx. 100ng / kg body weight / day ~ approx. 100 mg / kg body weight / day, approx. 100ng / kg body weight / day ~ approx. 10 mg / kg body weight / day, approx. 100ng / kg body weight / day ~ approx. kg body weight / day ~ approximately 100 μg / kg body weight / day, approximately 100 ng / kg body weight / day ~ approximately 10 μg / kg body weight / day, approximately 100 ng / kg body weight / day ~ approximately 1 μg / kg body weight / day, approximately 1 μg / kg body weight / day ~ approximately 100 mg / kg body weight / day, approximately 1 μg / kg body weight / day ~ approximately 10 mg / day kg body weight / day, approximately 1 μg / kg body weight / day to approximately 1 mg / kg body weight / day, approximately 1 μg / kg body weight / day to approximately 100 μg / kg body weight / day, approximately 1 μg / kg body weight / day to approximately 10 μg / kg body weight / day, approximately 10 μg / kg body weight / day to approximately 100 mg / kg body weight / day, approximately 10 μg / kg body weight The dosage range is about 10 mg / 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 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 1 mg / kg body weight / day to about 10 mg / kg body weight / day, about 10 mg / kg body weight / day to about 100 mg / kg body weight / day.

[0353] In some other embodiments, the dosage of the A2aR 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.

[0354] In some other embodiments, each A2aR antibody and / or active agent is administered in a dose 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 1 00ng to about 1μg, about 100ng to about 10μg, about 100ng to about 100μg, about 100ng to about 1mg, about 100ng to about 10mg, about 100ng to about 100mg, about 100ng ~about 1000mg, about 100ng to about 10,000mg, about 1μg to about 10μg, about 1μg to about 100μg, about 1μg to about 1mg, about 1μg to about 10mg, about 1μg to about 100mg, about 1μg~about 1000mg, about 1μg~about 10,000mg, about 10μg~about 100μg, about 10μg~about 1mg, about 10μg~about 10mg, about 10μg~about 100mg, about 10μg~ Approximately 1000mg, approximately 10μg to approximately 10,000mg, approximately 100μg to approximately 1mg, approximately 100μg to approximately 10mg, approximately 100μg to approximately 100mg, approximately 100μg to approximately 1000mg, approximately 100μ The antibody of the present disclosure may be administered daily, every 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, or 4 weeks.

[0355] In some other specific embodiments, the dosage of each A2aR antibody or active agent may be about 0.0006 mg / day, 0.001 mg / day, 0.003 mg / day, 0.006 mg / day, 0.01 mg / day, 0.03 mg / day, 0.06 mg / day, 0.1 mg / day, 0.3 mg / day, 0.6 mg / day, 1 mg / day, 3 mg / day, 6 mg / day, 10 mg / day, 30 mg / day, 60 mg / day, 100 mg / day, 300 mg / day, 600 mg / day, 1000 mg / day, 2000 mg / day, 5000 mg / day, or 10,000 mg / day.

[0356] In certain embodiments, the coding sequence for the A2aR 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 A2aR antibody or other active agent in a subject in need of treatment according to the methods described above. For example, in certain embodiments involving administration of one or more recombinant AAV (rAAV) viruses, the pharmaceutical composition comprises at least 10 10 , at least 10 11 , at least 10 12 , at least 10 13 , or at least 10 14 In one embodiment, the pharmaceutical composition can contain at least 10 genome copies (GC) or recombinant viral particles of the rAAV per subject, or any range thereof. 10 , at least 10 11 , at least 10 12 , at least 10 13 , at least 10 14 , at least 10 15 The recombinant virus, such as an rAAV, may comprise up to 100 genomic copies or up to 100 genomic copies of the recombinant virus particle, or any range thereof.

[0357] Dosage can be determined in one or more art-recognized animal models appropriate for any particular cell proliferative or immunodeficiency disease state.

[0358] Delivery methods also include polycation-condensed DNA with or without linkage to killed viruses, ligand-linked DNA, liposomes, eukaryotic delivery vehicle cells, deposition of photopolymerized hydrogel materials, use of handheld gene transfer particle guns, ionizing radiation, neutralization of nuclear charges or fusion with cell membranes, particle-mediated gene transfer, and the like.

[0359] 8. Diagnostic uses of antibodies

[0360] The antigen-binding molecules (e.g., antibodies or antigen-binding fragments thereof) of the present invention can also be used to detect and / or measure human or cynomolgus A2aR, or human or cynomolgus A2aR-expressing cells in a sample, e.g., for diagnostic purposes. For example, anti-A2aR antibodies or antigen-binding fragments thereof can be used to diagnose disorders or diseases characterized by aberrant expression (e.g., overexpression, underexpression, lack of expression, etc.) of A2aR. An exemplary diagnostic assay for A2aR includes, for example, contacting a sample taken from a patient with an antibody of the present invention that is labeled with a detectable label or reporter molecule. Alternatively, unlabeled antibodies can be used for diagnostic purposes in combination with a secondary antibody that is itself detectably labeled. The detectable label or reporter molecule can be, for example, 3 H, 14 C. 18 F, 32 p, 35 S or 125The A2aR may be a radioisotope such as I; a fluorescent or chemiluminescent moiety such as fluorescein isothiocyanate or rhodamine; or an enzyme such as alkaline phosphatase, β-galactosidase, horseradish peroxidase, or luciferase. Specific exemplary assays that can be used to detect or measure A2aR in a sample include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence-activated cell sorting (FACS). Samples that can be used in the A2aR diagnostic assays described in the present invention include any tissue or body fluid sample taken from a patient that contains a detectable amount of A2aR protein or a fragment thereof under normal or pathological conditions. In general, the level of A2aR in a particular sample taken from a healthy patient (e.g., a patient not suffering from a disease or disorder associated with abnormal A2aR levels or activity) is measured to first establish a baseline or standard level of A2aR. This baseline level of A2aR can then be compared to the level of A2aR measured in a sample taken from an individual suspected of having an A2aR-related disease or disorder.

[0361] Additionally, the anti-A2aR antibodies described herein can be used to purify human A2aR through immunoaffinity purification.

[0362] 9. Kit

[0363] Any of the compositions described herein, e.g., an anti-A2aR antigen binding molecule of the invention, and / or an additional therapeutic agent, may be included in a kit. In a non-limiting example, the kit includes an antigen binding molecule, e.g., an antibody or antigen-binding fragment thereof. In certain embodiments, the kit further includes an additional therapeutic agent described herein.

[0364] The kit may further include an agent or instructions for treating a disease or disorder, and may also include one or more buffers.

[0365] The components in the kits may be packaged in either aqueous media or lyophilized form. The container means of the kits will generally include at least one vial, test tube, flask, bottle, syringe or other container means into which the components may be placed, and preferably suitably dispensed. Where there is more than one component in the kit (labeling reagents and labels may be packaged together), the kits may generally include a second, third or other container for separately placing the other components. The kits may also include a second container means for containing a sterile, pharma- ceutically acceptable buffer and / or other diluent. Additionally, vials may contain various combinations of components. Kits according to the invention may also typically include a means for containing the compositions according to the invention (e.g., anti-A2aR antigen binding molecules and / or additional therapeutic agents) and any other reagent containers in close confinement for commercial sale.

[0366] When the above components of the kit are provided as one and / or more liquid solutions, the liquid solution is an aqueous solution, with a sterile aqueous solution being particularly preferred.Furthermore, the components of the kit can be provided as dry powder.When reagents and / or components are provided as dry powder, the powder can be reconstituted by adding a suitable solvent.It is also envisioned that the solvent can be provided in a separate container means.

[0367] The present invention is further illustrated, but not limited, by the following examples. All references, patents, published patent application contents, and figures and tables cited throughout this application are hereby incorporated by reference. EXAMPLES

[0368] Example 1: Generation of exemplary humanized anti-A2aR monoclonal antibodies Three mouse anti-human A2aR monoclonal antibodies 1B5-3D7, 3F6-9G5, and 3F8-12E9 shown in the '601 disclosure were used to generate exemplary humanized anti-A2aR monoclonal antibodies. Based on sequence similarity between the mouse anti-A2aR monoclonal antibodies and the racial systems, the closest human heavy chain variable region (VH) and light chain variable region (VL) sequences were selected using the IgBlast algorithm. CDR regions from the antibodies shown in the '601 disclosure were identified and defined based on a combination of Kabat and IMGT numbering schemes (see Tables 1 and 2). The frameworks of the selected racial systems were used as acceptors for the CDRs from the antibodies shown in the '601 disclosure. Vernier zone residues are known to be involved in antigen binding by either directly contacting the antigen or by influencing CDR loops or chain-docking at the VH / VL critical interface. Multiple combinations of Vernier zone residues were backmutated to the murine-derived residues of the parent antibody shown in the '601 disclosure.

[0369] To confirm that the humanized antibodies retain one or more functions or characteristics of the parent antibodies shown in the '601 disclosure (e.g., specific binding to human A2aR or inhibition of the adenosine pathway), CRO BonOpus Bioscience or BioIntron synthesized each humanized VH domain and VL domain and cloned them in frame into HC plasmid containing human IgG1 FES (L234F, L235E, P331S) isotype constant domain sequence and LC plasmid containing human IgK isotype constant domain sequence, respectively. Each plasmid was subjected to sequencing and restriction enzyme digestion to confirm the sequence and orientation of the inserted humanized VH or VL domain. Then, Expi293F cells (Gibco) were transfected with different matrix combinations of HC and LC plasmids in pairs using Expi293 Transfection Kit (Gibco, Catalog No.: A14524) according to the manufacturer's manual and instructions. Five days after transfection, the supernatant was collected. Each supernatant sample was added to a 384-well plate (Corning, Cat#: 3575) at 90μl / well, and antibody concentrations were quantified by ProA probe using Gator Plus (Gator Bio) according to the manufacturer's recommendations. Antibody concentrations in each supernatant sample were calculated based on a standard curve with Durvalumab (Imfinzi, a commercial anti-PD-L1 antibody drug), and the supernatants were normalized and used for further characterization.

[0370] To generate purified recombinant antibodies, transfection grade plasmids of each heavy-light chain pair of each humanized antibody variant or its derived parent clones (1B5-3D7, 3F6-9G5, 3F8-12E9) were generated. ExpiCHO cells were used for transient transfection. Supernatants were collected 5-7 days after transfection, and IgG1 FES (L234F, L235E, P331S) antibodies were purified by CRO BioIntron.

[0371] In certain assays, the parent antibodies set forth in the '601 disclosure were converted to chimeric antibodies. To generate the chimeric antibodies, the VH domain of the parent antibody was synthetically cloned in frame into a HC plasmid containing human IgG1 FES (L234F, L235E, P331S) isotype constant domain sequences, and the VL domain was synthetically cloned in frame into a LC plasmid containing human IgK isotype constant domain sequences. Antibodies produced in this manner are referred to as chimeric antibodies. For example, a chimeric antibody derived from clone 3F6-9G5 may be referred to as "chimeric 3F6" or "chimeric 3F6-9G5."

[0372] Example 2: Binding of exemplary humanized anti-A2aR monoclonal antibodies to human A2aR This study was aimed at determining whether exemplary humanized anti-A2aR monoclonal antibodies of the invention bind to human A2aR expressed on the cell surface.

[0373] (1) Binding of exemplary humanized anti-A2aR monoclonal antibodies to human A2aR in transfected Expi293F cell supernatants

[0374] Briefly, HEK293 cells stably overexpressing human A2aR (HEK-hA2aR OCL, BPS Bioscience, Cat. No.: 79381) were harvested from fresh culture. The cells were then cultured for 10 6The cells were resuspended in FACS buffer (1 x DPBS / 1% FBS) at a concentration of 100 μg / ml. 100 μl of cells were dispensed in equal amounts into each well of a 96-well plate. After centrifugation, the cell pellet of each well was resuspended with 100 μl of normalized supernatant sample containing 1 μg / ml of humanized anti-A2aR monoclonal antibody variants collected from transfected Expi293 cell cultures. The supernatant was mixed with the cells and incubated on ice for 1 hour. After washing the mixture of supernatant containing the expressed antibody and A2aR-expressing cells with FACS buffer, 100 μl of PE-conjugated anti-human Fc secondary antibody (Jackson Immuno Research, Catalog No.: 109-116-098) diluted 1:400 was added to each well and then incubated on ice for 30 minutes. After washing with FACS buffer, stained cells were resuspended in 150 μl of 7-AAD (Biolegend, Cat. No.: 420404) diluted in 1xDPBS buffer (1:100) and incubated for 5 min at room temperature in the dark before FACS analysis. Mean fluorescence intensity (MFI) was determined using Guava 11HT or 5HT (Luminex). Data were analyzed using Flowjo software.

[0375] The MFI values ​​measured above are summarized in Tables 22 to 24 below. As shown in Tables 22 to 24, the exemplary humanized anti-A2aR antibodies bound to A2aR expressed on the cell surface. A higher MFI value indicated stronger binding between the exemplary antibody and A2aR expressed on the cell surface. [Table 22]

[0376] [Table 23]

[0377] [Table 24]

[0378] * : Average value of four-point measurements (quadruplicate)

[0379] (2) Binding of Exemplary Humanized Anti-A2aR Monoclonal Antibodies Expressed by Purified ExpiCHO Cells

[0380] Briefly, HEK293 cells stably overexpressing human A2aR (HEK-hA2aR OCL, BPS Bioscience, Cat. No.: 79381) were harvested from fresh culture. The cells were then cultured at 2 × 10 6 The cells were resuspended in FACS buffer (1 x DPBS / 2% FBS) at a concentration of 1.5 μg / ml. 100 μl of cells were dispensed in equal amounts into each well of a 96-well plate. After centrifugation, the cell pellet in each well was resuspended in a 1:3 serial dilution (starting concentration: 3.3 μg / mL, diluted in FACS buffer (1 x PBS, 2% FBS)) of purified exemplary antibody (expressed by ExpiCHO cells and purified by CRO BioIntron). The serially diluted antibody was mixed with the cells and incubated on ice for 30 minutes. After washing with FACS buffer, 100 μl of PE-conjugated anti-human Fc secondary antibody (diluted 1:400, Jackson Immuno Research, Cat. No.: 109-116-098) was added to each well, followed by incubation on ice for 30 minutes. After washing with FACS buffer, stained cells were suspended in 200 μl of 7-AAD (Biolegend, Cat. No.: 420404, diluted 1:100 in 1xDPBS buffer) and incubated for 5 min at room temperature in the dark, after which flow cytometry analysis was performed on Guava 11HT or 5HT (Luminex). Mean fluorescence intensity (MFI) was acquired and data was analyzed using Flowjo software.

[0381] Table 25 below summarizes the EC50 values ​​measured above. The exemplary humanized anti-A2aR antibodies of the present disclosure were confirmed to specifically bind to A2aR expressed on the cell surface. The parental 3F6-9G5 and 3F8-12E9 antibodies served as positive controls. Anti-HEL isotype control (BioIntron) served as a negative control.

[0382] [Table 25] Example 3: In vitro blocking activity of humanized anti-human A2aR monoclonal antibodies

[0383] The activity of A2aR is mediated by Gs protein, which activates adenylyl cyclase to produce the synthesis of intracellular cAMP. The level of cAMP is related to the respective adenosine (agonist) level. cAMP can be detected using various commercially available cAMP assay kits. This study aims to determine whether several exemplary humanized antibodies can block A2aR activity.

[0384] (1) In vitro blocking activity of humanized anti-human A2aR monoclonal antibodies in the supernatant of transfected Expi293F cells

[0385] Briefly, 96-well tissue culture clear flat-bottom plates (Fisher Scientific) were coated with 50 μg / ml poly-D-lysine (Gibco Cat. No.: A3890401) solution at 50 μl / well and incubated overnight at 4 °C. After washing three times with endotoxin-free sterile water, the coated culture plates (without caps) were placed in a biosafety cabinet to dry. HEK293 cells (BPS Bioscience) stably overexpressing human A2aR were seeded at 5000 cells / well in 200 μl starvation medium (MEM (Hyclone Cat. No.: SH30024.01) + 2% charcoal-stripped serum (Thermo Fisher Cat. No.: A3382101) + 1% penicillin / streptomycin) and incubated overnight at 37 °C, 5% CO2. The next day, the coated cells were washed three times with 200 μl warm 1xPBS. Three-fold serial dilutions of normal supernatant containing 1 or 2 μg / ml humanized anti-A2aR antibody (see Example 1) were then mixed 1:1 with induction buffer (1x PBS containing 1000 μM 3-isobutyl-1-methylxanthine (IBMX) (Millipore Sigma, Cat. No.: I7018) + 200 μM Ro 20-1724 (Millipore Sigma, Cat. No.: B8279)). 10 μl of the supernatant / induction buffer mixture was then added to each well and incubated at 37°C for 15 min, followed by the addition of NECA (Eurofins Scientific, Cat. No.: 92-1191) (a stable adenosine receptor agonist) at a final concentration of 300 nM. The cells were then incubated at 37°C, 5% CO2 for 1 h. cAMP-Glo TM Cell lysis and cAMP detection were performed using an assay kit (Promega, Cat. No.: V1501) according to the manufacturer's manual and instructions. Results (Relative Light Units, RLU) were read using a luminometer. RLU can be converted to cAMP (nM) using a cAMP standard curve and GraphPad Prism software. The corresponding parent antibodies 1B5-3D7, 3F6-9G5 and 3F8-12E9 served as positive controls.

[0386] As shown in Tables 26-27, exemplary humanized anti-A2aR antibody variants block the activity of human A2aR expressed on the cell surface and have IC 50 The values ​​were found to be about 0.7 nM to about 20 nM.

[0387] [Table 26]

[0388] [Table 27]

[0389] (2) In Vitro Blocking Activity of Exemplary Humanized Anti-A2aR Monoclonal Antibodies Expressed by Purified ExpiCHO Cells

[0390] Briefly, 50 μg / ml poly-D-lysine (Gibco Cat. No.: A3890401) solution was coated at 50 μl / well onto 96-well tissue culture clear flat-bottom plates (Fisher Scientific) and placed at 4 °C overnight. After washing three times with endotoxin-free sterile water, the coated culture plates (without caps) were placed in a biosafety cabinet to dry. Fresh, thawed HEK293 cells overexpressing A2aR (BPS Bioscience) were plated at 10 in 100 μl of medium onto the poly-D-lysine-coated plates. 4Cells / well were seeded and incubated at 37°C, 5% CO2 in an incubator for 24 hours. After washing once with warm PBS, 100 μl of starvation medium (MEM (Hyclone Catalog No.: SH30024.01) + 2% carbon-adsorbed serum (Thermo Fisher Catalog No.: A3382101) + 1% penicillin / streptomycin) was added and incubated overnight at 37°C, 5% CO2 in an incubator. The next day, starved coated cells were washed once with 200 μl of warm 1x PBS. Serial dilutions of humanized anti-A2aR antibodies (produced in ExpiCHO cells by CRO BioIntron), A2aR antagonist small molecule inhibitors (SMIs) CPI-444 (MedChemExpress, Catalog No. HY-101978), and AB928 (MedChemExpress, Catalog No. HY-129393) were prepared. 30 μl of humanized anti-A2aR antibodies, parental 3F6-9G5 or SMI were added to each well and incubated for 105 min in a 37°C incubator. During the last 15 minutes of incubation, 4x induction buffer (10 μl of 2 mM IBMX 3-isobutyl-1-methylxanthine (IBMX) (Millipore Sigma, Catalog No. I7018) and 400 μM Ro20-1724 (Millipore Sigma, Catalog No. B8279)) was added to each well. After incubation, 30 μl of medium was removed from each well with a dropper and 10 μl of 10 μM adenosine (Alfa aesar, Catalog No. A10781) or 2.5 μM NECA (Eurofins Scientific, Catalog No. 92-1191) was added to each well and incubated for 1 hour at 37°C in an incubator with 5% CO2. cAMP-Glo TMCell lysis and cAMP detection were performed using an assay kit (Promega, Cat. No.: V1501) according to the manufacturer's manual and instructions. Plates were read using a SpectraMax 5 plate reader (Molecular Device) to obtain relative light units (RLU). Data were analyzed using Prism software. Corresponding parental antibodies of the same isotype, 3F6-9G5 or 3F8-12E9, were used as positive controls.

[0391] As shown in Figures 1A, 1B and Table 28, the exemplary humanized anti-A2aR antibody variants and the parent 3F6-9G5 have similar IC 50 Although both antibodies blocked the activity of cell surface-expressed human A2aR at IC values, SMI CPI-444 and AB928 had much higher IC values. 50 values ​​and was found to have a lower blocking capacity in that assay. [Table 28] Example 4: Endocytosis of humanized anti-human A2aR monoclonal antibodies

[0392] This example aims to confirm endocytosis of a complex consisting of an exemplary humanized anti-human A2aR monoclonal antibody of the present disclosure and A2aR expressed on the cell surface.

[0393] Endocytosis assay by flow cytometry

[0394] 2×10 5Freshly cultured HEK293 cells expressing human A2aR (BPS Bioscience) were incubated with the test antibodies at specific concentrations (1, 0.5 and 0.25 μg / ml) per well for 45 min on ice. Parental 3F6 was used as a positive control. After washing with FACS buffer, the samples were divided into four groups. One group of samples was kept on ice. Another three groups of samples were further incubated in a 37°C, 5% CO2 incubator for 1, 2 and 4 h, respectively. All four groups of samples were stained with PE-conjugated goat-anti-human Fc antibody (1:400, Jackson Immuno Research, Cat. No.: 109-116-098) for 30 min, followed by washing with frozen FACS buffer. Cells were then stained with 7-AAD (Biolegend) diluted 1:100 and then fixed with 2% paraformaldehyde (diluted from 10% paraformaldehyde (MilliporeSigma, Cat. No. R04586)). Both samples were analyzed on Guava 11HT or 5HT (Luminex). Mean fluorescence intensity (MFI) was acquired and data was analyzed using Flowjo software. Endocytosis rate was calculated by the following formula:

[0395] % Endocytosis = ((MFI of sample on ice)-(MFI of sample incubated at 37°C for a specific time) / (MFI of sample on ice)) x 100

[0396] As shown in Figure 2, the two exemplary humanized anti-human A2aR monoclonal antibodies induced the highest levels of endocytosis (shown as percent endocytosis) when incubated with HEK293 cells expressing human A2aR for 4 hours at 37°C. Under the same experimental conditions (e.g., incubation time), the levels of internalization induced by the two exemplary humanized anti-A2aR monoclonal antibodies were found to be similar to that of the parental 3F6-9G5 antibody.

[0397] (2) Endocytosis assay using pH-sensitive dyes

[0398] Invitrogen TM Zenon TM pHrodo TM iFL RED human IgG labeling reagent (catalog number: Z25612) is a low pH sensitive dye and was used to determine the endocytosis properties of the exemplary anti-A2aR antibodies according to the manufacturer's instructions. TM pHrodo TM Endocytosis of antibodies conjugated to the iFL RED human IgG labeling agent enhances the fluorescent signal in the low pH intracellular region of the cells, thereby providing a way to visualize endocytosis under a fluorescent microscope.

[0399] Briefly, a solution (4x) of 160 nM antibody (approximately 24 μg / ml, 25 μl) was prepared in FreeStyle CHO expression medium (Gibco, Cat. No. 12451-014) and then added to each well of a 96-well plate. Zenon was then incubated in FreeStyle CHO expression medium. TM pHrodo TM 25 μL of a 4× working solution of iFL IgG labeling agent was prepared and then added to each well of the 96-well plate containing the antibody. The solutions in the 96-well plate were mixed and incubated at room temperature for 5 minutes to form the labeling complex. CHO cells (2×10 650 μl of 3H 2+ / ml was added to each well containing the labeled antibody. The cells were then incubated with the labeled complex for 24 hours under standard cell culture conditions. The cells were analyzed by immunofluorescence microscopy (Invitrogen EVOS Imaging System M5000) to visualize the endocytosis of the antibody through the cells. The antibody endocytosis was imaged by EVOS Imaging System (Invitrogen). As shown in Figure 3, the induction of endocytosis was visualized in a representative figure, which showed that parental 3F6, humanized antibody variants Hu3F6-H3 / Hu3F6-L5 and Hu3F6-H5 / Hu3F6-L5 could induce endocytosis, but the control antibody of anti-HEL isotype could not. Hoechst 33342 (Invitrogen, Cat. No.: H3570) is a common cell-permeable nuclear counterstain that emits blue fluorescence and was used to stain condensed nuclei in the cells. In that assay, SMI CPI-444 and AB928 were found not to induce endocytosis (data not shown). Example 5: Binding of humanized anti-human A2aR antibodies to human and cynomolgus monkey primary cells and cross-reactivity with non-human primates

[0400] Human A2aR has been reported to be expressed by T cells and NK cells. This study aimed to confirm that exemplary humanized anti-A2aR monoclonal antibodies of the invention bind to A2aR on the cell surface of human and cynomolgus monkey PBMCs.

[0401] Human and cynomolgus monkey PBMCs were purchased from Cytologics LLC (San Diego, CA) and iQ Biosciences (Berkeley, CA), respectively. 10 6Human and / or cynomolgus PBMCs were incubated with anti-A2aR antibody for 30 min at 4° C. After washing, samples were stained with PE-conjugated goat-anti-human Fc antibody (1:400, Jackson ImmunoResearch Labs, Cat. No. 109-116-098) and other surface markers with cynomolgus cross-reactive APC / cy7-anti-CD3 (BD ​​Bioscience, Cat. No. 557757), BV421-anti-CD4 (Biolegend, Cat. No. 317433), BV650-anti-CD8 (Biolegend, Cat. No. 301042) and BV711-anti-CD56 (BD Bioscience, Cat. No. 742661). Zombie Green Fixable Viability Dye (Biolegend, Cat. No. 423111) was used to define viable immune cell subsets according to the manufacturer's instructions. After washing with FACS buffer, cells were analyzed on a FACSAria (BD). Data were analyzed with Flowjo (Becton Dickinson).

[0402] As shown in Figure 4 and Tables 29-32, the parental 3F6-9G5 and the two exemplary humanized anti-human A2aR antibodies were found to bind to small subsets of human T cells (CD3+CD8-CD4+CD4 T cells and CD3+CD4-CD8+CD8 T cells, Figure 4A), CD3-CD56+NK cells and CD3+CD56+NKT cells, whereas the anti-HEL isotype control (Biointron) did not bind to the above cells. They were further found to cross-react with cynomolgus monkey T cells (CD4+ and CD8+, Figure 4B), NK cells and NKT cells. [Table 29] [Table 30] [Table 31] [Table 32] Example 6: Restoration of T cell function by humanized anti-human A2aR monoclonal antibodies

[0403] This study aims to confirm that humanized antibodies can restore T cell activation and cytokine production.

[0404] 200,000 (2 x 10) in complete IMDM medium 5 ) human peripheral blood mononuclear cells (PBMCs) were dispensed into each well of a 96-well plate in equal amounts, and the cells in the 96-well plate were pre-incubated with specific concentrations of antibodies or small molecule inhibitors for 90 min. Then, anti-CD3 / 28 dynabeads (Gibco, Catalog No.: 11132D) for T cell proliferation stimulation were added at a ratio of 1:4 (Beads:PBMCs, v / v), and NECA (DiscoverX, Catalog No.: 92-1191, final concentration 0.1 μM) was added. The cells were cultured in a 37°C, 5% CO2 incubator for 24 h. After that, the cells were harvested and GolgiPlug was added. TMCells were stimulated with anti-CD3 / 28 Dynabeads for 4 h in the presence of protein transport inhibitors (BD Biosciences, Cat. No. 555028) according to the manufacturer's instructions. Cells were then incubated with human TruStain FcX blocker (Biolegend, Cat. No. 422302) and True-Stain mononuclear cell blocker (Biolegend, Cat. No. 426103) for 15 min according to the manufacturer's instructions, followed by PE-anti-CD4 (Biolegend, Cat. No. 300550), PerCP-cy5.5-anti-CD8 (Biolegend, Cat. No. 301032), APC / cy7-anti-CD3 (Biolegend, Cat. No. 301033), and 10% chromatin (100% chromatin). Cells were stained for surface markers with BV421 anti-IFN-γ (BD Bioscience, Catalog No.: 562988) and APC anti-IL-2 (BD Bioscience, Catalog No.: 551383) using an intracellular cytokine staining kit (BD Biosciences, Catalog No.: 555028) according to the manufacturer's instructions. Stained samples were analyzed on a FACSAria III instrument. Data were analyzed using Flowjo software.

[0405] As shown in Figure 5A, compared to the small molecule inhibitors AB928, AZD4635 and CPI-444, the exemplary anti-A2aR antibodies Hu3F6-VH3 / Hu3F6-VL5, Hu3F6-VH5 / Hu3F6-VL5 and the parental 3F6-9G5 antibody were found to exhibit greater potency in restoring IFN-γ production by NECA-suppressed human CD4 T. Additionally, as shown in Figure 5B, the anti-A2aR antibodies were found to be more potent than the small molecule inhibitors AZD4635 and CPI-444 in restoring IL-2 production in NECA-suppressed human CD4 T.

[0406] As shown in Table 33, the exemplary anti-A2aR antibodies Hu3F6-VH3 / Hu3F6-VL5, Hu3F6-VH5 / Hu3F6-VL, and the parental 3F6-9G5 antibody were able to not only restore IFN-γ and IL-2 production by NECA-suppressed CD4 T cells ( FIG. 5 ), but also restore IFN-γ and IL-2 production by NECA-suppressed CD8 T cells with greater potency than the small molecules AB928, CPI-444, and AZD4635. Moreover, the antibodies were able to restore IFN-γ and IL-2 production by NECA-suppressed NK cells with greater potency than the small molecule inhibitors tested. [Table 33]

[0407] As shown in Table 34, the exemplary anti-A2aR antibodies Hu3F6-VH3 / Hu3F6-VL5, Hu3F6-VH5 / Hu3F6-VL5, and the parental 3F6-9G5 antibody were found to restore activation of CD25 and CD69 surface markers of NECA-suppressed CD4 and CD8 T cells with potency similar to that of the small molecules AB928 used at 1 μg / ml and CPI-444 and AZD4635 used at up to 2 μg / ml. [Table 34]

[0408] The above description is intended to teach those skilled in the art how to carry out the invention, and is not intended to detail all obvious modifications and variations that would become apparent to those skilled in the art upon reading the description. However, all such obvious modifications and variations are intended to be included within the scope of the invention as defined by the following claims. The claims are intended to cover the recited elements and steps in any order that is effective to achieve the intended purpose, unless the context specifically indicates to the contrary.

Claims

1. 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 region (VL) domain comprising, from N-terminus to C-terminus, three light chain complementarity determining regions (CDRs): LCDR1, LCDR2, and LCDR3, (a) the HCDR1 is GFX １ FTX ２ X ３ WMN (sequence number) (where X １ is A or T, and X ２ is R or S, and X ３ is F or Y; (b) the HCDR2 is RIDPX ４ D.S.E.X. ５ X ６ YX ７ X ８ KFWX ９ (Sequence number) (where X ４ is S or Y, and X ５ is A or T, and X ６ is H or Q, and X ７ is A, H, or N; X ８ is A or H, and X ９ is D or G; (c) the HCDR3 comprises an amino acid sequence represented by GRSLYGKGDY (SEQ ID NO: 10) or LRSLYGKGDY (SEQ ID NO: 11); (d) the LCDR1 is RSSQSX １１ VHX １２ NGNTYLE (sequence number) (where X １１ is L or I, X １２ is R or S; (e) the LCDR2 comprises the amino acid sequence represented by KVSNRFS (SEQ ID NO: 14); (f) the LCDR3 comprises the amino acid sequence represented by FQGSHVPLT (SEQ ID NO: 15) or YQGSHVPLT (SEQ ID NO: 16); An isolated antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises a light chain framework region 4 (LFR4) comprising the amino acid sequence represented by FGGGTKVEIK (sequence number 50).

2. (a) the HCDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, and 3; (b) the HCDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 4-9 and 89; (c) the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 10 or 11; (d) the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 12 or 13; (e) the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 14; (f) The isolated antibody or antigen-binding fragment thereof of claim 1, wherein the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 15 or 16.

3. The antibody, (a) an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO:1, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO:4, an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO:10, an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO:12, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO:14, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO:15; (b) HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 5, HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 10, LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 12, LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 14, and LCDR3 comprising the amino acid sequence shown in SEQ ID NO: 15; (c) an HCDR1 comprising the amino acid sequence shown in SEQ ID NO:2, an HCDR2 comprising the amino acid sequence shown in SEQ ID NO:6, an HCDR3 comprising the amino acid sequence shown in SEQ ID NO:11, an LCDR1 comprising the amino acid sequence shown in SEQ ID NO:13, an LCDR2 comprising the amino acid sequence shown in SEQ ID NO:14, and an LCDR3 comprising the amino acid sequence shown in SEQ ID NO:16; (d) an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO:2, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO:7, an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO:11, an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO:13, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO:14, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO:16; (e) HCDR1 comprising the amino acid sequence shown in SEQ ID NO:3, HCDR2 comprising the amino acid sequence shown in SEQ ID NO:8, HCDR3 comprising the amino acid sequence shown in SEQ ID NO:11, LCDR1 comprising the amino acid sequence shown in SEQ ID NO:12, LCDR2 comprising the amino acid sequence shown in SEQ ID NO:14, and LCDR3 comprising the amino acid sequence shown in SEQ ID NO:16; (f) an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO:3, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO:9, an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO:11, an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO:12, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO:14, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO:16, or (g) An isolated antibody or antigen-binding fragment thereof described in claim 2, comprising an HCDR1 having an amino acid sequence set forth in SEQ ID NO: 3, an HCDR2 having an amino acid sequence set forth in SEQ ID NO: 89, an HCDR3 having an amino acid sequence set forth in SEQ ID NO: 11, an LCDR1 having an amino acid sequence set forth in SEQ ID NO: 12, an LCDR2 having an amino acid sequence set forth in SEQ ID NO: 14, and an LCDR3 having an amino acid sequence set forth in SEQ ID NO:

16.

4. 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 that includes, from N-terminus to C-terminus, three light chain complementarity determining regions (CDRs): LCDR1, LCDR2, and LCDR3; (a) the HCDR1 is GFX １ FTX ２ X ３ WMN (sequence number) (where X １ is A or T, and X ２ is R or S, and X ３ is F or Y; (b) the HCDR2 comprises an amino acid sequence selected from the group consisting of RIDPYDSETQYAHKFWD (SEQ ID NO: 5), RIDPSDSEAHYAHKFWG (SEQ ID NO: 7), RIDPSDSETHYNAKFWG (SEQ ID NO: 9), and RIDPSDSETHYAHKFWG (SEQ ID NO: 89); (c) the HCDR3 comprises the amino acid sequence shown in GRSLYGKGDY (SEQ ID NO: 10) or LRSLYGKGDY (SEQ ID NO: 11); (d) the LCDR1 is RSSQSX １１ VHX １２ NGNTYLE (sequence number) (where X １１ is L or I, and X １２ is R or S; (e) the LCDR2 comprises the amino acid sequence set forth in KVSNRFS (SEQ ID NO: 14); (f) An isolated antibody or antigen-binding fragment thereof, wherein the LCDR3 comprises the amino acid sequence shown in FQGSHVPLT (sequence number 15) or YQGSHVPLT (sequence number 16).

5. (a) the HCDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, and 3; (b) the HCDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 7, 9, and 89; (c) the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 10 or 11; (d) the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 12 or 13; (e) the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 14; (f) The isolated antibody or antigen-binding fragment thereof of claim 4, wherein the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 15 or 16.

6. The antibody (a) an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5, an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 10, an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 12, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 14, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 15; (b) HCDR1 comprising the amino acid sequence shown in SEQ ID NO:2, HCDR2 comprising the amino acid sequence shown in SEQ ID NO:7, HCDR3 comprising the amino acid sequence shown in SEQ ID NO:11, LCDR1 comprising the amino acid sequence shown in SEQ ID NO:13, LCDR2 comprising the amino acid sequence shown in SEQ ID NO:14, and LCDR3 comprising the amino acid sequence shown in SEQ ID NO:16; (c) an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO:3, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO:9, an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO:11, an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO:12, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO:14, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO:16, or (d) An isolated antibody or antigen-binding fragment thereof described in claim 4, comprising an HCDR1 having an amino acid sequence set forth in SEQ ID NO: 3, an HCDR2 having an amino acid sequence set forth in SEQ ID NO: 89, an HCDR3 having an amino acid sequence set forth in SEQ ID NO: 11, an LCDR1 having an amino acid sequence set forth in SEQ ID NO: 12, an LCDR2 having an amino acid sequence set forth in SEQ ID NO: 14, and an LCDR3 having an amino acid sequence set forth in SEQ ID NO:

16.

7. 7. The isolated antibody or antigen-binding fragment thereof of claim 5 or 6, further comprising a light chain framework region 4 (LFR4) comprising the amino acid sequence set forth in FGGGTKVEIK (SEQ ID NO:50).

8. (a) a heavy chain framework region 1 (HFR1) comprising the amino acid sequence shown in X14-V-Q-L-V-Q-S-G-A-E-V-K-K-P-G-A-S-V-K-V-S-C-K-X15-S (SEQ ID NO: 1) or E-V-Q-L-V-Q-S-G-A-E-V-K-K-P-G-E-S-L-R-I-S-C-K-X16-S (SEQ ID NO: 1) (wherein X14 is E or Q, X15 is A or T, and X16 is A or T); (b) a heavy chain framework region 2 (HFR2) comprising the amino acid sequence shown in W-V-R-Q-X17-P-G-X18-G-L-E-W-X19-G (SEQ ID NO:) (wherein X17 is A or M, X18 is K or Q, and X19 is I or M); (c) a heavy chain framework region 3 (HFR3) comprising the amino acid sequence shown in R-X20-T-X21-T-X22-D-X23-S-X24-S-T-V-Y-M-E-L-S-S-L-R-S-E-D-T-A-V-Y-Y-C (SEQ ID NO: 29) (wherein X20 is A or V, X21 is L or M, X22 is R or V, X23 is K or T, and X24 is S); (d) a heavy chain framework region 4 (HFR4) comprising the amino acid sequence shown in WGQGTTVTVSS (SEQ ID NO: 32) or WGHGTTVTVSS (SEQ ID NO: 33).

9. (a) the HFR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 17 to 21; (b) the HFR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 22, 23, and 24; (c) the HFR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 25-31; (d) The isolated antibody or antigen-binding fragment thereof of claim 8, wherein the HFR4 comprises an amino acid sequence selected from the group consisting of those shown in SEQ ID NO: 32 or 33.

10. the heavy chain variable region comprises, from N-terminus to C-terminus, the structure: HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4; The HFR1, HFR2, HFR3, and HFR4 are each (a) sequences shown in SEQ ID NO: 17, SEQ ID NO: 22, SEQ ID NO: 25, and SEQ ID NO: 32; (b) sequences shown in SEQ ID NO: 18, SEQ ID NO: 22, SEQ ID NO: 26, and SEQ ID NO: 32; (c) sequences shown in SEQ ID NO: 18, SEQ ID NO: 23, SEQ ID NO: 26, and SEQ ID NO: 32; (d) sequences shown in SEQ ID NO: 18, SEQ ID NO: 23, SEQ ID NO: 27, and SEQ ID NO: 32; (e) sequences shown in SEQ ID NO: 18, SEQ ID NO: 23, SEQ ID NO: 28, and SEQ ID NO: 32; (f) sequences shown in SEQ ID NO: 19, SEQ ID NO: 24, SEQ ID NO: 29, and SEQ ID NO: 32; (g) sequences shown in SEQ ID NO: 17, SEQ ID NO: 22, SEQ ID NO: 25, and SEQ ID NO: 33; (h) sequences shown in SEQ ID NO: 18, SEQ ID NO: 22, SEQ ID NO: 26, and SEQ ID NO: 33; (i) sequences shown in SEQ ID NO: 18, SEQ ID NO: 23, SEQ ID NO: 27, and SEQ ID NO: 33; (j) sequences shown in SEQ ID NO: 18, SEQ ID NO: 23, SEQ ID NO: 28, and SEQ ID NO: 33; (k) sequences shown in SEQ ID NO: 20, SEQ ID NO: 24, SEQ ID NO: 29, and SEQ ID NO: 33; (l) sequences shown in SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:26, and SEQ ID NO:32; (m) a sequence as set forth in SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:30, and SEQ ID NO:32; (n) a sequence as set forth in SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 31, and SEQ ID NO: 32; or (o) The isolated antibody or antigen-binding fragment thereof of claim 9, comprising the sequences set forth in SEQ ID NO:20, SEQ ID NO:24, SEQ ID NO:29, and SEQ ID NO:

32.

11. (a) a light chain framework region 1 (LFR1) comprising the amino acid sequence shown in D-X26-V-M-T-Q-X27-P-X28-S-L-X29-V-X30-X31-G-X32-P-A-S-I-S-C (sequence number) or DIVMTQSPDSLAVSLGERATINC (sequence number) (wherein X26 is I or V, X27 is S or T, X28 is D, L, or P, X29 is A, P, or S, X30 is N, S, or T, X31 is L or P, and X32 is E or Q); (b) a light chain framework region 2 (LFR2) comprising the amino acid sequence shown in W-X33-X34-Q-X35-P-G-Q-X36-P-X37-X38-L-I-Y (sequence number) (wherein X33 is F or Y, X34 is L or Q, X35 is K or R, X36 is P or S, X37 is K, Q or R, and X38 is L or R); and (c) a light chain framework region 3 (LFR3) comprising the amino acid sequence shown in SEQ ID NO: G-V-P-D-R-F-S-G-S-G-S-G-X39-D-F-T-L-X40-I-S-X41-X42-X43-A-E-D-V-X44-V-Y-Y-C (SEQ ID NO:), wherein X39 is S or T, X40 is K or T, X41 is R, S or W, X42 is L or V, X43 is E or Q, and X44 is A or G. The isolated antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, further comprising:

12. (a) the LFR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 34 to 41; (b) the LFR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 42 to 46; (c) the LFR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 47, 48 and 49, the isolated antibody or antigen-binding fragment thereof of claim 11 .

13. the light chain variable region comprises, from N-terminus to C-terminus, the structure LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4; The LFR1, LFR2, LFR3 and LFR4 are each (a) sequences as set forth in SEQ ID NO: 34, SEQ ID NO: 42, SEQ ID NO: 47 and SEQ ID NO: 50; (b) sequences shown in SEQ ID NO: 34, SEQ ID NO: 43, SEQ ID NO: 47 and SEQ ID NO: 50; (c) sequences shown in SEQ ID NO: 34, SEQ ID NO: 44, SEQ ID NO: 47 and SEQ ID NO: 50; (d) sequences shown in SEQ ID NO: 35, SEQ ID NO: 45, SEQ ID NO: 47 and SEQ ID NO: 50; (e) sequences shown in SEQ ID NO: 36, SEQ ID NO: 46, SEQ ID NO: 48 and SEQ ID NO: 50; (f) sequences shown in SEQ ID NO: 37, SEQ ID NO: 45, SEQ ID NO: 47 and SEQ ID NO: 50; (g) sequences shown in SEQ ID NO: 38, SEQ ID NO: 45, SEQ ID NO: 47 and SEQ ID NO: 50; (h) sequences shown in SEQ ID NO: 39, SEQ ID NO: 45, SEQ ID NO: 49 and SEQ ID NO: 50; (i) a sequence as set forth in SEQ ID NO: 40, SEQ ID NO: 45, SEQ ID NO: 49 and SEQ ID NO: 50; or (j) The isolated antibody or antigen-binding fragment thereof of claim 12, comprising the sequences set forth in SEQ ID NO:41, SEQ ID NO:45, SEQ ID NO:47 and SEQ ID NO:

50.

14. The antibody, (a) a heavy chain variable region (HCVR) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 51 to 70; (b) a light chain variable region (LCVR) comprising an amino acid sequence selected from the group consisting of those set forth in SEQ ID NOs: 71 to 88. The isolated antibody or antigen-binding fragment thereof according to any one of claims 1 to 13.

15. a heavy chain variable (VH) domain comprising, from N-terminus to C-terminus, three heavy chain complementarity determining regions (CDRs): HCDR1, HCDR2, and HCDR3; and a light chain variable (VL) domain comprising, from N-terminus to C-terminus, three light chain complementarity determining regions (CDRs): LCDR1, LCDR2, and LCDR3; and (a) the HCDR1 comprises an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identity to an amino acid sequence selected from the group consisting of the amino acid sequences set forth in SEQ ID NOs: 1, 2 and 3; (b) the HCDR2 comprises an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identity to an amino acid sequence selected from the group consisting of the amino acid sequences set forth in SEQ ID NOs: 4-9 and 89; (c) the HCDR3 comprises an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identity to the amino acid sequence set forth in SEQ ID NO: 10 or 11; (d) the LCDR1 comprises an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identity to an amino acid sequence selected from the group consisting of the amino acid sequences set forth in SEQ ID NOs: 12 and 13; (e) the LCDR2 comprises an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identity to the amino acid sequence set forth in SEQ ID NO:14; (f) the LCDR3 comprises an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identity to an amino acid sequence selected from the group consisting of the amino acid sequences set forth in SEQ ID NOs: 15 and 16; An isolated antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises a light chain framework region 4 (FR4) comprising an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identity to the amino acid sequence set forth in FGGGTKVEIK (SEQ ID NO:50).

16. The heavy chain variable (VH) domain comprises, from the N-terminus to the C-terminus, four framework regions (FRs): HFR1, HFR2, HFR3, and HFR4; The light chain variable region (VL) domain comprises, from the N-terminus to the C-terminus, four framework regions (FRs): LFR1, LFR2, LFR3, and LFR4; (a) the HFR1 comprises an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identity to an amino acid sequence selected from the group consisting of the amino acid sequences set forth in SEQ ID NOs: 17 to 21; (b) the HFR2 comprises an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identity to an amino acid sequence selected from the group consisting of the amino acid sequences set forth in SEQ ID NOs: 22, 23 and 24; (c) the HFR3 comprises an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identity to an amino acid sequence selected from the group consisting of the amino acid sequences set forth in SEQ ID NOs:25-31; (d) the HFR4 comprises an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identity to an amino acid sequence selected from the group consisting of the amino acid sequences set forth in SEQ ID NOs: 32 and 33; (e) the LFR1 comprises an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identity to an amino acid sequence selected from the group consisting of the amino acid sequences set forth in SEQ ID NOs: 34 to 41; (f) the LFR2 comprises an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identity to an amino acid sequence selected from the group consisting of the amino acid sequences set forth in SEQ ID NOs: 42 to 46; (g) The isolated antibody or antigen-binding fragment thereof of claim 15, wherein the LFR3 comprises an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identity to an amino acid sequence selected from the group consisting of the amino acid sequences shown in SEQ ID NOs: 47, 48 and 49.

17. (a) a heavy chain variable region (HCVR) comprising an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identity to an amino acid sequence selected from the group consisting of the amino acid sequences set forth in SEQ ID NOs: 51 to 70; (b) a light chain variable region (LCVR) comprising an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identity to an amino acid sequence selected from the group consisting of amino acid sequences set forth in SEQ ID NOs: 71 to 88, and an isolated antibody or antigen-binding fragment thereof that binds to the human adenosine A2A receptor (A2aR).

18. (a) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 60 and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 80; (b) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 62 and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 78; (c) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 62 and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 80; (d) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 62 and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 83; (e) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 67 and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 86; or (f) An isolated antibody or antigen-binding fragment thereof that binds to the human adenosine A2A receptor (A2aR), comprising a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:67 and a light chain comprising the amino acid sequence set forth in SEQ ID NO:

87.

19. 19. The isolated antibody or antigen-binding fragment thereof of any one of claims 1 to 18, wherein the N-terminus of the heavy and / or light chain is a pyroglutamic acid (pE) residue.

20. (i) the antibody binds to human A2aR in competition with a monoclonal antibody selected from the group consisting of 1B5-3D7, 3F6-9G5, and 3F8-12E9; (ii) the antibody inhibits activity of A2aR; (iii) the antibody improves an immune response. (iv) the antibody specifically binds to human A2aR on the cell surface; (v) the antibody reduces cAMP concentration in a tissue; (vi) the antibody reduces the activity of protein kinase A. (vii) the antibody reduces phosphorylation of a cAMP response element in the A2aR signaling pathway. (viii) the antibody specifically binds to human and / or cynomolgus A2aR; or (viii) The isolated antibody or antigen-binding fragment thereof of any one of claims 1 to 19, wherein the antibody induces internalization of A2aR.

21. An isolated antibody or antigen-binding fragment thereof, which binds to human A2aR in competition with the antibody or antigen-binding fragment thereof according to any one of claims 1 to 20.

22. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 21, wherein the antibody is a humanized antibody or a chimeric antibody.

23. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 22, wherein the antibody comprises a heavy chain constant region selected from the group consisting of classes IgA, IgD, IgE, IgG and IgM.

24. The antibody or antigen-binding fragment thereof of claim 23, wherein the heavy chain constant region is a constant region of an IgG class selected from the group consisting of IgG1, IgG2, IgG3 and IgG4.

25. 25. The antibody or antigen-binding fragment thereof of claim 23 or 24, wherein the heavy chain constant region is a human heavy chain constant region.

26. The antibody or antigen-binding fragment thereof of any one of claims 1 to 25, wherein the antibody or antigen-binding fragment thereof comprises a human light chain constant region.

27. 26. An isolated polynucleotide encoding the antibody or antigen-binding fragment thereof, its HCVR, its LCVR, its light chain, its heavy chain, or its antigen-binding fragment thereof according to any one of claims 1 to 25.

28. 28. An expression vector comprising the polynucleotide of claim 27.

29. 29. A recombinant cell comprising the polynucleotide of claim 27 or the expression vector of claim 28.

30. A method for producing a polypeptide selected from the group consisting of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 26, its HCVR, its LCVR, its light chain, its heavy chain, and its antigen-binding fragment, comprising:

30. The method comprising expressing the polypeptide in a recombinant cell of claim 29, and optionally further comprising isolating the expressed polypeptide.

31. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 26 and a pharma- ceutically acceptable carrier or diluent.

32. 32. The pharmaceutical composition of claim 31, wherein the antibody or antigen-binding fragment thereof is in an amount effective to: (a) specifically bind to human or cynomolgus monkey A2aR on the cell surface; (b) reduce cAMP concentration in tissue; (c) inhibit activity of human A2aR; (d) reduce phosphorylation of cAMP responsive elements in the A2aR signaling pathway; (e) improve immune response of immune cells; (f) reduce activity of protein kinase A; or (g) any combination of (a)-(f).

33. 33. A method for inhibiting activity of A2aR expressed on the surface of a cell, comprising inhibiting said A2aR activity in said cell by contacting said cell with an isolated antibody or antigen-binding fragment thereof of any one of claims 1 to 26, or a pharmaceutical composition of claim 31 or 32.

34. 33. A method for enhancing an immune response in a subject, comprising administering to the subject an isolated antibody or antigen-binding fragment thereof of any one of claims 1 to 26, or a pharmaceutical composition of claim 31 or 32, thereby enhancing the immune response in the subject.

35. 33. A method for inhibiting the growth of a tumor in a subject, comprising administering to the subject an isolated antibody or antigen-binding fragment thereof of any one of claims 1 to 26, or a pharmaceutical composition of claim 31 or 32, thereby inhibiting the growth of the tumor.

36. 34. A method for treating cancer in a subject, comprising administering to the subject an isolated antibody or antigen-binding fragment thereof of any one of claims 1 to 26, or a pharmaceutical composition of claim 31 or 32, thereby treating the cancer.

37. The method according to any one of claims 33 to 36, wherein said method activates and induces T cells to kill tumor target cells.

38. 38. The method of any one of claims 33 to 37, further comprising administering to the subject an additional therapeutic agent.

39. 39. The method of claim 38, wherein the additional therapeutic agent comprises an anti-tumor agent, radiation therapy, a chemotherapeutic agent, surgery, a cancer vaccine, an agonist for a stimulatory receptor on an immune cell, a cytokine, cell therapy, or a checkpoint inhibitor.

40. The checkpoint inhibitor is selected from the group consisting of CTLA-4, PD-1, PD-L1, PD-L2, TIGIT, LAG-3, TIM-3, B7-H3, B7-H4, CD73, PVRIG / PVRL2, neuritin, BTLA, CECAM-1, CECAM-5, CECAM6, IL-1R8, VISTA, LAIR1, LILRB1, LILRB2, LILRB3, and LI 40. The method of claim 39, wherein the agent inhibits LRB4, LILRB5, CD47, SIRPa, CD200R, CD96, CD112R, 2B4, TGFβ-R, KIR, NKG2A, SEMA4D, Axl, MerTK, GAS6, TNFR2, GARP, CCR8, IDO, NOX2, SIGLEC7, SIGLEC15, and / or combinations thereof.

41. 41. The method of claim 40, wherein the agent inhibits the interaction between PD-1 and PD-L1 and is selected from the group consisting of pembrolizumab, nivolumab, atezolizumab, avelumab, durvalumab, BMS-936559, sintilimab, toripalimab, ticilelitumab, camrelizumab, embafolimab, sugemalimab, penprimab, cadnilimab, sulfamonomethoxine, and sulfamethizole.

42. The method of claim 40, wherein the CTLA-4 inhibitor is ipilimumab, kadonilimab, YH001 (Eucure Biopharma), ADG116, or ADG126 (Tianyan Pharmaceuticals).

43. 40. The method of claim 39, wherein the additional therapeutic agent is an agonist for an immune cell stimulatory receptor selected from the group consisting of OX40, CD2, CD27, CD3, ICAM-1, LFA-1, ICOS (CD278), 4-1BB (CD137), GITR, CD28, CD30, CD40, BAFFR, HVEM, CD7, LIGHT, NKG2C, NKG2D, SLAMF7, NKp46, NKp80, CD160, and combinations thereof.

44. The method of any one of claims 38 to 43, wherein the additional therapeutic agent is formulated together with the antibody or antigen-binding fragment thereof in the same pharmaceutical composition.

45. The method of any one of claims 38 to 43, wherein the additional therapeutic agent is formulated in a separate pharmaceutical composition from the antibody or antigen-binding fragment thereof.

46. The method of any one of claims 38 to 43 and 45, wherein the additional therapeutic agent is administered before and / or after administration of the antibody or antigen-binding fragment thereof.

47. The method of any one of claims 38 to 46, wherein the additional therapeutic agent is administered simultaneously with the antibody or antigen-binding fragment thereof.

48. A kit comprising the isolated antibody or antigen-binding fragment thereof of any one of claims 1 to 26, or the pharmaceutical composition of claim 31 or 32.

49. 49. The kit of claim 48, further comprising an additional therapeutic agent.

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