Anti-adenosine receptor (A2RA) antibodies and uses thereof

Abstract

The invention provides anti-A2aR antigen-binding molecules, including antibodies and antigen-binding fragments thereof, and methods of using the molecules to treat various diseases associated with aberrant adenosine signaling, including cancer, chronic diseases, chronic infections, autoimmune diseases, inflammatory diseases, neurodegenerative diseases, and fibrotic diseases.

Description

[0001] This application claims priority to U.S. Provisional Application No. 63 / 325,578, filed on 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 on September 30, 2021. The entire contents of the above application are expressly incorporated herein by reference. Technical Field

[0003] The present invention relates to antibodies, and in particular to antibodies against adenosine receptor A2aR that can be used to treat a variety of diseases, including cancer. Background Art

[0004] The immune system plays an important role in the recognition and elimination of tumor cells. Tumor cells use various mechanisms to evade immune-mediated tumor cell destruction. Among these pathways, tumor cells use adenosine (a highly potent inhibitor of effector T cell function) receptor signaling pathways to increase adenosine levels and responsiveness to adenosine to evade immune defense.

[0005] Adenosine is a purine nucleoside produced by the degradation of adenosine triphosphate (ATP). Under adverse conditions (such as hypoxia, ischemia, inflammation or cancer), the level of adenosine outside the cell increases significantly. Once released, adenosine activates cell signaling pathways by interacting with four known G protein-coupled receptors, namely 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. CD39 and CD73 are two extracellular enzymes that work synergistically in a two-step reaction to convert pro-inflammatory ATP into immunosuppressive adenosine. CD39 hydrolyzes ATP into AMP, which is further hydrolyzed by CD73 into adenosine, which can easily enter most cells. In addition, when tumor cells undergo cell death due to metabolic or hypoxic stress, they release intracellular stored ATP, which is normally impermeable to the cell, into the extracellular space.

[0007] In the tumor microenvironment, adenosine produced by CD73 promotes tumor cell growth and survival while suppressing anti-tumor immune responses. Cancer cells express high levels of CD73 in tumor tissues, and the accumulation of CD73 is 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, as well as the metastasis and survival of cancer cells. Adenosine binds to A2A (or A2A) on T cells. 2A) receptor (A2aR) and activates intracellular signaling cascades, thereby inhibiting T cell activation and function. A2aR is a member of the adenosine receptor group of G protein-coupled receptors and is an anti-inflammatory effector of extracellular adenosine. It is mainly expressed in brain and lymphoid tissue cells. This group of receptors also includes A1R, A2bR and A3R.

[0008] Abnormally high concentrations of adenosine in the tumor microenvironment activate A2aR and trigger a negative feedback loop that allows tumors to evade immune recognition. Specifically, adenosine-mediated activation of A2aR enables tumors to escape immune surveillance by inhibiting the production of IFNγ, suppressing the expression of multiple anti-tumor immune cells (including CD8 + T cells, dendritic cells, natural killer cells, and M1 macrophages), while enhancing the activity of immunosuppressive cell types (including myeloid-derived suppressor cells (MDSCs) and T regulatory (T reg ) cells). Activation of A2aRs on tumor cells is also believed to promote tumor cell metastasis.

[0009] Although some small molecule antagonists of the A2A receptor have entered clinical trials for the treatment of Parkinson's disease and cancer, there is still a lack of biopharmaceutical candidates to block A2aR in cancer treatment. Mice treated with A2aR antagonists (such as ZM241385) showed a significant delay in tumor growth due to reduced immunosuppression of effector T cells. This was further highlighted by the fact that A2aR knockout mice showed stronger tumor rejection. In addition, compared with blocking a single PD-1 / PD-L1 or CTLA-4 pathway, combining small molecule antagonists to block A2aR with monoclonal antibodies to inhibit PD-1 / PD-L1 or CTLA-4 had a synergistic effect in improving the immune response.

[0010] Therefore, regulating the activity of A2aR, the concentration of adenosine and / or the expression of CD39 / CD73 and activating effector immune cells in the tumor microenvironment is an attractive therapeutic strategy that can limit tumor progression, improve anti-tumor immune responses, avoid treatment-induced immune deviations, and potentially limit toxicity to normal tissues. There is a need in the art for compositions and methods for treating cancer by regulating (e.g., inhibiting) the A2aR activity of immune cells. Summary of the invention

[0011] The present invention provides an antigen binding molecule (e.g., an anti-A2aR antibody or an antigen binding fragment thereof) for regulating (e.g., enhancing or inhibiting) the activity of A2aR by specifically binding to A2aR. The A2aR may be on the surface of a cell, such as a mammalian cell (e.g., a mammalian immune cell (e.g., a mouse immune cell, a cynomolgus monkey immune cell, or a human immune cell)). The present invention also provides a method for using the antigen binding molecule (e.g., an anti-A2aR antibody or an antigen binding fragment thereof) of the present invention to regulate (e.g., inhibit) the activity of A2aR, or a method for treating a subject (e.g., a subject suffering from or susceptible to an A2aR-related disease) that benefits from regulating (e.g., inhibiting) the activity of A2aR. In certain embodiments, the antigen binding molecule (e.g., an anti-A2aR antibody or an antigen binding fragment thereof) of the present invention is humanized.

[0012] Therefore, in one aspect, the present invention provides an isolated antigen-binding molecule, such as an antibody or an antigen-binding fragment thereof, that binds to a human adenosine A2A receptor (A2aR). The antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region comprises three heavy chain complementarity-determining regions (CDRs) from N-terminus to C-terminus: HCDR1, HCDR2 and HCDR3; and the light chain variable region comprises three light chain complementarity-determining regions (CDRs) from N-terminus to C-terminus: LCDR1, LCDR2 and LCDR3. Wherein, (a) HCDR1 comprises the amino acid sequence GFX1FTX2X3WMN (SEQ ID NO:), wherein X1 is A or T, X2 is R or S; and X3 is F or Y; (b) HCDR2 comprises the amino acid sequence RIDPX4DSEX5X6YX7X8KFWX9 (SEQ ID NO:), 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) HCDR3 comprises the amino acid sequence GRSLYGKGDY (SEQ ID NO: 10) or LRSLYGKGDY (SEQ ID NO: 11); (d) LCDR1 comprises the amino acid sequence RSSQSX 11 VX 12 NGNTYLE (SEQ ID NO: ), wherein X 11 is L or I, and X 12is R or S; (e) LCDR2 comprises the amino acid sequence KVSNRFS (SEQ ID NO: 14); and (f) LCDR3 comprises the amino acid sequence FQGSHVPLT (SEQ ID NO: 15) or YQGSHVPLT (SEQ ID NO: 16); wherein the antibody or antigen-binding fragment thereof comprises a light chain framework region 4 (LFR4) comprising the amino acid sequence FGGGTKVEIK (SEQ ID NO: 50). In one embodiment, (a) HCDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, and 3; (b) HCDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 4-9 and 89; (c) HCDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 10 or 11; (d) LCDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 12 or 13; (e) LCDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 14; and (f) LCDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 15 or 16.

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

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

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

[0016] In one embodiment of the various aspects of the present invention and any embodiment 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 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 2 (HFR2) comprising the amino acid sequence WVRQ-X17-PG-X18-GLEW-X19-G (SEQ ID NO:). 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 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 K or T, and X24 is S; and (d) a heavy chain framework region 4 (HFR4) comprising the amino acid sequence WGQGTTVTVSS (SEQ ID NO:32) or WGHGTTVTVSS (SEQ ID NO:33). In another embodiment, (a) HFR1 comprises an amino acid sequence selected from SEQ ID NOs:17-21; (b) HFR2 comprises an amino acid sequence selected from SEQ ID NOs:22, 23 and 24; (c) HFR3 comprises an amino acid sequence selected from SEQ ID NOs:26-31; and (d) HFR4 comprises an amino acid sequence shown in SEQ ID NOs:32 or 33.

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

[0018] In one embodiment of the various aspects of the present invention and any 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 D-X26-VMTQ-X27-P-X28-SL-X29-V-X30-X31-G-X32-PASISC (SEQ ID NO:) 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; (b) a light chain framework region 2 (LFR2) comprising the amino acid sequence W-X33-X34-Q-X35-PGQ-X36-P-X37-X38-LIY (SEQ ID NO:) NO:), 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) light chain framework region 3 (LFR3), which comprises the amino acid sequence GVPDRFSGSGSG-X39-DFTL-X40-IS-X41-X42-X43-AEDV-X44-VYYC (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. In one embodiment, (a) LFR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 34-41; (b) LFR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 42-46; and (c) LFR3 comprises an amino acid sequence selected from the group consisting of 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, which comprises a structure of LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4 from N-terminus to C-terminus, wherein LFR1, LFR2, LFR3 and LFR4 respectively comprise: (a) the sequence shown in SEQ ID NO:34, SEQ ID NO:42, SEQ ID NO:47 and SEQ ID NO:50; (b) the sequence shown in SEQ ID NO:34, SEQ ID NO:43, SEQ ID NO:47 and SEQ ID NO:50; (c) the sequence shown in SEQ ID NO:34, SEQ ID NO:44, SEQ ID NO:47 and SEQ ID NO:50; (d) the sequence shown in SEQ ID NO: 35, SEQ ID NO: 45, SEQ ID NO: 47 and SEQ ID NO: 50; (e) the sequence shown in SEQ ID NO: 36, SEQ ID NO: 46, SEQ ID NO: 48 and SEQ ID NO: (i) the sequence shown in SEQ ID NO:40, SEQ ID NO:45, SEQ ID NO:49 and SEQ ID NO:50; or (j) the sequence shown in SEQ ID NO:41, SEQ ID NO:45, SEQ ID NO:47 and SEQ ID NO:50.

[0020] In one embodiment, the isolated antibody or antigen-binding fragment thereof comprises: (a) a heavy chain variable region (HCVR), which comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 51-70; and (b) a light chain variable region (LCVR), which comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 71-88.

[0021] In one aspect, the invention provides antigen binding molecules, such as isolated antibodies or antigen binding fragments thereof, that bind to human adenosine A2A receptor (A2aR). The antibody or antigen-binding fragment thereof comprises a heavy chain variable (VH) domain, which comprises three heavy chain complementary determining regions (CDRs) from N-terminus to C-terminus: HCDR1, HCDR2 and HCDR3; and a light chain variable (VL) domain, which comprises three light chain complementary determining regions (CDRs) from N-terminus to C-terminus: LCDR1, LCDR2 and LCDR3; wherein (a) HCDR1 comprises an amino acid sequence that is about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2 and 3; (b) HCDR2 comprises an amino acid sequence that is about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 4-9 and 89; (c) HCDR3 comprises an amino acid sequence that is about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 4-9 and 89; NO:10 or 11 has an amino acid sequence that is about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identical; (d) LCDR1 comprises an amino acid sequence that is about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identical to the amino acid sequence selected from SEQ ID NOs:12 and 13; (e) LCDR2 comprises an amino acid sequence that is about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identical to the amino acid sequence selected from SEQ ID NOs:14; and (f) LCDR3 comprises an amino acid sequence that is about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identical to the amino acid sequence selected from SEQ ID NOs:15 and 16. The amino acid sequences shown in NOs:15 and 16 have amino acid sequences that are about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identical; and wherein the antibody or antigen-binding fragment thereof comprises a light chain framework region 4 (FR4), which comprises an amino acid sequence that is about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identical to the sequence FGGGTKVEIK (SEQ ID NO:50).In one embodiment, the heavy chain variable (VH) domain comprises four framework regions (FRs) from N-terminus to C-terminus: HFR1, HFR2, HFR3, and HFR4; and the light chain variable (VL) domain comprises four framework regions (FRs) from N-terminus to C-terminus: LFR1, LFR2, LFR3, and LFR4, wherein (a) HFR1 comprises an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 17-21; (b) HFR2 comprises an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 22, 23, and 24; (c) HFR3 comprises an amino acid sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 23, 24, and 25; The amino acid sequences shown in SEQ ID NOs:25-31 have an amino acid sequence that is about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identical; (d) HFR4 comprises an amino acid sequence that is about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs:32 and 33; (e) LFR1 comprises an amino acid sequence that is about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs:34-41; (f) LFR2 comprises an amino acid sequence that is about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: The amino acid sequences shown in NOs:42-46 have an amino acid sequence that is about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identical; and (g) LFR3 comprises an amino acid sequence that is about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identical to an amino acid sequence selected from SEQ ID NOs:47, 48 and 49.

[0022] In one aspect, the present invention provides an antigen binding molecule, such as an isolated antibody or antigen binding fragment thereof that binds to a 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 about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identical to an amino acid sequence selected from SEQ ID NOs: 51-70; and (b) a light chain variable region (LCVR) comprising an amino acid sequence about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identical to an amino acid sequence selected from SEQ ID NOs: 71-88.

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

[0024] In one embodiment of the various aspects of the present invention and any of its embodiments, the N-terminus of the heavy chain and / or light chain of the isolated antibody or antigen-binding fragment thereof is a pyroglutamic acid (pE) residue. In another embodiment, A2aR is human A2aR.

[0025] In another embodiment, the isolated antibody or antigen-binding fragment thereof (i) competes with a monoclonal antibody selected from 1B5-3D7, 3F6-9G5 and 3F8-12E9 for binding to human A2aR; (ii) inhibits the 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 the activity of protein kinase A; (vii) reduces the phosphorylation of cAMP response elements in the A2aR signaling pathway; (viii) specifically binds to human and / or cynomolgus monkey A2aR; (ix) induces A2aR endocytosis; or (x) achieves any combination of (i)-(ix).

[0026] In one embodiment, the binding of the antibody to A2aR or cell surface A2aR is determined using a flow cytometry assay described in Examples 2 and 5 or an assay substantially similar thereto. In another embodiment, the competitive binding of the antibody to A2aR or cell surface A2aR is determined using an assay known in the art, including, for example, an assay described by Harms et al. in 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 an assay substantially similar thereto. In yet another embodiment, the reduction in cAMP concentration is determined using an assay described in Example 3 or an assay substantially similar thereto. In yet another embodiment, the inhibition of A2aR activity is determined using an assay described in Example 4 or an assay substantially similar thereto. In one embodiment, the reduction of protein kinase A activity and / or the reduction of phosphorylation of the cAMP response element of the A2aR signaling pathway are determined by the method described in Karege et al. in Anon-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 substantially similar method thereof. In another embodiment, the enhancement of the immune response is determined by methods well known in the art. For example, the increase in inflammatory cytokine production by activated immune cells, the increase in the concentration of inflammatory cytokines in tissues, and the activation of CD25 + and / or CD69 + CD4 + T cells and cytotoxic CD8 + Increase in the number of T cells.

[0027] In one aspect, the present invention provides an antigen binding molecule, such as an isolated antibody or antigen binding fragment thereof, which competes for binding to human A2aR with the antibody or antigen binding fragment thereof according to any of the preceding aspects of the present invention and any embodiments thereof.

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

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

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

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

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

[0033] In another embodiment, the present invention provides a pharmaceutical composition. The pharmaceutical composition comprises the antibody and antigen-binding fragment thereof of the various aspects of the present invention and any embodiment thereof, and a pharmaceutically acceptable carrier or diluent. In one embodiment, the pharmaceutical composition comprises an effective amount of the antibody or antigen-binding fragment thereof, so that it (a) specifically binds to the human or cynomolgus monkey A2aR on the cell surface; (b) reduces the concentration of cAMP in the tissue; (c) inhibits the activity of human A2aR; (d) reduces the phosphorylation of the cAMP response element of the A2aR signaling pathway; (e) improves the immune response of immune cells; (f) reduces the activity of protein kinase A; or (g) achieves any combination of (a)-(f).

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

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

[0036] In yet another aspect, the present invention provides a method for inhibiting tumor growth in a subject. The method comprises administering to the subject an isolated antibody or antigen-binding fragment thereof of various aspects of the present invention and any embodiment thereof, or a pharmaceutical composition of various aspects of the present invention and any embodiment thereof, thereby inhibiting the growth of the tumor. 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 present invention provides a method for treating cancer in a subject, comprising administering to the subject an isolated antibody or antigen-binding fragment thereof according to various aspects of the present invention and any embodiment thereof, or a pharmaceutical composition according to various aspects of the present invention and any embodiment thereof, thereby treating cancer.

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

[0039] In one embodiment of the various 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-neoplastic agent, radiation therapy, a chemotherapeutic agent, surgery, a cancer vaccine, an agonist of an immune cell stimulating receptor, a cytokine, a cell therapy, or a checkpoint inhibitor.

[0041] In another embodiment, the checkpoint inhibitor is selected from inhibitors of CTLA-4, PD-1, PD-L1, PD-L2, TIGIT, LAG-3, TIM-3, B7-H3, B7-H4, CD73, PVRIG / PVRL2, mutexin, BTLA, CECAM-1, CECAM-5, CECAM6, IL-1R8, VISTA, LAIR1, LILRB1, LILRB2, LILRB3, LILRB4, LILRB5, CD47, SIRPα, CD200R, CD96, CD112R, 2B4, TGFβ-R, KIR, NKG2A, SEMA4D, Axl, MerTK, GAS6, TNFR2, GARP, CCR8, IDO, NOX2, SIGLEC7, SIGLEC15, and / or any combination thereof.

[0042] In another embodiment, the inhibitor inhibits the interaction between PD-1 and PD-L1, and the inhibitor is selected from pembrolizumab, nivolumab, atezolizumab, avelumab, durvalumab, BMS-936559, sintilimab, toripalimab, tislelizumab, carrelizumab, envolimab, sugemalimab, penampalimumab, candidizumab, sulfamethoxazole and sulfamethoxazole.

[0043] In yet another embodiment, the CTLA4 inhibitor is selected from ipilimumab, cadunolimab, YH001 (Euhe Pharmaceuticals), ADG116 and ADG126 (Tianyan Pharmaceuticals).

[0044] In one embodiment, the additional therapeutic agent is an agonist of 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 any combination thereof.

[0045] In another embodiment, the other therapeutic agent is formulated in the same pharmaceutical composition as the antibody or antigen-binding fragment thereof.

[0046] In yet another embodiment, the other therapeutic agent and the antibody or antigen-binding fragment thereof are formulated in different pharmaceutical compositions.

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

[0048] In one aspect, the present invention provides a kit. The kit comprises an isolated antibody or antigen-binding fragment thereof according to various aspects of the present invention and any embodiment thereof, or a pharmaceutical composition according to various aspects of the present invention and any embodiment thereof. In one embodiment, the kit comprises other therapeutic agents. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1A and 1B Shows that exemplary humanized anti-A2aR antibodies of the present invention and 3F6-9G5 antibody ('601 publication) block the activity of human A2aR expressed on the cell surface. RLU (Relative Light Unit): Relative Light Unit. Hu3F6-VH3 / VL5: Hu3F6-H3 / Hu3F6-L5, Hu3F6-VH5 / VL5: Hu3F6-H5 / Hu3F6-L5.

[0050] Figure 2 Exemplary humanized anti-A2aR antibodies of the invention and the 3F6-9G5 antibody ('601 publication) are shown to induce endocytosis after binding to cell surface A2aR. MFI (Mean Fluorescence Intensity): Mean fluorescence intensity. Hu3F6-VH3 / VL5: Hu3F6-H3 / Hu3F6-L5, Hu3F6-VH5 / VL5: Hu3F6-H5 / Hu3F6-L5.

[0051] Figure 3 Exemplary humanized anti-A2aR antibodies of the invention and the 3F6-9G5 antibody ('601 publication) were shown to be localized inside cells after incubation with cells expressing human A2aR. Hu3F6-VH3 / VL5: Hu3F6-H3 / Hu3F6-L5; Hu3F6-VH5 / VL5: Hu3F6-H5 / Hu3F6-L5.

[0052] Figure 4A and 4B Exemplary humanized anti-A2aR antibodies of the invention and the 3F6-9G5 antibody ('601 publication) were shown to bind to a variety of human and cynomolgus CD4+ and CD8+ T cells. Hu3F6-VH3 / VL5: Hu3F6-H3 / Hu3F6-L5; Hu3F6-VH5 / VL5: Hu3F6-H5 / Hu3F6-L5.

[0053] Figure 5A and 5B It was shown that in the presence of 0.1 μM NECA, the exemplary humanized anti-A2aR antibodies of the invention and the 3F6-9G5 antibody ('601 publication) can restore intracellular IFNγ and IL2 production by CD4+ T cells activated by immunomagnetic beads (Dynabeads). NECA is an A2aR agonist that can induce immunosuppression, such as inhibition of T cell function. Hu3F6-VH3 / VL5: Hu3F6-H3 / Hu3F6-L5; Hu3F6-VH5 / VL5: Hu3F6-H5 / Hu3F6-L5. DETAILED DESCRIPTION

[0054] The present invention and the accompanying drawings are now described to enable those skilled in the art to practice the invention. However, it should be understood by those skilled in the art that the invention described below may be practiced without the use of these specific details, or that it may be used for purposes other than those described herein. Indeed, in view of this disclosure, it may be modified and used in conjunction with products and techniques known to those skilled in the art. The accompanying drawings and descriptions are intended to illustrate various aspects of the present invention and are not intended to narrow the scope of the claims. In addition, it should be understood that the accompanying drawings may show various aspects of the present invention individually, and that elements in one figure may be used in conjunction with elements shown in other figures.

[0055] It should be understood that references to aspects, features, advantages, or similar language throughout this specification do not mean that all aspects and advantages can be achieved by the present invention, nor do they mean that they should or have been achieved in any single embodiment of the present invention. Instead, language referencing aspects and advantages should be understood as meaning that the specific aspects, features, advantages, or properties described in connection with an embodiment should be included in at least one embodiment of the present invention. Therefore, discussion of aspects and advantages and similar language throughout this specification may, but do not necessarily, refer to the same embodiment.

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

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

[0058] Furthermore, any cited reference, any issued patent, or patent application publication described in this application is expressly incorporated herein by reference for its teachings of the present invention.

[0059] I. Definition

[0060] In order that the present invention may be more readily understood, certain terms are first defined. In addition, it should be noted that whenever a parameter value or a range of values ​​is listed, it is intended to indicate that the values ​​and ranges intermediate to the listed values ​​are also part of the present invention.

[0061] The terms "a" and "an" and "the" and similar designators used in the context of describing the invention (particularly in the context of the claims) should be interpreted as covering both the singular and the plural (i.e., one or more), unless otherwise indicated herein or clearly contradicted by context. The terms "comprising," "having," "including," and "containing" should be interpreted as open-ended terms (i.e., meaning "including, but not limited to"), unless otherwise indicated herein. Recitation of numerical ranges herein is intended merely to serve as a shorthand method of referring individually to each individual value cited or to each individual value falling within the range, and each individual value is incorporated into the specification as if it were individually recited, unless otherwise indicated herein.

[0062] When the phrases "in one embodiment," "in another embodiment," "in other embodiments," "in some embodiments," or "in certain embodiments" are used, the disclosure should be understood to include any combination of features that define the different embodiments described herein, unless these features cannot be combined with each other, or these features are mutually exclusive or are explicitly disclaimed herein.

[0063] The term "approximately" or "about" refers to a value similar to the reference value when applied to one or more values ​​provided herein. In some embodiments, unless otherwise specified or otherwise apparent from the context, the term "approximately" or "about" refers to a range of values ​​falling within (greater than or less than) 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction of the reference value. In some embodiments, "approximately" or "about" may be understood as differing from the mean by about 2 standard deviations. In some embodiments, "approximately" or "about" refers to up to and including ± 10% (e.g., ± 10%, ± 9%, ± 8%, ± 7%, ± 6%, ± 5%, ± 4%, ± 3%, ± 2%, ± 1% or less). In some embodiments, "approximately" or "about" means ± 5%. When “about” or “approximately” precedes a series of numbers or ranges, it should be understood that “about” can modify each number in the series or range.

[0064] Ranges may be expressed herein as from "about" a particular value, and / or to "about" another particular value. When such a range is expressed, another embodiment includes from one particular value and / or to another particular value. Similarly, when a value is expressed as an approximation by using the antecedent "about", it is understood that the particular value constitutes another embodiment. It should also be understood that the endpoints of each range are both significant and independent relative to the other endpoint. It should also be understood that a large number of values ​​are disclosed herein, and each value also includes the "about" value of the particular value in addition to the value itself. For example, if the value "10" is disclosed, "about 10" is also disclosed. It is also understood that when a value is disclosed, "less than or equal to" (the value), "greater than or equal to" (the value) and the possible range between the values ​​are also disclosed, as appropriately understood by those skilled in the art. For example, if the value "10" is disclosed, "less than or equal to 10" and "greater than or equal to 10" are also disclosed.

[0065] The term "administering" as used herein refers to providing a medicament, such as an antibody, to a subject. In some embodiments, "administering" refers to providing an antibody or antibody conjugate to a subject in a physiologically and / or (e.g., and) pharmacologically useful manner (e.g., to treat a condition of the 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.

[0066] The term "agent" as used herein refers to any substance, compound (e.g., molecule), supramolecular complex, material, or combination or mixture thereof. A compound may be any agent represented by a chemical formula, chemical structure, or sequence. Examples of agents include, for example, small molecules, polypeptides, nucleic acids (e.g., RNAi agents, antisense oligonucleotides, nucleic acid aptamers), lipids, polysaccharides, etc. Generally, agents may be obtained using any suitable method known in the art. A person of ordinary skill will select a suitable method based on, for example, the properties of the agent. The agent may be at least partially purified. In some embodiments, the agent may be provided as part of a composition, and in various embodiments, in addition to the agent, the composition may include, for example, counterions, aqueous or non-aqueous diluents or carriers, buffers, preservatives, or other ingredients. In some embodiments, the agent may be provided in the form of a salt, ester, hydrate, or solvate. In some embodiments, the agent is permeable to cells, within the range of typical agents that are, for example, absorbed by cells and act intracellularly (e.g., act intracellularly to produce biological effects). Certain compounds may exist in specific geometric or stereoisomeric forms. Such compounds include cis and trans isomers, E- and Z-isomers, R- and S-enantiomers, diastereomers, (D)-isomers, (L)-isomers, (-)- and (+)-isomers, racemic mixtures thereof and other mixtures thereof, unless otherwise indicated, and such compounds are included in various embodiments of the present disclosure. Certain compounds may exist in multiple or protonated states, may have multiple configurations, may exist as solvates (e.g., with water (i.e., hydrates) or common solvents) and / or may have different crystalline forms (e.g., polymorphs) or different tautomeric forms. The present disclosure includes embodiments exhibiting such alternative protonation states, configurations, solvates, and forms, where applicable.

[0067] In certain embodiments, "medicament" also includes treatment methods, such as radiation therapy, chemotherapy, or surgery, as understood in context.

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

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

[0070] The term "agonist" refers to a substance that promotes (i.e., induces, causes, enhances, or improves) the biological activity or action of another molecule. The term agonist includes substances that bind to receptors (e.g., antibodies) as well as substances that promote receptor function but do not bind to it (e.g., promote receptor function by activating related proteins).

[0071] As used herein, the term "antibody" refers to any antigen-binding molecule or molecular complex comprising at least one complementary determining region (CDR) that specifically binds to or interacts with a specific antigen (e.g., A2aR). The term "antibody" includes immunoglobulin molecules comprising four polypeptide chains (i.e., two heavy (H) chains and two light (L) chains interconnected by disulfide bonds), and multimers thereof (e.g., IgM). Each heavy chain comprises a heavy chain variable region (abbreviated herein as HCVR, VH, or VH). H ) and the heavy chain constant region. The heavy chain constant region contains three domains, namely, C H 1 or CH1, C H 2 or CH2, and C H 3 or CH3. Each light chain comprises a light chain variable region (abbreviated herein as LCVR, VL or V L ) and the light chain constant region. The light chain constant region consists of a domain (CL or C L ). H and V L The regions can be further subdivided into hypervariable regions, 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 It consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In different embodiments of the present invention, the FRs of the anti-A2aR antibody (or its antigen-binding fragment) may be identical to the mouse or human germline sequence, or may be naturally or artificially modified. The amino acid consensus sequence may be defined based on a side-by-side analysis of two or more CDRs.

[0072] The term "antibody" as used herein also includes the antigen binding fragment of a complete antibody molecule. The term "antigen binding portion" of an antibody, "antigen binding fragment" of an antibody, etc. as used herein include any naturally occurring, enzymatically obtainable, synthetic or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. Any suitable standard technique, such as proteolysis or recombinant genetic engineering techniques (including manipulation and expression of DNA encoding antibody variable regions and optional constant regions), can be used to derive an antigen binding fragment of an antibody from a complete antibody molecule. Such DNA is known and / or readily available, such as commercial sources, DNA libraries (including, for example, phage-antibody libraries), or can be synthesized. The DNA can be sequenced and manipulated by chemical or molecular biological techniques, for example, one or more variable regions and / or constant regions are arranged in a suitable configuration, or codons are introduced, cysteine ​​residues are produced, amino acids are modified, added or deleted, etc.

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

[0074] The antigen-binding fragment of an antibody generally comprises at least one variable domain. The variable domain may be of any size or any amino acid composition and generally comprises at least one CDR adjacent to or in frame with one or more framework sequences. H Domain and V L In the antigen-binding fragment with connected domains, V H and V L The domains may be positioned relative to each other in any suitable arrangement. For example, the variable region may be a dimer and contain V H -V H 、V H -V L or V L -V LAlternatively, the antigen-binding fragment of an antibody may contain a monomeric VH or VL domain.

[0075] In certain embodiments, the antigen-binding fragment of an antibody may contain at least one variable domain covalently linked to at least one constant domain. Non-limiting exemplary configurations of variable and constant domains comprised by the antigen-binding fragment of an antibody of the invention include: (i) V H -C H 1; (ii) V H -C H 2; (iii) V H -C H 3; (iv) V H -C H 1-C H 2; (v) V H -C H 1-C H 2-C H 3; (vi) V H -C H 2-C H 3; (vii) V H -C L ; (viii) V L -C H 1; (ix) V L -C H 2; (x) V L -C H 3; (xi) V L -C H 1-C H 2; (xii) V L -C H 1-C H 2-C H 3; (xiii) V L -C H 2-C H 3; and (xiv) V L -C L . In any configuration of variable domains and constant domains (including any of the exemplary configurations listed above), the variable domains and constant domains may be directly connected to each other or may be connected by a complete or partial hinge or linker region. The hinge region may contain at least 2 (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids, thereby forming a flexible or semi-flexible connection between adjacent variable and / or constant domains in a single polypeptide molecule. In addition, the antigen-binding fragment may contain homodimers or heterodimers (or other multimers) of any of the variable domain and constant domain configurations listed above, which are non-covalently connected to each other and / or to one or more monomer V H or VL The domains are non-covalently linked (eg, via disulfide bonds). When hyphens are used between individual domains (eg, CH2-CH3) to describe a polypeptide domain arrangement, it is understood that the order in which the domains are listed is from N-terminus to C-terminus.

[0076] As with complete antibody molecules, antigen-binding fragments can be monospecific or multispecific (e.g., bispecific). Multispecific antigen-binding fragments of antibodies typically contain at least two different variable domains, each of which is capable of specifically binding to a separate antigen or a different epitope on the same antigen. By using conventional techniques available in the art, any multispecific antibody format (including the exemplary bispecific antibody formats disclosed herein) can be applied to the case of antigen-binding fragments of the antibodies of the present invention.

[0077] Antibodies of the present invention can be separated antibodies. "Separated" molecules used herein, such as separated antibodies or separated polypeptides, refer to molecules, such as antibodies, that have been identified and separated and / or recovered from at least one component of their natural environment. For example, molecules (such as antibodies) separated or removed from at least one component of an organism, or from tissues or cells that naturally exist or naturally produce antibodies are "separated" molecules (such as antibodies) for the purposes of the present invention. Separated molecules (such as antibodies) also include in situ molecules (such as antibodies) in recombinant cells. In certain embodiments, separated molecules (such as antibodies) are molecules (such as antibodies) that have been through at least one purification or separation step. According to certain embodiments, separated molecules (such as antibodies) may be substantially free of other cell materials and / or chemicals.

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

[0079] The anti-A2aR antibodies or antigen-binding domains thereof described herein may include one or more amino acid substitutions, insertions and / or deletions in the framework regions and / or CDR regions of the heavy and light chain variable domains thereof compared to the corresponding germline sequences from which the antigen-binding molecule or antigen-binding domain is derived. Such mutations can be easily determined by comparing the amino acid sequences disclosed herein with germline sequences that can be obtained, for example, from public antibody sequence databases. The present invention includes antibodies and antigen-binding domains thereof obtained from any amino acid sequence disclosed herein, wherein one or more amino acids within one or more framework regions and / or CDR regions are mutated to the corresponding residues in the germline sequence from which the antibody is derived, or the corresponding residues of another human germline sequence, or conservative amino acid substitutions of the corresponding germline residues (such sequence changes are collectively referred to herein as "germline mutations"). A person of ordinary skill in the art can easily prepare a large number of antibodies and antigen-binding fragments starting with the heavy and light chain variable region sequences disclosed herein, which contain one or more individual germline mutations or combinations thereof. In certain embodiments, V H and / or V L All framework regions and / or CDR residues in the domain are mutated back to the residues in the original germline sequence of the antibody source. In other embodiments, only some residues are mutated back to the original germline sequence, for example, only in the first 8 amino acids of FR1 or in the last 8 amino acids of FR4, or only in CDR1, CDR2 or CDR3. In other embodiments, one or more framework regions and / or CDR residues are mutated to the corresponding residues of different germline sequences (i.e., germline sequences different from the germline sequences of the antibody source). In addition, the antibody of the present invention or its antigen-binding domain can comprise any combination of two or more germline mutations in the framework region and / or CDR region, for example, wherein some individual residues are mutated to the corresponding residues of a specific germline sequence, and some other residues different from the original germline sequence are retained or mutated to the corresponding residues of different germline sequences. Once an antibody or its antigen-binding fragment containing one or more germline mutations is obtained, it can be easily tested for one or more desired properties, such as improved binding specificity, increased binding affinity, improved or enhanced antagonism or exciting biological properties (as the case may be), reduced immunogenicity, etc. Antibodies or antigen-binding fragments thereof obtained in this general manner are encompassed by the present invention.

[0080] The invention also includes anti-A2aR antibodies comprising variants of any HCVR, LCVR and / or CDR amino acid sequences disclosed herein. Exemplary variants encompassed by this aspect of the invention include variants of any HCVR, LCVR and / or CDR amino acid sequences disclosed herein having one or more conservative substitutions. For example, the invention includes an anti-A2aR antibody and antigen binding protein having a HCVR, LCVR and / or CDR amino acid sequence comprising 10 or fewer, 8 or fewer, 6 or fewer, 4 or fewer, etc., conservative amino acid substitutions relative to any HCVR, LCVR and / or CDR amino acid sequence listed in the Tables herein.

[0081] Light chains are classified as either kappa or lambda (κ, λ). Each heavy chain class can be combined with a kappa or lambda light chain. Generally, light and heavy chains are covalently bonded to each other, and when immunoglobulins are produced by hybridomas, B cells, or genetically engineered host cells, the "tails" of the two heavy chains are bonded to each other by covalent disulfide bonds or non-covalent bonds. In the heavy chain, its amino acid sequence is from the N-terminus at the end of the Y-configuration bifurcation to the C-terminus at the bottom of each chain.

[0082] As used herein, the terms "light chain constant region" and "CL" are used interchangeably herein to refer to an amino acid sequence derived from an antibody light chain. Preferably, the light chain constant region comprises at least one of a constant kappa domain or a constant lambda domain.

[0083] The term "heavy chain constant region" as used herein includes an amino acid sequence derived from an immunoglobulin heavy chain. The polypeptide comprising the heavy chain constant region includes at least one of the following groups: a CH1 domain, a hinge (e.g., the upper, middle and / or lower part of the hinge region) domain, a CH2 domain, a CH3 domain, or a variant or fragment thereof. For example, the antigen-binding polypeptide used in the present disclosure may include a polypeptide chain comprising a CH1 domain; a polypeptide chain comprising a CH1 domain, at least a portion of a hinge domain and a CH2 domain; a polypeptide chain comprising a CH1 domain and a CH3 domain; a polypeptide chain comprising a CH1 domain, at least a portion of a hinge domain and a CH3 domain, or a polypeptide chain comprising a CH1 domain, at least a portion of a hinge domain, a CH2 domain and a CH3 domain. In some embodiments, the polypeptide of the present invention includes a polypeptide chain comprising a CH3 domain. In addition, the antibodies used in the present disclosure may lack at least a portion of a CH2 domain (e.g., all or part of a CH2 domain). It should be understood that the heavy chain constant region may be modified so that it differs from a naturally occurring immunoglobulin molecule in terms of amino acid sequence.

[0084] The heavy chain constant region of the antibodies disclosed herein may be derived from different immunoglobulin molecules. For example, the heavy chain constant region of the polypeptide may include 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 may include a hinge region that is partially derived from an IgG1 molecule and partially derived from an IgG3 molecule. In another example, the heavy chain portion may include a chimeric hinge that is partially derived from an IgG1 molecule and partially derived from an IgG4 molecule.

[0085] A "light chain-heavy chain pair" refers to a collection of light chains and heavy chains, wherein the light chains and heavy chains can form a dimer via a disulfide bond 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. The term "VH domain" as used herein includes the N-terminal variable domain of an immunoglobulin heavy chain, and the term "CH1 domain" includes the first (most N-terminal) constant region domain of an immunoglobulin heavy chain. The CH1 domain is adjacent to the VH domain and is the N-terminal of the hinge region of an immunoglobulin heavy chain molecule.

[0087] The term "CH2 domain" as used herein includes the portion of the heavy chain molecule extending from, for example, about residue 244 to residue 360 ​​of an antibody, wherein the numbering uses the conventional numbering scheme (residues 244 to 360, Kabat numbering system; or residues 231-340, EU numbering system). The uniqueness of the CH2 domain is that it is not closely paired with another domain. In contrast, there are two N-linked branched sugar chains inserted between the two CH2 domains of a complete native IgG molecule. The CH3 domain extends from the CH2 domain to the C-terminus of the IgG molecule and comprises about 108 residues.

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

[0089] As used herein, the term "disulfide bond" includes a covalent bond formed between two sulfur atoms. Cysteine ​​contains a thiol group that can form a disulfide bond or bridge with a second thiol group. In most naturally occurring IgG molecules, the CH1 and CL regions are linked by disulfide bonds, and the two heavy chains are linked by two disulfide bonds corresponding to positions 239 and 242 (Kabat numbering system), which are positions 226 or 229 in the EU numbering system.

[0090] The term "antibody" includes various broad classes of polypeptides that can be distinguished biochemically. It will be appreciated by those skilled in the art that heavy chains are classified as alpha, delta, epsilon, gamma and mu (or α, δ, ε, γ and μ) and some subclasses therein (e.g., γ1-γ4). The nature of this chain determines the "class" of the antibody, IgG, IgM, IgA, IgD or IgE, respectively. Immunoglobulin subclasses (isotypes) such as IgG1, IgG2, IgG3, IgG4, etc. have been well characterized and are known to confer functional specialization. In view of the present disclosure, modified forms of each of these classes and isotypes are readily discernible to those skilled in the art and are therefore included within the scope of the present disclosure. All immunoglobulin classes are within the scope of the present disclosure, and the following discussion is generally directed to immunoglobulin molecules of the IgG class.

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

[0092] The term "humanized antibody" as used herein refers to a genetically engineered non-human antibody comprising a human antibody constant domain and a modified non-human variable domain with a high degree of sequence homology to a human variable domain. This can be achieved by transplanting the six non-human antibody complementary determining regions (CDRs) that form an antigen binding site onto a homologous human receptor framework region (FR). In order to rebuild the binding affinity and specificity of the parental antibody, the framework region residues of the parental antibody (i.e., non-human antibody) need to be replaced with human framework region residues (reverse mutations). Structural homology modeling can help identify amino acid residues in the framework region that are important to antibody binding properties. Therefore, a humanized antibody can include a non-human CDR sequence, mainly a human framework region that optionally includes one or more amino acid reverse mutations to a non-human amino acid sequence, and a fully human constant region. Optionally, additional amino acids (which are not necessarily reverse mutations) can be applied to modify to obtain a humanized antibody with preferred features such as affinity and biochemical properties.

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

[0094] "Variable single-chain fragment" or "scFv" refers to a fusion protein of the variable region of the immunoglobulin heavy chain (VH) and the variable region of the light chain (VL). In some aspects, the regions are connected by a short connecting peptide of 10 to about 25 amino acids. The connecting peptide can be rich in glycine for flexibility, rich in serine or threonine for solubility, and can connect the N-terminus of VH to the C-terminus of VL, and vice versa. Although the constant region is removed and the connecting peptide is introduced in the protein, it still retains the specificity of the original immunoglobulin.

[0095] Regarding IgG, a standard immunoglobulin molecule comprises two identical light chain polypeptides of approximately 23,000 Da, and two identical heavy chain polypeptides of 53,000-70,000 Da. These four chains are usually linked by disulfide bonds in a "Y" configuration, with the light chain surrounding the heavy chain, 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 present in vivo or provided or achieved by recombinant means). Biological activities include, but are not limited to, binding to receptors, inducing cell proliferation, inhibiting cell growth, inducing other cytokines, inducing apoptosis, and enzymatic activity.

[0097] The term "conjugate" or "antibody conjugate" refers to an antibody linked to one or more agents. The antibody can be covalently linked to the agent by a covalent bond or a linker. In certain embodiments, the linker is covalently bound to the antibody and also covalently bound to the agent. In certain embodiments, the linker is non-covalently linked to the antibody and / or the agent. In certain embodiments, the linker is covalently linked to the agent and is linked to the antibody by specific binding. In certain embodiments, the linker is a portion that can specifically bind to the antibody, for example, an antibody that binds to the Fc region of an antibody. In certain embodiments, the conjugate comprises an antibody non-covalently linked to one or more agents.

[0098] The term "control" or "reference" when referring to a substance refers to a composition used as a standard or comparison point, and other test results are measured against this composition. In certain embodiments, a "control" or "reference" is a composition known to be free of analyte ("negative control") or containing analyte ("positive control"). A positive control may contain an analyte of known concentration. "Control" and "positive control" may refer to a composition containing an analyte of known concentration. "Positive control" can be used to establish a measurement performance characteristic and is a usable indicator of the integrity of a pharmaceutical agent (e.g., an analyte). In certain embodiments, an appropriate "control" or "reference" refers to one in which only one element is changed to determine the effect of the element. In certain embodiments, a control refers to the level of a target gene (e.g., in a cell or object) before treatment (e.g., with an RNAi agent described herein).

[0099] The term "control" or "reference" also means a baseline level of measurement, depending on the context in which the term is used. The baseline level of measurement is a standard or comparison point, and the measurement result is compared with it. In certain embodiments, "control" or "reference" refers to the measured level of certain biological activities or substances in cells, tissues, organs or objects without the treatment of pharmaceutical agents (e.g., RNAi agents), such as the expression level of a gene, the number of mRNA copies of the gene, or the level of protein encoded by the gene. In some embodiments, "control" or "reference" refers to the average measured level of a certain biological activity or substance (e.g., a certain enzyme activity of the liver) in a group of healthy subjects (e.g., within certain geographic or population restrictions or any other restrictions that may be suitable for studying certain diseases or conditions, and the population does not suffer from certain diseases or conditions, such as liver disease).

[0100] The term "control" may be used for a "control individual," which is an individual with a similar condition, e.g., an individual with the same cell proliferative disorder as the individual being treated, who is about the same age as the individual being treated (to ensure that the stage of the disease is comparable in the treated and control individuals). The individual being treated (also referred to as a "patient" or "subject") can be a fetus, infant, child, adolescent, or adult with a cell proliferative disorder.

[0101] The term "reference" may also be used for a "reference sequence." The term "reference sequence" refers to a sequence, such as a nucleic acid sequence or an amino acid sequence, used as a 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 a paratope. A single antigen may have more than one epitope. Thus, different antibodies may bind to different regions of the antigen and may have different biological effects. Epitopes may be conformational or linear. Conformational epitopes are produced by the juxtaposition of amino acids in space in different segments of a linear polypeptide chain. Linear epitopes are produced by adjacent amino acid residues in a polypeptide chain. In some cases, an epitope may include a sugar, phosphoryl, or sulfonyl moiety on the antigen.

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

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

[0105] As used herein, the terms "improve," "increase," or "decrease" refer to a value or parameter relative to a baseline / control / reference measurement, such as measurements in cells or tissues before initiation of a treatment described herein, or measurements in cells or tissues in the absence of a treatment described herein, measurements in the same individual before initiation of a treatment described herein, or measurements in a control individual in the absence of a treatment described herein (or a standard measurement derived from multiple control individuals, such as an average of multiple control individuals).

[0106] In the context of describing the level of activity of an agent, the terms "inhibit," "reduce," and "reduced" refer to a statistically significant reduction in such level. The reduction may be, for example, at least 10%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or below the level of detection by a detection method.

[0107] The term "prevention" refers to reducing the occurrence of disease symptoms in a subject. As described above, the prevention can be complete (no detectable symptoms) or partial, such that fewer symptoms are observed than would occur without treatment.

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

[0109] The phrase "small molecule drug" refers to a molecular entity, usually organic or organometallic, that is not a polymer, has pharmaceutical activity, and has a molecular weight of less than about 2 kDa, less than about 1 kDa, less than about 900 Da, less than about 800 Da, or less than about 700 Da. The term includes most of the pharmaceutical compounds referred to as "drugs" other than proteins or nucleic acids, but small peptides or nucleic acid analogs may be considered small molecule drugs. Examples include chemotherapeutic anticancer drugs and enzyme inhibitors. Small molecule drugs can be obtained by synthesis, semi-synthesis (i.e., from naturally occurring precursors) or biological methods.

[0110] The term "specific binding" or "specifically binding" as used herein refers to the ability to distinguish possible binding objects in the environment in which the binding occurs. In some embodiments, when other potential antibodies are present, an antibody that interacts with a specific antigen, such as a preferential interaction, is referred to as "specifically binding" the antigen that interacts with it. In some embodiments, specific binding is assessed by detecting or determining the degree of binding between an antibody and its targeted antigen. In some embodiments, specific binding is assessed by detecting or determining the degree of dissociation of an antibody-antigen complex. In some embodiments, specific binding is assessed by detecting or determining the ability of an antibody to compete with another antibody for an alternative interaction with its target. In some embodiments, specific binding is assessed by performing such detection or determination over a certain concentration range. Typically, an antibody binds to an epitope through its antigen binding domain, and the binding requires a certain complementarity between the antigen binding domain and the epitope. Therefore, when an antibody binds to an epitope through its antigen binding domain more easily than it binds to a random, unrelated epitope, the antibody is referred to as "specifically binding" the epitope. The term "specificity" is used herein to define the relative affinity of a specific antibody to bind to a specific epitope. For example, antibody "A" may be considered to have a higher specificity for a given epitope than antibody "B," or antibody "A" may be considered to have a higher specificity for binding epitope "C" than for the related epitope "D." In some embodiments, if the dissociation constant (Kd) of the complex formed by the antibody or antigen-binding fragment thereof with the antigen is 10 -6 M or smaller, 10 -7 M or smaller, 10 -8 M or smaller, 10 -9 M or smaller, or 10 -10 M or less, then the antibody or antibody fragment is "specific for the antigen." In certain embodiments, specific binding of an antigen-binding molecule (e.g., an anti-human A2aR antibody or antigen-binding fragment thereof) is demonstrated by measuring the preferential binding of the antigen-binding molecule to human A2aR expressed on the surface of a cell using the assays described in Examples 4-7, or substantially similar methods.

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

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

[0113] When applied to polypeptides, the terms "substantially identical," "substantially similar," "substantially similar," or "substantially similar" mean that two peptide sequences share at least 95% sequence identity, even more preferably at least 98% or 99% sequence identity, when optimally aligned by a program such as GAP or BESTFIT using default gap weights. Preferably, residue positions that are not identical differ by conservative amino acid substitutions. "Conservative amino acid substitutions" refer to substitutions of one amino acid residue by another amino acid residue having a side chain (R group) of 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 the case where two or more amino acid sequences differ from each other by conservative substitutions, the sequence identity percentage or similarity may be adjusted upward to correct for the conservative nature of the substitution. Methods for making such adjustments are well known to those skilled 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, and isoleucine; (2) aliphatic hydroxyl side chains: serine and threonine; (3) amide-containing side chains: asparagine and glutamine; (4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; (5) basic side chains: lysine, arginine, and histidine; (6) acidic side chains: aspartic acid and glutamic acid, (7) sulfur-containing side chains: cysteine ​​and methionine. Preferred conservative amino acid substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine. Alternatively, a conservative substitution is any change that has a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256:1443-1445. A "moderately conservative" replacement is any change that has a non-negative value in the PAM250 log-likelihood matrix.

[0114] The sequence similarity of polypeptides, also referred to as sequence identity, is usually determined using sequence analysis software. Protein analysis software uses similarity metrics assigned to various substitutions, deletions and other modifications (including conservative amino acid substitutions) to match similar sequences. For example, GCG software includes programs such as Gap and Bestfit, which can use default parameters to determine the sequence homology or sequence identity between closely related polypeptides (e.g., homologous polypeptides from different biological species, or homologous polypeptides of wild-type proteins and their mutant proteins). See, for example, GCG version 6.1. Polypeptide sequences can also be compared using default or recommended parameters by FASTA, which is a program in GCG version 6.1. FASTA (e.g., FASTA2 and FASTA3) provides comparison and sequence identity percentages of the best overlapping regions between query and search sequences (Pearson (2000) supra). When comparing the sequence of the present invention with a database containing a large number of sequences from different organisms, another preferred algorithm is a computer program BLAST using default parameters, especially BLASTP or TBLASTN. See, e.g., Altschul et al., (1990) J. Mol. Biol. 215:403-410 and Altschul et al., (1997) Nucleic Acids Res. 25:3389-402. The term "sequence identity" also refers to a comparison between a pair of nucleic acid or polypeptide molecules, e.g., the relatedness between two nucleotide sequences. Typically, sequences are aligned to obtain the highest order match.

[0115] The terms "treat" and "treat" refer to ameliorating one or more symptoms associated with a disease or condition. Thus, these terms refer to any indication of success in treating or ameliorating an injury, disease, pathology, or condition, including preventing or delaying the onset of one or more symptoms of a disease or condition; reducing the severity or frequency of one or more symptoms of a disease or condition; any objective or subjective parameter, such as reduction; remission; a reduction in symptoms or a better ability of the patient to tolerate the injury, pathology, or condition; a slowing of the rate of degeneration or decline; a reduction in frailty at the degenerative endpoint; and / or improving the patient's physical or mental well-being. "Treatment" may also include prophylactic treatment.

[0116] The phrases "to a patient in need thereof," "to a patient in need of treatment," or "a subject in need of treatment" include subjects (eg, mammalian subjects) who would benefit from administration of an antibody of the disclosure in the treatment of a cell proliferative disorder.

[0117] The terms "therapeutically effective amount", "pharmacologically effective amount", "physiologically effective amount" or "effective amount" are used interchangeably and refer to the amount of an active agent sufficient to improve at least one symptom of a disease or condition. For example, for a given parameter, a therapeutically effective amount will show an increase or decrease of at least 5%, 10%, 15%, 20%, 25%, 40%, 50%, 60%, 75%, 80%, 90%, 95%, 99% or at least 100%. The therapeutic effect can also be expressed as an increase or decrease "fold". For example, a therapeutically effective amount can have an effect of at least 1.2 times, 1.5 times, 2 times, 5 times or more than a control. The exact amount will depend on many factors, such as the specific active agent, the components and physical properties of the composition, the expected patient population, patient considerations, including weight, sex, etc., and can be easily determined by those skilled in the art based on the information provided herein or other information provided in the relevant literature.

[0118] As used herein, the term "variant" refers to a variant that is altered from a precursor polypeptide or polynucleotide (e.g., A polypeptide (e.g., an antibody) or polynucleotide obtained by combining the insertion, substitution, or deletion of one or more amino acids or nucleotides in a "parent" polypeptide or polynucleotide. In certain embodiments, a variant polypeptide or polynucleotide has at least about 85% of the same amino acid or nucleotide sequence as the full amino acid or nucleotide sequence of a parent polypeptide or polynucleotide, such as about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% of the same amino acid or nucleotide sequence. The variant of a protein or peptide substantially retains the structure, function or activity of the protein. For example, a variant of an antibody retains the function or activity of specifically binding to its antigen and / or regulates, such as inhibiting antigen activity. In the case of a polynucleotide, its variant retains the function or activity of its parent polynucleotide. For example, a variant polynucleotide can encode a protein or peptide having a similar function or activity to a polypeptide encoded by a parent polynucleotide.

[0119] As used herein, the term "A2aR" refers to the adenosine A2A receptor. Unless otherwise indicated (e.g., specific reference to human A2aR), the term "A2aR" includes native A2aR from all mammalian species, such as humans, primates, rodents, canines, felines, equines, and bovines. The nucleotide and amino acid sequences of A2aR are known and can be found, 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] The following is an exemplary human A2aR amino acid sequence: MPIMGSSVYITVELAIAVLAILGNVLVCWAVWLNSNLQNVTNYFVVSL AAADIAVGVLAIPFAITISTGFCAACHGCLFIACFVLVLTQSSIFSLLAIAI DRYIAIRIPLRYNGLVTGTRAKGIIAICWVLSFAIGLTPMLGWNNCGQPK EGKNHSQGCGEGQVACLFEDVVPMNYMVYFNFFACVLVPLLLMLGV YLRIFLAARRQLKQMESQPLPGERARSTLQKEVHAAKSLAIIVGLFALC WLPLHIINCFTFFCPDCSHAPLWLMYLAIVLSHTNSVVNPFIYAYRIREF RQTFRKIIRSHVLRQQEPFKAAGTSARVLAAHGSDGEQVSLRLNGHPP GVWANGSAPHPERRPNGYALGLVSGGSAQESQGNTGLPDVELLSHELKGVCPEPPGLDDPLAQDGAGVS(SEQ ID NO:)

[0121] The exemplary human A2aR amino acid sequence is shown below: VPIMGSSVYITVELAIAVLAILGNVLVCWAVWLNSNLQNVTNYFVV SLAAADIAVGVLAIPFAITISTGFCAACHGCLFIACFVLVLTQSSIFSLL AIAIDRYIAIRIPLRYNGLVTGTRAKGIIAICWVLSFAIGLTPMLGWNN CGQPKEGKNHSQGCGEGQVACLFEDVVPMNYMVYFNFFACVLVPL LLMLGVYLRIFLAARRQLKQMESQPLPGERARSTLQKEVHAAKSL AIIVGLFALCWLPLHIINCFTFFCPDCNHAPLWLMYLAIVLSHTNSVV NPFIYAYRIREFRQTFRKIIRSHVLRQQEPFKAAGTSARVLAAHGSDG EQVSLRLNGHPPGVWANGSAPHPERRPNGYALGLVSGGSTQESQGN TSLPDVELLSHELKGVCPEPPGLDDPLAQGGAGVS (SEQ ID NO:)

[0122] The term "anti-A2aR antibody" or "A2aR antibody" refers to an antibody or polypeptide that specifically binds to A2aR. In certain embodiments, the anti-A2aR antibody is capable of inhibiting A2aR biological activity and / or inhibiting downstream signaling pathways mediated by A2aR. Anti-A2aR antibodies include antibodies or polypeptides containing 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 downstream events mediated by A2aR, such as A2aR binding and downstream signaling, stimulation of tumor growth, inhibition of anti-tumor immune responses, and immunosuppression in immunocompromised disease states.

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

[0124] 2. 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 specifically binds to a specific antigen, which contains or consists of at least one complementary determining region (CDR), which exists alone or in combination with one or more other CDRs and / or framework regions (FRs). As the term is defined elsewhere herein, in certain embodiments, the antigen binding molecule is an antibody or an antigen binding fragment thereof. In certain embodiments, the antigen binding molecule described in the present invention inhibits one or more biological functions of 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 that can specifically bind to A2aR. In certain embodiments, 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. A number of methods for humanizing non-human antibodies are known in the art. For example, a humanized antibody may have one or more amino acids introduced from a non-human source. These non-human amino acid residues are often referred to as "import" residues, and are typically from an "import" variable domain. Humanization can be performed essentially according to 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) method, that is, the hypervariable region sequence is replaced with the corresponding sequence of a human antibody. Therefore, such "humanized" antibodies are chimeric antibodies (U.S. Patent No. 4,816,567), in which a portion substantially less than the entire human variable domain is replaced by the corresponding sequence from a non-human species. In practice, humanized antibodies are typical human antibodies in which some hypervariable region residues and possibly some FR residues are replaced by residues from analogous sites in rodent antibodies.

[0129] The selection of human variable domains (light and heavy chains) for making humanized antibodies is important for reducing 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. The human sequence closest to the rodent sequence is regarded 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 of the consensus sequence of all human antibodies derived from a specific subgroup of light or heavy chains. The same framework can be used for several different 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] It is also generally expected that antibodies are humanized while retaining their high affinity for antigens and other favorable biological properties. In order to achieve this goal, humanized antibodies are prepared according to the following method, i.e., utilizing the three-dimensional model of the parent and humanized sequences, and preparing humanized antibodies by analyzing the process of the parental sequence and various conceptual humanized products. The three-dimensional immunoglobulin model is generally available and is familiar to those skilled in the art. It is also available to illustrate and display the computer program of the possible three-dimensional structural structure of the selected candidate immunoglobulin sequence. The detection of these displays can analyze the possible effect of the residue in the candidate immunoglobulin sequence function, i.e., the residues affecting the candidate immunoglobulin binding antigen ability are analyzed. In this way, it is possible to select and combine the receiving sequence and the importing sequence to obtain FR residues, to achieve the desired antibody characteristics, for example, to increase the affinity to (multiple) target antigens. Generally speaking, the hypervariable region residues directly and most substantially affect the combination of the antigen.

[0131] The present invention encompasses chimeric antibodies with different "levels" or "degrees" of humanization. In certain embodiments, the chimeric antibodies of the present invention are "fully" humanized, i.e., all FR regions in the heavy and light chain variable regions comprise sequences that are substantially identical or identical to corresponding human FR sequences. In certain embodiments, the chimeric antibodies of the present invention are "partially" humanized, e.g., 1, 2, 3, 4, 5, 6, or 7 FR regions in the heavy and light chain variable regions comprise sequences that are substantially identical or identical to corresponding human FR sequences, while the remaining FR regions comprise sequences that are substantially identical or identical to corresponding FR sequences of other species, e.g., FR sequences of mouse, rat, rabbit, chicken, and Chinese hamster.

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

[0133] 2. Sequences of Exemplary Antigen Binding Molecules

[0134] The A2aR antigen binding molecule can be in the form of a monoclonal antibody; in the form of one or more polypeptide fragments containing one or more A2aR antigen binding domains; or in the form of one or more nucleic acids encoding one or more A2aR binding domains. In certain embodiments, the present invention provides a humanized A2aR antibody or an antigen binding fragment thereof. The humanized antibody is humanized from the antibody disclosed in International Patent Publication No. WO2022 / 072601 (601 publication of International Patent Application PCT / US21 / 52819, filed on September 30, 2021). The heavy chain variable region and light chain variable region ("variable region") of the humanized antibody are named as follows. The variable regions derived from clone 1B5-3D7 in the 601 publication are referred to as "Hu1B5-Hn" and "Hu1B5-Ln" (representing the heavy chain variable region and the light chain variable region, respectively). The variable regions derived from clone 3F6-9G5 in the 601 publication 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 publication are referred to as "Hu3F8-Hn" and "Hu3F8-Ln" (representing the heavy chain variable region and the light chain variable region, respectively). The letter "n" in "Hn" and "Ln" in the above nomenclature refers to a number. The number "n" at the end of each heavy chain and light chain designation is for identification purposes only. Therefore, if a heavy chain and a light chain have identical other designations except for "H" and "L" (symbolizing the heavy chain and the light chain, respectively), then the designation has no meaning for any function or property of the antibody formed by these two specific heavy chains and light chains. For example, except for "H" and "L", the designations of Hu1B5-H1 and Hu1B5-L1 are exactly the same, and the number "1" at the end of these designations is for identification purposes only. The antibody formed by Hu1B5-H1 and Hu1B5-L1 is equivalent to the antibody formed by Hu1B5-H1 and other light chains (e.g., Hu1B5-L2). Antibodies can be represented by their heavy and light chains in the format of "clone-Hm / clone-Ln", where "m" can be the same as or different from "n". For example, an antibody represented by Hu3F6-H3 / Hu3F6-L5 means that it is composed of heavy chain Hu3F6-H3 and light chain Hu3F6-L5, both of which are derived from clone 3F6-9G5 in the 601 publication.

[0135] In various exemplary embodiments of the present invention, an antigen binding molecule, such as an anti-A2aR antibody or an antigen binding fragment thereof, comprises (1) a heavy chain variable region and (2) a light chain variable region, wherein the heavy chain variable region comprises three complementarity determining regions (HCDRs): HCDR1, HCDR2, and HCDR3, wherein HCDR1 has an amino acid sequence that is about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, and 3; HCDR2 has an amino acid sequence that is about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 4 to 9 and 89; and HCDR3 has an amino acid sequence that is about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 4 to 9 and 89. NO:10 or 11 has an amino acid sequence that is about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identical to the amino acid sequence shown in; the light chain variable region comprises three complementary determining regions (LCDR): LCDR1, LCDR2 and LCDR3, wherein LCDR1 has an amino acid sequence that is about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identical to the amino acid sequence selected from SEQ ID NOs:12 and 13; LCDR2 has an amino acid sequence that is about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identical to the amino acid sequence shown in SEQ ID NO:14; and LCDR3 has an amino acid sequence that is about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identical to the amino acid sequence selected from SEQ ID NOs:15 The amino acid sequence of IDNOs: 15 or 16 has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identical amino acid sequence; wherein the antigen binding molecule specifically binds to human A2aR. The amino acid sequences of exemplary HCDRs and LCDRs corresponding to 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 a CDR can be determined by one of skill in the art using any of a variety of known numbering schemes, including the scheme described by Kabat et al., supra ("Kabat" numbering scheme); Al-Lazikani et al., 1997, J. Mol. Biol., 273:927-948 ("Chothia" numbering scheme); MacCallum et al., 1996, J. Mol. Biol. 262:732-745 ("Contact" numbering scheme); Lefranc et al., Dev. Comp. Immunol., 2003, 27:55-77 ("IMGT" numbering scheme); and Honegge and Pluckthun, J. Mol. Biol, 2001, 309:657-70 ("AHo" numbering scheme); each of which is incorporated herein by reference in its entirety. The amino acid sequence boundaries of a CDR can also be determined by combining two numbering schemes (e.g., the Kabat and IMGT numbering schemes). Table 1 shows the heavy chain CDR sequences of exemplary antibodies of the present invention, wherein 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 present invention, wherein 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 refer to the CDRs derived from the clone 1B5-3D7 published in '601. 3F6 and 3F6-2 refer to the CDRs derived from the clone 3F6-9G5 published in '601. 3F8 and 3F8-2 refer to the CDRs derived from the clone 3F8-12E9 published in '601.

[0137] In certain embodiments, the present invention relates to an antigen binding molecule, such as an anti-A2aR antibody or an antigen binding fragment thereof, comprising CDRs defined based on the Kabat and IMGT numbering schemes or a combination thereof. Thus, in certain embodiments, the present invention relates to an antigen binding molecule, such as an anti-A2aR antibody or an antigen binding fragment thereof, comprising (1) a HCDR1 having an amino acid sequence of about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identical to, or comprising 1, 2 or 3 mutations compared to, an amino acid sequence selected from the HCDR1 sequences listed in Table 1; (2) a HCDR2 having an amino acid sequence of about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identical to, or comprising 1, 2, 3, 4, 5 or 6 mutations compared to, an amino acid sequence selected from the HCDR2 sequences listed in Table 1; (3) a HCDR3 having an amino acid sequence that is about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identical to, or comprising 1 or 2 mutations compared to, an amino acid sequence selected from the HCDR3 sequences listed in Table 1; (4) a LCDR1 having an amino acid sequence that is about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identical to, or comprising 1 or 2 mutations compared to, an amino acid sequence selected from the LCDR1 sequences listed in Table 2; 5) LCDR2 having an amino acid sequence that is about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identical to, or comprising 1 or 2 mutations compared to, an amino acid sequence selected from the LCDR2 sequences listed in Table 2; and (6) LCDR3 having an amino acid sequence that is about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identical to, or comprising 1 or 2 mutations compared to, an amino acid sequence selected from the LCDR3 sequences listed in Table 2. 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 certain 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 of HCDR1 based on a numbering scheme that combines the Kabat and IMGT numbering schemes is glycine (G).

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

[0140] Table 1: Amino acid sequences of heavy chain CDRs of exemplary antibodies (combination of Kabat and IMGT numbering schemes)

[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 It is G or L.

[0142] Table 2: Amino acid sequences of light chain CDRs of exemplary antigen binding molecules (combination of Kabat and IMGT numbering schemes)

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

[0144] In certain embodiments, the present invention provides humanized antibodies. 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: Amino acid sequences of the heavy chain variable region domains of antibodies derived from 1B5-3D7 Table 4: Amino acid sequences of the light chain variable region domains of antibodies derived from 1B5-3D7 Table 5: Amino acid sequences of the heavy chain variable region domains of antibodies derived from 3F6-9G5 Table 6: Amino acid sequences of the light chain variable region domains of antibodies derived from 3F6-9G5 Table 7: Amino acid sequences of the heavy chain variable region domains of antibodies derived from 3F8-12E9 Table 8: Amino acid sequences of the light chain variable region domains of antibodies derived from 3F8-12E9

[0145] Tables 9 to 16 show the FR sequences of some exemplary antibodies of the invention. Table 9: Amino acid sequence of heavy chain FR1

[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: Amino acid sequence of heavy chain FR2 Heavy chain sequence SEQ ID NO <![CDATA[Hu1B5-H1 1 ]]> WVRQAPGQGLEWMG 22 <![CDATA[Hu1B5-H2.1 2 ]]> WVRQAPGQGLEWIG 23 <![CDATA[Hu1B5-H6 3 ]]> WVRQMPGKGLEWIG 24 WVRQ-X17-PG-X18-GLEW-X19-G

[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: Amino acid sequence of heavy chain FR3

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

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

[0157] 3 Stands for Hu3F6-H3.

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

[0159] 5 Represents 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: Amino acid sequence of heavy chain FR4

[0162] 1 Represents 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: Amino acid sequence of light chain FR1

[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: Amino acid sequence of light chain FR2

[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: Amino acid sequence of light chain FR3

[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: Amino acid sequence of light chain FR4 Light chain sequence SEQ ID NO: <![CDATA[Hu1B5-L1 1 ]]> FGGGTKVEIK 50

[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 about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identical to an amino acid sequence selected from SEQ ID NOs: 51 to 70; and (2) a light chain variable region (LCVR) comprising an amino acid sequence about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identical to an amino acid sequence selected from SEQ ID NOs: 71 to 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 herein. Tables 19 and 20 show exemplary nucleotide sequences of DNA encoding HCVRs and LCVRs of exemplary anti-human A2aR antibodies of the present invention, respectively. Table 17: Amino acid sequences of HCVRs of exemplary antigen binding molecules Table 18: Amino acid sequences of pyroglutamylated HCVRs of exemplary antigen binding molecules Table 19: Amino Acid Sequences of LCVRs of Exemplary Antigen Binding Molecules Table 20: Nucleotide sequences encoding HCVRs of exemplary antigen binding molecules Table 21: Nucleotide sequences encoding LCVRs of exemplary antigen binding molecules

[0178] In certain embodiments, the antigen binding molecules of the present invention, such as antibodies or their antigen binding fragments, are modified after translation. Examples of post-translational modifications include cleavage of lysine at the C-terminus of the heavy chain by carboxypeptidase; modification of glutamine or glutamic acid at the N-terminus of the heavy chain and light chain to pyroglutamic acid by the following: pyroglutamylation, glycosylation, oxidation, deamidation, and saccharification, which are known to occur in various antibodies (see Journal of Pharmaceutical Sciences, 2008, Vol. 97, p. 2426-2447, which is incorporated herein by reference in its entirety). Examples of antigen binding molecules such as antibodies or their antigen binding fragments that undergo post-translational modifications include antigen binding molecules, such as antibodies or their antigen binding fragments, in which the N-terminus of the heavy chain variable region has been pyroglutamylated and / or the lysine at the C-terminus of the heavy chain has been missing. The sequences of exemplary antigen binding molecules with pyroglutamylated N-terminals are listed in Table 7. When used to represent amino acids in polypeptides, "pE" as used herein refers to pyroglutamic acid.

[0179] 2. Variants of Antigen Binding Molecules

[0180] In certain embodiments, the A2aR antigen binding molecules described herein, such as the anti-A2aR antibodies, can be monoclonal antibodies, chimeric antibodies, humanized antibodies, Fab, F(ab')2, scFv, or multispecific antibodies comprising additional binding specificities as described herein.

[0181] Thus, in certain embodiments, the anti-A2aR antibodies described herein may be linked to an Fc comprising one or more modifications, typically to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and / or antigen-dependent cellular cytotoxicity. In addition, the antibodies described herein may be chemically modified (e.g., one or more chemical moieties are attached to the antibody), or modified to alter its glycosylation to alter one or more functional properties of the antibody. More specifically, in certain embodiments, the antibodies described herein may comprise modifications in the Fc region to generate Fc variants having the following characteristics: (a) increased or decreased antibody-dependent cell-mediated cytotoxicity (ADCC), (b) increased or decreased complement-mediated cytotoxicity (CDC), (c) increased or decreased affinity for C1q, and / or (d) increased or decreased affinity for Fc receptors relative to the parent Fc. Such Fc region variants typically comprise at least one amino acid modification in the Fc region. Combinations of amino acid modifications are highly desirable. For example, an Fc region variant may comprise two, three, four, five, etc. substitutions therein, e.g., of specific Fc region sites identified herein.

[0182] For purposes requiring effector functions, certain effector function-enhanced Fcs may 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 may be used. In certain embodiments, in order to enhance the ADCC of a "Y"-shaped bivalent antibody, substitutions such as L234Y, L235Q, G236W, S239M, H268D, D270E, S298A are introduced into one heavy chain, and substitutions such as D270E, K326D, A330M, K334E are introduced into another heavy chain.

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

[0184] For uses where effector function needs to be avoided completely, for example, when antigen binding alone is sufficient to produce the desired therapeutic effect and effector function only causes (or increases the risk of) undesirable side effects, IgG4 antibodies or IgG1 L234F, L235E, P331S or L234A, L235A and P239G without ADCC effect can be used, or antibodies or fragments lacking the Fc region or a large part thereof can be designed, or the Fc can be mutated to completely eliminate glycosylation (e.g., N297A, N297Q or N297G). Alternatively, a hybrid construct of human IgG2 (CH1 domain and hinge region) and human IgG4 (CH2 and CH3 domains) can be generated, which lacks effector function and lacks binding to FcγR (e.g., IgG2) and the ability to activate complement (e.g., IgG4). When using an IgG4 constant domain, it is generally preferred to include an S228P substitution that mimics the IgG1 hinge sequence and an R409K mutation that prevents Fab arm exchange and thus stabilizes the IgG4 molecule, thereby reducing Fab arm exchange between the therapeutic antibody and endogenous IgG4 in the patient being treated.

[0185] In certain embodiments, the anti-A2aR antibody or fragment thereof may be modified to provide an extended biological half-life. Various methods may be employed, including, for example, methods for increasing the binding affinity of the Fc region to FcRn. In one embodiment, the CH1 or CL region of the antibody is altered to include salvage receptor binding epitopes from two loops of the CH2 domain of the Fc region of IgG, as described in U.S. Pat. Nos. 5,869,046 and 6,121,022. The residue numbering of the Fc region is the residue numbering of the EU index of Kabat. The sequence variants disclosed herein are provided with reference to the residue numbering, which is followed by the amino acid that replaces the naturally occurring amino acid, optionally preceded by the naturally occurring residue at that site. Where multiple amino acids may be present at a given site, for example, if the sequence differs between naturally occurring isoforms, or if there are multiple mutations that can be replaced at that site, they are separated by slashes (e.g., "X / Y / Z").

[0186] Exemplary Fc variants that enhance 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 enhance binding of Fc to FcRn include: 250E, 250Q, 428L, 428F, 250Q / 428L (Hinton et al., 2004, J. Biol. Chem. 279(8):6213-6216, Hinton et al., 2006 Journal of Immunology 176:346-356), 256A, 272A, 305A, 307A, 311A, 312A, 378Q, 380A, 382A, 434A (Shields et al., (2001) J. Biol. Chem., 27 6(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 US Pat. No. 8,367,805).

[0187] Modification of certain conserved residues in IgG Fc (I253, H310, Q311, H433, N434), such as the N434A variant (Yeung et al., (2009) J. Immunol. 182: 7663), can be used as a way to increase affinity with FcRn, thereby extending the half-life of the antibody in circulation (WO 98 / 023289). Combination Fc variants comprising M428L and N434S have been shown to increase binding to FcRn and extend serum half-life by five times (Zalevsky et al., (2010) Nat. Biotechnol. 28: 157). Fc variants comprising a combination of T307A, E380A and N434A modifications also extend the half-life of IgG1 antibodies (Petkova et al., (2006) Int. Immunol. 18: 1759). In addition, Fc variants comprising a combination of M252Y-M428L, M428L-N434H, M428L-N434F, M428L-N434Y, M428L-N434A, M428L-N434M and M428L-N434S can also extend the half-life ((US2006 / 173170). In addition, it is reported that an Fc variant comprising a combination of M252Y, S254T and T256E can extend the half-life by nearly 4 times (Dall'Acqua et al., (2006) J. Biol. Chem. 281:23514).

[0188] In certain embodiments, the A2aR antigen binding molecule described in the present invention is a bispecific antibody, which comprises: a first targeting domain that specifically binds to A2aR and a second targeting domain that specifically binds to another epitope in A2aR or another protein. In some embodiments, the first targeting domain comprises an antigen-binding fragment of any A2aR antibody described in the present invention.

[0189] In certain embodiments, the antigen binding molecules (e.g., anti-A2aR antibodies or antigen binding fragments thereof) described herein are chemically coupled to one or more therapeutically active peptides and / or small molecule drugs. The peptides or small molecule drugs can be linked to, for example, reduced SH groups and / or carbohydrate side chains. Methods for preparing covalent or non-covalent conjugates of peptides or small molecule drugs with antibodies are known in the art and any such known methods can be used.

[0190] In some embodiments, the peptide or small molecule drug is connected to the hinge region of the reduced antibody component via a disulfide bond. Alternatively, such an agent can be connected using a heterobifunctional cross-linker (e.g., N-succinyl 3-(2-pyridyldithio) propionate (SPDP)). The general techniques for this coupling are well known in the art. In some embodiments, the peptide or small molecule drug is coupled via a carbohydrate moiety in the Fc region of the antibody. The carbohydrate group can be used to increase the load of the same agent bound to the thiol group, or the carbohydrate moiety can be used to bind different therapeutic agents or diagnostic agents. Methods for coupling peptide inhibitors or small molecule drugs to antibodies via antibody carbohydrate moieties are well known to those skilled in the art. For example, in one embodiment, the method involves reacting an antibody component having an oxidized carbohydrate moiety with a carrier polymer having at least one free amine functional group. The reaction causes an initial Schiff base (imine) connection, which can be stabilized by reduction to a secondary amine to form a final conjugate. Exemplary methods for coupling small molecule drugs and peptides to antibodies are described in U.S. Patent Publication 2014 / 0356385.

[0191] The A2aR antibodies (including fragments thereof and multispecific forms thereof) may range in size from 50 kD to 300 kD, 50 kD to 250 kD, 60 kD to 250 kD, 80 kD to 250 kD, 100 kD to 250 kD, 125 kD to 250 kD, 150 kD to 250 kD, 60 kD to 225 kD, 75 kD to 225 kD, 100 kD to 225 kD, 125 kD to 225 kD, 150 kD to 225 kD, 60 kD to 2 kD to 200 kD, 75 kD to 200 kD, 100 kD to 200 kD, 125 kD to 200 kD, 150 kD to 200 kD, 60 kD to 150 kD, 75 kD to 150 kD, 100 kD to 150 kD, 60 kD to 125 kD, 75 kD to 125 kD, 75 kD to 100 kD, or any range encompassed by any combination of integers listed in the above ranges, or any range specified 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 monkey A2aR.

[0194] The present invention includes A2aR antigen binding molecules, such as A2aR antibodies or antigen binding fragments thereof, which are capable of specifically binding to human and cynomolgus monkey A2aR expressed on the cell surface and inhibiting A2aR activity or function. According to certain embodiments, the antigen binding molecules block the interaction between human A2aR expressed on the cell surface and A2aR agonists. The extent to which the A2aR antigen binding protein (e.g., A2aR antibody or antigen binding fragment thereof) inhibits A2aR activity can be assessed by the assay described in Example 3 or a substantially similar assay. The present invention includes antigen binding molecules, such as antibodies, which block the interaction between human A2aR expressed on the cell surface and A2aR agonists, and the IC is measured by the assay shown in Example 3 or a substantially similar assay. 50 The value is 0.7 nM to about 20 nM, or lower.

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

[0196] According to certain embodiments, the invention provides antigen binding molecules that bind to human A2aR but not to A2aR from other species. The invention also 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, provided antigen binding molecules can bind to human A2aR and can bind or not bind (as the case may be) to one or more A2aR from mice, rats, guinea pigs, hamsters, gerbils, pigs, cats, dogs, rabbits, goats, sheep, cattle, horses, camels, cynomolgus monkeys, marmosets, rhesus monkeys and chimpanzees. For example, in a particular exemplary embodiment of the present invention, the present invention provides antigen binding molecules comprising antigen binding domains that bind 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 (including antibodies, antigen binding fragments thereof, and multispecific antibodies thereof) of the present invention have several in vitro, in vivo, and ex vivo uses related to enhancing immune responses in cancer treatment by blocking signal transduction through adenosine and other signaling pathways. Without wishing to be bound by any theory, it is hypothesized that the antigen binding molecules (e.g., anti-A2aR antibodies or antigen binding fragments thereof) of the present invention bind to A2aR expressed on the cell surface and inhibit its activity, and the inhibition of A2aR activity leads to, for example, a decrease in intracellular cAMP concentration. Therefore, the antigen binding molecules (and therapeutic compositions comprising the same) of the present invention are particularly useful for treating any disease or condition that can benefit from inhibition of A2aR activity (e.g., stimulation and / or activation of an immune response). In view of 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) of the present invention can be used alone or in combination with a variety of active agents to treat a wide range of diseases or conditions, including a variety of cancers.

[0200] Thus, the present invention provides a method for reducing the concentration of intracellular cAMP, comprising contacting a cell with an antigen binding molecule (e.g., an anti-A2aR antibody or antigen binding fragment thereof) of the present invention. The reduction in the concentration of intracellular cAMP can be measured by the method described in Example 4 or a substantially similar method. In certain embodiments, the methods described herein reduce the concentration of intracellular cAMP by at least about 10%, about 20%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or more compared to the baseline level.

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

[0202] In one aspect, a method of enhancing an immune response (e.g., a T cell response) in a subject comprises administering an anti-A2aR antibody described herein to a subject to enhance an immune response (e.g., a T cell response) in the subject. In some embodiments, the subject is a tumor-bearing subject whose immune response to the tumor is enhanced. The tumor may be a solid tumor or a liquid tumor, such as a hematological malignancy. In certain embodiments, the tumor is an immunogenic tumor. In other embodiments, the tumor is non-immunogenic. In other embodiments, the subject is a pathogen-bearing subject whose immune response to the pathogen is enhanced by administering 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) enhancing 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 need of enhancing immune response. The method is particularly suitable for treating human patients with conditions that can be treated by enhancing immune response (e.g., T cell-mediated immune response). The method is particularly suitable for treating cancer, chronic infection and chronic inflammatory diseases. Preferably, the antibodies used in the methods of the present disclosure described herein are human antibodies or humanized antibodies.

[0205] In one embodiment, the method of inhibiting tumor cell growth in a subject comprises administering to the subject an anti-A2aR antibody described herein, thereby inhibiting the growth of a tumor in the subject. Inhibition of tumor growth can be determined by a variety of methods. Inhibition of tumor growth can be measured by a variety of methods. Tumor growth can be measured using the following methods: for example, the method described in Talkington, A and Durrett, R, Estimating Tumor Growth Rates in vivo, Bull Math Biol., 2015 Oct.: 77(10): 1934-54 (available in https: / / www.ncbi.nlm.nih.gov / pmc / articles / PMC4764475 / acquired, the entire contents of which are incorporated herein by reference). Inhibition of tumor growth can also be measured by a reduction in tumor size. In certain embodiments, the methods 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 certain embodiments, the antigen binding molecules described herein, such as anti-A2aR antibodies or antigen binding fragments thereof, can be used to reduce immunosuppression in the tumor microenvironment. This reduction can be measured by a variety of methods. For example, the level of immunosuppression in the tumor microenvironment can be measured by the presence and / or abundance of certain biomarkers (e.g., PD-L1, CD73, IL-10, or TGF-β) in the tumor. It can also be measured by the presence and / or abundance of CD8+ cytotoxic T cells and regulatory T (Treg) in the tumor. reg The level of immunosuppression is measured by the ratio of CD8+ cytotoxic T cells to T cells. reg In certain embodiments, the antigen binding molecules described herein, such as anti-A2aR antibodies or antigen binding fragments thereof, reduce the level of immunosuppression in the tumor microenvironment by at least about 10%, about 20%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95% or more compared to the baseline level.

[0207] Also included herein is the elimination of T cells from the tumor microenvironment of a subject having a tumor (e.g., a cancerous tumor). reg A method for stimulating the clearance of T cells in a tumor microenvironment, comprising administering to the subject a therapeutically effective amount of an anti-A2aR antibody described herein. reg For example, the Fc may be an Fc with suitable effector function or enhanced effector function conferred by one or more activated Fc receptors.

[0208] In certain preferred embodiments, T cells are generated in the tumor microenvironment. reg Clear and T eff There was no significant clearance or inhibition of T cells outside the tumor microenvironment. eff and T reg In certain embodiments, the subject has T cells in the tumor microenvironment. reg The level of A2aR on T cells is higher than that on Teff cells. In certain embodiments, anti-A2aR antibodies can eliminate T cells in tumors. regs and / or T cells in tumor-infiltrating lymphocytes (TILs) regs .

[0209] In certain preferred embodiments, the subject suffers from a cell proliferative disease or cancer. Blocking adenosine signaling through A2aR with the antigen binding molecules of the present invention (e.g., anti-A2aR antibodies or antigen binding fragments thereof) can enhance the immune response against cancer cells in the subject. Therefore, the present invention provides a method for treating a subject suffering from cancer, comprising administering an anti-A2aR antigen binding molecule, such as an antibody or antigen binding fragment thereof, described herein to the subject, so that the subject is treated, for example, to inhibit or reduce the growth of cancerous tumors and / or to cause tumor regression. The anti-A2aR antibody can be used alone to inhibit the growth of cancerous tumors. Alternatively, the anti-A2aR antibody can be used in combination with one or more other targeted active agents, such as other anti-cancer targets, immunogenic agents, standard cancer treatments or other antibodies, as described below. The antigen binding molecules of the present invention can be used to treat, for example, primary and / or metastatic tumors. The present invention also includes a method for treating residual cancer in a subject. The term "residual cancer" as used herein refers to one or more cancer cells that exist or persist in a subject after treatment with an anti-cancer therapy.

[0210] Thus, in one aspect, a method for 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. In addition, the anti-A2aR antigen binding molecule (eg, antibody) used in the method may include a chimeric or humanized non-human anti-A2aR antibody thereof. The efficacy of treating cancer can be determined by a variety of methods. For example, the efficacy of treating cancer can be determined by an improvement in survival rate or a reduction in tumor size. In certain embodiments, the methods described herein increase the efficacy of treating cancer by at least about 10%, about 20%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 1-fold, about 2-fold, about 4-fold or more compared to baseline levels. In the context of cancer treatment, if the A2aR antigen binding molecule of the present invention is the only therapeutic agent, the baseline level refers to the therapeutic effect of using a placebo; if the A2aR antigen binding molecule of the present invention is used in combination with other therapeutic agents, the baseline level refers to the therapeutic effect of using a placebo or other therapeutic agents.

[0211] Cancers whose growth can be inhibited by the antibodies of the present invention include a variety of cancers, especially those that are unresponsive or prone to unresponsiveness to monotherapy with other antibodies or chemotherapeutic agents. Non-limiting examples of cancers for treatment include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, squamous non-small cell lung cancer (NSCLC), non-NSCLC, glioma, gastrointestinal cancer, renal cancer (e.g., clear cell carcinoma), ovarian cancer, liver cancer, colorectal cancer, endometrial cancer, renal cancer (e.g., renal cell carcinoma (RCC)), prostate cancer (e.g., hormone-refractory prostate cancer), thyroid cancer, neuroblastoma, pancreatic cancer, glioblastoma (glioblastoma multiforme), cervical cancer, gastric cancer, bladder cancer, hepatoma, breast cancer, colon cancer and head and neck cancer, gastric cancer, germ cell tumor, pediatric Sarcoma, sinonasal natural killer cells, melanoma (e.g., metastatic malignant melanoma (e.g., skin or intraocular malignant melanoma)), bone cancer, skin cancer, uterine cancer, cancer of the anal region, testicular cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, esophageal cancer, small intestine cancer, endocrine system cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, solid tumors in children, ureteral cancer, renal pelvis cancer, central nervous system (CNS) tumors, primary CNS lymphoma, tumor angiogenesis, spinal cord axis tumors, brain stem glioma, pituitary adenoma, Kaposi's sarcoma (Kaposi's sarcoma), epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers (including asbestos-induced cancers, virus-related cancers (e.g., human papillomavirus (HPV)-related tumors), and hematological malignancies (which arise from either of the two major blood cell lineages, the myeloid lineage (which produces granulocytes, erythrocytes, platelets, macrophages, and mast cells) or the lymphoid lineage (which produces B, T, NK, and plasma cells), such as all types of leukemias, lymphomas, and myelomas (e.g., acute, chronic, lymphocytic and / or myeloid leukemias, such as acute leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CIVIL), undifferentiated AML (MO), myeloblastic leukemia (M1), myeloblastic leukemia (M2; cell maturity), promyelocytic leukemia (M3 or M3 variant [M3V]), myelomonocytic leukemia (M4 or M4 variant with eosinophilia [M4E]), monocytic leukemia (M5), erythroleukemia (M6), megakaryocytic leukemia (M7), solitary granulocytic sarcoma, and chloroma;Lymphoma (e.g., Hodgkin lymphoma (HL), non-Hodgkin lymphoma (NEIL), B-cell lymphoma, T-cell lymphoma, lymphoplasmacytoid lymphoma, monocytoid B-cell lymphoma, mucosa-associated lymphoid tissue (MALT) lymphoma, anaplastic (e.g., Ki 1+) Large cell lymphoma, adult T-cell lymphoma / leukemia, mantle cell lymphoma, angioimmunoblastic T-cell lymphoma, angiocentric lymphoma, intestinal T-cell lymphoma, primary mediastinal B-cell lymphoma, precursor T-lymphoblastic lymphoma, T-lymphoblastic lymphoma / leukemia (T-Lbly / T-ALL), peripheral T-cell lymphoma, lymphoblastic lymphoma, post-transplant lymphoproliferative disorder, true histiocytic lymphoma, primary central nervous system lymphoma, primary effusion lymphoma, lymphoblastic lymphoma (LBL), lymphoid hematopoietic neoplasms, acute lymphoblastic leukemia, diffuse large B-cell lymphoma, Burkitt's lymphoma (Burkitt's The main types of lymphoma include myeloma, follicular lymphoma, diffuse histiocytic lymphoma (DHL), immunoblastic large cell lymphoma, precursor B lymphoblastic lymphoma, cutaneous T-cell lymphoma (CTLC) (also known as mycosis fungoides or Sezary syndrome), and lymphoplasmacytoid lymphoma (LPL) with Waldenstrom's macroglobulinemia); myeloma (e.g., IgG myeloma, light chain myeloma, nonsecretory myeloma, smoldering myeloma (also known as indolent myeloma), solitary plasmacytoma, and multiple myeloma), chronic lymphocytic leukemia (CLL), hairy cell lymphoma; myeloid hematopoietic tumors, tumors of mesenchymal origin (including fibrosarcoma and rhabdomyosarcoma); seminoma, teratoma, central and peripheral nerve tumors (including astrocytic mesenchymal origin tumors (including fibrosarcoma, rhabdomyosarcoma and osteosarcoma); and other tumors (including melanoma, xeroderma pigmentosum, keratoacanthoma, seminoma, thyroid follicular carcinoma and teratocarcinoma), lymphoid lineage hematopoietic tumors (such as T-cell and B-cell tumors, including but not limited to T-cell diseases, such as T-prolymphocytic leukemia (T-PLL), including small cell and brain-like cell types; large granular lymphocytic leukemia (LGL) of preferred T-cell type; a / d T-NHL hepatosplenic lymphoma; peripheral / post-thymic T-cell lymphoma (polymorphic and immunoblastic subtypes); angiocentric (nasal) T-cell lymphoma); head and neck cancer, kidney cancer, colorectal cancer, thyroid cancer; acute myeloid lymphoma, and any combination of the above cancers. The methods described herein may also be used to treat metastatic cancers, refractory cancers (e.g., cancers that are refractory to previous immunotherapy (e.g., using antibodies that block CTLA-4 or PD-1)), and recurrent cancers. ;

[0212] In some embodiments, treatment of cancer patients with the anti-A2aR antibodies and / or other agents described herein can produce long-term durable responses, including long-term survival of at least 1, 2, 3, 4, 5, 10 years or more and / or recurrence-free survival of at least 1, 2, 3, 4, 5, 10 years or more, relative to the current standard of care. In certain embodiments, treatment of cancer subjects with the anti-A2aR antibodies and / or other agents described herein can prevent cancer recurrence or delay cancer recurrence by, for example, 1, 2, 3, 4, 5, 10 years or more. The anti-A2aR treatment can be used as a primary or second-line treatment.

[0213] Bone marrow transplantation is currently used to treat a variety of hematopoietic tumors. This treatment is associated with graft-versus-host disease, and inhibition of A2aR can reduce graft-versus-tumor responses, thereby increasing the effectiveness of donor-transplanted tumor-specific T cells.

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

[0215] Suitable routes for administering the antigen-binding molecules of the invention, 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 as described herein are well known in the art and can be selected by a person of ordinary skill. For example, the antibody composition can be administered by parenteral injection (e.g., intravenous or subcutaneous). As further described below, the appropriate dose depends on the age and weight of the subject and the concentration and / or formulation of the antibody composition.

[0216] Neurodegenerative diseases are associated with increased A2aR activity. Therefore, in certain embodiments, the present invention provides a method for treating a neurodegenerative disease, comprising administering an antigen binding molecule of the present invention, such as an anti-A2aR antibody or an antigen binding fragment thereof, to a subject in need thereof, thereby treating the neurodegenerative disease.

[0217] The term "cell proliferative disorder" refers to a disorder characterized by abnormal proliferation of cells. A proliferative disease does not imply any restriction on the rate of cell growth, but only a loss of normal control over growth and cell division. Thus, in some embodiments, cells of a proliferative disorder may have the same rate of cell division as normal cells but do not respond to signals that restrict such growth. The scope of "cell proliferative disorder" includes neoplasms, cancers, or tumors.

[0218] The term "cancer" refers to any of a variety of malignancies characterized by cell proliferation with the ability to invade surrounding tissues and / or metastasize to new colonization sites, including carcinomas, sarcomas, adenocarcinomas, melanomas, leukemias, lymphomas, germ cell tumors, and blastomas, including solid cancers and lymphoid cancers. Exemplary cancers that can be treated according to the compositions and methods of the present invention include brain cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, head and neck cancer, kidney cancer, lung cancer, non-small cell lung cancer, mesothelioma, ovarian cancer, prostate cancer, gastric and uterine cancer, leukemia, and medulloblastoma.

[0219] The term "carcinoma" refers to a malignant growth of epithelial cells that tends to infiltrate surrounding tissues and give rise to metastases. Exemplary carcinomas include, e.g., acinar carcinoma, acinous carcinoma, adenocystic carcinoma, adenoid cystic carcinoma, carcinoma adenomatosum, adrenocortical carcinoma, alveolar carcinoma, alveolar cell carcinoma, basal cell carcinoma, carcinoma basocellulare, basaloid carcinoma, basosquamous cell carcinoma, bronchioalveolar carcinoma, bronchiolar carcinoma, bronchogenic carcinoma, cerebriform carcinoma, cholangiocellular carcinoma, chorionic carcinoma, colloid carcinoma, comedo carcinoma, and the like. carcinoma, corpus carcinoma, cribriform carcinoma, carcinoma en cuirasse, carcinoma cutaneum, cylindrical carcinoma, cylindrical cell carcinoma, ductal carcinoma, carcinoma durum, embryonal carcinoma, encephaloid carcinoma, epiennoid carcinoma, carcinoma epitheliale adenoides, exophytic carcinoma, carcinoma exulcere, carcinoma fibrosum, gelatiniform carcinoma, gelatinous carcinoma, giant cell carcinomacarcinoma, giant cell carcinoma (carcinomagigantocellulare), glandular carcinoma, granular cell carcinoma, hair-matrix carcinoma, hematoid carcinoma, hepatocellular carcinoma, Hurthle cell carcinoma, hyaline carcinoma, hypemephroid carcinoma, infantile embryonal carcinoma, carcinoma in situ, intraepidermal carcinoma, intraepithelial carcinoma, Krompecher's carcinoma, Kulchitzky cell carcinoma, large-cell carcinoma, lenticular carcinoma, carcinoma lenticulare, lipomatous carcinoma carcinoma, lymphoepithelial carcinoma, carcinoma medullare, medullary carcinoma, melanotic carcinoma, carcinoma molle, mucinous carcinoma, carcinoma muciparum, carcinomamucocellulare, mucoepidermoid carcinoma, carcinomamucosum, mucous carcinoma, carcinoma myxomatodes, naspharyngeal carcinoma, oat cell carcinoma, carcinomaossificans, osteoid carcinoma, pancreatic ductaladenocarcinoma, papillary carcinomacarcinoma, periportal carcinoma, preinvasive carcinoma, prickle cell carcinoma, pultaceous carcinoma, renal cell carcinoma of kidney, reserve cell carcinoma, carcinoma sarcomatodes, schneiderian carcinoma, scirrhous carcinoma, carcinoma scroti, signet-ring cell carcinoma, carcinoma simplex, small-cell carcinoma, solanoid carcinoma, spheroidal cell carcinoma, spindle cell carcinoma, carcinoma spongiosum, squamous carcinoma, squamous cell carcinoma, string carcinoma carcinoma, carcinoma telangiectaticum, carcinoma telangiectodes, transitional cell carcinoma, carcinoma tuberosum, tuberous carcinoma, verrucous carcinoma, and carcinoma villosum.

[0220] The term "sarcoma" refers to tumors composed of material like embryonic connective tissue, usually consisting of tightly packed cells embedded in a fibrous or homogeneous material. Exemplary sarcomas include, for example, chondrosarcoma, fibrosarcoma, lymphosarcoma, melanosarcoma, myxosarcoma, osteosarcoma, Abemethy's sarcoma, adiposesarcoma, 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, giant cell sarcoma, granulocytic sarcoma, 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 cellsarcoma, angiosarcoma, leukosarcoma, malignant mesenchymoma sarcoma, parosteal sarcoma, reticulocytic sarcoma, Rous sarcoma, sarcoma), serocystic sarcoma, synovial sarcoma, and telangiectatic sarcoma.

[0221] The term "melanoma" refers to a tumor arising from the melanocyte system of the skin and other organs. Melanomas include, for example, acral-lentiginous melanoma, amelanotic melanoma, benign juvenile melanoma, Cloudman's melanoma, S91 melanoma, Harding-Passey melanoma, juvenile melanoma, lentigomaligna melanoma, malignant melanoma, nodular melanoma, subungal melanoma, and superficial spreading melanoma.

[0222] The term "lymphoma" refers to cancers that affect the hematopoietic and lymphoid tissues and arise from lymphocytes (blood cells found primarily in the lymph nodes, spleen, thymus, and bone marrow). The two main types of lymphoma are non-Hodgkin lymphoma and Hodgkin's disease. Hodgkin's disease accounts for about 15% of all diagnosed lymphomas. It is a cancer related to Reed-Sternberg malignant B lymphocytes. Non-Hodgkin lymphoma (NHL) can be classified based on how fast the cancer grows and the types of cells involved. There are aggressive (high-grade) and indolent (low-grade) types of NHL. Based on the types of cells involved, there are B-cell and T-cell NHL. Exemplary B-cell lymphomas include, but are not limited to, small lymphocytic lymphoma, mantle cell lymphoma, follicular lymphoma, marginal zone lymphoma, extranodal (MALT) lymphoma, nodal monocytic B-cell lymphoma, The present invention relates to 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, usually characterized by abnormal proliferation and development of leukocytes and their precursors in the blood and bone marrow. Exemplary leukemias include, for example, acute non-lymphocytic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, chronic myeloid leukemia, acute promyelocytic leukemia, adult T-cell leukemia, aleukemic leukemia, aleukocythemic leukemia, basophylic leukemia, blast cell leukemia, bovine leukemia, chronic myeloid leukemia, leukemia cutis, embryonal leukemia, eosinophilicleukemia, Gross' leukemia, hairy cell leukemia, hemoblasticleukemia, 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 leukemia), plasmacytic leukemia, promyelocytic leukemia, Riedercell leukemia, Schilling's leukemia, stem cell leukemia, subleukemic leukemia and undifferentiated cell leukemia.

[0224] Other cancers include, for example, multiple myeloma, neuroblastoma, breast cancer, ovarian cancer, lung cancer, rhabdomyosarcoma, essential thrombocythemia, essential macroglobulinemia, small cell lung cancer, primary brain tumors, gastric cancer, colon cancer, malignant pancreatic insular tumors, malignant carcinoids, precancerous skin lesions, testicular cancer, thyroid cancer, neuroblastoma, esophageal cancer, genitourinary tract cancer, hypercalcemia of malignancy, cervical cancer, endometrial cancer, and adrenocortical carcinoma.

[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 slowing tumor growth in a subject. The present invention includes compositions and therapeutic preparations comprising any (e.g., herein) exemplary antigen binding molecules in combination with one or more other therapeutic agents, and treatment methods comprising administering such compositions to a subject in need thereof. The term "other therapeutic agent" as used herein refers to any agent that can be used to treat a disease or condition, and any method for treating certain diseases or conditions. For example, when radiotherapy and surgery are used in combination with the antigen binding molecules described in the present invention, such as anti-A2aR antibodies or antigen-binding fragments thereof, they are considered "other therapeutic agents".

[0227] In certain embodiments, the other therapeutic agent may be an A2aR antagonist that is different from the antigen binding molecule described herein (e.g., an anti-A2aR antibody or antigen binding fragment thereof). 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 other therapeutic agent can be administered in the form of an antibody or an antibody fragment thereof, directed against another member of the adenosine signaling pathway, 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 their antigen binding sites (described in U.S. Patents 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 patents 10,766,966, 10,584,169, 10,556,968, and 10,167,343.

[0229] In some embodiments, the anti-A2aR antigen binding molecules described herein, such as anti-A2aR antibodies or antigen binding fragments thereof, are co-administered with one or more other therapeutic agents, wherein the other therapeutic agents are in an amount effective to stimulate immune response and / or apoptosis, to further enhance, stimulate or upregulate immune response and / or apoptosis in the subject. In addition, the one or more other 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 co-administered or co-administered with one or more other active agents (e.g., anti-cancer antibodies or polypeptides, chemotherapeutic agents, and radiotoxic agents). In other embodiments, the anti-A2aR antibodies described herein are co-administered or co-administered with standard cancer treatments (e.g., surgery or radiation).

[0231] Co-administration of the anti-A2aR antibodies with these active agents or treatment modalities may address the following clinical drawbacks: drug resistance, changes in antigenicity that render tumor cells unreactive with the antibody, and toxicity (by administering lower doses of one or more of the agents). A2aR inhibition is particularly suitable for use in combination with chemotherapeutic regimens that are otherwise refractory. In these cases, it is possible to achieve enhanced efficacy while reducing the dose of the chemotherapeutic agent administered (Mokyr et al., (1998) Cancer Research 58:5301-5304). The rationale for inhibiting A2aR with radiation therapy or chemotherapy is that the cytotoxic effects of radiation therapy and most chemotherapeutic compounds promote cell death, which can further lead to elevated levels of tumor antigens in the antigen presentation pathway. Other combination therapies that act additively or synergistically with A2aR inhibition through cell death include surgery and hormone deprivation or suppression. Each of these regimens further creates a 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 immune complex, immunoconjugate, or fusion protein. Alternatively, the anti-A2aR antibodies can be administered separately from the other active agents. In this case, the anti-A2aR antibodies and other antagonists can be administered before, after, or simultaneously with the other active agents, or can be co-administered with other known treatments (e.g., other anticancer agents, radiation, etc.). Thus, the present invention provides compositions and methods for providing two or more anticancer agents that act additively or synergistically through different mechanisms to beneficially provide cytotoxic and immunoprotective effects in human cancer cells.

[0233] For example, in some embodiments, the anti-A2aR antibodies described herein can be combined with anti-cancer agents such as alkylating agents; anthracycline antibiotics; antimetabolites; antidotes; interferons; polyclonal or monoclonal antibodies; EGFR inhibitors; HER2 inhibitors; histone deacetylase inhibitors; hormones; mitotic inhibitors; phosphatidylinositol-3-kinase (PI3K) inhibitors; Akt inhibitors; mammalian target of rapamycin (mTOR) inhibitors; proteasome inhibitors; poly (ADP-ribose) polymerase (PARP) inhibitors; Ras / MAPK pathway inhibitors; centrosome declustering agents; multikinase inhibitors; serine / threonine kinase inhibitors; tyrosine kinase inhibitors; VEGF / VEGFR inhibitors; taxanes or taxane derivatives, aromatase inhibitors, anthracyclines, microtubule-targeted drugs, topoisomerase-toxic drugs, inhibitors of molecular targets or enzymes (e.g., kinases or protein methyltransferases), cytidine analogs, or a combination thereof.

[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), bis(chloromethyldiethylamine) (Mustargen), busulfan (Myleran), carboplatin (Paraplatin), cisplatin (CDDP, Platinol), temozolomide (Temodar), thiotepa (Thioplex), bendamustine (Treanda), or streptozotocin (Zanosar).

[0235] Exemplary anthracyclines include, but are not limited to, doxorubicin (Adriamycin), liposomal doxorubicin (Doxil), mitoxantrone (Novantrone), 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, but are not limited to, fluorouracil (Adrucil), capecitabine (Xeloda), hydroxyurea (Hydrea), mercaptopurine (Purinethol), pemetrexed (Alimta), fludarabine (Fludara), nelarabine (Arranon), cladribine (Cladribine Novaplus), clofarabine (Clolar), cytarabine (Cytosar-U), decitabine (Dacogen), cytarabine liposomal (DepoCyt), hydroxyurea (Droxia); pralatrexate (Folotyn), floxuridine (FUDR), gemcitabine (Gemzar), cladribine (Leustatin), fludarabine (Oforta), methotrexate (MTX, Rheumatrex), methotrexate (Trexall), thioguanine (Tabloid), TS-1, or cytarabine (Tarabine PFS).

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

[0238] Exemplary interferons include, but are not limited to, interferon alfa(α)-2b (Intron A) or interferon α-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 tiuxetan (Zevalin; In-111; Y-90Zevalin); 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, but are not limited to, tamoxifen (Soltamox; Nolvadex); raloxifene (Evista); megestrol acetate (Megace); leuprolide (Lupron; Lupron Depot; Eligard; Viadur); fulvestrant (Faslodex); letrozole (Femara); triptorelin (Trelstar LA; Trelstar Depot; exemestane (Aromasin); goserelin (Zoladex); bicalutamide (Casodex); anastrozole (Arimidex); fluoxymesterone (Androxy; Halotestin); medroxyprogesterone (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; epothilone; 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 and gamma inhibitor); apelisib (BYL719, an alpha-specific PI3K inhibitor); TGR 1202 (formerly known as RP5264, an oral PI3K delta inhibitor); and cupanisib (BAY 80-6946, an inhibitor of predominantly 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, veliparib, 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 centrosome declustering agents include, but are not limited to, griseofulvin; narcotine, narcotine derivatives, such as brominated narcotine (e.g., 9-bromonacotine), reduced bromonacotine (RBN), N-(3-bromobenzyl)narcotine, aminonarcotine, and water-soluble derivatives thereof; CW069; phenanthrene-derived poly (ADP-ribose) polymerase inhibitor, PJ-34; N2-(3-pyridylmethyl)-5-nitro-2-furanamide, N2-(2-thienylmethyl)-5-nitro-2-furanamide, and N2-benzyl-5-nitro-2-furanamide.

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

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

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

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

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

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

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

[0259] Exemplary general chemotherapeutic agents, anti-tumor agents, anti-proliferative agents include, but are not limited to, hexamethylmelamine (Hexalen); isotretinoin (Accutane; Amnesteem; Claravis; Sotret); tretinoin (Vesanoid); azacitidine (Vidaza); bortezomib (Velcade); asparaginase (Elspar); levamisole (Ergamisol); mitotane (Lysodren); procarbazine (Matulane); pegaspargase (O ncaspar); denileukin diftitox (Ontak); porfimer (Photofrin); aldesleukin (Proleukin); lenalidomide (Revlimid); bexarotene (Targretin); thalidomide (Thalomid); temsirolimus (Torisel); arsenic trioxide (Trisenox); verteporfin (Visudyne); mimosapinol (Leucenol); (1M tegafur-0.4M 5-chloro-2,4-dihydroxypyrimidine-1M potassium oxide) or lovastatin.

[0260] In certain embodiments, A2aR inhibition is combined with CD3 stimulation (e.g., by co-incubation with cells expressing membrane CD3), and CD3 stimulation is performed before, simultaneously with, or after anti-A2aR antibody treatment. For example, in one embodiment, a method of enhancing an antigen-specific T cell response comprises contacting T cells with an anti-A2aR antibody described herein and cells expressing CD3, such that the antigen-specific T cell response is enhanced and A2aR-mediated immunosuppression is reduced. Any suitable indicator of an antigen-specific T cell response can be used to measure the antigen-specific T cell response. Non-limiting examples of such suitable indicators include increased T cell proliferation in the presence of an antibody and / or increased cytokine production in the presence of an antibody. In a preferred embodiment, the production of interleukin-2 and / or interferon-γ 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 target effector cells expressing Fcα or Fcγ receptors to tumor cells (see, e.g., U.S. Patents 5,922,845 and 5,837,243). Such bispecific antibodies can be used to target two different antigens. For example, anti-Fc receptor / anti-tumor antigen (e.g., Her-2 / neu) bispecific antibodies have been used to target macrophages to tumor sites. This targeting can more effectively activate tumor-specific responses. The T cell arm of these responses will be enhanced by the inhibition of A2aR. Alternatively, bispecific antibodies that bind to tumor antigens and dendritic cell-specific cell surface markers can be used to deliver antigens directly to DCs.

[0262] In all of the above approaches, 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 that utilizes two different binding specificities to provide enhanced tumor antigen presentation.

[0263] In some embodiments, the other therapeutic agent used in any of the aforementioned treatment methods, antigen binding molecules or pharmaceutical compositions is an immunostimulatory agent selected from the following: (a) an agent that blocks immune cell inhibitory receptor (immune checkpoint) signaling or its ligand (immune checkpoint inhibitor) or a nucleic acid encoding such an agent; (b) an agonist of an immune cell stimulating receptor or a nucleic acid encoding such an agonist; (c) a cytokine or a nucleic acid encoding a cytokine; (d) an oncolytic virus or a nucleic acid encoding an oncolytic virus; (e) a T cell expressing a chimeric antigen receptor; (f) a bispecific or multispecific T cell targeting antibody or a nucleic acid encoding such an antibody; (g) an anti-TGF-β antibody or a nucleic acid encoding such an antibody; (h) a TGF-β trap or a nucleic acid encoding such a trap; (i) vaccines for cancer-associated antigens, including such antigens or nucleic acids encoding such antigens, (j) cell therapy, and (k) combinations of the above. In some embodiments, the other therapeutic agent is an agent that blocks the signaling of an inhibitory receptor or its ligand of an immune cell or a nucleic acid encoding such an agent, wherein the inhibitory receptor or its ligand is selected from PD-1, PD-L1, TIGIT, CTLA-4, PD-L2, LAG-3, TIM-3, B7-H3, B7-H4, CD73, PVRIG / PVRL2, neurite protein, 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 combinations thereof. In some embodiments, the other therapeutic agents are agonists of immune cell stimulating receptors or nucleic acids encoding such agonists, the agonists are selected from 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 other therapeutic agents are cytokines or nucleic acids encoding cytokines selected from IL-2, IL-5, IL-7, IL-12, IL-15, IL-2I and combinations thereof. In some embodiments, the other therapeutic agents are oncolytic viruses or nucleic acids encoding oncolytic viruses selected from herpes simplex virus, vesicular stomatitis virus, adenovirus, Newcastle disease virus, vaccinia virus, Maraba virus and combinations thereof. In some embodiments, the other therapeutic agents are cell therapy. Cell therapy may include T cells, NK cells or macrophages with chimeric antigen receptors (CAR). In some embodiments, the cell therapy includes bispecific or multispecific T cell targeting antibodies.

[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 a ligand to a receptor can reduce or inhibit the immune response of an immune cell. An immune checkpoint inhibitor is an agent that reduces or inhibits the binding of a ligand to a receptor.

[0265] As used herein, the term "stimulatory receptor of an immune cell" refers to a receptor on an immune cell that, after binding to its ligand, can enhance the immune response of the immune cell.

[0266] In certain embodiments, the present invention provides a method for treating a disease or condition (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 one or more other therapeutic agents for a second time to a subject, wherein the subject has previously been treated with one or more other therapeutic agents for the first time. In certain embodiments, low efficacy may be exhibited when treating a disease in a subject with one or more therapeutic agents for the first time. For example, treatment with one or more therapeutic agents for the first time may be treatment with an anti-PD1 antibody, to which the subject may show resistance. In some embodiments, the one or more other therapeutic agents for the second time are the same as the one or more other therapeutic agents for the first time. In some embodiments, the one or more other therapeutic agents for the second time are different from the one or more other therapeutic agents for the first time.

[0267] In certain embodiments, the immune checkpoint inhibitor is an antibody that specifically interacts with an immune checkpoint. In some embodiments, the other therapeutic agents include immunostimulants. In some aspects, the immune checkpoint inhibitor is selected from anti-PD-1 antibodies (e.g., pembrolizumab or nivolumab), and anti-PD-L antibodies (e.g., atezolizumab), and anti-CTLA-4 antibodies (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 other therapeutic agent is an agent that inhibits the interaction between PD-1 and PD-L1. In some aspects, the other therapeutic agents that inhibit the interaction between PD-1 and PD-L1 are selected from antibodies, peptidomimetics, and small molecules. In some aspects, the other therapeutic agents that inhibit the interaction between PD-1 and PD-L1 are selected from pembrolizumab, nivolumab, atezolizumab, avelumab, durvalumab, BMS-936559, sindilimab, toripalizumab, tislelizumab, carrelizumab, envolizumab, sugemalimab, penampalimumab, cardunilizumab, sulfamethoxazole and sulfamethoxazole.

[0269] In some embodiments, the anti-A2aR antibody is combined with or administered simultaneously with an immunogenic agent. Non-limiting examples of immunogenic agents include cancer cells, tumor vaccines, and purified tumor antigens (including recombinant proteins, peptides, and carbohydrate molecules); oncolytic viruses; cells transfected with genes encoding immunostimulatory cytokines, etc.

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

[0271] In certain embodiments, an antigen-specific immune response can be enhanced by co-administering one or more of any of the anti-A2aR antibodies described herein 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) an antigen; and (ii) an A2aR-based antibody, thereby enhancing the subject's immune response to the antigen. The antigen can be, for example, a tumor antigen, a viral antigen, a bacterial antigen, or an antigen from a pathogen. Non-limiting examples of such antigens include the antigens described in the above section, such as the tumor antigens (or tumor vaccines) discussed above, or antigens from viruses, bacteria, or other pathogens described above.

[0272] In view of the synergistic benefits associated with the combination of active agents, in certain embodiments, the anti-A2aR antibody and the other active agent are each administered to a subject in need thereof at subtherapeutic doses relative to the doses used in their monotherapy.

[0273] In certain embodiments, A2aR inhibition is combined with standard cancer treatments (e.g., surgery, radiation therapy, and chemotherapy). In these cases, it is possible to reduce the dose of chemotherapeutic agents administered. It is believed that the combined use of A2aR inhibition and chemotherapy can exacerbate apoptosis and enhance tumor antigen presentation to achieve cytotoxic immunity. Other synergistic combination therapies include A2aR inhibition combined with radiation therapy, surgery, or hormone deprivation or suppression. Each of these regimens produces a source of tumor antigens in the host.

[0274] The other therapeutic agent may be administered before, simultaneously with, or after administration of the antigen binding molecules described herein; (for the purposes of this disclosure, such an administration regimen is considered to be administration of the antigen binding molecules "in combination" with the additional therapeutically active ingredient).

[0275] The present invention includes pharmaceutical compositions in which the antigen binding molecules described herein are co-formulated with one or more other therapeutic agents described herein.

[0276] 5. Nucleic Acids and Host Cells for Expression of Anti-A2aR Antibodies

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

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

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

[0280] When one nucleic acid sequence is placed in a functional relationship with another nucleic acid sequence, it is said that one nucleic acid sequence is "operably linked" to another nucleic acid sequence. For example, if the DNA of the presequence or signal peptide expresses a preprotein that participates in the secretion of the polypeptide, the DNA of the presequence or signal peptide is operably linked to the DNA of the polypeptide; if a promoter or enhancer affects the transcription of the sequence, the promoter or enhancer is operably linked to the coding sequence; or if a ribosome binding site is positioned to facilitate translation, the ribosome binding site is operably linked to the coding sequence. Generally, "operably linked" means that the DNA sequences being linked are continuous, and in the presence of a signal peptide, they are continuous and in the reading phase. However, enhancers do not have to be continuous. Connection is accomplished by connecting at convenient restriction sites. If such sites do not exist, synthetic oligonucleotide adapters or joints can be used according to conventional practices.

[0281] The nucleic acid sequence used to express the antibodies described in the present disclosure generally comprises an N-terminal signal peptide sequence removed from the mature protein. Since the signal peptide sequence can affect the expression level, the polynucleotide can encode any one of a variety of different N-terminal signal peptide sequences. It will be appreciated by those skilled in the art that the design of the expression vector may depend on the following factors: the choice of the host cell to be transformed, the expression level of the target protein, etc.

[0282] The above-mentioned "regulatory sequence" refers to a DNA sequence necessary for expressing an operably linked coding sequence in one or more host organisms. The term "regulatory sequence" is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Regulatory sequences include regulatory sequences that direct constitutive expression of nucleotide sequences in many types of host cells, or regulatory sequences that direct expression of nucleotide sequences only in certain host cells (e.g., tissue-specific regulatory sequences). Expression vectors typically contain sequences for transcription termination, and may additionally contain one or more elements that have a positive effect on mRNA stability.

[0283] The expression vector comprises one or more transcriptional regulatory elements, including a promoter and / or an enhancer, for directing the expression of the antibodies disclosed herein. The promoter comprises a DNA sequence, the function of which is to initiate transcription from a relatively fixed position relative to the transcription start site. The promoter comprises core elements required for the basic interaction of RNA polymerase and transcription factors, and can operate with other upstream elements and response elements.

[0284] The term "promoter" as used herein should be broadly interpreted to include, for example, transcriptional regulatory elements (TREs) from genomic genes or chimeric TREs thereof, including TATA boxes or initiation elements for precise transcription initiation, with or without other TREs (i.e., upstream activation sequences, transcription factor binding sites, enhancers and silencers), which regulate activation or repression of operably linked genes in response to development and / or external stimuli, and trans-acting regulatory proteins or nucleic acids. A promoter may comprise a genomic fragment or it may comprise a chimera of one or more TREs combined together.

[0285] Preferred promoters are promoters that can direct high-level expression in target target cells. The promoter may include constitutive promoters (e.g., HCMV, SV40, elongation factor-1α (EF-1α)), or promoters that show preferential expression in specific target cell types. An enhancer generally refers to a DNA sequence that functions away from the transcription start site, which may be located at the 5' or 3' end of the transcription unit. In addition, enhancers may fall within introns and within coding sequences. Its length is generally between 10 and 300 bp, and it functions in a cis manner. The function of an enhancer is to increase and / or regulate transcription of nearby promoters. Preferred enhancers include enhancers that direct high-level expression in cells that produce antibodies. Cell or tissue-specific transcriptional regulatory elements (TREs) may be incorporated into expression vectors to limit expression to desired cell types. Pol III promoters (H1 or U6) are particularly useful for expressing shRNAs that can express siRNAs. Expression vectors may be designed to promote expression of antibodies described in the present disclosure in one or more cell types.

[0286] siRNA is a double-stranded RNA that can be modified to induce gene silencing after sequence-specific transcription of mRNA. Synthetic siRNA structurally simulates the type of siRNA that is usually produced by Dicer enzyme processing in cells. When expressed with an expression vector, the expression vector is modified to transcribe the processed short double-stranded hairpin RNA (shRNA) into a targeted siRNA in the cell. siRNA and shRNA can be synthesized using a conventional DNA / RNA synthesizer and designed using a known algorithm.

[0287] In order to carry out the co-expression of the independent chains of the antibody disclosed herein, suitable splice donor and splice acceptor sequences can be incorporated to express two products. Alternatively, an internal ribosome binding sequence (IRES) or a 2A peptide sequence can be used to express multiple products from one promoter. IRES provides a structure to which the ribosome can bind, and it does not need to be located at the 5' end of the mRNA. Therefore, it can guide the ribosome to start translation at the second start codon in the mRNA, so that a single mRNA produces more than one polypeptide. The 2A peptide comprises a short sequence that mediates the co-translational self-cleavage of the upstream and downstream peptides of the 2A site, so that a single transcript of equimolar amounts produces two different proteins. CHYSEL is a non-limiting example of a 2A peptide, which allows the eukaryotic ribosome in translation to release the growing polypeptide chain it is synthesizing without dissociating from the mRNA. The ribosome continues to translate, thereby producing a second polypeptide.

[0288] Expression vectors may include viral vectors or non-viral vectors. Viral vectors may be derived from adeno-associated virus (AAV), adenovirus, herpes virus, vaccinia virus, polio virus, poxvirus, retrovirus (including lentivirus, such as HIV-1 and HIV-2), Sindbis virus and other RNA viruses, alphavirus, astrovirus, coronavirus, orthomyxovirus, papovavirus, paramyxovirus, parvovirus, picornavirus, togavirus, etc. Non-viral vectors are simply "naked" expression vectors that are not packaged with virus-derived components (e.g., capsid and / or envelope).

[0289] In some cases, these vectors can be engineered to target certain diseases or cell populations by using targeting properties inherent to or engineered into viral vectors. Specific cells can be "targeted" for delivery as well as expression of polynucleotides. In this context, the term "targeting" therefore refers to delivery to specific cells using endogenous or heterologous binding agents in the form of capsids, envelope proteins, antibodies, or to the use of tissue-specific regulatory elements to restrict expression to specific cell subsets, or both.

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

[0291] Non-viral expression vectors can be used for non-viral gene transfer, either by direct injection of naked DNA or by encapsulating the polynucleotide encoding the antibody in liposomes, microparticles, microcapsules, virus-like particles or erythrocyte ghosts. Such compositions can be further linked to targeting domains by chemical coupling to facilitate targeted delivery of nucleic acids to and / or into desired target cells. In addition, plasmid vectors can be incubated with synthetic gene transfer molecules (e.g., polymeric DNA binding cations such as polylysine, protamine, and albumin) and linked to cell targeting ligands (e.g., asialomucoids, insulin, galactose, lactose, or transferrin).

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

[0293] VI. Methods for producing anti-A2aR antibodies

[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. The host cell can be any bacterial or eukaryotic cell capable of expressing a nucleic acid or expression vector encoding an anti-A2aR HCVR and / or LCVR, or any other co-administered antibody or antagonist described herein.

[0295] In another aspect, a method of making an antibody of the present disclosure comprises culturing a host cell transformed with a nucleic acid or expression vector encoding an anti-A2a RHCVR and / or LCVR under conditions that permit production of the antibody or fragment thereof, and purifying the antibody from the cell.

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

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

[0298] Examples of selectable markers suitable for mammalian cells include dihydrofolate reductase (DHFR), thymidine kinase, neomycin, neomycin analog G418, hydromycin and puromycin. When such a selection marker is successfully transferred into a mammalian host cell, the transformed mammalian host cell can survive under the selection pressure. There are two widely used different selection schemes. The first category is based on the metabolism of the cell and the use of mutant cell lines that lack the ability to grow independently of the supplemented medium. Two examples are CHO DHFR- cells and mouse LTV cells. These cells cannot grow without the addition of nutrients such as thymidine or hypoxanthine. Because these cells lack certain genes necessary for the complete nucleotide synthesis pathway, they cannot survive unless the missing nucleotides are provided in the supplemented medium. Another method of supplementing the medium is to introduce a complete DHFR or TK gene into cells lacking the corresponding gene, thereby changing their growth requirements. Individual cells that are not transformed with DHFR or TK genes will not survive in unsupplemented medium.

[0299] The second category is dominant selection, which refers to a selection scheme that can be used in any cell type without the need to use mutant cell lines. In these schemes, drugs are usually used to prevent the growth of host cells. Cells with new genes will express proteins that transmit drug resistance and will survive the selection. Examples of such dominant selection include the use of drugs neomycin, mycophenolic acid or hygromycin. In these three examples, bacterial genes under the control of eukaryotic organisms transmit resistance to suitable drugs G418 or neomycin (Geneticin), xgpt (mycophenolic acid) or hygromycin, respectively. Other drugs include neomycin analogs G418 and puromycin.

[0300] Exemplary cells expressing antibodies include human T lymphocyte leukemia cells (Jurkat), human embryonic kidney (HEK) 293 cells, Chinese hamster ovary (CHO) cells, mouse WEHI fibrosarcoma cells, and single-cell protozoan species, such as Leishmania tarentolae. In addition, primary cells immortalized with c-myc or other immortalizing agents can be used to generate stable transformed antibody-producing cell lines.

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

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

[0303] The antibodies of the present invention can be cultured in any suitable culture medium (e.g., RPMI, DMEM and ) and then separated from the antibody expressing cells. Conventional protein purification methods (e.g., affinity purification, chromatography, etc.) can be used to purify the antibody, including the use of protein-A or protein-G immunoaffinity purification. In some embodiments, the antibody is engineered to be secreted into the culture supernatant for separation therefrom.

[0304] VII. Pharmaceutical Composition and Method of Administration

[0305] In one aspect, the pharmaceutical composition of the present invention comprises an antigen binding molecule described herein (e.g., an A2aR antibody or an antigen binding fragment thereof), and a pharmaceutically acceptable carrier. In other embodiments, the A2aR antibody or antigen binding fragment thereof is administered in combination with a pharmaceutically acceptable carrier. As described herein, the anti-A2aR composition may comprise 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 pharmaceutical compositions comprising the antigen binding molecules of the present invention. The pharmaceutical compositions of the present invention are formulated with suitable carriers, excipients and other agents that can improve transfer, delivery, tolerance, etc. A variety of suitable formulations can be found in the formulary known to pharmaceutical chemists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, vesicles containing lipids (cations or anions) (e.g., LIPOFECTIN TM , Life Technologies, Carlsbad, CA), DNA conjugates, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, carbowax emulsions (polyethylene glycol of various molecular weights), semisolid gels, and semisolid mixtures containing carbowax. See also Powell et al., "Compendium of excipients for parenteral formulations" PDA (1998) JPharm Sci Technol 52:238-311.

[0307] The dosage of the antigen binding molecules applied to the patient may vary according to the patient's age and body shape, target disease, illness, route of administration, etc. The preferred dosage is usually calculated according to body weight or body surface area. When the bispecific antigen binding molecules of the present invention are used for adult patients for the purpose of treatment, a favorable scheme is to administer the bispecific antigen binding molecules of the present invention intravenously in a single dose of about 0.01 to about 20 mg / kg body weight, more preferably about 0.02 to about 7, about 0.03 to about 5 or about 0.05 to about 3 mg / kg body weight. Depending on the severity of the disease, the frequency and duration of treatment can be adjusted. The effective dose and time schedule for administering the bispecific antigen binding molecules can be determined empirically; for example, the patient's disease progression can be monitored by regular assessment, and the dosage can be adjusted accordingly. In addition, interspecies scaling of dosage can be performed using methods known in the art (e.g., Mordenti et al., 1991, Pharmaceut. Res. 8: 1351).

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

[0309] The pharmaceutical composition of the present invention can be delivered subcutaneously or intravenously using a standard needle and syringe. In addition, with respect to subcutaneous delivery, pen delivery devices are readily applicable to the delivery of the pharmaceutical composition of the present invention. Such pen delivery devices can be reusable or disposable. Reusable pen delivery devices typically use a replaceable cartridge containing the pharmaceutical composition. Once all of the pharmaceutical composition in the cartridge has been administered and the cartridge is empty, the empty cartridge can be easily discarded and replaced with a new cartridge containing the pharmaceutical composition. The pen delivery device can then be reused. In a disposable pen delivery device, there is no replaceable cartridge. Instead, the disposable pen delivery device is pre-filled with the pharmaceutical composition, which is stored in a reservoir within the device. Once the pharmaceutical composition in the reservoir is emptied, the entire device is discarded.

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

[0311] The injection can include dosage forms for intravenous, subcutaneous, intradermal and intramuscular injection, infusion, etc. These injections can be prepared by known methods. For example, the above-mentioned antibody or its salt can be dissolved, suspended or emulsified in a sterile aqueous medium or oily medium conventionally used for injection to prepare an injectable preparation. The aqueous medium for injection includes, for example, physiological saline, isotonic solution containing glucose and other adjuvants, etc., which can be used in combination with the following appropriate solubilizers: for example, alcohols (such as ethanol), polyols (such as propylene glycol, polyethylene glycol), nonionic surfactants (such as polysorbate 80, HCO-50 (adduct of polyoxyethylene (50 mol) hydrogenated castor oil)), etc. The oily medium includes, for example, sesame oil, soybean oil, etc., which can be used in combination with solubilizers such as benzyl benzoate and benzyl alcohol. The prepared injection is preferably contained in a suitable ampoule.

[0312] Advantageously, the pharmaceutical composition for oral or parenteral use is prepared into a unit dosage form suitable for the dosage of the active ingredient. Such unit dosage forms include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc. The content of the above-mentioned antibody is usually about 5 to about 500 mg per dosage form in a unit dose; in particular, in the form of an injection, it is preferably about 5 to about 100 mg of the above-mentioned antibody, and for other dosage forms, it is preferably about 10 to about 250 mg.

[0313] In another aspect, a method for treating a cell proliferative disorder (e.g., cancer, chronic infection, or immunocompromised disease state) 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 pharmaceutically acceptable carrier. In some embodiments, the method restores, potentiates, or enhances lymphocyte activity in a subject in need thereof. In certain preferred embodiments, the antibody or fragment is a human or humanized anti-A2aR antibody that reduces or eliminates signaling of A2aR.

[0314] In some embodiments, administration of the pharmaceutical composition increases the activity of lymphocytes (e.g., T cells) in a patient with a disease, wherein the increased lymphocyte activity is beneficial, or the disease is caused or characterized by immunosuppression, immunosuppressive cells, or adenosine produced by, for example, CD4 T cells, CD8 T cells, B cells. The methods described herein are particularly useful, for example, in patients with solid tumors, where the tumor microenvironment (and the adenosine produced therein) can help reduce recognition by the immune system (immune escape). For example, a tumor can be characterized by immune cells that express (or overexpress) A2aR, such as CD4 T cells, CD8 T cells, T-regs, B cells.

[0315] In certain embodiments, the methods and compositions are used to treat a variety of cancers and other proliferative diseases. Because these methods can be used to reduce adenosine levels that can inhibit the anti-tumor activity of lymphocytes, they are applicable to a very wide range of cancers, particularly solid tumors, where adenosine in the tumor microenvironment is known to inhibit anti-tumor immune responses.

[0316] Non-limiting cancers for which the antigen-binding molecules described herein (e.g., anti-A2aR antibodies or antigen-binding fragments thereof) can be used to treat 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, solid tumors in children, lymphocytic lymphoma, bladder cancer, kidney or ureter, renal pelvis cancer, central nervous system cancer, system (CNS) tumors, primary CNS lymphomas, tumor angiogenesis, spinal axis tumors, brain stem gliomas, pituitary adenomas, Kaposi's sarcoma, epidermoid carcinomas, squamous cell carcinomas, environmentally induced cancers (including cancers induced by asbestos), hematological malignancies, including, for example, multiple myeloma, B-cell lymphomas, Hodgkin's lymphoma / primary mediastinal B-cell lymphomas, non-Hodgkin's lymphomas, acute myeloid lymphomas, chronic myeloid leukemias, chronic lymphatic leukemias, follicular lymphomas, diffuse large B-cell lymphomas, Burkitt's lymphomas, immunoblastic large cell lymphomas, precursor B-lymphoblastic lymphomas, mantle cell lymphomas, acute lymphocytic leukemias, mycosis fungoides, anaplastic large cell lymphomas, T-cell lymphomas, and precursor T-lymphoblastic lymphomas, or any combination of these cancers. The present disclosure is also applicable to the treatment of metastatic cancers. Patients may be tested or selected for one or more of the above clinical attributes before, during or after treatment.

[0317] In one embodiment, an effective amount of the anti-A2aR antibody is administered to achieve and / or maintain (e.g., for 1, 2, 3, 4 weeks, and / or until subsequent administration of an antigen binding compound) a blood concentration of at least EC 50 , optionally EC 70 , optionally substantially EC 100 , for neutralizing the enzymatic activity of A2aR. In one embodiment, the active amount of the anti-A2aR antibody is an EC of 1.5 to 1.5% to neutralize the enzymatic activity of A2aR in the extravascular tissue of the individual. 50 , optionally EC 70 , optionally substantially EC 100 In one embodiment, the active amount of the anti-A2aR antibody is an amount that achieves (or maintains) inhibition of neutralizing A2aR enzyme activity EC 50 , optionally EC 70 , optionally substantially EC 100 An effective amount.

[0318] Optionally, in one embodiment, in contrast to some antibodies that eliminate A2aR-expressing tumor cells by ADCC (which may, for example, be fully effective at concentrations equal to or significantly below those that provide receptor saturation), the anti-A2aR antibody is primarily a blocking agent (without substantial Fcγ receptor-mediated activity) and is administered in an amount effective to neutralize the enzymatic activity of A2aR for a desired period of time (e.g., 1 week, 2 weeks, a month) until the next consecutive administration of the anti-A2aR antibody.

[0319] In one embodiment, an effective amount of the anti-A2aR antibody is administered to achieve and / or maintain (e.g., for 1, 2, 3, 4 weeks, and / or until subsequent administration of the anti-A2aR antibody) an inhibition of A2aR-mediated catabolism of AMP to adenosine by at least an EC of 1.5. 50 , optionally EC 70 , optionally substantially EC 100 In one embodiment, the amount of the anti-A2aR antibody is an amount that achieves (or maintains) its EC of inhibiting A2aR-mediated catabolism of AMP to adenosine in the extravascular tissue of the individual. 50 , optionally EC 70 , optionally substantially EC 100 An effective amount.

[0320] In one embodiment, the invention provides a method for treating or preventing cancer in an individual, the method comprising administering to an individual with the disease an amount of an anti-A2aR antibody that achieves or maintains a circulating concentration for a specified period of time, optionally in a target extravascular tissue (e.g., a tumor or tumor environment) at a concentration above the concentration at which 50%, 70% or complete (e.g., 90%) receptor saturation of circulating A2aR expressing cells is achieved (e.g., as assessed in PBMCs). Optionally, the concentration achieved is at least 20%, 50% or 100% higher than the concentration at which a particular receptor is saturated.

[0321] In one embodiment, the invention provides a method for treating or preventing cancer in an individual, the method comprising administering to the individual an amount of an anti-A2aR antibody that achieves or maintains a concentration in the circulation, optionally in a target extravascular tissue (e.g., a tumor or tumor environment) for a specific period of time, that is above the EC for binding to cells expressing A2aR. 50 , optionally EC 70 , or optionally EC 100 Optionally, the concentration achieved is less than the EC for binding to cells expressing A2aR. 50 , optionally EC 70 , or optionally EC 100At least 20%, 50% or 100% higher.

[0322] In any embodiment, for example, the antibody is used to bind to ECs of A2aR expressing cells in human PBMCs. 50 , optionally EC 70 , or optionally EC 100 Between 0.5-100 ng / ml, optionally 1-100 ng / ml, optionally 30-100 ng / ml, for example, about 30-90 ng / ml. For example, the EC 50 It can be about 30, 37, 39, 43, 57, 58, 61, 62, 90, 95, 143 ng / ml.

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

[0324] When targeting tissues outside the vasculature (tumor environment, e.g., in the treatment of solid tumors), a dose about 10-fold higher than that which provides a corresponding concentration in the circulation is generally required. An amount of anti-A2aR antibody administered to achieve (and / or maintain) a concentration of about 1 μg / ml, 2 μg / ml, 10 μg / ml, or 20 μg / ml in the circulation (blood) is expected to achieve (and / or maintain) an extravascular tissue (e.g., tumor tissue) concentration of about 0.1 μg / ml, 0.2 μg / ml, 1 μg / ml, 2 μg / ml, respectively.

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

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

[0327] EC of a given A2aR antibody 50 ,EC 70 and EC 100 The value of can be assessed, for example, in a cell-based assay for neutralizing the enzymatic activity of A2aR. 50 " refers to the concentration of anti-A2aR antibody that can produce 50% of the maximum reaction or effect of neutralizing enzyme activity. The "EC 70 " refers to the concentration of anti-A2aR antibody that produces 70% of the maximal response or effect. The "EC 100 " refers to the effective concentration of anti-A2aR antibody that can produce such a maximum response or effect of neutralizing enzyme activity. In certain embodiments, depending on the context, EC 50 ,EC 70 or EC 100 Can be referred to as IC 50 、IC 70 or IC 100 ICxx refers to the concentration of drug required to inhibit xx% of a biological process.

[0328] In some embodiments, particularly for the treatment of solid tumors, the concentrations achieved are designed to achieve concentrations in tissues (outside the vasculature, e.g., in a tumor or tumor environment) that correspond to at least an EC for neutralization of enzyme activity. 50 , optionally about or at least about EC 100 .

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

[0330] In one embodiment, a method of treating cancer in a subject in need thereof comprises administering to the individual an effective amount of an anti-A2aR antibody of the present disclosure for at least one administration cycle (optionally at least 2, 3, 4 or more administration cycles), wherein the cycle is eight weeks or less, wherein for each of at least one cycle, one, two, three or four doses of the anti-A2aR antibody are administered at a dose of 1-20 mg / kg body weight. In one embodiment, the anti-A2aR antibody is administered by intravenous infusion.

[0331] Suitable treatment regimens for treating, for example, human subjects include, for example, administering an anti-A2aR antibody to a patient in an amount disclosed herein, wherein the method comprises at least one administration cycle, wherein at least one dose of the anti-A2aR antibody is administered in one administration cycle. Optionally, at least 2, 3, 4, 5, 6, 7 or 8 doses of the anti-A2aR antibody are administered. In one embodiment, the administration cycle is 2 weeks to 8 weeks.

[0332] In one embodiment, a method for treating or preventing a disease (e.g., cancer, a solid tumor, a hematological tumor) in an individual comprises administering to the individual an anti-A2aR antibody that neutralizes the enzymatic activity of A2aR for at least one administration cycle, the administration cycle comprising at least a first and a second (and optionally a 3rd, 4th, 5th, 6th, 7th and / or 8th or more) administration of the anti-A2aR antibody, wherein the anti-A2aR antibody is administered in an effective amount to achieve or maintain a blood (serum) concentration of the anti-A2aR antibody between two consecutive administrations in an amount of at least 0.1 μg / ml, at least 0.2 μg / ml, at least 1 μg / ml, at least 2 μg / ml, at least 10 μg / ml, at least 20 μg / ml, 1-100 μg / ml, 1-50 μg / ml, 1-20 μg / ml, 1-10 μg / ml, or a range between any of the foregoing concentrations.

[0333] In one embodiment, a specified continuous blood concentration is maintained, wherein the blood concentration does not significantly fall below a specified blood concentration within a specified time period (e.g., between two antibody administrations, weeks, 1 week, 2 weeks, 3 weeks, 4 weeks). In other words, although the blood concentration may change within a specified time period, the maintained specified blood concentration represents a minimum or "trough" concentration.

[0334] In one embodiment, a therapeutically active amount of an anti-A2aR antibody is one that is able to provide (at least) EC 50 concentration, optionally EC 70 concentration, optionally EC 100 The concentration is in an amount that neutralizes the enzymatic activity of A2aR for a period of time after administration of the antibody, the period of time being at least about 1 week, about 2 weeks, or about 1 month.

[0335] Prior to or during treatment with an anti-A2aR antibody as described herein, the expression level of A2aR, CD39, and / or CD73 in cells within and / or near a patient's tumor; the percentage of A2aR-expressing cells, CD39-expressing cells, and / or CD73-expressing cells; and / or the level of adenosine, ADP, and / or AMP can be assessed to assess whether the patient is suitable for treatment and whether the patient is likely to respond to treatment. An increase in the foregoing levels or expression may indicate that the individual is suitable for (e.g., may benefit from) treatment with an anti-A2aR antibody as described herein.

[0336] In some embodiments, the step of assessing the expression level of A2aR, CD39 and / or CD73 and the concentration of adenosine, ADP and / or AMP in and / or near a patient's tumor tissue sample comprises obtaining a biological sample of human tissue from a subject, wherein the human tissue is selected from a tissue of a cancer patient, such as cancer tissue, tissue near or surrounding cancer, cancer adjacent tissue, adjacent non-tumor tissue or normal adjacent tissue, and the expression level of A2aR, CD39 and / or CD73 and the concentration of adenosine, ADP and / or AMP in the tissue. The expression level or nucleotide concentration in the patient can be compared with a reference level (e.g., the level of a healthy individual).

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

[0338] In view of the foregoing, in certain embodiments, the method comprises the steps of: (a) determining the expression level of A2aR, CD39 and / or CD73 and / or the concentration of adenosine, ADP and / or AMP in the tumor environment, optionally within the tumor and / or adjacent tissue, and upon determining that the level of A2aR, CD39, CD73, adenosine, ADP and / or AMP in the tumor environment is increased compared to its corresponding reference level, (b) administering an anti-A2aR antibody to the individual.

[0339] In certain embodiments, determining the level of A2aR, CD39, CD73, adenosine, ADP and / or AMP in the tumor environment comprises obtaining a biological sample containing cancer tissue and / or tissue close to or located around the tumor (e.g., cancer adjacent tissue, adjacent non-tumor tissue or normal adjacent tissue) from a subject, and detecting the level and / or relative percentage of A2aR expressing cells, CD39 expressing cells and / or CD73 expressing cells, and / or the level 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 level of A2aR, CD39, CD73 can be determined by evaluating the mRNA expression (e.g., by RT-PCR) or polypeptide expression (e.g., by Western blotting, immunofluorescence staining) of A2aR, CD39, CD73 compared to a reference level of a healthy subject or to a reference level before treatment using techniques known to those of ordinary skill in the art.

[0340] Anti-A2aR antibodies can be used to treat subjects having cancer with or without assessing A2aR, CD39, CD73, adenosine, ADP and / or AMP levels in the tumor microenvironment (e.g., tumor cells, CD4 T cells, CD8 T cells, B cells).

[0341] Determination that a biological sample includes cells that overexpress A2aR, CD39 and / or CD73, and / or cells that contain elevated concentrations of adenosine, ADP and / or AMP, as compared to a reference, indicates that the subject suffers from a cancer that may benefit from treatment with an A2aR inhibitor. In some embodiments, the term "overexpression" refers to expression of an A2aR, CD39 and / or CD73 polypeptide in a large number of cells taken from a given patient, such as at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or more of tumor cells or lymphocytes taken from a subject.

[0342] In one embodiment, a method for treating or preventing cancer in a subject in need thereof comprises the steps of: (a) detecting the percentage of cells and / or extent of expression of A2aR, CD39 and / or CD73 in a tumor environment, optionally within a tumor and / or adjacent tissue, and after determining that the tumor environment includes cells that overexpress A2aR, CD39 and / or CD73, optionally with elevated levels of A2aR, CD39 and / or CD73 compared to a suitable reference level, (b) administering an anti-A2aR antibody to the subject. In one embodiment, the cells are tumor cells. In another embodiment, the cells within the tumor environment, tumor and / or adjacent tissue are non-malignant immune cells, such as T cells.

[0343] In some embodiments, determining the expression level of A2aR, CD39 and / or CD73 in the tumor environment comprises obtaining a biological sample from an individual, the biological sample comprising cancer tissue and / or tissue close to or surrounding the cancer (e.g., cancer adjacent tissue, adjacent non-tumor tissue, or normal adjacent tissue); contacting cells with antibodies that bind to A2aR polypeptides, CD39 polypeptides, and / or CD73 polypeptides, and detecting the percentage and / or expression level of cells of A2aR, CD39, and / or CD73. In certain embodiments, the expression of A2aR, CD39, and / or CD73 is assessed by their cell surface expression using immunohistochemistry.

[0344] The antibody compositions can be used as a monotherapy or in combination therapy with one or more other therapeutic agents, including agents that are commonly used for the specific therapeutic purpose for which the antibody is administered. See "Combination Therapy" above. The agent is typically administered in an amount and treatment regimen commonly used in monotherapy with other therapeutic agents to treat a specific disease or condition. Such therapeutic agents include, but are not limited to, anticancer agents and chemotherapeutic agents.

[0345] As described above, the methods of using the pharmaceutical compositions described herein include administering to a subject in need thereof an effective amount of the pharmaceutical composition described herein.

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

[0347] The pharmaceutical composition comprising the anti-A2aR antibody described in the present disclosure may be formulated in any pharmaceutically acceptable carrier or excipient. The term "pharmaceutically acceptable carrier" as used herein includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents and absorption delaying agents that are physiologically compatible. The pharmaceutical composition may contain a suitable solid or gel phase carrier or excipient. Exemplary carriers or excipients include, but are not limited to, calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycol. Exemplary pharmaceutically acceptable carriers include one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, and the like, and combinations thereof. In many cases, it is preferred to include an isotonic agent, such as a sugar, a polyol such as mannitol, sorbitol, or sodium chloride in the composition. A pharmaceutically acceptable carrier may also contain a small amount of auxiliary substances, such as a wetting agent or emulsifier, a preservative, or a buffer, which may increase the shelf life or effectiveness of the therapeutic agent.

[0348] In certain preferred embodiments, the therapeutically active agent may be incorporated into a pharmaceutical composition suitable for parenteral administration.The pharmaceutical composition may be formulated for parenteral administration by injection, for example, by bolus injection or continuous infusion.

[0349] Suitable buffers include, but are not limited to, sodium succinate, sodium citrate, sodium phosphate or potassium phosphate. Sodium chloride at a concentration of 0-300 mM (optimal concentration of 150 mM for liquid dosage forms) can be used to change the toxicity of the solution. Lyophilized dosage forms may include cryoprotectants, mainly 0-10% sucrose (optimal 0.5-1.0%). Other suitable cryoprotectants include trehalose and lactose. Lyophilized dosage forms may include fillers, mainly 1-10% mannitol (optimal 2-4%). ​​Stabilizers can be used for liquid dosage forms and lyophilized dosage forms, mainly 1-50 mM L-methionine (optimal 5-10 mM). Other suitable fillers include glycine, arginine, and 0-0.05% polysorbate-80 (optimal 0.005-0.01%). Other surfactants include, but are not limited to polysorbate 20 and BRIJ surfactants.

[0350] The therapeutic agent preparation can be lyophilized and stored as a sterile powder, preferably stored under vacuum, and then reconstituted in bacteriostatic water (including, for example, benzyl alcohol preservative) or sterile water before injection. The therapeutic agent in the pharmaceutical composition can be formulated with a "therapeutically effective amount" or a "preventive effective amount". "Therapeutically effective amount" refers to the amount that effectively achieves the desired therapeutic result within the necessary dosage and time period. The therapeutic effective amount of an antibody or an active agent can vary according to the following circumstances: the disease to be treated, the severity and course of the disease, the mode of administration, whether the antibody or active agent is administered for preventive purposes or therapeutic purposes, the bioavailability of a specific agent, the ability of the antibody to cause a desired response in an individual, previous treatment, the age, weight and sex of the patient, the clinical history of the patient and the response to the antibody, the type of antibody used, the discretion of the attending physician, etc. The therapeutic effective amount is also the amount in which the therapeutic beneficial effect is greater than any toxic or harmful effect of the recombinant vector. "Preventive effective amount" refers to the amount that effectively achieves the desired preventive result within the necessary dosage and time period.

[0351] Preferably, the polypeptide domains used in the antibodies or other active agents described herein are derived from the same host in which they are administered to reduce the inflammatory response caused by administration of the therapeutic agent. As suggested above, the therapeutic agent is suitably administered to the subject at one time or over a series of treatments, and may be administered to the patient at any time from diagnosis. The A2aR antibody may be administered as a sole therapy or in combination with other active agents or treatments useful for treating the disorder in question.

[0352] Whether administered once or multiple times, it is generally recommended that a therapeutically or prophylactically effective amount of an A2aR antibody (or other active agent) be administered in a range of about 1 ng / kg body weight / day to about 100 mg / kg body weight / day.In a specific embodiment, each A2aR antibody or active agent is administered in a range of about 1 ng / kg body weight / day to about 10 mg / kg body weight / day, about 1 ng / kg body weight / day to about 1 mg / kg body weight / day, about 1 ng / kg body weight / day to about 100 μg / kg body weight / day, about 1 ng / kg body weight / day to about 10 μg / kg body weight / day, about 1 ng / kg body weight / day to about 1 μg / kg body weight / day, about 1 ng / kg body weight / day to about 100 ng / kg body weight / day, about 1 ng / kg body weight / day to about 10 ng / kg body weight / day, about 10 ng / kg body weight / day to about 100 mg / kg body weight / day, about 10 ng / kg body weight / day to about 10 mg / kg body weight / day, about 10 ng / kg body weight / day to about 100 μg / kg body weight / day, about 10 ng / kg body weight / day to about 100 μg / kg body weight / day. 0 μg / kg body weight / day, about 10 ng / kg body weight / day to about 1 μg / kg body weight / day, 10 ng / kg body weight / day to about 100 ng / kg body weight / day, about 100 ng / kg body weight / day to about 100 mg / kg body weight / day, about 100 ng / kg body weight / day to about 10 mg / kg body weight / day, about 100 ng / kg body weight / day to about 1 mg / kg body weight / day, about 100 ng / kg body weight / day to about 100 μg / kg body weight / day, about 100 ng / kg body weight / day to about 10 μg / kg body weight / day, about 100 ng / kg body weight / day to about 1 μg / kg body weight / day, about 1 μg / kg body weight / day to about 100 mg / kg body weight / day, about 1 μg / kg body weight / day to about 100 μg / kg body weight / day, about 1 μg / kg body weight / day to about 10 μg / kg body weight / day, about 10 μg / kg body weight / day to about 100 mg / kg body weight / day, about 10 μg / kg body weight / day to about 10 mg / kg body weight / day, about 10 μg / kg body weight / day to about 1 mg / kg body weight / day, about 10 μg / kg body weight / day to about 100 μg / kg body weight / day, about 100 μg / kg body weight / day to about 100 mg / kg body weight / day, about 100 μg / kg body weight / day to about 10 mg / kg body weight / day, about 100 μg / kg body weight / day to about 10 mg / kg body weight / day, about 100 μg / kg body weight / day to about 10 mg / kg body weight / day.

[0353] In other embodiments, 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 other embodiments, each A2aR antibody and / or active agent is administered in a range of about 10 ng to about 100 ng, about 10 ng to about 1 μg, about 10 ng to about 10 μg, about 10 ng to about 100 μg, about 10 ng to about 1 mg, about 10 ng to about 10 mg, about 10 ng to about 100 mg, about 10 ng to about 1000 mg, about 10 ng to about 10,000 mg, about 100 ng to about 10,000 mg, about 100 ng to about 100 mg. 1 μg, about 100 ng to about 10 μg, about 100 ng to about 100 μg, about 100 ng to about 1 mg, about 100 ng to about 10 mg, about 100 ng to about 100 mg, about 100 ng to about 1000 mg, about 100 ng to about 10,000 mg, about 1 μg to about 10 μg, about 1 μg to about 100 μg, about 1 μg to about 1 mg, about 1 μg to about 10 mg, about 1 μg to about 100 mg, about 1 μg to about 1000 mg, about 1 μg to about 10,000 mg, about 10 μg to about 100 μg, about 10 μg to about 1 mg, about 10 μg to about 10 mg, about 10 μg to about 100 mg, about 10 μg to about 1000 mg, about 10 μg to about 10,000 mg, about 100 μg to about 1 mg, about 100 μg to about 10 mg, about 100 μg to about 100 mg, about 100 μg to about 1000 mg, about 100 μg to about 10,000 mg, about 1 mg to about 10 mg, about 1 mg to about 100 mg, about 1 mg to about 1000 mg, about 1 mg to about 10,000 mg, about 10 mg to about 100 mg, about 10 mg to about 1000 mg, about 10 mg to about 10,000 mg, about 100 mg to about 1000 mg, about 100 mg to about 10,000 mg, or about 1000 mg to about 10,000 mg. The antibodies described in the present disclosure can be administered every day, every 2, 3, 4, 5, 6 or 7 days, or every 1, 2, 3 or 4 weeks.

[0355] In other specific embodiments, the amount of each A2aR antibody or active agent administered can be a dosage of 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 of 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 above methods. In certain embodiments comprising administration of, for example, one or more recombinant AAV (rAAV) viruses, the pharmaceutical composition may include an amount of rAAVs containing at least 10 10 , at least 10 11 , at least 10 12 , at least 10 13 , or at least 10 14 genome copies (GC) or recombinant virus particles, or any range thereof. In certain embodiments, the pharmaceutical composition comprises an effective amount of the recombinant virus, such as rAAV, in an amount of at least 10 GC per subject. 10 , at least 10 11 , at least 10 12 , at least 10 13 , at least 10 14 , at least 10 15 genome copies or recombinant viral particle genome copies, or any range thereof.

[0357] Doses can be tested in one or several art-accepted animal models appropriate for any particular cell proliferative disorder or immunocompromised disease state.

[0358] Delivery methods may also include the use of polycation-concentrated DNA linked or unlinked to killed viruses, ligand-linked DNA, liposomes, eukaryotic cell delivery vector cells, deposition of photopolymerized hydrogel materials, use of handheld gene transfer particle guns, ionizing radiation, neutralization or fusion of nuclear charges with cell membranes, particle-mediated gene transfer, and the like.

[0359] 8. Diagnostic Uses of Antibodies

[0360] The antigen binding molecules (e.g., antibodies or antigen binding fragments thereof) described in the present invention can also be used to detect and / or measure human or cynomolgus monkey A2aR, or human or cynomolgus monkey A2aR expressing cells in a sample, for example, for diagnostic purposes. For example, anti-A2aR antibodies or antigen binding fragments thereof can be used to diagnose conditions or diseases characterized by abnormal expression of A2aR (e.g., overexpression, underexpression, lack of expression, etc.). Exemplary diagnostic assays for A2aR include, for example, contacting a sample obtained from a patient with an antibody described in the present invention, wherein the antibody is labeled with a detectable marker or reporter molecule. Alternatively, an unlabeled antibody can be combined with a secondary antibody that is itself detectably labeled for diagnostic applications. The detectable marker or reporter molecule can be a radioactive isotope, such as 3 H. 14 C. 18 F. 32 p. 35 S or 125 I; 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 for the A2aR diagnostic assay described in the present invention include any tissue or fluid sample that can be obtained from a patient, which contains a detectable amount of A2aR protein or fragments thereof under normal or pathological conditions. Typically, the level of A2aR in a specific sample obtained from a healthy patient (e.g., a patient who does not suffer from a disease or condition associated with abnormal A2aR levels or activity) is measured to preliminarily establish a baseline or standard level of A2aR. The baseline level of A2aR can then be compared with the level of A2aR measured in a sample obtained from an individual suspected of having an A2aR-related disease or condition.

[0361] Furthermore, human A2aR can be purified by immunoaffinity purification using the anti-A2aR antibodies described herein.

[0362] 9. Medicine Box

[0363] Any composition described herein, such as the anti-A2aR antigen binding molecules described herein, and / or other therapeutic agents, can be included in a kit. In a non-limiting example, the kit includes an antigen binding molecule, such as an antibody or an antigen binding fragment thereof. In certain embodiments, the kit further includes other therapeutic agents described herein.

[0364] The kit may also include an agent or instructions for treating a disease or condition. It may also include one or more buffers.

[0365] The ingredients of the kit can be packaged in aqueous medium or lyophilized form. The container device of the kit generally includes at least one vial, test tube, flask, bottle, syringe or other container device, in which the components can be placed, preferably, appropriately subpackaged in the container device. If there is more than one component in the kit (the labeled agent and the label can be packaged together), the kit generally also includes a second, third or other container, and the additional components can be placed separately in the other containers. The kit may also include a second container device for containing a sterile pharmaceutically acceptable buffer and / or other diluent. In addition, a variety of combinations of components may be contained in the vial. The kit of the present invention generally also includes a device for containing the composition of the present invention (e.g., anti-A2aR antigen binding molecules and / or other therapeutic agents), as well as any other sealed pharmaceutical container for commercial sale.

[0366] When the components of the kit are provided in the form of one and / or more liquid solutions, the liquid solution is an aqueous solution, particularly preferably a sterile aqueous solution. In addition, the components of the kit can be provided in dry powder form. When the medicament and / or the components are provided in dry powder form, the dry powder can be reconstituted by adding a suitable solvent. It is assumed that the solvent can also be provided in another container device.

[0367] The present invention is further illustrated by the following examples, which should not be construed as limiting the present invention.The contents of all references, patents and published patent applications cited throughout this application, as well as the figures and tables, are incorporated herein by reference. Example Example 1: Generation of Exemplary Humanized Anti-A2aR Monoclonal Antibodies

[0368] Three mouse anti-human A2aR monoclonal antibodies 1B5-3D7, 3F6-9G5 and 3F8-12E9 disclosed in the '601 publication were used to generate exemplary humanized anti-A2aR monoclonal antibodies. Based on the sequence identity between the mouse anti-A2aR monoclonal antibodies and the human germline, the IgBlast algorithm was used to select the closest human heavy chain variable region (VH) germline and light chain variable region (VL) germline. The CDR regions from the antibodies disclosed in the '601 publication were identified and defined based on a combination of Kabat numbering and IMGT numbering schemes (see Tables 1 and 2). The frameworks of the selected human germline were used as acceptors for the CDRs of the antibodies disclosed in the '601 publication. Vernier region residues are known to participate in antigen binding by: direct contact with the antigen, affecting the CDR loops, or affecting the chain-docking at the VH / VL interface. Multiple combinations of vernier region residues were reverse mutated to the murine residues of the parent antibody disclosed in the '601 publication.

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

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

[0371] In certain tests, the parent antibody in the '601 publication was converted into a chimeric antibody. To generate a chimeric antibody, the VH domain and VL domain of the parent antibody were synthesized and cloned in-frame into a HC plasmid containing a human IgG1 FES (L234F, L235E, P331S) isotype constant domain sequence and a LC plasmid containing a human IgK isotype constant domain sequence, respectively. The antibodies generated in this way 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". Example 2: Binding of Exemplary Humanized Anti-A2aR Monoclonal Antibodies to Human A2aR

[0372] This study was designed to confirm whether the exemplary humanized anti-A2aR monoclonal antibodies of the present invention bind to human A2aR expressed on the cell surface.

[0373] (1) Binding of Exemplary Humanized Anti-A2aR Monoclonal Antibodies to Human A2aR in the Supernatant of Transfected Expi293F Cells

[0374] Briefly, HEK293 cells stably overexpressing human A2aR (HEK-hA2aROCL, BPS Bioscience, Cat. No. 79381) were collected from fresh culture. 6 / ml concentration was resuspended in FACS buffer (1xDPBS / 1% FBS). 100 microliters (μl) of cells were aliquoted into each well of a 96-well plate. After centrifugation, the cell pellet in each well was resuspended in 100 μl of normalized supernatant sample, wherein the normalized supernatant sample contained 1 μg / ml humanized anti-A2aR monoclonal antibody variant, which was collected from a transfected Expi293 cell culture. The supernatant was mixed with the cells and incubated on ice for 1 hour. After washing the mixture of the supernatant (containing the expressed antibody) and the A2aR expressing cells with FACS buffer, 100 microliters (μl) of PE-conjugated anti-human Fc secondary antibody (Jackson ImmunoResearch, Cat. No.: 109-116-098) diluted 1:400 was added to each well, followed by incubation on ice for 30 minutes. After washing with FACS buffer, the stained cells were resuspended in 150 μl of 7-AAD (Biolegend, Cat. No. 420404) diluted (1:100) in 1xDPBS buffer and incubated at room temperature for 5 minutes in the dark before FACS analysis. Guava11HT or 5HT (Luminex) was used to obtain mean fluorescence intensity (MFI). Flowjo software was used to analyze the data.

[0375] Tables 22 to 24 below summarize the MFI values ​​determined above. As shown in Tables 22 to 24, the exemplary humanized anti-A2aR antibodies bind to A2aR expressed on the cell surface. The larger the MFI value, the stronger the binding of the exemplary antibody to A2aR expressed on the cell surface. Table 22: Binding of exemplary humanized anti-A2aR to human A2aR on the cell surface (derived from 1B5-3D7)

[0376] *: Average value of three parallel tests Table 23: Binding of exemplary humanized anti-A2aR to human A2aR on the cell surface (3F6-9G5 derived)

[0377] *: Average value of three parallel tests Table 24: Binding of exemplary humanized anti-A2aR to human A2aR on the cell surface (3F8-12E9 derived)

[0378] *: Average value of four parallel tests

[0379] (2) Binding of purified exemplary humanized anti-A2aR monoclonal antibodies expressed in ExpiCHO cells

[0380] Briefly, HEK293 cells stably overexpressing human A2aR (HEK-hA2aROCL, BPS Bioscience, Cat. No. 79381) were collected from fresh culture. The cells were then plated at 2 × 10 6 The concentration of 1xDPBS / 2% FBS was resuspended in FACS buffer (1xDPBS / 2% FBS) at 400 μl / ml. 100 μl of cells were aliquoted into each well of a 96-well plate. After centrifugation, the cell pellets in each well were resuspended in an exemplary antibody (expressed by ExpiCHO cells and purified by CRO BioIntron) purified by 1:3 serial dilution (starting concentration: 3.3 μg / mL, diluted in FACS buffer (1xPBS, 2% FBS)). The serially diluted antibodies were mixed with cells and incubated on ice for 30 minutes. After washing with FACS buffer, 100 μl of PE-coupled anti-human Fc secondary antibodies (diluted at 1:400, Jackson Immuno Research, Cat. No.: 109-116-098) were added to each well and then incubated on ice for 30 minutes. After washing with FACS buffer, the stained cells were resuspended in 200 μl of 7-AAD (Biolegend, Cat. No. 420404, diluted 1:100 in 1xDPBS buffer) and incubated at room temperature for 5 minutes in the dark before flow cytometry analysis on Guava 11HT or 5HT (Luminex). The mean fluorescence intensity (MFI) was obtained and the data were analyzed using Flowjo software.

[0381] Table 25 below summarizes the EC50 values ​​of the above assays. It was confirmed that the exemplary humanized anti-A2aR antibodies of the present disclosure specifically bind to A2aR expressed on the cell surface. Parental 3F6-9G5 and 3F8-12E9 antibodies were used as positive controls. Anti-HEL isotype control (BioIntron) was used as a negative control. Table 25: Binding of exemplary humanized anti-A2aR to cell surface human A2aR

[0382] *: Average value of 7 parallel tests Example 3: In vitro blocking activity of humanized anti-human A2aR monoclonal antibodies

[0383] The activity of A2aR is mediated by the Gs protein, which activates adenylate cyclase, leading to the synthesis of cAMP in the cell. The level of cAMP is related to the level of adenosine (agonist) of each. cAMP can be detected by a variety of commercial cAMP assay kits. This study aims to confirm whether several exemplary humanized antibodies can block A2aR activity.

[0384] (1) In vitro blocking activity of humanized anti-human A2aR monoclonal antibody 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 Catalog No.: A3890401) solution at 50 μl / well at 4°C overnight. After washing three times with sterile endotoxin-free water, the coated plates (uncovered) 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 Catalog No.: SH30024.01) + 2% charcoal-stripped serum (Thermo Fisher Catalog No.: A3382101) + 1% penicillin / streptomycin) and cultured overnight at 37°C, 5% CO2. On the next day, the coated cells were washed three times with 200 μl warm 1xPBS. Normal supernatant containing 1 or 2 μg / ml humanized anti-A2aR antibody (see Example 1) was diluted 3-fold serially and then mixed with induction buffer (1x PBS containing 1000 μM 3-isobutyl-1-methylxanthine (IBMX) ((MilliporeSigma, Catalog No.: I7018) + 200 μM Ro 20-1724 (Millipore Sigma, Catalog No.: B8279)) at a ratio of 1:1. Then 10 microliters (μl) of the supernatant / induction buffer mixture was added to each well and incubated at 37°C for 15 minutes, followed by the addition of NECA (Eurofins Scientific, Catalog No.: 92-1191) (stable adenosine receptor agonist) at a final concentration of 300 nM. The cells were then incubated at 37°C, 5% CO2 for 1 hour. cAMP-Glo ​​was used to detect the expression of NECA. TM The assay kit (Promega, Cat. No.: V1501) was used to perform cell lysis and cAMP detection according to the manufacturer's manual and instructions. Results (Relative Light Unit, RLU) were read using a photometer. RLU was converted to cAMP (nM) using a cAMP standard curve and GraphPad Prism software. The corresponding parent antibodies 1B5-3D7, 3F6-9G5 and 3F8-12E9 were used as positive controls.

[0386] As shown in Tables 26 to 27, the exemplary humanized anti-A2aR antibody variants blocked the activity of human A2aR expressed on the cell surface with an IC 50 The values ​​range from about 0.7 nM to about 20 nM. Table 26: IC of blocking activity of exemplary humanized anti-A2aR variants (3F6-9G5 derived) 50 Value (nM) Hu3F6-L3 Hu3F6-L4 Hu3F6-L5 Hu3F6-L8 3F6-9G5 LC Hu3F6-H2 na na na na Hu3F6-H3 2.0 19.5 5.3 na Hu3F6-H5 1.3 7.8 0.9 1.1 3F6-9G5 HC 1.3*

[0387] *: Average value of three parallel tests Table 27: IC of blocking activity of exemplary humanized anti-A2aR variants (3F8-12E9 derived) 50 Value (nM)

[0388] *: Average value of three parallel tests

[0389] (2) In vitro blocking activity of purified exemplary humanized anti-A2aR monoclonal antibodies expressed by ExpiCHO cells

[0390] Briefly, 96-well tissue culture clear flat-bottom plates (Fisher Scientific) were coated with 50 μg / ml poly-D-lysine (Gibco Catalog No. A3890401) solution at 50 μl / well at 4°C overnight. After washing three times with sterile endotoxin-free water, the coated plates (without covers) were placed in a biosafety cabinet to dry. Freshly thawed HEK293 cells overexpressing A2aR (BPS Bioscience) were incubated at 10 °C for 24 h on the poly-D-lysine-coated plates. 4Cells / well were seeded in 100 μl culture medium and cultured in a 37°C, 5% CO2 incubator for 24 hours. After washing once with warm PBS, 100 μl of starvation medium (MEM (Hyclone catalog number: SH30024.01) + 2% carbon-adsorbed serum (Thermo Fisher catalog number: A3382101) + 1% penicillin / streptomycin) was added and cultured overnight in an incubator at 37°C, 5% CO2. The next day, the starved coated cells were washed once with 200 μl warm 1xPBS. Serial dilutions of humanized anti-A2aR antibody (produced by CRO BioIntron in ExpiCHO cells) and A2aR antagonist small molecule inhibitor (SMI) CPI-444 (MedChemExpress, Cat. No.: HY-101978) and AB928 (MedChemExpress, Cat. No.: HY-129393) were prepared. 30 microliters (μL) of humanized anti-A2aR antibody, parental 3F6-9G5 or SMI was added to each well and incubated in a 37°C incubator for 105 minutes. During the last 15 minutes of incubation, 10 microliters (μl) of 4× induction buffer (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 microliters (μl) of medium was pipetted from each well 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 in an incubator at 37°C, 5% CO2 for 1 hour. Using cAMP-Glo TM Assay kit (Promega, Cat. No.: V1501), cell lysis and cAMP detection were performed according to the manufacturer's manual and instructions. SpectraMax 5 microplate reader (Molecular Device) was used to read the plate to obtain relative light units (RLU). Data were analyzed using Prism software. The corresponding parental antibodies 3F6-9G5 or 3F8-12E9 of the same isotype were used as positive controls.

[0391] like Figure 1A , Figure 1B As shown in Table 28, the exemplary humanized anti-A2aR antibody variants and the parent 3F6-9G5 have similar IC 50The IC values ​​of 1.5 and 1.5 were higher for blocking the activity of human A2aR expressed on the cell surface, while SMI CPI-444 and AB928 showed much higher IC values. 50 values, indicating that its blocking efficacy in this test is much lower. Table 28: IC of blocking activity of exemplary humanized anti-A2aR variants and SMI 50 Value (nM) IC50(nM) 3F6-9G5 Hu3F6-H3 / Hu3F6-L5 Hu3F6-H5 / Hu3F6-L5 CPI-444 AB928 5μM Adenosine 0.93 0.6 0.8 8 2.13 1.25 μM NECA 0.53 0.8 0.6 11.06 4.27 Example 4: Internalization of humanized anti-human A2aR monoclonal antibody

[0392] This example aims to confirm the internalization of the complex formed by the exemplary humanized anti-human A2aR monoclonal antibody of the present disclosure and A2aR expressed on the cell surface.

[0393] (1) Endocytosis assay based on flow cytometry

[0394] 2x10 5 Freshly cultured HEK293 cells expressing human A2aR (BPS Bioscience) were incubated on ice for 45 minutes with the test antibodies at the specified concentrations (1, 0.5 and 0.25 μg / ml) per well. Parental 3F6 was used as a positive control. After washing with FACS buffer, the samples were divided into 4 groups. One group of samples was maintained on ice. The other three groups of samples were further incubated in a 37°C, 5% CO2 incubator for 1, 2 and 4 hours, 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 minutes and then washed with cold FACS buffer. The cells were then treated with 7-AAD diluted 1:100. (Biolegend) staining, followed by fixation with 2% paraformaldehyde (diluted from 10% paraformaldehyde (MilliporeSigma, Cat. No. R04586)). All samples were analyzed on Guava 11HT or 5HT (Luminex). The mean fluorescence intensity (MFI) was obtained, and the data were analyzed using Flowjo software. The endocytosis rate was calculated as follows:

[0395] % internalization = ((MFI of sample on ice) - (MFI of sample incubated at 37°C for specified time) / (MFI of sample on ice)) × 100

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

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

[0398] Invitrogen TM Zenon TM pHrodo TM iFL RED Human IgG Labeling Reagent (Cat. No. Z25612) is a low pH sensitive dye that was used to determine the internalization properties of an exemplary anti-A2aR antibody according to the manufacturer's instructions. TM pHrodo TM Internalization of antibodies conjugated with iFL RED Human IgG Labeling Reagent can increase the fluorescent signal in the low pH intracellular region of the cell, which provides a method to visualize endocytosis under fluorescence microscopy.

[0399] Briefly, a 160 nM antibody (approximately 24 μg / ml, 25 μl) solution (4×) was prepared in FreeStyle CHO Expression Medium (Gibco, Cat. No. 12451-014) and then added to each well of a 96-well plate. Then, 25 microliters (μL) of Zenon TM pHrodo TM The iFLIgG labeling reagent working solution (4×) was then added to each well of the 96-well plate containing the antibody. The solution in the 96-well plate was mixed and incubated at room temperature for 5 minutes to form the labeling complex. Then, 50 microliters (μl) of CHO cells expressing human A2aR (2x10 6 / ml) was added to each well containing the labeled antibody. The cells were then incubated with the labeled complexes for 24 hours under standard cell culture conditions. The internalized antibodies were imaged and analyzed under an immunofluorescence microscope (Invitrogen EVOS Cell Imaging System M5000). The internalization of antibodies was imaged using the EVOS imaging system (Invitrogen). Figure 3As shown, endocytosis induction is visualized in representative figures, which shows that both parental 3F6 and humanized antibody variants Hu3F6-H3 / Hu3F6-L5 and Hu3F6-H5 / Hu3F6-L5 can induce endocytosis, while the anti-HEL isotype control antibody cannot. Hoechst 33342 (Invitrogen, Cat. No. H3570), a popular cell-permeable nuclear counterstain dye that emits blue fluorescence, was used to stain condensed nuclei inside cells. In this assay, SMI CPI-444 and AB928 did not 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] A2aR in humans is reported to be expressed by T cells and NK cells. This study was designed to confirm that the exemplary humanized anti-A2aR monoclonal antibodies of the present invention bind to A2aR on the surface of human and cynomolgus monkey PBMC cells.

[0401] Human and cynomolgus monkey PBMCs were purchased from Cytologics LLC (San Diego, CA) and iQ Biosciences (Berkeley, CA), respectively. 6 Human and / or cynomolgus PBMC 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 cynomolgus surface markers 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). Live immune cell subsets were defined using Zombie Green fixable viability dye (Biolegend, Cat. No.: 423111) according to the manufacturer's instructions. After washing with FACS buffer, cells were analyzed on a FACSAria (BD). Data were analyzed using Flowjo (Becton Dickinson).

[0402] As shown in Figure 4 and Tables 29 to 32, the parental 3F6-9G5 and two exemplary humanized anti-human A2aR antibodies bound to the following cells: a small subset 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; while the anti-HEL isotype control (Biointron) did not bind to the above cells. Parental 3F6-9G5 and two exemplary humanized anti-human A2aR antibodies also bound to cynomolgus monkey T cells (both CD4+ and CD8+, Figure 4B ), NK cells and NKT cells cross-react. Table 29: Binding of exemplary humanized anti-A2aR antibodies to CD4+ T cells Table 30: Binding of exemplary humanized anti-A2aR antibodies to CD8+ T cells Table 31: Binding of exemplary humanized anti-A2aR antibodies to NK cells Table 32: Binding of exemplary humanized anti-A2aR antibodies to NKT cells Example 6: Restoration of T cell function by humanized anti-human A2aR monoclonal antibody

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

[0404] 200,000 (2 × 10 5 ) Peripheral blood mononuclear cells (PBMC) were aliquoted into each well of a 96-well plate, and the cells in the 96-well plate were pre-incubated with the specified concentration of antibodies or small molecule inhibitors for 90 minutes. T cell activator anti-CD3 / 28 immunomagnetic beads (dynabeads) (Gibco, catalog number: 11132D) were then added at a volume ratio of 1:4 (Beads:PBMC, v / v) and NECA (DiscoverX, catalog number: 92-1191, final concentration of 0.1 μM) was added. The cells were cultured in an incubator at 37°C and 5% CO2 for 24 hours. The cells were then collected and placed in a GolgiPlug TMThe cells were stimulated with anti-CD3 / 28 immunomagnetic beads for 4 h in the presence of a protein transport inhibitor (BD Biosciences, Cat. No. 555028) according to the manufacturer’s instructions. After this, the cells were incubated with human TruStain FcX blocker (Biolegend, cat. no.: 422302) and True-Stain monocyte blocker (Biolegend, cat. no.: 426103) were incubated for 15 minutes, and then the cells were stained with the following surface markers: PE-anti-CD4 (Biolegend, cat. no.: 300550), PerCP-cy5.5-anti-CD8 (Biolegend, cat. no.: 301032), APC / cy7-anti-CD3 (Biolegend, cat. no.: 300426), PE / cy7-anti-CD56 (Biolegend, cat. no.: 362509), PE / cy7-anti-CD56 (Biolegend, cat. no.: 362509), BV711-anti-CD25 (Biolegend, cat. no.: 302636) and BV605-anti-CD69 (Biolegend, cat. no.: 31938). After washing with FACS buffer, cells were stained with BV421 anti-IFN-γ (BD Bioscience, Cat. No. 562988) and APC anti-IL-2 (BD Bioscience, Cat. No. 551383) according to the manufacturer's instructions using an intracellular cytokine staining kit (BD Biosciences, Cat. No. 555028). The stained samples were analyzed on a FACSAria III machine. Data were analyzed using Flowjo software.

[0405] like Figure 5A As shown, it was demonstrated that the exemplary anti-A2aR antibodies Hu3F6-VH3 / Hu3F6-VL5, Hu3F6-VH5 / Hu3F6-VL5 and the parental 3F6-9G5 antibody exhibited higher potency in restoring IFN-γ production by human CD4 T cells inhibited by NECA compared to the small molecule inhibitors AB928, AZD4635 and CPI-444. Figure 5B As shown, anti-A2aR antibodies were also more potent than the small molecule inhibitors AZD4635 and CPI-444 in restoring IL-2 production by human CD4 T cells suppressed by NECA.

[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 can not only restore the IFN-γ and IL-2 production of CD4 T cells inhibited by NECA (Figure 5), but also restore the IFN-γ and IL-2 production of CD8 T cells inhibited by NECA; its potency is higher than that of small molecules AB928, CPI-444 and AZD4635. In addition, these antibodies can also restore the IFN-γ and IL-2 production of NK cells inhibited by NECA, and their potency is higher than that of the small molecule inhibitors tested. Table 33: IFN and IL2 secreted by CD4+ T cells, CD8+ T cells and NK cells

[0407] As shown in Table 34, exemplary anti-A2aR antibodies Hu3F6-VH3 / Hu3F6-VL5, Hu3F6-VH5 / Hu3F6-VL5 and parental 3F6-9G5 antibody restored CD25 and CD69 surface marker activation of CD4 and CD8 T cells inhibited by NECA with potency similar to that of the small molecule AB928 used at 1 μg / ml and CPI-444 and AZD4635 used at 2 μg / ml. Table 34: Activation of CD4+ T cells and CD8+ T cells

[0408] The above description is intended to guide those of ordinary skill in the art in practicing the present invention, rather than to describe in detail all obvious modifications and variations, which will become apparent to those skilled in the art after reading this specification. However, all such obvious modifications and variations should be included within the scope of the present invention, which is defined by the appended claims. Unless the context specifically indicates otherwise, the claims are intended to cover the claimed components and steps in any order that is effective to meet their intended objectives.

Claims

1. An isolated antibody or antigen-binding fragment thereof that binds to a human adenosine A2A receptor (A2aR), comprising a heavy chain variable (VH) domain comprising, from N-terminus to C-terminus, three heavy chain complementarity-determining regions (CDRs): HCDR1, HCDR2, and HCDR3; and A light chain variable (VL) domain comprising three light chain complementarity determining regions (CDRs) from N-terminus to C-terminus: LCDR1, LCDR2 and LCDR3; wherein (a) HCDR1 comprises the amino acid sequence GFX1FTX2X3WMN (SEQ ID NO: ), wherein X1 is A or T, X2 is R or S; and X3 is F or Y; (b) HCDR2 comprises the amino acid sequence RIDPX4DSEX5X6YX7X8KFWX9 (SEQ ID NO:), 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) HCDR3 comprises the amino acid sequence GRSLYGKGDY (SEQ ID NO: 10) or LRSLYGKGDY (SEQ ID NO: 11); (d) LCDR1 contains the amino acid sequence RSSQSX 11 VX 12 NGNTYLE (SEQ ID NO: ), wherein X 11 is L or I, and X 12 is R or S; (e) LCDR2 comprises the amino acid sequence KVSNRFS (SEQ ID NO: 14); and (f) LCDR3 comprises the amino acid sequence FQGSHVPLT (SEQ ID NO: 15) or YQGSHVPLT (SEQ ID NO: 16); The antibody or antigen-binding fragment thereof comprises a light chain framework region 4 (LFR4), which comprises the amino acid sequence FGGGTKVEIK (SEQ ID NO: 50).

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

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

4. An isolated antibody or antigen-binding fragment thereof that binds to a human adenosine A2A receptor (A2aR), comprising a heavy chain variable (VH) domain comprising, from N-terminus to C-terminus, three heavy chain complementarity determining regions (CDRs): HCDR1, HCDR2, and HCDR3; and A light chain variable (VL) domain comprising three light chain complementarity determining regions (CDRs) from N-terminus to C-terminus: LCDR1, LCDR2 and LCDR3; wherein (a) HCDR1 comprises the amino acid sequence GFX1FTX2X3WMN (SEQ ID NO: ), wherein X1 is A or T, X2 is R or S; and X3 is F or Y; (b) 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) HCDR3 comprises the amino acid sequence GRSLYGKGDY (SEQ ID NO: 10) or LRSLYGKGDY (SEQ ID NO: 11); (d) LCDR1 contains the amino acid sequence RSSQSX 11 VX 12 NGNTYLE (SEQ ID NO: ), wherein X 11 is L or I, and X 12 is R or S; (e) LCDR2 comprises the amino acid sequence KVSNRFS (SEQ ID NO: 14); and (f) LCDR3 comprises the amino acid sequence FQGSHVPLT (SEQ ID NO: 15) or YQGSHVPLT (SEQ ID NO: 16).

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

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

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

8. The isolated antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, further comprising: (a) a heavy chain framework region 1 (HFR1) comprising the amino acid sequence 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 2 (HFR2) comprising the amino acid sequence 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 3 (HFR3) comprising the amino acid sequence 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 WGQGTTVTVSS (SEQ ID NO: 32) or WGHGTTVTVSS (SEQ ID NO: 33).

9. The isolated antibody or antigen-binding fragment thereof according to claim 8, wherein (a) HFR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 17-21; (b) HFR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 22, 23 and 24; (c) HFR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 25-31; and (d) HFR4 comprises the amino acid sequence shown in SEQ ID NOs: 32 or 33.

10. The isolated antibody or antigen-binding fragment thereof according to claim 9, wherein the heavy chain variable region comprises a structure of HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4 from N-terminus to C-terminus, wherein HFR1, HFR2, HFR3 and HFR4 respectively comprise: (a) sequences shown in SEQ ID NO:17, SEQ ID NO:22, SEQ ID NO:25, and SEQ ID NO:32; (b) the sequences shown in SEQ ID NO:18, SEQ ID NO:22, SEQ ID NO:26, and SEQ ID NO:32; (c) the sequences shown in SEQ ID NO:18, SEQ ID NO:23, SEQ ID NO:26, and SEQ ID NO:32; (d) the sequences shown in SEQ ID NO:18, SEQ ID NO:23, SEQ ID NO:27, and SEQ ID NO:32; (e) the sequences shown in SEQ ID NO:18, SEQ ID NO:23, SEQ ID NO:28, and SEQ ID NO:32; (f) the sequences shown in SEQ ID NO:19, SEQ ID NO:24, SEQ ID NO:29, and SEQ ID NO:32; (g) the sequences shown in SEQ ID NO:17, SEQ ID NO:22, SEQ ID NO:25, and SEQ ID NO:33; (h) the sequences shown in SEQ ID NO:18, SEQ ID NO:22, SEQ ID NO:26, and SEQ ID NO:33; (i) the sequences shown in SEQ ID NO:18, SEQ ID NO:23, SEQ ID NO:27, and SEQ ID NO:33; (j) the sequences shown in SEQ ID NO:18, SEQ ID NO:23, SEQ ID NO:28, and SEQ ID NO:33; (k) the sequences shown in SEQ ID NO:20, SEQ ID NO:24, SEQ ID NO:29, and SEQ ID NO:33; (1) the sequences shown in SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:26, and SEQ ID NO:32; (m) the sequences shown in SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:30, and SEQ ID NO:32; (n) the sequences shown in SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:31, and SEQ ID NO:32; or (o) The sequences shown in SEQ ID NO:20, SEQ ID NO:24, SEQ ID NO:29, and SEQ ID NO:

32.

11. The isolated antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, further comprising: (a) a light chain framework region 1 (LFR1) comprising the amino acid sequence D-X26-VMTQ-X27-P-X28-SL-X29-V-X30-X31-G-X32-PASISC (SEQ ID NO:) or DIVMTQSPDSLAVSLGERATINC (SEQ ID NO:), 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 W-X33-X34-Q-X35-PGQ-X36-P-X37-X38-LIY (SEQ ID NO:), 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) light chain framework region 3 (LFR3), which comprises the amino acid sequence GVPDRFSGSGSG-X39-DFTL-X40-IS-X41-X42-X43-AEDV-X44-VYYC (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.

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

13. The isolated antibody or antigen-binding fragment thereof according to claim 12, wherein the light chain variable region comprises a structure of LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4 from N-terminus to C-terminus, wherein LFR1, LFR2, LFR3 and LFR4 respectively comprise: (a) sequences shown in SEQ ID NO:34, SEQ ID NO:42, SEQ ID NO:47 and SEQ ID NO:50; (b) the 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) the sequences shown in SEQ ID NO:37, SEQ ID NO:45, SEQ ID NO:47 and SEQ ID NO:50; (g) the sequences shown in SEQ ID NO:38, SEQ ID NO:45, SEQ ID NO:47 and SEQ ID NO:50; (h) the sequences shown in SEQ ID NO:39, SEQ ID NO:45, SEQ ID NO:49 and SEQ ID NO:50; (i) the sequences shown in SEQ ID NO:40, SEQ ID NO:45, SEQ ID NO:49 and SEQ ID NO:50; or (j) The sequences shown in SEQ ID NO:41, SEQ ID NO:45, SEQ ID NO:47 and SEQ ID NO:

50.

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

15. An isolated antibody or antigen-binding fragment thereof that binds to human adenosine A2A receptor (A2aR), comprising: a heavy chain variable (VH) domain comprising, from N-terminus to C-terminus, three heavy chain complementarity determining regions (CDRs): HCDR1, HCDR2, and HCDR3; and A light chain variable (VL) domain comprising three light chain complementarity determining regions (CDRs) from N-terminus to C-terminus: LCDR1, LCDR2 and LCDR3; wherein (a) HCDR1 comprises an amino acid sequence that is about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, and 3; (b) HCDR2 comprises an amino acid sequence that is about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 4-9 and 89; (c) HCDR3 comprises an amino acid sequence that is about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identical to the amino acid sequence set forth in SEQ ID NO: 10 or 11; (d) LCDR1 comprises an amino acid sequence that is about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 12 and 13; (e) LCDR2 comprises an amino acid sequence that is about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identical to the amino acid sequence set forth in SEQ ID NO:14; and (f) LCDR3 comprises an amino acid sequence that is about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 15 and 16; and wherein the antibody or antigen-binding fragment thereof comprises a light chain framework region 4 (FR4) comprising an amino acid sequence that is about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to about 100% identical to the sequence FGGGTKVEIK (SEQ ID NO: 50).

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

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

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

87.

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

20. The isolated antibody or antigen-binding fragment thereof according to any one of claims 1 to 19, wherein (i) the antibody competes for binding to human A2aR with a monoclonal antibody selected from 1B5-3D7, 3F6-9G5 and 3F8-12E9; (ii) the antibody inhibits the activity of A2aR; (iii) the antibody improves the 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 cAMP response elements in the A2aR signaling pathway; (viii) the antibody specifically binds to human and / or cynomolgus monkey A2aR; or (viii) The antibody induces A2aR endocytosis.

21. An isolated antibody or antigen-binding fragment thereof that competes with the antibody or antigen-binding fragment thereof of any one of claims 1 to 20 for binding to human A2aR.

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 class IgA, IgD, IgE, IgG and IgM.

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

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

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

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

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

30. A method for preparing a polypeptide, comprising expressing the polypeptide in the recombinant cell of claim 29, optionally further comprising isolating the expressed polypeptide, wherein the polypeptide is selected from the antibody or antigen-binding fragment thereof, its HCVR, its LCVR, its light chain, its heavy chain and its antigen-binding fragment as described in any one of claims 1 to 26.

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

32. The pharmaceutical composition according to claim 31, wherein the pharmaceutical composition comprises an effective dose of the antibody or antigen-binding fragment thereof, so that it (a) specifically binds to human or cynomolgus monkey A2aR on the cell surface; (b) reduces the cAMP concentration in the tissue; (c) inhibits the activity of human A2aR; (d) reduces the phosphorylation of the cAMP response element of the A2aR signaling pathway; (e) improves the immune response of immune cells; (f) reduces the activity of protein kinase A; or (g) achieves any combination of (a)-(f).

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

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

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

36. A method in a subject, comprising administering to the subject 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, thereby treating cancer.

37. The method of any one of claims 33 to 36, wherein the method activates T cells and directs them to kill tumor target cells.

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

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

40. The method of claim 39, wherein the checkpoint inhibitor is selected from the group consisting of inhibitors of CTLA-4, PD-1, PD-L1, PD-L2, TIGIT, LAG-3, TIM-3, B7-H3, B7-H4, CD73, PVRIG / PVRL2, mutexin, BTLA, CECAM-1, CECAM-5, CECAM6, IL-1R8, VISTA, LAIR1, LILRB1, LILRB2, LILRB3, LILRB4, LILRB5, CD47, SIRPα, CD200R, CD96, CD112R, 2B4, TGFβ-R, KIR, NKG2A, SEMA4D, Axl, MerTK, GAS6, TNFR2, GARP, CCR8, IDO, NOX2, SIGLEC7, SIGLEC15, and / or any combination thereof.

41. The method of claim 40, wherein the inhibitor inhibits the interaction between PD-1 and PD-L1, and the inhibitor is selected from pembrolizumab, nivolumab, atezolizumab, avelumab, durvalumab, BMS-936559, sintilimab, toripalimab, tislelizumab, carrelizumab, envolimab, sugemalimab, penampalimumab, candidizumab, sulfamethoxazole and sulfamethoxazole.

42. The method of claim 40, wherein the CTLA4 inhibitor is selected from ipilimumab, cadunimumab, YH001 (Euhe Pharmaceuticals), ADG116 and ADG126 (Tianyan Pharmaceuticals).

43. The method of claim 39, wherein the other therapeutic agent is an agonist of an immune cell stimulating 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 any combination thereof.

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

45. The method of any one of claims 38 to 43, wherein the other therapeutic agent and the antibody or antigen-binding fragment thereof are formulated in different pharmaceutical compositions.

46. ​​The method of any one of claims 38 to 43 and 45, wherein the other 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 other 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. The kit of claim 48 further comprising an additional therapeutic agent.

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