Anti-adenosine receptor (A2aR) antibody

Abstract

We offer anti-adenosine receptor (A2aR) antibodies. [Solution] The present invention provides an anti-A2aR antigen-binding molecule comprising an antibody and its antigen-binding fragment, and a method for using it to treat various diseases associated with abnormal adenosine signaling, including cancer, chronic diseases, chronic infections, autoimmune diseases, inflammatory diseases, neurodegenerative diseases, and fibrotic diseases. The present invention provides an antigen-binding molecule, such as an anti-A2aR antibody or its antigen-binding fragment, for modulating (e.g., enhancing or inhibiting) the activity of A2aR by specifically binding to A2aR.

Description

[Technical Field]

[0001] Related applications This application relates to and claims priority to U.S. Provisional Patent Application No. 63 / 085,612 filed on 30 September 2020. The entire contents of the aforementioned application are expressly incorporated herein by reference.

[0002] field The present invention relates to antibodies for cancer treatment, and more particularly to adenosine receptor A2aR antibodies for cancer immunotherapy. [Background technology]

[0003] background The immune system plays a crucial role in identifying and eliminating neoplastic cells. Tumor cells employ various mechanisms to evade immune-mediated destruction. Among these pathways, tumor cells leverage the adenosine signaling pathway to evade immune defense by increasing the levels of adenosine and the responsiveness to adenosine, a highly effective inhibitor of effector T cell function.

[0004] Adenosine is a purine nucleoside derived from the breakdown of adenosine triphosphate (ATP). Extracellular levels of adenosine significantly increase under adverse conditions, including hypoxia, ischemia, inflammation, or cancer. Upon release, adenosine activates cellular signaling pathways through the association of four known G protein-coupled receptors: adenosine A1 receptor subtype (A1R), adenosine A2A receptor subtype (A2aR), adenosine A2B receptor subtype (A2bR), and adenosine A3 receptor subtype (A3R).

[0005] Adenosine levels are primarily regulated by the activity of CD39 and CD73. CD39 and CD73 are two ectoenzymes that work together in a two-step reaction to convert pro-inflammatory ATP into immunosuppressive adenosine. CD39 hydrolyzes ATP to AMP, which is further hydrolyzed by CD73 to adenosine, which can easily enter most cells. Furthermore, as tumor cells undergo cell death as a result of metabolic or hypoxic stress, tumor cells release their intracellular storage of ATP (to which cells are generally impermeable) into the extracellular space.

[0006] Within the tumor microenvironment, adenosine produced by CD73 suppresses the anti-tumor immune response while promoting tumor cell growth and survival. Cancer cells exhibit high levels of CD73 expression in tumor tissue, and their accumulation is associated with poor overall survival and poor recurrence-free survival in patients with breast and ovarian cancer. CD73 and adenosine support growth-promoting neoangiogenesis, metastasis, and survival in cancer cells. Adenosine is involved in A2A (or A) on T cells. 2A It binds to the A2aR receptor and activates an intracellular signaling cascade that leads to T cell activation and suppression of function. A2aR is a member of the adenosine receptor group of G protein-coupled receptors, which also includes A1R, A2bR, and A3R, and is an anti-inflammatory effector of extracellular adenosine through its dominant expression on cells in brain and lymphoid tissues.

[0007] The presence of abnormally high concentrations of adenosine in the immune microenvironment leads to activation of A2aRs, exhibiting a negative feedback loop that allows tumors to thereby evade immune recognition. In particular, adenosine-mediated activation of A2aRs is linked to the regulation of myeloid-derived suppressor cells (MDSCs) and T13 (T13). reg ) enhances the activity of immunosuppressive cell types, including CD8 cells, while inhibiting IFNγ production. +By suppressing the activity of multiple anti-tumor immune cells, including T cells, dendritic cells, natural killer cells, and M1 macrophages, tumors can evade immune surveillance. Activation of A2aR on tumor cells has also been suggested to promote tumor cell metastasis.

[0008] While several A2A receptor small molecule antagonists are progressing to clinical trials for the treatment of Parkinson's disease and cancer, A2aR blockade by biopharmaceutical drug candidates in the context of cancer treatment remains lacking. Mice treated with an A2aR antagonist, e.g., ZM241385, showed a significant delay in tumor growth, resulting from reduced immunosuppression against effector T cells. This was further highlighted by A2aR knockout mice, which showed increased tumor rejection. Furthermore, A2aR blockade by small molecule antagonists has been shown to have a synergistic effect on increasing the immune response when combined with PD-1 / PD-L1 or CTLA-4 inhibition by monoclonal antibodies, compared to blockade of a single PD-1 / PD-L1 or CTLA-4 pathway alone. [Overview of the Initiative] [Problems that the invention aims to solve]

[0009] As a result, modulating A2aR activity, adenosine concentration, and / or CD39 / CD73 expression and activation of effector immune cells in the tumor microenvironment presents an attractive therapeutic strategy for limiting tumor progression, improving the anti-tumor immune response, avoiding treatment-induced immune deviations, and potentially limiting normal tissue toxicity. Compositions and methods for treating cancer by modulating, for example, inhibiting, the A2aR activity of immune cells are needed in the field. [Means for solving the problem]

[0010] overview The present invention provides antigen-binding molecules, such as anti-A2aR antibodies or their antigen-binding fragments, for modulating (e.g., enhancing or inhibiting) the activity of A2aR by specifically binding to A2aR. A2aR may be present on the surface of cells, such as mammalian cells, such as mammalian immune cells, such as mouse immune cells, cynomolgus monkey immune cells, or human immune cells. The present invention also provides methods of using the antigen-binding molecules of the present invention, such as anti-A2aR antibodies or their antigen-binding fragments, to modulate, for example, inhibit the activity of A2aR, or to treat subjects who would benefit from modulating, for example, inhibiting the activity of A2aR, such as subjects suffering from or prone to suffering from A2aR-related diseases.

[0011] Accordingly, in one embodiment, the present invention provides an isolated antigen-binding molecule, such as an antibody or an antigen-binding fragment thereof, that binds to the human adenosine A2A receptor (A2aR). The antibody comprises a heavy chain variable (VH) domain from the N-terminus to the C-terminus containing three heavy chain complementarity-determining regions (CDRs): HCDR1, HCDR2, and HCDR3; and a light chain variable (VL) domain from the N-terminus to the C-terminus containing three light chain complementarity-determining regions (CDRs): LCDR1, LCDR2, and LCDR3; (a) HCDR1 comprises the amino acid sequence X1-X2-WMN (SEQ ID NO: 8), where X1 is S or R, and X2 (b) HCDR2 comprises the amino acid sequence RIDP-X3-DSE-X4-X5-Y-X6-HKFW-X7 (SEQ ID NO: 9), where X3 is S or Y, X4 is A or T, X5 is H or Q, X6 is H or N, and X7 is D or G; (c) HCDR3 comprises the amino acid sequence SLYGKGDY (SEQ ID NO: 3); (d) LCDR1 comprises the amino acid sequence RSSQSX 17 -VHX 18 -Includes NGNTYLE (SEQ ID NO: 30), in the formula, X 17 is L or I, and X 18is R or S; (e) LCDR2 comprises the amino acid sequence K-V-S-N-R-F-S (SEQ ID NO: 26); (f) LCDR3 comprises the amino acid sequence X 19 -Q-G-S-H-V-P-L-T (SEQ ID NO: 31), wherein X 19 is Y or F.

[0012] In various aspects and embodiments of the present invention, the antibody is an antigen-binding fragment of the antibody. In various aspects and embodiments of the present invention, human A2aR comprises the sequence shown in SEQ ID NO: 50.

[0013] In one embodiment, (a) HCDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 4 and 6; (b) HCDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 5 and 7; (c) HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 3; (d) LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 25 or 28; (e) LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 26; (f) LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 27 or 29.

[0014] In another embodiment, the isolated antigen-binding molecule (e.g., antibody) comprises (a) HCDR1 containing the amino acid sequence shown in SEQ ID NO: 1, HCDR2 containing the amino acid sequence shown in SEQ ID NO: 2, HCDR3 containing the amino acid sequence shown in SEQ ID NO: 3, LCDR1 containing the amino acid sequence shown in SEQ ID NO: 25, LCDR2 containing the amino acid sequence shown in SEQ ID NO: 26, and LCDR3 containing the amino acid sequence shown in SEQ ID NO: 27; (b) HCDR1 containing the amino acid sequence shown in SEQ ID NO: 4, HCDR2 containing the amino acid sequence shown in SEQ ID NO: 5, HCDR3 containing the amino acid sequence shown in SEQ ID NO: 3, LCDR1 containing the amino acid sequence shown in SEQ ID NO: 28, LCDR2 containing the amino acid sequence shown in SEQ ID NO: 26, and LCDR3 containing the amino acid sequence shown in SEQ ID NO: 29; or (c) HCDR1 containing the amino acid sequence shown in SEQ ID NO: 6, HCDR2 containing the amino acid sequence shown in SEQ ID NO: 7, HCDR3 containing the amino acid sequence shown in SEQ ID NO: 3, LCDR1 containing the amino acid sequence shown in SEQ ID NO: 25, LCDR2 containing the amino acid sequence shown in SEQ ID NO: 26, and LCDR3 containing the amino acid sequence shown in SEQ ID NO: 29

[0015] In yet another embodiment, (a) HCDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 21, 22, 23, and 24; and (b) HCDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 15, and 20.

[0016] In another embodiment, the antigen-binding molecule (e.g., an antibody) includes (a) a heavy chain variable region (HCVR) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs. 35, 36, 37, 38, 39, and 40; and (b) a light chain variable region (LCVR) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs. 41, 42, and 43.

[0017] In one embodiment, the antigen-binding molecule (e.g., an antibody) comprises (a) an HCVR containing the amino acid sequence shown in SEQ ID NO: 35 or 38 and an LCVR containing the amino acid sequence shown in SEQ ID NO: 41; (b) an HCVR containing the amino acid sequence shown in SEQ ID NO: 36 or 39 and an LCVR containing the amino acid sequence shown in SEQ ID NO: 42; or (c) an HCVR containing the amino acid sequence shown in SEQ ID NO: 37 or 40 and an LCVR containing the amino acid sequence shown in SEQ ID NO: 43.

[0018] In another embodiment, the present invention provides an isolated antigen-binding molecule (e.g., an antibody) that binds to the human adenosine A2A receptor (A2aR). This antigen-binding molecule (e.g., an antibody) comprises a heavy chain variable (VH) domain from the N-terminus to the C-terminus containing three heavy chain complementarity-determining regions (CDRs): HCDR1, HCDR2, and HCDR3; and a light chain variable (VL) domain from the N-terminus to the C-terminus containing three light chain complementarity-determining regions (CDRs): LCDR1, LCDR2, and LCDR3; (a) HCDR1 contains an amino acid sequence that is approximately 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, to approximately 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 4, and 6; (b) HCDR2 contains an amino acid sequence that is approximately 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, to approximately 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 5, and 7; (c) HCD (d) R3 contains an amino acid sequence that is approximately 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, to approximately 100% identical to the amino acid sequence shown in SEQ ID NO: 3; (d) LCDR1 contains an amino acid sequence that is approximately 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, to approximately 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 25 and 28; (e) LCDR2 contains an amino acid sequence that is approximately 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, to approximately 100% identical to the amino acid sequence shown in SEQ ID NO: 26; (f) LCDR3 contains an amino acid sequence that is approximately 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, to approximately 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 27 and 29.

[0019] In one embodiment, (a) HCDR1 includes a sequence containing one or two amino acid substitutions from SEQ ID NOs: 1, 4, or 6; (b) HCDR2 includes a sequence containing one, two, three, four, or five amino acid substitutions from SEQ ID NOs: 2, 5, or 7; (c) HCDR3 includes a sequence containing one or two amino acid substitutions from the sequence shown in SEQ ID NOs: 3 or from SEQ ID NOs: 3; (d) LCDR1 includes a sequence containing one, two, three, or four amino acid substitutions from SEQ ID NOs: 25 or 28; (e) LCDR2 includes a sequence containing one or two amino acid substitutions from the sequence shown in SEQ ID NOs: 26 or from SEQ ID NOs: 26; (f) LCDR3 includes a sequence containing one or two amino acid substitutions from SEQ ID NOs: 27 or 29. In another embodiment, the amino acid substitutions are conservative substitutions. In another embodiment, (a) HCDR1 includes an amino acid substitution at position 1 or 2 of HCDR1; (b) HCDR2 includes an amino acid substitution at position 5, 9, 10, 12, or 17 of HCDR2; (c) LCDR1 includes an amino acid substitution at position 6 or 9 of LCDR1; or (d) LCDR3 includes an amino acid substitution at position 1 of LCDR3. In one embodiment, the amino acid substitution is a conservative substitution. In another embodiment, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined based on the Kabat numbering scheme.

[0020] In one embodiment, (a) HCDR1 includes a sequence containing one, two, or three amino acid substitutions from SEQ ID NOs: 10, 13, or 16; (b) HCDR2 includes a sequence containing one, two, or three amino acid substitutions from SEQ ID NOs: 11, 14, or 17; (c) HCDR3 includes a sequence containing one, two, or three amino acid substitutions from SEQ ID NOs: 12 or 15; (d) LCDR1 includes a sequence containing one, two, three, or four amino acid substitutions from SEQ ID NOs: 32 or 33; and (e) LCDR3 includes a sequence containing one or two amino acid substitutions from SEQ ID NOs: 27 or 29. In another embodiment, the amino acid substitutions are conservative substitutions. In another embodiment, (a) HCDR1 includes an amino acid substitution at position 3, 6, or 7 of HCDR1; (b) HCDR2 includes an amino acid substitution at position 4 or 8 of HCDR2; (c) HCDR3 includes an amino acid substitution at position 1 of HCDR3; (d) LCDR1 includes an amino acid substitution at position 3 or 6 of LCDR1; or (e) LCDR3 includes an amino acid substitution at position 1 of LCDR3. In one embodiment, the amino acid substitution is a conservative substitution. In another embodiment, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined based on the IMGT numbering scheme.

[0021] In another embodiment, the present invention provides an antigen-binding molecule, for example, an antibody. This antibody comprises (a) a heavy chain variable region (HCVR) containing an amino acid sequence that is approximately 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to approximately 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs. 35, 36, and 37; and (b) a light chain variable region (LCVR) containing an amino acid sequence that is approximately 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to approximately 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs. 41, 42, and 43.

[0022] In one embodiment, (a) HCVR comprises a sequence containing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acid substitutions from SEQ ID NOs. 35, 36, and 37; and (b) LCVR comprises a sequence containing 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions from SEQ ID NOs. 41, 42, and 43. In another embodiment, the amino acid substitutions are conservative substitutions.

[0023] In one embodiment, the present invention provides an antigen-binding molecule, such as an antibody, that binds to the human adenosine A2A receptor (A2aR). This antibody comprises (a) a heavy chain variable region (HCVR) containing the amino acid sequence shown in SEQ ID NO: 35 or 38; and (b) a light chain variable region (LCVR) containing the amino acid sequence shown in SEQ ID NO: 41.

[0024] In another embodiment, the present invention provides an antigen-binding molecule, such as an antibody, that binds to the human adenosine 2A receptor (human A2aR). The antibody comprises a heavy chain variable region (HCVR) containing the amino acid sequence shown in SEQ ID NO: 36 or 39; and (b) a light chain variable region (LCVR) containing the amino acid sequence shown in SEQ ID NO: 42.

[0025] In another embodiment, the present invention provides an antigen-binding molecule that binds to the human adenosine 2A receptor (human A2aR), such as an antibody. The antibody comprises a heavy chain variable region (HCVR) containing the amino acid sequence shown in SEQ ID NO: 37 or 40; and a light chain variable region (LCVR) containing the amino acid sequence shown in SEQ ID NO: 43.

[0026] In one embodiment of various aspects of the present invention, the N-terminus of the heavy and / or light chain of an antigen-binding molecule, such as an antibody, is a pyroglutamic acid (pE) residue.

[0027] In another embodiment, (i) the antibody competes for binding to human A2aR with a monoclonal antibody selected from the group consisting of 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 cell surface human A2aR; (v) the antibody reduces cAMP concentration in tissues; (vi) the antibody reduces protein kinase A activity; (vii) the antibody reduces phosphorylation of the cAMP response element of the A2aR signaling pathway; or (viii) any combination of (i) to (vii).

[0028] In one embodiment, antibody binding to A2aR or cell surface A2aR is determined using a flow cytometry-based assay, or a substantially similar assay, as described in Examples 5, 6, and 7. In another embodiment, competition for antibody binding to A2aR or cell surface A2aR is determined using an assay known in the art, for example, the assay described in Harms, et al., Microtiter plate-based antibody-competition assay to determine binding affinities and plasma / blood stability of CXCR4 ligands, Scientific Reports, 2020:10:16036, doi.org / 10.1038 / s41598-020-73012-4. or substantially similar assays are used. In another embodiment, inhibition of A2aR activity is determined using the assay described in Example 4 or substantially similar assays. In yet another embodiment, reduction of cAMP concentration is determined using the assay described in Example 4 or substantially similar assays. In one embodiment, reduction in protein kinase A activity and / or reduction in phosphorylation of the cAMP response element of the A2aR signaling pathway is determined using the method described in Karage et al., A non-radioactive assay for the cAMP-dependent protein kinase activity in rat brain homogenates and age-related changes in hippocampus and cortex, Brain Res., 2001 Jun 8;903(1-2):86-93, doi: 10.1016 / s0006-8993(01)02409-x or substantially similar assays. In another embodiment, the enhancement of the immune response is determined using methods well known in the art, such as an increase in the concentration of inflammatory cytokines in the tissue or an increase in the number of cytotoxic CD8+ T cells.

[0029] In one embodiment, the antibody inhibits, for example, reducing the activity of A2aR by at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or about 100%. In another embodiment, the antibody enhances the immune response by at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 100%, about 1.5 times, about 2 times, about 4 times, or more. In yet another embodiment, the antibody reduces the concentration of cAMP in tissue by at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or about 100%. In yet another embodiment, the antibody reduces protein kinase A activity by at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or about 100%. In one embodiment, the antibody reduces phosphorylation of the cAMP response element of the A2aR signaling pathway by at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or about 100%.

[0030] In another embodiment, the antibody specifically binds to human A2aR and / or cynomolgus monkey A2aR. In yet another embodiment, the antibody specifically binds to human A2aR and / or cynomolgus monkey A2aR with similar affinity. In one embodiment, the antibody does not bind to non-primate A2aR, or binds to non-primate A2aR with an affinity significantly lower than that of human A2aR and / or cynomolgus monkey A2aR. In another embodiment, the antibody reduces intracellular cAMP production. In one embodiment, the antibody reduces the concentration of cAMP in tissue. In one embodiment, the antibody reduces the intracellular concentration of cAMP. In another embodiment, the antibody reduces the extracellular concentration of cAMP in tissue. In various aspects and embodiments of the present invention, cynomolgus monkey A2aR includes the sequence shown in SEQ ID NO: 51.

[0031] In one embodiment, the present invention provides an isolated antigen-binding molecule, such as an antibody, that competes with any embodiment of an antibody for binding to human A2aR.

[0032] In one embodiment, the antigen-binding molecule, for example, an antibody, is a humanized antibody or a chimeric antibody. In another embodiment, the antibody comprises a heavy chain constant region of a class selected from IgA, IgD, IgE, IgG, or IgM. In yet another embodiment, the antibody comprises a heavy chain constant region of class IgG, and IgG is selected from the group consisting of IgG4, IgG1, IgG2, and IgG3.

[0033] In another aspect, the present invention provides isolated polynucleotides encoding antigen-binding molecules of any aspect and various embodiments thereof, such as antibodies, their HCVR, their LCVR, their light chain, their heavy chain, or their antigen-binding fragments.

[0034] In another embodiment, the present invention includes comprising the We provide polynucleotide expression vectors.

[0035] In yet another aspect, the present invention provides recombinant cells comprising polynucleotides or expression vectors.

[0036] In one embodiment, the present invention provides a method for producing antigen-binding molecules, such as antibodies, in any embodiment and various embodiments thereof. The method includes the steps of expressing antibodies in recombinant cells and isolating the expressed antibodies.

[0037] In one embodiment, the present invention provides a pharmaceutical composition comprising an antigen-binding molecule, such as an antibody, in any embodiment and in various embodiments thereof, and a pharmaceutically acceptable carrier or diluent.

[0038] In one embodiment, the antibody in the pharmaceutical composition is effective in (a) specifically binding to human or cynomolgus monkey A2aR on the cell surface; (b) reducing the cAMP concentration in the tissue; (c) inhibiting the activity of human A2aR; (d) reducing the phosphorylation of the cAMP response element of the A2aR signaling pathway; (e) improving the immune response of immune cells; (f) reducing protein kinase A activity; and (g) any combination of (a) to (f). In another embodiment, the reduction of the cAMP concentration in the tissue is This is achieved by reducing intracellular cAMP production and / or intracellular and / or extracellular concentrations of cAMP compared to baseline levels. In another embodiment, inhibition of A2aR activity is achieved by inhibiting the physiological activity of adenosine.

[0039] In one embodiment, the present invention provides a method for inhibiting the activity of A2aR expressed on a cell surface, comprising the step of contacting cells with an isolated antibody of any embodiment or a pharmaceutical composition of any embodiment, thereby inhibiting A2aR activity in the cells. In one embodiment, inhibition of A2aR results in a reduction of cAMP concentration in tissue by at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or about 100%. In another embodiment, the method is used when treating cancer or neurodegenerative diseases. In yet another embodiment, an antigen-binding molecule, such as an antibody, inhibits A2aR activity by at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or about 100%. In another embodiment, the method is used when treating cancer or neurodegenerative diseases.

[0040] In another embodiment, the present invention provides a method for enhancing an immune response in a subject. The method comprises the step of administering an isolated antibody or a pharmaceutical composition of any embodiment to a subject, thereby enhancing an immune response in the subject. In one embodiment, the immune response includes a) promoting effector T cell function; b) Treg To reduce activity; c)T reg The present invention includes, but is not limited to, preventing proliferation; d) enhancing NK cell function; e) promoting type 1 activation of antigen-presenting cells; or f) reducing immunosuppression in the tumor microenvironment. In certain embodiments, the present invention's methods increase the immune response by at least about 10%, about 20%, about 50%, about 60%, about 70%, about 80%, about 90%, about 1x, about 2x, about 4x, or more, compared to baseline levels.

[0041] In another embodiment, the present invention provides a method for inhibiting tumor growth in a subject. The method comprises the step of administering an isolated antibody or a pharmaceutical composition of any embodiment to a subject, thereby inhibiting tumor growth.

[0042] In yet another embodiment, the present invention provides a method for treating cancer in a subject, comprising the step of administering an isolated antibody or a pharmaceutical composition of any embodiment, thereby treating the cancer. In one embodiment, the cancer is any cancer described herein. In a particular embodiment, the cancer is selected from the group consisting of triple-negative breast cancer (TNBC), pancreatic ductal adenocarcinoma (PDAC), metastatic castration-resistant prostate cancer (mCRPC), renal cell carcinoma (RCC), multiple myeloma, colorectal cancer (CRC), and diffuse large B-cell lymphoma (DLBCL).

[0043] In yet another aspect, the present invention provides a method for treating a neurodegenerative disease in a subject, comprising the step of administering an isolated antibody or a pharmaceutical composition of any aspect, thereby treating the neurodegenerative disease.

[0044] In one embodiment, any of the methods described above results in activating T cells and directing them to kill tumor target cells.

[0045] In another embodiment, any of the above embodiments of the method further includes the step of administering a further therapeutic agent. In one embodiment, the further therapeutic agent includes any therapeutic agent described herein. In another embodiment, the further therapeutic agent includes an antitumor agent, radiotherapy, chemotherapeutic agent, surgery, cancer vaccine, agonist, cytokine, cell therapy, or checkpoint inhibitor against stimulating receptors of immune cells. In one embodiment, the further therapeutic agent is an antibody that includes a multispecific antibody, for example, a bispecific antibody.

[0046] In another embodiment, the checkpoint inhibitors are drugs that inhibit PD-1, PD-L1, TIGIT, CTLA-4, PD-1, PD-L1, PD-L2, LAG-3, TIM-3, Neuritin, BTLA, CECAM-1, CECAM-5, IL-1R8, VISTA, LAIR1, LILRB1, LILRB2, LILRB3, LILRB4, LILRB5, CD96, CD112R, CD160, 2B4, TGFβ-R, KIR, NKG2A, and any combination thereof. In yet another embodiment, the checkpoint inhibitor is a drug selected from the group consisting of pembrolizumab, nivolumab, atezolizumab, avelumab, durvalumab, BMS-936559, sintilimab, tripalimab, tislerizumab, camrelizumab, sugemalimab, penpulimab, cadnilimab, sulfamonomethoxine 1, and sulfamethisole 2. In one embodiment, the CTLA inhibitor is ipilimumab, cadnilimab, or YH001 (Encure Biopharma).

[0047] In another embodiment, further therapeutic agents include agonists for immune cell stimulatory receptors selected from OX40, CD2, CD27, CDS, ICAM-1, LFA-1, ICOS (CD278), 4-1 BB (CD137), GITR, CD28, CD30, CD40, BAFFR, HVEM, CD7, LIGHT, NKG2C, NKG2D, SLAMF7, NKp46, NKp80, CD160, B7-H3, CD83 ligands, and any combination thereof.

[0048] In one embodiment, the further therapeutic agent is formulated in the same pharmaceutical composition as the antibody. In another embodiment, the further therapeutic agent is formulated in a pharmaceutical composition different from that of the antibody.

[0049] In another embodiment, further therapeutic agents are administered before antigen-binding molecules of various forms, such as antibodies. In yet another embodiment, further therapeutic agents are administered after the antibody administration step, such as after the antigen-binding molecule, such as antibodies. In another embodiment, further therapeutic agents are administered in combination with antigen-binding molecules, such as antibodies.

[0050] In one embodiment, the present invention provides a kit. The kit comprises a pharmaceutical composition comprising a pharmaceutical composition of any embodiment. In one embodiment, the pharmaceutical composition further comprises one or more of the further therapeutic agents described herein. [Brief explanation of the drawing]

[0051] [Figure 1] Figure 1 is a graph showing antibody titers against human A2aR in mouse serum induced by human A2aR-coding DNA immunization (each curve represents one mouse serum sample). Serum from eight mice (M1–M8) was tested for antibody titers determined via binding to human A2aR-overexpressing Expi293 cells by flow cytometry assay. MFI: Mean fluorescence intensity.

[0052] [Figure 2] Figure 2 includes fluorescence-activated cell sorting (FACS) plots showing the binding of anti-A2aR antibodies isolated from hybridoma cell culture media of four hybridoma clones: 1B5-3D7, 3F6-9G5, 3F8-12E9, and 8D5-16E2. Where used herein, the clone names, i.e., 1B5-3D7, 3F6-9G5, 3F8-12E9, and 8D5-16E2, also refer, depending on the context, to the monoclonal antibodies isolated from these clones. Expi293: Expi293 cells; A2aR / Expi293: Expi293 cells transfected with a vector expressing human A2aR.

[0053] [Figure 3] Figure 3 is a graph showing the in vitro blocking activity of anti-human A2aR mAbs 1B5-3D7, 3F6-9G5, 3F8-12E9, and 8D5-16E2 in a cell-based cAMP assay using exemplary antibodies of the present invention for whole IgG molecular types purified from hybridoma medium. RLU: relative light units; ZM241385: small molecule A2aR antagonist (Sigma, Cat.Z0153).

[0054] [Figure 4] Figure 4 is a graph showing the in vitro blocking activity of the exemplary antibodies of the present invention in a cell-based cAMP assay using whole recombinant IgG molecular types purified from the culture supernatant of Expi293 cells transiently transfected with a vector encoding the sequence of the exemplary antibody of the present invention.

[0055] [Figure 5]Figure 5 is a graph showing that exemplary antibodies 1B5-3D7 and 3F6-9G5 of the present invention specifically bind to human A2aR expressed on the cell surface. The graph also shows that the binding of anti-human A2aR antibodies from other sources to human A2aR expressed on the cell surface is undetectable or weak. 1B5:1B5-3D7;3F6:3F6-9G5;MAB9497:Human adenosine A2aR antibody, R&D Systems, Cat.MAB9497;SDIX-10:Human adenosine A2aR antibody disclosed in U.S. Patent Application Publication 2014 / 0322236A1, clone 864H10;SDIX-14:Human adenosine A2aR antibody disclosed in U.S. Patent Application Publication 2014 / 0322236A1, clone 864H14;mIgG2a iso:Mouse IgG2a isotype control;hIgG1 iso:Human IgG1 isotype control.

[0056] [Figure 6-1] Figure 6 includes a flow cytometry assay dot plot showing that the exemplary antibody 3F6-9G5 of the present invention binds to human A2aR and cynomolgus monkey A2aR expressed on the cell surface of human and cynomolgus monkey peripheral blood mononuclear cells (PBMCs) CD8+CD3+CD8 T cells and CD8-CD3+CD4 T cells. GMI: Geometric mean fluorescence intensity. [Figure 6-2] Figure 6 includes a flow cytometry assay dot plot showing that the exemplary antibody 3F6-9G5 of the present invention binds to human A2aR and cynomolgus monkey A2aR expressed on the cell surface of human and cynomolgus monkey peripheral blood mononuclear cells (PBMCs) CD8+CD3+CD8 T cells and CD8-CD3+CD4 T cells. GMI: Geometric mean fluorescence intensity. [Modes for carrying out the invention]

[0057] Detailed explanation The present invention and the accompanying drawings are considered herein to enable those skilled in the art to carry it out. However, those skilled in the art will understand that the invention described below may be carried out without using these specific details, or may be used for purposes other than those described herein. In fact, the present invention may be modified in consideration of this disclosure and used in conjunction with products and techniques known to those skilled in the art. The drawings and description are intended to illustrate various aspects of the invention and are not intended to limit the scope of the accompanying claims. Furthermore, it will be understood that the drawings may show aspects of the invention in isolation, and that elements in one drawing may be used in conjunction with elements shown in other drawings.

[0058] The references throughout this specification to aspects, features, advantages, or similar terms should not be understood as meaning that all aspects and advantages that can be realized using the present invention must be present in or within any single embodiment of the present invention. Rather, the terms referring to aspects and advantages should be understood as meaning that the specific aspects, features, advantages, or characteristics described in conjunction with the embodiments are included in at least one embodiment of the present invention. Accordingly, the discussions of aspects and advantages throughout this specification, as well as similar terms, may, but do not necessarily, refer to the same embodiment.

[0059] The described aspects, features, advantages, and characteristics of the present invention may be combined in any suitable manner in one or more further embodiments. Furthermore, those skilled in the art will recognize that the present invention may be carried out without one or more specific aspects or advantages of a particular embodiment. In other examples, further aspects, features, and advantages may be claimed in a particular embodiment that is recognized and may not be present in all embodiments of the present invention.

[0060] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which this application belongs. Those skilled in the art will recognize many techniques and materials similar or equivalent to those described herein that may be used in carrying out aspects and embodiments of the present invention. The aspects and embodiments described herein are not limited to the methods and materials described herein.

[0061] Furthermore, with respect to teachings in this invention, any cited references, any published patents or patent application publications described herein are expressly incorporated herein by reference.

[0062] I. Definition To make the present invention easier to understand, certain terms are defined first. Furthermore, when parameter values ​​or ranges of values ​​are enumerated, it should be noted that intermediate values ​​and ranges of the enumerated values ​​are also intended to be part of the present invention.

[0063] In situations describing the present invention (particularly in relation to the following claims), the use of the terms “a,” “an,” and “the,” and similar referents should be interpreted as covering both singular and plural forms (i.e., one or more), unless otherwise indicated herein or unless clearly inconsistent with the context. The terms “comprising,” “having,” “including,” and “containing” should be interpreted as open-ended terms (i.e., “including but not limited to”) unless otherwise noted herein. Enumerations of value ranges herein are merely intended to function as abbreviations to refer individually to each separate value enumerated or falling within its range, unless otherwise indicated herein, and each separate value is incorporated herein as if it were individually enumerated.

[0064] Where the phrases “in one embodiment,” “in another embodiment,” “in a different embodiment,” “in another embodiment,” “in some embodiment,” or “in a particular embodiment” are used, this disclosure should be construed as encompassing any combination of features defining the different embodiments described herein, unless the features are incomparable to each other, mutually exclusive, or expressly waived herein.

[0065] The terms “about” or “approximately” usually mean within 10%, preferably within 5%, or more preferably within 1%, of a given value or range.

[0066] A range may be expressed herein as "about" one particular value and / or "about" another particular value. Where such a range is expressed, another embodiment includes one particular value and / or another particular value. Similarly, where a value is expressed as an approximation by the preceding use of "about", it is understood that a particular value forms another embodiment. It is further understood that each endpoint of a range is important both in relation to and independently of the other endpoints. It is also understood that several values ​​are disclosed herein, and each value is disclosed herein not only as the value itself but also "about" its particular value. For example, where the value "10" is disclosed, "about 10" is also disclosed. Where a value is disclosed, it is also understood, as will be well understood by those skilled in the art, that "less than or equal to" (i.e., that value), "greater than or equal to" (i.e., that value), and possible ranges between values ​​are also disclosed. For example, where the value "10" is disclosed, "less than or equal to" and "greater than or equal to" are also disclosed.

[0067] As used herein, the term “agent” is used in reference to any substance, compound (e.g., molecule), supramolecular complex, material, or combination or mixture thereof. A compound can be any agent that can be represented by a chemical formula, chemical structure, or sequence. Examples of agents include, for example, small molecules, polypeptides, nucleic acids (e.g., RNAi agents, antisense oligonucleotides, aptamers), lipids, polysaccharides, and the like. Generally, agents can be obtained using any suitable method known in the art. Those skilled in the art will select a suitable method, for example, based on the properties of the agent. Agents can be purified at least partially. In some embodiments, the agent may be provided as part of a composition that, in various embodiments, may also contain, for example, a counterion, an aqueous or non-aqueous diluent or carrier, a buffer, a preservative, or other component. In some embodiments, the agent may be provided as a salt, ester, hydrate, or solvate. In some embodiments, the agent is cell-permeable, for example, within the range of typical agents taken up by cells, and acts intracellularly, for example, within mammalian cells, to produce a biological effect. Certain compounds may exist in specific geometric or stereoisomeric forms. Such compounds, including cis- and trans-isomers, E- and Z-isomers, R- and S-enantiomers, diastereomers, (D)-isomers, (L)-isomers, (-)- and (+)-isomers, racemic mixtures thereof, and other mixtures thereof, are encompassed by various embodiments of this disclosure unless otherwise indicated. Certain compounds may exist in various protonation states, have various configurations, exist as solvates (e.g., with water (i.e., hydrates) or common solvents), and / or have different crystalline forms (e.g., polymorphs) or different tautomeric forms. Embodiments exhibiting such alternative protonation states, configurations, solvates, and forms are encompassed by this disclosure where applicable.

[0068] In certain embodiments, depending on the context, “medicine” may also include methods of treatment, such as radiotherapy, chemotherapy, or surgery.

[0069] The term "amino acid" refers to 20 common naturally occurring amino acids. These naturally occurring 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).

[0070] The term "antagonist" or "inhibitor" refers to a substance that prevents, blocks, inhibits, neutralizes, or reduces the biological activity or effect of another molecule, such as a receptor.

[0071] The term "agonist" refers to a substance that promotes (i.e., induces, causes, enhances, or increases) the biological activity or effect of another molecule. The term agonist encompasses substances that bind to receptors, such as antibodies, and substances that promote receptor function without binding to receptors (for example, by activating related proteins).

[0072] The term “antibody,” as used herein, means any antigen-binding molecule or molecular complex containing at least one complementarity-determining region (CDR) that specifically binds to or interacts with a particular antigen (e.g., A2aR). The term “antibody” includes immunoglobulin molecules comprising four polypeptide chains interconnected by disulfide bonds: two heavy (H) chains and two light (L) chains, as well as their multimers (e.g., IgM). Each heavy chain comprises a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region comprises three domains: C H 1. C H 2 and C HIncludes 3. Each light chain includes a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region consists of one domain (C L Includes 1). V H and V L The region can be further subdivided into a hyper-variable region called the Complementarity Determination Region (CDR), which is interspersed with more conserved regions called the Framework Region (FR). H and V L It consists of three CDRs and four FRs aligned from the amino terminus to the carboxyl terminus in the following order: 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 those of mouse or human germline sequences, or may be naturally or artificially modified. The amino acid consensus sequence may be defined based on parallel analysis of two or more CDRs.

[0073] The term “antibody,” as used herein, also includes the antigen-binding fragment of a complete antibody molecule. Terms such as “antigen-binding portion” and “antigen-binding fragment” of an antibody, as used herein, include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. Antigen-binding fragments of antibodies may be derived, for example, from a complete antibody molecule using any suitable standard technique, e.g., proteolytic digestion, or recombinant genetic engineering techniques, including the manipulation and expression of DNA encoding the antibody variable domain and, optionally, the constant domain. Such DNA is known and / or readily available, for example, from commercial sources, DNA libraries (e.g., including phage-antibody libraries), or can be synthesized. DNA may be sequenced, manipulated, for example, by chemical or molecular biological techniques to align one or more variable and / or constant domains into appropriate positions, or to introduce codons, create cysteine ​​residues, or modify, add, or delete amino acids.

[0074] Non-limiting examples of antigen-binding fragments include (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv(scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units consisting of amino acid residues mimicking the hypervariable region of an antibody (e.g., an isolated complementarity-determining region (CDR), e.g., a CDR3 peptide), or a constrained FR3-CDR3-FR4 peptide. Other manipulated molecules, such as domain-specific antibodies, single-domain antibodies, domain deletion antibodies, chimeric antibodies, CDR graft antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains, are also included within the expression “antigen-binding fragment” as used herein.

[0075] The antigen-binding fragment of an antibody typically contains at least one variable domain. The variable domain can be of any size or amino acid composition and generally contains at least one CDR adjacent to or in-frame with one or more framework sequences. L V associated with the domain H In antigen-binding fragments having a domain, V H Domain and V L Domains can be placed relative to each other in any appropriate alignment. For example, a variable region is a dimer, and V H -V H , V H -V L or V L -V L It may contain a dimer. Alternatively, the antigen-binding fragment of the antibody may contain monomer V H Domain or V L It may contain a domain.

[0076] In certain embodiments, the antigen-binding fragment of the antibody may contain at least one variable domain covalently linked to at least one constant domain. Non-limiting exemplary arrangements of variable and constant domains that may be found in the antigen-binding fragment of the antibody of the present 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 LThis includes: In any configuration of variable and constant domains, including any of the exemplary configurations listed above, the variable and constant domains may be directly linked to each other or linked by a complete or partial hinge or linker region. A hinge region may consist of at least two (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids that result in a flexible or semi-flexible linkage between adjacent variable and / or constant domains in a single polypeptide molecule. Furthermore, antigen-binding fragments may be linked to each other and / or one or more monomeric V H Domain or V L It may include a homodimer or heterodimer (or other multimer) of any of the variable domain and constant domain configurations listed above, associated with the domain by non-covalent bonds (e.g., by disulfide bonds).

[0077] Similar to complete antibody molecules, antigen-binding fragments can be monospecific or polyspecific (e.g., bispecific). A polyspecific antigen-binding fragment of an antibody typically contains at least two distinct variable domains, each capable of specifically binding to a separate antigen or to a different epitope on the same antigen. Any polyspecific antibody type, including the exemplary bispecific antibody types disclosed herein, can be adapted for use relating to the antigen-binding fragments of the present invention using conventional techniques available in the art.

[0078] The antibodies of the present invention may be isolated antibodies. “Isolated” molecule, e.g., isolated antibody or isolated polypeptide, as used herein, means a molecule that has been identified and separated and / or recovered from at least one component of its natural environment, e.g., an antibody. For example, a molecule separated or extracted from at least one component of an organism, or from a tissue or cell in which antibodies naturally exist or are naturally produced, e.g., an antibody, is an “isolated” molecule, e.g., an antibody, for the purposes of the present invention. Isolated molecules, e.g., antibodies, also include in situ molecules within recombinant cells, e.g., antibodies. In certain embodiments, an isolated molecule, e.g., an antibody, is a molecule subjected to at least one purification or isolation step, e.g., an antibody. According to certain embodiments, an isolated molecule, e.g., an antibody, may substantially contain other cellular material and / or chemicals.

[0079] The present invention also includes a one-arm antibody that binds to A2aR. As used herein, “one-arm antibody” means an antigen-binding molecule comprising a single antibody heavy chain and a single antibody light chain. The one-arm antibodies of the present invention may comprise any of the HCVR / LCVR or CDR amino acid sequences shown in Tables 1-9.

[0080] The anti-A2aR antibodies or their antigen-binding domains herein may contain one or more amino acid substitutions, insertions, and / or deletions in the framework and / or CDR region of the heavy and light chain variable domains compared to the corresponding germline sequence from which the antigen-binding molecule or antigen-binding domain originates. Such mutations can be readily elucidated by comparing the amino acid sequences disclosed herein with germline sequences available, for example, from public antibody sequence databases. The present invention comprises an antibody and its antigen-binding domain derived from any of the amino acid sequences disclosed herein, wherein one or more amino acids in the framework and / or CDR region are mutated to the corresponding residue(s) of the germline sequence from which the antibody originates, or to the corresponding residue(s) of another human germline sequence, or to a conserved amino acid substitution of the corresponding germline residue(s) (such sequence changes are collectively referred to herein as “germline mutations”). Starting with the heavy and light chain variable region sequences disclosed herein, those skilled in the art can readily produce a number of antibodies and antigen-binding fragments containing one or more individual germline mutations or combinations thereof. In certain embodiments, V H Domain and / or V LAll framework and / or CDR residues within the domain are reverted to residues found in the original germline sequence from which the antibody originated. In other embodiments, only certain residues, for example, only mutated residues found within the first 8 amino acids of FR1 or the last 8 amino acids of FR4, or only mutated residues found within CDR1, CDR2, or CDR3, are reverted to the original germline sequence. In other embodiments, one or more framework and / or CDR residues are mutated to corresponding residues in a different germline sequence (i.e., a germline sequence different from the germline sequence from which the antibody originally originated). Furthermore, the antibody or its antigen-binding domain may contain any combination of two or more germline mutations within the framework and / or CDR region, for example, certain individual residues may be mutated to corresponding residues in a particular germline sequence, while certain other residues different from the original germline sequence are maintained or mutated to corresponding residues in a different germline sequence. Once obtained, antibodies or antigen-binding fragments containing one or more germline mutations can be readily tested for one or more desired properties, such as improved binding specificity, increased binding affinity, improved or enhanced antagonistic or agonist biological properties (if applicable), or reduced immunogenicity. Antibodies or antigen-binding fragments obtained by this general method are incorporated within the scope of the present invention.

[0081] The present invention also includes anti-A2aR antibodies comprising variants of any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein. Exemplary variants included in this aspect of the present invention include variants of any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein having one or more conservative substitutions. For example, the present invention includes anti-A2aR antibodies and antigen-binding proteins having HCVR, LCVR, and / or CDR amino acid sequences having, for example, 10 or fewer, 8 or fewer, 6 or fewer, 4 or fewer, or more conservative amino acid substitutions compared to any of the HCVR, LCVR, and / or CDR amino acid sequences shown in the table herein.

[0082] The light chain is classified as either kappa or lambda (lambdamko) (K, λ). Each heavy chain class can be bound to either a kappa light chain or a lambda light chain. Generally, the light and heavy chains are covalently bonded to each other, and the "tail" portions of the two heavy chains are linked to each other by covalent disulfide bonds or non-covalent bonds when the immunoglobulin is produced by a hybridoma, B cell, or genetically engineered host cell. In the heavy chains, the amino acid sequence extends from the N-terminus of a Y-configuration fork to the C-terminus at the bottom of each chain.

[0083] As used herein, the terms “light chain constant region” or “CL” are interchangeable herein with respect to the amino acid sequence derived from the antibody light chain. Preferably, the light chain constant region comprises at least one of a constant kappa domain or a constant lambda domain.

[0084] As used herein, the term “heavy chain constant region” includes an amino acid sequence derived from an immunoglobulin heavy chain. A polypeptide comprising a heavy chain constant region comprises at least one of the following: a CH1 domain, a hinge (e.g., upper, central, and / or lower hinge region) domain, a CH2 domain, a CH3 domain, or a variant or fragment thereof. For example, antigen-binding polypeptides for use in this 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 polypeptides of this disclosure include a polypeptide chain comprising a CH3 domain. Furthermore, antibodies for use in this 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 can be modified so that its amino acid sequence differs from that of naturally occurring immunoglobulin molecules.

[0085] The heavy chain constant regions of antibodies disclosed herein may originate from different immunoglobulin molecules. For example, the heavy chain constant region of a polypeptide may include a CH1 domain derived from the IgG1 molecule and a hinge region derived from the IgG3 molecule. In another example, the heavy chain constant region may include a hinge region partially derived from the IgG1 molecule and partially derived from the IgG3 molecule. In yet another example, the heavy chain portion may include a chimeric hinge partially derived from the IgG1 molecule and partially derived from the IgG4 molecule.

[0086] A "light-heavy chain pair" refers to an aggregate of light and heavy chains that can form a dimer via a disulfide bond between the CL domain of the light chain and the CH1 domain of the heavy chain.

[0087] The subunit structures and three-dimensional arrangements of the constant regions of various immunoglobulin classes are well known. As used herein, the term "VH domain" refers to the N-terminal variable domain of the immunoglobulin heavy chain, and the term "CH1 domain" refers to the first (most N-terminal) constant region domain of the immunoglobulin heavy chain. The CH1 domain is adjacent to the VH domain and is N-terminal relative to the hinge region of the immunoglobulin heavy chain molecule.

[0088] As used herein, the term “CH2 domain” includes, for example, the portion of the heavy chain molecule extending from approximately residues 244 to 360 of the antibody using conventional numbering schemes (residues 244–360, Kabat numbering system; and residues 231–340, EU numbering system). The CH2 domain is unique in that it does not closely pair with another domain. Rather, two N-linked branched carbohydrate chains are sandwiched between the two CH2 domains of the intact native IgG molecule. The CH3 domain extends from the CH2 domain to the C-terminus of the IgG molecule and contains approximately 108 residues.

[0089] As used herein, the term “hinge region” includes the portion of the heavy chain molecule that connects the CH1 domain to the CH2 domain. This hinge region comprises approximately 25 residues and is flexible, thereby allowing the two N-terminal antigen-binding regions to move independently. The hinge region can be subdivided into three distinct domains: the upper, middle, and lower hinge domains.

[0090] As used herein, the term “disulfide bond” includes a covalent bond formed between two sulfur atoms. The amino acid cysteine ​​contains a thiol group that can form a disulfide bond or bridge with a second thiol group. In most naturally occurring IgG molecules, the CH1 and CL regions are linked by a disulfide bond, and the two heavy chains are linked by two disulfide bonds at positions 239 and 242 (226 or 229, EU numbering system) using the Kabat numbering system.

[0091] The term "epitope" refers to an antigenic determinant that interacts with a specific antigen-binding site in the variable region of an antibody molecule, known as a paratope. A single antigen may have more than one epitope. Therefore, different antibodies may bind to different areas on an antigen and have different biological effects. Epitopes can be either conformational or linear. Conformational epitopes are produced by spatially juxtaposed amino acids from different segments of a linear polypeptide chain. Linear epitopes are epitopes produced by adjacent amino acid residues in a polypeptide chain. In certain circumstances, epitopes may include saccharide, phosphoryl, or sulfonyl group portions on an antigen.

[0092] The term “substantial identity” or “substantially identical,” when referring to a nucleic acid or fragment thereof, means that nucleotide sequence identity exists in at least about 95%, more preferably at least about 96%, 97%, 98%, or 99% of the nucleotide bases, when optimally aligned with another nucleic acid (or its complementary strand) with appropriate nucleotide insertions or deletions, as measured by any well-known algorithm for sequence identity, e.g., FASTA, BLAST, or Gap, as discussed below. A nucleic acid molecule having substantial identity with respect to a reference nucleic acid molecule may, in certain cases, encode a polypeptide having the same or substantially similar amino acid sequence as the polypeptide encoded by the reference nucleic acid molecule.

[0093] When applied to polypeptides, the term “substantial similarity” or “substantially identical” means that two peptide sequences share at least 95% sequence identity, and more preferably at least 98% or 99%, when optimally aligned using default gap weights, for example by programmed GAP or BESTFIT. Preferably, non-identical residue positions differ by conserved amino acid substitutions. A “conservative amino acid substitution” is a substitution in which one amino acid residue is replaced by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). Generally, conservative amino acid substitutions do not substantially alter the functional properties of the protein. If two or more amino acid sequences differ from each other by conservative substitutions, the percentage sequence identity, or degree of similarity, can be adjusted upward to compensate for the conservative nature of the substitutions. Means for making this adjustment are well known to those skilled in the art. See, for example, Pearson (1994) Methods Mol. BioI. 24: 307-331. Similarity Examples of amino acids having side chains with the following 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; and (7) sulfur-containing side chains: cysteine ​​and methionine. Preferred conserved 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 can be applied to the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256: 1443-1445. A "moderately conservative" permutation is any change that has a positive value in the PAM250 log-likelihood matrix.

[0094] Sequence similarity for polypeptides, also known as sequence identity, is typically measured using sequence analysis software. Protein analysis software matches similar sequences using similarity measures assigned to various substitutions, deletions, and other modifications, including conserved amino acid substitutions. For example, GCG software includes programs, e.g., Gap and Bestfit, which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, e.g., homologous polypeptides from different species, or between a wild-type protein and its mutaine. See, for example, GCG version 6.1. Polypeptide sequences can also be compared using the FASTA program in GCG version 6.1 with default or recommended parameters. FASTA (e.g., FASTA2 and FASTA3) provides alignment of the best overlap region and percent sequence identity between the query sequence and the search sequence (Pearson (2000) above). Another preferred algorithm for comparison with databases containing numerous sequences of origin is the computer program BLAST, specifically BLASTP or TBLASTN, which uses default parameters. For example, Altschul et al. (1990) J. Mol. See Biol. 215:403-410 and Altschul et al. (1997) Nucleic Acids Res. 25:3389-402.

[0095] The term “antibody” encompasses a broad range of polypeptides that can be biochemically distinguished. Those skilled in the art will understand that heavy chains, along with some subclasses within them (e.g., γ1-γ4), are classified as alpha, delta, epsilon, gamma, and mu, or α, δ, ε, γ, and μ. The properties of this chain determine the “class” of the antibody, such as IgG, IgM, IgA, IgG, or IgE, respectively. Immunoglobulin subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgG5, etc., are well-characterized and known to confer functional specialization. Modified versions of each of these classes and isotypes are readily identifiable to those skilled in the art in light of this disclosure and are therefore within the scope of this disclosure. All immunoglobulin classes are within the scope of this disclosure, and the following discussion generally focuses on immunoglobulin molecules of the IgG class.

[0096] The antibodies disclosed herein include, but are not limited to, polyclonal, monoclonal, multispecific, bispecific, trispecific, human, humanized, primated, chimeric, and single-chain antibodies. The antibodies disclosed herein may be of any animal origin, including birds and mammals. Preferably, the antibodies are human, mouse, donkey, rabbit, goat, guinea pig, camel, llama, horse, or chicken antibodies. In some embodiments, the variable region may be condricthoid in origin (e.g., shark-derived).

[0097] The term "humanized antibody," as used herein, refers to a genetically engineered non-human antibody containing a human antibody constant domain and a non-human variable domain modified to contain a high level of sequence homology to the human variable domain. This can be achieved by grafting six non-human antibody complementarity-determining regions (CDRs), which together form an antigen-binding site, onto a homologous human acceptor framework region (FR). To reconstitute the binding affinity and specificity of the parental antibody, substitution (reverse mutation) of framework residues from the parental antibody (i.e., the non-human antibody) into the human framework region may be required. Structural homology modeling can help identify amino acid residues in the framework region that are important for the antibody's binding properties. Thus, a humanized antibody may contain a non-human CDR sequence, a predominantly human framework region optionally containing one or more amino acid reverse mutations into the non-human amino acid sequence, and a fully human constant region. Further amino acid modifications, not necessarily reverse mutations, may be applied as needed to obtain a humanized antibody with desirable characteristics, such as affinity and biochemical properties.

[0098] As used herein, the term “chimeric antibody” means an antibody in which an immunoreactive region or site is obtained from or derived from a first species, and a constant region (which may be intact, partial, or modified in accordance with this disclosure) is obtained from a second species. In certain embodiments, the target-binding region or site is derived from a non-human source (e.g., mouse or primate), and the constant region is human.

[0099] "Single-chain fragment variable" or "scFv" refers to a fusion protein of the variable region (VH) of the heavy chain and the variable region (VL) of the light chain of an immunoglobulin. In some embodiments, the regions are linked using a short linker peptide of 10 to about 25 amino acids. The linker may be rich in glycine for flexibility and serine or threonine for solubility, and the N-terminus of the VH may be linked to the C-terminus of the VL, and vice versa. This protein retains the specificity of the original immunoglobulin despite the removal of the constant region and the introduction of the linker.

[0100] Regarding IgG, a standard immunoglobulin molecule contains two identical light-chain polypeptides with a molecular weight of approximately 23,000 daltons, and two identical heavy-chain polypeptides with a molecular weight of 53,000–70,000. The four chains are typically linked by disulfide bonds in a "Y" configuration, where the light chains flank the heavy chains, which begin at the mouth of the "Y" and continue through a variable region.

[0101] The term “variant,” as used herein, refers to a polypeptide, e.g., an antibody, or a polynucleotide induced by the insertion, substitution, or deletion of one or more amino acids or nucleotides into a precursor polypeptide or polynucleotide (e.g., a “parent” polypeptide or polynucleotide). In certain embodiments, the variant polypeptide or polynucleotide has at least about 85% amino acid or nucleotide sequence identity with respect to the entire amino acid or nucleotide sequence of the parent polypeptide or polynucleotide, e.g., about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% amino acid or nucleotide sequence identity. A variant of a protein or peptide substantially maintains the structure, function, or activity of the protein. For example, a variant of an antibody maintains the function or activity of specifically binding to and / or modulating, e.g., inhibiting the activity of its antigen. In the case of a polynucleotide, its variant maintains its function or activity of the parent polynucleotide. For example, a variant polynucleotide may encode a protein or peptide that has similar function or activity to the polypeptide encoded by the parent polynucleotide. The term “sequence identity,” as used herein, refers to the comparison between pairs of nucleic acid or polypeptide molecules, i.e., the relationship between two amino acid sequences or two nucleotide sequences. Generally, sequences are aligned to obtain the highest-order match. Methods for determining sequence identity are known and can be determined by commercially available computer programs capable of calculating the percentage of identity between two or more sequences. Typical examples of such computer programs are BLAST or CLUSTAL.

[0102] As used herein, the term “specific binding” or “specifically binding” refers to the ability to distinguish between possible binding partners in the environment in which binding occurs. In some embodiments, an antibody that interacts with one particular antigen in the presence of other potential antibodies, for example, an antibody that preferentially interacts with it, is said to “specifically bind” to the antigen it interacts with. In some embodiments, specific binding is assessed by detecting or determining the degree of association between the antibody and its targeted antigen; in some embodiments, specific binding is assessed by detecting or determining the degree of dissociation of the antibody-antigen complex; in some embodiments, specific binding is assessed by detecting or determining the ability of an antibody to compete with alternative interactions between its target and another antibody; in some embodiments, specific binding is assessed by performing such detection or determination over a range of concentrations. Generally, an antibody binds to an epitope via its antigen-binding domain, and this binding inevitably involves some complementarity between the antigen-binding domain and the epitope. Therefore, an antibody is said to "specifically bind" to an epitope if it binds to that epitope more easily via its antigen-binding domain than it would if it bound to a random, unrelated epitope. The term "specificity" is used herein to qualify the relative affinity with which a particular antibody binds to a particular epitope. For example, antibody "A" may be considered to have higher specificity to a given epitope than antibody "B", or antibody "A" may be said to bind to epitope "C" with higher specificity than it has to related epitope "D". In some embodiments, the antibody or its antigen-binding fragment is 10 -6 M or less, 10 -7 M or less, 10 -8 M or less, 10 -9 M or less, or 10 -10 M or a dissociation constant less than (K) dWhen an antibody or antibody fragment forms a complex with an antigen, it "has specificity for the antigen." In certain embodiments, the specific binding of an antigen-binding molecule, e.g., an anti-human A2aR antibody or its antigen-binding fragment, may be demonstrated by the preferential binding of the antigen-binding molecule to human A2aR expressed on the cell surface, using the assays described in Examples 4-7, or substantially similar methods.

[0103] As used herein, the term “A2aR” refers to the adenosine A2A receptor. Unless otherwise indicated by a specific reference to human A2aR, for example, the term “A2aR” includes native A2aRs from all mammalian species, such as humans, primates, rodents, dogs, cats, horses, and cattle. The nucleotide and amino acid sequences of A2aR are publicly known and can be found, for example, in GenBank accessions 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. The following is an exemplary human A2aR amino acid sequence: MPIMGSSVYITVELAIAVLAILGNVLVCWAVWLNSNLQNVTNYFVVSLAAADIAVGVLAIPFAITISTGFCAACHGCLFIACFVLVLTQSSIFSLLAIAIDRYIAIRIPLRYNGLVTGTRAKGIIAICWVLSFAIGLTPMLGWNNCGQPKEGKNHSQGCGEGQVACLFEDVVPMNYMVYFNFFACVLVPLLLMLGVYLRIFLAARRQLKQMESQPLPGERARSTLQKEVHAAKSLAIIVGLFALCWLPLHIINCFTFFCPDCSHAPLWLMYLAIVLSHTNSVVNPFIYAYRIREFRQTFRKIIRSHVLRQQEPFKAAGTSARVLAAHGSDGEQVSLRLNGHPPGVWANGSAPHPERRPNGYALGLVSGGSAQESQGNTGLPDVELLSHELKGVCPEPPGLDDPLAQDGAGVS (Sequence ID 50)

[0104] An example of the A2aR amino acid sequence for cynomolgus monkeys is shown below: VPIMGSSVYITVELAIAVLAILGNVLVCWAVWLNSNLQNVTNYFVVSLAAADIAVGVLAIPFAITISTGFCAACHGCLFIACFVLVLTQSSIFSLLAIAIDRYIAIRIPLRYNGLVTGTRAKGIIAICWVLSFAIGLTPMLGWNNCGQPKEGKNHSQGCGEGQVACLFEDVVPMNYMVYFNFFACVLVPLLLMLGVYLRIFLAARRQLKQMESQPLPGERARSTLQKEVHAAKSLAIIVGLFALCWLPLHIINCFTFFCPDCNHAPLWLMYLAIVLSHTNSVVNPFIYAYRIREFRQTFRKIIRSHVLRQQEPFKAAGTSARVLAAHGSDGEQVSLRLNGHPPGVWANGSAPHPERRPNGYALGLVSGGSTQESQGNTSLPDVELLSHELKGVCPEPPGLDDPLAQGGAGVS(Sequence ID 51)

[0105] The term “anti-A2aR antibody” or “A2aR antibody” refers to an antibody or polypeptide that specifically binds to A2aR. In certain embodiments, an anti-A2aR antibody can inhibit A2aR biological activity and / or downstream signaling pathways mediated by A2aR. Anti-A2aR antibodies encompass antibodies or polypeptides containing one or more antigen-binding domains in the form of a CDR or variable region. In certain embodiments, the anti-A2aR antibody of the present invention blocks, antagonistizes, suppresses or reduces (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 antitumor immune responses, and immunosuppression in immunocompromised disease states.

[0106] The term "small molecule drug" refers to molecular entities that are not polymers, possess pharmaceutically active properties, and have molecular weights less than approximately 2 kDa, less than approximately 1 kDa, less than approximately 900 Da, less than approximately 800 Da, or less than approximately 700 Da, often being organic or organometallic. While the term encompasses most pharmaceutical compounds called "drugs" other than proteins or nucleic acids, small peptides or nucleic acid analogs may also be considered small molecule drugs. Examples include chemotherapeutic anticancer drugs and enzyme inhibitors. Small molecule drugs can be synthesized, semi-synthesized (i.e., from naturally occurring precursors), or biologically derived.

[0107] As used herein, the term “recombinant” means a polypeptide or polynucleotide that can be produced by combining polynucleotides or polypeptides in an alignment that does not exist in nature and does not normally occur together.

[0108] When describing polypeptide domain alignment using hyphens between individual domains (e.g., CH2-CH3), it should be understood that the order of the listed domains is N-terminus to C-terminus.

[0109] The term "immunoconjugate" refers to an antibody fused to a peptide or small molecule drug by covalent bond. The peptide or small molecule drug may be fused to the C-terminus of the constant heavy chain or to the variable light chain and / or the N-terminus of the heavy chain.

[0110] With respect to the level of A2aR activity, the terms “inhibit,” “inhibit,” “reduce,” and “reduce” refer to a statistically significant decrease at such a level. The decrease may be, for example, at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or may fall below the detection level of the detection method. For example, inhibition or reduction of A2aR activity may be determined by a decrease in intracellular cAMP concentration using the method described in Example 4.

[0111] The terms “to treat” and “treatment” refer to the remission of one or more symptoms associated with a disease or disorder. Thus, these terms refer to any sign of success in treating or relieving an injury, disease, disorder or condition, including: prevention or delay of the onset of one or more symptoms of a disease or disorder; reduction of the severity or frequency of one or more symptoms of a disease or disorder; any objective or subjective parameter, e.g., reduction; improvement; attenuation of symptoms, or making the injury, condition or state more tolerable to the patient; slowing the rate of degeneration or decline; making the final stage of degeneration less debilitating; and / or improving the patient’s physical or mental health. “To treat” and “treatment” may also include preventive treatment.

[0112] The phrases “to patients who need it,” “to patients who need treatment,” or “subjects who need treatment” include subjects who would benefit from the administration of the antibodies of this disclosure for the treatment of cytoproliferative disorders, such as mammalian subjects.

[0113] The term "preventive" refers to a reduction in the occurrence of disease symptoms (e.g., related to A2aR activity or its function) in a patient. As shown above, prevention can be complete (no detectable symptoms) or partial, resulting in fewer symptoms being observed than might occur without treatment.

[0114] The terms “therapeutic effective dose,” “pharmacological effective dose,” and “physiological effective dose” are used interchangeably to mean the amount of active agent sufficient to alleviate at least one symptom of a disease or disorder. For example, for a given parameter, a therapeutic effective dose represents an increase or decrease of at least 5%, 10%, 15%, 20%, 25%, 40%, 50%, 60%, 75%, 80%, 90%, 95%, 99%, or at least 100%. Therapeutic effectiveness can also be expressed as an increase or decrease of “~ times.” For example, a therapeutic effective dose may have an effect of at least 1.2 times, 1.5 times, 2 times, 5 times, or greater than that of the control. The exact amount can be readily determined by a person skilled in the art based on information provided herein or otherwise available in the relevant literature, depending on a number of factors, including patient considerations such as the specific active agent of the composition, its components and physical characteristics, the intended patient population, weight, sex, etc.

[0115] When used in this context, the terms “improve,” “increase,” or “decrease” refer to values ​​or parameters compared to baseline / control / reference values, such as values ​​in cells or tissues before the commencement of the treatment described herein, or values ​​in cells or tissues in the absence of the treatment described herein, values ​​in the same individual before the commencement of the treatment described herein, or values ​​in a control individual in the absence of the treatment described herein (or standard values ​​derived from multiple control individuals, such as the mean value of multiple control individuals).

[0116] A “control individual” is an individual with a similar condition, for example, an individual with the same cytoproliferative disorder as the treated individual, or an individual of a similar age to the treated individual (to ensure that the disease stage is comparable in the treated individual and the control individual). The treated individual (also called the “patient” or “subject”) may be a fetus, infant, child, adolescent, or adult human with a cytoproliferative disorder.

[0117] The term “proliferative disorder” refers to a disorder characterized by abnormal cell proliferation. Proliferative disorders do not imply any limitation on the rate of cell growth, but merely indicate a loss of normal controls that affect growth and cell division. Therefore, in some embodiments, cells with proliferative disorders may have the same rate of cell division as normal cells, but they do not respond to signals that limit such growth. Neoplasms, cancers, or tumors fall within the scope of “proliferative disorders.”

[0118] The term “cancer” refers to any one of the various malignant neoplasms characterized by the proliferation of cells that have the ability to invade surrounding tissues and / or metastasize to new colony-forming sites, and includes cancers of both the solid and lymphoid systems, including carcinoma, sarcoma, adenocarcinoma, melanoma, leukemia, lymphoma, germ cell tumors and blastomas. Exemplary cancers that can be treated according to the compositions and methods of the present invention include cancers of the brain, bladder, breast, cervix, colon, head and neck, kidney, lung, non-small cell lung, mesothelioma, ovarian, prostate, stomach and uterine cancers, leukemia, and medulloblastoma.

[0119] The term "cancer" refers to the malignant growth of epithelial cells that tend to invade surrounding tissues and metastasize. Exemplary cancers include, for example, acinar carcinoma, adenoid cystic carcinoma, adenomatous carcinoma (carcinoma adenomatosum), adrenal cortical carcinoma, and alveolar carcinoma. Carcinoma, alveolar carcinoma, basal cell carcinoma, carcinoma basocellulare, basaloid carcinoma, basal squamous cell carcinoma, bronchioloalveolar epithelial carcinoma, bronchiolar carcinoma, bronchogenic carcinoma, cerebriform carcinoma, cholangiocarcinoma, chorionic carcinoma colloid carcinoma, comedocarcinoma, endometrial carcinoma, cribriform carcinoma, armor-like carcinoma, skin cancer, columnar carcinoma, columnar cell carcinoma, ductal carcinoma, carcinoma durum, embryonal carcinoma, encephaloid carcinoma, epidermoid carcinoma, carcinoma epitheliale adenoides, exogenous carcinoma, ulcerative carcinoma, fibrous carcinoma, gelatiniform carcinoma, gelatinous carcinoma, giant cell carcinoma, gigantocellular carcinoma, glandular carcinoma, granular membrane cell carcinoma, pilonidal carcinoma, hematoid carcinoma, hepatocellular carcinoma, heart cell carcinoma, hyporhizoma, hypodermal carcinoma, microembryonic carcinoma, intraepithelial carcinoma, intraepithelial carcinoma, Krompecher carcinoma, Kulchitzky cell carcinoma, large cell carcinoma, lenticular carcinoma, lenticular carcinoma lenticular carcinoma, lipomatous carcinoma, medullary carcinoma, medullary carcinoma, black carcinoma, soft carcinoma, mucinous carcinoma, muciparum carcinoma, mucocellular carcinoma, mucoepidermal carcinoma, mucosal carcinoma, mucous carcinoma, myxomatous carcinoma Myxomatodes, nasopharyngeal carcinoma, oat cell carcinoma, ossifying carcinoma (c Arcinoma ossificans), osteoid carcinoma, pancreatic ductal adenocarcinoma, papillary carcinoma, periportal carcinoma, pre-invasive carcinoma, squamous cell carcinoma, atherosclerotic carcinoma (pultaceous carcinoma), renal cell carcinoma of the kidney, reserve cell carcinoma, sarcomatodes, Schneiderian carcinoma, scirrhous carcinoma, scrotal carcinoma, signet ring cell carcinoma, simple carcinoma, small cell carcinoma, solanoid carcinoma, spheroidal cell carcinoma, spindle cell carcinoma, cavernous carcinoma, squamous cell carcinoma, squamous cell carcinoma, string carcinoma, carcinoma telangiectaticum, carcinoma telangiectodes, transitional cell carcinoma, carcinoma tuberosum, tuberous carcinoma This includes carcinoma, wart-like carcinoma, and choriocarcinoma (carcinoma villosum).

[0120] The term "sarcoma" refers to a tumor composed of materials such as embryonic connective tissue, and generally consists of densely packed cells embedded in fibrous or homogeneous material. Exemplary sarcomas include, for example, chondrosarcoma, fibrosarcoma, lymphosarcoma, melanosarcoma, myxosarcoma, osteosarcoma, Abemethy's sarcoma, adipose sarcoma, liposarcoma, alveolar soft tissue sarcoma, ameloblastic sarcoma, staphyloid sarcoma, green sarcoma, choriocarcinoma, embryonic sarcoma, Wilms tumor sarcoma, endometrial sarcoma, stromal sarcoma, Ewing's sarcoma, fascial sarcoma, fibroblastic sarcoma, giant cell sarcoma, granulocytic sarcoma, Hodgkin's sarcoma, and idiopathic multiple pigmented hemorrhagic sarcoma. This includes B-cell immunoblastic sarcomas, lymphomas (e.g., non-Hodgkin lymphoma), T-cell immunoblastic sarcomas, Jensen sarcomas, Kaposi's sarcomas, Kupffer cell sarcomas, angiosarcomas, leukemosarcomas, malignant mesenchymal sarcomas, paraosteal sarcomas, reticulocyte sarcomas, Rous sarcomas, serocystic sarcomas, synovial sarcomas, and telangiectatic sarcomas.

[0121] The term "melanoma" refers to tumors arising from the melanocyte system of the skin and other organs. Melanomas include, for example, acral lentiginous melanoma, achromatic melanoma, benign juvenile melanoma, Cloudmann melanoma, S91 melanoma, Harding-Passey melanoma, juvenile melanoma, lentigo malignant melanoma, malignant melanoma, nodular melanoma, subungual melanoma, and superficial spreading melanoma.

[0122] The term "lymphoma" refers to a group of cancers that affect hematopoietic and lymphatic tissues, beginning with lymphocytes, which are blood cells primarily found 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 approximately 15% of all diagnosed lymphomas. It is a cancer associated with Reed-Sternberg malignant B lymphocytes. Non-Hodgkin lymphoma (NHL) can be classified based on the rate at which the cancer grows and the type of cells involved. High-grade and low-grade NHLs exist. Based on the type of cells involved, B-cell and T-cell NHLs exist. 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, splenic lymphoma, diffuse large B-cell lymphoma, Burkitt lymphoma, lymphoblastic lymphoma, immunoblastic large B-cell lymphoma, or progenitor 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 progenitor T-lymphoblastic lymphoma.

[0123] The term "leukemia" refers to a progressive malignant disease of the hematopoietic organs, generally characterized by the distorted proliferation and development of white blood cells and their precursors in the blood and bone marrow. Exemplary leukemias include, for example, acute nonlymphocytic leukemia, chronic lymphocytic leukemia, acute granulocytic leukemia, chronic granulocytic leukemia, acute promyelocytic leukemia, adult T-cell leukemia, non-leukemia, leukocythemic leukemia, basophilic leukemia, blast cell leukemia, bovine leukemia, chronic myelocytic leukemia, cutaneous leukemia, embryocellular leukemia, eosinophilic leukemia, Gross leukemia, hairy cell leukemia, hemoblastic leukemia, and hemocytoblastic leukemia. Leukemia includes histiocytic leukemia, stem cell leukemia, acute monocytic leukemia, leukopenic leukemia, lymphocytic leukemia, lymphoblastic leukemia, lymphocytic leukemia, lymphogenous leukemia, lymphoid leukemia, lymphosarcoma cell leukemia, mast cell leukemia, megakaryocytic leukemia, micromyeloblastic leukemia, monocytic leukemia, myeloblastic leukemia, myeloid leukemia, myeloid granulocytic leukemia, myelomonocytic leukemia, Naegeli's leukemia, plasma cell leukemia, plasma cell leukemia, promyelocytic leukemia, leader cell leukemia, Schilling's leukemia, stem cell leukemia, subleukemia, and anaplastic cell leukemia.

[0124] Further cancers include, for example, multiple myeloma, neuroblastoma, breast cancer, ovarian cancer, lung cancer, rhabdomyosarcoma, primary thrombocytosis, primary macroglobulinemia, small cell lung tumor, primary brain tumor, gastric cancer, colon cancer, malignant pancreatic insulinoma, malignant carcinoid, pre-malignant skin lesions, testicular cancer, thyroid cancer, neuroblastoma, esophageal cancer, genitourinary cancer, malignant hypercalcemia, cervical cancer, endometrial cancer, and adrenocortical carcinoma.

[0125] II. A2aR antibody and antigen-binding protein The present invention provides an A2aR antigen-binding molecule that specifically binds to A2aR. As used herein, the term “antigen-binding molecule” refers to a protein, polypeptide, or molecular complex comprising or consisting of at least one complementarity-determining region (CDR), either alone or in combination with one or more further CDRs and / or framework regions (FRs) that specifically bind to a particular antigen. In certain embodiments, these terms mean that the antigen-binding molecule is an antibody or an antigen-binding fragment thereof, as defined elsewhere herein. In certain embodiments, the antigen-binding molecule of the present invention inhibits one or more of the biological functions of A2aR.

[0126] In certain embodiments, A2aR is human A2aR. An exemplary human A2aR has the amino acid sequence shown in SEQ ID NO: 50. In some embodiments, A2aR is cynomolgus monkey A2aR. An exemplary cynomolgus monkey A2aR has the amino acid sequence shown in SEQ ID NO: 51.

[0127] 1. Example sequence of antigen-binding molecule The A2aR antigen-binding molecule may be in the form of a monoclonal antibody; one or more polypeptide fragments containing one or more A2aR antigen-binding domains; or one or more nucleic acids encoding one or more A2aR-binding domains.

[0128] In various exemplary embodiments of the present invention, an antigen-binding molecule, for example, an anti-A2aR antibody or its antigen-binding fragment, is (1) a heavy chain variable region comprising three complementarity-determining regions (HCDRs): HCDR1, HCDR2, and HCDR3, wherein HCDR1 has an amino acid sequence that is approximately 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to approximately 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 4, and 6; HCDR2 has an amino acid sequence that is approximately 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to approximately 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 5, and 7; and HCDR3 has an amino acid sequence that is approximately 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to approximately 100% identical to the amino acid sequence shown in SEQ ID NO: 3. (2) a light chain variable region comprising three complementarity-determining regions (LCDRs): LCDR1, LCDR2, and LCDR3, wherein LCDR1 has an amino acid sequence that is approximately 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, to approximately 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs. 25 and 28; LCDR2 has an amino acid sequence that is approximately 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, to approximately 100% identical to the amino acid sequence shown in SEQ ID NOs. 26; and LCDR3 has an amino acid sequence that is approximately 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, to approximately 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs. 27 and 29; the antigen-binding molecule specifically binds to human A2aR. Exemplary HCDR and LCDR amino acid sequences corresponding to the exemplary anti-human A2aR monoclonal antibodies disclosed in this invention are shown in Tables 1-5.

[0129] The amino acid sequence boundaries of CDRs are described by Kabat et al. (as described above, using the "Kabat" numbering scheme); by Al-Lazikani et al., 1997, J. Mol. Biol., 273:927-948 (using the "Chothia" numbering scheme); and by MacCallum et al., 1996, J. Mol. Biol. 262:732-745 (using the "Cont" numbering scheme). The numbering schemes may be determined by those skilled in the art using any of several known numbering schemes, including the “act” numbering scheme; described by Lefranc et al., Dev. Comp. Immunol., 2003, 27:55-77 (the “IMGT” numbering scheme); and described by Honegge and Pluckthun, J. Mol. Biol, 2001, 309:657-70 (the “AHo” numbering scheme); each of which is incorporated in whole by reference. Tables 1 and 2 show the heavy chain CDR sequences of exemplary antibodies of the present invention according to the Kabat numbering scheme and the IMGT numbering scheme, respectively. Table 3 shows the heavy chain CDR sequences of exemplary antibodies of the present invention, where the CDR sequence is defined by combining CDRs based on the Kabat and IMGT numbering schemes. Tables 4 and 5 show the light chain CDR sequences of exemplary antibodies of the present invention according to the Kabat numbering scheme and the IMGT numbering scheme.

[0130] In certain embodiments, the present invention includes an antigen-binding molecule comprising a CDR defined based on the Kabat and IMGT numbering scheme, or a combination thereof, for example, an anti-A2aR antibody or its antigen-binding fragment. Accordingly, in certain embodiments, the present invention includes an antigen-binding molecule comprising (1) HCDR1 having an amino acid sequence selected from the HCDR1 sequences listed in Table 2; (2) HCDR2 having an amino acid sequence selected from the HCDR2 sequences listed in Table 2; (3) HCDR3 having an amino acid sequence selected from the HCDR3 sequences listed in Table 2; (4) LCDR1 having an amino acid sequence selected from the LCDR1 sequences listed in Table 5; (5) LCDR2 having an amino acid sequence selected from the LCDR2 sequences listed in Table 5; and (6) LCDR3 having an amino acid sequence selected from the LCDR3 sequences listed in Table 5, for example, an anti-A2aR antibody or its antigen-binding fragment.

[0131] In certain embodiments, the present invention includes an antigen-binding molecule, such as an anti-A2aR antibody or an antigen-binding fragment thereof, comprising: (1) HCDR1 having an amino acid sequence selected from the HCDR1 sequences listed in Table 3; (2) HCDR2 having an amino acid sequence selected from the HCDR2 sequences listed in Table 3; (3) HCDR3 having an amino acid sequence selected from the HCDR3 sequences listed in Table 3; (4) LCDR1 having an amino acid sequence selected from the LCDR1 sequences listed in Table 4; (5) LCDR2 having an amino acid sequence selected from the LCDR2 sequences listed in Table 4; and (6) LCDR3 having an amino acid sequence selected from the LCDR3 sequences listed in Table 4.

[0132] As used herein, “position” in a CDR refers to the amino acid counted from the N-terminus of the CDR. For example, position 1 in HCDR1 refers to the first amino acid of HCDR1. Therefore, in mAB 1B5-3D7, position 1 of HCDR1 according to the Kabat numbering scheme is arginine (R).

[0133] [Table 1]

[0134] As used herein, X1 is S or R, X2 is Y or F, X3 is S or Y, X4 is A or T, X5 is H or Q, X6 is H or N, and X7 is D or G.

[0135] [Table 2]

[0136] As used herein, X8 is A or T, X9 is R or S, and X 10 is F or Y, and X 15 is S or Y, and X 16 It is either G or L.

[0137] [Table 3]

[0138] When used herein, X 14 is A or T, and X 15 is R or S, X 16 It is either F or Y.

[0139] [Table 4]

[0140] When used herein, X 17 is L or I, and X 18 is R or S, X 19 It is either Y or F.

[0141] [Table 5]

[0142] When used herein, X20 is L or I, and X 21 It is either R or S.

[0143] In some embodiments, the antibody or its antigen-binding fragment comprises (1) a heavy chain variable region (HCVR) having an amino acid sequence that is approximately 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to approximately 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs. 35, 36, and 37; and (2) a light chain variable region (LCVR) having an amino acid sequence that is approximately 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to approximately 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs. 41, 42, and 43, and the antibody or its antigen-binding fragment specifically binds to human A2aR. Exemplary HCVR and LCVR amino acid sequences corresponding to the exemplary anti-human A2aR monoclonal antibodies disclosed in the present invention are shown in Tables 6-8. Tables 9 and 10 show exemplary nucleotide sequences of the DNA encoding the HCVR and LCVR, respectively, of the exemplary anti-human A2aR antibodies of the present invention.

[0144] [Table 6]

[0145] [Table 7]

[0146] [Table 8]

[0147] [Table 9]

[0148] [Table 10]

[0149] In certain embodiments, the antigen-binding molecule of the present invention, for example, an antibody or its antigen-binding fragment, is modified after translation. Examples of post-translational modifications include cleavage of lysine by carboxypeptidase at the C-terminus of the heavy chain; modification of glutamine or glutamic acid to pyroglutamic acid by pyroglutamylation at the N-terminus of the heavy and light chains; glycosylation; oxidation; deamide; and glycation, and such post-translational modifications are known to be present in various antibodies (see the journal of Pharmaceutical Sciences, 2008, Vol. 97, pp. 2426-2447, which is incorporated by reference in its entirety). Antigen-binding molecule that has undergone post-translational modification, For example, examples of antibodies or their antigen-binding fragments include antigen-binding molecules that undergo pyroglutamylation at the N-terminus of the heavy chain variable region and / or deletion of lysine at the C-terminus of the heavy chain, e.g., antibodies or their antigen-binding fragments. Examples of sequences of antigen-binding molecules that undergo pyroglutamylation at the N-terminus are listed in Table 7. As used herein, "pE" refers to pyroglutamic acid when used to indicate an amino acid in a polypeptide.

[0150] 2. Variants of antigen-binding molecules In certain embodiments, the A2aR antigen-binding molecule of the present invention, for example, an anti-A2aR antibody, may be a monoclonal antibody, a chimeric antibody, a humanized antibody, Fab, (Fab)2, scFv, or a polyspecific antibody comprising further binding specificities as described herein.

[0151] Accordingly, in certain embodiments, the anti-A2aR antibody described herein may be linked to an Fc containing one or more modifications to typically alter one or more functional properties of the antibody, such as serum half-life, complement binding, Fc receptor binding, and / or antigen-dependent cell-mediated cytotoxicity. Furthermore, the antibodies described herein may be chemically modified (e.g., one or more chemical moieties may be linked to the antibody) or modified to alter its glycosylation in order to alter one or more functional properties of the antibody. More specifically, in certain embodiments, the antibody in the present invention may contain modifications in the Fc region to produce an Fc variant having (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 the Fc receptor compared to the parent Fc. Such an Fc region variant generally contains at least one amino acid modification in the Fc region. Combining amino acid modifications is considered particularly desirable. For example, a variant Fc region may contain, for example, two, three, four, five, or more substitutions of specific Fc region positions identified herein.

[0152] In cases where effector function should be avoided entirely, for example, when antigen binding alone is sufficient to produce the desired therapeutic benefit and effector function only results in (or increases the risk of) undesirable side effects, IgG4 antibodies or ADCC-null versions of IgG1 L234F, L235E, P331S may be used, or antibodies or fragments lacking the Fc region or substantial parts thereof may be devised, or the Fc may be mutated to completely eliminate glycosylation (e.g., N297A). Alternatively, hybrid constructs of human IgG2 (CH1 domain and hinge region) and human IgG4 (CH2 domain and CH3 domain) lacking effector function, lacking the ability to bind to FcγR (similar to IgG2) and activate complement (similar to IgG4), may be generated. When using the constant domain of IgG4, it is generally preferable to reduce Fab-arm exchange between the therapeutic antibody and endogenous IgG4 in the treated patient by including the substitution S228P, which mimics the hinge sequence in IgG1, and the R409K mutation, which prevents Fab-arm exchange and thereby stabilizes the IgG4 molecule.

[0153] In certain embodiments, an anti-A2aR antibody or fragment(s) thereof may be modified to provide an increased biological half-life. For example, various approaches may be employed, including those that increase the binding affinity of the Fc region to FcRn. In one embodiment, the antibody is modified within the CH1 or CL region to contain a salvage receptor-binding epitope taken from two loops of the CH2 domain of the Fc region of IgG, as described in U.S. Patents No. 5,869,046 and No. 6,121,022. The numbering of residues in the Fc region is the numbering of the Kabat EU index. Sequence variants disclosed herein are provided by reference to residue numbers followed by the amino acid to be substituted in place of a naturally occurring amino acid, preceded by the naturally occurring residue at that position as necessary. If multiple amino acids may be present at a given position, for example, if the sequence differs between naturally occurring isotypes, or if multiple mutations may be substituted at that position, they are separated by a slash (e.g., "X / Y / Z").

[0154] Exemplary Fc variants that increase binding to FcRn and / or improve pharmacokinetic properties include, for example, 2591, 308F, 428L, 428M, 434S, 434H, 434F, 434Y, and 434M, including substitutions at positions 259, 308, and 434. Other variants that increase Fc binding to FcRn include: 250E, 250Q, 428L, 428F, 250Q / 428L (Hinton et al., 2004, J. Biol. Chem. 279(8): 6213-6216, Hinton et al. 2006 Journal of Immunology 176:346-356), 256A, 272A, 305A, 307A, 31IA, 312A, 378Q, 380A, 382A, 434A (Shields et al. (2001) J. Biol. Chem., 276(9):6591-6604), 252F, 252Y, 252W, 254T, 25 6Q, 256E, 256D, 433R, 434F, 434Y, 252Y / 254T / 256E, 433K / 434F / 436H (Dall'Acqua et al. (2002) J. Immunol., 169:5171-5180, Dall'Acqua et al. (2006) J. Biol. Chem., 281:23514-23524 and U.S. Patent No. 8,367,805).

[0155] Modifications of certain conserved residues in IgG Fc (1253, H310, Q311, H433, N434), such as the N434A variant (Yeung et al. (2009) J. Immunol. 182:7663), have been proposed as a method to increase FcRn affinity, thereby increasing the half-life of the antibody in circulation (WO98 / 023289). Combination Fc variants including M428L and N434S have been shown to increase FcRn binding and extend the serum half-life by up to five times (Zalevsky et al. (2010) Nat. Biotechnol. 28:157). Combination Fc variants including T307A, E380A, and N434A modifications also extend the half-life of IgG1 antibodies (Petkova et al. (2006) Int. Immunol. 18:1759). Furthermore, M252Y-M428L, M428L-N434H, Combination Fc variants, including the M428L-N434F, M428L-N434Y, M428L-N434A, M428L-N434M, and M428L-N434S variants, have also been shown to extend the half-life (US2006 / 173170). Furthermore, combination Fc variants including M252Y, S254T, and T256E have been reported to increase the half-life by nearly four times. Dall'Acqua et al. (2006) J. Biol. Chem. 281:23514.

[0156] In certain embodiments, the A2aR antigen-binding molecule of the present invention is a bispecific antibody comprising a first targeting domain that specifically binds to A2aR and a second targeting domain that specifically binds to A2aR or another epitope in another protein. In some embodiments, the first targeting domain comprises an antigen-binding fragment derived from one of the A2aR antibodies of the present invention.

[0157] In certain embodiments, the antigen-binding molecule of the present invention, for example, an anti-A2aR antibody or its antigen-binding fragment, is chemically conjugated to one or more therapeutically active peptides and / or small molecule drugs. The peptide or small molecule drug may be conjugated, for example, to a reduced SH group and / or a carbohydrate side chain. Methods for producing covalent or non-covalent conjugates of peptides or small molecule drugs with antibodies are known in the art, and any such known method may be utilized.

[0158] In some embodiments, peptides or small molecule drugs are conjugated to the hinge region of a reduced antibody component via disulfide bond formation. Alternatively, such drugs may be conjugated using a heterobifunctional crosslinker, such as N-succinyl 3-(2-pyridyldithio)propionate (SPDP). General techniques for such conjugations are well known in the art. In some embodiments, peptides or small molecule drugs are conjugated via a carbohydrate moiety in the Fc region of the antibody. The carbohydrate group may be used to increase the loading of the same drug conjugated to the thiol group, or the carbohydrate moiety may be used to conjugate different therapeutic or diagnostic agents. Methods for conjugating 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. This reaction produces an initial Schiff base (imine) bond, which can be stabilized by reduction to a secondary amine to form the final conjugate. Exemplary methods for conjugating small molecule drugs and peptides to antibodies are described in U.S. Patent Application Publication No. 2014 / 0356385.

[0159] The A2aR antibodies that include the fragment and its multispecific forms have a size that is included in the range of 50 kD to 300 kD, 50 kD to 250 kD, 60 kD to 250 kD, 80 kDa 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 200 kD, 75 kD to 200 kD, 100 kD to 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 the recited integers in the ranges cited above, or any range specified by any combination of integers between any of the ranges cited above.

[0160] 3. Biological Characteristics of Antibodies and Antigen-Binding Molecules The present invention includes antibodies that bind to human and cynomolgus A2aR and antigen-binding fragments thereof.

[0161] The present invention includes A2aR antigen-binding molecules, such as A2aR antibodies or antigen-binding fragments thereof, that are capable of specifically binding to human and cynomolgus A2aR expressed on the cell surface and inhibiting the activity or function of A2aR. According to certain embodiments, the antigen-binding molecule blocks the interaction between human A2aR expressed on the cell surface and an A2aR agonist. The degree to which an A2aR antigen-binding protein, such as an A2aR antibody or antigen-binding fragment thereof, inhibits the activity of A2aR can be evaluated by the assay described in Example 4 or an assay substantially similar thereto. The present invention includes antigen-binding molecules, such as antibodies, that block the interaction between human A2aR expressed on the cell surface and an A2aR agonist with an IC -9 value of 4.5×10 -9 M to about 1.5×10 50 M or less, when determined using the assay shown in Example 4 or an assay substantially similar thereto.

[0162] The present invention includes an A2aR antigen-binding molecule, such as an A2aR antibody, that specifically binds to human A2aR expressed on the cell surface. In certain embodiments, the binding of the antigen-binding molecule of the present invention to A2aR or certain non-human mammalian A2aR, such as human adenosine receptors other than mouse A2aR, is either undetectable or weakly detected when determined using the assay shown in Example 5 or a substantially similar assay.

[0163] The present invention includes an A2aR antigen-binding molecule, such as an A2aR antibody or its antigen-binding fragment, that specifically binds to non-human primate A2aR expressed on the cell surface, such as cynomolgus monkey A2aR. Therefore, in certain embodiments, the A2aR antigen-binding molecule, such as an A2aR antibody or antigen-binding fragment, binds to non-human primate A2aR with similar affinity when determined using the assay shown in Example 7 or a substantially similar assay.

[0164] The present invention includes an A2aR antigen-binding molecule, such as an A2aR antibody or its antigen-binding fragment, that specifically binds to endogenous human and non-human primate A2aR expressed on the surface of human or non-human primate immune cells, such as cynomolgus monkey A2aR. Thus, in certain embodiments, the A2aR antigen-binding molecule, such as an A2aR antibody or antigen-binding fragment, binds to human or non-human primate A2aR expressed on the surface of immune cells in peripheral blood mononuclear cells (PBMCs), as determined using the assay shown in Example 7 or a substantially similar assay.

[0165] 4. Species selectivity and species cross-reactivity According to certain embodiments, the present invention provides an antigen-binding molecule that binds to human A2aR but not to A2aR from other species. The present invention also includes antigen-binding molecules that bind to human A2aR and A2aR from one or more non-human species, for example, non-human primates.

[0166] According to certain exemplary embodiments of the present invention, an antigen-binding molecule is provided that binds to human A2aR, which may or may not bind to one or more A2aRs of mouse, rat, guinea pig, hamster, gerbil, pig, cat, dog, rabbit, goat, sheep, cattle, horse, camel, cynomolgus macaque, marmoset, rhesus macaque, or chimpanzee. For example, certain exemplary embodiments of the present invention provide an antigen-binding molecule comprising an antigen-binding domain that binds to human A2aR and a non-human primate, such as cynomolgus macaque A2aR.

[0167] III. Therapeutic use of anti-A2aR antigen-binding molecules The anti-A2aR antigen-binding molecule of the present invention, which comprises an antibody, its antigen-binding fragment, and the polyspecific antibody, is associated with enhancing the immune response in numerous in vitro and in vitro studies in cancer treatment by blocking adenosine-mediated signaling and other signaling pathways. It has both vivo and ex vivo utility. While we do not wish to be bound by any particular theory, it is hypothesized that the antigen-binding molecules of the present invention, e.g., anti-A2aR antibodies or their antigen-binding fragments, bind to A2aR expressed on the cell surface and inhibit its activity, for example, by reducing intracellular cAMP concentration as a result of inhibiting A2aR activity. Therefore, the antigen-binding molecules of the present invention (and therapeutic compositions containing them) are particularly useful for treating any disease or disorder in which inhibition of A2aR activity, e.g., stimulation and / or activation of the immune response, is beneficial. Considering the widespread expression of A2aR and the pleiotropic effects mediated by adenosine and A2aR, the anti-A2aR antigen-binding molecules of the present invention, e.g., antibodies or their antigen-binding fragments, can be used individually or in combination with various active agents for treating a wide range of diseases or disorders, including various cancers.

[0168] Accordingly, the present invention provides a method for reducing intracellular cAMP concentration in cells, comprising the steps of contacting cells with an antigen-binding molecule of the present invention, such as an anti-A2aR antibody or its antigen-binding fragment. The reduction in intracellular cAMP concentration can be measured by the method described in Example 4, or by a substantially similar method. In certain embodiments, the method of the present invention reduces the intracellular cAMP concentration by at least about 10%, about 20%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or more, compared to a baseline level.

[0169] In some embodiments, the antigen-binding molecules of the present invention, such as anti-A2aR antibodies or their antigen-binding fragments, are administered to cells in culture (in vitro) or to human subjects in vivo or ex vivo to enhance immunity in various diseases. Accordingly, in one embodiment, a method for stimulating an immune response in a subject that requires stimulation of an immune response comprises the step of administering to a subject an anti-A2aR antibody, its antigen-binding fragment (e.g., anti-A2aR HCVR and LCVR) or a multispecific anti-A2aR antibody as described herein, such that the immune response is enhanced, stimulated, or upregulated in the subject to, for example, inhibit tumor growth, stimulate anti-tumor T cell immunity, and / or stimulate antibacterial immunity.

[0170] In one embodiment, a method for enhancing an immune response (e.g., a T-cell response) in a subject comprises the step of administering an anti-A2aR antibody described herein to the subject such that an immune response (e.g., a T-cell response) is enhanced in the subject. In some embodiments, the subject is a tumor-carrier subject in which an immune response to the tumor is enhanced. The tumor may be a solid tumor or a liquid tumor, e.g., a hematological malignancy. In certain embodiments, the tumor is an immunogenic tumor. In other embodiments, the tumor is non-immunogenic. In other embodiments, the subject is a pathogen-carrier subject in which an immune response to the pathogen is enhanced as a result of administration of an anti-A2aR antibody described herein. The immune response includes, but is not limited to, a) promoting effector T-cell function; b) reducing Treg activity; c) preventing Treg proliferation; d) enhancing NK-cell function; or e) promoting type 1 activation of antigen-presenting cells.

[0171] In certain embodiments, the methods of the present invention increase the immune response by at least about 10%, about 20%, about 50%, about 60%, about 70%, about 80%, about 90%, about 1x, about 2x, about 4x, or more, compared to baseline levels.

[0172] Preferred subjects include human patients for whom an enhancement of the immune response is desired. The method is particularly suitable for treating human patients with disorders that can be treated by increasing the immune response (e.g., T cell-mediated immune response). The method is particularly suitable for treating cancer, chronic infections, and chronic inflammatory disease conditions. Preferably, the antibody for use in the disclosed method described herein is human or a humanized antibody.

[0173] In one embodiment, a method for inhibiting tumor cell growth in a subject includes the step of administering an anti-A2aR antibody described herein to a subject such that tumor growth is inhibited in the subject. Inhibition of tumor growth can be measured by various methods. Tumor growth can be measured, for example, using 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 at https: / / www.ncbi.nlm.nih.gov / pmc / articles / PMC4764475 / , which is incorporated herein by reference in its entirety. Inhibition of tumor growth can also be measured by a reduction in tumor size. In certain embodiments, the methods of the present invention 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.

[0174] In certain embodiments, the antigen-binding molecules of the present invention, such as an anti-A2aR antibody or its antigen-binding fragment, may be used in a method for reducing immunosuppression in the tumor microenvironment. Such reduction may be measured by various methods. For example, the level of immunosuppression in the tumor microenvironment may be measured by the presence and / or amount of certain biomarkers in the tumor, such as PD-L1, CD73, IL-10, or TGF-β. The level of immunosuppression may also be measured by the regulatory T(T) levels of CD8+ cytotoxic T cells in the tumor. reg It can be measured by the ratio of CD8+ cytotoxic T cells to T cells. In general, immunosuppression is measured by the ratio of CD8+ cytotoxic T cells to T cells. reg The ratio (ration) is reduced. In certain embodiments, the antigen-binding molecules of the present invention, such as an anti-A2aR antibody or its antigen-binding fragment, 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 baseline levels.

[0175] A method for depleting T cells from the tumor microenvironment of a subject having a tumor, such as a cancerous tumor, the method comprising administering to the subject a therapeutically effective amount of an anti-A2aR antibody described herein that includes an Fc that stimulates depletion of T cells in the tumor microenvironment is also encompassed herein. The Fc can be, for example, an Fc having an appropriate effector function or an enhanced effector function conferred by one or more activating Fc receptors. reg A method for depleting T cells from the tumor microenvironment of a subject having a tumor, such as a cancerous tumor, the method comprising administering to the subject a therapeutically effective amount of an anti-A2aR antibody described herein that includes an Fc that stimulates depletion of T cells in the tumor microenvironment is also encompassed herein. The Fc can be, for example, an Fc having an appropriate effector function or an enhanced effector function conferred by one or more activating Fc receptors. reg A method for depleting T cells from the tumor microenvironment of a subject having a tumor, such as a cancerous tumor, the method comprising administering to the subject a therapeutically effective amount of an anti-A2aR antibody described herein that includes an Fc that stimulates depletion of T cells in the tumor microenvironment is also encompassed herein. The Fc can be, for example, an Fc having an appropriate effector function or an enhanced effector function conferred by one or more activating Fc receptors.

[0176] In certain preferred embodiments, the depletion of T cells occurs without significant depletion or inhibition of T cells in the tumor microenvironment and without significant depletion or inhibition of T cells outside the tumor microenvironment. In certain embodiments, the subject has a higher level of A2aR on T cells than on T cells in the tumor microenvironment. In certain embodiments, the anti-A2aR antibody can deplete T cells in the tumor and / or T cells in tumor infiltrating lymphocytes (TILs). reg In certain preferred embodiments, the depletion of T cells occurs without significant depletion or inhibition of T cells in the tumor microenvironment and without significant depletion or inhibition of T cells outside the tumor microenvironment. In certain embodiments, the subject has a higher level of A2aR on T cells than on T cells in the tumor microenvironment. In certain embodiments, the anti-A2aR antibody can deplete T cells in the tumor and / or T cells in tumor infiltrating lymphocytes (TILs). eff In certain preferred embodiments, the depletion of T cells occurs without significant depletion or inhibition of T cells in the tumor microenvironment and without significant depletion or inhibition of T cells outside the tumor microenvironment. In certain embodiments, the subject has a higher level of A2aR on T cells than on T cells in the tumor microenvironment. In certain embodiments, the anti-A2aR antibody can deplete T cells in the tumor and / or T cells in tumor infiltrating lymphocytes (TILs). eff In certain preferred embodiments, the depletion of T cells occurs without significant depletion or inhibition of T cells in the tumor microenvironment and without significant depletion or inhibition of T cells outside the tumor microenvironment. In certain embodiments, the subject has a higher level of A2aR on T cells than on T cells in the tumor microenvironment. In certain embodiments, the anti-A2aR antibody can deplete T cells in the tumor and / or T cells in tumor infiltrating lymphocytes (TILs). reg In certain preferred embodiments, the depletion of T cells occurs without significant depletion or inhibition of T cells in the tumor microenvironment and without significant depletion or inhibition of T cells outside the tumor microenvironment. In certain embodiments, the subject has a higher level of A2aR on T cells than on T cells in the tumor microenvironment. In certain embodiments, the anti-A(2a)R antibody can deplete T cells in the tumor and / or T cells in tumor infiltrating lymphocytes (TILs). eff In certain preferred embodiments, the depletion of T cells occurs without significant depletion or inhibition of T cells in the tumor microenvironment and without significant depletion or inhibition of T cells outside the tumor microenvironment. In certain embodiments, the subject has a higher level of A2aR on T cells than on T cells in the tumor microenvironment. In certain embodiments, the anti-A(2a)R antibody can deplete T cells in the tumor and / or T cells in tumor infiltrating lymphocytes (TILs). reg In certain preferred embodiments, the depletion of T cells occurs without significant depletion or inhibition of T cells in the tumor microenvironment and without significant depletion or inhibition of T cells outside the tumor microenvironment. In certain embodiments, the subject has a higher level of A2aR on T cells than on T cells in the tumor microenvironment. In certain embodiments, the anti-A(2a)R antibody can deplete T cells in the tumor and / or T cells in tumor infiltrating lymphocytes (TILs). reg In certain preferred embodiments, the depletion of T cells occurs without significant depletion or inhibition of T cells in the tumor microenvironment and without significant depletion or inhibition of T cells outside the tumor microenvironment. In certain embodiments, the subject has a higher level of A2aR on T cells than on T cells in the tumor microenvironment. In certain embodiments, the anti-A(2a)R antibody can deplete T cells in the tumor and / or T cells in tumor infiltrating lymphocytes (TILs). reg In certain preferred embodiments, the depletion of T cells occurs without significant depletion or inhibition of T cells in the tumor microenvironment and without significant depletion or inhibition of T cells outside the tumor microenvironment. In certain embodiments, the subject has a higher level of A2aR on T cells than on T cells in the tumor microenvironment. In certain embodiments, the anti-A(2a)R antibody can deplete T cells in the tumor and / or T cells in tumor infiltrating lymphocytes (TILs).

[0177] In certain preferred embodiments, the subject has a cell proliferation disorder or cancer. Blocking of A2aR-mediated adenosine signaling using the antigen-binding molecules of the present invention, e.g., an anti-A2aR antibody or its antigen-binding fragment, can enhance the immune response against cancer cells in a patient. Accordingly, the present invention provides a method for treating a subject having cancer, comprising the step of administering an anti-A2aR antigen-binding molecule described herein, e.g., an antibody or its antigen-binding fragment, to the subject so that the subject is treated, e.g., so that the growth of a cancerous tumor is inhibited or reduced and / or the tumor regresses. The anti-A2aR antibody may be used alone to inhibit the growth of a cancerous tumor. Alternatively, the anti-A2aR antibody may be used in combination with one or more other active agents, e.g., other anti-cancer targets, immunogenic agents, standard cancer treatments or other antibodies, as described below. The antigen-binding molecules of the present invention may be used, for example, to treat primary and / or metastatic tumors. The present invention also includes a method for treating residual cancer in a subject. As used herein, the term “residual cancer” means the presence or persistence of one or more cancerous cells in a subject after treatment with anticancer therapy.

[0178] Accordingly, in one embodiment, a method for treating cancer comprises the step of administering a therapeutically effective dose of an anti-A2aR antibody as described herein to a subject in need of treatment for cancer. Preferably, the antibody inhibits the activity of human anti-A2aR and comprises one or more HCVRs and LCVRs as described herein. Furthermore, anti-A2aR antigen-binding molecules for use in this method, e.g., antibodies, may comprise chimeric or humanized non-human anti-A2aR antibodies derived therefrom. The effectiveness of treating cancer can be measured by various methods. For example, the effectiveness of treating cancer can be measured by improvement in survival or reduction in tumor size. In certain embodiments, the method of the present invention increases the effectiveness of treating cancer by at least about 10%, about 20%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 1x, about 2x, about 4x, or more, compared to baseline levels. When used in the context of cancer treatment, the baseline level refers to the efficacy with placebo if the A2aR antigen-binding molecule of the present invention is the sole therapeutic agent, or the efficacy with placebo or the additional therapeutic agent if the A2aR antigen-binding molecule of the present invention is used in combination with an additional therapeutic agent.

[0179] Cancers whose growth can be inhibited using the antibodies of the present invention include a wide range of cancers, particularly cancers that are unresponsive to monotherapy with other antibodies or chemotherapeutic agents, or that tend to become unresponsive. Non-limiting examples of cancers for treatment include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, squamous cell non-small cell lung cancer (NSCLC), non-NSCLC, glioma, gastrointestinal cancer, kidney cancer (e.g., clear cell carcinoma), ovarian cancer, liver cancer, colorectal cancer, endometrial cancer, kidney cancer (e.g., renal cell carcinoma (RCC)), prostate cancer (e.g., hormone-refractory adenocarcinoma of the prostate), thyroid cancer, neuroblastoma, pancreatic cancer, glioblastoma (glioblastoma multiforme), cervical cancer, stomach cancer, bladder cancer, Hepatoma, breast cancer, colon cancer, and head and neck cancer (or cancer), gastric cancer, germ cell tumors, pediatric sarcomas, sinus natural killer cancers, melanoma (e.g., metastatic melanoma, e.g., cutaneous or intraocular melanoma), bone cancer, skin cancer, uterine cancer, anal cancer, testicular cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, esophageal cancer, small intestine cancer, endocrine cancer, parathyroid cancer, adrenal gland cancer, soft tissue sarcoma, urethral cancer, penile cancer, This includes solid tumors in children, cancers of the ureter, cancers of the renal pelvis, neoplasms of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axial tumors, brainstem gliomas, pituitary adenomas, Kaposi's sarcoma, epidermal carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers including asbestos-induced cancers, virus-associated cancers (e.g., human papillomavirus (HPV)-associated tumors), and two major hematological lineages, namely the myeloid cell lineage (which includes granulocytes, erythrocytes, platelets, macrophages, and mass cells). Hematological malignancies originating from either the B cell-producing or lymphoid cell lineage (which produces B, T, NK, and plasma cells), such as all types of leukemia, lymphoma, and myeloma, such as acute, chronic, lymphocytic, and / or myeloid leukemia, such as acute leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), and chronic myeloid leukemia (CIVIL), undifferentiated AML (MO), myeloblastic leukemia (M1), and myeloblastic leukemia (M2);(with cell maturation), promyelocytic leukemia (M3 or M3 variant [M3V]), myelomonocytic leukemia (M4, or M4 variant with eosinophilia [M4E]), monocytic leukemia (M5), erythroleukemia (M6), megakaryoblastic leukemia (M7), solitary granulocytic sarcoma, and chloroplasma; lymphomas, e.g., Hodgkin lymphoma (HL), non-Hodgkin lymphoma (NEIL), B-cell lymphoma, T-cell lymphoma, lymphoplasmacytic lymphoma, monocytic B-cell lymphoma, mucosa-associated lymphoid tissue (MALT) lymphoma, undifferentiated (e.g., Ki 1+) Large cell lymphoma, adult T-cell lymphoma / leukemia, mantle cell lymphoma, angioimmunoblastic T-cell lymphoma, vascular central lymphoma, enteric T-cell lymphoma, mediastinal primary B-cell lymphoma, progenitor T-lymphoblastic lymphoma, T-lymphoblastic lymphoma; and lymphoma / leukemia (T-Lbly / T-ALL), peripheral T-cell lymphoma, lymphoblastic lymphoma, post-transplant lymphoproliferative disorder, histiocytic lymphoma, primary central nervous system lymphoma, primary exudative lymphoma, lymphoblastic lymphoma (LBL), hematopoietic malignancies of the lymphoid system, acute lymphoblastic lymphoma Lymphocytic leukemia, diffuse large B-cell lymphoma, Burkitt lymphoma, follicular lymphoma, diffuse histiocytic lymphoma (DHL), immunoblastic large B-cell lymphoma, progenitor B-lymphoblastic lymphoma, cutaneous T-cell lymphoma (CTLC) (also known as mycosis fungoides or Sézary syndrome), and lymphoplasmacytic lymphoma with Waldenström macroglobulinemia (LPL); myeloma, e.g., IgG myeloma, light chain myeloma, non-secretory myeloma, smoldering myeloma (also known as asymptomatic myeloma), solitary plasmacytoma, and multiple myeloma, chronic lymphocytic leukemia (CLL), hairy cell lymphoma; mesenchymal tumors, including myeloid hematopoietic malignancies, fibrosarcomas, and rhabdomyoscarcomas; seminos; Tumors of the central and peripheral nervous system, including teratomas, astrocytomas, and schwann cell tumors; tumors of mesenchymal origin, including fibrosarcomas, rhabdomyoscaromas, and osteosarcomas; as well as other tumors, including melanoma, xeroderma pigmentosum, keratoscanthoma, seminoma, follicular thyroid carcinoma and teratocarcinoma, hematopoietic malignancies of the lymphoid system, such as small cell and cerebral-like cell types; T-cell disorders, such as pre-T lymphocytic leukemia (T-PLL), but not limited to T-cell tumors and B-cell tumors; preferably large granular lymphocytic leukemia (LGL) of the T-cell type; a / d T-NHL hepatosplenic lymphoma; peripheral / postthymic T-cell lymphoma (pleomorphic and immunoblastic subtypes); vascular central (nasal) T-cell lymphoma; cancers of the head and neck, kidney cancer, rectal cancer, thyroid cancer; acute myeloid lymphoma, and any combination of the aforementioned cancers. The methods described herein may also be used for the treatment of metastatic cancer, refractory cancer (for example, cancer that is refractory to prior immunotherapy with, for example, blockade CTLA-4 or PD-1 antibodies), and recurrent cancer.

[0180] In some embodiments, treatment of cancer patients with anti-A2aR antibodies and / or other active agents according to the present invention may result in a long-term, sustained response compared to current standard treatment, including long-term survival of at least 1, 2, 3, 4, 5, or 10 years or longer, and / or recurrence-free survival of at least 1, 2, 3, 4, 5, or 10 years or longer. In certain embodiments, treatment of cancer patients with anti-A2aR antibodies and / or other active agents according to the present invention may prevent or delay cancer recurrence for, for example, 1, 2, 3, 4, 5, or 10 years or longer. Anti-A2aR treatment may be used as a first-line or second-line treatment.

[0181] Bone marrow transplantation is currently used to treat various tumors of hematopoietic origin. Graft-versus-host disease is a consequence of this procedure, but A2aR inhibition may be used to increase the effectiveness of donor-grafted tumor-specific T cells by reducing the graft-versus-tumor response.

[0182] In some embodiments, ex vivo activation and proliferation of antigen-specific T cells in the presence of an anti-A2aR antibody, as well as adoptive transfer of these cells into a recipient, may be used to stimulate antigen-specific T cells against cancer or viral infection by increasing the frequency and activity of the adopted T cells.

[0183] Suitable routes for administering the antigen-binding molecules of the present invention described herein, such as anti-A2aR antibodies or their antigen-binding fragments (e.g., humanized monoclonal antibodies, multispecific antibodies, and immunoconjugates), in vivo, ex vivo, or in vitro are well known in the art and can be selected by those skilled in the art. For example, antibody compositions may be administered by parenteral injection (e.g., intravenously or subcutaneously). Appropriate dosages depend on the age and weight of the subject, as well as the concentration and / or formulation of the antibody composition, as will be further described below.

[0184] Increased A2aR activity is associated with neurodegenerative diseases. Therefore, in certain embodiments, the present invention provides a method for treating a neurodegenerative disease, comprising the step of administering an antigen-binding molecule of the present invention, for example, an anti-A2aR antibody or an antigen-binding fragment thereof, to a subject requiring treatment for a neurodegenerative disease, thereby providing a method for treating a neurodegenerative disease.

[0185] IV. Combination Therapy In another embodiment, the present invention provides therapeutic compositions and combination therapies for enhancing antigen-specific T cell responses, reducing immunosuppression, and / or reducing tumor growth in a subject. The present invention includes compositions and therapeutic formulations comprising, for example, any of the exemplary antigen-binding molecules herein in combination with one or more further therapeutic agents, and methods of treatment comprising the step of administering such combination to a subject requiring treatment. The term “further therapeutic agent” as used herein means any agent that may be used to treat a disease or disorder, and any method of treatment for a particular disease or disorder. For example, radiotherapy and surgery are considered “further therapeutic agents” when used in combination with the antigen-binding molecules of the present invention, for example, an anti-A2aR antibody or its antigen-binding fragment.

[0186] In certain embodiments, further therapeutic agents may be A2aR antagonists different from the antigen-binding molecules of the present invention, such as anti-A2aR antibodies or their antigen-binding fragments. 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).

[0187] In one embodiment, further therapeutic agents may be administered in the form of antibodies or antibody fragments(s) against other adenosine signaling pathway members, such as A1aR, A2bR, A3R, CD39, CD73 antagonists, or combinations thereof. Exemplary CD39 antagonists include, but are not limited to, exemplary anti-CD39 antibodies, whose antigen-binding sites are 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 U.S. Patents 10,766,966, 10,584,169, 10,556,968, and 10,167,343.

[0188] In some embodiments, the anti-A2aR antigen-binding molecule of the present invention, for example, an anti-A2aR antibody or its antigen-binding fragment, is co-administered with one or more additional therapeutic agents in an effective amount for stimulating the immune response and / or apoptosis, in order to further enhance, stimulate, or upmodulate the immune response and / or apoptosis in a subject. Furthermore, one or more additional therapeutically active agents are administered before or after treatment with the anti-A2aR antibody.

[0189] In certain embodiments, the anti-A2aR antibody described herein is administered in combination with, or in combination with, one or more other active agents, such as anti-cancer antibodies or polypeptides, chemotherapeutic agents, and radiotoxic agents. In other embodiments, the anti-A2aR antibody described herein is administered in combination with, or in combination with, standard cancer treatments, such as surgery or radiation.

[0190] Co-administration of anti-A2aR antibodies with these active agents or treatment modalities can address clinical deficits related to drug resistance, antigenic changes in tumor cells that prevent them from reacting with the antibody, and toxicity (by administering lower doses of one or more agents). A2aR inhibition is particularly well-suited for use in combination with chemotherapy regimens that are otherwise refractory. In these cases, it may be possible to achieve enhanced efficacy while reducing the dose of the chemotherapy reagents administered (Mokyr et al.). (1998) Cancer Research 58: 5301-5304). The rationale for A2aR inhibition in combination with radiotherapy or chemotherapy is based on the promotion of cell death as a result of the cytotoxic effects of radiotherapy and most chemotherapy compounds, which can further increase the levels of tumor antigens in the antigen presentation pathway. Other combination therapies that may act additively or synergistically with A2aR inhibition via cell death include surgery and hormone deficiency or inhibition. Each of these protocols further creates a source of tumor antigens in the host.

[0191] In some embodiments, the anti-A2aR antibody described herein is conjugated to another active agent in the form of an immune complex, immunoconjugate, or fusion protein. Alternatively, the anti-A2aR antibody may be administered separately from the other active agent. In this case, the anti-A2aR antibody and the other antagonist may be administered before, after, or in combination with the other active agent, or co-administered with other known treatments, such as other anticancer agents or radiation. Accordingly, 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 both cytotoxic and immunoprotective effects in human cancer cells.

[0192] For example, in some embodiments, the anti-A2aR antibodies described herein may be combined with anticancer 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 (mTOR) inhibitors of rapamycin; proteasome inhibitors; poly(ADP-ribose) polymerase (PARP) inhibitors; Ras / MAPK pathway inhibitors; centrosome cluster separators; multikinase inhibitors; serine / threonine kinase inhibitors; tyrosine kinase inhibitors; VEGF / VEGFR inhibitors; taxanes or taxane derivatives; aromatase inhibitors; anthracyclines; microtubule-targeting drugs; topoisomerase toxins; inhibitors of molecular targets or enzymes (e.g., kinases or protein methyltransferases); cytidine analogs or combinations thereof.

[0193] Exemplary alkylating agents include, but are not limited to, cyclophosphamide (Cytoxan; Neosar); chlorambucil (Leukeran); melphalan (Alkeran); carmustine (BiCNU); busulfen (Busulfex); lomustine (CeeNU); dacarbazine (DTIC-Dome); oxaliplatin (Eloxatin); carmustine (Gliadel); ifosfamide (Ifex); mechloretamine (Mustargen); busulfen (Myleran); carboplatin (Paraplatin); cisplatin (CDDP; Platinol); temozolomide (Temodar); thiotepa (Thioplex); bendamustine (Treanda); or streptozocin (Zanosar).

[0194] Exemplary anthracycline antibiotics include, but are not limited to, doxorubicin (Adriamycin), doxorubicin liposome (Doxil), mitoxantrone (Novantrone), bleomycin (Blenoxane), daunorubicin (Cerubidine), daunorubicin liposome (DaunoXome), dactinomycin (Cosmegen), epirubicin (Ellence), idarubicin (Idamycin), plicamycin (Mithracin), mitomycin (Mutamycin), pentostatin (Nipent), or valrubicin (Valstar).

[0195] Examples of antimetabolites include, but are not limited to, fluorouracil (Adrucil), capecitabine (Xeloda), hydroxyurea (Hydrea), mercaptopurine (Purinethol), pemetrexed (Alimta), fludarabine (Fludara), nerarabine (Arranon), cladribine (Cladribine Novaplus), clofarabine (Clolar), cytarabine (Cytosar-U), decitabine (Dacogen), cytarabine liposome (DepoCyt), hydroxyurea (Droxia), pralatrexate (Folotyn), phloxuridine (FUDR), gemcitabine (Gemzar), cladribine (Leustatin), fludarabine (Oforta), methotrexate (MTX; Rheumatrex), methotrexate (Trexall), thioguanine (Tabloid), TS-1, or cytarabine (Tarabine PFS).

[0196] Examples of antidotes include, but are not limited to, amiphostine (Ethyol) or mesna (Mesnex).

[0197] Exemplary interferons include, but are not limited to, interferon alpha-2b (Intron A) or interferon alpha-2a (Roferon-A).

[0198] Examples of 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 (odine131) tositumomab (Bexxar); alemtuzumab (Campath); ibritumomab (Zevalin; In-111; Y-90 Zevalin); gemtuzumab (Mylotarg); eculizumab (Soliris); or denosumab (ordenosumab).

[0199] Examples of 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.

[0200] Exemplary HER2 inhibitors include, but are not limited to, trastuzumab (Herceptin), lapatinib (Tykerb), or AC-480.

[0201] Exemplary histone deacetylase inhibitors include, but are not limited to, vorinostat (Zolinza), valproic acid, romidepsin, entinostat, avexinostat, gibinostat, and mosetinostat.

[0202] Exemplary hormones include, but are not limited to, tamoxifen (Soltamox; Nolvadex); raloxifene (Evista); megestrol (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).

[0203] 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).

[0204] Exemplary phosphatidylinositol-3 kinase (PI3K) inhibitors include wortmannin, an irreversible inhibitor of PI3K; demethoxybirubin, a derivative of wortmannin; LY294002, a reversible inhibitor of PI3K; BKM120 (buparlisib); idelalisib (a PI3K delta inhibitor); duvelisib (IPI-145, an inhibitor of PI3K delta and gamma); alpelisib (BYL719), an alpha-specific PI3K inhibitor; TGR 1202 (formerly known as RP5264), an oral PI3K delta inhibitor; and copanlisib (BAY 80-6946), an inhibitor mainly of PI3Kα and δ isoforms.

[0205] Exemplary Akt inhibitors include, but are not limited to, miltefosine, AZD5363, GDC-0068, MK2206, perifosine, RX-0201, PBI-05204, GSK2141795, and SR13668.

[0206] Exemplary mTOR inhibitors include everolimus (Afinitor) or temsirolimus (Torisel); rapamycin (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, but are not limited thereto.

[0207] Exemplary proteasome inhibitors include bortezomib (PS-341), ixazomib (MLN2238), MLN 9708, delanzomib (CEP-18770), carfilzomib (PR-171), YU101, oprozomib (ONX-0912), marizomib (NPI-0052), and disulfiram, but are not limited thereto.

[0208] Exemplary PARP inhibitors include, but are not limited to, olaparib, iniparib, velaparib, BMN-673, BSI-201, AG014699, ABT-888, GPI21016, MK4827, INO-1001, CEP-9722, PJ-34, Tiq-A, Phen, PF-01367338, and combinations thereof.

[0209] Exemplary Ras / MAPK pathway inhibitors include, but are not limited to, trametinib, selumetinib, cobimetinib, CI-1040, PD0325901, AS703026, R04987655, R05068760, AZD6244, GSK1120212, TAK-733, U0126, MEK162, and GDC-0973.

[0210] Exemplary centriole cluster separators include, but are not limited to, griseofulvin; noscapine, noscapine derivatives, e.g., brominated noscapine (e.g., 9-bromonoscapine), reduced bromonoscapine (RBN), N-(3-bromobenzyl)noscapine, aminonoscapine and their water-soluble derivatives; CW069; phenanthridene-derived poly(ADP-ribose) polymerase inhibitors; PJ-34; N2-(3-pyridylmethyl)-5-nitro-2-flamid, N2-(2-thienylmethyl)-5-nitro-2-flamid and N2-benzyl-5-nitro-2-flamid.

[0211] Exemplary multikinase inhibitors include, but are not limited to, regorafenib; sorafenib (Nexavar); sunitinib (Sutent); BIBW 2992; E7080; Zd6474; PKC-412; motesanib; or AP24534.

[0212] Exemplary serine / threonine kinase inhibitors include, but are not limited to, ruboxystaurine; eril / easudil hydrochloride; flavopyridol; seliciclib (CYC202; Roscovitrine); SNS-032 (BMS-387032); Pkc412; briostatin; KAI-9803; SF1126; VX-680; Azd1152; Arry-142886 (AZD-6244); SCIO-469; GW681323; CC-401; CEP-1347 or PD 332991.

[0213] Examples of tyrosine kinase inhibitors include erlotinib (Tarceva), gefitinib (Iressa), imatinib (Gleevec), sorafenib (Nexavar), sunitinib (Sutent), trastuzumab (Herceptin), bevacizumab (Avastin), rituximab (Rituxan), lapatinib (Tykerb), cetuximab (Erbitux), panitumumab (Vectibix), and evero. This includes, but is not limited to, limus (Afinitor), alemtuzumab (Campath), gemtuzumab (Mylotarg), temsirolimus (Torisel), pazopanib (Votrient), dasatinib (Sprycel), nilotinib (Tasigna), batalanib (Ptk787; ZK222584), CEP-701; SU5614; MLN518; XL999; VX-322; Azd0530; BMS-354825; SKI-606; CP-690; AG-490; WHI-P154; WHI-P131; AC-220; or AMG888.

[0214] Examples of VEGF / VEGFR inhibitors include, but are not limited to, bevacizumab (Avastin), sorafenib (Nexavar), sunitinib (Sutent), ranibizumab, pegaptanib, or vandetinib.

[0215] Exemplary microtubule-targeting drugs include, but are not limited to, paclitaxel, docetaxel, vincristine, vinblastine, nocodazole, epotilon, and navelbine.

[0216] Examples of topoisomerase-toxic drugs include, but are not limited to, teniposide, etoposide, adriamycin, camptothecin, daunorubicin, dactinomycin, mitoxantrone, amsacrin, epirubicin, and idarubicin.

[0217] Exemplary taxanes or taxane derivatives include, but are not limited to, paclitaxel and docetaxol.

[0218] Examples of common chemotherapeutic agents, antineoplastic agents, and antiproliferative agents include altretamine (Hexalen); isotretinoin (Accutane; Amnesteem; Claravis; Sotret); tretinoin (Vesanoid); azacitidine (Vidaza); bortezomib (Velcade); asparaginase (Elspar); levamisol (Ergamisol); mitotane (Lysodren); procarbazine (Matulane); pegaspar gauze (Oncaspar); denileukin difutitox (Ontak); porfimer (Photofrin); aldesleukin (Proleukin); lenalidomide (Revlimid); bexarotene (Targretin); thalidomide (Thalomid); temsirolimus (Torisel); arsenic trioxide (Trisenox); verteporfin (Visudyne); mimosine (Leucenol); (1M tegafur - This includes, but is not limited to, 0.4M 5-chloro-2,4-dihydroxypyrimidine (1M potassium oxonate) or lovastatin.

[0219] In certain embodiments, A2aR inhibition is performed in combination with CD3 stimulation (e.g., by co-incubation with cells expressing membrane CD3) either before, simultaneously with, or after treatment with an anti-A2aR antibody. For example, in one embodiment, a method for enhancing antigen-specific T cell response includes the step of contacting T cells with anti-A2aR antibody and CD3-expressing cells as described herein, such that the antigen-specific T cell response is enhanced and A2aR-mediated immunosuppression is reduced. Any suitable indicator of antigen-specific T cell response may 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 preferred embodiments, interleukin-2 and / or interferon-γ production by antigen-specific T cells is enhanced.

[0220] In some embodiments, the anti-A2aR antibodies described herein may also be used in combination with bispecific antibodies that direct Fcα or Fcγ receptor-expressing effector cells to tumor cells (see, for example, U.S. Patents 5,922,845 and 5,837,243). Such bispecific antibodies may be used to target two separate antigens. For example, anti-Fc receptor / antitumor antigen (e.g., Her-2 / neu) bispecific antibodies have been used to direct macrophages to tumor sites. This targeting may more effectively activate tumor-specific responses. The T cell arms of these responses are enhanced by A2aR inhibition. Alternatively, antigens may be delivered directly to DCs by using bispecific antibodies that bind to tumor antigens and dendritic cell-specific cell surface markers.

[0221] In all of the above methods, A2aR inhibition may be combined with other forms of immunotherapy, such as cytokine treatment (e.g., interferon, GM-CSF, G-CSF, IL-2), or bispecific antibody therapy using two different binding specificities, to provide enhanced presentation of tumor antigens.

[0222] In some embodiments, further therapeutic agents for use in any of the methods of treatment described above, for use of antigen-binding molecules, or for use in pharmaceutical compositions are immunostimulants selected from: (a) agents that block signaling of inhibitory receptors (immune checkpoints) or their ligands on immune cells (immune checkpoint inhibitors), or nucleic acids encoding such agents; (b) agonists against stimulating receptors on immune cells, or nucleic acids encoding such agonists; (c) cytokines, or nucleic acids encoding cytokines; (d) oncolytic viruses, or nucleic acids encoding oncolytic viruses; (e) T cells expressing chimeric antigen receptors; (f) bispecific or polyspecific antibodies against T cells, or nucleic acids encoding such antibodies; (g) anti-TGF-β antibodies, or nucleic acids encoding such antibodies; (h) TGF-β traps, or nucleic acids encoding such traps; (i) vaccines against cancer-associated antigens comprising such antigens, or nucleic acids encoding such antigens; (j) cell therapies; and (k) combinations thereof. In some embodiments, further therapeutic agents are agents that block the signaling of inhibitory receptors or their ligands on immune cells, or nucleic acids encoding such agents, the inhibitory receptors or their ligands being selected from PD-1, PD-L1, TIGIT, CTLA-4, PD-1, PD-L1, PD-L2, LAG-3, TIM-3, Neuritin, BTLA, CECAM-1, CECAM-5, IL-1R8, VISTA, LAIR1, LILRB1, LILRB2, LILRB3, LILRB4, LILRB5, CD96, CD112R, CD160, 2B4, TGFβ-R, KIR, NKG2A, and combinations thereof.In some embodiments, further therapeutic agents are agonists to immune cell stimulatory receptors, or nucleic acids encoding such agonists, where the immune cell stimulatory receptors are selected from OX40, CD2, CD27, CDS, ICAM-1, LFA-1 (CD1a / CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD28, CD30, CD40, BAFFR, HVEM, CD7, LIGHT, KG2C, SLAMF7, NKG2C, NKG2D, NKp46, NKp80, CD160, B7-H3, CD83 ligands, and combinations thereof. In some embodiments, further 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, further therapeutic agents are oncolytic viruses selected from herpes simplex virus, varicella stomatitis virus, adenovirus, Newcastle disease virus, vaccinia virus, maraba virus, and combinations thereof, or nucleic acids encoding oncolytic viruses. In some embodiments, further therapeutic agents are cell therapies. Cell therapies may include T cells, NK cells, or macrophages having chimeric antigen receptors (CARs). In some embodiments, cell therapies include bispecific or polyspecific antibodies against T cells.

[0223] In certain embodiments, the present invention provides a method for treating a disease or disorder, such as cancer, in a subject. The method comprises the step of administering an antigen-binding molecule of the present invention, such as an anti-A2aR antibody or its antigen-binding fragment, to a subject, either alone or in combination with a second or more further therapeutic agent, wherein the subject has previously been treated with a first or more further therapeutic agent. In certain embodiments, the treatment with the first or more further therapeutic agents may exhibit low efficacy in treating the disease in the subject. For example, the treatment with the first or more therapeutic agents may be treatment with an anti-PD1 antibody to which the subject may develop resistance. In some embodiments, the second or more further therapeutic agent is the same as the first or more further therapeutic agent. In some embodiments, the second or more further therapeutic agent is different from the first or more further therapeutic agent.

[0224] In certain embodiments, the immune checkpoint inhibitor is an antibody that specifically interacts with an immune checkpoint. In some embodiments, further therapeutic agents include an immunostimulant. In some embodiments, the immune checkpoint inhibitor is selected from anti-PD-1 antibodies (e.g., pembrolizumab or nivolumab), anti-PD-L antibodies (e.g., atezolizumab), anti-CTLA-4 antibodies (e.g., ipilimumab), and combinations thereof. In some embodiments, the immune checkpoint inhibitor is pembrolizumab. In some embodiments, the immune checkpoint inhibitor is nivolumab. In some embodiments, the immune checkpoint inhibitor is atezolizumab.

[0225] In some embodiments, the additional therapeutic agent is an agent that inhibits the interaction between PD-1 and PD-L1. In some aspects, the additional therapeutic agent that inhibits the interaction between PD-1 and PD-L1 is selected from antibodies, peptidomimetics, and small molecules. In some aspects, the additional therapeutic agent that inhibits the interaction between PD-1 and PD-L1 is selected from pembrolizumab, nivolumab, atezolizumab, avelumab, durvalumab, BMS-936559, sulfamethoxine 1, and sulfamethizole 2.

[0226] In some embodiments, the anti-A2aR antibody is administered in combination with, or together 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 a gene encoding an immunostimulatory cytokine, and the like.

[0227] 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 being treated (e.g., a tumor-bearing or virus-bearing subject), in order to enhance antigen-specific immunity. If the anti-A2aR antibody is administered together with another agent, the two may be administered separately or simultaneously.

[0228] In certain embodiments, the anti-A2aR antibodies described herein may be used to enhance an antigen-specific immune response by co-administration (e.g., vaccine) of one or more of these antibodies with an antigen of interest. Thus, in one embodiment, a method for enhancing an immune response to an antigen in a subject includes the steps of administering to a subject (i) an antigen; and (ii) an A2aR-based antibody, such that an immune response to the antigen is enhanced in the subject. The antigen may be, for example, a tumor antigen, a viral antigen, a bacterial antigen, or an antigen derived from a pathogen. Non-limiting examples of such antigens include those discussed in the above section, e.g., the tumor antigens (or tumor vaccines) discussed above, or antigens derived from the aforementioned viruses, bacteria, or other pathogens.

[0229] Considering the benefits associated with synergistic active agent compositions, in certain embodiments, each of the anti-A2aR antibody and the other active agent is administered to the target in need at a dose below the therapeutic dose compared to the dose used in monotherapy with the same agent.

[0230] In certain embodiments, A2aR inhibition is combined with standard cancer treatments (e.g., surgery, radiation, and chemotherapy). In these cases, it may be possible to reduce the dose of chemotherapy reagents administered. The combined use of A2aR inhibition with chemotherapy is thought to enhance apoptosis and increase tumor antigen presentation for cytotoxic immunity. Other synergistic combination therapies include A2aR inhibition combined with radiation, surgery, or hormone deficiency or inhibition. Each of these protocols creates a source of tumor antigens in the host.

[0231] Further therapeutic agents may be administered before, in conjunction with, or after the administration of the antigen-binding molecule of the present invention (for the purposes of this disclosure, such administration regimens are considered to be administration of the antigen-binding molecule "in combination with" further therapeutically active components).

[0232] The present invention includes a pharmaceutical composition in which the antigen-binding molecule of the present invention is co-formulated with one or more further therapeutic agents, as described elsewhere herein.

[0233] V. Nucleic acids and host cells for expressing anti-A2aR antibodies In another embodiment, the present invention provides an antigen-binding molecule of the present invention, for example, a nucleic acid encoding an anti-A2aR antibody or its antigen-binding fragment, and an expression vector comprising such nucleic acid. In some embodiments, the nucleic acid encodes an HCVR and / or LCVR fragment of an antibody or fragment according to the embodiments described herein, or any other antibody and antibody fragment described herein.

[0234] The DNA encoding the antigen-binding site in a monoclonal antibody can be isolated and sequenced from hybridoma cells using conventional procedures (e.g., by using oligonucleotide probes capable of specifically binding to the genes encoding the heavy and light chains of the monoclonal antibody). Alternatively, the amino acid sequence derived from the immunoglobulin of interest can be determined by direct protein sequencing, and the appropriate coding nucleotide sequence can be designed according to the universal codon table. In other cases, the nucleotide and amino acid sequences of the antigen-binding site, or other immunoglobulin sequences containing constant regions, hinge regions, etc., can be obtained from publicly available sources well known in the art.

[0235] Expression vectors may be used to synthesize antibodies of the present disclosure in vitro in cultured cells, or they may be administered directly to a patient to express antibodies of the present disclosure in vivo or ex vivo. As used herein, “expression vector” refers to a viral or nonviral vector containing polynucleotides encoding one or more antibodies of the present disclosure in a form suitable for expression from polynucleotides in host cells, for the purpose of antibody preparation or direct administration as a therapeutic agent.

[0236] Nucleic acid sequences are "operably ligated" to another nucleic acid sequence if the former is placed in a functional relationship with the latter. For example, DNA for a pre-sequence or signal peptide is operably ligated to DNA for a polypeptide if it is expressed as a preprotein involved in polypeptide secretion; a promoter or enhancer is operably ligated to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably ligated to a coding sequence if it occupies a position to facilitate translation. Generally, "operably ligated" means that the ligated DNA sequences are contiguous, and in the case of signal peptides, contiguous and in the reading phase. Enhancers, however, do not need to be contiguous. Ligation is achieved by ligation at a convenient restriction site. If such a site is not present, synthetic oligonucleotide adapters or linkers may be used according to conventional practice.

[0237] The nucleic acid sequences for expressing the antibodies of this disclosure typically include an N-terminal signal peptide sequence that is removed from the mature protein. Since the signal peptide sequence can affect the level of expression, a polynucleotide may encode any one of several different N-terminal signal peptide sequences. It will be understood by those skilled in the art that the design of the expression vector may depend on factors such as the selection of the host cells to be transformed and the desired level of protein expression.

[0238] The term "regulatory sequence" above refers to a DNA sequence required for the expression of an operablely linked coding sequence in one or more host organisms. The term "regulatory sequence" is intended to include promoters, enhancers, and other expression regulatory elements (e.g., polyadenylation signals). Regulatory sequences may include those that direct the constitutive expression of a nucleotide sequence in many types of host cells, or those that direct the expression of a nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). Expression vectors generally contain sequences for transcription termination and may further contain one or more elements that positively affect mRNA stability.

[0239] The expression vector contains one or more transcriptional regulatory elements, including a promoter and / or enhancer, for directing the expression of the antibody of this disclosure. The promoter contains a DNA sequence that functions to initiate transcription from a position fixed relative to the transcription start site. The promoter contains core elements required for the fundamental interaction between RNA polymerase and transcription factors and may work in conjunction with other upstream and response elements.

[0240] As used herein, the term “promoter” should be broadly interpreted to include a genomic gene-derived transcriptional regulatory element (TRE) or a chimeric TRE derived therefrom, which contains a TATA box or initiator element for precise transcription initiation, with or without further TREs (i.e., upstream activating sequences, transcription factor binding sites, enhancers, and silencers), which modulate the activation or repression of a gene operably linked to it in response to developmental and / or external stimuli, as well as trans-acting regulatory proteins or nucleic acids. A promoter may contain a genomic fragment or a chimera of one or more TREs combined together.

[0241] A preferred promoter is one capable of directing high-level expression in the target cells of interest. Promoter types may include constitutive promoters (e.g., HCMV, SV40, elongation factor-1α (EF-1α)) or promoters that indicate preferential expression in a specific cell type of interest. Enhancers generally refer to DNA sequences that function a distance from the transcription start site and can be either 5' or 3' relative to the transcription unit. Furthermore, enhancers can be located within coding sequences as well as introns. These are typically between 10 and 300 bp in length and function in cis. Enhancers function to increase and / or regulate transcription from nearby promoters. A preferred enhancer is one that directs high-level expression in antibody-producing cells. Cell or tissue-specific transcriptional regulatory elements (TREs) may be incorporated into expression vectors to restrict expression to a desired cell type. Pol III promoters (H1 or U6) are particularly useful for expressing shRNAs on which siRNA is expressed. Expression vectors may be designed to facilitate the expression of antibodies of this disclosure in one or more cell types.

[0242] siRNA is double-stranded RNA that can be engineered to induce sequence-specific post-transcriptional gene silencing of mRNA. Synthetically produced siRNA structurally mimics the type of siRNA normally processed in cells by the enzyme Dicer. When expressed from an expression vector, the expression vector is engineered to transcribe short double-stranded hairpin-like RNA (shRNA) that is processed into targeted siRNA inside the cell. Synthetic siRNA and shRNA can be designed using well-known algorithms and synthesized using conventional DNA / RNA synthesizers.

[0243] To co-express individual chains of the antibodies of this disclosure, appropriate splice donor and splice acceptor sequences may be incorporated to express both products. Alternatively, an internal ribosome-binding sequence (IRES) or a 2A peptide sequence may be used to express multiple products from a single promoter. An IRES provides a structure to which a ribosome can bind, which does not need to be located at the 5' end of the mRNA. Thus, an IRES can instruct a ribosome to initiate translation at a second start codon in the mRNA, allowing more than one polypeptide to be produced from a single mRNA. A 2A peptide contains a short sequence that mediates the co-translational autocleavage of upstream and downstream peptides from a 2A site, allowing the production of two different proteins in equimolar amounts from a single transcript. CHYSEL is a non-limiting example of a 2A peptide that causes a eukaryotic ribosome during translation to release a growing polypeptide chain being synthesized without dissociating from the mRNA. The ribosome continues translation, thereby producing a second polypeptide.

[0244] Expression vectors may include viral or non-viral vectors. Viral vectors may be derived from adeno-associated viruses (AAV), adenoviruses, herpesviruses, vaccinia viruses, polioviruses, poxviruses, retroviruses (including lentiviruses, e.g., HIV-1 and HIV-2), Sindbis and other RNA viruses, alphaviruses, astroviruses, coronaviruses, orthomyxoviruses, papovaviruses, paramyxoviruses, parvoviruses, picornaviruses, togaviruses, etc. Non-viral vectors are simply "naked" expression vectors that do not package viral components (e.g., capsid and / or envelope).

[0245] In certain cases, these vectors may be engineered to target a particular disease or cell population by using targeting properties inherent to the viral vector or engineered within the viral vector. Specific cells may be “targeted” for the delivery and expression of polynucleotides. Therefore, the term “targeting” in this case refers to the delivery of the capsid to specific cells. This may be based on the use of endogenous or heterologous binders in the form of envelope proteins or antibodies, the use of tissue-specific regulatory elements to restrict expression to a specific subset(s) of cells, or both.

[0246] In some embodiments, antibody chain expression is under the control of regulatory elements, such as tissue-specific or ubiquitous promoters. In some embodiments, ubiquitous promoters are used to control the expression of specific antibody heavy or light chains or single-chain derivatives thereof, such as the CMV promoter, the CMV-chicken beta-actin hybrid (CAG) promoter, or tissue-specific or tumor-specific promoters.

[0247] Nonviral expression vectors can be used for nonviral gene transfer by either direct injection of naked DNA or by encapsulating antibody-coding polynucleotides in liposomes, microparticles, microcapsules, virus-like particles, or erythrocyte ghosts. Such compositions may be further ligated to a targeting domain by chemical conjugation to facilitate targeted delivery and / or entry of nucleic acids into the desired cells of interest. Furthermore, plasmid vectors may be incubated with synthetic gene transfer molecules, such as polymeric DNA-binding cations like polylysine, protamine, and albumin, and ligated to cell-targeting ligands, such as asialorosomucoids, insulin, galactose, lactose, or transferrin.

[0248] Alternatively, naked DNA may be used. The uptake efficiency of naked DNA can be improved by compaction or by using biodegradable latex beads. Such delivery can be further improved by treating the beads to increase their hydrophobicity, thereby facilitating endosome breakdown and release of DNA into the cytoplasm.

[0249] VI. Methods for producing anti-A2aR antibodies In another embodiment, the present invention provides host cells transformed with nucleic acids or expression vectors encoding anti-A2aR HCVR and / or LCVR. The host cells are anti-A2aR This may be any bacterial or eukaryotic cell capable of expressing a nucleic acid or expression vector encoding HCVR and / or LCVR, or any other co-administered antibody or antagonist described herein.

[0250] In another embodiment, a method for producing an antibody according to the present disclosure includes the steps of culturing host cells transformed with one or an anti-A2aR HCVR and / or LCVR nucleic acid or expression vector under conditions that enable the production of an antibody or fragment, and purifying the antibody from the cells.

[0251] In further embodiments, the present invention provides a method for producing an antibody, comprising the steps of: culturing cells transiently or stably expressing one or more constructs encoding one or more polypeptide chains in an antibody; and purifying the antibody from the cultured cells. Any cells capable of producing a functional antibody may be used. In preferred embodiments, the antibody-expressing cells are of eukaryotic or mammalian origin, preferably human cells. Cells derived from various tissue cell types may be used to express the antibody. In other embodiments, the cells are yeast cells, insect cells, or bacterial cells. Preferably, the antibody-producing cells are stably transformed with a vector expressing the antibody.

[0252] One or more expression vectors encoding antibody heavy or light chains can be introduced into cells by any conventional method, for example, by naked DNA techniques, cationic lipid-mediated transfection, polymer-mediated transfection, peptide-mediated transfection, virus-mediated infection, physical or chemical agents or treatments, electroporation, etc. Furthermore, cells can be transfected with one or more expression vectors expressing antibodies along with selectable markers to facilitate the selection of stably transformed clones expressing the antibodies. Antibodies produced by such cells can be collected and / or purified according to techniques known in the art, for example, by centrifugation, chromatography, etc.

[0253] Examples of suitable selectable markers for mammalian cells include dihydrofolate reductase (DHFR), thymidine kinase, neomycin, neomycin analog G418, hygromycin, and puromycin. If such selectable markers are successfully transferred into mammalian host cells, the transformed mammalian host cells can survive under selective pressure. Two distinct categories of widely used selection regimens exist. The first category is based on the use of mutant cell lines lacking cellular metabolism and the ability to grow independently of supplemented medium. Two examples are CHO DHFR - These are cells and mouse LTV cells. These cells lack the ability to grow without the addition of nutrients such as thymidine or hypoxanthine. These cells lack certain genes necessary for the complete nucleotide synthesis pathway and therefore cannot survive unless the missing nucleotides are provided in a supplemented medium. An alternative to supplementing the medium is to introduce intact DHFR or TK genes into the cells lacking the respective genes, thereby altering their growth requirements. Individual cells that have not been transformed with either the DHFR or TK gene cannot survive in an unsupplemented medium.

[0254] The second category refers to selection schemes used in any cell type and does not require the use of mutant cell lines; these schemes typically involve using drugs to halt the growth of host cells. Cells with the novel gene express proteins that transmit drug resistance and survive the selection. Examples of such dominant selection use the drugs neomycin, mycophenolate, or hygromycin. Three examples use bacterial genes under eukaryotic control to transmit resistance to the appropriate drugs, G418 or neomycin (geneticin), xgpt (mycophenolate), or hygromycin, respectively. Other drugs include the neomycin analog G418 and puromycin.

[0255] Exemplary antibody-expressing cell lines include human Jurkat cells, human fetal kidney (HEK) 293 cells, Chinese hamster ovary (CHO) cells, mouse WEHI fibrosarcoma cells, and single-cell protozoan species, such as Leishmania tarentolae. Furthermore, stably transformed antibody-producing cell lines can be produced using primary cells immortalized with c-myc or other immortalizing agents.

[0256] In one embodiment, the cell line comprises a stably transformed Leishmania cell line, e.g., Leishmania tarentolae. Leishmania is known to provide a robust, rapidly growing single-cell host for high-level expression of eukaryotic proteins exhibiting mammalian glycosylation patterns. A commercially available Leishmania eukaryotic expression kit is available (Jena Bioscience GmbH, Jena, Germany).

[0257] 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 antibody per liter of culture.

[0258] The antibodies in this invention can be isolated from antibody-expressing cells after culturing and maintenance in any suitable culture medium, such as RPMI, DMEM, and AIM V®. The antibodies can be purified using conventional protein purification procedures (e.g., affinity purification, chromatography, etc.), which include the use of protein A or protein G immunoaffinity purification. In some embodiments, the antibodies are manipulated for secretion into the culture supernatant for isolation therefrom.

[0259] VII. Pharmaceutical Compositions and Administration Procedures In one embodiment, the pharmaceutical composition of the present invention comprises, in combination with a pharmaceutically acceptable carrier, an antigen-binding molecule described herein, for example, an A2aR antibody or its antigen-binding fragment(s). In other embodiments, the A2aR antibody or its antigen-binding fragment(s) is administered in combination with a pharmaceutically acceptable carrier. The anti-A2aR composition may comprise, as described herein, one or more different antibodies, one or more polyspecific antibodies, one or more fusion proteins, one or more immunoconjugates, or a combination thereof.

[0260] This invention provides a pharmaceutical composition comprising the antigen-binding molecule of the present invention. The pharmaceutical composition of the present invention is formulated using suitable carriers, excipients, and other agents that provide improved transfer, delivery, tolerability, etc. Numerous suitable formulations can be found in the prescription collection known to all pharmacists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. These formulations may include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic)-containing vesicles (e.g., LIPOFECTIN®, Life Technologies, Carlsbad, CA), DNA conjugates, anhydrous absorbent pastes, oil-in-water and water-in-oil emulsions, emulsion carbowaxes (polyethylene glycol of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowaxes. See also Powell et al. "Compendium of excipients for parenteral formulations" PDA (1998) J Pharm Sci Technol 52:238-311.

[0261] The dose of the antigen-binding molecule administered to a patient may vary depending on the patient's age and size, target disease, condition, and route of administration. Preferred doses are typically calculated according to body weight or body surface area. When the bispecific antigen-binding molecule of the present invention is used for therapeutic purposes in adult patients, it may be beneficial to administer it intravenously in a single dose of approximately 0.01 to 20 mg / kg body weight, more preferably 0.02 to 7, 0.03 to 5, or 0.05 to 3 mg / kg body weight. The frequency and duration of treatment may be adjusted depending on the severity of the condition. Effective doses and schedules for administering the bispecific antigen-binding molecule can be determined empirically; for example, patient progression may be monitored by periodic assessments and doses may be adjusted accordingly. Furthermore, interspecies scaling of doses may be carried out using methods well known in the art (e.g., Mordenti). et al., 1991, Pharmaceut. Res. 8:1351).

[0262] Various delivery systems are known, and for example, liposomes, microparticles, encapsulation in microcapsules, recombinant cells capable of expressing mutant viruses, and receptor-mediated endocytosis may be used to administer the pharmaceutical composition of the present invention (see, for example, Wu et al., 1987, J. Biol. Chem. 262:4429-4432). Methods of delivery include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The composition may be administered by any convenient route, for example, by injection or bolus injection, by absorption via the epithelium or inner layers of the skin mucosa (e.g., oral mucosa, rectal and intestinal mucosa), and may be administered together with other bioactive agents. Administration may be systemic or topical.

[0263] The pharmaceutical composition of the present invention can be delivered subcutaneously or intravenously using a standard needle and syringe. Furthermore, with respect to subcutaneous delivery, a pen-type delivery device is readily applicable to the delivery of the pharmaceutical composition of the present invention. Such a pen-type delivery device may be reusable or disposable. Reusable pen-type delivery devices generally utilize replaceable cartridges 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-type delivery device can then be reused. In disposable pen-type delivery devices, there are no replaceable cartridges. Rather, disposable pen-type delivery devices are pre-filled with the pharmaceutical composition held in a reservoir within the device. Once the pharmaceutical composition in the reservoir is empty, the entire device is discarded.

[0264] In certain circumstances, pharmaceutical compositions may be delivered using a controlled-release system. In one embodiment, a pump may be used (see Langer, above; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201). In another embodiment, a polymeric material may 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 may be placed in close proximity to the target of the composition, and therefore only a small portion of the systemic dose is required (see, for example, Goodson, 1984, Medical Applications of Controlled Release, above, vol. 2, pp. 115-138). Other controlled-release systems This is discussed in the review article by Langer, 1990, Science 249:1527-1533.

[0265] Injectable preparations may include dosage forms for intravenous, subcutaneous, intradermal, and intramuscular injection, as well as intravenous infusion. These injectable preparations may be prepared by known methods. For example, an injectable preparation may be prepared by dissolving, suspending, or emulsifying the antibody or a salt thereof in a sterile aqueous or oily medium conventionally used for injection. Aqueous mediums for injection may be used in combination with a suitable solubilizer, such as an isotonic solution containing physiological saline, glucose, and other adjuvants, e.g., saline, glucose, and other adjuvants. Oily mediums may be used in combination with a solubilizer, such as benzyl benzoate or benzyl alcohol, e.g., sesame oil or soybean oil. The injection solution thus prepared is preferably filled into a suitable ampoule.

[0266] Advantageously, the above-mentioned pharmaceutical compositions for oral or parenteral use are prepared into dosage forms in unit doses adapted to the dose of the active ingredient. Such dosage forms in unit doses include, for example, tablets, pills, capsules, injections (ampoules), and suppositories. The amount of the above-mentioned antibody contained is generally about 5 to about 500 mg per unit dose of dosage form; in particular, it is preferable that the above-mentioned antibody be contained in about 5 to about 100 mg in the form of an injection, and about 10 to about 250 mg for other dosage forms.

[0267] In another embodiment, a method for treating a disease state of impaired cell proliferation, such as cancer, a chronic infection, or an immunologically depressed state, comprises the step of administering to a subject in need of such treatment a pharmaceutical composition containing an anti-A2aR antibody or antigen-binding fragment described herein, in combination with a pharmaceutically acceptable carrier. In some embodiments, the method restores, enhances, or enhances lymphocyte activity in a subject in need of such restoration, enhancement, or enhancement. In certain preferred embodiments, the antibody or fragment is a human or humanized anti-A2aR antibody that reduces or inactivates A2aR-mediated signaling.

[0268] In some embodiments, administration of a pharmaceutical composition increases the activity of lymphocytes (e.g., T cells) in patients with diseases in which increased lymphocyte activity is beneficial, or diseases caused by or characterized by immunosuppression, immunosuppressive cells, or adenosine produced by (e.g., CD4 T cells, CD8 T cells, B cells). The methods described herein are particularly useful, for example, in patients with solid tumors in which the tumor microenvironment (and adenosine production therein) is suspected to contribute to a lack of recognition by the immune system (immune evasion). Tumors may be characterized, for example, by A2aR-expressing (or overexpressing) immune cells, such as CD4 T cells, CD8 T cells, T-regs, and B cells.

[0269] In certain embodiments, the methods and compositions are used for the treatment of various cancers and other proliferative disorders. These methods function to reduce adenosine levels that can inhibit the antitumor activity of lymphocytes, and are therefore applicable to a very wide range of cancers, particularly solid tumors in which adenosine in the tumor microenvironment is known to suppress the antitumor immune response.

[0270] The antigen-binding molecule of the present invention, for example, an anti-A2aR antibody or its antigen-binding fragment, is not limited to cancers for treatment, including, 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 lymphoma, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal gland cancer, soft tissue sarcoma, urethral cancer, penile cancer, pediatric solid tumors, lymphocytic lymphoma, bladder cancer, kidney or ureteral cancer, renal pelvis cancer, central nervous system (CNS) neoplasms, progenitor cancers This disclosure includes environmentally induced cancers, such as asbestos-induced cancers, including primary CNS lymphoma, tumor angiogenesis, spinal axial tumors, brainstem gliomas, pituitary adenomas, Kaposi's sarcoma, epidermal carcinoma, squamous cell carcinoma, and hematological malignancies, such as multiple myeloma, B-cell lymphoma, Hodgkin lymphoma / mediastinal primary B-cell lymphoma, non-Hodgkin lymphoma, acute myeloid lymphoma, chronic myeloid leukemia, chronic lymphocytic leukemia, follicular lymphoma, diffuse large B-cell lymphoma, Burkitt lymphoma, immunoblastic large B-cell lymphoma, progenitor B-lymphoblastic lymphoma, mantle cell lymphoma, acute lymphoblastic leukemia, mycosis fungoides, anaplastic large cell lymphoma, T-cell lymphoma and progenitor T-lymphoblastic lymphoma, or any combination of these cancers. This 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 the procedure.

[0271] In one embodiment, the anti-A2aR antibody neutralizes the enzymatic activity of A2aR at least EC 50 EC as needed 70 , effectively EC as needed 100 The blood concentration of is administered in an amount effective to achieve and / or maintain in the individual (e.g., over 1, 2, 3, or 4 weeks and / or until subsequent administration of the antigen-binding compound). In one embodiment, the activity of the anti-A2aR antibody is such that, for neutralization of the enzymatic activity of A2aR, the EC 50 EC as needed 70 , effectively EC as needed 100 This is an effective amount to achieve in the extravascular tissue of an individual. In one embodiment, the activity level of the anti-A2aR antibody is for inhibition of neutralizing the enzymatic activity of A2aR. A2aR activity, EC 50 EC as needed 70 , effectively EC as needed 100 This is an effective amount for achieving (or maintaining) this in an individual.

[0272] In one embodiment, as needed, an anti-A2aR antibody is primarily an inhibitor (having virtually no Fcγ receptor-mediated activity) and is administered in an amount effective enough to neutralize the enzymatic activity of A2aR over a desired period, for example, one week, two weeks, or one month, until the next consecutive dose of the anti-A2aR antibody.

[0273] In one embodiment, the anti-A2aR antibody inhibits the A2aR-mediated catabolism of AMP to adenosine, at least EC 50 EC as needed 70 , effectively EC as needed 100The blood concentration of is administered in an amount effective to achieve and / or maintain in the individual (e.g., over 1, 2, 3, or 4 weeks and / or until subsequent administration of anti-A2aR antibody). In one embodiment, the amount of anti-A2aR antibody is such that it inhibits the A2aR-mediated catabolism of AMP to adenosine. 50 EC as needed 70 , effectively EC as needed 100 This is an effective amount for achieving (or maintaining) this in the extravascular tissues of an individual.

[0274] In one embodiment, a method is provided for treating or preventing cancer in an individual, comprising the step of administering to an individual having the disease an amount of anti-A2aR antibody that achieves or maintains a concentration higher than the concentration required for 50%, 70%, or complete (e.g., 90%) receptor-saturated A2aR-expressing cells (e.g., as assessed in PBMCs) in circulation, or as necessary, in extravascular tissue of interest (e.g., tumor or tumor environment), for a specified period of time. If necessary, the concentration achieved is at least 20%, 50%, or 100% higher than the concentration required for the specified receptor saturation.

[0275] In one embodiment, a method for treating or preventing cancer in an individual, wherein, regarding binding to A2aR-expressing cells, in circulation, and as necessary, in the extravascular tissue of interest (e.g., tumor or tumor environment), EC 50 EC as needed 70 Or, if necessary, e-commerce 100 A method is provided comprising the step of administering to an individual an amount of anti-A2aR antibody that achieves or maintains a higher concentration than specified over a specified period. If necessary, the concentration achieved is EC for binding to A2aR-expressing cells. 50 EC as needed 70 Or, if necessary, e-commerce 100 It is at least 20%, 50%, or 100% higher than [the other value].

[0276] In any embodiment, the antibody is used for binding to A2aR-expressing cells in human PBMCs at concentrations between 0.5 and 100 ng / ml, optionally between 1 and 100 ng / ml, optionally between 30 and 100 ng / ml, for example, at an EC of approximately 30 to 90 ng / ml. 50 EC as needed 70 Or, if necessary, e-commerce 100 It may have. For example, EC 50 The values ​​can be approximately 30, 37, 39, 43, 57, 58, 61, 62, 90, 95, and 143 ng / ml.

[0277] EC on the neutralization of A2aR enzymatic activity by anti-A2aR antibodies 50 For example, this could be between approximately 0.01 μg / ml and 1 μg / ml, between 0.1 μg / ml and 10 μg / ml as needed, or between 0.1 μg / ml and 1 μg / ml as needed. For example, EC 50 The amount may be approximately 0.1 μg / ml, approximately 0.2 μg / ml, or approximately 0.3 μg / ml. Therefore, the amount of this anti-A2aR antibody is, for example, 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 (so at It is administered to the patient.

[0278] When tissues outside vascular structures (e.g., the tumor environment in the treatment of solid tumors) are targeted, typically a dose approximately 10 times higher is considered necessary compared to the dose that provides the corresponding concentration in circulation. The amount of anti-A2aR antibody administered to achieve (and / or maintain) circulating (blood) concentrations of approximately 1 μg / ml, 2 μg / ml, 10 μg / ml, or 20 μg / ml is expected to achieve (and / or maintain) extravascular tissue (e.g., tumor tissue) concentrations of approximately 0.1 μg / ml, 0.2 μg / ml, 1 μg / ml, and 2 μg / ml, respectively.

[0279] In one embodiment, the anti-A2aR antibody is administered in an amount to achieve and / or maintain a tissue (e.g., tumor environment) concentration of, for example, at least 0.1 μg / ml, optionally at least 0.2 μg / ml, optionally at least 1 μg / ml, or optionally at least 2 μg / ml. The antibody may be administered in an amount to achieve and / or maintain blood concentrations between, for example, 1-100 μg / ml, 2 μg / ml, 10 μg / ml, or 20 μg / ml, e.g., between 1-100 μg / ml, 10-100 μg / ml, 1-50 μg / ml, 1-20 μg / ml, or 1-10 μg / ml. The amount administered may be adjusted to provide maintenance of the desired concentration over a specified period after administration (e.g., 1, 2, 3, 4 weeks, etc.).

[0280] In some embodiments, the amount of anti-A2aR antibody is such that it has at least one EC (Energy Concentration) to neutralize the enzymatic activity of A2aR in blood (serum) or extravascular tissue (e.g., tumor environment). 70 or EC 100 The antibody is administered to obtain the corresponding concentration. The antibody may be administered in amounts to achieve and / or maintain blood concentration or extravascular tissue (e.g., tumor environment) of, for example, at least about 1 μg / ml, 2 μg / ml, 10 μg / ml, or 20 μg / ml.

[0281] EC for a given A2aR antibody 50 , EC 70 and EC 100 The value can be evaluated in a cell assay, for example, regarding the neutralization of A2aR enzyme activity. 50 "EC" refers to the concentration of anti-A2aR antibody that produces 50% of the maximum response or effect in neutralizing the enzyme activity of A2aR. 70 " refers to the concentration of anti-A2aR antibody that produces 70% of the maximum response or effect in neutralizing the enzymatic activity of A2aR. 100" refers to the efficient concentration of anti-A2aR antibody that produces its maximum response or effect with respect to the neutralization of the enzymatic activity of A2aR. In certain embodiments, depending on the context, EC 50 , EC 70 or EC 100 IC, respectively, reflects the fact that antigen-binding molecules, such as anti-A2aR antibodies or their antigen-binding fragments, inhibit the activity of A2aR. 50 ,I C 70 or IC 100 It can be called IC. xx This refers to the concentration of a drug required to inhibit a biological process by xx%.

[0282] In some embodiments, particularly for the treatment of solid tumors, the concentration achieved is at least EC for neutralizing enzyme activity in the tissue (outside the vascular structure, e.g., in the tumor or tumor environment). 50 To accommodate, approximately EC as needed. 100 or at least about EC 100 It is designed to produce the corresponding concentration.

[0283] In one embodiment, the dose of anti-A2aR antibody is between 1 and 20 mg / kg body weight. In one embodiment, the dose is administered to the individual weekly, every two weeks, monthly, or every two months.

[0284] In one embodiment, a method for treating cancer in a subject requiring treatment of cancer comprises the step of administering an effective dose of the anti-A2aR antibody of the Disclosure to an individual over at least one administration cycle (or, if necessary, at least two, three, four or more administration cycles), the cycle being for a period of eight weeks or less, and for each of at least one cycle, one, two, three, or four doses of the anti-A2aR antibody being administered at a dose of 1 to 20 mg / kg body weight. In one embodiment, the anti-A2aR antibody is administered by intravenous infusion.

[0285] For example, a suitable treatment protocol for treating a human subject includes, for example, the step of administering to a patient an amount of anti-A2aR antibody disclosed herein, the method comprising at least one administration cycle in which at least one dose of anti-A2aR antibody is administered. If necessary, at least two, three, four, five, six, seven, or eight doses of anti-A2aR antibody are administered. In one embodiment, the administration cycle is between two and eight weeks.

[0286] In one embodiment, a method for treating or preventing a disease (e.g., cancer, solid tumor, hematological malignancy) in an individual comprises the step of administering to the individual an anti-A2aR antibody that neutralizes the enzymatic activity of A2aR over at least one administration cycle, the administration cycle comprising at least first and second (and optionally third, fourth, fifth, sixth, seventh and / or eighth or further) administrations of the anti-A2aR antibody, the amount administered being effective in achieving or maintaining between two consecutive administrations a blood (serum) concentration of the anti-A2aR antibody in the range of at least 0.1 μg / ml, at least 0.2 μg / ml, at least 1 μg / ml, at least 2 μg / ml, at least 10 μg / ml, at least 20 μg / ml, between 1 and 100 μg / ml, between 1 and 50 μg / ml, between 1 and 20 μg / ml, between 1 and 10 μg / ml, or any of the concentrations described above.

[0287] In one embodiment, a specified, continuous blood concentration is maintained, and the blood concentration does not substantially fall below the specified blood concentration over the duration of a specified period (e.g., between two doses of the antibody, weeks, 1 week, 2 weeks, 3 weeks, 4 weeks). In other words, the blood concentration may fluctuate during the specified period, but the maintained specified blood concentration represents the minimum or "trough" concentration.

[0288] In one embodiment, a therapeutically active amount of anti-A2aR antibody is present in the blood and / or tissues for a period of at least about 1 week, about 2 weeks, or about 1 month after administration of the antibody, with respect to (at least) EC for neutralizing the enzymatic activity of A2aR. 50Element, EC as needed 70 Element, EC as needed 100 This is the amount of such antibody that can provide a certain concentration.

[0289] Prior to or during treatment with the anti-A2aR antibody of this disclosure, the expression levels of A2aR, CD39, and / or CD73 in cells within and / or adjacent to the patient's tumor; the percentage of A2aR-expressing cells, CD39-expressing cells, and / or CD73-expressing cells; and / or levels of adenosine, ADP, and / or AMP may be evaluated to assess whether the patient is suitable for treatment and is likely to respond to treatment. Increased levels or expressions as described above may indicate that the individual is suitable for treatment with the anti-A2aR antibody of this disclosure (e.g., likely to benefit from the anti-A2aR antibody of this disclosure).

[0290] In some embodiments, evaluating the expression levels of A2aR, CD39, and / or CD73, as well as the concentrations of adenosine, ADP, and / or AMP, in and / or adjacent to a tissue sample of a patient's tumor includes the step of obtaining from a subject a biological sample of human tissue selected from the group consisting of cancer tissue, tissue proximal to or periphery of the tumor, adjacent tissue, adjacent non-tumorous tissue, or adjacent normal tissue, as well as the expression levels of A2aR, CD39, and / or CD73, and the concentrations of adenosine, ADP, and / or AMP in the tissue. The expression levels or nucleotide concentrations from the patient may be compared to, for example, a reference level corresponding to a healthy individual.

[0291] A decrease in the levels of adenosine, ADP, and / or AMP after administration (or administration of the antibody) compared to the levels before treatment (or administration of the antibody) may indicate that the individual is benefiting from treatment with the anti-A2aR antibody of this disclosure (including, but not limited to, an antibody that inhibits substrate-bound A2aR). If the patient is benefiting from treatment with the anti-A2aR antibody, the method may further include the step of administering a further dose of the anti-A2aR antibody to the patient, either alone or in combination with another active agent (e.g., the step of continuing the treatment).

[0292] In consideration of the foregoing, in a particular embodiment, the method comprises (a) determining the expression levels of A2aR, CD39, and / or CD73, and / or the concentrations of adenosine, ADP, and / or AMP in the tumor environment, and optionally in the tumor and / or adjacent tissues, and (b) administering an anti-A2aR antibody to the individual when it is determined that the tumor environment shows increased levels of A2aR, CD39, CD73, adenosine, ADP, and / or AMP compared to their corresponding reference levels.

[0293] In certain embodiments, determining the levels of A2aR, CD39, CD73, adenosine, ADP, and / or AMP in the tumor environment includes the steps of obtaining a biological sample from a subject containing tissue proximal to or periphery of cancer tissue and / or cancer tissue (e.g., adjacent cancer tissue, adjacent non-tumorous tissue, or adjacent normal tissue), and detecting the levels and / or relative percentages of A2aR-expressing cells, CD39-expressing cells, and / or CD73-expressing cells, and / or the levels of adenosine, ADP, and / or AMP. A2aR-expressing cells, CD39-expressing cells, and / or CD73-expressing cells may include, for example, tumor cells, CD4 T cells, CD8 T cells, B cells, and combinations thereof. The expression levels of A2aR, CD39, and CD73 can be determined by evaluating their mRNA expression (e.g., by RT-PCR) or polypeptide expression (e.g., by Western blotting, immunofluorescence staining) using techniques well known to those skilled in the art, compared to a reference level corresponding to a healthy subject or to a pre-treatment reference level.

[0294] Subjects with cancer have a tumor microenvironment (e.g., tumor cells, CD4 T cells, CD8 T cells and B cells may be treated with an anti-A2aR antibody, with or without a step to assess A2aR, CD39, CD73, adenosine, ADP, and / or AMP levels.

[0295] The determination that a biological sample contains cells that overexpress A2aR, CD39, and / or CD73, and / or contain high concentrations of adenosine, ADP, and / or AMP, compared to a reference, indicates that the subject has cancer that may benefit from treatment with an A2aR inhibitor. In some embodiments, the term “overexpressed” is used with respect to A2aR, CD39, and / or CD73 polypeptides expressed in a significant number of cells taken from a given patient, for example, in at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or more of tumor cells or lymphocytes taken from the subject.

[0296] In one embodiment, a method for treating or preventing cancer in a subject requiring treatment or prevention of cancer comprises (a) detecting the percentage and / or degree of expression of cells corresponding to A2aR, CD39, and / or CD73 in the tumor environment, and optionally in the tumor and / or adjacent tissue, and (b) administering an anti-A2aR antibody to the subject when it is determined that the tumor environment contains cells overexpressing A2aR, CD39, and / or CD73 at, optionally, increased levels compared to appropriate reference levels. In one embodiment, the cells are tumor cells. In another embodiment, the cells in the tumor environment, tumor, and / or adjacent tissue are non-malignant immune cells, e.g., T cells.

[0297] In some embodiments, determining the degree of A2aR, CD39, and / or CD73 expression within the tumor environment includes the steps of obtaining a biological sample from an individual containing tissue proximal to or periphery of cancer tissue and / or cancer tissue (e.g., adjacent cancer tissue, adjacent non-tumorous tissue, or adjacent normal tissue), contacting cells with antibodies that bind to the A2aR polypeptide, CD39 polypeptide, and / or CD73 polypeptide, and detecting the percentage of cells and / or the degree of expression corresponding to A2aR, CD39, and / or CD73. In certain embodiments, the expression of A2aR, CD39, and / or CD73 is assessed by their cell surface expression using an immunohistochemical assay.

[0298] Antibody compositions may be used as monotherapy or in combination with one or more other therapeutic agents, including drugs typically used for the specific therapeutic purpose in which the antibody is administered. See "Combination Therapy" above. Further therapeutic agents are usually administered in the amounts and treatment regimens typically used for that agent in monotherapy for the specific disease or condition being treated. Such therapeutic agents include, but are not limited to, anticancer agents and chemotherapeutic agents.

[0299] As described above, a method for using the pharmaceutical compositions described herein includes the step of administering an effective amount of the pharmaceutical composition according to this disclosure to a subject requiring administration.

[0300] Any appropriate route or mode of administration may be used to deliver a therapeutic or prophylactic effective dose of the antibody to the patient. Exemplary routes or modes of administration include parenteral (e.g., intravenous, intra-arterial, intramuscular, subcutaneous, intratumoral), oral, topical (nasal, percutaneous, intradermal, or intraocular), mucosal (e.g., nasal, sublingual, buccal, rectal, vaginal), inhalation, intralymphatic, intraspinal, intracranial, intraperitoneal, intratracheal, intravesical, intrabladderal, intrathecal, intraintestinal, intrapulmonary, intralymphatic, intracavitary, intraorbital, intracapsular, and transurethral, ​​as well as local delivery by catheter or stent.

[0301] A pharmaceutical composition comprising an anti-A2aR antibody according to this disclosure may be formulated in any pharmaceutically acceptable carrier(s) or excipient(s). As used herein, the term “pharmaceutically acceptable carrier” includes any and all physiologically compatible solvents, dispersions, coatings, antibacterial and antifungal agents, isotonic agents and absorption retarders, etc. The pharmaceutical composition may include a suitable solid or gel phase carrier or excipient. Exemplary carriers or excipients include, but are not limited to, calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers, such as polyethylene glycol. Exemplary pharmaceutically acceptable carriers include one or more of water, saline, phosphate-buffered saline, dextrose, glycerol, ethanol, etc., and combinations thereof. In many cases, it is preferable to include an isotonic agent in the composition, such as sugars, polyalcohols, such as mannitol, sorbitol, or sodium chloride. A pharmaceutically acceptable carrier may further contain trace amounts of auxiliary substances that enhance the duration or efficacy of the therapeutic agent, such as wetting agents or emulsifiers, preservatives, or buffers.

[0302] In certain preferred embodiments, a therapeutically active agent may be incorporated into a pharmaceutical composition suitable for parenteral administration. The pharmaceutical composition for parenteral administration may be formulated by injection, for example, by bolus injection or continuous infusion.

[0303] Suitable buffers include, but are not limited to, sodium succinate, sodium citrate, sodium phosphate, or potassium phosphate. Sodium chloride may be used to modify the toxicity of the solution at concentrations of 0–300 mM (optimally 150 mM for liquid dosage forms). For lyophilized dosage forms, cryoprotectants may basically consist of 0–10% sucrose (optimally 0.5–1.0%). Other suitable cryoprotectants include trehalose and lactose. For lyophilized dosage forms, expanders may basically consist of 1–10% mannitol (optimally 2–4%). For both liquid and lyophilized dosage forms, stabilizers may basically consist of 1–50 mM L-methionine (optimally 5–10 mM). Other suitable expanders include glycine and arginine, which may be included as 0–0.05% polysorbate-80 (optimally 0.005–0.01%). Further surfactants include, but are not limited to, polysorbate 20 and BRIJ® surfactants.

[0304] The therapeutic preparation is freeze-dried and preferably stored as a sterile powder under vacuum, and then, before injection, is treated with bacteriostatic water (e.g., benzyl alcohol preservative). It can be reconstituted in (containing) or sterile water. The therapeutic agent in the pharmaceutical composition may be formulated in a “therapeutic effective dose” or a “preventive effective dose.” A “therapeutic effective dose” refers to the dose required to achieve the desired therapeutic outcome and the amount that is effective over such a period of time. The therapeutic effective dose of an antibody or active agent may vary depending on the condition being treated, the severity and course of the condition, the mode of administration, whether the antibody or agent is administered for preventive or therapeutic purposes, the bioavailability of the particular agent(s), the ability of the antibody to elicit the desired response in the individual, previous treatments, the patient’s age, weight and sex, the patient’s clinical history and response to the antibody, the type of antibody used, and the discretion of the attending physician. The therapeutic effective dose is also the amount in which the therapeutically beneficial effect outweighs any toxicity or adverse effects of the recombinant vector. A “preventive effective dose” refers to the dose required to achieve the desired preventive effect and the amount that is effective over such a period of time.

[0305] Preferably, the polypeptide domains utilized in the antibodies or other active agents described herein are derived from the same host from which they are administered in order to reduce the inflammatory response to the administered therapeutic agent. As suggested above, the therapeutic agent(s) may be administered to the subject appropriately, either as a single treatment or over a series of treatments, and may be administered to the patient at any time after diagnosis. The A2aR antibody may be administered as a sole treatment or in combination with other active agents or treatments useful in treating the condition in question.

[0306] As a general suggestion, the therapeutic or prophylactic effective dose of A2aR antibody (or other active agent) is administered in the range of approximately 1 ng / kg body weight / day to approximately 100 mg / kg body weight / day, whether by a single dose or multiple doses.In a particular embodiment, each A2aR antibody or active agent is administered in doses of approximately 1 ng / kg body weight / day to approximately 10 mg / kg body weight / day, approximately 1 ng / kg body weight / day to approximately 1 mg / kg body weight / day, approximately 1 ng / kg body weight / day to approximately 100 μg / kg body weight / day, approximately 1 ng / kg body weight / day to approximately 10 μg / kg body weight / day, approximately 1 ng / kg body weight / day to approximately 1 μg / kg body weight / day, approximately 1 ng / kg body weight / day to approximately 100 ng / kg body weight / day, approximately 10 ng / kg body weight / day to approximately 100 mg / kg body weight / day, and approximately 10 ng / kg Body weight / day ~ approx. 10 mg / kg body weight / day, approx. 10 ng / kg body weight / day ~ approx. 1 mg / kg body weight / day, approx. 10 ng / kg body weight / day ~ approx. 100 μg / kg body weight / day, approx. 10 ng / kg body weight / day ~ approx. 10 μg / kg body weight / day, approx. 10 ng / kg body weight / day ~ approx. 1 μg / kg body weight / day, 10ng / kg body weight / day ~ approx. 100ng / kg body weight / day, approx. 100ng / kg body weight / day ~ approx. 100 mg / kg body weight / day, approx. 100ng / kg body weight / day ~ approx. 10 mg / kg body weight / day, approx. g body weight / day ~ approx. 100 μg / kg body weight / day, approx. 100 ng / kg body weight / day ~ approx. 10 μg / kg body weight / day, approx. 100 ng / kg body weight / day ~ approx. 1 μg / kg body weight / day, approx. 1 μg / kg body weight / day ~ approx. 100 mg / kg body weight / day, approx. 1 μg / kg body weight / day ~ approx. 10 mg / k g body weight / day, approximately 1 μg / kg body weight / day to approximately 1 mg / kg body weight / day, approximately 1 μg / kg body weight / day to approximately 100 μg / kg body weight / day, approximately 1 μg / kg body weight / day to approximately 10 μg / kg body weight / day, approximately 10 μg / kg body weight / day to approximately 100 mg / kg body weight / day, approximately 10 μg / kg body weight The dosage is administered in the following ranges: approximately 10 mg / kg body weight / day, approximately 10 μg / kg body weight / day to approximately 1 mg / kg body weight / day, approximately 10 μg / kg body weight / day to approximately 100 μg / kg body weight / day to approximately 100 mg / kg body weight / day, approximately 100 μg / kg body weight / day to approximately 10 mg / kg body weight / day, approximately 100 μg / kg body weight / day to approximately 1 mg / kg body weight / day to approximately 1 mg / kg body weight / day to approximately 100 mg / kg body weight / day, and approximately 10 mg / kg body weight / day to approximately 100 mg / kg body weight / day.

[0307] In other embodiments, the A2aR antibody and / or active agent is administered at doses of 500 μg to 20 g every three days, or at 25 mg / kg body weight every three days.

[0308] In other embodiments, each A2aR antibody and / or active agent is administered in doses of approximately 10 ng to 100 ng, approximately 10 ng to 1 μg, approximately 10 ng to 10 μg, approximately 10 ng to 100 μg, approximately 10 ng to 1 mg, approximately 10 ng to 10 mg, approximately 10 ng to 100 mg, approximately 10 ng to 1000 mg per injection, approximately 10 ng to 10,000 mg per injection, approximately 100 ng to 1 μg, and approximately 100 ng per injection. g to approximately 10 μg, approximately 100 ng to approximately 100 μg per dose, approximately 100 ng to approximately 1 mg per dose, approximately 100 ng to approximately 10 mg per dose, approximately 100 ng to approximately 100 mg per dose, approximately 100 ng to approximately 1000 mg per injection, approximately 100 ng to approximately 10,000 mg per dose, approximately 1 μg to approximately 10 μg per dose, approximately 1 μg to approximately 100 μg per dose, approximately 1 μg to approximately 1 mg per dose, approximately 1 μg to approximately 10 mg per dose, approximately 1 μg to approximately 100 mg per dose, approximately 1 μg to approximately 1 μg per injection 1000mg, approximately 1μg to approximately 10,000mg per dose, approximately 10μg to approximately 100μg per dose, approximately 10μg to approximately 1mg per dose, approximately 10μg to approximately 10mg per dose, approximately 10μg to approximately 100mg per dose, approximately 10μg to approximately 1000mg per injection, approximately 10μg to approximately 10,000mg per dose, approximately 100μg to approximately 1mg per dose, approximately 100μg to approximately 10mg per dose, approximately 100μg to approximately 100mg per injection, per dose It is administered in the range of approximately 100 μg to approximately 10,000 mg, approximately 1 mg to approximately 10 mg per individual dose, approximately 1 mg to approximately 100 mg per individual dose, approximately 1 mg to approximately 1000 mg per injection, approximately 1 mg to approximately 10,000 mg per individual dose, approximately 10 mg to approximately 100 mg per individual dose, approximately 10 mg to approximately 10,000 mg per injection, approximately 10 mg to approximately 10,000 mg per individual dose, approximately 100 mg to approximately 10,000 mg per injection, approximately 100 mg to approximately 10,000 mg per individual dose, and approximately 1000 mg to approximately 10,000 mg per individual dose.The antibodies described herein may be administered daily, every 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, or 4 weeks.

[0309] In other specific embodiments, the amount of each A2aR antibody or active agent may be administered in doses of approximately 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.

[0310] In certain embodiments, the coding sequence of an A2aR antibody and / or other active agent(s) 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 requiring treatment according to the above method. For example, in certain embodiments involving the administration of one or more recombinant AAV (rAAV) viruses, the pharmaceutical composition is at least 10 per kg 10 , at least 10 11 , at least 10 12 , at least 10 13 Or at least 10 14 The rAAV may be contained in an amount comprising genome copies (GC) or recombinant virus particles, or any range thereof. In certain embodiments, the pharmaceutical composition contains at least 10 per subject. 10 , at least 10 11 , at least 10 12 , at least 10 13 , at least 10 14 , at least 10 15 The amount includes an effective amount of recombinant virus, such as rAAV, in the form of a genome copy or a recombinant viral particle genome copy, or any range thereof.

[0311] The dosage may be tested in one or more accepted animal models in the field, appropriate for any specific disease state of cytoproliferative disorder or immunosuppression.

[0312] Delivery methodologies may also include the use of polycationic condensed DNA linked or unlinked to a killing virus, ligand-linked DNA, liposomes, eukaryotic cell delivery vehicle cells, deposition of photopolymerized hydrogel materials, use of portable gene transfer particle guns, ionizing radiation, nuclear charge neutralization or fusion with cell membranes, and particle-mediated gene transfer.

[0313] VIII. Diagnostic Use of Antibodies The antigen-binding molecules of the present invention, for example, antibodies or their antigen-binding fragments, may also be used, for example, to detect and / or measure human or cynomolgus monkey A2aR, or human or cynomolgus monkey A2aR-expressing cells, in a sample for diagnostic purposes. For example, an anti-A2aR antibody or its antigen-binding fragment may be used to diagnose a condition or disease characterized by abnormal expression of A2aR (e.g., overexpression, underexpression, absence of expression, etc.). For example, an exemplary diagnostic assay for A2aR involves contacting a sample obtained from a patient with the antibody of the present invention, where the antibody is labeled with a detectable label or reporter molecule. Alternatively, an unlabeled antibody may be used in a diagnostic application in combination with a secondary antibody that is itself detectably labeled. The detectable label or reporter molecule may be a radioisotope, for example, 3 H, 14 C, 18 F, 32 P, 35 S or 125I; a fluorescent or chemiluminescent moiety, e.g., fluorescein isothiocyanate or rhodamine; or an enzyme, e.g., alkaline phosphatase, beta-galactosidase, horseradish peroxidase, or luciferase. Specific exemplary assays that may 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 may be used in an A2aR diagnostic assay according to the present invention include any tissue or body fluid sample containing a detectable amount of A2aR protein or fragments, obtainable from a patient under normal or pathological conditions. Generally, the level of A2aR in a specific sample obtained from a healthy patient (e.g., a patient without any disease or condition associated with abnormal A2aR levels or activity) is measured to first establish a baseline or standard level of A2aR. This baseline level of A2aR can then be compared to the level of A2aR measured in a sample obtained from an individual suspected of having an A2aR-related disease or condition.

[0314] Furthermore, the anti-A2aR antibodies described herein may be used to purify human A2aR via immunoaffinity purification.

[0315] IX. Kit Any of the compositions described herein, for example, the anti-A2aR antigen-binding molecules of the present invention, and / or further therapeutic agents, may be included in the kit. In non-limiting examples, the kit includes an antigen-binding molecule, for example, an antibody or an antigen-binding fragment thereof. In certain embodiments, the kit further includes further therapeutic agents described herein.

[0316] The kit may further contain reagents or instructions for treating a disease or disorder. The kit may also contain one or more buffers.

[0317] The components of the kit may be packaged either in an aqueous medium or in a lyophilized form. The kit's container means generally include at least one vial, test tube, flask, bottle, syringe or other container means in which the components are placed, preferably appropriately aliquoted. If there are more than one component in the kit (labeled reagents and labels may be packaged together), the kit also generally includes a second, third or further container in which the further components may be placed separately. The kit may also include a second container means for containing sterile, pharmaceutically acceptable buffers and / or other diluents. However, various combinations of components may be contained in vials. The kit of the present invention also typically includes means for containing the composition of the present invention, e.g., an anti-A2aR antigen-binding molecule and / or further therapeutic agents, as well as any other reagent containers tightly sealed for commercial sale.

[0318] If the components of the kit are provided in one and / or more liquid solutions, the liquid solutions are aqueous solutions, and sterile aqueous solutions are particularly preferred. However, the components of the kit may be provided as dry powders. If the reagents and / or components are provided as dry powders, the powders may be reconstituted by the addition of a suitable solvent. The solvent may be provided in a separate container.

[0319] The present invention is further illustrated by the following embodiments, which should not be construed as limiting. All references, patents and published patent applications, as well as figures and tables, cited throughout this application are incorporated herein by reference. [Examples]

[0320] (Example 1) Generation of anti-A2aR monoclonal antibodies Mouse immunity Six- to eight-week-old C57BL / 6 mice were immunized by injection of a plasmid encoding human A2aR (human A2aR: SEQ ID NO: 50) under an IACUC-approved protocol. Briefly, the human A2aR gene insert was cloned into a modified expression vector. The DNA plasmid was then produced from Escherichia coli (HB101 strain) using the Mega Purification Kit (Qiagen, Cat. 12981). Six- to eight-week-old C57 / B6 mice (Taconic Farms) were each immunized with multiple rounds of human A2aR-coding plasmids delivered either by a gene gun (Bio-rad) system or by intradermal (ID) injection followed by electroporation (BTX-Harvard Apparatus). Serum samples were collected before the first immunization and 7 days after the last immunization.

[0321] Serum titration was performed using fluorescence-activated cell sorting (FACS) according to standard procedures. Human A2aR-expressing Expi293 cells were initially added to a 96-well plate (1 × 10⁶ cells per well). 5 Cells were incubated with 50 μl of serially diluted serum (1:50, 1:150, 1:450, 1:1350, 1:4053, 1:12150) derived from each individual mouse for 30 minutes on ice. After washing with FACS buffer (2% fetal bovine serum in PBS, 2 mM EDTA), Alex Fluor 647 conjugate anti-mouse IgG was added and incubated on ice for 20 minutes. The cells were resuspended in 100 μl of FACS buffer after two washes and prepared for FACS analysis (BD LSR II flow cytometer, HTS). As shown in Figure 1, several mice showed a strong A2aR-specific antibody response after A2aR-coding DNA immunization, and no binding from pre-collected serum was observed (MFI: mean fluorescence intensity).

[0322] Next, mice with high specific titers were selected for euthanasia and aseptic isolation of their spleens.

[0323] Fusion of Sp2 / 0 cells and spleen cells Spleen tissue was homogenized. A single-cell suspension was prepared from the spleen and then fused with SP2 / 0 myeloma cells in a 1:1 ratio by electrofusion (BTX-Harvard Apparatus). The cells were then subjected to hypoxanthine-aminopteri Cells were plated in 96-well plates containing thymidine (HAT) medium (Millipore-Sigma, Cat.H0262). The cells were cultured for 12–14 days with two medium changes to reduce nonspecific background for screening in the next step.

[0324] Initial screening of hybridomas Initial screening of hybridoma supernatant in 96-well plates was performed using the same FACS procedure as described for serum titration, with the following modifications: 1) Human A2aR-expressing Expi293 cells (expressing GFP) were mixed with parental Expi293 cells (not expressing A2aR) in a 1:1 ratio and added to 96-well plates (2 × 10⁶ cells per well). 5 1) 50 μl of hybridoma supernatant, transferred from each well of a 96-well plate, was incubated with the cells on ice for 30 minutes. Wells that were positive for human A2aR-expressing cells but not positive for parental Expi293 cells were selected and processed for subcloning.

[0325] Subcloning Subcloning was performed using the limiting dilution method. Briefly, positive wells identified by FACS were seeded into 96-well plates at an average density of 1 cell / well. The 96-well plates were incubated for 7–10 days to allow cells to grow. Cell growth was monitored periodically. On approximately day 4, wells on the plates were observed under a phase-contrast microscope, and wells that appeared to have a single growing colony (i.e., only one hybridoma aggregate) were recorded. Cells from single-colony wells were screened by FACS using the same procedure as described for initial hybridoma screening when the cell confluence had decreased to approximately 50%. As shown in Figure 2, antibodies isolated from hybridoma clones 1B5-3D7, 3F6-9G5 and 3F8-12E9, 8D5-16E2 showed specific binding to human A2aR-expressing cells but not to parental Expi293 cells.

[0326] Hybridoma cell culture and antibody purification Hybridoma cells were grown in BD Cell mAb Quantum yield medium (Thermo Fisher, Cat. 220511) supplemented with low-IgG fetal bovine serum (Millipore Sigma, Cat. F1283). The supernatant was collected when cell viability decreased to about 50%. Antibodies were purified using Protein G resin according to the manufacturer's protocol.

[0327] (Example 2) Cloning of antibody variable regions from anti-A2aR mouse hybridomas Using the SMARTer RACE kit (TaKaRa, Cat.634858), DNA fragments encoding the variable regions (i.e., HCVR and LCVR) of exemplary antibodies were cloned from four mouse hybridomas. Each of the four mouse hybridomas was cultured and confirmed via subclass assay to produce monoclonal antibodies with H and L chains consisting of γ2a and κ chains, respectively. Total RNA was analyzed using the Qiagen RNA kit at a rate of 3 × 10⁶. 6 It was prepared from cells.

[0328] Following the protocol for the SMARTer RACE cDNA amplification kit, the first strand of cDNA was initially synthesized with the addition of the SMART sequence at the 5' end, enabling the use of this site for downstream amplification and cloning using total RNA as a template. For each sample, two PCR reactions were performed simultaneously for the H and L strands, respectively, using universal forward primers (provided in the kit) and reverse primers (designed based on the constant region sequences of mouse IgG2a and mouse kappa classes registered in the NCBI nucleotide database).

[0329] The PCR product amplified from the above reaction was purified by agarose gel electrophoresis followed by gel extraction using the NuceloSpin Gel and PCR Clean-Up Kit (Qiagen, Cat.740609), and cloned into a linearized pRACE vector (provided in the SMARTer RACE kit) using In-Fusion HD Cloning (provided in the SMARTer RACE kit). The H and L variable region sequences were analyzed and determined by Sanger sequencing.

[0330] The amino acid sequences of the CDR, HCVR, and LCVR of the exemplary antibodies 1B5-3D7, 3F6-9G5, and 3F8-12E9 are defined using the Kabat and IMGT numbering schemes and are shown in Tables 1-5. Tables 10 and 11 show the exemplary nucleic acid sequences encoding the exemplary antibodies 1B5-3D7, 3F6-9G5, and 3F8-12E9.

[0331] (Example 3) Recombinant expression and purification of mouse IgG antibodies For expression in mammalian cell lines, antibody genes encoding 1B5-3D7 and 3F6-9G5 were first re-cloned into appropriate expression vectors. After small-scale preparation of plasmid DNA, the Expi293 cell line was used for transient transfection. The supernatant was collected 5–7 days after transfection, and IgG-type antibodies were purified using Protein G resin according to the manufacturer's protocol (GE, Cat.GE17-0618-01).

[0332] (Example 4) In vitro blocking activity of anti-human A2aR mAbs 8D5-16E2, 3F6-9G5, 1B5-3D7, and 3F8-12E9 A2aR activity is mediated by Gαs proteins that activate adenylyl cyclase, leading to intracellular cAMP synthesis. cAMP levels correlate with the levels of their respective adenosine (agonist) components. cAMP can be detected using various commercially available cAMP assay kits.

[0333] HEK293 cells (BPS Bioscience, Cat.79381) that stably express human A2aR were seeded at 5000 cells / well in 200 μl of starvation medium (MEM (Hyclone Cat.SH30024.01) + serum stripped with 2% charcoal (Thermo Fisher Cat.A3382101)) and cultured overnight at 37°C. The following day, after washing three times with 200 μl of warm PBS, the cells were pre-incubated at 37°C for 15 minutes with either purified A2aR mAbs (1B5-3D7, 3F6-9G5, and 3F8-12E9) or ZM241385 control (a standard small molecule antagonist for A2aR) in induction buffer (PBS containing 500 μM 3-isobutyl-1-methylxanthine (IBMX) (Millipore Sigma, Cat. I7018) + 100 μM Ro 20-1724, Millipore Sigma, Cat. B8279) at 2-fold or 3-fold serial dilutions. After incubation with antibody or ZM241385 (Millipore Sigma, Z0153), the stable adenosine agonist NECA (Millipore Sigma, E2387) was subsequently added to a final concentration of 300 nM or 500 nM. The cells were then incubated at 37°C for another hour. Cell lysis was performed using the cAMP-Glo® assay kit (Promega, Cat.V1501) to detect cAMP. The results (relative luminal units, RLU) were read using a luminometer. RLU can be converted to cAMP (nM) using the cAMP standard curve and Prism software.

[0334] As shown in Figure 3, the exemplary antibodies of the present invention, anti-human A2aR mAb 1B5-3D7, 3F6-9G5, and 3F8-12E9, purified from hybridoma supernatant, yielded approximately 4.5 × 10⁶ units. -9 M~approx. 1.5×10 -9 IC between M 50The values ​​indicated that the activity of human A2aR expressed on the cell surface was blocked. Table 11 shows the IC values ​​of exemplary antibodies that block cAMP production induced upon NECA binding to human A2aR on the cell surface. 50 The values ​​are shown. IC of the exemplary antibody. 50 The value is the IC for the small molecule inhibitor (SMI) ZM241385. 50 This value is 1 / 100th, indicating that the in vitro inhibition of the exemplary antibody is at least 100 times stronger than that of SMI ZM241385.

[0335] [Table 11]

[0336] As shown in Figure 4, the exemplary antibodies of the present invention, anti-human A2aR mAb 1B5-3D7, 3F6-9G5, purified from Expi293 cells transiently transfected with a recombinant expression vector expressing the antibody gene, block the activity of human A2aR expressed on the cell surface, similar to IC. 50 This was shown.

[0337] (Example 5) Determination of the specificity of anti-human A2aR antibodies The objective of this evaluation was to determine the specificity of exemplary anti-human A2aR antibodies (clones 1B5-3D7 and 3F6-9G5) against adenosine receptor family members, including human A1R, A2bR, and A3R, as well as their cross-reactivity with mouse A2aR.

[0338] method To test the specificity of the antigen-binding molecule of the present invention, e.g., an anti-A2aR antibody or its antigen-binding fragment, against human A2aR, rather than against any of the other three adenosine receptor family members, a flow cytometry-based cell binding assay was performed by Multispan Inc (Hayward, CA). HEK293T cells and HEK293T parental cell lines, which stably express various adenosine receptors generated by Multispan Inc., namely human A1R, A2bR, and A3R, as well as mouse A2R, were used in the assay. Briefly, adenosine receptor overexpressing cell lines and HEK293T parental cell lines were incubated in the dark at 4°C for 60 minutes with 2, 10, and 50 nM anti-human A2aR monoclonal antibodies (clones 1B5-3D7 and 3F6-9G5) and 10 and 50 nM mouse IgG2a isotype controls. Anti-FLAG antibodies (Abcam, ab72469, 2 μg / mL) were used as positive controls for various cell lines overexpressing different adenosine receptors. After three washes with FACS buffer (PBS + 0.1% BSA and 0.2% sodium azide), cells were stained with anti-mouse-IgG-PE (Invitrogen, cat.P852) detection antibody at 4°C for 45 minutes. Subsequently, cells were washed three times with FACS buffer and analyzed using FACSort (Becton Dickinson). Data were analyzed using CellQuest Pro (Becton Dickinson).

[0339] result Two exemplary antibodies of the present invention, 1B5-3D7 and 3F6-9G5, specifically bound to human A2aR. Binding to other human adenosine receptors or mouse A2aR was either similar to the binding levels detected in the negative control (HEK293T) or significantly weaker than binding to human A2aR (HEK293T-A1) (Table 12).

[0340] Table 12 summarizes the binding signals of exemplary antibodies 1B-3D7 and 3F6-9G5 to parental HEK293 T cells or HEK293 cells expressing different adenosine receptors, performed in a dual-chain FACS assay.

[0341] [Table 12]

[0342] (Example 6) Comparison of binding affinity between exemplary anti-human A2aR antibodies of the present invention and other anti-human A2aR antibodies. Human A2aR-expressing Expi293 cells or parental Expi293 cells were initially added to a 96-well plate (1 × 10⁶ cells per well). 5 Cells). 50 microliters (50 μl) of 12-point 1:2 serial dilutions of each antibody, starting at 7.5 ug / ml, were incubated with cells on ice for 30 minutes. After washing with FACS buffer (2% fetal bovine serum in DPBS), 100 μl of 1 μg / ml Alex Fluor 633 conjugate anti-mouse IgG (Life Technology, Cat.A21050) or Alex Fluor 647 conjugate anti-human IgG was added and incubated on ice for 30 minutes. Cells were resuspended in 100 μl of FACS buffer after two washes. 100 microliters (100 μl) of 1:50 dilution of 7-AAD was added to the cell suspension for vi / death staining, and FACS analysis (BD Accuri) was performed. The C6 plus or LSR II flow cytometer (HTS) is ready.

[0343] As shown in Figure 5, exemplary mIgG2a antibodies 1B5-3D7 and 3F6-9G5 showed strong dose-dependent specific binding to hA2aR-Expi293 but not to parental Expi293. Surprisingly, the absence of binding to hA2aR-Expi293 cells was detected by staining with the anti-A2aR monoclonal antibody MAB9497R (R&D Systems, clone 599717R) or SDIX-14 (US Patent Application Publication 2014 / 0322236A1, clone 864H14). Furthermore, the anti-hA2aR monoclonal antibody SDIX-10 (US Patent Application Publication No. 2014 / 0322236A1, clone 864H10) showed nonspecific binding to both hA2aR-Expi293 cells and parental Expi293 cells with similar but lower GMI (<2000 at a concentration of 50 nM) (GMI: geometric mean fluorescence intensity).

[0344] (Example 7) Determination of anti-human A2aR antibody binding to human and cynomolgus monkey primary cells, and cross-reactivity with non-human primates. In humans, A2aR has been reported to be expressed by T cells. We tested the potential binding of an adenosine-blocking anti-human A2aR antibody (clone 3F6-9G5) to human and cynomolgus monkey PBMCs.

[0345] method Human and cynomolgus monkey PBMCs were purchased from Cytologics LLC (San Diego, CA) and iQ Biosciences (Berkeley, CA), respectively. Primary cell binding was determined using rabbit Fc-type anti-human A2aR clone 3F6-9G5. Briefly, 10 in 100 μl of FACS buffer. 6Human and / or cynomolgus monkey PBMCs were incubated with 3F6-9G5 (2 μg / ml) at 4°C for 60 minutes. A subset of viable immune cells was defined using cynomolgus monkey cross-reactive anti-human CD3 (clone SP34, BD), anti-human CD8 (clone RPA-T8, Biolegend), and Zombie Green fixable viability dye (Biolegend, Cat. 423111) according to manufacturer's instructions. After three washes with FACS buffer, cells were stained with PE conjugate donkey anti-rabbit Fc detection antibody (1:200, Biolegend, Cat. 406421) at 4°C for 30 minutes. After three washes with FACS buffer, cells were analyzed using LSRII (Becton Dickinson). Data were analyzed using FlowJo (Becton Dickinson).

[0346] result The anti-human A2aR clone 3F6-9G5 bound to a small subset of human T cells (both CD4+ and CD8+) but not to the anti-HEL rabbit Fc isotype control (Biointron, Cat.B730001) (Figure 6). It also cross-reacted with cynomolgus monkey T cells (both CD4+ and CD8+) (Figure 6).

[0347] The above description is intended to instruct those skilled in the art on how to carry out the invention and is not intended to detail all obvious modifications and variations that would be apparent to those skilled in the art upon reading this description. However, all such obvious modifications and variations are intended to fall within the scope of the invention as defined by the following claims. The claims are intended to cover the claimed components and steps in any order that is effective in achieving the intended objective, unless the context specifically indicates otherwise. In certain embodiments, for example, the following are provided: (Item 1) An isolated antibody or its antigen-binding fragment that binds to the human adenosine A2A receptor (A2aR), From the N-terminus to the C-terminus, there are three heavy chain complementarity-determining regions (CDRs): HCDR1, HCDR2, and HCDR3, which constitute a heavy chain variable (VH) domain; and From the N-terminus to the C-terminus, the light chain variable (VL) domain contains three light chain complementarity-determining regions (CDRs): LCDR1, LCDR2, and LCDR3. Includes; (a) The HCDR1 comprises the amino acid sequence X1-X2-WMN (SEQ ID NO: 8), where X1 is S or R and X2 is Y or F; (b) The HCDR2 comprises the amino acid sequence RIDP-X3-DSE-X4-X5-Y-X6-HKFW-X7 (SEQ ID NO: 9), where X3 is S or Y, X4 is A or T, X5 is H or Q, X6 is H or N, and X7 is D or G; (c) The HCDR3 comprises the amino acid sequence SLYGKGDY (SEQ ID NO: 3); (d) The LCDR1 is an amino acid sequence RSSQSX 17 -VHX 18 -Includes NGNTYLE (SEQ ID NO: 30), in the formula, X 17 is L or I, and X 18 is either R or S; (e) The LCDR2 comprises the amino acid sequence KVSNRFS (SEQ ID NO: 26); (f) The above LCDR3 is an amino acid sequence X 19 -QGSHVPLT (Sequence ID 31) is included in the formula, X 19 This refers to an isolated antibody or its antigen-binding fragment, which is either Y or F. (Item 2) (a) The HCDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 4, and 6; (b) The HCDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 5, and 7; (c) The HCDR3 comprises the amino acid sequence shown in Sequence ID No. 3; (d) The LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 25 or 28; (e) The LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 26; (f) The isolated antibody or antigen-binding fragment according to item 1, wherein the LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 27 or 29. (Item 3) The aforementioned antibody (a) HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 2, HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 3, LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 25, LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 26, and LCDR3 comprising the amino acid sequence shown in SEQ ID NO: 27; (b) HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 4, HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 5, HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 3, LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 28, LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 26, and LCDR3 comprising the amino acid sequence shown in SEQ ID NO: 29; or (c) HCDR1 containing the amino acid sequence shown in SEQ ID NO: 6, HCDR2 containing the amino acid sequence shown in SEQ ID NO: 7, HCDR3 containing the amino acid sequence shown in SEQ ID NO: 3, LCDR1 containing the amino acid sequence shown in SEQ ID NO: 25, LCDR2 containing the amino acid sequence shown in SEQ ID NO: 26, and LCDR3 containing the amino acid sequence shown in SEQ ID NO: 29 The isolated antibody or its antigen-binding fragment as described in item 2, including the following. (Item 4) (a) The HCDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 21, 22, 23, and 24; (b) The HCDR3 is selected from the group consisting of sequence numbers 12, 15 and 20. An isolated antibody or its antigen-binding fragment, as described in item 1, containing a minoic acid sequence. (Item 5) The aforementioned antibody (a) Heavy chain variable region (HCVR) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs. 35, 36, 37, 38, 39, and 40; and (b) Light chain variable region (LCVR) containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 41, 42, and 43. An isolated antibody or antigen-binding fragment thereof, as described in any one of items 1 to 4, including the following: (Item 6) The aforementioned antibody (a) The HCVR comprising the amino acid sequence shown in SEQ ID NO: 35 or 38 and the LCVR comprising the amino acid sequence shown in SEQ ID NO: 41; (b) the HCVR comprising the amino acid sequence shown in SEQ ID NO: 36 or 39 and the LCVR comprising the amino acid sequence shown in SEQ ID NO: 42; or (c) The HCVR comprising the amino acid sequence shown in SEQ ID NO: 37 or 40 and the LCVR comprising the amino acid sequence shown in SEQ ID NO: 43 The isolated antibody or its antigen-binding fragment as described in item 5, including the isolated antibody or antigen-binding fragment described in item 5. (Item 7) An isolated antibody or its antigen-binding fragment that binds to the human adenosine A2A receptor (human A2aR), From the N-terminus to the C-terminus, there are three heavy chain complementarity-determining regions (CDRs): HCDR1, HCDR2, and HCDR3, which constitute a heavy chain variable (VH) domain; and From the N-terminus to the C-terminus, the light chain variable (VL) domain contains three light chain complementarity-determining regions (CDRs): LCDR1, LCDR2, and LCDR3. Includes; (a) The HCDR1 contains an amino acid sequence that is approximately 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to approximately 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 4, and 6; (b) The HCDR2 contains an amino acid sequence that is approximately 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to approximately 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 5, and 7; (c) The HCDR3 contains an amino acid sequence that is approximately 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to approximately 100% identical to the amino acid sequence shown in SEQ ID NO: 3; (d) The LCDR1 contains an amino acid sequence that is approximately 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to approximately 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs. 25 and 28; (e) The LCDR2 contains an amino acid sequence that is approximately 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to approximately 100% identical to the amino acid sequence shown in SEQ ID NO: 26, (f) An isolated antibody or antigen-binding fragment thereof, wherein LCDR3 contains an amino acid sequence that is approximately 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to approximately 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs. 27 and 29. (Item 8) An isolated antibody or its antigen-binding fragment that binds to the human adenosine A2A receptor (human A2aR), (a) Heavy chain variable regions (HCVRs) containing amino acid sequences that are approximately 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to approximately 100% identical to amino acid sequences selected from the group consisting of SEQ ID NOs. 35, 36, and 37; and (b) For an amino acid sequence selected from the group consisting of SEQ ID NOs: 41, 42, and 43 Light chain variable regions (LCVRs) containing approximately 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% to approximately 100% identical amino acid sequences. An isolated antibody or its antigen-binding fragment, containing [the specified substance]. (Item 9) An isolated antibody or its antigen-binding fragment that binds to the human adenosine A2A receptor (human A2aR), (a) Heavy chain variable region (HCVR) comprising the amino acid sequence shown in SEQ ID NO: 35 or 38; and (b) Light chain variable region (LCVR) containing the amino acid sequence shown in SEQ ID NO: 41 An isolated antibody or its antigen-binding fragment, containing [the specified substance]. (Item 10) An isolated antibody or its antigen-binding fragment that binds to the human adenosine A2A receptor (human A2aR), (a) Heavy chain variable region (HCVR) containing the amino acid sequence shown in SEQ ID NO: 36 or 39; and (b) Light chain variable region (LCVR) containing the amino acid sequence shown in SEQ ID NO: 42 An isolated antibody or its antigen-binding fragment, containing [the specified substance]. (Item 11) An isolated antibody or antigen-binding fragment according to any one of items 1 to 10, wherein the N-terminus of the heavy chain and / or light chain is a pyroglutamic acid (pE) residue. (Item 12) (i) The antibody competes for binding to human A2aR with monoclonal antibodies selected from the group consisting of 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 the concentration of cAMP in the tissue; (vi) The antibody reduces protein kinase A activity; (vii) The antibody reduces the phosphorylation of the cAMP response element of the A2aR signaling pathway; or (viii) An isolated antibody or antigen-binding fragment according to any one of items 1 to 11, wherein the antibody specifically binds to human and / or cynomolgus monkey A2aR. (Item 13) An isolated antibody or its antigen-binding fragment that competes with any of the antibodies described in item 1 to 12 for binding to human A2aR. (Item 14) The antibody or its antigen-binding fragment according to any one of items 1 to 13, wherein the antibody is a humanized antibody or a chimeric antibody. (Item 15) The antibody or antigen-binding fragment described in any one of items 1 to 14, wherein the antibody comprises a heavy chain constant region of a class selected from IgA, IgD, IgE, IgG, or IgM. (Item 16) The antibody or antigen-binding fragment according to item 15, wherein the antibody comprises a heavy chain constant region of class IgG, and the IgG is selected from the group consisting of IgG4, IgG1, IgG2, and IgG3. (Item 17) An isolated polynucleotide encoding an antibody or its antigen-binding fragment, its HCVR, its LCVR, its light chain, its heavy chain, or its antigen-binding fragment, as described in any one of items 1 through 16. (Item 18) An expression vector containing the polynucleotides described in item 17. (Item 19) Recombinant cells containing the polynucleotides described in item 17 or the expression vectors described in item 18. (Item 20) A method for producing an antibody or an antigen-binding fragment thereof as described in any one of items 1 to 16, comprising the steps of expressing the antibody in recombinant cells as described in item 17 and isolating the expressed antibody. (Item 21) A pharmaceutical composition comprising an antibody or antigen-binding fragment described in any one of items 1 to 16, and a pharmaceutically acceptable carrier or diluent. (Item 22) The pharmaceutical composition according to item 19, wherein the antibody or antigen-binding fragment in the pharmaceutical composition is in an effective amount for (a) specifically binding to human or cynomolgus monkey A2aR on the cell surface; (b) reducing the concentration of cAMP in tissue; (c) inhibiting the activity of human A2aR; (d) reducing the phosphorylation of the cAMP response element of the A2aR signaling pathway; (e) improving the immune response of immune cells; (f) reducing protein kinase A activity; and (g) any combination of (a) to (f). (Item 23) A method for inhibiting the activity of A2aR expressed on the surface of a cell, comprising the step of contacting the cell with an isolated antibody or antigen-binding fragment thereof as described in any one of items 1 to 16 or a pharmaceutical composition as described in item 21 or 22, thereby inhibiting the A2aR activity in the cell. (Item 24) A method for enhancing an immune response in a subject, comprising the step of administering to the subject an isolated antibody or an antigen-binding fragment thereof as described in any one of items 1 to 16 or a pharmaceutical composition as described in item 21 or 22, thereby enhancing the immune response in the subject. (Item 25) A method for inhibiting tumor growth in a subject, comprising the step of administering to the subject an isolated antibody or antigen-binding fragment thereof as described in any one of items 1 to 16 or a pharmaceutical composition as described in item 21 or 22, thereby inhibiting tumor growth. (Item 26) A method for treating cancer in a subject, comprising the step of administering an isolated antibody or an antigen-binding fragment thereof as described in any one of items 1 to 16 or a pharmaceutical composition as described in item 21 or 22, thereby treating the cancer. (Item 27) The method according to any one of items 23 to 26, which results in activating T cells and directing the T cells to kill tumor target cells. (Item 28) The method according to any one of items 23 to 27, further comprising the step of administering an additional therapeutic agent. (Item 29) The method according to item 28, wherein the further therapeutic agent includes an antitumor agent, radiotherapy, chemotherapeutic agent, surgery, cancer vaccine, agonist, cytokine, cell therapy, or checkpoint inhibitor for stimulating receptors of immune cells. (Item 30) The aforementioned checkpoint inhibitors include PD-1, PD-L1, TIGIT, CTLA-4, The method described in item 29, which involves drugs that inhibit PD-1, PD-L1, PD-L2, LAG-3, TIM-3, Neuritin, BTLA, CECAM-1, CECAM-5, IL-1R8, VISTA, LAIR1, LILRB1, LILRB2, LILRB3, LILRB4, LILRB5, CD96, CD112R, CD160, 2B4, TGFβ-R, KIR, NKG2A, and any combination thereof. (Item 31) The method according to item 30, wherein the drug inhibits the interaction between PD-1 and PD-L1, and the drug is selected from the group consisting of pembrolizumab, nivolumab, atezolizumab, avelumab, durvalumab, BMS-936559, cintilimab, tripalimab, tislerizumab, camrelizumab, sugemalimab, penprimab, cadnilimab, sulfamonomethoxine 1, and sulfamethizol 2. (Item 32) The method according to item 30, wherein the CTLA4 inhibitor is ipilimumab, cadnilimub, or YH001 (Encure Biopharma). (Item 33) The method according to item 29, wherein the further therapeutic agent is an agonist to an immune cell stimulatory receptor selected from OX40, CD2, CD27, CDS, ICAM-1, LFA-1, ICOS (CD278), 4-1 BB (CD137), GITR, CD28, CD30, CD40, BAFFR, HVEM, CD7, LIGHT, NKG2C, NKG2D, SLAMF7, NKp46, NKp80, CD160, B7-H3, CD83 ligands, and any combination thereof. (Item 34) The method according to any one of items 28 to 33, wherein the further therapeutic agent is formulated in the same pharmaceutical composition as the antibody. (Item 35) The method according to any one of items 28 to 33, wherein the further therapeutic agent is formulated in a pharmaceutical composition different from the antibody. (Item 36) The method according to any one of items 28 to 33 and 35, wherein the further therapeutic agent is administered before the step of administering the antibody and / or after the step of administering the antibody. (Item 37) The method according to any one of items 28 to 35, wherein the further therapeutic agent is administered in combination with the antibody or its antigen-binding fragment. (Item 38) A kit comprising the pharmaceutical composition described in item 21 or 22. (Item 39) The kit described in item 38, which further contains additional therapeutic agents.

Claims

[Claim 1] The invention described in the specification.

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