Anti-cd39 antibodies and uses thereof

Abstract

Provided herein are anti-CD39 antibodies that inhibit the enzymatic activity of human CD39 and methods of using the same.

Description

[0001] This application is a divisional application of the application filed on March 2, 2023, with the Chinese application number 202380024645.6, and the title of “Anti-CD39 Antibodies and Uses Thereof”.

[0002] Cross-reference Declaration

[0003] This application claims priority to International Application PCT / CN2022 / 079021 filed on March 3, 2022, and International Application PCT / CN2022 / 126070 filed on October 19, 2022, the disclosures of which are incorporated herein by reference. TECHNICAL FIELD

[0004] Provided herein are anti-CD39 antibodies that inhibit the enzymatic activity of human CD39 and methods of use thereof. BACKGROUND

[0005] In healthy conditions, ATP is released extracellularly by dying or stressed cells at negligible concentrations (10-100 nM). Once released, extracellular ATP is signaled via type 2 purinergic (P2) receptors to provide an inflammatory signal critical for effective innate and adaptive immune responses. CD39 is a rate-limiting ectoenzyme in the hydrolysis of extracellular ATP. By catabolizing the conversion of extracellular ATP to AMP, CD39 is a key regulator of extracellular ATP levels.

[0006] CD39 is also an important contributor to extracellular adenosine levels. Adenosine is an immunosuppressive metabolite and thus has an opposite relationship to extracellular ATP. CD39 contributes to increased extracellular adenosine levels by hydrolyzing ATP to AMP (which is converted to adenosine by CD73). Extracellular adenosine then signals via type 1 purinergic receptors to create an immunosuppressive environment.

[0007] Thus, CD39’s hydrolysis of extracellular ATP can affect immune responses via two different signaling pathways at opposite ends of a signaling axis. Notably, dysregulation of this signaling axis, referred to herein as the ATP-adenosine signaling axis, has been observed in several diseases. Accordingly, there is a need in the art for agents that target CD39 and inhibit its enzymatic activity for beneficial therapeutic purposes. SUMMARY

[0008] In various aspects of the disclosure, anti-CD39 antibodies are provided. The anti-CD39 antibodies are optionally (i) labeled with one or more detectable signals, including but not limited to fluorescent molecules, spin-label molecules, enzymes, or radioisotopes, and / or (ii) conjugated to another therapeutic agent, including but not limited to a chemotherapeutic agent, a radioisotope, or a nucleic acid. In some embodiments, the anti-CD39 antibodies of the disclosure are provided in isolated antibody form. In some embodiments, the anti-CD39 antibodies of the disclosure are provided in a composition. In some embodiments, the anti-CD39 antibodies of the disclosure are provided in a composition further comprising a pharmaceutically acceptable excipient.

[0009] In one embodiment, the disclosure provides an anti-CD39 antibody that specifically binds to human CD39, comprising: a heavy chain variable region comprising a complementarity determining region 1 (HI) having at least 80% sequence identity to SEQ ID NO: 10, a complementarity determining region 2 (H2) having at least 80% sequence identity to SEQ ID NO: 11, and a complementarity determining region 3 (H3) having at least 80% sequence identity to SEQ ID NO: 12; and a light chain variable region comprising a complementarity determining region 1 (LI) having at least 80% sequence identity to SEQ ID NO: 14, a complementarity determining region 2 (L2) having at least 80% sequence identity to SEQ ID NO: 15, and a complementarity determining region 3 (L3) having at least 80% sequence identity to SEQ ID NO: 16. In another embodiment, the disclosure provides an anti-CD39 antibody that specifically binds to human CD39, comprising: a heavy chain variable region comprising an HI having at least 90% sequence identity to SEQ ID NO: 10, an H2 having at least 90% sequence identity to SEQ ID NO: 11, and an H3 having at least 90% sequence identity to SEQ ID NO: 12; and a light chain variable region comprising an LI having at least 90% sequence identity to SEQ ID NO: 14, an L2 having at least 90% sequence identity to SEQ ID NO: 15, and an L3 having at least 90% sequence identity to SEQ ID NO: 16. In some of the foregoing embodiments, the heavy chain variable region of the antibody has at least 90% sequence identity to SEQ ID NO: 9, and the light chain variable region has at least 90% sequence identity to SEQ ID NO: 13. In some embodiments, the heavy chain variable region of the antibody has at least 95% sequence identity to SEQ ID NO: 9, and the light chain variable region has at least 95% sequence identity to SEQ ID NO: 13.

[0010] In one embodiment, the present disclosure provides an anti-CD39 antibody that specifically binds to human CD39, comprising: a heavy chain variable region comprising a H1 having at least 80% sequence identity to SEQ ID NO: 18, a H2 having at least 80% sequence identity to SEQ ID NO: 19, and a H3 having at least 80% sequence identity to SEQ ID NO: 20; and a light chain variable region comprising a L1 having at least 80% sequence identity to SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24, a L2 having at least 80% sequence identity to SEQ ID NO: 25, and a L3 having at least 80% sequence identity to SEQ ID NO: 26. In another embodiment, the present disclosure provides an anti-CD39 antibody that specifically binds to human CD39, comprising: a heavy chain variable region comprising a H1 having at least 90% sequence identity to SEQ ID NO: 18, a H2 having at least 80% sequence identity to SEQ ID NO: 19, and a H3 having at least 90% sequence identity to SEQ ID NO: 20; and a light chain variable region comprising a L1 having at least 90% sequence identity to SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24, a L2 having at least 90% sequence identity to SEQ ID NO: 25, and a L3 having at least 80% sequence identity to SEQ ID NO: 26. In some of the foregoing embodiments, the heavy chain variable region and the light chain variable region of the antibody have at least 90% sequence identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) or at least 95% sequence identity to: SEQ ID NO: 17 and SEQ ID NO: 21, respectively; or SEQ ID NO: 43 and SEQ ID NO: 45, respectively; or SEQ ID NO: 43 and SEQ ID NO: 46, respectively; or SEQ ID NO: 43 and SEQ ID NO: 47, respectively; or SEQ ID NO: 43 and SEQ ID NO: 48, respectively; or SEQ ID NO: 43 and SEQ ID NO: 49, respectively; or SEQ ID NO: 44 and SEQ ID NO: 48, respectively; or SEQ ID NO: 44 and SEQ ID NO: 49, respectively.

[0011] In one embodiment, the present disclosure provides an anti-CD39 antibody that specifically binds to human CD39, comprising a heavy chain variable region comprising a H1 having at least 80% sequence identity to SEQ ID NO: 28, a H2 having at least 80% sequence identity to SEQ ID NO: 29, and a H3 having at least 80% sequence identity to SEQ ID NO: 30; and a light chain variable region comprising a L1 having at least 80% sequence identity to SEQ ID NO: 32, a L2 having at least 80% sequence identity to SEQ ID NO: 33, and a L3 having at least 80% sequence identity to SEQ ID NO: 34. In some of the foregoing embodiments, the heavy chain variable region and the light chain variable region of the antibody have at least 90% sequence identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) or at least 95% sequence identity to: SEQ ID NO: 27 and SEQ ID NO: 31; or SEQ ID NO: 58 and SEQ ID NO: 60; or SEQ ID NO: 58 and SEQ ID NO: 61; or SEQ ID NO: 58 and SEQ ID NO: 62; or SEQ ID NO: 58 and SEQ ID NO: 63; or SEQ ID NO: 59 and SEQ ID NO: 61; or SEQ ID NO: 59 and SEQ ID NO: 60; or SEQ ID NO: 59 and SEQ ID NO: 62; or SEQ ID NO: 59 and SEQ ID NO: 63.

[0012] For any of the disclosed antibodies of the present disclosure, the anti-CD39 antibody can be a monoclonal antibody or an antigen-binding fragment thereof; a chimeric, humanized, or veneered antibody or an antigen-binding fragment thereof; or a human antibody or an antigen-binding fragment thereof. Additionally, the anti-CD39 antibody can further comprise a heavy chain constant region selected from human IgGl, human IgG2, human IgG3, or human IgG4, and optionally a human light chain constant region. The variant heavy chain constant region can be a wild-type heavy chain constant region, or can be a heavy chain constant region having enhanced or reduced effector function relative to the wild-type heavy chain constant region. In various embodiments, the IgG heavy chain constant region can comprise SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5; and the human light chain kappa constant region can comprise SEQ ID NO: 6.

[0013] In one embodiment, the present disclosure provides an anti-CD39 antibody comprising a heavy chain variable region and a light chain comprising a light chain variable region, wherein (a) the heavy chain has an amino acid sequence comprising SEQ ID NO: 50 and the light chain has an amino acid sequence comprising SEQ ID NO: 53; (b) the heavy chain has an amino acid sequence comprising SEQ ID NO: 50 and the light chain has an amino acid sequence comprising SEQ ID NO: 54; (c) the heavy chain has an amino acid sequence comprising SEQ ID NO: 50 and the light chain has an amino acid sequence comprising SEQ ID NO: 55; (d) the heavy chain has an amino acid sequence comprising SEQ ID NO: 50 and the light chain has an amino acid sequence comprising SEQ ID NO: 56; (e) the heavy chain has an amino acid sequence comprising SEQ ID NO: 51 and the light chain has an amino acid sequence comprising SEQ ID NO: 56; (f) the heavy chain has an amino acid sequence comprising SEQ ID NO: 50 and the light chain has an amino acid sequence comprising SEQ ID NO: 57; (g) the heavy chain has an amino acid sequence comprising SEQ ID NO: 52 and the light chain has an amino acid sequence comprising SEQ ID NO: 56; or (h) the heavy chain has an amino acid sequence comprising SEQ ID NO: 52 and the light chain has an amino acid sequence comprising SEQ ID NO: 57.

[0014] In one embodiment, the present disclosure provides an anti-CD39 antibody comprising a heavy chain variable region and a light chain comprising a light chain variable region, wherein (a) the heavy chain has an amino acid sequence comprising SEQ ID NO: 64 and the light chain has an amino acid sequence comprising SEQ ID NO: 67; (b) the heavy chain has an amino acid sequence comprising SEQ ID NO: 64 and the light chain has an amino acid sequence comprising SEQ ID NO: 68; (c) the heavy chain has an amino acid sequence comprising SEQ ID NO: 64 and the light chain has an amino acid sequence comprising SEQ ID NO: 69; (d) the heavy chain has an amino acid sequence comprising SEQ ID NO: 64 and the light chain has an amino acid sequence comprising SEQ ID NO: 70; (e) the heavy chain has an amino acid sequence comprising SEQ ID NO: 65 and the light chain has an amino acid sequence comprising SEQ ID NO: 68; (f) the heavy chain has an amino acid sequence comprising SEQ ID NO: 65 and the light chain has an amino acid sequence comprising SEQ ID NO: 67; (g) the heavy chain has an amino acid sequence comprising SEQ ID NO: 66 and the light chain has an amino acid sequence comprising SEQ ID NO: 67; (h) the heavy chain has an amino acid sequence comprising SEQ ID NO: 65 and the light chain has an amino acid sequence comprising SEQ ID NO: 69; or (i) the heavy chain has an amino acid sequence comprising SEQ ID NO: 65 and the light chain has an amino acid sequence comprising SEQ ID NO: 70.

[0015] In another aspect, the present disclosure provides any of the antibodies disclosed herein for use as a medicament. In some embodiments, an anti-CD39 antibody is provided for use in treating cancer. In some embodiments, an anti-CD39 antibody is provided for use in preventing cancer.

[0016] In another aspect, the present disclosure provides any of the antibodies disclosed herein for use as a medicament in combination with an additional therapy. In some embodiments, the foregoing anti-CD39 antibody is provided for use in treating cancer. In some embodiments, the foregoing anti-CD39 antibody is provided for use in preventing cancer. In certain embodiments, the additional therapy can be an immune checkpoint inhibitor, an immunogenic cell death inducing therapy, an ATP-adenosine axis targeting agent, a HIF-2a inhibitor, an arginase inhibitor, an AXL inhibitor, or a PI3K inhibitor. In other embodiments, the additional therapy can be a chemotherapy, a radiotherapy, durvalumab, zimberelimab, domvanalimab, AB308, AB521, or quemliclustat.

[0017] In another aspect, the present disclosure provides any of the antibodies disclosed herein for use as a medicament in combination with two or more additional therapies. In some embodiments, the foregoing anti-CD39 antibodies are provided for use in treating cancer. In some embodiments, the foregoing anti-CD39 antibodies are provided for use in preventing cancer. In certain embodiments, each additional therapy can be a chemotherapeutic agent, an immune checkpoint inhibitor, an immunogenic cell death inducing therapy, an ATP-adenosine axis targeting agent, a HIF-2a inhibitor, an arginase inhibitor, an AXL inhibitor, or a PI3K inhibitor. In other embodiments, the additional therapy can be chemotherapy, radiotherapy, durvalumab, sevipramab, domvanalib, AB308, AB521, or quilizalumab. In some embodiments, the one or more additional therapeutic agents comprise (a) pemetrexed, carboplatin, and an antagonist anti-PD-1 antibody or an antagonist anti-PD-L1 antibody, or (b) FOLFOX and an antagonist anti-PD-1 antibody or an antagonist anti-PD-L1 antibody.

[0018] In another aspect, the present disclosure provides a method for treating or preventing cancer, the method comprising administering to a subject in need thereof any of the antibodies disclosed herein. In some embodiments, the method further comprises administering one or more additional therapies. In certain embodiments, each additional therapy can be an immune checkpoint inhibitor, an immunogenic cell death inducing therapy, an ATP-adenosine axis targeting agent, a HIF-2a inhibitor, an arginase inhibitor, an AXL inhibitor, or a PI3K inhibitor. In other embodiments, the additional therapy can be chemotherapy, radiotherapy, durvalumab, sevipramab, domvanalib, AB308, AB521, or quilizalumab.

[0019] In each of the above aspects, the cancer can be breast cancer, gastrointestinal cancer, genitourinary cancer, head and neck cancer, kidney cancer, lung cancer, lymphoma, ovarian cancer, pancreatic cancer, skin cancer, or thyroid cancer. In some embodiments, the cancer can be acute myeloid lymphoma, colorectal cancer, gastric cancer, esophageal cancer, castration-resistant prostate cancer, non-small cell lung cancer, ovarian cancer, pancreatic cancer, triple-negative breast cancer, head and neck squamous cell carcinoma, pancreatic ductal adenocarcinoma, clear cell kidney cancer, or melanoma.

[0020] In some embodiments, the cancer is non-small cell lung cancer, and the method can further comprise administering an anti-CD39 antibody in combination with pemetrexed, carboplatin, and an antagonist anti-PD-1 antibody or an antagonist anti-PD-L1 antibody. In some embodiments, the cancer is gastric or gastroesophageal cancer, and the method can further comprise administering an anti-CD39 antibody in combination with FOLFOX and an antagonist anti-PD-1 antibody or an antagonist anti-PD-L1 antibody. These embodiments can further comprise administering one or more additional agents selected from the group consisting of an antagonistic anti-TIGIT antibody, A 2a R antagonist, A 2b R antagonist, A 2a / 2b R antagonist, and a CD73 inhibitor, optionally wherein the additional agent is selected from the group consisting of domvanalib, AB308, etrumadenant, and quilizalib.

[0021] Other aspects and repeated explanations of the disclosure are provided in greater detail below. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 is a plot depicting the binding of several antibodies to human CD39 expressed on the surface of B cells from peripheral blood mononuclear cells (PBMCs). The plot shows representative data from a single human donor. Mean fluorescence intensity (MFI) is on the y-axis and antibody concentration (M) is on the x-axis.

[0023] Figure 2 is a plot depicting the inhibition of the enzymatic activity of human CD39 expressed on the surface of human monocytes in the presence of 20 or 400 micromolar ATP. The plot shows representative data from a single human donor. Percent maximum inhibition (%) is on the y-axis and antibody concentration is on the x-axis.

[0024] Figure 3 is a schematic depicting key steps in the macrophage cytokine release assay described in Example 6. Mφ = macrophage.

[0025] Figure 4A , Figure 4B , Figure 4C , Figure 4D , Figure 4E , Figure 4F Depicts the results of the macrophage cytokine release assay. The relative amount of cytokines in the supernatant of macrophages treated with 10 nM Figure 4A ) or 100 nM Figure 4B -F) anti-CD39 antibodies or isotype control. Fold change was normalized to isotype control for each donor and each test was performed in duplicate Figure 4C Figure 4D ​) or three ( Figure 4A , Figure 4B , Figure 4E , Figure 4F ) donors. Error bars are SEM.

[0026] Figure 5A , Figure 5B and Figure 5C depicts results of a monocyte-derived dendritic cell (moDC) assay. Extracellular markers CD83 ( Figure 5A ), CD86 ( Figure 5B ), and CD14 ( Figure 5C ) were measured on the surface of moDCs in the presence or absence of ATP ± anti-CD39 or isotype control. Each line represents one biological donor, where the connector number is from the same donor, N=4.

[0027] Figure 6 is a plot depicting results of a competition flow cytometry assay between a fluorescently labeled reference antibody hu39.1_IGG4.P-AF647 and unlabeled test antibodies. Mean fluorescence intensity (MFI) is on the y-axis and test antibody concentration (nM) is on the x-axis.

[0028] Figure 7A is a plot depicting binding of hu39.1_IGG4.P and isotype control antibodies to human CD39 expressed on MOLP-8 human myeloma cells.

[0029] Figure 7B is a plot depicting enzymatic inhibition of human CD39 expressed on MOLP-8 human myeloma cells by hu39.1_IGG4.P and isotype control antibodies.

[0030] Figure 8A depicts enzymatic activity (ATP consumption) on splenocytes from hCD39 KI mouse model.

[0031] Figure 8B depicts inhibition of splenocyte enzymatic activity by hu39.1_IGG4.P.

[0032] Figure 9 depicts confirmation that hCD39 KI mouse model expresses human, not murine CD39.

[0033] Figure 10A is a plot depicting mean tumor volume of hCD39 KI mice treated as indicated. Points represent the mean and error bars represent the standard error of the mean (SEM), * = p < 0.5.

[0034] Figure 10BFigure depicting percent change in body weight of hCD39 KI mice treated as indicated.

[0035] Figure 11 Low magnification images showing tumors from hCD39 KI mice analyzed for human CD39 expression by immunohistochemistry (panels A and C) and for evidence of anti-CD39 enzymatic blockade activity by enzymatic histochemistry (panels B and D).

[0036] Figure 12 High magnification images showing tumors from hCD39 KI mice analyzed for human CD39 expression by immunohistochemistry (panels A and C) and for evidence of anti-CD39 enzymatic blockade activity by enzymatic histochemistry (panels B and D). Figure 12 Figure in Figure 11 High resolution image of the boxed area in

[0037] Figure 13 Low magnification images showing spleens from hCD39 KI mice analyzed for human CD39 expression by immunohistochemistry (panels A and C) and for evidence of anti-CD39 enzymatic blockade activity by enzymatic histochemistry (panels B and D).

[0038] Figure 14 High magnification images showing spleens from hCD39 KI mice analyzed for human CD39 expression by immunohistochemistry (panels A and C) and for evidence of anti-CD39 enzymatic blockade activity by enzymatic histochemistry (panels B and D). Figure 14 Figure in Figure 13 High resolution image of the boxed area in

[0039] Figure 15A , Figure 15B and Figure 15C Figure depicting ATP levels detected in supernatants of single cell suspensions derived from tumors (Figure Figure 15A : MC38 tumors; Figure 15B : 4T1 tumors) or peripheral whole blood cells (Figure Figure 15C ) obtained from hCD39 KI mice treated as indicated along the x-axis. ATP levels were measured by detecting relative luminescence units (RLU).

[0040] Figure 16Plots depicting cell surface CD39 and receptor occupancy in hCD39 KI mice treated with anti-CD39. The plot in the upper panel depicts the reduction in cell surface CD39 expression as determined by binding of the non-competitive anti-CD39 clone A1-PE in multiple cell types following in vivo anti-CD39 treatment. The plot in the lower panel depicts the binding of the competitive antibody observed on all CD39 (A1) expressing cells in in vivo isotype control treated mice, but not in in vivo anti-CD39 treated mice, indicating full or near full target coverage. Data for populations of >100 CD39 (A1) + Statistical analysis was performed with One-way ANOVA (Dunnett's multiple comparison test with single pooled variance), *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0041] Figure 17A Depicted are flow cytometry scatter plots showing expression levels of human CD39 (x-axis) and mouse CD73 (y-axis) on immune subsets from tumor-derived lymph nodes in vehicle treated hCD39 KI mice. Gating was set on isotype controls (hCD39 isotype control in gray) and applied to each group (black).

[0042] Figure 17B Depicted are flow cytometry scatter plots showing expression levels of human CD39 (x-axis) and mouse P2X7 (y-axis) on immune subsets from tumor-derived lymph nodes in vehicle treated hCD39 KI mice. Gating was set on isotype controls (hCD39 isotype control in gray) and applied to each group (black).

[0043] Figure 17C Depicted are plots showing the percentage of immune subsets observed following treatment as indicated along the x-axis. No significant changes in the percentage of immune subsets were observed in the case of anti-CD39 treatment. Statistical analysis was performed with One-way ANOVA (Dunnett's multiple comparison test with single pooled variance), *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, each symbol represents an individual mouse, bar height is the mean and error bars + / - SEM.

[0044] Figure 17DPlots depicting CD39 cell surface expression observed after treatment as indicated along the x-axis. A significant decrease in CD39 cell surface expression was observed in almost all immune subsets with anti-CD39 treatment. Statistical analysis was performed with one-way ANOVA (Dunnett's multiple comparison test with single pooled variance), *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, each symbol represents an individual mouse, bar height is the mean and error bars are + / - SEM.

[0045] Figure 17E Histogram overlay for each immune subset showing reduction in CD39 cell surface expression on a representative animal from each treatment group (isotype + saline (gray open histogram), isotype + oxaliplatin (black open histogram), anti-CD39 + saline (gray solid histogram), and anti-CD39 + oxaliplatin (black solid histogram)).

[0046] Figure 18 Plots depicting peripheral cytokine levels in plasma derived from hCD39 KI mice bearing MC38 tumors at the end of the experiment described in Example 9. Data presented as mean ± SEM with a total of 11-12 mice per treatment group. Statistical analysis was performed with ANOVA by Tukey's multiple comparison test.

[0047] Figure 19A Plots depicting cell viability of CT26 cells, MC38 cells, SK-MEL-5 cells and isotype control antibody, and SK-MEL-5 cells and hu39.5_IGG1.AA antibody after 24 hours following treatment with vehicle control (Veh), 100 μΜ oxaliplatin (OXA.L), or 250 μΜ oxaliplatin (OXA.H).

[0048] Figure 19B Plots depicting HMGB1 levels in culture supernatant of CT26 cells, MC38 cells, SK-MEL-5 cells and isotype control antibody, and SK-MEL-5 cells and hu39.5_IGG1.AA antibody after 24 hours following treatment with vehicle control (Veh), 100 μΜ oxaliplatin (OXA.L), or 250 μΜ oxaliplatin (OXA.H).

[0049] Figure 19C Plots depicting extracellular ATP release by CT26 cells, MC38 cells, SK-MEL-5 cells and isotype control antibody, and SK-MEL-5 cells and hu39.5_IGG1.AA antibody after 8 hours following treatment with vehicle (PBS), 100 μΜ oxaliplatin, or 250 μΜ oxaliplatin.

[0050] Figure 20A Graph depicting showing cell survival rate of SK-MEL-5 cells after 48 hours of treatment with the indicated chemotherapeutic agents.

[0051] Figure 20B Graph depicting showing levels of HMGB1 in culture supernatant of SK-MEL-5 cells after 48 hours of treatment with the indicated chemotherapeutic agents.

[0052] Figure 20C Graph depicting showing extracellular ATP release by SK-MEL-5 cells after 8 or 32 hours of treatment with the indicated chemotherapeutic agents.

[0053] Figure 21 Graph depicting showing relative expression of CD39 on peripheral immune cells in humans (top row) and C57BL / 6hCK39KI MC38 mice (bottom row).

[0054] Figure 22A Flow cytometry scatter plots of a representative donor depicting distribution of CD39 and CD73 on different myeloid cell types. CD14 + Monocytes were isolated directly from peripheral human blood. DC enriched from peripheral human blood were further subdivided into pDC, cDC1, cDC2 and other DC. M0, M1, M2 and moDC subsets were derived from CD14 + monocytes as described in the Examples. CD14

[0055] Figure 22B Graph depicting showing CD73 positive percentage (left panel), and CD39 positive percentage (middle panel) and CD39 protein expression (right panel) as determined by flow cytometry on human peripheral blood CD14 + monocytes; subsets of primary dendritic cells (DCs) including plasmacytoid DCs (pDCs, defined as HLA-DR + CD11c 阴性 CD123 + ), conventional DC 1 s (cDC1s, defined as HLA-DR + CD11c + CD141 + Clec9a + ), conventional DC 2 s (cDC2s, defined as HLA-DR + CD11c + CD1c + CD141 阴性 Clec9a 阴性 ), and other DCs (defined as HLA-DR + CD11c+ CD1c 阴性 CD141 阴性 Clec9a 阴性 ) and in vitro differentiated monocyte-derived myeloid cells, including macrophages (M0) polarized macrophages (M1, M2) and DCs (moDCs). For CD39 protein expression levels, CD39 + Mean fluorescence intensity of cells divided by mean fluorescence intensity of isotype control. Each symbol is a unique donor, bar height is median and error bars are range.

[0056] Figure 22C and Figure 22D Graph depicting relative induction of cell surface marker expression (compared to DMSO) after 1 hour of treatment with indicated inhibitors followed by 18 hours of treatment with 0 or 300 µM ATP. Cell surface expression of CD86 (top panel), CD83 (middle panel) and CD80 (bottom panel) was assessed by flow cytometry. Induction of activation marker expression was compared between inhibitor-treated cells and DMSO-treated cells according to 0 to 300 µM ATP. Bar height is mean and error bars are SEM. Each dot represents one donor. Statistical analysis was performed using ANOVA by Tukey’s multiple comparison test, *P≤0.05, ***P≤0.001. + Graphs depicting mRNA expression of ENTPD1 (CD39) (top panel) and NT5E (CD73) (bottom panel) evaluated on enriched populations of monocytes, primary dendritic cell subsets (enriched DCs) and in vitro differentiated monocyte-derived myeloid cells, including macrophages (M0) polarized macrophages (M1, M2) and DCs (moDCs). Figure 22C Figure 22D

[0057] Figure 22E Heatmap depicting unsupervised clustering of housekeeping gene normalized expression of CD39, adenosine and inflammasome pathway genes using custom NanoString panels in cell populations from four healthy donors described in 279. Low quality wells or abnormal control probes marked by low counts were removed according to this analysis. Specific donor ID is attached at the end of the cell type label. Dashed horizontal lines in the top dendrogram show sample split but do not affect the unsupervised nature of the clustering.

[0058] Figure 23 Graph depicting relative induction of cell surface marker expression (compared to DMSO) after 1 hour of treatment with indicated inhibitors followed by 18 hours of treatment with 0 or 300 µM ATP. Cell surface expression of CD86 (top panel), CD83 (middle panel) and CD80 (bottom panel) was assessed by flow cytometry. Induction of activation marker expression was compared between inhibitor-treated cells and DMSO-treated cells according to 0 to 300 µM ATP. Bar height is mean and error bars are SEM. Each dot represents one donor. Statistical analysis was performed using ANOVA by Tukey’s multiple comparison test, *P≤0.05, ***P≤0.001.

[0059] Figure 24A ​​To show a heatmap of TCGA RNA expression analysis in tumors (kidney renal clear cell carcinoma (KIRC), lung squamous cell carcinoma (LUSC), lung adenocarcinoma (LUAD), head and neck squamous cell carcinoma (HNSC), esophageal carcinoma (ESCA), stomach adenocarcinoma (STAD), and pancreatic adenocarcinoma (PAAD)) normalized on a per-gene basis. The proteins encoded by the genes are indicated in parentheses. The observed proteins and genes include: ALPL (TNAP), P2RX4 (P2X4), ENTPD1 (CD39), ENPP1, NT5E (CD73), P2RX5 (P2X5), P2RY2 (P2Y2), and P2RY11 (P2Y11). The distribution is shown in the histogram on the right.

[0060] Figure 24B A violin plot is depicted. The violin plot summarizes the kernel probability density of normalized log2 gene expression (y-axis) for the following genes (proteins) among a subset of TCGA samples: ENTPD1 (CD39), NT5E (CD73), P2RX7 (P2X7), and P2RY11 (P2Y11). Along the x-axis, the violin plots are grouped by specific TCGA subtype: esophageal carcinoma (ESCA), head and neck squamous cell carcinoma (HNSC), kidney renal clear cell carcinoma (KIRC), lung adenocarcinoma (LUAD), lung squamous cell carcinoma (LUSC), pancreatic adenocarcinoma (PAAD), and stomach adenocarcinoma (STAD). The pathway enrichment scores (y-axis) for the bone marrow signature are also shown for the same subset of TCGA cancer subtypes. DETAILED DESCRIPTION

[0061] The present disclosure provides antibodies that specifically bind to human CD39, more particularly to an epitope within the extracellular domain of human CD39, and inhibit the enzymatic activity of human CD39. Encompassed are methods for using the anti-CD39 antibodies disclosed herein to bind to cells expressing human CD39 and to inhibit the enzymatic activity of soluble and cell surface expressed human CD39. The present disclosure demonstrates the use of the anti-CD39 antibodies disclosed herein to inhibit the enzymatic activity of human CD39 and thereby affect one or both ends of the ATP-adenosine signaling axis. Accordingly, the present disclosure also provides medical uses of the anti-CD39 antibodies disclosed herein for therapeutic and diagnostic purposes.

[0062] I. DEFINITIONS

[0063] Unless otherwise defined, all technical terms, symbols and other scientific or professional terms or acronyms used herein have the meaning commonly understood by one of ordinary skill in the art to which this disclosure pertains. It will be understood that the aspects of the application described herein include “comprising,” “consisting of,” and “consisting essentially of” aspects. The terms “comprise,” “comprising,” “include,” “including,” and “have” are intended to be inclusive and permitting of other items, components, elements or steps in addition to those listed.

[0064] The term “about” as used herein, unless otherwise indicated, refers to the ordinary error range of an individual value as understood by persons of ordinary skill in the art in the field of technology to which this disclosure pertains. For the avoidance of doubt, reference herein to “about” one value or parameter includes (and describes) aspects that are directed to that value or parameter itself.

[0065] Also as used herein, “and / or” refers to and encompasses any and all possible combinations of the associated listed items, as well as the items in the alternative (“or”). Similarly, the phrase in the form “at least one of A / B” means (A) or (B) or (A and B). Likewise, the phrase in the form “at least one of A, B and C” means (A) or (B) or (C) or (A and B) or (A and C) or (B and C) or (A and B and C).

[0066] Unless otherwise indicated, the term “CD39” refers to human CD39. An exemplary human CD39 sequence is SEQ ID NO: 7 (UniProtKB Reference: P49961). CD39 is a dual transmembrane protein. The extracellular domain of CD39 comprises residues 38-478 of SEQ ID NO: 7. An exemplary cynomolgus monkey CD39 sequence is SEQ ID NO: 8. The term “CD39 enzymatic activity” refers to the hydrolysis of ATP to ADP or AMP.

[0067] The terms “anti-CD39 antibody” and “antibody that binds to CD39” are used interchangeably herein to refer to an antibody that is capable of binding human CD39 with sufficient affinity that the antibody is useful as a diagnostic and / or therapeutic agent in targeting human CD39 and inhibiting human CD39 enzymatic activity. Anti-CD39 antibodies of the present disclosure can have a dissociation constant (KD) for CD39 of 10 -6 M or lower. Anti-CD39 antibodies of the present disclosure can be monospecific antibodies or multispecific antibodies, and in some examples can be a multi-epitope antibody.

[0068] The term "antibody" as used herein is used in the broadest sense and encompasses various antibodies and antibody-like structures that specifically bind to a single antigen or multiple antigens, including but not limited to full-length antibodies, antigen-binding fragments, heavy chain antibodies, single chain antibodies, and higher order variants of single chain antibodies. Thus, unless the context requires otherwise, any reference to an antibody is understood to refer to an antibody in its intact form or an antigen-binding fragment. Preferably, but not necessarily, the antibodies useful herein are isolated and can be produced in recombinant fashion.

[0069] The terms "full-length antibody," "intact antibody," and "whole antibody" are used herein interchangeably to refer to an antibody having a structure substantially similar to a native antibody structure or an antibody having heavy chains with Fc regions.

[0070] A "native antibody" is a naturally occurring immunoglobulin molecule having different structures. For example, a native IgG antibody is a heterotetrameric glycoprotein of about 150,000 Da that is composed of two identical light chains (each about 25 kDa) and two identical heavy chains (each about 50-70 kDa) that are linked via disulfide bonds. From N- to C-terminus, each heavy chain has a variable region (VH), also called a variable heavy chain domain or a heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3). Similarly, from N- to C-terminus, each light chain has a variable region (VL), also called a variable light chain domain or a light chain variable domain, followed by a constant light (CL) domain. The light chains of an antibody can be assigned to one of two types, called kappa (K) and lambda (l), based on the amino acid sequence of their constant domains. The heavy chains are classified as gamma, mu, alpha, delta, or epsilon, and define the antibody's isotype as IgG, IgM, IgA, IgD, and IgE, respectively. The amino-terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The carboxy-terminal portion of each chain defines a constant region primarily responsible for effector function. Within light and heavy chains, the variable and constant regions are joined by a "J" region of about 12 or more amino acids, with the heavy chain also including a "D" region of about 10 more amino acids.

[0071] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to an antigen. The variable domains of antibody heavy and light chains generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three hypervariable regions (CDRs). (See, e.g., Kindt et al., Kuby Immunology, 6th Ed., W.H. Freeman and Co., page 91 (2007).) A single VHor VLdomain can be sufficient to confer antigen-binding specificity. Furthermore, a VHor VLdomain can be used to isolate an antibody that binds a particular antigen from antibodies that bind antigen, to screen libraries of complementary VLor VHdomains, respectively. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0072] A "framework region" or "FR" refers to variable domain residues other than hypervariable region residues. The FRs of a variable domain generally consist of four FR domains: FR1, FR2, FR3, and FR4. Thus, CDR and FR sequences generally appear in the following sequence: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The FR domains of the heavy and light chains can be different, as is known in the art.

[0073] The terms“hypervariable region” or“HVR” (also referred to as“complementarity determining region” or“CDR”), as used herein, are used interchangeably and refer to the regions of a variable domain in which sequence hypervariability and / or the formation of structurally defined loops (“hypervariable loops”) and / or the containment of antigen contact residues (“antigen contacts”) occur. Generally, an antibody comprises six CDRs: three in the VH (HI, H2, H3), and three in the VL (LI, L2, L3). As used herein, a“CDR derived from a variable region” refers to a CDR having no more than two amino acid substitutions compared to the corresponding CDR from the original variable region. Exemplary CDRs herein include: (a) the hypervariable loops that occur at amino acid residues 26-32 (LI), 50-52 (L2), 91-96 (L3), 26-32 (HI), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)); (b) the CDRs that occur at amino acid residues 24-34 (LI), 50-56 (L2), 89-97 (L3), 31-35b (HI), 50-65 (H2), and 95-102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)); (c) the antigen contacts that occur at amino acid residues 27c-36 (LI), 46-55 (L2), 89-96 (L3), 30-35b (HI), 47-58 (H2), and 93-101 (H3) (MacCallum et al., J. Mol. Biol. 262:732-745 (1996)); and (d) combinations of (a), (b), and / or (c), as defined below for the various antibodies of the disclosure. Unless otherwise indicated, CDR residues in a variable domain and other residues (e.g., FR residues) are numbered herein in accordance with Eu numbering as in Kabat et al., supra.

[0074] The term“isolated antibody” refers to an antibody that has been separated from a component of its natural environment. In some embodiments, an isolated antibody is purified to greater than 95% or 99% purity as determined by, for example, electrophoretic or chromatographic methods (e.g., ion exchange or reverse phase HPLC).

[0075] The term“chimeric” antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, while the remaining portion of the heavy and / or light chain is derived from a different source or species.

[0076] A "human antibody" is one whose amino acid sequence corresponds to that of an antibody produced by a human or a human cell or derived from a non-human source that utilizes a human antibody repertoire or other human antibody-encoding sequences. This definition of a human antibody specifically excludes humanized antibodies comprising non-human antigen binding residues. Human antibodies can also be produced using various techniques known in the art, including phage display libraries. Hoogenboom and Winter, J. Mol. Biol. 227:381, 1991; Marks et al., J. Mol. Biol. 222:581, 1991. The methods described by Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boerner et al., J. Immunol., 147(l):86-95, 1991, can also be used to produce human monoclonal antibodies. See also van Dijk and van de Winkel. Curr. Opin. Pharmacol. 5:368-74, 2001.

[0077] A "humanized" antibody refers to an antibody that contains amino acid residues from non-human CDRs and amino acid residues from human FRs. In certain aspects, a humanized antibody will comprise variable domains in which all or substantially all of the CDRs correspond to those of a non-human antibody, and all or substantially all of the FRs correspond to those of a human antibody. In certain aspects, all or substantially all of the FRs of a humanized antibody correspond to those of a human antibody, wherein any one of the FRs of the humanized antibody can contain one or more amino acid substitutions at one or more "Vernier" position residues, and / or at one or more other selected residues, e.g., as compared to the corresponding FR of the human antibody. A humanized antibody optionally can comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody, e.g., a non-human antibody, refers to an antibody that has undergone humanization. A humanized antibody retains the binding specificity and affinity of the starting non-human antibody.

[0078] The term "monoclonal antibody" refers to an antibody derived from a single copy or clone, including, e.g., any eukaryotic, prokaryotic, or phage clone. The term "monoclonal antibody" is not limited to antibodies produced through hybridoma technology. Monoclonal antibodies can be produced using hybridoma technology as well as recombinant technologies, phage display technologies, synthetic technologies, or combinations of such technologies and other techniques readily known in the art.

[0079] The term "epitope" refers to the specific site or sites on an antigen to which an antibody binds. The specific site or sites on an antigen to which an antibody binds can be determined, for example, by crystallography. Hydroxyl radical protein footprinting analysis and alanine scanning mutagenesis induced methods can also be used, although they can provide less resolution.

[0080] The term "monospecific antibody" refers to an antibody that specifically binds to only one antigen. A monospecific antibody can bind to only one epitope of an antigen or can bind to two or more epitopes of an antigen. A monospecific antibody that binds to two or more epitopes of an antigen is a monospecific, polyepitopic antibody.

[0081] The term "multispecific antibody" refers to an antibody that specifically binds to two or more antigens (e.g., bispecific antibodies, trispecific antibodies, etc.). Non-limiting examples of multispecific antibodies include, but are not limited to: antibodies comprising a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein the VH / VL unit has polyepitopic specificity; antibodies having two or more VL and VH domains, wherein each VH / VL unit binds to a different epitope; antibodies having two or more single variable domains, wherein each single variable domain binds to a different epitope; diabodies; triabodies; etc., as well as full-length antibodies and / or antibody fragments that have been covalently or non-covalently linked.

[0082] The terms "polyepitopic antibody" and "antibody having polyepitopic specificity" are used interchangeably herein to refer to an antibody that binds to two or more epitopes on the same or different antigens.

[0083] The term "Fc region" is used herein to define a C-terminal region of an immunoglobulin heavy chain, including native-sequence Fc regions and variant Fc regions. While the boundaries of the Fc region of an immunoglobulin heavy chain might vary, the human IgG heavy chain Fc region is usually defined to stretch from an amino acid residue at position Cys226, or from Pro230, to the carboxyl terminus. The C-terminal lysine (residue 447 according to Eu numbering system) of the Fc region can be removed, e.g., during production or purification of the antibody, or by engineering the nucleic acid encoding the antibody heavy chain in a recombinant manner. Accordingly, the composition of a whole antibody can include a population of antibodies in which all Lys447 residues are removed, a population of antibodies in which Lys447 residues are not removed, and a population of antibodies having a mixture of antibodies with and without Lys447 residues.

[0084] A "functional Fc region" has an effector function of a native sequence Fc region. Exemplary effector functions include Clq binding; complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g. B cell receptor; BCR), etc. Such effector functions generally require the Fc region to be combined with a binding domain (e.g. an antibody variable domain) and can be assessed using various assays disclosed herein or otherwise known in the art. The functional Fc region can have an effector function substantially similar to that of a wild-type IgG, decreased effector function compared to a wild-type IgG, or enhanced effector function compared to a wild-type IgG. For antibodies comprising a human Fc region, comparisons are typically made relative to a wild-type human IgGl.

[0085] A "native sequence Fc region" comprises an amino acid sequence identical to the amino acid sequence of a Fc region found in nature. Native sequence human Fc regions include a native sequence human IgGl Fc region (non-A and A allotypes); native sequence human lgG2 Fc region; native sequence human lgG3 Fc region; and native sequence human lgG4 Fc region, as well as naturally occurring variants thereof.

[0086] A "variant Fc region" comprises an amino acid sequence that differs from that of a native sequence Fc region by one or more amino acid modifications, e.g., about one to about ten amino acid modifications, and in some embodiments, about one to about five amino acid modifications, preferably one or more amino acid substitutions. A variant Fc region herein will preferably have at least about 80% identity, preferably at least about 90% identity, or preferably at least about 95% identity with a native sequence Fc region and / or the Fc region of the parent polypeptide. In some embodiments, a variant Fc region can have decreased or enhanced effector function compared to a wild-type IgG. For antibodies comprising a human Fc region, comparisons are typically made relative to a wild-type human IgGl.

[0087] As used herein, an "Fc component" refers to the hinge region, CH2 domain, or CH3 domain of an Fc region.

[0088] A "hinge region" is generally defined as extending from about residues 216-230 of IgG (Eu numbering), about residues 226-243 of IgG (Kabat numbering), or about residues 1-15 of IgG (IMGT unique numbering).

[0089] The term "antibody fragment" refers to a molecule other than a whole antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds. Examples of antigen-binding fragments include but are not limited to diabodies, Fab, Fab', F(ab')2, Fv), and complementarity determining region (CDR) fragments. cFv fragment, disulfide-stabilized Fv fragment (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized diabody (ds diabody), triabody, tetrabody, single-chain antibody, scFv, scFv dimer, single domain antibody, single-domain antibody, and multivalent domain antibody. Generally, a binding fragment competes with the intact antibody from which it was derived for specific binding. Binding fragments can be produced by recombinant DNA techniques or by enzymatic or chemical separation of intact immunoglobulins.

[0090] The term "Fab" refers to a portion of an antibody that consists of a single light chain (both variable and constant regions) bound to the variable region and first constant region of a single heavy chain by disulfide bonds.

[0091] The term "Fab'" refers to a Fab fragment that includes a portion of the hinge region.

[0092] The term "F(ab')2" refers to a dimer of Fab'. F(ab')2 antibody fragments are originally produced as a pair of Fab' fragments having a hinge cysteine between them. Other chemical couplings of antibody fragments are also known.

[0093] The term "Fv" refers to the minimum fragment of an antibody that carries the complete antigen binding site. An Fv fragment consists of the variable region of a single light chain bound to the variable region of a single heavy chain.

[0094] The term "single-chain antibody" refers to an antibody that consists of a heavy chain variable region and a light chain variable region connected by a linker. In most, but not all cases, the linker can be a peptide. The length of the linker varies depending on the type of single-chain antibody. Covalently or non-covalently linking two or more single-chain antibodies together produces higher order forms. Single-chain antibodies and higher order forms thereof can include, but are not limited to, single domain antibodies, multivalent domain antibodies, single-chain variable fragment (scFv), bivalent scFv (di-scFv), trivalent scFv (tri-scFv), tetravalent scFv (tetra-scFv), diabodies, and triabodies and tetrabodies.

[0095] The terms "single-chain Fv antibody" and "scFv" are used interchangeably herein to refer to a single-chain antibody composed of a heavy chain variable region and a light chain variable region connected by a linker. In most, but not all cases, the linker can be a peptide. The length of the linking peptide is preferably about 5 to 30 amino acids, or about 10 to 25 amino acids in length. Typically, the linker allows for stabilization of the variable domains without interfering with proper folding and production of the active binding site. In preferred embodiments, the linker peptide is rich in glycine and either serine or threonine. Two or more scFvs can be linked together in covalent or non-covalent fashion to produce higher order forms two-scFv, three-scFv, four-scFv, etc. The antigen binding site of each scFv in higher order forms can target the same or different antigens or epitopes.

[0096] The term "single-chain Fv-Fc antibody" or "scFv-Fc" refers to a full-length antibody composed of a scFv linked to an Fc region.

[0097] A "diabody" is a higher order single-chain antibody variant composed of two single-chain antibodies. For each single-chain antibody, a linker that is too short to allow pairing between the two domains on the same chain forces the domains to pair with the complementary domains of the other chain, resulting in two antigen binding sites. In most, but not all cases, the linker can be a peptide. The antigen binding sites can target the same or different antigens or epitopes. Triabodies (three single-chain antibodies assembled to form three antigen binding sites), tetrabodies (four single-chain antibodies assembled to form four antigen binding sites), and higher order variants can be produced in a similar fashion. See, e.g., Holliger P. et al., Proc Natl Acad Sci USA. July 15; 90(14):6444-8 (1993); EP 404097; WO 93 / 11161.

[0098] A "single-domain antibody" refers to an antibody fragment containing only a heavy chain variable region or a light chain variable region. In certain cases, two or more V H domains are covalently joined by a peptide linker to produce a multivalent domain antibody. The two or more V H domains of a multivalent domain antibody can target the same or different antigens or epitopes.

[0099] The term "heavy chain antibody" refers to an antibody composed of two heavy chains. Heavy chain antibodies can be IgG-like antibodies from camels, llamas, alpacas, sharks, etc., or IgNAR from cartilaginous fish. See, e.g., Riechmann L. and Muyldermans S., J Immunol Methods. Dec 10;231(1-2):25-38 (1999); Muyldermans S., J Biotechnol. Jun;74(4):277-302 (2001); WO 94 / 04678; WO 94 / 25591; or U.S. Patent No. 6,005,079. Heavy chain antibodies were originally derived from Camelidae (camels, dromedaries, and llamas). Despite the absence of light chains, camelized antibodies have a true antigen-binding repertoire (Hamers-Casterman C. et al., Nature. Jun 3;363(6428):446-8 (1993); Nguyen V.K. et al., "Heavy-chain antibodies in Camelidae; a case of evolutionary innovation", Immunogenetics. Apr;54(1):39-47 (2002); Nguyen V.K. et al., Immunology. May;109(1):93-101 (2003)). The variable domain of heavy chain antibodies (VHH domain) represents the smallest known antigen-binding unit produced by the adaptive immune response (Koch-Nolte F. et al., FASEB J. Nov;21(13):3490-8. Epub 2007 Jun 15 (2007)).

[0100] "Nanobody" refers to an antibody composed of a VHH domain from a heavy chain antibody and two constant domains CH2 and CH3.

[0101] "Percent (%) identity" with respect to a reference amino acid sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, or CLUSTAL. Those skilled in the art will appreciate that appropriate parameters will be determined using criteria well within the skill in the art, including any algorithm used for optimal alignment of the sequences being compared. Generally, the % sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B is calculated as: 100 times the fraction X / Y, where X is the number of amino acid residues scored as identical matches in the program alignment of A and B, and where Y is the total number of amino acid residues in B. It will be appreciated that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A.

[0102] The terms "patient" and "subject" are used interchangeably herein to refer to a human or non-human animal (e.g., a mammal) that expresses human CD39.

[0103] The terms "treat," "treating," "treatment," and the like, refer to the process of elminating, reducing, suppressing, alleviating, or ameliorating, or preventing the worsening of, a disease, disorder, or condition to which such terms apply, or at least one symptom associated therewith. As an example, treatment includes inhibiting (e.g., arresting the development or further development of a disease, disorder, or condition, or clinical symptoms associated therewith), improving quality of life, and / or prolonging survival of a subject.

[0104] The term "in need of treatment," as used herein, refers to a judgment by a physician or other caregiver that a subject needs or will benefit from treatment. This judgment is made based on a variety of factors that are within the purview of a physician's or caregiver's expertise.

[0105] The terms "prevent," "preventing," "prevention," "prophylaxis," and the like, refer to an inhibitory process (e.g., prior to the onset of a disease, disorder, condition, or symptoms thereof) that results in a subject temporarily or permanently avoiding, suppressing, inhibiting, or reducing the risk of, or delaying the onset of, a disease, disorder, condition, etc. (as determined by, e.g., the absence of clinical symptoms), typically in a subject predisposed to a particular disease, disorder, or condition. In certain instances, the terms also refer to slowing the progression of, or inhibiting the progression to, a harmful or other undesirable state. Prevention also refers to an inhibitory process initiated in a subject after the subject has been treated for a disease, disorder, condition, or symptoms related thereto, in order to prevent the recurrence of the disease, disorder, condition, or symptoms.

[0106] The term "in need of prevention" as used herein refers to a judgment by a physician or other caregiver that a subject requires or would benefit from prophylactic care. This judgment is made based on a variety of factors that are within the purview of a physician's or caregiver's expertise.

[0107] II. Antibodies of the Disclosure

[0108] The present disclosure provides antibodies that specifically bind to human CD39, and more particularly to the extracellular domain of human CD39. The antibodies of the present disclosure can specifically bind to soluble and / or membrane-bound human CD39. In some embodiments, the anti-CD39 antibodies of the present disclosure are monospecific. In other embodiments, the anti-CD39 antibodies of the present disclosure are multispecific. Additional antigen binding specificities encompassed in the context of the present disclosure include, but are not limited to, TGFβ. The anti-CD39 antibodies of the present application can be isolated or can be part of a composition.

[0109] The anti-CD39 antibodies of the present disclosure, whether monospecific or multispecific, specifically bind to human CD39, meaning that the equilibrium dissociation constant (K D ) of the antibody for CD39 is 10 -6 M or less, as measured by surface plasmon resonance (SPR). In certain embodiments, the K D of the anti-CD39 antibodies of the present disclosure for human CD39 is 10 -8 M or less (e.g., 10 -8 , 10 -9 , 10 -10 , etc.), as measured by SPR. For detailed methods, see, e.g., Example 3. In various embodiments, the K D of the anti-CD39 antibodies of the present disclosure for human CD39 is about 1 x 10 -9 M to about 1 x 10-14 M, or about 1 x 10 -9 M to about 1 x 10 -13 M, or about 1 x 10 -9 M to about 1 x 10 -12 M, or about 1 x 10 -9 M to about 1 x 10 -11 M. In some embodiments, an anti-CD39 antibody of the present disclosure has a K D of about 1 x 10 -10 M to about 1 x 10 -14 M, or about 1 x 10 -10 M to about 1 x 10 -13 M, or about 1 x 10 -10 M to about 1 x 10 -12 M. In some embodiments, an anti-CD39 antibody of the present disclosure has a K D of about 1 x 10 -11 M to about 1 x 10 -14 M, or about 1 x 10 -11 M to about 1 x 10 -13 M. In some embodiments, an anti-CD39 antibody of the present disclosure has a K D of about 1 x 10 -12 M to about 1 x 10 -14 M, or about 1 x 10 -12 M to about 1 x 10 -13 M. In some embodiments, an anti-CD39 antibody of the present disclosure has a K D of about 1 x 10 -10 M to about 1 x 10 -11 M, or about 1 x 10 -11 M to about 1 x 10 -12 M.

[0110] Anti-CD39 antibodies of the present disclosure also inhibit CD39 enzyme activity. In some embodiments, an anti-CD39 antibody of the present disclosure inhibits human CD39 enzyme activity with an IC 50 value of about 5 nM or less, as measured in Example 5. For example, an anti-CD39 antibody can inhibit recombinant human CD39 enzyme activity with an IC 50 value of about 5 nM, about 4 nM, about 3 nM, about 2 nM, about 1 nM, or less, as measured in Example 5. As another example, an anti-CD39 antibody can inhibit recombinant human CD39 enzyme activity with an IC 50inhibits recombinant human CD39 enzyme activity. As another example, the anti-CD39 antibody can have an IC50of about 0.5 nM or less, e.g., about 0.5 nM, about 0.4 nM, about 0.3 nM, about 0.2 nM, about 0.1 nM, about 0.09 nM, about 0.08 nM, about 0.07 nM, about 0.06 nM, about 0.05 nM or less, as measured in Example 5. 50 inhibits recombinant human CD39 enzyme activity. The above IC50values can also be expressed as individual values or ranges. For example, the anti-CD39 antibody can have an IC50of about 0.05 nM to about 5 nM, about 0.05 nM to about 1 nM, 0.05 nM to about 0.5 nM, about 1 nM to about 5 nM, about 0.5 nM to about 1.0 nM, or subranges thereof. 50 inhibits recombinant human CD39 enzyme activity. The above IC50values can also be expressed as individual values or ranges. For example, the anti-CD39 antibody can have an IC50of about 0.05 nM to about 5 nM, about 0.05 nM to about 1 nM, 0.05 nM to about 0.5 nM, about 1 nM to about 5 nM, about 0.5 nM to about 1.0 nM, or subranges thereof. 50 inhibits recombinant human CD39 enzyme activity. In one embodiment, the anti-CD39 antibody of the present disclosure has an IC50of about 0.5 nM to about 1.0 nM or about 0.6 nM to about 1.0 nM. 50 inhibits human CD39 enzyme activity. In one embodiment, the anti-CD39 antibody of the present disclosure has an IC50of about 0.05 nM to about 0.5 nM, about 0.05 nM to about 0.4 nM, or about 0.05 nM to about 0.3 nM. 50 inhibits human CD39 enzyme activity. In one embodiment, the anti-CD39 antibody of the present disclosure has an IC50of about 0.07 nM to about 0.5 nM, about 0.07 nM to about 0.4 nM, about 0.07 nM to about 0.3 nM, or about 0.07 nM to about 0.2 nM. 50 inhibits human CD39 enzyme activity. In the foregoing embodiments, (i) the human CD39 can be recombinant, soluble CD39 or can be cell surface-expressed CD39, and / or (ii) the inhibition can be assessed in the presence of low ATP (e.g., 20 µM) and / or high ATP (e.g., 400 µM).

[0111] A. Exemplary Variable Regions

[0112] The present disclosure provides anti-CD39 antibodies comprising a VH, or a VH and a VL, as described herein. In the following embodiments, “at least X% sequence identity” encompasses individual values and ranges thereof. For example, “at least 90% sequence identity” includes at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity, as well as individual values (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity) and ranges thereof.

[0113] In one example, an anti-CD39 antibody of the present disclosure comprises a heavy chain variable region (VH) having one or more CDRs derived from SEQ ID NO: 9 and optionally a light chain variable region (VL) having one or more CDRs derived from SEQ ID NO: 13. The CDRs derived from SEQ ID NO: 9 can be H1, H2, H3, or any combination thereof. In certain embodiments, the VH can comprise H1 having at least 90% sequence identity to SEQ ID NO: 10, H2 having at least 90% sequence identity to SEQ ID NO: 11, H3 having at least 90% sequence identity to SEQ ID NO: 12, or any combination thereof. The CDRs derived from SEQ ID NO: 13 can be L1, L2, L3, or any combination thereof. In certain embodiments, the VL can comprise L1 having at least 90% sequence identity to SEQ ID NO: 14, L2 having at least 90% sequence identity to SEQ ID NO: 15, L3 having at least 90% sequence identity to SEQ ID NO: 16, or any combination thereof. An antibody comprising one or more CDRs derived from SEQ ID NO: 9 can also comprise a VL comprising one or more CDRs derived from SEQ ID NO: 13. The CDRs can be L1, L2, L3, or any combination thereof. In one preferred embodiment, the VL can comprise L1 having an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 14, L2 having an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 15, L3 having an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 16, or any combination thereof. In one particular example, an anti-CD39 antibody of the present disclosure comprises a VH comprising H1 having an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 10, H2 having an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 11, H3 having an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 12, and a VL comprising L1 having an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 14, L2 having an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 15, and L3 having an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 16.In another particular example, an anti-CD39 antibody of the present disclosure comprises: a VH comprising a H1 having an amino acid sequence comprising SEQ ID NO: 10, a H2 having an amino acid sequence comprising SEQ ID NO: 11, a H3 having an amino acid sequence comprising SEQ ID NO: 12; and a VL comprising a L1 having an amino acid sequence comprising SEQ ID NO: 14, a L2 having an amino acid sequence comprising SEQ ID NO: 15, and a L3 having an amino acid sequence comprising SEQ ID NO: 16.

[0114] In some of the foregoing embodiments, the antibody can further comprise (i) a mature VH having an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 9 and / or a mature VL having an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 13. In one exemplary embodiment, the antibody comprises a mature VH and a mature VL, and is an antibody of Table A.

[0115] Table A

[0116] Antibody Mature VH Mature VL A SEQ ID NO:9 SEQ ID NO:13

[0117] In each of the foregoing embodiments, the anti-CD39 antibody can be (i) an intact antibody or antigen binding fragment and / or (ii) a chimeric, humanized, or human antibody. Suitable chimeric, humanized, and human antibodies are further described in Sections IIC, IID, and IIE. In certain embodiments, the anti-CD39 antibody can optionally comprise one or more constant regions or portions of constant regions that are substantially human. Suitable constant regions are further described in detail in Section II(F).

[0118] In another exemplary embodiment, the antibody is an antibody of Table B.

[0119] Table B

[0120] Antibody Mature HC Mature LC ch19_IGG4.P SEQ ID NO:35 SEQ ID NO:36

[0121] In another example, an anti-CD39 antibody of the present disclosure comprises a heavy chain variable region (VH) having one or more CDRs derived from SEQ ID NO: 17, SEQ ID NO: 43, or SEQ ID NO: 44, and optionally a light chain variable region (VL) having one or more CDRs derived from SEQ ID NO: 21, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, or SEQ ID NO: 49. The CDRs derived from SEQ ID NO: 17, SEQ ID NO: 43, or SEQ ID NO: 44 can be H1, H2, H3, or any combination thereof. In certain embodiments, the VH can comprise H1 having at least 90% sequence identity to SEQ ID NO: 18, H2 having at least 90% sequence identity to SEQ ID NO: 19, H3 having at least 90% sequence identity to SEQ ID NO: 20, or any combination thereof. The CDRs derived from SEQ ID NO: 21, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, or SEQ ID NO: 49 can be L1, L2, L3, or any combination thereof. In certain embodiments, the VL can comprise L1 having at least 90% sequence identity to SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24, L2 having at least 90% sequence identity to SEQ ID NO: 25, L3 having at least 90% sequence identity to SEQ ID NO: 26, or any combination thereof. An antibody comprising one or more CDRs derived from SEQ ID NO: 17, SEQ ID NO: 43, or SEQ ID NO: 44 can also comprise a VL comprising one or more CDRs derived from SEQ ID NO: 21, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, or SEQ ID NO: 49. The CDRs can be L1, L2, L3, or any combination thereof. In a preferred embodiment, the VL can comprise L1 having at least 90% sequence identity to SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24, L2 having at least 90% sequence identity to SEQ ID NO: 25, L3 having at least 90% sequence identity to SEQ ID NO: 26, or any combination thereof.In one particular example, an anti-CD39 antibody of the present disclosure comprises: a VH comprising a H1 having an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 18, a H2 having an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 19, a H3 having an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 20; and a VL comprising a L1 having an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24, a L2 having an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 25, and a L3 having an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 26. In another particular example, an anti-CD39 antibody of the present disclosure comprises: a VH comprising a H1 having an amino acid sequence comprising SEQ ID NO: 18, a H2 having an amino acid sequence comprising SEQ ID NO: 19, a H3 having an amino acid sequence comprising SEQ ID NO: 20; and a VL comprising a L1 having an amino acid sequence comprising SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24, a L2 having an amino acid sequence comprising SEQ ID NO: 25, and a L3 having an amino acid sequence comprising SEQ ID NO: 26.

[0122] In some of the foregoing embodiments, the antibody can further comprise (i) a VH having an amino acid sequence with at least 80% sequence identity to SEQ ID NO: 17 and / or a VL having an amino acid sequence with at least 80% sequence identity to SEQ ID NO: 21; or (ii) a VH having an amino acid sequence with at least 80% sequence identity to SEQ ID NO: 43 or SEQ ID NO: 44 and / or a VL having an amino acid sequence with at least 80% sequence identity to SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, or SEQ ID NO: 49. In one exemplary embodiment, the antibody comprises a VH and a VL, and is an antibody of Table C.

[0123] Table C

[0124] Antibody Mature VH Mature VL B SEQ ID NO:17 SEQ ID NO:21 C SEQ ID NO:43 SEQ ID NO:45 D SEQ ID NO:43 SEQ ID NO:46 E SEQ ID NO:43 SEQ ID NO:47 F SEQ ID NO:43 SEQ ID NO:48 G SEQ ID NO:43 SEQ ID NO:49 H SEQ ID NO:44 SEQ ID NO:48 I SEQ ID NO:44 SEQ ID NO:49

[0125] In each of the foregoing embodiments, the anti-CD39 antibody can be (i) an intact antibody or antigen-binding fragment and / or (ii) a chimeric, humanized, or human antibody. Suitable chimeric, humanized, and human antibodies are further described in Sections IIC, IID, and IIE. In certain embodiments, the anti-CD39 antibody can optionally comprise one or more constant regions or portions of constant regions that are substantially human. Suitable constant regions are further described in detail in Section II(F).

[0126] In another exemplary embodiment, the antibody is an antibody of Table D.

[0127] Table D

[0128]

[0129]

[0130] In another example, an anti-CD39 antibody of the present disclosure comprises a heavy chain variable region (VH) having one or more CDRs derived from SEQ ID NO: 27, SEQ ID NO: 58, or SEQ ID NO: 59, and optionally a light chain variable region (VL) having one or more CDRs derived from SEQ ID NO: 31, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, or SEQ ID NO: 63. The CDRs derived from SEQ ID NO: 58 or SEQ ID NO: 59 can be H1, H2, H3, or any combination thereof. In certain embodiments, the VH can comprise H1 having at least 90% sequence identity to SEQ ID NO: 28, H2 having at least 90% sequence identity to SEQ ID NO: 29, H3 having at least 90% sequence identity to SEQ ID NO: 30, or any combination thereof. The CDRs derived from SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63 can be L1, L2, L3, or any combination thereof. In certain embodiments, the VL can comprise L1 having at least 90% sequence identity to SEQ ID NO: 32, L2 having at least 90% sequence identity to SEQ ID NO: 33, L3 having at least 90% sequence identity to SEQ ID NO: 34, or any combination thereof. An antibody comprising one or more CDRs derived from SEQ ID NO: 58 or SEQ ID NO: 59 can also comprise a VL comprising one or more CDRs derived from SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63. The CDRs can be L1, L2, L3, or any combination thereof. In a preferred embodiment, the VL can comprise L1 having an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 32, L2 having an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 33, L3 having an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 34, or any combination thereof.In one particular example, an anti-CD39 antibody of the present disclosure comprises: a VH comprising a H1 having an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 28, a H2 having an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 29, and a H3 having an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 30; and a VL comprising a L1 having an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 32, a L2 having an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 33, and a L3 having an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 34. In another particular example, an anti-CD39 antibody of the present disclosure comprises: a VH comprising a H1 having an amino acid sequence comprising SEQ ID NO: 28, a H2 having an amino acid sequence comprising SEQ ID NO: 29, and a H3 having an amino acid sequence comprising SEQ ID NO: 30; and a VL comprising a L1 having an amino acid sequence comprising SEQ ID NO: 32, a L2 having an amino acid sequence comprising SEQ ID NO: 33, and a L3 having an amino acid sequence comprising SEQ ID NO: 34.

[0131] In some of the foregoing embodiments, the antibody can further comprise (i) a VH having an amino acid sequence with at least 80% sequence identity to SEQ ID NO: 27 and / or a VL having an amino acid sequence with at least 80% sequence identity to SEQ ID NO: 31; or (ii) a VH having an amino acid sequence with at least 80% sequence identity to SEQ ID NO: 58 or SEQ ID NO: 59 and / or a VL having an amino acid sequence with at least 80% sequence identity to SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, or SEQ ID NO: 63. In one exemplary embodiment, the antibody comprises a VH and a VL, and is an antibody of Table E.

[0132] Table E

[0133] Antibody Mature VH Mature VL J SEQ ID NO:27 SEQ ID NO:31 K SEQ ID NO:58 SEQ ID NO:60 L SEQ ID NO:58 SEQ ID NO:61 M SEQ ID NO:58 SEQ ID NO:62 N SEQ ID NO:59 SEQ ID NO:61 O SEQ ID NO:59 SEQ ID NO:60 P SEQ ID NO:59 SEQ ID NO:62 Q SEQ ID NO:59 SEQ ID NO:63 R SEQ ID NO:58 SEQ ID NO:63

[0134] In each of the foregoing embodiments, the anti-CD39 antibody can be (i) an intact antibody or antigen binding fragment and / or (ii) a chimeric antibody, a humanized antibody, or a human antibody. Suitable chimeric, humanized, and human antibodies are further described in Sections IIC, IID, and IIE, respectively. In certain embodiments, the anti-CD39 antibody can optionally comprise one or more constant regions or portions of constant regions that are substantially human. Suitable constant regions are described in further detail in Section II(F).

[0135] In another exemplary embodiment, the antibody is an antibody of Table F.

[0136] Table F

[0137] Antibody Mature HC Mature LC ch39_IGG4.P SEQ ID NO:39 SEQ ID NO:40 ch39_mIGG2A.AAG SEQ ID NO:41 SEQ ID NO:42 hu39.1_IGG4.P SEQ ID NO:64 SEQ ID NO:67 hu39.2_IGG4.P SEQ ID NO:64 SEQ ID NO:68 hu39.3_IGG4.P SEQ ID NO:64 SEQ ID NO:69 hu39.4_IGG4.P SEQ ID NO:65 SEQ ID NO:68 hu39.5_IGG4.P SEQ ID NO:65 SEQ ID NO: 67 hu39.5_IGG1.AA SEQ ID NO: 66 SEQ ID NO: 67 hu39.6_IGG4.P SEQ ID NO: 65 SEQ ID NO: 69 hu39.7_IGG4.P SEQ ID NO: 65 SEQ ID NO: 70 hu39.8_IGG4.P SEQ ID NO: 64 SEQ ID NO: 70

[0138] B. Antibodies with Similar Binding Specificity

[0139] The present disclosure also provides anti-CD39 antibodies that bind to the same or overlapping epitope as the antibody referred to above as 19 or chl9_IGG4.P, or the antibody referred to above as 31, ch31_IGG4.P, hu31.1_IGG4.P, hu31.2_IGG4.P, hu31.3_IGG4.P, hu31.4_IGG4.P, hu31.4_IGG1.AA, hu31.5_IGG4.P, hu31.6_IGG4.P, or hu31.7_IGG4.P, or the antibody referred to above as 39, ch39_IGG4.P, ch39_mIGG2A.AA G, hu39.1_IGG4.P, hu39.2_IGG4.P, hu39.3_IGG4.P, hu39.4_IGG4.P, hu39.5_IGG4.P, hu39.5_IGG1.AA, hu39.6_IGG4.P, hu39.7_IGG4.P, or hu39.8_IGG4.P. Epitopes can be identified by methods known in the art, such as X-ray crystallography of antibodies bound to the antigen to identify contacting residues. Competition assays can be used to identify such antibodies. For example, an anti-CD39 antibody can competitively inhibit the binding of a reference antibody to human CD39, the reference antibody selected from 19, chl9_IGG4.P, 31, ch31_IGG4.P, hu31.1_IGG4.P, hu31.2_IGG4.P, hu31.3_IGG4.P, hu31.4_IGG4.P, hu31.4_IGG1.AA, hu31.5_IGG4.P, hu31.6_IGG4.P, or hu31.7_IGG4.P, 39, ch39_IGG4.P, ch39_mIGG2A.AAG, hu39.1_IGG4.P, hu39.2_IGG4.P, hu39.3_IGG4.P, hu39.4_IGG4.P, hu39.5_IGG4.P, hu39.5_IGG1.AA, hu39.6_IGG4.P, hu39.7_IGG4.P, or hu39.8_IGG4.P. An antibody is said to competitively inhibit the binding of a reference antibody to human CD39 if the antibody blocks the binding of the reference antibody to human CD39 by at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%. Competitive inhibition can be determined, for example, by a competition flow assay, as described in Example 7.

[0140] In other embodiments, an antibody that competitively inhibits the binding of a reference antibody to human CD39 comprises a VH, or a VH and a VL, as described in Section II(A).

[0141] Other antibodies having such binding specificity can be generated by immunizing a mouse with human CD39 or a portion thereof including a desired epitope; and screening the resulting antibodies for binding to the extracellular domain of human CD39, optionally in competition with antibody 19 or a variant thereof (e.g., chl9_IGG4.P, etc.), antibody 31 or a variant thereof (e.g., ch31_IGG4.P, hu31.1_IGG4.P, hu31.2_IGG4.P, hu31.3_IGG4.P, hu31.4_IGG4.P, hu31.4_IGG1.AA, hu31.5_IGG4.P, hu31.6_IGG4.P, hu31.7_IGG4.P, etc.), or antibody 39 or a variant thereof (e.g., ch39_IGG4.P, ch39_mIGG2A.AAG, hu39.1_IGG4.P, hu39.2_IGG4.P, hu39.3_IGG4.P, hu39.4_IGG4.P, hu39.5_IGG4.P, hu39.5_IGG1.AA, hu39.6_IGG4.P, hu39.7_IGG4.P, hu39.8_IGG4.P, etc.). Antibodies can also be screened against a mutagenized version of human CD39 to identify antibodies that show the same or similar binding profile to the following antibodies for a set of mutations: 19, chl9_IGG4.P, 31, ch31_IGG4.P, hu31.1_IGG4.P, hu31.2_IGG4.P, hu31.3_IGG4.P, hu31.4_IGG4.P, hu31.4_IGG1.AA, hu31.5_IGG4.P, hu31.6_IGG4.P, hu31.7_IGG4.P, 39, ch39_IGG4.P, ch39_mIGG2A.AAG, hu39.1_IGG4.P, hu39.2_IGG4.P, hu39.3_IGG4.P, hu39.4_IGG4.P, hu39.5_IGG4.P, hu39.5_IGG1.AA, hu39.6_IGG4.P, hu39.7_IGG4.P, or hu39.8_IGG4.P. The mutations can be systematic substitution of one residue with alanine (or serine if alanine is already present) at time intervals simultaneously or more widely spaced throughout the extracellular domain of the CD39 antibody or portion thereof known to be an epitope.

[0142] Other antibodies can be obtained by mutagenesis of the cDNA encoding the heavy and light chains of an exemplary antibody, such as 19, chl9_IGG4.P, 31, ch31_IGG4.P, hu31.1_IGG4.P, hu31.2_IGG4.P, hu31.3_IGG4.P, hu31.4_IGG4.P, hu31.4_IGG1.AA, hu31.5_IGG4.P, hu31.6_IGG4.P, hu31.7_IGG4.P, 39, ch39_IGG4.P, ch39_mIGG2A.AAG, hu39.1_IGG4.P, hu39.2_IGG4.P, hu39.3_IGG4.P, hu39.4_IGG4.P, hu39.5_IGG4.P, hu39.5_IGG1.AA, hu39.6_IGG4.P, hu39.7_IGG4.P, or hu39.8_IGG4.P. The present disclosure also includes antibodies that are at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any of the foregoing in the amino acid sequence of the mature heavy and / or light chain variable region and retain the functional properties thereof, and / or differ from the corresponding antibody by a small number of functionally inconsequential amino acid substitutions (e.g., conservative substitutions), deletions, or insertions. Amino acids in the variable region framework that can be important for binding can be identified as described below in the section on humanization.

[0143] Variations of the phage display method can also be used to generate antibodies having the binding specificity of a selected rodent antibody (e.g., 19, 31, or 37) or a selected humanized antibody (e.g., hu31.1_IGG4.P, hu31.2_IGG4.P, hu31.3_IGG4.P, hu31.4_IGG4.P, hu31.4_IGG1.AA, hu31.5_IGG4.P, hu31.6_IGG4.P, hu31.7_IGG4.P, hu39.1_IGG4.P, hu39.2_IGG4.P, hu39.3_IGG4.P, hu39.4_IGG4.P, hu39.5_IGG4.P, hu39.5_IGG1.AA, hu39.6_IGG4.P, hu39.7_IGG4.P, or hu39.8_IGG4.P). See Winter, WO 92 / 20791. This method is particularly suitable for generating human antibodies. In this method, the heavy or light chain variable region of the selected antibody is used as starting material. For example, if the light chain variable region is selected as starting material, a phage library is constructed in which members display the same light chain variable region (i.e., that of the starting material) and different heavy chain variable regions. The heavy chain variable regions can be obtained, for example, from a library of rearranged human heavy chain variable regions. Phage displaying strong specific binding (e.g., at least 10 8 or at least 10 9 M -1 ) for human CD39 are selected. The heavy chain variable region from this phage then serves as starting material for construction of another phage library. In this library, each phage displays the same heavy chain variable region (i.e., that identified from the first display library) and a different light chain variable region. The light chain variable regions can be obtained, for example, from a library of rearranged human variable light regions. Again, phage showing strong specific binding for human CD39 are selected. The resulting antibody typically has the same or similar epitope specificity as the murine starting material.

[0144] In addition to the antibodies described herein, it can be possible to design antibody mimetics or aptamers with the same binding specificity as the antibodies of the application that function essentially the same as the antibodies of the application using methods known in the art. By "antibody mimetic" is meant a polypeptide or protein that can specifically bind to an antigen but is not structurally related to an antibody. Antibody mimetics range in mass from about 3 kDa to about 20 kDa. Non-limiting examples of antibody mimetics are affibody molecules, affilins, affimers, alphabodies, anticalins, avimers, DARPins, and unibodies. Aptamers are a class of small nucleic acid ligands composed of RNA or single-stranded DNA oligonucleotides and have high specificity and affinity for their targets. Aptamers interact with and bind to their targets via structural recognition, a process similar to the antigen-antibody reaction. Aptamers have a lower molecular weight than antibodies, typically about 8-25 kDa.

[0145] C. Chimeric and veneered antibodies

[0146] As described above, the present disclosure provides chimeric and veneered forms of non-human antibodies, including but not limited to chimeric anti-CD39 antibodies of antibodies, referred to herein as antibody 19, antibody 31, or antibody 39.

[0147] In certain embodiments, a chimeric antibody is an antibody in which the mature variable regions of the light and heavy chains of a non-human antibody (e.g., mouse, rat, etc.) are combined with human light and heavy chain constant regions. Such antibodies substantially or completely retain the binding specificity of the non-human antibody, and are about two-thirds human sequence.

[0148] A veneered antibody is a type of humanized antibody that retains some, and usually all, of the CDRs and some of the non-human variable region framework residues of the non-human antibody, but replaces other variable region framework residues (e.g., exposed residues) that can contribute to B- or T-cell epitopes with residues from the corresponding positions of a human antibody sequence (Padlan, Mol. Immunol. 28:489, 1991). The result is an antibody in which the CDRs are completely or substantially from the non-human antibody and the variable region framework of the non-human antibody is made more human by substitutions. Veneered forms of anti-CD39 antibodies are included in the present disclosure.

[0149] In some embodiments, the anti-CD39 chimeric antibody is a rat-human chimera having rat variable domains and human IgGl and kappa constant domains (or variants thereof) or human IgG4 and kappa constant domains (or variants thereof). Suitable human constant domains are known in the art and are further described in Section II(F). In one particular embodiment, the anti-CD39 chimeric antibody is ch19_IGG4.P, ch31_IGG4.P, and ch39_IGG4.P.

[0150] D. Humanized Antibodies

[0151] As described herein, the present disclosure provides humanized antibodies of the antibodies referred to herein as 19, 31, or 39, optionally wherein the humanized antibody inhibits human CD39 enzymatic activity with an IC50 of about 5 nM or less, about 0.05 nM to about 5 nM, about 1 nM or less, about 0.05 nM to about 1 nM, about 0.5 nM or less, or about 0.05 nM to about 0.5 nM, as measured in Example 5. Typically, a non-human antibody is humanized to reduce immunogenicity to humans, while retaining the specificity and affinity of the parental non-human antibody. A humanized antibody comprises one or more variable domains in which CDRs or portions thereof are derived from a non-human antibody, and FRs or portions thereof are derived from human antibody sequences. A humanized antibody optionally can also comprise at least a portion of a human constant region. In some embodiments, some FR residues in a humanized antibody are substituted with corresponding residues from the non-human antibody (e.g., the antibody from which the CDR residues are derived), for example, to restore or improve antibody specificity or affinity. 50 As described herein, the present disclosure provides humanized antibodies of the antibodies referred to herein as 19, 31, or 39, optionally wherein the humanized antibody inhibits human CD39 enzymatic activity with an IC50 of about 5 nM or less, about 0.05 nM to about 5 nM, about 1 nM or less, about 0.05 nM to about 1 nM, about 0.5 nM or less, or about 0.05 nM to about 0.5 nM, as measured in Example 5. Typically, a non-human antibody is humanized to reduce immunogenicity to humans, while retaining the specificity and affinity of the parental non-human antibody. A humanized antibody comprises one or more variable domains in which CDRs or portions thereof are derived from a non-human antibody, and FRs or portions thereof are derived from human antibody sequences. A humanized antibody optionally can also comprise at least a portion of a human constant region. In some embodiments, some FR residues in a humanized antibody are substituted with corresponding residues from the non-human antibody (e.g., the antibody from which the CDR residues are derived), for example, to restore or improve antibody specificity or affinity.

[0152] Accordingly, a humanized antibody is an antibody having some or all of the CDRs and variable region framework sequences from a donor antibody and constant regions, if present, that are completely or substantially from human antibody sequences. For example, a humanized heavy chain has at least one, two, and usually all three CDRs that are completely or substantially from a CDR of a heavy chain of a donor antibody, as well as heavy chain variable region framework sequences and heavy chain constant regions, if present, that are substantially from human heavy chain variable region framework and constant region sequences. Similarly, a humanized light chain has at least one, two, and usually all three CDRs that are completely or substantially from a CDR of a light chain of a donor antibody, as well as light chain variable region framework sequences and light chain constant regions, if present, that are substantially from human light chain variable region framework and constant region sequences. Here, as elsewhere in this application, a CDR in a subject antibody is substantially from a corresponding CDR in a reference antibody when at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the corresponding residues (as defined by Kabat) between the corresponding CDRs are identical; however, a CDR H2 in a subject antibody as defined by Kabat is substantially from a corresponding CDR in a reference antibody when at least about 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the corresponding residues (as defined by Kabat) between the corresponding CDRs are identical. A variable region framework sequence of an antibody chain or a constant region of an antibody chain is substantially from a human variable region framework sequence or a human constant region, respectively, when at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the corresponding residues defined by Kabat are identical.

[0153] Generally, a humanized antibody is a genetically engineered antibody in which CDRs from a non-human "donor" antibody are grafted into a human "acceptor" antibody sequence (see, e.g., Queen, US 5,530,101 and 5,585,089; Winter, US 5,225,539, Carter, US 6,407,213, Adair, US 5,859,205 6,881,557, Foote, US 6,881,557). The acceptor antibody sequence can be, for example, a mature human antibody sequence, a composite of such sequences, a consensus sequence or a germline region sequence of human antibodies. Although a humanized antibody can incorporate all six CDRs from a non-human (e.g., mouse, rat, etc.) antibody, it can also be made with fewer than all CDRs (e.g., at least 3, 4, or 5) from a non-human antibody (e.g., Pascalis et al., J. Immunol. 169:3076, 2002; Vajdos et al., Journal of Molecular Biology, 320:415-428, 2002; Iwahashi et al., Mol. Immunol. 36:1079-1091, 1999; Tamura et al., Journal of Immunology, 164:1432-1441, 2000).

[0154] In some antibodies, only a portion of the CDRs in a humanized antibody is needed to retain binding, i.e., a subset of the CDR residues required for binding, referred to as SDRs. CDR residues that do not contact the antigen and that are not in the SDRs can be identified based on prior studies (e.g., residues H60-H65 in CDR H2 are often not needed), from regions of Kabat CDRs that are outside of Chothia hypervariable loops (Chothia, J. Mol. Biol. 196:901, 1987), by molecular modeling and / or empirically, or as described in Gonzales et al., Mol. Immunol. 41:863, 2004. In such humanized antibodies, at positions where one or more donor CDR residues are absent or an entire donor CDR is omitted, the amino acid occupying the position can be the amino acid occupying the corresponding position (by Kabat numbering) in the acceptor antibody sequence. In some antibodies, potential sites for post-translational modification (e.g., glycosylation) in the CDRs can be substituted to eliminate post-translational modification. The positions within the CDRs for substitution and the amino acids to be substituted can also be chosen empirically.

[0155] While the sequence of the acceptor can be identical to the selected human framework sequence, whether it is from a human immunoglobulin or a human consensus framework, the present disclosure contemplates that the acceptor sequence can include pre-existing amino acid substitutions relative to the human immunoglobulin sequence or the human consensus framework sequence. These pre-existing substitutions can be minimal; typically only four, three, two, or one amino acid difference relative to the human immunoglobulin sequence or the consensus framework sequence. The human acceptor antibody sequence can optionally be selected from among a number of known human antibody sequences, resulting in a high degree of sequence identity (e.g., 65-85% identity) between the human acceptor sequence variable region framework and the corresponding variable region framework of the donor antibody chain.

[0156] Certain amino acids from the human variable region framework residues can be selected for substitution based on their potential impact on CDR conformation and / or binding to antigen. Investigation of such potential impacts can be carried out by modeling, inspection of the characteristics of the amino acids at particular positions, or empirical observation of the effects induced by substitution or mutation of particular amino acids. For example, when the amino acid between a non-human variable region framework residue and a selected human variable region framework residue is different, the human framework amino acid can be substituted with the equivalent framework amino acid from the non-human antibody when the amino acid is reasonably expected to: (1) directly non-covalently bind antigen, (2) be proximal to a CDR region, (3) otherwise interact with a CDR region (e.g., within about 5 A of a CDR region). Other substitution candidates at the position are acceptor human framework amino acids that are unusual for human immunoglobulins. These amino acids can be substituted with the amino acid from the equivalent position of the non-human donor antibody or the amino acid from the equivalent position of a more typical human immunoglobulin.

[0157] In some embodiments, the humanized anti-CD39 antibody has a mature VH comprising: H1 comprising the amino acid sequence of SEQ ID NO: 10 with zero to two amino acid substitutions or deletions, H2 comprising the amino acid sequence of SEQ ID NO: 11 with zero to two amino acid substitutions or deletions, H3 comprising the amino acid sequence of SEQ ID NO: 12 with zero to two amino acid substitutions or deletions, and a framework region that is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an acceptor human framework region; and a mature VL comprising: L1 comprising the amino acid sequence of SEQ ID NO: 14 with zero to two amino acid substitutions or deletions, L2 comprising the amino acid sequence of SEQ ID NO: 15 with zero to two amino acid substitutions or deletions, L3 comprising the amino acid sequence of SEQ ID NO: 16 with zero to two amino acid substitutions or deletions, and a framework region that is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an acceptor human framework region. The framework regions can be determined according to the Rabat definition.

[0158] In some embodiments, the humanized anti-CD39 antibody has a mature VH comprising: an H1 comprising the amino acid sequence of SEQ ID NO: 18 with zero to two amino acid substitutions or deletions, an H2 comprising the amino acid sequence of SEQ ID NO: 19 with zero to two amino acid substitutions or deletions, an H3 comprising the amino acid sequence of SEQ ID NO: 20 with zero to two amino acid substitutions or deletions, and a framework region that is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an acceptor human framework region; and a mature VL comprising: an L1 comprising the amino acid sequence of SEQ ID NO: 22 with zero to two amino acid substitutions or deletions, an L2 comprising the amino acid sequence of SEQ ID NO: 25 with zero to two amino acid substitutions or deletions, an L3 comprising the amino acid sequence of SEQ ID NO: 26 with zero to two amino acid substitutions or deletions, and a framework region that is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an acceptor human framework region. For example, the humanized anti-CD39 antibody referred to above can have an L1 comprising SEQ ID NO: 23 or SEQ ID NO: 24. The framework regions can be determined according to the Kabat definition. In some embodiments, the VH human acceptor is M99642 or KF698734 and / or the VL human acceptor is X12682 or Z00023.

[0159] In some embodiments, the humanized anti-CD39 antibody has a mature VH comprising: H1 comprising the amino acid sequence of SEQ ID NO: 28 with zero to two amino acid substitutions or deletions, H2 comprising the amino acid sequence of SEQ ID NO: 29 with zero to two amino acid substitutions or deletions, H3 comprising the amino acid sequence of SEQ ID NO: 30 with zero to two amino acid substitutions or deletions, and a framework region that is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an acceptor human framework region; and a mature VL comprising: L1 comprising the amino acid sequence of SEQ ID NO: 32 with zero to two amino acid substitutions or deletions, L2 comprising the amino acid sequence of SEQ ID NO: 33 with zero to two amino acid substitutions or deletions, L3 comprising the amino acid sequence of SEQ ID NO: 34 with zero to two amino acid substitutions or deletions, and a framework region that is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an acceptor human framework region. The framework regions can be determined according to the Kabat definition. In some embodiments, the VH human acceptor is M99651 or M77327 and / or the VL human acceptor is X12682 or M23090.

[0160] In the foregoing embodiments, the mature heavy chain variable region can be linked to at least a portion of a heavy chain constant region and the mature light chain variable region can be linked to at least a portion of a light chain constant region. In some embodiments, the mature heavy chain variable region is linked to a heavy chain constant region and the mature light chain variable region is linked to a light chain constant region. Suitable constant regions are described in further detail in Section II(F). In certain embodiments, the heavy chain constant region has effector function substantially similar to wild-type human IgGl. In other embodiments, the heavy chain constant region reduces or enhances effector function compared to wild-type human IgGl. In one particular example, the heavy chain constant region comprises or consists of SEQ ID NO: 1 and the light chain constant region comprises or consists of SEQ ID NO: 6. In one particular example, the heavy chain constant region comprises or consists of amino acids 1 to 329 of SEQ ID NO: 1 and the light chain constant region comprises or consists of SEQ ID NO: 6. In another particular example, the heavy chain constant region comprises or consists of SEQ ID NO: 4 and the light chain constant region comprises or consists of SEQ ID NO: 6. In another particular example, the heavy chain constant region comprises or consists of amino acids 1 to 326 of SEQ ID NO: 4 and the light chain constant region comprises or consists of SEQ ID NO: 6. In another particular example, the heavy chain constant region comprises or consists of SEQ ID NO: 2 and the light chain constant region comprises or consists of SEQ ID NO: 6. In another particular example, the heavy chain constant region comprises or consists of amino acids 1 to 329 of SEQ ID NO: 2 and the light chain constant region comprises or consists of SEQ ID NO: 6. In another particular example, the heavy chain constant region comprises or consists of SEQ ID NO: 5 and the light chain constant region comprises or consists of SEQ ID NO: 6. In another particular example, the heavy chain constant region comprises or consists of amino acids 1 to 326 of SEQ ID NO: 5 and the light chain constant region comprises or consists of SEQ ID NO: 6. In another particular example, the heavy chain constant region comprises or consists of SEQ ID NO: 3 and the light chain constant region comprises or consists of SEQ ID NO: 6. In another particular example, the heavy chain constant region comprises or consists of amino acids 1 to 329 of SEQ ID NO: 3 and the light chain constant region comprises or consists of SEQ ID NO: 6.

[0161] E. Human Antibodies

[0162] As described herein, the present disclosure also provides a human antibody having the binding specificity of an antibody referred to herein as 19, chl9_IGG4.P, 31, ch31_IGG4.P, hu31.1_IGG4.P, hu31.2_IGG4.P, hu31.3_IGG4.P, hu31.4_IGG4.P, hu31.4_IGG1.AA, hu31.5_IGG4.P, hu31.6_IGG4.P, hu31.7_IGG4.P, 39, ch39_IGG4.P, ch39_mIGG2A.AAG, hu39.1_IGG4.P, hu39.2_IGG4.P, hu39.3_IGG4.P, hu39.4_IGG4.P, hu39.5_IGG4.P, hu39.5_IGG1.AA, hu39.6_IGG4.P, hu39.7_IGG4.P, or hu39.8_IGG4.P, optionally wherein the human antibody inhibits human CD39 enzyme activity with an IC50of about 5 nM or less, about 0.05 nM to about 5 nM, about 1 nM or less, about 0.05 nM to about 1 nM, about 0.5 nM or less, or about 0.05 nM to about 0.5 nM, as measured in Example 5. Human antibodies can be selected to have the same epitope specificity as a particular rodent antibody (e.g., an antibody referred to herein as 19, 31, or 39), or the same epitope specificity as a particular humanized antibody (e.g., an antibody referred to herein as chl9_IGG4.P, ch31_IGG4.P, hu31.1_IGG4.P, hu31.2_IGG4.P, hu31.3_IGG4.P, hu31.4_IGG4.P, hu31.4_IGG1.AA, hu31.5_IGG4.P, hu31.6_IGG4.P, hu31.7_IGG4.P, ch39_IGG4.P, ch39_mIGG2A.AAG, hu39.1_IGG4.P, hu39.2_IGG4.P, hu39.3_IGG4.P, hu39.4_IGG4.P, hu39.5_IGG4.P, hu39.5_IGG1.AA, hu39.6_IGG4.P, hu39.7_IGG4.P, or hu39.8_IGG4.P) by competitive binding experiments, the phage display method of Winter, WO 92 / 20791, or otherwise. Human antibodies can also be screened for specificities to particular epitopes by using only fragments of CD39 as target antigens, and / or by screening antibodies against a collection of deletion mutants of CD39. 50 50

[0163] Methods for generating human antibodies include the trioma methods of Oestberg et al., Hybridoma 2:361-367 (1983); Oestberg, U.S. Patent No. 4,634,664; and Engleman et al., U.S. Patent No. 4,634,666, using transgenic mice comprising human immunoglobulin genes (see, e.g., Lonberg et al., WO 93 / 12227 (1993); US 5,877,397, US 5,874,299, US 5,814,318, US 5,789,650, US 5,770,429, US 5,661,016, US 5,633,425, US 5,625,126, US 5,569,825, US 5,545,806; Nature 148, 1547-1553 (1994); Nature Biotechnology 14, 826 (1996); Kucherlapati, WO 91 / 10741 (1991)) and phage display methods (see, e.g., Dower et al., WO 91 / 17271 and McCafferty et al., WO 92 / 01047; US 5,877,218, US 5,871,907, US 5,858,657, US 5,837,242, US 5,733,743, and US 5,565,332).

[0164] The human antibodies of the present disclosure can comprise a mature heavy chain variable region linked to at least a portion of a heavy chain constant region and a mature light chain variable region linked to at least a portion of a light chain constant region. In some embodiments, the mature heavy chain variable region is linked to a heavy chain constant region and the mature light chain variable region is linked to a light chain constant region. Suitable constant regions are described in further detail in Section II(F). In certain embodiments, the heavy chain constant region has effector function substantially similar to wild-type human IgGl. In other embodiments, the heavy chain constant region reduces or enhances effector function compared to wild-type human IgGl. In one particular example, the heavy chain constant region comprises or consists of SEQ ID NO: 1 and the light chain constant region comprises or consists of SEQ ID NO: 6. In another particular example, the heavy chain constant region comprises or consists of SEQ ID NO: 4 and the light chain constant region comprises or consists of SEQ ID NO: 6. In another particular example, the heavy chain constant region comprises or consists of SEQ ID NO: 2 and the light chain constant region comprises or consists of SEQ ID NO: 6. In another particular example, the heavy chain constant region comprises or consists of SEQ ID NO: 5 and the light chain constant region comprises or consists of SEQ ID NO: 6. In another particular example, the heavy chain constant region comprises or consists of SEQ ID NO: 3 and the light chain constant region comprises or consists of SEQ ID NO: 6.

[0165] F. Selection of Constant Region

[0166] The heavy and light chain variable regions of a chimeric antibody, humanized (including veneered) antibody, or human antibody can each be linked to at least a portion of a human constant region. In some embodiments, the heavy chain variable domain described in the above sections is linked to a portion of a human heavy chain constant region and the light chain variable domain described in the above sections is linked to a portion of a human light chain constant region. In some embodiments, the heavy chain variable domain described in the above sections is linked to a portion of a human heavy chain constant region and the light chain variable domain described in the above sections is linked to a full-length human light chain constant region. In some embodiments, the heavy chain variable domain described in the above sections is linked to a full-length human heavy chain constant region and the light chain variable domain described in the above sections is linked to a full-length human light chain constant region.

[0167] The choice of constant region (or truncation thereof) depends in part on whether effector function is desired, or even if enhancement is desired. "Effector function" refers to a biological activity attributable to the light chain or heavy chain constant region of an antibody and varies with the antibody isotype. Non-limiting examples of antibody effector functions include: Clq binding C1 complex and complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g., B cell receptor); and B cell activation. Human antibodies are classified into five isotypes (IgM, IgD, IgG, IgA, and IgE) depending on their heavy chains, each of which provides different functions. IgG consists of four human subclasses (IgGl, IgG2, IgG3, and IgG4) each containing a different heavy chain. They are highly homologous and differ primarily in the hinge region and the extent to which they activate the host immune system. For example, human isotypes IgGl and IgG3 can mediate complement-mediated cytotoxicity and human isotypes IgG2 and IgG4 mediate or do not mediate complement-mediated cytotoxicity at very low levels. The light chain constant region can be of subclass lambda or kappa.

[0168] Antibodies of the disclosure comprising a human constant region or portion thereof are typically IgG antibodies, preferably IgGl or IgG4 antibodies. Human constant regions show isoallotypic variation and isoallotypic variation between different individuals, i.e., the constant region can differ in different individuals at one or more polymorphic positions. Isoallotypic variation differs from allotypic variation in that serum recognizing the isoallotype binds to non-polymorphic regions of one or more other isotypes. Reference to a human constant region includes constant regions having any arrangement of residues of any natural allotype or occupying polymorphic positions in a natural allotype.

[0169] One or several amino acids at the amino- or carboxy-terminus of the light chain and / or heavy chain, such as the C-terminal lysine of the heavy chain, can be missing or derivatized in a proportion or all molecules. The N-terminal glutamine of the heavy or light chain can be substituted with a glutamic acid residue to prevent the formation of pyroglutamate.

[0170] In some embodiments, the antibodies of the disclosure are IgG4 antibodies. With respect to human IgG4, an engineered mutation including S228P (Eu numbering) on the heavy chain can be used to prevent Fab arm exchange. Sequences suitable for human IgG4 include, but are not limited to, SEQ ID NO: 4 and SEQ ID NO: 5. In some embodiments, the C-terminal lysine of the IgG4 antibody is absent.

[0171] In some embodiments, the antibodies of the present disclosure are IgGl or IgG3 antibodies. Sequences suitable for human IgGl or IgG3 are known in the art and include, but are not limited to, SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and human IgG3 disclosed in US 5,624,821. In some embodiments, the C-terminal lysine of the IgGl or IgG3 antibody is absent.

[0172] In certain embodiments, substitutions can be made in the constant region to extend half-life in humans (see, e.g., Hinton et al., J. Biol. Chem. 279:6213, 2004). Exemplary substitutions include Gln at position 250 and / or Leu at position 428 (Eu numbering) for increasing the half-life of the antibody.

[0173] Alternatively or additionally, substitutions can be made in the constant region to decrease or increase effector function, e.g., complement-mediated cytotoxicity (CDC) or antibody-dependent cell-mediated cytotoxicity (ADCC) (see, e.g., Winter et al., U.S. Patent No. 5,624,821; Tso et al., U.S. Patent No. 5,834,597; and Lazar et al., Proc. Natl. Acad. Sci. USA, 103:4005, 2006).

[0174] Some of the antibodies of the present disclosure are engineered to have reduced Fc effector functions, e.g., CDC, ADCC, and antibody-dependent cellular phagocytosis (ADCP), by introducing constant region mutations as compared to the same antibody without the mutations. In some embodiments, each or all of these effector functions are reduced by at least 50%, 75%, 90%, or 95% as compared to the antibody without the mutations. Effector functions can be determined as described in the Examples. Other assays are described by Shields et al., 2001 J. Biol. Chem., pp. 6591-6604; Chappel et al., 1993 J. Biol. Chem., vol. 268, pp. 25124-25131; Lazar et al., 2006 PNAS, 103; 4005-4010.

[0175] Substitutions at any or all of positions 234, 235, 236, and / or 237 reduce the affinity for Fcy receptors, in particular for the FcyRI receptor (see, e.g., US 6,624,821). In some embodiments, alanine residues are used for substitution, e.g., L234A / L235A double mutation to reduce effector function. Other combinations of mutations with reduced effector function include L234A / L235A / G237A, E233P / L234V / L235A / AG236, A327G / A330S / P331S, K322A, L234A / L235A, L234F / L235E / P331S, and L234A / L235E / G237A / A330S / P331S (Eu numbering). Optionally, positions 234, 236, and / or 237 in human IgG2 are substituted with alanine and position 235 is substituted with glutamine. (See, e.g., US 5,624,821.) Two amino acid substitutions in the complement Clq binding site at positions 330 and 331 of the Eu index reduce complement binding (see Tao et al., J. Exp. Med. 178:661 (1993) and Canfield and Morrison, J. Exp. Med. 173:1483 (1991)). Substitution of IgG2 residues for positions 233-236 of human IgGl and IgG4 residues for positions 327, 330 and 331 reduce ADCC and CDC (see, e.g., Armour KL. et al., 1999 Eur J Immunol. 29(8):2613-24; and Shields RL. et al., 2001. J Biol Chem. 276(9):6591-604). N297A, N297Q, or N297G (Eu numbering) mutations reduce glycosylation and thereby reduce effector function.

[0176] Some antibodies of the present disclosure are engineered to have enhanced Fc effector function by introducing constant region mutations. For example, FcyR binding can be enhanced by amino acid engineering. In some embodiments, this can be done by substituting one or more amino acids in the Fc region. Desirable mutations can be determined by, e.g., alanine scanning or rational design and library screening. IgG variants with enhanced binding to FcyR and enhanced effector function can be identified using these techniques. Alternatively, several mutations of the Fc receptor region are known in the art, e.g., as described in Smith P. et al. (2012) PNAS 6181-6186.

[0177] In some embodiments, the antibodies described herein include modified IgGl constant domains that increase the ability of the antibody to mediate ADCC compared to unmodified wild-type IgGl. The modified IgGl domains can be characterized by amino acid substitutions at one or more of L235V, S239D, F243L, R292P, A330L, I332E, P396L (Eu numbering). In other embodiments, the modified IgGl domains are characterized by substitutions at S239D, A330L, and I332E (Eu numbering).

[0178] Alternatively, glycoform perturbations can be used to enhance Fc-mediated therapeutic antibody function. N-linked Fc glycosylation on IgGl antibodies is critical for effector function. Sialylation, galactosylation, bisecting, and fucosylation can all influence IgG molecule binding and activity. Controlling the glycosylation pattern on therapeutic antibodies can be done in many different ways. The cell type in which the recombinant antibody is produced and its culture conditions can influence the glycosylation and activity of the therapeutic antibody. In addition, bioreactor conditions and downstream processing can also influence glycan microheterogeneity. Low or no fucosylated antibodies have been shown to enhance Fc-mediated properties. Many ways to achieve this reduction in fucose levels through glycoengineering are well known in the art. One way is to manipulate enzymes involved in post-translational modification of the antibody. This can involve overexpression of glucosidases, such as beta-1-4-N-acetylglucosaminyltransferase III, genetic knockout of fucosyltransferases, or use of cell lines that naturally lack fucose or have been mutated to express low levels of fucosylation. In addition, inhibitors of N-linked glucosidases, such as castanospermine, can also be used to obtain low fucose with IgG molecules.

[0179] In some embodiments, amino acid engineered variants can have more broad enhanced affinity for multiple FcyRs, while glycoengineered antibodies can typically have more specific affinity for enhanced FcyRIIIa binding. Glycoforms interact with proximal amino acids on the Fc portion and substitution of amino acids in contact with the Ig oligosaccharide can yield different glycoform structures.

[0180] G. Expression of recombinant antibodies

[0181] Chimeric antibodies, humanized (including veneered) antibodies, and human antibodies are typically produced by recombinant expression. Accordingly, the present disclosure also provides polynucleotides encoding the anti-CD39 antibodies of the IIA-F portion, vectors comprising the polynucleotides, and host cells comprising the vectors.

[0182] Polynucleotides encoding the anti-CD39 antibodies of the present disclosure can be inserted into vectors for amplification, expression, or further optimization. Numerous vectors are available. In some embodiments, the vector system includes mammalian, bacterial, yeast systems, etc., and includes plasmids such as, but not limited to, pALTER, pBAD, pcDNA, pCal, pL, pET, pGEMEX, pGEX, pCI, pCMV, pEGFP, pEGFT, pSV2, pFUSE, pVITRO, pVIVO, pMAL, pMONO, pSELECT, pUNO, pDUO, Psg5L, pBABE, pWPXL, pBI, p15TV-L, pPro18, pTD, pRS420, pLexA, pACT2.2, and the like, as well as other laboratory and commercially available vectors. Suitable vectors can include plasmid or viral vectors (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses). Vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, enhancer elements, a promoter (e.g., SV40, CMV, EF-1a), and a transcription termination sequence. For expression, recombinant polynucleotide constructs generally include expression control sequences operably linked to the coding sequences for the antibody chains, including naturally associated or heterologous promoter regions. In some embodiments, the expression control sequences are eukaryotic promoter systems capable of

[0183] Vectors comprising polynucleotide sequences encoding the anti-CD39 antibodies of the present disclosure can be introduced into host cells for cloning or gene expression. Host cells suitable for cloning or expression of polynucleotide sequences in vectors herein include prokaryotic and eukaryotic cells. Non-limiting examples of suitable prokaryotes include eubacteria, such as Gram-negative or Gram-positive organisms, for example, Enterobacteriaceae, such as Escherichia, e.g., E. coli; Enterobacter; Erwinia; Klebsiella; Proteus; Salmonella, e.g., S. typhimurium; Serratia, e.g., Serratia marcescans; and Shigella; and Bacilli, such as B. subtilis and B. licheniformis; Pseudomonas, such as P. aeruginosa; and Streptomyces. In addition to prokaryotes, eukaryotes, for example, fungi or yeasts, are suitable hosts for cloning or expression of vectors encoding the anti-CD39 antibodies.Non-limiting examples include Saccharomyces cerevisiae, Schizosaccharomyces pombe; Kluyveromyces hosts, such as K. lactis, K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906), K. thermotolerans, and K. marxianus; Yarrowia (EP 402,226); Pichia pastoris (EP 183,070); Candida; Trichoderma reesia (EP 244,234); Neurospora crassa; Schwanniomyces, such as S. occidentalis; and filamentous fungi, such as Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts, such as A. nidulans and A. niger. Suitable host cells can also be derived from multicellular organisms. Examples of invertebrate cells include plant and insect cells. A number of baculoviral strains and variants and corresponding permissive insect host cells from, for example, Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruit fly), and Bombyx mori have been identified. A number of viral strains for transfection are publicly available, for example, the L-l variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV, and such viruses are available for use as viral vectors in the present application, particularly for transfection of Spodoptera frugiperda cells.Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco can also be utilized as hosts. In some embodiments, mammalian cells are host cells for expressing nucleotide segments encoding immunoglobulins or fragments thereof. See Winnacker, From Genes to Clones, (VCH Publishers, NY, 1987). A number of suitable host cell lines capable of secreting intact heterologous proteins have been developed in the art and include CHO cell lines, various COS cell lines, HeLa cells, HEK293 cells, L cells, and non-antibody-producing myeloma lines including Sp2 / 0 and NS0. In some embodiments, the cells are non-human. Expression vectors for these cells can include, for example, expression control sequences, such as origins of replication, promoters, enhancers (Queen et al., Immunol. Rev. 89:49 (1986)), and necessary processing information sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcriptional terminator sequences. In some embodiments, the expression control sequences are promoters derived from endogenous genes, cytomegalovirus, SV40, adenovirus, bovine papilloma virus, and the like. See Co et al., J. Immunol. 148:1149 (1992).

[0184] Host cells are transformed with the above-described expression or cloning vectors for producing anti-CD39 antibodies and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences. Once expressed, the antibodies can be purified based on standard procedures in the art including HPLC purification, column chromatography, gel electrophoresis, and the like (see generally Scopes, Protein Purification (Springer-Verlag, NY, 1982)).

[0185] H. Labeled Antibodies

[0186] The disclosed anti-CD39 antibodies can be optionally labeled with one or more detectable signals, including but not limited to fluorescent molecules, spin-label molecules, enzymes, or radioisotopes. Such antibodies can be used to detect or isolate human CD39 in any number of in vitro, in vivo, or ex vivo assays.

[0187] For example, when any of the disclosed anti-CD39 antibodies are labeled with a detectable signal, such as a fluorescent molecule, spin-label molecule, enzyme, or radioisotope, such detectable antibodies can be used in pharmacodynamic assays, immunohistochemistry, receptor occupancy assays, ELISA (enzyme-linked immunosorbent assay), EIA (enzyme immunoassay), RIA (radioimmunoassay), and the like.

[0188] III. Methods of Use

[0189] The present disclosure provides methods for using the anti-CD39 antibodies described herein to prepare a medicament for inhibiting CD39 enzymatic activity. As used herein, the terms "inhibit," "inhibition," and the like refer to the ability of an antagonist to decrease the function or activity of a particular target, e.g., CD39. The decrease is preferably at least 50% and can be, for example, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95%. The present disclosure also encompasses the use of the anti-CD39 antibodies described herein to prepare a medicament for treating or preventing a disease, disorder, and / or condition that would benefit from inhibiting CD39 enzymatic activity. As one example, the present disclosure encompasses the use of the anti-CD39 antibodies described herein to prepare a medicament for treating a disease characterized by (i) high extracellular ATP (as compared to non-diseased tissue), (ii) expression of ENTPD1, P2RX7, P2RY11, or a combination thereof in the diseased tissue or increased expression thereof, (iii) expression of CD39, P2X7, P2Y11, or a combination thereof in the diseased tissue or increased expression thereof, (iv) bone marrow infiltration into the diseased tissue, or any combination of (i) to (iv). In another example, the present disclosure encompasses the use of the anti-CD39 antibodies described herein to prepare a medicament for treating a cancer. Optionally, the cancer can be characterized by (i) high extracellular ATP (as compared to non-diseased sample), (ii) expression of ENTPD1, P2RX7, P2RY11, or a combination thereof in the diseased tissue or increased expression thereof, (iii) expression of CD39, P2X7, P2Y11, or a combination thereof in the diseased tissue or increased expression thereof, (iv) bone marrow infiltration into the diseased tissue, or any combination of (i) to (iv). In some embodiments of the foregoing methods, the anti-CD39 antibodies described herein are used in combination with at least one additional therapy, examples of which are set forth elsewhere herein.

[0190] Extracellular ATP is present in negligible concentrations (e.g., about 10-100 nM) under healthy conditions, but rapidly increases in response to tissue injury, stress, hypoxia, and agents used to treat cancer, and can be found in high concentrations in tumors. ATP released by dying or stressed cells, including but not limited to cancer cells and other cells in the tumor microenvironment, provides an inflammatory signal important for effective innate and adaptive immune responses. Conversely, hydrolysis of extracellular ATP to adenosine in, for example, the tumor microenvironment, limits immune responses. CD39 is a rate-limiting ectoenzyme in the hydrolysis of extracellular ATP. By catabolizing the conversion of extracellular ATP to AMP, CD39 also increases extracellular adenosine production via CD73 (ecto-5'-nucleotidase), a rate-limiting ectoenzyme in the hydrolysis of extracellular AMP. Adenosine signals via type A 2a and A 2b receptors expressed on the surface of immune cells and has opposite effects from those mediated by ATP receptors. In particular, A 2a and A 2b receptors on tumor infiltrating immune cells in the tumor microenvironment elicit immune suppressive effects. Opposing effects of adenosine and ATP are reviewed in Chiarella et al., “Extracellular ATP and Adenosine in Cancer Pathogenesis and Treatment”, Trends in Cancer, 2021, 7(8):731-750. As demonstrated herein, using anti-CD39 antibodies of the present disclosure potently inhibits CD39 enzymatic activity, resulting in immune stimulation from ATP accumulation and preventing the formation of immune suppressive adenosine. Diseases, disorders, and / or conditions that would benefit from inhibiting CD39 enzymatic activity can include diseases, disorders, and / or conditions in which release of extracellular ATP can be higher, for example due to tissue injury, stress, hypoxia treatment by additional therapies, or any combination thereof. Additional diseases, disorders, and / or conditions that would benefit from inhibiting CD39 enzymatic activity can include additional diseases, disorders, and / or conditions in which extracellular adenosine levels are higher in diseased tissue samples, for example due to increased ATP hydrolysis compared to healthy controls and / or due to increased AMP hydrolysis compared to healthy controls (e.g., as measured by increased CD39 enzymatic activity in peripheral blood samples or tissue (e.g., tumor) samples), and / or in which CD39 and / or CD73 expression is detectable, and optionally higher compared to healthy controls, for example as measured by immunohistochemistry, immunophenotyping, RNA sequencing, or other clinically validated methods in peripheral blood samples or tissue (e.g., tumor) samples.

[0191] Accordingly, in some embodiments, an anti-CD39 antibody described herein is administered to a subject in need thereof in an amount effective to inhibit CD39 enzymatic activity. CD39 enzymatic activity can be assessed using a peripheral blood sample or a tissue (e.g., tumor) sample obtained from the subject, or both. As a non-limiting example, CD39 activity in the cellular and acellular compartments of a peripheral blood sample or a tumor sample can be measured using a commercial assay such as or Alternatively or additionally, CD39 enzymatic activity in tumor tissue can be measured by enzyme histochemistry. Inhibition can be determined, for example, by comparison to a prior sample obtained from the subject (i.e., prior to administration of the anti-CD39 antibody) or by comparison to a reference sample or reference value of a control group (e.g., subjects administered a control antibody, an anti-CD39 antibody that binds but does not inhibit CD39 enzymatic activity, a standard of care, a placebo, etc.). In some embodiments, an anti-CD39 antibody described herein is administered to a subject in need thereof in an amount effective to inhibit CD39 enzymatic activity in the tumor microenvironment. In some embodiments, an anti-CD39 antibody described herein is administered to a subject in need thereof in an amount effective to inhibit CD39 enzymatic activity in the tumor periphery. In some embodiments, an anti-CD39 antibody described herein is administered to a subject in need thereof in an amount effective to inhibit CD39 enzymatic activity in or on immune cells. In some embodiments, an anti-CD39 antibody described herein is administered to a subject in need thereof in an amount effective to inhibit CD39 enzymatic activity in or on cells of myeloid lineage. Myeloid lineage cells that express CD39 include those detailed in the Examples. In some embodiments, an anti-CD39 antibody described herein is administered to a subject in need thereof in an amount effective to inhibit CD39 enzymatic activity in or on stroma and / or blood vessels.

[0192] Alternatively or additionally, in some embodiments, an anti-CD39 antibody described herein is administered to a subject in need thereof in an amount effective to increase ATP-mediated immune stimulation compared to an appropriate control (e.g., a subject administered an isotype control antibody, a subject administered an anti-CD39 antibody that binds to but does not inhibit CD39 enzymatic activity, a subject receiving standard of care, a subject not receiving treatment). ATP-mediated immune stimulation can be assessed using a peripheral blood sample and / or a tissue (e.g., tumor) sample obtained from the subject. ATP-mediated immune stimulation can be identified, for example, by: (i) measuring an increase in ATP-dependent signaling via one or more type 2 purinergic (P2) receptors (e.g., P2Y G protein-coupled receptors or P2X cation-selective channel receptors); (ii) measuring an increase in NLRP3 inflammasome activation; (iii) measuring an increase in cell surface markers of dendritic cells; (iv) measuring an increase in CD4+ and / or CD8+ T cell activity and / or proliferation, and / or (V) measuring an increase in activation, maturation, cytokine secretion, or any combination thereof in one or more myeloid lineage cell types. In one example, an anti-CD39 antibody described herein is administered to a subject in need thereof in an amount effective to increase ATP-dependent signaling via one or more P2X receptors or P2Y receptors selected from P2X4, P2X5, P2X7, P2Y2, or P2Y11 compared to an appropriate control (e.g., a subject administered an isotype control antibody, a subject administered an anti-CD39 antibody that binds to but does not inhibit CD39 enzymatic activity, a subject receiving standard of care, a subject not receiving treatment). In one example, an anti-CD39 antibody described herein is administered to a subject in need thereof in an amount effective to increase ATP-dependent signaling via P2X7 compared to an appropriate control (e.g., a subject administered an isotype control antibody, a subject administered an anti-CD39 antibody that binds to but does not inhibit CD39 enzymatic activity, a subject receiving standard of care, a subject not receiving treatment). In one example, an anti-CD39 antibody described herein is administered to a subject in need thereof in an amount effective to increase ATP-dependent signaling via P2Y11 compared to an appropriate control (e.g., a subject administered an isotype control antibody, a subject administered an anti-CD39 antibody that binds to but does not inhibit CD39 enzymatic activity, a subject receiving standard of care, a subject not receiving treatment). In one example, an anti-CD39 antibody described herein is administered to a subject in need thereof in an amount effective to increase ATP-dependent signaling via P2X7 and / or P2Y11 compared to an appropriate control (e.g., a subject administered an isotype control antibody, a subject administered an anti-CD39 antibody that binds to but does not inhibit CD39 enzymatic activity, a subject receiving standard of care, a subject not receiving treatment).In one example, an anti-CD39 antibody described herein is administered to a subject in need thereof in an amount effective to increase NLRP3 inflammasome activation. Measures of NLRP3 inflammasome activation are known in the art and include those detailed in the Examples (e.g., IL-1β and IL-18 secretion). In one embodiment, an anti-CD39 antibody described herein is administered to a subject in need thereof in an amount effective to increase myeloid cell activation, maturation, cytokine secretion, or any combination thereof. Measures of myeloid cell activation, maturation, cytokine secretion are known in the art and include those detailed in the Examples.

[0193] Alternatively or in addition to the above, in some embodiments, an anti-CD39 antibody described herein is administered to a subject in need thereof in an amount effective to reduce or prevent adenosine-mediated immunosuppression as compared to a suitable control (e.g., a subject administered an isotype control antibody, a subject administered an anti-CD39 antibody that binds but does not inhibit CD39 enzyme activity, a subject receiving standard of care, a subject not receiving treatment). Adenosine-mediated immunosuppression can be identified, for example, by measuring an increase in adenosine signaling via A 2a R and / or A 2b R. Adenosine-mediated immunosuppression also includes adenosine-mediated suppression of lymphoid (e.g., T cells, B cells) and / or myeloid (e.g., monocytes, macrophages, dendritic cells, NK cells) cell activity. For example, one measure of adenosine-mediated immunosuppression can be NECA-induced pCREB activation in CD8+ T cells in human blood.

[0194] Alternatively or in addition to the above, in some embodiments, an anti-CD39 antibody described herein is administered to a subject in need thereof in an amount effective to achieve at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% target (i.e., CD39) engagement as measured by a receptor occupancy assay. In some embodiments, an anti-CD39 antibody described herein is administered to a subject in need thereof in an amount effective to achieve at least at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99 target (i.e., CD39) engagement as measured by a receptor occupancy assay. Exemplary receptor occupancy assays are detailed in the Examples.

[0195] Alternatively or in addition to the above, in some embodiments, an anti-CD39 antibody described herein is administered to a subject in need thereof in an amount effective to reduce CD39 expression. In some embodiments, an anti-CD39 antibody described herein is administered to a subject in need thereof in an amount effective to reduce cell surface CD39 expression. CD39 expression (intracellular and / or extracellular) can be assessed, for example, by immunohistochemistry or immunophenotyping, including by those methods further detailed in the Examples.

[0196] Alternatively or in addition to the above, in some embodiments, an anti-CD39 antibody described herein is administered to a subject in need thereof in an amount effective to treat or prevent cancer. Without wishing to be bound by theory, Applicants believe that increasing ATP-mediated immune stimulation and / or decreasing (or preventing) adenosine-mediated immune suppression can result in tumor death and can result in improved outcomes.

[0197] A. Oncology and Oncology-Related Disorders

[0198] In one or more embodiments, the antibodies described herein are useful for treating and / or preventing cancer (e.g., carcinoma, sarcoma, leukemia, lymphoma, myeloma, etc.). In certain embodiments, the cancer can be locally advanced and / or unresectable, metastatic, or at risk of becoming metastatic. Alternatively or additionally, the cancer can be relapsed or no longer responsive to treatment, e.g., standard of care or checkpoint inhibitors. Exemplary types of cancer encompassed by the present disclosure include genitourinary tract (e.g., bladder, kidney, renal cell, penis, prostate, testicle, Von Hippel-Lindau disease, etc.), uterine, cervix, ovary, breast, gastrointestinal tract (e.g., esophagus, oropharynx, stomach, small intestine or large intestine, colon or rectum), bone, bone marrow, skin (e.g., melanoma), head and neck, liver, gall bladder, bile duct, heart, lung, pancreas, salivary gland, adrenal gland, thyroid, brain (e.g., glioma), ganglia, central nervous system (CNS), peripheral nervous system (PNS), hematopoietic system (i.e., hematological malignancies), and immune system (e.g., spleen or thymus).

[0199] In some embodiments, the antibodies according to the present disclosure are useful for treating and / or preventing hematological malignancies. Exemplary types of cancers affecting the hematopoietic system include leukemias, lymphomas, and myelomas, including acute myeloid leukemia, adult T-cell leukemia, T-cell large granular lymphocyte leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, chronic myelocytic leukemia, acute monocytic leukemia, Hodgkin's and non-Hodgkin's lymphoma, diffuse large B-cell lymphoma, and multiple myeloma.

[0200] In another embodiment, the antibodies according to the present disclosure are useful for the treatment and / or prevention of a solid tumor. The solid tumor can be, for example, ovarian cancer, endometrial cancer, breast cancer, lung cancer (small cell or non-small cell), colon cancer, prostate cancer, cervical cancer, cholangiocarcinoma, pancreatic cancer, gastric cancer, esophageal cancer, liver cancer (hepatocellular carcinoma), kidney cancer (renal cell carcinoma), head and neck tumor, mesothelioma, melanoma, sarcoma, central nervous system (CNS) hemangioblastoma, and brain tumor (e.g., a glioma such as astrocytoma, oligodendroglioma, and glioblastoma).

[0201] In another embodiment, the antibodies according to the present disclosure are useful for the treatment and / or prevention of lung cancer, urogenital cancer, gastrointestinal cancer, or a combination thereof. In another embodiment, the antibodies according to the present disclosure are useful for the treatment and / or prevention of lung cancer, urogenital cancer, gastrointestinal cancer, skin cancer, or a combination thereof.

[0202] In some embodiments, the antibodies according to the present disclosure are useful for the treatment and / or prevention of skin cancer. In other embodiments, the skin cancer is melanoma.

[0203] In some embodiments, the antibodies according to the present disclosure are useful for the treatment and / or prevention of breast cancer. In other embodiments, the breast cancer is hormone receptor positive (e.g., ERa positive breast cancer, PR positive breast cancer, ERa positive and PR positive breast cancer), HER2 positive breast cancer, HER2 overexpressing breast cancer, or any combination thereof. In yet other embodiments, the breast cancer is triple negative breast cancer.

[0204] In some embodiments, the antibodies according to the present disclosure are useful for the treatment and / or prevention of kidney cancer. In other embodiments, the kidney cancer is renal cell carcinoma. In yet other embodiments, the renal cell carcinoma is clear cell renal carcinoma.

[0205] In some embodiments, the antibodies according to the present disclosure are useful for the treatment and / or prevention of lung cancer. In other embodiments, the lung cancer is non-small cell lung cancer (NSCLC). In yet other embodiments, the NSCLC is lung squamous cell carcinoma or lung adenocarcinoma.

[0206] In some embodiments, the disclosed methods of treating non-small cell lung cancer can further comprise administering any of the disclosed anti-CD39 antibodies in combination with pemetrexed, carboplatin, and an antagonist anti-PD-1 antibody or an antagonist anti-PD-L1 antibody. Such methods can also comprise: comprising administering one or more additional agents selected from the group consisting of: an antagonist anti-TIGIT antibody, an A2aR antagonist, an A2bR antagonist, an A2a / 2bR antagonist, and a CD73 inhibitor, optionally wherein the additional agent is selected from the group consisting of: domvanalib, AB308, itevelorle, and quilizalib.

[0207] In some embodiments, the antibodies according to the present disclosure are useful for the treatment and / or prevention of pancreatic cancer. In other embodiments, the pancreatic cancer is a pancreatic neuroendocrine tumor or a pancreatic adenocarcinoma.

[0208] In some embodiments, the antibodies according to the present disclosure are useful for the treatment and / or prevention of a neuroendocrine tumor. In other embodiments, the neuroendocrine tumor is a pancreatic neuroendocrine tumor, a pheochromocytoma, a paraganglioma, or an adrenal tumor.

[0209] In some embodiments, the antibodies according to the present disclosure are useful for the treatment and / or prevention of a brain cancer. In other embodiments, the brain cancer is a glioma. In yet other embodiments, the glioma is a astrocytoma, oligodendroglioma, or a glioblastoma.

[0210] In some embodiments, the antibodies according to the present disclosure are useful for the treatment and / or prevention of an upper GI cancer, such as esophageal cancer or gastric cancer. In other embodiments, the upper GI cancer is an adenocarcinoma, a squamous cell carcinoma, or any combination thereof. In yet other embodiments, the upper GI cancer is an esophageal adenocarcinoma (EAC), an esophageal squamous cell carcinoma (ESCC), a gastroesophageal junction adenocarcinoma (GEJ), a gastric adenocarcinoma (also referred to herein as “gastric cancer”), or any combination thereof.

[0211] In some embodiments, the disclosed methods of treating gastric or gastroesophageal cancer can further comprise administering any of the disclosed anti-CD39 antibodies in combination with FOLFOX and an antagonist anti-PD-1 antibody or an antagonist anti-PD-L1 antibody. Such methods can also comprise: comprising administering one or more additional agents selected from the group consisting of: an antagonist anti-TIGIT antibody, an A2aR antagonist, an A2bR antagonist, an A2a / 2bR antagonist, and a CD73 inhibitor, optionally wherein the additional agent is selected from the group consisting of: domvanalib, AB308, iteplidemstat, and quilizact.

[0212] In some embodiments, the antibodies according to the present disclosure are useful for the treatment and / or prevention of a lymphoma. In other embodiments, the hematological malignancy is acute myeloid lymphoma.

[0213] In some embodiments, the antibodies according to the present disclosure are useful for the treatment of breast cancer, gastrointestinal cancer, genitourinary cancer, lung cancer, lymphoma, or ovarian cancer. In other embodiments, the antibodies according to the present disclosure are useful for the treatment of acute myeloid lymphoma, colorectal cancer, gastric cancer, esophageal cancer, castration-resistant prostate cancer, non-small cell lung cancer, ovarian cancer, pancreatic cancer, or triple-negative breast cancer.

[0214] In some embodiments, the antibodies according to the present disclosure are suitable for treating lung cancer, head and neck cancer, thyroid cancer, pancreatic cancer, kidney cancer, or skin cancer. In yet other embodiments, the antibodies according to the present disclosure are suitable for treating non-small cell lung cancer, head and neck squamous cell carcinoma, pancreatic ductal adenocarcinoma, clear cell kidney cancer, melanoma.

[0215] In some embodiments, the antibodies according to the present disclosure are suitable for treating lower GI cancer, upper GI cancer, head and neck cancer, kidney cancer, lung cancer, or pancreatic cancer. In other embodiments, the antibodies according to the present disclosure are suitable for treating esophageal cancer, head and neck squamous cell carcinoma, kidney renal clear cell carcinoma, lung adenocarcinoma, lung squamous cell carcinoma, pancreatic adenocarcinoma, stomach (gastric) adenocarcinoma.

[0216] In the foregoing embodiments, the methods of the present disclosure can be practiced in an adjuvant setting or a neoadjuvant setting. The methods described herein can be indicated as first line, second line, third line, or higher line treatment. In some embodiments, the methods of the present disclosure can be practiced in a second line or higher line treatment, where the earlier line of treatment included a checkpoint inhibitor (i.e., the subject was subjected to a checkpoint inhibitor (CPI) treatment). In other embodiments, the checkpoint inhibitor is a CTLA-4 antagonist, a PD-1 antagonist, a PD-L1 antagonist, a TIM-3 antagonist, or a TIGIT antagonist.

[0217] The present disclosure also provides methods of treating or preventing other cancer-related diseases, disorders, or conditions. The use of the term cancer-related diseases, disorders, and conditions is intended to broadly refer to conditions that are directly or indirectly related to cancer and non-cancer proliferative diseases, and includes, for example, angiogenesis, precancerous conditions (e.g., dysplasia), and non-cancer proliferative diseases, disorders, or conditions (e.g., benign proliferative breast disease and papillomas). For the sake of clarity, the term cancer-related diseases, disorders, and conditions does not include cancer itself.

[0218] In general, the disclosed methods for treating or preventing cancer or a cancer-related disease, disorder, or condition in a subject in need thereof include administering to the subject an anti-CD39 antibody of Section II. The administration of the anti-CD39 antibody of Section II can comprise one or more (e.g., one, two, or three or more) dosing cycles. In some embodiments, the present disclosure provides methods of treating or preventing cancer or a cancer-related disease, disorder, or condition with an anti-CD39 antibody of Section II and at least one additional therapy, examples of which are set forth elsewhere herein.

[0219] Patient selection.In some cases, the methods according to the present disclosure can be provided in selected patients, e.g., patients identified as having, e.g., detectable PD-L1, CD73, and / or CD39 expression, having high microsatellite instability, having a high tumor mutational burden, or any combination thereof. In some cases, the patient is identified as having an oncogenic driver cancer with a mutation in at least one gene associated with cancer. In some cases, the patient is selected by assaying the patient for P2X7 variants. Generally expressed P2X7 splice variants, different SNPs, and post-translational receptor modifications can affect the function of P2X7. For example, certain modifications have been shown to cause partial or complete loss of P2X7 function, and other modifications have been shown to increase P2X7 function. See, e.g., Lara et al., Front Pharmacol, 2020, 11:793.

[0220] In some embodiments, a patient is selected by measuring PD-L1 (CD274), P2X7 (P2RX7), P2Y11 (P2RY11), IL-2, CXCL1 (CXCL1), MIP-2a (CXCL2), CXCL3 (CXCL3), IL-8 (CXCL8), COX2 (PTGS2), CD73 (NT5E), and / or CD39 (ENTPD1) expression in a relevant sample, e.g., a peripheral blood sample or a tumor sample. Expression can be measured by quantifying nucleic acids or proteins using methods known in the art, including but not limited to immunohistochemistry, immunophenotyping, RNA sequencing, gene expression analysis, single molecule imaging, or other clinically validated assays. In some embodiments, a patient is selected by measuring PD-L1, CD73, and / or CD39 expression (at the nucleic or protein level) in a relevant sample, e.g., a peripheral blood sample or a tumor sample, using immunohistochemistry, immunophenotyping, RNA sequencing, gene expression analysis, single molecule imaging, or other clinically validated assays. Alternatively or additionally, a patient can be selected by measuring CD39 enzymatic activity, e.g., in a peripheral blood sample or a tumor sample. In one embodiment, the present disclosure provides a method of treating a cancer in a patient by administering an anti-CD39 antibody described herein, the patient having (i) detectable PD-L1, P2X7, P2Y11, IL-2, CXCL1, CXCL2, CXCL3, CXCL8, COX2, CD73, and / or CD39 expression, e.g., as measured by immunohistochemistry, immunophenotyping, or other clinically validated tests, (ii) elevated PD-L1, P2X7, P2Y11, IL-2, CXCL1, CXCL2, CXCL3, CXCL8, COX2, CD73, and / or CD39 expression, e.g., as measured by immunohistochemistry, immunophenotyping, or other clinically validated tests, (iii) detectable CD39 enzymatic activity, or (iv) any combination of (i) to (iii). In another embodiment, the present disclosure provides a method of treating a cancer in a patient by administering a therapeutically effective amount of an anti-CD39 antibody described herein, the patient having (i) detectable PD-L1, P2X7, P2Y11, IL-2, CXCL1, CXCL2, CXCL3, CXCL8, COX2, CD73, and / or CD39 expression, e.g., as measured by immunohistochemistry, immunophenotyping, or other clinically validated tests, (ii) elevated PD-L1, CD73, and / or CD39 expression, e.g., as measured by immunohistochemistry, immunophenotyping, or other clinically validated tests, (iii) detectable CD39 enzymatic activity, or (iv) any combination of (i) to (iii).In another embodiment, the present disclosure provides a method of administering to an individual a therapeutically effective amount of an anti-CD39 antibody described herein to treat cancer, the method comprising measuring PD-L1, P2X7, P2Y11, IL-2, CXCL1, CXCL2, CXCL3, CXCL8, COX2, CD73, and / or CD39 expression. In another embodiment, the present disclosure provides a method of administering to an individual a therapeutically effective amount of an anti-CD39 antibody described herein to treat cancer, the method comprising measuring PD-L1, P2X7, P2Y11, IL-2, CXCL1, CXCL2, CXCL3, CXCL8, COX2, CD73, and / or CD39 expression in a sample obtained from the individual, e.g., by immunohistochemistry, immunophenotyping, or other clinically validated test, and administering to an individual whose sample contains detectable PD-L1 (CD274), P2X7 (P2RX7), P2Y11 (P2RY11), IL-2, CXCL1 (CXCL1), MIP-2a (CXCL2), CXCL3 (CXCL3), IL-8 (CXCL8), COX2 (PTGS2), CD73 (NT5E), and / or CD39 (ENTPD1) expression a therapeutically effective amount of the antibody.

[0221] B. Routes of Administration

[0222] In some embodiments, the pharmaceutical compositions containing the antibodies according to the present disclosure can be in a form suitable for oral administration. Oral administration can involve ingesting the formulation, thereby allowing the antibodies to be absorbed into the bloodstream in the gastrointestinal tract. Alternatively, oral administration can involve buccal, lingual, or sublingual administration, thereby allowing the antibodies to be absorbed into the bloodstream via the oral mucosa.

[0223] In another embodiment, the pharmaceutical compositions containing the antibodies according to the present disclosure can be in a form suitable for parenteral administration. Forms of parenteral administration include, but are not limited to, intravenous, intraarterial, intramuscular, intradermal, intraperitoneal, intrathecal, intracisternal, intracerebral, intraventricular, and subcutaneous. The pharmaceutical compositions suitable for parenteral administration can be formulated using suitable aqueous or nonaqueous carriers. Depot injection formulations, which are generally administered subcutaneously or intramuscularly, can also be used to release the antibodies disclosed herein over a defined period of time.

[0224] Other routes of administration are also contemplated by the present disclosure, including, but not limited to, nasal, vaginal, intraocular, rectal, topical (e.g., transdermal), and inhalation.

[0225] Particular embodiments of the present disclosure contemplate oral administration or parenteral administration.

[0226] C. Pharmaceutical Compositions

[0227] The anti-CD39 antibodies of the present disclosure can be in the form of a composition suitable for administration to a subject. Generally, such compositions are pharmaceutical compositions comprising an anti-CD39 antibody according to the present disclosure and one or more pharmaceutically acceptable excipients. In certain embodiments, the anti-CD39 antibody can be present in an effective amount. The pharmaceutical compositions can be used in the methods of the present disclosure; thus, for example, a pharmaceutical composition comprising an anti-CD39 antibody according to the present disclosure can be administered to a subject in order to practice the therapeutic and prophylactic methods and uses described herein.

[0228] The pharmaceutical compositions of the present disclosure can be formulated to be compatible with the intended method or route of administration. The route of administration can include those known in the art. Exemplary routes of administration are oral and parenteral. Further, the pharmaceutical compositions can be used in combination with one or more other therapies as described herein in order to treat or prevent diseases, disorders, and conditions as encompassed by the present disclosure. In one embodiment, one or more other additional therapeutic agents encompassed by the present disclosure are included in the same pharmaceutical composition comprising an anti-CD39 antibody according to the present disclosure. In another embodiment, one or more other therapeutic agents are in a composition separate from the pharmaceutical composition comprising an anti-CD39 antibody according to the present disclosure.

[0229] In one aspect, the anti-CD39 antibodies described herein can be administered orally. Oral administration can be via, for example, a capsule or tablet. In making the pharmaceutical compositions that include the anti-CD39 antibodies, the tablets or capsules typically include at least one pharmaceutically acceptable excipient. Non-limiting examples of pharmaceutically acceptable excipients include lactose, dextrose, sucrose, sorbitol, mannitol, starches, acacia, calcium phosphate, alginic acid, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, polyethylene glycol, cellulose, sterile water, syrup, and methyl cellulose. Additional pharmaceutically acceptable excipients include lubricating agents such as talc, magnesium stearate, and mineral oil; wetting agents; emulsifying agents and suspending agents; and preservatives such as methyl and propylhydroxy-benzoates. Some oral administration forms include a taste masking agent, a sweetener, or a flavoring agent. Oral dosage forms can be formulated as solutions or suspensions.

[0230] In another aspect, the antibodies described herein can be administered parenterally, for example, by intravenous injection. Pharmaceutical compositions suitable for parenteral administration can be formulated in a solution or can be reconstituted in a suitable system, such as a physiological solution, for injection. Such solutions can include sterile water, salts, buffers, and tonicity excipients in amounts appropriate to achieve isotonicity with appropriate physiology.

[0231] The pharmaceutical compositions described herein can be stored in one or more suitable sterile containers. In some embodiments, the containers are designed to maintain the stability of the pharmaceutical composition over a given period of time.

[0232] D. Administration

[0233] In general, the disclosed methods include administering to a subject in need thereof an effective amount of an anti-CD39 antibody or composition thereof described herein. An "effective amount" of an anti-CD39 antibody of the disclosure means an amount of antibody sufficient to engage a target (e.g., by binding to and inhibiting CD39 enzymatic activity) at a level indicative of antibody efficacy. For CD39, target engagement can be determined by one or more biochemical or cellular assays that produce EC 50 , ED 50 , EC 90 , IC 50 or similar values that can be used as an assessment of efficacy of the antibody. Assays for determining target engagement include, but are not limited to, those described in the Examples. An effective amount can be administered in a single dose or in multiple smaller doses (e.g., as one tablet with an "x" amount, as two tablets each with an "x / 2" amount, etc.).

[0234] In some embodiments, the disclosed methods include administering to a subject in need thereof a therapeutically effective amount of an anti-CD39 antibody described herein. As used herein, the phrase "therapeutically effective amount" in reference to an anti-CD39 antibody means a dosage regimen (i.e., amount and interval over time) of an antibody that provides a particular pharmacological effect for which the antibody is administered to a subject in need of such treatment. For prophylactic use, a therapeutically effective amount can be effective to eliminate or reduce the risk of, lessen the severity of, or delay the onset of a disease, including biochemical, histological, and / or behavioral indicators or symptoms of disease. For therapy, a therapeutically effective amount can be effective to reduce, ameliorate, or eliminate one or more signs or symptoms associated with a disease, delay disease progression, prolong survival, reduce the dosage of other medications required to treat the disease, or a combination thereof. In particular, in reference to cancer, a therapeutically effective amount can result in, for example, cancer cell killing, reduction in cancer cell count, reduction in tumor burden, elimination of a tumor or cancer metastasis, or reduction in metastatic spread. A therapeutically effective amount of an anti-CD39 antibody is not necessarily effective in treating every individual subject, but is considered a therapeutically effective amount by those of skill in the art. A therapeutically effective amount can vary based on, for example, one or more of the following: age and weight of the subject, overall health status of the subject, stage of disease in the subject, route of administration, and prior or concurrent treatments.

[0235] In certain embodiments, the anti-CD39 antibodies encompassed by the present disclosure can be administered (e.g., orally, parenterally, etc.) once or more times a day, week or month at about 0.01 mg / kg to about 50 mg / kg, or about 1 mg / kg to about 25 mg / kg of the subject’s body weight to achieve the desired effect. In some embodiments, a suitable weight-based dose of an anti-CD39 antibody encompassed by the present disclosure is used to determine a dose that is administered independent of the subject’s body weight (i.e., a fixed dose). In certain embodiments, the anti-CD39 antibodies of the present disclosure can be administered (e.g., orally, parenterally, etc.) once or more times a day, week or month at a fixed dose level of about 1 mg to about 1000 mg, in particular 1, 3, 5, 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 300, 400, 500, 600, 750, 800, 900, or 1000 mg, to achieve the desired effect. In some embodiments, the anti-CD39 antibodies of the present disclosure can be administered (e.g., orally, parenterally, etc.) once or more times a day, week or month at a fixed dose level of about 3 mg to about 3000 mg, in particular 3, 5, 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 300, 400, 500, 600, 750, 800, 900, 1000 mg, 1100 mg, 1200 mg, 1300 mg, 1400 mg, 1500 mg, 1600 mg, 1700 mg, 1800 mg, 1900 mg, 2000 mg, 2100 mg, 2200 mg, 2300 mg, 2400 mg, 2500 mg, 2600 mg, 2700 mg, 2800 mg, 2900 mg, or 3000 mg, to achieve the desired effect. The term “about” when used in reference to a dose herein means the stated numerical value and ± 10% of the stated numerical value. For example, “about 10” is understood to mean “10” and “9-11.”

[0236] In certain embodiments, the anti-CD39 antibodies of the present disclosure are included in “unit dosage form.” The phrase “unit dosage form” refers to physically discrete units suitable for dosing an individual, each unit containing a predetermined quantity of an anti-CD39 antibody, alone or in combination with one or more additional pharmaceutical agents, sufficient to produce the desired effect. It is understood that the parameters of a unit dosage form will depend on the particular agent and the effect to be achieved.

[0237] IV. Combinations with disclosed anti-CD39 antibodies

[0238] The present disclosure encompasses the use of a Part II anti-CD39 antibody alone or in combination with one or more additional therapies. Each additional therapy can be a therapeutic agent or another therapeutic modality. In embodiments comprising one or more additional therapeutic agents, each agent can target a different but complementary mechanism of action. The additional therapeutic agent can be a small chemical molecule; a macromolecule, such as a protein, antibody, peptibody, peptide, DNA, RNA, or a fragment of such a macromolecule; or a cell or gene therapy. Non-limiting examples of additional therapeutic modalities include surgical resection of a tumor, bone marrow transplantation, radiation therapy, and photodynamic therapy. The use of a Part II anti-CD39 antibody in combination with one or more additional therapies can have a synergistic or additive therapeutic or prophylactic effect on the underlying disease, disorder, or condition. Additionally or alternatively, the combination therapy can allow for a reduction in the dose of one or more of the therapies, thereby improving, reducing, or eliminating adverse effects associated with one or more of the agents.

[0239] In embodiments comprising one or more additional therapeutic modalities, a Part II anti-CD39 antibody can be administered prior to, after, or during treatment with the additional therapeutic modalities. In embodiments comprising one or more additional therapeutic agents, the therapeutic agents used in such combination therapy can be formulated as a single composition or as separate compositions. If administered separately, each therapeutic agent in the combination can be given at the same or about the same time or at different times. Further, the therapeutic agents are administered in “combination,” even if they have different forms of administration (e.g., oral capsule and intravenous), are given at different dosing intervals, one is given on a constant dosing regimen while the other is titrated up, titrated down, or discontinued, or each therapeutic agent in the combination is independently titrated up, titrated down, dosed up or down, or discontinued and / or resumed during the course of treatment of the patient. If the combination is formulated as separate compositions, in some embodiments, the separate compositions are provided together in a kit.

[0240] Cancer therapy

[0241] The present disclosure encompasses use of an anti-CD39 antibody of Section II in combination with one or more additional therapies useful for treating a cancer or a cancer-related disease, disorder, or condition. In some embodiments, one or more of the additional therapies is an additional treatment modality. Exemplary treatment modalities include, but are not limited to, surgical resection of a tumor, bone marrow transplantation, radiation therapy, and photodynamic therapy. In some embodiments, one or more of the additional therapies is a therapeutic agent. Exemplary therapeutic agents include chemotherapeutic agents, radiopharmaceuticals, hormone therapies, epigenetic modulators, ATP-adenosine axis targeting agents, targeted therapies, signal transduction inhibitors, RAS signaling inhibitors, PI3K inhibitors, arginase inhibitors, HIF inhibitors, AXL inhibitors, PAK4 inhibitors, immunotherapeutic agents, cellular therapies, gene therapies, immune checkpoint inhibitors, and agonists of stimulatory or costimulatory immune checkpoints.

[0242] In some embodiments, one or more of the additional therapeutic agents is a chemotherapeutic agent. Examples of chemotherapeutic agents include, but are not limited to, alkylating agents such as thiotepa and cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methyl triazenes including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphaoramide and triethylenethamine; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine;antibiotics such as aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, calicheamicin, carabicin, carminomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin, olivomycin, pomalidomide, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folate analogues such as denopterin, methotrexate, pemetrexed, pteropterin, trimetrexate; purine analogues such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifuridine, edoxudine, emitefur, floxuridine, 5- fluorouracil (5-FU); androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as canrenone, mitotane, trilostane; chlorambucil; chlormethine; melphalan; nitrosurea; nitrogen mustards such as chloromethyl-methyl-methylamine and methyl-methanesulfonate;pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenal agents such as aminoglutethimide, mitotane, trilostane; folate supplements such as folinic acid; acetylornidylate; alfosfosfosamide; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamide; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin; podophyllinic acid; 2-ethylhydrazide; procarbazine; razoxane; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"-trichlorotriethylamine; urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman;Gacytosine; arabinoside (Ara-C); cyclophosphamide; thiotepa; taxoids, e.g. paclitaxel, albumin-bound paclitaxel, and docetaxel; chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum and platinum coordination complexes (collectively "platinum agents") such as cisplatin, carboplatin and oxaliplatin; vinblastine; etoposide (VP- 16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT11 ; proteasome inhibitors such as bortezomib, carfilzomib, and ixazomib; topoisomerase inhibitors such as irinotecan, topotecan, etoposide, mitoxantrone, teniposide; difluoromethylornithine (DMFO); retinoic acid; esperamicin; capecitabine;an anthracycline and a pharmaceutically acceptable salt, acid, or derivative of any of the above. In certain embodiments, the combination therapy comprises a chemotherapy regimen comprising one or more chemotherapeutic agents. In one embodiment, the combination therapy comprises a chemotherapy regimen comprising FOLFOX (leucovorin, fluorouracil, and oxaliplatin), FOLFIRI (leucovorin, fluorouracil, and irinotecan), a taxane (e.g., docetaxel, paclitaxel, nab-paclitaxel, etc.), CAPOX (capecitabine and oxaliplatin), XELOX (capecitabine and oxaliplatin), irinotecan, a platinum-based chemotherapeutic agent, or gemcitabine. In another embodiment, the combination therapy comprises a chemotherapy regimen comprising an alkylating agent (e.g., cyclophosphamide), an anthracycline (e.g., doxorubicin, epirubicin, edotecarin, mitoxantrone), a platinum agent (e.g., oxaliplatin), a proteasome inhibitor (e.g., bortezomib), or any combination thereof. In another embodiment, the combination therapy comprises a chemotherapy regimen comprising an alkylating agent (e.g., cyclophosphamide), an anthracycline (e.g., doxorubicin, epirubicin, edotecarin, mitoxantrone), a platinum agent (e.g., carboplatin, cisplatin, oxaliplatin), a taxane (e.g., docetaxel, paclitaxel), a proteasome inhibitor (e.g., bortezomib), or any combination thereof. In another embodiment, the combination therapy comprises a chemotherapy regimen comprising bortezomib, cyclophosphamide, doxorubicin, epirubicin, edotecarin, mitoxantrone, oxaliplatin, or any combination thereof.

[0243] In some embodiments, one or more of the additional therapeutic agents is a radiopharmaceutical. A radiopharmaceutical is a form of internal radiotherapy in which a source of radiation (i.e., one or more radionuclides) is placed within the body of a subject. The source of radiation can be in solid or liquid form. Non-limiting examples of radiopharmaceuticals include sodium iodide I-131, radium dichloride-223, iobenguane iodine-131, radiolabeled vesicles (e.g., sphingolipid-activated protein C-dioleoyl phosphatidylserine (SapC-DOPS) nanovesicles), various forms of brachytherapy, and various forms of targeted radionuclides. A targeted radionuclide comprises a radionuclide associated (e.g., through covalent or ionic interactions) with a molecule that specifically binds to a target on a cell (typically a cancer cell or an immune cell) (“targeting agent”). The targeting agent can be a small molecule, a saccharide (including oligosaccharides and polysaccharides), an antibody, a lipid, a protein, a peptide, a non-natural polymer, or an aptamer. In some embodiments, the targeting agent is a saccharide (including oligosaccharides and polysaccharides), a lipid, a protein, or a peptide, and the target is a tumor-associated antigen (enriched but not specific to cancer cells), a tumor-specific antigen (expressed minimally to not at all in normal tissue), or a neoantigen (an antigen specific to the genome of a cancer cell resulting from a non-synonymous mutation or gene fusion in the genome of the tumor cell). In some embodiments, the targeting agent is an antibody and the target is a tumor-associated antigen (i.e., an antigen enriched but not specific to cancer cells), a tumor-specific antigen (i.e., an antigen with minimal expression to no expression in normal tissue), or a neoantigen (i.e., an antigen specific to the genome of a cancer cell resulting from a non-synonymous mutation or gene fusion in the genome of the tumor cell). Non-limiting examples of targeted radionuclides include radionuclides linked to somatostatin or a peptide analog thereof (e.g., 177Lu-Dotatate, etc.); prostate-specific membrane antigen or a peptide analog thereof (e.g., 177Lu-PSMA-617, 225Ac-PSMA-617, 177Lu-PSMA-I&T, 177Lu-MIP-1095, etc.); receptor homologous ligands, peptides derived from ligands, or variants thereof (e.g., 188Re-labeled VEGF 125-136 or variants thereof with higher affinity to VEGF receptors, etc.); antibodies targeting tumor antigens (e.g., 131I-tositumomab, 90Y-ibritumomab tiuxetan, CAM-H2-I131 (Precirix NV), I131-omburtamab, etc.).

[0244] In some embodiments, one or more of the additional therapeutic agents is a hormone therapy. Hormone therapy is used to modulate or inhibit the effects of hormones on tumors. Examples of hormone therapy include, but are not limited to: selective estrogen receptor degraders, such as fulvestrant, GDC-9545, SAR439859, RG6171, AZD9833, rintodestrant, ZN-c5, LSZ102, D-0502, LY3484356, SHR9549; selective estrogen receptor modulators, such as tamoxifen, raloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, toremifene; aromatase inhibitors, such as anastrozole, exemestane, letrozole, and other aromatase-inhibitory 4(5)-imidazoles; gonadotropin-releasing hormone agonists, such as nafarelin, triptorelin, goserelin; gonadotropin-releasing hormone antagonists, such as degarelix; antiandrogens, such as abiraterone, enzalutamide, apalutamide, darolutamide, flutamide, nilutamide, bicalutamide, leuprolide; 5a-reductase inhibitors, such as finasteride, dutasteride; and the like. In certain embodiments, the combination therapy comprises administration of a hormone or related hormonal agent. In one embodiment, the combination therapy comprises administration of enzalutamide.

[0245] In some embodiments, one or more of the additional therapeutic agents is an epigenetic modulator. Epigenetic modulators alter epigenetic mechanisms that control gene expression, and can be, for example, inhibitors or activators of epigenetic enzymes. Non-limiting examples of epigenetic modulators include DNA methyltransferase (DNMT) inhibitors, hypomethylating agents, and histone deacetylase (HDAC) inhibitors. In one or more embodiments, the anti-CD39 antibodies of Part II can be combined with a DNA methyltransferase (DNMT) inhibitor or a hypomethylating agent. Exemplary DNMT inhibitors include decitabine, zebularine, and azacitadine. In one or more embodiments, combinations of the anti-CD39 antibodies of Part II with a histone deacetylase (HDAC) inhibitor are also contemplated. Exemplary HDAC inhibitors include vorinostat, givinostat, abexinostat, panobinostat, belinostat, and trichostatin A.

[0246] In some embodiments, one or more of the additional therapeutic agents is an ATP- adenosine axis targeting agent. ATP-adenosine axis targeting agents alter signaling mediated by adenine nucleosides and nucleotides (e.g., adenosine, AMP, ADP, ATP), for example, by modulating adenosine levels or targeting adenosine receptors. Adenosine and ATP, which act at different classes of receptors, generally have opposite effects on inflammation, cell proliferation, and cell death. For example, ATP and other adenine nucleotides have anti-tumor effects via activation of the P2Y1 receptor subtype, while accumulation of adenosine in the tumor microenvironment has been shown to suppress the anti-tumor functions of various immune cells and enhance the immunosuppressive activity of myeloid and regulatory T cells by binding to cell surface adenosine receptors. In certain embodiments, the ATP-adenosine axis targeting agent is an inhibitor of ectonucleotidases involved in the conversion of ATP to adenosine or an antagonist of adenosine receptors. Ectonucleotidases involved in the conversion of ATP to adenosine include ecto-nucleoside triphosphate diphosphohydrolase 1 (ENTPD1, also known as CD39 or cluster of differentiation 39) and ecto-5'-nucleotidase (NT5E or 5NT, also known as CD73 or cluster of differentiation 73). Exemplary small molecule CD73 inhibitors include CB-708, ORIC-533, LY3475070, and AB680. Exemplary anti-CD73 antibodies include CPI-006, oleclumab (MEDI9447), NZV930, IPH5301, GS-1423, uliledlimab (TJD5, TJ004309), and BMS-986179. In one embodiment, the disclosure encompasses an anti-CD39 antibody of Part II in combination with a CD73 inhibitor, such as those described in WO 2017 / 120508, WO 2018 / 067424, WO 2018 / 094148, and WO 2020 / 046813. In other embodiments, the CD73 inhibitor is quinaparant. 2A R, A 2B R, and A3R. A2R antagonists include itrukumab, inupadenant, taminadenant, citicoline, NUV-1182, TT-702, DZD-2269, INCB-106385, EVOEXS-21546, AZD-4635, imaradenant, RVU-330, ciforadenant, PBF-509, PBF-999, PBF-1129, and CS-3005. In some embodiments, the disclosure encompasses an anti-CD39 antibody of Part II in combination with an A 2A R antagonist, A2B R antagonists or A 2A R and A 2B combinations of antagonists of R. In some embodiments, the present disclosure encompasses an anti-CD39 antibody of Section II in combination with an adenosine receptor antagonist described in WO 2018 / 136700, WO 2018 / 204661, WO 2018 / 213377, or WO 2020 / 023846, WO 2020 / 102646. In one embodiment, the adenosine receptor antagonist is itacumumab.

[0247] In some embodiments, one or more of the additional therapeutic agents is a targeted therapy. In one aspect, the targeted therapy can comprise a chemotherapeutic agent, a radionuclide, a hormonal therapy, or another small molecule drug linked to a targeting agent. The targeting agent can be a small molecule, a saccharide (including oligosaccharides and polysaccharides), an antibody, a lipid, a protein, a peptide, a non-natural polymer, or an aptamer. In some embodiments, the targeting agent is a saccharide (including oligosaccharides and polysaccharides), a lipid, a protein, or a peptide, and the target is a tumor-associated antigen (enriched but not specific to cancer cells), a tumor-specific antigen (expressed minimally to no expression in normal tissue), or a neoantigen (an antigen specific to the genome of a cancer cell resulting from a non-synonymous mutation in the genome of the tumor cell). In some embodiments, the targeting agent is an antibody, and the target is a tumor-associated antigen (enriched but not specific to cancer cells), a tumor-specific antigen (expressed minimally to no expression in normal tissue), or a neoantigen (an antigen specific to the genome of a cancer cell resulting from a non-synonymous mutation in the genome of the tumor cell). In some embodiments, the targeted therapy is an antibody-drug conjugate comprising an antibody and a drug, wherein the antibody specifically binds to HER2, HER3, nectin-4, or Trop-2. Particular examples of targeted therapies comprising an antibody and a drug include, but are not limited to, patritumab deruxtecan, sacituzumab govitecan-hziy, telisotuzumab vedotin, and trastuzumab deruxtecan. In other aspects, the targeted therapy can inhibit or interfere with a particular protein that helps a tumor to survive, grow, and / or spread. Non-limiting examples of such targeted therapies include signal transduction inhibitors, RAS signaling inhibitors, inhibitors of oncogenic transcription factors, activators of inhibitors of oncogenic transcription factors, angiogenesis inhibitors, immunotherapeutic agents, ATP-adenosine axis targeting agents, AXL inhibitors, PARP inhibitors, PAK4 inhibitors, PI3K inhibitors, HIF2a inhibitors, CD73 inhibitors, A2R antagonists, TIGIT antagonists, and PD-1 antagonists. ATP-adenosine axis targeting agents are described above, and other agents are described in further detail below.

[0248] In some embodiments, one or more of the additional therapeutic agents are signal transduction inhibitors. Signal transduction inhibitors are agents that selectively inhibit one or more steps in a signaling pathway. Signal transduction inhibitors (STIs) covered by this disclosure include, but are not limited to: (i) BCR-ABL kinase inhibitors (e.g., imatinib); (ii) epidermal growth factor receptor tyrosine kinase inhibitors (EGFR TKIs), including small molecule inhibitors (e.g., CLN-081, gefitinib, erlotinib, afatinib, icotinib, and osimertinib) and anti-EGFR antibodies; (iii) inhibitors of the human epidermal growth factor (HER) family of transmembrane tyrosine kinases, such as HER-2 / neu receptor inhibitors (e.g., trastuzumab and HER-3 receptor inhibitors); (iv) vascular endothelial growth factor receptor (VEGF) inhibitors; and (v) other transmembrane tyrosine kinase inhibitors. VEGFR inhibitors include small molecule inhibitors (such as axitinib, regorafenib, sunitinib, and sorafenib), VEGF kinase inhibitors (such as lenvatinib, cabozantinib, pazopanib, tivozanib, XL092, etc.), anti-VEGF antibodies (such as bevacizumab), and anti-VEGFR antibodies. (v) Inhibitors of AKT family kinases or the AKT pathway (e.g., ramucirumab); (vi) Inhibitors of serine / threonine-protein kinase BRAF (e.g., vemurafenib, dabrafenib, and encorafenib); (vii) Inhibitors of transfection rearrangement (RET), including, for example, selpercatinib and pralsetinib. (i) ib); (viii) tyrosine-protein kinase Met (MET) inhibitors (e.g., tepotinib, tivantinib, cabozantinib, and crizotinib); (ix) anaplastic lymphoma kinase (ALK) inhibitors (e.g., ensartinib, ceritinib, lorlatinib, crizotinib, and brigatinib);(x) inhibitors of the RAS signaling pathway (e.g., inhibitors of KRAS, HRAS, RAF, MEK, ERK), as described elsewhere herein; (xi) FLT-3 inhibitors (e.g., gilteritinib); (xii) inhibitors of Trop-2; (xiii) inhibitors of the JAK / STAT pathway, e.g., JAK inhibitors including tofacitinib and ruxolitinib, or STAT inhibitors such as napabucasin; (xiv) inhibitors of NF-kB; (xv) cell cycle kinase inhibitors (e.g., flavopiridol); (xvi) phosphatidylinositol kinase (PI3K) inhibitors; (xix) protein kinase B (AKT) inhibitors (e.g., capivasertib, miransertib); (xx) platelet-derived growth factor receptor (PDGFR) inhibitors (e.g., imatinib, sunitinib, regorafenib, avapritinib, Lenvatinib, nintedanib, famitinib, ponatinib, axitinib, repertinib, etc.); and (xxi) insulin growth factor receptor (IGFR) inhibitors (e.g., erlotinib, afatinib, gefitinib, o simertinib, dacomitinib). In one or more embodiments, the additional therapeutic agent comprises an inhibitor of EGFR, VEGFR, HER-2, HER-3, BRAF, RET, MET, ALK, RAS (e.g., KRAS, MEK, ERK), FLT-3, JAK, STAT, NF-kB, PI3K, AKT, or any combination thereof.

[0249] In some embodiments, one or more of the additional therapeutic agents is a RAS signaling inhibitor. Oncogenic mutations in the RAS family of genes, such as HRAS, KRAS, and NRAS, are associated with a variety of cancers. For example, G12C, G12D, G12V, G12A, G13D, Q61H, G13C, and G12S, among other mutations in KRAS family genes have been observed in multiple tumor types. Direct and indirect inhibition strategies have been investigated for inhibiting mutant RAS signaling. Indirect inhibitors target effectors in the RAS signaling pathway other than RAS, and include, but are not limited to, inhibitors of RAF, MEK, ERK, PI3K, PTEN, SOS (e.g., SOS1), mTORCl, SHP2 (PTPN11), and AKT. Non-limiting examples of indirect inhibitors in development include RMC-4630, RMC-5845, RMC-6291, RMC-6236, JAB-3068, JAB-3312, TNO155, RLY-1971, BI1701963. Direct inhibitors of RAS mutants have also been explored, and generally target the KRAS-GTP complex or the KRAS-GDP complex. Exemplary direct RAS inhibitors in development include, but are not limited to, sotorasib (AMG510), MRTX849, mRNA-5671, and ARS1620. In some embodiments, one or more RAS signaling inhibitors are selected from the group consisting of a RAF inhibitor, a MEK inhibitor, an ERK inhibitor, a PI3K inhibitor, a PTEN inhibitor, a SOS1 inhibitor, a mTORCl inhibitor, a SHP2 inhibitor, and an AKT inhibitor. In other embodiments, one or more RAS signaling inhibitors directly inhibit a RAS mutant.

[0250] In some embodiments, one or more of the additional therapeutic agents is an inhibitor of phosphoinositide 3-kinase (PI3K), particularly an inhibitor of the PI3K gamma isoform. PI3K gamma inhibitors can reduce cancer development and spread via modulating myeloid cell stimulation of anti-cancer immune responses, for example by inhibiting suppressive myeloid cells, attenuating immunosuppressive tumor infiltrating macrophages, or by stimulating macrophages and dendritic cells to make cytokines that contribute to effective T cell responses. Exemplary PI3K gamma inhibitors include copanlisib, duvelisib, AT-104, ZX-101, tenalisib, eganelisib, SF-1126, AZD3458, and pictilisib. In some embodiments, the anti-CD39 antibody of Part II can be combined with one or more PI3K gamma inhibitors described in WO 2020 / 0247496A1.

[0251] In some embodiments, one or more of the additional therapeutic agents is an arginase inhibitor. Arginase has been shown to be responsible for or involved in immune dysfunction that triggers inflammation, tumor immune escape, immunosuppression, and immunopathology of infectious diseases. Exemplary arginase compounds include CB-1158 and OAT-1746. In some embodiments, the anti-CD39 antibodies of Part II can be combined with one or more arginase inhibitors described in WO / 2019 / 173188 and WO 2020 / 102646.

[0252] In some embodiments, one or more of the additional therapeutic agents is an inhibitor of oncogenic transcription factors or activators of oncogenic transcription factor inhibitors. Suitable agents can act at the expression level (e.g., RNAi, siRNA, etc.), via physical degradation, at the protein / protein level, at the protein / DNA level, or by binding in the activation / inhibition pocket. Non-limiting examples include inhibitors of one or more subunits of the MLL complex (e.g., HDAC, DOT1L, BRD4, Menin, LEDGF, WDR5, KDM4C (JMJD2C), and PRMT1), inhibitors of hypoxia-inducible factor (HIF) transcription factors, etc.

[0253] In some embodiments, one or more of the additional therapeutic agents is an inhibitor of hypoxia-inducible factor (HIF) transcription factors, particularly HIF-2a. Exemplary HIF-2a inhibitors include belzutifan, ARO-HIF2, PT-2385, AB521, and those described in WO 2021113436 and WO 2021188769. In some embodiments, the anti-CD39 antibodies of Part II can be combined with one or more HIF-2a inhibitors described in WO 2021188769.

[0254] In some embodiments, one or more of the additional therapeutic agents is an inhibitor of anexelekto (AXL). The AXL signaling pathway is associated with tumor growth and cancer metastasis and is thought to mediate resistance to multiple cancer therapies. Multiple AXL inhibitors in development also inhibit other kinases in the TAM family (i.e., TYRO3, MERTK), as well as other receptor tyrosine kinases, including MET, FLT3, RON, and AURORA, as well as other kinases. Exemplary multi-kinase inhibitors include sitravatinib, rebastinib, glesatinib, gilteritinib, merestinib, cabozantinib, foretinib, BMS777607, LY2801653, S49076, GSK1363089, and RXDX-106. AXL-specific inhibitors have also been developed, such as small molecule inhibitors, including DS-1205, SGI-7079, SLC-391, TP-0903 (i.e., dubermatinib), BGB324 (i.e., bemcentinib), and DP3975; anti-AXL antibodies, such as ADCT-601; and antibody drug conjugates (ADCs), such as BA3011. Another strategy to inhibit AXL signaling involves targeting the ligand for AXL, GAS6. For example, AVB-500 is in development as an Fc fusion protein that binds the GAS6 ligand thereby inhibiting AXL signaling.

[0255] In some embodiments, one or more of the additional therapeutic agents is an inhibitor of p21-activated kinase 4 (PAK4). PAK4 overexpression has been demonstrated in multiple cancer types, including those that are resistant to PD-1 therapy. While PAK4 inhibitors have not yet been approved, some are in development and exhibit dual PAK4 / NAMPT inhibitor activity, such as ATG-019 and KPT-9274. In some embodiments, an antibody according to the present disclosure is combined with a PAK4-selective inhibitor. In some embodiments, an antibody according to the present disclosure is combined with a PAK4 / NAMPT dual inhibitor, such as ATG-019 or KPT-9274.

[0256] In some embodiments, one or more of the additional therapeutic agents is (i) an agent that inhibits the enzyme poly(ADP-ribose) polymerase (e.g., olaparib, niraparib, and rucaparib, etc.); (ii) an inhibitor of Bcl-2 family proteins (e.g., venetoclax, navitoclax, etc.); (iii) an inhibitor of MCL-1; (iv) an inhibitor of the CD47-SIRPa pathway (e.g., an anti-CD47 antibody, magrolimab, etc.); (v) an isocitrate dehydrogenase (IDH) inhibitor, e.g., an IDH-1 or IDH-2 inhibitor (e.g., ivosidenib, enasidenib, etc.).

[0257] In some embodiments, one or more of the additional therapeutic agents is an immunotherapeutic agent. Immunotherapeutic agents suitable for treating cancer generally elicit or augment an immune response against cancer cells. Non-limiting examples of suitable immunotherapeutic agents include: immunomodulatory agents; cellular immunotherapies; vaccines; gene therapies; ATP-adenosine axis targeting agents; immune checkpoint modulators; and certain signal transduction inhibitors. ATP-adenosine axis targeting agents are described above. Immunomodulatory agents, signal transduction inhibitors, cellular immunotherapies, vaccines, gene therapies, and immune checkpoint modulators are further described below.

[0258] In some embodiments, one or more of the additional therapeutic agents is an immunotherapeutic agent, more particularly a cytokine or chemokine, such as IL1, IL2, IL12, IL18, ELC / CCL19, SLC / CCL21, MCP-1, IL-4, IL-18, TNF, IL-15, MDC, IFNa / b, M-CSF, IL-3, GM-CSF, IL-13, and anti-IL-10; bacterial lipopolysaccharide (LPS); organic or inorganic adjuvants that activate antigen presenting cells and promote the presentation of antigenic epitopes on major histocompatibility complex molecules, including but not limited to Toll-like receptor (TLR) agonists, antagonists of the mevalonate pathway, agonists of STING; indoleamine 2,3-dioxygenase 1 (IDO1) inhibitors, and immunostimulatory oligonucleotides, and other T cell adjuvants.

[0259] In some embodiments, one or more of the additional therapeutic agents is an immunotherapeutic agent, more particularly a cell therapy. Cell therapy is a form of treatment in which live cells are administered to a subject. In certain embodiments, one or more of the additional therapeutic agents is a cellular immunotherapy that activates or suppresses cells of the immune system. Cellular immunotherapies suitable for treating cancer generally induce or expand an immune response. The cells can be autologous or allogeneic immune cells (e.g., monocytes, macrophages, dendritic cells, NK cells, T cells, etc.) collected from one or more subjects. Alternatively, the cells can be “(re)programmed” allogeneic immune cells generated from immune precursor cells (e.g., lymphoid progenitor cells, myeloid progenitor cells, common dendritic cell precursor cells, stem cells, induced pluripotent stem cells, etc.). In some embodiments, such cells can be an expanded subset of cells with different effector functions and / or markers of maturity (e.g., adaptive memory NK cells, tumor infiltrating lymphocytes, immature dendritic cells, monocyte-derived dendritic cells, plasmacytoid dendritic cells, conventional dendritic cells (sometimes referred to as classic dendritic cells), Ml macrophages, M2 macrophages, etc.), can be genetically modified to target the cells to specific antigens and / or to enhance the anti-tumor effects of the cells (e.g., engineered T cell receptor (TCR) cell therapy, chimeric antigen receptor (CAR) cell therapy, antigen-loaded dendritic cell lymph node homing, etc.), can be engineered to express or increase expression of tumor-associated antigens, or can be any combination thereof. Non-limiting types of cell therapy include CAR-T cell therapy, CAR-NK cell therapy, TCR therapy, and dendritic cell vaccines. Exemplary cellular immunotherapies include sipuleucel-T, tisagenlecleucel, lisocabtagene maraleucel, idecabtagene vicleucel, brexucabtagene autoleucel, and axicabtagene ciloleucel, as well as CTX110, JCAR015, JCAR017, MB-CART19.1, MB-CART20.1, MB-CART2019.1, UniCAR02-T-CD123, BMCA-CAR-T, JNJ-68284528, BNT211, and NK-92 / 5.28.z.

[0260] In some embodiments, one or more of the additional therapeutic agents is an immunotherapeutic agent, more particularly a gene therapy. Gene therapy includes the administration to a subject or ex vivo administration to a subject’s cells of a recombinant nucleic acid (e.g., a small interfering RNA (siRNA) agent, a double-stranded RNA (dsRNA) agent, a microRNA (miRNA) agent, a viral or bacterial gene delivery, etc.) to modify the expression of an endogenous gene or to cause the heterologous expression of a protein, as well as gene editing therapies that can or can not include a nucleic acid component (e.g., meganucleases, zinc finger nucleases, TAL nucleases, CRISPR / Cas nucleases, etc.), oncolytic viruses, etc. Non-limiting examples of gene therapies that can be suitable for cancer treatment include (rAd-p53), (rAD5-H101), talimogene laherparepvec, Mx-dnG1, ARO-HIF2 (Arrowhead), quaratusugene ozeplasmid (Immunogene), CTX110 (CRISPR Therapeutics), CTX120 (CRISPR Therapeutics), and CTX130 (CRISPR Therapeutics).

[0261] In some embodiments, one or more of the additional therapeutic agents is an immunotherapeutic agent, more particularly an agent that modulates an immune checkpoint. Immune checkpoints are a group of inhibitory and stimulatory pathways that directly influence the function of immune cells (e.g., B cells, T cells, NK cells, etc.). Immune checkpoints are engaged when a protein on the surface of an immune cell recognizes and binds to its cognate ligand. The present disclosure encompasses the use of an anti-CD39 antibody of Part II in combination with an agonist of a stimulatory or co-stimulatory pathway and / or an antagonist of an inhibitory pathway. The agonist of a stimulatory or co-stimulatory pathway and the antagonist of an inhibitory pathway can have utility as agents that overcome different immunosuppressive pathways within the tumor microenvironment, inhibit T regulatory cells, reverse / prevent T cell anergy or exhaustion, trigger innate immune activation and / or inflammation at the site of the tumor, or a combination thereof.

[0262] In some embodiments, one or more of the additional therapeutic agents is an immune checkpoint inhibitor. As used herein, the term “immune checkpoint inhibitor” refers to an antagonist of an inhibitory or co-inhibitory immune checkpoint. The terms “immune checkpoint inhibitor,” “checkpoint inhibitor,” and “CPI” are used interchangeably herein. Immune checkpoint inhibitors can antagonize inhibitory or co-inhibitory immune checkpoints by interfering with receptor-ligand binding and / or altering receptor signaling. Examples of immune checkpoints (ligands and receptors) that can be antagonized, some of which are selectively upregulated in various types of cancer cells, include PD-1 (programmed cell death protein 1); PD-L1 (PD-1 ligand); BTLA (B and T Lymphocyte Attenuator); CTLA-4 (cytotoxic T-lymphocyte-associated antigen 4); TIM-3 (T-cell immunoglobulin and mucin-domain containing-3); LAG-3 (lymphocyte activation gene 3); TIGIT (T-cell immunoreceptor with Ig and ITIM domains); CD276 (B7-H3), PD-L2, Galectin 9, CEACAM-1, BTLA, CD69, Galectin-1, CD113, GPR56, VISTA, 2B4, CD48, GARP, PD1H, LAIR1, TIM-1, and TIM-4, and killer inhibitory receptors, which can be grouped into two classes based on their structural features: i) killer-cell immunoglobulin-like receptors (KIRs), and ii) C-type lectin receptors (members of the family of type II transmembrane receptors). Other less well-defined immune checkpoints described in the literature are also contemplated, including receptors (such as the 2B4 (also known as CD244) receptor) and ligands (such as certain B7 family inhibitory ligands, such as B7-H3 (also known as CD276) and B7-H4 (also known as B7-S1, B7x, and VCTN1)).

[0263] In some embodiments, the immune checkpoint inhibitor is a CTLA-4 antagonist. In other embodiments, the CTLA-4 antagonist can be an antagonistic CTLA-4 antibody. Suitable antagonistic CTLA-4 antibodies include, for example, monospecific antibodies, such as ipilimumab or tremelimumab, and bispecific antibodies, such as MEDI5752 and KN046.

[0264] In some embodiments, the immune checkpoint inhibitor is a PD-1 antagonist. In other embodiments, the PD-1 antagonist can be an antagonistic PD-1 antibody, a small molecule, or a peptide. Suitable antagonistic PD-1 antibodies include, for example, monospecific antibodies, such as balstilimab, budigalimab, camrelizumab, cosibelimab, dostarlimab, cemiplimab, ezabenlimab, MEDI-0680 (AMP-514; WO2012 / 145493), nivolumab, pembrolizumab, pidilizumab, pimivalimab, retifanlimab, sasanlimab, spartalizumab, sintilmab, tislelizumab, toripalimab, and zimberelimab; and bispecific antibodies, such as LY3434172. In yet other embodiments, the PD-1 antagonist can be a recombinant protein composed of the extracellular domain of PD-L2 (B7-DC) fused to the Fc portion of IgGl (AMP-224). In certain embodiments, the immune checkpoint inhibitor is zimberelimab.

[0265] In some embodiments, the immune checkpoint inhibitor is a PD-L1 antagonist. In other embodiments, the PD-L1 antagonist can be an antagonistic PD-L1 antibody. Suitable antagonistic PD-Ll antibodies include, for example, monospecific antibodies, such as avelumab, atezolizumab, durvalumab, BMS-936559, and envafolimab, and bispecific antibodies, such as LY3434172 and KN046.

[0266] In some embodiments, the immune checkpoint inhibitor is a TIGIT antagonist. In other embodiments, the TIGIT antagonist can be an antagonistic TIGIT antibody. Suitable antagonistic anti-TIGIT antibodies include monospecific antibodies such as AGEN1327, AB308 (WO2021247591), BMS 986207, COM902, domvanalib, EOS-448, etigilimab, IBI-929, JS006, M6223, ociperlimab, SEA-TGT, tiragolumab, vibostolimab; and bispecific antibodies such as AGEN1777 and AZD2936. In certain embodiments, the immune checkpoint inhibitor is an antagonistic anti-TIGIT antibody disclosed in WO2017152088 or WO2021247591. In certain embodiments, the immune checkpoint inhibitor is domvanalib or AB308.

[0267] In some embodiments, the immune checkpoint inhibitor is a LAG-3 antagonist. In other embodiments, the LAG-3 antagonist can be an antagonistic LAG-3 antibody. Suitable antagonistic LAG-3 antibodies include, for example, BMS-986016 (WO10 / 19570, WO14 / 08218) or IMP-731 or IMP-321 (WO08 / 132601, WO09 / 44273).

[0268] In some embodiments, the immune checkpoint inhibitor is a B7-H3 antagonist. In other embodiments, the B7-H3 antagonist is an antagonistic B7-H3 antibody. Suitable antagonistic B7-H3 antibodies include, for example, MGA271 (WO11 / 109400), omburtumab, enoblituzumab, DS-7300a, ABBV-155, and SHR-A1811.

[0269] In some embodiments, the immune checkpoint inhibitor is a TIM-3 antagonist. In other embodiments, the TIM-3 antagonist can be an antagonistic TIM-3 antibody. Suitable antagonistic TIM-3 antibodies include, for example, motolimab, sabatolimab, BMS-986258. and RG7769 / RO7121661.

[0270] In some embodiments, one or more of the additional therapeutic agents activates a stimulatory or costimulatory immune checkpoint. Examples of stimulatory or costimulatory immune checkpoints (ligands and receptors) include B7-1, B7-2, CD28, 4-1BB (CD137), 4-1BBL, ICOS, ICOS-L, OX40, OX40L, GITR, GITRL, CD70, CD27, CD40, DR3, and CD2.

[0271] In some embodiments, the agent that activates a stimulatory or costimulatory immune checkpoint is a CD137 (4-1BB) agonist. In other embodiments, the CD137 agonist can be an agonistic CD137 antibody. Suitable CD137 antibodies include, for example, urelumab and PF-05082566 (WO12 / 32433). In some embodiments, the agent that activates a stimulatory or costimulatory immune checkpoint is a GITR agonist. In other embodiments, the GITR agonist can be an agonistic GITR antibody. Suitable GITR antibodies include, for example, BMS-986153, BMS-986156, TRX-518 (WO06 / 105021, WO09 / 009116), and MK-4166 (WO11 / 028683). In some embodiments, the agent that activates a stimulatory or costimulatory immune checkpoint is an OX40 agonist. In other embodiments, the OX40 agonist can be an agonistic OX40 antibody. Suitable OX40 antibodies include, for example, MEDI-6383, MEDI-6469, MEDI-0562, PF-04518600, GSK3174998, BMS-986178, and MOXR0916. In some embodiments, the agent that activates a stimulatory or costimulatory immune checkpoint is a CD40 agonist. In other embodiments, the CD40 agonist can be an agonist CD40 antibody, such as dacetuzumab, selicrelumab, APX005M, ADC-1013, or CDX-1140. In some embodiments, the agent that activates a stimulatory or costimulatory immune checkpoint is a CD27 agonist. In other embodiments, the CD27 agonist can be an agonistic CD27 antibody. Suitable CD27 antibodies include, for example, varlilumab.

[0272] In some embodiments, one or more of the additional therapies is an immunotherapeutic agent, more particularly a signal transduction inhibitor. Intracellular signaling molecules that affect immune cell function can also be targets for improving anti-tumor immunity. For example, one or more of the additional therapies can be an inhibitor of an intracellular signaling molecule, an inhibitor of hematopoietic progenitor kinase 1 (HPK1). HPK1 is a serine / threonine kinase that acts as a negative regulator of activation signals generated by T cell antigen receptors. As another example, one or more of the additional therapies can be an inhibitor of Cbl-b, an E3 ubiquitin ligase involved in the regulation of TCR signaling (e.g., AP401). As another example, one or more of the additional therapies can be an inhibitor of diacylglycerol kinase (DGK). In some embodiments, the inhibitor is a small molecule. Non-limiting examples of small molecule HPK1 inhibitors in clinical development include CFI-402411 and BGB-15025; non-limiting examples of Cbl-b inhibitors in clinical development include AP401. Non-limiting examples of small molecule DAG inhibitors include those described in WO2020006016A1 and WO2021130638.

[0273] In some embodiments, one or more of the additional therapeutic agents is an agent that inhibits or depletes immunosuppressive immune cells. For example, to inhibit or deplete immunosuppressive macrophages or monocytes, the agent can be a CSF-1R antagonist, e.g., a CSF-1R antagonist antibody, including RG7155 (WO11 / 70024, WO11 / 107553, WO11 / 131407, W013 / 87699, W013 / 119716, W013 / 132044) or FPA-008 (WO11 / 140249; W013169264) or antibodies disclosed in WO14 / 036357. As another example, to inhibit or deplete Tregs, the agent can be anti-CD25 beads for ex vivo depletion of Tregs.

[0274] In some embodiments, the present disclosure encompasses the use of an anti-CD39 antibody of Part II in combination with an immunogenic cell death (ICD) inducing therapy. ICD can be defined as a functionally unique, regulated subtype of cell death that is sufficient to induce adaptive immunity specific to antigens derived from the “corpse” of the cell. Criteria for identifying ICD inducing therapies can include those described in Vanmeerbeek et al., OncoImmunology, 2020, 9: 1, DOI: 10.1080 / 2162402X.2019.1703449; Keep et al., OncoImmunology, 2014, 3(9): e955691. Non-limiting examples of ICD inducing therapies are described in Galluzzi et al., Nature Reviews Clinical Oncology, 2020 17:725-742. In some embodiments, the ICD inducing therapy can be radiation therapy, photodynamic therapy, extracorporeal photochemotherapy, oncolytic virus therapy, bortezomib, cyclophosphamide, doxorubicin, epirubicin, edaravone, mitoxantrone, cetuximab, crizotinib, or oxaliplatin. In some embodiments, the ICD inducing therapy can be radiation therapy, photodynamic therapy, extracorporeal photochemotherapy, oncolytic virus therapy, bleomycin, bortezomib, carboplatin, cetuximab, crizotinib, cyclophosphamide, docetaxel, doxorubicin, epirubicin, gemcitabine, edaravone, irinotecan, mitoxantrone, oxaliplatin, paclitaxel, vemurafenib, or vorinostat.

[0275] In some embodiments, each additional therapy can independently be a radiation therapy, a chemotherapeutic agent, a radiopharmaceutical, a hormonal therapy, an epigenetic modulator, a targeted agent, an immunotherapeutic agent, a cellular therapy, a gene therapy, or an ICD-inducing therapy. For example, in one embodiment, the present disclosure encompasses the use of an anti-CD39 antibody of Part II in combination with one or more ICD-inducing therapies and optionally one or more additional therapies, wherein each additional therapy is independently selected from a radiation therapy, a radiopharmaceutical, a chemotherapeutic agent, a hormonal therapy, a targeted agent, an immunotherapeutic agent, a cellular therapy, or a gene therapy. In another example, in one embodiment, the present disclosure encompasses the use of an anti-CD39 antibody of Part II in combination with one or more chemotherapeutic agents and optionally one or more additional therapies, wherein each additional therapy is independently selected from a radiation therapy, a radiopharmaceutical, a hormonal therapy, a targeted agent, an immunotherapeutic agent, a cellular therapy, or a gene therapy. In another embodiment, the present disclosure encompasses the use of an anti-CD39 antibody of Part II in combination with one or more chemotherapeutic agents and one or more tyrosine kinase inhibitors and optionally one or more additional therapies, wherein each additional therapy is independently a targeted agent, an immunotherapeutic agent, or a cellular therapy. In another embodiment, the present disclosure encompasses the use of an anti-CD39 antibody of Part II in combination with one or more chemotherapeutic agents and one or more inhibitors independently selected from: (i) a BCR-ABL kinase inhibitor; (ii) an EGFR inhibitor (e.g., an EGFR TKI or an anti-EGFR antibody); (iii) a HER-2 / neu receptor inhibitor; (iv) an anti-angiogenic agent (e.g., an anti-VEGF antibody, a VEGF RTKI, a VEGF kinase inhibitor, etc.); (V) an AKT inhibitor; (vi) a BRAF inhibitor; (vii) a RET inhibitor; (viii) a MET inhibitor; (ix) a RAS inhibitor; and (x) an ALK inhibitor, and optionally one or more additional therapies, wherein each additional therapy is independently selected from a radiation therapy, a radiopharmaceutical, a targeted agent, an immunotherapeutic agent, or a cellular therapy. In another embodiment, the present disclosure encompasses the use of an anti-CD39 antibody of Part II in combination with one or more immunotherapeutic agents and optionally one or more additional therapies, wherein each additional therapy is independently selected from a radiation therapy, a radiopharmaceutical, a hormonal therapy, a targeted agent, a chemotherapeutic agent, a cellular therapy, or a gene therapy. In another embodiment, the present disclosure encompasses the use of an anti-CD39 antibody of Part II in combination with one or more immunotherapeutic agents and one or more chemotherapeutic agents and optionally one or more additional therapies, wherein each additional therapy is independently selected from a radiation therapy, a radiopharmaceutical, a hormonal therapy, a targeted agent, a cellular therapy, or a gene therapy.In another embodiment, the present disclosure encompasses the use of an anti-CD39 antibody of Part II in combination with one or more immunotherapeutics and one or more radiation therapy or radiopharmaceuticals and optionally one or more additional therapies, wherein each additional therapy is independently selected from a chemotherapeutic agent, a hormonal therapy, a targeted agent, a cellular therapy, or a gene therapy. In another embodiment, the present disclosure encompasses the use of an anti-CD39 antibody of Part II of the present disclosure in combination with one or more immune checkpoint inhibitors and / or one or more ATP-adenosine axis targeting agents and optionally one or more additional therapies, wherein each additional therapy is independently selected from a radiation therapy, a radiopharmaceutical, a chemotherapeutic agent, a targeted agent, an immunotherapeutic, or a cellular therapy. In another embodiment, the present disclosure encompasses the use of an anti-CD39 antibody of Part II in combination with one or more immune checkpoint inhibitors and / or one or more ATP-adenosine axis targeting agents and / or one or more chemotherapeutic agents, radiopharmaceuticals, or radiation therapies. In another embodiment, the present disclosure encompasses the use of an anti-CD39 antibody of Part II in combination with one or more immune checkpoint inhibitors and / or one or more ATP-adenosine axis targeting agents and one or more inhibitors independently selected from: (i) a BCR-ABL kinase inhibitor; (ii) an EGFR inhibitor (e.g., an EGFR TKI or an anti-EGFR antibody); (iii) a HER-2 / neu receptor inhibitor; (iv) an anti-angiogenic agent (e.g., an anti-VEGF antibody, a VEGFR TKI, a VEGF kinase inhibitor, etc.); (V) an AKT inhibitor; (vi) a BRAF inhibitor; (vii) a RET inhibitor; (viii) a MET inhibitor; (ix) a RAS inhibitor; and (x) an ALK inhibitor. In another embodiment, the present disclosure encompasses the use of an anti-CD39 antibody of Part II in combination with one or more immune checkpoint inhibitors and / or one or more ATP-adenosine axis targeting agents and one or more ICD-inducing therapies. In other embodiments of the above, (a) the targeted agent can be a PI3K inhibitor, an arginase inhibitor, a HIF2a inhibitor, an AXL inhibitor, a PAK4 inhibitor, or an anti-angiogenic agent; (b) the immunotherapeutic is an ATP-adenosine axis targeting agent, a cytokine therapy, an immune checkpoint inhibitor, or a combination thereof; (c) the ATP-adenosine axis targeting agent is A. 2A R and / or A 2BR antagonist or CD73 inhibitor; (d) the ATP-adenosine axis targeting agent is itrukuma denan or quelicruzastat; (e) the immunotherapeutic agent is an anti-PD-Ll antagonist antibody or an anti-PD-1 antagonist antibody, which is optionally selected from the group consisting of: batabulimab, camrelizumab, cosibelimab, domvanalimab, cemiplimab, emibetuzumab, nivolumab, pembrolizumab, pidilizumab, pimonidazole, rafewizumab, savolitinib, spartalizumab, sindigimab, tislelizumab, tremikimab, seviprotmab, LY3434172, avelumab, atezolizumab, batatabimab, durvalumab, envafolimab, LY3434172, and KN046; (f) the immunotherapeutic agent is an anti-TIGIT antagonist antibody, which is optionally selected from the group consisting of: AGEN1327, AB308 (WO2021247591), BMS 986207, COM902, domvanalimab, EOS-448, eftilagimod, IBI-929, JS006, M6223, osemitirumab, SEA-TGT, tiragolumab, vibostolimab; and bispecific antibodies such as AGEN1777 and AZD2936; (g) the immunotherapeutic agent is domvanalimab, AB308, seviprotmab, tiragolumab, pembrolizumab, nivolumab, atezolizumab, or durvalumab; (h) the anti-angiogenic agent is pazopanib, sorafenib, sunitinib, bevacizumab, axitinib, lenvatinib, tevosvertib, or cabozantinib; (i) the ICD-inducing therapy is radiation therapy (including both external beam therapy and internal radiation therapy), photodynamic therapy, extracorporeal photochemotherapy, oncolytic viral therapy, bortezomib, cyclophosphamide, doxorubicin, epirubicin, edelfosidine, mitoxantrone, or oxaliplatin; or (j) any combination thereof. In still other embodiments of the above, the present disclosure encompasses the use of an anti-CD39 antibody of Part II in combination with domvanalimab, AB308, itrukuma denan, quelicruzastat, seviprotmab, AB521, or any combination thereof. In still other embodiments of the above, the present disclosure encompasses the use of an anti-CD39 antibody of Part II in combination with itrukuma denan, quelicruzastat, or a combination of itrukuma denan and quelicruzastat. In still other embodiments of the above, the present disclosure encompasses the use of an anti-CD39 antibody of Part II in combination with itrukuma denan, AB521, or a combination of itrukuma denan and AB521. In still other embodiments of the above, the present disclosure encompasses the use of an anti-CD39 antibody of Part II in combination with quelicruzastat, AB521, or a combination of quelicruzastat and AB521.

[0276] The choice of additional therapeutic agent can be informed by the current standard of care for the particular cancer and / or the mutation status and / or disease stage of the subject’s cancer. Detailed guidelines for the standard of care are publicly available, for example, through the National Comprehensive Cancer Network (NCCN). See, e.g., NCCN Colon Cancer v3.2021, NCCN Hepatobiliary Cancer v5.2021, NCCN Kidney Cancer, v3.2022, NCCN NSCLC v7.2021, NCCN Pancreatic Adenocarcinoma v2.2021, NCCN Esophageal and Esophagogastric Junction Cancers v4.2021, NCCN Gastric Cancer v5.2021, Ovarian Cancer / Fallopian Tube Cancer / Primary Peritoneal Cancer v3.2021, Prostate Cancer v3.2022, Head and Neck Cancers v1.2022, Melanoma: Cutaneous v1.2022, Acute Myeloid Leukemia, v1.2022.

[0277] Experiments

[0278] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present disclosure, and are not intended to limit the scope of what the inventors regard as their application. Additional antibodies within the scope of the present disclosure can be made using methods based on those illustrated in these examples or based on other methods described herein. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.) but some experimental errors and deviations should be accounted for.

[0279] Unless otherwise indicated, temperatures are in degrees Celsius (°C) and pressures are at or near atmospheric pressure. Standard abbreviations are used, including the following: rt or r.t. = room temperature; min = minute(s); h or hr = hour(s); ng = nanogram; pg = microgram; mg = milligram; g = gram; kg = kilogram; pl or pL = microliter; ml or mL = milliliter; 1 or L = liter; pM = micromolar; mM = millimolar; M = molar; mol = mole; mmol = millimole; nM = nanomolar.

[0280] Example 1: Generation of Anti-CD39 Antibodies

[0281] CHO-K1 cell lines expressing human CD39, HEK-293 cell pools expressing cynomolgus CD39, and HEK-293 cell pools expressing murine CD39 were generated. Briefly, CHO-K1 cells or HEK-293 cells (Flp-In TM -293 cells) were transfected with pcDNA3.3 expression vectors encoding full-length human, cynomolgus, or murine CD39 (SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 71, respectively) using Lipofectamine 2000 transfection kit according to the manufacturer’s protocol 48-72 hours post transfection, transfected cells were cultured in media containing blasticidin for selection and testing for CD39 expression, then CD39-expressing cell pools were obtained. Cell lines or cell pools expressing human, cynomolgus, and murine CD39 were obtained by limiting dilution and used to screen hybridoma supernatants as described below.

[0282] To generate anti-CD39 antibodies, four 6-8 week old Sprague Dawley rats were immunized with 30-200 mg / animal of human CD39 antigen (protein or plasmid DNA). Adjuvant mixtures included Adju-Phos, CpG-ODN, or Titer-Max. Animals were injected every other week via footpad, subcutaneously, intraperitoneally, intramuscularly, and intradermally. Serum titers were measured by ELISA or fluorescence activated cell sorting (FACS). ELISA for measuring serum antibody titers against a given antigen was performed as follows. Plates (Nunc) were coated with 100 μL of human CD39 antigen at 1 μg / mL overnight at 4°C and then blocked with blocking buffer (1x PBS / 2% BSA) for 1 hour at ambient temperature. Rat serum was started at a 1: 100 dilution in blocking buffer with 3-fold serial dilutions and incubated for 1 hour at ambient temperature. Wells without serum sample added were used as negative controls. Plates were then washed and subsequently incubated with secondary antibody goat anti-rat IgG-Fc-HRP (Bethyl) for 1 hour. After washing, tetramethylbenzidine (TMB) substrate was added and the interaction was stopped by adding 2 M HC1. Absorbance at 450 nm was read using a microplate reader (Molecular Device). FACS assay for measuring serum antibody titers was performed as generally described for screening hybridoma supernatants (described below) with the exception that serially diluted serum was used. Serum titer was determined as the final dilution that gave a positive signal (i.e., >3-fold over negative control).

[0283] When serum titers were high enough (>1 :24,300), animals were given a final boost with protein and cell membrane lysate in sterile PBS without adjuvant. After 48-96 hours, animals were euthanized and lymph nodes and spleen were used for cell fusion.

[0284] Lymph nodes and spleen from immunized animals were homogenized and filtered to remove blood clots and cell debris. Logarithmically growing Sp2 / 0 myeloma cells were collected and centrifuged. B cells and Sp2 / 0 myeloma cells were treated separately with a solution of pronase and the reaction was stopped with 100% FBS. Cells were washed and counted. B cells were fused with Sp2 / 0 myeloma cells at a 1 : 1 ratio in an electrofusion solution following a general electrofusion procedure. The fused cells were resuspended in DMEM medium supplemented with 20% FBS and lx HAT and then transferred to a 96-well plate. The fused cells were kept in an incubator set at 37°C and 5% CO2 for 10-14 days.

[0285] Hybridoma cells were harvested and 150-200 cells were added to 1.5 mL of semi-solid HAT medium. The cells were gently mixed in a vortex shaker for 5-10 seconds and then seeded in a 6-well plate. The plate was kept in an incubator set at 37°C, 5% CO2 for 7-8 days. Each visible single colony was picked into a 96-well plate with DMEM medium supplemented with 10% FBS. After 2-3 days, the cell supernatants were collected and screened.

[0286] Four rounds of hybridoma production and antibody screening were performed, with the order of the assay cascade in the screening paradigm varying. To meet screening criteria, antibodies produced from a given hybridoma clone needed to bind to human and cynomolgus CD39 with high affinity, with no measurable binding to murine CD39 or to any of the other CD39 family members (CD39-L1, CD39-L2, CD39-L3, or CD39-L4). Antibodies also needed to exhibit potent inhibition of cellular human CD39 enzyme activity in both cell lines engineered to overexpress CD39 and human cell lines that naturally express CD39 (e.g., THP-1, SK-MEL-5, and MOLP-8). In the first round of fusion, a human CD39 ELISA based on CHO-K1 cells was the primary screen, and binding to cellular overexpression of human, cynomolgus, and murine CD39, as measured by FACS (using the cell lines described previously), and both the in vitro soluble human CD39 enzyme assay and the cellular enzyme assay were secondary screens. The second round of fusion failed to produce suitable hybridoma material. In the third round of fusion, the soluble human CD39 enzyme assay was the primary screen, and binding to cellular overexpression of human and murine CD39 as measured by FACS, the human CD39-L1 binding counter screen, and the confirmatory soluble human CD39 enzyme assay were secondary screens. In the fourth round of fusion, the THP-1 cell enzyme assay was used as the primary screen, and binding to cellular overexpression of human and cynomolgus CD39 as measured by FACS and the confirmatory THP-1 cell enzyme assay were secondary screens. Clone 39 from the first round, and clones 19 and 31 from the third round were selected for further characterization. No clones were selected from the fourth round.

[0287] For cell-based ELISAs, which are used to evaluate binding to CD39 expressed on the surface of cells, the assay is typically performed as follows. Plates (384 well) are pre-coated with 3-5 x 104cells / well and incubated in an incubator set at 37 °C, 5% CO2for 2 days. Plates are blocked with blocking buffer (lx PBS / 5% milk) for 1 hour at ambient temperature. Thirty μL of hybridoma supernatant is then added to the plates and incubated for 1 hour at ambient temperature. Plates are washed three times with PBS and then incubated with secondary antibody goat anti-rat IgG-Fc-HRP (1:500) for 1 hour at ambient temperature. After washing, TMB substrate is added to each well and the plates are incubated in the dark at ambient temperature for 3-5 minutes, and the reaction is stopped by the addition of 2M HC1. Absorbance at 450 nm is read using a microplate reader (Molecular Device). 4

[0288] ​FACS was also used to detect binding of anti-CD39 antibodies to CD39 in supernatants using the previously described CHO-K1 cell line expressing human CD39, HEK-293 cell line expressing cynomolgus CD39, and HEK-293 cell pool expressing murine CD39. Unlabeled cells were used as a control to set a threshold before detection, then the percent change of each group that exceeded the fluorescence intensity threshold was analyzed. 1 x 105cells / well were incubated with hybridoma supernatants in a 100 μL volume for 1 hour at 4°C. An anti-human CD39 reference antibody was used as a positive control. A human / rat IgG isotype antibody was used as a negative control. After washing the cells with 1 x PBS / 1% BSA, Alexa647-labeled goat anti-human antibody or goat anti-rat antibody was added (diluted 1:500 in FACS staining buffer). The cells were incubated for 1 hour at 4°C in the dark. The mean fluorescence intensity (MFI) of the cells was measured by flow cytometry and analyzed using FlowJo software. 5

[0289] Anti-CD39 antibodies were tested for their ability to block the enzymatic activity of soluble CD39 extracellular domain (ECD) antigen by measuring the inhibitory effect on the catalytic function of human CD39 for hydrolyzing ATP to AMP + inorganic phosphate (coupled to luciferase activity and light emission as a signal). Enzyme assays were performed in 96-well multiwell plates in a final volume of 120 μL. The assay mixture was incubated in an incubator set to 37°C for the indicated incubation period. Tris magnesium (TM) buffer contains 25 mM Tris, 5 mM MgCl2, and 0.01% BSA. Serial dilutions of inhibitory anti-CD39 antibodies were prepared starting with a 20 nM solution in TM buffer containing soluble human CD39 (Sino Biological, Catalog # 16020-H08B). Dilutions were made with or without ATP to determine the effect of pre-mixing antibody and enzyme, then substrate (ATP) was added. As an isotype control, an unrelated antibody was similarly diluted to test the specificity of antibody-mediated CD39 inhibition. The antibody / enzyme preparation was incubated in an incubator set to 37°C for 1 hour, then ATP was added at a final concentration of 20 μm and incubated at 37°C for 1 hour. Enzyme activity was quantified using CellTiter-Glo (CTG). The amount of remaining ATP was measured using CTG luciferase reagent from the CTG Luminescent Cell Viability Assay (Promega Corporation).

[0290] ​The functional activity of anti-CD39 antibodies was determined by measuring CD39 catalyzed ATP hydrolysis using the human myeloid monocytic leukemia cell line (THP-1). THP-1 cells were resuspended in TM buffer and 80 μL / well cells were seeded in a 96-well U plate at a density of 4 x 10 4 cells / well, then 40 μL / well of antibody CD39 solution (various concentrations, 5-fold serial dilutions from 100 nM to 1.28 pM in TM buffer) was added to the plate. Antibodies and cells were incubated for 1 hour in an incubator set to 37 °C. After incubation, ATP (50 μM in TM buffer) was added to the plate in a volume of 80 μL / well and kept in an incubator set to 37 °C for 1 hour. The plate was placed in a centrifuge set to 1500 rpm for 5 minutes, and 50 μL / well of supernatant was transferred to a white 96-well plate (Corning, Cat. No. 3903). CellTiter-Glo (50 μL / well) was added to the respective wells and the wells were mixed. Cell enzyme inhibition on CD39 was measured on a multi-label reader (Perkin-Elmer Envision Workstation).

[0291] To test whether the anti-CD39 antibodies specifically bind to human CD39 but not cross-react with other CD39 family members (CD39-L1, CD39-L2, CD39-L3, CD39-L4), the binding of anti-CD39 antibodies to human CD39-L1, CD39-L2, CD39-L3, and CD39-L4 was determined by ELISA. Briefly, 96-well high protein binding ELISA plates (Nunc MaxiSorp, Thermo Fisher) were used for pre-coating with the following His-tagged human CD39 cross-family proteins in carbonate-bicarbonate buffer (20 mM Na2CO3, 180 mM NaHCO3, pH 9.2): CD39-L1, CD39-L2, CD39-L3, or CD39-L4 (0.5 pg / mL) at 4 °C overnight. The next day, the plates were washed with 300 pL / well of PBS / 0.5% (v / v) Tween-20 three times and then blocked with PBS / 2% BSA for 1 h. After blocking, the plates were washed three times. After washing, various concentrations (6-fold serial dilutions from 100 nM to 0.36 pM in 2% BSA / PBS) of test antibodies were added to the plates and incubated in an incubator set to room temperature for 2 h. An in-house CD39-L1 binding antibody was used as a positive control and a human IgG4 isotype antibody was used as a negative control. After washing three times, 100 pL / well goat anti-human IgG Fc-HRP antibody (1:5000) was added and the plates were placed in an incubator set to room temperature for 1 h. For color development, 100 pL / well TMB substrate solution was added to the plates. After 3-5 min, the reaction was stopped by 100 pL / well of 2 M HC1. The absorbance was read at 450 nm using a M5e microplate spectrophotometer. EC values were obtained from four-parameter nonlinear regression analysis using GraphPad Prism software. 50

[0292] Antibodies from hybridoma clone 19, hybridoma clone 31, and hybridoma clone 39 with rat Fc domains were purified from the hybridoma supernatants. These antibodies are referred to herein as antibodies 19, 31, and 39, respectively. The harvested hybridoma supernatants were loaded onto a protein A column after adjustment to pH 7.0. The bound antibodies were eluted by glycine-HCl (pH 3.5) followed by immediate neutralization with 0.1 M Tris (pH 9.0). The proteins were dialyzed against PBS and filtered with a 0.22 pm membrane filter. Antibody concentrations were measured by NanoDrop. The purity of the antibodies was evaluated by SDS-PAGE and HPLC-SEC.

[0293] ​Total RNA was isolated from hybridoma clones 19, 31 and 39 using the RNeasy Plus Mini Kit (Qiagen). First strand cDNA was reverse transcribed using oligo dT. The VH and VL genes of the antibodies were amplified from the cDNA using 3’ constant region degenerate primers and 5’ degenerate primer sets. The 5’ degenerate primers were designed based on the upstream signal sequence encoding region of Ig variable sequences. The PCR products were then ligated into pMD18-T vector and 10 μL of the ligation product was transformed into Top10 competent cells. The transformed cells were plated on 2x YT plates with carbocinin and incubated in an incubator set to 37 °C overnight. Twelve positive colonies were randomly picked for DNA sequencing by Biosune.

[0294] The cloned genes were codon optimized for mammalian expression and then synthesized by GENEWIZ (Suzhou, China). The resulting plasmids contained either a gene encoding a heavy chain comprising the VH domain of antibody 19, 31 or 39 fused to human IgG4 CH1, hinge, CH2 and CH3 segments (including the hinge mutation S228P), or a gene encoding a light chain comprising the VL domain fused to the human Ig kappa CK domain in the light chain.

[0295] To produce antibodies ch19_IGG4.P, ch31_IGG4.P and ch39_IGG4.P, plasmids containing the VH and VL genes described above were co-transfected into Expi293F cells. 2.94x10 6 / mL Expi293F cells in 40 mL cell culture medium with greater than 95% viability were prepared for each antibody. Plasmid DNA at a final concentration of 1 μg / mL was mixed with ExpiFectamine TM 293 transfection reagent and then added to the cell culture medium. The cell culture was incubated in a platform rocker at a rotation rate of 150 rpm. The temperature was maintained in an incubator set to 37 °C and 8% CO2 level. After six days of incubation, the cells were pelleted using centrifugation at 4000 rpm, 25 °C for 10 minutes. The supernatant was collected as necessary for gel electrophoresis, SPR k off sorting and purification. Following the instructions of NuPAGE TM 4-12% Bis-Tris Protein Gels (Thermo Fisher), the supernatant was loaded on SDS-PAGE gels. The PageRuler TM Unstained Protein Ladder (Thermo Fisher) was used to determine the molecular weight of the antibodies along with the antibody samples.

[0296] To purify the antibodies, a 1 mL MabSelect Sure resin pre-packed Protein A column was used. The column was equilibrated with five column volumes (CV) of equilibration buffer (0.1 M Tris, pH 7.0) before loading with cell culture fluid. After loading, the column was washed with 15 CV of 0.1 M Tris (pH 7.0) followed by elution with 8 CV of 0.1 M glycine (pH 3.5). The buffer of the eluted sample was exchanged to PBS buffer using a desalting column. Finally, the purified antibodies were analyzed by SDS-PAGE and SEC-HPLC and then stored at -80 °C.

[0297] FACS analysis was used to measure the binding of the recombinant anti-CD39 antibodies to CHO-K1 cells overexpressing human CD39 and HEK293 cells overexpressing cynomolgus or murine CD39 previously described. The ability of these antibodies to inhibit the enzymatic activity of cellular CD39 was also determined. Table 1 shows that all three selected antibodies have high affinity for cellular human CD39 and potently inhibit the enzymatic activity of cellular human CD39.

[0298] The kinetic binding of these recombinant anti-CD39 antibodies to human CD39 was determined by surface plasmon resonance (SPR) using a Biacore 8K instrument. A goat anti-human Fc IgG coated CM5 sensor chip (GE, Cat. No. 29-1496-03) was used to immobilize the recombinant antibodies and soluble His-tagged CD39 ECD protein SEQ ID NO: 72 (SinoBiological, Cat. No. 16020-H08B) was used as the analyte. The binding constants were determined at 25 °C. As shown in Table 2, all three antibodies have similar high binding affinities as measured by slow off-rate (koff) and sub-nanomolar equilibrium dissociation constant (Kd). d ) and sub-nanomolar equilibrium dissociation constant (K D ) as measured.

[0299] Table 1: Recombinant anti-CD39 rat / human chimeric antibodies binding to CD39 overexpressed on the surface of CHO-K1 or HEK293 cells, and their blocking activity to inhibit the enzymatic activity of the cells.

[0300]

[0301] Table 2: Kinetics of recombinant rat / human chimeric anti-CD39 antibodies binding to His-tagged human CD39 ECD.

[0302] Anti-CD39 antibodies k a (M -1 s -1 )]]> k d (s -1 )]]> K D (M) ch19_IGG4.P 9.02E+05 4.00E-04 4.43E-10 ch31_IGG4.P 1.10E+06 1.25E-04 1.13E-10 ch39_IGG4.P 3.48E+05 7.45E-05 2.14E-10

[0303] Example 2: Generation of humanized anti-CD39 antibodies

[0304] Rat antibodies 31 and 39 were selected for humanization using CDR grafting techniques (Queen et al., Proc. Natl. Acad. Sci. USA. 86: 10029-10033, 1989). The closest human germlines for each chain were identified using the variable heavy (VH) and variable light (VL) sequences of antibodies 31 and 39. Human acceptors for the VH and VL frameworks were retrieved within the GenBank database (Benson et al., Nucleic Acids Res. 2005, 33, D34-D38). The frameworks were defined using extended CDR definitions, where Kabat CDR1 is extended at the N-terminus by 5 amino acids. The humanized VH and VL gene sequences were deduced using the top three hits, and from each of these sequences two were selected for expression of the humanized antibodies. For antibody 31 VH, IGHV4-24*01 with 64.6% sequence identity and IGHV1-69-2*01 with 63.4% identity were selected, and for VL, IGKV7-3*01 with 84.8% sequence identity and IGKV4-1*01 with 77.2% identity were selected (Table 3). For antibody 39 VH, IGHV3-9*01 with 87.8% sequence identity and IGHV3-30*15 with 85.4% identity were selected, and for VL, IGKV7-3*01 with 83.5% sequence identity and IGKV3-15*01 with 72.2% identity were selected (Table 4).

[0305] Table 3: Identification of human germlines and acceptors for clone 31.

[0306] Variable chain Human germline Human / rat identity (%) Human acceptor VH1 IGHV1-24*01 64.6％ M99642 VH2 IGHV1-69-2*01 63.4％ KF698734 VL1 IGKV7-3*01 84.8％ X12682 VL2 IGKV4-1*01 77.2％ Z00023

[0307] Table 4: Identification of human germlines and acceptors for clone 39.

[0308] Variable chain Human germline Human / rat identity (%) Human acceptor VH1 IGHV3-9*01 87.8％ M99651 VH3 IGHV3-30*15 85.4％ M77327 VL1 IGKV7-3*01 83.5％ X12682 VL3 IGKV3-15*01 72.2％ M23090

[0309] CDR grafting was performed for each human acceptor. For antibody 31, HC-CDR1 (SEQ ID NO: 18), HC-CDR2 (SEQ ID NO: 19), and HC-CDR3 (SEQ ID NO: 20) were used for the VH acceptor, and LC-CDR1 (SEQ ID NO: 22), LC-CDR2 (SEQ ID NO: 25), and LC-CDR3 (SEQ ID NO: 26) were used for the VL acceptor. For antibody 39, HC-CDR1 (SEQ ID NO: 28), HC-CDR2 (SEQ ID NO: 29), and HC-CDR3 (SEQ ID NO: 30) were used for the VH acceptor, and LC-CDR1 (SEQ ID NO: 32), LC-CDR2 (SEQ ID NO: 33), and LC-CDR3 (SEQ ID NO: 34) were used for the VL acceptor. The resulting sequences were checked for the introduction of any potential post-translational modification (PTM) sites (e.g., isomerization, deamination, glycosylation, and oxidation). Putative residues suitable for rat sequence back-mutations were also identified using antibody homology graphical modeling. After removal of PTM sites, the antibodies were analyzed to determine if the changes affected antigen binding compared to the parental antibody.

[0310] Humanized genes were reverse translated, codon optimized for mammalian expression, and synthesized by GENEWIZ. The synthesized genes were re-cloned into in-house IgG expression vectors, expressed, and purified.

[0311] Binding kinetics of purified antibodies to antigen were determined using surface plasmon resonance (SPR) and used to rank order the antibodies. Biacore 8K instrument was used to measure the affinity of anti-CD39 antibodies to human CD39 ECD His-tagged antigen (SEQ ID NO: 72). An activation reagent was prepared by mixing 400 mM EDC and 100 mM NHS (GE) immediately prior to injection. A CM5 sensor chip (GE, Cat. No. 29-1496-03) was activated with the activation reagent for 420 s. Goat anti-human Fc IgG (30 pg / mL in 10 mM NaAc, pH 4.5) was then injected to the channel at a flow rate of 10 pL / min for 420 s. The chip was inactivated by 1 M ethanolamine hydrochloride. Anti-CD39 antibodies were diluted to 4 pg / mL in running buffer (1 x HBS-EP+) and injected to the channel at a flow rate of 10 pL / min for 15 s. Six concentrations (8, 4, 2, 1, 0.5, and 0.25 nM) of CD39 antigen analyte were injected sequentially to the channel at a flow rate of 30 pL / min for an association phase of 180 s followed by a dissociation phase of 2400 s. Glycine (10 mM, pH 1.5) was injected as a regeneration buffer after the dissociation phase. Sensograms for reference and buffer channels were subtracted from the test sensograms. Experimental data were fitted with a 1:1 binding model steady state affinity / heterogeneous ligand. The molecular weights of CD39 antigen and anti-CD39 antibodies used for calculation were 52 and 145 kDa, respectively.

[0312] Humanized variants of antibody 39 K94R rat sequence backmutation was introduced into the VH1 chain (SEQ ID NO: 58) in order to maintain the binding affinity of ch39_IGG4.P, but VH3 did not require an equivalent backmutation due to the presence of R94 (SEQ ID NO: 59). The human framework of VL1 also introduced a glycosylation PTM site 81 NDT 83 which was removed by introducing an additional rat sequence backmutation N81D (SEQ ID NO: 61). VL3 (SEQ ID NO: 62) did not require a rat sequence backmutation.

[0313] VH1 (SEQ ID NO:58) or VH3 (SEQ ID NO:59) of antibody 39 were then expressed with VL1 (SEQ ID NO:61) or VL3 (SEQ ID NO:62) in all four VH / VL combinations, however VH1 + VL3 did not express well. The genes for the following variants were codon optimized for mammalian expression and then synthesized by GENEWIZ: a plasmid containing a VH domain fused to human IgG4 CH1, hinge, CH2, and CH3 regions in a heavy chain including the hinge mutation S228P; and a plasmid containing a VL domain fused to human IgK CK domain in a light chain.

[0314] The humanized IgG4 (S228P) variants of antibody 39 and the chimeric rVH + rVL / human IgG4 (S228P) variants of antibody 39 were analyzed by SPR as described above. The results are shown in Table 5.

[0315] Table 5: Kinetic binding affinity data for humanized IgG4 (S228P) variants of antibody 39.

[0316] Anti-CD39 antibodies VH+VL k a (M -1 s -1 )]]> k d (s -1 )]]> K D (M) ch39_IGG4.P rVH+rVL 2.80E+05 9.40E-05 3.36E-10 hu39.2_IGG4.P VH1+VL1 5.35E+05 5.01E-05 9.36E-11 hu39.3_IGG4.P VH1+VL3 4.55E+05 1.16E-04 2.54E-10 hu39.4_IGG4.P VH3+VL1 2.79E+05 5.15E-05 1.85E-10 hu39.6_IGG4.P VH3+VL3 2.70E+05 9.38E-05 3.47E-10

[0317] Additional rat sequence back mutations (I2T) were introduced into the VL1 framework (SEQ ID NO:60) and VL3 framework (SEQ ID NO:63) and antibodies were expressed and analyzed, however the VH1 + VL3 pair did not express well. Table 6 shows the SPR data for these antibody variants.

[0318] Table 6: Kinetic binding affinity data for humanized IgG4 antibodies of clone 39.

[0319] Anti-CD39 antibodies VH+VL k a (M -1 s -1 )]]> k d (s -1 )]]> K D (M)]]> hu39.1_IGG4.P VH1+VL1 3.35E+05 4.59E-05 1.37E-10 hu39.8_IGG4.P VH1+VL3 5.67E+05 8.50E-05 1.50E-10 hu39.5_IGG4.P VH3+VL1 3.11E+05 3.75E-05 1.21E-10 hu39.7_IGG4.P VH3+VL3 3.08E+05 5.87E-05 1.91E-10

[0320] The humanized variants of antibody 39 showed high affinity for cellular human CD39 and potently inhibited the enzymatic activity of cellular human CD39 (Table 7).

[0321] Table 7: Humanized IgG4 anti-CD39 antibodies binding to CD39 overexpressed on the surface of CHO-K1 or HEK293 cells, and their blocking activity to inhibit the cellular enzymatic activity.

[0322]

[0323] Humanized variants of antibody 31The following rat sequence back mutations were introduced into the VH1 chain: E71S, D76N, and A78V (SEQ ID NO: 43); and the back mutations D76N and A78V were introduced into the VH2 chain (SEQ ID NO: 44) in order to maintain the binding affinity of ch31_IGG4.P. Antibody 31 has a glycosylation PTM site sequence in the LC CDR1 26 NQT 28 ). This was mutated to Q27P (SEQ ID NO: 45) or N26Q (SEQ ID NO: 46) in VL1 and N26Q (SEQ ID NO: 49) in VL2. The human framework of VL1 also introduced a glycosylation PTM site 81 NDT 83 ), which was removed by introducing the additional rat sequence back mutation N81D (SEQ ID NO: 47 and SEQ ID NO: 48, respectively). VL2 did not require a rat sequence back mutation.

[0324] All four VH / VL combinations of VH1 (SEQ ID NO: 43) or VH2 (SEQ ID NO: 44) with VL1 (SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, or SEQ ID NO: 48) or VL2 (SEQ ID NO: 49) of the humanized variants of antibody 31 were expressed, however the VH1 + VL2 pair did not express well. The genes of the following variants were codon optimized for mammalian expression and then synthesized by GENEWIZ: a plasmid containing a VH domain fused to human IgG4 CH1, hinge, CH2, and CH3 regions in a heavy chain (including hinge mutation S228P); and a plasmid containing a VL domain fused to a human Ig kappa CK domain in a light chain.

[0325] The humanized IgG4 (S228P) variants of antibody 31 and the chimeric rVH+rVL / human IgG4 (S228P) variants of antibody 31 were analyzed by SPR as described above. The results are shown in Table 8.

[0326] Table 8: Kinetic binding affinity data for clone 31 humanized IgG4 antibodies.

[0327] Anti-CD39 antibodies VH+VL k a (M -1 s -1 )]]> k d (s -1 )]]> K D (M) ch31_IGG4.P rVH+rVL 1.16E+06 1.50E-04 1.29E-10 hu31.4_IGG4.P VH1+VL1 1.11E+06 1.96E-04 1.76E-10 hu31.6_IGG4.P VH2+VL1 1.12E+06 6.84E-04 6.13E-10 hu31.7_IGG4.P VH2+VL2 1.24E+06 6.15E-04 4.96E-10

[0328] The humanized variants of antibody 31 showed high affinity for cellular human CD39 and potently inhibited the enzymatic activity of cellular human CD39 (Table 9).

[0329] Table 9: Humanized IgG4 anti-CD39 antibodies that bind to CD39 overexpressed on the surface of CHO-K1 or HEK293 cells, and their blocking activity to inhibit the enzymatic activity of the enzyme.

[0330]

[0331] Humanized variants of both antibody 31 and antibody 39 were obtained via application of CDR grafting followed by a small number of rat sequence back mutations to the human variable domain framework. These antibodies retained or improved the binding affinity and inhibitory potency of their corresponding rat / human chimeric antibodies.

[0332] Example 3: Full kinetic binding characterization by SPR

[0333] Genes for the codon-optimized VH and VL domains of hu31.4_IGG4.P and hu39.5_IGG4.P humanized antibodies for mammalian expression were used to generate human IgG1 variants. A plasmid containing the VH domain fused to the human IgG1 CH1, hinge, CH2, and CH3 constant domain segments in the heavy chain (HC) including L234A and L235A (Eu numbering) mutations (SEQ ID NO: 2); and a plasmid containing the VL domain fused to the human IgK CK constant domain in the light chain (LC) (SEQ ID NO: 6) were transfected into CHO-K1 cells. Antibodies hu31.4_IGG1.AA (HC with SEQ ID NO: 51 and LC with SEQ ID NO: 56) and hu39.5_IGG1.AA (HC with SEQ ID NO: 66 and LC with SEQ ID NO: 67) were expressed using the fast stable pool protocol and purified to >95% purity by protein A affinity chromatography followed by size exclusion chromatography (SEC), confirmed by SEC-HPLC analysis.

[0334] The antibodies hu31.4_IGG1.AA and hu39.5_IGG1.AA were analyzed by SPR using a Biacore 4000 instrument. Kinetic binding characterization was performed in two modes. In the "monovalent affinity mode", the test antibodies were captured using a CM3 sensor chip (GE, Cat. No. 29-1049-90) coated with amine-coupled goat anti-human IgG coated CM3 sensor chip at seven different densities. Soluble human CD39 ECD His-tagged antigen (Sino Biological, Cat. No. 16020-H08B) was the analyte; a stock solution prepared at 33.3 μΜ was diluted to 33 nM as the highest concentration and tested in triplicate over the antibody surface in a three-fold dilution series. In the "bivalent avidity mode", soluble human CD39 ECD His-tagged antigen was amine-coupled to a CM3 sensor chip at six different densities. The test antibodies for the analyte were prepared as a 33.3 μΜ stock solution, diluted to 100 nM as the highest concentration and tested in triplicate over the CD39 surface in a three-fold dilution series. The running buffer contained 10 mM HEPES, 150 mM NaCl, 0.05% tween-20 and 0.2 mg / mL BSA. Data were collected using single-cycle kinetics, where multiple injections were performed with increasing concentrations of analyte and a 1 h dissociation phase was monitored. All data were collected at 25 °C. Reaction data for all six or seven surface densities were fitted to a 1 : 1 interaction model using local Rmax (the maximum amount of complex formed in response). Results of the experiments performed in monovalent affinity mode are shown in Table 10 and for bivalent avidity mode in Table 11.

[0335] The antibodies hu31.4_IGG1.AA and hu39.5_IGG1.AA have similar equilibrium dissociation constants (K D ) when analyzed in monovalent affinity mode, where the antibody is captured on the sensor chip and soluble CD39 ECD is the analyte. However, when analyzed in a mode that allows bivalent antibody binding to CD39 ECD captured on the sensor chip and soluble antibody as the analyte, hu31.4_IGG1.AA shows a K D very similar to the one obtained when analyzed in monovalent mode. In contrast, hu39.5_IGG1.AA exhibits up to three orders of magnitude lower K D , depending on the antigen surface density. This indicates that this antibody is able to bind to more than one immobilized CD39 molecule on the chip surface in bivalent mode and thus shows an enhanced kinetic binding affinity due to avidity. D

[0336] ​Table 10: Kinetic data for hu31.4_IGG1.AA and hu39.5_IGG1.AA from SPR with immobilized antibody and soluble CD39 ECD analyte.

[0337]

[0338] Table 11: Kinetic data for hu31.4_IGG1.AA and hu39.5_IGG1.AA from SPR with immobilized CD39 ECD and soluble antibody analyte.

[0339]

[0340]

[0341] Example 4: Binding of Anti-CD39 Antibodies to Endogenous Human CD39

[0342] Variants of antibodies 19, 31, and 39 were tested for binding to the surface of primary human cells. Binding was assessed in the absence of ATP and in the presence of high ATP (400 mM).

[0343] Purified monocyte binding: Antibodies were tested for binding to human CD39 expressed on the cell surface of primary human monocytes (CD14 + ) using EasySep Human CD14 Positive Selection Kit II (Stem Cell, Cat# 17858) from peripheral blood (leukopak or LRS chamber), frozen in Bambanker (Wako, Cat# 302-14681), and stored in liquid nitrogen for future use. On the day of assay, frozen monocytes were thawed, washed, and resuspended in 1:25 Fc Block (BD, Cat# 564220) and 1:250 Aqua Viability Dye (Invitrogen, Cat# L34966A) in DPBS and incubated at room temperature for 15 minutes in the dark. In polypropylene v-bottom 96-well plates, 0.2-0.5 x 10 6Cells were plated at 1 cell / well. Zero or 400 mM ATP was added and incubated for 30 minutes at 4°C, followed by addition of test antibody and incubation for an additional 30 minutes at 4°C. After washing twice with DPBS, 1 pg / mL of PE-labeled mouse anti-human IgG4 secondary antibody (Southern Biotech, Catalog #9200-09) was added for 30 minutes at 4°C for antibody detection. After washing twice in DPBS, cells were fixed with 50 pL IC Fixation Buffer (Invitrogen, Catalog #00-8222-49) for 20 minutes at room temperature. Cells were then washed once and resuspended in DPBS and flow cytometry was performed using a BD LSR Fortessa X-20 cell analyzer. The geometric mean of PE fluorescence intensity of the live single cell population was obtained. EC 50 values (Table 12).

[0344] Table 12: EC 50 (nM) values. Data in this table represent one biological donor.

[0345] Anti-CD39 antibodies 0 µM ATP 400 µM ATP ch19_IGG4.P 61 49 ch31_IGG4.P 27 28 hu31.2_IGG4.P 15 25 hu31.3_IGG4.P 7.7 26 hu31.4_IGG4.P 16 26 ch39_IGG4.P 0.69 1.3 hu39.1_IGG4.P 1.2 1.4 hu39.4_IGG4.P 1.4 10 hu39.5_IGG4.P 3.9 10 hu39.6_IGG4.P 3.5 6.5 hu39.7_IGG4.P 5.7 6.9

[0346] PBMC binding: Antibodies 31 and 39 IgGl isotype variants were also tested for binding to human CD39 expressed on the cell surface of PBMCs gated on CD14 + (monocytes) and CD19 + (B cells) populations by flow cytometry. In this experiment, an antibody reported to bind and inhibit human CD39, referred to herein as huBMK2_IGG1.AEASS, was used as a comparator. The amino acid sequence of the mature heavy chain of huBMK2_IGG1.AEASS is SEQ ID NO:73. The amino acid sequence of the mature light chain of huBMK2_IGG1.AEASS is SEQ ID NO:74. Previously purified frozen PBMCs were thawed, rinsed, and resuspended in 1 :25 Fc Block (BD, Catalog #564220) and 1 :250 Live / Dead® Far Red Vital Dye (Invitrogen, Catalog #L34966A) in DPBS and incubated for 15 minutes at room temperature in the dark. Cells were then washed in DPBS and resuspended in filtered staining buffer: DPBS supplemented with 5% goat serum (Gibco, Catalog #16210-064). PBMCs were plated at 0.5 x 10 6Cells were plated at 1 cell / well in staining buffer. Zero or 400 μΜ ATP was added and incubated for 30 min at 4°C, followed by addition of test antibody and incubation for another 30 min at 4°C. After two washes in staining buffer, 0.8 μg / test CD14 FITC (Invitrogen, Cat# 11-0149-42, clone 61D3) and 0.5 μg / test CD19 eFluor450 (eBioscience, Cat# 48-0199-42, clone HIB19) were added to PBMCs. CD39 binding by test antibody was detected with 0.5 μg / mL PE-labeled goat anti-human IgG (Southern Biotech, Cat# 2048-09) for 30 min at 4°C. Cells were then washed and resuspended in DPBS and flow cytometry was performed using a BD LSR Fortessa X-20 cell analyzer. The geometric mean of PE fluorescence intensity of the live single cell population gated on monocytes (CD14 + ) or B cells (CD19 + ) was obtained. EC 50 values were calculated using standard 4-parameter curve fit in GraphPad Prism (Table 13). A representative plot of one donor is shown in Figure 1

[0347] Table 13: EC 50 (nM) of antibodies binding to human CD39 on monocytes and B cells in human PBMCs. Reported error is standard error of the mean (SEM). N is the number of different biological donors.

[0348]

[0349] In general, the variants of antibodies 19, 31, 39 showed potent binding to the surface of primary cells. Potency was measured as 39 variant > 31 variant > 19 variant. The potency ranking of variants of 31 and 39 was maintained by isotype switching from IGG4.P to IGG1.AA and potency was maintained in the presence of high ATP. In comparison to the previously described anti-CD39 antibodies, the humanized IgG1 variant of antibody 31, hu31.4_IGG1.AA, and the humanized IgG1 variant of antibody 39, hu39.5_IGG1.AA, showed strong binding, with hu39.5_IGG1.AA > huBMK2_IGG1.AEASS > hu31.4_IGG1.AA.

[0350] Example 5: Inhibition of human CD39 enzymatic activity

[0351] ​Variants of test antibodies 19, 31 and 39 were tested for inhibition of human CD39 enzymatic activity. Inhibition was assessed using soluble recombinant CD39 and CD39 expressed on the surface of primary human cells or human cell lines derived from patient tumor samples. Inhibition was assessed in the presence of low and high ATP (20 mM and 400 mM, respectively).

[0352] Recombinant CD39 Inhibition: The potency of the antibodies described in the previous examples to inhibit CD39 enzymatic activity was first assessed using recombinant human CD39. The recombinant human CD39 biochemical assay was performed in an assay buffer consisting of 25 mM Tris-HCl, pH 7.4, 5 mM MgCl2, and 0.01% BSA. A 14-point 1:3 master serial dilution of test antibody was prepared in assay buffer to span a final concentration range of 200 nM to 0.125 pM. Five microliters of test antibody at 5-fold final concentration in assay buffer was added to each well of a 384-well plate. Ten microliters of 1.56 nM recombinant human CD39 resuspended in assay buffer was added to each well, and the plate was incubated for an additional 60 minutes at 37°C before adding 10 pL of 50 pM ATP in assay buffer. The final assay conditions included 0.625 nM recombinant human CD39 and 20 pM ATP substrate. After an additional 30 minutes of incubation at 37°C, 25 pL of Kinase Glo Plus reagent was added to each well of the 384-well plate. The reaction was measured using the Kinase-Glo kit (Promega, Cat# V3771) according to the manufacturer’s protocol. The amount of ATP remaining after CD39 inhibition was assessed as a function of luminescence produced and quantified using an Envision 2102 multi-label reader equipped with a luminescence filter. CD39 enzymatic activity was assessed as a function of the remaining ATP level. The percent of maximum activity in each test well was calculated based on the assay buffer (maximum activity, 0% inhibition) and enzyme-free control wells (baseline activity, 100% inhibition). The IC50 value of the test antibody was determined from the dose response curve fitted using a standard four-parameter fitting equation. Data are shown in Table 14. 50

[0353] Table 14: Potency of antibodies against soluble recombinant CD39.

[0354] Anti-CD39 antibodies IC 50 (nM) Maximum inhibition (%) ch19_IGG4.P 1.8 100％ ch31_IGG4.P 1.3 100％ hu31.4_IGG1.AA 0.30 86％ ch39_IGG4.P 2.3 100％ hu39.1_IGG4.P 0.26 92％ hu39.5_IGG1.AA 0.19 80％

[0355] ​SK-MEL-5 CD39 Inhibition: SK-MEL-5 cells, a melanoma cell line established from a patient-derived tumor sample, were also used to assess inhibition of CD39 enzyme activity. On the day of the experiment, a vial of previously frozen SK-MEL-5 cells was thawed and the cells were resuspended in 10 mL of assay buffer consisting of 20 mM HEPES (pH 7.4), 137 mM NaCl, 5.4 mM KCl, 1.3 mM CaCl2, 4.2 mM NaHCO3, and 0.1% glucose. A 14-point 1:3 master serial dilution of anti-CD39 antibodies was prepared in assay buffer to span a final concentration range of 100 nM to 0.063 pM. Twenty microliters of test antibody at 5x final concentration in assay buffer was added to each well of a 96-well round-bottom polypropylene plate. Forty microliters of SK-MEL-5 cells was added to each well and the plate was incubated for an additional 60 minutes at 37°C before adding 40 pL of 50 pM ATP in assay buffer. The final assay conditions included 10,000 cells / well and 20 pM ATP substrate. After incubation for an additional 60 minutes at 37°C and 5% CO2, the plate was centrifuged at 225 x g for 10 minutes. Thirty microliters of supernatant was transferred to a 96-well assay plate (Corning, catalog # 3912) and the amount of ATP remaining in the reaction was measured using the Kinase-Glo kit (Promega, catalog # V3771) according to the manufacturer’s protocol. The amount of ATP remaining after CD39 inhibition was assessed as a function of luminescence produced and quantified using an Envision 2102 multi-label reader equipped with a luminescence filter. CD39 enzyme activity was assessed in relation to the level of ATP remaining. The percent of maximum activity in each test well was calculated based on the assay buffer (maximum activity, 0% inhibition) and cell-free control wells (baseline activity, 100% inhibition). The IC50 value for the test antibody was determined from the dose response curve fitted using a standard four-parameter fitting equation. Data are shown in Table 15. 50 Table 15: Potency of antibodies against CD39 expressed on the surface of SK-MEL-5 cells.

[0356] Table 15: Potency of antibodies against CD39 expressed on the surface of SK-MEL-5 cells.

[0357] Anti-CD39 antibodies IC 50 (nM) Maximum inhibition (%) ch19_IGG4.P 5.4 55％ ch31_IGG4.P 2.4 63％ hu31.3_IGG4.P 0.61 71％ hu31.4_IGG4.P 1.1 71％ ch39_IGG4.P 0.56 63％ hu39.1_IGG4.P 0.16 65％ hu39.4_IGG4.P 0.13 60％ hu39.5_IGG4.P 0.14 66％ hu39.6_IGG4.P 0.12 55％ hu39.7_IGG4.P 0.20 65％

[0358] THP-1 CD39 Inhibition: Inhibition of CD39 enzyme activity was assessed in a second tumor cell line, specifically, a human monocytic cell line derived from a patient with acute monocytic leukemia. On the day of the experiment, one vial of previously frozen THP-1 cells was thawed and the cells were resuspended in 10 mL of assay buffer consisting of 20 mM HEPES (pH 7.4), 137 mM NaCl, 5.4 mM KC1, 1.3 mM CaCl2, 4.2 mM NaHC03, and 0.1% glucose. A 14-point 1:3 master serial dilution of anti-CD39 antibodies was prepared in assay buffer to span a final concentration range of 100 nM to 0.063 pM. Twenty microliters of test antibody at 5x final concentration was added to each well of a 96-well round-bottom polypropylene plate. Forty microliters of THP-1 cells was added to each well and the plate was incubated for an additional 60 minutes at 37°C before adding 40 pL of 50 pM ATP in assay buffer. The final assay conditions included 40,000 cells / well and 20 pM ATP substrate. After incubation for 60 minutes at 37°C and 5% C02, the plate was centrifuged at 225 x g for 10 minutes. Thirty microliters of supernatant was transferred to a 96-well assay plate (Corning, Catalog # 3912) and the amount of ATP remaining in the reaction was measured using the Kinase-Glo kit (Promega, Catalog # V3771) according to the manufacturer’s protocol. The amount of ATP remaining after CD39 inhibition was assessed as a function of luminescence produced and quantified using an Envision 2102 multi-label reader equipped with a luminescence filter. CD39 enzyme activity was assessed as a function of the remaining ATP level. The percent of maximum activity in each test well was calculated based on the assay buffer (maximum activity, 0% inhibition) and cell-free control well (baseline activity, 100% inhibition). The IC50 value for the test antibody was determined from the dose response curve fitted using a standard four-parameter fitting equation. Data are shown in Table 16. 50 Table 16: Potency of antibodies against CD39 expressed on the surface of THP-1 cells.

[0359] Table 16: Potency of antibodies against CD39 expressed on the surface of THP-1 cells.

[0360] Anti-CD39 antibodies IC 50 (nM) Maximal inhibition (%) ch19_IGG4.P 0.75 75％ ch31_IGG4.P 0.14 86％ hu31.3_IGG4.P 0.080 79％ hu31.4_IGG4.P 0.095 73％ hu31.4_IGG1.AA 0.53 62％ ch39_IGG4.P 0.11 67％ hu39.1_IGG4.P 0.077 78％ hu39.4_IGG4.P 0.064 76％ hu39.5_IGG4.P 0.062 83％ hu39.6_IGG4.P 0.058 79％ hu39.7_IGG4.P 0.071 77％ hu39.5_IGG1.AA 0.075 55％

[0361] Purified Monocyte CD39 Inhibition: Potency was additionally assessed using primary human monocytes. On the day of the experiment, two vials of previously frozen CD14 +Vials of forward selected human monocyte (donor 1003773) cells were thawed and the cells were resuspended in 10 ml of assay buffer consisting of 20 mM HEPES, pH 7.4, 137 mM NaCl, 5.4 mM KC1, 1.3 mM CaCl2, 4.2 mM NaHC03, and 0.1% glucose. A 14-point 1:3 master serial dilution of test antibody was prepared in assay buffer to span a final concentration range of 200 nM to 0.13 pM. Twenty microliters of test antibody at 5x final concentration in assay buffer was added to each well of a 96-well round-bottom polypropylene plate. Forty microliters of human monocytes were added to each well and the plate was incubated for an additional 60 minutes at 37°C before adding 40 μL of 50 μM or 1000 μM ATP in assay buffer. Final assay conditions included 20,000 cells / well and 20 μM or 400 μM ATP substrate. After incubation for 60 minutes at 37°C and 5% CO2, the plate was centrifuged at 225 x g for 10 minutes. Fifteen microliters of supernatant was transferred to a 96-well assay plate (Corning catalog number 3912) and the amount of AMP produced in the reaction was measured using the AMP-Glo kit (Promega, catalog number 5012) according to the manufacturer's protocol. Supernatant from the reaction plate with 400 μM ATP substrate was diluted 1:8 with assay buffer prior to transfer and addition of AMP-Glo reagent. The amount of AMP produced following CD39 inhibition was assessed as a function of luminescence produced and quantified using an Envision 2102 Multilabel Reader equipped with a luminescence filter. CD39 enzyme activity was assessed as a function of AMP levels. The percent of maximum activity in each test well was calculated based on assay buffer (maximum activity, 0% inhibition) and cell-free control wells (baseline activity, 100% inhibition). IC50values for test antibodies were determined from dose response curves fitted using a standard four-parameter fitting equation. Data are presented in Tables 17 and 18 and Figures 17 and 18. 50 Figure 2

[0362]

[0363]

[0364]

[0365] Table 18: Potency of antibodies against CD39 expressed on the surface of human monocytes in the presence of 400 μM ATP.

[0366] Anti-CD39 antibodies IC 50 (nM) Maximal inhibition (%) hu31.2_IGG4.P 0.87 71％ hu39.1_IGG4.P 0.33 76％ hu39.2_IGG4.P 0.14 80％ hu39.4_IGG4.P 0.45 73％ hu39.5_IGG4.P 0.20 60％ hu39.6_IGG4.P 0.38 73％ hu39.7_IGG4.P 0.39 70％ hu39.5_IGG1.AA 0.083 62％ hu31.4_IGG1.AA 0.70 63％ huBMK2_IGG1.AEASS 1.40 62％

[0367] ​​​Overall, the variants of antibodies 19, 31, 39 demonstrated potent inhibition of soluble and surface human CD39 enzymatic activity. Importantly, potency was maintained in the presence of high ATP.

[0368] Example 6: Immune stimulatory effects of CD39 inhibition

[0369] To evaluate the effects of the anti-CD39 antibodies generated in Examples 1-3, the downstream functional consequences of inhibiting CD39 enzymatic activity at the surface of bone marrow cells were evaluated in vitro.

[0370] Macrophage IL-1 β / IL-18 release assay: The NLRP3 inflammasome is a multi-protein, cytosolic complex that, once oligomerized, allows for the proteolytic cleavage and activation of procaspase-1 to caspase-1. Caspase-1 then promotes IL-1 β and IL-18 maturation via its cleavage of their inactive pro-isomers (pro-IL-1 β and pro-IL-18) to their active and secreted forms. These cytokines participate in the innate immune response, creating a generalized pro-inflammatory environment.

[0371] The ability of anti-CD39 antibodies to activate the NLRP3 inflammasome on macrophages was tested by measuring the secretion of mature IL-1 β and IL-18. A schematic of the assay is shown in Figure 3 Pre-isolated, positively selected CD14 + Monocytes (see Example 4) were thawed and plated at approximately 15 x 10 6 CD14 + Monocytes were cultured at a density of 0.125 x 10 6Density plated at 1 cell / well. Cells were then incubated overnight to allow adhesion. The next day, cells were treated with 1 ng / mL LPS (InvivoGen, Cat# tlrl-peklps), followed by treatment with 10 or 100 nM of test antibody or IgG control and incubated at 37°C, 5% CO2 for 3 hours. Cells were then treated with 500 μΜ ATP (Life Technologies, Cat# R0441) and incubated at 37°C, 5% CO2 for an additional 2-4 hours. Supernatants were collected and analyzed for IL-1 β levels by CBA (BD, Cat# 558279) or ELISA (R&D, Cat# QK201) according to manufacturer's instructions. IL-18 levels in supernatants were analyzed by ELISA (R&D, Cat# DL180) according to manufacturer's instructions. Experiments were performed in technical quadruplicate per donor, averaged, and then normalized to isotype control for each donor. As shown in Table 1, ch39_IGG4.P, hu39.1_IGG4.P, hu39.5_IGG1.AA, and hu31.4_IGG1.AA increased IL-1 β and IL-18 release from in vitro derived macrophages relative to isotype control. Figures 4A-4F

[0372] Monocyte-derived dendritic cell assay: Antibodies were also tested for their ability to mature monocyte-derived dendritic cells (moDCs) in the presence of ATP. On day 0, positively selected monocytes were resuspended in RPMI supplemented with 10% heat inactivated FBS, 1% Glutamax, 1% penicillin / streptomycin, 100 ng / mL recombinant human GM-CSF (R&D, Cat# 215-GM / CF), and 100 ng / mL IL-4 (Peprotech, Cat# 200-04) and seeded at 4 x 10 6 cells / well in 6-well plates (Falcon, Cat# 353046). On day 6, moDCs were collected and seeded at 0.5 x 10 6 ​Cells were plated at 1 cell / well in 24-well Upcell plates (Thermo Fisher, Cat# 174899) and treated with 10 pg / mL isotype control or anti-CD39 antibody for 1 hour at 37°C, 5% CO2, followed by 0 or 300 pM ATP for 18 hours at 37°C, 5% CO2. On day 7, moDCs were transferred to polypropylene V-bottom 96-well plates for staining. All wells were resuspended in 1 :25 Fc Block (BD, Cat# 564220) and 1 :250 Live / Dead® Far Red Vital Dye (Invitrogen, Cat# L34966A) in DPBS and incubated for 15 minutes at room temperature in the dark. After one wash with DPBS, cells were stained with the following antibodies [1 pg / test CD83 PE-Cy7 (BD, Cat# 561132), 1 pg / test CD86 BV605 (BD, Cat# 562999), 0.8 pg / test CD14 FITC (Invitrogen, Cat# 11-0149-42)] or the corresponding isotype controls [PE-Cy7 Mouse IgG1 kappa (BD, Cat# 565573), BV605 Mouse IgG1 kappa (BD, Cat# 562652), FITC Mouse IgG1 kappa (eBiosciences, Cat# 11-4714-41)] for 30 minutes at 4°C. Surface levels of the dendritic cell maturation markers CD83 and CD86 should increase with dendritic cell maturation, while CD14 (a monocyte marker) should decrease. After one wash in DPBS, cells were fixed with 50 pL IC Fixation Buffer (Invitrogen, Cat# 00-8222-49) for 20 minutes at room temperature. Cells were then washed and resuspended in DPBS and flow cytometry was performed using a BD LSR Fortessa X-20 Cell Analyzer. For CD83 and CD14, the percentage of the parent gate was set on the isotype control (<5% of total events) for each donor at 0 pM ATP condition. For CD86, >90% of cells were positive compared to the isotype control at the control-treated condition, so in practice, the geometric mean of fluorophore fluorescence intensity was reported for the viable single cell population.

[0373] As shown in Figure 5A and Figure 5B ATP matured moDCs as found by the increase in the dendritic cell maturation markers CD83 and CD86. Inhibition of CD39 enzymatic activity by the addition of anti-CD39 antibody, but not isotype control, enhanced the maturation effect, further increasing the percentage of CD83 expressing cells and the surface amount of CD86 per cell. The percentage of cells expressing the monocyte marker CD14 was also decreased upon the addition of anti-CD39 in the presence of ATP (Figure 5C ). The effects on CD83, CD86 and CD14 were ATP-dependent effects, except for hu39.6_IGG4.P.

[0374] Overall, the above data indicate that the enzymatic inhibition of human CD39 by the antibodies of the present disclosure has an immune stimulatory effect on different in vitro myeloid populations.

[0375] Example 7: Antibody characterization by competition flow

[0376] This example describes a competition flow assay that is suitable for determining whether two antibodies (a test antibody and a reference antibody) compete for binding to human CD39 expressed on the surface of cells. The selection of test antibody, reference antibody, and cell type can be modified.

[0377] Antibody hu39.1_IGG4.P and antibody IGG4.P, IgG4 (S228P) isotype control (CrownVivo) were directly conjugated to Alexa Fluor 647 (“AF647”) using the Alexa Fluor 647 conjugation kit - Lightning-Link (Abeam, Cat# ab269823). The conjugated antibodies are referred to as “hu39.1_IGG4.P-AF647” and “IGG4.P-AF647” in this example. Validation of binding was performed using the human multiple myeloma cell line MOLP-8 that expresses CD39. On the day of the assay, frozen MOLP-8 cells were thawed, rinsed, and resuspended in 1:25 Fc Block (BD, Cat# 564220) and 1:250 Live / Dead® Far Red Vital Dye (Invitrogen, Cat# L34966A) in DPBS and incubated at room temperature for 15 minutes in the dark. Cells were plated at 0.2 x 10 6 cells / well in a polypropylene v-bottom 96-well plate. An 11-point dose response curve was prepared by 3-fold serial dilutions in DPBS, with a concentration range of 833-0.0141 nM for each antibody. The twelfth point contained only DPBS and no antibody. Diluted antibodies from the dose response were added to the seeded cells at a 1:1 ratio and incubated at 4°C for 30 minutes. Cells were then washed three times and resuspended in DPBS and flow cytometry was performed using a BD LSR Fortessa X-20 cell analyzer. The mean fluorescence intensity of AF647 was obtained for the live single cell population. EC 50 and EC 95 values were calculated using standard 4-parameter nonlinear regression analysis in GraphPad Prism software.

[0378] Antibody hu39.1_IGG4.P-AF647 was then used as the reference antibody in competition flow-through assays with unlabelled test antibodies (hu31.1_IGG1.AA, hu39.1_IGG4.P, hu39.5_IGG1.AA, IGG1.AA isotype control, and IGG4.P isotype control (CrownVivo)). On the day of the assay, frozen MOLP-8 cells were thawed, rinsed, and resuspended in 1:25 Fc Block (BD, Cat# 564220) and 1:250 Live / Dead® Far Red Vital Dye (Invitrogen, Cat# L34966A) in DPBS and incubated at room temperature for 15 minutes in the dark. An 11-point dose response curve was prepared by performing 3-fold serial dilutions in DPBS, with a concentration range of 833-0.0141 nM for each test antibody. The twelfth point contained only DPBS and no antibody. 12.5 μL was added to each well of a 96-well staining plate. 12.5 μL of reference antibody hu39.1_IGG4.P-AF647 at a concentration of 93.7 nM was added to each well and mixed gently three times. Cells were plated at 0.2 x 10 6 50 EC50s were calculated using standard 4-parameter nonlinear regression analysis in GraphPad Prism software. Maximum competition (%) was calculated using the following formula: Maximum Competition = 1 - (MFI at highest concentration of test antibody / MFI at matching concentration of only hu39.1_IGG4.P-AF647). Only a single concentration of reference antibody hu39.1_IGG4.P-AF647 was used.

[0379] As Figure 6 and shown in Table 19, test antibody hu39.5_IGG1.AA competed with reference antibody hu39.1_IGG4.P-AF647 with a maximum competition of 93%. Similar levels of competition were observed for hu39.1_IGG4 (unlabelled reference antibody). In contrast, no competition was observed between test antibody hu31.4_IGG1.AA and reference antibody hu39.1_IGG4.P-AF647. These data indicate that antibodies 31 and 39, and their variants, bind to different, non-overlapping epitopes.

[0380] Table 19: Antibody Competition for CD39 Binding.

[0381]

[0382] ​Example 8: Efficacy in Xenograft Models

[0383] Anti-CD39 antibodies generated as described in Example 1 and Example 2 bind human but not murine CD39. Thus, to assess efficacy in vivo, a xenograft mouse model can be used. Five hundred thousand to ten million human cancer cells expressing human CD39 are injected subcutaneously or orthotopically into immunocompromised mice (e.g., SCID or nude mice) and tumor growth is monitored. Anti-CD39 antibodies can bind to and inhibit CD39 activity on several human cancer cell lines including, but not limited to, MOLP-8 myeloma (Example 8), THP-1 leukemia (Table 16), SK-MEL-5 melanoma (Table 15), and OAW42 ovarian cancer cells. Figure 7A and Figure 7B ). When tumors are less than 150 mm 3 , mice are treated with 0.1-30 mg / kg of an anti-CD39 antibody of the disclosure or isotype control BIWx 4 intraperitoneally (i.p.) or until tumor volume reaches 2,000 mm 3 . Treatment of human CD39-expressing MOLP-8 tumors with an anti-CD39 antibody of the disclosure results in tumor growth inhibition compared to mice treated with an isotype control. Tumor growth inhibition is also expected after treatment of other human CD39-expressing tumor types with an anti-CD39 antibody of the disclosure compared to mice treated with an isotype control.

[0384] Example 9: Efficacy in Human CD39 Knock-in Models

[0385] To evaluate anti-CD39 antibodies of the disclosure in an immunocompetent murine model, antibodies can be tested in a human CD39 knock-in (hCD39KI) mouse model, in which fully immunocompetent C57BL / 6 or BALB / c mice express human CD39 and do not express murine CD39 Figure 9 ). In this mouse model, expression of human CD39 is similar to expression of murine CD39 in wild-type mice. This mouse model allows for in vivo testing, in which anti-CD39 inhibition can act on host cells such as stromal cells, blood vessels, and immune cells. Mice are inoculated subcutaneously or orthotopically with murine wild-type or engineered tumor cell lines including, but not limited to, MCA-205 fibrosarcoma, B16-F10 melanoma, MC38 colon adenocarcinoma, ID8 ovarian cancer, 4T1 breast cancer, or CT-26 colon carcinoma cells. Five hundred thousand to ten million murine cancer cells are injected and tumor growth is monitored. When tumors are less than 150 mm 3 , mice are treated with 0.1-30 mg / kg ch39_mIGG2A.AAG or isotype control BIWx 4 intraperitoneally (i.p.) or until tumor volume reaches 2,000 mm 3Treatment of tumors with anti-CD39 as a single agent or in combination with one or more additional therapies (e.g., ICD inducers, immunotherapies, etc.) will result in tumor growth inhibition compared to mice treated with isotype control.

[0386] In one experiment, C57BL / 6 hCD39 KI mice were inoculated with 1 million MC38 cells on day 0. When tumors reached 80 mm 3 , mice were given a loading dose of 20 mg / kg anti-CD39 or isotype control antibody (vehicle: PBS) intraperitoneally (i.p.) and then subsequently given 10 mg / kg anti-CD39 or isotype control antibody 2 times / week (BIW). Each group consisted of 11-12 mice. In these experiments, the anti-CD39 antibody refers to ch39_mIGG2A.AAG. Oxaliplatin (OXA) treatment was initiated the day after the anti-CD39 or isotype control loading dose, 5 mg / kg i.p. (vehicle: physiological saline), and dosed 1 time / week. In a similarly designed experiment, BALB / c hCD39 KI mice were inoculated with 4T1 cells on day 0 and treated with OXA or anti-CD39 antibody as described for the MC38 model. Results from these experiments are described in the following paragraphs.

[0387] Efficacy of anti-CD39 in hCD39 KI model To evaluate the effectiveness of anti-CD39 treatment, tumor volume, intratumoral and peripheral CD39 enzymatic activity, and CD39 expression were evaluated.

[0388] Tumor volume and body weight were measured approximately twice a week (BIW) and are shown in Figure 10A and Figure 10B , respectively, where points represent the mean of the respective measure for the mice in each group and error bars represent the standard error of the mean (SEM). Significance was calculated using mixed effects models with multiple comparisons. As shown in Figure 10A , treatment with anti-CD39 and oxaliplatin was statistically significant (p = 0.0217) compared to treatment with single agents alone (p = 0.0372 for isotype + OXA vs. anti-CD39 + OXA, and p = 0.0217 for anti-CD39 + physiological saline vs. anti-CD39 + OXA). Body weight was stable throughout the experiment, indicating that anti-CD39 treatment was well-tolerated Figure 10B .

[0389] At the end of the study, tumors and spleens from C57BL / 6hCD39KI MC38 mice were additionally analyzed for human CD39 expression by immunohistochemistry (IHC) and evidence of anti-CD39 antibody enzyme-blocking activity was analyzed by enzyme histochemistry (EHC). Briefly, frozen OCT-embedded tumor or spleen samples were sectioned at 4 μm thickness, mounted on positively charged microscope slides, air-dried for 15 min, and then placed in 4% paraformaldehyde (PFA) fixative for 20 min. After fixation, the slides were washed in Tris-buffered saline and IHC was performed on an automated Leica Bond Rx platform, starting with blocking endogenous peroxidase, followed by incubation with rabbit anti-CD39 mAb (Abcam, EPR20627), and detected with an anti-rabbit HRP polymer system, then with diaminobenzidine (DAB) brown pigment, hematoxylin nucleus counterstaining, and then dehydrated and mounted. For EHC assays, after fixation in 4% PFA for 5 minutes, sections were washed in Tris-cis-butenedioate buffer and incubated at room temperature for 30 minutes in a solution of 50 mM Tris-cis-butenedioate, 250 mM sucrose, and 2 mM MgCl2. The sections were then incubated at 37°C for 2 hours in a solution of 100 mM Tris-cis-butenedioate containing 1 mM ATP, 2.5 mM levamisole, 2 mM lead nitrate, 0.25 mM sucrose, and 5 mM MnCl2. Levamisole is used to inhibit tissue-specific alkaline phosphatase (TNAP) activity; other inhibitors may also be used. The sections were then developed in 1% ammonium sulfide solution for 5 minutes, washed, counterstained with hematoxylin, dehydrated, and mounted. Representative images are shown below. Figures 11-14 Chinese (tumor, Figures 11-12 ;spleen, Figures 13-14 In tumors treated with isotype () Figure 11 Figure A and Figure 12 Figure A shows low and high magnification (representing both low and high magnification) and tumors treated with anti-CD39 (). Figure 11 Figure C and Figure 11 Human CD39 expression was detected in both low and high magnification (Figure C, representing low and high magnification respectively), located in the stromal components of vascular endothelium, infiltrating immune cells, and the tumor microenvironment. In isotype-treated spleens (… Figure 13 Figure A and Figure 14 Figure A shows the spleen treated with anti-CD39 at low and high magnification, respectively. Figure 13 Figure C and Figure 14 Human CD39 expression levels were observed in both low and high magnification (Figure C, showing low and high magnification respectively), located in the red pulp and to a lesser extent in the white pulp. Lead phosphate deposition (tumor-) was observed by enzyme histochemistry in tumors and spleens treated with isotype in vivo. Figure 11 Figure B andFigure 12 B, low and high magnification, respectively; spleen, Figure 13 B, and Figure 14 B, low and high magnification, respectively) In tumors and spleens treated in vivo with anti-CD39 antibody, no lead phosphate deposition was observed by enzyme histochemistry, indicating that enzyme activity had been blocked (tumor- Figure 11 D, and Figure 12 D, low and high magnification, respectively; spleen, Figure 13 D, and Figure 14 D, low and high magnification, respectively).

[0390] Intratumoral and peripheral CD39 enzyme activity was also evaluated ex vivo. Upon completion of the study, tumor samples obtained after sacrifice of C57BL / 6 hCD39 KI MC38 and BALB / c hCD39 KI 4T1 mice were placed in RPMI + 20% FBS + 250 pg / ml Collagenase D (Millipore Sigma, Cat# 11088858001) + 100 KU / ml DNAse 1 (Millipore Sigma, Cat# D5025-150KU) and enzymatically digested using gentleMACS TM Octo Dissociator to obtain a single cell suspension. Cells were then seeded at 2.5 x 10 4 cells / well in 96-well plates and treated with 100 nM of hu39.5_IGG1.AA or IgG1 Fc silent isotype control antibody (Absolute Antibody, Cat# Ab00178-10.3) and incubated for 1 hour at 37°C, 5% CO2. Cells were then treated with 20 mM ATP and incubated for an additional 30 minutes at 37°C, 5% CO2. At the end of the incubation period, 96-well plates were spun at 1000 rpm for 3 minutes and supernatant was collected. 2X Plus (Promega, Cat# V3771) was added to the supernatant. ATP levels in the supernatant were then measured by detecting luminescence using a 3 microplate reader. Data were presented as relative luminescence units (RLU). No difference between RLU in ex vivo hu39.5_IGG1.AA and isotype treated groups indicated complete enzyme activity inhibition by in vivo treatment. Anti-CD39 treatment inhibited enzyme activity in tumors Figure 15A and Figure 15B ).

[0391] To determine peripheral CD39 enzyme activity, peripheral white blood cells (WBCs) were isolated from terminal blood samples of hCD39 KI mice and assayed as generally described for tumor cell suspensions. Briefly, whole blood samples were mixed with 1X Pharm Lyse red blood cell lysis solution (BD Biosciences, Cat# 555899). WBC pellets were then collected after washing with 1X PBS solution and stored in BamBanker cell freezing media (Bulldog Bio, Cat# BB01). For enzyme inhibition assays, samples were thawed and then cells were plated at 2.5 x 10 4 cells / well in a 96-well plate. Each sample was incubated at room temperature for 1 hour with the following two treatment conditions: 100 nM of hu39.5_IGG1.AA or IgG1 Fc silent isotype control antibody (Absolute Antibody, Cat# Ab00178-10.3). All wells were then treated with 20 µM ATP and incubated at room temperature for 2 hours. At the end of the incubation period, the 96-well plate was spun at 1000 rpm for 3 minutes and the supernatant was collected. Then 2X Plus (Promega, Cat# V3771) was added to the supernatant. ATP levels in the supernatant were then measured by detecting luminescence using a 3 microplate reader. Data were presented as relative luminescence units (RLU). No difference between RLU in ex vivo hu39.5_IGG1.AA and isotype treated groups indicates complete enzyme activity inhibition by in vivo treatment. Anti-CD39 treatment inhibits enzyme activity in the periphery of 4T1 tumor bearing mice Figure 15C

[0392] Reduction of cell surface CD39 and complete or near complete coverage by anti-CD39 treatment in peripheral immune populations of hCD39 KI mice Figure 16 Terminal bled whole blood samples from C57BL / 6 hCD39 KI MC38 mice were evaluated by flow cytometry to assess target coverage and changes in CD39 expression on peripheral immune cell types including monocytes (CD3 - CD19 - CD11b + Ly6c ++ ), granulocytes (CD3 - CD19 - CD11b + Ly6c + ), T cells (CD3 + CD19 - ), B cells (CD3 - CD19 + ), and other lineage negative (Lin - ​lymphocytes. Target engagement was determined using hu39.5_IGG1.AA-AF647, a reagent that binds competitively with ch39_mIGG2A.AAG. hu39.5_IGG1.AA was conjugated to AF647 using the Abcam AF647 Lightning Link Conjugation Kit. Whole blood samples from 5-6 mice per treatment group were stained with a cocktail of extracellular antibodies including: CD39 hu39.5_IGG1.AA-AF647 (competes with anti-CD39), CD39 A1 PE (does not compete with anti-CD39), CD3 17A2 PerCP-Cy5.5, CD11b M1 / 70 FITC, CD19 6D5 BV421, and Ly6c HK1.4 PE-Cy7. Antibodies were incubated in whole blood for 30 minutes in the dark on ice, then samples were fixed / lysed using 1X BD FACS Lysing Solution for 20 minutes in the dark on ice. Samples were then centrifuged at 400xg for 5 minutes and washed once in 1X PBS buffer, resuspended in 1% paraformaldehyde solution, and filtered through a 20 pm filter before data was acquired. Samples were run on a BD FACSCanto Special Order cytometer and analyzed using Flow Jo and Prism software. Unbound CD39 was detected by the competing clone hu39.5_IGG1.AA-AF647 (measured by MFI of hu39.5_IGG1.AA-AF647 signal relative to its isotype control IgG1.FcS-AF647) across cell types in all animals treated with isotype antibody. Significant competitive binding of hu39.5_IGG1.AA-AF647 was observed on all CD39 (A1) expressing cells in animals treated with anti-CD39, indicating complete or near complete target coverage (Figure 3, lower panel). Total CD39 surface protein was detected by non-competitive CD39 antibody (clone A1 PE). Reduction in surface CD39 protein was observed across multiple peripheral immune cell types in mice treated with anti-CD39 (Figure 3, upper panel). Figure 16 Reduction of cell surface CD39 in tumor-draining lymph nodes of hCD39 KI mice with anti-CD39 treatment

[0393] Figure 17A ​​Tumor draining lymph nodes (TLDN) (right inguinal lymph nodes) were also collected at the end of the C57BL / 6 hCD39 KI MC38 mouse study described above. Lymph nodes were dissociated in a 37 °C water bath in RPMI 1640 (Gibco, Cat# 11875119) containing 0.6 mg / mL Collagenase P (Roche, Cat# 11213865001), 2.4 mg / mL Dispase II (Sigma, Cat# D4693), and 0.3 mg / mL DNAse I (Thermo Scientific, Cat# J62229MB) under gentle agitation for 30 minutes. Dissociated lymph nodes were counted and 2 x 10 5 cells / well were plated in 96-well polypropylene V-bottom plates. Cells were washed in PBS (Gibco, Cat# 14190-144) and resuspended in PBS containing 1:25 Fc Block (BD, Cat# 564220) and 1:250 Near IR fluorescent reactive dye (Invitrogen, Cat# L34976A) for 15 minutes in the dark. Following incubation, cells were stained with antibody cocktails (Table 20) and incubated for 30 minutes at 4 °C. Following incubation, cells were pelleted, supernatant removed and 100 μΐ^ of Fixation Buffer (eBioscience, Cat# 00-8222-49) was added for 1 hour. Cells were washed, resuspended in 200 μΐ^ PBS, and filtered with a 40 μιη filter plate (Fisher Scientific, Cat# NC0726512) before acquisition on a BD LSR Fortessa X-20 cell analyzer and analyzed using FlowJo TM v10.8 software (BD Life Sciences) to define immune subsets. Human CD39, mouse CD73, and mouse P2X7 expression on defined immune subsets are shown in Figure 17B and Figure 17C Anti-CD39 antibody ch39_mIgG2A.AAG did not cause significant changes in the percentage of immune subsets in tumor draining lymph nodes of hCD39 KI mice inoculated with MC38 tumor cells as a single agent or in combination with oxaliplatin treatment Figure 17D Although the immune population was not changed by anti-CD39 treatment, anti-CD39 treatment caused a significant decrease in surface human CD39 for nearly all immune subsets identified from tumor draining lymph nodes Figure 17E Representative histograms are shown in Anti-CD39 treatment does not change peripheral cytokine levels

[0394] Table 20. Flow cytometry staining information

[0395] ​

[0396]

[0397] Figure 18 Terminal bleeds from mice bearing MC38 tumors were collected into BD heparin tubes (BD Biosciences, Cat# 367871) and centrifuged at 2000xg for 15 minutes and the supernatant plasma samples were collected. Plasma samples were analyzed for cytokine levels using MSD following manufacturer’s instructions. No elevation in peripheral cytokine levels was observed in anti-CD39 treated mice. Data presented as mean ± SEM with a total of 11-12 mice per treatment group Figure 19A ). Statistical analysis was performed using ANOVA by Tukey’s multiple comparison test.

[0398] Example 10: Use of anti-CD39 antibodies in combination with immunogenic cell death (ICD) inducing therapies

[0399] The ICD inducing potential of chemotherapeutic agents was characterized in the absence or presence of anti-CD39 antibodies. This study used the following three metrics to measure ICD inducing potential: (1) cytotoxicity, (2) extracellular HMGB1 release and (3) extracellular ATP release.

[0400] To evaluate cell ATP release following oxaliplatin treatment, murine colon carcinoma cell lines CT26 and MC38 and human melanoma cell line SK-MEL-5 were thawed, rinsed and resuspended in their respective culture media. The culture media for CT26 and MC38 was RPMI + 10% FBS + 1% glutamax + 1% penicillin-streptomycin + 1% sodium pyruvate. The culture media for SK-MEL-5 was EMEM + 10% FBS + 1% glutamax + 1% penicillin-streptomycin + 1% sodium pyruvate. Cells were seeded at 2.5 x 10 4 cells / well in 96 well plates and incubated overnight at 37°C, 5% CO2. The next day, cells were treated with vehicle (PBS), 100 µM or 250 µM oxaliplatin. SK-MEL-5 cells received additional treatment with 100 nM hu39.5_IGG1.AA or IgG1 Fc silent isotype control antibody (Absolute Antibodies, Cat# Ab00178-10.3). To determine cell viability, cells were incubated with treatment agents for 24 hours at 37°C, 5% CO2. At the end of the incubation period, viability was determined using the luminescence-based Cell Titer Glo 2.0 assay (Promega, Cat# G9242). Data presented as viability normalized to untreated vehicle control group Figure 19B). To determine HMGB1 release, cells were incubated with treatment agents for 24 hours at 37°C, 5% C02. At the end of the incubation period, plates were centrifuged at 400 x g for 5 minutes and supernatant was collected. Lumit HMGB1 kit (Promega, Cat. No. CS3030B01) was then used to determine HMGB1 levels in culture supernatant following manufacturer’s instructions Figure 19C ). To examine ATP release, cells were then incubated with treatment agents for 8 hours at 37°C, 5% C02. At the end of the incubation period, 4x RealTime-Glo TM Extracellular ATP Assay (Promega Cat. No. GA5010) was added to cells. Luminescence was detected kinetically by using a 3 microplate reader at 15 minute intervals over a 6 hour period. All three cell lines exhibited ATP release following treatment with 250 mM oxaliplatin 3 microplate reader at 15 minute intervals over a 6 hour period. All three cell lines exhibited ATP release following treatment with 250 mM oxaliplatin Figure 19C ) in SK-MEL-5 cells, hu39.5_IGG1.AA treatment resulted in greater extracellular ATP accumulation compared to cells treated with isotype control Figure 20A

[0401] The ICD-inducing potential of additional chemotherapeutic agents was characterized on the human melanoma cell line SK-MEL-5. Cells were thawed, rinsed, and resuspended in culture medium EMEM + 10% FBS + 1% glutamax + 1% penicillin-streptomycin + 1% sodium pyruvate. Cells were seeded at 2.5 x 10 4 cells / well in 96-well plates and incubated overnight at 37°C, 5% C02. The next day, cells were treated with the chemotherapeutic agents doxorubicin (vehicle: DMSO), irinotecan (vehicle: DMSO), gemcitabine (vehicle: PBS), and cisplatin (vehicle: PBS) at the indicated concentrations and incubated for 48 hours at 37°C, 5% C02. (1) To assess cytotoxicity, at the end of the incubation period, cell viability was determined using the luminescence-based Cell Titer Glo 2.0 assay (Promega, Cat. No. G9242). Data are presented as viability normalized to untreated vehicle control groups Figure 20B ) (2) To measure HMGB1 release, at the end of the incubation period, plates were centrifuged at 400 x g for 5 minutes and supernatant was collected. Lumit HMGB1 kit (Promega, Cat. No. CS3030B01) was then used to determine HMGB1 levels in culture supernatant following manufacturer’s instructions Figure 20C ​)(3) Quantification of extracellular ATP release with chemotherapeutic agents with or without the addition of 100 nM hu39.5_IGG1.AA or IgG1 Fc silent isotype control antibody (Absolute Antibodies, Cat# Ab00178-10.3) and incubation for 8 hours or 32 hours. At the end of the incubation period, 4x RealTime-Glo TM Extracellular ATP assay (Promega Cat# GA5010) was added to cells. Luminescence was then detected kinetically by using a 3 microplate reader to measure luminescence at 15 minute intervals over a 10 hour period. All chemotherapeutic treated groups showed an increase in ATP release relative to untreated cells. The addition of hu39.5_IGG1.AA resulted in higher extracellular ATP levels Figure 21 ).

[0402] Example 11: Characterization of CD39 and CD39-related markers in human cells

[0403] A CD39 whole blood (WB) receptor occupancy (RO) flow cytometry assay was used to assess CD39 expression on various cell types in human, cynomolgus monkey, and hCD39 KI mouse WB. Instructions for the hCD39 KI RO assay are described in Example 9. Human whole blood samples were analyzed by the same method described for hCD39 KI mouse samples, but incorporating species-specific cell lineage markers for monocytes and B cells. The human CD39 WB RO panel consisted of the following antibodies: hu39.5_IGG1.AA-AF647 (competes with anti-CD39), anti-CD39 A1 PE (does not compete with anti-CD39), anti-CD14 61D3 FITC, and anti-CD19 H1B19 eFluor450. As shown in Figure 22A The relative expression of CD39 on peripheral immune cells varied between humans and C57BL / 6 hCD39 KI MC38 mice, as shown in Figure 11. While monocytes had the highest CD39 expression level among the cell types assayed in human blood, granulocytes had the highest CD39 expression in hCD39 KI mice. B cell expression varied most significantly between species. Human peripheral B cells expressed very high levels of CD39, while hCD39 KI mouse B cells had low to no CD39 expression.

[0404] CD39 and CD39-related markers were also characterized in human in vitro differentiated bone marrow cells such as M0-, M1-, M2-polarized macrophages and monocyte-derived dendritic cells (moDCs), as well as primary human bone marrow cells such as CD14 + ​Monocytes and bone marrow dendritic cells (DC) subsets. To generate in vitro differentiated macrophages, positively selected CD14 + Monocytes were resuspended in RPMI supplemented with 10% heat inactivated FBS, 1% GlutaMax, 1% penicillin / streptomycin and 50 ng / mL recombinant human M-CSF (R&D, Cat# 216-MCC / CF) and seeded at 4 x 10 6 cells / well in 6-well plates (Falcon, Cat# 353046) in a total of 2 mL. On day 4, cells were supplemented with 2 mL of fresh RPMI supplemented media containing 2X M-CSF. On day 6, cells were polarized into M1 macrophages using 100 ng / mL LPS (Invivogen, Cat# tlr-pek-lps) and 20 ng / mL recombinant human IFN-g (Peprotech, Cat# 300-02) while M2 macrophages were polarized with 20 ng / mL recombinant human IL-4 (Peprotech, Cat# 200-04). M0 macrophages were not added additional cytokines. To generate moDCs, positively selected CD14 + Monocytes were resuspended in RPMI supplemented with 10% heat inactivated FBS, 1% GlutaMax, 1% penicillin / streptomycin and 100 ng / mL recombinant human GM-CSF (R&D, Cat# 215-GM / CF) and 100 ng / mL recombinant human IL-4 and seeded at 4 x 10 6 cells / well in 6-well plates (Falcon, Cat# 353046) in a total of 2 mL. On day 4, cells were supplemented with 2 mL of fresh RPMI supplemented media containing 2X GM-CSF / IL-4. On day 7, M0-, M1-, M2-polarized macrophages and moDCs were collected, counted and aliquoted into U-bottom 96-well plates for flow cytometry or into 1.5 mL Eppendorf tubes for RNA. Primary CD14 + Monocytes and bone marrow DCs enriched using the negative selection kit were aliquoted into U-bottom 96-well plates for flow cytometry or into 1.5 mL Eppendorf tubes for RNA.

[0405] Flow cytometry quality control staining was performed to determine the purity of primary CD14 + Purity of monocyte isolation, purity of primary bone marrow dendritic cells (DC) enrichment and distribution of DC subsets resulting from primary bone marrow DC enrichment in cells. Monocytes were positively selected for CD14 + Expression definition. CD14 +The purity of monocyte isolation was about 99%. Enrichment of primary dendritic cells from peripheral blood of four human donors showed an enriched population of 48.5% dendritic cells defined as HLA-DR+CD11c+and pDC. Enriched DCs were composed of 2.8% plasmacytoid DCs (pDCs, defined as HLA - DR + CD11c 阴性 CD123 + ), 3.0% conventional DC1 (cDC1, defined as HLA - DR + CD11c + CD141 + Clec9a + ), 30.4% conventional DC2 (cDC2, defined as HLA - DR + CD11c + CD1c + CD141 阴性 Clec9a 阴性 ) and 12.0% other DCs (defined as HLA - DR + CD11c + CD1c 阴性 CD141 阴性 Clec9a 阴性 ).

[0406] For flow cytometry evaluation of CD39 and CD73 (Figure Figure 22B and Figure 22A ), CD14 +Monocytes, enriched bone marrow DCs, M0, M1, M2 macrophages, and moDCs were aliquoted into 96 well plates and resuspended in 50 μΐ / well of 1:25 Fc Block (BD, Cat# 564220) and 1:250 Live / Dead® Far Red Vital Dye (Invitrogen, Cat# L34966A) in DPBS and incubated for 15 minutes at room temperature in the dark. Cells were then stained with 50 μΐ / well of the following antibodies [0.25 μg / test CD39 PE-Cy7 (eBiosciences, Cat# 25-0399-42) and 0.5 μg / test CD73 BUV737 (BD, Cat# 612812) or the appropriate isotype controls, Mouse IgGl PE-Cy7 (BD, Cat# 557647) and Mouse IgGl BUV737 (BD, Cat# 612758)] for 30 minutes at 4°C. After one wash in DPBS, cells were fixed with 50 μΐ of IC Fixation Buffer (Invitrogen, Cat# 00-8222-49) for 20 minutes at room temperature. Cells were then washed and resuspended in DPBS and flow cytometry was performed using a BD LSR Fortessa X-20 Cell Analyzer. CD14 + The percentage of positive CD39 and CD73 on monocytes, enriched bone marrow DC subsets, M0, M1, M2 macrophages, and moDCs were assessed by comparison to isotype controls. CD14 + Monocytes, all subsets derived in vitro (M0, M1, M2 macrophages, and moDCs), and the primary enriched DC subsets cDC1, cDC2, and other DCs all had high percentages of positive CD39 and low percentages of positive CD73 Figure 22B and Figure 22B A relatively small number (15%) of pDCs were positive for CD39. Given that the majority of cell types had >90% positive for CD39, CD39 expression levels were further explored by observing the intensity of CD39 staining compared to isotype controls Figure 22C Although variable between donors, CD14 + CD39 expression was higher on monocytes, macrophages derived in vitro, moDCs. Among the primary DCs, cDC1 had the highest cell surface CD39 expression.

[0407] ENTPD1 (CD39) and NT5E (CD73) gene expression was analyzed by qPCR using standard methods Figure 22D and Figure 22E ENTPD1 gene expression levels were greater than NT5E gene expression levels in all cell types. The enriched DC population had the lowest relative expression of ENTPD1 and the highest relative expression of NT5E.

[0408] To evaluate a broad range of CD39-associated gene expression in these human bone marrow subsets, a custom NanoString panel was designed and utilized. RNA from each of the bone marrow cell subsets was isolated using Qiagen RNeasy Mini Kit (Cat# 74106) following the manufacturer’s instructions. RNA was then quantified on a Nanodrop and 70 ng of RNA was used for NanoString. To hybridize the RNA to the custom NanoString code set, 70 μΐ^ of hybridization buffer was added to the reporter code set and 8 μΐ^ was added to 5 μΐ^ of RNA per sample. Two microliters of capture probe set was then added to each sample and incubated at 65 °C for 16 hours in a thermocycler. Samples were then topped up to 30 μΐ^ with RNase-free water and loaded into the sample loading port of the nCounter SPRINT cartridge (NanoString, Cat# SPRINT-CAR-1.0). The cartridge was then run on the NanoString nCounter SPRINT profiler. Quality control metrics were run on the data from each sample and samples that passed the quality control test were normalized to 3 housekeeping genes included in the custom NanoString panel to evaluate gene expression.

[0409] Figure 23 A heatmap is shown in FIG. 12, showing unsupervised clustering of housekeeping normalized expression of CD39, adenosine, and inflammasome pathway genes using the custom NanoString panel. Low quality wells or abnormal control probes marked by low counts were removed from this analysis. Specific donor IDs are attached at the end of the cell type labels. The dotted horizontal line in the highest dendrogram shows sample splitting, but does not affect the unsupervised nature of the clustering. CD39 is expressed in all of the immune populations profiled. Given the consistent expression of genes in these pathways within each cell type, the cell types form different clusters. Monocytes, Ml macrophages, and enriched primary DCs cluster together, driven by expression on IL-1 β, P2X7, and A2AR. M0, Ml macrophages, and moDCs form another cluster with higher CD206, A2BR, and P2Y11 expression.

[0410] Example 12: P2Y11 antagonism reduces ATP-driven dendritic cell activation

[0411] To test the effect of P2X1, P2X7 and P2Y11 inhibitors on the activation of monocyte-derived dendritic cells (MoDCs). MoDCs were generated from positively selected human monocytes cultured with GM-CSF and IL-4 as described in Example 11. At day 7, 6-well plates were incubated on ice for 5 min and scraped to detach and count cells. Cells were resuspended in fresh media with GM-CSF and IL-4 and 125,000 cells in 100 pL were plated per well in 96-well flat bottom Upcell plates (Nunc, cat# 174897). Ten micromolar of each compound [P2X1 inhibitor, NF023, Millipore Sigma, cat# 104869-31-0; P2X7 inhibitor, A-740003, Millipore Sigma, cat# 861393-28-4; P2Y11 inhibitor NF340, APExBIO, cat# B7508; P2Y11 inhibitor NF157, APExBIO cat# B7060] or vehicle control (0.1% DMSO) was added, mixed gently and incubated at 37°C, 5% CO2 for 1 h. Subsequently, 0 or 300 pM ATP was added, mixed gently, and incubated at 37°C, 5% CO2 for 18 h. For flow cytometry evaluation, cells were detached from the plates and transferred to 96-well polypropylene v-bottom plates. Cells were washed in PBS (Gibco, cat# 14190-144) and resuspended in PBS containing 2% FBS, viability dye (LD-NIR, Invitrogen, cat# L34994), Fc blocker and staining antibodies [BV510 anti-CD80 (BD Horizon, cat# 563084), PE-Cy7 anti-CD83 (BD Horizon, cat# 561132) and BV605 anti-CD86 (BD Horizon, cat# 562999)]. Cells were incubated at room temperature for 60 min, washed to remove the dyes, and cells were fixed with BD Phosphoflow FIX Buffer I (BD, cat# 557870) and incubated for 15 min. Fixation buffer was removed, cells were washed with PBS and stored at 4°C until acquisition on a BD LSR Fortessa X-20 cell analyzer and analyzed with FlowJo TM v10.8 software (BD Life Sciences).

[0412] Within each inhibitor group, fold change was calculated between ATP and non-ATP treated groups to calculate the induction of each surface marker in the presence of ATP. The induction levels of the inhibitor treated groups were then normalized to the DMSO treated group. The results show that P2Y11 inhibitors inhibit CD86 induction by ATP but not CD80 or CD83 (Figure 24A ).

[0413] Example 13: Mechanism of response of solid tumors expressing elevated ATP due to CD39 inhibition

[0414] The ATP response mechanism in a subset of solid tumors was evaluated. Briefly, TCGA RNA expression was analyzed in the tumor type of interest and expression was normalized on a per gene basis Figure 24B ). Figure 24B The violin plots in FIG. 13 summarize the kernel probability density of normalized log2 gene expression (y-axis) of the following genes (proteins) among a subset of TCGA samples: ENTPD1 (CD39), NT5E (CD73), P2RX7 (P2X7), and P2RY11 (P2Y11). Along the x-axis, the violin plots are grouped by specific TCGA subtype: esophageal carcinoma (ESCA), head and neck squamous cell carcinoma (HNSC), kidney renal clear cell carcinoma (KIRC), lung adenocarcinoma (LUAD), lung squamous cell carcinoma (LUSC), pancreatic adenocarcinoma (PAAD), and stomach adenocarcinoma (STAD). Examples of the disclosed embodiments The pathway enrichment scores (y-axis) for the myeloid gene signature (IL-6, CXCL2, CXCL2, CXCL3, CXCL8, and PTGS2) calculated using gene set enrichment analysis for the same subset of TCGA cancer subtypes are also shown in FIG. 13. Using this metric, values > 0 indicate upregulation and values < 0 indicate downregulation. All tumors have high levels of CD39 and myeloid infiltration. All tumors also share similar levels of P2X7 and P2Y11, indicating that they can respond to elevated ATP in the tumor microenvironment.

[0415] SEQUENCE LISTING

[0416] Embodiment 1: An anti-CD39 antibody that specifically binds to human C...

Claims

1. An anti-CD39 antibody that specifically binds to human CD39, the anti-CD39 antibody comprising: a heavy chain variable region comprising a complementarity determining region 1 (HCDR1) consisting of SEQ ID NO: 28, a complementarity determining region 2 (HCDR2) consisting of SEQ ID NO: 29, and a complementarity determining region 3 (HCDR3) consisting of SEQ ID NO: 30; and a light chain variable region comprising a complementarity determining region 1 (LCDR1) consisting of SEQ ID NO: 32, a complementarity determining region 2 (LCDR2) consisting of SEQ ID NO: 33, and a complementarity determining region 3 (LCDR3) consisting of SEQ ID NO:

34.

2. The anti-CD39 antibody of claim 1, wherein the antibody has a heavy chain variable region and a light chain variable region according to claim 1, and the heavy chain variable region has at least 90% sequence identity to SEQ ID NO: 27 and the light chain variable region has at least 90% sequence identity to SEQ ID NO: 31; the heavy chain variable region has at least 90% sequence identity to SEQ ID NO: 58 and the light chain variable region has at least 90% sequence identity to SEQ ID NO: 60; the heavy chain variable region has at least 90% sequence identity to SEQ ID NO: 58 and the light chain variable region has at least 90% sequence identity to SEQ ID NO: 61; the heavy chain variable region has at least 90% sequence identity to SEQ ID NO: 58 and the light chain variable region has at least 90% sequence identity to SEQ ID NO: 62; the heavy chain variable region has at least 90% sequence identity to SEQ ID NO: 58 and the light chain variable region has at least 90% sequence identity to SEQ ID NO: 63; the heavy chain variable region has at least 90% sequence identity to SEQ ID NO: 59 and the light chain variable region has at least 90% sequence identity to SEQ ID NO: 61; the heavy chain variable region has at least 90% sequence identity to SEQ ID NO: 59 and the light chain variable region has at least 90% sequence identity to SEQ ID NO: 60; the heavy chain variable region has at least 90% sequence identity to SEQ ID NO: 59 and the light chain variable region has at least 90% sequence identity to SEQ ID NO: 62; or the heavy chain variable region has at least 90% sequence identity to SEQ ID NO: 59 and the light chain variable region has at least 90% sequence identity to SEQ ID NO:

63.

3. The anti-CD39 antibody of claim 1 or 2, wherein the anti-CD39 antibody is a human chimeric or humanized antibody or antigen binding fragment thereof.

4. The anti-CD39 antibody of claim 1 or 2, wherein the anti-CD39 antibody is a veneered antibody or antigen-binding fragment thereof.

5. The anti-CD39 antibody of claim 1 or 2, wherein the antibody further comprises a variant heavy chain constant region selected from a variant human IgGl constant region, a variant human IgG2 constant region, a variant human IgG3 constant region, or a variant human IgG4 constant region.

6. The anti-CD39 antibody of claim 5, wherein the variant human IgG heavy chain constant region comprises SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO:

5.

7. The anti-CD39 antibody of claim 1 or 2, wherein the antibody further comprises a wild-type human IgG heavy chain constant region.

8. The anti-CD39 antibody of claim 7, wherein the wild-type human IgG heavy chain constant region comprises SEQ ID NO: 1 or SEQ ID NO:

4.

9. The anti-CD39 antibody of claim 5, comprising a human light chain kappa constant region.

10. The anti-CD39 antibody of claim 7, comprising a human light chain kappa constant region.

11. The anti-CD39 antibody of claim 1 or 2, wherein the antibody has a heavy chain comprising the heavy chain variable region and a light chain comprising the light chain variable region, wherein: (a) the heavy chain comprises SEQ ID NO: 64 and the light chain comprises SEQ ID NO: 67; (b) the heavy chain comprises SEQ ID NO: 64 and the light chain comprises SEQ ID NO: 68; (c) the heavy chain comprises SEQ ID NO: 64 and the light chain comprises SEQ ID NO: 69; (d) the heavy chain comprises SEQ ID NO: 64 and the light chain comprises SEQ ID NO: 70; (e) the heavy chain comprises SEQ ID NO: 65 and the light chain comprises SEQ ID NO: 68; (f) the heavy chain comprises SEQ ID NO: 65 and the light chain comprises SEQ ID NO: 67; (g) the heavy chain comprises SEQ ID NO: 66 and the light chain comprises SEQ ID NO: 67; (h) the heavy chain comprises SEQ ID NO: 65 and the light chain comprises SEQ ID NO: 69; or (i) the heavy chain comprises SEQ ID NO: 65 and the light chain comprises SEQ ID NO:

70.

12. The anti-CD39 antibody of claim 1 or 2, wherein the antibody has a heavy chain variable region and a light chain variable region according to any one of claims 1-10, and the heavy chain variable region has at least 95% sequence identity to SEQ ID NO: 27 and the light chain variable region has at least 95% sequence identity to SEQ ID NO: 31; the heavy chain variable region has at least 95% sequence identity to SEQ ID NO: 58, and the light chain variable region has at least 95% sequence identity to SEQ ID NO: 60; the heavy chain variable region has at least 95% sequence identity to SEQ ID NO: 58, and the light chain variable region has at least 95% sequence identity to SEQ ID NO: 61; the heavy chain variable region has at least 95% sequence identity to SEQ ID NO: 58, and the light chain variable region has at least 95% sequence identity to SEQ ID NO: 62; the heavy chain variable region has at least 95% sequence identity to SEQ ID NO: 58, and the light chain variable region has at least 95% sequence identity to SEQ ID NO: 63; the heavy chain variable region has at least 95% sequence identity to SEQ ID NO: 59, and the light chain variable region has at least 95% sequence identity to SEQ ID NO: 61; the heavy chain variable region has at least 95% sequence identity to SEQ ID NO: 59, and the light chain variable region has at least 95% sequence identity to SEQ ID NO: 60; the heavy chain variable region has at least 95% sequence identity to SEQ ID NO: 59, and the light chain variable region has at least 95% sequence identity to SEQ ID NO: 62; or the heavy chain variable region has at least 95% sequence identity to SEQ ID NO: 59, and the light chain variable region has at least 95% sequence identity to SEQ ID NO:

63.

13. The anti-CD39 antibody of claim 1 or 2, wherein the antibody has a heavy chain variable region and a light chain variable region according to any one of claims 1-11, and the heavy chain variable region has at least 99% sequence identity to SEQ ID NO: 27, and the light chain variable region has at least 99% sequence identity to SEQ ID NO: 31; the heavy chain variable region has at least 99% sequence identity to SEQ ID NO: 58, and the light chain variable region has at least 99% sequence identity to SEQ ID NO: 60; the heavy chain variable region has at least 99% sequence identity to SEQ ID NO: 58, and the light chain variable region has at least 99% sequence identity to SEQ ID NO: 61; the heavy chain variable region has at least 99% sequence identity to SEQ ID NO: 58, and the light chain variable region has at least 99% sequence identity to SEQ ID NO: 62; the heavy chain variable region has at least 99% sequence identity to SEQ ID NO: 58, and the light chain variable region has at least 99% sequence identity to SEQ ID NO:

63. the heavy chain variable region has at least 99% sequence identity to SEQ ID NO: 59, and the light chain variable region has at least 99% sequence identity to SEQ ID NO: 61; the heavy chain variable region has at least 99% sequence identity to SEQ ID NO: 59, and the light chain variable region has at least 99% sequence identity to SEQ ID NO: 60; the heavy chain variable region has at least 99% sequence identity to SEQ ID NO: 59, and the light chain variable region has at least 99% sequence identity to SEQ ID NO: 62; or the heavy chain variable region has at least 99% sequence identity to SEQ ID NO: 59, and the light chain variable region has at least 99% sequence identity to SEQ ID NO:

63.

14. A pharmaceutical composition comprising the antibody of any one of claims 1-13 and a pharmaceutically acceptable carrier.

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