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 same.

Description

[Technical field]

[0001] Cross-reference statement This application claims priority to international application PCT / CN2022 / 079021, filed March 3, 2022, and international application PCT / CN2022 / 126070, filed October 19, 2022, the disclosures of which are incorporated herein by reference.

[0002] Technical Field Provided herein are anti-CD39 antibodies that inhibit the enzymatic activity of human CD39 and methods of using same. [Background technology]

[0003] Dying or stressed cells release ATP extracellularly, which is present at negligible concentrations (10-100 nM) under healthy conditions. Once released, extracellular ATP signals through type 2 purinergic (P2) receptors, providing inflammatory signals essential for effective innate and adaptive immune responses. CD39 is the rate-limiting ectoenzyme in the hydrolysis of extracellular ATP. CD39 is a key regulator of extracellular ATP levels by catabolism, converting extracellular ATP to AMP.

[0004] CD39 is also an important contributor to extracellular adenosine levels. Adenosine is an immunosuppressive metabolite and thus has a conversion relationship with extracellular ATP. CD39 contributes to the increase in 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.

[0005] Therefore, the hydrolysis of extracellular ATP by CD39 may affect immune response through two different signaling pathways at opposite ends of the signaling axis.Notably, dysregulation of this signaling axis, referred to herein as the ATP-adenosine signaling axis, has been observed in several diseases.Thus, the art needs agents that target CD39 and inhibit its enzymatic activity for beneficial therapeutic purposes. Summary of the Invention

[0006] Among various aspects of the present disclosure, there is provided an anti-CD39 antibody. The anti-CD39 antibody is optionally (i) labeled with one or more detectable signals, including but not limited to a fluorescent molecule, a spin-label molecule, an enzyme, or a radioisotope, and / or (ii) conjugated to one or more therapeutic agents, including but not limited to a chemotherapeutic agent, a radioisotope, or a nucleic acid. In some embodiments, the anti-CD39 antibody of the present disclosure is provided as an isolated antibody. In some embodiments, the anti-CD39 antibody of the present disclosure is provided in a composition. In some embodiments, the anti-CD39 antibody of the present disclosure is provided in a composition further comprising a pharma-ceutically acceptable excipient.

[0007] 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 (H1) 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 (L1) 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 H1 having at least 90% sequence identity to SEQ ID NO: 10, H2 having at least 90% sequence identity to SEQ ID NO: 11, and H3 having at least 90% sequence identity to SEQ ID NO: 12; and a light chain variable region comprising L1 having at least 90% sequence identity to SEQ ID NO: 14, L2 having at least 90% sequence identity to SEQ ID NO: 15, and 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.

[0008] In one embodiment, the disclosure provides an anti-CD39 antibody that specifically binds to human CD39, comprising a heavy chain variable region comprising H1 having at least 80% sequence identity to SEQ ID NO:18, H2 having at least 80% sequence identity to SEQ ID NO:19, and H3 having at least 80% sequence identity to SEQ ID NO:20; and a light chain variable region comprising L1 having at least 80% sequence identity to SEQ ID NO:22, SEQ ID NO:23, or SEQ ID NO:24, L2 having at least 80% sequence identity to SEQ ID NO:25, and L3 having at least 80% sequence identity to SEQ ID NO:26. In another embodiment, the disclosure provides an anti-CD39 antibody that specifically binds to human CD39, comprising a heavy chain variable region comprising H1 having at least 90% sequence identity to SEQ ID NO:18, H2 having at least 80% sequence identity to SEQ ID NO:19, and H3 having at least 90% sequence identity to SEQ ID NO:20; and a light chain variable region comprising 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, and L3 having at least 80% sequence identity to SEQ ID NO:26. In some of the foregoing embodiments, the heavy and light chain variable regions 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 with SEQ ID NO:17 and SEQ ID NO:21, respectively, or with SEQ ID NO:43 and SEQ ID NO:45, respectively, or with SEQ ID NO:43 and SEQ ID NO:46, respectively, or with SEQ ID NO:43 and SEQ ID NO:47, respectively, or with SEQ ID NO:43 and SEQ ID NO:48, respectively, or with SEQ ID NO:43 and SEQ ID NO:49, respectively, or with SEQ ID NO:44 and SEQ ID NO:48, respectively, or with SEQ ID NO:44 and SEQ ID NO:49, respectively.

[0009] In one embodiment, the disclosure provides an anti-CD39 antibody that specifically binds to human CD39, comprising a heavy chain variable region comprising H1 having at least 80% sequence identity to SEQ ID NO:28, H2 having at least 80% sequence identity to SEQ ID NO:29, and H3 having at least 80% sequence identity to SEQ ID NO:30; and a light chain variable region comprising L1 having at least 80% sequence identity to SEQ ID NO:32, L2 having at least 80% sequence identity to SEQ ID NO:33, and L3 having at least 80% sequence identity to SEQ ID NO:34. In some of the foregoing embodiments, the heavy and light chain variable regions 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 with SEQ ID NO:27 and SEQ ID NO:31, or with SEQ ID NO:58 and SEQ ID NO:60, or with SEQ ID NO:58 and SEQ ID NO:61, or with SEQ ID NO:58 and SEQ ID NO:62, or with SEQ ID NO:58 and SEQ ID NO:63, or with SEQ ID NO:59 and SEQ ID NO:61, or with SEQ ID NO:59 and SEQ ID NO:60, or with SEQ ID NO:59 and SEQ ID NO:62, or with SEQ ID NO:59 and SEQ ID NO:63.

[0010] For any of the disclosed antibodies of the present disclosure, the anti-CD39 antibody may 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. In addition, the anti-CD39 antibody may further comprise a heavy chain constant region selected from human IgG1, human IgG2, human IgG3, or human IgG4, and optionally a human light chain constant region. The variant heavy chain constant region may be a wild-type heavy chain constant region, or a heavy chain constant region with enhanced or reduced effector function relative to the wild-type heavy chain constant region. In various embodiments, the IgG heavy chain constant region may comprise SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5; the human light chain kappa constant region may comprise SEQ ID NO:6.

[0011] In one embodiment, the disclosure provides an anti-CD39 antibody comprising a light chain comprising a heavy chain variable region and 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.

[0012] In one embodiment, the disclosure provides an anti-CD39 antibody comprising a light chain comprising a heavy chain variable region and 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.

[0013] In another aspect, the present disclosure provides any of the antibodies disclosed herein for use as a medicament. In some embodiments, the anti-CD39 antibody is provided for use in the treatment of cancer. In some embodiments, the anti-CD39 antibody is provided for use in the prevention of cancer.

[0014] In another aspect, the present disclosure provides any of the antibodies disclosed herein for use as a medicament in combination with additional therapy. In some embodiments, the aforementioned anti-CD39 antibodies are provided for use in the treatment of cancer. In some embodiments, the aforementioned anti-CD39 antibodies are provided for use in the prevention of cancer. In certain embodiments, the additional therapy can be an immune checkpoint inhibitor, an immunogenic cell death-inducing therapeutic agent, an ATP-adenosine axis targeting agent, a HIF-2α inhibitor, an arginase inhibitor, an AXL inhibitor, or a PI3K inhibitor. In further embodiments, the additional therapy can be chemotherapy, radiation therapy, durvalumab, zimbelelimab, domvanalimab, AB308, AB521, or quemlicustat.

[0015] 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 aforementioned anti-CD39 antibodies are provided for use in the treatment of cancer. In some embodiments, the aforementioned anti-CD39 antibodies are provided for use in the prevention of cancer. In certain embodiments, each additional therapy can be a chemotherapeutic agent, an immune checkpoint inhibitor, an immunogenic cell death-inducing therapeutic agent, an ATP-adenosine axis targeting agent, an HIF-2α inhibitor, an arginase inhibitor, an AXL inhibitor, or a PI3K inhibitor. In further embodiments, the additional therapy can be chemotherapy, radiation therapy, durvalumab, zimbelelimab, domvanalimab, AB308, AB521, or quemlicustat. In some embodiments, the one or more additional therapeutic agents include (a) pemetrexed, carboplatin, and either an antagonist anti-PD-1 antibody or an antagonist anti-PD-L1 antibody, or (b) FOLFOX and either an antagonist anti-PD-1 antibody or an antagonist anti-PD-L1 antibody.

[0016] In another aspect, the present disclosure provides a method for treating or preventing cancer, comprising administering any of the antibodies disclosed herein to a subject in need of cancer treatment or prevention.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 therapeutic agent, an ATP-adenosine axis targeting agent, a HIF-2α inhibitor, an arginase inhibitor, an AXL inhibitor, or a PI3K inhibitor.In further embodiments, the additional therapy can be chemotherapy, radiation therapy, durvalumab, zimbelelimab, domvanalimab, AB308, AB521, or quemliclustat.

[0017] In each of the above aspects, the cancer may be breast cancer, gastrointestinal cancer, genitourinary cancer, head and neck cancer, renal cancer, lung cancer, lymphoma, ovarian cancer, pancreatic cancer, skin cancer, or thyroid cancer. In some embodiments, the cancer may 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 renal carcinoma, or melanoma.

[0018] In some embodiments, the cancer is non-small cell lung cancer, and the method may further comprise administering the anti-CD39 antibody in combination with pemetrexed, carboplatin, and either an antagonistic anti-PD-1 antibody or an antagonistic anti-PD-L1 antibody. In some embodiments, the cancer is gastric cancer or gastroesophageal cancer, and the method may further comprise administering the anti-CD39 antibody in combination with FOLFOX and either an antagonistic anti-PD-1 antibody or an antagonistic anti-PD-L1 antibody. These embodiments include the use of antagonistic anti-TIGIT antibodies, A, and / or cyclosporine. 2a R antagonist, A 2b R antagonist, A 2a / 2b The method may further include administering one or more additional agents selected from the group consisting of R antagonists and CD73 inhibitors, and optionally, the additional agents are selected from the group consisting of domvanalimab, AB308, etormadenant, and quemliculstat.

[0019] Other aspects and iterations of the present disclosure are provided in more detail below. [Brief description of the drawings]

[0020] [Figure 1] Graph showing binding of several antibodies to human CD39 expressed on the surface of B cells derived from peripheral blood mononuclear cells (PBMC). The graph 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. [Diagram 2]Graph showing inhibition of enzymatic activity of human CD39 expressed on the surface of human monocytes in the presence of 20 or 400 micromolar ATP. The graph shows representative data from a single human donor. Percentage (%) of maximum inhibition is on the y-axis and antibody concentration is on the x-axis. [Diagram 3] FIG. 1 is a schematic diagram showing key steps of the macrophage cytokine release assay described in Example 6. MΦ=macrophage. [Figure 4A] Results of a macrophage cytokine release assay. Relative amounts of cytokines in the supernatants of macrophages treated with 10 nM anti-CD39 antibody or isotype control. Fold changes were normalized to isotype control per donor, and 3 donors were run per experiment. Bar heights are the mean normalized fold change between donors, and error bars are SEM. [Figure 4B] Results of a macrophage cytokine release assay are shown. Relative amounts of cytokines in the supernatants of macrophages treated with 100 nM anti-CD39 antibody or isotype control. Fold changes were normalized to isotype control per donor, and 3 donors were run per experiment. Bar heights are the mean normalized fold change between donors, and error bars are SEM. [Figure 4C] Figure 1 shows the results of a macrophage cytokine release assay. Relative amounts of cytokines in the supernatants of macrophages treated with 100 nM anti-CD39 antibody or isotype control. Fold changes were normalized to isotype control per donor, with two donors run per experiment. Bar heights are the mean normalized fold change between donors, error bars are SEM. [Figure 4D] Figure 1 shows the results of a macrophage cytokine release assay. Relative amounts of cytokines in the supernatants of macrophages treated with 100 nM anti-CD39 antibody or isotype control. Fold changes were normalized to isotype control per donor, with two donors run per experiment. Bar heights are the mean normalized fold change between donors, error bars are SEM. [Figure 4E] Results of a macrophage cytokine release assay are shown. Relative amounts of cytokines in the supernatants of macrophages treated with 100 nM anti-CD39 antibody or isotype control. Fold changes were normalized to isotype control per donor, and 3 donors were run per experiment. Bar heights are the mean normalized fold change between donors, and error bars are SEM. [Figure 4F] Results of a macrophage cytokine release assay are shown. Relative amounts of cytokines in the supernatants of macrophages treated with 100 nM anti-CD39 antibody or isotype control. Fold changes were normalized to isotype control per donor, and 3 donors were run per experiment. Bar heights are the mean normalized fold change between donors, and error bars are SEM. [Figure 5A] Results of a monocyte-derived dendritic cell (moDC) assay are shown. The extracellular marker CD83 was measured on the surface of moDC in the presence or absence of ATP±anti-CD39 or isotype control. Each line represents one biological donor, and connected symbols are from the same donor, N=4. [Figure 5B] Figure 1 shows the results of a monocyte-derived dendritic cell (moDC) assay. The extracellular marker CD86 was measured on the surface of moDC in the presence or absence of ATP ± anti-CD39 or isotype control. Each line represents one biological donor, and connected symbols are from the same donor, N=4. [Figure 5C] Results of a monocyte-derived dendritic cell (moDC) assay are shown. The extracellular marker CD14 (Figure 5C) was measured on the surface of moDCs in the presence or absence of ATP ± anti-CD39 or isotype control. Each line represents one biological donor, and connected symbols are from the same donor, N=4. [Figure 6] Graph showing the results of a competition flow cytometry assay between 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. [Figure 7A]Figure 13 is a graph showing binding of hu39.1_IGG4.P and isotype control antibodies to human CD39 expressed on MOLP-8 human myeloma cells. [Figure 7B] FIG. 13 is a graph showing enzymatic inhibition of human CD39 expressed on human myeloma cells, MOLP-8, by hu39.1_IGG4.P and isotype control antibodies. [Figure 8A] Shows enzyme activity (ATP consumption) in splenocytes from hCD39KI mouse model. [Figure 8B] 1 shows inhibition of splenocyte enzyme activity by hu39.1_IGG4.P. [Figure 9] 1 shows confirmation that the hCD39KI mouse model expresses human, but not mouse, CD39. [Figure 10A] Graph showing mean tumor volume in hCD39KI mice receiving the indicated treatments. Points represent the mean, error bars represent standard error of the mean (SEM), *=p<0.5. [Figure 10B] Graph showing percent change in body weight in hCD39KI mice receiving the indicated treatments. [Figure 11] Shown are low magnification images of tumors from hCD39KI mice analyzed for human CD39 expression by immunohistochemistry (Panels A and C), as well as evidence of anti-CD39 enzyme blocking activity by enzyme histochemistry (Panels B and D). [Figure 12] High magnification images of tumors from hCD39KI mice analyzed for human CD39 expression by immunohistochemistry (Panels A and C), and evidence of anti-CD39 enzyme blocking activity by enzyme histochemistry (Panels B and D) are shown. The images in Figure 12 are high resolution images of the boxed area in Figure 11. [Figure 13] Shown are low magnification images of spleens from hCD39KI mice analyzed for human CD39 expression by immunohistochemistry (Panels A and C) and evidence of anti-CD39 enzyme blocking activity by enzyme histochemistry (Panels B and D). [Figure 14]High magnification images of spleens from hCD39KI mice analyzed for human CD39 expression by immunohistochemistry (Panels A and C), and evidence of anti-CD39 enzyme blocking activity by enzyme histochemistry (Panels B and D) are shown. The images in Figure 14 are high resolution images of the boxed areas in Figure 13. [Figure 15A] Graph showing ATP levels detected in supernatants obtained from single cell suspensions of tumors (MC38 tumors) obtained from hCD39KI mice treated as indicated along the x-axis. ATP levels were measured by detecting relative luminescence units (RLU). [Figure 15B] Graph showing ATP levels detected in supernatants obtained from single cell suspensions of tumors (4T1 tumors) obtained from hCD39KI mice treated as indicated along the x-axis. ATP levels were measured by detecting relative luminescence units (RLU). [Figure 15C] Graph showing ATP levels detected in supernatants obtained from single cell suspensions of peripheral whole blood cells obtained from hCD39KI mice treated as indicated along the x-axis. ATP levels were measured by detecting relative luminescence units (RLU). [Figure 16] Graphs showing cell surface CD39 and receptor occupancy in hCD39KI mice treated with anti-CD39. The graph in the top panel shows the reduction in cell surface CD39 expression as determined by binding of non-competitive anti-CD39 clone A1-PE in multiple cell types following in vivo anti-CD39 treatment. The graph in the bottom panel shows that binding of the competing antibody was observed on all CD39(A1) expressing cells in in vivo isotype control treated mice, but not on CD39(A1) expressing cells in in vivo anti-CD39 treated mice, indicating complete or near complete target coverage. Data are shown for populations in which >100 CD39(A1)+ events were collected. Statistical analysis was performed by one-way ANOVA (with Dunnett's multiple comparison test, single pooled variance), *P≦0.05, **P≦0.01, ***P≦0.001, ****P≦0.0001. [Figure 17A] Flow cytometry scatter plots showing expression levels of human CD39 (x-axis) and mouse CD73 (y-axis) in immune subpopulations from tumor-derived lymph nodes of vehicle-treated hCD39KI mice. Gates were set on the isotype control (hCD39 isotype control in grey) and applied to each group (black). [Figure 17B] Flow cytometry scatter plots showing expression levels of human CD39 (x-axis) and mouse P2X7 (y-axis) in immune subpopulations from tumor-derived lymph nodes of vehicle-treated hCD39KI mice. Gates were set on isotype controls (hCD39 isotype controls in grey) and applied to each group (black). [Figure 17C] Graph showing percentages of immune subpopulations observed following treatment, indicated along the x-axis. No significant changes in immune subpopulation percentages were observed with anti-CD39 treatment. Statistical analysis was performed by 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, error bars are + / - SEM. [Figure 17D] A graph showing CD39 cell surface expression observed following treatment is shown along the x-axis. A significant decrease in CD39 cell surface expression was observed with anti-CD39 treatment in almost all immune subpopulations. Statistical analysis was performed by 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. [Figure 17E] Histogram overlay of each immune subpopulation showing the reduction in CD39 cell surface expression in representative animals from each treatment group: isotype + saline (grey open histograms), isotype + oxaliplatin (black open histograms), anti-CD39 + saline (grey filled histograms), and anti-CD39 + oxaliplatin (black filled histograms). [Figure 18] Figure 13 is a graph showing peripheral cytokine levels in plasma obtained from hCD39KI mice bearing MC38 tumors at the end of the experiment described in Example 9. Data are presented as mean ± SEM for a total of 11-12 mice per treatment group. Statistical analysis was performed using ANOVA and Tukey's multiple comparison test. [Figure 19A] Graphs showing cell viability 24 hours after treatment with vehicle control (Veh), 100 μM oxaliplatin (OXA.L), or 250 μM oxaliplatin (OXA.H) of CT26 cells, MC38 cells, SK-MEL-5 cells and isotype control antibody, and SK-MEL-5 cells and hu39.5_IGG1.AA antibody. [Figure 19B] A graph showing HMGB1 levels in culture supernatants of CT26 cells, MC38 cells, SK-MEL-5 cells and an isotype control antibody, and SK-MEL-5 cells and hu39.5_IGG1.AA antibody 24 hours after treatment with vehicle control (Veh), 100 μM oxaliplatin (OXA.L), or 250 μM oxaliplatin (OXA.H). [Figure 19C] Graphs are shown showing 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 following 8 hour treatment with vehicle (PBS), 100 μM oxaliplatin, or 250 μM oxaliplatin. [Figure 20A] 2 shows a graph depicting cell viability of SK-MEL-5 cells following 48 hour treatment with the indicated chemotherapeutic agents. [Figure 20B] A graph showing HMGB1 levels in culture supernatants of SK-MEL-5 cells following 48 hour treatment with the indicated chemotherapeutic agents is shown. [Figure 20C] Graphs showing extracellular ATP release by SK-MEL-5 cells following treatment with the indicated chemotherapeutic agents for 8 or 32 hours. [Figure 21]Graphs showing relative expression of CD39 on peripheral immune cells in humans (top) and C57BL / 6hCK39KI MC38 mice (bottom). [Figure 22A] Flow cytometry scatter plots from a representative donor are shown, showing the distribution of CD39 and CD73 in different myeloid cell types. CD14+ monocytes were isolated directly from human peripheral blood. DCs enriched from peripheral human blood were further subtyped into pDC, cDC1, cDC2, and other DCs. M0, M1, M2, and moDC subpopulations were obtained from CD14+ monocytes as described in the Examples. [Figure 22B] Graphs are shown showing the CD73 positive percentage (left panel) and CD39 positive percentage (middle panel) and CD39 protein expression (right panel) assessed by flow cytometry for human peripheral blood CD14+ monocytes, primary dendritic cell (DC) subsets, such as plasmacytoid DC (pDC defined as HLA-DR+CD11cnegCD123+), conventional DC1 (cDC1 defined as HLA-DR+CD11c+CD141+Clec9a+), conventional DC2 (cDC2 defined as HLA-DR+CD11c+CD1c+CD141negClec9aneg), and other DC (defined as HLA-DR+CD11c+CD1cneg CD141neg Clec9aneg), as well as in vitro differentiated monocyte-derived myeloid cells, such as macrophage (M0), polarized macrophages (M1, M2) and DC (moDC). For CD39 protein expression levels, the mean fluorescence intensity of CD39+ cells was divided by the mean fluorescence intensity of the isotype control. Each symbol is a unique donor, bar height is the median, and error bars are the range. [Figure 22C] Graph showing mRNA expression of ENTPD1 (CD39) assessed in human peripheral blood CD14+ monocytes, enriched populations of primary dendritic cell subsets (enriched DCs), and in vitro differentiated monocyte-derived bone marrow cells including macrophages (M0), polarized macrophages (M1, M2), and DCs (moDCs) from four donors. [Figure 22D]Graph showing mRNA expression of NT5E (CD73) assessed in human peripheral blood CD14+ monocytes, enriched populations of primary dendritic cell subsets (enriched DCs), and in vitro differentiated monocyte-derived bone marrow cells including macrophages (M0), polarized macrophages (M1, M2), and DCs (moDCs) from four donors. [Figure 22E] Heatmap showing unsupervised clustering of housekeeping gene-normalized expression of CD39, adenosine, and inflammasome pathway genes using a custom NanoString panel in cell populations from four healthy donors listed in 279. Low quality wells flagged by low counts or aberrant control probes were excluded from this analysis. Specific donor IDs are appended to the end of cell type labels. The dotted horizontal lines in the top dendrogram indicate where samples are split, but do not affect the unsupervised nature of the clustering. [Figure 23] Graphs showing the relative induction of cell surface marker expression (relative to DMSO) after 1 h treatment with the indicated inhibitors followed by 18 h 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 by 0-300 μM ATP was compared between inhibitor-treated and DMSO-treated cells. Bar heights are means and error bars are SEM. Each point represents one donor. Statistical analysis was performed using ANOVA and Tukey's multiple comparison test, *P ≤ 0.05, ***P ≤ 0.001. [Figure 24A]Heatmap showing TCGA RNA expression analysis in tumors (renal clear cell carcinoma (KIRC), lung squamous cell carcinoma (LUSC), lung adenocarcinoma (LUAD), head and neck squamous cell carcinoma (HNSC), esophageal carcinoma (ESCA), gastric adenocarcinoma (STAD), and pancreatic adenocarcinoma (PAAD)) normalized by gene. Proteins encoded by the genes are indicated in brackets. Observed proteins and genes include ALPL (TNAP), P2RX4 (P2X4), ENTPD1 (CD39), ENPP1, NT5E (CD73), P2RX5 (P2X5), P2RY2 (P2Y2), and P2RY11 (P2Y11). Distribution is shown in the histogram on the right. [Figure 24B] Violin plots are shown. The violin plots outline the kernel probability density (width of the shaded area represents the percentage of data located there) of normalized log2 gene expression (y-axis) of the following genes (proteins): ENTPD1 (CD39), NT5E (CD73), P2RX7 (P2X7), and P2RY11 (P2Y11) in a subset of TCGA samples. Along the x-axis, the violin plots are grouped by specific TCGA subtypes: esophageal cancer (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 gastric adenocarcinoma (STAD). Also shown are pathway enrichment scores (y-axis) of myeloid signatures for the same subset of TCGA cancer subtypes. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] The present disclosure provides antibodies that specifically bind to human CD39, more specifically to an epitope within the extracellular domain of human CD39, and inhibit human CD39 enzymatic activity. Also contemplated are methods of using the anti-CD39 antibodies disclosed herein to bind to cells expressing human CD39 and inhibit soluble and cell surface expressed human CD39 enzymatic activity. The present disclosure demonstrates the use of the anti-CD39 antibodies disclosed herein to inhibit human CD39 enzymatic activity, thereby affecting one or both ends of the ATP-adenosine signaling axis. Thus, the present disclosure also provides medical uses of the anti-CD39 antibodies disclosed herein for therapeutic and diagnostic purposes.

[0022] I. Definition Unless otherwise defined, all technical terms, notations, and other scientific or technical terms used herein are intended to have the meaning commonly understood by one of ordinary skill in the art to which this disclosure pertains. It is understood that aspects of the invention described herein include the aspects "comprising," "consisting of," and "consisting essentially of." The terms "comprising," "including," and "having" are intended to be inclusive and mean that there may be additional elements other than the listed elements.

[0023] Unless otherwise specified, the term "about" as used herein refers to the normal error range for the respective value as would be understood by a person of ordinary skill in the art. For the avoidance of doubt, reference herein to "about" a value or parameter includes (and describes) aspects that are directed to that value or parameter itself.

[0024] Also, as used herein, "and / or" refers to and includes all possible combinations of one or more of the associated listed items, including but not limited to combinations when interpreted in the alternative ("or"). Similarly, a phrase in the form "A / B" or "A and / or B" means (A), (B), or (A and B); a phrase in the form "at least one of A, B, and C" means (A), (B), (C), (A and B), (A and C), (B and C), or (A, B, and C).

[0025] The term "CD39" refers to human CD39 unless otherwise specified. An exemplary human CD39 sequence is SEQ ID NO: 7 (UniProtKB Reference: P49961). CD39 is a two-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.

[0026] The terms "anti-CD39 antibody" and "antibody that binds to CD39" are used interchangeably herein and refer to an antibody that can bind to human CD39 with sufficient affinity so as to be useful as a diagnostic and / or therapeutic agent that targets human CD39 and inhibits human CD39 enzymatic activity. The anti-CD39 antibodies of the present disclosure bind to CD39 with a binding affinity of 10 to 15%. -6 The anti-CD39 antibodies of the present disclosure may be monospecific or multispecific, and in some instances, polyepitopic, antibodies.

[0027] The term "antibody" as used herein is used in the broadest sense and encompasses a variety of 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, reference to an antibody should be understood to refer to the antibody in its intact form or to an antigen-binding fragment, unless the context requires otherwise. Preferably, but not necessarily, the antibodies useful herein are isolated and may be recombinantly produced.

[0028] The terms "full length antibody," "intact antibody," and "whole antibody" are used interchangeably herein to refer to an antibody having a structure substantially similar to a native antibody structure or having a heavy chain that contains an Fc region.

[0029] "Native antibodies" are naturally occurring immunoglobulin molecules with a variety of structures. For example, native IgG antibodies are heterotetrameric glycoproteins of about 150,000 daltons composed of two identical light chains (about 25 kDa each) and two identical heavy chains (about 50-70 kDa each) that are disulfide-bonded. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH), also called the variable heavy domain or heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3). Similarly, from the N-terminus to the C-terminus, each light chain has a variable region (VL), also called the variable light domain or light chain variable domain, followed by a constant light (CL) domain. The light chain of an antibody can be assigned one of two types, called kappa (κ) and lambda (λ), based on the amino acid sequence of its constant domain. 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 light and heavy chain contains a variable region (VL and VH, respectively) of about 100-110 or more amino acids that is primarily responsible for antigen recognition. The carboxy-terminal portion of each chain defines a constant region that is primarily responsible for effector function. Within the 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 containing a "D" region of about 10 more amino acids.

[0030] 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 the heavy and light chains of an antibody generally have a similar structure, with each domain containing four conserved framework regions (FR) and three hypervariable regions (CDR). (See, e.g., Kindt et al. Kuby Immunology, 6th ed., WH Freeman and Co., page 91 (2007).) A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, a VH or VL domain from an antibody that binds to an antigen may be used to screen a library of complementary VL or VH domains, respectively, to isolate an antibody that binds to a particular antigen. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0031] "Framework region" or "FR" refers to variable domain residues other than the hypervariable region residues. The FR of a variable domain generally consists of four FR domains, FR1, FR2, FR3, and FR4. Thus, the 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 may differ as known in the art.

[0032] As used herein, the term "hypervariable region" or "HVR", also commonly referred to as "complementarity determining region" or "CDR", is used interchangeably and refers to each of the regions of the variable domain that are hypervariable in sequence and / or form structurally defined loops ("hypervariable loops") and / or contain antigen contact residues ("antigen contacts"). Generally, an antibody comprises six CDRs, three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). As used herein, "CDRs derived from a variable region" refer to CDRs that have no more than two amino acid substitutions compared to the corresponding CDR from the original variable region. Exemplary CDRs herein include, for various antibodies of the present disclosure, (a) hypervariable loops occurring at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901 -917 (1987)); (b) CDRs occurring at amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 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) antigen contacts occurring at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 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). Unless otherwise specified, CDR residues and other residues in the variable domain (e.g., FR residues) are numbered in Eu according to Kabat et al., supra, herein.

[0033] 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 greater than 99% purity, for example, as determined by electrophoresis or chromatography (e.g., ion exchange or reverse phase HPLC).

[0034] 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 remainder of the heavy and / or light chain is derived from a different source or species.

[0035] A "human antibody" is one that possesses an amino acid sequence that corresponds to the amino acid sequence of an antibody produced by a human or human cell, or from a non-human source that utilizes a human antibody repertoire, or other human antibody coding sequence. This definition of a human antibody specifically excludes humanized antibodies that contain non-human antigen-binding residues. Human antibodies can be produced using various techniques known in the art, including phage display libraries. The methods described in Hoogenboom and Winter. J. Mol. Biol. 227:381, 1991; Marks et al. J. Mol. Biol. 222:581, 1991. Cole et al. Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boerner et al. J. Immunol., 147(1):86-95,1991 can also be used to prepare human monoclonal antibodies. See also van Dijk and van de Winkel. Curr. Opin. Pharmacol. 5:368-74, 2001.

[0036] A "humanized" antibody refers to an antibody that comprises amino acid residues from non-human CDRs and amino acid residues from human FRs. In certain aspects, a humanized antibody will comprise a variable domain in which all or substantially all of its CDRs correspond to the CDRs of a non-human antibody and all or substantially all of its FRs correspond to the FRs of a human antibody. In certain aspects in which all or substantially all of the FRs of a humanized antibody correspond to the FRs of a human antibody, any of the FRs of the humanized antibody can contain one or more amino acid residues of the non-human FR(s), e.g., at one or more veneer position residues of the FR and / or at one or more other selected residues. A humanized antibody may optionally 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 similar binding specificity and affinity as the starting non-human antibody.

[0037] The term "monoclonal antibody" refers to an antibody derived from a single copy or clone, including, for example, any eukaryotic, prokaryotic, or phage clone. The term "monoclonal antibody" is not limited to antibodies produced by hybridoma technology. Monoclonal antibodies can be produced using hybridoma technology well known in the art, as well as recombinant, phage display, synthetic, or a combination of such technologies with other techniques readily known in the art.

[0038] The term "epitope" refers to the specific site on an antigen to which an antibody binds. The specific site on an antigen to which an antibody binds can be determined, for example, by crystallography. Methods such as hydroxyl radical protein footprinting and alanine scanning mutagenesis can also be used, but may have lower resolution.

[0039] 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 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.

[0040] 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), where the VH / VL unit has polyepitopic specificity, antibodies having two or more VL and VH domains, where each VH / VL unit binds to a different epitope, antibodies having two or more single variable domains, where each single variable domain binds to a different epitope, diabodies, triabodies, etc., as well as full length antibodies and / or covalently or non-covalently linked antibody fragments.

[0041] The terms "polyepitopic antibody" and "antibody with polyepitopic specificity" are used interchangeably herein and refer to an antibody that binds to two or more epitopes on the same or different antigens.

[0042] The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain, including native sequence Fc regions and variant Fc regions. Although the boundaries of an immunoglobulin heavy chain Fc region may vary, the human IgG heavy chain Fc region is usually defined as extending from an amino acid residue at position Cys226, or from Pro230, to its carboxyl terminus. The C-terminal lysine (residue 447 according to the Eu numbering system) of the Fc region may be removed, for example, during production or purification of the antibody, or by recombinantly engineering the nucleic acid encoding the antibody heavy chain. Thus, a composition of intact antibodies may include an antibody population with all Lys447 residues removed, an antibody population without the Lys447 residue removed, and an antibody population with a mixture of antibodies with and without the Lys447 residue.

[0043] A "functional Fc region" has the "effector functions" of a native sequence Fc region. Exemplary "effector functions" include C1q binding; complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor; BCR), and the like. Effector functions such as these generally require the combination of an Fc region 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. A functional Fc region can have effector functions substantially similar to wild-type IgG, reduced effector functions compared to wild-type IgG, or enhanced effector functions compared to wild-type IgG. For antibodies that include a human Fc region, the comparison is typically made with wild-type human IgG1.

[0044] A "native sequence Fc region" comprises an amino acid sequence identical to the amino acid sequence of an Fc region found in nature. Native sequence human Fc regions include native sequence human IgG1 Fc regions (non-A and A allotypes), native sequence human IgG2 Fc regions, native sequence human IgG3 Fc regions, and native sequence human IgG4 Fc regions, as well as naturally occurring variants thereof.

[0045] A "variant Fc region" comprises an amino acid sequence that differs from that of a native sequence Fc region by at least one amino acid modification (e.g., about 1 to about 10 amino acid modifications, and in some embodiments about 1 to about 5 amino acid modifications), preferably by one or more amino acid substitution(s). A variant Fc region herein preferably has at least about 80% homology to a native sequence Fc region and / or the Fc region of a parent polypeptide, preferably at least about 90% homology thereto, or preferably at least about 95% homology thereto. In some embodiments, a variant Fc region may have reduced or enhanced effector function compared to wild-type IgG. In the case of antibodies that comprise a human Fc region, the comparison is typically made to wild-type human IgG1.

[0046] As used herein, "Fc component" refers to the hinge region, CH2 domain, or CH3 domain of the Fc region.

[0047] The "hinge region" is generally defined as stretching from about residue 216 to about residue 230 of IgG (Eu numbering), from about residue 226 to about residue 243 of IgG (Kabat numbering), or from about residue 1 to about residue 15 of IgG (IMGT specific numbering).

[0048] The term "antibody fragment" refers to a molecule other than an intact antibody that contains a portion of an intact antibody that binds to 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 , F(ab)c , Fv fragment, disulfide stabilized Fv fragment (dsFv), (dsFv) 2 , bispecific dsFv (dsFv-dsFv'), disulfide-stabilized diabodies (ds diabodies), triabodies, tetrabodies, single chain antibodies, scFv, scFv dimers, single domain antibodies, single-domain antibodies, and multivalent domain antibodies. Typically, binding fragments compete for specific binding with the intact antibody from which they are derived. Binding fragments can be produced by recombinant DNA techniques or by enzymatic or chemical separation of intact immunoglobulins.

[0049] The term "Fab" refers to a portion of an antibody consisting of a single light chain (both variable and constant regions) linked by disulfide bonds to the variable region and first constant region of a single heavy chain.

[0050] The term "Fab" refers to a Fab fragment that includes a portion of the hinge region.

[0051] "F(ab') 2 The term "refers to a dimer of Fab'. F(ab')2 antibody fragments originally were produced as pairs of Fab' fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0052] The term "Fv" refers to the minimum fragment of an antibody that retains a 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.

[0053] The term "single chain antibody" refers to an antibody consisting 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. Two or more single chain antibodies can be covalently or non-covalently linked to obtain higher forms. Single chain antibodies and their higher forms can include, but are not limited to, single domain antibodies, multivalent domain antibodies, single chain variant fragments (scFv), bivalent scFv (di-scFv), trivalent scFv (tri-scFv), tetravalent scFv (tetra-scFvs), diabodies, and triabodies, and tetrabodies.

[0054] The terms "single chain Fv antibody" and "scFv" are used interchangeably herein and refer to a single chain antibody consisting 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 linker peptide is preferably about 5-30 amino acids long, or about 10-25 amino acids long. Typically, the linker allows for stabilization of the variable domains without preventing proper folding and creation of an active binding site. In a preferred embodiment, the linker peptide is rich in glycine, as well as serine or threonine. Two or more scFvs can be covalently or non-covalently linked to obtain higher order forms such as di-scFv, tri-scFv, tetra-scFv, etc. The antigen binding sites of each scFv in the higher order form can target the same or different antigens or epitopes.

[0055] The term "single chain Fv-Fc antibody" or "scFv-Fc" refers to a full length antibody consisting of an scFv connected to an Fc region.

[0056] A "diabody" is a higher variant of a single-chain antibody consisting of two single-chain antibodies. Each single-chain antibody can pair with a complementary domain of another chain, using a linker that is too short to allow pairing between the two domains on the same chain, thereby creating two antigen-binding sites. In most cases, but not all, 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 variants can be generated as well. See, for example, Holliger P. et al., Proc Natl Acad Sci USA. July 15;90(14):6444-8 (1993);EP404097;WO93 / 11161.

[0057] A "single domain antibody" refers to an antibody fragment that contains only the variable region of a heavy chain or the variable region of a light chain. In certain instances, two or more V H The domains are covalently linked with a peptide linker to create a multivalent domain antibody. H The domains can target the same or different antigens or epitopes.

[0058] The term "heavy chain antibody" refers to an antibody that consists of two heavy chains. Heavy chain antibodies can be IgG-like antibodies from camels, llamas, alpacas, sharks, etc., or IgNAR from cartilaginous fish. See, for example, Riechmann L. and Muyldermans S., J Immunol Methods. December 10;231(1-2): 25-38 (1999);Muyldermans S., J Biotechnol. June;74(4):277-302 (2001);WO94 / 04678;WO94 / 25591; or U.S. Patent No. 6,005,079. Heavy chain antibodies were originally derived from camelids (camels, dromedaries, and llamas). Although lacking light chains, camelized antibodies have a bona fide antigen-binding repertoire (see Hamers-Casterman C. et al., Nature. June 3;363(6428):446-8 (1993); Nguyen VK et al. "Heavy-chain antibodies in Camelidae; a case of evolutionary innovation," Immunogenetics. April;54(1):39-47 (2002); Nguyen VK et al. Immunology. May;109(1):93-101 (2003)). The variable domain of heavy chain antibodies (VHH domains) represents the smallest known antigen-binding unit generated by the adaptive immune response (Koch-Nolte F. et al., FASEB J. November;21(13):3490-8. Epub 2007 Jun. 15 (2007)).

[0059] "Nanobody" refers to an antibody that consists of a VHH domain derived from a heavy chain antibody and two constant domains, CH2 and CH3.

[0060] "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 to those in the reference sequence, without taking into account any conservative substitutions as part of sequence identity, after aligning the sequences and introducing gaps if necessary to obtain the maximum percent sequence identity. Alignment for the purpose of determining percent amino acid sequence identity can be achieved in various ways that are within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, or CLUSTAL software. Those skilled in the art can determine the appropriate parameters for aligning sequences, including any algorithms required to obtain maximum alignment over the entire length of the sequences being compared. Generally speaking, the sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B is calculated as follows: 100 x fraction X / Y, where X is the number of amino acid residues scored as perfect matches by a sequence alignment program in that program's alignment of A and B, and Y is the total number of amino acid residues in B. It is understood that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, then the % amino acid sequence identity of A to B is not equal to the % amino acid sequence identity of B to A.

[0061] The terms "patient" or "subject" are used interchangeably herein and refer to a human or non-human animal (eg, a mammal) that expresses human CD39.

[0062] The terms "treat," "treating," "treatment," and the like refer to a course of action that eliminates, alleviates, inhibits, relieves, or improves, or prevents the worsening of, the disease, disorder, or condition to which the term applies, or at least one symptom associated therewith, either temporarily or permanently. Treatment includes, by way of example, inhibiting active disease (e.g., preventing the onset or further development of the disease, disorder, or condition, or clinical symptoms associated therewith), improving quality of life, and / or prolonging the survival of a subject.

[0063] As used herein, the term "in need of treatment" refers to a judgment made by a physician or other caregiver that a subject requires or would benefit from treatment. This judgment is made based on a variety of factors that are within the physician's or caregiver's area of ​​expertise.

[0064] The terms "prevent", "preventing", "prevention", "prophylaxis", and the like generally refer to a course of action initiated in some manner (e.g., before the onset of a disease, disorder, condition, or symptoms thereof) in the context of a subject prone to having a particular disease, disorder, or condition, to temporarily or permanently prevent, suppress, inhibit, or reduce the risk of the subject developing the disease, disorder, condition, or the like (e.g., as determined by the absence of clinical symptoms), or to delay its onset. In certain instances, these terms also refer to slowing the progression of a disease, disorder, or condition, or inhibiting its progression to a harmful or otherwise undesirable state. Prevention also refers to a course of action initiated in a subject after the subject has been treated for a disease, disorder, condition, or symptoms associated therewith, to prevent the recurrence of the disease, disorder, condition, or symptoms.

[0065] As used herein, the term "in need of prevention" refers to a judgment made by a physician or other caregiver that a subject needs or would benefit from preventative care. This judgment is made based on a variety of factors that are within the physician's or caregiver's area of ​​expertise.

[0066] II. Antibodies of the Present Disclosure The present disclosure provides an antibody that specifically binds to human CD39, more specifically to the extracellular domain of human CD39. The antibody of the present disclosure may specifically bind to soluble and / or membrane-bound human CD39. In some embodiments, the anti-CD39 antibody of the present disclosure is monospecific. In other embodiments, the anti-CD39 antibody of the present disclosure is multispecific. Additional antigen binding specificities contemplated in the context of the present disclosure include, but are not limited to, TGFβ. The anti-CD39 antibody of the present application may be isolated or part of a composition.

[0067] The anti-CD39 antibodies of the present disclosure, whether monospecific or multispecific, specifically bind to human CD39, meaning that the antibodies bind to CD39 with a 10 -6 The equilibrium dissociation constant (K D In certain embodiments, the anti-CD39 antibodies of the disclosure have a K D But, 10 -8 M or less (e.g., 10 -8 , 10 -9 , 10 -10 For example, see Example 3 for detailed methodology. In various embodiments, the anti-CD39 antibodies of the present disclosure have a K D Approximately 1x10 -9 M ~ approx. 1x10 -14 M, or about 1x10 -9 M ~ approx. 1x10 -13 M, or about 1x10 -9 M ~ approx. 1x10 -12 M, or about 1x10 -9 M ~ approx. 1x10 -11In some embodiments, the anti-CD39 antibodies of the present disclosure have a K D Approximately 1x10 -10 ~ approx. 1x10 -14 M, or about 1x10 -10 M ~ approx. 1x10 -13 M, or about 1x10 -10 M ~ approx. 1x10 -12 In some embodiments, the anti-CD39 antibodies of the present disclosure have a K D Approximately 1x10 -11 M ~ approx. 1x10 -14 M, or about 1x10 -11 ~ approx. 1x10 -13 In some embodiments, the anti-CD39 antibodies of the present disclosure have a K D Approximately 1x10 -12 M ~ approx. 1x10 -14 M, or about 1x10 -12 M ~ approx. 1x10 -13 In some embodiments, the anti-CD39 antibodies of the present disclosure have a K D Approximately 1x10 -10 M ~ approx. 1x10 -11 M, or about 1x10 -11 M ~ approx. 1x10 -12 It's M.

[0068] The anti-CD39 antibodies of the disclosure also inhibit CD39 enzymatic activity. In some embodiments, the anti-CD39 antibodies of the disclosure have an IC of about 5 nM or less, measured as in Example 5. 50 For example, the anti-CD39 antibody may have an IC value of about 5 nM, about 4 nM, about 3 nM, about 2 nM, about 1 nM, or less, as measured in Example 5. 50 In another example, the anti-CD39 antibody can inhibit recombinant human CD39 enzyme activity with an IC value of about 1 nM or less, e.g., about 1.0 nM, about 0.9 nM, about 0.8 nM, about 0.7 nM, about 0.6 nM, about 0.5 nM, or less. 50As another example, the anti-CD39 antibody can inhibit recombinant human CD39 enzyme activity with an IC value of 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 as in Example 5. 50 The IC values ​​above are capable of inhibiting recombinant human CD39 enzyme activity. 50 Values ​​may also be expressed as individual values ​​or ranges. For example, an anti-CD39 antibody may inhibit recombinant human CD39 enzyme activity with an IC of 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 any subrange thereof. 50 In one embodiment, the anti-CD39 antibodies of the disclosure can inhibit human CD39 enzyme activity with an IC value of about 0.5 nM to about 1.0 nM, or about 0.6 nM to about 1.0 nM. 50 In one embodiment, the anti-CD39 antibodies of the disclosure inhibit human CD39 enzyme activity with an IC value of 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 In one embodiment, the anti-CD39 antibody of the disclosure inhibits human CD39 enzyme activity with an IC value of 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 In the foregoing embodiments, (i) human CD39 may be recombinant soluble CD39 or cell surface expressed CD39, and / or (ii) inhibition may be assessed in the presence of low ATP (e.g., 20 μM) and / or high ATP (e.g., 400 μM).

[0069] A. Exemplary Variable Regions 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" encompasses 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.

[0070] 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 a light chain variable region (VL) having, optionally, one or more CDRs derived from SEQ ID NO:13. The CDRs derived from SEQ ID NO:9 may be H1, H2, H3, or any combination thereof. In certain embodiments, the VH may 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 may be L1, L2, L3, or any combination thereof. In certain embodiments, the VL may 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 from SEQ ID NO:9 may further comprise a VL comprising one or more CDRs from SEQ ID NO:13. The CDRs may be L1, L2, L3, or any combination thereof. In a preferred embodiment, the VL may comprise an L1 having an amino acid sequence having at least 90% sequence identity to SEQ ID NO:14, an L2 having an amino acid sequence having at least 90% sequence identity to SEQ ID NO:15, an L3 having an amino acid sequence having at least 90% sequence identity to SEQ ID NO:16, or any combination thereof. In a specific example, an anti-CD39 antibody of the disclosure comprises a VH comprising an H1 having an amino acid sequence having at least 90% sequence identity to SEQ ID NO:10, an H2 having an amino acid sequence having at least 90% sequence identity to SEQ ID NO:11, an H3 having an amino acid sequence having at least 90% sequence identity to SEQ ID NO:12, and a VL comprising an L1 having an amino acid sequence having at least 90% sequence identity to SEQ ID NO:14, an L2 having an amino acid sequence having at least 90% sequence identity to SEQ ID NO:15, and an L3 having an amino acid sequence having at least 90% sequence identity to SEQ ID NO:16.In another specific example, an anti-CD39 antibody of the disclosure comprises a VH comprising an H1 having an amino acid sequence comprising SEQ ID NO:10, an H2 having an amino acid sequence comprising SEQ ID NO:11, an H3 having an amino acid sequence comprising SEQ ID NO:12, and a VL comprising an L1 having an amino acid sequence comprising SEQ ID NO:14, an L2 having an amino acid sequence comprising SEQ ID NO:15, and an L3 having an amino acid sequence comprising SEQ ID NO:16.

[0071] In some of the foregoing embodiments, the antibody may 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 an exemplary embodiment, the antibody comprises a mature VH and a mature VL and is an antibody of Table A. JPEG2025508956000001.jpg1892

[0072] In each of the foregoing embodiments, the anti-CD39 antibody may be (i) an intact antibody or an 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 may optionally include one or more constant regions, or portions of a constant region, that are substantially human. Suitable constant regions are described in further detail in Section II(F).

[0073] In another exemplary embodiment, the antibody is an antibody of Table B. JPEG2025508956000002.jpg18106

[0074] In another example, an anti-CD39 antibody of the 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 may be H1, H2, H3, or any combination thereof. In certain embodiments, the VH may comprise an H1 having at least 90% sequence identity to SEQ ID NO:18, an H2 having at least 90% sequence identity to SEQ ID NO:19, an 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 may be an L1, L2, L3, or any combination thereof. In certain embodiments, the VL may comprise an L1 having at least 90% sequence identity to SEQ ID NO:22, SEQ ID NO:23, and SEQ ID NO:24, an L2 having at least 90% sequence identity to SEQ ID NO:25, an L3 having at least 90% sequence identity to SEQ ID NO:26, or any combination thereof. An antibody comprising one or more CDRs from SEQ ID NO:17, SEQ ID NO:43, or SEQ ID NO:44 may further comprise a VL comprising one or more CDRs 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 may be L1, L2, L3, or any combination thereof. In preferred embodiments, the VL may comprise an L1 having at least 90% sequence identity to SEQ ID NO:22, SEQ ID NO:23, or SEQ ID NO:24, an L2 having at least 90% sequence identity to SEQ ID NO:25, an L3 having at least 90% sequence identity to SEQ ID NO:26, or any combination thereof.In a specific example, an anti-CD39 antibody of the disclosure includes a VH comprising an H1 having an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 18, an H2 having an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 19, an H3 having an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 20, and a VL comprising an L1 having an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24, an L2 having an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 25, and an L3 having an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 26. In another specific example, an anti-CD39 antibody of the disclosure includes a VH comprising an H1 having an amino acid sequence comprising SEQ ID NO: 18, an H2 having an amino acid sequence comprising SEQ ID NO: 19, an H3 having an amino acid sequence comprising SEQ ID NO: 20, and a VL comprising an L1 having an amino acid sequence comprising SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24, an L2 having an amino acid sequence comprising SEQ ID NO: 25, and an L3 having an amino acid sequence comprising SEQ ID NO: 26.

[0075] In some of the foregoing embodiments, the antibody may further comprise (i) a VH having an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 17, and / or a VL having an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 21; or (ii) a VH having an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 43 or SEQ ID NO: 44, and / or a VL having an amino acid sequence having 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 an exemplary embodiment, the antibody comprises a VH and a VL and is an antibody of Table C. JPEG2025508956000003.jpg5996

[0076] In each of the foregoing embodiments, the anti-CD39 antibody may be (i) an intact antibody or an 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 may optionally include one or more constant regions, or portions of a constant region, that are substantially human. Suitable constant regions are described in further detail in Section II(F).

[0077] In another exemplary embodiment, the antibody is an antibody of Table D. JPEG2025508956000004.jpg65111

[0078] In another example, an anti-CD39 antibody of the 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 a light chain variable region (VL) optionally 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 may be H1, H2, H3, or any combination thereof. In certain embodiments, the VH may comprise an H1 having at least 90% sequence identity to SEQ ID NO:28, an H2 having at least 90% sequence identity to SEQ ID NO:29, an 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 may be an L1, L2, L3, or any combination thereof. In certain embodiments, the VL may comprise an L1 having at least 90% sequence identity to SEQ ID NO:32, an L2 having at least 90% sequence identity to SEQ ID NO:33, an L3 having at least 90% sequence identity to SEQ ID NO:34, or any combination thereof. An antibody comprising one or more CDRs from SEQ ID NO:58 or SEQ ID NO:59 may further comprise a VL comprising one or more CDRs from SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63. The CDRs may be L1, L2, L3, or any combination thereof. In a preferred embodiment, the VL may comprise an L1 having an amino acid sequence having at least 90% sequence identity to SEQ ID NO:32, an L2 having an amino acid sequence having at least 90% sequence identity to SEQ ID NO:33, an L3 having an amino acid sequence having at least 90% sequence identity to SEQ ID NO:34, or any combination thereof.In a specific example, an anti-CD39 antibody of the disclosure comprises a VH comprising an H1 having an amino acid sequence having at least 90% sequence identity to SEQ ID NO:28, an H2 having an amino acid sequence having at least 90% sequence identity to SEQ ID NO:29, an H3 having an amino acid sequence having at least 90% sequence identity to SEQ ID NO:30, and a VL comprising an L1 having an amino acid sequence having at least 90% sequence identity to SEQ ID NO:32, an L2 having an amino acid sequence having at least 90% sequence identity to SEQ ID NO:33, and an L3 having an amino acid sequence having at least 90% sequence identity to SEQ ID NO:34. In another specific example, an anti-CD39 antibody of the disclosure comprises a VH comprising an H1 having an amino acid sequence comprising SEQ ID NO:28, an H2 having an amino acid sequence comprising SEQ ID NO:29, an H3 having an amino acid sequence comprising SEQ ID NO:30, and a VL comprising an L1 having an amino acid sequence comprising SEQ ID NO:32, an L2 having an amino acid sequence comprising SEQ ID NO:33, and an L3 having an amino acid sequence comprising SEQ ID NO:34.

[0079] In some of the foregoing embodiments, the antibody may further comprise (i) a VH having an amino acid sequence having at least 80% sequence identity to SEQ ID NO:27, and / or a VL having an amino acid sequence having at least 80% sequence identity to SEQ ID NO:31; or (ii) a VH having an amino acid sequence having at least 80% sequence identity to SEQ ID NO:58 or SEQ ID NO:59, and / or a VL having an amino acid sequence having at least 80% sequence identity to SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, or SEQ ID NO:63. In an exemplary embodiment, the antibody comprises a VH and a VL and is an antibody of Table E. JPEG2025508956000005.jpg6697

[0080] In each of the foregoing embodiments, the anti-CD39 antibody may be (i) an intact antibody or an 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, respectively. In certain embodiments, the anti-CD39 antibody may optionally include one or more constant regions, or portions of a constant region, that are substantially human. Suitable constant regions are described in further detail in Section II(F).

[0081] In another exemplary embodiment, the antibody is an antibody of Table F. JPEG2025508956000006.jpg77114

[0082] B. Antibodies with Similar Binding Specificities The present disclosure relates to an antibody designated as 19 or ch19_IGG4.P above, or 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 above, or as 39, ch39_I Also provided are anti-CD39 antibodies that bind to the same or overlapping epitopes as the antibodies designated as GG4.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. Epitopes may be identified by methods known in the art, such as X-ray crystallography of the antibody bound to its antigen to identify contact residues. Competitive assays may be used to identify such antibodies. For example, the anti-CD39 antibody can competitively inhibit binding of a reference antibody to human CD39, wherein the reference antibody is 19, ch19_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 hu u31.7_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 binding of a reference antibody to human CD39 if the antibody blocks 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 competitive flow assay, as described in Example 7.

[0083] In a further embodiment, an antibody that competitively inhibits binding of a reference antibody to human CD39, as described above, comprises a VH, or a VH and a VL, as described in Section II(A).

[0084] Other antibodies having such binding specificity can be produced by immunizing mice with human CD39, or a portion thereof containing the desired epitope, and optionally detecting the resulting antibody, such as antibody 19 or a variant thereof (such as, for example, ch19_IGG4.P), antibody 31, or a variant thereof (such as, for example, 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, hu31.8_IGG4.P, hu31.9_IGG4.P, hu31.10_IGG4.P, hu31.11_IGG4.P, hu31.12_IGG4.P, hu31.13_IGG4.P, hu31.14_IGG4.P, hu31.15_IGG4.P, hu31.16_IGG4.P, hu31.17_IGG4.P, hu31.18_IGG4.P, hu31.19_IGG4.P, hu31.20_IGG4.P, hu31.21_IGG4.P, hu31.22_IGG4.P, hu31.23_IGG4.P, hu31.24_IGG4.P, hu31.25_IGG4.P, hu31.26_IGG4.P, hu31.27_IGG4.P, hu31.28_IGG4.P, hu31.29_IGG4.P, hu31.30 _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 were also screened against mutagenized forms of human CD39, resulting in: 19, ch19_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, Antibodies can be identified that exhibit the same or similar binding profile as a collection of mutational changes, such as 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 replacement substitutions with alanine (or serine, if alanine is already present), one residue at a time, or at more widely spaced intervals, throughout the entire extracellular domain of the CD39 antibody, or the section thereof where the epitope is known to reside.

[0085] Other antibodies are exemplary antibodies, e.g., 19, ch19_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, ch3 9_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, can be obtained by mutagenesis of cDNA encoding the heavy and light chains of In the amino acid sequence of the mature heavy and / or light chain variable region, 19, ch19_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, hu39.6_IGG4.P, hu39.7_IGG4.P, hu39.8_IGG4.P, hu39.9_IGG4.P, hu39.10_IGG4.P, hu39.11_IGG4.P, hu39.12_IGG4.P, hu39.13_IGG4.P, hu39.14_IGG4.P, hu39.15_IGG4.P, hu39.16_IGG4.P, hu39.17_IGG4.P, hu39.18_IGG4.P, hu39.19_IGG4.P, hu39.20_IGG4.P, hu39.21_IGG4.P, hu39.22_IGG4.P, hu39.23_IGG4.P, hu39.24_IGG4.P, hu39 Also included in the disclosure are 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 IGG4.P, hu39.5_IGG1.AA, hu39.6_IGG4.P, hu39.7_IGG4.P or hu39.8_IGG4.P and retain their functional properties and / or differ from the respective antibodies by a small number of amino acid substitutions (e.g., conservative substitutions), deletions or insertions that are not functionally significant. Amino acids within the variable region frameworks that are likely to be important for binding can be identified as described in the section on humanization below.

[0086] 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. Antibodies having the binding specificity of 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) can also be produced using a variation of the phage display method. See Winter, WO 92 / 20791. This method is particularly suitable for producing human antibodies. In this method, either the heavy chain variable region or the light chain variable region of a selected antibody is used as starting material. For example, if a 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., the starting light chain) and different heavy chain variable regions. The heavy chain variable region can be obtained, for example, from a library of rearranged human heavy chain variable regions. Phages that exhibit strong specific binding to human CD39 (e.g., at least 10 8 Or at least 10 9 M -1 ) is selected. The heavy chain variable region from this phage then serves as starting material for constructing a further phage library, in which each phage displays the same heavy chain variable region (i.e., the region identified from the first display library) and a different light chain variable region. The light chain variable region can be obtained, for example, from a library of rearranged human light chain variable regions. Again, phage that show strong specific binding to human CD39 are selected. The resulting antibodies usually have the same or similar epitope specificity as the murine starting material.

[0087] In addition to the antibodies described herein, it may be possible to design antibody mimetics or aptamers with substantially the same function and binding specificity as the antibodies of the present invention using methods known in the art. An "antibody mimetic" refers to a polypeptide or protein that can specifically bind to an antigen but is not structurally related to an antibody. Antibody mimetics have a mass of about 3 kDa to about 20 kDa. Non-limiting examples of antibody mimetics are affibody molecules, affilins, affimers, alphabodies, anticalins, avimers, DARPins, and monobodies. 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 through structural recognition, a process similar to antigen-antibody reactions. Aptamers are smaller in molecular weight than antibodies, typically about 8 to 25 kDa.

[0088] C. Chimeric and Veneered Antibodies As mentioned above, the present disclosure provides chimeric and veneered forms of non-human antibodies, including, but not limited to, chimeric anti-CD39 antibodies of the antibodies designated herein as Antibody 19, Antibody 31, or Antibody 39.

[0089] 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 contain approximately two-thirds human sequences.

[0090] A veneered antibody is a type of humanized antibody that retains some, and usually all, of the CDRs of a non-human antibody and some of the non-human variable region framework residues, but replaces other variable region framework residues that may contribute to B-cell or T-cell epitopes, such as exposed residues (Padlan, Mol. Immunol. 28:489, 1991), with residues at the corresponding positions in a human antibody sequence. The result is an antibody whose CDRs are entirely or substantially derived from a non-human antibody and whose variable region framework is rendered more human-like by the substitutions. Veneered forms of anti-CD39 antibodies are included in the present disclosure.

[0091] In some embodiments, the anti-CD39 chimeric antibodies are rat-human chimeras having rat variable domains and human IgG1 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 specific embodiments, the anti-CD39 chimeric antibodies are ch19_IGG4.P, ch31_IGG4.P, and ch39_IGG4.P.

[0092] D. Humanized Antibodies As described herein, the disclosure provides a humanized antibody of an antibody designated herein as 19, 31 or 39, optionally wherein the humanized antibody has an IC 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 as in Example 5. 50Humanized antibodies inhibit human CD39 enzyme activity at a low level. Typically, non-human antibodies are humanized to reduce immunogenicity to humans while retaining the specificity and affinity of the parent non-human antibody. A humanized antibody comprises one or more variable domains in which the CDRs or portions thereof are derived from a non-human antibody and the FRs or portions thereof are derived from a human antibody sequence. A humanized antibody may also optionally comprise at least a portion of a human constant region. In some embodiments, some FR residues in a humanized antibody are replaced with corresponding residues from a non-human antibody (e.g., the antibody from which the CDR residues are derived), e.g., to restore or improve antibody specificity or affinity.

[0093] Thus, a humanized antibody is an antibody in which some or all CDRs are fully or substantially derived from a donor antibody, and the variable region framework sequences and constant regions, if present, are fully or substantially derived from human antibody sequences. For example, a humanized heavy chain has at least one, two, and usually all three CDRs that are fully or substantially derived from a donor antibody heavy chain, and heavy chain variable region framework sequences and heavy chain constant regions, if present, that are substantially derived 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 fully or substantially derived from a donor antibody light chain, and light chain variable region framework sequences and light chain constant regions, if present, that are substantially derived from human light chain variable region framework and constant region sequences. Again, as elsewhere in this application, a CDR of a subject antibody is substantially derived from a corresponding CDR of a reference antibody if 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) are identical between the respective CDRs; however, it is understood that a CDR as defined by Kabat in the subject antibody may be substantially derived from a corresponding CDR of a reference antibody if 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) are identical between the respective CDRs; H2 is substantially derived from a corresponding CDR of a reference antibody if 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) are identical between the respective CDRs. A variable region framework sequence of an antibody chain or a constant region of an antibody chain is substantially derived from a human variable region framework sequence or a human constant region, respectively, if 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 are identical.

[0094] Generally speaking, a humanized antibody is a genetically engineered antibody in which CDRs from a non-human "donor" antibody are grafted onto human "acceptor" antibody sequences (see, e.g., Queen, US 5,530,101 and 5,585,089; Winter, US 5,225,539; Carter, US 6,407,213; Adair, US 5,859,2056,881,557; Foote, US 6,881,557). The acceptor antibody sequences can be, for example, mature human antibody sequences, composites of such sequences, consensus sequences of human antibody sequences, or germline region sequences. A humanized antibody can incorporate all six CDRs from a non-human (e.g., mouse, rat, etc.) antibody, but can also be made using fewer than all CDRs (e.g., at least three, four, or five) from the 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).

[0095] In some antibodies, only a portion of the CDRs, i.e., a subset of the CDR residues required for binding, called SDRs, are required to retain binding in humanized antibodies. CDR residues that do not contact the antigen and are not included in the SDRs can be identified based on previous studies (e.g., residues H60-H65 of CDR H2 are often not required), from regions of the Kabat CDRs outside the 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, in positions where one or more donor CDR residues are absent, or where the entire donor CDR is omitted, the amino acid occupying that position can be the amino acid occupying the corresponding position (according to Kabat numbering) in the acceptor antibody sequence. In some antibodies, potential sites of post-translational modification (e.g., glycosylation) within the CDRs can be substituted to eliminate post-translational modification. The substitution positions within the CDRs and the amino acids to be substituted can also be selected empirically.

[0096] Although the acceptor may be identical in sequence to the selected human framework sequence, whether it is derived from a human immunoglobulin or a human consensus framework, the present disclosure contemplates that the acceptor sequence may include pre-existing amino acid substitutions relative to the human immunoglobulin sequence or the human consensus framework sequence. These pre-existing substitutions may be minimal; generally, there are only four, three, two, or one amino acid differences relative to the human immunoglobulin sequence or the consensus framework sequence. The human acceptor antibody sequence may be optionally selected from among many known human antibody sequences to provide 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.

[0097] Certain amino acids from human variable region framework residues can be selected for substitution based on their potential effect on CDR conformation and / or binding to antigen. Examination of such potential effects can be done by modeling, examining the characteristics of the amino acid at a particular position, or empirically observing the effect of substitution or mutagenesis of a particular amino acid. For example, if an amino acid differs between a non-human variable region framework residue and a selected human variable region framework residue, the human framework amino acid can be replaced with the equivalent framework amino acid from the non-human antibody if that amino acid is reasonably expected to: (1) directly bind non-covalently to the antigen, (2) be adjacent to the CDR region, or (3) otherwise interact with the CDR region (e.g., within about 6 Å of the CDR region). Other candidates for substitution are acceptor human framework amino acids that are rare in human immunoglobulins at that position. These amino acids can be replaced with the amino acid at the equivalent position of the non-human donor antibody, or with the amino acid at the equivalent position of a more typical human immunoglobulin.

[0098] In some embodiments, the humanized anti-CD39 antibody comprises an H1 comprising the amino acid sequence of SEQ ID NO: 10 with 0-2 amino acid substitutions or deletions, an H2 comprising the amino acid sequence of SEQ ID NO: 11 with 0-2 amino acid substitutions or deletions, an H3 comprising the amino acid sequence of SEQ ID NO: 12 with 0-2 amino acid substitutions or deletions, and a framework region having 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% identity to an acceptor human framework region. and a mature VL comprising an L1 comprising the amino acid sequence of SEQ ID NO: 14 with 0-2 amino acid substitutions or deletions, an L2 comprising the amino acid sequence of SEQ ID NO: 15 with 0-2 amino acid substitutions or deletions, an L3 comprising the amino acid sequence of SEQ ID NO: 16 with 0-2 amino acid substitutions or deletions, and a framework region having 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% identity to an acceptor human framework region. Framework regions may be determined according to the definition of Kabat.

[0099] In some embodiments, the humanized anti-CD39 antibody comprises an H1 comprising the amino acid sequence of SEQ ID NO: 18 with 0-2 amino acid substitutions or deletions, an H2 comprising the amino acid sequence of SEQ ID NO: 19 with 0-2 amino acid substitutions or deletions, an H3 comprising the amino acid sequence of SEQ ID NO: 20 with 0-2 amino acid substitutions or deletions, and a framework region having 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% identity to an acceptor human framework region. and a mature VL comprising an L1 comprising the amino acid sequence of SEQ ID NO:22 with 0-2 amino acid substitutions or deletions, an L2 comprising the amino acid sequence of SEQ ID NO:25 with 0-2 amino acid substitutions or deletions, an L3 comprising the amino acid sequence of SEQ ID NO:26 with 0-2 amino acid substitutions or deletions, and a framework region having 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% identity to the acceptor human framework regions. For example, the humanized anti-CD39 antibody referenced above may have an L1 comprising SEQ ID NO:23 or SEQ ID NO:24. Framework regions may be determined according to the definition of Kabat. In some embodiments, the VH human acceptor is M99642 or KF698734 and / or the VL human acceptor is X12682 or Z00023.

[0100] In some embodiments, the humanized anti-CD39 antibody comprises an H1 comprising the amino acid sequence of SEQ ID NO:28 with 0-2 amino acid substitutions or deletions, an H2 comprising the amino acid sequence of SEQ ID NO:29 with 0-2 amino acid substitutions or deletions, an H3 comprising the amino acid sequence of SEQ ID NO:30 with 0-2 amino acid substitutions or deletions, and a framework region having 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% identity to an acceptor human framework region. and a mature VL comprising an L1 comprising the amino acid sequence of SEQ ID NO: 32 with 0-2 amino acid substitutions or deletions, an L2 comprising the amino acid sequence of SEQ ID NO: 33 with 0-2 amino acid substitutions or deletions, an L3 comprising the amino acid sequence of SEQ ID NO: 34 with 0-2 amino acid substitutions or deletions, and a framework region having 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% identity to an acceptor human framework region. Framework regions may 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.

[0101] In the foregoing embodiments, the mature heavy chain variable region can be linked to at least a portion of the heavy chain constant region and the mature light chain variable region can be linked to at least a portion of the 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 IgG1. In other embodiments, the heavy chain constant region has reduced or enhanced effector function compared to wild-type human IgG1. In a specific 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 a specific example, the heavy chain constant region comprises or consists of amino acids 1-329 of SEQ ID NO:1 and the light chain constant region comprises or consists of SEQ ID NO:6. In another specific 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 specific 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 specific 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 specific 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 specific 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 specific 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 specific 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 specific 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.

[0102] E. Human antibodies As described herein, the present disclosure provides a method for the preparation of 19, ch19_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, and optionally the human antibody has an IC 50and inhibit human CD39 enzymatic activity with a CpG value 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. The human antibodies have the same epitope specificity as certain rodent antibodies, e.g., antibodies designated herein as 19, 31, or 39, or have the same epitope specificity as certain humanized antibodies, e.g., ch19_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, ch40_IGG4.P, ch41_IGG4.P, ch42_IGG4.P, ch43_IGG4.P, ch44_IGG4.P, ch45_IGG4.P, ch46_IGG4.P, ch47_IGG4.P, ch48_IGG4.P, ch49 ... Human antibodies can be selected by competitive binding experiments, the phage display method of Winter, WO 92 / 20791, or other methods to have the same epitope specificity as an antibody designated h39_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. Human antibodies can also be screened for a particular epitope specificity by using only a fragment of CD39 as the target antigen and / or by screening antibodies against a collection of deletion mutants of CD39.

[0103] Methods for producing human antibodies include the trioma method of Oestberg et al., Hybridoma 2:361-367 (1983); Oestberg, U.S. Pat. No. 4,634,664; and Engleman et al., U.S. Pat. No. 4,634,666, the use of transgenic mice containing human immunoglobulin genes (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 149, 1636-1638 (1995), Nature 15, 1636-1638 (1996), Nature 15, 1636-1638 (1997), Nature 15, 1636-1638 (1998), Nature 15, 1636-1638 (1999), Nature 15, 1636-1638 (20 ... Biotechnology 14, 826 (1996), Kucherlapati, WO 91 / 10741 (1991) and phage display methods (see, e.g., Dower et al., WO91 / 17271 and McCafferty et al., WO92 / 01047, US5,877,218, US5,871,907, US5,858,657, US5,837,242, US5,733,743 and US5,565,332).

[0104] A human antibody of the present disclosure may 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 IgG1. In other embodiments, the heavy chain constant region has reduced or enhanced effector function compared to wild-type human IgG1. In a specific 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 specific 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 specific 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 specific 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 specific 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. F. Selection of Constant Region(s)

[0105] The heavy and light chain variable regions of a chimeric, humanized (including veneered), or human antibody may each be linked to at least a portion of a human constant region. In some embodiments, the heavy chain variable domain described in the section above is linked to a portion of a human heavy chain constant region and the light chain variable domain described in the section above is linked to a portion of a human light chain constant region. In some embodiments, the heavy chain variable domain described in the section above is linked to a portion of a human heavy chain constant region and the light chain variable domain described in the section above is linked to a full-length human light chain constant region. In some embodiments, the heavy chain variable domain described in the section above is linked to a full-length human heavy chain constant region and the light chain variable domain described in the section above is linked to a full-length human light chain constant region.

[0106] The choice of constant region (or its truncation) depends in part on whether effector functions are desired or need to be further enhanced. "Effector function" refers to the biological activity attributable to the light or heavy chain constant region of an antibody and varies depending on the antibody isotype. Non-limiting examples of antibody effector functions include C1q binding to the C1 complex and complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation 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) according to their heavy chains, each of which serves a different function. IgG consists of four human subclasses (IgG1, IgG2, IgG3, and IgG4), each of which contains a different heavy chain. They are highly homologous and differ mainly in the hinge region and the extent to which they activate the host immune system. For example, human isotypes IgG1 and IgG3 can mediate complement-mediated cytotoxicity, whereas human isotypes IgG2 and IgG4 do not, or do so at very low levels. The light chain constant region can be of the subclass lambda or kappa.

[0107] The antibodies of the present disclosure that comprise a human constant region or a portion thereof are typically IgG antibodies, preferably IgG1 or IgG4 antibodies. Human constant regions exhibit allotypic and isoallotypic variation among different individuals, i.e., the constant region may differ at one or more polymorphic positions in different individuals. Isoallotypes differ from allotypes in that sera that recognize an isoallotype bind to non-polymorphic regions of one or more other isotypes. Reference to a human constant region includes constant regions that include any naturally occurring allotype or any permutation of residues that occupy polymorphic positions in naturally occurring allotypes.

[0108] One or several amino acids at the amino or carboxy termini of the light and / or heavy chains, e.g., the lysine at the C-terminus of the heavy chain, may be missing or derivatized in part or in whole of the molecule. The N-terminal glutamine of the heavy or light chain may be substituted with a glutamic acid residue to prevent the formation of pyroglutamic acid.

[0109] In some embodiments, the antibody of the present disclosure is an IgG4 antibody. For human IgG4, the incorporation of S228P (Eu numbering) engineered mutation in the heavy chain can be used to prevent Fab arm exchange. Suitable sequences 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 IgG4 antibodies is absent.

[0110] In some embodiments, the antibody of the present disclosure is an IgG1 or IgG3 antibody. Suitable sequences for human IgG1 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 the human IgG3 disclosed in U.S. Patent No. 5,624,821. In some embodiments, the C-terminal lysine of an IgG1 or IgG3 antibody is absent.

[0111] In certain embodiments, substitutions can be made in the constant region to increase 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) to increase the half-life of the antibody.

[0112] Alternatively, or additionally, substitutions can be made in the constant region to reduce or enhance effector functions such as complement-mediated cytotoxicity (CDC) or antibody-dependent cell-mediated cytotoxicity (ADCC) (see, e.g., Winter et al., U.S. Pat. No. 5,624,821; Tso et al., U.S. Pat. No. 5,834,597; and Lazar et al., Proc. Natl. Acad. Sci. USA, 103:4005, 2006).

[0113] Some antibodies of the present disclosure are engineered to have reduced Fc effector functions, such as CDC, ADCC, and antibody-dependent cellular phagocytosis (ADCP), by introducing constant region mutation(s) compared to the same antibody without the mutation(s). In some embodiments, each or all of these effector functions are reduced by at least 50%, 75%, 90%, or 95% compared to the antibody without the mutation. Effector functions can be assayed as described in the Examples. Other assays are described in Shields et al, 2001 J. Biol. Chem., Vol. 276, p 6591-6604;Chappel et al, 1993 J. Biol. Chem., Vol 268, p 25124-25131;Lazar et al, 2006 PNAS, 103;4005-4010.

[0114] Substitutions at any or all of positions 234, 235, 236, and / or 237 reduce affinity for Fcγ receptors, particularly FcγRI receptors (see, e.g., US 6,624,821). In some embodiments, alanine residues are used for substitutions such as the L234A / L235A double mutation to reduce effector function. Other combinations of mutations that reduce effector function include L234A / L235A / G237A, E233P / L234V / L235A / ΔG236, A327G / A330S / P331S, K322A, L234A / L235A, L234F / L235E / P331S, and L234A / L235E / G237A / A330S / P331S (Eu numbering). Optionally, positions 234, 236, and / or 237 of 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 C1q 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)). Substitutions at human IgG1 or IgG2 residues 233-236 and IgG4 residues 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). Mutations N297A, N297Q, or N297G (Eu numbering) reduce glycosylation and thereby effector function.

[0115] Some antibodies of the present disclosure are engineered to have enhanced Fc effector function by introducing constant region mutation(s). For example, FcγR binding can be enhanced by amino acid engineering. In some embodiments, this can be done by substitution of one or more amino acids in the Fc region. Desired mutations can be determined, for example, by either alanine scanning or rational design and library screening. These techniques can be used to identify IgG variants with enhanced binding to FcγR and enhanced effector function. Alternatively, some mutations to the Fc receptor region are known in the art and described, for example, in Smith P.et al (2012)PNAS 6181-6186.

[0116] In some embodiments, the antibodies described herein comprise a modified IgG1 constant domain that enhances the ability of the antibody to mediate ADCC compared to a wild-type IgG1 lacking the modification. The modified IgG1 domain 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 IgG1 domain is characterized by substitutions at S239D, A330L, and I332E (Eu numbering).

[0117] Alternatively, glycoform perturbation can be used to enhance Fc-mediated therapeutic antibody function. N-linked Fc glycosylation of IgG1 antibodies is important for effector function. Sialylation, galactosylation, bisecting sugar, and fucosylation can all affect the binding and activity of IgG molecules. Control of the glycosylation pattern of therapeutic antibodies can be achieved in several different ways. The type of cell producing the recombinant antibody and its culture conditions can affect the glycosylation and activity of the therapeutic antibody. In addition, bioreactor conditions and downstream processing can also affect the glycan microheterogeneity. Hypofucosylated or defucosylated antibodies have been shown to enhance Fc-mediated properties. Numerous methods are known in the art to achieve this reduction in fucose levels by glycoengineering. One method is to manipulate the enzymes involved in the post-translational modification of antibodies. This can include overexpressing glucosidases such as β-1-4-N-acetylglucosaminyltransferase III, knocking out fucose transferases, or using cell lines that are naturally deficient in fucose or mutated to express low fucosylation levels. Additionally, inhibitors of N-linked glucosidases such as castanospermine can also be used to obtain low fucose-bearing IgG molecules.

[0118] In some embodiments, amino acid engineered variants may have a broader enhanced affinity for multiple FcγRs, while glycoform engineered antibodies may generally have a more specific affinity for enhanced FcγRIIIa binding. Glycoforms interact with proximal amino acids in the Fc portion, and substitution of amino acids that contact Ig oligosaccharides may result in different glycoform structures.

[0119] G. Expression of Recombinant Antibodies Chimeric, humanized (including veneered) and human antibodies are typically produced by recombinant expression. Accordingly, the disclosure also provides polynucleotides encoding the anti-CD39 antibodies of Sections IIA-F, vectors containing the polynucleotides, and host cells containing the vectors.

[0120] The polynucleotide encoding the anti-CD39 antibody of the present disclosure can be inserted into a vector for amplification, expression, or further optimization. Many vectors are available. In some embodiments, the vector system includes mammalian, bacterial, yeast systems, etc., including 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 other laboratory-prepared and commercially available vectors. Suitable vectors can include plasmids, 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, an enhancer element, a promoter (e.g., SV40, CMV, EF-1α), and a transcription termination sequence. For expression, the recombinant polynucleotide construct typically contains expression control sequences operably linked to the coding sequence of the antibody chain, including naturally associated or heterologous promoter regions. In some embodiments, the expression control sequences are eukaryotic promoter systems in vectors capable of transforming or transfecting eukaryotic host cells. Once the vector has been incorporated into an appropriate host, the host is maintained under conditions suitable for high level expression of the nucleotide sequences, and the collection and purification of the recombinant antibody.

[0121] The vectors containing the polynucleotide sequences encoding the anti-CD39 antibodies of the present disclosure can be introduced into host cells for cloning or gene expression. Suitable host cells for cloning or expressing the polynucleotide sequences in the vectors herein include prokaryotic and eukaryotic cells. Non-limiting examples of suitable prokaryotes include eubacteria, such as gram-negative or gram-positive organisms, such as Enterobacteriaceae, such as Escherichia, such as E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, such as Salmonella typhimurium, Serratia, such as Serratia marcescans, and Shigella, as well as Bacilli, such as B. subtilis and B. licheniformis, Pseudomonas, such as P. aeruginosa, and Streptomyces. In addition to prokaryotes, eukaryotic microbes such as filamentous fungi and yeast are also suitable as cloning or expression hosts for vectors encoding 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 Schwanniomyces occidentalis, and filamentous fungi, such as Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts, including A. nidulans and A. niger. Suitable host cells may also be derived from multicellular organisms. Examples of invertebrate cells include plant cells and insect cells. Numerous baculovirus strains and variants have been identified, as well as corresponding permissive insect host cells derived from hosts, including Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruit fly), and Bombyx mori. Various virus strains for transfection, such as the L-1 variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV, are publicly available, and such viruses can be used as the viruses herein according to the present invention, 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 used as 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, including CHO cell lines, various COS cell lines, HeLa cells, HEK293 cells, L cells, and non-antibody producing myelomas such as Sp2 / 0 and NS0. In some embodiments, the cells are non-human. Expression vectors for these cells can include 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 transcription termination 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).

[0122] Host cells are transformed with the above-described expression or cloning vectors for anti-CD39 antibody production and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying genes encoding the desired sequences. After expression, antibodies can be purified according to standard procedures in the art, including HPLC purification, column chromatography, gel electrophoresis, and the like (see generally, Scopes, Protein Purification (Springer-Verlag, NY, 1982)).

[0123] H. Labeled antibody The disclosed anti-CD39 antibodies can be optionally labeled with one or more detectable signals, including, but not limited to, fluorescent molecules, spin-labeled 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.

[0124] For example, if any one of the disclosed anti-CD39 antibodies is labeled with a detectable signal, such as a fluorescent molecule, a spin-labeled molecule, an enzyme, or a radioisotope, such detectable antibody can be used in pharmacodynamic assays, immunohistochemistry, receptor occupancy assays, ELISA (enzyme-linked immunosorbent assay), EIA (enzyme immunoassay), RIA (radioimmunoassay), and the like.

[0125] III.How to use The present disclosure provides a method of using an anti-CD39 antibody described herein in the preparation of a medicament for the inhibition of CD39 enzymatic activity. As used herein, the terms "inhibit", "inhibition" and the like refer to the ability of an antagonist to reduce the function or activity of a particular target, e.g., CD39. The reduction is preferably at least 50%, and may 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 an anti-CD39 antibody described herein in the preparation of a medicament for the treatment or prevention of a disease, disorder, and / or condition that would benefit from the inhibition of CD39 enzymatic activity. As an example, the disclosure encompasses the use of an anti-CD39 antibody described herein in the preparation of a medicament for the treatment of a disease characterized by (i) high extracellular ATP (relative to non-diseased tissue), (ii) expression or increased expression of ENTPD1, ​​P2RX7, P2RY11, or a combination thereof in diseased tissue, (iii) expression or increased expression of CD39, P2X7, P2Y11, or a combination thereof in diseased tissue, (iv) bone marrow infiltration into diseased tissue, or any combination of (i)-(iv). In another example, the disclosure encompasses the use of an anti-CD39 antibody described herein in the preparation of a medicament for the treatment of cancer. Optionally, the cancer can be characterized by (i) high extracellular ATP (compared to non-diseased samples), (ii) expression or increased expression of ENTPD1, ​​P2RX7, P2RY11, or a combination thereof in diseased tissue, (iii) expression or increased expression of CD39, P2X7, P2Y11, or a combination thereof in diseased tissue, (iv) bone marrow infiltration into diseased tissue, or any combination of (i)-(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 described elsewhere herein.

[0126] Extracellular ATP is present at negligible concentrations (e.g., about 10-100 nM) in healthy conditions, but can be rapidly increased in response to tissue injury, stress, hypoxia, and drugs used to treat cancer, and can be found at 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 inflammatory signals important for effective innate and adaptive immune responses. Conversely, immune responses are limited when extracellular ATP is hydrolyzed to adenosine, for example in the tumor microenvironment. CD39 is the rate-limiting ectoenzyme in the hydrolysis of extracellular ATP. By its degradative action of converting extracellular ATP to AMP, CD39 also increases extracellular adenosine production via CD73 (ecto-5'-nucleotidase), a rate-limiting ectoenzyme in extracellular AMP hydrolysis. Adenosine signals through type 1 purinergic receptors, with effects opposite to those mediated by the ATP receptor. In particular, A expressed on the surface of immune cells is involved in the production of adenosine. 2a and A 2b Receptors help mediate the immunosuppressive effects of adenosine. For example, in the tumor microenvironment, adenosine binds to Aβ on tumor-infiltrating immune cells. 2a Receptor and A 2bIt induces immunosuppressive effects through direct interaction with receptors. The opposing roles 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, the use of anti-CD39 antibodies of the present disclosure potently inhibits CD39 enzyme activity, resulting in immune stimulation through ATP accumulation and preventing the formation of immunosuppressive adenosine. Diseases, disorders, and / or conditions that would benefit from inhibition of CD39 enzyme activity may include, for example, those in which the release of extracellular ATP is high due to tissue injury, stress, hypoxia treatment with additional therapy, or any combination thereof. Additional diseases, disorders and / or conditions that would benefit from inhibition of CD39 enzyme activity include those in which extracellular adenosine levels are high in diseased tissue samples, e.g., due to increased hydrolysis of ATP compared to healthy controls and / or due to increased hydrolysis of AMP to adenosine compared to healthy controls, as measured, e.g., by increased CD39 enzyme activity in peripheral blood or tissue (e.g., tumor) samples, and / or those in which expression of CD39 and / or CD73 is detectable in peripheral blood or tissue (e.g., tumor) samples, optionally higher compared to healthy controls, as measured, e.g., by immunohistochemistry, immunophenotyping, RNA sequencing, or other clinically validated methods.

[0127] Thus, in some embodiments, the anti-CD39 antibodies described herein are administered to a subject in need of inhibition of CD39 enzymatic activity in an amount effective to inhibit CD39 enzymatic activity. CD39 enzymatic activity can be assessed using peripheral blood samples or tissue (e.g., tumor) samples, or both, obtained from the subject. As a non-limiting example, commercially available assays such as Kinase-Glo® or AMP-Glo® can be used to measure CD39 activity in the cellular and non-cellular compartments of peripheral blood samples or tumor samples. Alternatively or additionally, CD39 enzymatic activity in tumor tissue can be measured by enzyme histochemistry. Inhibition can be determined, for example, by comparison with a prior sample obtained from the subject (i.e., before administration of the anti-CD39 antibody) or by comparison with a reference sample or reference value from a control group (e.g., subjects administered an isotype control antibody, an anti-CD39 antibody that binds to CD39 but does not inhibit CD39 enzymatic activity, a standard of care, a placebo, etc.). In some embodiments, the anti-CD39 antibodies described herein are administered to a subject in need of inhibition of CD39 enzymatic activity in the tumor microenvironment in an amount effective to inhibit CD39 enzymatic activity in the tumor microenvironment. In some embodiments, the anti-CD39 antibodies described herein are administered to a subject in need of inhibition of CD39 enzymatic activity in the peri-tumor area in an amount effective to inhibit CD39 enzymatic activity in the peri-tumor area. In some embodiments, the anti-CD39 antibodies described herein are administered to a subject in need of inhibition of CD39 enzymatic activity in or on immune cells in an amount effective to inhibit CD39 enzymatic activity in or on immune cells. In some embodiments, the anti-CD39 antibodies described herein are administered to a subject in need of inhibition of CD39 enzymatic activity in or on myeloid cells in an amount effective to inhibit CD39 enzymatic activity in or on myeloid cells. Myeloid cells expressing CD39 include those detailed in the Examples.In some embodiments, the anti-CD39 antibodies described herein are administered to a subject in need of inhibition of interstitial and / or intravascular or interstitial and / or vascular CD39 enzymatic activity in an amount effective to inhibit interstitial and / or intravascular or interstitial and / or vascular CD39 enzymatic activity.

[0128] Alternatively or additionally, in some embodiments, the anti-CD39 antibodies described herein are administered to a subject in need thereof in an amount effective to enhance ATP-mediated immune stimulation compared to a suitable control (e.g., a subject administered an isotype control antibody, a subject administered an anti-CD39 antibody that binds to CD39 but does not inhibit CD39 enzymatic activity, a subject receiving standard therapy, a subject not receiving therapy). ATP-mediated immune stimulation can be assessed using peripheral blood samples and / or tissue (e.g., tumor) samples obtained from the subject. ATP-mediated immune stimulation can be identified, for example, by (i) measuring an increase in ATP-dependent signaling through one or more type 2 purinergic (P2) receptors, such as P2YG 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 activity and / or proliferation of CD4+ and / or CD8+ T cells, and / or (v) measuring an increase in activation, maturation, cytokine secretion, or a combination thereof, in one or more cell types of myeloid cells. In one example, the anti-CD39 antibody described herein is administered to a subject in need thereof (e.g., a subject administered an isotype control antibody, a subject administered an anti-CD39 antibody that binds to CD39 but does not inhibit CD39 enzymatic activity, a subject receiving standard therapy, a subject not receiving therapy) in an amount effective to increase ATP-dependent signaling through one or more P2X or P2Y receptors selected from P2X4, P2X5, P2X7, P2Y2, or P2Y11, as compared to a suitable control. In one example, the anti-CD39 antibody described herein is administered to a subject in need thereof (e.g., a subject administered an isotype control antibody, a subject administered an anti-CD39 antibody that binds to CD39 but does not inhibit CD39 enzymatic activity, a subject receiving standard therapy, a subject not receiving therapy) in an amount effective to increase ATP-dependent signaling through P2X7, as compared to a suitable control.In one example, the anti-CD39 antibody described herein is administered to a subject in need thereof (e.g., a subject administered an isotype control antibody, a subject administered an anti-CD39 antibody that binds to CD39 but does not inhibit CD39 enzymatic activity, a subject receiving standard therapy, a subject not receiving therapy) in an amount effective to increase ATP-dependent signaling through P2Y11 compared to a suitable control. In one example, the anti-CD39 antibody described herein is administered to a subject in need thereof (e.g., a subject administered an isotype control antibody, a subject administered an anti-CD39 antibody that binds to CD39 but does not inhibit CD39 enzymatic activity, a subject receiving standard therapy, a subject not receiving therapy) in an amount effective to increase ATP-dependent signaling through P2X7 and / or P2Y11 compared to a suitable control. In one example, the anti-CD39 antibody described herein is administered to a subject in need thereof in an amount effective to increase activation of the NLRP3 inflammasome. Metrics for 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. Metrics for myeloid cell activation, maturation, cytokine secretion are known in the art and include those detailed in the Examples.

[0129] Alternatively, or in addition, 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 relative to a suitable control (e.g., a subject administered an isotype control antibody, a subject administered an anti-CD39 antibody that binds to CD39 but does not inhibit CD39 enzymatic activity, a subject receiving standard of care, a subject not receiving care). Adenosine-mediated immunosuppression can be, for example, 2a R and / or A 2bAdenosine-mediated immunosuppression can be identified by measuring the increase in adenosine signaling through R. Adenosine-mediated immunosuppression also includes adenosine-mediated inhibition of lymphoid (e.g., T cells, B cells) and / or myeloid (e.g., monocytes, macrophages, dendritic cells, NK cells) cell activity. For example, one metric of adenosine-mediated immunosuppression can be NECA-induced pCREB activation in CD8+ T cells in human blood.

[0130] Alternatively or in addition to the above, in some embodiments, the anti-CD39 antibodies described herein are 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) binding as measured by a receptor occupancy assay. In some embodiments, the anti-CD39 antibodies described herein are administered to a subject in need thereof in an amount effective to achieve 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) binding as measured by a receptor occupancy assay. Exemplary receptor occupancy assays are detailed in the Examples.

[0131] Alternatively, or in addition, 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 methods further detailed in the Examples.

[0132] Alternatively, or in addition, in some embodiments, the anti-CD39 antibodies described herein are 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 may result in tumor death and improve outcome.

[0133] A. Oncology and Oncology-Related Disorders In one or more embodiments, the antibodies described herein are useful for the treatment and / or prevention of cancer (e.g., carcinoma, sarcoma, leukemia, lymphoma, myeloma, etc.). In certain embodiments, the cancer may progress locally and / or be unresectable, metastatic, or at risk of becoming metastatic. Alternatively or additionally, the cancer may recur or become unresponsive to standard therapy or treatment, such as a checkpoint inhibitor. Examples of types of cancer contemplated by the present disclosure include cancer of the genitourinary tract (e.g., bladder, kidney, renal cell, penis, prostate, testis, von Hippel-Lindau disease, etc.), cancer of the uterus, cancer of the cervix, cancer of the ovary, cancer of the breast, cancer of the gastrointestinal tract (e.g., esophagus, oropharynx, stomach, small or large intestine, colon, or rectum), cancer of bone, bone marrow, skin (e.g., melanoma), cancer of the head and neck, cancer of the liver, cancer of the gallbladder, cancer of the bile duct, cancer of the heart, cancer of the lung, cancer of the pancreas, cancer of the salivary glands, cancer of the adrenal gland, cancer of the thyroid gland, cancer of the brain (e.g., glioma), cancer of the ganglia, cancer of the central nervous system (CNS), cancer of the peripheral nervous system (PNS), cancer of the hematopoietic system (i.e., hematologic malignancies), and cancer of the immune system (e.g., spleen or thymus).

[0134] In some embodiments, the antibodies according to the present disclosure are useful for the treatment and / or prevention of hematological malignancies. Examples of cancer types that affect the hematopoietic system include leukemias, lymphomas, and myelomas, including acute myeloid leukemia, adult T-cell leukemia, T-cell large granular lymphocytic leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, acute monocytic leukemia, Hodgkin's and non-Hodgkin's lymphomas, diffuse large B-cell lymphoma, and multiple myeloma.

[0135] In another embodiment, the antibodies according to the present disclosure are useful for the treatment and / or prevention of solid tumors, which may be, for example, ovarian cancer, endometrial cancer, breast cancer, lung cancer (small cell or non-small cell), colon cancer, prostate cancer, cervical cancer, biliary tract cancer, pancreatic cancer, gastric cancer, esophageal cancer, liver cancer (hepatocellular carcinoma), kidney cancer (renal cell carcinoma), head and neck tumors, mesothelioma, melanoma, sarcoma, central nervous system (CNS) hemangioblastoma, and brain tumors (e.g., gliomas, including astrocytoma, oligodendroglioma, and glioblastoma).

[0136] In another embodiment, the antibodies according to the present disclosure are useful for the treatment and / or prevention of lung cancer, genitourinary 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, genitourinary cancer, gastrointestinal cancer, skin cancer, or a combination thereof.

[0137] In some embodiments, antibodies according to the present disclosure are useful for the treatment and / or prevention of skin cancer, hi further embodiments, the skin cancer is melanoma.

[0138] In some embodiments, the antibodies according to the present disclosure are useful for treating and / or preventing breast cancer. In further 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 further embodiments, the breast cancer is triple negative breast cancer.

[0139] In some embodiments, the antibodies according to the present disclosure are useful for treating and / or preventing renal cancer. In further embodiments, the renal cancer is renal cell carcinoma. In yet further embodiments, the renal cell carcinoma is clear cell renal cell carcinoma.

[0140] In some embodiments, the antibodies according to the present disclosure are useful for treating and / or preventing lung cancer. In further embodiments, the lung cancer is non-small cell lung cancer (NSCLC). In yet further embodiments, the NSCLC is lung squamous cell carcinoma or lung adenocarcinoma.

[0141] In some embodiments, the disclosed methods of treating non-small cell lung cancer may further comprise administering any of the disclosed anti-CD39 antibodies in combination with pemetrexed, carboplatin, and any of the antagonistic anti-PD-1 or antagonistic anti-PD-L1 antibodies.Such methods may further comprise administering one or more additional agents selected from the group consisting of an antagonistic 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 domvanalimab, AB308, etormadenant, and quemliclustat.

[0142] In some embodiments, antibodies according to the present disclosure are useful for the treatment and / or prevention of pancreatic cancer, hi further embodiments, the pancreatic cancer is pancreatic neuroendocrine tumor or pancreatic adenocarcinoma.

[0143] In some embodiments, antibodies according to the present disclosure are useful for the treatment and / or prevention of neuroendocrine tumors, hi further embodiments, the neuroendocrine tumor is a pancreatic neuroendocrine tumor, a pheochromocytoma, a paraganglioma, or a tumor of the adrenal gland.

[0144] In some embodiments, the compounds according to the present disclosure are useful for treating and / or preventing brain tumors. In further embodiments, the brain tumor is a glioma. In yet further embodiments, the glioma is an astrocytoma, an oligodendroglioma, or a glioblastoma.

[0145] In some embodiments, the antibodies according to the present disclosure are useful for the treatment and / or prevention of upper GI cancer, such as esophageal or gastric cancer. In further embodiments, the upper GI cancer is adenocarcinoma, squamous cell carcinoma, or any combination thereof. In further embodiments, the upper GI cancer is esophageal adenocarcinoma (EAC), esophageal squamous cell carcinoma (ESCC), esophagogastric junction adenocarcinoma (GEJ), gastric adenocarcinoma (also referred to herein as "gastric cancer"), or any combination thereof.

[0146] In some embodiments, the disclosed methods of treating gastric or gastroesophageal cancer may further comprise administering any of the disclosed anti-CD39 antibodies in combination with FOLFOX and any of the antagonistic anti-PD-1 or antagonistic anti-PD-L1 antibodies.Such methods may further comprise administering one or more additional agents selected from the group consisting of an antagonistic 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 domvanalimab, AB308, etremadenant, and quemliclustat.

[0147] In some embodiments, antibodies according to the present disclosure are useful for the treatment and / or prevention of lymphoma, hi further embodiments, the hematological malignancy is acute myeloid lymphoma.

[0148] In some embodiments, antibodies according to the present disclosure are useful for treating breast cancer, gastrointestinal cancer, urogenital cancer, lung cancer, lymphoma, or ovarian cancer, hi further embodiments, antibodies according to the present disclosure are useful for treating 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.

[0149] In some embodiments, antibodies according to the present disclosure are useful for treating lung cancer, head and neck cancer, thyroid cancer, pancreatic cancer, renal cancer, or skin cancer, hi yet further embodiments, antibodies according to the present disclosure are useful for treating non-small cell lung cancer, head and neck squamous cell carcinoma, pancreatic ductal adenocarcinoma, clear cell renal carcinoma, melanoma.

[0150] In some embodiments, antibodies according to the present disclosure are useful for treating lower GI cancer, upper GI cancer, head and neck cancer, renal cancer, lung cancer, or pancreatic cancer. In further embodiments, antibodies according to the present disclosure are useful for treating esophageal cancer, head and neck squamous cell carcinoma, kidney renal clear cell carcinoma, lung adenocarcinoma, lung squamous cell carcinoma, pancreatic adenocarcinoma, gastric (stomach) adenocarcinoma.

[0151] In all described embodiments, the methods of the disclosure can be performed in an adjuvant setting or a neoadjuvant setting. The methods described herein may be indicated as first line, second line, third line, or higher line treatment. In some embodiments, the methods of the disclosure can be performed in a second or higher line treatment, where the previous line treatment included a checkpoint inhibitor (i.e., the subject is checkpoint inhibitor (CPI)-experienced). In further 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.

[0152] The present disclosure also provides methods for treating or preventing other cancer-related diseases, disorders, or conditions. The use of the term(s) cancer-related diseases, disorders, and conditions is meant to broadly refer to conditions directly or indirectly related to cancer and non-cancerous proliferative diseases, including, for example, pre-cancerous conditions such as angiogenesis, dysplasia, and non-cancerous proliferative diseases, disorders, or conditions such as benign proliferative breast disease and papilloma. For clarity, the term(s) cancer-related diseases, disorders, and conditions does not include cancer itself.

[0153] Generally, methods of the disclosure 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. Administration of the anti-CD39 antibody of Section II can include one or more (e.g., one, two, or three or more) dosing cycles. In some embodiments, the disclosure provides methods of treating or preventing cancer, or a cancer-related disease, disorder, or condition, using an anti-CD39 antibody of Section II and at least one additional therapy, examples of which are described elsewhere herein.

[0154] Patient Selection. In some cases, the methods according to the present disclosure can be provided to selected patients, for example, patients identified as having detectable PD-L1, CD73, and / or CD39 expression, high microsatellite instability, high tumor mutation burden, or any combination thereof. In some cases, the patient is identified as having a cancer gene-driven cancer with a mutation in at least one gene associated with the cancer. In some cases, the patient is selected by determining the P2X7 variant of the patient. Commonly 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 result in partial or complete loss of function of P2X7, while other modifications have been shown to increase the function of P2X7. See, e.g., Lara et al., Front Pharmacol, 2020, 11:793.

[0155] In some embodiments, patients are selected by measuring expression of 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) in a peripheral blood sample or a relevant sample, such as 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, patients are selected by measuring expression of PD-L1, CD73, and / or CD39 (at the nucleic acid or protein level) in a relevant sample, such as 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, patients can be selected by measuring, for example, CD39 enzyme activity in a peripheral blood sample or a tumor sample. In one embodiment, the disclosure provides a method of treating cancer in a patient with (i) detectable PD-L1, P2X7, P2Y11, IL-2, CXCL1, CXCL2, CXCL3, CXCL8, COX2, CD73, and / or CD39 expression, e.g., 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., by immunohistochemistry, immunophenotyping, or other clinically validated tests; (iii) detectable CD39 enzyme activity; or (iv) any combination of (i)-(iii), by administering an anti-CD39 antibody described herein.In another embodiment, the disclosure provides a method of treating cancer in a patient with (i) detectable PD-L1, P2X7, P2Y11, IL-2, CXCL1, CXCL2, CXCL3, CXCL8, COX2, CD73, and / or CD39 expression, e.g., by immunohistochemistry, immunophenotyping, or other clinically validated tests; (ii) elevated PD-L1, CD73, and / or CD39 expression, e.g., by immunohistochemistry, immunophenotyping, or other clinically validated tests; (iii) detectable CD39 enzyme activity; or (iv) any combination of (i)-(iii), by administering a therapeutically effective amount of an anti-CD39 antibody described herein. In another embodiment, the disclosure provides a method of administering a therapeutically effective amount of an anti-CD39 antibody described herein to an individual for the treatment of cancer, comprising assaying based on determination of PD-L1, P2X7, P2Y11, IL-2, CXCL1, CXCL2, CXCL3, CXCL8, COX2, CD73, and / or CD39 expression. In yet another embodiment, the disclosure provides a method of administering a therapeutically effective amount of an anti-CD39 antibody described herein to an individual for the treatment of 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 a therapeutically effective amount of the antibody to an individual comprising 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 in the sample.

[0156] B. Route of Administration In some embodiments, pharmaceutical compositions containing an antibody according to the present disclosure may be in a form suitable for oral administration. Oral administration involves swallowing the formulation, thereby allowing the antibody to be absorbed into the bloodstream from the gastrointestinal tract. Alternatively, oral administration involves buccal, lingual, or sublingual administration, thereby allowing the antibody to be absorbed through the oral mucosa into the bloodstream.

[0157] In another embodiment, pharmaceutical compositions containing antibodies according to the present disclosure may 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, intraventricular, and subcutaneous. Pharmaceutical compositions suitable for parenteral administration can be formulated using suitable aqueous or non-aqueous carriers. Depot injections, generally administered subcutaneously or intramuscularly, can also be utilized to release the antibodies disclosed herein over a defined period of time.

[0158] Other routes of administration are also contemplated by the present disclosure, including, but not limited to, nasal, vaginal, ocular, rectal, topical (eg, transdermal), and inhalation.

[0159] Certain embodiments of the present disclosure contemplate oral or parenteral administration.

[0160] C. Pharmaceutical Compositions The anti-CD39 antibody of the present disclosure may be in the form of a composition suitable for administration to a subject. In general, such a composition is a pharmaceutical composition comprising an anti-CD39 antibody according to the present disclosure and one or more pharma- ceutically acceptable excipients. In certain embodiments, the anti-CD39 antibody may be present in an effective amount. The pharmaceutical composition may 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 may be administered to a subject to practice the therapeutic and prophylactic methods and uses described herein.

[0161] The pharmaceutical compositions of the present disclosure can be formulated to be compatible with the intended method or route of administration. The routes of administration can include those known in the art. Exemplary routes of administration are oral and parenteral. Furthermore, the pharmaceutical compositions can be used in combination with one or more other therapeutic agents described herein to treat or prevent diseases, disorders and conditions contemplated by the present disclosure. In one embodiment, one or more other additional therapeutic agents contemplated by the present disclosure are included in the same pharmaceutical composition comprising an anti-CD39 antibody according to the present disclosure. In another embodiment, the one or more other therapeutic agents are in a separate composition from the pharmaceutical composition comprising an anti-CD39 antibody according to the present disclosure.

[0162] In one aspect, the anti-CD39 antibodies described herein can be administered orally. Oral administration can be performed, for example, via capsules or tablets. In preparing pharmaceutical compositions comprising 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, starch, gum acacia, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, polyethylene glycol, cellulose, sterile water, syrup, and methylcellulose. Additional pharmaceutically acceptable excipients include lubricants such as talc, magnesium stearate, and mineral oil; wetting agents; emulsifying and suspending agents; preserving agents such as methyl and propylhydroxybenzoates. Some oral dosage forms include taste masking agents, sweeteners, or flavoring agents. Oral dosage forms can be formulated as solutions or suspensions.

[0163] 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 for injection or reconstituted for injection in a suitable system, such as a physiological solution. Such a solution can contain sterile water for injection, salts, buffers, and isotonic excipients in an appropriate amount to achieve isotonicity at the appropriate physiological level.

[0164] The pharmaceutical compositions described herein may be stored in one or more suitable sterile containers. In some embodiments, the containers are designed to maintain the stability of the pharmaceutical composition for a given period of time.

[0165] D. Medication In general, the disclosed methods include administering an effective amount of an anti-CD39 antibody, or composition thereof, described herein to a subject in need thereof. An "effective amount" with respect to an anti-CD39 antibody of the present disclosure means an amount of antibody sufficient to bind to the target (e.g., bind to CD39 and inhibit CD39 enzymatic activity) at a level indicative of the efficacy of the antibody. For CD39, target binding can be determined by one or more biochemical or cellular assays, and these assays can provide an EC value that can be used as one assessment of the efficacy of the antibody. 50 , E.D. 50 , E.C. 90 ,I C 50 or a similar value is obtained. Assays for determining target binding include, but are not limited to, those described in the Examples. An effective amount can be administered as a single dose or as multiple smaller doses (e.g., as one tablet containing an "x" amount, as two tablets each containing an "x / 2" amount, etc.).

[0166] In some embodiments, the disclosed methods include administering a therapeutically effective amount of an anti-CD39 antibody described herein to a subject in need thereof. As used herein, the phrase "therapeutically effective amount" with respect to an anti-CD39 antibody refers to a dosing regimen (i.e., amount and interval) of the antibody that results in the specific pharmacological effect of administering the antibody to a subject in need of such treatment. In the case of prophylactic use, a therapeutically effective amount may be effective to eliminate or reduce the risk, reduce the severity, or delay the onset of a disease, including biochemical, histological, and / or behavioral signs or symptoms of the disease. In the case of treatment, a therapeutically effective amount may be effective to reduce, ameliorate, or eliminate one or more signs or symptoms associated with the disease, slow the progression of the disease, extend survival, reduce the dose of other pharmaceutical agent(s) required to treat the disease, or a combination thereof. With particular reference to cancer, a therapeutically effective amount may result, for example, in the death of cancer cells, a reduction in the number of cancer cells, a reduction in tumor burden, elimination of tumors or metastases, or a reduction in metastatic spread. A therapeutically effective amount of an anti-CD39 antibody need not necessarily be effective for treating every individual subject to be considered a therapeutically effective amount by one of skill in the art. A therapeutically effective amount may vary based on, for example, one or more of the subject's age and weight, the subject's overall health, the stage of the subject's disease, the route of administration, and previous or concurrent treatments.

[0167] In certain embodiments, anti-CD39 antibodies contemplated by the present disclosure can be administered (e.g., orally, parenterally, etc.) at about 0.01 mg / kg to about 50 mg / kg, or about 1 mg / kg to about 25 mg / kg of subject body weight per day, one or more times per day, week, or month to achieve the desired effect. In some embodiments, a suitable weight-based dose of an anti-CD39 antibody contemplated by the present disclosure is used to determine a dose to be administered independent of the subject's body weight (i.e., a fixed dose). In certain embodiments, the anti-CD39 antibodies of the disclosure can be administered (e.g., orally, parenterally, etc.) at fixed dosage levels of about 1 mg to about 1000 mg, particularly 1, 3, 5, 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 300, 400, 500, 600, 750, 800, 900, or 1000 mg, one or more times daily, weekly, or monthly to achieve the desired effect. In some embodiments, the anti-CD39 antibodies of the disclosure are administered at a dose of about 3 mg to about 3000 mg, particularly 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, 3000 mg, 3500 mg, 3600 mg, 3700 mg, 3800 mg, 3900 mg, 4000 mg, 4000 mg, 4500 mg, 5000 mg, 5000 mg, 6000 mg, 7500 mg, 8000 mg, 9000 mg, 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, 3000 mg, 3500 mg, 3600 mg, 3700 mg, 3800 mg, 3900 mg, 4000 mg, 4000 mg, 4000 mg, The compound may be administered (e.g., orally, parenterally, etc.) at fixed dosage levels of 00 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. The term "about" as used herein in relation to dosage means the stated numerical value, as well as plus or minus 10% of that numerical value. For example, "about 10" should be understood as both "10" and "9 to 11."

[0168] In certain embodiments, the anti-CD39 antibodies of the present disclosure are contained in a "unit dosage form." The phrase "unit dosage form" refers to physically discrete units, each unit containing a predetermined amount of an anti-CD39 antibody, alone or in combination with one or more additional agents, sufficient to produce a desired effect. It will be understood that the parameters of a unit dosage form will depend on the particular agent and the effect to be achieved.

[0169] IV. Combinations with Disclosed Anti-CD39 Antibodies The present disclosure contemplates the use of the anti-CD39 antibodies of Section II alone or in combination with one or more additional therapies. Each additional therapy may be a therapeutic agent or another therapeutic modality. In embodiments including one or more additional therapeutic agents, each agent may target a different but complementary mechanism of action. The additional therapeutic agent may be a small chemical molecule; a macromolecule such as a protein, an antibody, a peptibody, a peptide, DNA, RNA, or a fragment of such a macromolecule; or a cellular or genetic therapeutic agent. Non-limiting examples of additional therapeutic modalities include surgical resection of the tumor, bone marrow transplantation, radiation therapy, and photodynamic therapy. The anti-CD39 antibodies of Section II, when used in combination with one or more additional therapeutic agents, may have a synergistic or additive therapeutic or prophylactic effect on the underlying disease, disorder, or condition. Additionally or alternatively, the combination therapy may allow for a reduction in the dose of one or more therapeutic agents, thereby improving, reducing, or eliminating adverse effects associated with one or more agents.

[0170] In embodiments that include one or more additional therapeutic modalities, the anti-CD39 antibody of Section II can be administered before, after, or during treatment with the additional therapeutic modality. In embodiments that include 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. When administered separately, each therapeutic agent in the combination can be given at or near the same time, or at different times. Furthermore, therapeutic agents can be "administered in combination" even in different dosage forms (e.g., oral capsules and intravenous), given at different dosing intervals, one therapeutic agent is given in a fixed dosing regimen while another is titrated, tapered, or discontinued, or each therapeutic agent in the combination is titrated, tapered, dose-increased or reduced, or discontinued and / or resumed independently during the course of treatment of the patient. When the combination is formulated as separate compositions, in some embodiments, the separate compositions are provided together in a kit.

[0171] Cancer Therapy The present disclosure contemplates the use of the anti-CD39 antibodies of Section II in combination with one or more additional therapies useful in the treatment of cancer or cancer-related diseases, disorders, or conditions. In some embodiments, one or more of the additional therapies are additional therapeutic modalities. Exemplary therapeutic modalities include, but are not limited to, surgical resection of the tumor, bone marrow transplantation, radiation therapy, and photodynamic therapy. In some embodiments, the one or more additional therapeutic agents are targeted therapeutic agents. Exemplary therapeutic agents include chemotherapeutic agents, radiopharmaceuticals, hormonal therapeutic agents, epigenetic modulating agents, ATP-adenosine axis targeting agents, targeted therapies, signal transduction inhibitors, RAS signal transduction inhibitors, PI3K inhibitors, arginase inhibitors, HIF inhibitors, AXL inhibitors, PAK4 inhibitors, immunotherapeutic agents, cell therapy agents, gene therapy agents, immune checkpoint inhibitors, and agonists of stimulatory or costimulatory immune checkpoints.

[0172] In some embodiments, the one or more additional therapeutic agents are chemotherapeutic agents. Examples of chemotherapeutic agents include alkylating agents, such as thiotepa and cyclophosphamide; alkylsulfonates, such as busulfan, improsulfan, and piposulfan; aziridines, such as benzodopa, carboquone, meturedopa, and uredopa; ethyleneimines and methylamelamines, such as altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolmelamine; Trogen mustards, such as chlorambucil, chlornaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichine, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics, such as , aclacinomycins, actinomycin, ausramycin, azaserine, bleomycin, cactinomycin, calicheamicin, carabicin, caminomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin, mycophenolic acid, nogalamycin, olivomycin, pomalidomycin, Domide, peplomycin, potfilomycin, puromycin, queramycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites, e.g., methotrexate and 5-fluorouracil (5-FU); folic acid analogues, e.g., denopterin, methotrexate, pemetrexed, pteropterin, trimetrexate; purine analogues, e.g., fludarabine, 6-mercaptopurine, thiamiprine, thioguanine;Pyrimidine analogues, such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU; androgens, such as calsterone, dromostanolone propionate, epithiostanol, mepitiostane, testolactone; antiadrenal agents, such as aminoglutethimide, mitotane, trilostane; folic acid replenishers, such as folinic acid; aceglatone; aldophosphamide glycosides; aminolevulinic acid. Phosphate;Amsacrine;Bestravcil;Bisantrene;Edatraxate;Defofamine;Demecolcine;Diaziquone;Elformithine;Elliptinium acetate;Etoglucide;Gallium nitrate;Hydroxyurea;Lentinan;Lonidamine;Mitoguazone;Mitoxantrone;Mopidamol;Nitracrine;Pentostatin;Fenamet;Pirarubicin;Podophyllic acid;2-Ethylhydrazide;Procarbazine;Razoxane;Sizofiran;Spirogermanium;Tenuazonic acid;Triazicon;2, 2',2''-Trichlorotriethylamine;Urethane;Vindesine;Dacarbazine;Mannomustine;Mitobronitol;Mitolactol;Pipobroman;Gacytosine;Arabinoside (Ara-C);Cyclophosphamide;Thiotepa;Taxoids, such as paclitaxel, nab-paclitaxel, and docetaxel;Chlorambucil;Gemcitabine;6-Thioguanine;Mercaptopurine;Methotrexate;Platinum and platinum coordination complexes (collectively referred to as "platinum agents"), such as cisplatin, carboplatin, oxaliplatin;Vinbratamine, stine; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT11; proteasome inhibitors, e.g., bortezomib, carfilzomib, ixazomib; topoisomerase inhibitors, e.g., irinotecan, topotecan, etoposide, mitoxantrone, teniposide; difluoromethylornithine (DMFO); retinoic acid; esperamycin; capecitabine;These include, but are not limited to, anthracyclines and any of the above pharmacologic acceptable salts, acids or derivatives. In certain embodiments, the combination therapy comprises a chemotherapy regimen comprising one or more chemotherapy agents. In one embodiment, the combination therapy comprises a chemotherapy regimen comprising FOLFOX (folinic acid, fluorouracil, and oxaliplatin), FOLFIRI (folinic acid, fluorouracil, and irinotecan), taxane (e.g., docetaxel, paclitaxel, nab-paclitaxel, etc.), CAPOX (capecitabine and oxaliplatin), XELOX (capecitabine and oxaliplatin), platinum-based chemotherapy agent irinotecan, 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, idarubicin, 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, idarubicin, 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, idarubicin, mitoxantrone, oxaliplatin, or any combination thereof;

[0173] In some embodiments, the one or more additional therapeutic agents are radiopharmaceuticals. Radiopharmaceuticals are a form of internal radiation therapy in which a radioactive source (i.e., one or more radionuclides) is placed inside the subject's body. The radioactive source may be in solid or liquid form. Non-limiting examples of radiopharmaceuticals include sodium iodide I-131, radium-223 dichloride, lobenguane iodine-131, radioactive iodine vesicles (e.g., saposin C-dioleoylphosphatidylserine (SapC-DOPS) nanovesicles), various forms of brachytherapy, and various forms of targeted radionuclides. Targeted radionuclides include radionuclides associated (e.g., by covalent bonds or ionic interactions) with a molecule ("targeting agent") that specifically binds to a target on a cell, typically a cancer cell or immune cell. The targeting agent may be a small molecule, a sugar (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), lipid, protein, or peptide, and the target is a tumor-associated antigen (enriched but not specific to cancer cells), a tumor-specific antigen (minimal or no expression in normal tissues), or a neo-antigen (an antigen specific to the genome of cancer cells, generated by nonsynonymous mutations or gene fusions in the tumor cell genome). 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 or no expression in normal tissues), or a neo-antigen (i.e., an antigen specific to the genome of cancer cells, generated by nonsynonymous mutations or gene fusions in the tumor cell genome).Non-limiting examples of targeted radionuclides include radionuclides conjugated to somatostatin or its peptide analogues (e.g., 177Lu-Dotatate, etc.); prostate-specific membrane antigen or its peptide analogues (e.g., 177Lu-PSMA-617, 225Ac-PSMA-617, 177Lu-PSMA-I&T, 177Lu-MIP-1095, etc.); cognate ligands of receptors, peptides derived from said ligands, or variants thereof (e.g., 188Re-labeled VEGF125-136 or variants thereof with higher affinity for VEGF receptors, etc.); antibodies targeting tumor antigens (e.g., 131I-tositumomab, 90Y-ibritumomab tiuxetan, CAM-H2-I131 (Precirix NV), I131-omburtamab, etc.).

[0174] In some embodiments, one or more additional therapeutic agents are hormonal therapeutic agents.Hormonal therapeutic agents act to regulate or inhibit hormone action on tumors.Examples of hormonal therapeutic agents include selective estrogen receptor degraders, such as fulvestrant, GDC-9545, SAR439859, RG6171, AZD9833, linddestran, ZN-c5, LSZ102, D-0502, LY3484356, SHR9549; selective estrogen receptor modulators, such as tamoxifen, raloxifene, 4-hydroxytamoxifen, trioxyphene, keoxifene, toremifene; aromatase inhibitors, such as anastrozole, exemestane. , letrozole, and other aromatase inhibiting 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; 5α-reductase inhibitors, such as finasteride, dutasteride; and the like. In certain embodiments, the combination therapy includes the administration of a hormone or related hormone agent. In one embodiment, the combination therapy includes the administration of enzalutamide.

[0175] In some embodiments, the one or more additional therapeutic agents are epigenetic modulators. Epigenetic modulators alter the 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 Section II can be combined with DNA methyltransferase (DNMT) inhibitors or hypomethylating agents. Exemplary DNMT inhibitors include decitabine, zebularine, and azacitadine. In one or more embodiments, combinations of the anti-CD39 antibodies of Section II with histone deacetylase (HDAC) inhibitors are also contemplated. Exemplary HDAC inhibitors include vorinostat, divinostat, abexinostat, panobinostat, belinostat, and trichostatin A.

[0176] In some embodiments, the one or more additional therapeutic agents are ATP-adenosine axis targeting agents. ATP-adenosine axis targeting agents alter signal transduction mediated by adenine nucleosides and nucleotides (e.g., adenosine, AMP, ADP, ATP), for example, by regulating adenosine levels or targeting adenosine receptors. Adenosine and ATP act on different classes of receptors, often resulting in opposing effects on inflammation, cell proliferation, and cell death. For example, ATP and other adenine nucleotides have been shown to have antitumor effects through activation of the PS2Y1 receptor subtype, while accumulation of adenosine in the tumor microenvironment inhibits the antitumor function of various immune cells and enhances 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 ectonucleotidase, which is involved in the conversion of ATP to adenosine, or an antagonist of adenosine receptors. Ectonucleotidases involved in the conversion of ATP to adenosine include ectonucleoside 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, oleculab (MEDI9447), NZV930, IPH5301, GS-1423, uriledoriimab (TJD5, TJ004309), and BMS-986179. In one embodiment, the present disclosure contemplates a combination of an anti-CD39 antibody of Section II with a CD73 inhibitor, such as those described in WO2017 / 120508, WO2018 / 067424, WO2018 / 094148, and WO2020 / 046813. In a further embodiment, the CD73 inhibitor is quemlicustat. Adenosine binds to four different G protein-coupled receptors: A 1 R,A 2A R,A 2B R and A3 R, and activate them. 2 R antagonists include etrumadenant, inupadenant, taminadenant, caffeine citrate, 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 relates to a combination of an anti-CD39 antibody of Section II and an ACR antagonist. 2A R antagonist, A 2B R antagonist, or A 2A R and A 2B In some embodiments, the present disclosure contemplates a combination of an anti-CD39 antibody of Section II with an adenosine receptor antagonist described in WO2018 / 136700, WO2018 / 204661, WO2018 / 213377, or WO2020 / 023846, WO2020 / 102646. In one embodiment, the adenosine receptor antagonist is etremadenant.

[0177] In some embodiments, the one or more additional therapeutic agents are targeted therapeutic agents. In one aspect, the targeted therapeutic agent may include a chemotherapeutic agent, a radionuclide, a hormonal therapeutic agent, or another small molecule drug bound to the targeting agent. The targeting agent may be a small molecule, a sugar (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 sugar (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 (minimal or no expression in normal tissues), or a neo-antigen (an antigen specific to the genome of cancer cells, generated by non-synonymous mutations in the tumor cell genome). 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 (minimal or no expression in normal tissues), or a neo-antigen (an antigen specific to the genome of cancer cells, generated by non-synonymous mutations in the tumor cell genome). 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. Specific examples of targeted therapy comprising an antibody and a drug include, but are not limited to, patritumab deruxtecan, sacituzumab govitecan-hziy, terisotuzumab vedotin, and trastuzumab deruxtecan. In other aspects, the targeted therapy may inhibit or interfere with certain proteins that aid in tumor survival, growth, and / or spread. Non-limiting examples of such targeted therapy include signal transduction inhibitors, RAS signal transduction inhibitors, inhibitors of oncogenic transcription factors, activators of oncogenic transcription factor repressors, angiogenesis inhibitors, immunotherapeutic agents, ATP-adenosine axis targeting agents, AXL inhibitors, PARP inhibitors, PAK4 inhibitors, PI3K inhibitors, HIF2α inhibitors, CD73 inhibitors, A 2 R antagonists, TIGIT antagonists, and PD-1 antagonists. ATP-adenosine axis targeting agents are described above, while other agents are described in more detail below.

[0178] In some embodiments, the one or more additional therapeutic agents are signal transduction inhibitors. Signal transduction inhibitors are agents that selectively inhibit one or more steps in a signal transduction pathway. Signal transduction inhibitors (STIs) contemplated by the present 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). (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 (VEGFR) inhibitors, including small molecule inhibitors (e.g., axitinib, regorafenib, sunitinib, and sorafenib), VEGF kinase inhibitors (e.g., lenvatinib, cabozantinib, pazopanib, tivozanib, XL092, etc.), anti-VEGF antibodies (e.g., bevacizumab), and anti-VEGFR antibodies (e.g., ramucirumab); (v) inhibitors of the AKT family kinases or AKT pathway (e.g., rapamycin); (vi) inhibitors of the serine / threonine protein kinase B-Raf (BRAF), such as vemurafenib, dabrafenib, and encorafenib. (vii) rearrangement in transfection (RET) inhibitors, including, for example, selpercatinib and pralsetinib; (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 described elsewhere herein (e.g., inhibitors of KRAS, HRAS, RAF, MEK, ERK); (xi) FLT-3 inhibitors (e.g., gilteritinib); (xii) inhibitors of Trop-2; (xiii) inhibitors of the JAK / STAT pathway, including, for example, JAK inhibitors including tofacitinib and ruxolitinib, or STAT inhibitors such as napabucasin; (xiv) inhibitors of NF-κB;(xv) cell cycle kinase inhibitors (e.g., flavopiridol); (xvi) phosphatidylinositol kinase (PI3K) inhibitors; (xix) protein kinase B (AKT) inhibitors (e.g., capivasertib, milansertib), (xx) platelet-derived growth factor receptor (PDGFR) inhibitors (e.g., imatinib, sunitinib, regorafenib, avapritinib, lenvatinib, nintedanib, famitinib, ponatinib, axitinib, ripretinib, etc.); and (xxi) insulin-like growth factor receptor (IGFR) inhibitors (e.g., erlotinib, afatinib, gefitinib, osimertinib, dacomitinib). In one or more embodiments, the additional therapeutic agent comprises 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;

[0179] In some embodiments, one or more additional therapeutic agents are RAS signaling inhibitors. Oncogenic mutations in RAS family genes, such as HRAS, KRAS, and NRAS, are associated with various cancers. For example, among KRAS family genes, G12C, G12D, G12V, G12A, G13D, Q61H, G13C, and G12S mutations have been observed in multiple tumor types. Direct and indirect inhibition strategies have been investigated for the inhibition of mutant RAS signaling. Indirect inhibitors target effectors other than RAS in the RAS signaling pathway, and include, but are not limited to, inhibitors of RAF, MEK, ERK, PI3K, PTEN, SOS (e.g., SOS1), mTORC1, 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 are also being investigated, generally targeting 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, the one or more RAS signaling inhibitors are selected from the group consisting of RAF inhibitors, MEK inhibitors, ERK inhibitors, PI3K inhibitors, PTEN inhibitors, SOS1 inhibitors, mTORC1 inhibitors, SHP2 inhibitors, and AKT inhibitors. In other embodiments, the one or more RAS signaling inhibitors directly inhibit RAS mutants.

[0180] In some embodiments, one or more of the additional therapeutic agents are inhibitors of phosphatidylinositol 3-kinase (PI3K), particularly inhibitors of the PI3Kγ isoform. PI3Kγ inhibitors can stimulate anti-cancer immune responses through modulation of myeloid cells, for example by inhibiting suppressive myeloid cells, by attenuating immunosuppressive tumor-infiltrating macrophages, or by stimulating macrophages and dendritic cells to produce cytokines that contribute to effective T cells, thereby reducing the development and spread of cancer. Examples of PI3Kγ inhibitors include copanlisib, duvelisib, AT-104, ZX-101, tenalisib, eganelisib, SF-1126, AZD3458, and pictilisib. In some embodiments, the anti-CD39 antibodies of Section II can be combined with one or more PI3Kγ inhibitors described in WO2020 / 0247496A1.

[0181] In some embodiments, the one or more additional therapeutic agents are inhibitors of arginase. Arginase has been shown to be responsible for or involved in inflammation-induced immune dysfunction, tumor immune evasion, immunosuppression and immunopathology of infectious diseases. Exemplary arginase compounds include CB-1158 and OAT-1746. In some embodiments, the anti-CD39 antibodies of Section II can be combined with one or more arginase inhibitors described in WO / 2019 / 173188 and WO2020 / 102646.

[0182] In some embodiments, one or more additional therapeutic agents are inhibitors of oncogenic transcription factors or activators of oncogenic transcription factor repressors. Suitable agents may act at the expression level (e.g., RNAi, siRNA, etc.), through 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, and the like.

[0183] In some embodiments, one or more of the additional therapeutic agents is an inhibitor of hypoxia inducible factor (HIF) transcription factor, particularly HIF-2α. Exemplary HIF-2α inhibitors include velzutifan, ARO-HIF2, PT-2385, AB521, and those described in WO2021113436 and WO2021188769. In some embodiments, the anti-CD39 antibodies of Section II can be combined with one or more HIF-2α inhibitors described in WO2021188769.

[0184] In some embodiments, one or more of the additional therapeutic agents are inhibitors of anexelekto (AXL). The AXL signaling pathway is associated with tumor growth and metastasis and is believed to mediate resistance to various cancer treatments. Various AXL inhibitors are in development that also inhibit other kinases of the TAM family (i.e., TYRO3, MERTK), as well as other receptor tyrosine kinases, including MET, FLT3, RON, and AURORA, among others. Examples of multikinase inhibitors include sitravatinib, revastinib, glesatinib, gilteritinib, merestinib, cabozantinib, foretinib, BMS777607, LY2801653, S49076, GSK1363089, and RXDX-106. AXL-specific inhibitors, such as small molecule inhibitors including DS-1205, SGI-7079, SLC-391, TP-0903 (i.e., duvelmatinib), BGB324 (i.e., bemcentinib), and DP3975; anti-AXL antibodies such as ADCT-601; and antibody-drug conjugates (ADCs) such as BA3011, have also been developed. Another strategy to inhibit AXL signaling involves targeting GAS6, the ligand of AXL. For example, AVB-500 is in development, as is an Fc fusion protein that binds to the GAS6 ligand and inhibits AXL signaling.

[0185] In some embodiments, the one or more additional therapeutic agents are inhibitors of p21-activated kinase 4 (PAK4). Overexpression of PAK4 has been shown across a variety of cancer types, including cancers that are particularly resistant to PD-1 therapy. PAK4 inhibitors have not yet been approved, but are in development, some of which 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.

[0186] 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 the Bcl-2 protein family (e.g., venetoclax, navitoclax, etc.); (iii) an inhibitor of MCL-1; (iv) an inhibitor of the CD47-SIRPα pathway (e.g., anti-CD47 antibodies, magrolimab, etc.); (v) an isocitrate dehydrogenase (IDH) inhibitor, e.g., an IDH-1 inhibitor or an IDH-2 inhibitor (e.g., ivosidenib, enasidenib, etc.).

[0187] In some embodiments, one or more of the additional therapeutic agents are immunotherapeutic agents.Immunotherapeutic agents useful for treating cancer typically induce or amplify immune response against cancer cells.Non-limiting examples of suitable immunotherapeutic agents include immunomodulators; cellular immunotherapeutic agents; vaccines; gene therapy agents; ATP-adenosine axis targeting agents; immune checkpoint modulators; and certain signal transduction inhibitors.ATP-adenosine axis targeting agents are described above.Immunomodulators, signal transduction inhibitors, cellular immunotherapeutic agents, vaccines, gene therapy agents, and immune checkpoint modulators are further described below.

[0188] In some embodiments, one or more of the additional therapeutic agents is an immunotherapeutic agent, more specifically 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 presentation of antigen epitopes on major histocompatibility complex molecules, agonists 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, as well as other T cell adjuvants.

[0189] In some embodiments, the one or more additional therapeutic agents are immunotherapeutic agents, more specifically cellular therapeutic agents. Cellular therapeutic agents are a form of therapy in which living cells are administered to a subject. In certain embodiments, the one or more additional therapeutic agents are cellular immunotherapeutic agents that activate or suppress the immune system. Cellular immunotherapeutic agents useful in the treatment of cancer typically induce or amplify an immune response. The cells can be autologous or allogeneic immune cells (e.g., monocytes, macrophages, dendritic cells, NK cells, T cells, etc.) taken from one or more subjects. Alternatively, the cells can be "(re)programmed" allogeneic immune cells generated from immune precursor cells (e.g., lymphoid precursor cells, myeloid precursor cells, dendritic cell common precursor cells, stem cells, induced pluripotent stem cells, etc.). In some embodiments, such cells may be expanded subsets of cells with distinct effector functions and / or maturation markers (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 classical dendritic cells), M1 macrophages, M2 macrophages, etc.), may be genetically modified to target the cells to specific antigens and / or to enhance the anti-tumor effect of the cells (e.g., engineered T cell receptor (TCR) cell therapies, chimeric antigen receptor (CAR) cell therapies, lymph node homing of antigen-loaded dendritic cells, etc.), may be engineered to express or increase the expression of tumor-associated antigens, or 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 immunotherapeutics include sipuleucel-T, tisagenlecleucel, lysocabtagene maraleucel, idecbutagen bicelucel, brexcabtagene 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.

[0190] In some embodiments, one or more additional therapeutic agents are immunotherapeutic agents, more specifically gene therapy agents.Gene therapy agents include recombinant nucleic acids that are administered to subjects or cells of subjects ex vivo to cause heterologous expression of protein (e.g., small interfering RNA (siRNA) agents, double-stranded RNA (dsRNA) agents, microRNA (miRNA) agents, viral or bacterial gene delivery, etc.) to modify the expression of endogenous genes, and gene editing therapeutic agents that may or may not include nucleic acid components (e.g., meganucleases, zinc finger nucleases, TAL nucleases, CRISPR / Cas nucleases, etc.), oncolytic viruses, etc. Non-limiting examples of gene therapy agents that may be useful in cancer treatment include Gendicine® (rAd-p53), Oncorine® (rAD5-H101), talimogene laherparepvec, Mx-dnG1, ARO-HIF2 (Arrowhead), quaratusugene ozeplasmid (Immunogene), CTX110 (CRISPR Therapeutics), CTX120 (CRISPR Therapeutics), and CTX130 (CRISPR Therapeutics).

[0191] In some embodiments, the one or more additional therapeutic agents are immunotherapeutic agents, more specifically agents that modulate immune checkpoints. Immune checkpoints are a series of inhibitory and stimulatory pathways that directly affect the function of immune cells (e.g., B cells, T cells, NK cells). Immune checkpoints function when proteins on the surface of immune cells recognize and bind to their cognate ligands. The present invention contemplates the use of anti-CD39 antibodies of Section II in combination with agonists of stimulatory or costimulatory pathways and / or antagonists of inhibitory pathways. Agonists of stimulatory or costimulatory pathways and antagonists of inhibitory pathways, or combinations thereof, may be useful as agents to overcome different immune suppressive pathways within the tumor microenvironment, inhibit regulatory T cells, reverse / prevent T cell anergy or exhaustion, and induce innate immune activation and / or inflammation at the tumor site.

[0192] In some embodiments, the one or more additional therapeutic agents are immune checkpoint inhibitors. As used herein, the term "immune checkpoint inhibitor" refers to an inhibitory or co-inhibitory immune checkpoint antagonist. The terms "immune checkpoint inhibitor", "checkpoint inhibitor" and "CPI" can be used interchangeably herein. Immune checkpoint inhibitors can antagonize inhibitory or co-inhibitory immune checkpoints by preventing receptor-ligand binding and / or altering receptor signaling. Examples of immune checkpoints (ligands and receptors), some of which are selectively upregulated and can be antagonized 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 protein 3); LAG-3 (lymphocyte activation gene 3); TIGIT (Ig and ITIM domain-containing protein 3); and PD-L1 (PD-1 ligand). These include T cell immunoreceptors with a CD4+ domain; 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 divided into two classes based on their structural features: i) killer cell immunoglobulin-like receptors (KIRs), and ii) C-type lectin receptors (members of the type II transmembrane receptor family). Other less well-defined immune checkpoints described in the literature are also contemplated, including both receptors (e.g., the 2B4 (also known as CD244) receptor) and ligands (e.g., certain B7 family inhibitory ligands, such as B7-H3 (also known as CD276) and B7-H4 (also known as B7-S1, B7x, and VCTN1)).

[0193] In some embodiments, the immune checkpoint inhibitor is a CTLA-4 antagonist. In further 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.

[0194] In some embodiments, the immune checkpoint inhibitor is a PD-1 antagonist. In further 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, dostallimab, cemiplimab, ezabenlimab, MEDI-0680 (AMP-514; WO2012 / 145493), nivolumab, pembrolizumab, pidilizumab, pimivalimab, retifanlimab, sasanlimab, spartalizumab, sintilimab, tislelizumab, toripalimab, and zimberelimab; and bispecific antibodies, such as LY3434172. In yet a further embodiment, the PD-1 antagonist may be a recombinant protein composed of the extracellular domain of PD-L2 (B7-DC) fused to the Fc portion of IgG1 (AMP-224). In certain embodiments, the immune checkpoint inhibitor is zimberelimab.

[0195] In some embodiments, the immune checkpoint inhibitor is a PD-L1 antagonist. In further embodiments, the PD-L1 antagonist can be an antagonistic PD-L1 antibody. Suitable antagonistic PD-L1 antibodies include, for example, monospecific antibodies, such as avelumab, atezolizumab, durvalumab, BMS-936559, and embafolimab, and bispecific antibodies, such as LY3434172 and KN046.

[0196] In some embodiments, the immune checkpoint inhibitor is a TIGIT antagonist. In further 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, domvanalimab, EOS-448, etigilimab, IBI-929, JS006, M6223, osipelimbab, 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 domvanalimab or AB308.

[0197] In some embodiments, the immune checkpoint inhibitor is a LAG-3 antagonist. In further 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).

[0198] In some embodiments, the immune checkpoint inhibitor is a B7-H3 antagonist. In further embodiments, the B7-H3 antagonist is an antagonistic B7-H3 antibody. Suitable antagonistic B7-H3 antibodies include, for example, MGA271 (WO11 / 109400), omburtumab, enoblituumab, DS-7300a, ABBV-155, and SHR-A1811.

[0199] In some embodiments, the immune checkpoint inhibitor is a TIM-3 antagonist. In further embodiments, the TIM-3 antagonist can be an antagonistic TIM-3 antibody. Suitable antagonistic TIM-3 antibodies include, for example, dostallimab, sabatolimab, BMS-986258, and RG7769 / RO7121661.

[0200] In some embodiments, the one or more additional therapeutic agents activate stimulatory or costimulatory immune checkpoints. 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.

[0201] In some embodiments, the agent that activates the stimulatory or costimulatory immune checkpoint is a CD137 (4-1BB) agonist. In further 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 the stimulatory or costimulatory immune checkpoint is a GITR agonist. In further 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 stimulatory or costimulatory immune checkpoints is an OX40 agonist. In further 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 stimulatory or costimulatory immune checkpoints is a CD40 agonist. In further embodiments, the CD40 agonist can be an agonistic CD40 antibody, such as dacetuzumab, celicrelumab, APX005M, ADC-1013, or CDX-1140. In some embodiments, the agent that activates stimulatory or costimulatory immune checkpoints is a CD27 agonist. In further embodiments, the CD27 agonist may be an agonistic CD27 antibody. Suitable CD27 antibodies include, for example, varlilumab.

[0202] In some embodiments, one or more of the additional therapeutic agents are immunotherapeutic agents, more specifically signal transduction inhibitors. Intracellular signal transduction molecules that affect immune cell function may also be suitable targets for improving antitumor immunity. For example, one or more of the additional therapeutic agents may be inhibitors of intracellular signal transduction molecules, and are inhibitors of hematopoietic progenitor kinase 1 (HPK1). HPK1 is a serine / threonine kinase that functions as a negative regulator of activation signals generated by the T cell antigen receptor. As another example, one or more of the additional therapeutic agents may be inhibitors of Cbl-b, an E3 ubiquitin ligase involved in regulating TCR signal transduction (e.g., AP401). As another example, one or more of the additional therapeutic agents may be inhibitors of diacylglycerol kinase (DGK). In some embodiments, the inhibitors are small molecules. Non-limiting examples of small molecule HPK1 inhibitors in clinical development include CFI-402411 and BGB-15025, and 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.

[0203] In some embodiments, one or more of the additional therapeutic agents are agents that inhibit or deplete immunosuppressive immune cells.For example, to inhibit or deplete immunosuppressive macrophages or monocytes, the agent can be a CSF-1R antagonist, for example a CSF-1R antagonist antibody, including RG7155 (WO11 / 70024, WO11 / 107553, WO11 / 131407, WO13 / 87699, WO13 / 119716, WO13 / 132044) or FPA-008 (WO11 / 140249; WO13169264), or the antibody disclosed in WO14 / 036357.As another example, to inhibit or deplete Treg, the agent can be anti-CD25 beads, which are used to deplete Treg ex vivo.

[0204] In some embodiments, the present disclosure contemplates the use of anti-CD39 antibodies of section II in combination with immunogenic cell death (ICD)-inducing therapy. ICD can be defined as a functionally unique regulated cell death subtype sufficient to induce adaptive immunity specifically directed to antigens derived from cell "corpses". Criteria for identifying ICD-inducing therapy may 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 therapy are described in Galluzzi et al., Nature Reviews Clinical Oncology, 2020 17: 725-742. In some embodiments, the ICD-guided therapy can be radiation therapy, photodynamic therapy, extracorporeal photochemotherapy, oncolytic virus therapy, bortezomib, cyclophosphamide, doxorubicin, epirubicin, idarubicin, mitoxantrone, cetuximab, crizotinib, or oxaliplatin. In some embodiments, the ICD-guided therapy can be radiation therapy, photodynamic therapy, extracorporeal photochemotherapy, oncolytic virus therapy, bleomycin, bortezomib, carboplatin, cetuximab, crizotinib, cyclophosphamide, docetaxel, doxorubicin, epirubicin, gemcitabine, idarubicin, irinotecan, mitoxantrone, oxaliplatin, paclitaxel, vemurafenib, or vorinostat.

[0205] In some embodiments, each additional therapy may be independently a radiation therapy, a chemotherapeutic agent, a radiopharmaceutical agent, a hormonal therapy, an epigenetic modulator, a targeting agent, an immunotherapeutic agent, a cellular therapy, a gene therapy, or an ICD-induced therapy. For example, in one embodiment, the present disclosure contemplates the use of an anti-CD39 antibody of Section II in combination with one or more ICD-induced therapies and optionally one or more additional therapies, each additional therapy being independently selected from a radiation therapy, a radiopharmaceutical agent, a chemotherapeutic agent, a hormonal therapy, a targeting agent, an immunotherapeutic agent, a cellular therapy, or a gene therapy. In another example, in one embodiment, the present disclosure contemplates the use of an anti-CD39 antibody of Section II in combination with one or more chemotherapeutic agents and optionally one or more additional therapies, each additional therapy being independently selected from a radiation therapy, a radiopharmaceutical agent, a hormonal therapy, a targeting agent, an immunotherapeutic agent, a cellular therapy, or a gene therapy. In another embodiment, the present disclosure contemplates the use of an anti-CD39 antibody of Section II in combination with one or more chemotherapeutic agents and one or more tyrosine kinase inhibitors, and optionally one or more additional therapies, each of which is independently a targeted agent, an immunotherapeutic agent, or a cellular therapeutic agent. In another embodiment, the disclosure contemplates the use of an anti-CD39 antibody of Section 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 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, and optionally one or more additional therapies, wherein each additional therapy is independently selected from radiation therapy, a radiopharmaceutical, a targeted agent, an immunotherapeutic agent, or a cellular therapy agent.In another embodiment, the disclosure contemplates the use of an anti-CD39 antibody of Section II in combination with one or more immunotherapeutic agents and optionally one or more additional therapies, each of which is independently selected from radiation therapy, radiopharmaceuticals, hormonal therapy, targeted agents, chemotherapeutic agents, cellular therapy, or genetic therapy agents. In another embodiment, the disclosure contemplates the use of an anti-CD39 antibody of Section II in combination with one or more immunotherapeutic agents and one or more chemotherapeutic agents, and optionally one or more additional therapies, each of which is independently selected from radiation therapy, radiopharmaceuticals, hormonal therapy, targeted agents, cellular therapy, or genetic therapy agents. In another embodiment, the disclosure contemplates the use of an anti-CD39 antibody of Section II in combination with one or more immunotherapeutic agents and one or more radiation therapy or radiopharmaceuticals, and optionally one or more additional therapies, each of which is independently selected from chemotherapeutic agents, hormonal therapy, targeted agents, cellular therapy, or genetic therapy agents. In another embodiment, the disclosure contemplates the use of an anti-CD39 antibody of Section II 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, each of which is independently selected from radiation therapy, radiopharmaceuticals, chemotherapeutic agents, targeted agents, immunotherapeutic agents, or cellular therapeutic agents. In another embodiment, the disclosure contemplates the use of an anti-CD39 antibody of Section 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 therapy.In another embodiment, the present disclosure contemplates the use of an anti-CD39 antibody of Section 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 KRAS inhibitor, and (x) an ALK inhibitor. In another embodiment, the present disclosure contemplates the use of an anti-CD39 antibody of Section 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 further embodiments of the above, (a) the targeting agent can be a PI3K inhibitor, an arginase inhibitor, a HIF2α inhibitor, an AXL inhibitor, a PAK4 inhibitor, or an anti-angiogenic agent; (b) the immunotherapy agent can be 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 2B(d) the ATP-adenosine axis targeting agent is etormadenant or quemlicustat; (e) the immunotherapeutic agent is an anti-PD-L1 antagonist antibody or an anti-PD-1 antagonist antibody, optionally selected from the group consisting of budigalimab, camrelizumab, cosibelimab, dostallimab, cemiplimab, ezabenlimab, nivolumab, pembrolizumab, pidilizumab, pimivalimab, retifanlimab, sasanlimab, sulphosphamide ... (f) the immunotherapeutic agent is an anti-TIGIT antagonist antibody, optionally selected from the group consisting of AGEN1327, AB308 (WO2021247591), BMS986207; (g) the immunotherapeutic agent is selected from the group consisting of domvanalimab, AB308, zimberelimab, tiragolumab, pembrolizumab, nivolumab, atezolizumab, or durvalumab; (h) the antiangiogenic agent is pazopa. (i) the ICD induction therapy is radiation therapy (including both external and internal radiation therapy), photodynamic therapy, extracorporeal photochemotherapy, oncolytic virus therapy, bortezomib, cyclophosphamide, doxorubicin, epirubicin, idarubicin, mitoxantrone, or oxaliplatin; or (j) any combination thereof. In yet further embodiments of the above, the present disclosure contemplates the use of the anti-CD39 antibody of section II in combination with dombanalimab, AB308, etormadenant, quemriculistat, zimbelelimab, AB521, or any combination thereof.In yet further embodiments of the above, the disclosure contemplates the use of an anti-CD39 antibody of Section II in combination with etremadenant, quemlicustat, or etremadenant and quemlicustat. In yet further embodiments of the above, the disclosure contemplates the use of an anti-CD39 antibody of Section II in combination with etremadenant, AB521, or etremadenant and AB521. In yet further embodiments of the above, the disclosure contemplates the use of an anti-CD39 antibody of Section II in combination with quemlicustat, AB521, or quemlicustat and AB521.

[0206] The selection of additional therapeutic agent(s) may be informed by the current standard of care for a particular cancer and / or the mutational status and / or stage of the cancer of interest. Detailed standard of care guidelines have been published, for example, by the National Comprehensive Cancer Network (NCCN). See, for example, NCCN Colon Cancer v3.2021, NCCN Hepatobiliary Cancer v5.2021, NCCN Renal Cancer v3.2022, NCCN NSCLC v7.2021, NCCN Pancreatic Adenocarcinoma v2.2021, NCCN Esophageal and Esophagogastric Junction Cancer v4.2021, NCCN Gastric Cancer v5.2021, Ovarian / Fallopian Tube / Primary Peritoneal Cancer v3.2021, Prostate Cancer v3.2022, Head and Neck Cancer v1.2022, Melanoma: Skin v1.2022, Acute Myeloid Leukemia v1.2022. EXAMPLES

[0207] experiment The following examples are provided 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 invention. Additional antibodies within the scope of the present disclosure can be made using methods based on the methods shown in these examples, or other methods described herein. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should be accounted for.

[0208] Unless otherwise specified, temperatures are in degrees Celsius (°C) and pressure is at or near atmospheric pressure. Standard abbreviations are used, including: rt or rt = room temperature, min = minute(s); h or hr = hour(s); ng = nanogram; μg = microgram; mg = milligram; g = gram; kg = kilogram; μl or μL = microliter; ml or mL = milliliter; l or L = liter; μM = micromole; mM = millimole; M = molar; mol = mole; mmol = millimole; nM = nanomole.

[0209] Example 1: Generation of anti-CD39 antibodies Human CD39-expressing CHO-K1 cell lines, cynomolgus monkey CD39-expressing HEK-293 cell pools, and mouse CD39-expressing HEK-293 cell pools were generated. Briefly, CHO-K1 cells or HEK-293 cells (Flp-In™-293 cells) were transfected with pcDNA3.3 expression vectors encoding full-length human, cynomolgus monkey, or mouse CD39 (SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 71, respectively) using a Lipofectamine 2000 transfection kit according to the manufacturer's protocol. 48-72 hours after transfection, transfected cells were cultured in medium containing blasticidin for selection and CD39 expression was tested, after which CD39-expressing cell pools were obtained. Human, cynomolgus monkey, and mouse CD39-expressing cell lines or cell pools were obtained by limiting dilution and used to screen hybridoma supernatants as described below.

[0210] To generate anti-CD39 antibodies, four 6-8 week old Sprague Dawley rats were immunized with 30-200 mg / animal of human CD39 antigen, either protein or plasmid DNA. The adjuvant mixture included Adju-Phos, CpG-ODN, or Titer-Max. Animals were injected once every other week via plantar, subcutaneous, intraperitoneal, intramuscular, and intradermal routes. Serum titers were measured by ELISA or fluorescence activated cell sorting (FACS). ELISAs used to measure serum antibody titers against a given antigen were performed as follows: Plates (Nunc) were coated overnight with 100 μL of 1 μg / mL human CD39 antigen at 4°C and then blocked with blocking buffer (1xPBS / 2%BSA) for 1 h at ambient temperature. Rat sera were serially diluted 3-fold starting at a 1:100 dilution in blocking buffer and incubated for 1 h at ambient temperature. Wells without serum samples were used as negative controls. Plates were then washed and then incubated with the secondary antibody goat anti-rat IgG-Fc-HRP (Bethyl) for 1 h. After washing, tetramethylbenzidine (TMB) substrate was added and the interaction was stopped by adding 2 M HCl. Absorbance was read at 450 nm using a microplate reader (Molecular Device). The FACS assay used to measure serum antibody titers was performed as generally described for screening of hybridoma supernatants (see below), except that serially diluted sera were used. Serum titers were determined as the last dilution giving a positive signal (i.e., more than 3-fold higher than the negative control).

[0211] When serum titers were sufficiently high (>= 1:24,300), animals were given a final boost with protein and cell membrane lysate in sterile PBS without adjuvant. After 48-96 hours, the animals were euthanized and lymph nodes and spleens were used for cell fusion.

[0212] Lymph nodes and spleens from immunized animals were homogenized and filtered to remove blood clots and cell debris. Logarithmically growing Sp2 / 0 myeloma cells were harvested and centrifuged. B cells and Sp2 / 0 myeloma cells were treated separately with pronase solution 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 electrofusion solution according to general electrofusion procedures. The fused cells were resuspended in DMEM medium supplemented with 20% FBS and 1x HAT and then transferred to a 96-well plate. The fused cells were incubated at 37°C and 5% CO 2 The cells were then kept in an incubator set at 5 °C for 10 to 14 days.

[0213] Hybridoma cells were harvested and 150-200 cells were added to 1.5 mL of semi-solid HAT medium. The cells were mixed gently with a vortex oscillator for 5-10 seconds and then seeded into a 6-well plate. The plate was incubated at 37°C and 5% CO 2 The cells were kept in an incubator set at 4°C for 7–8 days. Each visible single colony was placed into a 96-well plate containing DMEM medium supplemented with 10% FBS. After 2–3 days, cell supernatants were collected and screened.

[0214] Four rounds of hybridoma generation and antibody screening were performed, varying the order of the assay cascade in the screening paradigm. To meet the screening criteria, antibodies produced from a given hybridoma clone had to bind with high affinity to both human and cynomolgus cellular CD39, without measurable binding to either mouse CD39 or other CD39 family members, i.e., CD39-L1, CD39-L2, CD39-L3, or CD39-L4. Antibodies also had to show potent inhibition of cellular human CD39 enzymatic activity in both cell lines engineered to overexpress CD39 and in human cell lines that naturally express CD39, such as THP-1, SK-MEL-5, and MOLP-8. In the first round of fusion, CHO-K1 cell-based human CD39 ELISA was the primary screen, binding to cell-overexpressed human, cynomolgus, and mouse CD39 was measured by FACS (using the cell lines mentioned above), and in vitro soluble human CD39 enzyme assay and cell-based enzyme assay were the secondary screens. The second round of fusion failed to produce suitable hybridoma material. In the third round of fusion, soluble human CD39 enzyme assay was the primary screen, binding to cell-overexpressed human and mouse CD39 was measured by FACS, human CD39-L1 binding counterscreen, and soluble human CD39 enzyme assay for confirmation were the secondary screens. In the fourth round of fusion, THP-1 cell-based enzyme assay was used as the primary screen, binding to cell-overexpressed human and cynomolgus CD39 was measured by FACS, and THP-1 cell-based enzyme assay for confirmation were the 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.

[0215] For cell-based ELISAs used to assess binding to cell surface expressed CD39, assays were generally performed as follows: 3–5 × 10 cells per well of a 384-well plate were added.4 Precoat the cells and incubate at 37°C, 5% CO 2 The plates were cultured for 2 days in an incubator set at 4°C. The plates were blocked with blocking buffer (1xPBS / 5% milk) for 1 h at ambient temperature. Then, 30 μL of hybridoma supernatant was added to the plate and incubated for 1 h at ambient temperature. The plates were washed three times with PBS and then incubated with the secondary antibody goat anti-rat IgG-Fc-HRP (1:500) for 1 h at ambient temperature. After washing, TMB substrate was added to each well and the plates were incubated for 3–5 min at ambient temperature in the dark, and the reaction was stopped by adding 2 M HCl. The absorbance at 450 nm was read using a microplate reader (Molecular Device).

[0216] FACS was also used to detect binding of anti-CD39 antibodies to CD39 in the supernatants using the human CD39-expressing CHO-K1 cell line, the cynomolgus monkey CD39-expressing HEK-293 cell line, and the mouse CD39-expressing HEK-293 cell pool described above. Unlabeled cells were used as a control to set a threshold before detection, and then the percentage change of each group above the fluorescence intensity threshold was analyzed. 5 Cells / well were incubated with hybridoma supernatant in a volume of 100 μL for 1 h at 4° C. Anti-human CD39 reference antibody was used as a positive control. Human / rat IgG isotype antibody was used as a negative control. After washing the cells with 1×PBS / 1% BSA, Alexa647-labeled goat anti-human or goat anti-rat antibodies (diluted 1:500 in FACS staining buffer) were added. Cells were incubated in the dark at 4° C. for 1 h. The mean fluorescence intensity (MFI) of the cells was measured by flow cytometer and analyzed using FlowJo software.

[0217] Anti-CD39 antibodies were tested for their ability to block the enzymatic activity of soluble CD39 extracellular domain (ECD) antigen by measuring inhibition of human CD39 catalytic function to hydrolyze ATP to AMP+inorganic phosphate, in conjunction with luciferase activity and luminescence as the signal. Enzyme assays were performed in 96-well multiwell plates in a final volume of 120 μL. The assay mixtures were incubated for the indicated incubation periods in an incubator set at 37°C. Tris Magnesium (TM) buffer was 25 mM Tris, 5 mM MgCl 2 , and 0.01% BSA. Serial dilutions of inhibitory anti-CD39 antibodies were prepared starting at 20 nM in TM buffer containing soluble human CD39 (Sino Biological, Cat. No. 16020-H08B). Dilutions were performed either with or without ATP to determine the effect of premixing the antibody and enzyme before the addition of substrate (ATP). As an isotype control, an irrelevant antibody was similarly diluted to examine the specificity of antibody-mediated CD39 inhibition. The antibody / enzyme preparations were incubated for 1 hour in an incubator set at 37°C, followed by addition of ATP to a final concentration of 20 μM and incubation 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).

[0218] The functional activity of anti-CD39 antibodies was determined by measuring CD39-catalyzed ATP hydrolysis using a human myeloid leukemia mononuclear cell line (THP-1). THP-1 cells were resuspended in TM buffer and 80 μL / well of cells were plated in a 96-well U-plate, with 4 × 10 4Cells were seeded at a density of 10 ...

[0219] To test whether anti-CD39 antibodies specifically bind human CD39 but do 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, ThermoFisher) were pre-coated with His-tagged human CD39 cross-family proteins (CD39-L1, CD39-L2, CD39-L3, or CD39-L4 (0.5 μg / mL)) and incubated in carbonate-bicarbonate buffer (20 mM NaCl). 2 CO 3 , 180 mM NaHCO 3The plates were incubated overnight at 4°C in PBS / 0.5% (v / v) Tween-20 (pH 9.2). The next day, the plates were washed three times with 300 μL / well of PBS / 0.5% (v / v) Tween-20 and then blocked with PBS / 2% BSA for 1 h. After blocking, the plates were washed three times. After washing, test antibodies at various concentrations (6-fold serial dilutions from 100 nM to 0.36 pM in 2% BSA / PBS) were added to the plates and left in an incubator set at 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 μL / well of goat anti-human IgG Fc-HRP antibody (1:5000) was added and the plates were placed in an incubator set at room temperature for 1 h. For color development, 100 μL / well of TMB substrate solution was added to the plates. After 3-5 min, the reaction was stopped with 100 μL / well of 2 M HCl. Absorbance was read at 450 nm using a SpectraMax® M5e microplate spectrophotometer. EC 50 Values ​​were obtained from four-parameter nonlinear regression analysis using GraphPad Prism software.

[0220] Antibodies from hybridoma clone 19, hybridoma clone 31, and hybridoma clone 39 with rat Fc domain were purified from hybridoma supernatants. These antibodies are referred to herein as antibodies 19, 31, and 39, respectively. The collected hybridoma supernatants were adjusted to pH 7.0 and then loaded onto a Protein A column. Bound antibodies were eluted with glycine-HCl (pH 3.5) and immediately neutralized with 0.1 M Tris (pH 9.0). Proteins were dialyzed against PBS and filtered through a 0.22 μm membrane filter. Antibody concentrations were measured with Nano Drop. Purity of the antibodies was assessed by SDS-PAGE and HPLC-SEC.

[0221] Total RNA was isolated from hybridoma clones 19, 31, and 39 using 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 cDNA using a 3'-constant region degenerate primer and a 5'-degenerate primer set. The 5'-degenerate primer was designed based on the upstream signal sequence coding region of the Ig variable sequence. 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 2xYT plates containing carbocinine and incubated overnight in an incubator set at 37°C. Twelve positive colonies were randomly selected for DNA sequencing by Biosune.

[0222] The cloned genes were codon-optimized for mammalian expression and then synthesized by GENEWIZ (Suzhou, China). The resulting plasmids contained genes encoding heavy chains containing the VH domains of antibodies 19, 31, or 39 fused with human IgG4 CH1, hinge, CH2, and CH3 segments (including the hinge mutation S228P), or genes encoding light chains containing the VL domains fused with the human Ig kappa CK domain of the light chain.

[0223] Plasmids containing the VH and VL genes described above were co-transfected into Expi293F cells to produce antibodies ch19_IGG4.P, ch31_IGG4.P, and ch39_IGG4.P. For each antibody, 2.94x10 cells with viability of over 95% were cultured in 40 mL of cell culture medium. 6 A final concentration of 1 μg / mL of Expi293F cells was prepared. Plasmid DNA and ExpiFectamine™ 293 transfection reagent at a final concentration of 1 μg / mL were mixed and then added to the cell culture medium. The cell culture was incubated in a platform shaker with a rotation speed of 150 rpm. The temperature was maintained at 37° C. and CO 2The cells were maintained in an incubator set at a concentration of 8%. After 6 days of incubation, the cells were pelleted using centrifugation at 4000 rpm for 10 minutes at 25° C. The supernatant was collected and analyzed by gel electrophoresis, SPR analysis, etc., as required. off The supernatants were loaded onto SDS-PAGE gels according to the NuPAGE™ 4-12% Bis-Tris Protein Gels (Thermo Fisher) instructions. The molecular weight of the antibodies was determined using PageRuler™ Unstained Protein Ladder (Thermo Fisher) with the antibody samples.

[0224] To purify the antibodies, a Protein A column was pre-loaded with 1 mL of MabSelect Sure resin. The column was equilibrated with 5 column volumes (CV) of equilibration buffer (0.1 M Tris, pH 7.0) before loading with cell culture medium. 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 eluted sample was buffer exchanged with PBS buffer using a desalting column. Finally, the purified antibodies were analyzed by SDS-PAGE and SEC-HPLC, and then stored at -80°C.

[0225] FACS analysis was used to measure the binding of recombinant anti-CD39 antibodies to CHO-K1 cells overexpressing human CD39 and HEK293 cells overexpressing cynomolgus or mouse CD39 as described above. The ability of these antibodies to inhibit cellular CD39 enzymatic activity 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.

[0226] The kinetic binding of these recombinant anti-CD39 antibodies to human CD39 was determined by surface plasmon resonance (SPR) using a Biacore 8K instrument. The recombinant antibodies were immobilized using a goat anti-human FcIgG coated CM5 sensor chip (GE, Cat. No. 29-1496-03) and soluble His-tagged CD39 ECD protein SEQ ID NO:72 (Sino Biological, 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 exhibited slow off-rates (k d ) and the subnanomolar equilibrium dissociation constant (K D ) showed similarly high binding affinity.

[0227] [Table 1]

[0228] [Table 2]

[0229] Example 2: Generation of humanized anti-CD39 antibodies 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 rat variable heavy (VH) and variable light (VL) sequences of antibodies 31 and 39 were used to identify the closest human germline for each chain. Human acceptors for the VH and VL frameworks were searched in the GenBank database (Benson et al., Nucleic Acids Res. 2005, 33, D34-D38). The frameworks were defined using the extended CDR definition in which Kabat CDR1 was extended by 5 amino acids at the N-terminus. The top three hits were used to derive the sequences of the humanized VH and VL genes, and from each of these, two were selected for expressing 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).

[0230] [Table 3]

[0231] [Table 4]

[0232] 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 as 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 as 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 as 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 as the VL acceptor. The resulting sequences were examined for the introduction of any potential post-translational modification (PTM) sites such as isomerization, deamination, glycosylation, and oxidation. Putative residues suitable for back mutation of the rat sequence were also identified using antibody homology graphic modeling. Following removal of the PTM sites, the antibody was assayed to determine whether the change(s) affected antigen binding in comparison to the parent antibody.

[0233] The humanized genes were back-translated, codon-optimized for mammalian expression, and synthesized by GENEWIZ. The synthetic genes were recloned into an in-house IgG expression vector, expressed, and purified.

[0234] The kinetics of binding of purified antibodies to antigen was determined using surface plasmon resonance (SPR) and used to rank the antibodies. The affinity of anti-CD39 antibodies to human CD39 ECD His-tagged antigen (SEQ ID NO: 72) was measured using a Biacore 8K instrument. Activator was prepared by mixing 400 mM EDC and 100 mM NHS (GE) immediately before injection. A CM5 sensor chip (GE, Cat. No. 29-1496-03) was activated with activator for 420 seconds. Goat anti-human Fc IgG (30 μg / mL in 10 mM NaAc, pH 4.5) was then injected into the channel at a flow rate of 10 μL / min for 420 seconds. The chip was inactivated with 1 M ethanolamine hydrochloride. Anti-CD39 antibodies were diluted to 4 μg / mL in running buffer (1xHBS-EP+) and injected into the channel at a flow rate of 10 μL / min for 15 seconds. Six concentrations of CD39 antigen analyte (8, 4, 2, 1, 0.5, 0.25 nM) were injected sequentially into the channel at a flow rate of 30 μL / min for a 180 s association phase followed by a 2400 s dissociation phase. Glycine (10 mM, pH 1.5) as regeneration buffer was injected following the dissociation phase. The sensorgrams of the reference and buffer channels were subtracted from the test sensorgrams. The experimental data were fitted with a 1:1 binding model steady-state affinity / heterogeneous ligand. The molecular weights of the CD39 antigen and anti-CD39 antibody used in the calculations were 52 kDa and 145 kDa, respectively.

[0235] To maintain the binding affinity of the humanized variant of antibody 39: ch39_IGG4.P, a K94R rat sequence back mutation was introduced into the VH1 chain (SEQ ID NO: 58), but an equivalent back mutation was not required in VH3 due to the presence of R94 (SEQ ID NO: 59). The human framework of VL1 also contains a glycosylation PTM site ( 81 NDT 83 ), which was removed by introducing an additional rat sequence backmutation N81D (SEQ ID NO:61). For VL3 (SEQ ID NO:62), no rat sequence backmutation was required.

[0236] Next, all four VH / VL combinations of VH1 (SEQ ID NO: 58) or VH3 (SEQ ID NO: 59) with VL1 (SEQ ID NO: 61) or VL3 (SEQ ID NO: 62) were expressed for the 39 humanized variants, but the VH1+VL3 pair was not well expressed. The genes of the 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 segments of the heavy chain (including hinge mutation S228P); and a plasmid containing a VL domain fused to human Ig kappa CK domain of the light chain.

[0237] The humanized IgG4(S228P) variant of antibody 39 was analyzed by SPR as described above, along with the chimeric rVH+rVL / human IgG4(S228P) variant of antibody 39. The results are shown in Table 5.

[0238] [Table 5]

[0239] An additional rat sequence back mutation (I2T) was introduced into the VL1 (SEQ ID NO:60) and VL3 (SEQ ID NO:63) frameworks and antibodies were expressed and analyzed, but the VH1+VL3 pair did not express well. Table 6 shows the SPR data for these antibody variants.

[0240] [Table 6]

[0241] Humanized variants of antibody 39 exhibit high affinity for cellular human CD39 and potently inhibit the enzymatic activity of cellular human CD39 (Table 7).

[0242] [Table 7]

[0243] Humanized variant of antibody 31: The following rat sequence back-mutations were introduced into the VH1 chain: E71S, D76N, and A78V (SEQ ID NO: 43). Additionally, D76N and A78V back-mutations were introduced into the VH2 chain (SEQ ID NO: 44) to maintain the binding affinity of ch31_IGG4.P. Antibody 31 contains a glycosylation PTM site sequence ( 26 NQT 28 ), which was mutated to either Q27P (SEQ ID NO: 45) or N26Q (SEQ ID NO: 46) in VL1 and to N26Q (SEQ ID NO: 49) in VL2. The human framework of VL1 also contains a glycosylation PTM site ( 81 NDT 83 ) was introduced, which was eliminated by introducing an additional rat sequence backmutation N81D (SEQ ID NO: 47 and SEQ ID NO: 48, respectively). For VL2, no rat sequence backmutation was required.

[0244] For the humanized variants of antibody 31, 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) were expressed, but the VH1+VL2 pair was not expressed well. The genes of the 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 segments of the heavy chain (including hinge mutation S228P); and a plasmid containing a VL domain fused to human Ig kappa CK domain of the light chain.

[0245] The humanized IgG4(S228P) variant of antibody 31 was analyzed by SPR as described above along with the chimeric rVH+rVL / human IgG4(S228P) variant of antibody 31. The results are shown in Table 8.

[0246] [Table 8]

[0247] Humanized variants of antibody 31 exhibit high affinity for cellular human CD39 and potently inhibit the enzymatic activity of cellular human CD39 (Table 9).

[0248] [Table 9]

[0249] For both antibody 31 and antibody 39, the application of CDR grafting followed by a small number of rat sequence backmutations into the human variable domain frameworks yielded humanized variants that retained or improved the binding affinity and inhibitory potency of the respective rat / human chimeric antibodies.

[0250] Example 3: Characterization of complete kinetic binding by SPR Human IgG1 variants were generated using the VH and VL domain genes of the hu31.4_IGG4.P and hu39.5_IGG4.P humanized antibodies, codon-optimized for mammalian expression. A plasmid containing a VH in which the VH domain is fused to a human IgG1 CH1, hinge, CH2, and CH3 constant domain segment of the heavy chain (HC) containing the L234A and L235A (Eu numbering) mutations (SEQ ID NO:2), and a plasmid containing a VL in which the VL domain is fused to a human Ig kappa CK constant domain of the light chain (LC) (SEQ ID NO:6) were transfected into CHO-K1 cells. Antibodies hu31.4_IGG1.AA (HC is sequence number 51, LC is sequence number 56) and hu39.5_IGG1.AA (HC is sequence number 66, LC is sequence number 67) were expressed using the high speed stable pool protocol and purified by Protein A affinity chromatography followed by size exclusion chromatography (SEC) to a purity of >95% as confirmed by SEC-HPLC analysis.

[0251] 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", a CM3 sensor chip (GE, Cat. No. 29-1049-90) coated with amine-coupled goat anti-human IgG was used to capture the test antibodies at seven different densities. Soluble human CD39 ECD His-tagged antigen (Sino Biological, Cat. No. 16020-H08B) was analyzed: a stock solution prepared at 33.3 μM was diluted to 33 nM as the highest concentration and tested in triplicate in a 3-fold dilution series on the antibody surface. In the "bivalent avidity mode", soluble human CD39 ECD His-tagged antigen was amine-coupled to a CM3 sensor chip at six different densities. Test antibodies against the analytes were prepared as 33.3 μM stock solutions, diluted to 100 nM as the highest concentration, and tested in triplicate in a 3-fold dilution series on the CD39 surface. 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 with multiple injections of increasing concentrations of analyte, and the dissociation phase was monitored for 1 hour. All data were collected at 25° C. Response data from all six or seven surface densities were fitted to a 1:1 interaction model using a local Rmax, which is the response to the maximum amount of complex formed. Results from experiments performed in monovalent affinity mode are shown in Table 10, and results from bivalent avidity mode are shown in Table 11.

[0252] Antibodies hu31.4_IGG1.AA and hu39.5_IGG1.AA had similar equilibrium dissociation constants (K DHowever, when analyzed in a mode allowing bivalent antibody binding using CD39 ECD captured on a sensor chip and soluble antibody as analyte, hu31.4_IGG1.AA had a K very similar to that obtained when analyzed in monovalent mode, independent of antigen surface density. D In contrast, hu39.5_IGG1.AA showed up to 3 orders of magnitude lower K depending on antigen surface density. D , indicating that this antibody can bind to multiple immobilized CD39 molecules on the chip surface in a bivalent mode, thus improving the kinetic binding affinity with avidity.

[0253] [Table 10]

[0254] [Table 11]

[0255] Example 4: Binding of anti-CD39 antibodies to endogenous human CD39 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 μM).

[0256] Purified monocyte binding: The antibody was bound to primary human monocytes (CD14 +) to human CD39 expressed on the cell surface. Monocytes were positively selected from peripheral blood (leukopak or LRS chamber) using the EasySep Human CD14 Positive Selection Kit II (Stem Cell, Cat. No. 17858), frozen in Bambanker (Wako, Cat. No. 302-14681), and stored in liquid nitrogen for future use. On the day of the assay, frozen monocytes were thawed, rinsed, resuspended in 1:25 Fc block (BD, Cat. No. 564220) and 1:250 Aqua viability dye (Invitrogen, Cat. No. L34966A) in DPBS, and incubated for 15 min at room temperature in the dark. Cells were plated at 0.2–0.5 × 10 per well in a polypropylene v-bottom 96-well plate. 6 Cells were plated with 100 μM PE. Zero or 400 μM ATP was added and incubated at 4°C for 30 min, followed by the addition of test antibodies and further incubation at 4°C for 30 min. After washing twice with DPBS, 1 μg / mL PE-labeled mouse anti-human IgG4 secondary (Southern Biotech, Cat. No. 9200-09) was added for 30 min at 4°C for antibody detection. After washing twice with DPBS, cells were fixed with 50 μL IC fixation buffer (Invitrogen, Cat. No. 00-8222-49) for 20 min at room temperature. Cells were then washed once, resuspended in DPBS, and subjected to flow cytometry using a BD LSR FORTESSA X-20 Cell Analyzer. The geometric mean of PE fluorescence intensity was obtained for the live single-cell population. EC was calculated using standard 4-parameter curve fitting in GraphPad Prism. 50 The values ​​were calculated (Table 12).

[0257] [Table 12]

[0258] PBMC binding: Recombinant and IgG1 isotype variants of antibody 39 also bind to CD14 +(monocytes) and CD19 + PBMCs gated on the (B cell) population were tested for binding to human CD39 expressed on the cell surface. In this experiment, an antibody reported to bind and inhibit human CD39, referred to herein as huBMK2_IGG1.AEASS, was used as a comparison. 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 and frozen PBMCs were thawed, rinsed, and resuspended in 1:25 Fc block (BD, Cat. No. 564220) and 1:250 Aqua Viability Dye (Invitrogen, Cat. No. L34966A) in DPBS and incubated for 15 minutes at room temperature in the dark. Cells were then washed with DPBS and resuspended in filtered staining buffer: DPBS supplemented with 5% goat serum (Gibco, Cat. No. 16210-064). In a polypropylene v-bottom 96-well plate, cells were plated at 0.5 x 10 cells per well in staining buffer. 6 Cells were plated with 100 μM ATP. Zero or 400 μM ATP was added and incubated at 4° C. for 30 min, followed by the addition of test antibodies and further incubation at 4° C. After two washes with staining buffer, 0.8 μg / test CD14 FITC (Invitrogen, Cat. No. 11-0149-42, clone 61D3) and 0.5 μg / test CD19 eFluor 450 (eBioscience, Cat. No. 48-0199-42, clone HIB19) were added to the PBMCs. CD39 binding by the test antibodies was detected with 0.5 μg / mL PE-labeled goat anti-human IgG (Southern Biotech, Cat. No. 2048-09) for 30 min at 4° C. Cells were then washed, resuspended in DPBS, and subjected to flow cytometry using a BD LSR FORTESSA X-20 Cell Analyzer. The geometric mean of the PE fluorescence intensity was calculated as the mean of the monocyte (CD14 + ) or B cells (CD19 + ) were obtained for live single-cell populations gated on either EC50 Values ​​were calculated (Table 13). A representative graph from one donor is shown in FIG.

[0259] [Table 13]

[0260] Overall, antibody 19, 31, and 39 variants showed strong binding to the surface of primary cells. Potency was determined to be 39 variant > 31 variant > 19 variant. The potency ranking of 31 and 39 variants was maintained with an isotype switch from IGG4.P to IGG1.AA, and potency was maintained in the presence of high ATP. The humanized IgG1 variant of antibody 31, hu31.4_IGG1.AA, and the humanized IgG1 variant of antibody 39, hu39.5_IGG1.AA, showed stronger binding compared to the previously mentioned anti-CD39 antibodies, with hu39.5_IGG1.AA > huBMK2_IGG1.AEASS > hu31.4_IGG1.AA.

[0261] Example 5: Inhibition of human CD39 enzymatic activity Variants of 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 cell surface of primary human cells derived from patient tumor samples or human cell lines. Inhibition was assessed in the presence of low and high ATP (20 μM and 400 μM, respectively).

[0262] Recombinant CD39 Inhibition: The ability of the antibodies described in the previous examples to inhibit CD39 enzymatic activity was first assessed using recombinant human CD39. Recombinant human CD39 biochemical assays were performed in 25 mM Tris-HCl, pH 7.4, 5 mM MgCl 2The assay was performed in assay buffer consisting of 0.625 nM recombinant human CD39 and 20 μM ATP substrate. After incubation at 37° C. for an additional 30 minutes, 25 μL of KinaseGlo Plus reagent was added to each well of the 384-well plate. Reactions were measured using the Kinase-Glo reagent kit (Promega, catalog number V3771) according to the manufacturer's protocol. The amount of ATP remaining after CD39 inhibition was assessed and quantified as a function of the luminescence generated using an Envision 2102 Multilabel reader equipped with an emission filter. CD39 enzyme activity was assessed as a function of remaining ATP levels. The percentage of maximum activity in each test well was calculated based on the assay buffer (maximum activity, 0% inhibition) and control wells without enzyme (baseline activity, 100% inhibition). IC of test antibodies 50 Values ​​were determined from dose-response curves fitted using a standard four-parameter fit equation. The data are shown in Table 14.

[0263] [Table 14]

[0264] SK-MEL-5 CD39 Inhibition: Inhibition of CD39 enzyme activity was also assessed using SK-MEL-5 cells, a melanoma cell line established from a patient-derived tumor sample. On the day of the experiment, one vial of previously frozen SK-MEL-5 cells was thawed and resuspended in 20 mM HEPES, pH 7.4, 137 mM NaCl, 5.4 mM KCl, 1.3 mM CaCl. 2, 4.2 mM NaHCO 3 Cells were resuspended in 10 mL of assay buffer consisting of 100 mM cDNA, 0.1% glucose, and 0.1% ATP. A 14-point, 1:3 master serial dilution of anti-CD39 antibody was prepared in assay buffer to span the final concentration range of 100 nM to 0.063 pM. Twenty microliters of test antibody at 5x the 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 were added to each well and the plate was incubated at 37°C for an additional 60 minutes before adding 40 μL of 50 μM ATP in assay buffer. Final assay conditions consisted of 10,000 cells per well and 20 μM ATP substrate. Incubate at 37°C and 5% CO 2 After further incubation at 4°C for 60 min, the plates were centrifuged at 225×g for 10 min. Thirty microliters of the supernatant was transferred to a 96-well assay plate (Corning, Cat. No. 3912) and the amount of ATP remaining in the reaction was measured using a Kinase-Glo reagent kit (Promega, Cat. No. V3771) according to the manufacture's protocol. The amount of ATP remaining after CD39 inhibition was assessed and quantified as a function of the luminescence generated using an Envision 2102 Multilabel reader equipped with an emission filter. CD39 enzyme activity was assessed as a function of the remaining ATP levels. The percentage of maximum activity in each test well was calculated based on the assay buffer (maximum activity, 0% inhibition) and the cell-free control wells (baseline activity, 100% inhibition). IC of test antibodies 50 Values ​​were determined from dose-response curves fitted using a standard four-parameter fit equation. Data are shown in Table 15.

[0265] [Table 15]

[0266] THP-1 CD39 Inhibition: Inhibition of CD39 enzymatic activity was evaluated 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 resuspended in 20 mM HEPES, pH 7.4, 137 mM NaCl, 5.4 mM KCl, 1.3 mM CaCl. 2 , 4.2 mM NaHCO 3 Cells were resuspended in 10 mL of assay buffer consisting of 0.1% ATP, 0.02% ATP, and 0.1% glucose. A 14-point, 1:3 master serial dilution of anti-CD39 antibody was prepared in assay buffer to span the final concentration range of 100 nM to 0.063 pM. Twenty microliters of test antibody at 5x the final concentration in assay buffer was added to each well of a 96-well round-bottom polypropylene plate. Forty microliters of THP-1 cells were added to each well and the plate was incubated at 37°C for an additional 60 minutes before adding 40 μL of 50 μM ATP in assay buffer. Final assay conditions consisted of 40,000 cells per well and 20 μM ATP substrate. Incubate at 37°C and 5% CO 2 After further incubation at 4°C for 60 min, the plates were centrifuged at 225×g for 10 min. Thirty microliters of the supernatant was transferred to a 96-well assay plate (Corning, Cat. No. 3912) and the amount of ATP remaining in the reaction was measured using a Kinase-Glo reagent kit (Promega, Cat. No. V3771) according to the manufacture's protocol. The amount of ATP remaining after CD39 inhibition was assessed and quantified as a function of the luminescence generated using an Envision 2102 Multilabel reader equipped with an emission filter. CD39 enzyme activity was assessed as a function of the remaining ATP levels. The percentage of maximum activity in each test well was calculated based on the assay buffer (maximum activity, 0% inhibition) and the cell-free control wells (baseline activity, 100% inhibition). IC of test antibodies 50 Values ​​were determined from dose-response curves fitted using a standard four-parameter fit equation. Data are shown in Table 16.

[0267] [Table 16]

[0268] Purified monocyte CD39 inhibition: Potency was further evaluated using primary human monocytes. + Thaw two vials of positively selected human monocyte (donor 1003773) cells and culture in 20 mM HEPES, pH 7.4, 137 mM NaCl, 5.4 mM KCl, 1.3 mM CaCl 2 , 4.2 mM NaHCO 3 Cells were resuspended in 10 ml of assay buffer consisting of 0.1% glucose, 0.01% ATP, and 0.1% glucose. A 14-point, 1:3 master serial dilution of the test antibody was prepared in assay buffer to span the final concentration range of 200 nM to 0.13 pM. Twenty microliters of test antibody at 5x the final concentration in assay buffer was added to each well of a 96-well round-bottom polypropylene plate. Forty microliters of human monocytic cells were added to each well and the plate was incubated at 37°C for an additional 60 minutes before adding 40 μL of 50 μM or 1000 μM ATP in assay buffer. Final assay conditions consisted of 20,000 cells per well and 20 μM or 400 μM ATP substrate. Incubate at 37°C and 5% CO 2After further incubation at 4°C for 60 min, the plates were centrifuged at 225×g for 10 min. Fifteen microliters of the supernatant was transferred to a 96-well assay plate (Corning, Cat. No. 3912) and the amount of AMP generated in the reaction was measured using the AMP-Glo ​​Reagent Kit (Promega, Cat. No. 5012) according to the manufacture's protocol. Supernatants from the reaction plates containing 400 μM ATP substrate were transferred after dilution 1:8 with assay buffer and AMP-Glo ​​Reagent was added. The amount of AMP generated after CD39 inhibition was assessed and quantified as a function of luminescence generated using an Envision 2102 Multilabel reader equipped with an emission filter. CD39 enzyme activity was assessed as a function of AMP levels. The percentage 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). IC of test antibodies 50 Values ​​were determined from dose-response curves fitted using a standard four parameter fitting equation. The data are shown in Tables 17 and 18 and in FIG.

[0269] [Table 17]

[0270] [Table 18]

[0271] Overall, antibody variants 19, 31, and 39 demonstrated potent inhibition of soluble and surface human CD39 enzymatic activity. Importantly, potency was maintained in the presence of high ATP.

[0272] Example 6: Immunostimulatory effects of CD39 blockade To assess the efficacy of the anti-CD39 antibodies produced in Examples 1-3, the downstream functional consequences of inhibiting CD39 enzymatic activity on the surface of bone marrow cells were evaluated in vitro.

[0273] Macrophage IL-1β / IL-18 release assay: The NLRP3 inflammasome is a multiprotein cytosolic complex that upon oligomerization can cleave and activate the pro-caspase-1 protein to become caspase-1. Caspase-1 then promotes the maturation of IL-1β and IL-18 by cleaving their inactive pro-isomers (pro-IL-1β and pro-IL-18) to their active and secreted forms. These cytokines are involved in the innate immune response and create a general pro-inflammatory environment.

[0274] Anti-CD39 antibodies were tested for their ability to activate the NLRP3 inflammasome on macrophages by measuring secretion of mature IL-1β and IL-18. A schematic of the assay is shown in Figure 3. On day 0, previously isolated and positively selected CD14 + Monocytes (see Example 4) were thawed and plated at approximately 15 × 10 cells per 10 cm dish (Thermo Scientific Nunclon Delta Surface, Catalog No. 150464) in RPMI medium containing 10% heat-inactivated FBS, 1% Glutamax, 1% penicillin / streptomycin, and 50 ng / mL recombinant human M-CSF (R&D, Catalog No. 216MCC / CF). 6 CD14 + On day 5, non-adherent cells were washed away and adherent macrophages were collected by scraping and plated in 24-well plates at a density of 0.125 × 10 per well. 6 The cells were plated at a density of 10 x 100 cells / well. The cells were then incubated overnight to allow attachment. The next day, the cells were treated with 1 ng / mL LPS (InvivoGen, Cat# tlrl-peklps) followed by 10 or 100 nM of test antibody or IgG control and incubated at 37°C, 5% CO 2 The cells were then treated with 500 μM ATP (Life Technologies, Cat. No. R0441) and incubated at 37° C., 5% CO 2The cells were incubated for an additional 2–4 h at 4°C for 1 h. Supernatants were collected and IL-1β levels were analyzed by CBA (BD, Cat. No. 558279) or ELISA (R&D, Cat. No. QK201) according to the manufacturer's instructions. IL-18 levels in the supernatants were analyzed by ELISA (R&D, Cat. No. DL180) according to the manufacturer's instructions. Experiments were performed in technical quadruplicates per donor, averaged, and then normalized to isotype controls for each donor. As shown in Figure 4, ch39_IGG4.P, hu39.1_IGG4.P, hu39.5_IGG1.AA, and hu31.4_IGG1.AA increased the release of IL-1β and IL-18 from in vitro-derived macrophages compared to isotype controls.

[0275] 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. No. 215-GM / CF), and 100 ng / mL IL-4 (Peprotech, Cat. No. 200-04) and plated at 4 x 10 cells per well in a 6-well plate (Falcon, Cat. No. 353046). 6 On day 6, moDCs were harvested and plated in 24-well Upcell plates (Thermo Fisher, Cat. No. 174899) at 0.5 × 10 cells per well. 6 Cells were seeded at 10 μg / mL and incubated with either isotype control or anti-CD39 antibody at 37°C, 5% CO 2 After treatment with 0 or 300 μM ATP for 1 h, the cells were incubated at 37 °C in 5% CO 2moDCs were treated with 100 mM EDTA for 18 hours. 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 Aqua viability dye (Invitrogen, Cat# L34966A) in DPBS and incubated for 15 minutes at room temperature in the dark. After washing once with DPBS, cells were stained with the following antibodies [1 μg / test CD83 PE-Cy7 (BD, Cat. No. 561132), 1 μg / test CD86 BV605 (BD, Cat. No. 562999), 0.8 μg / test CD14 FITC (Invitrogen, Cat. No. 11-0149-42)] or corresponding isotype controls [PE-Cy7 Mouse IgG1 Kappa (BD, Cat. No. 565573), BV605 Mouse IgG1 Kappa (BD, Cat. No. 562652), FITC Mouse IgG1 Kappa (eBiosciences, Cat. No. 11-4714-41)] for 30 min at 4° C. Surface levels of dendritic cell maturation markers CD83 and CD86 should increase with dendritic cell maturation, whereas monocyte marker CD14 should decrease. After one wash with DPBS, cells were fixed with 50 μL IC Fixation Buffer (Invitrogen, Cat. No. 00-8222-49) for 20 min at room temperature. Cells were then washed, resuspended in DPBS, and subjected to flow cytometry using a BD LSR FORTESSA X-20 Cell Analyzer. For CD83 and CD14, the percentage of the parent gate was set to the isotype control (<5% of total events) for each donor in the 0 μM ATP condition. For CD86, >90% of cells were positive compared to the isotype control in the control treatment condition, so instead the geometric mean of the fluorescence intensity of the fluorophore was reported for the live single cell population.

[0276] As shown in Figures 5A and 5B, ATP matured moDCs as evidenced by an increase in dendritic cell maturation markers CD83 and CD86. Inhibition of CD39 enzymatic activity by addition of anti-CD39 antibody promoted maturation, but not addition of an isotype control, which further increased the percentage of cells expressing CD83 and the surface amount of CD86 per cell. Addition of anti-CD39 in the presence of ATP also decreased the percentage of cells expressing the monocyte marker CD14 (Figure 5C). With the exception of hu39.6_IGG4.P, the effects on CD83, CD86, and CD14 were ATP-dependent effects.

[0277] Overall, the above data suggest that enzymatic inhibition of human CD39 by the antibodies of the present disclosure has immunostimulatory effects on different myeloid populations in vitro. Example 7: Antibody characterization by competitive flow

[0278] This example describes a competition flow assay that is useful for determining whether two antibodies (a test antibody and a reference antibody) compete for binding to human CD39 expressed on the surface of a cell. The choice of test antibody, reference antibody, and cell type can be varied.

[0279] Antibody hu39.1_IGG4.P and antibody IGG4.P, IgG4 (S228P) isotype control (CrownVivo) were directly conjugated to Alexa Fluor 647 ("AF647") using AlexaFluor647 Conjugation Kit-Lightning-Link (Abcam, Catalog No. ab269823). The conjugated antibodies are referred to in this example as "hu39.1_IGG4.P-AF647" and "IGG4.P-AF647". Binding validation was performed using MOLP-8, a human multiple myeloma, CD39 expressing cell line. On the day of the assay, frozen MOLP-8 cells were thawed, rinsed, and resuspended in 1:25 Fc Block (BD, Cat. No. 564220) and 1:250 Aqua Viability Dye (Invitrogen, Cat. No. L34966A) in DPBS and incubated for 15 min at room temperature in the dark. Cells were plated at 0.2 × 10 per well in a polypropylene v-bottom 96-well plate. 6 Cells were plated with AF647. An 11-point dose-response curve was prepared for each antibody by 3-fold serial dilution in DPBS, with concentrations ranging from 833 to 0.0141 nM. The 12th point contained only DPBS and no antibody. Diluted antibodies from the dose-response were added to the plated cells in a 1:1 ratio and incubated at 4°C for 30 min. Cells were then washed three times, resuspended in DPBS, and subjected to flow cytometry using a BD LSR FORTESSA X-20 Cell Analyzer. Mean fluorescence intensity of AF647 was obtained for the live single-cell population. EC 50 Value and EC 95 Values ​​were calculated using standard 4-parameter nonlinear regression analysis in GraphPad Prism software.

[0280] Antibody hu39.1_IGG4.P-AF647 was then used as a reference antibody in a competition flow assay with unlabeled 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, resuspended in 1:25 Fc block (BD, Cat. No. 564220) and 1:250 Aqua viability dye (Invitrogen, Cat. No. L34966A) in DPBS and incubated for 15 min at room temperature in the dark. An 11-point dose-response curve was prepared for each test antibody by 3-fold serial dilution in DPBS, with concentrations ranging from 833 to 0.0141 nM. The 12th 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 the reference antibody hu39.1_IGG4.P-AF647 at a concentration of 93.7 nM was added to each well and mixed gently three times. 0.2 × 10 cells per well 6 Cells were plated with AF647 and incubated at 4°C for 60 min. Cells were then washed three times, resuspended in DPBS, and subjected to flow cytometry 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 Values ​​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 hu39.1_IGG4.P-AF647 only). Only a single concentration of the reference antibody hu39.1_IGG4.P-AF647 was used.

[0281] As shown in Figure 6 and Table 19, the test antibody hu39.5_IGG1.AA competes with the reference antibody hu39.1_IGG4.P-AF647, with a maximum competition of 93%. A similar level of competition was observed with hu39.1_IGG4 (unlabeled reference antibody). In contrast, no competition was observed between the test antibody hu31.4_IGG1.AA antibody and the reference antibody hu39.1_IGG4.P-AF647. These data suggest that antibodies 31 and 39, and their variants, bind to different non-overlapping epitopes.

[0282] [Table 19]

[0283] Example 8: Efficacy in xenograft models Anti-CD39 antibodies produced as described in Examples 1 and 2 bind to human CD39 but not to mouse CD39. Therefore, xenograft mouse models can be used to assess efficacy in vivo. 0.5-10 million human cancer cells expressing human CD39 are subcutaneously or orthotopically injected into immunodeficient mice, such as SCID or nude mice, and tumor growth is monitored. Anti-CD39 antibodies can bind to and inhibit the activity of CD39 in several human cancer cell lines, including but not limited to MOLP-8 myeloma (Figure 7), THP-1 leukemia (Table 16), SK-MEL-5 melanoma (Table 15), and OAW42 ovarian cancer cells. When tumors reach 150 mm 3 If the tumor volume is less than 2,000 mm, the mice are treated with 0.1-30 mg / kg of an anti-CD39 antibody of the present disclosure or isotype control BIWx4, or 3 The tumors are treated intraperitoneally (ip) until a total of 100% of the tumors expressing human CD39 are reached. Treatment of MOLP-8 tumors expressing human CD39 with the anti-CD39 antibodies of the present disclosure resulted in tumor growth inhibition compared to isotype control treated mice. Tumor growth inhibition compared to isotype control treated mice is also expected following treatment of other tumor types expressing human CD39 with the anti-CD39 antibodies of the present disclosure.

[0284] Example 9: Efficacy in human CD39 knock-in model To evaluate anti-CD39 antibodies of the present disclosure in immune-competent mouse models, the antibodies can be tested in a human CD39 knock-in (hCD39KI) mouse model, where fully immune-competent C57BL / 6 or BALB / c mice express human CD39 but not mouse CD39 (Figure 9). In this mouse model, expression of human CD39 parallels that of mouse CD39 in wild-type mice. This mouse model allows for in vivo testing where anti-CD39 inhibition may act on host cells such as stromal cells, vasculature, and immune cells. Mice are inoculated subcutaneously or orthotopically with mouse wild-type or engineered tumor cell lines, including but not limited to MCA-205 fibrosarcoma, B16-F10 melanoma, MC38 colon adenocarcinoma, ID8 ovarian carcinoma, 4T1 breast carcinoma, or CT-26 colon carcinoma cells. 500,000 to 10 million mouse cancer cells are injected and tumor growth is monitored. The tumor is 150 mm 3 Mice were treated with 0.1–30 mg / kg ch39_mIGG2A.AAG or isotype control BIWx4 or with tumors with a volume of 2,000 mm 3 Treatment of tumors with anti-CD39 as a single agent or in combination with one or more additional therapies (e.g., ICD inducers, immunotherapies, etc.) inhibits tumor growth compared to isotype control treated mice.

[0285] In one experiment, C57BL / 6 hCD39KI mice were inoculated with 1 million MC38 cells on day 0. Tumors grew to 80 mm 3At the time of reaching 100 mg / kg, mice were administered a loading dose of 20 mg / kg anti-CD39 or isotype control antibody (vehicle: PBS) intraperitoneally (ip) followed by 10 mg / kg anti-CD39 or isotype control antibody twice / week (BIW). Each group consisted of 11–12 mice. In these experiments, anti-CD39 antibody refers to ch39_mIGG2A.AAG. Oxaliplatin (OXA) treatment at 5 mg / kg ip (vehicle: saline) was started the day after the loading dose of anti-CD39 or isotype control and was administered once / week. In a similarly designed experiment, BALB / c hCD39KI 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.

[0286] Anti-CD39 shows efficacy in the hCD39KI model To assess the efficacy of anti-CD39 treatment, tumor volume, intratumoral and peripheral CD39 enzyme activity, and CD39 expression were assessed.

[0287] Tumor volumes and body weights were measured approximately twice weekly (BIW) and are shown in Figure 10A and Figure 10B, respectively, where points represent the mean and error bars represent the standard error of the mean (SEM) of each metric from each group of mice. Significance was calculated using a mixed-effects model with multiple comparisons. As shown in Figure 10A, treatment with anti-CD39 and oxaliplatin was statistically significant (p=0.0217) compared to treatment with either single agent alone (p=0.0372 for isotype+OXA vs. anti-CD39+OXA, and p=0.0217 for anti-CD39+saline vs. anti-CD39+OXA). Body weights remained stable throughout the course of the experiment, and anti-CD39 treatment was well tolerated (Figure 10B).

[0288] Tumors and spleens from C57BL / 6 hCD39KI MC38 mice at the end of the study were further analyzed for evidence of human CD39 expression by immunohistochemistry (IHC) and anti-CD39 antibody enzyme inhibitory activity by enzyme histochemistry (EHC). Briefly, frozen OCT-embedded tumor or spleen samples were sectioned at a thickness of 4um, mounted on positively charged microscope slides, air dried for 15 minutes, and then placed in 4% paraformaldehyde (PFA) fixative for 20 minutes. After fixation, slides were washed with Tris-buffered saline and IHC was performed on an automated Leica Bond Rx platform, beginning with blocking of endogenous peroxidase, followed by incubation with rabbit anti-CD39 mAb (Abcam, EPR20627), detection with anti-rabbit HRP polymer system, followed by diaminobenzidine (DAB) brown chromogen, hematoxylin nuclear counterstain, and then dehydrated and mounted. For the EHC assay, after fixation with 4% PFA for 5 min, sections were washed with Tris-maleate buffer and then resuspended in 50 mM Tris-maleate, 250 mM sucrose, and 2 mM MgCl 2 The sections were then incubated at room temperature for 30 min in a solution of 1 mM ATP, 2.5 mM levamisole, 2 mM lead nitrate, 0.25 mM sucrose, and 5 mM MnCl 2The sections were incubated for 2 hours at 37°C in a solution of 100 mM Tris maleate containing 100 mM acetylated ketone. Levamisole was used to inhibit tissue non-specific alkaline phosphatase (TNAP) activity, but other inhibitors can also be used. Sections were then developed for 5 minutes in 1% ammonium sulfide solution, washed, counterstained with hematoxylin, dehydrated, and mounted. Representative images are shown in Figures 11-14 (tumor, Figures 11-12; spleen, Figures 13-14). Human CD39 expression was detected in both isotype-treated tumors (Figure 11, Panel A and Figure 12, Panel A, low and high power magnification, respectively) and anti-CD39-treated tumors (Figure 11, Panel C and Figure 11, Panel C, low and high power magnification, respectively) and was localized to vascular endothelium, infiltrating immune cells, and stromal components of the tumor microenvironment. Human CD39 expression levels were observed in both isotype-treated spleens (Figure 13, Panel A and Figure 14, Panel A, low and high power magnification, respectively) and anti-CD39-treated spleens (Figure 13, Panel C and Figure 14, Panel C, low and high power magnification, respectively) and were localized to the red pulp and, to a lesser extent, the white pulp. Lead phosphate deposition is observed by enzyme histochemistry in tumors and spleens treated in vivo with isotype (tumors-Figure 11, Panel B and Figure 12, Panel B, low and high power magnification, respectively; spleens-Figure 13, Panel B and Figure 14, Panel B, low and high power magnification, respectively). In tumors and spleens treated in vivo with anti-CD39 antibody, no lead phosphate deposits were observed by enzyme histochemistry, indicating that enzyme activity was blocked (tumors--FIG. 11, panel D and FIG. 12, panel D, low and high power magnification, respectively; spleens--FIG. 13, panel D and FIG. 14, panel D, low and high power magnification, respectively).

[0289] Intratumoral and peripheral CD39 enzymatic activity was also assessed ex vivo. After completion of the study, C57BL / 6hCD39KI MC38 mice and BALB / c hCD39KI 4T1 mice were sacrificed and tumor samples obtained were placed in RPMI+20%FBS+250μg / ml collagenase D (Millipore Sigma, Cat. No. 11088858001)+100KU / ml DNAse1 (MilliporeSigma Cat. No. D5025-150KU) and enzymatically digested using a gentleMACS™ Octo Dissociator to obtain a single cell suspension. Cells were plated at 2.510 μg / ml per well of a 96-well plate. 4 Cells were seeded and treated with 100 nM of hu39.5_IGG1.AA or IgG1 Fc silent isotype control antibody (Absolute Antibody, Cat. No. Ab00178-10.3) at 37°C and 5% CO 2 The cells were then treated with 20 μM ATP and incubated at 37° C., 5% CO 2 The 96-well plates were incubated at 37° C. for an additional 30 minutes. At the end of the incubation period, the 96-well plates were spun at 1000 rpm for 3 minutes to collect the supernatant. 2X Kinase-Glo® Plus (Promega, Cat. No. V3771) was then added to the supernatant. ATP levels in the supernatant were then measured by detecting luminescence using a Flexstation® 3 microplate reader. Data are presented as relative luminescence units (RLU). The lack of difference between RLU in the ex vivo hu39.5_IGG1.AA and isotype treatment groups indicates complete enzyme activity inhibition by in vivo treatment. Anti-CD39 treatment inhibited enzyme activity in the tumors (FIGS. 15A and 15B).

[0290] To assay peripheral CD39 enzyme activity, peripheral white blood cells (WBCs) were isolated from terminal blood samples of hCD39KI 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. No. 555899). Then, after washing with 1X PBS solution, the WBC pellet was collected and stored in BamBanker cell freezing medium (Bulldog Bio, Cat. No. BB01). To perform the enzyme inhibition assay, samples were thawed and cells were then frozen at 2.5 × 10 per well of a 96-well plate. 4 Cells were plated with 100 nM hu39.5_IGG1.AA. Each sample was incubated for 1 hour at room temperature with two treatment conditions: 100 nM hu39.5_IGG1.AA or IgG1 Fc silent isotype control antibody (Absolute Antibody, Cat. No. Ab00178-10.3). All wells were then treated with 20 μM ATP and incubated for 2 hours at room temperature. At the end of the incubation period, the 96-well plate was spun at 1000 rpm for 3 minutes to collect the supernatant. 2X Kinase-Glo® Plus (Promega, Cat. No. V3771) was then added to the supernatant. ATP levels in the supernatant were then measured by detecting luminescence using a Flexstation® 3 microplate reader. Data are presented as relative luminescence units (RLU). The lack of difference between the RLU in the ex vivo hu39.5_IGG1.AA and isotype treatment groups indicates complete enzyme activity inhibition by the in vivo treatment. Anti-CD39 treatment inhibited enzyme activity in the periphery of 4T1 tumor-bearing mice (FIG. 15C).

[0291] Reduction of cell surface CD39 in peripheral immune populations in hCD39KI mice, and complete or near complete coverage by anti-CD39 treatment. Whole blood samples from terminal bleeds of C57BL / 6 hCD39KI MC38 mice were assessed by flow cytometry to determine the number of monocytes (CD3 - CD19 - CD11b + Ly6c ++ ), granulocytes (CD3- CD19 - CD11b + Ly6c + ), T cells (CD3 + CD19 - ), B cells (CD3 - CD19 + ), and other lineage-negative (Lin -) Target coverage and changes in CD39 expression were assessed in peripheral immune cell types that express CD39, including lymphocytes. Target binding was measured using hu39.5_IGG1.AA-AF647, a reagent that competitively binds 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 (competing for anti-CD39), CD39A1 PE (non-competing for 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 on ice in the dark, and then samples were fixed / lysed for 20 minutes on ice in the dark using 1X BD FACSLysing solution. Samples were then centrifuged at 400 x g for 5 minutes, washed once with 1X PBS buffer, resuspended in 1% paraformaldehyde solution, and filtered through a 20 μm strainer before data acquisition. Samples were run on a BD FACSCanto SpecialOrder cytometer and analyzed using FlowJo and Prism software. Unbound CD39 was detected by competitive clone hu39.5_IGG1.AA-AF647 (MFI of hu39.5_IGG1.AA-AF647 signal measured against its isotype control IgG1.FcS-AF647) across cell types in all animals treated with isotype antibodies. Significant competitive binding of hu39.5_IGG1.AA-AF647 was observed on all CD39(A1) expressing cells in animals treated with anti-CD39, showing complete or near complete target coverage (Figure 16, bottom panel). Total CD39 surface protein was detected by a non-competitive CD39 antibody (clone A1 PE). Reduction of surface CD39 protein was observed on multiple types of peripheral immune cells in mice treated with anti-CD39 (Figure 16, top panel).

[0292] Cell surface CD39 is reduced by anti-CD39 treatment in tumor-draining lymph nodes of hCD39KI mice. Tumor-draining lymph nodes (TLDN) (right inguinal lymph nodes) were also collected at the end of the C57BL / 6 hCD39KI MC38 mouse study described above. Lymph nodes were dissociated in RPMI 1640 (Gibco, Catalog No. 11875119) containing 0.6 mg / mL collagenase P (Roche, Catalog No. 11213865001), 2.4 mg / mL dispase II (Sigma, Catalog No. D4693), and 0.3 mg / mL DNAse I (Thermo Scientific, Catalog No. J62229MB) in a 37°C water bath for 30 minutes with gentle agitation. Dissociated lymph nodes were counted and 2 x 10 per well were collected. 5of cells were plated in 96-well polypropylene v-bottom plates. Cells were washed with PBS (Gibco, Catalog No. 14190-144) and resuspended in PBS solution containing 1:25 Fc Block (BD, Catalog No. 564220) and 1:250 Near-infrared fluorescent reactive dye (Invitrogen, Catalog No. L34976A) for 15 minutes in the dark. After incubation, cells were stained with antibody cocktail (Table 20) and incubated at 4°C for 30 minutes. After incubation, cells were pelleted, the supernatant was removed, and 100 μL of fixation buffer (eBioscience, Catalog No. 00-8222-49) was added for 1 hour. Cells were washed, resuspended in 200 μL PBS, filtered through a 40 μm filter plate (Fisher Scientific, Catalog No. NC0726512), acquired on a BD LSR Fortessa X-20 Cell Analyzer, and analyzed with FlowJo™ v10.8 software (BD Life Sciences) to define immune subpopulations. Expression of human CD39, mouse CD73, and mouse P2X7 in the defined immune subpopulations is shown in Figures 17A and 17B. Anti-CD39 antibody ch39_mIGG2A.AAG, either as a single agent or in combination with oxaliplatin treatment, did not significantly alter the percentage of immune subpopulations in the tumor-draining lymph nodes of hCD39KI mice inoculated with MC38 tumor cells (Figure 17C). Despite the lack of change in this immune population, anti-CD39 treatment significantly reduced human CD39 on the surface of nearly all immune subpopulations identified from tumor-draining lymph nodes (Figure 17D). A representative histogram is shown in Figure 17E.

[0293] [Table 20]

[0294] Anti-CD39 treatment does not alter peripheral cytokine levels. Terminal bleeds of mice bearing MC38 tumors were collected in BD Vacutainer® heparin tubes (BD Biosciences, Cat. No. 367871), centrifuged at 2000xg for 15 min, and supernatant plasma samples were collected. Cytokine levels in plasma samples were analyzed using MSD according to the manufacturer's instructions. No increase in peripheral cytokine levels was observed in anti-CD39 treated mice. Data are presented as mean ± SEM with a total of 11-12 mice per treatment group (Figure 18). Statistical analysis was performed using ANOVA and Tukey's multiple comparison test.

[0295] Example 10: Use of anti-CD39 antibodies in combination with immunogenic cell death (ICD)-inducing therapy The ICD-inducing potential of chemotherapeutic agents was characterized in the absence or presence of anti-CD39 antibodies. Three metrics of ICD-inducing potential were used in this study: (1) cytotoxicity, (2) extracellular HMGB1 release, and (3) extracellular ATP release.

[0296] To assess cellular ATP release following oxaliplatin treatment, mouse colon cancer cell lines CT26 and MC38 and human melanoma cell line SK-MEL-5 were thawed, rinsed, and resuspended in their respective culture media. Culture media for CT26 and MC38 is RPMI+10% FBS+1% glutamax+1% penicillin-streptomycin+1% sodium pyruvate. Culture media for SK-MEL-5 is EMEM+10% FBS+1% glutamax+1% penicillin-streptomycin+1% sodium pyruvate. Cells were plated at 2.5×10 per well of a 96-well plate. 4 Plate the cells and incubate at 37°C, 5% CO 2The cells were incubated at 4°C overnight. The next day, cells were treated with vehicle (PBS), 100 μM or 250 μM oxaliplatin. SK-MEL-5 cells received an additional treatment of 100 nM hu39.5_IGG1.AA or IgG1 Fc silent isotype control antibody (Absolute Antibody, Cat. No. Ab00178-10.3). To determine cell viability, cells were incubated at 37°C, 5% CO 2 Cells were treated and incubated with 5% CO for 24 hours. At the end of the incubation period, 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 (FIG. 19A). To determine HMGB1 release, cells were incubated at 37° C., 5% CO 2 The cells were treated with and incubated with 5% CO for 24 hours. At the end of the incubation period, the plates were centrifuged at 400×g for 5 minutes and the supernatants were collected. HMGB1 levels in the culture supernatants were then determined using the Lumit HMGB1 kit (Promega, Cat. No. CS3030B01) according to the manufacturer's instructions ( FIG. 19B ). To examine ATP release, the cells were then incubated at 37° C., 5% CO 2 Cells were treated with hu39.5_IGG1.AA and incubated for 8 hours. At the end of the incubation period, 4x RealTime-Glo™ Extracellular ATP Assay (Promega Catalog No. GA5010) was added to the cells. Extracellular ATP was measured kinetically by detecting luminescence at 15 minute intervals over 6 hours using a Flexstation® 3 microplate reader. All three cell lines showed ATP release upon treatment with 250 μM oxaliplatin (FIG. 19C). In SK-MEL-5 cells, hu39.5_IGG1.AA treatment resulted in greater accumulation of extracellular ATP compared to isotype control treated cells (FIG. 19C).

[0297] The ICD-inducing potential of additional chemotherapeutic agents was characterized in 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 plated at 2.5 × 10 per well of a 96-well plate. 4 Plate the cells and incubate at 37°C, 5% CO 2 The following day, cells were treated with the chemotherapy agents doxorubicin (vehicle: DMSO), irinotecan (vehicle: DMSO), gemcitabine (vehicle: PBS), and cisplatin (vehicle: PBS) at the indicated concentrations and incubated at 37°C, 5% CO 2 The plates were incubated at 400×g for 48 h. (1) To assess cytotoxicity, cell viability was determined at the end of the incubation period using the luminescence-based Cell Titer Glo 2.0 assay (Promega, Cat. No. G9242). Data are presented as viability normalized to untreated vehicle control (FIG. 20A). (2) To measure HMGB1 release, the plates were centrifuged at 400×g for 5 min at the end of the incubation period and the supernatants were collected. HMGB1 levels in the culture supernatants were then determined using the Lumit HMGB1 kit (Promega, Cat. No. CS3030B01) according to the manufacturer's instructions (FIG. 20B). (3) Extracellular ATP release was quantified with chemotherapy, with and without 100 nM hu39.5_IGG1.AA or IgG1 Fc-silent isotype control antibody (Absolute Antibody, Cat. No. Ab00178-10.3), and incubated for 8 or 32 hours. At the end of the incubation period, 4x RealTime-Glo™ Extracellular ATP Assay (Promega Cat. No. GA5010) was added to the cells. Extracellular ATP release was then measured kinetically by detecting luminescence at 15-minute intervals over a 10-hour period using a Flexstation® 3 microplate reader. All chemotherapy treatment groups show increased ATP release compared to untreated cells. Addition of hu39.5_IGG1.AA results in higher levels of extracellular ATP (Figure 20C).

[0298] Example 11: Characterization of CD39 and CD39-related markers in human cells A CD39 whole blood (WB) receptor occupancy (RO) flow cytometry assay was used to evaluate CD39 expression on various cell types in human, cynomolgus monkey, and hCD39KI mouse WB. A description of the hCD39KI RO assay is described in Example 9. Human whole blood samples were analyzed by the same method described for hCD39KI mouse samples, but incorporating species-specific cell line markers for monocytes and B cells. The human CD39 WB RO panel consisted of the following antibodies: hu39.5_IGG1.AA-AF647 (competing with anti-CD39), anti-CD39A1 PE (non-competing with anti-CD39), anti-CD14 61D3 FITC, and -CD19 H1B19 eFluor450. As shown in Figure 21, the relative expression of CD39 on peripheral immune cells differs between humans and C57BL / 6 hCD39KI MC38 mice. Monocytes have the highest expression of CD39 among the cell types assayed in human blood, while granulocytes had the highest expression of CD39 in hCD39KI mice. B cell expression differs most significantly between species: human peripheral B cells express very high levels of CD39, whereas hCD39KI mouse B cells have low to no expression of CD39.

[0299] CD39 and CD39-related markers were also expressed in human in vitro differentiated bone marrow cells, such as M0-, M1-, M2-polarized macrophages, and monocyte-derived dendritic cells (moDCs), as well as in primary human bone marrow cells, such as CD14 cells from peripheral blood of four healthy donors. + The monocytic and myeloid dendritic cell (DC) subsets were characterized. 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, catalog no. 216-MCC / CF) and plated at 4 × 10 cells per well in a total of 2 mL in a 6-well plate (Falcon, catalog no. 353046). 6 On day 4, cells were replenished with 2 mL of fresh RPMI supplemented medium containing 2× M-CSF. On day 6, cells were polarized into M1 macrophages using 100 ng / mL LPS (Invivogen, Cat. No. tlr-peklps) and 20 ng / mL recombinant human IFN-γ (Peprotech, Cat. No. 300-02), while M2 macrophages were polarized with 20 ng / mL recombinant human IL-4 (Peprotech, Cat. No. 200-04). No additional cytokines were added to M0 macrophages. 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, catalog no. 215-GM / CF), and 100 ng / mL recombinant human IL-4, and plated in a 6-well plate (Falcon, catalog no. 353046) at 4 × 10 cells per well in a total of 2 mL. 6 On day 4, cells were replenished with 2 mL of fresh RPMI supplemented medium 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 cells isolated using a positive selection kit were + Monocytes and myeloid DCs enriched using a negative selection kit were also dispensed into U-bottom 96-well plates for flow cytometry or 1.5 mL Eppendorf tubes for RNA.

[0300] Perform flow cytometry quality control staining of primary CD14 +The purity of monocyte isolation, the purity of primary myeloid dendritic cell (DC) enrichment, and the distribution of DC subsets within cells resulting from the primary myeloid dendritic cell enrichment were determined. Monocytes were CD14 + It was defined by expression of CD14 + The purity of monocyte isolation was approximately 99%. Enrichment of primary dendritic cells from peripheral blood of four human donors demonstrated a 48.5% enriched population of dendritic cells, defined as HLA-DR+ CD11c+ and pDC. The enriched DC consisted of 2.8% plasmacytoid DC (HLA - DR + CD11c neg CD123 + pDC, defined as DC1 (HLA - DR + CD11c + CD141 + Clec9a + cDC1), 30.4% conventional DC2HLA - DR + CD11c + CD1c + CD141 neg Clec9a neg cDC2), and 12.0% of other DCs (HLA - DR + CD11c + CD1c neg CD141 neg Clec9a neg The 1990s was composed of a set of 1990s and 1991s.

[0301] For flow cytometric assessment of CD39 and CD73 (Figures 22A and 22B), CD14 +Monocytes, enriched myeloid DCs, M0, M1, M2 macrophages and moDCs were dispensed into 96-well plates and resuspended in 50 μL / well of 1:25 Fc block (BD, Cat. No. 564220) and 1:250 aqua viability dye (Invitrogen, Cat. No. L34966A) in DPBS and incubated in the dark at room temperature for 15 minutes. Cells were then stained with 50 μL / well of the following antibodies [CD39 PE-Cy7 (eBiosciences, Cat. No. 25-0399-42) at 0.25 μg / test, and CD73 BUV737 (BD, Cat. No. 612812) at 0.5 μg / test, or the corresponding isotype controls, Mouse IgG1 PE-Cy7 (BD, Cat. No. 557647) and Mouse IgG1 BUV737 (BD, Cat. No. 612758)] for 30 minutes at 4°C. After one wash with DPBS, cells were fixed with 50 μL IC Fixation Buffer (Invitrogen, Cat. No. 00-8222-49) for 20 minutes at room temperature. Cells were then washed, resuspended in DPBS, and subjected to flow cytometry using a BD LSR FORTESSA X-20 Cell Analyzer. CD14 + The positivity of CD39 and CD73 on monocytes, enriched myeloid DC subtypes, M0, M1, M2 macrophages, and moDCs was assessed by comparison with isotype controls. + Monocytes, all in vitro derived subsets (M0, M1, M2 macrophages, and moDC), as well as primary enriched DC subsets cDC1, cDC2, and other DCs all showed high percent positivity for CD39 and low percent positivity for CD73 (Figures 22A and 22B). Relatively few (15%) pDCs were CD39 positive. Given that the majority of cell types showed a percent positivity of CD39 >90%, CD39 expression levels were further investigated by examining the intensity of CD39 staining compared to isotype controls (Figure 22B). Although variability exists between donors, CD39 expression was significantly higher than CD14 compared to primary DCs. + It was high in monocytes, in vitro-derived macrophages, and moDCs. Among primary DCs, cDC1s had the highest expression of cell surface CD39.

[0302] ENTPD1 (CD39) and NT5E (CD73) gene expression was analyzed by qPCR using standard methods (Figures 22C and 22D). The level of ENTPD1 gene expression was higher than that of NT5E gene expression across all cell types. Enriched DC populations had the lowest relative expression of ENTPD1 and the highest relative expression of NT5E.

[0303] To assess a broader range of CD39-related gene expression in these human myeloid subsets, a custom NanoString panel was designed and utilized. RNA from each of the myeloid cell subsets was isolated using the Qiagen RNeasy Mini kit (catalog number 74106) according to the manufacturer's instructions. RNA was then quantified on a NanoDrop and 70ng of RNA was utilized for NanoString. To hybridize the RNA to the custom NanoString code sets, 70μL of hybridization buffer was added to the reporter code set and 8μL was added to 5μL of RNA for each sample. Two microliters of capture probe set was then added to each sample and incubated at 65°C for 16 hours in a thermocycler. The samples were then topped up to 30μL with RNase-free water, which was loaded into the sample loading port of the nCounter SPRINT cartridge (NanoString, catalog number 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 quality control tests were normalized to three housekeeping genes included within the custom NanoString panel to assess gene expression.

[0304] A heatmap showing unsupervised clustering of housekeeping gene-normalized expression of CD39, adenosine, and inflammasome pathway genes using a custom NanoString panel is shown in Figure 22E. Low quality wells flagged by low counts or aberrant control probes were excluded from this analysis. Specific donor IDs are appended to the end of the cell type labels. The dotted horizontal lines in the top dendrogram indicate where samples are split, but do not affect the unsupervised nature of the clustering. CD39 is expressed across all immune populations profiled. Cell types form distinct clusters, giving consistent expression of genes in these pathways within each cell type. Monocytes, M1 macrophages, and enriched primary DCs cluster together, driven by expression at IL-1β, P2X7, and A2AR. M0, M1 macrophages, and moDCs form other clusters with high expression of CD206, A2BR, and P2Y11.

[0305] Example 12: P2Y11 antagonism reduces ATP-driven dendritic cell activation The effects of P2X1, P2X7, and P2Y11 inhibitors on the activation of monocyte-derived dendritic cells (MoDCs) were examined. MoDCs were generated from positively selected human monocytes cultured with GM-CSF and IL-4 as described in Example 11. On day 7, 6-well plates were incubated on ice for 5 minutes, scraped to separate cells, and counted. Cells were resuspended in fresh medium containing GM-CSF and IL-4, and 125,000 cells in 100 μL were plated into each well of a 96-well flat-bottom Upcell plate (Nunc, Cat. No. 174897). Ten micromoles of each compound [P2X1 inhibitor, NF023, Millipore Sigma, Catalog No. 104869-31-0; P2X7 inhibitor, A-740003, Millipore Sigma, Catalog No. 861393-28-4; P2Y11 inhibitor NF340, APExBIO, Catalog No. B7508; P2Y11 inhibitor NF157, APExBIO Catalog No. B7060] or vehicle control (0.1% DMSO) was added, mixed gently, and incubated at 37°C with 5% CO. 2 Then, 0 or 300 μM ATP was added, mixed gently, and incubated at 37°C with 5% CO 2The cells were incubated at 4°C for 18 hours. For flow cytometry evaluation, the cells were detached from the plate and transferred to a 96-well polypropylene v-bottom plate. The cells were washed with PBS (Gibco, Cat. No. 14190-144) and resuspended in PBS solution containing 2% FBS, a viability dye (LD-NIR, Invitrogen, Cat. No. L34994), Fc block, and staining antibodies [BV510 anti-CD80 (BD Horizon, Cat. No. 563084), PE-Cy7 anti-CD83 (BD ​​Horizon, Cat. No. 561132), and BV605 anti-CD86 (BD Horizon, Cat. No. 562999)]. The cells were incubated at room temperature for 60 minutes, washed off the staining solution, and fixed with BD Phosphoflow FIX Buffer I (BD, Cat. No. 557870) and incubated for 15 minutes. Fixation buffer was removed and cells were washed with PBS and stored at 4°C until acquired on a BD LSR FORTESSA X-20 cell analyzer and analyzed with FLOWJO™ v10.8 software (BD Life Sciences).

[0306] Within each inhibitor group, the fold change between ATP-treated group and ATP-untreated group was calculated, and the induction of each surface marker in the presence of ATP was calculated.Then, the induction level of the inhibitor-treated group was normalized to the DMSO-treated group.The results show that P2Y11 inhibitors suppress CD86 induction by ATP, but not CD80 or CD83 (Figure 23). Example 13: Solid tumors express mechanisms for responding to ATP elevation as a result of CD39 inhibition

[0307] ATP response mechanisms were evaluated in a subset of solid tumors. Briefly, TCGA RNA expression was analyzed by tumor type of interest and expression was normalized per gene (Figure 24A). The violin plots in Figure 24B outline the kernel probability density (width of the shaded area represents the percentage of data located there) of normalized log2 gene expression (y-axis) of the following genes (proteins): ENTPD1 (CD39), NT5E (CD73), P2RX7 (P2X7), and P2RY11 (P2Y11) in a subset of TCGA samples. Along the x-axis, the violin plots are grouped by specific TCGA subtypes: 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 gastric adenocarcinoma (STAD). Also shown in Figure 24B is the pathway enrichment score (y-axis) of myeloid gene signatures (IL-6, CXCL2, CXCL2, CXCL3, CXCL8, and PTGS2) calculated using gene set enrichment analysis for the same subset of TCGA cancer subtypes. 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 may respond to elevated ATP in the tumor microenvironment. Examples of Disclosed Embodiments

[0308] Embodiment 1: An anti-CD39 antibody that specifically binds to human CD39, comprising: (a) a heavy chain variable region comprising complementarity determining region 1 (H1) having at least 80% sequence identity with SEQ ID NO:10, complementarity determining region 2 (H2) having at least 80% sequence identity with SEQ ID NO:11, and complementarity determining region 3 (H3) having at least 80% sequence identity with SEQ ID NO:12; and a light chain variable region comprising complementarity determining region 1 (L1) having at least 80% sequence identity with SEQ ID NO:14, complementarity determining region 2 (L2) having at least 80% sequence identity with SEQ ID NO:15, and complementarity determining region 3 (L3) having at least 80% sequence identity with SEQ ID NO:16; (b) a light chain variable region comprising H1 having at least 80% sequence identity with SEQ ID NO:18, H2 having at least 80% sequence identity with SEQ ID NO:19, and complementarity determining region 3 (H3) having at least 80% sequence identity with SEQ ID NO:20. an anti-CD39 antibody, comprising: a heavy chain variable region comprising an H3 having at least 80% sequence identity to SEQ ID NO:22, SEQ ID NO:23, or SEQ ID NO:24; and a light chain variable region comprising an L1 having at least 80% sequence identity to SEQ ID NO:25, and an L3 having at least 80% sequence identity to SEQ ID NO:26; or (c) a heavy chain variable region comprising an H1 having at least 80% sequence identity to SEQ ID NO:28, an H2 having at least 80% sequence identity to SEQ ID NO:29, and an H3 having at least 80% sequence identity to SEQ ID NO:30; and a light chain variable region comprising an L1 having at least 80% sequence identity to SEQ ID NO:32, an L2 having at least 80% sequence identity to SEQ ID NO:33, and an L3 having at least 80% sequence identity to SEQ ID NO:34.

[0309] Embodiment 2: The anti-CD39 antibody of embodiment 1, comprising: (a) a heavy chain variable region comprising: H1 having at least 90% sequence identity or at least 95% sequence identity to SEQ ID NO: 10, H2 having at least 90% sequence identity or at least 95% sequence identity to SEQ ID NO: 11, and H3 having at least 90% sequence identity or at least 95% sequence identity to SEQ ID NO: 12; and L1 having at least 90% sequence identity or at least 95% sequence identity to SEQ ID NO: 14, L2 having at least 90% sequence identity or at least 95% sequence identity to SEQ ID NO: 15, and (b) a light chain variable region comprising: (a) an H1 having at least 90% sequence identity or at least 95% sequence identity to SEQ ID NO:18, (b) an H2 having at least 90% sequence identity or at least 95% sequence identity to SEQ ID NO:19, and (c) an H3 having at least 90% sequence identity or at least 95% sequence identity to SEQ ID NO:20; and a light chain variable region comprising an L1 having at least 90% sequence identity or at least 95% sequence identity to SEQ ID NO:22, SEQ ID NO:23, or SEQ ID NO:24, an L2 having at least 90% sequence identity or at least 95% sequence identity to SEQ ID NO:25, and an L3 having at least 90% sequence identity or at least 95% sequence identity to SEQ ID NO:26; or (c) an H1 having at least 90% sequence identity or at lea...

Claims

1. An anti-CD39 antibody that specifically binds to human CD39, (a) a heavy-chain variable region comprising a complementarity determining region 1 (H1) having an amino acid sequence comprising SEQ ID NO:10, a complementarity determining region 2 (H2) having an amino acid sequence comprising SEQ ID NO:11, and a complementarity determining region 3 (H3) having an amino acid sequence comprising SEQ ID NO:12; and a light-chain variable region comprising a complementarity determining region 1 (L1) having an amino acid sequence comprising SEQ ID NO:14, a complementarity determining region 2 (L2) having an amino acid sequence comprising SEQ ID NO:15, and a complementarity determining region 3 (L3) having an amino acid sequence comprising SEQ ID NO:16; (b) a heavy chain variable region comprising H1 having an amino acid sequence comprising SEQ ID NO: 18, H2 having an amino acid sequence comprising SEQ ID NO: 19, and H3 having an amino acid sequence comprising SEQ ID NO: 20; and a light chain variable region comprising L1 having an amino acid sequence comprising SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24, L2 having an amino acid sequence comprising SEQ ID NO: 25, and L3 having an amino acid sequence comprising SEQ ID NO: 26; or (c) the anti-CD39 antibody, comprising a heavy chain variable region comprising H1 having an amino acid sequence comprising SEQ ID NO:28, H2 having an amino acid sequence comprising SEQ ID NO:29, and H3 having an amino acid sequence comprising SEQ ID NO:30; and a light chain variable region comprising L1 having an amino acid sequence comprising SEQ ID NO:32, L2 having an amino acid sequence comprising SEQ ID NO:33, and L3 having an amino acid sequence comprising SEQ ID NO:

34.

2. 2. The anti-CD39 antibody of claim 1 , the antibody has a heavy chain variable region and a light chain variable region according to claim 1(a), wherein the heavy chain variable region 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; or The antibody has a heavy chain variable region and a light chain variable region according to claim 1(b), wherein the heavy chain variable region has at least 90% sequence identity to SEQ ID NO: 17 and the light chain variable region has at least 90% sequence identity to SEQ ID NO: 21; the heavy chain variable region has at least 90% sequence identity to SEQ ID NO: 43 and the light chain variable region has at least 90% sequence identity to SEQ ID NO: 45; the heavy chain variable region has at least 90% sequence identity to SEQ ID NO: 43 and the light chain variable region has at least 90% sequence identity to SEQ ID NO: 46; the heavy chain variable region has at least 90% sequence identity to SEQ ID NO: 43 and the light chain variable region has at least 90% sequence identity to SEQ ID NO:

47. the heavy chain variable region has at least 90% sequence identity to SEQ ID NO:43 and the light chain variable region has at least 90% sequence identity to SEQ ID NO:48; the heavy chain variable region has at least 90% sequence identity to SEQ ID NO:43 and the light chain variable region has at least 90% sequence identity to SEQ ID NO:49; the heavy chain variable region has at least 90% sequence identity to SEQ ID NO:44 and the light chain variable region has at least 90% sequence identity to SEQ ID NO:48; or the heavy chain variable region has at least 90% sequence identity to SEQ ID NO:44 and the light chain variable region has at least 90% sequence identity to SEQ ID NO:49; The antibody has a heavy chain variable region and a light chain variable region according to claim 1(c), wherein 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 % 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. 3. The anti-CD39 antibody of claim 1 or 2, (a) Approximately 0.001 x 10 measured by surface plasmon resonance -11 M ~ approx. 100 x 10 -11 M, approximately 0.01×10 -11 M ~ approx. 100 x 10 -11 M, approximately 0.1 x 10 -11 M ~ approx. 100 x 10 -11 M, approximately 0.1 x 10 -11 M ~ approx. 10 x 10 -11 M, about 1 x 10 -11 M ~ approx. 100 x 10 -11 , or about 1×10 -11 M ~ approx. 10 x 10 -11 The equilibrium binding constant (K D ) or (b) inhibiting human CD39 enzyme activity with an IC 50 and optionally (i) inhibition is measured in the presence of 20 μM ATP or 400 μM ATP, and / or (ii) said CD39 is expressed on the surface of a human cell, and optionally said human cell is a primary monocyte as described in Example 5; or (c) any combination of (a) or (b).

4. 4. The anti-CD39 antibody of claim 3, having an IC of about 0.05 nM to about 0.5 nM, or about 0.05 nM to about 0.4 nM, or about 0.05 nM to about 0.3 nM. 50 and optionally (i) inhibition is measured in the presence of 20 μM ATP or 400 μM ATP, and / or (ii) said CD39 is expressed on the surface of a human cell, and optionally said human cell is a primary monocyte as described in Example 5.

5. 4. The anti-CD39 antibody of claim 3, having an IC of about 0.3 nM to about 1 nM, or about 0.3 nM to 1 nM, or about 0.5 nM to about 1 nM, or about 0.5 nM to 1 nM. 50 and optionally (i) inhibition is measured in the presence of 20 μM ATP or 400 μM ATP, and / or (ii) said CD39 is expressed on the surface of a human cell, and optionally said human cell is a primary monocyte as described in Example 5.

6. The anti-CD39 antibody of any one of claims 1 to 5, which is a monoclonal antibody or an antigen-binding fragment thereof.

7. 7. The anti-CD39 antibody of any one of claims 1 to 6, which is a chimeric, humanized, or veneered antibody, or an antigen-binding fragment thereof.

8. The anti-CD39 antibody of any one of claims 1 to 6, which is a human antibody or an antigen-binding fragment thereof.

9. 10. The anti-CD39 antibody of any one of the preceding claims, further comprising a variant heavy chain constant region selected from a variant human IgG1, a variant human IgG2, a variant human IgG3, or a variant human IgG4, and optionally a human light chain constant region.

10. The anti-CD39 antibody of claim 9 , wherein the variant heavy chain constant region has enhanced or reduced effector function relative to the wild-type heavy chain constant region.

11. 10. The anti-CD39 antibody of claim 9, wherein the variant human IgG heavy chain constant region comprises SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:

5.

12. 9. The anti-CD39 antibody of any one of claims 1 to 8, further comprising a wild-type human IgG heavy chain constant region, and optionally a human light chain constant region.

13. The anti-CD39 antibody of claim 12, wherein the wild-type human IgG heavy chain constant region comprises SEQ ID NO: 1 or SEQ ID NO:

4.

14. 14. The anti-CD39 antibody of any one of claims 9 to 13, comprising a human light chain kappa constant region, optionally wherein the human light chain constant region comprises SEQ ID NO:

6.

15. 2. The anti-CD39 antibody of claim 1, comprising a heavy chain comprising the heavy chain variable region and a light chain comprising the light chain variable region, (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 antibody, wherein 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.

16. 2. The anti-CD39 antibody of claim 1, comprising a heavy chain comprising the heavy chain variable region and a light chain comprising the light chain variable region, (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 antibody, wherein 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.

17. 17. The anti-CD39 antibody of claim 15 or 16, (a) Approximately 0.001 x 10 measured by surface plasmon resonance -11 M ~ approx. 100 x 10 -11 M, approximately 0.01×10 -11 M ~ approx. 100 x 10 -11 M, approximately 0.1 x 10 -11 M ~ approx. 100 x 10 -11 M, approximately 0.1 x 10 -11 M ~ approx. 10 x 10 -11 M, about 1 x 10 -11 M ~ approx. 100 x 10 -11 , or about 1×10 -11 M ~ approx. 10 x 10 -11 The equilibrium binding constant (K D ) or (b) inhibiting human CD39 enzyme activity with an IC 50 and optionally (i) inhibition is measured in the presence of 20 μM ATP or 400 μM ATP, and / or (ii) said CD39 is expressed on the surface of a human cell, and optionally said human cell is a primary monocyte as described in Example 5; or (c) any combination of (a) or (b).

18. 18. The anti-CD39 antibody of claim 17, having an IC of about 0.05 nM to about 0.5 nM, or about 0.05 nM to about 0.4 nM, or about 0.05 nM to about 0.3 nM. 50 and optionally (i) inhibition is measured in the presence of 20 μM ATP or 400 μM ATP, and / or (ii) said CD39 is expressed on the surface of a human cell, and optionally said human cell is a primary monocyte as described in Example 5.

19. 18. The anti-CD39 antibody of claim 17, having an IC of about 0.3 nM to about 1 nM, or about 0.3 nM to 1 nM, or about 0.5 nM to about 1 nM, or about 0.5 nM to 1 nM. 50 and optionally (i) inhibition is measured in the presence of 20 μM ATP or 400 μM ATP, and / or (ii) said CD39 is expressed on the surface of a human cell, and optionally said human cell is a primary monocyte as described in Example 5.

20. 20. The anti-CD39 antibody of any one of claims 1 to 19, which competes with a reference antibody for binding to human CD39 by at least about 55%, 60%, 65%, 70%, 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% as measured in a competition flow assay, wherein the reference antibody (a) the antibody of claim 17; (b) the antibody of claim 18; (c) an antibody comprising a heavy chain having an amino acid sequence comprising SEQ ID NO: 37 and a light chain having an amino acid sequence comprising SEQ ID NO: 38; or (d) the anti-CD39 antibody, which comprises a heavy chain having an amino acid sequence comprising SEQ ID NO: 39 and a light chain having an amino acid sequence comprising SEQ ID NO:

40.

21. 13. A method of inhibiting CD39 enzymatic activity comprising contacting CD39 with an anti-CD39 antibody according to any one of the preceding claims.

22. 21. A method for increasing extracellular ATP or activating the immune system in a subject in need thereof, comprising administering to the subject an effective regimen or a therapeutically effective amount of any one of the anti-CD39 antibodies of any one of claims 1 to 20.

23. 21. A method of treating or preventing cancer, comprising administering to a subject having or at risk of cancer an effective regimen or a therapeutically effective amount of any one of the anti-CD39 antibodies of any one of claims 1-20.

24. 24. The method of claim 23, wherein the cancer is a hematological malignancy or a solid tumor.

25. 24. The method of claim 23, wherein the cancer is breast cancer, gastrointestinal cancer, genitourinary cancer, lung cancer, lymphoma, or ovarian cancer.

26. 26. The method of claim 25, wherein the cancer is 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.

27. 24. The method of claim 23, wherein the cancer is non-small cell lung cancer, gastric cancer, or esophagogastric cancer.

28. 24. The method of claim 23, wherein the cancer is lung cancer, head and neck cancer, thyroid cancer, pancreatic cancer, kidney cancer, or skin cancer.

29. 29. The method of claim 28, wherein the cancer is non-small cell lung cancer, head and neck squamous cell carcinoma, pancreatic ductal adenocarcinoma, clear cell renal carcinoma, or melanoma.

30. The method of any one of claims 22 to 29, further comprising administering to the subject one or more additional therapeutic agents.

31. A combination comprising an anti-CD39 antibody according to any one of claims 1 to 20 and one or more additional therapeutic agents.

32. 32. The method of claim 30 or the combination of claim 31, wherein each additional therapeutic agent is independently selected from the group consisting of a chemotherapeutic agent, a radiopharmaceutical, an agent that induces immunogenic cell death, an inhibitory or co-inhibitory immune checkpoint antagonist, a stimulatory or co-stimulatory immune checkpoint agonist, and an ATP adenosine axis targeting agent.

33. 32. The method of claim 30 or the combination of claim 31, at least one additional therapeutic agent is a chemotherapeutic agent or an agent that induces immunogenic cell death; The above methods or the above combinations, wherein at least one additional therapeutic agent is an inhibitory or co-inhibitory immune checkpoint antagonist or an ATP-adenosine axis targeting agent.

34. 32. The method of claim 30 or the combination of claim 31, at least one additional therapeutic agent is an inhibitory or co-inhibitory immune checkpoint antagonist; The above methods or the above combinations, wherein at least one additional therapeutic agent is an ATP-adenosine axis targeting agent.

35. The method or combination of any one of claims 32 to 34, wherein the agent that induces immunogenic cell death is radiation therapy.

36. 35. The method or combination of any one of claims 32 to 34, wherein the agent that induces immunogenic cell death is (a) a chemotherapeutic agent selected from the group consisting of bortezomib, cyclophosphamide, doxorubicin, epirubicin, idarubicin, mitoxantrone, oxaliplatin, and any combination thereof; or (b) bleomycin, bortezomib, carboplatin, cetuximab, crizotinib, cyclophosphamide, docetaxel, doxorubicin, epirubicin, gemcitabine, idarubicin, irinotecan, mitoxantrone, oxaliplatin, paclitaxel, vemurafenib, vorinostat, and any combination thereof.

37. 37. The method or combination of any one of claims 32 to 36, wherein each chemotherapeutic agent is selected from the group consisting of alkylating agents, anthracyclines, platinum agents, taxanes, and proteasome inhibitors.

38. 38. The method or combination of any one of claims 32 to 37, wherein the inhibitory or co-inhibitory immune checkpoint antagonist blocks the activity of at least one of PD-1, PD-L1, BTLA, LAG-3, a B7 family member, TIM-3, TIGIT, or CTLA-4.

39. 39. The method or combination of any one of claims 32 to 38, wherein the inhibitory or co-inhibitory immune checkpoint antagonist blocks at least one activity of PD-1, PD-L1, or TIGIT.

40. 40. The method or combination of any one of claims 32 to 39, wherein the inhibitory or co-inhibitory immune checkpoint antagonist is an antagonistic anti-PD-1 antibody, an antagonistic anti-PD-L1 antibody, or an antagonistic anti-TIGIT antibody.

41. 41. The method or combination of any one of claims 32 to 40, wherein each inhibitory or co-inhibitory immune checkpoint antagonist is independently selected from the group consisting of: zimberelimab, domvanalimab, and AB308.

42. Each ATP-adenosine axis targeting agent is 2a R antagonist, A 2b R antagonist, A 2a / 2b The method or combination of any one of claims 32 to 41, wherein said compound is independently selected from the group consisting of: an R antagonist, and a CD73 inhibitor.

43. 43. The method or combination of any one of claims 32 to 42, wherein each ATP-adenosine axis targeting agent is independently selected from the group consisting of etrumadenant and quemliclustat.

44. 24. The method of claim 23, wherein the cancer is non-small cell lung cancer, and further comprising administering the anti-CD39 antibody in combination with pemetrexed, carboplatin, and either an antagonistic anti-PD-1 antibody or an antagonistic anti-PD-L1 antibody.

45. 24. The method of claim 23, wherein the cancer is gastric or gastroesophageal cancer, and further comprising administering the anti-CD39 antibody in combination with FOLFOX and either an antagonistic anti-PD-1 antibody or an antagonistic anti-PD-L1 antibody.

46. Antagonistic anti-TIGIT antibody, A 2a R antagonist, A 2b R antagonist, A 2a / 2b 46. ​​The method of claim 44 or 45, further comprising administering one or more additional agents selected from the group consisting of R antagonists and CD73 inhibitors, optionally wherein the additional agents are selected from the group consisting of domvanalimab, AB308, etormadenant, and quemlicustat.

47. 32. The combination of claim 31 , wherein the one or more additional therapeutic agents include (a) pemetrexed, carboplatin, and either an antagonist anti-PD-1 antibody or an antagonist anti-PD-L1 antibody, or (b) FOLFOX and either an antagonist anti-PD-1 antibody or an antagonist anti-PD-L1 antibody.

48. A pharmaceutical composition comprising the antibody of any one of claims 1 to 20 and a pharma- ceutically acceptable carrier.

49. An isolated polynucleotide encoding the antibody of any one of claims 1 to 20.

50. 50. A vector comprising the isolated polynucleotide of claim 49.

51. A host cell comprising the vector of claim 50.

52. A method for expressing an antibody according to any one of claims 1 to 20, comprising culturing a host cell according to claim 51 under conditions in which the polynucleotide according to claim 49 is expressed.

53. A kit comprising the antibody according to any one of claims 1 to 20.

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