Anti-CD39 antibodies and uses thereof

Abstract

To provide anti-CD39 antibodies that inhibit enzymatic activity of human CD39 and use methods thereof.SOLUTION: An anti-CD39 antibody that specifically binds to human CD39 is provided. The anti-CD39 antibody is, optionally, (i) labeled with one or more detectable signals including a fluorescent molecule, spin labeling molecule, enzyme, or radioisotope but not limited thereto; and / or (ii) conjugated with one or more therapeutic agents including a chemotherapeutic agent, radioisotope, or nucleic acid but not limited thereto. In some embodiments, the disclosed anti-CD39 antibody is provided as an isolated antibody. In some embodiments, the disclosed anti-CD39 antibody is provided as a composition. In some embodiments, the disclosed anti-CD39 antibody is provided as a composition further comprising a pharmaceutically acceptable excipient.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Cross-reference statements This application is a continuation of international application PCT / CN2022 / 079021 filed on March 3, 2022, and and international application PCT / CN2022 / 126070 filed on October 19, 2022 Priority is claimed, the disclosures of which are incorporated herein by reference.

[0002] Technical Field The present specification describes an anti-CD39 antibody that inhibits the enzymatic activity of human CD39 and a method for using the same. A method is provided. [Background technology]

[0003] It is present in negligible concentrations (10-100 nM) under normal conditions, but Cells or cells under stress release ATP into the extracellular space. ATP signals through type 2 purinergic (P2) receptors, promoting effective innate immunity. CD39 provides inflammatory signals essential for the immune and adaptive immune response. CD39 is a rate-limiting ectoenzyme in water splitting. It converts extracellular ATP into AMP. It is an important regulator of extracellular ATP levels through its oxidative action.

[0004] CD39 is also an important contributor to extracellular adenosine levels. CD39 converts ATP into A AMP is hydrolyzed to MP, which is converted to adenosine by CD73, resulting in cellular This contributes to an increase in extracellular adenosine levels, which in turn activates type 1 purinergic receptors. It transmits signals through receptors to create an immunosuppressive environment.

[0005] Thus, the hydrolysis of extracellular ATP by CD39 is at the opposing end of a signaling axis. It may affect immune responses via two distinct signaling pathways at its edge. In particular, this signaling, referred to herein as the ATP-adenosine signaling axis, Dysregulation of the axis has been observed in several diseases. Therefore, drugs that target CD39 and inhibit its enzymatic activity are needed for beneficial therapeutic purposes. It has been done. Summary of the Invention

[0006] Among various aspects of the present disclosure are provided anti-CD39 antibodies. optionally comprising (i) a fluorescent molecule, a spin-label molecule, an enzyme, or a radioisotope; is labeled with one or more detectable signals, including but not limited to (ii) ) one or more therapeutic agents, including but not limited to chemotherapeutic agents, radioisotopes, or nucleic acids In some embodiments, the anti-CD39 antibodies of the present disclosure are conjugated to a therapeutic agent. In some embodiments, the anti-CD39 antibodies of the present disclosure are provided as compositions. In some embodiments, the anti-CD39 antibodies of the present disclosure are provided in a pharmaceutically acceptable form. The composition further comprises an excipient.

[0007] In one embodiment, the present disclosure provides a homologue having at least 80% sequence identity to SEQ ID NO: 10. Complementarity determining region 1 (H1), complementary to SEQ ID NO: 11 with at least 80% sequence identity Determinant region 2 (H2), and complementary to SEQ ID NO: 12 with at least 80% sequence identity a heavy chain variable region containing the nucleotide determining region 3 (H3); and a heavy chain variable region having at least 80% sequence identity with SEQ ID NO: 14. Complementarity determining region 1 (L1) with sequence identity, at least 80% sequence identity with SEQ ID NO: 15 Complementarity determining region 2 (L2) has at least 80% sequence identity with SEQ ID NO: 16. and a light chain variable region containing complementarity-determining region 3 (L3) having the same identity as human CD39. In another embodiment, the present disclosure provides an anti-CD39 antibody that binds to SEQ ID NO: 10. H1, having at least 90% sequence identity with SEQ ID NO: 11 H2 having identity to SEQ ID NO: 12, and H3 having at least 90% sequence identity to SEQ ID NO: 12. a heavy chain variable region comprising: an L1 having at least 90% sequence identity with SEQ ID NO: 14; L2, which has at least 90% sequence identity with SEQ ID NO: 15, and L3, which has at least 90% sequence identity with SEQ ID NO: 16. a light chain variable region containing L3 having at least 90% sequence identity with human CD39; In some of the foregoing embodiments, the heavy chain of the antibody is heterologously linked to the CD39 antibody. The variable region has at least 90% sequence identity with SEQ ID NO: 9, and the light chain variable region has SEQ ID NO: 13. In some embodiments, the heavy chain of the antibody has at least 90% sequence identity with The variable region has at least 95% sequence identity with SEQ ID NO: 9, and the light chain variable region has SEQ ID NO: 1 3 has at least 95% sequence identity.

[0008] In one embodiment, the present disclosure provides a H having at least 80% sequence identity to SEQ ID NO: 18. H1, H2, which has at least 80% sequence identity with SEQ ID NO: 19, and H3, which has at least 80% sequence identity with SEQ ID NO: 20. a heavy chain variable region comprising H3 with at least 80% sequence identity; and SEQ ID NO: 22, L1 having at least 80% sequence identity with SEQ ID NO: 23, or SEQ ID NO: 24, L2 having at least 80% sequence identity with SEQ ID NO: 25, and L3 having at least 80% sequence identity with SEQ ID NO: 26. A light chain variable region containing L3 with 80% sequence identity, specific for human CD39. In another embodiment, the present disclosure provides an anti-CD39 antibody that binds to at least SEQ ID NO: 18. H1 with at least 90% sequence identity, H2 with at least 80% sequence identity with SEQ ID NO: 19 and H3 having at least 90% sequence identity with SEQ ID NO: 20. chain variable region; and SEQ ID NO:22, SEQ ID NO:23, or SEQ ID NO:24 and at least 9 L1, which has 0% sequence identity, and L2, which has at least 90% sequence identity with SEQ ID NO: 25 a light chain variable region comprising L2 and L3 having at least 80% sequence identity with SEQ ID NO:26; The present invention provides an anti-CD39 antibody that specifically binds to human CD39, comprising a region In some embodiments, the heavy and light chain variable regions of the antibody are SEQ ID NO: 17 and SEQ ID NO: 18, respectively. with SEQ ID NO: 21, or with SEQ ID NO: 43 and SEQ ID NO: 45, respectively, or with SEQ ID NO: 22, or with SEQ ID NO: 23 and SEQ ID NO: 24, respectively SEQ ID NO: 43 and SEQ ID NO: 46, or 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: 48, respectively with SEQ ID NO: 49, or with SEQ ID NO: 44 and SEQ ID NO: 48, respectively, or With SEQ ID NO: 44 and SEQ ID NO: 49: at least 90% sequence identity (e.g., 90%, 9 1%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 10 0%) or at least 95% sequence identity.

[0009] In one embodiment, the present disclosure provides a H having at least 80% sequence identity to SEQ ID NO:28. H1, H2, which has at least 80% sequence identity with SEQ ID NO:29, and H3, which has at least 80% sequence identity with SEQ ID NO:30. a heavy chain variable region comprising H3 with at least 80% sequence identity; and SEQ ID NO: 32 L1 with at least 80% sequence identity, L2 with at least 80% sequence identity with SEQ ID NO: 33 L2 having at least 80% sequence identity with SEQ ID NO: 34, and L3 having at least 80% sequence identity with SEQ ID NO: 34. The present invention provides an anti-CD39 antibody that specifically binds to human CD39, comprising a light chain variable region comprising: In some of the foregoing embodiments, the heavy and light chain variable regions of the antibody are selected from the group consisting of SEQ ID NO: 27 and SEQ ID NO: 31, or SEQ ID NO: 58 and SEQ ID NO: 60, or SEQ ID NO: 58 and SEQ ID NO: 61, or SEQ ID NO: 58 and SEQ ID NO: 62, or SEQ ID NO: 58 and SEQ ID NO: 63, or SEQ ID NO: 59 and SEQ ID NO: 61, or SEQ ID NO: 59 and SEQ ID NO: 60, or SEQ ID NO: 59 and SEQ ID NO: 62, or SEQ ID NO: 59 and SEQ ID NO: 63: at least 90% sequence identity (e.g., 90%, 91%, 92% , 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) or It has at least 95% sequence identity.

[0010] For any of the disclosed antibodies of the present disclosure, the anti-CD39 antibody may be a monoclonal antibody. or antigen-binding fragment thereof; chimeric, humanized, or veneered antibody or antigen-binding or a human antibody or antigen-binding fragment thereof. In addition, the anti-CD39 antibody may be a heavy chain selected from human IgG1, human IgG2, human IgG3, or human IgG4 The variant heavy chain constant region may further comprise a human light chain constant region. The region may be a wild-type heavy chain constant region or may be an endogenous region relative to the wild-type heavy chain constant region. In various embodiments, the heavy chain constant region may have enhanced or reduced effector function. The IgG heavy chain constant region is SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: the human light chain kappa constant region may comprise SEQ ID NO:6;

[0011] In one embodiment, the present disclosure provides an anti-CD3 antibody comprising a light chain comprising a heavy chain variable region and a light chain variable region. 9 antibody, wherein (a) the heavy chain has an amino acid sequence comprising SEQ ID NO: 50 and the light chain (b) the heavy chain has an amino acid sequence comprising SEQ ID NO: 50; (c) the heavy chain has an amino acid sequence comprising SEQ ID NO: 50; (d) the heavy chain has an amino acid sequence comprising SEQ ID NO: 55; The light chain has an amino acid sequence comprising SEQ ID NO: 56, and the light chain has an amino acid sequence comprising SEQ ID NO: 57. (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: 51; (g) the heavy chain has an amino acid sequence comprising SEQ ID NO: 52; (h) the heavy chain has an amino acid sequence comprising SEQ ID NO: 56; or (i) the light chain has an amino acid sequence comprising SEQ ID NO: 57. The light 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 present disclosure provides an anti-CD3 antibody comprising a light chain comprising a heavy chain variable region and a light chain variable region. 9 antibody, wherein (a) the heavy chain has an amino acid sequence comprising SEQ ID NO: 64 and the light chain (b) the heavy chain has an amino acid sequence comprising SEQ ID NO: 64; (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: 68; (d) the heavy chain has an amino acid sequence comprising SEQ ID NO: 69; and the light chain has an amino acid sequence comprising SEQ ID NO: 69; The light 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: 66; (g) the heavy chain has an amino acid sequence comprising SEQ ID NO: 66; (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 It has an array.

[0013] In another aspect, the 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 treating cancer. In some embodiments, the anti-CD39 antibodies are provided for use in the prevention of cancer. It is provided for

[0014] In another aspect, the present disclosure provides a compound according to the present disclosure for use as a pharmaceutical in combination with an additional therapy. In some embodiments, the antibody is any of the antibodies disclosed herein. Antibodies are provided for use in treating cancer. In some embodiments, the aforementioned anti-C D39 antibodies are provided for use in the prevention of cancer. In certain embodiments, additional Therapies include immune checkpoint inhibitors, immunogenic cell death-inducing therapeutics, and ATP-adenosine monophosphate (ATP). axis targeting agents, HIF-2α inhibitors, arginase inhibitors, AXL inhibitors, or PI3 In a further embodiment, the additional therapy may be chemotherapy, radiation therapy, dextromethorphan D-cell agonist, or a combination of both. urvalumab, zimberelimab, dombanalimab, AB308, AB521, or Que It may be muriclustat.

[0015] In another aspect, the present disclosure provides a compound for use as a pharmaceutical in combination with two or more additional therapies. In some embodiments, the antibody is any of the antibodies disclosed herein. In some embodiments, the anti-CD39 antibodies are provided for use in treating cancer. The aforementioned anti-CD39 antibodies are provided for use in the prevention of cancer. In this setting, each additional therapy may be a chemotherapeutic agent, immune checkpoint inhibitor, or immunogenic cell death inducer. Inducing therapeutic agents, ATP-adenosine axis targeting agents, HIF-2α inhibitors, arginase inhibitors, In a further embodiment, the additional therapy may be an AXL inhibitor, or a PI3K inhibitor. , chemotherapy, radiation therapy, durvalumab, zimberelimab, domvanalimab, AB30 8, AB521, or quemliculstat. The one or more additional therapeutic agents include (a) pemetrexed, carboplatin, and antagonizumab. (b) either an antagonistic anti-PD-1 antibody or an antagonistic anti-PD-L1 antibody; or OLFOX and antagonistic anti-PD-1 antibody or antagonistic anti-PD-L1 antibody Contains one of the following:

[0016] In another aspect, the disclosure provides a method for treating or preventing cancer, comprising administering to a subject a therapeutically effective amount of a compound as defined herein. Administering any of the disclosed antibodies 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 further comprises an immune checkpoint inhibitor. Anti-inflammatory agents, immunogenic cell death-inducing therapeutic agents, ATP-adenosine axis targeting agents, HIF-2α inhibitors , an arginase inhibitor, an AXL inhibitor, or a PI3K inhibitor. In this form, additional therapies include chemotherapy, radiation therapy, durvalumab, zimberelimab, and doxorubicin. It can be mubanalimab, AB308, AB521, or quemliculstat.

[0017] In each of the above aspects, the cancer is selected from the group consisting of breast cancer, gastrointestinal cancer, urogenital cancer, head and neck cancer, renal cancer, The cancer may be lung cancer, lymphoma, ovarian cancer, pancreatic cancer, skin cancer, or thyroid cancer. In terms of morphology, cancers include acute myeloid lymphoma, colorectal cancer, gastric cancer, esophageal cancer, and castration-resistant prostate cancer. Cancer, non-small cell lung cancer, ovarian cancer, pancreatic cancer, triple-negative breast cancer, head and neck squamous cell carcinoma, It may be pancreatic ductal adenocarcinoma, clear cell renal carcinoma, or melanoma.

[0018] In some embodiments, the cancer is non-small cell lung cancer and the method comprises administering to a subject an anti-CD39 antibody pemetrexed, carboplatin, and an antagonist anti-PD-1 antibody or The present invention may further comprise administering the compound in combination with any of the agonistic anti-PD-L1 antibodies. In some embodiments, the cancer is gastric cancer or gastroesophageal cancer, and the method comprises administering an anti-CD39 antibody. The body is treated with FOLFOX and an antagonistic anti-PD-1 antibody or an antagonistic anti-PD-L antibody. These embodiments may further comprise administering the compound in combination with any of the following antibodies: Antagonistic anti-TIGIT antibody, A 2a R antagonist, A 2b R antagonist, A 2a / 2b R antagonist, and one or more additional and optionally, the additional agent is dombanalima. AB308, etremadenant, and quemliculstat do.

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

[0020] [Figure 1]

[0023] Figure 1 is a graph showing the 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. [Figure 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. Percent maximal inhibition (%) is on the y-axis and antibody concentration is on the x-axis. [Figure 3] FIG. 1 is a schematic diagram showing key steps of the macrophage cytokine release assay described in Example 6. MΦ = macrophage. [Figure 4A] Figure 1 shows the 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 the isotype control per donor, with three donors run per experiment. Bar heights represent the normalized mean 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 the isotype control per donor, with three donors run per experiment. Bar heights represent the normalized mean fold change between donors, and error bars are SEM. [Figure 4C] 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 the isotype control per donor, with two donors run per experiment. Bar heights represent the mean normalized fold change between donors, and error bars are SEM. [Figure 4D] 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 the isotype control per donor, with two donors run per experiment. Bar heights represent the mean normalized fold change between donors, and 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 the isotype control per donor, with three donors run per experiment. Bar heights represent the normalized mean 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 the isotype control per donor, with three donors run per experiment. Bar heights represent the normalized mean 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 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 5B]Results of a monocyte-derived dendritic cell (moDC) assay are shown. The extracellular marker CD86 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 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] 1 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] 1 is a graph showing enzymatic inhibition of human CD39 expressed on human myeloma cells, MOLP-8, by hu39.1_IGG4.P and an isotype control antibody. [Figure 8A] Enzyme activity (ATP consumption) in splenocytes derived from hCD39KI mouse model. [Figure 8B] 1 shows inhibition of splenocyte enzyme activity by hu39.1_IGG4.P. [Figure 9] Figure 1 shows confirmation that the hCD39KI mouse model expresses human, but not murine, 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 of 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), and evidence of anti-CD39 enzyme blocking activity by enzyme histochemistry (Panels B and D). [Figure 12] Higher 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 area in Figure 13. [Figure 15A] 1 is a 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] 1 is a 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]Figure 1 shows graphs depicting 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 the non-competing anti-CD39 clone A1-PE, on multiple cell types after 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 gray) 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 the isotype control (hCD39 isotype control in gray) and applied to each group (black). [Figure 17C] Graph showing the percentage of immune subpopulations observed after treatment, indicated along the x-axis. No significant changes in the percentage of immune subpopulations were observed with anti-CD39 treatment. 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. Each symbol represents an individual mouse, bar height is the mean, and error bars are + / - SEM. [Figure 17D]A graph showing the CD39 cell surface expression observed after treatment is shown along the x-axis. A significant decrease in CD39 cell surface expression was observed in almost all immune subpopulations with anti-CD39 treatment. 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. 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 of CD39 cell surface expression in representative animals from each treatment group: isotype + saline (gray open histograms), isotype + oxaliplatin (black open histograms), anti-CD39 + saline (gray filled histograms), and anti-CD39 + oxaliplatin (black filled histograms). [Figure 18] Figure 12 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] A graph showing cell viability 24 hours after treatment with vehicle control (Veh), 100 μM oxaliplatin (OXA.L), or 250 μM oxaliplatin (OXA.H) is shown for CT26 cells, MC38 cells, SK-MEL-5 cells and an isotype control antibody, and SK-MEL-5 cells and the 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 the 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]A graph showing extracellular ATP release by CT26 cells, MC38 cells, SK-MEL-5 cells and an isotype control antibody, and SK-MEL-5 cells and the hu39.5_IGG1.AA antibody after 8 hours of treatment with vehicle (PBS), 100 μM oxaliplatin, or 250 μM oxaliplatin is shown. [Figure 20A] 1 shows a graph depicting cell viability of SK-MEL-5 cells following 48 hour treatment with the indicated chemotherapeutic agents. [Figure 20B] Graph showing HMGB1 levels in culture supernatants of SK-MEL-5 cells after 48 hours of treatment with the indicated chemotherapeutic agents. [Figure 20C] 1 shows a graph depicting extracellular ATP release by SK-MEL-5 cells following treatment with the indicated chemotherapeutic agents for 8 hours or 32 hours. [Figure 21] Graphs showing the relative expression of CD39 on peripheral immune cells in humans (top row) and C57BL / 6hCK39KI MC38 mice (bottom row). [Figure 22A] Flow cytometry scattergrams from a representative donor are shown, demonstrating 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 pDCs, 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 (center panel), and CD39 protein expression (right panel), as assessed by flow cytometry, for human peripheral blood CD14+ monocytes, primary dendritic cell (DC) subsets, such as plasmacytoid DCs (pDCs, defined as HLA-DR+CD11c+CD141+Clec9a+), conventional DC1s (cDC1s, defined as HLA-DR+CD11c+CD1c+CD141negClec9aneg), conventional DC2s (cDC2s, defined as HLA-DR+CD11c+CD1cnegCD141negClec9aneg), and other DCs, as well as in vitro differentiated monocyte-derived myeloid cells, such as macrophage (M0), polarized macrophages (M1, M2), and DCs (moDCs). 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 myeloid cells, including macrophages (M0), polarized macrophages (M1, M2), and DCs (moDCs), obtained 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 myeloid cells, including macrophages (M0), polarized macrophages (M1, M2), and DCs (moDCs), obtained from four donors. [Figure 22E]This heatmap shows 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 sample separation but do not affect the unsupervised nature of the clustering. [Figure 23] Graphs depicting the relative induction of cell surface marker expression (relative to DMSO) after 1 hour of treatment with the indicated inhibitors, followed by 18 hours of treatment with 0 or 300 μM ATP. Cell surface expression of CD86 (top panel), CD83 (middle panel), and CD80 (bottom panel) was assessed by flow cytometry. Induction of activation marker expression by 0 to 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 gene are indicated in parentheses. 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 plots outline the kernel probability density (y-axis) of the 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 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 the myeloid signature for the same subset of TCGA cancer subtypes. DETAILED DESCRIPTION OF THE INVENTION

[0021] The present disclosure relates to human CD39, and more specifically to epitopes within the extracellular domain of human CD39. The present invention also provides an antibody that specifically binds to human CD39 and inhibits human CD39 enzyme activity. The anti-CD39 antibody disclosed in and methods for inhibiting cell surface-expressed human CD39 enzymatic activity are also contemplated. inhibits human CD39 enzyme activity, thereby disrupting the ATP-adenosine signaling axis The use of anti-CD39 antibodies disclosed herein that affect one or both ends Thus, the present disclosure provides a method for the preparation of anti-CD3 antibodies disclosed herein for therapeutic and diagnostic purposes. 9 Medical uses of antibodies are also provided.

[0022] I. Definition Unless otherwise defined, all technical terms, notations, and other technical terms used herein are Scientific or technical terms have the meanings commonly understood by those of ordinary skill in the art to which this disclosure pertains. It is intended that the embodiments of the invention described herein "comprise" embodiments. "sing," "consisting of," and "consisting essentially of" It is understood that the term "(consisting essentially of)" includes "(consisting essentially of)". "comprising," "including," and "ha The term "having" is intended to be inclusive and includes more than just the listed elements. This means that there may be additional elements outside of the

[0023] Unless otherwise specified, the term "about" as used herein means any amount that is within the skill of the art. To avoid any misunderstanding, Reference herein to "about" a value or parameter refers to that value or parameter itself. This invention includes (and describes) aspects directed to:

[0024] Also, as used herein, "and / or" refers to any combination of the associated listed items. All possible combinations of one or more of the following are interpreted in alternative form ("or"). When a combination is used, it includes the combination as well as the exclusion of the combination. Similarly, a phrase in the form "A / B" or "A and / or B" means (A), means (A), (B), or (A and B); means "at least one of A, B, and C." Phrases of the form (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. The CD39 sequence is SEQ ID NO: 7 (UniProtKB Reference: P49961) CD39 is a two-transmembrane protein. The extracellular domain of CD39 is represented by SEQ ID NO: 7 It comprises residues 38 to 478. 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. It is used as a diagnostic agent and / or a therapeutic agent that targets human CD39 and inhibits human CD39 enzyme activity. or capable of binding to human CD39 with sufficient affinity to be useful as a therapeutic agent. The anti-CD39 antibody of the present disclosure refers to an antibody that can bind to CD39. -6 Equilibrium dissociation below M The anti-CD39 antibodies of the present disclosure may be monospecific or multispecific antibodies. The antibody may be a monoclonal antibody, and in some instances may be a polyepitopic antibody.

[0027] As used herein, the term "antibody" is used in the broadest sense and refers to a single antigen or The term encompasses a variety of antibodies and antibody-like structures that specifically bind to multiple antigens, including full-length antibodies, antigen-binding proteins, and These include, but are not limited to, recombinant fragments, heavy chain antibodies, single chain antibodies, and higher order variants of single chain antibodies. Therefore, references to antibodies are to antibodies in their intact form unless the context requires otherwise. It should be understood that the term refers to an antibody or antigen-binding fragment. Although antibodies useful herein are not limited to those described herein, they can be isolated and recombinantly produced. do.

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

[0029] "Native antibodies" are naturally occurring immunoglobulin molecules with diverse structures. For example, a natural IgG antibody consists of two identical disulfide-bonded light chains (each It consists of a single chain (approximately 25 kDa each) and two identical heavy chains (approximately 50-70 kDa each) , a heterotetrameric glycoprotein of approximately 150,000 daltons. Each heavy chain contains a variable heavy domain or heavy The variable region (VH), also called the chain variable domain, is followed by three constant domains (CH1, C Similarly, from the N-terminus to the C-terminus, each light chain contains a variable light domain (H2, and CH3). Also called variable light domain or light chain variable domain The light chain of an antibody contains a variable region (VL) followed by a constant light (CL) domain. Based on the amino acid sequence of the common domain, two types of ribosomal proteins are known as kappa (κ) and lambda (λ). Heavy chains can be assigned one of three types: gamma, mu, alpha, delta, or epsilon, and the antibody isotype is classified as IgG, IgM, IgA, The amino-terminal portions of the light and heavy chains are mainly responsible for antigen recognition. The variable regions (VL and VH, respectively) of approximately 100 to 110 amino acid sequences involved in recognition The carboxy-terminal portion of each chain contains a constant region primarily responsible for effector function. Within the light and heavy chains, the variable and constant regions are defined as sequences of about 12 or more amino acids. The heavy chains are joined by a "J" region of about 10 amino acids, and the heavy chains also have a "D" region of about 10 amino acids. Included.

[0030] The term "variable region" or "variable domain" refers to the molecules that are involved in binding an antibody to an antigen. The variable domains of the heavy and light chains of an antibody are the domains of the heavy and light chains of an antibody. Generally, they have a similar structure, with each domain containing four conserved framework regions (FRs). and three hypervariable regions (CDRs). (See, e.g., Kindt et al., Kub y Immunology, 6th ed., W. H. Freeman and Co., page 91 (2007).) A single VH or VL domain The antibody may be sufficient to confer antigen-binding specificity. The VH or VL domain of each antibody can be used to ligate a complementary VL or VH domain, respectively. The libraries may be screened to isolate antibodies that bind to a particular antigen. ortolano et al., J. Immunol. 150:880-887 (1993);Clarkson et al., Nature 352:624- See 628 (1991).

[0031] "Framework Regions" or "FR" refer 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 and FR4. Thus, the CDR and FR sequences generally appear in the following order: : FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Heavy and light chain F The R domains may vary as known in the art.

[0032] As used herein, the term "hypervariable region" or "HVR" generally refers to a Also called "complementarity determining regions" or "CDRs," which are used interchangeably, and are hypervariable in sequence. and / or form a structurally defined loop ("hypervariable loop"), and / or refers to each of the regions of the variable domain that contain antigen contact residues ("antigen contacts"). In general, an antibody contains six CDRs, three in the VH (H1, H2, H3) and three in the VL ( As used herein, "CDRs derived from a variable region" refers to CDRs in the L1, L2, and L3 regions. , CDRs with no more than two amino acid substitutions compared to the corresponding CDRs from the original variable region Exemplary CDRs herein refer to the following for various antibodies of the present disclosure: (a) amino acid residues 26-32 (L1), 50-52 (L2), and 91-9 6 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) Hypervariable loops (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987); (b) amino acid residues 24-34 (L1 ), 50~56(L2), 89~97(L3), 31~35b(H1), 50~65(H 2), and the CDR occurring at 95–102 (H3) (Kabat et al., Se Quences of Proteins of Immunological Int erest, 5th Ed. Public Health Service, Na tional Institutes of Health, Bethesda, M. D (1991 )); (c) Amino acid residues 27c~36 (L1), 46~55 (L2) , 89–96 (L3), 30–35b (H1), 47–58 (H2), and 93–101 Antigen contact occurs at (H3) (MacCallum et al. J. Mol. B iol. 262: 732-745 (1996)); and (d)(a), (b), and and / or a combination of (c). Unless otherwise specified, the variable domain The CDR residues and other residues (e.g., FR residues) in Eu numbering is according to at et al.

[0033] The term "isolated antibody" refers to an antibody that has been separated from a component of its natural environment. In this embodiment, the isolated antibody can be isolated by, for example, electrophoresis or chromatography (e.g., immunoprecipitation). Purified to >95% or >99% purity as determined by ion-exchange or reversed-phase HPLC will be done.

[0034] The term "chimeric" antibody refers to an antibody in which a portion of the heavy and / or light chains are derived from a particular source or species. an antibody derived from one of the heavy and / or light chains, while the remainder of the heavy and / or light chains are derived from a different source or species. Refers to...

[0035] A "human antibody" is an antibody produced by a human or human cells, or derived from the human antibody repertoire. The amino acid sequence of an antibody, or other human antibody coding sequence, derived from a non-human source utilizing This definition of a human antibody is one that possesses an amino acid sequence corresponding to a non-human antigen. Humanized antibodies containing binding residues are specifically excluded. Human antibodies can be generated using phage display libraries. These can be produced using a variety of techniques known in the art, including by ELISA. enboom and Winter. J. Mol. Biol. 227:381 ,1991;Marks et al. J. Mol. Biol. 222:58 1, 1991. Cole et al. Monoclonal Antibodie s and Cancer Therapy, Alan R. Liss, p. 7 7 (1985);Boerner et al. J. Immunol., 147 (1):86-95, 1991, is also used for the preparation of human monoclonal antibodies. van Dijk and van de Winkel. Cu See also RR. Opin. Pharmacol. 5:368-74, 2001. and.

[0036] A "humanized" antibody is one that contains amino acid residues from non-human CDRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody comprises all or substantially all of its CDRs. substantially all of the CDRs correspond to those of a non-human antibody, and all or substantially all of the FRs correspond to those of a human antibody. The humanized antibody contains variable domains corresponding to the FRs of the antibody. In certain embodiments, substantially all of the FRs of a human antibody correspond to the FRs of a humanized antibody. Either one or more of the veneer position residues of the FR, and / or one or more other selected The selected residues may contain one or more amino acid residues of the non-human FR(s). A humanized antibody can optionally contain at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody, e.g., a non-human antibody, refers to an antibody that has undergone humanization. A humanized antibody retains the same binding specificity and affinity as the starting non-human antibody.

[0037] The term "monoclonal antibody" refers to any antibody, e.g., eukaryotic, prokaryotic, or phagocytic. "Monoclonal" refers to an antibody derived from a single copy or clone, including a multi-copy clone. The term "clonal antibody" is not limited to antibodies produced by hybridoma technology. Monoclonal antibodies can be produced using hybridoma technology, as well as recombinant technology, which are well known in the art. techniques, phage display techniques, synthetic techniques, or techniques readily available in the art. It can be produced using a combination of other known techniques.

[0038] The term "epitope" refers to the specific site on an antigen to which an antibody binds. The specific site on the antigen where the hydroxyl group binds can be determined, for example, by crystallography. Radical protein footprinting and alanine scanning mutagenesis Any method can be used, but may result in lower resolution.

[0039] The term "monospecific antibody" refers to an antibody that specifically binds to only one antigen. A monospecific antibody may bind to only one epitope of an antigen or to two or more epitopes of an antigen. Monospecific antibodies that bind to more than one epitope of an antigen. is a monospecific polyepitope antibody.

[0040] The term "multispecific antibody" refers to an antibody that specifically binds to more than one antigen (e.g., For example, bispecific antibodies, trispecific antibodies, etc. Non-limiting examples of multispecific antibodies include: , a heavy chain variable domain (VH) and a light chain variable domain (VL), and the VH / VL unit is poly(amino acid). Antibodies with epitope specificity, having two or more VL and VH domains, each VH / Antibodies that bind to different epitopes in the VL units have two or more single variable domains, each of which antibodies, diabodies, triabodies, etc., in which one variable domain binds to a different epitope; as well as full length antibodies and / or covalently or non-covalently linked antibody fragments. Examples include, but are not limited to:

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

[0042] The term "Fc region" includes native sequence Fc regions and variant Fc regions. Used herein to define the C-terminal region of an immunoglobulin heavy chain. Although the boundaries of the Fc region of a globulin heavy chain can vary, the human IgG heavy chain Fc region typically From the amino acid residue at position ys226 or from Pro230 to its carboxyl terminus The C-terminal lysine of the Fc region (residue 44 according to the EU numbering system) is defined as the extension. 7) is used, for example, during the production or purification of an antibody, or to synthesize a nucleic acid encoding the heavy chain of an antibody. Therefore, the intact antibody composition may be removed by recombinant engineering. The composition includes an antibody population in which all Lys447 residues have been removed, an antibody population in which no Lys447 residues have been removed, Antibody populations, and antibody populations having a mixture of antibodies with and without the Lys447 residue. It may also include a group.

[0043] A "functional Fc region" possesses an "effector function" of a native sequence Fc region. Typical "effector functions" include C1q binding; complement-dependent cytotoxicity (CDC); Fc receptor antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; cell surface receptors (e.g. These effects include downregulation of the B cell receptor (BCR). The receptor function is generally achieved by combining the Fc region with a binding domain (e.g., an antibody variable domain). Various methods disclosed herein or otherwise known in the art may be used in combination. A functional Fc region has substantially the same structure as wild-type IgG. effector function similar to that of wild-type IgG, reduced effector function compared to wild-type IgG, or It may have enhanced effector functions compared to wild-type IgG. In the case of antibodies, the comparison is typically made with wild-type human IgG1.

[0044] A "native sequence Fc region" is an Fc region having an amino acid sequence identical to that of an Fc region found in nature. The native sequence human Fc region contains the amino acid sequence of native sequence human IgG1 F c region (non-A and A allotypes), native sequence human IgG2 Fc region, native Native sequence human IgG3 Fc region, and native sequence human IgG4 Fc region, and These include naturally occurring variants.

[0045] A "variant Fc region" is a variant Fc region that contains at least one amino acid modification (e.g., about 1 to about 10 amino acids). amino acid modifications, and in some embodiments from about 1 to about 5 amino acid modifications), preferably is a sequence of a native sequence Fc region comprising one or more amino acid substitutions The variant Fc region herein preferably comprises an amino acid sequence different from that of the The Fc region of the parent polypeptide is at least about 80% identical to the native sequence Fc region and / or the Fc region of the parent polypeptide. % homology thereto, preferably at least about 90% homology thereto, or preferably In some embodiments, the variant Fc The region may have reduced or enhanced effector function compared to wild-type IgG. For antibodies containing a human Fc region, the comparison is typically made with wild-type human IgG1. do.

[0046] As used herein, "Fc component" refers to the hinge region, CH2 domain, and Or it refers to the CH3 domain.

[0047] The "hinge region" generally extends from about residues 216 to 230 of IgG (Eu number (Kabat numbering), from about residue 226 to about residue 243 of IgG (Kabat numbering), or IgG residues 1 to 15 (IMGT specific numbering). It is justified.

[0048] The term "antibody fragment" refers to an intact antibody that binds to the antigen that the intact antibody binds. Antigen-binding fragments refer to molecules other than intact antibodies that contain a portion of an antibody. Examples of antigen-binding fragments include, but are not limited to, Not limited to, but includes diabodies, Fab, Fab', F(ab')2, F(ab) c , Fv disconnection fragment, disulfide-stabilized Fv fragment (dsFv), (dsFv)2, bispecific dsFv ( dsFv-dsFv'), disulfide-stabilized diabodies (ds diabodies), tri abody, tetrabody, single chain antibody, scFv, scFv dimer, single domain antibody, single These include single-domain antibodies and multivalent domain antibodies. Typically, a binding fragment is The binding fragments compete for specific binding with the intact antibody from which they are derived. by DNA techniques or by enzymatic or chemical separation of intact immunoglobulins It can be generated by

[0049] The term "Fab" refers to the variable region of a single heavy chain and a first The part of an antibody consisting of a single light chain (both variable and constant regions) bound to a constant region Point.

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

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

[0052] The term "Fv" refers to the minimum fragment of an antibody that retains a complete antigen-binding site. A fragment consists of the variable region of a single light chain linked to the variable region of a single heavy chain.

[0053] The term "single chain antibody" refers to a heavy chain variable region and a light chain variable region connected by a linker. In most, but not all, cases, the linker may be a peptide. The length of the linker varies depending on the type of single-chain antibody. Higher order forms can be obtained by covalently or non-covalently linking the antibodies. and their higher forms, such as single domain antibodies, multivalent domain antibodies, single chain variants, fragments (scFv), bivalent scFv (di-scFv), trivalent scFv (tri-scFv), Tetravalent scFvs (tetra-scFvs), diabodies, triabodies, and tetrabodies Examples of suitable materials include, but are not limited to,

[0054] The terms "single chain Fv antibody" and "scFv" are used interchangeably herein and It refers to a single-chain antibody consisting of a heavy chain variable region and a light chain variable region connected by a linker. In most cases, but not all, the linker may be a peptide. Linker peptides are preferred. or about 5 to 30 amino acids in length, or about 10 to 25 amino acids in length. Carriers stabilize variable domains without interfering with proper folding and creation of active binding sites. In a preferred embodiment, the linker peptide comprises glycine, as well as serine. Two or more scFvs can be covalently or non-covalently linked. By linking to the above, higher order Fvs such as di-scFv, tri-scFv, and tetra-scFv can be produced. The antigen binding site of each scFv in the higher order forms can bind to the same or different antigens. Or an epitope can be targeted.

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

[0056] "Diabodies" are higher-order variants of single-chain antibodies that consist of two single-chain antibodies. Multichain antibodies use a linker that is too short to allow pairing between the two domains on the same chain. This allows the domains to pair with complementary domains on another chain, thereby creating two antigen-binding sites. In most, but not all, cases, the linker can be a peptide. The antigen-binding sites can target the same or different antigens or epitopes. Triabodies (three single-chain antibodies assembled to form three antigen-binding sites) , tetrabodies (four single-chain antibodies assembled to form four antigen-binding sites), and and higher variants can be generated as well. For example, Holliger P et al., Proc Natl Acad Sci USA. July 1 5;90(14):6444-8 (1993);EP404097;WO93 / 111 See 61.

[0057] A "single domain antibody" is an antibody fragment containing 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. Multivalent domain antibodies are created by combining two or more V H The domain is The same or different antigens or epitopes can be targeted.

[0058] The term "heavy chain antibody" refers to an antibody that consists of two heavy chains. Heavy chain antibodies are found in camels, lambdas, and other mammals. It may be an IgG-like antibody from a macaque, alpaca, shark, etc., or an IgNAR from a cartilaginous fish. For example, Riechmann L. and Muyldermans S., J Immunol Methods. December 10;231(1-2): 25-38 (1999);Muyldermans S., J Biotechno l. June;74(4):277-302 (2001);WO94 / 04678; See WO94 / 25591; or U.S. Patent No. 6,005,079. Heavy Chain Antibodies originally derived from the Camelidae family (camels, dromedaries, and llamas). However, camelized antibodies have a genuine antigen-binding repertoire (Hamers-Ca sterman C. et al., Nature. June 3;363(64 28):446-8 (1993);Nguyen VK et al. avy-chain antibodies in Camelidae;a case of evolutionary innovation,” Immunogene tics. April;54(1):39-47 (2002);Nguyen V. K. et al. Immunology. May;109(1):93-101 (2003)). The variable domains of heavy chain antibodies (VHH domains) are represents the smallest known antigen-binding unit produced by the immune response (Koch-Nolte F et al., FASEB J. November;21(13):3490- 8. Epub 2007 Jun. 15 (2007)).

[0059] "Nanobodies" are composed of a VHH domain derived from a heavy chain antibody and two constant domains, C This refers to an antibody consisting of H2 and CH3.

[0060] "Percent identity (%)" with respect to a reference amino acid sequence is determined by aligning the sequences and The percentage of sequence identity is calculated after introducing gaps if necessary to achieve the maximum percent sequence identity. A sequence that is identical to an amino acid residue in a reference sequence, without taking into account any conservative substitutions. The amino acid sequence is defined as the percentage of amino acid residues in a candidate sequence that are Alignment for purposes of determining percent sequence identity is performed according to techniques known in the art. In various formats within the scope, e.g., BLAST, BLAST-2, or CLUSTAL achieved using publicly available computer software, such as Those skilled in the art will find that maximal alignment over the entire length of the sequences being compared is achieved. A suitable program for aligning sequences, including any algorithms required for Generally speaking, the amino acid sequence A can be determined by to, with, or against the amino acid sequence B ainst) sequence identity is calculated as follows: 100 × fraction X / Y, where X is , by a sequence alignment program, alignment of A and B in that program is the number of amino acid residues scored as perfect matches in B, and Y is the number of amino acid residues scored as perfect matches in B. The total number of amino acid residues. If the length of amino acid sequence A is not equal to the length of amino acid sequence B, The % amino acid sequence identity of A to B is equal to the % amino acid sequence identity of B to A. It is understood that this is not the case.

[0061] The terms "patient" or "subject" are used interchangeably herein and refer to a subject who is a patient with a human CD39 "A" refers to a human or non-human animal (e.g., a mammal) that expresses

[0062] "treat," "treating," "treatment" Terms such as "death" and "fever" refer to the illness or condition to which the term applies, whether temporary or permanent. Eliminate, reduce, or suppress at least one of the disorders or conditions or symptoms associated therewith Treatment refers to a course of action to alleviate, improve, or prevent the condition from worsening. , inhibition of active disease (e.g., a disease, disorder, or condition, or clinical symptoms associated therewith) preventing the onset or further onset of a disease), improving quality of life, and / or prolonging survival of a subject The length is mentioned.

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

[0064] "prevent," "preventing," "prevention" Terms such as "prevention" and "prophylaxis" are used interchangeably. Generally, in the context of a subject who is prone to having a particular disease, disorder, or condition, Temporarily or permanently prevent, suppress, inhibit, or reduce the risk of developing a disease, disorder, condition, etc. to reduce (e.g., as determined by the absence of clinical symptoms) or in some manner (e.g., prior to the onset of a disease, disorder, condition, or symptoms thereof) to delay the onset of In certain instances, these terms also refer to diseases, disorders, or slowing the progression of a condition or its harmful or otherwise undesirable effects. Prevention also refers to inhibiting the progression of an undesirable condition. or the recurrence of a disease, disorder, condition, or symptom after receiving treatment for that disease, disorder, condition, or symptom related thereto. It refers to a course of action initiated in a subject to prevent an outbreak.

[0065] As used herein, the term "in need of prevention" refers to a condition in which a subject is in need of preventative care. or by a physician or other caregiver that a patient would benefit from preventative care. This judgment is based on a variety of factors that are within the expertise of the physician or caregiver. This is based on the following:

[0066] II. Antibodies of the Present Disclosure The present disclosure provides a method for the production of antibodies that specifically bind to human CD39, more specifically to the extracellular domain of human CD39. The antibodies of the present disclosure are specific for soluble and / or membrane-bound human CD39. In some embodiments, the anti-CD39 antibodies of the present disclosure can bind monospecifically. In other embodiments, the anti-CD39 antibodies of the present disclosure are multispecific. Additional antigen binding specificities contemplated in the present invention include, but are not limited to, TGFβ. The anti-CD39 antibodies of the present application may be isolated or part of a composition. It is possible.

[0067] The anti-CD39 antibodies of the present disclosure, whether monospecific or multispecific, bind to human CD3 9, which is measured by surface plasmon resonance (SPR). 10 for CD39 -6 The equilibrium dissociation constant (K D ) means to have In certain embodiments, the anti-CD39 antibodies of the present disclosure exhibit a high affinity for human CD39 as measured by SPR. K for CD39 D But, 10 -8 M or less (e.g., 10 -8 , 10 -9 , 10 -10 etc.) See, e.g., Example 3 for detailed methodology. In various embodiments, the present disclosure The anti-CD39 antibody is a K antibody against human CD39. D is about 1x10 -9M ~ approx. 1x10 -14 M, Ma or approximately 1x10 -9 M ~ approx. 1x10 -13 M, or approximately 1x10 -9 M ~ approx. 1x10 -12 M, also is approximately 1x10 -9 M ~ approx. 1x10 -11 In some embodiments, the anti-CD31 inhibitor of the present disclosure is The 39 antibody is a K antibody against human CD39. D is about 1x10 -10 ~approx. 1x10 -14 M, or about 1 x10 -10 M ~ approx. 1x10 -13 M, or approximately 1x10 -10 M ~ approx. 1x10 -12 I'm M. In some embodiments, the anti-CD39 antibodies of the present disclosure have K D About 1x1 0 -11 M ~ approx. 1x10 -14 M, or approximately 1x10 -11 ~approx. 1x10 -13 M. Some In embodiments, the anti-CD39 antibodies of the present disclosure have a K D is about 1x10 -12 M ~approx. 1x10 -14 M, or approximately 1x10 -12 M ~ approx. 1x10 -13 M. Some implementations In one embodiment, the anti-CD39 antibodies of the present disclosure have a K D is about 1x10 -10 M ~ approx. 1x10 -11 M, or approximately 1x10 -11 M ~ approx. 1x10 -12 I am M.

[0068] The anti-CD39 antibodies of the present disclosure also inhibit CD39 enzymatic activity. The anti-CD39 antibodies of the present disclosure have an IC of about 5 nM or less, as measured as in Example 5.50 value For example, anti-CD39 antibodies inhibit the enzymatic activity of human CD39. IC50 of about 5 nM, about 4 nM, about 3 nM, about 2 nM, about 1 nM, or less 50 As another example, recombinant human CD39 can inhibit enzymatic activity at low levels. The antibody may have a concentration of about 1 nM or less, for example, about 1.0 nM, about 0.9 nM, about 0.8 nM, about 0.7 nM, M, about 0.6 nM, about 0.5 nM, or lower IC 50 Recombinant human CD39 enzyme As another example, anti-CD39 antibodies can inhibit the activity of the enzyme measured as in Example 5. The concentration of about 0.5 nM or less, for example, about 0.5 nM, about 0.4 nM, about 0.3 nM, about 0 .2nM, about 0.1nM, about 0.09nM, about 0.08nM, about 0.07nM, about 0.0 IC of 6 nM, approximately 0.05 nM, or less 50 Recombinant human CD39 enzyme activity It can inhibit the above IC 50 Values ​​may also be expressed as individual values ​​or ranges. For example, anti-CD39 antibodies inhibit recombinant human CD39 enzyme activity at concentrations ranging from about 0.05 nM to about 5 nM, about 0.05nM to about 1nM, 0.05nM to about 0.5nM, about 1nM to about 5nM, IC of about 0.5 nM to about 1.0 nM, or any subrange thereof 50 can be inhibited by In one embodiment, the anti-CD39 antibody of the present disclosure inhibits human CD39 enzymatic activity by about 0. IC of 5 nM to about 1.0 nM, or about 0.6 nM to about 1.0 nM 50 Inhibit by value. 1 In embodiments, the anti-CD39 antibodies of the disclosure inhibit human CD39 enzymatic activity by about 0.05 nM up to about 0.5 nM, about 0.05 nM to about 0.4 nM, or about 0.05 nM to about 0.3 nM IC50 In one embodiment, the anti-CD39 antibody of the present disclosure inhibits human CD39 The enzyme activity was adjusted to about 0.07 nM to about 0.5 nM, about 0.07 nM to about 0.4 nM, and about 0.0 IC of 7 nM to about 0.3 nM, or about 0.07 nM to about 0.2 nM 50 Inhibit by value. In the above-described embodiment, (i) the human CD39 may be recombinant soluble CD39, or or cell surface expressed CD39, and / or (ii) the inhibition is due to low ATP ( have been evaluated in the presence of high ATP (e.g., 20 μM) and / or high ATP (e.g., 400 μM). Good too.

[0069] A. Exemplary Variable Regions The present disclosure provides anti-CD39 antibodies comprising a VH, or a VH and a VL, described herein. In the following embodiments, "at least X% sequence identity" refers to each of these individual values ​​and ranges. For example, "at least 90% sequence identity" includes at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 9 6%, at least 97%, at least 98%, at least 99% sequence identity, and Individual values ​​(e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% , 98%, 99%, 100% sequence identity) and ranges therein.

[0070] In one example, an anti-CD39 antibody of the disclosure has one or more CDRs derived from SEQ ID NO:9. and optionally one or more CDRs from SEQ ID NO: 13. The CDRs derived from SEQ ID NO: 9 are H1, H2, H3, and H4. or any combination thereof. In certain embodiments, the VH may be H1 having at least 90% sequence identity with SEQ ID NO: 10, H2 having at least 90% sequence identity with SEQ ID NO: 11 H2, which has 0% sequence identity, and H3, which has at least 90% sequence identity with SEQ ID NO: 12 H3, or any combination thereof. CDRs derived from SEQ ID NO: 13 may be L1, L2, L3, or any combination thereof. In an embodiment, the VL comprises an L1 having at least 90% sequence identity to SEQ ID NO: 14, an L2 having at least 90% sequence identity to SEQ ID NO: 15, an L3 having at least 90% sequence identity to SEQ ID NO: 16, an L4 having at least 90% sequence identity to SEQ ID NO: 17, an L5 having at least 90% sequence identity to SEQ ID NO: 18, an L6 having at least 90 L2 having at least 90% sequence identity with SEQ ID NO: 15, L3 having at least 90% sequence identity with SEQ ID NO: 16 % sequence identity, or any combination thereof. An antibody comprising one or more CDRs from SEQ ID NO: 9 may also comprise one or more CDRs from SEQ ID NO: 13. The CDRs may further comprise a VL comprising R. The CDRs may be L1, L2, L3, or any of the above. In a preferred embodiment, the VL may be a combination of at least SEQ ID NO: 14. L1, which has an amino acid sequence with at least 90% sequence identity with SEQ ID NO: 15; L2, having an amino acid sequence with 0% sequence identity to SEQ ID NO: 16, at least 90% L3 having an amino acid sequence with sequence identity of In a specific example, the anti-CD39 antibody of the present disclosure may comprise a sequence identical to SEQ ID NO: 10 and at least 9 H1, having an amino acid sequence with 0% sequence identity, at least 90% with SEQ ID NO: 11 H2, having an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 12; VH comprising an H3 having an amino acid sequence with sequence identity to SEQ ID NO: 14, L1, which has an amino acid sequence with at least 90% sequence identity with SEQ ID NO: 15 L2, which has an amino acid sequence with 90% sequence identity, and SEQ ID NO: 16, and a VL comprising an L3 having an amino acid sequence with 90% sequence identity. In examples, the anti-CD39 antibodies of the disclosure include H1, having an amino acid sequence comprising SEQ ID NO: 10; H2 having an amino acid sequence comprising SEQ ID NO: 11, H3 having an amino acid sequence comprising SEQ ID NO: 12 VH comprising H3, and L1 having an amino acid sequence comprising SEQ ID NO: 14, SEQ ID NO: 15 L2 having an amino acid sequence comprising SEQ ID NO: 16, Contains VL.

[0071] In some of the foregoing embodiments, the antibody comprises: (i) a sequence that is at least 80% identical to SEQ ID NO: 9; a mature VH having an amino acid sequence identical to, and / or at least identical to, SEQ ID NO: 13; It may further comprise a mature VL having an amino acid sequence with 80% sequence identity. In embodiments, the antibody comprises a mature VH and a mature VL and is an antibody of Table A. JPEG2025148452000001.jpg1892

[0072] In each of the foregoing embodiments, the anti-CD39 antibody is (i) an intact antibody or and / or (ii) a chimeric, humanized, or human antibody. Suitable chimeric, humanized, and human antibodies are described in Sections IIC, IID, and and IIE. In certain embodiments, the anti-CD39 antibody is substantially The antibody may optionally include one or more constant regions, or portions of constant regions, that are fully 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. JPEG2025148452000002.jpg18106

[0074] In another example, the anti-CD39 antibody of the disclosure may be SEQ ID NO: 17, SEQ ID NO: 43, or SEQ ID NO: a heavy chain variable region (VH) having one or more CDRs from SEQ ID NO:44 and optionally No. 21, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, or SEQ ID NO: 4 and a light chain variable region (VL) having one or more CDRs from SEQ ID NO: 17, The CDRs derived from SEQ ID NO: 43 or SEQ ID NO: 44 may be H1, H2, H3, or any of the above. In certain embodiments, the VH may be any combination of SEQ ID NO: 18. H1, having at least 90% sequence identity with SEQ ID NO: 19 H2 having identity to SEQ ID NO: 20; H3 having at least 90% sequence identity to SEQ ID NO: 20; may include any combination thereof. 6, SEQ ID NO: 47, SEQ ID NO: 48, or SEQ ID NO: 49, the CDRs of which are L1, L2 , L3, or any combination thereof. L has at least 90% sequence identity with SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 24. L1 having at least 90% sequence identity with SEQ ID NO: 25; L2 having at least 90% sequence identity with SEQ ID NO: 2 L3 having at least 90% sequence identity with 6, or any combination thereof. One or more C sequences from SEQ ID NO: 17, SEQ ID NO: 43, or SEQ ID NO: 44 may be used. Antibodies containing DR include those listed in 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 a preferred embodiment, the VL is at least one of SEQ ID NO:22, SEQ ID NO:23, or SEQ ID NO:24. L1, having at least 90% sequence identity with SEQ ID NO: 25 L2 having at least 90% sequence identity to SEQ ID NO: 26, or In a specific example, the anti-CD39 antibodies of the present disclosure may comprise any combination of the following: H1, having an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 18; H2, SEQ ID NO: 2, having an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 19 VH comprising an H3 having an amino acid sequence with at least 90% sequence identity to and at least 90% sequence identity with SEQ ID NO:22, SEQ ID NO:23, or SEQ ID NO:24. L1 having an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 25 L2 having an amino acid sequence identical to SEQ ID NO:26, and L3 having an amino acid sequence identical to SEQ ID NO:27. In another specific example, the anti-CD31 antibody of the present disclosure includes a VL comprising an L3 having an amino acid sequence The 39 antibody includes H1 having an amino acid sequence comprising SEQ ID NO: 18, H2 having an amino acid sequence comprising SEQ ID NO: 19, VH comprising H2 having the amino acid sequence SEQ ID NO: 20, H3 having the amino acid sequence SEQ ID NO: 21, and 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 VL including L3 having

[0075] In some of the foregoing embodiments, the antibody has (i) at least 80% sequence identity with SEQ ID NO: 17. VH having an amino acid sequence with at least 8 sequences identical to SEQ ID NO: 21 or (ii) a VL having an amino acid sequence with SEQ ID NO: 43 or or a VH having an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 44; and / or SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, or the sequence VL having an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 49, In an exemplary embodiment, the antibody comprises a VH and a VL and is an antibody of Table C. . JPEG2025148452000003.jpg5996

[0076] In each of the foregoing embodiments, the anti-CD39 antibody is (i) an intact antibody or and / or (ii) a chimeric, humanized, or human antibody. Suitable chimeric, humanized, and human antibodies are described in Sections IIC, IID, and and IIE. In certain embodiments, the anti-CD39 antibody is substantially The antibody may optionally include one or more constant regions, or portions of constant regions, that are fully 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. JPEG2025148452000004.jpg65111

[0078] In another example, the anti-CD39 antibody of the disclosure may be SEQ ID NO: 27, SEQ ID NO: 58, or SEQ ID NO: a heavy chain variable region (VH) having one or more CDRs from SEQ ID NO:59 and optionally SEQ ID NO:6 SEQ ID NO: 31, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, or SEQ ID NO: 63 and a light chain variable region (VL) having at least one CDR. The CDRs from 9 may be H1, H2, H3, or any combination thereof. In certain embodiments, the VH has at least 90% sequence identity to SEQ ID NO:28. H1 having at least 90% sequence identity with SEQ ID NO: 29; H2 having at least 90% sequence identity with SEQ ID NO: 3 0, H3 with at least 90% sequence identity, or any combination thereof. CDs derived from SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, and SEQ ID NO: 63 may also be used. R may be L1, L2, L3, or any combination thereof. In embodiments, the VL comprises an L1 having at least 90% sequence identity to SEQ ID NO: 32; L2, which has at least 90% sequence identity with SEQ ID NO: 33; L3 having 0% sequence identity, or any combination thereof. Antibodies comprising one or more CDRs from SEQ ID NO: 58 or SEQ ID NO: 59 include SEQ ID NO: 60, Further, VLs comprising one or more CDRs from SEQ ID NO: 61, SEQ ID NO: 62, and SEQ ID NO: 63 are also included. The CDRs may be L1, L2, L3, or any combination thereof. In a preferred embodiment, the VL has at least 90% sequence identity with SEQ ID NO: 32. L1, having an amino acid sequence with at least 90% sequence identity with SEQ ID NO: 33 L2 having an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 34 L3 having an amino acid sequence corresponding to the amino acid sequence of the nucleotide ... In a typical example, the anti-CD39 antibody of the disclosure has at least 90% sequence identity with SEQ ID NO:28. H1 having an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 29 H2, having an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 30 VH comprising H3 having an amino acid sequence at least 90% identical to SEQ ID NO: 32. L1 having an amino acid sequence with identity, at least 90% sequence identity to SEQ ID NO: 33 L2 having an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 34 In another specific example, the VL comprises an L3 having an amino acid sequence having the same structure as that of the VL of the present disclosure. Anti-CD39 antibodies include H1 having an amino acid sequence comprising SEQ ID NO:28, SEQ ID NO:29 VH comprising H2 having an amino acid sequence, H3 having an amino acid sequence comprising SEQ ID NO: 30; and L1 having an amino acid sequence comprising SEQ ID NO: 32, L2 having an amino acid sequence comprising SEQ ID NO: 33. and VL comprising L2 having the amino acid sequence comprising SEQ ID NO: 34.

[0079] In some of the foregoing embodiments, the antibody has (i) at least 80% sequence identity with SEQ ID NO: 27. VH having an amino acid sequence with at least 8 sequences identical to SEQ ID NO: 31, and / or or (ii) a VL having an amino acid sequence with SEQ ID NO: 58 or or a VH having an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 59; and / or at least SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, or SEQ ID NO: 63 The VL may further comprise a VL having an amino acid sequence with at least 80% sequence identity. In an exemplary embodiment, the antibody comprises a VH and a VL and is an antibody of Table E. JPEG2025148452000005.jpg6697

[0080] In each of the foregoing embodiments, the anti-CD39 antibody is (i) an intact antibody or and / or (ii) a chimeric, humanized, or human antibody. Suitable chimeric, humanized, and human antibodies are described in Sections IIC, II, III, and II-IV, respectively. IID, and IIE. In certain embodiments, anti-CD39 antibodies optionally comprising one or more constant regions, or portions of constant regions, that are substantially human. Suitable constant regions are described in further detail in Section II(F).

[0081] In another exemplary embodiment, the antibody is an antibody of Table F. JPEG2025148452000006.jpg77114

[0082] B. Antibodies with similar binding specificities The present disclosure relates to an antibody designated as 19 or ch19_IGG4.P above, or Note 31, ch31_IGG4.P, hu31.1_IGG4.P, hu31.2_IGG 4.P, hu31.3_IGG4.P, hu31.4_IGG4.P, hu31.4_I GG1.AA, hu31.5_IGG4.P, hu31.6_IGG4.P, or h Antibodies designated as u31.7_IGG4.P, or above 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, hu Antibodies designated as 39.7_IGG4.P or hu39.8_IGG4.P, Also provided are anti-CD39 antibodies that bind to the same or overlapping epitopes as , X-ray crystallography of an antibody bound to its antigen to identify contact residues. Such antibodies may be identified by known methods. Competitive assays may be used to identify such antibodies. For example, an anti-CD39 antibody may competitively inhibit the binding of a reference antibody to human CD39. The reference antibodies are 19, ch19_IGG4.P, 31, ch31_IGG 4.P, hu31.1_IGG4.P, hu31.2_IGG4.P, hu31.3_I GG4.P, hu31.4_IGG4.P, hu31.4_IGG1.AA, hu31. 5_IGG4.P, hu31.6_IGG4.P, or hu31.7_IGG4.P, 39, ch39_IGG4.P, ch39_mIGG2A.AAG, hu39.1_IG G4.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 antibody inhibits the binding of the reference antibody to human CD39 by at least 50%, or At least 60%, at least 70%, at least 80%, or at least 90% blocked If so, the antibody is said to competitively inhibit the binding of the reference antibody to human CD39. The inhibition is determined by a competitive flow assay, e.g., as described in Example 7. It is possible.

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

[0084] Other antibodies with such binding specificity may be used, for example, antibodies containing human CD39 or a desired epitope. The resulting antibodies are then immunized with a portion of the antibody, optionally designated Antibody 19 or its derivatives. riants (e.g., ch19_IGG4.P), antibody 31, or its variants (e.g., ch31_IGG4.P, hu31.1_IGG4.P, hu31.2_IG G4.P, hu31.3_IGG4.P, hu31.4_IGG4.P, hu31.4_ IGG1.AA, hu31.5_IGG4.P, hu31.6_IGG4.P, hu31 .7_IGG4.P, etc.), or antibody 39 or a variant thereof (e.g., ch3 9_IGG4.P, ch39_mIGG2A.AAG, hu39.1_IGG4.P, h u39.2_IGG4.P, hu39.3_IGG4.P, hu39.4_IGG4.P , hu39.5_IGG4.P, hu39.5_IGG1.AA, hu39.6_IGG 4.P, hu39.7_IGG4.P, hu39.8_IGG4.P, etc.), which compete with generated by screening for binding to the extracellular domain of human CD39 Antibodies can also be screened against mutagenized forms of human CD39. te, 19, ch19_IGG4.P, 31, ch31_IGG4.P, hu31.1_I GG4.P, hu31.2_IGG4.P, hu31.3_IGG4.P, hu31.4 _IGG4.P, hu31.4_IGG1.AA, hu31.5_IGG4.P, hu3 1.6_IGG4.P, hu31.7_IGG4.P, 39, ch39_IGG4.P, ch39_mIGG2A.AAG, hu39.1_IGG4.P, hu39.2_IGG 4.P, hu39.3_IGG4.P, hu39.4_IGG4.P, hu39.5_I GG4.P, hu39.5_IGG1.AA, hu39.6_IGG4.P, hu39. The same as a collection of mutations, such as 7_IGG4.P or hu39.8_IGG4.P Antibodies that exhibit the same or similar binding profile can be identified. The entire extracellular domain of the antibody, or a section of it where the epitope is known to reside, Alanine (or alanine) residues are introduced one residue at a time or at more widely spaced intervals across the Systematic replacement of hydroxylase with hydroxylase (serine if hydroxylase is already present) institution).

[0085] Other antibodies include exemplary antibodies such as 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, h u31.5_IGG4.P, hu31.6_IGG4.P, hu31.7_IGG4.P , 39, ch39_IGG4.P, ch39_mIGG2A.AAG, hu39.1_I GG4.P, hu39.2_IGG4.P, hu39.3_IGG4.P, hu39.4 _IGG4.P, hu39.5_IGG4.P, hu39.5_IGG1.AA, hu3 9.6_IGG4.P, hu39.7_IGG4.P, or hu39.8_IGG4. The mature heavy chain of P can be obtained by mutagenesis of the cDNA encoding the heavy and light chains of P. 19, ch19_IGG4.P, 3 in the amino acid sequence of the chain and / or light chain variable region 1, ch31_IGG4.P, hu31.1_IGG4.P, hu31.2_IGG4. P, hu31.3_IGG4.P, hu31.4_IGG4.P, hu31.4_IGG 1.AA, hu31.5_IGG4.P, hu31.6_IGG4.P, hu31.7_ IGG4.P, 39, ch39_IGG4.P, ch39_mIGG2A.AAG, hu 39.1_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 at least about 80%, 81%, 82%, 83%, 84%, 85%, 8 6%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 9 6%, 97%, 98%, 99%, or 100% identity and its functional characteristics Antibodies that maintain their nucleotide sequence and / or have a small number of amino acid substitutions that are functionally insignificant (e.g. Antibodies that differ from the respective antibodies by deletions, conserved substitutions, deletions, or insertions are also disclosed herein. The amino acids in the variable region framework that are likely to be important for binding are: They can be identified as described in the section on humanization below.

[0086] The binding specificity or selection of the selected rodent antibody (e.g., 19, 31, or 37) Selected humanized antibodies (e.g., hu31.1_IGG4.P, hu31.2_IGG4. P, hu31.3_IGG4.P, hu31.4_IGG4.P, hu31.4_IGG 1.AA, hu31.5_IGG4.P, hu31.6_IGG4.P, hu31.7_ IGG4.P, hu39.1_IGG4.P, hu39.2_IGG4.P, hu39. 3_IGG4.P, hu39.4_IGG4.P, hu39.5_IGG4.P, hu3 9.5_IGG1.AA, hu39.6_IGG4.P, hu39.7_IGG4.P, Antibodies having the binding specificity of hu39.8_IGG4.P or hu39.8_IGG4.P can also be used in phage dimerization. It can be produced using a modification of the spray method. See 1. This method is particularly suitable for producing human antibodies. Either the heavy or light chain variable region of the antibody is used as the starting material. For example, if a light chain variable region is selected as the starting material, the members may be selected from the same light chain variable region (all That is, a phage library displaying the starting light chain and different heavy chain variable regions is constructed. The heavy chain variable region is constructed, 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 Tomo10 8 or at least 10 9 M -1 ) and then select the heavy chain variable fragment from this phage. This region serves as the starting material for constructing further phage libraries. In the library, each phage contains the same heavy chain variable region (i.e., the first display line). The light chain variable regions are shown in Table 1. The light chain variable regions are shown in Table 1. For example, they can be obtained from a library of rearranged human light chain variable regions. Phages that show strong specific binding to CD39 are selected. The resulting antibodies are usually It has the same or similar epitope specificity as the starting material.

[0087] In addition to the antibodies described herein, methods known in the art can be used to synthesize antibodies of the invention. Design antibody mimics or aptamers with the same binding specificity, with substantially the same function. An "antibody mimetic" is a compound that can specifically bind to an antigen but does not have the same structure as an antibody. refers to a polypeptide or protein that is not structurally related. Antibody mimics are those with a molecular mass of approximately 3 kDa. Non-limiting examples of antibody mimetics include affibody molecules, affibody molecules, and affibody molecules. Antibody, Avimer, Alphabody, Anticalin, Avimer, DARPin, and Molecular Biology Aptamers are composed of RNA or single-stranded DNA oligonucleotides. A class of small nucleic acid ligands with high specificity and affinity for their targets Aptamers bind to their targets through structural recognition, a process similar to that of antigen-antibody reactions. Aptamers have a lower molecular weight than antibodies, typically about 8 ~25kDa.

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

[0089] In certain embodiments, chimeric antibodies are antibodies derived from non-human antibodies (e.g., mouse, rat, etc.). The mature variable regions of the light and heavy chains of the antibody 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 have a binding specificity of about 3 Two-thirds are human sequences.

[0090] A veneered antibody is a non-human antibody that combines some, and usually all, of the CDRs with a non-human variable region frame. retain some of the framework residues but may contribute to B- or T-cell epitopes. Other variable region framework residues that may be present, such as exposed residues (Padlan, Mol. Im munol. 28:489, 1991) to the residue at the corresponding position in the human antibody sequence. A type of humanized antibody is one in which the CDRs are replaced with those of a fully or substantially non-human antibody. The variable region framework of a non-human antibody is made more human-like through substitutions. Veneered forms of anti-CD39 antibodies are included in the present disclosure.

[0091] In some embodiments, the anti-CD39 chimeric antibody comprises a rat variable domain and a human Ig G1 and kappa constant domains (or variants thereof) or human IgG4 and kappa constant domains A preferred human chimera is a rat-human chimera having a common domain (or a variant thereof). Common domains are known in the art and are further described in Section II(F). In a preferred embodiment, the anti-CD39 chimeric antibody is ch19_IGG4.P, ch31_IG G4.P, and ch39_IGG4.P.

[0092] D. Humanized Antibodies As described herein, the present disclosure provides antibodies designated herein as 19, 31 or 39. and optionally the humanized antibody has a molecular weight of about 5n, as measured as in Example 5. M or less, about 0.05nM to about 5nM, about 1nM or less, about 0.05nM to about 1nM, about 0. IC of 5 nM or less, or about 0.05 nM to about 0.5 nM 50 Human CD39 enzyme activity was measured using Typically, the non-human antibody inhibits the specificity and affinity of the parent non-human antibody while retaining the specificity and affinity of the parent non-human antibody. Humanized antibodies are antibodies that have CDRs or other fragments that are not specifically targeted to the human genome. A portion of these is derived from a non-human antibody, and the FRs or portions thereof are derived from human antibody sequences. Humanized antibodies optionally contain at least one human constant region. In some embodiments, some FR residues in a humanized antibody may also comprise a portion thereof. The group may be used to modify non-human antibodies, for example, to restore or improve antibody specificity or affinity. (eg, the antibody from which the CDR residues are derived).

[0093] Thus, a humanized antibody has some or all of its CDRs completely or substantially identical to those of the donor. The variable region framework sequences and constant region sequences, if present, are derived from an antibody. is an antibody that is substantially derived from human antibody sequences. For example, a humanized heavy chain may be derived from a donor antibody heavy chain. at least one, two, and usually all three CDRs derived entirely or substantially from and, if present, substantially derived from human heavy chain variable region framework and constant region sequences. The humanized light chain has a heavy chain variable region framework sequence and a heavy chain constant region corresponding to the humanized light chain. , at least one, two, and usually three, light chains derived entirely or substantially from the donor antibody light chain. All CDRs, if present, and substantially human light chain variable region framework and constant sequences. It has a light chain variable region framework sequence and a light chain constant region derived from a constant region sequence. However, as elsewhere in this application, the CDRs of the subject antibodies are identified by corresponding residues (as defined by Kabat). at least about 80%, 81%, 82%, 83%, 84%, 85%, 86% of the total 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, If there is 97%, 98%, 99%, or 100% identity between the respective CDRs, the reference Substantially derived from the corresponding CDRs of an antibody; but The CDR H2 to be used should have at least about 65% of the corresponding residues (as defined by Kabat) , 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% identity between the respective CDRs In this case, the variable region framework of the antibody chain is substantially derived from the corresponding CDR of the reference antibody. The constant region of the sequence or antibody chain has at least about 8 of the corresponding residues defined by Kabat. 0%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 9 0%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or If the human variable region framework sequences are 100% identical, or 100% identical, respectively, Substantially derived from the common region.

[0094] Generally speaking, a humanized antibody is one in which the CDRs from a non-human "donor" antibody are ligated to a human "acceptor" antibody. These are genetically engineered antibodies grafted onto a target antibody sequence (e.g., Queen, U S5,530,101 and 5,585,089; Winter, US5,225,539 , Carter, US6,407,213, Adair, US5,859,2056,8 81,557; Foote, U.S. Pat. No. 6,881,557). Acceptor antibody The sequences may be, for example, mature human antibody sequences, composites of such sequences, consensus sequences of human antibody sequences, or the like. The sequences may be either nucleotide sequences or germline region sequences. It is possible to incorporate all six CDRs from a non-human antibody (mouse, rat, etc.), but and using fewer than all CDRs (e.g., at least 3, 4, or 5) from a single host. Alternatively, the antibody can be prepared by the method described in, for example, Pascalis et al., J. Immunol. nol. 169:3076, 2002;Vajdos et al., Journ al of Molecular Biology, 320: 415-428, 2 002;Iwahashi et al., Mol. Immunol. 36:10 79-1091, 1999;Tamura et al, Journal of I mmunology, 164:1432-1441, 2000).

[0095] In some antibodies, a portion of the CDR, i.e., the SD, is removed to retain binding in the humanized antibody. Only a subset of CDR residues, called R, are required for binding. The CDR residues not included in the SDRs were determined based on previous studies (e.g., residues in CDR H2). Groups H60-H65 are often not required), outside the Chothia hypervariable loops from the Kabat CDR region in (Chothia, J. Mol. Biol. 196:901, 1987), by molecular modeling and / or empirically, or Gonzales et al., Mol. Immunol. 41: 863 Such humanized antibodies can be identified as described in [PubMed], [Chem.], [Google Scholar], 2004. indicates a position where one or more donor CDR residues are absent, or where the entire donor CDR is omitted. For positions where the amino acid occupying that position is the same as the amino acid at the corresponding position in the acceptor antibody sequence, (according to Kabat numbering). Substitute potential sites of post-translational modifications (e.g., glycosylation) within R to eliminate post-translational modifications. The substitution positions within the CDRs and the amino acids to be substituted can also be selected empirically. It is possible.

[0096] The acceptor is designed to match the selected human framework sequence with the human immunoglobulin. The sequences are identical, whether derived from the human consensus framework or the human Although the acceptor sequence may be a single sequence, in this disclosure, the acceptor sequence may include a human immunoglobulin sequence or a human immunoglobulin sequence. It is possible to include existing amino acid substitutions to the consensus framework sequence. It is contemplated that these existing substitutions may be minimal; generally, human immunoglobulin Four, three, two, or one sequence for the sequence or consensus framework sequence Only amino acid differences exist. The human acceptor antibody sequence may be High-quality inter-chain structure between the variable region framework and the corresponding variable region framework of the donor antibody chain To provide sequence identity (e.g., 65-85% identity), many known human antibodies The sequence can be selected arbitrarily.

[0097] Certain amino acids from the human variable region framework residues are essential for CDR conformation and / or or may be selected for substitution based on their potential effect on binding to the antigen. Investigation of such potential effects can be performed through modelling, location-specific network analysis and by examining the characteristics of amino acids or by empirical observation of the effects of specific amino acid substitutions or mutagenesis. For example, the non-human variable region framework residues can be combined with the selected human variable region framework residues. If an amino acid differs between a variable region framework residue and the amino acid is: (1) an antigen (2) directly non-covalently linked to the CDR region; (3) otherwise adjacent to the CDR region; regions reasonably expected to interact with the CDR region (e.g., within about 6 Å of the CDR region). Where the human framework amino acid is a non-human antibody, the human framework amino acid may be replaced by the equivalent framework amino acid from the non-human antibody. Other candidates for substitution are those rare in human immunoglobulins at that position. These amino acids are the acceptor human framework amino acids that are present in the non-human donor. The amino acids at the equivalent positions in antibodies, or the amino acids at the equivalent positions in more typical human immunoglobulins, It may be substituted with an amino acid.

[0098] In some embodiments, the humanized anti-CD39 antibody contains 0 to 2 amino acid substitutions or deletions. H1, including the amino acid sequence of SEQ ID NO: 10 having a deletion, 0 to 2 amino acid substitutions or deletions H2 comprising the amino acid sequence of SEQ ID NO: 11 having 0 to 2 amino acid substitutions or deletions H3 comprising the amino acid sequence of SEQ ID NO: 12, and an acceptor human framework region Area and at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, a mature VH comprising framework regions with 98%, 99%, or 100% identity and the amino acid sequence of SEQ ID NO: 14 having 0 to 2 amino acid substitutions or deletions. L1 containing the sequence, the amino acid sequence of SEQ ID NO: 15 with 0 to 2 amino acid substitutions or deletions L2 containing the sequence, the amino acid sequence of SEQ ID NO: 16 having 0 to 2 amino acid substitutions or deletions and an acceptor human framework region and 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% The mature VL comprises framework regions having the same identity. , may be determined according to the definition of Kabat.

[0099] In some embodiments, the humanized anti-CD39 antibody contains 0 to 2 amino acid substitutions or deletions. H1, including the amino acid sequence of SEQ ID NO: 18 having a deletion, 0 to 2 amino acid substitutions or deletions H2 comprising the amino acid sequence of SEQ ID NO: 19 having 0 to 2 amino acid substitutions or deletions H3 comprising the amino acid sequence of SEQ ID NO: 20, and an acceptor human framework region Area and at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, a mature VH comprising framework regions with 98%, 99%, or 100% identity and the amino acid sequence of SEQ ID NO: 22 having 0 to 2 amino acid substitutions or deletions. L1 containing the sequence, the amino acid sequence of SEQ ID NO: 25 with 0 to 2 amino acid substitutions or deletions L2 containing the sequence, the amino acid sequence of SEQ ID NO: 26 having 0 to 2 amino acid substitutions or deletions and an acceptor human framework region and 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% For example, the above-referenced The resulting humanized anti-CD39 antibody may have an L1 comprising SEQ ID NO:23 or SEQ ID NO:24. Framework regions may be determined according to the Kabat definition. wherein the VH human acceptor is M99642 or KF698734, and / or Or the VL human acceptor is X12682 or Z00023.

[0100] In some embodiments, the humanized anti-CD39 antibody contains 0 to 2 amino acid substitutions or deletions. H1, including the amino acid sequence of SEQ ID NO: 28 having a deletion, 0 to 2 amino acid substitutions or deletions H2 comprising the amino acid sequence of SEQ ID NO: 29 having 0 to 2 amino acid substitutions or deletions H3 comprising the amino acid sequence of SEQ ID NO: 30, and an acceptor human framework region Area and at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, a mature VH comprising framework regions with 98%, 99%, or 100% identity and the amino acid sequence of SEQ ID NO: 32 having 0 to 2 amino acid substitutions or deletions. L1 containing the sequence, the amino acid sequence of SEQ ID NO: 33 with 0 to 2 amino acid substitutions or deletions L2 containing the sequence, the amino acid sequence of SEQ ID NO: 34 having 0 to 2 amino acid substitutions or deletions and an acceptor human framework region and 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% The mature VL comprises framework regions having the same identity. In some embodiments, the VH human accession number may be determined according to the definition of Kabat. The VL acceptor is M99651 or M77327, and / or the VL human acceptor is The options are X12682 or M23090.

[0101] In the foregoing embodiments, the mature heavy chain variable region is linked to at least a portion of a heavy chain constant region. and the mature light chain variable region can be linked to at least a portion of a light chain constant region. In some embodiments, the mature heavy chain variable region is linked to a heavy chain constant region, The mature light chain variable region is linked to a light chain constant region. A suitable constant region is described in Section II. (F) In certain embodiments, the heavy chain constant region is a wild-type In another embodiment, the heavy chain constant has effector functions substantially similar to those of human IgG1. The common region may reduce or enhance 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: 1. The heavy chain constant region comprises or consists of SEQ ID NO: 6. In a specific example, The region includes amino acids 1 to 329 of SEQ ID NO: 1, or and the light chain constant region comprises or consists of SEQ ID NO:6. In an 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: 4. In another specific example, the heavy chain constant region comprises or consists of SEQ ID NO: 6. containing or consisting of amino acids 1 to 326 of SEQ ID NO: 4 In another specific example, the light chain constant region comprises or consists of SEQ ID NO:6. 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: 2. In another specific example, the heavy chain constant region comprises or consists of SEQ ID NO: 2. or consisting of amino acids 1 to 329 of SEQ ID NO: 2, The constant region comprises or consists of SEQ ID NO: 6. In another specific example, the heavy chain constant region The region comprises or consists of SEQ ID NO:5, and the light chain constant region comprises SEQ ID NO:6. or SEQ ID NO: 6. In another specific example, the heavy chain constant region consists of amino acid sequence of SEQ ID NO: 5. the light chain constant region comprises or consists of amino acids 1 to 326 of SEQ ID NO: 5, In another specific example, the heavy chain constant region comprises or consists of SEQ ID NO: 6. comprising or consisting of SEQ ID NO: 3, and the light chain constant region comprises SEQ ID NO: 6, or In another specific example, the heavy chain constant region consists of amino acids 1 to 3 of SEQ ID NO: 3. 29 or consisting of amino acids 1 to 329 of SEQ ID NO: 3, and the light chain constant region is 6 or consisting of SEQ ID NO:6.

[0102] E. Human antibodies As described herein, the present disclosure relates to: 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, hu 31.5_IGG4.P, hu31.6_IGG4.P, hu31.7_IGG4.P, 39, ch39_IGG4.P, ch39_mIGG2A.AAG, hu39.1_IG G4.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 a human antibody having the binding specificity of the antibodies specified herein, such as , human antibodies were measured in Example 550 Values ​​are approximately 5 nM or less, approximately 0.05 nM to approximately 5 nM M, about 1 nM or less, about 0.05 nM to about 1 nM, about 0.5 nM or less, or about 0.05 nM Human antibodies inhibit human CD39 enzyme activity at concentrations of approximately 0.5 nM. Human antibodies inhibit the activity of certain rodent antibodies. , e.g., antibodies designated herein as 19, 31, or 39. isomeric or certain humanized antibodies, e.g., ch19_IGG4.P herein; , 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_I GG4.P, ch39_IGG4.P, ch39_mIGG2A.AAG, hu39.1 _IGG4.P, hu39.2_IGG4.P, hu39.3_IGG4.P, hu39 .4_IGG4.P, hu39.5_IGG4.P, hu39.5_IGG1.AA, h u39.6_IGG4.P, hu39.7_IGG4.P, or hu39.8_IGG 4. Competitive binding experiments, W, to ensure that the antibody designated P has the same epitope specificity by the phage display method of inter, WO92 / 20791, or other methods. Human antibodies can be selected by using only a fragment of CD39 as a target antigen. and / or by screening antibodies against a collection of CD39 deletion mutants. It is also possible to screen for particular epitope specificity by performing

[0103] Methods for producing human antibodies include those described by Oestberg et al., Hybrid oma 2:361-367 (1983); Oestberg, U.S. Patent No. 4,634 ,664; and Engleman et al., U.S. Pat. No. 4,634,666. Trioma method, use of transgenic mice containing human immunoglobulin genes (e.g. For example, Lonberg et al., WO93 / 12227(1993); US5,877 7,397, US5,874,299, US5,814,318, US5,789,65 0, US5,770,429, US5,661,016, US5,633,425, US 5,625,126, US5,569,825, US5,545,806, Nature 148, 1547-1553 (1994), Nature Biotechnology 14, 826 (1996), Kucherlapati, WO 91 / 10741 (1991) and phage display methods (e.g., Dower et al., WO9 1 / 17271 and McCafferty et al., WO92 / 01047, US 5,877,218, US5,871,907, US5,858,657, US5,83 7,242, US 5,733,743 and US 5,565,332), It can be obtained.

[0104] The human antibodies of the present disclosure 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 heavy chain constant region. The region is linked to a light chain constant region. Suitable constant regions are further described in Section II(F). In certain embodiments, the heavy chain constant region is a wild-type human IgG 1. In another embodiment, the heavy chain constant region has effector functions substantially similar to those of the heavy chain constant region of the Compared to native human IgG1, the effector function was either reduced or enhanced. In an 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: 1. In another specific example, the heavy chain constant region comprises or consists of SEQ ID NO: 6. comprising or consisting of SEQ ID NO: 4, and the light chain constant region comprises SEQ ID NO: 6, or In another specific example, the heavy chain constant region comprises or consists of SEQ ID NO: 2. 2, 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 SEQ ID NO:5, and the light chain constant region comprises or consists of SEQ ID NO:5. The constant region comprises or consists of SEQ ID NO: 6. In another specific example, the heavy chain constant region The region comprises or consists of SEQ ID NO: 3, and the light chain constant region comprises SEQ ID NO: 6. or consisting of SEQ ID NO:6. F. Selection of Constant Region(s)

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

[0106] The choice of constant region (or truncation thereof) may depend on whether effector function is desired or further The effector functions of an antibody depend in part on whether it needs to be enhanced. Refers to the biological activity attributable to the light or heavy chain constant region and varies depending on the antibody isotype Non-limiting examples of antibody effector functions include C1q binding to the C1 complex and complementation. Body-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADC) C); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptors); and Human antibodies are classified into five isotypes (Ig) according to their heavy chains. IgM, IgD, IgG, IgA, and IgE, each of which serves a different function. IgG is classified into four human subclasses (IgG1, IgG2, IgG3, IgG4, IgG5), each containing a different heavy chain. IgG2, IgG3, and IgG4) which are highly homologous, mainly in the hinge region. and the degree to which they activate the host immune system. For example, human isoform IgG1 and IgG3 can mediate complement-mediated cytotoxicity, whereas human isotypes IgG2 and IgG4 and IgG5 do not or do so at very low levels. It can be class Lambda or Kappa.

[0107] Antibodies of the present disclosure that include a human constant region or a portion thereof are typically IgG antibodies, preferably The human constant region is an allotype that varies between different individuals. The constant regions exhibit allotypic and isoallotypic variation. Different individuals may differ at one or more polymorphic positions. Sera that recognize an isoallotype bind to non-polymorphic regions of one or more other isotypes Reference to a human constant region includes any naturally occurring allotype or or constant regions containing any permutation of residues occupying polymorphic positions in natural allotypes. do.

[0108] One or several amino acids at the amino or carboxy termini of the light and / or heavy chains The amino acids, e.g., the C-terminal lysine of the heavy chain, are either partially or completely missing from the molecule or The N-terminal glutamine of the heavy or light chain may be pyroglutamine. To prevent the formation of amino acids, glutamic acid residues can be substituted.

[0109] In some embodiments, the antibodies of the present disclosure are IgG4 antibodies. Incorporation of the S228P (Eu numbering) engineered mutation in the heavy chain to prevent Fab arm exchange Suitable sequences for human IgG4 include SEQ ID NO: 4 and SEQ ID NO: In some embodiments, the antibody is an IgG4 antibody. There is no C-terminal lysine.

[0110] In some embodiments, the antibodies of the present disclosure are IgG1 or IgG3 antibodies. Suitable sequences for IgG1 or IgG3 are known in the art and include SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and human IgG3 as disclosed in U.S. Pat. No. 5,624,821. In some embodiments, the antibody may be an IgG1 antibody or an IgG3 antibody. The C-terminal lysine of the antibody is absent.

[0111] In certain embodiments, substitutions are 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 those that increase the half-life of an antibody. Gln at position 250 and / or Leu at position 428 (Eu numbering) to increase Examples include:

[0112] Alternatively or additionally, substitutions can be made in the constant region to enhance complement-mediated cytotoxicity (CDC) or 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 may be modified by the introduction of constant region mutation(s). CDC, ADCC, and antibody-dependent cellular phagocytosis (ADCP) compared to the same antibody In some embodiments, the antibody is engineered to reduce Fc effector functions such as Each or all of these effector functions may be reduced compared to the unmutated antibody. The effector function is reduced by 50%, 75%, 90%, or 95%. Other assays can be performed as described in Shields et al. al, 2001 J. Biol. Chem., Vol. 276, p 65 91-6604;Chappel et al, 1993 J. Biol. Che m., Vol 268, p 25124-25131;Lazar et al, This is described in 2006 PNAS, 103;4005-4010.

[0114] Substitutions at any or all of positions 234, 235, 236, and / or 237 , which have reduced affinity for Fcγ receptors, particularly FcγRI receptors (e.g., US6,6 24,821). In some embodiments, an alanine residue is added to the effector Used for substitutions such as the L234A / L235A double mutation to reduce function. Another mutation combination that reduces effector function is L234A / L235A / G2 37A, E233P / L234V / L235A / ΔG236, A327G / A330S / P331S, K322A, L234A / L235A, L234F / L235E / P331 S and L234A / L235E / G237A / A330S / P331S (Eu numbering) Optionally, positions 234, 236, and / or 237 of human IgG2 are and position 235 is substituted with glutamine. (See, e.g., US Pat. No. 5,624,828.) 21) in the complement C1q binding site at positions 330 and 331 of the EU index Two amino acid substitutions reduce complement binding (Tao et al., J.E. xp. Med. 178:661 (1993) and Canfield and Morrison, J. Exp. Med. 173:1483 (1991) (See references for more information.) Human IgG1 or IgG2 residues 233-236 and IgG4 residues Substitutions at positions 327, 330, and 331 of the ribozyme reduce ADCC and CDC (e.g., Armor KL. et al., 1999 Eur J Immunol. 29(8):2613-24; and Shields RL. et al., 200 1. J Biol Chem. 276(9):6591-604). Mutations 297A, N297Q, or N297G (Eu numbering) reduce glycosylation. This reduces effector function.

[0115] Some antibodies of the disclosure may be modified with constant regions such that they have enhanced Fc effector function. For example, FcγR binding can be engineered by introducing a mutation(s) in the FcγR domain. In some embodiments, this can be enhanced by one or more of the following in the Fc region: The above amino acid substitutions can be used. Determined either by scanning or rational design and library screening These techniques can be used to enhance binding to FcγRs and enhance effector function. Alternatively, enhanced IgG variants can be identified. Several mutations are known in the art, for example, Smith P. et al. (20 12) Described in PNAS 6181-6186.

[0116] In some embodiments, the antibodies described herein have a specific activity compared to wild-type IgG1 without the modification. and a modified IgG1 constant domain that enhances the ability of the antibody to mediate ADCC. The domains are L235V, S239D, F243L, R292P, A330L, I332 E, characterized by one or more amino acid substitutions at P396L (Eu numbering) In other embodiments, the modified IgG1 domain may comprise S239D, A330L, and I332E (Eu numbering) substitutions.

[0117] Alternatively, glycoform perturbation can be used to enhance the function of Fc-mediated therapeutic antibodies. N-linked Fc glycosylation of IgG1 antibodies is important for effector function. Sialylation, galactosylation, bisecting sugars, and fucosylation can all affect the binding and activity of IgG molecules. Control of the glycosylation pattern of antibodies for use can be achieved in several different ways. The type of antibody-producing cells and their culture conditions affect the glycosylation and activity of therapeutic antibodies. Additionally, bioreactor conditions and downstream processing may also affect the This may affect the glycan microheterogeneity. Glycoengineering has been shown to enhance the fucose level of antibodies. Many methods are known in the art for achieving this reduction in ions. The goal is to manipulate enzymes involved in post-translational modifications of the enzyme. Overexpression of glucosidases such as glucosaminyltransferase III, fucosaminyltransferase III, knockout of fucose transferase or naturally deficient in fucose, or This may involve the use of cell lines that have been mutated to express low fucosylation levels. Furthermore, inhibitors of N-linked glucosidases, such as castanospermine, also inhibit the activity of low-fucose Ig can be used to obtain G molecules.

[0118] In some embodiments, the amino acid engineered variants have a broader range of activity against multiple FcγRs. Although glycoform-engineered antibodies can have enhanced affinity to a range of It may have a more specific affinity for FcγRIIIa binding, which is enhanced. The oligoform interacts with proximal amino acids in the Fc portion and contacts Ig oligosaccharides. Acid substitution can result in different glycoform structures.

[0119] G. Expression of Recombinant Antibodies Chimeric antibodies, humanized antibodies (including veneered antibodies), and human antibodies are typically recombinantly expressed. Thus, the present disclosure provides the anti-CD39 antibodies of Sections IIA-F. Polynucleotides encoding the polynucleotides, vectors containing the polynucleotides, and host cells containing the vectors. The chief cells are also provided.

[0120] Polynucleotides encoding the anti-CD39 antibodies of the disclosure may be amplified, expressed, or further processed. The fragment can be inserted into a vector for optimization. Many vectors are available. In some embodiments, vector systems include mammalian, bacterial, yeast systems, etc., and include pALT ER, pBAD, pcDNA, pCal, pL, pET, pGEMEX, pGEX, pC I, pCMV, pEGFP, pEGFT, pSV2, pFUSE, pVITRO, pVI VO, pMAL, pMONO, pSELECT, pUNO, pDUO, Psg5L, pB ABE, pWPXL, pBI, p15TV-L, pPro18, pTD, pRS420, Plasmids such as, but not limited to, pLexA, pACT2.2, and other laboratory Suitable vectors include prepared and commercially available vectors. Suitable vectors include plasmids, or Viral vectors (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated Vector components generally include: a signaling vector; a replication origin, one or more marker genes, an enhancer element, a promoter (e.g., For example, SV40, CMV, EF-1α), and a transcription termination sequence. Recombinant polynucleotide constructs for expression include, but are not limited to: is typically located in the coding sequence of an antibody chain, including a naturally associated or heterologous promoter region. In some embodiments, the expression control sequence comprises: eukaryotic vectors capable of transforming or transfecting eukaryotic host cells When the vector is introduced into a suitable host, the host expresses the nucleoside and maintained under conditions suitable for high level expression of the peptide sequence and for harvesting and purifying the recombinant antibody. will be done.

[0121] Vectors containing polynucleotide sequences encoding the anti-CD39 antibodies of the present disclosure can be cloned. The vectors of the present invention can be introduced into host cells for cloning or gene expression. Suitable host cells for cloning or expressing the polynucleotide sequences in Suitable prokaryotes include, but are not limited to, prokaryotic cells and eukaryotic cells. , eubacteria, e.g., gram-negative or gram-positive organisms, e.g., Enterobacteriaceae cteriaceae, e.g., Escherichia, e.g., E. coli, En terobacter, Erwinia, Klebsiella, Proteus, Sa Salmonella, e.g., Salmonella typhimurium, Ser atia, e.g., Serratia marcescans, and Shigella and Bacilli, e.g., B. subtilis and B. licheniformis is, Pseudomonas, e.g., P. aeruginosa, and Strept In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi and yeasts also produce antibiotics. It is suitable as a cloning or expression host for vectors encoding CD39 antibodies. Non-specific examples include Saccharomyces cerevisiae, Schizophyllum cerevisiae, Zosaccharomyces pombe; Kluyveromyces host, e.g. For example, K. lactis, K. fragilis (ATCC12,424), K. bul garicus (ATCC16,045), K. wickeramii (ATCC24, 178), K. waltii (ATCC 56,500), K. drosophilar um (ATCC 36,906), K. thermotolerans, and K. mar xianus, etc.; yarrowia (EP402,226); Pichia pas toris (EP183,070);Candida;Trichoderma re esia(EP244,234);Neurospora crassa;Schwan niomyces, e.g., Schwanniomyces occidentalis and filamentous fungi, such as Neurospora, Penicillium, To lypocladium, and Aspergillus hosts, e.g., A. nidula Suitable host cells are also derived from multicellular organisms. Examples of invertebrate cells include plant cells and insect cells. rovirus strains and variants, and corresponding permissive insect host cells derived from hosts, e.g. Spodoptera frugiperda (caterpillar), Aedes aegypti ( Aedes albopictus (mosquito), Drosophila melano gaster (Drosophila melanogaster), and Bombyx mori have been identified. Various virus strains for transfection, e.g., Autographa ca L-1 variant of Bombyx lifornica NPV and Bombyx mori NPV The Bm-5 strain is publicly available, and such viruses are particularly For transfection of frugiperda cells, the virus It can be used as a seed stalk for cotton, corn, potatoes, soybeans, and petunias. Plant cell cultures of cereals, tomatoes, and tobacco can also be used as hosts. In some embodiments, the mammalian cells are transfected with nucleic acids encoding immunoglobulins or fragments thereof. It is used as a host cell for expressing the octide segment. From Genes to Clones, (VCH Publishers, N See, for example, Y, 1987. A number of suitable vectors capable of secreting intact heterologous proteins are available. Host cell lines have been developed in the art and include CHO cell lines, various COS cell lines, HeL a cells, HEK293 cells, L cells, and non-antibody-producing bone marrow cells such as Sp2 / 0 and NS0 In some embodiments, the cells are non-human. The expression vector contains expression control sequences, such as an origin of replication, a promoter, an enhancer (Q ueen et al., Immunol. Rev. 89:49(1986)), and necessary processing information sites, e.g., ribosome binding sites, RNA splicing sites The sequence may include a transcription site, a polyadenylation site, and a transcription termination sequence. In this state, the expression control sequences are those of endogenous genes, cytomegalovirus, SV40, adenovirus, These promoters are derived from viruses such as rabies and bovine papillomavirus. Co et al. , J. Immunol. 148:1149 (1992).

[0122] The host cells are transformed with the expression or cloning vectors described above for the production of anti-CD39 antibodies. The gene encoding the desired sequence is then transformed, allowing for induction of the promoter, selection of transformants, or The cells are cultured in conventional nutrient media modified as appropriate for gene amplification. can be purified by techniques known in the art, including HPLC purification, column chromatography, gel electrophoresis, etc. It can be purified according to standard procedures (generally, Scopes, Protein Purification (Springer-Verlag, NY, 1982 ) for more information.

[0123] H. Labeled antibody The disclosed anti-CD39 antibodies can be linked to fluorescent molecules, spin-labeled molecules, enzymes, or radioisotopes. Optionally labeled with one or more detectable signals, including but not limited to Such antibodies can be used in any number of in vitro, in vivo, or ex vivo assays. It can be used in assays to detect or isolate human CD39.

[0124] For example, any one of the disclosed anti-CD39 antibodies may be conjugated to a fluorescent molecule, a spin-label molecule, or a If it is labeled with a detectable signal, such as a molecule, enzyme, or radioisotope, Such detectable antibodies are useful in pharmacodynamic assays, immunohistochemistry, receptor occupancy assays, EL ISA (Enzyme-linked Immunosorbent Assay), EIA (Enzyme Immunoassay), RIA (Radio It can be used in immunoassays, etc.

[0125] III.How to use The present disclosure provides a method for the preparation of a medicament for the inhibition of CD39 enzymatic activity using an antibody as described herein. Methods of using CD39 antibodies are provided. " and the like terms refer to antagonists that decrease the function or activity of a particular target, e.g., CD39. The reduction is preferably at least 50%, e.g., 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 less The present disclosure also provides methods for treating rheumatoid arthritis that benefit from inhibition of CD39 enzyme activity, and the methods may be at least about 95%. in the preparation of a medicament for the treatment or prevention of a disease, disorder, and / or condition described herein. The present disclosure also encompasses the use of anti-CD39 antibodies described herein. ATP (compared to non-diseased tissue), (ii) ENTPD1, ​​P2RX7 in diseased tissue (iii) expression or increased expression of P2RY11, P2RY21, or a combination thereof; Expression of CD39, P2X7, P2Y11, or a combination thereof in tissues (iv) bone marrow infiltration into diseased tissue, or any combination of (i) to (iv). an anti-CD39 antibody as described herein in the preparation of a medicament for the treatment of a disease characterized by In another example, the present disclosure encompasses the use of the present invention in the preparation of a medicament for the treatment of cancer. The present invention encompasses the use of anti-CD39 antibodies as described herein. Optionally, the cancer is characterized by: (i) high cell proliferation; extracellular ATP (compared to non-diseased samples), (ii) ENTPD1, ​​P2RX in diseased tissues 7, P2RY11, or a combination thereof; (iii) disease Expression or function of CD39, P2X7, P2Y11, or a combination thereof in tissues (iv) bone marrow infiltration into diseased tissue, or any combination of (i) to (iv). In some embodiments of the foregoing method, the method may be characterized by the combination of the The anti-CD39 antibody is used in combination with at least one additional therapy, and these Examples are provided elsewhere herein.

[0126] Extracellular ATP is present at negligible concentrations (e.g., approximately 10–100 nM) under normal conditions. However, it rapidly increases in response to tissue injury, stress, hypoxia, and drugs used in the treatment of cancer. They rapidly increase in number and can be found in high concentrations in tumors. to dying or stressed cells, including but not limited to cells The ATP released by this process is an inflammatory signal that is important for effective innate and adaptive immune responses. Conversely, for example, extracellular ATP hydrolyzed to adenosine in the tumor microenvironment provides CD39 is the rate-limiting enzyme in the hydrolysis of extracellular ATP. CD39 also acts as a catalytic enzyme by converting extracellular ATP into AMP. CD73 (ecto-5'-nucleotidyl AMP), a rate-limiting ectoenzyme in extracellular AMP hydrolysis Increases extracellular adenosine production via type 1 purinergic receptors (ATPases). It transmits signals through receptors with effects opposite to those mediated by ATP receptors. In particular, A expressed on the surface of immune cells 2a and A 2b The receptors for adenosine For example, in the tumor microenvironment, adenosine helps mediate immunosuppressive effects. A on the cell 2a Receptor and A 2b It induces immunosuppressive effects through direct interaction with receptors. Regarding the opposing roles of adenosine and ATP, Chiarella et al. “Extracellular ATP and Adenosine in Canc er Pathogenesis and Treatment,” Trends i Reviewed in Cancer, 2021, 7(8): 731-750. As demonstrated herein, the use of anti-CD39 antibodies of the present disclosure enhances CD39 enzymatic activity. inhibits the immune stimulation caused by ATP accumulation and prevents the formation of immunosuppressant adenosine. Diseases, disorders, and / or conditions that would benefit from inhibition of CD39 enzymatic activity include: For example, tissue damage, stress, hypoxia treatment with additional therapy, or any combination thereof. The CD39 enzyme may be used in combination to increase the release of extracellular ATP. Additional diseases, disorders, and / or conditions that would benefit from inhibition of activity include, for example, peripheral blood samples. or as measured by increased CD39 enzyme activity in tissue (e.g., tumor) samples, e.g., For example, due to increased ATP hydrolysis compared to healthy controls and / or In affected tissues due to increased hydrolysis of AMP to adenosine compared with normal controls Those with high extracellular adenosine levels in the sample and / or those with high levels of adenosine, e.g., immunohistochemistry by immunology, immunophenotyping, RNA sequencing, or other clinically validated methods Measured to detect CD39 and / or CD49 in peripheral blood samples or tissue (e.g., tumor) samples. D73 expression is detectable and optionally elevated compared to healthy controls. do.

[0127] Thus, in some embodiments, the anti-CD39 antibodies described herein are The method is administered to a subject in need of inhibition of enzymatic activity in an amount effective to inhibit CD39 enzymatic activity. CD39 enzyme activity can be measured in peripheral blood samples or tissue (e.g., tumor) samples obtained from a subject. The test can be performed using a sample, or both. Use commercially available assays such as se-Glo® or AMP-Glo®. to identify cellular and non-cellular compartments of peripheral blood or tumor samples. Alternatively or additionally, CD39 activity in tumor tissue can be measured. CD39 enzyme activity can be measured by enzyme histochemistry. by comparison with a prior sample obtained from the elephant (i.e., before administration of anti-CD39 antibody), or A control (e.g., an isotype control antibody, which binds to CD39 but inhibits CD39 enzymatic activity) Reference samples or subjects receiving anti-CD39 antibodies, standard of care, placebo, etc. In some embodiments, the amount of the ATP that is present in the blood can be determined by comparison with a reference value. The anti-CD39 antibody described above targets subjects who require inhibition of CD39 enzymatic activity in the tumor microenvironment. The compound is administered in an amount effective to inhibit CD39 enzymatic activity in the tumor microenvironment. In some embodiments, the anti-CD39 antibodies described herein inhibit CD39 enzymatic activity around the tumor. administered to a subject in need of inhibition in an amount effective to inhibit peritumoral CD39 enzymatic activity. In some embodiments, the anti-CD39 antibodies described herein are administered intracellularly or intracellularly. is administered intracellularly or intracellularly to a subject in need of inhibition of CD39 enzymatic activity on immune cells. In some embodiments, the compound is administered in an amount effective to inhibit CD39 enzymatic activity on the target cell. The anti-CD39 antibodies described herein inhibit CD39 enzymatic activity in or on myeloid cells. In subjects requiring inhibition of CD39 enzyme activity in or on myeloid cells, The CD39-expressing myeloid cells are administered in an amount effective to inhibit the proliferation of the myeloid cells. In some embodiments, the anti-CD39 antibodies described herein include Inhibition of CD39 enzyme activity in or on the interstitium and / or blood vessels is required. The subject is administered with the CD39 enzyme in the interstitium and / or blood vessels or on the interstitium and / or blood vessels. It is administered in an amount effective to inhibit activity.

[0128] Alternatively or additionally, in some embodiments, the anti-CD39 antibodies described herein are and a suitable control (e.g., a subject administered an isotype control antibody, which binds to CD39) Subjects receiving anti-CD39 antibodies that do not inhibit CD39 enzyme activity, subjects receiving standard treatment an amount effective to enhance ATP-mediated immune stimulation compared to a control (a non-treated control) ATP-mediated immune stimulation is achieved by administering the resulting ATP to a subject in need thereof. Peripheral blood samples and / or tissue (e.g., tumor) samples can be used to assess ATP-mediated immunity. The immunostimulation can be, for example, (i) a P2YG protein-coupled receptor or a P2X cation-selective channel. ATP-dependent signaling is mediated by one or more type 2 purinergic (P2) receptors, such as the purinergic receptor (ii) measuring an increase in NLRP3 inflammasome activation. (iii) measuring the increase in cell surface markers of dendritic cells; (iv) C measuring an increase in CD4+ and / or CD8+ T cell activity and / or proliferation; and and / or (v) activation, maturation, or cytokine secretion in one or more myeloid cell types. or a combination thereof. In one example, the anti-CD39 antibodies described herein inhibit the expression of P2X4, P2 one or more P2X receptors selected from P2X5, P2X7, P2Y2, or P2Y11 and in an amount effective to increase ATP-dependent signaling through a P2Y receptor or a P2Y receptor, subjects in need thereof (e.g., subjects administered an isotype control antibody, Subjects receiving anti-CD39 antibodies that inhibit CD39 enzyme activity but do not inhibit CD39 enzyme activity, and subjects receiving standard treatment In one example, the anti-CD31 antibody described herein is administered to a subject (e.g., a subject undergoing treatment, a subject not undergoing treatment). 39 antibodies increase ATP-dependent signaling through P2X7 compared to appropriate controls a subject in need thereof (e.g., a subject administered an isotype control antibody) in an amount effective to induce subjects administered an anti-CD39 antibody that binds to CD39 but does not inhibit CD39 enzymatic activity. In one example, the drug is administered to subjects receiving standard therapy, subjects receiving standard therapy, and subjects not receiving therapy. The anti-CD39 antibodies described herein inhibit P2Y11-mediated Aβ expression compared to a suitable control. administering to a subject in need thereof (e.g., Subjects treated with an isotype control antibody, which binds to CD39 but inhibits CD39 enzymatic activity Subjects receiving anti-CD39 antibodies that do not contain steroids, subjects receiving standard therapy, and subjects not receiving therapy In one example, the anti-CD39 antibodies described herein are administered to a subject (e.g., a non-human subject) compared to a suitable control. Increased ATP-dependent signaling via P2X7 and / or P2Y11 compared with and administering to a subject in need thereof (e.g., a subject who has received an isotype control antibody) in an amount effective to Subjects were administered an anti-CD39 antibody that binds to CD39 but does not inhibit CD39 enzymatic activity. In one example, the drug is administered to a patient (e.g., a subject receiving standard of care, a subject receiving no standard of care, or a subject not receiving treatment). The anti-CD39 antibodies described herein increase activation of the NLRP3 inflammasome The compound is administered to a subject in need thereof in an amount effective to inhibit NLRP3 inflammasome activity. Metrics for IL-1 activation are known in the art and are detailed in the Examples (e.g., IL-1 activation). In one embodiment, the anti-CD39 antibodies described herein are Increase myeloid cell activation, maturation, cytokine secretion, or any combination thereof The compound is administered to a subject in need thereof in an amount effective to activate, mature, and stimulate myeloid cells. Metrics for cytokine secretion are known in the art and are detailed in the Examples. Includes.

[0129] Alternatively, or in addition to the above, in some embodiments, an anti-CD39 antibody as described herein The antibody is administered to a suitable control (e.g., a subject administered an isotype control antibody, which binds to CD39). Subjects who received anti-CD39 antibodies that inhibit CD39 enzyme activity but do not inhibit CD39 enzyme activity, and subjects who received standard treatment reduced or no adenosine-mediated immunosuppression compared to subjects receiving treatment or untreated subjects is administered to a subject in need thereof in an amount effective to prevent adenosine-mediated immunity. Suppression can be achieved, for example, by 2a R and / or A 2b Increased R-mediated adenosine signaling Adenosine-mediated immunosuppression can also be identified by measuring lymphocyte activity. Myeloid (e.g., T cells, B cells) and / or myeloid (e.g., monocytes, macrophages, Also included are adenosine-mediated inhibition of cellular activity (dendritic cells, NK cells). One measure of immune suppression is the NECA-induced p-regulation of CD8+ T cells in human blood. It could be CREB activation.

[0130] Alternatively, or in addition, in some embodiments, an anti-CD3 antibody as described herein 9. The antibody has a receptor occupancy of at least 60%, but not less than 10%, as measured by a receptor occupancy assay. At least 65%, at least 70%, at least 75%, at least 80%, at least 85% %, at least 90%, at least 95%, or 100% of the targets (i.e., CD39 ) is administered to a subject in need thereof in an amount effective to achieve binding. In some embodiments, the anti-CD39 antibodies described herein may be used to detect receptor occupancy as measured by a receptor occupancy assay. 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 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 achieved The receptor occupancy assay is administered to a subject in need thereof in an amount effective to (i) will be described in detail in the Examples.

[0131] Alternatively, or in addition, in some embodiments, an anti-CD3 antibody as described herein The antibody is administered to a subject in need thereof in an amount effective to reduce CD39 expression. In some embodiments, the anti-CD39 antibodies described herein inhibit cell surface CD39 expression. The compound is administered to a subject in need thereof in an amount effective to reduce CD39 expression (cell Intracellular and / or extracellular) can be obtained by, for example, immunohistochemistry, including methods further detailed in the Examples. It can be assessed by chemical or immunophenotyping.

[0132] Alternatively, or in addition, in some embodiments, an anti-CD3 antibody as described herein The antibody is administered to a subject in need thereof in an amount effective to treat or prevent cancer. Without wishing to be bound by theory, the applicant believes that ATP-mediated immune stimulation Increase and / or decrease (or prevent) adenosine-mediated immunosuppression ) may result in tumor death and improve outcomes.

[0133] A. Oncology and Oncology-Related Disorders In one or more embodiments, the antibodies described herein are used to treat cancer (e.g., carcinoma, sarcoma, leukemia, These are useful in the treatment and / or prevention of cancers such as rhesus mammary gland tumors, lymphomas, myelomas, and the like. In the Alternatively or additionally, there may be a risk that the cancer will recur or that standard treatment will Alternatively, the patient may become unresponsive to treatments such as checkpoint inhibitors. Examples of contemplated cancer types include cancers of the genitourinary tract (e.g., bladder, kidney, renal cell , penile, prostate, testicular, von Hippel-Lindau disease, etc.), cancer of the uterus, cervical cancer of the ovaries, cancer of the breast, cancer of the digestive tract (e.g., esophagus, oropharynx, stomach, small intestine, or large intestine, colon, or rectum), bone, bone marrow, skin cancer (e.g., melanoma), head and neck cancer, Liver cancer, gallbladder cancer, bile duct cancer, heart cancer, lung cancer, pancreatic cancer, salivary gland cancer 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 Cancers of the central nervous system (CNS), cancers of the peripheral nervous system (PNS), cancers of the hematopoietic system (i.e., blood malignancies) cancers of the immune system (e.g., spleen or thymus).

[0134] In some embodiments, antibodies according to the present disclosure are used to treat and / or prevent hematological malignancies. Examples of cancer types that affect the hematopoietic system include leukemia, lymphoma, and These include acute myeloid leukemia, adult T-cell leukemia, and T-cell large granular lymphocyte leukemia. acute lymphoblastic leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, acute monocytic leukemia Hodgkin's and non-Hodgkin's lymphoma, diffuse large B-cell lymphoma, and Multiple myeloma is an example.

[0135] In another embodiment, the antibodies according to the present disclosure are useful for the treatment and / or prevention of solid tumors. Solid tumors include, 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, stomach cancer, esophageal cancer, liver cancer (hepatocellular carcinoma), kidney cancer Pancreatic cancer (renal cell carcinoma), head and neck tumors, mesothelioma, melanoma, sarcoma, central nervous system (CNS) hemangioblastoma , and brain tumors (e.g., gliomas, including astrocytomas, oligodendrogliomas, and glioblastomas) It could be.

[0136] In another embodiment, the antibody according to the present disclosure is directed to a cancer, such as lung cancer, genitourinary cancer, gastrointestinal cancer, or a combination thereof. In another embodiment, the combination of the present disclosure is useful for the treatment and / or prevention of The body is used to treat and / or treat lung cancer, genitourinary cancer, gastrointestinal cancer, skin cancer, or a combination thereof. It is useful for prevention or treatment.

[0137] In some embodiments, antibodies according to the present disclosure are useful for treating and / or preventing skin cancer. In a further embodiment, the skin cancer is melanoma.

[0138] In some embodiments, antibodies according to the present disclosure are useful for treating and / or preventing breast cancer. In a further embodiment, the breast cancer is hormone receptor positive (e.g., ERα positive). Breast cancer, PR-positive breast cancer, ERα-positive and PR-positive breast cancer), HER 2-positive breast cancer, HER2-overexpressing breast cancer, or any combination thereof. In a further embodiment, the breast cancer is triple-negative breast cancer.

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

[0140] In some embodiments, antibodies according to the present disclosure are useful for treating and / or preventing lung cancer. In a further embodiment, the lung cancer is non-small cell lung cancer (NSCLC). In 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 include administering a disclosed anti-C D39 antibody in combination with pemetrexed, carboplatin, and antagonist anti-PD-L1 in combination with either the -1 antibody or an antagonist anti-PD-L1 antibody Such methods may further comprise administering to a subject an antagonistic anti-TIGIT antibody, an A2aR antibody, antagonist, A2bR antagonist, A2a / 2bR antagonist, and CD73 and administering one or more additional agents selected from the group consisting of inhibitors of Frequently, the optional additional agents include domvanalimab, AB308, etormadenant, and and quemliculstat.

[0142] In some embodiments, antibodies according to the present disclosure are useful for treating and / or preventing pancreatic cancer. In a further embodiment, the pancreatic cancer is a pancreatic neuroendocrine tumor or a pancreatic adenocarcinoma.

[0143] In some embodiments, antibodies according to the present disclosure are useful in the treatment and / or prognosis of neuroendocrine tumors. In a further embodiment, the neuroendocrine tumor is a pancreatic neuroendocrine tumor, a brown cell tumor, or a thyroid tumor. Cysts, paragangliomas, or tumors of the adrenal glands.

[0144] In some embodiments, compounds according to the present disclosure are useful in the treatment and / or prevention of brain tumors. In a further embodiment, the brain tumor is a glioma. The glioma is an astrocytoma, an oligodendroglioma, or a glioblastoma.

[0145] In some embodiments, antibodies according to the present disclosure are directed to the treatment of upper GI cancers, such as esophageal or gastric cancer. In a further embodiment, the upper GI cancer is an adenocarcinoma, a squamous cell carcinoma, or a pancreatic cancer. epithelial cell carcinoma, or any combination thereof. Cancers include esophageal adenocarcinoma (EAC), esophageal squamous cell carcinoma (ESCC), and esophageal gastric junction adenocarcinoma (GE J), gastric adenocarcinoma (also referred to herein as "gastric cancer"), or any combination thereof is.

[0146] In some embodiments, the disclosed methods of treating gastric or gastroesophageal cancer include Either of the anti-CD39 antibodies was combined with FOLFOX and an antagonist anti-PD-1 antibody or and / or an antagonist anti-PD-L1 antibody. Such methods include the use of antagonistic anti-TIGIT antibodies, A2aR antagonists, Consists of an A2bR antagonist, an A2a / 2bR antagonist, and a CD73 inhibitor The method may further comprise administering one or more additional agents selected from the group The additional drugs are dombanalimab, AB308, etormadenant, and quemricle. Stats.

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

[0148] In some embodiments, antibodies according to the present disclosure are directed to cancers including breast cancer, gastrointestinal cancer, genitourinary cancer, lung cancer, lymphoma, and the like. In a further embodiment, the antibodies according to the present disclosure are useful for treating leukemia, leukemia, or ovarian cancer. , 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 directed to the treatment of lung cancer, head and neck cancer, thyroid cancer, pancreatic cancer, In yet further embodiments, the presently disclosed anti-cancer agents are useful for treating kidney cancer, or skin cancer. The body is effective in treating non-small cell lung cancer, head and neck squamous cell carcinoma, pancreatic ductal adenocarcinoma, clear cell renal carcinoma, and melanoma. It is useful.

[0150] In some embodiments, antibodies according to the present disclosure are used to treat lower GI cancer, upper GI cancer, head and neck cancer, In a further embodiment, the present disclosure provides a method for treating renal, lung, or pancreatic cancer. The antibody has been used in the treatment of esophageal cancer, head and neck squamous cell carcinoma, and kidney clear cell carcinoma. lear cell carcinoma), lung adenocarcinoma, lung squamous cell carcinoma, pancreatic adenocarcinoma, stomach It is useful in the treatment of gastric (stomach) adenocarcinoma.

[0151] In all described embodiments, the methods of the present disclosure are administered in an adjuvant setting. setting) or neoadjuvant setting The methods described herein can be carried out by a first selection, a second selection, It may be indicated as first-line, third-line, or higher-line treatment. In embodiments, the methods of the present disclosure can be practiced with second or more lines of therapy. , previous treatment of choice included a checkpoint inhibitor (i.e., subject had a checkpoint inhibitor In a further embodiment, the patient is a patient with a checkpoint inhibitor (CPI)-experienced patient. The inhibitors include CTLA-4 antagonists, PD-1 antagonists, and PD-L1 antagonists. antagonist, TIM-3 antagonist, or 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 cancer-related diseases, disorders and conditions includes both cancer and non-cancerous proliferative disorders. It is meant to broadly refer to conditions directly or indirectly related to disease, such as vascular Precancerous conditions such as neoplasia and dysplasia, and noncancerous growths such as benign proliferative breast disease and papillomas For clarity, cancer-related diseases, disorders and conditions are included. The term(s) does not include cancer itself.

[0153] Generally, a person in need of treatment or prevention of cancer or a cancer-related disease, disorder or condition To treat or prevent cancer or a cancer-related disease, disorder, or condition in a subject The method of the present disclosure includes administering to a subject an anti-CD39 antibody of Section II. The administration of anti-CD39 antibody in Phase II may be administered one or more times (e.g., one, two, or three or more times). In some embodiments, the present disclosure provides an anti-CD40 inhibitor as described in Section II. 39 antibodies and at least one additional therapeutic agent, examples of which are described elsewhere herein. methods for treating or preventing cancer or cancer-related diseases, disorders, or conditions using provide.

[0154] Patient Selection. In some cases, the methods according to the present disclosure involve selecting patients, e.g., competent PD-L1, CD73, and / or CD39 expression, high microsatellite instability Patients identified as having high tumor mutation burden, high tumor mutation burden, or any combination thereof In some cases, the patient may have a mutation in at least one gene associated with cancer. In some cases, the patient is identified as having a cancer gene-driven cancer that has a mutation. The P2X7 variants are selected by determining the commonly expressed P2X7 sequences. Price variants, distinct SNPs, and post-translational receptor modifications affect P2X7 function. For example, certain modifications may impart partial or complete function to P2X7. Other modifications have been shown to increase P2X7 function. For example, Lara et al., Front Pharmacol, See 2020, 11:793.

[0155] In some embodiments, the patient has PD in a relevant sample, such as a peripheral blood sample or tumor sample. -L1(CD274), P2X7(P2RX7), P2Y11(P2RY11), IL- 2, CXCL1 (CXCL1), MIP-2a (CXCL2), CXCL3 (CXCL3 ), IL-8 (CXCL8), COX2 (PTGS2), CD73 (NT5E) and / or The expression of CD39 (ENTPD1) or CD39 (ENTPD1) was measured. Histochemistry, immunophenotyping, RNA sequencing, gene expression analysis, single molecule imaging assays, including but not limited to, ELISA, or other clinically validated assays. Quantitative determination of nucleic acids or proteins using methods known in the art. In some embodiments, immunohistochemistry, immunophenotyping, RNA sequencing, Sensing, gene expression analysis, single molecule imaging, or other clinically validated The assay can be used to detect PD-L1, CD7 in peripheral blood samples or relevant samples such as tumor samples. 3, and / or measuring CD39 expression (at the nucleic acid or protein level) Alternatively or additionally, patients may be selected by, for example, a peripheral blood sample or The selection can be made by measuring CD39 enzyme activity in a tumor sample. In embodiments, the present disclosure provides a method for treating or preventing atopic dermatitis by administering an anti-CD39 antibody described herein, which can: (i) for example, by immunohistochemistry, immunophenotyping, or other clinically validated tests; Detectable PD-L1, P2X7, P2Y11, IL-2, CXCL1, CXCL2, C (ii) XCL3, CXCL8, COX2, CD73, and / or CD39 expression, e.g., Elevated serum creatinine, e.g., by immunohistochemistry, immunophenotyping, or other clinically validated tests PD-L1, P2X7, P2Y11, IL-2, CXCL1, CXCL2, CXCL 3, CXCL8, COX2, CD73, and / or CD39 expression, (iii) detectable or (iv) any combination of (i) to (iii). In another embodiment, the present disclosure provides a method for treating cancer in a patient. Administration of a therapeutically effective amount of an anti-CD39 antibody can result in: (i) a number of immunoreactive proteins, e.g., immunohistochemistry, immunophenotype, Detectable PD-L1, P2 by phenotyping or other clinically validated tests X7, P2Y11, IL-2, CXCL1, CXCL2, CXCL3, CXCL8, CO X2, CD73, and / or CD39 expression; (ii) e.g., immunohistochemistry, immunophenotyping Elevated PD-L1, CD73, or other clinically validated tests and / or CD39 expression, (iii) detectable CD39 enzyme activity, or (iv) The present invention provides a method for treating cancer in a patient having any combination of i) to (iii). In another embodiment, the present disclosure provides a method for the treatment of cancer using an anti-CD39 antibody described herein. A method for administering to an individual a therapeutically effective amount of PD-L1, P2X7, P2Y11, IL- 2, CXCL1, CXCL2, CXCL3, CXCL8, COX2, CD73, and / or or determining CD39 expression. In embodiments, the present disclosure provides therapeutic uses of the anti-CD39 antibodies described herein for the treatment of cancer. methods of administering an effective amount to an individual, e.g., immunohistochemistry, immunophenotyping, or the like; Other clinically validated tests have demonstrated the expression of PD-L1, P2X7, and P2Y11, IL-2, CXCL1, CXCL2, CXCL3, CXCL8, COX2, Measuring CD73 and / or CD39 expression and detecting PD in the sample -L1(CD274), P2X7(P2RX7), P2Y11(P2RY11), IL- 2, CXCL1 (CXCL1), MIP-2a (CXCL2), CXCL3 (CXCL3 ), IL-8 (CXCL8), COX2 (PTGS2), CD73 (NT5E) and / or or CD39(ENTPD1)-expressing individual, comprising administering a therapeutically effective amount of the antibody. Well, we provide a method.

[0156] B. Route of Administration In some embodiments, pharmaceutical compositions containing antibodies according to the present disclosure are suitable for oral administration. Oral administration involves swallowing the formulation, thereby delivering the antibody to the stomach. Alternatively, oral administration may be by buccal, lingual, or It involves sublingual administration, which allows the antibody to be absorbed through the oral mucosa into the bloodstream. do.

[0157] In another embodiment, a pharmaceutical composition containing an antibody according to the present disclosure is in a form suitable for parenteral administration. The parenteral administration form may be intravenous, intraarterial, intramuscular, intradermal, or intraperitoneal. Intrathecal, intracisternal, intracerebral, intraventricular, intraventricular, and subcutaneous. Pharmaceutical compositions suitable for parenteral administration are formulated using a suitable aqueous or non-aqueous carrier. Depot injections, generally administered subcutaneously or intramuscularly, can also be used to provide relief over a defined period of time. The antibodies disclosed herein can be used to deliver the antibodies over a wide range of time.

[0158] Other routes of administration are also contemplated by the present disclosure, including, but not limited to, intranasal, intravenous, These include 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 antibodies of the present disclosure may be in the form of a composition suitable for administration to a subject. Such compositions typically comprise an anti-CD39 antibody according to the present disclosure and one or more pharmaceutically acceptable salts of an anti-CD39 antibody. In certain embodiments, the anti-CD39 antibody is administered in an effective amount. The pharmaceutical compositions can be used in the methods of the present disclosure; For example, pharmaceutical compositions comprising anti-CD39 antibodies according to the present disclosure may be used in the treatment methods described herein. and can be administered to a subject to practice the prophylactic methods and uses.

[0161] Pharmaceutical compositions of the present disclosure are formulated to be compatible with the intended method or route of administration. Routes of administration may include those known in the art. Routes of administration are oral and parenteral. Additionally, pharmaceutical compositions contemplated by the present disclosure one or more of the other compounds described herein for treating or preventing diseases, disorders and conditions. In one embodiment, the compounds contemplated by the present disclosure may be used in combination with therapeutic agents. The one or more other additional therapeutic agents may be present 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 may be administered in combination with an anti-CD39 antibody according to the present disclosure. It is in a composition separate from the pharmaceutical composition in which it is contained.

[0162] In one aspect, the anti-CD39 antibodies described herein can be administered orally. Administration can be via, for example, a capsule or tablet. In preparing a pharmaceutical composition, a tablet or capsule typically contains at least one pharmaceutical Non-limiting examples of pharmaceutically acceptable excipients include lactose, lactic acid bacteria, and the like. Dextrose, sucrose, sorbitol, mannitol, starch, acacia Gum, calcium phosphate, alginate, tragacanth, gelatin, calcium silicate, Microcrystalline cellulose, polyvinylpyrrolidone, polyethylene glycol, cellulose, sterilized Additional pharmaceutically acceptable excipients include water, syrup, and methylcellulose. lubricants such as talc, magnesium stearate, and mineral oil; wetting agents; emulsifiers and suspending agents; preservatives such as methyl and propylhydroxybenzoates. Some oral dosage forms include taste-masking agents, sweeteners, or flavoring agents. can be formulated as a solution or a suspension.

[0163] In another embodiment, the antibodies described herein are administered parenterally, for example, by intravenous injection. Pharmaceutical compositions suitable for parenteral administration may be formulated in solutions for injection. The drug may be reconstituted for injection in an appropriate system, such as a physiological solution. The formulation contains sterile water for injection, salts, buffers, and the like in amounts appropriate to achieve isotonicity at the appropriate physiological Tonicity excipients may also be included.

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

[0165] D. Medication In general, the disclosed methods involve administering an anti-CD39 antibody, or composition thereof, described herein to a subject in need thereof. The present disclosure relates to an anti-CD39 antibody, and the present disclosure relates to a method for treating an inflammatory bowel disease. An "effective amount" is an amount that binds to a target at a level that demonstrates the efficacy of the antibody (e.g., binds to CD39). The term "antibody" refers to an amount of antibody sufficient to inhibit the enzymatic activity of CD39. Binding can be determined by one or more biochemical or cellular assays, The EC assay can be used as one assessment of antibody potency. 50 , ED 50 , E.C. 90 ,I C 50 or a similar value can be obtained. Examples of effective doses include, but are not limited to, those described in the Examples. as a single dose or as multiple smaller doses (e.g., as one tablet containing "x" amount, each It can be administered as two tablets containing the "x / 2" dose.

[0166] In some embodiments, the disclosed methods involve treatment with an anti-CD39 antibody described herein. As used herein, the term "antibody" includes administering an effective amount of an anti-cancer drug to a subject in need thereof. The phrase "therapeutically effective amount" with respect to a CD39 antibody refers to a therapeutically effective amount administered to a subject in need of such treatment. The administration regimen (i.e., amount and duration) of the antibody that results in a specific pharmacological effect is determined. For prophylactic use, a therapeutically effective amount refers to a dose that is sufficient to achieve biochemical, histological, and physiological improvements in disease. Eliminate or reduce the risk and / or severity of disease, including behavioral signs or symptoms In the case of treatment, a therapeutically effective amount is a dose that is effective to prevent or delay the onset of a disease. reduce, improve, or eliminate one or more signs or symptoms of the disease, slow the progression of the disease, and improve survival To extend the duration and reduce the dose of other medicine(s) needed to treat the disease, or With particular reference to cancer, a therapeutically effective amount may be, for example, cell death, reduction in cancer cell count, reduction in tumor burden, elimination of tumor or metastasis, or metastatic A therapeutically effective amount of an anti-CD39 antibody can be determined by one skilled in the art. To be considered a therapeutically effective amount, it need not necessarily be effective in treating every individual subject. A therapeutically effective amount may be determined based on, for example, the age and weight of the subject, the overall health of the subject, the Varies based on one or more of the following: stage of disease, route of administration, and previous or concurrent treatment. This may result in

[0167] In certain embodiments, the anti-CD39 antibodies contemplated by the present disclosure may have a desired effect. To achieve this, a daily dose of about 0.01 mg / kg to about 50 mg / kg of subject body weight, or about 1 mg / kg to about 25 mg / kg (e.g., orally, parenterally, etc.) for one day, one week, or The compound may be administered one or more times per month. In some embodiments, the compound contemplated by the present disclosure The preferred weight-based dose of the anti-CD39 antibody is administered independently of the subject's body weight. In certain embodiments, the dose is used to determine the desired dose (i.e., a fixed dose). The anti-CD39 antibodies of the present disclosure may be administered daily, weekly, or monthly to achieve the desired effect. More than once, about 1 mg to about 1000 mg, especially 1, 3, 5, 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 300, 400, 500, 600, 750, 8 It can be administered at fixed dose levels of 0, 900, or 1000 mg (e.g. In some embodiments, the anti-CD39 antibodies of the present disclosure are administered intravenously to achieve the desired effect. To achieve the desired results, take approximately 3 mg to 3,000 mg at least once a day, week, or month. Especially 3, 5, 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 300, 400, 500, 600, 750, 800, 900, 1000mg, 1100m g, 1200mg, 1300mg, 1400mg, 1500mg, 1600mg, 170 0mg, 1800mg, 1900mg, 2000mg, 2100mg, 2200mg, 2 300mg, 2400mg, 2500mg, 2600mg, 2700mg, 2800mg , 2900 mg, or 3000 mg fixed dose levels (e.g., For example, oral, parenteral, etc. The term "about" when used herein in relation to dosages , means the stated value as well as plus or minus 10% of that 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 providing a desired effect. a predetermined amount of anti-CD39 antibody, alone or in combination with one or more additional agents, sufficient to The parameters of the unit dosage form may depend on the particular drug and the effect to be achieved. It will be understood that

[0169] IV. Combinations with Disclosed Anti-CD39 Antibodies The present disclosure provides for the use of the anti-CD39 antibodies of Section II alone or in combination with one or more additional therapies. Each additional therapy is intended to be used in combination with a therapeutic agent or another treatment modality. In embodiments that include one or more additional therapeutic agents, each agent may be a different but complementary Additional therapeutic agents may target specific mechanisms of action. Macromolecules such as petibodies, peptides, DNA, RNA, or fragments of such macromolecules or a cell therapy or gene therapy agent. Typical examples include surgical removal of the tumor, bone marrow transplantation, radiation therapy, and photodynamic therapy. The anti-CD39 antibodies of Section II may be used in combination with one or more additional therapeutic agents. This results in a synergistic or additive therapeutic or preventative effect on the underlying disease, disorder, or condition. Additionally or alternatively, combination therapy can be used to treat a variety of conditions, including: This allows for a reduction in the dose of therapeutic drugs, thereby ameliorating adverse effects associated with one or more drugs. can be improved, mitigated, or eliminated.

[0170] In embodiments that include one or more additional therapeutic modalities, the anti-CD39 antibody of Section II The body may be administered before, after, or during treatment with additional therapeutic modalities In embodiments that include one or more additional therapeutic agents, the therapeutic agents used in such combination therapy The compounds can be formulated as a single composition or as separate compositions. When administered, each therapeutic agent in the combination may be administered simultaneously, at about the same time, or at different times. Additionally, the therapeutic agent may be administered in different dosage forms (e.g., oral capsules and Even if the drugs are administered intravenously, they are often given in combination and at different dosage intervals, resulting in a single treatment. An agent is given on a fixed dosing regimen while another therapeutic agent is titrated up, tapered, or discontinued Alternatively, each therapeutic agent in the combination may be titrated up, down, or administered independently over the course of a patient's treatment. The dose is increased or decreased, or discontinued and / or restarted. When formulated as separate compositions, in some embodiments the separate compositions are included in a kit It is served together with.

[0171] Cancer therapy The present disclosure provides one or more additional compounds useful in the treatment of cancer or a cancer-related disease, disorder, or condition. It is contemplated to use the anti-CD39 antibodies of Section II in combination with additional therapies. In some embodiments, one or more of the additional therapies is an additional treatment modality Exemplary treatment modalities include surgical resection of the tumor, bone marrow transplantation, radiation therapy, and phototherapy. In some embodiments, one or more The additional therapeutic agent is a targeted therapeutic agent. Exemplary therapeutic agents include chemotherapeutic agents, radiopharmaceuticals, and the like. Products, hormone therapeutic agents, epigenetic regulators, ATP-adenosine axis targeting agents, targets Therapy, Signal Transduction Inhibitors, RAS Signal Transduction Inhibitors, PI3K Inhibitors, Arginase inhibitors, HIF inhibitors, AXL inhibitors, PAK4 inhibitors, immunotherapy agents, cell therapy agents, genetic Drugs, immune checkpoint inhibitors, and stimulatory or costimulatory immune checkpoint inhibitors Examples include agonists of

[0172] In some embodiments, the one or more additional therapeutic agents is a chemotherapeutic agent. Examples of such agents include alkylating agents, e.g., thiotepa and cyclophosphamide; alkylating agents, e.g., thiotepa and cyclophosphamide; sulfonates, e.g., busulfan, improsulfan and piposulfan; azines, e.g., benzodopa, carboquone, meturedopa, and uredopa; ethyleneimines and methylmelamines, e.g., altretamine, triethylenemelamine, triethylenephosphazene sulforamide, triethylenethiophosphoramide, and trimethylolmelamine (tri methylolomelamime); nitrogen mustards, e.g. chloramphenicol Bucil, chlornaphazine, chlorophosphamide , estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride , melphalan, novembichine, fenesterine, prednisolone Mustard, trofosfamide, uracil mustard; nitrosoureas, e.g., carm chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; Bioactive substances, e.g., aclacinomycins, actinomas Isin, ausramycin, azaserine, bleomycin, cactinomycin, calicare mycin, carabicin, caminomycin, carzinophilin, chromomycin, dactinomycin mycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine , doxorubicin, epirubicin, esorubicin, idarubicin, marcelomycin, Itomycin, mycophenolic acid, nogalamycin, olivomycin, pomalidomide, Puromycin, potfilomycin, puromycin, queramycin, rodrubicin , 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-methylpropional Pyrimidine analogues, e.g., ansita azacytidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine androgens, e.g., doxifluridine, enocitabine, floxuridine, 5-FU; As, calsterone, dromostanolone propionate, epithiostanol, mepitiostanol antiadrenal drugs, e.g., aminoglutethimide, mitotane, trimethicone; Rostan; folic acid replenishers, e.g., folinic acid; Aceglatone; Aldophosphamide glycoside; Aminolevulinic acid; Amsacrine; Bess Trabucil; Bisantren; Edatraxate; Defofamine; Demecolcine; Diazide Quon; Elformithine; Elliptinium acetate; E Toglucide; Gallium nitrate; Hydroxyurea; Lentinan; Lonidamine; Mitoguazone ;Mitoxantrone;Mopidamol;Nitracrine;Pentostatin;Fenamet;Pi Larubicin; Podophyllic acid; 2-ethylhydrazide; Procarbazine; Razoxane; Cysteine Zofiran;Spirogermanium;Tenuazonic acid;Triazicon;2,2',2''-Triazicon Trichlorotriethylamine; Urethane; Vindesine; Dacarbazine; Mannomustine; Mi Tobronitol; Mitolactol; Pipobroman; Gacytosine; Arabinoside (Ara- C); cyclophosphamide; thiotepa; taxoids, e.g., paclitaxel, nabpac rituximab, and docetaxel; chlorambucil; gemcitabine; 6-thioguanine; methotrexate; platinum and platinum coordination complexes (collectively referred to as "platinum drugs"); cisplatin, carboplatin, oxaliplatin; vinblastine 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 , for example, irinotecan, topotecan, etoposide, mitoxantrone, teniposide; Difluoromethylornithine (DMFO); Retinoic acid; Esperamicin; Capecitabine anthracyclines and pharmaceutically acceptable salts, acids or derivatives of any of the above In certain embodiments, the combination therapy includes, but is not limited to, one In one embodiment, the combination therapy comprises a chemotherapy regimen comprising the above chemotherapeutic agents. FOX (folinic acid, fluorouracil, and oxaliplatin), FOLFIRI (folinic acid, fluorouracil, and oxaliplatin) phospholipid, fluorouracil, and irinotecan), taxanes (e.g., docetaxel, Paclitaxel, nab-paclitaxel, CAPOX (capecitabine and oxaliplatin), XELOX (capecitabine and oxaliplatin) platinum-based chemotherapy drugs In another embodiment, the chemotherapy regimen includes irinotecan, or gemcitabine. In , combination therapy includes alkylating agents (e.g., cyclophosphamide), anthracyclines (e.g., doxorubicin, epirubicin, idarubicin, mitoxantrone), platinum agents (e.g., oxaliplatin), proteasome inhibitors (e.g., bortezomib), and In another embodiment, the combination therapy includes a chemotherapy regimen comprising any combination thereof. The methods include alkylating agents (e.g., cyclophosphamide), anthracyclines (e.g., doxorubicin), and xorubicin, epirubicin, idarubicin, mitoxantrone), platinum agents (e.g. carboplatin, cisplatin, oxaliplatin), taxanes (e.g., docetaxel cerebrospinal fluid (e.g., cerebrospinal fluid, paclitaxel), proteasome inhibitors (e.g., bortezomib), or In another embodiment, the combination therapy includes a chemotherapy regimen comprising any combination of: , bortezomib, cyclophosphamide, doxorubicin, epirubicin, idarubicin, Chemotherapy containing mitoxantrone, oxaliplatin, or any combination thereof Includes regimen.

[0173] In some embodiments, the one or more additional therapeutic agents is a radiopharmaceutical. The product is an internal radiation therapy method in which a radiation 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 iodides include sodium iodide I-131, radium-223 dichloride, iodine Lobenguane Iodine-131, radioactive iodide vesicles (e.g., saposin SapC-dioleoylphosphatidylserine (SapC-DOPS) nanovesicles, various shapes These include various forms of brachytherapy, and targeted radionuclides. a molecule (a "targeting agent") that specifically binds to a target on a cell, typically a cancer cell or immune cell ) and a radionuclide associated (e.g., by covalent or ionic interactions). Targeting agents include small molecules, sugars (including oligosaccharides and polysaccharides), antibodies, lipids, proteins, and peptides. , a non-natural polymer, or an aptamer. The agent is a sugar (including oligosaccharides and polysaccharides), lipid, protein, or peptide, and the target is Tumor-associated antigens (enriched but not specific to cancer cells), tumor-specific antigens (normal minimal or no expression in the tissue), or neoantigens (nonsynonymous mutations in the tumor cell genome) or antigens specific to the genome of cancer cells, generated by gene fusion. In some embodiments, the targeting agent is an antibody and the target is a tumor-associated antigen (i.e., an enriched tumor-specific antigens (i.e., antigens that are present in normal tissues but are not specific to cancer cells); antigens with minimal or no expression in the tumor cell genome), or neoantigens (i.e., antigens with minimal or no expression in the tumor cell genome) specific to the genome of cancer cells, generated by nonsynonymous mutations or gene fusions in Non-limiting examples of targeted radionuclides include somatostatin or peptides thereof. Radionuclides bound to analogues (e.g., 177Lu-Dotatate); prostate-specific specific membrane antigens or their peptide analogs (e.g., 177Lu-PSMA-617, 225 Ac-PSMA-617, 177Lu-PSMA-I&T, 177Lu-MIP-109 5, etc.); the cognate ligand of the receptor, a peptide derived from that ligand, or a combination thereof riants (e.g., 188Re-labeled VEGF125-136 or VEGF receptor-specific antibodies targeting tumor antigens (e.g., antibodies having a higher affinity for the target antigen); 131I-tositumomab, 90Y-ibritumomab tiuxetan, CAM-H2-I13 1 (Precirix NV), I131-omburtamab, etc.

[0174] In some embodiments, the one or more additional therapeutic agents is a hormone therapy. Therapeutic agents act to regulate or block hormone action on tumors. Examples of therapeutic agents include selective estrogen receptor degraders, such as fulvestrant and GDC- 9545, SAR439859, RG6171, AZD9833, Lint Destrant, ZN-c5, LSZ102, D-0502, LY3484356, SHR9549;Select selective estrogen receptor modulators, e.g., tamoxifen, raloxifene, 4- Hydroxytamoxifen, trioxifen, keoxifen, toremifene; Aroma Tase inhibitors, such as anastrozole, exemestane, letrozole, and 4(5 )-imidazoles inhibit other aromatase; gonadotropin-releasing hormone agonists nafarelin, triptorelin, goserelin; gonadotropin-releasing hormone a antagonists, e.g., degarelix; antiandrogens, e.g., abiraterone, Zalutamide, apalutamide, darolutamide, flutamide, nilutamide, bicalutamide, lo iprolide; 5α-reductase inhibitors, e.g., finasteride, dutasteride; In certain embodiments, the combination therapy includes, but is not limited to, hormone therapy. In one embodiment, the combination therapy includes administration of enzalutamide or a related hormonal agent. This includes administering

[0175] In some embodiments, the one or more additional therapeutic agents are epigenetic modulators Epigenetic modulators are molecules that regulate epigenetic expression. altering the locking mechanism, e.g., by inhibiting or activating epigenetic enzymes Non-limiting examples of epigenetic modulators include DNA membrane modulators. DNMT inhibitors, hypomethylating agents, and histone deacetylases In one or more embodiments, the anti-CD3 inhibitors described in Section II are included. 39 antibodies are DNA methyltransferase (DNMT) inhibitors or hypomethylating agents. Exemplary DNMT inhibitors include decitabine, zebularine, and and azacitadine. In one or more embodiments, the anti-CD39 antibodies of Section II Combinations of with histone deacetylase (HDAC) inhibitors are also contemplated. Useful HDAC inhibitors include vorinostat, divinostat, abexinostat, and panobiostat. These include benzodiazepine, 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 may, for example, regulate adenosine levels or By targeting adenosine receptors, adenine nucleosides and nucleotides (e.g. alters signal transduction mediated by adenosine, AMP, ADP, and ATP Adenosine and ATP act on different classes of receptors and mediate inflammation, cell proliferation, and cellular For example, ATP and other adenine Nucleotides exert antitumor effects through activation of the PS2Y1 receptor subtype. The accumulation of adenosine in the tumor microenvironment inhibits the antitumor functions of various immune cells and inhibits cellular functions. The immunosuppressive activity of myeloid and regulatory T cells is enhanced by binding to adenosine receptors on the cell surface. In certain embodiments, the ATP-adenosine axis targeting The inhibitors are inhibitors of ectonucleotidases, which are involved in the conversion of ATP to adenosine, and is an adenosine receptor antagonist. It is involved in the conversion of ATP to adenosine. Ectonucleotidases include ectonucleoside triphosphate diphosphohydrolase 1 (E NTPD1, ​​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, ORI Exemplary anti-CD73 antibodies include C-533, LY3475070, and AB680. These include CPI-006, Olecurumb (MEDI9447), NZV930, and IPH5 301, GS-1423, uriledolimab (TJD5, TJ004309), and BMS In one embodiment, the present disclosure provides an anti-CD39 antibody of Section II. Antibodies and WO2017 / 120508, WO2018 / 067424, WO2018 / 0 94148, and combinations with CD73 inhibitors such as those described in WO2020 / 046813 In a further embodiment, the CD73 inhibitor is quemliculstat. Adenosine binds to four different G protein-coupled receptors: A1R, A 2A R.A. 2B R A2R antagonists can bind to and activate A2R and A3R. Denant, inupadenant, taminadenant, potassium citrate Fein, NUV-1182, TT-702, DZD-2269, INCB-10638 5, EVOEXS-21546, AZD-4635, Imaradenant, RVU-330, Siphoradenant, PBF-509, PBF-999, PBF-1129, and CS-3 In some embodiments, the present disclosure provides an anti-CD39 antibody of Section II. Body and A 2A R antagonist, A 2B R antagonist, or A 2A R and A 2B R's Anta In some embodiments, the disclosure contemplates combinations with agonists. I, and the anti-CD39 antibody of WO2018 / 136700, WO2018 / 204661, W O2018 / 213377, or WO2020 / 023846, WO2020 / 102 646. In , the adenosine receptor antagonist is etormadenant.

[0177] In some embodiments, the one or more additional therapeutic agents are targeted therapeutic agents. Targeted therapeutic agents are chemotherapeutic agents, radionuclides, hormonal therapeutic agents, or other therapeutic agents attached to a targeting agent. Targeting agents may include small molecules, sugars (including oligosaccharides and polysaccharides), antibodies, It may be 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 or peptides, and the target is a tumor-associated antigen (enriched but not specific to cancer cells). ), tumor-specific antigens (with minimal or no expression in normal tissues), or neoantigens ( (An antigen specific to the genome of cancer cells, generated by nonsynonymous mutations in the tumor cell genome) In some embodiments, the targeting agent is an antibody and the target is a tumor-associated antigen (enriched tumor-specific antigens (expressed minimally in normal tissues but not specific to cancer cells) or none at all), or neoantigens (generated by nonsynonymous mutations in the tumor cell genome, In some embodiments, the targeted therapeutic agent is an antigen specific to the genome of a cancer cell. and a drug, wherein the antibody is a HER2, HER3, or necrotic Targeted therapeutic agents, including antibodies and drugs, specifically bind to Tropin-4 or Trop-2. Examples include patritumab deruxtecan, sacituzumab govitecan-hziy, and teri These include, but are not limited to, sotuzumab vedotin, and trastuzumab deruxtecan. In other embodiments, the targeted therapeutic agent has specific properties that aid in tumor survival, growth, and / or spread. Non-limiting examples of such targeted therapeutic agents include: Signal transduction inhibitors, RAS signal transduction inhibitors, and inhibitors of oncogenic transcription factors , activators of oncogenic transcription factor repressors, angiogenesis inhibitors, immunotherapeutic agents, ATP-adenosine Synaptic targeting agents, AXL inhibitors, PARP inhibitors, PAK4 inhibitors, PI3K inhibitors, H IF2α inhibitor, CD73 inhibitor, A2R antagonist, TIGIT antagonist, and PD-1 antagonists. ATP-adenosine axis targeting agents include those described above. Other drugs 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 drugs 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: These include, but are not limited to: (i) BCR-ABL kinase inhibitors (e.g., imatinib); (ii) small molecule inhibitors (e.g., CLN-081, gefitinib, erlotinib, afatinib, epidermal growth factor receptor tyrosine kinase inhibitors, including cinib, icotinib, and osimertinib inhibitors (EGFR TKIs) and anti-EGFR antibodies; (iii) transmembrane tyrosine kinase Inhibitors of the human epidermal growth factor (HER) family, e.g., the HER-2 / neu receptor inhibitors (e.g., trastuzumab and HER-3 receptor inhibitors); (iv) small molecule inhibitors (e.g., axitinib, regorafenib, sunitinib, and sorafenib), VEGF Kinase inhibitors (e.g., lenvatinib, cabozantinib, pazopanib, tivozanib, X L092, etc.), anti-VEGF antibodies (e.g., bevacizumab), and anti-VEGFR antibodies (e.g., (v) vascular endothelial growth factor receptor (VEGFR) inhibitors, including, for example, ramucirumab; (vi) inhibitors of T family kinases or the AKT pathway (e.g., rapamycin); Inhibitors of the phospho / threonine protein kinase B-Raf (BRAF), e.g., BamHI (vii) for example, serperca Reconstitution during transfection (RET) inhibitors, including tinib 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 (x) inhibitors of the RAS signaling pathway as described elsewhere herein; (xi) inhibitors (e.g., inhibitors of KRAS, HRAS, RAF, MEK, ERK); (xii) T-3 inhibitors (e.g., gilteritinib); (xii) Trop-2 inhibitors; i) Inhibitors of the JAK / STAT pathway, including, for example, tofacitinib and ruxolitinib JAK inhibitors or STAT inhibitors such as napabucasin; (xiv) NF-κB inhibition (xv) cell cycle kinase inhibitors (e.g., flavopiridol); (xvi) phosphatase inhibitors (xix) Protein kinase B ( AKT) inhibitors (e.g., capivasertib, milansertib), (xx) platelet-derived proliferation inhibitors PDGFR inhibitors (e.g., imatinib, sunitinib, regorafenib, and Bapritinib, lenvatinib, nintedanib, famitinib, ponatinib, axitinib , repretinib, etc.); and (xxi) insulin-like growth factor inhibitors. Inflammatory Response Factor (IGFR) inhibitors (e.g., erlotinib, afatinib, gefitinib, In one or more embodiments, the additional therapeutic agent is an EGFR , VEGFR, HER-2, HER-3, BRAF, RET, MET, ALK, RAS( For example, KRAS, MEK, ERK), FLT-3, JAK, STAT, NF-kB, P I3K, AKT, or any combination thereof.

[0179] In some embodiments, the one or more additional therapeutic agents is a RAS signaling inhibitor. Oncogenic mutations in RAS family genes, such as HRAS, KRAS, and NRAS, , which are associated with various cancers. For example, among the KRAS family genes, G12 C, G12D, G12V, G12A, G13D, Q61H, G13C, and G12S variants These differences have been observed in multiple tumor types. Direct and indirect inhibitory strategies are being investigated. Indirect inhibitors inhibit the RAS signaling pathway. It targets effectors other than RAS, including RAF, MEK, ERK, PI3K, and P TEN, SOS (e.g., SOS1), mTORC1, SHP2 (PTPN11), and Non-limiting examples of indirect inhibitors under development include, but are not limited to, inhibitors of AKT. RMC-4630, RMC-5845, RMC-6291, RMC-6236, JA B-3068, JAB-3312, TNO155, RLY-1971, BI170196 Direct inhibitors of RAS mutants have also been investigated, including KRAS-GTP complexes. Exemplary direct RAS inhibitors in development are: The inhibitors include sotorasib (AMG510), MRTX849, mRNA-5671, and AR In some embodiments, one or more RAS signaling inhibitors include RAF inhibitors, MEK inhibitors, ERK inhibitors, and PI3K inhibitors. inhibitors, PTEN inhibitors, SOS1 inhibitors, mTORC1 inhibitors, SHP2 inhibitors, and In another embodiment, one or more RAS signaling inhibitors are selected from the group consisting of KT inhibitors. The inhibitor directly inhibits the RAS mutant.

[0180] In some embodiments, one or more of the additional therapeutic agents is phosphatidylinositol 3 -Inhibitors of kinase (PI3K), particularly the PI3Kγ isoform. 3Kγ inhibitors can inhibit suppressor myeloid cells through the regulation of myeloid cells, e.g. by attenuating immunosuppressive tumor-infiltrating macrophages or by inhibiting macrophages By stimulating lymphocytes and dendritic cells to produce cytokines that contribute to effective T cell proliferation. stimulates an anti-cancer immune response, thereby reducing the incidence and spread of cancer Examples of PI3Kγ inhibitors include copanlisib, duvelisib, AT-104, and Z X-101, tenalisib, eganelisib, SF-1126, AZD3458, and picrine In some embodiments, the anti-CD39 antibody of Section II is a vasodilator. In combination with one or more PI3Kγ inhibitors described in O2020 / 0247496A1 This can be done.

[0181] In some embodiments, the one or more additional therapeutic agents is an inhibitor of arginase. Ruginase is a key regulator of inflammation-induced immune dysfunction, tumor immune evasion, immunosuppression in infectious diseases, and immune responses. It has been shown to be a cause of or involved in pathologies. Compounds include CB-1158 and OAT-1746. In some embodiments, the anti-CD39 antibodies of Section II are described in WO / 2019 / 173188 and WO2 and one or more arginase inhibitors described in US Pat. No. 5,920,466. do.

[0182] In some embodiments, the one or more additional therapeutic agents are inhibitors of oncogenic transcription factors or is an activator of the oncogenic transcription factor repressor. Suitable agents include those that increase expression levels (e.g. , RNAi, siRNA, etc.) through physical degradation at the protein / protein level at the protein / DNA level or by binding in activation / inhibition pockets. Non-limiting examples include inhibitors of one or more subunits of the MLL complex (e.g., For example, HDAC, DOT1L, BRD4, menin, LEDGF, WDR5, KDM4C ( JMJD2C) and PRMT1), inhibitors of hypoxia-inducible factor (HIF) transcription factors, etc. Examples include:

[0183] In some embodiments, one or more of the additional therapeutic agents inhibits hypoxia inducible factor (HIF) transcription. Exemplary HIF-2α inhibitors include bells Chifan, ARO-HIF2, PT-2385, AB521, and WO202111 3436 and WO2021188769. In embodiments, the anti-CD39 antibody of Section II is a CD39 antibody described in WO2021188769. It may be combined with one or more HIF-2α inhibitors.

[0184] In some embodiments, one or more of the additional therapeutic agents is anexselect The AXL signaling pathway mediates tumor growth and metastasis. It is associated with cancer migration and is thought to mediate resistance to various cancer treatments. Other kinases of the M family (i.e., TYRO3, MERTK), as well as ME Also inhibits other receptor tyrosine kinases, including T, FLT3, RON, and AURORA A variety of AXL inhibitors are currently under development. Examples of multikinase inhibitors include Citraxon Vatinib, rebastinib, glesatinib, gilteritinib, merestinib, cabozantinib B, foretinib, BMS777607, LY2801653, S49076, GSK1 363089, and RXDX-106. AXL-specific inhibitors, such as DS -1205, SGI-7079, SLC-391, TP-0903 (i.e., Duvel Subdivisions including rituximab, BGB324 (i.e., bemcentinib), and DP3975 AXL inhibitors; anti-AXL antibodies such as ADCT-601; and antibody drug conjugates such as BA3011. Another strategy to inhibit AXL signaling is ADCs. For example, AVB-500 targets GAS6, a ligand for G Similar to Fc fusion proteins, which bind to AS6 ligands and inhibit AXL signaling, It is currently in development.

[0185] In some embodiments, the one or more additional therapeutic agents is p21-activated kinase 4 (PAK4). PAK4 is an inhibitor of PD-1. Overexpression of PAK4 is particularly prevalent in cancers resistant to PD-1 therapy. PAK4 inhibitors have not yet been approved. However, there are currently several PAK4 / NAMPT inhibitors under development, such as ATG-019 and KPT-9274. In some embodiments, antibodies according to the present disclosure are administered as P In some embodiments, an antibody according to the present disclosure is combined with an AK4 selective inhibitor. In combination with AK4 / NAMPT dual inhibitors, e.g., ATG-019 or KPT-9274 match.

[0186] In some embodiments, one or more of the additional therapeutic agents is (i) the enzyme poly(ADP -ribose) polymerase inhibitors (e.g., olaparib, niraparib, and ruca (ii) inhibitors of the Bcl-2 protein family (e.g., venetoclave, (iii) MCL-1 inhibitors; (iv) CD47-SI Inhibitors of the RPα pathway (e.g., anti-CD47 antibodies, magrolimab, etc.); (v) isoquen Acid dehydrogenase (IDH) inhibitors, e.g., IDH-1 inhibitors or IDH-2 inhibitors agents (e.g., ivosidenib, enasidenib, etc.).

[0187] In some embodiments, one or more of the additional therapeutic agents is an immunotherapeutic agent. Immunotherapeutic agents useful in treating cancer typically induce or amplify an immune response against cancer cells. Non-limiting examples of suitable immunotherapeutic agents include immunomodulatory agents; cellular immunotherapeutic agents; vaccines; Gene therapy drugs; ATP-adenosine axis targeting agents; immune checkpoint modulators and certain signal transduction inhibitors. ATP-adenosine axis targeting agents Immunomodulators, signal transduction inhibitors, cellular immunotherapeutics, vaccines, genetic Gene therapy drugs and immune checkpoint modulators are discussed further below. do.

[0188] In some embodiments, one or more of the additional therapeutic agents is an immunotherapeutic agent, more particularly, Cytokines or chemokines, e.g., IL1, IL2, IL12, IL18, EL C / CCL19, SLC / CCL21, MCP-1, IL-4, IL-18, TNF, I L-15, MDC, IFNa / b, M-CSF, IL-3, GM-CSF, IL-13, and anti-IL-10; bacterial lipopolysaccharide (LPS); activates antigen-presenting cells and inhibits major histocompatibility Tol, an organic or inorganic adjuvant that enhances the presentation of antigenic epitopes on the soluble complex molecule TLR agonist, mevalonate pathway antagonist, STING antagonist agonists, including but not limited to indoleamine 2,3-dioxygenase agonists; Inhibitors of IDO1 and immunostimulatory oligonucleotides, as well as other T A cellular adjuvant.

[0189] In some embodiments, the one or more additional therapeutic agents are immunotherapeutic agents, more particularly cytotoxic agents. Cell therapy is a form of treatment in which living cells are administered to a subject. In certain embodiments, the one or more additional therapeutic agents are cellular immune regulators that activate or suppress the immune system. Cellular immunotherapeutics useful in the treatment of cancer typically induce or The cells may be autologous or allogeneic immune cells obtained from one or more subjects. (e.g., monocytes, macrophages, dendritic cells, NK cells, T cells, etc.). Alternatively, the cells may be immune progenitor cells (e.g., lymphoid progenitor cells, myeloid progenitor cells, dendritic cells, etc.). (Re)programmed cells generated from various types of cells (e.g., progenitor cells, stem cells, induced pluripotent stem cells) In some embodiments, such cells may be allogeneic immune cells. Expanded subsets of cells (e.g., pluripotent stem cells) with effector functions and / or maturation markers For example, adaptive memory NK cells, tumor-infiltrating lymphocytes, immature dendritic cells, monocyte-derived dendritic cells, plasma cells follicular dendritic cells, conventional dendritic cells (sometimes called classical dendritic cells), M1 macrophage phage, M2 macrophage, etc.) to target cells to specific antigens. and / or may be genetically modified (e.g., engineered) to enhance the anti-tumor effect of the cells. T cell receptor (TCR) cell therapy, chimeric antigen receptor (CAR) cell therapy, and lymph node homing of antigen-loaded dendritic cells) to express tumor-associated antigens or or any combination thereof. Non-limiting types of cell therapy include CAR-T cell therapy, CAR-NK cell therapy, and the like. Exemplary cellular immunotherapeutics include cellular immunotherapeutics, TCR therapeutics, and dendritic cell vaccines. These include sipuleucel-T, tisagenlecleucel, lysocabtagene maraleucel, Idecabtagene-biculeucel, brexcabtagene-autoleucel, and axica Butagene ciloleucel, as well as CTX110, JCAR015, JCAR017, and M B-CART19.1, MB-CART20.1, MB-CART2019.1, Uni CAR02-T-CD123, BMCA-CAR-T, JNJ-68284528, BN T211, and NK-92 / 5.28.z.

[0190] In some embodiments, the one or more additional therapeutic agents are immunotherapeutic agents, more particularly immunotherapeutic agents. Gene therapy drugs are drugs that modify the expression of endogenous genes by transfecting proteins. Heterologous expression of (e.g., small interfering RNA (siRNA) agents, double-stranded RNA (dsRNA) agents, microRNA (miRNA) agents, viral or bacterial gene delivery, etc. recombinant nucleic acid administered ex vivo to a subject or cells of a subject to induce Nucleic acid components (e.g., meganucleases, zinc finger nucleases, TAL nucleases) These may include enzymes such as CRISPR / Cas nucleases, oncolytic viruses, etc. including gene editing therapeutics that may be useful in cancer treatment. Non-limiting examples include Gendicine® (rAd-p53), Oncor ine® (rAD5-H101), talimogene laherparepvec, Mx-dn G1, ARO-HIF2 (Arrowhead), quoratusgenose plasmid (qu aratusugene ozeplasmid) (Immunogene), CTX1 10 (CRISPR Therapeutics), CTX120 (CRISPR Th erapeutics), and CTX130 (CRISPR Therapeutics ) are listed.

[0191] In some embodiments, the one or more additional therapeutic agents are immunotherapeutic agents, more particularly immunotherapeutic agents. Immune checkpoints regulate immune cells (e.g. a series of inhibitory and stimulatory pathways that directly affect the function of immune cells (e.g., B cells, T cells, and NK cells) Immune checkpoints are activated when proteins on the surface of immune cells recognize their cognate ligands. The present invention relates to agonists and / or co-stimulatory pathways. The use of anti-CD39 antibodies in Section II in combination with antagonists of inhibitory pathways Agonists of stimulatory or costimulatory pathways and antagonists of inhibitory pathways, or These or their combinations overcome different immunosuppressive pathways within the tumor microenvironment and suppress regulatory T cells. inhibiting T cells, reversing / preventing T cell anergy or exhaustion, and activating and inhibiting innate immunity at tumor sites These compounds may be useful as agents that induce inflammation and / or inflammation.

[0192] In some embodiments, the one or more additional therapeutic agents is an immune checkpoint inhibitor. As used herein, the term "immune checkpoint inhibitor" refers to an inhibitory or co-inhibitory immune checkpoint antagonists. The terms "checkpoint inhibitor," "checkpoint inhibitor," and "CPI" are used interchangeably herein. Immune checkpoint inhibitors can be used in a variety of ways, including blocking receptor-ligand binding. Inhibitory or co-inhibitory effects by interfering with and / or altering receptor signaling It can antagonize immune checkpoints (ligands and Examples of receptors (e.g., ribosomal receptors) include those that are selectively upregulated in various types of cancer cells, Some can be antagonized, examples of which include PD-1 (programmed cell death protein). 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 immunity Globulin and mucin domain-containing protein 3; LAG-3 (lymphocyte activation gene 3); TIGIT (T cell immunoreceptor with Ig and ITIM domains); CD276 (B7-H3);PD-L2, Galectin 9, CEACAM-1, BTLA, CD69, Galectin-1, CD113, GPR56, VISTA, 2B4, CD48, GARP, Based on the structural characteristics of PD1H, LAIR1, TIM-1, and TIM-4 There are two classes: i) killer cell immunoglobulin-like receptors (KIRs), and ii) C-type receptors. Killer receptors (members of the type II transmembrane receptor family) Inhibitory receptors, such as 2B4 (also known as CD244) ) receptor) and ligands (e.g., B7-H3 (also known as CD276) and B Certain proteins, such as 7-H4 (also known as B7-S1, B7x, and VCTN1) The B7 family of inhibitory ligands (B7 family inhibitors) have been described in the literature, but less clearly Other immune checkpoints, as yet undefined, are also contemplated.

[0193] In some embodiments, the immune checkpoint inhibitor is a CTLA-4 antagonist. In a further embodiment, the CTLA-4 antagonist is an antagonistic CTLA-4 antagonist. Suitable antagonistic CTLA-4 antibodies include, for example, monospecific CTLA-4 antibodies. Heterologous antibodies, e.g., ipilimumab or tremelimumab, as well as MEDI5752 and and bispecific antibodies such as KN046.

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

[0195] In some embodiments, the immune checkpoint inhibitor is a PD-L1 antagonist. In a further embodiment, the PD-L1 antagonist is an antagonistic PD-L1 Suitable antagonistic PD-L1 antibodies include, for example, monospecific antibodies Examples include avelumab, atezolizumab, durvalumab, BMS-936559, and emvafolimab, as well as bispecific antibodies such as LY3434172 and KN046. Body included.

[0196] In some embodiments, the immune checkpoint inhibitor is a TIGIT antagonist. In a further embodiment, the TIGIT antagonist is an antagonistic TIG Suitable antagonistic anti-TIGIT antibodies include monospecific antibodies. Enzymes, for example, AGEN1327, AB308 (WO2021247591), BMS 986207, COM902, domvanalimab, EOS-448, etigilimab, IBI -929, JS006, M6223, osipelimab, SEA-TGT, tiragolumab, and bispecific antibodies such as AGEN1777 and AZD2936. In certain embodiments, the immune checkpoint inhibitor is 2088 or the antagonistic anti-TIGI disclosed in WO2021247591 In certain embodiments, the immune checkpoint inhibitor is a domban limpet antibody. AB or AB308.

[0197] In some embodiments, the immune checkpoint inhibitor is a LAG-3 antagonist. In a further embodiment, the LAG-3 antagonist is an antagonistic LAG Suitable antagonistic LAG-3 antibodies include, for example, BMS-9 86016 (WO10 / 19570, WO14 / 08218), or IMP-731 Examples include IMP-321 (WO08 / 132601, WO09 / 44273).

[0198] In some embodiments, the immune checkpoint inhibitor is a B7-H3 antagonist. In a further embodiment, the B7-H3 antagonist is an antagonistic B7-H3 antagonist. Suitable antagonist B7-H3 antibodies include, for example, MGA271 (W O11 / 109400), omburtumab, enoblitzumab , DS-7300a, ABBV-155, and SHR-A1811.

[0199] In some embodiments, the immune checkpoint inhibitor is a TIM-3 antagonist. In a further embodiment, the TIM-3 antagonist is an antagonistic TIM Suitable antagonistic TIM-3 antibodies include, for example, TIM-3 antibodies. Lulimab, sabatolimab, BMS-986258, and RG7769 / RO712166 1 is one example.

[0200] In some embodiments, the one or more additional therapeutic agents are stimulatory or costimulatory immune chain Activates stimulatory or costimulatory immune checkpoints (ligands and Examples of receptors include B7-1, B7-2, CD28, 4-1BB (CD137), 4 -1BBL, ICOS, ICOS-L, OX40, OX40L, GITR, GITRL, These include CD70, CD27, CD40, DR3, and CD2.

[0201] In some embodiments, drugs that activate stimulatory or costimulatory immune checkpoints In a further embodiment, the agent is a CD137 (4-1BB) agonist. The agonist can be an agonistic CD137 antibody. Suitable CD137 antibodies include, for example, Examples include urelumab and PF-05082566 (WO12 / 32433). In some embodiments, the agent that activates a stimulatory or costimulatory immune checkpoint is In a further embodiment, the GITR agonist is an agonist. Suitable GITR antibodies include, for example, BMS-9861. 53, BMS-986156, TRX-518(WO06 / 105021, WO09 / 0 09116) and MK-4166 (WO11 / 028683). In embodiments, the agent that activates a stimulatory or costimulatory immune checkpoint is OX4 In a further embodiment, the OX40 agonist is an agonistic OX40 agonist. Suitable OX40 antibodies include, for example, MEDI-6383, MEDI-6383, MEDI-6384, MEDI-6385, MEDI-6386, MEDI-6387, MEDI-6388, MEDI-6389 ... DI-6469, MEDI-0562, PF-04518600, GSK3174998 , BMS-986178, and MOXR0916. Agents that activate stimulatory or costimulatory immune checkpoints are CD40 agonists. In a further embodiment, the CD40 agonist is dacetuzumab, cericlerumab agonistic CDs such as APX005M, ADC-1013, or CDX-1140 40 antibody. In some embodiments, the antibody may be a stimulatory or costimulatory immune checkpoint inhibitor. In a further embodiment, the agent that activates the CD27 receptor is a CD27 agonist. The agonist can be an agonistic CD27 antibody. Suitable CD27 antibodies include, for example, , and varlilumab.

[0202] In some embodiments, one or more of the additional therapeutic agents is an immunotherapeutic agent, more particularly a synergistic agent. Intracellular signaling molecules that affect immune cell function are also may be a suitable target for improving anti-tumor immunity. For example, one or more of the additional therapeutic agents may be It can be an inhibitor of intracellular signaling molecules and inhibits hematopoietic progenitor kinase 1 (HPK1). HPK1 is a negative regulator of the activation signal generated by the T cell antigen receptor. Another example is the use of a serine / threonine kinase that functions as a blocking factor. One or more of these proteins are Cbl-b, an E3 ubiquitin ligand involved in regulating TCR signaling. In another example, one of the additional therapeutic agents may be an inhibitor of a steroid hormone (e.g., AP401). The above 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: Examples include CFI-402411 and BGB-15025, and Cbl Non-limiting examples of small molecule DAG inhibitors include AP401. Non-limiting examples include those described in WO2020006016A1 and WO2021130638. The following are listed:

[0203] In some embodiments, one or more of the additional therapeutic agents inhibits immunosuppressive immune cells. For example, agents that inhibit immunosuppressive macrophages or monocytes. To inhibit or deplete CSF-1R, the agent may be a CSF-1R antagonist, e.g., RG7 155(WO11 / 70024, WO11 / 107553, WO11 / 131407, W O13 / 87699, WO13 / 119716, WO13 / 132044) or FP CSF-1R anions containing A-008 (WO11 / 140249; WO13169264) The antibody may be an antagonist antibody, or an antibody disclosed in WO14 / 036357. For example, to inhibit or deplete Tregs, agents can be used to deplete Tregs ex vivo. The antibodies may be anti-CD25 beads used for blotting.

[0204] In some embodiments, the present disclosure provides a method for treating or preventing atherosclerosis in combination with immunogenic cell death (ICD)-inducing therapy. Also contemplated is the use of anti-CD39 antibodies in Section II. functional antibodies sufficient to induce adaptive immunity specifically directed against antigens derived from It can be defined as a regulated cell death subtype specific to ICD. The criteria for distinguishing between these two conditions are as follows: Vanmeerbeek et al., OncoImm unology, 2020, 9:1, DOI: 10.1080 / 2162402 X.2019.1703449;Keep et al., OncoImmunolo gy, 2014, 3(9): :e955691. Non-limiting examples of ICD-guided therapies are described in Galluzzi et al., Nature Reviews Clinical Oncology, 2020 17: 725- In some embodiments, the ICD-guided therapy is radiation therapy, phototherapy, or other therapy. Radiodynamic therapy, extracorporeal photochemotherapy, oncolytic virus therapy, bortezomib, cyclophosphamide Sulfamide, doxorubicin, epirubicin, idarubicin, mitoxantrone, ceftazidime In some embodiments, the medicament may be cimargin, crizotinib, or oxaliplatin. ICD-guided therapies include radiation therapy, photodynamic therapy, extracorporeal photochemotherapy, and oncolytic viruses. rus 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 is independently selected from the group consisting of radiation therapy, chemotherapy, radiation therapy, and chemotherapy alone. Sexual medicines, hormone therapy drugs, epigenetic modulators, targeted agents, immunotherapy drugs , cell therapy, gene therapy, or ICD-guided therapy. For example, in one embodiment, The present disclosure also provides a method for administering an anti-CD39 antibody of Section II to a patient in need of one or more ICD-inducing therapies and any It is intended to be used in combination with one or more additional therapies of choice, and each additional therapy are radiation therapy, radiopharmaceuticals, chemotherapy, hormonal therapy, targeted agents, immunotherapy, In another example, in one embodiment, The present disclosure also provides a method for treating a patient with an anti-CD39 antibody of Section II in combination with one or more chemotherapeutic agents and optionally It is intended for use in combination with one or more additional therapies, each of which may be Radiation therapy, radiopharmaceuticals, hormone therapy, targeted agents, immunotherapy, cellular therapy, or In another embodiment, the present disclosure provides a method for treating a genetic disorder comprising administering to a subject a genetically modified organism, ... a gene therapy agent, or a gene therapy product. an anti-CD39 antibody, one or more chemotherapeutic agents and one or more tyrosine kinase inhibitors; and optionally one or more additional therapies, wherein each therapy is independently a targeted agent, an immunotherapeutic agent, or a cellular therapy agent. In accordance with the present disclosure, the present disclosure provides a method for treating a patient with an anti-CD39 antibody of Section II in combination with one or more chemotherapeutic agents, comprising: (i) administering to a patient a therapeutically effective amount of the anti-CD39 antibody of Section II in combination with one or more chemotherapeutic agents; (ii) BCR-ABL kinase inhibitors; (iii) EGFR inhibitors (e.g., EGFR TKIs or or anti-EGFR antibodies); (iii) HER-2 / neu receptor inhibitors; (iv) anti-angiogenic Biologic agents (e.g., anti-VEGF antibodies, VEGFR TKIs, VEGF kinase inhibitors, etc.); (v) AKT inhibitors; (vi) BRAF inhibitors; (vii) RET inhibitors; (viii) (ix) a MET inhibitor; (ix) a RAS inhibitor; and (x) an ALK inhibitor, and optionally one or more additional therapies, wherein each additional therapy is selected from the group consisting of radiation therapy, radiopharmaceuticals, targeted agents, In another embodiment, the present disclosure provides a method for treating a leukemia, comprising administering to a subject a therapeutic agent, a medicament for treating a leukemia, or a cancer, the method ... the cancer, or a cancer, the The anti-CD39 antibody of Section II is administered in combination with one or more immunotherapeutic agents and optionally one or more additional Therapeutic options are intended for use in combination with additional therapies, each of which may include radiation therapy, radiotherapy, or chemotherapy. Injectable, hormonal, targeted, chemotherapy, cell therapy, or gene therapy drugs In another embodiment, the present disclosure provides an anti-CD39 antibody of Section II. The body is treated with one or more immunotherapeutic agents and one or more chemotherapeutic agents, and optionally one or more It is intended for use in combination with additional therapies, each of which may be radiation therapy, Independent of radiopharmaceuticals, hormone therapy drugs, targeted agents, cell therapy drugs, or gene therapy drugs In another embodiment, the present disclosure provides an anti-CD39 antibody of Section II selected from one or more of the following: or more immunotherapeutic agents and one or more radiotherapeutic or radiopharmaceutical agents, and optionally one and / or Independently selected from therapeutic, hormonal, targeted, cellular, or gene therapy drugs In another embodiment, the disclosure provides an anti-CD39 antibody of Section II, comprising one or more Immune checkpoint inhibitors and / or one or more ATP-adenosine axis targeting agents, and optionally one or more additional therapies, Each therapy may be radiotherapy, radiopharmaceuticals, chemotherapy, targeted agents, immunotherapy, or cytotoxic agents. In another embodiment, the present disclosure provides an anti-C1 inhibitor of Section II. The D39 antibody was administered in combination with one or more immune checkpoint inhibitors and / or one or more ATP- an adenosine axis targeting agent and / or one or more chemotherapeutic agents, radiopharmaceuticals, or radiotherapy In another embodiment, the present disclosure is intended to be used in conjunction with the method of Phase II anti-CD39 antibodies in combination with one or more immune checkpoint inhibitors and / or The above ATP-adenosine axis targeting agents and (i) BCR-ABL kinase inhibitors; (ii) EGFR inhibitors (e.g., EGFR TKIs or anti-EGFR antibodies); (iii) HER -2 / neu receptor inhibitors; (iv) antiangiogenic agents (e.g., anti-VEGF antibodies, VEGF (v) AKT inhibitors; (vi) BRAF inhibitors; (vii) RET inhibitors; (viii) MET inhibitors; (ix) KRAS inhibitors, and (x) an ALK inhibitor. In another embodiment, the present disclosure contemplates the use of anti-CD39 antibodies of Section II. The antibody is administered in combination with one or more immune checkpoint inhibitors and / or one or more ATP-adenosine monophosphate (ATP)- Intended for use in combination with a synaxial targeting agent and one or more ICD-guided therapies In a further embodiment of the above, (a) the targeting agent is a PI3K inhibitor, arginine enzyme inhibitor, HIF2α inhibitor, AXL inhibitor, PAK4 inhibitor, or anti-angiogenic agent (b) the immunotherapeutic agent can be an ATP adenosine axis targeting agent, a cytokine therapy, an immunotherapy (c) ATP-adenosine monophosphate (ATP) inhibitors, or combinations thereof; Synaxial targeting agents are 2A R and / or A 2B R antagonist or CD73 inhibitor (d) the ATP-adenosine axis targeting agent is etormadenant or quemri (e) the immunotherapeutic agent is an anti-PD-L1 antagonist antibody or an anti PD-1 antagonist antibodies, optionally including budigalimab, camrelizumab, Sibelimab, dostallimab, cemiplimab, ezabenlimab, nivolumab, pembrolizumab Izumab, pidilizumab, pimivalimab, retifanlimab, sasanlimab, spartalis Mab, sintilimab, tislelizumab, toripalimab, zimbele Limab, LY3434172, avelumab, atezolizumab, balstilimab, Durva lumab, envafolimab, LY3434172 and KN0 (f) the immunotherapeutic agent is optionally selected from the group consisting of an anti-TIGIT antagonist and optionally AGEN1327, AB308 (WO2021247591 ), BMS986207, COM902, domvanalimab, EOS-448, etigilima IBI-929, JS006, M6223, osipelimab, SEA-TGT, Chirago lumab, vibostolimab, and bispecifics such as AGEN1777 and AZD2936 (g) the immunotherapeutic agent is selected from the group consisting of domvanalimab, AB308, diclofenac, ribozyme, ribozyme-binding protein (RIP), ... mbelelimab, tiragolumab, pembrolizumab, nivolumab, atezolizumab, or (h) antiangiogenic agents include pazopanib, sorafenib, and sunitinib. bevacizumab, axitinib, lenvatinib, tivozanib, or cabozantinib (i) ICD-guided therapy is not related to radiation therapy (both external and internal radiation therapy). including steroid therapy, photodynamic therapy, extracorporeal photochemotherapy, oncolytic virus therapy, bortezomib cyclophosphamide, doxorubicin, epirubicin, idarubicin, mitoxanthin or (j) any combination thereof. In still further embodiments of the above, the present disclosure provides a method for treating rheumatoid arthritis comprising administering to a subject a therapeutically effective amount of domvanalimab, AB308, etol. Madenant, quemliculstat, zimberelimab, AB521, or any of these The present invention contemplates the use of an anti-CD39 antibody of Section II in combination with a combination of the above. In still further embodiments, the present disclosure provides a compound selected from the group consisting of ethlomadenant, quemliculstat, and methlosporin. or in combination with etremadenant and quemliculstat, In yet further embodiments of the above, the present disclosure contemplates the use of the D39 antibody. in combination with Nantes, AB521, or Etruscan Nantes and AB521, In yet further embodiments of the above, the present disclosure contemplates the use of anti-CD39 antibodies of type II. , Kemlikrustat, AB521, or a combination of Kemlikrustat and AB521 The use of the anti-CD39 antibodies of Section II in combination with the anti-CD39 antibodies of Section II is contemplated.

[0206] The selection of additional therapeutic agent(s) may depend on the particular cancer and / or mutational status of the cancer of interest, and / or may be informed by the current standard of care for the stage of the disease. The line is, for example, the National Comprehensive Cancer Network For example, NCCN Colon Cancer v3.20 21, NCCN Hepatobiliary Cancer v5.2021, NCCN Kidney Cancer v3.2022, NCCN N SCLC v7.2021, NCCN pancreatic adenocarcinoma v2.2021, NCCN esophagus and gastroesophageal Junctional cancer v4.2021, NCCN gastric cancer v5.2021, ovarian cancer / fallopian tube cancer / primary peritoneal cancer v3.2021, prostate cancer v3.2022, head and neck cancer v1.2022, melanoma: skin v1 .2022, Acute Myeloid Leukemia v1.2022. [Example]

[0207] experiment The following examples provide one of ordinary skill in the art with a complete disclosure and description of how to make and use the present disclosure. It is set forth for the purpose of illustration only and is not intended to limit the scope of what the inventors regard as their invention. Additional antibodies within the scope of this disclosure may be synthesized based on the methods shown in these examples. 2. Making a compound using a method based on the method described herein or other methods described herein. No effort is made to ensure accuracy of the values ​​used (e.g., amounts, temperatures, etc.). Although the results are accurate, some experimental error and deviation should be considered.

[0208] Unless otherwise specified, temperatures are in degrees Celsius (°C) and pressures are in atmospheric or 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 = microgram chlorliter; ml or mL = milliliter; l or L = liter; μM = microliter Molar; mM = millimole; M = molar; mol = mole; mmol = millimole; nM = nano Mole.

[0209] Example 1: Generation of anti-CD39 antibodies Human CD39-expressing CHO-K1 cell line, cynomolgus monkey CD39-expressing HEK-293 cells A pool of mouse CD39-expressing HEK-293 cells was generated. and CHO-K1 cells or HEK-293 cells (Flp-In™-293 cells). Then, Lipofectamine 2000 transfection kit was used to transfect Full-length human, cynomolgus monkey, or mouse CD39 (respectively) was isolated according to the manufacturer's protocol. pcDNA3.3 expression vectors encoding the following: SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 71 48-72 hours after transfection, The cells were cultured in medium containing blasticidin for selection and tested for CD39 expression. Afterwards, we obtained a pool of CD39-expressing cells. Human, cynomolgus monkey, and mouse CD39-expressing cells Lines or cell pools were obtained by limiting dilution and hybridoma supernatants were screened as described below. Used for cleaning.

[0210] To generate anti-CD39 antibodies, 6- to 8-week-old Sprague Dawley rats were cultured. Four mice were treated with either protein or plasmid DNA of the human CD39 antigen 30-2 The adjuvant mixture consisted of Adju-Phos, CpG- The animals were given either plantar, subcutaneous, intraperitoneal, or intramuscular injections of ODN or Titer-Max. The animals were injected once every two weeks via the intramuscular and intradermal routes. Serum titers were measured by ELISA or fluorescent activity. Serum antibody titers against a given antigen were measured by fluorescently activated cell sorting (FACS). The ELISA used to determine the marker was performed as follows: Plates (Nunc) were The cells were coated with 100 μL of 1 μg / mL human CD39 antigen at ℃ overnight, and then blotted. The rats were blocked with blocking buffer (1xPBS / 2% BSA) for 1 hour at ambient temperature. Serum was serially diluted 3-fold in blocking buffer starting at 1:100 and incubated at ambient temperature. The wells to which no serum samples were added were used as negative controls. Next, the plate was washed, and then the secondary antibody, goat anti-rat IgG-Fc-HRP, was added. After washing, the cells were incubated with tetramethylbenzidine (Bethyl) for 1 hour. (TMB) substrate was added and the interaction was stopped by adding 2M HCl. The absorbance was read at 450 nm using a reader (Molecular Device). The FACS assay used to measure serum antibody titers used serially diluted sera. Except for the following, the procedure was performed as generally described for screening hybridoma supernatants (see below). Serum titers were determined as the final titer that gave a positive signal (i.e., more than three times the negative control). Determined as a dilution.

[0211] When serum titers were sufficiently high (≥1:24,300), the patient was given an adjuvant-free antiseptic solution. Animals were given a final boost with protein and cell membrane lysate in bacterial PBS. After a period of time, the animals were euthanized and the lymph nodes and spleens were used for cell fusion.

[0212] Lymph nodes and spleens from immunized animals are homogenized and filtered to remove blood clots and cellular debris. Sp2 / 0 myeloma cells in logarithmic growth were collected and centrifuged. Myeloma cells were treated separately with pronase solution and the reaction was stopped with 100% FBS. B cells were washed and counted. B cells were electrofused with Sp2 / 0 myeloma cells according to standard electrofusion procedures. The fused cells were then fused at a 1:1 ratio in an electrofusion solution containing 20% ​​FBS and 1x HA. The fused cells were resuspended in DMEM medium supplemented with T and then transferred to a 96-well plate. The cells were then kept in an incubator set at 37°C and 5% CO2 for 10 to 14 days.

[0213] Harvest hybridoma cells and inoculate 150-200 cells in 1.5 mL of semi-solid HAT medium. The cells were mixed gently on a vortex oscillator for 5-10 seconds, then 6 w The plates were placed in an incubator set at 37°C and 5% CO2. Each visible single colony was cultured in DME supplemented with 10% FBS for 7-8 days. After 2-3 days, the cell supernatant was collected and used for screening. I did it.

[0214] The order of the assay cascade in the screening paradigm was changed to four rounds of hybridization. Lipoblast generation and antibody screening were performed. The antibody produced by a given hybridoma clone may be directed against mouse CD39 or other CD39 Family members, namely CD39-L1, CD39-L2, CD39-L3, if There was no measurable binding to either CD39-L4 or CD39-L4, and both human and cynomolgus cells The antibody had to bind with high affinity to CD39 in cells that overexpress CD39. These cell lines include THP-1, SK-MEL-5, and MOLP-8. potently inhibited cellular human CD39 enzymatic activity in both human cell lines naturally expressing CD39. In the first round of fusion, CHO-K1 cell-based human CD39 ELISA was the primary screening method, and cells overexpressing human, cynomolgus, and Binding to mouse CD39 was measured by FACS (using the aforementioned cell lines) and in vitro Soluble human CD39 enzyme assay and cellular enzyme assay were used as secondary screening. A second round of fusion failed to produce suitable hybridoma material. In the third round of fusion, a soluble human CD39 enzyme assay was used as the primary screen. Binding to cells overexpressing human and mouse CD39 was measured by FACS. Soluble human CD39 for CD39-L1 binding counterscreening and confirmation Enzyme assays were the secondary screen. In the fourth round of fusion, THP-1 cells were The cytoplasmic enzyme assay was used as a primary screen to identify cells overexpressing human and cynomolgus monkeys. Binding to monkey CD39 was measured by FACS, as well as THP-1 cell binding for confirmation. The cytoplasmic enzyme assay was the secondary screen. Clone 39 from the first round, Clones 19 and 31 from the third round were selected for further characterization. No clones were selected from the fourth round.

[0215] Cell-based ELI used to assess binding to cell surface-expressed CD39 For SA, the assay was generally performed as follows: 3–5 × 10 per well 4 The cells were pre-coated and incubated at 37°C and 5% CO2. The plates were incubated in an incubator for 2 days. The plates were then incubated in blocking buffer for 1 hour at ambient temperature. (1x PBS / 5% milk) and then blocked with 30 μL of hybridoma supernatant. was added to the plate and incubated at ambient temperature for 1 hour. The plate was washed three times with PBS. and then with the secondary antibody goat anti-rat IgG-Fc-HRP (1:500). After 1 hour of incubation at ambient temperature, TMB substrate was added to each well and the plate was Incubate in the dark at ambient temperature for 3-5 minutes and stop the reaction by adding 2M HCl. A microplate reader (Molecular Device) was used to measure the The absorbance was read at 50 nm.

[0216] FACS also detects the CD39-expressing CHO-K1 cell line, the cynomolgus monkey CD39 cell line, and We used a mouse CD39-expressing HEK-293 cell line and a mouse CD39-expressing HEK-293 cell pool. It was also used to detect the binding of anti-CD39 antibodies to CD39 in the supernatant. Cells were used as controls to set the threshold before detection, and then each cell that exceeded the fluorescence intensity threshold was counted. The percentage change of the groups was analyzed. 5 Cells / well were plated in a volume of 100 μL. The hybridoma supernatant was incubated for 1 hour at 4°C. A human / rat IgG isotype antibody was used as a negative control. The cells were washed with 1x PBS / 1% BSA and then incubated with Alexa647-labeled goat anti-human Antibody or goat anti-rat antibody (diluted 1:500 in FACS staining buffer) was added to the cells. The cells were incubated in the dark at 4°C for 1 hour. The mean fluorescence intensity (MFI) of the cells was measured using a flow cytometer. Measurements were performed on a cytometer and analyzed using FlowJo software.

[0217] Anti-CD39 antibody was used to detect AT cells in association with luciferase activity and luminescence as a signal. by measuring the inhibition of the catalytic function of human CD39, which hydrolyzes AMP to inorganic phosphate. and their ability to block the enzymatic activity of soluble CD39 extracellular domain (ECD) antigen. The enzyme assay was performed in a 96-well multiwell plate in a final volume of 120 μL. The assay mixture was incubated at the indicated incubation time in an incubator set at 37°C. The incubation period was 10 min. The Tris Magnesium (TM) buffer solution contained 25 mM Tris Magnesium. The antibody contained 0.1% BSA, 5 mM MgCl2, and 0.01% BSA. Serial dilutions of the antibody were prepared using soluble human CD39 (Sino Biological, catalog no. The substrate (A) was prepared in TM buffer containing 1000 nM of ATP starting from 20 nM. ATP is used to determine the effect of premixing antibodies and enzymes before addition of ATP. Dilutions were performed either with or without an irrelevant antibody as an isotype control. The antibodies were diluted similarly to test the specificity of antibody-mediated CD39 inhibition. Incubate for 1 hour in an incubator set at 37°C, then add 20 μL of M ATP was added and incubated at 37°C for 1 hour. Enzyme activity was quantified using the CTG luminescent cell viability assay (Promega). Residual ATP was measured using CTG luciferase reagent from the Corporation. The amount was measured.

[0218] The functional activity of anti-CD39 antibodies was assessed using a human myeloid leukemia mononuclear cell line (THP-1). The activity of THP-1 was determined by measuring CD39-catalyzed ATP hydrolysis. Resuspend the cells in TM buffer and add 80 µL of cells per well to a 96-well U-plate (4x). 10 4 Cells were seeded at a density of 100 cells / well, followed by 40 μL / well of antibody-CD39 solution (various Concentrations (5-fold serial dilutions from 100 nM to 1.28 pM in TM buffer) were added to the plate. The antibody and cells were incubated for 1 hour in an incubator set at 37°C. After incubation, ATP (50 μM in TM buffer) was plated at a volume of 80 μL / well. The plate was then placed in an incubator at 37°C for 1 hour. Place in a centrifuge set at 0 rpm for 5 minutes and dispense 50 μL / well of the supernatant into a white 96-well plate. The cells were transferred to a CellTiter-G plate (Corning, Cat. No. 3903). 10 μL / well was added to the corresponding wells and mixed well. The inhibition of the ATP was measured using a multilabel reader (Perkin-Elmer Envision Work Measurements were taken using a rkstation.

[0219] Anti-CD39 antibodies bind specifically to human CD39 but not to other CD39 family members. -Cross-reacts with (CD39-L1, CD39-L2, CD39-L3, CD39-L4) To test whether this antibody does not bind to human CD39-L1 and CD39-L1, Binding to CD39-L2, CD39-L3, and CD39-L4 was determined by ELISA. Then, a 96-well high protein binding ELISA plate (Nunc MaxiSor p, ThermoFisher) was used to identify His-tagged human CD39 cross-family proteins. Proteins (CD39-L1, CD39-L2, CD39-L3, or CD39-L4) Pre-coated with 0.5 μg / mL of carbonate-bicarbonate buffer (20 mM Na The cells were incubated overnight at 4°C in a 1000 mM NaHCO3 solution (180 mM NaHCO3, pH 9.2). Wash plates three times with 300 μL / well of PBS / 0.5% (v / v) Tween-20 Then, the plate was blocked with PBS / 2% BSA for 1 hour. After blocking, the plate was washed three times. After washing, various concentrations (100 nM to 0.36 pM, 6-fold increase in 2% BSA / PBS) were added. Add the test antibody (serial dilutions) to the plate and incubate at room temperature for 2 hours. Our own CD39-L1 binding antibody was used as a positive control, and human IgG4 isoform was used. After washing three times, 100 μL / well of goat anti-human IgG antibody was added. Add the IgG Fc-HRP antibody (1:5000) and place the plate in an incubator at room temperature. The plates were placed in an incubator for 1 hour. For color development, 100 μL / well of TMB substrate solution was added. After 3-5 minutes, the reaction was stopped with 100 μL / well of 2 M HCl. Absorbance was measured using a SpectraMax® M5e microplate spectrophotometer. The EC was read at 450 nm. 50 Values ​​were calculated using GraphPad Prism software. The data were obtained from a four-parameter nonlinear regression analysis using the software.

[0220] Hybridoma clone 19 and hybridoma clone 3 containing rat Fc domain Antibodies derived from hybridoma clone 1 and hybridoma clone 39 were purified from hybridoma supernatants. These antibodies are referred to herein as antibodies 19, 31, and 39, respectively. The resulting hybridoma supernatant was adjusted to pH 7.0 and then loaded onto a Protein A column. The combined antibody was eluted with glycine-HCl (pH 3.5) and immediately washed with 0.1 M Tris (pH The protein was dialyzed against PBS and filtered through a 0.22 μm membrane. The antibody concentration was measured using Nano Drop. The purity of the antibody was measured using SD The results were evaluated by S-PAGE and HPLC-SEC.

[0221] Total RNA was extracted using the RNeasy Plus Mini Kit (Qiagen). First strand cDNA was isolated from hybridoma clones 19, 31, and 39. The VH and VL genes of the antibody were reverse transcribed using 3'-constant region degenerate primers. The cDNA was amplified using a 5'-degenerate primer set. A PCR product was then designed based on the upstream signal sequence coding region of the Ig variable sequence. The product was ligated into the pMD18-T vector, and 10 μL of the ligation product was transferred to the To The transformed cells were cultured in 2xYT medium containing carboxime. Plate on a plate and incubate overnight in an incubator set at 37°C. Twelve positive colonies were selected for DNA sequencing by Biosune. Selected at random.

[0222] The cloned genes were codon-optimized for mammalian expression and then delivered to GENEWIZ (Su The resulting plasmid was synthesized by the company Sigma-Aldrich Co., Ltd. (Dongzhou, China). 1, in which the hinge, CH2, and CH3 segments (including the hinge mutation S228P) were fused A gene encoding a heavy chain containing the VH domain of antibody 19, 31, or 39, or a light chain A gene encoding a light chain containing a VL domain fused to the human Ig kappa CK domain of the chain It included:

[0223] Antibodies ch19_IGG4.P, ch31_IGG4.P, and ch39_IGG4.P To produce the vector, a plasmid containing the VH and VL genes described above was used as Expi293F. For each antibody, cells were co-transfected with 40 mL of cell culture medium to obtain >95% with a survival rate of 2.94x10 6 Expi293F cells were prepared at 1 μg / mL. Plasmid DNA and ExpiFectamine™ 293 were added to a final concentration of 1 / mL. The transfection reagent was mixed and then added to the cell culture medium. The cell culture was incubated at a rotation speed of Incubated in a platform shaker at 150 rpm. Temperature: 37°C. The cells were maintained in an incubator set at a temperature of 8% and a CO2 level of 8%. After incubation, pellet the cells using centrifugation at 4000 rpm at 25 °C for 10 min. The supernatant was collected and analyzed by gel electrophoresis, SPR, etc. off Ranking and refinement The supernatant was then analyzed by NuPAGE™ 4-12% Bis-Tris protein gel (Th The gels were loaded onto SDS-PAGE gels according to the manufacturer's instructions (Fermo Fisher). ageRuler™ Unstained Protein Ladder (Thermo Fisher) was used as an anti- Used with body samples to determine the molecular weight of the antibody.

[0224] To purify the antibody, a Protein A column was loaded onto a 1 mL MabSelect Sure The resin was pre-packed. The column was then loaded with 5 column volumes (CV) of equilibration buffer (0.1 M Tris The column was equilibrated with ethanol (pH 7.0) and then loaded with cell culture medium. After loading, the column was Wash with 8 CV of 0.1 M Tris (pH 7.0), followed by 8 CV of 0.1 M glycine (pH 3 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 analyzed at -80 Stored at ℃.

[0225] Using FACS analysis, the above-mentioned human CD39-overexpressing CHO-K1 cells and Recombinant anti-CD3 transfection into HEK293 cells overexpressing monkey or mouse CD39 The binding of nine antibodies was measured. 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 show that it potently inhibits the enzymatic activity of cellular human CD39.

[0226] The kinetic binding of these recombinant anti-CD39 antibodies to human CD39 was analyzed by Biacore The recombinant antibodies were determined by surface plasmon resonance (SPR) using an 8K instrument. , goat anti-human FcIgG coated CM5 sensor chip (GE, Cat. No. 29-1 496-03) and soluble His-tagged CD39 ECD protein. SEQ ID NO: 72 (Sino Biological, Catalog No. 16020-H08B) was used as the analyte. The binding constants were determined at 25°C. As shown in Table 2, the three antibodies All of the molecules have a slow off-rate (k d ) and sub-nanomolar average 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 technology. (Queen et al, Proc. Natl. Acad. Sci. U SA. 86:10029-10033, 1989). Rat variants of antibodies 31 and 39 The heavy chain (VH) and variable light chain (VL) sequences were used to identify the closest human germline region for each chain. The human acceptors for the VH and VL frameworks were identified using the GenBank database. The search was performed within the Nucleic Acids Res. 2005,33,D34-D38). The framework is composed of Kabat CDR1 at the N-terminus. The top three hits were defined using the extended CDR definition, which was extended by 5 amino acids. The sequences of the humanized VH and VL genes are derived from each of these using Two were selected for the expression of humanized antibodies. For antibody 31VH, 64.6% IGHV4-24*01 has sequence identity with IGHV1, which has 63.4% identity. -69-2*01 was selected, and for VL, it has 84.8% sequence identity with IGKV IGKV4-1*01, which has 77.2% identity with 7-3*01, was selected (Table 3). The antibody 39VH has 87.8% sequence identity with IGHV3-9*01 and IGHV3-30*15, which has 85.4% identity with the VL, was selected. IGKV7-3*01, which has 0.5% sequence identity, and IGKV7-3*01, which has 72.2% identity. KV3-15*01 was selected (Table 4).

[0230] [Table 3]

[0231] [Table 4]

[0232] CDR grafting was performed for each human acceptor. For antibody 31: As VH acceptors, HC-CDR1 (SEQ ID NO: 18) and HC-CDR2 (SEQ ID NO: 1 9), and HC-CDR3 (SEQ ID NO: 20) and LC-C as the VL acceptor. LC-CDR1 (SEQ ID NO: 22), LC-CDR2 (SEQ ID NO: 25), and LC-CDR3 (SEQ ID NO: For antibody 39, HC-CDR1 ( SEQ ID NO: 28), HC-CDR2 (SEQ ID NO: 29), and HC-CDR3 (SEQ ID NO: 30 ) was used, and LC-CDR1 (SEQ ID NO: 32) and LC-CDR2 were used as VL acceptors. (SEQ ID NO: 33), and LC-CDR3 (SEQ ID NO: 34) were used. The resulting sequences were Any potential post-translational modifications (PTMs) such as isomerization, deamination, glycosylation, and oxidation The putative residues suitable for backmutation in the rat sequence were also examined in the antibody homology graph. After removal of the PTM site, the antibody was assayed and It was determined whether the change(s) affected antigen binding compared to the parent antibody.

[0233] The humanized genes are reverse translated, codon optimized for mammalian expression, and manufactured by GENEWIZ. The synthetic gene was recloned into our own IgG expression vector, expressed, and purified. did.

[0234] The binding kinetics of purified antibodies to antigens was determined using surface plasmon resonance (SPR). and used to rank the antibodies. Human CD39 ECD His-tagged antigen The affinity of anti-CD39 antibodies to (SEQ ID NO: 72) was determined using a Biacore 8K instrument. The activator was 400 mM EDC and 100 mM NHS (GE ) and a CM5 sensor chip (GE, catalog number 29- 1496-03) was activated with an activator for 420 seconds. IgG (30 μg / mL in 10 mM NaAc, pH 4.5) at a flow rate of 10 μL / min for 4 h. The channel was injected for 20 seconds. The chip was deactivated with 1 M ethanolamine hydrochloride. Anti-CD39 antibody was diluted to 4 μg / mL in electrophoresis buffer (1x HBS-EP+) and Six concentrations (800 μL / min) of CD39 antigen analyte were injected into the channel for 15 seconds at a flow rate of 0 μL / min. , 4, 2, 1, 0.5, 0.25 nM) for a 180 s binding phase followed by a 2400 s During the dissociation phase, the channels were sequentially injected with HCl at a flow rate of 30 μL / min. Glycine (10 mM, pH 1.5) was injected following the dissociation phase. The sensorgram of the liquid channel was subtracted from the test sensorgram. The experimental data were: The binding model was fitted with a steady-state affinity / heterogeneous ligand of 1. The molecular weights of the CD39 antigen and anti-CD39 antibody were 52 kDa and 145 kDa, respectively. there were.

[0235] Humanized variant of antibody 39: ch39_IGG4.P to maintain binding affinity A K94R rat sequence back mutation was introduced into the VH1 chain (SEQ ID NO: 58), but R94 (SEQ ID NO: 59) No equivalent back mutation was required in VH3 because VL1 (number 59) was present. The human framework also contains glycosylation PTM sites ( 81 NDT 83 ) was introduced, and this was removed by introducing an additional rat sequence reversion mutation, N81D (SEQ ID NO: 61). For VL3 (SEQ ID NO: 62), no backmutation of the rat sequence was required.

[0236] Next, for the 39 humanized variants, VH1 (SEQ ID NO: 58) or VH3 (SEQ ID NO: 59) were analyzed. No. 59) and VL1 (SEQ ID NO: 61) or VL3 (SEQ ID NO: 62) All VL combinations were expressed, but the VH1+VL3 pair was not expressed sufficiently. The mutant gene was codon-optimized for mammalian expression and then purified by GENEWIZ. Synthesized: VH domain of human IgG4 heavy chain CH1, hinge, CH2, and CH3 segments A plasmid containing VH fused to menthol (containing hinge mutation S228P); and VL A plasmid containing the VL domain fused to the human Ig kappa CK domain of the light chain.

[0237] The humanized IgG4(S228P) variant of antibody 39 was synthesized using the chimeric rVH+r Together with the VL / human IgG4(S228P) variant, the IgG4A variant was analyzed by SPR as described above. The results are shown in Table 5.

[0238] [Table 5]

[0239] An additional rat sequence back mutation (I2T) was added to the VL1 framework (SEQ ID NO: 60) and V The antibody was expressed and analyzed. The VL3 pair did not express well. Table 6 shows the SPR data for these antibody variants. show.

[0240] [Table 6]

[0241] The humanized variant of antibody 39 exhibits high affinity for cellular human CD39 and It potently inhibits the enzymatic activity of 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: E7 1S, D76N, and A78V (SEQ ID NO: 43). Also, binding of ch31_IGG4.P To maintain affinity, the VH2 chain (SEQ ID NO: 44) was modified with D76N and A78V. Antibody 31 contained a glycosylation PTM site sequence ( 26 N QT 28 ) in VL1, which has Q27P (SEQ ID NO: 45) or N26Q (SEQ ID NO: 46). In VL1, the VL2 was mutated to either N26Q (SEQ ID NO: 46) or N26Q (SEQ ID NO: 49). The human framework of 1 also contains glycosylation PTM sites ( 81 NDT 83 ) was introduced, These contain the additional rat sequence reversion mutation N81D (SEQ ID NO: 47 and SEQ ID NO: 48, respectively). For VL2, backmutation of the rat sequence was not necessary. It was.

[0244] For the humanized variant of antibody 31, VH1 (SEQ ID NO: 43) or VH2 (SEQ ID NO: No. 44) and VL1 (SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, or SEQ ID NO: 48) or VL2 (SEQ ID NO: 49), all four VH / VL combinations were expressed, but V The H1+VL2 pair was not well expressed. The variant genes were coded for mammalian expression. Human IgG heavy chain VH domain was optimized and then synthesized by GENEWIZ. 4. Plasmids containing VH fused to CH1, hinge, CH2, and CH3 segments (including hinge mutation S228P); and the VL domain is a light chain human Ig kappa CK domain. A plasmid containing a VL fused to a nucleotide.

[0245] The humanized IgG4(S228P) variant of antibody 31 was synthesized using the chimeric rVH+r Together with the VL / human IgG4(S228P) variant, the IgG4A variant was analyzed by SPR as described above. The results are shown in Table 8.

[0246] [Table 8]

[0247] The humanized variant of antibody 31 exhibits high affinity for cellular human CD39 and It potently inhibits the enzymatic activity of human CD39 (Table 9).

[0248] [Table 9]

[0249] For both antibody 31 and antibody 39, CDR grafting and subsequent humanization were performed. A small number of rat sequence backmutations into the variable domain framework yielded humanized variants. These antibodies retained the binding affinity and inhibitory potency of the respective rat / human chimeric antibodies. maintained or improved.

[0250] Example 3: Characterization of complete kinetic binding by SPR hu31.4_IGG4.P and hu39.5_I codon-optimized for mammalian expression The VH and VL domain genes of the GG4.P humanized antibody were used to generate a human IgG1 barrier The VH domain contained the L234A and L235A (Eu numbering) mutations. The heavy chain (HC) of human IgG1 contains CH1, hinge, CH2, and CH3 constant domain segments. A plasmid containing a VH domain fused to a nucleotide sequence (SEQ ID NO: 2), and a VL domain The VL comprises a light chain (LC) fused to a human Ig kappa CK constant domain (SEQ ID NO: 6). The plasmid carrying the antibody hu31.4_I was transfected into CHO-K1 cells. GG1.AA (HC is SEQ ID NO: 51, LC is SEQ ID NO: 56) and hu39.5_IGG1 AA (HC is SEQ ID NO: 66, LC is SEQ ID NO: 67) using a rapid stable pooling protocol. The resulting product was expressed using a protein A affinity chromatography followed by size exclusion chromatography. Purity >99% was confirmed by SEC-HPLC analysis. It was purified to 5%.

[0251] Antibodies hu31.4_IGG1.AA and hu39.5_IGG1.AA were purchased from Biosciences. The kinetic binding was analyzed by SPR using a 2000 instrument. In the "monovalent affinity mode," the antibody was coated with amine-conjugated goat anti-human IgG. The CM3 sensor chip (GE, catalog number 29-1049-90) was used. Test antibodies were captured at seven different densities using soluble human CD39 ECD His tag. The antigen (Sino Biological, Cat. No. 16020-H08B) was identified. The target of analysis was a stock solution prepared at 33.3 μM diluted to 33 nM as the highest concentration. The antibody surface was tested in triplicate at 3-fold dilutions. Soluble human CD39 ECD His-tagged antigen was applied to the CM3 sensor at six different densities. Test antibodies against the analytes were amine-coupled to the chip. The highest concentration was 100 nM, and the antibody was applied to the CD39 surface in triplicate in 3-fold dilutions. The running buffer contained 10 mM HEPES, 150 mM NaCl, 0.0 5% Tween-20 and 0.2 mg / mL BSA were included. Single cycle kinetics with multiple injections ) and the dissociation phase was monitored for 1 hour. All data were collected at 25°C. The response data from all six or seven surface densities indicated the maximum amount of complex formed. The local Rmax, which is the response to The results of experiments performed in monovalent affinity mode are shown in Table 10, and those in bivalent avidity mode are shown in Table 10. The results of the single mode are shown in Table 11.

[0252] The antibodies hu31.4_IGG1.AA and hu39.5_IGG1.AA were used for the sensor chip. Monovalent affinity chromatography was performed using antibodies captured on a capillary plate and soluble CD39 ECD as the analyte. When analyzed in the ion exchange mode, similar equilibrium dissociation constants (K D However, the sensor chip Bivalent antibody binding was determined using CD39 ECD captured on a plate and soluble antibody as the analyte. When analyzed in a mode that allows for the detection of hu31.4_IGG1.AA, the antigen surface density is Regardless, K values ​​very similar to those obtained when analyzed in monovalent mode were obtained. D showed. In contrast, hu39.5_IGG1.AA exhibits up to three orders of magnitude lower K depending on antigen surface density. D Show This is because the antibody binds to multiple immobilized CD39 molecules on the chip surface in a bivalent mode. Therefore, avidity can improve kinetic binding affinity. This indicates that

[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 binds to primary human monocytes (CD14 + )of Binding to human CD39 expressed on the cell surface was tested. using the D14 Positive Selection Kit II (Stem Cell, Cat. No. 17858) Monocytes were positively selected from peripheral blood (leukopak or LRS chamber) and then cultured in a Bam and freeze in an anchor (Wako, Cat. No. 302-14681) for future use. On the day of the assay, frozen monocytes were thawed, rinsed, and stored in liquid nitrogen for 24 h in DPBS. 1:25 Fc Block (BD, Cat. No. 564220) and 1:250 Aqu aResuspended in viability dye (Invitrogen, Cat. No. L34966A) Incubate in the dark at room temperature for 15 minutes. Polypropylene v-bottom 96-well plate 0.2-0.5 x 10 cells per well 6 Cells were plated with zero or Add 400 μM ATP and incubate at 4° C. for 30 minutes, followed by addition of test antibody. After further incubation at 4°C for 30 minutes, the plate was washed twice with DPBS and then incubated for antibody detection. 1 μg / mL of PE-labeled mouse anti-human IgG4 secondary 4 secondary) (Southern Biotech, Catalog No. 920 After washing twice with DPBS, the cells were incubated with 50 μL of IC fixation solution. The mixture was incubated at room temperature for 20 minutes in constant buffer (Invitrogen, Cat. No. 00-8222-49). The cells were then washed once, resuspended in DPBS, and fixed in BD LSR FORT Subjected to flow cytometry using an ESSA X-20 Cell Analyzer The geometric mean of PE fluorescence intensity was obtained for the live single-cell population. Using standard 4-parameter curve fitting in Prism, EC 50 Calculate the value (Table 12).

[0257] [Table 12]

[0258] PBMC binding: Recombinant and IgG1 isotype variants of antibody 39 also bind to flow Cytometry revealed that CD14 + (monocytes) and CD19 + Gated on the (B cell) population Binding to human CD39 expressed on the cell surface of PBMC was tested. It has been reported that huBMK2_IGG1.A binds to and inhibits human CD39. An antibody called EASS was used as a comparison. huBMK2_IGG1.AEA The amino acid sequence of the mature heavy chain of SS is SEQ ID NO: 73. huBMK2_IGG1.AE The amino acid sequence of the mature light chain of ASS is SEQ ID NO: 74. Previously purified and frozen PBM Thaw, rinse, and add 1:25 Fc block (BD, Cat. No. 564) in DPBS. 220) and 1:250 Aqua Viability Dye (Invitrogen, catalog no. The cells were resuspended in PBS (product number L34966A) and incubated in the dark at room temperature for 15 minutes. The cells were washed with DPBS and resuspended in filtered staining buffer: 5% goat serum (Gibco, catalog no. The cells were resuspended in DPBS supplemented with 16210-064. Plate 0.5 x 10 cells per well in staining buffer. 6 Plating with cells Zero or 400 μM ATP was added, and the mixture was incubated at 4°C for 30 minutes. Test antibodies were added and incubated for another 30 minutes at 4°C. After washing twice with staining buffer, , 0.8 μg / test CD14 FITC (Invitrogen, Cat. No. 11-0 149-42, clone 61D3) and 0.5 μg / test CD19 eFluor 45 0 (eBioscience, Catalog No. 48-0199-42, Clone HIB19 ) was added to PBMCs. CD39 binding by test antibodies was assessed using 0.5 μg / mL PE-labeled Goat anti-human IgG (Southern Biotech, catalog number 2048-09) The cells were then washed, resuspended in DPBS, and incubated with BD Flow cytometry was performed using the LSR FORTESSA X-20 Cell Analyzer. The geometric mean of the PE fluorescence intensity was determined as the percentage of monocytes (CD14 + ) or B cells (CD19 + ) were obtained for live single-cell populations gated on either Grap Using standard 4-parameter curve fitting in hPad Prism, EC 50 value was calculated (Table 13). A representative graph from one donor is shown in Figure 1.

[0259] [Table 13]

[0260] Overall, variants of antibodies 19, 31, and 39 exhibited strong binding to the surface of primary cells. Efficacy was determined when 39 variants > 31 variants > 19 variants The potency ranking of variants 31 and 39 was from IGG4.P to IGG1.A. Antibody 3 maintained its potency upon isotype switching to A and maintained its potency in the presence of high ATP. 1, a humanized IgG1 variant of antibody 39, hu31.4_IGG1.AA, and The humanized IgG1 variant, hu39.5_IGG1.AA, is a humanized IgG1 variant of the previously described anti-CD39 The antibody showed stronger binding than the hu39.5_IGG1.AA antibody, and the hu39.5_IGG1.AA antibody showed stronger binding than the huBMK2_IG antibody. G1.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 observed in primary human cells derived from patient tumor samples or human cell lines expressed on the cell surface. Inhibition was assessed using soluble recombinant CD39 and CD39. The assay was performed in the presence of high ATP (20 μM and 400 μM, respectively).

[0262] Recombinant CD39 Inhibition: The efficacy of the antibodies described in the previous examples in inhibiting CD39 enzymatic activity. The potency of recombinant human CD39 was first assessed using recombinant human CD39 biochemically. Assays were performed in 25 mM Tris-HCl, pH 7.4, 5 mM MgCl, and 0.0 Assays were performed in assay buffer consisting of 1% BSA. 14-point, 1:3 grid of test antibodies. Dilute serially in assay buffer to cover a final concentration range of 200 nM to 0.125 pM. Five microliters of test antibody at 5x final concentration in assay buffer was added to 3 Ten microliters resuspended in assay buffer were added to each well of an 84-well plate. A 1.56 nM solution of recombinant human CD39 was added to each well, and the plate was incubated at 37°C. After a further 60 min incubation, 10 μL of 50 μM ATP in assay buffer was added. The final assay conditions were 0.625 nM recombinant human CD39 and 20 μM A After a further 30 min incubation at 37 °C, 25 μL of TP substrate was added. Kinase Glo Plus reagent was added to each well of a 384-well plate. The Nase-Glo Reagent Kit (Promega, Cat. No. V3771) was used to prepare The amount of ATP remaining after CD39 inhibition was measured using a luminescence filter. Using the Envision 2102 Multilabel reader with CD39 enzyme activity was assessed and quantified as a function of the luminescence produced. The percentage of maximum activity in each test well was calculated as a function of activity. Assay buffer (maximum activity, 0% inhibition) and control wells without enzyme (baseline activity, 1 The IC of the test antibody was calculated based on the 100% inhibition. 50 The values ​​are given by the standard four-parameter fitting equation The data were determined from dose-response curves fitted using the .Data are shown in Table 14.

[0263] [Table 14]

[0264] SK-MEL-5 CD39 inhibition: Inhibition of CD39 enzyme activity was also observed in patient-derived tumor samples. The evaluation was carried out using SK-MEL-5 cells, a melanoma cell line established by the University of Tokyo. Thaw one vial of pre-frozen SK-MEL-5 cells and add 20 mM HEPE S, pH7.4, 137mM NaCl, 5.4mM KCl, 1.3mM CaCl2 10 mL of assay buffer consisting of 4.2 mM NaHCO3, and 0.1% glucose The cells were resuspended in a 14-point, 1:3 master serial dilution of anti-CD39 antibody. The final concentrations ranged from 100 nM to 0.063 pM in assay buffer. Twenty microliters of test antibody at 5x final concentration in assay buffer was added to a 96-well round-bottom plate. Forty microliters of SK-MEL-5 solution was added to each well of the polypropylene plate. Cells were added to each well and the plate was incubated at 37°C for an additional 60 minutes before assay. 40 μL of 50 μM ATP in buffer was added. The final assay conditions were: The incubation consisted of 10,000 cells and 20 μM ATP substrate at 37°C and 5% CO After an additional 60 min incubation at 2°C, the plate was centrifuged at 225 x g for 10 min. Thirty microliters of the supernatant was placed in a 96-well assay plate (Corning, Cat. Log number 3912) and prepare the Kinase-Glo Reagent Kit according to the manufacturer's protocol. (Promega, Cat. No. V3771) to determine the amount of ATP remaining in the reaction. The amount of ATP remaining after CD39 inhibition was measured using EnviroLab equipped with an emission filter. Function of generated luminescence using the sion 2102 Multilabel reader CD39 enzyme activity was assessed and quantified as a function of residual ATP levels. The percentage of maximum activity in each test well was calculated using assay buffer (maximum activity The calculations were based on the activity (0% inhibition) and cell-free control wells (baseline activity, 100% inhibition). The IC of the test antibody was calculated. 50 Values ​​were fitted using the standard four-parameter fitting equation The data were determined from dose-response curves in which the agonist was administered intravenously. The data are shown in Table 15.

[0265] [Table 15]

[0266] THP-1 CD39 Inhibition: Inhibition of CD39 enzymatic activity was performed on a second tumor cell line, specifically The effect of thrombin time on the leukemia-associated leukemia was evaluated using a human monocytic cell line derived from a patient with acute monocytic leukemia. Thaw one vial of lysed THP-1 cells and resuspend in 20 mM HEPES, pH 7.4, 137mM NaCl, 5.4mM KCl, 1.3mM CaCl2, 4.2mM N Resuspend the cells in 10 mL of assay buffer consisting of aHCO3, HCl, and 0.1% glucose. A 14-point, 1:3 master serial dilution of anti-CD39 antibody was prepared with a final concentration range of 1 The assay buffer was prepared to range from 0.00 nM to 0.063 pM. Twenty microliters of test antibody at 5x final concentration was added to a 96-well round-bottom polypropylene plate. Forty microliters of THP-1 cells were added to each well. The plates were incubated for an additional 60 min at 37 °C, then 40 μL of the plate in assay buffer was added. 50 μM ATP was added. The final assay conditions were 40,000 cells per well, and The incubation consisted of 20 μM ATP substrate. The incubation was continued for an additional 60 min at 37°C and 5% CO2. After incubation, the plate was centrifuged at 225 x g for 10 minutes. The supernatant was transferred to a 96-well assay plate (Corning, Cat. No. 3912). and Kinase-Glo Reagent Kit (Promega, Cat. No. 100144) according to the manufacturer's protocol. The amount of ATP remaining in the reaction was measured using a log number V3771. The amount of ATP remaining after the assay was measured using an Envision 2102 M equipped with a luminescence filter. was assessed and quantified as a function of the luminescence produced using an Ultilabel reader CD39 enzyme activity was assessed as a function of the remaining ATP levels in each test well. The percentage of maximal activity in the assay buffer (maximal activity, 0% inhibition) and in the absence of cell The IC of the test antibody was calculated based on cell control wells (baseline activity, 100% inhibition). 50Values ​​were obtained from dose-response curves fitted using a standard four-parameter fitting equation. The data are shown in Table 16.

[0267] [Table 16]

[0268] Purified monocyte CD39 inhibition: Potency was further evaluated using primary human monocytes. Pre-frozen CD14 + Two biopsies of positively selected human monocyte (donor 1003773) cells Thaw Al, thaw 20 mM HEPES, pH 7.4, 137 mM NaCl, 5.4 mM KCl, 1.3 mM CaCl2, 4.2 mM NaHCO3, and 0.1% The cells were resuspended in 10 ml of assay buffer consisting of glucose. nt, 1:3 master serial dilutions were made to cover the final concentration range of 200 nM to 0.13 pM. Prepared in assay buffer at 5x the final concentration in assay buffer, 20 microliters of test antibody was added to each well of a 96-well round-bottom polypropylene plate. A small number of human monocyte cells were added to each well and the plate was incubated at 37°C for an additional 60 minutes. After incubation, 40 μL of 50 μM or 1000 μM ATP in assay buffer was added. The final assay conditions were 20,000 cells per well and 20 μM or 40 μM of IgG. The plate consisted of 0 μM ATP substrate. The plate was incubated for an additional 60 min at 37°C and 5% CO2. After bating, the plate was centrifuged at 225 x g for 10 minutes. The supernatant was transferred to a 96-well assay plate (Corning, Cat. No. 3912). AMP-Glo ​​reagent kit (Promega, catalog no. 5) was prepared according to the manufacture's protocol. The amount of AMP produced in the reaction was measured using 400 μM ATP substrate. The supernatant from the reaction plate containing AMP-G was transferred after dilution 1:8 with assay buffer. The amount of AMP produced after CD39 inhibition was measured using an emission filter. Generated using the Envision 2102 Multilabel reader CD39 enzyme activity was assessed and quantified as a function of luminescence. The percentage of maximum activity in each test well was determined by adding 100 mg of the assay buffer (maximum activity, 0% inhibition) and cell-free control wells (baseline activity, 100% inhibition). The IC of the test antibody was calculated. 50 The values ​​were fitted using a standard four-parameter fitting equation. The dose-response curves were determined using a 100-mg / mL ethanol-containing 100 mg ...

[0269] [Table 17]

[0270] [Table 18]

[0271] Overall, the variants of antibodies 19, 31, and 39 bind to soluble and surface human CD39 enzymes. These results demonstrated potent inhibition of the enzyme's activity. Importantly, the potency was maintained in the presence of high ATP.

[0272] Example 6: Immunostimulatory effects of CD39 blockade To evaluate the effectiveness of the anti-CD39 antibodies produced in Examples 1 to 3, the surface of bone marrow cells was analyzed. The downstream functional consequences of inhibiting CD39 enzymatic activity were assessed in vitro.

[0273] Macrophage IL-1β / IL-18 release assay: NLRP3 inflammasome , a multiprotein cytosolic complex that oligomerizes to procaspase-1 proteins The protein is cleaved and activated, allowing it to become caspase 1. -1 cleaves their inactive pro-isomers (proIL-1β and proIL-18) promotes the maturation of IL-1β and IL-18 by converting them into their active and secreted forms. These cytokines are involved in the innate immune response and create a general pro-inflammatory environment. Put out.

[0274] Anti-CD39 antibody was used to measure the secretion of mature IL-1β and IL-18. We tested the ability of NLRP3-inflammasome-activating phage markers on phage cultures. A schematic diagram of this process is shown in Figure 3. On day 0, previously isolated and positively selected CD14 + Monocytes (See Example 4) was thawed and mixed with 10% heat-inactivated FBS, 1% Glutamax, 1% penicillin / streptomycin, and 50 ng / mL recombinant human M-CSF ( R&D, Cat. No. 216MCC / CF) in RPMI medium. (Thermo Scientific Nunclon Delta Surface Approximately 15 x 10 per (Cat. No. 150464) 6 CD14 + Culture at a density of monocytes On day 5, non-adherent cells were washed away, and adherent macrophages were collected by scraping and then incubated for 2 h. 0.125 x 10 cells per well in a 4-well plate 6 The cells were plated at a density of 1000 cells / well. The cells were incubated overnight to allow adhesion. The next day, the cells were treated with 1 ng / mL LPS (I nvivoGen, Catalog No. tlrl-peklps), followed by 10 or The cells were treated with 100 nM of test antibody or IgG control and incubated at 37°C, 5% CO2 for 3 hours. The cells were then incubated with 500 μM ATP (Life Technologies (Cat. No. R0441) and incubated at 37°C, 5% CO2 for an additional 2-4 hours. The supernatant was collected and IL-1β levels were measured using CBA (B D, Catalog No. 558279) or ELISA (R&D, Catalog No. QK201) IL-18 levels in the supernatants were analyzed by ELISA ( R&D, Cat. No. DL180). Experiments were performed in technical quadruplicate for each donor. The results were run at 100 bp, averaged, and then normalized to the isotype control for each donor. As shown, ch39_IGG4.P, hu39.1_IGG4.P, hu39.5_IG G1.AA and hu31.4_IGG1.AA showed significant improvement compared to the isotype control. Increased the release of IL-1β and IL-18 from in vitro-derived macrophages.

[0275] Monocyte-derived dendritic cell assay: Antibodies were incubated with monocyte-derived dendritic cells (moDCs) in the presence of ATP. On day 0, positively selected monocytes were incubated with 10% heat-inactivated monocytes in 10% PBS. Activated FBS, 1% Glutamax, 1% penicillin / streptomycin, 100n g / mL recombinant human GM-CSF (R&D, Cat. No. 215-GM / CF), and and 100 ng / mL IL-4 (Peprotech, Cat. No. 200-04). Resuspend in RPMI filled with 100 mM MgCl and plate in a 6-well plate (Falcon, Cat. No. 35304 6) 4 x 10 cells per well 6On day 6, moDCs were harvested and plated in 24 well plates. Upcell plates (Thermo Fisher, catalog number 174899) , 0.5 x 10 per well 6 Cells were seeded at 10 μg / mL and treated with 10 μg / mL isotype control or After treatment with anti-CD39 antibody at 37°C and 5% CO2 for 1 hour, the cells were incubated at 0 μM or 300 μM. The cells were treated with 1 μM ATP for 18 hours at 37°C and 5% CO2. On the 7th day, the cells were cultured in a medium for staining. DCs were transferred to a polypropylene v-bottom 96-well plate. All wells were filled with DPBS 1:25 Fc Block (BD, Cat. No. 564220) and 1:250 Aqu aResuspended in viability dye (Invitrogen, Cat. No. L34966A) The cells were incubated in the dark at room temperature for 15 minutes. After washing once with DPBS, the cells were Antibodies [1 μg / test CD83 PE-Cy7 (BD, Cat. No. 561132), 1 μ g / test CD86 BV605 (BD, Catalog No. 562999), 0.8 μg / test Experimental CD14 FITC (Invitrogen, Cat. No. 11-0149-42)] , or the corresponding isotype control [PE-Cy7 mouse IgG1 κ (BD, catalog No. 565573), BV605 Mouse IgG1 κ (BD, Cat. No. 562652 ), FITC mouse IgG1 κ (eBiosciences, Cat. No. 11-47 14-41)] for 30 minutes at 4°C. Surface levels of 86 should increase with dendritic cell maturation, whereas monocyte markers After one wash with DPBS, cells were incubated in 50 μL of IC Fixation buffer (Invitrogen, Cat. No. 00-8222-49) at room temperature for 2 The cells were then washed, resuspended in DPBS, and resuspended in BD LSR FORTE Flow cytometry was performed using an SSA X-20 Cell Analyzer. For CD83 and CD14, the parental group was detected for each donor under the 0 μM ATP condition. The percentage of events was set to the isotype control (<5% of total events). In the control treatment condition, >90% of the cells were positive compared to the isotype control, indicating that Instead, the geometric mean of the fluorescence intensity of the fluorophore was reported for a population of live single cells. .

[0276] As shown in Figures 5A and 5B, the increase in dendritic cell maturation markers CD83 and CD86 As can be seen, ATP caused moDC maturation, which did not occur with the addition of an isotype control. However, inhibition of CD39 enzyme activity by addition of anti-CD39 antibody promoted maturation, which The percentage of cells expressing CD83 and the surface amount of CD86 per cell were significantly higher. The addition of anti-CD39 in the presence of ATP resulted in the expression of the monocyte marker CD14. The percentage of cells with IgG4 also decreased (Fig. 5C). The effects on CD83, CD86, and CD14 were ATP-dependent.

[0277] Overall, the above data demonstrate that enzymatic inhibition of human CD39 by the antibodies of the present disclosure is These results suggest that IL-1 has immunostimulatory effects on different myeloid populations in vitro. Example 7: Antibody characterization by competitive flow

[0278] In this example, two antibodies (a test antibody and a reference antibody) were tested against human IgG1 expressed on the surface of cells. A competitive flow assay useful for determining whether cells compete for binding to CD39 The selection of test antibody, reference antibody and cell type can be varied.

[0279] Antibody hu39.1_IGG4.P and antibody IGG4.P, IgG4(S228P) A type control (CrownVivo) was used as a control for AlexaFluor647 conjugation. Link Kit - Lightning-Link (Abcam, Cat. No. ab269823 ) directly conjugated to Alexa Fluor 647 ("AF647"). The conjugated antibody was designated "hu39.1_IGG4. The binding was verified using human multiple myeloma antigens. The assay was performed using MOLP-8, a myeloma, CD39-expressing cell line. Thaw frozen MOLP-8 cells, rinse, and incubate in 1:25 Fc block (BD, Catalog No. 564220) and 1:250 Aqua Viability Dye (Invitro Resuspend in 100% Fibroblast Growth Regulator (Gen, Catalog No. L34966A) and incubate at room temperature in the dark for 15 minutes. Cells were plated in a polypropylene v-bottom 96-well plate at 0.2x per well. 10 6 An 11-point dose-response curve was performed for each antibody. Concentrations ranging from ∼0.0141 nM were prepared by three-fold serial dilutions in DPBS. The 12th point contained only DPBS and no antibody. The diluted antibodies were added to the plated cells at a 1:1 ratio and incubated at 4°C for 30 minutes. The cells were then washed three times, resuspended in DPBS, and incubated with BD LSR FORT Subjected to flow cytometry using an ESSA X-20 Cell Analyzer The mean fluorescence intensity of AF647 was obtained for the live single-cell population. 50 Value and EC 95 Values ​​were calculated using standard 4-parameter nonlinear regression analysis in GraphPad Prism software. was calculated using the analysis.

[0280] Antibody hu39.1_IGG4.P-AF647 was then incubated with an unlabeled test antibody (hu31. 1_IGG1.AA, hu39.1_IGG4.P, hu39.5_IGG1.AA, I GG1.AA isotype control, and IGG4.P isotype control (CrownViv o)) was used as a reference antibody in a competitive flow assay. Thaw P-8 cells, rinse, and incubate in 1:25 Fc block (BD, catalog no. 10001) in DPBS. No. 564220) and 1:250 Aqua Viability Dye (Invitrogen, The cells were resuspended in PBS (catalog number L34966A) and incubated in the dark at room temperature for 15 minutes. An 11-point dose-response curve was performed for each test antibody at concentrations ranging from 833 to 0.0141 nM. The 12th point was prepared by serial 3-fold dilution with DPBS. Each well of a 96-well staining plate contained only S and no antibody. 1 μL of the reference antibody hu39.1_IGG4.P-AF647 was added at a concentration of 93.7 nM. 12.5 μL was added to each well and mixed gently three times. Cells were diluted at 0.2× per well. 10 6 The cells were plated and incubated at 4°C for 60 minutes. The cells were then washed three times. Wash and resuspend in DPBS and use BD LSR FORTESSA X-20 Cells Flow cytometry was performed using an analyzer. Mean fluorescence intensity of AF647 was obtained for live single cell populations. 50 The values ​​were calculated using GraphPad Prism software. The variance was calculated using Ware's standard 4-parameter nonlinear regression analysis using the following equation: Maximum competition (%) was calculated: Maximum competition = 1 - (MFI at highest concentration of antibody tested / hu39 .1_IGG4.P-AF647 alone at matched concentrations). Reference antibody hu39.1_ Only a single concentration of IGG4.P-AF647 was used.

[0281] As shown in Figure 6 and Table 19, the test antibody hu39.5_IGG1.AA was significantly higher than the reference antibody h It competes with u39.1_IGG4.P-AF647, with a maximum competition of 93%. hu39. Similar levels of competition were observed with 1_IGG4 (unlabeled reference antibody). In contrast, the test antibodies hu31.4_IGG1.AA antibody and reference antibody hu39.1_IGG4.P-AF64 No competition was observed between antibodies 31 and 39, and 7. These data support the use of antibodies 31 and 39, as well as This suggests that the 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 However, it does not bind to mouse CD39. Therefore, to assess its efficacy in vivo, For this purpose, a xenograft mouse model can be used. 500,000 to 1000 cells expressing human CD39 can be used. Million human cancer cells were injected subcutaneously into immunodeficient mice, such as SCID or nude mice. The anti-CD39 antibody inhibits MOLP-8 myeloma (Figure 1). 7), THP-1 leukemia (Table 16), SK-MEL-5 melanoma (Table 15), and OAW4 2. Binds to several human cancer cell lines, including but not limited to ovarian cancer cells, and CD It can inhibit the activity of 39. 3 If less than 0.1 ~30 mg / kg of an anti-CD39 antibody of the present disclosure or isotype control BIWx4 or tumor volume 2,000 mm 3 The injection is given intraperitoneally (ip) until the Treatment of MOLP-8 tumors expressing anti-CD39 with the anti-CD39 antibodies of the present disclosure resulted in This resulted in tumor growth inhibition compared to isotype control treated mice. The tumor growth inhibition compared to treated mice also demonstrated the efficacy of other tumor types expressing human CD39. This is also expected after treatment with the disclosed anti-CD39 antibodies.

[0284] Example 9: Efficacy in human CD39 knock-in model To evaluate the anti-CD39 antibodies of the present disclosure in an immune-competent mouse model, The antibody can be tested in a human CD39 knock-in (hCD39KI) mouse model. where fully immune-competent C57BL / 6 mice or BALB / c mice are , expresses human CD39 but not mouse CD39 (Figure 9). In this study, human CD39 expression paralleled that of mouse CD39 in wild-type mice. This mouse model allows us to determine whether anti-CD39 blockade affects stromal cells, the vasculature, and host cells, such as immune cells. This will enable in vivo testing of the potential effects on primary cells. fibrosarcoma, B16-F10 melanoma, MC38 colon adenocarcinoma, ID8 ovarian cancer, 4T1 breast cancer, or Mouse wild-type or engineered colon cancer cells, including but not limited to CT-26 colon cancer cells Tumor cell lines are inoculated subcutaneously or orthotopically. 500,000 to 10 million tumor cells are injected into the mouse. The tumor growth was monitored. 3 If less than 0.1-30 mg / kg of ch39_mIGG2A.AAG or isotype control BIWx4 or tumor volume is 2,000 mm 3 The patient is treated intraperitoneally (ip) until the patient reaches 100 mg / kg. Anti-CD39 may be administered as a single agent or with one or more additional therapies (e.g., ICD inducers, immunotherapies, etc.). Treatment of tumors in combination with ribozymes (e.g., ribozymes) significantly increased tumor growth compared to isotype control-treated mice. is inhibited.

[0285] In one experiment, C57BL / 6 hCD39KI mice were inoculated with 1 million MC3s on day 0. 8 cells were inoculated. The tumor was 80 mm 3 Once this had occurred, mice were given a 20 mg / kg loading dose. 100 mg of anti-CD39 or isotype control antibody (vehicle: PBS) was administered intraperitoneally (ip) followed by 2 doses of 10 mg / kg anti-CD39 or isotype control antibody Each group consisted of 11 to 12 mice. In the above, the anti-CD39 antibody refers to ch39_mIGG2A.AAG. 5 mg / kg Oxaliplatin (OXA) treatment, i.p. (vehicle: saline), was administered in combination with anti-CD39 or The doses were administered once a week starting the day after the loading dose of either the IV or isotype control. In the experiment, BALB / c hCD39KI mice were inoculated with 4T1 cells on day 0 and then treated with MC. 38 model, treated with OXA or anti-CD39 antibody as described. The results from the experiment are described in the following paragraphs.

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

[0287] Tumor volume and body weight were measured approximately twice weekly (BIW) and are shown in Figure 10A and Figure 10B, respectively. 10B, where points represent the mean and error bars represent the respective mean values ​​from each group of mice. The standard error of the mean (SEM) of the metric is shown. Significance is based on the mixed-effects model including multiple comparisons. As shown in Figure 10A, the anti-CD39 and oxaliplatin-induced Treatment was statistically significant (p=0.0217) compared with treatment with either drug alone. (p=0.0372 for isotype + OXA vs. anti-CD39 + OXA, and p=0.0372 for anti-CD (p=0.0217 for CD39+saline vs. anti-CD39+OXA). Body weight was maintained throughout the course of the experiment. Anti-CD39 treatment was well tolerated, with the tumor growth rate remaining stable over time (Fig. 10B).

[0288] At the end of the study, tumors and spleens from C57BL / 6 hCD39KI MC38 mice were Human CD39 expression by immunohistochemistry (IHC) and enzyme histochemistry (EHC) Further analysis was performed for evidence of CD39 antibody enzyme inhibitory activity. Briefly, frozen OC T-embedded tumor or spleen samples were sectioned at 4 μm thickness and microdissected onto positively charged microscope slides. After drying in air for 15 minutes, the sections were fixed in 4% paraformaldehyde (PFA) solution for 2 h. After fixation, slides were washed in Tris-buffered saline and subjected to IHC using an automated Lei ca Bond Rx platform and blocking of endogenous peroxidase First, we performed immunofluorescence staining with rabbit anti-CD39 mAb (Abcam, EPR20627). Incubation and detection with anti-rabbit HRP polymer system followed by diaminobenzidine (DA B) Brown chromogen and hematoxylin nuclear counterstaining were performed, followed by dehydration and mounting. For the EHC assay, sections were fixed in 4% PFA for 5 min, and then resuspended in Tris-maleate buffer. Washed with 50 mM Tris maleate, 250 mM sucrose, and 2 mM MgCl The sections were then incubated in a solution of 1 mM ATP for 30 minutes at room temperature. , 2.5 mM levamisole, 2 mM lead nitrate, 0.25 mM sucrose, and 5 mM The cells were incubated in a solution of 100 mM Tris-maleate containing MnCl2 for 2 hours at 37°C. Levamisole inhibits tissue nonspecific alkaline phosphatase (TNAP) Although other inhibitors can be used to inhibit the activity of The sections were developed in 1% ammonium sulfide solution for 5 minutes, washed, counterstained with hematoxylin, and then deconvoluted. The tissue was washed and mounted. Representative images are shown in Figures 11-14 (tumor, Figures 11-12, spleen). , Figures 13-14). Human CD39 expression was significantly higher in isotype-treated tumors (Figure 11, Panel A and Figure 12, Panel A, low and high power magnification, respectively) and anti-CD39-treated tumors (Figure 1 1, Panel C and Figure 11, Panel C, low and high power magnification, respectively). It was localized in vascular endothelium, infiltrating immune cells, and stromal components of the tumor microenvironment. 9 expression levels were significantly higher in isotype-treated spleens (Figure 13, Panel A and Figure 14, Panel A, respectively). low and high power magnification, respectively) and anti-CD39-treated spleen (Figure 13, panel C and Figure 14 and C, low and high power magnification, respectively), localized in the red pulp and The tumors and spleens treated in vivo with isotype In the liver, enzyme histochemistry revealed the deposition of lead phosphate (tumor - Figure 11, Panel B and Figure 12, Panel B, low and high power magnification, respectively; spleen - Figure 13, Panel B and Figure 14, Panel B, low and high power magnification, respectively). In the tumors and spleens treated with PEG, no lead phosphate deposition was observed by enzyme histochemistry, and the enzyme This indicates that the enzyme activity is blocked (tumor - Figure 11, panel D and Figure 12, panel E). Panel D, low and high power magnification, respectively; spleen - Figure 13, Panel D and Figure 14, Panel D, low and high power magnification, respectively).

[0289] Intratumoral and peripheral CD39 enzyme activity was also assessed ex vivo. 7BL / 6hCD39KI MC38 mice and BALB / c hCD39KI 4T1 The tumor samples obtained after sacrificing the mice were then resuspended in RPMI + 20% FBS + 250 μg / ml Collagenase D (Millipore Sigma, Cat. No. 110888 58001) + 100KU / ml DNAse 1 (MilliporeSigma catalog) (Product ID: D5025-150KU) and gentleMACS™ Octo Di The cells were then enzymatically digested using a ssociator to obtain a single cell suspension. 2.510 per well of the 4Cells were seeded with 100 nM hu39.5_IGG1. AA or IgG1 Fc silent isotype control antibody (Absolute Antibody The mice were treated with 100 μg / kg of ibody (Cat. No. Ab00178-10.3) and incubated at 37°C in 5% CO The cells were then incubated for 1 hour. Then, the cells were treated with 20 μM ATP and incubated at 37°C, 5% CO The plate was incubated at 2°C for an additional 30 minutes. At the end of the incubation period, the 96-well plate was The plate was spun at 1000 rpm for 3 minutes and the supernatant was collected. Glo® Plus (Promega, Cat. No. V3771) was added to the supernatant. Next, the Flexstation® 3 microplate reader was used to ATP levels in the supernatant were measured by detecting luminescence. Data were expressed in relative luminescence units ( The ex vivo hu39.5_IGG1.AA and isotype treatment groups were compared. The lack of difference between RLU in the 1000 mg / kg bwt. Anti-CD39 treatment inhibited enzyme activity in tumors (Figures 15A and 15B).

[0290] To assay peripheral CD39 enzyme activity, peripheral white blood cells (WBCs) were transfected with hCD39KI. Tumor cell suspensions isolated from terminal blood samples of mice were prepared as described commonly. Briefly, whole blood samples were lysed in 1X Pharm Lyse red blood cell lysis solution. (BD Biosciences, Catalog No. 555899). After washing with PBS solution, the WBC pellet was collected and frozen in BamBanker cell freezing medium ( The enzyme inhibition assay was performed using a 100% ethanol-free ... To line, thaw the samples and then plate 2.5 x 10 cells per well of a 96-well plate.4 Each sample was incubated at room temperature for 1 hour under two treatment conditions: 100 nM hu 39.5_IGG1.AA or IgG1 Fc silent isotype control antibody (Ab Solute Antibody, Catalog No. Ab00178-10.3) Then, all wells were treated with 20 μM ATP and incubated at room temperature for 2 hours. At the end of the incubation period, the 96-well plate was incubated at 1000°C. The supernatant was collected after spinning at 400 rpm for 3 minutes. us (Promega, Cat. No. V3771) was added to the supernatant. by detecting luminescence using a ation®3 microplate reader. ATP levels in the supernatant were measured. Data are presented as relative luminescence units (RLU). There was no difference between RLU in the Xvivo hu39.5_IGG1.AA and isotype-treated groups. The fact that anti-CD39 treatment inhibited the enzyme activity completely in vivo indicates that the treatment It inhibited enzyme activity in the periphery of T1 tumor-bearing mice (Fig. 15C).

[0291] Reduction of cell surface CD39 and anti-CD3 in peripheral immune populations of hCD39KI mice Complete or near complete coverage with 9 treatments. C57BL / 6 hCD39KI MC Whole blood samples from terminal bleeding of 38 mice were evaluated by flow cytometry to identify monocytes (CD 3 - CD19 - CD11b + Ly6c ++ ), granulocytes (CD3 - CD19 - CD11b + Ly6 c + ), T cells (CD3+ CD19 - ), B cells (CD3 - CD19 + ), and other strains Negative (Lin - ) Target coverage in peripheral immune cell types expressing CD39, including lymphocytes Changes in CD39 expression and IL-1 expression were assessed. Target binding was assessed by ch39_mIGG2A.AA Using hu39.5_IGG1.AA-AF647, a reagent that competitively binds to G hu39.5_IGG1.AA was measured using Abcam AF647 Lightni Conjugated to AF647 using the ng Link conjugation kit. Whole blood samples from 5–6 mice per treatment group were cultured using CD39 hu39.5_I GG1.AA-AF647 (competes with anti-CD39), CD39A1 PE (anti-CD39 non-competitive), CD3 17A2 PerCP-Cy5.5, CD11b M1 / 70 F Contains ITC, CD19 6D5 BV421, and Ly6c HK1.4 PE-Cy7. The antibodies were incubated in whole blood on ice in the dark for 30 minutes. The samples were then incubated in the dark using 1X BD FACSLysing solution. The samples were then centrifuged at 400 x g for 5 minutes and resuspended in 1X PB. Wash once with S buffer, resuspend in 1% paraformaldehyde solution, and then suspend for 20 minutes before data acquisition. The samples were filtered through a BD FACSCanto SpecialOr Run on a cytometer and analyze using FlowJo and Prism software. Unbound CD39 was expressed across cell types in all animals treated with isotype antibodies. The competitor clone hu39.5_IGG1.AA-AF647 (hu39.5_IG The MFI of the G1.AA-AF647 signal was compared with that of its isotype control, IgG1.Fc All animals treated with anti-CD39 were detected by immunohistochemistry (measured against S-AF647). Significant competition of hu39.5_IGG1.AA-AF647 with CD39(A1)-expressing cells Binding was observed, 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). The reduction of surface CD39 protein was observed in multiple types of mice treated with anti-CD39. was observed in peripheral immune cells (Fig. 16, upper panel).

[0292] Cell surface CD39 was expressed in tumor-draining lymph nodes of hCD39KI mice treated with anti-CD39. The tumor-draining lymph nodes (TLDN) (right inguinal lymph nodes) were also decreased by the above procedure. Lymph nodes were collected from C57BL / 6 hCD39KI MC38 mice at the end of the study. , 0.6 mg / mL Collagenase P (Roche, Cat. No. 112138650 01), 2.4 mg / mL Dispase II (Sigma, Cat. No. D4693) , and 0.3 mg / mL DNAse I (Thermo Scientific, Cat. RPMI 1640 (Gibco, catalog no. J62229MB) containing The cells were dissociated in a 37°C water bath (Product No. 11875119) for 30 minutes with gentle agitation. Count the dissociated lymph nodes and measure 2 x 10 cells per well. 5 The cells were plated in a 96-well polypropylene Cells were plated in a v-bottom Renn plate. Cells were incubated in PBS (Gibco, Cat. No. 1 4190-144) and washed with 1:25 Fc block (BD, Cat. No. 56422 0) and 1:250 near-infrared fluorescent reactive dye (Invitrogen, Cat. No. L3 The cells were resuspended in PBS containing 4976A for 15 minutes in the dark. Afterwards, cells were stained with an antibody cocktail (Table 20) and incubated at 4°C for 30 minutes. After incubation, the cells were pelleted, the supernatant removed, and 100 μL of fixation buffer (eB Cells were washed with PBS (Cell Signaling Technology, Inc., Cat. No. 00-8222-49) for 1 hour. The cells were then resuspended in 200 μL of PBS and filtered through a 40 μm filter plate (Fisher Sc After filtering with BD LSR F Acquired on an Ortessa X-20 Cell Analyzer and FlowJo (trademark ) v10.8 software (BD Life Sciences) and analyzed for immunosuppression. Human CD39, mouse CD73, and mouse CD81 were expressed in defined immune subpopulations. The expression of mouse P2X7 is shown in Figures 17A and 17B. G2A.AAG was administered as a single agent or in combination with oxaliplatin treatment in MC38 Immune subpopulation profiles in tumor-draining lymph nodes of tumor-inoculated hCD39KI mice This did not result in a significant change in the percentage of immune cells (Figure 17C). Nevertheless, anti-CD39 treatment suppressed nearly all immune responses identified in tumor-draining lymph nodes. Human CD39 on the surface of the immune subpopulation was significantly reduced (Figure 17D). Representative histograms is shown in Figure 17E.

[0293] [Table 20]

[0294] Anti-CD39 treatment does not alter peripheral cytokine levels. Mice were terminally bled using BD Vacutainer® heparin tubes (BD Vacutainer®). D Biosciences, Cat. No. 367871) at 2000 x g. After centrifugation for 15 minutes, supernatant plasma samples were collected. Cytokine levels in the plasma samples were measured using the same method as in Example 1. Anti-CD39 treated mice were analyzed using MSD according to the manufacturer's instructions. No increase in steroid levels was observed. Data were collected from a total of 11–12 animals per treatment group. The results are shown as mean ± SEM using 16 mice (Figure 18). Statistical analysis was performed using ANOVA and Theu's method. The results were performed using Key's multiple comparison test.

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

[0296] To assess cellular ATP release after oxaliplatin treatment, the mouse colon cancer cell line CT 26 and MC38, and the human melanoma cell line SK-MEL-5 were thawed, rinsed, and then The culture medium for CT26 and MC38 was RPMI + 10% FB. S + 1% glutamax + 1% penicillin-streptomycin + 1% sodium pyruvate The culture medium for SK-MEL-5 is EMEM + 10% FBS + 1% glutathione. amax + 1% penicillin-streptomycin + 1% sodium pyruvate. Cells were cultured at 2.5 x 10 per well of a 96-well plate.4 Cells were plated at 37 The cells were incubated overnight at 100°C and 5% CO2. The next day, the cells were resuspended in 100% PBS, ... and 100% PBS. SK-MEL-5 cells were treated with 100 μM or 250 μM oxaliplatin. nM hu39.5_IGG1.AA or IgG1 Fc silent isotype control Further treatment with antibody (absolute antibody, catalog number Ab00178-10.3) was performed. To determine cell viability, cells were incubated at 37°C and 5% CO2 for 24 hours. At the end of the incubation period, luminescence-based Cell Titer G assay was performed. Viability was measured using the lo 2.0 assay (Promega, Cat. No. G9242). was determined. Data are presented as percent survival normalized to untreated vehicle controls. To determine HMGB1 release, cells were incubated at 37°C, 5% CO for 24 hours (Fig. 19A). At the end of the incubation period, the plates were incubated for 400 min. The mixture was centrifuged at 37 × g for 5 minutes and the supernatant was collected. Culture media was prepared using a 100% ethanol-free medium (Omega, Cat. No. CS3030B01) according to the manufacturer's instructions. HMGB1 lev...

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; or (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. The anti-CD39 antibody comprising:

2. 2. The anti-CD39 antibody of claim 1, the antibody comprises 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.

3. 2. The anti-CD39 antibody of claim 1, which is a monoclonal antibody or an antigen-binding fragment thereof.

4. 2. The anti-CD39 antibody of claim 1, which is a human chimeric, humanized, or veneered antibody, or an antigen-binding fragment thereof.

5. An anti-CD39 antibody as described in claim 1, further comprising a variant heavy chain constant region selected from a variant human IgG1 constant region, a variant human IgG2 constant region, a variant human IgG3 constant region, or a variant human IgG4 constant region.

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

5.

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

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

4.

9. 8. The anti-CD39 antibody of claim 5 or 7, comprising a human light chain kappa constant region.

10. The anti-CD39 antibody described in Claim 9, wherein the human light chain kappa constant region comprises sequence number 6.

11. The anti-CD39 antibody of claim 1, the antibody comprises 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 95% sequence identity or at least 99% sequence identity to SEQ ID NO:9 and the light chain variable region has at least 95% sequence identity or at least 99% 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 95% sequence identity or at least 99% sequence identity to SEQ ID NO: 17 and the light chain variable region has at least 95% sequence identity or at least 99% sequence identity to SEQ ID NO: 21; the heavy chain variable region has at least 95% sequence identity or at least 99% sequence identity to SEQ ID NO: 43 and the light chain variable region has at least 95% sequence identity or at least 99% sequence identity to SEQ ID NO: 45; the heavy chain variable region has at least 95% sequence identity or at least 99% sequence identity to SEQ ID NO: 43 and the light chain variable region has at least 95% sequence identity or at least 99% sequence identity to SEQ ID NO: 46; the heavy chain variable region has at least 95% sequence identity or at least 99% sequence identity to SEQ ID NO: 43 and the light chain variable region has at least 95% sequence identity or at least 99% sequence identity to SEQ ID NO:

47. the heavy chain variable region has at least 95% sequence identity or at least 99% sequence identity to SEQ ID NO:43 and the light chain variable region has at least 95% sequence identity or at least 99% sequence identity to SEQ ID NO:48; the heavy chain variable region has at least 95% sequence identity or at least 99% sequence identity to SEQ ID NO:43 and the light chain variable region has at least 95% sequence identity or at least 99% sequence identity to SEQ ID NO:49; the heavy chain variable region has at least 95% sequence identity or at least 99% sequence identity to SEQ ID NO:44 and the light chain variable region has at least 95% sequence identity or at least 99% sequence identity to SEQ ID NO:48; or the heavy chain variable region has at least 95% sequence identity or at least 99% sequence identity to SEQ ID NO:44 and the light chain variable region has at least 95% sequence identity or at least 99% sequence identity to SEQ ID NO:

49.

12. 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.

13. A pharmaceutical composition comprising an anti-CD39 antibody described in any one of claims 1 to 12 and a pharmaceutically acceptable carrier. (a) a method for inhibiting CD39 enzymatic activity, comprising contacting CD39 with an anti-CD39 antibody according to any one of claims 1 to 12; or (b) A method for increasing extracellular ATP or activating the immune system in a subject in need thereof, the method 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 12. A composition comprising the anti-CD39 antibody of any one of claims 1 to 12 for use in

15. A pharmaceutical composition comprising an anti-CD39 antibody described in any one of claims 1 to 12.

16. 16. The pharmaceutical composition of claim 15, for use in a method of treating or preventing cancer, the method 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 1-12.

17. The pharmaceutical composition of claim 16, the cancer is non-small cell lung cancer, and the method further comprises 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; or The pharmaceutical composition, wherein the cancer is gastric cancer or gastroesophageal cancer, and the method further comprises administering the anti-CD39 antibody in combination with FOLFOX and either an antagonistic anti-PD-1 antibody or an antagonistic anti-PD-L1 antibody.

18. A combination comprising an anti-CD39 antibody according to any one of claims 1 to 12 and one or more additional therapeutic agents.

19. The combination of claim 18, 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.

20. An isolated polynucleotide encoding the anti-CD39 antibody of any one of claims 1 to 12.

21. 21. A host cell comprising a vector comprising the isolated polynucleotide of claim 20.

22. A kit comprising the anti-CD39 antibody of any one of claims 1 to 12.

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