Anti-CD39 antibodies and uses thereof

Abstract

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

Description

[0001] This application is a divisional application of the invention application with a filing date of March 2, 2023, Chinese application number 202380024645.6, and invention name “Anti-CD39 antibodies and their uses”.

[0002] Cross-reference statement

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

[0004] Provided herein are anti-CD39 antibodies that inhibit the enzymatic activity of human CD39 and methods of using the same. Background Art

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

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

[0007] Therefore, the hydrolysis of extracellular ATP by CD39 may affect the immune response via two different signaling pathways at opposite ends of the signaling axis. It is worth noting that dysregulation of this signaling axis (referred to herein as the ATP-adenosine signaling axis) has been observed in several diseases. Therefore, there is a need in the art for agents that target CD39 and inhibit its enzymatic activity for beneficial therapeutic purposes. Summary of the invention

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

[0009] In one embodiment, the present disclosure provides an anti-CD39 antibody that specifically binds to human CD39, comprising: a heavy chain variable region, the heavy chain variable region comprising a complementary determining region 1 (H1) having at least 80% sequence identity with SEQ ID NO:10, a complementary determining region 2 (H2) having at least 80% sequence identity with SEQ ID NO:11, and a complementary determining region 3 (H3) having at least 80% sequence identity with SEQ ID NO:12; and a light chain variable region, the light chain variable region comprising a complementary determining region 1 (L1) having at least 80% sequence identity with SEQ ID NO:14, a complementary determining region 2 (L2) having at least 80% sequence identity with SEQ ID NO:15, and a complementary determining region 3 (L3) having at least 80% sequence identity with SEQ ID NO:16. In another embodiment, the present disclosure provides an anti-CD39 antibody that specifically binds to human CD39, comprising: a heavy chain variable region comprising H1 having at least 90% sequence identity to SEQ ID NO: 10, H2 having at least 90% sequence identity to SEQ ID NO: 11, and H3 having at least 90% sequence identity to SEQ ID NO: 12; and a light chain variable region comprising L1 having at least 90% sequence identity to SEQ ID NO: 14, L2 having at least 90% sequence identity to SEQ ID NO: 15, and L3 having at least 90% sequence identity to SEQ ID NO: 16. In some of the foregoing embodiments, the heavy chain variable region of the antibody has at least 90% sequence identity to SEQ ID NO: 9, and the light chain variable region has at least 90% sequence identity to SEQ ID NO: 13. In some embodiments, the heavy chain variable region of the antibody has at least 95% sequence identity to SEQ ID NO:9, and the light chain variable region has at least 95% sequence identity to SEQ ID NO:13.

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

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

[0012] For any of the disclosed antibodies of the present disclosure, the anti-CD39 antibody may be a monoclonal antibody or an antigen-binding fragment thereof; a chimeric, humanized or veneered antibody or an antigen-binding fragment thereof; or a human antibody or an antigen-binding fragment thereof. In addition, the anti-CD39 antibody may also include a heavy chain constant region selected from human IgG1, human IgG2, human IgG3 or human IgG4, and optionally a human light chain constant region. The variant heavy chain constant region may be a wild-type heavy chain constant region, or may be a heavy chain constant region with enhanced or reduced effector function relative to a wild-type heavy chain constant region. In various embodiments, the IgG heavy chain constant region may include SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5; and the human light chain kappa constant region may include SEQ ID NO: 6.

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

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

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

[0016] On the other hand, the present disclosure provides any one of the antibodies disclosed herein for use as a drug in combination with additional therapies. In some embodiments, the aforementioned anti-CD39 antibodies are provided for use in the treatment of cancer. In some embodiments, the aforementioned anti-CD39 antibodies are provided for the prevention of cancer. In certain embodiments, the additional therapy may be an immune checkpoint inhibitor, an immunogenic cell death induction therapy, an ATP-adenosine axis targeting agent, a HIF-2α inhibitor, an arginase inhibitor, an AXL inhibitor, or a PI3K inhibitor. In other embodiments, the additional therapy may be chemotherapy, radiotherapy, durvalumab, zimberelimab, domvanalimab, AB308, AB521, or quemliclustat.

[0017] On the other hand, the present disclosure provides any one of the antibodies disclosed herein for use as a drug in combination with two or more additional therapies. In some embodiments, the aforementioned anti-CD39 antibodies are provided for use in treating cancer. In some embodiments, the aforementioned anti-CD39 antibodies are provided for use in preventing cancer. In certain embodiments, each additional therapy may be a chemotherapeutic agent, an immune checkpoint inhibitor, an immunogenic cell death induction therapy, an ATP-adenosine axis targeting agent, a HIF-2α inhibitor, an arginase inhibitor, an AXL inhibitor, or a PI3K inhibitor. In other embodiments, the additional therapy may be chemotherapy, radiotherapy, durvalumab, sepali monoclonal antibody, dovanali monoclonal antibody, AB308, AB521, or quercetin. In some embodiments, one or more additional therapeutic agents include (a) pemetrexed, carboplatin, and antagonist anti-PD-1 antibodies or antagonist anti-PD-L1 antibodies, or (b) FOLFOX and antagonist anti-PD-1 antibodies or antagonist anti-PD-L1 antibodies.

[0018] On the other hand, the present disclosure provides a method for treating or preventing cancer, the method comprising administering any of the antibodies disclosed herein to a subject in need thereof. In some embodiments, the method further comprises administering one or more additional therapies. In certain embodiments, each additional therapy may be an immune checkpoint inhibitor, an immunogenic cell death induction therapy, an ATP-adenosine axis targeting agent, a HIF-2α inhibitor, an arginase inhibitor, an AXL inhibitor, or a PI3K inhibitor. In other embodiments, the additional therapy may be chemotherapy, radiotherapy, durvalumab, sepali monoclonal antibody, dovanali monoclonal antibody, AB308, AB521, or quercetin.

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

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

[0021] Other aspects and repetitive descriptions of the present disclosure are provided in greater detail below. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

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

[0025] Figure 4A , Figure 4B , Figure 4C , Figure 4D , Figure 4E , Figure 4F Depicts the results of a macrophage cytokine release assay. Figure 4A ) or 100nM( Figure 4B -F) Relative amounts of cytokines in the supernatants of macrophages treated with anti-CD39 antibody or isotype control. The fold change for each donor was normalized to the isotype control and two ( Figure 4C , Figure 4D) or three ( Figure 4A , Figure 4B , Figure 4E , Figure 4F ) donors. Bar height is the normalized mean fold change between donors and error bars are SEM.

[0026] Figure 5A , Figure 5B and Figure 5C Depicts the results of a monocyte-derived dendritic cell (moDC) assay. The extracellular marker CD83 ( Figure 5A )、CD86( Figure 5B ) and CD14( Figure 5C ). Each line represents a biological donor, where the linker numbers are from the same donor, N=4.

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

[0028] Fig. 7A Graph depicting the binding of hu39.1_IGG4.P and isotype control antibodies to human CD39 expressed on MOLP-8 human myeloma cells.

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

[0030] Fig. 8A Enzyme activity (ATP consumption) on splenocytes from the hCD39KI mouse model is depicted.

[0031] Figure 8B Depicted is the inhibition of splenocyte enzyme activity by hu39.1_IGG4.P.

[0032] Fig. 9 Depicts confirmation that the hCD39KI mouse model expresses human but not murine CD39.

[0033] Fig. 10A Graph depicting mean tumor volume of hCD39KI mice with indicated treatments. Points represent mean and error bars represent standard error of the mean (SEM), *=p<0.5.

[0034] Fig. 10BGraph depicting percent body weight change in hCD39KI mice with the indicated treatments.

[0035] Fig.11 Shown are low magnification images of tumors from hCD39KI mice analyzed by immunohistochemistry for human CD39 expression (Panels A and C) and by enzyme histochemistry for evidence of anti-CD39 enzymatic blocking activity (Panels B and D).

[0036] Fig.12 High magnification images of tumors from hCD39KI mice analyzed by immunohistochemistry for human CD39 expression (Panels A and C) and by enzyme histochemistry for evidence of anti-CD39 enzyme blocking activity (Panels B and D) are shown. Fig.12 The image in Fig.11 High-resolution image of the framed area in .

[0037] Fig.13 Shown are low magnification images of spleens from hCD39KI mice analyzed by immunohistochemistry for human CD39 expression (Panels A and C) and by enzyme histochemistry for evidence of anti-CD39 enzymatic blocking activity (Panels B and D).

[0038] Fig.14 Shown are high magnification images of spleens from hCD39KI mice analyzed by immunohistochemistry for human CD39 expression (Panels A and C) and by enzyme histochemistry for evidence of anti-CD39 enzyme blocking activity (Panels B and D). Fig.14 The image in Fig.13 High-resolution image of the framed area in .

[0039] Fig.15A , Fig. 15B and Fig. 15C Figure 3 is a graph depicting tumors derived from hCD39KI mice treated as indicated along the x-axis ( Fig.15A : MC38 tumor; Fig. 15B : 4T1 tumor) or peripheral whole blood cells ( Fig. 15C ) in the supernatant of a single cell suspension of . ATP levels were measured by detecting relative luminescence units (RLU).

[0040] Fig.16Depicts graphs showing cell surface CD39 and receptor occupancy in hCD39KI mice treated with anti-CD39. The graphs in the upper graph show reduction in cell surface CD39 expression as measured by binding of the non-competitive anti-CD39 clone A1-PE in multiple cell types following in vivo anti-CD39 treatment. The graphs in the lower graph show 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. Graphs showing the expression of >100 CD39(A1) cells collected + Data of the event group. Statistical analysis was performed using One-way ANOVA (Dunnett's multiple comparison test with a single pooled variance), *P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001.

[0041] Fig.17A Depicted are flow cytometry dot plots showing the expression levels of human CD39 (x-axis) and mouse CD73 (y-axis) on immune subsets from tumor-derived lymph nodes in vehicle-treated hCD39KI mice. Gates were set on isotype controls (hCD39 isotype controls in grey) and applied to each group (black).

[0042] Fig. 17B Depict flow cytometry dot plots showing expression levels of human CD39 (x-axis) and mouse P2X7 (y-axis) on immune subsets from tumor-derived lymph nodes in vehicle-treated hCD39KI mice. Gates were set on isotype controls (hCD39 isotype controls in grey) and applied to each group (black).

[0043] Fig. 17C Describe and be shown in the figure of the percentage of immune subgroup observed after treatment as indicated along the x-axis.Under the situation of anti-CD39 treatment, no significant change of immune subgroup percentage was observed.Statistical analysis was performed with one-way ANOVA (Dunnett's multiple comparison test, with single pooled variance), *P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001, each symbol represents individual mice, and bar height is mean value and error bar is + / -SEM.

[0044] Fig.17DDescribe and show the figure of CD39 cell surface expression observed after treatment as indicated along x-axis.In almost all immune subgroups, the significant reduction of CD39 cell surface expression was observed under anti-CD39 treatment.Statistical analysis was performed with one-way ANOVA (Dunnett's multiple comparison test, with single pooled variance), *P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001, each symbol represents individual mice, and bar height is mean value and error bar is + / -SEM.

[0045] Fig.17E Overlay of histograms for each immune subset showing reduction in CD39 cell surface expression on a representative animal from each treatment group (isotype + saline (grey open histograms), isotype + oxaliplatin (black open histograms), anti-CD39 + saline (grey solid histograms), and anti-CD39 + oxaliplatin (black solid histograms)).

[0046] Fig.18 Depicts a graph showing peripheral cytokine levels in plasma derived from hCD39KI mice bearing MC38 tumors at the end of the experiment described in Example 9. Data are presented as mean ± SEM, with a total of 11-12 mice per treatment group. Statistical analysis was performed using ANOVA with Tukey's multiple comparison test.

[0047] Fig.19A Depicted are graphs showing cell viability of CT26 cells, MC38 cells, SK-MEL-5 cells and isotype control antibody, and SK-MEL-5 cells and hu39.5_IGG1.AA antibody 24 hours after treatment with vehicle control (Veh), 100 μM oxaliplatin (OXA.L), or 250 μM oxaliplatin (OXA.H).

[0048] Fig.19B Depicted are graphs showing HMGB1 levels in culture supernatants of CT26 cells, MC38 cells, SK-MEL-5 cells and isotype control antibody, and SK-MEL-5 cells and hu39.5_IGG1.AA antibody 24 hours after treatment with vehicle control (Veh), 100 μM oxaliplatin (OXA.L), or 250 μM oxaliplatin (OXA.H).

[0049] Fig.19C Depicted are graphs showing extracellular ATP release by CT26 cells, MC38 cells, SK-MEL-5 cells and isotype control antibody, and SK-MEL-5 cells and hu39.5_IGG1.AA antibody after 8 hours of treatment with vehicle (PBS), 100 μM oxaliplatin, or 250 μM oxaliplatin.

[0050] Fig. 20A Depicted are graphs showing cell viability of SK-MEL-5 cells after 48 hours of treatment with the indicated chemotherapeutic agents.

[0051] Fig. 20B Depicted are graphs showing HMGB1 levels in culture supernatants of SK-MEL-5 cells after 48 hours of treatment with the indicated chemotherapeutic agents.

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

[0053] Fig.21 Depicted are graphs showing the relative expression of CD39 on peripheral immune cells in humans (top row) and C57BL / 6hCK39KI MC38 mice (bottom row).

[0054] Fig.22A Flow cytometry dot plots of representative donors are depicted, showing the distribution of CD39 and CD73 on different myeloid cell types. + Monocytes were isolated directly from peripheral human blood. DCs enriched from peripheral human blood were further subdivided into pDCs, cDC1, cDC2, and other DCs. M0, M1, M2, and moDC subsets were derived from CD14 as described in the Examples. + Monocytes.

[0055] Fig. 22B Depicts a graph showing the percentage of CD73 positivity (left graph) and CD39 positivity (middle graph) and CD39 protein expression (right graph) as measured by flow cytometry in human peripheral blood CD14 + Monocytes; primary dendritic cell (DC) subsets, including plasmacytoid DC (pDC, defined as HLA-DR + CD11c 阴性 CD123 + ), conventional DC 1s (cDC1, defined as HLA-DR + CD11c + CD141 + Clec9a + ), conventional DC 2s (cDC2, defined as HLA-DR + CD11c + CD1c + CD141 阴性 Clec9a 阴性 ) and other DCs (defined as HLA-DR + CD11c+ CD1c 阴性 CD141 阴性 Clec9a 阴性 ); and in vitro differentiated monocyte-derived bone marrow cells, including macrophages (M0), polarized macrophages (M1, M2) and DCs (moDCs). + The mean fluorescence intensity of the cells was divided by the mean fluorescence intensity of the isotype control. Each symbol is a unique donor, the bar height is the median and the error bars are the range.

[0056] Fig. 22C and Fig.22D Figure 3 shows the expression of CD14 in human peripheral blood obtained from four donors. + ENTPD1 (CD39) was evaluated on monocytes, enriched populations of primary dendritic cell subsets (enriched DCs), and in vitro differentiated monocyte-derived bone marrow cells, including macrophages (M0), polarized macrophages (M1, M2), and DCs (moDCs) ( Fig. 22C ) and NT5E(CD73)( Fig.22D )'s mRNA expression.

[0057] Fig.22E Heatmap showing unsupervised clustering of normalized expression of housekeeping genes for CD39, adenosine, and inflammasome pathway genes in cell populations from four healthy donors described in 279 using a custom NanoString panel. Low quality wells or aberrant control probes marked by low counts were removed based on this analysis. Specific donor IDs are attached at the end of the cell type labels. Dashed horizontal lines in the highest dendrogram show sample splits but do not affect the unsupervised nature of the clustering.

[0058] Fig.23 Depict the relative induction (compared to DMSO) of cell surface marker expression after treatment with indicated inhibitors for 1 hour followed by treatment with 0 or 300 μM ATP for 18 hours. The cell surface expression of CD86 (upper graph), CD83 (middle graph) and CD80 (lower graph) was assessed by flow cytometry. Induction of activation marker expression according to 0 to 300 μM ATP was compared between inhibitor-treated cells and DMSO-treated cells. Bar heights are mean values ​​and error bars are SEM. Each point represents a donor. Statistical analysis was performed using ANOVA by Tukey's multiple comparison test, *P≤0.05, ***P≤0.001.

[0059] Fig.24AHeatmap showing TCGA RNA expression analysis in tumors (kidney renal clear cell carcinoma (KIRC), lung squamous cell carcinoma (LUSC), lung adenocarcinoma (LUAD), head and neck squamous cell carcinoma (HNSC), esophageal cancer (ESCA), gastric adenocarcinoma (STAD), and pancreatic adenocarcinoma (PAAD)) normalized on a per gene basis. Proteins encoded by genes 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). The distribution is shown in the histogram on the right.

[0060] Fig. 24B Violin plots are depicted. The violin plots summarize the core probability density of the normalized log2 gene expression (y-axis) of the following genes (proteins) among the TCGA sample subsets (the width of the shaded area indicates the proportion of the data located there): ENTPD1 (CD39), NT5E (CD73), P2RX7 (P2X7) and P2RY11 (P2Y11). Along the x-axis, the violin plots are grouped by specific TCGA subtypes: esophageal cancer (ESCA), head and neck squamous cell carcinoma (HNSC), kidney renal clear cell carcinoma (KIRC), lung adenocarcinoma (LUAD), lung squamous cell carcinoma (LUSC), pancreatic adenocarcinoma (PAAD) and gastric adenocarcinoma (STAD). The pathway enrichment scores (y-axis) of the bone marrow labels for the same subset of TCGA cancer subtypes are also shown. DETAILED DESCRIPTION

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

[0062] I. Definitions

[0063] Unless otherwise defined, all technical terms, symbols and other scientific terms or technical nouns used herein are intended to have the meanings commonly understood by those skilled in the art to which the present disclosure belongs. It should be understood that aspects of the present invention described herein include "comprising" aspects, "consisting of" aspects, and "consisting essentially of" aspects. The terms "comprising", "including" and "having" are intended to be inclusive and mean that there may be other elements in addition to the listed elements.

[0064] Unless otherwise indicated, the term "about" as used herein refers to the typical error range for individual values ​​as understood by those skilled in the art. For the avoidance of doubt, reference to "about" a value or parameter herein includes (and describes) aspects directed to the value or parameter itself.

[0065] Also as used herein, "and / or" refers to and encompasses any one and all possible combinations of one or more of the associated listed items, as well as no combinations when interpreted in the alternative ("or"). Similarly, phrases in the form "A / B" or in the form "A and / or B" mean (A), (B), or (A and B); phrases in the form "at least one of A, B, and C" mean (A), (B), (C), (A and B), (A and C), (B and C), or (A, B, and C).

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

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

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

[0069] The terms "full length antibody," "intact antibody," and "complete antibody" are used interchangeably herein to refer to an antibody that has a structure substantially similar to a native antibody structure or has heavy chains that contain an Fc region.

[0070] "Natural antibodies" are naturally occurring immunoglobulin molecules with different structures. For example, natural IgG antibodies are heterotetrameric glycoproteins of about 150,000 daltons, which are composed of two identical light chains (each about 25 kDa) and two identical heavy chains (each about 50-70 kDa) bonded by disulfide bonds. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH), also referred to as a variable heavy chain domain or a heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3). Similarly, from the N-terminus to the C-terminus, each light chain has a variable region (VL), also referred to as a variable light chain domain or a light chain variable domain, followed by a constant light chain (CL) domain. The light chain of an antibody can be classified into one of two types based on the amino acid sequence of its constant domain, referred to as kappa (κ) and lambda (λ). The heavy chain is classified as γ, μ, α, δ, or ε, and the antibody isotype is defined as IgG, IgM, IgA, IgD, and IgE, respectively. The amino terminal portion of each light chain and heavy chain includes about 100 to 110 or more variable regions (VL and VH, respectively) of the amino acid sequences that are primarily responsible for antigen recognition. The carboxyl terminal portion of each chain defines the constant region that is primarily responsible for effector function. Within the light chain and heavy chain, the variable region and constant region are joined by the "J" region of about 12 or more amino acid sequences, wherein the heavy chain also includes about 10 or more "D" regions of amino acid sequences.

[0071] The term "variable region" or "variable domain" refers to a domain in an antibody heavy chain or light chain that is involved in the binding of an antibody to an antigen. The variable domains of antibody heavy and light chains generally have a similar structure, wherein each domain comprises four conserved framework regions (FRs) and three hypervariable regions (CDRs). (See, e.g., Kindt et al., Kuby Immunology, 6th edition, WH Freeman and Co., p. 91 (2007).) A single VH or VL domain may be sufficient to confer antigen binding specificity. In addition, VH or VL domains can be used to separate antibodies that bind to a specific antigen from antibodies that bind to an antigen to screen libraries of complementary VL or VH domains, respectively. See, e.g., Portolano et al., J. Immunol. 150: 880-887 (1993); Clarkson et al., Nature 352: 624-628 (1991).

[0072] "Framework region" or "FR" refers to the variable domain residues other than the hypervariable region residues. The FR of the variable domain is usually composed of four FR domains: FR1, FR2, FR3 and FR4. Therefore, the CDR and FR sequences usually appear in the following sequence: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. As known in the art, the FR domains of the heavy chain and light chain can be different.

[0073] As used herein, the terms "hypervariable region" or "HVR" (also often referred to as "complementarity determining region" or "CDR") are used interchangeably and refer to each region of a variable domain that is hypervariable in sequence and / or forms structurally defined loops ("hypervariable loops") and / or contains antigen contact residues ("antigen contacts"). Typically, an antibody comprises six CDRs: three in VH (H1, H2, H3) and three in VL (L1, L2, L3). As used herein, a "CDR derived from a variable region" refers to a CDR that has no more than two amino acid substitutions compared to the corresponding CDR from the original variable region. Exemplary CDRs herein include: (a) hypervariable loops occurring at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)); (b) CDRs occurring at amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health Health, Bethesda, MD (1991)); (c) antigen contacts occurring at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum et al., J. Mol. Biol. 262:732-745 (1996)); and (d) combinations of (a), (b), and / or (c), as defined below for various antibodies of the present disclosure. Unless otherwise indicated, CDR residues and other residues (e.g., FR residues) in the variable domain are numbered herein according to Kabat et al., supra, by Eu.

[0074] The term "isolated antibody" refers to an antibody that has been separated from a component of its natural environment. In some embodiments, the isolated antibody is purified to greater than 95% or 99% purity as determined by, for example, electrophoresis or chromatography (eg, ion exchange or reverse phase HPLC).

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

[0076] "Human antibodies" are antibodies whose amino acid sequences correspond to those of antibodies produced by humans or human cells or derived from non-human sources using human antibody repertoires or other human antibody encoding sequences. This definition of human antibodies specifically excludes humanized antibodies comprising non-human antigen binding residues. Human antibodies can also be prepared using various techniques known in the art, including phage display libraries. Hoogenboom and Winter. J. Mol. Biol. 227: 381, 1991; Marks et al., J. Mol. Biol. 222: 581, 1991. Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boerner et al., J. Immunol., 147 (1): 86-95, 1991. The methods described in can also be used to prepare human monoclonal antibodies. See also van Dijk and van de Winkel. Curr. Opin. Pharmacol. 5: 368-74, 2001.

[0077] "Humanized" antibody refers to an antibody comprising amino acid residues from non-human CDR and amino acid residues from human FR. In some aspects, humanized antibody will include variable domains, wherein all or substantially all CDRs correspond to the CDRs of non-human antibodies, and all or substantially all FRs correspond to the FRs of human antibodies. In some aspects where all or substantially all FRs of humanized antibodies correspond to the FRs of human antibodies, any one of the FRs of humanized antibodies may, for example, contain one or more amino acid residues from non-human FRs at one or more cursor (Vernier) position residues of FR, and / or at one or more other selected residues. Humanized antibodies optionally may include at least a portion of an antibody constant region derived from human antibodies. For example, a "humanized form" of an antibody of a non-human antibody refers to an antibody that has undergone humanization. Humanized antibodies retain binding specificity and affinity similar to the starting non-human antibodies.

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

[0079] The term "epitope" refers to a specific site on an antigen to which an antibody binds. The specific site on an antigen to which an antibody binds can be determined, for example, by crystallography. Methods such as hydroxyl radical protein footprinting and alanine scanning mutagenesis can also be used, but the methods may provide less resolution.

[0080] 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 the antigen or may bind to two or more epitopes of the antigen. A monospecific antibody that binds to two or more epitopes of an antigen is a monospecific multi-epitope antibody.

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

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

[0083] The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain, including a native sequence Fc region and a variant Fc region. Although the boundaries of the Fc region of an immunoglobulin heavy chain may vary, the human IgG heavy chain Fc region is generally defined as extending from the amino acid residue at position Cys226 or from Pro230 to its carboxyl terminus. The C-terminal lysine (residue 447, according to the Eu numbering system) in the Fc region may be removed, for example during the preparation or purification of the antibody, or by recombinantly engineering the nucleic acid encoding the heavy chain of the antibody. Therefore, the composition of a complete antibody may include an antibody group in which all Lys447 residues are removed, an antibody group in which Lys447 residues are not removed, and an antibody group with a mixture of antibodies containing and not containing Lys447 residues.

[0084] A "functional Fc region" has the effector functions of a native sequence Fc region. Exemplary effector functions include C1q binding; complement dependent cytotoxicity (CDC); Fc receptor binding; antibody dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptors; BCR), etc. Such effector functions generally require an Fc region in combination with a binding domain (e.g., an antibody variable domain) and can be assessed using various assays disclosed herein or otherwise known in the art. A functional Fc region may have effector functions that are substantially similar to wild-type IgG, effector functions that are reduced compared to wild-type IgG, or effector functions that are enhanced compared to wild-type IgG. For antibodies comprising a human Fc region, comparisons are generally made relative to wild-type human IgG1.

[0085] A "native sequence Fc region" comprises an amino acid sequence identical to the amino acid sequence of an Fc region found in nature. Native sequence human Fc regions include native sequence human IgG1 Fc regions (non-A and A allotypes); native sequence human IgG2 Fc regions; native sequence human IgG3 Fc regions; and native sequence human IgG4 Fc regions, as well as naturally occurring variants thereof.

[0086] A "variant Fc region" comprises an amino acid sequence that differs from the amino acid sequence of a native sequence Fc region by at least one amino acid modification (e.g., about one to about ten amino acid modifications, and in some embodiments, about one to about five amino acid modifications), preferably one or more amino acid substitutions. The variant Fc region herein will preferably have at least about 80% homology with the native sequence Fc region and / or the Fc region of the parent polypeptide, preferably at least about 90% homology therewith, or preferably at least about 95% homology therewith. In some embodiments, the variant Fc region may have reduced or enhanced effector function compared to wild-type IgG. For antibodies comprising a human Fc region, comparisons are typically made relative to wild-type human IgG1.

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

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

[0089] The term "antibody fragment" refers to a molecule that is different from an intact antibody, contains a portion of an intact antibody, and binds to the antigen that the intact antibody binds to. Examples of antigen-binding fragments include, but are not limited to, bifunctional antibodies, Fab, Fab', F(ab')2, F(ab) c, Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized bifunctional antibodies (ds bifunctional antibodies), trifunctional antibodies, tetrafunctional antibodies, single-chain antibodies, scFv, scFv dimers, single-domain antibodies, single-domain antibodies, and multivalent domain antibodies. Typically, the binding fragment competes with the intact antibody from which it is derived for specific binding. The binding fragment can be produced by recombinant DNA technology or by enzymatic or chemical separation of intact immunoglobulins.

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

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

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

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

[0094] The term "single-chain antibody" refers to an antibody consisting of a heavy chain variable region and a light chain variable region connected by a joint. In most cases, but not all cases, the joint may be a peptide. The length of the joint varies according to the type of the single-chain antibody. Two or more single-chain antibodies are linked together in a covalent or non-covalent manner to produce a high-order form. Single-chain antibodies and their high-order forms may include but are not limited to single-domain antibodies, multivalent domain antibodies, single-chain variant fragments (scFv), divalent scFv (two-scFv), trivalent scFv (three-scFv), tetravalent scFv (four-scFv), bifunctional antibodies, and trifunctional antibodies and tetrafunctional antibodies.

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

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

[0097] "Bifunctional antibodies" are high-order single-chain antibody variants consisting of two single-chain antibodies. For each single-chain antibody, a linker that is too short to allow pairing between two domains on the same chain is used, forcing the domain to pair with the complementary domain of another chain, thereby generating two antigen-binding sites. In most cases, but not all cases, the linker can be a peptide. The antigen-binding site can target the same or different antigens or epitopes. Trifunctional antibodies (three single-chain antibodies assembled to form three antigen-binding sites), tetrafunctional antibodies (four single-chain antibodies assembled to form four antigen-binding sites) and high-order variants can be produced in a similar manner. See, for example, Holliger P. et al., Proc Natl Acad Sci USA. July 15; 90 (14): 6444-8 (1993); EP404097; WO93 / 11161.

[0098] "Single domain antibody" refers to an antibody fragment containing only the heavy chain variable region or the light chain variable region. H The domains are covalently joined by a peptide linker to produce a multivalent domain antibody. H The domains may target the same or different antigens or epitopes.

[0099] The term "heavy chain antibody" refers to an antibody consisting of two heavy chains. Heavy chain antibodies may be IgG-like antibodies from camels, llamas, alpacas, sharks, etc., or IgNARs from cartilaginous fish. See, for example, Riechmann L. and Muyldermans S., J Immunol Methods. Dec. 10; 231(1-2): 25-38 (1999); Muyldermans S., J Biotechnol. Jun; 74(4): 277-302 (2001); WO94 / 04678; WO94 / 25591; or U.S. Pat. No. 6,005,079. Heavy chain antibodies were originally derived from Camelidae (camels, dromedaries and llamas). Despite lacking light chains, camelid antibodies have a bona fide antigen-binding repertoire (Hamers-Casterman C. et al., Nature. June 3; 363(6428): 446-8 (1993); Nguyen VK et al., "Heavy-chain antibodies in Camelidae; a case of evolutionary innovation", Immunogenetics. April; 54(1): 39-47 (2002); Nguyen VK et al., Immunology. May; 109(1): 93-101 (2003)). The variable domain of heavy-chain antibodies (VHH domain) represents the smallest known antigen-binding unit produced by the adaptive immune response (Koch-Nolte F. et al., FASEB J. November; 21(13): 3490-8. Epub 2007 June 15 (2007)).

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

[0101] "Percentage (%) identity" relative to a reference amino acid sequence is defined as the percentage of amino acid residues in the candidate sequence that are identical to the amino acid residues in the reference sequence after aligning the reference sequence with the candidate sequence and introducing gaps, if necessary, to achieve the maximum percentage of sequence identity, and without considering any conservative substitutions as part of the sequence identity. Alignment for the purpose of determining the percentage of amino acid sequence identity can be achieved in various ways within the skill range of the art, such as using publicly available computer software, such as BLAST, BLAST-2 or CLUSTAL software. Those skilled in the art can determine the appropriate parameters for aligning sequences, including any algorithm required to achieve maximum alignment within the full length of the compared sequences. In general, the % sequence identity of a given amino acid sequence A relative to, with or for a given amino acid sequence B is calculated as follows: 100 multiplied by a fraction X / Y, wherein X is the number of amino acid residues scored as identical matches in the program alignment of A and B by a sequence alignment program, and wherein Y is the total number of amino acid residues in B. It will be appreciated that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B and the % amino acid sequence identity of B to A will not be equal.

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

[0103] The terms "treat", "treating", "treatment" and the like refer to a process of action that temporarily or permanently eliminates, reduces, inhibits, alleviates, or improves or prevents the worsening of the disease, disorder or condition to which the term applies, or at least one symptom associated therewith. As an example, treatment includes inhibiting (e.g., curbing the development or further development of a disease, disorder or condition or clinical symptoms associated therewith) active disease, improving the quality of life and / or prolonging the survival of a subject.

[0104] As used herein, the term "in need of treatment" refers to a judgment made by a physician or other health care provider that a subject requires or will benefit from treatment. This judgment is made based on a variety of factors within the physician's or health care provider's expertise.

[0105] The terms "prevent," "preventing," "prevention," "prophylaxis," and the like refer to a process of action initiated in a manner (e.g., prior to the onset of a disease, disorder, condition, or symptom thereof) so as to temporarily or permanently prevent, inhibit, suppress, or reduce a subject's risk of developing a disease, disorder, condition, or the like (as determined, for example, by the absence of clinical symptoms) or delay its onset, usually in a subject susceptible to a particular disease, disorder, or condition. In certain instances, the terms also refer to slowing the progression of a disease, disorder, or condition or inhibiting its progression to a harmful or other undesirable state. Prevention also refers to a process of action initiated in a subject after the subject has been treated for a disease, disorder, condition, or symptom associated therewith, so as to prevent recurrence of the disease, disorder, condition, or symptom.

[0106] As used herein, the term "in need of prevention" refers to a judgment made by a physician or other caregiver that a subject requires or will benefit from preventive healthcare. This judgment is made based on a variety of factors within the expertise of the physician or caregiver.

[0107] II. Antibodies of the Disclosure

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

[0109] The anti-CD39 antibodies disclosed herein, whether monospecific or multispecific, specifically bind to human CD39, which means that the equilibrium dissociation constant (K D ) is 10 -6 M or less, as measured by surface plasmon resonance (SPR). In certain embodiments, the anti-CD39 antibodies of the present disclosure have a K of 1.5 M or less for human CD39 as measured by SPR. D For 10 -8 M or lower (e.g. 10 -8 , 10 -9 , 10 -10 For detailed methods, see, e.g., Example 3. In various embodiments, the anti-CD39 antibodies of the present disclosure are resistant to the K of human CD39. D About 1×10 -9 M is about 1×10-14 M, or about 1×10 -9 M is about 1×10 -13 M, or about 1×10 -9 M is about 1×10 -12 M, or about 1×10 -9 M is about 1×10 -11 M. In some embodiments, the anti-CD39 antibodies of the present disclosure have a K of human CD39. D About 1×10 -10 M is about 1×10 -14 M, or about 1×10 -10 M is about 1×10 -13 M, or about 1×10 -10 M is about 1×10 -12 M. In some embodiments, the anti-CD39 antibodies of the present disclosure have a K of human CD39. D About 1×10 -11 M is about 1×10 -14 M, about 1×10 -11 M is about 1×10 -13 M. In some embodiments, the anti-CD39 antibodies of the present disclosure have a K of human CD39. D About 1×10 -12 M is about 1×10 -14 M or about 1×10 -12 M is about 1×10 -13 M. In some embodiments, the anti-CD39 antibodies of the present disclosure have a K of human CD39. D About 1×10 -10 M is about 1×10 -11 M or about 1×10 -11 M is about 1×10 -12 M.

[0110] The anti-CD39 antibodies of the present disclosure also inhibit CD39 enzymatic activity. In some embodiments, the anti-CD39 antibodies of the present disclosure have an IC of about 5 nM or less as measured in Example 5. 50 For example, as measured in Example 5, the anti-CD39 antibody can inhibit human CD39 enzymatic activity at an IC value of about 5 nM, about 4 nM, about 3 nM, about 2 nM, about 1 nM or less. 50 As another example, the anti-CD39 antibody can have an IC of about 1 nM or less, such as about 1.0 nM, about 0.9 nM, about 0.8 nM, about 0.7 nM, about 0.6 nM, about 0.5 nM or less. 50As another example, as measured in Example 5, the anti-CD39 antibody may have an IC of about 0.5 nM or less, such as about 0.5 nM, about 0.4 nM, about 0.3 nM, about 0.2 nM, about 0.1 nM, about 0.09 nM, about 0.08 nM, about 0.07 nM, about 0.06 nM, about 0.05 nM or less. 50 The above IC 50 Values ​​may also be expressed as individual values ​​or as ranges. For example, an anti-CD39 antibody may have an IC of about 0.05 nM to about 5 nM, about 0.05 nM to about 1 nM, 0.05 nM to about 0.5 nM, about 1 nM to about 5 nM, about 0.5 nM to about 1.0 nM, or subranges thereof. 50 In one embodiment, the anti-CD39 antibodies of the present disclosure inhibit the enzymatic activity of recombinant human CD39 with an IC value of about 0.5 nM to about 1.0 nM or about 0.6 nM to about 1.0 nM. 50 In one embodiment, the anti-CD39 antibodies of the present disclosure inhibit human CD39 enzymatic activity with an IC of about 0.05 nM to about 0.5 nM, about 0.05 nM to about 0.4 nM, or about 0.05 nM to about 0.3 nM. 50 In one embodiment, the anti-CD39 antibodies of the present disclosure inhibit human CD39 enzymatic activity with an IC of about 0.07 nM to about 0.5 nM, about 0.07 nM to about 0.4 nM, about 0.07 nM to about 0.3 nM, or about 0.07 nM to about 0.2 nM. 50 In the foregoing embodiments, (i) human CD39 may be recombinant, soluble CD39 or may be CD39 expressed on the surface of cells, and / or (ii) inhibition may be assessed in the presence of low ATP (e.g., 20 μM) and / or high ATP (e.g., 400 μM).

[0111] A. Exemplary variable regions

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

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

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

[0115] Table A

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

[0117] In each of the foregoing embodiments, the anti-CD39 antibody may be (i) a complete antibody or an antigen binding fragment and / or (ii) a chimeric antibody, a humanized antibody or a human antibody. Suitable chimeric, humanized and human antibodies are further described in sections IIC, IID and IIE. In certain embodiments, the anti-CD39 antibody may optionally include one or more constant regions or a portion of a constant region that is substantially human. Suitable constant regions are further described in detail in section II(F).

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

[0119] Table B

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

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

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

[0123] Table C

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

[0125] In each of the foregoing embodiments, the anti-CD39 antibody may be (i) a complete antibody or an antigen binding fragment and / or (ii) a chimeric antibody, a humanized antibody or a human antibody. Suitable chimeric, humanized and human antibodies are further described in sections IIC, IID and IIE. In certain embodiments, the anti-CD39 antibody may optionally include one or more constant regions or a portion of a constant region that is substantially human. Suitable constant regions are further described in detail in section II(F).

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

[0127] Table D

[0128]

[0129]

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

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

[0132] Table E

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

[0134] In each of the foregoing embodiments, the anti-CD39 antibody may be (i) a complete antibody or an antigen binding fragment and / or (ii) a chimeric antibody, a humanized antibody or a human antibody. Suitable chimeric, humanized and human antibodies are further described in sections IIC, IID and IIE, respectively. In certain embodiments, the anti-CD39 antibody may optionally include one or more constant regions or a portion of a constant region that is substantially human. Suitable constant regions are further described in detail in section II(F).

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

[0136] Table F

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

[0138] B. Antibodies with Similar Binding Specificities

[0139] The present disclosure also provides anti-CD39 antibodies that bind to the same or overlapping epitope as the following antibodies: the antibody referred to as 19 or ch19_IGG4.P above, or the antibody referred to as 31, ch31_IGG4.P, hu31.1_IGG4.P, hu31.2_IGG4.P, hu31.3_IGG4.P, hu31.4_IGG4.P, hu31.4_IGG1.AA, hu31.5_IGG4.P, hu31.6_IGG4.P or hu31.7_IGG4.P above, or the antibody referred to as 39, ch39_IGG4.P, ch39_mIGG2A.AA above The epitope can be identified by methods known in the art, such as X-ray crystallography of the antibody bound to the antigen to identify contact residues. Competition assays can be used to identify such antibodies. For example, the anti-CD39 antibody can competitively inhibit the binding of a reference antibody to human CD39, wherein the reference antibody is selected from 19, ch19_IGG4.P, 31, ch31_IGG4.P, hu31.1_IGG4.P, hu31.2_IGG4.P, hu31.3_IGG4.P, hu31.4_IGG4.P, hu31.4_IGG1.AA, hu31.5_IGG4.P, hu31.6_IGG4.P, or h 39, ch39_IGG4.P, ch39_mIGG2A.AAG, hu39.1_IGG4.P, hu39.2_IGG4.P, hu39.3_IGG4.P, hu39.4_IGG4.P, hu39.5_IGG4.P, hu39.5_IGG1.AA, hu39.6_IGG4.P, hu39.7_IGG4.P or hu39.8_IGG4.P. An antibody competitively inhibits binding of a reference antibody to human CD39 if the antibody blocks binding of the reference antibody to human CD39 by at least 50%, at least 60%, at least 70%, at least 80% or at least 90%. Competitive inhibition can be determined, for example, by a competitive flow assay, as described in Example 7.

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

[0141] Other antibodies with such binding specificity can be generated by immunizing mice with human CD39 or a portion thereof comprising a desired epitope; and screening the resulting antibodies for binding to the extracellular domain of human CD39, wherein the antibodies are optionally combined with antibody 19 or a variant thereof (e.g., ch19_IGG4.P, etc.), antibody 31 or a variant thereof (e.g., ch31_IGG4.P, hu31.1_IGG4.P, hu31.2_IGG4.P, hu31.3_IGG4.P, hu31.4_IGG4.P, hu31.4_IGG1.AA, hu31.5_ IGG4.P, hu31.6_IGG4.P, hu31.7_IGG4.P, etc.), or antibody 39 or its variants (e.g., ch39_IGG4.P, ch39_mIGG2A.AAG, hu39.1_IGG4.P, hu39.2_IGG4.P, hu39.3_IGG4.P, hu39.4_IGG4.P, hu39.5_IGG4.P, hu39.5_IGG1.AA, hu39.6_IGG4.P, hu39.7_IGG4.P, hu39.8_IGG4.P, etc.) compete. Antibodies can also be screened against mutation-induced forms of human CD39 to identify antibodies that show the same or similar binding profiles as the following antibodies for the set of mutation changes: 19, ch19_IGG4.P, 31, ch31_IGG4.P, hu31.1_IGG4.P, hu31.2_IGG4.P, hu31.3_IGG4.P, hu31.4_IGG4.P, hu31.4_IGG1.AA, hu31.5_IGG4.P, hu31.6_ IGG4.P, hu31.7_IGG4.P, 39, ch39_IGG4.P, ch39_mIGG2A.AAG, hu39.1_IGG4.P, hu39.2_IGG4.P, hu39.3_IGG4.P, hu39.4_IGG4.P, hu39.5_IGG4.P, hu39.5_IGG1.AA, hu39.6_IGG4.P, hu39.7_IGG4.P or hu39.8_IGG4.P. The mutation may be a systematic replacement of one residue with alanine (or serine if alanine is already present) at the same time or at more extensive intervals throughout the extracellular domain of the CD39 antibody or via a portion thereof where a known epitope is present.

[0142] Other antibodies can be obtained by inducing mutations in cDNA encoding the heavy and light chains of 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, hu31.5_IGG4.P, hu31.6_IGG4.P, hu31.7_IGG4.P, 39, ch39_IGG4.P, ch39_mIGG2A.AAG, hu39.1_IGG4.P, hu39.2_IGG4.P, hu39.3_IGG4.P , hu39.4_IGG4.P, hu39.5_IGG4.P, hu39.5_IGG1.AA, hu39.6_IGG4.P, hu39.7_IGG4.P or hu39.8_IGG4.P. The present disclosure also includes 19, ch19_IGG4.P, 31, ch31_IGG4.P, hu31.1_IGG4.P, hu31.2_IGG4.P, hu31.3_IGG4.P, hu31.4_IGG4.P, hu31.4_IGG1.AA, hu31.5_IGG4.P, hu31.6_IGG4.P, hu31.7_IGG4.P, 39, ch39_IGG4.P, ch39_mIGG2A.AAG, hu39.1_IGG4.P, hu39.2_IGG4.P, hu39.3_IGG4.P, hu39.4_IGG4.P, h 39.5_IGG4.P, hu39.5_IGG1.AA, hu39.6_IGG4.P, hu39.7_IGG4.P or hu39.8_IGG4.P are about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical in terms of the amino acid sequence of the mature heavy chain and / or light chain variable region and retain their functional properties, and / or differ from the corresponding antibody by a small number of amino acid substitutions (e.g., conservative substitutions), deletions or insertions that have no effect on function. Amino acids in the variable region framework that may be important for binding can be identified as described below in the section on humanization.

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

[0144] In addition to the antibodies described herein, it may be possible to design antibody mimetics or aptamers with the same binding specificity that are substantially the same as the antibody functions of the present invention using methods known in the art. "Antibody mimetics" refers to a polypeptide or protein that can specifically bind to an antigen but is structurally unrelated to an antibody. The mass of an antibody mimetics is about 3 kDa to about 20 kDa. Non-limiting examples of antibody mimetics are affibody molecules, affilins, affimers, alphabodies, anticalins, avimers, DARPins, and monofunctional antibodies. An aptamer is a class of small nucleic acid ligands that are composed of RNA or single-stranded DNA oligonucleotides and have high specificity and affinity for their targets. An aptamer interacts and binds to its target via structural recognition (a method similar to an antigen-antibody reaction). An aptamer has a molecular weight lower than that of an antibody, typically about 8-25 kDa.

[0145] C. Chimeric and masked antibodies

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

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

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

[0149] In some embodiments, the anti-CD39 chimeric antibodies are rat-human chimeras having rat variable domains and human IgG1 and kappa constant domains (or variants thereof) or human IgG4 and kappa constant domains (or variants thereof). Suitable human constant domains are known in the art and are further described in Section II (F). In a specific embodiment, the anti-CD39 chimeric antibodies are ch19_IGG4.P, ch31_IGG4.P, and ch39_IGG4.P.

[0150] D. Humanized Antibodies

[0151] As described herein, the present disclosure provides humanized antibodies of antibodies referred to herein as 19, 31, or 39, optionally wherein the humanized antibodies have an IC of about 5 nM or less, about 0.05 nM to about 5 nM, about 1 nM or less, about 0.05 nM to about 1 nM, about 0.5 nM or less, or about 0.05 nM to about 0.5 nM. 50 The value inhibits human CD39 enzymatic activity, as measured in Example 5. Typically, non-human antibodies are humanized to reduce immunogenicity to humans while retaining the specificity and affinity of the parent non-human antibody. Humanized antibodies include one or more variable domains, wherein CDR or a portion thereof is derived from a non-human antibody, and FR or a portion thereof is derived from a human antibody sequence. Humanized antibodies may also optionally include at least a portion of a human constant region. In some embodiments, some FR residues in humanized antibodies are replaced by corresponding residues from non-human antibodies (e.g., antibodies derived from CDR residues), for example, to restore or improve antibody specificity or affinity.

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

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

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

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

[0156] Certain amino acids from human variable region framework residues may be selected for substitution based on their possible effects on CDR conformation and / or binding to antigen. Studies of such possible effects may be conducted by modeling, examination of the characteristics of amino acids at specific positions, or empirical observation of the effects induced by substitution or mutation of specific amino acids. For example, when the amino acid difference between the non-human variable region framework residues and the selected human variable region framework residues is present, the human framework amino acids may be selected where it is reasonable to expect the amino acids to: (1) directly non-covalently bind to the antigen, (2) be adjacent to the CDR region, (3) otherwise interact with the CDR region (e.g., within about the CDR region), or (4) interact with the CDR region in other ways. In some embodiments, the present invention relates to a human immunoglobulin (H) antibody or a human immunoglobulin (I) antibody. ...

[0157] In some embodiments, the humanized anti-CD39 antibody has a mature VH comprising: H1 comprising the amino acid sequence of SEQ ID NO: 10 with zero to two amino acid substitutions or deletions, H2 comprising the amino acid sequence of SEQ ID NO: 11 with zero to two amino acid substitutions or deletions, H3 comprising the amino acid sequence of SEQ ID NO: 12 with zero to two amino acid substitutions or deletions, and framework regions having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the acceptor human framework regions; and a mature VL comprising: L1 comprising the amino acid sequence of SEQ ID NO: 14 with zero to two amino acid substitutions or deletions, L2 comprising the amino acid sequence of SEQ ID NO: 15 with zero to two amino acid substitutions or deletions, L3 comprising the amino acid sequence of SEQ ID NO: 16 with zero to two amino acid substitutions or deletions, L4 comprising the amino acid sequence of SEQ ID NO: 17 with zero to two amino acid substitutions or deletions, L5 comprising the amino acid sequence of SEQ ID NO: 18 with zero to two amino acid substitutions or deletions, L6 comprising the amino acid sequence of SEQ ID NO: 19 with zero to two amino acid substitutions or deletions, L7 comprising the amino acid sequence of SEQ ID NO: 20 with zero to two amino acid substitutions or deletions, L8 comprising the amino acid sequence of SEQ ID NO: 21 with zero to two amino acid substitutions or deletions, L9 comprising the amino acid sequence of SEQ ID NO: 22 with zero to two amino acid substitutions or deletions, L1 NO: 15 amino acid sequence, L2, L3 comprising the amino acid sequence of SEQ ID NO: 16 with zero to two amino acid substitutions or deletions, and a framework region having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the acceptor human framework region. The framework region can be determined according to the Kabat definition.

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

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

[0160] In the aforementioned embodiments, the mature heavy chain variable region may be connected to at least a portion of the heavy chain constant region and the mature light chain variable region may be connected to at least a portion of the light chain constant region. In some embodiments, the mature heavy chain variable region is connected to the heavy chain constant region and the mature light chain variable region is connected to the light chain constant region. Suitable constant regions are further described in detail in Section II (F). In certain embodiments, the heavy chain constant region has an effector function substantially similar to that of wild-type human IgG1. In other embodiments, the heavy chain constant region reduces or enhances effector function compared to wild-type human IgG1. In a specific example, the heavy chain constant region comprises SEQ ID NO: 1 or is composed of it, and the light chain constant region comprises SEQ ID NO: 6 or is composed of it. In a specific example, the heavy chain constant region comprises SEQ ID NO: 1 amino acids 1 to 329 or is composed of it, and the light chain constant region comprises SEQ ID NO: 6 or is composed of it. In another specific example, the heavy chain constant region comprises SEQ ID NO: 4 or is composed of it, and the light chain constant region comprises SEQ ID NO: 6 or is composed of it. In another specific example, the heavy chain constant region comprises SEQ ID NO:4 amino acids 1 to 326 or consists thereof, and the light chain constant region comprises SEQ ID NO:6 or consists thereof. In another specific example, the heavy chain constant region comprises SEQ ID NO:2 or consists thereof, and the light chain constant region comprises SEQ ID NO:6 or consists thereof. In another specific example, the heavy chain constant region comprises SEQ ID NO:2 amino acids 1 to 329 or consists thereof, and the light chain constant region comprises SEQ ID NO:6 or consists thereof. In another specific example, the heavy chain constant region comprises SEQ ID NO:5 or consists thereof, and the light chain constant region comprises SEQ ID NO:6 or consists thereof. In another specific example, the heavy chain constant region comprises SEQ ID NO:5 amino acids 1 to 326 or consists thereof, and the light chain constant region comprises SEQ ID NO:6 or consists thereof. In another specific example, the heavy chain constant region comprises SEQ ID NO:3 or consists thereof, and the light chain constant region comprises SEQ ID NO:6 or consists thereof. In another specific example, the heavy chain constant region comprises or consists of amino acids 1 to 329 of SEQ ID NO:3, and the light chain constant region comprises or consists of SEQ ID NO:6.

[0161] E. Human Antibodies

[0162] As described herein, the present disclosure also provides human antibodies having the binding specificity of the antibodies referred to herein 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, hu31.5_IGG4.P, hu31.6_IGG4.P, hu31.7_IGG4.P, 39, ch39_IGG4.P, ch39_mIGG2A. AAG, hu39.1_IGG4.P, hu39.2_IGG4.P, hu39.3_IGG4.P, hu39.4_IGG4.P, hu39.5_IGG4.P, hu39.5_IGG1.AA, hu39.6_IGG4.P, hu39.7_IGG4.P or hu39.8_IGG4.P, optionally wherein the human antibody has an IC of about 5 nM or less, about 0.05 nM to about 5 nM, about 1 nM or less, about 0.05 nM to about 1 nM, about 0.5 nM or less, or about 0.05 nM to about 0.5 nM. 50 The value of inhibiting human CD39 enzymatic activity as measured in Example 5. Human antibodies can be selected by competitive binding experiments, phage display methods of Winter, WO 92 / 20791, or otherwise to have the same epitope specificity as a particular rodent antibody (e.g., antibodies referred to herein as 19, 31, or 39), or to have the same epitope specificity as a particular humanized antibody (e.g., referred to herein as ch19_IGG4.P, ch31_IGG4.P, hu31.1_IGG4.P, hu31.2_IGG4.P, hu31.3_IGG4.P, hu31.4_IGG4.P, hu31.4_IGG1.AA, hu31.5_IGG4.P). .P, hu31.6_IGG4.P, hu31.7_IGG4.P, ch39_IGG4.P, ch39_mIGG2A.AAG, hu39.1_IGG4.P, hu39.2_IGG4.P, hu39.3_IGG4.P, hu39.4_IGG4.P, hu39.5_IGG4.P, hu39.5_IGG1.AA, hu39.6_IGG4.P, hu39.7_IGG4.P or hu39.8_IGG4.P). Human antibodies can also be screened for specific epitopes by using only fragments of CD39 as target antigens, and / or by screening antibodies against a collection of deletion mutants of CD39.

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

[0164] The human antibodies of the present disclosure may include a mature heavy chain variable region connected to at least a portion of the heavy chain constant region and a mature light chain variable region connected to at least a portion of the light chain constant region. In some embodiments, the mature heavy chain variable region is connected to the heavy chain constant region and the mature light chain variable region is connected to the light chain constant region. Suitable constant regions are further described in detail in Section II (F). In certain embodiments, the heavy chain constant region has an effector function substantially similar to that of wild-type human IgG1. In other embodiments, the heavy chain constant region reduces or enhances effector function compared to wild-type human IgG1. In a specific example, the heavy chain constant region comprises SEQ ID NO: 1 or is composed of it, and the light chain constant region comprises SEQ ID NO: 6 or is composed of it. In another specific example, the heavy chain constant region comprises SEQ ID NO: 4 or is composed of it, and the light chain constant region comprises SEQ ID NO: 6 or is composed of it. In another specific example, the heavy chain constant region comprises SEQ ID NO: 2 or is composed of it, and the light chain constant region comprises SEQ ID NO: 6 or is composed of it. In another specific example, the heavy chain constant region comprises or consists of SEQ ID NO: 5, and the light chain constant region comprises or consists of SEQ ID NO: 6. In another specific example, the heavy chain constant region comprises or consists of SEQ ID NO: 3, and the light chain constant region comprises or consists of SEQ ID NO: 6.

[0165] F. Selection of constant regions

[0166] The heavy chain and light chain variable regions of chimeric antibodies, humanized (including face-up) antibodies or human antibodies can each be connected to at least a portion of a human constant region. In some embodiments, the heavy chain variable domains described in the above section are connected to a portion of a human heavy chain constant region and the light chain variable domains described in the above section are connected to a portion of a human light chain constant region. In some embodiments, the heavy chain variable domains described in the above section are connected to a portion of a human heavy chain constant region and the light chain variable domains described in the above section are connected to a full-length human light chain constant region. In some embodiments, the heavy chain variable domains described in the above section are connected to a full-length human heavy chain constant region and the light chain variable domains described in the above section are connected to a full-length human light chain constant region.

[0167] The choice of constant region (or its truncation) depends in part on whether effector function is needed, or even whether enhancement is needed. "Effector function" refers to biological activities that can be attributed to the light chain or heavy chain constant region of the antibody and vary according to the antibody isotype. Non-limiting examples of antibody effector functions include: C1q binding to C1 complex and complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; cell surface receptors (e.g., B cell receptor) downregulation; and B cell activation. Human antibodies are divided into five isotypes (IgM, IgD, IgG, IgA, and IgE) according to their heavy chains, each of which provides different functions. IgG consists of four human subclasses (IgG1, IgG2, IgG3, and IgG4), each containing different heavy chains. It is highly homologous and differs mainly in the hinge region and the extent to which it activates the host immune system. For example, human isotypes IgGl and IgG3 can mediate complement-mediated cytotoxicity and human isotypes IgG2 and IgG4 mediate complement-mediated cytotoxicity at very low levels or not.The light chain constant region may be of subclass λ or κ.

[0168] The disclosed antibodies comprising a human constant region or a portion thereof are typically IgG antibodies, preferably IgG1 or IgG4 antibodies. Human constant regions show allotypic variation and isoallotypic variation between different individuals, i.e., the constant region may differ in one or more polymorphic positions in different individuals. An isoallotypic is different from an isotype in that sera that recognize an isoallotypic is bound to a non-polymorphic region of one or more other isotypes. Reference to a human constant region includes a constant region having any natural allotype or any arrangement of residues that occupy polymorphic positions in a natural allotype.

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

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

[0171] In some embodiments, the antibodies of the present disclosure are IgG1 or IgG3 antibodies. Sequences suitable for human IgG1 or IgG3 are known in the art and include, but are not limited to, SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and human IgG3 disclosed in US 5,624,821. In some embodiments, the C-terminal lysine of the IgG1 antibody or IgG3 antibody is absent.

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

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

[0174] Some antibodies of the present disclosure are engineered to have reduced Fc effector functions, such as CDC, ADCC, and antibody-dependent cellular phagocytosis (ADCP), compared to the same antibody without mutations by introducing constant region mutations. In some embodiments, each or all of these effector functions are reduced by at least 50%, 75%, 90%, or 95% compared to antibodies without mutations. Effector functions can be determined as described in the Examples. Other assays are described by Shields et al., 2001 J. Biol. Chem., Vol. 276, pp. 6591-6604; Chappel et al., 1993 J. Biol. Chem., Vol. 268, pp. 25124-25131; Lazar et al., 2006 PNAS, 103; 4005-4010.

[0175] Substitution of any or all positions in position 234, 235, 236 and / or 237 reduces affinity to Fcγ receptors, particularly to FcγRI receptors (see, e.g., US 6,624,821). In some embodiments, alanine residues are used to replace, such as L234A / L235A double mutations to reduce effector function. Other mutation combinations with reduced effector function include L234A / L235A / G237A, E233P / L234V / L235A / ΔG236, A327G / A330S / P331S, K322A, L234A / L235A, L234F / L235E / P331S and L234A / L235E / G237A / A330S / P331S (Eu numbering). Optionally, positions 234, 236 and / or 237 in human IgG2 are substituted with alanine and position 235 is substituted with glutamine. (See, e.g., US5,624,821.) Two amino acid substitutions in the complement C1q binding site at Eu index positions 330 and 331 reduce complement binding (see Tao et al., J. Exp. Med. 178: 661 (1993) and Canfield and Morrison, J. Exp. Med. 173: 1483 (1991)). Substitutions to IgG2 residues at positions 233-236 of human IgG1 and IgG4 residues at positions 327, 330 and 331 reduce ADCC and CDC (see, e.g., Armour KL. et al., 1999 Eur J Immunol. 29 (8): 2613-24; and Shields RL. et al., 2001. J Biol Chem. 276 (9): 6591-604). The N297A, N297Q or N297G (Eu numbering) mutations reduce glycosylation and thereby reduce effector function.

[0176] Some antibodies disclosed herein are engineered to have enhanced Fc effector functions by introducing constant region mutations. For example, FcγR binding can be enhanced by amino acid engineering. In some embodiments, this can be performed by replacing one or more amino acids in the Fc region. Desirable mutations can be determined by, for example, alanine screening or rational design and library screening. These techniques can be used to identify IgG variants with enhanced binding to FcγR and enhanced effector functions. Alternatively, several mutations in the Fc receptor region are known in the art, such as described in Smith P. et al. (2012) PNAS 6181-6186.

[0177] In some embodiments, the antibodies described herein include a modified IgG1 constant domain that increases the ability of the antibody to mediate ADCC compared to a wild-type IgG1 without modification. The modified IgG1 domain can be characterized by amino acid substitutions at one or more of L235V, S239D, F243L, R292P, A330L, I332E, P396L (Eu numbering). In other embodiments, the modified IgG1 domain is characterized by substitutions at S239D, A330L, and I332E (Eu numbering).

[0178] Alternatively, glycoform perturbations can be used to enhance Fc-mediated therapeutic antibody function. N-linked Fc glycosylation on IgG1 antibodies is essential for effector function. Sialylation, galactosylation, bisecting sugars, and fucosylation can all affect the binding and activity of IgG molecules. Controlling the glycosylation pattern on therapeutic antibodies can be done in many different ways. The cell type and its culture conditions that produce recombinant antibodies can affect the glycosylation and activity of therapeutic antibodies. In addition, bioreactor conditions and downstream processing can also affect glycan micro-heterogeneity. Low or no fucosylated antibodies have been shown to enhance Fc-mediated properties. Many ways to achieve this reduction in fucose levels through glycoengineering are well known in the art. One way is to manipulate enzymes involved in antibody post-translational modification. This can involve overexpression of glucosidases, such as β-1-4-N-acetylglucosamine transferase III, knocking out fucosyltransferases, or using cell lines that naturally lack fucose or have been mutated to express low fucosylation levels. In addition, inhibitors of N-linked glucosidases, such as castanospermine, can also be used to obtain low-fucose IgG molecules.

[0179] In some embodiments, amino acid engineered variants may have a more broadly enhanced affinity for a variety of FcγRs, while glycoform engineered antibodies may generally have a more specific affinity for enhanced FcγRIIIa binding. Glycoforms interact with proximal amino acids on the Fc portion and replacing amino acids that contact Ig oligosaccharides can produce different glycoform structures.

[0180] G. Expression of recombinant antibodies

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

[0182] The polynucleotide encoding the anti-CD39 antibody of the present disclosure can be inserted into a vector for amplification, expression or further optimization. Many vectors are available. In some embodiments, the vector system includes mammals, bacteria, yeast systems, etc., and includes, for example, but not limited to, the following plasmids: pALTER, pBAD, pcDNA, pCal, pL, pET, pGEMEX, pGEX, pCI, pCMV, pEGFP, pEGFT, pSV2, pFUSE, pVITRO, pVIVO, pMAL, pMONO, pSELECT, pUNO, pDUO, Psg5L, pBABE, pWPXL, pBI, p15TV-L, pPro18, pTD, pRS420, pLexA, pACT2.2, etc., as well as other laboratories and commercially available vectors. Suitable vectors may include plasmids or viral vectors (e.g., replication-deficient retroviruses, adenoviruses, and adeno-associated viruses). The vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter (e.g., SV40, CMV, EF-1α), and a transcription termination sequence. For expression, the recombinant polynucleotide construct generally includes an expression control sequence operably connected to the coding sequence of the antibody chain, including a naturally associated or heterologous promoter region. In some embodiments, the expression control sequence is a eukaryotic promoter system in the vector that can transform or transfect a eukaryotic host cell. Once the vector has been incorporated into an appropriate host, the host is maintained under conditions suitable for high-level expression of the nucleotide sequence and for collecting and purifying the recombinant antibody.

[0183] The vector containing the polynucleotide sequence encoding the anti-CD39 antibody of the present disclosure can be introduced into the host cell for cloning or gene expression. The host cells suitable for cloning or expressing the polynucleotide sequence in the vector herein include prokaryotes and eukaryotic cells. Non-limiting examples of suitable prokaryotes include eubacteria, such as Gram-negative or Gram-positive organisms, such as Enterobacteriaceae, such as Escherichia, such as Escherichia coli (E.coli); Enterobacter; Erwinia; Klebsiella; Proteus; Salmonella, such as Salmonella typhimurium; Serratia, such as Serratia marcescens. marcescans); and Shigella; and Bacilli, such as B. subtilis and B. licheniformis; Pseudomonas, such as Pseudomonas aeruginosa; and Streptomyces. In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are suitable cloning or expression hosts for vectors encoding anti-CD39 antibodies.Non-limiting examples include Saccharomyces cerevisiae, Schizosaccharomyces pombe; Kluyveromyces hosts, such as K. lactis, K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906), K. thermotolerans, and K. marxianus; Yarrowia (EP 402,226); Pichia pastoris (EP 402,226); 183,070); Candida; Trichoderma reesia (EP 244,234); Neurosporacrassa; Schwanniomyces, such as Schwanniomyces occidentalis; and filamentous fungi, such as Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts, such as A. nidulans and A. niger. Suitable host cells can also be derived from multicellular organisms. Examples of invertebrate cells include plant and insect cells. A variety of baculovirus strains and variants have been identified, as well as corresponding permissive insect host cells from hosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruit fly), and Bombyx mori. A variety of virus strains for transfection are publicly available, such as the L-1 variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV, and such viruses can be used as viruses herein according to the present invention, particularly for transfecting Spodoptera frugiperda cells.Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato and tobacco can also be used as hosts. In some embodiments, mammalian cells are host cells for expressing nucleotide segments encoding immunoglobulins or their fragments. See Winnacker, From Genes to Clones, (VCH Publishers, NY, 1987). Multiple suitable host cell lines capable of secreting complete heterologous proteins have been developed in the art, and include CHO cell lines, various COS cell lines, HeLa cells, HEK293 cells, L cells and non-antibody-producing myeloma, including Sp2 / 0 and NS0. In some embodiments, cells are non-human. The expression vectors of these cells may include, for example, expression control sequences of replication origins, promoters, enhancers (Queen et al., Immunol. Rev. 89: 49 (1986)), and, for example, ribosome binding sites, RNA splicing sites, polyadenylation sites, and necessary processing information sites, and transcription terminator sequences. In some embodiments, the expression control sequence is a promoter derived from an endogenous gene, cytomegalovirus, SV40, adenovirus, bovine papilloma virus, etc. See Co et al., J. Immunol. 148: 1149 (1992).

[0184] Host cells are transformed with the above-mentioned expression or cloning vectors used to produce anti-CD39 antibodies and cultured in conventional nutrient media adjusted as appropriate to induce promoters, select transformants or amplify genes encoding the desired sequences. Once expressed, antibodies can be purified according to standard procedures in the art, including HPLC purification, column chromatography, gel electrophoresis, etc. (see generally Scopes, Protein Purification (Springer-Verlag, NY, 1982)).

[0185] H. Labeled Antibodies

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

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

[0188] III. How to use

[0189] The present disclosure provides methods for preparing drugs for inhibiting CD39 enzyme activity using the anti-CD39 antibodies described herein. As used herein, the terms "inhibit", "inhibition", etc. refer to the ability of an antagonist to reduce the function or activity of a specific target (e.g., CD39). The reduction is preferably at least 50% and may be, for example, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95%. The present disclosure also encompasses the use of the anti-CD39 antibodies described herein for the preparation of drugs for treating or preventing diseases, disorders, and / or conditions that would benefit from inhibiting CD39 enzyme activity. As an example, the present disclosure encompasses the use of an anti-CD39 antibody described herein for the preparation of a medicament for treating a disease characterized by: (i) high extracellular ATP (compared to non-diseased tissue), (ii) expression of ENTPD1, ​​P2RX7, P2RY11, or a combination thereof, or increased expression in diseased tissue, (iii) expression of CD39, P2X7, P2Y11, or a combination thereof, or increased expression in diseased tissue, (iv) bone marrow infiltration into diseased tissue, or any combination of (i) to (iv). In another example, the present disclosure encompasses the use of an anti-CD39 antibody described herein for the preparation of a medicament for treating cancer. Optionally, the cancer may be characterized by: (i) high extracellular ATP (compared to non-lesional samples), (ii) expression of ENTPD1, ​​P2RX7, P2RY11, or a combination thereof, or increased expression in diseased tissue, (iii) expression of CD39, P2X7, P2Y11, or a combination thereof, or increased expression in diseased tissue, (iv) bone marrow infiltration into diseased tissue, or any combination of (i) to (iv). In some embodiments of the foregoing methods, an anti-CD39 antibody described herein is used in combination with at least one additional therapy, examples of which are described elsewhere herein.

[0190] Extracellular ATP exists in negligible concentrations (e.g., about 10-100 nM) in healthy conditions, but increases rapidly in response to tissue damage, stress, hypoxia, and agents used to treat cancer, and can be found in tumors at high concentrations. ATP released by stained or stressed cells (including but not limited to cancer cells and other cells in the tumor microenvironment) provides inflammatory signals important for effective innate and adaptive immune responses. Conversely, for example, hydrolysis of extracellular ATP to adenosine in the tumor microenvironment limits immune responses. CD39 is the rate-limiting extracellular enzyme in the hydrolysis of extracellular ATP. By catabolizing the conversion of extracellular ATP to AMP, CD39 also increases extracellular adenosine production via CD73 (extracellular-5'-nucleotidase), the rate-limiting extracellular enzyme in the hydrolysis of extracellular AMP. Adenosine is signaled via type 1 purinergic receptors and has effects opposite to those mediated by ATP receptors. In particular, A expressed on the surface of immune cells 2a and A 2b Receptors help mediate the immunosuppressive effects of adenosine. For example, in the tumor microenvironment, adenosine binds to A on tumor-infiltrating immune cells. 2a and A 2b Receptor direct interaction triggers immunosuppression. The opposite effects of adenosine and ATP are reviewed in Chiarella et al., "Extracellular ATP and Adenosine in Cancer Pathogenesis and Treatment", Trends in Cancer, 2021, 7 (8): 731-750. As demonstrated herein, the anti-CD39 antibody disclosed herein is used to strongly inhibit CD39 enzyme activity, thereby leading to immune stimulation from ATP accumulation and preventing immunosuppressive adenosine formation. Diseases, disorders and / or conditions that will benefit from inhibiting CD39 enzyme activity may include diseases, disorders and / or conditions in which the release of extracellular ATP may be higher, for example due to tissue damage, stress, hypoxia treatment by additional therapy, or any combination thereof. Additional diseases, disorders, and / or conditions that would benefit from inhibition of CD39 enzymatic activity may include additional diseases, disorders, and / or conditions in which extracellular adenosine levels are higher in diseased tissue samples, e.g., due to increased ATP hydrolysis compared to healthy controls and / or due to increased AMP hydrolysis compared to healthy controls (e.g., as measured by increased CD39 enzymatic activity in a peripheral blood sample or a tissue (e.g., tumor) sample), and / or in which CD39 and / or CD73 expression is detectable, and optionally higher, compared to healthy controls, e.g., as measured in a peripheral blood sample or a tissue (e.g., tumor) sample by immunohistochemistry, immunophenotype, RNA sequencing or other clinically validated method.

[0191] Thus, in some embodiments, an anti-CD39 antibody described herein is administered to a subject in need thereof in an amount effective to inhibit CD39 enzymatic activity. CD39 enzymatic activity can be assessed using a peripheral blood sample or a tissue (e.g., tumor) sample obtained from the subject, or both. As a non-limiting example, for example, or Commercial assays are used to measure CD39 activity in cells and non-cellular compartments of peripheral blood samples or tumor samples. Alternatively or in addition, CD39 enzyme activity in tumor tissues can be measured by enzyme histochemistry. Inhibition can be determined, for example, by comparing with a previous sample obtained from a subject (i.e., before administering an anti-CD39 antibody) or by comparing with a reference sample or reference value of a control group (e.g., a subject administered an isotype control antibody, an anti-CD39 antibody that binds but does not inhibit CD39 enzyme activity, standard care, placebo, etc.). In some embodiments, the anti-CD39 antibodies described herein are administered to subjects in need in an amount that effectively inhibits CD39 enzyme activity in the tumor microenvironment. In some embodiments, the anti-CD39 antibodies described herein are administered to subjects in need in an amount that effectively inhibits CD39 enzyme activity in the periphery of the tumor. In some embodiments, the anti-CD39 antibodies described herein are administered to subjects in need in an amount that effectively inhibits CD39 enzyme activity in or on immune cells. In some embodiments, the anti-CD39 antibodies described herein are administered to a subject in need in an amount effective to inhibit CD39 enzymatic activity in or on cells of the myeloid lineage. Myeloid lineage cells expressing CD39 include those cells described in detail in the Examples. In some embodiments, the anti-CD39 antibodies described herein are administered to a subject in need in an amount effective to inhibit CD39 enzymatic activity in or on the matrix and / or blood vessels.

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

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

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

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

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

[0197] A. Oncology and Oncology-Related Conditions

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

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

[0200] In another embodiment, the antibodies according to the present disclosure are suitable for treating and / or preventing solid tumors. Solid tumors can be, for example, ovarian cancer, endometrial cancer, breast cancer, lung cancer (small cell or non-small cell), colon cancer, prostate cancer, cervical cancer, bile duct cancer, pancreatic cancer, gastric cancer, esophageal cancer, liver cancer (hepatocellular carcinoma), kidney cancer (renal cell carcinoma), head and neck tumors, mesothelioma, melanoma, sarcoma, central nervous system (CNS) hemangioblastoma and brain tumors (e.g., gliomas, such as astrocytomas, oligodendrogliomas and glioblastomas).

[0201] In another embodiment, the antibodies according to the present disclosure are suitable for treating and / or preventing lung cancer, urogenital cancer, gastrointestinal cancer, or a combination thereof. In another embodiment, the antibodies according to the present disclosure are suitable for treating and / or preventing lung cancer, urogenital cancer, gastrointestinal cancer, skin cancer, or a combination thereof.

[0202] In some embodiments, the antibodies according to the present disclosure are suitable for treating and / or preventing skin cancer. In other embodiments, the skin cancer is melanoma.

[0203] In some embodiments, antibodies according to the present disclosure are suitable for treating and / or preventing breast cancer. In other embodiments, breast cancer is hormone receptor positive (e.g., ERα-positive breast cancer, PR-positive breast cancer, ERα-positive and PR-positive breast cancer), HER2-positive breast cancer, HER2-overexpressing breast cancer, or any combination thereof. In other embodiments, breast cancer is triple-negative breast cancer.

[0204] In some embodiments, antibodies according to the present disclosure are suitable for treating and / or preventing renal cancer. In other embodiments, the renal cancer is renal cell carcinoma. In yet other embodiments, the renal cell carcinoma is clear cell renal carcinoma.

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

[0206] In some embodiments, the disclosed methods of treating non-small cell lung cancer may further include administering any of the disclosed anti-CD39 antibodies in combination with pemetrexed, carboplatin, and an antagonist anti-PD-1 antibody or an antagonist anti-PD-L1 antibody. Such methods may further include administering one or more additional agents selected from the group consisting of: antagonist anti-TIGIT antibodies, A2aR antagonists, A2bR antagonists, A2a / 2bR antagonists, and CD73 inhibitors, optionally wherein the additional agent is selected from the group consisting of: dovanalimumab, AB308, etelumadram, and quetiapine.

[0207] In some embodiments, the antibodies according to the present disclosure are suitable for treating and / or preventing pancreatic cancer. In other embodiments, the pancreatic cancer is a pancreatic neuroendocrine tumor or a pancreatic adenocarcinoma.

[0208] In some embodiments, the antibodies according to the present disclosure are suitable for treating and / or preventing neuroendocrine tumors. In other embodiments, the neuroendocrine tumor is a pancreatic neuroendocrine tumor, a pheochromocytoma, a paraganglioma, or an adrenal tumor.

[0209] In some embodiments, antibodies according to the present disclosure are suitable for treating and / or preventing brain cancer. In other embodiments, the brain cancer is a glioma. In yet other embodiments, the glioma is an astrocytoma, an oligodendroglioma, or a glioblastoma.

[0210] In some embodiments, antibodies according to the present disclosure are suitable for treating and / or preventing upper GI cancers, such as esophageal cancer or gastric cancer. In other embodiments, the upper GI cancer is adenocarcinoma, squamous cell carcinoma, or any combination thereof. In yet other embodiments, the upper GI cancer is esophageal adenocarcinoma (EAC), esophageal squamous cell carcinoma (ESCC), gastroesophageal junction adenocarcinoma (GEJ), gastric adenocarcinoma (also referred to herein as "gastric cancer"), or any combination thereof.

[0211] In some embodiments, the disclosed methods of treating gastric cancer or gastroesophageal cancer may further include administering any of the disclosed anti-CD39 antibodies in combination with FOLFOX and an antagonist anti-PD-1 antibody or an antagonist anti-PD-L1 antibody. Such methods may also include administering one or more additional agents selected from the group consisting of: antagonist anti-TIGIT antibodies, A2aR antagonists, A2bR antagonists, A2a / 2bR antagonists, and CD73 inhibitors, optionally wherein the additional agent is selected from the group consisting of: dovanalimumab, AB308, etelumadram, and quetiapine.

[0212] In some embodiments, the antibodies according to the present disclosure are suitable for treating and / or preventing lymphoma.In other embodiments, the hematological malignancy is acute myeloid lymphoma.

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

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

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

[0216] In the aforementioned embodiments, the method of the present disclosure can be practiced in an auxiliary situation or a neo-adjuvant situation. The method described herein may be indicated as a first-line, second-line, third-line or higher-line treatment. In some embodiments, the method of the present disclosure may be practiced in a second-line or higher-line treatment, wherein the early treatment line includes a checkpoint inhibitor (ie, the subject has been treated with a checkpoint inhibitor (CPI)). In other embodiments, the checkpoint inhibitor is a CTLA-4 antagonist, a PD-1 antagonist, a PD-L1 antagonist, a TIM-3 antagonist or a TIGIT antagonist.

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

[0218] In general, the disclosed methods for treating or preventing cancer or cancer-related diseases, disorders or conditions in subjects in need thereof include administering an anti-CD39 antibody of Part II to the subject. Administration of the anti-CD39 antibody of Part II may comprise one or more (e.g., one, two, or three or more) dosing cycles. In some embodiments, the disclosure provides methods for treating or preventing cancer or cancer-related diseases, disorders or conditions with an anti-CD39 antibody of Part II and at least one additional therapy, examples of which are described elsewhere herein.

[0219] Patient selection.In some cases, methods according to the present disclosure may be provided in selected patients, such as patients identified as having, for example, detectable PD-L1, CD73 and / or CD39 expression, high microsatellite instability, high tumor mutation load, or any combination thereof. In some cases, patients are identified as having an oncogene-driven cancer with a mutation in at least one gene associated with cancer. In some cases, patients are selected by determining the patient's P2X7 variant. Normally expressed P2X7 splice variants, different SNPs, and post-translational receptor modifications may affect the function of P2X7. For example, it has been shown that certain modifications can cause partial or complete loss of P2X7 function, and other modifications have been shown to increase P2X7 function. See, for example, Lara et al., Front Pharmacol, 2020, 11: 793.

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

[0221] B. Route of Administration

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

[0223] In another embodiment, a pharmaceutical composition containing an antibody according to the present disclosure may be in a form suitable for parenteral administration. Forms of parenteral administration include, but are not limited to, intravenous, intraarterial, intramuscular, intradermal, intraperitoneal, intrathecal, intracisternal, intracerebral, intraventricular, intraventricular, and subcutaneous. Pharmaceutical compositions suitable for parenteral administration may be formulated using suitable aqueous or non-aqueous carriers. Reservoir injections, which are typically administered subcutaneously or intramuscularly, may also be used to release the antibodies disclosed herein over a defined period of time.

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

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

[0226] C. Pharmaceutical Compositions

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

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

[0229] In one aspect, the anti-CD39 antibodies described herein can be administered orally. Oral administration can be via, for example, capsules or tablets. In the preparation of a pharmaceutical composition comprising an anti-CD39 antibody, the tablet or capsule typically includes at least one pharmaceutically acceptable excipient. Non-limiting examples of pharmaceutically acceptable excipients include lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum arabic, calcium phosphate, alginate, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinyl pyrrolidone, polyethylene glycol, cellulose, sterile water, syrup and methylcellulose. Additional pharmaceutically acceptable excipients include: lubricants such as talc, magnesium stearate and mineral oil; wetting agents; emulsifiers and suspending agents; preservatives such as methyl hydroxybenzoate and propyl hydroxybenzoate. Some oral administration forms include taste masking agents, sweeteners or flavoring agents. Oral dosage forms can be formulated as solutions or suspensions.

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

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

[0232] D.Administration

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

[0234] In some embodiments, the disclosed methods include administering a therapeutically effective amount of an anti-CD39 antibody described herein to a subject in need thereof. As used herein, the phrase "therapeutically effective amount" with respect to an anti-CD39 antibody means providing a dosage regimen (i.e., amount and time interval) of an antibody for administering a specific pharmacological effect of an antibody to a subject in need of such treatment. For preventive use, a therapeutically effective amount can effectively eliminate or reduce the risk of a disease, reduce the severity of the disease, or postpone the onset of the disease, including biochemical, histological, and / or behavioral signs or symptoms of the disease. For treatment, a therapeutically effective amount can effectively reduce, improve, or eliminate one or more signs or symptoms associated with the disease, postpone disease progression, prolong survival, reduce the dose of other drugs required to treat the disease, or a combination thereof. Specifically, with respect to cancer, a therapeutically effective amount can, for example, lead to cancer cell killing, reduce cancer cell counts, reduce tumor load, eliminate tumors or cancer metastasis, or reduce metastatic spread. A therapeutically effective amount of an anti-CD39 antibody may not always be effective in treating each individual subject, but is considered a therapeutically effective amount by those skilled in the art. The therapeutically effective amount may vary based on, for example, one or more of the subject's age and weight, the subject's overall health, the subject's disease stage, route of administration, and prior or concomitant treatment.

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

[0236] In certain embodiments, the anti-CD39 antibodies of the present disclosure are contained in a "unit dosage form". The phrase "unit dosage form" refers to physically discrete units, each unit containing a predetermined amount of an anti-CD39 antibody sufficient to produce the desired effect, alone or in combination with one or more additional agents. It will be appreciated that the parameters of the unit dosage form will depend on the specific agent and the effect to be achieved.

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

[0238] The present disclosure encompasses the use of the anti-CD39 antibodies of Part II alone or in combination with one or more additional therapies. Each additional therapy may be a therapeutic agent or another therapeutic modality. In embodiments comprising one or more additional therapeutic agents, each agent may target different but complementary mechanisms of action. Additional therapeutic agents may be small chemical molecules; macromolecules, such as proteins, antibodies, peptidomimetics, peptides, DNA, RNA, or fragments of such macromolecules; or cell or gene therapy. Non-limiting examples of additional therapeutic modalities include surgical removal of tumors, bone marrow transplantation, radiotherapy, and photodynamic therapy. The use of the anti-CD39 antibodies of Part II in combination with one or more additional therapies may have a synergistic or additive therapeutic or preventive effect on underlying diseases, disorders, or conditions. Additionally or alternatively, combination therapy may allow a reduction in the dose of one or more of the therapies, thereby improving, reducing, or eliminating adverse effects associated with one or more of the agents.

[0239] In the embodiment comprising one or more additional treatment modalities, the anti-CD39 antibody of Part II can be used before, after or during treatment with the additional treatment modality. In the embodiment comprising one or more additional therapeutic agents, the therapeutic agent used in such combination therapy can be formulated as a single composition or as a separate composition. If applied separately, each therapeutic agent in the combination can be given at the same or approximately the same time or at different times. In addition, the "combination" administration of therapeutic agents, even if it has different forms of administration (e.g., oral capsules and intravenous), is given at different dosing intervals, a therapeutic agent is given with a constant dosing regimen, and titration increases, titration decreases or suspends another therapeutic agent, or titration increases, titration decreases, dosage increases or decreases, or suspends and / or restores each therapeutic agent in the combination during the course of treatment of the patient. If the combination is formulated as a separate composition, then in some embodiments, the separate composition is provided together in a medicine box.

[0240] Cancer Therapy

[0241] The present disclosure encompasses the use of the combination of anti-CD39 antibodies of Part II with one or more additional therapies suitable for treating cancer or cancer-related diseases, disorders or illnesses. In some embodiments, one or more of the additional therapies are additional treatment modalities. Exemplary treatment modalities include, but are not limited to, surgical removal of tumors, bone marrow transplantation, radiotherapy, and photodynamic therapy. In some embodiments, one or more of the additional therapies are therapeutic agents. Exemplary therapeutic agents include chemotherapeutics, radiopharmaceuticals, hormone therapy, epigenetic regulators, ATP-adenosine axis targeting agents, targeted therapies, signal transduction inhibitors, RAS signaling inhibitors, PI3K inhibitors, arginase inhibitors, HIF inhibitors, AXL inhibitors, PAK4 inhibitors, immunotherapeutics, cell therapy, gene therapy, immune checkpoint inhibitors, and stimulatory or costimulatory immune checkpoint agonists.

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

[0243] In some embodiments, one or more of the additional therapeutic agents is a radiopharmaceutical. Radiopharmaceuticals are a form of internal radiotherapy in which a radiation source (i.e., one or more radionuclides) is placed in the subject's body. The radiation source may be in solid or liquid form. Non-limiting examples of radiopharmaceuticals include sodium iodine I-131, radium dichloride-223, iodobenzylguanidine iodine-131, radioiodine-labeled vesicles (e.g., saposin C-dioleoylphosphatidylserine (SapC-DOPS) nanovesicles), various forms of close-up therapy, and various forms of targeted radionuclides. Targeted radionuclides include molecules ("targeting agents") that are specifically bound to a target on a cell (usually a cancer cell or an immune cell) and are associated (e.g., by covalent or ionic interactions) with a radionuclide. Targeting agents may be small molecules, sugars (including oligosaccharides and polysaccharides), antibodies, lipids, proteins, peptides, non-natural polymers, or aptamers. In some embodiments, the targeting agent is a sugar (including oligosaccharides and polysaccharides), a lipid, a protein, or a peptide, and the target is a tumor-associated antigen (enriched but not specific to cancer cells), a tumor-specific antigen (minimal to no expression in normal tissues), or a neoantigen (an antigen specific to the genome of cancer cells generated by non-synonymous mutations or gene fusions in the genome of tumor cells). In some embodiments, the targeting agent is an antibody and the target is a tumor-associated antigen (i.e., an antigen enriched but not specific to cancer cells), a tumor-specific antigen (i.e., an antigen with minimal to no expression in normal tissues), or a neoantigen (i.e., an antigen specific to the genome of cancer cells generated by non-synonymous mutations or gene fusions in the genome of tumor cells). Non-limiting examples of targeting radionuclides include radionuclides attached to: somatostatin or its peptide analogs (e.g., 177Lu-Dotatate, etc.); prostate specific membrane antigen or its peptide analogs (e.g., 177Lu-PSMA-617, 225Ac-PSMA-617, 177Lu-PSMA-I&T, 177Lu-MIP-1095, etc.); receptor cognate ligands, peptides derived from ligands, or variants thereof (e.g., 188Re-labeled VEGF 125-136 or variants thereof with higher affinity to VEGF receptors, etc.); antibodies targeting tumor antigens (e.g. 131I-tositumomab, 90Y-ibritumomab tiuxetan, CAM-H2-I131 (Precirix NV), I131-omburtamab, etc.).

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

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

[0246] In some embodiments, one or more of the additional therapeutic agents is an ATP-adenosine axis targeting agent. The ATP-adenosine axis targeting agent changes the signal transduction mediated by adenosine nucleosides and nucleotides (e.g., adenosine, AMP, ADP, ATP), for example, by regulating adenosine levels or targeting adenosine receptors. Adenosine and ATP, which act at different classes of receptors, generally have opposite effects on inflammation, cell proliferation, and cell death. For example, ATP and other adenine nucleotides have anti-tumor effects via activation of the PS2Y1 receptor subtype, while the accumulation of adenosine in the tumor microenvironment has been shown to inhibit the anti-tumor function of various immune cells and enhance the immunosuppressive activity of myeloid and regulatory T cells by binding to cell surface adenosine receptors. In certain embodiments, the ATP-adenosine axis targeting agent is an inhibitor of an ectonucleotidase involved in the conversion of ATP to adenosine or an antagonist of an adenosine receptor. Ecto-nucleotidases involved in the conversion of ATP to adenosine include ectonucleoside triphosphate diphosphohydrolase 1 (ENTPD1, ​​also known as CD39 or cluster of differentiation 39) and ecto-5'-nucleotidase (NT5E or 5NT, also known as CD73 or cluster of differentiation 73). Exemplary small molecule CD73 inhibitors include CB-708, ORIC-533, LY3475070, and AB680. Exemplary anti-CD73 antibodies include CPI-006, oleclumab (MEDI9447), NZV930, IPH5301, GS-1423, uliledlimab (TJD5, TJ004309), and BMS-986179. In one embodiment, the present disclosure encompasses a combination of an anti-CD39 antibody of Part II with a CD73 inhibitor, such as the inhibitors described in WO 2017 / 120508, WO 2018 / 067424, WO 2018 / 094148, and WO 2020 / 046813. In other embodiments, the CD73 inhibitor is quiliclusstat. Adenosine can bind to and activate four different G protein-coupled receptors: A1R, A2R, A3R, A4R, A5R, A6R, A7R, A8R, A9R, A10R, A20R, A11R, A21R, A31R, A41R, A51R, A6R, A73R, A8R, A9R, A11R, A12R, A13R, A14R, A15R, A16R, A17R, A18R, A19R, A20R, A21R, A31R, A19R, A21R, A31R, A4 2A R.A. 2B R and A3R. A2R antagonists include itrumadan, inupadenant, taminadenant, caffeine citrate, NUV-1182, TT-702, DZD-2269, INCB-106385, EVOEXS-21546, AZD-4635, imaradenant, RVU-330, ciforadenant, PBF-509, PBF-999, PBF-1129 and CS-3005. In some embodiments, the present disclosure encompasses the anti-CD39 antibodies of Part II and A 2A R antagonist, A2B R antagonist or A 2A R and A 2B In some embodiments, the present disclosure encompasses a combination of an anti-CD39 antibody of Part II with an adenosine receptor antagonist described in WO 2018 / 136700, WO 2018 / 204661, WO 2018 / 213377 or WO 2020 / 023846, WO 2020 / 102646. In one embodiment, the adenosine receptor antagonist is etrumadan.

[0247] In some embodiments, one or more of the additional therapeutic agents is targeted therapy. In one aspect, targeted therapy may include chemotherapeutic agents, radionuclides, hormone therapy, or another small molecule drug connected to a targeting agent. The targeting agent may be a small molecule, a sugar (including oligosaccharides and polysaccharides), an antibody, a lipid, a protein, a peptide, a non-natural polymer, or an aptamer. In some embodiments, the targeting agent is a sugar (including oligosaccharides and polysaccharides), a lipid, a protein, or a peptide, and the target is a tumor-associated antigen (enriched but not specific to cancer cells), a tumor-specific antigen (expressed minimally to no expression in normal tissues), or a new antigen (specific antigens for the genome of cancer cells produced by non-synonymous mutations in the tumor cell genome). In some embodiments, the targeting agent is an antibody, and the target is a tumor-associated antigen (enriched but not specific to cancer cells), a tumor-specific antigen (expressed rarely to no expression in normal tissues), or a new antigen (specific antigens for the genome of cancer cells produced by non-synonymous mutations in the tumor cell genome). In some embodiments, targeted therapy is an antibody-drug conjugate comprising an antibody and a drug, wherein the antibody specifically binds to HER2, HER3, nectin-4 or Trop-2. Specific examples of targeted therapy comprising antibodies and drugs include, but are not limited to, patritumab deruxtecan, sacituzumab govitecan-hziy, telisotuzumab vedotin and trastuzumab deruxtecan. In other aspects, targeted therapy can inhibit or interfere with specific proteins that help tumors survive, grow and / or spread. Non-limiting examples of such targeted therapies include signal transduction inhibitors, RAS signaling inhibitors, inhibitors of oncogenic transcription factors, activators of oncogenic transcription factor inhibitors, angiogenesis inhibitors, immunotherapeutics, ATP-adenosine axis targeting agents, AXL inhibitors, PARP inhibitors, PAK4 inhibitors, PI3K inhibitors, HIF2α inhibitors, CD73 inhibitors, A2R antagonists, TIGIT antagonists, and PD-1 antagonists. ATP-adenosine axis targeting agents are described above, while other agents are described in further detail below.

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

[0249] In some embodiments, one or more of the additional therapeutic agents are RAS signaling inhibitors.RAS gene families, such as oncogenic mutations in HRAS, KRAS and NRAS are associated with a variety of cancers.For example, G12C, G12D, G12V, G12A, G13D, Q61H, G13C and G12S and other mutations in KRAS family genes have been observed in multiple tumor types.Direct and indirect inhibition strategies have been studied for inhibiting mutant RAS signaling.Indirect inhibitors target effectors other than RAS in the RAS signaling pathway, and include but are not limited to inhibitors of RAF, MEK, ERK, PI3K, PTEN, SOS (such as SOS1), mTORC1, SHP2 (PTPN11) and AKT. Non-limiting examples of indirect inhibitors under development include RMC-4630, RMC-5845, RMC-6291, RMC-6236, JAB-3068, JAB-3312, TNO155, RLY-1971, BI1701963. Direct inhibitors of RAS mutants have also been explored, and KRAS-GTP complexes or KRAS-GDP complexes are generally targeted. Exemplary direct RAS inhibitors under development include but are not limited to sotorasib (AMG510), MRTX849, mRNA-5671 and ARS1620. In some embodiments, one or more RAS signaling inhibitors are selected from the group consisting of: RAF inhibitors, MEK inhibitors, ERK inhibitors, PI3K inhibitors, PTEN inhibitors, SOS1 inhibitors, mTORC1 inhibitors, SHP2 inhibitors and AKT inhibitors. In other embodiments, one or more RAS signaling inhibitors directly inhibit RAS mutants.

[0250] In some embodiments, one or more of the additional therapeutic agents are inhibitors of phosphatidylinositol 3-kinase (PI3K), particularly inhibitors of PI3Kγ isoforms.PI3Kγ inhibitors can stimulate anti-cancer immune responses via regulating myeloid cells, for example, by suppressing suppressive myeloid cells, weakening immunosuppressive tumor-infiltrating macrophages, or by stimulating macrophages and dendritic cells to manufacture cytokines that facilitate effective T cell responses, thereby reducing cancer development and spread.Exemplary PI3Kγ inhibitors include copanlisib, duvelisib, AT-104, ZX-101, tenalisib, eganelisib, SF-1126, AZD3458, and pictilisib.In some embodiments, the anti-CD39 antibodies of Part II can be combined with one or more PI3Kγ inhibitors described in WO 2020 / 0247496A1.

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

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

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

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

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

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

[0257] In some embodiments, one or more of the additional therapeutic agents is an immunotherapeutic agent. Immunotherapeutics suitable for treating cancer generally induce or amplify an immune response against cancer cells. Non-limiting examples of suitable immunotherapeutics include: immunomodulators; cellular immunotherapy; vaccines; gene therapy; ATP-adenosine axis targeting agents; immune checkpoint regulators; and certain signal transduction inhibitors. ATP-adenosine axis targeting agents are described above. Immunomodulators, signal transduction inhibitors, cellular immunotherapy, vaccines, gene therapy, and immune checkpoint regulators are further described below.

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

[0259] In some embodiments, one or more of the additional therapeutic agents are immunotherapeutics, more specifically cell therapy. Cell therapy is a form of treatment in which living cells are administered to a subject. In certain embodiments, one or more of the additional therapeutic agents are cell immunotherapies that activate or suppress the immune system. Cell immunotherapies suitable for treating cancer generally induce or amplify immune responses. Cells may be autologous or allogeneic immune cells (e.g., monocytes, macrophages, dendritic cells, NK cells, T cells, etc.) collected from one or more subjects. Alternatively, cells may be "(re)programmed" allogeneic immune cells produced by immune precursor cells (e.g., lymphocyte progenitors, bone marrow progenitors, common dendritic cell precursors, stem cells, induced pluripotent stem cells, etc.). In some embodiments, such cells may be an amplicon set of cells with different effector functions and / or maturation markers (e.g., adaptive memory NK cells, tumor infiltrating lymphocytes, immature dendritic cells, monocyte-derived dendritic cells, plasmacytoid dendritic cells, conventional dendritic cells (sometimes referred to as typical dendritic cells), M1 macrophages, M2 macrophages, etc.), may be genetically modified to target cells to specific antigens and / or enhance the anti-tumor effects of cells (e.g., engineered T cell receptor (TCR) cell therapy, chimeric antigen receptor (CAR) cell therapy, lymph node homing of antigen-loaded dendritic cells, etc.), may be engineered to express tumor-associated antigens or increase their expression, or may be any combination thereof. Non-limiting types of cell therapy include CAR T cell therapy, CAR-NK cell therapy, TCR therapy, and dendritic cell vaccines. Exemplary cellular immunotherapies include sipuleucel-T, tisagenlecle ucel, lisocabtagene maraleucel, idecabtagene vi cleucel, brexucabtagene autoleucel, and axicabtagene ciloleucel, as well as CTX110, JCAR015, JCAR017, MB-CART19.1, MB-CART20.1, MB-CART2019.1, UniCAR02-T-CD123, BMCA-CAR T, JNJ-68284528, BNT211, and NK-92 / 5.28.z.

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

[0261] In some embodiments, one or more of the additional therapeutic agents are immunotherapeutics, more specifically, agents that regulate immune checkpoints. Immune checkpoints are a group of inhibitory and stimulatory pathways that directly affect the functions of immune cells (e.g., B cells, T cells, NK cells, etc.). When proteins on the surface of immune cells recognize and bind to their cognate ligands, immune checkpoints engage. The present disclosure encompasses the use of anti-CD39 antibodies in Part II in combination with agonists of stimulatory or costimulatory pathways and / or antagonists of inhibitory pathways. Agonists of stimulatory or costimulatory pathways and antagonists of inhibitory pathways may have the utility of agents that overcome different immunosuppressive pathways within the tumor microenvironment, inhibit T regulatory cells, reverse / prevent T cell incapacity or exhaustion, trigger innate immune activation and / or inflammation at the tumor site, or a combination thereof.

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

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

[0264] In some embodiments, the immune checkpoint inhibitor is a PD-1 antagonist. In other embodiments, the PD-1 antagonist may be an antagonist PD-1 antibody, a small molecule, or a peptide. Suitable antagonist PD-1 antibodies include, for example, monospecific antibodies such as balstilimab, budigalimab, camrelizumab, cosibelimab, dostarlimab, cemiplimab, ezabenlimab, MEDI-0680 (AMP-514; WO2012 / 145493), nivolumab, pembrolizumab, and sirolimab. Monoclonal antibody (pembrolizumab), pidilizumab, pimivalimab, retifanlimab, sasanlimab, spartalizumab, sintilmab, tislelizumab, toripalimab and zimberelimab; and bispecific antibodies, such as LY3434172. In other embodiments, the PD-1 antagonist may be a recombinant protein (AMP-224) composed of the extracellular domain of PD-L2 (B7-DC) and the Fc portion of IgG1 fused. In certain embodiments, the immune checkpoint inhibitor is sepalimonab.

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

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

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

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

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

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

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

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

[0273] In some embodiments, one or more of the additional therapeutic agents is an agent that inhibits or depletes immunosuppressive immune cells. For example, to inhibit or deplete immunosuppressive macrophages or monocytes, the agent may be a CSF-1R antagonist, such as a CSF-1R antagonist antibody, including RG7155 (WO11 / 70024, WO11 / 107553, WO11 / 131407, WO13 / 87699, WO13 / 119716, WO13 / 132044) or FPA-008 (WO11 / 140249; WO13169264) or an antibody disclosed in WO14 / 036357. As another example, to inhibit or deplete Tregs, the agent may be an anti-CD25 bead for ex vivo depletion of Tregs.

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

[0275] In some embodiments, each additional therapy may be independently radiotherapy, chemotherapeutic agent, radiopharmaceutical, hormone therapy, epigenetic regulator, targeting agent, immunotherapeutic agent, cell therapy, gene therapy or ICD induction therapy. For example, in one embodiment, the present disclosure encompasses the use of the anti-CD39 antibody of Part II with one or more ICD induction therapies and optionally one or more additional therapies, wherein each additional therapy is independently selected from radiotherapy, radiopharmaceutical, chemotherapeutic agent, hormone therapy, targeting agent, immunotherapeutic agent, cell therapy or gene therapy. In another example, in one embodiment, the present disclosure encompasses the use of the anti-CD39 antibody of Part II with one or more chemotherapeutic agents and optionally one or more additional therapies, wherein each additional therapy is independently selected from radiotherapy, radiopharmaceutical, hormone therapy, targeting agent, immunotherapeutic agent, cell therapy or gene therapy. In another embodiment, the present disclosure encompasses the use of the anti-CD39 antibody of Part II with one or more chemotherapeutic agents and one or more tyrosine kinase inhibitors and optionally one or more additional therapies, wherein each additional therapy is independently targeted agent, immunotherapeutic agent or cell therapy. In another embodiment, the present disclosure encompasses the use of an anti-CD39 antibody of Part II with one or more chemotherapeutic agents and one or more inhibitors independently selected from the following: (i) BCR-ABL kinase inhibitors; (ii) EGFR inhibitors (e.g., EGFR TKI or anti-EGFR antibodies); (iii) HER-2 / neu receptor inhibitors; (iv) anti-angiogenic agents (e.g., anti-VEGF antibodies, VEGFRTKI, VEGF kinase inhibitors, etc.); (V) AKT inhibitors; (vi) BRAF inhibitors; (vii) RET inhibitors; (viii) MET inhibitors; (ix) RAS inhibitors; and (x) ALK inhibitors, and optionally one or more additional therapies in combination, wherein each additional therapy is independently selected from radiation therapy, radiopharmaceuticals, targeted agents, immunotherapeutic agents, or cell therapy. In another embodiment, the present disclosure encompasses the use of an anti-CD39 antibody of Part II with one or more immunotherapeutic agents and optionally one or more additional therapies in combination, wherein each additional therapy is independently selected from radiation therapy, radiopharmaceuticals, hormone therapy, targeted agents, chemotherapeutic agents, cell therapy, or gene therapy. In another embodiment, the disclosure encompasses the use of an anti-CD39 antibody of Part II in combination with one or more immunotherapeutic agents and one or more chemotherapeutic agents, and optionally one or more additional therapies, wherein each additional therapy is independently selected from radiation therapy, a radiopharmaceutical, a hormone therapy, a targeted agent, a cell therapy, or a gene therapy.In another embodiment, the present disclosure encompasses the use of an anti-CD39 antibody of Part II with one or more immunotherapeutics and one or more radiotherapies or radiopharmaceuticals and optionally one or more additional therapies, wherein each additional therapy is independently selected from a chemotherapeutic agent, a hormone therapy, a targeted agent, a cell therapy, or a gene therapy. In another embodiment, the present disclosure encompasses the use of an anti-CD39 antibody of Part II of the present disclosure with one or more immune checkpoint inhibitors and / or one or more ATP-adenosine axis targeting agents and optionally one or more additional therapies, wherein each additional therapy is independently selected from radiotherapy, radiopharmaceuticals, chemotherapeutics, targeted agents, immunotherapeutics, or cell therapy. In another embodiment, the present disclosure encompasses the use of an anti-CD39 antibody of Part II with one or more immune checkpoint inhibitors and / or one or more ATP-adenosine axis targeting agents and / or one or more chemotherapeutics, radiopharmaceuticals, or radiotherapy. In another embodiment, the present disclosure encompasses the use of an anti-CD39 antibody of Part II in combination with one or more immune checkpoint inhibitors and / or one or more ATP-adenosine axis targeting agents and one or more inhibitors independently selected from the following: (i) BCR-ABL kinase inhibitors; (ii) EGFR inhibitors (e.g., EGFR TKI or anti-EGFR antibodies); (iii) HER-2 / neu receptor inhibitors; (iv) anti-angiogenic agents (e.g., anti-VEGF antibodies, VEGFR TKI, VEGF kinase inhibitors, etc.); (V) AKT inhibitors; (vi) BRAF inhibitors; (vii) RET inhibitors; (viii) MET inhibitors; (ix) RAS inhibitors; and (x) ALK inhibitors. In another embodiment, the present disclosure encompasses the use of an anti-CD39 antibody of Part II in combination with one or more immune checkpoint inhibitors and / or one or more ATP-adenosine axis targeting agents and one or more ICD induction therapies. In other embodiments of the above, (a) the targeting agent may be a PI3K inhibitor, an arginase inhibitor, a HIF2α inhibitor, an AXL inhibitor, a PAK4 inhibitor or an anti-angiogenic agent; (b) the immunotherapeutic agent is an ATP-adenosine axis targeting agent, a cytokine therapy, an immune checkpoint inhibitor or a combination thereof; (c) the ATP-adenosine axis targeting agent is A. 2A R and / or A 2B(d) the ATP-adenosine axis targeting agent is etelumadnam or quetiapine; (e) the immunotherapeutic agent is an anti-PD-L1 antagonist antibody or an anti-PD-1 antagonist antibody, which is optionally selected from the group consisting of budigolimab, carrelizumab, cosibellimab, dotalimumab, cemiplizumab, ebenlizumab, nivolumab, pembrolizumab, pidilizumab, picromumab, rivulizumab, saxanthate monoclonal antibody, spartalizumab, sintilimab, tislelizumab, toripalimab, sepalimab, LY3434172, avelumab, atezolizumab, batilizumab, durvalumab, envolizumab, LY3434172 and KN046; (f) the immunotherapeutic agent is an anti-TIGIT antagonist antibody, which is optionally selected from the group consisting of: AGEN1327, AB308 (WO2021247591), BMS 986207, COM902, dovanalimumab, EOS-448, eitinib, IBI-929, JS006, M6223, osperlimumab, SEA-TGT, tisleliumab, and vembrolizumab; and bispecific antibodies, such as AGEN1777 and AZD2936; (g) immunotherapeutic agents are dovanalimumab, AB308, sepalizumab, tisleliumab, pembrolizumab, nivolumab, eitinib, and vembrolizumab. (h) the anti-angiogenic agent is pazopanib, sorafenib, sunitinib, bevacizumab, axitinib, lenvatinib, tivozanib or cabozantinib; (i) ICD induction therapy is radiation therapy (including both external beam therapy and internal radiation therapy), photodynamic therapy, extracorporeal photochemotherapy, oncolytic virus therapy, bortezomib, cyclophosphamide, doxorubicin, epirubicin, adamicin, mitoxantrone or oxaliplatin; or (j) any combination thereof. In other embodiments of the above, the present disclosure encompasses the use of the anti-CD39 antibody of Part II in combination with dovanalimab, AB308, itrumadan, queliclostra, sepalitumab, AB521 or any combination thereof. In other embodiments of the above, the present disclosure encompasses the use of the anti-CD39 antibody of Part II in combination with itrumadan, queliclostra, or itrumadan and queliclostra. In still other embodiments of the above, the present disclosure encompasses the use of an anti-CD39 antibody of Part II in combination with etrumadin, AB521, or etrumadin and AB521. In still other embodiments of the above, the present disclosure encompasses the use of an anti-CD39 antibody of Part II in combination with quiliclurestat, AB521, or quiliclurestat and AB521.

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

[0277] experiment

[0278] The following examples are presented to provide a person of ordinary skill in the art with a complete disclosure and description of how to make and use the present disclosure, and are not intended to limit the scope of what the inventors regard as their invention. Additional antibodies within the scope of the present disclosure may be made using methods based on the methods described in these examples or based on other methods described herein. Efforts have been made to ensure accuracy with respect to the quantities (e.g., amounts, temperatures, etc.) used, but it should be noted that there are some experimental errors and deviations.

[0279] Unless otherwise indicated, temperature is in degrees Celsius (° C.), and pressure is at or near atmospheric pressure. Standard abbreviations are used, including the following: rt or rt = room temperature; min = minute; h or hr = hour; ng = nanogram; μg = microgram; mg = milligram; g = gram; kg = kilogram; μl or μL = microliter; ml or mL = milliliter; l or L = liter; μM = micromolar; mM = millimolar; M = molar; mol = mole; mmol = millimolar; nM = nanomolar.

[0280] Example 1: Generation of anti-CD39 antibodies

[0281] A CHO-K1 cell line expressing human CD39, a HEK-293 cell pool expressing cynomolgus CD39, and a HEK-293 cell pool expressing murine CD39 were generated. Briefly, CHO-K1 cells or HEK-293 cells (Flp-In 2479) were transfected with a pcDNA3.3 expression vector encoding full-length human, cynomolgus, or murine CD39 (SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 71, respectively) using the Lipofectamine 2000 transfection kit according to the manufacturer's protocol. TM -293 cells). 48-72 hours after transfection, the transfected cells were cultured in medium containing blasticidin for selection and testing for CD39 expression, and then a pool of cells expressing CD39 was obtained. Cell lines or pools expressing human, cynomolgus monkey and murine CD39 were obtained by limiting dilution and used to screen hybridoma supernatants as described below.

[0282] In order to produce anti-CD39 antibodies, four 6-8 week old Sprague Dawley rats were immunized with 30-200 mg / animal of human CD39 antigen (protein or plasmid DNA). The adjuvant mixture included Adju-Phos, CpG-ODN or Titer-Max. Animals were injected once every other week via the paw, subcutaneous, intraperitoneal, intramuscular and intradermal routes. Serum titers were measured by ELISA or fluorescence activated cell sorting (FACS). ELISA for measuring serum antibody titers against a given antigen was performed as follows. The plate (Nunc) was coated overnight with 100 μL human CD39 antigen at 1 μg / mL at 4°C, and then blocked with blocking buffer (1x PBS / 2% BSA) for 1 hour at ambient temperature. Rat serum was diluted 3 times in blocking buffer at a dilution of 1:100, and incubated for 1 hour at ambient temperature. Wells without added serum samples were used as negative controls. The plate was then washed and incubated with secondary antibody goat anti-rat IgG-Fc-HRP (Bethyl) for 1 hour. After washing, tetramethylbenzidine (TMB) substrate was added, and the interaction was stopped by adding 2M HCl. The absorbance at 450nm was read using a microplate reader (Molecular Device). The FACS assay for measuring serum antibody titers was performed as described generally for screening hybridoma supernatants (described below), except that serially diluted serum was used. Serum titer was determined as final dilution, and positive signal was obtained (i.e. relative to negative control>3 times).

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

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

[0285] Harvest hybridoma cells and add 150-200 cells to 1.5mL semisolid HAT culture medium. Cells are gently mixed for 5-10 seconds in a vortex oscillator and then inoculated in a 6-well plate. Plates are maintained in an incubator set to 37°C, 5% CO2 for 7-8 days. Each visible single colony is picked into a 96-well plate with a DMEM culture medium supplemented with 10% FBS. After 2-3 days, collect and screen cell supernatants.

[0286] Four rounds of hybridoma production and antibody screening were performed, wherein the order of the assay cascade in the screening paradigm was different. In order to meet the screening criteria, the antibodies produced by the established hybridoma clones were required to bind to humans and cynomolgus monkey cell CD39 with high affinity, and no measurable binding with murine CD39 or with any of other CD39 family members (CD39-L1, CD39-L2, CD39-L3 or CD39-L4). It was also necessary to show antibodies that strongly inhibited the activity of human CD39 enzymes in cells engineered to overexpress CD39 and human cell lines (such as THP-1, SK-MEL-5 and MOLP-8) that naturally express CD39. In the first round of fusion, the human CD39 ELISA based on CHO-K1 cells was a primary screening, and the combination of overexpression of cells with humans, cynomolgus monkeys and murine CD39 measured by FACS (using previously described cell lines) and in vitro soluble human CD39 enzyme assays and cell enzyme assays were secondary screenings. The second round of fusion failed to produce suitable hybridoma material. In the third round of fusion, soluble human CD39 enzyme determination was a primary screening, and the combination of overexpression of cells with humans and murine CD39 measured by FACS, human CD39-L1 combined with reverse screening and confirmatory soluble human CD39 enzyme determination was a secondary screening. In four rounds of fusion, THP-1 cell enzyme determination was used as a primary screening, and the combination of overexpression of cells with humans and cynomolgus CD39 measured by FACS and confirmatory THP-1 cell enzyme determination was a secondary screening. Clone 39 from the first round and clones 19 and 31 from the third round were selected for further characterization. No clone was selected from the fourth round.

[0287] For cell-based ELISA, which is used to evaluate binding to CD39 expressed on the cell surface, the assay is generally performed as follows. Plates (384 wells) are filled with 3-5×10 4 The plate was pre-coated with 1x PBS / 5% milk and incubated for 1 hour at ambient temperature. The plate was washed three times with PBS and then incubated with secondary antibody goat anti-rat IgG-Fc-HRP (1:500) for 1 hour at ambient temperature. After washing, TMB substrate was added to each well and the plate was incubated for 3-5 minutes in the dark at ambient temperature, and the reaction was stopped by adding 2M HCl. The absorbance at 450nm was read using a microplate reader (Molecular Device).

[0288] FACS was also used to detect binding of anti-CD39 antibodies to CD39 in supernatants using a CHO-K1 cell line expressing human CD39, a HEK-293 cell line expressing cynomolgus CD39, and a pool of HEK-293 cells expressing murine CD39 as described previously. Unlabeled cells were used as controls to set thresholds prior to detection, and the percent change in each group that exceeded the fluorescence intensity threshold was then analyzed. 1x10 5 Cells / well were incubated with hybridoma supernatant in a volume of 100 μL for 1 hour. Anti-human CD39 reference antibody was used as a positive control. Human / rat IgG isotype antibody was used as a negative control. After washing the cells with 1×PBS / 1% BSA, goat anti-human antibody or goat anti-rat antibody labeled with Alexa647 was added (diluted 1:500 in FACS staining buffer). The cells were incubated in the dark at 4°C for 1 hour. The mean fluorescence intensity (MFI) of the cells was measured by flow cytometry and analyzed using FlowJo software.

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

[0290] The functional activity of anti-CD39 antibodies was determined by measuring CD39-catalyzed ATP hydrolysis using a human myeloid leukemia monocytic cell line (THP-1). THP-1 cells were resuspended in TM buffer and 80 μL / well of cells were plated at 4 × 10 4 The density of individual cells / well was inoculated in a 96-well U plate, and then 40 μL / well antibody CD39 solution (various concentrations, 5-fold serial dilutions from 100nM to 1.28pM in TM buffer) was added to the plate. Antibodies and cells were incubated in an incubator set to 37°C for 1 hour. After incubation, ATP (50 μM in TM buffer) was added to the plate with a volume of 80 μL / well and maintained in an incubator set to 37°C for 1 hour. The plate was placed in a centrifuge set to 1500rpm for 5 minutes, and the supernatant of 50 μL / well was transferred to a white 96-well plate (Corning, catalog number 3903). CellTiter-Glo (50 μL / well) was added to the corresponding wells and mixed wells. Cytoenzyme inhibition of CD39 was measured on a multi-label reader (Perkin-Elmer Envision Workstation).

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

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

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

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

[0295] To produce antibodies ch19_IGG4.P, ch31_IGG4.P and ch39_IGG4.P, plasmids containing the VH and VL genes described above were co-transfected into Expi293F cells. For each antibody, 2.94×10 6 / mL Expi293F cells. Mix plasmid DNA with ExpiFectamine at a final concentration of 1μg / mL TM 293 transfection reagent was then added to the cell culture medium. The cell culture was incubated in a platform shaker at a rotation rate of 150 rpm. The temperature was maintained in an incubator set to 37°C and 8% CO2. After six days of incubation, the cells were pelleted using centrifugation at 4000 rpm, 25°C for 10 minutes. The supernatant was collected for gel electrophoresis, SPR, and PCR, if necessary. off Sequencing and purification. Follow NuPAGE TM The supernatant was loaded on an SDS-PAGE gel following the instructions for a 4-12% Bis-Tris protein gel (Thermo Fisher). TM Unstained protein ladder (Thermo Fisher) was used together with antibody samples to determine the molecular weight of the antibodies.

[0296] In order to purify the antibody, a protein A column was pre-filled with 1 mL MabSelect Sure resin. The column was balanced with five column volumes (CV) of equilibrium buffer (0.1 M Tris, pH 7.0) before loading with cell culture fluid. After loading, the column was washed with 15 CV of 0.1 M Tris (pH 7.0), followed by 8 CV of 0.1 M glycine (pH 3.5). Desalting columns were used to exchange the buffer of the eluted sample with PBS buffer. Finally, the purified antibody was analyzed by SDS-PAGE and SEC-HPLC, and then stored at -80 °C.

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

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

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

[0300]

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

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

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

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

[0305] Table 3: Identification of the human germline and recipient of clone 31.

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

[0307] Table 4: Identification of the human germline and recipient of clone 39.

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

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

[0310] The humanized gene was reverse translated, codon optimized for mammalian expression, and synthesized by GENEWIZ. The synthetic gene was recloned into an in-house IgG expression vector, expressed, and purified.

[0311] Surface plasmon resonance (SPR) was used to measure the binding kinetics of purified antibodies to antigens and to sort the antibodies. Anti-CD39 antibodies were measured for affinity to the antigen (SEQ ID NO:72) of human CD39 ECD His markers using Biacore 8K instrument. Activators were prepared by mixing 400mM EDC and 100mM NHS (GE) immediately before injection. Activation of CM5 sensor chip (GE, catalog number 29-1496-03) with activator continued for 420s. Then goat anti-human Fc IgG (30 μg / mL in 10mM NaAc, pH 4.5) was injected into the channel at a flow rate of 10 μL / min for 420s. The chip was inactivated by 1M ethanolamine hydrochloric acid. Anti-CD39 antibodies were diluted to 4 μg / mL in electrophoresis buffer (1x HBS-EP+) and injected into the channel at a flow rate of 10 μL / min for 15s. Six concentrations (8, 4, 2, 1, 0.5 and 0.25 nM) of CD39 antigen analyte were sequentially injected into the channel at a flow rate of 30 μL / min for an association period of 180 s, followed by a 2400 s dissociation period. Glycine (10 mM, pH 1.5) was injected as a regeneration buffer after the dissociation period. The sensing patterns for the reference channel and the buffer channel were subtracted from the test sensing patterns. The experimental data were fitted with a 1:1 binding model steady-state affinity / heterogeneous ligand. The molecular weights of the CD39 antigen and anti-CD39 antibody used for calculation were 52 and 145 kDa, respectively.

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

[0313] Then express VH1 (SEQ ID NO: 58) or VH3 (SEQ ID NO: 59) of the humanized variant of 39 and all four VH / VL combinations of VL1 (SEQ ID NO: 61) or VL3 (SEQ ID NO: 62), however, VH1+VL3 was not well expressed. The genes of the following variants were codon-optimized for mammalian expression and then synthesized by GENEWIZ: a plasmid containing VH in which the VH domain was fused to the human IgG4CH1, hinge, CH2, and CH3 segments in the heavy chain (including the hinge mutation S228P); and a plasmid containing VL in which the VL domain was fused to the human IgκCK domain in the light chain.

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

[0315] Table 5: Kinetic binding affinity data for the humanized IgG4 (S228P) variant of Antibody 39.

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

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

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

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

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

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

[0322]

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

[0324] All four VH / VL combinations of VH1 (SEQ ID NO: 43) or VH2 (SEQ ID NO: 44) 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) of the humanized variants of antibody 31 were expressed, however, the VH1 + VL2 pair was not well expressed. The genes of the following variants were codon-optimized for mammalian expression and then synthesized by GENEWIZ: a plasmid containing VH in which the VH domain was fused to the human IgG4 CH1, hinge, CH2, and CH3 segments in the heavy chain (including the hinge mutation S228P); and a plasmid containing VL in which the VL domain was fused to the human IgκCK domain in the light chain.

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

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

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

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

[0329] Table 9: Humanized IgG4 anti-CD39 antibodies binding to CD39 overexpressed on the surface of CHO-K1 or HEK293 cells, and their blocking activity in inhibiting cellular enzyme activities.

[0330]

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

[0332] Example 3: Complete Kinetic Binding Characterization by SPR

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

[0334] Antibodies hu31.4_IGG1.AA and hu39.5_IGG1.AA were analyzed by SPR using a Biacore 4000 instrument. Kinetic binding characterization was performed in two modes. In the "monovalent affinity mode", a CM3 sensor chip (GE, catalog number 29-1049-90) was used to capture the test antibody, which was coated with an amine-coupled goat anti-human IgG coated CM3 sensor chip at seven different densities. Soluble human CD39 ECD His-labeled antigen (Sino Biological, catalog number 16020-H08B) was the analyte; a stock solution prepared at 33.3 μM was diluted to 33 nM as the highest concentration and tested in triplicate on the antibody surface with a three-fold dilution series. In the "bivalent affinity mode", soluble human CD39ECDHis-labeled antigen was amine-coupled to the CM3 sensor chip at six different densities. The test antibody for analyte was prepared as 33.3 μM stock solution, diluted to 100 nM as the highest concentration and tested in triplicate with a three-fold dilution series on the CD39 surface. The electrophoresis buffer contained 10 mM HEPES, 150 mM NaCl, 0.05% tween-20 and 0.2 mg / mL BSA. Single cycle kinetics were used to collect data, wherein multiple injections were performed in the case of increasing concentrations of analyte and the dissociation phase was monitored for 1 h. All data were collected at 25 ° C. The reaction data of all six or seven surface densities were fitted to a 1: 1 interaction model using local Rmax (the maximum amount of reaction of the complex formed). The results of the experiments performed in the monovalent affinity mode are shown in Table 10, and for the divalent affinity mode, they are shown in Table 11.

[0335] Antibodies hu31.4_IGG1.AA and hu39.5_IGG1.AA had similar equilibrium dissociation constants (K D ), where the antibody was captured on the sensor chip and soluble CD39 ECD was used as the analyte. However, when analyzed in a mode that allows the bivalent antibody to bind to the CD39 ECD captured on the sensor chip and the soluble antibody as the analyte, hu31.4_IGG1.AA showed a K value similar to that obtained when analyzed in the monovalent mode. D Very similar to K D , which is independent of antigen surface density. In contrast, hu39.5_IGG1.AA exhibits up to three orders of magnitude lower K depending on antigen surface density. D , indicating that this antibody is able to bind to more than one immobilized CD39 molecule on the chip surface in a bivalent mode and therefore displays enhanced kinetic binding affinity due to avidity.

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

[0337]

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

[0339]

[0340]

[0341] Example 4: Binding of anti-CD39 antibodies to endogenous human CD39

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

[0343] Purified monocyte binding: Antibodies were tested by flow cytometry for binding to proteins expressed on primary human monocytes (CD14 + ) on the cell surface of human CD39. Monocytes were positively selected from peripheral blood (leukopak or LRS chamber) using EasySep Human CD14 Positive Selection Kit II (Stem Cell, catalog number 17858), frozen in Bambanker (Wako, catalog number 302-14681), and stored in liquid nitrogen for future use. On the day of the assay, frozen monocytes were thawed, rinsed and resuspended in 1:25 Fc blocker (BD, catalog number 564220) and 1:250 aqua viability dye (Invitrogen, catalog number L34966A) in DPBS and incubated in the dark at room temperature for 15 minutes. In a polypropylene v-bottom 96-well plate, 0.2-0.5×10 6Cells were plated in 400 μM ATP wells. Zero or 400 μM ATP was added and incubated for 30 minutes at 4 ° C, followed by the addition of the test antibody and incubation for another 30 minutes at 4 ° C. After washing twice with DPBS, 1 μg / mL of PE-labeled mouse anti-human IgG4 secondary antibody (Southern Biotech, catalog number 9200-09) was added at 4 ° C for 30 minutes for antibody detection. After washing twice in DPBS, the cells were fixed at room temperature with 50 μL IC fixation buffer (Invitrogen, catalog number 00-8222-49) for 20 minutes. The cells were then washed once and resuspended in DPBS and flow cytometry was performed using a BD LSR Fortessa X-20 cell analyzer. The geometric mean of the PE fluorescence intensity of a live single cell population was obtained. EC was calculated using the standard 4-parameter curve fit in GraphPad Prism 50 Values ​​(Table 12).

[0344] Table 12: EC of antibodies binding to human CD39 on purified human monocytes 50 (nM) values. The data in this table represent one biological donor.

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

[0346] PBMC binding: IgG1 isotype variants of Antibody 31 and Antibody 39 were also tested by flow cytometry for binding to CD14 + (monocytes) and CD19 + (B cell) population gated on the human CD39 expressed on the cell surface of PBMC. In this experiment, antibodies reported to bind and inhibit human CD39 (referred to herein as huBMK2_IGG1.AEASS) were used as comparators. The amino acid sequence of the mature heavy chain of huBMK2_IGG1.AEASS is SEQ ID NO: 73. The amino acid sequence of the mature light chain of huBMK2_IGG1.AEASS is SEQ ID NO: 74. The previously purified frozen PBMCs were thawed, rinsed and resuspended in 1:25 Fc blocker (BD, catalog number 564220) and 1:250 light green viability dye (Invitrogen, catalog number L34966A) in DPBS and incubated in the dark at room temperature for 15 minutes. The cells were then washed in DPBS and resuspended in filtered staining buffer: DPBS supplemented with 5% goat serum (Gibco, catalog number 16210-064). In a polypropylene v-bottom 96-well plate, 0.5 x 10 6Cells / well were plated in staining buffer. Zero or 400 μM ATP was added and incubated at 4 ° C for 30 minutes, followed by the addition of test antibodies and incubation at 4 ° C for another 30 minutes. After washing twice in staining buffer, 0.8 μg / test CD14 FITC (Invitrogen, catalog number 11-0149-42, clone 61D3) and 0.5 μg / test CD19 eFluor450 (eBioscience, catalog number 48-0199-42, clone HIB19) were added to PBMC. At 4 ° C for 30 minutes, 0.5 μg / mL of PE-labeled goat anti-human IgG (Southern Biotech, catalog number 2048-09) was detected by CD39 binding of the test antibody. The cells were then washed and resuspended in DPBS and flow cytometry was performed using a BD LSR Fortessa X-20 cell analyzer. Obtained in monocytes (CD14 + ) or B cells (CD19 + ) was used to calculate the geometric mean of the PE fluorescence intensity of the live single cell population gated on . EC was calculated using a standard 4-parameter curve fit in GraphPad Prism 50 Values ​​(Table 13). A representative curve graph of one donor is shown in Figure 1 middle.

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

[0348]

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

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

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

[0352] Recombinant CD39 Inhibition: The potency of the antibodies described in the previous examples to inhibit CD39 enzymatic activity was first assessed using recombinant human CD39. The recombinant human CD39 biochemical assay was performed in an assay buffer consisting of 25 mM Tris-HCl (pH 7.4), 5 mM MgCl2, and 0.01% BSA. A 14-point 1:3 master serial dilution of the test antibody was prepared in assay buffer to span a final concentration range of 200 nM to 0.125 pM. Five microliters of the test antibody at 5 times the final concentration in assay buffer was added to each well of a 384-well plate. Ten microliters of 1.56 nM recombinant human CD39 resuspended in assay buffer was added to each well, and the plate was incubated at 37°C for an additional 60 minutes before adding 10 μL of 50 μM ATP in assay buffer. The final assay conditions contained 0.625 nM recombinant human CD39 and 20 μM ATP substrate. After incubation for another 30 minutes at 37°C, 25 μL of Kinase Glo Plus reagent was added to each well of the 384-well plate. Reactants were measured using the Kinase-Glo kit (Promega, catalog number V3771) according to the manufacturing protocol. The amount of ATP remaining after CD39 inhibition was assessed as a function of the luminescence generated and quantified using an Envision2102 multi-label reader equipped with a luminescence filter. CD39 enzyme activity was evaluated as being associated with the remaining ATP level. The maximum percentage of activity in each test well was calculated based on the assay buffer (maximum activity, 0% inhibition) and the enzyme-free control wells (baseline activity, 100% inhibition). The IC of the test antibody was determined by a dose-response curve fitted using a standard four-parameter fitting equation 50 The data are shown in Table 14.

[0353] Table 14: Antibody potency against soluble recombinant CD39.

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

[0355] SK-MEL-5CD39 inhibition: Inhibition of CD39 enzymatic activity was also evaluated using SK-MEL-5 cells, a melanoma cell line established from patient-derived tumor samples. On the day of the experiment, a vial with previously frozen SK-MEL-5 cells was thawed and the cells were resuspended in 10 mL of an assay buffer consisting of 20 mM HEPES (pH 7.4), 137 mM NaCl, 5.4 mM KCl, 1.3 mM CaCl2, 4.2 mM NaHCO3, and 0.1% glucose. 14-point 1:3 master serial dilutions of anti-CD39 antibodies were prepared in assay buffer to span a final concentration range of 100 nM to 0.063 pM. Twenty microliters of test antibody at 5 times the final concentration in assay buffer was added to each well of a 96-well round-bottom polypropylene plate. Forty microliters of SK-MEL-5 cells are added to each well and the plate is incubated for another 60 minutes at 37°C, followed by the addition of 40 μL of 50 μM ATP in assay buffer. Final assay conditions include 10,000 cells / well and 20 μM ATP substrate. After incubation for another 60 minutes at 37°C and 5% CO2, the plate is centrifuged at 225 x g for 10 minutes. Thirty microliters of supernatant are transferred to a 96-well assay plate (Corning, catalog number 3912) and the amount of ATP remaining in the reaction is measured according to the manufacturing protocol using Kinase-Glo kit (Promega, catalog number V3771). The amount of ATP remaining after CD39 inhibition is assessed as a function of the luminescence produced and is quantitatively measured using an Envision 2102 multi-label reader equipped with a luminescent filter. CD39 enzyme activity is evaluated as being associated with the remaining ATP level. The percentage of maximum activity in each test well was calculated based on the assay buffer (maximum activity, 0% inhibition) and the no-cell control wells (baseline activity, 100% inhibition). The IC of the test antibody was determined from the dose-response curves fitted using the standard four-parameter fitting equation 50 The data are shown in Table 15.

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

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

[0358] THP-1CD39 inhibition: Inhibition of CD39 enzymatic activity was assessed in a second tumor cell line (specifically, a human monocytic cell line derived from an acute monocytic leukemia patient). On the day of the experiment, a vial with previously frozen THP-1 cells was thawed and the cells were resuspended in 10 mL of an assay buffer consisting of 20 mM HEPES (pH 7.4), 137 mM NaCl, 5.4 mM KCl, 1.3 mM CaCl2, 4.2 mM NaHCO3, and 0.1% glucose. 14 points of 1:3 master serial dilutions of anti-CD39 antibodies were prepared in assay buffer to span a final concentration range of 100 nM to 0.063 pM. Twenty microliters of test antibody at 5 times the final concentration in assay buffer were added to each well of a 96-well round-bottom polypropylene plate. Forty microliters of THP-1 cells are added to each well and the plate is incubated for another 60 minutes at 37°C, followed by the addition of 40 μL of 50 μM ATP in assay buffer. Final assay conditions include 40,000 cells / well and 20 μM ATP substrates. After incubation for another 60 minutes at 37°C and 5% CO2, the plate is centrifuged at 225 x g for 10 minutes. Thirty microliters of supernatant are transferred to a 96-well assay plate (Corning, catalog number 3912) and the amount of ATP remaining in the reaction is measured according to the manufacturing protocol using Kinase-Glo kit (Promega, catalog number V3771). The amount of ATP remaining after CD39 inhibition is assessed as a function of the luminescence produced and is quantitatively measured using an Envision 2102 multi-label reader equipped with a luminescent filter. CD39 enzyme activity is evaluated as being related to the remaining ATP level. The percentage of maximum activity in each test well was calculated based on the assay buffer (maximum activity, 0% inhibition) and the no-cell control wells (baseline activity, 100% inhibition). The IC of the test antibody was determined from the dose-response curves fitted using the standard four-parameter fitting equation 50 The data are shown in Table 16.

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

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

[0361] Purified monocyte CD39 inhibition: Primary human monocytes were also used to evaluate efficacy. On the day of the experiment, two previously frozen CD14 +A vial of positively selected human monocyte (donor 1003773) cells was thawed and the cells were resuspended in 10 ml of assay buffer consisting of 20 mM HEPES (pH 7.4), 137 mM NaCl, 5.4 mM KCl, 1.3 mM CaCl2, 4.2 mM NaHCO3, and 0.1% glucose. A 14-point 1:3 master serial dilution of the test antibody was prepared in assay buffer to span a final concentration range of 200 nM to 0.13 pM. Twenty microliters of the test antibody at 5 times the final concentration in assay buffer was added to each well of a 96-well round-bottom polypropylene plate. Forty microliters of human monocytes were added to each well and the plates were incubated at 37°C for another 60 minutes before adding 40 μL of 50 μM or 1000 μM ATP in assay buffer. Final assay conditions included 20,000 cells / well and 20 μM or 400 μM ATP substrate. After incubation for another 60 minutes at 37°C and 5% CO2, the plate was centrifuged at 225xg for 10 minutes. Fifteen microliters of supernatant was transferred to a 96-well assay plate (Corning catalog number 3912) and the amount of AMP produced in the reaction was measured using an AMP-Glo ​​kit (Promega, catalog number 5012) according to the manufacturing protocol. The supernatant with 400 μM ATP substrate from the reaction plate was diluted 1:8 times with assay buffer before transfer and addition of AMP-Glo ​​reagent. The amount of AMP produced after CD39 inhibition was assessed as a function of the luminescence produced and quantified using an Envision 2102 multi-label reader equipped with a luminescence filter. CD39 enzyme activity was evaluated as being associated with AMP levels. The maximum activity percentage in each test well was calculated based on the assay buffer (maximum activity, 0% inhibition) and the cell-free control well (baseline activity, 100% inhibition). The IC of the test antibody was determined by a dose-response curve fitted using a standard four-parameter fitting equation 50 The data are shown in Tables 17 and 18 and Figure 2 middle.

[0362] Table 17: Potency of antibodies against CD39 expressed on the surface of human monocytes in the presence of 20 μM ATP.

[0363]

[0364]

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

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

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

[0368] Example 6: Immunostimulatory effects of CD39 inhibition

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

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

[0371] The ability of anti-CD39 antibodies to activate the NLRP3 inflammasome on macrophages was tested by measuring the secretion of mature IL-1β and IL-18. A schematic diagram of the assay is shown in Figure 3 On day 0, the pre-isolated positively selected CD14 + Monocytes (see Example 4) were thawed and plated at approximately 15 x 10 per 10 cm dish (Thermo Scientific Nunclon Delta Surface, catalog number 150464). 6 CD14 + Monocytes were cultured at a density of 10% inactivated FBS, 1% Glutamax, 1% penicillin / streptomycin, and 50 ng / mL recombinant human M-CSF (R&D, catalog number 216MCC / CF) in RPMI medium. On day 5, non-adherent cells were washed off and adherent macrophages were collected by scraping and plated in 24-well plates at 0.125×10 6The cells were then plated at a density of 1 cell / well. The cells were then incubated overnight to allow adhesion. The next day, the cells were treated with 1 ng / mL LPS (InvivoGen, catalog number tlrl-peklps), followed by 10 or 100 nM of test antibody or IgG control and incubated for 3 hours at 37 ° C, 5% CO2. The cells were then treated with 500 μM ATP (Life Technologies, catalog number R0441) and incubated for another 2-4 hours at 37 ° C, 5% CO2. The supernatant was collected and analyzed for IL-1β levels by CBA (BD, catalog number 558279) or ELISA (R&D, catalog number QK201) according to the manufacturer's instructions. The IL-18 levels in the supernatant were analyzed by ELISA (R&D, catalog number DL180) according to the manufacturer's instructions. The experiment was performed in quadruplicate per donor technically, the average was taken, and then normalized for each donor relative to the isotype control. As Figure 4A-4F As shown in , ch39_IGG4.P, hu39.1_IGG4.P, hu39.5_IGG1.AA, and hu31.4_IGG1.AA increased IL-1β and IL-18 release from in vitro derived macrophages relative to isotype controls.

[0372] Monocyte-derived dendritic cell assay: The antibodies were also tested for their ability to mature monocyte-derived dendritic cells (moDCs) in the presence of ATP. On day 0, positively selected monocytes were resuspended in RPMI supplemented with 10% heat-inactivated FBS, 1% Glutamax, 1% penicillin / streptomycin, 100 ng / mL recombinant human GM-CSF (R&D, catalog number 215-GM / CF) and 100 ng / mL IL-4 (Peprotech, catalog number 200-04) and cultured at 4 x 10 6 Cells / well were seeded in 6-well plates (Falcon, catalog number 353046). On day 6, moDCs were collected and cultured at 0.5 x 10 6Cells / well were seeded in 24-well Upcell plates (Thermo Fisher, catalog number 174899) and treated with 10 μg / mL isotype control or anti-CD39 antibody for 1 hour at 37°C, 5% CO2, followed by 0 μM or 300 μM ATP for 18 hours at 37°C, 5% CO2. On day 7, moDCs were transferred to polypropylene V-bottom 96-well plates for staining. All wells were resuspended in 1:25 Fc blocker (BD, catalog number 564220) and 1:250 light green viability dye (Invitrogen, catalog number L34966A) in DPBS and incubated in the dark at room temperature for 15 minutes. After washing once with DPBS, cells were stained with the following antibodies [1 μg / test CD83 PE-Cy7 (BD, catalog number 561132), 1 μg / test CD86 BV605 (BD, catalog number 562999), 0.8 μg / test CD14 FITC (Invitrogen, catalog number 11-0149-42)] or corresponding isotype controls [PE-Cy7 mouse IgG1κ (BD, catalog number 565573), BV605 mouse IgG1κ (BD, catalog number 562652), FITC mouse IgG1κ (eBiosciences, catalog number 11-4714-41)] for 30 minutes at 4°C. The surface levels of dendritic cell maturation markers CD83 and CD86 should increase as dendritic cells mature, while CD14 (monocyte marker) should decrease. After washing once in DPBS, cells were fixed at room temperature with 50 μL IC fixation buffer (Invitrogen, catalog number 00-8222-49) for 20 minutes. The cells were then washed and resuspended in DPBS and flow cytometry was performed using a BD LSR Fortessa X-20 cell analyzer. For CD83 and CD14, the percentage of the parent gate was set on the isotype control (<5% of total events) for each donor under 0 μM ATP conditions. For CD86, under control-treated conditions, >90% of cells were positive compared to the isotype control, so in fact, the geometric mean of the fluorophore fluorescence intensity of the live single cell population was reported.

[0373] like Figure 5A and Figure 5B As shown in , ATP matured moDCs as detected by increases in the dendritic cell maturation markers CD83 and CD86. Inhibition of CD39 enzymatic activity by adding anti-CD39 antibodies, but not isotype controls, enhanced maturation, further increasing the percentage of cells expressing CD83 and the surface amount of CD86 per cell. The percentage of cells expressing the monocyte marker CD14 was also reduced after adding anti-CD39 in the presence of ATP ( Figure 5C ). Except for hu39.6_IGG4.P, the effects on CD83, CD86 and CD14 were ATP-dependent.

[0374] Overall, the above data demonstrate that enzymatic inhibition of human CD39 by the disclosed antibodies has an immunostimulatory effect on different myeloid populations in vitro.

[0375] Example 7: Antibody Characterization by Competitive Flow

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

[0377] Alexa Fluor 647 conjugation kit-Lightning-Link (Abcam, catalog number ab269823) is used to make antibody hu39.1_IGG4.P and antibody IGG4.P, IgG4 (S228P) isotype control (CrownVivo) directly conjugate with Alexa Fluor647 (" AF647 "). The conjugated antibody is referred to as "hu39.1_IGG4.P-AF647" and "IGG4.P-AF647" in this embodiment. The cell line MOLP-8 expressing CD39 of human multiple myeloma is used to verify the combination. On the day of determination, frozen MOLP-8 cells were thawed, rinsed and resuspended in 1:25Fc blocker (BD, catalog number 564220) and 1:250 light green vigor dye (Invitrogen, catalog number L34966A) in DPBS and incubated in the dark for 15 minutes at room temperature. In a polypropylene v-bottom 96-well plate, 0.2 x 10 6 Cells were plated at 10 cells / well. An 11-point dose response curve was prepared by 3-fold serial dilution in DPBS, with the concentration range of each antibody being 833-0.0141 nM. The twelfth point contained only DPBS and no antibody. The diluted antibodies from the dose response 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 and resuspended in DPBS and flow cytometry was performed using a BD LSR Fortessa X-20 cell analyzer. The mean fluorescence intensity of AF647 was obtained for the live single cell population. EC was calculated using standard 4-parameter nonlinear regression analysis in GraphPad Prism software 50 and EC 95 value.

[0378] Antibody hu39.1_IGG4.P-AF647 is then used as a reference antibody in a competitive flow assay with unlabeled test antibodies (hu31.1_IGG1.AA, hu39.1_IGG4.P, hu39.5_IGG1.AA, IGG1.AA isotype control and IGG4.P isotype control (CrownVivo)). On the day of determination, frozen MOLP-8 cells were thawed, rinsed and resuspended in 1:25Fc blocker (BD, catalog number 564220) and 1:250 light green vigor dye (Invitrogen, catalog number L34966A) in DPBS and incubated in the dark for 15 minutes at room temperature. 11-point dose-response curves were prepared by 3 times of serial dilution in DPBS, wherein the concentration range of each test antibody was 833-0.0141nM. The twelfth point contained only DPBS and no antibody. 12.5 μL was added to each well of a 96-well staining plate. 12.5 μL of reference antibody hu39.1_IGG4.P-AF647 at a concentration of 93.7 nM was added to each well and mixed gently three times. Cells were plated at 0.2 x 10 6 Cells / well were plated and incubated at 4°C for 60 minutes. The cells were then washed three times and resuspended in DPBS and flow cytometry was performed using a BD LSR Fortessa X-20 cell analyzer. The mean fluorescence intensity of AF647 was obtained for the live single cell population. EC was calculated using standard 4-parameter nonlinear regression analysis in GraphPad Prism software. 50 The maximum competition (%) was calculated using the following formula: Maximum competition = 1 - (MFI at highest concentration of test antibody / MFI at matching concentration of hu39.1_IGG4.P-AF647 alone). Only a single concentration of the reference antibody hu39.1_IGG4.P-AF647 was used.

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

[0380] Table 19: Antibody competition for CD39 binding.

[0381]

[0382] Example 8: Efficacy in a xenograft model

[0383] Anti-CD39 antibodies produced as described in Examples 1 and 2 bind to human but not murine CD39. Therefore, in order to evaluate in vivo efficacy, a xenograft mouse model can be used. Five hundred thousand to ten million human cancer cells expressing human CD39 are injected subcutaneously or orthotopically into immunocompromised mice (e.g., SCID or nude mice), and tumor growth is monitored. Anti-CD39 antibodies can bind to and inhibit several human cancer cell lines (including but not limited to MOLP-8 myeloma ( Fig. 7A and Figure 7B ), THP-1 leukemia (Table 16), SK-MEL-5 melanoma (Table 15), and OAW42 ovarian cancer cells). 3 Mice were treated intraperitoneally (ip) with 0.1-30 mg / kg of the disclosed anti-CD39 antibody or isotype control BIW x 4, or until the tumor volume reached 2,000 mm 3 Treatment of MOLP-8 tumors expressing human CD39 with the anti-CD39 antibodies of the present disclosure resulted in tumor growth inhibition compared to mice treated with isotype controls. Tumor growth inhibition compared to mice treated with isotype controls is also expected following treatment of other tumor types expressing human CD39 with the anti-CD39 antibodies of the present disclosure.

[0384] Example 9: Efficacy in a human CD39 knock-in model

[0385] To evaluate the anti-CD39 antibodies of the present disclosure in an immunocompetent murine model, the antibodies can be tested in a human CD39 knock-in (hCD39KI) mouse model, in which fully immunocompetent C57BL / 6 or BALB / c mice express human CD39 and do not express murine CD39 ( Fig. 9 ). In this mouse model, the expression of human CD39 is similar to the expression of murine CD39 in wild-type mice. This mouse model allows in vivo testing, where anti-CD39 inhibition can act on host cells such as stromal cells, blood vessels, and immune cells. Mice are inoculated subcutaneously or orthotopically with murine wild-type or engineered tumor cell lines, including but not limited to MCA-205 fibrosarcoma, B16-F10 melanoma, MC38 colon adenocarcinoma, ID8 ovarian cancer, 4T1 breast cancer, or CT-26 colon carcinoma cells. Five hundred thousand to ten million murine cancer cells are injected and tumor growth is monitored. When the tumor is less than 150 mm 3 Mice were treated intraperitoneally (ip) with 0.1-30 mg / kg ch39_mIGG2A.AAG or isotype control BIWx4 at 4 °C or until tumor volume reached 2,000 mm 3Treatment of tumors with anti-CD39 as a single agent or in combination with one or more additional therapies (eg, ICD inducers, immunotherapy, etc.) will result in inhibition of tumor growth compared to mice treated with isotype controls.

[0386] In one experiment, C57BL / 6hCD39KI mice were inoculated with 1 million MC38 cells on day 0. When tumors reached 80 mm 3 When, mice were given a loading dose of 20mg / kg anti-CD39 or isotype control antibody (vehicle: PBS) via intraperitoneal (ip), and then subsequently given 10mg / kg anti-CD39 or isotype control antibody 2 times / week (BIW). Each group consists of 11-12 mice. In these experiments, anti-CD39 antibody refers to ch39_mIGG2A.AAG. Oxaliplatin (OXA) treatment was started on the same day after anti-CD39 or isotype control loading dose, 5mg / kg ip (vehicle: saline), and administered 1 time / week. In an experiment of similar design, BALB / c hCD39KI mice were inoculated with 4T1 cells at day 0 and treated with OXA or anti-CD39 antibodies as described for the MC38 model. The results from these experiments are described in the following paragraphs.

[0387] Anti-CD39 demonstrates efficacy in the hCD39KI model To evaluate the effectiveness of anti-CD39 treatment, tumor volume, intratumoral and peripheral CD39 enzyme activity, and CD39 expression were assessed.

[0388] Tumor volume and body weight were measured approximately twice a week (BIW) and are shown in Fig. 10A and Fig. 10B In the table, the midpoints represent the mean of the corresponding measurements of each group of mice and the error bars represent the standard error of the mean (SEM). A mixed effects model with multiple comparisons was used to calculate significance. Fig. 10A As shown in , treatment with anti-CD39 and oxaliplatin was statistically significant (p=0.0217) compared to treatment with a single agent alone (p=0.0372 for isotype+OXA vs. anti-CD39+OXA, and p=0.0217 for anti-CD39+saline vs. anti-CD39+OXA). Body weight was stable during the experiment, indicating that anti-CD39 treatment was well tolerated ( Fig. 10B ).

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

[0390] Intratumoral and peripheral CD39 enzyme activity was also evaluated ex vivo. After the study was completed, tumor samples obtained after sacrificing C57BL / 6hCD39KIMC38 and BALB / c hCD39KI 4T1 mice were placed in RPMI+20% FBS+250μg / ml collagenase D (Millipore Sigma, catalog number 11088858001)+100KU / ml DNAse 1 (Millipore Sigma, catalog number D5025-150KU) and gentleMACS TM Octo dissociation reagent was used to enzymatically digest the cells to obtain a single cell suspension. The cells were then plated at 2.5 x 10 4 Cells / well were seeded in a 96-well plate and treated with 100 nM hu39.5_IGG1.AA or IgG1Fc silent isotype control antibody (Absolute Antibody, catalog number Ab00178-10.3) and incubated at 37°C, 5% CO2 for 1 hour. The cells were then treated with 20 μM ATP and incubated for another 30 minutes at 37°C, 5% CO2. At the end of the incubation period, the 96-well plate was spun at 1000 rpm for 3 minutes and the supernatant was collected. Then 2X Plus (Promega, catalog number V3771) was added to the supernatant. 3 microplate reader to detect luminescence to measure ATP levels in the supernatant. Data are presented in relative luminescence units (RLU). No difference in RLU between the ex vivo hu39.5_IGG1.AA and isotype treated groups indicates complete enzyme activity inhibition by in vivo treatment. Anti-CD39 treatment inhibited enzyme activity in tumors ( Fig.15A and Fig. 15B ).

[0391] To determine peripheral CD39 enzyme activity, peripheral leukocytes (WBC) were isolated from terminal blood samples of hCD39KI mice and measured as generally described for tumor cell suspensions. In brief, whole blood samples were mixed with 1X Pharm Lyse red blood cell lysis solution (BD Biosciences, catalog number 555899). WBC pellets were then collected and stored in BamBanker cell freezing medium (Bulldog Bio, catalog number BB01) after washing with 1X PBS solution. In order to perform enzyme inhibition assays, samples were thawed and cells were then plated at 2.5 x 10 4 Cells / well were plated in a 96-well plate. Each sample was incubated for 1 hour at room temperature with the following two treatment conditions: 100 nM hu39.5_IGG1.AA or IgG1 Fc silent isotype control antibody (Absolute Antibody, catalog number Ab00178-10.3). All wells were then treated with 20 μM ATP and incubated for 2 hours at room temperature. At the end of the incubation period, the 96-well plate was rotated at 1000 rpm for 3 minutes and the supernatant was collected. Then 2X Plus (Promega, catalog number V3771) was added to the supernatant. 3 microplate reader to detect luminescence to measure ATP levels in the supernatant. Data are presented in relative luminescence units (RLU). No difference in RLU between the ex vivo hu39.5_IGG1.AA and isotype treated groups indicates complete enzyme activity inhibition by in vivo treatment. Anti-CD39 treatment inhibited enzyme activity in the periphery of mice bearing 4T1 tumors ( Fig. 15C ).

[0392] In the peripheral immune population of hCD39KI mice, anti-CD39 treatment reduced cell surface CD39 and completely or nearly complete coverage Whole blood samples from terminal bleeds of C57BL / 6hCD39KI MC38 mice were evaluated by flow cytometry to assess target coverage and changes in CD39 expression on CD39-expressing peripheral immune cell types, including monocytes (CD3 - CD19 - CD11b + Ly6c ++ ), granulocytes (CD3 - CD19 - CD11b + Ly6c + ), T cells (CD3 + CD19 - ), B cells (CD3 - CD19 + ) and other lineage negative (Lin -) lymphocytes. Target engagement was determined using hu39.5_IGG1.AA-AF647, a reagent that competitively binds to ch39_mIGG2A.AAG. hu39.5_IGG1.AA was conjugated to AF647 using the AbcamAF647 Lightning Link Binding Kit. Whole blood samples from 5-6 mice per treatment group were stained with a cocktail of extracellular antibodies including: CD39 hu39.5_IGG1.AA-AF647 (competes with anti-CD39), CD39 A1 PE (does not compete with anti-CD39), CD3 17A2 PerCP-Cy5.5, CD11b M1 / 70FITC, CD19 6D5 BV421, and Ly6c HK1.4 PE-Cy7. The antibody was incubated in whole blood on ice in the dark for 30 minutes, and then the sample was fixed / lysed on ice in the dark using 1X BD FACSLysing solution for 20 minutes. The sample was then centrifuged at 400xg for 5 minutes and washed once in 1X PBS buffer, resuspended in 1% paraformaldehyde solution, and filtered through a 20 μm filter, and then the data was acquired. The sample was run on a BD FACSCanto Special Order cell counter and analyzed using Flow Jo and Prism software. Unbound CD39 was detected by competitive clone hu39.5_IGG1.AA-AF647 (measured by the MFI of hu39.5_IGG1.AA-AF647 signal relative to its isotype control IgG1.FcS-AF647) in cell types in all animals treated with isotype antibodies. Significant competitive binding of hu39.5_IGG1.AA-AF647 was observed on all CD39(A1) expressing cells in animals treated with anti-CD39, indicating complete or near complete target coverage ( Fig.16 , lower panel). Total CD39 surface protein was detected by non-competitive CD39 antibody (clone A1 PE). Reduction of surface CD39 protein was observed in multiple peripheral immune cell types of mice treated with anti-CD39 ( Fig.16 , upper figure).

[0393] Cell surface CD39 is reduced in tumor-draining lymph nodes of hCD39KI mice following anti-CD39 treatmentTumor draining lymph nodes (TLDN) (right inguinal lymph nodes) were also collected at the end of the C57BL / 6hCD39KI MC38 mouse study described above. The lymph nodes were dissociated in RPMI 1640 (Gibco, catalog number 11875119) containing 0.6 mg / mL collagenase P (Roche, catalog number 11213865001), 2.4 mg / mL dispase II (Sigma, catalog number D4693) and 0.3 mg / mL DNAse I (ThermoScientific, catalog number J62229MB) in a 37°C water bath for 30 minutes under gentle stirring. The dissociated lymph nodes were counted and 2x10 5 Cells / well were plated in 96-well polypropylene V bottom plates. The cells were washed in PBS (Gibco, catalog number 14190-144) and resuspended in a PBS solution containing 1:25Fc blocker (BD, catalog number 564220) and 1:250Near IR fluorescent reactive dye (Invitrogen, catalog number L34976A) in the dark for 15 minutes. After incubation, the cells were stained with antibody mixture (Table 20) and incubated at 4 ° C for 30 minutes. After incubation, the cells were pelleted, the supernatant was removed and 100 μL of fixed buffer (eBioscience, catalog number 00-8222-49) was added for 1 hour. The cells were washed, resuspended in 200 μL PBS, and filtered with a 40 μm filter plate (Fisher Scientific, catalog number NC0726512), then acquired on a BD LSR Fortessa X-20 cell analyzer and used FlowJo TM v10.8 software (BD Life Sciences) to define immune subsets. The expression of human CD39, mouse CD73, and mouse P2X7 on the defined immune subsets is shown in Fig.17A and Fig. 17B The anti-CD39 antibody ch39_mIGG2A.AAG as a single agent or in combination with oxaliplatin treatment did not result in significant changes in the percentage of immune subsets in the tumor-draining lymph nodes of hCD39KI mice inoculated with MC38 tumor cells ( Fig. 17C Although immune populations did not change, anti-CD39 treatment resulted in a significant reduction in human CD39 on the surface of nearly all immune subsets identified in tumor-draining lymph nodes ( Fig.17D ). Representative histograms are shown in Fig.17E middle.

[0394] Table 20. Flow cytometry staining information

[0395]

[0396]

[0397] Anti-CD39 treatment does not alter peripheral cytokine levels Terminal bleeding of MC38 tumor-bearing mice was collected into BD Heparin tubes (BD Biosciences, catalog number 367871) were centrifuged at 2000 x g for 15 minutes, and supernatant plasma samples were collected. Cytokine levels in plasma samples were analyzed using MSD following the manufacturer's instructions. No increase in peripheral cytokine levels was observed in mice treated with anti-CD39. Data are presented as mean ± SEM, with a total of 11-12 mice ( Fig.18 ). Statistical analysis was performed using ANOVA with Tukey's multiple comparison test.

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

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

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

[0401] The ICD-inducing potential of additional chemotherapeutic agents was characterized on the human melanoma cell line SK-MEL-5. Cells were thawed, rinsed and resuspended in EMEM + 10% FBS + 1% glutamax + 1% penicillin-streptomycin + 1% sodium pyruvate. Cells were plated at 2.5 x 10 4 Cells / well were seeded in 96-well plates and incubated overnight at 37°C, 5% CO2. The next day, cells were treated with chemotherapeutic agents doxorubicin (vehicle: DMSO), irinotecan (vehicle: DMSO), gemcitabine (vehicle: PBS) and cisplatin (vehicle: PBS) at the indicated concentrations and incubated for 48 hours at 37°C, 5% CO2. (1) To assess cytotoxicity, cell viability was determined at the end of the incubation period using the luminescence-based Cell Titer Glo 2.0 assay (Promega, catalog number G9242). Data are presented as viability normalized to the untreated vehicle control group ( Fig. 20A (2) To measure HMGB1 release, at the end of the incubation period, the plates were centrifuged at 400 x g for 5 minutes and the supernatant was collected. The HMGB1 level in the culture supernatant was then determined using the Lumit HMGB1 kit (Promega, catalog number CS3030B01) following the manufacturer's instructions. Fig. 20B(3) Extracellular ATP release was quantified with chemotherapeutic agents with or without the addition of 100 nM hu39.5_IGG1.AA or IgG1 Fc silent isotype control antibody (Absolute Antibody, catalog number Ab00178-10.3) and incubated for 8 hours or 32 hours. At the end of the incubation period, 4xRealTime-Glo TM Extracellular ATP assay (Promega Cat. No. GA5010) was added to the cells. Extracellular ATP release was measured kinetically using a 3 microplate reader with luminescence detected at 15-minute intervals over a 10-hour period. All chemotherapy-treated groups showed an increase in ATP release relative to untreated cells. Addition of hu39.5_IGG1.AA resulted in higher extracellular ATP levels ( Fig. 20C ).

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

[0403] A CD39 whole blood (WB) receptor occupancy (RO) flow cytometry assay was used to assess CD39 expression on various cell types in human, cynomolgus monkey, and hCD39KI mouse WB. A description of the hCD39KI RO assay is described in Example 9. Human whole blood samples were analyzed by the same method as described for hCD39KI mouse samples, but species-specific cell lineage markers for monocytes and B cells were incorporated. The human CD39 WB RO panel consisted of the following antibodies: hu39.5_IGG1.AA-AF647 (competing with anti-CD39), anti-CD39A1 PE (not competing with anti-CD39), anti-CD14 61D3 FITC, and anti-CD19 H1B19eFluor450. As Fig.21 As shown in , the relative expression of CD39 on peripheral immune cells varies between humans and C57BL / 6hCD39KI MC38 mice. While monocytes have the highest CD39 expression levels among the cell types measured in human blood, granulocytes have the highest CD39 expression in hCD39KI mice. B cell expression varies most significantly between species. Human peripheral B cells express very high levels of CD39, while hCD39KI mouse B cells have low to no CD39 expression.

[0404] CD39 and CD39-related markers were also characterized in human in vitro differentiated bone marrow cells such as M0-, M1-, and M2-polarized macrophages and monocyte-derived dendritic cells (moDCs), as well as primary human bone marrow cells from peripheral blood of four healthy donors such as CD14 +In order to generate in vitro differentiated macrophages, positive selection of CD14 + Monocytes were resuspended in RPMI supplemented with 10% heat-inactivated FBS, 1% GlutaMax, 1% penicillin / streptomycin, and 50 ng / mL recombinant human M-CSF (R&D, catalog number 216-MCC / CF) and cultured at 4 x 10 6 Cells / well were seeded in a total of 2 mL in a 6-well plate (Falcon, catalog number 353046). On day 4, the cells were supplemented with 2 mL of fresh RPMI supplemented medium containing 2X M-CSF. On day 6, 100 ng / mL LPS (Invivogen, catalog number tlr-peklps) and 20 ng / mL recombinant human IFN-γ (Peprotech, catalog number 300-02) were used to polarize the cells into M1 macrophages, while 20 ng / mL recombinant human IL-4 (Peprotech, catalog number 200-04) was used to polarize M2 macrophages. No additional cytokines were added to M0 macrophages. To generate moDC, positively selected CD14 + Monocytes were resuspended in RPMI supplemented with 10% heat-inactivated FBS, 1% GlutaMax, 1% penicillin / streptomycin, and 100 ng / mL recombinant human GM-CSF (R&D, catalog number 215-GM / CF) and 100 ng / mL recombinant human IL-4 and cultured at 4 x 10 6 Cells / well were seeded in a total of 2 mL in a 6-well plate (Falcon, catalog number 353046). On day 4, cells were supplemented with 2 mL of fresh RPMI supplemented medium containing 2X GM-CSF / IL-4. On day 7, M0-, M1-, M2-polarized macrophages and moDCs were collected, counted and aliquoted into U-bottom 96-well plates for flow cytometry or into 1.5 mL Eppendorf tubes for RNA. Primary CD14 isolated using a positive selection kit was also used. + Monocytes and bone marrow DCs enriched using the negative selection kit were aliquoted into U-bottom 96-well plates for flow cytometry or into 1.5 mL Eppendorf tubes for RNA.

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

[0406] Flow cytometric evaluation of CD39 and CD73 ( Fig.22A and Fig. 22B ), CD14 +Monocytes, enriched bone marrow DCs, M0, M1, M2 macrophages, and moDCs were aliquoted into 96-well plates and resuspended in 1:25 Fc blocker (BD, catalog number 564220) and 1:250 light green viability dye (Invitrogen, catalog number L34966A) in DPBS at 50 μL / well and incubated in the dark for 15 minutes at room temperature. The cells were then stained with the following antibodies [0.25 μg / test of CD39 PE-Cy7 (eBiosciences, catalog number 25-0399-42) and 0.5 μg / test of CD73 BUV737 (BD, catalog number 612812) or corresponding isotype controls, mouse IgG1 PE-Cy7 (BD, catalog number 557647) and mouse IgG1 BUV737 (BD, catalog number 612758)] at 4°C for 30 minutes. After washing once in DPBS, cells were fixed with 50 μL IC fixation buffer (Invitrogen, catalog number 00-8222-49) for 20 minutes at room temperature. Cells were then washed and resuspended in DPBS and flow cytometry was performed using a BD LSR Fortessa X-20 cell analyzer. CD14 + The percentage of CD39 and CD73 positivity on monocytes, enriched myeloid DC subsets, M0, M1, and M2 macrophages, and moDCs was assessed by comparison with isotype controls. + Monocytes, all in vitro derived subsets (M0, M1, M2 macrophages and moDCs) and primary enriched DC subsets cDC1, cDC2 and other DCs all had high CD39 positive percentages and low CD73 positive percentages ( Fig.22A and Fig. 22B A relatively small number (15%) of pDCs were positive for CD39. Given that most cell types had >90% CD39 positivity, CD39 expression levels were further explored by observing CD39 staining intensity compared to isotype controls ( Fig. 22B Although variable between donors, CD14 + CD39 expression was higher on monocytes, in vitro derived macrophages, and moDCs. Among primary DCs, cDC1 had the highest cell surface CD39 expression.

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

[0408] In order to evaluate the CD39 related gene expression of the wider range in these human bone marrow subsets, design and use custom NanoString panels. Qiagen RNeasy micro kit (catalog number 74106) is used to follow the manufacturer's instructions to separate the RNA of each person in the bone marrow cell subset. Then quantitative RNA is used on Nanodrop and 70ng RNA is used for NanoString. To make RNA hybridize to the custom NanoString code set, 70 μL hybridization buffer is added to the reporter code set and 8 μL is added to the 5 μL RNA of each sample. Then two microliters of capture probe sets are added to each sample and incubated at 65 ° C for 16 hours in a thermal cycler. Then the sample is filled to 30 μL with RNase-free water and loaded into the sample loading port of nCounter SPRINT tube (NanoString, catalog number SPRINT-CAR-1.0). Then the tube is run on NanoString nCounter SPRINT profiler. Quality control metrics were run on the data from each sample, and samples that passed the quality control test were normalized to the 3 housekeeping genes included in the custom NanoString panel to assess gene expression.

[0409] Fig.22E A heat map is shown in Figure 2, which shows unsupervised clustering of normalized expression of housekeeping genes for CD39, adenosine, and inflammasome pathway genes using a custom NanoString panel. Low-quality wells or aberrant control probes marked by low counts were removed based on this analysis. Specific donor IDs are attached at the end of the cell type labels. The dashed horizontal lines in the highest dendrites show sample splits but do not affect the unsupervised nature of the clustering. CD39 is expressed in all profiled immune populations. Given the consistent expression of genes in these pathways within each cell type, the cell types formed distinct clusters. Monocytes, M1 macrophages, and enriched primary DC clusters together, driven by expression on IL-1β, P2X7, and A2AR. M0, M1 macrophages, and moDCs form another cluster with higher expression of CD206, A2BR, and P2Y11.

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

[0411] The effects of P2X1, P2X7 and P2Y11 inhibitors on the activation of monocyte-derived dendritic cells (MoDC) were examined. MoDC was produced by positively selected human monocytes cultured with GM-CSF and IL-4 as described in Example 11. On the 7th day, 6-well plates were incubated on ice for 5 minutes and scraped to separate and count cells. The cells were resuspended in fresh culture medium with GM-CSF and IL-4 and 125,000 cells per well in 100 μL were plated in 96-well flat-bottom Upcell plates (Nunc, catalog number 174897). Ten micromolar of each compound [P2X1 inhibitor, NF023, MilliporeSigma, catalog number 104869-31-0; P2X7 inhibitor, A-740003, Millipore Sigma, catalog number 861393-28-4; P2Y11 inhibitor NF340, APExBIO, catalog number B7508; P2Y11 inhibitor NF157, APExBIO catalog number B7060] or vehicle control (0.1% DMSO) was added, mixed gently and incubated for 1 h at 37° C., 5% CO2. Subsequently, 0 or 300 μM ATP was added, mixed gently, and incubated for 18 h at 37° C., 5% CO2. For flow cytometry evaluation, cells were detached from the plate and transferred to 96-well polypropylene v-bottom plates. Cells were washed in PBS (Gibco, catalog number 14190-144) and resuspended in PBS solution containing 2% FBS, viability dye (LD-NIR, Invitrogen, catalog number L34994), Fc blocker and staining antibodies [BV510 anti-CD80 (BD Horizon, catalog number 563084), PE-Cy7 anti-CD83 (BD ​​Horizon, catalog number 561132) and BV605 anti-CD86 (BD Horizon, catalog number 562999)]. Cells were incubated at room temperature for 60 min, the dye was washed off, and the cells were fixed with BD Phosphoflow FIX buffer I (BD, cat...

Claims

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

34.

2. The anti-CD39 antibody according to claim 1, wherein the antibody has a heavy chain variable region and a light chain variable region according to claim 1(a), and the heavy chain variable region has at least 90% sequence identity with SEQ ID NO:9, and the light chain variable region has at least 90% sequence identity with SEQ ID NO:13; or wherein the antibody has a heavy chain variable region and a light chain variable region according to claim 1(b), and the heavy chain variable region has at least 90% sequence identity with SEQ ID NO: 17, and the light chain variable region has at least 90% sequence identity with SEQ ID NO: 21; the heavy chain variable region has at least 90% sequence identity with SEQ ID NO: 43, and the light chain variable region has at least 90% sequence identity with SEQ ID NO: 45; the heavy chain variable region has at least 90% sequence identity with SEQ ID NO: 43, and the light chain variable region has at least 90% sequence identity with SEQ ID NO: 46; the heavy chain variable region has at least 90% sequence identity with SEQ ID NO: 43, and the light chain variable region has at least 90% sequence identity with SEQ ID NO: 47; the heavy chain variable region has at least 90% sequence identity with SEQ ID NO: 43, and the light chain variable region has at least 90% sequence identity with SEQ ID NO: 48; the heavy chain variable region has at least 90% sequence identity with SEQ ID NO: 49; NO:43 has at least 90% sequence identity, and the light chain variable region has at least 90% sequence identity with SEQ ID NO:49; the heavy chain variable region has at least 90% sequence identity with SEQ ID NO:44, and the light chain variable region has at least 90% sequence identity with SEQ ID NO:48; or the heavy chain variable region has at least 90% sequence identity with SEQ ID NO:44, and the light chain variable region has at least 90% sequence identity with SEQ ID NO:49; wherein the antibody has a heavy chain variable region and a light chain variable region according to claim 1(c), and the heavy chain variable region has at least 90% sequence identity with SEQ ID NO:27, and the light chain variable region has at least 90% sequence identity with SEQ ID NO:31; the heavy chain variable region has at least 90% sequence identity with SEQ ID NO:58, and the light chain variable region has at least 90% sequence identity with SEQ ID NO:60; the heavy chain variable region has at least 90% sequence identity with SEQ ID NO:58, and the light chain variable region has at least 90% sequence identity with SEQ ID NO:61; the heavy chain variable region has at least 90% sequence identity with SEQ ID NO:58, and the light chain variable region has at least 90% sequence identity with SEQ ID NO:62; the heavy chain variable region has at least 90% sequence identity with SEQ ID NO:58, and the light chain variable region has at least 90% sequence identity with SEQ ID NO:63; the heavy chain variable region has at least 90% sequence identity with SEQ ID NO:58, and the light chain variable region has at least 90% sequence identity with SEQ ID NO:64; NO:59 has at least 90% sequence identity, and the light chain variable region has at least 90% sequence identity with SEQ ID NO:61; the heavy chain variable region has at least 90% sequence identity with SEQ ID NO:59, and the light chain variable region has at least 90% sequence identity with SEQ ID NO:60; the heavy chain variable region has at least 90% sequence identity with SEQ ID NO:59, and the light chain variable region has at least 90% sequence identity with SEQ ID NO:62; or the heavy chain variable region has at least 90% sequence identity with SEQ ID NO:59, and the light chain variable region has at least 90% sequence identity with SEQ ID NO:

63.

3. The anti-CD39 antibody according to claim 1 or 2, wherein the anti-CD39 antibody: (a) has a surface plasmon resonance of about 0.001 × 10 -11 M to about 100×10 -11 M, about 0.01×10 -11 M to about 100×10 -11 M, about 0.1×10 -11 M to about 100×10 -11 M, about 0.1×10 -11 M is about 10×10 -11 M, about 1×10 -11 M to about 100×10 -11 M or about 1×10 -11 M is about 10×10 -11 The equilibrium binding constant (K D ); (b) with an IC of at least about 1 nM or less 50 Inhibiting human CD39 enzymatic activity, optionally wherein (i) the inhibition is measured in the presence of 20 μM ATP or 400 μM ATP, and / or (ii) said CD39 is expressed on the surface of human cells, optionally wherein said human cells are primary monocytes as described in Example 5; or (c) Any combination of (a) or (b).

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

5. The anti-CD39 antibody of claim 3, wherein the anti-CD39 antibody has an IC of about 0.3 nM to about 1 nM, or about 0.3 nM to 1 nM, or about 0.5 nM to about 1 nM, or about 0.5 nM to 1 nM. 50 Inhibiting CD39 enzymatic activity, optionally wherein (i) the inhibition is measured in the presence of 20 μM ATP or 400 μM ATP, and / or (ii) said CD39 is expressed on the surface of human cells, optionally wherein said human cells are primary monocytes as described in Example 5.

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

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

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

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

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

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