Methods and compositions for promoting and enhancing T cell mediated immune response by ADCC targeting of CD39 expressing cells

Abstract

In combination with conventional therapies (e.g., targeted therapies, chemotherapies, angiogenesis inhibitors, etc.), immunotherapies targeting checkpoint molecules have shown promising in the treatment of solid or liquid tumors. However, the action of non-tumor cells in the intratumoral microenvironment has indicated that elimination of these cells can be critical to the generation of effective immune responses against the tumor, including tumor infiltration of cytotoxic T cells and other anti-tumor cells of the immune system. The present invention is not focused on the exonucleotidase activity attempting to inhibit CD39 as an adenosine producing enzyme, but utilizes CD39 expression to achieve intra-tumor cell elimination by CD39-dependent ADCC.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority to U.S. Provisional Application Serial No. 62 / 885,509, filed on August 12, 2019; the entire contents of which are incorporated herein by reference in their entirety. Technical Field

[0003] The present invention relates to a novel anti-CD39 antibody or an antigen-binding fragment thereof and use thereof, belonging to the technical field of immunotherapy. Background Art

[0004] For people with advanced cancer, hope can be a valuable but rare commodity. In recent years, a new class of drugs called immune checkpoint inhibitors have shown remarkable promise, stalling tumors and stopping them from growing, and allowing some people who receive treatment to recover virtually. But these groundbreaking therapies come with a host of challenges. Despite the success of immunotherapies based on inhibitory antibodies against programmed cell death protein 1 (PD1), PD1 ligand 1 (PDL1), and cytotoxic T-lymphocyte antigen 4 (CTLA4) therapies in advanced cancer, a significant percentage of patients still do not respond to these treatments.

[0005] With increasing attention to the immunosuppressive tumor microenvironment as a major driver of resistance, and the characterization of "hot" and "cold" tumors based on the degree of immune cell infiltration, researchers have discovered several different mechanisms underlying the lack of effective response to checkpoint monotherapy. Immunologically "hot" tumors contain high levels of infiltrating T cells and more antigens, making them more easily recognized by the immune system and more likely to trigger a strong immune response. Cancers that are considered immunologically hot are bladder cancer, head and neck cancer, kidney cancer, melanoma, and non-small cell lung cancer. However, even in these immunologically "hot" cancers, only a minority of patients benefit from immunotherapy. In contrast, immunologically "cold" tumors contain almost no infiltrating T cells for a variety of reasons and do not appear to be recognized.

[0006] Cancers that are foreign and do not provoke a strong response from the immune system make these cancers difficult to treat with current immunotherapies. Traditionally immunologically "cold" cancers include glioblastoma, as well as ovarian, prostate, pancreatic and most breast cancers.

[0007] In addition to cancer cells, the microenvironment of the tumor also contains many cell types, including bone marrow-derived inflammatory cells, lymphocytes, blood vessels, fibroblasts, and extracellular matrix (ECM) containing collagen and proteoglycans. In fact, tumor drug response is not determined solely by the inherent characteristics of tumor cells, because tumor-associated stromal cells, including fibroblasts, mesenchymal stromal cells (MSC), immune inflammatory cells, vascular endothelial cells, and ECM are combined to respond to anticancer treatment. In many cases, whether the tumor has T cell infiltration or lack of T cell infiltration, resistance to checkpoint therapy or no response are all the results of the inhibitory effects of other cells present in the tumor, and the range of the inhibitory effect ranges from the downregulation of cytotoxic T cells already present in the tumor or the inhibition of intratumoral signaling to the development of a tumor microenvironment that completely excludes cytotoxic T cells by reducing the ability of those cells to infiltrate the tumor from surrounding blood vessels.

[0008] Therefore, there is a great need in the art to identify alternative mechanisms for enhancing T cell responses. Summary of the invention

[0009] The ectonucleotidase CD39 has been targeted in order to produce a reduction in intratumoral levels of the enzymatic activity associated with the protein, and thus reduce intratumoral levels of the immunosuppressant (adenosine). The present invention is based, at least in part, on the additional discovery that CD39 is expressed by a range of cells in the tumor microenvironment, such as stromal cells, type II NKT cells, and tumor-associated macrophages (TAMs), which function to create an immunosuppressive or immunorejective environment, and that targeting those cells using certain antibody-dependent cellular cytotoxicity (ADCC) active anti-CD39 antibodies for elimination in tumors can be used to increase the infiltration of cytotoxic T cells and, in effect, convert a "cold" tumor to an immunologically "hot" tumor.

[0010] For example, in one aspect, an anti-CD39 antibody or an antigen-binding fragment thereof is provided, comprising (i) at least one antigen-binding domain that binds to ectonucleoside triphosphate diphosphohydrolase-1 (CD39) at one site such that the anti-CD39 antibody forms a stable immune complex; and (ii) an FcγRIIIa binding portion that binds to the FcγRIIIa receptor and confers ADCC activity to the anti-CD39 antibody against CD39+ cells.

[0011] Many embodiments are further provided, which can be applied to any aspect of the invention and / or combined with any other embodiment described herein. For example, in one embodiment, the anti-CD39 antibody or antigen-binding fragment thereof promotes: (i) stable immune complex formation when incubated with HCC1739BL cells, as characterized by less than 40% loss of the immune complex after 24 hours, or less than 35%, less than 30%, less than 25%, less than 20%, less than 15% or even less than 10% loss of the immune complex after 24 hours, optionally wherein the immune complex formation is detected by fluorescence intensity using a fluorescently labeled secondary antibody (e.g., for illustration only, immune complex formation with anti-CD39 antibody) The stability of the complex can be determined by incubating anti-CD39 monoclonal antibodies (mAbs) (such as at 2 μg / ml or greater) with HCC1739BL cells for different times and then detecting the presence of the immune complex by a fluorescently conjugated secondary antibody); (ii) complement-dependent cytotoxicity (CDC) activity against CD39+ cells; (iii) antibody-mediated target endocytosis of CD39 on CD45+ immune cells; (iv) antibody-mediated target endocytosis of CD39 from tumors with endothelial damage or vascular network collapse in tumors; (v) binding to a protein selected from the group consisting of Fig.33 ; and / or (vi) binds to CD39 in a manner that non-competitively or only partially competitively binds to CD39 with monoclonal antibody clone A1.

[0012] In another embodiment, the FcγRIIIa binding portion is selected from the group consisting of: an Fc domain, an antibody or fragment thereof that binds to FcγRIIIa, and an FcγRIIIa binding peptide. In yet another embodiment, the antigen binding domain is selected from the group consisting of: Fab, Fab′, F(ab′)2, Fv or single-chain Fv (scFv), Fav, dsFv, sc(Fv)2, Fde, sdFv, single domain antibody (dAb) and bifunctional antibody (diabodies) fragments, and / or wherein the anti-CD39 antibody or antigen binding fragment is monoclonal. In yet another embodiment, the anti-CD39 antibody or antigen binding fragment thereof is conjugated to an agent, optionally wherein the agent is selected from the group consisting of: a binding protein, an enzyme, a drug, a chemotherapeutic agent, a biological agent, a toxin, a radionuclide, an immunomodulator, a detectable portion, and a label. In another embodiment, the anti-CD39 antibody or antigen-binding fragment thereof has a VH domain having an amino acid sequence encoded by a nucleic acid sequence that can hybridize to the nucleic acid of SEQ ID No. 1 under stringent conditions; and a VL domain having an amino acid sequence encoded by a nucleic acid sequence that can hybridize to the nucleic acid of SEQ ID No. 3 under stringent conditions (such as hybridizing in 6x sodium chloride / sodium citrate (SSC) at 45°C and washing in 0.2xSSC / 0.1% SDS at 50°C to 65°C). In yet another embodiment, the anti-CD39 antibody or antigen-binding fragment thereof comprises a heavy chain having a CDR that is at least 60% identical (e.g., at least 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater) to the CDR of SEQ ID No. 2, 6, 10, 14, 18, 22, 26, 42, 46, 50 or 54; and a light chain having a CDR that is at least 60% identical (e.g., at least 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater) to the CDR of SEQ ID No. 4, 8, 12, 16, 20, 24, 28, 44, 48, 52 or 56 is at least 60% identical (e.g., at least 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater) to a CDR.In yet another embodiment, the anti-CD39 antibody or antigen-binding fragment thereof comprises a variable heavy (VH) chain that is at least 60% identical (e.g., at least 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater) to SEQ ID No. 2, 6, 10, 14, 18, 22, 26, 42, 46, 50 or 54, and a VH chain that is at least 60% identical (e.g., at least 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater) to SEQ ID No. No. 4, 8, 12, 16, 20, 24, 28, 44, 48, 52 or 56 is at least 60% identical (e.g., at least 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) to a variable light (VL) chain.In another embodiment, the anti-CD39 antibody or antigen-binding fragment thereof comprises: (i) a heavy chain having a CDR1 amino acid sequence that is at least 80% identical (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater) to SEQ ID No. 29, a CDR2 amino acid sequence that is at least 80% identical (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater) to SEQ ID No. 30, and a CDR3 amino acid sequence that is at least 80% identical (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater) to SEQ ID No. 31 is at least 80% identical (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater) to a CDR3 amino acid sequence of SEQ ID No. 31; and (ii) a light chain having a CDR1 amino acid sequence at least 80% identical (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater) to SEQ ID No. 32, a CDR2 amino acid sequence at least 80% identical (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater) to SEQ ID No. No.33 has a CDR2 amino acid sequence that is at least 80% identical (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater) and a CDR3 amino acid sequence that is at least 80% identical (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater) to SEQ ID No.34. In yet another embodiment, the anti-CD39 antibody or antigen-binding fragment thereof comprises a heavy chain having CDRs selected from the group consisting of CDRs of SEQ ID No. 6, 10, 14, 18, 22, 26, 42, 46, 50 and 54; and a light chain having CDRs selected from the group consisting of CDRs of SEQ ID No. 8, 12, 16, 20, 24, 28, 44, 48, 52 or 56; and human framework sequences to form humanized heavy and light chains having an antigen binding site capable of specifically binding to human CD39.In yet another embodiment, the anti-CD39 antibody or its antigen-binding fragment comprises an Fc domain of an IgG1 or IgG3 isotype, optionally wherein the Fc domain is human. In another embodiment, the anti-CD39 antibody or its antigen-binding fragment is low-fucosylated or afucosylated. In yet another embodiment, the anti-CD39 antibody or its antigen-binding fragment is human or humanized. In yet another embodiment, the anti-CD39 antibody or its antigen-binding fragment is bispecific, including at least one additional antigen binding site for a tumor antigen, an immune checkpoint, or a co-stimulatory receptor, wherein if the additional antigen binding site is for an immune checkpoint, it is used as a checkpoint inhibitor, and wherein if the additional antigen binding site is for a co-stimulatory receptor, it is used as a co-stimulatory agonist. In another embodiment, the additional antigen binding site binds to a checkpoint protein selected from the group consisting of: PD-1, PD-L1, CTLA-4 / B7-1 / B7-2, PD-L2, NKG2A, KIR, LAG-3, TIM-3, CD96, VISTA, TIGIT, and Siglec-15. In yet another embodiment, the additional antigen binding site binds to a checkpoint protein that is upregulated on T cells and associated with T cell exhaustion. In yet another embodiment, the additional antigen binding site binds to an immune co-stimulatory receptor selected from the group consisting of: MHCI molecules, BTLA receptors, OX40, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), and 4-1BB (CD137). In another embodiment, the additional antigen binding site binds to CD47, SIRPα (, CD24, or Siglec-10.

[0013] In another aspect, a pharmaceutical preparation is provided, comprising a therapeutically effective amount of at least one anti-CD39 antibody or antigen-binding fragment thereof described herein and one or more pharmaceutically acceptable excipients, buffers or solutions. For example, the pharmaceutical preparation can be used to improve anti-tumor T cell immunity and is suitable for administration to a subject with a tumor, comprising an effective amount of an anti-CD39 antibody or antigen-binding fragment thereof and one or more pharmaceutically acceptable excipients, buffers or solutions, wherein administration of the anti-CD39 antibody to the subject reduces the number of CD39 high cells in the tumor, and enhances T cell infiltration into the tumor or reduces T cell exhaustion in the tumor, or both.

[0014] In yet another aspect, an isolated nucleic acid molecule is provided, which i) hybridizes under stringent conditions with the complement of a nucleic acid encoding an immunoglobulin heavy chain and / or light chain polypeptide of an anti-CD39 antibody or antigen-binding fragment thereof described herein; ii) has a sequence that is at least about 90% identical over its entire length to a nucleic acid encoding an immunoglobulin heavy chain and / or light chain polypeptide of an anti-CD39 antibody or antigen-binding fragment thereof described herein; or iii) encodes an immunoglobulin heavy chain and / or light chain polypeptide of an anti-CD39 antibody or antigen-binding fragment thereof described herein.

[0015] In yet another aspect, an isolated immunoglobulin heavy chain and / or light chain polypeptide encoded by a nucleic acid described herein is provided.

[0016] In another aspect, a vector comprising the isolated nucleic acid described herein is provided, optionally wherein the vector is an expression vector.

[0017] In yet another aspect, a host cell comprising the isolated nucleic acid described herein is provided, which host cell: a) expresses the anti-CD39 antibody or antigen-binding fragment thereof described herein; b) comprises the immunoglobulin heavy chain and / or light chain polypeptide described herein; or c) comprises the vector described herein.

[0018] In yet another aspect, a device or kit is provided, comprising at least one anti-CD39 antibody or antigen-binding fragment thereof described herein. The device or kit optionally comprises a label to detect at least one anti-CD39 antibody or antigen-binding fragment thereof, or a complex comprising the anti-CD39 antibody or antigen-binding fragment thereof.

[0019] In another aspect, a device or kit is provided, comprising a pharmaceutical composition, an isolated nucleic acid molecule, an isolated immunoglobulin heavy chain and / or light chain polypeptide, a vector and / or a host cell as described herein.

[0020] In yet another aspect, a method of producing at least one anti-CD39 antibody or antigen-binding fragment thereof as described in any one of claims 1 to 18, comprising the steps of: (i) culturing a transformed host cell that has been transformed with a nucleic acid comprising a sequence encoding at least one anti-CD39 antibody or antigen-binding fragment thereof under conditions suitable to allow expression of the anti-CD39 antibody or antigen-binding fragment thereof; and (ii) recovering the expressed anti-CD39 antibody or antigen-binding fragment thereof.

[0021] In yet another aspect, a method for detecting the presence or level of a CD39 polypeptide is provided, the method comprising obtaining a sample, and detecting the polypeptide in the sample by using at least one anti-CD39 antibody or antigen-binding fragment thereof as described herein. For example, the at least one anti-CD39 antibody or antigen-binding fragment thereof can form a complex with the CD39 polypeptide, and the complex can be detected in the form of an enzyme-linked immunosorbent assay (ELISA), a radioimmunoassay (RIA), an immunochemical assay, a Western blot, a mass spectrometry assay, a nuclear magnetic resonance assay, or using a cell-intracellular flow assay.

[0022] In another aspect, a method for depleting CD39 in a tumor is provided. 高 A method for improving anti-tumor T cell immunity by targeting cells, the method comprising administering an effective amount of a pharmaceutical composition of an anti-CD39 antibody or an antigen-binding fragment thereof described herein to a subject having a tumor, wherein the administration of the anti-CD39 antibody or the antigen-binding fragment thereof results in the inhibition of CD39 in the tumor. 高 The number of cells is reduced and T cell infiltration into the tumor is enhanced or T cell exhaustion in the tumor is reduced, or both.

[0023] In yet another aspect, a method for promoting the infiltration of immune cells into a tumor is provided, the method comprising administering an effective amount of a pharmaceutical composition of an anti-CD39 antibody or an antigen-binding fragment thereof described herein to a subject having a tumor, wherein administration of the anti-CD39 antibody or an antigen-binding fragment thereof eliminates and reduces CD39+CD45-SCA-1+ stromal cells in the tumor and increases the infiltration of cytotoxic T cells into the tumor.

[0024] In yet another aspect, a method for reducing the inhibition of intratumoral immune cell function by type II NKT cells is provided, the method comprising administering an effective amount of a pharmaceutical composition of an anti-CD39 antibody or an antigen-binding fragment thereof described herein to a subject having a tumor, wherein administration of the anti-CD39 antibody or an antigen-binding fragment thereof results in the elimination and reduction of type II NKT cells in the tumor.

[0025] In another aspect, a method for reducing the suppression of intratumoral immune cell function by regulatory T cells (Tregs) is provided, the method comprising administering to a subject having a tumor an effective amount of a pharmaceutical composition of an anti-CD39 antibody or an antigen-binding fragment thereof described herein, wherein the administration of the anti-CD39 antibody or antigen-binding fragment thereof results in a decrease in the expression of CD39 in the tumor. 高 Treg elimination and reduction.

[0026] In yet another aspect, a method for reducing the inhibition of tumor-associated macrophages (TAMs) on the function of intratumoral immune cells is provided, the method comprising administering to a subject having a tumor an effective amount of a pharmaceutical composition of an anti-CD39 antibody or an antigen-binding fragment thereof described herein, wherein the administration of the anti-CD39 antibody or antigen-binding fragment thereof results in the inhibition of CD39 in the tumor. 高 TAM elimination and reduction.

[0027] In yet another aspect, a method for promoting an anti-tumor immune response is provided, the method comprising administering to a subject having a tumor an amount of an anti-CD39 antibody or antigen-binding fragment thereof described herein sufficient to reduce CD39-expressing cells in the tumor.

[0028] In yet another aspect, a method for promoting T cell-mediated immune function in a subject's tumor is provided, the method comprising: (i) identifying a cancer subject having a tumor-infiltrating level of tumor-reactive lymphocytes below a predetermined threshold, characterized as a non-infiltrated or under-infiltrated tumor phenotype; and (ii) administering to the subject an anti-CD39 antibody or antigen-binding fragment thereof described herein in an amount that increases tumor-reactive T cells infiltrating the tumor.

[0029] As described above, many embodiments are further provided that can be applied to any aspect of the present invention and / or combined with any other embodiment described herein. For example, in one embodiment, the intratumoral CD39 high cells are selected from hematopoietic stem cells or progenitor cells (CD45-Sca-1+), CD39+NKT cells, CD39+ macrophages, CD39+ cancer cells, CD39+ endothelial cells, or a combination thereof. In another embodiment, the anti-CD39 antibody or antigen-binding fragment thereof reduces the CD39 present in one or more hematopoietic compartments. 高The level of cells, optionally wherein the one or more hematopoietic compartments are selected from the group consisting of blood, spleen and liver. In yet another embodiment, the anti-CD39 antibody or its antigen-binding fragment is administered as part of an anti-tumor therapy. In yet another embodiment, the anti-CD39 antibody or its antigen-binding fragment is administered as part of an anti-infective therapy, optionally wherein the anti-infective therapy is antiviral therapy (including HIV and HBV infection and COVID-19 infection treatment), for the treatment of Mycobacterium tuberculosis and for the treatment of visceral leishmaniasis. In another embodiment, the anti-CD39 antibody or its antigen-binding fragment is administered as part of an anti-tumor therapy for the treatment of solid tumors, optionally wherein the solid tumor is pancreatic cancer, liver cancer, lung cancer, gastric cancer, esophageal cancer, head and neck squamous cell carcinoma, prostate cancer, colon cancer, breast cancer, lymphoma, gallbladder cancer, kidney cancer, multiple myeloma, ovarian cancer, cervical cancer or glioma. In yet another embodiment, the anti-CD39 antibody or its antigen binding fragment is administered as part of an anti-tumor therapy for treating a liquid tumor, optionally wherein the liquid tumor is a leukemia. In yet another embodiment, the anti-CD39 antibody or its antigen binding fragment is administered as part of a therapy involving one or more chemotherapeutic agents, anti-angiogenic agents, immuno-oncology agents and / or radiation. In another embodiment, the therapy includes administering one or more inhibitors (antagonists) of one or more checkpoint molecules, optionally wherein the one or more checkpoint molecules are selected from the group consisting of: PD-1 antagonists, CTLA-4 antagonists, LAG-3 antagonists, TIM-3 antagonists, TIGIT antagonists, and Siglec-15 antagonists. In yet another embodiment, the therapy includes administering one or more activators (agonists) of one or more costimulatory molecules, optionally wherein the one or more costimulatory molecules are selected from the group consisting of: GITR agonists, CD27 agonists, 4-1BB agonists, OX40 agonists, CD137 agonists, ICOS agonists, and CD28 agonists.In yet another embodiment, the therapy comprises administering one or more of the following: a VEGFR or VEGF antagonist, an EGFR or EGF antagonist, an IDO inhibitor, an IDO1 inhibitor, an HDAC inhibitor, a PI3Kδ inhibitor, an IL-15 agonist, a CXCR4 antagonist, a CXCL12 antagonist, a DNMT inhibitor, interleukin-21, an anti-KIR antibody, an anti-CSF-1R antibody, an anti-CCR4 antibody, GMCSF, an anti-PS antibody, an anti-CD30 antibody-aurstatin E conjugate, an anti-CD19 antibody, an anti-CEA IL-2 antibody, an anti-NY-ESO-1 antibody, an anti-NKG2A antibody, a STING agonist, a TRL7 / 8 agonist, a RIG-1 agonist and / or an NRLP3 inhibitor, an anti-CD73 antibody (such as MEDI9447), a P2X7 antagonist, or an adenosine A2A receptor antagonist. In another embodiment, the treatment includes administering one or more innate immune inducers, optionally wherein the one or more innate immune inducers are selected from the group consisting of: inhibitors of the CD47-SIRPα axis (e.g., antibodies or other binding portions that bind to CD47 or SIRPα and inhibit the interaction of the two molecules), inhibitors of the CD24-Siglec-10 axis (e.g., antibodies or other binding portions that bind to CD24 or Siglec-10 and inhibit the interaction of the two molecules), NGK2A checkpoint inhibitors that block HLA-E-driven inhibition of NK and CD8+ cells, STING agonists, TLR7 / 8 agonists, and RIG-I agonists. In yet another embodiment, the anti-CD39 antibody or its antigen-binding fragment is administered as part of a therapy including tumor vaccines, adoptive cell therapy (including CAR-T and ACTR therapy), anti-tumor gene therapy, inhibitory nucleic acid therapy (such as siRNA, shRNA, antisense, CRISPR, and TALEN therapy), and / or oncolytic virus therapy. In yet another embodiment, the subject is an animal model of cancer. In another embodiment, the subject is a mammal, optionally wherein the mammal is a human or a rodent. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 . Affinity of Ig39-21 measured by flow cytometry using human CD39hi human B lymphoblastoid (HCC1739BL) cells. Fully human anti-CD39 antibody clone Ig39-21 produced by transient transfection was serially diluted as indicated and incubated with HCC1739BL cells at 4°C for 30 minutes followed by flow cytometry analysis. Kd was calculated to be 0.412 nM.

[0031] Figure 2.Ig39-21 demonstrated ADCC activity against HCC1739BL cells: Luc-reporter assay. HCC1739BL cells were used as target cells. Jurkat cells stably expressing luciferase and hCD16a-158V were used as effector cells. Target cells were pre-incubated with Ig39-21 serially diluted as indicated for 30 minutes at 37°C in 5% CO2, followed by co-culture with effector cells (T:E=1:6) for 6 hours. ADCC activity was indicated by an increase in luciferase activity over background. RLU: relative luminescence unit. EC50 was calculated to be 0.014 μg / mL.

[0032] Figure 3 .Ig39-21 demonstrated ADCC activity selectively against human CD39hi Raji cells: Luc-reporter assay. Various Raji cell lines with different human CD39 expression levels, including Raji cells (Raji-hCD39neg), hCD39-transfected Raji cells that highly expressed human CD39 (Raji-hCD39hi), or hCD39-transfected Raji cells that expressed low levels of human CD39 (Raji-hCD39lo) were used as target cells. Jurkat cells stably expressing luciferase and hCD16a-158V were used as effector cells. Target cells were pre-incubated with serially diluted Ig39-21 as indicated for 30 minutes at 37°C in 5% CO2, followed by co-culture with effector cells (T:E=1:6) for 6 hours. ADCC activity is indicated by the increase in luciferase activity over background (RLU). RLU: relative luminescence unit.

[0033] Figure 4 .Ig39-21 does not exert ADCC activity against CD39 low normal endothelial cells (HUVEC). Human melanoma cells (SK-MEL-28) and human umbilical vein endothelial cells (HUVEC) were used as target cells. Jurkat cells stably expressing luciferase and hCD16a-158V were used as effector cells. Target cells were pre-incubated with Ig39-21 serially diluted as indicated for 30 minutes at 37°C in 5% CO2, followed by co-culture with effector cells (T:E=1:6) for 6 hours. ADCC activity is indicated by an increase in luciferase activity over background. RLU: relative luminescence unit. This data indicates the safety of Ig39-21, which avoids potential systemic side effects.

[0034] Figure 5.CD39 confers resistance to NK cytotoxicity on target Raji-hCD3ghi cells. CFSE-labeled Raji-hCD3gneg or Raji-hCDSghi cells were used as target cells and co-cultured with NK-92-CD16V / V effector cells at different ratios (as indicated) for 6 hours at 37°C in 5% CO2. Target cell death was then analyzed by flow cytometry by propidium iodide (P / I) uptake. Results are expressed as CFSE + P / I + % of cells.

[0035] Figure 6 .Ig39-21 enhances NK cytotoxicity against Raji-hCD3ghi cells. CFSE-labeled Raji-hCD3ghi target cells were incubated with or without Ig39-21 antibody (10 μg / mL) for 30 minutes at 37°C in 5% CO2, followed by co-culture with NK-92-CD16 V / V effector cells at different ratios (as indicated) for 6 hours at 37°C. Target cell death was analyzed by flow cytometry, and CFSE + P / I + % of cells.

[0036] Figure 7 .Ig39-21 enhances NK cytotoxicity against hCD3ghi human B lymphoblastoid (HCC1739BL) cells. CFSE-labeled HCC1739BL target cells were incubated with or without Ig39-21 antibody (10 μg / mL) for 30 minutes at 37°C in 5% CO2, followed by co-culture with NK-92-CD16 V / V effector cells at different ratios (as indicated) for 6 hours at 37°C. Target cell death was analyzed by flow cytometry, and CFSE + P / I + % of cells.

[0037] Figure 8 . No fucosylation enhances Ig39-21-mediated ADCC against HCC1739BL cells: Luc-reporter assay. HCC1739BL target cells were pre-incubated with serially diluted Ig39-21 produced by transient transfection at 37°C for 30 minutes in the absence (Ig39-21WT) or presence of a fucosylation inhibitor (Ig39-21AF), and further co-cultured with Jurkat effector cells (T:E=1:6) for 6 hours. ADCC activity is indicated by the increase in luciferase activity over background. RLU: relative luminescence unit. EC50 was calculated to be 0.02 μg / mL (Ig39-21WT) and 0.0008 μg / mL (Ig39-21AF).

[0038] Fig. 9 . Afucosylation enhances Ig39-21-mediated ADCC against HCC1739BL cells: NK cytotoxicity assay. CFSE-labeled HCC1739BL target cells were incubated with serially diluted Ig39-21WT or Ig39-21AF as indicated for 30 minutes at 37°C in 5% CO2. Cells were then co-cultured with NK-92-CD16 V / V effector cells (E:T=1:8) for 6 hours at 37°C. Target cell death was analyzed by flow cytometry, and CFSE + P / I + % of cells (% of cytotoxicity). EC50 was calculated to be 0.04 μg / mL (Ig39-21WT) and 0.0006 μg / mL (Ig39-21AF).

[0039] Fig.10 . Optimized Ig39-21 (NP501-BK), Ig39-21WT and Ig39-21AF showed similar binding affinity using human CD39 positive CHO cells (CHO-hCD39). Ig39-21WT, Ig39-21AF or NP501-BK (produced by using stably transfected cells, an optimized version of Ig39-21) were serially diluted as indicated and incubated with CHO-hCD39 cells at 4°C for 30 minutes. The cells were then incubated with secondary antibodies (anti-human IgG (Fc specific), Alexa Fluor 500 antibody) at 4°C. 488) staining for 30 minutes and analyzed by flow cytometry. A human IgG1 isotype control antibody (KLH-hIgG1) was used in parallel. Kd was calculated to be 0.48 nM (NP501-BK), 0.52 nM (Ig39-21WT) and 0.51 nM (Ig39-21AF).

[0040] Fig.11 NP501-BK, Ig39-21WT, and Ig39-21AF showed similar binding affinity using CD39-expressing HCC1739BL cells. Ig39-21WT, Ig39-21AF, or NP501-BK was serially diluted as indicated and incubated with HCC1739BL cells at 4°C for 30 minutes. The cells were then incubated with a secondary antibody (anti-human IgG (Fc-specific antibody) Alexa Fluor 501-400 ng / mL) at 4°C. 488) staining for 30 minutes and analyzed by flow cytometry. A human IgG1 isotype control antibody (KLH-hIgG1) was used in parallel. Kd was calculated to be 0.16 nM (NP501-BK), 0.29 nM (Ig39-21WT) and 0.35 nM (Ig39-21AF).

[0041] Fig.12 . Optimized Ig39-21 (NP501-BK) and afucosylated Ig39-21 exert similar ADCC activity: Luc-reporter assay using HCC1739BL cells. HCC1739BL target cells were pre-incubated with serially diluted human IgG1 isotype control antibody (KLH-hIgG1) or fully human anti-CD39 monoclonal antibody (Ig39-21AF or NP501-BK) for 30 minutes at 37°C and further co-cultured with Jurkat effector cells (T:E=1:6) for 6 hours. ADCC activity is indicated by the increase in luciferase activity over background. RLU: relative luminescence unit. EC50 was calculated to be 0.0017 μg / mL (NP501-BK) and 0.00086 μg / mL (Ig39-21AF).

[0042] Fig.13 .NP501-BK and Ig39-21AF exert much higher ADCC activity than Ig39-21WT: NK cytotoxicity against HCC1739BL cells. CFSE-labeled HCC1739BL target cells were incubated with serially diluted human IgG1 isotype control antibody (KLH-hIgG1) or fully human anti-CD39 monoclonal antibody (Ig39-21WT, Ig39-21AF or NP501-BK) as indicated for 30 minutes at 37°C in 5% CO2. The cells were then co-cultured with NK-92-CD16 V / V effector cells (E:T=1:8) for 6 hours at 37°C. Target cell death was analyzed by flow cytometry and the % of CFSE+P / I+ cells (% of cytotoxicity) was calculated. The EC50 was calculated to be 1.58E-04 μg / mL (NP501-BK), 4.22E-03 μg / mL (Ig39-21WT), and 4.75E-05 μg / mL (Ig39-21AF).

[0043] Fig.14.Fully human anti-CD39 antibodies showed similar CDC activity against Raji-hCD39hi cells. Raji-hCD39hi target cells were pre-incubated with serially diluted human IgG1 isotype control (KLH-hIgG1) or fully human anti-CD39 monoclonal antibodies (Ig39-21WT, Ig39-21AF or NP501-BK) at 37°C for 30 minutes and further exposed to 10% normal human serum (NHS) for 2 hours. Target cell lysis was analyzed by flow cytometry and %P / I was calculated. + Cells (% of cytotoxicity). EC50 was calculated to be 0.31 μg / mL (NP501-BK), 0.38 μg / mL (Ig39-21WT), and 0.25 μg / mL (Ig39-21AF).

[0044] Fig.15 .NP501-BK and Ig39-21AF exert similar anti-tumor efficacy in vivo. C57BL6 humanized CD39 mice (hCD39 KI) implanted subcutaneously with MC38 cells were treated with 5 mg / kg human IgG1 isotype control antibody (KLH-hIgG1) or fully human anti-CD39 monoclonal antibody (Ig39-21AF or NP501-BK) on days 8, 11, 14 and 17 after tumor stimulation. Tumor length (L) and width (W) were measured twice a week using a digital caliper. Tumor volume (mm3) was determined to be L*W*W*0.52. n=5 per group.

[0045] Fig.16 NP501-BK treatment resulted in reduced hCD39 expression on CD39hi tumor-infiltrating lymphocytes. MC38 tumor-bearing hCD39 KI mice were treated with three doses of KLH-hlIG1 (5 mg / kg) or NP501-BK (5 mg / kg) on ​​days 8, 11, and 14 after tumor inoculation. On day 15, splenocytes and tumor-infiltrating lymphocytes were purified from these mice, stained with the indicated cell surface markers, and analyzed by flow cytometry as described in Materials and Methods. n = 6 to 8 per group.

[0046] Fig.17 NP501-BK exerts antitumor activity in the CD39+SK-MEL-28 xenograft model. NU / J nude mice implanted subcutaneously with SK-MEL-28 xenografts were used to evaluate the in vivo efficacy of NP501-BK. When tumors reached approximately 500 mm 3When the mean volume of 100 μg / kg NP501-BK was 40 μg / kg (considered as day 0 of treatment), mice were treated with two doses of 300 μl saline or 10 mg / kg NP501-BK via intraperitoneal route on days 0 and 3. Tumor length (L) and width (W) were measured every three days using a digital caliper. Tumor volume (mm 3 ) was determined to be L*W*W*0.52. n=6 to 7 per group.

[0047] Fig.18 .Epitope competition assay of 18 human / rabbit chimeric anti-human CD39 monoclonal antibodies against reference anti-hCD39 monoclonal antibody clone A1 on HCC1739BL cells. HCC1739BL cells were cultured with a panel of 18 anti-hCD39 monoclonal antibodies (human / rabbit chimeric clones; unconjugated, 2 μg / ml) for 30 minutes at 4°C. The cells were then washed twice and stained with mouse anti-hCD39 monoclonal antibody clone A1 conjugated to PE for 30 minutes at 4°C, followed by flow cytometry analysis. Cells incubated without chimeric antibodies were used as controls.

[0048] Fig.19 .Human / rabbit chimeric clone 9B6 and Ig39-21 exert similar ADCC activity: Luc-reporter assay using HCC1739BL cells. HCC1739BL cells were used as target cells. Jurkat cells stably expressing luciferase and hCD16a-158V were used as effector cells. Target cells were pre-incubated with serially diluted Ig39-21WT or human / rabbit chimeric clone 9B6 (Hu / Ra 9B6; among all chimeric clones, a rabbit anti-hCD39 monoclonal antibody chimeric with human IgG1 Fc with the highest ADCC activity) at 37°C for 30 minutes and further co-cultured with Jurkat effector cells (T:E=1:6) for 6 hours. ADCC activity is indicated by an increase in luciferase activity over background (RLU). RLU: relative luminescence unit. The EC50 was calculated to be 0.014 μg / mL (Ig39-21 WT) and 0.022 μg / mL (Hu / Ra 9B6).

[0049] Fig. 20.ADCC activity of 18 human / rabbit chimeric antibodies: Luc-reporter assay using HCC1739BL cells. HCC1739BL cells were used as target cells. Jurkat cells stably expressing luciferase and hCD16a-158V were used as effector cells. As described above, a panel of 18 human / rabbit chimeric antibodies was examined for ADCC activity. Briefly, target cells were pre-incubated with serially diluted antibodies for 30 minutes at 37°C and further co-cultured with Jurkat effector cells (T:E=1:6) for 6 hours. ADCC activity is indicated by the increase in luciferase activity over background (RLU). RLU is relative luminescence unit. Ten of the 18 clones showed positive ADCC activity.

[0050] Fig.21 Human / rabbit chimeric antibody with high ADCC activity: Luc-reporter gene assay using HCC1739BL cells. Luc-reporter gene ADCC assay as Fig. 20 Six clones with high ADCC activity were summarized. Five of the six clones (except 65H5) did not compete with the epitope of clone A1 (see Fig.18 ).

[0051] Fig. 22 Human / rabbit chimeric clone with low ADCC activity: Luc-reporter gene assay using HCC1739BL cells. Luc-reporter gene ADCC assay as Fig. 20 Four clones with low ADCC activity were pooled, all of which competed with the epitope of clone A1 (see Fig.18 ). 9B6 served as a positive control.

[0052] Fig.23 Human / rabbit chimeric antibody without ADCC activity: Luc-reporter gene assay using HCC1739BL cells. Luc-reporter gene ADCC assay as Fig. 20 Eight clones without ADCC activity were pooled, all of which fully competed with the epitope of clone A1 (see Fig.18 ). 9B6 served as a positive control.

[0053] Fig.24 .Select human / rabbit chimeric antibodies for NK cytotoxicity against HCC1739BL cells. CFSE-labeled HCC1739BL target cells were incubated with serially diluted human / rabbit chimeric clones as indicated at 37°C in 5% CO2 for 30 minutes. Then, the cells were co-cultured with NK-92-CD16 V / V effector cells (E:T=1:8) at 37°C for 6 hours. Target cell death was analyzed by flow cytometry and CFSE was calculated. + P / I+ The % of cells (% of cytotoxicity). The calculated EC50 is listed. The % of maximum cytotoxicity exceeding the background is determined as: for each clone, the % of maximum cytotoxicity at 1 μg / mL-the % of background cytotoxicity of each clone (at 10-4 μg / mL). Exemplary chimeric clones (8C11, 8D8, 9B6, 9C10, 48F10 and 65H5) from the luc-reporter gene high ADCC group show high NK killing activity. In contrast, chimeric clones (59B6, 60D9, 62G12 and 62H10) from the luc-reporter gene ADCC negative group show low NK killing activity.

[0054] Fig.25 . The reference anti-human CD39 monoclonal antibody (hCD39 Ref) inhibited hCD39 ATPase activity on CHO cell membranes. CHO-hCD39 cells were incubated with 10 μg / mL human IgG1 isotype Ultra-LEAF antibody or anti-hCD39 Ref antibody (hCD39 Ref) for 30 minutes at 37°C, followed by incubation with ATP (250 μM) for 15 minutes at room temperature. The supernatant was then collected and analyzed by luminescence using Detection of ATP levels. Cells without antibody (cells+ATP) or ATP alone in the absence of cells were also detected in parallel to calculate the % inhibition of enzyme activity as described in Materials and Methods.

[0055] Fig.26 .The reference antibody (hCD39 Ref) and Ig39-21WT containing the same human IgG1 Fc part exert similar ADCC activity: NK cytotoxicity against HCC1739BL cells. CFSE-labeled HCC1739BL target cells were incubated with serially diluted anti-hCD39 monoclonal antibodies (hCD39 Ref, NP501-BK, Ig39-21WT or Ig39-21AF) as indicated for 30 minutes at 37°C in 5% CO2. The cells were then co-cultured with NK-92-CD16 V / V effector cells (E:T=1:8) for 6 hours at 37°C. Target cell death was analyzed by flow cytometry and CFSE was calculated + P / I + % of cells (% of cytotoxicity). EC50 was calculated to be 2.62E-03 μg / mL (hCD39 Ref), 5.14E-05 μg / mL (NP501-BK), 1.98E-03 μg / mL (Ig39-21 WT), and 7.94E-06 μg / mL (Ig39-21 AF).

[0056] Fig. 27Epitope competition of matrix-I versus anti-hCD39 reference antibody (hCD39 Ref) on HCC1739BL cells. HCC1739BL cells were incubated with 10 μg / ml of unconjugated human IgG1 isotype Ultra-LEAF antibody or hCD39 Ref antibody for 30 min at 4°C. Cells were then exposed to Alexa Fluor 500 for 1 h at 4°C. AF647-conjugated antibodies (8C11, 8D8, 8E9, 9B6 and Ig39-21WT) or PE-conjugated clone A1 were added for 30 minutes, followed by two washes and flow cytometric analysis. The fold change of AF647 or PE MFI detection relative to the isotype control was calculated (no epitope overlap = 1). Ra: rabbit antibody; Hu / Ra: human / rabbit chimeric antibody.

[0057] Fig.28 Epitope competition of matrix-II versus anti-hCD39 reference antibody (hCD39 Ref) on HCC1739BL cells. HCC1739BL cells were incubated with 10 μg / ml unconjugated human IgG1 isotype Ultra-LEAF antibody or hCD39 Ref antibody for 30 minutes at 4°C. Cells were then exposed to unconjugated rabbit or human / rabbit chimeric antibodies (2A11, 2G12, 5F1, 9C10, 48F10, 52G4, 59B6, 65H5, and 67C1) for 30 minutes at 4°C, washed twice, and incubated with secondary antibodies (anti-rabbit IgG (H+L), Alexa Fluor 500, 400 μg / ml) at 4°C. 488) staining for 30 minutes. Finally, cells were washed and analyzed by flow cytometry. The fold change of AF488 MFI detection relative to isotype control was calculated (no epitope overlap = 1).

[0058] Fig.29 . Stability of Antibody: Antigen Immune Complexes on HCC1739BL Cells. Anti-human CD39 antibody (2 μg / ml) was incubated with HCC1739BL cells at 37°C in 5% CO2 for 24 hours or at 4°C for 20 minutes, followed by secondary antibody staining at 4°C (anti-human IgG (Fc specific), Alexa Fluor 488) for 30 minutes. The cells were then washed and analyzed by flow cytometry. The difference in AF488 MFI between 20 minutes and 24 hours of treatment represents the loss of human CD39 on the cell membrane, which is calculated as described in Materials and Methods. After 24 hours, the exemplary chimeric clones from the Iuc-reporter ADCC negative group (8E9, 59B6 and 67C1) and the low ADCC group (52G4) did not form stable immune complexes on the cell membrane (e.g., CD39 loss was greater than 40%). Hu / Ra: human / rabbit chimeric antibody; hlgG1: humanized rabbit antibody, IgG1 isotype; hIgG4: humanized rabbit antibody, IgG4 isotype.

[0059] Fig.30 Affinity of humanized rabbit antibodies measured by flow cytometry using HCC1739BL cells. Human / rabbit chimeric clones (Hu / Ra 8C11, 8D8, and 9C10) and their corresponding humanized clones (IgG1 or IgG4 isotype) were serially diluted as indicated and incubated with HCC1739BL cells for 30 minutes at 4°C. The cells were then incubated with secondary antibodies (anti-human IgG (Fc specific), Alexa Fluor 500, 100 μg / mL) at 4°C. 488) were stained for 30 min and analyzed by flow cytometry. Kd was calculated and indicated next to the graphs.

[0060] Fig.31 NK cytotoxicity of humanized rabbit antibodies against HCC1739BL cells. Human / rabbit chimeric clones (Hu / Ra8C11, 8D8, and 9C10) and their corresponding humanized clones (IgG1 or IgG4 isotype) were serially diluted as indicated and incubated with CFSE-labeled HCC1739BL target cells at 37°C in 5% CO2 for 30 minutes. The cells were then co-cultured with NK-92-CD16 V / V effector cells (E:T=1:8) for 6 hours at 37°C. Target cell death was analyzed by flow cytometry and CFSE was calculated + P / I + % of cells (% cytotoxicity). Calculated EC50 is listed.

[0061] Fig.32 . CDC activity of humanized rabbit antibodies against Raji-hCD39hi cells. Human / rabbit chimeric clones (Hu / Ra8C11, 8D8 and 9C10) and their corresponding humanized clones (IgG1 or IgG4 isotype) were serially diluted as indicated and pre-incubated with Raji-hCD39hi target cells at 37°C for 30 minutes, followed by incubation with 10% normal human serum (NHS) for 2 hours. Target cell lysis was analyzed by flow cytometry and %P / I was calculated. +Cells (% cytotoxicity). Calculated EC50s are listed.

[0062] Fig.33 . Conformational epitope mapping. A list of major putative CD39 epitope candidates is shown. Exemplary, representative CD39 extracellular domain sequences are provided for reference CD39 epitope sequences, as well as a homology model of a human CD39 dimer for data visualization. DETAILED DESCRIPTION

[0063] I. Overview

[0064] Extracellular adenosine is known to be an inhibitor of immune function. Although intracellular adenosine is involved in energy metabolism, nucleic acid metabolism and methionine cycle, in the tumor microenvironment, extracellular adenosine plays an important role in inhibiting immune signaling. The immunosuppressive adenosine 3′5′-monophosphate (cAMP)-mediated pathway through adenosine A2A receptor (A2AR) signaling can inhibit T lymphocytes and natural killer (NK) cells in hypoxia, inflammation and cancerous microenvironments (Ohta et al. (2006) Proc Natl Acad Sci USA, 103: 13132-7). Preclinical evidence, together with recent and evolving positive clinical trial data, confirms that the administration of A2AR inhibitors can be a potential novel strategy for immunotherapy. In addition, in experimental animal models, blocking the adenosine production pathway involving CD39 / CD73 also induced regression of breast cancer, colorectal cancer and melanoma. In the case of anti-CD39 and anti-CD73 antibody therapies, the focus has been primarily on inhibiting or reducing the catabolism of ATP and derived nucleotides to (ultimately) adenosine by binding to these cell surface enzymes that produce adenosine ("ectonucleotidases") and inhibiting the enzymatic activity or removing the enzymatic activity from the cell surface.

[0065] The present invention is based, at least in part, on the discovery that certain antibodies against CD39 are able to selectively target and eliminate (such as by antibody-dependent cellular cytotoxicity) CD39-expressing cells in the tumor microenvironment (more effectively than prior art anti-CD39 antibodies), including CD39+CD45-SCA-1+ stromal cells (such as hematopoietic progenitor cells), CD39+NKT cells, CD39+ macrophages and CD39+ endothelial cells, as well as CD39+ cancer cells. 高 A reduction in cell numbers can result in such changes in the inflammatory phenotype of the tumor, such as enhanced T cell infiltration into the tumor, reduced T cell exhaustion in the tumor, reduced type II NKT cell suppression of intratumoral immune cell function, and / or reduced regulatory T cell (Treg) suppression of intratumoral immune cell function, and / or reduced tumor-associated macrophage (TAM) suppression of intratumoral immune cell function.

[0066] Although not wishing to be bound by any particular theory, certain antibodies generated by the inventors are able to form more stable immune complexes with CD39 to produce more effective ADCC killing efficacy. The inventors have observed that antibodies that are not able to form immune complexes as stable as those encompassed by the present invention with CD39 lead to a reduction in CD39 on the surface, but by mechanisms that increase CD39 shedding or endocytosis (internalization), and do not have the same efficacy in terms of being able to eliminate CD39 expressing cells by antibody-dependent cellular toxicity. In certain embodiments, certain antibodies encompassed by the present invention have been shown to bind to an epitope on CD39 that is non-competitive or only partially competitive with monoclonal antibody clone A1 to bind to CD39.

[0067] As described in more detail in the exemplary methods and illustrated in the accompanying figures, in contrast to, for example, direct inhibition of CD39 NTPase activity by all anti-CD39 therapeutic antibodies in the prior art (Perrot et al., 2019, Cell Reports 27: 2411-2425; Li et al., 2020, Cancer Discovery 9(12): CD-19-0541; and PCT publication WO 2017 / 089334), our anti-CD39 antibodies are specifically designed to have a human constant region comprising an IgG1 Fc domain. This design confers FcγRIIIa receptor-dependent cellular activity to the anti-CD39 antibodies of the present invention, such as antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) against CD39+ cells and / or antibody-mediated target endocytosis of CD39+ cells within tumors. Thus, such cellular activity results in CD39 in tumors. 高 Elimination and reduction of cells.

[0068] Exemplary features of the subject anti-CD39 monoclonal antibodies, which are taught away from use in therapeutic anti-CD39 antibodies described in the references, are summarized below.

[0069] The subject antibodies target CD39+ cells in tumors via FcγRIIIa receptor-dependent activities (eg, ADCC).

[0070] As an example, Fig.13 and Fig.26 It was shown that reduced fucosylation (also known as hypofucosylation or afucosylation) of the leading clone Ig39-21 (a fully human anti-CD39 monoclonal antibody) by using a fucosylation inhibitor (Ig39-21AF) or by optimizing the production method (NP501-BK) significantly enhanced its ADCC activity against CD39+ cells in vitro. This was paralleled by an enhancement of the antitumor activity of these afucosylated antibodies in vivo ( Fig.15), while the fully glycosylated form (Ig39-21WT) showed no antitumor activity in the same tumor model (data not shown).

[0071] like Fig.13 and 26 The in vitro maximum effective dose (MaxED) of these differently fucosylated Ig39-21 antibodies predicted by the NK cytotoxicity assay further illustrates their differential in vivo anti-tumor activity. For example, afucosylation enhances the MaxED of Ig39-21WT from 0.1 μg / ml up to 0.001 μg / ml. When translated into the clinic, this 100-fold increase will be high efficacy, favorable safety profile, good tolerability and low cost.

[0072] In contrast, the reference hCD39 antibody (hCD39 Ref) used herein shares an antigen binding site with an antibody in the art. However, in contrast to the Ref antibody used in the current example, the prior art antibody was produced with an Fc portion specifically designed to have abrogated ADCC function (i.e., it was taught to have been specifically produced to bind CD39 and inhibit NTPase activity without causing CD39-dependent ADCC cell killing).

[0073] The ADCC activity of the subject anti-CD39 antibody is selective for CD39 高 cell.

[0074] As an example, Figure 3 and Figure 4 The ADCC activity of Ig39-21 was shown to be selective for CD39 高 Cells (i.e. Figure 3 Raji-hCD39hi cells and Figure 4 SK-MEL-28 cells in vitro).

[0075] Relevance of these in vitro data to the in vivo tumor microenvironment: Fig.16 hCD39 was confirmed to be highly upregulated within tumors of hCD39KI mice bearing MC38, including CD45+ tumor-infiltrating lymphocytes (i.e., CD3+CD11b- T cells, CD3-CD11b+ myeloid cells, and F4 / 80hiGr-1 tumor-associated macrophages) and tumor-associated vascular endothelial cells (data not shown). NP501-BK treatment resulted in the elimination and reduction of CD39 high cells in tumors ( Fig.16 and data not shown).

[0076] This functional feature should confer tumor specificity to the antibody, thereby avoiding systemic side effects that should result in safer anti-CD39 antibodies.

[0077] Anti-CD39 antibodies form stable immune complexes with antigens on the target cell membrane, giving the antibodies high ADCC activity.

[0078] As Fig.29 In one example shown in Figure 1, the stability of the antibody: antigen immune complex on the target cell surface was examined using antibodies selected from the following three groups: high ADCC (i.e., NP501-BK, hCD39 Ref, and human / rabbit chimeric clones 8C11, 8D8, 9C10, and 48F10), low ADCC (human / rabbit chimeric clone 52G4), or ADCC negative (human / rabbit chimeric clones 8E9, 59B6, and 67C1). The strong and positive correlation between the stability of this immune complex and the ADCC activity of the antibody is clearly visible. The more stable the antibody: antigen immune complex, the higher the ADCC activity of the antibody.

[0079] Different epitopes of anti-CD39 antibodies are directly related to the ADCC activity of the antibodies.

[0080] As an example, by comparing the epitope of the subject human / rabbit chimeric anti-hCD39 antibody with the epitope of the commercially available anti-hCD39 monoclonal antibody clone A1, Fig.18 and 21 23 showed that anti-CD39 antibodies that bind to anti-CD39 in a manner that does not compete or only partially competes with clone A1 for binding to CD39 are likely to have high ADCC activity, that is, except for 65H5, five of the six high ADCC antibodies (2G12, 8C11, 8D8, 9B6, and 9C10) showed this feature ( Fig.21 In contrast, all antibodies in the low ADCC (2A11, 5F1, 52G4, and 63B1) and ADCC-negative (8E9, 59B6, 60D9, 62G12, 62H10, 65E10, 67A8, and 67C1) groups showed epitopes that completely overlapped with that of clone A1 ( Fig. 22 and 23 ).

[0081] Multiple CD39 in tumors with subject anti-CD39 antibodies 高 Cellular targets.

[0082] As an example, the CD39-MC38 colorectal cancer model in hCD39 KI mice ( Fig.15 and 16 ). The antitumor activity of NP501-BK in this model is due to its inhibition of CD39 高 Results of the targeting effect on tumor infiltrating lymphocytes and tumor-associated vascular endothelial cells (data not shown).

[0083] Another example is a xenograft tumor model using NU / ] nude mice implanted with CD39+ human SK-MEL-28 melanoma cells ( Fig.17 ). These homozygous athymic nude mice lack T cells and also have partial defects in B cell development, while their NK cells are functionally competent. Therefore, in this xenograft tumor model, the target cells killed by NP501-BK-mediated ADCC are CD39+SK-MEL-28 tumor cells, which is consistent with the in vitro ADCC activity of the antibody against SK-MEL-28 cells ( Figure 4 ).

[0084] II. Definitions

[0085] To facilitate understanding of the present invention, a number of terms and phrases are defined below.

[0086] "CD39," also known as "cluster of differentiation 39," "ectonucleoside triphosphate diphosphohydrolase-1," or (gene) "ENTPD1," and (protein) "NTPDase 1" is a cell surface-localized ectonucleotidase with an extracellular-facing catalytic site that catalyzes the hydrolysis of the γ- and β-phosphate residues of triphosphate and diphosphate nucleosides to monophosphate nucleoside derivatives (enzyme entry: EC 3.6.1.5), such as hydrolyzing P2 receptor ligands such as ATP, ADP, UTP, and UDP (Junger et al. (2011) Nat. Rev. Immunol. 11: 201-212). A representative human NTPDase 1 protein sequence is provided in the UniProtKB entry "P49961 (ENTP1_human)," and a representative human coding sequence for the enzyme is provided in GenBank Accession No. S73813. Pericellular adenosine can modulate pro-inflammatory or pro-inhibitory signals in immune cells by binding to various adenosine receptors (Emst et al. (2010) J. Immunol. 185: 1993-1998; Antonioli et al. (2013) Trends Mol. Med. 19: 355-367; Parodi et al. (2013) Cancer Immunol. Immunother. 62: 851-862; Boer et al. (2013) Eur. J. Immunol. 43: 1925-1932; Xu et al. (2013) Neuro-Oncol. 15: 1160-1172; U.S. Patent Publication No. 2013 / 0123345). For example, adenosine binds to the A2A receptor expressed by lymphocytes, causing accumulation of intracellular cAMP, thereby preventing T cell activation and NK cell cytotoxicity (Zarek et al. (2008) Blood 111: 251-259; Lokshin et al. (2006) Canc. Res. 66: 7758-7765). CD39 was originally identified as an activation marker on human lymphocytes, but has subsequently been shown to be a hallmark characteristic of regulatory T cells (Kansas et al. (1991) J. Immunol. 146: 2235-2244; Deaglio et al. (2007) J. Exp. Med. 204: 1257-1265; Borsellino et al. (2007) Blood 110: 1225-1232). Loss of CD39 in Tregs significantly impairs their ability to suppress T cell activation, suggesting that the juxtacrine activity of CD39 acts to negatively regulate T cell function (Deaglio et al. (2007) J. Exp. Med. 204: 1257-1265).In general, CD8+ T cells have been reported to be CD39- (Kansas et al. (1991) J. Immunol. 146:2235-2244; Moncrieffe et al. (2010) J. Immunol. 185:134-143; Pulte et al. (2011) Clin. Lymph. Myeloma Leuk. 11:367-372; Boer et al. (2013) Eur. J. Immunol. 43:1925-1932). However, upregulation of this marker on exhausted T cells has recently been noted in the context of tumors and chronic viral infections, such as HCV and HIV, and coronaviruses such as SARS-COV2 (COVID-19) (Canale et al. (2017) Cancer Res. 78(1): 115-28; Gupta et al. (2015) PLoS Pathog. 11(10): e1005177; Mathew et al. (2020) Science 10.1126 / science.abc8511).

[0087] The structure-function relationship of the CD39 protein is well known in the art (reviewed, for example, by Antonioli et al. (2013) Trends Mol. Med. 19:355-367; Wang and Guidotti (1996) J. Biol. Chem. 271:9898-9901; Kaczmarek et al. (1996) J. Biol. Chem. 271:33116-33122). For example, human CD39 is a protein of approximately 500 amino acids with approximately seven potential N-linked glycosylation sites, eleven Cys residues, and two transmembrane regions (Maliszewski et al. (1994) J. Immunol. 153: 3574-3583) organized in the form of two transmembrane domains, a small cytoplasmic domain containing N-terminal and C-terminal segments, and a large extracellular hydrophobic domain consisting of five highly conserved domains, referred to as apyrase conserved regions (ACRs) 1 to 5, which are required for the enzyme's catabolic activity (Heine et al. (2001) Eur. J. Biochem. 268: 364-373). The amino acid sequences of ACR 1 and ACR 5 contain a phosphate binding motif (DXG) that is important for stabilizing the interaction between the enzyme and its nucleotide substrate during phosphate cleavage. In addition, two ACR residues are required for enzymatic activity, Glu 174 in ACR 3 and Ser 218 in ACR 4 (Heine et al. (2001) Eur. J. Biochem. 268: 364-373; Smith et al. (1998) Biochim. Biophys. Acta 1386: 65-78). After expression on the cell surface, CD39 becomes catalytically active (Smith et al. (1998) Biochim. Biophys. Acta 1386: 65-78).

[0088] Representative human CD39 cDNA and protein sequences are well known in the art and are publicly available from the National Center for Biotechnology Information (NCBI). For example, at least seven human CD39 transcript variants are known to encode six different human CD39 isoforms. Human CD39 isoform 1 is available under accession numbers NM_001776.5 and NP_001767.3. Transcript variants represent the longest transcript and encode isoform 1. Compared to transcript variant 1, human CD39 isoform 2, which can be obtained under accession numbers NM_001098175.1 and NP_001091645.1, uses an alternative 5' exon, which results in a different 5' untranslated region (UTR) and causes translation initiation at an alternative start codon, resulting in a longer and different N-terminus. Compared to transcript variant 1, human CD39 isoform 3, which can be obtained under accession numbers NM_001164178.1 and NP_001157650.1, uses an alternative 5' exon, which results in a different 5'UTR and causes translation initiation at an alternative start codon, resulting in a longer and different N-terminus. Relative to transcript variant 1, which produces a shorter isoform, human CD39 isoform 4, which can be obtained under accession numbers NM_001164179.1 and NP_001157651.1, uses an alternative in-frame splice site. Relative to transcript variant 1, which produces a shorter isoform, human CD39 isoform 5, which can be obtained under accession numbers NM_001164181.1 and NP_001157653.1, uses an alternative exon in the 5' region, resulting in a different 5'UTR and translation initiation at the downstream start codon. Relative to transcript variant 1 that produces a shorter isoform, human CD39 isoform 6, available at accession numbers NM_001164182.1 and NP_001157654.1, lacks alternative exons, resulting in a different 5'UTR at the downstream start codon and causing translation initiation. Relative to transcript variant 1 that produces a shorter isoform, human CD39 isoform 6, also encoded by another transcript variant, available at accession numbers NM_001164183.1 and NP_001157655.1, lacks two alternative internal exons, resulting in a different 5'UTR at the downstream start codon and causing translation initiation.

[0089] The nucleic acid and polypeptide sequences of CD39 orthologs in organisms other than humans are well known and include, for example, mouse CD39 (NM_009848.3 and NP_033978.1), rat CD39 (NM_022587.1 and NP_072109.1), cow CD39 (NM_174536.2 and NP_776961.1), frog CD39 (NM_001006795.1 and NP_001006796.1), and zebrafish CD39 (NM_001003545.1 and NP_001003545.1).

[0090] Extensive glycosylation of CD39 is associated with its cell surface expression and activity, such that deletion or mutation of glycosylated residues to non-glycosylated residues results in significantly reduced CD39 activity (see, e.g., deletion or mutation of glycosylated residue 73 at the N-terminus, residue 333 in the middle, and / or residue 429 and / or residue 458 at the C-terminus of rat CD39 or corresponding residues in their orthologs; Wu et al. (2005) Mol. Biol. Cell. 16: 1661-1672). Similarly, mutation of conserved residues in the apyrase conserved region (ACR) of any one or more of ACRs 1 to 5 results in a reduction in CD39 activity (Schulte am Esch et al. (1999) Biochem. 38:2248-2258; Yang et al. (2001) Biochem. 40:3943-4940; Wang and Guidotti (1998) J. Biol. Chem. 273:11392-11399).

[0091] The regulation (e.g., reduction) of CD39 activity can be measured in many ways (e.g., according to the measurements described herein, including the use of controls, ratios, comparisons with baselines, etc.). For example, a CD39 activity regulator can reduce the catalytic activity of the ectonucleotidase or the overall CD39 activity compared to the level of this ectonucleotidase in the presence of a test agent. In one embodiment, CD39 activity is determined by analyzing the concentration of adenosine in a sample. The concentration can be assessed over time. In another embodiment, ATP is added to the sample tested and the concentration of the remaining ATP, AMP, or adenosine is determined or assessed. Regulation (such as reduction) in this context can mean a reduction of 1%, 5%, 10%>, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 120%, 150%, 200%, 500%, 1000% or more. In one embodiment, the increase is detected over time.

[0092] "CD39 antibody" (or "anti-CD39 antibody") refers to an antibody that selectively binds to one or more epitopes of the NTPD enzyme 1 protein, and includes monoparatopic antibodies, as well as biparatopic antibodies and other multiparatopic forms of antibodies.

[0093] a. Antibodies and other peptides

[0094] The term "antibody" as used herein refers to an immunoglobulin molecule that recognizes and specifically binds to a target (such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or a combination of any of the foregoing) through at least one antigen binding site, wherein the antigen binding site is generally within the variable region of the immunoglobulin molecule. As used herein, the term encompasses intact polyclonal antibodies, intact monoclonal antibodies, antibody fragments (such as Fab, Fab', F(ab')2, and Fv fragments), single-chain Fv (scFv) antibodies (provided that those fragments have been formatted to contain Fc or other FcγRIII binding domains), multispecific antibodies, bispecific antibodies, monospecific antibodies, monovalent antibodies, chimeric antibodies, humanized antibodies, human antibodies, fusion proteins comprising an antigen binding site of an antibody (which is formatted to contain Fc or other FcγRIII binding domains), and any other modified immunoglobulin molecules comprising an antigen binding site, as long as the antibody exhibits the desired biological activity.

[0095] In the context of the present invention, "antibody-mediated target endocytosis" refers to antibody-mediated depletion of CD39 from the surface of CD45+ immune cells without a substantial decrease in the number of CD45+ immune cells, ie, by processes other than inducing CD45+ cell death.

[0096] As used herein, the term "antigen-binding portion" or "antigen-binding fragment" of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., human CD39). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term "antigen-binding portion" of an antibody (e.g., an anti-CD39 antibody described herein) include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (v) a dAb fragment consisting of the VH domain (Ward et al. (1989) Nature 341: 544-546); and (vi) an isolated complementarity determining region (CDR) or (vii) a combination of two or more isolated CDRs, which may be optionally linked by a synthetic linker. Such single-chain antibodies are also intended to be encompassed within the term "antigen-binding portion" of an antibody. These and other potential constructs are described in Chan and Carter (2010) Nat. Rev. Immunol. 10: 301. These antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened for utility in the same manner as intact antibodies. Antigen binding portions can be produced by recombinant DNA technology, or by enzymatic or chemical cleavage of intact immunoglobulins.

[0097] The term "variable region" of an antibody refers to the variable region of the antibody light chain or the variable region of the antibody heavy chain, alone or in combination. In general, the variable regions of the heavy and light chains are each composed of four framework regions (FRs) and three complementarity determining regions (CDRs) (also called "hypervariable regions"). The CDRs in each chain are tightly bound together by the framework regions and, together with the CDRs from the other chain, contribute to the formation of the antigen binding site of the antibody. There are at least two techniques for determining CDRs: (1) methods based on cross-species sequence variability (i.e., Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th ed., Bethesda Md.), and (2) methods based on crystallographic studies of antigen-antibody complexes (Al Lazikani et al., 1997, J. Mol. Biol., 273:927-948). In addition, a combination of these two methods is sometimes used in the art to determine CDRs.

[0098] Although antibodies can be any of the five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses (isotypes) thereof (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), which are designated α, δ, ε, γ, and μ, respectively, based on the identity of their heavy chain constant domains, preferred CD39 antibodies are of the IgG1 and IgG3 isotypes in order to most effectively engage FcγRIII (i.e., at 10 -7 or smaller Kd).

[0099] In certain embodiments, the antibodies are "hypofucosylated" and may even be "afucosylated". A "hypofucosylated" antibody preparation refers to an antibody preparation in which less than 50% of the oligosaccharide chains contain α-1,6-fucose. Typically, less than about 40%, less than about 30%, less than about 20%, less than about 10%, or less than 5%, or less than 1% of the oligosaccharide chains in a "hypofucosylated" antibody preparation contain α-1,6-fucose. An "afucosylated" antibody lacks α-1,6-fucose in the carbohydrate attached to the CH2 domain of the IgG heavy chain.

[0100] As used herein, the term "monoclonal antibody" refers to an antibody that displays a single binding specificity and affinity for a specific epitope or an antibody composition in which all antibodies display a single binding specificity and affinity for a specific epitope. Typically, such monoclonal antibodies will be derived from a single cell or nucleic acid encoding the antibody, and will be bred without intentionally introducing any sequence changes. Therefore, the term "human monoclonal antibody" refers to a monoclonal antibody with a variable and optional constant region derived from a human germline immunoglobulin sequence. In one embodiment, human monoclonal antibodies are produced by hybridomas, for example, by fusing B cells obtained from transgenic or transchromosomal non-human animals (e.g., transgenic mice with a genome comprising human heavy chain transgenes and light chain transgenes) to immortalized cells.

[0101] As used herein, the term "humanized antibody" refers to the form of non-human (e.g., murine) antibodies, which are specific immunoglobulin chains, chimeric immunoglobulins, or fragments thereof, which contain minimal non-human sequences. Typically, humanized antibodies are human immunoglobulins, in which the residues of CDR are replaced by residues of CDRs from non-human species (e.g., mice, rats, rabbits, or hamsters) with desired specificity, affinity, and / or binding ability. In some instances, the Fv framework region residues of human immunoglobulins are replaced by corresponding residues in antibodies from non-human species. Humanized antibodies can be further modified by replacing additional residues in the Fv framework region and / or in the replaced non-human residues to improve and optimize antibody specificity, affinity, and / or binding ability. Humanized antibodies may include variable domains containing all or substantially all CDRs corresponding to non-human immunoglobulins, and all or substantially all framework regions are those of human immunoglobulin sequences. In some embodiments, the variable domains include the framework region of human immunoglobulin sequences. In some embodiments, the variable domains comprise a framework region of a human immunoglobulin consensus sequence. The humanized antibody may also comprise at least a portion of an immunoglobulin constant region or domain, typically a constant region or domain (Fc) of a human immunoglobulin. It is generally believed that humanized antibodies are different from chimeric antibodies.

[0102] The term "human antibody" as used herein refers to an antibody produced by a human or an antibody having an amino acid sequence corresponding to an antibody produced by a human, made using any technique known in the art.

[0103] The term "chimeric antibody" as used herein refers to an antibody in which the amino acid sequence of the immunoglobulin molecule is derived from two or more species. Typically, the variable regions of the light and heavy chains correspond to the variable regions of an antibody having the desired specificity, affinity and / or binding ability derived from one species of mammal (e.g., mouse, rat, rabbit, etc.), while the constant regions are homologous to sequences in antibodies derived from another species (usually human) to avoid eliciting an immune response in the species.

[0104] "Fc receptor" or "FcR" is a receptor that binds to the Fc region of an immunoglobulin. FcRs that bind to IgG antibodies include receptors of the FcγR family, including allelic variants and alternatively spliced ​​forms of these receptors. The FcγR family consists of three activating (FcγRI, FcγRIII, and FcγRIV in mice; FcγRIA, FcγRIIA, and FcγRIIIA in humans) and one inhibitory (FcγRIIB) receptor.

[0105] "FcγRIIi binding portion" is a peptide, protein, nucleic acid or other portion that can bind to FcγRIII (CD16) and mediate antibody-dependent cellular cytotoxicity (ADCC) when bound to the antigen binding site of an anti-CD39 antibody. The heavy chain Fc fragment containing the CH2 and CH3 domains of the IgG1 and IgG3 isotypes is an FcγRIII binding portion.

[0106] The terms "epitope" and "antigenic determinant" are used interchangeably herein and refer to the portion of an antigen that can be recognized and specifically bound by a particular antibody. When the antigen is a polypeptide, the epitope can be formed by continuous amino acids and non-continuous amino acids juxtaposed by the tertiary folding of the protein. Epitopes formed by continuous amino acids (also called linear epitopes) are usually retained when the protein is denatured, while epitopes formed by tertiary folding (also called conformational epitopes) are usually lost when the protein is denatured. An epitope typically comprises at least 3, and more typically at least 5, 6, 7, or 8 to 10 amino acids in different spatial conformations.

[0107] As used herein, the term "specifically binds to" or "specific for..." refers to a measurable and reproducible interaction, such as binding between a target and an antibody, which determines the presence of a target in the presence of a heterogeneous population of molecules (including biomolecules). For example, an antibody that specifically binds to a target (which may be an epitope) binds to the antibody of this target with greater affinity, binding, greater ease, and / or greater duration than it binds to other targets. In one embodiment, the extent to which an antibody binds to an unrelated target is less than about 10% of the binding of the antibody to the target, as measured, for example, by radioimmunoassay (RIA). In certain embodiments, an antibody that specifically binds to a target has a dissociation constant (Kd) of less than or equal to 1 μM, 100 nM, 10 nM, 1 nM, or even 0.1 nM. In certain embodiments, an antibody specifically binds to an epitope on a protein that is conserved in proteins from different species. In another embodiment, specific binding may include but need not be exclusive binding.

[0108] The terms "polypeptide" and "peptide" and "protein" are used interchangeably herein and refer to polymers of amino acids of any length. The polymer may be linear or branched, it may contain modified amino acids, and it may be interrupted by non-amino acids. The term also encompasses amino acid polymers that have been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within the definition are, for example, polypeptides containing one or more analogs of amino acids (including, for example, non-natural amino acids), as well as other modifications known in the art. It should be understood that because the polypeptides encompassed by the present invention may be based on antibodies or other members of the immunoglobulin superfamily, in certain embodiments, the polypeptides may exist as single chains or as related chains.

[0109] In the context of two or more nucleic acids or polypeptides, the term "identity" or "identity" percentage refers to when comparing and aligning (if necessary, introducing spaces) to achieve maximum correspondence, without considering any conservative amino acid substitutions as part of sequence identity, two or more sequences or subsequences are identical or have a specified percentage of identical nucleotides or identical amino acid residues. The identity percentage can be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software that can be used to obtain the alignment of amino acid or nucleotide sequences are well known in the art. These include but are not limited to BLAST, ALIGN, Megalign, BestFit, GCG Wisconsin Package and variants thereof. In some embodiments, two nucleic acids or polypeptides encompassed by the present invention are substantially identical, meaning that when compared and aligned for maximum correspondence, they have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, and in some embodiments, at least 95%, 96%, 97%, 98%, 99% nucleotide or amino acid residue identity. In some embodiments, the identity exists in a region of an amino acid sequence of at least about 10 residues, at least about 20 residues, at least about 40 to 60 residues, at least about 60 to 80 residues, or any integer value therebetween. In some embodiments, the identity exists in a region longer than 60-80 residues (such as at least about 80-100 residues), and in some embodiments, the sequence is substantially identical over the full length of the compared sequence, such as the coding region of a target protein or antibody. In some embodiments, the identity exists in a region of a nucleotide sequence of at least about 10 bases, at least about 20 bases, at least about 40 to 60 bases, at least about 60-80 bases, or any integer value therebetween. In some embodiments, the identity exists in a region longer than 60-80 bases (such as at least about 80-1000 bases or more), and in some embodiments, the sequence is substantially identical over the full length of the compared sequence, such as the nucleotide sequence encoding the target protein.

[0110] "Conservative amino acid substitution" is a substitution in which one amino acid residue is replaced by another amino acid residue with a similar side chain. Families of amino acid residues with similar side chains have been generally defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). For example, substitution of tyrosine with phenylalanine is a conservative substitution. In general, conservative substitutions in the sequences of polypeptides, soluble proteins, and / or antibodies encompassed by the present invention do not eliminate binding of the polypeptide, soluble protein, or antibody containing the amino acid sequence to the target binding site. Methods for identifying conservative substitutions of amino acids that do not eliminate binding are well known in the art.

[0111] An "isolated" polypeptide, soluble protein, antibody, polynucleotide, vector, cell, or composition is a polypeptide, soluble protein, antibody, polynucleotide, vector, cell, or composition that is in a form not found in nature. Isolated polypeptides, soluble proteins, antibodies, polynucleotides, vectors, cells, or compositions include those that have been purified to the extent that they are no longer in a form found in nature. In some embodiments, an isolated polypeptide, soluble protein, antibody, polynucleotide, vector, cell, or composition is substantially pure.

[0112] As used herein, the term "substantially pure" refers to a material that is at least 50% pure (ie, free of contaminants), at least 90% pure, at least 95% pure, at least 98% pure, or at least 99% pure.

[0113] As used herein, the term "fusion protein" or "fusion polypeptide" refers to a hybrid protein expressed by a nucleic acid molecule comprising the nucleotide sequences of at least two genes.

[0114] As used herein, the term "linker" or "linker region" refers to a linker inserted between a first polypeptide (e.g., an anti-CD39 antibody) and a second polypeptide (e.g., an Fc or other FcγRIII binding portion; a scFV, Vhh domain, or the like, which binds to different proteins to produce a bispecific antibody format that maintains bivalence against CD39). In some embodiments, the linker is a peptide linker. The linker should not adversely affect the expression, secretion, or biological activity of the polypeptide. Preferably, the linker is not antigenic and does not elicit an immune response.

[0115] b. Nucleic Acids

[0116] The terms "polynucleotide" and "nucleic acid" and "nucleic acid molecule" are used interchangeably herein and refer to polymers of nucleotides of any length, and include DNA and RNA. The nucleotides may be deoxyribonucleic acids, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into a polymer by a DNA or RNA polymerase.

[0117] As used herein, the terms "nucleic acid molecule encoding", "DNA sequence encoding" and "DNA encoding" refer to the order or sequence of nucleotides along a deoxyribonucleotide chain of deoxyribonucleic acid. The order of these deoxyribonucleotides determines the order of amino acids along a polypeptide (protein) chain. Thus, a nucleic acid sequence encodes the amino acid sequence.

[0118] As used herein, the term "sequence", grammatical and other forms of the term, when used in reference to a nucleotide sequence, may comprise DNA or RNA, and may be single-stranded or double-stranded. A nucleic acid sequence may be mutated.

[0119] As used herein, the term "vector" means a construct that is capable of delivering and typically expressing one or more target genes or sequences in a host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, cosmids or phage vectors, DNA or RNA expression vectors associated with a cationic condensing agent, and DNA or RNA expression vectors encapsulated in liposomes.

[0120] As used herein, the term "transfection" refers to the entry of exogenous nucleic acids into eukaryotic cells. Transfection can be achieved by various methods known in the art, including calcium phosphate-DNA coprecipitation, DEAE-dextran-mediated transfection, polybrene-mediated transfection, electroporation, microinjection, liposome fusion, lipofection, protoplast fusion, retroviral infection, and gene gun technology (gene gun).

[0121] The term "vector" as used herein is an isolated nucleic acid containing an isolated nucleic acid, which can be used to deliver the composition to the interior of a cell. Many vectors are known in the art, including but not limited to linear polynucleotides, polynucleotides associated with ions or amphiphilic compounds, plasmids and viruses. Therefore, the term "vector" includes autonomously replicating plasmids or viruses. The term should also be interpreted as including promoting nucleic acid transfer to cells of non-plasmid and non-viral compounds (e.g., polylysine compounds, liposomes and the like). Examples of viral vectors include but are not limited to adenoviral vectors, adeno-associated viral vectors, retroviral vectors and the like.

[0122] As used herein, the term "expression vector" refers to a vector comprising a recombinant polynucleotide comprising an operably linked control sequence and a nucleotide sequence to be expressed. The expression vector comprises sufficient cis-acting elements (cis-acting elements) for expression; other elements for expression may be provided by a host cell or an in vitro expression system. Expression vectors include all vectors known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses).

[0123] As used herein, the term "operably linked" refers to the functional linkage between a regulatory sequence and a heterologous nucleic acid sequence connected to a linker resulting in the expression of the latter. For example, when a first nucleic acid sequence and a second nucleic acid sequence are in a functional relationship, the first nucleic acid sequence and the second nucleic acid sequence are operably linked. For example, if a promoter affects the transcription or expression of a coding sequence, the promoter is operably linked to a coding sequence. Typically, operably linked DNA sequences are continuous and, where necessary, two protein coding regions are linked in the same reading frame.

[0124] As used herein, the term "promoter" is defined as a promoter DNA sequence that is recognized or introduced by the synthetic machinery required for cell-specific transcription of a polynucleotide sequence.

[0125] As used herein, the term "constitutive expression" refers to expression under physiological conditions.

[0126] As used herein, the term "inducible expression" refers to expression under certain conditions, such as that occurring upon T cell antigen binding. How to get familiar with conventional "inducible expression".

[0127] The term "electroporation" refers to the use of transmembrane electric field pulses to induce microscopic pathways (pores) in biological membranes; the presence of which allows biomolecules (such as plasmids or other oligonucleotides) to pass from one side of the cell membrane to the other.

[0128] c. Checkpoint inhibitors, co-stimulatory agonists, innate immunity inducers, and chemotherapeutic agents

[0129] "Checkpoint molecules" refer to proteins that are expressed by tissues and / or immune cells and that reduce the efficacy of the immune response in a manner that depends on the degree of expression of the checkpoint molecule. When these proteins are blocked, the "brakes" of the immune system are released, and, for example, T cells are able to kill cancer cells more effectively. Examples of checkpoint proteins found on T cells or cancer cells include PD-1 / PD-L1 and CTLA-4 / B7-1 / B7-2, PD-L2, NKG2A, KIR, LAG-3, TIM-3, CD96, VISTA, TIGIT, and Siglec-15.

[0130] "Checkpoint inhibitors" refer to drug entities that reverse immunosuppressive signaling from checkpoint molecules.

[0131] "Costimulatory molecules" refer to immune cells such as T cell homologous binding partners, which specifically bind to costimulatory ligands, thereby mediating co-stimulation, such as but not limited to proliferation. Costimulatory molecules are cell surface molecules other than antigen receptors or ligands that promote effective immune responses. Costimulatory molecules include but are not limited to MHCI molecules, BTLA receptors and Toll ligands, and OX40, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278) and 4-1BB (CD137). Examples of co-stimulatory molecules include, but are not limited to, CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD 11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG 2D, NKG2C, TNFR2, TRANCE / RANKL, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRTAM, Ly9 CD96(CD229), CD160(BY55), PSGL1, CD100(SEMA4D), CD69, SLAMF6(NTB-A, Ly108), SLAM(SLAMF1, C D150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a and CD83 ligands.

[0132] A "costimulatory agonist" refers to a pharmaceutical entity that activates (agonizes) a co-stimulatory molecule (such as a co-stimulatory ligand would do) and produces an immunostimulatory signal or otherwise increases the potency or efficacy of an immune response.

[0133] "Innate immunity inducers" are agents that simulate innate immune responses (including activation of inflammatory activity of macrophages, NK cells, dendritic cells, monocytes, neutrophils and the like and / or deactivation of anti-inflammatory activity). Innate immunity inducers include inhibitors of the CD47-SIRPα axis, such as antibodies or other binding moieties that bind to CD47 or SlRPα and inhibit the interaction of the two molecules to promote anti-tumor macrophage activity. Innate immunity inducers include inhibitors of the CD24-Siglec-10 axis, such as antibodies or other binding moieties that bind to CD24 or Siglec-10 and inhibit the interaction of the two molecules to promote anti-tumor macrophage activity. In other embodiments, the innate immunity activator may be an NGK2A checkpoint inhibitor that blocks HLA-E-driven inhibition of NK and CD8+ cells. Small molecule inducers of innate immunity include agents such as STING agonists, TLR7 / 8 agonists, and RIG-I agonists.

[0134] "Chemotherapeutic agents" are chemical compounds that may be useful in treating cancer. Examples of chemotherapeutic agents include alkylating agents, such as thiotepa and cyclophosphamide (CYTOXAN); alkyl sulfonates, such as busulfan, improsulfan, and piposulfan; aziridines, such as benzodopa, carboquone, meturedopa, and uredopa; ethyleneimines and methylamelamines, including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide, and trimethylolomelamine; polyacetylenes (especially bullatacin and bullatacinone); delta-9-tetrahydrocannabinol (dronabinol, MARINOL); β-lapachone; lapachol; colchicine; betulinic acid; camptothecins (including synthetic analogs of HYCAMTIN, CPT-11 (irinotecan, CAMPTOSAR, acetylcamptothecin, scopolectin and 9-aminocamptothecin); bryostatin; pemetrexed; callystatin; CC-1065 (including its synthetic analogs of adozelesin, carzelesin and bizelesin); podophyllotoxin; podophyllinic acid acid; teniposide; cryptophycin (especially cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including synthetic analogs KW-2189 and CB1-TM1); eleutherobin; pancratistatin; TLK-286; CDP323, oral alpha-4 integrin inhibitors; sarcodictyin; spongistatin;Nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine tine), lomustine, nimustine and ranimnustine; antibiotics such as enediyne antibiotics (e.g., calicheamicins, especially calicheamicin gamma 1I and calicheamicin omega 1I (see, e.g., Nicolaou et al., Angew. Chem Intl. Ed. Engl. 33: 183-186 (1994)); dynemicins, including dynemicin A; esperamicin;and neocarzinostatin chromophores and related chromoprotein enediyne antibiotic chromophores), aclacinomysin, actinomycin, authramycin, azaserine, bleomycin, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, dimethoate, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including ADRIAMYCIN, morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrroline-doxorubicin, doxorubicin HCl liposome injection (DOXIL) and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid (mycophenolic acid), acid, nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin n), zorubicin; antimetabolites such as methotrexate, gemcitabine (GEMZAR), tegafur (UFTORAL), capecitabine (XELODA), epothilone, and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate;Purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytara bine, dideoxyuridine, doxifluridine, enocitabine, floxuridine and imatinib (2-phenylaminopyrimidine derivative) and other c-Kit inhibitors; antiadreners such as aminoglutethimide, mitotane, trilostane; folic acid supplements such as folinic acid; aceglatone; aldophosphamide glycosides; glycoside; aminolevulinic acid; enniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfornithine; elliptinium acetate; etoglucid; gallium nitrate nitrate; hydroxyurea; lentinan; lonidainine; maytansine alkaloids, such as maytansine and ansamitocin; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; 2-ethylhydrazide; procarbazine; PSK polysaccharide complex (JHS Natural Products Eugene Oreg.);razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2′,2″-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A) A) and anguidine); urethan; vindesine (ELDISINE, FILDESIN); dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; cytarabine ("Ara-C"); thiotepa; taxanes, such as paclitaxel (TAXOL), albumin-engineered paclitaxel nanoparticle formulations (ABRAXANE), and doxetaxel (TAXOTERE); chlorambucil; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs, such as cisplatin and carboplatin ; vinblastine (VELBAN); platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine (ONCOVIN); oxaliplatin; leucovovin; vinorelbine (NAVELBINE); mitoxantrone hydrochloride (novantrone); edatrexate; daunomycin; aminopterin; ibandronate; topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO); retinoids, such as retinoic acid; any of the above pharmaceutically acceptable salts, acids or derivatives;and a combination of two or more of the above such as CHOP (abbreviation for combination therapy of cyclophosphamide, doxorubicin, vincristine and prednisolone) and FOLFOX (abbreviation for a treatment regimen using a combination of oxaliplatin (ELOXATIN) with 5-FU and leucovovin). ;

[0135] Also included in this definition are anti-hormonal drugs, which act to modulate, reduce, block or inhibit the effects of hormones that can promote cancer growth, and are usually in the form of systemic or systemic treatments. They can themselves be hormones. Examples include antiestrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including NOLVADEX tamoxifen), raloxifene (EVISTA), droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene (FARESTON); antiprogestins; estrogen receptor downregulators (ERDs); estrogen receptor antagonists, such as fulvestrant (FASLODEX); agents that act to inhibit or shut down the ovaries, such as leucine releasing hormone (LHRH) agonists, such as leuprolide acetate (LUPRON and ELIGARD), goserelin acetate, buserelin acetate (BUSERELIN); acetate) and triptorelin; antiandrogens such as flutamide, nilutamide, and bicalutamide; and aromatase inhibitors that inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands, such as, for example, 4(5)-imidazole, aminoglutethimide, megestrol acetate (MEGASE), exemestane (AROMASIN), formestanie, fadrozole, vorozole (RIVISOR), letrozole (FEMARA), and anastrozole (ARIMIDEX).In addition, this definition of chemotherapeutic agents includes bisphosphonates, such as clodronate (e.g., BONEFOS or OSTAC), etidronate (DIDROCAL), NE-58095, zoledronic acid / zoledronate (ZOMETA), alendronate (FOSAMAX), pamidronate (AREDIA), tiludronate (SKELID), or risedronate (ACTONEL); and troxacitabine (1,3-dioxolane nucleoside cytosine analog); antisense oligonucleotides, especially those that inhibit the expression of genes in signal transduction pathways involved in cell proliferation, such as, for example, PKC-α, Raf, H-Ras, and epidermal growth factor receptor (EGF-R); vaccines, such as TH ERATOPE vaccines and gene therapy vaccines, such as ALLOVECTIN vaccines, LEUVECTIN vaccines and VAXID vaccines; topoisomerase 1 inhibitors (e.g., LURTOTECAN); antiestrogens, such as fulvestrant; Kit inhibitors, such as imatinib or EXEL-0862 (tyrosine kinase inhibitors); EGFR inhibitors, such as erlotinib or cetuximab; anti-VEGF inhibitors, such as bevacizumab; arinotecan; rmRH (e.g., ABARELIX); lapatinib and lapatinib ditosylate (ErbB-2 and EGFR dual tyrosine kinase small molecule inhibitors, also known as GW572016); 17AAG (geldanamycin derivative of heat shock protein (Hsp) 90 poison) and pharmaceutically acceptable salts, acids or derivatives of any of the above.

[0136] As used herein, the term "cytokine" generally refers to a protein released by one cell population that acts on another cell as an intercellular mediator or has an autocrine effect on the cell that produces the protein. Examples of such cytokines include lymphokines, monokines; interleukins ("IL"), such as IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL10, IL-11, IL-12, IL-13, IL-15, IL-17A-F, IL-18 to IL-29 (such as IL-23), IL-31, including PROLEUKIN rlL-2; tumor necrosis factors, such as TNF-α or TNF-β, TGF-β1-3; and other polypeptide factors, including leukemia inhibitory factor ("LIF"), ("ciliary neurotrophic factor" CNTF), CNTF-like cytokine ("CLC"), cardiotrophin ("CT") and Kit ligand ("KL").

[0137] As used herein, the term "chemokine" refers to a soluble factor (such as a cytokine) that has the ability to selectively induce chemotaxis and activation of leukocytes. They also trigger the processes of angiogenesis, inflammation, wound healing, and tumorigenesis. Examples of chemokines include IL-8, the human homolog of murine keratinocyte chemotactic factor (KC).

[0138] d. Treatment

[0139] In the context of immune dysfunction, the term "dysfunction" refers to a state of reduced immune responsiveness to antigenic stimulation. The term includes common elements in which exhaustion and / or anergy of antigen recognition can occur, but the immune response is then ineffective in controlling infection or tumor growth.

[0140] As used herein, "dysfunctional" also includes refractory or unresponsive to antigen recognition, specifically, impaired ability to translate antigen recognition into downstream T cell effector functions, such as proliferation, cytokine production (e.g., IL-2), and / or target cell killing.

[0141] The term "anergy" refers to a state of no response to antigenic stimulation caused by incomplete or insufficient signals delivered by the T cell receptor (e.g., an increase in intracellular Ca+2 in the absence of ras activation). T cell anergy can also be produced when stimulated with antigens in the absence of costimulation, resulting in cells becoming refractory to subsequent activation by antigens even in the case of costimulation. The anergy state can usually be overturned by the presence of interleukin-2. Anergic T cells do not undergo clonal expansion and / or acquire effector functions.

[0142] The term "exhaustion" refers to T cell exhaustion, a state of T cell dysfunction due to persistent TCR signaling that occurs during many chronic infections and cancers. It differs from anergy in that it is not caused by incomplete or insufficient signaling, but rather by persistent signaling. It is defined as poor effector function, persistent expression of inhibitory receptors, and a transcriptional state that is different from that of functional effector or memory T cells. Exhaustion prevents optimal control of infection and tumors.

[0143] "Enhancing T cell function" means inducing, causing or stimulating T cells to have a continuous or amplified biological function, or renewing or reactivating exhausted or inactive T cells. Examples of enhancing T cell function include: relative to such levels before intervention, increased secretion, increased proliferation, and increased antigenic reactivity of gamma interferon from CD8+T cells (e.g., virus, pathogen or tumor clearance). In one embodiment, the enhanced level is at least 50%, or 60%, 70%, 80%, 90%, 100%, 120%, 150%, 200%. The method of measuring this enhancement is known to those of ordinary skill in the art.

[0144] A "T cell dysfunction disorder" is a disorder or condition characterized by T cells with reduced responsiveness to antigenic stimulation. In a specific embodiment, a T cell dysfunction disorder is a disorder that is particularly associated with inappropriately increased levels of CD39. In another embodiment, a T cell dysfunction disorder is a disorder in which T cells are anergic or have a reduced ability to secrete cytokines, proliferate, or perform cytolytic activity. In a specific aspect, the reduced responsiveness results in ineffective control of pathogens or tumors expressing the immunogen. Examples of T cell dysfunction disorders characterized by T cell dysfunction include unresolved acute infections, chronic infections, and tumor immunity.

[0145] "Tumor immunity" refers to the process by which a tumor evades immune recognition and clearance. Thus, as a therapeutic concept, when this evasion is attenuated, tumor immunity is "cured" and the tumor is recognized and attacked by the immune system. Examples of tumor recognition include tumor binding, tumor shrinkage, and tumor clearance.

[0146] A "sustained response" refers to a persistent effect of reducing tumor growth after treatment has stopped. For example, the tumor size may remain the same or smaller than the size at the start of the administration period. In some embodiments, a sustained response has a duration at least as long as the duration of treatment, at least 1.5x, 2.0x, 2.5x, or 3.0x the length of the duration of treatment.

[0147] As used herein, the terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals in which a population of cells is characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinomas, blastomas, sarcomas, and hematological cancers, such as lymphomas and leukemias.

[0148] As used herein, the terms "tumor" and "neoplasm" refer to any mass of tissue caused by excessive cell growth or proliferation, benign (non-cancerous) or malignant (cancerous), including precancerous lesions. Tumor growth is generally unregulated and progressive, and does not induce or inhibit the proliferation of normal cells. Tumors can affect various cells, tissues or organs, including but not limited to those selected from the bladder, bones, brain, breast, cartilage, glial cells, esophagus, fallopian tubes, gallbladder, heart, intestine, kidney, liver, lung, lymph nodes, nervous tissue, ovary, pancreas, prostate, skeletal muscle, skin, spinal cord, spleen, stomach, testicles, thymus, thyroid, trachea, urethra, ureter, urethra, uterus, vaginal organs or tissues or corresponding cells. Tumors include cancers such as sarcomas, carcinomas, plasmacytomas or (malignant plasma cells). Tumors encompassed by the present invention may include, but are not limited to, leukemias (e.g., acute leukemias, acute lymphoblastic leukemias, acute myeloid leukemias, acute myeloid leukemias, acute promyelocytic leukemias, acute myelomonocytic leukemias, acute monocytic leukemias, acute leukemias, chronic leukemias, chronic myeloid leukemias, chronic lymphocytic leukemias, polycythemia vera), lymphomas (Hodgkin's disease, non-Hodgkin's disease), primary macroglobulinemia, heavy chain diseases, and solid tumors, such as sarcoma cancers (e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, endotheliosarcoma, lymphangiosarcoma, angiosarcoma, lymphangioendothelioma sarcoma, synovialoma, mesothelioma, Ewing's tumor, tumora), leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, carcinoma, bronchial carcinoma, medullary carcinoma, renal cell carcinoma, liver cancer, Nile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, uterine cancer, testicular cancer, lung cancer, small cell lung cancer, bladder cancer, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, schwannoma, meningioma, melanoma, neuroblastoma, retinoblastoma), esophageal cancer, gallbladder cancer, kidney cancer, multiple myeloma. Preferably, "tumor" includes, but is not limited to, pancreatic cancer, liver cancer, lung cancer, gastric cancer, esophageal cancer, head and neck squamous cell carcinoma, prostate cancer, colon cancer, breast cancer, lymphoma, gallbladder cancer, kidney cancer, leukemia, multiple myeloma, ovarian cancer, cervical cancer and glioma.

[0149] As used herein, the term "metastasis" refers to the process by which cancer spreads or metastasizes from a site of origin in the body to other areas of the body and develops similar cancerous lesions in the new location. A "metastatic" or "metastatic" cell is one that has lost adhesive contacts with neighboring cells and migrates from the primary site of the disease via the bloodstream or lymph to invade adjacent body structures.

[0150] The terms "cancer cell" and "tumor cell" refer to the total population of cells derived from cancer or a tumor or precancerous lesion, including non-tumorigenic cells, which comprise the majority of the cancer cell population, and tumorigenic stem cells (cancer stem cells). As used herein, the terms "cancer cell" or "tumor cell" are modified by the term "non-tumorigenic" when referring only to those cells that lack renewal and differentiation capabilities to distinguish those tumor cells from cancer stem cells.

[0151] As used herein, the term "effective amount" refers to an amount that provides a therapeutic or prophylactic benefit.

[0152] As used herein, "complete response" or "CR" refers to the disappearance of all target lesions; "partial response" or "PR" refers to a decrease of at least 30% in the sum of the longest diameters (SLD) of the target lesions, with reference to the baseline SLD; and "stable disease" or "SD" refers to neither a sufficient reduction in target lesions to meet the criteria for PR nor an adequate increase to meet the criteria for PD, with reference to the smallest SLD since the start of treatment.

[0153] As used herein, "progressive disease" or "PD" refers to an increase in the SLD of a target lesion by at least 20%, referenced to the smallest SLD documented since the start of treatment or the presence of one or more new lesions.

[0154] As used herein, "progression-free survival" (PFS) refers to the length of time during and after treatment during which the disease being treated (such as cancer) does not get worse. Progression-free survival can include the amount of time a patient has experienced a complete response or a partial response, and the amount of time a patient has experienced stable disease.

[0155] As used herein, "overall response rate" (ORR) refers to the sum of the complete response (CR) rate and the partial response (PR) rate.

[0156] As used herein, "overall survival rate" refers to the percentage of subjects in a group who are likely to be alive after a specific period of time.

[0157] As used herein, the term "treatment" refers to a process or treatment in which a subject attempts to change a clinical disease caused by cell intervention, which may be a preventive intervention process for clinical pathology. It includes but is not limited to preventing the occurrence or recurrence of a disease, alleviating symptoms, reducing the direct or indirect pathological consequences of any disease, preventing metastasis, slowing the rate of disease progression, improving or alleviating a disease, or improving prognosis.

[0158] The term "subject" refers to any animal (e.g., mammal), including but not limited to humans, non-human primates, canines, felines, rodents, and the like, that is the recipient of a particular treatment. Generally, with respect to human subjects, the terms "subject" and "patient" are used interchangeably herein.

[0159] As used herein, the terms "agonist" and "agonism" refer to or describe a therapeutic moiety that can directly or indirectly substantially induce, activate, promote, increase or enhance the biological activity of a target and / or pathway. The term "agonist" is used herein to include any agent that partially or completely induces, activates, promotes, increases or enhances the activity of a protein or other target of interest.

[0160] As used herein, the terms "antagonist" and "antagonism" refer to or describe a therapeutic moiety that can directly or indirectly partially or completely block, inhibit, reduce or neutralize the biological activity of a target and / or pathway. The term "antagonist" is used herein to include any agent that partially or completely blocks, inhibits, reduces or neutralizes the activity of a protein or other target of interest.

[0161] As used herein, the terms "modulation" and "modulate" refer to a change or alteration in biological activity. Modulation includes, but is not limited to, stimulating activity or inhibiting activity. Modulation can be an increase in activity or a decrease in activity, a change in binding characteristics, or any other change in a biological, functional, or immunological property associated with the activity of a protein, pathway, system, or other biological target of interest.

[0162] As used herein, the term "immune response" includes responses from the innate immune system and the adaptive immune system. It includes cell-mediated and / or humoral immune responses. It includes T cell and B cell responses, and responses from other cells of the immune system such as natural killer (NK) cells, monocytes, macrophages, etc.

[0163] The term "pharmaceutically acceptable" means a material approved or approvable by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, including humans.

[0164] The term "pharmaceutically acceptable excipient, carrier or adjuvant" or "acceptable pharmaceutical carrier" refers to an excipient, carrier or adjuvant that can be administered to a subject together with at least one agent of the present invention without destroying its pharmaceutical activity and is non-toxic when administered in an amount sufficient to deliver a therapeutic effect. In general, those skilled in the art and the U.S. FDA consider pharmaceutically acceptable excipients, carriers or adjuvants to be inactive ingredients of any formulation.

[0165] The term "effective amount" or "therapeutically effective amount" or "therapeutic effect" refers to an amount of an anti-CD39 antibody that is effective to "treat" a disease or condition in a subject, such as a mammal. In the case of cancer or tumors, a therapeutically effective amount of an anti-CD39 antibody has a therapeutic effect and thus can enhance the immune response, enhance the anti-tumor response, increase the cytolytic activity of immune cells, increase tumor killing by immune cells, reduce the number of tumor cells; reduce tumorigenicity, tumorigenic frequency or tumorigenic capacity; reduce the number or frequency of cancer stem cells; reduce tumor size; reduce the cancer cell population; inhibit or prevent cancer cell infiltration into surrounding organs, including, for example, cancer spread to soft tissue and bone; inhibit and prevent tumor or cancer cell metastasis; inhibit and prevent tumor or cancer cell growth; alleviate one or more of the symptoms associated with cancer to some extent; reduce morbidity and mortality; improve quality of life; or a combination of these effects.

[0166] The terms "treating" or "treatment" or "to treat" or "aleviating" or "alleviate" all refer to (1) therapeutic measures that cure, slow, reduce, and / or halt the progression of a diagnosed pathological condition or disorder, and (2) prophylactic or preventative measures that prevent or slow the development of a targeted pathological condition or disorder. Thus, those in need of treatment include those already suffering from the disorder; those susceptible to the disorder; and those in whom the disorder is to be prevented. In the case of cancer or tumors, a subject is successfully "treated" according to the methods encompassed by the invention if the patient exhibits one or more of the following: an increased immune response, an increased anti-tumor response, an increased cytolytic activity of immune cells, an increased killing of tumor cells by immune cells, a decrease in the number of cancer cells or the complete absence of cancer cells, a decrease in tumor size; inhibition of or absence of cancer cell infiltration into surrounding organs, including the spread of cancer cells into soft tissue and bone; inhibition of or absence of tumor or cancer cell metastasis; inhibition of or absence of cancer growth; alleviation of one or more symptoms associated with a particular cancer; reduced morbidity and mortality; improvement in quality of life; reduction in tumorigenicity; reduction in the number or frequency of cancer stem cells; or some combination of effects.

[0167] e. Miscellaneous

[0168] It should be understood that whenever an embodiment is described herein with the language "comprising," similar embodiments described in terms of "consisting of" and / or "consisting essentially of" are also provided. It should also be understood that whenever an embodiment is described herein with the language "consisting essentially of," similar embodiments described in terms of "consisting of" are also provided.

[0169] As used herein, reference to "about" or "approximately" a value or parameter includes (and describes) embodiments for that value or parameter. For example, description referring to "about X" includes description of "X".

[0170] The term "and / or" as used in phrases such as "A and / or B" herein is intended to include A and B; A or B; A (alone); and B (alone). Likewise, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0171] III. Anti-CD39 Antibodies

[0172] a. Monoclonal Antibodies

[0173] The anti-CD39 antibody may be a monoclonal antibody. Monoclonal antibodies may be prepared using a hybridoma method, such as those described by Kohler and Milstein, Nature, 256:495 (1975). In the hybridoma method, mice, hamsters or other appropriate host animals are typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, the lymphocytes may be immunized in vitro.

[0174] The immunizing agent will generally include a CD39 polypeptide or a fusion protein thereof. In general, if cells of human origin are required, peripheral blood lymphocytes ("PBL") are used, or if non-human mammalian sources are required, spleen cells or lymph node cells are used. The lymphocytes are then fused with immortalized cells using a suitable fusion agent (such as polyethylene glycol) to form hybridoma cells [Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986), pages 59 to 103]. Immortalized cell lines are generally transformed mammalian cells, especially myeloma cells of rodent, bovine and human origin. Typically, rat or mouse myeloma cell lines are used. Hybridoma cells can be cultured in a suitable culture medium, the culture medium preferably containing one or more substances that inhibit the growth or survival of unfused, immortalized cells. For example, if the parental cells lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine ("HAT medium"), which substances prevent the growth of HGPRT-deficient cells.

[0175] Preferred immortalized cell lines are those that fuse efficiently, support stable high levels of expression of the antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. More preferred immortalized cell lines are murine myeloma lines, which can be obtained, for example, from the Salk Institute Cell Distribution Center, San Diego, Calif. and the American Type Culture Collection, Manassas, Va. Human myeloma and mouse human heteromyeloma cell lines have also been described for the production of human monoclonal antibodies [Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, (1987), pp. 51-63].

[0176] The culture medium in which the hybridoma cells are cultured can then be analyzed for the presence of anti-polypeptide monoclonal antibodies. Preferably, the binding specificity of the monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). Such techniques and assays are known in the art. The binding affinity of the monoclonal antibodies can be determined, for example, by Scatchard analysis of Munson and Pollard, Anal. Biochem., 107: 220 (1980).

[0177] After identification of the desired hybridoma cells, clones can be subcloned by limiting dilution procedures and grown by standard methods [Goding, supra]. Suitable culture media for this purpose include, for example, Dulbecco's modified Eagle's medium and RPMI-1640 medium. Alternatively, hybridoma cells can be grown in vivo in mammals as ascites.

[0178] The monoclonal antibodies secreted by the subclones can be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0179] Monoclonal antibodies can also be manufactured by recombinant DNA methods, such as those described in U.S. Patent No. 4,816,567. The DNA encoding the monoclonal antibodies encompassed by the present invention can be easily separated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that can specifically bind to genes encoding murine antibody heavy and light chains). The hybridoma cells encompassed by the present invention serve as a preferred source of such DNA. Once separated, the DNA can be placed in an expression vector and then transfected into a host cell such as a monkey COS cell, a Chinese hamster ovary (CHO) cell, or a myeloma cell that does not otherwise produce immunoglobulins to obtain the synthesis of monoclonal antibodies in recombinant host cells. DNA can also be, for example, by replacing homologous murine sequences with coding sequences of human heavy and light chain constant domains [U.S. Patent No. 4,816,567; Morrison et al., supra], or by covalently linking to all or part of the immunoglobulin coding sequence of the coding sequence of a non-immunoglobulin polypeptide. Such non-immunoglobulin polypeptides can be substituted for the constant domains of an antibody encompassed by the invention, or can be substituted for the variable domains of one antigen combining site of an antibody encompassed by the invention to create a chimeric bivalent antibody.

[0180] b. Human and humanized antibodies

[0181] The anti-CD39 antibodies encompassed by the present invention may also include humanized antibodies or human antibodies. The humanized form of a non-human (such as a murine) antibody is a chimeric immunoglobulin, an immunoglobulin chain or fragment thereof (such as Fv, Fab, Fab', F(ab')2 or other antigen-binding subsequences of an antibody), which contains minimal sequences derived from non-human immunoglobulins. Humanized antibodies include human immunoglobulins (receptor antibodies), wherein residues from the complementary determining regions (CDRs) of the receptor are replaced by residues from non-human species (donor antibodies) such as mice, rats or rabbits with the desired specificity, affinity and ability CDRs. In some instances, the Fv framework residues of human immunoglobulins are replaced by corresponding non-human residues. Humanized antibodies may also include residues not found in the receptor antibody or in the imported CDR or framework sequences. In general, a humanized antibody will comprise at least one, and typically all or substantially all of two variable domains, wherein all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin, and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody will optimally also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin [Jones et al., Nature, 321: 522-525 (1986); Riechmann et al., Nature, 332: 323-329 (1988) and Presta, Curr. Op. Struct. Biol., 2: 593-596 (1992)].

[0182] Methods for humanizing non-human antibodies are well known in the art. In general, humanized antibodies have one or more amino acid residues of non-human origin introduced therein. These non-human amino acid residues are often referred to as "import" residues, which are usually taken from the "import" variable domain. Humanization can generally follow the method of Winter and colleagues [Jones et al., Nature, 321: 522-525 (1986); Riechmann et al., Nature, 332: 323-327 (1988); Verhoeyen et al., Science, 239: 1534-1536 (1988)], by replacing the corresponding sequence of a human antibody with a rodent CDR or CDR sequence. Therefore, these "humanized" antibodies are chimeric antibodies (U.S. Patent No. 4,816,567), in which substantially less than the entire human variable domain has been replaced by the corresponding sequence from a non-human species. In practice, humanized antibodies are human antibodies in which typically some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.

[0183] Human antibodies can also be produced using various techniques known in the art, including phage display libraries [Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991)]. The techniques of Cole et al. and Boerner et al. are also applicable to the preparation of human monoclonal antibodies [(Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985) and Boerner et al., J. Immunol., 147(1): 86-95 (1991)]. Similarly, human antibodies can be made by introducing human immunoglobulin loci into transgenic animals (e.g., mice in which endogenous immunoglobulin genes have been partially or completely inactivated). Upon stimulation, human antibody production is observed that is very similar to those seen in humans in all aspects, including gene rearrangement, assembly, and antibody repertoire. This method is described, for example, in U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, and the following scientific publications: Marks et al., Bio / Technology. 10, 779-783 (1992); Lonberg et al., Nature 368, 856-859 (1994); Morrison, Nature 368, 812-13 (1994); Fishwild et al., Nature Biotechnology 14, 845-51 (1996); Neuberger, Nature Biotechnology 14, 826 (1996); Lonberg and Huszar, Intern. Rev. Immunol. 1365-93 (1995).

[0184] The antibody affinity maturation can also be performed using known selection and / or mutagenesis methods as described above. Preferred affinity matured antibodies have an affinity that is five times greater, more preferably 10 times greater, and even more preferably 20 or 30 times greater than the starting antibody (usually murine, humanized or human) from which the mature antibody is prepared.

[0185] c. Bispecific Antibodies

[0186] The anti-CD39 antibodies described herein include bispecific molecules.Anti-CD39 antibodies or antigen-binding portions thereof may be derived or connected to another functional molecule, for example, another peptide or protein (e.g., a ligand of another antibody or receptor) to produce bispecific molecules that bind to at least two different binding sites or target molecules. In fact, the antibodies described herein may be derived or connected to more than one other functional molecule to produce multispecific molecules that bind to more than two different binding sites and / or target molecules; such multispecific molecules are also intended to be covered by the term "bispecific molecule" as used herein. To produce bispecific molecules described herein, the antibodies described herein may be functionally connected (e.g., by chemical coupling, gene fusion, non-covalent association or other means) to one or more other binding molecules such as another antibody, antibody fragment, peptide or binding mimetic to produce bispecific molecules.

[0187] Thus, provided herein are bispecific molecules comprising at least a first binding specificity for CD39 and a second binding specificity for a second target epitope. In one embodiment described herein wherein the bispecific molecule is multispecific, the molecule may further include a third binding specificity.

[0188] In certain embodiments, the subject bispecific (or multispecific as the case may be) includes one or more binding domains of immune checkpoints, for example, they are checkpoint inhibitors, such as PD-1, PD-L1, CTLA-4 / B7-1 / B7-2, PD-L2, KIR, LAG-3, TIM-3, CD96, VISTA, TIGIT and / or Siglec-15. In certain embodiments, the multispecific includes binding domains for checkpoint proteins on T cells (especially checkpoints such as LAG-3, TIM-3 or TIGIT associated with T cell exhaustion). In certain embodiments, the multispecific binds to CD39 and one or more other T cells associated with the checkpoint, and causes antibody-dependent cellular toxicity of cells expressing CD39 and each or both of the other checkpoint proteins to which it binds.

[0189] In certain embodiments, the subject bispecifics (or multispecifics, as the case may be) comprise one or more binding domains of immune co-stimulatory receptors, e.g., co-stimulatory agonists (activators), such as MHCl molecules, BTLA receptors and toluidine ligands, and agonists of OX40, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), and 4-1BB (CD137). Examples of co-stimulatory molecules that can be included in the multispecific include, but are not limited to, CDS, lCAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE / RANKL, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRTAM, Ly9(CD229), CD160(BY55), PS GL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, lPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a and CD83 ligands.

[0190] In certain embodiments, a subject bispecific (or multispecific as the case may be) comprises one or more binding domains that act as activators of innate immunity, such as a binding portion of CD47, SIRPα, CD24, Siglec-10, or NKG2A.

[0191] In one embodiment, the bispecific molecules described herein comprise at least one antibody or antibody fragment thereof (including, for example, Fab, Fab', F(ab')2, FV or single-chain Fv) as a binding specificity. The antibody may also be a light chain or heavy chain dimer, or any minimal fragment thereof, such as an Fv or single chain (scFv) construct.

[0192] Binding of the bispecific molecules to their specific targets can be confirmed using art-recognized methods such as enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), FACS analysis, bioassays (e.g., growth inhibition), or Western blot assays. Each of these assays generally detects the presence of a specific target protein-antibody complex by employing a labeled reagent (e.g., antibody) specific for the target complex.

[0193] Methods for preparing bispecific antibodies are known in the art. Traditionally, the recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy chain / light chain pairs, wherein the two heavy chains have different specificities [Milstein and Cuello, Nature, 305: 537-539 (1983)]. Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a possible mixture of ten different antibody molecules, of which only one has the correct bispecific structure. Purification of the correct molecule is usually carried out by an affinity chromatography step. Similar procedures are disclosed in WO 93 / 08829 published on May 13, 1993 and Traunecker et al., EMBO J., 10: 3655-3659 (1991).

[0194] The antibody variable domains (antibody-antigen combining sites) with the desired binding specificity can be fused to immunoglobulin constant domain sequences. The fusion is preferably fused to an immunoglobulin heavy chain constant domain, which comprises at least a portion of the hinge, CH2, and CH3 regions. It is preferred to have a first heavy chain constant region (CH1) containing a site necessary for light chain binding present in at least one of the fusions. DNA encoding the immunoglobulin heavy chain fusion and (if necessary) the immunoglobulin light chain are inserted into separate expression vectors and co-transfected into a suitable host organism. For additional detailed descriptions of the production of bispecific antibodies, see, e.g., Suresh et al., Methods in Enzymology, 121: 210 (1986).

[0195] According to another method described in WO 96 / 27011, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers recovered from recombinant cell culture.Preferred interfaces include at least a portion of the CH3 region of antibody constant domains.In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced by larger side chains (e.g., tyrosine or tryptophan).Compensation "cavities" of the same or similar size as larger side chains are produced on the interface of the second antibody molecule by replacing large amino acid side chains with smaller amino acid side chains (e.g., alanine or threonine).This is provided for increasing the yield of heterodimers to exceed the mechanism of other non-desired final products (such as homodimers).

[0196] Bispecific antibodies are prepared as full-length antibodies or antibody fragments (e.g., F(ab')2 bispecific antibodies). Techniques for producing bispecific antibodies from antibody fragments have been described in the literature. For example, bispecific antibodies can be prepared using chemical linkage. Brennan et al., Science 229: 81 (1985) describe a procedure in which intact antibodies are proteolytically cleaved to produce F(ab')2 fragments. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize ortho-dithiols and prevent intermolecular disulfide bond formation. The resulting Fab' fragments are then converted to thionitrobenzoate (TNB) derivatives. Then, one of the Fab'-TNB derivatives is converted back to Fab'-thiol by reduction with mercaptoethylamine and mixed with an equimolar amount of the other Fab'-TNB derivatives to form a bispecific antibody. The resulting bispecific antibodies can be used as agents for selective immobilization of enzymes.

[0197] Fab' fragments can be directly recovered from E. coli and chemically coupled to form bispecific antibodies. Shalaby et al., J. Exp. Med. 175: 217-225 (1992) describes the production of fully humanized bispecific antibody F(ab')2 molecules. Each Fab' fragment is secreted from E. coli and chemically coupled in vitro to form a bispecific antibody. The bispecific antibody thus formed can bind to cells overexpressing the ErbB2 receptor and normal human T cells, as well as trigger the lytic activity of human cytotoxic lymphocytes against human breast tumor targets.

[0198] Various techniques for preparing and isolating bispecific antibody fragments directly from recombinant cell culture have also been described. For example, bispecific antibodies have been produced using leucine zippers (Kostelny et al., J. Immunol. 148(5): 1547-1553 (1992)). Leucine zipper peptides from Fos and Jun proteins are linked to the Fab' portions of two different antibodies by gene fusion. Antibody homodimers are reduced at the hinge region to form monomers and then reoxidized to form antibody heterodimers. This method can also be used to produce antibody homodimers. The "bifunctional antibody" technology described by Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993) has provided an alternative mechanism for making bispecific antibody fragments. The fragments comprise a heavy chain variable domain (VH) connected to a light chain variable domain (VL) by a linker that is too short to allow pairing between the two domains on the same chain. Thus, the VH and VL domains of one fragment are forced to pair with the complementary VL and VH domains of another fragment, thereby forming two antigen binding sites. Another strategy for making bispecific antibody fragments by using single-chain Fv (sFv) dimers has also been reported. See, Gruber et al., J. Immunol. 152: 5368 (1994).

[0199] Antibodies with more than two valencies are contemplated. As a non-limiting example, trispecific antibodies can be prepared. See, e.g., Tutt et al., J. Immunol. 147:60 (1991).

[0200] d. Heteroconjugate Antibodies

[0201] Heteroconjugate antibodies are also within the scope of the present invention. Heteroconjugate antibodies comprise two covalently linked antibodies. For example, such antibodies have been proposed to target immune system cells to non-desirable cells [U.S. Patent No. 4,676,980], and for the treatment of HIV infection [WO 91 / 00360; WO 92 / 200373; EP 03089]. It is contemplated that the antibodies may be prepared in vitro using known methods in synthetic protein chemistry, including those involving cross-linking agents. For example, immunotoxins may be constructed using disulfide exchange reactions or by forming thioether bonds. Examples of reagents suitable for this purpose include iminothiol esters and methyl-4-mercaptobutyrylimidate, and those disclosed, for example, in U.S. Patent No. 4,676,980.

[0202] e. Effector function engineering

[0203] It may be desirable to modify the effector functions of the antibodies encompassed by the invention to enhance, for example, the effectiveness of anti-CD39 antibodies in treating cancer. For example, cysteine ​​residues may be introduced into the Fc region, thereby allowing interchain disulfide bonds to form in this region. The homodimeric antibodies thus produced may have improved internalization capability and / or enhanced complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC). See Caron et al., J. Exp Med., 176: 1191-1195 (1992) and Shopes, J. Immunol., 148: 2918-2922 (1992). In certain preferred embodiments, the engineered effector function is the ability of the anti-CD39 antibody to induce FcγRIII binding-dependent removal (such as by anti-CD39 antibody-mediated target endocytosis) of CD39 from immune cells, i.e., the ability to deplete immune cell populations without cell killing.

[0204] Homodimeric antibodies with enhanced anti-tumor activity can also be prepared using heterobifunctional cross-linkers as described in WoIff et al., Cancer Research, 53: 2560-2565 (1993). Alternatively, antibodies can be engineered to have dual Fc regions and thus have enhanced CD39 trogocytosis. See Stevenson et al., Anti-Cancer Drug Design, 3: 219-230 (1989).

[0205] f. Representative anti-CD39 antibody sequences

[0206] In certain embodiments, the anti-CD39 antibody is a fully human antibody, such as produced from a human antibody library. An exemplary fully human anti-CD39 antibody is clone Ig39-21, and the heavy and light variable domain (VH and VL) sequences are provided below:

[0207] Nucleic acid sequence Amino acid sequence VH domain SEQ ID NO.1 (VH) SEQ ID NO.2 (VH) VL domain SEQ ID NO.3 (VL) SEQ ID NO.4 (VL)

[0208] For the Ig39-21 clone, the CDRs for each of the VH and VL domains are:

[0209] CDR1 CDR2 CDR3 VH SEQ ID NO.29 SEQ ID NO.30 SEQ ID NO.31 V L SEQ ID NO.32 SEQ ID NO.33 SEQ ID NO.34

[0210] The sequences of exemplary full length antibodies and exemplary single chain antibodies (scFV) utilizing the above VH and VL domains are provided below:

[0211] Nucleic acid sequence Amino acid sequence Full length heavy chain SEQ ID NO.35 SEQ ID NO.36 Full length light chain SEQ ID NO.37 SEQ ID NO.38 scFV SEQ ID NO.39 SEQ ID NO.40

[0212] In some embodiments, the anti-CD39 antibody or antigen-binding fragment thereof comprises at least one heavy chain variable domain that is at least 60% identical to a VH domain sequence described herein (such as SEQ ID No. 2), and even more preferably at least 65%, 70%, 75%, 80%, 85% or even 90% identical to a VH domain sequence described herein (such as SEQ ID No. 2), and is capable of specifically binding to human CD39.

[0213] In some embodiments, the anti-CD39 antibody or antigen-binding fragment thereof comprises at least one light chain variable domain that is at least 60% identical to a VL domain sequence described herein (such as SEQ ID No. 4), and even more preferably at least 65%, 70%, 75%, 80%, 85% or even 90% identical to a VL domain sequence described herein (such as SEQ ID No. 4), and is capable of specifically binding to human CD39.

[0214] In certain embodiments, the anti-CD39 antibody is a humanized antibody comprising a VH domain having a human framework sequence associated with the CDRs of the VH domain shown in SEQ ID Nos. 29, 30 and 31 and the CDRs of the corresponding VL domain shown in SEQ ID Nos. 32, 33 and 34. The CDRs of the anti-CD39 antibodies described herein are preferably the same as the CDRs described herein, but may vary by 1, 2 or 3 amino acids in each CDR, as long as the resulting antibody specifically binds to human CD39.

[0215] In certain embodiments, the heavy and light chains of the anti-CD39 antibodies have variable domains that may be encoded by nucleic acids that are identical to the VH and VL domain (respectively) encoding sequences described herein, such as those shown in SEQ ID No. 1 (VH) and SEQ ID No. 3 (VL), or that hybridize under stringent conditions (such as 6x sodium chloride / sodium citrate (SSC) at 45°C, and washing in 0.2xSSC / 0.1% SDS at 50°C to 65°C).

[0216] In some embodiments, anti-CD39 antibodies are produced in rabbits, and the variable domains of the heavy and light chains of these antibodies are rabbit sequences, while the constant domains are human sequences. Exemplary sequences of the VH and VL domains of rabbit anti-CD39 antibodies are:

[0217]

[0218]

[0219] In some embodiments, the anti-CD39 antibody is generated in rabbits and then humanized by CDR grafting. Exemplary sequences of the VH and VL domains of humanized rabbit anti-CD39 antibodies are:

[0220]

[0221] In some embodiments, the anti-CD39 antibodies provided herein promote: (i) stable immune complex formation when incubated with HCC1739BL cells, as characterized by less than 30% loss of the immune complex after 24 hours, optionally wherein the immune complex formation is detected by fluorescence intensity using a fluorescently labeled secondary antibody; (ii) complement dependent cytotoxicity (CDC) activity against CD39+ cells; (iii) antibody-mediated target endocytosis of CD39 on CD45+ immune cells; (iv) antibody-mediated target endocytosis of CD39 from tumor vascular endothelial destruction or vascular distribution network collapse in tumors; (v) (optionally) binding to a protein selected from Fig.33 or (vi) a CD39 epitope that binds to a sequence of the group of CD39 amino acid epitope sequences listed in (e.g., binds to one or more linear or conformational CD39 epitopes, such as selected from the group consisting of: 1) IYLTDCMERAR, 2) LRMESEELADR, 3) RVKGPGISKFV, 4) DCMERAREVIPR, 5) LTDCMERAREVIPR, 6) SLSNYPFDFQGAR, 7) CRVKGPGISKF, 8) GAYGWITINYLLGKFSQK, 9) ILRDPCFHPGYKK, and any combination thereof, such as RVKGPGISKFV and DCMERAREVIPR, LTDCMERAREVIPR and SLSNYPFDFQGAR or CRVKGPGISKF, GAYGWITINYLLGKFSQK, and / or ILRDPCFHPGYKK); and / or (vi) (optionally) binds to CD39 in a manner that is non-competitive or only partially competitive with monoclonal antibody clone A1 for binding to CD39.

[0222] The representative anti-CD39 antibody sequences described above, numbered according to sequence identification, correspond to the following:

[0223] SEQ ID No.1 (clone IG39-21 vH nucleic acid sequence)

[0224]

[0225] SEQ ID No.2 (clone IG39-21vH amino acid sequence)

[0226]

[0227] SEQ ID No.3 (clone IG39-21vL domain nucleic acid sequence)

[0228]

[0229] SEQ ID No.4 (clone IG39-21 vL domain amino acid sequence)

[0230]

[0231] SEQ ID No.5 (clone 9B6 vH domain nucleic acid sequence)

[0232]

[0233] SEQ ID No.6 (clone 9B6 vH domain amino acid sequence)

[0234]

[0235] SEQ ID No.7 (clone 9B6 vL domain nucleic acid sequence)

[0236]

[0237] SEQ ID No.8 (clone 9B6 vL domain amino acid sequence)

[0238]

[0239] SEQ ID No.9 (clone 8C11 vH domain nucleic acid sequence)

[0240]

[0241] SEQ ID No.10 (clone 8C11 vH domain amino acid sequence)

[0242]

[0243] SEQ ID No.11 (clone 8C11 vL domain nucleic acid sequence)

[0244]

[0245] SEQ ID No.12 (clone 8C11 vL domain amino acid sequence)

[0246]

[0247] SEQ ID No.13 (clone 8D8 vH domain nucleic acid sequence)

[0248]

[0249] SEQ ID No.14 (clone 8D8 vH domain amino acid sequence)

[0250]

[0251] SEQ ID No.15 (clone 8D8 vL domain nucleic acid sequence)

[0252]

[0253] SEQ ID No.16 (clone 8D8 vL domain amino acid sequence)

[0254]

[0255] SEQ ID No.17 (clone 9C10 vH domain nucleic acid sequence)

[0256]

[0257] SEQ ID No.18 (clone 9C10 vH domain amino acid sequence)

[0258]

[0259] SEQ ID No.19 (clone 9C10 vL domain nucleic acid sequence)

[0260]

[0261] SEQ ID No.20 (clone 9C10 vL domain amino acid sequence)

[0262]

[0263] SEQ ID No.21 (clone 65H5 vH domain nucleic acid sequence)

[0264]

[0265] SEQ ID No.22 (clone 65H5 vH domain amino acid sequence)

[0266]

[0267] SEQ ID No.23 (clone 65H5 vL domain nucleic acid sequence)

[0268]

[0269] SEQ ID No.24 (clone 65H5 vL domain amino acid sequence)

[0270]

[0271] SEQ ID No.25 (clone 2G12 vH domain nucleic acid sequence)

[0272]

[0273] SEQ ID No.26 (clone 2G12 vH domain amino acid sequence)

[0274]

[0275] SEQ ID No.27 (clone 2G12 vL domain nucleic acid sequence)

[0276]

[0277] SEQ ID No.28 (clone 2G12 vL domain amino acid sequence)

[0278]

[0279] SEQ ID No.29 (clone IG39-21vH domain CDR1 amino acid sequence)

[0280]

[0281] SEQ ID No.30 (clone IG39-21vH domain CDR2 amino acid sequence)

[0282]

[0283] SEQ ID No.31 (clone IG39-21 vH domain CDR3 amino acid sequence)

[0284]

[0285] SEQ ID No.32 (clone IG39-21 vL domain CDR1 amino acid sequence)

[0286]

[0287] SEQ ID No.33 (clone IG39-21vL domain CDR2 amino acid sequence)

[0288]

[0289] SEQ ID No.34 (clone IG39-21vL domain CDR3 amino acid sequence)

[0290]

[0291] SEQ ID No.35 (clone IG39-21 full-length vH chain nucleic acid sequence)

[0292]

[0293]

[0294] SEQ ID No.36 (Clone IG39-21 full-length vH chain amino acid sequence)

[0295]

[0296]

[0297]

[0298] SEQ ID No.37 (clone IG39-21 full-length vL chain nucleic acid sequence)

[0299]

[0300] SEQ ID No.38 (Clone IG39-21 full-length vL chain amino acid sequence)

[0301]

[0302] SEQ ID No.39 (clone IG39-21 scFv nucleic acid sequence)

[0303]

[0304] SEQ ID No.40 (clone IG39-21 scFv amino acid sequence)

[0305]

[0306] SEQ ID No.41

[0307]

[0308] SEQ ID No.42

[0309]

[0310] SEQ ID No.43

[0311]

[0312] SEQ ID No.44

[0313]

[0314] SEQ ID No.45

[0315]

[0316] SEQ ID No.46

[0317]

[0318] SEQ ID No.47

[0319]

[0320] SEQ ID No.48

[0321]

[0322] SEQ ID No.49

[0323]

[0324] SEQ ID No.50

[0325]

[0326] SEQ ID No.51

[0327]

[0328] SEQ ID No.52

[0329]

[0330] SEQ ID No.53

[0331]

[0332] SEQ ID No.54

[0333]

[0334] SEQ ID No.55

[0335]

[0336] SEQ ID No.56

[0337]

[0338] For use in human patients, it will be desirable to humanize these antibodies, replacing the constant regions of the heavy and light chains with human constant regions, and replacing the framework regions of the variable regions with human antibody framework regions. In some embodiments, the anti-CD39 antibody or antigen-binding fragment thereof is a humanized version of a rabbit antibody.

[0339] In some embodiments, the anti-CD39 antibody or antigen-binding fragment thereof comprises at least one heavy chain variable that is at least 60% identical to SEQ ID No. 6, 10, 14, 18, 22, 26, 42, 46, 50 or 54, and even more preferably at least 65%, 70%, 75%, 80%, 85% or even 90% identical to SEQ ID No. 6, 10, 14, 18, 22, 26, 42, 46, 50 and 54, and is capable of specifically binding to human CD39.

[0340] In some embodiments, the anti-CD39 antibody or antigen-binding fragment thereof comprises at least one light chain variable that is at least 60% identical to SEQ ID No. 8, 12, 16, 20, 24, 28, 44, 48, 52 or 56, and even more preferably at least 65%, 70%, 75%, 80%, 85% or even 90% identical to SEQ ID No. 8, 12, 16, 20, 24, 28, 44, 48, 52 or 56, and is capable of specifically binding to human CD39.

[0341] In certain embodiments, chimeric antibodies are humanized antibodies. Typically, non-human antibodies are humanized to reduce immunogenicity to people while retaining the specificity and affinity of the parent non-human antibodies. Generally speaking, humanized antibodies include one or more variable domains, wherein HVR, such as CDR (or a portion thereof) are derived from non-human antibodies, and FR (or a portion thereof) are derived from human antibody sequences. Humanized antibodies will optionally also 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 from which HVR residues are derived), for example, to restore or improve antibody specificity or affinity.

[0342] In certain embodiments, the anti-CD39 antibody is a humanized antibody comprising a VH domain having a human framework sequence associated with a CDR of a VH domain selected from SEQ ID No. 6, 10, 14, 18, 22, 26, 42, 46, 50, or 54, and a CDR of a corresponding VL domain selected from SEQ ID No. 8, 12, 16, 20, 24, 28, 44, 48, 52, or 56. The CDRs are preferably identical, but may vary by 1, 2, or 3 amino acids in each CDR, as long as the resulting antibody specifically binds to human CD39.

[0343] Humanized antibodies and methods of making them are reviewed in, e.g., Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and further described in, e.g., Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989); U.S. Pat. Nos. 5,821.337, 7.527.791, 6,982,321, and 7,087,409; Kashmiri et al., Methods 36:25-34 (2005) (describing specificity determining region (SDR) grafting); Padlan, Mol. Immunol. 28:489-498 (1991) (describing "surface resurfacing"); Dall'Acqua et al., Methods 36:43-60 (2005) (describing "FR shuffling"); and Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer, 83:252-260 (2000) (describing the "directed selection" method of FR shuffling).

[0344] Human framework regions that can be used for humanization include, but are not limited to, framework regions selected using the "best fit" method (see, e.g., Sims et al., J. Immunol., 151:2296 (1993)); framework regions derived from the consensus sequence of human antibodies of a particular subgroup of light or heavy chain variable regions (see, e.g., Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta et al., J. Immunol., 151:2623 (1993)). 993)); human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and framework regions derived from screening FR libraries (see, e.g., Baca et al., J. Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J Biol. Chem. 271:22611-22618 (1996)).

[0345] In certain embodiments, the anti-CD39 antibodies provided herein are human antibodies. Human antibodies can be produced using various techniques known in the art. Human antibodies are generally described in van Dijk and Van de Winkel, Curr. Opin. Pharmacol. 5: 368-74 (2001) and Lonberg, Curr. Opin. Immunol. 20: 450-459 (2008).

[0346] For example, human antibodies can be prepared by administering an immunogen to a transgenic animal that has been modified to produce complete human antibodies or complete antibodies with human variable regions in response to antigenic stimulation. Such animals typically contain all or part of the human immunoglobulin loci, which replace endogenous immunoglobulin loci, or they are present outside the chromosomes or randomly integrated into the chromosomes of the animal. In such transgenic mice, the endogenous immunoglobulin loci are generally inactivated. For a review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23: 1117-1125 (2005). See also, for example, U.S. Patent Nos. 6,075,181 and 6,150,584 describing XENOMOUSE technology; U.S. Patent No. 5,770,429 describing HuMAB technology; U.S. Patent No. 7,041,870 describing K-MMOUSE technology, and U.S. Patent Application Publication No. US 2007 / 0061900 describing VELOCIMOUSE technology). Human variable regions from intact antibodies produced by such animals can be further modified, for example, by combining with different human constant regions.

[0347] Human antibodies can also be prepared by hybridoma-based methods. Human myeloma and mouse human heteromyeloma cell lines for producing human monoclonal antibodies have been described. (See, e.g., Kozbor J. Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51 to 63 (Marcel Dekker, inc., New York, 1987); and Boerner et al., J. Immunol., 147: 86 (1991)). Human antibodies produced via human B cell hybridoma technology are also described in Li et al., Proc. Natl. Acad. Sci USA, 103: 3557-3562 (2006). Additional methods include those described in, for example, U.S. Pat. No. 7,189,826 (describing the production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4):265-268 (2006) (describing human-human hybridomas). Human hybridoma technology (trioma technology) is also described in Vollmers and Brandlein, Histology and Histopathology, 20(3):927-937 (2005) and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27(3):185-91 (2005).

[0348] Human antibodies can also be produced by isolating the FV clone variable domain sequence selected from the phage, yeast or bacterial display library of human origin. Such variable domain sequences can then be combined with desired human constant domains. The following describes the technology for selecting human antibodies from antibody libraries.

[0349] To illustrate, anti-CD39 antibodies encompassed by the present invention can be isolated by screening combinatorial libraries for antibodies with the desired activity. For example, various methods are known in the art for generating phage or yeast display libraries and screening these libraries for antibodies with the desired binding characteristics. These methods are reviewed, for example, in Hoogenboom et al., Methods in Molecular Biology 178: 1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, 2001) and further described, for example, in McCafferty et al., Nature 348: 552-554; Clackson et al., Nature 352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597 (1992); Marks and Bradbury, Methods in Molecular Biology 248: 161-175 (Lo, ed., Human Press, Totowa, NJ, 2001). Press, Totowa, NJ, 2003); Sidhu et al., J. Mol. Biol. 338(2):299-310 (2004); Lee et al., J. Mol. Biol. 340(5):1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34):12467-12472 (2004); and Lee et al., J. Immunol. Methods 284(1-2):119-132 (2004).

[0350] As an example of a phage display method, the VH and VL gene repertoires are cloned separately by polymerase chain reaction (PCR) and randomly recombined in a phage library, which can then be screened for antigen binding phage as described in Winter et al., Ann. Rev. Immunol., 12: 433-455 (1994). Phage typically display antibody fragments, either in the form of single-chain Fv (scFv) fragments or in the form of Fab fragments. Libraries from immune sources provide high-affinity antibodies to the immunogen without the need to construct hybridomas. Alternatively, as described by Griffiths et al., EMBO J, 12: 725-734 (1993), the original library can be cloned (e.g., from humans) to provide a single source of antibodies to a wide range of non-self antigens and also self-antigens without any immunization. Finally, as described by Hoogenboom and Winter, J.Mol.Biol., 227:381-388 (1992), the original library can also be prepared by cloning unrearranged V gene segments from stem cells and using PCR primers containing random sequences to encode highly variable CDR3 regions and performing in vitro rearrangement synthesis. Patents describing human antibody phage libraries are published, for example: U.S. Patent No. 5,750,373 and U.S. Patent Publication Nos. 2005 / 0079574, 2005 / 0119455, 2005 / 0266000, 2007 / 0117126, 2007 / 0160598, 2007 / 0237764, 2007 / 0292936 and 2009 / 0002360.

[0351] Antibodies or antibody fragments isolated from human antibody libraries are considered human antibodies or human antibody fragments herein.

[0352] FcγRIII binding can also be increased by methods according to the prior art, for example, by modifying the amino acid sequence of the Fc part of the antibody or the glycosylation of the Fc part of the antibody (see, for example, EP2235061). In certain embodiments, a subject antibody is produced by a cell in which, when glycosylated, less than 50% of the oligosaccharide chains on the antibody contain α(-1,6-fucose. Typically, in a "hypofucosylated" antibody preparation, less than about 40%, less than about 30%, less than about 20%, less than about 10%, or less than 5%, or less than 1% of the oligosaccharide chains contain α(-1,6-fucose. "Afucosylated" antibodies lack α-1,6-fucose in the carbohydrate attached to the CH2 domain of the IgG heavy chain. Mori, K et al., Cytotechnology 55 (2007) 109 and Satoh M et al., Expert Opin Biol Ther. 6 (2006) 1161-1173 relate to the use of a FUT8 (α-1,6-fucosyltransferase) knockout CHO line for the production of afucosylated antibodies.

[0353] IV. Expression Vectors

[0354] In certain embodiments, a recombinant expression vector is used to amplify and express the DNA encoding the anti-CD39 antibodies described herein. For example, a recombinant expression vector may be a replicable DNA construct having a synthetic or cDNA-derived DNA fragment encoding a polypeptide chain of an anti-CD39 antibody operably linked to a suitable transcription and / or translation regulatory element derived from a mammalian, microbial, viral or insect gene. A transcription unit generally comprises the following assembly: (1) one or more genetic elements that have a regulatory effect in gene expression, such as a transcription promoter or enhancer, (2) a structural or coding sequence that is transcribed into mRNA and translated into protein, and (3) appropriate transcription and translation start and stop sequences. A regulatory element may include an operator sequence to control transcription. The ability to replicate in a host is generally conferred by the origin of replication, and a selection gene that promotes the recognition of transformants may be additionally incorporated. When DNA regions are functionally related to each other, they are "operably linked". For example, if the DNA of the signal peptide is expressed as a precursor that participates in the secretion of the polypeptide, it (the secretory leader sequence) is operably connected to the DNA of the polypeptide; if the promoter controls the transcription of the sequence, it is operably connected to the coding sequence; or if the ribosome binding site is placed so as to allow translation, it is operably connected to the coding sequence. In some embodiments, the structural elements intended for use in yeast expression systems include leader sequences that allow proteins that can be translated by extracellular secretion of host cells. In other embodiments, when recombinant proteins are expressed in the absence of a leader sequence or a transport sequence, it may include an N-terminal methionine residue. This residue may then optionally be cleaved from the expressed recombinant protein to provide a final product.

[0355] The selection of expression control sequences and expression vectors depends on the selection of host cells. Various expression host / vector combinations can be adopted. Expression vectors suitable for eukaryotic hosts include, for example, vectors comprising expression control sequences from SV40, bovine papilloma virus, adenovirus and cytomegalovirus. Expression vectors suitable for bacterial hosts include known bacterial plasmids, such as plasmids from Escherichia coli, including pCR1, pBR322, pMB9 and their derivatives, and more extensive host range plasmids, such as M13 and other filamentous single-stranded DNA phages.

[0356] Host cells suitable for expressing the polypeptide chains of anti-CD39 antibodies (or proteins used as targets) include prokaryotes, yeast cells, insect cells or higher eukaryotic cells under the control of appropriate promoters. Prokaryotes include gram-negative or gram-positive organisms, such as Escherichia coli or Bacillus. Higher eukaryotic cells include established mammalian cell lines as described below. Cell-free translation systems can also be used. Cloning and expression vectors suitable for use with bacteria, fungi, yeast and mammalian cell hosts are well known to those skilled in the art.

[0357] Various mammalian cell culture systems are used to express recombinant polypeptides. The expression of recombinant proteins in mammalian cells may be preferred because such proteins are generally correctly folded, appropriately modified, and have biological functions. Examples of suitable mammalian host cell lines include COS-7 (monkey kidney derived), L-929 (mouse fibroblast derived), C127 (mouse breast tumor derived), 3T3 (mouse fibroblast derived), CHO (Chinese hamster ovary derived), HeLa (human cervical cancer derived), BHK (hamster kidney fibroblast derived) and HEK-293 (human embryonic kidney derived) cell lines and variants thereof. Mammalian expression vectors may include non-transcriptional elements, such as replication origins, promoters and enhancers suitable for connection to the gene to be expressed, and other 5' or 3' flanking non-transcribed sequences and 5' or 3' non-translated sequences, such as necessary ribosome binding sites, polyadenylation sites, splicing donor and acceptor sites, and transcription termination sequences.

[0358] The expression of recombinant proteins in insect cell culture systems (eg, baculovirus) also provides a reliable method for producing correctly folded and biologically functional proteins. The baculovirus system for producing heterologous proteins in insect cells is well known to those skilled in the art.

[0359] In certain embodiments, the polynucleotide comprises a polynucleotide encoding an antibody light chain comprising a variable region that is at least 60% identical to SEQ ID No. 1, and even more preferably at least 65%, 70%, 75%, 80%, 85% or even 90% identical to SEQ ID No. 1, and is capable of specifically binding to human CD39.

[0360] In certain embodiments, the polynucleotide comprises a polynucleotide encoding an antibody heavy chain comprising a variable region that is at least 60% identical to SEQ ID No. 2, and even more preferably at least 65%, 70%, 75%, 80%, 85% or even 90% identical to SEQ ID No. 2, and is capable of specifically binding to human CD39.

[0361] V. Encoded anti-CD39 antibodies for in vivo delivery

[0362] Therapeutic vectors used to deliver the coding sequence of the anti-CD39 antibody to be expressed in a patient can be viral, non-viral or physical. See, e.g., Rosenberg et al., Science, 242: 1575-1578, 1988, and Wolff et al., Proc. Natl. Acad. Sci. USA 86: 9011-9014 (1989). Discussions of methods and compositions for use in gene therapy include Eck et al., Goodman & Gilman's The Pharmacological Basis of Therapeutics, 9th edition, Hardman et al., ed., McGraw-Hill, New York, (1996), Chapter 5, pp. 77 to 101; Wilson, Clin. Exp. Immunol. 107 (Suppl. 1): 31-32, 1997; Wivel et al., Hematology / Oncology Clinics of North America, Gene Therapy, SLEck, ed., 12 (3): 483-501, 1998; Romano et al., Stem Cells, 18: 19-39, 2000, and references cited therein. U.S. Pat. No. 6,080,728 also provides a discussion of various gene delivery methods and compositions. Delivery routes include, for example, systemic administration and in situ administration. Well-known viral delivery techniques include the use of adenoviral, retroviral, lentiviral, foamy viral, herpes simplex viral, vaccinia viral and adeno-associated viral vectors.

[0363] a. Viral vector

[0364] Preferred viral vectors are based on non-cytopathic eukaryotic viruses in which non-essential genes have been replaced by nucleic acid constructs carrying nucleic acid sequences encoding epitopes and targeting target sequences. Preferred viruses for certain embodiments encompassed by the present invention are adenoviruses and adeno-associated viruses (AAV), which are double-stranded DNA viruses that have been approved for human use in gene therapy. In addition, preferred vectors for tolerance do not contain immunostimulatory sequences.

[0365] Adenoviral vector

[0366] An illustrative method for delivering one or more nucleic acid sequences in vivo involves the use of an adenoviral expression vector. "Adenoviral expression vector" means those constructs including adenoviral sequences sufficient to (a) support packaging constructs and (b) express polynucleotides cloned therein in a sense or antisense direction. Of course, in the context of antisense constructs, expression does not require that the gene product be synthetic. In a specific embodiment, the delivery vector relates to a commercially available ORF of cytochrome b5 reductase 3 (CYB5R3), a transcript variant 1 in the adenoviral vector pAd, with a C-terminal Flag and His tag, (Vigene Biosciences product code AH889428). WIPO patent application WO / 2015 / 050364 also teaches a vector having an expression construct comprising a Cyb5r3 gene.

[0367] Adenoviral vectors are highly immunogenic and are therefore not preferred for inducing tolerance by presenting antigens or for administration in the setting of autoimmune disease. However, these vectors can be used to induce immunity, such as for the treatment of infectious diseases, including, for example, influenza, HBV, HCV, and HIV.

[0368] Adeno-associated virus (AAV)

[0369] AAV is a good choice for delivery vehicles due to its safety, i.e., it is not integrated into the host genome through genetic engineering (recombination). Likewise, AAV is not pathogenic and is not associated with any disease. The removal of viral coding sequences minimizes the immune response to viral gene expression, and therefore, rAAV does not cause an inflammatory response. According to a specific embodiment, an AAV vector comprising an epitope sequence containing a nucleic acid construct described herein is suitable for transducing APCs.

[0370] Typically, a viral vector comprising an epitope containing a nucleic acid construct is assembled from a polynucleotide encoding the desired epitope, a suitable regulatory element, and an element necessary for epitope expression for mediating cell transduction. In one embodiment, an adeno-associated virus (AAV) vector is used. In a more specific embodiment, the AAV vector is AAV1, AAV6, or AAV8.

[0371] AAV expression vectors having a DNA molecule of interest bound by an AAV ITR can be constructed by inserting the selected sequence directly into the AAV genome from which the major AAV open reading frame ("ORF") has been excised. Examples of constitutive promoters that may be included in the AAV of the present invention include, but are not limited to, the exemplary CMV immediate early enhancer / chicken β-actin (CBA) promoter.

[0372] For eukaryotic cells, expression control sequences typically include promoters, enhancers, such as those derived from immunoglobulin genes, SV40, cytomegalovirus, and polyadenylation sequences that may include splice donor and acceptor sites. The polyadenylation sequence is generally inserted after the transgenic sequence and before the 3'ITR sequence. In one embodiment, bovine growth hormone polyA can be used.

[0373] The selection of these and other common vectors and regulatory elements is routine, and many of these sequences are available. See, e.g., Sambrook et al. and references cited therein, e.g., at pages 3.18-3.26 and 16.17-16.27 and Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1989). Of course, not all vectors and expression control sequences will work equally well to express all transgenes of the present invention. However, those skilled in the art can make a selection among these expression control sequences without departing from the scope of the present invention. Suitable promoter / enhancer sequences can be selected by those skilled in the art using the guidance provided by the present application. This selection is a routine matter and is not a limitation to the molecule or construct.

[0374] Retroviral vectors

[0375] In certain embodiments, the viral vector may be a retroviral vector. A "retrovirus" is a virus with an RNA genome. In specific embodiments, the retroviral vector contains all cis-acting sequences necessary for packaging and integration of the viral genome, i.e., (a) long terminal repeats (LTRs) or portions thereof at each end of the vector; (b) primer binding sites for negative and positive strand DNA synthesis; and (c) packaging signals necessary for incorporating genomic RNA into the virion. For more details on retroviral vectors, see Boesen et al., 1994, Biotherapy 6:291-302; Clowes et al., 1994, J. Clin. Invest. 93:644-651; Kiem et al., 1994, Blood 83:1467-1473; Salmons and Gunzberg, 1993, Human Gene Therapy 4:129-141; Miller et al., 1993, Meth. Enzymol. 217:581-599; and Grossman and V Vilson, 1993, Curr. Opin. in Genetics and Devel. 3:110-114.

[0376] "Gammaretrovirus" refers to a genus of the Retroviridae family. Exemplary gammaretroviruses include mouse stem cell virus, murine leukemia virus, feline leukemia virus, feline sarcoma virus, and avian reticuloendotheliosis virus.

[0377] Widely used retroviral vectors include those based on murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), simian immunodeficiency virus (SIV), human immunodeficiency virus (HIV), and combinations thereof (see, e.g., Buchscher et al., J. Virol. 66:2731-2739, 1992; Johann et al., J. Virol. 66:1635-1640, 1992; Sommerfelt et al., Virol. 176:58-59, 1990; Wilson et al., J. Virol. 63:2374-2378, 1989; Miller et al., J. Virol. 65:2220-2224, 1991; and PCT / US94 / 05700).

[0378] Lentiviral vectors refer to a genus of retroviruses that can infect both dividing and non-dividing cells and usually produce high viral titers. Several examples of lentiviruses include HIV (human immunodeficiency virus: including HIV type 1 and HIV type 2); equine infectious anemia virus; feline immunodeficiency virus (FIV); bovine immunodeficiency virus (BIV); and simian immunodeficiency virus (SIV).

[0379] In certain embodiments, other retroviral vectors can be used. These include, for example, vectors based on human foamy virus (HFV) or other viruses in the genus Foamyvirus. Foamy virus (FVes) is the largest retrovirus known today, and is widely distributed in different mammals, including all non-human primate species, but not in humans. This complete lack of pathogenicity makes FV vectors an ideal gene transfer vehicle for human gene therapy, and clearly distinguishes FV vectors as gene delivery systems from HIV sources and also from gamma retrovirus sources.

[0380] Non-cytopathic viruses include retroviruses (e.g., lentiviruses), whose life cycle involves reverse transcription of genomic viral RNA into DNA, and subsequent integration of proviruses into host cell DNA. Retroviruses have been approved for human gene therapy trials. The most useful are those retroviruses that are replication-deficient (i.e., capable of directed synthesis of desired proteins, but unable to produce infectious particles). Such genetically altered retroviral expression vectors have general utility for efficient transduction of genes in vivo. Standard protocols for producing replication-deficient retroviruses (including incorporating exogenous genetic material into plasmids, transfecting packaging cells lined with plasmids, producing recombinant retroviruses by packaging cell lines, collecting viral particles from tissue culture media, and infecting target cells with viral particles) are known to those skilled in the art.

[0381] The retroviral genome contains three genes gag, pol and env that encode capsid protein, polymerase and envelope components respectively. The sequence found from the upstream of the gag gene contains a signal for packaging the genome into the virus body. Retroviral vectors are gene transfer plasmids, in which heterologous nucleic acid residues are located between two retroviral LTRs. Retroviral vectors usually contain appropriate packaging signals, which allow retroviral vectors or RNA transcribed using retroviral vectors as templates to be packaged into the viral virions in appropriate packaging cell lines (see, e.g., U.S. Patent number 4,650,764). These two long terminal repeat (LTR) sequences are present at the 5' and 3' ends of the viral genome. These contain strong promoter and enhancer sequences, and also need to be integrated into the host cell genome (Coffin, 1990). To construct a retroviral vector, a nucleic acid encoding one or more target oligonucleotides or polynucleotide sequences is inserted into the viral genome to replace certain viral sequences to produce a replication-defective virus. Also included are free or non-integrated forms of retroviral vectors based on slow viruses (e.g., a type of retrovirus).

[0382] Lentiviral vectors are useful when stable expression is required, but lentiviral vectors can be immunogenic and may have other undesirable effects. Therefore, although lentiviral vectors are convenient for research, they should be used with caution when used for human administration, especially when tolerance rather than immunity needs to be induced. Although mRNA electroporation is safer, lentivirus is suitable for engineering T cells or dendritic cells or other antigen-presenting cells in vitro for cancer therapy. However, two recent advances have made the use of lentivirus safer and more clinically translatable. First, the co-expression of suicide genes together with antigens, the products of which become active when the drug is administered. A typical example is herpes simplex virus thymidine kinase (HSV-Tk). Cells expressing these genes can metabolize the drug ganciclovir into cytotoxic products that induce cell death. Therefore, in the case where some transduced cells become malignant, they can be eradicated. There are about a dozen such systems (Duarte et al., Cancer Letters, 324: 160-170, 2012). Second, non-integrating lentiviral vectors are being developed and are therefore non-oncogenic (Nightingale et al., 2006, Mol. Ther., 13: 1121-1132). These methods can be used with the present invention according to the judgment of one skilled in the art.

[0383] Retroviral vectors suitable for use herein are described in, for example, U.S. Pat. Nos. 5,399,346 and 5,252,479; and WIPO publications WO 92 / 07573, WO 90 / 06997, WO 89 / 05345, WO 92 / 05266, and WO 92 / 14829, which provide descriptions of methods for effectively introducing nucleic acids into human cells using these retroviral vectors. Other retroviral vectors include, for example, mouse mammary tumor virus vectors (e.g., Shackleford et al., Proc. Natl. Acad. Sci. USA 85: 9655-9659, 1998), lentiviruses, and the like. An exemplary viral vector is plentilox-IRES-GFP.

[0384] Additional retroviral delivery systems that can be readily adapted to deliver a transgene encoding an anti-CD39 antibody agent include, by way of illustration only, published PCT applications WO / 2010 / 045002, WO / 2010 / 148203, WO / 2011 / 126864, WO / 2012 / 058673, WO / 2014 / 066700, WO / 2015 / 021077, WO / 2015 / 148683, WO / 2017 / 040815, the specifications and figures of each of which are incorporated herein by reference.

[0385] In certain embodiments, the retrovirus is a retrovirus with recombinant replication capacity comprising: a nucleic acid sequence encoding a retroviral GAG protein; a nucleic acid sequence encoding a retroviral POL protein; a nucleic acid sequence encoding a retroviral envelope; and an oncoretroviral polynucleotide sequence comprising long terminal repeat (LTR) sequences at the 5′ and 3′ ends of the oncoretroviral polynucleotide sequence; a box comprising an internal ribosome entry site (IRES) operably linked to the coding sequence of the anti-CD39 antibody agent, wherein the box is located 5′ of the U3 region of the 3′ LTR and 3′ of the sequence encoding the retroviral envelope; and a cis-acting sequence for reverse transcription, packaging and integration in target cells.

[0386] In certain embodiments, the retrovirus is a retrovirus with recombinant replication capacity comprising: a retroviral GAG protein; a retroviral POL protein; a retroviral envelope; a retroviral polynucleotide comprising a long terminal repeat (LTR) sequence at the 3′ end of the retroviral polynucleotide sequence, a promoter sequence at the 5′ end of the retroviral polynucleotide (the promoter is suitable for expression in mammalian cells), a gag nucleic acid domain, a pol nucleic acid domain, and an env nucleic acid domain; a box comprising an anti-CD39 antibody agent encoding sequence operably linked to a heterologous polynucleotide, wherein the box is located 5′ to 3′ LTR and is operably linked, and 3′ to the env nucleic acid domain encoding the retroviral envelope; and cis-acting sequences necessary for reverse transcription, packaging and integration in target cells.

[0387] In certain preferred embodiments of the recombinant replication-competent retrovirus, the envelope is selected from one of the following: amphoteric, polyoepic, heterologous, 10A1, GALV, baboon endogenous virus, RD114, rhabdovirus, alphavirus, measles, or influenza virus envelopes.

[0388] In certain preferred embodiments of the retrovirus having recombinant replication capability, the retroviral polynucleotide sequence is engineered from a virus selected from the group consisting of: murine leukemia virus (MLV), Moloney murine leukemia virus (MoMLV), feline leukemia virus (FeLV), baboon endogenous retrovirus (BEV), porcine endogenous virus (PERV), feline retrovirus RD114, squirrel monkey retrovirus, xenogeneic murine leukemia virus-related virus (XMRV), avian reticuloendothelial disease virus (REV) or gibbon ape leukemia virus (GALV).

[0389] In certain preferred embodiments of the recombinant replication-competent retrovirus, the retrovirus is a gammaretrovirus.

[0390] In certain preferred embodiments of retroviruses with recombinant replication capability, there is a second box, which comprises a coding sequence of a second therapeutic protein, such as another checkpoint inhibitor polypeptide, a co-stimulatory polypeptide and / or an immunostimulatory cytokine (as an example only), for example, downstream of the box. In certain examples, the second box may comprise an internal ribosome entry site (lRES) or a mini-promoter or poIII promoter operably connected to the coding sequence of the second therapeutic protein.

[0391] In certain preferred embodiments of the recombinant replication-competent retrovirus, it is a non-lytic, amphipathic retroviral replicating vector, which preferably selectively infects and replicates in cells of the tumor microenvironment.

[0392] Other viral vectors as expression constructs

[0393] Other viral vectors can be used as expression constructs for delivering oligonucleotides or polynucleotide sequences to host cells in the present invention. Viral vectors derived from, for example, vaccinia virus, polio virus, and herpes virus can be used. They provide several attractive features for various mammalian cells. Hepatitis B virus is also included.

[0394] b. Non-viral vectors

[0395] Plasmid vector

[0396] Other vectors include plasmid vectors. Plasmid vectors have been widely described in the art and are well known to those skilled in the art. See, for example, Sambrook et al., 1989 cited above. In the past few years, plasmid vectors have been used as DNA vaccines for delivering antigen encoding genes to cells. They are particularly advantageous for this because they do not have many of the same safety issues as viral vectors. However, these plasmids with promoters compatible with host cells can express peptide epitopes encoded by nucleic acids in the plasmid. Other plasmids are well known to those of ordinary skill in the art. In addition, plasmids can be custom designed using restriction enzymes and ligation reactions to remove and add specific fragments of DNA. Plasmids can be delivered by various parenteral, mucosal and local routes. For example, DNA plasmids can be injected intramuscularly, intradermally, subcutaneously or by other routes. It can also be administered by intranasal sprays or drops, rectal suppositories and oral administration. It can also be administered to the epidermis or mucosal surface using a gene gun. The plasmids may be administered in aqueous solution, dried on gold particles, or combined with another DNA delivery system including but not limited to liposomes, dendrimers, intracellular and microencapsulation.

[0397] Therefore, in one aspect, plasmid is provided for expressing the nucleic acid construct containing epi-position, and the nucleic acid construct comprises expression cassette; Also referred to as transcription unit.When the plasmid is placed in the environment suitable for epi-position expression, the transcription unit will express polynucleotide, and the polynucleotide comprises the sequence of encoding epi-position, ETS and MHC I1 promoter sequence or the sequence of encoding epi-position and secretion signal sequence, and any other sequence encoded in the construct.The transcription unit comprises transcription control sequence, and it is transcribed and connected with the cellular immune response element coding sequence.The transcription control sequence may comprise promoter / enhancer sequence, such as cytomegalovirus (CMV) promoter / enhancer sequence.However, those skilled in the art will know that various other promoter sequences suitable for expression in eukaryotic cells are known and can be used in construct disclosed herein equally.The expression level of nucleic acid product will depend on the existence and activation of associated promoter and associated enhancer element.

[0398] In certain embodiments, the sequences encoding the desired epitopes and targeting sequences can be cloned into expression plasmids containing regulatory elements for transcription, translation, RNA stability and replication (i.e., including transcription control sequences). Such expression plasmids are well known in the art and one of ordinary skill in the art will be able to design appropriate expression constructs using polynucleotides comprising sequences encoding cellular immune response elements or fragments thereof in a manner in which the cellular immune response elements can be expressed. There are many examples of suitable expression plasmids in which the polynucleotides comprising the sequences can be cloned, such as pCI-neo, pUMVC, or pcDNA3.

[0399] Large quantities of bacterial hosts carrying plasmids for expressing cellular immune response elements or fragments thereof can be fermented and the plasmids can be purified for subsequent use. Current human clinical trials using plasmids utilize this approach. Recombinant DNA Advisory Committee Data Management Report, Human Gene Therapy 6:535-548, 1994. Current DNA isolation methods known in the art include the removal of lipopolysaccharides (endotoxins), which are contaminants from the bacteria used to propagate the plasmids. This step is most preferred for use with tolerogenic DNA vaccines, as endotoxins act as strong adjuvants and can produce undesirable immune stimulation.

[0400] The purpose of the plasmid is to effectively deliver the nucleic acid sequence to a cell or tissue and express a therapeutic epitope in the cell or tissue. In particular, the purpose of the plasmid can be to achieve a high copy number, avoid potential causes of plasmid instability, and provide a means of plasmid selection. As for expression, the nucleic acid cassette contains the necessary elements for expressing the nucleic acid in the cassette. Expression includes effectively transcribing the inserted gene, nucleic acid sequence, or nucleic acid cassette with a plasmid. The expression product can be a protein, polypeptide, or RNA. The nucleic acid cassette can contain a nucleic acid sequence. The expression of nucleic acids can be continuous or regulated.

[0401] Microring

[0402] Embodiments of the nucleic acid constructs described herein can be processed in the form of mini-ring DNA. Mini-ring DNA is a small (2 to 4 kb) circular plasmid derivative that has been partially released from all prokaryotic vectors. Since mini-ring DNA vectors do not contain bacterial DNA sequences, they are unlikely to be considered foreign and destroyed. (Typical transgenic delivery methods involve plasmids containing exogenous DNA). Therefore, compared to conventional plasmids (several days to weeks), these vectors can express longer time periods (on the order of weeks or months). Smaller-sized mini-rings also expand their cloning capabilities and facilitate their delivery into cells. Kits for producing mini-ring DNA are known in the art and are commercially available (System Biosciences, Inc., Palo Alto, Calif.). Information on minicircle DNA is provided in Dietz et al., Vector Engineering and Delivery Molecular Therapy (2013); 218, 1526-1535 and Hou et al., Molecular Therapy-Methods & Clinical Development, article number: 14062 (2015) doi: 10.1038 / mtm.2014.62. More information on minicircles is provided in Chen ZY, He CY, Ehrhardt A, Kay MA. Mol Ther. 2003 Sep; 8(3): 495-500, and minicircle DNA vectors achieve sustained expression reflected by active chromatin and transcription levels. Gracey Maniar LE, Maniar JM, Chen ZY, Lu J, Fire AZ, Kay MA. Mol Ther. 2013 Jan; 21(1): 131-8.

[0403] As an initial step in a method that ultimately obtains expression of a product encoded by a nucleic acid, it is to achieve uptake of the nucleic acid by a cell. Uptake of nucleic acids by cells depends on many factors, one of which is the length of time the nucleic acid is in proximity to the cell surface. For example, after intramuscular (im) administration of plasmid DNA in a buffer, a significant decrease in gene expression is observed if the muscle is massaged, possibly due to leakage of the DNA from the muscle directly or via lymphatic vessels (Human Gene Therapy 4: 151-159; 1993). Therefore, it may be necessary to formulate nucleic acids with compounds that delay the rate at which the nucleic acid diffuses or is carried away from the site where cellular uptake of the nucleic acid is desired. In addition, these compounds may be suitable for administration to an organism by means such as injection while maintaining or restoring the physical characteristics necessary to increase cellular uptake of the nucleic acid.

[0404] To achieve expression of the oligonucleotide or polynucleotide sequence, the expression construct must be delivered into the cell.In certain embodiments encompassed by the present invention, the expression construct comprising one or more oligonucleotide or polynucleotide sequences may consist solely of naked recombinant DNA or a plasmid.

[0405] To induce immunity, any type of DNA vaccine vector is preferably engineered to be CpG-rich (to stimulate TLR9 on immune cells) or conversely engineered to remove CpG and, where possible, replace CpG motifs with GpG motifs (Ho et al., J. Immunol. 71(9):4920-6, 2003; Ho et al., J. Immunol. 175(9):6226-34, 2005). DNA vaccines can be engineered to contain antigens / epitopes and may also contain additional genes co-expressed with the antigen to act as adjuvants or immunomodulators (multiple promoter vectors). These DNA vaccines have been found to be clinically safe, for example in T1D patients (Roep et al., Sci. Transl. Med. 5(191):191ra82, 2013).

[0406] Mechanical delivery system

[0407] Additional non-viral delivery methods include, but are not limited to, mechanical delivery systems that can be used in vitro, such as the method described in Woffendin et al., Proc. Natl. Acad. Sci. USA 91 (24): 11581, 1994; deposition of photopolymerized hydrogel materials or use of ionizing radiation (see, e.g., U.S. Pat. No. 5,206,152 and WO 92 / 11033); use of a handheld gene transfer particle gun (see, e.g., U.S. Pat. No. 5,149,655); and use of ionizing radiation to activate the transferred gene (see, e.g., U.S. Pat. No. 5,206,152 and WO 92 / 11033). The delivery device may also be biocompatible and may also be biodegradable. The formulation preferably provides a relatively constant level of release of the active ingredient. On the other hand, a faster rate of immediate release may be desired after administration. The preparation of such compositions is well within the level of ordinary skill in the art using known techniques.

[0408] Physical methods of enhanced delivery include electroporation (where short pulses of high voltage carry nucleic acids across membranes), gene guns (where DNA is loaded onto gold particles and forced to penetrate into cells), sonoporation, magnetofection, hydrodynamic delivery, and the like, all of which are known to those skilled in the art. DNA may also be encapsulated in liposomes, preferably cationic liposomes, or polymers (synthetic liposomes) that can interact with cell membranes and fuse or undergo endocytosis to effect transfer of the DNA into cells. DNA may also form complexes with polymers (polyplexes) or with dendrimers that can release the payload directly into the cytoplasm of the cell.

[0409] Illustrative carriers suitable for use in this regard include microparticles of poly(lactide-co-glycolide), polyacrylates, latex, starch, cellulose, dextran, and the like. Other illustrative delayed release carriers include supramolecular biocarriers comprising a non-liquid hydrophilic core (e.g., a cross-linked polysaccharide or oligosaccharide) and optionally an outer layer of an amphiphilic compound (such as a phospholipid) (see, e.g., U.S. Pat. No. 5,151,254 and PCT Applications WO 94 / 20078, WO / 94 / 23701, and WO 96 / 06638). The amount of active agent contained in the sustained release formulation depends on the site of implantation, the rate and expected duration of release, and the nature of the condition to be treated or prevented.

[0410] Biodegradable microspheres (e.g., polylactic acid polyglycolate) can be used as carriers of the composition. Suitable biodegradable microspheres are disclosed in, for example, U.S. Pat. Nos. 4,897,268; 5,075,109; 5,928,647; 5,811,128; 5,820,883; 5,853,763; 5,814,344; 5,407,609 and 5,942,252. Modified hepatitis B nucleoprotein carrier systems such as those described in WO / 9940934 and references cited therein will also be suitable for many applications. Another illustrative carrier / delivery system employs a carrier comprising a microparticle-protein complex, such as those described in U.S. Pat. No. 5,928,647, which may have additional benefits when used intratumorally to deliver the coding sequence of an anti-CD39 antibody agent, which is capable of inducing an MHCl-restricted cytotoxic T lymphocyte response targeting the patient's tumor tissue.

[0411] Biodegradable polymer nanoparticles promote nonviral nucleic acid transfer into N cells. Small (approximately 200 nm), positively charged (approximately 10 mV) particles are formed by self-assembly of cationic, hydrolytically degradable poly(β-amino esters) and plasmid DNA.

[0412] The polynucleotides can also be administered to cells by direct microinjection, temporary cell permeabilization (eg, co-administration of a repressor and / or activator with a cell permeabilizing agent), fusion to a membrane translocating peptide, and the like.

[0413] In certain specific embodiments of the present invention, gene constructs are introduced into target cells via electroporation. Electroporation involves exposing cells (or tissues) and DNA (or DNA complexes) to high voltage discharges. In vivo electroporation has been successfully used to effectively deliver plasmid DNA to gene delivery techniques of many different tissues. Studies have reported the use of in vivo electroporation to deliver plasmid DNA to B16 melanoma and other tumor tissues. Systemic and local expression of genes or cDNA encoded by plasmids can be obtained by using in vivo electroporation. In vivo electroporation is used to enhance the uptake of plasmid DNA in tumor tissue, resulting in expression in the tumor, and plasmids are delivered to muscle tissue, resulting in systemic expression of secreted proteins such as cytokines (see, e.g., US8026223). Exemplary techniques, vectors and devices for in vivo electroporation of anti-CD39 antibody agents into cells include PCT publications WO / 2017 / 106795, WO / 2016 / 161201, WO / 2016 / 154473, WO / 2016 / 112359 and WO / 2014 / 066655.

[0414] U.S. Patent No. 7,245,963 describes modular electrode systems and their use for facilitating the introduction of biomolecules into cells of selected tissues in the body or plants. The modular electrode system comprises a plurality of needle electrodes; a hypodermic needle; an electrical connector providing a conductive connection of a programmable constant current pulse controller to the plurality of needle electrodes; and a power source. An operator can grasp the plurality of needle electrodes mounted on a support structure and firmly insert them into a selected tissue in the body or plant. The biomolecules are then delivered into the selected tissue via the hypodermic needle. The programmable constant current pulse controller is activated, and a constant current electrical pulse is applied to the plurality of needle electrodes. The applied constant current electrical pulse facilitates the introduction of biomolecules into cells between the plurality of electrodes. The entire contents of U.S. Patent No. 7,245,963 are hereby incorporated by reference.

[0415] U.S. Patent Publication 2005 / 0052630 describes an electroporation device that can be used to effectively facilitate the introduction of biomolecules into cells of selected tissues in the body or plants. The electroporation device includes an electrokinetic device ("EKD device"), the operation of which is dictated by software or firmware. The EKD device generates a series of programmable constant current pulse patterns between electrodes in an array based on user control and input of pulse parameters, and allows storage and retrieval of current waveform data. The electroporation device also includes a replaceable electrode tray having an array of needle electrodes, a central injection channel for an injection needle, and a removable guide tray (see, e.g., U.S. Patent Publication 2005 / 0052630), which is hereby incorporated by reference.

[0416] The electrode arrays and methods described in U.S. Pat. No. 7,245,963 and U.S. Pat. Publication No. 2005 / 0052630 are suitable for deep penetration not only into tissues such as muscle, but also into other tissues or organs. Because of the configuration of the electrode array, the injection needle (to deliver the selected biomolecule) is also fully inserted into the target organ, and the injection is administered perpendicular to the target tissue in the area pre-delineated by the electrodes.

[0417] Generally, the magnitude of the electric field required for in vivo cell electroporation is generally similar to the electric field required for in vitro cells. In one embodiment, the magnitude of the electric field is about 10V / cm to about 1500V / cm, preferably about 300V / cm to 1500V / cm, and preferably in the range of about 1000V / cm to 1500V / cm. Or, the pulse length of lower field intensity (about 10V / cm to 100V / cm, and more preferably about 25V / cm to 75V / cm) is longer. For example, when the nominal electric field is about 25 to 75V / cm, the pulse length is preferably about 10msec.

[0418] The pulse length may be from about 10 s to about 100 ms. There may be any desired number of pulses, typically 1 to 100 pulses per second. The delay between pulses may be any desired time, such as one second. The waveform, electric field strength, and pulse duration may also depend on the type of cell and the type of molecule entering the cell via electroporation.

[0419] Also contemplated are electroporation devices incorporating electrochemical impedance spectroscopy ("EIS"). These devices provide real-time information about in vivo (particularly, intratumoral electroporation efficiency), thereby allowing optimization of conditions. Examples of electroporation devices incorporating EIS can be found, for example, in WO2016 / 161201, which is hereby incorporated by reference.

[0420] The uptake of non-viral delivery vectors encompassed by the present invention can also be enhanced by plasma electroporation (also known as avalanche transfection). In short, microsecond discharges generate cavitation microbubbles on the electrode surface. Compared to the diffusion-mediated transport associated with conventional electroporation, the combination of mechanical forces generated by ruptured microbubbles and magnetic fields acts to increase the efficiency of transport across cell membranes. The technology of plasma electroporation is described in U.S. Patent Nos. 7,923,251 and 8,283,171. This technology can also be used in vivo for the transformation of cells. Chaiberg et al. (2006) Investigative Ophthalmology & Visual Science 47: 4083-4090; Chaiberg et al., U.S. Patent No. 8,101169, issued January 24, 2012.

[0421] Other alternative electroporation techniques are also contemplated. In vivo plasmid delivery can also be performed using cold plasma. Plasma is one of the four fundamental states of matter, the other states being solid, liquid, and gas. Plasma is an electrically neutral medium of unbound positive and negative particles (i.e., the total charge of the plasma is approximately zero). Plasma can be generated by heating a gas or subjecting it to a strong electromagnetic field (applied with a laser or microwave generator). This reduces or increases the number of electrons, produces positively or negatively charged particles called ions (Luo et al. (1998) Phys. Plasma 5: 2868-2870), and, if present, is accompanied by the dissociation of molecular bonds.

[0422] Cold plasma (i.e., non-thermal plasma) is generated by delivering a pulsed high voltage signal to a suitable electrode. A cold plasma device may take the form of a gas jet device or a dielectric barrier discharge (DBD) device. Low temperature plasmas have attracted great enthusiasm and attention by providing plasma at relatively low gas temperatures. Providing plasma at such temperatures is of interest for various applications, including wound healing, antibacterial methods, various other medical therapies, and sterilization. As previously indicated, cold plasma (i.e., non-thermal plasma) is generated by delivering a pulsed high voltage signal to a suitable electrode. A cold plasma device may take the form of a gas jet device, a dielectric barrier discharge (DBD) device, or a multi-frequency rich harmonic power supply.

[0423] Dielectric barrier discharge devices rely on different methods to generate cold plasma. A dielectric barrier discharge (DBD) device contains at least one conductive electrode covered by a dielectric layer. The electrical circuit is formed by grounding, which can be provided by a target substrate undergoing cold plasma treatment or by providing a built-in ground for the electrode. The energy of the dielectric barrier discharge device can be provided by a high voltage power supply such as those mentioned above. More generally, energy is input into the dielectric barrier discharge device in the form of a pulsed DC voltage to form a plasma discharge. Through the dielectric layer, the discharge is separated from the conductive electrode and reduces electrode etching and gas heating. The pulsed DC voltage can be varied in amplitude and frequency to achieve varying operating schemes. Any device (e.g., a DBD electrode device) incorporating this principle of cold plasma generation falls within the scope of the various embodiments covered by the present invention.

[0424] Cold plasma has been used to transfect cells with foreign nucleic acids. In particular, transfection of tumor cells (see, e.g., Connolly et al. (2012) Human Vaccines & Immunotherapy 8: 1729-1733; and Connolly et al. (2015) Bioelectrochemistry 103: 15-21).

[0425] In certain illustrative embodiments, a transgenic construct encoding an anti-CD39 antibody agent encompassed by the present invention is delivered using an electroporation device comprising: an applicator; a plurality of electrodes extending from the applicator, the electrodes being associated with a coverage area; a power source electrically connected to the electrodes, the power source being configured to generate one or more electroporation signals to cells within the coverage area; and a guide member coupled to the electrodes, wherein the guide member is configured to adjust the coverage area of ​​the electrodes. At least a portion of the electrodes may be placed in the applicator in a conical arrangement. One or more electroporation signals may each be associated with an electric field. The device may also include a potentiometer coupled to the power source and the electrodes. The potentiometer may be configured to maintain the electric field substantially within a predetermined range.

[0426] The one or more electroporation signals may each be associated with an electric field. The device may also include a potentiometer coupled to the power source and the electrode. The potentiometer may be configured to maintain the electric field within a predetermined range so as to substantially prevent permanent damage to cells within the coverage area and / or substantially minimize pain. For example, the potentiometer may be configured to maintain the electric field to about 1300 V / cm.

[0427] A power supply may provide a first electrical signal to the first electrode and a second electrical signal to the second electrode. The first electrical signal and the second electrical signal may be combined to generate an electrical wave having a beat frequency. The first electrical signal and the second electrical signal may each have at least one of a unipolar waveform and a bipolar waveform. The first electrical signal may have a first frequency and a first amplitude. The second electrical signal may have a second frequency and a second amplitude. The first frequency may be different from or the same as the second frequency. The first amplitude may be different from or the same as the second amplitude.

[0428] In certain embodiments, the present invention provides a method for treating a subject with a tumor, the method comprising: injecting an effective dose of a plasmid encoding an anti-CD39 antibody agent into the tumor; and applying electroporation therapy to the tumor. In certain embodiments, the electroporation therapy also includes applying at least one voltage pulse of about 200V / cm to about 1500V / cm at a pulse width of about 100 microseconds to about 20 milliseconds.

[0429] In certain embodiments, the plasmid (or second electroporation plasmid) further encodes at least one immunostimulatory cytokine, such as selected from the group encoding IL-12, IL-15, and a combination of IL-12 and IL-15.

[0430] Lipid and polycationic molecules for delivery of nuclear constructs encoding anti-CD39 antibodies

[0431] Lipid-mediated nucleic acid delivery and expression of foreign nucleic acids (including mRNA) in vitro and in vivo have been very successful. Lipid-based non-viral formulations provide an alternative to adenoviral gene therapy. Current in vivo lipid delivery methods use subcutaneous, intradermal, intratumoral or intracranial injections. The progress of lipid formulations improves the efficiency of in vivo gene transfer (see PCT application WO98 / 07408). For example, lipid formulations containing equimolar ratios of l, 2-bis (oleoyloxy) -3- (trimethylammonio) propane (DOTAP) and cholesterol can significantly enhance systemic in vivo gene transfer. DOTAP: cholesterol lipid formulations form unique structures, called "sandwich liposomes". This formulation is reported as a "sandwich" DNA between invaginated bilayers or "vase" structures. The beneficial features of these lipid structures include positive p, colloidal stabilization of cholesterol, two-dimensional nucleic acid packaging and increased serum stability.

[0432] Cationic liposome technology is based on the ability of amphiphilic lipids with positively charged head groups and hydrophobic lipid tails to bind to negatively charged DNA or RNA and form particles that enter cells, generally via endocytosis. Some cationic liposomes also contain neutral co-lipids, which are believed to enhance liposome uptake by mammalian cells. Similarly, other polycations such as poly-l-lysine and polyethyleneimine complex with nucleic acids via charge interactions and help condense the DNA or RNA into nanoparticles, which are then substrates for endosomal-mediated uptake. [8] Several of these cation-nucleic acid complexation technologies have been developed into potential clinical products, including complexes with plasmid DNA (pDNA), oligodeoxynucleotides, and various forms of synthetic RNA.

[0433] The nucleic acid construct disclosed herein can be associated with a polycationic molecule that acts to enhance uptake into the cell. The complex of the nucleic acid construct and the polycationic molecule also helps to package the construct, so that they are reduced in size, which is believed to help cellular uptake. Once inside the endosome, the complex dissociates due to the lower pH, and the polycationic molecule can destroy the membrane of the endosome to promote DNA escape into the cytoplasm, which can then be degraded. Preliminary data show that when complexed with the polycationic molecule polylysine or polyethyleneimine, the nucleic acid construct embodiment has been enhanced to uptake in the SC compared to the DC.

[0434] One example of a polycationic molecule suitable for complexing with a nucleic acid construct includes a cell permeating peptide (CPP), examples include polylysine (described above), polyarginine and Tat peptides. Cell permeating peptides (CPP) are small peptides that can bind to DNA and, once released, permeate the cell membrane to facilitate the escape of DNA from endosomes to the cytoplasm. Another example of a CPP involves a 27-residue chimeric peptide, called MPG, which has been shown some time ago to bind ss and ds oligonucleotides in a stable manner, thereby generating a non-covalent complex that protects nucleic acids from DNA enzyme degradation and effectively delivers oligonucleotides to cells in vitro (Mahapatro A et al., Nanobiotechnol, 2011, 9: 55). When different peptide: DNA ratios and 10: 1 and 5: 1 ratios (150 nm and 1 um, respectively) were examined, the complex formed small particles of approximately 150 nm to 1 um. Another CPP involves a modified tetrapeptide [tetralysine containing a guanidinocarbonylpyrrole (GCP) group (TL-GCP)], which was reported to bind to 6.2 kb plasmid DNA with high affinity, resulting in 700 to 900 nm positively charged aggregates (Li et al., Agnew Chem Int Ed Enl 2015;54(10):2941-4). RNA can also be complexed by such polycationic molecules for in vivo delivery.

[0435] Other examples of polycationic molecules that can be complexed with the nucleic acid constructs described herein include polycationic polymers commercially available as TRANSFERR® and InVivo JET (Polypus-transfection, SA, Illkirch, France).

[0436] VI. Methods of Use and Pharmaceutical Compositions

[0437] The anti-CD39 antibodies encompassed by the present invention are suitable for various applications, including but not limited to therapeutic treatment methods, such as immunotherapy for cancer. In certain embodiments, the anti-CD39 antibodies described herein are suitable for activating, promoting, increasing and / or enhancing immune responses, inhibiting tumor growth, reducing tumor volume, inducing tumor regression, increasing tumor cell apoptosis and / or reducing the tumorigenicity of tumors. In certain embodiments, the anti-CD39 antibodies encompassed by the present invention are also suitable for immunotherapy for pathogens (such as viruses). In certain embodiments, the anti-CD39 antibodies described herein are suitable for inhibiting viral infection, reducing viral infection, increasing virally infected cell apoptosis and / or increasing the killing of virally infected cells. The method of use may be an in vitro, ex vivo or in vivo method.

[0438] The present invention provides methods for activating an immune response of a subject using an anti-CD39 antibody as described herein. In some embodiments, the present invention provides methods for promoting an immune response of a subject using an anti-CD39 antibody as described herein. In some embodiments, the present invention provides methods for increasing an immune response of a subject using an anti-CD39 antibody as described herein. In some embodiments, the present invention provides methods for enhancing an immune response of a subject using an anti-CD39 antibody as described herein. In some embodiments, the activation, promotion, increase and / or enhancement of an immune response includes increasing cell-mediated immunity. In some embodiments, the activation, promotion, increase and / or enhancement of an immune response includes increasing Th1 type responses. In some embodiments, the activation, promotion, increase and / or enhancement of an immune response includes increasing T cell activity. In some embodiments, the activation, promotion, increase and / or enhancement of an immune response includes increasing CD4+T cell activity. In some embodiments, the activation, promotion, increase and / or enhancement of an immune response includes increasing CD8+T cell activity. In some embodiments, the activation, promotion, increase and / or enhancement of an immune response includes increasing CTL activity. In some embodiments, the activation, promotion, increase and / or enhancement of an immune response includes increasing NK cell activity. In some embodiments, the activation, promotion, increase and / or enhancement of an immune response include increasing T cell activity and increasing NK cell activity. In some embodiments, the activation, promotion, increase and / or enhancement of an immune response include increasing CU activity and increasing NK cell activity. In some embodiments, the activation, promotion, increase and / or enhancement of an immune response include suppressing or reducing the inhibitory activity of Treg cells. In some embodiments, the activation, promotion, increase and / or enhancement of an immune response include suppressing or reducing the inhibitory activity of MDSC. In some embodiments, the activation, promotion, increase and / or enhancement of an immune response include increasing the number of percentages of memory T cells. In some embodiments, the activation, promotion, increase and / or enhancement of an immune response include increasing long-term immune memory function. In some embodiments, the activation, promotion, increase and / or enhancement of an immune response include increasing long-term memory. In some embodiments, the activation, promotion, increase and / or enhancement of an immune response does not include evidence of substantial side effects and / or immune-based toxicity. In some embodiments, the activation, promotion, increase and / or enhancement of an immune response does not include evidence of cytokine release syndrome (CRS) or cytokine storm. In some embodiments, an immune response is the result of antigen stimulation. In some embodiments, the antigenic stimulus is a tumor cell. In some embodiments, the antigenic stimulus is a cancer. In some embodiments, the antigenic stimulus is a pathogen. In some embodiments, the antigenic stimulus is a virus-infected cell.

[0439] In vivo and in vitro assays for determining whether an anti-CD39 antibody modulates, activates, or inhibits an immune response are known in the art or are being developed.

[0440] In certain embodiments of the methods described herein, the method of inducing durable or long-term immunity that inhibits tumor recurrence or tumor regrowth comprises administering to a subject a therapeutically effective amount of an anti-CD39 antibody.

[0441] In some embodiments, the tumor is a solid tumor. In some embodiments, the tumor is a tumor selected from the group consisting of: colorectal tumors, pancreatic tumors, lung tumors, ovarian tumors, liver tumors, breast tumors, kidney tumors, prostate tumors, neuroendocrine tumors, gastrointestinal tumors, melanomas, cervical tumors, bladder tumors, glioblastomas, lymphomas, and head and neck tumors. In some embodiments, the tumor is a colorectal tumor. In some embodiments, the tumor is an ovarian tumor. In some embodiments, the tumor is a lung tumor. In some embodiments, the tumor is a pancreatic or pancreatic islet tumor. In some embodiments, the tumor is a melanoma tumor. In some embodiments, the tumor is a bladder or urothelial tumor.

[0442] In some embodiments, the tumor is a liquid tumor. In certain embodiments, the tumor is a leukemia, such as myeloid or granulocytic leukemia, lymphoid, lymphocytic or lymphoblastic leukemia, and polycythemia vera or polycythemia.

[0443] In some embodiments, the tumor expresses or overexpresses a tumor antigen that is targeted by an anti-CD39 antibody (such as a bispecific agent comprising an antigen binding site that specifically binds to the tumor antigen).

[0444] The present invention further provides a method for treating cancer in a subject, the method comprising administering to the subject a therapeutically effective amount of an anti-CD39 antibody described herein. In some embodiments, the anti-CD39 antibody inhibits or reduces the growth of cancer.

[0445] The present invention provides methods for treating cancer, comprising administering to a subject (e.g., a subject in need of treatment) a therapeutically effective amount of an anti-CD39 antibody described herein. In certain embodiments, the subject is a human. In certain embodiments, the subject has a cancerous tumor. In certain embodiments, the subject has had a tumor removed.

[0446] In certain embodiments, the cancer is a cancer selected from the group consisting of colorectal cancer, pancreatic cancer, lung cancer, ovarian cancer, liver cancer, breast cancer, kidney cancer, prostate cancer, gastrointestinal cancer, melanoma, cervical cancer, neuroendocrine cancer, bladder cancer, brain cancer, glioblastoma, and head and neck cancer. In certain embodiments, the cancer is pancreatic cancer. In certain embodiments, the cancer is ovarian cancer. In certain embodiments, the cancer is colorectal cancer. In certain embodiments, the cancer is breast cancer. In certain embodiments, the cancer is prostate cancer. In certain embodiments, the cancer is lung cancer. In certain embodiments, the cancer is melanoma. In some embodiments, the cancer is bladder cancer.

[0447] The present invention provides compositions comprising anti-CD39 antibodies described herein. The present invention also provides pharmaceutical compositions comprising anti-CD39 antibodies described herein and a pharmaceutically acceptable vehicle. In some embodiments, the pharmaceutical composition is suitable for use in immunotherapy. In some embodiments, the pharmaceutical composition is suitable for use in immuno-oncology. In some embodiments, the composition is suitable for use in inhibiting tumor growth. In some embodiments, the pharmaceutical composition is suitable for use in inhibiting tumor growth in a subject (e.g., a human patient). In some embodiments, the composition is suitable for use in treating cancer. In some embodiments, the pharmaceutical composition is suitable for use in treating cancer in a subject (e.g., a human patient).

[0448] The preparation is prepared by combining the purifying agent covered by the present invention with a pharmaceutically acceptable vehicle (e.g., carrier or excipient) for storage and use. Those skilled in the art generally regard pharmaceutically acceptable carriers, excipients and / or stabilizers as inactive ingredients of the preparation or pharmaceutical composition.

[0449] In some embodiments, the anti-CD39 antibody is lyophilized and / or stored in lyophilized form. In some embodiments, the formulations comprising the anti-CD39 antibodies described herein are lyophilized.

[0450] Suitable pharmaceutically acceptable vehicles include, but are not limited to, nontoxic buffers such as phosphates, citrates and other organic acids; salts such as sodium chloride; antioxidants including ascorbic acid and methionine; preservatives such as octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl alcohol or benzyl alcohol, alkyl parabens (such as methyl or propyl paraben), catechol, m-hydroxybenzoate, and 1,2-diol. Hydroquinone, cyclohexanol, 3-pentanol and m-cresol; low molecular weight polypeptides (e.g., less than about 10 amino acid residues); proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; carbohydrates such as monosaccharides, disaccharides, glucose, mannose or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes such as Zn-protein complexes; and nonionic surfactants such as TWEEN or polyethylene glycol (PEG). (Remington: The Science and Practice of Pharmacy, 22nd Supplement, 2012, Pharmaceutical Press, London.)

[0451] The pharmaceutical compositions encompassed by the present invention can be administered in many ways for local or systemic treatment. Administration can be topical, by epidermal or transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders; pulmonary, by inhalation or insufflation of powders or aerosols, including by nebulizer, intratracheal and intranasal; oral; or parenteral, including intravenous, intraarterial, intratumoral, subcutaneous, intraperitoneal, intramuscular (e.g., injection or infusion) or intracranial (e.g., intrathecal or intraventricular).

[0452] The therapeutic preparation may be in unit dosage form. Such preparations include tablets, pills, capsules, powders, granules, solutions or suspensions in water or non-aqueous media, or suppositories. In solid compositions such as tablets, the main active ingredient is mixed with a pharmaceutical carrier. Conventional tableting ingredients include corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate or gum and diluents (e.g., water). These can be used to form a solid preformulated composition containing a homogeneous mixture of the compound encompassed by the present invention or its nontoxic pharmaceutically acceptable salt. The solid preformulated composition is then subdivided into unit dosage forms of the type described above. Tablets, pills, etc. of the preparation or composition may be coated or otherwise mixed to provide a dosage form that provides the advantage of extended action. For example, the tablet or pill may include an internal composition covered by an external component. In addition, the two components may be separated by an enteric layer that acts to resist disintegration and allows the internal component to pass through the stomach intact or to delay release. A variety of materials can be used for such enteric layers or coatings, such materials including a number of polymeric acids and mixtures of polymeric acids with such materials as shellac, cetyl alcohol, and cellulose acetate.

[0453] Anti-CD39 antibodies can also be embedded in microcapsules. Such microcapsules are prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin microcapsules and poly-(methyl methacrylate) microcapsules in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in coarse emulsions as described in Remington: The Science and Practice of Pharmacy, 22nd Supplement, 2012, Pharmaceutical Press, London.

[0454] In certain embodiments, the pharmaceutical preparation comprises an anti-CD39 antibody complexed with a liposome. Methods for producing liposomes are known to those skilled in the art. For example, some liposomes can be produced by reverse phase evaporation using a lipid composition comprising phosphatidylethanolamine (PEG-PE) derived from lecithin, cholesterol and PEG. Liposomes can be extruded through a filter that determines the pore size to produce liposomes with a desired diameter.

[0455] In certain embodiments, a sustained release formulation comprising anti-CD39 antibodies can be produced.Sustained release formulations include semi-permeable matrix containing a solid hydrophobic polymer of anti-CD39 antibodies, wherein the matrix is ​​in the form of a formed article (e.g., film or microcapsule).Examples of sustained release matrix include polyesters, hydrogels (such as poly (2-hydroxyethyl-methacrylate) or poly (vinyl alcohol)), polylactic acid, copolymers of L-glutamic acid and 7 ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers (such as LUPRONDEPOT (injectable microspheres comprising lactic acid-glycolic acid copolymers and leuprolide acetate)), sucrose acetate isobutyrate and poly-D-(-)-3-hydroxybutyric acid.

[0456] In certain embodiments, in addition to administering anti-CD39 antibodies, methods or treatments also include administering at least one additional immune response stimulant. In some embodiments, the additional immune response stimulant includes but is not limited to colony stimulating factors (e.g., granulocyte-macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M-CSF), granulocyte colony stimulating factor (G-CSF), stem cell factor (SCF)), interleukins (e.g., IL-1, IL2, IL-3, IL-7, IL-12, IL-15, IL-18), checkpoint inhibitors, antibodies that block immunosuppressive functions (e.g., anti-CTLA-4 antibodies, anti-CD28 antibodies, anti-CD3 antibodies), toll-like receptors (e.g., TLR4, TLR7, TLR9) or members of the B7 family (e.g., CD80, CD86). Additional immune response stimulants may be administered before, simultaneously and / or after administering anti-CD39 antibodies. A pharmaceutical composition comprising anti-CD39 antibodies and immune response stimulants is also provided. In some embodiments, the immune response stimulator comprises 1, 2, 3 or more immune response stimulators.

[0457] In certain embodiments, in addition to administering an anti-CD39 antibody, the method or treatment further comprises administering at least one additional therapeutic agent. The additional therapeutic agent may be administered before, simultaneously with, and / or after the administration of the anti-CD39 antibody. A pharmaceutical composition comprising an anti-CD39 antibody and an additional therapeutic agent is also provided. In some embodiments, the at least one additional therapeutic agent comprises 1, 2, 3 or more additional therapeutic agents.

[0458] Although this is not necessary, combination therapy using two or more therapeutic agents generally uses agents that work through different mechanisms of action. Combination therapy using agents with different mechanisms of action can produce cumulative or synergistic effects. Compared to the dosage of each agent used in a monotherapy, combination therapy can allow a lower dose of each agent, thereby reducing toxic side effects and / or increasing the therapeutic index of anti-CD39 antibodies. Combination therapy can reduce the possibility of drug-resistant cancer cell development. In some embodiments, combination therapy includes a therapeutic agent that affects an immune response (e.g., enhances or activates the response) and a therapeutic agent that affects (e.g., inhibits or kills) tumor / cancer cells.

[0459] In some embodiments of the methods described herein, the combination of an anti-CD39 antibody and at least one additional therapeutic agent produces an additive or synergistic result. In some embodiments, the combination therapy increases the therapeutic index of the anti-CD39 antibody. In some embodiments, the combination therapy increases the therapeutic index of the additional therapeutic agent. In some embodiments, the combination therapy reduces the toxicity and / or side effects of the anti-CD39 antibody. In some embodiments, the combination therapy reduces the toxicity and / or side effects of the additional therapeutic agent.

[0460] Useful classes of therapeutic agents include, for example, anti-tubulin agents, auristatins, DNA minor groove binders, DNA replication inhibitors, alkylating agents (e.g., platinum complexes such as cisplatin, mono(platinum), bis(platinum) and trinuclear platinum complexes and carboplatin), anthracyclines, antibiotics, antifolates, antimetabolites, chemotherapy sensitizers, duocarmycins, etoposide, fluorinated pyrimidines, ionophores, lexitropsin, nitrosoureas, cisplatinol, purine antimetabolites, puromycin, radiosensitizers, steroids, taxanes, topoisomerase inhibitors, vinca alkaloids, etc. In certain embodiments, the second therapeutic agent is an alkylating agent, antimetabolite, antimitotic agent, topoisomerase inhibitor, or an angiogenesis inhibitor.

[0461] Therapeutic agents that can be used in combination with the anti-CD39 antibodies described herein include chemotherapeutic agents. Therefore, in some embodiments, the method or treatment involves the use of a combination of an anti-CD39 antibody and a chemotherapeutic agent or a combination of a mixture of a chemotherapeutic agent. Treatment using anti-CD39 antibodies can occur before, simultaneously, or after the administration of chemotherapy. Combination administration can include co-administration, with a single drug formulation or using different formulations, or in any order but generally in a continuous administration within a certain period of time, so that all active agents can exert their biological activities at the same time. The preparation and administration schedule of such chemotherapeutic agents can be used according to the manufacturer's instructions or as determined empirically by a skilled practitioner. The preparation and administration schedule of this chemotherapy are also described in The Chemotherapy Source Book, the 4th supplementary edition, 2008, compiled by MCPerry, Lippincott, Williams & Wilkins, Philadelphia, Pa.

[0462] Chemotherapeutic agents suitable for use in the present invention include, but are not limited to, alkylating agents such as thiotepa and cyclophosphamide (CYTOXAN); alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquinone, mitodopa, and euridopa; ethyleneimines and methylmelamines, including hexamethylmelamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trihydroxymethylmelamine; nitrogen mustards such as benzyl alcohol, benzophenone, and benzophenone. Nitrogen mustard, naphthalene nitrogen mustard, chlorphosphamide, estramustine, ifosfamide, nitrogen mustard, nitrogen mustard oxide hydrochloride, melphalan, nembixin, phenamic acid, prednimustine, trofosfamide, uracil nitrogen mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics such as aclarubicin, actinomycin, anthramycin, azaserine, bleomycin, actinomycin C, calicheamicin , carrubicin, caminomycin, carcinophilin, chromomycin, dactinomycin, daunomycin, detoxibicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, mexilomycin, mitomycin, mycophenolic acid, noramycin, olivemycin, peplomycin, porfiromycin, puromycin, triferric doxorubicin, rhodorubicin, streptozotocin, streptozotocin, tuberculocidin, ubenimex, netastatin, doxycycline; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as dimethoate, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiopurine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytosine arabinoside (cytosine arabinoside), cytosine ... arabinoside), dideoxyuridine, deoxyfluridine, enocitabine, floxuridine, 5-FU; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitioStane, testolactone; antiadreners such as aminoglutethimide, mitotane, trilostane; folic acid supplements such as folinic acid; aceglucuronolide; aldophosphamide glycoside; aminolevulinic acid ; amsacrine; betribucil; bisantrene; edatrexadate; defofamin; colcemid; diazocone; eflornithine; elliptonium acetate; etoglu; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguanidine; mitoxantrone; mopidamol; nitracrine; pentostatin; methamine; pirarubicin; podophyllic acid; 2-ethylhydrazide; procarbazine; PSK; razoxane; sizofuran; spirogermanium; tenuisporic acid; triazoquinone; 2,2′,2″-trichlorotriethylamine; urethane; vindesine;Dacarbazine; mannomustine; dibromomannitol; dibromodulanol; pipobroman; garcitocin; cytarabine (Ara-C); taxanes, such as paclitaxel (TAXOL) and docetaxel (TAXOTERE); chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; platinum analogs, such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide ; Mitomycin C; Mitoxantrone; Vincristine; Vinorelbine; Navelbine; Mitoxantrone hydrochloride; Teniposide; Daunomycin; Aminopterin; Ibandronate; CPT11; Topoisomerase inhibitor RFS2000; Difluoromethylornithine (DMFO); Retinoic acid; Esperamycin; Capecitabine (XELODA); and a pharmaceutically acceptable salt, acid or derivative of any one of the above. Chemotherapeutic agents also include antihormonal drugs that act to regulate or inhibit hormonal effects on tumors, such as antiestrogens, including, for example, tamoxifen, raloxifene, 4 (5) -imidazoles that inhibit aromatase, 4-hydroxytamoxifen, troxifene, xifen, LY117018, onapristone, and toremifene (FARESTON); and antiandrogens, such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptable salts, acids, or derivatives of any of the above. In certain embodiments, the additional therapeutic agent is cisplatin. In certain embodiments, the additional therapeutic agent is carboplatin. ;

[0463] In certain embodiments of the methods described herein, the chemotherapeutic agent is a topoisomerase inhibitor. A topoisomerase inhibitor is a chemotherapeutic agent that interferes with the effect of a topoisomerase (e.g., topoisomerase I or II). Topoisomerase inhibitors include, but are not limited to, doxorubicin hydrochloride, daunomycin citrate, mitoxantrone hydrochloride, actinomycin D, etoposide, topotecan hydrochloride, teniposide (VM-26), and irinotecan, and any of these pharmaceutically acceptable salts, acids, or derivatives.

[0464] In certain embodiments, the chemotherapeutic agent is an antimetabolite. Antimetabolites are chemical substances that are similar in structure to the metabolites required for normal biochemical reactions, but are sufficiently different to interfere with one or more normal functions (such as cell division) of cells. Antimetabolites include but are not limited to gemcitabine, fluorouracil, capecitabine, methotrexate sodium, ralitrexed, pemetrexed, tegafur, cytosine arabinoside, thioguanine, 5-azacytidine, 6-mercaptopurine, thioazapurine, 6-thioguanine, pentostatin, fludarabine phosphate (fludarabine phosphate) and cladribine (cladribine), and any of these pharmaceutically acceptable salts, acids or derivatives.

[0465] In certain embodiments of the methods described herein, the chemotherapeutic agent is an antimitotic agent, including but not limited to agents that bind to tubulin. In some embodiments, the agent is a taxane. In certain embodiments, the agent is paclitaxel or docetaxel, or a pharmaceutically acceptable salt, acid or derivative of paclitaxel or docetaxel. In certain embodiments, the agent is paclitaxel (TAXOL), docetaxel (TAXOTERE), albumin-bound paclitaxel (nab-paclitaxel; ABRAXANE), DHA-paclitaxel or PG-paclitaxel. In certain alternative embodiments, the antimitotic agent comprises a vinca alkaloid, such as vincristine, vinblastine, vinorelbine or vindesine, or a pharmaceutically acceptable salt, acid or derivative thereof. In some embodiments, the antimitotic agent is an inhibitor of kinesin Eg5 or an inhibitor of mitotic kinases such as Aurora A or Plk1.

[0466] In certain embodiments of the methods described herein, it is expected that the subject anti-CD39 antibodies will have a greater combined effect (perhaps even a synergistic effect) with those chemotherapeutic agents that induce the release of ATP in tumors and / or cause upregulation of intratumoral CD39 or CD73. There is a wide range of chemotherapeutic agents that cause ATP to be released into the extracellular space because they induce tumor cell death, such as, but not limited to, anthracyclines (such as doxorubicin, daunomycin, epirubicin, and idarubicin), platinum-based drugs (such as cisplatin, carboplatin, and oxaliplatin), and proteasome inhibitors (such as bortezomib). Radiotherapy and photodynamic therapy (PDT) can also result in ATP release and / or upregulation of intratumoral levels of CD39 and / or CD73.

[0467] In some embodiments of the methods described herein, additional therapeutic agents include agents such as small molecules. For example, treatment may involve the combined administration of anti-CD39 antibodies and small molecules acting as inhibitors for tumor-associated antigens (including but not limited to EGFR, HER2 (ErbB2) and / or VEGF). In some embodiments, anti-CD39 antibodies are administered in combination with protein kinase inhibitors selected from the group consisting of: gefitinib (IRESSA), erlotinib (TARCEVA), sunitinib (sunitinib) (SUTENT), lapatinib (lapatanib), vandetanib (vandetanib) (ZACTIMA), AEE788, CI-1033, cediranib (cediranib) (RECENTIN), sorafenib (sorafenib) (NEXAVAR) and pazopanib (pazopanib) (GW786034B). In some embodiments, additional therapeutic agents include mTOR inhibitors.

[0468] In certain embodiments of the methods described herein, the additional therapeutic agent is a small molecule that inhibits a cancer stem cell pathway. In some embodiments, the additional therapeutic agent is an inhibitor of the Notch pathway. In some embodiments, the additional therapeutic agent is an inhibitor of the Wnt pathway. In some embodiments, the additional therapeutic agent is an inhibitor of the BMP pathway. In some embodiments, the additional therapeutic agent is an inhibitor of the Hippo pathway. In some embodiments, the additional therapeutic agent is an inhibitor of the mTOR / AKR pathway. In some embodiments, the additional therapeutic agent is an inhibitor of the RSPO / LGR pathway.

[0469] In some embodiments of the methods described herein, the additional therapeutic agent comprises a biomolecule, such as an antibody. For example, treatment may involve the combined administration of an anti-CD39 antibody and an antibody against a tumor-associated antigen, including but not limited to antibodies that bind EGFR, HER2 / ErbB2 and / or VEGF. In certain embodiments, the additional therapeutic agent is an antibody specific for a cancer stem cell marker. In some embodiments, the additional therapeutic agent is an antibody that binds a component of the Notch pathway. In some embodiments, the additional therapeutic agent is an antibody that binds a component of the Wnt pathway. In certain embodiments, the additional therapeutic agent is an antibody that inhibits cancer stem cell pathways. In some embodiments, the additional therapeutic agent is an inhibitor of the Notch pathway. In some embodiments, the additional therapeutic agent is an inhibitor of the Wnt pathway. In some embodiments, the additional therapeutic agent is an inhibitor of the BMP pathway. In some embodiments, the additional therapeutic agent is an antibody that inhibits β-catenin signaling. In certain embodiments, the additional therapeutic agent is an antibody (e.g., anti-VEGF or VEGF receptor antibody) to an angiogenesis inhibitor. In certain embodiments, the additional therapeutic agent is bevacizumab (AVASTIN), ramucirumab, trastuzumab (HERCEPTIN), pertuzumab (OMNITARG), panitumumab (VECTIBIX), nimotuzumab, zalutumumab, or cetuximab (ERBITUX).

[0470] I / O Portfolio - Representative Checkpoint Inhibitors and Co-stimulatory Agonists

[0471] In some embodiments of the methods described herein, the additional therapeutic agent is an antibody that modulates an immune response. In some embodiments, the additional therapeutic agent is an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-CTLA-4 antibody, an anti-LAG-3 antibody, an anti-T1M-3 antibody, an anti-TIGIT antibody, and / or an anti-Siglec-15 antibody.

[0472] For example, treatment may also include administering an inhibitor of an immune checkpoint molecule or an activator of a co-stimulatory molecule, or a combination thereof. Exemplary inhibitors of immune checkpoints include inhibitors of one or more of the following: PD-1, CTLA-4, TIM-3, LAG-3, CEACAM, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, NLRP1, NRLP3, STING, TGFRβ or Siglec-15. Exemplary activators of co-stimulatory molecules include agonists of one or more of the following: OX40, CD2, CD27, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD30, CD40, BAFFR, HVEM, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160, B7-H3 or CD83 ligands. Exemplary inhibitors of immune checkpoints and exemplary activators of co-stimulatory molecules can be found in PCT Publication WO 2016 / 054555, which is incorporated herein by reference.

[0473] PD-1 antagonists

[0474] The PD-1 gene is a 55 kDa type I transmembrane protein that is part of the Ig gene superfamily (Agata et al. (1996) Int Immunol 8: 765-72). PD-1 contains a membrane-proximal immunoreceptor tyrosine-based inhibitory motif (ITIM) and a membrane-remote tyrosine-based switch motif (ITSM) (Thomas, ML (1995) J Exp Med 181: 1953-6; Vivier, E and Daeron, M (1997) Immunol Today 18: 286-91). Two ligands for PD-1 have been identified, PD-L1 and PD-L2, which have been shown to downregulate T cell activation after binding to PD-1 (Freeman et al. (2000) J Exp Med 192: 1027-34; Latchman et al. (2001) Nat Immunol 2: 261-8; Carter et al. (2002) Eur J Immunol 32: 634-43). Both PD-L1 and PD-L2 are B7 homologs that bind to PD-1 but not to other CD28 family members. PD-L1 is abundant in various human cancers (Dong et al. (2002) Nat. Med. 8: 787-9). The interaction between PD-1 and PD-L1 leads to a decrease in tumor-infiltrating lymphocytes, a decrease in T cell receptor-mediated proliferation, and immune evasion of cancer cells (Dong et al. (2003) J. Mol. Med. 81: 281-7; Blank et al. (2005) Cancer Immunol. Immunother. 54: 307-314; Konishi et al. (2004) Clin. Cancer Res. 10: 5094-100). Immunosuppression can be reversed by inhibiting the local interaction between PD-1 and PD-L1, and the effect is additive when the interaction between PD-1 and PD-L2 is also blocked (Iwai et al. (2002) Proc. Nat'l. Acad. Sci. USA 99: 12293-7; Brown et al. (2003) J. Immunol. 170: 1257-66).

[0475] As used herein, the terms "programmed death 1", "programmed cell death 1", "protein PD-1", "PD-1", PD1, "PDCD1", "hPD-1" and "hPD-I" are used interchangeably and include variants, isoforms, species homologs of human PD-1, and analogs that have at least one common epitope with human PD-1. The complete human PD-1 sequence can be found under GenBank Accession No. U64863.

[0476] As used herein, the terms "programmed cell death 1 ligand 1", "PD-L1", "PDL1", "PDCD1L1", "PDCD1LG1", "CD274", "B7 homolog 1", "B7-H1", "B7-H" and "B7H1" are used interchangeably and include variants, isoforms, species homologs of human PDL-1, and analogs that have at least one common epitope with human PDL-1. The complete human PD-L1 amino acid sequence - isoform a precursor - can be found under GenBank Accession No. NP_054862.1. The complete human PD-L1 amino acid sequence - isoform b precursor - can be found under GenBank Accession No. NP_001254635.1.

[0477] The term "PD-1 axis binding antagonist" is a molecule that inhibits the interaction of a PD-1 axis binding partner with one or more of its binding partners, so as to remove T cell dysfunction caused by signal transduction on the PD-1 signal transduction axis, thereby restoring or enhancing T cell function (e.g., proliferation, cytokine production, target cell killing). As used herein, PD-1 axis binding antagonists include PD-1 binding antagonists, PD-L1 binding antagonists, and PD-L2 binding antagonists.

[0478] The term "PD-1 binding antagonist" is a molecule that reduces, blocks, inhibits, eliminates or interferes with signal transduction caused by the interaction of one or more of PD-1 and its binding partners (such as PD-L1, PD-L2). In some embodiments, the PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 to its binding partner. In a specific aspect, the PD-1 binding antagonist inhibits the binding of PD-1 to PD-L1 and / or PD-L2. For example, PD-1 binding antagonists include anti-PD-1 antibodies, antigen-binding fragments thereof, immunoadhesins, fusion proteins, oligopeptides, and other molecules that reduce, block, inhibit, eliminate or interfere with signal transduction caused by the interaction of PD-1 with PD-L1 and / or PD-L2. In one embodiment, the PD-1 binding antagonist reduces negative co-stimulatory signals mediated by or through cell surface proteins expressed on T lymphocytes mediated by PD-1 signaling, so as to make dysfunctional T cells less dysfunctional (e.g., enhancing effector responses to antigen recognition). In some embodiments, the PD-1 binding antagonist is an anti-PD-1 antibody. In one specific aspect, the PD-1 binding antagonist is MDX-1106 described herein. In another specific aspect, the PD-1 binding antagonist is Merck3745 described herein. In another specific aspect, the PD-1 binding antagonist is CT-011 described herein.

[0479] The term "PD-L1 binding antagonist" is a molecule that reduces, blocks, inhibits, eliminates or interferes with the signal transduction caused by the interaction of PD-L1 with one or more of its binding partners (such as PD-1, B7-1). In some embodiments, the PD-L1 binding antagonist is a molecule that inhibits the binding of PD-L1 to its binding partner. In a specific aspect, the PD-L1 binding antagonist inhibits the binding of PD-L1 to PD-1 and / or B7-1. In some embodiments, the PD-L1 binding antagonist includes anti-PD-L1 antibodies, antigen-binding fragments thereof, immunoadhesins, fusion proteins, oligopeptides, and other molecules that reduce, block, inhibit, eliminate or interfere with the signal transduction caused by the interaction of PD-L1 with one or more of its binding partners (such as PD-1, B7-1). In one embodiment, the PD-L1 binding antagonist reduces negative co-stimulatory signals mediated by or through cell surface proteins expressed on T lymphocytes mediated by PD-L1 signaling, so as to make dysfunctional T cells less dysfunctional (e.g., enhance effector responses to antigen recognition). In some embodiments, the PD-L1 binding antagonist is an anti-PD-L1 antibody. In a specific aspect, the anti-PD-L1 antibody is YW243.55.S70 described herein. In another specific aspect, the anti-PD-L1 antibody is MDX-1105 described herein. In yet another specific aspect, the anti-PD-L1 antibody is MPDL3280A described herein.

[0480] The term "PD-L2 binding antagonist" is a molecule that reduces, blocks, inhibits, eliminates or interferes with the signal transduction caused by the interaction of one or more of PD-L2 and its binding partners (such as PD-1). In some embodiments, the PD-L2 binding antagonist is a molecule that inhibits the binding of PD-L2 to its binding partner. In a specific aspect, the PD-L2 binding antagonist inhibits the binding of PD-L2 to PD-1. In some embodiments, the PD-L2 antagonist includes anti-PD-L2 antibodies, antigen-binding fragments thereof, immunoadhesins, fusion proteins, oligopeptides, and other molecules that reduce, block, inhibit, eliminate or interfere with the signal transduction caused by the interaction of one or more of PD-L2 and its binding partners (such as PD-1). In one embodiment, the PD-L2 binding antagonist reduces the negative co-stimulatory signal mediated by or through the cell surface protein mediated by PD-L2 signaling on T lymphocytes, so as to make the dysfunctional T cells less dysfunctional (e.g., enhancing the effector response to antigen recognition). In some embodiments, the PD-L2 binding antagonist is an immunoadhesin.

[0481] PD-1 pathway: The members of the PD-1 pathway are all proteins associated with PD-1 signaling. On the one hand, these may be proteins that induce PD-1 signaling upstream of PD-1, such as, for example, ligands of PD-1 PD-L1 and PD-L2 and signaling receptor PD-1. On the other hand, these may be signaling proteins downstream of the PD-1 receptor. In the context encompassed by the present invention, it is particularly preferred that the members of the PD-1 pathway are PD-1, PD-L1 and PD-L2.

[0482] PD-1 pathway inhibitors: In the context encompassed by the present invention, PD-1 pathway inhibitors are preferably defined herein as compounds capable of impairing PD-1 pathway signaling, preferably signaling mediated by the PD-1 receptor. Thus, the PD-1 pathway inhibitor may be any inhibitor directed against any member of the PD-1 pathway that is capable of antagonizing PD-1 pathway signaling. In this context, the inhibitor may be an antagonistic antibody as defined herein, targeting any member of the PD-1 pathway, preferably against the PD-1 receptor PD-L1 or PD-L2. Such antagonistic antibodies may also be encoded by nucleic acids. Such encoded antibodies are also referred to as "intracellular antibodies" as defined herein. In addition, the PD-1 pathway inhibitor may be a fragment of the PD-1 receptor or a PD1-receptor that blocks the activity of a PD1 ligand. B7-1 or a fragment thereof may also serve as a PD1 inhibitory ligand. In addition, the PD-1 pathway inhibitor may be an siRNA (small interfering RNA) or antisense RNA directed against a member of the PD-1 pathway (preferably PD-1, PD-L1 or PD-L2). In addition, the PD-1 pathway inhibitor may be a protein comprising an amino acid sequence capable of binding to PD-1 but preventing PD-1 signaling, for example, by inhibiting the interaction between PD-1 and B7-H1 or B7-DL (or a nucleic acid encoding an amino acid sequence capable of binding to PD-1 but preventing PD-1 signaling, for example, by inhibiting the interaction between PD-1 and B7-H1 or B7-DL). In addition, the PD-1 pathway inhibitor may be a small molecule inhibitor capable of inhibiting PD-1 pathway signaling, for example, a PD-1 binding peptide or a small organic molecule.

[0483] In certain embodiments, the PD-1 antagonists encompassed by the present invention include agents that bind to PD-1 ligands and interfere with, reduce or inhibit the binding of one or more ligands to PD-1 receptors, or directly bind to the PD-1 receptors without participating in signal transduction through the PD-1 receptors. In one embodiment, the PD-1 antagonist directly binds to PD-1 and blocks PD-1 inhibitory signal transduction. In another embodiment, the PD-1 antagonist binds to one or more ligands of PD-1 (e.g., PD-L1 and PD-L2), and reduces or inhibits ligands from triggering inhibitory signal transduction through PD-1. In one embodiment, the PD-1 antagonist directly binds to PD-L1, inhibits or prevents PD-L1 from binding to PD-1, thereby blocking PD-1 inhibitory signal transduction.

[0484] The PD-1 antagonists used in the methods and compositions encompassed by the present invention include PD-1 binding scaffold proteins, and include, but are not limited to, PD-ligands, antibodies, and multivalent agents. In a specific embodiment, the antagonist is a fusion protein, such as AMP-224. In another embodiment, the antagonist is an anti-PD-1 antibody ("PD-1" antibody). Anti-human PD-1 antibodies (or VH and / or VL domains derived therefrom) suitable for use in the present invention can be produced using methods well known in the art. Alternatively, recognized anti-PD-1 antibodies can be used. For example, antibodies MK-3475 or CT-011 can be used. In addition, monoclonal antibodies 5C4, 17D8, 2D3, 4H1, 4A11, 7D3, and 5F4 described in WO 2006 / 121168 can be used, and their teachings are incorporated herein by reference. Antibodies that compete with any of these recognized antibodies for binding to PD-1 can also be used.

[0485] In another embodiment, the PD-1 antagonist is an anti-PD-L1 antibody. Anti-human PD-L1 antibodies suitable for use in the present invention (or VH and / or VL domains derived therefrom) can be produced using methods well known in the art. Alternatively, recognized anti-PD-L1 antibodies can be used. For example, MEDI4736 (also known as anti-B7-HI) or MPDL3280A (also known as RG7446) can be used. In addition, monoclonal antibodies 12A4, 3G10, 10A5, 5F8, 10H10, 1B12, 7H1, 11E6, 12B7, and 13G4 described in WO 2007 / 005874 and U.S. Patent No. 7,943,743 can be used, and their teachings are incorporated herein by reference. Antibodies that compete with any of these recognized antibodies for binding to PD-L1 can also be used.

[0486] An exemplary anti-PD-L1 antibody is 12A4 described in WO 2007 / 005874 and U.S. Pat. No. 7,943,743. In one embodiment, the antibody comprises the heavy chain and light chain CDRs or VRs of 12A4. In another embodiment, the antibody competes with the antibodies mentioned above for binding to the same epitope on PD-L1 and / or binds to the same epitope on PD-L1. In another embodiment, the antibody has at least about 90% variable region amino acid sequence identity with the antibodies mentioned above.

[0487] The anti-PD-1 or anti-PD-L1 antibody may bind to PD-1 or PD-L1 with a KD of 10-7 M, 5x10-8 M, 10-8 M, 5x10-9 M, 10-9 M, 5x10-10 M, 10-10 M or less, respectively.

[0488] In one embodiment, the PD-1 inhibitor is an anti-PD-1 antibody selected from Nivolumab, Pembrolizumab or PiCilizumab. The preferred PD-1 inhibitor is Nivolumab.

[0489] In some embodiments, the anti-PD-1 antibody is nivolumab. Alternative names for nivolumab include MDX-1106, MDX-1106-04, ONO-4538, or BMS-936558. In some embodiments, the anti-PD-1 antibody is nivolumab (CAS registration number: 946414-94-4). Nivolumab is a fully human IgG4 monoclonal antibody that specifically blocks PDI. Nivolumab (clone 5C4) and other human monoclonal antibodies that specifically bind to PD1 are disclosed in US 8,008,449 (incorporated by reference) and WO 2006 / 121168 (incorporated by reference). In other embodiments, the anti-PD-1 antibody is pembrolizumab. Pembrolizumab (trade name formerly known as Lambrolizumab, also known as Merck 3745, MK-3475 or SCH-900475) is a humanized IgG4 monoclonal antibody that binds to PD1. Pembrolizumab is disclosed in, for example, Hamid, O. et al. (2013) New England Journal of Medicine 369(2): 134-44, WO 2009 / 114335 (incorporated by reference), and US 8,354,509 (incorporated by reference).

[0490] In some embodiments, the anti-PD-1 antibody is pidilizumab. pidilizumab (CT-011; CureTech) is a humanized IgG1k monoclonal antibody that binds to PD1. pidilizumab and other humanized anti-PD-1 monoclonal antibodies are disclosed in WO2009 / 101611. Other anti-PDi antibodies are disclosed in US 8,609,089, US2010028330 and / or US20120114649. Other anti-PD1 antibodies include AMP 514 (Amplimmune).

[0491] In some embodiments, the PD-1 inhibitor is an immunoadhesin {e.g., an immunoadhesin comprising an extracellular or PD-1 binding portion of PD-L1 or PD-L2 fused to a constant region {e.g., an Fc region of an immunoglobulin sequence). In some embodiments, the PD-1 inhibitor is AMP-224. In some embodiments, the PD-L1 inhibitor is an anti-PD-L1 antibody. In some embodiments, the anti-PD-L1 inhibitor is YW243.55.S70, MPDL3280A, MEDI-4736, MSB-0010718C, or MDX-1105.

[0492] In one embodiment, the PD-L1 inhibitor is MDX-1105. MDX-1105 (also known as BMS-936559) is an anti-PD-L1 antibody described in WO2007 / 005874. In one embodiment, the PD-L1 inhibitor is YW243.55.S70. The YW243.55.S70 antibody is an anti-PD-L1 described in WO 2010 / 077634 (incorporated by reference) (in WO 2010 / 077634, the heavy chain and light chain variable region sequences shown in SEQ ID No. 20 and 21, respectively).

[0493] In one embodiment, the PD-L1 inhibitor is MDPL3280A (Genentech / Roche). MDPL3280A is an IgG1 monoclonal antibody optimized for human Fc binding to PD-L1. MDPL3280A and other human monoclonal antibodies for PD-L1 are disclosed in U.S. Patent No. 7,943,743 (incorporated by reference) and U.S. Publication No. 2012 / 0039906 (incorporated by reference). In other embodiments, the PD-L2 inhibitor is AMP-224. AMP-224 is a PD-L2 Fc fusion soluble receptor (B7-DCIg; Amplimmune; For example, disclosed in WO 2010 / 027827 (incorporated by reference) and WO 2011 / 066342 (incorporated by reference)) that blocks the interaction between PD1 and B7-H1.

[0494] In certain embodiments, the PD-1 pathway inhibitor is a small molecule antagonist of PD-1 pathway signaling. Such small molecule antagonists include those that bind to one or more of PD-1, PD-1L, and / or PD-1L2 and inhibit the interaction of PD-1 with PD-1L1 and / or PD-1L2.

[0495] Exemplary small molecule antagonists of PD-1 pathway signaling can be found, among others, in published US applications 2014 / 0294898 and 2014 / 0199334, and published PCT applications WO 2013 / 132317 and WO 2012 / 168944, each of which is incorporated herein by reference.

[0496] For illustration only, the subject combination therapy may be practiced as a small molecule antagonist selected from the group consisting of:

[0497]

[0498] In other embodiments, the small molecule antagonist is represented by the following general formula:

[0499]

[0500] in,

[0501] R1 is the free C-terminus or amidated C-terminus of Ser;

[0502] L is a linker selected from -NH(CH2)nNH- or -NH(CH2CH2O)nNH-;

[0503] R4 is selected from hydrogen, amino(C1-C20)alkyl, -NHCOCH3 or -NHCONH2;

[0504] or a reverse analog or a pharmaceutically acceptable stereoisomer or a pharmaceutically acceptable salt thereof.

[0505] In yet other embodiments, the small molecule antagonist is represented by the following general formula:

[0506]

[0507] in,

[0508] R1 is the N-terminus of Ser; or a (C1-C20) acyl group substituted by the hydroxyl or amino group of Ser;

[0509] L is a linker selected from -NH(CH2)nNH-, -NH(CH2)nCH(NH2)CO-, -OOC(CH2)mCOO-, -NH(CH2)nCO-, -NH(CH2CH2O)nNH-, -NH(CH2CH2O)nCO- or -CO(CH2CH2O)nCO-;

[0510] R2 is the free C-terminus, amidated C-terminus or N-terminus of Am2; or Y-R5;

[0511] Y is an optional linker selected from -OOC(CH2)mCOO-, -CO(CH2)nNH-, -CO(CH2CH2O)nNH- or -COCH2(OCH2CH2)nNH-;

[0512] R5 is an albumin binding moiety, such as maleimidopropionic acid;

[0513] R3 is OH or NH2;

[0514] R4 is a substituent on the phenyl group of Phe and is selected from hydrogen, amino(C1-C20)alkyl, -NHCOCH3 or -NHCONH2;

[0515] n is an integer having a value selected from 2 to 10, inclusive;

[0516] m is an integer having a value selected from 0 to 8, inclusive; and

[0517] One of the peptide bonds (-CONH-) of Ser-Asn, Asn-Thr or Thr-Ser may be replaced by a modified peptide bond of,

[0518] wherein Q is hydrogen, -CO(C1-C20)alkyl or -COO(C1-C20)alkyl; wherein one or more or all of the amino acids may be in the D-configuration;

[0519] or a reverse analog or a pharmaceutically acceptable stereoisomer or a pharmaceutically acceptable salt thereof.

[0520] For example, the small molecule antagonist may be selected from the group consisting of:

[0521]

[0522]

[0523]

[0524]

[0525]

[0526]

[0527]

[0528] CTLA-4 antagonists

[0529] In certain embodiments, the combinations described herein further include CTLA-4 inhibitors. Exemplary anti-CTLA-4 antibodies include Tremelimumab (IgG2 monoclonal antibody available from Pfizer, formerly known as ticilimumab, CP-675,206); and Ipilimumab (CTLA-4 antibody, also known as MDX-010, CAS No. 477202-00-9).

[0530] Information about tremelimumab (or its antigen-binding fragment) used in the methods provided herein can be found in US6,682,736 (incorporated by reference) (wherein it is referred to as 11.2.1), the disclosure of which is incorporated herein by reference in its entirety. Tremelimumab (also known as CP-675,206, CP-675, CP-675206 and tremelimumab) is a human IgG2 monoclonal antibody that is highly selective for CTLA-4 and blocks the binding of CTLA-4 to CD80 (B7.1) and CD86 (B7.2). It has been shown to cause in vitro immune activation, and some patients treated with tremelimumab have shown tumor regression.

[0531] The tremelimumab used in the method provided herein comprises a heavy chain and a light chain or a heavy chain variable region and a light chain variable region. In a specific aspect, the tremelimumab or its Fab used in the method provided herein comprises a light chain variable region containing the amino acid sequence shown above herein and a heavy chain variable region containing the amino acid sequence shown above herein. In a specific aspect, the tremelimumab or its Fab used in the method provided herein comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the CDR1, CDR2 and CDR3 sequences defined by Kabat shown above herein, and wherein the light chain variable region comprises the CDR1, CDR2 and CDR3 sequences defined by Kabat shown above herein. Those of ordinary skill in the art will be able to easily determine Chothia definitions, Abm definitions or other CDR definitions known to those of ordinary skill in the art. In a specific aspect, tremelimumab or an antigen-binding fragment thereof for use in the methods provided herein comprises the variable heavy chain and variable light chain CDR sequences of an antibody as disclosed in US Pat. No. 6,682,736, which is incorporated herein by reference in its entirety.

[0532] The present invention also contemplates the use of small molecule inhibitors of CTLA-4, such as described by Huxley et al., 2004 Cell Chemical Biology 11: 1651-1658, which include compounds of the formula:

[0533]

[0534]

[0535] Other small molecule CTLA-4 antagonists include

[0536]

[0537] In one embodiment, the combination includes an immuno-DASH inhibitor, an anti-PD-1 antibody molecule (e.g., as described herein), and an anti-CTLA-4 antibody (e.g., ipilimumab). Exemplary dosages that can be used include a dose of about 1 to 10 mg / kg (e.g., 3 mg / kg) of an anti-PD-1 antibody molecule, and a dose of about 3 mg / kg of an anti-CTLA-4 antibody (e.g., ipilimumab).

[0538] Other exemplary anti-CTLA-4 antibodies are disclosed, for example, in U.S. Pat. No. 5,811,097.

[0539] In some embodiments of the methods described herein, the additional therapeutic agent is an antibody that modulates an immune response. In some embodiments, the additional therapeutic agent is an anti-PD-1 antibody, an anti-LAG-3 antibody, an anti-CTLA-4 antibody, an anti-TIM-3 antibody, an anti-TIGIT antibody, or an anti-Siglec-15 antibody.

[0540] In some embodiments, the LAG3 antibody is IM P701, IM P731, BMS-986016, LAG 525, and GSK2831781. In some embodiments, the LAG3 antagonist comprises a soluble LAG3 receptor, for example, IMP321.

[0541] In some embodiments, the immune response stimulator is selected from the group consisting of: CD28 agonists, 4-1BB agonists, OX40 agonists, CD27 agonists, CD80 agonists, CD86 agonists, CD40 agonists, and GITR agonists. In some embodiments, the OX40 agonist includes an OX40 ligand or its OX40 binding portion. For example, the OX40 agonist may be MEDI6383. In some embodiments, the OX40 agonist is an antibody that specifically binds to OX40. In some embodiments, the antibody that binds to OX40 is MEDI6469, MEDI0562, or MOXR0916 (RG7888). In some embodiments, the OX40 agonist is a vector (e.g., an expression vector or virus, such as an adenovirus) capable of expressing an OX40 ligand. In some embodiments, the vector expressing OX40 is δ-24-RGDOX or DNX2401.

[0542] In some embodiments, the 4-1BB (CD137) agonist is a binding molecule, such as anticalin. In some embodiments, the anticalin is PRS-343. In some embodiments, the 4-1BB agonist is an antibody that specifically binds to 4-1BB. In some embodiments, the antibody that binds to 4-1BB is PF-2566 (PF-05082566) or urelumab (BMS-663513).

[0543] In some embodiments, the CD27 agonist is an antibody that specifically binds CD27. In some embodiments, the antibody that binds CD27 is varlilumab (CDX-1127).

[0544] In some embodiments, the GITR agonist comprises a GITR ligand or a GITR binding portion thereof. In some embodiments, the GITR agonist is an antibody that specifically binds to GITR. In some embodiments, the antibody that binds to GITR is TRX518, MK-4166, or INBRX-110.

[0545] In certain embodiments, an anti-CD39 antibody is combined with a STING agonist, preferably as part of a pharmaceutical composition. The cyclic dinucleotide (CDN) cyclic diAMP (produced by Listeria monocytogenes and other bacteria) and its analog cyclic diGMP, and cyclic GMP-AMP are recognized by host cells as pathogen-associated molecular patterns (PAMPs), which bind to a pathogen recognition receptor (PRR) called stimulator of interferon genes (STING). STING is an adaptor protein in the cytoplasm of host mammalian cells that activates the TANK binding kinase (TBK1)-IRF3 and NF-KB signaling axis, leading to the induction of IFN-β and other gene products that strongly activate innate immunity. It is now recognized that STING is a component of the host cytoplasmic surveillance pathway (Vance et al., 2009), which senses infection with intracellular pathogens and induces the production of IFN-β in response, leading to the development of an adaptive protective pathogen-specific immune response, which consists of antigen-specific CD4+ and CD8+ T cells and pathogen-specific antibodies. U.S. Patent Nos. 7,709,458 and 7,592,326; PCT Publication Nos. WO2007 / 054279, WO2014 / 093936, WO2014 / 179335, WO2014 / 189805, WO2015 / 185565, WO2016 / 096174, WO2016 / 145102, WO2017 / 027645, WO2017 / 027646, and WO2017 / 075477; and Yan et al., Bioorg. Med. Chem Lett. 18:5631-4, 2008.

[0546] Exemplary combinations

[0547] In a preferred embodiment, the present invention relates to a combination of an anti-CD39 antibody and an anti-tumor platinum coordination complex to treat cancer, and more particularly to treat a cancer selected from the group consisting of lung cancer, sarcoma, malignant melanoma, prostate cancer, pancreatic cancer, gastric cancer, ovarian cancer, liver cancer, breast cancer, colorectal cancer, kidney cancer, brain cancer, and lymphoma. This group of chemotherapeutic agents includes, but is not limited to, cisplatin, oxaliplatin, carboplatin, triplatin tetranitrate (BBR3464), satraplatin, tetraplatin, omiplatin, isoproplatin, nedaplatin, and lobaplatin. Particularly preferred is a combination of an anti-CD39 antibody and cisplatin, oxaliplatin, carboplatin, triplatin tetranitrate, satraplatin, tetraplatin, omiplatin, isoproplatin, nedaplatin, and lobaplatin, and even more preferred is a combination with cisplatin and oxaliplatin to treat cancer, and more particularly to treat a cancer selected from the group consisting of lung cancer, sarcoma, malignant melanoma, prostate cancer, pancreatic cancer, gastric cancer, ovarian cancer, liver cancer, breast cancer, colorectal cancer, kidney cancer, and brain cancer. In another preferred embodiment, the present invention relates to a combination of an anti-CD39 antibody and an antimetabolite to treat cancer, and more particularly to treat a cancer selected from the group consisting of lung cancer, sarcoma, malignant melanoma, bladder cancer, prostate cancer, pancreatic cancer, gastric cancer, ovarian cancer, liver cancer, breast cancer, colorectal cancer, kidney cancer, esophageal cancer, brain cancer, anal cancer, leukemia and lymphoma. This group of chemotherapeutic agents includes, but is not limited to, 5-fluorouracil, gemcitabine, cytarabine, capecitabine, decitabine, floxuridine, fludarabine, aminopterin, methotrexate, pemetrexed, raltitrexed, cladribine, clofarabine, mercaptopurine, pentostatin and thioguanine. Particularly preferred are combinations of anti-CD39 antibodies with 5-fluorouracil, gemcitabine, cytarabine, capecitabine, decitabine, floxuridine, fludarabine, aminopterin, methotrexate, pemetrexed, raltitrexed, cladribine, clofarabine, mercaptopurine, pentostatin and thioguanine, and even more preferred are combinations with 5-fluorouracil, gemcitabine, cytarabine and methotrexate for the treatment of cancer, and more particularly for the treatment of a cancer selected from the group consisting of lung cancer, sarcoma, malignant melanoma, prostate cancer, pancreatic cancer, gastric cancer, ovarian cancer, liver cancer, breast cancer, colorectal cancer, kidney cancer, brain cancer, leukemia and lymphoma.

[0548] In another preferred embodiment, the present invention relates to a combination of an anti-CD39 antibody and a mitotic inhibitor to treat cancer, and more particularly to treat a cancer selected from the group consisting of lung cancer, sarcoma, prostate cancer, gastric cancer, ovarian cancer, liver cancer, breast cancer, colorectal cancer, kidney cancer, brain cancer, leukemia and lymphoma. This group of chemotherapeutic agents includes, but is not limited to, paclitaxel, docetaxel, vinblastine, vincristine, vindesine and vinorelbine. Particularly preferred is a combination of an anti-CD39 antibody and paclitaxel, docetaxel, vinblastine, vincristine, vindesine and vinorelbine, and even more preferred is a combination of paclitaxel, docetaxel, vincristine and vinorelbine to treat cancer, and more particularly to treat a cancer selected from the group consisting of lung cancer, sarcoma, prostate cancer, gastric cancer, ovarian cancer, liver cancer, breast cancer, colorectal cancer, kidney cancer and brain cancer.

[0549] In another preferred embodiment, the present invention relates to a combination of an anti-CD39 antibody and an anticancer antibiotic to treat cancer, and more particularly to treat the following cancers: lung cancer, sarcoma, malignant melanoma, bladder cancer, prostate cancer, pancreatic cancer, thyroid cancer, gastric cancer, ovarian cancer, liver cancer, breast cancer, colorectal cancer, kidney cancer, neuroblastoma, brain cancer, anal cancer, testicular cancer, leukemia, multiple myeloma and lymphoma. This group of chemotherapeutic agents includes, but is not limited to, daunomycin, doxorubicin, epirubicin, idarubicin, mitoxantrone, pixantrone, valrubicin, mitomycin C, bleomycin, actinomycin A and mithramycin. Particularly preferred are combinations of anti-CD39 antibodies with daunomycin, doxorubicin, epirubicin, idarubicin, mitoxantrone, pixantrone, valrubicin, mitomycin C, bleomycin, actinomycin D and mithramycin, and even more preferred are combinations with daunomycin, doxorubicin, mitomycin C and actinomycin D for the treatment of cancer, and more particularly for the treatment of lung cancer, sarcoma, malignant melanoma, prostate cancer, pancreatic cancer, gastric cancer, ovarian cancer, liver cancer, breast cancer, colorectal cancer, kidney cancer, brain cancer, leukemia and lymphoma.

[0550] In another preferred embodiment, the invention relates to a combination of an anti-CD39 antibody and a topoisomerase I and / or II inhibitor to treat cancer, and more particularly to treat lung cancer, sarcoma, malignant melanoma, prostate cancer, pancreatic cancer, gastric cancer, ovarian cancer, liver cancer, breast cancer, colorectal cancer, kidney cancer, neuroblastoma, brain cancer, cervical cancer, testicular cancer, leukemia and lymphoma. This group of chemotherapeutic agents includes, but is not limited to, topotecan, SN-38, irinotecan, camptothecin, rubitecan, etoposide, amsacrine and teniposide. Particularly preferred are combinations of PM00104 or a pharmaceutically acceptable salt thereof with topotecan, SN-38, irinotecan, camptothecin, rubitecan, etoposide, amsacrine and teniposide, and even more preferred are combinations with topotecan, irinotecan and etoposide for the treatment of cancer, and more particularly for the treatment of lung cancer, sarcoma, malignant melanoma, prostate cancer, pancreatic cancer, gastric cancer, ovarian cancer, liver cancer, breast cancer, colorectal cancer, kidney cancer and brain cancer.

[0551] In another preferred embodiment, the present invention relates to a combination of an anti-CD39 antibody and a proteasome inhibitor to treat cancer, and more particularly to treat lung cancer, prostate cancer, pancreatic cancer, gastric cancer, liver cancer, colorectal cancer, brain cancer, multiple myeloma and lymphoma. This group of chemotherapeutic agents includes, but is not limited to, bortezomib, disulfiram, epigallocatechin gallate and salinosporamide A. Particularly preferred is a combination of an anti-CD39 antibody with bortezomib, disulfiram, epigallocatechin gallate and salinosporamide A, and even more preferred is a combination with bortezomib to treat cancer, and more particularly to treat lung cancer, prostate cancer, pancreatic cancer, gastric cancer, liver cancer, colorectal cancer and brain cancer.

[0552] In another preferred embodiment, the present invention relates to a combination of an anti-CD39 antibody and a histone deacetylase inhibitor to treat cancer, and more particularly to treat lung cancer, sarcoma, prostate cancer, pancreatic cancer, gastric cancer, ovarian cancer, breast cancer, colorectal cancer, kidney cancer, brain cancer and lymphoma. This group of chemotherapeutic agents includes, but is not limited to, romidepsin, panobinostat, vorinostat, mocetinostat, belinostat, entinostat, resminostat, PCI-24781, AR-42, CUDC-101 and valproic acid. Particularly preferred are combinations of anti-CD39 antibodies with romidepsin, panobinostat, vorinostat, moxistat, belinostat, entinostat, resminostat, PCI-24781, AR-42, CUDC-101 and valproic acid, and even more preferred are combinations with vorinostat for the treatment of cancer, and more particularly for the treatment of lung cancer, sarcoma, prostate cancer, pancreatic cancer, gastric cancer, ovarian cancer, breast cancer, colorectal cancer, kidney cancer and brain cancer.

[0553] In another preferred embodiment, the present invention relates to a combination of an anti-CD39 antibody and a nitrogen mustard alkylating agent to treat cancer, and more particularly to treat lung cancer, sarcoma, bladder cancer, gastric cancer, ovarian cancer, liver cancer, breast cancer, colorectal cancer, kidney cancer, leukemia, multiple myeloma and lymphoma. This group of chemotherapeutic agents includes, but is not limited to, melphalan, ifosfamide, chlorambucil, cyclophosphamide, mechlorethamine, uramustine, estramustine and bendamustine. Particularly preferred is a combination of an anti-CD39 antibody and melphalan, ifosfamide, chlorambucil, cyclophosphamide, mechlorethamine, uramustine, estramustine and bendamustine, and even more preferred is a combination with cyclophosphamide to treat cancer, and more particularly to treat lung cancer, sarcoma, gastric cancer, ovarian cancer, liver cancer, breast cancer, colorectal cancer and kidney cancer. In another preferred embodiment, the present invention relates to a combination of an anti-CD39 antibody and a nitrosourea alkylating agent to treat cancer, and more particularly to treat lung cancer, ovarian cancer, breast cancer, brain cancer, multiple myeloma and lymphoma. This group of chemotherapeutic agents includes, but is not limited to, lomustine, semustine, carmustine, fotemustine and streptozotocin. Particularly preferred is a combination of an anti-CD39 antibody and lomustine, semustine, carmustine, fotemustine and streptozotocin, and even more preferably a combination with carmustine to treat cancer, and more particularly to treat lung cancer, ovarian cancer and breast cancer.

[0554] In another preferred embodiment, the present invention relates to a combination of an anti-CD39 antibody and an atypical alkylating agent to treat cancer, and more particularly to treat lung cancer, sarcoma, malignant melanoma, pancreatic cancer, gastric cancer, ovarian cancer, breast cancer, colorectal cancer, kidney cancer, brain cancer, leukemia and lymphoma. This group of chemotherapeutic agents includes, but is not limited to, procarbazine, dacarbazine, temozolomide and altretamine. Particularly preferred is a combination of an anti-CD39 antibody with procarbazine, dacarbazine, temozolomide and altretamine, and even more preferably with dacarbazine and temozolomide to treat lung cancer, sarcoma, malignant melanoma, gastric cancer, ovarian cancer, breast cancer, colorectal cancer, kidney cancer and brain cancer. In another preferred embodiment, the present invention relates to a combination of an anti-CD39 antibody with an estrogen antagonist to treat cancer, and more particularly to treat breast cancer. This group of chemotherapeutic agents includes, but is not limited to, toremifene, fulvestrant, tamoxifen and nafoxidine. Particularly preferred is the combination of an anti-CD39 antibody with toremifene, fulvestrant, tamoxifen and nafoxidine, and even more preferred is the combination with tamoxifen for the treatment of breast cancer.

[0555] In another preferred embodiment, the present invention relates to a combination of an anti-CD39 antibody and an androgen antagonist to treat cancer, and more particularly to treat prostate cancer. This chemotherapeutic group includes, but is not limited to, bicalutamide, flutamide, MDV3100 and nilutamide. Particularly preferred is a combination of an anti-CD39 antibody with bicalutamide, flutamide, MDV3100 and nilutamide, and even more preferably a combination with flutamide to treat prostate cancer.

[0556] In another preferred embodiment, the present invention relates to a combination of an anti-CD39 antibody and an mTOR inhibitor to treat cancer, and more particularly to treat lung cancer, sarcoma, malignant melanoma, prostate cancer, pancreatic cancer, gastric cancer, ovarian cancer, breast cancer, colorectal cancer, kidney cancer and brain cancer. This group of chemotherapeutic agents includes, but is not limited to, sirolimus, temsirolimus, everolimus, ridaforolimus, KU-0063794 and WYE-354. Particularly preferred is a combination of an anti-CD39 antibody and sirolimus, temsirolimus, everolimus, ridaforolimus, KU-0063794 and WYE-354, and even more preferably a combination with temsirolimus to treat lung cancer, sarcoma, malignant melanoma, prostate cancer, pancreatic cancer, gastric cancer, ovarian cancer, breast cancer, colorectal cancer and brain cancer.

[0557] In another preferred embodiment, the invention relates to a combination of an anti-CD39 antibody and a tyrosine kinase inhibitor to treat cancer, and more particularly to treat a cancer selected from the group consisting of lung cancer, sarcoma, prostate cancer, pancreatic cancer, gastric cancer, ovarian cancer, liver cancer, breast cancer, colorectal cancer, kidney cancer and brain cancer. This group of chemotherapeutic agents includes, but is not limited to, erlotinib, sorafenib, axitinib, bosutinib, cediranib, crizotinib, dasatinib, gefitinib, imatinib, canertinib, lapatinib, lestaurtinib, neratinib, nilotinib, semaxanib, sunitinib, vatalanib and vandetanib. Particularly preferred are combinations of anti-CD39 antibodies with erlotinib, sorafenib, axitinib, bosutinib, cediranib, crizotinib, dasatinib, gefitinib, imatinib, canertinib, lapatinib, lestaurtinib, neratinib, nilotinib, semaxanib, sunitinib, vatalanib and vandetanib, and even more preferred are combinations with erlotinib for the treatment of cancer, and more particularly for the treatment of a cancer selected from the group consisting of lung cancer, sarcoma, prostate cancer, pancreatic cancer, gastric cancer, ovarian cancer, liver cancer, breast cancer, colorectal cancer, kidney cancer and brain cancer.

[0558] Another aspect contemplated by the invention relates to any of the above methods, further comprising administering to the patient a MAP kinase pathway inhibitor or a WNT pathway inhibitor.

[0559] In some embodiments, the MAP kinase pathway inhibitor is selected from the group consisting of a BRAF inhibitor, a MEK inhibitor, a PI3K inhibitor, and a c-KIT inhibitor.

[0560] In some embodiments, the BRAF inhibitor is selected from the group consisting of GDC-0879, PLX-4720, sorafenib tosylate, dabrafenib, and LGX818.

[0561] In some embodiments, the MEK inhibitor is selected from the group consisting of GSK1120212, selumetinib, and MEK162.

[0562] In some embodiments, the WNT pathway inhibitor is a β-catenin inhibitor or a frizzled inhibitor.

[0563] In some embodiments, the β-catenin inhibitor is selected from the group consisting of niclosamide, XAV-939, FH 535, and lCG 001.

[0564] Another aspect contemplated by the invention relates to any of the above methods, further comprising administering a cancer vaccine to the patient. In some embodiments, the cancer vaccine is a dendritic cell vaccine.

[0565] Another aspect contemplated by the invention relates to any of the above methods, further comprising administering adoptive cell transfer to the patient.

[0566] In some embodiments, the adoptive cell transfer is CAR-T cell therapy.

[0567] Another aspect contemplated by the invention relates to any of the above methods, further comprising administering the antibody therapy to the patient.

[0568] Another aspect contemplated by the invention relates to any of the above methods, wherein administration of an anti-CD39 antibody enhances antibody-dependent cell-mediated cytotoxicity of the antibody therapy.

[0569] In some embodiments, the antibody therapy is selected from the group consisting of trastuzamab, cetuximab, bevacizumab, and rituximab.

[0570] In addition, treatment with anti-CD39 antibodies may include combination therapy with other biomolecules such as one or more cytokines (e.g., lymphokines, interleukins, tumor necrosis factors, and / or growth factors) or may be accompanied by surgical removal of the tumor, removal of cancer cells, or any other therapy deemed necessary by the treating physician. In some embodiments, the additional therapeutic agent is an immune response stimulator.

[0571] In some embodiments of the methods described herein, the anti-CD39 antibody can be combined with a growth factor selected from the group consisting of adrenomedullin (AM), angiogenin (Ang), BMP, BDNF, EGF, erythropoietin (EPO), FGF, GDNF, G-CSF, GM-CSF, GDF9, HGF, HDGF, IGF, migration stimulating factor, myostatin (GDF-8), NGF, neurotrophin, PDGF, thrombopoietin, TGF-α, TGF-β, TNF-α, VEGF, P1GF, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-12, IL-15 and IL-18.

[0572] In some embodiments of the methods described herein, the additional therapeutic agent is an immune response stimulator. In some embodiments, the immune response stimulator is selected from the group consisting of granulocyte-macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M-CSF), granulocyte colony stimulating factor (G-CSF), interleukin-3 (IL-3), interleukin-12 (IL-12), interleukin-1 (IL-1) or interleukin-2 (IL-2).

[0573] Administration schedule

[0574] In certain embodiments of the methods described herein, treatment involves administering a combination of an anti-CD39 antibody and radiotherapy. Treatment with an anti-CD39 antibody may occur before, simultaneously with, or after radiotherapy. The dosing schedule for such radiotherapy may be determined by a skilled medical practitioner.

[0575] In certain embodiments of the methods described herein, treatment involves administering a combination of an anti-CD39 antibody and an antiviral therapy. Treatment with an anti-CD39 antibody can occur before, simultaneously with, or after the antiviral therapy. The antiviral drug used in the combination therapy will depend on the virus with which the subject is infected.

[0576] Combined administration may include co-administration, either in a single pharmaceutical formulation or using different formulations, or sequential administration in any order but generally over a period of time such that all active agents can simultaneously exert their biological activities.

[0577] It should be understood that the combination of anti-CD39 antibodies and at least one other therapeutic agent can be administered in any order or simultaneously. In some embodiments, the anti-CD39 antibody will be administered to a patient who has previously undergone treatment using a second therapeutic agent. In certain other embodiments, the anti-CD39 antibody and the second therapeutic agent will be administered substantially simultaneously or in parallel. For example, a subject may be given an anti-CD39 antibody while undergoing a treatment course using a second therapeutic agent (e.g., chemotherapy). In certain embodiments, the anti-CD39 antibody will be administered within 1 year of the treatment using the second therapeutic agent. In certain alternative embodiments, the anti-CD39 antibody will be administered within 10, 8, 6, 4 or 2 months of any treatment using the second therapeutic agent. In certain other embodiments, the anti-CD39 antibody will be administered within 4, 3, 2 or 1 week of any treatment using the second therapeutic agent. In some embodiments, the anti-CD39 antibody will be administered within 5, 4, 3, 2 or 1 day of any treatment using the second therapeutic agent. It should be further understood that two (or more) agents or treatments can be administered to a subject within hours or minutes (i.e., substantially simultaneously).

[0578] For the treatment of the disease, the appropriate dose of the anti-CD39 antibody depends on the type of disease to be treated, the severity and course of the disease, the responsiveness of the disease, whether the anti-CD39 antibody is administered for treatment or prevention, previous therapy, the patient's clinical history, etc., all of which are weighed at the discretion of the attending physician. The anti-CD39 antibody can be administered once or for a series of treatments for several days to several months, or until a cure is achieved or a reduction in the disease state (e.g., a reduction in tumor size) is achieved. The optimal dosing schedule can be calculated from the measured values ​​of drug accumulation in the patient's body and will vary depending on the relative potency of the individual agents. The administering physician can determine the optimal dose, administration method, and repetition rate. In certain embodiments, the dose is 0.01 μg to 100 mg / kg body weight, 0.1 μg to 100 mg / kg body weight, 1 μg to 100 mg / kg body weight, 1 mg to 100 mg / kg body weight, 1 mg to 80 mg / kg body weight, 10 mg to 100 mg / kg body weight, 10 mg to 75 mg / kg body weight, or 10 mg to 50 mg / kg body weight. In certain embodiments, the dose of the anti-CD39 antibody is about 0.1 mg to about 20 mg / kg body weight. In some embodiments, the dose of the anti-CD39 antibody is about 0.1 mg / kg body weight. In some embodiments, the dose of the anti-CD39 antibody is about 0.25 mg / kg body weight. In some embodiments, the dose of the anti-CD39 antibody is about 0.5 mg / kg body weight. In some embodiments, the dose of the anti-CD39 antibody is about 1 mg / kg body weight. In some embodiments, the dose of the anti-CD39 antibody is about 1.5 mg / kg body weight. In some embodiments, the dose of the anti-CD39 antibody is about 2 mg / kg body weight. In some embodiments, the dose of the anti-CD39 antibody is about 2.5 mg / kg body weight. In some embodiments, the dose of the anti-CD39 antibody is about 5 mg / kg body weight. In some embodiments, the dose of the anti-CD39 antibody is about 7.5 mg / kg body weight. In some embodiments, the dose of the anti-CD39 antibody is about 10 mg / kg body weight. In some embodiments, the dosage of the anti-CD39 antibody is about 12.5 mg / kg body weight. In some embodiments, the dosage of the anti-CD39 antibody is about 15 mg / kg body weight. In certain embodiments, the dosage can be given once or more every day, every week, every month or every year. In certain embodiments, the anti-CD39 antibody is given once a week, once every two weeks, once every three weeks, or once every four weeks.

[0579] In some embodiments, anti-CD39 antibodies can be administered with an initial higher "loading" dose, followed by one or more lower doses. In some embodiments, the frequency of administration may also be changed. In some embodiments, the dosing regimen may include administering an initial dose, followed by administering another dose (or "maintenance dose") once a week, once every two weeks, once every three weeks, or once a month. For example, a dosing regimen may include administering an initial loading dose, followed by administering a maintenance dose (e.g., half of the initial dose) weekly. Or a dosing regimen may include administering an initial loading dose, followed by administering a maintenance dose (e.g., half of the initial dose) every other week. Or a dosing regimen may include administering three initial doses for 3 weeks, followed by administering a maintenance dose (e.g., the same amount) every other week.

[0580] As known to those skilled in the art, the administration of any therapeutic agent may result in side effects and / or toxicity. In some instances, the side effects and / or toxicity are so severe that it is not possible to exclude the administration of a particular agent at a therapeutically effective dose. In some instances, drug therapy must be stopped, and other agents may be tried. However, many agents in the same therapeutic class typically show similar side effects and / or toxicity, meaning that the patient has to stop therapy, or, if possible, suffer from unpleasant side effects associated with the therapeutic agent.

[0581] In some embodiments, the dosing schedule may be limited to a specific number of administrations or "cycles". In some embodiments, the anti-CD39 antibody is administered for 3, 4, 5, 6, 7, 8 or more cycles. For example, the anti-CD39 antibody is administered every 2 weeks for 6 cycles, the anti-CD39 antibody is administered every 3 weeks for 6 cycles, the anti-CD39 antibody is administered every 2 weeks for 4 cycles, the anti-CD39 antibody is administered every 3 weeks for 4 cycles, etc. The dosing schedule may be determined and subsequently modified by those skilled in the art.

[0582] Therefore, the present invention provides a method for administering an anti-CD39 antibody described herein to a subject, the method comprising administering one or more agents using an intermittent dosing strategy, which can reduce the side effects and / or toxicity associated with the administration of anti-CD39 antibodies, chemotherapeutic agents, etc. In some embodiments, the method for treating cancer in a human subject comprises administering to the subject a therapeutically effective dose of an anti-CD39 antibody in combination with a therapeutically effective dose of a chemotherapeutic agent, wherein one or both of the agents are administered according to an intermittent dosing strategy. In some embodiments, the intermittent dosing strategy comprises administering an initial dose of an anti-CD39 antibody to the subject, and administering a subsequent dose of the anti-CD39 antibody approximately once every 2 weeks. In some embodiments, the intermittent dosing strategy comprises administering an initial dose of an anti-CD39 antibody to the subject, and administering a subsequent dose of the anti-CD39 antibody approximately once every 3 weeks. In some embodiments, the intermittent dosing strategy comprises administering an initial dose of an anti-CD39 antibody to the subject, and administering a subsequent dose of the anti-CD39 antibody approximately once every 4 weeks. In some embodiments, the anti-CD39 antibody is administered using an intermittent dosing strategy, and the chemotherapeutic agent is administered weekly.

[0583] Anti-infective therapy combination

[0584] In one embodiment, the invention provides a method of treating a subject using an anti-CD39 antibody, wherein the subject suffers from a viral infection. In one embodiment, the viral infection is a viral infection selected from the group consisting of human immunodeficiency virus (HIV), hepatitis virus (A, B or C), herpes virus (e.g., VZV, HSV-1, HAV-6, HSV-II and CMV, Epstein-Barr virus), adenovirus, influenza virus, flavivirus, echovirus, rhinovirus, coxsackievirus, coronavirus, respiratory syncytial virus, mumps virus, rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus, HTLV virus, dengue virus, papillomavirus, molluscum virus, poliovirus, rabies virus, JC virus or arboviral encephalitis virus.

[0585] In one embodiment, the invention provides a method of treating a subject with an anti-CD39 antibody, wherein the subject suffers from a bacterial infection. In one embodiment, the bacterial infection is a bacterial infection selected from the group consisting of Chlamydia, rickettsial bacteria, mycobacteria, staphylococci, streptococci, pneumonococci, meningococci and gonococci, klebsiella, proteus, serratia, pseudomonas, Legionella, Corynebacterium diphtheriae, Salmonella, bacilli, Vibrio cholerae, Clostridium tetan, Clostridium botulinum, Bacillus anthracis. anthricis, Yersinia pestis, My cobacterium le prae, My cobacterium lepromatosis, and Borriella.

[0586] In one embodiment, the invention provides a method of treating a subject with an anti-CD39 antibody, wherein the subject suffers from a fungal infection. In one embodiment, the fungal infection is a fungal infection selected from the group consisting of Candida (albicans, krusei, glabrata, tropicalis, etc.), Cryptococcus neoformans, Aspergillus (fumigatus, niger, etc.), Genus Mucorales (mucor, absidia, rhizopus), Sporothrix schenkii, Blastomyces dermatitidis, Paracoccidioides brasiliensis, Coccidioides immitis, and Histoplasma capsulatum.

[0587] In one embodiment, the invention provides a method of treating a subject with an anti-CD39 antibody, wherein the subject suffers from a parasitic infection. In one embodiment, the parasitic infection is a parasitic infection selected from the group consisting of Entamoeba histolytica, Balantidium coli, Naegleria fowleri, Acanthamoeba, Giardia lamblia, Cryptosporidium, Pneumocystis carinii, Plasmodium vivax, Babesia microti, Trypanosoma brucei, Trypanosoma cruzi, Leishmania donovani, Toxoplasma gondii, and Nippostrongylus brasiliensis.

[0588] VII. Anti-CD39 Antibody Conjugates

[0589] The anti-CD39 antibodies disclosed herein may also be conjugated to a chemical moiety. The chemical moiety may be, among other things, a polymer, a radionuclide, or a cytotoxic factor.

[0590] For example, the present invention provides an anti-CD39 antibody conjugated to a therapeutic moiety (i.e., a drug). The therapeutic moiety may be, for example, a cytotoxin, a chemotherapeutic agent, a cytokine, an immunosuppressant, an immunostimulant, a lytic peptide, or a radioisotope. Such conjugates are referred to herein as "antibody-drug conjugates" or "ADCs". Therefore, in one aspect, an anti-CD39 antibody according to any aspect or embodiment described above is conjugated to a therapeutic moiety. Exemplary therapeutic moieties include cytotoxic moieties, radioisotopes, cytokines, and lytic peptides.

[0591] In certain embodiments, the anti-CD39 antibodies are capable of inducing cytotoxicity in CD39 expressing cells by internalizing the antibody conjugated to or associated with a cytotoxic moiety. The cytotoxic moiety can, for example, be selected from the group consisting of: paclitaxel; cytochalasin B; gramicidin D; ethidium bromide; emetine; mitomycin; etoposide; tenoposide; vincristine; vinblastine; colchicine; doxorubicin; daunomycin; dihydroxyanthraquinone dione; microtubule inhibitors such as maytansine or an analog or derivative thereof; antimitotic agents such as monomethyl auristatin E or F or an analog or derivative thereof; dolastatin 10 or 15 or an analog thereof; irinotecan or an analog thereof; mitoxantrone; mithramycin; actinomycin D; 1-dehydrotestosterone; glucocorticoids; procaine; tetracaine; lidocaine; propranolol; puromycin; calicheamicin or an analog or derivative thereof; an antimetabolite such as methotrexate, 6-mercaptopurine, 6-thioguanine amphetamine, cytarabine, fludarabine, 5-fluorouracil, decarbazine, hydroxyurea, asparaginase, gemcitabine or cladribine; an alkylating agent such as nitrogen mustard, thiothion, chlorambucil, melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, dacarbazine (DTIC), procarbazine, mitomycin C; a platinum derivative such as cisplatin or carboplatin; duocarmycin A, duocarmycin SA, razemycin (CC-1065) or an analog or derivative thereof; an antibiotic such as actinomycin D, bleomycin, daunomycin, doxorubicin, idarubicin, mithramycin, mitomycin, mitoxantrone, plicamycin, anthramycin (AMC); pyrrolo[2,1-c][1,4]-benzodiazepine (PDB);Diphtheria toxin and related molecules, such as diphtheria A chain and active fragments and hybrid molecules thereof, ricin, such as ricin A or deglycosylated ricin A chain toxin, cholera toxin, Shiga-like toxins, such as SLT I, SLT II, ​​SLT IIV, LT toxin, C3 toxin, Shiga toxin, pertussis toxin, tetanus toxin, soybean Bowman-Birk protease inhibitor, Pseudomonas exotoxin, alorin, saporin, modeccin, gelanin, abrin A chain, modeccin A chain, αα-sarcin, Aleurites fordii protein, dianthin protein, Phytolacca americana protein protein) such as PAPI, PAPII and PAP-S, momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin and enomycin toxins; ribonucleases (RNases); DNase I, Staphylococcal enterotoxin A; pokeweed antiviral protein; diphtheria toxin toxin; and Pseudomonas endotoxin. ;

[0592] In one embodiment, the anti-CD39 antibody is conjugated to an auristatin or a peptide analog, derivative or prodrug thereof. Auristatins have been shown to interfere with microtubule dynamics, GTP hydrolysis, and nuclear and cell division (Woyke et al. (2001) Antimicrob. Agents and Chemother. 45(12): 3580-3584) and have anticancer activity (US5663149) and antifungal activity (Pettit et al. (1998) Antimicrob. Agents and Chemother. 42: 2961-2965. For example, auristatin E can be reacted with p-acetylbenzoic acid or benzoylvaleric acid to produce AEB and AEVB, respectively. Other typical auristatin derivatives include AFP, MMAF (monomethyl auristatin F) and MMAE (monomethyl auristatin E). Suitable auristatins and auristatin analogs, derivatives and prodrugs and linkers suitable for conjugating auristatins to Abs Described in, for example, U.S. Pat. Nos. 5,635,483, 5,780,588 and 6,214,345 and in International Patent Application Publications WO02088172, WO2004010957, WO2005081711, WO2005084390, WO2006132670, WO03026577, WO200700860, WO207011968 and WO205082023.

[0593] In another embodiment, the anti-CD39 antibody is conjugated to pyrrolo[2,1-c][1,4]-benzodiazepine (PDB) or an analog, derivative or prodrug thereof. Suitable PDB and PDB derivatives and related technologies are described, for example, in Hartley J.A. et al., Cancer Res 2010; 70(17): 6849-6858; Antonow D. et al., Cancer J 2008; 14(3): 154-169; Howard PW et al., Bioorg Med Chem Lett 2009; 19: 6463-6466 and Sagnou et al., Bioorg Med Chem Lett 2000; 10(18): 2083-2086.

[0594] In another embodiment, the anti-CD39 antibody is conjugated to a cytotoxic moiety selected from the group consisting of anthracyclines, maytansine, calicheamicin, duocarmycin, razimicin (CC-1065), Aplysia 10, Aplysia 15, irinotecan, monomethyl auristatin E, monomethyl auristatin F, PDB, or any analog, derivative, or prodrug thereof.

[0595] In a specific embodiment, the anti-CD39 antibody is conjugated to an anthracycline or an analog, derivative or prodrug thereof. In another specific embodiment, the antibody is conjugated to maytansine or an analog, derivative or prodrug thereof. In another specific embodiment, the antibody is conjugated to calicheamicin or an analog, derivative or prodrug thereof. In another specific embodiment, the antibody is conjugated to duocarmycin or an analog, derivative or prodrug thereof. In another specific embodiment, the antibody is conjugated to lacrimycin (CC-1065) or an analog, derivative or prodrug thereof. In another specific embodiment, the antibody is conjugated to Aplysia 10 or an analog, derivative or prodrug thereof. In another specific embodiment, the antibody is conjugated to Aplysia 15 or an analog, derivative or prodrug thereof. In another specific embodiment, the antibody is conjugated to monomethyl auristatin E or an analog, derivative or prodrug thereof. In another specific embodiment, the antibody is conjugated to monomethyl auristatin F or an analog, derivative or prodrug thereof. In another specific embodiment, the antibody is conjugated to a pyrrolo[2,1-c][1,4]-benzodiazepine or an analog, derivative or prodrug thereof. In another specific embodiment, the antibody is conjugated to irinotecan or an analog, derivative or prodrug thereof.

[0596] In one embodiment, the anti-CD39 antibodies encompassed by the present invention are conjugated to nucleic acids or nucleic acid-related molecules. In one such embodiment, the conjugated nucleic acid is a cytotoxic ribonuclease (RNase) or a deoxyribonuclease (e.g., DNase I), an antisense nucleic acid, an inhibitory RNA molecule (e.g., an siRNA molecule), or an immunostimulatory nucleic acid (e.g., a DNA molecule containing an immunostimulatory CpG motif). In another embodiment, the CD39-specific antibodies encompassed by the present invention are conjugated to an aptamer or a ribozyme.

[0597] In one embodiment, the anti-CD39 antibodies encompassed by the invention are conjugated to a lytic peptide (such as CLIP, Magainin 2, melittin, Cecropin, and P18), for example, as a fusion protein.

[0598] In one embodiment, the anti-CD39 antibody is conjugated to a cytokine, such as, for example, IL-2, IL-4, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, IL-18, IL-23, IL-24, IL-27, IL-28a, IL-28b, IL-29, KGF, IFNα, IFNβ, IFNγ, GM-CSF, CD40L, Flt3 ligand, stem cell factor, ancestim, and TNFα.

[0599] In certain embodiments, the chemical moiety is a polymer that increases the half-life of the antibody or fragment in a subject. Suitable polymers include, but are not limited to, hydrophilic polymers, which include, but are not limited to, polyethylene glycol (PEG) (e.g., PEG with a molecular weight of 2 kDa, 5 kDa, 10 kDa, 12 kDa, 20 kDa, 30 kDa, or 40 kDa), dextran, and monomethoxy polyethylene glycol (mPEG). Lee et al. (1999) (Bioconj. Chem. 10: 973-981) disclose PEG-conjugated single-chain antibodies. Wen et al. (2001) (Bioconj. Chem. 12: 545-553) disclose conjugating antibodies to PEG attached to a radioactive metal chelator (diethylene triamino pentaacetic acid (DTPA)).

[0600] Anti-CD39 antibodies can also be conjugated to labels such as 99Tc, 90Y, 111In, 32P, 14C, 1251, 3H, 131I, 11C, 15O, 13N, 18F, 35S, 51Cr, 57To, 226Ra, 60Co, 59Fe, 57Se, 152Eu, 67CU, 217Ci, 211At, 212Pb, 47Sc, 109Pd, 234Th, 40K, 157Gd, 55Mn, 52Tr and 56Fe.

[0601] Anti-CD39 antibodies can also be conjugated to fluorescent or chemiluminescent labels, including fluorophores such as rare earth chelates, fluorescein and its d...

Claims

1. An anti-CD39 antibody or an antigen-binding fragment thereof, comprising: (i) a light chain variable region comprising a complementarity determining region (CDR) L1 consisting of the amino acid sequence of SEQ ID No. 32, a CDR L2 consisting of the amino acid sequence of SEQ ID No. 33, and a CDR L3 consisting of the amino acid sequence of SEQ ID No. 34, and (ii) a heavy chain variable region comprising a CDR H1 consisting of the amino acid sequence of SEQ ID No.29, a CDR H2 consisting of the amino acid sequence of SEQ ID No.30, and a CDR H3 consisting of the amino acid sequence of SEQ ID No.

31.

2. The anti-CD39 antibody or antigen-binding fragment thereof according to claim 1, wherein the anti-CD39 antibody or antigen-binding fragment thereof comprises: a) a light chain variable region comprising the amino acid sequence of SEQ ID No. 4; and b) a heavy chain variable region comprising the amino acid sequence of SEQ ID No.

2.

3. The anti-CD39 antibody or antigen-binding fragment thereof according to claim 1, wherein the anti-CD39 antibody or antigen-binding fragment thereof comprises: a) a light chain comprising the amino acid sequence of SEQ ID No. 38; and b) a heavy chain comprising the amino acid sequence of SEQ ID No.

36.

4. The anti-CD39 antibody or antigen-binding fragment thereof according to claim 1, further comprising an FcγRIIIa binding portion, which binds to the FcγRIIIa receptor and confers antibody-dependent cellular cytotoxicity (ADCC) activity of the anti-CD39 antibody against CD39+ cells.

5. The anti-CD39 antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, wherein the anti-CD39 antibody or antigen-binding fragment thereof promotes: (i) stable immune complex formation when incubated with HCC1739BL cells, characterized by less than 30% loss of the immune complex after 24 hours; (ii) complement-dependent cytotoxicity (CDC) activity against CD39+ cells; (iii) antibody-mediated targeted endocytosis of CD39 on CD45+ immune cells; (iv) Antibody-mediated targeted endocytosis of CD39 from tumors with disrupted tumor vascular endothelium or collapsed vascular distribution network. 6 . The anti-CD39 antibody or antigen-binding fragment thereof according to claim 5 , wherein the immune complex formation is detected by fluorescence intensity using a fluorescently labeled secondary antibody.

7. The anti-CD39 antibody or antigen-binding fragment thereof according to claim 4, wherein the FcγRIIIa binding moiety is selected from the group consisting of an Fc domain, an antibody or fragment thereof that binds to FcγRIIIa, and an FcγRIIIa binding peptide.

8. An anti-CD39 antibody or antigen-binding fragment thereof as described in any one of claims 1 to 4, comprising at least one antigen-binding domain, and the antigen-binding domain is selected from the group consisting of: Fab, Fab', F(ab')2, Fv, dsFv, sc(Fv)2 and a bifunctional antibody fragment, and / or wherein the anti-CD39 antibody or antigen-binding fragment is monoclonal. 9 . The anti-CD39 antibody or antigen-binding fragment thereof according to claim 8 , wherein the Fv is a single-chain Fv (scFv). 10 . The anti-CD39 antibody or antigen-binding fragment thereof according to claim 9 , wherein the amino acid sequence of the scFv is shown in SEQ ID No.

40.

11. The anti-CD39 antibody or antigen-binding fragment thereof of any one of claims 1 to 4, wherein the anti-CD39 antibody or antigen-binding fragment thereof is conjugated to a binding protein, an enzyme, a drug, a chemotherapeutic agent, a biological agent, a toxin, a radionuclide, an immunomodulator, a detectable moiety or a label. 12 . The anti-CD39 antibody or antigen-binding fragment thereof according to claim 1 , wherein the anti-CD39 antibody or antigen-binding fragment thereof comprises an Fc domain of the IgG1 or IgG3 isotype.

13. The anti-CD39 antibody or antigen-binding fragment thereof of claim 12, wherein the Fc domain is human. 14 . The anti-CD39 antibody or antigen-binding fragment thereof according to claim 1 , wherein the anti-CD39 antibody or antigen-binding fragment thereof is hypofucosylated or afucosylated.

15. The anti-CD39 antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, wherein the anti-CD39 antibody or antigen-binding fragment thereof is human or humanized.

16. An anti-CD39 antibody or antigen-binding fragment thereof as described in any one of claims 1 to 4, wherein the anti-CD39 antibody or antigen-binding fragment thereof is bispecific, comprising at least one additional antigen binding site for a tumor antigen, an immune checkpoint or a co-stimulatory receptor, wherein if the additional antigen binding site is for an immune checkpoint, it is used as a checkpoint inhibitor, and wherein if the additional antigen binding site is for a co-stimulatory receptor, it is used as a co-stimulatory agonist.

17. The anti-CD39 antibody or antigen-binding fragment thereof of claim 16, wherein the additional antigen-binding site binds to one or more of: i) a checkpoint protein selected from the group consisting of PD-1, PD-L1, CTLA-4, B7-1, B7-2, PD-L2, NKG2A, KIR, LAG-3, TIM-3, CD96, VISTA, TIGIT, and Siglec-15; ii) checkpoint proteins that are upregulated on T cells and associated with T cell exhaustion; iii) an immune co-stimulatory receptor selected from the group consisting of: MHC I molecules, BTLA receptor, OX40, CD27, CD28, CDS, ICAM-1, LFA-1, CD11a, CD18, ICOS (CD278) and 4-1BB (CD137); or iv) CD47, SIRPα, CD24 or Siglec-10.

18. A pharmaceutical preparation comprising a therapeutically effective amount of at least one anti-CD39 antibody or antigen-binding fragment thereof according to any one of claims 1 to 17, and one or more pharmaceutically acceptable excipients or solutions.

19. The pharmaceutical formulation of claim 18, wherein the excipient is a buffer.

20. A use of an anti-CD39 antibody or an antigen-binding fragment thereof as described in any one of claims 1 to 17 or a pharmaceutical preparation as described in claim 18 in the preparation of a medicament for improving anti-tumor T cell immunity in a subject with a tumor, and suitable for administration to a subject with a tumor, the pharmaceutical preparation comprising an effective amount of the anti-CD39 antibody or an antigen-binding fragment thereof and one or more pharmaceutically acceptable excipients, buffers or solutions, wherein administration of the anti-CD39 antibody to the subject reduces the number of CD39 high cells in the tumor, enhances T cell infiltration into the tumor, and / or reduces T cell exhaustion in the tumor.

21. An isolated nucleic acid molecule encoding the anti-CD39 antibody or antigen-binding fragment thereof according to any one of claims 1 to 17.

22. A vector comprising the isolated nucleic acid of claim 21.

23. The vector of claim 22, wherein the vector is an expression vector.

24. A host cell comprising the isolated nucleic acid of claim 21, wherein: a) expressing the anti-CD39 antibody or antigen-binding fragment thereof according to any one of claims 1 to 17; b) expressing the heavy and light chain polypeptides of the anti-CD39 antibody or antigen-binding fragment thereof according to any one of claims 1 to 17; and / or c) comprising the vector according to claim 22.

25. A device or kit comprising at least one anti-CD39 antibody or antigen-binding fragment thereof according to any one of claims 1 to 17.

26. The device or kit of claim 25, comprising a label to detect at least one anti-CD39 antibody or antigen-binding fragment thereof, or a complex comprising said anti-CD39 antibody or antigen-binding fragment thereof.

27. A device or kit comprising the pharmaceutical formulation of any one of claims 18 to 24, an isolated nucleic acid molecule, an isolated heavy chain and light chain polypeptide comprising the anti-CD39 antibody or antigen-binding fragment thereof of claims 1-17, a vector and / or a host cell.

28. A method for producing at least one anti-CD39 antibody or antigen-binding fragment thereof according to any one of claims 1 to 17, the method comprising the following steps: (i) culturing a transformed host cell that has been transformed with a nucleic acid comprising a sequence encoding at least one anti-CD39 antibody or antigen-binding fragment thereof under conditions suitable for allowing expression of the anti-CD39 antibody or antigen-binding fragment thereof; and (ii) recovering the expressed anti-CD39 antibody or antigen-binding fragment thereof.

29. Use of at least one anti-CD39 antibody or antigen-binding fragment thereof according to any one of claims 1 to 17 in the preparation of a medicament for detecting the presence or concentration of a CD39 polypeptide, wherein the detection comprises obtaining a sample, and detecting the polypeptide in the sample by using the medicament.

30. The use according to claim 29, wherein at least one anti-CD39 antibody or antigen-binding fragment thereof forms a complex with the CD39 polypeptide, and the complex is detected in the form of enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunochemical assay, Western blot, mass spectrometry, nuclear magnetic resonance assay, or using a cell-intracellular flow assay.

31. A pharmaceutical composition of an anti-CD39 antibody or antigen-binding fragment thereof according to any one of claims 1 to 17 for use in the preparation of a pharmaceutical composition for use in a subject with a tumor by depleting CD39 in the tumor. 高 The invention relates to a drug for improving anti-tumor T cell immunity by targeting cells, wherein the improvement comprises administering the drug to a subject with a tumor, wherein the administration of the drug results in the inhibition of CD39 in the tumor. 高 The invention relates to reducing the number of T cells and enhancing T cell infiltration into the tumor and / or reducing T cell exhaustion in the tumor, or both.

32. Use of a pharmaceutical composition of an anti-CD39 antibody or an antigen-binding fragment thereof as described in any one of claims 1 to 17 in the preparation of a medicament for promoting the infiltration of immune cells into a tumor in a subject suffering from a tumor, wherein the promotion comprises administering the medicament to the subject suffering from the tumor, wherein the administration of the medicament reduces the number of CD39+CD45-SCA-1+ stromal cells in the tumor, and / or increases the infiltration of cytotoxic T cells into the tumor.

33. Use of a pharmaceutical composition of an anti-CD39 antibody or antigen-binding fragment thereof as described in any one of claims 1 to 17 in the preparation of a medicament for reducing the inhibition of the function of intratumoral immune cells by type II NKT cells in a subject suffering from a tumor, wherein the reduction comprises administering the medicament to a subject suffering from a tumor, wherein administration of the medicament reduces the number of type II NKT cells in the tumor.

34. Use of a pharmaceutical composition of an anti-CD39 antibody or antigen-binding fragment thereof according to any one of claims 1 to 17 in the preparation of a medicament for reducing the inhibition of intratumoral immune cell function by regulatory T cells (Treg) in a subject with a tumor, wherein the reduction comprises administering the medicament to a subject with a tumor, wherein the administration of the medicament results in the inhibition of CD39 in the tumor. 高 The number of Tregs decreased.

35. Use of a pharmaceutical composition of an anti-CD39 antibody or antigen-binding fragment thereof according to any one of claims 1 to 17 in the preparation of a medicament for reducing the inhibition of tumor-associated macrophages (TAMs) on the function of intratumoral immune cells in a subject with a tumor, wherein the reduction comprises administering the medicament to a subject with a tumor, wherein, Administration of the drug causes CD39 in the tumor to 高 The number of macrophages decreases.

36. Use of an anti-CD39 antibody or antigen-binding fragment thereof according to any one of claims 1 to 17 in the preparation of a medicament for promoting an anti-tumor immune response in a subject with a tumor, wherein the promotion comprises administering to the subject with the tumor an amount of the medicament sufficient to reduce the number of CD39-expressing cells in the tumor.

37. Use of the anti-CD39 antibody or antigen-binding fragment thereof according to any one of claims 1 to 17 in the preparation of a medicament for promoting T cell-mediated immune function in a tumor of a subject.

38. The use of claim 37, wherein said promoting comprises: (i) identifying cancer subjects having a tumor-infiltrating tumor-reactive lymphocyte level below a predetermined threshold to characterize a non-infiltrated or under-infiltrated tumor phenotype; and (ii) The drug increases the amount of tumor-reactive T cells infiltrating the tumor.

39. The use of claim 31, wherein the intratumoral CD39 high cells are selected from hematopoietic stem cells or progenitor cells CD45-Sca-1+, CD39+NKT cells, CD39+ macrophages, CD39+ cancer cells, CD39+ endothelial cells, or a combination thereof.

40. The use of claim 31, wherein the anti-CD39 antibody or antigen-binding fragment thereof reduces the level of CD39 high cells present in one or more hematopoietic compartments.

41. The use of claim 40, wherein the one or more hematopoietic compartments are selected from the group consisting of blood, spleen and liver.

42. Use of the anti-CD39 antibody or antigen-binding fragment thereof according to any one of claims 1 to 17 in the preparation of a medicament for treating tumors.

43. Use of the anti-CD39 antibody or antigen-binding fragment thereof according to any one of claims 1 to 17 in the preparation of a medicament for treating infection.

44. The use according to claim 43, wherein the infection is a viral infection, a Mycobacterium tuberculosis infection or a visceral leishmaniasis.

45. The use of claim 44, wherein the viral infection is HIV infection, HBV infection or COVID-19 infection.

46. ​​The use of claim 42, wherein the medicament is for treating solid tumors.

47. The method of claim 46, wherein the solid tumor is pancreatic cancer, liver cancer, lung cancer, gastric cancer, esophageal cancer, head and neck squamous cell carcinoma, prostate cancer, colon cancer, breast cancer, lymphoma, gallbladder cancer, kidney cancer, multiple myeloma, ovarian cancer, cervical cancer or glioma.

48. The use of claim 42, wherein the medicament is for treating liquid tumors.

49. The use according to claim 48, wherein the liquid tumor is leukemia.

50. The use of claim 42, wherein the medicament is used in combination with one or more chemotherapeutic agents, anti-angiogenic agents, immuno-oncology agents and / or radiation.

51. The use of claim 42, wherein the medicament is used in combination with one or more inhibitors of one or more checkpoint molecules.

52. The use of claim 51, wherein the one or more checkpoint molecules are selected from the group consisting of: a PD-1 antagonist, a CTLA-4 antagonist, a LAG-3 antagonist, a TIM-3 antagonist, a TIGIT antagonist, and a Siglec-15 antagonist.

53. The use of claim 42, wherein the medicament is used in combination with one or more activators of one or more co-stimulatory molecules.

54. The use of claim 53, wherein the one or more co-stimulatory molecules are selected from the group consisting of a GITR agonist, a CD27 agonist, a 4-1BB agonist, an OX40 agonist, a CD137 agonist, an ICOS agonist, and a CD28 agonist.

55. The use of claim 42, wherein the medicament is used in combination with one or more of the following: a VEGFR or VEGF antagonist, an EGFR or EGF antagonist, an IDO inhibitor, an IDO1 inhibitor, an HDAC inhibitor, a PI3Kδ inhibitor, an IL-15 agonist, a CXCR4 antagonist, a CXCL12 antagonist, a DNMT inhibitor, interleukin-21, an anti-KIR antibody, an anti-CSF-1R antibody, an anti-CCR4 antibody, GMCSF, an anti-PS antibody, an anti-CD30 antibody-auristatin E conjugate, an anti-CD19 antibody, an anti-CEAIL-2 antibody, an anti-NY-ESO-1 antibody, an anti-NKG2A antibody, a STING agonist, a TRL7 / 8 agonist, a RIG-1 agonist and / or an NRLP3 inhibitor, an anti-CD73 antibody, a P2X7 antagonist or an adenosine A2A receptor antagonist.

56. The use of claim 55, wherein the anti-CD73 antibody is MEDI9447.

57. The use of claim 42, wherein the medicament is used in combination with one or more innate immunity inducers.

58. The use of claim 57, wherein the one or more innate immunity inducers are selected from the group consisting of inhibitors of the CD47-SIRPα axis, inhibitors of the CD24-Siglec-10 axis, NGK2A checkpoint inhibitors that block HLA-E driven inhibition of NK and CD8+ cells, STING agonists, TLR7 / 8 agonists, and RIG-I agonists.

59. The use of claim 42, wherein the medicament is used in combination with one or more therapies selected from the group consisting of tumor vaccines, adoptive cell therapy, anti-tumor gene therapy, inhibitory nucleic acid therapy, and oncolytic virus therapy.

60. The use of claim 20, wherein the subject is an animal model of cancer.

61. The use of claim 20, wherein the subject is a mammal.

62. The use of claim 61, wherein the mammal is a human or a rodent.

63. The use of any one of claims 31-41, wherein the subject is an animal model for cancer.

64. The use of any one of claims 31-41, wherein the subject is a mammal.

65. The use of claim 64, wherein the mammal is a human or a rodent.

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