Methods and compositions for promoting and enhancing T cell-mediated immune responses through ADCC targeting of CD39 expressing cells

Abstract

In combination with conventional therapies (e.g., targeted therapy, chemotherapy, and angiogenesis inhibitors, etc.), immunotherapy targeting checkpoint molecules has shown promise in treating solid or liquid tumors. However, the role of non-tumor cells in the intratumoral microenvironment has indicated that the elimination of these cells may be key to generating an effective immune response against the tumor, including tumor infiltration of cytotoxic T cells and other anti-tumor cells of the immune system. The present invention does not focus on attempting to inhibit the ectonucleotidase activity of CD39 as an enzyme that produces adenosine, but rather utilizes CD39 expression to achieve intratumoral cell elimination through 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 August 12, 2019; the entire contents of which are incorporated herein by reference in their entirety. Background of the Invention

[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 has shown remarkable promise, stalling tumors and stopping their growth, and allowing some people who receive treatment to achieve near-complete cures. But these groundbreaking therapies come with numerous 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) in advanced cancer, a significant proportion of patients still do not respond to these treatments.

[0005] With increasing attention on 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 based on the lack of effective response to checkpoint monotherapy. Immunologically "hot" tumors contain high levels of infiltrating T cells and more antigens, making them easier for the immune system to recognize and more likely to trigger a strong immune response. Cancers that are considered hot immunologically are bladder cancer, head and neck cancer, kidney cancer, melanoma, and non-small cell lung cancer. However, even among these immunologically "hot" cancers, only a few patients benefit from immunotherapy. In contrast, immunologically "cold" tumors are cancers that, for various reasons, contain almost no infiltrating T cells, do not seem to be recognized as foreign and do not provoke a strong response from the immune system, making these cancers difficult to treat with current immunotherapies. Traditionally, immunologically "cold" cancers include glioblastoma, as well as ovarian cancer, prostate cancer, pancreatic cancer, and most breast cancers.

[0006] In addition to cancer cells, the tumor microenvironment also contains many cell types, including bone marrow-derived inflammatory cells, lymphocytes, blood vessels, fibroblasts, and an 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 combine to respond to anticancer treatment. In many cases, whether the tumor has T cell infiltration or lacks T cell infiltration, resistance to checkpoint therapy or no response is the result of the inhibitory effect of other cells present in the tumor, which ranges from causing downregulation of cytotoxic T cells already present in the tumor or inhibiting intratumoral signaling to developing a tumor microenvironment that completely excludes cytotoxic T cells by reducing the ability of those cells to infiltrate the tumor from surrounding blood vessels.

[0007] Therefore, there is a great need in the art to identify alternative mechanisms for enhancing T cell responses. Summary of the Invention

[0008] The ectonucleotidase CD39 has been targeted to produce a reduction in intratumoral levels of the enzymatic activity associated with the protein, and thus to 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 for elimination in tumors using certain antibody-dependent cellular cytotoxicity (ADCC)-active anti-CD39 antibodies can be used to increase the infiltration of cytotoxic T cells and, in effect, convert "cold" tumors to immunologically "hot" tumors.

[0009] For example, in one aspect, an anti-CD39 antibody or antigen-binding fragment thereof is provided, comprising (i) at least one antigen-binding domain that binds ectonucleoside triphosphate diphosphohydrolase-1 (CD39) at a 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.

[0010] A number of 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)

[0014] The present invention provides a method for the present invention to provide ... Figure 33 and / or (vi) binds to CD39 in a manner that non-competitively or only partially competes with monoclonal antibody clone A1 for binding to CD39.

[0011] 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 that can be hybridized to the nucleic acid of SEQ ID No. 1 under stringent conditions, or an amino acid sequence encoded by a nucleic acid; and a VL domain having an amino acid sequence that can be hybridized to the nucleic acid of SEQ ID No. 3 under stringent conditions (such as hybridization in 6x sodium chloride / sodium citrate (SSC) at 45° C. and washing in 0.2x SSC / 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 CDRs that are 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 CDRs of SEQ ID No. 2, 6, 10, 14, 18, 22, 26, 42, 46, 50 or 54; and a light chain having CDRs that are 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 CDRs 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 greater) 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. %, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater) of 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, 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) to SEQ ID No. 33 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 the 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, 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. 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.

[0012] In another aspect, a pharmaceutical formulation 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 formulation can be used to improve anti-tumor T cell immunity and is suitable for administration to a subject having 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 results in the inhibition of CD39 in the tumor. 高 The number of cells is reduced and either T cell infiltration into the tumor is enhanced or T cell exhaustion in the tumor is reduced, or both.

[0013] In yet another aspect, an isolated nucleic acid molecule is provided that i) hybridizes under stringent conditions to 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.

[0014] In yet another aspect, an isolated immunoglobulin heavy and / or light chain polypeptide encoded by a nucleic acid described herein is provided.

[0015] In another aspect, provided is a vector comprising the isolated nucleic acid described herein, optionally wherein the vector is an expression vector.

[0016] In yet another aspect, a host cell comprising the isolated nucleic acid described herein is provided, wherein the host cell: a) expresses an anti-CD39 antibody or antigen-binding fragment thereof described herein; b) comprises an immunoglobulin heavy chain and / or light chain polypeptide described herein; or c) comprises a vector described herein.

[0017] In yet another aspect, a device or kit is provided, comprising at least one anti-CD39 antibody or antigen-binding fragment thereof as described herein, optionally comprising 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.

[0018] 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 described herein.

[0019] In yet another aspect, a method of producing an anti-CD39 antibody or antigen-binding fragment thereof, the method 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.

[0020] 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 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 an intracellular flow assay.

[0021] 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 as described herein to a subject having a tumor, wherein administration of the anti-CD39 antibody or antigen-binding fragment thereof results in the inhibition of intratumoral CD39 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.

[0022] In yet another aspect, a method for promoting the infiltration of immune cells into a tumor is provided, 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 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.

[0023] In yet another aspect, a method for reducing the suppression of intratumoral immune cell function by type II NKT 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 administration of the anti-CD39 antibody or antigen-binding fragment thereof results in the elimination and reduction of type II NKT cells in the tumor.

[0024] 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 antigen-binding fragment thereof as described herein, wherein the administration of the anti-CD39 antibody or antigen-binding fragment thereof results in the suppression of CD39 in the tumor. 高 Treg elimination and reduction.

[0025] In yet another aspect, a method for reducing the suppression of intratumoral immune cell function by tumor-associated macrophages (TAMs) 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 antigen-binding fragment thereof as described herein, wherein 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.

[0026] In yet another aspect, a method for promoting an anti-tumor immune response is provided, 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.

[0027] In yet another aspect, a method for promoting T cell-mediated immune function in a tumor in a subject is provided, the method comprising: (i) identifying a cancer subject having a level of tumor-infiltrating 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 cell infiltration into the tumor.

[0028] As described above, further embodiments are 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, intratumoral CD39 高 The cells are selected from hematopoietic stem 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 expression of CD39 in one or more hematopoietic compartments. 高In another embodiment, the anti-CD39 antibody or its antigen-binding fragment is administered as a part of anti-tumor therapy, wherein the anti-infection therapy is antiviral therapy (including HIV and HBV infection and COVID-19 infection treatment), for the treatment of Mycobacterium tuberculosis (Mycobacterium tuberculosis) and for the treatment of visceral leishmaniasis. In another embodiment, the anti-CD39 antibody or its antigen-binding fragment is administered as a part of anti-tumor therapy for the treatment of solid tumors, 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 liquid tumors, optionally wherein the liquid tumor is 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

[0029] 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, generated by transient transfection, was serially diluted as indicated and incubated with HCC1739BL cells at 4°C for 30 minutes, followed by flow cytometric analysis. Kd was calculated to be 0.412 nM.

[0030] Figure 2Ig39-21 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 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 was indicated by an increase in luciferase activity over background. RLU: relative luminescence unit. EC50 was calculated to be 0.014 μg / mL.

[0031] Figure 3 .Ig39-21 demonstrated ADCC activity selectively against human CD39hi Raji cells: Luc-reporter gene assay. Various Raji cell lines with different human CD39 expression levels, including Raji cells (Raji-hCD39neg), hCD39-transfected Raji cells (Raji-hCD39hi) that highly express human CD39, or hCD39-transfected Raji cells (Raji-hCD39lo) that express low levels of human CD39 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 was indicated by the increase in luciferase activity over background (RLU). RLU: relative luminescence unit.

[0032] 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 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 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.

[0033] Figure 5CD39 confers resistance to NK cytotoxicity on target Raji-hCD39hi cells. CFSE-labeled Raji-hCD39neg or Raji-hCD39hi 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 using propidium iodide (P / I) uptake. Results are expressed as CFSE + P / I + % of cells.

[0034] Figure 6 Ig39-21 enhances NK cell cytotoxicity against Raji-hCD39hi cells. CFSE-labeled Raji-hCD39hi 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 expression was calculated. + P / I + % of cells.

[0035] Figure 7 Ig39-21 enhances NK cell cytotoxicity against hCD39hi human B lymphoblastoid cells (HCC1739BL). 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 expression was calculated. + P / I + % of cells.

[0036] Figure 8 Absence of fucosylation enhances Ig39-21-mediated ADCC against HCC1739BL cells: Luc-reporter assay. HCC1739BL target cells were preincubated with serially diluted Ig39-21 produced by transient transfection for 30 minutes at 37°C in the absence (Ig39-21 WT) or presence of a fucosylation inhibitor (Ig39-21 AF) and further co-cultured with Jurkat 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 0.02 μg / mL (Ig39-21 WT) and 0.0008 μg / mL (Ig39-21 AF).

[0037] Figure 9 Afucosylation enhances Ig39-21-mediated ADCC against HCC1739BL cells: NK cytotoxicity assay. CFSE-labeled HCC1739BL target cells were incubated with serially diluted Ig39-21 WT or Ig39-21 AF 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 expression was calculated. + P / I + % of cells (% of cytotoxicity). EC50 was calculated to be 0.04 μg / mL (Ig39-21 WT) and 0.0006 μg / mL (Ig39-21 AF).

[0038] Figure 10 Optimized Ig39-21 (NP501-BK), Ig39-21 WT, and Ig39-21 AF exhibited similar binding affinities using human CD39-positive CHO cells (CHO-hCD39). Ig39-21 WT, Ig39-21 AF, 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) and were stained 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 (Ig39-21 WT), and 0.51 nM (Ig39-21 AF).

[0039] Figure 11 NP501-BK, Ig39-21 WT, and Ig39-21 AF exhibited similar binding affinities using CD39-expressing HCC1739BL cells. Ig39-21 WT, Ig39-21 AF, 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 501-485 (Alexa Fluor 501-485) at 4°C. 488) were stained 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 (Ig39-21 WT), and 0.35 nM (Ig39-21 AF).

[0040] Figure 12 Optimized Ig39-21 (NP501-BK) and afucosylated Ig39-21 exhibit similar ADCC activity: A Luc-reporter assay was performed using HCC1739BL cells. HCC1739BL target cells were preincubated with serially diluted human IgG1 isotype control antibody (KLH-hIgG1) or fully human anti-CD39 monoclonal antibodies (Ig39-21 AF 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 was indicated by an increase in luciferase activity over background. RLU: relative luminescence unit. The EC50 was calculated to be 0.0017 μg / mL (NP501-BK) and 0.00086 μg / mL (Ig39-21 AF).

[0041] Figure 13 .NP501-BK and Ig39-21 AF exert much higher ADCC activity than Ig39-21 WT: NK cell 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 (Ig39-21 WT, Ig39-21 AF or NP501-BK) 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 1.58E-04 μg / mL (NP501-BK), 4.22E-03 μg / mL (Ig39-21 WT), and 4.75E-05 μg / mL (Ig39-21 AF).

[0042] Figure 14Fully human anti-CD39 antibodies exhibited similar CDC activity against Raji-hCD39hi cells. Raji-hCD39hi target cells were preincubated with serially diluted human IgG1 isotype control (KLH-hIgG1) or fully human anti-CD39 monoclonal antibodies (Ig39-21WT, Ig39-21 AF, or NP501-BK) for 30 minutes at 37°C 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 (Ig39-21 WT), and 0.25 μg / mL (Ig39-21 AF).

[0043] Figure 15 .NP501-BK and Ig39-21 AF 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 (Ig39-21 AF or NP501-BK) on days 8, 11, 14, and 17 after tumor challenge. Tumor length (L) and width (W) were measured twice a week using a digital caliper. Tumor volume (mm 3 ) was determined to be L*W*W*0.52. n=5 per group.

[0044] Figure 16 NP501-BK treatment reduces hCD39 expression on CD39hi tumor-infiltrating lymphocytes. MC38 tumor-bearing hCD39 KI mice were treated with three doses of KLH-hIgG1 (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.

[0045] Figure 17 NP501-BK exerts antitumor activity in a 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. 3When the mean volume of the tumor was 100 μg / kg (considered as day 0 of treatment), mice were treated intraperitoneally with two doses of 300 μl saline or 10 mg / kg NP501-BK 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.

[0046] Figure 18 Epitope competition assay of 18 human / rabbit chimeric anti-human CD39 monoclonal antibodies against the reference anti-hCD39 monoclonal antibody clone A1 on HCC1739BL cells. HCC1739BL cells were incubated 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 PE-conjugated mouse anti-hCD39 monoclonal antibody clone A1 for 30 minutes at 4°C, followed by flow cytometric analysis. Cells incubated without chimeric antibodies served as controls.

[0047] Figure 19 Human / rabbit chimeric clones 9B6 and Ig39-21 exhibit 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 preincubated with serially diluted Ig39-21 WT or human / rabbit chimeric clone 9B6 (Hu / Ra 9B6; among all chimeric clones, the rabbit anti-hCD39 monoclonal antibody chimeric with human IgG1 Fc had 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 was 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 / Ra9B6).

[0048] Figure 20.ADCC activity of 18 human / rabbit chimeric antibodies: Luc-reporter gene 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 at 37°C for 30 minutes and further co-cultured with Jurkat effector cells (T:E=1:6) for 6 hours. ADCC activity was indicated by the increase (RLU) in luciferase activity over background. RLU: relative luminescence unit. 10 of the 18 clones showed positive ADCC activity.

[0049] Figure 21 Human / rabbit chimeric antibody with high ADCC activity: Luc-reporter gene assay using HCC1739BL cells. Luc-reporter gene ADCC assay as Figure 20 The 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 Figure 18 ).

[0050] Figure 22 Human / rabbit chimeric clone with low ADCC activity: Luc-reporter gene assay using HCC1739BL cells. Luc-reporter gene ADCC assay as Figure 20 Four clones with low ADCC activity were pooled, all of which competed with the epitope of clone A1 (see Figure 18 ). 9B6 served as a positive control.

[0051] Figure 23 Human / rabbit chimeric antibody without ADCC activity: Luc-reporter gene assay using HCC1739BL cells. Luc-reporter gene ADCC assay as Figure 20 Eight clones without ADCC activity were collected, all of which fully competed with the epitope of clone A1 (see Figure 18 ). 9B6 served as a positive control.

[0052] Figure 24 .Select human / rabbit chimeric antibodies for NK cell cytotoxicity against HCC1739BL cells. CFSE-labeled HCC1739BL target cells were incubated with serially diluted human / rabbit chimeric clones as indicated for 30 minutes at 37°C in 5% CO2. 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 percentage of cells (% of cytotoxicity) is shown. The calculated EC50 is listed. The % of maximum cytotoxicity over background was determined as: % of maximum cytotoxicity at 1 μg / mL for each clone - % of background cytotoxicity for each clone (at 10 -4 10 μg / mL). Exemplary chimeric clones from the luc-reporter high ADCC group (8C11, 8D8, 9B6, 9C10, 48F10, and 65H5) exhibited high NK killing activity. In contrast, chimeric clones from the luc-reporter ADCC negative group (59B6, 60D9, 62G12, and 62H10) displayed low NK killing activity.

[0053] Figure 25 Reference anti-human CD39 monoclonal antibody (hCD39 Ref) inhibits 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 (hCD39Ref) 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. 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.

[0054] Figure 26 . The reference antibody (hCD39 Ref) and Ig39-21WT containing the same human IgG1 Fc portion exert similar ADCC activity: NK cell cytotoxicity against HCC1739BL cells. CFSE-labeled HCC1739BL target cells were incubated with serially diluted anti-hCD39 monoclonal antibodies (hCD39 Ref, NP501-BK, Ig39-21 WT, or Ig39-21 AF) as indicated 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 + 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).

[0055] Figure 27Epitope competition of matrix-I 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 Alexa Fluor 500 antibody at 4°C. AF647-conjugated antibodies (8C11, 8D8, 8E9, 9B6, and Ig39-21 WT) or PE-conjugated clone A1 were incubated for 30 minutes, followed by two washes and flow cytometric analysis. The fold change in MFI for AF647 or PE was calculated relative to the isotype control (no epitope overlap = 1). Ra: rabbit antibody; Hu / Ra: human / rabbit chimeric antibody.

[0056] Figure 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 576-114 antibody) 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 the isotype control was calculated (no epitope overlap = 1).

[0057] Figure 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 (anti-human IgG (Fc specific), Alexa Fluor 500 antibody) at 4°C. 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 was calculated as described in Materials and Methods. After 24 hours, exemplary chimeric clones from the luc-reporter gene 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; hIgG1: humanized rabbit antibody, IgG1 isotype; hIgG4: humanized rabbit antibody, IgG4 isotype.

[0058] Figure 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 at 4°C for 30 minutes. The cells were then incubated with secondary antibodies (anti-human IgG (Fc specific), Alexa Fluor 500 antibody, and Alexa Fluor 500 antibody) at 4°C for 30 minutes. 488) were stained for 30 minutes and analyzed by flow cytometry. Kd was calculated and indicated next to the graph.

[0059] Figure 31 NK cell 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.

[0060] Figure 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.

[0061] Figure 33 Conformational epitope mapping. A list of key 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

[0062] I. Overview

[0063] It is known that extracellular adenosine is 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 suppressing immune signal transduction. The immunosuppressive adenosine 3'5'-monophosphate (cAMP)-mediated pathway through adenosine A2A receptor (A2AR) signal transduction 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 ongoing positive clinical trial data, confirms that the administration of A2AR inhibitors may be a potential novel strategy for immunotherapy. In addition, in experimental animal models, blocking the adenosine production pathway involving CD39 / CD73 also induced the 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.

[0064] 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 through 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.

[0065] While 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 unable to form as stable immune complexes with CD39 as those encompassed by the present invention result in a reduction of 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 for binding to CD39.

[0066] As described in more detail in the exemplary methods and illustrated in the accompanying figures, compared 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 the inhibition of CD39 in tumors. 高 Elimination and reduction of cells.

[0067] 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.

[0068] The subject antibodies target CD39+ cells in tumors through FcγRIIIa receptor-dependent activities (eg, ADCC).

[0069] As an example, Figure 13 and Figure 26 showed 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 enhanced antitumor activity of these afucosylated antibodies in vivo ( Figure 15), while the fully glycosylated form (Ig39-21 WT) showed no antitumor activity in the same tumor model (data not shown).

[0070] like Figure 13 and 26 The in vitro maximum effective dose (MaxED) of these differently fucosylated Ig39-21 antibodies predicted by the NK cytotoxicity assay further explains their differential in vivo anti-tumor activity. For example, afucosylation enhanced the MaxED of Ig39-21 WT from 0.1 μg / ml to 0.001 μg / ml. When translated into the clinic, this 100-fold increase would translate into high efficacy, a favorable safety profile, good tolerability, and low cost.

[0071] 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 examples, the prior art antibody was generated with an Fc portion specifically designed to have ablated ADCC function (i.e., it was taught to have been specifically generated to bind CD39 and inhibit NTPase activity without causing CD39-dependent ADCC cell killing).

[0072] The ADCC activity of the subject anti-CD39 antibody is selective for CD39 高 cell.

[0073] 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).

[0074] Relevance of these in vitro data to the in vivo tumor microenvironment: Figure 16 hCD39 was confirmed to be highly upregulated in 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 expression of CD39 in tumors. 高 Elimination and reduction of cells ( Figure 16 and data not shown).

[0075] This functional feature should confer tumor specificity to the antibody, thereby avoiding the systemic side effects that should result in safer anti-CD39 antibodies.

[0076] Anti-CD39 antibodies form stable immune complexes with antigens on the target cell membrane, giving the antibodies high ADCC activity.

[0077] As Figure 29 In one example shown in Figure 2, 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.

[0078] Different epitopes of anti-CD39 antibodies are directly related to the ADCC activity of the antibodies.

[0079] 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, Figure 18 and 21 23 showed that anti-CD39 antibodies that bind to 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, five of the six high ADCC antibodies (2G12, 8C11, 8D8, 9B6, and 9C10) except 65H5 showed this feature ( Figure 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 ( Figure 22 and 23 ).

[0080] Multiple CD39 in tumors with the subject anti-CD39 antibody 高 Cellular targets.

[0081] As an example, the CD39-MC38 colorectal cancer model in hCD39 KI mice was used ( Figure 15 and 16 The anti-tumor 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).

[0082] Another example is a xenograft tumor model using NU / J nude mice implanted with CD39+ human SK-MEL-28 melanoma cells ( Figure 17 These homozygous athymic nude mice lack T cells and 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 ).

[0083] II. definition

[0084] To facilitate understanding of the present invention, a number of terms and phrases are defined below.

[0085] "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 nucleoside triphosphates and diphosphates to monophosphate nucleoside derivatives (enzyme entry: EC 3.6.1.5), such as hydrolysis of 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 Uni ProtKB entry "P49961 (ENTP1_homo)", and a representative human coding sequence for the enzyme is provided in GenBank Accession No. S73813. Pericellular adenosine can modulate proinflammatory or proinhibitory signals in immune cells by binding to various adenosine receptors (Ernst 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 the 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). The loss of CD39 in Tregs significantly impairs their ability to suppress T cell activation, suggesting that the juxtacrine activity of CD39 plays a role in negatively regulating T cell function (Deaglio et al. (2007) J. Exp. Med. 204: 1257-1265). + 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).

[0086] 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 as 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, termed 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), which 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).

[0087] 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 is available 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, which results in a shorter isoform, human CD39 isoform 6, available under 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, which results in a shorter isoform, human CD39 isoform 6, also encoded by another transcript variant, available under 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.

[0088] 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).

[0089] 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 decrease 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).

[0090] Modulation (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 to baseline, etc.). For example, a CD39 activity modulator can reduce the catalytic activity of the ectonucleotidase or overall CD39 activity compared to the level of such ectonucleotidase in the presence of a test agent. In one embodiment, CD39 activity is measured 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 being tested and the concentration of the remaining ATP, AMP, or adenosine is measured or assessed. Modulation (e.g., 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.

[0091] "CD39 antibody" (or "anti-CD39 antibody") refers to an antibody that selectively binds to one or more epitopes of the NTPD-1 protein, and includes monoparatopic antibodies, as well as biparatopic antibodies and other multiparatopic antibody formats.

[0092] a. Antibodies and other peptides

[0093] As used herein, the term "antibody" 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 an Fc or other FcγRIII binding domain), multispecific antibodies, bispecific antibodies, monospecific antibodies, monovalent antibodies, chimeric antibodies, humanized antibodies, human antibodies, fusion proteins comprising the antigen binding site of an antibody (which have been formatted to contain an Fc or other FcγRIII binding domain), and any other modified immunoglobulin molecule comprising an antigen binding site, so long as the antibody exhibits the desired biological activity.

[0094] 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.

[0095] 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., the anti-CD39 antibodies described herein) include (i) Fab fragments, which consist of V L 、V H (ii) a F(ab')2 fragment, a bivalent fragment consisting of two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a F(ab')2 fragment consisting of a V H and CH1 domains; (iv) a V fragment consisting of a single arm of the antibody L and V H Fv fragment composed of V HdAb fragments (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 optionally be connected 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.

[0096] 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 region of the heavy and light chains is composed of four framework regions (FR) 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 edition, National Institutes of Health, 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.

[0097] Although antibodies can be of 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), 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 for most efficient engagement of FcγRIII (i.e., at 10 -7 or smaller Kd).

[0098] 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.

[0099] 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 human germline immunoglobulin sequences. 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.

[0100] 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, containing minimal non-human sequences. Typically, humanized antibodies are human immunoglobulins, in which the residues of CDR are replaced by residues from non-human species (e.g., mouse, rat, rabbit, or hamster) with desired specificity, affinity, and / or binding capacity. In some instances, the Fv framework region residues of human immunoglobulin are replaced by corresponding residues from antibodies of 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 capacity. Humanized antibodies may comprise variable domains containing all or substantially all CDRs corresponding to non-human immunoglobulins and all or substantially all framework regions being those of human immunoglobulin sequences. In some embodiments, the variable domains comprise 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.

[0101] 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.

[0102] As used herein, the term "chimeric antibody" 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 those of an antibody derived from one species of mammal (e.g., mouse, rat, rabbit, etc.) with the desired specificity, affinity, and / or binding ability, while the constant regions are homologous to sequences in antibodies derived from another species (usually human) to avoid eliciting an immune response in that species.

[0103] "Fc receptor" or "FcR" is a receptor that binds to the Fc region of an immunoglobulin. The FcR that binds to IgG antibodies comprises the FcγR family of receptors, 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.

[0104] An "FcγRIII binding portion" is a peptide, protein, nucleic acid, or other portion that, when bound to the antigen-binding site of an anti-CD39 antibody, can bind to FcγRIII (CD16) and mediate antibody-dependent cellular cytotoxicity (ADCC). Heavy chain Fc fragments containing the CH2 and CH3 domains of IgG1 and IgG3 isotypes are FcγRIII binding portions.

[0105] 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 contiguous amino acids and non-contiguous amino acids juxtaposed by the tertiary folding of the protein. Epitopes formed by contiguous amino acids (also called linear epitopes) are generally retained upon protein denaturation, while epitopes formed by tertiary folding (also called conformational epitopes) are generally lost upon protein denaturation. 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.

[0106] 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 with greater duration than it binds to other targets. In one embodiment, the extent to which the 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 among proteins from different species. In another embodiment, specific binding may include but need not be exclusive binding.

[0107] 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 an amino acid (including, for example, non-natural amino acids), as well as other modifications known in the art. It will be appreciated 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.

[0108] In the context of two or more nucleic acids or polypeptides, the term "identical" or "identity" percentage refers to that when comparing and aligning (if necessary, introducing gaps) 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. Percent identity 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 is present in a region of an amino acid sequence that is 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 is present 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 total length of the compared sequence, such as the coding region of a target protein or antibody. In some embodiments, the identity is present in a region of a nucleotide sequence that is 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 is present 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 total length of the compared sequence, such as the nucleotide sequence encoding the target protein.

[0109] "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 are 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), beta-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 the binding of the polypeptide, soluble protein, or antibody containing the amino acid sequence to the target binding site. Methods for identifying conservative amino acid substitutions that do not eliminate binding are well known in the art.

[0110] An "isolated" polypeptide, soluble protein, antibody, polynucleotide, vector, cell, or composition is one that is not found in nature. Isolated polypeptides, soluble proteins, antibodies, polynucleotides, vectors, cells, or compositions include those that have been purified to an extent that they are no longer in a form found in nature. In some embodiments, the isolated polypeptide, soluble protein, antibody, polynucleotide, vector, cell, or composition is substantially pure.

[0111] 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.

[0112] 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.

[0113] 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; an scFV, Vhh domain, or the like, which binds to different proteins to produce a bispecific antibody format that maintains bivalency 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.

[0114] b. Nucleic acid

[0115] The terms "polynucleotide," "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 deoxyribonucleotides, 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.

[0116] As used herein, the terms "nucleic acid molecule code," "DNA sequence code," 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.

[0117] As used herein, the term "sequence," when used in reference to a nucleotide sequence, the term, grammatical and other forms, may encompass DNA or RNA, and may be single-stranded or double-stranded. A nucleic acid sequence may be mutated.

[0118] As used herein, the term "vector" refers to 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.

[0119] As used herein, the term "transfection" refers to the introduction 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).

[0120] As used herein, the term "vector" is an isolated nucleic acid comprising an isolated nucleic acid that can be used to deliver a 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 amphipathic 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 (such as 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.

[0121] 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 the 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).

[0122] As used herein, the term "operably linked" refers to a functional linkage between a regulatory sequence and a heterologous nucleic acid sequence connected to a linker that results in expression of the latter. For example, a first nucleic acid sequence and a second nucleic acid sequence are operably linked when they are in a functional relationship. For example, a promoter is operably linked to a coding sequence if it affects the transcription or expression of the coding sequence. Typically, operably linked DNA sequences are contiguous and, where necessary, link two protein coding regions in the same reading frame.

[0123] As used herein, the term "promoter" is defined as a promoter DNA sequence that is recognized or introduced by the synthetic machinery required for the cell-specific transcription of a polynucleotide sequence.

[0124] As used herein, the term "constitutive expression" refers to expression under physiological conditions.

[0125] As used herein, the term "inducible expression" refers to expression under certain conditions, such as that which occurs upon T cell antigen binding. How to become familiar with conventional "inducible expression".

[0126] 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.

[0127] c. Checkpoint inhibitors, co-stimulatory agonists, innate immunity inducers, and chemotherapeutic agents

[0128] "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.

[0129] "Checkpoint inhibitors" refer to pharmaceutical entities that reverse immunosuppressive signaling from checkpoint molecules.

[0130] "Costimulatory molecules" refer to immune cells such as T cell homologous binding partners, which specifically bind to costimulatory ligands, thereby mediating costimulation, 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 costimulatory 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.

[0131] 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.

[0132] "Innate immunity inducers" are agents that simulate innate immune responses (including activation of inflammatory activity and / or deactivation of anti-inflammatory activity of macrophages, NK cells, dendritic cells, monocytes, neutrophils and the like). Innate immunity inducers include inhibitors of the CD47-SIRPα axis, such as antibodies or other binding portions that bind to CD47 or SIRPα 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 portions 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.

[0133] A "chemotherapeutic agent" is a chemical compound that may be useful in treating cancer. Examples of chemotherapeutic agents include alkylating agents, such as thiotepa and cyclophosphamide; alkyl sulfonates, such as busulfan, improsulfan, and piposulfan; aziridines, such as benzodopa, carboquone, meturedopa, and uredopa; ethyleneimines and methylamelamines, including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide, and trimethylolomelamine; polyacetyl groups (especially bullatacin and bullatacinone); delta-9-tetrahydrocannabinol (dronabinol, MARINOL); beta-lapachone; lapachol; colchicine; betulinic acid; acid); camptothecins (including synthetic analogs of topotecan (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; cryptophycins (especially cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including synthetic analogs KW-2189 and CB1-TM1); eleutherobin; pancratistatin; TLK-286; CDP323, an oral alpha-4 integrin inhibitor; sarcodictyin; spongistatin;Nitrogen mustards, such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard, mustard); nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as enediyne antibiotics (e.g., calicheamicins, particularly calicheamicin gamma and calicheamicin omega (see, e.g., Nicolaou et al., Angew. Chem Intl. Ed. Engl. 33:183-186 (1994)); dynemicins, including dynemicin A; esperamicin;and neocarzinostatin chromophore 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-pyrrolino-doxorubicin, doxorubicin HCl liposome injection (DOXIL) and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, 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, and trimetrexate;Purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, and imatinib (a 2-phenylaminopyrimidine derivative) and other c-Kit inhibitors; antiadreners such as aminoglutethimide, mitotane, and trilostane; folic acid supplements such as folinic acid; aceglatone; aldophosphamide glycosides; glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfornithine; elliptinium acetate; etoglucid; gallium nitrate nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids, 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; edatrexate; daunomycin; aminopterin; ibandronate; the topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO); a retinoid, such as retinoic acid; a pharmaceutically acceptable salt, acid, or derivative of any of the foregoing;and combinations 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). ;

[0134] Also included in this definition are antihormonal drugs, which act to regulate, 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 suppress or shut down the ovaries, for example, leucine releasing hormone (LHRH) agonists, such as leuprorelin acetate (LUPRON and ELIGARD), goserelin acetate, buserelin acetate, and leuprorelin acetate. acetate) and triptorelin; antiandrogens such as flutamide, nilutamide, and bicalutamide; and aromatase inhibitors, which 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).Additionally, 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 (a 1,3-dioxolane nucleoside cytosine analog); antisense oligonucleotides, particularly those that inhibit the expression of genes in signaling pathways involved in cell proliferation, such as, for example, PKC-α, Raf, H-Ras, and epidermal growth factor receptor (EGF-R); vaccines, such as TH1 inhibitors. ERATOPE vaccines and gene therapy vaccines, such as ALLOVECTIN vaccine, LEUVECTIN vaccine and VAXID vaccine; 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 (a small molecule inhibitor of ErbB-2 and EGFR dual tyrosine kinases, also known as GW572016); 17AAG (a geldanamycin derivative of heat shock protein (Hsp) 90 poison) and pharmaceutically acceptable salts, acids or derivatives of any of the above.

[0135] As used herein, the term "cytokine" generally refers to a protein released by one cell population that acts as an intercellular mediator on another cell 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 rIL-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").

[0136] As used herein, the term "chemokine" refers to soluble factors (such as cytokines) that have 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 chemoattractant (KC).

[0137] d. Treatment

[0138] 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 then becomes ineffective in controlling infection or tumor growth.

[0139] 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.

[0140] The term "anergic" refers to a response that results from incomplete or insufficient signaling through the T cell receptor (e.g., intracellular Ca +2 T cell anergy can also occur when cells are stimulated with antigen in the absence of co-stimulation, resulting in cells becoming refractory to subsequent activation by the antigen even in the presence of co-stimulation. The anergic state can often be overturned by the presence of interleukin-2. Anergic T cells do not undergo clonal expansion and / or acquire effector functions.

[0141] The term "exhaustion" refers to T cell exhaustion, a state of T cell dysfunction caused by 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 distinct from that of functional effector or memory T cells. Exhaustion prevents optimal control of infection and tumors.

[0142] "Enhancing T cell function" means inducing, causing or stimulating T cells to have a sustained or amplified biological function, or renewing or reactivating exhausted or inactive T cells. Examples of enhancing T cell function include: increased secretion of gamma interferon from CD8+ T cells, increased proliferation, increased antigen reactivity (e.g., virus, pathogen or tumor clearance) relative to such levels before intervention. In one embodiment, the enhanced level is at least 50%, or 60%, 70%, 80%, 90%, 100%, 120%, 150%, 200%. The manner of measuring this enhancement is known to those of ordinary skill in the art.

[0143] A "T cell dysfunction disorder" is a disorder or condition characterized by T cells having 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 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 that express the immunogen. Examples of T cell dysfunction disorders characterized by T cell dysfunction include unresolved acute infections, chronic infections, and tumor immunity.

[0144] "Tumor immunity" refers to the process by which tumors evade immune recognition and clearance. Therefore, as a therapeutic concept, when this evasion is reduced, 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.

[0145] A "sustained response" refers to a persistent effect of reducing tumor growth after treatment has stopped. For example, the tumor size can remain the same or smaller than its size at the start of the administration period. In some embodiments, the sustained response has a duration that is at least as long as the duration of treatment, at least 1.5x, 2.0x, 2.5x, or 3.0x the duration of treatment.

[0146] As used herein, the terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals in which a cell population is characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinomas, blastomas, sarcomas, and hematologic cancers, such as lymphomas and leukemias.

[0147] 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 organs or tissues selected from the group consisting of bladder, bone, 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, vagina 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), essential macroglobulinemia, heavy chain diseases, and solid tumors such as sarcoma cancers (e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, endotheliosarcoma, lymphangiosarcoma, angiosarcoma, lymphangioendothelioma sarcoma, synovioma, mesothelioma, Ewing's tumor, and sarcoma). tumora), leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland cancer, sebaceous gland cancer, papillary carcinoma, papillary adenocarcinoma, carcinoma, bronchogenic 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, and 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.

[0148] As used herein, the term "metastasis" refers to the process by which cancer spreads or metastasizes from its site of origin 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.

[0149] The terms "cancer cell" and "tumor cell" refer to the total population of cells derived from cancer or tumor or precancerous lesions, 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 the ability to renew and differentiate to distinguish those tumor cells from cancer stem cells.

[0150] As used herein, the term "effective amount" refers to an amount that provides a therapeutic or prophylactic benefit.

[0151] 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, using the baseline SLD as the reference; and "stable disease" or "SD" refers to neither a sufficient decrease in target lesions to meet the criteria for a PR nor an increase in target lesions to meet the criteria for a PD, using the smallest SLD since the start of treatment as the reference.

[0152] 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.

[0153] 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 worsen. Progression-free survival can include the amount of time a patient has experienced a complete response or partial response, and the amount of time a patient has experienced stable disease.

[0154] As used herein, "overall response rate" (ORR) refers to the sum of the complete response (CR) rate and the partial response (PR) rate.

[0155] As used herein, "overall survival" refers to the percentage of subjects in a group who are likely to be alive after a specific period of time.

[0156] As used herein, the term "treatment" refers to an attempt by a subject to alter the course or treatment of a clinical disease caused by cellular intervention, which may be a preventive intervention process for clinical pathology. This includes, but is not limited to, treatments that prevent the occurrence or recurrence of a disease, alleviate symptoms, reduce any direct or indirect pathological consequences of a disease, prevent metastasis, slow the rate of disease progression, improve or alleviate the disease, or improve prognosis.

[0157] 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.

[0158] 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 fully induces, activates, promotes, increases, or enhances the activity of a protein or other target of interest.

[0159] As used herein, the terms "antagonist" and "antagonize" 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.

[0160] As used herein, the terms "modulation" and "modulate" refer to a change or alteration in a biological activity. Modulation includes, but is not limited to, stimulating activity or inhibiting activity. Modulation can be an increase or 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.

[0161] As used herein, the term "immune response" includes responses from both the innate and adaptive immune systems. 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, and the like.

[0162] The term "pharmaceutically acceptable" means a substance 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.

[0163] 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 along 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. Generally speaking, those skilled in the art and the U.S. FDA consider a pharmaceutically acceptable excipient, carrier or adjuvant to be an inactive ingredient of any formulation.

[0164] 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, the spread of cancer into soft tissue and bone; inhibit and prevent tumor or cancer cell metastasis; inhibit and prevent tumor or cancer cell growth; alleviate to some extent one or more of the symptoms associated with cancer; reduce morbidity and mortality; improve quality of life; or a combination of these effects.

[0165] The terms "treating" or "treatment" or "to treat" or "alleviating" 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, increased cytolytic activity of immune cells, 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 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.

[0166] e. Miscellaneous

[0167] 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.

[0168] As used herein, reference to "about" or "approximately" a value or parameter includes (and describes) embodiments for that value or parameter. For example, a description referring to "about X" includes a description of "X."

[0169] 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). Similarly, 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).

[0170] III. anti-CD39 antibody

[0171] a. Monoclonal Antibodies

[0172] Anti-CD39 antibodies may be monoclonal antibodies. Monoclonal antibodies can be prepared using hybridoma methods, 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, lymphocytes can be immunized in vitro.

[0173] Immunizing agents generally include CD39 polypeptide or its fusion protein. Generally speaking, if cells of human origin are needed, peripheral blood lymphocytes ("PBL") are used, or if non-human mammalian sources are needed, 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), pp. 59 to 103]. Immortalized cell lines are generally transformed mammalian cells, especially myeloma cells of rodent, cattle and human origin. Typically, rat or mouse myeloma cell lines are used. Hybridoma cells can be cultured in a suitable culture medium, which preferably contains 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 will typically include hypoxanthine, aminopterin, and thymidine ("HAT medium"), which substances prevent the growth of HGPRT-deficient cells.

[0174] Preferred immortalized cell lines are those that efficiently fuse, support stable high expression levels of the antibody by the selected antibody-producing cells, and are sensitive to culture media such as HAT culture media. A more preferred immortalized cell line is a murine myeloma line, 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].

[0175] The culture medium in which the hybridoma cells are cultured can then be analyzed for the presence of monoclonal antibodies against the polypeptide. 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 a radioimmunoassay (RIA) or an 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).

[0176] 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.

[0177] 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.

[0178] 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 isolated, 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 modified, for example, by replacing the homologous murine sequence with the coding sequence 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.

[0179] b. Human and humanized antibodies

[0180] 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 a fragment thereof (such as Fv, Fab, Fab', F(ab')2, or other antigen-binding subsequences of an antibody) that contains minimal sequences derived from a non-human immunoglobulin. Humanized antibodies include human immunoglobulins (recipient antibodies) in which residues from the complementary determining regions (CDRs) of the receptor are replaced with residues from the CDRs of non-human species (donor antibodies) such as mice, rats, or rabbits with the desired specificity, affinity, and capacity. In some instances, the Fv framework residues of the human immunoglobulin are replaced with 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)].

[0181] Methods for humanizing non-human antibodies are well known in the art. Generally speaking, humanized antibodies have one or more amino acid residues of non-human origin introduced therein. These non-human amino acid residues are generally referred to as "input" residues, which are generally taken from the "input" variable domains. 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 a complete human variable domain has been replaced by a 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.

[0182] 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 approach is described, for example, in U.S. Patent Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, and in 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. 13, 65-93 (1995).

[0183] Also can use known selection and / or mutagenesis method as described above to make antibody affinity maturation.Preferred affinity matured antibodies have than the starting antibody (usually murine, humanized or people) of preparing described mature antibody big five times, more preferably 10 times, even more preferably 20 or 30 times of affinity.

[0184] c. Bispecific Antibodies

[0185] The anti-CD39 antibodies described herein include bispecific molecules. Anti-CD39 antibodies or their antigen-binding portions can be derived or connected to another functional molecule, for example, another peptide or protein (e.g., a ligand for 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 can 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 can 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.

[0186] Thus, provided herein are bispecific molecules comprising at least one first binding specificity for CD39 and a second binding specificity for a second target epitope. In one embodiment described herein where the bispecific molecule is multispecific, the molecule can further comprise a third binding specificity.

[0187] In certain embodiments, the subject bispecific (or multispecific as the case may be) includes one or more binding domains of an immune checkpoint, 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 a binding domain that binds to a checkpoint protein on a T cell (especially a checkpoint 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 results in antibody-dependent cellular toxicity of cells expressing each or both of CD39 and the other checkpoint proteins to which it is bound.

[0188] 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 MHC I molecules, BTLA receptors and Toll ligands, and agonists of OX40, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), and 4-1BB (CD137). Examples of costimulatory molecules that can be included in the multispecific 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, 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, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a and CD83 ligands.

[0189] 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.

[0190] 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 can also be a light chain or heavy chain dimer, or any minimal fragment thereof, such as an Fv or single-chain (scFv) construct.

[0191] 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., an antibody) specific for the target complex.

[0192] Methods for preparing bispecific antibodies are known in the art. Traditionally, the recombinant production of bispecific antibodies is based on the coexpression 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 (quadroma) 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 and Traunecker et al., EMBO J., 10:3655-3659 (1991), published on May 13, 1993.

[0193] The antibody variable domains (antibody-antigen combination sites) with the desired binding specificity can be fused to the immunoglobulin constant domain sequence. The fusion is preferably fused to the 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 the light chain binding site necessary for at least one of the fusions. The 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 other detailed descriptions of the generation of bispecific antibodies, see, for example, Suresh et al., Methods in Enzymology, 121: 210 (1986).

[0194] According to another method described in WO 96 / 27011, the interface between a pair of antibody molecules can be through engineering approaches to maximize the percentage of the heterodimer recovered from recombinant cell culture.Preferred interface comprises at least a portion of the CH3 district of antibody constant domain.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 (such as tyrosine or tryptophan).Compensation " cavity " of the same or similar size as the larger side chain is produced on the interface of the second antibody molecule by replacing the large amino acid side chain with smaller amino acid side chains (such as alanine or threonine).This is provided for increasing the productive rate of heterodimer and exceeds the mechanism of other non-desired final products (such as homodimer).

[0195] Bispecific antibodies are prepared as full-length antibodies or antibody fragments (e.g., F(ab')2 bispecific antibodies). The technology for producing bispecific antibodies from antibody fragments has 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 antibody proteolytic cleavage is performed to produce F(ab')2 fragments. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize adjacent dithiols and prevent intermolecular disulfide bond formation. The resulting Fab' fragments are then converted into thionitrobenzoate (TNB) derivatives. One of the Fab'-TNB derivatives is then converted into Fab'-thiol by reduction with mercaptoethylamine and mixed with an equimolar amount of other Fab'-TNB derivatives to form bispecific antibodies. The resulting bispecific antibodies can be used as agents for selective immobilization of enzymes.

[0196] 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) describe 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, and trigger the lytic activity of human cytotoxic lymphocytes against human breast tumor targets.

[0197] Various techniques for preparing and isolating bispecific antibody fragments directly from recombinant cell cultures 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 connected to the Fab' portion of two different antibodies by gene fusion. Antibody homodimers are reduced to form monomers at the hinge region 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 fragment comprises a linker connected to the light chain variable domain (VLD) by a linker that is too short to allow pairing between the two domains on the same chain. L ) of the heavy chain variable domain (V H ). Therefore, forcing a fragment's V H and V L The complementary V domain of another segmentL and V H The domains pair to form two antigen-binding sites. Another strategy for making bispecific antibody fragments using single-chain Fv (sFv) dimers has also been reported. See Gruber et al., J. Immunol. 152: 5368 (1994).

[0198] 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).

[0199] d. Heteroconjugate Antibodies

[0200] 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 treating HIV infection [WO 91 / 00360; WO 92 / 200373; EP 03089]. It is contemplated that the antibodies can be prepared in vitro using known methods in synthetic protein chemistry, including those involving cross-linking agents. For example, immunotoxins can 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 in, for example, U.S. Patent No. 4,676,980.

[0201] e. Effector function engineering

[0202] It may be necessary to modify the effector functions of the antibodies encompassed by the present invention to enhance, for example, the effectiveness of anti-CD39 antibodies in treating cancer. For example, cysteine ​​residues can be introduced into the Fc region, thereby allowing the formation of interchain disulfide bonds in this region. The homodimeric antibodies thus produced may have improved internalization capacity 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 that the anti-CD39 antibody induces 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.

[0203] Homodimeric antibodies with enhanced anti-tumor activity can also be prepared using heterobifunctional cross-linkers as described in Wolff et al., Cancer Research, 53:2560-2565 (1993). Alternatively, antibodies can be engineered to have dual Fc regions and thereby have enhanced CD39 cytotoxicity. See Stevenson et al., Anti-Cancer Drug Design, 3:219-230 (1989).

[0204] f. Representative anti-CD39 antibody sequences

[0205] 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:

[0206] 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)

[0207] For the Ig39-21 clone, the CDRs for each of the VH and VL domains are:

[0208] CDR1 CDR2 CDR3 VH SEQ ID No. 29 SEQ ID No.30 SEQ ID No.31 VL SEQ ID No.32 SEQ ID No.33 SEQ ID No.34

[0209] The sequences of exemplary full-length antibodies and exemplary single-chain antibodies (scFV) utilizing the above VH and VL domains are provided below:

[0210] 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

[0211] 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.

[0212] 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.

[0213] 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 identical to 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.

[0214] 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 0.2x SSC / 0.1% SDS at 50° C. to 65° C.).

[0215] 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:

[0216]

[0217]

[0218] In some embodiments, anti-CD39 antibodies are generated in rabbits and then humanized by CDR grafting. Exemplary sequences of the VH and VL domains of humanized rabbit anti-CD39 antibodies are:

[0219]

[0220]

[0221] In some embodiments, the anti-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 tumors with damaged tumor vascular endothelium or collapsed vascular distribution network; (v) (optionally) binding to a protein selected from Figure 33and / or (vi) optionally binds to CD39 in a manner that is non-competitive or only partially competitive with binding to CD39 as with monoclonal antibody clone A1.

[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 (cloned IG39-21vL domain nucleic acid sequence)

[0228]

[0229] SEQ ID No. 4 (clone IG39-21vL 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]

[0244] SEQ ID No. 11 (clone 8C11 vL domain nucleic acid sequence)

[0245]

[0246] SEQ ID No. 12 (clone 8C11 vL domain amino acid sequence)

[0247]

[0248] SEQ ID No. 13 (clone 8D8 vH domain nucleic acid sequence)

[0249]

[0250]

[0251] SEQ ID No. 14 (clone 8D8 vH domain amino acid sequence)

[0252]

[0253] SEQ ID No. 15 (clone 8D8 vL domain nucleic acid sequence)

[0254]

[0255] SEQ ID No. 16 (clone 8D8 vL domain amino acid sequence)

[0256]

[0257] SEQ ID No. 17 (clone 9C10 vH domain nucleic acid sequence)

[0258]

[0259] SEQ ID No. 18 (clone 9C10 vH domain amino acid sequence)

[0260]

[0261] SEQ ID No. 19 (clone 9C10 vL domain nucleic acid sequence)

[0262]

[0263] SEQ ID No. 20 (clone 9C10 vL domain amino acid sequence)

[0264]

[0265]

[0266] SEQ ID No. 21 (clone 65H5 vH domain nucleic acid sequence)

[0267]

[0268] SEQ ID No. 22 (clone 65H5 vH domain amino acid sequence)

[0269]

[0270]

[0271] SEQ ID No. 23 (clone 65H5 vL domain nucleic acid sequence)

[0272]

[0273] SEQ ID No. 24 (clone 65H5 vL domain amino acid sequence)

[0274]

[0275]

[0276] SEQ ID No. 25 (clone 2G12 vH domain nucleic acid sequence)

[0277]

[0278] SEQ ID No. 26 (clone 2G12 vH domain amino acid sequence)

[0279]

[0280] SEQ ID No. 27 (clone 2G12 vL domain nucleic acid sequence)

[0281]

[0282] SEQ ID No. 28 (clone 2G12 vL domain amino acid sequence)

[0283]

[0284] SEQ ID No. 29 (clone IG39-21vH domain CDR1 amino acid sequence)

[0285]

[0286] SEQ ID No. 30 (clone IG39-21vH domain CDR2 amino acid sequence)

[0287]

[0288] SEQ ID No. 31 (clone IG39-21vH domain CDR3 amino acid sequence)

[0289]

[0290] SEQ ID No. 32 (clone IG39-21vL domain CDR1 amino acid sequence)

[0291]

[0292] SEQ ID No. 33 (clone IG39-21vL domain CDR2 amino acid sequence)

[0293]

[0294] SEQ ID No. 34 (clone IG39-21vL domain CDR3 amino acid sequence)

[0295]

[0296] SEQ ID No. 35 (clone IG39-21 full-length vH chain nucleic acid sequence)

[0297]

[0298]

[0299]

[0300] SEQ ID No. 36 (Clone IG39-21 full-length vH chain amino acid sequence)

[0301]

[0302]

[0303]

[0304] SEQ ID No. 37 (clone IG39-21 full-length vL chain nucleic acid sequence)

[0305]

[0306] SEQ ID No. 38 (Clone IG39-21 full-length vL chain amino acid sequence)

[0307]

[0308] SEQ ID No. 39 (clone IG39-21 scFv nucleic acid sequence)

[0309]

[0310] SEQ ID No. 40 (clone IG39-21 scFv amino acid sequence)

[0311]

[0312]

[0313] SEQ ID No.41

[0314]

[0315] SEQ ID No.42

[0316]

[0317]

[0318] SEQ ID No.43

[0319]

[0320] SEQ ID No.44

[0321]

[0322] SEQ ID No.45

[0323]

[0324]

[0325] SEQ ID No.46

[0326]

[0327] SEQ ID No.47

[0328]

[0329]

[0330] SEQ ID No.48

[0331]

[0332] SEQ ID No.49

[0333]

[0334]

[0335] SEQ ID No.50

[0336]

[0337] SEQ ID No.51

[0338]

[0339] SEQ ID No.52

[0340]

[0341] SEQ ID No.53

[0342]

[0343] SEQ ID No.54

[0344]

[0345]

[0346] SEQ ID No.55

[0347]

[0348] SEQ ID No.56

[0349]

[0350]

[0351] 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.

[0352] 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.

[0353] 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.

[0354] In certain embodiments, chimeric antibodies are humanized antibodies. Generally, non-human antibodies are humanized to reduce immunogenicity to people while retaining the specificity and affinity of the parent non-human antibody. Generally speaking, humanized antibodies include one or more variable domains, wherein HVR, such as CDR (or part thereof) are derived from non-human antibodies, and FR (or part thereof) are derived from human antibody sequences. Humanized antibodies optionally also will 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 (such as antibodies from which HVR residues are derived), for example, to restore or improve antibody specificity or affinity.

[0355] 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 a VH domain selected from SEQ ID No. 6, 10, 14, 18, 22, 26, 42, 46, 50, or 54, and the CDRs of the 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.

[0356] Humanized antibodies and methods of making them are reviewed, for example, in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and further described, for example, in Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989); U.S. Patent 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 "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).

[0357] 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. 151:2377 (1993)). Chem., 151:2623 (1993)); 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)).

[0358] 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).

[0359] 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 locus, which replaces the endogenous immunoglobulin locus, or they are present outside the chromosome or randomly integrated into the chromosome of the animal. In such transgenic mice, the endogenous immunoglobulin locus is 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; 5,770,429 describing HuMAB technology; 7,041,870 describing K-MMOUSE technology, and U.S. Patent Application Publication No. US2007 / 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.

[0360] 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, for example, 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).

[0361] Human antibodies can also be produced by isolating the Fv clone variable domain sequence from a phage, yeast or bacterial display library selected from 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.

[0362] To illustrate, the 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., eds., 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 eds., Human 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).

[0363] As an example of a phage display method, VH and VL gene libraries 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 immunogens 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 single-source antibodies to a wide range of non-self antigens as well as self-antigens without any immunization. Finally, as described by Hoogenboom and Winter, J.Mol.Biol., 227:381-388 (1992), primary libraries 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 to rearrange the libraries in vitro. Patents describing human antibody phage libraries are published, for example, including: 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.

[0364] Antibodies or antibody fragments isolated from human antibody libraries are considered herein to be human antibodies or human antibody fragments.

[0365] FcγRIII binding can also be increased by methods according to the prior art, for example, by modifying the amino acid sequence of the Fc portion of the antibody or the glycosylation of the Fc portion of the antibody (see, for example, EP2235061). In certain embodiments, the subject antibody is produced by cells in which, when glycosylated, less than 50% of the oligosaccharide chains on the antibody contain α-1,6-fucose. Typically, in a "low fucosylated" 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 carbohydrates 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 involve the use of FUT8 (α-1,6-fucosyltransferase) gene knockout CHO lines for the production of afucosylated antibodies.

[0366] IV. expression vector

[0367] In certain embodiments, recombinant expression vectors are used to amplify and express DNA encoding the anti-CD39 antibodies described herein. For example, a recombinant expression vector can 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 suitable transcriptional and / or translational regulatory elements derived from mammalian, microbial, viral, or insect genes. A transcription unit generally comprises the following assembly: (1) one or more genetic elements that have a regulatory role in gene expression, such as a transcriptional promoter or enhancer, (2) a structural or coding sequence that is transcribed into mRNA and translated into protein, and (3) appropriate transcriptional and translational initiation and termination sequences. Regulatory elements may include operator sequences to control transcription. The ability to replicate in a host is generally conferred by the origin of replication, and selection genes that facilitate recognition of transformants may be additionally incorporated. DNA regions are "operably linked" when they are functionally related to each other. For example, if the DNA of a signal peptide is expressed as a precursor that participates in the secretion of a polypeptide, it (a secretory leader sequence) is operably linked to the DNA of the polypeptide; if a promoter controls the transcription of a sequence, it is operably linked to a coding sequence; or if a ribosome binding site is placed so as to allow translation, it is operably linked to a coding sequence. In some embodiments, the structural elements intended for use in yeast expression systems include leader sequences that allow proteins translated to be secreted extracellularly by the host cell. In other embodiments, when recombinant proteins are expressed in the absence of a leader sequence or a transport sequence, they may include an N-terminal methionine residue. This residue may then optionally be cleaved from the expressed recombinant protein to provide a final product.

[0368] 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.

[0369] Suitable host cells 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 cell lines of mammalian origin as described below. Cell-free translation systems can also be used. Cloning and expression vectors suitable for use with bacterial, fungal, yeast and mammalian cell hosts are well known to those skilled in the art.

[0370] Various mammalian cell culture systems are used to express recombinant polypeptides. The expression of recombinant proteins in mammalian cells can 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 mammary 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 can include non-transcriptional elements, such as a replication origin, a promoter and enhancer suitable for being connected 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, splice donor and acceptor sites, and transcription termination sequences.

[0371] The expression of recombinant proteins in insect cell culture systems (e.g., 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.

[0372] 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.

[0373] 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.

[0374] V. Encoded anti-CD39 antibodies for in vivo delivery

[0375] Therapeutic vectors for delivering the coding sequence of the anti-CD39 antibody to be expressed in a patient can be viral, non-viral or physical. See, for example, 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 gene therapy include Eck et al., Goodman & Gilman's The Pharmacological Basis of Therapeutics, 9th ed., Hardman et al., ed., McGraw-Hill, New York, (1996), Chapter 5, pp. 77-101; Wilson, Clin. Exp. Immunol. 107 (Suppl. 1): 31-32, 1997; Wivel et al., Hematology / Oncology Clinics of North America, Gene Therapy, ed. SLE, 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.

[0376] a. Viral vectors

[0377] 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 approved for human use in gene therapy. Additionally, preferred vectors for tolerance do not contain immunostimulatory sequences.

[0378] Adenoviral vector

[0379] An illustrative method for delivering one or more nucleic acid sequences in vivo involves the use of an adenoviral expression vector. "Adenoviral expression vector" is meant to include constructs containing 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 the Cyb5r3 gene.

[0380] Adenoviral vectors are highly immunogenic and therefore not preferred for use in inducing tolerance by presenting antigens or in the setting of autoimmune diseases. However, these vectors can be used to induce immunity, for example, for the treatment of infectious diseases, including, for example, influenza, HBV, HCV, and HIV.

[0381] Adeno-associated virus (AAV)

[0382] 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.

[0383] Typically, a viral vector comprising an epitope containing a nucleic acid construct is assembled from a polynucleotide encoding the desired epitope, suitable regulatory elements, and elements necessary for epitope expression that mediates cell transduction. In one embodiment, an adeno-associated virus (AAV) vector is employed. In a more specific embodiment, the AAV vector is AAV1, AAV6, or AAV8.

[0384] AAV expression vectors having a DNA molecule of interest bound by an AAV ITR can be constructed by directly inserting the selected sequence into the AAV genome from which the major AAV open reading frame ("ORF") has been excised. Examples of constitutive promoters that can be included in the AAV of the present invention include, but are not limited to, the exemplary CMV immediate early enhancer / chicken beta-actin (CBA) promoter.

[0385] For eukaryotic cells, expression control sequences typically include promoters, enhancers, such as those derived from immunoglobulin genes, SV40, cytomegalovirus, and polyadenylation sequences that may contain 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.

[0386] The selection of these and other common vectors and regulatory elements is routine, and many of these sequences are available. See, for example, Sambrook et al. and the 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 function 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 herein. This selection is a matter of routine and is not a limitation to the molecule or construct.

[0387] Retroviral vectors

[0388] 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 the 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 the 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 Wilson, 1993, Curr. Opin. in Genetics and Devel. 3:110-114.

[0389] "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.

[0390] 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).

[0391] Lentiviral vectors refer to a genus of retroviruses that can infect both dividing and non-dividing cells and typically 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).

[0392] 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 absence 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 vectors derived from γ-retroviruses.

[0393] Non-cytopathic viruses include retroviruses (e.g., lentiviruses), whose life cycle involves reverse transcription of genomic viral RNA into DNA, and subsequent integration of provirus 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 medium, and infecting target cells with viral particles) are known to those skilled in the art.

[0394] The retroviral genome contains three genes, gag, pol, and env, that encode capsid protein, polymerase, and envelope components, respectively. The sequence found upstream of the gag gene contains a signal for packaging the genome into the virion. Retroviral vectors are gene transfer plasmids in which heterologous nucleic acid residues are located between two retroviral LTRs. Retroviral vectors typically contain appropriate packaging signals that allow the retroviral vector or RNA transcribed using the retroviral vector as a template to be packaged into viral virions in appropriate packaging cell lines (see, e.g., U.S. Patent No. 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 episomal or non-integrated forms of retroviral vectors based on lentiviruses (e.g., a type of retrovirus).

[0395] When stable expression is required, lentiviral vectors are useful, but lentiviral vectors can be immunogenic and may have other undesirable effects. Therefore, although lentiviral vectors are convenient for research, when they are used for human administration, care should be taken, especially when tolerance rather than immunity needs to be induced. Although mRNA electroporation is safer, lentivirus is suitable for in vitro engineering of T cells or dendritic cells or other antigen-presenting cells for cancer therapy. However, two recent advances have made the use of lentivirus safer and more clinically transformable. First, the co-expression of suicide genes together with antigens, when the drug is administered, the product of the antigen becomes active. 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 currently 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.

[0396] Retroviral vectors suitable for use herein are described in, for example, U.S. Patent 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 plentiox-IRES-GFP.

[0397] Additional retroviral delivery systems that can be readily adapted to deliver transgenes encoding anti-CD39 antibody agents 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.

[0398] In certain embodiments, the retrovirus is a recombinant replication-competent retrovirus 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 cassette comprising an internal ribosome entry site (IRES) operably linked to a coding sequence for an anti-CD39 antibody agent, wherein the cassette is located 5' of the U3 region of the 3'LTR and 3' of the sequence encoding the retroviral envelope; and cis-acting sequences for reverse transcription, packaging, and integration in target cells.

[0399] In certain embodiments, the retrovirus is a recombinant replication-competent retrovirus 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 being suitable for expression in mammalian cells), a gag nucleic acid domain, a pol nucleic acid domain, and an env nucleic acid domain; a cassette comprising an anti-CD39 antibody agent encoding sequence operably linked to a heterologous polynucleotide, wherein the cassette is located 5' to and operably linked to the 3' LTR, 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.

[0400] In certain preferred embodiments of the recombinant replication-competent retrovirus, the envelope is selected from one of an amphoteric, polyecious, heterologous, 10A1, GALV, baboon endogenous virus, RD114, rhabdovirus, alphavirus, measles, or influenza virus envelope.

[0401] In certain preferred embodiments of the recombinant replication-competent retrovirus, 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 retrovirus (PERV), feline retrovirus RD114, squirrel monkey retrovirus, xenogeneic murine leukemia virus-related virus (XMRV), avian reticuloendotheliosis virus (REV), or gibbon ape leukemia virus (GALV).

[0402] In certain preferred embodiments of the recombinant replication-competent retrovirus, the retrovirus is a gammaretrovirus.

[0403] In certain preferred embodiments of the retrovirus with recombinant replication capability, there is a second box comprising a coding sequence for 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 instances, the second box may comprise an internal ribosome entry site (IRES) or a mini-promoter or pol III promoter operably linked to the coding sequence for the second therapeutic protein.

[0404] In certain preferred embodiments of the recombinant replication-competent retrovirus, it is a non-lytic, amphoteric retroviral replicating vector, which preferably selectively infects and replicates in cells of the tumor microenvironment.

[0405] Other viral vectors as expression constructs

[0406] Other viral vectors can be used as expression constructs for delivering oligonucleotide or polynucleotide sequences to host cells in the present invention. Vectors derived from viruses such as 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.

[0407] b. Non-viral vectors

[0408] Plasmid vector

[0409] 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. Over the past few years, plasmid vectors have been used as DNA vaccines to deliver antigen-encoding genes into cells. They are particularly advantageous in this regard because they do not have many of the same safety issues as viral vectors. However, these plasmids, which have promoters compatible with the host cells, can express peptide epitopes encoded by nucleic acids within 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 through various parenteral, mucosal, and topical routes. For example, DNA plasmids can be injected intramuscularly, intradermally, subcutaneously, or by other routes. They can also be administered via intranasal sprays or drops, rectal suppositories, and orally. They can also be administered using a gene gun to the epidermis or mucosal surface. The plasmids can be administered in aqueous solution, dried onto gold particles, or combined with another DNA delivery system including but not limited to liposomes, dendrimers, cochlea, and microencapsulation.

[0410] Therefore, in one aspect, a plasmid is provided for expressing a nucleic acid construct containing an epitope, the nucleic acid construct comprising an expression cassette; also referred to as a transcription unit. When the plasmid is placed in an environment suitable for epitope expression, the transcription unit will express a polynucleotide comprising a sequence encoding the epitope, an ETS and MHCII promoter sequence or a sequence encoding the epitope and a secretion signal sequence, as well as any other sequences encoded in the construct. The transcription unit comprises a transcription control sequence that is transcriptionally linked to the cellular immune response element coding sequence. The transcription control sequence may comprise a promoter / enhancer sequence, such as a cytomegalovirus (CMV) promoter / enhancer sequence. However, those skilled in the art will appreciate that various other promoter sequences suitable for expression in eukaryotic cells are known and can be used in the constructs disclosed herein. The expression level of the nucleic acid product will depend on the presence and activation of the associated promoter and associated enhancer element.

[0411] 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 such that the cellular immune response elements can be expressed. There are many examples of suitable expression plasmids into which the polynucleotides comprising the sequences can be cloned, such as pCI-neo, pUMVC, or pcDNA3.

[0412] A large number 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 removing lipopolysaccharides (endotoxins), which are contaminants from the bacteria used to propagate the plasmids. This step is most preferably used for tolerogenic DNA vaccines because endotoxins act as strong adjuvants and can produce undesired immune stimulation.

[0413] The purpose of a plasmid is to effectively deliver a nucleic acid sequence to a cell or tissue and express a therapeutic epitope therein. In particular, the purpose of the plasmid may be to achieve high copy number, avoid potential causes of plasmid instability, and provide a means for plasmid selection. Regarding expression, a nucleic acid cassette contains the necessary elements for expressing a nucleic acid within the cassette. Expression involves the efficient transcription of an inserted gene, nucleic acid sequence, or nucleic acid cassette using a plasmid. The expression product may be a protein, polypeptide, or RNA. The nucleic acid cassette may contain a nucleic acid sequence. Expression of the nucleic acid may be continuous or regulated.

[0414] microring

[0415] The embodiments of the nucleic acid constructs described herein can be processed in the form of mini-ring DNA. Mini-ring DNA belongs to small (2 to 4 kb) circular plasmid derivatives that have been partially released from all prokaryotic vectors. Since mini-ring DNA vectors do not contain bacterial DNA sequences, it is unlikely that they will be considered foreign and destroyed. (Typical transgenic delivery methods involve plasmids containing exogenous DNA). Therefore, compared to conventional plasmids (several days to several weeks), these vectors can express longer time periods (on the order of several 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 about minicircle DNA is provided in Dietz et al., Vector Engineering and Delivery Molecular Therapy (2013); 21 8, 1526-1535 and Hou et al., Molecular Therapy-Methods & Clinical Development, article number: 14062 (2015) doi: 10.1038 / mtm.2014.62. More information about 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 as 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.

[0416] As the initial step in the method for finally obtaining the expression of the product encoded by nucleic acid, it is to realize the uptake of nucleic acid by cells. Uptake of nucleic acid by cells depends on many factors, one of which is the length of time that nucleic acid is close to the cell surface. For example, after intramuscular (im) administration of plasmid DNA in a buffer, if the muscle is massaged, a significant reduction in gene expression is observed, which may be due to DNA leaking from the muscle directly or via lymphatic vessels (Human Gene Therapy 4:151-159; 1993). Therefore, it may be necessary to prepare nucleic acids with a compound that delays the rate of nucleic acid diffusion or is taken away from the site where cells need to uptake nucleic acid. In addition, these compounds may be suitable for use in organisms by means such as injection, while maintaining or restoring the physical characteristics necessary for increasing cellular uptake of nucleic acid.

[0417] To achieve expression of an 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.

[0418] 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).

[0419] Mechanical delivery system

[0420] 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 photopolymerizable 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 release may be desired immediately after administration. The preparation of such compositions is well within the level of ordinary skill in the art using known techniques.

[0421] Physical methods of enhanced delivery include electroporation (where a short pulse of high voltage carries the nucleic acid across the membrane), gene guns (where the DNA is loaded onto gold particles and forced to penetrate into the cell), sonoporation, magnetofection, hydrodynamic delivery, and the like, all of which are known to those skilled in the art. DNA can 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 the cell. DNA can also form complexes with polymers (polyplexes) or dendrimers that can release the cargo directly into the cytoplasm of the cell.

[0422] 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. Patent No. 5,151,254 and PCT Application Nos. 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.

[0423] Biodegradable microspheres (e.g., polylactic acid polyglycolate) can be used as carriers for the compositions. Suitable biodegradable microspheres are disclosed, for example, in U.S. Patent 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 carriers comprising microparticle-protein complexes, such as those described in U.S. Pat. No. 5,928,647, which may have additional benefits when used intratumorally to deliver coding sequences for anti-CD39 antibody agents capable of inducing an MHC I-restricted cytotoxic T lymphocyte response targeted to a patient's tumor tissue.

[0424] Biodegradable polymeric nanoparticles facilitate the transfer of non-viral nucleic acids into cells. Small (approximately 200 nm), positively charged (approximately 10 mV) particles are formed by self-assembly of cationic, hydrolytically degradable poly(β-amino ester) and plasmid DNA.

[0425] 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.

[0426] 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 technologies 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 within the tumor, and plasmids are delivered to muscle tissue, resulting in systemic expression of secretory 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.

[0427] 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 plant. The modular electrode system comprises a plurality of needle electrodes; a hypodermic needle; an electrical connector that provides an electrically conductive connection from a programmable constant current pulse controller to the plurality of needle electrodes; and a power source. An operator grasps the plurality of needle electrodes mounted on a support structure and securely inserts 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 the biomolecules into cells between the plurality of electrodes. The entire contents of U.S. Patent No. 7,245,963 are hereby incorporated by reference.

[0428] U.S. Patent Publication No. 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 comprises 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 for the 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 No. 2005 / 0052630), which is hereby incorporated by reference.

[0429] The electrode arrays and methods described in U.S. Patent No. 7,245,963 and U.S. Patent 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, an injection needle (to deliver a 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.

[0430] In some embodiments, the electric field of the present invention is a field strength of about 1000V / cm to about 1500V / cm, preferably about 300V / cm to 1500V / cm, and preferably about 1000V / cm to 1500V / cm. Alternatively, the pulse length of relatively low 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.

[0431] The pulse length may be from about 10 seconds to about 100 milliseconds. 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 introduced into the cell via electroporation.

[0432] Also contemplated are electroporation devices that incorporate 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 that incorporate EIS can be found, for example, in WO2016 / 161201, which is hereby incorporated by reference.

[0433] The uptake of the non-viral delivery vector 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 the mechanical force generated by the ruptured microbubbles and the magnetic field plays a role in increasing the transport efficiency across the cell membrane. The technology of plasma electroporation is described in U.S. Patents 7,923,251 and 8,283,171. This technology can also be used for the transformation of cells in vivo. Chaiberg et al. (2006) Investigative Ophthalmology & Visual Science 47:4083-4090; Chaiberg et al., U.S. Patent No. 8,101 169, issued January 24, 2012.

[0434] Other alternative electroporation techniques are also contemplated. In vivo plasmid delivery can also be performed using cold plasma. Plasma is one of the four basic states of matter, with other states being solid, liquid, and gas. Plasma is an electrically neutral medium (i.e., the total charge of the plasma is approximately zero) of unbound positive and negative particles. Plasma can be produced 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 (Luo et al. (1998) Phys. Plasma 5: 2868-2870) called ions, and, if present, with the dissociation of molecular bonds.

[0435] Cold plasma (i.e., non-thermal plasma) is generated by delivering a pulsed high-voltage signal to a suitable electrode. A cold plasma device can take the form of a gas jet device or a dielectric barrier discharge (DBD) device. Low-temperature plasmas have attracted considerable interest and attention due to their ability to provide plasma at relatively low gas temperatures. Providing plasma at such temperatures is of interest for a variety of applications, including wound healing, antimicrobial 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 can take the form of a gas jet device, a dielectric barrier discharge (DBD) device, or a multi-frequency rich harmonic power supply.

[0436] 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 a ground, which can be provided by the target substrate undergoing the cold plasma treatment or by providing a built-in ground for the electrode. The energy for 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. The discharge is separated from the conductive electrode by the dielectric layer and reduces electrode etching and gas heating. The pulsed DC voltage can be varied in amplitude and frequency to achieve different operating schemes. Any device incorporating this principle of cold plasma generation (such as a DBD electrode device) falls within the scope of the various embodiments encompassed by the present invention.

[0437] 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 & Immune-therapeutics 8:1729-1733; and Connolly et al. (2015) Bioelectrochemistry 103:15-21).

[0438] 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 can be placed within the applicator in a conical arrangement. One or more electroporation signals can each be associated with an electric field. The device can also include a potentiometer coupled to the power source and the electrodes. The potentiometer can be configured to maintain the electric field substantially within a predetermined range.

[0439] The one or more electroporation signals may each be associated with an electric field. The device may further include a potentiometer coupled to the power supply and the electrodes. 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 at approximately 1300 V / cm.

[0440] A power supply may provide a first electrical signal to a first electrode and a second electrical signal to a second electrode. The first and second electrical signals may be combined to generate an electrical wave having a beat frequency. The first and second electrical signals 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.

[0441] In certain embodiments, the present invention provides a method for treating a subject having a tumor, the method comprising: injecting an effective dose of a plasmid encoding an anti-CD39 antibody agent into the tumor; and administering electroporation to the tumor. In certain embodiments, the electroporation further comprises administering at least one voltage pulse of about 200 V / cm to about 1500 V / cm at a pulse width of about 100 microseconds to about 20 milliseconds.

[0442] 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.

[0443] Lipid and polycationic molecules for delivery of nuclear constructs encoding anti-CD39 antibodies

[0444] 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. Advances in lipid formulations have improved the efficiency of in vivo gene transfer (see PCT application WO98 / 07408). For example, lipid formulations containing 1,2-bis(oleoyloxy)-3-(trimethylammonio)propane (DOTAP) and cholesterol in an equimolar ratio 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 an invaginated bilayer or "vase" structure. The beneficial features of these lipid structures include positive p, colloidal stabilization of cholesterol, two-dimensional nucleic acid packaging and increased serum stability.

[0445] 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 typically enter cells 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 DNA or RNA into nanoparticles, which are then substrates for endosomal-mediated uptake. [8] Several of these cationic-nucleic acid complexation technologies have been developed into potential clinical products, including complexes with plasmid DNA (pDNA), oligodeoxynucleotides, and various forms of synthetic RNA.

[0446] The nucleic acid constructs disclosed herein can be associated with polycationic molecules that act to enhance uptake into cells. The complexation of the nucleic acid constructs with the polycationic molecules also helps to package the constructs, reducing their size, which is believed to aid cellular uptake. Once within the endosome, the complex dissociates due to the lower pH, and the polycationic molecules can disrupt the membranes of the endosomes to facilitate escape of the DNA into the cytoplasm, where it can then be degraded. Preliminary data show that when complexed with the polycationic molecules polylysine or polyethyleneimine, nucleic acid construct embodiments have enhanced uptake into SCs compared to DCs.

[0447] An example of a polycationic molecule suitable for use with a nucleic acid construct includes a cell penetrating peptide (CPP), examples include polylysine (described above), polyarginine and Tat peptides. Cell penetrating peptides (CPP) are small peptides that can be bound to DNA and, once released, permeate the cell membrane to facilitate DNA escape from endosomes to the cytoplasm. Another example of CPP relates to a 27-residue chimeric peptide, referred to as MPG, which has been shown to bind ss and ds oligonucleotides in a stable manner to produce non-covalent complexes that protect nucleic acids from DNA enzymatic degradation and effectively deliver oligonucleotides to cells in vitro (Mahapatro A et al., J Nanobiotechnol, 2011, 9: 55). When different peptides: DNA ratios and 10: 1 and 5: 1 ratios (respectively 150 nm and 1 μm), the complex forms small particles of approximately 150 nm to 1 μm. 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 positively charged aggregates of 700 to 900 nm (Li et al., Agnew Chem Int Ed Enl 2015;54(10):2941-4). RNA can also be complexed with this type of polycationic molecule for in vivo delivery.

[0448] Other examples of polycationic molecules that can be complexed with the nucleic acid constructs described herein include and polycationic polymers commercially available from In Vivo JET (Polypus-transfection, SA, Illkirch, France).

[0449] VI. Methods of use and pharmaceutical compositions

[0450] 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 tumorigenicity. 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 in vitro, ex vivo or in vivo.

[0451] The present invention provides methods for activating an immune response in a subject using the anti-CD39 antibodies described herein. In some embodiments, the present invention provides methods for promoting an immune response in a subject using the anti-CD39 antibodies described herein. In some embodiments, the present invention provides methods for increasing an immune response in a subject using the anti-CD39 antibodies described herein. In some embodiments, the present invention provides methods for enhancing an immune response in a subject using the anti-CD39 antibodies described herein. In some embodiments, the activation, promotion, increase, and / or enhancement of an immune response comprises increasing cell-mediated immunity. In some embodiments, the activation, promotion, increase, and / or enhancement of an immune response comprises increasing a Th1-type response. In some embodiments, the activation, promotion, increase, and / or enhancement of an immune response comprises increasing T cell activity. In some embodiments, the activation, promotion, increase, and / or enhancement of an immune response comprises increasing CD4+ T cell activity. In some embodiments, the activation, promotion, increase, and / or enhancement of an immune response comprises increasing CD8+ T cell activity. In some embodiments, the activation, promotion, increase, and / or enhancement of an immune response comprises increasing CTL activity. In some embodiments, the activation, promotion, increase, and / or enhancement of an immune response comprises 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 do 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 do 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.

[0452] In vivo and in vitro assays for determining whether anti-CD39 antibodies modulate, activate, or inhibit immune responses are known in the art or are being developed.

[0453] 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.

[0454] In some embodiments, the tumor is a solid tumor. In certain 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 certain embodiments, the tumor is a colorectal tumor. In certain embodiments, the tumor is an ovarian tumor. In certain embodiments, the tumor is a lung tumor. In certain embodiments, the tumor is a pancreatic or pancreatic islet tumor. In certain embodiments, the tumor is a melanoma tumor. In certain embodiments, the tumor is a bladder or urothelial tumor.

[0455] 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.

[0456] In some embodiments, the tumor expresses or overexpresses a tumor antigen that is targeted by an anti-CD39 antibody, such as a bispecific agent that comprises an antigen binding site that specifically binds to the tumor antigen.

[0457] The present invention further provides a method for treating cancer in a subject, 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 the cancer.

[0458] 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 as 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.

[0459] 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 certain embodiments, the cancer is bladder cancer.

[0460] The present invention provides compositions comprising the anti-CD39 antibodies described herein. The present invention also provides pharmaceutical compositions comprising the 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).

[0461] The preparation is prepared by combining the purifying agent encompassed by the present invention with a pharmaceutically acceptable vehicle (e.g., carrier or excipient) for storage and use. Those skilled in the art generally consider pharmaceutically acceptable carriers, excipients, and / or stabilizers to be inactive ingredients of the preparation or pharmaceutical composition.

[0462] In some embodiments, the anti-CD39 antibodies are lyophilized and / or stored in lyophilized form. In some embodiments, the formulations comprising the anti-CD39 antibodies described herein are lyophilized.

[0463] Suitable pharmaceutically acceptable vehicles include, but are not limited to, nontoxic buffers such as phosphate, citrate, 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, and methylbenzyl ammonium chloride. chloride), phenol, butyl alcohol or benzyl alcohol, alkyl parabens (such as methyl paraben or propyl paraben), catechol, resorcinol, 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 polyvinyl pyrrolidone; 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 Supplementary Edition, 2012, Pharmaceutical Press, London.)

[0464] The pharmaceutical compositions encompassed by the present invention can be administered in a variety of ways for local or systemic treatment. Administration can be topical, via 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).

[0465] Therapeutic preparations can 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 a diluent (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. The tablets, pills, etc. of the preparation or composition can be coated or otherwise mixed to provide a dosage form that provides the advantage of a prolonged effect. For example, the tablet or pill can include an inner composition covered by an outer component. In addition, the two components can be separated by an enteric layer that acts to resist disintegration and allows the inner 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.

[0466] 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.

[0467] In certain embodiments, the pharmaceutical preparation comprises an anti-CD39 antibody complexed with liposomes. Methods for producing liposomes are well 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 to produce liposomes with the desired diameter by a filter that determines the pore size.

[0468] In certain embodiments, a sustained release formulation comprising anti-CD39 antibodies can be produced. Suitable examples of sustained release formulations include semipermeable matrices of solid hydrophobic polymers containing anti-CD39 antibodies, wherein the matrix is ​​in the form of formed articles (for example, film or microcapsules). The example of sustained release matrix includes polyester, hydrogel (such as poly (2-hydroxyethyl-methacrylate) or poly (vinyl alcohol)), polylactic acid, L-glutamic acid and 7 ethyl-L-glutamate copolymers, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers (such as LUPRON DEPOT (injectable microspheres comprising lactic acid-glycolic acid copolymers and leuprorelin acetate)), sucrose acetate isobutyrate and poly-D-(-)-3-hydroxybutyric acid.

[0469] 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 factor (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)), interleukin (e.g., IL-1, IL2, IL-3, IL-7, IL-12, IL-15, IL-18), checkpoint inhibitors, antibodies that block immunosuppressive function (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 can 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.

[0470] 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 can be administered before, simultaneously with, and / or after administering the anti-CD39 antibody. Pharmaceutical compositions comprising an anti-CD39 antibody and an additional therapeutic agent are also provided. In some embodiments, the at least one additional therapeutic agent comprises 1, 2, 3, or more additional therapeutic agents.

[0471] Although this is not necessary, the combination therapy using two or more therapeutic agents generally uses agents that play a role 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 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., enhancing or activating the reaction) and a therapeutic agent that affects (e.g., suppresses or kills) tumor / cancer cells.

[0472] 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.

[0473] 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, an antimetabolite, an antimitotic agent, a topoisomerase inhibitor, or an angiogenesis inhibitor.

[0474] Therapeutic agents that can be administered in combination with the anti-CD39 antibodies described herein include chemotherapeutic agents. Therefore, in some embodiments, the method or treatment involves administering a combination of an anti-CD39 antibody and a chemotherapeutic agent or a combination of a mixture of chemotherapeutic agents. 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 time period, so that all active agents can simultaneously exert their biological activity. The preparation and dosing schedule of such chemotherapeutic agents can be based on the manufacturer's instructions or as determined empirically by a skilled practitioner. The preparation and dosing schedule of such chemotherapy are also described in The Chemotherapy Source Book, 4th Supplementary Edition, 2008, compiled by M.C. Perry, Lippincott, Williams & Wilkins, Philadelphia, Pa.

[0475] 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, tebuconazole, and euridopa; ethyleneimines and methylmelamines, including hexamethylmelamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolmelamine; nitrogen mustards such as benzophenone, benzophenone, and benzophenone. Nitrogen mustard, naphthalene nitrogen mustard, clofosamide, estramustine, ifosfamide, nitrogen mustard, nitrogen mustard oxide hydrochloride, melphalan, nembice, phenylephrine, prednimustine, trofosamide, 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, carmomycin, chromomycin, dactinomycin, daunomycin, detopicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, mexicomycin, mitomycin, mycophenolic acid, noramycin, olive mycin, peplomycin, porphyromycin, puromycin, triferric doxorubicin, rhodorubicin, streptozotocin, streptozotocin, Tuberculocidin, ubenimex, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as dimethylfolate, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thioimidazole, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytosine arabinoside, dideoxyuridine, deoxyfluridine, enocitabine, floxuridine, 5-FU; androgens such as calusterone, dromostanolone propionate, propionate), epitiostanol, mepitiostane, testolactone; antiadreners such as aminoglutethimide, mitotane, and trilostane; folic acid supplements such as folinic acid; aceglucuronolide; aldophosphamide glycosides; aminolevulinic acid; amsacrine; betribucil; bisantrene; edatrexate; defofamin; colcemid; diazocone; eflornithine; and elytraxate. ammonium; etogluconol; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguanidine; mitoxantrone; mopidamol; nitracrine; pentostatin; methambucil; pirarubicin; podophyllic acid; 2-ethylhydrazide; procarbazine; PSK; razoxane; sizofuran; spirogermanium; tricholomanic acid; triazoline quinone; 2,2',2"-trichlorotriethylamine; urethane; vindesine;Dacarbazine; mannomustine; dibromomannitol; dibromodulcitol; 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 pharmaceutically acceptable salts, acids or derivatives 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, oxifene, 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. ;

[0476] 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 effects of topoisomerase (e.g., topoisomerase I or II). Topoisomerase inhibitors include, but are not limited to, doxorubicin hydrochloride, daunorubicin citrate, mitoxantrone hydrochloride, dactinomycin D, etoposide, topotecan hydrochloride, teniposide (VM-26), and irinotecan, and pharmaceutically acceptable salts, acids, or derivatives of any of these.

[0477] In certain embodiments, chemotherapeutic agent is antimetabolite.Antimetabolite is similar to the metabolite required for normal biochemical reaction, but different enough to interfere with one or more normal functions (such as cell division) of cell.Antimetabolite includes but is not limited to gemcitabine, fluorouracil, capecitabine, methotrexate sodium, ralitrexed (ralitrexed), pemetrexed, tegafur, cytosine arabinoside, thioguanine, 5-azacytidine, 6-mercaptopurine, azathioprine, 6-thioguanine, pentostatin, fludarabine phosphate (fludarabine phosphate) and cladribine (cladribine), and any one of these pharmaceutically acceptable salts, acids or derivatives.

[0478] 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.

[0479] 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 CD39 or CD73 within tumors. 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 upregulation of ATP release and / or intratumoral levels of CD39 and / or CD73.

[0480] 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 a protein kinase inhibitor selected from the group consisting of gefitinib (IRESSA), erlotinib (TARCEVA), sunitinib (sunitinib) (SUTENT), lapatinib (lapatanib), vandetanib (vandetanib) (ZACTIMA), AEE788, CI-1033, cediranib (RECENTIN), sorafenib (sorafenib) (NEXAVAR) and pazopanib (pazopanib) (GW786034B). In some embodiments, additional therapeutic agents include mTOR inhibitors.

[0481] 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.

[0482] 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 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 antibody that inhibits β-catenin signaling. In certain embodiments, the additional therapeutic agent is an antibody that inhibits angiogenesis inhibitor (e.g., an anti-VEGF or VEGF receptor antibody). In certain embodiments, the additional therapeutic agent is bevacizumab (AVASTIN), ramucirumab, trastuzumab (HERCEPTIN), pertuzumab (OMNITARG), panitumumab (VECTIBIX), nimotuzumab, zalutumumab, or cetuximab (ERBITUX).

[0483] I / O Portfolio - Representative Checkpoint Inhibitors and Co-stimulatory Agonists

[0484] 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-TIM-3 antibody, an anti-TIGIT antibody, and / or an anti-Siglec-15 antibody.

[0485] For example, treatment may also include administering an inhibitor of an immune checkpoint molecule or an activator of a costimulatory 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 costimulatory 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.

[0486] PD-1 antagonists

[0487] 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). Immune suppression can be reversed by inhibiting the local interaction between PD-1 and PD-L1, and the effects are 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).

[0488] As used herein, the terms "programmed death 1," "programmed cell death 1," "protein PD-1," "PD-1," PD1, "PDCD1," "hPD-1," and "hPD-1" are used interchangeably and include variants, isoforms, species homologs, and analogs of human PD-1 that share at least one common epitope with human PD-1. The complete human PD-1 sequence can be found under GenBank Accession No. U64863.

[0489] 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, and analogs of human PDL-1 that share at least one common epitope. 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.

[0490] 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 to facilitate the removal of T cell dysfunction caused by signal transduction on the PD-1 signaling 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.

[0491] The term "PD-1 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-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 the 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 the negative co-stimulatory signal mediated by or through cell surface proteins expressed on T lymphocytes that are mediated by PD-1 signaling, so as to make the dysfunctional T cells less dysfunctional (e.g., enhancing the effector response 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 Merck 3745 described herein. In another specific aspect, the PD-1 binding antagonist is CT-011 described herein.

[0492] 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 the negative co-stimulatory signal mediated by or through cell surface proteins expressed on T lymphocytes that are 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.

[0493] 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 partners. 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 cell surface proteins expressed on T lymphocytes that are mediated by PD-L2 signaling, so as to make dysfunctional T cells less dysfunctional (e.g., enhancing effector responses to antigen recognition). In some embodiments, the PD-L2 binding antagonist is an immunoadhesin.

[0494] PD-1 pathway: Members of the PD-1 pathway are all proteins associated with PD-1 signaling. In one aspect, these can be proteins that induce PD-1 signaling upstream of PD-1, such as, for example, the ligands for PD-1, PD-L1, and PD-L2, and the signaling receptor PD-1. In another aspect, these can be signaling proteins downstream of the PD-1 receptor. In the context of the present invention, particularly preferred members of the PD-1 pathway are PD-1, PD-L1, and PD-L2.

[0495] PD-1 pathway inhibitors: In the context encompassed by the present invention, PD-1 pathway inhibitors are preferably defined herein as compounds that are 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 receptors PD-L1 or PD-L2. Such antagonistic antibodies may also be encoded by a nucleic acid. Such encoded antibodies are also referred to as "intracellular antibodies" as defined herein. Furthermore, 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 act as a PD1 inhibitory ligand. Furthermore, 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.

[0496] 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 the PD-1 receptor, or directly bind to the PD-1 receptor without participating in signal transduction through the PD-1 receptor. In one embodiment, the PD-1 antagonist binds directly 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 binds directly to PD-L1, inhibits or prevents PD-L1 from binding to PD-1, thereby blocking PD-1 inhibitory signal transduction.

[0497] 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 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-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, the teachings of which are incorporated herein by reference. Antibodies that compete with any of these recognized antibodies for binding to PD-1 can also be used.

[0498] 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-H1) 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, the teachings of which are incorporated herein by reference. Antibodies that compete with any of these recognized antibodies for binding to PD-L1 can also be used.

[0499] An exemplary anti-PD-L1 antibody is 12A4, described in WO 2007 / 005874 and U.S. Patent No. 7,943,743. In one embodiment, the antibody comprises the heavy and light chain CDRs or VRs of 12A4. In another embodiment, the antibody competes for binding to the same epitope on PD-L1 and / or binds to the same epitope on PD-L1 as the antibodies mentioned above. In another embodiment, the antibody has at least about 90% variable region amino acid sequence identity with the antibodies mentioned above.

[0500] Anti-PD-1 or anti-PD-L1 antibodies can be used at 10 -7 M, 5x 10 -8 M, 10 -8 M, 5x 10 -9 M, 10 -9 M, 5x 10 -10 M, 10 -10 Binds to PD-1 or PD-L1 with a KD of M or less.

[0501] In one embodiment, the PD-1 inhibitor is an anti-PD-1 antibody selected from nivolumab, pembrolizumab, or pidilizumab. The preferred PD-1 inhibitor is nivolumab.

[0502] 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 Registry Number: 946414-94-4). Nivolumab is a fully human IgG4 monoclonal antibody that specifically blocks PD1. 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 Pembrolizumab (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, WO2009 / 114335 (incorporated by reference), and US 8,354,509 (incorporated by reference).

[0503] In some embodiments, the anti-PD-1 antibody is pidilizumab. pidilizumab (CT-011; CureTech) is a humanized IgG1 kb monoclonal antibody that binds to PD-1. pidilizumab and other humanized anti-PD-1 monoclonal antibodies are disclosed in WO2009 / 101611. Other anti-PD1 antibodies are disclosed in US 8,609,089, US2010028330, and / or US20120114649. Other anti-PD1 antibodies include AMP 514 (Amplimmune).

[0504] 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.

[0505] 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 antibody described in WO 2010 / 077634 (incorporated by reference) (in WO 2010 / 077634, the heavy and light chain variable region sequences are shown in SEQ ID Nos. 20 and 21, respectively).

[0506] In one embodiment, the PD-L1 inhibitor is MDPL3280A (Genentech / Roche). MDPL3280A is an IgG1 monoclonal antibody optimized for human Fc that binds to PD-L1. MDPL3280A and other human monoclonal antibodies to 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-fused soluble receptor (B7-DCIg; Amplimmune; e.g., disclosed in WO 2010 / 027827 (incorporated by reference) and WO 2011 / 066342 (incorporated by reference)) that blocks the interaction between PD1 and B7-H1.

[0507] 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.

[0508] 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.

[0509] For illustration only, the subject combination therapy may be practiced as a small molecule antagonist selected from the group consisting of:

[0510]

[0511] In other embodiments, the small molecule antagonist is represented by the following general formula:

[0512]

[0513] in,

[0514] R1 is the free C-terminus or amidated C-terminus of Ser;

[0515] L is selected from -NH(CH2) n NH- or -NH(CH2CH2O) n NH-linker;

[0516] R4 is selected from hydrogen, amino (C1-C 20) alkyl, -NHCOCH3 or -NHCONH2;

[0517] or a retro analog or a pharmaceutically acceptable stereoisomer or a pharmaceutically acceptable salt thereof.

[0518] In yet other embodiments, the small molecule antagonist is represented by the following general formula:

[0519]

[0520] in,

[0521] R1 is the N-terminus of Ser; or (C1-C 20 ) acyl group;

[0522] L is selected from -NH(CH2) n NH-, -NH(CH2) n CH(NH2)CO-, -OOC(CH2) m COO-, -NH(CH2) n CO-, -NH(CH2CH2O) n NH-, -NH(CH2CH2O) n CO- or -CO(CH2CH2O) n CO- connector;

[0523] R2 is the free C-terminus, amidated C-terminus or N-terminus of Am2; or Y-R5;

[0524] Y is selected from -OOC(CH2) m COO-, -CO(CH2) n NH-, -CO(CH2CH2O) n NH- or -COCH2(OCH2CH2) n Optional linker for NH-;

[0525] R5 is an albumin binding moiety, such as maleimidopropionic acid;

[0526] R3 is OH or NH2;

[0527] R4 is a substituent on the phenyl group of Phe and is selected from hydrogen, amino (C1-C 20 ) alkyl, -NHCOCH3 or -NHCONH2;

[0528] n is an integer having a value selected from 2 to 10, inclusive;

[0529] m is an integer having a value selected from 0 to 8, inclusive; and

[0530] One of the peptide bonds (-CONH-) of Ser-Asn, Asn-Thr or Thr-Ser may be The modified peptide bond replacement,

[0531] Where Q is hydrogen, -CO(C1-C 20 )alkyl or -COO(C1-C 20 ) alkyl; wherein one or more or all amino acids may be in the D-configuration;

[0532] or a retro analog or a pharmaceutically acceptable stereoisomer or a pharmaceutically acceptable salt thereof.

[0533] For example, the small molecule antagonist may be selected from the group consisting of:

[0534]

[0535]

[0536]

[0537]

[0538]

[0539]

[0540]

[0541] CTLA-4 antagonists

[0542] In certain embodiments, the combinations described herein further comprise a CTLA-4 inhibitor. Exemplary anti-CTLA-4 antibodies include Tremelimumab (an IgG2 monoclonal antibody available from Pfizer, formerly known as ticilimumab, CP-675,206); and Ipilimumab (a CTLA-4 antibody also known as MDX-010, CAS No. 477202-00-9).

[0543] Information about tremelimumab (or its antigen-binding fragment) for use in the methods provided herein can be found in US Pat. No. 6,682,736 (incorporated by reference) (referred to therein as 11.2.1), the disclosure of which is incorporated herein by reference in its entirety. Tremelimumab (also referred to 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 immune activation in vitro, and some patients treated with tremelimumab have shown tumor regression.

[0544] Tremelimumab for the method provided herein comprises heavy chain and light chain or heavy chain variable region and light chain variable region.In a specific aspect, tremelimumab for the method provided herein or its Fab comprises the light chain variable region containing the amino acid sequence shown above herein and the heavy chain variable region containing the amino acid sequence shown above herein.In a specific aspect, tremelimumab for the method provided herein or its Fab comprises heavy chain variable region and light chain variable region, wherein the heavy chain variable region comprises the CDR1, CDR2 and CDR3 sequences of the Kabat definition shown above herein, and wherein the light chain variable region comprises the CDR1, CDR2 and CDR3 sequences of the Kabat definition shown above herein. Those of ordinary skill in the art will be able to easily determine Chothia definition, Abm definition 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 6,682,736, which is herein incorporated by reference in its entirety.

[0545] 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:

[0546]

[0547]

[0548] Other small molecule CTLA-4 antagonists include

[0549]

[0550] 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 the anti-PD-1 antibody molecule and a dose of about 3 mg / kg of the anti-CTLA-4 antibody (e.g., ipilimumab).

[0551] Other exemplary anti-CTLA-4 antibodies are disclosed, for example, in U.S. Patent No. 5,811,097.

[0552] 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.

[0553] In some embodiments, the LAG3 antibodies are IMP701, IMP731, BMS-986016, LAG525, and GSK2831781. In some embodiments, the LAG3 antagonist comprises a soluble LAG3 receptor, for example, IMP321.

[0554] 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 an OX40 binding portion thereof. 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.

[0555] In some embodiments, the 4-1BB (CD137) agonist is a binding molecule, such as an 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).

[0556] 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).

[0557] 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.

[0558] In certain embodiments, anti-CD39 antibodies are combined with STING agonists, preferably as part of a pharmaceutical composition. Cyclic dinucleotides (CDNs) cyclic diAMP (produced by Listeria monocytogenes and other bacteria) and their analogs cyclic diGMP, and cyclic GMP-AMP are recognized by host cells as pathogen-associated molecular patterns (PAMPs), which bind to pathogen recognition receptors (PRRs) called stimulators of interferon genes (STING). STING is an adaptor protein in the cytoplasm of host mammalian cells that activates TANK binding kinase (TBK1)-IRF3 and NF-κB signaling axes, 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 adaptive protective pathogen-specific immune responses, which are composed 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.

[0559] Exemplary combinations

[0560] In a preferred embodiment, the present invention relates to a combination of an anti-CD39 antibody and an anti-tumor platinum coordination complex for the treatment of cancer, and more particularly for the treatment of cancers 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, iproplatin, nedaplatin, and lobaplatin. Particularly preferred are combinations of anti-CD39 antibodies with cisplatin, oxaliplatin, carboplatin, triplatin tetranitrate, satraplatin, tetraplatin, omiplatin, iproplatin, nedaplatin, and lobaplatin, and even more preferred are combinations with cisplatin and oxaliplatin for the treatment of cancer, and more particularly for the treatment of cancers 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 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, 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.

[0561] In another preferred embodiment, the present invention relates to a combination of an anti-CD39 antibody and a mitotic inhibitor for the treatment of cancer, and more particularly for the treatment of cancers 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 the combination of an anti-CD39 antibody with paclitaxel, docetaxel, vinblastine, vincristine, vindesine, and vinorelbine, and even more preferred is the combination of paclitaxel, docetaxel, vincristine, and vinorelbine for the treatment of cancer, and more particularly for the treatment of cancers 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.

[0562] In another preferred embodiment, the present invention relates to a combination of an anti-CD39 antibody and an anticancer antibiotic for the treatment of cancer, and more particularly for the treatment of the following cancers: lung cancer, sarcoma, malignant melanoma, bladder cancer, prostate cancer, pancreatic cancer, thyroid cancer, stomach 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, daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, pixantrone, valrubicin, mitomycin C, bleomycin, actinomycin A and mithramycin. Particularly preferred are combinations of anti-CD39 antibodies with daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, pixantrone, valrubicin, mitomycin C, bleomycin, actinomycin D and mithramycin, and even more preferred are combinations with daunorubicin, 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.

[0563] In another preferred embodiment, the present invention relates to a combination of an anti-CD39 antibody and a topoisomerase I and / or II inhibitor 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, 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.

[0564] In another preferred embodiment, the present invention relates to a combination of an anti-CD39 antibody and a proteasome inhibitor for the treatment of cancer, and more particularly for the treatment of 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 are combinations of anti-CD39 antibodies with bortezomib, disulfiram, epigallocatechin gallate, and salinosporamide A, and even more preferred are combinations with bortezomib for the treatment of cancer, and more particularly for the treatment of lung cancer, prostate cancer, pancreatic cancer, gastric cancer, liver cancer, colorectal cancer, and brain cancer.

[0565] In another preferred embodiment, the present invention relates to a combination of an anti-CD39 antibody and a histone deacetylase inhibitor 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, 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, sarcoma, prostate, pancreatic, gastric, ovarian, breast, colorectal, renal and brain cancers.

[0566] In another preferred embodiment, the present invention relates to a combination of an anti-CD39 antibody and a nitrogen mustard alkylating agent for the treatment of cancer, and more particularly for the treatment of 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 are combinations of anti-CD39 antibodies with melphalan, ifosfamide, chlorambucil, cyclophosphamide, mechlorethamine, uramustine, estramustine, and bendamustine, and even more preferred are combinations with cyclophosphamide for the treatment of cancer, and more particularly for the treatment of 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 are combinations of anti-CD39 antibodies with lomustine, semustine, carmustine, fotemustine, and streptozotocin, and even more preferably with carmustine to treat cancer, and more particularly to treat lung cancer, ovarian cancer, and breast cancer.

[0567] In another preferred embodiment, the present invention relates to a combination of an anti-CD39 antibody and an atypical alkylating agent for the treatment of cancer, and more particularly for the treatment of 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 are combinations of anti-CD39 antibodies with procarbazine, dacarbazine, temozolomide and altretamine, and even more preferred are combinations with dacarbazine and temozolomide for the treatment of 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 and an estrogen antagonist for the treatment of cancer, and more particularly for the treatment of breast cancer. This group of chemotherapeutic agents includes, but is not limited to, toremifene, fulvestrant, tamoxifen and nafoxidine. Particularly preferred are combinations of anti-CD39 antibodies with toremifene, fulvestrant, tamoxifen and nafoxidine, and even more preferred are combinations with tamoxifen for the treatment of breast cancer.

[0568] In another preferred embodiment, the present invention relates to a combination of an anti-CD39 antibody and an androgen antagonist for the treatment of cancer, and more particularly for the treatment of prostate cancer. This group of chemotherapeutic agents includes, but is not limited to, bicalutamide, flutamide, MDV3100, and nilutamide. Particularly preferred is the combination of an anti-CD39 antibody with bicalutamide, flutamide, MDV3100, and nilutamide, and even more preferred is the combination with flutamide for the treatment of prostate cancer.

[0569] In another preferred embodiment, the present invention relates to a combination of an anti-CD39 antibody and an mTOR inhibitor 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, 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 are combinations of anti-CD39 antibodies with sirolimus, temsirolimus, everolimus, ridaforolimus, KU-0063794, and WYE-354, and even more preferred are combinations with temsirolimus for the treatment of lung cancer, sarcoma, malignant melanoma, prostate cancer, pancreatic cancer, gastric cancer, ovarian cancer, breast cancer, colorectal cancer, and brain cancer.

[0570] In another preferred embodiment, the present invention relates to a combination of an anti-CD39 antibody and a tyrosine kinase inhibitor 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. 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.

[0571] Another aspect contemplated by the invention relates to any of the methods described above, further comprising administering to the patient a MAP kinase pathway inhibitor or a WNT pathway inhibitor.

[0572] 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.

[0573] In some embodiments, the BRAF inhibitor is selected from the group consisting of GDC-0879, PLX-4720, sorafenib tosylate, dabrafenib, and LGX818.

[0574] In some embodiments, the MEK inhibitor is selected from the group consisting of GSK1120212, selumetinib, and MEK162.

[0575] In some embodiments, the WNT pathway inhibitor is a β-catenin inhibitor or a frizzled inhibitor.

[0576] In some embodiments, the β-catenin inhibitor is selected from the group consisting of niclosamide, XAV-939, FH 535, and ICG 001.

[0577] Another aspect contemplated by the invention relates to any of the methods described above, further comprising administering a cancer vaccine to the patient. In some embodiments, the cancer vaccine is a dendritic cell vaccine.

[0578] Another aspect contemplated by the invention relates to any of the above methods, further comprising administering adoptive cell transfer to the patient.

[0579] In some embodiments, the adoptive cell transfer is CAR-T cell therapy.

[0580] Another aspect contemplated by the invention relates to any of the above methods, further comprising administering the antibody therapy to the patient.

[0581] 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.

[0582] In some embodiments, the antibody therapy is selected from the group consisting of trastuzamab, cetuximab, bevacizumab, and rituximab.

[0583] 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 factor, 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.

[0584] In some embodiments of the methods described herein, an anti-CD39 antibody can be combined with a growth factor selected from the group consisting of adrenomedullin (AM), angiopoietin (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.

[0585] 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).

[0586] Administration schedule

[0587] 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 can occur before, simultaneously with, or after radiotherapy. The dosing schedule for such radiotherapy can be determined by a skilled medical practitioner.

[0588] In certain embodiments of the methods described herein, treatment involves administering an anti-CD39 antibody in combination with 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.

[0589] Combined administration can 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.

[0590] It should be understood that the combination of an anti-CD39 antibody and at least one additional 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 with 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 course of treatment with a second therapeutic agent (e.g., chemotherapy). In certain embodiments, the anti-CD39 antibody will be administered within 1 year of treatment with 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 with 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 with 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 with 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).

[0591] 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 therapeutic or preventive purposes, 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 over 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 measurements 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, method of administration, 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 administered once a day, weekly, monthly, or annually or more frequently. In certain embodiments, the anti-CD39 antibody is administered once a week, once every two weeks, once every three weeks, or once every four weeks.

[0592] 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, a 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.

[0593] As is 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 administration of a particular agent at a therapeutically effective dose cannot be ruled out. In some instances, drug therapy must be discontinued and other agents may be tried. However, many agents in the same therapeutic class often exhibit similar side effects and / or toxicity, meaning that the patient has to discontinue therapy or, if possible, suffer from unpleasant side effects associated with the therapeutic agent.

[0594] 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, and the like. The dosing schedule can be determined and subsequently modified by one skilled in the art.

[0595] Thus, the present invention provides methods of administering an anti-CD39 antibody described herein to a subject, the methods comprising administering one or more agents using an intermittent dosing strategy, which can reduce side effects and / or toxicity associated with administering anti-CD39 antibodies, chemotherapeutic agents, and the like. In some embodiments, a 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 to the subject an initial dose of an anti-CD39 antibody and administering subsequent doses of the anti-CD39 antibody approximately once every 2 weeks. In some embodiments, the intermittent dosing strategy comprises administering to the subject an initial dose of an anti-CD39 antibody and administering subsequent doses of the anti-CD39 antibody approximately once every 3 weeks. In some embodiments, the intermittent dosing strategy comprises administering to the subject an initial dose of an anti-CD39 antibody and administering subsequent doses 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.

[0596] Anti-infective treatment combination

[0597] In one embodiment, the present invention provides a method of treating a subject with 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.

[0598] In one embodiment, the invention provides a method of treating a subject with an anti-CD39 antibody, wherein the subject has 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 tetanus, Clostridium botulinum, Bacillus anthracis. anthricis), Yersinia pestis, Mycobacterium leprae, Mycobacterium lepromatosis, and Borriella.

[0599] 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.

[0600] In one embodiment, the present 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 lambia, Cryptosporidium, Pneumocystis carinii, Plasmodium vivax, Babesia microti, Trypanosoma brucei, Trypanosoma cruzi, Leishmania donovani, Toxoplasma gondii, and Nippostrongylus brasiliensis.

[0601] VII. Anti-CD39 Antibody Conjugates

[0602] 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.

[0603] For example, the present invention provides anti-CD39 antibodies conjugated to a therapeutic moiety (i.e., a drug). The therapeutic moiety can be, for example, a cytotoxin, a chemotherapeutic agent, a cytokine, an immunosuppressant, an immunostimulatory agent, a lytic peptide, or a radioisotope. Such conjugates are referred to herein as "antibody-drug conjugates" or "ADCs." Thus, in one aspect, an anti-CD39 antibody according to any of the aspects or embodiments described above is conjugated to a therapeutic moiety. Exemplary therapeutic moieties include cytotoxic moieties, radioisotopes, cytokines, and lytic peptides.

[0604] 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; a microtubule inhibitor such as maytansine or an analog or derivative thereof; an antimitotic agent 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; a glucocorticoid; 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 mechlorethamine, thiothion, chlorambucil, melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, dacarbazine (DTIC), procarbazine, or mitomycin C; a platinum derivative such as cisplatin or carboplatin; duocarmycin A, duocarmycin SA, razepamycin (CC-1065), or an analog or derivative thereof; an antibiotic such as actinomycin D, bleomycin, daunomycin, doxorubicin, idarubicin, mithramycin, mitomycin, mitoxantrone, plicamycin, or 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, α-sarcin, Aleurites fordii protein, dianthin protein, Phytolacca americana protein proteins) 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; and Pseudomonas endotoxin.

[0605] In one embodiment, the anti-CD39 antibody is conjugated to auristatin or a peptide analog, derivative or prodrug thereof. Auristatin has 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 has 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, as well as linkers suitable for conjugating auristatin to Ab, are described in, for example, U.S. Patent Nos. 5,635,483, 5,780,588 and 6,214,345 and International Patent Application Publications WO02088172, WO2004010957, WO 2005081711, WO2005084390, WO2006132670, WO03026577, WO200700860, WO207011968 and WO205082023.

[0606] In another embodiment, the anti-CD39 antibody is conjugated to a 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 JA 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.

[0607] In another embodiment, the anti-CD39 antibody is conjugated to a cytotoxic moiety selected from the group consisting of anthracycline, maytansine, calicheamicin, duocarmycin, razetamicin (CC-1065), Aplysia 10, Aplysia 15, irinotecan, monomethyl auristatin E, monomethyl auristatin F, PDB, or any analog, derivative, or prodrug thereof.

[0608] 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 razetamicin (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.

[0609] In one embodiment, the anti-CD39 antibodies encompassed by the present invention are conjugated to a nucleic acid or nucleic acid-related molecule. In one such embodiment, the conjugated nucleic acid is a cytotoxic ribonuclease (RNase) or 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.

[0610] 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), e.g., as a fusion protein.

[0611] 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.

[0612] In certain embodiments, the chemical moiety is a polymer that increases the half-life of the antibody or fragment in the subject. Suitable polymers include, but are not limited to, hydrophilic polymers, including, but not limited to, polyethylene glycol (PEG) (e.g., a PEG with a molecular weight of 2kDa, 5kDa, 10kDa, 12kDa, 20kDa, 30kDa, or 40kDa), dextran, and monomethoxy polyethylene glycol (mPEG). Lee et al. (1999) (Bioconj.Chem.10:973-981) disclose single-chain antibodies that PEG is conjugated. Wen et al. (2001) (Bioconj.Chem.12:545-553) disclose conjugating antibodies to PEG attached to a radioactive metal chelator (diethylenetriaminepentaacetic acid (DTPA)).

[0613] Anti-CD39 antibodies can also be used in combination with 99 Tc, 90 Y. 111 In, 32 P. 14 C. 125 I. 3 H. 131 I. 11 C. 15 O. 13 N. 18 F. 35 S. 51 Cr, 57 To 226 Ra, 60 Co、 59 Fe, 57 Se, 152 Eu, 67 CU, 217 Ci、 211 At 212 Pb, 47 Sc, 109 Pd, 234 Th, 40 K. 157 Gd, 55 Mn, 52 Tr and 56 Fe label conjugation.

[0614] Anti-CD39 antibodies can also be conjugated to fluorescent or chemiluminescent labels, including fluorophores such as rare earth chelates, fluorescein and its derivatives, rhodamine and its derivatives, isothiocyanates, hemoglobin, phycocyanin, allophycocyanin, o-phthalaldehyde, fluorescamine, 152Eu, dansyl, umbelliferone, fluorescein, luminal labels, isoluminal labels, aromatic acridinium ester labels, imidazole labels, acridinium salt labels, oxalate labels, aequorin labels, 2,3-dihydrophthalazinedione, biotin / avidin, spin labels, and stable free radicals.

[0615] Any method known in the art for conjugating the antibodies and antigen-binding fragments thereof encompassed by the present invention to various moieties may be employed, including those described by Hunter et al. (1962) Nature 144:945; David et al. (1974) Biochemistry 13:1014; Pain et al. (1981) J. Immunol. Meth. 40:219; and Nygren, J., (1982) Histochem. and Cytochem. 30:407. Methods for conjugating antibodies and fragments are conventional and well known in the art.

[0616] VIII. Pharmaceutical composition

[0617] The anti-CD39 antibodies, antibody fragments, nucleic acids or vectors encompassed by the present invention can be formulated into compositions, particularly pharmaceutical compositions. Such compositions comprise a therapeutically or prophylactically effective amount of an anti-CD39 antibody, antibody fragment, nucleic acid or vector encompassed by the present invention, mixed with a suitable carrier (e.g., a pharmaceutically acceptable agent). Typically, the anti-CD39 antibodies, antibody fragment...

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; and / or (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, single-chain Fv (scFv) and bifunctional antibody fragments, 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 amino acid sequence of the scFv is shown in SEQ ID No.

40.

10. 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 biologic, a toxin, a detectable moiety or a label.

11. The anti-CD39 antibody or antigen-binding fragment thereof according to claim 10, wherein the drug is a chemotherapeutic agent or an immunomodulatory agent.

12. The anti-CD39 antibody or antigen-binding fragment thereof of claim 10, wherein the detectable moiety or label is a radionuclide.

13. 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 comprises an Fc domain of the IgG1 or IgG3 isotype.

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

16. 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.

17. 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.

18. The anti-CD39 antibody or antigen-binding fragment thereof of claim 17, 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.

19. 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 18, and one or more pharmaceutically acceptable excipients or solutions.

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

21. Use of an anti-CD39 antibody or antigen-binding fragment thereof according to any one of claims 1 to 18 or a pharmaceutical preparation according to claim 19 in the preparation of a medicament for improving anti-tumor T cell immunity in a subject with a tumor, wherein the intratumoral CD39 高 The number of T cells is reduced, and T cell infiltration into the tumor is enhanced, and / or T cell exhaustion in the tumor is reduced.

22. An isolated nucleic acid molecule encoding the anti-CD39 antibody or antigen-binding fragment thereof according to any one of claims 1 to 18.

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

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

25. A host cell comprising the isolated nucleic acid of claim 22, wherein: a) expressing the anti-CD39 antibody or antigen-binding fragment thereof according to any one of claims 1 to 18; 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 18; and / or c) comprising the vector according to claim 23.

26. A device or kit comprising at least one anti-CD39 antibody or antigen-binding fragment thereof according to any one of claims 1 to 18.

27. The device or kit of claim 26, 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.

28. A device or kit comprising the pharmaceutical formulation, isolated nucleic acid molecule, vector and / or host cell of any one of claims 19 to 25, or the heavy chain and light chain polypeptides of the anti-CD39 antibody or antigen-binding fragment thereof of any one of claims 1-18.

29. A method for producing at least one anti-CD39 antibody or antigen-binding fragment thereof according to any one of claims 1 to 18, 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.

30. Use of at least one anti-CD39 antibody or antigen-binding fragment thereof according to any one of claims 1 to 18 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.

31. The use according to claim 30, 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.

32. A pharmaceutical composition of an anti-CD39 antibody or antigen-binding fragment thereof according to any one of claims 1 to 18 for use in the preparation of a pharmaceutical composition for use in a subject with a tumor by depleting CD39 in the tumor. 高 Use of cells in a drug for improving anti-tumor T cell immunity, wherein CD39 高 The invention relates to a reduction in the number of T cells, an enhancement in the infiltration of T cells into the tumor, and / or a reduction in T cell exhaustion in 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 18 for the preparation of a medicament for promoting the infiltration of immune cells into a tumor in a subject suffering from a tumor, wherein the number of CD39+CD45-SCA-1+ stromal cells in the tumor is reduced, and / or the infiltration of cytotoxic T cells into the tumor is increased.

34. Use of a pharmaceutical composition of an anti-CD39 antibody or antigen-binding fragment thereof according to any one of claims 1 to 18 for the preparation of a medicament for reducing the inhibition of intratumoral immune cell function by type II NKT cells in a subject suffering from a tumor, wherein the number of type II NKT cells in the tumor is reduced.

35. Use of a pharmaceutical composition of an anti-CD39 antibody or antigen-binding fragment thereof according to any one of claims 1 to 18 in the preparation of a medicament for reducing the inhibition of the function of intratumoral immune cells by regulatory T cells (Treg) in a subject with a tumor, wherein the CD39 in the tumor 高 The number of Tregs decreased.

36. Use of a pharmaceutical composition of an anti-CD39 antibody or antigen-binding fragment thereof according to any one of claims 1 to 18 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 CD39 in the tumor 高 The number of macrophages decreases.

37. Use of the anti-CD39 antibody or antigen-binding fragment thereof according to any one of claims 1 to 18 in the preparation of a medicament for promoting an anti-tumor immune response in a subject having a tumor, wherein the number of CD39 expressing cells in the tumor is reduced.

38. Use of an anti-CD39 antibody or antigen-binding fragment thereof according to any one of claims 1 to 18 in the preparation of a medicament for promoting T cell-mediated immune function in a tumor of a subject, wherein the promotion 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 32, wherein the intratumoral CD39 高 The cells are selected from hematopoietic stem 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 32, wherein the anti-CD39 antibody or antigen-binding fragment thereof reduces CD39 expression in one or more hematopoietic compartments. 高 Cellular level.

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 18 in the preparation of a medicament for treating a tumor in a subject.

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

44. The method of claim 43, 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.

45. The use according to claim 42, wherein the medicament is for treating liquid tumors.

46. ​​The use according to claim 45, wherein the liquid tumor is leukemia.

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

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

49. The use of claim 48, 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.

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

51. The use of claim 50, 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.

52. 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.

53. The use of claim 52, wherein the anti-CD73 antibody is MEDI9447.

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

55. The use of claim 54, 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.

56. 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.

57. The use of claim 21, wherein the subject is an animal model of cancer.

58. The use of claim 21, wherein the subject is a mammal.

59. The use of claim 58, wherein the mammal is a human or a rodent.

60. The use of any one of claims 32 to 42, wherein the subject is an animal model for cancer.

61. The use of any one of claims 32 to 42, wherein the subject is a mammal.

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

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