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

Abstract

Immunotherapy targeting checkpoint molecules, in combination with conventional therapies (e.g., targeted therapy, chemotherapy, and angiogenesis inhibitors), has shown promise in the treatment of solid or liquid tumors. However, the role of non-tumor cells in the tumor microenvironment indicates that elimination of these cells may be key to initiating an effective immune response against tumors, including tumor infiltration of cytotoxic T cells and other anti-tumor cells of the immune system. Rather than focusing on inhibiting the ectonucleotidase activity of CD39 as an adenosine-generating enzyme, the present invention instead exploits CD39 expression to result in the elimination of intratumoral cells by CD39-dependent ADCC.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Application No. 62 / 885,509, filed August 12, 2019, the entire contents of which are incorporated herein by this reference in their entirety. [Background technology]

[0002] For people with advanced cancer, hope can be a rare, albeit precious, commodity. In recent years, a new class of drugs called immune checkpoint inhibitors has shown remarkable promise, potentially forestalling tumors, preventing tumor growth, and essentially curing some treated individuals. However, these breakthrough therapies present significant challenges. Despite the success of immunotherapies for advanced cancers based on inhibitory antibodies against programmed cell death protein 1 (PD1), PD1 ligand 1 (PDL1), and cytotoxic T-lymphocyte antigen 4 (CTLA4) therapy in advanced cancers, a significant proportion of patients still do not respond to these treatments.

[0003] With increasing attention focused on the immunosuppressive tumor microenvironment as a major driver of resistance, and the characterization of "hot" and "cold" tumors according to the level of immune cell infiltration, researchers have discovered several distinct mechanisms underlying the lack of effective responses to checkpoint monotherapy. Immunologically "hot" tumors contain high levels of infiltrating T cells and more antigens, making them more likely to be recognized by the immune system and provoke a strong immune response. Cancers considered immunologically hot include bladder cancer, head and neck cancer, renal carcinoma, melanoma, and non-small cell lung cancer. However, even among these immunologically "hot" cancers, only a minority of patients still benefit from immunotherapy. In contrast, immunologically "cold" tumors are cancers that, for various reasons, contain few infiltrating T cells and are therefore not recognized as foreign and do not provoke a strong immune system response, making them difficult to treat with current immunotherapies. Classically immunologically "cold" cancers include glioblastoma, as well as ovarian, prostate, pancreatic, and most breast cancers.

[0004] The tumor microenvironment contains many cell types in addition to cancer cells, including bone marrow-derived inflammatory cells, lymphocytes, blood vessels, fibroblasts, and the extracellular matrix (ECM) composed of collagen and proteoglycans. Indeed, tumor drug response is not solely determined by the intrinsic properties of tumor cells, as tumor-associated stromal cells, such as fibroblasts, mesenchymal stem cells (MSCs), immune-inflammatory cells, vascular endothelial cells, and ECM, combine in response to anticancer therapy. In many cases, resistance or non-responsiveness to checkpoint therapy, whether in a tumor with or without T cell infiltration, is the result of the inhibitory effects of other cells present in the tumor. These inhibitory effects range from intratumoral signaling that downregulates or inhibits cytotoxic T cells already present in the tumor to creating a tumor microenvironment that collectively eliminates cytotoxic T cells by reducing their ability to extravasate into the tumor from surrounding blood vessels.

[0005] Therefore, there is a great need in the art to identify alternative mechanisms for enhancing T cell responses. Summary of the Invention [Means for solving the problem]

[0006] The ectonucleotidase CD39 is intended to cause a decrease in intratumoral levels of the enzyme activity associated with that protein, thereby reducing intratumoral levels of the immunosuppressant adenosine. The present invention is based, at least in part, on the further discovery that expression of CD39 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 targeting these cells for tumor elimination using specific antibody-dependent cellular cytotoxicity (ADCC)-competent anti-CD39 antibodies, can be used to increase the infiltration of cytotoxic T cells, effectively converting a "cold" tumor into an immunologically "hot" tumor.

[0007] For example, in one aspect, an anti-CD39 antibody or antigen-binding fragment thereof is provided that comprises: (i) at least one antigen-binding domain that binds to 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 against CD39+ cells to the anti-CD39 antibody.

[0008] Numerous embodiments are further provided that 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 (i) upon incubation with HCC1739BL cells forms stable immune complexes characterized by loss of less than 40% of the immune complex after 24 hours, or optionally less than 35%, 30%, 25%, 20%, 15%, or even less than 10% after 24 hours, where the formation of the immune complex is detected by fluorescence intensity using a fluorescently labeled secondary antibody (e.g., for illustrative purposes only, the stability of immune complexes formed with an anti-CD39 antibody can be determined by incubating an anti-CD39 monoclonal antibody (mAb) (e.g., 2 μg / ml or greater) with HCC1739BL cells for different periods of time, and then measuring the fluorescence intensity. (ii) complement-dependent cytotoxicity (CDC) activity against CD39+ cells; (iii) antibody-mediated targeted cytosis of CD39 on CD45+ immune cells; (iv) antibody-mediated targeted cytosis of CD39 derived from tumor vascular endothelial disruption or vasculature network collapse in tumors; (v) binding to a CD39 epitope having a sequence selected from the group of CD39 amino acid epitope sequences set forth in Figure 33; and / or (vi) promoting binding to CD39 in a manner that is non-competitive or only partially competitive with monoclonal antibody clone A1 that binds to CD39.

[0009] 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 diabody fragment, and / or 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, the agent is selected from the group consisting of a binding protein, enzyme, drug, chemotherapeutic agent, biological agent, toxin, radionuclide, immunomodulatory agent, detectable moiety, and tag. In another embodiment, the anti-CD39 antibody or antigen-binding fragment thereof comprises a VH domain having an amino acid sequence that can be encoded by a nucleic acid sequence or a nucleic acid that hybridizes under stringent conditions to the nucleic acid of SEQ ID NO: 1, and a VL domain having an amino acid sequence that can be encoded by a nucleic acid sequence or a nucleic acid that hybridizes under stringent conditions to the nucleic acid of SEQ ID NO: 3 (e.g., hybridization in 6x sodium chloride / sodium citrate (SSC) at 45°C and washing in 0.2x SSC / 0.1% SDS at 50-65°C). In yet another embodiment, the anti-CD39 antibody or antigen-binding fragment thereof is at least 60% (e.g., at least 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical 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% (e.g., at least 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to the CDRs of SEQ ID NOs: 4, 8, 12, 16, 20, 24, 28, 44, 48, 52, or 56.In yet another embodiment, the anti-CD39 antibody or antigen-binding fragment thereof is at least 60% (e.g., at least 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to SEQ ID NO: 2, 6, 10, 14, 18, 22, 26, 42, 46, 50, or 54. and a variable light chain (VL) that is at least 60% (e.g., at least 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to SEQ ID NO: 4, 8, 12, 16, 20, 24, 28, 44, 48, 52, or 56.In another embodiment, the anti-CD39 antibody or antigen-binding fragment thereof comprises: (i) a CDR1 amino acid sequence that is at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to SEQ ID NO: 29; 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to SEQ ID NO: 31; and a CDR2 amino acid sequence that is at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to SEQ ID NO: 31. and (ii) a CDR1 amino acid sequence that is at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to SEQ ID NO: 32, and at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or more) identical to SEQ ID NO: 33. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to SEQ ID NO: 34, and a light chain having a CDR2 amino acid sequence that is at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical 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 NOs: 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 NOs: 8, 12, 16, 20, 24, 28, 44, 48, 52, or 56, and human framework sequences, forming 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 antigen-binding fragment thereof comprises an Fc domain of an IgG1 or IgG3 isotype, optionally, the Fc domain is human. In another embodiment, the anti-CD39 antibody or antigen-binding fragment thereof is hypofucosylated or defucosylated. In yet another embodiment, the anti-CD39 antibody or antigen-binding fragment thereof is human or humanized. In yet another embodiment, the anti-CD39 antibody or antigen-binding fragment thereof is bispecific and contains at least one additional antigen-binding site for a tumor antigen, an immune checkpoint, or a costimulatory receptor, and functions as a checkpoint inhibitor when the additional antigen-binding site is for an immune checkpoint, or as a costimulatory agonist when the additional antigen-binding site is for a costimulatory receptor. 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 an MHCI molecule, a BTLA receptor, 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.

[0010] In another aspect, a pharmaceutical preparation is provided that includes a therapeutically effective amount of at least one anti-CD39 antibody or antigen-binding fragment thereof described herein, and one or more pharmaceutically acceptable excipients, buffers, or solutions. For example, the pharmaceutical preparation can be for improving anti-tumor T-cell immunity and can be suitable for administration to a subject having a tumor, the pharmaceutical preparation including 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 increases intratumoral CD39. high This results in a reduction in the number of cells, enhancing T cell infiltration into tumors and / or reducing T cell exhaustion in tumors.

[0011] 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 having at least about 90% identity 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.

[0012] In yet another aspect, there are provided isolated immunoglobulin heavy and / or light chain polypeptides encoded by the nucleic acids described herein.

[0013] In another aspect, there is provided a vector comprising the isolated nucleic acid described herein, optionally wherein the vector is an expression vector.

[0014] In yet another aspect, there is provided a host cell comprising an isolated nucleic acid described herein, 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 of a polypeptide described herein; or c) comprises a vector described herein.

[0015] In yet another aspect, a device or kit is provided that comprises at least one anti-CD39 antibody or antigen-binding fragment thereof described herein, and optionally includes a label for detecting the at least one anti-CD39 antibody or antigen-binding fragment thereof, or a complex comprising the anti-CD39 antibody or antigen-binding fragment thereof.

[0016] In another aspect, a device or kit is provided that comprises the pharmaceutical compositions, isolated nucleic acid molecules, isolated immunoglobulin heavy and / or light chain polypeptides, vectors, and / or host cells described herein.

[0017] In yet another aspect, there is provided 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 steps of: (i) culturing a transformed host cell 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.

[0018] In yet another aspect, a method for detecting the presence or level of an anti-CD39 polypeptide is provided, comprising obtaining a sample and detecting the polypeptide in the sample by using at least one anti-CD39 antibody or antigen-binding fragment thereof 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 using an enzyme-linked immunosorbent assay (ELISA), a radioimmunoassay (RIA), an immunochemical assay, Western blot, mass spectrometry, nuclear magnetic resonance, or an intracellular flow assay.

[0019] In another aspect, the intratumoral CD39 high 1. A method for improving anti-tumor T-cell immunity by depleting cells, 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, wherein administering the anti-CD39 antibody or antigen-binding fragment thereof increases intratumoral CD39 high This results in a reduction in the number of cells, enhancing T cell infiltration into tumors and / or reducing T cell exhaustion in tumors.

[0020] In yet another aspect, a method is provided for promoting immune cell infiltration into a tumor, 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, wherein administration of the anti-CD39 antibody or antigen-binding fragment thereof results in the removal and reduction of CD39+CD45-SCA-1+ stromal cells within the tumor, resulting in increased infiltration of the tumor by cytotoxic T cells.

[0021] In yet another aspect, a method is provided for reducing type II NKT cell suppression of intratumoral immune cell function, 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 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 within the tumor.

[0022] In another aspect, a method for reducing regulatory T cell (Treg) suppression of intratumoral immune cell function 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, wherein administering the anti-CD39 antibody or antigen-binding fragment thereof reduces CD39 in the tumor. high Methods are provided that result in the removal and reduction of Tregs.

[0023] In yet another aspect, there is provided a method for reducing tumor-associated macrophage (TAM) suppression of intratumoral immune cell function, 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 described herein, wherein administering the anti-CD39 antibody or antigen-binding fragment thereof reduces tumor-associated macrophage (TAM) suppression of intratumoral immune cell function. high Methods are provided that result in the removal and reduction of TAMs.

[0024] In yet another aspect, a method is provided for promoting an anti-tumor immune response, the method comprising administering to a subject having a tumor an anti-CD39 antibody or antigen-binding fragment thereof described herein in an amount sufficient to result in a reduction in CD39-expressing cells in the tumor.

[0025] In yet another aspect, provided is a method for promoting T cell-mediated immune function in a tumor in a subject, the method comprising: (i) identifying a cancer subject having tumor-infiltrating tumor-reactive lymphocytes below a predetermined threshold, thereby characterized as having a non-invasive or less invasive 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 infiltration by tumor-reactive T cells.

[0026] As noted above, numerous embodiments are further provided that may be applied to any aspect of the invention and / or combined with any other embodiment described herein. For example, in one embodiment, intratumoral CD39 high 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 combinations thereof. In another embodiment, the anti-CD39 antibody or antigen-binding fragment thereof binds to CD39 cells that occur within one or more hematopoietic compartments. highand reducing the level of cells in one or more hematopoietic compartments selected from the group consisting of blood, spleen, and liver. In yet another embodiment, the anti-CD39 antibody or antigen-binding fragment thereof is administered as part of an anti-tumor therapy. In yet another embodiment, the anti-CD39 antibody or antigen-binding fragment thereof is administered as part of an anti-infective therapy, optionally, the anti-infective therapy is an antiviral therapy (treatment of HIV and HBV infections and COVID-19 infections), treatment of Mycobacterium tuberculosis, and treatment of visceral leishmaniasis. In another embodiment, the anti-CD39 antibody or antigen-binding fragment thereof is administered as part of an anti-tumor therapy to treat a solid tumor, optionally, the solid tumor is pancreatic cancer, liver cancer, lung cancer, gastric cancer, esophageal cancer, head and neck squamous cell carcinoma, prostate cancer, colorectal cancer, breast cancer, lymphoma, gallbladder cancer, renal cancer, multiple myeloma, ovarian cancer, cervical cancer, or glioma. In yet another embodiment, the anti-CD39 antibody or antigen-binding fragment thereof is administered as part of an anti-tumor therapy to treat a liquid tumor, optionally wherein the liquid tumor is leukemia. In yet another embodiment, the anti-CD39 antibody or antigen-binding fragment thereof 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 comprises 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 a PD-1 antagonist, a CTLA-4 antagonist, a LAG-3 antagonist, a TIM-3 antagonist, a TIGIT antagonist, and a Siglec-15 antagonist. In yet another embodiment, the treatment comprises 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 a GITR agonist, a CD27 agonist, a 4-1BB agonist, an OX40 agonist, a CD137 agonist, an ICOS agonist, and a CD28 agonist.In yet another embodiment, the therapy includes a VEGFR or VEGF antagonist, an EGFR or EGF antagonist, an IDO inhibitor, an IDO1 inhibitor, an HDAC inhibitor, a PI3K delta 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-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 (such as MEDI9447), a P2X7 antagonist, or an adenosine A2A receptor antagonist. In another embodiment, the treatment method comprises administering one or more innate immune inducers, optionally selected from the group consisting of an inhibitor of the CD47-SIRPα axis (e.g., an antibody or other binding moiety that binds to CD47 or SIRPα and inhibits the interaction of the two molecules), an inhibitor of the CD24-Siglec-10 axis (e.g., an antibody or other binding moiety that binds to CD24 or Siglec-10 and inhibits the interaction of the two molecules), an NGK2A checkpoint inhibitor that blocks HLA-E-driven inhibition of NK cells and CD8+ cells, a STING agonist, a TLR7 / 8 agonist, and a RIG-I agonist. In yet another embodiment, the anti-CD39 antibody or antigen-binding fragment thereof is administered as part of a tumor vaccine, adoptive cell therapy (such as 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, the mammal is a human or a rodent. [Brief explanation of the drawings]

[0027] [Figure 1]Affinity of Ig39-21 measured by flow cytometry using human CD39hi human B lymphoblastoid (HCC1739BL) cells. Fully human anti-CD39 antibody clone Ig39-21, produced by transient transfection, was serially diluted as indicated and incubated with HCC1739BL cells at 4°C for 30 minutes prior to flow cytometry analysis. The Kd was calculated to be 0.412 nM. [Figure 2] Ig39-21 exhibits 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 preincubated with serially diluted Ig39-21 as indicated for 30 minutes at 37°C in 5% CO2, and then co-cultured with effector cells (T:E = 1:6) for 6 hours. ADCC activity was indicated by an increase in luciferase activity above background. RLU: relative light units. EC50 was calculated as 0.014 μg / mL. [Figure 3] Ig39-21 selectively exhibits ADCC activity against human CD39hi Raji cells: Luc-reporter assay. Various Raji cell lines with different human CD39 expression levels were used as target cells, including Raji cells (Raji-hCD39neg), hCD39-transfected Raji cells expressing high levels of human CD39 (Raji-hCD39hi), or hCD39-transfected Raji cells expressing low levels of human CD39 (Raji-hCD39lo). Jurkat cells stably expressing luciferase and hCD16a-158V were used as effector cells. Target cells were preincubated with serially diluted Ig39-21 as indicated for 30 minutes at 37°C in 5% CO2, and then cocultured with effector cells (T:E = 1:6) for 6 hours. ADCC activity was indicated by an increase in luciferase activity (RLU) above background. RLU: Relative Light Units. [Figure 4]Ig39-21 did not exert ADCC activity against HUVEC, a CD39low normal endothelial cell line. Both 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 preincubated with serially diluted Ig39-21 as indicated for 30 minutes at 37°C in 5% CO2, followed by coculture with effector cells (T:E = 1:6) for 6 hours. ADCC activity was indicated by an increase in luciferase activity above background. RLU: relative light units. This data demonstrates the safety of Ig39-21, avoiding potential systemic side effects. [Figure 5-1] CD39 confers resistance of target Raji-hCD39hi cells to NK cytotoxicity. CFSE-labeled Raji-hCD39neg or Raji-hCD39hi cells were used as targets and cocultured with NK-92-CD16 V / V effector cells at various ratios (as indicated) for 6 hours at 37°C, 5% CO2. Target cell death was then analyzed by propidium iodide (P / I) uptake by flow cytometry. Results were expressed as the percentage of CFSE+P / I+ cells. [Figure 5-2] (continuation) [Figure 6] Ig39-21 enhances NK cytotoxicity against Raji-hCD39hi cells. CFSE-labeled Raji-hCD39hi target cells were incubated with or without Ig39-21 antibody (10 μg / mL) at 37°C, 5% CO for 30 min, followed by co-culture with NK-92-CD16 V / V effector cells at various ratios (as indicated) for 6 h at 37°C. Target cell death was analyzed by flow cytometry, and the % of CFSE+P / I+ cells was calculated. [Figure 7-1]Ig39-21 enhances NK cytotoxicity against hCD39hi human B lymphoblastoid (HCC1739BL) cells. CFSE-labeled HCC1739BL target cells were incubated with or without Ig39-21 antibody (10 μg / mL) at 37°C and 5% CO for 30 minutes, followed by co-culture with NK-92-CD16 V / V effector cells at various ratios (as indicated) for 6 hours at 37°C. Target cell death was analyzed by flow cytometry, and the % of CFSE+P / I+ cells was calculated. [Figure 7-2] (continuation) [Figure 8] Defucosylation enhances Ig39-21-mediated ADCC in HCC1739BL cells: Luc-reporter assay. HCC1739BL target cells were preincubated for 30 minutes at 37°C with serially diluted Ig39-21 produced by transient transfection in the absence (Ig39-21 WT) or presence (Ig39-21 AF) of fucosylation inhibitors, and then cocultured with Jurkat effector cells (T:E = 1:6) for 6 hours. ADCC activity was indicated by an increase in luciferase activity above background. RLU: relative light units. EC50 values ​​were calculated as 0.02 μg / mL (Ig39-21 WT) and 0.0008 μg / mL (Ig39-21 AF). [Figure 9-1] Defucosylation enhances Ig39-21-mediated ADCC against HCC1739BL cells: NK cytotoxicity assay. CFSE-labeled HCC1739BL target cells were incubated with serially diluted Ig39-21WT or Ig39-21AF as indicated for 30 minutes at 37°C in 5% CO2. The cells were then cocultured with NK-92-CD16 V / V effector cells (E:T = 1:8) for 6 hours at 37°C. Target cell death was analyzed by flow cytometry, and the percentage of CFSE+P / I+ cells (% cytotoxicity) was calculated. The EC50 was calculated as 0.04 μg / mL (Ig39-21WT) and 0.0006 μg / mL (Ig39-21AF). [Figure 9-2] (continuation) [Figure 9-3] (continuation) [Figure 10] The optimized Ig39-21 (NP501-BK), Ig39-21 WT, and Ig39-21 AF exhibit similar binding affinities using human CD39-positive CHO cells (CHO-hCD39). Ig39-21 WT, Ig39-21 AF, or NP501-BK (optimized versions of Ig39-21 produced using stably transfected cells) were serially diluted as indicated and incubated with CHO-hCD39 cells for 30 minutes at 4°C. The cells were then stained with a secondary antibody (anti-human IgG (Fc-specific), AlexaFluor® 488) for 30 minutes at 4°C and analyzed by flow cytometry. A human IgG1 isotype control antibody (KLH-hIgG1) was used in parallel. The Kd was calculated as 0.48 nM (NP501-BK), 0.52 nM (Ig39-21 WT), and 0.51 nM (Ig39-21AF). [Figure 11] NP501-BK, Ig39-21 WT, and Ig39-21 AF exhibit similar binding affinities using CD39-expressing HCC1739BL cells. Ig39-21 WT, Ig39-21 AF, or NP501-BK were serially diluted as indicated and incubated with HCC1739BL cells for 30 minutes at 4°C. The cells were then stained with a secondary antibody (anti-human IgG (Fc-specific), AlexaFluor® 488) for 30 minutes at 4°C and analyzed by flow cytometry. A human IgG1 isotype control antibody (KLH-hIgG1) was used in parallel. The Kd values ​​were calculated as 0.16 nM (NP501-BK), 0.29 nM (Ig39-21 WT), and 0.35 nM (Ig39-21 AF). [Figure 12]Optimized Ig39-21 (NP501-BK) and defucosylated Ig39-21 exert similar ADCC activity: Luc-reporter assay 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 then cocultured with Jurkat effector cells (T:E = 1:6) for 6 hours. ADCC activity was indicated by an increase in luciferase activity above background. RLU: relative light units. EC50 values ​​were calculated as 0.0017 μg / mL (NP501-BK) and 0.00086 μg / mL (Ig39-21 AF). [Figure 13] NP501-BK and Ig39-21AF exert significantly higher ADCC activity than Ig39-21 WT: NK cytotoxicity against HCC1739BL cells. CFSE-labeled HCC1739BL target cells were incubated with serially diluted human IgG1 isotype control antibody (KLH-hIgG1) or fully human anti-CD39 monoclonal antibodies (Ig39-21 WT, Ig39-21 AF, or NP501-BK) as indicated for 30 minutes at 37°C in 5% CO2. The cells were then co-cultured with NK-92-CD16 V / V effector cells (E:T = 1:8) for 6 hours at 37°C. Target cell death was analyzed by flow cytometry, and the percentage of CFSE+P / I+ cells (% cytotoxicity) was calculated. The EC50 was calculated as 1.58E-04 μg / mL (NP501-BK), 4.22E-03 μg / mL (Ig39-21 WT), and 4.75E-05 μg / mL (Ig39-21 AF). [Figure 14]Fully human anti-CD39 antibodies exhibit 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 (Ig39-21 WT, Ig39-21 AF, or NP501-BK) at 37°C for 30 min and then exposed to 10% normal human serum (NHS) for 2 h. Target cell lysis was analyzed by flow cytometry, and the P / I+ cell ratio (% cytotoxicity) was calculated. EC50 values ​​were calculated as 0.31 μg / mL (NP501-BK), 0.38 μg / mL (Ig39-21 WT), and 0.25 μg / mL (Ig39-21 AF). [Figure 15] NP501-BK and Ig39-21AF exert similar antitumor effects in vivo. C57BL6 humanized CD39 mice (hCD39 KI) subcutaneously implanted with MC38 cells were treated with 5 mg / kg of a human IgG1 isotype control antibody (KLH-hIgG1) or fully human anti-CD39 monoclonal antibodies (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 weekly using digital calipers. Tumor volume (mm3) was determined as L*W*W*0.52. n=5 / group. [Figure 16] NP501-BK treatment results in a decrease in hCD39 expression on CD39hi tumor-infiltrating lymphocytes. MC38 tumor-bearing hCD39KI 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 = 6–8 per group. [Figure 17]NP501-BK exerts antitumor activity in a CD39+SK-MEL-28 xenograft model. NU / J nude mice subcutaneously implanted with SK-MEL-28 xenografts were used to evaluate the in vivo efficacy of NP501-BK. When tumors reached a mean volume of approximately 500 mm3 (considered day 0 of treatment), mice were treated with two ip administrations of 300 μl of saline or 10 mg / kg of NP501-BK on days 0 and 3. Tumor length (L) and width (W) were measured every 3 days using digital calipers. Tumor volume (mm3) was determined as L*W*W*0.52. n = 6–7 per group. [Figure 18-1] 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. 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 cytometry analysis. Cell incubation without chimeric antibodies was used as a control. [Figure 18-2] (continuation) [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 / Ra9B6; a rabbit anti-hCD39 monoclonal antibody chimerized with human IgG1Fc, which has the highest ADCC activity among all chimeric clones) for 30 minutes at 37°C, and then cocultured with Jurkat effector cells (T:E = 1:6) for 6 hours. ADCC activity was indicated by an increase in luciferase activity above background (RLU). RLU: relative light units. The EC50 was calculated as 0.014 μg / mL (Ig39-21 WT) and 0.022 μg / mL (Hu / Ra 9B6). [Figure 20] ADCC activity of 18 human / rabbit chimeric antibodies: Luc-reporter assay using HCC1739BL cells. HCC1739BL cells were used as target cells. Jurkat cells stably expressing luciferase and hCD16a-158V were used as effector cells. A panel of 18 human / rabbit chimeric antibodies was tested for ADCC activity as described above. Briefly, target cells were preincubated with serially diluted antibodies at 37°C for 30 minutes and then co-cultured with Jurkat effector cells (T:E = 1:6) for 6 hours. ADCC activity was indicated by an increase in luciferase activity above background (RLU). RLU: relative light units. Ten of the 18 clones showed positive ADCC activity. [Figure 21] Human / rabbit chimeric antibodies with high ADCC activity: Luc-reporter assay using HCC1739BL cells. Luc-reporter ADCC assay was performed as shown in Figure 20 above. Six clones with high ADCC activity were summarized. Five of the six clones (except 65H5) do not compete with the epitope of clone A1 (see Figure 18). [Figure 22] Human / rabbit chimeric antibodies with low ADCC activity: Luc-reporter assay using HCC1739BL cells. Luc-reporter ADCC assay was performed as shown in Figure 20 above. Four clones with low ADCC activity were summarized, all of which fully compete with the epitope of clone A1 (see Figure 18). 9B6 served as a positive control. [Figure 23] Human / rabbit chimeric antibodies without ADCC activity: Luc-reporter assay using HCC1739BL cells. Luc-reporter ADCC assay was performed as shown in Figure 20 above. Eight clones without ADCC activity were summarized, all of which fully compete with the epitope of clone A1 (see Figure 18). 9B6 served as a positive control. [Figure 24-1] NK cytotoxicity of selected human / rabbit chimeric antibodies 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. The cells were then cocultured with NK-92-CD16 V / V effector cells (E:T = 1:8) for 6 hours at 37°C. Target cell death was analyzed by flow cytometry, and the percentage of CFSE+P / I+ cells (% cytotoxicity) was calculated. Calculated EC50 values ​​were listed. Maximum % cytotoxicity relative to background was determined as follows: maximum % cytotoxicity at 1 μg / mL minus background % cytotoxicity of each clone (10-4 μg / mL). Exemplary chimeric clones in the luc-reporter ADCC-high group (8C11, 8D8, 9B6, 9C10, 48F10, and 65H5) exhibit high NK killing activity. In contrast, chimeric clones from the luc-receptor ADCC-negative group (59B6, 60D9, 62G12, and 62H10) exhibit low NK killing activity. [Figure 24-2] (continuation) [Figure 25]The reference anti-human CD39 monoclonal antibody (hCD39 Ref) inhibits hCD39 ATPase activity against CHO cell membranes. CHO-hCD39 cells were incubated with 10 μg / mL of human IgG1 isotype Ultra-LEAF antibody or anti-hCD39Ref antibody (hCD39 Ref) at 37°C for 30 minutes, followed by incubation with ATP (250 μM) at room temperature for 15 minutes. The supernatant was then collected, and ATP levels were detected by luminescence using CellTiter-Glo®. Cells without antibody (cells + ATP) or ATP alone without cells were detected in parallel, as described in Materials and Methods, and the enzyme activity inhibition rate (%) was calculated. [Figure 26] The reference antibody (hCD39 Ref) and Ig39-21 WT, both containing the same human IgG1 Fc fraction, exert similar ADCC activity and NK cytotoxicity against HCC1739BL cells. CFSE-labeled HCC1739BL target cells were incubated with serially diluted anti-hCD39 monoclonal antibodies (hCD39 Ref, NP501-BK, Ig39-21WT, or Ig39-21AF) for 30 minutes at 37°C in 5% CO2. The cells were then co-cultured with NK-92-CD16 V / V effector cells (E:T = 1:8) for 6 hours at 37°C. Target cell death was analyzed by flow cytometry, and the percentage of CFSE+P / I+ cells (% cytotoxicity) was calculated. The EC50 was calculated as 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). [Figure 27]Figure 1 shows the epitope competition matrix-I for anti-hCD39 reference antibody (hCD39 Ref) on HCC1739BL cells. HCC1739BL cells were incubated with 10 μg / ml of unconjugated human IgG1 isotype Ultra-LEAF antibody or hCD39Ref antibody for 30 minutes at 4°C. Cells were then exposed to AlexaFluor® 647-conjugated antibodies (8C11, 8D8, 8E9, 9B6, and Ig39-21 WT) or PE-conjugated clone A1 for 30 minutes at 4°C, washed twice, and subjected to flow cytometry analysis. The fold change in AF647 or PE MFI detection relative to the isotype control was calculated (no epitope overlap = 1). Ra: rabbit antibody; Hu / Ra: human / rabbit chimeric antibody. [Figure 28] Figure 1 shows the epitope competition matrix-II for anti-hCD39 reference antibody (hCD39 Ref) on HCC1739BL cells. HCC1739BL cells were incubated with 10 μg / ml of unconjugated human IgG1 isotype Ultra-LEAF antibody or hCD39Ref antibody for 30 minutes at 4°C. Next, the cells were 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 stained with secondary antibody (anti-rabbit IgG (H+L), AlexaFluor® 488) for 30 minutes at 4°C. Finally, the cells were washed and analyzed by flow cytometry. The fold change in AF488 MFI detection relative to the isotype control was calculated (no epitope overlap = 1). [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 staining with a secondary antibody (anti-human IgG (Fc-specific), AlexaFluor® 488) for 30 minutes at 4°C. 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, calculated as described in Materials and Methods. Exemplary chimeric clones from the luc-reporter ADCC-negative group (8E9, 59B6, and 67C1) and the ADCC-low group (52G4) did not form stable immune complexes on the cell membrane after 24 hours (e.g., CD39 loss of more than 40%). Hu / Ra: human / rabbit chimeric antibody; hIgG1: humanized rabbit antibody, IgG1 isotype; hIgG4: humanized rabbit antibody, IgG4 isotype. [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 respective humanized clones (IgG1 or IgG4 isotype) were serially diluted as indicated and incubated with HCC1739BL cells for 30 minutes at 4°C. Cells were then stained with secondary antibody (anti-human IgG (Fc specific), AlexaFluor® 488) for 30 minutes at 4°C and analyzed by flow cytometry. Kd was calculated and is shown next to the figure. [Figure 31]NK cytotoxicity of humanized rabbit antibodies against HCC1739BL cells. Human / rabbit chimeric clones (Hu / Ra 8C11, 8D8, and 9C10) and their respective humanized clones (IgG1 or IgG4 isotype) were serially diluted as indicated and incubated with CFSE-labeled HCC1739BL target cells at 37°C in 5% CO for 30 minutes. The cells were then 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 the percentage of CFSE+P / I+ cells (% cytotoxicity) was calculated. The calculated EC50 values ​​are listed. [Figure 32] CDC activity of humanized rabbit antibodies against Raji-hCD39hi cells. Human / rabbit chimeric clones (Hu / Ra 8C11, 8D8, and 9C10) and their respective humanized clones (IgG1 or IgG4 isotype) were serially diluted as indicated and preincubated 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 the P / I+ cell ratio (% cytotoxicity) was calculated. The calculated EC50 values ​​are listed. [Figure 33-1] Conformational epitope mapping. A list of major putative CD39 epitope candidates is shown. An exemplary representative CD39 extracellular domain sequence is provided for reference of CD39 epitope sequences, and a homology model of the human CD39 dimer is also provided for data visualization. [Figure 33-2] (continuation) DETAILED DESCRIPTION OF THE INVENTION

[0028] I. Overview Extracellular adenosine is known to be an inhibitor of immune function. Intracellular adenosine is involved in energy metabolism, nucleic acid metabolism, and the methionine cycle, whereas in the tumor microenvironment, extracellular adenosine plays a crucial role in suppressing immune signaling. The immunosuppressive adenosine 3'5'-monophosphate (cAMP)-mediated pathway signals through the adenosine A2A receptor (A2AR) and can inhibit T lymphocytes and natural killer (NK) cells in hypoxic, inflammatory, and cancerous microenvironments (Ohta et al. (2006) Proc Natl Acad Sci USA, 103:13132-7). Along with recent and evolving positive clinical trial data, preclinical studies have demonstrated that administration of A2AR inhibitors could be a potential novel strategy for immunotherapy. Furthermore, blockade of the adenosine production pathway involving CD39 / CD73 also induces regression of breast cancer, colon cancer, and melanoma in experimental animal models. In the case of anti-CD39 and anti-CD73 antibody therapy, the focus is primarily on inhibiting or reducing the catabolism of ATP and derivative nucleotides, ultimately to adenosine, by binding to these cell surface adenosine-generating enzymes ("ectonucleotidases") and inhibiting their enzymatic activity or removing them from the cell surface.

[0029] The present invention is based, at least in part, on the discovery that certain antibodies against CD39 can selectively target and eliminate CD39+CD45-SCA-1+ stromal cells (e.g., hematopoietic progenitor cells), CD39+ NKT cells, CD39+ macrophages, and CD39+ endothelial cells, as well as CD39+ cancer cells, more efficiently than prior art anti-CD39 antibodies, by antibody-dependent cellular cytotoxicity, CD39-expressing cells in the tumor microenvironment, etc. The resulting intratumoral CD39 high A reduction in cell numbers can lead to 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.

[0030] Without wishing to be bound by any particular theory, some of the antibodies generated by the present inventors are able to form more stable immune complexes with CD39, resulting in more potent ADCC killing effects. The present inventors have observed that antibodies that are unable to form immune complexes with CD39 as stable as those encompassed by the present invention result in a reduction of CD39 through a mechanism that increases CD39 shedding or cytosis (internalization), albeit from the surface, but do not have the same effect in terms of being able to eliminate CD39-expressing cells by antibody-dependent cellular cytotoxicity. In certain embodiments, certain antibodies encompassed by the present invention have been shown to bind to epitopes on CD39 that are non-competitive or only partially competitive with the binding of monoclonal antibody clone A1 to CD39.

[0031] As described in more detail in the exemplary method and illustrated in the figures, rather than directly inhibiting 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 WO2017 / 089334), the inventors' anti-CD39 antibodies were specifically designed to have a human constant region with an lgG1 Fc domain. This design confers FcγRIIIa receptor-dependent cellular activity, such as antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) against CD39+ cells, and / or antibody-mediated targeted cytosis of intratumoral CD39+ cells. As a result, such cellular activity can inhibit the proliferation and proliferation of CD39+ cells within tumors. high This results in the removal and reduction of cells.

[0032] Summarized below are exemplary features of the subject anti-CD39 monoclonal antibodies that are differently taught for use in therapeutic anti-CD39 antibodies described in the literature.

[0033] The subject antibodies target CD39+ cells within tumors through FcγRIIIa receptor-dependent activity (eg, ADCC). As an example, Figures 13 and 26 show that reducing the fucosylation (also known as hypofucosylation or defucosylation) of the fully human anti-CD39 monoclonal antibody, lead clone Ig39-21, either by using a fucosylation inhibitor (Ig39-21 AF) or by optimizing the production process (NP501-BK) dramatically improved its ADCC activity against CD39+ cells in vitro. This was accompanied by enhanced antitumor activity of these defucosylated antibodies in vivo (Figure 15), whereas the fully glycosylated version (Ig39-21 WT) did not demonstrate antitumor activity in the same tumor model (data not shown).

[0034] As predicted by the NK cytotoxicity assays in Figures 13 and 26, the in vitro maximum effective doses (MaxED) of these different fucosylated Ig39-21 antibodies further illustrate their differences in in vivo antitumor activity. For example, defucosylation enhances the MaxED of Ig39-21WT from 0.1 μg / ml to 0.001 μg / ml. This 100-fold increase translates to high efficacy, a favorable safety profile, excellent tolerability, and low cost when translated into the clinic.

[0035] By comparison, the reference hCD39 antibody (hCD39 Ref) used herein shares an antigen-binding site with prior art antibodies. However, in contrast to the Ref antibody used in the current example, that prior art antibody was generated with an Fc portion specifically designed to have abrogated ADCC function (i.e., it was taught to be specifically generated to bind to CD39 and inhibit NTPase activity without causing CD39-dependent ADCC cell killing).

[0036] The ADCC activity of the subject anti-CD39 antibodies selectively inhibits CD39 highIt is directed at cells. For example, Figures 3 and 4 show that the ADCC activity of Ig39-21 is significantly enhanced by CD39 high These results demonstrate that the antibody is selective for the Raji-hCD39hi cells in FIG. 3 and the SK-MEL-28 cells in FIG. 4.

[0037] The relevance of these in vitro data to the in vivo tumor microenvironment: Figure 16 demonstrates that hCD39 is highly upregulated within tumors of MC38-bearing hCD39KI mice (e.g., 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 significantly increased intratumoral CD39 expression. high This results in cell elimination and depletion (Figure 16 and data not shown).

[0038] This functional feature confers tumor specificity to the antibody and avoids systemic side effects, making it a safer anti-CD39 antibody.

[0039] The formation of a stable immune complex between the anti-CD39 antibody and an antigen on the target cell membrane confers high ADCC activity to the antibody. As an example shown in Figure 29, the stability of antibody:antigen immune complexes on the surface of target cells was examined using antibodies selected from three groups: ADCC-high (i.e., NP501-BK, hCD39 Ref, and human / rabbit chimeric clones 8C11, 8D8, 9C10, and 48F10), ADCC-low (human / rabbit chimeric clone 52G4), or ADCC-negative (human / rabbit chimeric clones 8E9, 59B6, and 67C1). A strong positive correlation was clearly observed between the stability of such immune complexes and the ADCC activity of the antibodies. That is, the higher the stability of the antibody:antigen immune complex, the higher the ADCC activity of the antibody.

[0040] The different epitopes of an anti-CD39 antibody are directly related to the ADCC activity of the antibody. As an example, by comparing the epitope of the subject human / rabbit chimeric anti-hCD39 antibody with the commercially available anti-hCD39 monoclonal antibody clone A1, Figure 18 and Figures 21-23 show that the anti-CD39 antibodies bind to CD39 in a manner that is non-competitive or only partially competitive with clone A1, and in this case have a high probability of including high ADCC activity; i.e., of the six ADCC-high antibodies, five (2G12, 8C11, 8D8, 9B6, and 9C10), excluding 65H5, exhibit such properties (Figure 21). In contrast, all antibodies in the ADCC-low (2A11, 5F1, 52G4, and 63B1) and ADCC-negative (8E9, 59B6, 60D9, 62G12, 62H10, 65E10, 67A8, and 67C1) groups present epitopes that completely overlap with clone A1 (Figures 22 and 23).

[0041] Multiple CD39s of interest in the anti-CD39 antibody within the tumor high Cellular targets. As an example, we used the CD39-MC38 colon cancer model in hCD39 KI mice (Figures 15 and 16). The antitumor activity of NP501-BK in this model was confirmed by CD39 high This is a result of its targeting effect on tumor-infiltrating lymphocytes and tumor-associated vascular endothelial cells (data not shown).

[0042] 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 a partial defect in B cell development, but their NK cells are functionally competent. Therefore, the target cells for NP501-BK-mediated ADCC killing in this xenograft tumor model are CD39+ SK-MEL-28 tumor cells, which is consistent with the in vitro ADCC activity of antibodies against SK-MEL-28 cells (Figure 4).

[0043] II. Definition To facilitate the understanding of this invention, a number of terms and phrases are defined below.

[0044] CD39, also known as "cluster of differentiation 39," "ectonucleoside triphosphate diphosphohydrolase-1," or (gene) "ENTPD1" and (protein) "NTPDase1," is a cell surface-located ectonucleotidase with an extracellular-facing catalytic site. This catalytic site catalyzes the hydrolysis of γ- and β-phosphate residues of triphosphonucleosides and diphosphonucleosides to monophosphonucleoside derivatives (ENZYME entry: EC 3.6.1.5), including hydrolyzing P2 receptor ligands such as ATP, ADP, UTP, and UDP (Junger et al. (2011) Nat. Rev. Immunol. 11:201-212). A representative human NTPDase1 protein sequence is provided in UniProtKB entry "P49961 (ENTP1_HUMAN)," and a representative human coding sequence for the enzyme is provided in GenBank accession S73813. Pericellular adenosine can regulate proinflammatory or inhibitory signals within 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; US Pat. Publ. 2013 / 0123345). For example, adenosine binds to the A2A receptor expressed by lymphocytes, causing the accumulation of intracellular cAMP and preventing T cell activation and NK cytotoxicity (Zarek et al. (2008) Blood 111:251-259; Lokshin et al. (2006) Canc. Res. 66:7758-7765).CD39 was first identified as an activation marker for human lymphocytes but was later shown to be a characteristic of regulatory T cells (Kansas et al. (1991) J. Immunol. 146:2235-2244; Deaglio et al. (2007) J. Exp. Med. 204:1257-1265; Borsellino et al. (2007) Blood 110:1225-1232). Loss of CD39 in Tregs significantly impaired their ability to suppress T cell activation, suggesting that the juxtacrine activity of CD39 serves to negatively regulate T cell function (Deaglio et al. (2007) J. Exp. Med. 204:1257-1265). CD8 + T cells generally express CD39 - It has been reported that (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, in recent years, upregulation of this marker in exhausted T cells has been noted in the context of tumors and chronic viral infections (e.g., HCV and HIV, as well as 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).

[0045] The relationship between structure and function of the CD39 protein is well known in the art (e.g., reviewed 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, 11 Cys residues, and two transmembrane regions (Maliszewski et al. (1994) J. Immunol. 153:3574-3583). It is organized into two transmembrane domains, a small cytoplasmic domain containing N- and C-terminal segments, and a large extracellular hydrophobic domain consisting of five highly conserved domains known as apyrase conserved regions (ACR) 1-5, which are required for the enzyme's catabolic activity (Heine et al. (2001) Eur. J. Biochem. 268:364-373). The amino acid sequences of ACR1 and ACR5 contain a phosphate-binding motif (DXG) that is important for stabilizing the interaction of the enzyme with its nucleotide substrate during phosphate cleavage. Furthermore, two ACR residues, Glu174 in ACR3 and Ser218 in ACR4, are also required for enzymatic activity (Heine et al. (2001) Eur. J. Biochem. 268:364-373; Smith et al. (1998) Biochim. Biophys. Acta 1386:65-78). Upon cell surface expression, CD39 becomes catalytically active (Smith et al. (1998) Biochim. Biophys. Acta 1386:65-78).

[0046] Representative human CD39 cDNA and protein sequences are well known in the art and publicly available from the National Center for Biotechnology Information (NCBI). For example, at least seven human CD39 transcript variants are known to encode six distinct human CD39 isoforms. Human CD39 isoform 1 is available under accession numbers NM_001776.5 and NP_001767.3. The transcript variants represent the longest transcript and encode isoform 1. Human CD39 isoform 2, available under accession numbers NM_001098175.1 and NP_001091645.1, uses an alternative 5' exon than transcript variant 1, resulting in a distinct 5' untranslated region (UTR) and translation initiation at an alternative start codon, resulting in a longer, distinct N-terminus. Human CD39 isoform 3, available under accession numbers NM_001164178.1 and NP_001157650.1, uses an alternative 5' exon than transcript variant 1, resulting in a distinct 5' UTR and translation initiation at an alternative start codon, resulting in a longer, distinct N-terminus. Human CD39 isoform 4, available under accession numbers NM_001164179.1 and NP_001157651.1, uses an alternative in-frame splice site compared to transcript variant 1, resulting in a shorter isoform. Human CD39 isoform 5, available under accession numbers NM_001164181.1 and NP_001157653.1, uses an alternative exon in the 5' region, resulting in a distinct 5' UTR and translation initiation at a downstream start codon relative to transcript variant 1, resulting in a shorter isoform. Human CD39 isoform 6, available under accession numbers NM_001164182.1 and NP_001157654.1, does not contain the alternative exon, resulting in a distinct 5'UTR and translation initiation at a downstream start codon relative to transcript variant 1, resulting in a shorter isoform.Human CD39 isoform 6 is also encoded by another transcript variant available under accession numbers NM_001164183.1 and NP_001157655.1, which does not contain the two alternative internal exons, resulting in a distinct 5'UTR and translation initiation at a downstream start codon relative to transcript variant 1, resulting in a shorter isoform.

[0047] The nucleic acid and polypeptide sequences of CD39 orthologs in organisms other than humans are well known, including, for example, mouse CD39 (NM_009848.3 and NP_033978.1), rat CD39 (NM_022587.1 and NP_072109.1), bovine 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).

[0048] Extensive glycosylation of CD39 is associated with its cell surface expression and activity, such that deletion of glycosylated residues or mutation of glycosylated residues to non-glycosylated residues significantly reduces CD39 activity (e.g., deletion or mutation of glycosylated residues 73 at the N-terminus, 333 in the middle, and / or 429 and / or 458 at the C-terminus of rat CD39, or corresponding residues within its orthologs; see Wu et al. (2005) Mol. Biol. Cell. 16:1661-1672). Similarly, mutation of conserved residues in any one or more of the apyrase conserved regions (ACRs) 1 to 5 results in reduced 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).

[0049] Modulation (e.g., reduction) of CD39 activity can be measured in any number of ways (e.g., by measurements described herein, such as using a control, ratio, comparison to baseline, etc.). For example, a CD39 activity modulator can reduce the catalytic activity of an 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 determined by analyzing the concentration of adenosine in a sample. The concentration can be assessed over time. In another embodiment, ATP is added to the tested sample, and the remaining concentrations of ATP, AMP, or adenosine are determined or assessed. Modulation (e.g., decrease) in this context is 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.

[0050] "CD39 antibody" (or "anti-CD39 antibody") refers to an antibody that selectively binds to one or more epitopes of the NTPDase1 protein, and includes monoparatopic antibodies, as well as biparatopic and other multiparatopic antibodies.

[0051] Antibodies and other polypeptides As used herein, the term "antibody" refers to an immunoglobulin molecule that recognizes and specifically binds to a target, e.g., a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or any combination of the foregoing, via at least one antigen recognition site, which antigen recognition site is typically located within a variable region of the immunoglobulin molecule. As used herein, the term encompasses intact polyclonal antibodies, intact monoclonal antibodies, antibody fragments (e.g., Fab, Fab', F(ab')2, and Fv fragments), single-chain Fv (scFv) antibodies, multispecific antibodies, bispecific antibodies, monospecific antibodies, monovalent antibodies, chimeric antibodies, humanized antibodies, human antibodies, fusion proteins comprising the antigen-binding site of an antibody (formatted to include an Fc or other FcγRIII-binding domain), and any other modified immunoglobulin molecule containing an antigen-binding site, so long as the antibody exhibits the desired biological activity.

[0052] "Antibody-mediated targeted cytosis" in the context of the present invention refers to antibody-mediated depletion of CD39 from the surface of CD45+ immune cells without substantially reducing the number of CD45+ immune cells, i.e., through a process other than the induction of CD45+ cell death.

[0053] As used herein, the term "antigen-binding portion" or "antibody-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 by the term "antigen-binding portion" of an antibody, e.g., an anti-CD39 antibody described herein, include (i) Fab fragments, V L , V H (ii) a monovalent fragment consisting of the V, CL, and CH1 domains; (iii) a bivalent fragment comprising an F(ab')2 fragment and two Fab fragments linked by a disulfide bridge at the hinge region; H and an Fd fragment consisting of the CH1 domain; (iv) a V of a single arm of an antibody Land V H Fv fragment consisting of domains, (v) V H (vi) a dAb fragment consisting of a domain (Ward et al., (1989) Nature 341:544-546), and (vi) an isolated complementarity-determining region (CDR), or (vii) a combination of two or more isolated CDRs, optionally joined by a synthetic linker. Such single-chain antibodies are also intended to be encompassed by the term "antigen-binding portion" of an antibody. These and other potential configurations are described in Chan & Carter (2010) Nat. Rev. Immunol. 10:301. These antibody fragments are obtained using conventional techniques known to those of skill 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 techniques or by enzymatic or chemical cleavage of intact immunoglobulins.

[0054] 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. Generally, heavy and light chain variable regions each consist of four framework regions (FRs) and three complementarity-determining regions (CDRs), also known as "hypervariable regions." The CDRs within each chain are held together in close proximity by the framework regions and, together with the CDRs from the other chain, contribute to the formation of the antibody's antigen-binding site. There are at least two techniques for determining CDRs: (1) an approach based on interspecies sequence variability (i.e., Kabat, et al., Sequences of Proteins of Immunological Interest, 5th ed., 1991, National Institutes of Health, Bethesda, Md.), and (2) an approach based on crystallographic studies of antigen-antibody complexes (Al Lazikani, et al., 1997, J. Mol. Biol., 273:927-948). Additionally, a combination of these two approaches is sometimes used in the art to determine CDRs.

[0055] Antibodies can be of any of the five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or their subclasses (isotypes) (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), based on the identity of the heavy chain constant domains, designated alpha, delta, epsilon, gamma, and mu, respectively; preferred CD39 antibodies are of the IgG1 and IgG3 isotypes for most effective engagement with FcγRIII (i.e., Kd of 10 -7 (See below).

[0056] In certain embodiments, antibodies are "hypofucosylated" and may be "defucosylated." A "hypofucosylated" antibody preparation refers to an antibody preparation in which less than 50% of the oligosaccharide chains contain α-1,6-fucosyl. 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-fucosyl. A "defucosylated" antibody does not contain α-1,6-fucosyl in the carbohydrate attached to the CH2 domain of the IgG heavy chain.

[0057] As used herein, the term "monoclonal antibody" refers to an antibody displaying a single binding specificity and affinity for a particular epitope, or a composition of antibodies in which all of the antibodies display a single binding specificity and affinity for a particular epitope. Typically, such monoclonal antibodies are derived from a single cell or nucleic acid encoding the antibody and are propagated without the intentional introduction of any sequence modifications. Thus, the term "human monoclonal antibody" refers to a monoclonal antibody having variable and optional constant regions derived from human germline immunoglobulin sequences. In one embodiment, human monoclonal antibodies are produced by hybridomas obtained, for example, by fusing B cells obtained from a transgenic or transchromosomal non-human animal (e.g., a transgenic mouse having a genome containing human heavy chain and light chain transgenes) to immortalized cells.

[0058] As used herein, the term "humanized antibody" refers to forms of non-human antibodies that are specific immunoglobulin chains, chimeric immunoglobulins, or fragments thereof that contain minimal non-human (e.g., murine) sequences. Typically, humanized antibodies are human immunoglobulins in which residues of the CDRs are replaced by residues from the CDRs of a non-human species (e.g., mouse, rat, rabbit, or hamster) having the desired specificity, affinity, and / or binding function. In some cases, residues of the Fv framework regions of the human immunoglobulin are replaced with corresponding residues in an antibody from the non-human species. Humanized antibodies can be further modified by substitution of additional residues in the Fv framework regions and / or within the replaced non-human residues to improve and optimize the specificity, affinity, and / or binding function of the antibody. Humanized antibodies may comprise variable domains containing all or substantially all of the CDRs corresponding to the non-human immunoglobulin, while all or substantially all of the framework regions are those of human immunoglobulin sequences. In some embodiments, the variable domains comprise framework regions of human immunoglobulin sequences. In some embodiments, the variable domains comprise framework regions of human immunoglobulin consensus sequences. A humanized antibody can also comprise at least a portion of an immunoglobulin constant region or domain (Fc), typically that of a human immunoglobulin. Humanized antibodies are generally considered to be distinct from chimeric antibodies.

[0059] As used herein, the term "human antibody" 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.

[0060] As used herein, the term "chimeric antibody" refers to an antibody in which the amino acid sequences of the immunoglobulin molecule are derived from more than one species. Typically, the variable regions of both the light and heavy chains correspond to the variable regions of antibodies derived from one species of mammal (e.g., mouse, rat, rabbit, etc.) having the desired specificity, affinity, and / or binding capacity to an antigen, while the constant regions are homologous to the sequences of antibodies derived from that species to avoid eliciting an immune response in another species (usually human).

[0061] An "Fc receptor" or "FcR" is a receptor that binds to the Fc region of an immunoglobulin. FcRs that bind to IgG antibodies include receptors of the FcγR family, including allelic variants and alternatively spliced ​​forms of these receptors. The FcγR family consists of three activating receptors (FcγRI, FcγRIII, and FcγRIV in mice; FcγRIA, FcγRIIA, and FcγRIIIA in humans) and one inhibitory (FcγRIIB) receptor.

[0062] An "FcγRIII binding portion" is a peptide, protein, nucleic acid, or other moiety that, when associated with the antigen-binding site of an anti-CD39 antibody, is capable of binding to FcγRIII (CD16) and mediating antibody-dependent cellular cytotoxicity (ADCC). Heavy chain Fc fragments containing the CH2 and CH3 domains of IgG1 and IgG3 isotypes are FcγRIII binding portions.

[0063] The terms "epitope" and "antigenic determinant" are used interchangeably herein and refer to a portion of an antigen capable of being recognized and specifically bound by a particular antibody. When an antigen is a polypeptide, epitopes can be formed from both contiguous amino acids and noncontiguous amino acids juxtaposed by tertiary folding of the protein. Epitopes formed from contiguous amino acids (also called linear epitopes) are usually retained upon protein denaturation, whereas epitopes formed by tertiary folding (also called conformational epitopes) are usually lost upon protein denaturation. Epitopes typically include at least 3, and more usually at least 5, 6, 7, or 8-10 amino acids in a unique spatial conformation.

[0064] As used herein, the terms "specifically binds to" or "specific for" refer to a measurable and reproducible interaction, such as binding between a target and an antibody, that determines the presence of the 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) is an antibody that binds to this target with higher affinity, avidity, more readily, and / or with a longer duration than it binds to other targets. In one embodiment, the extent to which an antibody binds to an unrelated target is less than about 10% of the binding of the antibody to the target, as measured, for example, by radioimmunoassay (RIA). In certain embodiments, an antibody that specifically binds to a target has a dissociation constant (Kd) of 1 μM or less, 100 nM or less, 10 nM or less, 1 nM or less, or even 0.1 nM or less. 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 can include, but does not require, exclusive binding.

[0065] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. A polymer can be linear or branched, it can comprise modified amino acids, and it can be interrupted by non-amino acids. The terms also include amino acid polymers that are modified, either naturally or by intervention, such as by 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, unnatural amino acids), and other modifications known in the art. Because polypeptides encompassed by the present invention can be based on antibodies or other members of the immunoglobulin superfamily, it is understood that in certain embodiments the polypeptides can occur as single chains or linked chains.

[0066] The terms "identical" or percent "identity" in the context of two or more nucleic acids or polypeptides refer to two or more sequences or subsequences that are the same or have a specified proportion of nucleotides or amino acid residues that are the same when compared and aligned (with gaps introduced, if necessary) for maximum correspondence, without considering any conservative amino acid substitutions as part of the sequence identity. Percent identity may be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software that may be used to obtain 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 variations thereof. In some embodiments, two nucleic acids or polypeptides encompassed by the present invention are substantially identical, meaning that 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%, or 99% nucleotide or amino acid residue identity when compared and aligned for maximum correspondence, as determined using a sequence comparison algorithm or by visual inspection. In some embodiments, identity exists over a region of the amino acid sequence that is at least about 10 residues, at least about 20 residues, at least about 40-60 residues, at least about 60-80 residues in length, or any integer value therebetween. In some embodiments, identity exists over a region longer than 60-80 residues, such as at least about 80-100 residues, and in some embodiments, the sequences are substantially identical over the entire length of the sequences being compared, such as the coding regions of target proteins or antibodies. In some embodiments, the identity exists over a region of the nucleotide sequence that is at least about 10 bases, at least about 20 bases, at least about 40-60 bases, at least about 60-80 bases in length, or any integer value therebetween.In some embodiments, the identity exists over a region longer than 60-80 bases, such as at least about 80-1000 bases or more, and in some embodiments, the sequences are substantially identical over the entire length of the sequence being compared, such as the nucleotide sequence encoding the protein of interest.

[0067] A "conservative amino acid substitution" refers to a substitution of one amino acid residue with another amino acid residue having a similar side chain. A family of amino acid residues with similar side chains is 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), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). For example, the 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 abrogate binding of the polypeptide, soluble protein, or antibody containing that amino acid sequence to its target binding site. Methods for identifying conservative amino acid substitutions that do not eliminate binding are well known in the art.

[0068] An "isolated" polypeptide, soluble protein, antibody, polynucleotide, vector, cell, or composition is a polypeptide, soluble protein, antibody, polynucleotide, vector, cell, or composition that is in a form not found in nature. Isolated polypeptides, soluble proteins, antibodies, polynucleotides, vectors, cells, or compositions include those that have been purified to the extent that they are no longer in the form in which they are found in nature. In some embodiments, an isolated polypeptide, soluble protein, antibody, polynucleotide, vector, cell, or composition is substantially pure.

[0069] As used herein, the term "substantially pure" refers to a material that is at least 50% pure (i.e., free from contaminants), at least 90% pure, at least 95% pure, at least 98% pure, or at least 99% pure.

[0070] 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.

[0071] The term "linker" or "linker region," as used herein, 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 moiety; scFV, Vhh domain, etc.) that binds to different proteins to create a bispecific antibody format that maintains the bivalency of CD39. In some embodiments, the linker moiety 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.

[0072] b.Nucleic acid The terms "polynucleotide" and "nucleic acid" and "nucleic acid molecule" are used interchangeably herein to refer to a polymer of nucleotides of any length, including DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase.

[0073] As used herein, the terms "nucleic acid molecule encoding," "DNA sequence encoding," and "DNA encoding" refer to the order or sequence of nucleotides along a chain of deoxyribonucleic acid deoxyribonucleotides. The order of these deoxyribonucleotides determines the order of amino acids along a polypeptide (protein) chain. Thus, a nucleic acid sequence that encodes an amino acid sequence.

[0074] When used in reference to a nucleotide sequence, "sequence" as used herein, grammatical and other forms of the term may include DNA or RNA, and may be single-stranded or double-stranded. Nucleic acid sequences may be mutated.

[0075] The term "vector" as used herein refers to a construct that can deliver and typically express one or more genes or sequences of interest in a host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmid, cosmid, or phage vectors, DNA or RNA expression vectors associated with cationic condensing agents, and DNA or RNA expression vectors encapsulated in liposomes.

[0076] As used herein, the term "transfection" refers to the transfer of exogenous nucleic acid into a eukaryotic cell. Transfection can be achieved by various means known in the art, such as calcium phosphate-DNA co-precipitation, DEAE-dextran-mediated transfection, polybrene-mediated transfection, electroporation, microinjection, liposome fusion, lipofection, protoplast fusion, retroviral infection, and biolistic technology (particle bombardment).

[0077] As used herein, the term "carrier" refers to an isolated nucleic acid, such as an isolated nucleic acid, that can be used to deliver a composition to the interior of a cell. Several carriers are known in the art, including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term "vector" includes autonomously replicating plasmids or viruses. This term should also be interpreted to include non-plasmid and non-viral compounds, such as polylysine compounds and liposomes, that facilitate the transfer of nucleic acids into cells. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, and retroviral vectors.

[0078] As used herein, the term "expression vector" refers to a vector containing a recombinant polynucleotide comprising an expression control sequence and an operably linked nucleotide sequence to be expressed. An expression vector contains sufficient cis-acting elements used for expression; other elements for expression can be supplied by the host cell or an in vitro expression system. Expression vectors include all those 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).

[0079] As used herein, the term "operably linked" refers to a functional linkage between a regulatory sequence and a heterologous nucleic acid sequence, such that the latter is expressed by the connection. For example, a first nucleic acid sequence and a second nucleic acid sequence are operably linked when there is a functional relationship between the first and second nucleic acid sequences. 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, are those that join two protein-coding regions in the same reading frame.

[0080] As used herein, the term "promoter" is defined as a promoter DNA sequence that is recognized by or directed to the synthetic machinery required for cell-specific transcription of a polynucleotide sequence.

[0081] As used herein, the term "constitutive expression" refers to any expression under physiological conditions. As used herein, the term "inducible expression" refers to expression under specific conditions, such as those that occur upon T cell antigen binding. How do those skilled in the art "induce expression"?

[0082] The term "electroporation" refers to the use of transmembrane electric field pulses to induce microscopic pathways (pores) in biological membranes. These pores allow the passage of biomolecules, such as plasmids or other oligonucleotides, from one side of the cell membrane to the other.

[0083] c. Checkpoint inhibitors, costimulatory agonists, innate immune inducers, and chemotherapeutic agents "Checkpoint molecules" refer to proteins expressed by tissues and / or immune cells that reduce the effectiveness of the immune response depending on the level of expression of the checkpoint molecule. When these proteins are blocked, the "brakes" on the immune system are released, allowing, for example, T cells to more effectively kill cancer cells. 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.

[0084] "Checkpoint inhibitors" refer to drug entities that reverse immunosuppressive signaling from checkpoint molecules.

[0085] A "costimulatory molecule" refers to an immune cell, such as a T cell cognate binding partner, that specifically binds to a costimulatory ligand, thereby mediating costimulation, such as, but not limited to, proliferation. A costimulatory molecule is a cell surface molecule other than an antigen receptor or ligand that promotes an effective immune response. Costimulatory molecules include, but are not limited to, MHCI molecules, BTLA receptors and Toll ligands, as well as OX40, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), and 4-1BB (CD137). Examples of costimulatory molecules include 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, These include, but are not limited to, NKG2D, NKG2C, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactyl), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, 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 ligand.

[0086] "Costimulatory agonist" refers to a drug entity that activates (stimulates) a costimulatory molecule, such as a costimulatory ligand, to generate an immunostimulatory signal or otherwise increase the potency or effectiveness of an immune response.

[0087] An "innate immune inducer" is an agent that mimics the innate immune response, such as activating inflammatory and / or deactivating anti-inflammatory activities of macrophages, NK cells, dendritic cells, monocytes, neutrophils, etc. Innate immune inducers include inhibitors of the CD47-SIRPα axis, such as antibodies or other binding moieties that bind to CD47 or SIRPα and inhibit the interaction of the two molecules to promote anti-tumor macrophage activity. Innate immune inducers include inhibitors of the CD24-Siglec-10 axis, such as antibodies or other binding moieties that bind to CD24 or Siglec-10 and inhibit the interaction of the two molecules to promote anti-tumor macrophage activity. In another embodiment, the innate immune activator can be an NGK2A checkpoint inhibitor that blocks HLA-E-driven inhibition of NK and CD8+ cells. Small molecule inducers of innate immunity include STING agonists, TLR7 / 8 agonists, and RIG-I agonists.

[0088] A "chemotherapeutic agent" is a chemical compound useful in the treatment of cancer. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosifamide (CYTOXAN); alkylsulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylameramines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolmelamine; acetogenins (especially bullatacin and and blattacinone; delta-9-tetrahydrocannabinol (dronabinol, MARINOL; beta-lapachone; lapachol; colchicine; betulinic acid; camptothecin (including synthetic analogs topotecan (HYCAMTIN), CPT-11 (irinotecan, CAMPTOSAR), acetylcamptothecin, scopolecin, and 9-aminocamptothecin); bryostatin; pemetrexed; kallistatin; CC-1065 (including adzelesin, carzelesin, and bzelesin) including synthetic analogs); podophyllotoxin; podophyllic acid; teniposide; cryptophycins (especially cryptophycin 1 and cryptophycin 8); dolastatins; duocarmycins (including synthetic analogs, KW-2189 and CB1-TM1); eluterobin; pancratistatin; TLK-286; CDP323, an oral alpha-4 integrin inhibitor; sarcodictyin; spongistatin; chlorambucil, chlornaphazine, colofosfamide, estramustine, ifos nitrogen mustards such as famide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembicine, fenesterine, prednimustine, trofosfamide, and uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and laninustine; enediyne antibiotics (e.g., antibiotics such as calicheamicins, particularly calicheamicin gamma 11 and calicheamicin omega 11 (see, e.g., Nicolaou et al., Angew. Chem Intl. Ed. Engl., 33:183-186 (1994)); dynemicins, including dynemicin A; esperamicin;and neocarzinostatin chromophores and related chromoprotein enediyne antibiotic chromophores), aclacinomycin, actinomycin, authramycin, azaserine, bleomycin, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (ADRIAMYC IN, morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, doxorubicin HCl liposome injection (DOXIL), and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcelomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin, keramycin Antibiotics such as quelamycin, lodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, and zorubicin; antimetabolites such as methotrexate, gemcitabine (GEMZAR), tegafur (UFTORAL), capecitabine (XELODA), epothilones, and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, and trimetrexate; fludarabine, 6- Purine analogues such as mercaptopurine, thiamiprine, and thioguanine; pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, and imatinib (a 2-phenylaminopyrimidine derivative), as well as other c-Kit inhibitors; anti-adrenals such as aminoglutethimide, mitotane, and trilostane; folic acid supplements such as florinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestravcil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfornithine;Elliptinium acetate; etoglucide; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenameth; pirarubicin; losoxantrone; 2-ethylhydrazide; procarbazine; PSK polysaccharide complex (JHS Natural Products, Eugene, OR); razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triazicon; 2,2',2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, and roridin A) A), and anguidine); urethane; vindesine (ELDISINE, FILDESIN); dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); thiotepa; taxoids, such as paclitaxel (TAXOL), albumin-engineered nanoparticle formulation of paclitaxel (ABRAXANE), and doxetaxel (TAXOTERE); chlorambucil; 6-thioguaiamar nin; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine (VELBAN); platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine (ONCOVIN); oxaliplatin; leucovovin; vinorelbine (NAVELBINE); novantrone; edatrexate; daunomycin; aminopterin; ibandronate; the topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; pharmaceutically acceptable salts, acids, or derivatives of any of the above;and combinations of two or more of the above, such as CHOP (an abbreviation for the combination therapy of cyclophosphamide, doxorubicin, vincristine, and prednisolone), and FOLFOX (an abbreviation for a treatment regimen using oxaliplatin (ELOXATIN™) in combination with 5-FU and leucovorin);

[0089] Also included in this definition are "antihormonal agents," which act to regulate, reduce, block, or inhibit the effectiveness of hormones that can promote cancer growth, often in the form of systemic or body-wide treatments. They may 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, ketoxifene, LY117018, onapristone, and toremifene (FARESTON); antiprogesterone drugs; estrogen receptor downregulators (ERDs); estrogen receptor antagonists, such as fulvestrant (FASLODEX); agents that function to suppress or shut down the ovaries, such as leuprolide acetate (LUPRON and ELIGARD), gonococcal acetate, and steroids. These include luteinizing hormone-releasing hormone (LHRH) agonists such as serelin, buserelin acetate, and tripterelin; antiandrogens such as flutamide, nilutamide, and bicalutamide; and aromatase inhibitors that inhibit the enzyme aromatase, which controls estrogen production in the adrenal glands, such as 4(5)-imidazole, aminoglutethimide, megestrol acetate (MEGASE), exemestane (AROMASIN), formestani, 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 the proliferation of adherent cells, such as PKC-α, Raf, H-Ras, and epidermal growth factor receptor (EGF-R); vaccines, such as THERAPOPE vaccines and gene therapy vaccines, e.g., These include ALLOVECTIN vaccine, LEUVECTIN vaccine, and VAXID vaccine; topoisomerase 1 inhibitors (e.g., LURTOTECAN); anti-estrogens 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 (an ErbB-2 and EGFR dual tyrosine kinase small molecule inhibitor, also known as GW572016); 17AAG (a geldanamycin derivative, a heat shock protein (Hsp) 90 poison), and pharmaceutically acceptable salts, acids, or derivatives of any of the above.

[0090] As used herein, the term "cytokine" refers collectively to proteins released by one cell population that act as intercellular mediators on another cell or that have an autocrine effect on the cell producing 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, such as PROLEUKINrIL-2; tumor necrosis factors, such as TNF-α or TNF-β, TGF-β1-3; and other polypeptide factors, such as leukemia inhibitory factor ("LIF"), ciliary neurotrophic factor ("CNTF"), CNTF-like cytokine ("CLC"), cardiotrophin ("CT"), and Kit ligand ("KL").

[0091] As used herein, the term "chemokine" refers to soluble factors (e.g., cytokines) that have the ability to selectively induce leukocyte chemotaxis and activation. They also trigger the processes of angiogenesis, inflammation, wound healing, and tumorigenesis. Exemplary chemokines include IL-8, the human homolog of mouse keratinocyte chemoattractant (KC).

[0092] d.Treatment The term "dysfunction" in the context of immune dysfunction refers to a state of reduced immune responsiveness to antigenic stimulation. This term includes the common elements of both exhaustion and / or anergy, in which antigen recognition can occur but the subsequent immune response is ineffective in controlling infection or tumor growth.

[0093] As used herein, the term "dysfunctional" also includes refractoriness or unresponsiveness to antigen recognition, specifically, an 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.

[0094] The term "anergy" refers to the incomplete or insufficient signal delivered via the T cell receptor (e.g., intracellular Ca in the absence of ras activation). +2 T cell anergy refers to a state of unresponsiveness to antigenic stimulation due to an increase in T cell proliferation (increase in T cell proliferation). T cell anergy can also occur upon stimulation with antigen in the absence of costimulation, resulting in refractory T cells to subsequent activation by antigen, even in the context of costimulation. The unresponsive state can often be reversed by the presence of interleukin-2. Anergic T cells do not undergo clonal expansion and / or acquire effector function.

[0095] The term "exhaustion" refers to T cell exhaustion as a state of T cell dysfunction resulting from persistent TCR signaling, which occurs during many chronic infections and cancer development. It is distinct from anergy in that it results from persistent signaling rather than through defective or insufficient signaling. It is defined by defective effector function, persistent expression of inhibitory receptors, and a transcriptional state that differs from functional effector or memory T cells. Exhaustion prevents optimal control of infections and tumors.

[0096] "Enhancing T cell function" means inducing, causing, or stimulating T cells to have sustained or amplified biological function, or regenerating 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, and increased antigen responsiveness (e.g., viral, pathogen, or tumor clearance) compared to pre-intervention levels. In one embodiment, the level of enhancement is at least 50%, alternatively 60%, 70%, 80%, 90%, 100%, 120%, 150%, or 200%. Manners for measuring this enhancement are known to those skilled in the art.

[0097] A "T cell dysfunctional disorder" is a disorder or condition of T cells characterized by decreased responsiveness to antigenic stimulation. In a specific embodiment, the T cell dysfunctional disorder is a disorder particularly associated with inappropriately increased CD39 levels. In another embodiment, the T cell dysfunctional disorder is a disorder in which T cells are anergic or have a reduced ability to secrete cytokines, proliferate, or carry out cytolytic activity. In a specific aspect, the decreased responsiveness results in ineffective control of immunogen-expressing pathogens or tumors. Examples of T cell dysfunctional disorders characterized by a T cell dysfunctional disorder include unexplained acute infections, chronic infections, and tumor immunity.

[0098] "Tumor immunity" refers to the process by which tumors evade immune recognition and clearance. Thus, as a therapeutic concept, tumor immunity is "treated" when such evasion is attenuated and tumors are recognized and attacked by the immune system. Examples of tumor recognition include tumor binding, tumor shrinkage, and tumor clearance.

[0099] "Sustained response" refers to a sustained effect on tumor growth reduction after treatment is stopped. For example, the tumor size may remain the same or become smaller compared to the size at the beginning of the administration period. In some embodiments, the sustained response has a duration at least equal to the treatment period, or at least 1.5, 2.0, 2.5, or 3.0 times the treatment period.

[0100] As used herein, the terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals in which a population of cells is characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinoma, blastoma, sarcoma, and blood cancers (such as lymphoma and leukemia).

[0101] As used herein, the terms "tumor" and "neoplasm" refer to any mass of tissue resulting from excessive cell growth or proliferation, either benign (non-cancerous) or malignant (cancerous), including precancerous lesions. Tumor growth is generally uncontrolled, progressive, and does not induce or inhibit the growth of normal cells. Tumors can affect a variety of cells, tissues, or organs, including, but not limited to, the following organs or tissues or corresponding cells: bladder, bone, brain, breast, cartilage, glial cells, esophagus, fallopian tubes, gallbladder, heart, intestine, kidney, liver, lung, lymph nodes, nervous tissue, ovaries, pancreas, prostate, skeletal muscle, skin, spinal cord, spleen, stomach, testes, thymus, thyroid, trachea, urethra, ureter, urethra, uterus, and vagina. Tumors include cancers such as sarcoma, carcinoma, plasmacytoma, or (malignant plasma cell) cancers. Tumors encompassed by the present invention include, but are not limited to, leukemias (e.g., acute leukemia, acute lymphoblastic leukemia, acute myeloid leukemia, acute myeloid leukemia, acute promyelocytic leukemia, acute myeloid monocytic leukemia, acute monocytic leukemia, acute leukemia, chronic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, polycythemia vera), lymphomas (Hodgkin's disease, non-Hodgkin's disease), primary macroglobulinemia disease, heavy chain disease, and solid tumors such as sarcoma cancers (e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, endothelium sarcoma, lymphangiosarcoma, angiosarcoma, lymphangioendotheliosarcoma). sarcoma), synovial sarcoma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colorectal cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, carcinoma, bronchogenic carcinoma, medullary carcinoma, renal cell carcinoma, hepatocellular carcinoma, bile duct carcinoma (Nile duct carcinoma), choriocarcinoma, spermatocyte tumor, embryonal carcinoma, Wilms' tumor, cervical cancer, uterine cancer, testicular tumor, lung cancer, small cell lung cancer, bladder cancer, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pineal tumor, hemangioblastoma, acoustic neuroma, oligodendroglioma, neurilemmoma, meningioma, melanoma, neuroblastoma, retinoblastoma), esophageal cancer, gallbladder, 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.

[0102] 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, resulting in the development of similar cancerous lesions at the new location. "Metastatic" or "metastasizing" cells are cells that lose adhesive contacts with neighboring cells and migrate from the primary site of disease via the bloodstream or lymphatics to invade adjacent body structures.

[0103] The terms "cancer cells" and "tumor cells" refer to the total population of cells derived from a cancer or tumor or precancerous lesion, including both non-tumorigenic cells and tumorigenic stem cells (cancer stem cells), which make up the majority of the cancer cell population. As used herein, the terms "cancer cells" or "tumor cells" are modified by the term "non-tumorigenic" when referring only to cells that do not have the ability to reproduce and differentiate, which distinguishes them from cancer stem cells.

[0104] As used herein, the term "effective amount" refers to an amount to provide a therapeutic or prophylactic benefit.

[0105] As used herein, "complete response" or "CR" refers to the disappearance of all target lesions, "partial response" or "PR" refers to at least a 30% reduction in the sum of the longest diameters (SLD) of the target lesions, based on baseline SLD, and "stable disease" or "SD" refers to neither sufficient shrinkage of target lesions since initiating treatment to qualify for PR nor sufficient increase to qualify for PD, based on the smallest SLD,

[0106] As used herein, "progressive disease" or "PD" refers to at least a 20% increase in the SLD of a target lesion, referenced to the smallest SLD recorded since the start of treatment, or the presence of one or more new lesions.

[0107] As used herein, "progression-free survival" (PFS) refers to the length of time during and after treatment during which the disease being treated (e.g., cancer) does not worsen. Progression-free survival may include the amount of time a patient experiences a complete or partial response, as well as the amount of time a patient experiences stable disease. As used herein, "overall response rate" (ORR) refers to the sum of the complete response (CR) rate and the partial response (PR) rate.

[0108] As used herein, "overall survival rate" refers to the proportion of individuals in a group who are likely to be alive after a specified period of time.

[0109] As used herein, the term "treatment" refers to an individual attempting to alter the process or treatment of a clinical disease caused by cellular intervention, and may be any of the preventative intervention pathways for clinical pathology, including, but not limited to, treatment to prevent the onset or recurrence of disease, alleviate symptoms, reduce the direct or indirect pathological consequences of any disease, prevent metastasis, reduce the rate of disease progression, alleviate or ameliorate disease, or improve prognosis.

[0110] The term "subject" refers to any animal (e.g., mammal), which will be the recipient of a particular treatment, including, but not limited to, humans, non-human primates, dogs, cats, rodents, etc. Typically, the terms "subject" and "patient" are used interchangeably herein, e.g., with reference to a human subject.

[0111] As used herein, the terms "agonist" and "agonistic" 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.

[0112] As used herein, the terms "antagonist" and "antagonistic" 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.

[0113] As used herein, the terms "modulation" and "modulating" refer to a change or alteration in biological activity. Modulation includes, but is not limited to, stimulating activity or inhibiting activity. Modulation can be an increase in activity or a decrease in activity, a change in binding characteristics, or any other change in the biological, functional, or immunological properties associated with the activity of a protein, pathway, system, or other biological target of interest.

[0114] As used herein, the term "immune response" includes responses from both the innate and adaptive immune systems. It includes both cell-mediated and / or humoral immune responses. It includes both T cell and B cell responses, as well as responses from other cells of the immune system, such as natural killer (NK) cells, monocytes, macrophages, etc.

[0115] The term "pharmaceutically acceptable" refers to a substance that is approved or approvable by a regulatory agency of the federal or state government or listed in the United States Pharmacopoeia or other generally recognized pharmacopeia for use in animals, including humans.

[0116] The term "pharmaceutically acceptable excipient, carrier, or adjuvant" or "acceptable pharmaceutical carrier" refers to an excipient, carrier, or adjuvant that can be administered to a subject together with at least one agent of the present disclosure and that does not impair its pharmacological activity and is non-toxic when administered in a dose sufficient to deliver a therapeutic effect. Generally, those skilled in the art and the US FAD consider a pharmaceutically acceptable excipient, carrier, or adjuvant to be an inactive ingredient of any formulation.

[0117] The terms "effective amount" or "therapeutically effective amount" or "therapeutic effect" refer to an amount of an anti-CD39 antibody effective to "treat" a disease or disorder 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 such as enhancing the immune response, enhancing the anti-tumor response, improving the cytolytic activity of immune cells, increasing the killing of tumor cells by immune cells, reducing tumor cell count; reducing tumor formation, tumor frequency, or tumorigenicity; reducing the number or frequency of cancer stem cells; shrinking tumor size; reducing the cancer cell population; inhibiting or stopping cancer cell invasion to peripheral organs, e.g., spread of cancer to soft tissue and bone; inhibiting or stopping metastasis of tumors or cancer cells; inhibiting or stopping the growth of tumors or cancer cells; alleviating to some extent one or more symptoms associated with cancer; reducing morbidity and mortality; improving quality of life; or a combination of such effects.

[0118] The terms "treating" or "treatment" or "to treat" or "alleviating" or "to alleviate" refer to both (1) therapeutic measures that cure, slow, reduce symptoms, and / or halt the progression of a diagnosed pathological condition or disorder, and (2) prophylactic or preventative measures that prevent or slow the onset of the targeted pathological condition or disorder. Thus, those in need of treatment include those already with the disorder as well as those prone to have the disorder and those in whom the disorder is to be prevented. In the case of cancer or tumors, a subject has been "treated" successfully according to the methods encompassed by the present invention if the patient exhibits one or more of the following: an increased immune response, an increased anti-tumor response, an increased cytolytic activity of immune cells, an increased killing of tumor cells by immune cells, a reduction in the number or complete absence of cancer cells; a reduction in tumor size; an inhibition or absence of cancer cell invasion into peripheral organs, such as the spread of cancer cells to soft tissue and bone; an inhibition or absence of tumor or cancer cell metastasis; an inhibition or absence of cancer growth; a reduction in one or more symptoms associated with the particular cancer; a reduction in morbidity and mortality; an improvement in quality of life; a reduction in tumorigenicity; a reduction in the number or frequency of cancer stem cells; or some combination of effects.

[0119] e.Other Whenever an embodiment is described herein using the language "comprising," it is understood that analogous embodiments otherwise described with the terms "consisting of" and / or "consisting essentially of" are also provided. Whenever an embodiment is described herein using the language "consisting essentially of," it is also understood that analogous embodiments otherwise described with the term "consisting of" are also provided.

[0120] As used herein, reference to "about" or "approximately" a value or parameter includes (and describes) embodiments directed to that value or parameter. For example, a statement referring to "about X" includes a statement of "X."

[0121] Thus, the term "and / or" as used herein in phrases such as "A and / or B" is intended to include "both 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).

[0122] III. Anti-CD39 antibody A monoclonal antibody The anti-CD39 antibody may be a monoclonal antibody. Monoclonal antibodies may be prepared using a hybridoma method, such as that described by Kohler and Milstein, Nature, 256:495 (1975). In the hybridoma method, a mouse, hamster, or other suitable host animal is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that specifically bind to the immunizing agent. Alternatively, lymphocytes may be immunized in vitro.

[0123] The immunizing agent typically contains a CD39 polypeptide or a fusion protein thereof. Generally, either peripheral blood lymphocytes ("PBLs") are used if cells of human origin are desired, or spleen cells or lymph node cells are used if cells of non-human mammalian origin are desired. The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp. 59-103). The immortalized cell line is usually a transformed mammalian cell, particularly a myeloma cell of rodent, bovine, or human origin. Rat or mouse myeloma cell lines are usually used. The hybridoma cells may be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of unfused, immortalized cells. For example, if the parent cells are deficient in the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the culture medium for the hybridoma typically contains hypoxanthine, aminopterin, and thymidine ("HAT medium"), which substances prevent the growth of HGPRT-deficient cells.

[0124] Preferred immortalized cell lines are those that fuse efficiently, support stable, high-level antibody expression by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. More preferred immortalized cell lines are mouse myeloma lines, available, 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).

[0125] The culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against the polypeptide. Preferably, the binding specificity of monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). Such techniques and assays are readily known to those skilled in the art. The binding affinity of the monoclonal antibody can be determined, for example, by Scatchard analysis (Munson and Pollard, Anal. Biochem., 107:220 (1980)).

[0126] After the desired hybridoma cells are identified, the clones may 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, the hybridoma cells may be grown in vivo as ascites in a mammal.

[0127] 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.

[0128] Monoclonal antibodies may be produced by recombinant DNA methods, such as those described in U.S. Pat. No. 4,816,567. DNA encoding monoclonal antibodies encompassed by the invention can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). The hybridoma cells encompassed by the invention serve as a preferred source of such DNA. Once isolated, the DNA may be placed into an expression vector and then transfected into host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin proteins, resulting in the synthesis of monoclonal antibodies in the recombinant host cells. The DNA may also be modified, for example, by substituting coding sequences for human heavy and light chain constant domains for the homologous murine sequences [U.S. Pat. No. 4,816,567; Morrison et al., supra], or by covalently linking all or part of the coding sequence for a non-immunoglobulin polypeptide to the immunoglobulin coding sequence. Such non-immunoglobulin polypeptides can be substituted for the constant domains of the antibodies encompassed by the invention, or can be substituted for the variable domains of one antigen-combining site of the antibodies encompassed by the invention to create chimeric bivalent antibodies.

[0129] b. Human and humanized antibodies Anti-CD39 antibodies encompassed by the present invention may further include humanized or human antibodies. Humanized forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (such as Fv, Fab, Fab', F(ab')2, or other antigen-binding subsequences of antibodies) that contain minimal sequence derived from the non-human immunoglobulin. Humanized antibodies include human immunoglobulins (recipient antibodies) in which residues from the recipient's complementarity-determining regions (CDRs) are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat, or rabbit having the desired specificity, affinity, and capacity. In some cases, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies may also comprise residues found neither in the recipient antibody nor in the imported CDR or framework sequences. Generally, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which 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 optionally also comprises 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)).

[0130] Methods for humanizing non-human antibodies are well known in the art. Generally, a humanized antibody has one or more amino acid residues introduced into it from a non-human source. These non-human amino acid residues are often referred to as "import" residues, typically derived from an "import" variable domain. Humanization is essentially performed by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody according to the method of Winter et al. [Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)]. Such "humanized" antibodies are therefore chimeric antibodies (U.S. Pat. No. 4,816,567), in which substantially less intact human variable domain has been substituted by the corresponding sequences of a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.

[0131] Human antibodies can also be produced using various techniques known in the art, such as 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. can also be used to prepare human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985), and Boerner et al. 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 the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, 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 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).

[0132] Antibodies can also be affinity matured using known selection and / or mutagenesis methods, as described above. Preferred affinity-matured antibodies have 5-fold, more preferably 10-fold, and even more preferably 20 or 30-fold better affinity than the starting antibody (generally murine, humanized, or human) from which the mature antibody is prepared.

[0133] c. Bispecific antibodies The anti-CD39 antibodies described herein include bispecific molecules. Anti-CD39 antibodies, or antigen-binding portions thereof, may be derivatized or linked to another functional molecule, e.g., another peptide or protein (e.g., another antibody or ligand for a receptor), to generate a bispecific molecule that binds to at least two different binding sites or target molecules. The antibodies described herein may, in fact, be derivatized or linked to two or more other functional molecules to generate a multispecific molecule that binds to two or more different binding sites and / or target molecules. Such multispecific molecules are also intended to be encompassed by the term "bispecific molecule" as used herein. To create the bispecific molecules described herein, the antibodies described herein can be operatively linked (e.g., by chemical coupling, genetic fusion, noncovalent association, or otherwise) to one or more other binding molecules, such as another antibody, antibody fragment, peptide, or binding mimetic, thereby resulting in a bispecific molecule.

[0134] 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 embodiments described herein in which the bispecific molecule is multispecific, the molecule can further comprise a third binding specificity.

[0135] In certain embodiments, a bispecific (or possibly multispecific) of interest comprises one or more binding domains to an immune checkpoint, e.g., a checkpoint inhibitor 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 comprises binding domains that bind to a checkpoint protein on T cells, particularly a checkpoint associated with T cell exhaustion, such as LAG-3, TIM-3, or TIGIT. In certain embodiments, the multispecific binds to CD39 and one or more other T cell-associated checkpoints, resulting in antibody-dependent cellular cytotoxicity of cells expressing each or both of CD39 and the other checkpoint protein to which it binds.

[0136] In certain embodiments, the bispecific (or possibly multispecific) antibodies of interest comprise one or more binding domains for immune costimulatory receptors that are costimulatory agonists (activators), such as, for example, MHCI 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 multispecificity include 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 (Tactyl), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, 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 ligand.

[0137] In certain embodiments, the subject bispecifics (or in some cases may be multispecifics) comprise one or more binding domains that function as innate immune activators, such as binding moieties for CD47, SIRPα, CD24, Siglec-10, or NKG2A.

[0138] In one embodiment, the bispecific molecules described herein comprise as binding specificities at least one antibody or antibody fragment thereof, such as Fab, Fab', F(ab')2, Fv, or single-chain Fv. The antibody can also be a light or heavy chain dimer, or any smallest fragment thereof, such as an Fv or single-chain (scFv) construct.

[0139] Binding of 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 particular protein-antibody complex of interest by employing a labeled reagent (e.g., an antibody) that is specific for the complex of interest.

[0140] Methods for producing bispecific antibodies are known in the art. Traditional recombinant production of bispecific antibodies is based on the coexpression of two immunoglobulin heavy / light chain pairs, where the two heavy chains have different specificities [Milstein and Cuello, Nature, 305:537-539 (1983)]. Due to the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of 10 different antibody molecules, of which only one has the correct bispecific structure. Purification of the correct molecule is usually achieved by affinity chromatography steps. Similar techniques are disclosed in WO 93 / 08829, published May 13, 1993, and Traunecker et al., EMBOJ., 10:3655-3659 (1991).

[0141] Antibody variable domains with the desired binding specificities (antibody-antigen combining sites) can be fused to immunoglobulin constant domain sequences. Preferably, the fusion is with an immunoglobulin heavy-chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. It is preferred to have the first heavy-chain constant region (CH1) containing the site necessary for light-chain binding present in at least one of the fusions. DNAs encoding the immunoglobulin heavy-chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors and co-transfected into a suitable host organism. For further details on the generation of bispecific antibodies, see, e.g., Suresh et al., Methods in Enzymology, 121:210 (1986).

[0142] According to another approach described in WO 96 / 27011, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers recovered from recombinant cell culture. A preferred interface comprises at least a portion of the CH3 region of the antibody constant domain. In this method, one or more small amino acid side chains from the interface of a first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan). A compensatory "cavity" of identical or similar size to the large side chain(s) is created on the interface of a second antibody molecule by replacing the large amino acid side chain(s) with smaller ones (e.g., alanine or threonine). This provides a mechanism for increasing the yield of heterodimers over other unwanted end-products, such as homodimers.

[0143] Bispecific antibodies can be prepared as full-length antibodies or antibody fragments (e.g., F(ab')2 bispecific antibodies). Techniques for generating bispecific antibodies from antibody fragments have been described in the literature. For example, bispecific antibodies can be prepared using chemical linkage. Brennan et al., Science 229:81 (1985) describe a procedure in which intact antibodies are proteolytically cleaved to generate F(ab')2 fragments. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular disulfide formation. The generated Fab' fragments are then converted to thionitrobenzoate (TNB) derivatives. One of the Fab'-TNB derivatives is reconverted to the Fab'-thiol by reduction with mercaptoethylamine and mixed with an equimolar amount of the other Fab'-TNB derivative to form the bispecific antibody. The generated bispecific antibody can be used as an agent for the selective immobilization of enzymes.

[0144] 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) described the production of fully humanized bispecific antibody F(ab')2 molecules. Each Fab' fragment was separately secreted from E. coli and subjected to directed chemical coupling in vitro to form the bispecific antibody. The bispecific antibody thus formed was capable of binding to cells overexpressing the ErbB2 receptor and normal human T cells, as well as inducing the lytic activity of human cytotoxic lymphocytes against human breast tumor targets.

[0145] Various techniques for producing and isolating bispecific antibody fragments directly from recombinant cell culture have also been described. For example, bispecific antibodies have been produced using leucine zippers (Kostelny et al., J. Immunol. 148(5):1547-1553 (1992)). The leucine zipper peptides from the Fos and Jun proteins were linked to the Fab' portions of two different antibodies by gene fusion. Antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized to form the antibody heterodimers. This method can also be utilized for the production of antibody homodimers. The "diabody" technology described by Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993) provides an alternative mechanism for making bispecific antibody fragments. The fragments comprise a light-chain variable domain (V) connected by a linker that is too short to allow pairing between the two domains on the same chain. L ) linked to a heavy chain variable domain (V H ) is included. Therefore, the V of one fragment H and V L Domain complementary to another fragment V L and V H The Fv domains are paired together to form two antigen-binding sites. Another strategy for making bispecific antibody fragments by the use of single-chain Fv (sFv) dimers has also been reported. See Gruber et al., J. Immunol. 152:5368 (1994).

[0146] Antibodies with three or more valencies are contemplated. As one non-limiting example, trispecific antibodies can be prepared. See, e.g., Tutt et al., J. Immunol. 147:60 (1991).

[0147] d. Heteroconjugate antibodies Heteroconjugate antibodies are also within the scope of the present invention. Heteroconjugate antibodies are composed of two covalently linked antibodies. Such antibodies have been proposed, for example, to target immune system cells to unwanted cells (U.S. Pat. No. 4,676,980) and to treat HIV infection (WO 91 / 00360; WO 92 / 200373; EP 03089). It is contemplated that antibodies can be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents. For example, immunotoxins may be constructed using a disulfide exchange reaction or by forming a thioether bond. Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate, as well as those disclosed, for example, in U.S. Pat. No. 4,676,980.

[0148] e. Effector function operation For example, it may be desirable to modify antibodies encompassed by the present invention with respect to effector function to enhance the efficacy of the anti-CD39 antibody in treating cancer. For example, cysteine ​​residue(s) can be introduced into the Fc region, thereby allowing interchain disulfide bond formation in this region. The homodimeric antibody thus generated may have improved internalization capability and / or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC). See Caron et al., J. Exp Med., 176:1191-1195 (1992) and Shopes, J. Immunol., 148:2918-2922 (1992). In certain preferred embodiments, the engineered effector function is the ability of the anti-CD39 antibody to induce FcγRIII binding-dependent removal of CD39 from immune cells (e.g., by anti-CD39 antibody-mediated target cytosis) without depleting the immune cell population by cell killing. 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, an antibody can be engineered which has dual Fc regions and may thereby have enhanced CD39 trogocytosis capabilities. See Stevenson et al., Anti-Cancer Drug Design 3:219-230 (1989).

[0149] f. Representative anti-CD39 antibody sequences In certain embodiments, the anti-CD39 antibody is a fully human antibody, such as one generated from a human antibody library. An exemplary fully human anti-CD39 antibody is clone Ig39-21, the heavy and light chain variable domain (VH and VL) sequences of which are provided as follows: [Table A] In the Ig39-21 clone, the CDRs of each of the VH and VL domains are as follows: [Table B]

[0150] The sequences of exemplary full-length antibodies and exemplary single-chain antibodies (scFVs) utilizing the above VH and VL domains are provided below: [Table C]

[0151] 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, e.g., SEQ ID NO: 2, even more preferably at least 65%, 70%, 75%, 80%, 85%, or even 90% identical to a VH domain sequence described herein, e.g., SEQ ID NO: 2, and is capable of specifically binding to human CD39.

[0152] 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, e.g., SEQ ID NO: 4, even more preferably at least 65%, 70%, 75%, 80%, 85%, or even 90% identical to a VL domain sequence described herein, e.g., SEQ ID NO: 4, and is capable of specifically binding to human CD39.

[0153] In certain embodiments, the anti-CD39 antibody is a humanized antibody comprising a VH domain having human framework sequences related to the CDRs of the VH domain set forth in SEQ ID NOs: 29, 30, and 31, and the CDRs of the corresponding VL domain set forth 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 differ by one, two, or three amino acids in each CDR, so long as the resulting antibody specifically binds to human CD39.

[0154] In certain embodiments, the heavy and light chains of the anti-CD39 antibody have variable domains that can be encoded by nucleic acids that are identical to or hybridize to the VH and VL domain (corresponding) coding sequences described herein, such as those set forth in SEQ ID NO: 1 (VH) and SEQ ID NO: 3 (VL), under stringent conditions (e.g., 6x sodium chloride / sodium citrate (SSC) at 45°C, washed in 0.2x SSC / 0.1% SDS at 50-65°C).

[0155] In some embodiments, anti-CD39 antibodies are produced in rabbits, and the heavy and light chain variable domains 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 as follows: [Table D-1] [Table D-2]

[0156] In some embodiments, anti-CD39 antibodies were generated in rabbits and then humanized by CDR grafting. Exemplary sequences of the VH and VL domains of humanized rabbit anti-CD39 antibodies are as follows: [Table E]

[0157] In some embodiments, the anti-CD39 antibodies provided herein promote: (i) stable immune complex formation upon incubation with HCC1739BL cells characterized by less than 30% loss of immune complexes after 24 hours, where optionally, 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 targeted cytosis of CD39 on CD45+ immune cells; (iv) antibody-mediated targeted cytosis of CD39 from tumor vascular endothelial disruption or vasculature network collapse in tumors; (v) (optionally) binding to a CD39 epitope having a sequence selected from the group of CD39 amino acid epitope sequences listed in Figure 33 (e.g., 1) IYLTDCMERAR, 2) LRMESEE (e.g., binding to one or more linear or conformational CD39 epitopes, such as those selected from the group consisting of: LADR, 3) RVKGPGISKFV, 4) DCMERAREVIPR, 5) LTDCMERAREVIPR, 6) SLSNYPFDFQGAR, 7) CRVKGPGISKF, 8) GAYGWITINYLLGKFSQK, 9) ILRDPCFHPGYKK, and any combination thereof, e.g., RVKGPGISKFV and DCMERAREVIPR, LTDCMERAREVIPR and SLSNYPFDFQGAR, or CRVKGPGISKF, GAYGWITINYLLGKFSQK, and / or ILRDPCFHPGYKK); and / or (vi) (optionally) binding to CD39 in a manner that is non-competitive or only partially competitive with monoclonal antibody clone A1, which binds to CD39.

[0158] The representative anti-CD39 antibody sequences listed above by sequence identification number correspond to the following: [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka]

change

change

change

change

change

change

change

change

change

change

change

change

change

change

change

change

change

change

[0159] For use in human patients, it may be desirable to humanize these antibodies, replacing both the heavy and light chain constant regions with human constant regions and the variable region framework 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.

[0160] 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 SEQ ID NO: 6, 10, 14, 18, 22, 26, 42, 46, 50, or 54, 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.

[0161] 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, 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.

[0162] In certain embodiments, a chimeric antibody is a humanized antibody. Typically, a non-human antibody is humanized to reduce immunogenicity to humans while retaining the specificity and affinity of the parent non-human antibody. Generally, a humanized antibody comprises one or more variable domains in which the HVRs, e.g., CDRs (or portions thereof), are derived from a non-human antibody and the FRs (or portions thereof) are derived from human antibody sequences. Optionally, a humanized antibody also comprises at least a portion of a human constant region. In some embodiments, some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., the antibody from which the HVR residues are derived), e.g., to restore or improve antibody specificity or affinity.

[0163] In certain embodiments, the anti-CD39 antibody is a humanized antibody comprising a VH domain having human framework sequences associated with the CDRs of the VH domain selected from SEQ ID NOs: 6, 10, 14, 18, 22, 26, 42, 46, 50, or 54 and the CDRs of the corresponding VL domain selected from SEQ ID NOs: 8, 12, 16, 20, 24, 28, 44, 48, 52, or 56. The CDRs are preferably identical, but may differ by one, two, or three amino acids in each CDR, so long as the resulting antibody specifically binds to human CD39.

[0164] Humanized antibodies and methods for making them are reviewed, for example, in Almagro and Fransson, Front. Biosci. 13:1619-1633, (2008), and are also 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, Kashmir 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"), Osbourn et al., Methods 36:61-68 (2005), and Klimka et al., Br. J. Cancer, 83:252-260 (2000) (describing a "guided selection" approach to FR shuffling).

[0165] 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 consensus sequences of human antibodies of particular subpopulations of light or heavy chain variable regions (see, e.g., Carter et al. Proc. Natl. Acad. Sci. USA, 89:4285 (1992) and Presta et al. J. Immunol., 151:2623 (1993)), 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 of FR libraries (see, e.g., Baca et al. al., J. Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271:22611-22618 (1996).

[0166] 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).

[0167] For example, human antibodies can be prepared by administering immunogens to transgenic animals that have been modified to produce intact human antibodies or intact antibodies containing human variable regions in response to antigen challenge. Such animals typically contain all or part of human immunoglobulin loci that replace endogenous immunoglobulin loci or that are extrachromosomally present or randomly integrated into the animal's chromosomes. In such transgenic mice, endogenous immunoglobulin loci are generally inactivated. For a review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23:1117-1125 (2005). See also, for example, U.S. Patent Nos. 6,075,181 and 6,150,584 (describing XENOMOUSE technology), U.S. Patent No. 5,770,429 (describing HUMAB technology), U.S. Patent No. 7,041,870 (describing KM MOUSE technology), and U.S. Patent Application Publication No. US2007 / 0061900 (describing VELOCIMOUSE technology). The human variable regions from intact antibodies produced by such animals may be further modified, for example, by combining with different human constant regions.

[0168] Human antibodies can also be produced by hybridoma-based methods. Human myeloma cell lines and mouse-human heteromyeloma cell lines for the production of 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. 51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., (1991) J. Immunol., 147:86.) Human antibodies produced by 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, for example, in U.S. Patent 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).

[0169] Human antibodies may also be generated by isolating Fv clone variable domain sequences from human-derived phage, yeast, or bacterial display libraries. Such variable domain sequences may then be combined with desired human constant domains. Techniques for selecting human antibodies from antibody libraries are described below.

[0170] To illustrate, anti-CD39 antibodies encompassed by the invention can be isolated by screening combinatorial libraries for antibodies with the desired activity(ies). A variety of methods are known in the art for generating, for example, phage or yeast display libraries and screening such libraries for antibodies possessing the desired binding characteristics. Such methods are reviewed, for example, by Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, 2001), and further described, for example, by 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, in Methods in Molecular Biology 248:161-175 (Lo, ed., Human Press, Totowa, NJ, 2003); Sidhu et al., J. Mol. Biol. 338(2):299-310 (2004); Lee et al. 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).

[0171] As an example of phage display, repertoires of VH and VL genes can be cloned separately by polymerase chain reaction (PCR) and randomly recombined into phage libraries, which can then be screened for antigen-binding phage as described in Winter et al., Ann. Rev. Immunol., 12:433-455 (1994). The phage typically display antibody fragments, either as single-chain Fv (scFv) fragments or as Fab fragments. Libraries from immunized 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), naive repertoires can be cloned (e.g., from humans) to provide a single source of antibodies against a wide range of non-self or self antigens without any immunization. Finally, naive libraries can also be synthetically generated by cloning unrearranged V gene segments from stem cells and using PCR primers containing random sequences to encode hypervariable CDR3 regions and achieve rearrangement in vitro, as described in Hoogenboom and Winter, J. Mol. Biol., 227:381-388 (1992). Patent publications describing human antibody phage libraries include, for example, U.S. Patent No. 5,750,373, and U.S. Patent Publication Nos. 2005 / 0079574, 2005 / 0119455, 2005 / 0266000, 2007 / 0117126, 2007 / 0160598, 2007 / 0237764, 2007 / 0292936, and 2009 / 0002360.

[0172] Antibodies or antibody fragments isolated from a human antibody library are considered human antibodies or human antibody fragments herein.

[0173] FcyRIII binding can also be increased by state-of-the-art methods, such as modifying the amino acid sequence of the Fc portion of the antibody or the glycosylation of the Fc portion (see, e.g., 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-fucosyl. Typically, in "low-fucosylated" antibody preparations, 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-fucosyl. "Defucosylated" antibodies do not contain α-1,6-fucosyl in the carbohydrate attached to the CH2 domain of the IgG heavy chain. Mori, K et al., Cytotechnology 55 (2007) 109 and Satoh M, et al., Expert Opin Biol Ther. 6 (2006) 1161-1173 relate to a FUT8 (α-1,6-fucosyltransferase) gene knockout CHO line for producing defucosylated antibodies.

[0174] IV. Expression Vectors In certain embodiments, recombinant expression vectors are used to amplify and express DNA encoding the anti-CD39 antibodies described herein. For example, recombinant expression vectors can be replicable DNA constructs containing synthetic or cDNA-derived DNA fragments encoding the polypeptide chains of anti-CD39 antibodies operably linked to suitable transcriptional and / or translational regulatory elements derived from mammalian, microbial, viral, or insect genes. A transcription unit generally comprises an assembly of (1) genetic element(s) that play 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 can include operator sequences to control transcription. The ability to replicate in a host, usually conferred by an origin of replication, and a selection gene to facilitate recognition of transformants can also be incorporated. DNA regions are "operably linked" when they are functionally related to each other. For example, DNA for a signal peptide (secretory leader) is operably linked to DNA for a polypeptide if it is expressed as a precursor that participates in the secretion of the polypeptide; a promoter is operably linked to a coding sequence if it controls the transcription of that sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned to permit translation. In some embodiments, structural elements intended for use in yeast expression systems include a leader sequence that enables extracellular secretion of the translated protein by the host cell. In other embodiments in which the recombinant protein is expressed without a leader or transport sequence, an N-terminal methionine residue can be included. This residue can optionally be subsequently cleaved from the expressed recombinant protein to provide the final product.

[0175] The choice of expression control sequences and expression vectors will depend on the choice of host. A wide variety of expression host / vector combinations can be used. Useful expression vectors for eukaryotic hosts include, for example, vectors containing expression control sequences from SV40, bovine papilloma virus, adenovirus, and cytomegalovirus. Useful expression vectors for bacterial hosts include known bacterial plasmids, such as E. coli-derived plasmids, including pCR1, pBR322, pMB9, and their derivatives, as well as broad-host-range plasmids such as M13 and other filamentous single-stranded DNA phages.

[0176] Suitable host cells for expression of 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 an appropriate promoter. Prokaryotes include gram-negative or gram-positive organisms, such as E. coli or Bacillus. Higher eukaryotic cells include established cell lines of mammalian origin, as described below. Cell-free translation systems may also be used. Appropriate cloning and expression vectors for use in bacterial, fungal, yeast, and mammalian cell hosts are well known to those of skill in the art.

[0177] Various mammalian cell culture systems are used to express recombinant polypeptides. Expression of recombinant proteins in mammalian cells may be preferred because such proteins are generally correctly folded, appropriately modified, and biologically functional. Examples of suitable mammalian host cell lines include COS-7 (derived from monkey kidney), L-929 (derived from mouse fibroblast), C127 (derived from mouse mammary tumor), 3T3 (derived from mouse fibroblast), CHO (derived from Chinese hamster ovary), HeLa (derived from human cervical carcinoma), BHK (derived from hamster kidney fibroblast), and HEK-293 (derived from human embryonic kidney) cell lines and their variants. Mammalian expression vectors can include non-transcribed elements, such as an origin of replication, a suitable promoter and enhancer linked to the gene to be expressed, and other non-transcribed sequences flanking the 5' or 3' ends, as well as 5' or 3' non-translated sequences, such as necessary ribosome binding sites, polyadenylation sites, splice donor and splice acceptor sites, and transcription termination sequences.

[0178] Expression of recombinant proteins in insect cell culture systems (e.g., baculovirus) also provides a powerful method for producing correctly folded, biologically functional proteins. Baculovirus systems for producing heterologous proteins in insect cells are well known to those skilled in the art. In certain embodiments, the polynucleotide comprises a polynucleotide encoding an antibody light chain that comprises 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 that is capable of specifically binding to human CD39.

[0179] In certain embodiments, the polynucleotide comprises a polynucleotide encoding an antibody heavy chain that comprises 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 that is capable of specifically binding to human CD39.

[0180] V. Encoded Anti-CD39 Antibodies for In Vivo Delivery Therapeutic vectors for delivering the coding sequence of an anti-CD39 antibody to be expressed in a patient can be viral, non-viral, or physical. See, e.g., Rosenberg et al., Science, 242:1575-1578, 1988, and Wolff et al., Proc. Natl. Acad. Sci. USA 86:9011-9014 (1989). A discussion of methods and compositions for use in gene therapy can be found in Eck et al., in Goodman & Gilman's *The Pharmacological Basis of Therapeutics*, Ninth Edition, Hardman et al., eds., 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*, SLEck, ed., 12(3):483-501, 1998; Romano et al., *Stem Cells*, 18:19-39, 2000, and references cited therein. No. 6,080,728 also provides a discussion of a wide variety of 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 virus, herpes simplex virus, vaccinia virus, and adeno-associated viral vectors.

[0181] a. viral vectors Preferred viral vectors are based on non-cytopathic eukaryotic viruses in which non-essential genes have been replaced with nucleic acid constructs carrying nucleic acid sequences encoding epitopes and target sequences of interest. Preferred viruses for certain embodiments encompassed by the present invention are adenoviruses and adeno-associated (AAV) viruses, which are double-stranded DNA viruses already approved for human use in gene therapy. Furthermore, preferred vectors for conferring resistance do not contain immunostimulatory sequences.

[0182] Adenovirus vectors One exemplary method for in vivo delivery of one or more nucleic acid sequences involves the use of an adenoviral expression vector. "Adenoviral expression vector" is meant to include those constructs containing sufficient adenoviral sequences to (a) assist in packaging the construct and (b) express a polynucleotide cloned therein, in either a sense or antisense orientation. Of course, in the context of an antisense construct, expression does not require that the gene product be synthesized. In certain embodiments, the delivery vector relates to the commercially available ORF of cytochrome b5 reductase 3 (CYB5R3), 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 vectors with an expression construct containing the Cyb5r3 gene. Although adenoviral vectors are highly immunogenic and therefore less preferred for administration to induce tolerance by presenting antigens or in the case of autoimmune diseases, these vectors can be used to induce immunity in the treatment of infectious diseases such as influenza, HBV, HCV, and HIV.

[0183] Adeno-associated viral vector (AAV) AAV is a good choice of delivery vehicle due to its safety, i.e., the genetically engineered (recombinant) gene is not integrated into the host genome. Similarly, AAV is not pathogenic and is not associated with any disease. By removing the viral coding sequence, immune responses to viral gene expression are minimized, so rAAV does not induce inflammatory responses. According to certain embodiments, AAV vectors containing epitope sequences comprising the nucleic acid constructs described herein are useful for transducing APCs.

[0184] Typically, viral vectors containing epitopes containing nucleic acid constructs are assembled from polynucleotides encoding the desired epitopes, suitable regulatory elements, and elements required for epitope expression that mediate cell transduction. In one embodiment, adeno-associated virus (AAV) vectors are used. In more specific embodiments, the AAV vector is AAV1, AAV6, or AAV8.

[0185] AAV expression vectors carrying a DNA molecule of interest bounded by AAV ITRs can be constructed by directly inserting a selected sequence(s) into the AAV genome with the major AAV open reading frame ("ORF") excised. Examples of constitutive promoters that can be included in the AAVs of the present invention include, but are not limited to, the exemplified CMV immediate-early enhancer / chicken β-actin (CBA) promoter.

[0186] In eukaryotic cells, expression control sequences typically include a promoter, an enhancer such as those derived from immunoglobulin genes, SV40, cytomegalovirus, etc., and a polyadenylation sequence, which may contain splice donor and acceptor sites. The polyadenylation sequence is generally inserted after the transgene sequence and before the 3' ITR sequence. In one embodiment, bovine growth hormone polyA may be used.

[0187] The selection of these and other common vectors and regulatory elements is conventional, and many such sequences are available. See, e.g., Sambrook et al., and the references cited therein, e.g., 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. Naturally, not all vectors and expression control sequences function equally well to express all of the transgenes of the present invention. However, one of skill in the art can make a selection from among these expression control sequences without departing from the scope of the present invention. Suitable promoter / enhancer sequences can be selected by one of skill in the art using the guidance provided by this application. Such selection is a routine matter and is not intended to limit the molecules or constructs.

[0188] Retroviral vectors In certain embodiments, the viral vector may be a retroviral vector. A "retrovirus" is a virus having an RNA genome. In certain embodiments, a retroviral vector comprises all of the cis-acting sequences required for packaging and integration of the viral genome, namely, (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 required for incorporating genomic RNA into virions. Further details regarding retroviral vectors can be found in 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.

[0189] "Gammaretrovirus" refers to a genus of the Retroviridae family. Exemplary gammaretroviruses include murine stem cell virus, murine leukemia virus, feline leukemia virus, feline sarcoma virus, and avian reticuloendotheliosis virus.

[0190] 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., 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).

[0191] Lentiviral vectors are retroviral vectors that can infect dividing and non-dividing cells and typically produce high viral titers. Some 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).

[0192] In certain embodiments, other retroviral vectors can be used. These include, for example, vectors based on human foamy virus (HFV) or other viruses of the Spumavirus genus. Foamy virus (FVe) is the largest retrovirus known today and is widespread in a variety of mammals, including all non-human primate species, but absent from humans. This complete non-pathogenicity qualifies FV vectors as ideal gene transfer vehicles for human gene therapy and clearly distinguishes them as gene delivery systems from HIV- and gammaretrovirus-derived vectors.

[0193] Non-cytopathic viruses include retroviruses (e.g., lentiviruses), whose life cycle involves reverse transcription of genomic viral RNA into DNA and subsequent proviral integration into host cell DNA. Retroviruses have been approved for human gene therapy trials. The most useful retroviruses are replication-deficient (i.e., capable of directing the synthesis of desired proteins but unable to manufacture infectious particles). Such genetically modified retroviral expression vectors have general utility for highly efficient gene transduction in vivo. Standard protocols for producing replication-deficient retroviruses (such as incorporating exogenous genetic material into a plasmid, transfecting packaging cells with the plasmid, producing recombinant retrovirus using a packaging cell line, collecting viral particles from tissue culture medium, and infecting target cells with the viral particles) are known to those skilled in the art.

[0194] The retroviral genome contains three genes, gag, pol, and env, which encode capsid proteins, polymerase enzyme, and envelope components, respectively. A sequence found upstream of the gag gene contains a signal for packaging the genome into virions. Retroviral vectors are gene transfer plasmids in which a heterologous nucleic acid resides between two retroviral long terminal repeats (LTRs). Retroviral vectors typically contain appropriate packaging signals that enable the retroviral vector, or RNA transcribed using the retroviral vector as a template, to be packaged into viral virions in an appropriate packaging cell line (see, e.g., U.S. Pat. No. 4,650,764). These two long terminal repeat (LTR) sequences are located at the 5' and 3' ends of the viral genome. They contain strong promoter and enhancer sequences and are also required for integration into the host cell genome (Coffin, 1990). To construct a retroviral vector, a nucleic acid encoding one or more oligonucleotide or polynucleotide sequences of interest is inserted into the viral genome in place of specific viral sequences to produce a replication-deficient virus. Also included are episomal or non-integrating forms of retroviral vectors based on lentiviruses (eg, a type of retrovirus).

[0195] Lentiviral vectors are useful when stable expression is required, but they can be immunogenic and have other undesirable effects. Therefore, while lentiviral vectors are convenient for research, caution is required when using them in humans, especially when the goal is to induce tolerance rather than immunity. Lentiviruses are suitable for ex vivo manipulation of T cells or dendritic cells or other antigen-presenting cells for cancer therapy, but mRNA electroporation is safer. However, two recent advances have made the use of lentiviruses safer and more clinically translatable. First, coexpression of a suicide gene and an antigen whose product becomes functional upon drug administration. A classic 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, if some transduced cells become malignant, they can be eradicated. Approximately 12 such systems exist (Duarte et al., Cancer Letters, 324:160-170, 2012). Secondly, there are non-integrating lentiviral vectors currently under development, which 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 those skilled in the art.

[0196] Retroviral vectors suitable for use herein are described, for example, in U.S. Patent Nos. 5,399,346 and 5,252,479; WIPO publications WO92 / 07573, WO90 / 06997, WO89 / 05345, WO92 / 05266, and WO92 / 14829, which provide descriptions of methods for efficiently introducing nucleic acids into human cells using such retroviral vectors. Other retroviral vectors include, for example, mouse mammary tumor virus vectors (e.g., Shackleford et al., Proc. Natl. Acad. Sci. USA 85:9655-9659, 1998), lentiviruses, and the like. An exemplary viral vector is plentilox-IRES-GFP.

[0197] Additional retroviral delivery systems that can be readily adapted for delivery of transgenes encoding anti-CD39 antibody agents are included, by way of example only, in 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.

[0198] 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; 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 (the cassette is located 5' to the U3 region of the 3' LTR and 3' to the sequence encoding the retroviral envelope); and cis-acting sequences for reverse transcription, packaging and integration into a target cell.

[0199] 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 (a promoter suitable for expression in mammalian cells) at the 5' end of the retroviral polynucleotide, a gag nucleic acid domain, a pol nucleic acid domain, and an env nucleic acid domain; a cassette comprising the coding sequence of an anti-CD39 antibody agent operably linked to a heterologous polynucleotide (the cassette is positioned 5' to the 3' LTR and operably linked and 3' to the env nucleic acid domain encoding the retroviral envelope); and cis-acting sequences required for reverse transcription, packaging, and integration into a target cell.

[0200] In certain preferred embodiments of the recombinant replication-competent retrovirus, the envelope is selected from one of amphotropic, polytropic, xenotropic, 10A1, GALV, baboon endogenous virus, RD114, rhabdovirus, alphavirus, measles, or influenza virus envelopes.

[0201] 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 virus (PERV), feline-derived retrovirus RD114, squirrel monkey retrovirus, xenogeneic murine leukemia virus-related virus (XMRV), avian reticuloendotheliosis virus (REV), or gibbon ape leukemia virus (GALV). In certain preferred embodiments of the recombinant replication-competent retrovirus, the retrovirus is a gammaretrovirus.

[0202] In certain preferred embodiments of the recombinant, replication-competent retrovirus, a second cassette is present that includes the coding sequence of a second therapeutic protein, e.g., downstream of the cassette, such as another checkpoint inhibitor polypeptide, a costimulatory polypeptide, and / or an immunostimulatory cytokine (by way of example only). In certain examples, the second cassette can include an internal ribosome entry site (IRES) or a minipromoter or pol III promoter operably linked to the coding sequence of the second therapeutic protein.

[0203] In certain preferred embodiments of the recombinant replication-competent retrovirus, it is preferably a non-lytic, amphotropic retroviral replicating vector that selectively infects and replicates in cells of the tumor microenvironment.

[0204] Other viral vectors as expression constructs Other viral vectors can be used as expression constructs in the present invention to deliver oligonucleotide or polynucleotide sequences to host cells. Vectors derived from viruses such as vaccinia virus, poliovirus, and herpesvirus can be used. These offer several attractive functions in various mammalian cells. Hepatitis B virus is also included.

[0205] b. Non-viral vectors Plasmid vector Other vectors include plasmid vectors. Plasmid vectors have been widely described and are well known to those skilled in the art. See, for example, Sambrook et al., 1989, cited above. For the past several years, plasmid vectors have been used as DNA vaccines to deliver antigen-encoding genes to cells in vivo. Plasmid vectors are particularly advantageous for such delivery because they do not have the same safety concerns as many viral vectors. However, these plasmids have promoters compatible with the host cell and can express peptide epitopes encoded by the nucleic acid within the plasmid. Other plasmids are well known to those skilled in the art. Furthermore, plasmids can be custom designed using restriction enzymes and ligation reactions to remove and add specific fragments of DNA. Plasmids can be delivered via a variety of parenteral, mucosal, and topical routes. For example, DNA plasmids can be injected intramuscularly, intradermally, subcutaneously, or other routes. They can also be administered via nasal sprays or drops, rectal suppositories, and orally. They can also be administered to the epidermis or mucosal surfaces using a gene gun. The plasmid may be obtained in aqueous solution and dried onto gold particles, or may be obtained in association with another DNA delivery system, including, but not limited to, liposomes, dendrimers, cochleates, and microencapsulation.

[0206] Thus, in one aspect, a plasmid is provided for expression of an epitope-containing 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 expresses a polynucleotide comprising a sequence encoding the epitope, an ETS and an MHC II activator sequence, or a sequence encoding the epitope and a secretory signal sequence, and other elements encoded in the construct. The transcription unit comprises a transcriptional control sequence transcriptionally linked to a cellular immune response element-encoding sequence. The transcriptional control sequence may comprise a promoter / enhancer sequence, such as a cytomegalovirus (CMV) promoter / enhancer sequence. However, those skilled in the art will recognize that a variety of other promoter sequences suitable for expression in eukaryotic cells are known and can similarly be used in the constructs disclosed herein. The level of expression of the nucleic acid product will depend on the presence and activation of the associated promoter and associated enhancer elements.

[0207] In certain embodiments, sequences encoding the desired epitope and targeting sequence can be cloned into an expression plasmid containing regulatory elements (i.e., transcriptional control sequences, etc.) for transcription, translation, RNA stability, and replication. Such expression plasmids are well known in the art, and one skilled in the art can design an appropriate expression construct using a polynucleotide containing a sequence encoding a cellular immune response element or a fragment thereof so that the cellular immune response element can be expressed. There are many examples of suitable expression plasmids into which a polynucleotide containing any sequence can be cloned, such as pCI-neo, pUMVC, or pcDNA3.

[0208] Large quantities of bacterial hosts harboring plasmids for expression of cellular immune response elements or fragments thereof may 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 involve removing contaminating lipopolysaccharides (endotoxins) from the bacteria used to propagate the plasmids. This step is most preferably performed for the use of tolerogenic DNA vaccines, as endotoxins act as potent adjuvants and can cause unwanted immune stimulation.

[0209] The purpose of a plasmid is to efficiently deliver a nucleic acid sequence to a therapeutic epitope in a cell or tissue and express the therapeutic epitope. In particular, the purpose of a plasmid may be to achieve high copy number, avoid potential sources of plasmid instability, and provide a means for plasmid selection. With regard to expression, a nucleic acid cassette contains the elements necessary for expression of the nucleic acid within the cassette. Expression involves efficient transcription by the plasmid of the inserted gene, nucleic acid sequence, or nucleic acid cassette. The expression product may be a protein, polypeptide, or RNA. The nucleic acid sequence may be contained in the nucleic acid cassette. Expression of the nucleic acid may be continuous or regulated.

[0210] Mini Circle Nucleic acid construct embodiments described herein can be processed in the form of minicircle DNA. Minicircle DNA refers to small (2-4 kb) circular plasmid derivatives that are free of all prokaryotic vector components. Because minicircle DNA vectors do not contain bacterial DNA sequences, they are less likely to be recognized as foreign and destroyed (typical transgene delivery methods involve plasmids containing foreign DNA). As a result, these vectors can be expressed for longer periods (on the order of weeks or months) compared to traditional plasmids (days to weeks). The smaller size of minicircles expands cloning capacity and facilitates their delivery into cells. Kits for producing minicircle DNA are known in the art and are commercially available (System Biosciences, Inc., Palo Alto, Calif.). Information on minicircle DNA is provided in Dietz et al., Vector Engineering and Delivery Molecular Therapy (2013); 21 8, 1526-1535 and Hou et al., Molecular Therapy - Methods & Clinical Development, Article number: 14062 (2015) doi:10.1038 / mtm.2014.62. Further information on minicircles is provided in Chen ZY, He CY, Ehrhardt A, Kay M A. Mol Ther. 2003 September; 8(3): 495-500, which states that minicircle DNA vectors achieve sustained expression reflected by active chromatin and transcription levels. Gracey Maniar LE, Maniar JM, Chen ZY, Lu J, Fire AZ, Kay M A. Mol Ther. 2013 January; 21(1): 131-8.

[0211] The first step in ultimately achieving expression of a nucleic acid-encoded product is cellular uptake of the nucleic acid. Cellular uptake of nucleic acids depends on several factors, one of which is the length of time the nucleic acid remains in close proximity to the cell surface. For example, intramuscular (i.m.) administration of a buffer solution containing plasmid DNA followed by muscle massage resulted in a significant reduction in gene expression, likely due to leakage of DNA from the muscle directly or via lymphatics (Human Gene Therapy 4:151-159; 1993). Therefore, it may be desirable to formulate nucleic acids with compounds that slow the rate at which the nucleic acid diffuses or transport it away from the site where cellular uptake of the nucleic acid is desired. Furthermore, these compounds may be suitable for administration to an organism by means such as injection, while maintaining or restoring the physical properties necessary to increase cellular uptake of the nucleic acid.

[0212] To effect expression of the oligonucleotide or polynucleotide sequences, the expression construct must be delivered to a cell. In certain embodiments encompassed by the present invention, the expression construct containing one or more oligonucleotide or polynucleotide sequences may simply consist of naked recombinant DNA or a plasmid.

[0213] To prime the immune system, any type of DNA vaccine vector can be engineered to be CpG-rich (to stimulate TLR9 on immune cells) or, conversely, to remove CpGs, replacing CpG motifs with GpG motifs where possible (Ho et al., J. Immunol. 71(9):4920-6, 2003; Ho et al., J. Immunol. 175(9):6226-34, 2005). DNA vaccines can be designed to contain antigen(s) / epitope(s) and can also contain additional genes for coexpression with the antigen to act as adjuvants or immunomodulators (multiple promoter vectors). These DNA vaccines have been shown to be clinically safe, for example, in T1D patients (Roep et al., Sci. Transl. Med. 5(191):191ra82, 2013).

[0214] mechanical delivery system Additional non-viral delivery methods include, but are not limited to, mechanical delivery systems that can be used in vitro, such as the approach described in Woffendin et al., Proc. Proc. Natl. Acad. Sci. USA 91(24):11581, 1994; deposition of photopolymerized hydrogel materials or the use of ionizing radiation (see, e.g., U.S. Pat. No. 5,206,152 and WO92 / 11033); the use of handheld gene transfer particle guns (see, e.g., U.S. Pat. No. 5,149,655); and the use of ionizing radiation to activate transferred genes (see, e.g., U.S. Pat. No. 5,206,152 and WO92 / 11033). Delivery devices may also be biocompatible and biodegradable. The formulation preferably provides a relatively constant level of release of the active ingredient. On the other hand, a more rapid release rate immediately after administration may be desired. The formulation of such compositions is well within the level of one skilled in the art using known techniques.

[0215] Physical methods for enhancing delivery include electroporation (short pulses of high voltage carry nucleic acids across a membrane), gene guns (DNA is attached to gold particles, forcing the DNA to penetrate cells), sonoporation, magnetofection, and hydrodynamic delivery, all of which are known to those skilled in the art. DNA can also be encapsulated in liposomes, preferably cationic liposomes, or polymersomes (synthetic liposomes), which can interact with cell membranes and undergo fusion or endocytosis, resulting in DNA transfer into cells. DNA can also be complexed with polymers (polyplexes) or dendrimers, which can release the load directly into the cell cytoplasm.

[0216] Exemplary carriers useful in this regard include microparticles of poly(lactide-co-glycolide), polyacrylate, latex, starch, cellulose, dextran, and the like. Other exemplary delayed-release carriers include supramolecular biovectors comprising a non-liquid hydrophilic core (e.g., cross-linked polysaccharides or oligosaccharides) and, optionally, an outer layer comprising an amphiphilic compound such as a phospholipid (see, e.g., U.S. Pat. No. 5,151,254 and PCT applications WO94 / 20078, WO94 / 23701, and WO96 / 06638). The amount of active agent contained in a 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.

[0217] Biodegradable microspheres (e.g., polylactic acid polyglycolate) can be used as carriers for the composition.Suitable biodegradable microspheres are disclosed in, for example, 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 core protein carrier systems, such as those described in WO / 99 40934 and the references cited therein, may also be useful for many applications. Another exemplary carrier / delivery system uses a carrier comprising a particle-protein complex, such as those described in U.S. Pat. No. 5,928,647, which may have additional advantages when used intratumorally to deliver coding sequences for anti-CD39 antibody agents that can induce an MHC I-restricted cytotoxic T lymphocyte response targeted to the patient's tumor tissue.

[0218] Biodegradable polymeric nanoparticles facilitate the delivery of nonviral nucleic acids into cells. Small (approximately 200 nm), positively charged (approximately 10 mV) particles are formed by the self-assembly of cationic, hydrolyzable poly(beta-amino ester) and plasmid DNA.

[0219] Polynucleotides can also be administered to cells by direct microinjection, transient cell permeabilization (eg, co-administration of a repressor and / or activator with a cell permeabilizing agent), fusion to a membrane-translocating peptide, or the like.

[0220] In certain embodiments of the present disclosure, gene constructs are introduced into target cells via electroporation. Electroporation involves exposing cells (or tissues) and DNA (or DNA complexes) to a high-voltage electrical discharge. In vivo electroporation is a gene delivery technique that has been successfully used to efficiently deliver plasmid DNA to 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 cDNAs encoded by the plasmids can be achieved by performing in vivo electroporation. The use of in vivo electroporation has been shown to enhance the uptake of plasmid DNA in tumor tissue, resulting in intratumoral expression, and to deliver plasmids to muscle tissue, resulting in the systemic expression of secreted proteins such as cytokines (see, e.g., US8026223). Exemplary techniques, vectors, and devices for electroporating anti-CD39 antibody agent transgenes into cells in vivo include PCT Publications WO / 2017 / 106795, WO / 2016 / 161201, WO / 2016 / 154473, WO / 2016 / 112359, and WO / 2014 / 066655.

[0221] U.S. Patent No. 7,245,963 describes a modular electrode system and its use for facilitating the introduction of biomolecules into cells of selected tissues in a body or plant. The modular electrode system includes multiple needle electrodes, a hypodermic needle, an electrical connector that provides a conductive connection from a programmable constant electrode pulse controller to the multiple needle electrodes, and a power source. An operator can grasp the multiple needle electrodes mounted on a support structure and securely insert them into selected tissues in a body or plant. The biomolecules are then delivered into the selected tissue via the hypodermic needle. The programmable constant current controller is activated to apply constant current electrical pulses to the multiple needle electrodes. The applied constant current electrical pulses promote the introduction of biomolecules into cells between the multiple electrodes. The entire contents of U.S. Patent No. 7,245,963 are incorporated herein by reference.

[0222] U.S. Patent No. 2005 / 0052630 describes an electroporation device that can be used to effectively promote the introduction of biomolecules into cells of selected tissues within a body or plant. The electroporation device includes an electrokinetic device ("EKD device"), the operation of which is specified by software or firmware. The EKD device generates a series of programmable constant current pulse patterns between an array of electrodes 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 disk having a series of needle electrodes, a central injection channel for an injection needle, and a removable guide disk (see, e.g., U.S. Patent No. 2005 / 0052630), which is incorporated herein by reference.

[0223] The electrode arrays and methods described in U.S. Patent No. 7,245,963 and U.S. Patent Publication No. 2005 / 0052630 are adapted to penetrate deep into tissues such as muscle, as well as other tissues or organs. Due to the structure of the electrode array, the injection needle (for delivering the selected biomolecule) is also inserted completely into the target organ, and the injection is administered perpendicular to the target tissue, in the area pre-delineated by the electrodes.

[0224] Typically, the electric field required for cell electroporation in vivo is generally similar in magnitude to that required for cells in vitro. In one embodiment, the electric field magnitude ranges from about 10 V / cm to about 1500 V / cm, preferably from about 300 V / cm to 1500 V / cm, and preferably from about 1000 V / cm to 1500 V / cm. Alternatively, lower electric field strengths (about 10 V / cm to 100 V / cm, more preferably from about 25 V / cm to 75 V / cm) have longer pulse lengths. For example, when the nominal electric field is about 25 to 75 V / cm, a pulse length of about 10 milliseconds is preferred.

[0225] The pulse length can be from about 10 seconds to about 100 milliseconds. Any desired number of pulses can be used, typically 1 to 100 pulses per second. The delay between pulse sets can be any desired time, such as 1 second. The waveform, field strength, and pulse duration can also depend on the type of cell and the type of molecule to be introduced into the cell via electroporation.

[0226] Also included are electroporation devices that incorporate electrochemical impedance spectroscopy (" EIS "). Such devices provide real-time information in vivo, particularly intratumoral electroporation efficiency, allowing for optimization of conditions. Examples of electroporation devices that incorporate EIS can be found in, for example, WO2016 / 161201, which is incorporated herein by reference.

[0227] The uptake of non-viral delivery vectors encompassed by the present invention can also be enhanced by plasma electroporation, also known as avalanche transfection. Briefly, a microsecond discharge generates cavitation microbubbles on the electrode surface. The mechanical force created by the collapsing microbubbles combined with a magnetic field helps to increase the transport efficiency across the cell membrane compared to the diffusion-mediated transport associated with conventional electroporation. The plasma electroporation technique is described in U.S. Patent Nos. 7,923,251 and 8,283,171. This technique can also be used in vivo for cell transformation. Chaiberg, et al. (2006) Investigative Ophthalmology & Visual Science 47:4083-4090; Chaiberg, et al. United States Patent No. 8,101, 169, Issued January 24, 2012.

[0228] Other alternative electroporation techniques are also being considered. In vivo plasmid delivery can be performed using cold plasma. Plasma is one of the four fundamental states of matter; the others are solid, liquid, and gas. Plasma is an electrically neutral medium of unbound positive and negative particles (i.e., the overall charge of plasma is near zero). Plasma can be created by heating a gas or by exposing it to a strong electromagnetic field using a laser or microwave generator. This increases or decreases the number of electrons, generating positively or negatively charged particles called ions (Luo, et al. (1998) Phys. Plasma 5:2868-2870), accompanied by the dissociation of molecular bonds, if any exist.

[0229] Cold plasma (i.e., non-thermal plasma) is generated by delivering a pulsed, high-voltage signal to suitable electrodes. Cold plasma devices can take the form of gas jet devices or dielectric barrier discharge (DBD) devices. Cold plasma has attracted much enthusiasm and interest due to its ability to provide plasma at relatively low gas temperatures. Providing plasma at such temperatures is interesting for a variety of applications, such as wound healing, antibacterial processes, and various other medical treatments and sterilization. As previously mentioned, cold plasma (i.e., non-thermal plasma) is generated by delivering a pulsed, high-voltage signal to suitable electrodes. Cold plasma devices can take the form of gas jet devices, dielectric barrier discharge (DBD) devices, or multi-frequency harmonic-rich power sources.

[0230] Dielectric barrier discharge devices rely on a different process to generate cold plasma. Dielectric barrier discharge (DBD) devices include at least one conductive electrode covered with a dielectric layer. An electrical return path is formed by a ground, which may be provided by the target substrate undergoing cold plasma processing, or by providing a built-in ground to the electrode. Energy for a dielectric barrier discharge device can be provided by a high-voltage power supply, such as those described above. More commonly, energy is input to a dielectric barrier discharge device in the form of a pulsed DC voltage to form a plasma discharge. The dielectric layer isolates the discharge from the conductive electrode, reducing electrode etching and gas heating. The pulsed DC voltage can be varied in amplitude and frequency to achieve various operating regimes. Any device incorporating such principles of cold plasma generation (e.g., a DBD electrode device) is within the scope of various embodiments encompassed by the present invention.

[0231] Cold plasma has been used to transfect cells with foreign nucleic acids, particularly 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).

[0232] In certain exemplary embodiments, a transgene 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 associated with a coverage area; a power source in electrical communication with the electrodes, the power source configured to generate one or more electroporation signals to cells within the coverage area; and a guide member coupled to the electrodes, the guide member configured to adjust the coverage area of ​​the electrodes. At least a portion of the electrodes can be arranged within the applicator in a conical configuration. The one or more electroporation signals can each be associated with an electric field. The device can further comprise 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.

[0233] The one or more electroporation signals can each be associated with an electric field. The device can further include a potentiometer coupled to the power source and the electrodes. The potentiometer can be configured to maintain the electric field within a predetermined range to substantially prevent permanent damage to cells within the covered area and / or substantially minimize pain. For example, the potentiometer can be configured to maintain the electric field at approximately 1300 V / cm.

[0234] The power source may provide a first electrical signal to the first electrode and a second electrical signal to the second electrode. The first and second electrical signals may combine to generate a 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.

[0235] In certain embodiments, the present invention provides a method for treating a subject having a tumor, the method comprising injecting an effective amount of a plasmid encoding an anti-CD39 antibody agent into the tumor; and administering electroporation therapy to the tumor. In certain embodiments, the electroporation therapy further comprises administering at least one voltage pulse of about 200 V / cm to about 1500 V / cm with a pulse width of about 100 microseconds to about 20 milliseconds.

[0236] In certain embodiments, the plasmid (or the second electroporation plasmid) further encodes at least one immunostimulatory cytokine, such as those selected from the group encoding IL-12, IL-15, and a combination of IL-12 and IL-15.

[0237] Lipids and polycationic molecules for delivery of anti-CD39 antibody encoding nucleic acid constructs Lipid-mediated nucleic acid delivery and expression of exogenous nucleic acids, such as mRNA, have been highly successful in vitro and in vivo. Lipid-based nonviral formulations offer 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 composed of an equimolar ratio of 1,2-bis(oleoyloxy)-3-(trimethylammonio)propane (DOTAP) and cholesterol can significantly improve systemic in vivo gene transfer. DOTAP:cholesterol lipid formulations form unique structures called "sandwich liposomes." These formulations have been reported to "sandwich" DNA between an invaginated bilayer or "vase" structure. Beneficial features of these lipid structures include a positive pH, colloidal stabilization by cholesterol, two-dimensional nucleic acid packing, and increased serum stability.

[0238] Cationic liposome technology is based on the ability of amphipathic lipids, which possess positively charged head groups and hydrophobic lipid tails, to bind negatively charged DNA or RNA, forming particles that typically enter cells via endocytosis. Some cationic liposomes also contain neutral co-lipids, which are thought to enhance liposome uptake by mammalian cells. Similarly, other polycations, such as poly-l-lysine and polyethyleneimine, complex with nucleic acids through charge interactions and aid in the condensation of DNA or RNA into nanoparticles, which are then substrates for endosome-mediated uptake. [8] Several of these cationic nucleic acid complex technologies have been developed as potential clinical products, such as complexes with various forms of plasmid DNA (pDNA), oligodeoxynucleotides, and synthetic RNA.

[0239] The nucleic acid constructs disclosed herein may be associated with polycationic molecules, which help enhance cellular uptake. Complexing the nucleic acid construct with polycationic molecules also aids in packaging the construct, such as reducing its size, which is believed to aid cellular uptake. Upon entering the endosome, the low pH causes the complex to dissociate, allowing the polycationic molecules to disrupt the endosomal membrane, facilitating the escape of DNA into the cytoplasm before it can be degraded. Preliminary data indicate that embodiments of the nucleic acid construct, when complexed with polycationic molecules such as polylysine or polyethyleneimine, exhibit enhanced uptake in SCs compared to DCs.

[0240] One example of a polycationic molecule useful for complexing with nucleic acid constructs is a cell-penetrating peptide (CPP), such as polylysine (see above), polyarginine, or Tat peptide. Cell-penetrating peptides (CPPs) are small peptides that bind to DNA and, upon release, penetrate the cell membrane, facilitating DNA escape from endosomes to the cytoplasm. Another example of a CPP is a 27-residue chimeric peptide, termed MPG, which has recently been shown to bind ss- and ds-oligonucleotides in a stable manner, resulting in noncovalent complexes that protect the nucleic acid from DNase degradation and efficiently deliver the oligonucleotides to cells in vitro (Mahapatro A, et al., J Nanobiotechnol, 2011, 9:55). When different peptide:DNA ratios were tested, as well as ratios of 10:1 and 5:1 (150 nm and 1 um, respectively), the complexes formed small particles ranging from approximately 150 nm to 1 um. Another CPP is a modified tetrapeptide [tetralysine containing a guanidinocarbonylpyrrole (GCP) group (TL-GCP)], which has been reported to bind with high affinity to 6.2 kb of plasmid DNA, producing positively charged aggregates of 700–900 nm (Li et al., Agnew Chem Int Ed Enl 2015;54(10):2941–4). RNA can also be complexed with such polycationic molecules for in vivo delivery.

[0241] Other examples of polycationic molecules that may form complexes with the nucleic acid constructs described herein include the polycationic polymers commercially available as JETPRIME® and In Vivo JET (Polypus-transfection, SA, Illkirch, France).

[0242] VI. Methods of Use and Pharmaceutical Compositions The anti-CD39 antibodies encompassed by the present invention are useful in a variety of applications, including, but not limited to, therapeutic treatment methods such as immunotherapy for cancer. In certain embodiments, the anti-CD39 antibodies described herein are useful 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 tumor tumorigenicity. In certain embodiments, the anti-CD39 antibodies encompassed by the present invention are also useful in immunotherapy against pathogens such as viruses. In certain embodiments, the anti-CD39 antibodies described herein are useful for inhibiting viral infection, reducing viral infection, increasing apoptosis of virally infected cells, and / or increasing killing of virally infected cells. Methods of use can be in vitro, ex vivo, or in vivo.

[0243] 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 present invention provides methods for enhancing an immune response in a subject using the anti-CD39 antibodies described herein. In some embodiments, activating, promoting, increasing, and / or enhancing an immune response comprises increasing cell-mediated immunity. In some embodiments, activating, promoting, increasing, and / or enhancing an immune response comprises increasing a Th1-type response. In some embodiments, activating, promoting, increasing, and / or enhancing an immune response comprises increasing T cell activity. In some embodiments, activating, promoting, increasing, and / or enhancing an immune response comprises increasing CD4+ T cell activity. In some embodiments, activating, promoting, increasing, and / or enhancing an immune response comprises increasing CD8+ T cell activity. In some embodiments, activating, promoting, increasing, and / or enhancing the immune response comprises increasing CTL activity. In some embodiments, activating, promoting, increasing, and / or enhancing the immune response comprises increasing NK cell activity. In some embodiments, activating, promoting, increasing, and / or enhancing the immune response comprises increasing T cell activity and increasing NK cell activity. In some embodiments, activating, promoting, increasing, and / or enhancing the immune response comprises increasing CU activity and increasing NK cell activity. In some embodiments, activating, promoting, increasing, and / or enhancing the immune response comprises inhibiting or decreasing the suppressive activity of Treg cells. In some embodiments, activating, promoting, increasing, and / or enhancing the immune response comprises inhibiting or decreasing the suppressive activity of MDSCs.In some embodiments, activating, promoting, increasing, and / or enhancing the immune response comprises increasing the percentage or number of memory T cells. In some embodiments, activating, promoting, increasing, and / or enhancing the immune response comprises increasing long-term immune memory function. In some embodiments, activating, promoting, increasing, and / or enhancing the immune response comprises increasing long-term memory. In some embodiments, activating, promoting, increasing, and / or enhancing the immune response does not include evidence of substantial side effects and / or immune-based toxicity. In some embodiments, activating, promoting, increasing, and / or enhancing the immune response does not include evidence of cytokine release syndrome (CRS) or cytokine storm. In some embodiments, the immune response is the result of antigenic stimulation. In some embodiments, the antigenic stimulation is tumor cells. In some embodiments, the antigenic stimulation is cancer. In some embodiments, the antigenic stimulation is a pathogen. In some embodiments, the antigenic stimulation is virally infected cells.

[0244] In vivo and in vitro assays for determining whether an anti-CD39 antibody modulates, activates, or inhibits an immune response are known in the art or have been developed. In certain embodiments of the methods described herein, the method of inducing sustained 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.

[0245] In some embodiments, the tumor is a solid tumor. In certain embodiments, the tumor is a tumor selected from the group consisting of a colon tumor, a pancreatic tumor, a lung tumor, an ovarian tumor, a liver tumor, a breast tumor, a kidney tumor, a prostate tumor, a neuroendocrine tumor, a gastrointestinal tumor, a melanoma, a cervical tumor, a bladder tumor, a glioblastoma, a lymphoma, and a head and neck tumor. In certain embodiments, the tumor is a colon tumor. In certain embodiments, the tumor is an ovarian tumor. In some embodiments, the tumor is a lung tumor. In certain embodiments, the tumor is a pancreatic tumor or a pancreatic islet tumor. In certain embodiments, the tumor is a melanoma tumor. In some embodiments, the tumor is a bladder tumor or a urothelial tumor. In some embodiments, the tumor is a liquid tumor. In certain embodiments, the tumor is a leukemia (such as myeloid or granulocytic leukemia, lymphatic, lymphocytic, or lymphoblastic leukemia), and polycythemia vera or erythrocyte tumor.

[0246] 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. The present invention further provides methods 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.

[0247] The present invention provides methods of treating cancer, comprising administering to a subject (e.g., a subject in need of treatment) a therapeutically effective amount of an anti-CD39 antibody described herein. In certain embodiments, the subject is a human. In certain embodiments, the subject has a cancerous tumor. In certain embodiments, the subject has had the tumor removed.

[0248] In certain embodiments, the cancer is a cancer selected from the group consisting of colon cancer, pancreatic cancer, lung cancer, ovarian cancer, liver cancer, breast cancer, renal cancer, prostate cancer, gastric 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 colon cancer. In certain embodiments, the cancer is breast cancer. In certain embodiments, the cancer is prostate cancer. In certain embodiments, the cancer is lung cancer. In certain embodiments, the cancer is melanoma. In some embodiments, the cancer is bladder cancer.

[0249] The present invention also provides pharmaceutical compositions comprising an anti-CD39 antibody described herein. The present invention also provides pharmaceutical compositions comprising an anti-CD39 antibody described herein and a pharmaceutically acceptable vehicle. In some embodiments, the pharmaceutical compositions find use in immunotherapy. In some embodiments, the pharmaceutical compositions find use in immuno-oncology. In some embodiments, the compositions find use in inhibiting tumor growth. In some embodiments, the pharmaceutical compositions find use in inhibiting tumor growth in a subject (e.g., a human patient). In some embodiments, the compositions find use in treating cancer. In some embodiments, the pharmaceutical compositions find use in treating cancer in a subject (e.g., a human patient).

[0250] Formulations are prepared for storage and use by combining a purified agent encompassed by the present invention with a pharmaceutically acceptable vehicle (e.g., a carrier or excipient). Those skilled in the art generally consider pharmaceutically acceptable carriers, excipients, and / or stabilizers to be inactive ingredients of a formulation or pharmaceutical composition.

[0251] In some embodiments, the anti-CD39 antibodies are lyophilized and / or stored in lyophilized form, hi some embodiments, formulations comprising the anti-CD39 antibodies described herein are lyophilized.

[0252] Suitable pharmaceutically acceptable vehicles include non-toxic buffers, such as phosphate, citric acid, and other organic acids; salts, such as sodium chloride; antioxidants such as ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight polypeptides (fewer than about 10 amino acid residues); proteins, such as serum albumin. , gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates such as 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 (e.g., Zn-protein complexes); and nonionic surfactants such as TWEEN® or polyethylene glycol (PEG). (Remington: The Science and Practice of Pharmacy, 22nd Edition, 2012, Pharmaceutical Press, London.)

[0253] The pharmaceutical compositions encompassed by the present invention can be administered in any number of ways for either local or systemic treatment: topically, by epidermal or transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, and powders; pulmonary, by inhalation or insufflation of powders or aerosols, such as with a nebulizer, intratracheal, and intranasal; or parenterally, such as intravenously, intraarterially, intratumorally, subcutaneously, intraperitoneally, intramuscularly (e.g., by injection or infusion), or intracranially (e.g., intrathecally or intraventricularly).

[0254] Therapeutic formulations can be in unit dosage form. Such formulations 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 ingredient is mixed with a pharmaceutical carrier. Traditional tablet ingredients include corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate, or gums, and diluents (e.g., water). These can be used to form solid preformulation compositions containing a homogeneous mixture of the compounds encompassed by the present invention, or their non-toxic pharmaceutically acceptable salts. The solid preformulation composition is then subdivided into unit dosage forms of the type described above. Tablets or pills of the formulation or composition can be coated or otherwise compounded to provide a dosage form offering the advantage of prolonged action. For example, a tablet or pill can include an inner composition surrounded by an outer component. Moreover, the two components can be separated by an enteric layer, which serves to resist disintegration and permits the inner component to pass intact into the stomach or to be delayed in release. A variety of materials can be used for such enteric layers or coatings, such materials including polymeric acids and mixtures of polymeric acids with such materials as shellac, cetyl alcohol, and cellulose acetate.

[0255] Anti-CD39 antibodies can also be encapsulated in microcapsules, prepared, for example, by coacervation techniques or by interfacial polymerization, for example, in 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 macroemulsions, as described in Remington: The Science and Practice of Pharmacy, 22nd Edition, 2012, Pharmaceutical Press, London.

[0256] In certain embodiments, the pharmaceutical formulation comprises an anti-CD39 antibody complexed with a liposome. Methods for producing liposomes are known to those skilled in the art. For example, some liposomes can be produced by reverse phase evaporation using a lipid composition containing phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes can be extruded through filters of defined pore size to obtain liposomes with the desired diameter.

[0257] In certain embodiments, sustained-release preparations containing anti-CD39 antibodies can be produced. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing anti-CD39 antibodies, these matrices being in the form of shaped articles (e.g., films or microcapsules). Examples of sustained-release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate) or poly(vinyl alcohol)), polylactide, copolymers of L-glutamic acid and 7-ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as LUPRON DEPOT (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), sucrose acetate isobutyrate, and poly-D-(-)-3-hydroxybutyric acid.

[0258] In certain embodiments, in addition to administering an anti-CD39 antibody, the method or treatment further comprises administering at least one additional immune response stimulator. In some embodiments, the additional immune response stimulator includes, but is not limited to, a 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 inhibitor, antibody that blocks immunosuppressive function (e.g., anti-CTLA-4 antibody, anti-CD28 antibody, anti-CD3 antibody), toll-like receptor (e.g., TLR4, TLR7, TLR9), or member of the B7 family (e.g., CD80, CD86). The additional immune response stimulator can be administered before, simultaneously with, and / or after administration of the anti-CD39 antibody. Also provided are pharmaceutical compositions comprising an anti-CD39 antibody and an immune response stimulator(s). In some embodiments, the immune response stimulator comprises one, two, three, or more immune response stimulators.

[0259] 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 administration of the anti-CD39 antibody. Pharmaceutical compositions comprising an anti-CD39 antibody and additional therapeutic agent(s) are also provided. In some embodiments, the at least one additional therapeutic agent comprises one, two, three, or more additional therapeutic agents.

[0260] Combination therapy with two or more therapeutic agents often, but not necessarily, uses agents that act via different mechanisms of action. Combination therapy using agents with different mechanisms of action may result in additive or synergistic effects. Combination therapy may allow for lower doses of each agent than those used in monotherapy, thereby reducing toxic side effects and / or increasing the therapeutic index of the anti-CD39 antibody. Combination therapy may reduce the likelihood of resistant cancer cells developing. In some embodiments, combination therapy includes a therapeutic agent that affects the immune response (e.g., enhances or activates the response) and a therapeutic agent that affects tumor / cancer cells (e.g., inhibits or kills them).

[0261] In some embodiments of the methods described herein, the combination of the anti-CD39 antibody with at least one additional therapeutic agent results in additive or synergistic results. In some embodiments, the combination therapy results in an increased therapeutic index of the anti-CD39 antibody. In some embodiments, the combination therapy results in an increased therapeutic index of the additional therapeutic agent(s). In some embodiments, the combination therapy results in reduced toxicity and / or side effects of the anti-CD39 antibody. In some embodiments, the combination therapy results in reduced toxicity and / or side effects of the additional therapeutic agent(s). Useful classes of therapeutic agents include, for example, antitubulin agents, auristatins, DNA minor groove binders, DNA replication inhibitors, alkylating agents (e.g., cisplatin, mononuclear (platinum), dinuclear (platinum), and trinuclear platinum complexes, and platinum complexes such as carboplatin), anthracyclines, antibiotics, antifolates, antimetabolites, chemotherapy sensitizers, duocarmycins, etoposide, fluorinated pyrimidines, ionophores, lexitropsin, nitrosoureas, platinol, purine antimetabolites, puromycin, radiosensitizers, steroids, taxanes, topoisomerase inhibitors, vinca alkaloids, etc. In certain embodiments, the second therapeutic agent is an alkylating agent, antimetabolite, mitotic inhibitor, topoisomerase inhibitor, or angiogenesis inhibitor.

[0262] Therapeutic agents that may be administered in combination with the anti-CD39 antibodies described herein include chemotherapeutic agents. Thus, in some embodiments, a method or treatment involves administering an anti-CD39 antibody in combination with a chemotherapeutic agent or a cocktail of chemotherapeutic agents. Treatment with an anti-CD39 antibody can occur before, simultaneously with, or after chemotherapy. Combination administration herein can include coadministration, either in a single pharmaceutical formulation or in separate formulations, or in any order, generally sequentially within a time period that allows all active agents to simultaneously exert their biological activities. Preparation and dosing schedules for such chemotherapeutic agents can be used according to manufacturer's instructions or can be empirically determined by one of skill in the art. Preparation and dosing schedules for such chemotherapeutic agents are also described in The Chemotherapy Source Book, 4th Edition, 2008, M.C. Perry, Editor, Lippincott, Williams & Wilkins, Philadelphia, Pa.

[0263] Chemotherapeutic agents useful in the present invention include alkylating agents (e.g., thiotepa and cyclophosphamide (CYTOXAN)), alkylsulfonates (e.g., busulfan, improsulfan, and piposulfan), aziridines (e.g., benzodopa, carboquone, mesuredopa, and uredopa), ethylenimines and methylmelamines (e.g., altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphaolamide, and trimethylolmelamine), nitrogen mustards (e.g., chlorambucil, fluticasone, chlornaphazine, colofosfamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, nobuenbiquin, phenesterine, prednimustine, trofosfamide, uracil mustard), nitrosoureas (e.g., carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine), antibiotics (e.g., aclacinomycins, actinomycin, ausramycin, azaserine, bleomycins, cactinomycin, calicheamicin, carabicin, carminomicin), mycobacterium, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfilomycin, puromycin, queramycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin), antimetabolites (e.g., methotrexate) and 5-fluorouracil (5-FU)), folic acid analogues (e.g., denopterin, methotrexate, pteropterin, trimetrexate), purine analogues (e.g., fludarabine, 6-mercaptopurine, thiamiprine, thioguanine), pyrimidine analogues (e.g., ancitabine, azacitidine, 6-azauridine, carmofur, cytosine arabinoside, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU), androgens (e.g., calsterone, dromostanolone propionate, epithiostanol,mepitiostane, testolactone), antiadrenal agents (e.g., aminoglutethimide, mitotane, trilostane), folic acid supplements (e.g., folinic acid), aceglatone, aldophosphamide glycoside, aminolevulinic acid, amsacrine, bestravcil, bisantrene, edatraxate, defofamine, demecolcine, diaziquone, elformitin, elliptinium acetate, etoglucide, gallium nitrate, hydroxyurea, lentinan, lonidamine, mitoguazone, mitoxantrone, mopidamol, nitracrine, pentostatin, phenamt, pirarubicin, podophyllic acid, 2-ethylhydrazide, procarbazine, PSK, razoxyle San, sizofuran, spirogermanium, tenuazonic acid, triazicon, 2,2',2''-trichlorotriethylamine, urethane, vindesine, dacarbazine, mannomustine, mitobronitol, mitolactol, pipobroman, gacytosine, arabinoside ("Ara-C"), taxoids (e.g., paclitaxel (TAXOL), and docetaxel (TAXOTERE)), chlorambucil, gemcitabine, 6-thioguanine, mercaptopurine, platinum analogs (e.g., cisplatin), and carboplatin), vinblastine, platinum, etoposide (VP-16), ifosfamide, mitomycin C, mitoxantrone, vincristine, vinorelbine, navelbine, novantrone, teniposide, daunomycin, aminopterin, ibandronate, CPT-11, topoisomerase inhibitor RFS2000, difluoromethylornithine (DMFO), retinoic acid, esperamicins, capecitabine (XELODA), and any of the above. Chemotherapeutic agents include, but are not limited to, pharmaceutically acceptable salts, acids, or derivatives thereof. Chemotherapeutic agents also include antihormonal agents that act to regulate or inhibit hormone action on tumors, such as antiestrogens (e.g., tamoxifen, raloxifene, aromatase-inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, ketoxifene, LY117018, onapristone, and toremifene (FARESTON)), and antiandrogens (e.g.,In some embodiments, the additional therapeutic agent is cisplatin. In some embodiments, the additional therapeutic agent is carboplatin.

[0264] 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 action of a topoisomerase enzyme (e.g., topoisomerase I or II). Topoisomerase inhibitors include, but are not limited to, doxorubicin HCl, daunorubicin citrate, mitoxantrone HCl, actinomycin D, etoposide, topotecan HCl, teniposide (VM-26), and irinotecan, as well as pharmaceutically acceptable salts, acids, or derivatives of any of these.

[0265] In certain embodiments, the chemotherapeutic agent is an antimetabolite. An antimetabolite is a chemical compound that has a structure similar to a metabolite required for normal biochemical reactions, but differs sufficiently to interfere with one or more normal functions of a cell, such as cell division. Antimetabolites include, but are not limited to, gemcitabine, fluorouracil, capecitabine, methotrexate sodium, ralitrexed, pemetrexed, tegafur, cytosine arabinoside, thioguanine, 5-azacytidine, 6-mercaptopurine, azathioprine, 6-thioguanine, pentostat, fludarabine phosphate, and cladribine, as well as pharmaceutically acceptable salts, acids, or derivatives of any of these.

[0266] In certain embodiments of the methods described herein, the chemotherapeutic agent is a mitotic inhibitor, including but not limited to, an agent that binds 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 mitotic inhibitor comprises a vinca alkaloid, such as vincristine, vinblastine, vinorelbine, or vindesine, or a pharmaceutically acceptable salt, acid, or derivative thereof. In some embodiments, the mitotic inhibitor is an inhibitor of kinesin Eg5, or an inhibitor of a mitotic kinase, such as Aurora A or Plk1.

[0267] In certain embodiments of the methods described herein, the subject anti-CD39 antibodies are expected to have a greater combination effect (perhaps even synergistic effect) with chemotherapeutic agents that induce ATP release within tumors and / or cause upregulation of CD39 or CD73 within tumors. A wide range of chemotherapeutic agents exist that cause the release of ATP into the extracellular space when inducing tumor cell death, including (but not limited to) anthracyclines (such as doxorubicin, daunorubicin, epirubicin, and idarubicin), platinum-based agents (such as cisplatin, carboplatin, and oxaliplatin), and proteasome inhibitors (such as bortezomib). Radiation therapy and photodynamic therapy (PDT) can also cause ATP release and / or upregulation of intratumoral levels of CD39 and / or CD73.

[0268] In some embodiments of the methods described herein, the additional therapeutic agent comprises an agent such as a small molecule. For example, the treatment may include the combined administration of an anti-CD39 antibody and a small molecule that acts as an inhibitor of a tumor-associated antigen, including, but not limited to, EGFR, HER2 (ErbB2), and / or VEGF. In some embodiments, the anti-CD39 antibody is administered in combination with a protein kinase inhibitor selected from the group consisting of gefitinib (IRESSA), erlotinib (TARCEVA), sunitinib (SUTENT), lapatanib, vandetanib (ZACTIMA), AEE788, CI-1033, cediranib (RECENTIN), sorafenib (NEXAVAR), and pazopanib (GW786034B). In some embodiments, the additional therapeutic agent comprises an mTOR inhibitor.

[0269] 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.

[0270] In some embodiments of the methods described herein, the additional therapeutic agent comprises a biomolecule such as an antibody. For example, treatment may include the combined administration of an anti-CD39 antibody and an antibody against a tumor-associated antigen, including, but not limited to, an antibody that binds to 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 to a component of the Notch pathway. In some embodiments, the additional therapeutic agent is an antibody that binds to 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 is an 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).

[0271] I / O Combinations - Representative Checkpoint Inhibitors and Costimulatory Agonists In some embodiments of the methods described herein, the additional therapeutic agent is an antibody that modulates an immune response, hi 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.

[0272] For example, the treatment method can further 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 one or more inhibitors of PD-1, CTLA-4, TIM-3, LAG-3, CEACAM, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, NLRP1, NRLP3, STING, TGFRbeta, or Siglec-15. Exemplary activators of costimulatory molecules include one or more agonists of 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 ligand. Exemplary inhibitors of immune checkpoints and exemplary activators of costimulatory molecules can be found in PCT Publication WO2016 / 054555, which is incorporated herein by reference.

[0273] PD-1 antagonist 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-distal 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, PD-L1 and PD-L2, have been identified and shown to downregulate T cell activation when they bind 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). PD-L1 and PD-L2 are B7 homologs that bind to PD-1 but not other CD28 family members. PD-L1 is abundant in a variety of human cancers (2002) Nat. Med. 8:787-9). The interaction between PD-1 and PD-L1 reduces tumor-infiltrating lymphocytes, reduces T cell receptor-mediated proliferation, and leads to immune evasion by cancerous 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 effect is additive when the interaction between PD-1 and PD-L2 is also blocked (Iwai et al. (2002) Proc. Nat'l. Acad. Sci. USA 99:12293-7; Brown et al. (2003) J. Immunol. 170:1257-66).

[0274] As used herein, the terms "programmed death 1," "programmed cell death 1," "protein PD-1," "PD-1," "PD1," "PDCD1," "hPD-1," and "hPD-I" are used interchangeably and include variants, isoforms, species homologs of human PD-1, and analogs that share at least one epitope with human PD-1. The complete human PD-1 sequence can be found at GenBank Accession No. U64863.

[0275] As used herein, the terms "programmed cell death 1 ligand 1," "PD-L1," "PDL1," "PDCD1L1," "PDCD1LG1," "CD274," "B7 homolog 1," "B7-H1," "B7-H," and "B7H1" are used interchangeably and include variants, isoforms, species homologs of human PDL-1, and analogs that share at least one epitope with human PDL-1. The complete human PD-L1 amino acid sequence (isoform precursor) can be found at GenBank Accession No. NP_054862.1. The complete human PD-L1 amino acid sequence (isoform precursor) can be found at GenBank Accession No. NP_001254635.1.

[0276] The term "PD-1 axis binding antagonist" refers to a molecule that inhibits the interaction of a PD-1 axis binding partner with one or more of its binding partners, such that T cell dysfunction resulting from signaling in the PD-1 signaling axis is ablated, resulting in restoration or enhancement of 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.

[0277] The term "PD-1 binding antagonist" refers to a molecule that reduces, blocks, inhibits, abrogates, or interferes with signal transduction resulting from the interaction of PD-1 with one or more of its binding partners, such as PD-L1 and PD-L2. In some embodiments, a PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 to its binding partners. In a specific aspect, a 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, abrogate, or interfere with signal transduction resulting from the interaction of PD-1 with PD-L1 and / or PD-L2. In one embodiment, the PD-1 binding antagonist reduces negative costimulatory signals mediated by or through cell surface proteins expressed on T lymphocytes that mediated signaling through PD-1, rendering 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 a specific embodiment, the PD-1 binding antagonist is MDX-1106, as described herein. In another specific embodiment, the PD-1 binding antagonist is Merck3745, as described herein. In another specific embodiment, the PD-1 binding antagonist is CT-011, as described herein. The term "PD-L1 binding antagonist" refers to a molecule that reduces, blocks, inhibits, abrogates, or interferes with signal transduction resulting from the interaction of PD-L1 with one or more of its binding partners, such as PD-1 or B7-1. In some embodiments, a PD-L1 binding antagonist is a molecule that inhibits the binding of PD-L1 to its binding partners. In a specific aspect, a PD-L1 binding antagonist inhibits the binding of PD-L1 to PD-1 and / or B7-1. In some embodiments, PD-L1 binding antagonists include anti-PD-L1 antibodies, antigen-binding fragments thereof, immunoadhesins, fusion proteins, oligopeptides, and other molecules that reduce, block, inhibit, abrogate, or interfere with signal transduction resulting from the interaction of PD-L1 with one or more of its binding partners, e.g., PD-1, B7-1. In one embodiment, the PD-L1 binding antagonist reduces the negative costimulatory signal mediated by or through cell surface proteins expressed on T lymphocytes that mediated PD-L1-mediated signaling, rendering dysfunctional T cells less dysfunctional (e.g., enhancing the effector response 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, as described herein. In another specific aspect, the anti-PD-L1 antibody is MDX-1105, as described herein. In yet another specific aspect, the anti-PD-L1 antibody is MPDL3280A, as described herein.

[0278] The term "PD-L2 binding antagonist" refers to a molecule that reduces, blocks, inhibits, abrogates, or interferes with signal transduction resulting from the interaction of PD-L2 with one or more of its binding partners, such as PD-1. In some embodiments, a PD-L2 binding antagonist is a molecule that inhibits the binding of PD-L2 to its binding partners. In a specific aspect, a PD-L2 binding antagonist inhibits the binding of PD-L2 to PD-1. In some embodiments, PD-L2 antagonists include anti-PD-L2 antibodies, antigen-binding fragments thereof, immunoadhesins, fusion proteins, oligopeptides, and other molecules that reduce, block, inhibit, abrogate, or interfere with signal transduction resulting from the interaction of PD-L2 with any one or more of its binding partners, e.g., PD-1. In one embodiment, the PD-L2 binding antagonist reduces the negative costimulatory signal mediated by or through cell surface proteins expressed in T lymphocyte-mediated signaling via PD-L2, such that dysfunctional T cells are rendered less dysfunctional (e.g., enhance the effector response to antigen recognition). In some embodiments, the PD-L2 binding antagonist is an immunoadhesin. PD-1 pathway: Members of the PD-1 pathway are all proteins involved in PD-1 signal transduction. On the one hand, they may be proteins that induce PD-1 signal transduction upstream of PD-1, such as the ligands of PD-1 PD-L1 and PD-L2, and the signaling receptor PD-1. On the other hand, they may be signaling proteins downstream of the PD-1 receptor. Particularly preferred members of the PD-1 pathway in the context of the present invention are PD-1, PD-L1, and PD-L2.

[0279] PD-1 pathway inhibitor: In the context of the present invention, a PD-1 pathway inhibitor is preferably defined herein as a compound capable of impairing PD-1 pathway signaling, preferably signaling mediated by the PD-1 receptor. Therefore, a PD-1 pathway inhibitor can be any inhibitor directed against any member of the PD-1 pathway that can antagonize PD-1 pathway signaling. In this context, the inhibitor can be an antagonistic antibody, as defined herein, that targets any member of the PD-1 pathway, preferably directed against the PD-1 receptor, PD-L1, or PD-L2. This antagonistic antibody can also be encoded by a nucleic acid. Such an encoded antibody is also referred to as an "intrabody," as defined herein. The PD-1 pathway inhibitor can also be a fragment of the PD-1 receptor or a fragment of the PD1 receptor that blocks the activity of the PD-1 ligand. B7-1 or a fragment thereof can also act as a PD1 inhibitory ligand. Furthermore, the PD-1 pathway inhibitor may be a siRNA (small interfering RNA) or antisense RNA directed against a member of the PD-1 pathway, preferably PD-1, PD-L1, or PD-L2. Furthermore, the PD-1 pathway inhibitor may be a protein containing an amino acid sequence (or a nucleic acid encoding this amino acid sequence) that can bind to PD-1 and prevent PD-1 signaling, for example, by inhibiting the interaction of PD-1 and B7-H1 or B7-DL. Furthermore, the PD-1 pathway inhibitor may be a small molecule inhibitor, such as a PD-1-binding peptide or a small organic molecule, that can inhibit PD-1 pathway signaling.

[0280] In certain embodiments, PD-1 antagonists encompassed by the present invention include agents that bind to a ligand of PD-1 and interfere with, reduce, or inhibit the binding of one or more ligands to the PD-1 receptor, or that bind directly to the PD-1 receptor without participating in signaling through the PD-1 receptor. In one embodiment, the PD-1 antagonist binds directly to PD-1 and blocks PD-1 inhibitory signaling. 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 the ligand(s) from causing inhibitory signaling through PD-1. In one embodiment, the PD-1 antagonist binds directly to PD-L1 and inhibits or prevents PD-L1 from binding to PD-1, thereby blocking PD-1 inhibitory signaling.

[0281] PD-1 antagonists used in the methods and compositions encompassed by the present invention include PD-1-binding scaffold proteins, including, but not limited to, PD ligands, antibodies, and multivalent agents. In certain embodiments, the antagonist is a fusion protein such as AMP-224. In another embodiment, the antagonist is an anti-PD-1 antibody ("PD-1 antibody"). Anti-human PD-1 antibodies (or VH and / or VL domains derived therefrom) suitable for use in the present invention can be generated using methods well known in the art. Alternatively, art-recognized anti-PD-1 antibodies can be used. For example, antibodies MK-3475 or CT-011 can be used. Additionally, monoclonal antibodies 5C4, 17D8, 2D3, 4H1, 4A11, 7D3, and 5F4, described in WO2006 / 121168, the teachings of which are incorporated herein by reference, can be used. Antibodies that compete with any of these art-recognized antibodies for binding to PD-1 can also be used.

[0282] In another embodiment, the PD-L1 binding antagonist is an anti-PD-L1 antibody. Anti-human PD-L1 antibodies (or VH and / or VL domains derived therefrom) suitable for use in the present invention can be generated using methods well known in the art. Alternatively, art-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 art-recognized antibodies for binding to PD-L1 can also be used.

[0283] An exemplary anti-PD-L1 antibody is 12A4, which is 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 with the above antibodies and / or binds to the same epitope on PD-L1 as the above antibodies. In another embodiment, the antibody has at least about 90% variable region amino acid sequence identity with the above antibodies.

[0284] The anti-PD-1 antibody or anti-PD-L1 antibody may bind to PD-1 or PD-L1, respectively, and have a KD of 10 -7 Medium, 5x10 -8 M, 10 -8 Medium, 5x10 -9 M, 10 -9 Medium, 5x10 -10 M, 10 -10 M or less.

[0285] In one embodiment, the PD-1 inhibitor is an anti-PD-1 antibody selected from nivolumab, pembrolizumab, or pidilizumab. A preferred PD-1 inhibitor is nivolumab. 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 KEYTRUDA®, 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, for example, in 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). In some embodiments, the anti-PD-1 antibody is pidilizumab. Pidilizumab (CT-011; Cure Tech) is a humanized IgGlk monoclonal antibody that binds to PD1. Pidilizumab and other humanized anti-PD-1 monoclonal antibodies are disclosed in WO2009 / 101611. Other anti-PD1 antibodies are disclosed in US8,609,089, US2010028330, and / or US20120114649. Other anti-PD1 antibodies include AMP514 (Amplimmune).

[0286] 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.

[0287] 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 WO2010 / 077634 (incorporated by reference) (in WO2010 / 077634, the heavy and light chain variable regions are set forth in SEQ ID NOs: 20 and 21, respectively).

[0288] In one embodiment, the PD-L1 inhibitor is MDPL3280A (Genentech / Roche). MDPL3280A is a human Fc-optimized IgG1 monoclonal antibody that binds to PD-L1. MDPL3280A and other human monoclonal antibodies against 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 another embodiment, the PD-L2 inhibitor is AMP-224. AMP-224 is a PD-L2Fc fusion soluble receptor that blocks the interaction between PD1 and B7-H1 (B7-DCIg; Amplimmune; disclosed, for example, in WO2010 / 027827 (incorporated by reference) and WO2011 / 066342 (incorporated by reference)).

[0289] In certain embodiments, the PD-1 pathway inhibitor is a small molecule antagonist of PD-1 pathway signaling. Such small molecule antagonists include agents 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.

[0290] Exemplary small molecule antagonists of PD-1 pathway signaling can be found, inter alia, in U.S. Patent Application Publication Nos. 2014 / 0294898 and 2014 / 0199334, and PCT Published Applications WO2013 / 132317 and WO2012 / 168944, each of which is incorporated herein by reference.

[0291] For illustrative purposes only, a subject's combination therapy can be carried out with a small molecule antagonist selected from the group consisting of: [ka]

[0292] In other embodiments, the small molecule antagonist has the general formula: [ka] During the ceremony, R1 is the free or amidated C-terminus of Ser; L is -NH(CH2) n NH- or -NH(CH2CH2O) n is a linker selected from NH-; R4 is hydrogen, amino (C1-C 20 ) alkyl, -NHCOCH3 or -NHCONH2; or a retro analog or a pharmaceutically acceptable stereoisomer or a pharmaceutically acceptable salt thereof.

[0293] In yet other embodiments, the small molecule antagonist has the general formula: [ka] During the ceremony, R1 is the N-terminus of Ser; or substituted with either the hydroxyl or amino group of Ser (C1-C 20 ) is acyl; L is -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-; R2 is the free C-terminus, the amidated C-terminus, or the N-terminus of Am2; or Y-R5; Y is -OOC(CH2) m COO-, -CO(CH2) n NH-, -CO(CH2CH2O) n NH- or -COCH2(OCH2CH2) n is an optional linker selected from NH-; R5 is an albumin binding moiety such as maleimidopropionic acid; R3 is OH or NH2; R4 is a substituent of the phenyl group of Phe, and is selected from the group consisting of hydrogen, amino (C1-C 20 ) alkyl, -NHCOCH3 or -NHCONH2; n is an integer having a value selected from 2 to 10, inclusive; m is an integer having a value selected from 0 to 8, inclusive; One of the Ser-Asn, Asn-Thr, or Thr-Ser peptide bonds (-CONH-) may be replaced with the following modified peptide bond: [ka] In the formula, Q is hydrogen, —CO(C1 to C 20) alkyl or -COO(C1-C 20 ) alkyl groups; wherein one or more or all of the amino acids may be in the D-configuration; or a retro analog or a pharmaceutically acceptable stereoisomer or a pharmaceutically acceptable salt thereof.

[0294] For example, the small molecule antagonist can be selected from the group consisting of: [ka] [ka] [ka] [ka] [ka] [ka] [ka]

[0295] CTLA-4 antagonists In certain embodiments, the combinations described herein also include 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).

[0296] Information regarding tremelimumab (or its antigen-binding fragment) for use in the methods provided herein can be found in U.S. Pat. No. 6,682,736 (incorporated by reference) (where referred to as 11.2.1), the disclosure of which is incorporated herein by reference in its entirety. Tremelimumab (also known as CP-675,206, CP-675, CP-675206, and ticilimumab) 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 result in immune activation in vitro, and some patients treated with tremelimumab have shown tumor regression.

[0297] Tremelimumab for use in the methods provided herein comprises a heavy chain and a light chain, or a heavy chain variable region and a light chain variable region. In certain embodiments, tremelimumab or an antigen-binding fragment thereof for use in the methods provided herein comprises a light chain variable region comprising the amino acid sequence described above herein and a heavy chain variable region comprising the amino acid sequence described above herein. In certain embodiments, tremelimumab or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the Kabat-defined CDR1, CDR2, and CDR3 sequences described above herein, and the light chain variable region comprises the Kabat-defined CDR1, CDR2, and CDR3 sequences described above herein. Those of skill in the art will be able to readily identify Chothia-defined, Abm-defined, or other CDR definitions known to those of skill in the art. In certain aspects, tremelimumab or an antigen-binding fragment thereof for use in the methods provided herein comprises the variable heavy and variable light chain CDR sequences of the antibody disclosed in US Pat. No. 6,682,736, which is incorporated herein by reference in its entirety.

[0298] The present invention also contemplates utilizing small molecule inhibitors of CTLA-4, such as those described by Huxley et al. 2004 Cell Chemical Biology 11:1651-1658, including compounds of the formula: [ka]

[0299] Other small molecule CTLA-4 antagonists include: [ka]

[0300] In one embodiment, the combination includes an immune DASH inhibitor, e.g., an anti-PD-1 antibody molecule described herein, and an anti-CTLA-4 antibody, e.g., ipilimumab. Exemplary doses that can be used include a dose of about 1-10 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.

[0301] Other exemplary anti-CTLA-4 antibodies are disclosed, for example, in US Pat. No. 5,811,097.

[0302] In some embodiments of the methods described herein, the additional therapeutic agent is an antibody that modulates the immune response, ie, 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.

[0303] 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.

[0304] In some embodiments, the immune response stimulator is selected from the group consisting of a CD28 agonist, a 4-1BB agonist, an OX40 agonist, a CD27 agonist, a CD80 agonist, a CD86 agonist, a CD40 agonist, and a GITR agonist. In some embodiments, the OX40 agonist comprises an OX40 ligand, or an OX40-binding portion thereof. For example, the OX40 agonist can 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 a virus such as an adenovirus) capable of expressing an OX40 ligand. In some embodiments, the OX40 expression vector is Delta-24-RGDOX or DNX2401.

[0305] In some embodiments, the 4-1BB (CD137) agonist is a binding molecule such as anticalin. In some embodiments, the anticalin is PRS-343. In some embodiments, the 4-1BB agonist is an antibody that specifically binds to 4-1BB. In some embodiments, the antibody that binds to 4-1BB is PF-2566 (PF-05082566) or urelumab (BMS-663513).

[0306] In some embodiments, the CD27 agonist is an antibody that specifically binds to CD27. In some embodiments, the antibody that binds to CD27 is valilumab (CDX-1127).

[0307] In some embodiments, the GITR agonist comprises a GITR ligand or its GITR binding portion. 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.

[0308] In certain embodiments, an anti-CD39 antibody is combined with a STING agonist, preferably as part of a pharmaceutical composition. The cyclic-di-nucleotide (CDN) cyclic-di-AMP (produced by Listeria monocytogenes and other bacteria) and its analogs cyclic-di-GMP and cyclic-GMP-AMP are recognized by host cells as pathogen-associated molecular patterns (PAMPs) and bind to a pathogen recognition receptor (PRR) known as stimulator of interferon genes (STING). STING is an adaptor protein located in the cytoplasm of host mammalian cells that activates the TANK-binding kinase (TBK1)-IRF3 and NF-κB signaling axis, leading to the induction of IFN-β and other gene products that potently activate innate immunity. It is now recognized that STING is a component of the host cytotoxic surveillance pathway (Vance et al., 2009), sensing intracellular pathogen infection and inducing the production of IFN-β in response, which leads to the generation of an adaptive, protective, pathogen-specific immune response consisting of both 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.

[0309] Exemplary Combinations In a preferred embodiment, the present invention relates to the combination of an anti-CD39 antibody with an antitumor platinum coordination complex in the treatment of cancer, more particularly in the treatment of a cancer selected from lung cancer, sarcoma, melanoma, prostate cancer, pancreatic cancer, gastric cancer, ovarian cancer, hepatoma, breast cancer, colorectal cancer, renal cancer, brain cancer, and lymphoma, including, but not limited to, cisplatin, oxaliplatin, carboplatin, triplatin tetranitrate (BBR3464), satraplatin, tetraplatin, olmiplatin, iproplatin, nedaplatin, and lobaplatin. In the treatment of cancer, more particularly in the treatment of cancer selected from lung cancer, sarcoma, melanoma, prostate cancer, pancreatic cancer, gastric cancer, ovarian cancer, hepatoma, breast cancer, colorectal cancer, renal cancer, and brain cancer, particularly preferred is the combination of an anti-CD39 antibody with cisplatin, oxaliplatin, carboplatin, triplatin tetranitrate, satraplatin, tetraplatin, olmiplatin, iproplatin, nedaplatin, and lobaplatin, and even more preferred is the combination with cisplatin and oxaliplatin. In another preferred embodiment, the present invention relates to the combination of an anti-CD39 antibody with an antimetabolite in the treatment of cancer, more particularly in the treatment of cancer selected from lung cancer, sarcoma, melanoma, prostate cancer, pancreatic cancer, gastric cancer, ovarian cancer, hepatoma, breast cancer, colorectal cancer, renal cancer, esophageal cancer, brain cancer, anal cancer, leukemia, and lymphoma. This group of chemotherapy drugs includes, but is not limited to, 5-fluorouracil, gemcitabine, cytarabine, capecitabine, decitabine, floxuridine, fludarabine, aminopterin, methotrexate, pemetrexed, raltitrexed, cladribine, clofarabine, mercaptopurine, pentostatin, and thioguanine.In the treatment of cancer, more particularly in the treatment of cancer selected from lung cancer, sarcoma, malignant melanoma, prostate cancer, pancreatic cancer, gastric cancer, ovarian cancer, hepatoma, breast cancer, colorectal cancer, renal cancer, brain cancer, leukemia, and lymphoma, 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 of 5-fluorouracil, gemcitabine, cytarabine, and methotrexate.

[0310] In another preferred embodiment, the present invention relates to the combination of an anti-CD39 antibody with a mitotic inhibitor in the treatment of cancer, more particularly in the treatment of cancer selected from lung cancer, sarcoma, prostate cancer, gastric cancer, ovarian cancer, hepatoma, breast cancer, colorectal cancer, renal cancer, brain cancer, leukemia, and lymphoma. This chemotherapy group includes, but is not limited to, paclitaxel, docetaxel, vinblastine, vincristine, vindesine, and vinorelbine. In the treatment of cancer, more particularly in the treatment of cancer selected from lung cancer, sarcoma, malignant melanoma, prostate cancer, gastric cancer, ovarian cancer, hepatoma, breast cancer, colorectal cancer, renal cancer, and brain cancer, the combination of an anti-CD39 antibody with paclitaxel, docetaxel, vinblastine, vincristine, vindesine, and vinorelbine is particularly preferred, and the combination of paclitaxel, docetaxel, vincristine, and vinorelbine is even more preferred.

[0311] In another preferred embodiment, the present invention relates to the combination of an anti-CD39 antibody with anti-cancer antibiotics, including but not limited to daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, pixantrone, valrubicin, mitomycin C, bleomycin, actinomycin A, and mithramycin, in the treatment of cancer, more particularly lung cancer, sarcoma, melanoma, bladder cancer, prostate cancer, pancreatic cancer, thyroid cancer, gastric cancer, ovarian cancer, hepatoma, breast cancer, colorectal cancer, renal cancer, neuroblastoma, brain cancer, anal cancer, testicular cancer, leukemia, multiple myeloma, and lymphoma. In the treatment of cancer, more particularly in the treatment of lung cancer, sarcoma, malignant melanoma, prostate cancer, pancreatic cancer, gastric cancer, ovarian cancer, hepatoma, breast cancer, colorectal cancer, renal cancer, brain cancer, leukemia, and lymphoma, particularly preferred is a combination of an anti-CD39 antibody with daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, pixantrone, valrubicin, mitomycin C, bleomycin, actinomycin D, and mithramycin, and even more preferred is a combination of an anti-CD39 antibody with daunorubicin, doxorubicin, mitomycin C, and actinomycin D.

[0312] In another preferred embodiment, the present invention relates to the combination of an anti-CD39 antibody with a topoisomerase I and / or II inhibitor in the treatment of cancer, more particularly in the treatment of lung cancer, sarcoma, melanoma, prostate cancer, pancreatic cancer, gastric cancer, ovarian cancer, hepatoma, breast cancer, colorectal cancer, renal cancer, neuroblastoma, brain cancer, cervical cancer, testicular cancer, leukemia, and lymphoma. This group of chemotherapy includes, but is not limited to, topotecan, SN-38, irinotecan, camptothecin, rubitecan, etoposide, amsacrine, and teniposide. In the treatment of cancer, particularly in the treatment of lung cancer, sarcoma, malignant melanoma, prostate cancer, pancreatic cancer, gastric cancer, ovarian cancer, hepatoma, breast cancer, colon cancer, renal cancer, and brain cancer, particularly preferred is a combination of PM00104 or a pharmaceutically acceptable salt thereof with topotecan, SN-38, irinotecan, camptothecin, rubitecan, etoposide, amsacrine, and teniposide, and even more preferred is a combination of topotecan, irinotecan, and etoposide.

[0313] In another preferred embodiment, the present invention relates to the combination of an anti-CD39 antibody with a proteosome inhibitor in the treatment of cancer, more particularly in the treatment of lung cancer, prostate cancer, pancreatic cancer, gastric cancer, hepatoma, colorectal cancer, brain cancer, multiple myeloma, and lymphoma. This chemotherapy group includes, but is not limited to, bortezomib, disulfiram, epigallocatechin gallate, and salinosporamide A. In the treatment of cancer, more particularly in the treatment of lung cancer, prostate cancer, pancreatic cancer, gastric cancer, hepatoma, colorectal cancer, and brain cancer, the combination of an anti-CD39 antibody with bortezomib, disulfiram, epigallocatechin gallate, and salinosporamide A is particularly preferred, and the combination with bortezomib is even more preferred.

[0314] In another preferred embodiment, the present invention relates to the combination of an anti-CD39 antibody with a histone deacetylase inhibitor, including but not limited to romidepsin, panobinostat, vorinostat, mocetinostat, belinstat, entinostat, resminostat, PCI-24781, AR-42, CUDC-101, and valproic acid, in the treatment of cancer, more particularly lung cancer, sarcoma, prostate cancer, pancreatic cancer, gastric cancer, ovarian cancer, breast cancer, colorectal cancer, renal cancer, brain cancer, and lymphoma. In the treatment of cancer, more particularly in the treatment of lung cancer, sarcoma, prostate cancer, pancreatic cancer, gastric cancer, ovarian cancer, breast cancer, colorectal cancer, renal cancer, and brain cancer, particularly preferred is a combination of an anti-CD39 antibody with romidepsin, panobinostat, vorinostat, mocetinostat, belinostat, entinostat, resminostat, PCI-24781, AR-42, CUDC-101, and valproic acid, and even more preferred is a combination with vorinostat.

[0315] In another preferred embodiment, the present invention relates to the combination of an anti-CD39 antibody with a nitrogen mustard alkylating agent in the treatment of cancer, more particularly in the treatment of lung cancer, sarcoma, bladder cancer, gastric cancer, ovarian cancer, hepatoma, breast cancer, colorectal cancer, renal cancer, leukemia, multiple myeloma, and lymphoma. This chemotherapy group includes, but is not limited to, melphalan, ifosfamide, chlorambucil, cyclophosphamide, mechlorethamine, uramustine, estramustine, and bendamustine. In the treatment of cancer, particularly in the treatment of lung cancer, sarcoma, gastric cancer, ovarian cancer, hepatoma, breast cancer, colorectal cancer, and renal cancer, the combination of an anti-CD39 antibody with melphalan, ifosfamide, chlorambucil, cyclophosphamide, mechlorethamine, uramustine, estramustine, and bendamustine is particularly preferred, and the combination with cyclophosphamide is even more preferred. In another preferred embodiment, the present invention relates to the combination of an anti-CD39 antibody with a nitrosourea alkylating agent in the treatment of cancer, more particularly in the treatment of lung cancer, ovarian cancer, breast cancer, brain cancer, multiple myeloma, and lymphoma. This chemotherapy group includes, but is not limited to, lomustine, semustine, carmustine, fotemustine, and streptozotocin. In the treatment of cancer, more particularly in the treatment of lung cancer, ovarian cancer, and breast cancer, the combination of an anti-CD39 antibody with lomustine, semustine, carmustine, fotemustine, and streptozotocin is particularly preferred, and the combination with carmustine is even more preferred.

[0316] In another preferred embodiment, the present invention relates to the combination of an anti-CD39 antibody with a non-classical alkylating agent in the treatment of cancer, more particularly in the treatment of lung cancer, sarcoma, melanoma, pancreatic cancer, gastric cancer, ovarian cancer, breast cancer, colorectal cancer, renal cancer, brain cancer, leukemia, and lymphoma. This chemotherapy group includes, but is not limited to, procarbazine, dacarbazine, temozolomide, and altretamine. In the treatment of lung cancer, sarcoma, melanoma, gastric cancer, ovarian cancer, breast cancer, colorectal cancer, renal cancer, and brain cancer, the combination of an anti-CD39 antibody with procarbazine, dacarbazine, temozolomide, and altretamine is particularly preferred, and the combination of dacarbazine and temozolomide is even more preferred. In another preferred embodiment, the present invention relates to the combination of an anti-CD39 antibody with an estrogen antagonist in the treatment of cancer, more particularly in the treatment of breast cancer. This group of chemotherapy drugs includes, but is not limited to, toremifene, fulvestrant, tamoxifen, and nafoxidine. In the treatment of breast cancer, particularly preferred is a combination of an anti-CD39 antibody with toremifene, fulvestrant, tamoxifen, or nafoxidine, and even more preferred is a combination with tamoxifen.

[0317] In another preferred embodiment, the present invention relates to the combination of an anti-CD39 antibody with an androgen antagonist in the treatment of cancer, more particularly in the treatment of prostate cancer. This chemotherapy group includes, but is not limited to, bicalutamide, flutamide, MDV3100, and nilutamide. In the treatment of prostate cancer, the combination of an anti-CD39 antibody with bicalutamide, flutamide, MDV3100, and nilutamide is particularly preferred, and the combination with flutamide is even more preferred.

[0318] In another preferred embodiment, the present invention relates to the combination of an anti-CD39 antibody with an mTOR inhibitor in the treatment of cancer, more specifically in the treatment of lung cancer, sarcoma, melanoma, prostate cancer, pancreatic cancer, gastric cancer, ovarian cancer, breast cancer, colorectal cancer, renal cancer, and brain cancer. This chemotherapy group includes, but is not limited to, sirolimus, temsirolimus, everolimus, ridaforolimus, KU-0063794, and WYE-354. In the treatment of lung cancer, sarcoma, melanoma, prostate cancer, pancreatic cancer, gastric cancer, ovarian cancer, breast cancer, colorectal cancer, and brain cancer, the combination of an anti-CD39 antibody with sirolimus, temsirolimus, everolimus, ridaforolimus, KU-0063794, and WYE-354 is particularly preferred, and the combination with temsirolimus is even more preferred.

[0319] In another preferred embodiment, the present invention relates to the combination of an anti-CD39 antibody with a tyrosine kinase inhibitor in the treatment of cancer, more particularly in the treatment of cancer selected from lung cancer, sarcoma, prostate cancer, pancreatic cancer, gastric cancer, ovarian cancer, hepatoma, breast cancer, colorectal cancer, renal cancer, and brain cancer, including, but not limited to, erlotinib, sorafenib, axitinib, bosutinib, cediranib, crizotinib, dasatinib, gefitinib, imatinib, canertinib, lapatinib, lestaurtinib, neratinib, nilotinib, semaxanib, sunitinib, vatalanib, and vandetanib. In the treatment of cancer, more specifically in the treatment of cancer selected from lung cancer, sarcoma, prostate cancer, pancreatic cancer, gastric cancer, ovarian cancer, hepatoma, breast cancer, colorectal cancer, renal cancer, and brain cancer, particularly preferred is a combination of an anti-CD39 antibody with erlotinib, sorafenib, axitinib, bosutinib, cediranib, crizotinib, dasatinib, gefitinib, imatinib, canertinib, lapatinib, lestaurtinib, neratinib, nilotinib, semaxanib, sunitinib, vatalanib, and vandetanib, and more preferred is a combination with erlotinib.

[0320] Another embodiment encompassed by the present invention relates to any one of the aforementioned methods further comprising administering to the patient a MAP kinase pathway inhibitor or a WNT pathway inhibitor.

[0321] 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.

[0322] In some embodiments, the BRAF inhibitor is selected from the group consisting of GDC-0879, PLX-4720, sorafenib tosylate, dabrafenib, and LGX818. In some embodiments, the MEK inhibitor is selected from the group consisting of GSK1120212, selumetinib, and MEK162.

[0323] In some embodiments, the WNT pathway inhibitor is a β-catenin inhibitor or a frizzled inhibitor.

[0324] In some embodiments, the β-catenin inhibitor is selected from the group consisting of niclosamide, XAV-939, FH535, and ICG001.

[0325] Another aspect of the invention relates to any one of the aforementioned methods, further comprising administering to the patient a cancer vaccine. In some embodiments, the cancer vaccine is a dendritic cell vaccine. Another embodiment encompassed by the present invention relates to any one of the aforementioned methods, further comprising administering adoptive cell transfer to the patient.

[0326] In some embodiments, the adoptive cell transfer is a CAR-T cell therapy.

[0327] Another embodiment encompassed by the invention relates to any one of the aforementioned methods, further comprising administering antibody therapy to the patient.

[0328] Another embodiment encompassed by the invention relates to any one of the aforementioned methods, wherein administration of an anti-CD39 antibody enhances antibody-dependent cell-mediated cytotoxicity of the antibody therapy.

[0329] In some embodiments, the antibody therapy is selected from the group consisting of trastuzamab, cetuximab, bevacizumab, and rituximab.

[0330] Additionally, treatment with anti-CD39 antibodies may include combination therapy with other biological molecules, such as one or more cytokines (e.g., lymphokines, interleukins, tumor necrosis factors, and / or growth factors), or may involve surgical removal of tumors, removal of cancer cells, or other therapy as deemed necessary by the treating physician. In some embodiments, the additional therapeutic agent is an immune response stimulator.

[0331] In some embodiments of the methods described herein, the anti-CD39 antibody may 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, neurotrophins, 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.

[0332] In some embodiments of the methods described herein, the additional therapeutic agent is an immune response stimulator, hi 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).

[0333] Dosage schedule In certain embodiments of the methods described herein, the treatment comprises administering an anti-CD39 antibody in combination with radiation therapy. The anti-CD39 antibody treatment can be administered before, simultaneously with, or after radiation therapy. The dosing schedule for such radiation therapy can be determined by a skilled practitioner.

[0334] In certain embodiments of the methods described herein, the treatment comprises administering an anti-CD39 antibody in combination with antiviral therapy. The treatment with the anti-CD39 antibody can be administered before, simultaneously with, or after the antiviral therapy. The antiviral drug used in the combination therapy depends on the virus that the subject is infected with.

[0335] Concomitant administration herein includes co-administration using a single pharmaceutical formulation or separate formulations, and sequential administration in either order, generally with a period of time during which all active agents simultaneously exert their biological activity.

[0336] It will be understood that the combination of an anti-CD39 antibody and at least one additional therapeutic agent may be administered in any order or simultaneously. In some embodiments, the anti-CD39 antibody will be administered to a patient who has previously received 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 concurrently. For example, a subject may be given the 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 one 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 will further be understood that two (or more) agents or treatments may be administered to a subject within hours or minutes of each other (ie, substantially simultaneously).

[0337] For the treatment of disease, the appropriate dosage of an anti-CD39 antibody will depend on the type of disease being treated, the severity and course of the disease, the response of the disease, whether the anti-CD39 antibody is being administered for therapeutic or prophylactic purposes, previous therapy, and the patient's clinical history, all of which will be determined by the treating physician. The anti-CD39 antibody can be administered once or over a series of treatments lasting from several days to several months, or until a cure is effected or a reduction in disease status is achieved (e.g., reduction in tumor size). The optimal dosing schedule can be calculated from measurements of drug accumulation in the patient's body and will vary depending on the relative efficacy of individual agents. The administering physician will be able to determine the optimal dosage, dosing method, and repetition rate. In certain embodiments, the dosage ranges from 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 dosage of the anti-CD39 antibody is about 0.1 mg to about 20 mg / kg body weight. In some embodiments, the dosage of the anti-CD39 antibody is about 0.1 mg / kg body weight. In some embodiments, the dosage of the anti-CD39 antibody is about 0.25 mg / kg body weight. In some embodiments, the dosage of the anti-CD39 antibody is about 0.5 mg / kg body weight. In some embodiments, the dosage of the anti-CD39 antibody is about 1 mg / kg body weight. In some embodiments, the dosage of the anti-CD39 antibody is about 1.5 mg / kg body weight. In some embodiments, the dosage of the anti-CD39 antibody is about 2 mg / kg body weight. In some embodiments, the dosage of the anti-CD39 antibody is about 2.5 mg / kg body weight. In some embodiments, the dosage of the anti-CD39 antibody is about 5 mg / kg body weight. In some embodiments, the dosage of the anti-CD39 antibody is about 7.5 mg / kg body weight. In some embodiments, the dosage 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, dosages can be given one or more times daily, one or more times weekly, one or more times monthly, or one or more times yearly, hi certain embodiments, the anti-CD39 antibody is given once every week, once every two weeks, once every three weeks, or once every four weeks.

[0338] In some embodiments, the anti-CD39 antibody may be administered at an initial, higher "loading" dose, followed by one or more lower doses. In some embodiments, the frequency of administration may also vary. In some embodiments, a dosing regimen may include administering an initial dose, followed by additional doses (or "maintenance" doses) 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 weekly maintenance doses, e.g., half the initial dose. Alternatively, a dosing regimen may include administering an initial loading dose, followed by biweekly maintenance doses, e.g., half the initial dose. Alternatively, a dosing regimen may include administering three initial doses over three weeks, followed by maintenance doses of the same amount, e.g., every other week.

[0339] As known to those skilled in the art, the administration of any therapeutic agent can result in side effects and / or toxicity. In some cases, the side effects and / or toxicity are so severe that the administration of a particular agent at a therapeutically effective dose is impossible. In some cases, it may be necessary to discontinue drug therapy and try other agents. However, many agents in the same therapeutic class often exhibit similar side effects and / or toxicity. This means that patients may need to discontinue treatment or may experience unpleasant side effects associated with the therapeutic agent.

[0340] 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, or the anti-CD39 antibody is administered every 3 weeks for 4 cycles. The dosing schedule can be determined and subsequently modified by one of skill in the art.

[0341] Thus, the present invention provides methods of administering an anti-CD39 antibody described herein to a subject, comprising using an intermittent dosing strategy to administer one or more agents that can reduce side effects and / or toxicity associated with the administration of an anti-CD39 antibody, a chemotherapeutic agent, or 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 the intermittent dosing strategy. In some embodiments, the intermittent dosing strategy comprises administering to the subject an initial dose of the anti-CD39 antibody and administering subsequent doses of the anti-CD39 antibody approximately once every two weeks. In some embodiments, the intermittent dosing strategy comprises administering to the subject an initial dose of the anti-CD39 antibody and administering subsequent doses of the anti-CD39 antibody approximately once every three weeks. In some embodiments, the intermittent dosing strategy comprises administering to the subject an initial dose of the anti-CD39 antibody and administering subsequent doses of the anti-CD39 antibody approximately once every four weeks. In some embodiments, the anti-CD39 antibody is administered using an intermittent dosing strategy and the chemotherapeutic agent is administered weekly.

[0342] Anti-infective treatment combinations In one embodiment, the invention provides a method for treating a subject using an anti-CD39 antibody, wherein the subject is suffering from a viral infection. In one embodiment, the viral infection is an infection with a virus selected from the group consisting of human immunodeficiency virus (HIV), hepatitis virus (A, B, or C), herpesvirus (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 contagiosum virus, poliovirus, rabies virus, JC virus, or arboviral encephalitis virus. In one embodiment, the invention provides a method for treating a subject with an anti-CD39 antibody, wherein the subject is suffering from a bacterial infection. In one embodiment, the bacterial infection is an infection with a bacterium selected from the group consisting of Chlamydia, rickettsial bacteria, mycobacteria, staphylococci, streptococci, pneumonococci, meningococci and gonococcci, klebsiella, proteus, serratia, pseudomonas, Legionella, Corynebacterium diphtheriae, Salmonella, bacilli, Vibrio cholerae, Clostridium tetans, Clostridium botulinum, Bacillus anthricis, Yersinia pestis, Mycobacterium leprae, Mycobacterium lepromatosis, and Borriella.

[0343] In one embodiment, the invention provides a method for treating a subject with an anti-CD39 antibody, wherein the subject is suffering from a fungal infection. In one embodiment, the fungal infection includes infection with a fungus selected from the group consisting of Candida (e.g., albicans, krusei, glabrata, tropicalis), Cryptococcus neoformans, Aspergillus (e.g., fumigatus, niger), Mucorales (e.g., mucor, absidia, rhizopus), Sporothrix schenkii, Blastomyces dermatitidis, Paracoccidioides brasiliensis, Coccidioides immitis, and Histoplasma capsulatum.

[0344] In one embodiment, the invention provides a method for treating a subject with an anti-CD39 antibody, wherein the subject is suffering from a parasitic infection. In one embodiment, the parasitic infection is an infection with a parasite 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.

[0345] VII. Anti-CD39 antibody complex The anti-CD39 antibodies disclosed herein may also be conjugated to a chemical moiety, which may be, among other things, a polymer, a radionuclide, or a cytotoxic agent. For example, the present invention provides an anti-CD39 antibody 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 immunostimulant, 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 above aspects or embodiments is conjugated to a therapeutic moiety. Exemplary therapeutic moieties include cytotoxic moieties, radioisotopes, cytokines, and lytic peptides.

[0346] In certain embodiments, anti-CD39 antibodies can induce cytotoxicity in CD39-expressing cells by internalization of the antibody conjugated to or associated with a cytotoxic moiety. Cytotoxic moieties include, for example, taxol; cytochalasin B; gramicidin D; ethidium bromide; emetine; mitomycin; etoposide; tenoposide; vincristine; vinblastine; colchicine; doxorubicin; daunorubicin; dihydroxy anthracin dione; dione); tubulin inhibitors, for example, maytansine or an analogue or derivative thereof; mitotic inhibitors such as monomethyl auristatin E or F or an analogue or derivative thereof; dolastatin 10 or 15 or an analogue thereof; irinotecan or an analogue thereof; mitoxantrone; mithramycin; actinomycin D; 1-dehydrotestosterone; glucocorticoids; procaine; tetracaine; lidocaine; propranolol; puromycin; calicheamicin or an analogue or derivative thereof; methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, fludarabine, 5-fluorouracil, decarbazine, hydroxyurea, asparaginase, gemcitabine, or clarithromycin. antimetabolites such as dolivine; alkylating agents such as mechlorethamine, thioepa, chlorambucil, melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, dacarbazine (DTIC), procarbazine, and mitomycin C; platinum derivatives such as cisplatin or carboplatin; duocarmycin A, duocarmycin SA, rachelmycin (CC-1065), or their analogs or derivatives; antibiotics such as dactinomycin, bleomycin, daunorubicin, doxorubicin, idarubicin, mithramycin, mitomycin, mitoxantrone, plicamycin, and anthramycin (AMC); pyrrolo[2,1-c][1,4]-benzodiazepines (PDB);diphtheria toxins and related molecules such as diphtheria A chains and their active fragments and hybrid molecules, ricin toxins such as ricin A or deglycosylated ricin A chain toxins, cholerae toxins, Shiga-like toxins such as SLTI, SLT II, ​​SLT IIV, LT toxins, C3 toxins, Shiga toxins, pertussis toxins, tetanus toxins, soybean Bowman-Birk protease inhibitors, Pseudomonas exotoxins, allorin, saporin, modeccin, geranin, abrin A chain, modeccin A chain, alphasarcin, Aleurites fordii proteins, dianthin proteins, Phytolacca americana proteins such as PAPI, PAPII, and PAP-S, momordica charantia inhibitors, curcin, sablefish, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, and enomycin toxins; ribonuclease (RNase); DNase I, Staphylococcal enterotoxin A; pokeweed antiviral protein; diphtherin toxin; and Pseudomonas endotoxin.

[0347] In one embodiment, the anti-CD39 antibody is conjugated to an auristatin or its peptide analog, derivative, or prodrug. Auristatins interfere with microtubule dynamics, GTP hydrolysis, and nuclear and cell division (Woyke et al. (2001) Antimicrob. Agents and Chemother. 45(12):3580-3584), and have anticancer activity (US Pat. No. 5,663,149) and antifungal activity (Pettit et al., (1998) Antimicrob. Agents and Chemother. 42:2961-2965). For example, auristatin E can react with paraacetylbenzoic 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 suitable linkers for conjugating auristatins to Abs, are described, for example, in U.S. Pat. Nos. 5,635,483, 5,780,588, and 6,214,345 and International Patent Application Publication Nos. WO02088172, WO2004010957, WO2005081711, WO2005084390, WO2006132670, WO03026577, WO200700860, WO207011968, and WO205082023.

[0348] In another embodiment, the anti-CD39 antibody is conjugated to pyrrolo[2,1-c][1,4]-benzodiazepine (PDB) or an analog, derivative, or prodrug thereof. Suitable PDBs and PDB derivatives and related techniques 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 P Wet al., Bioorg Med Chem Lett 2009;19:6463-6466, and Sagnou et al., Bioorg Med Chem Lett 2000;10(18):2083-2086.

[0349] In another embodiment, the anti-CD39 antibody is conjugated to a cytotoxic moiety selected from the group consisting of an anthracycline, maytansine, calicheamicin, duocarmycin, rachelmycin (CC-1065), dolastatin 10, dolastatin 15, irinotecan, monomethyl auristatin E, monomethyl auristatin F, PDB, or an analog, derivative, or prodrug thereof.

[0350] In certain embodiments, 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 rachelmycin (CC-1065), or an analog, derivative, or prodrug thereof. In another specific embodiment, the antibody is conjugated to dolastatin 10, or an analog, derivative, or prodrug thereof. In another specific embodiment, the antibody is conjugated to dolastatin 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.

[0351] 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., siRNA molecule), or an immunostimulatory nucleic acid (e.g., immunostimulatory CpG motif-containing DNA molecule). In another embodiment, the CD39-specific antibodies encompassed by the present invention are conjugated to an aptamer or ribozyme.

[0352] In one embodiment, an anti-CD39 antibody encompassed by the present invention is conjugated to a lytic peptide, such as CLIP, magainin 2, melittin, cecropin, and P18, for example, as a fusion protein.

[0353] 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.

[0354] In certain embodiments, the chemical moiety is a polymer that increases the half-life of the antibody or fragment within the subject's body. Suitable polymers include, but are not limited to, hydrophilic polymers, including polyethylene glycol (PEG) (e.g., PEG with a molecular weight of 2 kDa, 5 kDa, 10 kDa, 12 kDa, 20 kDa, 30 kDa, or 40 kDa), dextran, and monomethoxypolyethylene glycol (mPEG). Lee, et al., (1999) (Bioconj. Chem. 10:973-981) disclose PEG-conjugated single-chain antibodies. Wen, et al., (2001) (Bioconj. Chem. 12:545-553) disclose conjugated antibodies with PEG attached to a radiometal chelator (diethylenetriaminepentaacetic acid (DTPA)).

[0355] Anti-CD39 antibodies are 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 It may be conjugated with a label such as Fe.

[0356] The anti-CD39 antibodies can also be conjugated with fluorescent or chemiluminescent labels, including fluorophores such as rare earth chelates, fluorescein and its derivatives, rhodamine and its derivatives, isothiocyanates, phycoerythrin, phycocyanin, allophycocyanin, o-phthalaldehyde, fluorescamine, 152 These include Eu, dansyl, umbelliferone, luciferin, luminal label, isoluminal label, aromatic acridinium ester label, imidazole label, acridimium salt label, oxalate ester label, aequorin label, 2,3-dihydrophthalazinedione, biotin / avidin, spin label, and stable free radicals.

[0357] 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 used, including those described in 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.

[0358] VIII. Pharmaceutical Compositions 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 the anti-CD39 antibodies, antibody fragments, nucleic acids, or vectors, mixed with a suitable carrier, e.g., a pharmaceutically acceptable agent. Typically, the anti-CD39 antibodies, antibody fragments, nucleic acids, or vectors encompassed by the present invention are sufficiently purified for administration to animals before being formulated into a pharmaceutical composition.

[0359] Pharmaceutically acceptable agents for use in the pharmaceutical compositions include carriers, excipients, diluents, antioxidants, preservatives, colorants, flavorings and diluents, emulsifiers, suspending agents, solvents, fillers, extenders, buffers, delivery vehicles, tonicity agents, co-solvents, wetting agents, complexing agents, buffering agents, antibacterial agents, and surface active agents.

[0360] Neutral buffered saline or saline mixed with serum albumin are exemplary suitable carriers. Pharmaceutical compositions may include antioxidants, including ascorbic acid; low-molecular-weight polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, such as glucose, mannose, or dextrin; chelating agents, such as EDTA; sugar alcohols, such as mannitol or sorbitol; salt-forming counterions, such as sodium; and / or nonionic surfactants, such as Tween®, Pluronics®, and polyethylene glycol (PEG). Suitable tonicity enhancers include, for example, alkali metal halides (preferably sodium chloride or potassium chloride), mannitol, sorbitol, and the like. Suitable preservatives include benzalkonium chloride, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, and the like. Hydrogen peroxide may also be used as a preservative. Suitable cosolvents include glycerin, propylene glycol, and PEG. Suitable complexing agents include caffeine, polyvinylpyrrolidone, beta-cyclodextrin, or hydroxypropyl-beta-cyclodextrin. Suitable surfactants or wetting agents include sorbitan esters, polysorbates such as polysorbate 80, tromethamine, lecithin, cholesterol, tyloxapal, and the like. The buffer may be a conventional buffer such as acetate, borate, citrate, phosphate, bicarbonate, or Tris-HCl. Acetate buffers may have a pH of about 4-5.5, and Tris buffers may have a pH of about 7-8.5. Additional pharmaceutical agents are described in Remington's Pharmaceutical Sciences, 18th Edition, A.R. Gennaro, ed., Mack Publishing Company, 1990.

[0361] The composition may be in liquid or lyophilized or freeze-dried form and may include one or more cryoprotectants, excipients, surfactants, high molecular weight structural additives, and / or bulking agents (see, e.g., U.S. Patent Nos. 6,685,940, 6,566,329, and 6,372,716). In one embodiment, a cryoprotectant is included, and the cryoprotectant is a non-reducing sugar, such as sucrose, lactose, or trehalose. The amount of cryoprotectant typically included is such that, upon reconstitution, the resulting formulation is isotonic, although hypertonic or slightly hypotonic formulations may also be suitable. Furthermore, the amount of cryoprotectant should be sufficient to prevent unacceptable amounts of protein degradation and / or aggregation upon lyophilization. Exemplary cryoprotectant concentrations of sugars (e.g., sucrose, lactose, trehalose) in the pre-lyophilized formulation are from about 10 mM to about 400 mM.In another embodiment, the surfactant may be, for example, a non-ionic surfactant or an ionic surfactant, such as a polysorbate (e.g., polysorbate 20 or 80); a poloxamer (e.g., poloxamer 188); a poly(ethylene glycol) phenyl ether (e.g., Triton®); sodium dodecyl sulfate (SDS); sodium lauryl sulfate; sodium octyl glucoside; lauryl sulfobetaine, myristyl sulfobetaine, linoleyl sulfobetaine, or stearyl sulfobetaine; lauryl sarcosine, myristyl sarcosine, linoleyl sarcosine, or stearyl sarcosine; linoleic acid, methyl sarcosine ... Examples of surfactants that may be present in the pre-lyophilized formulation include yl betaine, myristyl betaine, or cetyl betaine; lauroamidopropyl betaine, cocamidopropyl betaine, linoleamidopropyl betaine, myristamidopropyl betaine, palmidopropyl betaine, or isostearamidopropyl betaine (e.g., lauroamidopropyl); myristamidopropyl dimethylamine, palmidopropyl dimethylamine, or isostearamidopropyl dimethyla...

Claims

1. An anti-CD39 antibody or antigen-binding fragment thereof, a) a light chain variable domain comprising a complementarity determining region (CDR) L1 comprising the sequence of SEQ ID NO: 32, a CDRL2 comprising the sequence of SEQ ID NO: 33, and a CDRL3 comprising the sequence of SEQ ID NO: 34, and a heavy chain variable domain comprising a CDRH1 comprising the sequence of SEQ ID NO: 29, a CDRH2 comprising the sequence of SEQ ID NO: 30, and a CDRH3 comprising the sequence of SEQ ID NO: 31; b) a light chain variable domain comprising a CDRL1 comprising the sequence QNIYSN, a CDRL2 comprising the sequence RAS, and a CDRL3 comprising the sequence QQGFDSSNIDNT, and a heavy chain variable domain comprising a CDRH1 comprising the sequence GFSLSAYG, a CDRH2 comprising the sequence IYSSGRT, and a CDRH3 comprising the sequence ARSRAGISSGDGFDS; c) a light chain variable domain comprising a CDRL1 comprising the sequence QSVLLNNQ, a CDRL2 comprising the sequence DAS, and a CDRL3 comprising the sequence LGGYSGNLYA, and a heavy chain variable domain comprising a CDRH1 comprising the sequence GFSLSKSI, a CDRH2 comprising the sequence IGSSGST, and a CDRH3 comprising the sequence ARGLLYSGNKS; d) a light chain variable domain comprising a CDRL1 comprising the sequence QNIYSN, a CDRL2 comprising the sequence RAS, and a CDRL3 comprising the sequence QQGFSSNNVDNT, and a heavy chain variable domain comprising a CDRH1 comprising the sequence GFSLSSYA, a CDRH2 comprising the sequence INSYGTT, and a CDRH3 comprising the sequence ARGDSYGSGVGLGL; e) a light chain variable domain comprising a CDRL1 comprising the sequence EIIYSN, a CDRL2 comprising the sequence GAS, and a CDRL3 comprising the sequence QQSFSSNNVGNI, and a heavy chain variable domain comprising a CDRH1 comprising the sequence GFSLSSYA, a CDRH2 comprising the sequence ISSSGST, and a CDRH3 comprising the sequence ARDRVIYSIGPYYFNL; f) a light chain variable domain comprising a CDRL1 comprising the sequence QNINTW, a CDRL2 comprising the sequence RAS, and a CDRL3 comprising the sequence QQYDASINIDNA, and a heavy chain variable domain comprising a CDRH1 comprising the sequence GFSLSTHA, a CDRH2 comprising the sequence TYASGRT, and a CDRH3 comprising the sequence ARNGADETFYYFDL; g) a light chain variable domain comprising a CDRL1 comprising the sequence ERIYSN, a CDRL2 comprising the sequence YAS, and a CDRL3 comprising the sequence QQGYSNNNVDNT, and a heavy chain variable domain comprising a CDRH1 comprising the sequence GIDLSSNA, a CDRH2 comprising the sequence IRNNDIT, and a CDRH3 comprising the sequence ARGGGSYSIVFWNL; h) a light chain variable domain comprising a CDRL1 comprising the sequence ERIYSN, a CDRL2 comprising the sequence YTS, and a CDRL3 comprising the sequence QQGYSSSNVDNT, and a heavy chain variable domain comprising a CDRH1 comprising the sequence GIDLSNNA, a CDRH2 comprising the sequence IRSSGST, and a CDRH3 comprising the sequence ARGGGSYSIVFWNL; i) a light chain variable domain comprising a CDRL1 comprising the sequence QNIYSN, a CDRL2 comprising the sequence RAS, and a CDRL3 comprising the sequence QQGFSSNNVDNT, and a heavy chain variable domain comprising a CDRH1 comprising the sequence GFSLSSYA, a CDRH2 comprising the sequence INSYGTT, and a CDRH3 comprising the sequence ARGDSYGSGVGLGL; j) a light chain variable domain comprising a CDRL1 comprising the sequence QSVLLNNQ, a CDRL2 comprising the sequence DAS, and a CDRL3 comprising the sequence LGGYSGNLYA, and a heavy chain variable domain comprising a CDRH1 comprising the sequence GFSLSKSI, a CDRH2 comprising the sequence IGSSGST, and a CDRH3 comprising the sequence ARGLLYSGNKS; or k) a light chain variable domain comprising a CDRL1 comprising the sequence EIIYSN, a CDRL2 comprising the sequence GAS, and a CDRL3 comprising the sequence QQSFSSNNVGNI; and a heavy chain variable domain comprising a CDRH1 comprising the sequence GFSLSSYA, a CDRH2 comprising the sequence ISSSGST, and a CDRH3 comprising the sequence ARDRVIYSIGPYYFNL. An anti-CD39 antibody or antigen-binding fragment thereof comprising:

2. a) a light chain variable domain having at least 90% identity with the sequence of SEQ ID NO: 4, and a heavy chain variable domain having at least 90% identity with the sequence of SEQ ID NO: 2; b) a light chain variable domain having at least 90% identity with the sequence of SEQ ID NO: 8 and a heavy chain variable domain having at least 90% identity with the sequence of SEQ ID NO: 6; c) a light chain variable domain having at least 90% identity with the sequence of SEQ ID NO: 12, and a heavy chain variable domain having at least 90% identity with the sequence of SEQ ID NO: 10; d) a light chain variable domain having at least 90% identity with the sequence of SEQ ID NO: 16, and a heavy chain variable domain having at least 90% identity with the sequence of SEQ ID NO: 14; e) a light chain variable domain having at least 90% identity with the sequence of SEQ ID NO: 20, and a heavy chain variable domain having at least 90% identity with the sequence of SEQ ID NO: 18; f) a light chain variable domain having at least 90% identity with the sequence of SEQ ID NO: 24, and a heavy chain variable domain having at least 90% identity with the sequence of SEQ ID NO: 22; g) a light chain variable domain having at least 90% identity with the sequence of SEQ ID NO: 28 and a heavy chain variable domain having at least 90% identity with the sequence of SEQ ID NO: 26; h) a light chain variable domain having at least 90% identity to the sequence of SEQ ID NO: 44, and a heavy chain variable domain having at least 90% identity to the sequence of SEQ ID NO: 42; i) a light chain variable domain having at least 90% identity to the sequence of SEQ ID NO: 48, and a heavy chain variable domain having at least 90% identity to the sequence of SEQ ID NO: 46; j) a light chain variable domain having at least 90% identity to the sequence of SEQ ID NO: 52 and a heavy chain variable domain having at least 90% identity to the sequence of SEQ ID NO: 50; or k) a light chain variable domain having at least 90% identity with the sequence of SEQ ID NO: 56, and a heavy chain variable domain having at least 90% identity with the sequence of SEQ ID NO:

54. The anti-CD39 antibody or antigen-binding fragment thereof of claim 1, comprising:

3. a) a light chain variable domain comprising the sequence of SEQ ID NO: 4, and a heavy chain variable domain comprising the sequence of SEQ ID NO: 2; b) a light chain variable domain comprising the sequence of SEQ ID NO: 8, and a heavy chain variable domain comprising the sequence of SEQ ID NO: 6; c) a light chain variable domain comprising the sequence of SEQ ID NO: 12, and a heavy chain variable domain comprising the sequence of SEQ ID NO: 10; d) a light chain variable domain comprising the sequence of SEQ ID NO: 16, and a heavy chain variable domain comprising the sequence of SEQ ID NO: 14; e) a light chain variable domain comprising the sequence of SEQ ID NO: 20, and a heavy chain variable domain comprising the sequence of SEQ ID NO: 18; f) a light chain variable domain comprising the sequence of SEQ ID NO: 24, and a heavy chain variable domain comprising the sequence of SEQ ID NO: 22; g) a light chain variable domain comprising the sequence of SEQ ID NO: 28, and a heavy chain variable domain comprising the sequence of SEQ ID NO: 26; h) a light chain variable domain comprising the sequence of SEQ ID NO: 44, and a heavy chain variable domain comprising the sequence of SEQ ID NO: 42; i) a light chain variable domain comprising the sequence of SEQ ID NO: 48, and a heavy chain variable domain comprising the sequence of SEQ ID NO: 46; j) a light chain variable domain comprising the sequence of SEQ ID NO: 52, and a heavy chain variable domain comprising the sequence of SEQ ID NO: 50; or k) a light chain variable domain comprising the sequence of SEQ ID NO: 56, and a heavy chain variable domain comprising the sequence of SEQ ID NO: 54 The anti-CD39 antibody or antigen-binding fragment thereof of claim 1 or 2, comprising:

4. a) a light chain variable domain comprising a complementarity determining region (CDR) L1 comprising the sequence of SEQ ID NO: 32, a CDRL2 comprising the sequence of SEQ ID NO: 33, and a CDRL3 comprising the sequence of SEQ ID NO: 34, and a heavy chain variable domain comprising a CDRH1 comprising the sequence of SEQ ID NO: 29, a CDRH2 comprising the sequence of SEQ ID NO: 30, and a CDRH3 comprising the sequence of SEQ ID NO:

31. The anti-CD39 antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, comprising:

5. 5. The anti-CD39 antibody or antigen-binding fragment thereof of claim 4, comprising a light chain variable domain comprising the sequence of SEQ ID NO: 4 and a heavy chain variable domain comprising the sequence of SEQ ID NO:

2.

6. 6. The anti-CD39 antibody or antigen-binding fragment thereof according to any one of claims 4 to 5, comprising a light chain comprising the sequence of SEQ ID NO: 38 and a heavy chain comprising the sequence of SEQ ID NO:

36.

7. The anti-CD39 antibody or antigen-binding fragment thereof of any one of claims 1 to 6, wherein the anti-CD39 antibody or antigen-binding fragment thereof comprises at least one antigen-binding domain that binds to ectonucleoside triphosphate diphosphohydrolase-1 (CD39) at a site such that the anti-CD39 antibody forms a stable immune complex.

8. The anti-CD39 antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, wherein the anti-CD39 antibody or antigen-binding fragment thereof comprises an FcγRIIIa-binding portion that binds to an FcγRIIIa receptor and confers antibody-dependent cellular cytotoxicity (ADCC) activity against CD39+ cells to the anti-CD39 antibody.

9. the anti-CD39 antibody or antigen-binding fragment thereof (i) complement-dependent cytotoxicity (CDC) activity against CD39+ cells; (ii) antibody-mediated targeted cytosis of CD39 on CD45+ immune cells; (iii) antibody-mediated targeted cytosis of CD39 from tumor vascular endothelial disruption or disruption of the vasculature network in tumors; and / or (iv) binding to a CD39 epitope having a sequence selected from IYLTDCMERAR, LRMESEELADR, RVKGPGISKFV, DCMERAREVIPR, LTDCMERAREVIPR, SLSNYPFDFQGAR, CRVKGPGISKF, GAYGWITINYLLGKFSQK, or ILRDPCFHPGYKK. The anti-CD39 antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, which promotes

10. 9. The anti-CD39 antibody or antigen-binding fragment thereof of claim 8, wherein the FcγRIIIa-binding portion is selected from the group consisting of an Fc domain, an antibody that binds to FcγRIIIa, and an FcγRIIIa-binding peptide.

11. The antigen-binding domain is Fab, Fab', F(ab') ２ 8. The anti-CD39 antibody or antigen-binding fragment thereof of claim 7, wherein the antibody or antigen-binding fragment thereof is selected from the group consisting of a Fv or single-chain Fv (scFv), a dsFv, a sc(Fv)2, and a diabody fragment, and / or the anti-CD39 antibody or antigen-binding fragment thereof is monoclonal.

12. The anti-CD39 antibody or antigen-binding fragment thereof of any one of claims 1 to 11, wherein the anti-CD39 antibody or antigen-binding fragment thereof is conjugated to an agent.

13. 13. The anti-CD39 antibody or antigen-binding fragment thereof of claim 12, 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 immunomodulatory agent, a detectable moiety, and a tag.

14. The anti-CD39 antibody or antigen-binding fragment thereof according to any one of claims 1 to 13, wherein the anti-CD39 antibody or antigen-binding fragment thereof comprises an Fc domain of the IgG1 or IgG3 isotype.

15. The anti-CD39 antibody or antigen-binding fragment thereof of claim 14, wherein the Fc domain is a human Fc domain.

16. The anti-CD39 antibody or antigen-binding fragment thereof according to any one of claims 1 to 15, wherein the anti-CD39 antibody or antigen-binding fragment thereof is hypofucosylated or defucosylated.

17. The anti-CD39 antibody or antigen-binding fragment thereof according to any one of claims 1 to 16, wherein the anti-CD39 antibody or antigen-binding fragment thereof is a human antibody or a humanized antibody.

18. The anti-CD39 antibody or antigen-binding fragment thereof according to any one of claims 1 to 17, 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 costimulatory receptor, and functions as a checkpoint inhibitor when the additional antigen-binding site is for an immune checkpoint, or functions as a costimulatory agonist when the additional antigen-binding site is for a costimulatory receptor.

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

20. 20. The anti-CD39 antibody or antigen-binding fragment thereof of claim 18 or 19, wherein the additional antigen-binding site binds to a checkpoint protein that is upregulated on T cells and is associated with T cell exhaustion.

21. 19. The anti-CD39 antibody or antigen-binding fragment thereof of claim 18, wherein the additional antigen-binding site binds to an immune co-stimulatory receptor selected from the group consisting of an MHCI molecule, a BTLA receptor, OX40, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), and 4-1BB (CD137).

22. 19. The anti-CD39 antibody or antigen-binding fragment thereof of claim 18, wherein the additional antigen-binding site binds to CD47, SIRPα, CD24, or Siglec-10.

23. 23. 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 22, and one or more pharmaceutically acceptable excipients, buffers or solutions.

24. 24. The pharmaceutical preparation of claim 23, wherein the pharmaceutical preparation is for improving anti-tumor T cell immunity and is suitable for administration to a subject having a tumor, the pharmaceutical preparation 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 improves intratumoral CD39. ｈｉｇｈ The pharmaceutical preparation results in a reduction in the number of cells, enhancing T cell infiltration into the tumor and / or reducing T cell exhaustion in the tumor.

25. An isolated nucleic acid molecule encoding the immunoglobulin heavy and light chain polypeptides of the anti-CD39 antibody or antigen-binding fragment thereof of any one of claims 1 to 22.

26. 26. Isolated immunoglobulin heavy and light chain polypeptides encoded by the isolated nucleic acid molecule of claim 25.

27. 26. A vector comprising the isolated nucleic acid molecule of claim 25.

28. 28. The vector of claim 27, which is an expression vector.

29. 26. A host cell comprising the isolated nucleic acid molecule of claim 25, a) expressing the anti-CD39 antibody or antigen-binding fragment thereof of any one of claims 1 to 22; b) comprising immunoglobulin heavy and light chain polypeptides according to claim 26; and / or c) A host cell comprising the vector of claim 27 or 28.

30. A kit for detecting CD39, comprising at least one anti-CD39 antibody or antigen-binding fragment thereof according to any one of claims 1 to 22.

31. The kit of claim 30, wherein the kit comprises at least one label for detecting the anti-CD39 antibody or antigen-binding fragment thereof of any one of claims 1 to 22, or a complex comprising the anti-CD39 antibody or antigen-binding fragment thereof.

32. 23. A method for producing at least one anti-CD39 antibody or antigen-binding fragment thereof according to any one of claims 1 to 22, comprising the steps of: (i) culturing a transformed host cell 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.

33. 23. A method for detecting the presence or level of a CD39 polypeptide, the method comprising detecting said polypeptide in a sample by use of at least one anti-CD39 antibody or antigen-binding fragment thereof of any one of claims 1 to 22.

34. 34. The method of claim 33, wherein at least one anti-CD39 antibody or antigen-binding fragment thereof of any one of claims 1 to 22 forms a complex with the CD39 polypeptide, and the complex is detected using an enzyme-linked immunosorbent assay (ELISA), a radioimmunoassay (RIA), an immunochemical assay, Western blot, mass spectrometry assay, nuclear magnetic resonance assay, or an intracellular flow assay.

35. Intratumoral CD39 ｈｉｇｈ 23. A method for improving anti-tumor T-cell immunity by depleting cells, the method comprising administering the agent to a subject having a tumor, the agent comprising the anti-CD39 antibody or antigen-binding fragment thereof according to any one of claims 1 to 22, wherein the administration of the anti-CD39 antibody or antigen-binding fragment thereof improves intratumoral CD39 T-cell immunity. ｈｉｇｈ The agent results in a reduction in the number of cells, enhancing T cell infiltration into the tumor, or reducing T cell exhaustion in the tumor, or both.

36. 23. A drug for use in a method for promoting immune cell infiltration into a tumor, the drug comprising the anti-CD39 antibody or antigen-binding fragment thereof of any one of claims 1 to 22, the method comprising administering the drug to a subject having a tumor, wherein administration of the anti-CD39 antibody or antigen-binding fragment thereof results in the removal and reduction of CD39+CD45-SCA-1+ stromal cells in the tumor, and results in increased infiltration of the tumor by cytotoxic T cells.

37. 23. A method for reducing type II NKT cell suppression of intratumoral immune cell function, the method comprising administering the agent to a subject having a tumor ... The agent, 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.

38. 23. A method for reducing regulatory T cell (Treg) suppression of intratumoral immune cell function, the method comprising administering the agent to a subject having a tumor ... The administration of the anti-CD39 antibody or antigen-binding fragment thereof increases the CD39 in the tumor. ｈｉｇｈ The agent results in the removal and reduction of Tregs.

39. 23. A method for reducing tumor-associated macrophage (TAM) suppression of intratumoral immune cell function, the method comprising administering the agent to a subject having a tumor, the method comprising administering the agent to the subject, the method comprising administering the anti-CD39 antibody or antigen-binding fragment thereof to the subject, the method comprising administering the anti-CD39 antibody or antigen-binding fragment thereof to the subject, the method comprising administering the agent ... ｈｉｇｈ The agent results in the removal and reduction of macrophages.

40. 23. A method for promoting an anti-tumor immune response, comprising administering to a subject having a tumor the anti-CD39 antibody or antigen-binding fragment thereof of any one of claims 1 to 22, in an amount sufficient to result in a reduction in CD39-expressing cells in the tumor.

41. 23. A method for promoting T cell-mediated immune function in a tumor in a subject, the method comprising the steps of: (i) identifying cancer subjects with tumor-infiltrating tumor-reactive lymphocytes below a predetermined threshold, thereby characterizing the subject as having a non-invasive or less invasive tumor phenotype; (ii) administering the agent to the subject in an amount that increases tumor infiltration by tumor-reactive T cells.

42. the intratumoral CD39 ｈｉｇｈ 36. The method of claim 35, wherein 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 combinations thereof.

43. The anti-CD39 antibody or antigen-binding fragment thereof is capable of targeting CD39 expressed in one or more hematopoietic compartments. ｈｉｇｈ 40. The agent of claim 35, 36 or 39, which reduces the level of cells.

44. 44. The method of claim 43, wherein the one or more hematopoietic compartments are selected from the group consisting of blood, spleen, and liver.

45. The agent of any one of claims 35 to 44, wherein the anti-CD39 antibody or antigen-binding fragment thereof is administered as part of an anti-tumor therapy.

46. The method of any one of claims 35 to 45, wherein the anti-CD39 antibody or antigen-binding fragment thereof is administered as part of an anti-infective therapy.

47. 47. The method of claim 46, wherein the anti-infective therapy is an antiviral therapy (treatment of HIV and HBV infections and COVID-19 infections), treatment of Mycobacterium tuberculosis, and treatment of visceral leishmaniasis.

48. The agent of any one of claims 35 to 47, wherein the anti-CD39 antibody or antigen-binding fragment thereof is administered as part of an anti-tumor therapy for treating a solid tumor.

49. The method of claim 48, 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, renal cancer, multiple myeloma, ovarian cancer, cervical cancer, or glioma.

50. The agent of any one of claims 35 to 49, wherein the anti-CD39 antibody or antigen-binding fragment thereof is administered as part of an anti-tumor therapy for treating a liquid tumor.

51. 51. The method of any one of claims 50, wherein the liquid tumor is leukemia.

52. The agent of any one of claims 35 to 51, wherein the anti-CD39 antibody or antigen-binding fragment thereof is administered as part of a therapy involving one or more chemotherapeutic agents, anti-angiogenic agents, immuno-oncology agents and / or radiation.

53. 53. The agent of claim 52, wherein the therapy comprises administering one or more inhibitors (antagonists) of one or more checkpoint molecules.

54. 54. The method of claim 53, wherein the one or more checkpoint molecules are selected from the group consisting of PD-1, CTLA-4, LAG-3, TIM-3, TIGIT, and Siglec-15.

55. 53. The agent of claim 52, wherein the treatment comprises administering one or more activators (agonists) of one or more costimulatory molecules.

56. 56. The method of claim 55, wherein the one or more costimulatory molecules are selected from the group consisting of GITR, CD27, 4-1BB, OX40, CD137, ICOS, and CD28.

57. The treatments include VEGFR or VEGF antagonists, EGFR or EGF antagonists, IDO inhibitors, IDO1 inhibitors, HDAC inhibitors, PI3K delta inhibitors, IL-15 agonists, CXCR4 antagonists, CXCL12 antagonists, DNMT inhibitors, interleukin-21, anti-KIR antibodies, anti-CSF-1R antibodies, anti-CCR4 antibodies, GMCSF, anti-PS antibodies, anti-CD30 antibodies, and the like.

53. The method of claim 52, comprising administering one or more of a medicament for treating rheumatoid arthritis, a rheumatoid arthritis, a rheumatoid arthritis recurrence ...

58. 53. The method of claim 52, wherein the treatment comprises administering one or more innate immune inducers.

59. 59. The agent of claim 58, wherein the one or more innate immune 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 cells and CD8+ cells, STING agonists, TLR7 / 8 agonists, and RIG-I agonists.

60. The agent of any one of claims 35 to 57, wherein the anti-CD39 antibody or antigen-binding fragment thereof is administered as part of a tumor vaccine, adoptive cell therapy, anti-tumor gene therapy, inhibitory nucleic acid therapy, and / or oncolytic virus therapy.

61. 61. The pharmaceutical preparation or medicament of any one of claims 35 to 60, wherein the subject is an animal model of cancer.

62. 62. The pharmaceutical preparation or medicament of any one of claims 35 to 61, wherein the subject is a mammal.

63. 63. The pharmaceutical preparation or medicament of claim 62, wherein the mammal is a human or a rodent.

Images