HK40062439A - Methods and compositions for promoting and potentiating t-cell mediated immune responses through adcc targeting of cd39 expressing cells - Google Patents

Description

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

Cross Reference to Related Applications

This application claims priority to U.S. provisional application serial No. 62/885,509 filed on 12.8.2019; the entire contents of said application are incorporated herein by reference in their entirety.

Background

For people with advanced cancer, a commodity that may be valuable but rare is desirable. In recent years, a novel class of drugs known as immune checkpoint inhibitors has shown great promise, can harbor tumors and prevent their growth, and allow some people receiving treatment to substantially heal. But these invasive therapies have a number of challenges. Despite the success of immunotherapy based on inhibitory antibodies in advanced cancers against programmed cell death protein 1(PD1), PD1 ligand 1(PDL1) and cytotoxic T lymphocyte antigen 4(CTLA4) therapies in advanced cancers, a significant proportion of patients still do not respond to these treatments.

With increasing interest in immunosuppressing the tumor microenvironment as a major driver of resistance and characterizing "hot" and "cold" tumors according to the degree of immune cell infiltration, researchers have discovered several different mechanisms based on the lack of an effective response to checkpoint monotherapy. Immunologically "hot" tumors contain high levels of infiltrating T cells and more antigens, making them more readily recognized by the immune system and more likely to trigger a strong immune response. Cancers that are immunologically considered hot are bladder cancer, head and neck cancer, renal cancer, melanoma, and non-small cell lung cancer. However, even in these immunologically "hot" cancers, only a few patients benefit from immunotherapy. In contrast, immunologically "cold" tumors are cancers that for various reasons contain few infiltrating T cells, do not appear to be recognized as foreign and do not elicit a strong response from the immune system, making these cancers difficult to treat with current immunotherapy. Traditionally, "cold" cancers immunologically included glioblastoma, as well as ovarian, prostate, pancreatic and most breast cancers.

In addition to cancer cells, the microenvironment of a tumor contains many cell types, including myeloid-derived inflammatory cells, lymphocytes, blood vessels, fibroblasts, and extracellular matrix (ECM) containing collagen and proteoglycans. Indeed, tumor drug response is not solely determined by the intrinsic characteristics of tumor cells, as tumor-associated stromal cells, including fibroblasts, Mesenchymal Stromal Cells (MSCs), immunoinflammatory cells, vascular endothelial cells, and ECMs, combine to respond to anti-cancer therapy. In many cases, whether a tumor has T cell infiltration or lacks T cell infiltration, resistance or unresponsiveness to checkpoint therapy is the result of inhibitory effects of other cells present in the tumor, ranging from intratumoral signaling leading to down-regulation or inhibition of cytotoxic T cells already present in the tumor to the development of a tumor microenvironment that completely excludes cytotoxic T cells by reducing the ability of those cells to infiltrate into the tumor from the surrounding blood vessels.

Therefore, there is a great need in the art to identify alternative mechanisms for enhancing T cell responses.

Disclosure of Invention

The ectonucleotidase CD39 has been targeted in order to produce a reduction in the intratumoral level of enzymatic activity associated with the protein, and thus a reduction in the intratumoral level of immunosuppressive agents (adenosine). The present invention is based, at least in part, on the following additional findings: CD39 is expressed by a range of cells in the tumor microenvironment, such as stromal cells, type II NKT cells, and tumor-associated macrophages (TAMs), which function to create an immunosuppressive or immunorejection environment, and those cells are targeted with certain antibody-dependent cellular cytotoxicity (ADCC) activity anti-CD 39 antibodies for eliminating infiltrates in the tumor that can be used to increase cytotoxic T cells, and in fact convert "cold" tumors to immunologically "hot" tumors.

For example, in one aspect, an anti-CD 39 antibody or antigen-binding fragment thereof is provided, the anti-CD 39 antibody or antigen-binding fragment thereof comprising (i) at least one antigen-binding domain that binds ectonucleoside triphosphate diphosphohydrolase-1 (CD39) at one site such that the anti-CD 39 antibody forms a stable immune complex; and (ii) an Fc γ RIIIa binding moiety that binds to an Fc γ RIIIa receptor and confers ADCC activity of the anti-CD 39 antibody against CD39+ cells.

Further provided are a number of embodiments 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-CD 39 antibody or antigen-binding fragment thereof facilitates: (i) stable immune complex formation when incubated with HCC1739BL cells, as characterized by less than 40% loss of the immune complex after 24 hours, or less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, or even less than 10% loss of the immune complex after 24 hours, optionally wherein the immune complex formation is detected by fluorescence intensity using a fluorescently labeled secondary antibody (e.g., for illustration only, the stability of an immune complex formed with anti-CD 39 antibody can be determined by incubating an anti-CD 39 monoclonal antibody (mAb), such as at 2 μ g/ml or greater, with HCC1739BL cells for different times and then detecting the presence of the immune complex by a fluorescently conjugated secondary antibody); (ii) complement Dependent Cytotoxicity (CDC) activity against CD39+ cells; (iii) antibody-mediated target endocytosis of CD39 on CD45+ immune cells; (iv) antibody-mediated target endocytosis of CD39 from tumor vascular endothelial destruction or collapse of the vasculoprostrial network in a tumor; (v) (ii) binds to a CD39 amino acid epitope having a sequence selected from the group consisting of the CD39 amino acid epitope sequences set forth in figure 33; and/or (vi) binds to CD39 in a manner that non-competes or only partially competes for binding to CD39 with monoclonal antibody clone a 1.

In another implementationIn this embodiment, the Fc γ RIIIa binding moiety is selected from the group consisting of: an Fc domain, an antibody or fragment thereof that binds to Fc γ RIIIa, and an Fc γ RIIIa binding peptide. In yet another embodiment, the antigen binding domain is selected from the group consisting of: fab, Fab ', F (ab')₂Fv or single chain Fv (scFv), Fav, dsFv, sc (Fv)2, Fde, sdFv, single domain antibody (dAb), and bifunctional antibody (diabodies) fragments, and/or wherein the anti-CD 39 antibody or antigen-binding fragment is monoclonal. In yet another embodiment, the anti-CD 39 antibody or antigen-binding fragment thereof is conjugated to an agent, optionally wherein the agent is selected from the group consisting of: binding proteins, enzymes, drugs, chemotherapeutic agents, biological agents, toxins, radionuclides, immunomodulators, detectable moieties and tags. In another embodiment, the anti-CD 39 antibody or antigen-binding fragment thereof has a VH domain having an amino acid sequence that is encoded by a nucleic acid sequence or 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 is encoded by a nucleic acid sequence or nucleic acid that hybridizes under stringent conditions to the nucleic acid of SEQ ID No.3, such as 6x sodium chloride/sodium citrate (SSC) at 45 ℃ and washed in 0.2 xSSC/0.1% SDS at 50 ℃ to 65 ℃. In yet another embodiment, the anti-CD 39 antibody or antigen-binding fragment thereof comprises a heavy chain having a CDR that is at least 60% identical (e.g., at least 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater) to a CDR of SEQ ID No.2, 6, 10, 14, 18, 22, 26, 42, 46, 50 or 54; and a light chain having a CDR that is at least 60% identical (e.g., at least 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater) to a CDR of SEQ ID No.4, 8, 12, 16, 20, 24, 28, 44, 48, 52 or 56. In yet another embodiment, the anti-CD 39 antibody or antigen-binding fragment thereof comprises a heavy chain variable region that hybridizes to SEQ ID Nos. 2, 6, 10, 14, 18, 22. 26, 42, 46, 50, or 54 Variable Heavy (VH) chains that are at least 60% identical (e.g., at least 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) and Variable Light (VL) chains that are at least 60% identical (e.g., at least 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) to SEQ ID nos. 4, 8, 12, 16, 20, 24, 28, 44, 48, 52, or 56. In another embodiment, the anti-CD 39 antibody or antigen-binding fragment thereof comprises: (i) a heavy chain having a CDR1 amino acid sequence that is at least 80% identical (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater) to SEQ ID No.29, a CDR2 amino acid sequence that is at least 80% identical (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater) to SEQ ID No.30 and a CDR3 amino acid sequence that is at least 80% identical (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater) to SEQ ID No. 31; and (ii) a light chain having a CDR1 amino acid sequence that is at least 80% identical (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater) to SEQ ID No.32, a CDR2 amino acid sequence that is at least 80% identical (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater) to SEQ ID No.33, and a CDR2 amino acid sequence that is at least 80% identical (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, "o, 95%, 96%, 97%, 98%, 99% or more) of the CDR3 amino acid sequence. In yet another embodiment, the anti-CD 39 antibody or antigen-binding fragment thereof comprises a heavy chain having CDRs selected from the group consisting of the 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 the CDRs of SEQ ID nos. 8, 12, 16, 20, 24, 28, 44, 48, 52 or 56; and human framework sequences to form humanized heavy and light chains having an antigen binding site capable of specifically binding to human CD 39. In yet another embodiment, the anti-CD 39 antibody or antigen-binding fragment thereof comprises an Fc domain of the IgG1 or IgG3 isotype, optionally wherein the Fc domain is human. In another embodiment, the anti-CD 39 antibody or antigen-binding fragment thereof is low fucosylated or afucosylated. In yet another embodiment, the anti-CD 39 antibody or antigen-binding fragment thereof is human or humanized. In yet another embodiment, the anti-CD 39 antibody or antigen-binding fragment thereof is bispecific, comprising at least one additional antigen-binding site for a tumor antigen, an immune checkpoint or a co-stimulatory receptor, wherein if the additional antigen-binding site is for an immune checkpoint it serves as a checkpoint inhibitor and wherein if the additional antigen-binding site is for a co-stimulatory receptor it serves as a co-stimulatory agonist. In another embodiment, the additional antigen binding site binds to a checkpoint protein selected from the group consisting of: PD-1, PD-L1, CTLA-4/B7-1/B7-2, PD-L2, NKG2A, KIR, LAG-3, TIM-3, CD96, VISTA, TIGIT and Siglec-15. In yet another embodiment, the additional antigen binding site binds to a checkpoint protein on a T cell that is upregulated and associated with T cell depletion. In yet another embodiment, the additional antigen binding site binds to an immune co-stimulatory receptor selected from the group consisting of: MHCI molecules, BTLA receptor, OX40, CD27, CD28, CDS, ICAM-1, LFA-1(CD11a/CD18), ICOS (CD278) and 4-1BB (CD 137). In another embodiment, the additional antigen binding site binds to CD47, sirpa, CD24, or Siglec-10.

In another aspect, aProvided is a pharmaceutical formulation comprising a therapeutically effective amount of at least one anti-CD 39 antibody or antigen-binding fragment thereof described herein and one or more pharmaceutically acceptable excipients, buffers, or solutions. For example, the pharmaceutical formulation may be used to improve anti-tumor T cell immunity and is suitable for administration to a subject having a tumor, comprising an effective amount of an anti-CD 39 antibody or antigen-binding fragment thereof and one or more pharmaceutically acceptable excipients, buffers, or solutions, wherein administration of the anti-CD 39 antibody to the subject results in intratumoral CD39^{Height of}The number of cells is reduced and infiltration of T cells into the tumor is enhanced or T cell depletion in the tumor is reduced or both.

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 and/or light chain polypeptide of an anti-CD 39 antibody or antigen-binding fragment thereof described herein; ii) has a sequence that is at least about 90% identical over its full length to a nucleic acid encoding an immunoglobulin heavy and/or light chain polypeptide of an anti-CD 39 antibody or antigen-binding fragment thereof described herein; or iii) an immunoglobulin heavy and/or light chain polypeptide encoding an anti-CD 39 antibody or antigen-binding fragment thereof described herein.

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

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

In yet another aspect, there is provided a host cell comprising an isolated nucleic acid described herein, the host cell: a) expressing an anti-CD 39 antibody or antigen-binding fragment thereof described herein; b) comprises an immunoglobulin heavy chain and/or light chain polypeptide as described herein; or c) comprises a vector as described herein.

In yet another aspect, a device or kit is provided comprising at least one anti-CD 39 antibody or antigen-binding fragment thereof described herein. The device or kit optionally comprises a label to detect at least one anti-CD 39 antibody or antigen-binding fragment thereof, or a complex comprising the anti-CD 39 antibody or antigen-binding fragment thereof.

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

In yet another aspect, a method of producing at least one anti-CD 39 antibody or antigen-binding fragment thereof of any one of claims 1-18, the method comprising the steps of: (i) culturing a transformed host cell that has been transformed by a nucleic acid comprising a sequence encoding at least one anti-CD 39 antibody or antigen-binding fragment thereof under conditions suitable to allow expression of the anti-CD 39 antibody or antigen-binding fragment thereof; and (ii) recovering the expressed anti-CD 39 antibody or antigen-binding fragment thereof.

In yet another aspect, a method of detecting the presence or level of a CD39 polypeptide is provided, the method comprising obtaining a sample, and detecting the polypeptide in the sample by using at least one anti-CD 39 antibody or antigen-binding fragment thereof described herein. For example, the at least one anti-CD 39 antibody or antigen-binding fragment thereof can form a complex with CD39 polypeptide, and the complex can be detected in the form of an enzyme-linked immunosorbent assay (ELISA), Radioimmunoassay (RIA), immunochemical assay, western blot, mass spectrometry, nuclear magnetic resonance assay, or using an intracellular flow assay.

In another aspect, a method for reducing intratumoral CD39 by depletion is provided ^{Height of}A method of cytologically improving anti-tumor T cell immunity, the method comprising administering to a subject having a tumor an effective amount of a pharmaceutical composition of an anti-CD 39 antibody or antigen-binding fragment thereof described herein, wherein the administration of the anti-CD 39 antibody or antigen-binding fragment thereof results in intratumoral CD39^{Height of}The number of cells is reduced and infiltration of T cells into the tumor is enhanced or T cell depletion in the tumor is reduced or both.

In yet another aspect, there is provided a method for promoting infiltration of immune cells into a tumor, the method comprising administering to a subject having a tumor an effective amount of a pharmaceutical composition of an anti-CD 39 antibody or antigen-binding fragment thereof described herein, wherein the administration of the anti-CD 39 antibody or antigen-binding fragment thereof results in the elimination and reduction of CD39+ CD45-SCA-1+ stromal cells in the tumor and increased infiltration of cytotoxic T cells into the tumor.

In yet another aspect, there is provided a method for reducing inhibition of immune cell function in a tumor by type II NKT cells, the method comprising administering to a subject having a tumor an effective amount of a pharmaceutical composition of an anti-CD 39 antibody or antigen-binding fragment thereof described herein, wherein administration of the anti-CD 39 antibody or antigen-binding fragment thereof results in elimination and reduction of type II NKT cells in the tumor.

In another aspect, there is provided a method for reducing suppression of immune cell function within a tumor by regulatory T cells (tregs), the method comprising administering to a subject having a tumor an effective amount of a pharmaceutical composition of an anti-CD 39 antibody or antigen-binding fragment thereof described herein, wherein administration of the anti-CD 39 antibody or antigen-binding fragment thereof results in CD39 in the tumor^{Height of}Treg elimination and reduction.

In yet another aspect, there is provided a method for reducing inhibition of immune cell function within a tumor by tumor-associated macrophages (TAMs), the method comprising administering to a subject having a tumor an effective amount of a pharmaceutical composition of an anti-CD 39 antibody or antigen-binding fragment thereof described herein, wherein the administration of the anti-CD 39 antibody or antigen-binding fragment thereof results in CD39 in the tumor^{Height of}TAM elimination and reduction.

In yet another aspect, there is provided a method for promoting an anti-tumor immune response, the method comprising administering to a subject having a tumor an anti-CD 39 antibody or antigen-binding fragment thereof described herein in an amount sufficient to cause a reduction in CD 39-expressing cells in the tumor.

In yet another aspect, there is provided a method for promoting T cell-mediated immune function in a tumor of a subject, the method comprising: (i) identifying a cancer subject having a degree of tumor-reactive lymphocytes less than a predetermined threshold of tumor infiltration, for a tumor phenotype characterized as non-infiltrated or under-infiltrated (under-infiltrated); and (ii) administering to the subject an anti-CD 39 antibody or antigen-binding fragment thereof described herein in an amount that increases tumor-reactive T cell infiltration into the tumor.

As described above, further provided are a number of embodiments that can be applied to any aspect of the present invention and/or combined with any other embodiment described herein. For example, in one embodiment, intratumoral CD39^{Height of}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-CD 39 antibody or antigen-binding fragment thereof reduces the presence of CD39 within one or more hematopoietic compartments^{Height of}(ii) a level of cells, optionally wherein the one or more hematopoietic compartments are selected from the group consisting of blood, spleen and liver. In yet another embodiment, the anti-CD 39 antibody or antigen-binding fragment thereof is administered as part of an anti-tumor therapy. In yet another embodiment, the anti-CD 39 antibody or antigen-binding fragment thereof is administered as part of an anti-infection therapy, optionally wherein the anti-infection therapy is an antiviral therapy (including treatment of HIV and HBV infections and COVID-19 infections), for the treatment of Mycobacterium tuberculosis (Mycobacterium tuberculosis), and for the treatment of visceral leishmaniasis. In another embodiment, the anti-CD 39 antibody or antigen-binding fragment thereof is administered as part of an anti-tumor therapy for the treatment of a solid tumor, optionally wherein the solid tumor is pancreatic cancer, liver cancer, lung cancer, gastric cancer, esophageal cancer, head and neck squamous cell carcinoma, prostate cancer, colon cancer, breast cancer, lymphoma, gallbladder cancer, renal cancer, multiple myeloma, ovarian cancer, cervical cancer, or glioma. In yet another embodiment, the anti-CD 39 antibody or antigen-binding fragment thereof is administered as part of an anti-tumor therapy for treating a liquid tumor, optionally wherein the liquid tumor is leukemia. In yet another embodiment, the anti-CD 39 antibody or antigen-binding fragment thereof is administered as a pharmaceutical composition comprising one or more chemotherapeutic agents, Anti-angiogenic agents, immunooncology agents and/or part of a radiation therapy. 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: PD-1 antagonists, CTLA-4 antagonists, LAG-3 antagonists, TIM-3 antagonists, TIGIT antagonists, and Siglec-15 antagonists. In yet another embodiment, the therapy comprises administering one or more activators (agonists) of one or more co-stimulatory molecules, optionally wherein the one or more co-stimulatory molecules are selected from the group consisting of: GITR agonist, CD27 agonist, 4-1BB agonist, OX40 agonist, CD137 agonist, ICOS agonist, and CD28 agonist. In yet another embodiment, the therapy comprises administering one or more of: VEGFR or VEGF antagonists, EGFR or EGF antagonists, IDO inhibitors, IDO1 inhibitors, HDAC inhibitors, PI3K δ inhibitors, IL-15 agonists, CXCR4 antagonists, CXCL12 antagonists, DNMT inhibitors, interleukin-21, anti-KIR antibodies, anti-CSF-1R antibodies, anti-CCR 4 antibodies, GMCSF, anti-PS antibodies, anti-CD 30 antibody-auristatin (auristatin) E conjugates, anti-CD 19 antibodies, anti-CEA IL-2 antibodies, anti-NY-ESO-1 antibodies, anti-NKG 2A antibodies, STING agonists, TRL7/8 agonists, RIG-1 agonists and/or NRLP3 inhibitors, anti-CD 73 antibodies (such as MEDI9447), P2X7 antagonists or adenosine A2A receptor antagonists. In another embodiment, the treatment comprises administering one or more innate immunity inducing agents, optionally wherein the one or more innate immunity inducing agents are selected from the group consisting of: inhibitors of the CD 47-sirpa axis (e.g., antibodies or other binding moieties that bind to CD47 or sirpa and inhibit the interaction of the two molecules), inhibitors of the CD24-Siglec-10 axis (e.g., antibodies or other binding moieties that bind to CD24 or Siglec-10 and inhibit the interaction of the two molecules), NGK2A checkpoint inhibitors that block HLA-E driven inhibition of NK and CD8+ cells, STING agonists, TLR7/8 agonists, and RIG-I agonists. In yet another embodiment, the anti-CD 39 antibody or antigen-binding fragment thereof is administered as a vaccine including tumor, adoptive cell therapy (including C) AR-T and ACTR therapies), anti-tumor gene therapy, inhibitory nucleic acid therapy (such as siRNA, shRNA, antisense, CRISPR, and TALEN therapy), and/or therapy of oncolytic virus therapy. In yet another embodiment, the subject is an animal model of cancer. In another embodiment, the subject is a mammal, optionally wherein the mammal is a human or a rodent.

Drawings

FIG. 1. affinity of Ig39-21 as measured by flow cytometry using human CD39hi human B lymphoblastoid cells (HCC1739 BL). The fully human anti-CD 39 antibody clone Ig39-21 produced by transient transfection was serially diluted as indicated and incubated with HCC1739BL cells for 30 minutes at 4 ℃ followed by flow cytometry analysis. Kd was calculated to be 0.412 nM.

Figure 2 Ig39-21 demonstrated ADCC activity against HCC1739BL cells: luc-reporter assay. HCC1739BL cells were used as target cells. Jurkat cells stably expressing luciferase and hCD16a-158V were used as effector cells. At 37 ℃ in 5% CO₂Target cells were preincubated with serial dilutions of Ig39-21 as indicated for 30 min, followed by co-culture with effector cells (T: E ═ 1:6) for 6 hours. ADCC activity is indicated by an increase in luciferase activity over background. RLU: relative luminescence units. EC50 was calculated to be 0.014 μ g/mL.

Figure 3 Ig39-21 demonstrates ADCC activity selectively against human CD39hi Raji cells: luc-reporter assay. Various Raji cell lines with different expression levels of human CD39, including Raji cells (Raji-hCD39neg), Raji cells transfected with hCD39 highly expressing human CD39 (Raji-hCD39hi), or Raji cells transfected with hCD39 expressing low levels of human CD39 (Raji-hCD39lo) were used as target cells. Jurkat cells stably expressing luciferase and hCD16a-158V were used as effector cells. At 37 ℃ in 5% CO₂Target cells were preincubated with serial dilutions of Ig39-21 as indicated for 30 min, followed by co-culture with effector cells (T: E ═ 1:6) for 6 hours. ADCC activity is indicated by an increase in luciferase activity over background (RLU). RLU: relative luminescence units.

FIG. 4 Ig39-21 directed against CD39 hyponormal endotheliumCells (HUVEC) did not exert ADCC activity. 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. At 37 ℃ in 5% CO₂Target cells were preincubated with serial dilutions of Ig39-21 as indicated for 30 min, followed by co-culture with effector cells (T: E ═ 1:6) for 6 hours. ADCC activity is indicated by an increase in luciferase activity over background. RLU: relative luminescence units. This data indicates the safety of Ig39-21, which avoids potential systemic side effects.

Figure 5 CD39 confers resistance to NK cytotoxicity on target Raji-hCD39hi cells. CFSE-labeled Raji-hCD39neg or Raji-hCD39hi cells were used as target cells and at 37 ℃ in 5% CO₂In various ratios (as indicated) with NK-92-CD16V/V effector cells for 6 hours. Target cell death was then analyzed by flow cytometry by propidium iodide (P/I) uptake. The results are expressed as CFSE⁺P/I⁺% of cells.

FIG. 6 Ig39-21 enhanced NK cytotoxicity against Raji-hCD39hi cells. At 37 ℃ in 5% CO₂In (c) CFSE-labeled Raji-hCD39hi target cells were incubated with or without Ig39-21 antibody (10. mu.g/mL) for 30 minutes, followed by co-culture with NK-92-CD16V/V effector cells at 37 ℃ for 6 hours at various ratios as indicated. Analysis of target cell death by flow cytometry, and calculation of CFSE⁺P/I⁺% of cells.

Figure 7 Ig39-21 potentiates NK cytotoxicity against hCD39hi human B lymphoblastoid cells (HCC1739 BL). At 37 ℃ in 5% CO₂The CFSE-labeled HCC1739BL target cells were incubated with or without Ig39-21 antibody (10. mu.g/mL) for 30 minutes, followed by co-culture with NK-92-CD16V/V effector cells at 37 ℃ for 6 hours at various ratios as indicated. Analysis of target cell death by flow cytometry, and calculation of CFSE ⁺P/I⁺% of cells.

Figure 8 afucosylation enhances Ig39-21 mediated ADCC against HCC1739BL cells: luc-reporter assay. HCC1739BL target cells were preincubated at 37 ℃ for 30 min with serially diluted Ig39-21 produced by transient transfection in the absence (Ig39-21 WT) or in the presence of fucosylation inhibitors (Ig39-21 AF) and further co-cultured with Jurkat effector cells (T: E ═ 1:6) for 6 hours. ADCC activity is indicated by an increase in luciferase activity over background. RLU: relative luminescence units. EC50 was calculated to be 0.02. mu.g/mL (Ig39-21 WT) and 0.0008. mu.g/mL (Ig39-21 AF)

Figure 9 afucosylation enhances Ig39-21 mediated ADCC against HCC1739BL cells: NK cytotoxicity assay. At 37 ℃ in 5% CO₂The CFSE-labeled HCC1739BL target cells were incubated with serial dilutions of Ig39-21 WT or Ig39-21 AF as indicated for 30 minutes. The cells were then co-cultured with NK-92-CD 16V/V effector cells (E: T ═ 1:8) for 6 hours at 37 ℃. Analysis of target cell death by flow cytometry, and calculation of CFSE⁺P/I⁺% of cells (% of cytotoxicity). EC50 was calculated to be 0.04. mu.g/mL (Ig39-21 WT) and 0.0006. mu.g/mL (Ig39-21 AF).

FIG. 10 optimized Ig39-21(NP501-BK), Ig39-21 WT, and Ig39-21AF display similar binding affinities using human CD39 positive CHO cells (CHO-hCD 39). Ig39-21 WT, Ig39-21AF or NP501-BK (produced by using stably transfected cells, an optimized version of Ig 39-21) were serially diluted as indicated and incubated with CHO-hCD39 cells for 30 minutes at 4 ℃. Then, the cells were treated with a secondary antibody (anti-human IgG (Fc-specific), Alexa) at 4 ℃488) Staining was performed for 30 min and analyzed by flow cytometry. In parallel, a human IgG1 isotype control antibody (KLH-hIgG1) was used. Kd was calculated to be 0.48nM (NP501-BK), 0.52nM (Ig39-21 WT) and 0.51nM (Ig39-21 AF).

FIG. 11 NP501-BK, Ig39-21 WT, and Ig39-21AF showed similar binding affinities using HCC1739BL cells expressing CD 39. Ig39-21 WT, Ig39-21AF or NP501-BK were serially diluted as indicated and incubated with HCC1739BL cells for 30 minutes at 4 ℃. Then, the cells were treated with a secondary antibody (anti-human IgG (Fc-specific antibody) Alexa at 4 ℃488) Staining was performed for 30 min and analyzed by flow cytometry. In parallel, a human IgG1 isotype control antibody (KLH-hIgG1) was used. Kd was calculated to be 0.16nM (NP501-BK), 0.29nM (Ig39-21 WT) and 0.35nM (Ig39-21 AF).

FIG. 12 optimized Ig39-21(NP501-BK) and afucosylated Ig39-21 exert similar ADCC activities: 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-CD 39 monoclonal antibody (Ig39-21 AF or NP501-BK) for 30 min at 37 ℃ and further co-incubated with Jurkat effector cells (T: E ═ 1:6) for 6 hours. ADCC activity is indicated by an increase in luciferase activity over background. RLU: relative luminescence units. EC50 was calculated to be 0.0017. mu.g/mL (NP501-BK) and 0.00086. mu.g/mL (Ig39-21 AF).

FIG. 13 NP501-BK and Ig39-21 AF exert far higher ADCC activity than Ig39-21 WT: NK cytotoxicity against HCC1739BL cells. At 37 ℃ in 5% CO₂The CFSE-labeled HCC1739BL target cells were incubated for 30 minutes with serially diluted human IgG1 isotype control antibody (KLH-hIgG1) or fully human anti-CD 39 monoclonal antibody (Ig39-21 WT, Ig39-21 AF or NP501-BK) as indicated. The cells were then co-cultured with NK-92-CD 16V/V effector cells (E: T ═ 1:8) for 6 hours at 37 ℃. Analysis of target cell death by flow cytometry and calculation of CFSE⁺P/I⁺% of cells (% of cytotoxicity). EC50 was calculated to be 1.58E-04. mu.g/mL (NP501-BK), 4.22E-03. mu.g/mL (Ig39-21 WT), and 4.75E-05. mu.g/mL (Ig39-21 AF).

FIG. 14 fully human anti-CD 39 antibody showed 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-CD 39 monoclonal antibody (Ig39-21WT, Ig39-21 AF or NP501-BK) for 30 minutes at 37 ℃ and further exposed to 10% Normal Human Serum (NHS) for 2 hours. Analysis of target cell lysis by flow cytometry and calculation of% P/I⁺Cells (% of cytotoxicity). EC50 was calculated to be 0.31. mu.g/mL (NP501-BK), 0.38. mu.g/mL (I)g39-21 WT) and 0.25. mu.g/mL (Ig39-21 AF).

FIG. 15 NP501-BK and Ig39-21 AF exert similar anti-tumor efficacy in vivo. C57BL6 humanized CD39 mice (hCD39 KI) subcutaneously implanted with MC38 cells were treated with 5mg/kg human IgG1 isotype control antibody (KLH-hIgG1) or fully human anti-CD 39 monoclonal antibody (Ig39-21 AF or NP501-BK) on days 8, 11, 14 and 17 after tumor challenge. Tumor length (L) and width (W) were measured twice weekly using digital calipers. Tumor volume (mm)³) Was determined as L W0.52. Each group n is 5.

Figure 16 NP501-BK treatment resulted in decreased expression of hCD39 on CD39hi tumor-infiltrating lymphocytes. On days 8, 11 and 14 post tumor inoculation, MC38 tumor-bearing hCD39 KI mice were treated with three doses of KLH-hIgG1(5mg/kg) or NP501-BK (5 mg/kg). 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. Each group n is 6 to 8.

FIG. 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 NP 501-BK. When the tumor reaches about 500mm³When considering the mean volume of treatment (day 0), mice were treated with two doses of 300 μ l saline or 10mg/kg NP501-BK intraperitoneally on day 0 and day 3. Tumor length (L) and width (W) were measured every three days using digital calipers. Tumor volume (mm)³) Was determined as L W0.52. Each group n is 6 to 7.

Figure 18.18 epitope competition assay of human/rabbit chimeric anti-human CD39 monoclonal antibody against HCC1739BL cells with reference anti-hCD 39 monoclonal antibody clone a 1. HCC1739BL cells were incubated with a panel of 18 anti-hCD 39 monoclonal antibodies (human/rabbit chimeric clones; unconjugated, 2. mu.g/ml) for 30 min at 4 ℃. The cells were then washed twice and stained with the mouse anti-hCD 39 monoclonal antibody clone a1 conjugated with PE for 30 minutes at 4 ℃ followed by flow cytometry analysis. Cells incubated without chimeric antibody were used as controls.

FIG. 19 human/rabbit chimeric clone 9B6 and Ig39-21 exert similar ADCC activities: 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. The target cells were preincubated with serial dilutions of Ig39-21 WT or human/rabbit chimeric clone 9B6(Hu/Ra 9B 6; in all chimeric clones, rabbit anti-hCD 39 monoclonal antibody chimeric with human IgG1 Fc with the highest ADCC activity) for 30 min at 37 ℃ and further co-cultured with Jurkat effector cells (T: E ═ 1:6) for 6 hours. ADCC activity is indicated by an increase in luciferase activity over background (RLU). RLU: relative luminescence units. EC50 was calculated to be 0.014. mu.g/mL (Ig39-21 WT) and 0.022. mu.g/mL (Hu/Ra 9B 6).

FIG. 20.18 ADCC Activity of 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 examined for ADCC activity as described above. Briefly, target cells were preincubated with serially diluted antibodies for 30 minutes at 37 ℃ and further co-cultured with Jurkat effector cells (T: E ═ 1:6) for 6 hours. ADCC activity is indicated by an increase in luciferase activity over background (RLU). RLU: relative luminescence units. 10 of the 18 clones showed positive ADCC activity.

FIG. 21 shows a human/rabbit chimeric antibody having high ADCC activity: luc-reporter assay using HCC1739BL cells. The Luc-reporter ADCC assay was performed as described above in figure 20. Six clones with high ADCC activity were pooled. Five of the six clones (except 65H 5) did not compete with the epitope of clone a1 (see fig. 18).

Figure 22 human/rabbit chimeric clones with low ADCC activity: luc-reporter assay using HCC1739BL cells. The Luc-reporter ADCC assay was performed as described above in figure 20. Four clones with low ADCC activity were pooled, all competing with the epitope of clone a1 (see figure 18). 9B6 served as a positive control.

Figure 23 human/rabbit chimeric antibody without ADCC activity: luc-reporter assay using HCC1739BL cells. The Luc-reporter ADCC assay was performed as described above in figure 20. A summary of eight clones without ADCC activity, all of which completely competed with the epitope of clone A1 (see FIG. 18). 9B6 served as a positive control.

Figure 24 NK cytotoxicity of selected human/rabbit chimeric antibodies against HCC1739BL cells. At 37 ℃ in 5% CO₂The CFSE-labeled HCC1739BL target cells were incubated with serial dilutions of human/rabbit chimeric clones as indicated for 30 minutes. Then, the cells were co-cultured with NK-92-CD 16V/V effector cells (E: T ═ 1:8) at 37 ℃ for 6 hours. Analysis of target cell death by flow cytometry and calculation of CFSE⁺P/I⁺% of cells (% of cytotoxicity). Enumerate the calculated EC 50. The% of maximal cytotoxicity over background was determined as: for each clone,% of maximal cytotoxicity at 1. mu.g/mL-background cytotoxicity for each clone (at 10%^-4μ g/mL). Exemplary chimeric clones from the luc-reporter high ADCC group (8C11, 8D8, 9B6, 9C10, 48F10 and 65H5) showed high NK killing activity. In contrast, chimeric clones from the luc-reporter ADCC negative panel (59B6, 60D9, 62G12 and 62H10) displayed low NK killing activity.

FIG. 25 reference anti-human CD39 monoclonal antibody (hCD39 Ref) inhibits hCD39 ATPase activity on CHO cell membranes. CHO-hCD39 cells were incubated with 10. mu.g/mL human IgG1 isotype Ultra-LEAF antibody or anti-hCD 39 Ref antibody (hCD39 Ref) for 30 minutes at 37 ℃ followed by 15 minutes at room temperature with ATP (250. mu.M). The supernatant is then collected and used by luminescenceATP levels are measured. Antibody-free cells (cells + ATP) or ATP alone in the absence of cells were also tested in parallel to calculate% inhibition of enzyme activity as described in materials and methods.

FIG. 26 reference antibody (hCD39 Ref) containing the same Fc portion of human IgG1 and Ig39-21 WT exerted similar ADCC activities: NK cytotoxicity against HCC1739BL cells. At 37 ℃ in 5% CO₂The CFSE-labeled HCC1739BL target cells were combined with serial dilutions of anti-hCD 39 monoclonal antibody (hCD39 Ref, NP501-BK, Ig39-21 WT or Ig 39-21) as indicatedAF) were incubated together for 30 minutes. The cells were then co-cultured with NK-92-CD 16V/V effector cells (E: T ═ 1:8) for 6 hours at 37 ℃. Analysis of target cell death by flow cytometry and calculation of CFSE⁺P/I⁺% of cells (% of cytotoxicity). EC50 was calculated to be 2.62E-03. mu.g/mL (hCD39 Ref), 5.14E-05. mu.g/mL (NP501-BK), 1.98E-03. mu.g/mL (Ig39-21 WT), and 7.94E-06. mu.g/mL (Ig39-21 AF).

FIG. 27 epitope competition matrix-I on HCC1739BL cells versus anti-hCD 39 reference antibody (hCD39 Ref). HCC1739BL cells were incubated with 10 μ g/ml of unconjugated human IgG1 isotype Ultra-LEAF antibody or hCD39 Ref antibody for 30 minutes at 4 ℃. Then, the cells were exposed to Alexa at 4 ℃647 conjugated antibody (8C11, 8D8, 8E9, 9B6 and Ig39-21 WT) or PE conjugated clone A1 for 30 minutes, followed by two washes and flow cytometry analysis. Fold change was calculated for AF647 or PE MFI assays relative to isotype controls (no epitope overlap-1). Ra: a rabbit antibody; Hu/Ra: human/rabbit chimeric antibodies.

FIG. 28 epitope competition matrix-II on HCC1739BL cells versus anti-hCD 39 reference antibody (hCD39 Ref). HCC1739BL cells were incubated with 10 μ g/ml of unconjugated human IgG1 isotype Ultra-LEAF antibody or hCD39 Ref antibody for 30 minutes at 4 ℃. Cells were then exposed to unconjugated rabbit or human/rabbit chimeric antibodies (2a11, 2G12, 5F1, 9C10, 48F10, 52G4, 59B6, 65H5, and 67C1) at 4 ℃ for 30 minutes, washed twice and treated with secondary antibodies (anti-rabbit IgG (H + L), Alexa, at 4 ℃)488) And dyeing for 30 minutes. Finally, the cells were washed and analyzed by flow cytometry. Fold change for AF488MFI assay relative to isotype control was calculated (no epitope overlap ═ 1).

FIG. 29 stability of antibody antigen immune complexes on HCC1739BL cells. At 37 ℃ in 5% CO₂In (2) the anti-human CD39 antibody (2. mu.g/ml) was incubated with HCC1739BL cells for 24 hours or at 4 ℃ for 20 minutesFollowed by secondary antibody staining (anti-human IgG (Fc specific), Alexa) at 4 ℃488) For 30 minutes. The cells were then washed and analyzed by flow cytometry. The difference in AF488MFI between 20 min and 24 hr treatment indicates loss of human CD39 on the cell membrane, calculated as described in materials and methods. After 24 hours, exemplary chimeric clones from the luc-reporter ADCC negative group (8E9, 59B6, and 67C1) and the low ADCC group (52G4) did not form stable immune complexes on the cell membrane (e.g., greater than 40% loss of CD 39). Hu/Ra: human/rabbit chimeric antibodies; hIgG 1: humanized rabbit antibody, IgG1 isotype; hIgG 4: 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 corresponding humanized clones (IgG1 or IgG4 isotype) were serially diluted as indicated and incubated with HCC1739BL cells for 30 min at 4 ℃. Then, the cells were treated with a secondary antibody (anti-human IgG (Fc-specific), Alexa) at 4 ℃ 488) Staining was performed for 30 min and analyzed by flow cytometry. Kd is calculated and indicated next to the graph.

Figure 31 NK cytotoxicity of humanized rabbit antibodies against HCC1739BL cells. Human/rabbit chimeric clones (Hu/Ra 8C11, 8D8, and 9C10) and their corresponding humanized clones (IgG1 or IgG4 isotype) were serially diluted as indicated and at 37 ℃ at 5% CO₂With CFSE-labeled HCC1739BL target cells for 30 minutes. The cells were then co-cultured with NK-92-CD 16V/V effector cells (E: T ═ 1:8) for 6 hours at 37 ℃. Analysis of target cell death by flow cytometry and calculation of CFSE⁺P/I⁺% of cells (% of cytotoxicity). Enumerate the calculated EC 50.

FIG. 32 CDC activity of humanized rabbit antibodies against Raji-hCD39hi cells. Human/rabbit chimeric clones (Hu/Ra 8C11, 8D8 and 9C10) and their corresponding humanizationsClones (IgG1 or IgG4 isotype) were serially diluted as indicated and preincubated with Raji-hCD39hi target cells for 30 minutes at 37 ℃ followed by incubation with 10% Normal Human Serum (NHS) for 2 hours. Analysis of target cell lysis by flow cytometry and calculation of% P/I⁺Cells (% of cytotoxicity). Enumerate the calculated EC 50.

FIG. 33 conformational epitope mapping. A list of predominantly putative CD39 epitope candidates is shown. An exemplary, representative CD39 extracellular domain sequence is provided for reference to the CD39 epitope sequence, and a homology model of the dimer of human CD39 is provided for data visualization.

Detailed Description

I.SUMMARY

Extracellular adenosine is known to be an inhibitor of immune function. Although intracellular adenosine is involved in energy metabolism, nucleic acid metabolism and methionine cycle, extracellular adenosine plays an important role in inhibiting immune signaling in the tumor microenvironment. Immunosuppressive adenosine 3'5' -monophosphate (cAMP) -mediated pathway through adenosine A2A receptor (A2AR) signaling 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). Preclinical evidence, along with recent and evolving active clinical trial data, confirms that administration of A2AR inhibitors can be a potential novel strategy for immunotherapy. In addition, blocking the adenosine production pathway involving CD39/CD73 also induced regression of breast, colorectal and melanoma cancers in experimental animal models. In the context of anti-CD 39 and anti-CD 73 antibody therapies, the emphasis is primarily on inhibiting or reducing the catabolism of ATP and derivative nucleotides to adenosine (ultimately) by binding to these cell surface adenosine producing enzymes ("ectonucletinases") and inhibiting or removing enzymatic activity from the cell surface.

The present invention is based, at least in part, on the following findings: certain antibodies directed to CD39 are capable of selectively targeting and eliminating (such as by antibody-dependent cytotoxicity) CD39 expressing cells (more potent than the anti-CD 39 antibodies of the prior art) in the tumor microenvironment, including CD39+ CD45-SCA-1+ stromal cells (such as hematopoietic progenitors), CD39+ NKT cells, CD39+ macrophages and CD39+ endothelial cells and CD39+ cancer cells. Resulting intratumoral CD39^{Height of}A reduction in cell number may lead to such changes in the inflammatory phenotype of the tumor, such as enhanced T cell infiltration into the tumor, reduced T cell depletion in the tumor, type II NKT cell suppression of reduced immune cell function within the tumor, and/or regulatory T cell (Treg) inhibition of reduced immune cell function within the tumor, and/or tumor-associated macrophage (TAM) inhibition of reduced immune cell function within the tumor.

While not wishing to be bound by any particular theory, certain antibodies produced by the inventors are able to form a more stable immune complex with CD39 to produce more effective ADCC killing efficacy. The inventors have observed that antibodies that are not able to form immune complexes with CD39 as stable as those encompassed by the present invention lead to a reduction in CD39 on the surface, but by increasing the mechanism of CD39 shedding or endocytosis (internalization), and do not have the same efficacy in terms of being able to eliminate CD39 expressing cells by antibody-dependent cytotoxicity. In certain embodiments, certain antibodies encompassed by the present invention have been shown to bind to an epitope on CD39 that is non-competitive or only partially competitive with monoclonal antibody clone a1 for binding to CD 39.

As described in more detail in the exemplary methods and illustrated in the figures, our anti-CD 39 antibodies are specifically designed to have human constant regions comprising IgG1 Fc domains, compared to, for example, direct inhibition of CD39 NTP enzymatic activity by all anti-CD 39 therapeutic antibodies of the prior art (Perrot et al, 2019, Cell Reports 27: 2411-. This design confers Fc γ RIIIa receptor-dependent cellular activity, e.g. antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) against CD39+ cells and/or antibody-mediated target endocytosis of intratumoral CD39+ cells, to the anti-CD 39 antibody of the invention. Thus, such cellular activity results in CD39 in tumors^{Height of}Elimination and reduction of cells.

Exemplary features of the subject anti-CD 39 monoclonal antibodies are summarized below, which features are taught away from use with therapeutic anti-CD 39 antibodies described in the references.

The subject antibodies target CD39+ cells in the tumor via Fc γ RIIIa receptor-dependent activity (e.g., ADCC).

As an example, fig. 13 and 26 show that the reduction in fucosylation (also referred to as low fucosylation or afucosylation) of the primary clone Ig39-21 (fully human anti-CD 39 monoclonal antibody) significantly enhances its ADCC activity against CD39+ cells in vitro, either by using fucosylation inhibitors (Ig39-21AF) or by optimizing the production method (NP 501-BK). This parallels the enhancement of the anti-tumor activity of these afucosylated antibodies in vivo (fig. 15), while the fully glycosylated form (Ig39-21 WT) showed no anti-tumor activity in the same tumor model (data not shown).

The maximal effective doses in vitro (MaxED) of these different fucosylated Ig39-21 antibodies, as predicted by the NK cytotoxicity assays in fig. 13 and 26, further illustrate their differential in vivo anti-tumor activity. For example, afucosylation enhances the MaxED of Ig39-21 WT from 0.1. mu.g/ml up to 0.001. mu.g/ml. When converted to the clinic, this 100-fold increase would be high efficacy, a favorable safety profile, good tolerability and low cost.

In contrast, the reference hCD39 antibody (hCD39 Ref) used herein shares an antigen binding site with antibodies in the art. However, in contrast to the Ref antibodies used in the current examples, prior art antibodies were produced with an Fc portion specifically designed to have abrogated ADCC function (i.e., taught to have been specifically produced to bind CD39 and inhibit NTP enzymatic activity without causing CD 39-dependent ADCC cell killing).

The ADCC activity of the subject anti-CD 39 antibodies is selective for CD39^{Height of}A cell.

As an example, FIGS. 3 and 4 show that the ADCC activity of Ig39-21 is selective against CD39^{Height of}Cells (i.e., Raji-hCD39hi cells in FIG. 3 and SK-MEL-28 cells in FIG. 4).

Correlation of these in vitro data with the tumor microenvironment in vivo: FIG. 16 demonstrates that hCD39 is highly upregulated within tumors of MC 38-bearing hCD39 KI mice, including CD45+ tumor infiltrating lymphocytes (i.e., CD3+ CD11b-T cells, CD3-CD11b + myeloid cells, and F4/80hiG r-1 tumor-associated macrophages) and tumor-associated vascular endothelial cells (data not shown). NP501-BK treatment resulted in CD39 in tumors^{Height of}Elimination and reduction of cells (figure 16 and data not shown).

This functional feature should confer tumor specificity to the antibody, thereby avoiding systemic side effects that should lead to safer anti-CD 39 antibodies.

The anti-CD 39 antibody forms a stable immune complex with the antigen on the target cell membrane, conferring high ADCC activity to the antibody.

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

The different epitopes of the anti-CD 39 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-hCD 39 antibody with the epitope of the commercial anti-hCD 39 monoclonal antibody clone a1, fig. 18 and 21 to 23 show that anti-CD 39 antibodies that bind to anti-CD 39 in a manner that does not compete with clone a1 or only partially competes for binding to CD39 likely contain high ADCC activity, i.e., five of the six high ADCC antibodies (2G12, 8C11, 8D8, 9B6, and 9C10) display this characteristic in addition to 65H5 (fig. 21). In contrast, all antibodies in the low ADCC (2a11, 5F1, 52G4 and 63B1) and ADCC negative (8E9, 59B6, 60D9, 62G12, 62H10, 65E10, 67A8 and 67C1) groups showed epitopes that completely overlapped the epitope of clone a1 (fig. 22 and 23).

Multiple CD39 of the subject anti-CD 39 antibody in tumors^{Height of}A cellular target.

As an example, the CD39-MC38 colorectal cancer model in hCD39 KI mice was employed (fig. 15 and 16). The antitumor activity of NP501-BK in the model is that the NP501-BK has CD39^{Height of}Tumor infiltrating lymphocytes and tumor phaseResults of targeting effects on vascular endothelial cells (data not shown).

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

II.Definition of

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

"CD 39," also known as "cluster of differentiation 39", "ectonucleoside triphosphate diphosphohydrolase-1" or (gene) "ENTPD 1" and (protein) "NTPD enzyme 1", is a cell surface-localized ectonucletidase which has the ability to catalyze the hydrolysis of the γ -and β -phosphate residues of nucleoside triphosphates and diphosphates to nucleoside monophosphate derivatives (enzyme entry: EC 3.6.1.5), and the extracellular-facing catalytic sites such as ligands of the P2 receptor, such as ATP, ADP, UTP and UDP (Junger et al (2011) nat. Rev. Immunol.11: 201-. A representative human NTPD enzyme 1 protein sequence is provided in the UniProtKB entry "P49961 (ENTP1_ human)" and a representative human coding sequence line for the enzyme is provided in GenBank accession number S73813. Intracellular adenosine can modulate pro-inflammatory or pro-inhibitory signals in immune cells by binding to various adenosine receptors (Ernst et al (2010) J.Immunol.185: 1993-1998; Antonioli et al (2013) Trends mol.Med.19: 355-367; Parodi et al (2013) Cancer Immunol.62: 851-862; Boer et al (2013) Eur.J.Immunol.43: 1925-1932; Xu et al (2013) Neuro-Oncol.15: 1160-1172; U.S. patent publication 2013/0123345). For example, adenosine binds to the A2A receptor expressed by lymphocytes, causing the accumulation of intracellular cAMP, thereby preventing T cell activation and NK cytotoxicity (Zarek et al (2008) Blood 111: 251-. CD39 was originally identified as an activation marker on human lymphocytes, but has subsequently been shown to be a marker feature of regulatory T cells (Kansas) Et al (1991) J.Immunol.146: 2235-2244; deaglio et al (2007) J.exp.Med.204: 1257-1265; borselino et al (2007) Blood 110: 1225-1232). Loss of CD39 in tregs significantly impairs its ability to inhibit T cell activation, suggesting that the near-secretory activity of CD39 acts to negatively regulate T cell function (Deaglio et al (2007) j.exp.med.204: 1257-. In general, CD8⁺T cells have been reported as CD39^–(Kansas et al (1991) J.Immunol.146: 2235-. However, up-regulation of this marker on depleted T-cells has recently been noted in the context of tumors and chronic viral infections (e.g., HCV and HIV as well as coronaviridae such as SARS-COV2 (COVID-19)) (Cancer et al (2017) Cancer Res.78(1): 115-28; Gupta et al (2015) PLoS Patholog.11 (10): e 1005177; Mathew et al (2020) Science 10.1126/Science. abc8511).

The structure-function relationships of CD39 proteins are well known in the art (reviewed, for example, by Antonioli et al (2013) Trends mol. Med.19: 355-367; Wang and Guidotti (1996) J.biol.chem.271: 9898-9901; Kaczmarek et al (1996) J.biol.chem.271: 33116-33122). For example, human CD39 is an approximately 500 amino acid protein with approximately seven potential N-linked glycosylation sites, eleven Cys residues, and two transmembrane regions organized as two transmembrane domains, a small cytoplasmic domain containing N-and C-terminal fragments, and a large extracellular hydrophobic domain consisting of five highly conserved domains, called Apyrase Conserved Regions (ACR) 1-5, required for catabolic activity of the enzyme (Heine et al (2001) Eur. J. biochem.268: 364-. The amino acid sequences of ACR 1 and ACR 5 contain a phosphate binding motif (DXG), which is important for stabilizing the interaction between the enzyme and its nucleotide substrates during phosphate cleavage. In addition, two ACR residues, Glu 174 in ACR 3 and Ser 218 in ACR 4, are required for enzymatic activity (Heine et al (2001) Eur.J.biochem.268: 364-373; Smith et al (1998) Biochim.Biophys.acta 1386: 65-78). CD39 became catalytically active after cell surface expression (Smith et al (1998) Biochim. Biophys. acta 1386: 65-78).

Representative human CD39 cDNA and protein sequences are well known in the art and are publicly available from the National Center for Biotechnology Information (NCBI). For example, at least seven human CD39 transcript variants are known to encode six different human CD39 isoforms. Human CD39 isoform 1 is available under accession numbers NM _001776.5 and NP _ 001767.3. Transcript variants represent the longest transcript and encode isoform 1. Compared to transcript variant 1, human CD39 isoform 2, available under accession numbers NM _001098175.1 and NP _001091645.1, uses an alternative 5 'exon, which results in a different 5' untranslated region (UTR) and causes translation initiation at the alternative start codon, resulting in a longer and different N-terminus. Human CD39 isoform 3, available under accession numbers NM _001164178.1 and NP _001157650.1, uses an alternative 5 'exon, which results in a different 5' UTR and causes translation initiation at the alternative start codon, resulting in a longer and different N-terminus, compared to transcript variant 1. The human CD39 isoform 4, available under accession numbers NM _001164179.1 and NP _001157651.1, uses alternative in-frame splice sites relative to transcript variant 1, which produces shorter isoforms. Human CD39 isoform 5, available under accession numbers NM _001164181.1 and NP _001157653.1, uses alternative exons in the 5 'region, resulting in a different 5' UTR and translation initiation at the downstream initiation codon relative to transcript variant 1, which produces shorter isoforms. The human CD39 isoform 6, available at accession nos. NM _001164182.1 and NP _001157654.1, lacks alternative exons, results in a different 5' UTR at the downstream initiation codon and causes translation initiation relative to transcript variant 1, which produces shorter isoforms. The human CD39 isoform 6, also encoded by another transcript variant, available under accession numbers NM _001164183.1 and NP _001157655.1, lacks two alternative internal exons, leads to a different 5' UTR at the downstream initiation codon and causes translation initiation relative to transcript variant 1, which results in a shorter isoform.

Nucleic acid and polypeptide sequences of CD39 orthologs in organisms other than humans are well known and include, for example, mouse CD39(NM _009848.3 and NP _033978.1), rat CD39(NM _022587.1 and NP _072109.1), cow CD39(NM _174536.2 and NP _776961.1), frog CD39(NM _001006795.1 and NP _001006796.1), and zebrafish CD39(NM _001003545.1 and NP _ 001003545.1).

Extensive glycosylation of CD39 was associated with its cell surface expression and activity, such that deletion or mutation of glycosylated residues to non-glycosylated residues resulted in significantly reduced CD39 activity (see, e.g., N-terminal glycosylated residues 73, middle residues 333, and/or residues 429 and/or C-terminal residues 458 of rat CD39 or deletion or mutation of the corresponding residues in orthologs thereof; Wu et al (2005) mol.biol.cell.16: 1661-1672). Similarly, mutation of conserved residues in the conserved region of Apyrase (ACR) of any one or more of ACR 1 to 5 results in a decrease in CD39 activity (Schulte am Esch et al (1999) biochem.38: 2248-.

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

A "CD 39 antibody" (or "anti-CD 39 antibody") refers to an antibody that selectively binds to one or more epitopes of NTPD enzyme 1 protein and includes single paratope antibodies, as well as biparatopic antibodies and other multiparatopic forms of antibodies.

a. Antibodies and other polypeptides

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

In the context of the present invention, "antibody-mediated target endocytosis" refers to antibody-mediated depletion of CD39 from the surface of CD45+ immune cells without substantial reduction in the number of CD45+ immune cells, i.e. by a process other than induction of CD45+ cell death.

As used herein, the term "antigen-binding portion" or "antigen-binding fragment" of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., human CD 39). It has been shown that the antigen binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term "antigen-binding portion" of an antibody (e.g., an anti-CD 39 antibody described herein) include (i) Fab fragments, consisting of V_L、V_HA monovalent fragment consisting of the CL and CH1 domains; (ii) f (ab')₂A fragment comprising a bivalent fragment of two Fab fragments linked by a disulfide bridge at the hinge region; (iii) from V_HAnd the CH1 domain; (iv) v from one arm of an antibody_LAnd V_H(iii) an Fv fragment consisting of a domain; (v) from V_HdAb fragments consisting of domains (Ward et al (1989) Nature 341: 544-546); and (vi) an isolated Complementarity Determining Region (CDR) or (vii) a combination of two or more isolated CDRs, which may optionally be joined by a synthetic linker. Such single chain antibodies are also intended to be encompassed within the term "antigen-binding portion" of an antibody. These and other potential constructs are described in Chan and Carter (2010) nat. rev. immunol.10: 301. These antibody fragments are obtained using conventional techniques known to those skilled in the art and the fragments are screened for utility in the same manner as intact antibodies. Antigen binding portions can be produced by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact immunoglobulins.

The term "variable region" of an antibody refers to the variable region of an antibody light chain or the variable region of an antibody heavy chain, alone or in combination. In general, the variable regions of the heavy and light chains each consist of four Framework Regions (FRs) and three Complementarity Determining Regions (CDRs) (also referred to as "hypervariable regions"). The CDRs in each chain are held together tightly by the framework regions and, together with the CDRs from the other chain, contribute to the formation of the antigen-binding site of the antibody. There are at least two techniques for determining CDRs: (1) methods based on cross-species sequence variability (i.e., Kabat et Al, 1991, Sequences of Proteins of Immunological Interest, 5 th edition, National Institutes of Health, Bethesda Md.), and (2) methods based on crystallographic studies of antigen-antibody complexes (Al Lazikani et Al, 1997, J.mol.biol.,273: 927-. In addition, a combination of these two methods is sometimes used in the art to determine CDRs.

Although antibodies can be any of the five major classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, or subclasses (isotypes) thereof (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2), but are referred to as α, δ, ε, γ and μ, respectively, based on the identity of their heavy chain constant domains, with the preferred CD39 antibodies being the IgG1 and IgG3 isotypes in order to most effectively bind Fc γ RIII (i.e., at 10 a) ^-7Or smaller Kd).

In certain embodiments, the antibody is "low fucosylated" and may even be "afucosylated". By "low fucosylation" antibody preparation is meant an antibody preparation in which less than 50% of the oligosaccharide chains contain alpha-1, 6-fucose. Typically, less than about 40%, less than about 30%, less than about 20%, less than about 10%, or less than 5%, or less than 1% of the oligosaccharide chains in a "low fucosylated" antibody preparation contain alpha-1, 6-fucose. An "afucosylated" antibody lacks alpha-1, 6-fucose in the carbohydrate attached to the CH2 domain of an IgG heavy chain.

As used herein, the term "monoclonal antibody" refers to an antibody that exhibits a single binding specificity and affinity for a particular epitope or an antibody composition in which all antibodies exhibit a single binding specificity and affinity for a particular epitope. Typically, such monoclonal antibodies will be derived from a single cell or nucleic acid encoding the antibody, and will be propagated without intentionally introducing any sequence changes. Thus, the term "human monoclonal antibody" refers to a monoclonal antibody having variable and optionally constant regions derived from human germline immunoglobulin sequences. In one embodiment, the human monoclonal antibodies are produced by hybridomas, e.g., obtained by fusing B cells obtained from transgenic or transchromosomal non-human animals (e.g., transgenic mice having a genome comprising a human heavy chain transgene and a light chain transgene) to immortalized cells.

The term "humanized antibody" as used herein refers to a form of non-human (e.g., murine) antibody which is a specific immunoglobulin chain, chimeric immunoglobulin or fragment thereof that contains minimal non-human sequences. Typically, humanized antibodies are human immunoglobulins in which residues from a CDR are replaced by residues from a CDR of a non-human species (e.g., mouse, rat, rabbit or hamster) having the desired specificity, affinity and/or binding capacity. In some examples, Fv framework region residues of the human immunoglobulin are replaced with corresponding residues in antibodies from non-human species. Humanized antibodies can be further modified by the substitution of additional residues in the Fv framework regions and/or within the substituted non-human residues to improve and optimize antibody specificity, affinity, and/or binding capacity. Humanized antibodies may comprise variable domains that comprise all or substantially all of the CDRs corresponding to a non-human immunoglobulin, and all or substantially all of the framework regions are human immunoglobulin sequences. In some embodiments, the variable domain comprises a framework region of a human immunoglobulin sequence. In some embodiments, the variable domain comprises a framework region of a human immunoglobulin consensus sequence. The humanized antibody may further comprise at least a portion of an immunoglobulin constant region or domain, typically a human immunoglobulin constant region or domain (Fc). Humanized antibodies are generally considered to be distinct from chimeric antibodies.

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

The term "chimeric antibody" as used herein refers to an antibody in which the amino acid sequences of immunoglobulin molecules are derived from two or more species. Typically, the variable regions of the light and heavy chains correspond to those of an antibody of one species (e.g., mouse, rat, rabbit, etc.) derived from a mammal with the desired specificity, affinity, and/or binding capacity, while the constant regions are homologous to sequences in an antibody derived from another species (typically human) to avoid eliciting an immune response in that species.

An "Fc receptor" or "FcR" is a receptor that binds to the Fc region of an immunoglobulin. FcR binding to IgG antibodies comprises receptors of the Fc γ R family, including allelic variants and alternatively spliced forms of these receptors. The Fc γ R family consists of three activating (Fc γ RI, Fc γ RIII and Fc γ RIV in mice; Fc γ RIA, Fc γ RIIA and Fc γ RIIIA in humans) and one inhibitory (Fc γ RIIB) receptor.

An "Fc γ RIII binding moiety" is a peptide, protein, nucleic acid or other moiety that, when bound to the antigen binding site of an anti-CD 39 antibody, can bind to Fc γ RIII (CD16) and mediate antibody-dependent cellular cytotoxicity (ADCC). The heavy chain Fc fragment containing the CH2 and CH3 domains of the IgG1 and IgG3 isotypes is an Fc γ RIII binding moiety.

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

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

The terms "polypeptide" and "peptide" and "protein" are used interchangeably herein and refer to a polymer of amino acids of any length. The polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids. The term also encompasses amino acid polymers that have been modified either naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation to 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), as well as other modifications known in the art. It is to be understood that because the polypeptides encompassed by the present invention may be based on antibodies or other members of the immunoglobulin superfamily, in certain embodiments, the polypeptides may exist as single chains or as related chains.

The term "identical" or percent "identity," in the context of two or more nucleic acids or polypeptides, refers to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned (introducing gaps, if necessary) to achieve maximum correspondence, without regard to any conservative amino acid substitutions as part of the sequence identity. Percent identity can be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software that can be used to obtain an alignment of amino acid or nucleotide sequences are well known in the art. These include, but are not limited to, BLAST, ALIGN, Megalign, BestFit, GCG Wisconsin Package, and variants thereof. In some embodiments, two nucleic acids or polypeptides encompassed by the 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%, 99% nucleotide or amino acid residue identity when compared and aligned for maximum correspondence as measured using a sequence comparison algorithm or by visual inspection. In some embodiments, identity exists over a region of an amino acid sequence that is at least about 10 residues, at least about 20 residues, at least about 40 to 60 residues, at least about 60 to 80 residues, or any integer value therebetween in length. In some embodiments, identity exists over a region that is 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 compared sequences, such as the coding region of a target protein or antibody. In some embodiments, identity exists over a region of a nucleotide sequence that is at least about 10 bases in length, at least about 20 bases in length, at least about 40 to 60 bases in length, at least about 60-80 bases in length, or any integer value therebetween. In some embodiments, identity exists over a region that is 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 compared sequences, such as nucleotide sequences encoding a protein of interest.

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

An "isolated" polypeptide, soluble protein, antibody, polynucleotide, vector, cell, or composition is a polypeptide, soluble protein, antibody, polynucleotide, vector, cell, or composition 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 found in nature. In some embodiments, the isolated polypeptide, soluble protein, antibody, polynucleotide, vector, cell, or composition is substantially pure.

The term "substantially pure" as used herein refers to a material that is at least 50% pure (i.e., free of contaminants), at least 90% pure, at least 95% pure, at least 98% pure, or at least 99% pure.

The term "fusion protein" or "fusion polypeptide" as used herein refers to a hybrid protein expressed from a nucleic acid molecule comprising the nucleotide sequences of at least two genes.

The term "linker" or "linker region" as used herein refers to a linker interposed between a first polypeptide (e.g., an anti-CD 39 antibody) and a second polypeptide (e.g., an Fc or other Fc γ RIII binding moiety; scFV, Vhh domain, or the like, which binds to a different protein to produce a bispecific antibody format that maintains a bivalent directed against CD 39). In some embodiments, the linker is a peptide linker. The linker should not adversely affect the expression, secretion or biological activity of the polypeptide. Preferably, the linker is not antigenic and does not elicit an immune response.

b. Nucleic acids

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

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

The term "sequence" as used herein when used in reference to a nucleotide sequence, may include DNA or RNA, and may be single-stranded or double-stranded. The nucleic acid sequence may be mutated.

The term "vector" as used herein means a construct capable of delivering and generally expressing 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.

As used herein, the term "transfection" refers to the entry of an exogenous nucleic acid into a eukaryotic cell. Transfection may be accomplished by a variety of methods known in the art, including calcium phosphate-DNA co-precipitation, DEAE-dextran mediated transfection, polybrene mediated transfection, electroporation, microinjection, liposome fusion, lipofection, protoplast fusion, retroviral infection, and gene gun technology (gene gun).

The term "vector" as used herein is an isolated nucleic acid comprising an isolated nucleic acid, which can be used to deliver a composition to the interior of a cell. Many vectors are known in the art, including but not limited to linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term "vector" includes an autonomously replicating plasmid or virus. The term should also be construed to include facilitating the transfer of nucleic acids into cells that are not plasmids and are not viral compounds (e.g., polylysine compounds, liposomes, and the like). Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, and the like.

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

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

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

The term "constitutive expression" as used herein means that all are expressed under physiological conditions.

The term "inducible expression" as used herein refers to expression under certain conditions, such as occurs upon T cell antigen binding. How to familiarize conventional "induction of expression".

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

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

By "checkpoint molecule" is meant a protein that is expressed by a tissue and/or immune cell and reduces the efficacy of an immune response in a manner that depends on the extent of expression of the checkpoint molecule. When these proteins are blocked, the "brake" of the immune system is released and, for example, T cells can kill cancer cells more effectively. Examples of checkpoint proteins found on T cells or cancer cells include PD-1/PD-L1 and CTLA-4/B7-1/B7-2, PD-L2, NKG2A, KIR, LAG-3, TIM-3, CD96, VISTA, TIGIT and Siglec-15.

By "checkpoint inhibitor" is meant a pharmaceutical entity that reverses immunosuppressive signaling from a checkpoint molecule.

"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. Costimulatory molecules are cell surface molecules other than antigen receptors or ligands that promote an effective immune response. Costimulatory molecules include, but are not limited to, MHCI molecules, BTLA receptors and Toll ligands, and OX40, CD27, CD28, CDS, ICAM-1, LFA-1(CD11a/CD18), ICOS (CD278), and 4-1BB (CD 137). Examples of co-stimulatory molecules include, but are not limited to: CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHT TR), SLAMF, NKp (KLRF), NKp, CD160, CD α, CD β, IL2 γ, IL7 α, ITGA, VLA, CD49, ITGA, IA, CD49, ITGA, VLA-6, CD49, ITGAD, CD11, ITGAE, CD103, ITGAL, CD11, LFA-1, ITGAM, CD11, ITGAX, CD11, ITGB, CD, LFA-1, ITGB, NKG2, TNFR, TRANCE/RANKL, DNAM (CD226), SLAMF (CD244, 2B), CD (Tactile), ACAM, CRTAM, Ly CD (CD229), CD160 (BY), CD100, SLGL (SLGL), SLAMF-14, SLAMF (SLAMF-14, SLAMBR), SLAMBR (CD-16, SLAMBR), SLAMBR, CD-16, CD-L-P (CD-16, CD-L), SLAMBR, CD-L-P, CD-L (CD-L), CD-L-P, CD-L (CD-L), CD-L (CD-L (CD-L (CD-L (CD-L (CD-L (CD-L (.

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

An "innate immunity inducer" is an agent that mimics the innate immune response, including activation of inflammatory activity and/or deactivation of anti-inflammatory activity of macrophages, NK cells, dendritic cells, monocytes, neutrophils, and the like. Innate immunity inducers include inhibitors of the CD 47-sirpa axis, such as antibodies or other binding moieties that bind to CD47 or sirpa and inhibit the interaction of the two molecules to promote anti-tumor macrophage activity. Innate immunity inducers include inhibitors of the CD24-Siglec-10 axis, such as antibodies or other binding moieties that bind to CD24 or Siglec-10 and inhibit the interaction of the two molecules to promote anti-tumor macrophage activity. In other embodiments, the innate immune activator may be a NGK2A checkpoint inhibitor that blocks HLA-E driven inhibition of NK and CD8+ cells. Small molecule inducers of innate immunity include agents such as STING agonists, TLR7/8 agonists, and RIG-I agonists.

A "chemotherapeutic agent" is a chemical compound that may be useful in the treatment of cancer. Examples of chemotherapeutic agents include alkylating agents such as thiotepa (thiotepa) and Cyclophosphamide (CYTOXAN); alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa (benzodopa), carboquone (carboquone), mitdopa (meteedopa), and ulidopa (uredopa); ethyleneimine and methylmelamine including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolmelamine; polyacetyl (especially buclatacin (bullatacin) and bullatacin (bullatacinone); delta-9-tetrahydrocannabinol (dronabinol), MARINOL); beta-lapachone (beta-lapachone); lapachol (lapachol); colchicine (colchicines), betulinic acid (betulinic acid); camptothecin (including the synthetic analogue topotecan (HYCAMTIN), CPT-11 (irinotecan), CAMPTOSAR, acetyl camptothecin, hyoscyamine (scopoletin) and 9-amino camptothecin); bryostatin (brystatin); pemetrexed (pemetrexed); spongetin (callystatin); CC-1065 (including adolesein, carbozelescinin) and bizephytin (kyropodophyllosine); (especially kytopropodophyllosin), and kytophilin (kytopirin) (including cryptophycin) (2188); and kytophilin (kytopirinotecan) (including cryptophytin) (myriocin) (especially kytopinin and cryptophytin) (2188); and myriophyllin) (including cryptophytin) (myriophyllin) (synthetic analogs thereof); and myriophyllin) (cynanchoricin) (cynanchorin) (2188);) and cryptophytin) (including cryptophytin) (cynanchorin) (cynanchoricin) (cynanchorin) (cynanchori) and cryptophytin) (cynanchorin) (cynanchori) and cryptophytin) (cynanchorin) (cynanchori (cynanchorin) (cynanchori) and cryptophytin) (cynanchorin) (cynanchori (cynanchorin) (cynanchori) and cryptophytin) (cynanchorin) (cynanchori) and cryptophytin) (cynanchorin) (cynanchin) and cryptophytin) (cynanchorin) (2) and cryptophytin) (cynanchorin) (cynanchin) and cryptophytin) (cynanchorin) (cynanchin) and cryptophytin) (cynanchin) -TM 1); eiscosahol (eleutherobin); coprinus atrata base (pancratistatin); TLK-286; CDP323, oral α -4 integrin inhibitors; sarcandra glabra alcohol (sarcodictyin); spongistatin (spongistatin); nitrogen mustards (nitrosgen mustards), such as chlorambucil (chlorambucil), chlorambucil (chlorenaphazine), chlorophosphamide (chlorophosphamide), estramustine (estramustine), ifosfamide (ifosfamide), mechlorethamine (mechlorethamine), mechlorethamine oxide hydrochloride (mechlorethamine oxide hydrochloride), melphalan (melphalan), neonebivoxin (novembichin), benzene mustard cholesterol (phenylesterine), prednimustine (prednimustine), triamcinolone (trofosfamide), uracil mustard (uracil mustard); nitrosoureas (nitrosureas) such as carmustine (carmustine), chlorouretocin (chlorozotocin), fotemustine (fotemustine), lomustine (lomustine), nimustine (nimustine) and ramustine (ranirnustine); antibiotics such as enediynes antibiotics (e.g., calicheamicin, especially calicheamicin gamma 1I and calicheamicin omega I1 (see, e.g., Nicolaou et al, Angew. chem. Ed. Engl.33:183-186(1994)), dalnamycin (dynemicin), including dalnamycin A (dynemicin A), esperamicin (esperamicin), and neocarzinostain chromophore (neocarzinostatin chromophoropterin) and related chromoproteenediynes antibiotics chromophore, aclacinomycin (acaromycin), actinomycin (actinomycin), ampamycin (actamycin), antromycin (aureomycin), azaserine (azaserine), bleomycin (bleomycin), actinomycin C (calcimycin), carzinomycin (caromycin), carminomycin (carotinomycin), carzinomycin (monocrotamycin), monocrotamycin (monocrotamycin-5-D), monocrotamycin (monocrotamycin-6-D), monocrotamycin (monocrotamycin-5-D, and D-, Doxorubicin (doxorubicin) (including ADRIAMYCIN, morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, doxorubicin HCl liposome injection (DOXIL) and deoxydoxorubicin (deoxydoxorubicin)), epirubicin (epirubicin), esorubicin (esorubicin), idarubicin (idarubicin), marijumycin (marcelomycin), mitomycin (mitomycin), such as mitomycin C, mycophenolic acid (mycophenolic acid), noramycin (nogalamycin), olivomycin (olivomycin), pelomomycin (polyplomycin), pofiomycin (potrofibriomycin), puromycin (puromycin), doxorubicin (queamycin), roxobicin (nigrocicin), streptozocin (streptozocin), tubercidin (zotocin), tubercidin (tubercidin); antimetabolites such as methotrexate (methotrexate), gemcitabine (gemcitabine) (GEMZAR), tegafur (uftoal), capecitabine (XELODA), epothilone (epothilone), and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine (fludarabine), 6-mercaptopurine, thiamiprine (thiamiprine), thioguanine (thioguanine); pyrimidine analogs such as ancitabine (ancitabine), azacitidine (azacitidine), 6-azauridine (6-azauridine), carmofur (carmofur), cytarabine (cytarabine), dideoxyuridine (dideoxyuridine), deoxyfluorouridine (doxifluridine), enocitabine (enocitabine), floxuridine (floxuridine), and imatinib (imatinib) (2-phenylamino pyrimidine derivatives), and other c-Kit inhibitors; anti-adrenaline such as aminoglutethimide (aminoglutethimide), mitotane (mitotane), trilostane (trilostane); folic acid supplements such as folinic acid (folinic acid); acetoglucurolactone (acegultone); (ii) an aldophosphamide glycoside; aminolevulinic acid (aminolevulinic acid); eniluracil (eniluracil); amsacrine (amsacrine); doubly-branched betuzucil; bisantrene; edatrexate (edatraxate); defluoromine (defofamine); colchicine (demecolcine); diazaquinone (diaziqutone); isoflurine (elfornithine); ammonium etitanium acetate; etoglut (etoglucid); gallium nitrate (gallium nitrate); hydroxyurea (hydroxyurea); lentinan (lentinan); lonidamine (lonidainine); maytansinoids such as maytansinoids (maytansine) and ansamitocins (ansamitocins); mitoguazone (mitoguzone); mitoxantrone (mitoxantrone); mopidanol (mopidanmol); diamine nitracridine (nitrarine); pentostatin (pentostatin); methionine mustard (phenamett); pirarubicin (pirarubicin); losoxantrone (losoxantrone); 2-ethyl hydrazide (2-ethyl hydrazide); procarbazine (procarbazine); PSK polysaccharide complex (JHS Natural Products Eugene Oreg.); razoxane (rizoxane); rhizomycin (rhizoxin); umirolium (sizofiran); germanium spiroamines (spirogyranium); tenuazonic acid (tenuazonic acid); triimine quinone (triaziquone); 2,2',2 "-trichlorotriethylamine (2,2', 2" -trichlorotriethylamine); trichothecene toxins (trichothecenes) (especially T-2 toxin, verrucin a (verrucin a), tuberculin a (roridin a), and serpentin (anguidine)); urethane (urethan); vindesine (vindesine, FILDESIN); dacarbazine (dacarbazine); mannomustine (mannomustine); dibromomannitol (mitobronitol); dibromodulcitol (mitolactol); pipobromane (pipobroman); gatifloxacin (gacytosine); cytarabine ("Ara-C"); thiotepa; taxanes such as paclitaxel (TAXOL), albumin-engineered paclitaxel nanoparticle formulation (ABRAXANE) and docetaxel (TAXOTERE); chlorambucil; 6-thioguanine; mercaptopurine (mercaptoprine); methotrexate; platinum analogs such as cisplatin (cissplatin) and carboplatin (carboplatin); vinblastine (vinblastine) (VELBAN); platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine (vincristine) (ONCOVIN); oxaliplatin (oxaliplatin); leucovorin (leucovovin); vinorelbine (vinorelbine) (NAVELBINE); mitoxantrone hydrochloride (novantrone); edatrexate (edatrexate); daunomycin (daunomycin); aminopterin (aminopterin); ibandronate (ibandronate); topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids (retinoids), such as retinoic acid; a pharmaceutically acceptable salt, acid or derivative of any of the above; and combinations of two or more of the above such as CHOP (abbreviation for combination therapy of cyclophosphamide, doxorubicin, vincristine and prednisolone) and FOLFOX (abbreviation for treatment regimen using oxaliplatin (ELOXATIN) in combination with 5-FU and leucovorin (leucovorin)).

Also included in this definition are anti-hormonal agents that act to modulate, reduce, block or inhibit the effects of hormones that can promote the growth of cancer, and are generally in the form of systemic or systemic treatment. They may themselves be hormones. Examples include antiestrogens and Selective Estrogen Receptor Modulators (SERMs), including, for example, tamoxifen (tamoxifen) (including NOLVADEX tamoxifen), raloxifene (raloxifene) (EVISTA), droloxifene (droloxifene), 4-hydroxytamoxifene, trioxifene (trioxifene), kexifene (keoxifene), LY117018, onapristone (onapristone), and toremifene (toremifene) (FARESTON); an antiprogestin; estrogen receptor down-regulator (ERD); estrogen receptor antagonists such as Fulvestrant (FASLODEX); agents that act to inhibit or close the ovary, for example leucine releasing hormone (LHRH) agonists such as leuprolide acetate (LUPRON and ELIGARD), goserelin acetate (goserelin acetate), buserelin acetate (buserelin acetate) and triptorelin (tripterelin); antiandrogens such as flutamide (flutamide), nilutamide (nilutamide), and bicalutamide (bicalutamide); and aromatase inhibitors which inhibit the enzyme aromatase which regulates estrogen production in the adrenal gland, such as, for example, 4(5) -imidazole, aminoglutethimide, megestrol acetate (megestro acetate) (MEGASE), exemestane (AROMASIN), formestane (formestanine), fadrozole (fadrozole), vorozole (rivarozole) (rimarovir), letrozole (letrozole) (FEMARA) and Anastrozole (ARIMIDEX). In addition, this definition of chemotherapeutic includes bisphosphonates, such as clodronate (e.g., BONEFOS or OSTAC), etidronate (DIDROCAL), NE-58095, zoledronic acid/Zoledronate (ZOMETA), alendronate (FOSAMAX), pamidronate (aridia), tiludronate (skeld), or risedronate (ACTONEL); and troxacitabine (troxacitabine) (1, 3-dioxolane nucleoside cytosine analogues); antisense oligonucleotides, particularly those that inhibit gene expression in signaling pathways involved in cell proliferation, such as, for example, PKC- α, Raf, H-Ras and epidermal growth factor receptor (EGF-R); vaccines, such as THERATOPE vaccines and gene therapy vaccines, such as ALLOVECTIN vaccine, LEUVECTIN vaccine and VAXID vaccine; topoisomerase 1 inhibitors (e.g., lutotecan); antiestrogens, such as fulvestrant; kit inhibitors such as imatinib or EXEL-0862 (tyrosine kinase inhibitors); EGFR inhibitors such as erlotinib (erlotinib) or cetuximab (cetuximab); anti-VEGF inhibitors such as bevacizumab (bevacizumab); alinotikang (arinotecan); rmRH (e.g., ABARELIX); lapatinib (lapatinib) and lapatinib ditosylate (ErbB-2 and EGFR dual tyrosine kinase small molecule inhibitor, also known as GW 572016); 17AAG (geldanamycin derivative of a heat shock protein (Hsp)90 poison) and a pharmaceutically acceptable salt, acid or derivative of any of the foregoing.

As used herein, the term "cytokine" generally refers to a protein released by one cell population that acts on another cell as an intercellular mediator or has an autocrine effect on the cell producing the protein. Examples of such cytokines include lymphokines, monokines; interleukins- ("IL"), such as IL-1, IL-1 α, 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 through IL-29 (such as IL-23), IL-31, including PROLEUKIN rIL-2; tumor necrosis factors such as TNF-alpha or TNF-beta, TGF-beta 1-3; and other polypeptide factors including leukemia inhibitory factor ("LIF"), ("ciliary neurotrophic factor" CNTF), CNTF-like cytokines ("CLC"), cardiotrophin ("CT"), and Kit ligand ("KL").

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

d. Treatment of

In the context of immune dysfunction, the term "dysfunction" refers to a state of reduced immune reactivity to antigen stimulation. The term includes the common elements in which depletion and/or unresponsiveness of antigen recognition can occur, but the ensuing immune response is ineffective in controlling infection or tumor growth.

As used herein, "hampering" function also includes being refractory or unresponsive to antigen recognition, specifically, translating antigen recognition into downstream T cell effector functions such as impaired ability to proliferate, cytokine production (e.g., IL-2), and/or target cell killing.

The term "anergy" refers to incomplete or insufficient signaling by T cell receptor delivery (e.g., intracellular Ca in the absence of ras activation⁺²Increase) resulting in a state of unresponsiveness to antigenic stimulation. T cell anergy can also be produced when stimulated with antigen in the absence of co-stimulation, resulting in thin cells even in the presence of co-stimulationThe cells also become refractory to subsequent activation by the antigen. The unresponsive state can be generally overridden by the presence of interleukin-2. Anergic T cells do not undergo clonal expansion and/or gain effector function.

The term "depletion" refers to T cell depletion, a state of T cell dysfunction due to sustained TCR signaling that occurs during many chronic infections and cancers. It differs from anergy in that it is not caused by incomplete or insufficient signaling, but rather by sustained signaling. It is defined as poor effector function, suppression of sustained receptor expression and transcriptional state distinct from functional effector or memory T cells. Depletion prevents optimal control of infection and tumors.

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

"T cell dysfunctional disorders" are characterized by disorders or conditions of T cells with reduced responsiveness to antigen stimulation. In a particular embodiment, the T cell dysfunctional disorder is a disorder that is particularly associated with an inappropriately increased level of CD 39. In another embodiment, a T cell dysfunctional disorder is one in which the T cell is anergic or has reduced ability to secrete cytokines, proliferate or perform cytolytic activity. In a particular aspect, the reduced reactivity results in ineffective control of the pathogen or tumor expressing the immunogen. Examples of T cell dysfunction conditions characterized by T cell dysfunction include non-resolved acute infection, chronic infection, and tumor immunity.

"tumor immunity" refers to a process in which a tumor evades immune recognition and clearance. Thus, as a therapeutic concept, when this escape is attenuated, tumor immunity is "treated" and the tumor is recognized and attacked by the immune system. Examples of tumor recognition include tumor binding, tumor shrinkage, and tumor clearance.

By "sustained response" is meant a sustained effect that reduces tumor growth after cessation of treatment. For example, the tumor size may remain the same or smaller than the size at the beginning of the administration phase. In some embodiments, the sustained response has a duration that is at least the same as the duration of treatment, at least 1.5x, 2.0x, 2.5x, or 3.0x length of the duration of treatment.

The terms "cancer" and "cancerous" as used herein refer to or describe a 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 hematological cancers, such as lymphoma and leukemia.

The terms "tumor" and "neoplasm", as used herein, refer to any mass of tissue resulting from excessive cell growth or proliferation, benign (non-cancerous) or malignant (cancerous), including precancerous lesions. Tumor growth is generally unregulated and progressive, and does not induce or inhibit proliferation of normal cells. Tumors can affect a variety of cells, tissues or organs, including but not limited to those selected from the group consisting of bladder, bone, brain, breast, cartilage, glial cells, esophagus, fallopian tube, gall bladder, heart, intestine, kidney, liver, lung, lymph node, neural tissue, ovary, pancreas, prostate, skeletal muscle, skin, spinal cord, spleen, stomach, testis, thymus, thyroid, trachea, urethra, ureter, urethra, uterus, vaginal organs or tissues or corresponding cells. Tumors include cancers such as sarcomas, carcinomas, plasmacytomas, or (malignant plasma cells). Tumors contemplated by the present invention may include, but are not limited to, leukemias (e.g., acute leukemia, acute lymphoblastic leukemia, acute myelogenous leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, acute monocytic leukemia, acute leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, polycythemia vera), lymphomas (Hodgkin's disease), non-Hodgkin's disease), primary macroglobulinemia, heavy chain diseases, and solid tumors, such as sarcoma cancers (e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, endothelioma, lymphosarcoma, angiosarcoma, lymphangioendothelioma sarcoma, synovioma, mesothelioma, Ewing's tumor), Leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary adenocarcinoma, carcinoma, bronchial carcinoma, medullary carcinoma, renal cell carcinoma, liver cancer, Nile river ductal carcinoma (nie duct carcinoma), choriocarcinoma, seminal cell tumor, embryonic carcinoma, Wilms' tumor, cervical cancer, uterine cancer, testicular cancer, lung cancer, small cell lung cancer, bladder cancer, epithelial cancer, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, schwannoma, meningioma, melanoma, neuroblastoma, retinoblastoma), esophageal cancer, gallbladder cancer, kidney cancer, multiple myeloma. Preferably, a "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, renal cancer, leukemia, multiple myeloma, ovarian cancer, cervical cancer, and glioma.

The term "metastasis" as used herein refers to the process by which cancer spreads or metastasizes from the site of origin of the body to other areas of the body and develops similar cancerous lesions at new locations. "metastatic" or "metastatic" cells are those that lose adhesive contact with adjacent cells and migrate from the primary site of disease via the bloodstream or lymph to invade adjacent bodily structures.

The terms "cancer cell" and "tumor cell" refer to the total population of cells derived from a cancer or tumor or precancerous lesion, including non-tumorigenic cells, which include the majority of the cancer cell population, and tumorigenic stem cells (cancer stem cells). As used herein, the term "cancer cell" or "tumor cell" when referring to only those cells lacking the ability to renew and differentiate is modified by the term "non-tumorigenic" to distinguish those tumor cells from cancer stem cells.

The term "effective amount" as used herein refers to an amount that provides a therapeutic or prophylactic benefit.

As used herein, "complete response" or "CR" refers to the disappearance of all target lesions; "partial response" or "PR" means that the sum of the longest diameters (SLDs) of the target lesions is reduced by at least 30%, referenced to the baseline SLD; and "stable disease" or "SD" neither means that the target lesion is sufficiently reduced to meet PR criteria, nor sufficiently increased to meet PD, with reference to the minimum SLD since the onset of treatment.

As used herein, "progressive disease" or "PD" refers to an increase in SLD of a target lesion of at least 20%, referenced to the minimum SLD recorded since the initiation of therapy or the presence of one or more new lesions.

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

As used herein, "overall reaction rate" (ORR) refers to the sum of the rate of Complete Reaction (CR) and the rate of Partial Reaction (PR).

As used herein, "overall survival" refers to the percentage of subjects in a group that are likely to survive a particular period of time.

The term "treatment" as used herein refers to the process or treatment of a subject attempting to alter a clinical disease caused by cellular intervention, which may be a preventive intervention process of clinical pathology. Including but not limited to treatment to prevent the occurrence or recurrence of disease, alleviation of symptoms, diminishment of the direct or indirect pathological consequences of any disease, preventing metastasis, slowing the rate of disease progression, amelioration or palliation of the disease, or improved prognosis.

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

The terms "agonist" and "agonism" as used herein refer to or describe a therapeutic moiety that can directly or indirectly substantially induce, activate, promote, increase or enhance a 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.

The terms "antagonist" and "antagonize" as used herein refer to or describe a therapeutic moiety that can directly or indirectly partially or completely block, inhibit, reduce, or neutralize a 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.

The terms "modulation" and "modulation" as used herein refer to a change or alteration in biological activity. Modulation includes, but is not limited to, stimulatory activity or inhibitory activity. Modulation may be an increase or decrease in activity, a change in binding characteristics, or any other change in biological, functional, or immunological properties associated with the activity of a protein, pathway, system, or other biological target of interest.

The term "immune response" as used herein includes responses from the innate immune system and the adaptive immune system. Which includes cell-mediated and/or humoral immune responses. It includes T cell and B cell responses, and responses from other cells of the immune system such as Natural Killer (NK) cells, monocytes, macrophages, and the like.

The term "pharmaceutically acceptable" refers to materials that are approved or approvable by a regulatory agency of the federal or a state government or that are listed in the U.S. pharmacopeia or other generally recognized pharmacopeia for use in animals, including humans.

The term "pharmaceutically acceptable excipient, carrier or adjuvant" or "acceptable pharmaceutical carrier" refers to an excipient, carrier or adjuvant that can be administered to a subject along with at least one agent of the invention and that does not destroy its pharmaceutical activity and is non-toxic when administered in an amount sufficient to deliver a therapeutic effect. In general, pharmaceutically acceptable excipients, carriers, or adjuvants are considered by those skilled in the art and the U.S. FDA to be inactive ingredients of any formulation.

The term "effective amount" or "therapeutically effective amount" or "therapeutic effect" refers to an amount of anti-CD 39 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-CD 39 antibody has a therapeutic effect and thus may enhance the immune response, enhance the anti-tumor response, increase the cytolytic activity of immune cells, increase the killing of tumors by immune cells, decrease the number of tumor cells; reducing tumorigenicity, tumorigenic frequency, or tumorigenic capacity; reducing the number or frequency of cancer stem cells; reducing tumor size; reducing the population of cancer cells; inhibiting or preventing cancer cell infiltration into peripheral organs, including, for example, the spread of cancer into soft tissue and bone; inhibiting and preventing metastasis of tumor or cancer cells; inhibiting and arresting tumor or cancer cell growth; relieving to some extent one or more of the symptoms associated with the cancer; reducing morbidity and mortality; improving the quality of life; or a combination of these effects.

The terms "treating" or "to treat" or "to alleviate" both refer to (1) a therapeutic measure that cures, slows, reduces the progression of, and/or halts the progression of a diagnosed pathological condition or disorder, and (2) a prophylactic or preventative measure that prevents or slows the development of a targeted pathological condition or disorder. Thus, those in need of treatment include those already suffering from the condition; those susceptible to such disorders; and those for which the disorder is to be prevented. In the case of a cancer or tumor, the subject is successfully "treated" according to the methods encompassed by the present invention if the patient shows one or more of: increased immune response, increased anti-tumor response, increased cytolytic activity of immune cells, increased killing of tumor cells by immune cells, reduction in the number of cancer cells or complete absence of cancer cells, reduction in tumor size; inhibition or absence of cancer cell infiltration into peripheral organs, including cancer cell spread into soft tissue and bone; inhibition or absence of metastasis of tumors or cancer cells; inhibition or absence of cancer growth; alleviation of one or more symptoms associated with a particular cancer; reduced morbidity and mortality; improvement in quality of life; a reduction in tumorigenicity; a reduction in the number or frequency of cancer stem cells; or some combination of effects.

e. Miscellaneous items

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

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

The term "and/or" as used in phrases such as "a and/or B" herein is intended to include a and B; a or B; a (alone); and B (alone). Also, 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).

III.anti-CD 39 antibodies

a. Monoclonal antibodies

The anti-CD 39 antibody can be a monoclonal antibody. Monoclonal antibodies can be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature,256:495 (1975). In the hybridoma method, a mouse, hamster, or other appropriate host animal, is typically immunized with an immunizing agent to elicit lymphocytes that produce, or are capable of producing, antibodies that will specifically bind to the immunizing agent. Alternatively, lymphocytes may be immunized in vitro.

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

Preferred immortalized cell lines are those that fuse efficiently, support a stably high degree of expression of the antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. More preferred immortalized Cell lines are murine myeloma lines, which are available, for example, from the Salk Institute Cell Distribution Center, San Diego, Calif., and 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 to 63 ].

Then, the medium in which the hybridoma cells are cultured can be analyzed for the presence of monoclonal antibodies against the polypeptide. Preferably, the binding specificity of a monoclonal antibody produced by a hybridoma cell is determined by immunoprecipitation or by an in vitro binding assay such as Radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). such techniques and assays are known in the art the binding affinity of the monoclonal antibody can be determined, for example, by Scatchard analysis of Munson and Pollard, anal.biochem.,107:220 (1980).

After identifying the desired hybridoma cells, the clones can be subcloned by limiting dilution procedures and grown by standard methods [ Goding, supra ]. Suitable 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 in a mammal as ascites fluid.

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, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

Monoclonal antibodies can also be made by recombinant DNA methods, such as those described in U.S. patent No. 4,816,567. DNA encoding a monoclonal antibody encompassed by the invention can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of specifically binding to genes encoding the heavy and light chains of murine antibodies). Hybridoma cells encompassed by the present invention serve as a preferred source of such DNA. Once isolated, the DNA can be placed into an expression vector and then transfected into a host cell such as a simian COS cell, a Chinese Hamster Ovary (CHO) cell, or a myeloma cell that does not otherwise produce immunoglobulin protein, to obtain synthesis of the monoclonal antibody in the recombinant host cell. DNA can also be obtained, for example, by substituting the coding sequence for the human heavy and light chain constant domains for homologous murine sequences [ U.S. patent No. 4,816,567; morrison et al, supra ], or by covalent linkage to all or part of the coding sequence for a non-immunoglobulin polypeptide. Such non-immunoglobulin polypeptides may replace the constant domains of an antibody encompassed by the invention, or may replace the variable domains of one antigen combining site of an antibody encompassed by the invention to produce a chimeric bivalent antibody.

b. Human and humanized antibodies

anti-CD encompassed by the invention39 the antibody may further comprise a humanized or human antibody. Humanized forms of non-human (such as murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab ', F (ab')₂Or other antigen binding subsequences of antibodies) that contain minimal sequences derived from non-human immunoglobulins. Humanized antibodies include human immunoglobulins (recipient antibody) in which residues from a Complementarity Determining Region (CDR) of the recipient 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 examples, Fv framework residues of the human immunoglobulin are replaced with corresponding non-human residues. Humanized antibodies may also comprise residues that are not found in either the recipient antibody or imported CDR or framework sequences. In general, a humanized antibody will comprise all or 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 will also optimally comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin [ Jones et al, Nature,321:522-525 (1986); riechmann et al, Nature,332:323-329(1988) and Presta, curr. Op. struct. biol.,2:593-596(1992) ]。

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

Human antibodies can also be produced using various techniques known in the art, including phage display libraries and Winter, j.mol.biol.,227:381 (1991); marks et al, J.mol.biol.,222:581(1991) ]. The techniques of Cole et al and Boerner et al are also applicable to the preparation of human Monoclonal Antibodies [ (Cole et al, Monoclonal Antibodies and Cancer Therapy, Alan R.Liss, p.77 (1985) and Boerner et al, J.Immunol.,147(1):86-95(1991) ]. similarly, human Antibodies can be prepared by introducing human immunoglobulin loci into transgenic animals (e.g., mice in which endogenous immunoglobulin genes have been partially or completely inactivated.) Once elicitation, i.e., human antibody production is observed, is quite similar in all respects to those seen in humans, including gene rearrangement, assembly and antibody repertoire, such methods are described, for example, in U.S. Pat. Nos. 5,545,807, 5,545,806; 5,569,88, 5,625,126; 5,633,425; 5,661,016; and the scientific publications: Marks et al, Bio/Technology 10,779 (1992), Lornber et al, 8556; Nature et al, Fisherd., 36 368,812; 1994; Fisherd., WO 24, nature Biotechnology 14,845-51 (1996); neuberger, Nature Biotechnology 14,826 (1996); lonberg and Huszar, Intern.Rev.Immunol.1365-93 (1995).

Antibody affinity maturation can also be achieved using known selection and/or mutagenesis methods as described above. Preferred affinity matured antibodies have an affinity five times, more preferably 10 times, even more preferably 20 or 30 times greater than the starting antibody (typically murine, humanized or human) from which the mature antibody was made.

c. Bispecific antibodies

The anti-CD 39 antibodies described herein include bispecific molecules. The anti-CD 39 antibody or antigen-binding portion thereof can be derivatized or linked to another functional molecule, e.g., another peptide or protein (e.g., another antibody or ligand of a receptor), to produce a bispecific molecule that binds to at least two different binding sites or target molecules. Indeed, the antibodies described herein may be derivatized or linked to more than one other functional molecule to produce multispecific molecules that bind to more than two different binding sites and/or target molecules; such multispecific molecules are also intended to be encompassed by the term "bispecific molecule" as used herein. To produce a bispecific molecule described herein, an antibody described herein can be functionally linked (e.g., by chemical coupling, genetic fusion, non-covalent association, or other means) to one or more other binding molecules such as another antibody, antibody fragment, peptide, or binding mimetic, such that a bispecific molecule is produced.

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

In certain embodiments, the subject bispecific (or as the case may be multispecific) include one or more binding domains of an immune checkpoint, e.g., they are checkpoint inhibitors such as PD-1, PD-L1, CTLA-4/B7-1/B7-2, PD-L2, KIR, LAG-3, TIM-3, CD96, VISTA, TIGIT, and/or Siglec-15. In certain embodiments, the multispecific includes binding domains that bind checkpoint proteins on T cells (particularly checkpoints associated with T cell depletion such as LAG-3, TIM-3 or TIGIT). In certain embodiments, the multispecific binding to CD39 and one or more other T cells associated with a checkpoint and results in antibody-dependent cytotoxicity of cells expressing each or both CD39 and the other checkpoint proteins to which it binds.

In certain embodiments, the subject bispecific (or as the case may be multispecific) includes one or more binding domains of immune co-stimulatory receptors, e.g., they are co-stimulatory agonists (activators) such as 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 (CD 137). Examples of co-stimulatory molecules that may be included in the multispecific include, but are not limited to: CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHT TR), SLAMF, NKp (KLRF), NKp, CD160, CD α, CD β, IL2 γ, IL7 α, ITGA, VLA, CD49, ITGA, IA, CD49, ITGA, VLA-6, CD49, ITGAD, CD11, ITGAE, CD103, ITGAL, CD11, LFA-1, ITGAM, CD11, ITGAX, CD11, ITGB, CD, LFA-1, ITGB, NKG2, TNFR, TRANCE/RANKL, DNAM (CD226), SLAMF (CD244, 2B), CD (Tactile), ACAM, CRTAM, Ly (CD229), CD160 (BY), CD100 (SLGL), SEGL 4, SLMF-150, SLAMF (SLAMBR), SLAMBR, SLAMF-16, CD-6, CD-1, CD-6, CD-TAM, CD-6, CD-SLAML, CD-L.

In certain embodiments, the subject bispecific (or as the case may be multispecific) includes one or more binding domains that act as innate immune activators, such as binding portions of CD47, sirpa, CD24, Siglec-10, or NKG 2A.

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

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, bioassay (e.g., growth inhibition), or western blot assay. Each of these assays generally detects the presence of a particular target protein-antibody complex by employing a labeled reagent (e.g., an antibody) specific for the target complex.

Methods for making bispecific antibodies are known in the art. Traditionally, recombinant production of bispecific antibodies has been based on the co-expression of two immunoglobulin heavy/light chain pairs, where the two heavy chains have different specificities [ Milstein and Cuello, Nature,305:537-539(1983) ]. Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a possible mixture of ten different antibody molecules, only one of which has the correct bispecific structure. The purification of the correct molecule is typically performed by an affinity chromatography step. Similar procedures are disclosed in WO 93/08829 and Traunecker et al, EMBO J.,10:3655-3659(1991), published 5, 13, 1993.

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

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. Preferred interfaces comprise 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 the first antibody molecule are replaced with a larger side chain (e.g., tyrosine or tryptophan). A compensatory "cavity" of the same or similar size to the larger side chain is created at the interface of the second antibody molecule by replacing the large amino acid side chain with a smaller amino acid side chain (e.g., alanine or threonine). This provides a mechanism for increasing the yield of heterodimers over other undesired end products (such as homodimers).

Bispecific antibodies are prepared as full length antibodies or antibody fragments (e.g., F (ab')₂Bispecific 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, Science229:81(1985) describe a method in which intact antibodies are proteolytically cleaved to yield F (ab')₂Program of fragments. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize the ortho-dithiols and prevent intermolecular disulfide formation. The resulting Fab' fragments are then converted to Thionitrobenzoate (TNB) derivatives. One of the Fab ' -TNB derivatives is then reconverted to Fab ' -thiol by reduction with mercaptoethylamine and mixed with an equimolar amount of the other Fab ' -TNB derivative to form the bispecific antibody. The bispecific antibodies produced can be used as agents for the selective immobilization of enzymes.

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

Various techniques for the preparation and isolation of bispecific antibody fragments directly from recombinant cell culture have also been described. For example, bispecific antibodies have been generated using leucine zippers (Kostelny et al, J.Immunol.148(5):1547-1553 (1992)). 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 antibody heterodimers. This method can also be used to generate antibody homodimers. The "bifunctional antibody" technique described by Hollinger et al, Proc. Natl. Acad. Sci. USA 90: 6444-. The fragments comprise a light chain variable domain (V) linked by a linker that is too short to allow pairing between the two domains on the same chain_L) Heavy chain variable domain of (V)_H). Thus, V of a segment is forced_HAnd V_LComplementarity of the Domain to another fragment V_LAnd V_HThe domains pair, thereby forming two antigen binding sites. Bispecific production by Using Single chain fv (sFv) dimersAnother strategy for antibody fragments has also been reported. See Gruber et al, J.Immunol.152:5368 (1994).

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

d. Heteroconjugate (Heteroconjugate) antibodies

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

e. Effect function engineering

It may be desirable to modify the effector function of antibodies encompassed by the present invention to enhance the effectiveness of, for example, an anti-CD 39 antibody in treating cancer. For example, cysteine residues may be introduced into the Fc region, thereby allowing interchain disulfide bonds to form in this region. The homodimeric antibody thus produced may have improved internalization capability and/or enhanced complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC). See Caron et al, J.Exp. Med.,176: 1191-. In certain preferred embodiments, the engineered effector function is the ability of the anti-CD 39 antibody to induce Fc γ RIII binding-dependent removal (such as target endocytosis mediated by anti-CD 39 antibody) of CD39 from the immune cell, i.e., not by cell killing of the depleted immune cell population.

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-. Alternatively, the antibody may be engineered to have dual Fc regions, and may thus have enhanced CD39 trogocytosis (trogocytosis) capabilities. See Stevenson et al, Anti-Cancer Drug Design,3:219-230 (1989).

f. Representative anti-CD 39 antibody sequences

In certain embodiments, the anti-CD 39 antibody is a fully human antibody, such as produced from a human antibody library. An exemplary fully human anti-CD 39 antibody is clone Ig39-21, with the heavy and light variable domain (VH and VL) sequences provided below:

	nucleic acid sequences	Amino acid sequence
			VH domains	SEQ ID No.1(VH)	SEQ ID No.2(VH)
VL domain	SEQ ID No.3(VL)	SEQ ID No.4(VL)

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

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

the sequences of exemplary full-length antibodies and exemplary single chain antibodies (scFV) utilizing the above VH and VL domains are provided as follows:

	nucleic acid sequences	Amino acid sequence
			Full length heavy chain	SEQ ID No.35	SEQ ID No.36
Full length light chain	SEQ ID No.37	SEQ ID No.38
			scFV	SEQ ID No.39	SEQ ID No.40

In some embodiments, the anti-CD 39 antibody or antigen-binding fragment thereof comprises at least one heavy chain variable domain that is at least 60% identical to a VH domain sequence described herein (such as SEQ ID No.2), and even more preferably at least 65%, 70%, 75%, 80%, 85% or even 90% identical to a VH domain sequence described herein (such as SEQ ID No.2), and is capable of specifically binding to human CD 39.

In some embodiments, the anti-CD 39 antibody or antigen-binding fragment thereof comprises at least one light chain variable domain that is at least 60% identical to a VL domain sequence described herein (such as SEQ ID No.4), and even more preferably at least 65%, 70%, 75%, 80%, 85%, or even 90% identical to a VL domain sequence described herein (such as SEQ ID No.4), and is capable of specifically binding to human CD 39.

In certain embodiments, the anti-CD 39 antibody is a humanized antibody comprising a VH domain having human framework sequences associated with the CDRs of the VH domains set forth in SEQ ID nos. 29, 30 and 31 and the CDRs of the corresponding VL domains set forth in SEQ ID nos. 32, 33 and 34. The CDRs of the anti-CD 39 antibodies described herein are preferably identical to the CDRs described herein, but may vary by 1, 2, or 3 amino acids on each CDR, so long as the resulting antibody specifically binds human CD 39.

In certain embodiments, the heavy and light chains of the anti-CD 39 antibody have variable domains that may be encoded by nucleic acids that are identical to the VH and VL domain (respectively) encoding sequences described herein, such as those shown in SEQ ID No.1(VH) and SEQ ID No.3(VL), or that hybridize under stringent conditions, such as 6x sodium chloride/sodium citrate (SSC) at 45 ℃, and washes in 0.2 xSSC/0.1% SDS at 50 ℃ to 65 ℃.

In some embodiments, the anti-CD 39 antibodies are produced in rabbits, and the variable domains of the heavy and light chains of these antibodies are rabbit sequences, while the constant domains are human sequences. Exemplary sequences for the VH and VL domains of the rabbit anti-CD 39 antibody are:

in some embodiments, the anti-CD 39 antibody is produced in rabbits and then humanized by CDR grafting. Exemplary sequences of the VH and VL domains of the humanized rabbit anti-CD 39 antibody are:

in some embodiments, the anti-CD 39 antibodies provided herein promote: (i) stable immune complex formation when incubated with HCC1739BL cells, as characterized by less than 30% loss of the immune complex after 24 hours, optionally wherein the immune complex formation is detected by fluorescence intensity using a fluorescently labeled secondary antibody; (ii) complement Dependent Cytotoxicity (CDC) activity against CD39+ cells; (iii) antibody-mediated target endocytosis of CD39 on CD45+ immune cells; (iv) antibody-mediated target endocytosis of CD39 from tumor vascular endothelial destruction or collapse of the vasculoprostrial network in a tumor; (v) (optionally) a CD39 epitope (e.g., binding to one or more linear or conformational CD39 epitopes, such as selected from the group consisting of 1) IYLTDCMERAR, 2) LRMESEELADR, 3) RVKGPGISKFV, 4) DCMERAREVIPR, 5) LTDCMERAREVIPR, 6) SLSNYPFDFQGAR, 7) CRVKGPGISKF, 8) GAYGWITINYLLGKFSQK, 9) ILRDPCFHPGYKK and any combination thereof, such as RVKGPGISKFV and DCMERAREVIPR, LTDCMERAREVIPR and SLSNYPFDFQGAR or CRVKGPGISKF, GAYGWITINYLLGKFSQK, and/or ILRDPCFHPGYKK), that binds to a sequence having a sequence selected from the group of CD39 amino acid epitope sequences listed in figure 33; and/or (vi) (optionally) binds to CD39 in a manner that non-competes or only partially competes for binding to CD39 with monoclonal antibody clone a 1.

The representative anti-CD 39 antibody sequences described above according to sequence identification numbers correspond to the following:

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

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

SEQ ID No.3 (clone IG39-21 vL Domain nucleic acid sequence)

SEQ ID No.4 (clone IG39-21 vL Domain amino acid sequence)

SEQ ID No.5 (clone 9B6 vH Domain nucleic acid sequence)

SEQ ID No.6 (clone 9B6 vH Domain amino acid sequence)

SEQ ID No.7 (clone 9B6 vL Domain nucleic acid sequence)

SEQ ID No.8 (clone 9B6 vL Domain amino acid sequence)

SEQ ID No.9 (clone 8C11 vH Domain nucleic acid sequence)

SEQ ID No.10 (clone 8C11 vH Domain amino acid sequence)

SEQ ID No.11 (clone 8C11 vL Domain nucleic acid sequence)

SEQ ID No.12 (clone 8C11 vL Domain amino acid sequence)

SEQ ID No.13 (clone 8D8 vH Domain nucleic acid sequence)

SEQ ID No.14 (clone 8D8 vH Domain amino acid sequence)

SEQ ID No.15 (clone 8D8 vL Domain nucleic acid sequence)

SEQ ID No.16 (clone 8D8 vL Domain amino acid sequence)

SEQ ID No.17 (clone 9C10 vH Domain nucleic acid sequence)

SEQ ID No.18 (clone 9C10 vH Domain amino acid sequence)

SEQ ID No.19 (clone 9C10 vL Domain nucleic acid sequence)

SEQ ID No.20 (clone 9C10 vL Domain amino acid sequence)

SEQ ID No.21 (clone 65H5 vH Domain nucleic acid sequence)

SEQ ID No.22 (clone 65H5 vH Domain amino acid sequence)

SEQ ID No.23 (clone 65H5 vL Domain nucleic acid sequence)

SEQ ID No.24 (clone 65H5 vL Domain amino acid sequence)

SEQ ID No.25 (clone 2G12 vH Domain nucleic acid sequence)

SEQ ID No.26 (clone 2G12 vH Domain amino acid sequence)

SEQ ID No.27 (clone 2G12 vL Domain nucleic acid sequence)

SEQ ID No.28 (clone 2G12 vL Domain amino acid sequence)

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

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

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

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

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

SEQ ID No.34 (clone IG39-21 vL Domain CDR3 amino acid sequence)

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

SEQ ID No.36 (clone IG39-21 full-length vH chain amino acid sequence)

SEQ ID No.37 (clone IG39-21 full-Length vL chain nucleic acid sequence)

SEQ ID No.38 (clone IG39-21 full-Length vL chain amino acid sequence)

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

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

SEQ ID No.41

SEQ ID No.42

SEQ ID No.43

SEQ ID No.44

SEQ ID No.45

SEQ ID No.46

SEQ ID No.47

SEQ ID No.48

SEQ ID No.49

SEQ ID No.50

SEQ ID No.51

SEQ ID No.52

SEQ ID No.53

SEQ ID No.54

SEQ ID No.55

SEQ ID No.56

For use in human patients, it will be desirable to humanize these antibodies, replace the constant regions of the heavy and light chains with human constant regions, and replace the framework regions of the variable regions with human antibody framework regions. In some embodiments, the anti-CD 39 antibody or antigen-binding fragment thereof is a humanized form of a rabbit antibody.

In some embodiments, the anti-CD 39 antibody or antigen-binding fragment thereof comprises at least one heavy chain variable that is at least 60% identical to SEQ ID nos. 6, 10, 14, 18, 22, 26, 42, 46, 50, or 54, and even more preferably at least 65%, 70%, 75%, 80%, 85%, or even 90% identical to SEQ ID nos. 6, 10, 14, 18, 22, 26, 42, 46, 50, and 54, and is capable of specifically binding to human CD 39.

In some embodiments, the anti-CD 39 antibody or antigen-binding fragment thereof comprises at least one light chain variable that is at least 60% identical to SEQ ID No.8, 12, 16, 20, 24, 28, 44, 48, 52, or 56, and even more preferably at least 65%, 70%, 75%, 80%, 85%, or even 90% identical to SEQ ID No.8, 12, 16, 20, 24, 28, 44, 48, 52, or 56, and is capable of specifically binding to human CD 39.

In certain embodiments, the chimeric antibody is a humanized antibody. Typically, non-human antibodies are 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 HVRs, e.g., CDRs (or portions thereof), are derived from a non-human antibody and FRs (or portions thereof) are derived from a human antibody sequence. The humanized antibody optionally will also comprise at least a portion of a human constant region. In some embodiments, some FR residues in the humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., an antibody in which HVR residues are derived), e.g., to restore or improve antibody specificity or affinity.

In certain embodiments, the anti-CD 39 antibody is a humanized antibody comprising a VH domain having human framework sequences associated with the CDRs of a VH domain selected from SEQ ID nos. 6, 10, 14, 18, 22, 26, 42, 46, 50 or 54, and the CDRs of a 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 vary by 1, 2 or 3 amino acids on each CDR, so long as the resulting antibody specifically binds human CD 39.

Humanized antibodies and methods for making them are reviewed, for example, in Almagro and Fransson, front.biosci.13:1619-1633(2008), and further described, for example, in Riechmann et al, Nature 332:323-329 (1988); queen et al, Proc.nat' l Acad. Sci. USA 86:10029-10033 (1989); U.S. Pat. nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; kashmiri et al, Methods 36:25-34(2005) (description Specificity Determination Region (SDR) grafting); padlan, mol.Immunol.28:489-498(1991) (description "resurfacing"); dall' Acqua et al, Methods 36:43-60(2005) (description "FR shuffling"); and Osbourn et al, Methods 36:61-68(2005) and Klimka et al, Br.J. cancer,83: 252-.

Human framework regions that may be used for humanization include, but are not limited to: framework regions selected using the "best fit" approach (see, e.g., Sims et al, J.Immunol.151:2296 (1993)); the framework regions of consensus sequences of human antibodies derived from a particular subset 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 the framework regions derived from screening FR libraries (see, e.g., Baca et al, J. biol. chem.272:10678-10684(1997) and Rosok et al, J biol. chem.271:22611-22618 (1996)).

In certain embodiments, the anti-CD 39 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-.

For example, human antibodies can be prepared by administering an immunogen to a transgenic animal that has been modified to produce fully human antibodies or fully antibodies with human variable regions in response to antigenic challenge. Such animals typically contain all or a portion of the human immunoglobulin loci that replace endogenous immunoglobulin loci, or that are present extrachromosomally or randomly integrated into the chromosomes of the animal. In such transgenic mice, the endogenous immunoglobulin loci have typically been inactivated. For an overview of the methods used to obtain human antibodies from transgenic animals, see Lonberg, nat. Biotech.23:1117-1125 (2005). See also, for example, U.S. Pat. nos. 6,075,181 and 6,150,584, which describe the xenomose technology; U.S. patent No. 5,770,429 describing the HuMAB technique; U.S. patent No. 7,041,870, which describes the K-MMOUSE technique, and U.S. patent application publication No. US 2007/0061900, which describes the velomiuse technique). The human variable regions from intact antibodies produced by such animals may be further modified, for example by combination with different human constant regions.

Human antibodies can also be made by hybridoma-based methods. Human myeloma and mouse human heteromyeloma cell lines for producing human monoclonal antibodies have been described. (see, e.g., Kozbor J.Immunol.,133:3001 (1984); Brodeur et al, Monoclonal Antibody Production Techniques and Applications, pp.51 to 63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al, J.Immunol.,147:86 (1991)). Human antibodies produced via human B-cell hybridoma technology are also described in Li et al, proc.natl.acad.sci USA,103:3557-3562 (2006). Additional methods include those described, for example, in U.S. Pat. No. 7,189,826 (describing the production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue,26(4):265-268(2006) (describing human-human hybridomas). Human hybridoma technology (trioma technology) is also described in Vollmers and Brandleins, Histology and Histopathology,20(3): 927-.

Human antibodies can also be produced by isolating Fv clone variable domain sequences selected from phage, yeast or bacterial display libraries of human origin. Such variable domain sequences can then be combined with the desired human constant domains. Techniques for selecting human antibodies from antibody libraries are described below.

To illustrate, the anti-CD 39 antibodies encompassed by the invention can be isolated by screening combinatorial libraries for antibodies having a desired activity. For example, various methods are known in the art for generating phage or yeast display libraries and screening these libraries for antibodies having desired binding characteristics. These Methods are reviewed, for example, in Hoogenboom et al, Methods in Molecular Biology 178:1-37(O' Brien et al, eds., Human Press, Totowa, N.J.,2001) and further described, for example, in McCafferty et al, Nature 348: 552-; clackson et al, Nature 352: 624-; marks et al, J.mol.biol.222:581-597 (1992); marks and Bradbury, Methods in Molecular Biology 248:161-175(Lo eds., Human Press, Totowa, N.J., 2003); sidhu et al, J.mol.biol.338(2):299-310 (2004); lee et al, J.mol.biol.340(5): 1073-; fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-; and Lee et al, J.Immunol.methods 284(1-2):119-132 (2004).

As an example of a phage display method, VH and VL gene libraries are separately cloned by Polymerase Chain Reaction (PCR) and randomly recombined into phage libraries, which can then be screened against antigen-binding phage as described in Winter et al, Ann. Rev. Immunol.,12:433-455 (1994). Phage typically display antibody fragments, either as single chain fv (scfv) fragments or as Fab fragments. Libraries from immune sources provide high affinity antibodies to the immunogen without the need to construct hybridomas. Alternatively, the original library can be cloned (e.g., from humans) to provide a single source of antibody against a wide range of non-self antigens as well as also self antigens without any immunization, as described by Griffiths et al, EMBO J,12: 725-. Finally, the original library can also be prepared synthetically by cloning unrearranged V gene fragments from stem cells and using PCR primers containing random sequences to encode the highly variable CDR3 regions and rearrangement in vitro, as described by Hoogenboom and Winter, J.Mol.biol.,227:381-388 (1992). Patent publications describing human antibody phage libraries include, for example: U.S. Pat. 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.

Antibodies or antibody fragments isolated from a human antibody library are considered herein to be human antibodies or human antibody fragments.

Fc γ RIII binding can also be increased by methods according to the prior art, e.g., by modifying the amino acid sequence of the Fc portion of an antibody or the glycosylation of the Fc portion of an antibody (see, e.g., EP 2235061). In certain embodiments, the subject antibodies are produced by cells in which less than 50% of the oligosaccharide chains on the antibody, when glycosylated, contain alpha-1, 6-fucose. Typically, less than about 40%, less than about 30%, less than about 20%, less than about 10%, or less than 5%, or less than 1% of the oligosaccharide chains contain alpha-1, 6-fucose in the "low fucosylated" antibody preparation. Among the carbohydrates attached to the CH2 domain of IgG heavy chains, an "afucosylated" antibody lacks alpha-1, 6-fucose. Mori, K et al, Cytotechnology 55(2007)109 and Satoh M et al, Expert Opin Biol ther.6(2006)1161-1173 involved the FUT8 (alpha-1, 6-fucosyltransferase) gene knockout CHO line for the production of afucosylated antibodies.

IV.Expression vector

In certain embodiments, recombinant expression vectors are used to amplify and express DNA encoding the anti-CD 39 antibodies described herein. For example, the recombinant expression vector can be a replicable DNA construct having a DNA segment of synthetic or cDNA origin encoding a polypeptide chain of an anti-CD 39 antibody operably linked to suitable transcriptional and/or translational regulatory elements derived from a mammalian, microbial, viral, or insect gene. The transcriptional unit generally comprises the following assembly: (1) one or more genetic elements that have a regulatory role in gene expression, such as transcriptional promoters or enhancers, (2) structural or coding sequences that are transcribed into mRNA and translated into protein, and (3) appropriate transcriptional and translational initiation and termination sequences. Regulatory elements may include operator sequences to control transcription. The ability to replicate in a host is often conferred by the origin of replication, and a selection gene may additionally be incorporated that facilitates the recognition of transformants. DNA regions are "operably linked" when they are functionally related to each other. For example, if the DNA for the signal peptide is expressed as a precursor involved in secretion of the polypeptide, it is operably linked (secretory leader sequence) to the DNA for the polypeptide; a promoter is operably linked to a coding sequence if it controls the transcription of that sequence; or operably linked to a coding sequence if a ribosome binding site is placed so as to allow translation. In some embodiments, the structural element intended for use in a yeast expression system comprises a leader sequence that allows for extracellular secretion of the translated protein by the host cell. In other embodiments, when the recombinant protein is expressed in the absence of a leader or transporter sequence, it may include an N-terminal methionine residue. This residue can then optionally be cleaved from the expressed recombinant protein to provide the final product.

The choice of expression control sequences and expression vectors depends on the choice of the host cell. Various expression host/vector combinations may be employed. Expression vectors suitable for eukaryotic hosts include, for example, vectors containing expression control sequences from SV40, bovine papilloma virus, adenovirus and cytomegalovirus. Expression vectors suitable for use in bacterial hosts include known bacterial plasmids such as those from e.coli, including pCR1, pBR322, pMB9 and their derivatives, and a broader host range plasmid such as M13 and other filamentous single stranded DNA phages.

Suitable host cells (or proteins for use as targets) for expressing the polypeptide chain of the anti-CD 39 antibody include prokaryotes, yeast cells, insect cells, or higher eukaryotes under the control of a suitable 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 employed. Cloning and expression vectors suitable for use with bacterial, fungal, yeast and mammalian cell hosts are well known to those skilled in the art.

Various mammalian cell culture systems are used to express recombinant polypeptides. Expression of recombinant proteins in mammalian cells may be preferred, as such proteins are generally correctly folded, appropriately modified, and biologically functional. Examples of suitable mammalian host cell lines include COS-7 (of monkey kidney origin), L-929 (of murine fibroblast origin), C127 (of murine breast tumor origin), 3T3 (of murine fibroblast origin), CHO (of Chinese hamster ovary origin), HeLa (of human cervical cancer origin), BHK (of hamster kidney fibroblast origin) and HEK-293 (of human embryonic kidney origin) cell lines and variants thereof. Mammalian expression vectors may contain non-transcriptional elements such as origins of replication, promoters and enhancers suitable for linkage to the gene to be expressed, and other 5 'or 3' flanking non-transcribed and 5 'or 3' non-translated sequences such as essential ribosome binding sites, polyadenylation sites, splice donor and acceptor sites, and transcriptional termination sequences.

Expression of recombinant proteins in insect cell culture systems (e.g., baculoviruses) also provides a reliable method for producing correctly folded and biologically functional proteins. Baculovirus systems for the production of 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 comprising a variable region that is at least 60% identical to SEQ ID No.1, and even more preferably at least 65%, 70%, 75%, 80%, 85%, or even 90% identical to SEQ ID No.1, and capable of specifically binding to human CD 39.

In certain embodiments, the polynucleotide comprises a polynucleotide encoding an antibody heavy chain comprising a variable region that is at least 60% identical to SEQ ID No.2, and even more preferably at least 65%, 70%, 75%, 80%, 85%, or even 90% identical to SEQ ID No.2, and capable of specifically binding to human CD 39.

V.Encoded anti-CD 39 antibodies for in vivo delivery

The therapeutic vector used to deliver the coding sequence of the anti-CD 39 antibody to be expressed in the patient may be viral, non-viral, or physical. See, for example, Rosenberg et al, Science,242: 1575-. A discussion of methods and compositions for use in gene therapy includes Eck et al, Goodman & Gilman's The pharmaceutical Basis of Therapeutics, ninth edition, compiled by Hardman et al, McGraw-Hill, New York, (1996), Chapter 5, pages 77 to 101; wilson, Clin. exp. Immunol.107 (supplement 1):31-32,1997; wivel et al, Hematology/Oncology Clinics of North America, Gene Therapy, S.L. Eck eds, 12(3):483-501, 1998; romano et al, Stem Cells,18:19-39,2000, and references cited therein. U.S. patent No. 6,080,728 also provides a discussion of various gene delivery methods and compositions. Routes of delivery include, for example, systemic administration and in situ administration. Well known viral delivery techniques include the use of adenovirus, retrovirus, lentivirus, foamy virus, herpes simplex virus, vaccinia virus and adeno-associated viral vectors.

a. Viral vectors

Preferred viral vectors are based on noncytopathic eukaryotic viruses in which a non-essential gene has been replaced with a nucleic acid construct carrying a nucleic acid sequence encoding an epitope and a targeting sequence. Preferred viruses for certain embodiments encompassed by the present invention are adenoviruses and adeno-associated viruses (AAV), which are double-stranded DNA viruses that have been approved for human use in gene therapy. In addition, preferred vectors for tolerance do not comprise immunostimulatory sequences.

Adenoviral vectors

One illustrative 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 comprising adenoviral sequences sufficient to (a) support the packaging construct and (b) express in sense or antisense orientation the polynucleotide into which it has been cloned. Of course, in the context of antisense constructs, expression does not require that the gene product be synthetic. In a specific embodiment, the delivery vector involves the commercially available ORF for cytochrome b5 reductase 3(CYB5R3), transcript variant 1 in adenoviral vector pAd, with a C-terminal Flag and a His tag, (gene Biosciences product code AH 889428). WIPO patent application WO/2015/050364 also teaches vectors having expression constructs comprising the Cyb5r3 gene.

Adenoviral vectors are highly immunogenic and therefore are not preferred for administration to induce tolerance by presenting antigen or in the case of autoimmune disease. However, these vectors can be used to induce immunity, e.g., for the treatment of infectious diseases and the like, including, for example, influenza, HBV, HCV, and HIV.

Adeno-associated virus vectors (AAV)

AAV is a good choice for delivery vehicles due to its safety, i.e., it is not genetically engineered (recombined) to integrate into the host genome. Likewise, AAV is not pathogenic and is not associated with any disease. Removal of viral coding sequences minimizes the immune response to viral gene expression, and thus, rAAV does not elicit an inflammatory response. According to a specific embodiment, an AAV vector comprising an epitope sequence comprising a nucleic acid construct described herein is suitable for transducing an APC.

Typically, a viral vector comprising an epitope comprising the nucleic acid construct is assembled from a polynucleotide encoding the desired epitope, appropriate regulatory elements, and elements necessary for expression of the epitope to mediate cell transduction. In one embodiment, an adeno-associated virus (AAV) vector is employed. In a more specific embodiment, the AAV vector is AAV1, AAV6, or AAV 8.

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

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

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

Retroviral vectors

In certain embodiments, the viral vector may be a retroviral vector. A "retrovirus" is a virus having an RNA genome. In particular embodiments, the retroviral vector contains all of the cis-acting sequences necessary for packaging and integration of the viral genome, i.e., (a) Long Terminal Repeats (LTRs) or portions thereof located at each end of the vector; (b) primer binding sites for negative and positive strand DNA synthesis; and (c) a packaging signal necessary for incorporation of the genomic RNA into the virion. For more details on retroviral vectors see Boesen et al, 1994, Biotherapy 6: 291-302; clowes et al, 1994, J.Clin.invest.93: 644-; kiem et al, 1994, Blood 83: 1467-; salmonos 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 Genetics and Devel.3: 110-114.

"Gamma retrovirus" refers to a genus of the family Retroviridae. Exemplary gamma retroviruses include mouse stem cell virus, murine leukemia virus, feline sarcoma virus, and avian reticuloendotheliosis virus.

Widely used retroviral vectors include those based on murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), Simian Immunodeficiency Virus (SIV), Human Immunodeficiency Virus (HIV) and combinations thereof (see, e.g., Buchscher et al, J.Virol.66:2731-2739, 1992; Johann et al, J.Virol.66:1635-1640, 1992; Sommerfelt et al, Virol.176:58-59,1990; Wilson et al, J.Virol.63:2374-2378, 1989; Miller et al, J.Virol.65:2220-2224, 1991; and PCT/US 94/05700).

Lentiviral vectors refer to the genus of retrovirus that infects both dividing and non-dividing cells and typically produces high viral titers. Several examples of lentiviruses include HIV (human immunodeficiency virus: including type 1 HIV and type 2 HIV); equine infectious anemia virus; feline Immunodeficiency Virus (FIV); bovine Immunodeficiency Virus (BIV); and Simian Immunodeficiency Virus (SIV).

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

Non-cellular viruses include retroviruses (e.g., lentiviruses), whose life cycle involves reverse transcription of genomic viral RNA into DNA, and subsequent integration of the provirus into host cell DNA. Retroviruses have been approved for human gene therapy trials. Most useful are those retroviruses that are replication defective (i.e., capable of targeted synthesis of the desired protein, but incapable of producing infectious particles). Such genetically altered retroviral expression vectors have general utility for the efficient transduction of genes in vivo. Standard protocols for the production of replication-defective retroviruses (including the steps of incorporating exogenous genetic material into a plasmid, transfecting packaging cells lined with the plasmid, producing recombinant retrovirus from 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.

The retroviral genome contains three genes gag, pol and env which encode the capsid protein, polymerase and envelope components, respectively. The sequence found upstream from the gag gene contains a signal for packaging the genome into the virion. Retroviral vectors are gene transfer plasmids in which heterologous nucleic acid residues are located between two retroviral LTRs. Retroviral vectors typically contain appropriate packaging signals that allow the retroviral vector, or RNA transcribed using the retroviral vector as a template, to be packaged into a viral virion in an appropriate packaging cell line (see, e.g., U.S. Pat. No. 4,650,764). These two Long Terminal Repeat (LTR) sequences are present at the 5 'and 3' ends of the viral genome. These contain strong promoter and enhancer sequences and also need to be integrated into the host cell genome (Coffin, 1990). To construct a retroviral vector, a nucleic acid encoding one or more target oligonucleotide or polynucleotide sequences is inserted into the viral genome in place of certain viral sequences to produce a replication-defective virus. Also included are episomal or non-integrative forms of retroviral vectors based on lentiviruses (e.g., one type of retrovirus).

Lentiviral vectors are useful when stable expression is desired, but lentiviral vectors can be immunogenic and may have other undesirable effects. Thus, although lentiviral vectors are convenient for research, care should be taken when using them for human administration, especially where tolerance is to be induced rather than immunity. Although mRNA electroporation is safer, lentiviruses are suitable for engineering T cells or dendritic cells or other antigen presenting cells ex vivo for cancer therapy. However, two recent advances have made the use of lentiviruses safer and more clinically transformable. First, the co-expression of a suicide gene together with an antigen, the product of which becomes functional when the drug is administered. A typical example is herpes simplex virus thymidine kinase (HSV-Tk). Cells expressing these genes metabolize the drug ganciclovir (ganciclovir) into cytotoxic products that induce cell death. Thus, in the event that some of the transduced cells become malignant, they can be eradicated. There is about a dozen such systems (Duarte et al, Cancer Letters,324: 160-. Second, non-integrating lentiviral vectors are now being developed, and therefore they are non-oncogenic (Nightingale et al, 2006, mol. ther.,13: 1121-. These methods may be used with the present invention, at the discretion of those skilled in the art.

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

Additional retroviral delivery systems that can be readily adapted to deliver transgenes encoding anti-CD 39 antibody agents include, by way of illustration only, published PCT applications WO/2010/045002, WO/2010/148203, WO/2011/126864, WO/2012/058673, WO/2014/066700, WO/2015/021077, WO/2015/148683, WO/2017/040815, the specification and figures of each of which are incorporated herein by reference.

In certain embodiments, the retrovirus is a retrovirus having recombinant replication capability comprising: a nucleic acid sequence encoding a retroviral GAG protein; a nucleic acid sequence encoding a retroviral POL protein; a nucleic acid sequence encoding a retroviral envelope; and an oncoretroviral polynucleotide sequence comprising Long Terminal Repeat (LTR) sequences at the 5 'and 3' ends of the oncoretroviral (oncoretroviral) polynucleotide sequence; a cassette comprising an Internal Ribosome Entry Site (IRES) operably linked to the coding sequence of an anti-CD 39 antibody agent, wherein 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 in target cells.

In certain embodiments, the retrovirus is a retrovirus having recombinant replication capability comprising: retroviral GAG proteins; a retroviral POL protein; a retroviral envelope; a retroviral polynucleotide comprising a Long Terminal Repeat (LTR) sequence 3 'to the retroviral polynucleotide sequence, a promoter sequence 5' to the retroviral polynucleotide, said promoter suitable for expression in a mammalian cell, a gag nucleic acid domain, a pol nucleic acid domain and an env nucleic acid domain; a cassette comprising an anti-CD 39 antibody agent coding sequence operably linked to a heterologous polynucleotide, wherein the cassette is located at and operably linked to a 5' to 3' LTR, and 3' to an env nucleic acid domain encoding a retroviral envelope; and reverse transcription, packaging and integration of the necessary cis-acting sequences in the target cell.

In certain preferred embodiments of the recombinant replication-competent retrovirus, the envelope is selected from one of an ampholytic, a pleiotropic, a xenobiotic, 10a1, GALV, baboon endogenous virus, RD114, rhabdovirus, alphavirus, measles, or influenza virus envelope.

In certain preferred embodiments of the retrovirus having recombinant replication capability, the retroviral polynucleotide sequence is engineered from a virus selected from the group consisting of: murine Leukemia Virus (MLV), Moloney murine leukemia virus (MoMLV), feline leukemia virus (FeLV), baboon endogenous retrovirus (BEV), porcine endogenous virus (PERV), feline-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 retrovirus having recombinant replication capacity, the retrovirus is a gammaretrovirus.

In certain preferred embodiments of the recombinant replication competent retrovirus, a second cassette is present comprising the coding sequence for a second therapeutic protein, such as another checkpoint inhibitor polypeptide, a co-stimulatory polypeptide and/or an immunostimulatory cytokine (as examples only), e.g. downstream of the cassette. In certain examples, the second cassette can comprise an Internal Ribosome Entry Site (IRES) or a mini-promoter or polIII promoter operably linked to the coding sequence of the second therapeutic protein.

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

Other viral vectors as expression constructs

Other viral vectors may be used as expression constructs in the present invention for delivery of oligonucleotide or polynucleotide sequences to host cells. Vectors derived from viruses such as vaccinia virus, poliovirus, and herpes virus may be used. They provide several attractive features for various mammalian cells. Also included are hepatitis B viruses.

b. Non-viral vectors

Plasmid vector

Other vectors include plasmid vectors. Plasmid vectors are widely described in the art and are well known to those skilled in the art. See, for example, Sambrook et al, 1989, cited above. Over the past several years, plasmid vectors have been used as DNA vaccines for the in vivo delivery of antigen-encoding genes to cells. They are particularly advantageous in this regard because they do not have the same safety issues as many of the viral vectors. However, these plasmids, which have promoters compatible with the host cell, can express peptide epitopes encoded by nucleic acids within the plasmid. Other plasmids are well known to those of ordinary skill in the art. In addition, plasmids can be custom designed using restriction enzymes and ligation reactions to remove and add specific fragments of DNA. Plasmids can be delivered by a variety of parenteral, mucosal and topical routes. For example, the DNA plasmid may be injected intramuscularly, intradermally, subcutaneously, or otherwise. It can also be administered by intranasal spray or drops, rectal suppository and orally. It can also be administered into the epidermal or mucosal surface using a gene gun. The plasmid can be administered in aqueous solution, dried on gold particles, or combined with another DNA delivery system (including but not limited to liposomes, dendrimers, cochlea, and microencapsulation).

Thus, in one aspect, a plasmid is provided for expressing an epitope-containing nucleic acid construct comprising an expression cassette; also known as transcription units. When the plasmid is placed in an environment suitable for expression of the epitope, the transcription unit will express a polynucleotide that includes the sequence encoding the epitope, the ETS and MHCII promoter sequences or the sequences encoding the epitope and secretion signal sequences, as well as any other sequences encoded in the construct. The transcription unit includes a transcription control sequence that is transcriptionally linked to a cellular immune response element coding sequence. The transcriptional control sequence may include a promoter/enhancer sequence, such as a Cytomegalovirus (CMV) promoter/enhancer sequence. However, one skilled in the art will know that various other promoter sequences suitable for expression in eukaryotic cells are known and may be equally 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.

In certain embodiments, sequences encoding the desired epitope and targeting sequences can be cloned into an expression plasmid containing regulatory elements for transcription, translation, RNA stability, and replication (i.e., including transcriptional control sequences). Such expression plasmids are well known in the art and one of ordinary skill in the art will be able to design appropriate expression constructs with polynucleotides comprising sequences encoding cellular immune response elements or fragments thereof in a manner that the cellular immune response elements are expressible. There are many examples of suitable expression plasmids in which a polynucleotide comprising a sequence may be cloned, such as pCI-neo, pUMV or pcDNA 3.

A large number of bacterial hosts carrying plasmids for expression of cellular immune response elements or fragments thereof may be fermented and the plasmids may be purified for subsequent use. Current human clinical trials using plasmids utilize this approach. Data management report of the recombinant DNA consultative Committee, Human Gene Therapy 6:535 and 548, 1994. Current DNA isolation methods known in the art include the removal of lipopolysaccharides (endotoxins), which are contaminants from the bacteria used to propagate the plasmid. This step is most preferred for use with tolerogenic DNA vaccines because endotoxins act as strong adjuvants and can produce undesirable immune stimulation.

The purpose of the plasmid is to efficiently deliver the nucleic acid sequence to a cell or tissue and to express the therapeutic epitope in the cell or tissue. In particular, the purpose of the plasmid may be to achieve high copy number, avoid potential causes of plasmid instability and provide a means of plasmid selection. With respect to expression, a nucleic acid cassette contains the necessary elements for expressing the nucleic acid within the cassette. Expression includes efficient transcription of the inserted gene, nucleic acid sequence or nucleic acid cassette with a plasmid. The expression product may be a protein, polypeptide or RNA. The nucleic acid cassette may contain nucleic acid sequences. Expression of the nucleic acid may be continuous or regulated.

Micro-ring

Embodiments of the nucleic acid constructs described herein can be processed in the form of minicircle DNA. Minicircle DNA belongs to the class of small (2 to 4kb) circular plasmid derivatives that have been released from all prokaryotic vector parts. Since the minicircle DNA vectors do not contain bacterial DNA sequences, they are unlikely to be considered foreign and destroyed. (typical transgene delivery methods involve plasmids containing foreign DNA). Thus, these vectors can be expressed for a longer period of time (in the order of weeks or months) than conventional plasmids (days to weeks). The smaller size of the micro-loops also expands their clonality and facilitates their delivery into the cell. 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); 218, 1526-: 14062(2015) doi: 10.1038/mtm.2014.62. More information about the micro-loops is provided in Chen Z Y, He C Y, ehrhhardt a, Kay M a. mol ther. 9 months 2003; 8(3) 495-500, and the minicircle DNA vector achieves sustained expression reflected by active chromatin and transcription levels. Gracey Maniar L E, Maniar J M, Chen Z Y, Lu J, Fire a Z, Kay M a. mol ther.2013, 1 month; 21(1):131-8.

As an initial step in the method of ultimately obtaining expression of the product encoded by the nucleic acid, to achieve uptake of the nucleic acid by the cell. The uptake of nucleic acids by cells depends on a number of factors, one of which is the length of time that the nucleic acid is in proximity to the cell surface. For example, following intramuscular (i.m.) administration of plasmid DNA in buffer, if the muscle is massaged, a significant reduction in Gene expression is observed, probably due to leakage of DNA from the muscle either directly or via lymphatic vessels (Human Gene Therapy 4: 151-159; 1993). Thus, it may be desirable to formulate nucleic acids at a rate that delays nucleic acid diffusion or by compounds that are carried away from the site where uptake of the nucleic acid by the cell is desired. In addition, these compounds may be suitable for administration to an organism by means such as injection, while maintaining or restoring the physical characteristics necessary to increase nucleic acid uptake by the cells.

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

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

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 methods described in Woffendin et al, proc.natl.acad.sci.usa 91(24):11581,1994; depositing a photopolymerizable hydrogel material or using ionizing radiation (see, e.g., U.S. patent nos. 5,206,152 and WO 92/11033); using a hand-held gene transfer particle gun (see, e.g., U.S. patent No. 5,149,655); and the use of ionizing radiation to activate the transferred gene (see, e.g., U.S. Pat. nos. 5,206,152 and WO 92/11033). The delivery device may also be biocompatible, and may also be biodegradable. The formulation preferably provides a relatively constant level of active ingredient release. On the other hand, a faster rate of immediate release after administration may be desired. The formulation of such compositions is well within the level of ordinary skill in the art using known techniques.

Physical methods to enhance delivery include electroporation (where a short pulse of high pressure carries the nucleic acid across the membrane), gene guns (where DNA is loaded onto gold particles and forced to infiltrate into the cell), sonoporation, magnetic transfection, hydrodynamic delivery, and the like, all of which are known to those skilled in the art. The DNA may also be encapsulated in liposomes, preferably cationic liposomes, or polymers that can interact with cell membranes and fuse or undergo endocytosis to effect transfer of the DNA into the cell (synthetic liposomes). The DNA may also form a complex with a polymer (polymeric complex) or with a dendrimer, which can directly release the load into the cytoplasm of the cell.

Illustrative carriers suitable for use in this aspect include the following microparticles: poly (lactide-co-glycolide), polyacrylate, latex, starch, cellulose, dextran, and the like. Other illustrative delayed release carriers include supramolecular biovectors comprising a non-liquid hydrophilic core (e.g., a cross-linked polysaccharide or oligosaccharide) 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 WO 94/20078, WO/94/23701 and WO 96/06638). The amount of active agent contained within 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.

Biodegradable microspheres (e.g., polylactic acid polyglycolide) can be used as carriers for the compositions. Suitable biodegradable microspheres are disclosed, for example, in U.S. patent nos. 4,897,268; 5,075,109; 5,928,647; 5,811,128, respectively; 5,820,883, respectively; 5,853,763, respectively; 5,814,344, respectively; 5,407,609 and 5,942,252. Modified hepatitis B nucleoprotein carrier systems such as those described in WO/9940934 and references cited therein will also be suitable for many applications. Another illustrative carrier/delivery system employs carriers comprising microparticle-protein complexes, such as those described in U.S. patent No. 5,928,647, which may have the additional benefit when used intratumorally to deliver coding sequences for anti-CD 39 antibody agents capable of inducing MHC I-restricted cytotoxic T lymphocyte responses targeted to a patient's tumor tissue.

The biodegradable polymeric nanoparticles facilitate transfer of non-viral nucleic acids to cells. Small (about 200nm), positively charged (about 10mV) particles are formed by the self-assembly of cations, hydrolytically degradable poly (. beta. -amino esters), and plasmid DNA.

The polynucleotide may also be administered to the cell by direct microinjection, temporary cell permeabilization (e.g., co-administration of a repressor and/or activator with a cell permeabilizer), fusion to a membrane translocation peptide, and the like.

In certain particular embodiments of the invention, the genetic construct is introduced into the target cell via electroporation. Electroporation involves exposing cells (or tissues) and DNA (or DNA complexes) to a high voltage discharge. In vivo electroporation has been successfully used in gene delivery techniques for the efficient delivery of plasmid DNA to many different tissues. Studies have reported administration of in vivo electroporation to deliver plasmid DNA to B16 melanoma and other tumor tissues. Systemic and local expression of the gene or cDNA encoded by the plasmid can be obtained by in vivo electroporation. In vivo electroporation is used to enhance plasmid DNA uptake in tumor tissue, resulting in expression within the tumor, and to deliver plasmids to muscle tissue, resulting in systemic expression of secreted proteins such as cytokines (see, e.g., US 8026223). Exemplary techniques, vectors and devices for in vivo electroporation of anti-CD 39 antibody agent transgenes into cells include PCT publications WO/2017/106795, WO/2016/161201, WO/2016/154473, WO/2016/112359 and WO/2014/066655.

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

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

The electrode arrays and methods described in U.S. Pat. No. 7,245,963 and U.S. patent publication No. 2005/0052630 are suitable for deep penetration not only into tissues such as muscle, but also into other tissues or organs. Because of the configuration of the electrode array, an injection needle (to deliver the selected biomolecules) is also inserted completely within the target organ, and the injection is applied perpendicular to the target tissue in the region pre-delineated by the electrodes.

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

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

Electroporation devices incorporating electrochemical impedance spectroscopy ("EIS") are also contemplated. These devices provide real-time information about the in vivo (in particular, the efficiency of the electroporation in the tumor), allowing conditions to be optimized. Examples of electroporation devices incorporating EIS can be found, for example, in WO2016/161201, which is hereby incorporated by reference.

Uptake of non-viral delivery vectors encompassed by the present invention can also be enhanced by plasma electroporation (also known as avalanche transfection). In short, the microsecond discharge generates cavitation microbubbles at the electrode surface. The combination of mechanical force generated by the ruptured microbubbles and the magnetic field acts to increase the efficiency of transport across the cell membrane compared to diffusion-mediated transport associated with conventional electroporation. Techniques for plasma electroporation are described in U.S. patent nos. 7,923,251 and 8,283,171. This technique can also be used for transformation of cells in vivo. Chaiberg et al (2006) Investigative opthalmology & Visual Science 47: 4083-; chaiberg et al, U.S. patent No. 8,101169 issued on 24/1/2012.

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

Cold plasma (i.e., non-thermal plasma) is generated by delivering a pulsed high voltage signal to a suitable electrode. The cold plasma device may take the form of a gas jet device or a Dielectric Barrier Discharge (DBD) device. Low temperature plasmas have attracted great enthusiasm and attention through their ability to provide plasmas at relatively low gas temperatures. Providing plasma at such temperatures is of interest for a variety of applications, including wound healing, antimicrobial methods, various other medical therapies, and sterilization. As previously indicated, cold plasma (i.e., non-thermal plasma) is generated by delivering a pulsed high voltage signal to a suitable electrode. The cold plasma device may take the form of a gas jet device, a Dielectric Barrier Discharge (DBD) device or a multi-frequency rich harmonic power supply.

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

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

In certain illustrative embodiments, the transgene construct encoding an anti-CD 39 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 footprint; a power source in electrical communication with the electrode, the power source configured to generate one or more electroporation signals to cells within the footprint; and a guide member coupled to the electrode, wherein the guide member is configured to adjust a coverage area of the electrode. At least a portion of the electrodes may be placed in a tapered arrangement within the applicator. The one or more electroporation signals may each be associated with an electric field. The device may also include a potentiometer coupled to the power source and the electrode. The potentiometer may be configured to substantially maintain an electric field within a predetermined range.

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

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 be combined to generate an electrical wave having a beat frequency. The first and second electrical signals may each have at least one of a unipolar waveform and a bipolar waveform. The first electrical signal may have a first frequency and a first amplitude. The second electrical signal may have a second frequency and a second amplitude. The first frequency may be different from or the same as the second frequency. The first amplitude may be different from or the same as the second amplitude.

In certain embodiments, the present invention provides a method for treating a subject having a tumor, the method comprising: injecting an effective dose of a plasmid encoding an anti-CD 39 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 200V/cm to about 1500V/cm over a pulse width of about 100 microseconds to about 20 milliseconds.

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

Lipid and polycationic molecules for delivery of nuclear constructs encoding anti-CD 39 antibodies

Lipid-mediated nucleic acid delivery and expression of foreign nucleic acids (including mRNA) both in vitro and in vivo has been very successful. Lipid-based non-viral formulations provide an alternative to adenovirus gene therapy. Current in vivo lipid delivery methods use subcutaneous, intradermal, intratumoral or intracranial injection. The development of lipid formulations improved the efficiency of gene transfer in vivo (see PCT application WO 98/07408). For example, a lipid formulation comprising an equimolar ratio of l, 2-bis (oleoyloxy) -3- (trimethylammonium) propane (DOTAP) and cholesterol can significantly enhance systemic in vivo gene transfer. DOTAP cholesterol lipid formulations form a unique structure called "sandwich liposomes". This formulation is reported as a "sandwich" DNA between an invaginated bilayer or "vase" structure. Beneficial features of these lipid structures include positive p, colloidal stabilization of cholesterol, two-dimensional nucleic acid packaging, and increased serum stability.

Cationic liposome technology is based on the ability of amphiphilic lipids with a positively charged head group and a hydrophobic lipid tail to bind to negatively charged DNA or RNA and form particles that generally enter the cell by endocytosis. Some cationic liposomes also contain neutral co-lipids, which are believed to enhance liposomal uptake by mammalian cells. Likewise, other polycations such as poly-l-lysine and polyethylene-imine complex with nucleic acids via charge interactions and help to condense DNA or RNA into nanoparticles, which are then substrates for endosome-mediated uptake. [8] Several of these cation-nucleic acid complexing technologies have evolved into potential clinical products, including complexes with plasmid dna (pdna), oligodeoxynucleotides, and various forms of synthetic RNA.

The nucleic acid constructs disclosed herein can be associated with polycationic molecules that function to enhance uptake into cells. Complexation of the nucleic acid constructs with polycationic molecules also aids in packaging the constructs so that they are reduced in size, which is believed to aid cellular uptake. Once inside the endosome, the complex dissociates due to the lower pH, and the polycationic molecule can disrupt the membrane of the endosome to facilitate the escape of DNA into the cytoplasm, which can then degrade. Preliminary data show that nucleic acid construct embodiments have enhanced uptake into SC compared to DC when complexed with the polycationic molecule polylysine or polyethyleneimine.

One example of a polycationic molecule suitable for complexing with a nucleic acid construct includes Cell Penetrating Peptide (CPP), examples include polylysine (described above), polyarginine, and Tat peptide. Cell Penetrating Peptides (CPPs) are small peptides that can bind to DNA and, once released, penetrate the cell membrane to facilitate escape of DNA from the endosome to the cytoplasm. Another example of a CPP involves a 27-residue chimeric peptide, called MPG, which has been shown in the previous paragraph to bind ss and ds oligonucleotides in a stable manner, thereby creating a non-covalent complex that protects nucleic acids from dnase degradation and efficiently delivers the oligonucleotides to cells in vitro (Mahapatro a et al, JNanobiotechnol,2011,9: 55). When examining different peptide to DNA ratios and 10:1 and 5:1 ratios (150 nm and 1um, respectively), the complexes formed small particles of approximately 150nm to 1 um. Another CPP involves a modified tetrapeptide [ tetrasysine containing a Guanidinocarbonylpyrrole (GCP) group (TL-GCP) ], which is reported to bind with high affinity to 6.2kb plasmid DNA, resulting in 700 to 900nm positively charged aggregates, Li et al, Agnew Chem Int el 2015; 54(10):2941-4). RNA can also be complexed with such polycationic molecules for in vivo delivery.

Other examples of polycationic molecules that can be complexed with the nucleic acid constructs described herein includeAnd In Vivo JET (Polypus-transformation, S.A., Illkirch, France) commercially available polycationic polymers.

VI.Methods of use and pharmaceutical compositions

The anti-CD 39 antibodies encompassed by the invention are suitable for use in a variety of applications, including but not limited to therapeutic treatment methods, such as immunotherapy against cancer. In certain embodiments, the anti-CD 39 antibodies described herein are useful for activating, promoting, increasing, and/or enhancing an immune response, inhibiting tumor growth, reducing tumor volume, inducing tumor regression, increasing tumor apoptosis, and/or reducing the tumorigenicity of a tumor. In certain embodiments, the anti-CD 39 antibodies encompassed by the invention are also suitable for use in immunotherapy against pathogens, such as viruses. In certain embodiments, the anti-CD 39 antibodies described herein are useful for inhibiting viral infection, reducing viral infection, increasing apoptosis of viral infected cells, and/or increasing killing of viral infected cells. The method of use may be in vitro, ex vivo or in vivo.

The present invention provides methods of activating an immune response in a subject using the anti-CD 39 antibodies described herein. In some embodiments, the invention provides methods of promoting an immune response in a subject using an anti-CD 39 antibody described herein. In some embodiments, the invention provides methods of increasing an immune response in a subject using an anti-CD 39 antibody described herein. In some embodiments, the invention provides methods for enhancing an immune response in a subject using an anti-CD 39 antibody described herein. In some embodiments, the activation, promotion, augmentation and/or enhancement of an immune response comprises increasing cell-mediated immunity. In some embodiments, the activation, promotion, increase and/or enhancement of an immune response comprises an increase in a Th 1-type response. In some embodiments, the activation, promotion, increase, and/or enhancement of an immune response comprises increasing T cell activity. In some embodiments, the activation, promotion, increase, and/or enhancement of an immune response comprises increasing CD4+ T cell activity. In some embodiments, the activation, promotion, increase, and/or enhancement of an immune response comprises increasing CD8+ T cell activity. In some embodiments, the activation, promotion, increase, and/or enhancement of an immune response comprises increasing CTL activity. In some embodiments, the activation, promotion, increase, and/or enhancement of an immune response comprises increasing NK cell activity. In some embodiments, the activation, promotion, increase, and/or enhancement of an immune response comprises increasing T cell activity and increasing NK cell activity. In some embodiments, the activation, promotion, increase, and/or enhancement of an immune response comprises increasing CU activity and increasing NK cell activity. In some embodiments, the activation, promotion, augmentation and/or enhancement of the immune response comprises inhibiting or reducing the suppressive activity of Treg cells. In some embodiments, the activation, promotion, increase, and/or enhancement of an immune response comprises inhibiting or reducing the inhibitory activity of MDSCs. In some embodiments, the activation, promotion, increase, and/or enhancement of an immune response comprises increasing the number of percentages of memory T cells. In some embodiments, the activation, promotion, augmentation and/or enhancement of an immune response comprises increasing long-term immune memory function. In some embodiments, the activation, promotion, augmentation, and/or enhancement of an immune response comprises increasing long-term memory. In some embodiments, activation, promotion, augmentation, and/or enhancement of an immune response does not include evidence of substantial side effects and/or immune-based toxicity. In some embodiments, the activation, promotion, augmentation, and/or enhancement of 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 antigen stimulation. In some embodiments, the antigenic stimulus is a tumor cell. In some embodiments, the antigenic stimulus is cancer. In some embodiments, the antigenic stimulus is a pathogen. In some embodiments, the antigenic stimulus is a virally infected cell.

In vivo and in vitro assays for determining whether an anti-CD 39 antibody modulates, activates or suppresses an immune response are known in the art or are being developed.

In certain embodiments of the methods described herein, the method of inducing persistent or long-term immunity that inhibits tumor recurrence or tumor regeneration comprises administering to a subject a therapeutically effective amount of an anti-CD 39 antibody.

In some embodiments, the tumor is a solid tumor. In certain embodiments, the tumor is a tumor selected from the group consisting of: colorectal, pancreatic, lung, ovarian, liver, breast, kidney, prostate, neuroendocrine, gastrointestinal, melanoma, cervical, bladder, glioblastoma, lymphoma, and head and neck tumors. In certain embodiments, the tumor is a colorectal 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 or islet tumor. In certain embodiments, the tumor is a melanoma tumor. In some embodiments, the tumor is a bladder or urothelial tumor.

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

In some embodiments, the tumor expresses or overexpresses a tumor antigen targeted by an anti-CD 39 antibody (such as a bispecific agent comprising an antigen binding site that specifically binds the tumor antigen).

The invention further provides a method for treating cancer in a subject, the method comprising administering to the subject a therapeutically effective amount of an anti-CD 39 antibody described herein. In some embodiments, the anti-CD 39 antibody inhibits or reduces the growth of a cancer.

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-CD 39 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 removed a tumor.

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

The present invention provides compositions comprising an anti-CD 39 antibody described herein. The invention also provides pharmaceutical compositions comprising the anti-CD 39 antibodies described herein and a pharmaceutically acceptable vehicle. In some embodiments, the pharmaceutical composition is suitable for use in immunotherapy. In some embodiments, the pharmaceutical composition is suitable for use in immunooncology. In some embodiments, the composition is suitable for inhibiting tumor growth. In some embodiments, the pharmaceutical composition is suitable for inhibiting tumor growth in a subject (e.g., a human patient). In some embodiments, the composition is suitable for use in the treatment of cancer. In some embodiments, the pharmaceutical composition is suitable for treating cancer in a subject (e.g., a human patient).

Formulations are prepared for storage and use by combining the purifying agents encompassed by the present invention with a pharmaceutically acceptable vehicle (e.g., carrier or excipient). Pharmaceutically acceptable carriers, excipients and/or stabilizers are generally considered by those skilled in the art as inactive ingredients of the formulation or pharmaceutical composition.

In some embodiments, the anti-CD 39 antibody is lyophilized and/or stored in lyophilized form. In some embodiments, a formulation comprising an anti-CD 39 antibody described herein is lyophilized.

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

The pharmaceutical compositions encompassed by the present invention can be administered in a number of ways for local or systemic treatment. Administration can be topical, by means of epidermal or transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders; pulmonary, by inhalation or insufflation of powders or aerosols, including by nebulizer, intratracheal, and intranasal; orally taking; or parenterally, including intravenously, intraarterially, intratumorally, subcutaneously, intraperitoneally, intramuscularly (e.g., by injection or infusion), or intracranially (e.g., intrathecally or intracerebroventricularly).

The therapeutic formulation may be in unit dosage form. Such formulations include tablets, pills, capsules, powders, granules, solutions or suspensions in aqueous or non-aqueous media, or suppositories. In solid compositions such as tablets, the principal active ingredient is mixed with a pharmaceutical carrier. Conventional tableting ingredients include corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate or 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 non-toxic pharmaceutically acceptable salts thereof. The solid preformulation composition is then subdivided into unit dosage forms of the type described above. Tablets, pills, etc. of the formulation or composition may be coated or otherwise compounded to provide a dosage form affording the advantage of prolonged action. For example, the tablet or pill can comprise an inner composition covered by an outer component. In addition, the two components may be separated by an enteric layer that acts to resist disintegration and allows the inner component to pass intact through the stomach or to be delayed in release. A variety of materials may be used for such enteric layers or coatings, such materials including a number of polymeric acids and mixtures of polymeric acids with such materials as shellac, cetyl alcohol and cellulose acetate.

anti-CD 39 antibodies may also be embedded in microcapsules. Such microcapsules are prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin microcapsules and poly- (methylmethacylate) microcapsules, respectively, 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, 22 th supplementary edition, 2012, Pharmaceutical Press, London.

In certain embodiments, the pharmaceutical formulation comprises an anti-CD 39 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 with a lipid composition comprising lecithin, cholesterol, and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes can be extruded through filters of defined pore size to produce liposomes of desired diameter.

In certain embodiments, sustained release formulations comprising the anti-CD 39 antibody can be produced. Suitable examples of sustained release formulations include semipermeable matrices of solid hydrophobic polymers containing the anti-CD 39 antibody, wherein the matrices are in the form of shaped articles (e.g., films or microcapsules). Examples of sustained release matrices include polyesters, hydrogels such as poly (2-hydroxyethyl-methacrylate) or poly (vinyl alcohol), polylactic acid, copolymers of L-glutamic acid and 7 ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as LUPRON DEPOT (injectable microspheres comprising lactic acid-glycolic acid copolymer and leuprolide acetate), sucrose acetate isobutyrate, and poly D- (-) -3-hydroxybutyric acid.

In certain embodiments, the method or treatment comprises administering at least one additional immune response stimulating agent in addition to the anti-CD 39 antibody. In some embodiments, the additional immune response stimulating agent includes, but is not limited to, colony stimulating factors (e.g., granulocyte-macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M-CSF), granulocyte colony stimulating factor (G-CSF), Stem Cell Factor (SCF)), interleukins (e.g., IL-1, IL2, IL-3, IL-7, IL-12, IL-15, IL-18), checkpoint inhibitors, antibodies that block immunosuppressive functions (e.g., anti-CTLA-4 antibodies, anti-CD 28 antibodies, anti-CD 3 antibodies), toll-like receptors (e.g., TLR4, TLR7, 9), or members of the B7 family (e.g., CD80, CD 86). Additional immune response stimulating agents may be administered prior to, concurrently with, and/or after administration of the anti-CD 39 antibody. Also provided are pharmaceutical compositions comprising an anti-CD 39 antibody and an immune response stimulating agent. In some embodiments, the immune response stimulating agent comprises 1, 2, 3, or more immune response stimulating agents.

In certain embodiments, the method or treatment comprises administering at least one additional therapeutic agent in addition to the anti-CD 39 antibody. The additional therapeutic agent may be administered prior to, concurrently with, and/or after administration of the anti-CD 39 antibody. Also provided are pharmaceutical compositions comprising an anti-CD 39 antibody and an additional therapeutic agent. In some embodiments, the at least one additional therapeutic agent comprises 1, 2, 3, or more additional therapeutic agents.

Although this is not required, combination therapies using two or more therapeutic agents typically use agents that act through different mechanisms of action. Combination therapy with agents having different mechanisms of action may produce additive or synergistic effects. Combination therapy may allow for lower doses of each agent compared to the doses of each agent used in monotherapy, thereby reducing toxic side effects and/or increasing the therapeutic index of the anti-CD 39 antibody. Combination therapy can reduce the likelihood of development of drug resistant cancer cells. In some embodiments, the combination therapy comprises a therapeutic agent that affects an immune response (e.g., enhances or activates the response) and a therapeutic agent that affects (e.g., inhibits or kills) a tumor/cancer cell.

In some embodiments of the methods described herein, the combination of the anti-CD 39 antibody and at least one additional therapeutic agent produces additive or synergistic results. In some embodiments, the combination therapy results in an increase in the therapeutic index of the anti-CD 39 antibody. In some embodiments, the combination therapy results in an increase in the therapeutic index of the additional therapeutic agent. In some embodiments, the combination therapy results in reduced toxicity and/or side effects of the anti-CD 39 antibody. In some embodiments, the combination therapy results in reduced toxicity and/or side effects of the additional therapeutic agent.

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

Therapeutic agents that may be administered in combination with the anti-CD 39 antibodies described herein include chemotherapeutic agents. Thus, in some embodiments, the methods or treatments involve administering an anti-CD 39 antibody in combination with a chemotherapeutic agent or in combination with a mixture of chemotherapeutic agents. Treatment with the anti-CD 39 antibody can occur before, concurrently with, or after administration of chemotherapy. Combined administration may include co-administration, either in a single pharmaceutical formulation or using different formulations, or sequential administration in any order, but generally over a period of time, such that all active agents can exert their biological activities simultaneously. The preparation and schedule of administration of such chemotherapeutic agents may be used according to the manufacturer's instructions or as determined empirically by the skilled practitioner. The preparation and schedule of administration of this Chemotherapy is also described in The Chemotherapy Source Book, 4 th supplementary edition, 2008, ed.c. perry, Lippincott, Williams & Wilkins, philiadelphia, Pa.

Chemotherapeutic agents suitable for use in the present invention include, but are not limited to, alkylating agents such as thiotepa and Cyclophosphamide (CYTOXAN); alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, midodopa, and ulidopa; ethyleneimine and methylmelamine including hexamethylmelamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolmelamine; nitrogen mustards such as chlorambucil, cyclophosphamide, estramustine, ifosfamide, mechlorethamine oxide hydrochloride, melphalan, neonebixin, benzene mustard cholesterol, prednimustine, trofosfamide, uracil mustard; nitrosoureas such as carmustine, chlorourethrin, fotemustine, lomustine, nimustine, ramustine; antibiotics, such as aclacinomycin, actinomycin, antrocin, azaserine, bleomycin, actinomycin C, calicheamicin, carminomycin (caminomycin), carcinomycin, tryptomycin, dactinomycin, daunomycin, ditobicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, sisomicin, mitomycin, mycophenolic acid, norubicin, olivomycin, pelomycin, posomycin, puromycin, doxorubicin, streptonigrin, streptozotocin, tubercidin, ubenimex, cistatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytosine arabinoside (cytosine arabine), dideoxyuridine, deoxyfluorouridine, enocitabine, fluorouridine, 5-FU; androgens such as testosterone carprofonate (calusterone), triamcinolone propionate (dromostanolone propionate), epithioandrostanol (epithiostanol), mepiquat (mepiquitane), testolactone (testolactone); anti-adrenaline such as aminoglutethimide, mitotane, trostane; folic acid replenisher such as folinic acid; acetic acid glucurolactone; an aldehydic phosphoramide glycoside; (ii) aminolevulinic acid; amsacrine; betrebuche; a bisantrene group; edatrexae; defu famine; colchicine; diazaquinone; eflornithine; ammonium etiolate; etoglut; gallium nitrate; a hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamol (mopidamol); diamine nitracridine (nitrarine); pentostatin; methionine; pirarubicin; podophyllinic acid; 2-ethyl hydrazide; procarbazine; PSK; lezoxan; umirolium (sizofuran); a germanium spiroamine; alternarionic acid; a tri-imine quinone; 2,2' -trichlorotriethylamine; uratan; vindesine; dacarbazine; mannomustine; dibromomannitol; dibromodulcitol; pipobroman; adding the star of tussingo; cytarabine (Ara-C); taxanes, such as paclitaxel (TAXOL) and docetaxel (TAXOTERE); chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine (navelbine); mitoxantrone hydrochloride; (ii) teniposide; daunomycin; aminopterin; ibandronate; CPT 11; topoisomerase inhibitor RFS 2000; difluoromethyl ornithine (DMFO); retinoic acid; an epothilone; capecitabine (XELODA); and a pharmaceutically acceptable salt, acid or derivative of any of the foregoing. Chemotherapeutic agents also include anti-hormonal agents that act to modulate or inhibit hormonal effects on tumors, such as anti-estrogens including, for example, tamoxifen, raloxifene, aromatase inhibiting 4(5) -imidazole, 4-hydroxyttamoxifen, trovaxifen, kexifene, LY117018, onapristone, and toremifene (FARESTON); and antiandrogens such as flutamide, nilutamide, bicalutamide, leuprolide and goserelin; and a pharmaceutically acceptable salt, acid or derivative of any of the foregoing. In certain embodiments, the additional therapeutic agent is cisplatin. In certain embodiments, the additional therapeutic agent is carboplatin.

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

In certain embodiments, the chemotherapeutic agent is an antimetabolite agent. Antimetabolites are chemical substances that are structurally similar to metabolites required for normal biochemical reactions, but differ enough 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, raltitrexed, pemetrexed, tegafur, cytosine arabinoside, thioguanine, 5-azacytidine, 6-mercaptopurine, azathioprine, 6-thioguanine, pentostatin, fludarabine phosphate (fludarabine phosphate), and cladribine (cladribine), as well as pharmaceutically acceptable salts, acids, or derivatives of any of these.

In certain embodiments of the methods described herein, the chemotherapeutic agent is an antimitotic agent, including but not limited to agents that bind tubulin. In some embodiments, the agent is a taxane. In certain embodiments, the agent is paclitaxel or docetaxel, or a pharmaceutically acceptable salt, acid or derivative of paclitaxel or docetaxel. In certain embodiments, the agent is paclitaxel (TAXOL), docetaxel (TAXOTERE), albumin-bound paclitaxel (nab-paclitaxel; ABRAXANE), DHA-paclitaxel, or PG-paclitaxel. In certain alternative embodiments, the antimitotic agent comprises a vinca alkaloid, such as vincristine, vinblastine, vinorelbine, or vindesine, or a pharmaceutically acceptable salt, acid, or derivative thereof. In some embodiments, the anti-mitotic agent is an inhibitor of kinesin Eg5 or an inhibitor of a mitotic kinase such as Aurora a or Plk 1.

In certain embodiments of the methods described herein, it is expected that the subject anti-CD 39 antibodies will have a greater combined effect (perhaps even a synergistic effect) with those chemotherapeutic agents that induce the release of ATP in tumors and/or cause upregulation of CD39 or CD73 within tumors. There is a wide range of chemotherapeutic agents that cause the release of ATP into the extracellular space because they induce tumor cell death, such as, but not limited to, anthracyclines (such as doxorubicin, daunorubicin, epirubicin, and idarubicin), platinum-based drugs (such as cisplatin, carboplatin, and oxaliplatin), and proteasome inhibitors (such as bortezomib). Radiotherapy and photodynamic therapy (PDT) can also lead to upregulation of ATP release and/or intra-tumor levels of CD39 and/or CD 73.

In some embodiments of the methods described herein, the additional therapeutic agent comprises an agent such as a small molecule. For example, treatment may involve administering an anti-CD 39 antibody in combination with a small molecule that acts as an inhibitor against tumor-associated antigens, including but not limited to EGFR, HER2(ErbB2), and/or VEGF. In some embodiments, the anti-CD 39 antibody is administered in combination with a protein kinase inhibitor selected from the group consisting of: gefitinib (IRESSA), erlotinib (TARCEVA), sunitinib (sunitinib) (SUTENT), lapatinib (lapatanib), vandetanib (vandetanib) (ZACTIMA), AEE788, CI-1033, cediranib (cediranib) (RECENTIN), sorafenib (NEXAVAR) and pazopanib (pazopanib) (GW 786034B). In some embodiments, the additional therapeutic agent comprises an mTOR inhibitor.

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.

In some embodiments of the methods described herein, the additional therapeutic agent comprises a biomolecule, such as an antibody. For example, treatment may involve administering an anti-CD 39 antibody in combination with an antibody directed against a tumor-associated antigen, including but not limited to antibodies that bind EGFR, HER2/ErbB2, and/or VEGF. In certain embodiments, the additional therapeutic agent is an antibody specific for a cancer stem cell marker. In some embodiments, the additional therapeutic agent is an antibody that binds 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 to an angiogenesis inhibitor (e.g., an anti-VEGF or VEGF receptor antibody). In certain embodiments, the additional therapeutic agent is bevacizumab (AVASTIN), ramucirumab (ramucirumab), trastuzumab (trastuzumab) (HERCEPTIN), pertuzumab (pertuzumab) (OMNITARG), panitumumab (vectib), nimotuzumab (nimotuzumab), zalutumumab (zalutumumab), or cetuximab (ERBITUX).

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. In some embodiments, the additional therapeutic agent is an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-CTLA-4 antibody, an anti-LAG-3 antibody, an anti-TIM-3 antibody, an anti-TIGIT antibody, and/or an anti-Siglec-15 antibody.

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

PD-1 antagonists

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

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

As used herein, the terms "programmed cell death 1 ligand 1", "PD-L1", "PDL 1", "PDCD 1L 1", "PDCD 1LG 1", "CD 274", "B7 homolog 1", "B7-H1", "B7-H", and "B7H 1" are used interchangeably and include variants, isoforms, species homologs of human PDL-1, and analogs having at least one common epitope with human PDL-1. The complete human PD-L1 amino acid sequence-isoform a precursor-can be found under GenBank accession No. NP _ 054862.1. The complete human PD-L1 amino acid sequence-isoform b precursor-can be found under GenBank accession No. NP _ 001254635.1.

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

The term "PD-1 binding antagonist" is a molecule that reduces, blocks, inhibits, eliminates, or interferes with signal transduction resulting from the interaction of PD-1 with one or more of its binding partners (such as PD-L1, PD-L2). In some embodiments, the PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 to its binding partner. In a particular aspect, the PD-1 binding antagonist inhibits the binding of PD-1 to PD-L1 and/or PD-L2. For example, PD-1 binding antagonists include anti-PD-1 antibodies, antigen-binding fragments thereof, immunoadhesins, fusion proteins, oligopeptides, and other molecules that reduce, block, inhibit, eliminate, or interfere with signal transduction caused by the interaction of PD-1 with PD-L1 and/or PD-L2. In one embodiment, the PD-1 binding antagonist reduces negative co-stimulatory signals mediated by or through cell surface proteins expressed on T lymphocytes mediated by PD-1 signaling, so as to render the dysfunctional T cells less dysfunctional (e.g., enhance effector responses to antigen recognition). In some embodiments, the PD-1 binding antagonist is an anti-PD-1 antibody. In a particular aspect, the PD-1 binding antagonist is MDX-1106 described herein. In another particular aspect, the PD-1 binding antagonist is Merck 3745 described herein. In another particular aspect, the PD-1 binding antagonist is CT-011 described herein.

The term "PD-L1 binding antagonist" is a molecule that reduces, blocks, inhibits, eliminates or interferes with signal transduction resulting from the interaction of PD-L1 with one or more of its binding partners (such as PD-1, B7-1). In some embodiments, a PD-L1 binding antagonist is a molecule that inhibits the binding of PD-L1 to its binding partner. In a particular aspect, the PD-L1 binding antagonist inhibits the binding of PD-L1 to PD-1 and/or B7-1. In some embodiments, the PD-L1 binding antagonist includes an anti-PD-L1 antibody, antigen-binding fragment thereof, immunoadhesin, fusion protein, oligopeptide and other molecules that reduce, block, inhibit, eliminate or interfere with signal transduction caused by the interaction of PD-L1 with one or more of its binding partners (such as PD-1, B7-1). In one embodiment, the PD-L1 binding antagonist reduces negative co-stimulatory signals mediated by or through cell surface proteins expressed on T lymphocytes mediated by PD-L1 signaling, so as to render the dysfunctional T cells less dysfunctional (e.g., enhance effector responses to antigen recognition). In some embodiments, the PD-L1 binding antagonist is an anti-PD-L1 antibody. In a particular aspect, the anti-PD-L1 antibody is yw243.55.s70 described herein. In another particular aspect, the anti-PD-L1 antibody is MDX-1105 as described herein. In yet another particular aspect, the anti-PD-L1 antibody is MPDL3280A described herein.

The term "PD-L2 binding antagonist" is a molecule that reduces, blocks, inhibits, eliminates, 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 partner. In a particular aspect, the PD-L2 binding antagonist inhibits the binding of PD-L2 to PD-1. In some embodiments, the PD-L2 antagonist includes an anti-PD-L2 antibody, antigen binding fragment thereof, immunoadhesin, fusion protein, oligopeptide and other molecules that reduce, block, inhibit, eliminate or interfere with signal transduction caused by the interaction of PD-L2 with one or more of its binding partners, such as PD-1. In one embodiment, the PD-L2 binding antagonist reduces negative co-stimulatory signals mediated by or through cell surface proteins expressed on T lymphocytes mediated by PD-L2 signaling, so as to render the dysfunctional T cells less dysfunctional (e.g., enhance effector responses 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 associated with PD-1 signaling. In one aspect, these may be proteins that induce PD-1 signaling upstream of PD-1, such as, for example, ligands and signaling receptor PD-1 of PD-1PD-L1 and PD-L2. In another aspect, these may be signal transduction proteins downstream of the PD-1 receptor. Particularly preferred members of the PD-1 pathway in the context encompassed by the present invention are PD-1, PD-L1 and PD-L2.

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

In certain embodiments, PD-1 antagonists encompassed by the present invention include agents that bind to a PD-1 ligand 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 signal transduction through the PD-1 receptor. In one embodiment, the PD-1 antagonist binds directly to PD-1 and blocks PD-1 inhibitory signal transduction. In another embodiment, the PD-1 antagonist binds to one or more ligands of PD-1 (e.g., PD-L1 and PD-L2) and reduces or inhibits ligand inhibition signaling by PD-1 triggering. In one embodiment, the PD-1 antagonist binds directly to PD-L1, inhibits or prevents PD-L1 from binding to PD-1, thereby blocking PD-1 inhibitory signaling.

PD-1 antagonists used in the methods and compositions encompassed by the present invention include PD-1 binding scaffold proteins, and include, but are not limited to, PD-ligands, antibodies, and multivalent agents. In a particular embodiment, the antagonist is a fusion protein, such as AMP-224. In another embodiment, the antagonist is an anti-PD-1 antibody ("PD-1" antibody). Anti-human PD-1 antibodies (or VH and/or VL domains derived therefrom) suitable for use in the present invention can be produced using methods well known in the art. Alternatively, recognized anti-PD-1 antibodies may be used. For example, the antibodies MK-3475 or CT-011 can be used. In addition, monoclonal antibodies 5C4, 17D8, 2D3, 4H1, 4a11, 7D3 and 5F4 described in WO 2006/121168, the teachings of which are incorporated herein by reference, may be used. Antibodies that compete for binding to PD-1 with any of these recognized antibodies can also be used.

In another embodiment, the PD-1 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 invention can be produced using methods well known in the art. Alternatively, a recognized anti-PD-L1 antibody may be used. For example, MEDI4736 (also known as anti-B7-Hl) or MPDL3280A (also known as RG7446) may 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, the teachings of which are incorporated herein by reference, may be used. Antibodies that compete with any of these recognized antibodies for binding to PD-L1 can also be used.

An exemplary anti-PD-L1 antibody is 12a4 described in WO 2007/005874 and U.S. patent No. 7,943,743. In one embodiment, the antibody comprises the heavy and light chain CDRs or VRs of 12a 4. In another embodiment, the antibody competes for binding to the same epitope on PD-L1 and/or to the same epitope on PD-L1 with an antibody as mentioned above. In another embodiment, the antibody has at least about 90% variable region amino acid sequence identity to an antibody mentioned above.

The anti-PD-1 or anti-PD-L1 antibodies may each be 10^-7M、5x10^-8M、10^-8M、5x10^-9M、10^-9M、5x10^-10M、10^-10M or less KD binds to PD-1 or PD-L1.

In one embodiment, the PD-1 inhibitor is an anti-PD-1 antibody selected from Nivolumab (Nivolumab), Pembrolizumab (Pembrolizumab), or Pidilizumab (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 PDl. Nivolumab (clone 5C4) and other human monoclonal antibodies that specifically bind to PDl 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)The original name ramulizumab (Lambrolizumab), also known as Merck 3745, MK-3475 or SCH-900475) is a humanized IgG4 monoclonal antibody that binds to PD 1. Pembrolizumab is disclosed, for example, in Hamid, O. et al (2013) New England Journal of Medicine 369(2):134-44, WO 2009/114335 (incorporated by reference) and US 8,354,509 (incorporated by reference).

In some embodiments, the anti-PD-1 antibody is pidilizumab. Pidilizumab (CT-011; Cure Tech) is a humanized IgGlk monoclonal antibody that binds to PD 1. Pidilizumab and other humanized anti-PD-1 monoclonal antibodies are disclosed in WO 2009/101611. Other anti-PDl antibodies are disclosed in US 8,609,089, US 2010028330 and/or US 20120114649. Other anti-PD 1 antibodies include AMP 514 (amplimune).

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., the 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.

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 WO 2007/005874. In one embodiment, the PD-L1 inhibitor is yw243.55. s70. The yw243.55.s70 antibody is anti-PD-L1 (in WO 2010/077634, the heavy and light chain variable region sequences shown in SEQ ID nos. 20 and 21, respectively) described in WO 2010/077634 (incorporated by reference).

In one embodiment, the PD-L1 inhibitor is MDPL3280A (Genentech/Roche). MDPL3280A is a human Fc optimized IgGl monoclonal antibody that binds to PD-L1. MDPL3280A and other human monoclonal antibodies to PD-L1 are disclosed in U.S. patent No. 7,943,743 (incorporated by reference) and U.S. publication No. 2012/0039906 (incorporated by reference). In other embodiments, the PD-L2 inhibitor is AMP-224. AMP-224 is a PD-L2 Fc fusion soluble receptor that blocks the interaction between PD1 and B7-H1 (B7-DCIg; Amplimmune; e.g., disclosed in WO 2010/027827 (incorporated by reference) and WO 2011/066342 (incorporated by reference)).

In certain embodiments, the PD-1 pathway inhibitor is a small molecule antagonist of PD-1 pathway signaling. Such small molecule antagonists include those 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-1L 2.

Exemplary small molecule antagonists of PD-1 pathway signaling can be found, inter alia, in: published U.S. applications 2014/0294898 and 2014/0199334, and published PCT applications WO 2013/132317 and WO 2012/168944, each of which is incorporated herein by reference.

For illustration only, the subject combination therapy may be practiced with small molecule antagonists selected from the group consisting of:

in other embodiments, the small molecule antagonist is represented by the general formula:

wherein the content of the first and second substances,

r1 is Ser free C-terminus or amidated C-terminus;

l is selected from-NH (CH)₂)_nNH-or-NH (CH)₂CH₂O)_nA linker of NH-;

r4 is selected from hydrogen, amino (C)₁-C₂₀) Alkyl, -NHCOCH₃or-NHCONH₂；

Or a retro-analogue or a pharmaceutically acceptable stereoisomer or a pharmaceutically acceptable salt thereof.

In yet other embodiments, the small molecule antagonist is represented by the general formula:

wherein the content of the first and second substances,

R₁is the N-terminus of Ser; or substituted by hydroxy or amino of Ser (C)₁-C₂₀) An acyl group;

l is selected from-NH (CH)₂)_nNH-、-NH(CH₂)_nCH(NH₂)CO-、-OOC(CH₂)_mCOO-、-NH(CH₂)_nCO-、-NH(CH₂CH₂O)_nNH-、-NH(CH₂CH₂O)_nCO-or-CO (CH)₂CH₂O)_nA linker for CO-;

R₂am is₂Free C-terminus, amidated C-terminus, or N-terminus of (a); or Y-R₅；

Y is selected from-OOC (CH)₂)_mCOO-、-CO(CH₂)_nNH-、-CO(CH₂CH₂O)_nNH-or-COCH₂(OCH₂CH₂)_nAn optional linker for NH-;

R₅is an albumin binding moiety such as maleimidopropionic acid;

R₃is OH or NH₂；

R₄Is a substituent on the phenyl group of Phe and is selected from hydrogen, amino (C)₁-C₂₀) Alkyl, -NHCOCH₃or-NHCONH₂；

n is an integer having a value selected from 2 to 10, including 2 and 10;

m is an integer having a value selected from 0 to 8, including 0 and 8; and is

One of peptide bonds (-CONH-) of Ser-Asn, Asn-Thr or Thr-Ser may be substituted The modified peptide bond of (a) is replaced,

wherein Q is hydrogen, -CO (C)₁-C₂₀) Alkyl or-COO (C)₁-C₂₀) An alkyl group; wherein one or more or all of the amino acids may be in the D-configuration;

or a retro-analogue or a pharmaceutically acceptable stereoisomer or a pharmaceutically acceptable salt thereof.

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

CTLA-4 antagonists

In certain embodiments, the combinations described herein further comprise a CTLA-4 inhibitor. Exemplary anti-CTLA-4 antibodies include Tremelimumab (Tremelimumab) (IgG 2 monoclonal antibody available from Pfizer, original name Tremelimumab (ticilimumab), CP-675,206); and Ipilimumab (Ipilimumab) (CTLA-4 antibody, also known as MDX-010, CAS number 477202-00-9).

Information on tramadol single antibody (or antigen-binding fragments thereof) for use in the methods provided herein can be found in US 6,682,736 (incorporated by reference) (where it is referred to as 11.2.1), the disclosure of which is incorporated herein by reference in its entirety. Tramelimumab (also known as CP-675,206, CP-675, CP-675206, and tikitamumumab) is a human IgG2 monoclonal antibody that is highly selective for CTLA-4 and blocks CTLA-4 binding to CD80(B7.1) and CD86 (B7.2). It has been shown to lead to immune activation in vitro, and some patients treated with tramadol have shown tumor regression.

The trametes single antibody used in the methods provided herein comprises heavy and light chains or a heavy chain variable region and a light chain variable region. In a particular aspect, 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 shown herein above and a heavy chain variable region comprising the amino acid sequence shown herein above. In a particular aspect, tremelimumab or 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 shown herein above, and wherein the light chain variable region comprises the Kabat-defined CDR1, CDR2 and CDR3 sequences shown herein above. One of ordinary skill in the art will be readily able to adhere to the Chothia definition, the Abm definition, or other CDR definitions known to those of ordinary skill in the art. In one particular aspect, tremelimumab or antigen-binding fragment thereof for use in the methods provided herein comprises the variable heavy and variable light chain CDR sequences of an antibody as disclosed in US6,682,736, which is incorporated herein by reference in its entirety.

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

other small molecule CTLA-4 antagonists include

In one embodiment, the combination comprises an immune-DASH inhibitor, an anti-PD-1 antibody molecule (e.g., as described herein), and an anti-CTLA-4 antibody (e.g., ipilimumab). Exemplary doses that can be used include doses of the anti-PD-1 antibody molecule of about 1 to 10mg/kg (e.g., 3mg/kg), and doses of the anti-CTLA-4 antibody (e.g., ipilimumab) of about 3 mg/kg.

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

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

In some embodiments, the LAG3 antibody is IMP701, IMP731, BMS-986016, LAG525, and GSK 2831781. In some embodiments, the LAG3 antagonist comprises a soluble LAG3 receptor, e.g., IMP 321.

In some embodiments, the immune response stimulating agent is selected from the group consisting of: CD28 agonists, 4-1BB agonists, OX40 agonists, CD27 agonists, CD80 agonists, CD86 agonists, CD40 agonists, and GITR agonists. In some embodiments, the OX40 agonist comprises an OX40 ligand or an OX40 binding portion thereof. For example, the OX40 agonist can be MEDI 6383. In some embodiments, the OX40 agonist is an antibody that specifically binds OX 40. In some embodiments, the antibody that binds OX40 is MEDI6469, MEDI0562, or MOXR0916(RG 7888). 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 vector expressing OX40 is delta-24-RGDOX or DNX 2401.

In some embodiments, the 4-1BB (CD137) agonist is a binding molecule, such as an anti-transporter (anticalin). In some embodiments, the antiporter protein is PRS-343. In some embodiments, the 4-1BB agonist is an antibody that specifically binds 4-1 BB. In some embodiments, the antibody that binds 4-1BB is PF-2566(PF-05082566) or Urelumab (ureluumab) (BMS-663513).

In some embodiments, the CD27 agonist is an antibody that specifically binds CD 27. In some embodiments, the antibody that binds CD27 is vallizumab (varluumab) (CDX-1127).

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

In certain embodiments, the anti-CD 39 antibody is combined with a STING agonist, preferably as part of a pharmaceutical composition. Cyclic di-nucleotides (CDNs) cyclic di-AMPs (produced by listeria monocytogenes and other bacteria) and their analogs cyclic di-GMPs, and cyclic GMP-AMP is recognized by host cells as pathogen-associated molecular patterns (PAMPs) that bind to Pathogen Recognition Receptors (PRRs) known as interferon gene Stimulators (STING). STING is an adaptor protein in the cytoplasm of host mammalian cells that activates the TANK binding kinase (TBK1) -IRF3 and NF- κ B signaling axes, leading to induction of IFN- β and other gene products that strongly activate innate immunity. STING is now recognized as a component of the host cytoplasmic surveillance pathway (Vance et al, 2009), which senses infection with intracellular pathogens and induces production of IFN- β in response, leading to the development of an adaptive protective pathogen-specific immune response consisting of antigen-specific CD4+ and CD8+ T cells and pathogen-specific antibodies. U.S. patent nos. 7,709,458 and 7,592,326; PCT publication Nos. WO2007/054279, WO2014/093936, WO2014/179335, WO2014/189805, WO2015/185565, WO2016/096174, WO2016/145102, WO2017/027645, WO2017/027646, and WO 2017/075477; and Yan et al, bioorg.Med.chem Lett.18:5631-4, 2008.

Exemplary combination

In a preferred embodiment, the invention relates to the combination of an anti-CD 39 antibody with an anti-tumor platinum coordination complex to treat cancer, and more particularly to treat a cancer selected from the group consisting of: lung cancer, sarcoma, malignant melanoma, prostate cancer, pancreatic cancer, gastric cancer, ovarian cancer, liver cancer, breast cancer, colorectal cancer, renal cancer, brain cancer, and lymphoma. This chemotherapeutic group includes, but is not limited to, cisplatin, oxaliplatin, carboplatin, triplatin tetranitrate (BBR3464), satraplatin, tetraplatin, ormiplatin, iproplatin, nedaplatin, and lobaplatin. Particularly preferred is the combination of an anti-CD 39 antibody with cisplatin, oxaliplatin, carboplatin, triplatin tetranitrate, satraplatin, tetraplatin, ormiplatin, iproplatin, nedaplatin and lobaplatin, and even more preferred with cisplatin and oxaliplatin for the treatment of cancer, and more particularly for the treatment of a cancer selected from: lung cancer, sarcoma, malignant melanoma, prostate cancer, pancreatic cancer, gastric cancer, ovarian cancer, liver cancer, breast cancer, colorectal cancer, renal cancer, and brain cancer. In another preferred embodiment, the invention relates to the combination of an anti-CD 39 antibody with an antimetabolite to treat cancer, and more particularly to treat cancer selected from the group consisting of: lung cancer, sarcoma, malignant melanoma, bladder cancer, prostate cancer, pancreatic cancer, gastric cancer, ovarian cancer, liver cancer, breast cancer, colorectal cancer, renal cancer, esophageal cancer, brain cancer, anal cancer, leukemia, and lymphoma. This chemotherapeutic group includes, but is not limited to, 5-fluorouracil, gemcitabine, cytarabine, capecitabine, decitabine (decitabine), floxuridine, fludarabine, aminopterin, methotrexate, pemetrexed, raltitrexed, cladribine, clofarabine (clofarabine), mercaptopurine, pentostatin, and thioguanine. Particularly preferred is the combination of an anti-CD 39 antibody with 5-fluorouracil, gemcitabine, cytarabine, capecitabine, decitabine, floxuridine, fludarabine, aminopterin, methotrexate, pemetrexed, raltitrexed, cladribine, clofarabine, mercaptopurine, pentostatin, and thioguanine, and even more preferred with 5-fluorouracil, gemcitabine, cytarabine, and methotrexate for the treatment of cancer, and more particularly for the treatment of cancer selected from the group consisting of: lung cancer, sarcoma, malignant melanoma, prostate cancer, pancreatic cancer, gastric cancer, ovarian cancer, liver cancer, breast cancer, colorectal cancer, renal cancer, brain cancer, leukemia, and lymphoma.

In another preferred embodiment, the invention relates to the combination of an anti-CD 39 antibody with a mitotic inhibitor for the treatment of cancer, and more particularly for the treatment of a cancer selected from the group consisting of: lung cancer, sarcoma, prostate cancer, gastric cancer, ovarian cancer, liver cancer, breast cancer, colorectal cancer, renal cancer, brain cancer, leukemia and lymphoma. This chemotherapeutic group includes, but is not limited to, paclitaxel, docetaxel, vinblastine, vincristine, vindesine, and vinorelbine. Particularly preferred is the combination of an anti-CD 39 antibody with paclitaxel, docetaxel, vinblastine, vincristine, vindesine and vinorelbine, and even more preferred with paclitaxel, docetaxel, vincristine and vinorelbine for the treatment of cancer, and more particularly for the treatment of a cancer selected from: lung cancer, sarcoma, prostate cancer, gastric cancer, ovarian cancer, liver cancer, breast cancer, colorectal cancer, renal cancer, and brain cancer.

In another preferred embodiment, the invention relates to the combination of an anti-CD 39 antibody with an anti-cancer antibiotic to treat cancer, and more particularly to treat the following cancers: lung cancer, sarcoma, malignant melanoma, bladder cancer, prostate cancer, pancreatic cancer, thyroid cancer, gastric cancer, ovarian cancer, liver cancer, breast cancer, colorectal cancer, kidney cancer, neuroblastoma, brain cancer, anal cancer, testicular cancer, leukemia, multiple myeloma, and lymphoma. This group of chemotherapeutic agents includes, but is not limited to, daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, pixantrone, valrubicin, mitomycin C, bleomycin, actinomycin A, and mithramycin. Particularly preferred is the combination of the anti-CD 39 antibody with daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, pixantrone (pixantrone), valrubicin, mitomycin C, bleomycin, actinomycin D and mithramycin, and even more preferred with daunorubicin, doxorubicin, mitomycin C and actinomycin D for the treatment of cancer, and more particularly for the treatment of lung cancer, sarcoma, malignant melanoma, prostate cancer, pancreatic cancer, gastric cancer, ovarian cancer, liver cancer, breast cancer, colorectal cancer, kidney cancer, brain cancer, leukemia and lymphoma.

In another preferred embodiment, the invention relates to the combination of an anti-CD 39 antibody with a topoisomerase I and/or II inhibitor for the treatment of cancer, and more particularly for the treatment of lung cancer, sarcoma, malignant melanoma, prostate cancer, pancreatic cancer, gastric cancer, ovarian cancer, liver cancer, breast cancer, colorectal cancer, kidney cancer, neuroblastoma, brain cancer, cervical cancer, testicular cancer, leukemia and lymphoma. This group of chemotherapeutic agents includes, but is not limited to, topotecan, SN-38, irinotecan, camptothecin, rubitecan, etoposide, amsacrine, and teniposide. Particularly preferred is the combination of PM00104 or a pharmaceutically acceptable salt thereof with topotecan, SN-38, irinotecan, camptothecin, rubitecan, etoposide, amsacrine and teniposide, and even more preferred with topotecan, irinotecan and etoposide for the treatment of cancer, and more particularly for the treatment of lung cancer, sarcoma, malignant melanoma, prostate cancer, pancreatic cancer, gastric cancer, ovarian cancer, liver cancer, breast cancer, colorectal cancer, renal cancer and brain cancer.

In another preferred embodiment, the invention relates to the combination of an anti-CD 39 antibody with a proteasome inhibitor for the treatment of cancer, and more particularly for the treatment of lung cancer, prostate cancer, pancreatic cancer, gastric cancer, liver cancer, colorectal cancer, brain cancer, multiple myeloma and lymphoma. This group of chemotherapeutic agents includes, but is not limited to, bortezomib, disulfiram, epigallocatechin gallate (epigallocatechin gallate) and salinosporamide a (salinosporamide a). Particularly preferred is the combination of an anti-CD 39 antibody with bortezomib, disulfiram, epigallocatechin gallate and salinospiramide a, and even more preferred with bortezomib for the treatment of cancer, and more particularly for the treatment of lung, prostate, pancreatic, gastric, liver, colorectal and brain cancer.

In another preferred embodiment, the invention relates to the combination of an anti-CD 39 antibody with a histone deacetylase inhibitor for the treatment of cancer, and more particularly for the treatment of lung cancer, sarcoma, prostate cancer, pancreatic cancer, gastric cancer, ovarian cancer, breast cancer, colorectal cancer, renal cancer, brain cancer and lymphoma. This group of chemotherapeutic agents includes, but is not limited to, romidepsin (romidepsin), panobinostat (panobinostat), vorinostat (vorinostat), moxystat (mocetinostat), belinostat (belinostat), entinostat (entinostat), reminostat (reminiostat), PCI-24781, AR-42, CUDC-101, and valproic acid. Particularly preferred is the combination of an anti-CD 39 antibody with romidepsin, panobinostat, vorinostat, moxisstat, belinostat, entinostat, reminostat, PCI-24781, AR-42, CUDC-101 and valproic acid, and even more preferred with vorinostat for the treatment of cancer, and more particularly for the treatment of lung, sarcoma, prostate, pancreatic, gastric, ovarian, breast, colorectal, kidney and brain cancers.

In another preferred embodiment, the invention relates to the combination of an anti-CD 39 antibody and a nitrogen mustard alkylating agent to treat cancer, and more particularly to treat lung cancer, sarcoma, bladder cancer, gastric cancer, ovarian cancer, liver cancer, breast cancer, colorectal cancer, renal cancer, leukemia, multiple myeloma and lymphoma. This group of chemotherapeutic agents includes, but is not limited to, melphalan, ifosfamide, chlorambucil, cyclophosphamide, mechlorethamine, uramustine (uramustine), estramustine, and bendamustine (bendamustine). Particularly preferred is the combination of an anti-CD 39 antibody with melphalan, ifosfamide, chlorambucil, cyclophosphamide, mechlorethamine, uramustine, estramustine and bendamustine, and even more preferred with cyclophosphamide for the treatment of cancer, and more particularly for the treatment of lung cancer, sarcoma, gastric cancer, ovarian cancer, liver cancer, breast cancer, colorectal cancer and kidney cancer. In another preferred embodiment, the invention relates to the combination of an anti-CD 39 antibody with a nitrosourea alkylating agent to treat cancer, and more particularly to treat lung cancer, ovarian cancer, breast cancer, brain cancer, multiple myeloma and lymphoma. This group of chemotherapeutic agents includes, but is not limited to, lomustine, semustine (semustine), carmustine, fotemustine, and streptozotocin. Particularly preferred is the combination of the anti-CD 39 antibody with lomustine, semustine, carmustine, fotemustine and streptozotocin, and even more preferred with carmustine for the treatment of cancer, and more particularly for the treatment of lung, ovarian and breast cancer.

In another preferred embodiment, the invention relates to the combination of an anti-CD 39 antibody with a non-classical alkylating agent for the treatment of cancer, and more particularly for the treatment of lung cancer, sarcoma, malignant melanoma, pancreatic cancer, gastric cancer, ovarian cancer, breast cancer, colorectal cancer, renal cancer, brain cancer, leukemia and lymphoma. This group of chemotherapeutic agents includes, but is not limited to, procarbazine, dacarbazine, temozolomide (temozolomide), and altretamine. Particularly preferred is the combination of an anti-CD 39 antibody with procarbazine, dacarbazine, temozolomide, and altretamine, and even more preferred is the combination with dacarbazine and temozolomide for the treatment of lung cancer, sarcoma, malignant melanoma, gastric cancer, ovarian cancer, breast cancer, colorectal cancer, renal cancer, and brain cancer. In another preferred embodiment, the invention relates to the combination of an anti-CD 39 antibody and an estrogen antagonist for the treatment of cancer, and more particularly for the treatment of breast cancer. This chemotherapeutic group includes, but is not limited to, toremifene, fulvestrant, tamoxifen, and nafoxidine (nafoxidine). Particularly preferred is the combination of an anti-CD 39 antibody with toremifene, fulvestrant, tamoxifen and nafoxidine, and even more preferred with tamoxifen for the treatment of breast cancer.

In another preferred embodiment, the invention relates to the combination of an anti-CD 39 antibody and an androgen antagonist to treat cancer, and more particularly to treat prostate cancer. This group of chemotherapeutic agents includes, but is not limited to, bicalutamide, flutamide, MDV3100, and nilutamide. Particularly preferred is the combination of an anti-CD 39 antibody with bicalutamide, flutamide, MDV3100 and nilutamide, and even more preferred with flutamide for the treatment of prostate cancer.

In another preferred embodiment, the invention relates to the combination of an anti-CD 39 antibody and an mTOR inhibitor for the treatment of cancer, and more particularly for the treatment of lung cancer, sarcoma, malignant melanoma, prostate cancer, pancreatic cancer, gastric cancer, ovarian cancer, breast cancer, colorectal cancer, renal cancer and brain cancer. This group of chemotherapeutic agents includes, but is not limited to, sirolimus (sirolimus), temsirolimus (temsirolimus), everolimus (everolimus), ridaforolimus (ridaforolimus), KU-0063794, and WYE-354. Particularly preferred is the combination of an anti-CD 39 antibody with sirolimus, temsirolimus, everolimus, diphospholimus, KU-0063794 and WYE-354, and even more preferred with temsirolimus for the treatment of lung cancer, sarcoma, malignant melanoma, prostate cancer, pancreatic cancer, gastric cancer, ovarian cancer, breast cancer, colorectal cancer and brain cancer.

In another preferred embodiment, the present invention relates to the combination of an anti-CD 39 antibody with a tyrosine kinase inhibitor for the treatment of cancer, and more particularly for the treatment of a cancer selected from the group consisting of: lung cancer, sarcoma, prostate cancer, pancreatic cancer, gastric cancer, ovarian cancer, liver cancer, breast cancer, colorectal cancer, renal cancer, and brain cancer. This group of chemotherapeutic agents includes, but is not limited to, erlotinib, sorafenib, axitinib, bosutinib (bosutinib), cediranib, crizotinib (crizotinib), dasatinib (dasatinib), gefitinib, imatinib, canertinib (canertinib), lapatinib (lapatinib), lestatinib (lestaurtinib), neratinib (neratinib), nilotinib (nilotinib), semaxanib (semaxanib), sunitinib, vatalanib (vatalanib), and vandetanib. Particularly preferred is the combination of an anti-CD 39 antibody with erlotinib, sorafenib, axitinib, bosutinib, cediranib, crizotinib, dasatinib, gefitinib, imatinib, canertinib, lapatinib, lestatinib, lenatinib, nilotinib, semaxanib, sunitinib, vatalanib and vandetanib, and even more preferred with erlotinib for the treatment of cancer, and more particularly for the treatment of a cancer selected from the group consisting of: lung cancer, sarcoma, prostate cancer, pancreatic cancer, gastric cancer, ovarian cancer, liver cancer, breast cancer, colorectal cancer, renal cancer, and brain cancer.

Another aspect encompassed by the present invention relates to any one of the above methods further comprising administering to the patient a MAP kinase pathway inhibitor or a WNT pathway inhibitor.

In some embodiments, the MAP kinase pathway inhibitor is selected from the group consisting of: BRAF inhibitors, MEK inhibitors, PI3K inhibitors, and c-KIT inhibitors.

In some embodiments, the BRAF inhibitor is selected from the group consisting of: GDC-0879, PLX-4720, sorafenib tosylate, dabrafenib (dabrafenib) and LGX 818.

In some embodiments, the MEK inhibitor is selected from the group consisting of: GSK1120212, semetinib (selumetinib), and MEK 162.

In some embodiments, the WNT pathway inhibitor is a beta-catenin inhibitor or a frizzled inhibitor (frizzled inhibitor).

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

Another aspect encompassed by the present invention relates to any one of the above methods further comprising administering a cancer vaccine to the patient. In some embodiments, the cancer vaccine is a dendritic cell vaccine.

Another aspect encompassed by the present invention relates to any one of the above methods further comprising administering adoptive cell transfer to the patient.

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

Another aspect encompassed by the present invention relates to any one of the above methods further comprising administering to the patient an antibody therapy.

Another aspect encompassed by the present invention relates to any one of the above methods, wherein administration of the anti-CD 39 antibody enhances antibody-dependent cell-mediated cytotoxicity of the antibody therapy.

In some embodiments, the antibody therapy is selected from the group consisting of: trastuzumab (trastuzamab), cetuximab, bevacizumab, and rituximab (rituximab).

In addition, treatment with anti-CD 39 antibodies may include combination therapy with other biomolecules such as one or more cytokines (e.g., lymphokines, interleukins, tumor necrosis factors, and/or growth factors) or any other therapy that may accompany surgical removal of tumors, removal of cancer cells, or deemed necessary by the treating physician. In some embodiments, the additional therapeutic agent is an immune response stimulating agent.

In some embodiments of the methods described herein, the anti-CD 39 antibody may be combined with a growth factor selected from the group consisting of: adrenomedullin (AM), angiogenin (Ang), BMP, BDNF, EGF, Erythropoietin (EPO), FGF, GDNF, G-CSF, GM-CSF, GDF9, HGF, HDGF, IGF, migration stimulating factor, myostatin (GDF-8), NGF, 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.

In some embodiments of the methods described herein, the additional therapeutic agent is an immune response stimulating agent. In some embodiments, the immune response stimulating agent 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).

Application time schedule

In certain embodiments of the methods described herein, the treatment involves administering an anti-CD 39 antibody in combination with radiation therapy. Treatment with the anti-CD 39 antibody can occur before, concurrently with, or after radiation therapy. The schedule of administration of such radiation therapy can be determined by a skilled medical practitioner.

In certain embodiments of the methods described herein, the treatment involves administering an anti-CD 39 antibody in combination with an antiviral therapy. Treatment with an anti-CD 39 antibody can occur before, concurrently with, or after antiviral therapy. The antiviral drug used in the combination therapy will depend on the virus with which the subject is infected.

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

It is to be understood that the combination of the anti-CD 39 antibody and the at least one additional therapeutic agent can be administered in any order or simultaneously. In some embodiments, the anti-CD 39 antibody will be administered to a patient who has previously undergone treatment with a second therapeutic agent. In certain other embodiments, the anti-CD 39 antibody and the second therapeutic agent will be administered substantially simultaneously or concurrently. For example, a subject may be administered an anti-CD 39 antibody while undergoing a course of treatment with a second therapeutic agent (e.g., chemotherapy). In certain embodiments, the anti-CD 39 antibody will be administered within 1 year of treatment with the second therapeutic agent. In certain alternative embodiments, the anti-CD 39 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-CD 39 antibody will be administered within 4, 3, 2, or 1 weeks of any treatment with the second therapeutic agent. In some embodiments, the anti-CD 39 antibody will be administered within 5, 4, 3, 2, or 1 days of any treatment with the second therapeutic agent. It is further understood that two (or more) agents or treatments may be administered to a subject within hours or minutes (i.e., substantially simultaneously).

For the treatment of disease, the appropriate dosage of anti-CD 39 antibody will depend on the type of disease to be treated, the severity and course of the disease, the responsiveness of the disease, whether the anti-CD 39 antibody is administered for therapeutic or prophylactic purposes, previous therapy, the patient's clinical history, and the like, as appropriate by the attending physician. The anti-CD 39 antibody can be administered once or over a series of treatments for days to months, or until a cure is achieved or a reduction in disease state (e.g., a reduction in tumor size) is achieved. The optimal dosing schedule may be calculated from measurements of drug accumulation in the patient and will vary depending on the relative potency of the individual agents. The administering physician can determine the optimal dosage, method of administration and repetition rate. In certain embodiments, the dose is 0.01 μ g to 100mg/kg body weight, 0.1 μ g to 100mg/kg body weight, 1mg to 80mg/kg body weight, 10mg to 100mg/kg body weight, 10mg to 75mg/kg body weight, or 10mg to 50mg/kg body weight. In certain embodiments, the dose of the anti-CD 39 antibody is from about 0.1mg to about 20mg/kg body weight. In some embodiments, the dose of the anti-CD 39 antibody is about 0.1mg/kg body weight. In some embodiments, the dose of the anti-CD 39 antibody is about 0.25mg/kg body weight. In some embodiments, the dose of the anti-CD 39 antibody is about 0.5mg/kg body weight. In some embodiments, the dose of the anti-CD 39 antibody is about 1mg/kg body weight. In some embodiments, the dose of the anti-CD 39 antibody is about 1.5mg/kg body weight. In some embodiments, the dose of the anti-CD 39 antibody is about 2mg/kg body weight. In some embodiments, the dose of the anti-CD 39 antibody is about 2.5mg/kg body weight. In some embodiments, the dose of the anti-CD 39 antibody is about 5mg/kg body weight. In some embodiments, the dose of the anti-CD 39 antibody is about 7.5mg/kg body weight. In some embodiments, the dose of the anti-CD 39 antibody is about 10mg/kg body weight. In some embodiments, the dose of the anti-CD 39 antibody is about 12.5mg/kg body weight. In some embodiments, the dose of the anti-CD 39 antibody is about 15mg/kg body weight. In certain embodiments, the dose may be administered one or more times per day, week, month, or year. In certain embodiments, the anti-CD 39 antibody is administered once a week, once every two weeks, once every three weeks, or once every four weeks.

In some embodiments, the anti-CD 39 antibody may be administered in 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 administration of an initial dose followed by administration of 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 comprise administration of an initial loading dose followed by weekly administration of a maintenance dose (e.g., half of the initial dose). Or a dosing regimen may comprise administration of an initial loading dose followed by administration of a maintenance dose every other week (e.g., half of the initial dose). Or a dosing regimen may comprise administration of three initial doses for 3 weeks followed by administration of a maintenance dose (e.g., the same amount) every other week.

As known to those skilled in the art, administration of any therapeutic agent can result in side effects and/or toxicity. In some instances, the side effects and/or toxicity are so severe as to preclude administration of a particular agent at a therapeutically effective dose. In some instances, drug therapy must be discontinued and other agents may be tried. However, many agents in the same treatment class often exhibit similar side effects and/or toxicity, meaning that the patient has to stop the therapy or, if possible, suffer from unpleasant side effects associated with the therapeutic agent.

In some embodiments, the dosing schedule may be limited to a particular number of administrations or "cycles". In some embodiments, the anti-CD 39 antibody is administered for 3, 4, 5, 6, 7, 8 or more cycles. For example, the anti-CD 39 antibody is administered every 2 weeks for 6 cycles, the anti-CD 39 antibody is administered every 3 weeks for 6 cycles, the anti-CD 39 antibody is administered every 2 weeks for 4 cycles, the anti-CD 39 antibody is administered every 3 weeks for 4 cycles, and so forth. The dosing schedule can be determined by one skilled in the art and subsequently modified.

Accordingly, the present invention provides methods of administering an anti-CD 39 antibody described herein to a subject, the methods comprising administering one or more agents using an intermittent dosing strategy, which can reduce side effects and/or toxicity associated with administration of anti-CD 39 antibodies, chemotherapeutic agents, and the like. In some embodiments, a method for treating cancer in a human subject comprises administering to the subject a therapeutically effective dose of an anti-CD 39 antibody in combination with a therapeutically effective dose of a chemotherapeutic agent, wherein one or both of the agents are administered according to an intermittent dosing strategy. In some embodiments, the intermittent dosing strategy comprises administering to the subject an initial dose of the anti-CD 39 antibody and administering subsequent doses of the anti-CD 39 antibody about once every 2 weeks. In some embodiments, the intermittent dosing strategy comprises administering to the subject an initial dose of the anti-CD 39 antibody and administering subsequent doses of the anti-CD 39 antibody about once every 3 weeks. In some embodiments, the intermittent dosing strategy comprises administering to the subject an initial dose of the anti-CD 39 antibody and administering subsequent doses of the anti-CD 39 antibody about once every 4 weeks. In some embodiments, the anti-CD 39 antibody is administered using an intermittent dosing strategy, and the chemotherapeutic agent is administered weekly.

Anti-infective therapeutic combinations

In one embodiment, the present invention provides a method of treating a subject with an anti-CD 39 antibody, wherein the subject is suffering from a viral infection. In one embodiment, the viral infection is a viral infection selected from the group consisting of: human Immunodeficiency Virus (HIV), hepatitis virus (A, B or C), herpes virus (e.g., VZV, HSV-1, HAV-6, HSV-II and CMV, Epstein-Barr virus), adenovirus, influenza virus, flavivirus, echovirus, rhinovirus, coxsackievirus, coronavirus, respiratory syncytial virus, mumps virus, rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus, HTLV virus, dengue virus, papilloma virus, molluscum virus, poliovirus, rabies virus, JC virus or arbovirus encephalitis virus.

In one embodiment, the present invention provides a method of treating a subject with an anti-CD 39 antibody, wherein the subject is suffering from a bacterial infection. In one embodiment, the bacterial infection is a bacterial infection selected from the group consisting of: chlamydia (Chlamydia), rickettsia (rickettsiae), Mycobacterium (mycobactria), staphylococcus (staphylococci), streptococcus (streptococci), pneumococcus (pneumococi), meningococcus (meningococci) and gonococcus (gonococci), klebsiella (klebsiella), proteus (proteus), serratia (serrata), pseudomonas (pseudomonad), Legionella (Legionella), Corynebacterium diphtheriae (Corynebacterium diphtheriae), Salmonella (Salmonella), Bacillus (Bacillus), Vibrio (Vibrio cholerae), Clostridium tetani (Clostridium tetani), Clostridium botulinum (Clostridium tetani), Bacillus anthracis (Bacillus anthracis), Bacillus subtilis (Yersinia), Yersinia (Yersinia), Mycobacterium tuberculosis (Mycobacterium tuberculosis), and Mycobacterium tuberculosis (Mycobacterium tuberculosis).

In one embodiment, the present invention provides a method of treating a subject with an anti-CD 39 antibody, wherein the subject is suffering from a fungal infection. In one embodiment, the fungal infection is a fungal infection selected from the group consisting of: candida (Candida) (Candida albicans), Candida krusei (Krusei), Candida glabrata (glabrata), Candida tropicalis (tropicalis), etc.), Cryptococcus neoformans (Cryptococcus neoformans), Aspergillus (Aspergillus) (Aspergillus fumigatus), Aspergillus niger (niger, etc.), Mucor (Genus Mucorales) (Mucor), Absidia (Absidia), Rhizopus (rhizopus), Trichosporon (Sporotrichi), Bacillus dermatitidis (Blastomyces dermatitidis), Paracoccus bractealis (Paracoccus brasiliensis), Coccidioides (Coccidioides immitis), and Histoplasma capsulata (Histoplasma).

In one embodiment, the present invention provides a method of treating a subject with an anti-CD 39 antibody, wherein the subject is suffering from a parasitic infection. In one embodiment, the parasitic infection is a parasitic infection selected from the group consisting of: entamoeba histolytica (Entamoeba histolytica), Microcystis coli (Ballantidia coli), Entamoeba freudenri (Naegleria foeteri), Acanthamoeba (Acanthamoeba), Giardia lamblia (Giardia lambia), Cryptosporidium (Cryptosporidium), Pneumocystis carinii (Pneumocystis carinii), Plasmodium vivax (Plasmodium vivax), Babesia micturi (Babesia microti), Trypanosoma brucei (Trypanosoma brucei), Trypanosoma cruzi (Trypanosoma cruzi), Leishmania dorovani, Toxoplasma gondii (Toxoa ndii) and Nippostrongoides brasiliensis (Nippongostilbenis brasiliensis).

anti-CD 39 antibody conjugates

The anti-CD 39 antibodies disclosed herein can also be conjugated to a chemical moiety. The chemical moiety may be, inter alia, a polymer, a radionuclide or a cytotoxic factor.

For example, the present invention provides anti-CD 39 antibodies conjugated to a therapeutic moiety (i.e., a drug). The therapeutic moiety can be, for example, a cytotoxin, a chemotherapeutic agent, a cytokine, an immunosuppressive agent, an immunostimulatory agent, a lytic peptide, or a radioisotope. Such conjugates are referred to herein as "antibody-drug conjugates" or "ADCs". Thus, in one aspect, an anti-CD 39 antibody according to any of the above-described aspects or embodiments is conjugated to a therapeutic moiety. Exemplary therapeutic moieties include cytotoxic moieties, radioisotopes, cytokines, and lytic peptides.

In certain embodiments, the anti-CD 39 antibody is capable of inducing cytotoxicity in a CD39 expressing cell by internalizing the antibody conjugated to or associated with a cytotoxic moiety. The cytotoxic moiety may for example be selected from the group consisting of: paclitaxel; cytochalasin B; gramicidin D; ethidium bromide; emetine (emetine); mitomycin; etoposide; teniposide (tenoposide); vincristine; vinblastine; colchicine; doxorubicin; daunomycin; dihydroxy anthraquinone diones; tubulin inhibitors such as maytansine or analogs or derivatives thereof; an antimitotic agent such as monomethyl auristatin E or F or an analog or derivative thereof; dolastatin 10 or 15 or an analog thereof; irinotecan or an analog thereof; mitoxantrone; mithramycin; actinomycin D; 1-dehydrotestosterone; a glucocorticoid; procaine; tetracaine; lidocaine; propranolol; puromycin; calicheamicin or an analog or derivative thereof; antimetabolites such as methotrexate, 6 mercaptopurine, 6 thioguanine, cytarabine, fludarabine, 5 fluorouracil, dacarbazine (decarbazine), hydroxyurea, asparaginase, gemcitabine, or cladribine; alkylating agents such as nitrogen mustards, thiothiothiofos, chlorambucil, melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, Dacarbazine (DTIC), procarbazine, mitomycin C; platinum derivatives such as cisplatin or carboplatin; duocarmycin a, duocarmycin SA, lacrimycin (CC-1065), or an analog or derivative thereof; antibiotics, such as actinomycin D, bleomycin, daunomycin, doxorubicin, idarubicin, mithramycin, mitomycin, mitoxantrone, plicamycin, Anthracycline (AMC)); pyrrolo [2,1-c ] [1,4] -benzodiazepine (PDB); diphtheria toxin and related molecules such as diphtheria A chain and active fragments and hybrids thereof, ricins such as ricin A or deglycosylated ricin A chain toxin, cholera toxin, shiga-like toxins such as SLT I, SLT II, SLT IIV, LT toxin, C3 toxin, Shiga toxin, pertussis toxin, tetanus toxin, soybean Bowman-Birk protease inhibitor, Pseudomonas exotoxin, aroline, saponin (saporin), Canagliflozin toxin (modescin), Canaglucon toxin (modescin), alpha-sarcina (alpha-sarcin), alexin (gelanin), abrin A chain (abrin A chain), Canaglucoxin A chain (modeccin A chain), alpha-sarcina (alpha-sarcin), Aleurites fordii protein (Aleurites fordii protein), carnation protein (dianthinprotein), Phytolacca americana protein (Phytolacca), such as Pacifera pi, PI-PI and PIS, Momordica charantia inhibitors, curcin (curcin), crotin (crotin), saponin inhibitors (sapaonaria officinalis inhibitor), gelonin (gelonin), mitrellin (mitogellin), restrictocin (restrictocin), phenomycin (phenomycin) and enomycin (enomycin) toxins; ribonucleases (rnases); DNase I, staphylococcal enterotoxin A; pokeweed antiviral protein; diphtheria toxin; and pseudomonas endotoxins.

In one embodiment, the anti-CD 39 antibody is conjugated to an auristatin or a peptide analog, derivative, or prodrug thereof. Auristatin has been shown to interfere with microtubule dynamics, GTP hydrolysis and nuclear and cell division (Woyke et al (2001) Antimicrob. Agents and Chemotherm.45 (12): 3580-353584) and has anticancer activity (US5663149) and antifungal activity (Pettit et al (1998) Antimicrorob. Agents and Chemotherm.42: 2961-2965. for example, auristatin E can be reacted with p-acetylbenzoic acid or benzoylvaleric acid to yield AEB and AEVB. other typical auristatin derivatives including MMAF, MMAF (monomethyl auristatin F) and MMAE (monomethyl auristatin E). suitable auristatin and analogs, derivatives and AFP, and linkers suitable for conjugating the auristatin prodrug to the Ab are described in, for example, U.S. Pat. Nos. 5,635,483, 5,780,588 and 6,214 and published International patent applications WO 2000202005057345, WO 2005057345, WO 200505771390, WO 200505746,881, WO 2005057390, WO 03057390, WO 20050578, WO 03057390, WO2005084390, WO 2005057390, and WO 20071390, WO 03057390, WO 20071390, and WO 2007160.

In another embodiment, the anti-CD 39 antibody is conjugated to pyrrolo [2,1-c ] [1,4] -benzodiazepine (PDB) or an analog, derivative or prodrug thereof. Suitable PDB and PDB derivatives and related techniques are described, for example, in Hartley j.a. et al, Cancer Res 2010; 70(17) 6849-6858; antonow d. et al, Cancer J2008; 14(3) 154-169; howard p.w. et al, Bioorg Med Chem Lett 2009; 6463-; 10(18) 2083 and 2086.

In another embodiment, the anti-CD 39 antibody is conjugated to a cytotoxic moiety selected from the group consisting of: anthracycline, maytansine, calicheamicin, duocarmycin, lacrimycin (CC-1065), urocerin 10, urocerin 15, irinotecan, monomethyl auristatin E, monomethyl auristatin F, PDB, or any analog, derivative, or prodrug thereof.

In a particular embodiment, the anti-CD 39 antibody is conjugated to an anthracycline or an analog, derivative, or prodrug thereof. In another particular embodiment, the antibody is conjugated to maytansine or an analogue, derivative or prodrug thereof. In another particular embodiment, the antibody is conjugated to calicheamicin or an analog, derivative or prodrug thereof. In another particular embodiment, the antibody is conjugated to duocarmycin or an analog, derivative or prodrug thereof. In another particular embodiment, the antibody is conjugated to calicheamicin (CC-1065) or an analog, derivative, or prodrug thereof. In another particular embodiment, the antibody is conjugated to dolastatin 10 or an analog, derivative, or prodrug thereof. In another particular embodiment, the antibody is conjugated to dolastatin 15 or an analog, derivative, or prodrug thereof. In another particular embodiment, the antibody is conjugated to monomethyl auristatin E or an analog, derivative, or prodrug thereof. In another particular embodiment, the antibody is conjugated to monomethyl auristatin F or an analog, derivative, or prodrug thereof. In another particular embodiment, the antibody is conjugated to pyrrolo [2,1-c ] [1,4] -benzodiazepine or an analog, derivative or prodrug thereof. In another particular embodiment, the antibody is conjugated to irinotecan, or an analog, derivative, or prodrug thereof.

In one embodiment, the anti-CD 39 antibodies encompassed by the invention are conjugated to a nucleic acid or nucleic acid-related molecule. In one such embodiment, the conjugated nucleic acid is a cytotoxic ribonuclease (rnase) or deoxyribonuclease (e.g., dnase I), an antisense nucleic acid, an inhibitory RNA molecule (e.g., an siRNA molecule), or an immunostimulatory nucleic acid (e.g., a DNA molecule containing an immunostimulatory CpG motif). In another embodiment, the CD 39-specific antibodies encompassed by the invention are conjugated to aptamers or ribozymes.

In one embodiment, the anti-CD 39 antibodies encompassed by the invention are conjugated to lytic peptides such as CLIP, bombesin 2(Magainin 2), melittin (mellitin), Cecropin (Cecropin) and P18, for example, as fusion proteins.

In one embodiment, the anti-CD 39 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, IFN α, IFN β, IFN γ, GM-CSF, CD40L, Flt3 ligand, stem cell factor, ansamifostin (ancetim), and TNF α.

In certain embodiments, the chemical moiety is a polymer that increases the half-life of the antibody or fragment in a subject. Suitable polymers include, but are not limited to, hydrophilic polymers including, but not limited to, polyethylene glycol (PEG) (e.g., PEG having a molecular weight of 2kDa, 5kDa, 10kDa, 12kDa, 20kDa, 30kDa, 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) (bioconnj. chem.12:545-553) discloses conjugating an antibody to PEG attached to a radiometal chelator, Diethylene Triamino Pentaacetic Acid (DTPA).

anti-CD 39 antibodies may also be used⁹⁹Tc、⁹⁰Y、¹¹¹In、³²P、¹⁴C、¹²⁵I、³H、¹³¹I、¹¹C、¹⁵O、¹³N、¹⁸F、³⁵S、⁵¹Cr、⁵⁷To、²²⁶Ra、⁶⁰Co、⁵⁹Fe、⁵⁷Se、¹⁵²Eu、⁶⁷CU、²¹⁷Ci、²¹¹At、²¹²Pb、⁴⁷Sc、¹⁰⁹Pd、²³⁴Th、⁴⁰K、¹⁵⁷Gd、⁵⁵Mn、⁵²Tr and⁵⁶labeling conjugation of Fe.

The anti-CD 39 antibody may also be conjugated to fluorescent or chemiluminescent labels including fluorophores such as rare earth chelates, fluorescein and its derivatives, rhodamine and its derivatives, isothiocyanates, hemoglobin, phycocyanin, allophycocyanin, o-phthaldehyde, fluorescamine, fluorescein, and fluorescein, and fluorescein, and the like,¹⁵²Eu, dansyl (dansyl), umbelliferone, fluorescein, luminol (luminal) tag, isoluminol (isoluminol) tag, aromatic acridinium ester tag, imidazole tag, acridinium salt tag, oxalate ester tag, aequorin tag, 2, 3-dihydrophthalazinedione, biotin/avidin, spin tag, and stable free radical.

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

VIII.Pharmaceutical composition

The anti-CD 39 antibodies, antibody fragments, nucleic acids, or vectors encompassed by the invention can be formulated into compositions, particularly pharmaceutical compositions. Such compositions comprise a therapeutically or prophylactically effective amount of an anti-CD 39 antibody, antibody fragment, nucleic acid, or vector encompassed by the invention, in admixture with a suitable carrier (e.g., a pharmaceutically acceptable agent). Typically, the anti-CD 39 antibodies, antibody fragments, nucleic acids, or vectors encompassed by the invention are sufficiently purified for administration to an animal prior to formulation into a pharmaceutical composition.

Pharmaceutically acceptable agents for use in the pharmaceutical compositions of the present invention include carriers, excipients, diluents, antioxidants, preservatives, coloring agents, flavoring agents and diluents, emulsifying agents, suspending agents, solvents, fillers, bulking agents, buffers, delivery vehicles, tonicity agents, co-solvents, wetting agents, complexing agents, buffering agents, antibacterial agents and surfactants.

Neutral buffered saline or saline mixed with serum albumin are exemplary suitable carriers. The pharmaceutical composition may include an antioxidant, such as ascorbic acid; a low molecular weight polypeptide; 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 including glucose, mannose, or dextrins; chelating agents, such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions, such as sodium; and/or non-ionic surfactants such as Tween, Pluronics or polyethylene glycol (PEG). Also exemplified are suitable tonicity enhancing agents including alkali metal halides (preferably sodium chloride or potassium chloride), mannitol, sorbitol and the like. Suitable preservatives include benzalkonium chloride, thimerosal, phenylethyl alcohol, methyl paraben, propyl paraben, chlorhexidine (chlorexidine), sorbic acid, and the like. Hydrogen peroxide may also be used as a preservative. Suitable co-solvents include glycerol, 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, tyloxapol (tyloxapal), and the like. The buffer may be a conventional buffer such as acetate, borate, citrate, phosphate, bicarbonate or Tris-HCl. The acetate buffer may be about pH 4 to 5.5, and the Tris buffer may be about pH 7 to 8.5. Additional Pharmaceutical agents are described in Remington's Pharmaceutical Sciences, 18 th edition, edited by a.r. gennaro, Mack publishing company, 1990.

The compositions may be in liquid form or in lyophilized or freeze-dried form and may include one or more lyoprotectants, excipients, surfactants, high molecular weight structural additives, and/or bulking agents (see, e.g., U.S. patents 6,685,940, 6,566,329, and 6,372,716). In one embodiment, a lyoprotectant is included that is a non-reducing sugar, such as sucrose, lactose, or trehalose. The lyoprotectant is generally included in an amount such that upon reconstitution, the resulting formulation will be isotonic, although highly viscousFormulations which are isotonic or slightly hypotonic may also be suitable. In addition, the amount of lyoprotectant should be sufficient to prevent degradation and/or accumulation of unacceptable amounts of protein upon lyophilization. Exemplary lyoprotectant concentrations of sugars (e.g., sucrose, lactose, trehalose) in the pre-lyophilized formulation are from about 10mM to about 400 mM. In another embodiment, surfactants, such as nonionic surfactants and ionic surfactants, such as polysorbates (e.g., polysorbate 20, polysorbate 80); poloxamers (e.g., poloxamer 188); poly (ethylene glycol) phenyl ethers (e.g., Triton); sodium Dodecyl Sulfate (SDS); sodium lauryl sulfate; sodium octyl glucoside; lauryl-, myristyl-, linoleyl-or stearyl-sulfobetaine; lauryl-, myristyl-, linoleyl-or stearyl-sarcosine; linoleyl-, myristyl-or cetyl-betaine; lauramidopropyl-, cocamidopropyl-, phenylacetamidopropyl-, myrimidopropyl-, palmitoamidopropyl-, or isostearamidopropyl-betaine (e.g., lauramidopropyl); myrimidopropyl-, palmitoamidopropyl-, or isosteamidopropyl-dimethylamine; sodium methyl cocoyl taurate or sodium methyl taurate; and MONAQUAT ^TMSeries (Mona Industries, inc., Paterson, NJ.), polyethylene glycol, polypropylene glycol, and copolymers of ethylene glycol and propylene glycol (e.g., Pluronics, PF68, etc.). An exemplary amount of surfactant that may be present in the pre-lyophilized formulation is about 0.001% to 0.5%. High molecular weight structural additives (e.g., fillers, binders) may include, for example, gum arabic, albumin, alginic acid, calcium phosphate (dibasic acid), cellulose, carboxymethylcellulose, sodium carboxymethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, microcrystalline cellulose, dextran, dextrin, dextrates (dextrates), sucrose, infiltrates (tylose), pregelatinized starch, calcium sulfate, amylose, glycine, bentonite, maltose, sorbitol, ethylcellulose, disodium hydrogen phosphate, disodium metabisulfite, polyvinyl alcohol, gelatin, glucose, guar gum, liquid glucose, compressible sugar, magnesium aluminum silicate, maltodextrin, polyethylene oxide, polygylcellulose, sodium phosphate, sodium metabisulfite, polyethylene glycol, sodium alginate, polyethylene oxide, polyethylene glycol, and mixtures of the likeMethacrylate, povidone, sodium alginate, tragacanth microcrystalline cellulose, starch and zein. Exemplary concentrations of the high molecular weight structure additive are 0.1 wt% to 10 wt%. In other embodiments, bulking agents (e.g., mannitol, glycine) may be included.

The compositions may be suitable for parenteral administration. Exemplary compositions are suitable for injection or infusion into an animal by any route available to the skilled artisan, such as intra-articular, subcutaneous, intravenous, intramuscular, intraperitoneal, intracerebral (intraparenchymal), intracerebroventricular, intramuscular, intraocular, intraarterial, or intralesional routes. Parenteral formulations will generally be sterile, pyrogen-free isotonic aqueous solutions, optionally containing a pharmaceutically acceptable preservative.

Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, ringer's dextrose, dextrose and sodium chloride, lactated ringer's solution, or fixed oils. Intravenous vehicles include liquid and nutritional supplements, electrolyte supplements such as those based on ringer's dextrose, and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, antioxidants, chelating agents, inert gases and the like. See, generally, Remington's Pharmaceutical Science, 16 th edition, Mack eds, 1980, which is incorporated herein by reference.

The pharmaceutical compositions described herein can be formulated for controlled or sustained delivery in a manner that provides local concentrations (e.g., bolus injection, long acting effect) and/or increased stability or half-life of the product in a particular local environment. The compositions may comprise particulate formulations of the anti-CD 39 antibodies, antibody fragments, nucleic acids or vectors contemplated by the present invention with polymeric compounds (such as polylactic acid, polyglycolic acid, etc.), as well as formulations of agents such as biodegradable matrices, injectable microspheres, microcapsule particles, microcapsules, bioerodible particulate beads, liposomes, and implantable delivery devices that provide controlled or sustained release of the active agent, which can then be delivered as a long acting injectable. Techniques for formulating such sustained-release or controlled-release delivery means are known and various polymers have been developed and will be used for the controlled release and delivery of drugs. Such polymers are typically biodegradable and biocompatible. Polymeric hydrogels (including those formed by the complexation of enantiomeric polymers or polypeptide fragments) and hydrogels with temperature or pH sensitive properties may be suitable for providing drug long-lasting effects due to the mild and aqueous conditions involved in capturing bioactive protein agents (e.g., antibodies). See, for example, PCT application publication WO 93/15722 for a description of controlled release porous polymeric microparticles for delivery of pharmaceutical compositions.

Materials suitable for this purpose include polylactide (see, for example, U.S. Pat. No. 3,773,919), polymers of poly (. alpha. -hydroxycarboxylic acids), such as poly-D- (-) -3-hydroxybutyric acid (EP133,988A), copolymers of L-glutamic acid and γ -ethyl-L-glutamate (Sidman et al, Biopolymers,22:547-556(1983)), poly (2-hydroxyethyl-methacrylate) (Langer et al, J.biomed.Mater.Res.,15:167-277(1981) and Langer, chem.Tech.,12:98-105(1982)), ethylene vinyl acetate or poly-D- (-) -3-hydroxybutyric acid. Other biodegradable polymers include poly (lactones), poly (acetals), poly (orthoesters), and poly (orthocarbonates). Sustained release compositions may also include liposomes, which can be prepared by any of several methods known in the art (see, e.g., Eppstein et al, proc.natl.acad.sci.usa,82:3688-92 (1985)). The vector itself or its degradation products should be non-toxic in the target tissue and should not further exacerbate the condition. This can be determined by routine screening in animal models of the target disorder or in normal animals if these models are not available. [00196] Microencapsulation of recombinant proteins for sustained release has been successfully performed with human growth hormone (rhGH), interferon (rhIFN) -, interleukin-2, and MNrgpl 20. Johnson et al, nat. Med.,2: 795-; yasuda, biomed.Ther.,27:1221-1223 (1993); hora et al, Bio/technology v.8: 755-; cleland, "Design and Production of Single Immunization Vaccines Using polylactic polyol microspheres Systems", in Vaccine Design The Subunit and Adjuvant Approach, ed by Powell and Newman, (Plenum Press: New York,1995), pages 439 to 462; WO 97/03692, WO 96/40072, WO 96/07399; and U.S. patent No. 5,654,010. Sustained release formulations of these proteins were developed using polylactic-glycolic acid (PLGA) polymers due to their biocompatibility and broad biodegradable properties. Degradation products of PLGA (lactic and glycolic acids) are rapidly cleared in the human body. In addition, the degradability of this polymer may depend on its molecular weight and composition. Lewis, "Controlled release of bioactive agents from cellulose/polysaccharide Polymers", in M.Chasin and R.Langer (eds.), Biodegradable Polymers as Drug Delivery Systems (Marcel Dekker: New York,1990), pages 1 to 41. Additional examples of sustained release compositions include, for example, EP 58,48IA, U.S. patent nos. 3,887,699, EP158,277A, canadian patent No. 1176565, u.sidman et al, Biopolymers 22,547[1983 ]; langer et al, chem.Tech.12,98[1982 ]; sinha et al, j.control.release 90,261[2003 ]; zhu et al, nat. Biotechnol.18,24[2000] and Dai et al, Colloids Surf B Biointerfaces 41,117[2005 ].

Bioadhesive polymers are also contemplated for use in or with the compositions encompassed by the present invention. Bioadhesives are synthetic and naturally occurring materials that are capable of adhering to biological substrates for extended periods of time. For example, carbomer (Carbopol) and Polycarbophil (Polycarbophil) are both synthetic crosslinked derivatives of poly (acrylic acid). Bioadhesive delivery systems based on naturally occurring substances include, for example, hyaluronic acid, also known as hyaluronic acid. Hyaluronic acid is a naturally occurring mucopolysaccharide consisting of D-glucuronic acid and N-acetyl-D-glucosamine. Hyaluronic acid is found in the extracellular tissue matrix of vertebrates, including in connective tissue, as well as in synovial fluid and in the vitreous of the eye and in the aqueous humor. Esterified derivatives of hyaluronic acid have been used to generate microspheres for use in biocompatible and biodegradable delivery (see, e.g., Cortivo et al, Biomaterials (1991)12: 727-141; European publication No. 517,565; International publication No. WO 96/29998; Ilium et al, J.controlled ReI. (1994)29: 133-141). Exemplary hyaluronic acid-containing compositions encompassed by the invention comprise the hyaluronic acid ester polymer in an amount of about 0.1% to about 40% (w/w) IL-1/3 binding antibody or fragment bound to the hyaluronic acid polymer. [00198] Both biodegradable and non-biodegradable polymeric matrices can be used to deliver the compositions contemplated by the present invention, and such polymeric matrices can comprise natural or synthetic polymers. Biodegradable matrices are preferred. The time period for which release occurs is a polymer-based selection. In general, release over a period ranging from several hours to three to twelve months is most desirable. Exemplary synthetic polymers that can be used to form biodegradable delivery systems include: polymers of lactic acid and glycolic acid, polyamides, polycarbonates, polyolefins, polyalkylglycols, polyalkylene oxides, polyalkylene terephthalates, polyvinyl alcohols, polyvinyl ethers, polyvinyl esters, polyvinyl halides, polyvinylpyrrolidone, polyglycolides, polysiloxanes, polyanhydrides, polyurethanes and copolymers thereof, poly (butyric acid), poly (valeric acid), alkyl celluloses, hydroxyalkyl celluloses, cellulose ethers, cellulose esters, nitrocellulose, polymers of acrylic acid and methacrylic acid esters, methyl cellulose, ethyl cellulose, hydroxypropyl cellulose, hydroxy-propyl methyl cellulose, hydroxybutyl methyl cellulose, cellulose acetate, cellulose propionate, cellulose acetate butyrate, cellulose acetate phthalate, carboxyethyl cellulose, cellulose triacetate, cellulose sulfate sodium salt, poly (methyl methacrylate), poly (ethylene glycol, Poly (ethyl methacrylate), poly (butyl methacrylate), poly (isobutyl methacrylate), poly (hexyl methacrylate), poly (isodecyl methacrylate), poly (lauryl methacrylate), poly (phenyl methacrylate), poly (methyl acrylate), poly (isopropyl acrylate), poly (isobutyl acrylate), poly (octadecyl acrylate), polyethylene, polypropylene, poly (ethylene glycol), polyethylene oxide, polyethylene terephthalate, poly (vinyl alcohol), polyvinyl acetate, polyvinyl chloride, polystyrene, and polyvinyl pyrrolidone. Exemplary natural polymers include alginates and other polysaccharides, including dextrose liver and cellulose, collagen, chemical derivatives thereof (substitution of chemical groups, addition, e.g., alkyl, alkylene, hydroxylation, oxidation, and other modifications routinely made by those skilled in the art), albumin and other hydrophilic proteins, zein and other prolamine and hydrophobic proteins, copolymers, and mixtures thereof. Generally, these materials degrade by enzymatic hydrolysis or in vivo exposure to water, by surface or bulk erosion. The polymers are optionally in the form of hydrogels (see, e.g., WO 04/009664, WO 05/087201, Sawhney et al, Macromolecules,1993,26, 581-.

Delivery systems also include non-polymeric systems, which are lipids, including sterols (such as cholesterol, cholesterol esters, and fatty acids) or neutral fats (such as mono-, di-, and triglycerides); a hydrogel release system; a silicone rubber system; a peptide-based system; coating with wax; compressed tablets using conventional binders and excipients; a partially fused implant; and the like. Specific examples include, but are not limited to: (a) aggressive systems in which the product is contained in a matrix, such as those described in U.S. Pat. nos. 4,452,775, 4,675,189, and 5,736,152, and (b) diffusive systems in which the product diffuses from the polymer at a controlled rate, such as those described in U.S. Pat. nos. 3,854,480, 5,133,974, and 5,407,686. Products containing liposomes can be prepared by known methods such as, for example, (DE 3,218,121; Epstein et al, Proc. Natl. Acad. Sci. USA,82:3688-3692 (1985); Hwang et al, Proc. Natl. Acad. Sci. USA,77:4030-4034 (1980); EP 52,322; EP 36,676; EP 88,046; EP 143,949; EP 142,641; Japanese patent application 83-118008; U.S. Pat. Nos. 4,485,045 and 4,544,545; and EP 102,324).

Alternatively or additionally, the compositions may be administered topically via implantation into the affected area of a membrane, sponge, or other suitable material into which an anti-CD 39 antibody, antibody fragment, nucleic acid, or vector encompassed by the invention has been absorbed or encapsulated. Where an implanted device is used, the device may be implanted within any suitable tissue or organ, and the delivery of the anti-CD 39 antibody, antibody fragment, nucleic acid, or vector encompassed by the invention may be administered directly by the device via bolus injection, or via continuous administration, or via a catheter using continuous infusion.

Pharmaceutical compositions comprising the anti-CD 39 antibodies, antibody fragments, nucleic acids, or vectors encompassed by the invention can be formulated for inhalation, such as, for example, as a dry powder. Inhalation solutions can also be formulated in liquefied propellants for aerosol delivery. In yet another formulation, the solution may be atomized. Additional pharmaceutical compositions for pulmonary administration include those described, for example, in PCT application publication WO 94/20069, which discloses pulmonary delivery of chemically modified proteins. For pulmonary delivery, the particle size should be suitable for delivery to the remote lung. For example, the particle size may be 1 μm to 5 μm; however, larger particles may be used, for example, if each particle is fairly porous.

Certain formulations containing the anti-CD 39 antibodies, antibody fragments, nucleic acids, or vectors encompassed by the present invention can be administered orally. Formulations administered in this manner may be formulated with or without those carriers typically used in compounding solid dosage forms such as tablets and capsules. For example, a capsule can be designed to release the active portion of the formulation at a point in the gastrointestinal tract where bioavailability is maximized and pre-systemic degradation is minimized. Additional agents may be included to facilitate absorption of the selective binding agent. Diluents, flavoring agents, low melting waxes, vegetable oils, lubricants, suspending agents, tablet disintegrating agents, and binding agents may also be used.

Another formulation may involve an effective amount of an anti-CD 39 antibody, antibody fragment, nucleic acid, or vector encompassed by the invention in admixture with non-toxic excipients suitable for the manufacture of tablets. Solutions may be prepared in unit dosage form by dissolving the tablets in sterile water or another suitable vehicle. Suitable excipients include, but are not limited to, inert diluents such as calcium carbonate, sodium carbonate or bicarbonate, lactose or calcium phosphate; or a binding agent such as starch, gelatin or gum arabic; or a lubricant such as magnesium stearate, stearic acid or talc.

IX.Exemplary method

Materials and methods

Reagent

Unless otherwise specified herein, the term "a", "an", "the" or "the" is used interchangeably,otherwise all chemicals were purchased from Sigma-Aldrich (St. Louis, Mo.), cell culture media from Life Technologies (Carlsbad, Calif.), and cell culture consumables from Life Technologies (Calif.), all chemicals were purchased from Sigma-Aldrich (St. Louis, Mo.), and all cell culture consumables were purchased from Carlsbad, CalifScientific Products (Shirley, MA) and commercial antibodies were purchased from Biolegend (San Diego, CA). Comprising Alexa488-conjugated AffiniPure donkey anti-human IgG (Fc specificity) (#709-545-098) and AlexaA second 488-conjugated anti-rabbit IgG (H + L) (#711-545-152) antibody was obtained from Jackson ImmunoResearch (West Grove, Pa.),(# G7571) and Bio-Glo^TM(# G7941) was obtained from Promega (Madison, Wis.). 2-deoxy-2-fluoro-L-fucose is purchased from BIOSYNTH Carbosynth (# MD06089), and normal human serum (# A113) is purchased from Quidel corporation (San Diego, Calif.). Anti-human CD39 reference antibody (hCD39 Ref) ExpicHO was used by transient transfection ^TMExpression system kits (# A29133; Thermo Fisher Scientific, Waltham, MA) were generated and antibody sequences were obtained as disclosed (Perrot et al, Cell Reports 27:2411-2425 (2019)). Both the hCD39 Ref antibody and the fully human anti-CD 39 monoclonal antibody (Ig39-21) contain the same human IgG1 Fc portion.

The hCD39 Ref antibody shares an antigen binding site with antibodies in the art. However, unlike Ref antibodies used in the current examples, prior art antibodies were produced with an Fc portion specifically designed to have abrogated ADCC function (i.e., taught to be specifically produced to bind CD39 and inhibit NTP enzyme activity without invoking CD 39-dependent ADCC cell killing).

Cell culture

Chinese hamster ovary cells (CHO-hCD39) stably transfected with human CD39 were maintained in a cell culture supplemented with 10% Fetal Bovine Serum (FBS), 1% penicillin-streptomycinF12K. Raji cells stably transfected with human B lymphoblastoid cells (HCC1739BL, ATCC # CRL-2334), Raji cells (Raji-hCD39neg) and human CD39 (Raji-hCD39hi) were cultured in RPMI 1640 supplemented with 10% FBS, 1% penicillin-streptomycin. Human melanoma cells (SK-MEL-28, ATCC # HTB-72) were grown in EMEM plus 10% FBS, 1% penicillin-streptomycin. Human natural killer cells (NK-92-CD 16V/V) (ATCC # PTA-6967) were cultured in MEM-alpha medium with IL-2(10 ng/ml). Human Umbilical Vein Endothelial Cells (HUVEC), Single Donor, EGM ^TM-2(Lonza # C2517A, Basel, Switzerland) in EGM^TMEndothelial cell growth medium set (Lonza # CC-3124). All cell lines were incubated at 37 ℃ in 5% CO₂The atmosphere was maintained in a culture flask at 100% humidity, except that Jurkat cells/NFAT-luc + Fc γ RIIIA (Promega Cat #: G7011) were thawed in a water bath at 37 ℃ prior to use in the experiment.

Production and afucosylation of fully human anti-CD 39 antibodies

The fully human anti-CD 39 antibody Ig39-21 was prepared by using FreeStyle in the absence (Ig39-21 WT) or in the presence of the fucosylation inhibitor 2-deoxy-2-fluoro-L-fucose (Ig39-21 AF)^TM293-F cells (Thermo Fisher Scientific # R79007). Optimized Ig39-21 (identified as clone NP501-BK) was produced from stably transfected CHO cells.

Monoclonal antibody affinity to cell line expressing human CD39

CHO-hCD39 cells or HCC1739BL cells (endogenously expressing high levels of hCD39) (1X 10)⁵Individual cells) were incubated with serially diluted monoclonal antibodies at 4 ℃ for 30 minutes. After washing twice with cell staining buffer, cells were incubated with anti-human IgG (Fc specific) Alexa488(1:5000) were incubated together at 4 ℃ for 30 minutes. Cells were then washed twice with cell staining buffer and passed through Cytek ^TMAnalysis was performed by Aurora flow cytometry (Cytek Biosciences, Fremont, CA). Detecting Alexa488 Median Fluorescence Intensity (MFI) and data analyzed by FCS Express 7 Software (De Novo Software, Los Angeles, CA).

Inhibition of human CD39 enzymatic activity on intact cells

CHO-hCD39 cells (8X 10) were trypsinized⁴Individual cells/well) were counted and plated into the bottom of a 96-well plate U, followed by two washes with modified Ringer Buffer (RB) (120mM NaCl, 5mM KCl, 2.5mM caci 2, 1.2mM MgSO4, 25mM NaHCO3, 10mM glucose, 80mM Tris-HCl, pH 7.4) and incubation with monoclonal antibody at 37 ℃ for 30 minutes. CHO-hCD39 cells were then exposed to ATP (250. mu.M) for 15 minutes at room temperature. The supernatant was finally collected into a 96-well opaque-wall multi-well plate (BRAND plate #781968) and used by luminescenceATP levels are measured. Luminescence value in Synergy^TMReadings on a Neo2 multimode reader (BioTeK Instruments inc., Winooski, VT) are directly related to ATP levels. Cells without antibody (cells + ATP) or ATP alone served as controls in the absence of cells. Results are expressed as% inhibition of enzyme activity calculated from: [ (cell + ATP + Ab) - (cell + ATP)/(ATP) - (cell + ATP) ]And (4) X100. All steps are performed in RB.

NK cell mediated antibody dependent cytotoxicity (NK cytotoxicity assay)

Target cells (expressing hCD39) were pre-labeled with CFSE (0.025. mu.M) in a water bath at 37 ℃ for 5 minutes. After washing twice with 1 × PBS, cells were plated in MEM-a medium (Thermo Fisher Scientific #32561037) containing 4% ultra-low IgG FBS (Thermo Fisher Scientific # a3381901) with or without monoclonal antibody at 5% CO₂And incubated for 30 minutes. Then, the target cells were compared with NK-92-CD 16V/V effector cells at different ratios at 37 ℃ in 5% CO₂The cells were cultured for 6 hours. After incubation, cells were stained with propidium iodide (P/I) (200ng/mL) for 10 min at room temperature and passed through Cytek^TMAurora flow cytometry (Cytek Biosciences) analyzed target cell death. The results are expressed as CFSE⁺P/I⁺% of cells or% of cytotoxicity.

NFAT luciferase reporter Jurkat System (ADCC assay)

The attached target cells (SK-MEL-28 melanoma cells endogenously expressing intermediate levels of hCD39 or HUVEC cells endogenously expressing low levels of hCD39) were seeded in 96-well plates (8X 10)³Individual cells/100 μ l/well) (BRAND plate #781965) and grown for 24 hours just before the experiment (5X 10) ⁵Individual cells/ml) were inoculated with suspension target cells (HCC1739 BL). Then, the cells were washed twice with ADCC assay buffer (DMEM or RPMI 1640 medium supplemented with 4% ultra low IgG serum) and incubated with serially diluted monoclonal antibodies for 30 minutes at 37 ℃. Then, effector cells (Jurkat cells/NFAT-luc + Fc. gamma. RIIIA) (3X 10)⁶Cells/ml) were added to the wells and the mixture (E: T ═ 1:6) was incubated at 37 ℃ for 6 hours. Finally, Bio-Glo^TMAdd to well and use Synergy^TMNeo2 multimode reader (BioTeK Instruments Inc.) read the luminescence values at 5, 15 and 30 minutes. ADCC activity is indicated by an increase in luciferase activity over background.

Complement Dependent Cytotoxicity (CDC) assay

Target Raji cells overexpressing human CD39 cells (Raji-CD39hi) were washed twice with serum-free RPMI 1640 medium at 2X10⁶The final concentration of/ml was resuspended in CDC assay buffer (RPMI 1640 medium with 4% ultra-low IgG FBS) and left on ice for 2-3 hours. Cells were then incubated with serially diluted monoclonal antibodies at 37 ℃ in 5% CO₂And incubated for 30 minutes. Normal human serum (NHS 10%) was then added to the cells and incubated at 37 ℃ in 5% CO₂And incubated for 2 hours. After incubation, cells were stained with propidium iodide (P/I) (200ng/mL) for 10 min at room temperature and passed through Cytek ^TMAurora flow cytometry (Cytek Biosciences) analyzed target cell death. Results are expressed as% of cytotoxicity (P/I)⁺A cell).

Epitope competition assay

Epitope-competing substrate-I (fig. 27): using the antibody labeling kit according to the manufacturer's instructions (Thermo Fisher Scientific # A20186)Anti-human CD39 monoclonal antibodies (clones 8C11, 8D8, 8E9, 9B6 and Ig39-21 WT) and Alexa647. Mouse anti-human CD39 clone A1-PE was purchased from Biolegend (# 328208). Unconjugated human IgG1 isotype Ultra-LEAF antibody (Biolegend #403502) or anti-human CD39 monoclonal antibody (10. mu.g/mL) was conjugated to HCC1739BL cells (1X 10)⁵Individual cells) were incubated together for 30 minutes. Then, Alexa is added647 conjugated (1. mu.g/mL) or PE conjugated (0.25. mu.g/mL) anti-human CD39 monoclonal antibody was added to each well and incubated for 30 minutes at 4 ℃. Cells were then washed twice with cell staining buffer and passed through Cytek^TMAurora flow cytometry was used for the analysis. Detecting Alexa647(AF647) or PE Median Fluorescence Intensity (MFI) and analysis of the data by FCS Express 7 Software (De Novo Software). Fold change was calculated for AF647 or PE MFI assays relative to isotype controls (no epitope overlap-1).

Epitope competition matrix-II (fig. 28): unconjugated human IgG1 isotype control or hCD39 Ref monoclonal antibody (10. mu.g/mL) was incubated with HCC1739BL cells (1X 10) ⁵Individual cells) were incubated together for 30 minutes. Subsequently, a rabbit or human/rabbit chimeric clone (1. mu.g/mL) was added to each well and incubated at 4 ℃ for 30 minutes. The cells were then washed twice with cell staining buffer and with Alexa at 4 deg.C488-conjugated anti-rabbit IgG (H + L) (1:5000) was stained for 30 min. Cells were washed twice again with cell staining buffer and passed through Cytek^TMAurora flow cytometry was used for the analysis. Detecting Alexa488(AF488) MFI, and by FCS Express 7 software (De Novo Softw)are) analysis of the data. Fold change for AF488MFI assay relative to isotype control was calculated (no epitope overlap ═ 1).

Conformational epitope mapping

This was done using The proprietary CLIPS technology of Pepscan (Lelystad, The Netherlands). The results of epitope mapping were visualized using a homology model established by the Schwanese model (Swiss-model) using the template PDB entry 3ZX3. PDB.

Stable immune complex assay

At 37 ℃ in 5% CO₂HCC1739BL cells (5X 10)⁵Individual cells/mL) were incubated with anti-human CD39 antibody (2 μ g/mL) or left untreated for 24 hours. The following day, untreated cells were exposed to the same set of monoclonal antibodies (2 μ g/ml), but at 4 ℃ for 20 minutes to obtain a basal level of CD39 expression. The cells were then washed twice with cell staining buffer and with anti-human IgG (Fc specific) Alexa at 4 ℃ 488(1:2000) stain for 30 minutes, followed by two additional washes, and fix with paraformaldehyde (PFA, 2%) for 10 minutes at room temperature. Finally, cells were washed twice and passed through Cytek^TMAnalysis was performed by Aurora flow cytometry (Cytek Biosciences). Detecting Alexa488(AF488) MFI, and data were analyzed by FCS Express 7 Software (De Novo Software). The percentage of human CD39 lost on the cell membrane at 24 hours was calculated as: [ (20min MFI-24h MFI/20min MFI)]X100。

Purification of lymphocytes from tumors and spleen

Spleens and tumors from tumor-bearing mice were excised at study termination. Single cell suspensions from the spleen were obtained by mechanical engagement of the spleen through a 70 μ M cell filter, followed by red blood cell lysis (# 555899; BD Biosciences, San Jose, Calif.). Tumor infiltrating lymphocytes were obtained in GentlemACS by using mouse tumor isolation kit (#130-^TMThe manufacturer's instructions (Miltenyi Biot) were followed on the separator (#130-ec, Bergisch Gladbach, Germany). Genotyping Using Cytek^TMAurora flow cytometry. The detection antibodies are listed in table 1.

Animal research

Immunocompetent syngeneic mouse models: c57BL6 hCD39 KI mice, in which the extracellular domain of mouse CD39 was replaced by the human counterpart, have been licensed from Beth Israel deacesses medical center. Female mice six to eight weeks old were used for tumor inoculation. Syngeneic murine MC38 colorectal cancer cells were maintained in RPMI 1640 medium supplemented with 10% FBS, penicillin (100 units/mL) and streptomycin (100. mu.g/mL). Harvesting of 1X10 by Trypsin digestion ⁵MC38 cells, and with 10% FBS supplemented 150 u l RPMI1640 resuspension for injection. MC38 cells were injected subcutaneously into the right flank of the mice. The mice were then randomized into three groups (n-5 per group). On days 8, 11, 14 and 17, tumor-bearing mice received 5mg/kg of human IgG1 isotype control antibody (KLH-hIgG1) or fully human anti-CD 39 monoclonal antibody (Ig39-21 AF or NP501-BK) intraperitoneally.

Xenograft tumor model: homozygous athymic nude mice (# 002019; NU/J) were purchased from The Jackson Laboratory (Bar Harbor, ME). Female mice five weeks old were used for tumor inoculation. SK-MEL-28 xenografts were prepared by suspending 4X10 in 200. mu.l EMEM mixed 1:1 with BD Matrigel matrix (BD Biosciences #354262)⁶Individual SK-MEL-28 cells were prepared by subcutaneous injection into the right flank of the mouse. When the tumor reaches about 500mm³The mice were randomized into two groups (each group n ═ 6 to 7) and treated intraperitoneally on day 0 and day 3 with two doses of 300 μ l saline or 10mg/kg NP 501-BK.

Tumor length (L) and width (W) were measured twice weekly using digital calipers. Tumor volume (mm)³) The assay was L W0.52.

Statistical analysis

Statistical analysis was performed using GraphPad Prism 8(GraphPad Software, San Diego, CA).

TABLE 1 detection of antibodies

Is incorporated by reference

All publications, patents, and patent applications mentioned herein are hereby incorporated by reference in their entirety to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control.

Also incorporated herein by reference in its entirety are any polynucleotide and polypeptide sequences referenced to accession numbers associated with entries in public databases, such as those maintained by the genome institute (TIGR) at the world wide web and/or the National Center for Biotechnology Information (NCBI) at the world wide web.

Equivalents and ranges

The details of one or more implementations are set forth in the description above. Although preferred materials and methods have been described above, any materials and methods similar or equivalent to those described herein can be used in the practice or testing of embodiments encompassed by the present invention. Other features, objects, and advantages associated with the invention will be apparent from the description. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present disclosure as provided above will be described as controlling.

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. The scope of coverage of this disclosure is not intended to be limited to the description provided herein and such equivalents are intended to be covered by the appended claims.

The articles "a" and "an" as used herein refer to one or to more than one (i.e., to at least one) of the grammatical object of the article, unless otherwise indicated to the contrary or otherwise apparent from the context. By way of example, "an element" means one element or more than one element. Claims or descriptions that include an "or" between one or more members of a group are deemed to be satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The invention includes embodiments in which exactly one member of the group is present in, taken from, or otherwise relevant to a given product or process. The invention also includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process.

It should also be noted that the term "comprising" is intended to be open-ended and allows, but does not require, the inclusion of additional elements or steps. When the term "comprising" is used herein, the term "consisting of" is also hereby encompassed and disclosed.

In the case of a given range, the endpoints are included. Further, it is to be understood that unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values expressed as ranges can assume any specific value or subrange within the stated range in different embodiments encompassed by the invention, unless the context clearly indicates otherwise, to one tenth of the unit of the lower limit of the range.

In addition, it should be understood that any particular implementation encompassed by the invention falling within the prior art may be explicitly excluded from any one or more of the claims. Since such embodiments are considered to be known to those of ordinary skill in the art, they may be excluded even if the exclusion is not explicitly set forth herein. Any particular embodiment of a composition encompassed by the present invention (e.g., any antibiotic, therapeutic or active ingredient; any method of production; any method of use; etc.) may be excluded from any one or more claims for any reason, whether or not relevant to the presence of prior art.

It is to be understood that the words which have been used are words of description rather than limitation, and that changes may be made within the scope of the appended claims without departing from the true scope and spirit of the invention in its broader aspects.

Although the present invention has been described in relation to several embodiments described herein, in certain length and with certain features, it is not intended to be limited to any such details or embodiments or any particular embodiments, but rather should be construed with reference to the appended claims so as to provide the broadest possible interpretation of such claims in view of the prior art and, therefore, to effectively encompass the intended scope encompassed by the invention.

Sequence listing

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Wu Yan (Wu, Yan)

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<220>

<223> derived from rabbit monoclonal

<220>

<221> CDS

<222> (1)..(345)

<400> 9

cag tcg gtg gag gag tcc ggg ggt cgc ctg gtc acg cct ggg aca cac 48

Gln Ser Val Glu Glu Ser Gly Gly Arg Leu Val Thr Pro Gly Thr His

1 5 10 15

ctg aca ctc acc tgc aca gtc tct gga ttc tcc ctc agt aag agt ata 96

Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Lys Ser Ile

20 25 30

ata agt tgg gtc cgc cag gct cca ggg aag ggg ctg gaa tac atc gga 144

Ile Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr Ile Gly

35 40 45

atc att ggt agt agt ggt agc aca tac tac gcg aac tgg gcg aaa ggc 192

Ile Ile Gly Ser Ser Gly Ser Thr Tyr Tyr Ala Asn Trp Ala Lys Gly

50 55 60

cga ttc acc atc tcc aaa acc tcg tcg acc acg gtg gat ctg aga atg 240

Arg Phe Thr Ile Ser Lys Thr Ser Ser Thr Thr Val Asp Leu Arg Met

65 70 75 80

acc agt ctg aca ccc gag gac acg gcc acc tat ttc tgt gcc aga gga 288

Thr Ser Leu Thr Pro Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg Gly

85 90 95

ctt ctt tat tct ggt aat aaa tcg tgg ggc ccg ggc acc ctg gtc acc 336

Leu Leu Tyr Ser Gly Asn Lys Ser Trp Gly Pro Gly Thr Leu Val Thr

100 105 110

gtc tcc tca 345

Val Ser Ser

115

<210> 10

<211> 115

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<400> 10

Gln Ser Val Glu Glu Ser Gly Gly Arg Leu Val Thr Pro Gly Thr His

1 5 10 15

Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Lys Ser Ile

20 25 30

Ile Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr Ile Gly

35 40 45

Ile Ile Gly Ser Ser Gly Ser Thr Tyr Tyr Ala Asn Trp Ala Lys Gly

50 55 60

Arg Phe Thr Ile Ser Lys Thr Ser Ser Thr Thr Val Asp Leu Arg Met

65 70 75 80

Thr Ser Leu Thr Pro Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg Gly

85 90 95

Leu Leu Tyr Ser Gly Asn Lys Ser Trp Gly Pro Gly Thr Leu Val Thr

100 105 110

Val Ser Ser

115

<210> 11

<211> 339

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> derived from rabbit monoclonal

<220>

<221> CDS

<222> (1)..(339)

<400> 11

gcc aca ttt gcc att gat atg acc cag act cca tcc tcc gtg tct gca 48

Ala Thr Phe Ala Ile Asp Met Thr Gln Thr Pro Ser Ser Val Ser Ala

1 5 10 15

gct gtg gga ggc aca gtc acc atc aac tgc cag tcc agt cag agt gtt 96

Ala Val Gly Gly Thr Val Thr Ile Asn Cys Gln Ser Ser Gln Ser Val

20 25 30

tta ctg aac aac caa tta tcc tgg ttt cag cag aaa cca ggg cag cct 144

Leu Leu Asn Asn Gln Leu Ser Trp Phe Gln Gln Lys Pro Gly Gln Pro

35 40 45

ccc aag ctc ctg atc tat gat gca tcc act ctg gaa tct ggg gtc cca 192

Pro Lys Leu Leu Ile Tyr Asp Ala Ser Thr Leu Glu Ser Gly Val Pro

50 55 60

tct cgg ttc aca ggc agt gga tct ggg aca cag ttc act ctc acc atc 240

Ser Arg Phe Thr Gly Ser Gly Ser Gly Thr Gln Phe Thr Leu Thr Ile

65 70 75 80

agc gac ctg gag tgt gac gat gct gcc act tac tat tgt tta ggc ggt 288

Ser Asp Leu Glu Cys Asp Asp Ala Ala Thr Tyr Tyr Cys Leu Gly Gly

85 90 95

tat agt ggg aac ctt tat gct ttc ggc gga ggg acc gag gtg cta gtc 336

Tyr Ser Gly Asn Leu Tyr Ala Phe Gly Gly Gly Thr Glu Val Leu Val

100 105 110

aaa 339

Lys

<210> 12

<211> 113

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<400> 12

Ala Thr Phe Ala Ile Asp Met Thr Gln Thr Pro Ser Ser Val Ser Ala

1 5 10 15

Ala Val Gly Gly Thr Val Thr Ile Asn Cys Gln Ser Ser Gln Ser Val

20 25 30

Leu Leu Asn Asn Gln Leu Ser Trp Phe Gln Gln Lys Pro Gly Gln Pro

35 40 45

Pro Lys Leu Leu Ile Tyr Asp Ala Ser Thr Leu Glu Ser Gly Val Pro

50 55 60

Ser Arg Phe Thr Gly Ser Gly Ser Gly Thr Gln Phe Thr Leu Thr Ile

65 70 75 80

Ser Asp Leu Glu Cys Asp Asp Ala Ala Thr Tyr Tyr Cys Leu Gly Gly

85 90 95

Tyr Ser Gly Asn Leu Tyr Ala Phe Gly Gly Gly Thr Glu Val Leu Val

100 105 110

Lys

<210> 13

<211> 351

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> derived from rabbit monoclonal

<220>

<221> CDS

<222> (1)..(351)

<400> 13

cag tcg gtg gag gag tcc ggg ggt cgc ctg gtc acg cct ggg aca ccc 48

Gln Ser Val Glu Glu Ser Gly Gly Arg Leu Val Thr Pro Gly Thr Pro

1 5 10 15

ctg aca ctc acc tgc aca gtc tct gga ttc tcc ctc agt agc tat gca 96

Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Ser Tyr Ala

20 25 30

ata agt tgg gtc cgc cag gct cca ggg aag ggg ctc gaa tat atc gcg 144

Ile Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr Ile Ala

35 40 45

atc att aat agt tat ggt acc aca tac tac gcg agc tgg gcg aaa ggc 192

Ile Ile Asn Ser Tyr Gly Thr Thr Tyr Tyr Ala Ser Trp Ala Lys Gly

50 55 60

cga gtc acc atc tcc aaa acc tcg agc acg gtg gat ctg aaa atc tcc 240

Arg Val Thr Ile Ser Lys Thr Ser Ser Thr Val Asp Leu Lys Ile Ser

65 70 75 80

agt ccg aca acc gag gac acg gcc acc tat ttc tgt gcc aga ggc gat 288

Ser Pro Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg Gly Asp

85 90 95

agt tat ggt agt ggt gtt ggt ttg ggc ttg tgg ggc cca ggc acc ctg 336

Ser Tyr Gly Ser Gly Val Gly Leu Gly Leu Trp Gly Pro Gly Thr Leu

100 105 110

gtc acc gtc tcc tca 351

Val Thr Val Ser Ser

115

<210> 14

<211> 117

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<400> 14

Gln Ser Val Glu Glu Ser Gly Gly Arg Leu Val Thr Pro Gly Thr Pro

1 5 10 15

Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Ser Tyr Ala

20 25 30

Ile Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr Ile Ala

35 40 45

Ile Ile Asn Ser Tyr Gly Thr Thr Tyr Tyr Ala Ser Trp Ala Lys Gly

50 55 60

Arg Val Thr Ile Ser Lys Thr Ser Ser Thr Val Asp Leu Lys Ile Ser

65 70 75 80

Ser Pro Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg Gly Asp

85 90 95

Ser Tyr Gly Ser Gly Val Gly Leu Gly Leu Trp Gly Pro Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 15

<211> 339

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> derived from rabbit monoclonal

<220>

<221> CDS

<222> (1)..(339)

<400> 15

gcc aga tgt gcc tat gat atg acc cag act cca gcc tct gtg gag gta 48

Ala Arg Cys Ala Tyr Asp Met Thr Gln Thr Pro Ala Ser Val Glu Val

1 5 10 15

gct gtg gga ggc aca gtc acc atc aag tgc cag gcc agt cag aac att 96

Ala Val Gly Gly Thr Val Thr Ile Lys Cys Gln Ala Ser Gln Asn Ile

20 25 30

tac agc aat tta gcc tgg tat cag cag aaa cca ggg cag cgt ccc aag 144

Tyr Ser Asn Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Arg Pro Lys

35 40 45

ctc ctg atc tac agg gca tcc agt ctg gca tct ggg gtc ccg tcg cgg 192

Leu Leu Ile Tyr Arg Ala Ser Ser Leu Ala Ser Gly Val Pro Ser Arg

50 55 60

ttc agt ggc agt gga tct ggg aca gag ttc act ctc acc atc agc ggt 240

Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Gly

65 70 75 80

gtg cag tgt gac gat gct gcc act tac tac tgt caa cag ggt ttt agt 288

Val Gln Cys Asp Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Gly Phe Ser

85 90 95

agt aat aat gtt gat aat act ttc ggc gga ggg acc gag gtg gtg gtc 336

Ser Asn Asn Val Asp Asn Thr Phe Gly Gly Gly Thr Glu Val Val Val

100 105 110

aaa 339

Lys

<210> 16

<211> 113

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<400> 16

Ala Arg Cys Ala Tyr Asp Met Thr Gln Thr Pro Ala Ser Val Glu Val

1 5 10 15

Ala Val Gly Gly Thr Val Thr Ile Lys Cys Gln Ala Ser Gln Asn Ile

20 25 30

Tyr Ser Asn Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Arg Pro Lys

35 40 45

Leu Leu Ile Tyr Arg Ala Ser Ser Leu Ala Ser Gly Val Pro Ser Arg

50 55 60

Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Gly

65 70 75 80

Val Gln Cys Asp Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Gly Phe Ser

85 90 95

Ser Asn Asn Val Asp Asn Thr Phe Gly Gly Gly Thr Glu Val Val Val

100 105 110

Lys

<210> 17

<211> 357

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> derived from rabbit monoclonal

<220>

<221> CDS

<222> (1)..(357)

<400> 17

cag tcg gtg gag gag tcc ggg ggt cgc ctg gtc acg cct ggg aca ccc 48

Gln Ser Val Glu Glu Ser Gly Gly Arg Leu Val Thr Pro Gly Thr Pro

1 5 10 15

ctg aca ctc acc tgc acc gtc tcc gga ttc tcc ctc agt agc tat gca 96

Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Ser Tyr Ala

20 25 30

atg agc tgg gtc cgc cag gct cca ggg aag ggg ctg gaa tac atc gga 144

Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr Ile Gly

35 40 45

atc att agt agt agt ggt agc aca tac tac gcg agc tgg gcg aaa ggc 192

Ile Ile Ser Ser Ser Gly Ser Thr Tyr Tyr Ala Ser Trp Ala Lys Gly

50 55 60

cga ttc acc atc tcc aaa acc tcg acc acg gtg gat ctg aaa atc tcc 240

Arg Phe Thr Ile Ser Lys Thr Ser Thr Thr Val Asp Leu Lys Ile Ser

65 70 75 80

agt ccg aca acc gag gac acg gcc acc tat ttc tgt gcc aga gat cgt 288

Ser Pro Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg Asp Arg

85 90 95

gtt att tat agt att ggt ccg tat tat ttt aat ttg tgg ggc cca ggc 336

Val Ile Tyr Ser Ile Gly Pro Tyr Tyr Phe Asn Leu Trp Gly Pro Gly

100 105 110

acc ctg gtc acc gtc tcc tca 357

Thr Leu Val Thr Val Ser Ser

115

<210> 18

<211> 119

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<400> 18

Gln Ser Val Glu Glu Ser Gly Gly Arg Leu Val Thr Pro Gly Thr Pro

1 5 10 15

Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Ser Tyr Ala

20 25 30

Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr Ile Gly

35 40 45

Ile Ile Ser Ser Ser Gly Ser Thr Tyr Tyr Ala Ser Trp Ala Lys Gly

50 55 60

Arg Phe Thr Ile Ser Lys Thr Ser Thr Thr Val Asp Leu Lys Ile Ser

65 70 75 80

Ser Pro Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg Asp Arg

85 90 95

Val Ile Tyr Ser Ile Gly Pro Tyr Tyr Phe Asn Leu Trp Gly Pro Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 19

<211> 339

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> derived from rabbit monoclonal

<220>

<221> CDS

<222> (1)..(339)

<400> 19

gcc aga tgt gcc tat gat atg acc cag act cca tcc tcc gtg tct gca 48

Ala Arg Cys Ala Tyr Asp Met Thr Gln Thr Pro Ser Ser Val Ser Ala

1 5 10 15

act gtg gga ggc aca gtc acc atc aat tgc cag gcc agt gag atc att 96

Thr Val Gly Gly Thr Val Thr Ile Asn Cys Gln Ala Ser Glu Ile Ile

20 25 30

tat agc aat tta gcc tgg tat cag cag aaa cca ggg cag cct ccc aag 144

Tyr Ser Asn Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys

35 40 45

ctc ctg atc tat ggc gca tcc act ctg gca tct ggg gtc cca tcg cgg 192

Leu Leu Ile Tyr Gly Ala Ser Thr Leu Ala Ser Gly Val Pro Ser Arg

50 55 60

ttc aaa ggc agt gga tct ggg aca gag tac act ctc acc atc agc gac 240

Phe Lys Gly Ser Gly Ser Gly Thr Glu Tyr Thr Leu Thr Ile Ser Asp

65 70 75 80

ctg cag tgt gac gat gct gcc act tac tac tgt caa cag agt ttt agt 288

Leu Gln Cys Asp Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Ser Phe Ser

85 90 95

agt aat aat gtt ggg aat att ttc ggc gga ggg acc gag gtg gtg gtc 336

Ser Asn Asn Val Gly Asn Ile Phe Gly Gly Gly Thr Glu Val Val Val

100 105 110

aaa 339

Lys

<210> 20

<211> 113

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<400> 20

Ala Arg Cys Ala Tyr Asp Met Thr Gln Thr Pro Ser Ser Val Ser Ala

1 5 10 15

Thr Val Gly Gly Thr Val Thr Ile Asn Cys Gln Ala Ser Glu Ile Ile

20 25 30

Tyr Ser Asn Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys

35 40 45

Leu Leu Ile Tyr Gly Ala Ser Thr Leu Ala Ser Gly Val Pro Ser Arg

50 55 60

Phe Lys Gly Ser Gly Ser Gly Thr Glu Tyr Thr Leu Thr Ile Ser Asp

65 70 75 80

Leu Gln Cys Asp Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Ser Phe Ser

85 90 95

Ser Asn Asn Val Gly Asn Ile Phe Gly Gly Gly Thr Glu Val Val Val

100 105 110

Lys

<210> 21

<211> 351

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> derived from rabbit monoclonal

<220>

<221> CDS

<222> (1)..(351)

<400> 21

cag tcg gtg gag gag tcc ggg ggt cgc ctg gtc acg cct ggg aca ccc 48

Gln Ser Val Glu Glu Ser Gly Gly Arg Leu Val Thr Pro Gly Thr Pro

1 5 10 15

ctg aca ctc acc tgc aca gcc tct gga ttc tcc ctc agt acc cat gca 96

Leu Thr Leu Thr Cys Thr Ala Ser Gly Phe Ser Leu Ser Thr His Ala

20 25 30

ata aac tgg gtc cgc cag gct cca ggg aag ggg ctg gaa tgg atc ggg 144

Ile Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile Gly

35 40 45

atc act tat gct agt ggt agg aca tat tac gcg agc tgg gcg aaa ggc 192

Ile Thr Tyr Ala Ser Gly Arg Thr Tyr Tyr Ala Ser Trp Ala Lys Gly

50 55 60

cga ttc acc atc tcc aaa acc tcg acc acg gtg gat ctg aaa atc acc 240

Arg Phe Thr Ile Ser Lys Thr Ser Thr Thr Val Asp Leu Lys Ile Thr

65 70 75 80

agt ccg aca acc gag gac acg gcc acc tat ttc tgt gcc aga aat ggg 288

Ser Pro Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg Asn Gly

85 90 95

gct gat gaa aca ttt tac tac ttt gac ttg tgg ggc cca ggc acc ctg 336

Ala Asp Glu Thr Phe Tyr Tyr Phe Asp Leu Trp Gly Pro Gly Thr Leu

100 105 110

gtc acc gtc tcc tca 351

Val Thr Val Ser Ser

115

<210> 22

<211> 117

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<400> 22

Gln Ser Val Glu Glu Ser Gly Gly Arg Leu Val Thr Pro Gly Thr Pro

1 5 10 15

Leu Thr Leu Thr Cys Thr Ala Ser Gly Phe Ser Leu Ser Thr His Ala

20 25 30

Ile Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile Gly

35 40 45

Ile Thr Tyr Ala Ser Gly Arg Thr Tyr Tyr Ala Ser Trp Ala Lys Gly

50 55 60

Arg Phe Thr Ile Ser Lys Thr Ser Thr Thr Val Asp Leu Lys Ile Thr

65 70 75 80

Ser Pro Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg Asn Gly

85 90 95

Ala Asp Glu Thr Phe Tyr Tyr Phe Asp Leu Trp Gly Pro Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 23

<211> 339

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> derived from rabbit monoclonal

<220>

<221> CDS

<222> (1)..(339)

<400> 23

gcc aga tgt gcc tat gat atg acc cag act cca gcc tcc gtg gag gca 48

Ala Arg Cys Ala Tyr Asp Met Thr Gln Thr Pro Ala Ser Val Glu Ala

1 5 10 15

gct gtg gga ggc aca gtc acc atc aag tgc cag gcc agt cag aat att 96

Ala Val Gly Gly Thr Val Thr Ile Lys Cys Gln Ala Ser Gln Asn Ile

20 25 30

aat act tgg tta tcc tgg tat cag cag aag gca ggg cag cct ccc aag 144

Asn Thr Trp Leu Ser Trp Tyr Gln Gln Lys Ala Gly Gln Pro Pro Lys

35 40 45

ctc ctg atc tac agg gca tcc act ctg gca tct ggg gtc tca tcg cgg 192

Leu Leu Ile Tyr Arg Ala Ser Thr Leu Ala Ser Gly Val Ser Ser Arg

50 55 60

ttc aaa ggc agt gga tct ggg aca cag ttc act ctc acc atc agc ggc 240

Phe Lys Gly Ser Gly Ser Gly Thr Gln Phe Thr Leu Thr Ile Ser Gly

65 70 75 80

gtg gag tgt gcc gat gct gcc act tac tac tgt caa caa tat gat gct 288

Val Glu Cys Ala Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Tyr Asp Ala

85 90 95

agt att aat att gat aat gct ttc ggc gga ggg acc gag gtg gtg gtc 336

Ser Ile Asn Ile Asp Asn Ala Phe Gly Gly Gly Thr Glu Val Val Val

100 105 110

aaa 339

Lys

<210> 24

<211> 113

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<400> 24

Ala Arg Cys Ala Tyr Asp Met Thr Gln Thr Pro Ala Ser Val Glu Ala

1 5 10 15

Ala Val Gly Gly Thr Val Thr Ile Lys Cys Gln Ala Ser Gln Asn Ile

20 25 30

Asn Thr Trp Leu Ser Trp Tyr Gln Gln Lys Ala Gly Gln Pro Pro Lys

35 40 45

Leu Leu Ile Tyr Arg Ala Ser Thr Leu Ala Ser Gly Val Ser Ser Arg

50 55 60

Phe Lys Gly Ser Gly Ser Gly Thr Gln Phe Thr Leu Thr Ile Ser Gly

65 70 75 80

Val Glu Cys Ala Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Tyr Asp Ala

85 90 95

Ser Ile Asn Ile Asp Asn Ala Phe Gly Gly Gly Thr Glu Val Val Val

100 105 110

Lys

<210> 25

<211> 351

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> derived from rabbit monoclonal

<220>

<221> CDS

<222> (1)..(351)

<400> 25

cag tcg gtg gag gag tcc ggg ggt cgc ctg gtc acg cct ggg aca ccc 48

Gln Ser Val Glu Glu Ser Gly Gly Arg Leu Val Thr Pro Gly Thr Pro

1 5 10 15

ctg aca ctc acc tgc aca gtc tct gga atc gac ctc agt agc aat gca 96

Leu Thr Leu Thr Cys Thr Val Ser Gly Ile Asp Leu Ser Ser Asn Ala

20 25 30

atg agc tgg gtc cgc cag gct cca ggg aag ggg ctg gaa tat atc gga 144

Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr Ile Gly

35 40 45

att att agg aat aat gat atc aca tac tac gcg agc tgg gcg aaa ggc 192

Ile Ile Arg Asn Asn Asp Ile Thr Tyr Tyr Ala Ser Trp Ala Lys Gly

50 55 60

cga ttc acc atc tcc aaa acc tcg acc acg gtg gat ctg ata atc acc 240

Arg Phe Thr Ile Ser Lys Thr Ser Thr Thr Val Asp Leu Ile Ile Thr

65 70 75 80

agt ccg aca acc gag gac acg gcc acc tat ttc tgt gcc aga ggg ggt 288

Ser Pro Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg Gly Gly

85 90 95

ggt tct tac agt att gtc ttc tgg aac tta tgg ggc cca ggc acc ctg 336

Gly Ser Tyr Ser Ile Val Phe Trp Asn Leu Trp Gly Pro Gly Thr Leu

100 105 110

gtc acc gtc tcc tca 351

Val Thr Val Ser Ser

115

<210> 26

<211> 117

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<400> 26

Gln Ser Val Glu Glu Ser Gly Gly Arg Leu Val Thr Pro Gly Thr Pro

1 5 10 15

Leu Thr Leu Thr Cys Thr Val Ser Gly Ile Asp Leu Ser Ser Asn Ala

20 25 30

Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr Ile Gly

35 40 45

Ile Ile Arg Asn Asn Asp Ile Thr Tyr Tyr Ala Ser Trp Ala Lys Gly

50 55 60

Arg Phe Thr Ile Ser Lys Thr Ser Thr Thr Val Asp Leu Ile Ile Thr

65 70 75 80

Ser Pro Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg Gly Gly

85 90 95

Gly Ser Tyr Ser Ile Val Phe Trp Asn Leu Trp Gly Pro Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 27

<211> 339

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> derived from rabbit monoclonal

<220>

<221> CDS

<222> (1)..(339)

<400> 27

gcc aga tgt gcc tat gat atg acc cag act cca gcc tct gtg gag gta 48

Ala Arg Cys Ala Tyr Asp Met Thr Gln Thr Pro Ala Ser Val Glu Val

1 5 10 15

gct gtg gga ggc aca gtc acc atc aat tgc cag gcc agt gag agg att 96

Ala Val Gly Gly Thr Val Thr Ile Asn Cys Gln Ala Ser Glu Arg Ile

20 25 30

tat agc aat tta gcc tgg tat cag cag aaa cca ggg cag cgt ccc aaa 144

Tyr Ser Asn Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Arg Pro Lys

35 40 45

ctc ctg atc tat tat gca tcc act ctg gca tct ggg gtc tca tcg cgg 192

Leu Leu Ile Tyr Tyr Ala Ser Thr Leu Ala Ser Gly Val Ser Ser Arg

50 55 60

ttc aaa ggc agt gga tct ggg aca cag ttc act ctc acc atc agc ggc 240

Phe Lys Gly Ser Gly Ser Gly Thr Gln Phe Thr Leu Thr Ile Ser Gly

65 70 75 80

gtg cag tgt gcc gat gct gcc act tac tac tgt cag cag ggt tat agt 288

Val Gln Cys Ala Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Gly Tyr Ser

85 90 95

aat aat aat gtt gac aat act ttc ggc gga ggg acc gag gtg gtg gtc 336

Asn Asn Asn Val Asp Asn Thr Phe Gly Gly Gly Thr Glu Val Val Val

100 105 110

aga 339

Arg

<210> 28

<211> 113

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<400> 28

Ala Arg Cys Ala Tyr Asp Met Thr Gln Thr Pro Ala Ser Val Glu Val

1 5 10 15

Ala Val Gly Gly Thr Val Thr Ile Asn Cys Gln Ala Ser Glu Arg Ile

20 25 30

Tyr Ser Asn Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Arg Pro Lys

35 40 45

Leu Leu Ile Tyr Tyr Ala Ser Thr Leu Ala Ser Gly Val Ser Ser Arg

50 55 60

Phe Lys Gly Ser Gly Ser Gly Thr Gln Phe Thr Leu Thr Ile Ser Gly

65 70 75 80

Val Gln Cys Ala Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Gly Tyr Ser

85 90 95

Asn Asn Asn Val Asp Asn Thr Phe Gly Gly Gly Thr Glu Val Val Val

100 105 110

Arg

<210> 29

<211> 8

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> derived from human VH Domain

<400> 29

Gly Phe Thr Phe Ser Ser Tyr Ala

1 5

<210> 30

<211> 8

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> derived from human VH Domain

<400> 30

Ile Ser Tyr Asp Val Ser Asn Lys

1 5

<210> 31

<211> 12

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> derived from human VH Domain

<400> 31

Ala Arg Ser Tyr Tyr Tyr Tyr Tyr Gly Met Asp Val

1 5 10

<210> 32

<211> 6

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> derived from human VL domain

<400> 32

Gln Ser Ile Ser Arg Tyr

1 5

<210> 33

<211> 3

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> derived from human VL domain

<400> 33

Asp Ala Ser

1

<210> 34

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> derived from human VL domain

<400> 34

Gln Gln Phe Gly Arg Ser Pro Arg Thr

1 5

<210> 35

<211> 1350

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> derived from human variable domains and human Ig domains

<220>

<221> CDS

<222> (1)..(1350)

<400> 35

gag gtg caa ctg gtg gag tct ggg gga ggt gtg gta agg cct ggg ggg 48

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Arg Pro Gly Gly

1 5 10 15

tcc ctg aga ctc tcc tgt gca gcc tct gga ttc acc ttc agt agc tat 96

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

gct atg cac tgg gtc cgc cag gct cca ggc aag ggg ctg gag tgg gtg 144

Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

gca gtt ata tca tat gat gta agc aat aaa tac tac gca gac tcc gtg 192

Ala Val Ile Ser Tyr Asp Val Ser Asn Lys Tyr Tyr Ala Asp Ser Val

50 55 60

aag ggc cga ttc acc atc tcc aga gac aat tcc aag aac acg ctg tat 240

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

ctg caa atg aac agc ctg aga gct gag gac acg gct gtg tat tac tgt 288

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

gcg aga tct tac tac tac tac tac ggt atg gac gtc tgg ggc caa ggg 336

Ala Arg Ser Tyr Tyr Tyr Tyr Tyr Gly Met Asp Val Trp Gly Gln Gly

100 105 110

acc acg gtc acc gtc tcc tca gcc tcc act aag ggc cca tcc gtg ttc 384

Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

cca ctg gca ccc tct agt aag agc aca tct ggg ggt act gcc gct ctg 432

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

gga tgt ctg gtg aag gat tac ttc cca gag cca gtc acc gtg tcc tgg 480

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

aac agc ggg gcc ctg act tcc ggt gtc cat acc ttt cca gct gtg ctg 528

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

cag tca tcc ggc ctg tac agc ctg agc tct gtg gtc acc gtc ccc agt 576

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

tca tcc ctg gga aca cag act tat atc tgc aac gtg aat cac aag cca 624

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

tcc aat aca aaa gtc gac aag aaa gtg gaa ccc aag agc tgt gat aaa 672

Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

acc cat aca tgc ccc cct tgt cct gct cca gag ctg ctg gga gga cca 720

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

225 230 235 240

tcc gtg ttc ctg ttt cca ccc aag cct aaa gac act ctg atg att tct 768

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

245 250 255

cga acc ccc gaa gtc aca tgc gtg gtc gtg gac gtg tcc cac gag gat 816

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

260 265 270

cct gaa gtc aag ttc aac tgg tac gtg gat ggc gtc gag gtg cat aat 864

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

gcc aag aca aaa cca cga gag gaa cag tac aac agt acc tat cgt gtc 912

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

290 295 300

gtg tca gtc ctg aca gtg ctg cac cag gac tgg ctg aac ggg aag gaa 960

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

tat aag tgc aaa gtg agc aat aag gca ctg ccc gcc cct atc gag aaa 1008

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

325 330 335

aca att tct aag gct aaa gga cag cct agg gaa cca cag gtg tac act 1056

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

340 345 350

ctg cct cca tca cgg gac gag ctg aca aag aac cag gtc agt ctg act 1104

Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr

355 360 365

tgt ctg gtg aaa ggg ttc tat cct tct gat atc gcc gtg gag tgg gaa 1152

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

agt aat ggt cag cca gag aac aat tac aag acc aca ccc cct gtc ctg 1200

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

gac tct gat ggg agt ttc ttt ctg tat tcc aag ctg acc gtg gat aaa 1248

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

405 410 415

agc cgg tgg cag cag ggt aat gtc ttt agt tgt tca gtg atg cac gag 1296

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

gca ctg cac aat cac tac acc cag aaa tca ctg tca ctg tca cca ggt 1344

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

aaa tga 1350

Lys

<210> 36

<211> 449

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<400> 36

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Arg Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Val Ile Ser Tyr Asp Val Ser Asn Lys Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Tyr Tyr Tyr Tyr Tyr Gly Met Asp Val Trp Gly Gln Gly

100 105 110

Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

260 265 270

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

340 345 350

Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr

355 360 365

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

Lys

<210> 37

<211> 645

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> derived from human variable domains and human Ig domains

<220>

<221> CDS

<222> (1)..(645)

<400> 37

gat gtt gtg atg acc cag tct cca tcc tcc ctg tct gca tct gta gga 48

Asp Val Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

gac aga gtc acc atc act tgc cgg gca agt cag agc att agc agg tac 96

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Arg Tyr

20 25 30

tta gcc tgg tac caa cag aaa cct ggc cag gct ccc agg ctc ctc atc 144

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

tat gat gca tcc aac agg gcc act ggc atc cca gtc agg ttc agt ggc 192

Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Val Arg Phe Ser Gly

50 55 60

agt ggg tct ggg aca gac ttc act ctc acc atc agc aga ctg gag cca 240

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro

65 70 75 80

gaa gat ttt gca gtg tat tac tgt cag cag ttt ggt agg tca cct cgg 288

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Phe Gly Arg Ser Pro Arg

85 90 95

acg ttc ggc caa ggg aca cga ctg gag att aaa cga act gtg gct gca 336

Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

cca tct gtc ttc atc ttc ccg cca tct gat gag cag ttg aaa tct gga 384

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

act gcc tct gtt gtg tgc ctg ctg aat aac ttc tat ccc aga gag gcc 432

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

aaa gta cag tgg aag gtg gat aac gcc ctc caa tcg ggt aac tcc cag 480

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

gag agt gtc aca gag cag gac agc aag gac agc acc tac agc ctc agc 528

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

agc acc ctg acg ctg agc aaa gca gac tac gag aaa cac aaa gtc tac 576

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

gcc tgc gaa gtc acc cat cag ggc ctg agc tcg ccc gtc aca aag agc 624

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

ttc aac agg gga gag tgt tag 645

Phe Asn Arg Gly Glu Cys

210

<210> 38

<211> 214

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<400> 38

Asp Val Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Arg Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Val Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Phe Gly Arg Ser Pro Arg

85 90 95

Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 39

<211> 732

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> derived from human variable domains

<220>

<221> CDS

<222> (1)..(732)

<400> 39

gat gtt gtg atg acc cag tct cca tcc tcc ctg tct gca tct gta gga 48

Asp Val Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

gac aga gtc acc atc act tgc cgg gca agt cag agc att agc agg tac 96

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Arg Tyr

20 25 30

tta gcc tgg tac caa cag aaa cct ggc cag gct ccc agg ctc ctc atc 144

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

tat gat gca tcc aac agg gcc act ggc atc cca gtc agg ttc agt ggc 192

Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Val Arg Phe Ser Gly

50 55 60

agt ggg tct ggg aca gac ttc act ctc acc atc agc aga ctg gag cca 240

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro

65 70 75 80

gaa gat ttt gca gtg tat tac tgt cag cag ttt ggt agg tca cct cgg 288

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Phe Gly Arg Ser Pro Arg

85 90 95

acg ttc ggc caa ggg aca cga ctg gag att aaa ggc gga tcc tct agg 336

Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys Gly Gly Ser Ser Arg

100 105 110

tca agt tcc agc ggc ggc ggt ggc agc gga ggc ggc ggt gag gtg caa 384

Ser Ser Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Glu Val Gln

115 120 125

ctg gtg gag tct ggg gga ggt gtg gta agg cct ggg ggg tcc ctg aga 432

Leu Val Glu Ser Gly Gly Gly Val Val Arg Pro Gly Gly Ser Leu Arg

130 135 140

ctc tcc tgt gca gcc tct gga ttc acc ttc agt agc tat gct atg cac 480

Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr Ala Met His

145 150 155 160

tgg gtc cgc cag gct cca ggc aag ggg ctg gag tgg gtg gca gtt ata 528

Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Val Ile

165 170 175

tca tat gat gta agc aat aaa tac tac gca gac tcc gtg aag ggc cga 576

Ser Tyr Asp Val Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys Gly Arg

180 185 190

ttc acc atc tcc aga gac aat tcc aag aac acg ctg tat ctg caa atg 624

Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met

195 200 205

aac agc ctg aga gct gag gac acg gct gtg tat tac tgt gcg aga tct 672

Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Ser

210 215 220

tac tac tac tac tac ggt atg gac gtc tgg ggc caa ggg acc acg gtc 720

Tyr Tyr Tyr Tyr Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val

225 230 235 240

acc gtc tcc tca 732

Thr Val Ser Ser

<210> 40

<211> 244

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<400> 40

Asp Val Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Arg Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Val Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Phe Gly Arg Ser Pro Arg

85 90 95

Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys Gly Gly Ser Ser Arg

100 105 110

Ser Ser Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Glu Val Gln

115 120 125

Leu Val Glu Ser Gly Gly Gly Val Val Arg Pro Gly Gly Ser Leu Arg

130 135 140

Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr Ala Met His

145 150 155 160

Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Val Ile

165 170 175

Ser Tyr Asp Val Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys Gly Arg

180 185 190

Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met

195 200 205

Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Ser

210 215 220

Tyr Tyr Tyr Tyr Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val

225 230 235 240

Thr Val Ser Ser

<210> 41

<211> 351

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> chimeric sequence

<220>

<221> CDS

<222> (1)..(351)

<400> 41

cag tcg gtg gag gag tcc ggg ggt cgc ctg gtc acg cct ggg aca ccc 48

Gln Ser Val Glu Glu Ser Gly Gly Arg Leu Val Thr Pro Gly Thr Pro

1 5 10 15

ctg aca ctc acc tgc aca gtc tct gga atc gac ctc agt aac aat gca 96

Leu Thr Leu Thr Cys Thr Val Ser Gly Ile Asp Leu Ser Asn Asn Ala

20 25 30

atg agc tgg gtc cgc cag gct cca ggg aag ggg ctg gaa tat atc gga 144

Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr Ile Gly

35 40 45

atc att agg agt agt ggt agt aca tat tac gcg aac tgg gca aaa ggc 192

Ile Ile Arg Ser Ser Gly Ser Thr Tyr Tyr Ala Asn Trp Ala Lys Gly

50 55 60

cgg ttc acc atc tcc aaa acc tcg acc acg gtg gat ctg ata atc acc 240

Arg Phe Thr Ile Ser Lys Thr Ser Thr Thr Val Asp Leu Ile Ile Thr

65 70 75 80

agt ccg aca acc gag gac acg gcc acc tat ttc tgt gcc aga ggg ggt 288

Ser Pro Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg Gly Gly

85 90 95

ggt tct tac agt att gtc ttc tgg aac ttg tgg ggc cca ggc acc ctg 336

Gly Ser Tyr Ser Ile Val Phe Trp Asn Leu Trp Gly Pro Gly Thr Leu

100 105 110

gtc acc gtc tcc tca 351

Val Thr Val Ser Ser

115

<210> 42

<211> 117

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<400> 42

Gln Ser Val Glu Glu Ser Gly Gly Arg Leu Val Thr Pro Gly Thr Pro

1 5 10 15

Leu Thr Leu Thr Cys Thr Val Ser Gly Ile Asp Leu Ser Asn Asn Ala

20 25 30

Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr Ile Gly

35 40 45

Ile Ile Arg Ser Ser Gly Ser Thr Tyr Tyr Ala Asn Trp Ala Lys Gly

50 55 60

Arg Phe Thr Ile Ser Lys Thr Ser Thr Thr Val Asp Leu Ile Ile Thr

65 70 75 80

Ser Pro Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg Gly Gly

85 90 95

Gly Ser Tyr Ser Ile Val Phe Trp Asn Leu Trp Gly Pro Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 43

<211> 342

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> chimeric sequence

<220>

<221> CDS

<222> (1)..(342)

<400> 43

gcc aga tgt gcc tat gat atg acc cag act cca gcc tct gtg gag gta 48

Ala Arg Cys Ala Tyr Asp Met Thr Gln Thr Pro Ala Ser Val Glu Val

1 5 10 15

gct gtg gga ggc aca gtc acc atc aat tgc cag gcc agt gag agg att 96

Ala Val Gly Gly Thr Val Thr Ile Asn Cys Gln Ala Ser Glu Arg Ile

20 25 30

tat agc aat tta gcc tgg tat cag cag aaa cca ggg cag cgt ccc aag 144

Tyr Ser Asn Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Arg Pro Lys

35 40 45

ctc ctg atc tat tat aca tcc act ctg gca tct ggg gtc tca tcg cgg 192

Leu Leu Ile Tyr Tyr Thr Ser Thr Leu Ala Ser Gly Val Ser Ser Arg

50 55 60

ttc aaa ggc agt gga tct ggg aca cag ttc act ctc acc atc agc ggc 240

Phe Lys Gly Ser Gly Ser Gly Thr Gln Phe Thr Leu Thr Ile Ser Gly

65 70 75 80

gtg gag tgt gcc gat gct gcc act tac tac tgt caa cag ggt tat agt 288

Val Glu Cys Ala Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Gly Tyr Ser

85 90 95

agt agt aat gtt gac aat act ttc ggc gga ggg acc gag gtg gtg gtc 336

Ser Ser Asn Val Asp Asn Thr Phe Gly Gly Gly Thr Glu Val Val Val

100 105 110

aaa ggt 342

Lys Gly

<210> 44

<211> 114

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<400> 44

Ala Arg Cys Ala Tyr Asp Met Thr Gln Thr Pro Ala Ser Val Glu Val

1 5 10 15

Ala Val Gly Gly Thr Val Thr Ile Asn Cys Gln Ala Ser Glu Arg Ile

20 25 30

Tyr Ser Asn Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Arg Pro Lys

35 40 45

Leu Leu Ile Tyr Tyr Thr Ser Thr Leu Ala Ser Gly Val Ser Ser Arg

50 55 60

Phe Lys Gly Ser Gly Ser Gly Thr Gln Phe Thr Leu Thr Ile Ser Gly

65 70 75 80

Val Glu Cys Ala Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Gly Tyr Ser

85 90 95

Ser Ser Asn Val Asp Asn Thr Phe Gly Gly Gly Thr Glu Val Val Val

100 105 110

Lys Gly

<210> 45

<211> 363

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> chimeric sequence

<220>

<221> CDS

<222> (1)..(363)

<400> 45

ggc gag cag cag ctg gtg gag agc ggc gga ggc ctg gtg cag cct gga 48

Gly Glu Gln Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly

1 5 10 15

gga agc ctg agg ctg agc tgc gcc gtg tcc ggc ttc agc ctg agc agc 96

Gly Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Ser Leu Ser Ser

20 25 30

tac gcc atc agc tgg gtg agg cag gcc ccc gga aag ggc ctg gag tac 144

Tyr Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr

35 40 45

atc gcc atc atc aac agc tac ggc acc acc tac tac gcc agc tgg gcc 192

Ile Ala Ile Ile Asn Ser Tyr Gly Thr Thr Tyr Tyr Ala Ser Trp Ala

50 55 60

aag ggc aga gtg acc atc tcc aag gat tcc tcc aag aac acc gtg tac 240

Lys Gly Arg Val Thr Ile Ser Lys Asp Ser Ser Lys Asn Thr Val Tyr

65 70 75 80

ctg cag atg ggc tcc ctg aga gcc gag gat atg gcc gtg tac ttt tgc 288

Leu Gln Met Gly Ser Leu Arg Ala Glu Asp Met Ala Val Tyr Phe Cys

85 90 95

gcc aga ggc gat tcc tac ggc tcc ggc gtg ggc ctg ggc ctg tgg gga 336

Ala Arg Gly Asp Ser Tyr Gly Ser Gly Val Gly Leu Gly Leu Trp Gly

100 105 110

cct gga acc ctg gtg aca gtg tcc tcc 363

Pro Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 46

<211> 121

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<400> 46

Gly Glu Gln Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly

1 5 10 15

Gly Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Ser Leu Ser Ser

20 25 30

Tyr Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr

35 40 45

Ile Ala Ile Ile Asn Ser Tyr Gly Thr Thr Tyr Tyr Ala Ser Trp Ala

50 55 60

Lys Gly Arg Val Thr Ile Ser Lys Asp Ser Ser Lys Asn Thr Val Tyr

65 70 75 80

Leu Gln Met Gly Ser Leu Arg Ala Glu Asp Met Ala Val Tyr Phe Cys

85 90 95

Ala Arg Gly Asp Ser Tyr Gly Ser Gly Val Gly Leu Gly Leu Trp Gly

100 105 110

Pro Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 47

<211> 333

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> chimeric sequence

<220>

<221> CDS

<222> (1)..(333)

<400> 47

gga gac tac cag atg aca cag tcc cct agc acc ctg tcc gcc tcc gtg 48

Gly Asp Tyr Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val

1 5 10 15

ggc gac aga gtg aca atc acc tgt cag gcc tcc cag aat atc tac agc 96

Gly Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Asn Ile Tyr Ser

20 25 30

aat ctg gcc tgg tac cag cag aag cct ggc aag agg ccc aag ctg ctg 144

Asn Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Arg Pro Lys Leu Leu

35 40 45

atc tac aga gcc agc tcc ctg gcc tcc ggc gtg cca tct aga ttt tcc 192

Ile Tyr Arg Ala Ser Ser Leu Ala Ser Gly Val Pro Ser Arg Phe Ser

50 55 60

ggc tcc ggc agc ggc aca gag ttt acc ctg aca atc agc agc ctg cag 240

Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln

65 70 75 80

ccc gat gat ttc gcc acc tac tac tgt cag cag ggc ttc agc agc aat 288

Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Phe Ser Ser Asn

85 90 95

aat gtg gac aat aca ttt ggc ggc ggc aca aag gtg gag atc aag 333

Asn Val Asp Asn Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 48

<211> 111

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<400> 48

Gly Asp Tyr Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val

1 5 10 15

Gly Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Asn Ile Tyr Ser

20 25 30

Asn Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Arg Pro Lys Leu Leu

35 40 45

Ile Tyr Arg Ala Ser Ser Leu Ala Ser Gly Val Pro Ser Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln

65 70 75 80

Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Phe Ser Ser Asn

85 90 95

Asn Val Asp Asn Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 49

<211> 354

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> chimeric sequence

<220>

<221> CDS

<222> (1)..(354)

<400> 49

ggc gag cag cag ctg gtg gag agc ggc gga ggc ctg gtg cag cct gga 48

Gly Glu Gln Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly

1 5 10 15

gga agc ctg agg ctg agc tgc gcc gtg tcc ggc ttt tcc ctg agc aag 96

Gly Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Ser Leu Ser Lys

20 25 30

agc atc atc agc tgg gtg agg cag gcc cct ggc aag ggc ctg gag tac 144

Ser Ile Ile Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr

35 40 45

atc ggc atc atc ggc agc agc ggc tcc acc tac tac gcc aac tgg gcc 192

Ile Gly Ile Ile Gly Ser Ser Gly Ser Thr Tyr Tyr Ala Asn Trp Ala

50 55 60

aag ggc aga ttc aca atc tcc aag gac tcc tcc aag aat acc gtg tac 240

Lys Gly Arg Phe Thr Ile Ser Lys Asp Ser Ser Lys Asn Thr Val Tyr

65 70 75 80

ctg cag atg ggc tcc ctg agg gcc gag gat atg gcc gtg tac ttt tgt 288

Leu Gln Met Gly Ser Leu Arg Ala Glu Asp Met Ala Val Tyr Phe Cys

85 90 95

gcc aga ggc ctg ctg tac tcc ggc aat aag tcc tgg ggc ccc ggc aca 336

Ala Arg Gly Leu Leu Tyr Ser Gly Asn Lys Ser Trp Gly Pro Gly Thr

100 105 110

ctg gtg acc gtg agc tcc 354

Leu Val Thr Val Ser Ser

115

<210> 50

<211> 118

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<400> 50

Gly Glu Gln Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly

1 5 10 15

Gly Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Ser Leu Ser Lys

20 25 30

Ser Ile Ile Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr

35 40 45

Ile Gly Ile Ile Gly Ser Ser Gly Ser Thr Tyr Tyr Ala Asn Trp Ala

50 55 60

Lys Gly Arg Phe Thr Ile Ser Lys Asp Ser Ser Lys Asn Thr Val Tyr

65 70 75 80

Leu Gln Met Gly Ser Leu Arg Ala Glu Asp Met Ala Val Tyr Phe Cys

85 90 95

Ala Arg Gly Leu Leu Tyr Ser Gly Asn Lys Ser Trp Gly Pro Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 51

<211> 333

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> chimeric sequence

<220>

<221> CDS

<222> (1)..(333)

<400> 51

ggc gac atc gtg atg acc cag tcc ccc gat tcc ctg gcc gtg tcc ctg 48

Gly Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu

1 5 10 15

ggc gag aga gcc aca atc aat tgt cag tcc tcc cag agc gtg ctg ctg 96

Gly Glu Arg Ala Thr Ile Asn Cys Gln Ser Ser Gln Ser Val Leu Leu

20 25 30

aac aat cag ctg tcc tgg ttc cag cag aag cct ggc cag cct ccc aag 144

Asn Asn Gln Leu Ser Trp Phe Gln Gln Lys Pro Gly Gln Pro Pro Lys

35 40 45

ctg ctg atc tac gac gcc tcc aca ctg gag tcc ggc gtg ccc gat agg 192

Leu Leu Ile Tyr Asp Ala Ser Thr Leu Glu Ser Gly Val Pro Asp Arg

50 55 60

ttc agc ggc tcc ggc agc ggc acc gac ttt acc ctg acc atc tcc agc 240

Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser

65 70 75 80

ctg cag gcc gag gat gtg gcc gtg tac tac tgc ctg ggc ggc tac agc 288

Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Leu Gly Gly Tyr Ser

85 90 95

ggc aac ctg tac gcc ttt ggc ggc ggc acc aag gtg gag atc aag 333

Gly Asn Leu Tyr Ala Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 52

<211> 111

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<400> 52

Gly Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu

1 5 10 15

Gly Glu Arg Ala Thr Ile Asn Cys Gln Ser Ser Gln Ser Val Leu Leu

20 25 30

Asn Asn Gln Leu Ser Trp Phe Gln Gln Lys Pro Gly Gln Pro Pro Lys

35 40 45

Leu Leu Ile Tyr Asp Ala Ser Thr Leu Glu Ser Gly Val Pro Asp Arg

50 55 60

Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser

65 70 75 80

Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Leu Gly Gly Tyr Ser

85 90 95

Gly Asn Leu Tyr Ala Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 53

<211> 369

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> chimeric sequence

<220>

<221> CDS

<222> (1)..(369)

<400> 53

ggc gag cag cag ctg gtg gag tcc ggc gga ggc ctg gtg cag cca gga 48

Gly Glu Gln Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly

1 5 10 15

gga agc ctg agg ctg tcc tgt gcc gtg agc ggc ttc tcc ctg agc tcc 96

Gly Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Ser Leu Ser Ser

20 25 30

tac gcc atg agc tgg gtg agg cag gcc ccc gga aag ggc ctg gag tac 144

Tyr Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr

35 40 45

atc ggc atc atc agc agc agc ggc agc aca tac tac gcc agc tgg gcc 192

Ile Gly Ile Ile Ser Ser Ser Gly Ser Thr Tyr Tyr Ala Ser Trp Ala

50 55 60

aag ggc agg ttc aca atc agc aag gat tcc tcc aag aat aca gtg tac 240

Lys Gly Arg Phe Thr Ile Ser Lys Asp Ser Ser Lys Asn Thr Val Tyr

65 70 75 80

ctg cag atg ggc tcc ctg agg gcc gag gac atg gcc gtg tac ttc tgt 288

Leu Gln Met Gly Ser Leu Arg Ala Glu Asp Met Ala Val Tyr Phe Cys

85 90 95

gcc aga gac agg gtc atc tat tcc atc ggc cct tac tac ttc aac ctg 336

Ala Arg Asp Arg Val Ile Tyr Ser Ile Gly Pro Tyr Tyr Phe Asn Leu

100 105 110

tgg ggc ccc ggc aca ctg gtg aca gtg tcc agc 369

Trp Gly Pro Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 54

<211> 123

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<400> 54

Gly Glu Gln Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly

1 5 10 15

Gly Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Ser Leu Ser Ser

20 25 30

Tyr Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr

35 40 45

Ile Gly Ile Ile Ser Ser Ser Gly Ser Thr Tyr Tyr Ala Ser Trp Ala

50 55 60

Lys Gly Arg Phe Thr Ile Ser Lys Asp Ser Ser Lys Asn Thr Val Tyr

65 70 75 80

Leu Gln Met Gly Ser Leu Arg Ala Glu Asp Met Ala Val Tyr Phe Cys

85 90 95

Ala Arg Asp Arg Val Ile Tyr Ser Ile Gly Pro Tyr Tyr Phe Asn Leu

100 105 110

Trp Gly Pro Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 55

<211> 333

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> chimeric sequence

<220>

<221> CDS

<222> (1)..(333)

<400> 55

ggc gat tac cag atg aca cag tcc ccc tcc tcc ctg agc gcc tcc gtg 48

Gly Asp Tyr Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val

1 5 10 15

gga gat agg gtg acc atc aca tgc cag gcc agc gag atc atc tac agc 96

Gly Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Glu Ile Ile Tyr Ser

20 25 30

aat ctg gcc tgg tac cag cag aag ccc ggc aag ccc ccc aag ctg ctg 144

Asn Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Pro Pro Lys Leu Leu

35 40 45

atc tac ggc gcc tcc aca ctg gcc agc ggc gtg cct agc aga ttc agc 192

Ile Tyr Gly Ala Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Ser

50 55 60

ggc agc ggc tcc ggc acc gat tac acc ctg aca atc tcc agc ctg cag 240

Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln

65 70 75 80

cct gag gat ttt gcc aca tac tac tgt cag cag tcc ttc agc tcc aat 288

Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Phe Ser Ser Asn

85 90 95

aac gtg ggc aac atc ttc ggc ggc ggc aca aag gtg gag atc aag 333

Asn Val Gly Asn Ile Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 56

<211> 111

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic construct

<400> 56

Gly Asp Tyr Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val

1 5 10 15

Gly Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Glu Ile Ile Tyr Ser

20 25 30

Asn Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Pro Pro Lys Leu Leu

35 40 45

Ile Tyr Gly Ala Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln

65 70 75 80

Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Phe Ser Ser Asn

85 90 95

Asn Val Gly Asn Ile Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

Claims (50)

1. An anti-CD 39 antibody or antigen-binding fragment thereof, the anti-CD 39 antibody or antigen-binding fragment thereof comprising:

(i) at least one antigen binding domain that binds ectonucleoside triphosphate diphosphohydrolase-1 (CD39) at a site such that the anti-CD 39 antibody forms a stable immune complex, and

(ii) an Fc γ RIIIa binding moiety that binds to an Fc γ RIIIa receptor and confers antibody-dependent cellular cytotoxicity (ADCC) activity of the anti-CD 39 antibody against CD39+ cells.

2. The anti-CD 39 antibody or antigen-binding fragment thereof of claim 1, wherein the anti-CD 39 antibody or antigen-binding fragment thereof facilitates:

(i) stable immune complex formation when incubated with HCC1739BL cells, as characterized by less than 30% loss of the immune complex after 24 hours, optionally wherein the immune complex formation is detected by fluorescence intensity using a fluorescently labeled secondary antibody;

(ii) complement Dependent Cytotoxicity (CDC) activity against CD39+ cells;

(iii) Antibody-mediated endocytosis of CD39 on CD45+ immune cells;

(iv) antibody-mediated target endocytosis of CD39 from tumor vascular endothelial destruction or collapse of the vasculoprostrial network in a tumor;

(v) (optionally) a CD39 epitope that binds to a sequence selected from the group of CD39 amino acid epitope sequences set forth in figure 33; and/or

(vi) (optionally) binds to CD39 in a manner that is non-competitive or only partially competitive with monoclonal antibody clone a1 for binding to CD 39.

3. The anti-CD 39 antibody or antigen-binding fragment thereof of claim 1 or 2, wherein the Fc γ RIIIa binding portion is selected from the group consisting of: an Fc domain, an antibody or fragment thereof that binds to Fc γ RIIIa, and an Fc γ RIIIa binding peptide.

4. The anti-CD 39 antibody or antigen-binding fragment thereof of any one of claims 1 to 3, wherein the antigen-binding domain is selected from the group consisting of: fab, Fab ', F (ab')₂Fv or single chain Fv (scFv), Fav, dsFv, sc (Fv)2, Fde, sdFv, single domain antibody (dAb), and diabody fragments, and/or wherein the anti-CD 39 antibody or antigen-binding fragment is monoclonal.

5. The anti-CD 39 antibody or antigen-binding fragment thereof of any one of claims 1 to 4, wherein the anti-CD 39 antibody or antigen-binding fragment thereof is conjugated to an agent, optionally wherein the agent is selected from the group consisting of: binding proteins, enzymes, drugs, chemotherapeutic agents, biological agents, toxins, radionuclides, immunomodulators, detectable moieties and tags.

6. The anti-CD 39 antibody or antigen-binding fragment thereof of any one of claims 1 to 5, wherein the anti-CD 39 antibody or antigen-binding fragment thereof has a VH domain having an amino acid sequence capable of being encoded by 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 capable of being encoded by a nucleic acid that hybridizes under stringent conditions to the nucleic acid of SEQ ID No. 3.

7. The anti-CD 39 antibody or antigen-binding fragment thereof of any one of claims 1-6, wherein the anti-CD 39 antibody or antigen-binding fragment thereof comprises a heavy chain having CDRs at least 60% identical to the CDRs of SEQ ID Nos. 2, 6, 10, 14, 18, 22, 26, 42, 46, 50, or 54; and a light chain having a CDR that is at least 60% identical to a CDR of SEQ ID No.4, 8, 12, 16, 20, 24, 28, 44, 48, 52 or 56.

8. The anti-CD 39 antibody or antigen-binding fragment thereof of any one of claims 1 to 7, wherein the anti-CD 39 antibody or antigen-binding fragment thereof comprises a Variable Heavy (VH) chain that is at least 60% identical to SEQ ID Nos. 2, 6, 10, 14, 18, 22, 26, 42, 46, 50, or 54, and a Variable Light (VL) chain that is at least 60% identical to SEQ ID Nos. 4, 8, 12, 16, 20, 24, 28, 44, 48, 52, or 56.

9. The anti-CD 39 antibody or antigen-binding fragment thereof of any one of claims 1-8, wherein the anti-CD 39 antibody or antigen-binding fragment thereof comprises:

(i) a heavy chain having a CDR1 amino acid sequence at least 80% identical to SEQ ID No.29, a CDR2 amino acid sequence at least 80% identical to SEQ ID No.30, and a CDR3 amino acid sequence at least 80% identical to SEQ ID No. 31; and

(ii) a light chain having a CDR1 amino acid sequence at least 80% identical to SEQ ID No.32, a CDR2 amino acid sequence at least 80% identical to SEQ ID No.33, and a CDR3 amino acid sequence at least 80% identical to SEQ ID No. 34.

10. The anti-CD 39 antibody or antigen-binding fragment thereof of any one of claims 1-8, wherein the anti-CD 39 antibody or antigen-binding fragment thereof comprises a heavy chain having CDRs selected from the group consisting of the 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 the CDRs of SEQ ID nos. 8, 12, 16, 20, 24, 28, 44, 48, 52 and 56 and human framework sequences to form a humanized heavy and light chain having an antigen-binding site capable of specifically binding to human CD 39.

11. The anti-CD 39 antibody or antigen-binding fragment thereof of any one of claims 1-10, wherein the anti-CD 39 antibody or antigen-binding fragment thereof comprises an Fc domain of the IgG1 or IgG3 isotype, optionally wherein the Fc domain is human.

12. The anti-CD 39 antibody or antigen-binding fragment thereof of any one of claims 1-11, wherein the anti-CD 39 antibody or antigen-binding fragment thereof is low fucosylated or afucosylated.

13. The anti-CD 39 antibody or antigen-binding fragment thereof of any one of claims 1-12, wherein the anti-CD 39 antibody or antigen-binding fragment thereof is human or humanized.

14. The anti-CD 39 antibody or antigen-binding fragment thereof of any one of claims 1-13, wherein the anti-CD 39 antibody or antigen-binding fragment thereof is bispecific, comprising at least one additional antigen-binding site for a tumor antigen, an immune checkpoint, or a co-stimulatory receptor, wherein if the additional antigen-binding site is for an immune checkpoint it serves as a checkpoint inhibitor, and wherein if the additional antigen-binding site is for a co-stimulatory receptor it serves as a co-stimulatory agonist.

15. The anti-CD 39 antibody or antigen-binding fragment thereof of claim 14, 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.

16. The anti-CD 39 antibody or antigen-binding fragment thereof of claim 14 or 15, wherein the additional antigen-binding site binds a checkpoint protein that is upregulated on T cells and associated with T cell depletion.

17. The anti-CD 39 antibody or antigen-binding fragment thereof of claim 14, wherein the additional antigen-binding site binds to an immune co-stimulatory receptor selected from the group consisting of: MHCI molecules, BTLA receptor, OX40, CD27, CD28, CDS, ICAM-1, LFA-1(CD11a/CD18), ICOS (CD278) and 4-1BB (CD 137).

18. The anti-CD 39 antibody or antigen-binding fragment thereof of claim 14, wherein the additional antigen-binding site binds to CD47, sirpa, CD24, or Siglec-10.

19. A pharmaceutical formulation comprising a therapeutically effective amount of at least one anti-CD 39 antibody or antigen-binding fragment thereof of any one of claims 1-18, and one or more pharmaceutically acceptable excipients, buffers, or solutions.

20. The pharmaceutical formulation of claim 19, for improving anti-tumor T cell immunity and suitable for administration to a subject having a tumor, comprising an effective amount of the anti-CD 39 antibody or antigen-binding fragment thereof and one or more pharmaceutically acceptable excipients, buffers, or solutions, wherein administration of the anti-CD 39 antibody to the subject results in intratumoral CD39^{Height of}The number of cells is reduced and infiltration of T cells into the tumor is enhanced or T cell depletion in the tumor is reduced or both.

21. An isolated nucleic acid molecule, said isolated nucleic acid molecule

i) Hybridizing under stringent conditions to the complement of a nucleic acid encoding an immunoglobulin heavy and/or light chain polypeptide of the anti-CD 39 antibody or antigen-binding fragment thereof of any one of claims 1-18;

ii) has a sequence that is at least about 90% identical over its full length to a nucleic acid encoding an immunoglobulin heavy and/or light chain polypeptide of the anti-CD 39 antibody or antigen-binding fragment thereof of any one of claims 1-18; or

iii) an immunoglobulin heavy and/or light chain polypeptide encoding the anti-CD 39 antibody or antigen-binding fragment thereof of any one of claims 1-18.

22. An isolated immunoglobulin heavy and/or light chain polypeptide encoded by the nucleic acid of claim 21.

23. A vector comprising the isolated nucleic acid of claim 21, optionally wherein the vector is an expression vector.

24. A host cell comprising the isolated nucleic acid of claim 21, the host cell:

a) expressing the anti-CD 39 antibody or antigen-binding fragment thereof of any one of claims 1-18;

b) comprises the immunoglobulin heavy and/or light chain polypeptide of claim 22; and/or

c) Comprising the vector of claim 23.

25. A device or kit comprising at least one anti-CD 39 antibody or antigen-binding fragment thereof of any one of claims 1-18, optionally comprising a label to detect at least one anti-CD 39 antibody or antigen-binding fragment thereof, or a complex comprising the anti-CD 39 antibody or antigen-binding fragment thereof.

26. A device or kit comprising the pharmaceutical composition, the isolated nucleic acid molecule, the isolated immunoglobulin heavy and/or light chain polypeptide, the vector and/or the host cell of any one of claims 19 to 24.

27. A method of producing at least one anti-CD 39 antibody or antigen-binding fragment thereof of any one of claims 1-18, the method comprising the steps of: (i) culturing a transformed host cell that has been transformed with a nucleic acid comprising a sequence encoding at least one anti-CD 39 antibody or antigen-binding fragment thereof under conditions suitable to allow expression of the anti-CD 39 antibody or antigen-binding fragment thereof; and (ii) recovering the expressed anti-CD 39 antibody or antigen-binding fragment thereof.

28. A method of detecting the presence or concentration of a CD39 polypeptide, the method comprising obtaining a sample, and detecting the polypeptide in the sample by using at least one anti-CD 39 antibody or antigen-binding fragment thereof of any one of claims 1-18.

29. The method of claim 28, wherein at least one anti-CD 39 antibody or antigen-binding fragment thereof forms a complex with the CD39 polypeptide and the complex is detected in an enzyme-linked immunosorbent assay (ELISA), Radioimmunoassay (RIA), immunochemical assay, western blot, mass spectrometry, nuclear magnetic resonance assay, or using an intracellular flow assay.

30. A method for treating tumor by depleting intratumoral CD39 ^{Height of}A method of improving anti-tumor T cell immunity in a cell, the method comprising administering to a subject having a tumor an effective amount of the anti-CD 39 antibody or antigen-binding fragment thereof pharmaceutical composition of any one of claims 1-18, wherein administration of the anti-CD 39 antibody or antigen-binding fragment thereof results in intratumoral CD39^{Height of}The number of cells is reduced and infiltration of T cells into the tumor is enhanced or T cell depletion in the tumor is reduced or both.

31. A method for promoting infiltration of immune cells into a tumor, the method comprising administering to a subject having a tumor an effective amount of the anti-CD 39 antibody or antigen-binding fragment thereof of any one of claims 1-18, wherein administration of the anti-CD 39 antibody or antigen-binding fragment thereof results in elimination and reduction of CD39+ CD45-SCA-1+ stromal cells in the tumor and increased infiltration of cytotoxic T cells into the tumor.

32. A method for reducing inhibition of immune cell function within a tumor by type II NKT cells, the method comprising administering to a subject having a tumor an effective amount of the anti-CD 39 antibody or antigen-binding fragment thereof of any one of claims 1 to 18,

Wherein administration of the anti-CD 39 antibody or antigen-binding fragment thereof eliminates and reduces type II NKT cells in the tumor.

33. A method for reducing suppression of immune cell function within a tumor by regulatory T cells (Tregs), the method comprising administering to a subject having a tumor an effective amount of the pharmaceutical composition of the anti-CD 39 antibody or antigen-binding fragment thereof of any one of claims 1-18,

wherein administration of the anti-CD 39 antibody or antigen-binding fragment thereof results in CD39 in the tumor^{Height of}Treg elimination and reduction.

34. A method for reducing inhibition of immune cell function within a tumor by tumor-associated macrophages (TAMs), the method comprising administering to a subject having a tumor an effective amount of the anti-CD 39 antibody or antigen-binding fragment thereof of any one of claims 1-18, wherein administration of the anti-CD 39 antibody or antigen-binding fragment thereof is such that CD39 in the tumor^{Height of}Macrophage elimination and reduction.

35. A method for promoting an anti-tumor immune response, the method comprising administering to a subject having a tumor the anti-CD 39 antibody or antigen-binding fragment thereof of any one of claims 1-18 in an amount sufficient to cause a reduction in CD 39-expressing cells in the tumor.

36. A method for promoting T cell-mediated immune function in a tumor in a subject, the method comprising:

(i) identifying a cancer subject having a degree of tumor-reactive lymphocytes less than a predetermined threshold of tumor infiltration to characterize a tumor phenotype that is non-infiltrated or insufficiently infiltrated; and

(ii) administering to the subject the anti-CD 39 antibody or antigen-binding fragment thereof of any one of claims 1-18 in an amount that increases tumor-reactive T-cell infiltrating tumor.

37. The method of claim 30, 31 or 34The method, wherein the intratumoral CD39^{Height of}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.

38. The method of claim 30, 31, or 34, wherein the anti-CD 39 antibody or antigen-binding fragment thereof reduces CD39 present within one or more hematopoietic compartments^{Height of}(ii) a level of cells, optionally wherein the one or more hematopoietic compartments are selected from the group consisting of blood, spleen and liver.

39. The method of any one of claims 30-38, wherein the anti-CD 39 antibody or antigen-binding fragment thereof is administered as part of an anti-tumor therapy.

40. The method of any one of claims 30 to 39, wherein the anti-CD 39 antibody or antigen-binding fragment thereof is administered as part of anti-infection therapy, optionally wherein the anti-infection therapy is antiviral therapy (including treatment of HIV and HBV infections and COVID-19 infections), for treatment of Mycobacterium tuberculosis, and for treatment of visceral leishmaniasis.

41. The method of any one of claims 30 to 40, wherein the anti-CD 39 antibody or antigen-binding fragment thereof is administered as part of an anti-tumor therapy for treating a solid tumor, optionally wherein the solid tumor is pancreatic cancer, liver cancer, lung cancer, gastric cancer, esophageal cancer, head and neck squamous cell carcinoma, prostate cancer, colon cancer, breast cancer, lymphoma, gallbladder cancer, renal cancer, multiple myeloma, ovarian cancer, cervical cancer, or glioma.

42. The method of any one of claims 30 to 41, wherein the anti-CD 39 antibody or antigen-binding fragment thereof is administered as part of an anti-tumor therapy for treating a liquid tumor, optionally wherein the liquid tumor is leukemia.

43. The method of any one of claims 30 to 42, wherein the anti-CD 39 antibody or antigen-binding fragment thereof is administered as part of a therapy involving one or more chemotherapeutic agents, anti-angiogenic agents, immunooncology agents, and/or radiation.

44. The method of any one of claims 30 to 43, wherein the therapy comprises administration of one or more inhibitors (antagonists) of one or more checkpoint molecules, optionally wherein the one or more checkpoint molecules are selected from the group consisting of: PD-1 antagonists, CTLA-4 antagonists, LAG-3 antagonists, TIM-3 antagonists, TIGIT antagonists, and Siglec-15 antagonists.

45. The method of any one of claims 30 to 44, wherein the therapy comprises administration of one or more activators (agonists) of one or more co-stimulatory molecules, optionally wherein the one or more co-stimulatory molecules are selected from the group consisting of: GITR agonist, CD27 agonist, 4-1BB agonist, OX40 agonist, CD137 agonist, ICOS agonist, and CD28 agonist.

46. The method of any one of claims 30 to 45, wherein the therapy comprises administration of one or more of: VEGFR or VEGF antagonists, EGFR or EGF antagonists, IDO inhibitors, IDO1 inhibitors, HDAC inhibitors, PI3K δ inhibitors, IL-15 agonists, CXCR4 antagonists, CXCL12 antagonists, DNMT inhibitors, interleukin-21, anti-KIR antibodies, anti-CSF-1R antibodies, anti-CCR 4 antibodies, GMCSF, anti-PS antibodies, anti-CD 30 antibody-auristatin E conjugates, anti-CD 19 antibodies, anti-CEA IL-2 antibodies, anti-NY-ESO-1 antibodies, anti-NKG 2A antibodies, STING agonists, TRL7/8 agonists, RIG-1 agonists and/or NRLP3 inhibitors, anti-CD 73 antibodies (such as MEDI9447), P2X7 antagonists or adenosine A2A receptor antagonists.

47. The method of any one of claims 30 to 46, wherein the therapy comprises administration of one or more innate immunity inducing agents, optionally wherein the one or more innate immunity inducing agents are selected from the group consisting of: inhibitors of the CD47-SIRP α axis, inhibitors of the CD24-Siglec-10 axis, NGK2A checkpoint inhibitors that block HLA-E driven inhibition of NK and CD8+ cells, STING agonists, TLR7/8 agonists, and RIG-I agonists.

48. The method of any one of claims 30-46, wherein the anti-CD 39 antibody or antigen-binding fragment thereof is administered as part of a therapy comprising a tumor vaccine, adoptive cell therapy, anti-tumor gene therapy, inhibitory nucleic acid therapy, and/or oncolytic virus therapy.

49. The pharmaceutical composition or method of any one of claims 20 or 30-48, wherein the subject is an animal model of cancer.

50. The pharmaceutical composition or method of any one of claims 20 or 30 to 49, wherein the subject is a mammal, optionally wherein the mammal is a human or a rodent.