Anti-CD39 Antibody and Its Use

Abstract

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

Description

Technical Field

[0001] Cross-reference statement This application claims priority to International Application PCT / CN2022 / 079021 filed on March 3, 2022 and International Application PCT / CN2022 / 126070 filed on October 19, 2022, the disclosures of which are incorporated herein by reference.

[0002] Technical Field This specification provides anti-CD39 antibodies that inhibit the enzymatic activity of human CD39 and methods of using the same.

Background Art

[0003] Dying or stressed cells, which are present at negligible concentrations (10 - 100 nM) under healthy conditions, release ATP extracellularly. Once released, extracellular ATP transmits signals via type 2 purinergic (P2) receptors, providing inflammatory signals essential for effective innate and adaptive immune responses. CD39 is the rate-limiting ectoenzyme in the hydrolysis of extracellular ATP. CD39 is an important regulator of extracellular ATP levels through its catabolic action of converting extracellular ATP to AMP.

[0004] CD39 is also an important contributor to extracellular adenosine levels. Since adenosine is an immunosuppressive metabolite, it is in a conversion relationship with extracellular ATP. CD39 contributes to the increase in extracellular adenosine levels by hydrolyzing ATP to AMP, which is then converted to adenosine by CD73. Extracellular adenosine then transmits signals via type 1 purinergic receptors, creating an immunosuppressive environment.

[0005] Therefore, the hydrolysis of extracellular ATP by CD39 may affect the immune response via two different signaling pathways at opposite ends of the signaling axis. In particular, dysregulation of this signaling axis, referred to herein as the ATP-adenosine signaling axis, has been observed in several diseases. Thus, there is a need in the art for agents that target CD39 and inhibit its enzymatic activity for beneficial therapeutic purposes. SUMMARY OF THE INVENTION

[0006] Among various aspects of the present disclosure, there is provided, inter alia, an anti-CD39 antibody. The anti-CD39 antibody is optionally labeled with one or more detectable signals including, but not limited to, (i) a fluorescent molecule, a spin-labeled molecule, an enzyme, or a radioisotope, and / or (ii) conjugated to one or more therapeutic agents including, but not limited to, a chemotherapeutic agent, a radioisotope, or a nucleic acid. In some embodiments, the anti-CD39 antibody of the present disclosure is provided as an isolated antibody. In some embodiments, the anti-CD39 antibody of the present disclosure is provided in a composition. In some embodiments, the anti-CD39 antibody of the present disclosure is provided in a composition further comprising a pharmaceutically acceptable excipient.

[0007] In one embodiment, the present disclosure provides an anti-CD39 antibody that specifically binds to human CD39 and comprises a heavy chain variable region comprising complementarity determining region 1 (H1) having at least 80% sequence identity with SEQ ID NO: 10, complementarity determining region 2 (H2) having at least 80% sequence identity with SEQ ID NO: 11, and complementarity determining region 3 (H3) having at least 80% sequence identity with SEQ ID NO: 12; and a light chain variable region comprising complementarity determining region 1 (L1) having at least 80% sequence identity with SEQ ID NO: 14, complementarity determining region 2 (L2) having at least 80% sequence identity with SEQ ID NO: 15, and complementarity determining region 3 (L3) having at least 80% sequence identity with SEQ ID NO: 16. In another embodiment, the present disclosure provides an anti-CD39 antibody that specifically binds to human CD39 and comprises a heavy chain variable region comprising H1 having at least 90% sequence identity with SEQ ID NO: 10, H2 having at least 90% sequence identity with SEQ ID NO: 11, and H3 having at least 90% sequence identity with SEQ ID NO: 12; and a light chain variable region comprising L1 having at least 90% sequence identity with SEQ ID NO: 14, L2 having at least 90% sequence identity with SEQ ID NO: 15, and L3 having at least 90% sequence identity with SEQ ID NO: 16. In some of the foregoing embodiments, the heavy chain variable region of the antibody has at least 90% sequence identity with SEQ ID NO: 9, and the light chain variable region has at least 90% sequence identity with SEQ ID NO: 13. In some embodiments, the heavy chain variable region of the antibody has at least 95% sequence identity with SEQ ID NO: 9, and the light chain variable region has at least 95% sequence identity with SEQ ID NO: 13.

[0008] In one embodiment, the present disclosure provides an anti-CD39 antibody that specifically binds to human CD39, comprising a heavy chain variable region comprising H1 having at least 80% sequence identity with SEQ ID NO: 18, H2 having at least 80% sequence identity with SEQ ID NO: 19, and H3 having at least 80% sequence identity with SEQ ID NO: 20; and a light chain variable region comprising L1 having at least 80% sequence identity with SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24, L2 having at least 80% sequence identity with SEQ ID NO: 25, and L3 having at least 80% sequence identity with SEQ ID NO: 26. In another embodiment, the present disclosure provides an anti-CD39 antibody that specifically binds to human CD39, comprising a heavy chain variable region comprising H1 having at least 90% sequence identity with SEQ ID NO: 18, H2 having at least 80% sequence identity with SEQ ID NO: 19, and H3 having at least 90% sequence identity with SEQ ID NO: 20; and a light chain variable region comprising L1 having at least 90% sequence identity with SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24, L2 having at least 90% sequence identity with SEQ ID NO: 25, and L3 having at least 80% sequence identity with SEQ ID NO: 26. In some of the foregoing embodiments, the heavy chain variable region and the light chain variable region of the antibody have at least 90% sequence identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) or at least 95% sequence identity with SEQ ID NO: 17 and SEQ ID NO: 21, respectively, or with SEQ ID NO: 43 and SEQ ID NO: 45, respectively, or with SEQ ID NO: 43 and SEQ ID NO: 46, respectively, or with SEQ ID NO: 43 and SEQ ID NO: 47, respectively, or with SEQ ID NO: 43 and SEQ ID NO: 48, respectively, or with SEQ ID NO: 43 and SEQ ID NO: 49, respectively, or with SEQ ID NO: 44 and SEQ ID NO: 48, respectively, or with SEQ ID NO: 44 and SEQ ID NO: 49, respectively.

[0009] In one embodiment, the present disclosure provides an anti-CD39 antibody that specifically binds to human CD39 and comprises a heavy chain variable region comprising H1 having at least 80% sequence identity with SEQ ID NO: 28, H2 having at least 80% sequence identity with SEQ ID NO: 29, and H3 having at least 80% sequence identity with SEQ ID NO: 30; and a light chain variable region comprising L1 having at least 80% sequence identity with SEQ ID NO: 32, L2 having at least 80% sequence identity with SEQ ID NO: 33, and L3 having at least 80% sequence identity with SEQ ID NO: 34. In some of the foregoing embodiments, the heavy chain variable region and the light chain variable region of the antibody have at least 90% sequence identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) or at least 95% sequence identity with SEQ ID NO: 27 and SEQ ID NO: 31, or SEQ ID NO: 58 and SEQ ID NO: 60, or SEQ ID NO: 58 and SEQ ID NO: 61, or SEQ ID NO: 58 and SEQ ID NO: 62, or SEQ ID NO: 58 and SEQ ID NO: 63, or SEQ ID NO: 59 and SEQ ID NO: 61, or SEQ ID NO: 59 and SEQ ID NO: 60, or SEQ ID NO: 59 and SEQ ID NO: 62, or SEQ ID NO: 59 and SEQ ID NO: 63.

[0010] For any of the disclosed antibodies of the present disclosure, the anti-CD39 antibody can be a monoclonal antibody or an antigen-binding fragment thereof; a chimeric, humanized, or veneered antibody or an antigen-binding fragment thereof; or a human antibody or an antigen-binding fragment thereof. Additionally, the anti-CD39 antibody may further comprise a heavy chain constant region selected from human IgG1, human IgG2, human IgG3, or human IgG4, and optionally a human light chain constant region. The variant heavy chain constant region may be a wild-type heavy chain constant region or a heavy chain constant region with enhanced or reduced effector function relative to the wild-type heavy chain constant region. In various embodiments, the IgG heavy chain constant region may comprise SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5; and the human light chain kappa constant region may comprise SEQ ID NO: 6.

[0011] In one embodiment, the present disclosure provides an anti-CD39 antibody comprising a light chain comprising a heavy chain variable region and a light chain variable region, wherein: (a) the heavy chain has an amino acid sequence comprising SEQ ID NO: 50 and the light chain has an amino acid sequence comprising SEQ ID NO: 53; (b) the heavy chain has an amino acid sequence comprising SEQ ID NO: 50 and the light chain has an amino acid sequence comprising SEQ ID NO: 54; (c) the heavy chain has an amino acid sequence comprising SEQ ID NO: 50 and the light chain has an amino acid sequence comprising SEQ ID NO: 55; (d) the heavy chain has an amino acid sequence comprising SEQ ID NO: 50 and the light chain has an amino acid sequence comprising SEQ ID NO: 56; (e) the heavy chain has an amino acid sequence comprising SEQ ID NO: 51 and the light chain has an amino acid sequence comprising SEQ ID NO: 56; (f) the heavy chain has an amino acid sequence comprising SEQ ID NO: 50 and the light chain has an amino acid sequence comprising SEQ ID NO: 57; (g) the heavy chain has an amino acid sequence comprising SEQ ID NO: 52 and the light chain has an amino acid sequence comprising SEQ ID NO: 56; or (h) the heavy chain has an amino acid sequence comprising SEQ ID NO: 52 and the light chain has an amino acid sequence comprising SEQ ID NO: 57.

[0012] In one embodiment, the present disclosure provides an anti-CD39 antibody comprising a light chain comprising a heavy chain variable region and a light chain variable region, wherein: (a) the heavy chain has an amino acid sequence comprising SEQ ID NO: 64 and the light chain has an amino acid sequence comprising SEQ ID NO: 67; (b) the heavy chain has an amino acid sequence comprising SEQ ID NO: 64 and the light chain has an amino acid sequence comprising SEQ ID NO: 68; (c) the heavy chain has an amino acid sequence comprising SEQ ID NO: 64 and the light chain has an amino acid sequence comprising SEQ ID NO: 69; (d) the heavy chain has an amino acid sequence comprising SEQ ID NO: 64 and the light chain has an amino acid sequence comprising SEQ ID NO: 70; (e) the heavy chain has an amino acid sequence comprising SEQ ID NO: 65 and the light chain has an amino acid sequence comprising SEQ ID NO: 68; (f) the heavy chain has an amino acid sequence comprising SEQ ID NO: 65 and the light chain has an amino acid sequence comprising SEQ ID NO: 67; (g) the heavy chain has an amino acid sequence comprising SEQ ID NO: 66 and the light chain has an amino acid sequence comprising SEQ ID NO: 67; (h) the heavy chain has an amino acid sequence comprising SEQ ID NO: 65 and the light chain has an amino acid sequence comprising SEQ ID NO: 69; or (i) the heavy chain has an amino acid sequence comprising SEQ ID NO: 65 and the light chain has an amino acid sequence comprising SEQ ID NO: 70.

[0013] In another aspect, the present disclosure provides any of the antibodies disclosed herein for use as a medicament. In some embodiments, the anti-CD39 antibody is provided for use in the treatment of cancer. In some embodiments, the anti-CD39 antibody is provided for use in the prevention of cancer.

[0014] In another aspect, the present disclosure provides any of the antibodies disclosed herein for use as a medicament in combination with an additional therapy. In some embodiments, the aforementioned anti-CD39 antibody is provided for use in the treatment of cancer. In some embodiments, the aforementioned anti-CD39 antibody is provided for use in the prevention of cancer. In certain embodiments, the additional therapy can be an immune checkpoint inhibitor, an immunogenic cell death inducer, an ATP-adenosine axis targeting agent, an HIF-2α inhibitor, an arginase inhibitor, an AXL inhibitor, or a PI3K inhibitor. In further embodiments, the additional therapy can be chemotherapy, radiotherapy, durvalumab, zimberelimab, domvanalimab, AB308, AB521, or quemriclustat.

[0015] In another aspect, the present disclosure provides any of the antibodies disclosed herein for use as a medicament in combination with two or more additional therapies. In some embodiments, the aforementioned anti-CD39 antibody is provided for use in the treatment of cancer. In some embodiments, the aforementioned anti-CD39 antibody is provided for use in the prevention of cancer. In certain embodiments, each additional therapy can be a chemotherapeutic agent, an immune checkpoint inhibitor, an immunogenic cell death-inducing therapeutic, an ATP-adenosine axis targeting agent, a HIF-2α inhibitor, an arginase inhibitor, an AXL inhibitor, or a PI3K inhibitor. In further embodiments, the additional therapy can be chemotherapy, radiation therapy, durvalumab, zimberelimab, domvanalimab, AB308, AB521, or quemriclustat. In some embodiments, one or more additional therapeutic agents include (a) pemetrexed, carboplatin, and either an antagonist anti-PD-1 antibody or an antagonist anti-PD-L1 antibody, or (b) FOLFOX and either an antagonist anti-PD-1 antibody or an antagonist anti-PD-L1 antibody.

[0016] In another aspect, the present disclosure provides a method for treating or preventing cancer, the method comprising administering any of the antibodies disclosed herein to a subject in need of treatment or prevention of cancer. In some embodiments, the method further comprises administering one or more additional therapies. In certain embodiments, each additional therapy can be an immune checkpoint inhibitor, an immunogenic cell death-inducing therapeutic, an ATP-adenosine axis targeting agent, a HIF-2α inhibitor, an arginase inhibitor, an AXL inhibitor, or a PI3K inhibitor. In further embodiments, the additional therapy can be chemotherapy, radiation therapy, durvalumab, zimberelimab, domvanalimab, AB308, AB521, or quemriclustat.

[0017] In each of the above aspects, the cancer can be breast cancer, gastrointestinal cancer, uro-genital cancer, head and neck cancer, kidney cancer, lung cancer, lymphoma, ovarian cancer, pancreatic cancer, skin cancer, or thyroid cancer. In some embodiments, the cancer can be acute myeloid lymphoma, colorectal cancer, gastric cancer, esophageal cancer, castration-resistant prostate cancer, non-small cell lung cancer, ovarian cancer, pancreatic cancer, triple-negative breast cancer, head and neck squamous cell carcinoma, pancreatic ductal adenocarcinoma, clear cell renal carcinoma, or melanoma.

[0018] In some embodiments, the cancer is non-small cell lung cancer, and the method can further include administering an anti-CD39 antibody in combination with either pemetrexed, carboplatin, and an antagonist anti-PD-1 antibody or an antagonist anti-PD-L1 antibody. In some embodiments, the cancer is gastric cancer or gastroesophageal cancer, and the method can further include administering an anti-CD39 antibody in combination with either FOLFOX and an antagonist anti-PD-1 antibody or an antagonist anti-PD-L1 antibody. These embodiments may further include administering one or more additional agents selected from the group consisting of an antagonist anti-TIGIT antibody, A 2a R antagonist, A 2b R antagonist, A 2a / 2b R antagonist, and a CD73 inhibitor, and optionally, the additional agent is selected from the group consisting of domvanalimab, AB308, etrumadenant, and quemriculstat.

[0019] Other aspects and iterations of the present disclosure are provided in more detail below.

Brief Description of the Drawings

[0020]

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Mode for Carrying Out the Invention

[0021] The present disclosure provides antibodies that specifically bind to epitopes within the extracellular domain of human CD39, more specifically human CD39, and inhibit human CD39 enzymatic activity. Also contemplated are methods of using the anti-CD39 antibodies disclosed herein to bind to cells expressing human CD39 and inhibit soluble and cell surface-expressed human CD39 enzymatic activity. The present disclosure demonstrates the use of the anti-CD39 antibodies disclosed herein to inhibit human CD39 enzymatic activity and thereby affect one or both ends of the ATP-adenosine signaling axis. Accordingly, the present disclosure also provides for the medical use of the anti-CD39 antibodies disclosed herein for therapeutic and diagnostic purposes.

[0022] I. Definitions Unless otherwise defined, all technical terms, notations, and other scientific or technical terms or terminology used herein are intended to have the meaning commonly understood by one of ordinary skill in the art to which this disclosure pertains. It is understood that the aspects of the invention described herein include the aspects “comprising,” “consisting of,” and “consisting essentially of.” The terms “comprising,” “including,” and “having” are intended to be inclusive and mean that additional elements other than the recited elements may exist.

[0023] Unless otherwise specified, the term “about” as used herein refers to the normal error range of each value as understood by one of ordinary skill in the art. To avoid misunderstanding, references to “about” values or parameters herein include (and describe) aspects directed to the value or parameter itself.

[0024] Also, as used herein, "and / or" refers to all of the combinations of one or more of the related listed items, and when interpreted in the alternative form ("or"), it means that the combinations are not included, while including these. Similarly, the phrase in the form of "A / B" or "A and / or B" means (A), (B), or (A and B); the phrase in the form of "at least one of A, B, and C" means (A), (B), (C), (A and B), (A and C), (B and C), or (A, B, and C).

[0025] The term "CD39" refers to human CD39 unless otherwise specified. An exemplary human CD39 sequence is SEQ ID NO: 7 (UniProtKB accession number: P49961). CD39 is a two-pass transmembrane protein. The extracellular domain of CD39 includes residues 38 to 478 of SEQ ID NO: 7. An exemplary cynomolgus monkey CD39 sequence is SEQ ID NO: 8. The term "CD39 enzymatic activity" refers to the hydrolysis of ATP to ADP or AMP.

[0026] The terms "anti-CD39 antibody" and "antibody that binds to CD39" are used interchangeably herein and refer to an antibody that can bind to human CD39 with sufficient affinity to be useful as a diagnostic agent and / or therapeutic agent that targets human CD39 and inhibits human CD39 enzymatic activity. The anti-CD39 antibodies of the present disclosure have an equilibrium dissociation constant (KD) of 10 -6 M or less. The anti-CD39 antibodies of the present disclosure can be monospecific antibodies or multispecific antibodies, and in some examples, can be polyepitope antibodies.

[0027] As used herein, the term "antibody" is used in the broadest sense and encompasses various antibodies and antibody-like structures that specifically bind to a single antigen or multiple antigens, including but not limited to full-length antibodies, antigen-binding fragments, heavy-chain antibodies, single-chain antibodies, and higher-order variants of single-chain antibodies. Thus, references to an antibody should be understood to refer to an antibody in intact form or an antigen-binding fragment, unless the context requires otherwise. Preferably, although not necessarily, the antibodies useful herein are isolated and can be produced recombinantly.

[0028] The terms "full-length antibody", "intact antibody", and "whole antibody" are used interchangeably herein to refer to an antibody having a structure substantially similar to a native antibody structure or having a heavy chain that contains an Fc region.

[0029] A "native antibody" is a naturally occurring immunoglobulin molecule having various structures. For example, a native IgG antibody is a heterotetrameric glycoprotein of approximately 150,000 daltons, composed of two identical light chains (each approximately 25 kDa) and two identical heavy chains (each approximately 50 - 70 kDa) linked by disulfide bonds. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH), also called the variable heavy domain, followed by three constant domains (CH1, CH2, and CH3). Similarly, from the N-terminus to the C-terminus, each light chain has a variable region (VL), also called the variable light domain, followed by a constant light (CL) domain. The light chains of an antibody can be assigned one of two types, called kappa (κ) and lambda (λ), based on the amino acid sequence of their constant domain. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, defining the isotypes of the antibody as IgG, IgM, IgA, IgD, and IgE, respectively. The amino-terminal portions of each light and heavy chain contain variable regions (VL and VH, respectively) of approximately 100 - 110 or more amino acid sequences that are mainly involved in antigen recognition. The carboxy-terminal portions of each chain define constant regions that are mainly involved in effector functions. Within the light and heavy chains, the variable and constant regions are joined by a "J" region of approximately 12 or more amino acid sequences, and the heavy chain also contains a "D" region of approximately 10 amino acid sequences.

[0030] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to an antigen. The variable domains of the antibody heavy and light chains generally have a similar structure, and each domain contains four conserved framework regions (FRs) and three hypervariable regions (CDRs). (See, e.g., Kindt et al. Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007).) A single VH or VL domain may be sufficient to confer antigen-binding specificity. Further, a library of complementary VL or VH domains may be screened using the VH or VL domain from an antibody that binds an antigen to isolate an antibody that binds a particular antigen. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0031] The term "framework region" or "FR" refers to the variable domain residues other than hypervariable region residues. The FRs of a variable domain generally consist of four FR domains, FR1, FR2, FR3, and FR4. Thus, CDR and FR sequences generally occur in the following sequence: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The FR domains of the heavy and light chains may differ as known in the art.

[0032] As used herein, the terms "hypervariable region" or "HVR" are generally also referred to as "complementary determining region" or "CDR", and are used interchangeably, and each refers to a region of a variable domain that is hypervariable and / or forms a structurally defined loop ("hypervariable loop") and / or contains antigen contact residues ("antigen contact"). Generally, an antibody contains six CDRs, three in VH (H1, H2, H3) and three in VL (L1, L2, L3). As used herein, "CDR derived from a variable region" refers to a CDR having no more than two amino acid substitutions compared to the corresponding CDR from the original variable region. Exemplary CDRs herein include, as defined below for various antibodies of the present disclosure, (a) hypervariable loops occurring at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)); (b) CDRs occurring at amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)); (c) antigen contacts occurring at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum et al. J. Mol. Biol. 262: 732-745 (1996)); and (d) combinations of (a), (b), and / or (c). Unless otherwise specified, CDR residues and other residues (e.g., FR residues) in the variable domain are Eu-numbered herein according to Kabat et al. above.

[0033] The term "isolated antibody" refers to an antibody that has been separated from the components of its natural environment. In some embodiments, the isolated antibody is purified to greater than 95% or greater than 99% purity as determined, for example, by electrophoresis or chromatography (e.g., ion exchange or reverse phase HPLC).

[0034] The term "chimeric" antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species while the remaining portion of the heavy and / or light chain is derived from a different source or species.

[0035] A "human antibody" is one that possesses an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human or human cell, or derived from a non-human source that utilizes a human antibody repertoire. This definition of a human antibody specifically excludes humanized antibodies that contain non-human antigen-binding residues. Human antibodies can be produced using a variety of techniques known in the art, including phage display libraries. Hoogenboom and Winter. J. Mol. Biol. 227:381, 1991; Marks et al. J. Mol. Biol. 222:581, 1991. The methods described in Cole et al. Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boerner et al. J. Immunol., 147(1):86-95, 1991 are also available for the preparation of human monoclonal antibodies. See also van Dijk and van de Winkel. Curr. Opin. Pharmacol. 5:368-74, 2001.

[0036] A "humanized" antibody refers to an antibody that contains amino acid residues from non-human CDRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody will contain a variable domain in which all or substantially all of its CDRs correspond to the CDRs of a non-human antibody and all or substantially all of its FRs correspond to the FRs of a human antibody. In certain embodiments where all or substantially all of the FRs of the humanized antibody correspond to the FRs of a human antibody, any of the FRs of the humanized antibody can contain one or more amino acid residues of a non-human FR(s) at, for example, one or more benign position residues of the FR and / or one or more other selected residues. A humanized antibody may optionally include at least a portion of an antibody constant region derived from a human antibody. The "humanized form" of an antibody, e.g., a non-human antibody, refers to an antibody that has been humanized. A humanized antibody retains the binding specificity and affinity similar to the starting non-human antibody.

[0037] The term "monoclonal antibody" refers to an antibody derived from a single copy or clone, including, for example, any eukaryotic, prokaryotic, or phage clone. The term "monoclonal antibody" is not limited to antibodies produced by hybridoma technology. Monoclonal antibodies can be produced using hybridoma technology well known in the art, as well as recombinant technology, phage display technology, synthetic technology, or combinations of such technologies with other technologies readily known in the art.

[0038] The term "epitope" refers to a specific site on an antigen to which an antibody binds. The specific site on an antigen to which an antibody binds can be determined, for example, by crystallography. Methods such as hydroxyl radical protein footprinting and mutagenesis by alanine scanning can also be used, but there is a possibility of lower resolution.

[0039] The term "monospecific antibody" refers to an antibody that specifically binds to only one antigen. A monospecific antibody can bind to only one epitope of an antigen or to two or more epitopes of an antigen. A monospecific antibody that binds to two or more epitopes of an antigen is a monospecific polyepitope antibody.

[0040] The term "multispecific antibody" refers to an antibody that specifically binds to two or more antigens (e.g., bispecific antibody, trispecific antibody, etc.). Non-limiting examples of multispecific antibodies include antibodies that contain heavy chain variable domains (VH) and light chain variable domains (VL), where the VH / VL units have polyepitope specificity; antibodies that have two or more VL and VH domains, where each VH / VL unit binds to a different epitope; antibodies that have two or more single variable domains, where each single variable domain binds to a different epitope; diabodies, tribodies, etc., as well as full-length antibodies and / or antibody fragments that are covalently or non-covalently linked, but are not limited thereto.

[0041] The terms "polyepitope antibody" and "antibody having polyepitope specificity" are used interchangeably herein and refer to an antibody that binds to two or more epitopes on the same or different antigens.

[0042] The term "Fc region" is used herein to define the C-terminal region of the immunoglobulin heavy chain, including the native sequence Fc region and variant Fc regions. The boundaries of the Fc region of the immunoglobulin heavy chain can vary, but the human IgG heavy chain Fc region is typically defined as extending from the amino acid residue at position Cys226 or Pro230 to its carboxyl terminus. The C-terminal lysine of the Fc region (residue 447 according to the EU numbering system) may be removed, for example, during antibody production or purification, or by recombinant manipulation of the nucleic acid encoding the antibody heavy chain. Thus, a composition of intact antibodies may include an antibody population in which all Lys447 residues have been removed, an antibody population without removed Lys447 residues, and an antibody population having a mixture of antibodies with and without the Lys447 residue.

[0043] A "functional Fc region" has the "effector functions" of the native sequence Fc region. Exemplary "effector functions" include C1q binding; complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor; BCR), etc. Such effector functions generally need to be combined with an Fc region and a binding domain (e.g., an antibody variable domain) and can be evaluated using various assays disclosed herein or otherwise known in the art. A functional Fc region can have effector functions substantially similar to wild-type IgG, reduced effector functions compared to wild-type IgG, or enhanced effector functions compared to wild-type IgG. In the case of antibodies containing a human Fc region, the comparison is typically made with wild-type human IgG1.

[0044] The "native sequence Fc region" includes an amino acid sequence identical to the amino acid sequence of the Fc region found in nature. Native sequence human Fc regions include the native sequence human IgG1 Fc region (non-A and A allotypes), the native sequence human IgG2 Fc region, the native sequence human IgG3 Fc region, and the native sequence human IgG4 Fc region, as well as naturally occurring variants thereof.

[0045] The "variant Fc region" includes an amino acid sequence that is different from that of the native sequence Fc region by at least one amino acid modification (e.g., about 1 to about 10 amino acid modifications, and in some embodiments about 1 to about 5 amino acid modifications), preferably one or more amino acid substitutions. Variant Fc regions herein preferably have at least about 80% homology, preferably at least about 90% homology, or preferably at least about 95% homology with the native sequence Fc region and / or the Fc region of the parent polypeptide. In some embodiments, the variant Fc region may have reduced or enhanced effector function compared to wild-type IgG. In the case of antibodies comprising a human Fc region, the comparison is typically made to wild-type human IgG1.

[0046] As used herein, "Fc component" refers to the hinge region, CH2 domain or CH3 domain of the Fc region.

[0047] The "hinge region" is generally defined as extending from around residue 216 to around residue 230 (Eu numbering) of IgG, from around residue 226 to around residue 243 (Kabat numbering) of IgG, or from around residue 1 to around residue 15 (IMGT unique numbering) of IgG.

[0048] The term "antibody fragment" refers to a molecule other than an intact antibody that includes a portion of an intact antibody and binds to an antigen to which the intact antibody binds. Examples of antigen-binding fragments include, but are not limited to, diabodies, Fab, Fab', F(ab')2, F(ab) c, Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv’), disulfide-stabilized diabodies (ds diabodies), tribodies, tetrabodies, single-chain antibodies, scFv, scFv dimers, single-domain antibodies, single-domain antibodies, and multivalent domain antibodies. Typically, binding fragments compete with intact antibodies from which they are derived for specific binding. Binding fragments can be produced by recombinant DNA techniques or by enzymatic or chemical separation of intact immunoglobulins.

[0049] The term "Fab" refers to a portion of an antibody consisting of a single light chain (both variable and constant regions) linked by a disulfide bond to the variable region and the first constant region of a single heavy chain.

[0050] The term "Fab" refers to a Fab fragment that includes a portion of the hinge region.

[0051] The term "F(ab’)2" refers to a dimer of Fab’. F(ab’)2 antibody fragments were originally generated as pairs of Fab’ fragments with an intervening hinge cysteine. Other chemical couplings of antibody fragments are also known.

[0052] The term "Fv" refers to the smallest fragment of an antibody that retains the complete antigen-binding site. The Fv fragment consists of the variable region of a single light chain linked to the variable region of a single heavy chain.

[0053] The term "single-chain antibody" refers to an antibody consisting of a heavy-chain variable region and a light-chain variable region connected by a linker. In most, but not all, cases, the linker can be a peptide. The length of the linker varies depending on the type of single-chain antibody. Higher-order forms can be obtained by covalently or non-covalently linking two or more single-chain antibodies. Examples of single-chain antibodies and their higher-order forms include, but are not limited to, single-domain antibodies, multivalent domain antibodies, single-chain variable fragments (scFv), bivalent scFv (di-scFv), trivalent scFv (tri-scFv), tetravalent scFv (tetra-scFvs), diabodies, and triabodies, and tetrabodies.

[0054] The terms "single-chain Fv antibody" and "scFv" are used interchangeably herein and refer to a single-chain antibody consisting of a heavy-chain variable region and a light-chain variable region connected by a linker. In most, but not all, cases, the linker can be a peptide. The linker peptide is preferably about 5 to 30 amino acids in length, or about 10 to 25 amino acids in length. Typically, the linker enables stabilization of the variable domains without interfering with proper folding and the creation of an active binding site. In a preferred embodiment, the linker peptide is rich in glycine, as well as serine or threonine. Higher-order forms such as di-scFv, tri-scFv, tetra-scFv can be obtained by covalently or non-covalently linking two or more scFv. The antigen-binding sites of each scFv in the higher-order forms can target the same or different antigens or epitopes.

[0055] The term "single-chain Fv-Fc antibody" or "scFv-Fc" refers to a full-length antibody consisting of an scFv connected to an Fc region.

[0056] "Diabody" is a higher-order variant of single-chain antibodies consisting of two single-chain antibodies. Each single-chain antibody uses a linker that is too short to allow pairing between two domains on the same chain, and the domains are paired with complementary domains on another chain, thereby creating two antigen-binding sites. In most but not all cases, the linker can be a peptide. The antigen-binding sites can target the same or different antigens or epitopes. Triabodies (formed by the assembly of three single-chain antibodies to form three antigen-binding sites), tetrabodies (formed by the assembly of four single-chain antibodies to form four antigen-binding sites), and higher-order variants can be generated in the same way. See, for example, Holliger P. et al., Proc Natl Acad Sci USA. July 15;90(14):6444-8 (1993); EP404097; WO93 / 11161.

[0057] "Single-domain antibody" refers to an antibody fragment containing only the variable region of the heavy chain or the variable region of the light chain. In one particular example, two or more V H domains are covalently linked with a peptide linker to create a multivalent domain antibody. Two or more V H domains of the multivalent domain antibody can target the same or different antigens or epitopes.

[0058] The term "heavy-chain antibody" refers to an antibody consisting of two heavy chains. Heavy-chain antibodies can be IgG-like antibodies from camels, llamas, alpacas, sharks, etc., or IgNAR from cartilaginous fish. See, for example, Riechmann L. and Muyldermans S., J Immunol Methods. December 10;231(1-2): 25-38 (1999); Muyldermans S., J Biotechnol. June;74(4):277-302 (2001); WO94 / 04678; WO94 / 25591; or U.S. Patent No. 6,005,079. Heavy-chain antibodies originally derived from the Camelidae family (camels, dromedaries, and llamas). Although lacking light chains, camelized antibodies have a genuine antigen-binding repertoire (see Hamers-Casterman C. et al., Nature. June 3;363(6428):446-8 (1993); Nguyen V. K. et al. “Heavy-chain antibodies in Camelidae; a case of evolutionary innovation,” Immunogenetics. April;54(1):39-47 (2002); Nguyen V. K. et al. Immunology. May;109(1):93-101 (2003)). The variable domain (VHH domain) of the heavy-chain antibody represents the smallest known antigen-binding unit generated by the adaptive immune response (see Koch-Nolte F. et al., FASEB J. November;21(13):3490-8. Epub 2007 Jun. 15 (2007)).

[0059] "Nanobody" refers to an antibody consisting of a VHH domain derived from a heavy-chain antibody and two constant domains, CH2 and CH3.

[0060] The "percent identity (%)" with respect to a reference amino acid sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in the reference sequence, after aligning the sequences and introducing gaps if necessary to obtain the maximum percent sequence identity, and without considering any conservative substitutions as part of the sequence identity. Alignments for the purpose of determining amino acid sequence identity percentages can be achieved in a variety of ways within the skill of the art, using publicly available computer software such as, for example, BLAST, BLAST-2, or CLUSTAL software. One of ordinary skill in the art can determine appropriate parameters for aligning the sequences, including any algorithms necessary to obtain the maximum alignment over the full length of the sequences being compared. Generally speaking, the sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B is calculated as follows: 100× fraction X / Y, where X is the number of amino acid residues scored as exact matches in the alignment of A and B by the sequence alignment program, and Y is the total number of amino acid residues in B. It is understood that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, the percent amino acid sequence identity of A to B will not be equal to the percent amino acid sequence identity of B to A.

[0061] The terms "patient" or "subject" are used interchangeably herein and refer to a human or non-human animal (e.g., a mammal) that expresses human CD39.

[0062] The terms “treat,” “treating,” “treatment,” etc. refer to a series of actions that temporarily or permanently remove, reduce, suppress, alleviate, or improve at least one of the diseases, disorders, or conditions to which the term applies, or the symptoms associated therewith, or prevent their worsening. Treatments include, by way of example, inhibition of an active disease (e.g., prevention of the onset or further onset of a disease, disorder or condition, or the clinical symptoms associated therewith), improvement of quality of life, and / or extension of the lifespan of a subject.

[0063] As used herein, the term “in need of treatment” refers to a determination made by a physician or other caregiver that a subject needs treatment or would benefit from treatment. This determination is made based on a variety of factors within the realm of the physician's or caregiver's expertise.

[0064] The terms “prevent,” “preventing,” “prevention,” “prophylaxis,” etc. generally refer to a series of actions that are initiated in one manner in order to temporarily or permanently prevent, suppress, inhibit, or reduce the risk that a subject having a tendency to have a particular disease, disorder, or condition will develop the disease, disorder, condition, etc. (e.g., as determined by the absence of clinical symptoms), or to delay its onset (e.g., prior to the onset of the disease, disorder, condition, or its symptoms). In certain specific examples, these terms also refer to slowing the progression of a disease, disorder, or condition, or inhibiting their progression to a harmful or otherwise undesirable state. Prevention also refers to a series of actions that are initiated in a subject in order to prevent recurrence of the disease, disorder, condition, or symptoms associated therewith after the subject has received treatment for the disease, disorder, condition, or symptoms.

[0065] As used herein, the term "in need of prophylaxis" refers to a determination made by a physician or other caregiver that a subject requires prophylactic care or would benefit from prophylactic care. This determination is made based on a variety of factors within the realm of the physician's or caregiver's expertise.

[0066] II. Antibodies of the Disclosure The present disclosure provides antibodies that specifically bind to human CD39, more specifically to the extracellular domain of human CD39. The antibodies of the present disclosure can specifically bind to soluble and / or membrane-bound human CD39. In some embodiments, the anti-CD39 antibodies of the present disclosure are monospecific. In other embodiments, the anti-CD39 antibodies of the present disclosure are multispecific. Additional antigen-binding specificities contemplated in the context of the present disclosure include, but are not limited to, TGFβ. The anti-CD39 antibodies of the present application can be isolated or can be part of a composition.

[0067] Whether monospecific or multispecific, the anti-CD39 antibodies of the present disclosure specifically bind to human CD39, which means that the antibody has an equilibrium dissociation constant (K -6 ) of 10 D M or less as measured by surface plasmon resonance (SPR). In certain embodiments, the anti-CD39 antibodies of the present disclosure have a K D for human CD39 of 10 -8 M or less (e.g., 10 -8 , 10 -9 , 10 -10 , etc.) as measured by SPR. For example, see Example 3 for detailed methodology. In various embodiments, the anti-CD39 antibodies of the present disclosure have a K D for human CD39 of about 1x10 -9 M to about 1x10 -14 M, or about 1x10 -9 M to about 1x10 -13 M, or about 1x10 -9 M to about 1x10 -12 M, or about 1x10 -9 M to about 1x10 -11It is M. In some embodiments, the anti-CD39 antibody of the present disclosure has a K for human CD39 D of about 1x10 -10 to about 1x10 -14 M, or about 1x10 -10 M to about 1x10 -13 M, or about 1x10 -10 M to about 1x10 -12 M. In some embodiments, the anti-CD39 antibody of the present disclosure has a K for human CD39 D of about 1x10 -11 M to about 1x10 -14 M, or about 1x10 -11 to about 1x10 -13 M. In some embodiments, the anti-CD39 antibody of the present disclosure has a K for human CD39 D of about 1x10 -12 M to about 1x10 -14 M, or about 1x10 -12 M to about 1x10 -13 M. In some embodiments, the anti-CD39 antibody of the present disclosure has a K for human CD39 D of about 1x10 -10 M to about 1x10 -11 M, or about 1x10 -11 M to about 1x10 -12 M.

[0068] The anti-CD39 antibody of the present disclosure also inhibits CD39 enzyme activity. In some embodiments, the anti-CD39 antibody of the present disclosure inhibits human CD39 enzyme activity at an IC 50 value of about 5 nM or less, as measured in Example 5. For example, the anti-CD39 antibody can inhibit recombinant human CD39 enzyme activity at an IC 50 value of about 5 nM, about 4 nM, about 3 nM, about 2 nM, about 1 nM, or less, as measured in Example 5. As another example, the anti-CD39 antibody has an IC 50Values can inhibit recombinant human CD39 enzyme activity. As another example, an anti-CD39 antibody has an IC 50 value that can inhibit recombinant human CD39 enzyme activity. The above IC 50 value may also be expressed as individual values or ranges. For example, an anti-CD39 antibody can inhibit recombinant human CD39 enzyme activity at an IC 50 value of about 0.05 nM to about 5 nM, about 0.05 nM to about 1 nM, 0.05 nM to about 0.5 nM, about 1 nM to about 5 nM, about 0.5 nM to about 1.0 nM, or a sub-range thereof. In one embodiment, the anti-CD39 antibody of the present disclosure inhibits human CD39 enzyme activity at an IC 50 value of about 0.5 nM to about 1.0 nM, or about 0.6 nM to about 1.0 nM. In one embodiment, the anti-CD39 antibody of the present disclosure inhibits human CD39 enzyme activity at an IC 50 value of about 0.05 nM to about 0.5 nM, about 0.05 nM to about 0.4 nM, or about 0.05 nM to about 0.3 nM. In one embodiment, the anti-CD39 antibody of the present disclosure inhibits human CD39 enzyme activity at an IC 50 value of about 0.07 nM to about 0.5 nM, about 0.07 nM to about 0.4 nM, about 0.07 nM to about 0.3 nM, or about 0.07 nM to about 0.2 nM. In the foregoing embodiments, (i) human CD39 may be recombinant soluble CD39 or cell surface-expressed CD39, and / or (ii) inhibition may be evaluated in the presence of low ATP (e.g., 20 μM) and / or high ATP (e.g., 400 μM).

[0069] A. Exemplary variable regions The present disclosure provides anti-CD39 antibodies comprising VH, or VH and VL, as described herein. In the following embodiments, "at least X% sequence identity" encompasses their individual values and ranges. For example, "at least 90% sequence identity" includes at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity, as well as the individual values (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity) and ranges thereof.

[0070] In one example, the anti-CD39 antibody of the present disclosure comprises a heavy chain variable region (VH) having one or more CDRs derived from SEQ ID NO: 9 and a light chain variable region (VL) optionally having one or more CDRs derived from SEQ ID NO: 13. The CDRs derived from SEQ ID NO: 9 may be H1, H2, H3, or any combination thereof. In certain embodiments, VH may comprise H1 having at least 90% sequence identity with SEQ ID NO: 10, H2 having at least 90% sequence identity with SEQ ID NO: 11, H3 having at least 90% sequence identity with SEQ ID NO: 12, or any combination thereof. The CDRs derived from SEQ ID NO: 13 may be L1, L2, L3, or any combination thereof. In certain embodiments, VL may comprise L1 having at least 90% sequence identity with SEQ ID NO: 14, L2 having at least 90% sequence identity with SEQ ID NO: 15, L3 having at least 90% sequence identity with SEQ ID NO: 16, or any combination thereof. An antibody comprising one or more CDRs derived from SEQ ID NO: 9 may further comprise a VL comprising one or more CDRs derived from SEQ ID NO: 13. The CDRs may be L1, L2, L3, or any combination thereof. In preferred embodiments, VL comprises L1 having an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 14, L2 having an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 15, L3 having an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 16, or any combination thereof. In a specific example, the anti-CD39 antibody of the present disclosure comprises a VH comprising H1 having an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 10, H2 having an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 11, H3 having an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 12, and a VL comprising L1 having an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 14, L2 having an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 15, and L3 having an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 16.In another specific example, the anti-CD39 antibody of the present disclosure includes a VH containing H1 having an amino acid sequence including SEQ ID NO: 10, H2 having an amino acid sequence including SEQ ID NO: 11, and H3 having an amino acid sequence including SEQ ID NO: 12, and a VL including L1 having an amino acid sequence including SEQ ID NO: 14, L2 having an amino acid sequence including SEQ ID NO: 15, and L3 having an amino acid sequence including SEQ ID NO: 16.

[0071] In some of the foregoing embodiments, the antibody may further include (i) a mature VH having an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 9, and / or (ii) a mature VL having an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 13. In an exemplary embodiment, the antibody includes a mature VH and a mature VL and is the antibody of Table A. JPEG0007712496000001.jpg1892

[0072] In each of the foregoing embodiments, the anti-CD39 antibody can be (i) an intact antibody or an antigen-binding fragment, and / or (ii) a chimeric antibody, a humanized antibody, or a human antibody. Suitable chimeric antibodies, humanized antibodies, and human antibodies are further described in Sections IIC, IID, and IIE. In certain embodiments, the anti-CD39 antibody can optionally include one or more constant regions, or a portion of a constant region, that is substantially human. Suitable constant regions are described in more detail in Section II(F).

[0073] In another exemplary embodiment, the antibody is the antibody of Table B. JPEG0007712496000002.jpg18106

[0074] In another example, the anti-CD39 antibody of the present disclosure comprises a heavy chain variable region (VH) having one or more CDRs derived from SEQ ID NO: 17, SEQ ID NO: 43, or SEQ ID NO: 44, and optionally a light chain variable region (VL) having one or more CDRs derived from SEQ ID NO: 21, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, or SEQ ID NO: 49. The CDRs derived from SEQ ID NO: 17, SEQ ID NO: 43, or SEQ ID NO: 44 may be H1, H2, H3, or any combination thereof. In certain embodiments, VH may comprise H1 having at least 90% sequence identity with SEQ ID NO: 18, H2 having at least 90% sequence identity with SEQ ID NO: 19, H3 having at least 90% sequence identity with SEQ ID NO: 20, or any combination thereof. The CDRs derived from SEQ ID NO: 21, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, or SEQ ID NO: 49 may be L1, L2, L3, or any combination thereof. In certain embodiments, VL may comprise L1 having at least 90% sequence identity with SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 24, L2 having at least 90% sequence identity with SEQ ID NO: 25, L3 having at least 90% sequence identity with SEQ ID NO: 26, or any combination thereof. An antibody comprising one or more CDRs derived from SEQ ID NO: 17, SEQ ID NO: 43, or SEQ ID NO: 44 may further comprise a VL comprising one or more CDRs derived from SEQ ID NO: 21, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, or SEQ ID NO: 49. The CDRs may be L1, L2, L3, or any combination thereof. In preferred embodiments, VL may comprise L1 having at least 90% sequence identity with SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24, L2 having at least 90% sequence identity with SEQ ID NO: 25, L3 having at least 90% sequence identity with SEQ ID NO: 26, or any combination thereof.In a specific example, the anti-CD39 antibody of the present disclosure includes VH having an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 18, H2 having an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 19, H3 having an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 20, and VL including L1 having an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24, L2 having an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 25, and L3 having an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 26. In another specific example, the anti-CD39 antibody of the present disclosure includes VH including an amino acid sequence containing SEQ ID NO: 18, H2 including an amino acid sequence containing SEQ ID NO: 19, H3 including an amino acid sequence containing SEQ ID NO: 20, and VL including L1 having an amino acid sequence containing SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24, L2 having an amino acid sequence containing SEQ ID NO: 25, and L3 having an amino acid sequence containing SEQ ID NO: 26.

[0075] In some of the foregoing embodiments, the antibody may further include (i) VH having an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 17 and / or VL having an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 21; or (ii) VH having an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 43 or SEQ ID NO: 44 and / or VL having an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, or SEQ ID NO: 49. In an exemplary embodiment, the antibody includes VH and VL and is the antibody of Table C. JPEG0007712496000003.jpg5996

[0076] In each of the foregoing embodiments, the anti-CD39 antibody can be (i) an intact antibody or antigen-binding fragment, and / or (ii) a chimeric antibody, humanized antibody, or human antibody. Suitable chimeric antibodies, humanized antibodies, and human antibodies are further described in Sections IIC, IID, and IIE. In certain embodiments, the anti-CD39 antibody can optionally include one or more substantially human constant regions, or portions of constant regions. Suitable constant regions are described in further detail in Section II(F).

[0077] In another exemplary embodiment, the antibody is the antibody of Table D. JPEG0007712496000004.jpg65111

[0078] In another example, an anti-CD39 antibody of the present disclosure comprises a heavy chain variable region (VH) having one or more CDRs derived from SEQ ID NO: 27, SEQ ID NO: 58, or SEQ ID NO: 59, and optionally a light chain variable region (VL) having one or more CDRs derived from SEQ ID NO: 31, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, or SEQ ID NO: 63. The CDRs derived from SEQ ID NO: 58 or SEQ ID NO: 59 may be H1, H2, H3, or any combination thereof. In certain embodiments, VH may comprise H1 having at least 90% sequence identity with SEQ ID NO: 28, H2 having at least 90% sequence identity with SEQ ID NO: 29, H3 having at least 90% sequence identity with SEQ ID NO: 30, or any combination thereof. The CDRs derived from SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63 may be L1, L2, L3, or any combination thereof. In certain embodiments, VL may comprise L1 having at least 90% sequence identity with SEQ ID NO: 32, L2 having at least 90% sequence identity with SEQ ID NO: 33, L3 having at least 90% sequence identity with SEQ ID NO: 34, or any combination thereof. An antibody comprising one or more CDRs derived from SEQ ID NO: 58 or SEQ ID NO: 59 may further comprise a VL comprising one or more CDRs derived from SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63. The CDRs may be L1, L2, L3, or any combination thereof. In a preferred embodiment, VL may comprise L1 having an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 32, L2 having an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 33, L3 having an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 34, or any combination thereof.In a specific example, the anti-CD39 antibody of the present disclosure includes VH having an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 28, H2 having an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 29, H3 having an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 30, and VL including L1 having an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 32, L2 having an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 33, and L3 having an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 34. In another specific example, the anti-CD39 antibody of the present disclosure includes VH including an amino acid sequence containing SEQ ID NO: 28, H2 including an amino acid sequence containing SEQ ID NO: 29, H3 including an amino acid sequence containing SEQ ID NO: 30, and VL including L1 having an amino acid sequence containing SEQ ID NO: 32, L2 having an amino acid sequence containing SEQ ID NO: 33, and L3 having an amino acid sequence containing SEQ ID NO: 34.

[0079] In some of the foregoing embodiments, the antibody may further include (i) VH having an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 27 and / or VL having an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 31; or (ii) VH having an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 58 or SEQ ID NO: 59 and / or VL having an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, or SEQ ID NO: 63. In an exemplary embodiment, the antibody includes VH and VL and is the antibody in Table E. JPEG0007712496000005.jpg6697

[0080] In each of the foregoing embodiments, the anti-CD39 antibody can be (i) an intact antibody or antigen-binding fragment, and / or (ii) a chimeric antibody, humanized antibody, or human antibody. Suitable chimeric antibodies, humanized antibodies, and human antibodies are further described in Sections IIC, IID, and IIE, respectively. In certain embodiments, the anti-CD39 antibody can optionally include one or more constant regions, or portions of constant regions, that are substantially human. Suitable constant regions are described in more detail in Section II(F).

[0081] In another exemplary embodiment, the antibody is the antibody of Table F. JPEG0007712496000006.jpg77114

[0082] B. Antibodies Having Similar Binding Specificity The present disclosure also provides anti-CD39 antibodies that bind to the same or overlapping epitopes as the antibodies designated as 19 or ch19_IGG4.P above, or the antibodies designated as 31, ch31_IGG4.P, hu31.1_IGG4.P, hu31.2_IGG4.P, hu31.3_IGG4.P, hu31.4_IGG4.P, hu31.4_IGG1.AA, hu31.5_IGG4.P, hu31.6_IGG4.P, or hu31.7_IGG4.P above, or the antibodies designated as 39, ch39_IGG4.P, ch39_mIGG2A.AAG, hu39.1_IGG4.P, hu39.2_IGG4.P, hu39.3_IGG4.P, hu39.4_IGG4.P, hu39.5_IGG4.P, hu39.5_IGG1.AA, hu39.6_IGG4.P, hu39.7_IGG4.P, or hu39.8_IGG4.P above. The epitope may be identified by methods known in the art, such as X-ray crystallography of the antibody bound to its antigen, to identify the contacting residues. Such antibodies may be identified using a competition assay. For example, the anti-CD39 antibody can competitively inhibit the binding of a reference antibody to human CD39, and the reference antibody is selected from 19, ch19_IGG4.P, 31, ch31_IGG4.P, hu31.1_IGG4.P, hu31.2_IGG4.P, hu31.3_IGG4.P, hu31.4_IGG4.P, hu31.4_IGG1.AA, hu31.5_IGG4.P, hu31.6_IGG4.P, or hu31.7_IGG4.P, 39, ch39_IGG4.P, ch39_mIGG2A.AAG, hu39.1_IGG4.P, hu39.2_IGG4.P, hu39.3_IGG4.P, hu39.4_IGG4.P, hu39.5_IGG4.P, hu39.5_IGG1.AA, hu39.6_IGG4.P, hu39.7_IGG4.P, or hu39.8_IGG4.P. An antibody is said to competitively inhibit the binding of a reference antibody to human CD39 if it blocks the binding of the reference antibody to human CD39 by at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%.Competitive inhibition can be determined, for example, by a competitive flow assay as described in Example 7.

[0083] In a further embodiment, as described above, antibodies that competitively inhibit the binding of a reference antibody to human CD39 include the VH, or VH and VL, described in Section II(A).

[0084] Other antibodies having such binding specificities can be produced by immunizing mice with human CD39 or a portion thereof containing the desired epitope, and screening the resulting antibodies for binding to the extracellular domain of human CD39 that competes with antibody 19 or its variant (e.g., ch19_IGG4.P, etc.), antibody 31, or its variant (e.g., ch31_IGG4.P, hu31.1_IGG4.P, hu31.2_IGG4.P, hu31.3_IGG4.P, hu31.4_IGG4.P, hu31.4_IGG1.AA, hu31.5_IGG4.P, hu31.6_IGG4.P, hu31.7_IGG4.P, etc.), or antibody 39 or its variant (e.g., ch39_IGG4.P, ch39_mIGG2A.AAG, hu39.1_IGG4.P, hu39.2_IGG4.P, hu39.3_IGG4.P, hu39.4_IGG4.P, hu39.5_IGG4.P, hu39.5_IGG1.AA, hu39.6_IGG4.P, hu39.7_IGG4.P, hu39.8_IGG4.P, etc.). The antibodies can also be screened against mutagenized forms of human CD39 to identify antibodies that exhibit a binding profile the same as or similar to that of the set of mutagenic changes, such as 19, ch19_IGG4.P, 31, ch31_IGG4.P, hu31.1_IGG4.P, hu31.2_IGG4.P, hu31.3_IGG4.P, hu31.4_IGG4.P, hu31.4_IGG1.AA, hu31.5_IGG4.P, hu31.6_IGG4.P, hu31.7_IGG4.P, 39, ch39_IGG4.P, ch39_mIGG2A.AAG, hu39.1_IGG4.P, hu39.2_IGG4.P, hu39.3_IGG4.P, hu39.4_IGG4.P, hu39.5_IGG4.P, hu39.5_IGG1.AA, hu39.6_IGG4.P, hu39.7_IGG4.P, or hu39.8_IGG4.P.The mutation can be a systematic replacement substitution with alanine (or serine if alanine is already present), one residue at a time or at wider intervals, across the entire extracellular domain of the CD39 antibody or the section in which the epitope is known to be present.

[0085] Other antibodies can be obtained by mutagenesis of the cDNA encoding the heavy and light chains of exemplary antibodies such as 19, ch19_IGG4.P, 31, ch31_IGG4.P, hu31.1_IGG4.P, hu31.2_IGG4.P, hu31.3_IGG4.P, hu31.4_IGG4.P, hu31.4_IGG1.AA, hu31.5_IGG4.P, hu31.6_IGG4.P, hu31.7_IGG4.P, 39, ch39_IGG4.P, ch39_mIGG2A.AAG, hu39.1_IGG4.P, hu39.2_IGG4.P, hu39.3_IGG4.P, hu39.4_IGG4.P, hu39.5_IGG4.P, hu39.5_IGG1.AA, hu39.6_IGG4.P, hu39.7_IGG4.P, or hu39.8_IGG4.P. Antibodies having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to 19, ch19_IGG4.P, 31, ch31_IGG4.P, hu31.1_IGG4.P, hu31.2_IGG4.P, hu31.3_IGG4.P, hu31.4_IGG4.P, hu31.4_IGG1.AA, hu31.5_IGG4.P, hu31.6_IGG4.P, hu31.7_IGG4.P, 39, ch39_IGG4.P, ch39_mIGG2A.AAG, hu39.1_IGG4.P, hu39.2_IGG4.P, hu39.3_IGG4.P, hu39.4_IGG4.P, hu39.5_IGG4.P, hu39.5_IGG1.AA, hu39.6_IGG4.P, hu39.7_IGG4.P, or hu39.8_IGG4.P in the amino acid sequence of the mature heavy and / or light chain variable regions and maintaining their functional properties, and / or antibodies that differ from each respective antibody by a small number of amino acid substitutions (e.g., conservative substitutions), deletions, or insertions that are not functionally important are also included in the present disclosure. Amino acids within the variable region framework that are likely to be important for binding can be identified as described in the section on humanization below.

[0086] Antibodies having the binding specificity of a selected rodent antibody (e.g., 19, 31, or 37) or a selected humanized antibody (e.g., hu31.1_IGG4.P, hu31.2_IGG4.P, hu31.3_IGG4.P, hu31.4_IGG4.P, hu31.4_IGG1.AA, hu31.5_IGG4.P, hu31.6_IGG4.P, hu31.7_IGG4.P, hu39.1_IGG4.P, hu39.2_IGG4.P, hu39.3_IGG4.P, hu39.4_IGG4.P, hu39.5_IGG4.P, hu39.5_IGG1.AA, hu39.6_IGG4.P, hu39.7_IGG4.P, or hu39.8_IGG4.P) can also be produced using a modification of the phage display method. See Winter's WO92 / 20791. This method is particularly suitable for the production of human antibodies. In this method, either the heavy chain variable region or the light chain variable region of the selected antibody is used as the starting material. For example, if the light chain variable region is selected as the starting material, a phage library is constructed in which the members display the same light chain variable region (i.e., the light chain of the starting material) and different heavy chain variable regions. The heavy chain variable regions can be obtained, for example, from a library of rearranged human heavy chain variable regions. Phages that show strong specific binding to human CD39 (e.g., at least 10 8 or at least 10 9 M -1 ) are selected. Next, the heavy chain variable region from this phage functions as the starting material for constructing a further phage library. In this library, each phage displays the same heavy chain variable region (i.e., the region identified from the first display library) and different light chain variable regions. The light chain variable regions can be obtained, for example, from a library of rearranged human light chain variable regions. Again, phages that show strong specific binding to human CD39 are selected. The resulting antibodies usually have the same or similar epitope specificity as the murine starting material.

[0087] In addition to the antibodies described herein, it may be possible to design antibody mimetics or aptamers having the same binding specificity and substantially the same function as the antibodies of the present invention using methods known in the art. An "antibody mimetic" refers to a polypeptide or protein that can specifically bind to an antigen but is not structurally related to an antibody. Antibody mimetics have a mass of about 3 kDa to about 20 kDa. Non-limiting examples of antibody mimetics are affibody molecules, affilins, affimers, alphabodies, anticalins, avimers, DARPins, and monobodies. An aptamer is a class of small nucleic acid ligands composed of RNA or single-stranded DNA oligonucleotides that have high specificity and affinity for their targets. Aptamers interact and bind to their targets through structure recognition, a process similar to the antigen-antibody reaction. Aptamers have a lower molecular weight than antibodies and are typically about 8 - 25 kDa.

[0088] C. Chimeric Antibodies and Humanized Antibodies As described above, the present disclosure provides chimeric and humanized forms of non-human antibodies, including but not limited to chimeric anti-CD39 antibodies of the antibodies designated herein as antibody 19, antibody 31, or antibody 39.

[0089] In certain embodiments, a chimeric antibody is an antibody in which the mature variable regions of the light and heavy chains of a non-human antibody (e.g., mouse, rat, etc.) are combined with human light and heavy chain constant regions. Such antibodies substantially or completely retain the binding specificity of the non-human antibody and are about two-thirds human sequence.

[0090] A veneered antibody is a type of humanized antibody that retains some, and usually all, of the CDRs of a non-human antibody and some of the non-human variable region framework residues, but replaces other variable region framework residues that may contribute to B cell or T cell epitopes, such as exposed residues (Padlan, Mol. Immunol. 28:489, 1991), with residues at the corresponding positions of a human antibody sequence. As a result, the CDRs are fully or substantially derived from the non-human antibody, and the variable region framework of the non-human antibody becomes more human-like by substitution. Veneered forms of anti-CD39 antibodies are included in the present disclosure.

[0091] In some embodiments, the anti-CD39 chimeric antibody is a rat-human chimera having a rat variable domain and a human IgG1 and kappa constant domain (or a variant thereof) or a human IgG4 and kappa constant domain (or a variant thereof). Suitable human constant domains are known in the art and are further described in Section II(F). In a specific embodiment, the anti-CD39 chimeric antibodies are ch19_IGG4.P, ch31_IGG4.P, and ch39_IGG4.P.

[0092] D. Humanized Antibodies As described herein, the present disclosure provides humanized antibodies of the antibodies designated 19, 31, or 39 herein, and optionally, the humanized antibodies have an IC as measured as in Example 5 of about 5 nM or less, about 0.05 nM to about 5 nM, about 1 nM or less, about 0.05 nM to about 1 nM, about 0.5 nM or less, or about 0.05 nM to about 0.5 nM. 50Inhibits human CD39 enzyme activity by a value. Typically, non-human antibodies are humanized to reduce immunogenicity in humans while retaining the specificity and affinity of the parental non-human antibody. A humanized antibody contains one or more variable domains in which the CDRs or portions thereof are derived from a non-human antibody and the FRs or portions thereof are derived from a human antibody sequence. The humanized antibody may optionally also include at least a portion of a human constant region. In some embodiments, some FR residues in the humanized antibody are replaced with corresponding residues from a non-human antibody (e.g., the antibody from which the CDR residues are derived) to, for example, restore or improve antibody specificity or affinity.

[0093] Accordingly, a humanized antibody is an antibody in which some or all of the CDRs are derived entirely or substantially from a donor antibody and, where present, the variable region framework sequences and constant regions are derived entirely or substantially from human antibody sequences. For example, a humanized heavy chain has at least one, two, and usually all three CDRs that are derived entirely or substantially from a donor antibody heavy chain, and, where present, a heavy chain variable region framework sequence and heavy chain constant region that are derived substantially from a human heavy chain variable region framework and constant region sequence. Similarly, a humanized light chain has at least one, two, and usually all three CDRs that are derived entirely or substantially from a donor antibody light chain, and, where present, a light chain variable region framework sequence and light chain constant region that are derived substantially from a human light chain variable region framework and constant region sequence. Here too, as in other places in this application, the CDRs of the subject antibody are substantially derived from the corresponding CDRs of a reference antibody when at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the corresponding residues (as defined by Kabat) are identical between the respective CDRs; however, CDR H2 as defined by Kabat in the subject antibody is substantially derived from the corresponding CDR of a reference antibody when at least about 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the corresponding residues (as defined by Kabat) are identical between the respective CDRs. The variable region framework sequence of an antibody chain or the constant region of an antibody chain is substantially derived from a human variable region framework sequence or human constant region, respectively, when at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the corresponding residues as defined by Kabat are identical.

[0094] Generally speaking, a humanized antibody is a genetically engineered antibody in which the CDRs derived from a non-human "donor" antibody are grafted onto a human "acceptor" antibody sequence (see, for example, Queen, US5,530,101 and 5,585,089; Winter, US5,225,539, Carter, US6,407,213, Adair, US5,859,2056,881,557, Foote, US6,881,557). The acceptor antibody sequence can be, for example, a mature human antibody sequence, a complex of such sequences, a consensus sequence of human antibody sequences, or a germline region sequence. A humanized antibody can incorporate all six CDRs from a non-human (e.g., mouse, rat, etc.) antibody, but can also be made using fewer CDRs (e.g., at least 3, 4, or 5) than all from a non-human antibody (see, for example, Pascalis et al., J. Immunol. 169:3076, 2002; Vajdos et al., Journal of Molecular Biology, 320: 415-428, 2002; Iwahashi et al., Mol. Immunol. 36:1079-1091, 1999; Tamura et al, Journal of Immunology, 164:1432-1441, 2000).

[0095] For some antibodies, only a portion of the CDR, i.e., a subset of the CDR residues necessary for binding, called the SDR, is required to retain binding in a humanized antibody. CDR residues that do not contact the antigen and are not included in the SDR can be identified from regions of the Kabat CDR outside the Chothia hypervariable loops (Chothia, J. Mol. Biol. 196:901, 1987), by molecular modeling, and / or empirically, and / or as described in Gonzales et al., Mol. Immunol. 41: 863, 2004, based on previous studies (e.g., residues H60 - H65 of CDR H2 are often not required). In such humanized antibodies, at positions where one or more donor CDR residues are absent, or where the entire donor CDR is omitted, the amino acid occupying that position can be the amino acid occupying the corresponding position (by Kabat numbering) in the acceptor antibody sequence. In some antibodies, potential sites of post-translational modification (e.g., glycosylation) within the CDR can be substituted to eliminate post-translational modification. The substitution positions within the CDR and the amino acids to be substituted can also be selected empirically.

[0096] The acceptor can have the same sequence for a selected human framework sequence, regardless of whether it is from a human immunoglobulin or a human consensus framework, but in the present disclosure, it is contemplated that the acceptor sequence can include existing amino acid substitutions relative to the human immunoglobulin sequence or the human consensus framework sequence. These existing substitutions can be minimal; generally, there are only 4, 3, 2, or 1 amino acid differences relative to the human immunoglobulin sequence or the consensus framework sequence. The human acceptor antibody sequence can be arbitrarily selected from among many known human antibody sequences to provide a high degree of sequence identity (e.g., 65 - 85% identity) between the human acceptor sequence variable region framework and the corresponding variable region framework of the donor antibody chain.

[0097] Certain amino acids from human variable region framework residues can be selected for substitution based on their potential influence on the CDR conformation and / or binding to an antigen. Investigation of such potential influence can be done by modeling, examining the characteristics of the amino acids at specific positions, or by empirical observation of the effects of substitution or mutagenesis of specific amino acids. For example, if an amino acid differs between a non-human variable region framework residue and a selected human variable region framework residue, and that amino acid is reasonably predicted to: (1) bind directly to the antigen by non-covalent bonding, (2) be adjacent to a CDR region, (3) otherwise, interact with the CDR region (e.g., be within about 6 Å of the CDR region), then the human framework amino acid can be substituted with the equivalent framework amino acid from a non-human antibody. Other candidates for substitution are acceptor human framework amino acids that are rare in human immunoglobulins at that position. These amino acids can be substituted with the amino acids at the equivalent position of a non-human donor antibody, or the amino acids at the equivalent position of a more typical human immunoglobulin.

[0098] In some embodiments, the humanized anti-CD39 antibody has a mature VH comprising H1 comprising the amino acid sequence of SEQ ID NO: 10 having 0 to 2 amino acid substitutions or deletions, H2 comprising the amino acid sequence of SEQ ID NO: 11 having 0 to 2 amino acid substitutions or deletions, H3 comprising the amino acid sequence of SEQ ID NO: 12 having 0 to 2 amino acid substitutions or deletions, and a framework region having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with an acceptor human framework region; and a mature VL comprising L1 comprising the amino acid sequence of SEQ ID NO: 14 having 0 to 2 amino acid substitutions or deletions, L2 comprising the amino acid sequence of SEQ ID NO: 15 having 0 to 2 amino acid substitutions or deletions, L3 comprising the amino acid sequence of SEQ ID NO: 16 having 0 to 2 amino acid substitutions or deletions, and a framework region having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with an acceptor human framework region. The framework region may be determined according to the definition of Kabat.

[0099] In some embodiments, the humanized anti-CD39 antibody has a mature VH comprising an amino acid sequence of SEQ ID NO: 18 having 0 to 2 amino acid substitutions or deletions (H1), an amino acid sequence of SEQ ID NO: 19 having 0 to 2 amino acid substitutions or deletions (H2), an amino acid sequence of SEQ ID NO: 20 having 0 to 2 amino acid substitutions or deletions (H3), and a framework region having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with an acceptor human framework region; and a mature VL comprising an amino acid sequence of SEQ ID NO: 22 having 0 to 2 amino acid substitutions or deletions (L1), an amino acid sequence of SEQ ID NO: 25 having 0 to 2 amino acid substitutions or deletions (L2), an amino acid sequence of SEQ ID NO: 26 having 0 to 2 amino acid substitutions or deletions (L3), and a framework region having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with an acceptor human framework region. For example, the humanized anti-CD39 antibody referred to above may have an L1 comprising SEQ ID NO: 23 or SEQ ID NO: 24. The framework region may be determined according to the Kabat definition. In some embodiments, the VH human acceptor is M99642 or KF698734, and / or the VL human acceptor is X12682 or Z00023.

[0100] In some embodiments, the humanized anti-CD39 antibody has a mature VH comprising H1 comprising the amino acid sequence of SEQ ID NO: 28 having 0 to 2 amino acid substitutions or deletions, H2 comprising the amino acid sequence of SEQ ID NO: 29 having 0 to 2 amino acid substitutions or deletions, H3 comprising the amino acid sequence of SEQ ID NO: 30 having 0 to 2 amino acid substitutions or deletions, and a framework region having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to an acceptor human framework region; and a mature VL comprising L1 comprising the amino acid sequence of SEQ ID NO: 32 having 0 to 2 amino acid substitutions or deletions, L2 comprising the amino acid sequence of SEQ ID NO: 33 having 0 to 2 amino acid substitutions or deletions, L3 comprising the amino acid sequence of SEQ ID NO: 34 having 0 to 2 amino acid substitutions or deletions, and a framework region having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to an acceptor human framework region. The framework region may be determined according to Kabat's definition. In some embodiments, the VH human acceptor is M99651 or M77327, and / or the VL human acceptor is X12682 or M23090.

[0101] In the foregoing embodiments, the mature heavy chain variable region can be linked to at least a portion of the heavy chain constant region, and the mature light chain variable region can be linked to at least a portion of the light chain constant region. In some embodiments, the mature heavy chain variable region is linked to the heavy chain constant region, and the mature light chain variable region is linked to the light chain constant region. Suitable constant regions are described in more detail in Section II(F). In certain embodiments, the heavy chain constant region has effector functions substantially similar to wild-type human IgG1. In other embodiments, the heavy chain constant region has decreased or enhanced effector functions as compared to wild-type human IgG1. In specific examples, the heavy chain constant region comprises or consists of SEQ ID NO: 1, and the light chain constant region comprises or consists of SEQ ID NO: 6. In specific examples, the heavy chain constant region comprises or consists of amino acids 1-329 of SEQ ID NO: 1, and the light chain constant region comprises or consists of SEQ ID NO: 6. In another specific example, the heavy chain constant region comprises or consists of SEQ ID NO: 4, and the light chain constant region comprises or consists of SEQ ID NO: 6. In another specific example, the heavy chain constant region comprises or consists of amino acids 1-326 of SEQ ID NO: 4, and the light chain constant region comprises or consists of SEQ ID NO: 6. In another specific example, the heavy chain constant region comprises or consists of SEQ ID NO: 2, and the light chain constant region comprises or consists of SEQ ID NO: 6. In another specific example, the heavy chain constant region comprises or consists of amino acids 1-329 of SEQ ID NO: 2, and the light chain constant region comprises or consists of SEQ ID NO: 6. In another specific example, the heavy chain constant region comprises or consists of SEQ ID NO: 5, and the light chain constant region comprises or consists of SEQ ID NO: 6. In another specific example, the heavy chain constant region comprises or consists of amino acids 1-326 of SEQ ID NO: 5, and the light chain constant region comprises or consists of SEQ ID NO: 6. In another specific example, the heavy chain constant region comprises or consists of SEQ ID NO: 3, and the light chain constant region comprises or consists of SEQ ID NO: 6.In another specific example, the heavy chain constant region comprises or consists of amino acids 1 to 329 of SEQ ID NO: 3, and the light chain constant region comprises or consists of SEQ ID NO: 6.

[0102] E. Human antibody As described herein, the present disclosure provides human antibodies having the binding specificities of the antibodies specified herein, such as 19, ch19_IGG4.P, 31, ch31_IGG4.P, hu31.1_IGG4.P, hu31.2_IGG4.P, hu31.3_IGG4.P, hu31.4_IGG4.P, hu31.4_IGG1.AA, hu31.5_IGG4.P, hu31.6_IGG4.P, hu31.7_IGG4.P, 39, ch39_IGG4.P, ch39_mIGG2A.AAG, hu39.1_IGG4.P, hu39.2_IGG4.P, hu39.3_IGG4.P, hu39.4_IGG4.P, hu39.5_IGG4.P, hu39.5_IGG1.AA, hu39.6_IGG4.P, hu39.7_IGG4.P, or hu39.8_IGG4.P. Optionally, the human antibody has an IC measured in Example 5 50Inhibits human CD39 enzyme activity at a value of about 5 nM or less, about 0.05 nM to about 5 nM, about 1 nM or less, about 0.05 nM to about 1 nM, about 0.5 nM or less, or about 0.05 nM to about 0.5 nM. The human antibody has the same epitope specificity as a specific rodent antibody, such as the antibodies designated as 19, 31, or 39 herein, or a specific humanized antibody, such as ch19_IGG4.P, ch31_IGG4.P, hu31.1_IGG4.P, hu31.2_IGG4.P, hu31.3_IGG4.P, hu31.4_IGG4.P, hu31.4_IGG1.AA, hu31.5_IGG4.P, hu31.6_IGG4.P, hu31.7_IGG4.P, ch39_IGG4.P, ch39_mIGG2A.AAG, hu39.1_IGG4.P, hu39.2_IGG4.P, hu39.3_IGG4.P, hu39.4_IGG4.P, hu39.5_IGG4.P, hu39.5_IGG1.AA, hu39.6_IGG4.P, hu39.7_IGG4.P, or hu39.8_IGG4.P herein, and can be selected by competitive binding experiments, Winter's phage display method, WO92 / 20791, or other methods. The human antibody can also be screened for specific epitope specificity by using only fragments of CD39 as the target antigen and / or by screening antibodies against a collection of deletion mutants of CD39.

[0103] Methods for producing human antibodies include the trioma method of Oestberg et al., Hybridoma 2:361-367 (1983); Oestberg, U.S. Patent No. 4,634,664; and Engleman et al., U.S. Patent No. 4,634,666, the use of transgenic mice containing human immunoglobulin genes (e.g., Lonberg et al., WO93 / 12227 (1993); US5,877,397, US5,874,299, US5,814,318, US5,789,650, US5,770,429, US5,661,016, US5,633,425, US5,625,126, US5,569,825, US5,545,806, Nature148, 1547-1553 (1994), Nature Biotechnology 14, 826 (1996), Kucherlapati, WO 91 / 10741 (1991) and phage display methods (see, for example, Dower et al, WO91 / 17271 and McCafferty et al., WO92 / 01047, US5,877,218, US5,871,907, US5,858,657, US5,837,242, US5,733,743 and US5,565,332).

[0104] The human antibodies of the present disclosure can include a mature heavy chain variable region linked to at least a portion of the heavy chain constant region and a mature light chain variable region linked to at least a portion of the light chain constant region. In some embodiments, the mature heavy chain variable region is linked to the heavy chain constant region and the mature light chain variable region is linked to the light chain constant region. Suitable constant regions are described in more detail in Section II(F). In certain embodiments, the heavy chain constant region has effector functions substantially similar to wild-type human IgG1. In other embodiments, the heavy chain constant region has reduced or enhanced effector functions as compared to wild-type human IgG1. In a specific example, the heavy chain constant region comprises or consists of SEQ ID NO: 1, and the light chain constant region comprises or consists of SEQ ID NO: 6. In another specific example, the heavy chain constant region comprises or consists of SEQ ID NO: 4, and the light chain constant region comprises or consists of SEQ ID NO: 6. In another specific example, the heavy chain constant region comprises or consists of SEQ ID NO: 2, and the light chain constant region comprises or consists of SEQ ID NO: 6. In another specific example, the heavy chain constant region comprises or consists of SEQ ID NO: 5, and the light chain constant region comprises or consists of SEQ ID NO: 6. In another specific example, the heavy chain constant region comprises or consists of SEQ ID NO: 3, and the light chain constant region comprises or consists of SEQ ID NO: 6. F. Selection of Constant Region(s)

[0105] The heavy and light chain variable regions of a chimeric antibody, humanized (including veneered), or human antibody can each be linked to at least a portion of a human constant region. In some embodiments, the heavy chain variable domain described in the above section is linked to a portion of a human heavy chain constant region, and the light chain variable domain described in the above section is linked to a portion of a human light chain constant region. In some embodiments, the heavy chain variable domain described in the above section is linked to a portion of a human heavy chain constant region, and the light chain variable domain described in the above section is linked to the full-length human light chain constant region. In some embodiments, the heavy chain variable domain described in the above section is linked to the full-length human heavy chain constant region, and the light chain variable domain described in the above section is linked to the full-length human light chain constant region.

[0106] The choice of constant region (or its truncation) depends in part on whether effector function is desired or needs to be enhanced further. "Effector function" refers to the biological activities resulting from the constant region of the antibody's light or heavy chain and varies according to the antibody isotype. Non-limiting examples of antibody effector functions include C1q binding to the C1 complex and complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptors); and B cell activation. Human antibodies are classified into five isotypes (IgM, IgD, IgG, IgA, and IgE) according to their heavy chains, each providing different functions. IgG consists of four human subclasses (IgG1, IgG2, IgG3, and IgG4), each containing a different heavy chain. They are highly homologous and mainly differ in the hinge region and the degree to which they activate the host immune system. For example, human isotypes IgG1 and IgG3 can mediate complement-mediated cytotoxicity, while human isotypes IgG2 and IgG4 do not or do so at very low levels. The light chain constant region can be of the subclass lambda or kappa.

[0107] The antibodies of the present disclosure that include a human constant region or a portion thereof are typically IgG antibodies, preferably IgG1 or IgG4 antibodies. The human constant region exhibits allotype variations and isotype allotype variations among different individuals, i.e., the constant region may differ at one or more polymorphic positions in different individuals. Isotype allotypes differ from allotypes in that sera that recognize an isotype allotype bind to non-polymorphic regions of one or more other isotypes. References to human constant regions include constant regions that include any natural allotype or any permutation of residues occupying polymorphic positions in a natural allotype.

[0108] One or several amino acids at the amino terminus or carboxy terminus of the light chain and / or heavy chain, e.g., the lysine at the C terminus of the heavy chain, may be partially or completely deleted or derivatized. The N-terminal glutamine of the heavy chain or light chain can be substituted with a glutamate residue to prevent the formation of pyroglutamic acid.

[0109] In some embodiments, the antibodies of the present disclosure are IgG4 antibodies. In the case of human IgG4, the incorporation of the S228P (Eu numbering) engineered mutation into the heavy chain can be used to prevent Fab arm exchange. Suitable sequences for human IgG4 include, but are not limited to, SEQ ID NO: 4 and SEQ ID NO: 5. In some embodiments, the C-terminal lysine of the IgG4 antibody is absent.

[0110] In some embodiments, the antibodies of the present disclosure are IgG1 or IgG3 antibodies. Suitable sequences for human IgG1 or IgG3 are known in the art and include, but are not limited to, SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and the human IgG3 disclosed in U.S. Patent No. 5,624,821. In some embodiments, the C-terminal lysine of the IgG1 antibody or IgG3 antibody is absent.

[0111] In certain embodiments, the constant region can be modified to extend the half-life in humans (see, e.g., Hinton et al., J. Biol. Chem. 279:6213, 2004). Exemplary modifications include Gln at position 250 and / or Leu at position 428 (Eu numbering) to increase the half-life of the antibody.

[0112] Alternatively or additionally, the constant region can be modified to reduce or increase effector functions such as complement-mediated cytotoxicity (CDC) or antibody-dependent cell-mediated cytotoxicity (ADCC) (see, e.g., Winter et al., U.S. Patent No. 5,624,821; Tso et al., U.S. Patent No. 5,834,597; and Lazar et al., Proc. Natl. Acad. Sci. USA, 103:4005, 2006).

[0113] Some antibodies of the present disclosure are engineered to have reduced Fc effector functions, such as CDC, ADCC, and antibody-dependent cell phagocytosis (ADCP), by introduction of constant region mutations (s), as compared to the same antibody without the mutation (s). In some embodiments, each or all of these effector functions are reduced by at least 50%, 75%, 90%, or 95% as compared to the antibody without the mutation. Effector functions can be assayed as described in the Examples. Other assays are described in Shields et al, 2001 J. Biol. Chem., Vol. 276, p 6591-6604;Chappel et al, 1993 J. Biol. Chem., Vol 268, p 25124-25131;Lazar et al, 2006 PNAS, 103;4005-4010.

[0114] Substitution of any or all of positions 234, 235, 236, and / or 237 results in a decrease in affinity for Fcγ receptors, particularly the FcγRI receptor (see, e.g., US 6,624,821). In some embodiments, alanine residues are used for substitutions such as the L234A / L235A double mutation to reduce effector function. Other combinations of mutations that reduce effector function include L234A / L235A / G237A, E233P / L234V / L235A / ΔG236, A327G / A330S / P331S, K322A, L234A / L235A, L234F / L235E / P331S, and L234A / L235E / G237A / A330S / P331S (Eu numbering). Optionally, positions 234, 236, and / or 237 of human IgG2 are substituted with alanine and position 235 is substituted with glutamine. (See, e.g., US 5,624,821.) Two amino acid substitutions at the complement C1q binding sites at positions 330 and 331 of the Eu index reduce complement binding (see Tao et al., J. Exp. Med. 178:661 (1993) and Canfield and Morrison, J. Exp. Med. 173:1483 (1991)). Substitutions at positions 233-236 of human IgG1 or IgG2 residues and positions 327, 330, and 331 of IgG4 residues reduce ADCC and CDC (see, e.g., Armour KL. et al., 1999 Eur J Immunol. 29(8):2613-24; and Shields RL. et al., 2001. J Biol Chem. 276(9):6591-604). Mutations of N297A, N297Q, or N297G (Eu numbering) reduce glycosylation and thereby reduce effector function.

[0115] Some antibodies of the present disclosure are engineered by introduction of constant region mutation(s) to have enhanced Fc effector function. For example, FcγR binding can be enhanced by amino acid engineering. In some embodiments, this can be done by substitution of one or more amino acids in the Fc region. Desirable mutations can be determined, for example, by either alanine scanning or rational design and library screening. Using these techniques, IgG variants with enhanced binding to FcγR and enhanced effector function can be identified. Alternatively, some mutations to the Fc receptor region are known in the art and are described, for example, in Smith P. et al (2012) PNAS 6181-6186.

[0116] In some embodiments, the antibodies described herein include a modified IgG1 constant domain that enhances the antibody's ADCC-mediated ability as compared to wild-type IgG1 without modification. The modified IgG1 domain can be characterized by amino acid substitution at one or more of L235V, S239D, F243L, R292P, A330L, I332E, P396L (Eu numbering). In other embodiments, the modified IgG1 domain is characterized by substitution at S239D, A330L, and I332E (Eu numbering).

[0117] Alternatively, glycoform perturbation can be used to enhance the function of Fc-mediated therapeutic antibodies. N-linked Fc glycosylation of IgG1 antibodies is important for effector function. Sialylation, galactosylation, bisecting sugar, and fucosylation can all potentially affect the binding and activity of IgG molecules. Control of the glycosylation pattern of therapeutic antibodies can be achieved in multiple different ways. The type of cell producing the recombinant antibody and its culture conditions can potentially affect the glycosylation and activity of the therapeutic antibody. Additionally, bioreactor conditions and downstream processing can also potentially affect the microheterogeneity of the glycans. Hypofucosylated or defucosylated antibodies have been shown to enhance Fc-mediated properties. Numerous methods for achieving this reduction in fucose level by glycan engineering are well known in the art. One approach is to manipulate the enzymes involved in the post-translational modification of the antibody. This can include overexpression of glucosidases such as β-1-4-N-acetylglucosaminyltransferase III, knockout of fucosyltransferase, or use of cell lines that are naturally fucose-deficient or mutated to express low fucosylation levels. Additionally, inhibitors of N-linked glucosidases such as castanospermine can also be used to obtain low-fucose-bearing IgG molecules.

[0118] In some embodiments, amino acid engineered variants can have enhanced affinity over a broader range for multiple FcγRs, while glycoform engineered antibodies can generally have a more specific affinity for enhanced FcγRIIIa binding. Glycoforms can interact with proximal amino acids of the Fc portion and substitutions of amino acids that contact the Ig oligosaccharide can result in different glycoform structures.

[0119] G. Expression of Recombinant Antibodies Chimeric antibodies, humanized antibodies (including veneered antibodies), and human antibodies are typically produced by recombinant expression. Accordingly, the present disclosure also provides polynucleotides encoding the anti-CD39 antibodies of Sections IIA-F, vectors containing the polynucleotides, and host cells containing the vectors.

[0120] The polynucleotides encoding the anti-CD39 antibodies of the present disclosure can be inserted into vectors for amplification, expression, or further optimization. Many vectors are available. In some embodiments, the vector systems include mammalian, bacterial, yeast systems, etc., and include plasmids such as pALTER, pBAD, pcDNA, pCal, pL, pET, pGEMEX, pGEX, pCI, pCMV, pEGFP, pEGFT, pSV2, pFUSE, pVITRO, pVIVO, pMAL, pMONO, pSELECT, pUNO, pDUO, Psg5L, pBABE, pWPXL, pBI, p15TV-L, pPro18, pTD, pRS420, pLexA, pACT2.2, etc., but are not limited thereto, and other laboratory-prepared vectors and commercially available vectors. Suitable vectors can include plasmids or viral vectors (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses). Vector components generally include one or more of the following: signal sequences, origins of replication, one or more marker genes, enhancer elements, promoters (e.g., SV40, CMV, EF-1α), and transcription termination sequences, but are not limited thereto. For expression, the recombinant polynucleotide construct typically includes an expression control sequence operably linked to the coding sequence of the antibody chain, including a naturally associated or heterologous promoter region. In some embodiments, the expression control sequence is a eukaryotic promoter system in a vector capable of transforming or transfecting eukaryotic host cells. When the vector is incorporated into a suitable host, the host is maintained under conditions suitable for high-level expression of the nucleotide sequence and for collection and purification of the recombinant antibody.

[0121] A vector containing a polynucleotide sequence encoding an anti-CD39 antibody of the present disclosure can be introduced into a host cell for cloning or gene expression. Suitable host cells for cloning or expressing the polynucleotide sequence in the vectors herein include prokaryotic cells and eukaryotic cells. Non-limiting examples of suitable prokaryotes include eubacteria, such as gram-negative or gram-positive organisms, for example, Enterobacteriaceae, such as Escherichia, for example, E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, for example, Salmonella typhimurium, Serratia, for example, Serratia marcescens, and Shigella, as well as Bacilli, such as B. subtilis and B. licheniformis, Pseudomonas, such as P. aeruginosa, and Streptomyces. In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi and yeasts are also suitable as cloning or expression hosts for vectors encoding anti-CD39 antibodies.Non-limiting examples include Saccharomyces cerevisiae, Schizosaccharomyces pombe; Kluyveromyces hosts such as K. lactis, K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906), K. thermotolerans, and K. marxianus; Yarrowia (EP 402,226); Pichia pastoris (EP 183,070); Candida; Trichoderma reesia (EP 244,234); Neurospora crassa; Schwanniomyces such as Schwanniomyces occidentalis, and filamentous fungi such as Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts such as A. nidulans and A. niger. Suitable host cells can also be derived from multicellular organisms. Examples of invertebrate cells include plant cells and insect cells. A number of baculovirus strains and variants, as well as the corresponding permissive insect host cells derived from the host, such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruit fly), and Bombyx mori, have been identified. Various virus strains for transfection, such as the L-1 variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV, are publicly available, and such viruses can be used as the viruses herein according to the present invention, particularly for transfection of Spodoptera frugiperda cells. Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco can also be utilized as hosts.In some embodiments, mammalian cells are used as host cells for expressing a nucleotide segment encoding an immunoglobulin or fragment thereof. See Winnacker, From Genes to Clones, (VCH Publishers, NY, 1987). Numerous suitable host cell lines capable of secreting intact heterologous proteins have been developed in the art, including CHO cell lines, various COS cell lines, HeLa cells, HEK293 cells, L cells, and non-antibody-producing myelomas such as Sp2 / 0 and NS0. In some embodiments, the cells are non-human. Expression vectors for these cells can include expression control sequences such as an origin of replication, a promoter, an enhancer (Queen et al., Immunol. Rev. 89:49 (1986)), and the necessary processing information sites such as a ribosome binding site, an RNA splice site, a polyadenylation site, and a transcription termination sequence. In some embodiments, the expression control sequences are promoters derived from endogenous genes, cytomegalovirus, SV40, adenovirus, bovine papillomavirus, etc. See Co et al., J. Immunol. 148:1149 (1992).

[0122] The host cells are transformed with the above-described expression or cloning vector for anti-CD39 antibody production and cultured in a conventional nutrient medium modified as appropriate for promoter induction, selection of transformants, or amplification of the gene encoding the desired sequence. After expression, the antibody can be purified according to standard procedures in the art, including HPLC purification, column chromatography, gel electrophoresis, etc. (generally, see Scopes, Protein Purification (Springer-Verlag, NY, 1982)).

[0123] H. Labeled Antibodies The disclosed anti-CD39 antibodies can optionally be labeled with one or more detectable signals, including but not limited to fluorescent molecules, spin-labeled molecules, enzymes, or radioisotopes. Such antibodies can be used to detect or isolate human CD39 in any number of in vitro, in vivo, or ex vivo assays.

[0124] For example, if any one of the disclosed anti-CD39 antibodies is labeled with a detectable signal such as a fluorescent molecule, spin-labeled molecule, enzyme, or radioisotope, such a detectable antibody can be used in pharmacodynamic assays, immunohistochemistry, receptor occupancy assays, ELISA (enzyme-linked immunosorbent assay), EIA (enzyme immunoassay), RIA (radioimmunoassay), and the like.

[0125] III. Methods of Use The present disclosure provides methods of using the anti-CD39 antibodies described herein in the preparation of a medicament for inhibiting CD39 enzyme activity. As used herein, terms such as "inhibit," "inhibition," etc. refer to the ability of an antagonist to reduce the function or activity of a particular target, e.g., CD39. The reduction is preferably at least 50%, and may be, for example, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95%. The present disclosure also encompasses the use of the anti-CD39 antibodies described herein in the preparation of a medicament for the treatment or prevention of a disease, disorder, and / or condition that would benefit from inhibition of CD39 enzyme activity. By way of example, the present disclosure encompasses the use of the anti-CD39 antibodies described herein in the preparation of a medicament for the treatment of a disease characterized by (i) high extracellular ATP (compared to non-diseased tissue), (ii) expression or increased expression of ENTPD1, P2RX7, P2RY11, or combinations thereof in diseased tissue, (iii) expression or increased expression of CD39, P2X7, P2Y11, or combinations thereof in diseased tissue, (iv) bone marrow infiltration into diseased tissue, or any combination of (i)-(iv). In another example, the present disclosure encompasses the use of the anti-CD39 antibodies described herein in the preparation of a medicament for the treatment of cancer. Optionally, the cancer can be characterized by (i) high extracellular ATP (compared to non-diseased samples), (ii) expression or increased expression of ENTPD1, P2RX7, P2RY11, or combinations thereof in diseased tissue, (iii) expression or increased expression of CD39, P2X7, P2Y11, or combinations thereof in diseased tissue, (iv) bone marrow infiltration into diseased tissue, or any combination of (i)-(iv). In some embodiments of the methods described above, the anti-CD39 antibodies described herein are used in combination with at least one additional therapy, examples of which are described elsewhere in this specification.

[0126] Extracellular ATP is present at negligible concentrations (e.g., about 10 - 100 nM) in the healthy state but rapidly increases in response to tissue damage, stress, hypoxia, and drugs used in cancer treatment and may be found at high concentrations in tumors. ATP released by dying or stressed cells, including but not limited to cancer cells and other cells in the tumor microenvironment, provides important inflammatory signals for effective innate and adaptive immune responses. Conversely, for example, when extracellular ATP is hydrolyzed to adenosine in the tumor microenvironment, the immune response is restricted. CD39 is the rate-limiting ectoenzyme in the hydrolysis of extracellular ATP. By its degradative action of converting extracellular ATP to AMP, CD39 also increases extracellular adenosine production via CD73 (ecto-5'-nucleotidase), the rate-limiting ectoenzyme in extracellular AMP hydrolysis. Adenosine signals through type 1 purinergic receptors and has effects opposite to those mediated by ATP receptors. In particular, the A 2a and A 2b receptors on the surface of immune cells help mediate the immunosuppressive effects of adenosine. For example, in the tumor microenvironment, adenosine binds to A 2a receptors and A 2bIt induces an immunosuppressive effect through direct interaction with the receptor. The opposing roles of adenosine and ATP are reviewed in Chiarella et al., “Extracellular ATP and Adenosine in Cancer Pathogenesis and Treatment,” Trends in Cancer, 2021, 7(8): 731-750. As demonstrated herein, the use of the anti-CD39 antibodies of the present disclosure potently inhibits CD39 enzyme activity, resulting in immune stimulation by ATP accumulation and prevention of the formation of immunosuppressive adenosine. Diseases, disorders, and / or conditions that would benefit from inhibition of CD39 enzyme activity may include, for example, those in which extracellular ATP release can be increased by tissue damage, stress, hypoxic treatment by additional therapies, or any combination thereof. Additional diseases, disorders and / or conditions that would benefit from inhibition of CD39 enzyme activity include, for example, those in which increased CD39 enzyme activity in a peripheral blood sample or tissue (e.g., tumor) sample is measured, such that, for example, hydrolysis of ATP is increased as compared to healthy controls, and / or hydrolysis of AMP to adenosine is increased as compared to healthy controls, resulting in high extracellular adenosine levels in an affected tissue sample, and / or those in which the expression of CD39 and / or CD73 is detectable in a peripheral blood sample or tissue (e.g., tumor) sample, optionally at a higher level as compared to healthy controls, as measured by, for example, immunohistochemistry, immunophenotyping, RNA sequencing, or other clinically validated methods.

[0127] Thus, in some embodiments, the anti-CD39 antibodies described herein are administered in an amount effective to inhibit CD39 enzyme activity to a subject in need thereof. CD39 enzyme activity can be evaluated using a peripheral blood sample and / or a tissue (e.g., tumor) sample obtained from the subject. As a non-limiting example, commercially available assays such as Kinase-Glo® or AMP-Glo® can be used to measure CD39 activity in the cellular and acellular compartments of a peripheral blood sample or a tumor sample. Alternatively or additionally, CD39 enzyme activity in tumor tissue can be measured by enzyme histochemistry. Inhibition can be determined, for example, by comparison to a prior sample obtained from the subject (i.e., prior to administration of the anti-CD39 antibody), or by comparison to a reference sample or reference value of a control group (e.g., a subject administered an isotype control antibody, an anti-CD39 antibody that binds to CD39 but does not inhibit CD39 enzyme activity, standard of care, placebo, etc.). In some embodiments, the anti-CD39 antibodies described herein are administered in an amount effective to inhibit CD39 enzyme activity in the tumor microenvironment to a subject in need thereof. In some embodiments, the anti-CD39 antibodies described herein are administered in an amount effective to inhibit CD39 enzyme activity in the peritumoral area to a subject in need thereof. In some embodiments, the anti-CD39 antibodies described herein are administered in an amount effective to inhibit CD39 enzyme activity within or on immune cells to a subject in need thereof. In some embodiments, the anti-CD39 antibodies described herein are administered in an amount effective to inhibit CD39 enzyme activity within or on myeloid cells to a subject in need thereof. Myeloid cells expressing CD39 include those detailed in the Examples.In some embodiments, the anti-CD39 antibodies described herein are administered in an amount effective to inhibit CD39 enzyme activity in the stroma and / or intravascularly or on the stroma and / or vasculature to a subject in need of inhibition of CD39 enzyme activity in the stroma and / or intravascularly or on the stroma and / or vasculature.

[0128] Alternatively or additionally, in some embodiments, the anti-CD39 antibodies described herein are administered to a subject in need thereof in an amount effective to enhance ATP-mediated immune stimulation as compared to a suitable control (e.g., a subject administered an isotype control antibody, a subject administered an anti-CD39 antibody that binds to CD39 but does not inhibit CD39 enzyme activity, a subject receiving standard treatment, a non-treated subject). ATP-mediated immune stimulation can be evaluated using peripheral blood samples and / or tissue (e.g., tumor) samples obtained from the subject. ATP-mediated immune stimulation can be identified, for example, by (i) measuring an increase in ATP-dependent signaling through one or more type 2 purinergic (P2) receptors such as P2YG protein-coupled receptors or P2X cation-selective channel receptors; (ii) measuring an increase in NLRP3 inflammasome activation; (iii) measuring an increase in cell surface markers of dendritic cells; (iv) measuring an increase in the activity and / or proliferation of CD4+ and / or CD8+ T cells; and / or (v) measuring an increase in activation, maturation, cytokine secretion, or a combination thereof in one or more cell types of myeloid cells. In one example, the anti-CD39 antibodies described herein are administered to a subject in need thereof (e.g., a subject administered an isotype control antibody, a subject administered an anti-CD39 antibody that binds to CD39 but does not inhibit CD39 enzyme activity, a subject receiving standard treatment, a non-treated subject) in an amount effective to increase ATP-dependent signaling through one or more P2X or P2Y receptors selected from P2X4, P2X5, P2X7, P2Y2, or P2Y11 as compared to a suitable control. In one example, the anti-CD39 antibodies described herein are administered to a subject in need thereof (e.g., a subject administered an isotype control antibody, a subject administered an anti-CD39 antibody that binds to CD39 but does not inhibit CD39 enzyme activity, a subject receiving standard treatment, a non-treated subject) in an amount effective to increase ATP-dependent signaling through P2X7 as compared to a suitable control.In one example, the anti-CD39 antibody described herein is administered to a subject in need thereof (e.g., a subject administered an isotype control antibody, a subject administered an anti-CD39 antibody that binds to CD39 but does not inhibit CD39 enzyme activity, a subject receiving standard treatment, a subject not receiving treatment) in an amount effective to increase P2Y11-mediated ATP-dependent signaling as compared to a suitable control. In one example, the anti-CD39 antibody described herein is administered to a subject in need thereof (e.g., a subject administered an isotype control antibody, a subject administered an anti-CD39 antibody that binds to CD39 but does not inhibit CD39 enzyme activity, a subject receiving standard treatment, a subject not receiving treatment) in an amount effective to increase P2X7- and / or P2Y11-mediated ATP-dependent signaling as compared to a suitable control. In one example, the anti-CD39 antibody described herein is administered to a subject in need thereof in an amount effective to increase the activation of the NLRP3 inflammasome. Assays for measuring NLRP3 inflammasome activation are known in the art and include those detailed in the Examples (e.g., IL-1β and IL-18 secretion). In one embodiment, the anti-CD39 antibody described herein is administered to a subject in need thereof in an amount effective to increase the activation, maturation, cytokine secretion, or any combination thereof of myeloid cells. Assays for measuring the activation, maturation, cytokine secretion of myeloid cells are known in the art and include those detailed in the Examples.

[0129] Alternatively, or in addition to the above, in some embodiments, the anti-CD39 antibody described herein is administered to a subject in need thereof in an amount effective to reduce or prevent adenosine-mediated immunosuppression as compared to a suitable control (e.g., a subject administered an isotype control antibody, a subject administered an anti-CD39 antibody that binds to CD39 but does not inhibit CD39 enzyme activity, a subject receiving standard treatment, a subject not receiving treatment). Adenosine-mediated immunosuppression is, for example, A 2a R and / or A 2bIt can be identified by measuring the increased signaling of adenosine via R. Adenosine-mediated immunosuppression also includes adenosine-mediated inhibition of lymphocyte lineage (e.g., T cells, B cells) and / or myeloid lineage (e.g., monocytes, macrophages, dendritic cells, NK cells) cell activities. For example, one measure of adenosine-mediated immunosuppression can be NECA-induced pCREB activation in CD8+ T cells in human blood.

[0130] Alternatively, or in addition to the above, in some embodiments, the anti-CD39 antibodies described herein are administered to a subject in need thereof in an amount effective to achieve at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% target (i.e., CD39) binding as measured by a receptor occupancy assay. In some embodiments, the anti-CD39 antibodies described herein are administered to a subject in need thereof in an amount effective to achieve at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99 target (i.e., CD39) binding as measured by a receptor occupancy assay. Exemplary receptor occupancy assays are detailed in the Examples.

[0131] Alternatively, or in addition to the above, in some embodiments, the anti-CD39 antibodies described herein are administered to a subject in need thereof in an amount effective to reduce CD39 expression. In some embodiments, the anti-CD39 antibodies described herein are administered to a subject in need thereof in an amount effective to reduce cell surface CD39 expression. CD39 expression (intracellular and / or extracellular) can be evaluated by immunohistochemistry or immunophenotyping, including, for example, the methods further detailed in the Examples.

[0132] Alternatively, or in addition to the above, in some embodiments, the anti-CD39 antibodies described herein are administered to a subject in need thereof in an amount effective to treat or prevent cancer. Without wishing to be bound by theory, the Applicants believe that it may be possible to effect tumor death and potentially improve outcomes by increasing ATP-mediated immune stimulation and / or decreasing (or preventing) adenosine-mediated immunosuppression.

[0133] A. Oncology and Oncology-Related Disorders In one or more embodiments, the antibodies described herein are useful for the treatment and / or prevention of cancer (e.g., carcinoma, sarcoma, leukemia, lymphoma, myeloma, etc.). In certain embodiments, the cancer is locally advanced and / or inoperable, metastatic, or at risk of becoming metastatic. Alternatively or additionally, the cancer may recur or become refractory to treatment such as standard therapy or checkpoint inhibitors. Examples of types of cancer contemplated by the present disclosure include genitourinary cancers (e.g., bladder, kidney, renal cell, penis, prostate, testis, von Hippel-Lindau disease, etc.), uterine cancer, cervical cancer, ovarian cancer, breast cancer, gastrointestinal cancers (e.g., esophagus, oropharynx, stomach, small or large intestine, colon, or rectum), bone, bone marrow, skin cancers (e.g., melanoma), head and neck cancers, liver cancer, gallbladder cancer, bile duct cancer, heart cancer, lung cancer, pancreatic cancer, salivary gland cancer, adrenal cancer, thyroid cancer, brain cancer (e.g., glioma), ganglionic cancer, central nervous system (CNS) cancers, peripheral nervous system (PNS) cancers, hematopoietic cancers (i.e., blood malignancies), and cancers of the immune system (e.g., spleen or thymus).

[0134] In some embodiments, the antibodies according to the present disclosure are useful for the treatment and / or prevention of hematological malignancies. Examples of types of cancer that affect the hematopoietic system include leukemia, lymphoma, and myeloma, such as acute myeloid leukemia, adult T-cell leukemia, T-cell large granular lymphocyte leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, acute monocytic leukemia, Hodgkin lymphoma and non-Hodgkin lymphoma, diffuse large B-cell lymphoma, and multiple myeloma.

[0135] In another embodiment, the antibodies according to the present disclosure are useful for the treatment and / or prevention of solid tumors. Solid tumors can be, for example, ovarian cancer, endometrial cancer, breast cancer, lung cancer (small cell or non-small cell), colorectal cancer, prostate cancer, cervical cancer, biliary tract cancer, pancreatic cancer, gastric cancer, esophageal cancer, liver cancer (hepatocellular carcinoma), kidney cancer (renal cell carcinoma), head and neck tumors, mesothelioma, melanoma, sarcoma, central nervous system (CNS) hemangioblastoma, and brain tumors (e.g., gliomas, such as astrocytomas, oligodendrogliomas, and glioblastomas).

[0136] In another embodiment, the antibodies according to the present disclosure are useful for the treatment and / or prevention of lung cancer, genitourinary cancer, gastrointestinal cancer, or combinations thereof. In another embodiment, the antibodies according to the present disclosure are useful for the treatment and / or prevention of lung cancer, genitourinary cancer, gastrointestinal cancer, skin cancer, or combinations thereof.

[0137] In some embodiments, the antibodies according to the present disclosure are useful for the treatment and / or prevention of skin cancer. In a further embodiment, the skin cancer is melanoma.

[0138] In some embodiments, the antibodies according to the present disclosure are useful for the treatment and / or prevention of breast cancer. In a further embodiment, the breast cancer is hormone receptor positive (e.g., ERα-positive breast cancer, PR-positive breast cancer, ERα-positive and PR-positive breast cancer), HER2-positive breast cancer, HER2-overexpressing breast cancer, or any combination thereof. In still a further embodiment, the breast cancer is triple-negative breast cancer.

[0139] In some embodiments, the antibodies according to the present disclosure are useful for the treatment and / or prevention of renal cancer. In further embodiments, the renal cancer is renal cell carcinoma. In still further embodiments, the renal cell carcinoma is clear cell renal cell carcinoma.

[0140] In some embodiments, the antibodies according to the present disclosure are useful for the treatment and / or prevention of lung cancer. In further embodiments, the lung cancer is non-small cell lung cancer (NSCLC). In still further embodiments, the NSCLC is squamous cell lung cancer or adenocarcinoma of the lung.

[0141] In some embodiments, the disclosed methods of treating non-small cell lung cancer may further comprise administering any of the disclosed anti-CD39 antibodies in combination with any of pemetrexed, carboplatin, and either an antagonist anti-PD-1 antibody or an antagonist anti-PD-L1 antibody. Such methods may further comprise administering one or more additional agents selected from the group consisting of antagonist anti-TIGIT antibodies, A2aR antagonists, A2bR antagonists, A2a / 2bR antagonists, and CD73 inhibitors, and optionally, the additional agent is selected from the group consisting of domvanalimab, AB308, etrumadenant, and quemriculstat.

[0142] In some embodiments, the antibodies according to the present disclosure are useful for the treatment and / or prevention of pancreatic cancer. In further embodiments, the pancreatic cancer is pancreatic neuroendocrine tumor or pancreatic adenocarcinoma.

[0143] In some embodiments, the antibodies according to the present disclosure are useful for the treatment and / or prevention of neuroendocrine tumors. In further embodiments, the neuroendocrine tumor is a pancreatic neuroendocrine tumor, pheochromocytoma, paraganglioma, or adrenal tumor.

[0144] In some embodiments, the compounds according to the present disclosure are useful for the treatment and / or prevention of brain tumors. In further embodiments, the brain tumor is glioma. In still further embodiments, the glioma is astrocytoma, oligodendroglioma, or glioblastoma.

[0145] In some embodiments, the antibodies according to the present disclosure are useful for the treatment and / or prevention of upper GI cancers such as esophageal cancer or gastric cancer. In further embodiments, the upper GI cancer is adenocarcinoma, squamous cell carcinoma, or any combination thereof. In further embodiments, the upper GI cancer is esophageal adenocarcinoma (EAC), esophageal squamous cell carcinoma (ESCC), gastroesophageal junction adenocarcinoma (GEJ), gastric adenocarcinoma (also referred to herein as "gastric cancer"), or any combination thereof.

[0146] In some embodiments, the disclosed methods of treating gastric cancer or gastroesophageal cancer may further comprise administering any of the disclosed anti-CD39 antibodies in combination with either FOLFOX and an antagonist anti-PD-1 antibody or an antagonist anti-PD-L1 antibody. Such methods may further comprise administering one or more additional agents selected from the group consisting of antagonist anti-TIGIT antibodies, A2aR antagonists, A2bR antagonists, A2a / 2bR antagonists, and CD73 inhibitors, and optionally, the additional agent is selected from the group consisting of domvanalimab, AB308, etrumadenant, and quemriculstat.

[0147] In some embodiments, the antibodies according to the present disclosure are useful for the treatment and / or prevention of lymphoma. In further embodiments, the hematological malignancy is acute myeloid lymphoma.

[0148] In some embodiments, the antibodies according to the present disclosure are useful for the treatment of breast cancer, gastrointestinal cancer, uro-genital cancer, lung cancer, lymphoma, or ovarian cancer. In further embodiments, the antibodies according to the present disclosure are useful for the treatment of acute myeloid lymphoma, colorectal cancer, gastric cancer, esophageal cancer, castration-resistant prostate cancer, non-small cell lung cancer, ovarian cancer, pancreatic cancer, or triple-negative breast cancer.

[0149] In some embodiments, the antibodies according to the present disclosure are useful for the treatment of lung cancer, head and neck cancer, thyroid cancer, pancreatic cancer, kidney cancer, or skin cancer. In further embodiments, the antibodies according to the present disclosure are useful for the treatment of non-small cell lung cancer, head and neck squamous cell carcinoma, pancreatic ductal adenocarcinoma, clear cell renal carcinoma, melanoma.

[0150] In some embodiments, the antibodies according to the present disclosure are useful for the treatment of lower GI cancer, upper GI cancer, head and neck cancer, kidney cancer, lung cancer, or pancreatic cancer. In further embodiments, the antibodies according to the present disclosure are useful for the treatment of esophageal cancer, head and neck squamous cell carcinoma, kidney renal clear cell carcinoma, lung adenocarcinoma, lung squamous cell carcinoma, pancreatic adenocarcinoma, gastric (stomach) adenocarcinoma of the fundus.

[0151] In all of the above-described embodiments, the methods of the present disclosure can be carried out in an adjuvant setting or a neoadjuvant setting. The methods described herein may be presented as a first choice, second choice, third choice, or more choices of treatment. In some embodiments, the methods of the present disclosure can be carried out as a second or more choices of treatment, where the previous choice of treatment included a checkpoint inhibitor (i.e., the subject has experience with a checkpoint inhibitor (CPI)). In further embodiments, the checkpoint inhibitor is a CTLA-4 antagonist, a PD-1 antagonist, a PD-L1 antagonist, a TIM-3 antagonist, or a TIGIT antagonist.

[0152] The present disclosure also provides methods for treating or preventing other cancer-related diseases, disorders, or conditions. The use of the term(s) "cancer-related disease, disorder, and condition" is meant to broadly refer to conditions directly or indirectly related to cancer and non-cancerous proliferative diseases, such as pre-cancerous conditions such as angiogenesis, dysplasia, and non-cancerous proliferative diseases, disorders, or conditions such as benign proliferative breast diseases and papillomas. For clarity, the term(s) "cancer-related disease, disorder, and condition" does not include cancer itself.

[0153] Generally, the methods of the present disclosure for treating or preventing cancer, or a cancer-related disease, disorder, or condition, in a subject in need thereof, comprise administering to the subject an anti-CD39 antibody of Section II. Administration of the anti-CD39 antibody of Section II can include one or more (e.g., 1, 2, or more than 3) dosing cycles. In some embodiments, the present disclosure provides methods for treating or preventing cancer, or a cancer-related disease, disorder, or condition, using an anti-CD39 antibody of Section II and at least one additional therapy, examples of which are described elsewhere herein.

[0154] Patient selection. In some cases, the methods according to the present disclosure can be provided to selected patients, such as patients identified as having detectable PD-L1, CD73, and / or CD39 expression, high microsatellite instability, high tumor mutational burden, or any combination thereof. In some cases, the patient is identified as having a cancer gene-driven cancer having a mutation in at least one gene related to cancer. In some cases, the patient is selected by determining the patient's P2X7 variant. Commonly expressed P2X7 splice variants, different SNPs, and post-translational receptor modifications can affect the function of P2X7. For example, certain modifications have been shown to result in partial or complete loss of P2X7 function, and other modifications have been shown to increase P2X7 function. See, for example, Lara et al., Front Pharmacol, 2020, 11:793.

[0155] In some embodiments, patients are selected by measuring the expression of PD-L1 (CD274), P2X7 (P2RX7), P2Y11 (P2RY11), IL-2, CXCL1 (CXCL1), MIP-2a (CXCL2), CXCL3 (CXCL3), IL-8 (CXCL8), COX2 (PTGS2), CD73 (NT5E), and / or CD39 (ENTPD1) in a relevant sample such as a peripheral blood sample or a tumor sample. Expression can be measured by quantifying nucleic acids or proteins using methods known in the art, including but not limited to immunohistochemistry, immunophenotyping, RNA sequencing, gene expression analysis, single molecule imaging, or other clinically validated assays. In some embodiments, patients are selected by measuring the expression of PD-L1, CD73, and / or CD39 (at the nucleic acid or protein level) in a relevant sample such as a peripheral blood sample or a tumor sample using immunohistochemistry, immunophenotyping, RNA sequencing, gene expression analysis, single molecule imaging, or other clinically validated assays. Alternatively or additionally, patients can be selected, for example, by measuring the CD39 enzyme activity in a peripheral blood sample or a tumor sample. In one embodiment, the present disclosure provides a method of treating cancer in a patient having (i) detectable PD-L1, P2X7, P2Y11, IL-2, CXCL1, CXCL2, CXCL3, CXCL8, COX2, CD73, and / or CD39 expression, (ii) elevated PD-L1, P2X7, P2Y11, IL-2, CXCL1, CXCL2, CXCL3, CXCL8, COX2, CD73, and / or CD39 expression, (iii) detectable CD39 enzyme activity, or (iv) any combination of (i)-(iii), by administering an anti-CD39 antibody described herein.In another embodiment, the present disclosure provides a method for treating cancer in a patient having (i) detectable PD-L1, P2X7, P2Y11, IL-2, CXCL1, CXCL2, CXCL3, CXCL8, COX2, CD73, and / or CD39 expression, (ii) elevated PD-L1, CD73, and / or CD39 expression, (iii) detectable CD39 enzyme activity, or (iv) any combination of (i)-(iii), by administering a therapeutically effective amount of an anti-CD39 antibody described herein. In another embodiment, the present disclosure provides a method for administering to an individual a therapeutically effective amount of an anti-CD39 antibody described herein for the treatment of cancer, the method comprising measuring based on the determination of PD-L1, P2X7, P2Y11, IL-2, CXCL1, CXCL2, CXCL3, CXCL8, COX2, CD73, and / or CD39 expression. In yet another embodiment, the present disclosure provides a method for administering to an individual a therapeutically effective amount of an anti-CD39 antibody described herein for the treatment of cancer, the method comprising measuring PD-L1, P2X7, P2Y11, IL-2, CXCL1, CXCL2, CXCL3, CXCL8, COX2, CD73, and / or CD39 expression in a sample obtained from the individual by, for example, immunohistochemistry, immunophenotyping, or other clinically validated tests, and administering to the individual a therapeutically effective amount of the antibody, wherein the sample contains detectable PD-L1 (CD274), P2X7 (P2RX7), P2Y11 (P2RY11), IL-2, CXCL1 (CXCL1), MIP-2a (CXCL2), CXCL3 (CXCL3), IL-8 (CXCL8), COX2 (PTGS2), CD73 (NT5E) and / or CD39 (ENTPD1) expression.

[0156] B. Route of Administration In some embodiments, a pharmaceutical composition containing an antibody according to the present disclosure may be in a form suitable for oral administration. Oral administration involves swallowing the formulation, thereby enabling the antibody to be absorbed into the bloodstream of the gastrointestinal tract. Alternatively, oral administration may involve buccal, lingual, or sublingual administration, thereby enabling the antibody to be absorbed into the bloodstream through the oral mucosa.

[0157] In another embodiment, a pharmaceutical composition containing an antibody according to the present disclosure may be in a form suitable for parenteral administration. Forms of parenteral administration include, but are not limited to, intravenous, intraarterial, intramuscular, intradermal, intraperitoneal, intrathecal, intracisternal, intracerebral, intraventricular, intracardiac, and subcutaneous. A pharmaceutical composition suitable for parenteral administration can be formulated using a suitable aqueous or non-aqueous carrier. Depot injections, generally administered subcutaneously or intramuscularly, can also be utilized to release the antibodies disclosed herein over a defined period.

[0158] Other routes of administration are contemplated by the present disclosure and include, but are not limited to, nasal, vaginal, intraocular, rectal, topical (e.g., transdermal), and inhalation.

[0159] Certain embodiments of the present disclosure contemplate oral or parenteral administration.

[0160] C. Pharmaceutical Composition The anti-CD39 antibody of the present disclosure may be in the form of a composition suitable for administration to a subject. Generally, such a composition is a pharmaceutical composition comprising an anti-CD39 antibody according to the present disclosure and one or more pharmaceutically acceptable excipients. In certain embodiments, the anti-CD39 antibody may be present in an effective amount. The pharmaceutical composition can be used in the methods of the present disclosure; thus, for example, a pharmaceutical composition comprising an anti-CD39 antibody according to the present disclosure can be administered to a subject to practice the therapeutic, prophylactic methods, and uses described herein.

[0161] The pharmaceutical compositions of the present disclosure can be formulated to be compatible with the intended method or route of administration. Routes of administration can include those known in the art. Exemplary routes of administration are oral and parenteral. Further, the pharmaceutical compositions can be used in combination with one or more other therapeutic agents described herein to treat or prevent the diseases, disorders, and conditions contemplated by the present disclosure. In one embodiment, one or more other additional therapeutic agents contemplated by the present disclosure are included in the same pharmaceutical composition containing the anti-CD39 antibody according to the present disclosure. In another embodiment, one or more other therapeutic agents are in a composition separate from the pharmaceutical composition containing the anti-CD39 antibody according to the present disclosure.

[0162] In one aspect, the anti-CD39 antibodies described herein can be administered orally. Oral administration can be effected, for example, via capsules or tablets. When manufacturing a pharmaceutical composition containing an anti-CD39 antibody, tablets or capsules typically contain at least one pharmaceutically acceptable excipient. Non-limiting examples of pharmaceutically acceptable excipients include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, polyethylene glycol, cellulose, sterile water, syrup, and methylcellulose. Additional pharmaceutically acceptable excipients include lubricants such as talc, magnesium stearate, and mineral oil; wetting agents; emulsifying and suspending agents; and preservatives such as methyl and propyl hydroxybenzoates. Some oral dosage forms include taste masking agents, sweetening agents, or flavoring agents. Oral dosage forms can be formulated as solutions or suspensions.

[0163] In another aspect, the antibodies described herein can be administered parenterally, for example, by intravenous injection. Pharmaceutical compositions suitable for parenteral administration may be formulated as solutions for injection or may be reconstituted for injection in a suitable system such as a physiological solution. Such solutions may include an appropriate amount of sterile water for injection, salts, buffers, and isotonic excipients suitable to achieve appropriate physiological isotonicity.

[0164] The pharmaceutical compositions described herein may be stored in one or more suitable sterile containers. In some embodiments, the containers are designed to maintain the stability of the pharmaceutical composition over a given period of time.

[0165] D. Dosage Generally, the disclosed methods include administering an effective amount of an anti-CD39 antibody, or a composition thereof, described herein, to a subject in need thereof. The "effective amount" with respect to the anti-CD39 antibodies of the present disclosure means an amount of the antibody sufficient to bind to the target at a level indicative of the antibody's potency (e.g., bind to CD39 and inhibit CD39 enzymatic activity). For CD39, target binding can be determined by one or more biochemical or cellular assays, and EC values that can be used as one measure of the antibody's potency can be obtained from these assays. 50 ED 50 EC 90 IC 50 or similar values. Assays for determining target binding include, but are not limited to, those described in the Examples. The effective amount can be administered as a single dose or as multiple smaller doses (e.g., as one tablet containing an amount "x" or as two tablets each containing an amount "x / 2").

[0166] In some embodiments, the disclosed method includes administering a therapeutically effective amount of an anti-CD39 antibody described herein to a subject in need thereof. As used herein, the phrase "therapeutically effective amount" with respect to an anti-CD39 antibody means a dosing regimen (i.e., amount and interval) of the antibody that produces a specific pharmacological effect of administering the antibody to a subject in need of such treatment. In the case of prophylactic use, a therapeutically effective amount may be effective to eliminate or reduce the risk of a disease, reduce its severity, or delay its onset, including biochemical, histological, and / or behavioral signs or symptoms of the disease. In the case of treatment, a therapeutically effective amount may be effective to reduce, ameliorate, or eliminate one or more signs or symptoms associated with the disease, slow the progression of the disease, extend survival, reduce the dosage of other pharmaceuticals (if any) required for treatment of the disease, or combinations thereof. Particularly with respect to cancer, a therapeutically effective amount may result in, for example, the death of cancer cells, a decrease in the number of cancer cells, a decrease in tumor mass, the elimination of a tumor or metastasis, or a decrease in metastatic spread. A therapeutically effective amount of an anti-CD39 antibody need not necessarily be effective for the treatment of every individual subject in order to be considered a therapeutically effective amount by one of ordinary skill in the art. A therapeutically effective amount may vary, for example, based on one or more of the age and weight of the subject, the overall health of the subject, the stage of the disease in the subject, the route of administration, and previous or concurrent treatments.

[0167] In certain embodiments, the anti-CD39 antibodies contemplated by the present disclosure can be administered one or more times per day, per week, or per month (e.g., orally, parenterally, etc.) at about 0.01 mg / kg to about 50 mg / kg of subject body weight, or about 1 mg / kg to about 25 mg / kg of subject body weight, to obtain the desired effect. In some embodiments, a suitable weight-based dosage of the anti-CD39 antibodies contemplated by the present disclosure is used to determine a dosage (i.e., a fixed dosage) that is administered independent of the subject's body weight. In certain embodiments, the anti-CD39 antibodies of the present disclosure can be administered one or more times per day, per week, or per month at a fixed dosage level of about 1 mg to about 1000 mg, particularly 1, 3, 5, 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 300, 400, 500, 600, 750, 800, 900, or 1000 mg (e.g., orally, parenterally, etc.) to obtain the desired effect. In some embodiments, the anti-CD39 antibodies of the present disclosure can be administered one or more times per day, per week, or per month at a fixed dosage level of about 3 mg to about 3000 mg, particularly 3, 5, 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 300, 400, 500, 600, 750, 800, 900, 1000 mg, 1100 mg, 1200 mg, 1300 mg, 1400 mg, 1500 mg, 1600 mg, 1700 mg, 1800 mg, 1900 mg, 2000 mg, 2100 mg, 2200 mg, 2300 mg, 2400 mg, 2500 mg, 2600 mg, 2700 mg, 2800 mg, 2900 mg, or 3000 mg (e.g., orally, parenterally, etc.) to obtain the desired effect. As used herein with respect to dosages, the term "about" means the recited numerical value, as well as plus or minus 10% of that numerical value. For example, "about 10" should be understood to mean both "10" and "9 - 11".

[0168] In certain embodiments, the anti-CD39 antibodies of the present disclosure are contained in a "unit dosage form". The phrase "unit dosage form" refers to physically discrete units, each of which contains a predetermined amount of the anti-CD39 antibody sufficient to produce the desired effect, alone or in combination with one or more additional agents. It will be understood that the parameters of the unit dosage form will depend on the particular agent and the effect to be achieved.

[0169] IV. Combinations with the Disclosed Anti-CD39 Antibodies The present disclosure contemplates using the anti-CD39 antibodies of Section II alone or in combination with one or more additional therapies. Each additional therapy can be a therapeutic agent or another therapeutic modality. In embodiments that include one or more additional therapeutic agents, each agent can target different but complementary mechanisms of action. The additional therapeutic agents can be small chemical molecules; macromolecules such as proteins, antibodies, peptibodies, peptides, DNA, RNA, or fragments of such macromolecules; or cell therapy agents or gene therapy agents. Non-limiting examples of additional therapeutic modalities include surgical resection of tumors, bone marrow transplantation, radiation therapy, and photodynamic therapy. When the anti-CD39 antibodies of Section II are used in combination with one or more additional therapeutic agents, they can have a synergistic or additive therapeutic or prophylactic effect against the underlying disease, disorder, or condition. Additionally or alternatively, combination therapy can allow for a reduction in the dosage of one or more therapeutic agents, thereby improving, reducing, or eliminating the adverse effects associated with one or more of the agents.

[0170] In embodiments that include one or more additional therapeutic modalities, the anti-CD39 antibodies of Section II can be administered before, after, or during treatment with the additional therapeutic modality. In embodiments that include one or more additional therapeutic agents, the therapeutic agents used in such combination therapies can be formulated as a single composition or as separate compositions. When administered separately, each therapeutic agent in the combination can be administered simultaneously, substantially simultaneously, or at different times. Further, the therapeutic agents can be administered "in combination" even if they are in different dosage forms (e.g., oral capsules and intravenous), are administered at different dosing intervals, one therapeutic agent is administered according to a fixed dosing regimen while another is increased, decreased, or discontinued, or each therapeutic agent in the combination independently increases, decreases, has its dosage increased or decreased, or is discontinued and / or restarted during the course of treatment of the patient. When the combination is formulated as separate compositions, in some embodiments, the separate compositions are provided together in a kit.

[0171] Cancer therapy The present disclosure contemplates using the anti-CD39 antibodies of Section II in combination with one or more additional therapies useful for the treatment of cancer, or cancer-related diseases, disorders, or conditions. In some embodiments, one or more of the additional therapies are additional therapeutic modalities. Exemplary therapeutic modalities include, but are not limited to, surgical resection of a tumor, bone marrow transplantation, radiation therapy, and photodynamic therapy. In some embodiments, one or more additional therapeutic agents are targeted therapeutic agents. Exemplary therapeutic agents include chemotherapeutic agents, radiopharmaceuticals, hormonal therapeutic agents, epigenetic modulators, ATP-adenosine axis targeting agents, targeted therapies, signal transduction inhibitors, RAS signal transduction inhibitors, PI3K inhibitors, arginase inhibitors, HIF inhibitors, AXL inhibitors, PAK4 inhibitors, immunotherapeutic agents, cell therapy agents, gene therapy agents, immune checkpoint inhibitors, and agonists of stimulatory or co-stimulatory immune checkpoints.

[0172] In some embodiments, one or more additional therapeutic agents are chemotherapeutic agents. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carbocone, meturedopa, and uredopa; ethyleneimine and methylamelamine such as altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolomelamime; nitrogen mustards such as chlorambucil, chloronaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics such as aclacinomysins, actinomycin, aclarubicin, azaserine, bleomycin, cactinomycin, calicheamicin, carabicin, caminomycin, cardifilin, chromomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin, mycophenolic acid, nogalamycin, olivomycin, pomalidomide, peplomycin, potfiromycin, puromycin, quelamycin, rhodrubicin, streptozocin, tubercidin, ubenimex, dinostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pemetrexed, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiampurine, thioguanine;Pyrimidine analogs, such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, didoxyridine, doxifluridine, enocitabine, floxuridine, 5-FU; androgens, such as calusterone, drostanolone propionate, epithiostanol, mepitiostane, testolactone; anti-adrenal agents, such as aminoglutethimide, mitotane, trilostane; folic acid replenisher, such as folic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demeclocycline; diaziquone; elformithine; elliptinium acetate; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin; podophyllinic acid; 2-ethylhydrazide; procarbazine; razoxane; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2’,2’’-trichloroethylamine; urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside (Ara-C); cyclophosphamide; thiotepa; taxoids, such as paclitaxel, nab-paclitaxel, and docetaxel; chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum and platinum coordination complexes (collectively referred to as "platinum agents"), such as cisplatin, carboplatin, oxaliplatin; vinblastine; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; zeloda; ibandronate; CPT11; proteasome inhibitors, such as bortezomib, carfilzomib, ixazomib; topoisomerase inhibitors, such as irinotecan, topotecan, etoposide, mitoxantrone, teniposide; difluoromethylornithine (DMFO); retinoic acid; esperamicin; capecitabine;Anthracycline and any of the above pharmaceutically acceptable salts, acids or derivatives, are included, but not limited thereto. In certain embodiments, the combination therapy includes a chemotherapy regimen comprising one or more chemotherapeutic agents. In one embodiment, the combination therapy includes a chemotherapy regimen comprising FOLFOX (folic acid, fluorouracil, and oxaliplatin), FOLFIRI (folic acid, fluorouracil, and irinotecan), taxane (e.g., docetaxel, paclitaxel, nab-paclitaxel, etc.), CAPOX (capecitabine and oxaliplatin), XELOX (capecitabine and oxaliplatin), irinotecan which is a platinum-based chemotherapeutic agent, or gemcitabine. In another embodiment, the combination therapy includes a chemotherapy regimen comprising an alkylating agent (e.g., cyclophosphamide), anthracycline (e.g., doxorubicin, epirubicin, idarubicin, mitoxantrone), platinum agent (e.g., oxaliplatin), proteasome inhibitor (e.g., bortezomib), or any combination thereof. In another embodiment, the combination therapy includes a chemotherapy regimen comprising an alkylating agent (e.g., cyclophosphamide), anthracycline (e.g., doxorubicin, epirubicin, idarubicin, mitoxantrone), platinum agent (e.g., e.g., carboplatin, cisplatin, oxaliplatin), taxane (e.g., docetaxel, paclitaxel), proteasome inhibitor (e.g., bortezomib), or any combination thereof. In another embodiment, the combination therapy includes a chemotherapy regimen comprising bortezomib, cyclophosphamide, doxorubicin, epirubicin, idarubicin, mitoxantrone, oxaliplatin, or any combination thereof.;

[0173] In some embodiments, one or more additional therapeutic agents are radiopharmaceuticals. A radiopharmaceutical is a form of internal radiotherapy that places a radiation source (i.e., one or more radionuclides) within the body of a subject. The radiation source may be in solid form or in liquid form. Non-limiting examples of radiopharmaceuticals include sodium iodide I-131, radium-223 dichloride, iodine-131 labeled metaiodobenzylguanidine (MIBG), radiolabeled vesicles (e.g., saposin C-dioleoylphosphatidylserine (SapC-DOPS) nanovesicles), various forms of brachytherapy, and various forms of targeted radionuclides. A targeted radionuclide includes a radionuclide associated (e.g., by covalent or ionic interaction) with a molecule (a "targeting agent") that specifically binds to a target on a cell, typically a cancer cell or an immune cell. The targeting agent may be a small molecule, a sugar (including oligosaccharides and polysaccharides), an antibody, a lipid, a protein, a peptide, a non-natural polymer, or an aptamer. In some embodiments, the targeting agent is a sugar (including oligosaccharides and polysaccharides), a lipid, a protein, or a peptide, and the target is a tumor-associated antigen (enriched but not specific to cancer cells), a tumor-specific antigen (minimally or not expressed in normal tissue), or a neoantigen (an antigen specific to the genome of a cancer cell generated by a non-synonymous mutation or gene fusion in the tumor cell genome). In some embodiments, the targeting agent is an antibody, and the target is a tumor-associated antigen (i.e., an antigen that is enriched but not specific to cancer cells), a tumor-specific antigen (i.e., an antigen that is minimally or not expressed in normal tissue), or a neoantigen (i.e., an antigen specific to the genome of a cancer cell generated by a non-synonymous mutation or gene fusion in the tumor cell genome).Non-limiting examples of target radionuclides include radionuclides conjugated to somatostatin or its peptide analogs (e.g., 177Lu-Dotatate, etc.); prostate-specific membrane antigen or its peptide analogs (e.g., 177Lu-PSMA-617, 225Ac-PSMA-617, 177Lu-PSMA-I&T, 177Lu-MIP-1095, etc.); receptor cognate ligands, peptides derived from such ligands, or their variants (e.g., 188Re-labeled VEGF125-136 or its variants with higher affinity for the VEGF receptor, etc.); antibodies targeting tumor antigens (e.g., 131I-tositumomab, 90Y-ibritumomab tiuxetan, CAM-H2-I131 (Precirix NV), I131-omburtamab, etc.).

[0174] In some embodiments, one or more additional therapeutic agents are hormonal therapeutics. Hormonal therapeutics act to modulate or inhibit the hormonal action on tumors. Examples of hormonal therapeutics include selective estrogen receptor degrader, such as fulvestrant, GDC-9545, SAR439859, RG6171, AZD9833, lintestrant, ZN-c5, LSZ102, D-0502, LY3484356, SHR9549; selective estrogen receptor modulator, such as tamoxifen, raloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, toremifene; aromatase inhibitor, such as anastrozole, exemestane, letrozole, and other aromatase that inhibits 4(5)-imidazole; gonadotropin-releasing hormone agonist, such as nafarelin, triptorelin, goserelin; gonadotropin-releasing hormone antagonist, such as degarelix; anti-androgen agent, such as abiraterone, enzalutamide, apalutamide, darolutamide, flutamide, nilutamide, bicalutamide, leuprolide; 5α-reductase inhibitor, such as finasteride, dutasteride; etc., but are not limited thereto. In certain embodiments, the combination therapy includes administration of a hormone or related hormonal agent. In one embodiment, the combination therapy includes administration of enzalutamide.

[0175] In some embodiments, one or more additional therapeutic agents are epigenetic modulators. An epigenetic modulator is one that alters the epigenetic mechanisms that control gene expression and can be, for example, an inhibitor or activator of an epigenetic enzyme. Non-limiting examples of epigenetic modulators include DNA methyltransferase (DNMT) inhibitors, hypomethylating agents, and histone deacetylase (HDAC) inhibitors. In one or more embodiments, the anti-CD39 antibody of Section II can be combined with a DNA methyltransferase (DNMT) inhibitor or a hypomethylating agent. Exemplary DNMT inhibitors include decitabine, zebularine, and azacitidine. In one or more embodiments, combinations of the anti-CD39 antibody of Section II with a histone deacetylase (HDAC) inhibitor are also contemplated. Exemplary HDAC inhibitors include vorinostat, givinostat, abexinostat, panobinostat, belinostat, and trichostatin A.

[0176] In some embodiments, one or more additional therapeutic agents are ATP-adenosine axis targeting agents. ATP-adenosine axis targeting agents alter signaling mediated by adenine nucleosides and nucleotides (e.g., adenosine, AMP, ADP, ATP) by, for example, modulating the levels of adenosine or targeting adenosine receptors. Adenosine and ATP act on different classes of receptors and often have opposing effects on inflammation, cell proliferation, and cell death. For example, ATP and other adenine nucleotides produce antitumor effects via activation of the P2Y1 receptor subtype, while the accumulation of adenosine in the tumor microenvironment inhibits the antitumor functions of various immune cells and has been shown to enhance the immunosuppressive activity of myeloid and regulatory T cells by binding to adenosine receptors on the cell surface. In certain embodiments, the ATP-adenosine axis targeting agent is an inhibitor of ectonucleotidase involved in the conversion of ATP to adenosine, or an antagonist of the adenosine receptor. Ectonucleotidases involved in the conversion of ATP to adenosine include ectonucleoside triphosphate diphosphohydrolase 1 (ENTPD1, also known as CD39 or cluster of differentiation 39) and ecto-5'-nucleotidase (NT5E or 5NT, also known as CD73 or cluster of differentiation 73). Exemplary small molecule CD73 inhibitors include CB-708, ORIC-533, LY3475070, and AB680. Exemplary anti-CD73 antibodies include CPI-006, oleclumab (MEDI9447), NZV930, IPH5301, GS-1423, uliledrimab (TJD5, TJ004309), and BMS-986179. In one embodiment, the present disclosure contemplates combinations of the anti-CD39 antibody of Section II with CD73 inhibitors such as those described in WO2017 / 120508, WO2018 / 067424, WO2018 / 094148, and WO2020 / 046813. In a further embodiment, the CD73 inhibitor is cemdisiran. Adenosine binds to four different G protein-coupled receptors: A1R, A 2A R, A 2BIt can bind to R and A3R and activate them. A2R antagonists include etomadenant, inupadenant, taminadenant, caffeine citrate, NUV-1182, TT-702, DZD-2269, INCB-106385, EVOEXS-21546, AZD-4635, imaladenant, RVU-330, siploradenant, PBF-509, PBF-999, PBF-1129, and CS-3005. In some embodiments, the present disclosure relates to the anti-CD39 antibody of Section II and A 2A R antagonist, A 2B R antagonist, or A 2A R and A 2B R antagonist in combination. In some embodiments, the present disclosure contemplates a combination of the anti-CD39 antibody of Section II and an adenosine receptor antagonist described in WO2018 / 136700, WO2018 / 204661, WO2018 / 213377, or WO2020 / 023846, WO2020 / 102646. In one embodiment, the adenosine receptor antagonist is etomadenant.

[0177] In some embodiments, one or more additional therapeutic agents are targeted therapeutic agents. In one aspect, the targeted therapeutic agent may include a chemotherapeutic agent, a radionuclide, a hormonal therapeutic agent, or another small molecule drug conjugated to a targeting agent. The targeting agent may be a small molecule, a sugar (including oligosaccharides and polysaccharides), an antibody, a lipid, a protein, a peptide, a non-natural polymer, or an aptamer. In some embodiments, the targeting agent is a sugar (including oligosaccharides and polysaccharides), a lipid, a protein, or a peptide, and the target is a tumor-associated antigen (concentrated but not specific to cancer cells), a tumor-specific antigen (minimally or not expressed in normal tissues), or a neoantigen (an antigen specific to the cancer cell genome generated by non-synonymous mutations in the tumor cell genome). In some embodiments, the targeting agent is an antibody, and the target is a tumor-associated antigen (concentrated but not specific to cancer cells), a tumor-specific antigen (minimally or not expressed in normal tissues), or a neoantigen (an antigen specific to the cancer cell genome generated by non-synonymous mutations in the tumor cell genome). In some embodiments, the targeted therapeutic agent is an antibody-drug conjugate comprising an antibody and a drug, and the antibody specifically binds to HER2, HER3, nectin-4, or Trop-2. Specific examples of targeted therapeutic agents comprising an antibody and a drug include, but are not limited to, patritumab deruxtecan, sacituzumab govitecan-hziy, telisotuzumab vedotin, and trastuzumab deruxtecan. In other aspects, the targeted therapeutic agent may inhibit or interfere with specific proteins that aid in tumor survival, growth, and / or spread. Non-limiting examples of such targeted therapeutic agents include signaling inhibitors, RAS signaling inhibitors, inhibitors of oncogenic transcription factors, activators of oncogenic transcription factor suppressors, angiogenesis inhibitors, immunotherapeutic agents, ATP-adenosine axis targeting agents, AXL inhibitors, PARP inhibitors, PAK4 inhibitors, PI3K inhibitors, HIF2α inhibitors, CD73 inhibitors, A2R antagonists, TIGIT antagonists, and PD-1 antagonists. ATP-adenosine axis targeting agents are described above, and other agents are described in further detail below.

[0178] In some embodiments, one or more additional therapeutic agents are signaling inhibitors. A signaling inhibitor is an agent that selectively inhibits one or more steps of a signaling pathway. Signal transduction inhibitors (STIs) contemplated by the present disclosure include, but are not limited to: (i) BCR-ABL kinase inhibitors (e.g., imatinib); (ii) epidermal growth factor receptor tyrosine kinase inhibitors (EGFR TKIs) and anti-EGFR antibodies, including small molecule inhibitors (e.g., CLN-081, gefitinib, erlotinib, afatinib, icotinib, and osimertinib); (iii) inhibitors of the human epidermal growth factor (HER) family of transmembrane tyrosine kinases, e.g., HER-2 / neu receptor inhibitors (e.g., trastuzumab) and HER-3 receptor inhibitors; (iv) vascular endothelial growth factor receptor (VEGFR) inhibitors, including small molecule inhibitors (e.g., axitinib, regorafenib, sunitinib, and sorafenib), VEGF kinase inhibitors (e.g., lenvatinib, cabozantinib, pazopanib, tivozanib, XL092, etc.), anti-VEGF antibodies (e.g., bevacizumab), and anti-VEGFR antibodies (e.g., ramucirumab); (v) inhibitors of the AKT family kinase or the AKT pathway (e.g., rapamycin); (vi) inhibitors of serine / threonine protein kinase B-Raf (BRAF), such as vemurafenib, dabrafenib, and encorafenib; (vii) rearrangement during transfection (RET) inhibitors, including, for example, selpercatinib and pralsetinib; (viii) tyrosine protein kinase Met (MET) inhibitors (e.g., tepotinib, crizotinib, cabozantinib, and crizotinib); (ix) anaplastic lymphoma kinase (ALK) inhibitors (e.g., ensartinib, ceritinib, lorlatinib, crizotinib, and brigatinib); (x) inhibitors of the RAS signaling pathway described elsewhere herein (e.g., inhibitors of KRAS, HRAS, RAF, MEK, ERK); (xi) FLT-3 inhibitors (e.g., gilteritinib); (xii) inhibitors of Trop-2; (xiii) inhibitors of the JAK / STAT pathway, e.g., JAK inhibitors including tofacitinib and ruxolitinib, or STAT inhibitors such as napabucasin; (xiv) inhibitors of NF-κB;(xv) Cell cycle kinase inhibitors (e.g., flavopiridol); (xvi) Phosphatidylinositol kinase (PI3K) inhibitors; (xix) Protein kinase B (AKT) inhibitors (e.g., capivasertib, miransertib), (xx) Platelet-derived growth factor receptor (PDGFR) inhibitors (e.g., imatinib, sunitinib, regorafenib, avapritinib, lenvatinib, nintedanib, famitinib, ponatinib, axitinib, repretinib, etc.); and (xxi) Insulin-like growth factor receptor (IGFR) inhibitors (e.g., erlotinib, afatinib, gefitinib, osimertinib, dacomitinib). In one or more embodiments, the additional therapeutic agent comprises EGFR, VEGFR, HER-2, HER-3, BRAF, RET, MET, ALK, RAS (e.g., KRAS, MEK, ERK), FLT-3, JAK, STAT, NF-kB, PI3K, AKT, or any combination thereof.;

[0179] In some embodiments, one or more additional therapeutic agents are RAS signaling inhibitors. Oncogenic mutations in RAS family genes such as HRAS, KRAS, and NRAS are associated with various cancers. For example, among the KRAS family genes, mutations such as G12C, G12D, G12V, G12A, G13D, Q61H, G13C, and G12S have been observed in multiple tumor types. For the inhibition of mutant RAS signaling, direct and indirect inhibition strategies have been investigated. Indirect inhibitors target effectors other than RAS in the RAS signaling pathway and include, but are not limited to, inhibitors of RAF, MEK, ERK, PI3K, PTEN, SOS (e.g., SOS1), mTORC1, SHP2 (PTPN11), and AKT. Non-limiting examples of indirect inhibitors in development include RMC-4630, RMC-5845, RMC-6291, RMC-6236, JAB-3068, JAB-3312, TNO155, RLY-1971, BI1701963. Direct inhibitors of RAS variants have also been studied and generally target the KRAS-GTP complex or the KRAS-GDP complex. Exemplary direct RAS inhibitors in development include, but are not limited to, sotorasib (AMG510), MRRTX849, mRNA-5671, and ARS1620. In some embodiments, one or more RAS signaling inhibitors are selected from the group consisting of RAF inhibitors, MEK inhibitors, ERK inhibitors, PI3K inhibitors, PTEN inhibitors, SOS1 inhibitors, mTORC1 inhibitors, SHP2 inhibitors, and AKT inhibitors. In other embodiments, one or more RAS signaling inhibitors directly inhibit RAS variants.

[0180] In some embodiments, one or more of the additional therapeutic agents are inhibitors of phosphatidylinositol 3-kinase (PI3K), particularly inhibitors of the PI3Kγ isoform. PI3Kγ inhibitors can stimulate the anti-cancer immune response by modulating myeloid cells, for example by inhibiting suppressive myeloid cells, by weakening immunosuppressive tumor-infiltrating macrophages, or by stimulating macrophages and dendritic cells to produce cytokines that contribute to effective T cells, thereby reducing cancer development and spread. Examples of PI3Kγ inhibitors include copanlisib, duvelisib, AT-104, ZX-101, tenalisib, eganelisib, SF-1126, AZD3458, and pictilisib. In some embodiments, the anti-CD39 antibody of Section II can be combined with one or more PI3Kγ inhibitors described in WO2020 / 0247496A1.

[0181] In some embodiments, one or more of the additional therapeutic agents are inhibitors of arginase. Arginase has been shown to be a cause of or involved in inflammatory-induced immunodeficiency, tumor immune evasion, immunosuppression in infectious diseases, and immunopathology. Exemplary arginase compounds include CB-1158 and OAT-1746. In some embodiments, the anti-CD39 antibody of Section II can be combined with one or more arginase inhibitors described in WO / 2019 / 173188 and WO2020 / 102646.

[0182] In some embodiments, one or more additional therapeutic agents are inhibitors of oncogenic transcription factors or activators of oncogenic transcription factor repressors. Suitable agents can act at the expression level (e.g., RNAi, siRNA, etc.), through physical degradation, at the protein / protein level, at the protein / DNA level, or by binding in the activation / inhibition pocket. Non-limiting examples include inhibitors of one or more subunits of the MLL complex (e.g., HDAC, DOT1L, BRD4, menin, LEDGF, WDR5, KDM4C (JMJD2C), and PRMT1), inhibitors of hypoxia-inducible factor (HIF) transcription factors, and the like.

[0183] In some embodiments, one or more of the additional therapeutic agents are inhibitors of hypoxia-inducible factor (HIF) transcription factors, particularly HIF-2α. Exemplary HIF-2α inhibitors include belzutifan, ARO-HIF2, PT-2385, AB521, and those described in WO2021113436 and WO2021188769. In some embodiments, the anti-CD39 antibody of Section II can be combined with one or more HIF-2α inhibitors described in WO2021188769.

[0184] In some embodiments, one or more of the additional therapeutic agents are inhibitors of anexelekto (AXL). The AXL signaling pathway is associated with tumor growth and metastasis and is thought to mediate resistance to various cancer therapies. Various AXL inhibitors are in development that also inhibit other kinases of the TAM family (i.e., TYRO3, MERTK), as well as other receptor tyrosine kinases including in particular MET, FLT3, RON, and AURORA. Examples of multi-kinase inhibitors include sitravatinib, levastinib, glesatinib, gilteritinib, merestinib, cabozantinib, foretinib, BMS777607, LY2801653, S49076, GSK1363089, and RXDX-106. Small molecule inhibitors including AXL-specific inhibitors such as DS-1205, SGI-7079, SLC-391, TP-0903 (i.e., duvelisib), BGB324 (i.e., bemcentinib), and DP3975; anti-AXL antibodies such as ADCT-601; and antibody-drug conjugates (ADCs) such as BA3011 are also in development. Another strategy for inhibiting AXL signaling involves targeting GAS6, the ligand of AXL. For example, AVB-500 is in development as an Fc fusion protein that binds to the GAS6 ligand and inhibits AXL signaling.

[0185] In some embodiments, one or more of the additional therapeutic agents are inhibitors of p21-activated kinase 4 (PAK4). Overexpression of PAK4 has been shown across various cancer types, including cancers that are resistant to PD-1 therapy in particular. PAK4 inhibitors are not yet approved but are in development, and some, such as ATG-019 and KPT-9274, exhibit dual activity as PAK4 / NAMPT inhibitors. In some embodiments, an antibody according to the present disclosure is combined with a PAK4-selective inhibitor. In some embodiments, an antibody according to the present disclosure is combined with a PAK4 / NAMPT dual inhibitor, such as ATG-019 or KPT-9274.

[0186] In some embodiments, one or more of the additional therapeutic agents are agents that (i) inhibit the enzyme poly(ADP-ribose) polymerase (e.g., olaparib, niraparib, rucaparib, etc.); (ii) inhibitors of the Bcl-2 protein family (e.g., venetoclax, navitoclax, etc.); (iii) inhibitors of MCL-1; (iv) inhibitors of the CD47-SIRPα pathway (e.g., anti-CD47 antibodies, magrolimab, etc.); (v) isocitrate dehydrogenase (IDH) inhibitors, e.g., IDH-1 inhibitors or IDH-2 inhibitors (e.g., ivosidenib, enasidenib, etc.).

[0187] In some embodiments, one or more of the additional therapeutic agents are immunotherapeutic agents. Immunotherapeutic agents useful for the treatment of cancer typically induce or amplify an immune response against cancer cells. Non-limiting examples of suitable immunotherapeutic agents include immunomodulators; cellular immunotherapeutics; vaccines; gene therapeutics; ATP-adenosine axis targeting agents; immune checkpoint modulators; and certain signal transduction inhibitors. ATP-adenosine axis targeting agents are described above. Immunomodulators, signal transduction inhibitors, cellular immunotherapeutics, vaccines, gene therapeutics, and immune checkpoint modulators are further described below.

[0188] In some embodiments, one or more of the additional therapeutic agents are immunotherapeutic agents, more specifically, cytokines or chemokines, such as, for example, IL1, IL2, IL12, IL18, ELC / CCL19, SLC / CCL21, MCP-1, IL-4, IL-18, TNF, IL-15, MDC, IFNa / b, M-CSF, IL-3, GM-CSF, IL-13, and anti-IL-10; bacterial lipopolysaccharide (LPS); organic or inorganic adjuvants that activate antigen-presenting cells and promote the presentation of antigen epitopes on major histocompatibility complex molecules, Toll-like receptor (TLR) agonists, antagonists of the mevalonate pathway, agonists including but not limited to agonists of STING; indoleamine 2,3-dioxygenase 1 (IDO1) inhibitors, and immunostimulatory oligonucleotides, and other T cell adjuvants.

[0189] In some embodiments, one or more additional therapeutic agents are immunotherapeutic agents, more specifically cell therapy agents. Cell therapy agents are a form of treatment administered to viable cells. In certain embodiments, one or more additional therapeutic agents are cellular immunotherapy agents that activate or suppress the immune system. Cellular immunotherapy agents useful for the treatment of cancer typically induce or amplify an immune response. The cells can be autologous immune cells or allogeneic immune cells (e.g., monocytes, macrophages, dendritic cells, NK cells, T cells, etc.) harvested from one or more subjects. Alternatively, the cells can be “(re)programmed” allogeneic immune cells generated from immune progenitor cells (e.g., lymphoid progenitor cells, myeloid progenitor cells, dendritic cell common progenitor cells, stem cells, induced pluripotent stem cells, etc.). In some embodiments, such cells are an expanded subset of cells having different effector functions and / or maturation markers (e.g., adaptive memory NK cells, tumor infiltrating lymphocytes, immature dendritic cells, monocyte-derived dendritic cells, plasmacytoid dendritic cells, conventional dendritic cells (also called classical dendritic cells), M1 macrophages, M2 macrophages, etc.), and may be genetically modified to target the cells to specific antigens and / or enhance the anti-tumor effect of the cells (e.g., engineered T cell receptor (TCR) cell therapy agents, chimeric antigen receptor (CAR) cell therapy agents, lymph node homing of antigen-loaded dendritic cells, etc.), engineered to express or increase the expression of tumor-associated antigens, or any combination thereof. Non-limiting types of cell therapy agents include CAR-T cell therapy agents, CAR-NK cell therapy agents, TCR therapy agents, and dendritic cell vaccines. Exemplary cellular immunotherapy agents include sipuleucel-T, tisagenlecleucel, lisocabtagene maraleucel, idecabtagene vicleucel, brexucabtagene autoleucel, and axicabtagene ciloleucel, as well as CTX110, JCAR015, JCAR017, MB-CART19.1, MB-CART20.1, MB-CART2019.1, UniCAR02-T-CD123, BMCA-CAR-T, JNJ-68284528, BNT211, and NK-92 / 5.28.z.

[0190] In some embodiments, one or more additional therapeutic agents are immunotherapeutic agents, more specifically gene therapy agents. Gene therapy agents are recombinant nucleic acids administered ex vivo to a subject or the subject's cells to modify the expression of endogenous genes, to effect heterologous expression of proteins (e.g., small interfering RNA (siRNA) agents, double-stranded RNA (dsRNA) agents, microRNA (miRNA) agents, viral or bacterial gene delivery, etc.), and gene editing therapeutic agents, which may or may not include nucleic acid components (e.g., meganucleases, zinc finger nucleases, TAL nucleases, CRISPR / Cas nucleases, etc.), oncolytic viruses, etc. Non-limiting examples of gene therapy agents that may be useful in cancer treatment include Gendicine® (rAd-p53), Oncorine® (rAD5-H101), talimogene laherparepvec, Mx-dnG1, ARO-HIF2 (Arrowhead), quarusugene ozeplasmid (Immunogene), CTX110 (CRISPR Therapeutics), CTX120 (CRISPR Therapeutics), and CTX130 (CRISPR Therapeutics).

[0191] In some embodiments, the one or more additional therapeutic agents are immunotherapeutic agents, more specifically agents that modulate immune checkpoints. Immune checkpoints are a series of inhibitory and stimulatory pathways that directly affect the function of immune cells (e.g., B cells, T cells, NK cells). Immune checkpoints function when a protein on the surface of an immune cell recognizes and binds its cognate ligand. The present invention contemplates the use of the anti-CD39 antibodies of Section II in combination with agonists of stimulatory or costimulatory pathways and / or antagonists of inhibitory pathways. Agonists of stimulatory or costimulatory pathways and antagonists of inhibitory pathways, or combinations thereof, may be useful as agents that overcome different immunosuppressive pathways within the tumor microenvironment, inhibit regulatory T cells, reverse / prevent T cell anergy or exhaustion, and induce innate immune activation and / or inflammation at the tumor site.

[0192] In some embodiments, one or more additional therapeutic agents are immune checkpoint inhibitors. As used herein, the term "immune checkpoint inhibitor" refers to an antagonist of an inhibitory or co-inhibitory immune checkpoint. The terms "immune checkpoint inhibitor", "checkpoint inhibitor", and "CPI" can be used interchangeably herein. Immune checkpoint inhibitors can antagonize inhibitory or co-inhibitory immune checkpoints by preventing receptor-ligand binding and / or altering receptor signaling. Examples of immune checkpoints (ligands and receptors) include those that are selectively upregulated in various types of cancer cells and can be antagonized, and these examples include PD-1 (programmed cell death protein 1); PD-L1 (PD-1 ligand); BTLA (B and T lymphocyte attenuator); CTLA-4 (cytotoxic T lymphocyte-associated antigen 4); TIM-3 (T cell immunoglobulin and mucin domain-containing protein 3); LAG-3 (lymphocyte activation gene 3); TIGIT (T cell immunoreceptor with Ig and ITIM domains); CD276 (B7-H3); PD-L2, galectin 9, CEACAM-1, BTLA, CD69, galectin-1, CD113, GPR56, VISTA, 2B4, CD48, GARP, PD1H, LAIR1, TIM-1, and TIM-4, and killer inhibitory receptors, which can be divided into two classes based on their structural characteristics: i) killer cell immunoglobulin-like receptors (KIR), and ii) C-type lectin receptors (members of the type II transmembrane receptor family). Also contemplated are other immune checkpoints that are less well-defined and described in the literature, including both receptors (e.g., the 2B4 (also known as CD244) receptor) and ligands (e.g., certain B7 family inhibitory ligands such as B7-H3 (also known as CD276) and B7-H4 (also known as B7-S1, B7x, and VCTN1)).

[0193] In some embodiments, the immune checkpoint inhibitor is a CTLA-4 antagonist. In further embodiments, the CTLA-4 antagonist can be an antagonistic CTLA-4 antibody. Suitable antagonistic CTLA-4 antibodies include, for example, monospecific antibodies such as ipilimumab or tremelimumab, and bispecific antibodies such as MEDI5752 and KN046.

[0194] In some embodiments, the immune checkpoint inhibitor is a PD-1 antagonist. In further embodiments, the PD-1 antagonist can be an antagonistic PD-1 antibody, a small molecule, or a peptide. Suitable antagonistic PD-1 antibodies include, for example, monospecific antibodies such as balstilimab, bdurigirimab, camrelizumab, cossirimumab, dostarlimab, semaprimab, ezabenlimab, MEDI-0680 (AMP-514; WO2012 / 145493), nivolumab, pembrolizumab, pidilizumab, pimivizumab, retifanlimab, sasanlimab, spartalizumab, sintilmab, tislelizumab, toripalimab, and zimberelimab; and bispecific antibodies such as LY3434172. In still further embodiments, the PD-1 antagonist can be a recombinant protein composed of the extracellular domain of PD-L2 (B7-DC) fused to the Fc portion of IgG1 (AMP-224). In certain embodiments, the immune checkpoint inhibitor is zimberelimab.

[0195] In some embodiments, the immune checkpoint inhibitor is a PD-L1 antagonist. In further embodiments, the PD-L1 antagonist can be an antagonistic PD-L1 antibody. Suitable antagonistic PD-L1 antibodies include, for example, monospecific antibodies such as avelumab, atezolizumab, durvalumab, BMS-936559, and enobafolimab, and bispecific antibodies such as LY3434172 and KN046.

[0196] In some embodiments, the immune checkpoint inhibitor is a TIGIT antagonist. In further embodiments, the TIGIT antagonist can be an antagonistic TIGIT antibody. Suitable antagonistic anti-TIGIT antibodies include monospecific antibodies such as AGEN1327, AB308 (WO2021247591), BMS 986207, COM902, domvanalimab, EOS-448, etigilimab, IBI-929, JS006, M6223, osperlimab, SEA-TGT, tirgolumab, vibostolimab; and bispecific antibodies such as AGEN1777 and AZD2936. In certain embodiments, the immune checkpoint inhibitor is an antagonistic anti-TIGIT antibody disclosed in WO2017152088 or WO2021247591. In certain embodiments, the immune checkpoint inhibitor is domvanalimab or AB308.

[0197] In some embodiments, the immune checkpoint inhibitor is a LAG-3 antagonist. In further embodiments, the LAG-3 antagonist can be an antagonistic LAG-3 antibody. Suitable antagonistic LAG-3 antibodies include, for example, BMS-986016 (WO10 / 19570, WO14 / 08218), or IMP-731 or IMP-321 (WO08 / 132601, WO09 / 44273).

[0198] In some embodiments, the immune checkpoint inhibitor is a B7-H3 antagonist. In further embodiments, the B7-H3 antagonist can be an antagonistic B7-H3 antibody. Suitable antagonistic B7-H3 antibodies include, for example, MGA271 (WO11 / 109400), omburtumab, enoblituzumab, DS-7300a, ABBV-155, and SHR-A1811.

[0199] In some embodiments, the immune checkpoint inhibitor is a TIM-3 antagonist. In further embodiments, the TIM-3 antagonist can be an antagonistic TIM-3 antibody. Suitable antagonistic TIM-3 antibodies include, for example, dostarlimab, sabatolimab, BMS-986258, and RG7769 / RO7121661.

[0200] In some embodiments, one or more additional therapeutic agents activate stimulatory or costimulatory immune checkpoints. Examples of stimulatory or costimulatory immune checkpoints (ligands and receptors) include B7-1, B7-2, CD28, 4-1BB (CD137), 4-1BBL, ICOS, ICOS-L, OX40, OX40L, GITR, GITRL, CD70, CD27, CD40, DR3, and CD2.

[0201] In some embodiments, the agent that activates a stimulatory or co-stimulatory immune checkpoint is a CD137 (4-1BB) agonist. In further embodiments, the CD137 agonist can be an agonistic CD137 antibody. Suitable CD137 antibodies include, for example, urelumab and PF-05082566 (WO12 / 32433). In some embodiments, the agent that activates a stimulatory or co-stimulatory immune checkpoint is a GITR agonist. In further embodiments, the GITR agonist can be an agonistic GITR antibody. Suitable GITR antibodies include, for example, BMS-986153, BMS-986156, TRX-518 (WO06 / 105021, WO09 / 009116), and MK-4166 (WO11 / 028683). In some embodiments, the agent that activates a stimulatory or co-stimulatory immune checkpoint is an OX40 agonist. In further embodiments, the OX40 agonist can be an agonistic OX40 antibody. Suitable OX40 antibodies include, for example, MEDI-6383, MEDI-6469, MEDI-0562, PF-04518600, GSK3174998, BMS-986178, and MOXR0916. In some embodiments, the agent that activates a stimulatory or co-stimulatory immune checkpoint is a CD40 agonist. In further embodiments, the CD40 agonist can be an agonistic CD40 antibody such as dacetuzumab, serplulimab, APX005M, ADC-1013, or CDX-1140. In some embodiments, the agent that activates a stimulatory or co-stimulatory immune checkpoint is a CD27 agonist. In further embodiments, the CD27 agonist can be an agonistic CD27 antibody. Suitable CD27 antibodies include, for example, balstilimab.

[0202] In some embodiments, one or more of the additional therapeutic agents are immunotherapeutic agents, more specifically, signal transduction inhibitors. Intracellular signal transduction molecules that affect immune cell function can also be suitable targets for improving anti-tumor immunity. For example, one or more of the additional therapeutic agents can be inhibitors of intracellular signal transduction molecules and inhibitors of hematopoietic progenitor kinase 1 (HPK1). HPK1 is a serine / threonine kinase that functions as a negative regulator of the activation signal generated by the T cell antigen receptor. As another example, one or more of the additional therapies can be inhibitors of Cbl-b, an E3 ubiquitin ligase (e.g., AP401) involved in the regulation of TCR signal transduction. As another example, one or more of the additional therapeutic agents can be inhibitors of diacylglycerol kinase (DGK). In some embodiments, the inhibitor is a small molecule. Non-limiting examples of small molecule HPK1 inhibitors in clinical development include CFI-402411 and BGB-15025, and a non-limiting example of a Cbl-b inhibitor in clinical development is AP401. Non-limiting examples of small molecule DAG inhibitors include those described in WO2020006016A1 and WO2021130638.

[0203] In some embodiments, one or more of the additional therapeutic agents are agents that inhibit or deplete immunosuppressive immune cells. For example, to inhibit or deplete immunosuppressive macrophages or monocytes, the agent can be a CSF-1R antagonist, such as an anti-CSF-1R antibody comprising RG7155 (WO11 / 70024, WO11 / 107553, WO11 / 131407, WO13 / 87699, WO13 / 119716, WO13 / 132044) or FPA-008 (WO11 / 140249; WO13169264), or an antibody disclosed in WO14 / 036357. As another example, to inhibit or deplete Tregs, the agent can be anti-CD25 beads used to deplete Tregs ex vivo.

[0204] In some embodiments, the present disclosure contemplates the use of the anti-CD39 antibodies of Section II in combination with immunogenic cell death (ICD)-inducing therapies. ICD can be defined as a functionally distinct and regulated subtype of cell death sufficient to elicit an adaptive immune response directed specifically to antigens derived from cell “corpses”. Criteria for identifying ICD-inducing therapies can include those described in Vanmeerbeek et al., OncoImmunology, 2020, 9:1, DOI: 10.1080 / 2162402X.2019.1703449; Keep et al., OncoImmunology, 2014, 3(9): :e955691. Non-limiting examples of ICD-inducing therapies are described in Galluzzi et al., Nature Reviews Clinical Oncology, 2020 17: 725-742. In some embodiments, the ICD-inducing therapy can be radiotherapy, photodynamic therapy, extracorporeal photopheresis, oncolytic virus therapy, bortezomib, cyclophosphamide, doxorubicin, epirubicin, idarubicin, mitoxantrone, cetuximab, crizotinib, or oxaliplatin. In some embodiments, the ICD-inducing therapy can be radiotherapy, photodynamic therapy, extracorporeal photopheresis, oncolytic virus therapy, bleomycin, bortezomib, carboplatin, cetuximab, crizotinib, cyclophosphamide, docetaxel, doxorubicin, epirubicin, gemcitabine, idarubicin, irinotecan, mitoxantrone, oxaliplatin, paclitaxel, vemurafenib, or vorinostat.

[0205] In some embodiments, each additional therapy can independently be radiotherapy, a chemotherapeutic agent, a radiopharmaceutical, a hormonal therapeutic agent, an epigenetic modulator, a targeting agent, an immunotherapeutic agent, a cell therapy agent, a gene therapy agent, or an ICD-inducing therapy. For example, in one embodiment, the present disclosure contemplates using the anti-CD39 antibody of Section II in combination with one or more ICD-inducing therapies and optionally one or more additional therapies, where each additional therapy is independently selected from radiotherapy, a radiopharmaceutical, a chemotherapeutic agent, a hormonal therapeutic agent, a targeting agent, an immunotherapeutic agent, a cell therapy agent, or a gene therapy agent. In another example, in one embodiment, the present disclosure contemplates using the anti-CD39 antibody of Section II in combination with one or more chemotherapeutic agents and optionally one or more additional therapies, where each additional therapy is independently selected from radiotherapy, a radiopharmaceutical, a hormonal therapeutic agent, a targeting agent, an immunotherapeutic agent, a cell therapy agent, or a gene therapy agent. In another embodiment, the present disclosure contemplates using the anti-CD39 antibody of Section II in combination with one or more chemotherapeutic agents and one or more tyrosine kinase inhibitors, and optionally one or more additional therapies, where each additional therapy is independently a targeting agent, an immunotherapeutic agent, or a cell therapy agent. In another embodiment, the present disclosure contemplates using the anti-CD39 antibody of Section II in combination with one or more chemotherapeutic agents and one or more inhibitors independently selected from (i) a BCR-ABL kinase inhibitor; (ii) an EGFR inhibitor (e.g., an EGFR TKI or an anti-EGFR antibody); (iii) a HER-2 / neu receptor inhibitor; (iv) an anti-angiogenic agent (e.g., an anti-VEGF antibody, a VEGFR TKI, a VEGF kinase inhibitor, etc.); (v) an AKT inhibitor; (vi) a BRAF inhibitor; (vii) a RET inhibitor; (viii) a MET inhibitor; (ix) a RAS inhibitor; and (x) an ALK inhibitor, and optionally one or more additional therapies, where each additional therapy is independently selected from radiotherapy, a radiopharmaceutical, a targeting agent, an immunotherapeutic agent, or a cell therapy agent.In another embodiment, the present disclosure contemplates using the anti-CD39 antibodies of Section II in combination with one or more immunotherapeutic agents and optionally one or more additional therapies, each additional therapy being independently selected from radiation therapy, radiopharmaceuticals, hormonal therapies, targeting agents, chemotherapeutic agents, cell therapies, or gene therapies. In another embodiment, the present disclosure contemplates using the anti-CD39 antibodies of Section II in combination with one or more immunotherapeutic agents and one or more chemotherapeutic agents, and optionally one or more additional therapies, each additional therapy being independently selected from radiation therapy, radiopharmaceuticals, hormonal therapies, targeting agents, cell therapies, or gene therapies. In another embodiment, the present disclosure contemplates using the anti-CD39 antibodies of Section II in combination with one or more immunotherapeutic agents and one or more radiation therapies or radiopharmaceuticals, and optionally one or more additional therapies, each additional therapy being independently selected from chemotherapeutic agents, hormonal therapies, targeting agents, cell therapies, or gene therapies. In another embodiment, the present disclosure contemplates using the anti-CD39 antibodies of Section II in combination with one or more immune checkpoint inhibitors and / or one or more ATP-adenosine axis targeting agents, and optionally one or more additional therapies, each additional therapy being independently selected from radiation therapy, radiopharmaceuticals, chemotherapeutic agents, targeting agents, immunotherapeutic agents, or cell therapies. In another embodiment, the present disclosure contemplates using the anti-CD39 antibodies of Section II in combination with one or more immune checkpoint inhibitors and / or one or more ATP-adenosine axis targeting agents and / or one or more chemotherapeutic agents, radiopharmaceuticals or radiation therapies.In another embodiment, the present disclosure contemplates using the anti-CD39 antibodies of Section II in combination with one or more immune checkpoint inhibitors and / or one or more ATP-adenosine axis targeting agents and one or more inhibitors independently selected from: (i) BCR-ABL kinase inhibitors; (ii) EGFR inhibitors (e.g., EGFR TKIs or anti-EGFR antibodies); (iii) HER-2 / neu receptor inhibitors; (iv) anti-angiogenic agents (e.g., anti-VEGF antibodies, VEGFR TKIs, VEGF kinase inhibitors, etc.); (v) AKT inhibitors; (vi) BRAF inhibitors; (vii) RET inhibitors; (viii) MET inhibitors; (ix) KRAS inhibitors; and (x) ALK inhibitors. In another embodiment, the present disclosure contemplates using the anti-CD39 antibodies of Section II in combination with one or more immune checkpoint inhibitors and / or one or more ATP-adenosine axis targeting agents, and one or more ICD-inducing therapies. In the further embodiments described above, (a) the targeting agent can be a PI3K inhibitor, an arginase inhibitor, an HIF2α inhibitor, an AXL inhibitor, a PAK4 inhibitor, or an anti-angiogenic agent; (b) the immunotherapeutic agent can be an ATP-adenosine axis targeting agent, cytokine therapy, an immune checkpoint inhibitor, or a combination thereof; (c) the ATP-adenosine axis targeting agent is A. 2A R and / or A 2Bis an R antagonist or a CD73 inhibitor; (d) the ATP-adenosine axis targeting agent is etomadenant or quemriculstat; (e) the immunotherapeutic agent is an anti-PD-L1 antagonist antibody or an anti-PD-1 antagonist antibody, optionally selected from the group consisting of budigalimab, camrelizumab, cossimbilumab, dostarlimab, semiprimab, ezabenlimab, nivolumab, pembrolizumab, pidilizumab, pimivaliumab, retifanlimab, sasanalimab, spartalizumab, sintilmab, tislelizumab, tripalizumab, zinberelimab, LY3434172, avelumab, atezolizumab, balsilimab, durvalumab, envafolimab, LY3434172 and KN046; (f) the immunotherapeutic agent is an anti-TIGIT antagonist antibody, optionally selected from the group consisting of AGEN1327, AB308 (WO2021247591), BMS986207, COM902, domvanalimab, EOS-448, etigilimab, IBI-929, JS006, M6223, osipelimab, SEA-TGT, tirgolimab, vibostrimab, and bispecific antibodies such as AGEN1777 and AZD2936; (g) the immunotherapeutic agent is domvanalimab, AB308, zinberelimab, tirgolimab, pembrolizumab, nivolumab, atezolizumab, or durvalumab; (h) the anti-angiogenic agent is pazopanib, sorafenib, sunitinib, bevacizumab, axitinib, lenvatinib, tivozanib, or cabozantinib; (i) the ICD induction therapy is radiotherapy (including both external radiotherapy and internal radiotherapy), photodynamic therapy, extracorporeal photochemotherapy, oncolytic virus therapy, bortezomib, cyclophosphamide, doxorubicin, epirubicin, idarubicin, mitoxantrone, or oxaliplatin; or (j) any combination thereof. In still further embodiments above, the disclosure contemplates the use of the anti-CD39 antibody of Section II in combination with domvanalimab, AB308, etomadenant, quemriculstat, zinberelimab, AB521, or any combination thereof.In still further embodiments, the present disclosure contemplates the use of the anti-CD39 antibodies of Section II in combination with etomadenant, quemriclustat, or etomadenant and quemriclustat. In still further embodiments, the present disclosure contemplates the use of the anti-CD39 antibodies of Section II in combination with etomadenant, AB521, or etomadenant and AB521. In still further embodiments, the present disclosure contemplates the use of the anti-CD39 antibodies of Section II in combination with quemriclustat, AB521, or quemriclustat and AB521.

[0206] The selection of additional therapeutic agent(s) may be informed by the particular cancer and / or the mutational status and / or stage of the subject's cancer and / or the current standard of care for the disease. Detailed standard of care guidelines are issued, for example, by the National Comprehensive Cancer Network (NCCN). See, for example, NCCN Colon Cancer v3.2021, NCCN Hepatobiliary Cancer v5.2021, NCCN Kidney Cancer v3.2022, NCCN NSCLC v7.2021, NCCN Pancreatic Adenocarcinoma v2.2021, NCCN Esophageal and Esophagogastric Junction Cancer v4.2021, NCCN Gastric Cancer v5.2021, Ovarian Cancer / Fallopian Tube Cancer / Primary Peritoneal Cancer v3.2021, Prostate Cancer v3.2022, Head and Neck Cancer v1.2022, Melanoma: Skin v1.2022, Acute Myeloid Leukemia v1.2022.

Example

[0207] Experiment The following examples are set forth to provide a complete disclosure and description of how to make and use the present disclosure to those skilled in the art and are not intended to limit the scope of what the inventors regard as their invention. Additional antibodies within the scope of the present disclosure can be made using methods based on the methods shown in these examples or other methods described herein. Efforts have been made to ensure accuracy with respect to the numerical values used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should be accounted for.

[0208] Unless otherwise specified, temperature is in degrees Celsius (°C) and pressure is atmospheric or near atmospheric pressure. Standard abbreviations are used, including: rt or r.t. = room temperature; min = minute(s); h or hr = hour(s); ng = nanogram; μg = microgram; mg = milligram; g = gram; kg = kilogram; μl or μL = microliter; ml or mL = milliliter; l or L = liter; μM = micromolar; mM = millimolar; M = molarity; mol = mole; mmol = millimole; nM = nanomolar.

[0209] Example 1: Generation of Anti-CD39 Antibodies Human CD39-expressing CHO-K1 cell lines, cynomolgus monkey CD39-expressing HEK-293 cell pools, and mouse CD39-expressing HEK-293 cell pools were generated. Briefly, full-length human, cynomolgus monkey, or mouse CD39 (SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 71, respectively) encoding pcDNA3.3 expression vectors were transfected into CHO-K1 cells or HEK-293 cells (Flp-In™-293 cells) using the Lipofectamine 2000 transfection kit according to the manufacturer's protocol. Forty-eight to seventy-two hours after transfection, the transfected cells were cultured in medium containing blasticidin for selection, and after testing for CD39 expression, CD39-expressing cell pools were obtained. Human, cynomolgus monkey, and mouse CD39-expressing cell lines or cell pools were obtained by limiting dilution and used to screen hybridoma supernatants as described below.

[0210] To generate anti-CD39 antibodies, four 6- to 8-week-old Sprague Dawley rats were immunized with 30 - 200 mg / animal of human CD39 antigen, which was either protein or plasmid DNA. The adjuvant mixture included Adju-Phos, CpG-ODN, or Titer-Max. The animals were injected once every two weeks via the plantar globe, subcutaneous, intraperitoneal, intramuscular, and intradermal routes. Serum titers were measured by ELISA or fluorescence-activated cell sorting (FACS). The ELISA used to measure the serum antibody titer against a given antigen was performed as follows. Plates (Nunc) were coated overnight at 4°C with 100 μL of 1 μg / mL human CD39 antigen and then blocked with blocking buffer (1xPBS / 2% BSA) for 1 hour at ambient temperature. Rat sera were serially diluted 3-fold starting from a 1:100 dilution in blocking buffer and incubated for 1 hour at ambient temperature. Wells without serum samples were used as negative controls. Next, the plates were washed and then incubated for 1 hour with the secondary antibody, goat anti-rat IgG-Fc-HRP (Bethyl). After washing, tetramethylbenzidine (TMB) substrate was added and 2M HCl was added to stop the interaction. Absorbance at 450 nm was read using a microplate reader (Molecular Device). The FACS assay used to measure serum antibody titers was performed as generally described for screening of hybridoma supernatants (see below), except that serial dilution sera were used. Serum titers were determined as the final dilution that gave a positive signal (i.e., more than 3-fold that of the negative control).

[0211] When the serum titers were sufficiently high (≥1:24,300), the animals were given a final boost with protein and cell lysates in sterile PBS without adjuvant. After 48 - 96 hours, these animals were euthanized and the lymph nodes and spleens were used for cell fusion.

[0212] Lymph nodes and spleens from immunized animals were homogenized and filtered to remove blood clots and cell debris. Logarithmically growing Sp2 / 0 myeloma cells were collected and centrifuged. B cells and Sp2 / 0 myeloma cells were separately treated with pronase solution, and the reaction was stopped with 100% FBS. The cells were washed and counted. B cells were fused with Sp2 / 0 myeloma cells at a ratio of 1:1 in an electrofusion solution according to a general electrofusion procedure. The fused cells were resuspended in DMEM medium supplemented with 20% FBS and 1×HAT, and then transferred to a 96-well plate. The fused cells were maintained in an incubator set at 37 °C and 5% CO2 for 10 - 14 days.

[0213] Hybridoma cells were recovered, and 150 - 200 cells were added to 1.5 mL of semi-solid HAT medium. The cells were gently mixed for 5 - 10 seconds with a vortex oscillator and then seeded into a 6-well plate. The plate was maintained in an incubator set at 37 °C, 5% CO2 for 7 - 8 days. Each visible single colony was placed into a 96-well plate containing DMEM medium supplemented with 10% FBS. After 2 - 3 days, the cell supernatant was collected and screened.

[0214] The order of the assay cascade of the screening paradigm was changed to perform four rounds of hybridoma generation and antibody screening. To meet the screening criteria, the antibody produced from a given hybridoma clone should have no measurable binding to mouse CD39 or other CD39 family members, namely CD39-L1, CD39-L2, CD39-L3, or CD39-L4, and should bind to cell CD39 of both human and cynomolgus monkeys with high affinity. The antibody should also show strong inhibition of cell human CD39 enzyme activity in both cell lines engineered to overexpress CD39 and human cell lines that naturally express CD39, such as THP-1, SK-MEL-5, and MOLP-8. In the first round of fusion, the CHO-K1 cell-based human CD39 ELISA was the primary screening, and the binding to cell-overexpressed human, cynomolgus monkey, and mouse CD39 was measured by FACS (using the aforementioned cell lines), and the in vitro soluble human CD39 enzyme assay and cell enzyme assay were the secondary screening. In the second round of fusion, suitable hybridoma material could not be produced. In the third round of fusion, the soluble human CD39 enzyme assay was the primary screening, and the binding to cell-overexpressed human and mouse CD39 was measured by FACS, and the human CD39-L1 binding counter-screening and the soluble human CD39 enzyme assay for confirmation were the secondary screening. In the fourth round of fusion, the THP-1 cell enzyme assay was used as the primary screening, and the binding to cell-overexpressed human and cynomolgus monkey CD39 was measured by FACS, and the THP-1 cell enzyme assay for confirmation was the secondary screening. Clone 39 from the first round, as well as clones 19 and 31 from the third round, were selected for further characterization. No clones were selected from the fourth round.

[0215] For the cell-based ELISA used to evaluate the binding to CD39 expressed on the cell surface, the assay was generally performed as follows. Plates (384-well) were seeded with 3 - 5×10 per well4 Individual cells were pre-coated and cultured for 2 days in an incubator set at 37 °C and 5% CO2. The plates were blocked with blocking buffer (1xPBS / 5% milk) for 1 hour at ambient temperature. Next, 30 μL of hybridoma supernatant was added to the plates and incubated for 1 hour at ambient temperature. The plates were washed 3 times with PBS and then incubated for 1 hour at ambient temperature with the secondary antibody, goat anti-rat IgG-Fc-HRP (1:500). After washing, TMB substrate was added to each well and the plates were incubated for 3 - 5 minutes at ambient temperature in the dark, and 2M HCl was added to stop the reaction. The absorbance at 450 nm was read using a microplate reader (Molecular Device).

[0216] FACS was also used to detect the binding of anti-CD39 antibodies in the supernatant to CD39 using the aforementioned human CD39-expressing CHO-K1 cell line, cynomolgus monkey CD39-expressing HEK-293 cell line, and mouse CD39-expressing HEK-293 cell pool. Unlabeled cells were used as a control for setting the threshold before detection, and then the percentage change in each group exceeding the fluorescence intensity threshold was analyzed. 1x10 5 Cells / well were incubated with hybridoma supernatant in a volume of 100 μL for 1 hour at 4 °C. Anti-human CD39 reference antibody was used as a positive control. Human / rat IgG isotype antibody was used as a negative control. After washing the cells with 1×PBS / 1% BSA, Alexa647-labeled goat anti-human antibody or goat anti-rat antibody (diluted 1:500 in FACS staining buffer) was added. The cells were incubated for 1 hour at 4 °C in the dark. The mean fluorescence intensity (MFI) of the cells was measured with a flow cytometer and analyzed using FlowJo software.

[0217] The ability of anti-CD39 antibodies to block the enzymatic activity of soluble CD39 extracellular domain (ECD) antigen was tested by measuring the inhibition of human CD39 catalytic function that hydrolyzes ATP to AMP + inorganic phosphate, in association with luciferase activity and luminescence as a signal. The enzyme assay was performed in a 96-well multiwell plate with a final volume of 120 μL. The assay mixture was incubated in an incubator set at 37 °C for the indicated incubation period. Tris magnesium (TM) buffer contained 25 mM Tris, 5 mM MgCl2, and 0.01% BSA. Serial dilutions of inhibitory anti-CD39 antibodies were prepared starting from 20 nM in TM buffer containing soluble human CD39 (Sino Biological, catalog number 16020-H08B). Dilutions were performed either with or without ATP to determine the effect of pre-mixed antibodies and enzyme before substrate (ATP) addition. As an isotype control, irrelevant antibodies were similarly diluted to examine the specificity of antibody-mediated CD39 inhibition. The antibody / enzyme preparations were incubated in an incubator set at 37 °C for 1 hour, then ATP at a final concentration of 20 μM was added and incubated at 37 °C for 1 hour. Enzyme activity was quantified using CellTiter-Glo (CTG). The amount of residual ATP was measured using the CTG luciferase reagent from the CTG luminescent cell viability assay (Promega Corporation).

[0218] The functional activity of anti-CD39 antibodies was determined by measuring CD39-catalyzed hydrolysis of ATP using the human myeloid leukemia monocytic cell line (THP-1). THP-1 cells were resuspended in TM buffer and 80 μL / well of cells were plated at 4×10 4Seeded at a cell / well density and then added 40 μL / well of an anti-CD39 antibody solution (various concentrations, 5-fold serial dilution from 100 nM to 1.28 pM in TM buffer) to the plate. Incubated the antibody and cells in an incubator set at 37 °C for 1 hour. After incubation, added ATP (50 μM in TM buffer) to the plate in an amount of 80 μL / well and held it in an incubator set at 37 °C for 1 hour. Placed the plate in a centrifuge set at 1500 rpm for 5 minutes and transferred 50 μL / well of the supernatant to a white 96-well plate (Corning, catalog number 3903). Added CellTiter-Glo (50 μL / well) to the corresponding wells and mixed well. Measured the cellular enzymatic inhibition of CD39 with a multi-label reader (Perkin-Elmer Envision Workstation).

[0219] To test whether the anti-CD39 antibody specifically binds to human CD39 but does not cross-react with other CD39 family members (CD39-L1, CD39-L2, CD39-L3, CD39-L4), the binding of the anti-CD39 antibody to human CD39-L1, CD39-L2, CD39-L3, and CD39-L4 was determined by ELISA. Briefly, 96-well high protein-binding ELISA plates (Nunc MaxiSorp, ThermoFisher) were pre-coated with His-tagged human CD39 cross-family proteins (CD39-L1, CD39-L2, CD39-L3, or CD39-L4 (0.5 μg / mL)) and left overnight at 4 °C in carbonate-bicarbonate buffer (20 mM Na2CO3, 180 mM NaHCO3, pH 9.2). The next day, the plates were washed three times with 300 μL / well of PBS / 0.5% (v / v) Tween-20 and then blocked with PBS / 2% BSA for 1 hour. After blocking, the plates were washed three times. After washing, test antibodies at various concentrations (6-fold serial dilution from 100 nM to 0.36 pM in 2% BSA / PBS) were added to the plates and left in an incubator set at room temperature for 2 hours. The company's CD39-L1 binding antibody was used as a positive control, and the human IgG4 isotype antibody was used as a negative control. After washing three times, 100 μL / well of goat anti-human IgG Fc-HRP antibody (1:5000) was added, and the plates were placed in an incubator set at room temperature for 1 hour. For color development, 100 μL / well of TMB substrate solution was added to the plates. After 3 - 5 minutes, the reaction was stopped with 100 μL / well of 2M HCl. Absorbance was read at 450 nm using a SpectraMax® M5e microplate spectrophotometer. EC 50 values were obtained from four-parameter non-linear regression analysis using GraphPad Prism software.

[0220] Antibodies derived from hybridoma clone 19, hybridoma clone 31, and hybridoma clone 39 having a rat Fc domain were purified from the hybridoma supernatant. These antibodies are referred to herein as antibody 19, 31, and 39, respectively. The recovered hybridoma supernatant was adjusted to pH 7.0 and then loaded onto a Protein A column. The bound antibodies were eluted with glycine-HCl (pH 3.5) and immediately neutralized with 0.1 M Tris (pH 9.0). The protein was dialyzed against PBS and filtered through a 0.22 μm membrane filter. The antibody concentration was measured with a Nano Drop. The purity of the antibody was evaluated by SDS-PAGE and HPLC-SEC.

[0221] Total RNA was isolated from hybridoma clones 19, 31, and 39 using the RNeasy Plus Mini Kit (Qiagen). First-strand cDNA was reverse-transcribed using oligo dT. The VH and VL genes of the antibody were amplified from the cDNA using a 3'-constant region degenerate primer and a 5'-degenerate primer set. The 5'-degenerate primer was designed based on the upstream signal sequence coding region of the Ig variable sequence. Next, the PCR product was ligated into the pMD18-T vector, and 10 μL of the ligation product was transformed into Top10 competent cells. The transformed cells were plated on 2xYT plates containing carbenicillin and incubated overnight in an incubator set at 37°C. Twelve positive colonies were randomly selected for DNA sequencing by Biosune.

[0222] The genes of the clones were codon-optimized for mammalian expression and then synthesized by GENEWIZ (Suzhou, China). The resulting plasmids contained a gene encoding a heavy chain containing the VH domain of antibody 19, 31, or 39 fused with the human IgG4 CH1, hinge, CH2, and CH3 segments (including the hinge mutation S228P), or a gene encoding a light chain containing the VL domain fused with the human Ig kappa CK domain.

[0223] To produce antibodies ch19_IGG4.P, ch31_IGG4.P, and ch39_IGG4.P, plasmids containing the above VH and VL genes were co-transfected into Expi293F cells. For each antibody, 2.94x10 6 / mL of Expi293F cells with a viability of over 95% were prepared in 40 mL of cell culture medium. Plasmid DNA and ExpiFectamine™ 293 transfection reagent at a final concentration of 1 μg / mL were mixed and then added to the cell culture medium. The cell culture was incubated in a platform shaker at a rotational speed of 150 rpm. The temperature was maintained in an incubator set at 37 °C and a CO2 level of 8%. After 6 days of incubation, the cells were pelleted using centrifugation at 4000 rpm for 10 minutes at 25 °C. The supernatant was collected and subjected to gel electrophoresis, SPR k off ranking, and purification as needed. The supernatant was loaded onto an SDS-PAGE gel according to the instructions for use of a NuPAGE™ 4–12% Bis-Tris protein gel (Thermo Fisher). A PageRuler™ Unstained Protein Ladder (Thermo Fisher) was used together with the antibody sample to determine the molecular weight of the antibody.

[0224] To purify the antibody, a protein A column was pre-packed with 1 mL of MabSelect Sure resin. The column was equilibrated with 5 column volumes (CV) of equilibration buffer (0.1 M Tris, pH 7.0) and then filled with the cell culture. After filling, the column was washed with 15 CV of 0.1 M Tris (pH 7.0) and then eluted with 8 CV of 0.1 M glycine (pH 3.5). The eluted sample was buffer-exchanged with PBS buffer using a desalting column. Finally, the purified antibody was analyzed by SDS-PAGE and SEC-HPLC and then stored at -80 °C.

[0225] Using FACS analysis, the binding of the recombinant anti-CD39 antibodies to CHO-K1 cells overexpressing the aforementioned human CD39 and HEK293 cells overexpressing cynomolgus or mouse CD39 was measured. The ability of these antibodies to inhibit cell CD39 enzyme activity was also determined. Table 1 shows that all three selected antibodies have high affinity for cell human CD39 and potently inhibit the enzyme activity of cell human CD39.

[0226] The kinetic binding of these recombinant anti-CD39 antibodies to human CD39 was determined by surface plasmon resonance (SPR) using a Biacore 8K instrument. The recombinant antibodies were immobilized using a goat anti-human FcIgG-coated CM5 sensor chip (GE, catalog number 29-1496-03), and the soluble His-tagged CD39 ECD protein sequence number 72 (Sino Biological, catalog number 16020-H08B) was used as the analyte. The binding constants were determined at 25°C. As shown in Table 2, all three antibodies showed similarly high binding affinities, as measured by a slow off-rate (k d ) and sub-nanomolar equilibrium dissociation constant (K D ).

[0227]

Table 1

[0228]

Table 2

[0229] Example 2: Generation of Humanized Anti-CD39 Antibodies Rat antibodies 31 and 39 were selected for humanization using CDR grafting technology (Queen et al, Proc. Natl. Acad. Sci. USA. 86:10029-10033, 1989). The rat variable heavy chain (VH) and variable light chain (VL) sequences of antibodies 31 and 39 were used to identify the human germline sequences closest to each chain. Human acceptors for the VH and VL frameworks were searched within the GenBank database (Benson et al., Nucleic Acids Res. 2005, 33, D34-D38). The frameworks were defined using the extended CDR definition where Kabat CDR1 was extended by 5 amino acids at the N-terminus. The top three hits were used to derive the sequences of the humanized VH and VL genes, and two were selected from each of these to express the humanized antibodies. For antibody 31 VH, IGHV4-24*01 with 64.6% sequence identity and IGHV1-69-2*01 with 63.4% identity were selected, and for VL, IGKV7-3*01 with 84.8% sequence identity and IGKV4-1*01 with 77.2% identity were selected (Table 3). For antibody 39 VH, IGHV3-9*01 with 87.8% sequence identity and IGHV3-30*15 with 85.4% identity were selected, and for VL, IGKV7-3*01 with 83.5% sequence identity and IGKV3-15*01 with 72.2% identity were selected (Table 4).

[0230]

Table 3

[0231]

Table 4

[0232] CDR grafting was performed for each human acceptor. For antibody 31, HC-CDR1 (SEQ ID NO: 18), HC-CDR2 (SEQ ID NO: 19), and HC-CDR3 (SEQ ID NO: 20) were used as VH acceptors, and LC-CDR1 (SEQ ID NO: 22), LC-CDR2 (SEQ ID NO: 25), and LC-CDR3 (SEQ ID NO: 26) were used as VL acceptors. For antibody 39, HC-CDR1 (SEQ ID NO: 28), HC-CDR2 (SEQ ID NO: 29), and HC-CDR3 (SEQ ID NO: 30) were used as VH acceptors, and LC-CDR1 (SEQ ID NO: 32), LC-CDR2 (SEQ ID NO: 33), and LC-CDR3 (SEQ ID NO: 34) were used as VL acceptors. The resulting sequences were examined for the introduction of any potential post-translational modification (PTM) sites such as isomerization, deamination, glycosylation, and oxidation. Putative residues suitable for back mutations in the rat sequences were also identified using antibody homology graphic modeling. After removal of the PTM sites, the antibodies were assayed to determine whether the change(s) affected antigen binding compared to the parental antibody.

[0233] The humanized gene was reverse translated, codon optimized for mammalian expression, and synthesized by GENEWIZ. The synthetic gene was recloned into the company's IgG expression vector, expressed, and purified.

[0234] The reaction rate of the binding of the purified antibody to the antigen was determined using surface plasmon resonance (SPR) and used to rank the antibodies. The affinity of the anti-CD39 antibodies for the human CD39 ECD His-tagged antigen (SEQ ID NO: 72) was measured using a Biacore 8K instrument. The activator was prepared by mixing 400 mM EDC and 100 mM NHS (GE) immediately before injection. A CM5 sensor chip (GE, catalog number 29-1496-03) was activated with the activator for 420 seconds. Next, goat anti-human Fc IgG (30 μg / mL in 10 mM NaAc, pH 4.5) was injected into the channel at a flow rate of 10 μL / min for 420 seconds. The chip was inactivated with 1 M ethanolamine hydrochloride. The anti-CD39 antibody was diluted to 4 μg / mL with running buffer (1xHBS-EP+) and injected into the channel at a flow rate of 10 μL / min for 15 seconds. Six concentrations (8, 4, 2, 1, 0.5, 0.25 nM) of the CD39 antigen analyte were injected into the channel in sequence at a flow rate of 30 μL / min during a 180-second binding phase followed by a 2400-second dissociation phase. Glycine (10 mM, pH 1.5) as the regeneration buffer was injected following the dissociation phase. The sensorgrams of the reference channel and the buffer channel were subtracted from the test sensorgram. The experimental data were fitted with a 1:1 binding model steady-state affinity / heterogeneous ligand. The molecular weights of the CD39 antigen and the anti-CD39 antibody used in the calculation were 52 kDa and 145 kDa, respectively.

[0235] To maintain the binding affinity of the humanized variant of antibody 39: ch39_IGG4.P, the K94R rat sequence revertant mutation was introduced into the VH1 chain (SEQ ID NO: 58), but since R94 (SEQ ID NO: 59) was present, an equivalent revertant mutation was not required in VH3. The human framework of VL1 also had a 81 NDT 83 glycosylation PTM site (

[0236] Next, for 39 humanized variants, all four VH / VL combinations of VH1 (SEQ ID NO: 58) or VH3 (SEQ ID NO: 59) and VL1 (SEQ ID NO: 61) or VL3 (SEQ ID NO: 62) were expressed, but the VH1+VL3 pair was not expressed well. The variant genes were codon-optimized for mammalian expression and then synthesized by GENEWIZ: a plasmid containing VH in which the VH domain was fused to the human IgG4 CH1, hinge, CH2, and CH3 segments of the heavy chain (including the hinge mutation S228P); and a plasmid containing VL in which the VL domain was fused to the human Ig kappa CK domain of the light chain.

[0237] The humanized IgG4 (S228P) variant of antibody 39 was analyzed by SPR as described above together with the chimeric rVH+rVL / human IgG4 (S228P) variant of antibody 39. The results are shown in Table 5.

[0238]

Table 5

[0239] Additional rat sequence revertant mutations (I2T) were introduced into the VL1 framework (SEQ ID NO: 60) and VL3 framework (SEQ ID NO: 63), and the antibodies were expressed and analyzed, but the VH1+VL3 pair was not expressed well. Table 6 shows the SPR data of these antibody variants.

[0240]

Table 6

[0241] The humanized variants of antibody 39 show high affinity for cell human CD39 and potently inhibit the enzymatic activity of cell human CD39 (Table 7).

[0242]

Table 7

[0243] Humanized variant of antibody 31: The following rat sequence revertant mutations were introduced into the VH1 chain: E71S, D76N, and A78V (SEQ ID NO: 43). Also, to maintain the binding affinity of ch31_IGG4.P, the revertant mutations D76N and A78V were introduced into the VH2 chain (SEQ ID NO: 44). Antibody 31 has a glycosylation PTM site sequence ( 26 NQT 28 ) in LC CDR1. This was mutated to either Q27P (SEQ ID NO: 45) or N26Q (SEQ ID NO: 46) in VL1, and to N26Q (SEQ ID NO: 49) in VL2. The human framework of VL1 also had a glycosylation PTM site ( 81 NDT 83 ) introduced, which was removed by introducing additional rat sequence revertant mutations N81D (SEQ ID NO: 47 and SEQ ID NO: 48 respectively). For VL2, no rat sequence revertant mutations were necessary.

[0244] For the humanized variant of antibody 31, all four VH / VL combinations of VH1 (SEQ ID NO: 43) or VH2 (SEQ ID NO: 44) and VL1 (SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, or SEQ ID NO: 48) or VL2 (SEQ ID NO: 49) were expressed, but the VH1+VL2 pair was not expressed sufficiently. The variant genes were codon-optimized for mammalian expression and then synthesized by GENEWIZ: a plasmid containing VH where the VH domain is fused to the human IgG4 CH1, hinge, CH2, and CH3 segments of the heavy chain (including the hinge mutation S228P); and a plasmid containing VL where the VL domain is fused to the human Ig kappa CK domain of the light chain.

[0245] The humanized IgG4 (S228P) variant of antibody 31 was analyzed by SPR as described above together with the chimeric rVH+rVL / human IgG4 (S228P) variant of antibody 31. The results are shown in Table 8.

[0246]

Table 8

[0247] The humanized variant of antibody 31 exhibits high affinity for cell human CD39 and potently inhibits the enzymatic activity of cell human CD39 (Table 9).

[0248]

Table 9

[0249] For both antibody 31 and antibody 39, humanized variants were obtained by applying CDR grafting followed by a small number of rat sequence revertant mutations to the human variable domain framework. These antibodies retained or improved the binding affinity and inhibitory potency of their respective rat / human chimeric antibodies.

[0250] Example 3: Characterization of Complete Kinetic Binding by SPR Using the genes of the VH and VL domains of the humanized antibodies hu31.4_IGG4.P and hu39.5_IGG4.P codon-optimized for mammalian expression, human IgG1 variants were generated. A plasmid containing a VH in which the VH domain is fused to the human IgG1 CH1, hinge, CH2, and CH3 constant domain segments (SEQ ID NO: 2) of the heavy chain (HC) containing the L234A and L235A (Eu numbering) mutations, and a plasmid containing a VL in which the VL domain is fused to the human Ig kappa CK constant domain (SEQ ID NO: 6) of the light chain (LC) were transfected into CHO-K1 cells. Antibodies hu31.4_IGG1.AA (HC is SEQ ID NO: 51, LC is SEQ ID NO: 56) and hu39.5_IGG1.AA (HC is SEQ ID NO: 66, LC is SEQ ID NO: 67) were expressed using a high-speed stable pool protocol and purified to >95% purity by protein A affinity chromatography followed by size exclusion chromatography (SEC), as confirmed by SEC-HPLC analysis.

[0251] The antibodies hu31.4_IGG1.AA and hu39.5_IGG1.AA were analyzed by SPR using a Biacore 4000 instrument. The kinetic binding properties were evaluated in two modes. In the "monovalent affinity mode", the test antibodies were captured at seven different densities using a CM3 sensor chip (GE, catalog number 29-1049-90) coated with amine-coupled goat anti-human IgG. The soluble human CD39 ECD His-tagged antigen (Sino Biological, catalog number 16020-H08B) was the analyte: a stock solution prepared at 33.3 μM was diluted to 33 nM as the highest concentration and tested in triplicate at a 3-fold dilution series on the antibody surface. In the "divalent avidity mode", the soluble human CD39 ECD His-tagged antigen was amine-coupled to the CM3 sensor chip at six different densities. The test antibodies against the analyte were prepared as a 33.3 μM stock solution, diluted to 100 nM as the highest concentration, and tested in triplicate at a 3-fold dilution series on the CD39 surface. The running buffer contained 10 mM HEPES, 150 mM NaCl, 0.05% tween-20, and 0.2 mg / mL BSA. Data were collected using single cycle kinetics with multiple injections by increasing the concentration of the analyte, and the dissociation phase was monitored for 1 hour. All data were collected at 25°C. Response data from all six or seven surface densities were fitted to a 1:1 interaction model using the local Rmax, which is the response to the maximum amount of complex formed. The results of the experiments performed in the monovalent affinity mode are shown in Table 10, and the results of the divalent avidity mode are shown in Table 11.

[0252] The antibodies hu31.4_IGG1.AA and hu39.5_IGG1.AA, when analyzed in the monovalent affinity mode using the captured antibody on the sensor chip and soluble CD39 ECD as the analyte, had similar equilibrium dissociation constants (K D) has. However, when analyzed in a mode that enables divalent antibody binding using CD39 ECD and soluble antibody captured on the sensor chip as analytes, hu31.4_IGG1.AA shows a K very similar to that obtained when analyzed in the monovalent mode, regardless of the antigen surface density. D was shown. In contrast, hu39.5_IGG1.AA shows a K up to three orders of magnitude lower depending on the antigen surface density. D This indicates that this antibody can bind to multiple immobilized CD39 molecules on the chip surface in a divalent mode, thus improving the kinetic binding affinity due to avidity.

[0253]

Table 10

[0254]

Table 11

[0255] Example 4: Binding of anti-CD39 antibodies to endogenous human CD39 Variants of antibodies 19, 31, and 39 were tested for binding to the surface of primary human cells. Binding was evaluated in the absence of ATP and in the presence of high ATP (400 μM).

[0256] Purified monocyte binding: Antibodies were analyzed by flow cytometry for binding to primary human monocytes (CD14 +The binding to human CD39 expressed on the cell surface of () was tested. Monocytes were positively selected from peripheral blood (leukopak or LRS chamber) using the EasySep Human CD14 Positive Selection Kit II (Stem Cell, catalog number 17858), frozen in Bambanker (Wako, catalog number 302-14681), and stored in liquid nitrogen for future use. On the day of the assay, the frozen monocytes were thawed, rinsed, and resuspended in 1:25 Fc block (BD, catalog number 564220) and 1:250 Aqua live / dead dye (Invitrogen, catalog number L34966A) in DPBS, and incubated for 15 minutes at room temperature in the dark. Cells were plated at 0.2 - 0.5×10 6 cells per well in a polypropylene v-bottom 96-well plate. Zero or 400 μM ATP was added, incubated at 4 °C for 30 minutes, followed by the addition of the test antibody and further incubation at 4 °C for 30 minutes. After washing twice with DPBS, 1 μg / mL of PE-labeled mouse anti-human IgG4 secondary (Southern Biotech, catalog number 9200-09) was added at 4 °C for 30 minutes for antibody detection. After two washes with DPBS, the cells were fixed with 50 μL IC fixation buffer (Invitrogen, catalog number 00-8222-49) for 20 minutes at room temperature. Next, the cells were washed once, resuspended in DPBS, and subjected to flow cytometry using a BD LSR FORTESSA X-20 Cell Analyzer. The geometric mean of the fluorescence intensity of PE was obtained for the live single-cell population. The EC 50 value was calculated using standard 4-parameter curve fitting in GraphPad Prism (Table 12).

[0257]

Table 12

[0258] PBMC binding: Recombinant and IgG1 isotype variant of antibody 39 were also tested by flow cytometry for CD14 +(Single cells) and CD19 + (B cells) were tested for their binding to human CD39 expressed on the cell surface of PBMCs gated in the (B cell) population. In this experiment, an antibody reported to bind to and inhibit human CD39, designated huBMK2_IGG1.AEASS herein, was used as a comparator. The amino acid sequence of the mature heavy chain of huBMK2_IGG1.AEASS is SEQ ID NO: 73. The amino acid sequence of the mature light chain of huBMK2_IGG1.AEASS is SEQ ID NO: 74. PBMCs previously purified and frozen were thawed, washed, and resuspended in 1:25 Fc block (BD, catalog number 564220) and 1:250 Aqua live / dead dye (Invitrogen, catalog number L34966A) in DPBS and incubated for 15 minutes at room temperature in the dark. Next, the cells were washed with DPBS and resuspended in filtered staining buffer: DPBS supplemented with 5% goat serum (Gibco, catalog number 16210-064). The cells were plated in a polypropylene V-bottom 96-well plate at 6 0.5×10 cells per well in staining buffer. Zero or 400 μM ATP was added and incubated at 4 °C for 30 minutes, followed by the addition of the test antibody and further incubation at 4 °C for 30 minutes. After washing twice with staining buffer, 0.8 μg / test CD14 FITC (Invitrogen, catalog number 11-0149-42, clone 61D3) and 0.5 μg / test CD19 eFluor 450 (eBioscience, catalog number 48-0199-42, clone HIB19) were added to the PBMCs. Binding of CD39 by the test antibody was detected using 0.5 μg / mL PE-labeled goat anti-human IgG (Southern Biotech, catalog number 2048-09) for 30 minutes at 4 °C. Next, the cells were washed and resuspended in DPBS and subjected to flow cytometry using a BD LSR FORTESSA X-20 Cell Analyzer. The geometric mean of the fluorescence intensity of PE was obtained for live single-cell populations gated on either + monocytes (CD14 + ) or B cells (CD19 + ). Standard 4-parameter curve fitting in GraphPad Prism was used to determine the EC50 Values were calculated (Table 13). Representative graphs from one donor are shown in Figure 1.

[0259]

Table 13

[0260] Overall, variants of antibodies 19, 31, and 39 showed strong binding to the surface of primary cells. Potency was determined to be variant of 39 > variant of 31 > variant of 19. The potency ranking of variants of 31 and 39 was maintained by the isotype switch from IGG4.P to IGG1.AA, and the potency was maintained in the presence of high ATP. Hu31.4_IGG1.AA, which is a humanized IgG1 variant of antibody 31, and hu39.5_IGG1.AA, which is a humanized IgG1 variant of antibody 39, showed strong binding compared to the aforementioned anti-CD39 antibodies, and hu39.5_IGG1.AA > huBMK2_IGG1.AEASS > hu31.4_IGG1.AA.

[0261] Example 5: Inhibition of Human CD39 Enzyme Activity Variants of antibodies 19, 31, and 39 were tested for inhibition of human CD39 enzyme activity. Inhibition was evaluated using soluble recombinant CD39 and CD39 expressed on the cell surface of primary human cells or human cell lines derived from patient tumor samples. Inhibition was evaluated in the presence of low ATP and high ATP (20 μM and 400 μM, respectively).

[0262] Recombinant CD39 Inhibition: The efficacy of the antibodies described in the previous examples to inhibit CD39 enzyme activity was first evaluated using recombinant human CD39. A recombinant human CD39 biochemical assay was performed in an assay buffer consisting of 25 mM Tris-HCl, pH 7.4, 5 mM MgCl2, and 0.01% BSA. A 14-point, 1:3 master dilution series of the test antibody was prepared in assay buffer to cover a final concentration range of 200 nM to 0.125 pM. Five microliters of the test antibody at a final concentration five-fold in assay buffer was added to each well of a 384-well plate. Ten microliters of 1.56 nM recombinant human CD39 resuspended in assay buffer was added to each well, and the plate was incubated at 37 °C for an additional 60 minutes, after which 10 μL of 50 μM ATP in assay buffer was added. The final assay conditions consisted of 0.625 nM recombinant human CD39 and 20 μM ATP substrate. After incubation at 37 °C for an additional 30 minutes, 25 μL of Kinase Glo Plus reagent was added to each well of the 384-well plate. The reaction was measured according to the manufacturer's protocol using the Kinase-Glo reagent kit (Promega, catalog number V3771). The amount of ATP remaining after CD39 inhibition was evaluated and quantified as a function of the luminescence generated using an Envision 2102 Multilabel reader equipped with a luminescence filter. CD39 enzyme activity was evaluated as the correlation of the remaining ATP levels. The percentage of maximum activity in each test well was calculated based on the assay buffer (maximum activity, 0% inhibition) and the control well without enzyme (baseline activity, 100% inhibition). The IC 50 values were determined from a dose-response curve fitted using a standard four-parameter fitting equation. The data are shown in Table 14.

[0263]

Table 14

[0264] SK-MEL-5 CD39 inhibition: Inhibition of CD39 enzyme activity was also evaluated using SK-MEL-5 cells, a melanoma cell line established from patient-derived tumor samples. On the day of the experiment, one vial of pre-frozen SK-MEL-5 cells was thawed and the cells were resuspended in 10 mL of assay buffer consisting of 20 mM HEPES, pH 7.4, 137 mM NaCl, 5.4 mM KCl, 1.3 mM CaCl2, 4.2 mM NaHCO3, and 0.1% glucose. A 14-point, 1:3 master dilution series of the anti-CD39 antibody was prepared in assay buffer such that the final concentration range spanned from 100 nM to 0.063 pM. Twenty microliters of the test antibody, at a final concentration five times that in the assay buffer, was added to each well of a 96-well round-bottom polypropylene plate. Forty microliters of SK-MEL-5 cells were added to each well and the plate was incubated at 37 °C for an additional 60 minutes, after which 40 μL of 50 μM ATP in assay buffer was added. The final assay conditions consisted of 10,000 cells per well and 20 μM of the ATP substrate. After incubation at 37 °C and 5% CO2 for an additional 60 minutes, the plate was centrifuged at 225 × g for 10 minutes. Thirty microliters of the supernatant was transferred to a 96-well assay plate (Corning, catalog number 3912) and the amount of ATP remaining during the reaction was measured using the Kinase-Glo reagent kit (Promega, catalog number V3771) according to the manufacturer's protocol. The amount of ATP remaining after CD39 inhibition was evaluated and quantified as a function of the luminescence generated using an Envision 2102 Multilabel reader equipped with a luminescence filter. CD39 enzyme activity was evaluated as the correlation of the remaining ATP levels. The percentage of maximum activity in each test well was calculated based on the assay buffer (maximum activity, 0% inhibition) and the cell-free control well (baseline activity, 100% inhibition). The IC 50 values of the test antibody were determined from a dose-response curve fitted using a standard four-parameter fitting equation. The data are shown in Table 15.

[0265] [Table 15]

[0266] Inhibition of THP-1 CD39: Inhibition of CD39 enzyme activity was evaluated in a second tumor cell line, specifically, a human monocyte cell line derived from a patient with acute monocytic leukemia. On the day of the experiment, one vial of pre-frozen THP-1 cells was thawed and the cells were resuspended in 10 mL of assay buffer consisting of 20 mM HEPES, pH 7.4, 137 mM NaCl, 5.4 mM KCl, 1.3 mM CaCl2, 4.2 mM NaHCO3, and 0.1% glucose. A 14-point, 1:3 master dilution series of the anti-CD39 antibody was prepared in assay buffer such that the final concentration range was from 100 nM to 0.063 pM. Twenty microliters of the test antibody, at a final concentration five times that in the assay buffer, was added to each well of a 96-well round-bottom polypropylene plate. Forty microliters of THP-1 cells were added to each well and the plate was incubated at 37 °C for an additional 60 minutes, after which 40 μL of 50 μM ATP in assay buffer was added. The final assay conditions consisted of 40,000 cells per well and 20 μM of the ATP substrate. After incubation at 37 °C and 5% CO2 for an additional 60 minutes, the plate was centrifuged at 225 × g for 10 minutes. Thirty microliters of the supernatant was transferred to a 96-well assay plate (Corning, catalog number 3912) and the amount of ATP remaining during the reaction was measured using the Kinase-Glo reagent kit (Promega, catalog number V3771) according to the manufacturer's protocol. The amount of ATP remaining after CD39 inhibition was evaluated and quantified as a function of the luminescence generated using an Envision 2102 Multilabel reader equipped with a luminescence filter. CD39 enzyme activity was evaluated as the correlation of the remaining ATP levels. The percentage of maximum activity in each test well was calculated based on the assay buffer (maximum activity, 0% inhibition) and the cell-free control well (baseline activity, 100% inhibition). The IC 50 values of the test antibody were determined from a dose-response curve fitted using a standard four-parameter fit equation. The data are shown in Table 16.

[0267]

Table 16

[0268] Purified Monocyte CD39 Inhibition: Potency was further evaluated using primary human monocytes. On the day of the experiment, two vials of pre-frozen CD14 + positive selected human monocyte (donor 1003773) cells were thawed and resuspended in 10 ml of assay buffer consisting of 20 mM HEPES, pH 7.4, 137 mM NaCl, 5.4 mM KCl, 1.3 mM CaCl2, 4.2 mM NaHCO3, and 0.1% glucose. A 14-point, 1:3 master dilution series of the test antibody was prepared in assay buffer to cover a final concentration range of 200 nM to 0.13 pM. Twenty microliters of the test antibody at 5 times the final concentration in assay buffer was added to each well of a 96-well round bottom polypropylene plate. Forty microliters of human monocyte cells were added to each well and the plate was incubated at 37 °C for an additional 60 minutes, after which 40 μL of 50 μM or 1000 μM ATP in assay buffer was added. The final assay conditions consisted of 20,000 cells per well and 20 μM or 400 μM ATP substrate. After incubation at 37 °C and 5% CO2 for an additional 60 minutes, the plate was centrifuged at 225 × g for 10 minutes. Fifteen microliters of the supernatant was transferred to a 96-well assay plate (Corning, catalog number 3912) and the amount of AMP generated in the reaction was measured using the AMP-Glo reagent kit (Promega, catalog number 5012) according to the manufacturer's protocol. The supernatant from the reaction plate containing 400 μM ATP substrate was diluted 1:8 with assay buffer before transfer and the AMP-Glo reagent was added. The amount of AMP generated after CD39 inhibition was evaluated and quantified as a function of the luminescence generated using an Envision 2102 Multilabel reader equipped with a luminescence filter. CD39 enzyme activity was evaluated as the correlation of AMP levels. The percentage of maximum activity in each test well was calculated based on the assay buffer (maximum activity, 0% inhibition) and the cell-free control well (baseline activity, 100% inhibition). The IC50 The value was determined from the dose-response curve fitted using the standard four-parameter fitting equation. The data are shown in Tables 17 and 18, and Figure 2.

[0269] [Table 17]

[0270] [Table 18]

[0271] Overall, variants of antibodies 19, 31, and 39 showed potent inhibition of soluble and surface human CD39 enzyme activity. Importantly, the efficacy was maintained in the presence of high ATP.

[0272] Example 6: Immunostimulatory Effect of CD39 Inhibition To evaluate the efficacy of the anti-CD39 antibodies produced in Examples 1-3, the downstream functional consequences of inhibiting CD39 enzyme activity on the surface of bone marrow cells were evaluated in vitro.

[0273] Macrophage IL-1β / IL-18 Release Assay: The NLRP3 inflammasome is a multi-protein cytosolic complex that, upon oligomerization, can cleave and activate the procaspase-1 protein to become caspase 1. Caspase-1 then promotes the maturation of IL-1β and IL-18 by cleaving their inactive pro-forms (pro-IL-1β and pro-IL-18) into their active and secreted forms. These cytokines are involved in the innate immune response and create a general pro-inflammatory environment.

[0274] Anti-CD39 antibodies were tested for their ability to activate the NLRP3 inflammasome on macrophages by measuring the secretion of mature IL-1β and IL-18. A schematic of the assay is shown in Figure 3. On day 0, previously isolated and positively selected CD14 +Thaw single cells (see Example 4), and culture them at a density of approximately 15×10 6 CD14 + single cells per 10 cm dish (Thermo Scientific Nunclon Delta Surface, catalog number 150464) in RPMI medium containing 10% heat-inactivated FBS, 1% Glutamax, 1% penicillin / streptomycin, and 50 ng / mL recombinant human M-CSF (R&D, catalog number 216MCC / CF). On day 5, wash away non-adherent cells, scrape and collect adherent macrophages, and plate them at a density of 0.125×10 6 cells per well in a 24-well plate. Next, incubate the cells overnight to allow them to adhere. The next day, treat the cells with 1 ng / mL LPS (InvivoGen, catalog number tlrl-peklps), followed by treatment with 10 or 100 nM of the test antibody or IgG control, and incubate at 37°C, 5% CO2 for 3 hours. Next, treat the cells with 500 μM ATP (Life Technologies, catalog number R0441), and incubate at 37°C, 5% CO2 for an additional 2 - 4 hours. Collect the supernatant, and analyze the IL-1β level by CBA (BD, catalog number 558279) or ELISA (R&D, catalog number QK201) according to the manufacturer's instructions. Analyze the IL-18 level in the supernatant by ELISA (R&D, catalog number DL180) according to the manufacturer's instructions. Perform the experiment in technical quadruplicates for each donor, take the average, and then normalize it to the isotype control for each donor. As shown in Figure 4, ch39_IGG4.P, hu39.1_IGG4.P, hu39.5_IGG1.AA, and hu31.4_IGG1.AA increased the release of IL-1β and IL-18 from in vitro-derived macrophages compared to the isotype control.

[0275] Monocyte-derived dendritic cell assay: Antibodies were also tested for their ability to mature monocyte-derived dendritic cells (moDCs) in the presence of ATP. On day 0, positively selected monocytes were resuspended in RPMI supplemented with 10% heat-inactivated FBS, 1% Glutamax, 1% penicillin / streptomycin, 100 ng / mL recombinant human GM-CSF (R&D, catalog number 215-GM / CF), and 100 ng / mL IL-4 (Peprotech, catalog number 200-04), and seeded at 4×10 6 cells per well in 6-well plates (Falcon, catalog number 353046). On day 6, moDCs were harvested and seeded at 0.5×10 6Seeded with individual cells and treated with 10 μg / mL isotype control or anti-CD39 antibody at 37°C and 5% CO2 for 1 hour, followed by treatment with 0 μM or 300 μM ATP at 37°C and 5% CO2 for 18 hours. On day 7, for staining, moDCs were transferred to polypropylene V-bottom 96-well plates. All wells were resuspended in 1:25 Fc block (BD, catalog number 564220) and 1:250 Aqua live / dead dye (Invitrogen, catalog number L34966A) in DPBS and incubated for 15 minutes at room temperature in the dark. After washing once with DPBS, the cells were stained with the following antibodies [1 μg / test CD83 PE-Cy7 (BD, catalog number 561132), 1 μg / test CD86 BV605 (BD, catalog number 562999), 0.8 μg / test CD14 FITC (Invitrogen, catalog number 11-0149-42)], or the corresponding isotype controls [PE-Cy7 mouse IgG1 κ (BD, catalog number 565573), BV605 mouse IgG1 κ (BD, catalog number 562652), FITC mouse IgG1 κ (eBiosciences, catalog number 11-4714-41)] at 4°C for 30 minutes. The surface levels of the dendritic cell maturation markers CD83 and CD86 should increase with dendritic cell maturation, while the monocyte marker CD14 should decrease. After washing once with DPBS, the cells were fixed with 50 μL IC fixation buffer (Invitrogen, catalog number 00-8222-49) for 20 minutes at room temperature. Next, the cells were washed and resuspended in DPBS and subjected to flow cytometry using a BD LSR FORTESSA X-20 Cell Analyzer. For CD83 and CD14, the percentage of the parent gate was set to the isotype control (<5% of total events) for each donor under the 0 μM ATP condition. For CD86, since >90% of the cells were positive compared to the isotype control under the control treatment condition, instead, the geometric mean of the fluorescence intensity of the fluorophore was reported for the live single-cell population.

[0276] As shown in FIGS. 5A and 5B, ATP matured moDCs, as evidenced by the increase in the dendritic cell maturation markers CD83 and CD86. Inhibition of CD39 enzymatic activity by addition of anti-CD39 antibody promoted maturation, which did not occur with addition of isotype control, resulting in a further increase in the percentage of cells expressing CD83 and the surface amount of CD86 per cell. Addition of anti-CD39 in the presence of ATP also decreased the percentage of cells expressing the monocyte marker CD14 (FIG. 5C). The effects on CD83, CD86, and CD14 were ATP-dependent effects, except for hu39.6_IGG4.P.

[0277] Overall, the above data suggest that enzymatic inhibition of human CD39 by the antibodies of the present disclosure has an immunostimulatory effect on different bone marrow populations in vitro. Example 7: Characterization of Antibodies by Competitive Flow

[0278] In this example, a competitive flow assay useful for determining whether two antibodies (a test antibody and a reference antibody) compete for binding to human CD39 expressed on the surface of cells is described. The selection of the test antibody, reference antibody, and cell type can be varied.

[0279] The antibodies hu39.1_IGG4.P and antibody IGG4.P, IgG4(S228P) isotype control (CrownVivo) were directly conjugated to Alexa Fluor 647 (“AF647”) using the AlexaFluor647 Conjugation Kit - Lightning-Link (Abcam, catalog number ab269823). The conjugated antibodies are referred to in this example as “hu39.1_IGG4.P-AF647” and “IGG4.P-AF647”. Verification of binding was performed using MOLP-8, a human multiple myeloma, CD39-expressing cell line. On the day of the assay, frozen MOLP-8 cells were thawed, rinsed, and resuspended in 1:25 Fc block (BD, catalog number 564220) and 1:250 Aqua live / dead dye (Invitrogen, catalog number L34966A) in DPBS and incubated for 15 minutes at room temperature in the dark. The cells were plated in a polypropylene v-bottom 96-well plate at 0.2×10 6 cells per well. An 11-point dose-response curve was prepared for each antibody by serial 3-fold dilutions in DPBS in the concentration range of 833 to 0.0141 nM. The 12th point contained only DPBS and no antibody. The diluted antibodies obtained from the dose-response were added to the plated cells at a 1:1 ratio and incubated at 4°C for 30 minutes. The cells were then washed three times, resuspended in DPBS, and subjected to flow cytometry using a BD LSR FORTESSA X-20 Cell Analyzer. The mean fluorescence intensity of AF647 was obtained for the live single-cell population. EC 50 values and EC 95 values were calculated using standard 4-parameter non-linear regression analysis of GraphPad Prism software.

[0280] Next, the antibody hu39.1_IGG4.P-AF647 was used as a reference antibody in a competitive flow assay with unlabeled test antibodies (hu31.1_IGG1.AA, hu39.1_IGG4.P, hu39.5_IGG1.AA, IGG1.AA isotype control, and IGG4.P isotype control (CrownVivo)). On the day of the assay, frozen MOLP-8 cells were thawed, washed, and resuspended in 1:25 Fc block (BD, catalog number 564220) and 1:250 Aqua live / dead dye (Invitrogen, catalog number L34966A) in DPBS and incubated for 15 minutes at room temperature in the dark. An 11-point dose-response curve was prepared for each test antibody by serial 3-fold dilution in DPBS at concentrations ranging from 833 to 0.0141 nM. The 12th point contained only DPBS and no antibody. 12.5 μL was added to each well of a 96-well staining plate. 12.5 μL of the reference antibody hu39.1_IGG4.P-AF647 at a concentration of 93.7 nM was added to each well and gently mixed 3 times. Cells were plated at 0.2×10 6 cells per well and incubated at 4°C for 60 minutes. Next, the cells were washed 3 times, resuspended in DPBS, and subjected to flow cytometry using a BD LSR FORTESSA X-20 Cell Analyzer. The mean fluorescence intensity of AF647 was obtained for the live single-cell population. EC 50 values were calculated using standard 4-parameter non-linear regression analysis of GraphPad Prism software. The maximum competition (%) was calculated using the following formula: maximum competition = 1 - (MFI at the highest concentration of the test antibody / MFI at the matching concentration of hu39.1_IGG4.P-AF647 only). Only a single concentration of the reference antibody hu39.1_IGG4.P-AF647 was used.

[0281] As shown in Figure 6 and Table 19, the test antibody hu39.5_IGG1.AA competes with the reference antibody hu39.1_IGG4.P-AF647, and the maximum competition is 93%. A similar competition level was also observed with hu39.1_IGG4 (unlabeled reference antibody). In contrast, no competition was observed between the test antibody hu31.4_IGG1.AA and the reference antibody hu39.1_IGG4.P-AF647. These data suggest that antibodies 31 and 39, and their variants, bind to different non-overlapping epitopes.

[0282]

Table 19

[0283] Example 8: Efficacy in Xenograft Models The anti-CD39 antibodies produced as described in Examples 1 and 2 bind to human CD39 but not to mouse CD39. Therefore, a xenograft mouse model can be used to evaluate in vivo efficacy. Five hundred thousand to ten million human cancer cells expressing human CD39 are injected subcutaneously or locally into immunodeficient mice such as SCID or nude mice, and tumor growth is monitored. Anti-CD39 antibodies can bind to and inhibit the activity of CD39 in several human cancer cell lines, including but not limited to MOLP-8 myeloma (Figure 7), THP-1 leukemia (Table 16), SK-MEL-5 melanoma (Table 15), and OAW42 ovarian cancer cells. When the tumor is less than 150 mm 3 in size, the mice are treated intraperitoneally (i.p.) with 0.1 - 30 mg / kg of the anti-CD39 antibody of the present disclosure or an isotype control BIWx4, or until the tumor volume reaches 2,000 mm 3 . Treatment of MOLP-8 tumors expressing human CD39 with the anti-CD39 antibody of the present disclosure resulted in inhibition of tumor growth compared to isotype control-treated mice. Inhibition of tumor growth compared to isotype control-treated mice is also expected after treatment of other tumor types expressing human CD39 with the anti-CD39 antibody of the present disclosure.

[0284] Example 9: Efficacy in a Human CD39 Knock-in Model To evaluate the anti-CD39 antibodies of the present disclosure in an immunocompetent mouse model, the antibodies can be tested in a human CD39 knock-in (hCD39KI) mouse model, where fully immunocompetent C57BL / 6 mice or BALB / c mice express human CD39 but not mouse CD39 (Figure 9). In this mouse model, the expression of human CD39 is parallel to the expression of mouse CD39 in wild-type mice. This mouse model enables in vivo testing of the potential effects of anti-CD39 inhibition on host cells such as stromal cells, the vascular system, and immune cells. Mice are inoculated subcutaneously or orthotopically with wild-type or engineered tumor cell lines of mice, including but not limited to MCA-205 fibrosarcoma, B16-F10 melanoma, MC38 colon adenocarcinoma, ID8 ovarian cancer, 4T1 breast cancer, or CT-26 colon cancer cells. 500,000 to 10 million mouse cancer cells are injected, and tumor growth is monitored. When the tumor is less than 150 mm 3 in size, the mice are treated intraperitoneally (i.p.) with 0.1 - 30 mg / kg of ch39_mIGG2A.AAG or an isotype control BIWx4 until the tumor volume reaches 2,000 mm 3 in size. Treating the tumors with anti-CD39 as a single agent or in combination with one or more additional therapies (e.g., ICD inducers, immunotherapies, etc.) inhibits tumor growth compared to isotype control-treated mice.

[0285] In one experiment, 1 million MC38 cells were inoculated into C57BL / 6 hCD39KI mice on day 0. When the tumor reached 80 mm 3When the mice reached that point, a loading dose of 20 mg / kg of anti-CD39 or isotype control antibody (vehicle: PBS) was administered intraperitoneally (i.p.) to the mice, followed by subsequent administration of 10 mg / kg of anti-CD39 or isotype control antibody twice a week (BIW). Each group consisted of 11 - 12 mice. In these experiments, the anti-CD39 antibody refers to ch39_mIGG2A.AAG. Oxaliplatin (OXA) treatment at 5 mg / kg i.p. (vehicle: saline) was initiated on the day following the loading dose of anti-CD39 or isotype control and administered once a week. In experiments designed similarly, 4T1 cells were inoculated into BALB / c hCD39KI mice on day 0, and they were treated with OXA or anti-CD39 antibody as described for the MC38 model. The results from these experiments are explained in the following paragraphs.

[0286] Anti-CD39 shows efficacy in the hCD39KI model. To evaluate the efficacy of anti-CD39 treatment, tumor volume, CD39 enzyme activity in the tumor and peripherally, and CD39 expression were evaluated.

[0287] Tumor volume and body weight were measured approximately twice a week (BIW), which are shown in Figures 10A and 10B respectively, where the dots represent the mean and the error bars represent the standard error of the mean (SEM) of each measurement criterion from each group of mice. Significance was calculated using a mixed effects model including multiple comparisons. As shown in Figure 10A, treatment with anti-CD39 and oxaliplatin was statistically significant (p = 0.0217) compared to treatment with either single agent alone (p = 0.0372 for isotype + OXA vs anti-CD39 + OXA, and p = 0.0217 for anti-CD39 + saline vs anti-CD39 + OXA). Body weight remained stable throughout the experiment, and anti-CD39 treatment showed good tolerance (Figure 10B).

[0288] Tumors and spleens from C57BL / 6 hCD39KI MC38 mice at the end of the study were further analyzed for evidence of human CD39 expression by immunohistochemistry (IHC) and anti-CD39 antibody enzyme inhibitory activity by enzyme histochemistry (EHC). Briefly, frozen OCT-embedded tumor or spleen samples were sectioned to a thickness of 4 μm, mounted on positively charged microscope slides, air-dried for 15 minutes, and then placed in 4% paraformaldehyde (PFA) fixative for 20 minutes. After fixation, the slides were washed with Tris-buffered saline, and IHC was performed on an automated Leica Bond Rx platform, starting with blocking of endogenous peroxidase, followed by incubation with rabbit anti-CD39 mAb (Abcam, EPR20627), detection with an anti-rabbit HRP polymer system, then diaminobenzidine (DAB) brown chromogen and hematoxylin nuclear counterstaining, and then dehydration and mounting. In the EHC assay, after fixation with 4% PFA for 5 minutes, the sections were washed with Tris maleate buffer and incubated for 30 minutes at room temperature in a solution of 50 mM Tris maleate, 250 mM sucrose, and 2 mM MgCl2. Then, the sections were incubated for 2 hours at 37°C in a solution of 100 mM Tris maleate containing 1 mM ATP, 2.5 mM levamisole, 2 mM lead nitrate, 0.25 mM sucrose, and 5 mM MnCl2. Levamisole was used to inhibit tissue-nonspecific alkaline phosphatase (TNAP) activity, but other inhibitors could also be used. Then, the sections were developed in 1% ammonium sulfide solution for 5 minutes, washed, counterstained with hematoxylin, dehydrated, and mounted. Representative images are shown in Figures 11-14 (tumors, Figures 11-12; spleens, Figures 13-14). Human CD39 expression was detected in both isotype-treated tumors (Figures 11, panel A and Figure 12, panel A, low-power and high-power magnifications, respectively) and anti-CD39-treated tumors (Figures 11, panel C and Figure 11, panel C, low-power and high-power magnifications, respectively) and was localized in vascular endothelium, infiltrating immune cells, and stromal components of the tumor microenvironment.Human CD39 expression levels were observed in both isotype-treated spleens (Figure 13, panel A and Figure 14, panel A, low and high power magnifications, respectively) and anti-CD39-treated spleens (Figure 13, panel C and Figure 14, panel C, low and high power magnifications, respectively), and were localized in the red pulp and to a lesser extent in the white pulp. In tumors and spleens treated in vivo with isotype, deposition of lead phosphate was observed by enzyme histochemistry (tumor - Figure 11, panel B and Figure 12, panel B, low and high power magnifications, respectively; spleen - Figure 13, panel B and Figure 14, panel B, low and high power magnifications, respectively). In tumors and spleens treated in vivo with anti-CD39 antibody, no deposition of lead phosphate was observed by enzyme histochemistry, indicating that enzyme activity was blocked (tumor - Figure 11, panel D and Figure 12, panel D, low and high power magnifications, respectively; spleen - Figure 13, panel D and Figure 14, panel D, low and high power magnifications, respectively).

[0289] CD39 enzyme activity in tumors and peripherally was also evaluated ex vivo. After completion of the study, tumor samples obtained after sacrificing C57BL / 6 hCD39KI MC38 mice and BALB / c hCD39KI 4T1 mice were placed in RPMI + 20% FBS + 250 μg / ml collagenase D (Millipore Sigma, catalog number 11088858001) + 100 KU / ml DNAse1 (MilliporeSigma catalog number D5025 - 150KU) and enzymatically digested using a gentleMACS™ Octo Dissociator to obtain a single cell suspension. Cells were plated at 2.5×10 4Cells were seeded and treated with 100 nM hu39.5_IGG1.AA or IgG1 Fc silent isotype control antibody (Absolute Antibody, catalog number Ab00178-10.3) and incubated at 37 °C, 5% CO2 for 1 hour. Next, the cells were treated with 20 μM ATP and incubated at 37 °C, 5% CO2 for an additional 30 minutes. At the end of the incubation period, the 96-well plate was spun at 1000 rpm for 3 minutes to collect the supernatant. Next, 2X Kinase-Glo® Plus (Promega, catalog number V3771) was added to the supernatant. Next, the ATP level in the supernatant was measured by detecting luminescence using a Flexstation® 3 microplate reader. Data are shown as relative light units (RLU). The absence of a difference in RLU between the ex vivo hu39.5_IGG1.AA and isotype treatment groups indicates complete inhibition of enzymatic activity by in vivo treatment. Anti-CD39 treatment inhibited enzymatic activity in tumors (Figures 15A and 15B).

[0290] To assay peripheral CD39 enzyme activity, peripheral white blood cells (WBCs) were isolated from terminal blood samples of hCD39KI mice and assayed as generally described for tumor cell suspensions. Briefly, whole blood samples were mixed with 1X Pharm Lyse red blood cell lysing solution (BD Biosciences, catalog number 555899). Next, after washing with 1× PBS solution, the WBC pellet was collected and stored in BamBanker cell freezing medium (Bulldog Bio, catalog number BB01). To perform the enzyme inhibition assay, the sample was thawed and then the cells were plated at 2.5×10 per well of a 96-well plate 4Plated with cells. Each sample was incubated for 1 hour at room temperature with two treatment conditions: 100 nM of hu39.5_IGG1.AA or IgG1 Fc silent isotype control antibody (Absolute Antibody, catalog number Ab00178-10.3). Next, all wells were treated with 20 μM ATP and incubated for 2 hours at room temperature. At the end of the incubation period, the 96-well plate was spun at 1000 rpm for 3 minutes to collect the supernatant. Next, 2X Kinase-Glo® Plus (Promega, catalog number V3771) was added to the supernatant. Next, the ATP level in the supernatant was measured by detecting luminescence using a Flexstation® 3 microplate reader. The data are shown as relative light units (RLU). The absence of a difference in RLU between the ex vivo hu39.5_IGG1.AA and isotype treatment groups indicates complete inhibition of enzyme activity by in vivo treatment. Anti-CD39 treatment inhibited enzyme activity in the periphery of 4T1 tumor-bearing mice (Figure 15C).

[0291] Decrease in cell surface CD39 in the peripheral immune population of hCD39KI mice and complete or nearly complete coverage by anti-CD39 treatment. Whole blood samples from the terminal bleeding of C57BL / 6 hCD39KI MC38 mice were evaluated by flow cytometry for monocytes (CD3 - CD19 - CD11b + Ly6c ++ ), granulocytes (CD3 - CD19 - CD11b + Ly6c + ), T cells (CD3 + CD19 - ), B cells (CD3 - CD19 + ), and other lineage negative (Lin -)The changes in target coverage and CD39 expression were evaluated in peripheral immune cell types expressing CD39 that include lymphocytes. Target binding was measured using hu39.5_IGG1.AA-AF647, a reagent that binds competitively with ch39_mIGG2A.AAG. hu39.5_IGG1.AA was conjugated to AF647 using the Abcam AF647 Lightning Link conjugation kit. Whole blood samples from 5 - 6 mice per treatment group were stained with a cocktail of extracellular antibodies that included CD39 hu39.5_IGG1.AA-AF647 (competitive with anti-CD39), CD39A1 PE (non-competitive with anti-CD39), CD3 17A2 PerCP-Cy5.5, CD11b M1 / 70 FITC, CD19 6D5 BV421, and Ly6c HK1.4 PE-Cy7. The antibodies were incubated in whole blood for 30 minutes on ice in the dark, and then the samples were fixed / lysed for 20 minutes on ice in the dark using 1X BD FACSLysing solution. Next, the samples were centrifuged at 400×g for 5 minutes, washed once with 1X PBS buffer, resuspended in 1% paraformaldehyde solution, and filtered through a 20 μm strainer prior to data acquisition. The samples were run on a BD FACSCanto SpecialOrder cytometer and analyzed using FlowJo and Prism software. Unbound CD39 was detected by the competitive clone hu39.5_IGG1.AA-AF647 (measuring the MFI of the hu39.5_IGG1.AA-AF647 signal against its isotype control IgG1.FcS-AF647) across cell types of all animals treated with isotype antibodies. Significant competitive binding of hu39.5_IGG1.AA-AF647 was observed in all CD39(A1)-expressing cells of animals treated with anti-CD39, indicating complete or nearly complete target coverage (Figure 16, lower panel). Total CD39 surface protein was detected by the non-competitive CD39 antibody (clone A1 PE). A decrease in surface CD39 protein was observed in multiple types of peripheral immune cells of mice treated with anti-CD39 (Figure 16, upper panel).

[0292] Cell surface CD39 is decreased by anti-CD39 treatment in the tumor-draining lymph nodes of hCD39KI mice. Tumor-draining lymph nodes (TLDNs) (right inguinal lymph nodes) were also collected at the end of the above C57BL / 6 hCD39KI MC38 mouse study. Lymph nodes were dissociated in a 37°C water bath of RPMI 1640 (Gibco, catalog number 11875119) containing 0.6 mg / mL collagenase P (Roche, catalog number 11213865001), 2.4 mg / mL dispase II (Sigma, catalog number D4693), and 0.3 mg / mL DNAse I (Thermo Scientific, catalog number J62229MB) for 30 minutes with gentle agitation. Dissociated lymph nodes were counted and 2×10 5The cells were plated in a 96-well polypropylene V-bottom plate. The cells were washed with PBS (Gibco, catalog number 14190-144) and resuspended in a PBS solution containing 1:25 Fc block (BD, catalog number 564220) and 1:250 near-infrared fluorescent reactive dye (Invitrogen, catalog number L34976A) for 15 minutes in the dark. After incubation, the cells were stained with the antibody cocktail (Table 20) and incubated at 4°C for 30 minutes. After incubation, the cells were pelleted, the supernatant was removed, and 100 μL of fixation buffer (eBioscience, catalog number 00-8222-49) was added for 1 hour. The cells were washed, resuspended in 200 μL of PBS, filtered through a 40-μm filter plate (Fisher Scientific, catalog number NC0726512), acquired on a BD LSR Fortessa X-20 Cell Analyzer, and analyzed with FlowJo™ v10.8 software (BD Life Sciences) to define immune subpopulations. The expression of human CD39, mouse CD73, and mouse P2X7 in the defined immune subpopulations is shown in FIGS. 17A and 17B. The anti-CD39 antibody ch39_mIGG2A.AAG did not cause a significant change in the percentage of immune subpopulations in the tumor-draining lymph nodes of hCD39KI mice inoculated with MC38 tumor cells, either as a single agent or in combination with oxaliplatin treatment (FIG. 17C). Despite no change in this immune population, anti-CD39 treatment significantly reduced human CD39 on the surface of almost all immune subpopulations identified from the tumor-draining lymph nodes (FIG. 17D). A representative histogram is shown in FIG. 17E.

[0293]

Table 20

[0294] Anti-CD39 treatment does not change peripheral cytokine levels. Terminal bleeding of mice bearing MC38 tumors was collected into BD Vacutainer® heparin tubes (BD Biosciences, catalog number 367871), centrifuged at 2000 x g for 15 minutes, and the supernatant plasma samples were collected. Cytokine levels in the plasma samples were analyzed using MSD according to the manufacturer's instructions. In anti-CD39 treated mice, no increase in peripheral cytokine levels was observed. Data are shown as mean ± SEM using a total of 11 - 12 mice per treatment group (Figure 18). Statistical analysis was performed using ANOVA and Tukey's multiple comparison test.

[0295] Example 10: Use of anti-CD39 antibody in combination with immunogenic cell death (ICD) inducing therapy The ICD inducing potential of chemotherapeutic agents was characterized in the absence or presence of anti-CD39 antibody. In this study, three criteria for measuring ICD inducing potential were used: (1) cytotoxicity, (2) extracellular HMGB1 release, and (3) extracellular ATP release.

[0296] To evaluate cellular ATP release after oxaliplatin treatment, mouse colon cancer cell lines CT26 and MC38 and human melanoma cell line SK-MEL-5 were thawed, rinsed, and resuspended in their respective culture media. The culture media for CT26 and MC38 were RPMI + 10% FBS + 1% glutamax + 1% penicillin-streptomycin + 1% sodium pyruvate. The culture media for SK-MEL-5 was EMEM + 10% FBS + 1% glutamax + 1% penicillin-streptomycin + 1% sodium pyruvate. Cells were plated at 2.5×10 per well of a 96-well plate 4Cells were plated and incubated overnight at 37°C with 5% CO2. The next day, the cells were treated with vehicle (PBS), 100 μM or 250 μM oxaliplatin. SK-MEL-5 cells were further treated with 100 nM hu39.5_IGG1.AA or IgG1 Fc silent isotype control antibody (Absolute Antibody, catalog number Ab00178-10.3). To determine cell viability, the cells were treated and incubated at 37°C with 5% CO2 for 24 hours. At the end of the incubation period, viability was determined using the luminescence-based Cell Titer Glo 2.0 assay (Promega, catalog number G9242). Data are presented as viability normalized to untreated vehicle control (Figure 19A). To determine HMGB1 release, the cells were treated and incubated at 37°C with 5% CO2 for 24 hours. At the end of the incubation period, the plates were centrifuged at 400 × g for 5 minutes and the supernatants were collected. Next, the Lumit HMGB1 kit (Promega, catalog number CS3030B01) was used according to the manufacturer's instructions to determine HMGB1 levels in the culture supernatants (Figure 19B). To assay for ATP release, the cells were then treated and incubated at 37°C with 5% CO2 for 8 hours. At the end of the incubation period, 4x RealTime-Glo™ Extracellular ATP Assay (Promega catalog number GA5010) was added to the cells. Extracellular ATP was measured kinetically by detecting luminescence at 15-minute intervals over 6 hours using a Flexstation® 3 microplate reader. All three cell lines showed ATP release when treated with 250 μM oxaliplatin (Figure 19C). In SK-MEL-5 cells, hu39.5_IGG1.AA treatment led to increased accumulation of extracellular ATP compared to isotype control-treated cells (Figure 19C).

[0297] The ICD-inducing potential of additional chemotherapeutic agents was characterized in the human melanoma cell line SK-MEL-5. Cells were thawed, rinsed, and resuspended in culture medium of EMEM + 10% FBS + 1% glutamax + 1% penicillin-streptomycin + 1% sodium pyruvate. Cells were plated at 2.5×10 4 cells per well of a 96-well plate and incubated overnight at 37 °C and 5% CO2. The next day, cells were treated with the chemotherapeutic agents doxorubicin (vehicle: DMSO), irinotecan (vehicle: DMSO), gemcitabine (vehicle: PBS), and cisplatin (vehicle: PBS) at the indicated concentrations and incubated at 37 °C and 5% CO2 for 48 h. (1) To evaluate cytotoxicity, at the end of the incubation period, cell viability was determined using the luminescence-based Cell Titer Glo 2.0 assay (Promega, catalog number G9242). Data are presented as viability normalized to untreated vehicle controls (Figure 20A). (2) To measure HMGB1 release, at the end of the incubation period, plates were centrifuged at 400×g for 5 min and the supernatant was collected. Next, the HMGB1 levels in the culture supernatant were determined using the Lumit HMGB1 kit (Promega, catalog number CS3030B01) according to the manufacturer's instructions (Figure 20B). (3) Extracellular ATP release was quantified with chemotherapeutic agents with and without the addition of 100 nM hu39.5_IGG1.AA or IgG1 Fc-silent isotype control antibody (Absolute Antibody, catalog number Ab00178-10.3) and incubated for 8 h or 32 h. At the end of the incubation period, 4x RealTime-Glo™ Extracellular ATP assay (Promega catalog number GA5010) was added to the cells. Next, extracellular ATP release was measured kinetically by detecting luminescence at 15-min intervals over 10 h using a Flexstation® 3 microplate reader. All chemotherapeutic treatment groups showed an increase in ATP release compared to untreated cells. The addition of hu39.5_IGG1.AA resulted in higher levels of extracellular ATP (Figure 20C).

[0298] Example 11: Characterization of CD39 and CD39-related markers in human cells Using a CD39 whole blood (WB) receptor occupancy (RO) flow cytometry assay, CD39 expression in various cell types of human, cynomolgus monkey, and hCD39KI mouse WB was evaluated. The description of the hCD39KI RO assay is provided in Example 9. Human whole blood samples were analyzed by the same method described for hCD39KI mouse samples, but with the incorporation of monocyte- and B cell-specific cell line markers. The human CD39 WB RO panel consisted of the following antibodies: hu39.5_IGG1.AA-AF647 (competitive for anti-CD39), anti-CD39A1 PE (non-competitive for anti-CD39), anti-CD14 61D3 FITC, and -CD19 H1B19 eFluor450. As shown in Figure 21, the relative expression of CD39 in peripheral immune cells differed between humans and C57BL / 6 hCD39KI MC38 mice. Monocytes had the highest level of CD39 expression among the cell types assayed in human blood, while granulocytes had the highest CD39 expression in hCD39KI mice. B cell expression differed most significantly between species. Human peripheral B cells express very high levels of CD39, while hCD39KI mouse B cells have low to no CD39 expression.

[0299] CD39 and CD39-related markers were also characterized in human in vitro differentiated bone marrow cells, such as M0-, M1-, M2-polarized macrophages, and monocyte-derived dendritic cells (moDC), as well as in primary human bone marrow cells, such as CD14 from the peripheral blood of 4 healthy donors + monocytes and bone marrow dendritic cell (DC) subsets. To generate in vitro differentiated macrophages, positively selected CD14 + monocytes were resuspended in RPMI supplemented with 10% heat-inactivated FBS, 1% Glutamax, 1% penicillin / streptomycin, and 50 ng / mL of recombinant human M-CSF (R&D, catalog number 216-MCC / CF) and plated at a total of 2 mL per well at 4×10 6Cells were seeded. On day 4, the cells were replenished with 2 mL of fresh RPMI supplemented medium containing 2×M-CSF. On day 6, the cells were polarized into M1 macrophages using 100 ng / mL LPS (Invivogen, catalog number tlr-peklps) and 20 ng / mL recombinant human IFN-γ (Peprotech, catalog number 300-02), while M2 macrophages were polarized with 20 ng / mL recombinant human IL-4 (Peprotech, catalog number 200-04). No additional cytokines were added to M0 macrophages. To generate moDC, positively selected CD14 + monocytes were resuspended in RPMI supplemented with 10% heat-inactivated FBS, 1% Glutamax, 1% penicillin / streptomycin, and 100 ng / mL of recombinant human GM-CSF (R&D, catalog number 215-GM / CF), and 100 ng / mL of recombinant human IL-4, and seeded at 4×10 6 cells per well in a total volume of 2 mL per well in 6-well plates (Falcon, catalog number 353046). On day 4, the cells were replenished with 2 mL of fresh RPMI supplemented medium containing 2X GM-CSF / IL-4. On day 7, M0-, M1-, M2-polarized macrophages and moDC were collected, counted, and aliquoted into U-bottom 96-well plates for flow cytometry or 1.5 mL Eppendorf tubes for RNA. Primary CD14 + monocytes isolated using a positive selection kit and bone marrow-derived DCs enriched using a negative selection kit were also aliquoted into U-bottom 96-well plates for flow cytometry or 1.5 mL Eppendorf tubes for RNA.

[0300] Flow cytometry quality control staining was performed to determine the purity of primary CD14 + monocyte isolation, the purity of primary bone marrow dendritic cell (DC) enrichment, and the distribution of DC subsets within the cells resulting from primary bone marrow dendritic cell enrichment. Monocytes were defined by CD14 + expression. CD14 +The purity of single cell isolation was approximately 99%. For the enrichment of primary dendritic cells from the peripheral blood of 4 human donors, an enriched population of dendritic cells defined as HLA-DR+ CD11c+ and pDC was shown, which was 48.5%. The enriched DCs consisted of 2.8% plasmacytoid DCs (HLA - DR + CD11c neg CD123 + and defined as pDC), 3.0% conventional DC1s (HLA - DR + CD11c + CD141 + Clec9a + and defined as cDC1), 30.4% conventional DC2s HLA - DR + CD11c + CD1c + CD141 neg Clec9a neg and defined as cDC2), and 12.0% other DCs (HLA - DR + CD11c + CD1c neg CD141 neg Clec9a neg and defined as).

[0301] For the flow cytometry evaluation of CD39 and CD73 (Figures 22A and 22B), CD14 +Single cells, enriched myeloid DCs, M0, M1, M2 macrophages, and moDCs were aliquoted into 96-well plates and resuspended in 50 μL / well of 1:25 Fc block (BD, catalog number 564220) and 1:250 aqua live / dead dye (Invitrogen, catalog number L34966A) in DPBS and incubated for 15 minutes at room temperature in the dark. Next, the cells were stained at 50 μL / well with the following antibodies [0.25 μg / test of CD39 PE-Cy7 (eBiosciences, catalog number 25-0399-42), and 0.5 μg / test of CD73 BUV737 (BD, catalog number 612812), or the corresponding isotype controls, mouse IgG1 PE-Cy7 (BD, catalog number 557647) and mouse IgG1 BUV737 (BD, catalog number 612758)] for 30 minutes at 4°C. After one wash with DPBS, the cells were fixed with 50 μL IC fixation buffer (Invitrogen, catalog number 00-8222-49) for 20 minutes at room temperature. Next, the cells were washed and resuspended in DPBS and subjected to flow cytometry using a BD LSR FORTESSA X-20 Cell Analyzer. CD14 + The positive rates of CD39 and CD73 in monocytes, enriched myeloid DC subtypes, M0, M1, M2 macrophages, and moDCs were evaluated by comparison with isotype controls. CD14 + Monocytes, all in vitro-derived subsets (M0, M1, M2 macrophages, and moDCs), as well as primary enriched DC subsets cDC1, cDC2, and other DCs, all showed a high positive percentage of CD39 and a low positive percentage of CD73 (Figures 22A and 22B). A relatively small number (15%) of pDCs were CD39 positive. Considering that the majority of cell types showed a >90% positive percentage of CD39, the CD39 expression levels were further investigated by examining the intensity of CD39 staining compared to isotype controls (Figure 22B). Although there was variation among donors, CD39 expression was higher in monocytes, in vitro-derived macrophages, and moDCs compared to primary DCs. CD14 + Among primary DCs, cDC1 had the highest expression of cell surface CD39.

[0302] The expression of ENTPD1 (CD39) and NT5E (CD73) genes was analyzed by qPCR using standard methods (Figure 22C and Figure 22D). The level of ENTPD1 gene expression was higher than that of NT5E gene expression across all cell types. The enriched DC population had the lowest relative expression of ENTPD1 and the highest relative expression of NT5E.

[0303] To evaluate the broader CD39-related gene expression in these human bone marrow subsets, a custom NanoString panel was designed and utilized. RNA from each bone marrow cell subset was isolated using the Qiagen RNeasy Mini kit (catalog number 74106) according to the manufacturer's instructions. Next, the RNA was quantified with a NanoDrop, and 70 ng of RNA was used for NanoString. To hybridize the RNA to the custom NanoString code set, 70 μL of hybridization buffer was added to the reporter code set, and 8 μL was added to 5 μL of RNA for each sample. Next, 2 microliters of the capture probe set was added to each sample, and the samples were incubated at 65 °C for 16 hours in a thermocycler. Next, the samples were supplemented with RNase-free water to 30 μL, and this was loaded into the sample loading port of an nCounter SPRINT cartridge (NanoString, catalog number SPRINT-CAR-1.0). Next, the cartridge was run on a NanoString nCounter SPRINT profiler. Quality control metrics were performed on the data from each sample, and samples passing the quality control test were normalized against three housekeeping genes included in the custom NanoString panel to evaluate gene expression.

[0304] Figure 22E shows a heatmap of unsupervised clustering of the expression of CD39, adenosine, and inflammasome pathway genes normalized by housekeeping genes using a custom NanoString panel. Wells of low quality flagged by low counts or abnormal control probes were excluded from this analysis. Specific donor IDs are appended at the end of the cell type labels. The dotted horizontal line in the upper dendrogram indicates that the samples are split, but does not affect the unsupervised nature of the clustering. CD39 is expressed across all immune populations profiled. Cell types form distinct clusters that give a consistent expression of genes in these pathways within each cell type. Monocytes, M1 macrophages, and enriched primary DCs are driven by expression of IL-1β, P2X7, and A2AR and cluster together. M0, M1 macrophages, and moDCs form another cluster with high expression of CD206, A2BR, and P2Y11.

[0305] Example 12: P2Y11 antagonism reduces the activation of ATP-driven dendritic cells The effects of P2X1, P2X7, and P2Y11 inhibitors on the activation of monocyte-derived dendritic cells (MoDCs) were investigated. MoDCs were generated from positively selected human monocytes cultured with GM-CSF and IL-4 as described in Example 11. On day 7, the 6-well plates were incubated on ice for 5 minutes, scraped to detach the cells, and counted. The cells were resuspended in fresh medium containing GM-CSF and IL-4, and 125,000 cells in 100 μL were plated into each well of a 96-well flat-bottom Upcell plate (Nunc, catalog number 174897). Each compound at 10 micromolar [P2X1 inhibitor, NF023, Millipore Sigma, catalog number 104869-31-0; P2X7 inhibitor, A-740003, Millipore Sigma, catalog number 861393-28-4; P2Y11 inhibitor NF340, APExBIO, catalog number B7508; P2Y11 inhibitor NF157, APExBIO catalog number B7060] or vehicle control (0.1% DMSO) was added, gently mixed, and incubated at 37 °C with 5% CO2 for 1 hour. Subsequently, 0 or 300 μM ATP was added, gently mixed, and incubated at 37 °C with 5% CO2 for 18 hours. For flow cytometry evaluation, the cells were detached from the plate and transferred to a 96-well polypropylene v-bottom plate. The cells were washed with PBS (Gibco, catalog number 14190-144) and resuspended in a PBS solution containing 2% FBS, a viability dye (LD-NIR, Invitrogen, catalog number L34994), an Fc block, and staining antibodies [BV510 anti-CD80 (BD Horizon, catalog number 563084), PE-Cy7 anti-CD83 (BD Horizon, catalog number 561132), and BV605 anti-CD86 (BD Horizon, catalog number 562999)]. The cells were incubated at room temperature for 60 minutes, the staining solution was washed away, and fixed with BD Phosphoflow FIX Buffer I (BD, catalog number 557870) and incubated for 15 minutes.The fixation buffer was removed, the cells were washed with PBS, acquired on a BD LSR FORTESSA X-20 cell analyzer, and stored at 4°C until analyzed with FLOWJO™ v10.8 software (BD Life Sciences).

[0306] Within each inhibitor group, the fold change between the ATP-treated and non-ATP-treated groups was calculated, and the induction of each surface marker in the presence of ATP was calculated. Next, the induction levels in the inhibitor-treated groups were normalized to the DMSO-treated group. The results show that the P2Y11 inhibitor suppresses CD86 induction by ATP, but not CD80 or CD83 (Figure 23). Example 13: Solid tumors express a mechanism for response to elevated ATP as a result of CD39 inhibition

[0307] The ATP response mechanism was evaluated in a subset of solid tumors. Briefly, TCGA RNA expression was analyzed in the tumor type of interest and expression was normalized gene-by-gene (Figure 24A). The violin plot in Figure 24B outlines the kernel probability density of the normalized log2 gene expression (y-axis) of the following genes (proteins): ENTPD1 (CD39), NT5E (CD73), P2RX7 (P2X7), and P2RY11 (P2Y11) in a subset of TCGA samples. Along the x-axis, the violin plot is grouped by specific TCGA subtypes: esophageal carcinoma (ESCA), head and neck squamous cell carcinoma (HNSC), kidney renal clear cell carcinoma (KIRC), lung adenocarcinoma (LUAD), lung squamous cell carcinoma (LUSC), pancreatic adenocarcinoma (PAAD), and stomach adenocarcinoma (STAD). Also shown in Figure 24B are the pathway enrichment scores (y-axis) of the myeloid gene signature (IL-6, CXCL2, CXCL2, CXCL3, CXCL8, and PTGS2) calculated using gene set enrichment analysis for the same subset of TCGA cancer subtypes. Using this metric, values >0 indicate upregulation and values <0 indicate downregulation. All tumors have high levels of CD39 and myeloid infiltration. Also, all tumors share similar levels of P2X7 and P2Y11, indicating that they may respond to elevated ATP in the tumor microenvironment. Examples of the disclosed embodiments

[0308] Embodiment 1: An anti-CD39 antibody that specifically binds to human CD39, comprising: (a) a heavy chain variable region comprising complementarity-determining region 1 (H1) having at least 80% sequence identity with SEQ ID NO: 10, complementarity-determining region 2 (H2) having at least 80% sequence identity with SEQ ID NO: 11, and complementarity-determining region 3 (H3) having at least 80% sequence identity with SEQ ID NO: 12; and a light chain variable region comprising complementarity-determining region 1 (L1) having at least 80% sequence identity with SEQ ID NO: 14, complementarity-determining region 2 (L2) having at least 80% sequence identity with SEQ ID NO: 15, and complementarity-determining region 3 (L3) having at least 80% sequence identity with SEQ ID NO: 16; (b) a heavy chain variable region comprising H1 having at least 80% sequence identity with SEQ ID NO: 18, H2 having at least 80% sequence identity with SEQ ID NO: 19, and H3 having at least 80% sequence identity with SEQ ID NO: 20; and a light chain variable region comprising L1 having at least 80% sequence identity with SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24, L2 having at least 80% sequence identity with SEQ ID NO: 25, and L3 having at least 80% sequence identity with SEQ ID NO: 26; or (c) a heavy chain variable region comprising H1 having at least 80% sequence identity with SEQ ID NO: 28, H2 having at least 80% sequence identity with SEQ ID...

Claims

1. An anti-CD39 antibody that specifically binds to human CD39, comprising a heavy chain variable region comprising complementarity-determining region 1 (H1) containing SEQ ID NO: 28, complementarity-determining region 2 (H2) containing SEQ ID NO: 29, and complementarity-determining region 3 (H3) containing SEQ ID NO: 30; and a light chain variable region comprising complementarity-determining region 1 (L1) containing SEQ ID NO: 32, complementarity-determining region 2 (L2) containing SEQ ID NO: 33, and complementarity-determining region 3 (L3) containing SEQ ID NO: 34, said anti-CD39 antibody.

2. The anti-CD39 antibody according to claim 1, wherein said antibody has the heavy chain variable region and the light chain variable region described in claim 1, wherein said heavy chain variable region has at least 90% sequence identity with SEQ ID NO: 27, and said light chain variable region has at least 90% sequence identity with SEQ ID NO: 31; wherein said heavy chain variable region has at least 90% sequence identity with SEQ ID NO: 58, and said light chain variable region has at least 90% sequence identity with SEQ ID NO: 60; wherein said heavy chain variable region has at least 90% sequence identity with SEQ ID NO: 58, and said light chain variable region has at least 90% sequence identity with SEQ ID NO: 61; wherein said heavy chain variable region has at least 90% sequence identity with SEQ ID NO: 58, and said light chain variable region has at least 90% sequence identity with SEQ ID NO: 62; wherein said heavy chain variable region has at least 90% sequence identity with SEQ ID NO: 58, and said light chain variable region has at least 90% sequence identity with SEQ ID NO: 63; wherein said heavy chain variable region has at least 90% sequence identity with SEQ ID NO: 59, and said light chain variable region has at least 90% sequence identity with SEQ ID NO: 61; wherein said heavy chain variable region has at least 90% sequence identity with SEQ ID NO: 59, and said light chain variable region has at least 90% sequence identity with SEQ ID NO: 60; wherein said heavy chain variable region has at least 90% sequence identity with SEQ ID NO: 59, and said light chain variable region has at least 90% sequence identity with SEQ ID NO: 62; or wherein said heavy chain variable region has at least 90% sequence identity with SEQ ID NO: 59, and said light chain variable region has at least 90% sequence identity with SEQ ID NO: 63, said anti-CD39 antibody.

3. The anti-CD39 antibody according to claim 1, which is a monoclonal antibody or an antigen-binding fragment thereof, said anti-CD39 antibody.

4. The anti-CD39 antibody according to claim 1, which is a human chimeric, humanized, or veneer antibody, or an antigen-binding fragment thereof.

5. The anti-CD39 antibody according to claim 1, which comprises a variant heavy chain constant region selected from a variant human IgG1 constant region, a variant human IgG2 constant region, a variant human IgG3 constant region, or a variant human IgG4 constant region.

6. The anti-CD39 antibody according to claim 5, wherein the variant heavy chain constant region comprises SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO:

5.

7. The anti-CD39 antibody according to claim 1, which comprises a wild-type human IgG heavy chain constant region.

8. The anti-CD39 antibody according to claim 7, wherein the wild-type human IgG heavy chain constant region comprises SEQ ID NO: 1 or SEQ ID NO:

4.

9. The anti-CD39 antibody according to claim 5, which comprises a human kappa light chain constant region.

10. The anti-CD39 antibody according to claim 1, which has a heavy chain comprising the heavy chain variable region and a light chain comprising the light chain variable region, (a) the heavy chain comprises SEQ ID NO: 64 and the light chain comprises SEQ ID NO: 67; (b) the heavy chain comprises SEQ ID NO: 64 and the light chain comprises SEQ ID NO: 68; (c) the heavy chain comprises SEQ ID NO: 64 and the light chain comprises SEQ ID NO: 69; (d) the heavy chain comprises SEQ ID NO: 64 and the light chain comprises SEQ ID NO: 70; (e) the heavy chain comprises SEQ ID NO: 65 and the light chain comprises SEQ ID NO: 68; (f) the heavy chain comprises SEQ ID NO: 65 and the light chain comprises SEQ ID NO: 67; (g) the heavy chain comprises SEQ ID NO: 66 and the light chain comprises SEQ ID NO: 67; (h) the heavy chain comprises SEQ ID NO: 65 and the light chain comprises SEQ ID NO: 69; or (i) the heavy chain comprises SEQ ID NO: 65 and the light chain comprises SEQ ID NO:

70.

11. The anti-CD39 antibody according to claim 1, the antibody has the heavy chain variable region and the light chain variable region according to claim 1, the heavy chain variable region has at least 95% sequence identity with SEQ ID NO: 27, and the light chain variable region has at least 95% sequence identity with SEQ ID NO: 31; the heavy chain variable region has at least 95% sequence identity with SEQ ID NO: 58, and the light chain variable region has at least 95% sequence identity with SEQ ID NO: 60; The heavy chain variable region has at least 95% sequence identity with SEQ ID NO: 58, and the light chain variable region has at least 95% sequence identity with SEQ ID NO: 61; The heavy chain variable region has at least 95% sequence identity with SEQ ID NO: 58, and the light chain variable region has at least 95% sequence identity with SEQ ID NO: 62; The heavy chain variable region has at least 95% sequence identity with SEQ ID NO: 58, and the light chain variable region has at least 95% sequence identity with SEQ ID NO: 63; The heavy chain variable region has at least 95% sequence identity with SEQ ID NO: 59, and the light chain variable region has at least 95% sequence identity with SEQ ID NO: 61; The heavy chain variable region has at least 95% sequence identity with SEQ ID NO: 59, and the light chain variable region has at least 95% sequence identity with SEQ ID NO: 60; The heavy chain variable region has at least 95% sequence identity with SEQ ID NO: 59, and the light chain variable region has at least 95% sequence identity with SEQ ID NO: 62; or The heavy chain variable region has at least 95% sequence identity with SEQ ID NO: 59, and the light chain variable region has at least 95% sequence identity with SEQ ID NO: 63, said anti-CD39 antibody.

12. The anti-CD39 antibody according to claim 1, wherein the antibody has the heavy chain variable region and the light chain variable region described in claim 1, the heavy chain variable region has at least 99% sequence identity with SEQ ID NO: 27, and the light chain variable region has at least 99% sequence identity with SEQ ID NO: 31; the heavy chain variable region has at least 99% sequence identity with SEQ ID NO: 58, and the light chain variable region has at least 99% sequence identity with SEQ ID NO: 60; the heavy chain variable region has at least 99% sequence identity with SEQ ID NO: 58, and the light chain variable region has at least 99% sequence identity with SEQ ID NO: 61; the heavy chain variable region has at least 99% sequence identity with SEQ ID NO: 58, and the light chain variable region has at least 99% sequence identity with SEQ ID NO: 62; the heavy chain variable region has at least 99% sequence identity with SEQ ID NO: 58, and the light chain variable region has at least 99% sequence identity with SEQ ID NO: 63; the heavy chain variable region has at least 99% sequence identity with SEQ ID NO: 59, and the light chain variable region has at least 99% sequence identity with SEQ ID NO: 61; The heavy chain variable region has at least 99% sequence identity with SEQ ID NO: 59, and the light chain variable region has at least 99% sequence identity with SEQ ID NO: 60; The heavy chain variable region has at least 99% sequence identity with SEQ ID NO: 59, and the light chain variable region has at least 99% sequence identity with SEQ ID NO: 62; or The anti-CD39 antibody, wherein the heavy chain variable region has at least 99% sequence identity with SEQ ID NO: 59, and the light chain variable region has at least 99% sequence identity with SEQ ID NO:

63. **Claim 13** A pharmaceutical composition comprising the antibody according to any one of claims 1 to 12 and a pharmaceutically acceptable carrier.

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