Anti-CD39 antibodies and their uses

Abstract

This disclosure provides an antibody or an antigen-binding fragment thereof that specifically binds to CD39 and inhibits the activity of soluble CD39 protease without inhibiting the activity of membrane-bound CD39 enzymes.

Description

[0001] This application is a divisional application of the invention patent application filed on December 27, 2023, with application number 202311834233.7 and invention title: Anti-CD39 antibody and its uses. Technical Field

[0002] This disclosure relates to the field of antibodies, and more specifically to an antibody against CD39. Background Technology

[0003] CD39, also known as exonucleoside triphosphate diphosphate hydrolase 1 (ENTPDase 1), converts ATP or ADP to AMP. CD73 then dephosphorylates AMP to adenosine, a potent immunosuppressant. Adenosine binds to adenosine receptors (e.g., A2A receptors) on the surface of CD4 and CD8 T cells and natural killer (NK) cells, inhibiting T cell and NK cell responses and thus suppressing the immune system. Adenosine also binds to A2A or A2B receptors on macrophages and dendritic cells, inhibiting phagocytosis and antigen presentation, and increasing the secretion of oncogenes (e.g., VEGF, TGFβ, and IL-6). Elevated adenosine levels mediated by CD39 and CD73 create an immunosuppressive environment, thereby promoting cancer development and progression. Furthermore, CD39 is also present as a soluble enzyme constitutive form in the blood of humans and mice and, along with other purinergic enzymes, promotes ADP metabolism.

[0004] CD39 is widely expressed in various tissues and organs, such as the bladder, brain, breast, colon, uterus, stomach, and prostate, and is mainly expressed on endothelial cells and immune cells. CD39 exhibits high expression in various human tumors, including lymphoma, sarcoma, lung cancer, pancreatic cancer, ovarian cancer, renal cell carcinoma, thyroid cancer, and testicular cancer. Besides effector T cells and NK cells mentioned above, CD39's regulatory function on the immune system is also manifested in its influence on macrophages, MDSCs, neutrophils, regulatory T cells, and antigen-presenting cells. CD39 is associated with immunosuppression and immune exhaustion in most immune cells, while knocking out CD39 can upregulate the secretion of immune factors, inhibit the activity of regulatory T cells, and stimulate the function of immune cells.

[0005] Given the crucial role of CD39 in the tumor microenvironment, it has become an emerging target for researchers developing tumor immunotherapies. In the tumor microenvironment, targeting CD39 to block adenosine-mediated immunosuppression can inhibit tumor growth.

[0006] Currently, publicly available information shows that antibodies targeting CD39 simultaneously inhibit both soluble CD39 and membrane-bound CD39. The antibody screened in this patent only inhibits the activity of the soluble CD39 protease, without inhibiting the activity of the membrane-bound CD39 enzyme. Because CD39 is widely expressed in tissues, CD39 antibodies, as antibody drugs, pose certain safety risks. The characteristics of the antibody screened in this patent eliminate this risk, improving the safety of antibody drugs. Summary of the Invention

[0007] To address the aforementioned problems, this disclosure provides antibodies, methods for their preparation, compositions, etc. The benefits provided by this disclosure are broadly applicable to the fields of antibody therapy and diagnostics, and can be used in combination with antibodies that respond to various targets. This invention provides an antibody capable of specifically binding to the human CD39 antigen.

[0008] This invention discloses an isolated antibody or its antigen-binding fragment thereof, wherein the antibody or its antigen-binding fragment specifically binds to CD39 and comprises a heavy chain variable region (VH) and a light chain variable region (VL).

[0009] The heavy chain variable region includes:

[0010] (i)HCDR1, comprising a sequence having at least 80%, at least 85%, at least 95%, or 100% sequence identity with one of SEQ ID NO: 21-24, or consisting of SEQ ID NO: 21-24; and

[0011] (ii) HCDR2, comprising or consisting of a sequence having at least 80%, at least 85%, at least 95%, or 100% sequence identity with one of SEQ ID NO: 25-28; and

[0012] (iii)HCDR3, which comprises a sequence having at least 80%, at least 85%, at least 95%, or 100% sequence identity with one of SEQ ID NO: 29-32 or is composed of SEQ ID NO: 29-32;

[0013] The light chain variable region includes:

[0014] (i) LCDR1, comprising or consisting of a sequence having at least 80%, at least 85%, at least 95%, or 100% sequence identity with one of SEQ ID NO: 33-36; and

[0015] (ii) LCDR2, comprising or consisting of a sequence having at least 60%, at least 65%, at least 75%, at least 95%, or 100% sequence identity with one of SEQ ID NO: 37-40; and

[0016] (iii) LCDR3, which comprises or consists of a sequence having at least 80%, at least 85%, at least 95%, or 100% sequence identity with one of SEQ ID NO: 41-44.

[0017] In some embodiments of the present invention, the antibody or fragment includes combinations of the following:

[0018] (i) HCDR1 shown in SEQ ID NO: 21, HCDR2 shown in SEQ ID NO: 25, and HCDR3 shown in SEQ ID NO: 29, LCDR1 shown in SEQ ID NO: 33, LCDR2 shown in SEQ ID NO: 37, and LCDR3 shown in SEQ ID NO: 41; or

[0019] (ii) HCDR1 shown in SEQ ID NO: 22, HCDR2 shown in SEQ ID NO: 26, and HCDR3 shown in SEQ ID NO: 30, LCDR1 shown in SEQ ID NO: 34, LCDR2 shown in SEQ ID NO: 38, and LCDR3 shown in SEQ ID NO: 42; or

[0020] (iii) HCDR1 shown in SEQ ID NO: 23, HCDR2 shown in SEQ ID NO: 27, and HCDR3 shown in SEQ ID NO: 31, LCDR1 shown in SEQ ID NO: 35, LCDR2 shown in SEQ ID NO: 39, and LCDR3 shown in SEQ ID NO: 43; or

[0021] (iv) HCDR1 shown in SEQ ID NO: 24, HCDR2 shown in SEQ ID NO: 28, and HCDR3 shown in SEQ ID NO: 32, LCDR1 shown in SEQ ID NO: 36, LCDR2 shown in SEQ ID NO: 40, and LCDR3 shown in SEQ ID NO: 44.

[0022] In some embodiments of the present invention, the antibody or fragment wherein the heavy chain variable region comprises a sequence having at least 80%, at least 85%, 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%, at least 99%, or 100% sequence identity with one of SEQ ID NO: 12-15, or is composed of one of SEQ ID NO: 12-15;

[0023] The light chain variable region comprises a sequence having at least 80%, at least 85%, 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%, at least 99%, or 100% sequence identity with one of SEQ ID NO: 17-20, or is composed of one of SEQ ID NO: 17-20.

[0024] In some embodiments of the present invention, the antibody or fragment includes combinations of the following:

[0025] (i) VH shown in SEQ ID NO: 12, VL shown in SEQ ID NO: 17; or

[0026] (ii) VH shown in SEQ ID NO: 13, VL shown in SEQ ID NO: 18; or

[0027] (iii) VH shown in SEQ ID NO: 14, VL shown in SEQ ID NO: 19; or

[0028] (iv) VH shown in SEQ ID NO: 15, VL shown in SEQ ID NO: 20.

[0029] The antibody or fragment as described in any of the preceding claims further comprises a heavy chain constant region and a light chain constant region, wherein the heavy chain constant region is selected from the IgG1, IgG2, IgG3, or IgG4 constant regions, and the light chain constant region is selected from the κ or λ light chain constant region, wherein the heavy chain constant region is preferably selected from the IgG1 constant region, and the light chain constant region is preferably selected from the κ chain constant region.

[0030] In some embodiments of the present invention, the antibody or fragment includes:

[0031] The heavy chain comprises a sequence having 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%, at least 99%, or 100% sequence identity with one of SEQ ID NO: 3, 5, 7, or 9, or is composed of one of SEQ ID NO: 3, 5, 7, or 9;

[0032] A light chain comprising or consisting of a sequence having 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%, at least 99%, or 100% sequence identity with one of SEQ ID NO: 4, 6, 8, or 10.

[0033] In some embodiments of the present invention, the antibody or fragment includes:

[0034] (i) the heavy chain shown in SEQ ID NO: 3 and the light chain shown in SEQ ID NO: 4; or

[0035] (ii) the heavy chain shown in SEQ ID NO: 5 and the light chain shown in SEQ ID NO: 6; or

[0036] (iii) the heavy chain shown in SEQ ID NO: 7 and the light chain shown in SEQ ID NO: 8; or (iv) the heavy chain shown in SEQ ID NO: 9 and the light chain shown in SEQ ID NO: 10.

[0037] In some embodiments of the present invention, the antibody or fragment is a whole antibody, a bispecific antibody, a monoclonal antibody, a chimeric antibody, a humanized antibody, or a fully human antibody.

[0038] In some embodiments of the present invention, the antibody or fragment, wherein the antigen-binding fragment is selected from the group consisting of: Fab fragment, Fab' fragment, F(ab)2 fragment, Fv fragment, and ScFv.

[0039] In some embodiments of the present invention, the antibody or fragment inhibits the activity of soluble CD39 protease but does not inhibit the activity of membrane-bound CD39.

[0040] On the other hand, the present invention also provides an isolated nucleic acid molecule containing a nucleic acid sequence encoding the antibody or fragment described above.

[0041] On the other hand, the present invention also provides a carrier containing the aforementioned nucleic acid molecules.

[0042] On the other hand, the present invention also provides a host cell containing the aforementioned nucleic acid molecule or the aforementioned vector.

[0043] On the other hand, the present invention also provides a conjugate comprising the antibody or fragment conjugated to at least one detectable marker.

[0044] On the other hand, the present invention also provides an antibody-drug conjugate comprising an antibody, including one or more drug portions which are directly or covalently linked to the antibody or fragment via a linker.

[0045] On the other hand, the present invention also provides a multispecific molecule comprising the antibody or antigen-binding fragment described above; preferably, the multispecific molecule specifically binds to CD39 and additionally specifically binds to one or more other targets; more preferably, the multispecific molecule further comprises at least one molecule having a second binding specificity against a second target.

[0046] On the other hand, the present invention also provides a pharmaceutical composition or kit comprising the antibody or fragment, or the nucleic acid molecule, or the carrier, or the host cell, or the conjugate, or the antibody-drug conjugate, or the multispecific molecule, and a pharmaceutically acceptable carrier.

[0047] On the other hand, the present invention also provides the use of the antibody or fragment, or the nucleic acid molecule, or the vector, or the host cell, or the conjugate, or the antibody-drug conjugate, or the multispecific molecule, or the pharmaceutical composition or kit in the preparation of a kit for diagnosing, detecting or monitoring diseases related to CD39 expression.

[0048] On the other hand, the present invention also provides the use of the antibody or fragment, or the nucleic acid molecule, or the vector, or the host cell, or the conjugate, or the antibody-drug conjugate, or the multispecific molecule, or the pharmaceutical composition or kit in the preparation of a medicament for treating diseases related to CD39 expression or determining their prognosis.

[0049] On the other hand, in the above-mentioned uses, the disease associated with CD39 expression is cancer, and the cancer is selected from the group consisting of lymphoma, sarcoma, lung cancer, pancreatic cancer, ovarian cancer, renal cell carcinoma, thyroid cancer, and testicular cancer, etc.

[0050] The antibody screened in this patent inhibits only the activity of soluble CD39 protease, without inhibiting the activity of membrane-bound CD39. Since CD39 is widely expressed in tissues, CD39 antibodies pose certain safety risks as antibody drugs. The characteristics of the antibody screened in this patent eliminate this risk, thus improving the safety of antibody drugs. Attached Figure Description

[0051] The accompanying drawings are provided to further understand this disclosure and form part of the specification. They are used together with the embodiments of this disclosure to explain this disclosure and do not constitute a limitation thereof.

[0052] Figure 1 The results of the binding activity of the chimeric antibody of the present invention to the Human CD39 protein are shown;

[0053] Figure 2 The results of the cellular-level binding activity of the chimeric antibody of the present invention are shown;

[0054] Figure 3 The results of the chimeric antibody protease activity inhibition activity of the present invention are shown. Detailed Implementation

[0055] To enable those skilled in the art to better understand the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are merely illustrative examples of some implementations of the present invention, and not all embodiments. Therefore, the present invention is not limited to the specific embodiments illustrated herein. Furthermore, any section headings used herein are not to be construed as limiting the described subject matter.

[0056] Unless otherwise defined herein, scientific and technical terms used in conjunction with this invention will have the meaning commonly understood by one of ordinary skill in the art. Furthermore, unless the context otherwise requires, singular terms shall include plural forms, and plural terms shall include singular forms. More specifically, as used in this specification and the appended claims, unless the context otherwise clearly indicates, the singular forms “a,” “an,” and “the” include plural indicators. In this application, unless otherwise stated, the use of “or” means “and / or.” Furthermore, the use of the term “comprising” and other forms such as “including” and “containing” is not limiting. Moreover, the scope provided in the specification and the appended claims includes all values ​​between endpoints.

[0057] definition

[0058] To better understand this invention, the definitions and explanations of relevant terms are provided below.

[0059] The term "antibody" or "Ab" generally refers to a Y-shaped tetrameric protein comprising two heavy chains (H) and two light chains (L) held together by covalent disulfide bonds and non-covalent interactions. The light chains of an antibody can be classified as κ or λ light chains. The heavy chains can be classified as μ, δ, γ, α, or ε, which define the antibody isotype as IgM, IgD, IgG, IgA, or IgE, respectively. In both the light and heavy chains, the variable region is linked to the constant region via a "J" region of about 12 or more amino acids, and the heavy chain also contains a "D" region of about 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains (CH1, CH2, and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The VH and VL regions can be further divided into hypervariable regions (called complementarity-determining regions, or CDRs) separated by relatively conserved regions (called framework regions, or FRs). Each VH and VL consists of three CDRs and four FRs in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 from the N-terminus to the C-terminus. The CDRs on VH are HCDR1, HCDR2, and HCDR3; the CDRs on VL are LCDR1, LCDR2, and LCDR3. The variable regions (VH and VL) of each heavy / light chain pair form antigen-binding sites / parts, respectively. The distribution of amino acids in various regions or domains follows the numbering definitions in common systems such as Kabat, IMGT, or Chothia. In the specific embodiments of this disclosure, the CDR sequence is determined using the numbering definition in the Kabat system.

[0060] In this disclosure, antibodies also include antigen-binding moieties (used interchangeably with the term "antigen-binding fragment"). An antigen-binding moiety is a polypeptide containing a fragment of a complete antibody that retains the ability to specifically bind to an antigen that binds specifically to a full-length or complete antibody, and / or competes with a full-length antibody for binding to the same antigen. Under certain conditions, antigen-binding moieties include Fab, Fab', F(ab')2, Fd, Fv, dAb, and complementarity-determining region (CDR) fragments, single-chain antibodies (e.g., scFv), chimeric antibodies, biantibodies, and antibodies containing at least a portion sufficient to confer specific antigen-binding ability to the polypeptide. The antigen-binding moieties of antibodies can be obtained from a given antibody using conventional techniques known to those skilled in the art (e.g., recombinant DNA techniques or enzymatic or chemical cleavage methods) and specificity can be screened in the same manner as for complete antibodies.

[0061] The term "isotype" refers to an antibody class (e.g., IgM or IgG1) encoded by a gene in the heavy chain constant region.

[0062] The term "monoclonal antibody" or "mAb" refers to an antibody molecule / formulation consisting of a single molecule. Monoclonal antibodies exhibit single binding specificity and affinity for a specific epitope. The antibodies of this invention can be derived from various species, including but not limited to mice, rats, rabbits, guinea pigs, and humans.

[0063] The term "epitope" refers to an antigenic determinant in a molecule, specifically a portion of the molecule that is recognized by the immune system (e.g., by antibodies), such as a discontinuous three-dimensional site on an antigen recognized by the immune system. In this invention, the epitope shown is, for example, the CD39 protein.

[0064] As used herein, the term "chimeric antibody" refers to an antibody whose variable region sequence is derived from one species and whose constant region sequence is derived from another species, such as an antibody whose variable region sequence is derived from a mouse antibody and whose constant region sequence is derived from a human antibody.

[0065] The term "humanized antibody" refers to an antibody in which a CDR sequence / antigen-binding portion or site derived from another mammalian species, such as a mouse, has been transplanted onto a human frame sequence. Furthermore, additional frame region modifications can be performed within the human frame sequence.

[0066] "Fully human" or "completely human" antibodies are created by transferring the entire human antibody-encoding gene into a genetically engineered animal lacking the antibody gene, using transgenic or transchromosomal techniques. This allows the animal to express human antibodies, achieving the goal of fully humanizing the antibody. Generally, it refers to antibodies containing therapeutic regions derived from fully human amino acid sequences, where antigen specificity has been selected in vivo using genetically modified mice or through antibody engineering methods with binding screening. Compared to humanized antibodies, fully human antibodies have a lower risk of inducing an immune response in the human body, stronger specific immune effects, and higher ADCC activity, CDC activity, and / or CD39 binding stability.

[0067] The term "KD value" refers to the equilibrium dissociation constant between an antibody and its antigen, specifically the koff / kon or kd / ka ratio (measured using SPR technology). Therefore, a lower KD value (lower concentration) indicates higher antibody affinity. Thus, the "KD value" can be used to measure the binding affinity between an antibody and its antigen.

[0068] The terms “CD39” and “CD39 antigen” are used interchangeably herein and include any variant, isotype, and species homologue of human CD39 expressed naturally in cells or on cells transfected with the CD39 gene. In some embodiments, the binding of the antibody of this disclosure to the CD39 antigen mediates the killing of CD39-expressing cells (e.g., tumor cells) by inactivating CD39. The killing of CD39-expressing cells can occur through one or more of the following mechanisms: cell death / apoptosis induction, ADCC, and CDC.

[0069] The term “anti-CD39 antibody” or “CD39 antibody” refers to an antibody, as defined herein, that is capable of binding to the CD39 antigen or to cells expressing CD39.

[0070] The term "specific binding" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and the antigen it targets.

[0071] The term "separated" refers to a state obtained artificially from the natural state. If a "separated" substance or component exists naturally, it may be due to changes in its natural environment, separation of the substance from its natural environment, or both. For example, an unseparated polynucleotide or polypeptide naturally exists within a living organism; a high-purity copy of the same polynucleotide or polypeptide separated from that natural state is called a separated polynucleotide or polypeptide. The term "separated" does not exclude the presence of artificial or synthetic substances, nor does it exclude other impurities that do not affect the activity of the separated substance. For example, a separated antibody may be substantially free of other cellular material and / or chemicals.

[0072] The term "vector" refers to a nucleic acid medium in which polynucleotides can be inserted. When a vector allows the expression of a protein encoded by the polynucleotide inserted therein, the vector is called an expression vector. This vector can be used to express the carried genetic material elements in host cells through transformation, transduction, or transfection. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids, bacteriophages, granules, artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and multivacuolar papillomaviruses (such as SV40). Vectors may contain multiple elements for controlling expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, vectors may contain an origin of replication. For the vector expressing the antibody, a vector type in which the antibody heavy chain and light chain exist in different vectors or a vector type in which the heavy chain and light chain exist in the same vector can be used.

[0073] The term "host cell" refers to a cellular system that can be engineered to produce a target protein, protein fragment, or peptide. Host cells include, but are not limited to, cultured cells, such as mammalian cultured cells derived from rodents (rats, mice, guinea pigs, or hamsters), such as CHO, BHK, NSO, SP2 / 0, YB2 / 0; or human tissue or hybridoma cells, yeast cells, and insect cells, as well as cells contained within transgenic animals or cultured tissues. The term covers not only the specific test cell but also its progeny. Because certain modifications can occur in subsequent generations due to mutations or environmental influences, such progeny may differ from the parent cell but are still included within the scope of the term "host cell."

[0074] The term "identity" refers to the relationship between the sequences of two or more polypeptide (or protein) molecules or two or more nucleic acid molecules, determined by alignment and comparison of sequences. "Percentage identity" refers to the percentage of identical residues among amino acids or nucleotides in the compared molecules, calculated based on the size of the smallest molecule being compared. For these calculations, gaps in the alignment (if any) are preferably addressed using a specific mathematical model or computer program (i.e., an "algorithm"). Methods that can be used to calculate the identity of aligned nucleic acids or peptides include those described in Computational Molecular Biology (Lesk, AM, ed.), 1988, New York: Oxford University Press; Biocomputing Informatics and Genome Projects (Smith, DW, ed.), 1993, New York: Academic Press; Computer Analysis of Sequence Data, Part I (Griffin, AM, and Griffin, HG, eds.), 1994, New Jersey: Humana Press; von Heinje, G., 1987, Sequence Analysis in Molecular Biology, New York: Academic Press; Sequence Analysis Primer (Gribskov, M. and Devereux, J., eds.), 1991, New York: M. Stockton Press; and Carillo et al., 1988, SIAM J. Applied Math. 48:1073.

[0075] The term "immunogenicity" refers to the ability of an organism to stimulate the formation of specific antibodies or sensitized lymphocytes. It refers not only to the property of an antigen to stimulate the activation, proliferation, and differentiation of specific immune cells to ultimately produce immune effector substances such as antibodies and sensitized lymphocytes, but also to the specific immune response of antibodies or sensitized T lymphocytes that can be formed in the organism's immune system after stimulation with an antigen. Immunogenicity is the most important characteristic of an antigen. Whether an antigen can successfully induce an immune response in the host depends on three factors: the nature of the antigen, the host's reactivity, and the immunization method.

[0076] The term “transfection” refers to the process of introducing nucleic acids into eukaryotic cells, particularly mammalian cells. Protocols and techniques used for transfection include, but are not limited to, lipid transfection and chemical and physical methods such as electroporation. Many transfection techniques are well known in the art and are disclosed herein. See, for example, Graham et al., 1973, Virology 52:456; Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual, ibid.; Davis et al., 1986, Basic Methods in Molecular Biology, Elsevier; Chu et al., 1981, Gene 13:197.

[0077] The terms "hybridoma" and "hybridoma cell line" are used interchangeably. When referring to the terms "hybridoma" and "hybridoma cell line," they also include subclones and progeny cells of the hybridoma.

[0078] The term "immune effector function" includes any function mediated by components of the immune system that results in the inhibition of tumor growth and / or tumorigenesis, as well as the inhibition of tumor dissemination and metastasis. Preferably, the immune effector function results in the killing of tumor cells. Preferably, the immune effector function in this invention is an antibody-mediated effector function. Such functions include complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), induction of apoptosis in cells carrying tumor-associated antigens (e.g., by binding of antibodies to surface antigens), and / or inhibition of the proliferation of cells carrying tumor-associated antigens, preferably ADCC and / or CDC. Antibodies can also exert their effects simply by binding to tumor-associated antigens on the surface of tumor cells. For example, antibodies can block the function of tumor-associated antigens or induce apoptosis simply by binding to tumor-associated antigens on the surface of tumor cells.

[0079] The term "cancer" refers to any tumor or malignant cell growth, proliferation, or metastasis that causes a medical condition, including solid tumors and non-solid tumors such as leukemia. For example, cancers associated with or caused by abnormal CD39 expression include, but are not limited to: lymphoma, sarcoma, lung cancer, pancreatic cancer, ovarian cancer, renal cell carcinoma, thyroid cancer, and testicular cancer.

[0080] The term "pharmaceutically acceptable" means that the carrier, diluent, excipient and / or salt thereof is chemically and / or physically compatible with other components in the formulation and physiologically compatible with the recipient.

[0081] The term "pharmaceutically acceptable carrier and / or excipient" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active agent, and is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19). th (ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to, pH adjusters, surfactants, adjuvants, and ionic strength enhancers. For example, pH adjusters include, but are not limited to, phosphate buffers; surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80; and ionic strength enhancers include, but are not limited to, sodium chloride.

[0082] The term "adjuvant" refers to a nonspecific immune enhancer that, when delivered to an organism along with or before an antigen, can enhance the organism's immune response to the antigen or alter the type of immune response. Various adjuvants exist, including but not limited to aluminum adjuvants (e.g., aluminum hydroxide), Freund's adjuvants (e.g., complete and incomplete Freund's adjuvants), Corynebacterium breve, lipopolysaccharides, and cytokines. Freund's adjuvant is currently the most commonly used adjuvant in animal experiments. Aluminum hydroxide adjuvant is more commonly used in clinical trials.

[0083] Anti-CD39 antibody

[0084] In some aspects, the present invention includes isolated antibodies or antigen-binding fragments thereof.

[0085] In the context of this application, "antibody" can include polyclonal antibodies, monoclonal antibodies, chimeric antibodies, humanized and primate-derived antibodies, CDR transplantation antibodies, human antibodies, recombinant antibodies, intracellular antibodies, bifunctional antibodies, multispecific antibodies, monovalent antibodies, multivalent antibodies, anti-idiotype antibodies, synthetic antibodies, including mutant proteins and variants thereof, modified antibodies; and derivatives thereof (including Fc fusion proteins and other modifications), as well as any other immunoreactive molecule that exhibits preferential association or binding to the CD39 protein. Furthermore, unless the context otherwise requires, the term also includes all classes of antibodies (i.e., IgA, IgD, IgE, IgG, and IgM) and all subclasses (i.e., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2). In a preferred embodiment, the antibody is a monoclonal antibody. In a more preferred embodiment, the antibody is a chimeric monoclonal antibody or a humanized monoclonal antibody or a modified chimeric monoclonal antibody.

[0086] Variable regions and CDRs in antibody sequences can be identified according to a numbering system based on general rules already developed in the art (as described above, such as Kabat) or by comparing the sequence with a database of known variable regions.

[0087] Regardless of how the antibody is produced, methods for testing the ability of an antibody to bind to an antigen (e.g., CD39) are known in the art and include any antibody-antigen binding assay, such as radioimmunoassay (RIA), ELISA, Western blotting, immunoprecipitation, SPR, and competitive inhibition assays (see, for example, Janeway et al., below and U.S. Patent Application Publication No. 2002 / 0197266 and the above sections concerning competitive assays).

[0088] According to the present invention, in a standard assay (e.g., the assay described herein), if an antibody has a significant affinity for a predetermined target (e.g., CD39 protein or cells expressing CD39), then the antibody is capable of binding to the predetermined target. Flow cytometry (FCM) can be used to test the binding of a monoclonal antibody to live cells expressing CD39. Preferably, in a flow cytometry fluorescence sorting (FACS) analysis, the binding of the antibody to a target expressed on the cell surface is measured. If the antibody detectably binds to the target (CD39 protein or cells expressing CD39), then the antibody is capable of binding to the target and has "affinity".

[0089] The CD39 specificity described in this invention refers to the ability to bind to one or more CD39 epitopes, especially CD39 epitopes in their natural conformation, particularly human CD39 specificity.

[0090] In this field, various methods are employed to modify antibodies without altering their desired properties, such as the recombination of the light and heavy chains of the antibody or the substitution of amino acids, as used in this disclosure. For example, the sequences in this invention, including chimeric antibody sequences or humanized antibody sequences, can be modified by making conserved amino acid substitutions.

[0091] Antibodies primarily interact with target antigens through amino acid residues located in the complementarity-determining regions (CDRs) of the six heavy and light chains. For this reason, the amino acid sequences of the CDRs are more diverse among antibodies than other sequences. Since the CDR sequence is responsible for most antibody-antigen interactions, it is possible to express recombinant antibodies that mimic the properties of a specific naturally occurring antibody by constructing expression vectors containing the CDR sequence from that specific naturally occurring antibody, which are then grafted onto frame sequences from different antibodies with different properties (see, for example, Riechmann, L. et al. (1998) Nature 332:323-327; Jones, P. et al. (1986) Nature 321:522-525; and Queen, C. et al. (1989) Proc. Natl. Acad. Sci. USA 86:10029-10033). Such frame sequences are available from public DNA databases that include germline antibody gene sequences. These germline sequences differ from the mature antibody gene sequences because they do not contain the fully assembled variable gene, which is formed during B cell maturation via V(D)J linkage. The germline gene sequences will also have sequences that differ from those of the high-affinity secondary repertoire antibody at individual locations that uniformly traverse the variable region.

[0092] Mouse antibodies exhibit high immunogenicity in humans, leading to decreased therapeutic efficacy with repeated administration. The primary immunogenicity is mediated by the heavy chain constant region. However, the immunogenicity of mouse antibodies in humans can be reduced or completely avoided by chimeric or humanized versions of the antibodies.

[0093] Chimeric antibodies are antibodies whose different parts originate from different animal species, for example, antibodies having a variable region derived from a mouse antibody and a constant region from a human immunoglobulin. Chimeric antibodies are obtained by linking the variable regions of the mouse antibody heavy and light chains to the constant regions of the human heavy and light chains (e.g., as described by Kraus et al., in Methods in Molecular Biology series, Recombinant antibodies for cancer therapy ISBN-0-89603-918-8). In a preferred embodiment, chimeric antibodies are generated by linking the constant region of the human κ light chain to the variable region of the mouse light chain. In another preferred embodiment, chimeric antibodies are generated by linking the constant region of the human λ light chain to the variable region of the mouse light chain.

[0094] Humanized antibodies are antibodies that have had their CDR sequences / antigen-binding portions or sites derived from another mammalian species, such as mice, transplanted onto a human frame sequence.

[0095] To reduce the immunogenicity of antibodies to humans, humanized anti-CD39 antibodies are produced using the CD39 antibody sequence of this disclosure. The CDR region of a mouse anti-CD39 antibody is combined with a human framework region (e.g., human immunoglobulin) to form the humanized anti-CD39 antibody of this disclosure. The humanized antibody is expected to retain the function of binding to human CD39 as well as the function of binding to monkey CD39.

[0096] Antibody preparation or production

[0097] The antibodies of this invention can be generated using various techniques, including conventional monoclonal antibody methods, such as the standard somatic cell hybridization technique described in Kohler and Milstein, Nature 256:495 (1975). While hybridoma technology is preferred, other techniques for generating monoclonal antibodies can be used in principle, such as viral or oncogene transformation of B lymphocytes or phage display using antibody gene libraries, somatic cell hybridization, and, for example, genetic engineering recombination techniques. For instance, DNA molecules encoding the heavy and light chain genes of the antibodies of this invention can be obtained through chemical synthesis or PCR amplification, the resulting DNA molecules can be inserted into an expression vector, and then transfected into host cells. The transfected host cells can then be cultured under specific conditions to express the antibodies of this invention.

[0098] Other preferred animal systems for preparing hybridomas that secrete monoclonal antibodies are the rat and rabbit systems (e.g., described in Spieker-Polet et al., Proc. Natl. Acad. Sci. USA 92: 9348 (1995), see also Rossie et al. Am. J. Clin. Pathol. 124: 295 (2005)). Hybridoma production in mice is a well-established method. Immunization protocols and techniques for isolating immunized spleen cells for fusion are known in the art. Fusion partners (e.g., mouse myeloma cells) and fusion methods are also known.

[0099] Monoclonal antibodies can be prepared using a variety of techniques known in the art, including hybridoma techniques, recombinant techniques, phage display techniques, transgenic animals, or combinations thereof. For example, monoclonal antibodies can be produced using hybridomas and well-established biochemical and genetic engineering techniques, as described in detail in An, Zhiqiang (ed.) Therapeutic Monoclonal Antibodies: From Bench to Clinic, John Wiley and Sons, 1st ed. 2009; Shire et al. (eds.) Current Trends in Monoclonal Antibody Development and Manufacturing, Springer Science+Business Media LLC, 1st ed. 2010; Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 2nd ed. 1988; Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, NY, 1981), each of which is incorporated herein by reference in its entirety.

[0100] It should be understood that the selected binding sequence can be further modified, for example, to increase the affinity for the target, humanize the target binding sequence, improve its production in cell cultures, reduce its immunogenicity in vivo, generate multispecific antibodies, etc., and antibodies containing modified target binding sequences are also antibodies of the present invention.

[0101] In some embodiments, a method for producing the antibodies or fragments described in this disclosure includes the following steps:

[0102] (i) expressing the antibody or fragment in a host cell; and optionally...

[0103] (ii) Isolate the antibody or its antigen-binding fragment from the host cell.

[0104] In a preferred embodiment, anti-CD39 monoclonal antibodies are prepared by using hybridomas.

[0105] To obtain hybridomas that produce the antibodies of the present invention, such as the human monoclonal antibodies of the present invention, spleen cells and / or lymph node cells from immunized mice can be isolated and fused into a suitable immortalized cell line, such as a mouse myeloma cell line. The resulting hybridomas are screened for the production of antigen-specific antibodies. The generation of hybridomas is well known in the art. See, for example, Harlow and Lane (1988), Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York.

[0106] The antibodies of the present invention can also be generated in host cells transfected with tumors using, for example, a combination of recombinant DNA techniques and gene transfection methods well known in the art (e.g., Morrison, S. (1985) Science 229:1202). In some embodiments, DNA encoding a portion or the full length of the light and heavy chains, obtained by standard molecular biology techniques, is inserted into one or more expression vectors such that the gene is operatively linked to transcriptional and translational regulatory sequences. In this context, the term "operatively linked" is intended to mean linking the antibody gene to a vector such that the transcriptional and translational control sequences within the vector perform their intended functions of regulating the transcription and translation of the antibody gene.

[0107] Antibody light chain genes and antibody heavy chain genes can be inserted into the same or different expression vectors. In some embodiments, the variable region is used to generate a full-length antibody gene of any antibody isotype by inserting it into an expression vector that already encodes the heavy chain constant region and light chain constant region of the desired isotype, such that the VH segment is operatively linked to the CH segment within the vector and the VL segment is operatively linked to the CL segment within the vector. Alternatively or additionally, the recombinant expression vector can encode a signal peptide that promotes the secretion of the antibody chain from the host cell. The antibody chain gene can be cloned into the vector such that the signal peptide is linked to the N-terminus of the antibody chain gene. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide derived from a non-immunoglobulin protein).

[0108] To express the light and heavy chains, expression vectors encoding the heavy and light chains are transfected into host cells using standard techniques. Various forms of the term "transfection" are intended to encompass a wide range of techniques commonly used to introduce exogenous DNA into prokaryotic or eukaryotic host cells, such as electroporation, calcium phosphate precipitation, DEAE-glucan transfection, etc. The antibodies of the present invention can be expressed in prokaryotic or eukaryotic host cells, such as mammalian host cells (which can assemble and secrete antibodies with appropriate folding and immunological activity).

[0109] Mammalian host cells used to express the recombinant antibodies of the present invention include Chinese hamster ovary cells (CHO cells) (including dhfr CHO cells described in Urlaub and Chasin, (1980) Proc. Natl. Acad. Sci. USA 77: 4216-4220) used with DHFR selection markers (e.g., as described in RJ Kaufman and PA Sharp (1982) J. MoI. Biol. 159: 601-621), NSO myeloma cells, COS cells, and SP2 cells. In particular, for use with NSO myeloma, another expression system is the GS gene expression system disclosed in WO 87 / 04462, WO 89 / 01036, and EP 338,841. When the recombinant expression vector encoding the antibody gene is introduced into mammalian host cells, the antibody is produced by culturing the host cells for a period sufficient to allow antibody expression in the host cells or by secreting the antibody into the culture medium in which the host cells grow. The antibody can be recovered from the culture medium using standard protein purification methods.

[0110] In another preferred embodiment, transgenic or transchromatic mice with a partial human immune system (rather than a mouse system) can be used to generate human monoclonal antibodies against CD39.

[0111] Another strategy for generating monoclonal antibodies is to directly isolate the antibody-encoding gene from the antibody-producing lymphocytes of a defined strategy, for example, see Babcocketal., 1996; A novel strategy for generating monoclonal antibodies from single, isolated lymphocytes producing antibodies of a defined strategy. For details on recombinant antibody engineering, see Welschof and Krau, Recombinant Antibodies for Cancer Therapy ISBN-0-89603-918-8 and Benny KCLo Antibody Engineering ISBN 1-58829-092-1.

[0112] To prepare chimeric antibodies, the variable region of mouse immunoglobulin can be ligated to the constant region of human immunoglobulin using methods known in the art (see, for example, US Patent 4,816,567, Cabilly et al.). A separate nucleic acid encoding the VH region can be converted into a full-length heavy chain gene by operably ligating a nucleic acid encoding VH to another DNA molecule encoding the heavy chain constant region (CH1, CH2, and CH3). Sequences of human heavy chain constant region genes are known in the art (see, for example, Kabat et al. (1991), Sequences Of Proteins of Immunological Interest, Fifth Edition, USDapartment of Health and Human Services, NIH Publication No. 91-3242). The heavy chain constant region can be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD constant region, but more preferably an IgG1 or IgG4 constant region. A separate nucleic acid encoding the VL region can be converted into a full-length light chain gene (and a Fab light chain gene) by operably ligating a DNA molecule encoding the light chain constant region CL to another DNA molecule encoding the light chain constant region CL. The sequences of human light chain constant region genes are known in the art (see, for example, Kabat et al., ibid.), and DNA fragments containing these regions can be obtained by standard PCR amplification. In a preferred embodiment, the light chain constant region may be a κ or λ constant region, but is generally preferred to be a κ constant region. Once the DNA fragments encoding the VH and VL regions are obtained, these DNA fragments can be further manipulated using standard recombinant DNA techniques, such as converting the variable region gene into a full-length antibody chain gene, a Fab fragment gene, or an scFv gene. In these manipulations, the DNA fragment encoding VL or VH is operatively ligated to another DNA fragment encoding a different protein, such as an antibody constant region or a flexible linker. The term “operatively ligated” as used herein is intended to mean that two DNA fragments are ligated such that the amino acid sequences encoded by both DNA fragments remain within the frame.

[0113] To prepare humanized antibodies, mouse CDR regions can be inserted into human framework sequences using methods known in the art (see Winter's US 5,225,539; Queen et al.'s US 5,530,101; US ​​5,585,089; US 5,693,762; and Lo, Benny, KC, editor, in Antibody Engineering: Methods and Protocols, volume 248, Humana Press, New Jersey, 2004). Alternatively, transgenic animals that do not produce endogenous immunoglobulins after immunization and can generate a complete human antibody library can be used. For example, it has been reported that homozygous deletion of the antibody heavy chain linker (JH) gene in chimeric and germline mutant mice can completely suppress the production of endogenous antibodies, and then transfer of human germline immunoglobulin gene arrays into said germline mutant mice will cause the mice to produce human antibodies upon encountering antigen stimulation (see, for example, Jakobovits et al., 1993, Proc. Natl. Acad. Sci. USA 90: 2551; Jakobovits et al., 1993, Nature 362: 255-258; Bruggermann et al., 1993, Year in Immunology 7: 33; and Duchosal et al., 1992, Nature 355: 258). Non-limiting examples of the aforementioned transgenic animals include the HuMAb mouse (Medarex, Inc.) containing a miniloci of the human immunoglobulin gene encoding unrearranged human heavy chain (μ and γ) and κ light chain immunoglobulin sequences, coupled with targeted mutations that inactivate the endogenous μ and κ chain loci (see, for example, Lonberg et al. (1994) Nature 368(6474):856-859); or the “KM mouse™” carrying human heavy chain transgenes and human light chain transchromosomes (see patent application WO02 / 43478). Other methods of antibody humanization include phage display technology (Hoogenboom et al., 1991, J. Mol. Biol. 227:381; Marks et al., J. Mol. Biol. 1991, 222:581-597; Vaughan et al., 1996, Nature Biotech 14:309).

[0114] Nucleic acid molecules encoding the antibodies of this invention

[0115] In some aspects, the present invention relates to isolated nucleic acid molecules comprising nucleic acid sequences encoding isolated antibodies or fragments thereof as disclosed herein.

[0116] The nucleic acids of this invention can be obtained using standard molecular biology techniques. For antibodies expressed by hybridomas (e.g., hybridomas prepared from transgenic mice carrying human immunoglobulin genes, as further described below), the light and heavy chains of the antibody prepared via hybridoma can be obtained by standard PCR amplification or cDNA cloning techniques. For antibodies obtained from immunoglobulin gene libraries (e.g., using phage display technology), the nucleic acid encoding such antibody can be recovered from the gene library.

[0117] To prepare chimeric antibodies, the variable region of a mouse immunoglobulin can be ligated to the constant region of a human immunoglobulin using methods known in the art (see, for example, U.S. Patent No. 4,816,567 to Cabilly et al.). By operatively ligating a nucleic acid encoding VH to another DNA molecule encoding the heavy chain constant region (CH1, CH2, and CH3), isolated nucleic acids encoding the VH region can be converted into a full-length heavy chain gene, and DNA fragments containing these regions can be obtained by standard PCR amplification. By operatively ligating DNA encoding VL to another DNA molecule encoding the light chain constant region CL, isolated nucleic acids encoding the VL region can be converted into a full-length light chain gene (and a Fab light chain gene). Once DNA fragments encoding the VH and VL regions are obtained, these DNA fragments can be further manipulated using standard recombinant DNA techniques, such as converting the variable region gene into a full-length antibody chain gene, a Fab fragment gene, or an scFv gene. In these operations, the DNA fragment encoding VL or VH is operatively ligated to another DNA fragment encoding a different protein, such as an antibody constant region or a flexible linker.

[0118] Conjugate

[0119] On one hand, this disclosure provides a conjugate comprising an antibody or fragment thereof as described above, conjugated to at least one detectable marker. The detectable marker includes, but is not limited to: (i) providing a detectable signal; (ii) interacting with a second marker to modify the detectable signal provided by the first or second marker, such as FRET (Fluorescence Resonance Energy Transfer); (iii) influencing mobility (e.g., electrophoretic mobility) through charge, hydrophobicity, shape, or other physical parameters; or (iv) providing a trapping motif, such as affinity, antibody / antigen, or ion complexation.

[0120] Suitable structures for labeling include fluorescent labels, luminescent labels, chromophore labels, radioisotope labels, isotope labels, preferably stable isotope labels, isobaric labels, enzyme labels (e.g., horseradish peroxidase, alkaline phosphatase, β-galactosidase, urease, glucose oxidase, etc.), particulate labels (especially metal particulate labels, magnetic particulate labels, polymer particulate labels), and small organic molecules (e.g., biotin, receptor ligands or binding molecules (e.g., cell adhesion proteins or lecithin), which can be bound by... The reagents detect marker sequences containing nucleic acid and / or amino acid residues. The markers, without limitation, include barium sulfate, iodopic acid, iodopic acid, calcium amiodarone propionate, sodium diatrizoate, meglumine diatrizoate, meglumine methyl diatrizoate, sodium caseinate, and radiodiagnostic agents (including positron emitters (e.g., fluorine-18 and carbon-11), gamma emitters (e.g., iodine-123, iodine-125, technetium-99m, iodine-131, and indium-111), nuclear magnetic resonance isotopes (e.g., fluorine and gadolinium)), luminescent substances (e.g., isoluminol and acridine ester), fluorescent substances (e.g., fluorescein and rhodamine), and colored substances (e.g., latex particles and colloidal gold).

[0121] The detectable markers described above can be detected by methods known in the art. For example, fluorescent markers can be detected using a photodetector to detect emitted light. Enzyme markers are generally detected by providing a substrate to an enzyme and detecting the reaction product produced by the enzyme's action on the substrate. In some embodiments, such markers can be used for immunological assays (e.g., enzyme-linked immunosorbent assay, radioimmunoassay, fluorescence immunoassay, chemiluminescence immunoassay, etc.). In some embodiments, the detectable markers described above can be linked to the antibodies or antigen-binding fragments of the present invention using linkers of varying lengths to reduce potential steric hindrance.

[0122] Antibody drug conjugates / immunoconjugates

[0123] On one hand, this disclosure provides an antibody-drug conjugate comprising one or more drug portions / therapeutic agents, said drug portions being directly or via a linker (e.g., covalently linked) to the antibody or fragment thereof as described above. In the antibody-drug conjugates of this application, there are no particular limitations on the linker structure for conjugating the anti-CD39 antibody to the drug, as long as the resulting antibody-drug conjugate can be used.

[0124] Because antibody-drug conjugates have the ability to selectively deliver one or more drugs to target tissues (e.g., tumor-associated antigens, such as tumors expressing CD39), antibody-drug conjugates can enhance the therapeutic efficacy of the antibodies or antigen-binding fragments of the present invention in treating diseases (e.g., cancer).

[0125] Multispecific molecules

[0126] The antibodies or antigen-binding fragments of the present invention can be used to form multispecific molecules (e.g., bispecific molecules). The antibodies or antigen-binding fragments of the present invention can be part of a multispecific molecule (e.g., a bispecific molecule) comprising a second functional module (e.g., a second antibody) or a third functional module (e.g., a third antibody) having a binding specificity different from that of the antibodies or antigen-binding fragments of the present invention, thereby enabling binding to at least two different binding sites and / or target molecules. For example, the antibodies or antigen-binding fragments of the present invention can be linked to a second antibody or antigen-binding fragment capable of specifically binding to any protein that can be used as a potential target for combination therapy. To generate said bispecific or multispecific molecules, the antibodies or antigen-binding fragments of the present invention can be linked (e.g., by chemical coupling, gene fusion, non-covalent association, or other means) to one or more other binding molecules (e.g., additional antibodies, antibody fragments, peptides, or binding mimics).

[0127] Therefore, in some aspects, the present invention provides a multispecific molecule comprising the antibody or antigen-binding fragment of the present invention.

[0128] In some preferred embodiments, the multispecific molecule specifically binds to CD39 (e.g., human CD39 or monkey CD39) and specifically binds to one or more other targets.

[0129] In some preferred embodiments, the multispecific molecule further comprises at least one molecule (e.g., a second antibody) having a second binding specificity against a second target.

[0130] In some preferred embodiments, the multispecific molecule is a bispecific antibody.

[0131] Pharmaceutical Composition

[0132] In some aspects, the present invention relates to pharmaceutical compositions, and this disclosure provides a pharmaceutical composition or kit comprising, as described above, an antibody or fragment, a nucleic acid molecule, a carrier, a host cell, a conjugate, an antibody-drug conjugate, a multispecific molecule, as described above; and a pharmaceutically acceptable carrier.

[0133] The pharmaceutical composition may optionally contain one or more additional pharmaceutically active ingredients, such as another antibody or drug. The pharmaceutical compositions of the present invention may also be administered in combination with, for example, another immunostimulant, anticancer agent, antiviral agent, or vaccine, such that the anti-CD39 antibody enhances the immune response to the vaccine. Pharmaceutically acceptable carriers may include, for example, pharmaceutically acceptable liquid, gel, or solid carriers, aqueous media, non-aqueous media, antimicrobial agents, isotonic agents, buffers, antioxidants, anesthetics, suspending / dispersing agents, chelating agents, diluents, adjuvants, excipients, or non-toxic excipients, combinations or more of various components known in the art.

[0134] Suitable components may include, for example, antioxidants, fillers, binders, disintegrants, buffers, preservatives, lubricants, flavorings, thickeners, colorants, emulsifiers, or stabilizers such as sugars and cyclodextrins. Suitable antioxidants may include, for example, methionine, ascorbic acid, EDTA, sodium thiosulfate, platinum, catalase, citric acid, cysteine, mercaptoglycerol, mercaptoacetic acid, mercaptosorbitol, butylated methyl anisole, butylated hydroxytoluene, and / or propyl arsenate. As disclosed in this invention, antibodies containing compositions disclosed herein can be oxidized in solvents containing one or more antioxidants such as methionine, which are reducing antibodies or antigen-binding fragments thereof. Redox reactions can prevent or reduce the decrease in binding affinity, thereby enhancing antibody stability and extending shelf life. Therefore, in some embodiments, this invention provides compositions comprising one or more antibodies or antigen-binding fragments thereof and one or more antioxidants such as methionine. The present invention further provides various methods in which an antibody or its antigen-binding fragment is mixed with one or more antioxidants such as methionine, thereby preventing the antibody or its antigen-binding fragment from oxidation, thereby extending its shelf life and / or increasing its activity.

[0135] To further illustrate, pharmaceutically acceptable carriers may include, for example, aqueous media such as sodium chloride injection, Ringer's injection, isotonic dextran injection, sterile water injection, or dextran and lactated Ringer's injection; non-aqueous media such as non-volatile plant-derived oils, cottonseed oil, corn oil, sesame oil, or peanut oil; antimicrobial agents at antibacterial or antifungal concentrations; isotonic agents such as sodium chloride or glucose; buffers such as phosphate or citrate buffers; antioxidants such as sodium bisulfate; local anesthetics such as procaine hydrochloride; suspending and dispersing agents such as sodium carboxymethyl cellulose, hydroxypropyl methylcellulose, or polyvinylpyrrolidone; emulsifiers such as polysorbate 80 (TWEEN-80); isolating agents or chelating agents such as EDTA (ethylenediaminetetraacetic acid) or EGTA (ethylene glycol tetraacetic acid); ethanol; polyethylene glycol; propylene glycol; sodium hydroxide; hydrochloric acid; citric acid; or lactic acid. Antimicrobial agents used as carriers can be added to pharmaceutical compositions containing phenols or cresols, mercury preparations, benzyl alcohol, chlorobutanol, methylparaben and propylparaben, thimerosal, benzalkonium chloride, and benzyl chloride in multi-dose containers. Suitable excipients may include, for example, water, saline, dextran, glycerol, or ethanol. Suitable non-toxic adjuvants may include, for example, wetting agents or emulsifiers, pH buffers, stabilizers, solubility enhancers, or agents such as sodium acetate, sorbitol monolaurate, triethanolamine oleate, or cyclodextrin.

[0136] Application, formulation and dosage

[0137] The pharmaceutical compositions of the present invention can be administered to subjects in need via various routes, including but not limited to oral, intravenous, intra-arterial, subcutaneous, parenteral, intranasal, intramuscular, intracranial, intracardiac, intraventricular, intratracheal, oral, rectal, intraperitoneal, intradermal, topical, percutaneous and intrathecal, or implantation or inhalation. The compositions of the present invention can be formulated into solid, semi-solid, liquid, or gaseous forms; including but not limited to tablets, capsules, powders, granules, ointments, solutions, suppositories, enemas, injections, inhalers, and aerosols. Appropriate formulations and routes of administration can be selected based on the intended application and treatment regimen.

[0138] Suitable formulations for enteral administration include hard or soft gelatin capsules, pills, tablets (including coated tablets), elixirs, suspensions, syrups, or inhalers and their controlled-release formulations.

[0139] Formulations suitable for parenteral administration (e.g., by injection) include aqueous or non-aqueous, isotonic, pyrogen-free, sterile liquids (e.g., solutions, suspensions) in which the active ingredient is dissolved, suspended, or otherwise provided (e.g., in liposomes or other microparticles). These liquids may additionally contain other pharmaceutically acceptable components, such as antioxidants, buffers, preservatives, stabilizers, antibacterial agents, suspending agents, thickeners, and solutes that make the formulation isotonic with the intended recipient's blood (or other relevant bodily fluids). Examples of excipients include, for example, water, alcohols, polyols, glycerol, vegetable oils, etc. Examples of isotonic carriers suitable for such formulations include sodium chloride injection, Ringer's solution, or lactated Ringer's solution. Similarly, specific dosing regimens (including dose, time, and repetition) will depend on the specific individual and their medical history, as well as empirical considerations such as pharmacokinetic parameters (e.g., half-life, clearance, etc.).

[0140] The requirements for effective drug carriers for injectable formulations / compositions are well known to those skilled in the art (see, for example, Pharmaceutics and Pharmacy Practice, JBLippincott Company, Philadelphia, PA, edited by Banker and Chalmers, pp. 238-250 (1982), and ASHP Handbook on Injectable Drugs, Toissel, 4th edition, pp. 622-630 (1986)).

[0141] The frequency of administration can be determined and adjusted during treatment, and is based on reducing the number of proliferating or tumorigenic cells, maintaining this reduction in tumor cells, reducing tumor cell proliferation, or delaying the development of metastasis. In some embodiments, the administered dose can be adjusted or reduced to control potential side effects and / or toxicity. Alternatively, a continuously releasing formulation of the therapeutic composition of the present invention may be suitable.

[0142] Those skilled in the art will understand that appropriate dosages can vary from patient to patient. Determining the optimal dosage typically involves balancing the level of therapeutic benefit with any risks or adverse side effects. The chosen dosage level will depend on a variety of factors, including, but not limited to, the activity of the specific compound, administration, timing of administration, compound clearance rate, duration of treatment, other drugs, compounds and / or materials used in combination, severity of the condition, and species, the patient's sex, age, weight, condition, general health status, and medical history. However, a dosage is generally chosen to achieve a local concentration at the site of action to achieve the desired effect without causing substantial harmful or adverse side effects.

[0143] Generally, the antibodies or antigen-binding fragments of the present invention can be applied in a variety of applications.

[0144] In some preferred embodiments, the treatment process involving the antibody or antigen-binding fragment thereof of the present invention will comprise multiple doses of the selected pharmaceutical product administered over a period of weeks or months. More specifically, the antibody or antigen-binding fragment thereof of the present invention may be administered daily, every two days, every four days, weekly, every ten days, every two weeks, every three weeks, monthly, every six weeks, every two months, every ten weeks, or every three months. In this regard, it is understood that the dosage or interval may be varied or adjusted based on patient response and clinical practice.

[0145] Compatible formulations intended for parenteral administration (e.g., intravenous injection) will contain an antibody or antigen-binding fragment thereof as disclosed herein at a concentration of about 5 μg / mL to about 100 mg / mL.

[0146] The antibody of the present invention can be co-administered with one or more therapeutic agents (e.g., cytotoxic agents, radiotoxic agents, antitumor agents, anti-angiogenic agents, or immunosuppressants) to reduce the induction of an immune response against the antibody of the present invention. The antibody can be conjugated to the therapeutic agent (as an immune complex) or administered separately from the therapeutic agent.

[0147] In the context of treatment administration, the terms "combination" or "co-administration" as used herein refer to the use of more than one treatment or therapeutic agent. The use of the term "combination" does not limit the order in which treatments or therapeutic agents are administered to the subject. Treatments or therapeutic agents may be administered before, simultaneously with, or after administering a second treatment or therapeutic agent to the patient. Preferably, treatments or therapeutic agents are administered to the subject in a specific order, amount, and / or at specific time intervals so that the treatments or therapeutic agents can work together. In one specific embodiment, treatments or therapeutic agents are administered to the subject in a specific order, amount, and / or at specific time intervals so that they provide an increased benefit compared to if administered in other ways (particularly independently of each other). Preferably, the increased benefit is a synergistic effect.

[0148] Medical Use

[0149] The antibodies, antibody compositions, and methods of the present invention have numerous in vitro and in vivo uses, including, for example, the detection of CD39 or the enhancement of immune responses. For example, these molecules can be administered in vitro or ex vivo to cultured cells, or, for example, in vivo to human subjects.

[0150] Preferred subjects include mammals, such as humans / patients. In the context of this invention, mammals include humans, non-human primates, domesticated animals such as dogs, cats, sheep, cattle, goats, pigs, horses, etc., laboratory animals such as mice, rats, rabbits, Guinea pigs, etc., and captive animals, such as zoo animals.

[0151] Treatment of diseases related to CD39 expression

[0152] In some aspects, the present invention provides a method for treating a disease in mammals, comprising administering a therapeutically effective amount of an antibody or antigen-binding fragment thereof disclosed herein to a subject (e.g., a human) requiring treatment.

[0153] As described herein, the disclosed antibodies possess one or more activities that can be therapeutically applied to kill and / or inhibit cell proliferation. In particular, they can be used to kill cells, inhibit cell proliferation, and / or inhibit cell colony formation for the treatment or prevention of cancer (including cancer metastasis). Inhibition of cell proliferation, colony formation, and / or metastasis can be applied, especially for the treatment or prevention of cancer metastasis and the metastatic spread of cancer cells.

[0154] In some aspects, this disclosure provides a method for treating a disease associated with CD39 expression or determining its prognosis in a subject, comprising administering an effective dose of the antibody or its antigen-binding fragment, the nucleic acid molecule, the vector, the host cell, the conjugate, the antibody-drug conjugate, the multispecific molecule, or the pharmaceutical composition or kit to the desired subject.

[0155] In some aspects, this disclosure provides an antibody or antigen-binding fragment thereof, a nucleic acid molecule, a vector, a host cell, a conjugate, an antibody-drug conjugate, a multispecific molecule, or a pharmaceutical composition or kit in a method for treating a disease associated with CD39 expression or determining its prognosis in a subject.

[0156] In some aspects, this disclosure provides the use of the antibody or its antigen-binding fragment, the nucleic acid molecule, the vector, the host cell, the conjugate, the antibody-drug conjugate, the multispecific molecule, or the pharmaceutical composition or kit in the preparation of reagents (or drugs) for treating diseases associated with CD39 expression or determining their prognosis.

[0157] In one implementation, diseases associated with CD39 expression include oncological diseases, such as cancer.

[0158] Antibodies or their antigen-binding fragments can be used alone as a monotherapy or in combination with chemotherapy or radiotherapy.

[0159] Antibodies or their antigen-binding fragments can be used in combination with anticancer agents, cytotoxic agents, or chemotherapy agents.

[0160] The terms "anticancer agent" or "antiproliferative agent" refer to any agent that can be used to treat cell-proliferating conditions such as cancer, and include, but are not limited to, cytotoxic agents, cell inhibitors, anti-angiogenic agents, radiotherapy and radiotherapy agents, targeted anticancer agents, BRMs, therapeutic antibodies, cancer vaccines, cytokines, hormone therapy, radiotherapy, antimetastatic agents, and immunotherapy agents. It should be understood that, in selected embodiments as described above, such anticancer agents may comprise conjugates and may be bound to a disclosed site-specific antibody prior to administration. More specifically, in some embodiments, a selected anticancer agent is linked to an unpaired cysteine ​​residue of an engineered antibody to provide an engineered conjugate as described herein. Therefore, such engineered conjugates are explicitly contemplated within the scope of this invention. In other embodiments, the disclosed anticancer agent is administered in combination with a site-specific conjugate comprising the various therapeutic agents described above.

[0161] diagnosis

[0162] This invention provides in vitro and in vivo methods for detecting, diagnosing, or monitoring proliferative disorders, as well as methods for screening cells from patients to identify tumor cells, including tumorigenic cells. Such methods include identifying an individual with cancer for treatment or monitoring cancer progression, including contacting the patient or a sample obtained from the patient (in vivo or in vitro) with an antibody as described herein and detecting the presence or absence of the antibody in the sample, or the binding level, of a bound or free target molecule. In some embodiments, the antibody will comprise a detectable marker or reported molecule as described herein.

[0163] In some aspects, this disclosure provides a method for diagnosing, detecting, or monitoring diseases associated with CD39 expression, comprising administering an effective dose of the antibody or its antigen-binding fragment, the nucleic acid molecule, the vector, the host cell, the conjugate, the antibody-drug conjugate, the multispecific molecule, or the pharmaceutical composition or kit to a desired subject.

[0164] In some aspects, this disclosure provides an antibody or antigen-binding fragment thereof, a nucleic acid molecule, a vector, a host cell, a conjugate, an antibody-drug conjugate, a multispecific molecule, or a pharmaceutical composition or kit in a method for diagnosing, detecting, or monitoring diseases associated with CD39 expression in a subject.

[0165] In another aspect, this disclosure provides the use of the antibody or its antigen-binding fragment, the nucleic acid molecule, the vector, the host cell, the conjugate, the antibody-drug conjugate, the multispecific molecule, or the pharmaceutical composition or kit in the preparation of reagents (or drugs) for diagnosing, detecting or monitoring diseases associated with CD39 expression.

[0166] Samples can be analyzed using a variety of assays, such as radioimmunoassay, enzyme immunoassay (e.g., ELISA), competitive binding assay, fluorescence immunoassay, immunoblotting, Western blot analysis, and flow cytometry. Compatible in vivo diagnostic or diagnostic assays may include imaging or monitoring techniques known in the art, such as magnetic resonance imaging, computed tomography (e.g., CAT scan), positron emission tomography (e.g., PET scan), radiography, ultrasound, etc., as known to those skilled in the art.

[0167] The method described in this invention for detecting or monitoring CD39 expression or the level of CD39-expressing cells in vitro can also be used for non-diagnostic purposes.

[0168] Preferred subjects include mammals, such as people / patients in need.

[0169] The subject's samples are blood, excrement (urine or feces), oral or nasal secretions, or bronchoalveolar lavage fluid, tissue fluid, sweat, or extracts thereof.

[0170] Drug packaging and reagent kits

[0171] Pharmaceutical packages and kits containing one or more containers of an antibody or its antigen-binding fragment thereof are also provided. In some embodiments, a unit dose is provided, wherein the unit dose contains a predetermined amount of a composition comprising, for example, an antibody or its antigen-binding fragment, with or without one or more other reagents. For other embodiments, such a unit dose is supplied in a single-use, pre-filled syringe. In other embodiments, the composition contained in the unit dose may comprise saline, sucrose, or the like; buffers, such as phosphates; and / or formulated within a stable and effective pH range. Alternatively, in some embodiments, the conjugate composition may be provided as a lyophilized powder, which can be reconstituted upon addition of a suitable liquid (e.g., sterile water or saline solution). In some preferred embodiments, the composition comprises one or more substances that inhibit protein aggregation, including but not limited to sucrose and arginine. Any label on or associated with the container indicates that the packaged conjugate composition is intended for the treatment of selected oncological conditions.

[0172] Such kits typically contain a pharmaceutically acceptable formulation of the engineered conjugate in a suitable container, and optionally contain one or more anticancer agents or other pharmaceutical agents in the same or different containers. The kits may also contain other pharmaceutically acceptable formulations for diagnostic or combination therapy.

[0173] More specifically, the kits may have a single container containing the antibody or antigen-binding fragment of the present disclosure, with or without additional components, or they may have different containers for each desired reagent. In the case of providing a combination therapeutic agent for conjugation, a single solution may be premixed in molar equivalents or in a manner where one component is more than another. Alternatively, the conjugates and any optional anticancer agents in the kit may be stored separately in different containers prior to administration to the patient. The kits may also contain a second / third container for containing sterile, pharmaceutically acceptable buffers or other diluents such as sterile water for injection (BWFI), phosphate-buffered saline (PBS), Ringer's solution, and glucose solution.

[0174] When the reagent kit components are provided as one or more liquid solutions, the liquid solutions are preferably aqueous solutions, particularly sterile aqueous solutions or saline solutions. However, the reagent kit components may also be provided as dry powders. When reagents or components are provided in dry powder form, the powder can be reconstituted by adding a suitable solvent. It is conceivable that the solvent may also be provided in a separate container.

[0175] Example

[0176] The invention generally described herein will be more readily understood by referring to the following examples, which are provided by way of illustration and are not intended to limit the invention. Furthermore, unless otherwise specified, the experimental methods in the following examples are conventional methods. Unless otherwise specified, the raw materials, reagents, and other materials used in the following examples are commercially available products.

[0177] Example 1: Generation and screening of immunogenicity and hybridoma

[0178] To generate antibodies against CD39, Balb / c mice were immunized with recombinant human CD39-ECD protein (purchased from ACRO, catalog number CD9-H52H4). Serum samples were collected via tail vein or retro-orbital blood collection during immunization to monitor the immune response. Mice with sufficient titers of anti-CD39 antibodies were used for hybridoma fusion experiments. Spleen cells and lymph node cells from immunized mice were isolated and fused with a mouse myeloma cell line (SP2 / 0). ELISA analysis was performed using the recombinant human CD39-ECD protein, and binding analysis was also performed using CHOZN-hCD39 cells (constructed by our company) stably expressing human CD39 (BD FACSLyric). TMHybridomas capable of producing CD39-specific antibodies were screened. The specificity of the hybridoma clones for Human CD39 was confirmed by FACS and enzyme activity inhibition assays, and they were subcloned to obtain stable hybridoma clones. Antibody-secreting hybridomas were subcloned using a limiting dilution method. After 1-2 rounds of subcloning, monoclonal hybridomas were amplified to produce antibodies. Stable subclones were cultured in vitro to produce antibodies for characterization in tissue culture medium. After approximately 14 days of culture, the hybridoma cell culture medium was collected and purified using a Protein A affinity chromatography (GE) column. The selected hybridoma clone anti-CD39 antibody was named 19.4.

[0179]

[0180] SEQ ID NO:3 19.4 Heavy Chain Full-Length Sequence

[0181] EVQLQQSGAELVRPGASVKLSCTASGFSFKNTYMHWMKQRPEQGLEWIGRIDPVNGNTKYAPKFQ

[0182] GKATITTDTSSNTAYLQLSSLTSEDTAVYFCARDYRFTYWGQGTLVTVSS

[0183] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSS

[0184] VVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL

[0185] MISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLN

[0186] GKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWE

[0187] SNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKSEQID NO:4 19.4 Light chain full-length sequence

[0188] DIQMTQSPSSSLSASLGGKVTITCKASQDINKNIAWYQHKPGKGPRLLIWYTSTLQPGIPSRFSGSGSG

[0189] RDYSFSISNLEPEDIATYYCLQYDNLPYTFGSGTKLEIK

[0190] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTY

[0191] SLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0192] I394 is the reference antibody, derived from patent WO2018167267A1. Its VH is shown in SEQ ID NO:11, its VL is shown in SEQ ID NO:16, the heavy chain sequence is shown in SEQ ID NO:1, and the light chain sequence is shown in SEQ ID NO:2.

[0193] Example 2: Binding affinity with Human CD39 protein

[0194] The binding affinity of the antibody to the Human CD39 protein was determined using an ELISA method.

[0195] Human CD39 protein diluted with coating buffer (0.05M carbonate, pH 9.6) to a final concentration of 0.5 μg / ml was added to each well of a 96-well plate and incubated overnight at 4°C. The plate was washed three times with PBST and blocked with 5% skim milk powder for 1 hour. After washing with PBST, 50 μl of anti-CD39 antibody serially diluted with PBS (initial concentration 3.75 μg / ml, 4-fold dilution, 7 concentration gradients) was added and incubated at room temperature for 1 hour. After washing with PBST, 50 μl of Goat Anti-Human IgG, Fcγ-HRP secondary antibody (purchased from Jackson ImmunoResearch, catalog number 109-035-170) diluted 1:10000 was added and incubated at room temperature for 1 hour. After washing with PBST, 50 μl of TMB was added and incubated at room temperature for 5 minutes. The reaction was terminated by adding stop solution, and the absorbance at OD450 was measured using a microplate reader.

[0196] Example 3: Binding affinity with CHOZN-hCD39

[0197] FACS was used to determine the binding of antibodies to cell lines expressing Human CD39 (CHOZN-hCD39) or, as a negative control, to cells lacking CD39 expression (CHOZN-blank).

[0198] CHOZN-hCD39 and CHOZN-blank cells were cultured in medium. Cells were collected and cultured at 2 × 10⁻⁶ cells / year. 5 Cells were resuspended in blocking buffer at a density of 10 cells / ml. Cells were transferred to 96-well FACS plates at 50 μl / well, centrifuged, and washed twice with FACS buffer (PBS, 1% FBS, 0.05% Tween-20). Serial dilutions of anti-CD39 antibody were prepared 4-fold with FACS buffer, starting at 30 μg / ml. Mouse IgG controls were used as negative controls. Cells were resuspended in 50 μL / well of the diluted antibody, and the plates were incubated at 4°C for 30 min. The plates were washed with FACS buffer, and FITC-labeled secondary antibody (1:1000 in FACS buffer) was added to each well, and the plates were incubated at 4°C for 30 min. The plates were washed with FACS buffer, and cells were resuspended in 50 μL / well of PBS. Then, the plates were treated with BD FACSLyric... TM Cells were analyzed, and mean fluorescence intensity was determined. Complete binding curves for CD39-expressing cells were generated by detecting the concentrations of a series of antibodies. The epigenetic affinity of each antibody was calculated.

[0199] Example 4: Detection of antibody protease activity and inhibitory activity

[0200] Human CD39 protein was diluted to 0.6 μg / mL with reaction solution (25 mM Tris, 5 mM MgCl2, pH 7.5) and added to a 96-well plate (Greiner, catalog number 655209). The control wells were empty of protein, with 30 μL of the solution added to each well. The anti-CD39 antibody was diluted with antibody diluent, starting at 30 μg / mL and serially diluted 4-fold to prepare 7 concentration gradients. The serially diluted antibody was then added to each well of the 96-well plate, 30 μL per well. Incubate at 37°C for 10 min; dilute 10 mM ATP (Sigma, catalog number: A7699-1G) to 150 μM with reaction solution, add 30 μl to each well of a 96-well plate (excluding NC wells), and incubate at 37°C for 30 min; add 50 μL of CTG detection reagent (Promega, catalog number: G7572) to each well, incubate for 5 min, and then measure the chemiluminescence value. Calculate the inhibition rate: Inhibition rate = (RLU - RLU) NC ) / (RLU Control -RLU NC )*100%.

[0201] Example 5 Hybridoma Sequencing

[0202] RNA was isolated from monoclonal hybridoma cells and reverse transcribed into cDNA using a commercial kit. The heavy and light chain variable regions were then amplified using Mouse Ig-Primers primers as a template. Correctly sized PCR products were collected and purified, and then ligated into appropriate plasmid vectors. The ligation products were transformed into DH5α competent cells. Clones were screened, and the inserted fragments were analyzed by DNA sequencing.

[0203] Example 6: Production and Characterization of Chimeric Antibodies

[0204] 6.1 Production of chimeric antibodies

[0205] DNA encoding the variable regions of four selected hybridoma antibodies (263.11, 383.16, 19.4, 105.1) was synthesized and subcloned into expression vectors pre-containing the human IgG1 constant region gene. The vectors were transfected into mammalian cells for recombinant protein expression, and the expressed chimeric antibodies were purified using a Protein A affinity column.

[0206] 6.2 Characterization of chimeric antibodies

[0207] The binding ability and protease inhibitory activity of the chimeric antibody were tested according to the methods described in Examples 2, 3, and 4.

[0208] The results showed that the protein and cellular binding affinity of the four anti-CD39 chimeric antibodies screened in this study were as follows: Figure 1 , Figure 2 As shown, all results were superior to the reference antibody I394. The results of protease activity inhibition are as follows: Figure 3 As shown, all four antibodies exhibited good protease inhibition activity, which was also superior to the reference antibody I394.

[0209] By incorporating references

[0210] The full contents of every patent and scientific document mentioned in this article are incorporated herein by reference for all purposes.

[0211] Equivalence

[0212] This invention may be embodied in other specific ways without departing from its spirit or essential characteristics. Therefore, the above embodiments should be considered illustrative in all cases, and not as limiting of the invention described herein. Consequently, the scope of the invention is defined by the appended claims rather than by the foregoing description, and is intended to be encompassed by all variations within the equivalent meaning and scope of the claims.

Claims

1. An isolated antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof specifically binds to CD39 and comprises a heavy chain variable region (VH) and a light chain variable region (VL). The heavy chain variable region and the light chain variable region include: HCDR1 shown in SEQ ID NO: 21, HCDR2 shown in SEQ ID NO: 25, and HCDR3 shown in SEQ ID NO: 29, LCDR1 shown in SEQ ID NO: 33, LCDR2 shown in SEQ ID NO: 37, and LCDR3 shown in SEQ ID NO:

41.

2. The antibody or antigen-binding fragment thereof as described in claim 1, comprising: VH is shown in SEQ ID NO: 12, and VL is shown in SEQ ID NO:

17.

3. The antibody or antigen-binding fragment thereof as claimed in claim 2, further comprising a heavy chain constant region and a light chain constant region, wherein the heavy chain constant region is selected from the constant regions of IgG1, IgG2, IgG3, or IgG4, and the light chain constant region is selected from the constant region of κ or λ light chains.

4. The antibody or its antigen-binding fragment as described in claim 3, wherein the heavy chain constant region is selected from the IgG1 constant region and the light chain constant region is selected from the κ chain constant region.

5. The antibody or antigen-binding fragment thereof as described in claim 4, comprising: The heavy chain shown in SEQ ID NO:3 and the light chain shown in SEQ ID NO:

4.

6. The antibody or its antigen-binding fragment as described in claim 5, wherein the antibody is a whole antibody, a bispecific antibody, a monoclonal antibody, or a chimeric antibody.

7. The antibody or antigen-binding fragment thereof as claimed in claim 6, wherein the antigen-binding fragment is selected from the group consisting of: Fab fragment, Fab' fragment, F(ab)2 fragment, Fv fragment, and ScFv.

8. The antibody or its antigen-binding fragment as described in claim 1, which inhibits the activity of soluble CD39 protease but does not inhibit the activity of membrane-bound CD39 enzyme.

9. An isolated nucleic acid molecule comprising a nucleic acid sequence encoding an antibody or an antigen-binding fragment thereof as described in any one of claims 1-8.

10. A vector comprising the nucleic acid molecule as described in claim 9.

11. A host cell comprising the nucleic acid molecule of claim 9 or the vector of claim 10.

12. A conjugate comprising an antibody or antigen-binding fragment thereof as described in any one of claims 1-8 conjugated to at least one detectable marker.

13. A multispecific molecule comprising the antibody or antigen-binding fragment thereof as described in any one of claims 1-8.

14. The multispecific molecule of claim 13, wherein the multispecific molecule specifically binds to CD39 and additionally specifically binds to one or more other targets.

15. The multispecific molecule of claim 14, wherein the multispecific molecule further comprises at least one molecule having a second binding specificity against a second target.

16. A pharmaceutical composition or kit comprising an antibody or antigen-binding fragment thereof as claimed in any one of claims 1-8, or a nucleic acid molecule as claimed in claim 9, or a vector as claimed in claim 10, or a host cell as claimed in claim 11, or a conjugate as claimed in claim 12, or a multispecific molecule as claimed in any one of claims 13-15, and a pharmaceutically acceptable carrier.

17. Use of the antibody or antigen-binding fragment thereof as described in any one of claims 1-8, or the nucleic acid molecule as described in claim 9, or the vector as described in claim 10, or the host cell as described in claim 11, or the conjugate as described in claim 12, or the multispecific molecule as described in claims 13-15, or the pharmaceutical composition or kit as described in claim 16 in the preparation of a kit for diagnosing, detecting or monitoring diseases associated with CD39 expression, wherein the diseases associated with CD39 expression are cancers selected from the group consisting of lymphoma, sarcoma, lung cancer, pancreatic cancer, ovarian cancer, renal cell carcinoma, thyroid cancer and testicular cancer.

18. Use of the antibody or antigen-binding fragment thereof as described in any one of claims 1-8, or the nucleic acid molecule as described in claim 9, or the vector as described in claim 10, or the host cell as described in claim 11, or the conjugate as described in claim 12, or the multispecific molecule as described in claims 13-15, or the pharmaceutical composition or kit as described in claim 16 in the preparation of a medicament for treating or determining the prognosis of a disease associated with CD39 expression, wherein the disease associated with CD39 expression is cancer selected from the group consisting of lymphoma, sarcoma, lung cancer, pancreatic cancer, ovarian cancer, renal cell carcinoma, thyroid cancer, and testicular cancer.

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