WO2024078381A1 - 结合人cd39的抗体或其抗原结合片段、其制备方法及用途 - Google Patents

结合人cd39的抗体或其抗原结合片段、其制备方法及用途 Download PDF

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WO2024078381A1
WO2024078381A1 PCT/CN2023/123176 CN2023123176W WO2024078381A1 WO 2024078381 A1 WO2024078381 A1 WO 2024078381A1 CN 2023123176 W CN2023123176 W CN 2023123176W WO 2024078381 A1 WO2024078381 A1 WO 2024078381A1
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seq
amino acid
acid sequence
variable region
chain variable
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French (fr)
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黄浩旻
张学赛
徐菲
邓岚
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三生国健药业（上海）股份有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • the present invention relates to the field of tumor treatment, and in particular to an antibody or an antigen-binding fragment thereof binding to human CD39, and a preparation method and use thereof.
  • Nucleotide hydrolase CD39 is the initial enzyme and rate-limiting enzyme that converts the immunostimulatory molecule ATP into immunosuppressive adenosine in the tumor microenvironment.
  • high levels of extracellular ATP enhance the inflammatory activity of dendritic cells, T cells and other bone marrow-derived cells, as well as the activation of cells related to adaptive immunity, while high levels of extracellular adenosine enhance the immunosuppressive activity of almost all immune cells in the microenvironment by binding to immune cell surface receptors;
  • CD39 is highly expressed in various human tumors and tumor-infiltrating leukocytes, thereby helping tumor cells achieve a certain degree of immune escape, including lymphoma, sarcoma, chronic lymphocytic leukemia, lung cancer, pancreatic cancer, ovarian cancer, kidney cancer, thyroid cancer and testicular cancer, and effector T cells, regulatory T cells (Treg), macrophages, etc.
  • Treg regulatory T cells
  • the inventors of the present invention conducted a large number of experiments, screened and obtained mouse antibodies that specifically bind to human CD39, and further constructed and obtained chimeric antibodies and humanized antibodies on this basis.
  • the object of the present invention is to provide a novel antibody or antigen-binding fragment thereof binding to human CD39, and a preparation method and use thereof.
  • an antibody or antigen-binding fragment thereof that binds to human CD39 wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein:
  • the heavy chain variable region comprises:
  • HCDR1 having an amino acid sequence as shown in SEQ ID NO: 1, 7, 13 or 19;
  • HCDR2 having an amino acid sequence as shown in SEQ ID NO: 2, 8, 14 or 20;
  • HCDR3 having an amino acid sequence as shown in SEQ ID NO: 3, 9, 15 or 21;
  • the light chain variable region comprises:
  • LCDR1 having an amino acid sequence as shown in SEQ ID NO: 4, 10, 16 or 22;
  • LCDR2 having an amino acid sequence as shown in SEQ ID NO: 5, 11, 17 or 23;
  • LCDR3 having an amino acid sequence as shown in SEQ ID NO: 6, 12, 18 or 24.
  • the heavy chain variable region comprises:
  • HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NOs: 1, 2 and 3 respectively; or
  • HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NOs: 7, 8 and 9 respectively; or
  • HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NOs: 13, 14 and 15 respectively; or
  • HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs: 19, 20 and 21, respectively;
  • the light chain variable region comprises:
  • LCDR1, LCDR2 and LCDR3 whose amino acid sequences are shown in SEQ ID NOs: 4, 5 and 6 respectively; or
  • LCDR1, LCDR2 and LCDR3 whose amino acid sequences are shown in SEQ ID NOs: 10, 11 and 12 respectively; or
  • LCDR1, LCDR2 and LCDR3 whose amino acid sequences are shown in SEQ ID NOs: 16, 17 and 18 respectively; or
  • LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO: 22, 23 and 24 respectively.
  • the antibodies of the present invention are composed of two identical light chains (L) and two identical heavy chains (H). Each light chain is connected to the heavy chain by a covalent disulfide bond, and the number of disulfide bonds between the heavy chains of different immunoglobulin isotypes is different. Each heavy chain and light chain also has regularly spaced intrachain disulfide bonds. One end of each heavy chain has a variable region (VH), followed by a constant region. One end of each light chain has a variable region (VL) and the other end has a constant region; the constant region of the light chain is opposite to the first constant region of the heavy chain, and the variable region of the light chain is opposite to the variable region of the heavy chain.
  • the antibodies of the present invention are selected from monoclonal antibodies, polyclonal antibodies, multispecific antibodies (such as bispecific antibodies) formed by at least two antibodies, etc., preferably mouse antibodies, chimeric antibodies or humanized antibodies.
  • the antigen-binding fragment of the present invention refers to a fragment of an antibody that can specifically bind to human CD39.
  • the antigen-binding fragment is selected from a Fab fragment, a F(ab') 2 fragment, a Fv fragment, and the like.
  • the antibody or antigen-binding fragment thereof that binds to human CD39 of the present invention comprises: a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 1, 2 and 3, respectively, and a light chain variable region comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 4, 5 and 6, respectively; or a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 7, 8 and 9, respectively, and a light chain variable region comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 10, 11 and 12, respectively.
  • the heavy chain variable region includes HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NOs: 13, 14 and 15, respectively, and the light chain variable region includes LCDR1, LCDR2 and LCDR3 whose amino acid sequences are shown in SEQ ID NOs: 16, 17 and 18, respectively; or the heavy chain variable region includes HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NOs: 19, 20 and 21, respectively, and the light chain variable region includes LCDR1, LCDR2 and LCDR3 whose amino acid sequences are shown in SEQ ID NOs: 22, 23 and 24, respectively.
  • the heavy chain variable region has an amino acid sequence as shown in SEQ ID NO: 25, 29, 33, 37, 43, 47, 50, 53 or 56.
  • the light chain variable region has an amino acid sequence as shown in SEQ ID NO: 27, 31, 35, 39, 59, 62, 65 or 68.
  • the antibody or antigen-binding fragment that binds to human CD39 of the present invention comprises: a heavy chain variable region selected from any one of the following amino acid sequences: SEQ ID NO: 25, 29, 33, 37, 43, 47, 50, 53, 56; and/or a light chain variable region selected from any one of the following amino acid sequences: SEQ ID NO: 27, 31, 35, 39, 59, 62, 65, 68.
  • the antibody or antigen-binding fragment thereof that binds to human CD39 of the present invention comprises: a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 25 and a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 27; or a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 29 and a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 31; or a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 33 and a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 35; or a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 37 and a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 39; or a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 43 and an amino acid sequence as shown in SEQ ID NO: 44.
  • the heavy chain of an antibody also includes a heavy chain constant region.
  • the heavy chain constant region is derived from IgG.
  • the heavy chain constant region is derived from a mammal; preferably, the heavy chain constant region is human or mouse. Further preferably, the heavy chain constant region is a human antibody heavy chain IgG4 (S228P) constant region.
  • the light chain of the antibody also includes a light chain constant region.
  • the light chain constant region is derived from IgG.
  • the light chain constant region is derived from a mammal; preferably, the light chain constant region is human or mouse. Further preferably, the light chain constant region is a human antibody light chain kappa constant region.
  • the antibody or antigen-binding fragment thereof that binds to human CD39 of the present invention comprises: a heavy chain selected from any one of the amino acid sequences shown in SEQ ID NOs: 44, 48, 51, 54, and 57, or a heavy chain formed by splicing a human IgG4 (S228P) constant region with an amino acid sequence as shown in SEQ ID NOs: 41 and a heavy chain variable region selected from any one of the amino acid sequences shown in SEQ ID NOs: 25, 29, 33, and 37; a light chain selected from any one of the amino acid sequences shown in SEQ ID NOs: 60, 63, 66, and 69, or a light chain formed by splicing a human kappa chain constant region with an amino acid sequence as shown in SEQ ID NOs: 42 and a light chain variable region selected from any one of the amino acid sequences shown in SEQ ID NOs: 27, 31, 35, and 39.
  • S228P human IgG4
  • the antibody or antigen-binding fragment thereof that binds to human CD39 of the present invention comprises: a heavy chain having an amino acid sequence as shown in SEQ ID NO: 44 and a light chain having an amino acid sequence as shown in SEQ ID NO: 60; or an amino acid sequence as shown in The heavy chain shown in SEQ ID NO:48 and the light chain shown in SEQ ID NO:60; or the heavy chain shown in SEQ ID NO:51 and the light chain shown in SEQ ID NO:63; or the heavy chain shown in SEQ ID NO:54 and the light chain shown in SEQ ID NO:66; or the heavy chain shown in SEQ ID NO:44 and the light chain shown in SEQ ID NO:69; or the heavy chain shown in SEQ ID NO:57 and the light chain shown in SEQ ID NO:66.
  • the antibody or antigen-binding fragment thereof that binds to human CD39 of the present invention comprises: (1) a heavy chain formed by splicing a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 25 and a human IgG4 (S228P) constant region with an amino acid sequence as shown in SEQ ID NO: 41, and a light chain formed by splicing a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 27 and a human kappa chain constant region with an amino acid sequence as shown in SEQ ID NO: 42; or (2) a heavy chain formed by splicing a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 29 and a human IgG4 (S228P) constant region with an amino acid sequence as shown in SEQ ID NO: 41, and a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 31 and a human kappa chain constant region with an amino acid sequence as shown in SEQ ID NO:
  • ppa chain constant region or (3) a heavy chain formed by splicing a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 33 and a human IgG4 (S228P) constant region with an amino acid sequence as shown in SEQ ID NO: 41, and a light chain formed by splicing a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 35 and a human kappa chain constant region with an amino acid sequence as shown in SEQ ID NO: 42; or (4) a heavy chain formed by splicing a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 37 and a human IgG4 (S228P) constant region with an amino acid sequence as shown in SEQ ID NO: 41, and a light chain formed by splicing a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 39 and a human kappa chain constant region with an amino acid sequence as shown in SEQ ID NO: 42.
  • the second aspect of the present invention provides a nucleotide molecule, the specific scheme is:
  • An isolated nucleotide molecule which encodes the above-mentioned antibody or antigen-binding fragment thereof that binds to human CD39.
  • nucleotide molecules of the present invention include nucleotide molecules capable of encoding heavy chain variable regions as shown in SEQ ID NOs: 26, 30, 34, and 38; and/or nucleotide molecules capable of encoding light chain variable regions as shown in SEQ ID NOs: 28, 32, 36, and 40.
  • the nucleotide molecules of the present invention include nucleotide molecules capable of encoding heavy chains as shown in SEQ ID NOs: 45, 46, 49, 52, and 55; and/or nucleotide molecules capable of encoding light chains as shown in SEQ ID NOs: 58, 61, 64, and 67.
  • the preparation method of the nucleotide molecule of the present invention is a conventional preparation method in the art, such as by gene cloning technology (PCR method) or artificial sequence synthesis.
  • PCR method gene cloning technology
  • the nucleotide sequence encoding the amino acid sequence of the above-mentioned antibody or antigen-binding fragment that binds to human CD39 can be appropriately introduced with substitution, deletion, change, insertion or addition to provide a polynucleotide homologue.
  • the polynucleotide homologue can be prepared by replacing, deleting or adding one or more bases of the gene encoding the antibody or antigen-binding fragment that binds to human CD39 within the range of maintaining the antibody activity.
  • the third aspect of the present invention provides an expression vector, the specific scheme is:
  • An expression vector comprising the above-mentioned nucleotide molecule.
  • the expression vector of the present invention is a conventional expression vector in the art, such as an expression vector comprising appropriate regulatory sequences such as a promoter sequence, a terminator sequence, etc.
  • the expression vector may be a virus or a plasmid.
  • the fourth aspect of the present invention provides a host cell, the specific scheme is:
  • a host cell comprising the above-mentioned expression vector.
  • the host cell of the present invention is any conventional host cell in the art, such as a prokaryotic expression cell and a eukaryotic expression cell, as long as the above-mentioned expression vector can stably replicate itself and the nucleotides carried can be effectively expressed.
  • a fifth aspect of the present invention provides a method for preparing the above-mentioned antibody or antigen-binding fragment thereof that binds to human CD39, the method comprising the following steps:
  • the host cell culture method and the antibody separation and purification method described in the present invention are conventional methods in the art.
  • the sixth aspect of the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising the above-mentioned antibody or antigen-binding fragment thereof that binds to human CD39 and a pharmaceutically acceptable carrier or excipient.
  • the antibody or antigen-binding fragment thereof binding to human CD39 provided by the present invention can be combined with a pharmaceutically acceptable carrier or excipient to form a pharmaceutical composition so as to exert the therapeutic effect more stably.
  • a pharmaceutically acceptable carrier or excipient can ensure the conformational integrity of the antibody or antigen-binding fragment thereof binding to human CD39 disclosed by the present invention, while also protecting the multifunctional groups of the protein to prevent its degradation (including but not limited to aggregation, deamination or oxidation).
  • the pharmaceutical composition is in the form of a suspension, water injection, freeze-dried preparation, etc. commonly used in the pharmaceutical field.
  • the seventh aspect of the present invention provides a use of the above-mentioned antibody or antigen-binding fragment thereof or pharmaceutical composition binding to human CD39 in the preparation of anti-tumor drugs.
  • the tumors include lymphoma, sarcoma, chronic lymphocytic leukemia, lung cancer, pancreatic cancer, ovarian cancer, kidney cancer, thyroid cancer and testicular cancer.
  • the eighth aspect of the present invention provides a method for treating tumors, comprising the following steps: administering to a subject in need thereof an antibody or antigen-binding fragment thereof that binds to human CD39 as described in the first aspect of the present invention, or a pharmaceutical composition as described in the sixth aspect of the present invention.
  • the tumor is a tumor with high expression of CD39.
  • the tumor includes lymphoma, sarcoma, chronic lymphocytic leukemia, lung cancer, pancreatic cancer, ovarian cancer, kidney cancer, thyroid cancer, melanoma or testicular cancer.
  • the present invention develops therapeutic antibodies that specifically target CD39 through mouse immunization, hybridoma cell preparation and screening, antibody humanization, and pharmacodynamics research.
  • the antibodies will inhibit the production of adenosine by maintaining the ATP concentration in the tumor microenvironment, thereby reducing immunosuppression, improving immune effects, and exerting a synergistic anti-tumor effect, and have good application prospects.
  • Figures 1 to 3 are the binding curves of different mouse antibodies and CD39-His protein in Example 3;
  • Figures 4 to 6 are test results of different mouse antibodies and control antibodies inhibiting the activity of CD39 protein on the surface of 293E-CD39 cells in Example 4;
  • FIG7 is a test result of the inhibitory effect of the chimeric antibody and the control antibody on the exogenous CD39 protease activity in Example 6;
  • FIG8 is a test result of the inhibitory effect of the chimeric antibody and the control antibody on the CD39 protease activity on the surface of SKMEL-28 cells in Example 7;
  • Figures 9 to 11 are the binding curves of the humanized antibody and the control antibody to CD39 protein in Example 9;
  • Figures 12 and 13 are test results of the inhibitory effect of the humanized antibody on exogenous CD39 protease activity in Example 10;
  • FIG14 is a test result showing the inhibitory effect of the humanized antibody and the control antibody on the CD39 protease activity on the surface of tumor cells SKMEL-28 in Example 11;
  • FIG15 is a test result showing the inhibitory effect of the humanized antibody and the control antibody on CD39 protease activity on the surface of MOLP-8 tumor cells in Example 11;
  • FIG16 is a test result showing the inhibitory effect of the humanized antibody and the control antibody on the CD39 protease activity on the surface of CD14+ monocytes in Example 12;
  • FIG17 is a test result showing the inhibitory effect of the humanized antibody and the control antibody on CD39 protease activity on the surface of B cells in Example 13;
  • FIG18 is a test result showing the inhibitory effect of the humanized antibody and the control antibody on the activity of CD39 protease on the surface of macrophages in Example 14;
  • FIG19 is a test result showing the promoting effect of the humanized antibody and the control antibody on the secretion of IL-1 ⁇ by macrophages in Example 14;
  • FIG20 is the detection result of the humanized antibody in Example 15 promoting the maturation of DC cells and promoting the secretion of IFN- ⁇ by CD4 + T cells;
  • FIG21 is the detection result of the humanized antibody in Example 15 promoting the maturation of DC cells and promoting the secretion of IFN- ⁇ by CD8 + T cells;
  • FIG. 22 and FIG. 23 are the results of cross-reaction experiments between the humanized antibody in Example 17 and CD39 proteins of different species.
  • the term "expression vector” is a conventional expression vector in the art, and refers to an expression vector containing appropriate regulatory sequences, such as promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes and/or sequences, and other appropriate sequences.
  • the expression vector can be a virus or a plasmid, such as an appropriate phage or phagemid.
  • a virus or a plasmid such as an appropriate phage or phagemid.
  • the expression vector of the present invention is preferably selected from pCGS3, pcDNA3.4, pDR1, pcDNA3.1(+), pcDNA3.1/ZEO(+), pDHFR or pTT5.
  • the term "host cell” includes the above-mentioned expression vector, which is any conventional host cell in the art, as long as the above-mentioned recombinant expression vector can stably replicate itself and the nucleotide carried can be effectively expressed.
  • the host cells include prokaryotic expression cells and eukaryotic expression cells, preferably including: COS, CHO (Chinese Hamster Ovary), NS0, sf9, sf21, DH5 ⁇ , BL21 (DE3) or TG1, more preferably E. coli TG1, BL21 (DE3) cells or CHO-K1 cells.
  • the antibody or antigen-binding fragment thereof that binds to human CD39 of the present invention can be obtained by transforming the aforementioned expression vector into a host cell.
  • the host cell of the present invention is a eukaryotic cell, preferably selected from CHO cells, 293T, 293E and Expi293F cells.
  • the term "pharmaceutical composition” refers to the antibody or antigen-binding fragment thereof that binds to human CD39 of the present invention and a pharmaceutically acceptable carrier or excipient that together constitute a pharmaceutical preparation composition so as to exert the therapeutic effect more stably.
  • These preparations can ensure the conformational integrity of the amino acid core sequence of the antibody or antigen-binding fragment thereof that binds to human CD39 disclosed in the present invention, while also protecting the multifunctional groups of the protein to prevent its degradation (including but not limited to aggregation, deamination or oxidation).
  • the amount or dosage that produces the expected effect in the treated individual, and the expected effect includes the improvement of the individual's symptoms.
  • the objects or subjects required include but are not limited to mammals, such as humans, non-human primates, rats and mice, etc.
  • the antibody or antigen-binding fragment thereof that binds to human CD39 of the present invention or its pharmaceutical composition can also be used with other therapeutic agents.
  • “Pharmaceutically acceptable” refers to molecular entities and compositions that do not produce adverse, allergic or other untoward reactions when appropriately administered to an animal or a human.
  • “Pharmaceutically acceptable carriers or excipients” should be compatible with the active ingredient, that is, they can be mixed with it without significantly reducing the efficacy of the drug under normal circumstances.
  • Specific examples of some substances that can be used as pharmaceutically acceptable carriers or excipients are sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium methylcellulose, ethyl cellulose and methyl cellulose; tragacanth powder; malt; gelatin; talc; solid lubricants, such as hard fatty acid and magnesium stearate; calcium sulfate; vegetable oils, such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil and cocoa butter; polyols, such as propylene glycol, glycerol, sorbitol, mannitol and polyethylene glycol; alginic acid; emulsifiers, such as Tween; wetting agents, such
  • the drug dosage form is not particularly limited, and can be made into injections, oral liquids, tablets, capsules, dripping pills, sprays and other dosage forms, and can be prepared by conventional methods.
  • the choice of drug dosage form should match the administration method.
  • Subjects in need include, but are not limited to, animals, preferably mammals; the mammals are preferably rodents, artiodactyls, perissodactyls, lagomorphs, primates, etc.
  • the mammals include, for example, humans, non-human primates (e.g., monkeys), mice, pigs, cows, goats, rabbits, rats, guinea pigs, hamsters, horses, monkeys, sheep, or other non-human mammals; non-mammals include, for example, non-mammalian vertebrates, such as birds (e.g., chickens or ducks) or fish, and non-mammalian invertebrates.
  • the subject in need may be a human.
  • Treatment includes preventing or alleviating a condition, reducing the rate at which a condition occurs or develops, reducing the risk of developing a condition, preventing or delaying the development of symptoms associated with a condition, reducing or stopping symptoms associated with a condition, producing complete or partial reversal of a condition, curing a condition, or a combination of the above.
  • the dosage When administered to a subject, the dosage varies depending on the patient's age and weight, disease characteristics and severity, and route of administration, and may refer to the results of animal experiments and various circumstances. The total dosage cannot exceed a certain range.
  • the methods described herein may further include administering one or more additional therapies (e.g., one or more additional therapeutic agents and/or one or more treatment regimens) in combination with other compounds.
  • additional therapies may include, but are not limited to, surgery, chemotherapy, and combinations thereof.
  • high expression of CD39 or “overexpression of CD39” means that the expression level of CD39 is higher than that of healthy cells.
  • the embodiments do not include a detailed description of conventional methods, such as those used to construct vectors and plasmids, insert protein-encoding genes into such vectors and plasmids, or introduce plasmids into host cells, or prepare antibodies using engineering methods based on knowledge of the antibody sequence, as such methods are routine techniques in the art.
  • CD4 MicroBeads human: purchased from Miltenyibiotec, catalog number 130-045-101
  • CD8 MicroBeads human: purchased from Miltenyibiotec, catalog number 130-097-057
  • LS Columns plus tubes purchased from Miltenyi Biotec, 130-122-7291.
  • the amino acid sequences of the heavy chain and light chain variable regions of the control antibody TTX-030 were derived from patent WO 2019027935A1.
  • the heavy chain variable regions were fused to the constant regions of human IgG4 (S228P) (amino acid sequence as shown in
  • the heavy chain plasmid TTX-030-HC and the light chain plasmid TTX-030-LC expressing TTX-030 were obtained respectively, and the heavy chain plasmid and the light chain plasmid were co-transfected into HEK-293F cells. After 5 days of expression, the cell culture supernatant was collected and purified by Protein A affinity chromatography column to obtain TTX-030 protein.
  • the heavy chain amino acid sequence of the control antibody TTX-030 is shown in SEQ ID NO: 70
  • the light chain amino acid sequence is shown in SEQ ID NO: 71.
  • the extracellular domain gene of CD39 (sequence from UniProt database, registration number P49961, CD39 extracellular domain amino acid sequence T38-V478 as shown in SEQ ID NO: 72) was constructed into the pcDNA 3.4 expression vector through conventional gene synthesis and molecular cloning methods, and a signal peptide sequence was added to its N-terminus and a 6 ⁇ His tag was added to the C-terminus.
  • HEK-293F cells were transfected and expressed for 5 days. The cell culture supernatant was collected and purified through a Ni+ affinity chromatography column to obtain CD39-His protein.
  • the human CD39-His protein (purity>95%) prepared as above was used to routinely immunize Balb/c mice.
  • soluble human CD39-His protein was emulsified with Freund's complete adjuvant and injected subcutaneously at multiple points into Balb/c mice (human CD39-His 100 ⁇ g/mouse/0.5 mL).
  • soluble human CD39-His protein was emulsified with Freund's incomplete adjuvant and injected subcutaneously into Balb/c mice (human CD39-His 50 ⁇ g/mouse/0.5 mL).
  • soluble human CD39-His protein was emulsified with Freund's incomplete adjuvant and injected subcutaneously into Balb/c mice (human CD39-His 50 ⁇ g/mouse/0.5 mL). Three weeks later, soluble human CD39-His protein, 50 ⁇ g/mouse/0.2 mL, was injected intraperitoneally for stimulation. After 3-4 days, the spleens of mice were taken for fusion experiments.
  • mouse spleen cells were PEG-fused with mouse myeloma cells SP2/0 using conventional hybridoma technology protocols.
  • the fused cells were evenly suspended in complete culture medium, which was a culture medium composed of RPMI1640-GLUMAX with 1% Penicillin-streptomycin, 20% FBS (fetal bovine serum), and 1*HAT.
  • the fused cells were cultured in 60 96-well culture plates at 3 ⁇ 10 4 cells/200 ⁇ L/well. After 7-12 days, the supernatant was harvested and the hybridoma wells with positive human CD39 binding activity were screened by ELISA.
  • the method of screening hybridoma wells with positive human CD39 binding activity by ELISA method is as follows: dilute CD39-His protein to 2 ⁇ g/mL with PBS buffer, add 100 ⁇ L/well to the plate, and culture at 4°C overnight; discard the supernatant the next day, add 5% skim milk powder to block at 37°C for 2 hours, wash the plate 3 times with PBST for standby use; add the collected hybridoma supernatant to the blocked plate in sequence, 100 ⁇ L/well, and place at 37°C for 1 hour; wash the plate 3 times with PBST, add HRP-labeled goat anti-mouse IgG secondary antibody, and place at 37°C for 30 minutes; after washing the plate 3 times with PBST, pat dry the residual droplets on absorbent paper as much as possible, add 100 ⁇ L of TMB to each well, and place at room temperature away from light for 5 minutes; Add 50 ⁇ L of 2M H 2 SO 4 stop solution to each well to terminate the
  • hybridoma cell lines obtained by amplification and screening in serum-containing complete medium were centrifuged and replaced with serum-free medium Hybridoma-SFM medium to a cell density of 1-2 ⁇ 10 7 /mL. They were cultured for 1 week under 8% CO 2 and 37°C, and the culture supernatant was obtained by centrifugation. Purification was performed by Protein G affinity chromatography to obtain a variety of mouse antibodies against human CD39 protein. A total of 28 hybridoma cell lines were obtained in this round of screening.
  • Example 2 Each mouse antibody obtained in Example 2 was named and its ability to bind to human CD39-His protein was determined using an indirect enzyme-linked immunosorbent assay (ELISA).
  • the specific determination method is as follows: CD39-His protein was diluted to 2 ⁇ g/mL with coating solution (50 mM carbonate coating buffer, pH 9.6), and coated on ELISA plate at 4°C overnight; then blocked with 5% skim milk powder, incubated at 37°C for 2 hours; after washing the plate 3 times with PBST, mouse antibody against human CD39 protein was graded diluted with 1% BSA buffer, added to the plate pre-coated with CD39-His protein at 100 ⁇ L/well, and incubated at 37°C for one hour; the plate was washed 3 times with PBST, HRP-labeled goat anti-mouse IgG secondary antibody was added, and placed at 37°C for 30 minutes; after washing the plate 3 times with PBST, the residual droplets were patted dry on absorbent paper as much as
  • the binding curves of each mouse antibody to CD39-His protein are shown in Figures 1 to 3, and the EC 50 and the high platform value (Top) of binding activity are shown in Tables 1 to 3.
  • the mouse antibodies 302B4H10, 317B1A5, 321D9E5, 339E8A9, 343E7G6, 344E2C6, 346A1B12, 355F9D6, 406A10E3, 439G11A9, 435H6H8, 453D9A3, 426F10E2, 403A1B3, 431H1F7, 438C1B6, and 52A12H7F1 have relatively good binding activity to the target antigen CD39-His.
  • the inhibitory effect of each mouse antibody and the control antibody TTX-030 on CD39 enzyme activity was determined by the ATP detection method.
  • the specific method is as follows: HEK293E cells (293E-CD39) overexpressing CD39 were collected, centrifuged at 300g to remove the cell culture fluid, and washed once with PBS buffer; counted and diluted to 1 ⁇ 10 4 /well with Tris buffer (25mM Tris, 5mM CaCl 2 , PH 7.5), and the cells were plated into a 96-well cell culture plate, centrifuged and the cell culture supernatant was discarded; each mouse antibody to be tested and the control antibody TTX-030 were diluted to 10 ⁇ g/mL with Tris buffer, 3-fold gradient dilution, 100uL/well, a total of 11 wells, added to the culture plate, and incubated at 37°C incubator for 60min; centrifuged and the supernatant was discarded, and ATP was added to the cell culture plate
  • mice antibodies 419H10A4, 439G11A9, 52A12H7F1, and 2D2G2F2 can significantly inhibit the activity of CD39 enzyme on the surface of 293E-CD39 cells, and the activities of 439G11A9 and 52A12H7F1 are significantly better than the control antibody TTX-030.
  • Table 4 IC 50 of mouse antibody inhibiting the enzymatic activity of CD39 protein on the surface of 293E-CD39 cells
  • Table 5 IC 50 of mouse antibodies inhibiting the enzymatic activity of CD39 protein on the surface of 293E-CD39 cells
  • Table 6 IC 50 of mouse antibodies inhibiting the enzymatic activity of CD39 protein on the surface of 293E-CD39 cells
  • Example 5 Obtaining candidate antibody variable region genes and preparing chimeric antibodies
  • 419H10A4 The heavy chain variable region and light chain variable region of 439G11A9, 52A12H7F1, and 2D2G2F2 are as follows:
  • RNA of each hybridoma cell was extracted by Trizol, and Oligo-dT primer was used to reverse transcribe mRNA to obtain cDNA. Then, cDNA was used as a template to perform PCR with degenerate primers of the heavy chain and light chain of mouse antibodies (Antibody Engineering Volume 1, Edited by Roland Kontermann and Stefan Dübel, the sequence of the combined primers is from page 323). The obtained PCR products were sequenced and analyzed through the kabat database to determine that the obtained sequences were the variable region sequences of mouse antibodies.
  • the specific information of each mouse antibody is as follows:
  • 419H10A4 The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 25, the amino acid sequences of HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NO: 1, 2 and 3, respectively, and the nucleotide sequence encoding the heavy chain variable region (full length is 369 bp, encoding 123 amino acid residues) is shown in SEQ ID NO: 26; the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 27, the amino acid sequences of LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO: 4, 5 and 6, respectively, and the nucleotide sequence encoding the light chain variable region (full length is 321 bp, encoding 107 amino acid residues) is shown in SEQ ID NO: 28.
  • the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 29, the amino acid sequences of HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs: 7, 8 and 9, respectively, and the nucleotide sequence encoding the heavy chain variable region (full length is 366 bp, encoding 122 amino acid residues) is shown in SEQ ID NO: 30; the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 31, the amino acid sequences of LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NOs: 10, 11 and 12, respectively, and the nucleotide sequence encoding the light chain variable region (full length is 333 bp, encoding 111 amino acid residues) is shown in SEQ ID NO: 32.
  • 52A12H7F1 The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 33, the amino acid sequences of HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs: 13, 14 and 15, respectively, and the nucleotide sequence encoding the heavy chain variable region (full length is 360 bp, encoding 120 amino acid residues) is shown in SEQ ID NO: 34; the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 35, the amino acid sequences of LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NOs: 16, 17 and 18, respectively, and the nucleotide sequence encoding the light chain variable region (full length is 336 bp, encoding 112 amino acid residues) is shown in SEQ ID NO: 36.
  • 2D2G2F2 The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 37, the amino acid sequences of HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs: 19, 20 and 21, respectively, and the nucleotide sequence encoding the heavy chain variable region (full length is 351 bp, encoding 117 amino acid residues) is shown in SEQ ID NO: 38; the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 39, the amino acid sequences of LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NOs: 22, 23 and 24, respectively, and the nucleotide sequence encoding the light chain variable region (full length is 318 bp, encoding 106 amino acid residues) is shown in SEQ ID NO: 40.
  • the heavy chain variable region sequences of the four candidate murine antibodies were spliced with the human IgG4 (S228P) constant region (amino acid sequence as shown in SEQ ID NO: 41), and the light chain variable region sequences were spliced with the human kappa chain constant region (amino acid sequence as shown in SEQ ID NO: 42).
  • the heavy chain and light chain of each chimeric antibody were constructed into the pcDNA3.4 expression vector and co-transfected into HEK-293F The chimeric antibodies were expressed and purified from the cells.
  • the chimeric antibodies corresponding to the mouse antibodies 419H10A4, 439G11A9, 52A12H7F1, and 2D2G2F2 were named 419H-ch, 439G-ch, 52A12-ch, and 2D2-ch, respectively.
  • the method of detecting ATP was used to determine the inhibitory effect of each chimeric antibody and the control antibody on the activity of exogenous free CD39 protease.
  • the specific determination method was as follows: the CD39-His protein prepared and stored above was resuspended in 0.01% BSA25mM Tris, 5mM CaCl2 , pH 7.5 (Tris buffer), and added to a white opaque 96-well plate at a concentration of 160nM, 25 ⁇ L per well; the antibody to be tested was diluted with Tris buffer in a 3-fold gradient, and each group was continuously diluted 12 times.
  • each antibody was 1 ⁇ M, and 25 ⁇ L of the diluted antibody to be tested was added to the above 96-well plate, and the final volume of each well was 50 ⁇ L; the 96-well plate was incubated at room temperature for 2 hours; after the incubation, ATP was diluted to 1000 ⁇ M with Tris buffer, and added to the 96-well plate, 50 ⁇ L/well, mixed, and incubated at 37°C for 60min; then Cell 1000 was directly added at a volume ratio of 1:1. Titer-Glo reagent was added to a 96-well plate, and the fluorescence intensity was read and the data were analyzed in a multi-function microplate reader.
  • the cell culture supernatant was discarded the next day, and the chimeric antibody to be tested and the control antibody were diluted to 1000nM (SKMEL-28) with Tris buffer, 3-fold gradient dilution, and then added to the above cell culture plate at 100 ⁇ L/well, and incubated at 37°C for 60 minutes; ATP with a final concentration of 50 ⁇ M was added, 50 ⁇ L/well, and incubated at 37°C for 60 minutes, 50 ⁇ L of the culture supernatant was taken into a 96-well white opaque detection plate, and 50 ⁇ L of Cell Titer-Glo detection reagent, incubate at room temperature for 5 min, read the fluorescence intensity and analyze in a multifunctional microplate reader.
  • the inhibition test curves of the chimeric antibody and the control antibody on the CD39 protease activity on the surface of SKMEL-28 cells are shown in FIG8 , and the corresponding enzyme activity IC 50 and the maximum inhibitory activity (Top) results are shown in Table 8 .
  • Table 8 IC 50 of each chimeric antibody against CD39 protease activity on the surface of SKMEL-28 cells
  • amino acid sequences of the light chain variable region (amino acid sequence as shown in SEQ ID NO: 31) and the heavy chain variable region (amino acid sequence as shown in SEQ ID NO: 29) of the mouse antibody 439G11A9 were analyzed, and the heavy chain and light chain antigen binding complementary determining regions (CDRs) and framework regions (FRs) of 439G11A9 were determined according to the Kabat rule.
  • the amino acid sequences of the complementary determining regions HCDR1, HCDR2, and HCDR3 of the heavy chain variable region are shown in SEQ ID NO: 7, 8, and 9, respectively, and the amino acid sequences of the complementary determining regions LCDR1, LCDR2, and LCDR3 of the light chain variable region are shown in SEQ ID NO: 10, 11, and 12, respectively.
  • the human IgG Germline sequence template that best matches the non-CDR sequence of the heavy and light chain variable regions of the 439G11A9 antibody was selected from the Germline database.
  • IGHV14-3*02 was selected as the humanization template for the heavy chain variable region
  • IGKV3-2*01 was selected as the humanization template for the light chain variable region.
  • the CDR regions of the heavy and light chains of the 439G11A9 antibody were transplanted to the selected human templates to replace the CDR regions in the humanized templates.
  • each humanized heavy chain variable region was recombined with the human IgG4 (S228P) constant region
  • each humanized light chain variable region was recombined with the human kappa chain constant region, and constructed into the pcDNA3.4 expression vector, expressed and purified by self-paired co-transfected HEK-293F cells to obtain each humanized antibody.
  • the names, nucleotide and amino acid sequences of each humanized antibody heavy and light chain of 439G11A9 are as follows:
  • 439G-HC-HuML The amino acid sequence is shown in SEQ ID NO: 57, the amino acid sequence of its variable region is shown in SEQ ID NO: 56, and the encoding nucleotide sequence is shown in SEQ ID NO: 55.
  • 439G-LC-G The amino acid sequence is shown in SEQ ID NO: 60, the amino acid sequence of its variable region is shown in SEQ ID NO: 59, and the encoding nucleotide sequence is shown in SEQ ID NO: 58.
  • (3)439G-LC-HuMut3 The amino acid sequence is shown in SEQ ID NO: 66, the amino acid sequence of its variable region is shown in SEQ ID NO: 65, and the encoding nucleotide sequence is shown in SEQ ID NO: 64.
  • (4)439G-LC-HuM85E The amino acid sequence is shown in SEQ ID NO: 69, the amino acid sequence of its variable region is shown in SEQ ID NO: 68, and the encoding nucleotide sequence is shown in SEQ ID NO: 67.
  • ELISA indirect enzyme-linked immunosorbent assay
  • the CD39-his protein was diluted to 0.5 ⁇ g/mL with coating solution (50 mM carbonate coating buffer, pH 9.6), coated on ELISA plate, 100 ⁇ L/well, placed in a wet box, 4°C, coated for 16 h; the ELISA plate was washed three times with PBST to remove unbound antigens, and the ELISA plate was patted dry on absorbent paper to remove excess liquid, and then blocked with 2% BSA prepared in PBS, 200 ⁇ L/well, at room temperature for 2 h; washed once with PBST to remove excess blocking solution, and the ELISA plate was patted dry to remove excess liquid, and each antibody was diluted 3-fold with 1% BSA (antibody diluent) prepared in PBST, with the highest concentration of 20 nM, 12 dilution gradients, added to ELISA wells, 100 ⁇ L/well, incubated at room temperature for 1 h; unbound or non-specifically bound primary antibody was washed away,
  • Table 10 EC 50 of humanized antibodies binding to CD39 protein
  • Table 11 EC 50 of humanized antibodies binding to CD39 protein
  • Table 12 EC 50 of humanized antibodies binding to CD39 protein
  • Example 6 the ability of each humanized antibody to inhibit exogenous CD39 protease activity was determined.
  • the experimental results are shown in Figures 12 to 13 and Tables 13 to 14.
  • the humanized antibodies 439G-HuG, 439G-HuG-LN85E, 439G-Hu-HMELW100Y, 439G-Hu-HMKLmut3, and 439G-Hu-HMLLmut3 have comparable inhibitory effects on the ATP degradation activity of exogenous free CD39 protein, and are significantly superior to the control antibody TTX-030.
  • Table 15 IC 50 of humanized antibodies inhibiting CD39 protease activity on the surface of tumor cells SKMEL-28
  • Table 16 IC 50 of humanized antibodies inhibiting CD39 protease activity on the surface of tumor cells MOLP-8
  • the humanized antibodies 439G-HuG, 439G-HuG-LN85E, 439G-Hu-HMELW100Y, 439G-Hu-HMKLmut3, and 439G-Hu-HMLLmut3 have comparable activities in inhibiting ATP degradation by CD39 protein on the surface of SKMEL-28 cells, and are significantly superior to the control antibody TTX-030.
  • the humanized antibodies 439G-HuG-LN85E, 439G-Hu-HMELW100Y, 439G-Hu-HMKLmut3, and 439G-Hu-HMLLmut3 have comparable activity in inhibiting ATP degradation by CD39 protein on the surface of MOLP-8, are slightly better than 439G-HuG, and are significantly better than the control antibody TTX-030.
  • the inhibitory effect of the antibody on the activity of CD39 protease on the surface of CD14 + monocytes in PBMC was determined by detecting ATP.
  • the specific method is as follows:
  • CD14 + monocytes were sorted from human PBMC (purchased from Shanghai Saili Biotechnology Co., Ltd.), the cell culture medium was removed by centrifugation, the cells were washed once with PBS buffer, counted and resuspended with complete culture medium (1640+10% FBS+1% Pen Strep+1% Sodium Pyruvate+1% GlutaMAX), and inoculated into a round-bottom 96-well plate at 1.0 ⁇ 10 5 cells/well, 100 ⁇ L/well, and cultured in a cell culture incubator at 37°C overnight; the next day, the cell culture supernatant was discarded, the antibody to be tested was diluted to 100 nM with Tris buffer, 3-fold gradient dilution, and then added at 100 ⁇ L/well.
  • the cells were incubated at 37°C for 60 min in the cell culture plate.
  • ATP was added at a final concentration of 20 ⁇ M, 50 ⁇ L/well, and incubated at 37°C for 60 min.
  • 50 ⁇ L of the culture supernatant was placed in a 96-well white opaque detection plate.
  • 50 ⁇ L of Cell Titer-Glo detection reagent was added and incubated at room temperature for 5 min. The fluorescence intensity was read and analyzed in a multifunctional microplate reader.
  • the experimental results are shown in Figure 16 and Table 17.
  • the humanized antibodies 439G-HuG-LN85E, 439G-Hu-HMELW100Y, and 439G-Hu-HMKLmut3 have comparable activity in inhibiting ATP degradation by CD39 protein on the surface of CD14 + monocytes, slightly better than 439G-HuG and 439G-Hu-HMLLmut3, and significantly better than the control antibody TTX-030.
  • Table 17 IC 50 of humanized antibodies inhibiting CD39 protease activity on CD14 + monocyte surface
  • B cells were sorted from human PBMC (purchased from Shanghai Saili Biotechnology Co., Ltd.) according to the CD19 MicroBeads manual, the cell culture medium was removed by centrifugation, and the cells were washed once with PBS buffer; the cells were counted and resuspended with complete culture medium (1640+10% FBS+1% Pen Strep+1% Sodium Pyruvate+1% GlutaMAX), and the cells were inoculated into a round-bottom 96-well plate at 5.0 ⁇ 10 4 cells/well, 100 ⁇ L/well, and cultured in a cell culture incubator at 37°C overnight; the next day, the cell culture supernatant was discarded, and the antibody to be tested was diluted to 100 nM with Tris buffer, diluted 3 times in a gradient, and then added to the above cell culture plate at 100 ⁇ L/well, and incubated at 37°C for 60 min; ATP with a final concentration of 50 ⁇ M was added, 50 ⁇ L/well, and
  • Table 18 IC 50 of humanized antibodies inhibiting CD39 protease activity on B cell surface
  • human PBMCs were sorted (purchased from Shanghai Saili Biotechnology Co., Ltd.) CD14 + monocytes. Resuspend the sorted CD14 + monocytes in RPMI-1640 complete medium containing 400ng/mL GM-CSF (RPMI-1640 medium containing 10% FBS), add 20mL of cell suspension to a T75 cell culture flask to induce differentiation, and the cell density is 1.5 ⁇ 10 5 /mL; on the seventh day, half of the medium is replaced with fresh complete medium containing 400ng/mL GM-CSF; on the tenth day, remove the cell culture supernatant, wash the cells once with serum-free medium RPMI-1640, add 3mL TrypLE Select to digest the above cells, wait for the cells to fall off, terminate the digestion with complete medium, centrifuge, and wash the cells twice with PBS.
  • RPMI-1640 complete medium containing 400ng/mL GM-CSF RPMI-1640 medium containing 10% FBS
  • the resulting cells are macrophages.
  • the macrophages were resuspended in complete medium, the cell density was adjusted to 5 ⁇ 10 5 /mL, 100 ⁇ L per well, and plated in a round-bottom 96-well plate; the antibody to be tested was gradiently diluted with complete medium, the highest concentration of the antibody was 1000 nM, and 12 gradients were diluted 3 times; the 96-well plate with macrophages was centrifuged, the cell supernatant was completely discarded, and the gradient diluted antibody to be tested was added, 50 ⁇ L of the antibody to be tested was added to each well, and the plate was placed in a cell culture incubator for incubation for 1 hour; the LPS solution with a final concentration of 10 ng/mL was prepared with complete medium, 50 ⁇ L per well was added to the 96-well plate, and the plate was placed in a cell culture incubator for incubation for 3 hours; the ATP solution with a final concentration of 1000 ⁇ M was prepared with complete medium, 100
  • Humanized antibodies 439G-HuG, 439G-Hu-HMKLmut3, and 439G-HuG-LN85E can effectively inhibit the activity of CD39 protease on the surface of macrophages to maintain the extracellular ATP concentration.
  • each humanized antibody in the presence of LPS, can effectively stimulate macrophages to secrete IL-1 ⁇ by inhibiting the activity of CD39 protease on the surface of macrophages and retaining ATP.
  • Table 19 IC 50 of humanized antibodies promoting IL-1 ⁇ secretion by macrophages
  • CD14 + monocytes were sorted from human PBMC (purchased from Shanghai Saili Biotechnology Co., Ltd.), washed once with PBS buffer and resuspended in 30 mL of 1640 complete medium containing 400 ng/mL GM-CSF and 20 ng/mL IL-4, and added to a T75 cell culture flask to induce differentiation, with a cell density of 1 ⁇ 10 5 cells/mL; on the seventh day, half of the cell culture medium supernatant was discarded and replaced with new 1640 complete medium containing 400 ng/mL GM-CSF and 20 ng/mL IL-4; on the tenth day, the suspended cells were collected and washed once with serum-free medium 1640; the number of cells and the viability were counted.
  • DC cells were plated in a 96-well round-bottom cell culture plate at 1 ⁇ 10 4 cells/well, 100 ⁇ L/well, and the antibody to be tested was gradiently diluted with 10% FBS 1640 medium and added to the above 96-well plate, 100 ⁇ L/well, and the 96-well plate was filled with 10% FBS 1640 medium.
  • the plate was placed in a cell culture incubator and incubated for 1 hour; 10% FBS 1640 medium was used to prepare an ATP solution with a final concentration of 1000 ⁇ M, 50 ⁇ L per well was added to the 96-well plate, and the 96-well plate was placed in a cell culture incubator and cultured for 48 hours. After the culture was completed, the cell supernatant was removed by centrifugation, and the DC cells were washed twice with PBS.
  • CD4 + and CD8 + T cells were sorted from another batch of human PBMC (purchased from Shanghai Saili Biotechnology Co., Ltd.).
  • the cell plate was placed in a 37°C cell culture incubator for 5 days, and the cell supernatant was collected for IFN- ⁇ detection.
  • 439G-HuG, 439G-Hu-HMKLmut3, and 439G-HuG-LN85E can significantly promote the maturation of DC cells and promote the secretion of IFN- ⁇ by CD4 + T cells in a dose-dependent manner.
  • 439G-HuG, 439G-Hu-HMKLmut3, and 439G-HuG-LN85E can also significantly promote the maturation of DC cells and promote the secretion of IFN- ⁇ by CD8 + T cells in a dose-dependent manner.
  • the antibody was captured using a chip covalently coupled with Protein A (purchased from GE Healthcare, catalog number 29139131-AA), and the relevant operating parameters were as follows: antibody concentration of 1 ⁇ g/mL, contact time of 60 s, flow rate of 10 ⁇ L/min, and regeneration contact time of 30 s; CD39 antigen was diluted with HBS-EP+pH7.4 buffer, with the highest concentration of 80 nM, and diluted 2.5 times to 0.8192 nM and 0 concentration point, and 6 M guanidine hydrochloride solution was used as the regeneration buffer.
  • the sample was injected on Biacore 8K according to the following parameters: binding time of 180 s, dissociation time of 720 s, flow rate of 30 L/min, and regeneration contact time of 30 s; data were collected and analyzed using Biacore 8K Evaluation Software.
  • CD39-His protein was diluted to 2 ⁇ g/mL, coated on the ELISA plate, 100 ⁇ L/well, placed in a wet box, 4°C, coated for 16 hours; the ELISA plate was washed three times with PBST to remove unbound antigens, and the ELISA plate was patted dry on absorbent paper to remove excess liquid, and then blocked with 2% BSA prepared in PBS, 200 ⁇ L/well, at room temperature for 2 hours; washed once with PBST to wash away excess blocking solution, and the ELISA plate was patted dry to remove excess liquid, and the antibody to be tested was diluted with 1% BSA prepared in PBST in a 3-fold gradient, with the highest concentration of 20 nM, 3-fold dilution of 11 gradients, added to the ELISA wells at 100 ⁇ L/well, incubated at room temperature for 1 hour, and 2 replicates were made for each sample in parallel; unbound or non-specifically bound primary antibody was
  • the humanized antibody 439G-Hu-HMKLmut3 cross-reacted with macaque CD39 protein (Maca CD39) with an EC50 of 0.145 nM, and bound to human CD39 protein (Human) with an EC50 of 0.172 nM.

Abstract

本发明属于肿瘤治疗技术领域，具体涉及一种结合人CD39的抗体或其抗原结合片段、其制备方法及用途。本发明的结合人CD39的抗体或其抗原结合片段能够有效结合人CD39，应用于制备治疗CD39过表达的疾病的药物，具有良好的临床前景。

Description

结合人CD39的抗体或其抗原结合片段、其制备方法及用途 技术领域
本发明涉及肿瘤治疗领域，特别是涉及结合人CD39的抗体或其抗原结合片段、其制备方法及用途。
背景技术
核苷酸水解酶CD39是肿瘤微环境中将免疫刺激分子ATP转化为免疫抑制性腺苷的初始酶和限速酶。在肿瘤微环境中，高水平的细胞外ATP会增强树突状细胞、T细胞和其它骨髓生成细胞的炎症活性，以及与适应性免疫能力相关的细胞的激活，而高水平的细胞外腺苷则会通过与免疫细胞表面受体结合增强微环境中几乎所有免疫细胞的免疫抑制活性；CD39在各种人类肿瘤和肿瘤浸润白细胞中均呈现高表达现象，从而帮助肿瘤细胞实现一定程度的免疫逃逸，前者包括淋巴瘤、肉瘤、慢性淋巴细胞白血病、肺癌、胰腺癌、卵巢癌、肾癌、甲状腺癌和睾丸癌等，后者如效应T细胞、调节性T细胞(Treg)、巨噬细胞等。目前，有关CD39抗体的免疫疗法研究相对较少，临床应用需求尚未得到满足。
发明内容
为解决上述问题，本发明的发明人进行了大量的试验，筛选获得了特异性结合人CD39的鼠源抗体，并在此基础上进一步构建获取嵌合抗体以及人源化抗体。
本发明的目的在于提供一种新型的结合人CD39的抗体或其抗原结合片段、其制备方法和用途。
为实现上述目的及其他相关目的，本发明采用了如下技术方案：
在本发明的第一方面，提供了一种结合人CD39的抗体或其抗原结合片段，该抗体或其抗原结合片段包括重链可变区和轻链可变区，其中，
所述重链可变区包括：
氨基酸序列如SEQ ID NO：1、7、13或19所示的HCDR1；
氨基酸序列如SEQ ID NO：2、8、14或20所示的HCDR2；
氨基酸序列如SEQ ID NO：3、9、15或21所示的HCDR3；
所述轻链可变区包括：
氨基酸序列如SEQ ID NO：4、10、16或22所示的LCDR1；
氨基酸序列如SEQ ID NO：5、11、17或23所示的LCDR2；
氨基酸序列如SEQ ID NO：6、12、18或24所示的LCDR3。
在本发明的某些实施方式中，所述重链可变区包括：
氨基酸序列分别如SEQ ID NO：1、2及3所示的HCDR1、HCDR2及HCDR3；或
氨基酸序列分别如SEQ ID NO：7、8及9所示的HCDR1、HCDR2及HCDR3；或
氨基酸序列分别如SEQ ID NO：13、14及15所示的HCDR1、HCDR2及HCDR3；或
氨基酸序列分别如SEQ ID NO：19、20及21所示的HCDR1、HCDR2及HCDR3；
所述轻链可变区包括：
氨基酸序列分别如SEQ ID NO：4、5及6所示的LCDR1、LCDR2及LCDR3；或
氨基酸序列分别如SEQ ID NO：10、11及12所示的LCDR1、LCDR2及LCDR3；或
氨基酸序列分别如SEQ ID NO：16、17及18所示的LCDR1、LCDR2及LCDR3；或
氨基酸序列分别如SEQ ID NO：22、23及24所示的LCDR1、LCDR2及LCDR3。
本发明的抗体由两个相同的轻链(L)和两个相同的重链(H)组成。每条轻链通过一个共价二硫键与重链相连，而不同免疫球蛋白同种型的重链间的二硫键数目不同。每条重链和轻链也有规则间隔的链内二硫键。每条重链的一端有可变区(VH)，其后是恒定区。每条轻链的一端有可变区(VL)，另一端有恒定区；轻链的恒定区与重链的第一个恒定区相对，轻链的可变区与重链的可变区相对。本发明的抗体选自单克隆抗体、多克隆抗体、由至少两种抗体形成的多特异性抗体(例如双特异性抗体)等，优选为鼠源抗体、嵌合抗体或人源化抗体。
本发明的抗原结合片段是指能够与人CD39特异性结合的抗体的片段。所述抗原结合片段选自Fab片段、F(ab’)2片段、Fv片段等。
优选的，本发明的结合人CD39的抗体或其抗原结合片段，包含：重链可变区包括氨基酸序列分别如SEQ ID NO：1、2及3所示的HCDR1、HCDR2及HCDR3，及轻链可变区包括氨基酸序列分别如SEQ ID NO：4、5及6所示的LCDR1、LCDR2及LCDR3；或重链可变区包括氨基酸序列分别如SEQ ID NO：7、8及9所示的HCDR1、HCDR2及HCDR3，及轻链可变区包括氨基酸序列分别如SEQ ID NO：10、11及12所示的LCDR1、LCDR2及LCDR3；或重链可变区包括氨基酸序列分别如SEQ ID NO：13、14及15所示的HCDR1、HCDR2及HCDR3，及轻链可变区包括氨基酸序列分别如SEQ ID NO：16、17及18所示的LCDR1、LCDR2及LCDR3；或重链可变区包括氨基酸序列分别如SEQ ID NO：19、20及21所示的HCDR1、HCDR2及HCDR3，及轻链可变区包括氨基酸序列分别如SEQ ID NO：22、23及24所示的LCDR1、LCDR2及LCDR3。
优选的，所述重链可变区具有如SEQ ID NO：25、29、33、37、43、47、50、53或56所示的氨基酸序列。优选的，所示轻链可变区具有如SEQ ID NO：27、31、35、39、59、62、65或68所示的氨基酸序列。
本发明的结合人CD39的抗体或抗原结合片段包含：选自以下氨基酸序列中任一氨基酸序列的重链可变区：SEQ ID NO：25、29、33、37、43、47、50、53、56；和/或选自以下氨基酸序列中任一氨基酸序列的轻链可变区：SEQ ID NO：27、31、35、39、59、62、65、68。
优选的，本发明的结合人CD39的抗体或其抗原结合片段包含：氨基酸序列如SEQ ID NO：25所示的重链可变区以及氨基酸序列如SEQ ID NO：27所示的轻链可变区；或氨基酸序列如SEQ ID NO：29所示的重链可变区以及氨基酸序列如SEQ ID NO：31所示的轻链可变区；或氨基酸序列如SEQ ID NO：33所示的重链可变区以及氨基酸序列如SEQ ID NO：35所示的轻链可变区；或氨基酸序列如SEQ ID NO：37所示的重链可变区以及氨基酸序列如SEQ ID NO：39所示的轻链可变区；或氨基酸序列如SEQ ID NO：43所示的重链可变区以及氨基酸序列如SEQ ID NO：59所示的轻链可变区；或氨基酸序列如SEQ ID NO：47所述式的重链可变区以及氨基酸序列如SEQ ID NO：59所示的轻链可变区；或氨基酸序列如SEQ ID NO：50所示的重链可变区以及氨基酸序列如SEQ ID NO：62所示的轻链可变区；或氨基酸序列如SEQ ID NO：53所示的重链可变区以及氨基酸序列如SEQ ID NO：65所示的轻链可变区；或氨基酸序列如SEQ ID NO：43所示重链可变区以及氨基酸序列如SEQ ID NO：68所示的轻链可变区；或氨基酸序列如SEQ ID NO：56所示的重链可变区以及氨基酸序列如SEQ ID NO：65所示的轻链可变区。
抗体的重链还包括重链恒定区。在本发明的某些实施例中，所述重链恒定区来源于IgG。
在本发明的某些实施例中，所述重链恒定区来源于哺乳动物；优选的，所述重链恒定区为人源或鼠源的。进一步优选的，所述重链恒定区为人源抗体重链IgG4(S228P)恒定区。
抗体的轻链还包括轻链恒定区。在本发明的某些实施例中，所述轻链恒定区来源于IgG。
在本发明的某些实施例中，所述轻链恒定区来源于哺乳动物；优选的，所述轻链恒定区为人源或鼠源的。进一步优选的，所述轻链恒定区为人源抗体轻链kappa恒定区。
优选的，本发明的结合人CD39的抗体或其抗原结合片段包含：选自如SEQ ID NO：44、48、51、54、57所示的任一氨基酸序列的重链，或者由氨基酸序列如SEQ ID NO：41所示的人IgG4(S228P)恒定区与选自如SEQ ID NO：25、29、33、37所示任一氨基酸序列的重链可变区拼接而成的重链；选自如SEQ ID NO：60、63、66、69所示的任一氨基酸序列的轻链，或者由氨基酸序列如SEQ ID NO：42所示的人kappa链恒定区与选自如SEQ ID NO：27、31、35、39所示的任一氨基酸序列的轻链可变区拼接而成的轻链。
进一步优选的，本发明的结合人CD39的抗体或其抗原结合片段，包含：氨基酸序列如SEQ ID NO：44所示的重链以及氨基酸序列如SEQ ID NO：60所示轻链；或氨基酸序列如 SEQ ID NO：48所示的重链以及氨基酸序列如SEQ ID NO：60所示的轻链；或氨基酸序列如SEQ ID NO：51所示的重链以及氨基酸序列如SEQ ID NO：63所示的轻链；或氨基酸序列如SEQ ID NO：54的重链以及氨基酸序列如SEQ ID NO：66所示的轻链；或氨基酸序列如SEQ ID NO：44所示的重链以及氨基酸序列如SEQ ID NO：69所示的轻链；或氨基酸序列如SEQ ID NO：57所示的重链以及氨基酸序列如SEQ ID NO：66所示的轻链。
进一步优选的，本发明的结合人CD39的抗体或其抗原结合片段含：(1)氨基酸序列如SEQ ID NO：25所示的重链可变区与氨基酸序列如SEQ ID NO：41所示的人IgG4(S228P)恒定区拼接而成的重链，和氨基酸序列如SEQ ID NO：27所示的轻链可变区以及氨基酸序列如SEQ ID NO：42所示的人kappa链恒定区拼接而成的轻链；或(2)氨基酸序列如SEQ ID NO：29所示的重链可变区与氨基酸序列如SEQ ID NO：41所示的人IgG4(S228P)恒定区拼接而成的重链，和氨基酸序列如SEQ ID NO：31所示的轻链可变区以及氨基酸序列如SEQ ID NO：42所示的人kappa链恒定区拼接而成的轻链；或(3)氨基酸序列如SEQ ID NO：33所示的重链可变区与氨基酸序列如SEQ ID NO：41所示的人IgG4(S228P)恒定区拼接而成的重链，和氨基酸序列如SEQ ID NO：35所示的轻链可变区以及氨基酸序列如SEQ ID NO：42所示的人kappa链恒定区拼接而成的轻链；或(4)氨基酸序列如SEQ ID NO：37所示的重链可变区与氨基酸序列如SEQ ID NO：41所示的人IgG4(S228P)恒定区拼接而成的重链，和氨基酸序列如SEQ ID NO：39所示的轻链可变区以及氨基酸序列如SEQ ID NO：42所示的人kappa链恒定区拼接而成的轻链。
本发明的第二方面提供了一种核苷酸分子，具体方案为：
一种分离的核苷酸分子，所述核苷酸分子编码上述结合人CD39的抗体或其抗原结合片段。
本发明的核苷酸分子包括能够编码重链可变区的核苷酸分子如SEQ ID NO：26、30、34、38所示；和/或能够编码轻链可变区的核苷酸分子如SEQ ID NO：28、32、36、40所示。
本发明的核苷酸分子包括能够编码重链的核苷酸分子如SEQ ID NO：45、46、49、52、55所示；和/或能够编码轻链的核苷酸分子如SEQ ID NO：58、61、64、67所示。
本发明的核苷酸分子的制备方法为本领域内常规的制备方法如通过基因克隆技术(PCR方法)或者人工序列合成。本领域技术人员知晓，编码上述结合人CD39的抗体或其抗原结合片段的氨基酸序列的核苷酸序列可以适当引入替换、缺失、改变、插入或增加来提供一个多聚核苷酸的同系物。多聚核苷酸的同系物可以通过对编码该结合人CD39的抗体或其抗原结合片段基因的一个或多个碱基在保持抗体活性范围内进行替换、缺失或增加来制得。
本发明的第三方面提供了一种表达载体，具体方案为：
一种表达载体，所述表达载体含有上述的核苷酸分子。
本发明的表达载体为本领域常规的表达载体，如包含适当的调控序列如启动子序列、终止子序列等的表达载体。所述表达载体可以是病毒或质粒。
本发明的第四方面提供了一种宿主细胞，具体方案为：
一种宿主细胞，所述宿主细胞含有上述的表达载体。
本发明的宿主细胞为本领域常规的各种宿主细胞如原核表达细胞和真核表达细胞，只要能满足使上述表达载体稳定地自行复制，且所携带的核苷酸可被有效表达即可。
本发明的第五方面提供了一种制备上述结合人CD39所述抗体或其抗原结合片段的方法，所述方法包括以下步骤：
a)在表达条件下，培养如上述的宿主细胞，从而表达所述的结合人CD39的抗体或其抗原结合片段；
b)分离并纯化a)所述的结合人CD39的抗体或其抗原结合片段。
本发明所述的宿主细胞的培养方法、所述抗体的分离和纯化方法为本领域常规方法。
本发明的第六方面提供了一种药物组合物，该药物组合物含有上述结合人CD39的抗体或其抗原结合片段和药学上可接受的载体或辅料。
本发明提供的结合人CD39的抗体或其抗原结合片段，可以和药学上可以接受的载体或辅料一起组成药物组合物从而更稳定地发挥疗效，这些制剂可以保证本发明公开的结合人CD39的抗体或其抗原结合片段的构像完整性，同时还保护蛋白质的多官能团防止其降解(包括但不限于凝聚、脱氨或氧化)。该药物组合物的形式为制药领域常用的混悬、水针、冻干等制剂。
本发明的第七方面提供了一种上述结合人CD39的抗体或其抗原结合片段或药物组合物在制备抗肿瘤药物中的应用。所述肿瘤包括淋巴瘤、肉瘤、慢性淋巴细胞白血病、肺癌、胰腺癌、卵巢癌、肾癌、甲状腺癌和睾丸癌等。
本发明的第八方面提供了一种肿瘤的治疗方法，包括以下步骤：给需要的对象施用如本发明的第一方面所述的结合人CD39的抗体或其抗原结合片段，或如本发明的第六方面所述的药物组合物。
所述肿瘤为CD39高表达的肿瘤。在本发明的某些实施方式中，所述肿瘤包括淋巴瘤、肉瘤、慢性淋巴细胞白血病、肺癌、胰腺癌、卵巢癌、肾癌、甲状腺癌、黑色素瘤或睾丸癌等。
有益效果：本发明通过小鼠免疫、杂交瘤细胞制备筛选、抗体人源化、药效学研究等开发特异性靶向CD39的治疗抗体，该抗体将通过维持肿瘤微环境中的ATP浓度，抑制腺苷的生成，以减少免疫抑制、提高免疫效应，发挥协同抗肿瘤作用，具有较好的应用前景。
附图说明
图1～图3为实施例3中不同鼠源抗体与CD39-His蛋白的结合曲线；
图4～图6为实施例4中不同鼠源抗体以及对照抗体抑制293E-CD39细胞表面CD39蛋白活性测试结果；
图7为实施例6中嵌合抗体以及对照抗体对外源CD39蛋白酶活性的抑制作用测试结果；
图8为实施例7中嵌合抗体以及对照抗体对SKMEL-28细胞表面CD39蛋白酶活性的抑制作用测试结果；
图9～图11为实施例9中人源化抗体以及对照抗体对CD39蛋白结合曲线；
图12～图13为实施例10中人源化抗体对外源CD39蛋白酶活性的抑制作用测试结果；
图14为实施例11中人源化抗体以及对照抗体对肿瘤细胞SKMEL-28表面CD39蛋白酶活性的抑制作用测试结果；
图15为实施例11中人源化抗体以及对照抗体对肿瘤细胞MOLP-8表面CD39蛋白酶活性的抑制作用测试结果；
图16为实施例12中人源化抗体以及对照抗体对CD14+单核细胞表面CD39蛋白酶活性的抑制作用测试结果；
图17为实施例13中人源化抗体以及对照抗体对B细胞表面CD39蛋白酶活性的抑制作用测试结果；
图18为实施例14中人源化抗体以及对照抗体对巨噬细胞表面CD39蛋白酶活性的抑制作用测试结果；
图19为实施例14中人源化抗体以及对照抗体对巨噬细胞分泌IL-1β的促进作用测试结果；
图20为实施例15中人源化抗体促进DC细胞的成熟并促进CD4+T细胞分泌IFN-γ的检测结果；
图21为实施例15中人源化抗体促进DC细胞的成熟并促进CD8+T细胞分泌IFN-γ的检测结果；
图22～图23为实施例17中人源化抗体与不同种属CD39蛋白的交叉反应实验结果。
具体实施方式
本发明中，术语“表达载体”为本领域常规的表达载体，是指包含适当的调控序列，例如启动子序列、终止子序列、多腺苷酰化序列、增强子序列、标记基因和/或序列以及其他适当的序列的表达载体。所述表达载体可以是病毒或质粒，如适当的噬菌体或者噬菌粒，更多技术细节请参见例如Sambrook等，Molecular Cloning：A Laboratory Manual，第二版，Cold Spring Harbor Laboratory Press，1989。许多用于核酸操作的已知技术和方案请参见Current Protocols in Molecular Biology，第二版，Ausubel等编著。
本发明所述表达载体较佳地为选自pCGS3，pcDNA3.4，pDR1，pcDNA3.1(+)，pcDNA3.1/ZEO(+)，pDHFR或pTT5。
本发明中，术语“宿主细胞”含有上述表达载体，为本领域常规的各种宿主细胞，只要能满足使上述重组表达载体稳定地自行复制，且所携带所述的核苷酸可被有效表达即可。其中所述宿主细胞包括原核表达细胞和真核表达细胞，较佳地包括：COS、CHO(中国仓鼠卵巢，Chinese Hamster Ovary)、NS0、sf9、sf21、DH5α、BL21(DE3)或TG1，更佳地为E.coli TG1、BL21(DE3)细胞或者CHO-K1细胞。将前述表达载体转化至宿主细胞中，即可得本发明的结合人CD39的抗体或其抗原结合片段。
作为优选的方案，本发明所述宿主细胞是真核细胞，优选自CHO细胞，293T、293E和Expi293F细胞。
本发明中，术语“药物组合物”是指本发明的结合人CD39的抗体或其抗原结合片段和药学上可以接受的载体或辅料一起组成药物制剂组合物从而更稳定地发挥疗效，这些制剂可以保证本发明公开的结合人CD39的抗体或其抗原结合片段的氨基酸核心序列的构象完整性，同时还保护蛋白质的多官能团防止其降解(包括但不限于凝聚、脱氨或氧化)。有效量的本发明的结合人CD39的抗体或其抗原结合片段或其药物组合物施用受试者后，在治疗的个体中产生预期效果的量或剂量，该预期效果包括个体病症的改善。其中需要的对象或受试者包括但不限于哺乳动物，例如人、非人灵长类动物、大鼠和小鼠等。此外，本发明的结合人CD39的抗体或其抗原结合片段或其药物组合物还可与其他治疗剂一起使用。
“药学上可接受的”是指当分子本体和组合物适当地给予动物或人时，它们不会产生不利的、过敏的或其它不良反应。
“药学上可接受的载体或辅料”应当与所述有效成分相容，即能与其共混而不会在通常情况下大幅度降低药物的效果。可作为药学上可接受的载体或辅料的一些物质的具体例子是糖类，如乳糖、葡萄糖和蔗糖；淀粉，如玉米淀粉和土豆淀粉；纤维素及其衍生物，如甲基纤维素钠、乙基纤维素和甲基纤维素；西黄蓍胶粉末；麦芽；明胶；滑石；固体润滑剂，如硬 脂酸和硬脂酸镁；硫酸钙；植物油，如花生油、棉籽油、芝麻油、橄榄油、玉米油和可可油；多元醇，如丙二醇、甘油、山梨糖醇、甘露糖醇和聚乙二醇；海藻酸；乳化剂，如Tween；润湿剂，如月桂基硫酸钠；着色剂；调味剂；压片剂、稳定剂；抗氧化剂；防腐剂；无热原水；等渗盐溶液；磷酸盐缓冲液等。这些物质根据需要用于帮助配方的稳定性或有助于提高活性或它的生物有效性或在口服的情况下产生可接受的口感或气味。
本发明中，除非特别说明，药物剂型并无特别限定，可以被制成针剂、口服液、片剂、胶囊、滴丸、喷剂等剂型，可通过常规方法进行制备。药物剂型的选择应与给药方式相匹配。
“需要的对象”包括但不限于动物，优选为哺乳动物；所述哺乳动物优选为啮齿目动物、偶蹄目动物、奇蹄目动物、兔形目动物、灵长目动物等。所述哺乳动物包括例如人、非人灵长类动物(例如猴)、小鼠、猪、牛、山羊、兔、大鼠、豚鼠、仓鼠、马、猴、绵羊或其他非人哺乳动物；非哺乳动物，包括例如非哺乳动物脊椎动物，例如鸟(例如鸡或鸭)或鱼，以及非哺乳动物无脊椎动物。需要的对象可以是人类。
“治疗”包括预防或减轻某种状态，降低某种状态发生或发展的速度，减少发展出某种状态的风险，预防或延迟与某种状态相关的症状发展，减少或终止与某种状态相关的症状，产生某种状态的完全或部分的逆转，治愈某种状态，或以上的组合。
在向受试者施用时，给药剂量因病人的年龄和体重，疾病特性和严重性，以及给药途径而异，可以参考动物实验的结果和种种情况，总给药量不能超过一定范围。
在一些实施方式中，本文描述的方法可以进一步包括与其他化合物联合给予一种或多种另外的疗法(例如，一种或多种另外的治疗剂和/或一种或多种治疗方案)。一种或多种另外的疗法可以包括但不限于：手术，化学疗法，以及它们的组合。
本发明中，“CD39高表达”或“CD39过表达”是指CD39的表达量高于健康细胞。
以下通过特定的具体实例说明本发明的实施方式，本领域技术人员可由本说明书所揭露的内容轻易地了解本发明的其他优点与功效。本发明还可以通过另外不同的具体实施方式加以实施或应用，本说明书中的各项细节也可以基于不同观点与应用，在没有背离本发明的精神下进行各种修饰或改变。
在进一步描述本发明具体实施方式之前，应理解，本发明的保护范围不局限于下述特定的具体实施方案；还应当理解，本发明实施例中使用的术语是为了描述特定的具体实施方案，而不是为了限制本发明的保护范围；在本发明说明书和权利要求书中，除非文中另外明确指出，单数形式“一个”、“一”和“这个”包括复数形式。
当实施例给出数值范围时，应理解，除非本发明另有说明，每个数值范围的两个端点以 及两个端点之间任何一个数值均可选用。除非另外定义，本发明中使用的所有技术和科学术语与本技术领域技术人员通常理解的意义相同。除实施例中使用的具体方法、设备、材料外，根据本技术领域的技术人员对现有技术的掌握及本发明的记载，还可以使用与本发明实施例中所述的方法、设备、材料相似或等同的现有技术的任何方法、设备和材料来实现本发明。
实施例不包括对传统方法的详细描述，如那些用于构建载体和质粒的方法，将编码蛋白的基因插入到这样的载体和质粒的方法或将质粒引入宿主细胞的方法，亦或是在知晓抗体序列的前提下利用工程化方法制备抗体，这样的方法为本领域常规技术。
以下实施例中使用的实验材料均为市售常规产品，具体来源说明如下：
(1)小鼠骨髓瘤细胞SP2/0：购自ATCC，货号CRL-1581；(2)Balb/c小鼠：购自上海灵畅生物科技有限公司；(3)SKMEL-28细胞：购自ATCC，货号HTB-72；(4)MOLP-8细胞：购自南京科佰生物，货号CBP60562；(5)CD39+-293细胞为抗体蛋白工程部实验室构建；(6)HRP-山羊抗人IgG Fc二抗：购自Sigma，货号A0170-1ML；(7)FITC标记山羊抗人IgG-Fc二抗：购自Abcam，货号97224；(8)TMB：购自KPL公司，货号52-00-03；(9)牛血清白蛋白(BSA)：购自生工，货号A600332-0100；(10)RPMI 1640 Medium：购自Gibco公司，货号61870127；(11)青霉素-链霉素(Penicillin-streptomycin)：购自Gibco公司，货号15140122；(12)胎牛血清(FBS)：购自Gibco公司，货号10091-148；(13)polyethylene glycol solution购自sigma公司，货号P7181；(14)Hybridoma-SFM购自life technologies，货号12045-076；(15)HAT：购自Gibco，货号21060017；(16)pcDNA 3.4：购自ThermoFisher，货号A14697；(17)HEK-293F：购自Thermo Fisher，货号A14527；(18)Streptavidin HRP:购自BD Pharmingen，货号554066(19)EZ-Link NHS-Biotin Reagent：购自Thermo Fisher，货号20217；(20)Cell Titer-Glo：购自Promega，货号G7570；(21)ATP：购自Sigma，货号A7655；(22)HBS-EP pH7.4缓冲液：购自GE Healthcare，货号BR-1006-69；(23)Protein A/G的芯片：购自GE Healthcare，货号BR-1005-30；(24)CD19 MicroBeads，human：购自Miltenyi Biotec，货号130-050-301；(25)CD14 MicroBeads，human：购自Miltenyi Biotec，货号130-050-201；(26)CD4 MicroBeads,human：购自Miltenyibiotec，货号130-045-101；(27)CD8 MicroBeads，human：购自Miltenyibiotec，货号130-097-057；(28)LS Columns plus tubes：购自Miltenyi Biotec，130-122-7291。
实施例1对照抗体的制备
对照抗体TTX-030的重链和轻链可变区氨基酸序列来自于专利WO 2019027935A1，利用常规的基因合成和分子克隆的方法将其重链可变区与人IgG4(S228P)恒定区(氨基酸序列如 SEQ ID NO：41所示)相连，轻链可变区与人kappa链(氨基酸序列如SEQ ID NO：42所示)恒定区相连，并在其N端加上信号肽序列构建到pcDNA 3.4表达载体中，分别获得表达TTX-030的重链质粒TTX-030-HC和轻链质粒TTX-030-LC，将其重链质粒、轻链质粒共转染HEK-293F细胞，表达5d后，收集细胞培养上清通过Protein A亲和层析柱纯化获得TTX-030蛋白。对照抗体TTX-030的重链氨基酸序列如SEQ ID NO：70所示，轻链氨基酸序列如SEQ ID NO：71所示。
实施例2抗原免疫动物以及杂交瘤的制备和筛选
(1)抗原的表达
通过常规的基因合成和分子克隆的方法将CD39的胞外区基因(序列来自UniProt数据库，登记号为P49961，CD39胞外区氨基酸序列T38-V478如SEQ ID NO：72所示)构建到pcDNA 3.4表达载体中，并在其N端加上信号肽序列，C末端加上6×His标签，转染HEK-293F细胞，表达5d后，收集细胞培养上清并通过Ni+亲和层析柱纯化获得CD39-His蛋白。
(2)抗原免疫小鼠
用上述制备的人CD39-His蛋白(纯度>95％)常规免疫Balb/c小鼠。第1天，可溶性人CD39-His蛋白与弗氏完全佐剂乳化后，对Balb/c小鼠进行皮下多点注射(人CD39-His 100μg/鼠/0.5mL)，第14天，可溶性人CD39-His蛋白与弗氏不完全佐剂乳化后，对Balb/c小鼠进行皮下注射(人CD39-His 50μg/鼠/0.5mL)，在第28天，可溶性人CD39-His蛋白与弗氏不完全佐剂乳化后，对Balb/c小鼠进行皮下注射(人CD39-His 50μg/鼠/0.5mL)，三周后可溶性人CD39-His蛋白，50μg/小鼠/0.2mL，腹腔内注射激发，3-4天后，取小鼠脾脏进行融合实验。
(3)杂交瘤细胞制备和筛选
在小鼠末次免疫后3-4天，使用常规的杂交瘤技术方案，将小鼠脾细胞与小鼠骨髓瘤细胞SP2/0进行PEG融合。融合后的细胞在完全培养基中悬浮均匀，完全培养基为将RPMI1640-GLUMAX加入1％Penicillin-streptomycin(青霉素-链霉素)，20％FBS(胎牛血清)，1*HAT组成的培养基。融合后的细胞按3×104个细胞/200μL/孔，共铺60块96孔培养板中培养。在7-12天后，收获上清液，通过ELISA方法筛选人CD39结合活性阳性的杂交瘤孔。
其中，ELISA方法筛选人CD39结合活性阳性的杂交瘤孔的方法如下：将CD39-His蛋白以PBS缓冲液稀释至2μg/mL，100μL/孔加入板中，4℃培养过夜；次日甩掉上清，加入5％脱脂奶粉37℃封闭2小时，PBST洗板3次待用；将收取的杂交瘤上清液依次加入封闭后的板中，100μL/孔，37℃放置1h；PBST洗板3次，加入HRP标记的羊抗鼠IgG二抗，37℃放置30min；PBST洗板3次后，在吸水纸上尽量拍干残留液滴，每孔加入100μL的TMB，室温避光放置5min； 每孔加入50μL 2M H2SO4终止液终止底物反应，于酶标仪450nm处读取OD值，分析待测抗体与靶抗原CD39结合能力。将含血清完全培养基中扩增筛选获得的杂交瘤细胞株，离心换液至无血清培养液Hybridoma-SFM培养基，使细胞密度为1～2×107/mL，在8％CO2、37℃条件下培养1周，离心获取培养上清，通过Protein G亲和层析进行纯化，得到多种抗人CD39蛋白的鼠源抗体。本轮筛选共计拿到28株杂交瘤细胞株。
实施例3鼠源抗体对人CD39-his蛋白的结合能力测试
对实施例2得到的各鼠源抗体进行命名后并利用间接酶联免疫吸附测定法(ELISA)测定其对人CD39-His蛋白的结合能力。具体测定方法如下：将CD39-His蛋白以包被液(50mM的碳酸盐包被缓冲液，pH 9.6)稀释至2μg/mL，4℃包被ELISA板过夜；再用5％的脱脂奶粉封闭，37℃孵育2小时；PBST洗板3次后，将抗人CD39蛋白的鼠源抗体以1％BSA缓冲液梯度稀释，按100μL/孔加入预包被CD39-His蛋白的板子中，37℃孵育一小时；PBST洗板3次，加入HRP标记的羊抗鼠IgG二抗，37℃放置30min；PBST洗板3次后，在吸水纸上尽量拍干残留液滴，每孔加入100μL的TMB，室温避光放置5min，每孔加入50μL 2M H2SO4终止液终止底物反应，酶标仪450nm处读取OD值，分析待测各鼠源抗体与抗原人CD39-His的结合能力。
各鼠源抗体与CD39-His蛋白结合曲线如图1～3所示，EC50和结合活性高平台值(Top)如表1～表3所示。由图1～图3可知，鼠源抗体302B4H10、317B1A5、321D9E5、339E8A9、343E7G6、344E2C6、346A1B12、355F9D6、406A10E3、439G11A9、435H6H8、453D9A3、426F10E2、403A1B3、431H1F7、438C1B6、52A12H7F1与靶抗原CD39-His结合活性相对较优。
表1各鼠源抗体与CD39-His蛋白结合的EC50
表2：鼠源抗体与CD39-His蛋白结合的EC50
表3：鼠源抗体与CD39-His蛋白结合的EC50
实施例4鼠源抗体抑制细胞表面CD39蛋白的酶活性检测
利用检测ATP的方法测定各鼠源抗体与对照抗体TTX-030对CD39酶活性的抑制作用，具体方法如下：收集过表达CD39的HEK293E细胞(293E-CD39)，300g离心去除细胞培养液，用PBS缓冲液洗涤1遍；计数并用Tris buffer(25mM Tris，5mM CaCl2，PH 7.5)稀释至1×104/孔，铺细胞至96孔细胞培养板中，离心弃细胞培养上清；将各待测鼠源抗体与对照抗体TTX-030用Tris buffer稀释至10μg/mL，3倍梯度稀释，100uL/孔，共11孔，加入培养板中，37℃培养箱孵育60min；离心弃上清，向细胞培养板中加入ATP终浓度50μM 200μL/孔，37℃培养箱孵育60min，离心取50μL培养上清于96孔白色不透光检测板中，加入50μL的Cell Titer-Glo检测试剂，室温孵育5min，于多功能酶标仪中读取荧光强度并分析。
各鼠源抗体的抑制酶活性曲线如图4～图6所示，对应的酶活性IC50以及最大抑制活性(Top)结果如表4～表6所示。由图4～图6可知，鼠源抗体419H10A4、439G11A9、52A12H7F1、2D2G2F2可以明显抑制293E-CD39细胞表面的CD39酶的活性，且439G11A9、52A12H7F1的活性明显优于对照抗体TTX-030。
表4：鼠源抗体抑制293E-CD39细胞表面CD39蛋白的酶活IC50
NA:数据不可用
表5：鼠源抗体抑制293E-CD39细胞表面CD39蛋白的酶活IC50
表6：鼠源抗体抑制293E-CD39细胞表面CD39蛋白的酶活IC50
实施例5候选抗体可变区基因获取及嵌合抗体的制备
(1)候选抗体可变区基因的获取
通过分子生物学的相关方法获取四种具有较好效果的候选鼠源抗体419H10A4、 439G11A9、52A12H7F1、2D2G2F2的重链可变区和轻链可变区，具体方案如下：
通过Trizol提取各杂交瘤细胞的RNA，并利用Oligo-dT引物进行mRNA反转录获取cDNA，随后以cDNA为模板，分别用鼠源抗体的重链和轻链简并引物(《Antibody Engineering》Volume 1，Editedby RolandKontermann and Stefan Dübel，组合引物的序列来自第323页)进行PCR，对所获得的PCR产物进行测序并通过kabat数据库分析，确定所获得的序列为鼠源抗体的可变区序列。各鼠源抗体的具体信息为：
419H10A4：重链可变区氨基酸序列如SEQ ID NO：25所示，HCDR1、HCDR2、HCDR3的氨基酸序列分别如SEQ ID NO：1、2、3所示，重链可变区编码核苷酸序列(全长为369bp，编码123个氨基酸残基)如SEQ ID NO：26所示；轻链可变区氨基酸序列如SEQ ID NO：27所示，LCDR1、LCDR2、LCDR3的氨基酸序列分别如SEQ ID NO：4、5、6所示，轻链可变区编码核苷酸序列(全长为321bp，编码107个氨基酸残基)如SEQ ID NO：28所示。
439G11A9：重链可变区氨基酸序列如SEQ ID NO：29所示，HCDR1、HCDR2、HCDR3的氨基酸序列分别如SEQ ID NO：7、8、9所示，重链可变区编码核苷酸序列(全长为366bp，编码122个氨基酸残基)如SEQ ID NO：30所示；轻链可变区氨基酸序列如SEQ ID NO：31所示，LCDR1、LCDR2、LCDR3的氨基酸序列分别如SEQ ID NO：10、11、12所示，轻链可变区编码核苷酸序列(全长为333bp，编码111个氨基酸残基)如SEQ ID NO：32所示。
52A12H7F1：重链可变区氨基酸序列如SEQ ID NO：33所示，HCDR1、HCDR2、HCDR3的氨基酸序列分别如SEQ ID NO：13、14、15所示，重链可变区编码核苷酸序列(全长为360bp，编码120个氨基酸残基)如SEQ ID NO：34所示；轻链可变区氨基酸序列如SEQ ID NO：35所示，LCDR1、LCDR2、LCDR3的氨基酸序列分别如SEQ ID NO：16、17、18，轻链可变区编码核苷酸序列(全长为336bp，编码112个氨基酸残基)如SEQ ID NO：36所示。
2D2G2F2：重链可变区氨基酸序列如SEQ ID NO：37所示，HCDR1、HCDR2、HCDR3的氨基酸序列分别如SEQ ID NO：19、20、21所示，重链可变区编码核苷酸序列(全长为351bp，编码117个氨基酸残基)如SEQ ID NO：38所示；轻链可变区氨基酸序列如SEQ ID NO：39所示，LCDR1、LCDR2、LCDR3的氨基酸序列分别如SEQ ID NO：22、23、24所示，轻链可变区编码核苷酸序列(全长为318bp，编码106个氨基酸残基)如SEQ ID NO：40所示。
(2)嵌合抗体的制备
将四种候选鼠源抗体的重链可变区序列分别与人的IgG4(S228P)恒定区(氨基酸序列如SEQ ID NO：41所示)拼接，轻链可变区序列与人的kappa链恒定区(氨基酸序列如SEQ ID NO：42所示)拼接，分别构建各嵌合抗体的重链和轻链至pcDNA3.4表达载体，共转染HEK-293F 细胞表达并纯化获得各嵌合抗体，鼠源抗体419H10A4、439G11A9、52A12H7F1、2D2G2F2对应的嵌合抗体分别命名为419H-ch、439G-ch、52A12-ch、2D2-ch。
实施例6嵌合抗体对外源CD39蛋白酶活性的抑制作用
本实施例采用检测ATP的方法测定各嵌合抗体以及对照抗体对外源游离CD39蛋白酶活性的抑制作用，具体测定方法如下：将上述制备存储的CD39-His蛋白重悬于0.01％BSA25mM Tris，5mM CaCl2，pH 7.5(Tris buffer)中，以160nM的浓度，每孔25μL加到白色不透光96孔板中；用Tris buffer按照3倍梯度稀释待测抗体，各组均连续稀释12梯度，各抗体最高工作浓度为1μM，加入25μL稀释后的待测抗体于上述96孔板，每孔终体积为50μL；将96孔板置于室温孵育2小时；孵育结束后，用Tris buffer稀释ATP至1000μM，并加入96孔板中，50μL/孔，混匀，置于37℃孵育60min；随后以1:1的体积比直接加入Cell Titer-Glo试剂至96孔板，于多功能酶标仪中读取荧光强度并分析数据。
各嵌合抗体对外源CD39蛋白酶活性的抑制测试结果如图7所示，对应的酶活性IC50以及最大抑制活性(Top)结果如表7所示。
表7各嵌合抗体抑制外源CD39蛋白酶活性的IC50
实施例7嵌合抗体抑制肿瘤细胞表面CD39酶活性检测
收集处于对数生长期的恶性黑色素瘤细胞SKMEL-28，离心去除细胞培养液，用PBS洗涤细胞1遍；计数并用完全培养基(1640+10％FBS+1％Pen Strep+1％Sodium Pyruvate+1％GlutaMAX)重悬，以3.5×104个细胞/孔接种到圆底96细胞培养孔板中，100μL/孔，于细胞培养箱37℃培养过夜，次日弃细胞培养上清，将待测嵌合抗体以及对照抗体用Tris buffer稀释至1000nM(SKMEL-28)，3倍梯度稀释，随后按照100μL/孔，加入上述细胞培养板中，于37℃孵育60min；再加入终浓度50μM的ATP，50μL/孔，37℃孵育60min，取50μL培养上清于96孔白色不透光检测板中，加入50μL的Cell Titer-Glo检测试剂，室温孵育5min，于多功能酶标仪中读取荧光强度并分析。
嵌合抗体以及对照抗体对SKMEL-28细胞表面CD39蛋白酶活性的抑制测试曲线如图8所示，对应的酶活性IC50以及最大抑制活性(Top)结果如表8所示。
表8：各嵌合抗体对SKMEL-28细胞表面CD39蛋白酶活抑制的IC50

实施例8人源化抗体的构建和制备
对鼠源抗体439G11A9轻链可变区(氨基酸序列如SEQ ID NO：31所示)和重链可变区(氨基酸序列如SEQ ID NO：29所示)的氨基酸序列进行分析，依据Kabat规则确定439G11A9的重链、轻链抗原结合互补决定区(CDR)和框架区(FR)。重链可变区氨基酸的互补决定区HCDR1、HCDR2、HCDR3的氨基酸序列分别如SEQ ID NO：7、8、9所示，轻链可变区的互补决定区LCDR1、LCDR2、LCDR3的氨基酸序列分别如SEQ ID NO：10、11、12所示。
在Germline数据库中选取与439G11A9抗体重、轻链可变区非CDR序列匹配最好的人IgG Germline序列模板，其中重链可变区选择IGHV14-3*02作为人源化模板，轻链可变区选择IGKV3-2*01作为人源化模板，然后将439G11A9抗体重、轻链的CDR区分别移植到所选择的人模板上，替换人源化模板中的CDR区，同时以该抗体的三维结构为基础，对包埋残基、与CDR区有直接相互作用的残基，以及对抗体的VL和VH的构象有重要影响的残基进行回复突变，最终获得多个人源化抗体的重、轻链可变区；将各人源化重链可变区分别与人IgG4(S228P)恒定区重组，各人源化轻链可变区与人的kappa链恒定区重组，并构建至pcDNA3.4表达载体中，给自配对共转染HEK-293F细胞表达并纯化以获得各人源化抗体。439G11A9的各人源化抗体重、轻链命名、核苷酸和氨基酸序列如下：
重链：
(1)439G-HC-Hu：氨基酸序列如SEQ ID NO：44所示，其可变区氨基酸序列如SEQ ID NO：43所示，编码核苷酸序列如SEQ ID NO：45所示。
(2)439G-HC-G：氨基酸序列如SEQ ID NO：48所示，其可变区氨基酸序列如SEQ ID NO：47所示，编码核苷酸序列如SEQ ID NO：46所示。
(3)439G-HC-HuME：氨基酸序列如SEQ ID NO：51所示，其可变区氨基酸序列如SEQ ID NO：50所示，编码核苷酸序列如SEQ ID NO：49所示。
(4)439G-HC-HuMK：氨基酸序列如SEQ ID NO：54所示，其可变区氨基酸序列如SEQ ID NO：53所示，编码核苷酸序列如SEQ ID NO：52所示。
(5)439G-HC-HuML：氨基酸序列如SEQ ID NO：57所示，其可变区氨基酸序列如SEQ ID NO：56所示，编码核苷酸序列如SEQ ID NO：55所示。
轻链：
(1)439G-LC-G：氨基酸序列如SEQ ID NO：60所示，其可变区氨基酸序列如SEQ ID NO：59所示，编码核苷酸序列如SEQ ID NO：58所示。
(2)439G-LC-HuM100Y：氨基酸序列如SEQ ID NO：63所示，其可变区氨基酸序列如SEQ ID NO：62所示，编码核苷酸序列如SEQ ID NO：61所示。
(3)439G-LC-HuMut3：氨基酸序列如SEQ ID NO：66所示，其可变区氨基酸序列如SEQ ID NO：65所示，编码核苷酸序列如SEQ ID NO：64所示。
(4)439G-LC-HuM85E：氨基酸序列如SEQ ID NO：69所示，其可变区氨基酸序列如SEQ ID NO：68所示，编码核苷酸序列如SEQ ID NO：67所示。
各人源化抗体重、轻链组合配对表达获得的抗体命名如表9所示。
表9各人源化抗体重、轻链组合配对表达获得的抗体
实施例9人源化抗体对人CD39-His蛋白的结合活性
在此采用间接酶联免疫吸附测定法(ELISA)测定各人源化抗体对人CD39-His蛋白的结合能力。具体方法如下：
用包被液(50mM的碳酸盐包被缓冲液，pH 9.6)将CD39-his蛋白稀释至0.5μg/mL，包被ELISA板，100μL/孔，置于湿盒中，4℃，包被16h；PBST洗涤ELISA板三次，去除未结合抗原，并将ELISA板于吸水纸上拍干，除去多余的液体，然后用PBS配制的2％BSA，200μL/孔，于室温封闭2h；用PBST洗涤一次，洗除多余的封闭液，并将ELISA板拍干，除去多余的液体，用PBST配制的1％BSA(抗体稀释液)按照3倍梯度稀释各抗体，最高浓度为20nM，稀释12个梯度，加入ELISA孔，100μL/孔，室温孵育1h；洗除未结合的或非特异性结合的一抗，用抗体稀释液将HRP标记的Anti-Human Fc(1:5000)(sigma，A0170)二抗稀释至合适浓度，加入ELISA板，100μL/孔，室温孵育1h；用PBST洗涤五次，并将ELISA板于吸水纸上拍干，除去多余的液体，加入TMB显色液，100μL/孔，显色至合适深浅，加入 2M H2SO4，50μL/孔，以终止显色，于多功能酶标仪450nm处读取OD值，收集数据并分析待测抗体与靶抗原人CD39-His的结合能力。
实验结果如图9～11和表10～12所示。由图9和表10可知，人源化抗体中439G-HuG的亲和力显著强于439G-GG和对照抗体TTX-030。由图10～11和表11～12可知，人源化抗体中439G-Hu-HMELW100Y、439G-Hu-HMKLmut3对CD39蛋白的亲和力最优，显著强于其他人源化抗体和对照抗体TTX-030。
表10：人源化抗体对CD39蛋白结合的EC50
表11：人源化抗体对CD39蛋白结合的EC50
表12：人源化抗体对CD39蛋白结合的EC50
实施例10人源化抗体对外源CD39蛋白酶活性的抑制作用
参照实施例6测定各人源化抗体对外源CD39蛋白酶活性的抑制能力。
实验结果如图12～13和表13～14所示，人源化抗体439G-HuG、439G-HuG-LN85E、439G-Hu-HMELW100Y、439G-Hu-HMKLmut3、439G-Hu-HMLLmut3对外源游离CD39蛋白降解ATP活性的抑制作用相当，均明显优于对照抗体TTX-030。
表13：人源化抗体抑制外源CD39蛋白酶活性的IC50
表14：人源化抗体抑制外源CD39蛋白酶活性的IC50

实施例11人源化抗体抑制肿瘤细胞表面CD39蛋白酶活性检测
参照实施例7测定各人源化抗体对恶性黑色素瘤SKMEL-28和多发性骨髓瘤MOLP-8细胞表面CD39酶活性抑制能力。测试结果分别如图14～15以及表15～16所示。
表15：人源化抗体抑制肿瘤细胞SKMEL-28表面CD39蛋白酶活性的IC50
表16：人源化抗体抑制肿瘤细胞MOLP-8表面CD39蛋白酶活性的IC50
由图14和表15可知，人源化抗体439G-HuG、439G-HuG-LN85E、439G-Hu-HMELW100Y、439G-Hu-HMKLmut3、439G-Hu-HMLLmut3抑制SKMEL-28细胞表面CD39蛋白降解ATP的活性相当，显著优于对照抗体TTX-030。
由图15和表16可知，人源化抗体439G-HuG-LN85E、439G-Hu-HMELW100Y、439G-Hu-HMKLmut3、439G-Hu-HMLLmut3抑制MOLP-8表面CD39蛋白降解ATP的活性相当，稍优于439G-HuG，显著优于对照抗体TTX-030。
实施例12人源化抗体抑制CD14+单核细胞表面CD39蛋白酶活性的检测
本实施例通过检测ATP的方法测定抗体对PBMC中CD14+单核细胞表面CD39蛋白酶活性的抑制作用。具体方法如下：
根据CD14 MicroBeads说明书从人PBMC(购自上海赛笠生物科技有限公司)中分选获得CD14+单核细胞，离心去除细胞培养液，用PBS缓冲液洗涤细胞1遍，计数并用完全培养基(1640+10％FBS+1％Pen Strep+1％Sodium Pyruvate+1％GlutaMAX)重悬，以1.0×105个细胞/孔接种到圆底96孔板中，100μL/孔，于细胞培养箱37℃培养过夜；次日，弃细胞培养上清，将待测抗体用Tris buffer稀释至100nM，3倍梯度稀释，随后按照100μL/孔，加入上 述细胞培养板中，于37℃孵育60min；再加入终浓度20μM的ATP，50μL/孔，37℃孵育60min，取50μL培养上清于96孔白色不透光检测板中，加入50μL的Cell Titer-Glo检测试剂，室温孵育5min，于多功能酶标仪中读取荧光强度并分析。
实验结果如图16和表17所示，人源化抗体439G-HuG-LN85E、439G-Hu-HMELW100Y、439G-Hu-HMKLmut3抑制CD14+单核细胞表面CD39蛋白降解ATP的活性相当，稍优于439G-HuG和439G-Hu-HMLLmut3，显著优于对照抗体TTX-030。
表17：人源化抗体对CD14+单核细胞表面CD39蛋白酶活抑制的IC50
实施例13人源化抗体抑制B细胞表面CD39蛋白酶活性的检测
根据CD19MicroBeads说明书从人PBMC(购自上海赛笠生物科技有限公司)中分选得到的B细胞，离心去除细胞培养液，用PBS缓冲液洗涤细胞1遍；计数并用完全培养基(1640+10％FBS+1％Pen Strep+1％Sodium Pyruvate+1％GlutaMAX)重悬，细胞以5.0×104个细胞/孔接种到圆底96孔板中，100μL/孔，于细胞培养箱37℃培养过夜；次日，弃细胞培养上清，将待测抗体用Tris buffer稀释至100nM，3倍梯度稀释，随后按照100μL/孔，加入上述细胞培养板中，于37℃孵育60min；再加入终浓度50μM的ATP，50μL/孔，37℃孵育60min，取50μL培养上清于96孔白色不透光检测板中，加入50μL的Cell Titer-Glo检测试剂，室温孵育5min，于多功能酶标仪中读取荧光强度并分析。
实验结果如图17和表18所示，各个人源化抗体对B细胞表面CD39蛋白降解ATP的活性抑制均显著强于对照抗体TTX-030，其中以439G-Hu-HMKLmut3稍优。
表18：人源化抗体对B细胞表面CD39蛋白酶活抑制的IC50
实施例14人源化抗体抑制巨噬细胞表面CD39蛋白酶活性的检测
根据CD14 MicroBeads说明书从人PBMC(购自上海赛笠生物科技有限公司)中分选 CD14+的单核细胞。将分选出的CD14+单核细胞重悬至含有400ng/mL GM-CSF的RPMI-1640完全培养基中(含10％FBS的RPMI-1640培养基)，添加20mL细胞悬液至T75细胞培养瓶中诱导分化，细胞密度为1.5×105个/mL；在第七天，用含有400ng/mL GM-CSF的新鲜完全培养基进行半换液；在第十天，移除细胞培养上清，用无血清培养基RPMI-1640洗涤细胞一遍，加入3mL TrypLE Select消化上述细胞，待细胞脱落，用完全培养基终止消化，离心，用PBS清洗细胞两遍，所得细胞即为巨噬细胞。将巨噬细胞用完全培养基重悬，调整细胞密度为5×105/mL，每孔100μL，铺于圆底96孔板中；用完全培养基梯度稀释待测抗体，抗体的最高浓度为1000nM，按照3倍梯度稀释12个梯度；将上述铺好巨噬细胞的96孔板离心，完全弃去细胞上清，再加入梯度稀释好的待测抗体，待测抗体每孔加入50μL，并置于细胞培养箱孵育1h；用完全培养基配置终浓度为10ng/mL的LPS溶液，每孔50μL加入到上述96孔板中，并置于细胞培养箱中培养3h；用完全培养基配置终浓度为1000μM的ATP溶液，每孔100μL加入到96孔板中，并置于细胞培养箱中培养2h；培养结束后，收集细胞上清，用于检测IL-1β，并取50μL上清加入50μL的Cell Titer-Glo检测试剂，室温孵育5min，于多功能酶标仪中读取荧光强度用于分析测定上清中ATP水平。
实验结果如图18所示，人源化抗体439G-HuG、439G-Hu-HMKLmut3、439G-HuG-LN85E可有效抑制巨噬细胞表面CD39蛋白酶活性以维持胞外的ATP浓度。
如图19和表19所示，在LPS存在情况下，各人源化抗体通过抑制巨噬细胞表面CD39蛋白酶活性保留的ATP可有效刺激巨噬细胞分泌IL-1β。
表19：人源化抗体促进巨噬细胞分泌IL-1β的IC50
实施例15人源化抗体对T细胞活化作用
根据CD14 MicroBeads说明书从人PBMC(购自上海赛笠生物科技有限公司)中分选获得CD14+单核细胞，用PBS缓冲液洗涤细胞1遍并重悬至30mL含有400ng/mLGM-CSF和20ng/mL IL-4的1640完全培养基中加入至T75细胞培养瓶中诱导分化，细胞密度为1×105个/mL；在第七天，将弃掉细胞培养基上清一半，换成新的含有400ng/mL GM-CSF和20ng/mL IL-4的1640完全培养基；在第十天，收取悬浮细胞，用无血清培养基1640清洗一遍；计数细胞数量和活率。将DC细胞按照1×104个细胞/孔铺制于96孔圆底细胞培养板中，100μL/孔，用10％FBS 1640培养基梯度稀释待测抗体，并加入上述96孔板中，100μL/孔，将96孔 板置于细胞培养箱中孵育1h；用10％FBS 1640培养基配置终浓度为1000μM的ATP溶液，每孔50μL加入到96孔板中，将96孔板置于细胞培养箱中培养48h，培养结束后，离心去除细胞上清，并用PBS洗涤DC细胞2次。
根据CD4 MicroBeads、CD8MicroBeads说明书从另一批人PBMC(购自上海赛笠生物科技有限公司)中分选分别获得CD4+和CD8+T细胞。将CD4+T和CD8+T细胞分别用1640完全培养基重悬，按照DC：T＝1：10的比例加入到上述含有1×104个DC细胞/孔的96孔板中，100μL/孔，同时所有孔均加入100IU/mL的IL-2，将细胞板置于37℃细胞培养箱中培养5天，收集细胞上清用于IFN-γ的检测。
实验结果如图20所示，439G-HuG、439G-Hu-HMKLmut3、439G-HuG-LN85E均能显著促进DC细胞的成熟并剂量依赖性的促进CD4+T细胞分泌IFN-γ。
如图21所示，439G-HuG、439G-Hu-HMKLmut3、439G-HuG-LN85E亦能显著促进DC细胞的成熟并剂量依赖性的促进CD8+T细胞分泌IFN-γ。
实施例16人源化抗体对CD39蛋白的结合动力学检测
利用共价偶联有Protein A的芯片(购自GE Healthcare，货号29139131-AA)捕获抗体，相关运行参数如下：抗体浓度为1μg/mL，接触时间60s，流速10μL/min，再生接触时间为30s；利用HBS-EP+pH7.4缓冲液稀释CD39抗原，最高浓度为80nM，按照2.5倍稀释至0.8192nM及0浓度点，采用6M盐酸胍溶液作为再生缓冲液，在Biacore 8K上按照如下参数进样，结合时间180s，解离时间720s，流速30L/min，再生接触时间为30s；收集数据，利用Biacore 8K Evaluation Software对数据进行分析。
结果如表20所示，各人源化抗体对CD39蛋白的结合动力学明显优于对照抗体TTX-030，主要体现在解离速度更慢，其中尤以439G-Hu-HMKLmut3更为突出，结合快，解离慢。
表20：人源化抗体对CD39蛋白的结合动力学
实施例17人源化抗体种属交叉反应
用ELISA包被液将人(Human)、猕猴(Maca，序列来自NCBI数据库，登记号为XP_015311944)、小鼠(Mouse，序列来自UniProt数据库，登记号为P55772)、大鼠(Rat，序列来自NCBI数据库，登记号为XP_032747831.1)、猪(Pig，序列来自UniProt数据库， 登记号为Q9MYU4)CD39-His蛋白稀释至2μg/mL,包被ELISA板，100μL/孔，置于湿盒中，4℃，包被16h；用PBST洗涤ELISA板三次，去除未结合抗原，并将ELISA板于吸水纸上拍干，除去多余的液体，然后用PBS配制的2％BSA，200μL/孔，于室温封闭2h；用PBST洗涤一次，洗除多余的封闭液，并将ELISA板拍干，除去多余的液体，用PBST配制的1％BSA按3倍梯度待测抗体，最高浓度为20nM，3倍稀释11个梯度，按照100μL/孔加入ELISA孔，室温孵育1h，每个样品平行做2个复孔；洗除未结合的或非特异性结合的一抗，用抗体稀释液将HRP标记的Anti-Human Fc二抗按照1:5000稀释，100μL/孔加入ELISA孔，室温孵育1h；用PBST洗涤三次，并将ELISA板于吸水纸上拍干，除去多余的液体，加入TMB显色液，100μL/孔，显色至合适深浅，加入2M H2SO4，50μL/孔，以终止显色，并于多功能酶标仪中在450nm波长处测定其吸光度，分析数据。
如图22和23所示，人源化抗体439G-Hu-HMKLmut3与猕猴CD39蛋白(Maca CD39)具有交叉反应，EC50为0.145nM，与人CD39蛋白(Human)结合的EC50为0.172nM。
以上的实施例是为了说明本发明公开的实施方案，并不能理解为对本发明的限制。此外，本文所列出的各种修改以及发明中方法的变化，在不脱离本发明的范围和精神的前提下对本领域内的技术人员来说是显而易见的。虽然已结合本发明的多种具体优选实施例对本发明进行了具体的描述，但应当理解，本发明不应仅限于这些具体实施例。事实上，各种如上所述的对本领域内的技术人员来说显而易见的修改来获取发明都应包括在本发明的范围内。

Claims (18)

  1. 一种结合人CD39的抗体或其抗原结合片段，包括重链可变区和轻链可变区，其特征在于，所述重链可变区包括：
    氨基酸序列如SEQ ID NO：1、7、13或19所示的HCDR1；
    氨基酸序列如SEQ ID NO：2、8、14或20所示的HCDR2；
    氨基酸序列如SEQ ID NO：3、9、15或21所示的HCDR3；
    所述轻链可变区包括：
    氨基酸序列如SEQ ID NO：4、10、16或22所示的LCDR1；
    氨基酸序列如SEQ ID NO：5、11、17或23所示的LCDR2；
    氨基酸序列如SEQ ID NO：6、12、18或24所示的LCDR3。
  2. 根据权利要求1所述的结合人CD39的抗体或其抗原结合片段，其特征在于，所述重链可变区包括：
    氨基酸序列分别如SEQ ID NO：1、2及3所示的HCDR1、HCDR2及HCDR3；或
    氨基酸序列分别如SEQ ID NO：7、8及9所示的HCDR1、HCDR2及HCDR3；或
    氨基酸序列分别如SEQ ID NO：13、14及15所示的HCDR1、HCDR2及HCDR3；或
    氨基酸序列分别如SEQ ID NO：19、20及21所示的HCDR1、HCDR2及HCDR3；
    所述轻链可变区包括：
    氨基酸序列分别如SEQ ID NO：4、5及6所示的LCDR1、LCDR2及LCDR3；或
    氨基酸序列分别如SEQ ID NO：10、11及12所示的LCDR1、LCDR2及LCDR3；或
    氨基酸序列分别如SEQ ID NO：16、17及18所示的LCDR1、LCDR2及LCDR3；或
    氨基酸序列分别如SEQ ID NO：22、23及24所示的LCDR1、LCDR2及LCDR3。
  3. 根据权利要求1所述的结合人CD39的抗体或其抗原结合片段，其特征在于，包含：
    重链可变区包括氨基酸序列分别如SEQ ID NO：1、2及3所示的HCDR1、HCDR2及HCDR3，及轻链可变区包括氨基酸序列分别如SEQ ID NO：4、5及6所示的LCDR1、LCDR2及LCDR3；
    或
    重链可变区包括氨基酸序列分别如SEQ ID NO：7、8及9所示的HCDR1、HCDR2及HCDR3，及轻链可变区包括氨基酸序列分别如SEQ ID NO：10、11及12所示的LCDR1、LCDR2及LCDR3；
    或
    重链可变区包括氨基酸序列分别如SEQ ID NO：13、14及15所示的HCDR1、HCDR2及 HCDR3，及轻链可变区包括氨基酸序列分别如SEQ ID NO：16、17及18所示的LCDR1、LCDR2及LCDR3；
    或
    重链可变区包括氨基酸序列分别如SEQ ID NO：19、20及21所示的HCDR1、HCDR2及HCDR3，及轻链可变区包括氨基酸序列分别如SEQ ID NO：22、23及24所示的LCDR1、LCDR2及LCDR3。
  4. 根据权利要求1或2所述的结合人CD39的抗体或其抗原结合片段，其特征在于，包含：
    选自以下任一氨基酸序列的重链可变区：SEQ ID NO：25、29、33、37、43、47、50、53、56；和/或
    选自以下任一氨基酸序列的轻链可变区：SEQ ID NO：27、31、35、39、59、62、65、68。
  5. 根据权利要求1或2所述的结合人CD39的抗体或其抗原结合片段，其特征在于，包含：
    氨基酸序列如SEQ ID NO：25所示的重链可变区以及氨基酸序列如SEQ ID NO：27所示的轻链可变区；或
    氨基酸序列如SEQ ID NO：29所示的重链可变区以及氨基酸序列如SEQ ID NO：31所示轻链可变区；或
    氨基酸序列如SEQ ID NO：33所示的重链可变区以及氨基酸序列如SEQ ID NO：35所示的轻链可变区；或
    氨基酸序列如SEQ ID NO：37所示的重链可变区以及氨基酸序列如SEQ ID NO：39所示的轻链可变区；或
    氨基酸序列如SEQ ID NO：43所示的重链可变区以及氨基酸序列如SEQ ID NO：59所示的轻链可变区；或
    氨基酸序列如SEQ ID NO：47所示的重链可变区以及氨基酸序列如SEQ ID NO：59所示的轻链可变区；或
    氨基酸序列如SEQ ID NO：50所示的重链可变区以及氨基酸序列如SEQ ID NO：62所示的轻链可变区；或
    氨基酸序列如SEQ ID NO：53所示的重链可变区以及氨基酸序列如SEQ ID NO：65所示的轻链可变区；或
    氨基酸序列如SEQ ID NO：43所示的重链可变区以及氨基酸序列如SEQ ID NO：68所示的轻链可变区；或
    氨基酸序列如SEQ ID NO：56所示的重链可变区以及氨基酸序列如SEQ ID NO：65所示 的轻链可变区。
  6. 根据权利要求1或2所述的结合人CD39的抗体或其抗原结合片段，其特征在于，包含：
    选自如SEQ ID NO：44、48、51、54、57所示的任一氨基酸序列的重链，或者由氨基酸序列如SEQ ID NO：41所示的人IgG4恒定区与选自如SEQ ID NO：25、29、33、37所示任一氨基酸序列的重链可变区拼接而成的重链；
    选自如SEQ ID NO：60、63、66、69所示的任一氨基酸序列的轻链，或者由氨基酸序列如SEQ ID NO：42所示的人kappa链恒定区与选自如SEQ ID NO：27、31、35、39所示的任一氨基酸序列的轻链可变区拼接而成的轻链。
  7. 根据权利要求1或2所述的结合人CD39的抗体或其抗原结合片段，其特征在于，包含：
    氨基酸序列如SEQ ID NO：44所示的重链以及氨基酸序列如SEQ ID NO：60所示的轻链；或
    氨基酸序列如SEQ ID NO：48所示的重链以及氨基酸序列如SEQ ID NO：60所示的轻链；或
    氨基酸序列如SEQ ID NO：51所示的重链以及氨基酸序列如SEQ ID NO：63所示的轻链；或
    氨基酸序列如SEQ ID NO：54所示的重链以及氨基酸序列如SEQ ID NO：66所示的轻链；或
    氨基酸序列如SEQ ID NO：44所示的重链以及氨基酸序列如SEQ ID NO：69所示的轻链；或
    氨基酸序列如SEQ ID NO：57所示的重链以及氨基酸序列如SEQ ID NO：66所示的轻链；或
    由氨基酸序列如SEQ ID NO：25所示的重链可变区与氨基酸序列如SEQ ID NO：41所示的人IgG4恒定区拼接而成的重链，和由氨基酸序列如SEQ ID NO：27所示的轻链可变区以及氨基酸序列如SEQ ID NO：42所示的人kappa链恒定区拼接而成的轻链；或
    由氨基酸序列如SEQ ID NO：29所示的重链可变区与氨基酸序列如SEQ ID NO：41所示的人IgG4恒定区拼接而成的重链，和由氨基酸序列如SEQ ID NO：31所示的轻链可变区以及氨基酸序列如SEQ ID NO：42所示的人kappa链恒定区拼接而成的轻链；或
    由氨基酸序列如SEQ ID NO：33所示的重链可变区与氨基酸序列如SEQ ID NO：41所示的人IgG4恒定区拼接而成的重链，和由氨基酸序列如SEQ ID NO：35所示的轻链可变区以及氨基酸序列如SEQ ID NO：42所示的人kappa链恒定区拼接而成的轻链；或
    由氨基酸序列如SEQ ID NO：37所示的重链可变区与氨基酸序列如SEQ ID NO：41所示的人IgG4恒定区拼接而成的重链，和由氨基酸序列如SEQ ID NO：39所示的轻链可变区以及氨基酸序列如SEQ ID NO：42所示的人kappa链恒定区拼接而成的轻链。
  8. 一种核苷酸分子，其特征在于，所述核苷酸分子编码如权利要求1～7任一项所述的结合人CD39的抗体或其抗原结合片段。
  9. 根据权利要求8所述的核苷酸分子，其特征在于，包括如SEQ ID NO：26、30、34、38所示的编码重链可变区的核苷酸分子；和/或，如SEQ ID NO：28、32、36、40所示的编码轻链可变区的核苷酸分子。
  10. 根据权利要求8所述的核苷酸分子，其特征在于，包括如SEQ ID NO：45、46、49、52、55所示的编码重链的核苷酸分子；和/或如SEQ ID NO：58、61、64、67所示的编码轻链的核苷酸分子。
  11. 一种表达载体，其特征在于，所述表达载体含有如权利要求8-10任一所述的核苷酸分子。
  12. 一种宿主细胞，其特征在于，所述宿主细胞含有如权利要求11所述的表达载体。
  13. 一种制备如权利要求1～7中任一项所述的结合人CD39所述抗体或其抗原结合片段的方法，所述方法包括以下步骤：
    a)在表达条件下，培养如权利要求12所述的宿主细胞，从而表达所述的结合人CD39的抗体或其抗原结合片段；
    b)分离并纯化a)所述的结合人CD39的抗体或其抗原结合片段。
  14. 一种药物组合物，其特征在于，所述药物组合物含有如权利要求1～7中任一项所述的结合人CD39的抗体或其抗原结合片段和药学上可接受的载体或辅料。
  15. 如权利要求1～7中任一项所述的结合人CD39的抗体或其抗原结合片段或如权利要求14所述的药物组合物在制备抗肿瘤药物中的应用。
  16. 一种肿瘤的治疗方法，其特征在于，包括以下步骤：给需要的对象施用如权利要求1～7中任一项所述的结合人CD39的抗体或其抗原结合片段，或如权利要求14所述的药物组合物。
  17. 根据权利要求16所述的治疗方法，其特征在于，所述肿瘤为CD39高表达的肿瘤。
  18. 根据权利要求16所述的治疗方法，其特征在于，所述肿瘤选自淋巴瘤、肉瘤、慢性淋巴细胞白血病、肺癌、胰腺癌、卵巢癌、肾癌、甲状腺癌、黑色素瘤或睾丸癌。
PCT/CN2023/123176 2022-10-10 2023-10-07 结合人cd39的抗体或其抗原结合片段、其制备方法及用途 WO2024078381A1 (zh)

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CN112714768A (zh) * 2019-08-27 2021-04-27 科望(苏州)生物医药科技有限公司 新型抗cd39抗体
CN114651013A (zh) * 2019-11-05 2022-06-21 北京加科思新药研发有限公司 对cd39具有特异性的结合分子及其用途
CN114729038A (zh) * 2019-09-25 2022-07-08 上海岸迈生物科技有限公司 Cd39的高亲和力抗体及其用途
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