WO2019174603A1 - 靶向ctla-4抗体、其制备方法和用途 - Google Patents

靶向ctla-4抗体、其制备方法和用途 Download PDF

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WO2019174603A1
WO2019174603A1 PCT/CN2019/078032 CN2019078032W WO2019174603A1 WO 2019174603 A1 WO2019174603 A1 WO 2019174603A1 CN 2019078032 W CN2019078032 W CN 2019078032W WO 2019174603 A1 WO2019174603 A1 WO 2019174603A1
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antibody
variable region
chain variable
seq
ctla
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French (fr)
Inventor
徐丽娜
刘礼乐
杨达志
韦雨杏
邵小慧
王倩
张洁
王美玲
张瑜
段清
宋宁宁
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Shanghai Pharmaexplorer Co Ltd
Pharmaexplorer Ltd
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Shanghai Pharmaexplorer Co Ltd
Pharmaexplorer Ltd
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Priority to EP19767333.8A priority Critical patent/EP3766901A4/en
Priority to US16/979,906 priority patent/US20210324080A1/en
Priority to JP2020549033A priority patent/JP2021526012A/ja
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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
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • 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
    • A61K39/44—Antibodies bound to carriers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61P35/00—Antineoplastic agents
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
    • 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
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/35—Valency
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/40—Immunoglobulins specific features characterized by post-translational modification
    • C07K2317/41—Glycosylation, sialylation, or fucosylation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/74—Inducing cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
    • G01N2333/70521—CD28, CD152

Definitions

  • the present invention relates to the field of biomedicine, and more particularly to a CTLA-4 antibody and a preparation method and application thereof.
  • monoclonal antibodies are one of the most successful and revolutionary treatments for cancer treatment over the past 20 years. Compared with traditional chemical drugs, antibody drugs have higher specificity and lower toxicity. Although the monoclonal antibody has achieved continuous success, it still faces many challenges.
  • murine monoclonal antibodies have great limitations in the diagnosis and treatment of diseases such as tumors and organ transplants; chimeric antibodies still retain 30% of the mouse sequence, which can cause different degrees of HAMA reaction. Clinically, different chimeric antibodies have different degrees of immunogenicity; humanized antibodies are also known as transplanted antibodies. Simple CDR grafting often results in a decrease in antigen-antibody affinity, and because it still has at least 10% heterologous protein, it is subject to varying degrees of limitations in clinical applications. Therefore, it is necessary to further develop a more complete therapeutic antibody-complete human antibody.
  • Cancer immunotherapy is the latest breakthrough in cancer treatment, using the patient's own immune system to attack tumor cells.
  • An immunological checkpoint refers to some inhibitory signaling pathways present in the immune system that prevent tissue damage by regulating the persistence and intensity of immune responses in peripheral tissues and participate in maintaining tolerance to autoantigens. Inhibition of T cell activity by the inhibitory signaling pathway of the immune checkpoint is an important mechanism for tumors to escape immune killing. Blocking against immune checkpoints is one of many effective strategies for activating anti-tumor immunity.
  • Inhibitors of immunological checkpoint proteins have the potential to treat various tumor types such as metastatic melanoma, lung cancer, breast cancer, renal cell carcinoma, and the like. Recent research on cancer immunotherapy methods has shown promising results, especially for metastatic cancer cases. In addition, cancer immunotherapy has great potential in the treatment of hematological cancer, including Hodgkin's lymphoma, multiple myeloma, myelodysplastic syndrome, non-Hodgkin's lymphoma and the like. The side effects caused by immunological checkpoint inhibitors are negligible, reversible and controllable, and effective immune checkpoint inhibitors can significantly improve the overall survival of cancer patients. Immunological checkpoint inhibitors can be used in combination with targeted or conventional radiation and chemotherapy, and such combination therapies are effective in treating many types of cancer.
  • CTLA-4 cytotoxic T lymphocyte-associated protein 4 or CD152
  • CD152 cytotoxic T lymphocyte-associated protein 4
  • CD152 cytotoxic T lymphocyte-associated protein 4
  • CD152 cytotoxic T lymphocyte-associated protein 4
  • CD152 cytotoxic T lymphocyte-associated protein 4
  • CTLA-4 competes with CD28 for ligand and inhibits TCR signaling pathway by itself.
  • the intensity of the T cell response to the antigen is modulated such that the body exhibits tolerance to recognition of the autoantigen or exogenous weak antigen.
  • CTLA-4/B7 ligand interaction can enhance the body's recognition activity against tumor antigens, stimulate the proliferation of antigen-specific T cells, thereby activating the immune system and enhancing the anti-tumor immune response, and has been in multiple In a homologous mouse tumor model, it was demonstrated that blocking of CTLA-4 promotes antitumor activity. Therefore, drugs blocking the CTLA-4/B7.1 and B7.2 pathways can provide new treatments for a variety of cancers and other immune diseases.
  • the present invention provides a CTLA-4 antibody having high affinity and specificity and a preparation method thereof.
  • a heavy chain variable region of an antibody comprising the following three complementarity determining region CDRs:
  • n is independently 0, 1, 2, 3 or 4;
  • any one of the above amino acid sequences further comprises a derivative sequence which optionally adds, deletes, modifies and/or substitutes at least one amino acid and is capable of retaining CTLA-4 binding affinity.
  • the heavy chain variable region has the amino acid sequence set forth in SEQ ID NO: 8n+1, wherein n is 0, 1, 2, 3 or 4.
  • a heavy chain of an antibody having the heavy chain variable region of claim 1 is provided.
  • a light chain variable region of an antibody comprising the following three complementarity determining region CDRs:
  • n is independently 0, 1, 2, 3 or 4;
  • any one of the above amino acid sequences further comprises a derivative sequence which optionally adds, deletes, modifies and/or substitutes at least one amino acid and is capable of retaining CTLA-4 binding affinity.
  • the light chain variable region has the amino acid sequence set forth in SEQ ID NO: 8n+5, wherein n is 0, 1, 2, 3 or 4.
  • a light chain of an antibody having the light chain variable region of claim 3 is provided.
  • an antibody having:
  • the antibody has: a heavy chain according to the second aspect of the invention; and/or a light chain according to the fourth aspect of the invention,
  • any one of the above amino acid sequences further comprises a derivative sequence which optionally adds, deletes, modifies and/or substitutes at least one amino acid and is capable of retaining CTLA-4 binding affinity.
  • the amino acid sequence of any of the above CDRs comprises a derivative CDR sequence that has been added, deleted, modified and/or substituted 1, 2 or 3 amino acids, and which results in VH and VL containing the derived CDR sequences.
  • the resulting derivative antibody is capable of retaining the affinity for binding to CTLA-4.
  • the ratio of the affinity F1 of the derivative antibody to CTLA-4 binding to the affinity of the corresponding non-derivatized antibody to CTLA-4 is 0.5-2, preferably 0.7-1.5, and more preferably 0.8-1.2.
  • the number of amino acids added, deleted, modified and/or substituted is from 1 to 5 (e.g., from 1 to 3, preferably from 1 to 2, more preferably 1).
  • the derived sequence that has been added, deleted, modified and/or substituted for at least one amino acid and is capable of retaining CTLA-4 binding affinity is an amino acid sequence having a homology of at least 96%.
  • the antibody further comprises a heavy chain constant region and/or a light chain constant region.
  • the heavy chain constant region is of human origin, and/or the light chain constant region is of human origin.
  • the antibody is selected from the group consisting of a chimeric antibody, a humanized antibody, a fully human antibody, or a combination thereof.
  • the ratio of the immunogenicity Z1 of the fully human antibody in human to the immunogenicity Z0 of the non-human antibody (such as a murine antibody) in human is (01/Z0) is 0. -0.5, preferably 0-0.2, more preferably 0-0.05 (e.g., 0.001-0.05).
  • the antibody is a partially or fully humanized, or fully human, monoclonal antibody.
  • the antibody is a diabody, or a single chain antibody.
  • the antibody is an antibody full length protein, or an antigen binding fragment.
  • the antibody is a bispecific antibody, or a multispecific antibody.
  • the antibody has one or more characteristics selected from the group consisting of:
  • the antibody has a heavy chain variable region according to the first aspect of the invention and a light chain variable region according to the third aspect of the invention;
  • the heavy chain variable region comprises the following three complementarity determining region CDRs:
  • the light chain variable region comprises the following three complementarity determining region CDRs:
  • any one of the above amino acid sequences further comprises a derivative sequence which optionally adds, deletes, modifies and/or substitutes at least one amino acid and is capable of retaining CTLA-4 binding affinity.
  • the heavy chain variable region of the antibody comprises the amino acid sequence set forth in SEQ ID NO: 8n+1; and/or the light chain variable region of the antibody comprises SEQ ID NO: 8n+5 The amino acid sequence shown, wherein each n is independently 0, 1, 2, 3 or 4.
  • the heavy chain variable region of the antibody comprises the amino acid sequence set forth in SEQ ID NO: 1.
  • the light chain variable region of the antibody comprises the amino acid sequence set forth in SEQ ID NO:5.
  • the heavy chain variable region of the antibody comprises the amino acid sequence set forth in SEQ ID NO: 1
  • the light chain variable region of the antibody comprises the amino acid sequence set forth in SEQ ID NO: 5.
  • the heavy chain variable region of the antibody comprises the amino acid sequence set forth in SEQ ID NO: 9
  • the light chain variable region of the antibody comprises the amino acid sequence set forth in SEQ ID NO: 13.
  • the heavy chain variable region of the antibody comprises the amino acid sequence set forth in SEQ ID NO: 17
  • the light chain variable region of the antibody comprises the amino acid sequence set forth in SEQ ID NO:21.
  • the heavy chain variable region of the antibody comprises the amino acid sequence set forth in SEQ ID NO: 25 and the light chain variable region of the antibody comprises the amino acid sequence set forth in SEQ ID NO:29.
  • the heavy chain variable region of the antibody comprises the amino acid sequence set forth in SEQ ID NO: 33
  • the light chain variable region of the antibody comprises the amino acid sequence set forth in SEQ ID NO:37.
  • the antibody is selected from the group consisting of 97A8D1, 92C8B6, 31C12F3, 40C4C697B8E1.
  • the amino acid sequence of the heavy chain variable region is at least as long as the amino acid sequence shown as SEQ ID NO: 1, SEQ ID NO: 9, SEQ ID NO: 17, SEQ ID NO: 25, SEQ ID NO: 33 in the Sequence Listing There are 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology.
  • amino acid sequence of the light chain variable region is at least as long as the amino acid sequence shown as SEQ ID NO: 5, SEQ ID NO: 13, SEQ ID NO: 21, SEQ ID NO: 29, SEQ ID NO: 37 in the Sequence Listing There are 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology.
  • the antibody is a defucosylated antibody.
  • the antibody is a defucosylated antibody 97A8D1.
  • a recombinant protein comprising:
  • the tag sequence comprises a 6His tag.
  • the recombinant protein comprises a fusion protein.
  • the recombinant protein is a monomer, a dimer, or a multimer.
  • the recombinant protein comprises:
  • the recombinant protein comprises:
  • a defucosylated antibody eg, defucosylated antibody 97A8D1;
  • a polynucleotide is provided, the polynucleotide encoding a polypeptide selected from the group consisting of:
  • polynucleotide encoding the heavy chain variable region is set forth in SEQ ID NO: 41, 43, 45, 47 or 49; and/or
  • the polynucleotide encoding the light chain variable region is shown as 42, 44, 46, 48 or 50.
  • polynucleotide encoding the heavy chain variable region sequence is set forth in SEQ ID NO: 41; and the polynucleotide encoding the light chain variable region sequence is set forth at 42.
  • polynucleotide encoding the heavy chain variable region is set forth in SEQ ID NO: 43; and the polynucleotide encoding the light chain variable region is set forth at 44.
  • polynucleotide encoding the heavy chain variable region is set forth in SEQ ID NO: 45; and the polynucleotide encoding the light chain variable region is set forth at 46.
  • polynucleotide encoding the heavy chain variable region is set forth in SEQ ID NO: 47; and the polynucleotide encoding the light chain variable region is set forth at 48.
  • polynucleotide encoding the heavy chain variable region is set forth in SEQ ID NO: 49; and the polynucleotide encoding the light chain variable region is set forth at 50.
  • the invention provides a vector comprising the polynucleotide of any one of the seventh aspect of the invention.
  • the vector comprises: a bacterial plasmid, a bacteriophage, a yeast plasmid, a plant cell virus, a mammalian cell virus such as an adenovirus, a retrovirus, or other vector.
  • a genetically engineered host cell comprising the vector or genome of the eighth aspect of the invention, wherein any one of the seventh aspects of the invention is integrated Polynucleotides as described.
  • composition comprising:
  • an active ingredient selected from the group consisting of a heavy chain variable region according to the first aspect of the invention, a heavy chain according to the second aspect of the invention, a third of the invention
  • the light chain variable region of the aspect, the light chain of the fourth aspect of the invention, or the antibody of any of the fifth aspect of the invention, according to the sixth aspect of the invention Recombinant protein, or a combination thereof;
  • the pharmaceutical composition is a liquid formulation.
  • the pharmaceutical composition is an injection.
  • the pharmaceutical composition comprises 0.01 to 99.99% of the antibody of any one of the fifth aspect of the invention, the recombinant protein of the sixth aspect of the invention, or Combinations and 0.01 to 99.99% of a pharmaceutically acceptable carrier, the percentage being the percentage by mass of the pharmaceutical composition.
  • an active ingredient selected from the group consisting of a heavy chain variable region according to the first aspect of the invention, such as the second aspect of the invention
  • the heavy chain, the light chain variable region according to the third aspect of the invention, the light chain according to the fourth aspect of the invention, or the fifth aspect of the invention The antibody, a recombinant protein according to the sixth aspect of the invention, or a combination thereof, wherein the active ingredient is used for the preparation of a medicament for preventing and/or treating a disease associated with a CTLA-4-associated tumor and/or a viral infection.
  • the CTLA-4 associated disease is selected from the group consisting of melanoma, mesothelioma, non-small cell lung cancer, breast cancer, liver cancer, synovial sarcoma, metastatic colon cancer, renal cancer, and bladder.
  • prostate cancer ovarian cancer, chronic hepatitis C virus infection, advanced solid cancer, digestive organ malignancy, metastatic non-small cell lung cancer, prostate tumor, endometrial cancer, endometrial carcinosarcoma, recurrent melanoma, head Squamous cell carcinoma of the neck, sarcoma, Merkel cell carcinoma, cutaneous T-cell lymphoma, fallopian tube cancer, peritoneal tumor, muscle invasive bladder cancer, extensive stage small cell lung cancer, adult acute myeloid leukemia, atypical chronic Myeloid leukemia, previously treated myelodysplastic syndrome, ovarian epithelial cell carcinoma, urinary malignancy, adult grade III lymphomatoid granuloma, B cell chronic lymphocytic leukemia, skin B cell non-Hodgkin's lymphoma, eye Endolymphoma, testicular choriocarcinoma, neuroblastoma, esophageal cancer, etc.
  • composition for detecting a CTLA-4 protein in a sample in vitro comprising the antibody of any one of the fifth aspect of the invention, according to the sixth aspect of the invention
  • the recombinant protein, or a combination thereof, is used as an active ingredient.
  • a method of treating a tumor or a viral infection comprising: using the antibody of any one of the fifth aspect of the invention, according to the sixth aspect of the invention Recombinant protein, or a combination thereof.
  • the tumor is cancer
  • Figure 2 Results of FACS detection of HEK293 cells transfected with CTLA-4 (Y201V) protein.
  • FIG. 3 ELISA detection of serum antibody titers in Harbour transgenic mice after HEK293-CTLA-4 cell immunization.
  • Figure 6 (A), (B) and (C) inhibition of binding of CTLA-4 protein to its receptor B7.1 by CTLA-4 antibody.
  • Figure 7 (A), (B) and (C) inhibition of binding of the CTLA-4 protein to its receptor B7.2 by the CTLA-4 antibody.
  • Figure 8 Effect of CTLA-4 antibody on IL-2 secretion in PBMC lymphocyte stimulation assay.
  • Figure 9 (A) and (B) Flow cytometry (FACS) assay for binding of antibodies to human CTLA-4 expressing cells.
  • Figure 10 (A) and (B) Flow cytometry (FACS) assay for binding of antibodies to monkey CTLA-4 expressing cells.
  • Figure 11 (A) and (B) Inhibition of binding of CTLA-4 protein to its receptor B7.1 by a fully human CTLA-4 antibody.
  • Figure 14 (A), (B) (C) and (D) Effect of CTLA-4 fully human antibody on IL-2 secretion in PHA-induced T lymphocyte activation assay.
  • Figure 16 (A), (B), (C) and (D) Determination of the molecular weight of the Fc segment of the defucosylated and fucosylated anti-CTLA-4 antibody by LC/MS.
  • Figure 17 Effect of defucosylation and fucosylated anti-CTLA-4 antibodies on IL-2 secretion in an SEB-dependent PBMC activation assay.
  • the present inventors have conducted extensive and intensive studies on transgenic mice using rat-human chimeric antibodies, and immunized transgenic mice with 293F-HuCTLA-4 cells (for example, recombinantly expressed 293F-HuCTLA4 cells), which was unexpectedly obtained.
  • the experimental results show that the CTLA-4 protein with human and monkey origin has high affinity (K D is 2.15E-09nM), which can inhibit the binding of CTLA-4 to its receptors B7.1 and B7.2. It can significantly increase IL-2 expression levels in PHA-induced human T lymphocytes or SEB-mediated activation of human peripheral blood mononuclear cells, and lacks cross-reactivity with similar protein antigens such as human CD28.
  • the obtained human anti-CTLA-4 antibody has the characteristics of higher affinity, stronger activity in vitro and in vivo. Moreover, the anti-tumor effect of the whole human CTLA-4 antibody (for example, 97A8D1) is superior to the antibody Ipilimumab in the prior art. The present invention has been completed on this basis.
  • CTLA-4 a cytotoxic T lymphocyte-associated antigen 4
  • CTLA-4 is structurally a CD28 immunoglobulin superfamily, expressed in an inducible form in activated T cells and constitutively expressed in regulatory T cells, as an immunological checkpoint protein, CD28 competitively binds to the ligand, thereby downregulating the activity of T cells.
  • Studies on transgenic mice and tumor-bearing mice have shown that CTLA-4 is involved in tumor escape from immune system recognition and killing, so blocking the activity of CTLA-4 may increase the body's inhibition of tumor growth.
  • antibody or "immunoglobulin” is an isotetrameric glycoprotein of about 150,000 daltons having the same structural features, consisting of two identical light chains (L) and two identical heavy chains. (H) Composition. Each light chain is linked 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 and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) at one end followed by a plurality of constant regions.
  • VH variable region
  • Each light chain has a variable region (VL) at one end and a constant region at the other end; the constant region of the light chain is opposite 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 .
  • Particular amino acid residues form an interface between the variable regions of the light and heavy chains.
  • variable means that certain portions of the variable regions of an antibody differ in sequence, which form the binding and specificity of various specific antibodies for their particular antigen. However, the variability is not evenly distributed throughout the variable region of the antibody. It is concentrated in three segments in the variable region of the light and heavy chains called the complementarity determining region (CDR) or hypervariable region. The more conserved portion of the variable region is referred to as the framework region (FR).
  • the variable regions of the native heavy and light chains each comprise four FR regions which are substantially in a beta-sheet configuration and are joined by three CDRs forming a linker, in some cases forming a partial beta sheet structure.
  • the CDRs in each chain are closely joined together by the FR region and together with the CDRs of the other chain form the antigen binding site of the antibody (see Kabat et al, NIH Publ. No. 91-3242, Vol. I, pp. 647-669). (1991)).
  • the constant regions are not directly involved in the binding of the antibody to the antigen, but they exhibit different effector functions, such as antibody-dependent cytotoxicity of the participating antibodies.
  • the "light chain" of a vertebrate antibody can be classified into one of two distinct classes (called kappa and lambda) depending on the amino acid sequence of its constant region.
  • Immunoglobulins can be classified into different classes based on the amino acid sequence of their heavy chain constant regions. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes) such as IgG1, IgG2, IgG3, IgG4, IgA, and IgA2.
  • the heavy chain constant regions corresponding to different classes of immunoglobulins are called ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ , respectively.
  • the subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known to those skilled in the art.
  • variable regions which are divided into four framework regions (FR), four
  • FR framework regions
  • the amino acid sequence of FR is relatively conservative and is not directly involved in the binding reaction.
  • CDRs form a cyclic structure in which the ⁇ -sheets formed by the FRs are spatially close to each other, and the CDRs on the heavy chain and the CDRs on the corresponding light chain constitute the antigen-binding site of the antibody.
  • the amino acid sequence of the same type of antibody can be compared to determine which amino acids constitute the FR or CDR regions.
  • the present invention encompasses not only intact antibodies, but also fragments of immunologically active antibodies or fusion proteins formed by antibodies with other sequences. Accordingly, the invention also includes fragments, derivatives and analogs of the antibodies.
  • antibodies include murine, chimeric, humanized or fully human antibodies prepared by techniques well known to those skilled in the art.
  • Recombinant antibodies such as chimeric and humanized monoclonal antibodies, including both human and non-human portions, can be obtained by standard DNA recombination techniques, all of which are useful antibodies.
  • a chimeric antibody is a molecule in which different portions are derived from different animal species, such as a variable region having a monoclonal antibody from a murine, and a chimeric antibody from a constant region of a human immunoglobulin (see, e.g., U.S. Patent 4,816,567 and U.S. Patent No. 4,816,397, incorporated herein by reference in its entirety herein.
  • a humanized antibody refers to an antibody molecule derived from a non-human species having one or more complementarity determining regions (CDRs) derived from a non-human species and a framework region derived from a human immunoglobulin molecule (see U.S. Patent 5,585,089, This article is hereby incorporated by reference in its entirety.
  • CDRs complementarity determining regions
  • These chimeric and humanized monoclonal antibodies can be prepared using recombinant DNA techniques well known in the art.
  • the antibody may be monospecific, bispecific, trispecific, or more multiple specificity.
  • the antibody of the present invention further includes a conservative variant thereof, which means that there are up to 10, preferably up to 8, more preferably up to 5, optimally compared to the amino acid sequence of the antibody of the present invention. Up to 3 amino acids are replaced by amino acids of similar or similar nature to form a polypeptide. These conservative variant polypeptides are preferably produced by amino acid substitution according to Table A.
  • the present invention provides a highly specific and high affinity antibody against CTLA-4 comprising a heavy chain and a light chain, the heavy chain comprising a heavy chain variable region (VH) amino acid sequence, the light chain comprising a light chain Variable region (VL) amino acid sequence.
  • VH heavy chain variable region
  • VL light chain Variable region
  • the heavy chain variable region (VH) comprises the following three complementarity determining region CDRs:
  • the light chain variable region includes the following three complementarity determining regions CDRs:
  • any one of the above amino acid sequences further comprises a derivative sequence which optionally adds, deletes, modifies and/or substitutes at least one amino acid and is capable of retaining CTLA-4 binding affinity.
  • the sequence formed by adding, deleting, modifying and/or substituting at least one amino acid sequence preferably has a homology or sequence identity of at least 80%, preferably at least 85%, more preferably At least 90%, optimally at least 95% of the amino acid sequence.
  • Methods for determining sequence homology or identity include, but are not limited to, Computational Molecular Biology, Lesk, AM, Oxford University Press, New York, 1988; Biocomputing: Information Biocomputing: Informatics and Genome Projects, Smith, DW, Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part 1, Griffin, AM and Griffin, HG , Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987 and Sequence Analysis Primer, Gribskov, M. and Devereux , J. M. Stockton Press, New York, 1991 and Carillo, H. and Lipman, D., SIAM J.
  • the preferred method of determining identity is to obtain the largest match between the sequences tested.
  • the method of determining identity is compiled in a publicly available computer program.
  • Preferred computer program methods for determining identity between two sequences include, but are not limited to, the GCG package (Devereux, J. et al., 1984), BLASTP, BLASTN, and FASTA (Altschul, S, F. et al., 1990).
  • the BLASTX program is available to the public from NCBI and other sources (BLAST Handbook, Altschul, S. et al, NCBI NLM NIH Bethesda, Md. 20894; Altschul, S. et al, 1990).
  • the well-known Smith Waterman algorithm can also be used to determine identity.
  • the antibody of the present invention may be a double-stranded or single-chain antibody, and may be selected from an animal-derived antibody, a chimeric antibody, a humanized antibody, more preferably a humanized antibody, a human-animal chimeric antibody, and more preferably a whole human. Sourced antibodies.
  • the antibody derivative of the present invention may be a single chain antibody, and/or an antibody fragment such as Fab, Fab', (Fab') 2 or other known antibody derivatives in the field, and IgA, IgD, IgE. Any one or more of IgG and IgM antibodies or antibodies of other subtypes.
  • the animal is preferably a mammal, such as a mouse.
  • the antibody of the invention may be a chimeric antibody, a humanized antibody, a CDR grafted and/or a modified antibody that targets human CTLA-4.
  • the number of amino acids added, deleted, modified and/or substituted is preferably not more than 40%, more preferably not more than 35%, more preferably 1-33% of the total amino acid number of the initial amino acid sequence. More preferably, it is 5-30%, more preferably 10-25%, and still more preferably 15-20%.
  • the number of amino acids added, deleted, modified and/or substituted may be 1-7, more preferably 1-5, more preferably 1-3, more preferably It is 1-2.
  • the antibody that targets CTLA-4 is 97A8D1, 92C8B6, 31C12F3, 40C4C6 or 97B8E1.
  • the heavy chain variable region (VH) amino acid sequence of the antibody 97A8D1 is the amino acid sequence set forth in SEQ ID NO.: 1.
  • the light chain variable region amino acid sequence of the antibody 97A8D1 is the amino acid sequence set forth in SEQ ID NO.: 5.
  • sequence of the DNA molecule of the antibody or fragment thereof of the present invention can be obtained by a conventional technique such as PCR amplification or genomic library screening.
  • the coding sequences of the light and heavy chains can also be fused together to form a single chain antibody.
  • the recombinant sequence can be used to obtain the relevant sequences in large quantities. This is usually done by cloning it into a vector, transferring it to a cell, and then isolating the relevant sequence from the proliferated host cell by conventional methods.
  • synthetic sequences can be used to synthesize related sequences, especially when the fragment length is short.
  • a long sequence of fragments can be obtained by first synthesizing a plurality of small fragments and then performing the ligation.
  • DNA sequence encoding the antibody (or a fragment thereof, or a derivative thereof) of the present invention completely by chemical synthesis.
  • the DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art.
  • mutations can also be introduced into the protein sequences of the invention by chemical synthesis.
  • the invention also relates to vectors comprising the appropriate DNA sequences described above, as well as appropriate promoters or control sequences. These vectors can be used to transform appropriate host cells to enable them to express proteins.
  • the host cell can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell.
  • Preferred animal cells include, but are not limited to, CHO-S, HEK-293 cells.
  • the resulting host cells are cultured under conditions suitable for expression of the antibody of the invention.
  • immunoglobulin purification steps such as protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography or affinity chromatography, etc.
  • the antibodies of the present invention are purified by conventional separation and purification means well known to those skilled in the art.
  • the resulting monoclonal antibodies can be identified by conventional means.
  • the binding specificity of a monoclonal antibody can be determined by immunoprecipitation or in vitro binding assays such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).
  • the binding affinity of a monoclonal antibody can be determined, for example, by the Scatchard analysis of Munson et al, Anal. Biochem., 107: 220 (1980).
  • the antibodies of the invention can be expressed intracellularly, or on the cell membrane, or secreted extracellularly.
  • the recombinant protein can be isolated and purified by various separation methods using its physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of such methods include, but are not limited to, conventional renaturation treatment, treatment with a protein precipitant (salting method), centrifugation, osmotic sterilizing, sonication, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • the invention also provides the use of an antibody of the invention, for example for the preparation of a medicament for the treatment of a CTLA-4 related disorder.
  • CTLA-4 related diseases include tumor growth, growth and/or metastasis, viral infection and the like.
  • tumors include, but are not limited to, breast cancer (eg, triple negative breast cancer), pancreatic cancer, lung cancer (eg, non-small cell lung cancer, extensive stage small cell lung cancer, metastatic non-small cell lung cancer), malignant colloid Tumor, digestive organ malignancy, gastric cancer, liver cancer, esophageal cancer, kidney cancer, colorectal cancer, metastatic colon cancer, bladder cancer, prostate cancer (such as prostate cancer), endometrial cancer, endometrial carcinosarcoma, cervical cancer , ovarian cancer, fallopian tube cancer, leukemia (such as adult acute myeloid leukemia, atypical chronic myeloid leukemia), bone marrow cancer, sarcoma (such as angiosarcoma, synovial sarcoma), melanoma, recurrent melanoma, mesothelioma, Advanced solid cancer, head and neck squamous cell carcinoma, Merkel cell
  • lung cancer eg, non-small cell lung cancer, extensive stage
  • viruses include, but are not limited to, hepatitis B virus, hepatitis C virus (chronic infection), and HIV.
  • the invention also provides a composition.
  • the composition is a pharmaceutical composition comprising the above antibody or active fragment thereof or a fusion protein thereof, and a pharmaceutically acceptable carrier.
  • these materials can be formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium wherein the pH is usually from about 5 to about 8, preferably from about 6 to about 8, although the pH may be The nature of the formulation and the condition to be treated vary.
  • the formulated pharmaceutical compositions can be administered by conventional routes including, but not limited to, intratumoral, intraperitoneal, intravenous, or topical administration.
  • the pharmaceutical composition of the present invention can be directly used for binding to CTLA-4 protein molecules, and thus can be used for the prevention and treatment of diseases such as tumors.
  • other therapeutic agents can be used simultaneously.
  • the pharmaceutical composition of the present invention contains a safe and effective amount (e.g., 0.001 to 99% by weight, preferably 0.01 to 90% by weight, more preferably 0.1 to 80% by weight) of the above monoclonal antibody of the present invention, and a pharmaceutically acceptable carrier or Shape agent.
  • a pharmaceutically acceptable carrier or Shape agent include, but are not limited to, saline, buffer, dextrose, water, glycerol, ethanol, and combinations thereof.
  • the pharmaceutical preparation should be matched to the mode of administration.
  • the pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injections and solutions are preferably prepared under sterile conditions.
  • the amount of active ingredient administered is a therapeutically effective amount, for example from about 1 microgram per kilogram body weight to about 5 milligrams per kilogram body weight per day.
  • the polypeptides of the invention
  • a safe and effective amount of the antibody is administered to a mammal, wherein the safe and effective amount is usually at least about 10 micrograms per kilogram of body weight, and in most cases no more than about 50 milligrams per kilogram of body weight, preferably The dose is from about 10 micrograms per kilogram of body weight to about 20 milligrams per kilogram of body weight.
  • specific doses should also consider factors such as the route of administration, the health of the patient, etc., which are within the skill of the skilled physician.
  • the transgenic mouse used in the present invention can more easily obtain a fully human antibody than a wild type mouse, thereby reducing the immunogenicity of the antibody; and the number of antibodies obtained is higher than that of the transgenic mouse of the whole human antibody. Strong affinity, good sequence diversity and high activity.
  • the present invention adopts hybridoma technology to obtain an antibody, and has higher antibody affinity and good sequence expression than the antibody obtained by the phage library.
  • the recombinant 293F-HuCTLA-4 cells of the present invention have a more conformation of the expressed protein expression than the protein or polypeptide immunogen; and the expression level is higher than that of other recombinant expression cells.
  • the present invention obtains antibodies having different sequences and is capable of specifically binding to a CTLA-4 antibody, and its binding activity is less than nanomolar (nM).
  • the antibody obtained by the present invention has a good T cell activating activity capable of blocking the binding of CTLA-4 to its two ligands (B7.1 and B7.2) by reversing CTLA-4 to T cells. Inhibition of activation activates T cells to secrete IL-2.
  • the antibody obtained by the present invention exhibits an activity of significantly inhibiting tumor growth and increasing survival rate of mice in a mouse (e.g., human CTLA-4 transgenic mouse).
  • the antibody obtained by the present invention has a series of excellent characteristics: the variable region sequence has low homology with the existing antibody (homology ⁇ 92%), and each activity is better than that of the antibody from the comparative document.
  • the room temperature described in the examples is room temperature conventional in the art, generally 10-30 °C.
  • the PBS described in the examples was a PBS phosphate buffer, pH 7.2.
  • H2L2 transgenic mice were obtained from (purchased from) and Platinum Pharmaceuticals (Shanghai) Co., Ltd., which produced a traditional tetrameric antibody consisting of two heavy chains and two light chains (H2L2) with a fully human variable region.
  • Genetically engineered mouse technology is one of the main tools for producing fully human antibodies [4] .
  • the present invention first prepares human CTLA-4 as an immunogen, and adopts human antibody transgenic mouse technology to prepare a fully human antibody [Lonberg, et al. 1994, Nature 368 (6474): 856-859, Lonberg, N. And Huszar, D. 1995, Intern. Rev. Immunol. 13: 65-93, and Harding, F. and Lonberg, N., 1995, Ann. NY Acad. Sci. 764: 536-546], obtaining CTLA-4 antibody Leading antibody.
  • the antibody has high affinity (affinity KD ⁇ 1*10 -9 M), effectively blocks the binding of CTLA-4 to the receptors B7.1 and B7.2, Peripheral blood mononuclear cells or T lymphocyte reactions significantly increase the expression level of IL-2, and the CTLA-4 antibody with excellent biological properties such as lack of cross-reaction with human CD28 and other protein antigens.
  • the amino acid sequences of the heavy chain variable region and the light chain variable region of the CTLA-4 antibody are then sequenced by molecular biological methods.
  • the human CTLA-4 protein extracellular domain amino acid sequence Lys36-Asp161 was cloned into the pCpC vector (purchased from Invitrogen, V044-50) carrying the human IgG Fc fragment (hFc) and the plasmid was prepared according to established standard molecular biology methods. For a specific method, see Sambrook, J., Fritsch, EF, and Maniatis T. (1989). Molecular Cloning: A Laboratory Manual, Second Edition (Plainview, New York: Cold Spring Harbor Laboratory Press). In HEK293 cells (purchased from Invitrogen) for transient transfection (PEI, Polysciences), using FreeStyle TM 293 (Invitrogen) were expanded at 37 °C.
  • the cell culture medium was collected, and the cell components were removed by centrifugation to obtain a culture supernatant containing the extracellular region of CTLA-4 protein.
  • the culture supernatant was applied to a Protein A affinity chromatography column (Mabselect Sure, available from GE Healthcare) while monitoring the change in ultraviolet absorption value (A280 nm) with an ultraviolet (UV) detector.
  • the protein A affinity column was washed with PBS phosphate buffer (pH 7.2) until the UV absorbance value returned to the baseline, and then eluted with 0.1 M glycine hydrochloride (pH 2.5) to collect affinity from protein A.
  • the hFc-tagged CTLA-4 protein (CTLA-4-hFc) eluted on the column was dialyzed against PBS phosphate buffer (pH 7.2) overnight at 4 ° C in a refrigerator.
  • the dialyzed protein was sterile-filtered at 0.22 ⁇ m and stored at -80 ° C to obtain purified immunogen A.
  • Immunogen A requires a series of quality control tests before use, such as detecting its protein concentration, purity, molecular weight and biological activity.
  • Table 1 shows that the binding of CTLA-4 to B7.1 or B7.2 at the protein level varies with the concentration of B7.1 or B7.2, wherein the control protein is a non-CTLA-4 fusion protein, and the data in the table is OD. 450 nm value.
  • the immunogenic A biological activity is detected by ELISA, specifically:
  • the hFc-tagged CTLA-4 protein (CTLA-4-hFc, immunogen A) was diluted to 1 ⁇ g/mL with PBS, added to an ELISA microplate at 100 ⁇ l/well, and incubated overnight at 4 °C. After blocking with ELISA blocking solution (containing 1% BSA, PBS phosphate buffer pH 7.4, the percentage is mass%) at 37 ° C for two hours, add a gradient dilution of biotinylated B7.1 or B7.2 – hFc was incubated for 1 hour at 37 °C.
  • ELISA blocking solution containing 1% BSA, PBS phosphate buffer pH 7.4, the percentage is mass
  • the biotin-labeled B7.1-hFc or B7.2-hFc can be prepared by reacting B7.1-hFc or B7.2-hFc with a biotinylation reagent.
  • the preparation method of the B7.1-hFc or B7.2-hFc is the same as the preparation method of the immunogen A, and the amino acid sequence information of the B7.1 extracellular domain protein (Val35-Asn242) can be found in the Uniprot database, No.
  • CTLA-4 The full-length amino acid sequence of human CTLA-4 was mutated to CTLA-4 (Y201V) and cloned into the pIRES vector (purchased from Clontech) and plasmids were prepared. Plasmid transfection of HEK293 cell line and CHOK1 cell line (both purchased from Invitrogen) (transfection using X-treme GENE HP DNA Transfection Reagent, available from Roche, Cat. No.
  • the specific selection results are shown in Table 2 and Figure 2.
  • the positive cells (%) in Table 2 refer to the percentage of positive cells in the total number of cells.
  • Figure 2 illustrates that HEK293 cells have higher levels of CTLA-4 expression and are suitable for use as immunogens and for antibody binding activity identification.
  • the harbor immunogenic mouse introduced the human immunoglobulin variable region gene and the rat immunoglobulin constant region gene, while the mouse itself's Ig expression was silenced (FGFrankl in, et al, patent#WO 2010/070263 Al) .
  • the transgenic mouse is immunized with an antigen to produce an immune response and antibody titer comparable to that of a normal mouse (e.g., Balb/c).
  • Immunogen A immunization 6 to 8 weeks old Harbour human antibody transgenic mice (purchased from Beijing Vital Lihua Co., Ltd.) were used, and the mice were raised under SPF conditions. At the time of primary immunization, the immunogen A protein was emulsified with Freund's complete adjuvant and intraperitoneally injected with 0.25 ml, that is, 100 ⁇ g of immunogen A protein was injected per mouse. When boosting the immunization, the immunogen A protein was emulsified with Freund's incomplete adjuvant and intraperitoneally injected with 0.25 ml, that is, 50 ⁇ g of immunogen A protein was injected per mouse.
  • the cells were washed twice with DMEM basal medium, and after cell counting, the cells were diluted with phosphate buffer (pH 7.2) to 2 ⁇ 10 7 cells per ml. Each mouse was intraperitoneally injected with 0.5 ml of cell suspension per immunization. The first and second immunizations were separated by 2 weeks, and each subsequent immunization interval was 3 weeks. In addition to the first immunization, blood was collected 1 week after each immunization, and the antibody titer and specificity in the serum were measured by ELISA. Table 3 and Figure 3 show the results of antibody titer detection by HEK-CTLA-4 cell immune serum by ELISA.
  • each mouse selected was intraperitoneally injected with 100 ⁇ g of purified CTLA-4-hFc (mouse against immunogen A and immunogen B) for immunological reaction or containing human source.
  • HEK293-hCTLA-4 stable cells of CTLA-4 (mouse reacted against immunogen B) mice were sacrificed 5 days later, and spleen cells were collected.
  • NH 4 OH was added to a final concentration of 1% (w/w), and the erythrocytes mixed in the spleen cells were lysed to obtain a spleen cell suspension.
  • the cells were washed three times with DMEM basal medium at 1000 rpm, then mixed with mouse myeloma cells SP2/0 (purchased from ATCC) at a 5:1 ratio of viable cells, using high-efficiency electrofusion or PEG method (see METHODS) IN ENZYMOLOGY, VOL. 220) performs cell fusion.
  • the fused cells were diluted into DMEM medium containing 20% fetal calf serum, 1 x HAT, the percentage being a mass percentage.
  • 1 ⁇ 10 5 / 20 ⁇ l per well was added to a 96-well cell culture plate and placed in a 5% CO 2 , 37 ° C incubator, the percentage being a volume percentage.
  • the cell fusion plates were screened by ELISA and Acumen (microplate assay), and positive clones with OD 450nm >1.0 in ELISA and MFI values >100 in Acumen were amplified into 24-well plates at 10% (in 10%).
  • w/w) fetal bovine serum in DMEM (Invitrogen) medium was expanded at 37 ° C under 5% (v/v) CO 2 conditions. After culturing for 3 days, the culture medium expanded in a 24-well plate was centrifuged, and the supernatant was collected, and the supernatant was subjected to antibody subtype analysis.
  • the binding activity of CTLA-4 protein and CTLA-4 positive cells was determined by ELISA and FACS, and the blocking activity of the antibody sample to CTLA-4 receptor was determined by ligand receptor binding assay.
  • the hybridoma cells are eligible positive clones. Selected hybridoma cells were subcloned in a 96-well plate by limiting dilution in DMEM medium containing 10% (w/w) FBS (purchased from Invitrogen) at 37 ° C, 5% (v/v) CO Culture under 2 conditions.
  • the positive clones were expanded in DMEM (purchased from Invitrogen) medium containing 10% (w/w) FBS, and cultured at 37 ° C under 5% (v/v) CO 2 .
  • the cells were suspended in a cryopreservation solution [DMEM containing 20% (w/w) FBS and 10% (w/w) DMSO], and the hybridoma cells of the present invention were obtained by routine freezing of liquid nitrogen, and can be used for subsequent Antibody production, purification and amino acid sequence determination.
  • the pIRES plasmid containing the full-length nucleotide sequence encoding human CTLA-4 was transfected into the 293F cell line in the step (2) of Example 1, and the 293F stably transfected cell line containing human CTLA-4 (herein referred to as HEK293) was obtained.
  • HEK293-cCTLA-4 stable cell line the pIRES plasmid carrying the full-length cDNA of monkey-derived CTLA-4, wherein the accession number of the monkey-derived CTLA-4 nucleotide acid sequence is XM_005574014.1, transfected into HEK293 cell line HEK293 stable cell line containing monkey CTLA-4 (herein referred to as HEK293-cCTLA-4 stable cell line).
  • the HEK293-hCTLA-4 stable cell line and the HEK293-cCTLA-4 stable cell line were expanded to a 90% confluence in a T-75 cell culture flask, and the medium was exhausted, and the HBSS buffer (Hanks Balanced Salt Solution, purchased) was used.
  • the cells were washed twice from Invitrogen and then treated and collected with an enzyme-free cell dissociation solution (Versene solution, purchased from Life Technology).
  • the cells were washed twice with HBSS buffer, and after cell counting, the cells were diluted with HBSS buffer to 2 ⁇ 10 6 cells per ml, and 1% goat serum blocking solution was added, the percentage being mass percentage.
  • the collected cells were suspended in FACS buffer (HBSS containing 1% BSA, the percentage is mass%) to 2 ⁇ 10 6 cells/mL, and added to a 96-well FACS reaction plate at 100 ⁇ L per well, and the addition was carried out.
  • the purified CTLA-4 antibody sample obtained in Example 2 was subjected to 100 ⁇ l per well and incubated on ice for 2 hours.
  • the cells were washed twice with FACS buffer, and 100 ⁇ l of a fluorescent (Alexa 488)-labeled secondary antibody (purchased from Invitrogen) per well was added and incubated on ice for 1 hour.
  • the cells were washed three times with FACS buffer, and 100 ⁇ l of fixative [4% (v/v) paraformaldehyde] suspension cells were added to each well, and after 10 minutes, the cells were washed twice with FACS buffer.
  • the cells were suspended in 100 ⁇ l of FACS buffer, and the results were detected and analyzed by FACS (FACS Cal ibur, available from BD).
  • the antibody to be tested can bind to the human or monkey CTLA-4 protein on the cell surface, and the activity of each antibody is equivalent, indicating that the antibody has strong binding ability to CTLA-4.
  • the IgG control is human IgG, and the data in the table is the average fluorescence intensity value of the cell population measured by MFI.
  • the receptor ligand binding assay of CTLA-4 protein detects that CTLA-4 antibody blocks the binding of CTLA-4 protein to its ligand B7.1 or B7.2.
  • CTLA-4 extracellular domain protein (CTLA-4-hFc) was diluted with PBS to a final concentration of 1.0 ⁇ g/mL, and then added to a 96-well ELISA plate at 100 ⁇ l per well. Incubate overnight at 4 °C with plastic film, wash plate twice with washing solution [PBS containing 0.01% (v/v) Tween20], add blocking solution [containing 0.01% (v/v) Tween20 and 1 % (w/w) BSA in PBS] was blocked at room temperature for 2 hours.
  • the blocking solution was poured out, and 50 ⁇ l of the purified CTLA-4 antibody sample obtained in Example 2 was added to each well, followed by biotin-labeled B7.1 extracellular domain protein (B7.1-hFc) or B7.2-. hFc, 100 ⁇ l per well, was incubated at 37 ° C after mixing. After 2 hours, the plate was washed 3 times with a washing solution [PBS containing 0.01% (v/v) Tween 20].
  • the results showed that the obtained antibody inhibited the binding of CTLA-4 protein to its ligand B7.1 or B7.2 to varying degrees, and the antibody activity was comparable.
  • the IgG control was human IgG, and the data in the table is the inhibition rate (%).
  • Lymphocyte stimulation assay detects that CTLA-4 antibody blocks the binding of CTLA-4 protein to its receptors B7.1 and B7.2, thereby relieving its inhibition of T lymphocyte activity, thereby stimulating T cell proliferation.
  • the freshly obtained whole blood was diluted with a phosphate buffered saline PBS in a volume ratio of 1:1 to obtain diluted whole blood, and the diluted whole blood was gently flattened on a Ficoll liquid surface (purchased from GE Healthcare) using a sterile pipette.
  • the volume ratio of Ficoll to diluted whole blood is 3:4, avoiding shaking and mixing, and centrifuging at 400g for 20 minutes at room temperature 20°C.
  • the centrifuge tube after centrifugation is divided into three layers, the upper layer is plasma, and the middle layer is milky white. It is a mononuclear lymphocyte.
  • the intermediate layer cells were gently aspirated with a sterile pipette, collected into a new centrifuge tube, diluted to three volumes with PBS phosphate buffer, centrifuged at 100 g for 10 minutes at room temperature, and the supernatant was discarded.
  • the lymphocytes were resuspended to 10 mL with PBS phosphate buffer, and the platelets were removed by repeating the previous procedure.
  • the lymphocytes were resuspended to 10 mL of multi-component RPMI 1640 medium (purchased from Invitrogen) containing 10% fetal calf serum for use as peripheral blood mononuclear lymphocyte PBMC, the percentage being mass percentage.
  • Example 2 Before the test, an equal volume ratio of the purified CTLA-4 antibody obtained in Example 2 was prepared to obtain a sample solution to be tested.
  • peripheral blood mononuclear lymphocyte PBMC was plated to a 96-well cell culture plate at 100 ⁇ l per well of 1 ⁇ 10 5 cells, and then the sample solution to be tested was added to the culture plate, and cultured at room temperature for 30 minutes. Finally, the superantigen SEB was added, and 50 ⁇ l of 100 ng/ml SEB was contained in each reaction well to ensure a volume of 250 ⁇ L per reaction well.
  • the reaction plate was cultured at 37 ° C in a 5% CO 2 incubator for 72 hours, and the supernatant was collected to obtain cells. The supernatant was frozen at -20 ° C and the percentage was a percentage by volume.
  • the cytokine interleukin IL-2 enzyme-linked immunosorbent assay in the cell supernatant was assayed using the R&D system-related detection kit Quantikine ELISA human IL-2 (S2050) and operated according to the instructions. All test reagents except the test antibody are provided by the test kit.
  • the enzyme-linked immunosorbent assay for the determination of the cytokine interleukin IL-2 content in the cell supernatant was performed using a double-antibody sandwich ELISA kit (purchased from R&D Systems, IL-2 Cat #S2050).
  • the experimental procedure is strictly in accordance with the instructions of the kit, and all test reagents are provided by the kit.
  • the specific experiment is briefly described as follows: IL-2 polyclonal antibody is coated on an ELISA microplate, and the cell supernatant obtained in the step 2. is used as a sample to be tested, and the standard sample and the sample to be tested are incubated at room temperature for 2 hours. Add 400 ⁇ l of wash solution to each well and wash the plate 4 times.
  • the activity of 97A8D1 is better than other antibodies.
  • the hIgG control was human IgG and the data in the table was IL-2 value (pg/mL).
  • RNA isolation The supernatant obtained from the subcloning culture of Example 1 was tested for antigen binding (ie, after the assay and activity assays of Examples 3-6), and 5 ⁇ 10 7 hybridoma cells were collected by centrifugation and added. 1 mL Trizol was mixed and transferred to a 1.5 mL centrifuge tube and allowed to stand at room temperature for 5 minutes. 0.2 mL of chloroform was added, shaken for 15 seconds, allowed to stand for 2 minutes, centrifuged at 12000 g for 5 minutes at 4 ° C, and the supernatant was transferred to a new 1.5 mL centrifuge tube. 0.5 mL of isopropanol was added, and the liquid in the tube was gently mixed.
  • Reverse transcription and PCR 1 ⁇ g of total RNA was taken, 20 ⁇ l of the system was placed, reverse transcriptase was added, and the reaction was carried out at 42 ° C for 60 minutes, and the reaction was terminated at 7 ° C for 10 minutes.
  • a 50 [mu]l PCR system was configured, including 1 [mu]l cDNA, 25 pmol of each primer, 1 [mu]l DNA polymerase, and a matching buffer system, 250 [mu]mol dNTPs.
  • the PCR program was set up, pre-denatured at 95 ° C for 3 minutes, denatured at 95 ° C for 30 seconds, annealed at 55 ° C for 30 seconds, extended at 72 ° C for 35 seconds, and further extended at 72 ° C for 5 minutes after 35 cycles to obtain a PCR product.
  • the kit for reverse transcription was PrimeScript RT Master Mix, purchased from Takara, catalog number RR036; the kit used for PCR was Q5 super-fidelity enzyme, purchased from NEB, catalog number M0492.
  • the ligation reaction was carried out: 50 ng of sample, 50 ng of T vector, 0.5 ⁇ l of ligase, 1 ⁇ l of buffer, 10 ⁇ l of reaction system, and reacted at 16 ° C for half an hour to obtain a ligation product, wherein the ligated kit was T4 DNA ligase, purchased from NEB, article number M0402 5 ⁇ l of the ligation product was added to 100 ⁇ l of competent cells (Ecos 101competent cells, available from Yeastern, Cat. No. FYE607), and ice bathed for 5 minutes.
  • T4 DNA ligase purchased from NEB
  • article number M0402 5 ⁇ l of the ligation product was added to 100 ⁇ l of competent cells (Ecos 101competent cells, available from Yeastern, Cat. No. FYE607), and ice bathed for 5 minutes.
  • the strain was preserved on an LB solid culture dish. After the end of the PCR reaction, 5 ⁇ l was taken for agarose gel electrophoresis detection, and the positive samples were sequenced. Among them, the steps of sequencing are described in Kabat, Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md. (1991).
  • nucleotide sequence of the CDR1 domain of the heavy chain protein variable region encoding 97A8D1 is from position 88 to position 105 of SEQ ID NO: 41;
  • the nucleotide sequence of the CDR2 domain of the heavy chain protein variable region encoding 97A8D1 is from positions 148 to 198 of SEQ ID NO: 41 of the Sequence Listing;
  • nucleotide sequence of the CDR3 domain of the heavy chain protein variable region encoding 97A8D1 is position 289 to 321 of SEQ ID NO: 41 of the Sequence Listing;
  • the nucleotide sequence of the CDR1 domain of the light chain protein variable region encoding 97A8D1 is from positions 73 to 108 of SEQ ID NO: 42 of the Sequence Listing;
  • the nucleotide sequence of the CDR2 domain in the light chain protein variable region encoding 97A8D1 is from positions 151 to 177 in SEQ ID NO: 42 of the Sequence Listing;
  • nucleotide sequence of the CDR3 domain of the light chain protein variable region encoding 97A8D1 is position 268 to 294 of SEQ ID NO: 42 of the Sequence Listing;
  • the nucleotide sequence of the CDR1 domain in the heavy chain protein variable region encoding 92C8B6 is the 88th to 105th positions in SEQ ID NO: 43 of the Sequence Listing;
  • the nucleotide sequence of the CDR2 domain of the heavy chain protein variable region encoding 92C8B6 is from positions 148 to 198 of SEQ ID NO: 43 of the Sequence Listing;
  • nucleotide sequence of the CDR3 domain of the heavy chain protein variable region encoding 92C8B6 is position 289 to 327 of SEQ ID NO: 43 of the Sequence Listing;
  • the nucleotide sequence of the CDR1 domain of the light chain protein variable region encoding 92C8B6 is the 73rd to 105th position in SEQ ID NO: 44 of the Sequence Listing;
  • the nucleotide sequence of the CDR2 domain of the light chain protein variable region encoding 92C8B6 is from positions 148 to 174 of SEQ ID NO: 44 of the Sequence Listing;
  • the nucleotide sequence of the CDR3 domain of the light chain protein variable region encoding 92C8B6 is from positions 265 to 294 of SEQ ID NO: 44 of the Sequence Listing;
  • the nucleotide sequence of the CDR1 domain in the heavy chain protein variable region encoding 31C12F3 is the 88th to 105th positions in SEQ ID NO: 45 of the Sequence Listing;
  • the nucleotide sequence of the CDR2 domain of the heavy chain protein variable region encoding 31C12F3 is from positions 148 to 198 of SEQ ID NO: 45 of the Sequence Listing;
  • nucleotide sequence of the CDR3 domain of the heavy chain protein variable region encoding 31C12F3 is position 286 to 327 of SEQ ID NO: 45 of the Sequence Listing;
  • the nucleotide sequence of the CDR1 domain in the light chain protein variable region encoding 31C12F3 is the 73rd to 105th positions in SEQ ID NO: 46 of the Sequence Listing;
  • the nucleotide sequence of the CDR2 domain in the light chain protein variable region encoding 31C12F3 is from positions 148. to 174 in SEQ ID NO: 46 of the Sequence Listing;
  • nucleotide sequence of the CDR3 domain of the light chain protein variable region encoding 31C12F3 is position 265 to 291 of SEQ ID NO: 46 of the Sequence Listing;
  • the nucleotide sequence of the CDR1 domain in the heavy chain protein variable region encoding 40C4C6 is from position 88 to position 105 in SEQ ID NO: 47 of the Sequence Listing;
  • the nucleotide sequence of the CDR2 domain of the heavy chain protein variable region encoding 40C4C6 is 148th to 198th in the SEQ ID NO: 47 of the Sequence Listing;
  • nucleotide sequence of the CDR3 domain of the heavy chain protein variable region encoding 40C4C6 is position 289 to 327 of SEQ ID NO: 47 of the Sequence Listing;
  • the nucleotide sequence of the CDR1 domain in the light chain protein variable region encoding 40C4C6 is the 73rd to 105th position in SEQ ID NO: 48 of the Sequence Listing;
  • the nucleotide sequence of the CDR2 domain in the light chain protein variable region encoding 40C4C6 is 148th to 174th in SEQ ID NO: 48 of the Sequence Listing;
  • the nucleotide sequence of the CDR3 domain in the light chain protein variable region encoding 40C4C6 is position 265 to 291 in SEQ ID NO: 48 of the Sequence Listing.
  • the nucleotide sequence of the CDR1 domain in the heavy chain protein variable region encoding 97B8E1 is from position 88 to position 105 in SEQ ID NO: 49 of the Sequence Listing;
  • the nucleotide sequence encoding the CDR2 domain of the heavy chain protein variable region of 97B8E1 is from positions 148 to 198 of SEQ ID NO: 49 of the Sequence Listing;
  • the nucleotide sequence of the CDR3 domain of the heavy chain protein variable region encoding 97B8E1 is from positions 289 to 330 of SEQ ID NO: 49 of the Sequence Listing;
  • the nucleotide sequence of the CDR1 domain of the light chain protein variable region encoding 97B8E1 is from positions 73 to 105 of SEQ ID NO: 50 of the Sequence Listing;
  • the nucleotide sequence of the CDR2 domain of the light chain protein variable region encoding 97B8E1 is from positions 148 to 174 of SEQ ID NO: 50 of the Sequence Listing;
  • the nucleotide sequence of the CDR3 domain in the light chain protein variable region encoding 97B8E1 is from positions 265 to 291 in SEQ ID NO: 50 of the Sequence Listing.
  • Example 2 has obtained purified CTLA-4 antibody from the culture supernatant of hybridoma cells, and according to the sequencing result of Example 7, it is confirmed that the heavy chain of the CTLA-4 antibody is variable. Region and light chain variable region sequences.
  • the heavy chain variable region sequence of the CTLA-4 antibody was recombined into an expression vector containing the signal peptide and the human heavy chain antibody IgG1 constant region (wherein the expression vector was purchased from Invitrogen), and the light chain variable region of the CTLA-4 antibody was The sequence was recombined into an expression vector containing the signal peptide and the human antibody light chain kappa constant region, and the recombinant plasmid was obtained and verified by sequencing (the sequencing method was the same as the sequencing method in Example 7).
  • the recombinant plasmid having a purity increased by mass spectrometry using an alkaline lysis kit was used at a mass of 500 ⁇ g or more, and filtered through a 0.22 ⁇ m filter (purchased from Millopore) for transfection.
  • Freestyle 293 expression medium was added with 10% (v/v) F68 (purchased from Invitrogen) to a final concentration of 0.1% (v/v) of F68 during transfection to obtain Freestyle 293 expression culture containing 0.1% (v/v) F68.
  • Base medium A.
  • Peptone was added the next day to a final concentration of 0.5% (w/v).
  • the antibody titer of the culture solution was measured.
  • the supernatant was collected by centrifugation (3500 RPM, 30 minutes), and filtered through a 0.22 ⁇ m filter to obtain a filtered cell supernatant for purification.
  • the concentration was measured using a spectrophotometer or a kit, the purity of the antibody was determined using HPLC-SEC, and the endotoxin content of the antibody was detected using an endotoxin test kit (purchased from Lonza).
  • the binding activity of the obtained human CTLA-4 antibody to the cell expression CTLA-4 was identified, and the detection results are shown in Fig. 9, Fig. 10, and Table 10 and Table 11, respectively.
  • the blocking activity of the obtained human CTLA-4 antibody was identified, and the detection results are shown in Fig. 11, Fig. 12, and Table 12 and Table 13, respectively.
  • Figure 11 Figure 12 and Table 12, Table 13 show that the whole human CTLA-4 antibody transformed by whole human IgG can block the binding of CTLA-4 to B7.1 and B7.2, and the antibody blocking activity is measured. quite.
  • Example 8 PHA-induced CD3+ T lymphocyte assay detects that CTLA-4 antibody blocks the activation of T lymphocytes after binding of B7.1 or B7.2 to CTLA-4.
  • CTLA-4 antibody blocked the activation of T lymphocytes by binding of B7.1 or B7.2 to CTLA-4.
  • T cells constitutively express CD28 PHA can non-specifically activate T cells, thereby inducing membrane expression of CTLA-4; Raji cells constitutively express CTLA-4/CD28 ligands B7.1 and B7.2; when activated T cells When incubated with Raji cells, CTLA-4 and CD28 competitively bind to the ligand. Since CTLA-4 has a higher affinity for ligand than CD28, when it is incubated with Raji cells, it shows a partial down-regulation of T cell activation when CTLA-4 When the ligand binding capacity is blocked, the down-regulation of T cell activation is reversed, which is manifested as an increase in IL-2 secretion.
  • PBMC cells were isolated from whole blood using the reagent FICOLL PAQUE PLUS and operated according to the instructions. The specific steps are as described in (5) of 4.4.2.
  • the obtained PBMC cells were manipulated according to the instructions using a human CD3+ cell extraction kit (MagCellectTM Human CD3+T Cell Isolation Kit) to obtain purified CD3+ cells, and human CD3+ T cells were seeded into a 6-well plate, and 10 ⁇ g/ was added. mL PHA was treated for 72 hours to obtain CTLA-4 overexpressing CD3+ T cell vesicles.
  • a human CD3+ cell extraction kit MagneticCellectTM Human CD3+T Cell Isolation Kit
  • Raji cells Prior to use, Raji cells were treated with 10 ⁇ g/mL mitomycin at 37 ° C for 1.5 hours and washed 3 times with PBS to remove residual mitomycin. At the same time, an equal volume ratio of the purified CTLA-4 antibody to be tested is prepared to obtain a sample solution to be tested.
  • the CD3+ T cell capsule obtained in step 1 was inoculated into a 96-well cell culture plate at a seeding density of 1 ⁇ 10 5 cells/well, and the inoculation volume was 50 ⁇ L per well, and the sample solution to be tested was added, 50 ⁇ L per well, and incubated for 30 minutes. Then, the Raji cells of step 2 were added, and 100 ⁇ L of 3 ⁇ 10 4 cells/well were plated to a 96-cell culture plate, and incubated at 37° C. in a 5% CO 2 incubator for 4 days. Cell supernatants were collected for cytokine detection, the percentage being volume percent.
  • the cytokine interleukin IL-2 in the cell supernatant obtained in the step 3 was subjected to enzyme-linked immunosorbent assay.
  • the R&D system related detection kit Quantikine ELISA human IL-2 (S2050) was used and operated according to the instructions. All test reagents except the test antibody are provided by the test kit.
  • Anti-human Fc IgG was immobilized on flow cells 1 and 2: HBS-EP+ (10 mmol/L HEPES, 150 mmol/L NaCl, 3 mM EDTA, 0.05% P20, pH 7.4) was used as running buffer, using a fixed guide template Immobilization of anti-human Fc IgG.
  • Flow cells 1 and 2 of the series S CM5 sensor chip were activated with freshly mixed 50 mmol/L NHS and 200 mmol/L EDC.
  • Anti-human Fc IgG was diluted to 20 ⁇ g/mL with 10 mmol/L NaAC (pH 4.5) and injected into activated flow cells 1 and 2. The remaining active coupling sites were blocked with 1 mol/L ethanolamine.
  • His-tagged hCTLA-4 ECD protein was diluted to 50 nmol/L, and then serially diluted 4 times in a 2-fold ratio with HBS-EP+ buffer. His-tagged hCTLA-4 ECD protein concentrations were 0 nmol/L, 3.125 nmol/L, 6.25 nmol/L, 12.5 nmol/L, 25 nmol/L and 50 nmol/L. KD measurements were performed using HBS-EP+ as the running buffer. Each antibody was injected onto the CM5 sensor flow cell 2 at a flow rate of 10 [mu]L/min to achieve a response of 230 RU.
  • the prepared His-tagged hCTLA-4 ECD protein was then injected into the flow cells 1 and 2 at a flow rate of 30 ⁇ L/min for 180 seconds.
  • the buffer flow was maintained for 400 seconds for dissociation measurement (30 ⁇ L/min).
  • 10 mmol/L glycine-HCl pH 1.5 was injected for 20 seconds (30 ⁇ L/min).
  • Flow cell 1 is used as a reference flow cell.
  • KD values for each antibody were assessed using Biacore T200 Evaluation Software 1.0 and the data was fitted using a 1:1 binding model. The results are shown in Table 15.
  • the KD values of the tested antibodies were at the nanomolar level and were comparable to the tool antibodies, indicating that these antibodies have good affinity for human CTLA-4 ECD.
  • the 97A8D1 antibody has the most affinity for human CTLA-4 ECD. Good, suitable for confirmation of in vivo activity as a candidate antibody.
  • the anti-tumor activity of the antibody in mice was evaluated using a human CTLA-4 knock-in C57BL/6 mouse using the MC38 syngeneic mouse model.
  • the experimental design was to select 50 human CTLA-4 knock-in C57BL/6 mice, which were divided into 6 groups, 10 in each group, using Ipilimumab and the isotype antibody hIgG1 as controls, and the samples were 92C8B6, 97A8D1 and 97B8E1.
  • the route of administration was intraperitoneal injection at a dose of 10 mg/kg, intraperitoneal injection at 0, 3, 6, and 10 days, and the mice were sacrificed on the 24th day, and tumor volume, mouse body weight, tumor weight, and mouse survival rate were measured.
  • the experimental results are shown in Figure 15.
  • Figure 15 shows that the antibodies of the present invention (including 92C8B6, 97A8D1 and 97B8E1) can significantly inhibit tumor growth, and the inhibitory effect is significantly better than the control antibody Ipilimumab (for example, the median survival time of 97A8D1 is 22.5 days, which is superior to the median survival of Ipilimumab. Period 19 days).
  • the fully human anti-CTLA-4 antibody 97A8D1 is expressed in a cell line lacking fucosyltransferase such that the cell line produces a defucosylated protein.
  • the molecular weight of the defucosylated antibody 97A8D1 and the fucosylated antibody 97A8D1 after Ide Z and EndoS digestion was analyzed by LC/MS to determine whether the Fc end of the antibody carries fucose. The results of the analysis are shown in Figure 16.
  • Figure 16 shows that after enzymatic treatment, the de-fucosylated antibody 97A8D1 has a Fc molecular weight of 23755 Da (Fig. 16A) and a non-degreasing Fc molecular weight of 23957 Da (Fig. 16B). The molecular weight difference is 202 Da (GlcNAc ). After enzymatic treatment, the fucosylated antibody 97A8D1 has a Fc molecular weight of 23,755 Da after de-glycanization (Fig. 16C), and a non-degreasing Fc molecular weight of 24,104 Da (Fig. 16D).
  • the molecular weight difference between the two is 349 Da (Fuc + GlcNAc), and because The Fc molecular weight of the defucosylated antibody (23957 Da) is 147 Da less than the Fc molecular weight of the fucosylated antibody (24104 Da), so it is speculated that the defucosylated antibody does not contain fucose at the molecular weight level, fucoal
  • the glycosylated antibody contains fucose.
  • Example 2 The experimental method is shown in Example 2 (3), and the test results are shown in Table 18 and Figure 17.
  • Antibodies can be obtained by immunizing wild-type mice, but humanized antibodies need to be humanized to obtain humanized antibodies.
  • the disadvantage is that the modified antibody may be more immunogenic and the antibody structure may be Changes result in loss of activity or poor productivity.
  • Full-body antibodies can be obtained by immunizing whole-human transgenic mice, but the amount or affinity of the obtained antibodies may be poor.
  • the expression and activity screening of antibodies can be performed by phage display technology by constructing an immunized mouse antibody library, but random recombination of the heavy and light chains of the antibody is involved, resulting in poor productivity of the formed antibody.
  • Antibodies can be expressed and screened by phage display technology by constructing a human antibody library, but the affinity of the obtained antibody is poor because it is not immunized.
  • the immunogen may be a polypeptide, a protein, other kinds of cells and genes, but there may be problems such as incorrect conformation, low expression, and poor immunogenicity.
  • sequence information related to the present invention is as follows:
  • nucleotide sequence corresponding to the amino acid sequence of the CTLA-4 antibody wherein the nucleotide sequences encoding CDR1, CDR2, CDR3, CDR1', CDR2', CDR3' are underlined:
  • CTLA-4 antibodies are as follows:

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Abstract

本发明公开了一种靶向CTLA-4的抗体、其制备方法和用途。具体地，本发明公开了一种新的靶向CTLA-4的全人单克隆抗体。本发明还公开了制备所述的单克隆抗体的方法。本发明的单克隆抗体能够高特异性地结合CTLA-4抗原，其具有很高的亲和力并且具有显著抗肿瘤等活性。

Description

靶向CTLA-4抗体、其制备方法和用途 技术领域
本发明涉及生物医药领域，更具体地涉及一种CTLA-4抗体及其制备方法和应用。
背景技术
单克隆抗体的应用是过去20年时间肿瘤治疗中最成功及最具变革意义的治疗手段之一。与传统化学药物相比，抗体药物具有更高的特异性及更低的毒性。虽然单抗药物取得了持续不断的成功，但仍面临诸多挑战。
鼠源单克隆抗体最大的缺陷则是它所诱发的HAMA(人抗鼠抗体)反应。因而，鼠单抗在肿瘤、器官移植等疾病的诊断和治疗上有较大的局限性；嵌合抗体仍保留着30％的鼠源序列，可引起不同程度的HAMA反应。临床显示不同的嵌合抗体有着不同程度的免疫原性；人源化抗体又称移植抗体。简单CDR移植常常导致抗原抗体亲和力下降，由于其仍至少具有10％的异源蛋白，在临床应用中还是受到不同程度的限制。因而有待于进一步研制更完善的治疗性抗体-完全人抗体。
1994年，美国Abgenix和Genpham两公司报告了利用转基因小鼠制备完全人抗体，从而解决了人体不能被随意免疫这一人抗体制备研究的难题。此后完全人抗体制备技术的不断发展成熟，所获人单克隆抗体已具有较强的抗肿瘤活性。
癌症免疫治疗是癌症治疗最新突破，利用患者自身免疫系统来攻击肿瘤细胞。
免疫检查点是指免疫系统中存在的一些抑制性信号通路，通过调节外周组织中免疫反应的持续性和强度避免组织损伤，并参与维持对于自身抗原的耐受。利用免疫检查点的抑制性信号通路抑制T细胞活性是肿瘤逃避免疫杀伤的重要机制。针对免疫检查点的阻断是众多激活抗肿瘤免疫的有效策略之一。
免疫检查点蛋白的抑制剂具有治疗各种肿瘤类型(如转移性黑素瘤，肺癌，乳腺癌，肾细胞癌等)的潜力。最近癌症免疫治疗方法的研究已经显示出可喜的成果，特别是对转移癌癌症病例。此外，癌症免疫治疗在治疗血液癌症方面具有巨大的潜力，包括霍奇金淋巴瘤，多发性骨髓瘤，骨髓发育不良综合征， 非霍奇金淋巴瘤等。免疫检查点抑制剂引起的副作用是可以忽略的，可逆的和可控的，有效的免疫检查点抑制剂可以显著提高癌症患者的总生存期。免疫检查点抑制剂可以与靶向治疗或常规放射治疗和化学疗法结合使用，并且这种组合疗法可有效治疗许多类型的癌症。
CTLA-4(细胞毒性T淋巴细胞相关蛋白4或CD152)是作为主要免疫检查点分子之一的I型跨膜蛋白，其包含细胞外Ig-V样结构域，Ig-C样结构域，跨膜结构域和胞内C-末端结构域。CTLA-4主要在调节性T细胞胞内为组成型表达，在活化的T细胞表面为诱导型表达，并与配体B7.1(CD80)和B7.2(CD82)相互作用诱导抑制性信号，导致T细胞活化、增值抑制和细胞因子产生减少，CTLA-4在Treg细胞表面呈组成型高表达，参与Treg对其他效应T细胞活性的下调。
CTLA-4在T细胞激活早期阶段活性的调节至关重要，从而成为外周淋巴系统免疫耐受的主要调控机制，CTLA-4通过与CD28竞争配体，及通过其自身对TCR信号通路的抑制，调节T细胞对抗原的反应强度，从而使机体表现出对自身抗原或外源弱抗原识别的耐受。
研究认为阻断CTLA-4/B7配体相互作用，可以提高机体对肿瘤抗原的识别活性，刺激抗原特异性T细胞的增值，从而达到激活免疫系统并增强抗肿瘤免疫应答，并且已在多个同系小鼠肿瘤模型中被证明，CTLA-4的阻断促进抗肿瘤活性。因此，药物阻断CTLA-4/B7.1及B7.2途径可以为各种癌症和其他免疫疾病提供新的治疗方法。
目前，本领域现有的CTLA-4抗体还有许多的不足，比如治疗窗口较小、适应症有限、治疗成本高等，所以开发CTLA-4的阻断型抗体是治疗多种癌的迫切需求。
发明内容
为了克服目前缺少全人CTLA-4抗体及现有CTLA-4抗体在活性及安全性方面的缺点，本发明提供一种亲和力高、特异性强的CTLA-4抗体及其制备方法。
在本发明的第一方面，提供了一种抗体的重链可变区，所述的重链可变区包括以下三个互补决定区CDR：
SEQ ID NO:8n+2所示的CDR1，
SEQ ID NO:8n+3所示的CDR2，和
SEQ ID NO:8n+4所示的CDR3；
其中，各n独立地为0、1、2、3或4；
其中，上述氨基酸序列中任意一种氨基酸序列还包括任选地经过添加、缺失、修饰和/或取代至少一个氨基酸的，并能够保留CTLA-4结合亲和力的衍生序列。
在另一优选例中，所述重链可变区具有SEQ ID NO:8n+1所示的氨基酸序列，其中，n为0、1、2、3或4。
在本发明的第二方面，提供了一种抗体的重链，所述的重链具有如权利要求1所述的重链可变区。
在本发明的第三方面，提供了一种抗体的轻链可变区，所述的轻链可变区包括以下三个互补决定区CDR：
SEQ ID NO:8n+6所示的CDR1'，
SEQ ID NO:8n+7所示的CDR2'，和
SEQ ID NO:8n+8所示的CDR3'；
其中，各n独立地为0、1、2、3或4；
其中，上述氨基酸序列中任意一种氨基酸序列还包括任选地经过添加、缺失、修饰和/或取代至少一个氨基酸的，并能够保留CTLA-4结合亲和力的衍生序列。
在另一优选例中，所述轻链可变区具有SEQ ID NO:8n+5所示的氨基酸序列，其中，n为0、1、2、3或4。
在本发明的第四方面，提供了一种抗体的轻链，所述的轻链具有如权利要求3所述的轻链可变区。
在本发明的第五方面，提供了一种抗体，所述抗体具有：
(1)如本发明的第一方面所述的重链可变区；和/或
(2)如本发明的第三方面所述的轻链可变区；
或者，所述抗体具有：如本发明的第二方面所述的重链；和/或如本发明的第四方面所述的轻链，
其中，上述氨基酸序列中任意一种氨基酸序列还包括任选地经过添加、缺失、修饰和/或取代至少一个氨基酸的，并能够保留CTLA-4结合亲和力的衍生序列。
在另一优选例中，上述任一CDR的氨基酸序列中包含经过添加、缺失、修饰和/或取代1、2或3个氨基酸的衍生CDR序列，并且使得含有所述衍生CDR序列的VH和VL所构成的衍生抗体能够保留与CTLA-4结合的亲和力。
在另一优选例中，所述的衍生抗体与CTLA-4结合的亲和力F1与相应非衍生的抗体与CTLA-4结合的亲和力F0之比(F1/F0)为0.5-2，较佳地为0.7-1.5，和更佳地0.8-1.2。
在另一优选例中，所述添加、缺失、修饰和/或取代的氨基酸数量为1-5个(如1-3个，较佳地1-2个，更佳地1个)。
在另一优选例中，所述的经过添加、缺失、修饰和/或取代至少一个氨基酸的，并能够保留CTLA-4结合亲和力的衍生序列为同源性为至少96％的氨基酸序列。
在另一优选例中，所述的抗体还包括重链恒定区和/或轻链恒定区。
在另一优选例中，所述的重链恒定区为人源的，和/或所述的轻链恒定区为人源的。
在另一优选例中，所述抗体选自下组：嵌合抗体、人源化抗体、全人抗体、或其组合。
在另一优选例中，所述的全人抗体在人中的免疫原性Z1与非全人的抗体(如鼠源抗体)在人中的免疫原性Z0之比(Z1/Z0)为0-0.5，较佳地0-0.2，更佳地0-0.05(如0.001-0.05)。
在另一优选例中，所述的抗体是部分或全人源化、或全人的单克隆抗体。
在另一优选例中，所述的抗体为双链抗体、或单链抗体。
在另一优选例中，所述抗体为抗体全长蛋白、或抗原结合片段。
在另一优选例中，所述抗体为双特异性抗体、或多特异性抗体。
在另一优选例中，所述抗体具有选自下组的一个或多个特性：
(a)抑制肿瘤细胞迁移或转移；
(b)抑制肿瘤生长。
在另一优选例中，所述的抗体具有如本发明的第一方面所述的重链可变区和如本发明的第三方面所述的轻链可变区；
其中，所述的重链可变区包括以下三个互补决定区CDR：
SEQ ID NO:2所示的CDR1，
SEQ ID NO:3所示的CDR2，和
SEQ ID NO:4所示的CDR3；
其中，所述的轻链可变区包括以下三个互补决定区CDR：
SEQ ID NO:6所示的CDR1'，
SEQ ID NO:7所示的CDR2'，和
SEQ ID NO:8所示的CDR3'；
其中，上述氨基酸序列中任意一种氨基酸序列还包括任选地经过添加、缺失、修饰和/或取代至少一个氨基酸的，并能够保留CTLA-4结合亲和力的衍生序列。
在另一优选例中，所述抗体的重链可变区含有SEQ ID NO:8n+1所示的氨基酸序列；和/或所述抗体的轻链可变区含有SEQ ID NO:8n+5所示的氨基酸序列，其中，各n独立地为0、1、2、3或4。
在另一优选例中，所述抗体的重链可变区含有SEQ ID NO:1所示的氨基酸序列。
在另一优选例中，所述抗体的轻链可变区含有SEQ ID NO:5所示的氨基酸序列。
在另一优选例中，所述抗体的重链可变区含有SEQ ID NO:1所示的氨基酸序列，并且所述抗体的轻链可变区含有SEQ ID NO:5所示的氨基酸序列。
在另一优选例中，所述抗体的重链可变区含有SEQ ID NO:9所示的氨基酸序列，并且所述抗体的轻链可变区含有SEQ ID NO:13所示的氨基酸序列。
在另一优选例中，所述抗体的重链可变区含有SEQ ID NO:17所示的氨基酸序列，并且所述抗体的轻链可变区含有SEQ ID NO:21所示的氨基酸序列。
在另一优选例中，所述抗体的重链可变区含有SEQ ID NO:25所示的氨基酸序列，并且所述抗体的轻链可变区含有SEQ ID NO:29所示的氨基酸序列。
在另一优选例中，所述抗体的重链可变区含有SEQ ID NO:33所示的氨基酸序列，并且所述抗体的轻链可变区含有SEQ ID NO:37所示的氨基酸序列。
在另一优选例中，所述的抗体选自下组：97A8D1、92C8B6、31C12F3、40C4C697B8E1。
所述重链可变区的氨基酸序列与如序列表中SEQ ID NO:1、SEQ ID NO:9、SEQ  ID NO:17、SEQ ID NO:25、SEQ ID NO:33所示的氨基酸序列至少有80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的序列同源性。
所述轻链可变区的氨基酸序列与如序列表中SEQ ID NO:5、SEQ ID NO:13、SEQ ID NO:21、SEQ ID NO:29、SEQ ID NO:37所示的氨基酸序列至少有80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的序列同源性。
在另一优选例中，所述的抗体为去岩藻糖基化抗体。
在另一优选例中，所述的抗体为去岩藻糖基化抗体97A8D1。
在本发明的第六方面，提供了一种重组蛋白，所述的重组蛋白包括：
(i)如本发明的第一方面所述的重链可变区、如本发明的第二方面所述的重链、如本发明的第三方面所述的轻链可变区、如本发明的第四方面所述的轻链、或如本发明的第五方面中任一项所述的抗体；以及
(ii)任选的协助表达和/或纯化的标签序列。
在另一优选例中，所述的标签序列包括6His标签。
在另一优选例中，所述的重组蛋白(或多肽)包括融合蛋白。
在另一优选例中，所述的重组蛋白为单体、二聚体、或多聚体。
在另一优选例中，所述重组蛋白包括：
(i)如本发明的第五方面所述的抗体，所述抗体的重链可变区含有SEQ ID NO:1所示的氨基酸序列，并且所述抗体的轻链可变区含有SEQ ID NO:5所示的氨基酸序列，以及
(ii)任选的协助表达和/或纯化的标签序列。
在另一优选例中，所述重组蛋白包括：
(i)去岩藻糖基化抗体(例如去岩藻糖基化抗体97A8D1)；
(ii)任选的协助表达和/或纯化的标签序列。
在本发明的第七方面，提供了一种多核苷酸，所述多核苷酸编码选自下组的多肽：
(1)如本发明的第一方面所述的重链可变区、如本发明的第二方面所述的重链、如本发明的第三方面所述的轻链可变区、如本发明的第四方面所述的轻链、或如本发明的第五方面中任一项所述的抗体；以及
(2)如本发明的第六方面所述的重组蛋白。
在另一优选例中，编码所述重链可变区的多核苷酸如SEQ ID NO:41、43、45、47或49所示；和/或，
编码所述轻链可变区的多核苷酸如42、44、46、48或50所示。
在另一优选例中，编码所述重链可变区序列的多核苷酸如SEQ ID NO:41所示；并且编码所述轻链可变区序列的多核苷酸如42所示。
在另一优选例中，编码所述重链可变区的多核苷酸如SEQ ID NO:43所示；并且编码所述轻链可变区的多核苷酸如44所示。
在另一优选例中，编码所述重链可变区的多核苷酸如SEQ ID NO:45所示；并且编码所述轻链可变区的多核苷酸如46所示。
在另一优选例中，编码所述重链可变区的多核苷酸如SEQ ID NO:47所示；并且编码所述轻链可变区的多核苷酸如48所示。
在另一优选例中，编码所述重链可变区的多核苷酸如SEQ ID NO:49所示；并且编码所述轻链可变区的多核苷酸如50所示。
在本发明的第八方面，提供了一种载体，所述载体含有本发明的第七方面中任一项所述的多核苷酸。
在另一优选例中，所述的载体包括：细菌质粒、噬菌体、酵母质粒、植物细胞病毒、哺乳动物细胞病毒如腺病毒、逆转录病毒、或其他载体。
在本发明的第九方面，提供了一种遗传工程化的宿主细胞，所述宿主细胞含有本发明的第八方面所述的载体或基因组中整合有本发明的第七方面中任一项所述的多核苷酸。
在本发明的第十方面，提供了一种药物组合物，所述药物组合物含有：
(i)活性成分，所述活性成分选自下组：如本发明的第一方面所述的重链可变区、如本发明的第二方面所述的重链、如本发明的第三方面所述的轻链可变区、如本发明的第四方面所述的轻链、或如本发明的第五方面中任一项所述的抗体、如本发明的第六方面所述的重组蛋白、或其组合；以及
(ii)药学上可接受的载体。
在另一优选例中，所述的药物组合物为液态制剂。
在另一优选例中，所述的药物组合物为注射剂。
在另一优选例中，所述的药物组合物包括0.01～99.99％的如本发明的第五方面中任一项所述的抗体、如本发明的第六方面所述的重组蛋白、或其组合和0.01～99.99％的药用载体，所述百分比为占所述药物组合物的质量百分比。
在本发明的第十一方面，提供了一种活性成分的用途，所述活性成分选自下组：如本发明的第一方面所述的重链可变区、如本发明的第二方面所述的重链、如本发明的第三方面所述的轻链可变区、如本发明的第四方面所述的轻链、或如本发明的第五方面中任一项所述的抗体、如本发明的第六方面所述的重组蛋白、或其组合，其中所述活性成分被用于制备预防和/或治疗CTLA-4相关肿瘤和/或病毒感染相关的疾病的药物。
在另一优选例中，所述CTLA-4相关的疾病选自下组：黑色素瘤，间皮瘤，非小细胞肺癌，乳腺癌，肝癌，滑膜肉瘤，转移性结肠癌，肾癌，膀胱癌，前列腺癌，卵巢癌，丙型肝炎病毒慢性感染，晚期实体癌，消化器官恶性肿瘤，转移性非小细胞肺癌，前列腺肿瘤，子宫内膜癌，子宫内膜癌肉瘤，复发黑色素瘤，头颈部鳞状细胞癌，肉瘤，默克尔细胞癌，皮肤T细胞淋巴瘤，输卵管癌，腹膜肿瘤，肌肉浸润性膀胱癌，广泛的阶段小细胞肺癌，成人急性髓细胞性白血病，非典型慢性粒细胞白血病，先前治疗的骨髓增生异常综合征，卵巢上皮细胞癌，泌尿系统恶性肿瘤，成人III级淋巴瘤样肉芽肿，B细胞慢性淋巴细胞白血病，皮肤B细胞非霍奇金淋巴瘤，眼内淋巴瘤，睾丸绒毛膜癌，神经母细胞瘤，食管癌等。
在本发明的第十二方面，提供了一种体外检测样品中CTLA-4蛋白的组合物，其包括如本发明的第五方面中任一项所述的抗体、如本发明的第六方面所述的重组蛋白、或其组合作为活性成分。
在本发明的第十三方面，提供了一种治疗肿瘤或者病毒感染疾病的方法，包括：使用如本发明的第五方面中任一项所述的抗体、如本发明的第六方面所述的 重组蛋白、或其组合。
在另一优选例中，所述肿瘤为癌症。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1 CTLA-4-hFc蛋白与生物素标记的配体B7.1-hFc或B7.1-hFc的结合活性。
图2 CTLA-4(Y201V)蛋白转染的HEK293细胞FACS检测结果。
图3 ELISA检测HEK293-CTLA-4细胞免疫后Harbour转基因小鼠血清抗体效价。
图4(A)和(B)FACS检测CTLA-4抗体与CHOK1-hCTLA-4的结合反应。
图5(A)和(B)FACS检测CTLA-4抗体与293F-cCTLA-4的结合反应。
图6(A)、(B)和(C)CTLA-4抗体对CTLA-4蛋白与其受体B7.1的结合的抑制。
图7(A)、(B)和(C)CTLA-4抗体对CTLA-4蛋白与其受体B7.2的结合的抑制。
图8 CTLA-4抗体在PBMC淋巴细胞刺激试验中对IL-2分泌的影响。图9(A)和(B)流式细胞实验(FACS)检测抗体与人CTLA-4表达细胞的结合。
图10(A)和(B)流式细胞实验(FACS)检测抗体与猴CTLA-4表达细胞的结合。
图11(A)和(B)全人CTLA-4抗体对CTLA-4蛋白与其受体B7.1的结合的抑制。
图12(A)和(B)全人CTLA-4抗体对CTLA-4蛋白与其受体B7.2的结合的抑制。
图13 CTLA-4全人源抗体在SEB依赖的PBMC激活试验中对IL-2分泌的影响。
图14(A)、(B)(C)和(D)CTLA-4全人源抗体在PHA诱导的T淋巴囊细胞激活试验中对IL-2分泌的影响。
图15 CTLA-4抗体对荷瘤小鼠存活率的影响。
图16(A)、(B)(C)和(D)用液质联用测定去岩藻糖基化和岩藻糖基化抗CTLA-4抗体Fc段分子量。
图17去岩藻糖基化和岩藻糖基化抗CTLA-4抗体在SEB依赖的PBMC激活试验中对IL-2分泌的影响。
具体实施方式
本发明人通过广泛而深入的研究，采用大鼠-人嵌合抗体的转基因小鼠，采用293F-HuCTLA-4细胞(例如重组表达的293F-HuCTLA4细胞)对转基因小鼠进行免疫，意外地获得高活性的抗CTLA-4的全人抗体。实验结果表明，所述的与人源和猴源的CTLA-4蛋白均具有高度亲和力(K D为2.15E-09nM)，能够抑制CTLA-4与其受体B7.1和B7.2的结合，能在PHA诱导的人T淋巴细胞或SEB介导激活的人外周血单核细胞中显著增加IL-2的表达水平，并且缺乏与人CD28等同类蛋白抗原的交叉反应。所获得全人抗CTLA-4抗体，具有亲和力更高、体外及体内活性更强等特点。并且所述全人CTLA-4抗体(例如97A8D1)体内抗肿瘤效果优于现有技术中的抗体Ipilimumab。在此基础上完成了本发明。
术语
CTLA-4
CTLA-4即细胞毒性T淋巴细胞相关抗原4，结构上属于CD28免疫球蛋白超家族，在激活的T细胞呈诱导型表达和在调节性T细胞组成型表达，作为免疫检验点蛋白，通过与CD28竞争性结合配体，从而下调T细胞的活性。对于转基因小鼠和荷瘤小鼠的研究表明，CTLA-4参与肿瘤对免疫系统识别和杀伤的逃避，所以阻断CTLA-4的活性，可能提高机体对肿瘤生长的抑制。
抗体
如本文所用，术语“抗体”或“免疫球蛋白”是有相同结构特征的约150000道尔顿的异四聚糖蛋白，其由两个相同的轻链(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)，其后是多个恒定区。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。特殊的氨基酸残基在轻链和重链的可变区之间形成界面。
如本文所用，术语“可变”表示抗体中可变区的某些部分在序列上有所不同，它形成了各种特定抗体对其特定抗原的结合和特异性。然而，可变性并不均匀地分布在整个抗体可变区中。它集中于轻链和重链可变区中称为互补决定区(CDR)或超变区中的三个片段中。可变区中较保守的部分称为构架区(FR)。天然重链和轻链的可变区中各自包含四个FR区，它们大致上呈β-折叠构型，由形成连接环的三个CDR相连，在某些情况下可形成部分β折叠结构。每条链中的CDR通过FR区紧密地靠在一起并与另一链的CDR一起形成了抗体的抗原结合部位(参见Kabat等,NIH Publ.No.91-3242,卷I，647-669页(1991))。恒定区不直接参与抗体与抗原的结合，但是它们表现出不同的效应功能，例如参与抗体的依赖于抗体的细胞毒性。
脊椎动物抗体(免疫球蛋白)的“轻链”可根据其恒定区的氨基酸序列归为明显不同的两类(称为κ和λ)中的一类。根据其重链恒定区的氨基酸序列，免疫球蛋白可以分为不同的种类。主要有5类免疫球蛋白：IgA、IgD、IgE、IgG和IgM，其中一些还可进一步分成亚类(同种型)，如IgG1、IgG2、IgG3、IgG4、IgA和IgA2。对应于不同类免疫球蛋白的重链恒定区分别称为α、δ、ε、γ、和μ。不同类免疫球蛋白的亚单位结构和三维构型是本领域人员所熟知的。
一般，抗体的抗原结合特性可由位于重链和轻链可变区的3个特定的区域来描述，称为可变区域(CDR)，将该段间隔成4个框架区域(FR)，4个FR的氨基酸序列相对比较保守，不直接参与结合反应。这些CDR形成环状结构，通过其间的FR形成的β折叠在空间结构上相互靠近，重链上的CDR和相应轻链上的CDR构成了抗体的抗原结合位点。可以通过比较同类型的抗体的氨基酸序列来确定是哪些氨基酸构成了FR或CDR区域。
本发明不仅包括完整的抗体，还包括具有免疫活性的抗体的片段或抗体与其他序列形成的融合蛋白。因此，本发明还包括所述抗体的片段、衍生物和类似物。
在本发明中，抗体包括用本领域技术人员熟知技术所制备的鼠的、嵌合的、人源化的或者全人的抗体。重组抗体，例如嵌合的和人源化的单克隆抗体，包 括人的和非人的部分，可以通过标准的DNA重组技术获得，它们都是有用的抗体。嵌合抗体是一个分子，其中不同的部分来自不同的动物种，例如具有来自鼠的单克隆抗体的可变区，和来自人免疫球蛋白的恒定区的嵌合抗体(见例如美国专利4,816,567和美国专利4,816,397,在此通过引用方式整体引入本文)。人源化的抗体是指来源于非人物种的抗体分子，具有一个或多个来源于非人物种的互补决定区(CDRs)和来源于人免疫球蛋白分子的框架区域(见美国专利5,585,089，在此通过引用方式整体引入本文)。这些嵌合和人源化的单克隆抗体可以采用本领域熟知的DNA重组技术制备。
在本发明中，抗体可以是单特异性、双特异性、三特异性、或者更多的多重特异性。
在本发明中，本发明的抗体还包括其保守性变异体，指与本发明抗体的氨基酸序列相比，有至多10个，较佳地至多8个，更佳地至多5个，最佳地至多3个氨基酸被性质相似或相近的氨基酸所替换而形成多肽。这些保守性变异多肽最好根据表A进行氨基酸替换而产生。
表A
最初的残基 代表性的取代 优选的取代
Ala(A) Val；Leu；Ile Val
Arg(R) Lys；Gln；Asn Lys
Asn(N) Gln；His；Lys；Arg Gln
Asp(D) Glu Glu
Cys(C) Ser Ser
Gln(Q) Asn Asn
Glu(E) Asp Asp
Gly(G) Pro；Ala Ala
His(H) Asn；Gln；Lys；Arg Arg
Ile(I) Leu；Val；Met；Ala；Phe Leu
Leu(L) Ile；Val；Met；Ala；Phe Ile
Lys(K) Arg；Gln；Asn Arg
Met(M) Leu；Phe；Ile Leu
Phe(F) Leu；Val；Ile；Ala；Tyr Leu
Pro(P) Ala Ala
Ser(S) Thr Thr
Thr(T) Ser Ser
Trp(W) Tyr；Phe Tyr
Tyr(Y) Trp；Phe；Thr；Ser Phe
Val(V) Ile；Leu；Met；Phe；Ala Leu
抗CTLA-4的抗体
本发明提供一种针对CTLA-4的高特异性和高亲和力的抗体，其包括重链和轻链，所述重链含有重链可变区(VH)氨基酸序列，所述轻链含有轻链可变区(VL)氨基酸序列。
优选地，重链可变区(VH)包括以下三个互补决定区CDR：
SEQ ID NO:2所示的CDR1，
SEQ ID NO:3所示的CDR2，和
SEQ ID NO:4所示的CDR3；
轻链可变区(VL)包括以下三个互补决定区CDR：
SEQ ID NO:6所示的CDR1'，
SEQ ID NO:7所示的CDR2'，和
SEQ ID NO:8所示的CDR3'；
其中，上述氨基酸序列中任意一种氨基酸序列还包括任选地经过添加、缺失、修饰和/或取代至少一个氨基酸的，并能够保留CTLA-4结合亲和力的衍生序列。
在另一优选例中，所述经过添加、缺失、修饰和/或取代至少一个氨基酸序列所形成的序列优选为同源性或序列相同性为至少80％，较佳地至少85％，更佳地至少为90％，最佳地至少95％的氨基酸序列。
本领域普通技术人员公知的测定序列同源性或相同性的方法包括但不限于：计算机分子生物学(Computational Molecular Biology)，Lesk，A.M.编，牛津大学出版社，纽约，1988；生物计算：信息学和基因组项目(Biocomputing:Informatics and Genome Projects)，Smith，D.W.编，学术出版社，纽约，1993；序列数据的计算机分析(Computer Analysis of Sequence Data)，第一部分，Griffin，A.M.和Griffin，H.G.编，Humana Press，新泽西，1994；分子生物学中的序列分析(Sequence Analysis in Molecular Biology)，von Heinje，G.，学术出版社，1987和序列分析引物(Sequence Analysis Primer)，Gribskov，M.与Devereux，J.编M Stockton Press，纽约，1991和Carillo，H.与Lipman，D.，SIAM J.Applied Math.，48:1073(1988)。测定相同性的优选方法要在测试的序列之间得到最大的匹配。测定相同性的方法编译在公众可获得的计算机程序中。优选的测定两条序列之间相同性的计算 机程序方法包括但不限于：GCG程序包(Devereux，J.等，1984)、BLASTP、BLASTN和FASTA(Altschul，S，F.等，1990)。公众可从NCBI和其它来源得到BLASTX程序(BLAST手册，Altschul，S.等，NCBI NLM NIH Bethesda，Md.20894；Altschul，S.等，1990)。熟知的Smith Waterman算法也可用于测定相同性。
本发明的抗体可以是双链或单链抗体，并且可以是选自动物源抗体、嵌合抗体、人源化抗体，更优选为人源化抗体、人-动物嵌合抗体，更优选为全人源化抗体。
本发明所述抗体衍生物可以是单链抗体、和/或抗体片段，如：Fab、Fab'、(Fab')2或该领域内其他已知的抗体衍生物等，以及IgA、IgD、IgE、IgG以及IgM抗体或其他亚型的抗体中的任意一种或几种。
其中，所述动物优选为哺乳动物，如鼠。
本发明抗体可以是靶向人CTLA-4的嵌合抗体、人源化抗体、CDR嫁接和/或修饰的抗体。
本发明上述内容中，所述添加、缺失、修饰和/或取代的氨基酸数量，优选为不超过初始氨基酸序列总氨基酸数量的40％，更优选为不超过35％，更优选为1-33％，更优选为5-30％，更优选为10-25％，更优选为15-20％。
本发明上述内容中，更优选地，所述添加、缺失、修饰和/或取代的氨基酸数量，可以是1-7个，更优选为1-5个，更优选为1-3个，更优选为1-2个。
在另一优选例中，所述靶向CTLA-4的抗体为97A8D1、92C8B6、31C12F3、40C4C6或97B8E1。
在另一优选例中，所述抗体97A8D1的重链可变区(VH)氨基酸序列为如SEQ ID NO.:1所示的氨基酸序列。
在另一优选例中，所述抗体97A8D1的轻链可变区氨基酸序列为如SEQ ID NO.:5所示的氨基酸序列。
抗体的制备
本发明抗体或其片段的DNA分子的序列可以用常规技术，比如利用PCR扩增或基因组文库筛选等方法获得。此外，还可将轻链和重链的编码序列融合在一起，形成单链抗体。
一旦获得了有关的序列，就可以用重组法来大批量地获得有关序列。这通常是将其克隆入载体，再转入细胞，然后通过常规方法从增殖后的宿主细胞中分离得到有关序列。
此外，还可用人工合成的方法来合成有关序列，尤其是片段长度较短时。通常，通过先合成多个小片段，然后再进行连接可获得序列很长的片段。
目前，已经可以完全通过化学合成来得到编码所述的本发明的抗体(或其片段，或其衍生物)的DNA序列。然后可将该DNA序列引入本领域中已知的各种现有的DNA分子(或如载体)和细胞中。此外，还可通过化学合成将突变引入本发明蛋白序列中。
本发明还涉及包含上述的适当DNA序列以及适当启动子或者控制序列的载体。这些载体可以用于转化适当的宿主细胞，以使其能够表达蛋白质。
宿主细胞可以是原核细胞，如细菌细胞；或是低等真核细胞，如酵母细胞；或是高等真核细胞，如哺乳动物细胞。优选的动物细胞包括(但并不限于)：CHO-S、HEK-293细胞。
通常，在适合本发明抗体表达的条件下，培养转化所得的宿主细胞。然后用常规的免疫球蛋白纯化步骤，如蛋白A-Sepharose、羟基磷灰石层析、凝胶电泳、透析、离子交换层析、疏水层析、分子筛层析或亲和层析等本领域技术人员熟知的常规分离纯化手段纯化得到本发明的抗体。
所得单克隆抗体可用常规手段来鉴定。比如，单克隆抗体的结合特异性可用免疫沉淀或体外结合试验(如放射性免疫测定(RIA)或酶联免疫吸附测定(ELISA))来测定。单克隆抗体的结合亲和力例如可用Munson等,Anal.Biochem.,107:220(1980)的Scatchard分析来测定。
本发明的抗体可在细胞内、或在细胞膜上表达、或分泌到细胞外。如果需要，可利用其物理的、化学的和其它特性通过各种分离方法分离和纯化重组的蛋白。这些方法是本领域技术人员所熟知的。这些方法的例子包括但并不限于：常规的复性处理、用蛋白沉淀剂处理(盐析方法)、离心、渗透破菌、超声处理、超离心、分子筛层析(凝胶过滤)、吸附层析、离子交换层析、高效液相层析(HPLC)和其它各种液相层析技术及这些方法的结合。
应用
本发明还提供了本发明抗体的用途，例如用于制备用于治疗CTLA-4相关的疾病的药物。所述CTLA-4相关的疾病包括肿瘤发生、生长和/或转移、病毒感染等相关疾病等。
本发明抗体的用途，包括(但并不限于)：
(i)治疗肿瘤发生、生长和/或转移。所述肿瘤包括(但并不限于)：乳腺癌(如三阴性乳腺癌)、胰腺癌、肺癌(如非小细胞肺癌、广泛的阶段小细胞肺癌、转移性非小细胞肺癌)、恶性胶质瘤、消化器官恶性肿瘤、胃癌、肝癌、食道癌、肾癌、结直肠癌、转移性结肠癌、膀胱癌、前列腺肿瘤(如前列腺癌)、子宫内膜癌、子宫内膜癌肉瘤、宫颈癌、卵巢癌、输卵管癌、白血病(如成人急性髓细胞性白血病、非典型慢性粒细胞白血病)、骨髓癌、肉瘤(如血管肉瘤、滑膜肉瘤)、黑色素瘤、复发黑色素瘤、间皮瘤、晚期实体癌、头颈部鳞状细胞癌、默克尔细胞癌、皮肤T细胞淋巴瘤、腹膜肿瘤、肌肉浸润性膀胱癌、先前治疗的骨髓增生异常综合征、卵巢上皮细胞癌、泌尿系统恶性肿瘤、成人III级淋巴瘤样肉芽肿、B细胞慢性淋巴细胞白血病、皮肤B细胞非霍奇金淋巴瘤、眼内淋巴瘤、睾丸绒毛膜癌、神经母细胞瘤、食管癌。尤其是三阴性乳腺癌、胰腺癌、恶性胶质瘤和肺癌、更优选为三阴性乳腺癌和/或胰腺癌；
(ii)治疗病毒感染性疾病。所述病毒包括(但并不限于)：乙型肝炎病毒、丙型肝炎病毒(慢性感染)、艾滋病毒。
药物组合物
本发明还提供了一种组合物。在优选例中，所述的组合物是药物组合物，它含有上述的抗体或其活性片段或其融合蛋白，以及药学上可接受的载体。通常，可将这些物质配制于无毒的、惰性的和药学上可接受的水性载体介质中，其中pH通常约为5-8，较佳地pH约为6-8，尽管pH值可随被配制物质的性质以及待治疗的病症而有所变化。配制好的药物组合物可以通过常规途径进行给药，其中包括(但并不限于)：瘤内、腹膜内、静脉内、或局部给药。
本发明的药物组合物可直接用于结合CTLA-4蛋白分子，因而可用于预防和治疗肿瘤等疾病。此外，还可同时使用其他治疗剂。
本发明的药物组合物含有安全有效量(如0.001-99wt％,较佳地0.01-90wt％，更佳地0.1-80wt％)的本发明上述的单克隆抗体以及药学上可接受的载体或赋形剂。这类载体包括(但并不限于)：盐水、缓冲液、葡萄糖、水、甘油、乙醇、及其组合。药物制剂应与给药方式相匹配。本发明的药物组合物可以被制成针剂形式，例如用生理盐水或含有葡萄糖和其他辅剂的水溶液通过常规方法进行制备。药物组合物如针剂、溶液宜在无菌条件下制造。活性成分的给药量是治疗有效量，例如每天约1微克/千克体重-约5毫克/千克体重。此外，本发明的多肽还可与其他治疗剂一起使用。
使用药物组合物时，是将安全有效量的抗体用于哺乳动物，其中该安全有效量通常至少约10微克/千克体重，而且在大多数情况下不超过约50毫克/千克体重，较佳地该剂量是约10微克/千克体重-约20毫克/千克体重。当然，具体剂量还应考虑给药途径、病人健康状况等因素，这些都是熟练医师技能范围之内的。
本发明的主要优点在于：
(1)本发明采用的转基因小鼠，与野生型小鼠比能够更容易获得全人源抗体，从而降低抗体的免疫原性；与全人抗体的转基因小鼠比，获得的抗体数量多、亲和力强，序列多样性好并且活性高。
(2)本发明采用杂交瘤技术获得抗体，与噬菌体库获得的抗体相比，抗体亲和力高，序列表达良好。
(3)本发明采用重组表达的293F-HuCTLA-4细胞，与蛋白或者多肽类免疫原相比，表达的目的蛋白构象更趋天然；与其他重组表达细胞相比，其表达量更高。
(4)本发明获得了序列不同的抗体，能够与CTLA-4抗体特异性结合，其结合活性低于纳摩尔(nM)。
(5)本发明所获得抗体具有很好的刺激T细胞激活活性，其能够阻断CTLA-4与其两个配体(B7.1和B7.2)的结合，通过逆转CTLA-4对T细胞激活活性的抑制，从而激活T细胞分泌IL-2。
(6)本发明所获得的抗体，在小鼠(例如人CTLA-4转基因小鼠)体内表 现出显著抑制肿瘤生长和提高小鼠存活率的活性。
(7)本发明所获得抗体具有一系列的优异特征：可变区序列与现有抗体同源性低(同源性<92％)，各项活性均好于来自对比文件的抗体。
下面结合具体实施例，进一步详陈本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明详细条件的实验方法，通常按照常规条件如美国Sambrook.J等著《分子克隆实验室指南》(黄培堂等译，北京：科学出版社，2002年)中所述的条件，或按照制造厂商所建议的条件(例如商品说明书)。除非另外说明，否则百分比和份数按重量计算。以下实施例中所用的实验材料和试剂如无特别说明均可从市售渠道获得。
实施例中所述的室温为本领域常规的室温，一般为10-30℃。
若无特别说明，实施例中所述的PBS为PBS磷酸缓冲液，pH7.2。
材料
H2L2转基因鼠获自(购自)和铂医药(上海)有限责任公司，该鼠产生具有完全人类可变区的由两条重链和两条轻链(H2L2)组成的传统四聚体抗体的小鼠。产生的抗体亲和力成熟，可变区完全人源化，具有优异的溶解性。基因工程小鼠技术是产生完全人类抗体的主要工具之一 [4]。
通用方法
本发明先制备人源CTLA-4作为免疫原，采用人源抗体转基因小鼠技术进行全人源抗体的制备[Lonberg,et al.1994，Nature 368(6474)：856-859，Lonberg,N.and Huszar,D.1995，Intern.Rev.Immunol.13:65-93,and Harding,F.and Lonberg,N.,1995，Ann.N.Y.Acad.Sci.764:536-546]，获得CTLA-4抗体的先导抗体。再通过对先导抗体的初步生产、纯化和检定，获得具备抗体亲和力高(亲和力KD＜1*10 -9M)、有效封闭CTLA-4与受体B7.1和B7.2的结合、在人外周血单核细胞或T淋巴细胞反应中显著增加IL-2的表达水平、与人CD28等同类蛋白抗原缺乏交叉反应等优异生物性特性的CTLA-4抗体。然后通过分子生物学方法测序获知CTLA-4抗体的重链可变区和轻链可变区的氨基酸序列。
实施例1 CTLA-4抗体的制备
(一)免疫原A的制备
将人源CTLA-4蛋白胞外区氨基酸序列Lys36-Asp161克隆到带有人IgG Fc片段(hFc)的pCpC载体(购自Invitrogen，V044-50)并按已建立的标准分子生物学方法制备质粒，具体方法参见Sambrook,J.,Fritsch,E.F.,and Maniatis T.(1989).Molecular Cloning:A Laboratory Manual,Second Edition(Plainview,New York:Cold Spring Harbor Laboratory Press)。对HEK293细胞(购自Invitrogen)进行瞬时转染(PEI，Polysciences)并使用FreeStyle  TM 293(Invitrogen)在37℃下进行扩大培养。4天后收集细胞培养液，离心去除细胞成分，得含CTLA-4蛋白胞外区的培养上清液。将培养上清液上样到蛋白A亲和层析柱(Mabselect Sure，购自GE Healthcare)，同时用紫外(UV)检测仪监测紫外吸收值(A280nm)的变化。上样后用PBS磷酸盐缓冲液(pH7.2)清洗蛋白A亲和层析柱直到紫外吸收值回到基线，然后用0.1M甘氨酸盐酸(pH2.5)洗脱，收集从蛋白A亲和层析柱上洗脱下来的带hFc标签的CTLA-4蛋白(CTLA-4-hFc)，用PBS磷酸盐缓冲液(pH7.2)在4℃冰箱透析过夜。透析后的蛋白经0.22微米无菌过滤后分装于-80℃保存，即获得纯化的免疫原A。免疫原A在使用前需要进行一系列质控检测，如检测其蛋白浓度、纯度、分子量和生物活性等，结果如图1和表1所示。表1说明CTLA-4与B7.1或B7.2在蛋白水平的结合随B7.1或B7.2的浓度变化而变化，其中对照蛋白为非CTLA-4融合蛋白，表中的数据为OD 450nm值。
其中，免疫原A生物活性采用ELISA检测，具体为：
将带hFc标签的CTLA-4蛋白(CTLA-4-hFc，即免疫原A)，用PBS稀释至1μg/mL，以100μl/孔加入ELISA微孔板，4℃孵育过夜。用ELISA封闭液(含1％BSA，pH7.4的PBS磷酸缓冲液，所述百分比为质量百分比)37℃封闭两小时后，再加入梯度稀释的生物素标记的B7.1或B7.2–hFc，37℃温育1小时。生物素标记的B7.1-hFc或B7.2-hFc的制备方法如下：将B7.1-hFc或B7.2-hFc与生物素化试剂反应即可。所述B7.1-hFc或B7.2-hFc的制备方法与免疫原A的制备方法相同，其中B7.1胞外区蛋白(Val35-Asn242)的氨基酸序列信息参见Uniprot数据库，编号P33681；B7.2胞外区蛋白(Ala24-Pro247)的氨基酸序列信息参见Uniprot 数据库，编号P42081；所述生物素化试剂购自Sigma，商品号B3295；与生物素化试剂反应的操作步骤参见该生物素化试剂的说明书。加入链霉亲和素标记的辣根过氧化物酶(购自Sigma商品号S2438)，室温孵育30分钟，用洗液将未结合的链霉亲和素标记的辣根过氧化物酶洗去，加入100微升/孔TMB显色液。室温孵育15分钟后，加入50微升1N盐酸终止显色反应，用ELISA读板机读取OD 450nm读数。
表1 CTLA-4-hFc蛋白与生物素标记的配体B7.1-hFc或B7.1-hFc的结合活性
Figure PCTCN2019078032-appb-000001
具体活性结果如表1和图1所示，表明表达的配体和受体蛋白有正确的构象，适合进行免疫、建立受体-配体结合阻断检测方法及进行抗体活性鉴定。
(二)免疫原B的制备
人源CTLA-4全长氨基酸序列被突变为CTLA-4(Y201V)，并克隆到pIRES载体(购自Clontech)并制备质粒。对HEK293细胞系和CHOK1细胞系(均购自Invitrogen)进行质粒转染(转染使用X-treme GENE HP DNA Transfection Reagent，购自Roche公司，货号Cat#06 366 236 001，并按说明书操作)后，在含0.5μg/ml嘌呤霉素的含10％(w/w)FBS的DMEM培养基中选择性培养2周，用有限稀释法在96孔培养板中进行亚克隆，并置于37℃，5％(v/v)CO 2培养。大约2周后选择部分单克隆孔扩增到6孔板中。对扩增后的克隆用已知的CTLA-4抗体染色，经流式细胞分析法进行筛选。选择长势较好、荧光强度较高、单克隆的细胞系继续扩大培养并液氮冻存，即获得免疫原B。具体选择结果如表2和图2所示，表2中阳性细胞(％)指阳性细胞占总细胞数目的百分比。图2说明，HEK293细胞有较高水平CTLA-4的表达，适合用做免疫原和进行抗体结合活性鉴定。
表2 CTLA-4(Y201V)蛋白转染的HEK293细胞FACS筛选检测结果
Figure PCTCN2019078032-appb-000002
(三)杂交瘤细胞的制备和抗体筛选
Harbour转基因小鼠引入了人免疫球蛋白可变区基因和大鼠免疫球蛋白恒定区基因，而小鼠本身的Ig表达则被沉默(F.G.Frankl in,et al,patent#WO 2010/070263 Al)。该转基因小鼠经抗原免疫后能产生与正常小鼠(如Balb/c)相当的免疫反应和抗体效价。
A、免疫原A免疫采用6～8周龄Harbour人源抗体转基因小鼠(购自北京维通利华公司)，小鼠在SPF条件下饲养。初次免疫时，免疫原A蛋白用弗氏完全佐剂乳化后腹腔注射0.25毫升，即每只小鼠注射100微克免疫原A蛋白。加强免疫时，免疫原A蛋白用弗氏不完全佐剂乳化后腹腔注射0.25毫升，即每只小鼠注射50微克免疫原A蛋白。初次免疫与第一次加强免疫之间间隔2周，以后每次加强免疫之间间隔3周。每次加强免疫1周后采血，用ELISA和FACS检测血清中免疫原A的抗体效价和特异性，结果如图3和表3所示。表3说明，经CTLA-4-hFc免疫的小鼠的免疫后血清对免疫原均有不同程度的结合，呈现抗原抗体反应，其中最高稀释度在一千(1000)左右。其中空白对照为1％(w/w)BSA，其中批次指第三次加强免疫后第七天的小鼠血清，表中的数据为OD 450nm值。
B、免疫原B免疫采用6～8周龄Harbour人源抗体转基因小鼠(购自北京维通利华公司)，小鼠在SPF条件下饲养。将按步骤(二)中的得到的含人源CTLA-4的HEK293-hCTLA-4(Y201V)稳定细胞系在T-75细胞培养瓶中扩大培养至90％汇合度，吸尽培养基。用DMEM基础培养基(购自Invitrogen)洗涤2次，然后用无酶细胞解离液(购自Invitrogen)37℃处理直至细胞从培养皿壁上可脱落，收集细胞。用DMEM基础培养基洗涤2次，进行细胞计数后将细胞用磷酸盐缓冲液(pH7.2)稀释至2×10 7细胞每毫升。每只小鼠每次免疫时腹腔注射0.5毫升细胞悬液。第一次与第二次免疫之间间隔2周，以后每次免疫间隔3周。除第一 次免疫以外，每次免疫1周后采血，用ELISA检测血清中抗体效价和特异性。表3和图3为HEK-CTLA-4细胞免疫血清，用ELISA进行抗体效价检测的结果。
表3 ELISA检测HEK293-CTLA-4细胞免疫后Harbour转基因小鼠血清抗体效价
Figure PCTCN2019078032-appb-000003
通常以免疫原A或B进行免疫，大部分小鼠经3次免疫后ELISA效价可达到1:1000以上，说明H2L2小鼠可对免疫原产生较好的体液免疫反应，其脾细胞可以用来进行杂交瘤细胞制备。
A或B步骤完成前，将所选择的每只小鼠最后一次免疫腹腔注射100微克纯化的CTLA-4-hFc(针对免疫原A和免疫原B)进行免疫反应的小鼠)或含人源CTLA-4的HEK293-hCTLA-4稳定细胞(针对免疫原B进行免疫反应的小鼠)，5天后处死小鼠，收集脾细胞。加入NH 4OH至终浓度1％(w/w)，裂解脾细胞中参杂的红细胞，获得脾细胞悬液。用DMEM基础培养基1000转每分钟离心清洗细胞3次，然后按活细胞数目5:1比率与小鼠骨髓瘤细胞SP2/0混合(购自ATCC)，采用高效电融合或PEG方法(参见METHODS IN ENZYMOLOGY,VOL.220)进行细胞融合。融合后的细胞稀释到含20％胎牛血清、1×HAT的DMEM培养基中，所述百分比为质量百分比。然后按1×10 5/20微升每孔加入到96孔细胞培养板中，放入5％CO 2、37℃培养箱中，所述百分比为体积百分比。14天后用ELISA和Acumen(微孔板细胞检测法)筛选细胞融合板上清，将ELISA中OD 450nm>1.0和Acumen中MFI值>100的阳性克隆扩增到24孔板，在含10％(w/w)胎牛血清的DMEM(Invitrogen)的培养基中，在37℃，5％(v/v)CO 2条件下扩大培养。培养3天后取24孔板中扩大培养的培养液进行离心，收集上清液，对上清液进行抗体亚型分析。用ELISA、FACS确定对CTLA-4蛋白和CTLA-4阳性细胞的结合活性， 配体受体结合实验确定抗体样品对CTLA-4受体的封闭活性。
根据24孔板筛选结果，挑选ELISA实验中OD 450nm>1.0、FACS实验中MFI值>50和配体受体结合实验中杂交瘤细胞培养上清对CTLA-4受体的封闭抑制率达到60％的杂交瘤细胞为符合条件的阳性克隆。选择符合条件的杂交瘤细胞用有限稀释法在96孔板进行亚克隆，在含10％(w/w)FBS的DMEM培养基中(购自Invitrogen)37℃，5％(v/v)CO 2条件下培养。亚克隆后10天用ELISA和Acumen进行初步筛选，挑选阳性单克隆扩增到24孔板继续培养。3天后用FACS确定抗原结合阳性并用CTLA-4受体配体结合实验评估生物活性(评估标准为ELISA实验中OD 450nm>1.0、FACS实验中MFI值>50和配体受体结合实验中杂交瘤细胞培养上清对B7.1配体的封闭抑制率达到60％)。
根据24孔板样品检测结果，阳性克隆在含10％(w/w)FBS的DMEM(购自Invitrogen)培养基中，在37℃，5％(v/v)CO 2条件下进行扩大培养，细胞悬浮于冻存液[含有20％(w/w)FBS和10％(w/w)DMSO的DMEM]中，按常规方法液氮冻存即得本发明杂交瘤细胞，并可用于后续的抗体生产、纯化和氨基酸序列测定。
实施例2 嵌合抗体的鉴定
(一)流式细胞实验(FACS)检测抗体与CTLA-4表达细胞的结合
将实施例1步骤(二)中所述含有编码人源CTLA-4全长核苷酸序列的pIRES质粒转染293F细胞株得含人CTLA-4的293F稳转细胞株(此处称为HEK293-hCTLA-4稳定细胞株)，将带有猴源CTLA-4全长基因的pIRES质粒，其中猴源CTLA-4核苷酸酸序列的数据库登录号为XM_005574014.1，转染HEK293细胞株的含猴CTLA-4的HEK293稳转细胞株(此处称为HEK293-cCTLA-4稳定细胞株)。将HEK293-hCTLA-4稳定细胞株和HEK293-cCTLA-4稳定细胞株在T-75细胞培养瓶中扩大培养至90％汇合度，吸尽培养基，用HBSS缓冲液(Hanks Balanced Salt Solution，购自Invitrogen)洗涤2次，然后用无酶细胞解离液(Versene solution，购自Life technology公司)处理和收集细胞。用HBSS缓冲液洗涤细胞2次，进行细胞计数后将细胞用HBSS缓冲液稀释至2×10 6细胞每毫升，加入1％山羊血清封闭液，所述百分比为质量百分比。冰上孵育30分钟，然后用HBSS 缓冲液离心洗涤2次。将收集的细胞用FACS缓冲液(含有1％BSA的HBSS，所述百分比为质量百分比)悬浮至2×10 6细胞/mL，按每孔100微升加入到96孔FACS反应板中，加入实施例2所得的纯化的CTLA-4抗体待测样品每孔100微升，冰上孵育2小时。用FACS缓冲液离心洗涤2次，加入每孔100微升荧光(Alexa 488)标记的二抗(购自Invitrogen)，冰上孵育1小时。用FACS缓冲液离心洗涤3次，加入每孔100微升固定液[4％(v/v)多聚甲醛]悬浮细胞，10分钟后用FACS缓冲液离心洗涤2次。用100微升FACS缓冲液悬浮细胞，用FACS(FACS Cal ibur，购自BD公司)检测和分析结果。
表4 FACS检测CTLA-4抗体与CHOK1-hCTLA-4的结合反应
Figure PCTCN2019078032-appb-000004
表5 FACS检测CTLA-4抗体与293F-cCTLA-4的结合反应
Figure PCTCN2019078032-appb-000005
结果如图4和图5，表4和表5所示，待测抗体可结合细胞表面的人或猴CTLA-4蛋白，各抗体活性相当，表明抗体与CTLA-4结合能力较强。其中IgG对照为人IgG，表中的数据为MFI所测细胞群的平均荧光强度值。
(二)检测CTLA-4抗体阻断CTLA-4蛋白与其配体B7.1或B7.2的结合
CTLA-4蛋白的受体配体结合试验检测CTLA-4抗体阻断CTLA-4蛋白与其配 体B7.1或B7.2的结合。
CTLA-4胞外区蛋白(CTLA-4-hFc)用PBS稀释到终浓度1.0μg/mL，然后以100μl每孔加到96孔ELISA板。用塑料膜封好4℃孵育过夜，第二天用洗板液[含0.01％(v/v)Tween20的PBS]洗板2次，加入封闭液[含0.01％(v/v)Tween20和1％(w/w)BSA的PBS]室温封闭2小时。倒掉封闭液，先加入实施例2所得的纯化的CTLA-4抗体待测样品50μl每孔，后加入生物素标记的B7.1胞外区蛋白(B7.1-hFc)或B7.2-hFc，每孔100微升，混匀后37℃孵育。2小时后，用洗板液[含0.01％(v/v)Tween20的PBS]洗板3次。加入HRP(辣根过氧化物酶)标记的亲和素稀释液(购自Sigma)每孔100微升，37℃孵育2小时后，用洗板液[含0.01％(v/v)Tween20的PBS]洗板3次。加入TMB底物100μl每孔，室温孵育30分钟后，加入终止液(1.0N HCl)100μl每孔。用ELISA读板机(SpectraMax 384plus,Molecular Device)读取A450nm数值，结果如图6，图7和表6、表7所示。
表6 CTLA-4抗体对CTLA-4蛋白与其受体B7.1的结合的抑制
Figure PCTCN2019078032-appb-000006
表7 CTLA-4抗体对CTLA-4蛋白与其受体B7.2的结合的抑制
Figure PCTCN2019078032-appb-000007
结果表明，所得抗体可不同程度的抑制CTLA-4蛋白与其配体B7.1或B7.2的结合，所测抗体活性相当。其中IgG对照为人IgG，表中的数据为抑制率(％)。
(三)淋巴细胞刺激实验检测CTLA-4抗体对淋巴细胞活性的影响
淋巴细胞刺激实验检测CTLA-4抗体阻断CTLA-4蛋白与其受体B7.1和B7.2 的结合从而解除其对T淋巴细胞活性的抑制，从而刺激T细胞的增殖。
1.Ficoll分离全血获取外周血单核淋巴细胞PBMC
将新鲜获取的全血用磷酸缓冲液PBS以1:1的体积比例稀释得稀释后的全血，用无菌吸管轻轻将稀释后的全血铺平在Ficoll液面(购自GE Healthcare)，Ficoll与稀释后的全血的体积比为3:4，避免震荡混匀，以400g转速室温20℃梯度离心30分钟，离心后的离心管分为三层，上层为血浆，中间乳白色分层即为单核淋巴细胞。用无菌吸管轻轻吸取中间层细胞，收集至新的离心管，用PBS磷酸缓冲液稀释至三倍体积，100g转速室温离心10分钟，弃上清。将淋巴细胞用PBS磷酸缓冲液重悬至10mL，重复前面步骤取出血小板。最后将淋巴细胞重悬至10mL含有10％胎牛血清的多组份RPMI1640培养基(购自Invitrogen)备用，即为外周血单核淋巴细胞PBMC，所述百分比为质量百分比。
2.SEB依赖的PBMC刺激实验
试验前，配制等体积比稀释的待测实施例2所得的纯化的CTLA-4抗体，得待测样品溶液。
将获得的外周血单核淋巴细胞PBMC以1×10 5个细胞100微升每孔铺至96孔细胞培养板，然后将所述的待测样品溶液加入培养板，室温培养30分钟。最后加入超抗原SEB，每反应孔中含有50微升100ng/ml SEB,保证每个反应孔250μL体积，将反应板于37℃、5％CO 2培养箱培养72小时后收集上清，得细胞上清液，于-20℃冻存，所述百分比为体积百分比。
3.细胞上清中细胞因子白介素IL-2酶联免疫吸附检测
细胞上清中细胞因子白介素IL-2酶联免疫吸附检测使用R&D system相关检测试剂盒Quantikine ELISA human IL-2(S2050)，并按照说明书操作。除检测抗体外的所有检测试剂均由检测试剂盒提供。
测定细胞上清中细胞因子白介素IL-2含量的酶联免疫吸附检测采用双抗夹心ELISA试剂盒(购自R&D Systems，IL-2Cat#S2050)。实验操作严格按照试剂盒说明书要求，所有检测试剂均由试剂盒提供。具体实验简述如下：将IL-2多克隆抗体包被于ELISA微孔板上，将步骤2.获得的细胞上清液作为待测 样品，加入标准品和待测样品室温孵育2小时。每孔加入400微升洗液，重复洗板4次；再加入抗人IL-2的辣根过氧化物酶标抗体，室温孵育2小时，与微孔板上的IL-2形成免疫复合物，清洗微孔；加入底物显色，避光室温30分钟，最终加入终止液，用酶标仪测定A450nm吸光度。检测CTLA-4抗体在步骤2.所述PBMC刺激实验中对IL-2分泌的影响。结果如图8，和表8所示。
表8 CTLA-4抗体在PBMC淋巴细胞刺激试验中对IL-2分泌的影响
Figure PCTCN2019078032-appb-000008
结果表明，在PBMC淋巴细胞刺激试验中待测抗体可使PBMC的IL-2分泌增强，并且激活作用呈浓度梯度依赖性，其中97A8D1活性率优于其他抗体。其中hIgG对照为人IgG，表中的数据为IL-2值(pg/mL)。
实施例3 轻重链可变区氨基酸序列测定
总RNA分离：将实施例1亚克隆培养所得的上清液检验过抗原结合后(即经过实施例3-6的检定和活性测定后)，通过离心搜集5×10 7个杂交瘤细胞，加入1mL Trizol混匀并转移到1.5mL离心管中，室温静置5分钟。加0.2mL氯仿，振荡15秒，静置2分钟后于4℃，12000g离心5分钟，取上清转移到新的1.5mL离心管中。加入0.5mL异丙醇，将管中液体轻轻混匀，室温静置10分钟后于4℃，12000g离心15分钟，弃上清。加入1mL 75％乙醇(所述百分比为体积百分比)，轻轻洗涤沉淀，4℃，12000g离心5分钟后弃上清，将沉淀物晾干，加入DEPC处理过的H 2O溶解(55℃水浴促溶10分钟)，即得总RNA。
逆转录与PCR：取1μg总RNA，配置20μl体系，加入逆转录酶后于42℃反应60分钟，于7℃反应10分钟终止反应。配置50μl PCR体系，包括1μl cDNA、每种引物25pmol、1μl DNA聚合酶以及相配的缓冲体系、250μmol dNTPs。设置PCR程序，预变性95℃3分钟，变性95℃30秒，退火55℃30秒，延伸72℃35秒，35 个循环后再额外于72℃延伸5分钟，得PCR产物。其中逆转录所用的试剂盒为PrimeScript RT Master Mix，购自Takara，货号RR036；PCR所用的试剂盒为Q5超保真酶，购自NEB，货号M0492。
克隆与测序：取5μl PCR产物进行琼脂糖凝胶电泳检测，将检测阳性样品使用柱回收试剂盒纯化，其中回收试剂盒为
Figure PCTCN2019078032-appb-000009
Gel&PCR Clean-up，购自MACHEREY-NAGEL，货号740609。进行连接反应：样品50ng，T载体50ng，连接酶0.5μl，缓冲液1μl，反应体系10μl，于16℃反应半小时得连接产物，其中连接的试剂盒为T4DNA连接酶，购自NEB，货号M0402；取5μl连接产物加入100μl的感受态细胞(Ecos 101competent cells，购自Yeastern，货号FYE607)中，冰浴5分钟。而后于42℃水浴热激1分钟，放回冰上1分钟后加入650μl无抗生素SOC培养基，于37℃摇床上以200RPM的速度复苏30分钟，取出200μl涂布于含抗生素的LB固体培养基上于37℃孵箱过夜培养。次日，使用T载体上引物M13F和M13R配置30μl PCR体系，进行菌落PCR，用移液器枪头蘸取菌落于PCR反应体系中吹吸，并吸出0.5μl点于另一块含100nM氨苄青霉素的LB固体培养皿上以保存菌株。PCR反应结束后，取出5μl进行琼脂糖凝胶电泳检测，将阳性样品进行测序。其中，测序的步骤参见Kabat，Sequences of Proteins of Immunological Interest，National Institutes of Health,Bethesda,Md.(1991)。
测序结果如表9所示：
表9 CTLA-4抗体基因序列编号
Figure PCTCN2019078032-appb-000010
Figure PCTCN2019078032-appb-000011
其中，表9中的数字即为序列表“SEQ ID NO:”编号，如编码97A8D1的重链蛋白可变区的核苷酸序列为序列表SEQ ID NO:41。
其中，编码97A8D1的重链蛋白可变区中CDR1域的核苷酸序列为序列表SEQ ID NO:41中的第88位至第105位；
编码97A8D1的重链蛋白可变区中CDR2域的核苷酸序列为序列表SEQ ID NO:41中的第148位至第198位；
编码97A8D1的重链蛋白可变区中CDR3域的核苷酸序列为序列表SEQ ID NO:41中的第289位至第321位；
编码97A8D1的轻链蛋白可变区中CDR1域的核苷酸序列为序列表SEQ ID NO:42中的第73位至第108位；
编码97A8D1的轻链蛋白可变区中CDR2域的核苷酸序列为序列表SEQ ID NO:42中的第151位至第177位；
编码97A8D1的轻链蛋白可变区中CDR3域的核苷酸序列为序列表SEQ ID NO:42中的第268位至第294位；
编码92C8B6的重链蛋白可变区中CDR1域的核苷酸序列为序列表SEQ ID NO:43中的第88位至第105位；
编码92C8B6的重链蛋白可变区中CDR2域的核苷酸序列为序列表SEQ ID NO:43中的第148位至第198位；
编码92C8B6的重链蛋白可变区中CDR3域的核苷酸序列为序列表SEQ ID NO:43中的第289位至第327位；
编码92C8B6的轻链蛋白可变区中CDR1域的核苷酸序列为序列表SEQ ID NO:44中的第73位至第105位；
编码92C8B6的轻链蛋白可变区中CDR2域的核苷酸序列为序列表SEQ ID NO:44中的第148位至第174位；
编码92C8B6的轻链蛋白可变区中CDR3域的核苷酸序列为序列表SEQ ID NO:44中的第265位至第294位；
编码31C12F3的重链蛋白可变区中CDR1域的核苷酸序列为序列表SEQ ID NO:45中的第88位至第105位；
编码31C12F3的重链蛋白可变区中CDR2域的核苷酸序列为序列表SEQ ID NO:45中的第148位至第198位；
编码31C12F3的重链蛋白可变区中CDR3域的核苷酸序列为序列表SEQ ID NO:45中的第286位至第327位；
编码31C12F3的轻链蛋白可变区中CDR1域的核苷酸序列为序列表SEQ ID NO:46中的第73位至第105位；
编码31C12F3的轻链蛋白可变区中CDR2域的核苷酸序列为序列表SEQ ID NO:46中的第148.位至第174位；
编码31C12F3的轻链蛋白可变区中CDR3域的核苷酸序列为序列表SEQ ID NO:46中的第265位至第291位；
编码40C4C6的重链蛋白可变区中CDR1域的核苷酸序列为序列表SEQ ID NO:47中的第88位至第105位；
编码40C4C6的重链蛋白可变区中CDR2域的核苷酸序列为序列表SEQ ID NO:47中的第148位至第198位；
编码40C4C6的重链蛋白可变区中CDR3域的核苷酸序列为序列表SEQ ID NO:47中的第289位至第327位；
编码40C4C6的轻链蛋白可变区中CDR1域的核苷酸序列为序列表SEQ ID NO:48中的第73位至第105位；
编码40C4C6的轻链蛋白可变区中CDR2域的核苷酸序列为序列表SEQ ID NO:48中的第148位至第174位；
编码40C4C6的轻链蛋白可变区中CDR3域的核苷酸序列为序列表SEQ ID NO:48中的第265位至第291位。
编码97B8E1的重链蛋白可变区中CDR1域的核苷酸序列为序列表SEQ ID NO:49中的第88位至第105位；
编码97B8E1的重链蛋白可变区中CDR2域的核苷酸序列为序列表SEQ ID NO:49中的第148位至第198位；
编码97B8E1的重链蛋白可变区中CDR3域的核苷酸序列为序列表SEQ ID NO:49中的第289位至第330位；
编码97B8E1的轻链蛋白可变区中CDR1域的核苷酸序列为序列表SEQ ID NO:50中的第73位至第105位；
编码97B8E1的轻链蛋白可变区中CDR2域的核苷酸序列为序列表SEQ ID NO:50中的第148位至第174位；
编码97B8E1的轻链蛋白可变区中CDR3域的核苷酸序列为序列表SEQ ID NO:50中的第265位至第291位。
实施例4 全人抗体IgG转化和制备
(一)质粒构建与准备：实施例2已从杂交瘤细胞的培养上清液中获得了纯化的CTLA-4抗体，并根据实施例7的测序结果明确了CTLA-4抗体的重链可变区和轻链可变区序列。将CTLA-4抗体的重链可变区序列重组到包含信号肽和人源重链抗体IgG1恒定区的表达载体(其中表达载体购买自Invitrogen)中，将CTLA-4抗体的轻链可变区序列重组到包含信号肽和人源抗体轻链kappa恒定区的表达载体当中，得重组质粒并经测序验证(测序方法与实施例7中测序方法相同)。使用碱裂解法试剂盒(购自MACHEREY-NAGEL)中量抽提高纯度的重组质粒，质量为500μg以上，经0.22μm滤膜(购自Millopore)过滤，供转染使用。
(二)细胞转染：在培养基Freestyle 293 expression medium(购自Invitrogen)培养293E细胞(购自Invitrogen)。摇床设置为37℃、130RPM，8％CO 2(v/v)浓度。
Freestyle 293 expression medium在转染时添加10％(v/v)F68(购自Invitrogen)至F68终浓度为0.1％(v/v)，得含0.1％(v/v)F68的Freestyle 293表达培养基，即培养基A。
取5mL培养基A和200μg/mL PEI(购自Sigma)混匀，得培养基B。取5mL培养基A和100μg/mL步骤(1)所得的重组质粒混匀，得培养基C。5分钟后将培养基B和培养基C合并混匀，静置15分钟，得混合液D。将10mL混合液D缓缓加入100mL含293E细胞的培养基Freestyle 293 expression medium中至293E的细胞密度为1.5×10 6/mL，边加边振荡，避免PEI过度集中，放入摇床培养。第二天加入蛋白胨至终浓度为0.5％(w/v)。第5～7天，测培养液抗体效价。第6～7天，离心(3500RPM，30分钟)收集上清，经0.22μm滤膜过滤，得滤好的细胞上清液，以供纯化。
(三)抗体纯化：对于连续生产的无内毒素的层析柱和Protein A填料(购自GE)，使用0.1M NaOH处理30分钟或者5个柱体积的0.5M NaOH冲洗。对于长 期未使用的柱料和层析柱至少使用1M NaOH浸泡1h，用无内毒的水冲洗至中性，用10倍柱体积的1％(v/v)Triton×100对柱料清洗。使用5个柱体积的PBS(PBS磷酸缓冲液，pH7.2)进行平衡，将步骤(2)所得过滤好的细胞上清液上柱，必要时收集流穿液。上柱完成后，使用5倍柱体积的PBS清洗。用5倍柱体积的0.1M pH3.0的Glycine-HCl进行洗脱，收集洗脱液，并用0.5倍柱体积洗脱液的pH8.5的1MTris-HCl(1.5M NaCl)中和，收获全人CTLA-4抗体。上述所用溶液均需要新配置。收获全人CTLA-4抗体后，在1×PBS中透析4小时，避免内毒素污染。透析结束后，使用分光光度或试剂盒测定浓度，使用HPLC-SEC测定抗体纯度，使用内毒素检测试剂盒(购自Lonza)检测抗体内毒素含量。
实施例5 流式细胞实验(FACS)检测全人抗体与CTLA-4表达细胞的结合
对所获全人CTLA-4抗体与细胞表达CTLA-4的结合活性进行鉴定，检测结果分别如图9、图10和表10、表11所示。
表10.流式细胞实验(FACS)检测抗体与人CTLA-4表达细胞的结合
Figure PCTCN2019078032-appb-000012
表11.流式细胞实验(FACS)检测抗体与猴CTLA-4表达细胞的结合
Figure PCTCN2019078032-appb-000013
结果表明，经过全人IgG转化并制备所得全人CTLA-4抗体能够以较高的活 性与人和猴的CTLA-4结合，抗体活性接近。
实施例6 检测CTLA-4抗体阻断CTLA-4蛋白与其配体B7.1或B7.2的结合
对所获全人CTLA-4抗体进行阻断活性鉴定，检测结果分别如图11、图12和表12、表13所示。
表12 全人CTLA-4抗体对CTLA-4蛋白与其受体B7.1的结合的抑制
Figure PCTCN2019078032-appb-000014
表13 全人CTLA-4抗体对CTLA-4蛋白与其受体B7.2的结合的抑制
Figure PCTCN2019078032-appb-000015
图11、图12和表12、表13表明，经过全人IgG转化并制备所得全人CTLA-4抗体能够阻断CTLA-4与B7.1和B7.2的结合，所测抗体阻断活性相当。
实施例7 淋巴细胞刺激实验检测CTLA-4抗体对淋巴细胞活性的影响
实验方法见实施例2(三)，检测结果见表14和图13。
表14 CTLA-4全人源抗体在SEB依赖的PBMC激活试验中对IL-2分泌的影响
Figure PCTCN2019078032-appb-000016
从表14、图13可见，全人源化抗体在SEB依赖的PBMC激活试验中，能够显著刺激IL-2分泌，并且该活性具有浓度梯度依赖效应，表明CTLA-4抗体能够逆转CTLA-4对T细胞激活的抑制作用，所测抗体活性水平相当。其中IgG对照为人IgG1(hIgG1)，表14中的数据为IL-2浓度。
实施例8 PHA诱导的CD3+T淋巴细胞实验检测CTLA-4抗体阻断B7.1或B7.2与CTLA-4结合后对T淋巴细胞的激活
检测CTLA-4抗体阻断B7.1或B7.2与CTLA-4结合后对T淋巴细胞的激活。
T细胞组成型表达CD28，PHA可非特异性激活T细胞，从而诱导CTLA-4的膜表达；Raji细胞组成型表达CTLA-4/CD28的配体B7.1和B7.2；当激活的T细胞与Raji细胞孵育时，CTLA-4和CD28竞争性结合配体，由于CTLA-4对配体的亲和力高于CD28，所以与Raji细胞孵育时，表现为T细胞激活的部分下调，当CTLA-4的配体结合能力被阻断时，T细胞激活的下调会被逆转，即表现为IL-2分泌的提高。
(一)PHA诱导人CD3+细胞的活化
从全血中用使用试剂FICOLL PAQUE PLUS，并按照其说明书操作分离PBMC细胞。具体步骤如4.4.2中(5)所述。
使用人CD3+细胞提取试剂盒(MagCellectTM Human CD3+T Cell Isolation Kit)对所得的PBMC细胞按说明书进行操作，得分离纯化的CD3+细胞，将人CD3+T 细胞接种到6孔板中，加入10μg/mL PHA，处理72小时，以获得CTLA-4过表达的CD3+T细胞囊。
(二)丝裂霉素处理Raji细胞
使用前，将Raji细胞用10μg/mL丝裂霉素，37℃处理1.5小时，用PBS洗3次以去除残留的丝裂霉素。同时配制等体积比稀释的待测的纯化CTLA-4抗体，得待测样品溶液。
(三)Raji介导的T细胞囊的激活
将步骤①所得的CD3+T细胞囊接种至96孔细胞培养板，接种密度分别为1×10 5cells/孔接种体积为每孔50μL，加入待测样品溶液，每孔50μL，共同孵育30分钟，然后加入步骤②的Raji细胞，以100μL 3×10 4cells/孔铺至96细胞培养板，于37℃，5％CO2培养箱孵育4天。收集细胞上清，用于细胞因子检测，所述百分比为体积百分比。
(四)细胞上清中细胞因子白介素IL-2酶联免疫吸附检测
将步骤③所得的细胞上清中细胞因子白介素IL-2进行酶联免疫吸附检测。使用R&D system相关检测试剂盒Quantikine ELISA human IL-2(S2050)，并按照说明书操作。除检测抗体外的所有检测试剂均由检测试剂盒提供。
CTLA-4全人源抗体在PHA诱导的T淋巴囊细胞激活试验中对IL-2分泌的影响见图14。结果表明，所测抗体均能刺激T细胞分泌IL2，并且该效应与抗体浓度呈剂量相关性，即随抗体浓度提高IL2水平也升高，其中92C8B6和97A8D1活性最好。
实施例9 抗CTLA-4抗体亲和力的分析测定
将抗人Fc IgG固定在流通池1和2上：将HBS-EP+(10mmol/L HEPES，150mmol/L NaCl，3mM EDTA，0.05％P20，pH 7.4)用作运行缓冲液，使用固定向导模板进行抗人Fc IgG的固定。串联S CM5传感器芯片的流通池1和2用新鲜混合的50mmol/L NHS和200mmol/L EDC活化。用10mmol/L NaAC(pH4.5)稀释抗人Fc IgG至20μg/mL，注射到活化的流通池1和2中。剩余的活性偶联位点用1mol/L乙醇胺封闭。
将重组His标记的hCTLA-4 ECD蛋白稀释至50nmol/L，随后用HBS-EP+缓冲 液以2倍比例连续稀释4次。His标记的hCTLA-4 ECD蛋白浓度为0nmol/L，3.125nmol/L，6.25nmol/L，12.5nmol/L，25nmol/L和50nmol/L。用HBS-EP+作为运行缓冲液进行KD测量。每个抗体以10μL/min的流速注射到CM5传感器流动池2上以达到响应230RU。然后将制备的His标记的hCTLA-4ECD蛋白以30μL/min的流速注入流通池1和2，持续180秒。缓冲液流动维持400秒以进行解离测量(30μL/min)。为了从表面除去测试的抗体，将10mmol/L甘氨酸-HCl pH1.5注射20秒(30μL/min)。流动池1用作参考流动池。对于每个浓度的连续稀释的His-标记的CTLA-4ECD蛋白重复上述步骤。使用Biacore T200评估软件1.0评估每种抗体的KD值，并且使用1:1结合模型拟合数据。结果见表15。
表15 抗CTLA-4抗体亲和力的分析测定
克隆ID K D(nM) k a(1/Ms) k d(1/s)
97A8D1 2.15E-09 4.05E+05 8.70E-04
92C8B6 2.52E-09 7.44E+05 0.001873
31C12F3 2.62E-09 4.50E+05 0.00118
40C4C6 2.36E-09 7.20E+05 0.0017
97B8E1 3.50E-09 4.31E+05 0.001506
结果表明，所测抗体的KD值均在纳摩水平，且与工具抗体相当，表明这些抗体对人CTLA-4 ECD均有较好的亲和力，其中，97A8D1抗体对人CTLA-4 ECD的亲和力最好，适合作为候选抗体进行体内活性的确认。
实施例10 抗CTLA-4抗体小鼠体内抗肿瘤活性评价
采用MC38同系小鼠模型，使用人CTLA-4基因敲入的C57BL/6小鼠评价抗体在小鼠体内的抗肿瘤活性。实验设计为，选择50只人CTLA-4基因敲入的C57BL/6小鼠，分为6组，每组10只，使用Ipilimumab和同型抗体hIgG1作为对照，样品为92C8B6、97A8D1和97B8E1。给药途径为腹腔注射，给药剂量为10mg/kg，在0、3、6、10天腹腔注射，第24天处死小鼠，测量肿瘤体积，小鼠体重，肿瘤重量及小鼠存活率。实验结果见图15。
图15结果表明，本发明的抗体(包括92C8B6、97A8D1和97B8E1)能够显著 抑制肿瘤生长，抑制效果显著优于对照抗体Ipilimumab(例如97A8D1的中位生存期为22.5天，优于Ipilimumab的中位生存期19天)。
实施例11 去岩藻糖抗CTLA-4抗体的制备和表征
在该实施例中，在缺乏岩藻糖基转移酶的细胞系中表达全人抗CTLA-4抗体97A8D1，使得该细胞系产生去岩藻糖基化的蛋白质。通过液质联用技术分析去岩藻糖基化抗体97A8D1和岩藻糖基化抗体97A8D1经过Ide Z和EndoS酶解后的分子量，确定抗体Fc端是否带有岩藻糖。分析结果见图16。
图16结果表明，经过酶处理后,去岩藻糖基化抗体97A8D1去糖后Fc分子量为23755Da(图16A),不去糖的Fc分子量为23957Da(图16B),两者分子量相差202Da(GlcNAc)。经过酶处理后，岩藻糖基化抗体97A8D1去糖后Fc分子量为23755Da(图16C)，不去糖的Fc分子量为24104Da(图16D)，两者分子量相差349Da(Fuc+GlcNAc)，又因为去岩藻糖基化抗体的Fc分子量(23957Da)比岩藻糖基化抗体的Fc分子量(24104Da)少147Da，所以在分子量水平上推测去岩藻糖基化抗体不含岩藻糖，岩藻糖基化抗体含有岩藻糖。
实施例12 淋巴细胞刺激实验检测去岩藻糖基化抗CTLA-4抗体对淋巴细胞活性的影响
实验方法见实施例2(三)，检测结果见表18和图17。
表18 去岩藻糖基化和岩藻糖基化抗CTLA-4抗体在SEB依赖的PBMC激活试验中对IL-2分泌的影响
Figure PCTCN2019078032-appb-000017
从表18、图17表去岩藻糖基化和岩藻糖基化抗CTLA-4抗体均能够逆转CTLA-4对T细胞激活的抑制作用，但去岩藻糖基化抗CTLA-4抗体活性要优于岩藻糖基化抗CTLA-4抗体。
讨论
(1)可以通过对野生型小鼠进行免疫获得抗体，但是需要对鼠源抗体进行人源化改造而获得人源化抗体，缺点在于改造后的抗体免疫原性可能更强、抗体结构可能会改变从而导致活性丢失或可生产性变差。
(2)可以通过对全人源转基因小鼠进行免疫获得全人抗体，但是所获得抗体的数量或亲和力会较差。
(3)可通过构建免疫小鼠抗体库用噬菌体展示技术对抗体进行表达和活性筛选，但是涉及到抗体重轻链的随机重新组合，会导致形成的抗体可生产性较差。
(4)可通过构建人源抗体库用噬菌体展示技术对抗体进行表达和活性筛选，但是因为未经免疫，所得抗体亲和力会较差。
(5)免疫原可以为多肽，蛋白，其他种类细胞和基因等，但是会存在构象不正确、表达量低、免疫原性差等问题。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。
本发明涉及的序列信息如下：
CTLA-4抗体的氨基酸序列编号对应的氨基酸序列，其中CDR1、CDR2、CDR3、CDR1'、CDR2'、CDR3'分别用下划线标示出来：
SEQ ID No.1>97A8D1_VH(mAb49)
Figure PCTCN2019078032-appb-000018
SEQ ID No.5>97A8D1_VL(mAb49)
Figure PCTCN2019078032-appb-000019
SEQ ID No.9>92C8B6_VH(mAb42)
Figure PCTCN2019078032-appb-000020
SEQ ID No.13>92C8B6_VL(mAb42)
Figure PCTCN2019078032-appb-000021
SEQ ID No.17>31C12F3_VH(mAb010)
Figure PCTCN2019078032-appb-000022
SEQ ID No.21>31C12F3_VL(mAb010)
Figure PCTCN2019078032-appb-000023
SEQ ID No.25>40C4C6_VH(mAb09)
Figure PCTCN2019078032-appb-000024
SEQ ID No.29>40C4C6_VL(mAb09)
Figure PCTCN2019078032-appb-000025
SEQ ID No.33>97B8E1_VH(mAb50)
Figure PCTCN2019078032-appb-000026
SEQ ID No.37>97B8E1_VL(mAb50)
Figure PCTCN2019078032-appb-000027
CTLA-4抗体的编码氨基酸序列对应的核苷酸序列，其中CDR1、CDR2、CDR3、CDR1'、CDR2'、CDR3'的编码核苷酸序列分别用下划线标示出来：
SEQ ID No.41_97A8D1_VH(mAb49)
Figure PCTCN2019078032-appb-000028
Figure PCTCN2019078032-appb-000029
SEQ ID No. 42_97A8D1_VL (mAb49) 
Figure PCTCN2019078032-appb-000030
SEQ ID No. 43_92C8B6_VH (mAb42) 
Figure PCTCN2019078032-appb-000031
SEQ ID No. 44_92C8B6_VL (mAb42) 
Figure PCTCN2019078032-appb-000032
SEQ ID No. 45_31C12F3_VH (mAb010) 
Figure PCTCN2019078032-appb-000033
SEQ ID No. 46_31C12F3_VL (mAb010) 
Figure PCTCN2019078032-appb-000034
SEQ ID No. 47_40C4C6_VH (mAb09) 
Figure PCTCN2019078032-appb-000035
SEQ ID No. 48_40C4C6_VL (mAb09) 
Figure PCTCN2019078032-appb-000036
SEQ ID No. 49_97B8E1_VH (mAb50) 
Figure PCTCN2019078032-appb-000037
SEQ ID No.50_97B8E1_VL(mAb50)
Figure PCTCN2019078032-appb-000038
表16 CTLA-4抗体的CDR区序列
Figure PCTCN2019078032-appb-000039
表17 CTLA-4抗体的CDR区序列编号如下：
Figure PCTCN2019078032-appb-000040
参考文献：
[4]任军,黄红艳,靶向免疫检查点的肿瘤免疫治疗现状与趋势[J].中国肿瘤临床,2014,41(7):415-419

Claims (17)

  1. 一种抗体的重链可变区，其特征在于，所述的重链可变区包括以下三个互补决定区CDR：
    SEQ ID NO:8n+2所示的CDR1，
    SEQ ID NO:8n+3所示的CDR2，和
    SEQ ID NO:8n+4所示的CDR3；
    其中，各n独立地为0、1、2、3或4；
    其中，上述氨基酸序列中任意一种氨基酸序列还包括任选地经过添加、缺失、修饰和/或取代至少一个氨基酸的，并能够保留CTLA-4结合亲和力的衍生序列。
  2. 一种抗体的重链，其特征在于，所述的重链具有如权利要求1所述的重链可变区。
  3. 一种抗体的轻链可变区，其特征在于，所述的轻链可变区包括以下三个互补决定区CDR：
    SEQ ID NO:8n+6所示的CDR1'，
    SEQ ID NO:8n+7所示的CDR2'，和
    SEQ ID NO:8n+8所示的CDR3'；
    其中，各n独立地为0、1、2、3或4；
    其中，上述氨基酸序列中任意一种氨基酸序列还包括任选地经过添加、缺失、修饰和/或取代至少一个氨基酸的，并能够保留CTLA-4结合亲和力的衍生序列。
  4. 一种抗体的轻链，其特征在于，所述的轻链具有如权利要求3所述的轻链可变区。
  5. 一种抗体，其特征在于，所述抗体具有：
    (1)如权利要求1所述的重链可变区；和/或
    (2)如权利要求3所述的轻链可变区；
    或者，所述抗体具有：如权利要求2所述的重链；和/或如权利要求4所述的轻链，
    其中，上述氨基酸序列中任意一种氨基酸序列还包括任选地经过添加、缺失、修饰和/或取代至少一个氨基酸的，并能够保留CTLA-4结合亲和力的衍生序列。
  6. 如权利要求5所述的抗体，其特征在于，所述的抗体具有如权利要求1所述的重链可变区和如权利要求3所述的轻链可变区；
    其中，所述的重链可变区包括以下三个互补决定区CDR：
    SEQ ID NO:2所示的CDR1，
    SEQ ID NO:3所示的CDR2，和
    SEQ ID NO:4所示的CDR3；
    其中，所述的轻链可变区包括以下三个互补决定区CDR：
    SEQ ID NO:6所示的CDR1'，
    SEQ ID NO:7所示的CDR2'，和
    SEQ ID NO:8所示的CDR3'；
    其中，上述氨基酸序列中任意一种氨基酸序列还包括任选地经过添加、缺失、修饰和/或取代至少一个氨基酸的，并能够保留CTLA-4结合亲和力的衍生序列。
  7. 如权利要求5所述的抗体，其特征在于，所述抗体的重链可变区含有SEQ ID NO:8n+1所示的氨基酸序列；和/或所述抗体的轻链可变区含有SEQ ID NO:8n+5所示的氨基酸序列，其中，各n独立地为0、1、2、3或4。
  8. 如权利要求6所述的抗体，其特征在于，所述抗体的重链可变区含有SEQ ID NO:1所示的氨基酸序列，并且所述抗体的轻链可变区含有SEQ ID NO:5所示的氨基酸序列。
  9. 一种重组蛋白，其特征在于，所述的重组蛋白包括：
    (i)如权利要求1所述的重链可变区、如权利要求2所述的重链、如权利要求3所述的轻链可变区、如权利要求4所述的轻链、或如权利要求5-8中任一项所述的抗体；以及
    (ii)任选的协助表达和/或纯化的标签序列。
  10. 一种多核苷酸，其特征在于，所述多核苷酸编码选自下组的多肽：
    (1)如权利要求1所述的重链可变区、如权利要求2所述的重链、如权利要求3所述的轻链可变区、如权利要求4所述的轻链、或如权利要求5-8中任一项所述的抗体；以及
    (2)如权利要求9所述的重组蛋白。
  11. 如权利要求10所述的多核苷酸，其特征在于，编码所述重链可变区的多核苷酸如SEQ ID NO:41、43、45、47或49所示；和/或，
    编码所述轻链可变区的多核苷酸如42、44、46、48或50所示。
  12. 如权利要求11所述的多核苷酸，其特征在于，编码所述重链可变区序列的多核苷酸如SEQ ID NO:41所示；并且编码所述轻链可变区序列的多核苷酸如42所示。
  13. 一种载体，其特征在于，所述载体含有本发明权利要求10-12中任一项所述的多核苷酸。
  14. 一种遗传工程化的宿主细胞，其特征在于，所述宿主细胞含有权利要求13所述的载体或基因组中整合有权利要求10-12中任一项所述的多核苷酸。
  15. 一种药物组合物，其特征在于，所述药物组合物含有：
    (i)活性成分，所述活性成分选自下组：如权利要求1所述的重链可变区、如权利要求2所述的重链、如权利要求3所述的轻链可变区、如权利要求4所述的轻链、或如权利要求5-8中任一项所述的抗体、如权利要求9所述的重组蛋白、或其组合；以及
    (ii)药学上可接受的载体。
  16. 一种活性成分的用途，其特征在于，所述活性成分选自下组：如权利要求1所述的重链可变区、如权利要求2所述的重链、如权利要求3所述的轻链可变区、如权利要求4所述的轻链、或如权利要求5-8中任一项所述的抗体、如权利要求9所述的重组蛋白、或其组合，其中所述活性成分被用于制备预防和/或治疗CTLA-4相关肿瘤和/或病毒感染相关的疾病的药物。
  17. 一种体外检测样品中CTLA-4蛋白的组合物，其特征在于，其包括如权利要求5-8中任一项所述的抗体、如权利要求9所述的重组蛋白、或其组合作为活性成分。
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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4816397A (en) 1983-03-25 1989-03-28 Celltech, Limited Multichain polypeptides or proteins and processes for their production
US4816567A (en) 1983-04-08 1989-03-28 Genentech, Inc. Recombinant immunoglobin preparations
US5585089A (en) 1988-12-28 1996-12-17 Protein Design Labs, Inc. Humanized immunoglobulins
WO2010070263A1 (en) 2008-12-18 2010-06-24 Erasmus University Medical Center Rotterdam Non-human transgenic animals expressing humanised antibodies and use therof
CN107406504A (zh) * 2015-11-19 2017-11-28 蔡则玲 Ctla‑4抗体及其用途

Family Cites Families (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1212422B1 (en) * 1999-08-24 2007-02-21 Medarex, Inc. Human ctla-4 antibodies and their uses
DK2439273T3 (da) * 2005-05-09 2019-06-03 Ono Pharmaceutical Co Humane monoklonale antistoffer til programmeret død-1(pd-1) og fremgangsmåder til behandling af cancer ved anvendelse af anti-pd-1- antistoffer alene eller i kombination med andre immunterapeutika
WO2013013029A1 (en) * 2011-07-19 2013-01-24 The Board Of Trustees Of The University Of Illinois Anti-clta4, anti-glut2 protein for the treatment of type 1 diabetes
CN104292334B (zh) * 2014-04-25 2018-11-27 河南省健康伟业生物医药研究股份有限公司 一种全人源抗ctla-4单克隆抗体、制备方法及应用
MA53355A (fr) * 2015-05-29 2022-03-16 Agenus Inc Anticorps anti-ctla-4 et leurs procédés d'utilisation
US20170233476A1 (en) * 2016-02-12 2017-08-17 Sorrento Therapeutics, Inc. Antibody Therapeutics That Bind CTLA4
NL2017270B1 (en) * 2016-08-02 2018-02-09 Aduro Biotech Holdings Europe B V New anti-hCTLA-4 antibodies

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4816397A (en) 1983-03-25 1989-03-28 Celltech, Limited Multichain polypeptides or proteins and processes for their production
US4816567A (en) 1983-04-08 1989-03-28 Genentech, Inc. Recombinant immunoglobin preparations
US5585089A (en) 1988-12-28 1996-12-17 Protein Design Labs, Inc. Humanized immunoglobulins
WO2010070263A1 (en) 2008-12-18 2010-06-24 Erasmus University Medical Center Rotterdam Non-human transgenic animals expressing humanised antibodies and use therof
CN107406504A (zh) * 2015-11-19 2017-11-28 蔡则玲 Ctla‑4抗体及其用途

Non-Patent Citations (13)

* Cited by examiner, † Cited by third party
Title
"Biocomputing: Informatics and Genome Projects", 1993, ACADEMIC PRESS
"Sequence Analysis in Molecular Biology", 1987, ACADEMIC PRESS
CARILLO, H.LIPMAN, D., SIAM J. APPLIED MATH., vol. 48, 1988, pages 1073
HARDING, F.LONBERG, N., ANN. NY ACAD. SCI., vol. 764, 1995, pages 536 - 546
KABAT: "Sequences of Proteins of Immunological Interest", 1991, NATIONAL INSTITUTES OF HEALTH
LONBERG ET AL., NATURE, vol. 368, no. 6474, 1994, pages 856 - 859
LONBERG, N.HUSZAR, D., INTERN. REV. IMMUNOL., vol. 13, 1995, pages 65 - 93
METHODS IN ENZYMOLOGY, vol. 220
MUNSON ET AL., ANAL. BIOCHEM., vol. 107, 1980, pages 220
REN JUNHUANG HONGYAN: "Current status and trend of tumor immunotherapy targeted at immune checkpoints [J", CHINESE CANCER CLINIC, vol. 41, no. 7, 2014, pages 415 - 419
SAMBROOK, J.FRITSCH, E.F.MANIATIS T.: "Molecular Cloning: A Laboratory Manual", 1989, COLD SPRING HARBOR LABORATORY PRESS
SAMBROOK. J ET AL.: "Guide to Molecular Cloning Laboratory", 2002, SCIENCE PRESS
See also references of EP3766901A4 *

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* Cited by examiner, † Cited by third party
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WO2021122866A1 (en) 2019-12-17 2021-06-24 Ose Immunotherapeutics Bifunctional molecules comprising an il-7 variant
WO2022017428A1 (zh) * 2020-07-21 2022-01-27 上海君实生物医药科技股份有限公司 抗ctla-4抗体及其用途
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