WO2019192493A1 - 抗人lag-3单克隆抗体及其应用 - Google Patents

抗人lag-3单克隆抗体及其应用 Download PDF

Info

Publication number
WO2019192493A1
WO2019192493A1 PCT/CN2019/081063 CN2019081063W WO2019192493A1 WO 2019192493 A1 WO2019192493 A1 WO 2019192493A1 CN 2019081063 W CN2019081063 W CN 2019081063W WO 2019192493 A1 WO2019192493 A1 WO 2019192493A1
Authority
WO
WIPO (PCT)
Prior art keywords
seq
antibody
variable region
chain variable
light chain
Prior art date
Application number
PCT/CN2019/081063
Other languages
English (en)
French (fr)
Inventor
宋宁宁
段清
邵晓慧
王鹏
卞晓娇
王倩
卫培培
黄亚珺
吴建
王美玲
王远东
徐丽娜
杨达志
刘礼乐
Original Assignee
上海开拓者生物医药有限公司
钜川生物医药
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 上海开拓者生物医药有限公司, 钜川生物医药 filed Critical 上海开拓者生物医药有限公司
Priority to EP19781983.2A priority Critical patent/EP3778632A4/en
Priority to US17/045,166 priority patent/US11685778B2/en
Priority to JP2020554402A priority patent/JP2021526013A/ja
Publication of WO2019192493A1 publication Critical patent/WO2019192493A1/zh

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • 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
    • C07K2317/24—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
    • C07K2317/50—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
    • C07K2317/76—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

Definitions

  • the invention relates to the field of biomedicine, in particular to a LAG-3 antibody and a preparation method and application thereof.
  • Cancer immunotherapy refers to the treatment of cancer through the immune system. Recently, cancer immunotherapy has attracted much attention. In addition to surgery, chemotherapy and radiotherapy, it has become a new means of cancer treatment.
  • 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. 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 also be used in combination with targeted therapies or conventional radiation and chemotherapy, and such combination therapies are effective in treating many types of cancer and may be a hope for treating or curing a variety of cancers.
  • the lymphocyte activating gene (LAG-3, CD223) is a type I membrane protein of 525 amino acids and is one of the known Immune Checkpoints. Studies have shown that LAG-3-/-C57BL/6 mice show relatively normal phenotype over a period of time, indicating that the regulation of LAG-3 in the immune system is quite subtle and may play a fine-tuning role in the immune response. Animal model experiments in vivo have shown that knockdown of the LAG-3 gene with an anti-LAG-3 antibody or genetically enhances the activity of antigen-specific CD8+ T cells at the tumor site, thereby blocking tumor growth.
  • LAG-3 and human programmed death receptor-1 are co-expressed in tolerant tumor-infiltrating lymphocytes, which together play a tumor-induced immunosuppressive effect. It has been demonstrated that in mice MC38 colon adenocarcinoma and Sa1N fibrosarcoma models, the combination of anti-LAG-3 and anti-PD-1 antibodies can cure most mice, and the therapeutic effect is better than that of the single drug.
  • the present invention provides a LAG-3 antibody having high affinity and high 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, 4 or 5;
  • 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 LAG-3 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, 4 or 5.
  • the heavy chain variable region has the amino acid sequence set forth in SEQ ID NO. 64, SEQ ID NO. 66, SEQ ID NO. 68, SEQ ID NO. 70, SEQ ID NO.
  • the heavy chain variable region has the amino acid sequence set forth in SEQ ID No. 87, SEQ ID No. 91, SEQ ID No. 92, and SEQ ID No. 93.
  • a heavy chain of an antibody having a heavy chain variable region according to the first aspect of the invention.
  • a light chain variable region of an antibody comprising the following three complementarity determining region CDRs:
  • n is independently 0, 1, 2, 3, 4 or 5;
  • 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 LAG-3 binding affinity.
  • the VL-CDR2 of the light chain variable region has the amino acid sequence set forth in SEQ ID NO. 84 (VL-CDR2 of 405B8H3-1 (D ⁇ E)).
  • 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, 4 or 5.
  • the light chain variable region has the amino acid sequence set forth in SEQ ID NO. 74, SEQ ID NO. 76, SEQ ID NO. 78, SEQ ID NO. 80 or SEQ ID NO.
  • the light chain variable region has the amino acid sequence of SEQ ID No. 89 and SEQ ID No. 95.
  • a light chain of an antibody having a light chain variable region according to the third aspect of the invention 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 LAG-3 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 LAG-3.
  • the ratio of the affinity F1 of the derivative antibody to LAG-3 binding to the affinity of the corresponding non-derivatized antibody to LAG-3 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 derivative sequence which is added, deleted, modified and/or substituted for at least one amino acid and which retains the LAG-3 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 murine origin and/or the light chain constant region is of murine origin.
  • the heavy chain constant region is of human origin, and/or the light chain constant region is of human origin.
  • the heavy chain variable region of the antibody further comprises a framework region of human origin, and/or the light chain variable region of the antibody further comprises a framework region of human origin.
  • the heavy chain variable region of the antibody further comprises a murine framework region, and/or the light chain variable region of the antibody further comprises a murine framework region.
  • the antibody is selected from the group consisting of a chimeric antibody, a humanized antibody, a fully human antibody, or a combination thereof.
  • the antibody is a mutated humanized antibody.
  • the antibody is a humanized antibody having a D ⁇ E mutation in the CDR region.
  • the antibody is a humanized antibody comprising a D ⁇ E mutation in VL-CDR2 or VH-CDR3.
  • 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:
  • CDR2' represented by SEQ ID NO: 7 or SEQ ID NO. 84, and
  • 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:
  • 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 LAG-3 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, 4 or 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: 1
  • the light chain variable region of the antibody comprises the amino acid sequence set forth in SEQ ID NO: or
  • the heavy chain variable region of the antibody comprises the amino acid sequence set forth in SEQ ID NO: 9, and 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, and 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 heavy chain variable region of the antibody comprises the amino acid sequence set forth in SEQ ID NO: 41
  • the light chain variable region of the antibody comprises the amino acid sequence set forth in SEQ ID NO:45.
  • the heavy chain variable region of the antibody comprises the amino acid sequence set forth in SEQ ID NO. 64
  • the light chain variable region of the antibody comprises the amino acid sequence set forth in SEQ ID NO.
  • the heavy chain variable region of the antibody comprises the amino acid sequence set forth in SEQ ID NO. 64
  • the light chain variable region of the antibody comprises the amino acid sequence set forth in SEQ ID NO.
  • the heavy chain variable region of the antibody comprises the amino acid sequence set forth in SEQ ID NO. 66
  • the light chain variable region of the antibody comprises the amino acid sequence set forth in SEQ ID NO.
  • the heavy chain variable region of the antibody comprises the amino acid sequence set forth in SEQ ID NO. 66
  • the light chain variable region of the antibody comprises the amino acid sequence set forth in SEQ ID NO.
  • the heavy chain variable region of the antibody comprises the amino acid sequence set forth in SEQ ID NO. 68
  • the light chain variable region of the antibody comprises the amino acid sequence set forth in SEQ ID NO.
  • the heavy chain variable region of the antibody comprises the amino acid sequence set forth in SEQ ID NO. 70
  • the light chain variable region of the antibody comprises the amino acid sequence set forth in SEQ ID NO.
  • the heavy chain variable region of the antibody comprises the amino acid sequence set forth in SEQ ID NO. 72
  • the light chain variable region of the antibody comprises the amino acid sequence set forth in SEQ ID NO.
  • the heavy chain variable region of the antibody comprises the amino acid sequence set forth in SEQ ID NO. 72
  • the light chain variable region of the antibody comprises the amino acid sequence set forth in SEQ ID NO.
  • the antibody is selected from the group consisting of 405B8H3, 556F6B8, 105F1E10, 409B11E12, 409D4E10, 553G8G8.
  • the antibody is selected from the group consisting of 405B8H3-1 (D ⁇ E), 405B8H3-1, 405B8H3-2, 405B8H3-6, 405B8H3-7, 556F6B8-3, 556F6B8-7, 556F6B8 -3 (D ⁇ E).
  • the amino acid sequence of the heavy chain variable region is as in the sequence listing, SEQ ID NO: 1, SEQ ID NO: 9, SEQ ID NO: 17, SEQ ID NO: 25, SEQ ID NO
  • the amino acid sequence of 33, SEQ ID NO: 41, SEQ ID NO. 64, SEQ ID NO. 66, SEQ ID NO. 68, SEQ ID NO. 70 or SEQ ID NO. 72 is at least 80%, 85%. , 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology or identity.
  • the amino acid sequence of the light chain variable region is as SEQ ID NO: 5, SEQ ID NO: 13, SEQ ID NO: 21, SEQ ID NO: 29, SEQ ID NO: 37.
  • the amino acid sequence of SEQ ID NO: 45, SEQ ID NO. 74, SEQ ID NO. 76, SEQ ID NO. 78, SEQ ID NO. 80 or SEQ ID NO. 82 is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology or identity.
  • 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:
  • a polynucleotide is provided, the polynucleotide encoding a polypeptide selected from the group consisting of:
  • polynucleotide encoding the heavy chain variable region is as set forth in SEQ ID NO: 49, 51, 53, 55, 57, 59, 65, 67, 69, 71 or 73; or,
  • the polynucleotide encoding the light chain variable region is shown as 50, 52, 54, 56, 58, 60, 75, 77, 79, 81 or 83.
  • polynucleotide encoding the heavy chain variable region sequence is set forth in SEQ ID NO: 49; and the polynucleotide encoding the light chain variable region sequence is as indicated at 50;
  • the polynucleotide encoding the heavy chain variable region is set forth in SEQ ID NO: 51; and the polynucleotide encoding the light chain variable region is set forth at 52;
  • the polynucleotide encoding the heavy chain variable region is set forth in SEQ ID NO: 53; and the polynucleotide encoding the light chain variable region is set forth at 54.
  • polynucleotide encoding the heavy chain variable region is set forth in SEQ ID NO: 55; and the polynucleotide encoding the light chain variable region is set forth at 56.
  • polynucleotide encoding the heavy chain variable region is set forth in SEQ ID NO: 57; and the polynucleotide encoding the light chain variable region is set forth at 58.
  • polynucleotide encoding the heavy chain variable region is set forth in SEQ ID NO: 59; and the polynucleotide encoding the light chain variable region is set forth at 60.
  • 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 LAG-3 related disease.
  • the LAG-3 related diseases include, but are not limited to, the following group: melanoma (such as metastatic malignant melanoma), kidney cancer, prostate cancer, breast cancer, colon cancer, lung cancer (if not Small cell lung cancer), uterine cancer, ovarian cancer, rectal cancer, gastric cancer, esophageal cancer, small intestine cancer, liver cancer, bladder cancer, oral cancer, brain cancer, testicular cancer, skin cancer, endocrine cancer, fallopian tube cancer, chronic or acute leukemia (including acute or chronic myeloid leukemia, acute or chronic lymphocytic leukemia), lymphocytic lymphoma, primary CNS lymphoma, T-cell lymphoma, advanced solid tumor.
  • melanoma such as metastatic malignant melanoma
  • kidney cancer such as metastatic malignant melanoma
  • prostate cancer such as metastatic malignant melanoma
  • breast cancer such as metastatic malignant melanoma
  • colon cancer lung cancer (if not
  • composition for detecting a LAG-3 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 for preparing a recombinant polypeptide comprising:
  • the recombinant polypeptide is isolated from the culture, the recombinant polypeptide according to any one of the fifth aspects of the invention or the recombinant protein according to the sixth aspect of the invention.
  • a method of treating a LAG-3-related disease comprising: using the antibody of any one of the fifth aspect of the invention, as in the sixth aspect of the invention A recombinant protein, a pharmaceutical composition according to the tenth aspect of the invention, or a combination thereof.
  • the LAG-3 related disease is cancer.
  • Antibody 2 is also included.
  • the antibody 2 is selected from the group consisting of a PD-1 antibody, a CTLA-4 antibody, and a PDL-1 antibody.
  • a pharmaceutical combination comprising:
  • the antibody according to any one of the fifth aspect of the invention, or the recombinant protein according to the sixth aspect of the invention, or the drug of the tenth aspect of the invention Use of a combination of a composition with another antibody 2 in the manufacture of a medicament for the treatment of a LAG-3 related disease.
  • Figure 4 ELISA detects serum antibody titer in mice after LAG-3-hFC protein immunization.
  • Figure 5a FACS detects the binding reaction of LAG-3 antibody to HEK293-hLAG-3.
  • Figure 5b FACS detects the binding reaction of LAG-3 antibody to HEK293-hLAG-3.
  • Figure 6a FACS detects the binding reaction of LAG-3 antibody to HEK293-cLAG-3.
  • Figure 6b FACS detects the binding reaction of LAG-3 antibody to HEK293-cLAG-3.
  • Figure 7a Inhibition of binding of the LAG-3 protein to its receptor MHC II by the LAG-3 antibody.
  • Figure 7b Inhibition of binding of the LAG-3 protein to its receptor MHC II by the LAG-3 antibody.
  • Figure 8a Effect of LAG-3 antibody on IL-2 secretion in an antigen-specific T lymphocyte stimulation assay.
  • Figure 8b Effect of LAG-3 antibody on IL-2 secretion in an antigen-specific T lymphocyte stimulation assay.
  • Figure 9 a LAG-3 antibody 105F1E10 heavy chain variable region protein and gene sequence.
  • FIG. 10 A LAG-3 antibody 405B8H3 heavy chain variable region protein and gene sequence.
  • FIG. 10b LAG-3 antibody 405B8H3 light chain variable region protein and gene sequence.
  • FIG. 11 LAG-3 antibody 556F6B8 heavy chain variable region protein and gene sequence.
  • FIG. 11b LAG-3 antibody 556F6B8 light chain variable region protein and gene sequence.
  • Figure 12 a LAG-3 antibody 409B11E12 heavy chain variable region protein and gene sequence.
  • Figure 12b LAG-3 antibody 409B11E12 light chain variable region protein and gene sequence.
  • Figure 13 a LAG-3 antibody 409D4E10 heavy chain variable region protein and gene sequence.
  • FIG. 13b LAG-3 antibody 409D4E10 light chain variable region protein and gene sequence.
  • Figure 14 a LAG-3 antibody 553G8G8 heavy chain variable region protein and gene sequence.
  • FIG. 14b LAG-3 antibody 553G8G8 light chain variable region protein and gene sequence.
  • Figure 15 Reactivity of LAG-3 murine-human chimeric antibody with human LAG-3 extracellular domain protein in an enzyme-linked immunosorbent assay.
  • Figure 16 Recombinant activity of LAG-3 murine-human chimeric antibody and monkey LAG-3 extracellular domain protein in an enzyme-linked immunosorbent assay.
  • Figure 17 Reactivity of LAG-3 murine-human chimeric antibody with murine LAG-3 extracellular domain protein in an enzyme-linked immunosorbent assay.
  • Figure 18 FACS detects the binding reaction of LAG-3 murine-human chimeric antibody to HEK293-hLAG-3.
  • Figure 19 FACS detects the binding reaction of LAG-3 murine-human chimeric antibody to HEK293-cLAG-3.
  • Figure 20 Inhibition of binding of the LAG-3 protein to its receptor MHC II by the LAG-3 murine-human chimeric antibody.
  • Figure 21 Inhibition of binding of the LAG-3 protein to its receptor LSECtin by a LAG-3 murine-human chimeric antibody.
  • Figure 22 Effect of LAG-3 murine-human chimeric antibody on IL-2 secretion in an antigen-specific T lymphocyte stimulation assay.
  • Figure 23 Effect of LAG-3 murine-human chimeric antibody on IL-2 secretion in an SEB-dependent PBMC stimulation assay.
  • Figure 24 FACS detection of the binding reaction of 405B8H3 humanized antibody to HEK293-hLAG-3.
  • Figure 25 FACS detection of the binding reaction of 556F6B8 humanized antibody to HEK293-hLAG-3.
  • Figure 26 FACS detects the binding reaction of a hot spot mutant antibody to HEK293-hLAG-3.
  • Figure 27 Inhibition of binding of the LAG-3 protein to its receptor MHC II by a hot mutant antibody, wherein the ordinate is % inhibition (blocking %).
  • Figure 28 Effect of hot mutant antibodies on IL-2 secretion in an antigen-specific T lymphocyte stimulation assay.
  • the present inventors have unexpectedly obtained a highly active specific monoclonal antibody (murine antibody) that binds to the human lymphocyte activation gene (LAG-3) by an extensive and intensive research using an immunized mouse/hybridoma technique.
  • the present invention antibody mouse - human chimeric antibody
  • the antigen protein with high affinity e.g., K D 405B8H3 to 1.96E-09M
  • K D 405B8H3 to 1.96E-09M capable of binding to the extracellular domain of LAG-3 receptor at the protein level
  • antibodies of the invention including but not limited to inhibition of negative regulation of LAG-3/MHC II and/or LAG-3/LSECtin-mediated signaling pathways, activation of tumor-specific immune responses, alone or in combination with anti-PD-1, CTLA- 4 monoclonal antibodies or other anti-tumor drugs are used in combination for tumor immunotherapy.
  • the antibody variable region of the present invention is combined with a human antibody constant region into a murine-human chimeric antibody molecule, or converted into a humanized antibody molecule by a humanization technique, or constructed into other molecular forms such as bispecific antibodies according to a specific use, Multispecific antibodies, single chain antibodies, single fragment antibodies, and the like.
  • the antibody of the present invention may be a full-length protein (e.g., IgG1, IgG2a, IgG2b or IgG2c), or may be a protein fragment (e.g., Fab, F(ab'), sdAb, ScFv fragment) comprising an antigen-antibody binding domain.
  • the antibody of the present invention may be a wild-type protein or a mutant protein which has achieved a certain specific effect by a specific mutation, for example, an effector function of eliminating an antibody by mutation.
  • the present invention has been completed on this basis.
  • the lymphocyte activating gene (LAG-3, CD223) is a type I membrane protein of 525 amino acids and is one of the known Immune Checkpoints. LAG-3 is mainly expressed in activated T lymphocytes, NK cells and dendritic cells. LAG-3 is structurally an immunoglobulin superfamily, which has four IgG-like domains extracellularly, which are similar to CD4 and require a major histocompatibility complex with their ligand-II class (MHC class). II) Binding exerts its biological activity, but its ability to bind to MHC II is stronger than that of CD4.
  • LAG-3 The binding of LAG-3 to its ligand MHC class II inhibits the activation and proliferation of CD4+ T lymphocytes, thereby downregulating the relevant cellular immune responses in vivo.
  • an anti-LAG-3 antibody to block the negative regulation of the LAG-3/MHC II signaling pathway leads to T cell proliferation and promotion of cytokine secretion and activation of the immune system.
  • LAG-3 is also expressed on regulatory T cells (Treg) by promoting Treg cell activity. This in turn negatively regulates T cell activation and proliferation as well as maturation of dendritic cells (DC cells) (Workman et al, 2005, Immunol, 174:688-695).
  • 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 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 LAG-3 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:
  • CDR2' represented by SEQ ID NO: 7 or SEQ ID NO. 84, and
  • 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 LAG-3 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 LAG-3.
  • 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 LAG-3 is 405B8H3, 556F6B8, 105F1E10, 409B11E12, 409D4E10 or 553G8G8.
  • the antibody is selected from the group consisting of 405B8H3-1 (D ⁇ E), 405B8H3-1, 405B8H3-2, 405B8H3-6, 405B8H3-7, 556F6B8-3, 556F6B8-7, 556F6B8 -3 (D ⁇ E).
  • the heavy chain variable region has the amino acid sequence set forth in SEQ ID NO. 64, SEQ ID NO. 66, SEQ ID NO. 68, SEQ ID NO. 70, SEQ ID NO.
  • the heavy chain variable region has the amino acid sequence set forth in SEQ ID No. 87, SEQ ID No. 91, SEQ ID No. 92, and SEQ ID No. 93.
  • the light chain variable region has the amino acid sequence set forth in SEQ ID NO. 74, SEQ ID NO. 76, SEQ ID NO. 78, SEQ ID NO. 80 or SEQ ID NO.
  • the light chain variable region has the amino acid sequence set forth in SEQ ID No. 89 and SEQ ID No. 95.
  • the heavy chain variable region (VH) amino acid sequence of the antibody 405B8H3 is the amino acid sequence set forth in SEQ ID NO.: 1.
  • the light chain variable region amino acid sequence of the antibody 405B8H3 is the amino acid sequence set forth in SEQ ID NO.: 5.
  • the heavy chain variable region (VH) amino acid sequence of the antibody 405B8H3-1 (D ⁇ E) is the amino acid sequence set forth in SEQ ID NO.
  • the light chain variable region (VH) amino acid sequence of the antibody 405B8H3-1 (D ⁇ E) is the amino acid sequence set forth in SEQ ID NO.
  • 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 diagnostic preparation, or for the preparation of a medicament for the prevention and/or treatment of a LAG-3 related disease.
  • the LAG-3 related diseases include related diseases including tumorigenesis, growth and/or metastasis related diseases, and the like.
  • melanoma such as metastatic malignant melanoma
  • kidney cancer such as metastatic malignant melanoma
  • breast cancer such as non-small cell lung cancer
  • lung cancer such as non-small cell lung cancer
  • uterine cancer ovarian cancer
  • rectal cancer gastric cancer, esophageal cancer, small intestine cancer, liver cancer, bladder cancer, oral cancer, brain cancer, testicular cancer, skin cancer, endocrine system cancer, fallopian tube cancer, chronic or acute leukemia (including acute or chronic myeloid leukemia, acute or chronic lymphatics) Cellular leukemia), lymphocytic lymphoma, primary CNS lymphoma, T-cell lymphoma, advanced solid tumor.
  • chronic or acute leukemia including acute or chronic myeloid leukemia, acute or chronic lymphatics
  • lymphocytic lymphoma primary CNS lymphoma
  • T-cell lymphoma advanced solid tumor.
  • 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 antibody of the present invention may also be a cell therapy for expression of a nucleotide sequence in a cell, for example, the antibody is used for chimeric antigen receptor T cell immunotherapy (CAR-T) and the like.
  • CAR-T chimeric antigen receptor T cell immunotherapy
  • the pharmaceutical composition of the present invention can be directly used for binding to a LAG-3 protein molecule, 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 immunoconjugate is administered to the mammal, wherein the safe and effective amount is typically 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 dosage 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 antibody of the present invention has a series of excellent characteristics:
  • variable region sequence is different from existing antibodies (homology ⁇ 92%)
  • the antibody of the invention has strong affinity with LAG-3 (for example, the KD value of 405B8H3 is 1.96 nM);
  • the antibody of the present invention has a good stimulation of T cell activation activity.
  • the present invention adopts hybridoma technology to obtain an antibody, and has higher antibody affinity and good sequence expression than the antibody obtained from the phage library.
  • the present invention obtains antibodies with different sequences, is capable of specifically binding to a LAG-3 antibody, has a binding activity lower than nanomolar, and is capable of blocking the binding of LAG-3 to its ligand MHCII/LSECtin by reversing LAG- 3 inhibition of T cell activation activity, thereby activating T cells to secrete IL-2, the activity is better.
  • the ability of the antibody of the present invention to bind to a cell surface of a monkey (e.g., Chinese monkey) LAG-3 protein is stronger than that of a control antibody.
  • the antibody of the present invention has a cross-binding reaction with a monkey antigen, and can be applied to an in vivo experiment of a primate for preclinical toxicology research and preclinical pharmacokinetic study.
  • the present invention uses a conventional hybridoma technique to prepare monoclonal antibodies.
  • Traditional hybridoma preparation technology was established 40 years ago by Kohler and Milstein and is now widely used in the preparation and production of many related monoclonal antibodies such as research, diagnosis and treatment.
  • its basic methods have been used up to now, there have been changes, improvements and innovations in many aspects, including the use of different strains of animals such as transgenic animals, the introduction of electrofusion technology, the application of high-efficiency screening technology equipment such as ClonePix equipment, etc.
  • the application of tumor technology is more diverse and efficient.
  • the monoclonal antibody prepared by a conventional animal such as a mouse can clone the antibody heavy chain variable region and the light chain variable region gene by conventional molecular biological methods, and the variable region gene can be grafted to the human antibody constant region gene to form a human mouse. Chimeric antibodies to greatly reduce immunogenicity in human use.
  • the CDR domain of the murine antibody variable region can be grafted onto the human antibody framework, thereby reducing the murine antibody component to less than 5%, greatly increasing the safety of the antibody for use in humans.
  • This pathway results in antibodies called humanized antibodies and is currently the main product of the antibody drug market.
  • the present invention employs optimized hybridoma technology to prepare the desired anti-LAG-3 antibodies.
  • An immunogen including an extracellular region LAG-3 protein, a LAG-3 recombinant cell strain, and an expression plasmid of a LAG-3 DNA vector was prepared.
  • HEK293 cells purchased from Invitrogen for transient transfection (PEI, Polysciences), using FreeStyle TM 293 (Invitrogen) were expanded at 37 °C. After 4 days, the cell culture medium was collected, and the cell components were removed by centrifugation to obtain a culture supernatant containing the extracellular region of the LAG-3 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.
  • PKI Protein A affinity chromatography column
  • the protein 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 the affinity layer from Protein A.
  • the hFc-tagged LAG-3 protein (LAG-3-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 a purified immunogen human LAG-3-hFc protein.
  • the immunogen LAG-3-hFc protein requires a series of quality control tests before use, such as detection of protein concentration, purity, molecular weight and biological activity.
  • the binding activity of the immunogen LAG-3-hFc to MHC II was detected by FACS, specifically:
  • the MHC II-expressing Raji cells were expanded to a 75-90% confluence in a T-175 cell culture flask, the medium was discarded by centrifugation, and the cells were washed 1-2 times with PBS buffer, and the cells were counted and closed.
  • the solution (PBS, 2% fetal bovine serum) was diluted to 1-2 x 10 6 cells per ml, incubated on ice for 20-30 minutes, and then washed twice with blocking solution (PBS, 2% fetal bovine serum).
  • the collected cells were suspended in a blocking solution (PBS, 2% fetal bovine serum) to 2 ⁇ 10 6 cells/ml, and added to a 96-well FACS reaction plate (2 ⁇ 10 5 cells per well) at 100 ⁇ L per well.
  • the medium was discarded by centrifugation, and the hFc-tagged LAG-3 protein was serially diluted, then added to Raji cells at 100 ⁇ L per well, and incubated on ice for 1-2 hours. Wash twice with blocking solution (PBS, 2% fetal bovine serum), add 100 ⁇ l of fluorescent (Alexa 488) labeled secondary antibody per well, and incubate for 0.5-1.0 hours on ice.
  • the cells were washed 2-3 times with a blocking solution (PBS, 2% fetal bovine serum), and 100 ⁇ l of PBS suspension cells per well were added, and the results were detected and analyzed by FACS (FACSVerse, BD).
  • the binding activity of the immunogen LAG-3-hFc to LSECtin was detected by ELISA, specifically:
  • the hFc-tagged LAG-3 protein (LAG-3-hFc, immunogen) 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 (1% BSA, PBS phosphate buffer pH 7.4, the percentage by mass) at 37 ° C for two hours, a gradient dilution of LSECtin-His tag was added and incubated for 1 hour at 37 °C.
  • the LSECtin-His was purchased from R&D system, product number 2947-CL.
  • Anti-His tagged horseradish peroxidase (purchased from GenScript product number A00612) was added, and after incubation for 30 minutes at room temperature, 100 ⁇ l/well of TMB color developing solution was added. After incubating for 15 minutes at room temperature, the color reaction was stopped by the addition of 50 ⁇ l of 1N hydrochloric acid, and the OD450nm reading was read using an ELISA plate reader. Washing is required after each step.
  • LAG-3 protein immunization was carried out using 6-8 week old BabL/C and SJL mice (provided by Shanghai Slack Animal Center), and the mice were reared under SPF conditions after receiving.
  • the primary immunized LAG-3 protein was emulsified with Freund's complete adjuvant and intraperitoneally injected with 0.25 ml of 50 ⁇ g protein per mouse.
  • the booster LAG-3 protein was emulsified with Freund's incomplete adjuvant and intraperitoneally injected with 0.25 ml of 50 ⁇ g protein per mouse.
  • the initial immunization was separated from the first booster by 2 weeks, and each subsequent immunization interval was 3 weeks. Blood was collected 7 days after each boost, and antibody titer and specificity in serum were measured by ELISA and FACS.
  • HEK293 cells have higher levels of LAG-3 expression and are suitable for use as immunogens and for antibody binding activity identification.
  • LAG-3 cell immunization was carried out using 6-8 week old BabL/C and SJL mice (provided by Shanghai Slack Animal Center), and the mice were reared under SPF conditions after receiving.
  • Human LAG-3 transfected HEK293 stable cell line was expanded to 75-90% confluence in T-75 cell culture flask, the medium was exhausted, washed with DMEM basal medium for 1-2 times, and then with no enzyme cells. Dissociation treatment and collection of cells. The cells were washed 1-2 times with DMEM basal medium, and after cell counting, the cells were diluted with PBS to 1-2 x 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. Blood was collected 7 days after each boost, and antibody titer and specificity in serum were measured by FACS.
  • LAG-3 full-length amino acid sequence cDNA was cloned into pCDNA3.1 vector and coated onto a 1.0 um gold colloidal bullet, and immunized with a Helios gene gun (Bio-rad). The detailed method was developed according to the instructions of the Helios gene gun.
  • mice 6-8 week old BabL/C and SJL mice (provided by Shanghai Slack Animal Center) were received and fed under SPF conditions. All mice were immunized 3-4 times with a gene gun through the abdomen, 3-4 shots each time, and 1.0 microgram of cDNA per shot. The initial immunization was separated from the first booster by 2 weeks, and each subsequent immunization interval was 3 weeks. Blood was collected 7 days after each boost, and the antibody titer in the serum was measured by ELISA. Usually, most mice can achieve an ELISA titer of 1:1000 or more after 2-3 immunizations. Table 4 and Figure 4 show the results of antibody titer detection by ELISA for LAG-3-hFC protein immunized serum.
  • mice that meet the requirements for potency can be selected for cell fusion and hybridoma preparation.
  • the mice Prior to cell fusion, the mice were immunized with 50-100 micrograms of purified LAG-3-hFc per patient for the last immunization. Mice were sacrificed 3-5 days later and spleen cells were collected. 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. Then, 1 ⁇ 10 5 /200 ⁇ 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. After 14 days, 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%).
  • 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 to LAG-3 protein and LAG-3 positive cells was determined by ELISA and FACS, and the blocking activity of the antibody sample to the LAG-3 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 LAG-3 was transfected into the 293F cell line of the preparation of immunogen 2 in Example 1 to obtain a 293F stably transfected cell line containing human LAG-3 (herein referred to as HEK293).
  • HEK293 human LAG-3
  • -hLAG-3 stable cell line, transfected pIRES plasmid carrying the full-length gene of monkey source transfected into HEK293 cell line to construct HEK293 stably transfected cell line containing monkey LAG-3 (herein referred to as HEK293-cLAG-3 stable cell) Strain).
  • the HEK293-hLAG-3 stable cell line and the HEK293-cLAG-3 stable cell line were expanded to a 90% confluence in a T-75 cell culture flask, and the medium was exhausted and washed with HBSS (Hanks' Balanced Salt Solution). -2 times, then the cells were treated and collected with Versene solution: Life technology. After the cells were washed with HBSS buffer 1-2 times, cell counting cells were diluted with HBSS to 1-2x10 6 cells per milliliter, 1% goat serum blocking solution, incubated on ice for 20-30 min and then centrifuged and washed with HBSS 2 times.
  • the collected cells were suspended in FACS buffer (HBSS+1% BSA) to 2 ⁇ 10 6 cells/ml, and added to a 96-well FACS reaction plate at 100 ⁇ L per well, and 100 ⁇ l per well of the antibody sample to be tested was added, ice. Incubate for 1-2 hours. Wash twice with FACS buffer, add 100 ⁇ l of fluorescent (Alexa 488) labeled secondary antibody per well, and incubate for 0.5-1.0 hours on ice. The cells were washed 2-3 times with FACS buffer, and the cells were suspended by adding 100 ⁇ l of a fixed solution (4% Paraformaldehyde) per well, and washed by 1-2 times with FACS buffer after 5-10 minutes.
  • FACS buffer HBSS+1% BSA
  • the cells were suspended in 100 ⁇ l of FACS buffer, and the results were detected and analyzed by FACS (FACSCalibur, BD). The results are shown in Table 5 and Table 6, Figure 5a, Figure 5b and Figure 6a, Figure 6b.
  • the antibody to be tested binds to the human or monkey LAG-3 protein on the cell surface, and the activity of each antibody is equivalent, indicating that the antibody binds to LAG-3. Strong ability.
  • the IgG control was mouse IgG, and the data in the table is the average fluorescence intensity value of the cell population measured by MFI.
  • the MHC II-expressing Raji cells were expanded to a 75-90% confluence in a T-175 cell culture flask, the medium was discarded by centrifugation, and the cells were washed 1-2 times with PBS buffer, and the cells were counted and closed.
  • the cells were washed twice with a blocking solution (PBS, 2% fetal bovine serum), and 100 ⁇ l of fluorescent (Alexa 488)-labeled secondary antibody per well was added and incubated on ice for 1.0 hour.
  • the cells were washed 2-3 times with a blocking solution (PBS, 2% fetal bovine serum), and 100 ⁇ l of PBS suspension cells per well were added, and the results were detected and analyzed by FACS (FACS Calibur, BD).
  • the results are shown in Table 7 and Figures 7a and 7b, wherein the IgG control is murine IgG, and the data in the table is the inhibition rate (%).
  • Antigen-specific T lymphocyte stimulation assay detects that LAG-3 antibody blocks the binding of LAG-3 to MHC II, thereby inhibiting the inhibition of T lymphocyte activity, thereby stimulating the proliferation of T cells.
  • CD4+ T cells were negatively screened from lymphocyte samples of OVA transgenic mice using immunomagnetic beads technology and combined antibodies using a mouse CD4 isolation kit to isolate antigens. Specific CD4+ T lymphocytes. The obtained T cells were mixed with the mouse thymoma cell line BW5147.G.1.4 at a ratio of 5:1, and cell fusion was carried out by a polyethylene glycol (PEG) cell fusion method.
  • PEG polyethylene glycol
  • the fused cells were selectively cultured in a medium of 1 ⁇ HAT containing hypoxanthine, aminopteridine and thymidine, and the obtained monoclonal was amplified into a 24-well plate for expansion culture, and the monoclonal was screened after 2-3 days. .
  • the clones were screened by in vitro antigen presentation experiments, and a mixture of spleen cells mixed with specific antigens OVA323-339 of the same strain of common C57BL/6 mice was added to the medium of the monoclonal cells, and the supernatant cells were incubated overnight to collect the supernatant (specifically See David H et al, Methods Mol Biol, 2013, 960: 297-307).
  • Enzyme-linked immunosorbent assay was used to detect the content of mouse IL2 (mIL2) in the supernatant, and to select the best monoclonal cell with good cell growth status, passage (at least a dozen generations), and high secretion of mIL-2. Enlarged culture and liquid nitrogen cryopreservation. The T lymphocyte hybridoma (8B2) was finally selected as the optimal clone.
  • T lymphocyte hybridoma cell line 8B2 was subjected to lentivirus infection, and the transfected cells were selectively cultured in an antibiotic-containing medium, and after 2 weeks, subcloned in a 96-well culture plate by limiting dilution. After the clones are grown, the monoclonal well cells are expanded into 6-well plates or culture flasks. The amplified clones were screened by flow cytometry using anti-LAG-3 specific antibodies. The cells with better growth and higher fluorescence intensity were selected, and the monoclonal cell lines continued to expand and cryopreserved. The T lymphocyte hybridoma (8B2)_hLAG-3 (3E4) was finally selected as the optimal clone.
  • T lymphocyte hybridoma (8B2)_hLAG-3 (3E4) was cultured in T-175 cell culture flask to 75-90% confluence, the medium was discarded, and PBS was used. Wash 1-2 times; after counting, pour the cells into 50-well cells of 1-2E5 cells into 96-well cell culture plates, then add twice the final concentration of the test antibody dilution to the culture plate, incubate for 30 min at room temperature.
  • the spleen cells of the same strain of common C57BL/6 mice were incubated with the specific antigen OVA 323-339 for 30 min at room temperature, and finally 50 ⁇ L of each mixture was added to the culture plate to ensure a volume of 200 ul per reaction well.
  • the plates were incubated overnight at 37 ° C in a 5% CO 2 incubator and the supernatants were collected and frozen below -20 ° for testing.
  • the cytokine interleukin IL-2 enzyme-linked immunosorbent assay in the cell supernatant was detected using the R&D system-related detection kit Mouse IL-2DuoSet ELISA (DY402) 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 in the cell supernatant was performed using a double-antibody sandwich ELISA kit (purchased from R&D Systems, IL-2Cat # DY402).
  • 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: the IL-2 polyclonal antibody was coated on an ELISA microplate, and the membrane was sealed with a plastic membrane at 4 ° C overnight, and the plate was washed 4 times with the washing solution the next day, and the blocking solution was added at room temperature to block 1 -2 hours.
  • the plate was washed 4 times with a washing solution, and the cell supernatant obtained in the step 2. was used as a sample to be tested, and the standard sample and the sample to be tested were incubated at room temperature for 2 hours. Add 400 ⁇ l of wash solution to each well and wash the plate 4 times. Add anti-human IL-2 horseradish peroxidase antibody and incubate for 2 hours at room temperature to form immune complex with IL-2 on the microplate. The micropores were cleaned; the substrate was added to develop color, and the room temperature was kept for 30 minutes in the dark. Finally, the stop solution was added, and the absorbance at A450nm was measured by a microplate reader.
  • the antibody to be tested in the antigen-specific T lymphocyte stimulation test can enhance the IL-2 secretion of T lymphocytes, and the activation is in a concentration-dependent manner, and the 405B8H3 activity rate is superior to other antibodies.
  • an exposed outer loop having the following amino acid sequence is included: GPPAAAPGHPLAPGPHP AAPSSWGPRRY.
  • ELISA enzyme-linked immunosorbent assay
  • streptavidin Sigma, Cat#M5432
  • the cells were incubated overnight at 4 ° C with a plastic film, and the plate was washed twice with a washing solution (PBS + 0.01% Tween 20) the next day, and blocked with a blocking solution (PBS + 0.01% Tween 20 + 1% BSA) for 1-2 hours at room temperature. The blocking solution was discarded, and the biotinylated peptide was added to a final concentration of 1 ug/ml. 100 ul per well was added to a 96-well ELISA plate, and incubated at 37 ° C for 1-2 hours, followed by washing (PBS + 0.01% Tween 20). Wash the plate 2-3 times.
  • RNA isolation Subcloned culture supernatants were tested for antigen binding, and 1-5 x 10 7 hybridoma cells were collected by centrifugation. Add 1 mL of Trizol and mix and transfer to a 1.5 ml centrifuge tube, let stand for 5 min at room temperature; add 0.2 ml of chloroform, shake for 15 s, let stand for 2 min, centrifuge at 4 ° C, 12000 g ⁇ 5 min, transfer the supernatant to a new 1.5 ml centrifuge.
  • Reverse transcription and PCR 1 ⁇ g of tRNA was taken, 20 ul of system was placed, reverse transcriptase was added, reacted at 42 ° C for 60 min, and reacted at 70 ° C for 10 min to terminate the reaction.
  • Configure 50 ⁇ l PCR system including 1 ⁇ l cDNA, 25pmol of each primer, 1 ⁇ l DNA polymerase and matching buffer system, 250 ⁇ mol dNTPs; set PCR program, pre-denatured 95°C for 3min, denatured 95°C for 30s, annealed 55°C for 30s, extended 72°C 35s, 35 cycles and then extended at 72 ° C for 5 min.
  • the extension temperature can be adjusted according to the actual situation.
  • 5 ⁇ l PCR product was detected by agarose gel electrophoresis, and the positive samples were purified by column recovery kit.
  • the ligation reaction was carried out: 50ng of sample, 50ng of T vector, 0.5 ⁇ l of ligase, 1 ⁇ l of buffer, 10 ⁇ l of reaction system , react at 16 ° C for half an hour; take 5 ⁇ l of the ligation product into 100 ⁇ l of competent cells, ice bath for 5 min, then heat shock for 1 min in a 42 ° C water bath, put back on ice for 1 min, then add 650 ⁇ l of antibiotic-free SOC medium at 37 ° C The shaker was resuscitated at 200 RPM for 30 min, and 200 ⁇ l of the solution was applied to an antibiotic-containing LB solid medium and cultured overnight at 37 ° C.
  • the heavy chain variable region protein and gene (DNA) sequences, light chain variable region proteins and gene sequences of the antibody products of the invention are as follows:
  • Antibody 105F1E10 is shown in Figure 9a, Figure 9b; Antibody 405B8H3 is shown in Figure 10a, Figure 10b; Antibody 556F6B8 is shown in Figure 11a, Figure 11b; Antibody 409B11E12 is shown in Figure 12a, Figure 12b; Antibody 409D4E10 is shown in Figure 13a, Figure 13b; Antibody 553G8G8 is shown in Figure 14a Figure 14b.
  • Plasmid construction and preparation The hybridoma antibody heavy chain variable region sequence was cloned into a pCP expression vector containing the signal peptide and the human heavy chain antibody IgG4 constant region, and the light chain variable region was recombined into a signal peptide and a human antibody.
  • the light chain kappa (lambda) constant region was expressed in the pCP vector and verified by sequencing.
  • the purity plasmid was increased by volume extraction using an alkali lysis kit, and filtered through a 0.22 ⁇ m filter for transfection.
  • Cell transfection Freestyle 293F cells were used, and the medium was Freestyle 293 expression medium, and 10% F68 was added at the final concentration of 0.1%.
  • the cell density was cultured to 1-1.5 x 10 6 cells per ml during transfection; the shaker was set to 37 ° C, 130 RPM, 8% CO 2 concentration.
  • Antibody purification For continuous production of endotoxin-free chromatography columns and Protein A packing, rinse with 0.1M NaOH for 30 min or 5 column volumes with 0.5 M NaOH; use at least 1 M NaOH for long-term unused column and column Soak for 1 h, rinse to neutral with non-endotoxic water, and clean the column with 10 column volumes of 1% Triton X100. Equilibration was performed using 5 column volumes of PBS, and the filtered cell supernatant was applied to the column, and the flow-through was collected as necessary. After the upper column was completed, it was washed with 5 column volumes of PBS.
  • Enzyme-linked immunosorbent assay for detection of binding of antibodies to LAG-3 protein
  • the amino acid sequence 23-450 of the human LAG-3 protein extracellular region described in the preparation of immunogen 1 was cloned into pCpC carrying the human IgG Fc fragment (hFc).
  • the vector was transfected into HEK293 cells, and the cell culture medium was collected to obtain the hFc-tagged human LAG-3 protein (herein referred to as hLAG-3-hFc protein); the amino acid sequence of the extracellular region of the monkey-derived LAG-3 protein was 18- 449 (shown in SEQ ID No.
  • the vector was transfected into HEK293 cells, and the cell culture medium was collected and purified to obtain a hFc-tagged mouse LAG-3 protein (herein referred to as mLAG-3-hFc protein).
  • mLAG-3-hFc protein Purified human, monkey, and murine LAG-3 extracellular domain proteins (hLAG-3-hFc, cLAG-3-hFc, mLAG-3-hFc) were diluted with PBS to a final concentration of 1.0 ⁇ g/ml, and then added at 100 ⁇ l per well. To a 96-well ELISA plate.
  • the cells were incubated overnight at 4 ° C with a plastic film, and the plate was washed twice with a washing solution (PBS + 0.01% Tween 20) the next day, and blocked with a blocking solution (PBS + 0.01% Tween 20 + 1% BSA) for 1-2 hours at room temperature.
  • the blocking solution was poured out, 50-100 ⁇ l of the antibody sample to be tested was added, and the cells were incubated at 37 ° C for 1-2 hours, and then washed with a washing solution (PBS + 0.01% Tween 20) for 2-3 times.
  • Example 2 The experimental method is shown in Example 2 (I), and the binding activity of the obtained mouse-human chimeric LAG-3 antibody to the cell expressing LAG-3 was identified. The results are shown in Fig. 18 and Fig. 19, Table 13 and Table 14.
  • 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 murine-human chimeric antibody obtained by the present invention can bind to human and monkey LAG-3 proteins on the cell surface. Moreover, the antibody of the present invention binds to the cell surface of the monkey LAG-3 protein more strongly than the control antibody (BMS986016).
  • the sequence of monkey LAG-3 in the experiment was obtained from tissue samples of Chinese monkeys.
  • the antibody of the present invention has a cross-binding reaction with a monkey antigen, and in vivo experiments of primates can be carried out for preclinical toxicology studies and preclinical pharmacokinetic studies.
  • LAG-3 receptor ligand binding assay detects LAG-3 antibody blocking the binding of LAG-3 to its ligands MHC II and LSECtin
  • LAG-3 receptor ligand binding assay detects LAG-3 antibody blocking the binding of LAG-3 to its ligand MHC II
  • Example 2 The experimental method is shown in Example 2 (b), and the obtained murine-human chimeric LAG-3 antibody was subjected to blocking activity identification, and the detection results are shown in Fig. 20 and Table 15, respectively.
  • LAG-3 receptor ligand binding assay detects that LAG-3 antibody blocks the binding of LAG-3 to its ligand LSECtin
  • the LAG-3 extracellular domain protein (LAG-3-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 each sample of the purified LAG-3 antibody obtained in Example 2 was added, and then LSECtin protein (LSECtin-His) was added thereto, 50 ⁇ L per well, and the mixture 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].
  • Example 2 The experimental procedure is shown in Example 2 (c). The results are shown in Table 17 and Figure 22, wherein the IgG control is human IgG (hIgG) and the data in the table is the concentration of murine IL-2.
  • Lymphocyte stimulation assay detects that LAG-3 antibody blocks the binding of LAG-3 protein to its receptor MHC II, 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.
  • an equal volume ratio of the murine-human chimeric antibody LAG-3 antibody to be tested 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 400 ng/ml SEB was contained in each reaction well to ensure a volume of 200 ⁇ 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 detected using the R&D system-related detection kit human IL-2 DuoSet ELISA (DY202) 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 in the cell supernatant was performed using a double-antibody sandwich ELISA kit (purchased from R&D Systems, IL-2Cat # DY202).
  • 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: the IL-2 polyclonal antibody was coated on an ELISA microplate, and the membrane was sealed with a plastic membrane at 4 ° C overnight, and the plate was washed 4 times with the washing solution the next day, and the blocking solution was added at room temperature to block 1 -2 hours.
  • the plate was washed 4 times with a washing solution, and the cell supernatant obtained in the step 2. was used as a sample to be tested, and the standard sample and the sample to be tested were incubated at room temperature for 2 hours. Add 400 ⁇ l of wash solution to each well and wash the plate 4 times. Add anti-human IL-2 horseradish peroxidase antibody and incubate for 2 hours at room temperature to form immune complex with IL-2 on the microplate. The micropores were cleaned; the substrate was added to develop color, and the room temperature was kept for 30 minutes in the dark. Finally, the stop solution was added, and the absorbance at A450nm was measured by a microplate reader.
  • the results indicate that in the SEB-dependent PBMC lymphocyte stimulation assay, the antibody obtained by the present invention can enhance the IL-2 secretion of PBMC, and the activity has a concentration gradient-dependent effect, indicating that the LAG-3 antibody can reverse the LAG-3 to T cells.
  • the inhibitory effect of activation as can be seen from the measured results, the level of antibody activity obtained in the present invention is comparable.
  • the hIgG control was human IgG and the data in the table was IL-2 value (pg/mL).
  • anti-human Fc IgG was prepared by immobilization on the surface of the CM5 chip to 6000-10000 RU by amino coupling method, and FC1 was used as a reference channel.
  • the coupling procedure was as follows: activation with a freshly configured mixture of 1:1 50 mM NHS and 200 mM EDC for 7 minutes followed by injection of 10-50 ug/ml anti-human Fc IgG diluted in 10 mM sodium acetate ph 5.0 buffer. The remaining activation sites were blocked with 1 M ethanolamine.
  • the antibody to be tested was diluted to 5 ug/ml with HBS-EP+ buffer (adjustable according to the capture level), and captured on the chip at a flow rate of 10 ul/min to obtain a response value of about 100 to 300 RU.
  • the antigenic protein was then diluted to 100 nM (maximum concentration tentatively 100 nM) and flowed through the surface of the chip at a flow rate of 30 ul/min. If sufficient signal values are obtained, the antigenic proteins are then diluted by several concentration gradients and flowed through the surface of the chip, respectively. At the end of each cycle, the surface of the chip was regenerated with 10 mM Glycine at pH 1.5.
  • the heavy chain, light chain variable region of clones 405B8H3 and 556F6B8 was used as a humanized template.
  • NCBI-Igblast The germline gene sequence with the highest homology of the light chain variable region of the candidate antibody 405B8H3 heavy chain variable region (NCBI-Igblast) was selected as the variable region transplantation backbone by sequence alignment (IGBI-Igblast): IGHV1-46*01 and IGKV1-16 *01. After the selection of the human antibody backbone, the key amino acids that may determine the structure in the murine anti-constant region were predicted by homology modeling, and the grafted framework region was subjected to a back mutation design.
  • variable region grafting framework by sequence alignment (NCBI-Igblast): IGHV4-59*01 and IGKV1-9 *01.
  • NCBI-Igblast sequence alignment
  • the key amino acids that may determine the structure in the murine anti-constant region were predicted by homology modeling, and the grafted framework region was subjected to a back mutation design.
  • Antibody purification Experimental procedure See Example 4, cell culture supernatants were purified using a endotoxin-free Protein A column, and antibodies were harvested. Then dialyzed overnight in 1 x PBS to avoid endotoxin contamination.
  • the obtained antibody was subjected to protein concentration and purity detection analysis. The yield and purity analysis of all antibodies were normal.
  • the affinity of the antibody after humanization was evaluated and the results are shown in Table 28.
  • the results showed that the KD values of the antibodies obtained in the present invention were all at the nanomolar (nM) level, and the affinity of the antibody after humanization was equivalent to that of the corresponding murine-human chimeric antibody.
  • Point mutations were made to the antibody 405B8H3-1 hot spot.
  • the 405B8H3-1 antibody has one mutated site, and the asparagine D at position 56 of the light chain is mutated to glutamic acid E.
  • Example 5 The method is the same as in Example 5. As a result, as shown in Table 29 and Figure 26, both the hot spot mutant antibody and the chimeric antibody bind to human LAG-3 on the cell surface.
  • 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.
  • LAG-3 receptor ligand binding assay detects antibody blocking LAG-3 binding to its ligand MHC II
  • Example 2 The experimental method is shown in Example 2 (2), and the test results are shown in Table 30 and Figure 27, respectively.
  • the results showed that both the antibody and the murine-human chimeric antibody could block the binding of LAG-3 to the ligand MHCII, and the activity was comparable.
  • Example 2 The experimental procedure is shown in Example 2 (c). The results are shown in Table 31 and Figure 28, wherein the IgG control is human IgG (hIgG) and the data in the table is the concentration of murine IL-2.
  • the method is the same as in Example 5.
  • the affinity of the hot mutant antibody was evaluated and the results are shown in Table 32.
  • the clinical research of Bristol-Myers Squibb's LAG-3 antibody BMS986016 is mainly used for the treatment of malignant solid tumors, and it is mainly focused on its combination with other therapies or target drugs, and the development of a wide range of antibodies to expand its Applicable clinical symptoms, including unresectable metastatic melanoma, advanced solid cancer, breast cancer, endometrial cancer, ovarian cancer, kidney cancer, pancreatic cancer, recurrent glioblastoma, head and neck cancer, bladder cancer, metastasis Straight colon cancer, gastrointestinal stromal tumor, acinar cell carcinoma, advanced malignant solid tumor, non-small cell lung cancer, and the like.
  • variable region sequence determines the determinant, binding affinity and rate of metabolism in the body, which affects the activity in vivo and even the clinical effects of individuals in different patients.
  • VH is a heavy chain variable region and VL is a light chain variable region.
  • VH-CDR1, VH-CDR2, VH-CDR3 are heavy chain variable region CDR1, CDR2, CDR3, respectively;
  • VL-CDR1, VL-CDR2, VL-CDR3 are light chain variable region CDR1', CDR2', CDR3', respectively .

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Biochemistry (AREA)
  • Genetics & Genomics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biomedical Technology (AREA)
  • Biophysics (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Cell Biology (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Pathology (AREA)
  • General Physics & Mathematics (AREA)
  • Food Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Veterinary Medicine (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Zoology (AREA)
  • Mycology (AREA)
  • Plant Pathology (AREA)
  • Epidemiology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Peptides Or Proteins (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

本发明公开了一种靶向LAG-3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LAG-3的单克隆抗体。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LAG-3抗原，其具有很高的亲和力并且具有显著抗肿瘤等活性。

Description

抗人LAG-3单克隆抗体及其应用 技术领域
本发明涉及生物医药领域，具体涉及一种LAG-3抗体及其制备方法和应用。
背景技术
癌症免疫治疗是指通过免疫系统达到对抗癌症目的的治疗方式。近来癌症免疫治疗备受关注，除了手术、化疗及放疗外，已成为癌症治疗的新手段。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。免疫检查点抑制剂还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，可能是治疗或者治愈多种癌症的希望。
淋巴细胞激活基因(LAG-3,CD223)是一种有525个氨基酸的I型膜蛋白，是已知的主要免疫检查点(Immune Checkpoint)之一。研究表明，LAG-3-/-C57BL/6小鼠在一段时间内表现相对正常的显型，说明LAG-3在免疫系统内的调节作用相当微妙，可能在免疫反应中发挥着微调的作用。在体内的动物模型试验表明用抗LAG-3抗体或者遗传学敲除LAG-3基因能够增强肿瘤发生点的抗原特异性CD8+T细胞的活性，进而阻断肿瘤的生长。Grosso JF等研究表明LAG-3和人程序性死亡受体-1(PD-1)共表达在耐受的肿瘤浸润淋巴细胞，他们共同发挥肿瘤诱导的免疫抑制作用。已经证明，在小鼠MC38结肠腺癌和Sa1N纤维肉瘤模型用抗LAG-3和抗PD-1的抗体联合应用可以治愈大多数小鼠，其治疗效果好于单独用药。
基于目前LAG-3抗体在研数量很少，急需开发出活性更好、适应症广和产 量高的LAG-3抗体，进一步以提高治疗和检测效果。
发明内容
为了开发活性好、适应症广和产量高的LAG-3抗体，本发明提供一种亲和力高、特异性强的LAG-3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、4或5；
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LAG-3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、4或5。
在另一优选例中，所述重链可变区具有SEQ ID NO.64、SEQ ID NO.66、SEQ ID NO.68、SEQ ID NO.70、SEQ ID NO.72所示的氨基酸序列。
在另一优选例中，所述重链可变区具有SEQ ID No.87、SEQ ID No.91、SEQ ID No.92、SEQ ID No.93所示的氨基酸序列。
在本发明的第二方面，提供了一种抗体的重链，所述的重链具有如本发明的第一方面所述的重链可变区。
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、4或5；
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LAG-3结合亲和力的衍生序列。
在另一优选例中，所述轻链可变区的VL-CDR2具有SEQ ID NO.84所示的氨基酸序列(405B8H3-1(D→E)的VL-CDR2)。
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、4或5。
在另一优选例中，所述轻链可变区具有SEQ ID NO.74、SEQ ID NO.76、SEQ ID NO.78、SEQ ID NO.80或SEQ ID NO.82所示的氨基酸序列。
在另一优选例中，所述轻链可变区具有SEQ ID No.89、SEQ ID No.95所示的氨基酸序列。
在本发明的第四方面，提供了一种抗体的轻链，所述的轻链具有如本发明的第三方面所述的轻链可变区。
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LAG-3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LAG-3结合的亲和力。
在另一优选例中，所述的衍生抗体与LAG-3结合的亲和力F1与相应非衍生的抗体与LAG-3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LAG-3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鼠源的，和/或所述的轻链恒定区为鼠源的。
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，所述抗体的重链可变区还包括人源的框架区，和/或所述抗体的轻链可变区还包括人源的框架区。
在另一优选例中，所述抗体的重链可变区还包括鼠源的框架区，和/或所述抗体的轻链可变区还包括鼠源的框架区。
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，所述抗体为突变的人源化抗体。
在另一优选例中，所述抗体为CDR区含有D→E突变的人源化抗体。
在另一优选例中，所述抗体为VL-CDR2或VH-CDR3含有D→E突变的人源化抗体。
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；
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或SEQ ID NO.84所示的CDR2'，和
SEQ ID NO:8所示的CDR3'；
或
所述的重链可变区包括以下三个互补决定区CDR：
SEQ ID NO:10所示的CDR1，
SEQ ID NO:11所示的CDR2，和
SEQ ID NO:12所示的CDR3；
所述的轻链可变区包括以下三个互补决定区CDR：
SEQ ID NO:14所示的CDR1'，
SEQ ID NO:15所示的CDR2'，和
SEQ ID NO:16所示的CDR3'；
或
所述的重链可变区包括以下三个互补决定区CDR：
SEQ ID NO:18所示的CDR1，
SEQ ID NO:19所示的CDR2，和
SEQ ID NO:20所示的CDR3；
所述的轻链可变区包括以下三个互补决定区CDR：
SEQ ID NO:22所示的CDR1'，
SEQ ID NO:23所示的CDR2'，和
SEQ ID NO:24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LAG-3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、4或5。
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:9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: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。
在另一优选例中，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: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；或
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；或
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，所述抗体的重链可变区含有SEQ ID NO:41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:45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.64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.74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.64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.76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.66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.76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.66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.78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.68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.78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.70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.80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.72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.80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.72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.82所示的氨基酸序列。
在另一优选例中，所述的抗体选自下组：405B8H3、556F6B8、105F1E10、409B11E12、409D4E10、553G8G8。
在另一优选例中，所述的抗体选自下组：405B8H3-1(D→E)、405B8H3-1、405B8H3-2、405B8H3-6、405B8H3-7、556F6B8-3、556F6B8-7、556F6B8-3(D→E)。
在另一优选例中，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、SEQ ID NO:41、SEQ ID NO.64、SEQ ID NO.66、SEQ ID NO.68、SEQ ID NO.70或SEQ ID NO.72所示的氨基酸序列至少有80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的序列同源性或相同性。
在另一优选例中，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、SEQ ID NO:45、SEQ ID NO.74、SEQ ID NO.76、SEQ ID NO.78、SEQ ID NO.80或SEQ ID NO.82所示的氨基酸序列至少有80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的序列同源性或相同性。
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，以及
(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:49、51、53、55、57、59、65、67、69、71或73所示；和/或，
编码所述轻链可变区的多核苷酸如50、52、54、56、58、60、75、77、79、81或83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:49所示；并且编码所述轻链可变区序列的多核苷酸如50所示；或
编码所述重链可变区的多核苷酸如SEQ ID NO:51所示；并且编码所述轻链可变区的多核苷酸如52所示；或
编码所述重链可变区的多核苷酸如SEQ ID NO:53所示；并且编码所述轻链可变区的多核苷酸如54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:55所示；并且编码所述轻链可变区的多核苷酸如56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:57所示；并 且编码所述轻链可变区的多核苷酸如58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:59所示；并且编码所述轻链可变区的多核苷酸如60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LAG-3相关疾病的药物。
在另一优选例中，所述LAG-3相关的疾病包括，但不限于下组：黑色素瘤(如转移性恶性黑色素瘤)，肾癌，前列腺癌，乳腺癌，结肠癌，肺癌(如非小细胞肺癌)，子宫癌，卵巢癌，直肠癌，胃癌，食道癌，小肠癌，肝癌，膀胱癌，口腔癌，脑癌，睾丸癌，皮肤癌，内分泌系统癌症，输卵管癌，慢性或急性白血病(包括急性或慢性髓样白血病、急性或慢性淋巴细胞性白血病)、淋巴细胞性淋巴瘤、原发性CNS淋巴瘤、T细胞淋巴瘤，晚期实体瘤。
在本发明的第十二方面，提供了一种体外检测样品中LAG-3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，提供了一种重组多肽的制备方法，该方法包括：
(a)在适合表达的条件下，培养如本发明的第九方面所述的宿主细胞；
(b)从培养物中分离出重组多肽，所述的重组多肽是如本发明的第五方面中任一项所述的抗体或如本发明的第六方面所述的重组蛋白。
在本发明的第十四方面，提供了一种治疗LAG-3相关的疾病的方法，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、如本发明的第十方面所述的药物组合物、或其组合。
在另一优选例中，所述LAG-3相关的疾病为癌症。
在另一优选例中，还包括使用抗体2。
在另一优选例中，所述抗体2选自下组：PD-1抗体、CTLA-4抗体、PDL-1抗体。
在本发明的第十五方面，提供了一种药物组合，包括：
(i)第一活性成分，所述第一活性成分为如本发明的第五方面中任一项所述的抗体；
(ii)第二活性成分，所述第二活性成分包括抗体2。
在本发明的第十六方面，提供了本发明的第五方面中任一项所述的抗体，或本发明的第六方面所述的重组蛋白、或本发明的第十方面所述的药物组合物与另外一种抗体2的组合在制备用于治疗LAG-3相关疾病的药物中的用途。
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 LAG-3-hFc蛋白与其配体MHCII的结合活性。
图2 LAG-3-hFc蛋白与其配体LSECtin的结合活性。
图3 LAG-3基因转染的HEK293细胞FACS检测结果。
图4 ELISA检测LAG-3-hFC蛋白免疫后小鼠血清抗体效价。
图5a FACS检测LAG-3抗体与HEK293-hLAG-3的结合反应。
图5b FACS检测LAG-3抗体与HEK293-hLAG-3的结合反应。
图6a FACS检测LAG-3抗体与HEK293-cLAG-3的结合反应。
图6b FACS检测LAG-3抗体与HEK293-cLAG-3的结合反应。
图7a LAG-3抗体对LAG-3蛋白与其受体MHC II的结合的抑制。
图7b LAG-3抗体对LAG-3蛋白与其受体MHC II的结合的抑制。
图8a LAG-3抗体在抗原特异性T淋巴细胞刺激试验中对IL-2分泌的影响。
图8b LAG-3抗体在抗原特异性T淋巴细胞刺激试验中对IL-2分泌的影响。
图9a LAG-3抗体105F1E10重链可变区蛋白和基因序列。
图9b LAG-3抗体105F1E10轻链可变区蛋白和基因序列。
图10a LAG-3抗体405B8H3重链可变区蛋白和基因序列。
图10b LAG-3抗体405B8H3轻链可变区蛋白和基因序列。
图11a LAG-3抗体556F6B8重链可变区蛋白和基因序列。
图11b LAG-3抗体556F6B8轻链可变区蛋白和基因序列。
图12a LAG-3抗体409B11E12重链可变区蛋白和基因序列。
图12b LAG-3抗体409B11E12轻链可变区蛋白和基因序列。
图13a LAG-3抗体409D4E10重链可变区蛋白和基因序列。
图13b LAG-3抗体409D4E10轻链可变区蛋白和基因序列。
图14a LAG-3抗体553G8G8重链可变区蛋白和基因序列。
图14b LAG-3抗体553G8G8轻链可变区蛋白和基因序列。
图15酶联免疫吸附实验中LAG-3鼠-人嵌合抗体与人LAG-3胞外区蛋白反应活性。
图16酶联免疫吸附实验中LAG-3鼠-人嵌合抗体与猴LAG-3胞外区蛋白反应活性。
图17酶联免疫吸附实验中LAG-3鼠-人嵌合抗体与鼠LAG-3胞外区蛋白反应活性。
图18 FACS检测LAG-3鼠-人嵌合抗体与HEK293-hLAG-3的结合反应。
图19 FACS检测LAG-3鼠-人嵌合抗体与HEK293-cLAG-3的结合反应。
图20 LAG-3鼠-人嵌合抗体对LAG-3蛋白与其受体MHC II的结合的抑制。
图21 LAG-3鼠-人嵌合抗体对LAG-3蛋白与其受体LSECtin的结合的抑制。
图22 LAG-3鼠-人嵌合抗体在抗原特异性T淋巴细胞刺激试验中对IL-2分泌的影响。
图23 LAG-3鼠-人嵌合抗体在SEB依赖的PBMC刺激试验中对IL-2分泌的影响。
图24 FACS检测405B8H3人源化抗体与HEK293-hLAG-3的结合反应。
图25 FACS检测556F6B8人源化抗体与HEK293-hLAG-3的结合反应。
图26 FACS检测热点突变抗体与HEK293-hLAG-3的结合反应。
图27热点突变抗体对LAG-3蛋白与其受体MHC II的结合的抑制，其中，纵坐标为抑制率％(blocking％)。
图28热点突变抗体在抗原特异性T淋巴细胞刺激试验中对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，采用免疫小鼠/杂交瘤技术，意外地获得高活性的与人淋巴细胞活化基因(LAG-3)相结合的特异性单克隆抗体(鼠源抗体)。实验结果表明，本发明抗体(鼠-人嵌合抗体)与抗原蛋白具有高度亲和力(例如405B8H3的K D为1.96E-09M)，能够结合LAG-3受体的胞外区并能够在蛋白水平和细胞水平有效封闭LAG-3与配体MHC class II、LSECtin的结合；抗原特异性T淋巴细胞刺激实验证明所得LAG-3抗体具有良好的生物活性。本发明抗体的用途，包括但不限于抑制LAG-3/MHC II和/或LAG-3/LSECtin介导的信号通路的负调控，激活肿瘤特异免疫反应，单独或与抗PD-1，CTLA-4单克隆抗体或其它抗肿瘤药物联合应用于用于肿瘤免疫治疗。
本发明抗体可变区与人源抗体恒定区组合为鼠-人嵌合抗体分子，或者通过 人源化技术转变为人源化抗体分子，或者根据特定用途构建成其它分子形式如双特异性抗体、多特异性抗体、单链抗体、单片段抗体等。
本发明中的抗体可以是全长蛋白(如IgG1,IgG2a,IgG2b或者IgG2c)，也可以是包含抗原抗体结合域的蛋白片段(例如Fab，F(ab’),sdAb,ScFv片段)。本发明中的抗体可以是野生型蛋白，也可以是经过特定突变已达到某种特定效果的突变型蛋白，例如利用突变消除抗体的效应子功能。
在此基础上完成了本发明。
术语
LAG-3
淋巴细胞激活基因(LAG-3,CD223)是一种有525个氨基酸的I型膜蛋白，是已知的主要免疫检查点(Immune Checkpoint)之一。LAG-3主要表达在激活的T淋巴细胞，NK细胞以及树突细胞。LAG-3在结构上属于免疫球蛋白超家族，它的胞外具有4个IgG样结构域，它与CD4类似，都需要和它们的配体-II类主要组织相容性复合体(MHC class II)结合发挥其生物学活性，但其与MHC II结合能力强于CD4。LAG-3与其配体MHC class II结合可以抑制CD4+T淋巴细胞的活化与增殖，进而下调相关的体内细胞免疫反应。在体外的抗原特异性T细胞反应研究中，加入抗LAG-3抗体阻断LAG-3/MHC II信号通路的负调控作用，可以导致T细胞增殖以及促进细胞因子的分泌，激活免疫系统。另外LAG-3还表达在调节性T细胞(Treg)上，通过促进Treg细胞活性。进而负调节T细胞活化和增殖以及树突状细胞(DC细胞)的成熟(Workman et al,2005,Immunol,174:688-695)。
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LAG-3的抗体
本发明提供一种针对LAG-3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或SEQ ID NO.84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LAG-3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LAG-3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LAG-3的抗体为405B8H3、556F6B8、105F1E10、409B11E12、409D4E10或553G8G8。
在另一优选例中，所述的抗体选自下组：405B8H3-1(D→E)、405B8H3-1、405B8H3-2、405B8H3-6、405B8H3-7、556F6B8-3、556F6B8-7、556F6B8-3(D→E)。
在另一优选例中，所述重链可变区具有SEQ ID NO.64、SEQ ID NO.66、SEQ ID NO.68、SEQ ID NO.70、SEQ ID NO.72所示的氨基酸序列。
在另一优选例中，所述重链可变区具有SEQ ID No.87、SEQ ID No.91、SEQ ID No.92、SEQ ID No.93所示的氨基酸序列。
在另一优选例中，所述轻链可变区具有SEQ ID NO.74、SEQ ID NO.76、SEQ ID NO.78、SEQ ID NO.80或SEQ ID NO.82所示的氨基酸序列。
在另一优选例中，所述轻链可变区具有SEQ ID No.89、SEQ ID No.95所示的氨 基酸序列。
在另一优选例中，所述抗体405B8H3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405B8H3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。
在另一优选例中，所述抗体405B8H3-1(D→E)的重链可变区(VH)氨基酸序列为如SEQ ID NO.64所示的氨基酸序列。
在另一优选例中，所述抗体405B8H3-1(D→E)的轻链可变区(VH)氨基酸序列为如SEQ ID NO.74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，例如用于制备诊断制剂、或制备用于预防和/或治疗LAG-3相关的疾病的药物。所述LAG-3相关的疾病包括相关的疾病包括肿瘤发生、生长和/或转移相关疾病等。
本发明抗体的用途，包括(但并不限于)：
(i)诊断、预防和/或治疗黑色素瘤(如转移性恶性黑色素瘤)，肾癌，前列腺癌，乳腺癌，结肠癌，肺癌(如非小细胞肺癌)，子宫癌，卵巢癌，直肠癌，胃癌，食道癌，小肠癌，肝癌，膀胱癌，口腔癌，脑癌，睾丸癌，皮肤癌，内分泌系统癌症，输卵管癌，慢性或急性白血病(包括急性或慢性髓样白血病、急性或慢性淋巴细胞性白血病)、淋巴细胞性淋巴瘤、原发性CNS淋巴瘤、T细胞淋巴瘤，晚期实体瘤。
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。
本发明所述抗体也可以是由核苷酸序列在细胞内表达用于的细胞治疗，比如，所述抗体用于嵌合抗原受体T细胞免疫疗法(CAR-T)等。
本发明的药物组合物可直接用于结合LAG-3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，是将安全有效量的免疫偶联物施用于哺乳动物，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)本发明抗体具有一系列的优异特征：
①可变区序列与现有抗体不同(同源性<92％)；
②与BMS986016结合的抗原表位不同；
③本发明抗体与LAG-3具有较强的亲和力(例如405B8H3的KD值为1.96nM)；
④本发明抗体具有很好的刺激T细胞激活活性。
(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)本发明获得了序列不同的抗体，能够与LAG-3抗体特异性结合，其结合活性低于纳摩尔，并且能够阻断LAG-3与其配体MHCII/LSECtin的结合，通过逆转LAG-3对T细胞激活活性的抑制，从而激活T细胞分泌IL-2，各项活性比较好。
(4)本发明抗体结合细胞表面的猴(例如中国猴)LAG-3蛋白能力强于对照抗体。本发明抗体与猴抗原具有交叉结合反应，可应用于进行灵长类动物的体内实验，用于进行临床前毒理学研究和临床前药代动力学研究。
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。
通用方法
本发明采用传统的杂交瘤技术制备单克隆抗体。传统的杂交瘤制备技术由Kohler and Milstein在40年前建立，现在已广泛应用于科研、诊断、治疗等许多相关的单克隆抗体的制备和生产之中。其基本方法虽然延用至今，但在许多方面都有所变化、改进和创新，包括不同品系动物如转基因动物的使用、电融合技术的引入、高效筛选技术设备的应用如ClonePix设备等，使杂交瘤技术的应用更多样化和高效化。常规动物如小鼠等制备的单抗，可以通过常规分子生物学方法克隆抗体重链可变区和轻链可变区基因，可变区基因可以嫁接到人源抗体恒定区基因从而形成人鼠嵌合抗体，以大大降低人体使用时的免疫原性。此外，鼠源抗体可变区的CDR结构域可以嫁接到人源抗体架构上，从而使鼠源抗体成分降低到5％以下，大大增加了抗体在人体内使用的安全性。这一途径得到抗体称为人源化抗体，并且是目前抗体药物市场的主要产品。根据目前最新的单抗技术进展，本发明采用优化的杂交瘤技术来制备所需的抗LAG-3抗体。
实施例1 LAG-3特异性抗体的制备
制备包括胞外区LAG-3蛋白、LAG-3重组细胞株、LAG-3DNA载体的表达质粒等免疫原。
免疫原1)，将人源LAG-3蛋白胞外区氨基酸序列23-450(如序列表SEQ ID No.61所示)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LAG-3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LAG-3蛋白(LAG-3-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人LAG-3-hFc蛋白。免疫原LAG-3-hFc蛋白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。
其中，免疫原LAG-3-hFc与MHC II的结合活性采用FACS检测，具体为：
将表达MHC II的Raji细胞在T-175细胞培养瓶中扩大培养至75-90％汇合度，离心弃去培养基，用PBS缓冲液洗涤细胞1-2次,进行细胞计数后将细胞用封闭液(PBS，2％胎牛血清)稀释至1-2×10 6细胞每毫升，冰上孵育20-30分钟，然后用封闭液(PBS，2％胎牛血清)洗涤2次。将收集的细胞用封闭液(PBS，2％胎牛血清)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(每孔2×10 5细胞)，离心弃去培养基，将带hFc标签的LAG-3蛋白梯度稀释,然后按照每孔100微升加入Raji细胞中，冰上孵育1-2小时。用封闭液(PBS，2％胎牛血清)离心洗涤2次，加入每孔100微升荧光(Alexa 488)标记的二抗，冰 上孵育0.5-1.0小时。用封闭液(PBS，2％胎牛血清)离心洗涤2-3次，加入每孔100微升PBS悬浮细胞，用FACS(FACSVerse,BD)检测和分析结果。
其中，免疫原LAG-3-hFc与LSECtin的结合活性采用ELISA检测，具体为：
将带hFc标签的LAG-3蛋白(LAG-3-hFc，即免疫原)，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LSECtin-His标签，37℃温育1小时。所述LSECtin-His购自R&D system，商品号2947-CL。加入抗His标签的辣根过氧化物酶(购自GenScript商品号A00612)，室温孵育30分钟后加入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OD450nm读数。每个步骤之后都需要洗板。
结果如图1、图2和表1、表2所示。
表1 LAG-3-hFc蛋白与其配体MHCII的结合活性
Figure PCTCN2019081063-appb-000001
表2 LAG-3-hFc蛋白与其配体LSECtin的结合活性
Figure PCTCN2019081063-appb-000002
Figure PCTCN2019081063-appb-000003
结果表明，LAG-3与MHCII在细胞水平的结合随LAG-3-hFC的浓度变化而变化，LAG-3与LSECtin在蛋白水平的结合随LAG-3-hFC的浓度变化而变化，其中对照蛋白为非LAG-3融合蛋白。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。
LAG-3蛋白免疫采用6-8周龄BabL/C和SJL小鼠(上海斯莱克动物中心繁殖提供)，小鼠接收后在SPF条件下饲养。初次免疫LAG-3蛋白用福氏完全佐剂乳化后腹腔注射0.25毫升，50微克蛋白每只小鼠。加强免疫LAG-3蛋白用福氏不完全佐剂乳化后腹腔注射0.25毫升，50微克蛋白每只小鼠。初次免疫与第一次加强免疫之间隔2周，以后每次免疫间隔3周。每次加强免疫7天后采血，用ELISA和FACS检测血清中抗体效价和特异性。
免疫原2)，人源LAG-3全长氨基酸序列克隆到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LAG-3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LAG-3重组细胞株。具体选择结果如表3和图3所示，表3中阳性细胞(％)指阳性细胞占总细胞数目的百分比，MFI是所测细胞群的平均荧光强度值。
表3 LAG-3基因转染的HEK293细胞FACS筛选检测结果
Figure PCTCN2019081063-appb-000004
结果表明，HEK293细胞有较高水平LAG-3的表达，适合用做免疫原和进行抗体结合活性鉴定。
LAG-3细胞免疫采用6-8周龄BabL/C和SJL小鼠(上海斯莱克动物中心繁殖提供)，小鼠接收后在SPF条件下饲养。人LAG-3转染的HEK293稳定细胞系在T-75细胞培养瓶中扩大培养至75-90％汇合度，吸尽培养基，用DMEM基础培养基洗涤1-2次，然后用无酶细胞解离液处理和收集细胞。用DMEM基础培养基洗涤1-2次,进行细胞计数后将细胞用PBS稀释至1-2x10 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隔2周，以后每次免疫间隔3周。每次加强免疫7天后采血，用FACS检测血清中抗体效价和特异性。
免疫原3)，LAG-3全长氨基酸序列cDNA被克隆到pCDNA3.1载体并包被到1.0um金胶体子弹上，用Helios基因枪(Bio-rad)免疫。详细方法根据Helios基因枪说明书进行制定。
6-8周龄BabL/C和SJL小鼠(上海斯莱克动物中心繁殖提供)接收后在SPF条件下饲养。所有小鼠经腹部用基因枪免疫3-4次，每次3-4枪，每枪1.0微克cDNA量。初次免疫与第一次加强免疫之间隔2周，以后每次免疫间隔3周。每次加强免疫7天后采血，用ELISA检测血清中抗体效价。通常大部分小鼠经2-3次免疫后ELISA效价可达到1:1000以上。表4和图4为LAG-3-hFC蛋白免疫血清，用ELISA进行抗体效价检测的结果。
表4 ELISA检测LAG-3-hFC蛋白免疫后小鼠血清抗体效价
Figure PCTCN2019081063-appb-000005
结果表明：以免疫原进行免疫，大部分小鼠经3次免疫后ELISA效价可达到1:100000以上，说明小鼠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。
效价符合要求的小鼠可选择进行细胞融合和杂交瘤制备。细胞融合前，小鼠最后一次免疫用每只50-100微克纯化的LAG-3-hFc腹腔注射。3-5天后处死小鼠，收集脾细胞。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 5/200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LAG-3蛋白和LAG-3阳性细胞的结合活性，配体受体结合实验确定抗体样品对LAG-3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LAG-3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LAG-3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MHCII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 纯化抗体的鉴定
(一)流式细胞实验(FACS)检测抗体与LAG-3表达细胞的结合
将实施例1制备免疫原2中所述含有编码人源LAG-3全长核苷酸序列的pIRES质粒转染293F细胞株得含人LAG-3的293F稳转细胞株(此处称为HEK293-hLAG-3稳定细胞株)，将带有猴源全长基因的pIRES质粒，转染HEK293细胞株构建含猴LAG-3的HEK293稳转细胞株(此处称为HEK293-cLAG-3稳定细胞株)。将HEK293-hLAG-3稳定细胞株和HEK293-cLAG-3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(Hanks'Balanced Salt Solution)洗涤1-2次，然后用无酶细胞解离液(Versene solution:Life technology)处理和收集细胞。用HBSS缓冲液洗涤细胞1-2次,进行细胞计数后将细胞用HBSS稀释至1-2x10 6细胞每毫升，加入1％山羊血清封闭液，冰上孵育20-30分钟，然后用HBSS离心洗涤2次。将收集的细胞用FACS缓冲液(HBSS+1％BSA)悬浮至2x10 6细胞/ml,按每孔100微升加入到96孔FACS反应板中，加入待测抗体样品每孔100微升，冰上孵育1-2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，冰上孵育0.5-1.0小时。用FACS缓冲液离心洗涤2-3次，加入每孔100微升固定液(4％Paraformaldehyde)悬浮细胞，5-10分钟后用FACS缓冲液离心洗涤1-2次。用100微升FACS缓冲液悬浮细胞，用FACS(FACSCalibur,BD)检测和分析结果。结果如表5和表6、图5a、图5b和图6a、图6b所示，待测抗体可结合细胞表面的人或猴LAG-3蛋白，各抗体活性相当，表明抗体与LAG-3结合能力较强。其中IgG对照为鼠源IgG，表中的数据为MFI所测细胞群的平均荧光强度值。
表5 FACS检测LAG-3抗体与HEK293-hLAG-3的结合反应
Figure PCTCN2019081063-appb-000006
Figure PCTCN2019081063-appb-000007
表6 FACS检测LAG-3抗体与HEK293-cLAG-3的结合反应
Figure PCTCN2019081063-appb-000008
(二)LAG-3受体配体结合实验检测LAG-3抗体阻断LAG-3与其配体MHC II的结合
将表达MHC II的Raji细胞在T-175细胞培养瓶中扩大培养至75-90％汇合度，离心弃去培养基，用PBS缓冲液洗涤细胞1-2次,进行细胞计数后将细胞用封闭液(PBS，2％胎牛血清)稀释至1-2×10 6细胞每毫升，按每孔100微升加入到96孔FACS反应板中(每孔1×10 5细胞)，冰上孵育20-30分钟，将待测抗体样品与1ug/ml的LAG-3-hFc蛋白等体积混合，并在室温孵育30分钟后，将孵育Raji细胞的FACS反应板离心弃掉上清，按照每孔100微升将上述的混合物加入Raji细胞中，冰上孵育1-2小时。用封闭液(PBS，2％胎牛血清)离心洗涤2次，加入每孔100微升荧光(Alexa 488)标记的二抗，冰上孵育1.0小时。用封闭液(PBS，2％胎牛血清)离心洗涤2-3次，加入每孔100微升PBS悬浮细胞，用FACS(FACS Calibur,BD)检测和分析结果。结果如表7和图7a、图7b所示，其中IgG对照为 鼠源IgG，表中的数据为抑制率(％)。
表7 LAG-3抗体对LAG-3蛋白与其受体MHC II的结合的抑制
Figure PCTCN2019081063-appb-000009
结果表明，所得抗体可不同程度的抑制LAG-3蛋白与其配体MHCII的结合，所测抗体活性相当。
(三)抗原特异性T淋巴细胞刺激实验检测LAG-3抗体对淋巴细胞活性的影响
抗原特异性T淋巴细胞刺激实验检测LAG-3抗体阻断LAG-3与MHC II的结合从而解除对T淋巴细胞活性的抑制，从而刺激T细胞的增殖。
1.抗原特异性T淋巴细胞的获得，首先，使用小鼠CD4分离试剂盒,利用免疫磁珠技术和联合抗体，从OVA转基因小鼠的淋巴细胞样品中阴性筛选CD4+T细胞，分离出抗原特异性的CD4+T淋巴细胞。将获得的T细胞与小鼠胸腺瘤细胞系BW5147.G.1.4按5:1的比率混合，采用聚乙二醇(PEG)细胞融合方法进行细胞融合。融合后的细胞用1×HAT含次黄嘌呤、氨基蝶啶和胸苷的培养基进行选择性培养，将获得的单克隆扩增到24孔板扩大培养，2-3天后对单克隆进行筛选。用体外抗原提呈实验筛选克隆，用同品系普通C57BL/6小鼠的脾细胞混合特异性抗原OVA323-339的混合物加入到单克隆细胞的培养基中，孵育单克隆细胞过夜收集上清(具体方法参见David H等，Methods Mol Biol，2013，960:297–307)。酶联免疫吸附检测检测上清中的小鼠白介素2(mouse IL2，mIL2)的含量，挑选细胞生长状态良好、传代(至少十几代)稳定，且分泌mIL-2量高的最优单克隆进行扩大培养、液氮冻存。最终选择T淋巴细胞杂交瘤(8B2)为 最优克隆。
过表达免疫抑制因子稳定细胞株的构建，将人LAG-3的全长基因序列克隆到pIRES表达载体上，并包装成慢病毒(上海吉玛)。对T淋巴细胞杂交瘤细胞株8B2进行慢病毒感染，转染后的细胞在含抗生素的培养基中选择性培养，2周后，用有限稀释法在96孔培养板中亚克隆。待克隆长大后，将单克隆孔细胞扩增到6孔板中或培养瓶中。对扩增后的克隆用抗LAG-3特异性抗体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。最终选择T淋巴细胞杂交瘤(8B2)_hLAG-3(3E4)为最优克隆。
2.抗原特异性T淋巴细胞刺激实验，将T淋巴细胞杂交瘤(8B2)_hLAG-3(3E4)在T-175细胞培养瓶中培养至75-90％汇合度，弃去培养基，用PBS洗涤1-2次；计数后将细胞以1-2E5个细胞50微升每孔铺至96孔细胞培养板，然后将两倍于终浓度的待测抗体稀释液加入培养板，室温孵育30min，同时用同品系普通C57BL/6小鼠的脾细胞与特异性抗原OVA 323-339混合室温孵育30min，最后将其混合物50微升每孔加入到培养板，保证每个反应孔200ul体积，将反应板于37℃5％CO2培养箱培养过夜后收集上清，低于-20°冻存待测。
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Mouse IL-2DuoSet ELISA(DY402)，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 # DY402)。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，用塑料膜封好4℃孵育过夜，第二天用洗板液洗板4次，加入封闭液室温封闭1-2小时。用洗板液洗板4次，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LAG-3抗体在步骤2.所述抗原特异性T淋巴细胞刺激实验中对IL-2分 泌的影响。结果如图8a、图8b)，和表8所示，其中mIgG对照为鼠IgG，表中的数据为IL-2值(pg/mL)。
表8 LAG-3抗体在抗原特异性T淋巴细胞刺激试验中对IL-2分泌的影响
Figure PCTCN2019081063-appb-000010
结果表明，在抗原特异性T淋巴细胞刺激试验中待测抗体可使T淋巴细胞的IL-2分泌增强，并且激活作用呈浓度梯度依赖性，其中405B8H3活性率优于其它抗体。
(四)抗原表位预测
在人LAG-3蛋白胞外区的包含一具有以下氨基酸序列的暴露的外环(eatra loop)：GPPAAAPGHPLAPGPHP AAPSSWGPRPRRY。为了检验纯化抗体与这一区域的结合并预测每个抗体所结合的表位，在整个这一区域内进行肽扫描实验。
制备了包括外环序列的全长在内的15种重叠肽并将在C端偶联生物素。用酶联免疫吸附实验(ELISA)检测抗体与这些肽段的结合，链霉亲和素(sigma,Cat#M5432)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(PBS+0.01％Tween20)洗板2次，加入封闭液(PBS+0.01％Tween20+1％BSA)室温封闭1-2小时。倒掉封闭液，加入生物素化的肽段，终浓度1ug/ml,以每孔100ul加到96孔ELISA板，37℃孵育1-2小时后，用洗板液(PBS+0.01％Tween20)洗板2-3次。然后将待测抗体样品100μl每孔,37℃孵育1-2小时后，用洗板液(PBS+0.01％Tween20)洗板2-3次。加入HRP(辣根过氧化物酶)标记的二抗，37℃孵育1-2小时后， 用洗板液(PBS+0.01％Tween20)洗板2-3次。加入TMB底物100μl每孔，室温孵育15-30分钟后，加入终止液(1.0N HCl)100μl每孔。用ELISA读板机(TiterMax384plus,Molecular Device)读取A450nm数值。肽扫描实验的结果总结如下表9所示。
表9 肽扫描实验的结果
Figure PCTCN2019081063-appb-000011
基于上述表9的结果，可确定553G8G8和556F6B8不识别该外环序列，405B8H3、409B11E12和409D4E10识别在包括氨基酸序列SSWGPRPR的外环内的一区域，105F1E10识别包括氨基酸序列APSSWGPR的外环内的一区域。
结果表明：本发明抗体与BMS的LAG-3抗体BMS986016结合的表位不一致，本发明抗体不会对BMS的专利CN102176921A所保护的抗原表位HPAAPSSW造成侵权。
实施例3 轻重链可变区氨基酸序列测定
总RNA分离：亚克隆培养的上清检验过抗原结合后，通过离心搜集1-5×10 7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5min；加0.2ml氯仿，振荡15s，静置2min后于4℃离心，12000g×5min，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10min后于4℃离心，12000g×15min，弃上清；加入1ml 75％乙醇，轻轻洗涤沉淀。4℃，12000g×5min，弃上清并晾干，加入适量的DEPC H 2O溶解(55℃水浴促溶10min)。
逆转录与PCR：取1μg tRNA，配置20ul体系，加入逆转录酶后于42℃反应60min，于70℃反应10min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℃3min，变性95℃30s，退火55℃30s，延伸72℃35s，35个循环后再额外于72℃延伸5min。注：延伸温度可根据实际情况有所调整。
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；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；取5μl连接产物加入100μl的感受态细胞中，冰浴5min，而后于42℃水浴热激1min，放回冰上1min后加入650μl无抗生素SOC培养基，于37℃摇床上以200RPM的速度复苏30min，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点于另一块含抗生素的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：本发明抗体产品的重链可变区蛋白和基因(DNA)序列、轻链可变区蛋白和基因序列如下：
抗体105F1E10见图9a、图9b；抗体405B8H3见图10a、图10b；抗体556F6B8见图11a、图11b；抗体409B11E12见图12a、图12b；抗体409D4E10见图13a、图13b；抗体553G8G8见图14a、图14b。
实施例4 鼠-人嵌合抗体构建、以及抗体的生产和纯化
质粒构建与准备：将杂交瘤抗体重链可变区序列克隆到包含信号肽和人源重链抗体IgG4恒定区的pCP表达载体当中，将轻链可变区重组到包含信号肽和人源抗体轻链kappa(lambda)恒定区的表达pCP载体当中，并经测序验证。使用碱裂解法试剂盒中量抽提高纯度质粒，经0.22μm滤膜过滤，供转染使用。
细胞转染：使用Freestyle 293F细胞，培养基为Freestyle 293表达培养基(Freestyle 293expression medium)，使用时添加10％F68指终浓度0.1％。转染时将细胞密度培养至每毫升1-1.5×10 6细胞；摇床设置为37℃，130RPM，8％CO 2浓度。取5毫升培养基和PEI混匀(200μg/ml)，取5毫升培养基和一定量质粒混匀(质粒用量为100μg/ml)，5min后合并混匀，静置15分钟；缓缓加到细胞中，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(sigma)至终浓度为0.5％；第5～7天，测培养液抗体效价，第6～7天，离心(3500RPM，30min)、过滤收集上清以供纯化。
抗体纯化：对于连续生产的无内毒素的层析柱和Protein A填料，使用0.1M NaOH处理30min或者5个柱体积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％Triton X100对柱料清洗。使用5个柱体积的PBS进行平衡，将过滤好的细胞上清上柱，必要时收集流穿液。上柱完成后，使用5倍柱体积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1/10体积的pH8.5的1M Tris-HCl(1.5M NaCl)中和。收获抗体后，在1×PBS中透析过夜，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检测抗体内毒素含量。
实施例5 鼠-人嵌合抗体的鉴定
(一)酶联免疫吸附实验(ELISA)检测抗体与LAG-3蛋白的结合
将实施例1制备免疫原1中所述的人源LAG-3蛋白胞外区氨基酸序列23-450(如序列表SEQ ID No.61所示)克隆到带有人IgG Fc片段(hFc)的pCpC载体，转染HEK293细胞，收集细胞培养液，纯化得到带hFc标签的人LAG-3蛋白(此处称为hLAG-3-hFc蛋白)；将猴源LAG-3蛋白胞外区氨基酸序列18-449(如序列 表SEQ ID No.62所示)克隆到带有人IgG Fc片段(hFc)的pCpC载体，转染HEK293细胞，收集细胞培养液，纯化得到带hFc标签的猴LAG-3蛋白(此处称为cLAG-3-hFc蛋白)；将鼠源LAG-3蛋白胞外区氨基酸序列24-442(如序列表SEQ ID No.63所示)克隆到带有人IgG Fc片段(hFc)的pCpC载体，转染HEK293细胞，收集细胞培养液，纯化得到带hFc标签的鼠LAG-3蛋白(此处称为mLAG-3-hFc蛋白)。纯化的人、猴、鼠LAG-3胞外区蛋白(hLAG-3-hFc、cLAG-3-hFc、mLAG-3-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(PBS+0.01％Tween20)洗板2次，加入封闭液(PBS+0.01％Tween20+1％BSA)室温封闭1-2小时。倒掉封闭液，加入待测抗体样品50-100μl每孔,37℃孵育1-2小时后，用洗板液(PBS+0.01％Tween20)洗板2-3次。加入HRP(辣根过氧化物酶)标记的二抗，37℃孵育1-2小时后，用洗板液(PBS+0.01％Tween20)洗板2-3次。加入TMB底物100μl每孔，室温孵育15-30分钟后，加入终止液(1.0N HCl)100μl每孔。用ELISA读板机(TiterMax384plus,Molecular Device)读取A450nm数值。结果如图15,图16和图17，表10,表11和表12所示。
表10 酶联免疫吸附实验中LAG-3鼠-人嵌合抗体与人LAG-3胞外区蛋白反应活性
Figure PCTCN2019081063-appb-000012
表11 酶联免疫吸附实验中LAG-3鼠-人嵌合抗体与猴LAG-3胞外区蛋白反应活性
Figure PCTCN2019081063-appb-000013
Figure PCTCN2019081063-appb-000014
表12 酶联免疫吸附实验中LAG-3鼠-人嵌合抗体与鼠LAG-3胞外区蛋白反应活性
Figure PCTCN2019081063-appb-000015
结果表明：本发明所得鼠-人嵌合抗体可结合人和猴LAG-3胞外区蛋白，且各抗体活性接近；不能结合鼠LAG-3蛋白。其中IgG对照为人IgG，表中的数据为A450nm数值。
(二)流式细胞实验(FACS)检测抗体与LAG-3表达细胞的结合
实验方法见实施例2(一)，对所获鼠-人嵌合LAG-3抗体与细胞表达LAG-3的结合活性进行鉴定,结果如图18和图19，表13和表14所示。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。
表13 FACS检测LAG-3鼠-人嵌合抗体与HEK293-hLAG-3的结合反应
Figure PCTCN2019081063-appb-000016
表14 FACS检测LAG-3鼠-人嵌合抗体与HEK293-cLAG-3的结合反应
Figure PCTCN2019081063-appb-000017
Figure PCTCN2019081063-appb-000018
结果表明：本发明所得鼠-人嵌合抗体可结合细胞表面的人和猴LAG-3蛋白。并且本发明抗体结合细胞表面的猴LAG-3蛋白能力强于对照抗体(BMS986016)。
实验中猴LAG-3的序列是从中国猴的组织样品中得到的。本发明抗体与猴抗原具有交叉结合反应，将来可进行灵长类动物的体内实验，用于进行临床前毒理学研究和临床前药代动力学研究。
(三)：LAG-3受体配体结合实验检测LAG-3抗体阻断LAG-3与其配体MHC II和LSECtin的结合
(1)LAG-3受体配体结合实验检测LAG-3抗体阻断LAG-3与其配体MHC II的结合
实验方法见实施例2(二)，对所获鼠-人嵌合LAG-3抗体进行阻断活性鉴定，检测结果分别如图20和表15所示。
表15 LAG-3鼠-人嵌合抗体对LAG-3蛋白与其受体MHC II的结合的抑制
Figure PCTCN2019081063-appb-000019
结果表明：本发明所得鼠-人嵌合抗体LAG-3抗体能够不同程度地阻断LAG-3与配体MHCII的结合。
(2)LAG-3受体配体结合实验检测LAG-3抗体阻断LAG-3与其配体LSECtin的结合
LAG-3胞外区蛋白(LAG-3-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LAG-3抗体待测样品50μl每孔，后加入LSECtin蛋白(LSECtin-His)，每孔50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。加入抗His标签的HRP(辣根过氧化物酶)稀释液(购自GenScript)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。检测结果分别如图21和表16所示。
表16 LAG-3鼠-人嵌合抗体对LAG-3蛋白与其受体LSECtin的结合的抑制
Figure PCTCN2019081063-appb-000020
结果表明：本发明所得鼠-人嵌合抗体LAG-3抗体能够不同程度地阻断LAG-3与配体LSECtin的结合。
(四)：抗原特异性T淋巴细胞刺激实验检测LAG-3抗体对淋巴细胞活性的影响
实验方法见实施例2(三)，结果见表17和图22，其中IgG对照为人IgG(hIgG)，表中的数据为鼠IL-2浓度。
表17 LAG-3鼠-人嵌合抗体在抗原特异性T淋巴细胞刺激试验中对IL-2分泌的影响
Figure PCTCN2019081063-appb-000021
结果表明：本发明所得鼠-人嵌合抗体在抗原特异性T淋巴细胞刺激试验中能够刺激IL-2分泌，并且该活性具有浓度梯度依赖效应，表明LAG-3抗体能够 逆转LAG-3对T细胞激活的抑制作用，从所测结果可以看出，本发明所得抗体活性水平相当。
(五)淋巴细胞刺激实验检测LAG-3抗体对淋巴细胞活性的影响
淋巴细胞刺激实验检测LAG-3抗体阻断LAG-3蛋白与其受体MHC II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的鼠-人嵌合抗体LAG-3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400ng/ml SEB,保证每个反应孔200μL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human IL-2 DuoSet ELISA(DY202)，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 # DY202)。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，用塑料膜封好4℃孵育过夜，第二天用洗板液洗板4次，加入封闭液室温封闭1-2小时。用洗板液洗板4次，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LAG-3抗体在步骤2.所述SEB依赖的PBMC刺激实验中对IL-2分泌的影响。结果如图23，和表18所示。
表18 LAG-3鼠-人嵌合抗体在SEB依赖的PBMC刺激试验中对IL-2分泌的影响
Figure PCTCN2019081063-appb-000022
结果表明，在SEB依赖的PBMC淋巴细胞刺激试验中，本发明所得抗体可使PBMC的IL-2分泌增强，并且该活性具有浓度梯度依赖效应，表明LAG-3抗体能够逆转LAG-3对T细胞激活的抑制作用，从所测结果可以看出，本发明所得抗体活性水平相当。其中hIgG对照为人IgG，表中的数据为IL-2值(pg/mL)。
(六)抗体亲和力检测试验
首先，准备用氨基偶联方法将anti-human Fc IgG固定在CM5芯片表面至6000-10000RU，FC1作为参比通道。偶联过程如下：用新鲜配置的1:1的50mM NHS和200mM EDC的混合物活化7分钟，然后注入10-50ug/ml稀释在10mM醋酸纳ph5.0缓冲液中的anti-human Fc IgG。剩余的活化位点用1M乙醇胺封闭。然后，用HBS-EP+缓冲液将待测抗体稀释至5ug/ml(可根据捕获水平适当调整)，以10ul/min的流速捕获到芯片上，得到大约100～300RU的响应值。接着将抗原蛋白稀释至100nM(最高浓度暂定100nM)，以30ul/min的流速流经芯片表面。 若得到足够的信号值，然后将抗原蛋白倍比稀释几个浓度梯度，分别流经芯片表面。在每个循环结束后，芯片表面用10mM，PH 1.5的Glycine进行再生。动力学速率常数需减去空白对照，用global fit分析方法1：1结合模型进行数据拟合。解离平衡速率常数(KD)用以下公式计算：KD＝kd/ka。结果见表19。
表19 抗LAG-3抗体亲和力的分析测定
克隆ID ka(1/Ms) kd(1/s) KD(M)
105F1E10 3.85E+04 9.66E-05 2.51E-09
405B8H3 8.04E+04 1.58E-04 1.96E-09
556F6B8 1.22E+05 4.59E-04 3.77E-09
结果表明，本发明所得抗体的KD值均在纳摩(nM)水平，且与工具抗体相当，表明这些抗体对人LAG-3ECD均有较好的亲和力，其中，405B8H3抗体对人LAG-3ECD的亲和力最好。
实施例6 人源化抗体的制备、鉴定和热点突变
(一)人源化抗体的制备
将克隆405B8H3和556F6B8的重链，轻链可变区作为人源化模板。
通过序列比对(NCBI-Igblast)选择与候选抗体405B8H3重链可变区，轻链可变区同源性最高的胚系基因序列作为可变区移植骨架：IGHV1-46*01和IGKV1-16*01。在选定人抗体骨架后，通过同源建模，预测在鼠抗恒定区中可能决定结构的关键氨基酸，对嫁接的骨架区进行回复突变设计。
根据以上原则，分别设计4个重链可变区序列(405B8H3 VH_g0，405B8H3 VH_g1，405B8H3 VH_g2，405B8H3 VH_g3)(见表20)和3个轻链可变区序列(405B8H3 VL_g0,405B8H3 VL_g1，405B8H3 VL_g2)(见表21)，随后做交叉组合进行表达，共12种表达组合，见表22。
表20 405B8H3重链可变区同源建模后回复突变设计
Figure PCTCN2019081063-appb-000023
Figure PCTCN2019081063-appb-000024
表21 405B8H3轻链可变区同源建模后回复突变设计
Figure PCTCN2019081063-appb-000025
表22 405B8H3人源化抗体表达组合
  405B8H3 VH.g0 405B8H3 VH.g1 405B8H3 VH.g2 405B8H3 VH.g3
405B8H3 VL.g0 405B8H3‐1 405B8H3‐2 405B8H3‐3 405B8H3‐4
405B8H3 VL.g1 405B8H3‐5 405B8H3‐6 405B8H3‐7 405B8H3‐8
405B8H3 VL.g2 405B8H3‐9 405B8H3‐10 405B8H3‐11 405B8H3‐12
通过序列比对(NCBI-Igblast)选择与候选抗体556F6B8重链可变区，轻链可变区同源性最高的胚系基因序列作为可变区移植骨架：IGHV4-59*01和IGKV1-9*01。在选定人抗体骨架后，通过同源建模，预测在鼠抗恒定区中可能决定结构的关键氨基酸,对嫁接的骨架区进行回复突变设计。
根据以上原则，分别设计4个重链可变区序列(556F6B8 VH_g0，556F6B8 VH_g1，556F6B8 VH_g2，556F6B8 VH_g3)(见表23)和3个轻链可变区序列(556F6B8 VL_g0,556F6B8 VL_g1，556F6B8 VL_g2)(见表24)，随后做交叉组合进行表达，共12种表达组合，见表25。
表23 556F6B8重链可变区同源建模后回复突变设计
Figure PCTCN2019081063-appb-000026
表24 556F6B8轻链可变区同源建模后回复突变设计
Figure PCTCN2019081063-appb-000027
表25 556F6B8人源化抗体表达组合
  556F6B8 VH.g0 556F6B8 VH.g1 556F6B8 VH.g2 556F6B8 VH.g3
556F6B8 VL.g0 556F6B8‐1 556F6B8‐2 556F6B8‐3 556F6B8‐4
556F6B8 VL.g1 556F6B8‐5 556F6B8‐6 556F6B8‐7 556F6B8‐8
556F6B8 VL.g2 556F6B8‐9 556F6B8‐10 556F6B8‐11 556F6B8‐12
载体构建：实验方法见实施例4，将重链可变区序列和轻链可变区序列克隆到包含信号肽和人源抗体IgG4恒定区的pCP表达载体当中，并经测序验证。
人源化抗体的制备：
细胞转染：实验方法见实施例4，使用Freestyle 293F细胞，将构建的质粒转染到细胞中，培养6-7天，过滤收集上清以供纯化。
抗体纯化：实验方法见实施例4，用无内毒素的Protein A层析柱纯化细胞培养上清液，收获抗体。然后在1×PBS中透析过夜，避免内毒素污染。
将所得抗体进行蛋白浓度、纯度检测分析。所有抗体的产量，纯度分析均表现正常。
(二)人源化抗体的鉴定
A.流式细胞实验(FACS)检测抗体与LAG-3表达细胞的结合，方法同实施例5。结果如表26和图24以及表27和图25所示，所得抗体均可结合细胞表面的人LAG-3。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。
表26 FACS检测405B8H3人源化抗体与HEK293-hLAG-3的结合反应
Figure PCTCN2019081063-appb-000028
表27 FACS检测556F6B8人源化抗体与HEK293-hLAG-3的结合反应
Figure PCTCN2019081063-appb-000029
Figure PCTCN2019081063-appb-000030
B.LAG-3抗体亲和常数的测定，方法同实施例5。
对人源化改造后抗体的亲和力进行评估，结果见表28。结果表明，本发明所得抗体的KD值均在纳摩(nM)水平，人源化改造后抗体与对应的鼠-人嵌合抗体的亲和力相当。
表28 抗LAG-3抗体亲和力的分析测定
克隆ID ka(1/Ms) kd(1/s) KD(M)
405B8H3 6.395E+04 1.652E-04 2.583E-09
405B8H3-1 1.001E+05 2.543E-04 2.539E-09
405B8H3-2 1.096E+05 1.844E-04 1.682E-09
405B8H3-6 9.488E+04 1.932E-04 2.037E-09
405B8H3-7 9.643E+04 3.165E-04 3.282E-09
556F6B8 1.341E+05 4.548E-04 3.392E-09
556F6B8-3 1.406E+05 4.648E-04 3.307E-09
556F6B8-7 1.393E+05 4.530E-04 3.252E-09
(三)人源化抗体的热点突变
对抗体405B8H3-1热点进行点突变。405B8H3-1抗体有1个可突变位点，轻链的第56位天冬酰胺D突变成谷氨酸E。
热点突变抗体的载体构建和制备，方法同实施例6中人源化抗体的载体构建和制备。将所得的热点突变抗体进行蛋白浓度、纯度检测分析。所有抗体的产量，纯度分析均表现正常。
热点突变抗体的活性鉴定
A.流式细胞实验(FACS)检测抗体与LAG-3表达细胞的结合
方法同实施例5。结果如表29和图26，热点突变抗体和嵌合抗体均可结合细胞表面的人LAG-3。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。
表29 FACS检测热点突变抗体与HEK293-hLAG-3的结合反应
Figure PCTCN2019081063-appb-000031
B.LAG-3受体配体结合实验检测抗体阻断LAG-3与其配体MHC II的结合
实验方法见实施例2(二)，检测结果分别如表30和图27所示。结果表明：热点突变后抗体和鼠-人嵌合抗体均能够阻断LAG-3与配体MHCII的结合,且活性相当。
表30 热点突变抗体对LAG-3蛋白与其受体MHC II的结合的抑制
Figure PCTCN2019081063-appb-000032
C.抗原特异性T淋巴细胞刺激实验检测LAG-3抗体对淋巴细胞活性的影响
实验方法见实施例2(三)，结果见表31和图28，其中IgG对照为人IgG(hIgG)，表中的数据为鼠IL-2浓度。
结果表明:热点突变的人源化抗体与对应的鼠-人嵌合抗体均在抗原特异性T淋巴细胞刺激试验中能够刺激IL-2分泌，且具有浓度梯度依赖效应，活性相当。
表31 热点突变抗体在抗原特异性T淋巴细胞刺激试验中对IL-2分泌的影响
Figure PCTCN2019081063-appb-000033
D.LAG-3抗体亲和常数的测定
方法同实施例5。对热点突变抗体的亲和力进行评估，结果见表32。
结果表明，本发明所得抗体的KD值均在纳摩(nM)水平，热点突变的人源化抗体405B8H3-1(D→E)与对应的鼠-人嵌合抗体405B8H3的亲和力提高1.5倍(3.11/2.03＝1.5)，而同时将人源化抗体556F6B8-3进行热点突变(重链的第100位天冬酰胺D突变成谷氨酸E)，与对应的鼠-人嵌合抗体405B8H3相比，亲和力降低，降低为原来的1/12.5(33.5/2.68＝12.5)。
表32 热点突变抗LAG-3抗体亲和力的分析测定
克隆ID ka(1/Ms) kd(1/s) KD(M)
405B8H3 5.28E+04 1.64E-04 3.11E-09
405B8H3-1(D→E) 7.75E+04 1.58E-04 2.03E-09
556F6B8 1.62E+05 4.33E-04 2.68E-09
556F6B8-3(D→E) 1.15E+05 0.003843 3.35E-08
讨论
目前百时美施贵宝公司的LAG-3抗体BMS986016的临床研究主要用于恶性实体瘤的治疗，而且也主要集中于它和其他疗法或靶点药物的联合使用，开发适应症广的抗体从而扩大其适用的临床症状，包括不可切除的转移性黑色素瘤、晚期实体癌、乳腺癌、子宫内膜癌、卵巢癌、肾癌、胰腺癌、复发性胶质母细胞瘤、头颈癌、膀胱癌、转移性直结肠癌、胃肠道间质肿瘤、腺泡细胞癌、高级恶性固体肿瘤、非小细胞肺癌等。
抗体本身的活性受可变区序列和恒定区结构的影响。抗体的可变区序列决定了识别抗原的决定簇、结合亲和力及在体内代谢速率，都会影响其体内活性，甚至不同病患个人的临床效果。
本领域亟待开发产量更高的LAG-3抗体以降低患者的治疗成本，惠及更多患者。目前肿瘤免疫治疗价格昂贵，急需发明并且生产新的抗体以降低成本。
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。
本发明的序列信息：
LAG-3抗体的重链可变区(VH)、轻链可变区(VL)的氨基酸序列和编码所述重链可变区(VH)、轻链可变区(VL)的核苷酸序列，其中CDR1、CDR2、CDR3、CDR1'、CDR2'、CDR3'分别用下划线标示出来：
mAb103-405B8H3-VH SEQ ID No.1
Figure PCTCN2019081063-appb-000034
mAb103-405B8H3-VH SEQ ID No.49
Figure PCTCN2019081063-appb-000035
mAb103-405B8H3-VL SEQ ID No.5
Figure PCTCN2019081063-appb-000036
mAb103-405B8H3-VL SEQ ID No.50
Figure PCTCN2019081063-appb-000037
mAb123-556F6B8-VH SEQ ID No.9
Figure PCTCN2019081063-appb-000038
mAb123-556F6B8-VH SEQ ID No. 51
Figure PCTCN2019081063-appb-000039
mAb123-556F6B8-VL SEQ ID No. 13
Figure PCTCN2019081063-appb-000040
mAb123-556F6B8-VL SEQ ID No. 52
Figure PCTCN2019081063-appb-000041
mAb009-105F1E10-VH SEQ ID No. 17
Figure PCTCN2019081063-appb-000042
mAb009-105F1E10-VH SEQ ID No. 53
Figure PCTCN2019081063-appb-000043
Figure PCTCN2019081063-appb-000044
mAb009-105F1E10-VL SEQ ID No. 21
Figure PCTCN2019081063-appb-000045
mAb009-105F1E10-VL SEQ ID No. 54
Figure PCTCN2019081063-appb-000046
mAb104-409B11E12-VH SEQ ID No. 25
Figure PCTCN2019081063-appb-000047
mAb104-409B11E12-VH SEQ ID No. 55
Figure PCTCN2019081063-appb-000048
mAb104-409B11E12-VL SEQ ID No. 29
Figure PCTCN2019081063-appb-000049
mAb104-409B11E12-VL SEQ ID No. 56
Figure PCTCN2019081063-appb-000050
mAb112-409D4E10-VH SEQ ID No. 33
Figure PCTCN2019081063-appb-000051
mAb112-409D4E10-VH SEQ ID No. 57
Figure PCTCN2019081063-appb-000052
mAb112-409D4E10-VL SEQ ID No. 37
Figure PCTCN2019081063-appb-000053
mAb112-409D4E10-VL SEQ ID No. 58
Figure PCTCN2019081063-appb-000054
Figure PCTCN2019081063-appb-000055
mAb120-553G8G8-VH SEQ ID No. 41
Figure PCTCN2019081063-appb-000056
mAb120-553G8G8-VH SEQ ID No. 59
Figure PCTCN2019081063-appb-000057
mAb120-553G8G8-VL SEQ ID No. 45
Figure PCTCN2019081063-appb-000058
mAb120-553G8G8-VL SEQ ID No. 60
Figure PCTCN2019081063-appb-000059
405B8H3-1-VH SEQ ID No. 64
Figure PCTCN2019081063-appb-000060
405B8H3-1-VH SEQ ID No. 65
Figure PCTCN2019081063-appb-000061
405B8H3-1-VL SEQ ID No. 76
Figure PCTCN2019081063-appb-000062
405B8H3-1-VL SEQ ID No. 77
Figure PCTCN2019081063-appb-000063
405B8H3-2-VH SEQ ID No. 66
Figure PCTCN2019081063-appb-000064
405B8H3-2-VH SEQ ID No. 67
Figure PCTCN2019081063-appb-000065
Figure PCTCN2019081063-appb-000066
405B8H3-2-VL 氨基酸序列 SEQ ID No.76
405B8H3-2-VL 核苷酸序列 SEQ ID No.77
405B8H3-6-VH 氨基酸序列 SEQ ID No.66
405B8H3-6-VH 核苷酸序列 SEQ ID No.67
405B8H3-6-VL SEQ ID No.78
Figure PCTCN2019081063-appb-000067
405B8H3-6-VL SEQ ID No.79
Figure PCTCN2019081063-appb-000068
405B8H3-7-VH SEQ ID No.68
Figure PCTCN2019081063-appb-000069
405B8H3-7-VH SEQ ID No.69
Figure PCTCN2019081063-appb-000070
Figure PCTCN2019081063-appb-000071
405B8H3-7-VL 氨基酸序列 SEQ ID No.78
405B8H3-7-VL 核苷酸序列 SEQ ID No.79
405B8H3-1(D→E)-VH 氨基酸序列 SEQ ID No.64
405B8H3-1(D→E)-VH 核苷酸序列 SEQ ID No.65
405B8H3-1(D→E)-VL SEQ ID No.74
Figure PCTCN2019081063-appb-000072
405B8H3-1(D→E)-VL SEQ ID No.75
Figure PCTCN2019081063-appb-000073
556F6B8-3-VH SEQ ID No.72
Figure PCTCN2019081063-appb-000074
556F6B8-3-VH SEQ ID No.73
Figure PCTCN2019081063-appb-000075
Figure PCTCN2019081063-appb-000076
556F6B8-3-VL SEQ ID No.80
Figure PCTCN2019081063-appb-000077
556F6B8-3-VL SEQ ID No.81
Figure PCTCN2019081063-appb-000078
556F6B8-7-VH 氨基酸序列 SEQ ID No.72
556F6B8-7-VH 核苷酸序列 SEQ ID No.73
556F6B8-7-VL SEQ ID No.82
Figure PCTCN2019081063-appb-000079
556F6B8-7-VL SEQ ID No.83
Figure PCTCN2019081063-appb-000080
Figure PCTCN2019081063-appb-000081
556F6B8-3(D→E)-VH SEQ ID No.70
Figure PCTCN2019081063-appb-000082
556F6B8-3(D→E)-VH SEQ ID No.71
Figure PCTCN2019081063-appb-000083
556F6B8-3(D→E)-VL 氨基酸序列 SEQ ID No.80
556F6B8-3(D→E)-VL 核苷酸序列 SEQ ID No.81
表20 LAG3-抗体的CDR区序列及其氨基酸序列编号(SEQ ID No.)如下：
Figure PCTCN2019081063-appb-000084
Figure PCTCN2019081063-appb-000085
Figure PCTCN2019081063-appb-000086
其中VH为重链可变区，VL为轻链可变区。VH-CDR1、VH-CDR2、VH-CDR3分别为重链可变区CDR1、CDR2、CDR3；VL-CDR1、VL-CDR2、VL-CDR3分别为轻链可变区CDR1'、CDR2'、CDR3'。
表33 人源化VH或VL汇总表
人源化VH或VL 氨基酸的SEQ ID No. 核苷酸的SEQ ID No.
人源化VH1 64 65
人源化VH2 66 67
人源化VH3 68 69
人源化VH4 70 71
人源化VH5 72 73
人源化VL1 74 75
人源化VL2 76 77
人源化VL3 78 79
人源化VL4 80 81
人源化VL5 82 83
表34 抗体的VH和VL汇总表
  VH的SEQ ID No. VL的SEQ ID No.
405B8H3 1 5
556F6B8 9 13
105F1E10 17 21
409B11E12 25 29
409D4E10 33 37
553G8G8 41 45
405B8H3-1(D→E) 64 74
405B8H3-1 64 76
405B8H3-2 66 76
405B8H3-6 66 78
405B8H3-7 68 78
556F6B8-3(D→E) 70 80
556F6B8-3 72 80
556F6B8-7 72 82
人LAG-3 蛋白氨基酸序列 SEQ ID No.61
Figure PCTCN2019081063-appb-000087
猴LAG-3 蛋白氨基酸序列 SEQ ID No.62
Figure PCTCN2019081063-appb-000088
鼠LAG-3 蛋白氨基酸序列 SEQ ID No.63
Figure PCTCN2019081063-appb-000089
Figure PCTCN2019081063-appb-000090

Claims (18)

  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或SEQ ID NO.85所示的CDR3；
    其中，各n独立地为0、1、2、3、4或5；
    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LAG-3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或SEQ ID NO.84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、4或5；
    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LAG-3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LAG-3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；
    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或SEQ ID NO.84所示的CDR2'，和
    SEQ ID NO:8所示的CDR3'；
    或
    所述的重链可变区包括以下三个互补决定区CDR：
    SEQ ID NO:10所示的CDR1，
    SEQ ID NO:11所示的CDR2，和
    SEQ ID NO:12所示的CDR3；
    所述的轻链可变区包括以下三个互补决定区CDR：
    SEQ ID NO:14所示的CDR1'，
    SEQ ID NO:15所示的CDR2'，和
    SEQ ID NO:16所示的CDR3'；
    或
    所述的重链可变区包括以下三个互补决定区CDR：
    SEQ ID NO:18所示的CDR1，
    SEQ ID NO:19所示的CDR2，和
    SEQ ID NO:20所示的CDR3；
    所述的轻链可变区包括以下三个互补决定区CDR：
    SEQ ID NO:22所示的CDR1'，
    SEQ ID NO:23所示的CDR2'，和
    SEQ ID NO:24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LAG-3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、SEQ ID NO.64、SEQ ID NO.66、SEQ ID NO.68、SEQ ID NO.70、SEQ ID NO.72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、SEQ ID NO.74、SEQ ID NO.76、SEQ ID NO.78、SEQ ID NO.80、SEQ ID NO.82所示的氨基酸序列，其中，各n独立地为0、1、2、3、4或5。
  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；或
    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；或
    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；或
    所述抗体的重链可变区含有SEQ ID NO:64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:74所示的氨基酸序列；或
    所述抗体的重链可变区含有SEQ ID NO:64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:76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:49、51、53、55、57、59、65、67、69、71或73所示；和/或，
    编码所述轻链可变区的多核苷酸如50、52、54、56、58、60、75、77、79、81或83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:49所示；并且编码所述轻链可变区序列的多核苷酸如50所示；或
    编码所述重链可变区的多核苷酸如SEQ ID NO:51所示；并且编码所述轻链可变区的多核苷酸如52所示；或
    编码所述重链可变区的多核苷酸如SEQ ID NO:53所示；并且编码所述轻链可变区的多核苷酸如54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LAG-3相关疾病的药物。
  17. 一种体外检测样品中LAG-3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。
  18. 一种重组多肽的制备方法，其特征在于，该方法包括：
    (a)在适合表达的条件下，培养如权利要求14所述的宿主细胞；
    (b)从培养物中分离出重组多肽，所述的重组多肽是如权利要求5-8中任一项 所述的抗体或如权利要求9所述的重组蛋白。
PCT/CN2019/081063 2018-04-03 2019-04-02 抗人lag-3单克隆抗体及其应用 WO2019192493A1 (zh)

Priority Applications (3)

Application Number Priority Date Filing Date Title
EP19781983.2A EP3778632A4 (en) 2018-04-03 2019-04-02 MONOCLONAL ANTI-HUMAN LAG-3 ANTIBODIES AND USES THEREOF
US17/045,166 US11685778B2 (en) 2018-04-03 2019-04-02 Anti-human LAG-3 monoclonal antibody and use thereof
JP2020554402A JP2021526013A (ja) 2018-04-03 2019-04-02 抗ヒトｌａｇ−３モノクローナル抗体とその応用

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201810290569.4A CN110343178B (zh) 2018-04-03 2018-04-03 抗人lag-3单克隆抗体及其应用
CN201810290569.4 2018-04-03

Publications (1)

Publication Number Publication Date
WO2019192493A1 true WO2019192493A1 (zh) 2019-10-10

Family

ID=68099836

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2019/081063 WO2019192493A1 (zh) 2018-04-03 2019-04-02 抗人lag-3单克隆抗体及其应用

Country Status (6)

Country Link
US (1) US11685778B2 (zh)
EP (1) EP3778632A4 (zh)
JP (1) JP2021526013A (zh)
CN (1) CN110343178B (zh)
TW (1) TW202003854A (zh)
WO (1) WO2019192493A1 (zh)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113348182B (zh) * 2019-12-30 2022-07-12 上海海路生物技术有限公司 Lag-3抗体及其医药用途
CN114621344B (zh) * 2020-12-10 2022-08-30 北京东方百泰生物科技股份有限公司 一种抗lag-3单克隆抗体的纯化方法

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4816567A (en) 1983-04-08 1989-03-28 Genentech, Inc. Recombinant immunoglobin preparations
US4816397A (en) 1983-03-25 1989-03-28 Celltech, Limited Multichain polypeptides or proteins and processes for their production
US5585089A (en) 1988-12-28 1996-12-17 Protein Design Labs, Inc. Humanized immunoglobulins
CN102176921A (zh) 2008-08-11 2011-09-07 梅达雷克斯股份有限公司 结合淋巴细胞活化基因3(lag-3)之人类抗体及其用途
WO2017037203A1 (en) * 2015-09-02 2017-03-09 Immutep S.A.S. Anti-LAG-3 Antibodies
WO2017149143A1 (en) * 2016-03-04 2017-09-08 Agency For Science, Technology And Research Anti-lag-3 antibodies

Family Cites Families (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1987839A1 (en) * 2007-04-30 2008-11-05 I.N.S.E.R.M. Institut National de la Sante et de la Recherche Medicale Cytotoxic anti-LAG-3 monoclonal antibody and its use in the treatment or prevention of organ transplant rejection and autoimmune disease
UY34887A (es) * 2012-07-02 2013-12-31 Bristol Myers Squibb Company Una Corporacion Del Estado De Delaware Optimización de anticuerpos que se fijan al gen de activación de linfocitos 3 (lag-3) y sus usos
CR20160425A (es) * 2014-03-14 2017-05-26 Novartis Ag Moléculas de anticuerpos que se unen a lag-3 y usos de las mismas
MA41463A (fr) * 2015-02-03 2017-12-12 Anaptysbio Inc Anticorps dirigés contre le gène d'activation 3 des lymphocytes (lag-3)
WO2017049143A1 (en) * 2015-09-18 2017-03-23 Dana-Farber Cancer Institute, Inc. Methods of reducing liver pd-1-expressing cd8+t cells using pd-1 fc fusion proteins that bind fc receptors
US11214620B2 (en) * 2016-06-20 2022-01-04 F-Star Therapeutics Limited Binding molecules binding PD-L1 and LAG-3
UA127048C2 (uk) * 2016-06-23 2023-03-29 Джянгсу Хенгруй Медісін Ко., Лтд. Lag-3 антитіло, його антигензв'язуючий фрагмент та його фармацевтичне застосування

Patent Citations (7)

* 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
CN102176921A (zh) 2008-08-11 2011-09-07 梅达雷克斯股份有限公司 结合淋巴细胞活化基因3(lag-3)之人类抗体及其用途
CN107474137A (zh) * 2008-08-11 2017-12-15 施贵宝有限责任公司 结合淋巴细胞活化基因3（lag‑3）之人类抗体及其用途
WO2017037203A1 (en) * 2015-09-02 2017-03-09 Immutep S.A.S. Anti-LAG-3 Antibodies
WO2017149143A1 (en) * 2016-03-04 2017-09-08 Agency For Science, Technology And Research Anti-lag-3 antibodies

Non-Patent Citations (13)

* Cited by examiner, † Cited by third party
Title
"Biocomputing: Informatics and Genome Projects", 1993, ACADEMIC PRESS
"Computer Analysis of Sequence Data", 1994, HUMANA PRESS
CARILLO, H.LIPMAN, D., SIAM J. APPLIED MATH., vol. 48, 1988, pages 1073
DAVID H ET AL., METHODS MOL BIOL, vol. 960, 2013, pages 297 - 307
KABAT ET AL., NIH PUBL. NO. 91-3242, 1991, pages 647 - 669
KABAT: "Sequences of Proteins of Immunological Interest", 1991, NATIONAL INSTITUTES OF HEALTH
METHODS IN ENZYMOLOGY, vol. 220
MUNSON ET AL., ANAL. BIOCHEM., vol. 107, 1980, pages 220
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 EP3778632A4
VON HEINJE, G.: "Sequence Analysis in Molecular Biology", 1987, ACADEMIC PRESS
WORKMAN ET AL., IMMUNOL, vol. 174, 2005, pages 688 - 695

Also Published As

Publication number Publication date
EP3778632A1 (en) 2021-02-17
EP3778632A4 (en) 2021-10-27
US11685778B2 (en) 2023-06-27
TW202003854A (zh) 2020-01-16
CN110343178B (zh) 2022-07-22
JP2021526013A (ja) 2021-09-30
CN110343178A (zh) 2019-10-18
US20210155689A1 (en) 2021-05-27

Similar Documents

Publication Publication Date Title
WO2019174603A1 (zh) 靶向ctla-4抗体、其制备方法和用途
TWI701259B (zh) ４﹘１ｂｂ抗體及其製備方法和應用
WO2021170082A1 (zh) 抗cd47/抗pd-l1抗体及其应用
CN113348180A (zh) Ox40抗体及其制备方法和应用
WO2020199860A1 (zh) 针对程序性死亡配体的结合物及其应用
WO2022171080A1 (zh) 抗cd112r抗体及其用途
WO2020108660A1 (zh) 抗人tim-3单克隆抗体及其应用
TW202246341A (zh) 針對NKp46的抗體及其應用
WO2019192493A1 (zh) 抗人lag-3单克隆抗体及其应用
WO2019238074A1 (zh) 一种高亲和力高生物活性的lag-3抗体及其应用
WO2020108636A1 (zh) 全人抗gitr抗体及其制备方法
KR20240004860A (ko) 디엘엘3에 대한 결합 분자 및 이의 용도
CN110563848A (zh) 一种双特异性抗体及其用途
TWI835166B (zh) 靶向pd-1和/或ox40的特異性結合蛋白及其應用
EP4410834A1 (en) Ctla-4 binding molecule and use thereof
WO2022068891A1 (zh) Pd-1抗体及其制备方法与应用
CN115947855B (zh) 抗cd24抗体的制备及其用途
WO2023236991A1 (zh) 靶向her2，pd-l1和vegf的三特异性抗体
WO2024078558A1 (zh) 抗cd100抗体及其用途
WO2023186113A1 (zh) 靶向pd-l1和cd40的抗原结合蛋白及其制备和应用
JP2024509369A (ja) 抗ｐｄ－ｌ１抗体及びその使用
TW202235436A (zh) Ｓｉｇｌｅｃ－１５結合蛋白的製備及其用途
IL304096A (en) PD-1 binding molecule and its application
TW202246340A (zh) 抗ctla-4抗體及其應用
CN117186235A (zh) 抗4-1BB/抗EGFRvⅢ的双特异抗体及其制备方法和应用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 19781983

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 2020554402

Country of ref document: JP

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 2019781983

Country of ref document: EP

Effective date: 20201103