WO2020094071A1 - Nkg2a抗体及其制备方法和应用 - Google Patents

Nkg2a抗体及其制备方法和应用 Download PDF

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Publication number
WO2020094071A1
WO2020094071A1 PCT/CN2019/116060 CN2019116060W WO2020094071A1 WO 2020094071 A1 WO2020094071 A1 WO 2020094071A1 CN 2019116060 W CN2019116060 W CN 2019116060W WO 2020094071 A1 WO2020094071 A1 WO 2020094071A1
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Prior art keywords
antibody
variable region
chain variable
light chain
heavy chain
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PCT/CN2019/116060
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English (en)
French (fr)
Inventor
段清
刘礼乐
杨达志
高婧
胡莉莉
随瑞瑞
王冬旭
韩烨
谢荣荣
陆妍
邵小慧
张洁
周文明
郭翠翠
童国珍
王荔娜
戴朝晖
王梦莹
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上海怀越生物科技有限公司
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Application filed by 上海怀越生物科技有限公司 filed Critical 上海怀越生物科技有限公司
Priority to US17/292,379 priority Critical patent/US20220259306A1/en
Priority to EP19881466.7A priority patent/EP3878869A4/en
Priority to JP2021524469A priority patent/JP2022512954A/ja
Publication of WO2020094071A1 publication Critical patent/WO2020094071A1/zh

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    • 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4613—Natural-killer cells [NK or NK-T]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464402—Receptors, cell surface antigens or cell surface determinants
    • A61K39/464429—Molecules with a "CD" designation not provided for elsewhere
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A—HUMAN NECESSITIES
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    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6849—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
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    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the cancer treated
    • A61K2239/48—Blood cells, e.g. leukemia or lymphoma
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    • 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
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
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    • 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
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    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • C07K2317/00—Immunoglobulins specific features
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    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
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    • 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
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    • C12N2510/00—Genetically modified cells

Definitions

  • the present invention belongs to the field of antibodies, and in particular relates to an NKG2A antibody and its preparation method and application.
  • NK cells Natural killer (NK) cells are a subpopulation of lymphocytes involved in non-traditional immunity. NK cells provide an effective immune surveillance mechanism, thereby eliminating undesirable cells such as tumor cells or virus-infected cells. NK cell activity is regulated by a complex mechanism containing two activation and inhibition signals.
  • NKp30, NKp46, and NKp44 are members of the Ig superfamily. Their cross-linking (induced by specific mAb) leads to strong NK cell activation, resulting in increased intracellular Ca ++ levels, triggering cytotoxicity and lymphokine release. Importantly, monoclonal antibody-mediated activation of NKp30, NKp46, and / or NKp44 leads to activation of NK cytotoxicity against a variety of target cells.
  • CD94-NKG2A another important inhibitory receptor on NK cells, interacts with the atypical MHC class I molecule HLA-E. Some of these receptors have the ability to regulate the threshold of T cell antigen receptor-dependent T cell activation. In the absence of rare inhibitory receptors, these activation isoforms may expand the function of T cell effectors and contribute to autoimmune pathology.
  • the amino acid sequence of NKG2A changes in mammals (including primates). For example, the human NKG2A protein has less than 90% homology to cynomolgus monkeys.
  • Efforts for therapies for modulating NKG2A have been focused on the study of atypical MHC class I molecules, HLA-E for human receptors and Qa-Ib for mouse receptors.
  • these MHC molecules preferably bind to peptides derived from signal peptides of other MHC class I molecules.
  • the expression of other Class I MHC molecules can regulate the expression of HLA-E, thus allowing NK cells to monitor the status of MHC Class I-dependent antigen presentation pathways in potential target cells.
  • the level of HLA-E on the cell surface is critical for NK cytotoxicity against tumor and virus-infected cells.
  • Therapeutic regimens used to modulate HLA-E expression or function generally focus on the use of HLA-I or HSP60 peptides to induce a protective state for preventing inflammation so that NK cells are not activated.
  • the invention discloses an NKG2A antibody, its preparation method and application.
  • the NKG2A antibody includes one or more of heavy chain CDR1 (VH-CDR1), heavy chain CDR2 (VH-CDR2) and heavy chain CDR3 (VH-CDR3) of NKG2A antibody, and NKG2A antibody
  • VH-CDR1 heavy chain CDR1
  • VL-CDR2 heavy chain CDR2
  • VL-CDR3 VL-CDR3
  • a heavy chain variable region of an antibody having a complementarity determining region CDR selected from the group consisting of:
  • VH-CDR1 shown in SEQ ID NO. 8n + 2
  • VH-CDR2 shown in SEQ ID NO. 8n + 3, and
  • VH-CDR3 shown in SEQ ID NO.8n + 4;
  • n is independently 0, 1, 2, 3, 4, 5, 6, 7 or 8;
  • any one of the above-mentioned amino acid sequences also includes a derivative sequence that is optionally added, deleted, modified, and / or substituted for at least one amino acid, and can retain the binding affinity of NKG2A.
  • the heavy chain variable region has the amino acid sequence shown in SEQ ID NO. 8n + 1, where n is 0, 1, 2, 3, 4, 5, 6, 7 or 8.
  • the heavy chain variable region has the amino acid sequence shown in SEQ ID NO. 1.
  • the heavy chain variable region has the amino acid sequence shown in SEQ ID NO. 9.
  • the heavy chain variable region has the amino acid sequence shown in SEQ ID NO.17.
  • the heavy chain variable region has the amino acid sequence shown in SEQ ID NO.25.
  • a heavy chain of an antibody having a heavy chain variable region as described in the first aspect of the present invention.
  • the heavy chain further includes a heavy chain constant region.
  • the heavy chain constant region is of human or murine origin.
  • a light chain variable region of an antibody having a complementarity determining region CDR selected from the group consisting of:
  • VL-CDR1 shown in SEQ ID NO. 8n + 6,
  • VL-CDR2 shown in SEQ ID NO. 8n + 7, and
  • VL-CDR3 shown in SEQ ID NO. 8n + 8;
  • n is independently 0, 1, 2, 3, 4, 5, 6, 7 or 8;
  • any one of the above-mentioned amino acid sequences also includes a derivative sequence that is optionally added, deleted, modified, and / or substituted for at least one amino acid, and capable of retaining the binding affinity of NKG2A.
  • the light chain variable region has the amino acid sequence shown in SEQ ID NO. 8n + 5, where n is 0, 1, 2, 3, 4, 5, 6, 7 or 8.
  • the light chain variable region has the amino acid sequence shown in SEQ ID NO.
  • the light chain variable region has the amino acid sequence shown in SEQ ID NO.13.
  • the light chain variable region has the amino acid sequence shown in SEQ ID NO.21.
  • the light chain variable region has the amino acid sequence shown in SEQ ID NO.29.
  • a light chain of an antibody having a light chain variable region as described in the third aspect of the present invention.
  • the light chain further includes a light chain constant region.
  • the light chain constant region is of human or murine origin.
  • 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-mentioned amino acid sequences also includes a derivative sequence that is optionally added, deleted, modified, and / or substituted for at least one amino acid, and capable of retaining the binding affinity of NKG2A.
  • the amino acid sequence of any of the above CDRs includes derived, deleted, modified, and / or substituted 1, 2 or 3 amino acid derived CDR sequences, and the VH and VL containing the derived CDR sequences are included
  • the derived antibody constructed can retain the affinity of binding to NKG2A.
  • the ratio (F1 / F0) of the affinity F1 of the derived antibody to NKG2A to the affinity of the corresponding non-derivatized antibody to NKG2A is 0.5-2, preferably 0.7-1.5, And better still 0.8-1.2.
  • the number of added, deleted, modified and / or substituted amino acids is 1-5 (such as 1-3, preferably 1-2, more preferably 1).
  • the derived sequence that has been added, deleted, modified, and / or substituted for at least one amino acid and can retain the binding affinity of NKG2A is an amino acid sequence with homology or sequence identity of at least 96%.
  • the antibody further includes a heavy chain constant region and / or a light chain constant region.
  • the heavy chain constant region is of human origin, and / or the light chain constant region is of human origin.
  • the heavy chain variable region of the antibody further includes a framework region of human origin, and / or the light chain variable region of the antibody further includes a framework region of human origin.
  • the heavy chain variable region of the antibody further includes a murine framework region, and / or the light chain variable region of the antibody further includes a murine framework region.
  • the antibody is selected from the group consisting of animal-derived antibodies, chimeric antibodies, humanized antibodies, fully human antibodies, or a combination thereof.
  • the ratio of the immunogenicity Z1 of the chimeric antibody in humans to the immunogenicity Z0 of non-chimeric antibodies (such as murine antibodies) in humans 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 double-chain antibody 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 is in the form of a drug conjugate.
  • 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 present invention and a light chain variable region according to the third aspect of the present invention;
  • heavy chain variable region and the light chain variable region include CDRs selected from the group consisting of:
  • any one of the above-mentioned amino acid sequences also includes a derivative sequence that is optionally added, deleted, modified, and / or substituted for at least one amino acid, and capable of retaining the binding affinity of NKG2A.
  • the antibody has a heavy chain variable region according to the first aspect of the present invention and a light chain variable region according to the third aspect of the present invention; wherein,
  • the heavy chain variable region includes the following three complementarity determining regions CDR:
  • VH-CDR1 shown in SEQ ID NO.2,
  • VH-CDR2 shown in SEQ ID NO. 3, and
  • VH-CDR3 shown in SEQ ID NO.4;
  • the light chain variable region includes the following three complementarity determining regions CDR:
  • VL-CDR1 shown in SEQ ID NO.6,
  • VL-CDR2 shown in SEQ ID NO.7, and
  • VL-CDR3 shown in SEQ ID NO.8;
  • the heavy chain variable region includes the following three complementarity determining regions CDR:
  • VH-CDR2 shown in SEQ ID NO.11, and
  • VH-CDR3 shown in SEQ ID NO.12;
  • the light chain variable region includes the following three complementarity determining regions CDR:
  • VL-CDR2 shown in SEQ ID NO.15, and
  • VL-CDR3 shown in SEQ ID NO.16;
  • the heavy chain variable region includes the following three complementarity determining regions CDR:
  • VH-CDR2 shown in SEQ ID NO.19, and
  • VH-CDR3 shown in SEQ ID NO.20;
  • the light chain variable region includes the following three complementarity determining regions CDR:
  • VL-CDR2 shown in SEQ ID NO.23, and
  • VL-CDR3 shown in SEQ ID NO.24;
  • the heavy chain variable region includes the following three complementarity determining regions CDR:
  • VH-CDR2 shown in SEQ ID NO.27, and
  • VH-CDR3 shown in SEQ ID NO.28;
  • the light chain variable region includes the following three complementarity determining regions CDR:
  • VL-CDR2 shown in SEQ ID NO.31, and
  • VL-CDR3 shown in SEQ ID NO.32.
  • the heavy chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO. 1, 9, 17, 25, 33, 41, 49, 57 or 65; and / or the antibody
  • the light chain variable region contains the amino acid sequence shown in SEQ ID NO. 5, 13, 21, 29, 37, 45, 53, 61 or 69.
  • the heavy chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO.1; and the light chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO.5.
  • the antibody heavy chain variable region contains the amino acid sequence shown in SEQ ID NO. 9; and the antibody light chain variable region contains the amino acid sequence shown in SEQ ID NO. 13.
  • the heavy chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO.17; and the light chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO.21.
  • the antibody heavy chain variable region contains the amino acid sequence shown in SEQ ID NO. 25; and the antibody light chain variable region contains the amino acid sequence shown in SEQ ID NO. 29.
  • the antibody is selected from the group consisting of:
  • amino acid sequence of the variable region of the heavy chain is at least as 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology or sequence identity.
  • the amino acid sequence of the light chain variable region has at least the amino acid sequence shown in SEQ ID NO. 5, 13, 21, 29, 37, 45, 53, 61 or 69 of the sequence listing 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology or sequence identity.
  • a recombinant protein in a sixth aspect of the present invention, includes:
  • the tag sequence includes a 6His tag.
  • the recombinant protein includes a fusion protein.
  • the recombinant protein is a monomer, dimer, or polymer.
  • the recombinant protein includes:
  • polynucleotide encoding the heavy chain variable region is shown in SEQ ID NO. 73, 75, 77, 79, 81, 83, 85, 87, or 89; and / or The polynucleotide of the light chain variable region is shown in SEQ ID NO. 74, 76, 78, 80, 82, 84, 86, 88 or 90.
  • polynucleotide encoding the heavy chain variable region sequence and the polynucleotide encoding the light chain variable region sequence are selected from the group consisting of:
  • the vector includes: bacterial plasmid, bacteriophage, yeast plasmid, plant cell virus, mammalian cell virus such as adenovirus, retrovirus, or other vectors.
  • a genetically engineered host cell containing the vector or genome of the eighth aspect of the present invention incorporating the polynucleoside of the seventh aspect of the present invention acid.
  • an antibody conjugate comprising:
  • a coupling portion coupled to the antibody portion is selected from the group consisting of a detectable label, a drug, a toxin, a cytokine, a radionuclide, an enzyme, or a combination thereof.
  • the antibody part and the coupling part are coupled by a chemical bond or a linker.
  • an immune cell which expresses or is exposed to the cell membrane the antibody of the fifth aspect of the present invention.
  • the immune cells include NK cells and T cells.
  • the immune cells are derived from human or non-human mammals (such as mice).
  • a pharmaceutical composition comprising:
  • an active ingredient selected from the group consisting of the heavy chain variable region according to the first aspect of the invention, the heavy chain according to the second aspect of the invention, and the third according to the invention
  • the pharmaceutical composition is a liquid preparation.
  • the pharmaceutical composition is an injection.
  • the pharmaceutical composition includes 0.01-99.99% of the antibody according to the fifth aspect of the invention, the recombinant protein according to the sixth aspect of the invention, and the tenth according to the invention
  • the antibody conjugate, the immune cell according to the eleventh aspect of the present invention, or a combination thereof and a pharmaceutical carrier of 0.01 to 99.99% the percentage is a mass percentage of the pharmaceutical composition.
  • a use of an active ingredient selected from the group consisting of a heavy chain variable region according to the first aspect of the present invention, and a second aspect of the present invention The heavy chain, the light chain variable region according to the third aspect of the present invention, the light chain according to the fourth aspect of the present invention, or the antibody according to the fifth aspect of the present invention, such as the present
  • preparation of diagnostic reagents or kits and / or (b) preparation of drugs for preventing and / or treating diseases related to abnormal expression or function of NKG2A.
  • the diagnostic reagent is a test piece or a test plate.
  • the diseases related to abnormal expression or function of NKG2A are selected from the group consisting of cancer, autoimmune diseases, and inflammatory diseases.
  • the diagnostic reagent or kit is used for:
  • the medicine is used to prevent and / or treat diseases related to abnormal expression or function of NKG2A.
  • diseases related to abnormal expression or function of NKG2A are cancer, autoimmune diseases, and inflammatory diseases.
  • the cancer is selected from the group consisting of lymphocytic leukemia, ovarian cancer, non-small cell lung cancer, and head and neck cancer.
  • the cancer is HLA-E overexpressed cancer.
  • the cancer is HLA-E overexpressing cancer, and the cancer is selected from the group consisting of lymphocytic leukemia, ovarian cancer, non-small cell lung cancer, and head and neck cancer.
  • the antibody is in the form of a drug conjugate (ADC).
  • ADC drug conjugate
  • the diagnostic reagent or kit is used to diagnose NKG2A-related diseases.
  • the diagnostic reagent or kit is used to detect NKG2A protein in the sample.
  • a fourteenth aspect of the present invention there is provided a method for in vitro detection (including diagnostic or non-diagnostic) of NKG2A protein in a sample, the method comprising the steps of:
  • composition for in vitro detection of NKG2A protein in a sample which comprises the antibody according to the fifth aspect of the invention, the recombinant protein according to the sixth aspect of the invention.
  • the antibody conjugate according to the tenth aspect of the present invention, the immune cell according to the eleventh aspect of the present invention, or a combination thereof is used as an active ingredient.
  • a detection board comprising: a substrate (supporting plate) and a test strip, the test strip contains the antibody according to the fifth aspect of the invention 2.
  • kit includes:
  • a first container containing the antibody of the present invention containing the antibody of the present invention.
  • the kit contains the detection plate according to the sixteenth aspect of the present invention.
  • a method for preparing a recombinant polypeptide comprising:
  • the recombinant polypeptide is isolated from the culture, and the recombinant polypeptide is the antibody according to the fifth aspect of the present invention or the recombinant protein according to the sixth aspect of the present invention.
  • a pharmaceutical combination including:
  • a first active ingredient comprising the antibody 1 according to the fifth aspect of the invention, or the recombinant protein according to the sixth aspect of the invention, or the tenth aspect according to the invention
  • a second active ingredient which includes a second antibody or a chemotherapeutic agent.
  • the second antibody is selected from the group consisting of CTLA4 antibody and PD-1 antibody.
  • the second antibody is PD-1 antibody.
  • the chemotherapeutic agent is selected from the group consisting of docetaxel, carboplatin, or a combination thereof.
  • the antibody according to the fifth aspect of the invention, or the recombinant protein according to the sixth aspect of the invention, or the antibody conjugate according to the tenth aspect of the invention Or the combination of the immune cell according to the eleventh aspect of the present invention, and / or the pharmaceutical composition according to the twelfth aspect of the present invention and the second antibody or chemotherapeutic agent is related to the preparation for the treatment of abnormal NKG2A expression or function Use of medicines for diseases.
  • the second antibody is selected from the group consisting of CTLA4 antibody and PD-1 antibody.
  • the second antibody is PD-1 antibody.
  • a method for treating a disease related to abnormal expression or function of NKG2A an effective amount of the antibody according to the fifth aspect of the present invention is administered to a subject in need thereof, or
  • the recombinant protein according to the sixth aspect of the invention, or the antibody conjugate according to the tenth aspect of the invention, or the immune cell according to the eleventh aspect of the invention, or the tenth according to the invention The pharmaceutical composition according to the second aspect, or a combination thereof.
  • the diseases related to abnormal expression or function of NKG2A are cancer, autoimmune diseases, and inflammatory diseases.
  • the cancer is selected from the group consisting of lymphocytic leukemia, ovarian cancer, non-small cell lung cancer, and head and neck cancer.
  • the cancer is HLA-E overexpressed cancer.
  • the cancer is HLA-E overexpressing cancer, and the cancer is selected from the group consisting of lymphocytic leukemia, ovarian cancer, non-small cell lung cancer, and head and neck cancer.
  • the method further includes: administering a safe and effective amount of the second antibody to the subject before, during and / or after administration of the first active ingredient.
  • the second antibody is selected from the group consisting of PD-1 antibody and CTLA4 antibody.
  • the second antibody is PD-1 antibody.
  • Figure 1 shows the binding activity of the control antibody to NKG2A / CD94 protein.
  • Figure 2a shows the serum antibody titer of Balb / c mice after immunization by ELISA.
  • Figure 2b shows the serum antibody titer of Balb / c mice after FACS detection.
  • Figure 3a is the serum antibody titer of SJL mice after immunization detected by ELISA.
  • Figure 3b is the FACS detection of serum antibody titers of SJL mice after immunization.
  • Figure 4 shows the technical process of animal immunization.
  • Figure 5a shows the serum antibody titer of Balb / c mice after immunization by ELISA.
  • Figure 5b shows the serum antibody titer of SJL mice after immunization by ELISA.
  • Fig. 6a is the serum antibody titer of Balb / c mice after FACS detection immunization, where cyno refers to cynomolgus monkey.
  • Figure 6b is the FACS detection of serum antibody titers of SJL mice after immunization.
  • Figure 7 is the technical process of animal immunization.
  • Figure 8a shows the serum antibody titer of Balb / c mice after immunization by ELISA.
  • Figure 8b shows the serum antibody titer of SJL mice after immunization by ELISA.
  • Figure 9a is the serum antibody titer of Balb / c mice after FACS detection.
  • Fig. 9b shows the serum antibody titer of SJL mice after FACS detection.
  • Figure 10 shows FACS detection of the binding reaction of NKG2A antibody with CHOK1-hNKG2A / CD94.
  • Figure 11 shows FACS detection of the binding reaction between NKG2A antibody and CHOK1-cNKG2A / CD94.
  • Figure 12 shows FACS detection of the binding reaction of NKG2A antibody with CHOK1-hNKG2C / CD94.
  • Fig. 13 shows the binding reaction of NKG2A antibody and NKG22E by ELISA.
  • Figure 14 shows the blocking activity of FACS detection antibodies.
  • Figure 15 shows the activity detection of NKG2A antibody in NK92 killing experiment (antibodies enhance the killing activity of NK92 cells).
  • Figure 16 shows the activity of NKG2A antibody in primary NK cell killing assay (PBMC donor-1).
  • the antibody enhances the killing activity of primary NK cells, donor 1.
  • Figure 17 is the activity detection of NKG2A antibody in primary NK cell killing experiment (PBMC donor-2).
  • the antibody enhances the killing activity of primary NK cells, donor 2.
  • Figure 18a is the FACS detection of the binding reaction of humanized NKG2A antibody with CHOK1-hNKG2A / CD94.
  • Fig. 18b shows the binding reaction between humanized NKG2A antibody and CHOK1-hNKG2A / CD94 by FACS.
  • Figure 19a shows FACS detection of the binding reaction of humanized NKG2A antibody with CHOK1-hNKG2C / CD94.
  • Figure 19b shows FACS detection of the binding reaction between humanized NKG2A antibody and CHOK1-hNKG2C / CD94.
  • Figure 20a shows FACS detection of the binding reaction of humanized NKG2A antibody with CHOK1-cNKG2A / CD94.
  • Fig. 20b shows the binding reaction between humanized NKG2A antibody and CHOK1-cNKG2A / CD94 by FACS.
  • Figure 21 shows the blocking activity of humanized NKG2A antibody by FACS, blocking the binding of HLA-E to NKG2A / CD94 cells.
  • Figure 22 Humanized NKG2A antibody enhances the killing activity of NK92.
  • the NKG2A antibody of the present invention can bind to human NKG2A and has high affinity (KD reaches 1.04 ⁇ 10 -11 ) (the affinity of the antibody obtained by the present invention is 18 times higher than that of the positive reference antibody); and the NKG2A antibody can bind The extracellular region of NKG2A protein receptor, and can effectively block NKG2A protein at the cellular level, preventing the binding of NKG2A protein to ligand HLA-E; the NKG2A antibody can significantly increase the killing effect of NK92 or human NK cells on target cells; Based on the primary NK cell activity test, its biological activity is significantly better than the reference antibody.
  • the NKG2A antibody of the present invention has high specificity and lacks cross-reactivity with human NKG2C and NKG2E homologous protein antigens.
  • the NKG2A antibody of the present invention can be used to block the negative regulation of NKG2A / HLA-E-mediated signaling pathways, activate the body's natural immune response to tumors, alone or in combination with anti-PD-1 monoclonal antibodies or other anti-tumor Drugs in cancer immunotherapy.
  • the antibody of the present invention can be used in the preparation of drugs for treating tumors, autoimmune diseases and the like. On this basis, the present invention has been completed.
  • Natural killer cells are a very important type of lymphocyte in the body, and play an important role in both innate immunity and acquired immunity.
  • NK cells There are two types of surface receptors on the surface of NK cells. According to their functions, they can be divided into two types: inhibitory and activated receptors, which mediate different recognition patterns of NK cells and transmit different activation and inhibition signals.
  • the CD94 / NKG2 family is a type of receptor family that has been studied a lot, mainly including NKG2A, NKG2B, NKG2C, NKG2D, NKG2E, NKG2F, NKG2H and other members.
  • NKG2A is an inhibitory receptor, and its ligand is a nonclassical major histocompatibility complex class I molecule-HLA-E.
  • the HLA-E molecule expressed on the target cells binds to NKG2A, which has an inhibitory effect on the killing function of NK cells. Therefore, antibodies that inhibit CD94 / NKG2A may increase the killing activity of tumor-specific lymphocytes on tumor cells.
  • certain lymphomas, such as NK-lymphoma are characterized by CD94 / NKG2A expression. In such patients, therapeutic antibodies that target and kill CD94 / NKG2A-expressing cells may be able to eradicate tumor cells.
  • antibody or "immunoglobulin” is a heterotetrameric glycoprotein of about 150,000 Daltons with the same structural characteristics, which is composed of two identical light chains (L) and two identical heavy chains (H) Composition. Each light chain is connected to the heavy chain through a covalent disulfide bond, and the number of disulfide bonds between heavy chains of different immunoglobulin isotypes is different. Each heavy and light chain also has regularly spaced disulfide bonds in the chain. Each heavy chain has a variable region (VH) at one end, followed by multiple 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 to the first constant region of the heavy chain, and the variable region of the light chain is opposite to the variable region of the heavy chain .
  • Special amino acid residues form an interface between the variable regions of the light chain and the heavy chain.
  • variable means that certain parts of the variable region of an antibody differ in sequence, which forms the binding and specificity of various specific antibodies for their specific antigens. However, the variability is not evenly distributed throughout the variable region of the antibody. It is concentrated in three segments called the complementarity determining region (CDR) or hypervariable region in the light chain and heavy chain variable regions. The more conserved part of the variable region is called the framework region (FR).
  • CDR complementarity determining region
  • FR framework region
  • the variable regions of the natural heavy and light chains each contain four FR regions, which are roughly in a ⁇ -sheet configuration, connected by three CDRs that form a connecting loop, and in some cases may form part of the ⁇ -sheet structure.
  • the CDRs in each chain are closely together through 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, Volume I, pages 647-669 (1991)).
  • the constant regions are not directly involved in the binding of antibodies to antigens, but they exhibit different effector functions, such as antibody-dependent cytotoxicity involved in antibodies.
  • immunoglobulins can be classified into one of two distinct classes (called kappa and lambda) based on the amino acid sequence of its constant region. According to the amino acid sequence of the constant region of their heavy chains, immunoglobulins can be divided into different types. There are five main types 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 divide this segment into 4 framework regions (FR)
  • FR framework regions
  • the amino acid sequence of FR is relatively conservative and does not directly participate in the binding reaction. These CDRs form a circular structure, and the ⁇ sheets formed by the FRs in between are close to each other in space structure, 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 sequences of antibodies of the same type can be compared to determine which amino acids constitute the FR or CDR regions.
  • the present invention includes not only whole antibodies, but also fragments of antibodies with immunological activity or fusion proteins formed by antibodies and other sequences. Therefore, the present 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 parts, can be obtained by standard DNA recombination techniques, and they are all useful antibodies.
  • a chimeric antibody is a molecule in which different parts come from different animal species, for example, a variable region with a monoclonal antibody from a mouse, and a chimeric antibody from a constant region of a human immunoglobulin (see, for example, U.S. Patent No. 4,816,567 and US Patent 4,816,397, hereby incorporated by reference in its entirety).
  • Humanized antibodies refer to antibody molecules derived from non-human species, having one or more complementarity determining regions (CDRs) derived from non-human species and framework regions derived from human immunoglobulin molecules (see US Patent 5,585,089, It is hereby incorporated by reference in its entirety). These chimeric and humanized monoclonal antibodies can be prepared using recombinant DNA techniques well known in the art.
  • CDRs complementarity determining regions
  • the antibody may be monospecific, bispecific, trispecific, or more multispecific.
  • the antibody of the present invention also includes conservative variants, which means that there are at most 10, preferably at most 8, more preferably at most 5, compared with the amino acid sequence of the antibody of the present invention, and most preferably Up to 3 amino acids are replaced by amino acids with similar or similar properties to form a polypeptide.
  • conservatively variant polypeptides are preferably produced by amino acid substitution according to Table 1.
  • substitution Ala 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; Thr
  • the antibody is an anti-NKG2A antibody.
  • the present invention provides a high specificity and high affinity antibody against NKG2A, which includes a heavy chain and a light chain, the heavy chain contains a heavy chain variable region (VH) amino acid sequence, and the light chain contains 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) has a complementarity determining region CDR selected from the group consisting of:
  • VH-CDR1 shown in SEQ ID NO. 8n + 2
  • VH-CDR2 shown in SEQ ID NO. 8n + 3, and
  • VH-CDR3 shown in SEQ ID NO.8n + 4;
  • n is independently 0, 1, 2, 3, 4, 5, 6, 7 or 8;
  • the light chain variable region (VL) has a complementarity determining region CDR selected from the group consisting of:
  • VL-CDR1 shown in SEQ ID NO. 8n + 6,
  • VL-CDR2 shown in SEQ ID NO. 8n + 7, and
  • VL-CDR3 shown in SEQ ID NO. 8n + 8;
  • n is independently 0, 1, 2, 3, 4, 5, 6, 7 or 8;
  • any one of the above-mentioned amino acid sequences also includes a derivative sequence that is optionally added, deleted, modified, and / or substituted for at least one amino acid, and capable of retaining the binding affinity of NKG2A.
  • the heavy chain variable region (VH) includes the following three complementarity determining regions CDR:
  • VH-CDR1 shown in SEQ ID NO. 8n + 2
  • VH-CDR2 shown in SEQ ID NO. 8n + 3, and
  • VH-CDR3 shown in SEQ ID NO.8n + 4;
  • the light chain variable region (VL) includes the following three complementarity determining regions CDR:
  • VL-CDR1 shown in SEQ ID NO. 8n + 6,
  • VL-CDR2 shown in SEQ ID NO. 8n + 7, and
  • VL-CDR3 shown in SEQ ID NO. 8n + 8;
  • n is independently 0, 1, 2, 3, 4, 5, 6, 7 or 8; preferably n is 0 or 1;
  • any one of the above-mentioned amino acid sequences further includes a derivative sequence optionally added, deleted, modified and / or substituted for at least one amino acid, and capable of retaining the binding affinity of NKG2A.
  • sequence formed by the addition, deletion, modification and / or substitution of at least one amino acid sequence is preferably homology or sequence identity of at least 80%, preferably at least 85%, more preferably The amino acid sequence is at least 90%, most preferably at least 95%.
  • the preferred method of determining identity is to obtain the greatest match between the sequences tested.
  • the method of determining identity is compiled in a publicly available computer program.
  • Preferred computer program methods for determining the identity between two sequences include, but are not limited to: 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 Manual, 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 antibodies described herein are full-length antibody antibodies, antigen-antibody binding domain protein fragments, bispecific antibodies, multispecific antibodies, single chain antibodies (single chain antibody, scFv), single domain antibodies (single domain antibody) , SdAb) and single-region antibodies (Signle-domain antibodies) one or more, and monoclonal antibodies or polyclonal antibodies prepared by the above antibodies.
  • the monoclonal antibody can be developed in various ways and technologies, including hybridoma technology, phage display technology, single lymphocyte gene cloning technology, etc. The mainstream is to prepare monoclonal antibodies from wild-type or transgenic mice through hybridoma technology.
  • the antibody full-length protein is a conventional antibody full-length protein in the art, which includes a heavy chain variable region, a light chain variable region, a heavy chain constant region, and a light chain constant region.
  • the heavy chain variable region and light chain variable region of the protein, the human heavy chain constant region and the human light chain constant region constitute a full human antibody full-length protein.
  • the full-length antibody is IgG1, IgG2, IgG3 or IgG4.
  • the antibody of the present invention may be a double-chain or single-chain antibody, and may be selected from animal-derived antibodies, chimeric antibodies, humanized antibodies, more preferably humanized antibodies, human-animal chimeric antibodies, more preferably fully human Source antibody.
  • the antibody derivative of the present invention may be a single chain antibody, and / or antibody fragments, such as: Fab, Fab ', (Fab') 2 or other known antibody derivatives in the art, as well as IgA, IgD, IgE , IgG and IgM antibodies or other subtypes of any one or several.
  • the single chain antibody is a conventional single chain antibody in the art, which includes a heavy chain variable region, a light chain variable region and a short peptide of 15-20 amino acids.
  • the animal is preferably a mammal, such as a rat.
  • the antibody of the present invention may be a chimeric antibody, humanized antibody, CDR grafted and / or modified antibody targeting NKG2A (eg, human NKG2A).
  • the number of added, deleted, modified and / or substituted amino acids is preferably not more than 40% of the total number of amino acids in the original amino acid sequence, more preferably not more than 35%, more preferably 1-33% , More preferably 5-30%, more preferably 10-25%, more preferably 15-20%.
  • the number of the added, deleted, modified and / or substituted amino acids may be 1-7, more preferably 1-5, more preferably 1-3, more preferably For 1-2.
  • the heavy chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO. 1, 9, 17, 25, 33, 41, 49, 57 or 65.
  • the light chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO. 5, 13, 21, 29, 37, 45, 53, 61 or 69.
  • the amino acid sequence of the heavy chain variable region (VH), and / or the light chain variable region of the antibody targeting NKG2A is shown in Table 2-1 below:
  • the antibodies targeting NKG2A are M15-5, Mpb416, Mab031, Mab032, Mab033, Mab036, 2F10, 9B10, 14G3.
  • the antibody targeting NKG2A is M15-5.
  • the antibody targeting NKG2A is Mpb416.
  • the present invention also provides a recombinant protein comprising one or more of heavy chain CDR1 (VH-CDR1), heavy chain CDR2 (VH-CDR2) and heavy chain CDR3 (VH-CDR3) of NKG2A antibody, and / or Or, one or more of the light chain CDR1 (VL-CDR1), light chain CDR2 (VL-CDR2) and light chain CDR3 (VL-CDR3) of the NKG2A antibody,
  • the sequence of the heavy chain CDR1-3 is as follows:
  • VH-CDR1 shown in SEQ ID NO. 8n + 2
  • VH-CDR2 shown in SEQ ID NO. 8n + 3,
  • VH-CDR3 shown in SEQ ID NO.8n + 4;
  • the sequence of the light chain CDR1-3 is as follows:
  • VL-CDR1 shown in SEQ ID NO. 8n + 6,
  • VL-CDR2 shown in SEQ ID NO. 8n + 7, and
  • VL-CDR3 shown in SEQ ID NO. 8n + 8;
  • n is independently 0, 1, 2, 3, 4, 5, 6, 7 or 8, preferably n is 0;
  • any one of the above-mentioned amino acid sequences also includes a derivative sequence that is optionally added, deleted, modified, and / or substituted for at least one amino acid, and capable of retaining the binding affinity of NKG2A.
  • sequence formed by the addition, deletion, modification and / or substitution of at least one amino acid sequence is preferably homology or sequence identity of at least 80%, preferably at least 85%, more preferably The amino acid sequence is at least 90%, most preferably at least 95%.
  • the recombinant protein of the present invention includes the heavy chain variable region of the NKG2A antibody and / or the light chain variable region of the NKG2A antibody.
  • the heavy chain variable region of the antibody contains SEQ ID NO.1 , 9, 17, 25, 33, 41, 49, 57 or 65; the light chain variable region of the antibody contains SEQ ID NO. 5, 13, 21, 29, 37, 45, 53, Amino acid sequence shown in 61 or 69.
  • the recombinant protein of the present invention includes the heavy chain variable region of the NKG2A antibody and the light chain variable region of the NKG2A antibody.
  • the heavy chain variable region of the antibody contains SEQ ID NO. 1, 9 , 17, 25, 33, 41, 49, 57 or 65, and the light chain variable region of the antibody contains SEQ ID NO. 5, 13, 21, 29, 37, 45, 53, 61 Or the amino acid sequence shown in 69.
  • sequence numbers of the amino acid sequences of the recombinant protein and the heavy chain CDR1-3 and light chain CDR1-3 included in the recombinant protein are shown in Table 2-2:
  • any one of the above-mentioned amino acid sequences also includes a derivative sequence that is optionally added, deleted, modified, and / or substituted for at least one amino acid, and capable of retaining the binding affinity of NKG2A.
  • the recombinant protein further includes an antibody heavy chain constant region and / or an antibody light chain constant region.
  • the antibody heavy chain constant region is conventional in the art, preferably a rat antibody heavy chain constant region Or a human antibody heavy chain constant region, more preferably a human antibody heavy chain constant region.
  • the antibody light chain constant region is conventional in the art, preferably a rat light chain antibody constant region or a human antibody light chain constant region, and more preferably a human antibody light chain constant region.
  • the recombinant protein is a conventional protein in the art, preferably, it is an antibody full-length protein, an antigen-antibody binding domain protein fragment, a bispecific antibody, a multispecific antibody, a single chain antibody (single chain antibody, scFv) ), Single domain antibody (single domain antibody, sdAb) and single domain antibody (Signle-domain antibody) one or more, and monoclonal antibody or polyclonal antibody prepared by the above antibody.
  • the monoclonal antibody can be developed in various ways and technologies, including hybridoma technology, phage display technology, single lymphocyte gene cloning technology, etc. The mainstream is to prepare monoclonal antibodies from wild-type or transgenic mice through hybridoma technology.
  • the antibody full-length protein is a conventional antibody full-length protein in the art, which includes a heavy chain variable region, a light chain variable region, a heavy chain constant region, and a light chain constant region.
  • the heavy chain variable region and light chain variable region of the protein, the human heavy chain constant region and the human light chain constant region constitute a full human antibody full-length protein.
  • the full-length antibody is IgG1, IgG2, IgG3 or IgG4.
  • the single chain antibody is a conventional single chain antibody in the art, which includes a heavy chain variable region, a light chain variable region and a short peptide of 15-20 amino acids.
  • the antigen-antibody binding domain protein fragments are conventional antigen-antibody binding domain protein fragments in the art, which include the light chain variable region, the light chain constant region, and the Fd segment of the heavy chain constant region.
  • the protein fragments of the antigen-antibody binding domain are Fab and F (ab ').
  • the single domain antibody is a conventional single domain antibody in the art, which includes a heavy chain variable region and a heavy chain constant region.
  • the single-region antibody is a conventional single-region antibody in the art, which includes only the heavy chain variable region.
  • the preparation method of the recombinant protein is a conventional preparation method in the art.
  • the preparation method is preferably obtained by separation from an expression transformant that recombinantly expresses the protein or by artificially synthesizing the protein sequence.
  • the method for separating and obtaining the recombinant transformant that expresses the protein is preferably as follows: clone the nucleic acid molecule encoding the protein and carrying a point mutation into a recombinant vector, and transform the resulting recombinant vector into a transformant to obtain recombinant expression
  • the transformant can be isolated and purified to obtain the recombinant protein by culturing the obtained recombinant expression transformant.
  • the present invention also provides a nucleic acid encoding the heavy chain variable region or light chain variable region of the above-mentioned antibody (eg, anti-NKG2A antibody) or recombinant protein or anti-NKG2A antibody.
  • a nucleic acid encoding the heavy chain variable region or light chain variable region of the above-mentioned antibody (eg, anti-NKG2A antibody) or recombinant protein or anti-NKG2A antibody.
  • nucleotide sequence of the nucleic acid encoding the variable region of the heavy chain is shown in SEQ ID NO. 73, 75, 77, 79, 81, 83, 85, 87 or 89 of the Sequence Listing; and / or, The nucleotide sequence of the nucleic acid encoding the light chain variable region is shown in the sequence listing SEQ ID NO. 74, 76, 78, 80, 82, 84, 86, 88 or 90.
  • nucleotide sequence of the nucleic acid encoding the heavy chain variable region is shown in SEQ ID NO. 73, 75, 77, 79, 81, 83, 85, 87 or 89 of the sequence listing; and encodes the The nucleotide sequence of the light chain variable region nucleic acid is shown in SEQ ID NO. 74, 76, 78, 80, 82, 84, 86, 88 or 90 of the Sequence Listing.
  • the preparation method of the nucleic acid is a conventional preparation method in the art, and preferably includes the following steps: obtaining a nucleic acid molecule encoding the above protein by gene cloning technology, or obtaining a nucleic acid molecule encoding the above protein by artificial full sequence synthesis .
  • the base sequence encoding the amino acid sequence of the above-mentioned protein may be appropriately substituted, deleted, altered, inserted, or added to provide a polynucleotide homolog.
  • the homologue of the polynucleotide in the present invention can be prepared by replacing, deleting, or adding one or more bases of the gene encoding the protein sequence within the range of maintaining antibody activity.
  • the invention also provides a recombinant expression vector containing the nucleic acid.
  • the recombinant expression vector can be obtained by a conventional method in the art, that is, it is constructed by connecting the nucleic acid molecule of the present invention to various expression vectors.
  • the expression vector is a variety of conventional vectors in the art, as long as it can contain the aforementioned nucleic acid molecule.
  • the vector preferably includes: various plasmids, cosmids, phage or viral vectors, and the like.
  • the present invention also provides a recombinant expression transformant comprising the above recombinant expression vector.
  • the preparation method of the recombinant expression transformant is a conventional preparation method in the art, preferably: it is prepared by transforming the above-mentioned recombinant expression vector into a host cell.
  • the host cell is a variety of conventional host cells in the art, as long as it can satisfy the above-mentioned recombinant expression vector to stably replicate itself, and the nucleic acid carried can be effectively expressed.
  • the host cell is E. coli TG1 or E. coli BL21 cells (expressing single chain antibody or Fab antibody), or HEK293 or CHO cells (expressing full-length IgG antibody).
  • the conversion method is a conventional conversion method in the art, preferably a chemical conversion method, a heat shock method or an electric conversion method.
  • sequence of the DNA molecule of the antibody or fragment thereof of the present invention can be obtained by conventional techniques, such as PCR amplification or genomic library screening.
  • the coding sequences of the light chain and the heavy chain can be fused together to form a single chain antibody.
  • the relevant sequence can be obtained in large quantities by the recombination method. This is usually done by cloning it into a vector, then transferring it into cells, and then isolating the relevant sequence from the proliferated host cells by conventional methods.
  • synthetic methods can be used to synthesize the relevant sequences, especially when the length of the fragments is short.
  • a long sequence can be obtained by synthesizing multiple small fragments and then connecting them.
  • the DNA sequence encoding the antibody (or fragment or derivative thereof) of the present invention can be obtained completely by chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or such as vectors) and cells known in the art. In addition, mutations can also be introduced into the protein sequence of the invention by chemical synthesis.
  • the present invention also relates to vectors containing the appropriate DNA sequence described above and an appropriate promoter or control sequence. These vectors can be used to transform appropriate host cells so that they can express proteins.
  • the host cell may 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 transformed host cells are cultured under conditions suitable for expression of the antibody of the present invention. Then use conventional 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.
  • 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 the personnel.
  • the obtained monoclonal antibody can be identified by conventional means.
  • the binding specificity of monoclonal antibodies can be determined by immunoprecipitation or in vitro binding tests (such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA)).
  • the binding affinity of the monoclonal antibody can be determined, for example, by Scatchard analysis of Munson et al., Anal. Biochem., 107: 220 (1980).
  • the antibody of the present invention can be expressed in a cell, on a cell membrane, or secreted out of a cell. If necessary, the recombinant protein can be isolated and purified by various separation methods using its physical, chemical and other characteristics. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional renaturation treatment, treatment with protein precipitant (salting method), centrifugation, osmotic disruption, ultrasonic treatment, 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.
  • conventional renaturation treatment treatment with protein precipitant (salting method), centrifugation, osmotic disruption, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography
  • ADC Antibody-drug conjugate
  • the invention also provides antibody-drug conjugate (ADC) based on the antibody of the invention.
  • ADC antibody-drug conjugate
  • the antibody-coupled drug includes the antibody and an effector molecule, and the antibody is coupled to the effector molecule, and is preferably chemically coupled.
  • the effector molecule is preferably a drug having therapeutic activity.
  • the effector molecule may be one or more of toxic protein, chemotherapeutic drug, small molecule drug or radionuclide.
  • the antibody of the present invention and the effector molecule may be coupled by a coupling agent.
  • the coupling agent may be any one or more of a non-selective coupling agent, a coupling agent using a carboxyl group, a peptide chain, and a coupling agent using a disulfide bond.
  • the non-selective coupling agent refers to a compound that forms a covalent bond between the effector molecule and the antibody, such as glutaraldehyde and the like.
  • the coupling agent using a carboxyl group may be any one or several of cis-aconitic anhydride type coupling agents (such as cis-aconitic anhydride) and acylhydrazone type coupling agents (coupling sites are acylhydrazones).
  • Antibodies can be coupled to functional agents to form antibody-functional agent conjugates.
  • Functional agents eg drugs, detection reagents, stabilizers
  • the functional agent may be directly or indirectly linked to the antibody via a linker.
  • Antibodies can be coupled to drugs to form antibody drug conjugates (ADCs).
  • ADC antibody drug conjugates
  • the ADC contains a linker between the drug and antibody.
  • the joint may be degradable or non-degradable.
  • the degradable linker is typically easily degraded in the intracellular environment, for example, the linker degrades at the target site, thereby releasing the drug from the antibody.
  • Suitable degradable linkers include, for example, enzymatically degraded linkers, including peptide group-containing linkers that can be degraded by intracellular proteases (such as lysosomal proteases or endosomal proteases), or sugar linkers, such as glucuronide Enzymatically degraded glucuronide-containing linker.
  • Peptidyl linkers may include, for example, dipeptides such as valine-citrulline, phenylalanine-lysine or valine-alanine.
  • Other suitable degradable linkers include, for example, pH sensitive linkers (such as linkers that hydrolyze at a pH less than 5.5, such as hydrazone linkers) and linkers that can degrade under reducing conditions (such as disulfide bond linkers).
  • Non-degradable linkers typically release the drug under conditions where the antibody is hydrolyzed by a protease.
  • the linker Before connecting to the antibody, the linker has an active reactive group capable of reacting with certain amino acid residues, and the connection is achieved through the active reactive group.
  • Mercapto-specific reactive reactive groups are preferred and include: for example maleimides, halogenated amides (eg iodine, bromine or chlorinated); halogenated esters (eg iodine, bromine or chlorinated) ); Halogenated methyl ketone (such as iodine, bromine or chloro), benzyl halide (such as iodine, bromine or chloro); vinyl sulfone, pyridyl disulfide; mercury derivatives such as 3,6- Di- (mercurymethyl) dioxane, and the counter ion is acetate, chloride or nitrate; and polymethylene dimethyl sulfide thiosulfonate.
  • the linker may include, for example, maleimide attached to the antibody through thios
  • the drug may be any drug that is cytotoxic, inhibits cell growth, or immunosuppresses.
  • the linker connects the antibody and the drug, and the drug has a functional group that can form a bond with the linker.
  • the drug may have an amino group, a carboxyl group, a mercapto group, a hydroxyl group, or a ketone group that can form a bond with the linker.
  • the drug is directly connected to the linker, the drug has a reactive active group before being connected to the antibody.
  • Useful drug classes include, for example, antitubulin drugs, DNA minor groove binding reagents, DNA replication inhibitors, alkylating agents, antibiotics, folic acid antagonists, antimetabolites, chemotherapy sensitizers, topoisomerase inhibitors , Vinca alkaloids, etc.
  • particularly useful cytotoxic drugs include, for example, DNA minor groove binding reagents, DNA alkylation reagents, and tubulin inhibitors
  • typical cytotoxic drugs include, for example, auristatins, camptothecin (camptothecins), dokamycin / duocarmycins, etoposides, maytansines and maytansinoids (e.g.
  • DM1 and DM4 taxanes
  • benzodiazepines or benzodiazepine containing drugs such as pyrrolo [1,4] benzodiazepines (PBDs), indoline benzodiazepines (Indolinobenzodiazepines) and oxazolidinobenzodiazepines (oxazolidinobenzodiazepines) and vinca alkaloids (vinca alkaloids).
  • PBDs pyrrolo [1,4] benzodiazepines
  • Indolinobenzodiazepines Indolinobenzodiazepines
  • oxazolidinobenzodiazepines oxazolidinobenzodiazepines
  • vinca alkaloids vinca alkaloids
  • the drug-linker can be used to form an ADC in a simple step.
  • the bifunctional linker compound can be used to form an ADC in a two-step or multi-step method.
  • the cysteine residue reacts with the reactive part of the linker in the first step, and in the subsequent step, the functional group on the linker reacts with the drug, thereby forming an ADC.
  • the functional group on the linker is selected to facilitate specific reaction with the appropriate reactive group on the drug moiety.
  • the azide-based moiety can be used to specifically react with a reactive alkynyl group on the drug moiety.
  • the drug is covalently bonded to the linker through 1,3-dipolar cycloaddition between the azide and the alkynyl group.
  • ketones and aldehydes suitable for reaction with hydrazides and alkoxyamines
  • phosphines suitable for reaction with azides
  • isocyanates and isothiocyanates suitable for reaction with amines
  • React with alcohols and activated esters, such as N-hydroxysuccinimide ester (suitable for reaction with amines and alcohols).
  • activated esters such as N-hydroxysuccinimide ester (suitable for reaction with amines and alcohols).
  • the present invention also provides a method for preparing an ADC, which may further include: combining the antibody and the drug-linker compound under conditions sufficient to form an antibody conjugate (ADC).
  • the methods of the invention include: binding the antibody to a bifunctional linker compound under conditions sufficient to form an antibody-linker conjugate.
  • the method of the present invention further includes: binding the antibody linker conjugate to the drug moiety under conditions sufficient to covalently link the drug moiety to the antibody via a linker.
  • the antibody drug conjugate ADC is represented by the following formula:
  • Ab is an antibody
  • D is a drug
  • the present invention also provides the use of the antibody, antibody conjugate ADC, recombinant protein, and / or immune cell of the present invention, for example, for preparing a diagnostic preparation or preparing a medicine.
  • the medicine is a medicine for preventing and / or treating diseases related to abnormal expression or function of NKG2A.
  • the diseases related to abnormal expression or function of NKG2A are conventional diseases related to abnormal expression or function of NKG2A in the art.
  • the diseases related to abnormal expression or function of NKG2A are cancer, autoimmune diseases, and inflammatory diseases.
  • the cancer is a conventional cancer in the art, preferably an HLA-E overexpressing cancer, such as HLA-E overexpressing lymphocytic leukemia, ovarian cancer, non-small cell lung cancer, head and neck cancer, and the like.
  • an HLA-E overexpressing cancer such as HLA-E overexpressing lymphocytic leukemia, ovarian cancer, non-small cell lung cancer, head and neck cancer, and the like.
  • the autoimmune disease is a conventional autoimmune disease in the art, preferably hemolytic anemia, pernicious anemia, polyarteritis nodosa, systemic lupus erythematosus, Alzheimer's disease, diabetes, etc.
  • Uses of the antibodies, ADCs, recombinant proteins, and / or immune cells of the present invention include (but are not limited to):
  • the tumors include (but are not limited to): preferably HLA-E overexpressing cancers, such as HLA-E overexpressing lymphocytic leukemia, ovarian cancer, non-small cell lung cancer, head and neck cancer, and the like.
  • autoimmune diseases including (but not limited to): preferably hemolytic anemia, pernicious anemia, polyarteritis nodosa, systemic lupus erythematosus, Alzheimer's disease, diabetes, etc.
  • Diagnosis, prevention and / or treatment of inflammatory diseases including (but not limited to): rheumatoid arthritis, glomerulonephritis, myasthenia gravis, multiple sclerosis, alopecia areata and the like.
  • the antibody of the present invention or its ADC can be used in a detection application, for example, to detect a sample, thereby providing diagnostic information.
  • the samples (samples) used include cells, tissue samples and biopsy specimens.
  • the term "biopsy” used in the present invention shall include all kinds of biopsies known to those skilled in the art. Therefore, the biopsy used in the present invention may include, for example, a resection sample of a tumor, a tissue sample prepared by an endoscopic method or an puncture or needle biopsy of an organ.
  • the samples used in the present invention include fixed or preserved cell or tissue samples.
  • the present invention also provides a kit containing the antibody (or fragment thereof) of the present invention.
  • the kit further includes a container, an instruction manual, a buffer, and the like.
  • the antibody of the present invention may be fixed to the detection plate.
  • the invention also provides a composition.
  • the composition is a pharmaceutical composition, which contains the above antibody or its active fragment or its fusion protein or its ADC or corresponding immune cells, and a pharmaceutically acceptable carrier.
  • these substances can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, where the pH is usually about 5-8, preferably about 6-8, although the pH can be The nature of the formulated substance and the condition to be treated vary.
  • the formulated pharmaceutical composition can be administered by conventional routes, including (but not limited to): intratumoral, intraperitoneal, intravenous, or topical administration.
  • the route of administration of the pharmaceutical composition of the present invention is preferably injection or oral administration.
  • the injection administration preferably includes intravenous injection, intramuscular injection, intraperitoneal injection, intradermal injection, or subcutaneous injection.
  • the pharmaceutical composition is a variety of conventional dosage forms in the art, preferably in the form of solid, semi-solid or liquid, which may be aqueous solution, non-aqueous solution or suspension, more preferably tablets, capsules, granules , Injections or infusions.
  • the antibody of the present invention may also be used for cell therapy by expressing 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 is a pharmaceutical composition for preventing and / or treating diseases related to abnormal expression or function of NKG2A.
  • the pharmaceutical composition of the present invention can be directly used to bind NKG2A protein molecules, and thus can be used to prevent and treat diseases such as tumors.
  • the pharmaceutical composition of the present invention contains a safe and effective amount (such as 0.001-99wt%, preferably 0.01-90wt%, more preferably 0.1-80wt%) of the above-mentioned monoclonal antibody (or its conjugate) of the present invention and pharmaceutical Acceptable carrier or excipient.
  • Such carriers include (but are not limited to): saline, buffer, glucose, water, glycerin, ethanol, and combinations thereof.
  • the pharmaceutical preparation should match the mode of administration.
  • the pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, prepared 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 manufactured under sterile conditions.
  • the amount of active ingredient administered is a therapeutically effective amount, for example, about 1 microgram / kg body weight to about 5 mg / kg body weight per day.
  • the polypeptide of the present invention can be used together with other
  • the pharmaceutical composition of the present invention further includes one or more pharmaceutically acceptable carriers.
  • the pharmaceutical carrier is a conventional pharmaceutical carrier in the art, and the pharmaceutical carrier may be any suitable physiological or pharmaceutically acceptable pharmaceutical excipient.
  • the pharmaceutical excipients are conventional pharmaceutical excipients in the art, and preferably include pharmaceutically acceptable excipients, fillers or diluents. More preferably, the pharmaceutical composition includes 0.01-99.99% of the aforementioned protein and 0.01-99.99% of a pharmaceutical carrier, and the percentage is a mass percentage of the pharmaceutical composition.
  • the administration amount of the pharmaceutical composition is an effective amount
  • the effective amount is an amount that can alleviate or delay the progression of a disease, degenerative or traumatic condition.
  • the effective amount can be determined on an individual basis and will be based in part on consideration of the symptoms to be treated and the results sought. Those skilled in the art can determine the effective amount by using the above factors such as individual basis and using no more than routine experimentation.
  • a safe and effective amount of an immunoconjugate is administered to a mammal, wherein the safe and effective amount is usually at least about 10 ⁇ g / kg body weight, and in most cases does not exceed about 50 mg / kg body weight, Preferably the dose is about 10 micrograms / kg body weight to about 20 mg / kg body weight.
  • the specific dosage should also consider factors such as the route of administration, the patient's health status, etc., which are within the skills of skilled physicians.
  • the present invention provides the use of the above pharmaceutical composition in the preparation of a medicament for the prevention and / or treatment of diseases related to abnormal expression or function of NKG2A.
  • diseases related to abnormal expression or function of NKG2A are cancer, autoimmune diseases, and inflammatory diseases.
  • the present invention also provides a method for detecting NKG2A protein in a sample (for example, detecting over-expressing NKG2A cells), which includes the steps of contacting the above-mentioned antibody with the test sample in vitro to detect whether the above-mentioned antibody binds to the test sample It is sufficient to form an antigen-antibody complex.
  • overexpression is conventional in the art, and refers to the overexpression of RNA or protein of the NKG2A protein in the sample to be tested (due to increased transcription, post-transcriptional processing, translation, post-translational processing, and protein degradation changes), and due to the protein Changes in delivery mode (increased nuclear localization) result in local overexpression and increased functional activity (eg in the case of increased enzymatic hydrolysis of the substrate).
  • the detection method of whether the above forms an antigen-antibody complex is a conventional detection method in the art, preferably a flow cytometry (FACS) test.
  • FACS flow cytometry
  • the invention provides a composition for detecting NKG2A protein in a sample, which comprises the above-mentioned antibody, recombinant protein, antibody conjugate, immune cell, or a combination thereof as an active ingredient.
  • a composition for detecting NKG2A protein in a sample which comprises the above-mentioned antibody, recombinant protein, antibody conjugate, immune cell, or a combination thereof as an active ingredient.
  • it also includes a compound composed of the functional fragments of the above-mentioned antibody as an active ingredient.
  • the NKG2A antibody of the present invention has a high affinity with NKG2A protein (affinity constant KD reaches 1.04 ⁇ 10 -11 );
  • the NKG2A antibody of the present invention can bind to the extracellular region of the NKG2A protein receptor, and can effectively block the NKG2A protein at the cellular level, preventing the binding of the NKG2A protein to the ligand HLA-E;
  • NKG2A antibody of the present invention has high specificity and lacks cross-reactivity with human NKG2C and NKG2E same family protein antigens;
  • the room temperature described in the examples is conventional room temperature in the art, and is generally 10-30 ° C.
  • nucleotide sequence containing the amino acid sequence Pro94-Leu233 (shown in SEQ ID NO.91 of the sequence listing) encoding the extracellular domain of human NKG2A protein was cloned into a pCpC vector (purchased from Invitrogen, V044-50) and prepare plasmids according to established standard molecular biology methods.
  • nucleotide sequence containing the amino acid sequence Lys32-Ile179 (shown in SEQ ID NO.92 of the sequence listing) encoding the extracellular domain of human CD94 protein was cloned into a pCpC vector (purchased from Invitrogen) with a human IgG Fc fragment (hFc) , V044-50, and according to the established standard molecular biology methods for the preparation of plasmids for specific methods, see Sambrook, J., Fritsch, EF, and Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual, Second Edition (Plainview, New York: Cold Spring Laboratory (Press).
  • HEK293 cells purchased from Invitrogen
  • PI Proliferative Inducible Cell
  • FreeStyle TM 293 Invitrogen
  • the cell culture fluid was collected, The cell components were removed by centrifugation to obtain a culture supernatant containing heterodimeric protein of NKG2A and CD94 protein extracellular domain.
  • the culture supernatant was loaded onto a protein A affinity chromatography column (MabselectSure, purchased from GE Healthcare), At the same time, the change of ultraviolet absorption value (A280nm) was monitored with an ultraviolet (UV) detector.
  • UV ultraviolet
  • the protein A affinity chromatography column was washed with PBS phosphate buffer (pH7.2) until the ultraviolet absorption value returned to the baseline, and then 0.1M Gan Hydrochloric acid (pH2.5) was eluted, and the hFc-tagged NKG2A / CD94 protein (NKG2A / CD94-hFc) eluted from the Protein A affinity chromatography column was collected using PBS phosphate buffer (pH 7. 2) Dialyze at 4 ° C overnight. The dialyzed protein is sterile filtered at 0.22 microns and stored at -80 ° C to obtain purified immunogen A. Immunogen A undergoes a series of quality control tests before use, such as Detection of protein concentration, purity, molecular weight, biological activity, etc.
  • the results are shown in Figure 1.
  • the binding of the NKG2A / CD94 protein to the control antibody at the protein level varies with the concentration of the antibody.
  • the control protein is a non-NKG2A / CD94 fusion protein.
  • the data in the table are OD450nm values.
  • the NKG2A full-length amino acid sequence cDNA (as shown in the sequence listing SEQ ID NO.93) and CD94 full-length amino acid were cloned from the DNA sequence (as shown in the sequence listing SEQ ID NO.94) into the Pcho1.0 vector (purchased from Invitrogen) , And coated onto 1.0um gold colloid bullets, immunized with Helios gene gun (Helios Gene Gun System, Bio-rad, Catalog No. 165-2431). Among them, the method of coating to 1.0 ⁇ m gold colloid bullets and immunization is shown in the Helios gene gun instruction manual. Immunogen B was obtained after immunization.
  • the prepared immunogen B was transfected into plasmids (PEI, purchased from Polysciences) of HEK293 cell line (both purchased from Invitrogen), the 10% (w / w) fetal bovine serum containing 0.5 ⁇ g / ml Selective culture in DMEM medium for 2 weeks, subcloning in 96-well culture plate by limiting dilution method, and incubation at 37 ° C, 5% (v / v) CO 2 , select some monoclonal wells after about 2 weeks Amplify into 6-well plates. The amplified clones were screened by flow cytometry using known NKG2A antibody (CP in house) and CD94 antibody (purchased from eBioscience).
  • the prepared immunogen B was subjected to plasmid transfection (PEI, purchased from Polysciences) on the CHOK1 cell line (both purchased from Invitrogen), the 10% (w / w) fetal bovine serum containing 0.5 ⁇ g / ml Selective culture in DMEM medium for 2 weeks, subcloning in 96-well culture plate by limiting dilution method, and incubation at 37 ° C, 5% (v / v) CO 2 , select some monoclonal wells after about 2 weeks Amplify into 6-well plates. The amplified clones were screened by flow cytometry using known NKG2A antibody (CP in house) and CD94 antibody (purchased from eBioscience).
  • the positive cells (%) in Table 4 refer to the percentage of positive cells in the total number of cells.
  • Table 4 shows that a series of CHOK1 cell lines with positive expression of both NKG2A and CD94 have been prepared.
  • the full-length amino acid sequence of cynomolgus monkey NKG2A (shown in SEQ ID NO. 95 in the sequence listing) and CD94 full-length amino acid (shown in SEQ ID NO. 96 in the sequence listing) were cloned into the Pcho1.0 vector (purchased from Invitrogen). After the prepared plasmid was subjected to plasmid transfection (PEI, purchased from Polysciences) of the 293 cell line (both purchased from Invitrogen), it was cultured in DMEM containing 10% (w / w) fetal bovine serum at 0.5 ⁇ g / ml.
  • PEI plasmid transfection
  • Immunogen A immunized with 6-8 weeks old Balb / c mice, and the mice were raised under SPF conditions. At the initial immunization, immunogen A protein was emulsified with Freund's complete adjuvant and injected intraperitoneally 0.25 ml, that is, each mouse was injected with 50 micrograms of immunogen protein A. During booster immunization, immunogen A protein was emulsified with Freund's incomplete adjuvant and injected intraperitoneally 0.25 ml, that is, each mouse was injected with 50 micrograms of immunogen A protein. The interval between the first immunization and the first booster immunization is 2 weeks, and the interval between subsequent booster immunizations is 3 weeks. Blood was collected one week after each booster immunization, and the antibody titer and specificity of immunogen A in the serum were detected by ELISA and FACS.
  • F12K basal medium purchased from Invitrogen
  • enzymatic cell dissociation solution purchased from Invitrogen
  • the interval between the first and second immunization is 2 weeks, and the interval between subsequent immunizations is 3 weeks. Except for the first immunization, blood was collected 1 week after each immunization, and the antibody titer and specificity in the serum were detected by ELISA and FACS.
  • FIG. 3a The results are shown in Figure 3a.
  • the mouse sera bind to the immunogen to varying degrees, showing antigen-antibody reactions, with the highest dilution of about 10,000.
  • Figure 3b shows that mouse serum can also specifically bind to human NKG2A on the cell surface, with the highest dilution at 1,000 times.
  • Immunogen E was immunized with 6-8 week old SJL and Balb / c mice, which were raised under SPF conditions. Immunogen E was expanded and cultivated to 90% confluence in T-75 cell culture flasks, the medium was exhausted, washed twice with DMEM basal medium (purchased from Invitrogen), and then with enzyme-free cell dissociation solution (purchased from Invitrogen) Treat at 37 ° C until the cells can detach from the wall of the culture dish, and collect the cells. After washing twice with DMEM basic medium, the cells were counted, and the cells were diluted with phosphate buffer (pH 7.2) to 2 ⁇ 10 7 cells per ml.
  • phosphate buffer pH 7.2
  • Each mouse was injected intraperitoneally with 0.5 ml of cell suspension at each immunization.
  • the interval between the first and second immunization is 2 weeks, and the interval between subsequent immunizations is 3 weeks.
  • blood was collected 1 week after each immunization, and antibody titer and specificity in serum were detected by FACS.
  • After the second immunization replace immunogen A for immunization.
  • immunogen A was emulsified with Freund's complete adjuvant and injected intraperitoneally with 0.25 ml, that is, each mouse was injected with 50 ⁇ g of immunogen A protein.
  • immunogen A protein was emulsified with Freund's incomplete adjuvant and injected intraperitoneally 0.25 ml, that is, each mouse was injected with 50 micrograms of immunogen protein A (Figure 4). Blood was collected 1 week after boosting immunization, and the antibody titer and specificity of immunogen A in the serum were detected by ELISA and FACS.
  • Immunogen B is immunized with 6-8 weeks old Balb / c or SJL mice raised under SPF conditions. All mice were immunized 4 times with Helios gene gun through the abdomen, 4 shots each time, 1.0 ⁇ g cDNA per shot. The interval between the first immunization and the first booster immunization is 2 weeks, and the interval between subsequent booster immunizations is 3 weeks. Blood was collected one week after each booster immunization, and the antibody titer in serum was detected by ELISA or FACS. After the second booster immunization, immunogen A was replaced for immunization.
  • immunogen A was emulsified with Freund's complete adjuvant and injected intraperitoneally with 0.25 ml, that is, each mouse was injected with 50 micrograms of immunogen A protein.
  • the immunogen A protein was emulsified with Freund's incomplete adjuvant and injected intraperitoneally with 0.25 ml, that is, each mouse was injected with 50 micrograms of the immunogen protein A (Figure 7). Blood was collected 1 week after booster immunization, and the antibody titer and specificity of immunogen A in the serum were detected by ELISA and FACS.
  • each selected mouse was immunized intraperitoneally with 100 ⁇ g of purified hNKG2A / CD94-ECD-Fc ) Or CHOK1 stable cell line containing human NKG2A and CD94 (mouse immunoreactive against immunogen B), the mice were sacrificed 3-4 days later, and spleen cells were collected. NH 4 OH was added to a final concentration of 1% (w / w) to lyse the red blood cells in the spleen cells to obtain a spleen cell suspension.
  • the cell fusion plate supernatant was screened by ELISA and Acumen (microplate cell detection method), and the positive clones with OD 450nm > 1.0 and Percentage% value> 15 in Acumen were amplified to 24 well plates, containing 10% (w / w) HT fetal bovine serum, DMEM (invitrogen) was expanded at 37 ° C, 5% (v / v) CO 2 conditions. After culturing for 3 days, the culture medium of the expanded culture in the 24-well plate was centrifuged, the supernatant was collected, the supernatant was analyzed for antibody subtypes, and the binding activity to NKG2A / CD94 positive cells was determined by FACS. (See Example 3A and Example 3B, respectively). The ligand receptor binding experiment determines the blocking activity of the antibody sample to the NKG2A receptor (for the detection method of binding activity, see Example 4).
  • the hybridoma clones that positively bind to CHOK1 cells expressing human NKG2A and CD94 and expressing monkey NKG2A and CD94 and negatively binding to CHOK1 cells expressing human NKG2C and CD94 were selected for cloning in FACS experiments.
  • the blocking effect of hybridoma cell culture supernatant on NKG2A receptor was detected as an alternative criterion for selecting clones.
  • the optimal clones were selected and placed in DMEM medium (purchased from invitrogen) containing 10% (w / w) FBS at 37 ° C, 5% (v / v) CO 2 conditions
  • DMEM medium purchased from invitrogen
  • the optimal clone is expanded and cultured, and the hybridoma cells of the present invention are obtained by cryopreservation in liquid nitrogen, and can be used for subsequent antibody production and purification.
  • the antibody concentration produced by the hybridoma cells is low, only about 1-10 ⁇ g / ml, and the concentration varies greatly.
  • various proteins produced by cell culture in the culture medium and fetal bovine serum components contained in the culture medium have different degrees of interference with many biological activity analysis methods. Therefore, small-scale (1-5 mg) antibody production and purification are required.
  • the hybridoma cells obtained in Example 1 were inoculated into T-75 cell culture flasks and domesticated and passaged for 3 generations with production medium (Hybridoma serum free medium, purchased from Invitrogen).
  • production medium Hybridoma serum free medium, purchased from Invitrogen.
  • 500 ml of production medium was added to each 2 liter culture flask, and the cell density was 1.0 ⁇ 10 5 / ml. Close the cap tightly and place the spinner bottle on the spinner in a 37 ° C incubator at a speed of 3 rpm.
  • the cell culture fluid was collected, filtered to remove the cells, and filtered with a 0.45 micron filter membrane until the culture supernatant was clear.
  • the clarified culture supernatant can be purified immediately or frozen at -30 °C.
  • the monoclonal antibody in the culture supernatant (300 mL) of the clarified hybridoma cells was purified with a 2 mL Protein G column (purchased from GE Healthcare).
  • the protein G column was first equilibrated with an equilibration buffer (PBS phosphate buffer, pH 7.2), and then the clarified culture supernatant was loaded onto the protein G column, and the flow rate was controlled at 3 mL / min. After loading the sample, wash the protein G column with equilibration buffer.
  • the volume of the equilibration buffer is 4 times the bed volume of the protein G column.
  • the NKG2A antibody bound to the protein G column was eluted with an eluent (0.1M glycine hydrochloric acid buffer, pH 2.5), and the elution was monitored with an ultraviolet detector (A280 ultraviolet absorption peak). Collect the eluted antibody, add 10% 1.0M Tris-HCl buffer to neutralize the pH, the percentage is the volume percentage, then immediately dialyze with PBS phosphate buffer overnight, change the fluid once the next day and continue dialysis for 3 hours.
  • the NKG2A antibody after dialysis is collected, sterile filtered with a 0.22 micron filter, and stored aseptically to obtain purified NKG2A antibody.
  • the purified NKG2A antibody was analyzed for protein concentration (A280 / 1.4) and purity. The results are shown in Table 5. The results showed that the endotoxin concentration of the final antibody product was within 1.0EU / mg.
  • RNA isolation After the supernatant obtained from the subcloning culture of Example 1 was tested for antigen binding (that is, after the verification and activity measurement of Examples 3 to 6), 5 ⁇ 10 7 hybridoma cells were collected by centrifugation and added Mix 1mL Trizol and transfer to a 1.5mL centrifuge tube, let stand at room temperature for 5 minutes; add 0.2mL chloroform, shake for 15 seconds, let stand for 2 minutes, centrifuge at 4 °C, 12000g for 5 minutes, take the supernatant and transfer to a new 1.5 In a centrifuge tube; add 0.5mL of isopropanol, gently mix the liquid in the tube, let stand at room temperature for 10 minutes, then centrifuge at 12,000g for 15 minutes at 4 ° C, discard the supernatant; add 1mL of 75% (v / v) ethanol , Wash the pellet gently, centrifuge at 12000g for 5 minutes at 4 ° C, discard the supernatant, dry the pellet, and add DE
  • Reverse transcription and PCR Take 1 ⁇ g of total RNA and configure 20 ⁇ l system. After adding reverse transcriptase, react at 42 ° C for 60 minutes and at 7 ° C for 10 minutes 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-denaturation 95 °C for 3 minutes, denaturation 95 °C for 30 seconds, annealing 55 °C for 30 seconds, After extension at 72 ° C for 35 seconds, after 35 cycles, additional extension at 72 ° C for 5 minutes to obtain PCR product.
  • the kit used for reverse transcription is PrimeScript RT Master, purchased from Takara, catalog number RR036; the kit used for PCR includes Q5 ultra-fidelity enzyme, purchased from NEB, catalog number M0492.
  • Cloning and sequencing take 5 ⁇ l PCR product for agarose gel electrophoresis detection, and use the column recovery kit to purify the positive test samples, of which the recovery kit is Gel & PCR Clean-up, purchased from MACHEREY-NAGEL, Catalog No. 740609.
  • Perform ligation reaction sample 50 ng, T vector 50 ng, ligase 0.5 ⁇ l, buffer 1 ⁇ l, reaction system 10 ⁇ l, and ligation product at 16 ° C for half an hour.
  • the ligation kit is T4 DNA ligase, purchased from NEB, Catalog No. ; Take 5 ⁇ l of ligation product into 100 ⁇ l of competent cells (Ecos 101competent cells, purchased from Yeasten, Catalog No.
  • mice immunized with HNKG2A / CD94-ECD-Fc and CHOK-1 / HNKG2A / CD94 as antigens sprint immunize with 293 / cynoNKG2A / CD94 cells, and isolate mouse spleen cells to prepare an immune library 3 days later .
  • the separated splenocytes were resuspended in DMEM medium, centrifuged at 2000 rpm and 4 ° C for 10 min, and the supernatant was discarded.
  • the cell pellet was lysed with RNAisoplus (purchased from Takara, Cat. No. 9108). The ratio of 1ml RNAisoplus was added to the spleen of a mouse. After incubation at room temperature for 5 minutes, it was stored at -80 °C.
  • RNA extraction Thaw the frozen mouse spleen cells at room temperature and vortex for 5 min. 0.2ml of 1-bromo-3-chloropropane (1-Bromo-3-chloropropane, purchased from Sigma, catalog number: B9673-200ml) was added to each 1ml of RNAiso plus sample, vigorously shaken for 15 seconds, and then incubated at room temperature for 5 minutes. Centrifuge the sample at 12,000g for 10min at 4 ° C, transfer the aqueous phase to a new tube, add 0.7ml of isopropanol to precipitate RNA, and incubate at room temperature for 10min. Centrifuge at 12000g for 10min at 4 ° C and discard the supernatant.
  • RNA pellet was washed once with 1 ml of 75% ethanol (without RNAase), centrifuged at 12000g for 5 min, 4 ° C, and the supernatant was discarded. After the dried RNA was precipitated for 15 min, the RNA was dissolved in 40 ul of DEPC-containing water (purchased from Invitrogen, 46-2224), gently mixed and left at room temperature for 5 min. The RNA of all samples was mixed in half, and the concentration of the obtained RNA library was measured. The result was 2175.6 ng / ul.
  • Preparation of cDNA library Refer to the reverse transcription kit 5 * PrimeScript TM RT Master Mix (purchased from Takara, catalog number RR036A) to prepare a reverse transcription reaction system as shown in the table below, and perform thermal cycling.
  • the setting conditions are 37 °C for 20min, 85 °C for 20s, and 4 °C for continuous.
  • the obtained reverse transcription products were mixed and divided into two parts. One was stored at -80 ° C and one was stored at 4 ° C for subsequent experiments.
  • VH and VL libraries For primer design for amplification, see Journal of Immunological Methods 201 (1997), 35-5. Mix the heavy chain and light chain forward and reverse amplification primers separately, and prepare the PCR reaction as follows:
  • the amplified products were gel purified.
  • the results showed that 230 ng / ul and 170 ng / ul VH and VL were obtained, respectively.
  • the obtained VH and VL libraries were assembled into scFvs by the method of SOE (splicing, overlap, extension) PCR, and the resulting PCR product was purified (QIAquick Gel) / PCR purification kit, purchased from QIAGEN, Catalog No. 28706), and finally the concentration of purified scFv was obtained It is 130ng / ul.
  • phagemids the pCAN vector and scFv were digested with Sfi enzyme (purchased from NEB, catalog number R0123S), and the digested product was recovered by gel, and the obtained pCAN vector and scFv were digested with T4 ligase (purchased from NEB)
  • Sfi enzyme purchased from NEB, catalog number R0123S
  • T4 ligase purchased from NEB
  • the ligation reaction was performed, and the resulting ligation product was purified using a purification kit (purchased from Qiagen, Catalog No. 28014), which was used to prepare an immunophage library.
  • Preparation of immunophage library 500 ng of the DNA purified above was mixed with 200 ul of TG1 competent E. coli (this competent state can be obtained by a conventional method) and electroporated. The electrotransformed product was cultured in 1ml YT culture with shaking for 1h at 37 ° C. After taking 10ul of cell suspension and diluting it with a 10-fold gradient (10 -4 , 10 -5 , 10 -6 ), spread the plate to check the storage capacity.
  • the bacterial pellet was collected by centrifugation, the excess bacterial solution was discarded, the bacterial pellet was resuspended and coated on a plate, and cultured overnight. The next day, the bacterial clones in the culture plate were scraped off, the pellet was collected by centrifugation, and resuspended in 4 ml of 2 * YT medium (containing 40% glycerol), and the resulting immune library was frozen at -80 ° C.
  • the ELISA method was used to determine the binding activity of the scFv antibodies screened to NKG2A / CD94-ECD-Fc and NKG2C / CD94-ECD-Fc.
  • the clones with an OD450nm reading ratio of more than 4 (NKG2A / CD94-ECD-Fc / NKG2C / CD94-ECD-Fc) were selected.
  • the binding activity of the scFv antibody screened to CHOK1 / NKG2A / CD94-ECD-Fc and CHOK1 / NKG2C / CD94 cells was determined by FACS method.
  • Amplification of heavy and light chain variable regions According to the sequencing results of positive clones, the variable regions of light and heavy chains were amplified by PCR, respectively.
  • Configure 50 ⁇ L reaction system including 0.5 ⁇ L plasmid containing the transfected positive clone E. coli TG1, 10pmol of each primer, 25 ⁇ L Q5 high-fidelity DNA polymerase and add water to make up to 50 ⁇ L.
  • the PCR program was set, pre-denatured at 95 ° C for 5 minutes, denatured at 95 ° C for 30 seconds, annealed at 55 ° C for 30 seconds, extended at 68 ° C for 30 seconds, and extended for an additional 68 ° C for 1 minute after 25 cycles to obtain PCR products.
  • the DNA polymerase used for PCR was purchased from NEB under the catalog number E0555L. Take 5 ⁇ L of PCR product for agarose gel electrophoresis detection, and purify the positive test sample using a recovery kit, of which the recovery kit is QIAquick Gel extraction kit, purchased from Qiagen, Catalog No. 28706.
  • Preparation of human IgG4 antibody Perform ligation reaction: insert 3 ⁇ L, digested expression vector 2 ⁇ L, recombinant enzyme Exnase 2 ⁇ L, buffer 4 ⁇ L, reaction system 20 ⁇ L, react at 37 ° C for half an hour to obtain ligation product, ie constructed Recombinant vector.
  • the recombinase was purchased from Vazyme, Catalog No.
  • the buffer was a buffer used to purchase the recombinase
  • the heavy chain variable region was directionally cloned into a heavy chain IgG4 (S228P) containing a signal peptide and a human antibody Constant region expression vector (in which the expression vector was purchased from Invitrogen, the recombination step is a routine step)
  • the light chain variable region was directional cloned into the expression vector containing the signal peptide and human antibody light chain Kappa constant region (where the expression vector was purchased from Invitrogen, the reorganization step is a routine step).
  • a 30 ⁇ L PCR system was configured to perform colony PCR.
  • the colony PCR system is: 1 ⁇ L of each primer, 10 ⁇ L of PCR premix (purchased from Novoprotein), and make up to 20 ⁇ L.
  • Use a pipette tip to dip the colony into the PCR reaction system and blow up, and suck out 0.5 ⁇ L spot on another LB solid petri dish containing 100 ⁇ g / mL ampicillin to preserve the strain.
  • the recombinant vector heavy and light chain expression vectors with correct sequences were transiently transfected into FreeStyle TM 293-F cells (purchased from Invitrogen) to produce antibodies.
  • the density of 293-F cells should be 1-1.5 ⁇ 10 6 cells / mL, and 100 ⁇ g of cells requires 100 ⁇ g of the recombinant vector constructed above (where the mass ratio of recombinant heavy chain vector to light chain vector is 2: 3) and 200 ⁇ g of transfection reagent polyethyleneimine (PEI).
  • Recombinant vector and PEI were added to 5 mL of medium, and allowed to stand at room temperature for 5 minutes.
  • the mixture of recombinant vector and PEI was allowed to stand at room temperature for 15 minutes. Then, the above mixture was slowly added to the cells, and cultured at 120 rpm in a 37 ° C, 8% (v / v) CO 2 incubator. After 7 days, the cell culture solution was centrifuged at 3500 g for 30 minutes, and the supernatant was collected and filtered with a 0.22 ⁇ m filter.
  • the monoclonal antibody in 200 mL of clear supernatant was purified with a 1 mL Protein A column (purchased from GE Healthcare).
  • the protein A column was first equilibrated with equilibration buffer (PBS phosphate buffer, pH 7.2), and then the supernatant was loaded onto the protein A column, and the flow rate was controlled at 3 mL / min. After loading the sample, wash the protein A column with equilibration buffer. The volume of the equilibration buffer is 20 times the volume of the protein A column bed.
  • the monoclonal antibody bound to the protein A column was eluted with an eluent (0.1 M glycine hydrochloric acid buffer, pH 3.0), and the elution status was monitored with an ultraviolet detector (A280 ultraviolet absorption peak).
  • the eluted antibody was collected and added to 10% (v / v) 1.0M Tris-HCl buffer to neutralize the pH. Then immediately dialyzed against PBS phosphate buffer overnight. The monoclonal antibodies after dialysis were collected, sterile filtered with a 0.22 ⁇ m filter, and stored aseptically to obtain purified NKG2A antibody as the lead antibody. The lead antibody was tested and analyzed for protein concentration (A280 / 1.4), purity, and endotoxin (Lonza kit). The results are shown in Table 9 below.
  • the pCHO1.0 plasmid containing the nucleotide sequence encoding the full-length amino acid sequence of human-derived NKG2A / CD94 as described in step (2) of Example 1 was transfected into the CHOK1 cell line to obtain the CHOK1 stable cell line containing human NKG2A and CD94 (this It is called CHOk1-hNKG2A / CD94 stable cell line), and the pCHO1.0 plasmid with the full-length genes of monkey-derived NKG2A and CD94 (the preparation method is the same as that in step 1 of Example 1 (1) "Preparation of immunogen A".
  • the method for preparing the pCpC vector of human IgG Fc fragment (hFc) is the same.
  • CHOk1-hNKG2A / CD94 stable cell line, CHOk1-cNKG2A / CD94 stable cell line and CHOK1-hNKG2C / CD94 stable cell line were expanded to 90% in T-75 cell culture flask Confluence, exhaust the medium, wash twice with PBS buffer (purchased from Invitrogen), and then treat and collect the cells with enzyme-free cell dissociation solution (Versene solution, purchased from Life technology). Wash the cells with PBS buffer 2 times, after counting the cells, dilute the cells to 2 with PBS buffer ⁇ 10 6 cells per ml, add 1% goat serum blocking solution, the percentage is the mass percentage, incubate on ice for 30 minutes, and then wash twice with PBS buffer.
  • PBS buffer purchased from Invitrogen
  • the collected cells are washed with FACS buffer (PBS + 1) % BSA, the percentage is the mass percentage) suspended to 2 ⁇ 10 6 cells / mL, added 100 ⁇ l per well to a 96-well FACS reaction plate, and added the purified NKG2A antibody test sample obtained in Example 2 per well 100 ⁇ l, incubate for 2 hours on ice. Wash twice with FACS buffer centrifugation, add 100 ⁇ l of fluorescent (Alexa 488) labeled secondary antibody (purchased from Invitrogen) per well, and incubate for 1 hour on ice.
  • FACS buffer PBS + 1 % BSA, the percentage is the mass percentage
  • FACS buffer Wash three times by centrifugation, then suspend the cells with 100 ⁇ l of FACS buffer, and use FACS (FACS Calibur, purchased from BD) to detect and analyze the results.
  • FACS Fluorescence Activated Cell Sorting
  • the Octet red96 instrument (purchased from Fortiebio) was used to determine the affinity constant. The specific operation and method are based on the instrument manual and the detailed method provided by the manufacturer. Specifically: Affinity sensor (SA sensor, purchased from Fortiebio) was used for affinity determination.
  • SA sensor purchased from Fortiebio
  • the biotinylated human-derived NKG2A / CD94-ECD-Fc i.e.
  • CHOk1-hNKG2A / CD94 stable cell line was expanded and cultured to 90% confluence in T-75 cell culture flasks, the culture medium was exhausted, washed twice with PBS buffer (purchased from Invitrogen), and then digested with enzyme-free cells
  • PBS buffer purchased from Invitrogen
  • the chaotropic liquid Versene solution, purchased from Life Technology
  • the collected cells were suspended in FACS buffer (PBS + 1% BSA, the percentage is the mass percentage) to 2 ⁇ 10 6 cells / mL, 100 ⁇ l per well was added to the 96-well FACS reaction plate, and washed once by centrifugation After that, the purified NKG2A antibody was diluted with FACS buffer, and the cells were resuspended at 50 ⁇ l per well. At the same time, diluted HLA-E-PE was added to each well, 50 ul per well; incubate 2.5-3 on ice in the dark hour.
  • FACS buffer PBS + 1% BSA, the percentage is the mass percentage
  • the cells were centrifugally washed three times with FACS buffer, and then the cells were suspended with 100 microliters of FACS buffer, and the results were detected and analyzed with FACS (FACS Calibur, purchased from BD). The results are shown in Figure 14.
  • FACS Fluorescence Activated Cell Sorting
  • Example 7 Detection of NKG2A antibody increases the killing effect of NK92 on target cells
  • the LCL721.221 cells were expanded and cultured to 90% confluence in T-75 cell culture flasks, the medium was aspirated, and washed twice with PBS buffer (purchased from Invitrogen). The cells were resuspended to 1 * 10 6 / 500ul in a medium containing 1 mM peptide, and after being cultured in a 26 ° C incubator overnight, they were washed twice with PBS. Resuspend LCL721.221 cells to 2 * 10 5 cells / ml with analysis buffer (1640 medium plus 5% FBS), and add 50ul to 96-well plates to ensure the number of cells per well is 10,000.
  • analysis buffer (1640 medium plus 5% FBS
  • NK92 cells 37 ° C 30min
  • NK92 cells 37 ° C 30min
  • target cell ratio of 2 1, mixed thoroughly with LCL721.221 cells, and centrifuged at 250g for 5 minutes.
  • the 96-well plate was placed in a 37 ° C incubator for 4h.
  • the volume ratio of Ficoll to diluted whole blood is 3: 4, avoid shaking and mixing, centrifuge at 400g at room temperature and 20 ° C for 30 minutes, the centrifuge tube is divided into three layers, the upper layer is plasma, and the middle milky white layer That is, mononuclear lymphocytes, gently absorb the middle layer cells with a sterile pipette, collect into a new centrifuge tube, dilute to three volumes with PBS phosphate buffer, centrifuge at 100g for 10 minutes at room temperature, and discard the supernatant.
  • NK primary NK was isolated from PBMC and cultured for 4 days in vitro.
  • the formula of 1640 medium was as follows: 0.01mm 2-me, 1 * L-Glu, 1 * NEAA, 10% FBS, 10ng / ml IL-2, 20ng / ml IL-12.
  • the LCL721.221 cells were expanded and cultured to 90% confluence in T-75 cell culture flasks, the medium was aspirated, and washed twice with PBS buffer (purchased from Invitrogen). The cells were resuspended to 1 * 10 6 / 500ul in a medium containing 1 mM peptide, and after being cultured in a 26 ° C incubator overnight, they were washed twice with PBS.
  • variable region graft skeleton by sequence alignment (NCBI-Igblast): IGHV1-46 * 01 (60.2%) and IGKV2-28 * 01 (80.0%).
  • the amplification primer was synthesized by Genewiz, and then the variable regions of the light chain and the heavy chain were amplified by PCR method, respectively.
  • Configure 50 ⁇ L reaction system including 50-100ng heavy chain variable region, light chain variable region, forward and reverse primers 1ul, 1ul pfxD enzyme (purchased from invitrogen, 12344-012), 10 * pfx buffer 5ul ( The supplier has the same enzyme as pfx) and water to make up to 50 ⁇ L.
  • the PCR program was set, pre-denatured at 95 ° C for 5 minutes, denatured at 95 ° C for 30 seconds, annealed at 56 ° C for 30 seconds, extended at 68 ° C for 30 seconds, and extended at an additional 68 ° C for 10 minutes after 25 cycles to obtain PCR products.
  • ligation reaction insert 20-40ng, digested expression vector 60-100ng, recombinant enzyme Exnase (purchased from Vazyme, Catalog No. C112-01 / 02) 1 ⁇ L, buffer 2 ⁇ L, reaction system 10 ⁇ L was reacted at 37 ° C for half an hour to obtain the ligation product, that is, the constructed recombinant vector.
  • the buffer is the buffer used to purchase the recombinase; the heavy chain variable region is directionally cloned into an expression vector containing a signal peptide and a human antibody heavy chain IgG4 (S228P) constant region (where the expression vector is purchased from Invitrogen, recombination step) Is a routine step), the light chain variable region is directionally cloned into an expression vector containing a signal peptide and a human antibody light chain kappa constant region (where the expression vector is purchased from Invitrogen, and the recombination step is a conventional step).
  • Add 10 ⁇ L of ligation product to 100 ⁇ L of competent cells (Ecos101 Competent Cells, purchased from Yeasten, Catalog No.
  • the recombinant antibody heavy and light chain expression vectors with correct sequence were amplified, and then FreeStyle TM 293-F cells (purchased from Invitrogen) were transiently transfected to produce antibodies.
  • the density of 293-F cells should be 1-1.2 ⁇ 10 6 cells / mL.
  • 100 ⁇ g of the recombinant vector constructed above and 200 ⁇ g of transfection reagent polyethyleneimine (PEI) are required.
  • Recombinant vector and PEI were added to 5mL medium, and allowed to stand at room temperature for 5 minutes. After filtering with 0.22 ⁇ m filter, the mixture of recombinant vector and PEI was allowed to stand at room temperature for 15 minutes.
  • the above mixture was then slowly added to the cells, and cultured at 130 rpm in a 37 ° C, 8% (v / v) CO 2 incubator.
  • the culture supernatant and cell pellet were taken every day to detect antibody expression.
  • the cell culture solution was centrifuged at 3000 g for 30 minutes, and the supernatant was collected and filtered with a 0.22 ⁇ m filter.
  • the monoclonal antibody in 200 mL of clear supernatant was purified with 1 mL MabSelect TM SuRe TM column (purchased from GE Healthcare).
  • MabSelect TM SuRe TM column is first equilibrated with equilibration buffer (PBS phosphate buffer, pH 7.2), MabSelect TM SuRe TM column. After loading the sample, wash the MabSelect TM SuRe TM column with equilibration buffer. The volume of the equilibration buffer is 5 times the volume of the protein A column bed.
  • the monoclonal antibody bound to the MabSelect TM SuRe TM column was eluted with an eluent (0.1M glycine hydrochloric acid buffer, pH 3.0). The eluted antibody was collected and added to 10% (v / v) 1.0M Tris-HCl buffer to neutralize the pH.
  • Flow cytometry (FACS) detection antibody binding to NKG2A / CD94 expressing cells the results are shown in Figure 18a, Figure 18b, Figure 19a, Figure 19b, Figure 20a, Figure 20b, the resulting antibodies can bind to the cell surface Human NKG2A and monkey NKG2A do not combine human NKG2C with human CD94.
  • 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.
  • NKG2A antibody blocks the binding of NKG2A to its ligand HLA-E.
  • the method is the same as in Example 6.
  • the results are shown in Figure 21.
  • the antibody to be tested can block HLA-E binding to NKG2A protein on the cell surface.
  • the isotype control is human IgG, and the data in the table is the average fluorescence intensity value of the cell population measured by MFI.
  • NKG2A antibody blocks the binding of NKG2A to its ligand HLA-E, in the same way as in Example 7. M15-5, M15-10, and M15-15 were tested. The results are shown in Figure 22. Antibodies can enhance the killing activity of NK92 against tumor cells.
  • NKG2A antibody affinity constant is the same as in Example 5B.

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Abstract

提供了一种靶向NKG2A的抗体、其制备方法和用途。具体地，提供了一种新的靶向NKG2A的鼠源或人源化单克隆抗体以及制备所述的单克隆抗体的方法。该单克隆抗体能够高特异性地结合NKG2A抗原，其具有很高的亲和力并且具有显著抗肿瘤等活性。

Description

NKG2A抗体及其制备方法和应用 技术领域
本发明属于抗体领域，具体地涉及一种NKG2A抗体及其制备方法和应用。
背景技术
保持有效的免疫监视而不引起自身免疫反应需要效应T细胞反应的准确性。当免疫系统针对自身抗原发起免疫反应时便出现自身免疫性疾病。尽管在引发和维持自身免疫性反应中涉及的机制还不清楚，但是可能涉及在次级淋巴器官中以前在免疫方面被忽视的抗原的出现。
自然杀伤(NK)细胞是包括在非传统免疫中涉及的淋巴细胞的亚种群。NK细胞提供一种有效的免疫监督机制，由此可消除不希望的细胞如肿瘤细胞或病毒感染的细胞。NK细胞活性是通过包含两种活化和抑制信号的复杂机制进行调控的。
已证实多种不同的NK特异性受体在NK细胞介导的对HLA I类缺陷型靶细胞的识别和杀伤中起重要作用。这些受体(称为NKp30、NKp46和NKp44)是Ig超家族的成员。它们的交联(通过特异性mAb诱导)导致強烈的NK细胞激活，致使细胞内Ca++水平升高，触发细胞毒性和淋巴因子释放。重要的是，单克隆抗体介导的NKp30、NKp46和/或NKp44的活化导致对于多种靶细胞的NK细胞毒性的激活。这些发现为这些受体在天然细胞毒性中的ー种中心性作用提供了证据。
NK细胞上的另一重要的抑制性受体CD94-NKG2A，其与非典型的MHC I类分子HLA-E相互作用。这些受体中的一些具有调节T细胞抗原受体-依赖性T细胞活化的阈值的能力。在罕见的抑制性受体缺乏的情况下，这些活化同类型(isoform)可能扩大T细胞效应器的功能并且促成自身免疫病理。NKG2A的氨基酸序列在哺乳动物(包括灵长类动物)中发生变化。例如，人NKG2A蛋白的和猕猴同源性少于90％。
对于用于调节NKG2A的疗法的努力(主要用于防止炎症)已集中在对非典型MHCI类分子、用于人类受体的HLA-E和用于小鼠受体的Qa-Ib的研究上。对于细胞表面表达，这些MHC分子优选与来源于其他MHC I类分子的信号肽的肽结合。其他I类MHC分子的表达可以调控HLA-E的表达，因此，允许NK细胞在 潜在的靶细胞中监控MHC I类依赖性抗原呈递途径的状态。细胞表面HLA-E的水平对于针对肿瘤和病毒感染细胞的NK细胞毒性是至关重要的。用于调节HLA-E表达或功能的治疗方案通常集中在利用HLA-I或HSP60肽来诱导用于防止发炎的保护状态，致使NK细胞不被活化。
尽管目前关于靶向NKG2A的抗体已经开展相关研究，但仍需获得活性更强，亲和力更高的特异性抗体。
发明内容
本发明公开了一种NKG2A抗体及其制备方法和应用。所述NKG2A抗体包括NKG2A抗体的重链可变区重链CDR1(VH-CDR1)、重链CDR2(VH-CDR2)和重链CDR3(VH-CDR3)中的一种或多种，和NKG2A抗体的轻链可变区轻链CDR1(VL-CDR1)、轻链CDR2(VL-CDR2)和轻链CDR3(VL-CDR3)中的一种或多种。
在本发明的第一方面，提供了一种抗体的重链可变区，所述的重链可变区具有选自下组的互补决定区CDR：
SEQ ID NO.8n+2所示的VH-CDR1，
SEQ ID NO.8n+3所示的VH-CDR2，和
SEQ ID NO.8n+4所示的VH-CDR3；
其中，各n独立地为0、1、2、3、4、5、6、7或8；
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NKG2A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、5、6、7或8。
在另一优选例中，所述重链可变区具有SEQ ID NO.1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.9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.17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.25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 第一方面述的重链可变区。
在另一优选例中，所述重链还包括重链恒定区。
在另一优选例中，所述重链恒定区为人源或鼠源的。
在本发明的第三方面，提供了一种抗体的轻链可变区，所述的轻链可变区具有选自下组的互补决定区CDR：
SEQ ID NO.8n+6所示的VL-CDR1，
SEQ ID NO.8n+7所示的VL-CDR2，和
SEQ ID NO.8n+8所示的VL-CDR3；
其中，各n独立地为0、1、2、3、4、5、6、7或8；
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NKG2A结合亲和力的衍生序列。
在另一优选例中，所述轻链可变区具有SEQ ID NO.8n+5所示的氨基酸序列，其中，n为0、1、2、3、4、5、6、7或8。
在另一优选例中，所述轻链可变区具有SEQ ID NO.5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.13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.21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.29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。
在另一优选例中，所述轻链还包括轻链恒定区。
在另一优选例中，所述轻链恒定区为人源或鼠源的。
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NKG2A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NKG2A结合的亲和力。
在另一优选例中，所述的衍生抗体与NKG2A结合的亲和力F1与相应非衍生的抗体与NKG2A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NKG2A结合亲和力的衍生序列为同源性或序列相同性为至少96％的氨基酸序列。
在另一优选例中，所述的抗体还包括重链恒定区和/或轻链恒定区。
在另一优选例中，所述的重链恒定区为人源的，和/或所述的轻链恒定区为人源的。
在另一优选例中，所述抗体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：动物源抗体、嵌合抗体、人源化抗体、全人抗体、或其组合。
在另一优选例中，所述的嵌合抗体在人中的免疫原性Z1与非嵌合的抗体(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，所述的抗体为药物偶联物形式。
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，所述的重链可变区和所述的轻链可变区包括选自下组的CDR：
Figure PCTCN2019116060-appb-000001
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NKG2A结合亲和力的衍生序列。
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VH-CDR1，
SEQ ID NO.3所示的VH-CDR2，和
SEQ ID NO.4所示的VH-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VL-CDR1，
SEQ ID NO.7所示的VL-CDR2，和
SEQ ID NO.8所示的VL-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VH-CDR1，
SEQ ID NO.11所示的VH-CDR2，和
SEQ ID NO.12所示的VH-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VL-CDR1，
SEQ ID NO.15所示的VL-CDR2，和
SEQ ID NO.16所示的VL-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VH-CDR1，
SEQ ID NO.19所示的VH-CDR2，和
SEQ ID NO.20所示的VH-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VL-CDR1，
SEQ ID NO.23所示的VL-CDR2，和
SEQ ID NO.24所示的VL-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.26所示的VH-CDR1，
SEQ ID NO.27所示的VH-CDR2，和
SEQ ID NO.28所示的VH-CDR3；
所述的轻链可变区包括以下三个互补决定区CDR：
SEQ ID NO.30所示的VL-CDR1，
SEQ ID NO.31所示的VL-CDR2，和
SEQ ID NO.32所示的VL-CDR3。
在另一优选例中，所述抗体的重链可变区含有SEQ ID NO.1、9、17、25、33、41、49、57或65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.5、13、21、29、37、45、53、61或69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；且所述抗体的轻链可变区含有SEQ ID NO.5所示的氨基酸序列。
在另一优选例中，所述抗体的重链可变区含有SEQ ID NO.9所示的氨基酸序列；且所述抗体的轻链可变区含有SEQ ID NO.13所示的氨基酸序列。
在另一优选例中，所述抗体的重链可变区含有SEQ ID NO.17所示的氨基酸序 列；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；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，所述的抗体选自下组：
抗体编号 克隆 VH序列编号 VL序列编号
1 M15-5 1 5
2 Mpb416 9 13
3 Mab031 17 21
4 Mab032 25 29
5 Mab033 33 37
6 Mab036 41 45
7 2F10 49 53
8 9B10 57 61
9 14G3 65 69
。
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、9、17、25、33、41、49、57或65所示的氨基酸序列至少有80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的序列同源性或序列相同性。
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、13、21、29、37、45、53、61或69所示的氨基酸序列至少有80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的序列同源性或序列相同性。
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)选自下组的抗体，
抗体编号 克隆 VH序列编号 VL序列编号
1 M15-5 1 5
2 Mpb416 9 13
3 Mab031 17 21
4 Mab032 25 29
5 Mab033 33 37
6 Mab036 41 45
7 2F10 49 53
8 9B10 57 61
9 14G3 65 69
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.73、75、77、79、81、83、85、87或89所示；和/或，编码所述轻链可变区的多核苷酸如SEQ ID NO.74、76、78、80、82、84、86、88或90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和编码所述轻链可变区序列的多核苷酸选自下组：
Figure PCTCN2019116060-appb-000002
Figure PCTCN2019116060-appb-000003
在本发明的第八方面，提供了一种载体，所述载体含有本发明的第七方面所述的多核苷酸。
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所述的多核苷酸。
在本发明的第十方面，提供了一种抗体偶联物，该抗体偶联物含有：
(a)抗体部分，所述抗体部分选自下组：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所述的抗体、或其组合；和
(b)与所述抗体部分偶联的偶联部分，所述偶联部分选自下组：可检测标记物、药物、毒素、细胞因子、放射性核素、酶、或其组合。
在另一优选例中，所述的抗体部分与所述的偶联部分通过化学键或接头进行偶联。
在本发明的第十一方面，提供了一种免疫细胞，所述免疫细胞表达或在细胞膜外暴露有本发明的第五方面所述的抗体。
在另一优选例中，所述的免疫细胞包括NK细胞、T细胞。
在另一优选例中，所述的免疫细胞来自人或非人哺乳动物(如鼠)。
在本发明的第十二方面，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抗体偶联物、本发明的第十一方面所述的免疫细胞、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抗体偶联物、本发明的第十一方面所述的免疫细胞、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。
在本发明的第十三方面，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抗体偶联物、本发明的第十一方面所述的免疫细胞、或其组合，其中所述活性成分被用于(a)制备诊断试剂或试剂盒；和/或(b)制备预防和/或治疗与NKG2A表达或功能异常相关的疾病的药物。
在另一优选例中，所述的诊断试剂为检测片或检测板。
在另一优选例中，所述NKG2A表达或功能异常相关的疾病选自下组：癌症、自身免疫疾病，炎性疾病。
在另一优选例中，所述诊断试剂或试剂盒用于：
(1)检测样品中NKG2A蛋白；和/或
(2)检测肿瘤细胞中内源性的NKG2A蛋白；和/或
(3)检测表达NKG2A蛋白的肿瘤细胞；
而所述药物用于预防和/或治疗与NKG2A表达或功能异常相关的疾病，所述与NKG2A表达或功能异常相关的疾病为癌症、自身免疫疾病，炎性疾病。
在另一优选例中，所述癌症选自下组：淋巴细胞白血病，卵巢癌，非小细胞 肺癌，头颈癌。
在另一优选例中，所述癌症为HLA-E过表达的癌症。
在另一优选例中，所述癌症为HLA-E过表达的癌症，所述癌症选自下组：淋巴细胞白血病，卵巢癌，非小细胞肺癌，头颈癌。
在另一优选例中，所述的抗体为药物偶联物(ADC)形式。
在另一优选例中，所述的诊断试剂或试剂盒用于诊断NKG2A相关疾病。
在另一优选例中，所述的诊断试剂或试剂盒用于检测样品中NKG2A蛋白。
在本发明的第十四方面，提供了一种体外检测(包括诊断性或非诊断性)样品中NKG2A蛋白的方法，所述方法包括步骤：
(1)在体外，将所述样品与如本发明的第五方面所述的抗体接触；
(2)检测是否形成抗原-抗体复合物，其中形成复合物就表示样品中存在NKG2A蛋白。
在本发明的第十五方面，提供了一种体外检测样品中NKG2A蛋白的组合物，其包括如本发明的第五方面所述的抗体、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抗体偶联物、本发明的第十一方面所述的免疫细胞、或其组合作为活性成分。
在本发明的第十六方面，提供了一种检测板，所述的检测板包括：基片(支撑板)和测试条，所述的测试条含有如本发明的第五方面所述的抗体、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抗体偶联物、本发明的第十一方面所述的免疫细胞、或其组合。
在本发明的第十七方面，提供了一种试剂盒，所述试剂盒中包括：
(1)第一容器，所述第一容器中含有本发明的抗体；和/或
(2)第二容器，所述第二容器中含有抗本发明抗体的二抗；
或者，
所述试剂盒含有如本发明的第十六方面所述的检测板。
在本发明的第十八方面，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。
在本发明的第十九方面，提供了一种药物组合，包括：
(i)第一活性成分，所述第一活性成分包括如本发明的第五方面所述的抗体1、或如本发明的第六方面所述的重组蛋白、或如本发明的第十方面所述的抗体偶联物、或如本发明的第十一方面所述的免疫细胞、或如本发明的第十二方面所述的药物组合物、或其组合；
(ii)第二活性成分，所述第二活性成分包括第二抗体、或化疗剂。
在另一优选例中，所述第二抗体选自下组：CTLA4抗体、PD-1抗体。
在另一优选例中，所述的第二抗体为PD-1抗体。
在另一优选例中，所述化疗剂选自下组：多西他赛、卡铂、或其组合。
在本发明的第二十方面，提供了本发明的第五方面所述的抗体，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抗体偶联物、或本发明的第十一方面所述的免疫细胞、和/或本发明的第十二方面所述的药物组合物与第二抗体或化疗剂的组合在制备用于治疗NKG2A表达或功能异常相关的疾病的药物中的用途。
在另一优选例中，所述第二抗体选自下组：CTLA4抗体、PD-1抗体。
在另一优选例中，所述的第二抗体为PD-1抗体。
在本发明的第二十一方面，提供了一种治疗与NKG2A表达或功能异常相关的疾病的方法，向有需要的对象施用有效量的如本发明的第五方面所述的抗体、或如本发明的第六方面所述的重组蛋白、或如本发明的第十方面所述的抗体偶联物、或如本发明的第十一方面所述的免疫细胞、或如本发明的第十二方面所述的药物组合物、或其组合。
在另一优选例中，所述与NKG2A表达或功能异常相关的疾病为癌症、自身免疫疾病，炎性疾病。
在另一优选例中，所述癌症选自下组：淋巴细胞白血病，卵巢癌，非小细胞肺癌，头颈癌。
在另一优选例中，所述癌症为HLA-E过表达的癌症。
在另一优选例中，所述癌症为HLA-E过表达的癌症，所述癌症选自下组：淋巴细胞白血病，卵巢癌，非小细胞肺癌，头颈癌。
在另一优选例中，所述的方法还包括：在施用第一活性成分之前、之中和/或之后，向所述对象施用安全有效量的第二抗体。
在另一优选例中，所述的第二抗体选自下组：PD-1抗体、CTLA4抗体。
在另一优选例中，所述的第二抗体为PD-1抗体。
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为对照抗体与NKG2A/CD94蛋白的结合活性。
图2a为ELISA检测免疫后Balb/c小鼠血清抗体效价。
图2b为FACS检测免疫后Balb/c小鼠血清抗体效价。
图3a为ELISA检测免疫后SJL小鼠血清抗体效价。
图3b为FACS检测免疫后SJL小鼠血清抗体效价。
图4为动物免疫的技术流程。
图5a为ELISA检测免疫后Balb/c小鼠血清抗体效价。
图5b为ELISA检测免疫后SJL小鼠血清抗体效价。
图6a为FACS检测免疫后Balb/c小鼠血清抗体效价，其中，cyno是指猕猴。
图6b为FACS检测免疫后SJL小鼠血清抗体效价。
图7为动物免疫的技术流程。
图8a为ELISA检测免疫后Balb/c小鼠血清抗体效价。
图8b为ELISA检测免疫后SJL小鼠血清抗体效价。
图9a为FACS检测免疫后Balb/c小鼠血清抗体效价。
图9b为FACS检测免疫后SJL小鼠血清抗体效价。
图10为FACS检测NKG2A抗体与CHOK1-hNKG2A/CD94的结合反应。
图11为FACS检测NKG2A抗体与CHOK1-cNKG2A/CD94的结合反应。
图12为FACS检测NKG2A抗体与CHOK1-hNKG2C/CD94的结合反应。
图13为ELISA检测NKG2A抗体与NKG22E的结合反应。
图14为FACS检测抗体的阻断活性。
图15为NKG2A抗体在NK92杀伤实验中的活性检测(抗体增强NK92细胞的杀伤活性)。
图16为NKG2A抗体在原代NK细胞杀伤实验中的活性检测(PBMC供体-1)抗体增强原代NK细胞的杀伤活性，供体1。
图17为NKG2A抗体在原代NK细胞杀伤实验中的活性检测(PBMC供体-2)抗体增强原代NK细胞的杀伤活性，供体2。
图18a为FACS检测人源化NKG2A抗体与CHOK1-hNKG2A/CD94的结合反应。
图18b为FACS检测人源化NKG2A抗体与CHOK1-hNKG2A/CD94的结合反应。
图19a为FACS检测人源化NKG2A抗体与CHOK1-hNKG2C/CD94的结合反应。
图19b为FACS检测人源化NKG2A抗体与CHOK1-hNKG2C/CD94的结合反应。
图20a为FACS检测人源化NKG2A抗体与CHOK1-cNKG2A/CD94的结合反应。
图20b为FACS检测人源化NKG2A抗体与CHOK1-cNKG2A/CD94的结合反应。
图21为FACS检测人源化NKG2A抗体的阻断活性，阻断HLA-E与NKG2A/CD94细胞的结合。
图22人源化NKG2A抗体增强NK92的杀伤活性。
具体实施方式
本发明人通过广泛而深入的研究，分别通过噬菌体展示技术和杂交瘤技术，意外地获得一组具有全新氨基酸序列的人鼠嵌合以及人源化的NKG2A抗体(例如M15-5、Mpb416)。本发明所述NKG2A抗体可以和人NKG2A结合，具有高 亲和力(KD达到1.04×10 -11)(本发明所获抗体较阳性参比抗体的亲和力高出18倍)；并且所述NKG2A抗体能够结合NKG2A蛋白受体的胞外区，并能够在细胞水平有效封闭NKG2A蛋白，阻止NKG2A蛋白与配体HLA-E的结合；所述NKG2A抗体能够显著增加NK92或人NK细胞对靶细胞的杀伤效果；基于原代NK细胞活性实验检测，其生物学活性显著优于参比抗体。此外，本发明所述的NKG2A抗体特异性高，缺乏与人NKG2C和NKG2E同家族蛋白抗原的交叉反应。本发明所述的NKG2A抗体能够用于阻断NKG2A/HLA-E介导的信号通路的负调控、激活机体对肿瘤的天然免疫反应、单独或与抗PD-1等单克隆抗体或其它抗肿瘤药物联合的肿瘤免疫治疗中。本发明抗体能够运用于治疗肿瘤、自身免疫性疾病等药物的制备中。在此基础上完成了本发明。
术语
NKG2A
自然杀伤细胞(NK)是体内一类十分重要的淋巴细胞，在天然免疫和获得性免疫中均发挥重要作用。在NK细胞表面存在两类表面受体，根据其功能可分为抑制型和激活型两类受体，分别介导NK细胞的不同识别模式，传递不同的活化信号和抑制信号。CD94/NKG2家族是研究较多的一类受体家族，主要包括NKG2A，NKG2B，NKG2C，NKG2D，NKG2E，NKG2F，NKG2H等成员。其中，NKG2A属抑制性受体，其配体是非经典主要组织相容性复合物I类分子-HLA-E。表达于靶细胞上的HLA-E分子与NKG2A结合后，对NK细胞的杀伤功能具有抑制作用。因此抑制CD94/NKG2A的抗体可能提高肿瘤特异性淋巴细胞对肿瘤细胞的杀伤活性。另外，某些淋巴瘤例如，NK-淋巴瘤，特征为CD94/NKG2A表达。在这样的患者中，靶向和杀死CD94/NKG2A-表达细胞的治疗性抗体可能能够根除肿瘤细胞。
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1进行氨基酸替换而产生。
表1
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NKG2A的抗体
本发明中，所述抗体为抗NKG2A的抗体。本发明提供一种针对NKG2A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，
所述的重链可变区(VH)具有选自下组的互补决定区CDR：
SEQ ID NO.8n+2所示的VH-CDR1，
SEQ ID NO.8n+3所示的VH-CDR2，和
SEQ ID NO.8n+4所示的VH-CDR3；
其中，各n独立地为0、1、2、3、4、5、6、7或8；
所述的轻链可变区(VL)具有选自下组的互补决定区CDR：
SEQ ID NO.8n+6所示的VL-CDR1，
SEQ ID NO.8n+7所示的VL-CDR2，和
SEQ ID NO.8n+8所示的VL-CDR3；
其中，各n独立地为0、1、2、3、4、5、6、7或8；
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NKG2A结合亲和力的衍生序列。
优选地，重链可变区(VH)包括以下三个互补决定区CDR：
SEQ ID NO.8n+2所示的VH-CDR1，
SEQ ID NO.8n+3所示的VH-CDR2，和
SEQ ID NO.8n+4所示的VH-CDR3；
轻链可变区(VL)包括以下三个互补决定区CDR：
SEQ ID NO.8n+6所示的VL-CDR1，
SEQ ID NO.8n+7所示的VL-CDR2，和
SEQ ID NO.8n+8所示的VL-CDR3；
各n独立地为0、1、2、3、4、5、6、7或8；较佳地n为0或1；
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NKG2A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。
较佳地，本文所述抗体为抗体全长蛋白、抗原抗体结合域蛋白质片段、双特异性抗体、多特异性抗体、单链抗体(single chain antibody fragment，scFv)、单域抗体(single domain antibody，sdAb)和单区抗体(Signle-domain antibody)中的一种或多种，以及上述抗体所制得的单克隆抗体或多克隆抗体。所述单克隆抗体可以由多种途径和技术进行研制，包括杂交瘤技术、噬菌体展示技术、单淋巴细胞基因克隆技术等，主流是通过杂交瘤技术从野生型或转基因小鼠制备单克隆抗体。
所述的抗体全长蛋白为本领域常规的抗体全长蛋白，其包括重链可变区、轻链可变区、重链恒定区和轻链恒定区。所述的蛋白质的重链可变区和轻链可变区与人源重链恒定区和人源轻链恒定区构成全人源抗体全长蛋白。较佳地，所述的抗体全长蛋白为IgG1、IgG2、IgG3或IgG4。
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。
所述的单链抗体为本领域常规的单链抗体，其包括重链可变区、轻链可变区和15～20个氨基酸的短肽。
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NKG2A(例如人NKG2A)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，所述抗体的重链可变区含有SEQ ID NO.1、9、17、25、33、41、49、57或65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、13、21、29、37、45、53、61或69所示的氨基酸序列。
在另一优选例中，所述靶向NKG2A的抗体的重链可变区(VH)氨基酸序列，和/或，轻链可变区氨基酸序列如下表2-1所示：
表2-1
Figure PCTCN2019116060-appb-000004
Figure PCTCN2019116060-appb-000005
在另一优选例中，所述靶向NKG2A的抗体为M15-5、Mpb416、Mab031、Mab032、Mab033、Mab036、2F10、9B10、14G3。
在另一优选例中，所述靶向NKG2A的抗体为M15-5。
在另一优选例中，所述靶向NKG2A的抗体为Mpb416。
重组蛋白
本发明还提供一种重组蛋白，其包括NKG2A抗体的重链CDR1(VH-CDR1)、重链CDR2(VH-CDR2)和重链CDR3(VH-CDR3)中的一种或多种，和/或，NKG2A抗体的轻链CDR1(VL-CDR1)、轻链CDR2(VL-CDR2)和轻链CDR3(VL-CDR3)中的一种或多种，
所述重链CDR1-3的序列如下：
SEQ ID NO.8n+2所示的VH-CDR1，
SEQ ID NO.8n+3所示的VH-CDR2，
SEQ ID NO.8n+4所示的VH-CDR3；
所述轻链CDR1-3的序列如下：
SEQ ID NO.8n+6所示的VL-CDR1，
SEQ ID NO.8n+7所示的VL-CDR2，和
SEQ ID NO.8n+8所示的VL-CDR3；
各n独立地为0、1、2、3、4、5、6、7或8、；较佳地n为0；
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NKG2A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。
在另一优选例中，本发明所述的重组蛋白包括NKG2A抗体的重链可变区和/或NKG2A抗体的轻链可变区，所述抗体的重链可变区含有SEQ ID NO.1、9、17、25、33、41、49、57或65所示的氨基酸序列；所述抗体的轻链可变区含有SEQ ID NO.5、13、21、29、37、45、53、61或69所示的氨基酸序列。
在另一优选例中，本发明所述的重组蛋白包括NKG2A抗体的重链可变区和NKG2A抗体的轻链可变区，所述抗体的重链可变区含有SEQ ID NO.1、9、17、25、33、41、49、57或65所示的氨基酸序列，且所述抗体的轻链可变区含有SEQ ID NO.5、13、21、29、37、45、53、61或69所示的氨基酸序列。
在另一优选例中，所述重组蛋白及其包括的重链CDR1-3、轻链CDR1-3的氨基酸序列的序列编号如表2-2所示：
表2-2重链CDR1-3、轻链CDR1-3的氨基酸序列的序列编号
Figure PCTCN2019116060-appb-000006
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NKG2A结合亲和力的衍生序列。
较佳地，所述的重组蛋白还包括抗体重链恒定区和/或抗体轻链恒定区，所述的抗体重链恒定区为本领域常规，较佳地为大鼠源抗体重链恒定区或人源抗体重链恒定区，更佳地为人源抗体重链恒定区。所述的抗体轻链恒定区为本领域常规，较佳地为大鼠源轻链抗体恒定区或人源抗体轻链恒定区，更佳地为人源抗体轻链恒定区。
所述的重组蛋白为本领域常规的蛋白质，较佳地，其为抗体全长蛋白、抗 原抗体结合域蛋白质片段、双特异性抗体、多特异性抗体、单链抗体(single chain antibody fragment，scFv)、单域抗体(single domain antibody，sdAb)和单区抗体(Signle-domain antibody)中的一种或多种，以及上述抗体所制得的单克隆抗体或多克隆抗体。所述单克隆抗体可以由多种途径和技术进行研制，包括杂交瘤技术、噬菌体展示技术、单淋巴细胞基因克隆技术等，主流是通过杂交瘤技术从野生型或转基因小鼠制备单克隆抗体。
所述的抗体全长蛋白为本领域常规的抗体全长蛋白，其包括重链可变区、轻链可变区、重链恒定区和轻链恒定区。所述的蛋白质的重链可变区和轻链可变区与人源重链恒定区和人源轻链恒定区构成全人源抗体全长蛋白。较佳地，所述的抗体全长蛋白为IgG1、IgG2、IgG3或IgG4。
所述的单链抗体为本领域常规的单链抗体，其包括重链可变区、轻链可变区和15～20个氨基酸的短肽。
所述的抗原抗体结合域蛋白质片段为本领域常规的抗原抗体结合域蛋白质片段，其包括轻链可变区、轻链恒定区和重链恒定区的Fd段。较佳地，所述的抗原抗体结合域蛋白质片段为Fab和F(ab’)。
所述的单域抗体为本领域常规的单域抗体，其包括重链可变区和重链恒定区。
所述的单区抗体为本领域常规的单区抗体，其仅包括重链可变区。
其中，所述重组蛋白的制备方法为本领域常规的制备方法。所述制备方法较佳地为：从重组表达该蛋白质的表达转化体中分离获得或者通过人工合成蛋白质序列获得。所述的从重组表达该蛋白质的表达转化体中分离获得优选如下方法：将编码所述蛋白质并且带有点突变的核酸分子克隆到重组载体中，将所得重组载体转化到转化体中，得到重组表达转化体，通过培养所得重组表达转化体，即可分离纯化获得所述重组蛋白。
核酸
本发明还提供一种核酸，其编码上述的抗体(例如抗NKG2A的抗体)或重组蛋白或抗NKG2A的抗体的重链可变区或轻链可变区。
较佳地，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID NO.73、 75、77、79、81、83、85、87或89所示；和/或，编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID NO.74、76、78、80、82、84、86、88或90所示。
更佳地，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID NO.73、75、77、79、81、83、85、87或89所示；且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID NO.74、76、78、80、82、84、86、88或90所示。
所述核酸的制备方法为本领域常规的制备方法，较佳地，包括以下的步骤：通过基因克隆技术获得编码上述蛋白质的核酸分子，或者通过人工全序列合成的方法得到编码上述蛋白质的核酸分子。
本领域技术人员知晓，编码上述蛋白质的氨基酸序列的碱基序列可以适当引入替换、缺失、改变、插入或增加来提供一个多聚核苷酸的同系物。本发明中多聚核苷酸的同系物可以通过对编码该蛋白序列基因的一个或多个碱基在保持抗体活性范围内进行替换、缺失或增加来制得。
载体
本发明还提供一种包含所述核酸的重组表达载体。
其中所述重组表达载体可通过本领域常规方法获得，即：将本发明所述的核酸分子连接于各种表达载体上构建而成。所述的表达载体为本领域常规的各种载体，只要其能够容载前述核酸分子即可。所述载体较佳地包括：各种质粒、粘粒、噬菌体或病毒载体等。
本发明还提供一种包含上述重组表达载体的重组表达转化体。
其中，所述重组表达转化体的制备方法为本领域常规的制备方法，较佳地为：将上述重组表达载体转化至宿主细胞中制得。所述的宿主细胞为本领域常规的各种宿主细胞，只要能满足使上述重组表达载体稳定地自行复制，且所携带所述的核酸可被有效表达即可。较佳地，所述宿主细胞为E.coli TG1或E.coli BL21细胞(表达单链抗体或Fab抗体)，或者HEK293或CHO细胞(表达全长IgG抗体)。将前述重组表达质粒转化至宿主细胞中，即可得本发明优选的重组表达转化体。其中所述转化方法为本领域常规转化方法，较佳地为化学转化法，热激法或电转法。
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。
抗体-药物偶联物(ADC)
本发明还提供了基于本发明抗体的抗体偶联药物(antibody-drug conjugate，ADC)。
典型地，所述抗体偶联药物包括所述抗体、以及效应分子，所述抗体与所述效应分子偶联，并优选为化学偶联。其中，所述效应分子优选为具有治疗活性的药物。此外，所述效应分子可以是毒蛋白、化疗药物、小分子药物或放射性核素中的一种或多种。
本发明抗体与所述效应分子之间可以是通过偶联剂进行偶联。所述偶联剂的例子可以是非选择性偶联剂、利用羧基的偶联剂、肽链、利用二硫键的偶联剂中的任意一种或几种。所述非选择性偶联剂是指使效应分子和抗体形成共价键连接的化合物，如戊二醛等。所述利用羧基的偶联剂可以是顺乌头酸酐类偶联剂(如顺乌头酸酐)、酰基腙类偶联剂(偶联位点为酰基腙)中的任意一种或几种。
抗体上某些残基(如Cys或Lys等)用于与多种功能基团相连，其中包括成像试剂(例如发色基团和荧光基团)，诊断试剂(例如MRI对比剂和放射性同位素)，稳定剂(例如乙二醇聚合物)和治疗剂。抗体可以被偶联到功能剂以形成抗体-功能剂的偶联物。功能剂(例如药物，检测试剂，稳定剂)被偶联(共价连接)至抗体上。功能剂可以直接地、或者是通过接头间接地连接于抗体。
抗体可以偶联药物从而形成抗体药物偶联物(ADCs)。典型地，ADC包含位于药物和抗体之间的接头。接头可以是可降解的或者是不可降解的接头。可降解的接头典型地在细胞内环境下容易降解，例如在目标位点处接头发生降解，从而使药物从抗体上释放出来。合适的可降解的接头包括，例如酶降解的接头，其中包括可以被细胞内蛋白酶(例如溶酶体蛋白酶或者内体蛋白酶)降解的含有肽基的接头，或者糖接头例如，可以被葡糖苷酸酶降解的含葡糖苷酸的接头。肽基接头可以包括，例如二肽，例如缬氨酸-瓜氨酸，苯丙氨酸-赖氨酸或者缬 氨酸-丙氨酸。其它合适的可降解的接头包括，例如，pH敏感接头(例如pH小于5.5时水解的接头，例如腙接头)和在还原条件下会降解的接头(例如二硫键接头)。不可降解的接头典型地在抗体被蛋白酶水解的条件下释放药物。
连接到抗体之前，接头具有能够和某些氨基酸残基反应的活性反应基团，连接通过活性反应基团实现。巯基特异性的活性反应基团是优选的，并包括：例如马来酰亚胺类化合物，卤代酰胺(例如碘、溴或氯代的)；卤代酯(例如碘、溴或氯代的)；卤代甲基酮(例如碘、溴或氯代)，苄基卤代物(例如碘、溴或氯代的)；乙烯基砜，吡啶基二硫化物；汞衍生物例如3,6-二-(汞甲基)二氧六环，而对离子是醋酸根、氯离子或者硝酸根；和聚亚甲基二甲基硫醚硫代磺酸盐。接头可以包括，例如，通过硫代丁二酰亚胺连接到抗体上的马来酰亚胺。
药物可以是任何细胞毒性，抑制细胞生长或者免疫抑制的药物。在实施方式中，接头连接抗体和药物，而药物具有可以和接头成键的功能性基团。例如，药物可以具有可以和连接物成键的氨基，羧基，巯基，羟基，或者酮基。在药物直接连接到接头的情况下，药物在连接到抗体之前，具有反应的活性基团。
有用的药物类别包括，例如，抗微管蛋白药物、DNA小沟结合试剂、DNA复制抑制剂、烷化试剂、抗生素、叶酸拮抗物、抗代谢药物、化疗增敏剂、拓扑异构酶抑制剂、长春花生物碱等。特别有用的细胞毒性药物类的例子包括，例如，DNA小沟结合试剂、DNA烷基化试剂、和微管蛋白抑制剂、典型的细胞毒性药物包括、例如奥瑞他汀(auristatins)、喜树碱(camptothecins)、多卡霉素/倍癌霉素(duocarmycins)、依托泊甙(etoposides)、美登木素(maytansines)和美登素类化合物(maytansinoids)(例如DM1和DM4)、紫杉烷(taxanes)、苯二氮卓类(benzodiazepines)或者含有苯二氮卓的药物(benzodiazepine containing drugs)(例如吡咯并[1,4]苯二氮卓类(PBDs)，吲哚啉苯并二氮卓类(indolinobenzodiazepines)和噁唑烷并苯并二氮卓类(oxazolidinobenzodiazepines))和长春花生物碱(vinca alkaloids)。
在本发明中，药物-接头可以用于在一个简单步骤中形成ADC。在其它实施方式中，双功能连接物化合物可以用于在两步或多步方法中形成ADC。例如，半胱氨酸残基在第一步骤中与接头的反应活性部分反应，并且在随后的步骤中，接头上的功能性基团与药物反应，从而形成ADC。
通常，选择接头上功能性基团，以利于特异性地与药物部分上的合适的反应活性基团进行反应。作为非限制性的例子，基于叠氮化合物的部分可以用于特异性地与药物部分上的反应性炔基基团反应。药物通过叠氮和炔基之间的1,3-偶极环加成，从而共价结合于接头。其它的有用的功能性基团包括，例如酮类和醛类(适合与酰肼类和烷氧基胺反应)，膦(适合与叠氮反应)；异氰酸酯和异硫氰酸酯(适合与胺类和醇类反应)；和活化的酯类，例如N-羟基琥珀酰亚胺酯(适合与胺类和醇类反应)。这些和其它的连接策略，例如在《生物偶联技术》，第二版(Elsevier)中所描述的，是本领域技术人员所熟知的。本领域技术人员能够理解，对于药物部分和接头的选择性反应，当选择了一个互补对的反应活性功能基团时，该互补对的每一个成员既可以用于接头，也可以用于药物。
本发明还提供了制备ADC的方法，可进一步地包括：将抗体与药物-接头化合物，在足以形成抗体偶联物(ADC)的条件下进行结合。
在某些实施方式中，本发明方法包括：在足以形成抗体-接头偶联物的条件下，将抗体与双功能接头化合物进行结合。在这些实施方式中，本发明方法还进一步地包括：在足以将药物部分通过接头共价连接到抗体的条件下，将抗体接头偶联物与药物部分进行结合。
在一些实施方式中，抗体药物偶联物ADC如下分子式所示：
Figure PCTCN2019116060-appb-000007
其中：
Ab是抗体，
LU是接头；
D是药物；
而且下标p是选自1到8的值。
应用
本发明还提供了本发明抗体、抗体偶联物ADC、重组蛋白、和/或免疫细胞的用途，例如用于制备诊断制剂或制备药物。
较佳地，所述的药物是用于预防和/或治疗与NKG2A表达或功能异常相关的疾病的药物。
本发明中，所述与NKG2A表达或功能异常相关的疾病是本领域常规的与NKG2A表达或功能异常相关的疾病。较佳地，所述与NKG2A表达或功能异常相关的疾病为癌症、自身免疫疾病，炎性疾病。
本发明中，所述癌症为本领域常规的癌症，较佳地为HLA-E过表达的癌症，例如HLA-E过表达的淋巴细胞白血病，卵巢癌，非小细胞肺癌，头颈癌等。
本发明中，所述自身免疫疾病为本领域常规的自身免疫疾病，较佳地为溶血性贫血，恶性贫血，结节性多动脉炎，系统性红斑狼疮，阿尔兹海默症，糖尿病等。
本发明抗体、ADC、重组蛋白、和/或免疫细胞的用途，包括(但并不限于)：
(i)诊断、预防和/或治疗肿瘤发生、生长和/或转移，尤其是NKG2A高表达的肿瘤。所述肿瘤包括(但并不限于)：较佳地为HLA-E过表达的癌症，例如HLA-E过表达的淋巴细胞白血病，卵巢癌，非小细胞肺癌，头颈癌等。
(ii)诊断、预防和/或治疗自身免疫疾病，所述自身免疫疾病包括(但并不限于)：较佳地为溶血性贫血，恶性贫血，结节性多动脉炎，系统性红斑狼疮，阿尔兹海默症，糖尿病等。
(iii)诊断、预防和/或治疗炎性疾病，所述炎性疾病包括(但并不限于)：类风湿关节炎，血管球性肾炎，重症肌无力，多发性硬化，斑秃等。
检测用途和试剂盒
本发明的抗体或其ADC可用于检测应用，例如用于检测样本，从而提供诊断信息。
本发明中，所采用的样本(样品)包括细胞、组织样本和活检标本。本发明使用的术语“活检”应包括本领域技术人员已知的所有种类的活检。因此本发明中使用的活检可以包括例如肿瘤的切除样本、通过内窥镜方法或器官的穿刺或针刺活检制备的组织样本。
本发明中使用的样本包括固定的或保存的细胞或组织样本。
本发明还提供了一种指含有本发明的抗体(或其片段)的试剂盒，在本发明 的一个优选例中，所述的试剂盒还包括容器、使用说明书、缓冲剂等。在优选例中，本发明的抗体可以固定于检测板。
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或其ADC或相应的免疫细胞，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。典型地，本发明所述的药物组合物的给药途径较佳地为注射给药或口服给药。所述注射给药较佳地包括静脉注射、肌肉注射、腹腔注射、皮内注射或皮下注射等途径。所述的药物组合物为本领域常规的各种剂型，较佳地为固体、半固体或液体的形式，可以为水溶液、非水溶液或混悬液，更佳地为片剂、胶囊、颗粒剂、注射剂或输注剂等。
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。
本发明所述的药物组合物是用于预防和/或治疗与NKG2A表达或功能异常相关的疾病的药物组合物。
本发明的药物组合物可直接用于结合NKG2A蛋白分子，因而可用于预防和治疗肿瘤等疾病。
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(或其偶联物)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。
本发明中，较佳地，本发明所述的药物组合物还包括一种或多种药用载体。所述的药用载体为本领域常规药用载体，所述的药用载体可以为任意合适的生理学或药学上可接受的药物辅料。所述的药物辅料为本领域常规的药物辅料，较佳的包括药学上可接受的赋形剂、填充剂或稀释剂等。更佳地，所述的药物组合物包括0.01～99.99％的上述蛋白质和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。
本发明中，较佳地，所述的药物组合物的施用量为有效量，所述有效量为能够缓解或延迟疾病、退化性或损伤性病症进展的量。所述有效量可以以个体基础来测定，并将部分基于待治疗症状和所寻求结果的考虑。本领域技术人员可以通过使用个体基础等上述因素和使用不超过常规的实验来确定有效量。
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。
本发明提供上述药物组合物在制备预防和/或治疗与NKG2A表达或功能异常相关的疾病的药物中的应用。较佳地，所述与NKG2A表达或功能异常相关的疾病为癌症、自身免疫疾病，炎性疾病。
检测样品中NKG2A蛋白的方法、组合物
本发明还提供一种检测样品中NKG2A蛋白(例如检测过表达NKG2A细胞)的方法，包括如下的步骤：上述的抗体与待检样品在体外接触，检测上述的抗体与所述待检样品是否结合形成抗原-抗体复合物即可。
所述的过表达的含义为本领域常规，指NKG2A蛋白在待检样品中的RNA或蛋白质的过表达(由于转录增加、转录后加工、翻译、翻译后加工以及蛋白质降解改变)，以及由于蛋白质运送模式改变(核定位增加)而导致的局部过表达和功能活性提高(如在底物的酶水解作用增加的情况下)。
本发明中，上述是否结合形成抗原-抗体复合物的检测方式是本领域常规的检测方式，较佳地为流式细胞实验(FACS)检测。
本发明提供一种检测样品中NKG2A蛋白的组合物，其包括上述的抗体、重组蛋白、抗体偶联物、免疫细胞、或其组合作为活性成分。较佳地，其还包括上述的抗体的功能片段组成的化合物作为活性成分。
在符合本领域常识的基础上，上述各优选条件，可任意组合，即得本发明各较佳实例。
本发明的主要优点在于：
(1)本发明所述的NKG2A抗体，其与NKG2A蛋白具有高度亲和力(亲和力常数KD达到1.04×10 -11)；
(2)本发明NKG2A抗体能够结合NKG2A蛋白受体的胞外区，并能够在细胞水平有效封闭NKG2A蛋白，阻止NKG2A蛋白与配体HLA-E的结合；
(3)本发明所述的NKG2A抗体特异性高，缺乏与人NKG2C和NKG2E同家族蛋白抗原的交叉反应；
(4)通过原代分离的NK细胞和NK92细胞系介导的杀伤实验证明，本发明抗体能显著增加两种细胞来源NK细胞对高表达HLA-E肿瘤细胞的裂解杀伤活性。
下面结合具体实施例，进一步详陈本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明详细条件的实验方法，通常按照常规条件如美国Sambrook.J等著《分子克隆实验室指南》(黄培堂等译，北京：科学出版社，2002年)中所述的条件，或按照制造厂商所建议的条件(例如商品说明书)。除非另外说明，否则百分比和份数按重量计算。以下实施例中所用的实验材料和试剂如无特别说明均可从市售渠道获得。
实施例中所述的室温为本领域常规的室温，一般为10～30℃。
实施例1 NKG2A抗体的制备
(一)、免疫原A的制备
将含有编码人源NKG2A蛋白胞外区氨基酸序列Pro94-Leu233(如序列表SEQ ID NO.91所示)的核苷酸序列克隆到带有人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。同时将含有编码人源CD94蛋白胞外区氨基酸序列Lys32-Ile179(如序列表SEQ ID NO.92所示)的核苷酸序列克隆到带有人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NKG2A和CD94蛋白胞外区异源二聚蛋白的培养上清液。将培养上清液上样到蛋白A亲和层析柱(Mabselect Sure，购自GE Healthcare)，同时用紫外(UV)检测仪监测紫外吸收值(A280nm)的变化。上样后用PBS磷酸盐缓冲液(pH7.2)清洗蛋白A亲和层析柱直到紫外吸收值回到基线，然后用0.1M甘氨酸盐酸(pH2.5)洗脱，收集从蛋白A亲和层析柱上洗脱下来的带hFc标签的NKG2A/CD94蛋白(NKG2A/CD94-hFc)，用PBS磷酸盐缓冲液(pH7.2)在4℃冰箱透析过夜。透析后的蛋白经0.22微米无菌过滤后分装于-80℃保存，即获得纯化的免疫原A。免疫原A在使用前进行一系列质控检测，如检测其蛋白浓度、纯度、分子量、生物活性等。
结果如图1所示。NKG2A/CD94蛋白与对照抗体在蛋白水平的结合随抗体的浓度变化而变化，其中对照蛋白为非NKG2A/CD94融合蛋白，表中的数据为OD450nm值。
(二)、免疫原B的制备
NKG2A全长氨基酸序列cDNA(如序列表SEQ ID NO.93所示)与CD94全长氨基酸从DNA序列(如序列表SEQ ID NO.94所示)被克隆到Pcho1.0载体(购自Invitrogen)，并包被到1.0um金胶体子弹上，用Helios基因枪(Helios Gene Gun System，Bio-rad，货号165-2431)免疫。其中，包被到1.0μm金胶体子弹和免疫的方法参见Helios基因枪说明书。免疫后即获得了免疫原B。
(三)、免疫原C的制备
将已制备好的免疫原B对HEK293细胞系(均购自Invitrogen)进行质粒转 染(PEI，购自Polysciences)后，在含0.5μg/ml的含10％(w/w)胎牛血清的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NKG2A抗体(CP in house)与CD94抗体(购自eBioscience)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C。具体选择结果如表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。即获得一系列NKG2A与CD94均阳性表达的HEK293细胞系。
表3 NKG2A/CD94蛋白转染的HEK293细胞FACS筛选检测结果
Figure PCTCN2019116060-appb-000008
(四)、免疫原D的制备
将已制备好的免疫原B对CHOK1细胞系(均购自Invitrogen)进行质粒转染(PEI，购自Polysciences)后，在含0.5μg/ml的含10％(w/w)胎牛血清的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NKG2A抗体(CP in house)与CD94抗体(购自eBioscience)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C。具体选择结果如表4所示，表4中阳性细胞(％)指阳性细胞占总细胞数目的百分比。
表4 NKG2A/CD94转染的CHOK1细胞FACS筛选检测结果
Figure PCTCN2019116060-appb-000009
表4说明，已经制得一系列NKG2A与CD94均阳性表达的CHOK1细胞系。
(五)、免疫原E的制备
将猕猴NKG2A全长氨基酸序列(如序列表SEQ ID NO.95所示)与CD94全长氨基酸(如序列表SEQ ID NO.96所示)被克隆到Pcho1.0载体(购自Invitrogen)。将已制备好的质粒对293细胞系(均购自Invitrogen)进行质粒转染(PEI，购自Polysciences)后，在含0.5μg/ml的含10％(w/w)胎牛血清的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NKG2A抗体(CP in house)与CD94抗体(购自eBioscience)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E。
(四)、杂交瘤细胞的制备和抗体筛选
A、免疫原A免疫采用6～8周龄Balb/c小鼠，小鼠在SPF条件下饲养。初次免疫时，免疫原A蛋白用弗氏完全佐剂乳化后腹腔注射0.25毫升，即每只小鼠注 射50微克免疫原A蛋白。加强免疫时，免疫原A蛋白用弗氏不完全佐剂乳化后腹腔注射0.25毫升，即每只小鼠注射50微克免疫原A蛋白。初次免疫与第一次加强免疫之间间隔2周，以后每次加强免疫之间间隔3周。每次加强免疫1周后采血，用ELISA和FACS检测血清中免疫原A的抗体效价和特异性。
结果如图2a,经人NKG2A/CD94ECD-Fc免疫的小鼠血清对免疫原均有不同程度的结合，呈现抗原抗体反应，其中最高稀释度在十万左右。图2b则显示小鼠血清对细胞表面人NKG2A也能够特异性结合，其中最高稀释度在1000倍。
B、免疫原D免疫采用6～8周龄SJL小鼠，小鼠在SPF条件下饲养。将免疫原D在T-75细胞培养瓶中扩大培养至90％汇合度，吸尽培养基，用F12K基础培养基(购自Invitrogen)洗涤2次，然后用无酶细胞解离液(购自Invitrogen)37℃处理直至细胞从培养皿壁上可脱落，收集细胞。用F12K基础培养基洗涤2次，进行细胞计数后将细胞用磷酸盐缓冲液(pH7.2)稀释至2×10 7细胞每毫升。每只小鼠每次免疫时腹腔注射0.5毫升细胞悬液。第一次与第二次免疫之间间隔2周，以后每次免疫间隔3周。除第一次免疫以外，每次免疫1周后采血，用ELISA和FACS检测血清中抗体效价和特异性。
结果如图3a,小鼠血清对免疫原均有不同程度的结合，呈现抗原抗体反应，其中最高稀释度在一万左右。图3b则显示小鼠血清对细胞表面人NKG2A也能够特异性结合，其中最高稀释度在1000倍。
C、免疫原E免疫采用6～8周龄SJL和Balb/c小鼠，小鼠在SPF条件下饲养。将免疫原E在T-75细胞培养瓶中扩大培养至90％汇合度，吸尽培养基，用DMEM基础培养基(购自Invitrogen)洗涤2次，然后用无酶细胞解离液(购自Invitrogen)37℃处理直至细胞从培养皿壁上可脱落，收集细胞。用DMEM基础培养基洗涤2次，进行细胞计数后将细胞用磷酸盐缓冲液(pH7.2)稀释至2×10 7细胞每毫升。每只小鼠每次免疫时腹腔注射0.5毫升细胞悬液。第一次与第二次免疫之间间隔2周，以后每次免疫间隔3周。除第一次免疫以外，每次免疫1周后采血，用FACS检测血清中抗体效价和特异性。在第二次免疫后，更换免疫原A进行免疫，初次免疫时，免疫原A用弗氏完全佐剂乳化后腹腔注射0.25毫升，即每只小鼠注射50微克免疫原A蛋白,第三次免疫时，免疫原A蛋白用弗氏不完全佐剂乳化后腹腔注射0.25毫升，即每只小鼠注射50微克免疫原A蛋白(图4)。加强免疫1 周后采血，用ELISA和FACS检测血清中免疫原A的抗体效价和特异性。
结果如图所示(图5a，图5b，图6a，图6b)，经NKG2A/CD94-ECD-Fc免疫的小鼠的免疫后血清对免疫原均有不同程度的结合，呈现抗原抗体反应，其中批次TB3指第三次加强免疫后第七天的小鼠血清。FACS检测血清抗体效价达到1:1000以上，ELISA效价在1:100，000以上。
D、免疫原B免疫采用6～8周龄的Balb/c或SJL小鼠在SPF条件下饲养。所有小鼠经腹部用Helios基因枪免疫4次，每次4枪，每枪1.0微克cDNA。初次免疫与第一次加强免疫之间隔2周，以后每次加强免疫间隔3周。每次加强免疫1周后采血，用ELISA或FACS检测血清中抗体效价。在第二次加强免疫后，更换免疫原A进行免疫，初次免疫时，免疫原A用弗氏完全佐剂乳化后腹腔注射0.25毫升，即每只小鼠注射50微克免疫原A蛋白,第三次免疫时，免疫原A蛋白用弗氏不完全佐剂乳化后腹腔注射0.25毫升，即每只小鼠注射50微克免疫原A蛋白(图7)。加强免疫1周后采血，用ELISA和FACS检测血清中免疫原A的抗体效价和特异性。
结果如图所示(图8a，图8b，图9a，图9b)，经NKG2A/CD94-ECD-Fc免疫的小鼠的免疫后血清对免疫原均有不同程度的结合，其中批次指第三次加强免疫后第七天的小鼠血清。FACS检测血清抗体效价达到1:1000以上，ELISA效价在1:100，000以上。
A～D步骤完成前，将所选择的每只小鼠最后一次免疫腹腔注射100微克纯化的hNKG2A/CD94-ECD-Fc(针对免疫原A，免疫原C和免疫原D进行免疫反应的小鼠)或含人NKG2A和CD94的CHOK1稳定细胞系(针对免疫原B进行免疫反应的小鼠)，3-4天后处死小鼠，收集脾细胞。加入NH 4OH至终浓度1％(w/w)，裂解脾细胞中的红细胞，获得脾细胞悬液。用DMEM基础培养基1000转每分钟离心清洗细胞3次，然后按活细胞数目5:1比率与小鼠骨髓瘤细胞SP2/0(购自ATCC)混合，采用高效电融合方法(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 Percentage％值>15的阳性克隆扩增到24孔板，在含10％(w/w)HT胎牛血清，DMEM(invitrogen)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FACS确定对NKG2A/CD94阳性细胞的结合活性(结合活性的检测方法请分别参见实施例3A和实施例3B)，配体受体结合实验确定抗体样品对NKG2A受体的封闭活性(结合活性的检测方法请分别参见实施例4)。
根据24孔板筛选结果，挑选FACS实验中与表达人NKG2A和CD94，表达猴NKG2A和CD94的CHOK1细胞结合呈阳性，且与表达人NKG2C和CD94的CHOK1细胞结合呈阴性的杂交瘤克隆进行克隆，同时检测杂交瘤细胞培养上清对NKG2A受体的封闭作用，作为挑选克隆的备选标准。
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，挑选单个阳性单克隆，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NKG2A受体配体结合实验评估生物活性。
根据24孔板样品检测结果，挑选出最优的克隆，并于含10％(w/w)FBS的DMEM培养基中(购自invitrogen)在37℃，5％(v/v)CO 2条件下将该最优的克隆进行扩大培养，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和纯化。
实施例2先导抗体的生产和纯化
杂交瘤细胞产生的抗体浓度较低，大约仅1-10μg/毫升，浓度变化较大。且培养基中细胞培养所产生的多种蛋白和培养基所含胎牛血清成分对很多生物活性分析方法都有不同程度的干扰，因此需要进行小规模(1-5毫克)抗体生产纯化。
将实施例1所得的杂交瘤细胞接种到T-75细胞培养瓶并用生产培养基(Hybridoma serum free medium，购自Invitrogen公司)驯化传代3代。待其生长状态良好，接种细胞培养转瓶。每个2升的培养转瓶中加入500毫升生产培养基，接种细胞密度为1.0×10 5/毫升。盖紧瓶盖，将转瓶置于37℃培养箱中的 转瓶机上，转速3转/分钟。连续旋转培养14天后，收集细胞培养液，过滤去除细胞，并用0.45微米的滤膜过滤至培养上清液澄清。澄清的培养上清液可马上进行纯化或-30℃冻存。
澄清的杂交瘤细胞的培养上清液(300mL)中的单克隆抗体用2mL蛋白G柱(购自GE Healthcare)纯化。蛋白G柱先用平衡缓冲液(PBS磷酸缓冲液，pH7.2)平衡，然后将澄清的培养上清液上样到蛋白G柱，控制流速在3mL/分钟。上样完毕后用平衡缓冲液清洗蛋白G柱，平衡缓冲液的体积为4倍蛋白G柱柱床体积。用洗脱液(0.1M甘氨盐酸缓冲液，pH2.5)洗脱结合在蛋白G柱上的NKG2A抗体，用紫外检测器监测洗脱情况(A280紫外吸收峰)。收集洗脱的抗体，加入10％ 1.0M Tris-HCl缓冲液中和pH，所述百分比为体积百分比，然后立即用PBS磷酸缓冲液透析过夜，第二天换液1次并继续透析3小时。收集透析后的NKG2A抗体，用0.22微米的滤器进行无菌过滤，无菌保存，即得纯化的NKG2A抗体。
将纯化的NKG2A抗体进行蛋白浓度(A280/1.4)、纯度等检测分析，结果如表5所示，结果发现，抗体最终产品内毒素浓度在1.0EU/毫克以内。
表5纯化抗体的性质分析
Figure PCTCN2019116060-appb-000010
实施例3轻重链可变区氨基酸序列测定
总RNA分离：将实施例1亚克隆培养所得的上清液检验过抗原结合后(即经过实施例3～6的检定和活性测定后)，通过离心搜集5×10 7个杂交瘤细胞，加入1mL Trizol混匀并转移到1.5mL离心管中，室温静置5分钟；加0.2mL氯仿，振荡15秒，静置2分钟后于4℃，12000g离心5分钟，取上清转移到新的1.5mL离心管中；加入0.5mL异丙醇，将管中液体轻轻混匀，室温静置10分钟后于4℃，12000g离心15分钟，弃上清；加入1mL 75％(v/v)乙醇，轻轻洗涤沉淀，4℃， 12000g离心5分钟后弃上清，将沉淀物晾干，加入DEPC处理过的H 2O溶解(55℃水浴促进溶剂10分钟)，即得总RNA。
逆转录与PCR：取1μg总RNA，配置20μl体系，加入逆转录酶后于42℃反应60分钟，于7℃反应10分钟终止反应。配置50μl PCR体系，包括1μl cDNA、每种引物25pmol、1μl DNA聚合酶以及相配的缓冲体系、250μmol dNTPs；设置PCR程序，预变性95℃3分钟，变性95℃30秒，退火55℃ 30秒，延伸72℃35秒，35个循环后再额外于72℃延伸5分钟，得PCR产物。其中逆转录所用的试剂盒为PrimeScript RT Master Mix，购自Takara，货号RR036；PCR所用的试剂盒包括Q5超保真酶，购自NEB，货号M0492。
克隆与测序：取5μl PCR产物进行琼脂糖凝胶电泳检测，将检测阳性样品使用柱回收试剂盒纯化，其中回收试剂盒为
Figure PCTCN2019116060-appb-000011
Gel&PCR Clean-up，购自MACHEREY-NAGEL，货号740609。进行连接反应：样品50ng，T载体50ng，连接酶0.5μl，缓冲液1μl，反应体系10μl，于16℃反应半小时得连接产物，其中连接的试剂盒为T4DNA连接酶，购自NEB，货号M0402；取5μl连接产物加入100μl的感受态细胞(Ecos 101competent cells,购自Yeastern，货号FYE607)中，冰浴5分钟，而后于42℃水浴热激1分钟，放回冰上1分钟后加入650μl无抗生素SOC培养基，于37℃摇床上以200RPM的速度复苏30分钟，取出200μl涂布于含抗生素的LB固体培养基上于37℃孵箱过夜培养；次日，使用T载体上引物M13F和M13R配置30μlPCR体系，进行菌落PCR，用移液器枪头蘸取菌落于PCR反应体系中吹吸，并吸出0.5μl点于另一块含100nM氨苄青霉素的LB固体培养皿上以保存菌株；PCR反应结束后，取出5μl进行琼脂糖凝胶电泳检测，将阳性样品进行测序和分析[参见Kabat，“Sequences of Proteins of Immunological Interest,”National Institutes of Health,Bethesda,Md.(1991)]。
测序结果见附录中本发明的序列信息。
实施例4噬菌体展示制备NKG2A抗体
获取脾脏细胞：分别将HNKG2A/CD94-ECD-Fc和CHOK-1/HNKG2A/CD94作为抗原免疫过的小鼠，用293/cynoNKG2A/CD94细胞做冲刺免疫，3天后分离小鼠脾脏细胞制备免疫库。将分离的脾细胞重悬到DMEM培养基中，2000rpm，4℃离心 10min，弃去上清。用RNAiso plus(购自Takara，货号：9108)裂解细胞沉淀，比例为1ml RNAiso plus加入一只小鼠的脾脏，室温孵育5min后，保存于-80℃。
抽提RNA:将冻存的小鼠脾脏细胞于室温解冻并涡旋5min。每1ml的RNAiso plus样品中加入0.2ml的1-溴-3-氯丙烷(1-Bromo-3-chloropropane，购自Sigma，货号：B9673-200ml)，剧烈震荡15seconds，然后于室温孵育5min。将样品4℃，12000g离心10min，将水相转移到一只新管，加入0.7ml异丙醇沉淀RNA,室温孵育10min后，12000g离心10min，4℃，弃去上清。将RNA沉淀用1ml 75％乙醇(无RNAase)洗涤一次，12000g离心5min，4℃，弃去上清。干燥RNA沉淀15min后，用40ul含DEPC的水(购自Invitrogen，46-2224)溶解RNA，轻柔混匀并于室温放置5min。将所有样品的RNA各取一半混合，并对所获得RNA库进行浓度测定，结果为2175.6ng/ul。
cDNA文库的制备：参照逆转录试剂盒5*PrimeScript TMRT Master Mix(购自Takara，货号RR036A)准备逆转录反应体系如下表，进行热循环。设定条件为37℃20min，85℃20s，4℃持续。将所获得逆转录产物混合，并分为两份。一份保存于-80℃，一份储存于4℃进行后续实验。
表6 PCR反应体系
试剂 体积(ul) Master Mix(*3)
5*Mix 20 60
RNA 6.9 21
H2O 补充至100  
VH和VL库的扩增和纯化：扩增用引物设计参见Journal of Immunological Methods 201(1997),35-5。分别混合重链，轻链的正向，反向扩增引物，并准备PCR反应如下：
表7-1 PCR反应
Figure PCTCN2019116060-appb-000012
Figure PCTCN2019116060-appb-000013
设定PCR程序如下：
表7-2 PCR程序
94℃ 1min 30s
94℃ 1min
63℃ 30s
58℃ 50s
72℃ 1min
94℃ 1min
63℃ 1min
72℃ 1min
72℃ 5min
4℃ 持续
PCR结束，对扩增产物进行凝胶纯化，结果显示分别获得230ng/ul，170ng/ul的VH和VL。将获得的VH和VL库通过SOE(splicing overlap extension)PCR的方法组装scFvs，将所得PCR产物进行纯化(QIAquick Gel)/PCR纯化试剂盒，购自QIAGEN,货号28706)，最终获得纯化scFv的浓度为130ng/ul。
制备噬菌粒：将pCAN载体与scFv用Sfi酶(购自NEB,货号R0123S)进行酶切，并胶回收酶切产物，对所获得pCAN载体和scFv用T4连接酶(购自NEB)进行酶连反应，并用纯化试剂盒(购自Qiagen，货号：28014)纯化所得连接产物，用于制备免疫噬菌体库。
免疫噬菌体库的制备：将500ng上述纯化所得DNA与200ul TG1感受态大肠杆菌(可通过常规方法获得该感受态)混合并进行电转。将电转产物于1ml YT培养中震荡培养1h，37℃。取10ul细胞悬液以10倍梯度稀释后(10 -4,10 -5,10 -6)，涂平板检测库容。
离心收集细菌沉淀，弃去多余菌液，将细菌沉淀重悬后涂平板，培养过夜。第二天刮下培养板中细菌克隆，离心收集沉淀，并重悬于4ml 2*YT培养基(medium)(含40％甘油)，将所得免疫库冻存于-80℃。
淘选：
方案一：将上述获得免疫库用CHOK1/NKG2C/CD94细胞进行负筛选。将收集的CHOK1/NKG2C/CD94细胞用10ml噬菌体液体重悬，4℃转动孵育1h，离心去除 细胞，收集上清中的噬菌体库进行下一步的淘选。将上述所得上清与CHOK1/hNKG2A/CD94和CHOK1细胞进行正筛选，等体积孵育细胞与上清，4℃转动孵育1.5h。用PBS重复洗涤细胞沉淀，并重悬至1ml Glycine(购自Shanghai Lingfeng化学，货号：56-40-6)洗脱缓冲液中(ph2.2)洗脱噬菌体，室温孵育15min，并轻柔颠倒混匀。18000g离心10min后，将上清转移至新的离心管，加入100ul 2M Tris(pH8)进行中和。将1mL的上述溶液加到4mL处于对数生长期的大肠杆菌TG1中，37℃孵育30分钟，得到TG1的培养液。将TG1的培养液梯度稀释，涂布平板，37℃培养过夜。计算所得的HNKG2A/CD94结合的和对照管的克隆数，并挑选30个克隆测序。第一轮淘选后共获得1300个克隆。
方案二：首先将上述获得免疫库用CD94-ECD-Fc与CHOK-1细胞进行负筛选。将免疫小管用40ug的Anti-hFc(购自ROCKLAND,货号009-1103)于2ml PBS中进行包被，随后加入23ug的CD94-Fc蛋白室温孵育1h。同时用3ml的2％MPBS(含2％脱脂奶粉(Non-fat Dry Milk)，购自Bio-Rad，货号170-6404)对phage于4℃封闭1h，将封闭好的phage加入偶联CD94蛋白的免疫小管，室温孵育1h，随后将封闭好的CHOK1细胞沉淀用phage上清重悬，补足2％MPBS至11ml，4℃转动孵育1h。对CD94和CHOK1细胞负筛选过的phage进行CHOK-hNKG2A/CD94细胞正筛选。用上述所得phage重悬，并于4℃孵育2h，并轻柔震动。用PBS重复洗涤细胞沉淀，并重悬至1ml Glycine(购自Shanghai Lingfeng化学，货号：56-40-6)洗脱缓冲液中(ph2.2)洗脱噬菌体，室温孵育15min，并轻柔颠倒混匀。18000g离心10min后，将上清转移至新的离心管，加入100ul 2M Tris(pH8)进行中和。将1mL的上述溶液加到4mL处于对数生长期的大肠杆菌TG1中，37℃孵育30分钟，得到TG1的培养液。将TG1的培养液梯度稀释，涂布平板，37℃培养过夜。计算所得的HNKG2A/CD94结合的和对照管的克隆数，并挑选30个克隆测序。第一轮淘选后共获得6000个克隆。
从上述两种淘选策略的平板中挑选单克隆于96孔板培养，每孔中含有200μL加抗生素的2YT培养基，37℃、1000rpm振荡培养过夜。取10μL过夜培养的上清加到4mL含抗生素培养基中，37℃、250rpm振荡培养1.5-2.5小时。添加终浓度为1mM的IPTG，30℃振荡培养16小时，4000rpm离心10分钟，上清即得到单链抗体。
首先用ELISA方法确定筛选得到的scFv抗体与NKG2A/CD94-ECD-Fc和NKG2C/CD94-ECD-Fc的结合活性。将二者(NKG2A/CD94-ECD-Fc/NKG2C/CD94-ECD-Fc)OD450nm读数比例大于4的克隆挑选出来。部分克隆用FACS方法确定筛选得到的scFv抗体与CHOK1/NKG2A/CD94-ECD-Fc和CHOK1/NKG2C/CD94细胞的结合活性。将特异的只与CHOK1/hNKG2A/CD94细胞结合的克隆挑选出来。将上述所有特异性的克隆通过FACS方法确定其与猕猴(cyno)NKG2A的结合活性，对仍旧呈现阳性的克隆进行测序，得到具有不同重链CDR3序列的克隆,共21个，见表8。
表8可变区测序结果
Figure PCTCN2019116060-appb-000014
Figure PCTCN2019116060-appb-000015
噬菌体来源的先导抗体的生产和纯化：扩增重链与轻链可变区：根据阳性克隆的测序结果，通过PCR方法分别扩增轻链和重链的可变区。配置50μL反应体系，包括0.5μL含有转染阳性克隆大肠杆菌TG1中提取的质粒、每种引物10pmol、25μL Q5高保真DNA聚合酶以及加水补足至50μL。设置PCR程序，预变性95℃5分钟，变性95℃30秒，退火55℃30秒，延伸68℃30秒，25个循环后再额外68℃延伸1分钟，得到PCR产物。其中PCR所用的DNA聚合酶，购自NEB，货号E0555L。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回收试剂盒纯化，其中回收试剂盒为QIAquick Gel extraction kit，购自Qiagen，货号28706。
人IgG4型抗体的制备：进行连接反应：插入片段3μL，酶切过的表达载体2μL，重组酶Exnase 2μL，缓冲液4μL，反应体系20μL，于37℃反应半小时得到连接产物，即构建好的重组载体。其中，重组酶购自Vazyme，货号C112-01/02；缓冲液为该重组酶配套购买使用的缓冲液；将重链可变区定向克隆到包含信号肽和人源抗体重链IgG4(S228P)恒定区的表达载体(其中表达载体购买自Invitrogen，重组步骤为常规步骤)，将轻链可变区定向克隆到包含信号肽和人源抗体轻链Kappa恒定区的表达载体(其中表达载体购买自Invitrogen，重组步骤为常规步骤)中。将10μL连接产物加入100μL的感受态细胞(Ecos 101competent cells，购自Yeastern，货号FYE607)中，冰浴30分钟。再于42℃水浴热激90秒，放回冰上2分钟后加入800μL无抗生素的2YT培养基，于37℃摇床上以200rpm培养45分钟，取出200μL涂布于含100μg/mL氨苄青霉素的LB固体培养基上，于37℃孵箱过夜培养。次日，使用表达载体上引物pTT-EF1a-F和pSV40(其核苷酸序列分别为序列表SEQ ID No.97～98所示)，配置30μL PCR体系，进行菌落PCR。菌落PCR的体系为：引物各1μL，10μL的PCR预混液(购自Novoprotein)，补足至20μL。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100μg/mL氨苄青霉素的LB固体培养皿上以保存菌株。PCR反应结束后，取出5μL进行琼脂糖凝胶电泳检测，将阳性样品进行测序和分析[参见Kabat,“Sequences of Proteins of  Immunological Interest,”National Institutes of Health,Bethesda,Md.(1991)]。
经过菌落PCR验证，将序列正确的重组抗体重、轻链的表达载体瞬时转染FreeStyle TM 293-F细胞(购自Invitrogen)以生产抗体。转染时，293-F细胞的密度应为1-1.5×10 6个/mL，100mL细胞需要100μg上述已构建好的重组载体(其中，重组重链载体和轻链载体的质量比为2:3)和200μg的转染试剂聚乙烯亚胺(PEI)。将重组载体和PEI分别加入到5mL培养基中，室温静置5分钟，0.22μm滤膜过滤后，将重组载体和PEI的混合物于室温静置15分钟。然后将上述混合物缓慢地加入到细胞中，在37℃、8％(v/v)CO 2培养箱中以120rpm的转速培养。7天后，3500g离心细胞培养液30分钟，收集上清液，0.22μm滤器过滤。用1mL蛋白A柱(购自GE Healthcare)纯化200mL澄清上清液中的单克隆抗体。蛋白A柱先用平衡缓冲液(PBS磷酸缓冲液，pH7.2)平衡，然后将上清液上样到蛋白A柱，控制流速在3mL/分钟。上样完毕后用平衡缓冲液清洗蛋白A柱，平衡缓冲液的体积为蛋白A柱柱床体积的20倍。用洗脱液(0.1M甘氨盐酸缓冲液，pH3.0)洗脱结合在蛋白A柱上的单克隆抗体，用紫外检测器监测洗脱情况(A280紫外吸收峰)。收集洗脱的抗体，加入10％(v/v)1.0M Tris-HCl缓冲液中和pH。然后立即用PBS磷酸缓冲液透析过夜。收集透析后的单克隆抗体，用0.22μm的滤器进行无菌过滤，无菌保存，即得纯化的NKG2A抗体作为先导抗体。将先导抗体进行蛋白浓度(A280/1.4)、纯度、内毒(Lonza试剂盒)等检测分析。结果如下表9所示。
表9噬菌体展示筛选抗体纯化结果
Figure PCTCN2019116060-appb-000016
Figure PCTCN2019116060-appb-000017
结果显示：11G5抗体的产量与纯度均较低，无法进行进一部分的纯化分析。其余抗体的产量，纯度，内毒素均分析均表现正常。
实施例5先导抗体的检定
A.流式细胞实验(FACS)检测抗体与NKG2A/CD94表达细胞的结合
将实施例1步骤(二)中所述含有编码人源NKG2A/CD94全长氨基酸序列的核苷酸序列的pCHO1.0质粒转染CHOK1细胞株得含人NKG2A和CD94的CHOK1稳定细胞株(此处称为CHOk1-hNKG2A/CD94稳定细胞株)，将带有猴源NKG2A和CD94全长基因的pCHO1.0质粒(其制备方法与实施例1步骤(一)“免疫原A的制备”中带有人源IgG Fc片段(hFc)的pCpC载体的制备方法相同，转染CHOK1细胞株得含猴NKG2A/CD94的CHOK1稳定细胞株(此处称为CHOk1-cNKG2A/CD94稳定细胞株)，用同样方法制备CHOK1-hNKG2C/CD94稳定细胞株。将CHOk1-hNKG2A/CD94稳定细胞株，CHOk1-cNKG2A/CD94稳定细胞株和CHOK1-hNKG2C/CD94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PBS缓冲液(购自Invitrogen)洗涤2次，然后用无酶细胞解离液(Versene solution,购自Life  technology公司)处理和收集细胞。用PBS缓冲液洗涤细胞2次，进行细胞计数后将细胞用PBS缓冲液稀释至2×10 6细胞每毫升，加入1％山羊血清封闭液，所述百分比为质量百分比，冰上孵育30分钟，然后用PBS缓冲液离心洗涤2次。将收集的细胞用FACS缓冲液(PBS+1％BSA，所述百分比为质量百分比)悬浮至2×10 6细胞/mL，按每孔100微升加入到96孔FACS反应板中，加入实施例2所得的纯化的NKG2A抗体待测样品每孔100微升，冰上孵育2小时。用FACS缓冲液离心洗涤2次，加入每孔100微升荧光(Alexa 488)标记的二抗(购自Invitrogen)，冰上孵育1小时。用FACS缓冲液离心洗涤3次，后用100微升FACS缓冲液悬浮细胞，用FACS(FACS Calibur，购自BD公司)检测和分析结果。结果如图10-13所示。结果表明：待测抗体可特异性结合细胞表面的人NKG2A(图10)，猕猴NKG2A(图11)，而不结合细胞表面NKG2C/CD94(图12)，与NKG2E蛋白也无交叉反应性(图13)。其中IgG对照为人IgG。
B.NKG2A抗体亲和常数的测定
使用Octet red96仪器(购自Fortiebio)进行亲和常数的测定。具体操作和方法根据仪器说明书和厂家提供的详细方法。具体为：用链霉素亲和素传感器(SA sensor，购自Fortiebio)进行亲和力测定。将生物素标记的人源NKG2A/CD94-ECD-Fc(即免疫原A)用含0.1％(w/w)BSA，0.02％(v/v)Tween，pH 7.4的PBS溶液稀释至10μg/ml，然后与链霉素亲和素传感器反应；结合免疫原A的传感器与五个不同浓度梯度稀释的NKG2A抗体在30℃下温育三分钟，再和含0.1％(w/v)BSA，0.02％(v/v)Tween，pH 7.4的PBS溶液30℃温育5分钟；通过Octet仪器检测干涉波长的变化来检测抗体与免疫原A结合与解离，然后用
Figure PCTCN2019116060-appb-000018
Software软件拟合得到解离常数与结合常数，亲和力常数为解离常数与结合常数的比值。结果如表10所示：
表10 NKG2A抗体对免疫原A的亲和常数
Figure PCTCN2019116060-appb-000019
实施例6检测NKG2A抗体阻断NKG2A与其配体HLA-E的结合
将CHOk1-hNKG2A/CD94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PBS缓冲液(购自Invitrogen)洗涤2次，然后用无酶细胞解离液(Versene solution,购自Life technology公司)处理和收集细胞。用PBS缓冲液洗涤细胞2次，进行细胞计数后将细胞用PBS缓冲液稀释至2×10 6细胞每毫升，加入1％山羊血清封闭液，所述百分比为质量百分比，冰上孵育30分钟，然后用PBS缓冲液离心洗涤2次。将收集的细胞用FACS缓冲液(PBS+1％BSA，所述百分比为质量百分比)悬浮至2×10 6细胞/mL，按每孔100微升加入到96孔FACS反应板中，离心洗涤一次后，将纯化所得NKG2A抗体用FACS缓冲液进行梯度稀释，按每孔50微升重悬细胞，同时每孔加入稀释好的HLA-E-PE，每孔50ul；避光冰上孵育2.5-3小时。用FACS缓冲液离心洗涤3次，后用100微升FACS缓冲液悬浮细胞，用FACS(FACS Calibur，购自BD公司)检测和分析结果。结果如图14所示,待测抗体可阻断HLA-E结合细胞表面的NKG2A蛋白。其中同型对照为人IgG，表中的数据为MFI所测细胞群的平均荧光强度值。
实施例7检测NKG2A抗体增加NK92对靶细胞的杀伤作用
将LCL721.221细胞在T-75细胞培养瓶中扩大培养至90％汇合度，吸尽培养基，用PBS缓冲液(购自Invitrogen)洗涤2次。用含有1mM peptide的培养基重悬细胞至1*10 6/500ul，在26℃培养箱中培养过夜后，用PBS洗涤2次。用分析缓冲液(1640培养基加入5％FBS)将LCL721.221细胞重悬至2*10 5cells/ml，取50ul加入96孔板中，确保每个孔细胞数为10000个。将已经与抗体孵育好的NK92细胞(37℃30min)按照效应细胞：靶细胞比例2:1，以体积100ul加入上述96孔板中，与LCL721.221细胞充分混合后，250g离心5分钟，将96孔板放置在37℃培养箱中4h。
加入75ul/孔AAF-Glo TM反应液，15分钟后读取荧光值，反映死细胞的数量。再加入75ul/孔的裂解液，15分钟后读取荧光值，反映总细胞的数量。
结果如图15所示，结果表明：所测抗体均能增强NK92细胞对LCL721.221的杀伤活性，并且表现优于参比抗体Z270。
实施例8检测NKG2A抗体原代NK细胞对靶细胞的杀伤作用
(一)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。
随后从PBMC中分离得到原代NK，在体外培养4天，1640培养基配方如下：0.01mm 2-me,1*L-Glu,1*NEAA,10％FBS,10ng/ml IL-2，20ng/ml IL-12。将LCL721.221细胞在T-75细胞培养瓶中扩大培养至90％汇合度，吸尽培养基，用PBS缓冲液(购自Invitrogen)洗涤2次。用含有1mM peptide的培养基重悬细胞至1*10 6/500ul，在26℃培养箱中培养过夜后，用PBS洗涤2次。用培养基(assay buffer)将LCL721.221细胞重悬至2*10 5cells/ml，取50ul加入96孔板中，确保每个孔细胞数为10000个。将已经与抗体孵育好的NK92细胞(37℃30min)按照效应细胞：靶细胞比例2:1，以体积100ul加入上述96孔板中，与LCL721.221细胞充分混合后，250g离心5分钟，将96孔板放置在37℃培养箱中4h。
加入75ul/孔AAF-Glo TM反应液，15分钟后读取荧光值，反映死细胞的数量。再加入75ul/孔的裂解液，15分钟后读取荧光值，反映总细胞的数量。
结果如图16、图17所示，结果表明：在不同的供体中，所测抗体均能增强原代NK细胞对LCL721.221的杀伤活性。
实施例9人源化抗体的制备
分析12G8即mpb416克隆的轻重链CDR区，其中CDRH2与CDRL1中的NG序列被突变为NA，获得M15抗体，将以下重链，轻链可变区作为人源化模板。
表11 M15的轻重链可变区氨基酸序列
Figure PCTCN2019116060-appb-000020
Figure PCTCN2019116060-appb-000021
通过序列比对(NCBI-Igblast)选择与候选抗体重链可变区，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(60.2％)和IGKV2-28*01(80.0％).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5个重链可变区序列(Mpb416-M15VH_g0，Mpb416-M15VH_g1，Mpb416-M15VH_g2，Mpb416-M15VH_g3，Mpb416-M15VH_g4)和3个轻链可变区序列(Mpb416-M15VL_g0,Mpb416-M15VL_g1，Mpb416-M15VL_g2)，随后做交叉组合进行表达，共15种表达组合，见表12。
表12人源化抗体表达组合
  M15 VH.g0 M15 VH.g1 M15 VH.g2 M15 VH.g3 M15 VH.g4
M15 VL.g0 M15-1 M15-2 M15-3 M15-4 M15-5
M15 VL.g1 M15-6 M15-7 M15-8 M15-9 M15-10
M15 VL.g2 M15-11 M15-12 M15-13 M15-14 M15-15
载体构建：扩增引物由Genewiz合成，随后通过PCR方法分别扩增轻链和重链的可变区。配置50μL反应体系，包括50-100ng的重链可变区，轻链可变区、正向反向引物各1ul、1ul pfxD酶(购自invitrogen，12344-012)、10*pfx缓冲液5ul(供应商同pfx同酶)以及加水补足至50μL。设置PCR程序，预变性95℃5分钟，变性95℃30秒，退火56℃30秒，延伸68℃30秒，25个循环后再额外68℃延伸10min，得到PCR产物。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回收试剂盒纯化，其中回收试剂盒为PureLink Quick Gel extraction kit，购自Qiagen，货号28706。
人源化抗体的制备：进行连接反应：插入片段20-40ng，酶切过的表达载体60-100ng，重组酶Exnase(购自Vazyme，货号C112-01/02)1μL，缓冲液2μL，反应体系10μL，于37℃反应半小时得到连接产物，即构建好的重组载体。缓冲液为该重组酶配套购买使用的缓冲液；将重链可变区定向克隆到包含信号肽和人源抗体重链IgG4(S228P)恒定区的表达载体(其中表达载体购买自Invitrogen，重组步骤为常规步骤)，将轻链可变区定向克隆到包含信号肽和 人源抗体轻链kappa恒定区的表达载体(其中表达载体购买自Invitrogen，重组步骤为常规步骤)中。将10μL连接产物加入100μL的感受态细胞(Ecos101competent cells，购自Yeastern，货号FYE607)中，42℃水浴热激60秒，放回冰上3分钟，取出80μL涂布于含氨苄青霉素的LB固体培养基上，于37℃孵箱过夜培养。次日，使用表达载体上引物pEF1A和pSV40，配置30μL PCR体系，进行菌落PCR。菌落PCR的体系为：引物各1μL，15μL的PCR预混液(购自Novoprotein)，补足至30μL。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100μg/mL氨苄青霉素的LB固体培养皿上以保存菌株。PCR反应结束后，取出4.5μL进行琼脂糖凝胶电泳检测，将阳性样品进行测序。
将序列正确的重组抗体重、轻链的表达载体进行扩增，随后瞬时转染FreeStyle TM 293-F细胞(购自Invitrogen)以生产抗体。转染时，293-F细胞的密度应为1-1.2×10 6个/mL，100mL细胞需要100μg上述已构建好的重组载体和200μg的转染试剂聚乙烯亚胺(PEI)。将重组载体和PEI分别加入到5mL培养基中，室温静置5分钟，0.22μm滤膜过滤后，将重组载体和PEI的混合物于室温静置15分钟。然后将上述混合物缓慢地加入到细胞中，在37℃、8％(v/v)CO 2培养箱中以130rpm的转速培养。每天采取培养上清和细胞沉淀检测抗体的表达。5天后，3000g离心细胞培养液30分钟，收集上清液，0.22μm滤器过滤。用1mL MabSelect TMSuRe TMcolumn(购自GE Healthcare)纯化200mL澄清上清液中的单克隆抗体。MabSelect TMSuRe TMcolumn先用平衡缓冲液(PBS磷酸缓冲液，pH7.2)平衡，MabSelect TMSuRe TMcolumn。上样完毕后用平衡缓冲液清洗MabSelect TMSuRe TMcolumn，平衡缓冲液的体积为蛋白A柱柱床体积的5倍。用洗脱液(0.1M甘氨盐酸缓冲液，pH3.0)洗脱结合在MabSelect TMSuRe TMcolumn上的单克隆抗体。收集洗脱的抗体，加入10％(v/v)1.0M Tris-HCl缓冲液中和pH。然后立即用PBS磷酸缓冲液透析过夜。收集透析后的单克隆抗体，用0.22μm的滤器进行无菌过滤，无菌保存，即得纯化的NKG2A人源化抗体。将所得抗体进行蛋白浓度、纯度检测分析。结果如下表13所示，结果显示，M15-3抗体的纯度均较低。剩余抗体的产量，纯度分析均表现正常。
表13人源化抗体纯化结果分析
Figure PCTCN2019116060-appb-000022
人源化抗体的活性鉴定(方法同实施例5)
A.流式细胞实验(FACS)检测抗体与NKG2A/CD94表达细胞的结合,结果如图18a,图18b,图19a,图19b,图20a,图20b所示，所得抗体均可结合细胞表面的人NKG2A，猴NKG2A，并不结合人NKG2C与人CD94。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。
B.检测NKG2A抗体阻断NKG2A与其配体HLA-E的结合，方法同实施例6。结果如图21所示,待测抗体可阻断HLA-E结合细胞表面的NKG2A蛋白。其中同型对照为人IgG，表中的数据为MFI所测细胞群的平均荧光强度值。
C.检测NKG2A抗体阻断NKG2A与其配体HLA-E的结合，方法同实施例7。M15-5，M15-10，M15-15进行检测，结果如图22所示，抗体均能加强NK92对肿瘤细胞的杀伤活性。
D.NKG2A抗体亲和常数的测定，方法同实施例5B。
对人源化改造后抗体的亲和力进行评估，结果显示其亲和力显著优于参比阳性抗体。结果见表14。
表14 NKG2A抗体对免疫原A的亲和常数
Figure PCTCN2019116060-appb-000023
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。
附录 本发明的序列信息
表17 NKG2A抗体氨基酸序列编号
Figure PCTCN2019116060-appb-000024
表18 NKG2A抗体基因序列编号
克隆号 重链可变区 轻链可变区
M15-5 73 74
Mpb416 75 76
Mab031 77 78
Mab032 79 80
Mab033 81 82
Mab036 83 84
2F10 85 86
9B10 87 88
14G3 89 90
M15-5
Figure PCTCN2019116060-appb-000025
Figure PCTCN2019116060-appb-000026
Mpb416
Figure PCTCN2019116060-appb-000027
Mab031
Figure PCTCN2019116060-appb-000028
Mab032
Figure PCTCN2019116060-appb-000029
Figure PCTCN2019116060-appb-000030
Mab033
Figure PCTCN2019116060-appb-000031
Mab036
Figure PCTCN2019116060-appb-000032
Figure PCTCN2019116060-appb-000033
2F10
Figure PCTCN2019116060-appb-000034
9B10
Figure PCTCN2019116060-appb-000035
Figure PCTCN2019116060-appb-000036
14G3
Figure PCTCN2019116060-appb-000037
M15-5
M15-5 heavy chain
Figure PCTCN2019116060-appb-000038
M15-5 light chain
Figure PCTCN2019116060-appb-000039
Mpb416 heavy chain
Figure PCTCN2019116060-appb-000040
Figure PCTCN2019116060-appb-000041
Mpb416 light chain
Figure PCTCN2019116060-appb-000042
Mab031 heavy chain
Figure PCTCN2019116060-appb-000043
Mab031 light chain
Figure PCTCN2019116060-appb-000044
Mab032 heavy chain
Figure PCTCN2019116060-appb-000045
Mab032 light chain
Figure PCTCN2019116060-appb-000046
Figure PCTCN2019116060-appb-000047
Mab033 heavy chain
Figure PCTCN2019116060-appb-000048
Mab033 light chain
Figure PCTCN2019116060-appb-000049
Mab036 heavy chain
Figure PCTCN2019116060-appb-000050
Mab036 light chain
Figure PCTCN2019116060-appb-000051
2F10 heavy chain
Figure PCTCN2019116060-appb-000052
2F10 light chain
Figure PCTCN2019116060-appb-000053
Figure PCTCN2019116060-appb-000054
9B10 heavy chain
Figure PCTCN2019116060-appb-000055
9B10 light chain
Figure PCTCN2019116060-appb-000056
14G3 heavy chain
Figure PCTCN2019116060-appb-000057
14G3 light chain
Figure PCTCN2019116060-appb-000058
SEQ ID NO.91(NKG2A ECD)
Figure PCTCN2019116060-appb-000059
SEQ ID NO.92(CD94 ECD)
Figure PCTCN2019116060-appb-000060
SEQ ID NO.93(NKG2A full length 核酸)
Figure PCTCN2019116060-appb-000061
SEQ ID NO.94(CD94 full length 核酸)
Figure PCTCN2019116060-appb-000062
SEQ ID No.95(猕猴NKG2A全长氨基酸序列)
Figure PCTCN2019116060-appb-000063
SEQ ID No.96(猕猴CD94全长氨基酸序列)
Figure PCTCN2019116060-appb-000064
SEQ ID No.97 pTT-EF1a-F
Figure PCTCN2019116060-appb-000065
SEQ ID No.98 pSV40
Figure PCTCN2019116060-appb-000066
SEQ ID No.99 M15重链可变区
Figure PCTCN2019116060-appb-000067
SEQ ID No.100M15轻链可变区
Figure PCTCN2019116060-appb-000068

Claims (17)

  1. 一种抗体的重链可变区，其特征在于，所述的重链可变区具有选自下组的互补决定区CDR：
    SEQ ID NO.8n+2所示的VH-CDR1，
    SEQ ID NO.8n+3所示的VH-CDR2，和
    SEQ ID NO.8n+4所示的VH-CDR3；
    其中，各n独立地为0、1、2、3、4、5、6、7或8；
    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NKG2A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，所述的轻链可变区具有选自下组的互补决定区CDR：
    SEQ ID NO.8n+6所示的VL-CDR1，
    SEQ ID NO.8n+7所示的VL-CDR2，和
    SEQ ID NO.8n+8所示的VL-CDR3；
    其中，各n独立地为0、1、2、3、4、5、6、7或8；
    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NKG2A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NKG2A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，所述的重链可变区和所述的轻链可变区包括选自下组的CDR：
    Figure PCTCN2019116060-appb-100001
    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NKG2A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.1、9、17、25、33、41、49、57或65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.5、13、21、29、37、45、53、61或69所示的氨基酸序列。
  8. 如权利要求6所述的抗体，其特征在于，所述的抗体选自下组：
    抗体编号 克隆 VH序列编号 VL序列编号 1 M15-5 1 5 2 Mpb416 9 13 3 Mab031 17 21 4 Mab032 25 29 5 Mab033 33 37 6 Mab036 41 45 7 2F10 49 53 8 9B10 57 61 9 14G3 65 69
    。
  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.73、75、77、79、81、83、85、87或89所示；和/或，编码所述轻链可变区的多核苷酸如SEQ ID NO.74、76、78、80、82、84、86、88或90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和编码所述轻链可变区序列的多核苷酸选自下组：
    Figure PCTCN2019116060-appb-100002
  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. 一种抗体偶联物，其特征在于，该抗体偶联物含有：
    (a)抗体部分，所述抗体部分选自下组：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、或其组合；和
    (b)与所述抗体部分偶联的偶联部分，所述偶联部分选自下组：可检测标记物、药物、毒素、细胞因子、放射性核素、酶、或其组合。
  16. 一种免疫细胞，其特征在于，所述免疫细胞表达或在细胞膜外暴露有权利 要求5-8中任一项所述的抗体。
  17. 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、如权利要求15所述的抗体偶联物、权利要求16所述的免疫细胞、或其组合；以及
    (ii)药学上可接受的载体。
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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2022509942A (ja) * 2018-11-16 2022-01-25 ブリストル－マイヤーズ スクイブ カンパニー 抗ｎｋｇ２ａ抗体およびその使用
WO2022074206A1 (en) 2020-10-08 2022-04-14 Affimed Gmbh Trispecific binders
WO2022093641A1 (en) * 2020-10-30 2022-05-05 BioLegend, Inc. Anti-nkg2a agents and compositions and methods for making and using the same
WO2022253805A1 (en) * 2021-06-01 2022-12-08 Symphogen A/S Anti-nkg2a antibodies and compositions
WO2023007023A1 (en) 2021-07-30 2023-02-02 Affimed Gmbh Duplexbodies
WO2024056862A1 (en) 2022-09-15 2024-03-21 Avidicure Ip B.V. Multispecific antigen binding proteins for tumor-targeting of nk cells and use thereof
EP4234580A4 (en) * 2020-11-23 2024-04-24 Bioheng Therapeutics Ltd ANTIBODIES TARGETING NKG2A AND ITS USE

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CN112552406B (zh) * 2021-02-24 2021-05-11 吴江近岸蛋白质科技有限公司 抗人cd73抗体
CN113278073B (zh) * 2021-06-21 2023-04-07 山西农业大学 一种nkg2a纳米抗体及其应用
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WO2024056010A1 (zh) * 2022-09-16 2024-03-21 北京三诺佳邑生物技术有限责任公司 特异性识别nkg2a的抗体及其应用

Citations (9)

* 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
WO2005105849A1 (en) * 2004-04-30 2005-11-10 Innate Pharma Compositions and methods for treating proliferative disorders such as nk-type ldgl
US20080118516A1 (en) * 2006-11-21 2008-05-22 Freishtat Robert J Method of treating respiratory virus-induced asthma with Th2 lymphocyte NKG2A receptor agonists
CN101484471A (zh) * 2006-06-30 2009-07-15 诺沃-诺迪斯克有限公司 抗-nkg2a抗体及其用途
CN101952317A (zh) * 2008-01-24 2011-01-19 诺沃-诺迪斯克有限公司 人化抗-人nkg2a单克隆抗体
CN102977213A (zh) * 2004-12-28 2013-03-20 依奈特制药公司 抗nkg2a 的单克隆抗体
CN107074950A (zh) * 2014-09-16 2017-08-18 依奈特制药公司 使用抗nkg2a抗体的治疗方案

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107106677A (zh) * 2014-08-28 2017-08-29 莱顿大学学术医院以Lumc的名义运作 Cd94/nkg2a和/或cd94/nkg2b抗体、疫苗组合
CA2959841A1 (en) * 2014-10-23 2016-04-28 Innate Pharma Treatment of cancers using anti-nkg2a agents

Patent Citations (9)

* 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
WO2005105849A1 (en) * 2004-04-30 2005-11-10 Innate Pharma Compositions and methods for treating proliferative disorders such as nk-type ldgl
CN102977213A (zh) * 2004-12-28 2013-03-20 依奈特制药公司 抗nkg2a 的单克隆抗体
CN101484471A (zh) * 2006-06-30 2009-07-15 诺沃-诺迪斯克有限公司 抗-nkg2a抗体及其用途
US20080118516A1 (en) * 2006-11-21 2008-05-22 Freishtat Robert J Method of treating respiratory virus-induced asthma with Th2 lymphocyte NKG2A receptor agonists
CN101952317A (zh) * 2008-01-24 2011-01-19 诺沃-诺迪斯克有限公司 人化抗-人nkg2a单克隆抗体
CN107074950A (zh) * 2014-09-16 2017-08-18 依奈特制药公司 使用抗nkg2a抗体的治疗方案

Non-Patent Citations (8)

* Cited by examiner, † Cited by third party
Title
"Biocomputing: Information Biocomputing: Informatics and Genome Projects", 1993, ACADEMIC PRESS
"Computational Molecular Biology", 1988, OXFORD UNIVERSITY PRESS
"Computer Analysis of Sequence Data", 1994, HUMANA PRESS
"Sequence Analysis in Molecular Biology", 1987, ACADEMIC PRESS
"Sequence Analysis Primer", 1991, M STOCKTON PRESS
MUNSON ET AL., ANAL. BIOCHEM., vol. 107, 1980, pages 220
REFERING TO JOURNAL OF IMMUNOLOGICAL METHODS, vol. 201, 1997, pages 35 - 5
See also references of EP3878869A4

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2022509942A (ja) * 2018-11-16 2022-01-25 ブリストル－マイヤーズ スクイブ カンパニー 抗ｎｋｇ２ａ抗体およびその使用
WO2022074206A1 (en) 2020-10-08 2022-04-14 Affimed Gmbh Trispecific binders
WO2022093641A1 (en) * 2020-10-30 2022-05-05 BioLegend, Inc. Anti-nkg2a agents and compositions and methods for making and using the same
EP4234580A4 (en) * 2020-11-23 2024-04-24 Bioheng Therapeutics Ltd ANTIBODIES TARGETING NKG2A AND ITS USE
WO2022253805A1 (en) * 2021-06-01 2022-12-08 Symphogen A/S Anti-nkg2a antibodies and compositions
WO2023007023A1 (en) 2021-07-30 2023-02-02 Affimed Gmbh Duplexbodies
WO2024056862A1 (en) 2022-09-15 2024-03-21 Avidicure Ip B.V. Multispecific antigen binding proteins for tumor-targeting of nk cells and use thereof
WO2024056861A1 (en) 2022-09-15 2024-03-21 Avidicure Ip B.V. Multispecific antigen binding proteins for stimulating nk cells and use thereof

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US20220259306A1 (en) 2022-08-18
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