WO2023208123A1 - 特异性结合金黄色葡萄球菌Hla毒素的全人源单克隆抗体 - Google Patents

特异性结合金黄色葡萄球菌Hla毒素的全人源单克隆抗体 Download PDF

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WO2023208123A1
WO2023208123A1 PCT/CN2023/091218 CN2023091218W WO2023208123A1 WO 2023208123 A1 WO2023208123 A1 WO 2023208123A1 CN 2023091218 W CN2023091218 W CN 2023091218W WO 2023208123 A1 WO2023208123 A1 WO 2023208123A1
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sed
seq
optionally
lcdr2
hcdr2
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PCT/CN2023/091218
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French (fr)
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郑伟宏
王孝丽
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珠海泰诺麦博制药股份有限公司
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Publication of WO2023208123A1 publication Critical patent/WO2023208123A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
    • C07K16/1267—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria
    • C07K16/1271—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria from Micrococcaceae (F), e.g. Staphylococcus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/40—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum bacterial
    • 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
    • A61P31/04—Antibacterial agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0684—Cells of the urinary tract or kidneys
    • C12N5/0686—Kidney cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • G01N33/56938—Staphylococcus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G01N2333/305—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Micrococcaceae (F)
    • G01N2333/31—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Micrococcaceae (F) from Staphylococcus (G)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2469/00—Immunoassays for the detection of microorganisms
    • G01N2469/10—Detection of antigens from microorganism in sample from host

Definitions

  • the present invention relates to the fields of medicine and immunology, specifically, to fully human antibodies that specifically bind to Staphylococcus aureus ⁇ -toxin and antigen-binding fragments thereof, including the anti- ⁇ -toxin antibody and its antigen-binding fragment.
  • Staphylococcus aureus is the second most common cause of human infection, after Enterobacteriaceae. Infections caused by Staphylococcus aureus can lead to skin, mucous membrane, deep tissue infections, endocarditis, pneumonia and other diseases. The fatality rate of severe infections and complications is as high as 20%.
  • Staphylococcus aureus hemolytic strains have four hemolysins: ⁇ , ⁇ , ⁇ , ⁇ , among which ⁇ -hemolysin (also known as ⁇ -toxin, Hla) is the most toxic and is the main cause of hemolysis, skin necrosis and death.
  • Hla ⁇ -hemolysin
  • the present invention meets this need.
  • the present invention provides fully human antibodies and antigen-binding fragments thereof that specifically bind to Hla.
  • the antibody can neutralize alpha-toxin, thereby preventing, treating Staphylococcus aureus infection, and diseases or conditions related to Staphylococcus aureus infection.
  • the invention provides a fully human antibody that specifically binds Hla and an antigen-binding fragment thereof, which includes:
  • the invention provides antibodies that specifically bind Hla and antigen-binding fragments thereof, comprising:
  • the invention provides antibodies that specifically bind Hla and antigen-binding fragments thereof, comprising:
  • HCDR1 shown in SEQ ID NO:47 HCDR2 shown in SED ID NO:4 and HCDR3 shown in SED ID NO:5; and/or, LCDR1 shown in SEQ ID NO:6, SED ID NO: LCDR2 shown in 7 and LCDR3 shown in SED ID NO:8;
  • HCDR1 shown in SEQ ID NO:48 HCDR2 shown in SED ID NO:4 and HCDR3 shown in SED ID NO:5; and/or, LCDR1 shown in SEQ ID NO:6, SED ID NO: LCDR2 shown in 7 and LCDR3 shown in SED ID NO:8;
  • HCDR1 shown in SEQ ID NO:49 HCDR2 shown in SED ID NO:4 and HCDR3 shown in SED ID NO:5; and/or, LCDR1 shown in SEQ ID NO:6, SED ID NO: LCDR2 shown in 7 and LCDR3 shown in SED ID NO:8;
  • HCDR1 shown in SEQ ID NO:50 HCDR2 shown in SED ID NO:4 and HCDR3 shown in SED ID NO:5; and/or, LCDR1 shown in SEQ ID NO:6, SED ID NO: LCDR2 shown in 7 and LCDR3 shown in SED ID NO:8;
  • HCDR1 shown in SEQ ID NO:51 HCDR2 shown in SED ID NO:4 and HCDR3 shown in SED ID NO:5; and/or, LCDR1 shown in SEQ ID NO:6, SED ID NO: LCDR2 shown in 7 and LCDR3 shown in SED ID NO:8;
  • HCDR1 shown in SEQ ID NO:52 HCDR2 shown in SED ID NO:4 and HCDR3 shown in SED ID NO:5; and/or, LCDR1 shown in SEQ ID NO:6, SED ID NO: LCDR2 shown in 7 and LCDR3 shown in SED ID NO:8;
  • HCDR1 shown in SEQ ID NO:53 HCDR2 shown in SED ID NO:4 and HCDR3 shown in SED ID NO:5; and/or, LCDR1 shown in SEQ ID NO:6, SED ID NO: LCDR2 shown in 7 and LCDR3 shown in SED ID NO:8;
  • HCDR1 shown in SEQ ID NO:54 HCDR2 shown in SED ID NO:4 and HCDR3 shown in SED ID NO:5; and/or, LCDR1 shown in SEQ ID NO:6, SED ID NO: LCDR2 shown in 7 and LCDR3 shown in SED ID NO:8;
  • HCDR1 shown in SEQ ID NO: 96 HCDR2 shown in SED ID NO: 4 and HCDR3 shown in SED ID NO: 5; and/or, LCDR1 shown in SEQ ID NO: 6, SED ID NO: LCDR2 shown in 7 and LCDR3 shown in SED ID NO:8;
  • HCDR1 shown in SEQ ID NO:47 HCDR2 shown in SED ID NO:55 and HCDR3 shown in SED ID NO:5; and/or, LCDR1 shown in SEQ ID NO:6, SED ID NO: LCDR2 shown in 7 and LCDR3 shown in SED ID NO:8;
  • HCDR1 shown in SEQ ID NO:102 HCDR2 shown in SED ID NO:4 and HCDR3 shown in SED ID NO:5; and/or, LCDR1 shown in SEQ ID NO:6, SED ID NO: LCDR2 shown in 7 and LCDR3 shown in SED ID NO:8;
  • HCDR1 shown in SEQ ID NO:50 HCDR2 shown in SED ID NO:58 and HCDR3 shown in SED ID NO:66; and/or, LCDR1 shown in SEQ ID NO:6, SED ID NO: LCDR2 shown in 7 and LCDR3 shown in SED ID NO:8;
  • HCDR1 shown in SEQ ID NO:102 HCDR2 shown in SED ID NO:4 and HCDR3 shown in SED ID NO:5; and/or, LCDR1 shown in SEQ ID NO:105, SED ID NO: LCDR2 shown in 7 and LCDR3 shown in SED ID NO:8;
  • HCDR1 shown in SEQ ID NO:102 HCDR2 shown in SED ID NO:4 and HCDR3 shown in SED ID NO:5; and/or, LCDR1 shown in SEQ ID NO:6, SED ID NO: LCDR2 shown in 7 and LCDR3 shown in SED ID NO:106;
  • HCDR1 shown in SEQ ID NO:50 HCDR2 shown in SED ID NO:58 and HCDR3 shown in SED ID NO:66; and/or, LCDR1 shown in SEQ ID NO:6, SED ID NO: LCDR2 shown in 7 and LCDR3 shown in SED ID NO:95;
  • HCDR1 shown in SEQ ID NO:50 HCDR2 shown in SED ID NO:58 and HCDR3 shown in SED ID NO:66; and/or, LCDR1 shown in SEQ ID NO:105, SED ID NO: LCDR2 shown in 7 and LCDR3 shown in SED ID NO:8;
  • HCDR1 shown in SEQ ID NO:50 HCDR2 shown in SED ID NO:58 and HCDR3 shown in SED ID NO:66; and/or, LCDR1 shown in SEQ ID NO:6, SED ID NO: LCDR2 shown in 7 and LCDR3 shown in SED ID NO:106;
  • HCDR1 shown in SEQ ID NO:102 HCDR2 shown in SED ID NO:4 and HCDR3 shown in SED ID NO:5; and/or, LCDR1 shown in SEQ ID NO:81, SED ID NO: LCDR2 shown in 85 and LCDR3 shown in SED ID NO:90;
  • the invention provides antibodies that specifically bind Hla and antigen-binding fragments thereof, which comprise variants of HCDR1 (SEQ ID NO:243) as shown in SEQ ID NO:3, SEQ ID NO:4 Variants of HCDR2 as set forth in SEQ ID NO:244 and variants of HCDR3 as set forth in SEQ ID NO:5 (SEQ ID NO:245); and variants of LCDR1 as set forth in SEQ ID NO:6 (SEQ ID NO:246), a variant of LCDR2 shown in SEQ ID NO:7 (SEQ ID NO:247), and a variant of LCDR3 shown in SEQ ID NO:8 (SEQ ID NO:248).
  • the invention provides antibodies that specifically bind Hla and antigen-binding fragments thereof, comprising heavy chain variable regions, wherein:
  • the heavy chain variable region includes the amino acid sequence shown in SEQ ID NO:33, or has at least 90%, 91%, 92%, 93%, 94%, or 95% of the amino acid sequence of SEQ ID NO:33 , an amino acid sequence that is 96%, 97%, 98% or 99% identical, or consisting of SEQ ID NO: 33;
  • the heavy chain variable region includes the amino acid sequence shown in SEQ ID NO:35, or has at least 90%, 91%, 92%, 93%, 94%, or 95% of the amino acid sequence of SEQ ID NO:35 , an amino acid sequence that is 96%, 97%, 98% or 99% identical, or consisting of SEQ ID NO: 35;
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 37, or has at least 90%, 91%, 92%, 93%, 94%, 95% with the amino acid sequence of SEQ ID NO: 37 , an amino acid sequence that is 96%, 97%, 98% or 99% identical, or consisting of SEQ ID NO: 37;
  • the heavy chain variable region includes the amino acid sequence shown in SEQ ID NO:39, or has at least 90%, 91%, 92%, 93%, 94%, or 95% of the amino acid sequence of SEQ ID NO:39 , an amino acid sequence that is 96%, 97%, 98% or 99% identical, or consisting of SEQ ID NO: 39; or
  • the heavy chain variable region includes the amino acid sequence shown in SEQ ID NO:41, or has at least 90%, 91%, 92%, 93%, 94%, or 95% of the amino acid sequence of SEQ ID NO:41 , an amino acid sequence that is 96%, 97%, 98% or 99% identical, or consists of SEQ ID NO: 41.
  • the invention provides antibodies that specifically bind Hla and antigen-binding fragments thereof, comprising light chain variable regions, wherein:
  • the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 34, or has at least 90%, 91%, 92%, 93%, 94%, 95% with the amino acid sequence of SEQ ID NO: 34 , an amino acid sequence that is 96%, 97%, 98% or 99% identical, or consisting of SEQ ID NO: 34;
  • the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 36, or has at least 90%, 91%, 92%, 93%, 94%, 95% with the amino acid sequence of SEQ ID NO: 36 , an amino acid sequence that is 96%, 97%, 98% or 99% identical, or consisting of SEQ ID NO: 36;
  • the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 38, or has at least 90%, 91%, 92%, 93%, 94%, 95% with the amino acid sequence of SEQ ID NO: 38 , an amino acid sequence that is 96%, 97%, 98% or 99% identical, or consisting of SEQ ID NO: 38;
  • the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:40, or has at least 90%, 91%, 92%, 93%, 94%, or 95% of the amino acid sequence of SEQ ID NO:40 , an amino acid sequence of 96%, 97%, 98% or 99% identity, or by SEQ ID NO: 40 composition; or
  • the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 42, or has at least 90%, 91%, 92%, 93%, 94%, 95% with the amino acid sequence of SEQ ID NO: 42 , an amino acid sequence that is 96%, 97%, 98% or 99% identical, or consisting of SEQ ID NO: 42.
  • the invention provides antibodies that specifically bind Hla and antigen-binding fragments thereof, which comprise a heavy chain variable region and a light chain variable region, wherein:
  • the heavy chain variable region includes the amino acid sequence shown in SEQ ID NO:33, or has at least 90%, 91%, 92%, 93%, 94%, or 95% of the amino acid sequence of SEQ ID NO:33 , 96%, 97%, 98% or 99% identical amino acid sequence, or consisting of SEQ ID NO:33, the light chain variable region comprising the amino acid sequence shown in SEQ ID NO:34, or with SEQ ID
  • the amino acid sequence of NO:34 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence, or is determined by SEQ ID NO:34 composition;
  • the heavy chain variable region includes the amino acid sequence shown in SEQ ID NO:35, or has at least 90%, 91%, 92%, 93%, 94%, or 95% of the amino acid sequence of SEQ ID NO:35 , 96%, 97%, 98% or 99% identical amino acid sequence, or consisting of SEQ ID NO:35, the light chain variable region comprising the amino acid sequence shown in SEQ ID NO:36, or with SEQ ID
  • the amino acid sequence of NO:36 has an amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or is represented by SEQ ID NO:36 composition;
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 37, or has at least 90%, 91%, 92%, 93%, 94%, 95% with the amino acid sequence of SEQ ID NO: 37 , 96%, 97%, 98% or 99% identical amino acid sequence, or consisting of SEQ ID NO:37, the light chain variable region comprising the amino acid sequence shown in SEQ ID NO:38, or with SEQ ID NO:37
  • the amino acid sequence of NO:38 has an amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or is represented by SEQ ID NO:38 composition;
  • the heavy chain variable region includes the amino acid sequence shown in SEQ ID NO:39, or has at least 90%, 91%, 92%, 93%, 94%, or 95% of the amino acid sequence of SEQ ID NO:39 , 96%, 97%, 98% or 99% identical amino acid sequence, or consisting of SEQ ID NO:39, the light chain variable region comprising the amino acid sequence shown in SEQ ID NO:40, or with SEQ ID
  • the amino acid sequence of NO:40 has an amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or is represented by SEQ ID NO:40 composition; or
  • the heavy chain variable region includes the amino acid sequence shown in SEQ ID NO:41, or has at least 90%, 91%, 92%, 93%, 94%, or 95% of the amino acid sequence of SEQ ID NO:41 , 96%, 97%, 98% or 99% identical amino acid sequence, or consisting of SEQ ID NO:41, the light chain variable region comprising the amino acid sequence shown in SEQ ID NO:42, or with SEQ ID
  • the amino acid sequence of NO:42 has an amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or is represented by SEQ ID NO:42 composition.
  • G in the TRN1029HCDR1 sequence can be conservatively substituted for A; F can be conservatively substituted for W, L, V, I, A, Y, preferably conservatively substituted for Y; T can be conservatively substituted for V, S, preferably Conservative substitution of S; S can be conservative substitution of T; Y can be conservative substitution of W, F, T, S, preferably conservative substitution of F; D can be conservative substitution of E, N, preferably conservative substitution of E; amino acid substitution can be one or more.
  • M in the TRN1029HCDR2 sequence can be conservatively substituted as L, F, I, preferably conservatively substituted as L; S can be conservatively substituted as T; Y can be conservatively substituted as W, F, T, S, preferably conservatively substituted as F; D can be conservatively substituted by E or N, preferably conservatively substituted by E; G can be conservatively substituted by A; K can be conservatively substituted by R, Q, N, H, preferably conservatively substituted by R; amino acid substitution can be one or more indivual.
  • a in the TRN1029HCDR3 sequence can be conservatively substituted as V, L, I, preferably conservatively substituted as V; K can be conservatively substituted as R, Q, N, H, preferably conservatively substituted as R; P can be conservatively substituted is A; R can be conservatively substituted by K, Q, N, H, preferably K; G can be conservatively substituted by A; S can be conservatively substituted by T; H can be conservatively substituted by N, Q, K, R, preferably Conservative substitution is R; Y can be conservatively substituted by W, F, T, S, preferably conservative substitution F; D can be conservatively substituted by E, N, preferably conservatively substituted by E; T can be conservatively substituted by V, S, preferably conservatively substituted is S; F can be conservatively substituted as W, L, V, I, A, Y, preferably conservatively Replace with Y; the amino acid substitution can be one or more.
  • Q in the TRN1029LCDR1 sequence can be conservatively substituted with N, E, preferably conservatively substituted with N; S can be conservatively substituted with T; I can be conservatively substituted with L, V, M, A, F, norleucine Acid, preferably conservatively substituted as L; T can be conservatively substituted as V, S, preferably conservatively substituted as S; N can be conservatively substituted as Q, H, D, K, R, preferably conservatively substituted as Q; amino acid substitution can be one or Multiple.
  • G in the TRN1029LCDR2 sequence can be conservatively substituted for A; A can be conservatively substituted for V, L, I, preferably conservatively substituted for V; S can be conservatively substituted for T; the amino acid substitution can be one or more.
  • Q in the TRN1029LCDR3 sequence can be conservatively substituted as N, E, preferably conservatively substituted as N; Y can be conservatively substituted as W, F, T, S, preferably conservatively substituted as F; H can be conservatively substituted as N , Q, K, R, preferably conservatively substituted to R; N can be conservatively substituted to Q, H, D, K, R, preferably conservatively substituted to Q; W can be conservatively substituted to Y, F, preferably conservatively substituted to Y; P Can be conservatively substituted as A; L can be conservatively substituted as I, V, M, A, F, norleucine, preferably conservatively substituted as I; T can be conservatively substituted as V, S, preferably conservatively substituted as S; amino acid substitutions can is one or more.
  • G in the TRN1030HCDR1 sequence can be conservatively substituted with A; T can be conservatively substituted with V, S, preferably conservatively substituted with S; F can be conservatively substituted with W, L, V, I, A, Y, preferably conservatively Substituted as Y; R can be conservatively substituted as K, Q, N, H, preferably conservatively substituted as K; Q can be conservatively substituted as N, E, preferably conservatively substituted as N; H can be conservatively substituted as N, Q, K, R , the preferred conservative substitution is R; A can be conservatively substituted as V, L, I, the preferred conservative substitution is V, and the amino acid substitution can be one or more.
  • I in the TRN1030HCDR2 sequence can be conservatively substituted with L, V, M, A, F, norleucine, preferably conservatively substituted with L; P can be conservatively substituted with A; D can be conservatively substituted with E, N, preferably conservatively substituted to E; L can be conservatively substituted to I, V, M, A, F, norleucine, preferably conservatively substituted to I; T can be conservatively substituted to V, S, preferably conservatively substituted to S; P It can be conservatively substituted as A, and the amino acid substitution can be one or more.
  • a in the TRN1030HCDR3 sequence can be conservatively substituted as V, L, I, preferably conservatively substituted as V; R can be conservatively substituted as K, Q, N, H, preferably conservatively substituted as K; D can be conservatively substituted is E, N, preferably conservatively substituted for E; P can be conservatively substituted for A; W can be conservatively substituted for Y, F, preferably conservatively substituted for Y; S can be conservatively substituted for T; A can be conservatively substituted for V, L, I , preferably conservatively substituted to V; D can be conservatively substituted to E, N, preferably conservatively substituted to E; I can be conservatively substituted to L, V, M, A, F, norleucine, preferably conservatively substituted to L; N can be conservatively substituted Conservative substitutions are Q, H, D, K, R, and the preferred conservative substitution is Q; V can be conservatively substituted as I, L, M, F, A, norleucine
  • Q in the TRN1030LCDR1 sequence can be conservatively substituted as N, E, preferably conservatively substituted as N; S can be conservatively substituted as T; V can be conservatively substituted as I, L, M, F, A, norleucine Acid, preferably conservatively substituted is L; R can be conservatively substituted into K, Q, N, H, preferably conservatively substituted into K; N can be conservatively substituted into Q, H, D, K, R, preferably conservatively substituted into Q, amino acid substitution Can be one or more.
  • G in the TRN1030LCDR2 sequence can be conservatively substituted for A; A can be conservatively substituted for V, L, I, preferably conservatively substituted for V; S can be conservatively substituted for T, and the amino acid substitution can be one or more.
  • Q in the TRN1030LCDR3 sequence can be conservatively substituted as N, E, preferably conservatively substituted as N; Y can be conservatively substituted as W, F, T, S, preferably conservatively substituted as F; N can be conservatively substituted as Q , H, D, K, R, preferably conservatively substituted as Q; D can be conservatively substituted as E, N, preferably conservatively substituted as E; W can be conservatively substituted as Y, F, preferably conservatively substituted as Y; P can be conservatively substituted as A; T can be conservatively substituted into V or S, preferably conservatively substituted into S, and the amino acid substitution can be one or more.
  • G in the TRN1031HCDR1 sequence can be conservatively substituted for A; F can be conservatively substituted for W, L, V, I, A, Y, preferably conservatively substituted for Y; S can be conservatively substituted for T; D can be conservatively substituted
  • the substitutions are E and N, and the preferred conservative substitution is E; Y can be conservatively substituted as W, F, T, and S, and the conservative substitution is F.
  • I in the TRN1031HCDR2 sequence can be conservatively substituted with L, V, M, A, F, norleucine, preferably conservatively substituted with L; Y can be conservatively substituted with W, F, T, S, preferably conservatively substituted Conservative substitution is F; P can be conservatively substituted for A; G can be conservatively substituted for A; E can be conservatively substituted for D, Q, preferably conservatively substituted for D; S can be conservatively substituted for T; A can be conservatively substituted for V, L, I is preferably conservatively substituted with V.
  • a in the TRN1031 HCDR3 sequence can be conservatively substituted to V, L, or I, preferably conservatively substituted to V; T can be conservatively substituted.
  • Conservative substitutions are V and S, preferably conservative substitutions are S; P can be conservatively substituted for A; D can be conservatively substituted for E, N, preferably conservative substitutions are E; D can be conservatively substituted for E, N, preferably conservative substitutions are E ; F can be conservatively substituted as W, L, V, I, A, Y, preferably conservatively substituted as Y; S can be conservatively substituted as T; H can be conservatively substituted as N, Q, K, R, preferably conservatively substituted as R; G can be conservatively substituted as A; Y can be conservatively substituted as W, F, T, S, preferably conservatively substituted as F; Q can be conservatively substituted as N, E, preferably conservatively substituted as N; L can be conservatively substituted as I, V, M, A, F
  • D in the TRN1031LCDR1 sequence can be conservatively substituted by E, N, preferably conservatively substituted by E;
  • A can be conservatively substituted by V, L, I, preferably conservatively substituted by V;
  • I can be conservatively substituted by L, V , M, A, F, norleucine, preferably conservatively substituted to L;
  • T can be conservatively substituted into V, S, preferably conservatively substituted into S;
  • S can be conservatively substituted into T;
  • N can be conservatively substituted into Q, H, D , K, R, preferably conservative substitution is Q.
  • G in the TRN1031LCDR2 sequence can be conservatively substituted by A; A can be conservatively substituted by V, L, I, preferably conservatively substituted by V; S can be conservatively substituted by T.
  • L in the TRN1031LCDR3 sequence can be conservatively substituted by I, V, M, A, F, norleucine, preferably conservatively substituted by I;
  • Q can be conservatively substituted by N, E, preferably conservatively substituted by N ;
  • D can be conservatively substituted by E, N, preferably conservatively substituted by E;
  • F can be conservatively substituted by W, L, V, I, A, Y, preferably conservatively substituted by Y;
  • R can be conservatively substituted by K, Q, N, H, preferably conservatively substituted with K;
  • P can be conservatively substituted with A;
  • T can be conservatively substituted with V, S, preferably conservatively substituted with S.
  • G in the TRN1032HCDR1 sequence can be conservatively substituted for A; F can be conservatively substituted for W, L, V, I, A, Y, preferably conservatively substituted for Y; S can be conservatively substituted for T; L can be conservatively substituted
  • the substitutions are I, V, M, A, F, norleucine, and the preferred conservative substitution is I; K can be conservatively substituted by R, Q, N, H, and the preferred conservative substitution is R; N can be conservatively substituted by Q, H , D, K, R, preferably conservatively substituted as Q; Y can be conservatively substituted as W, F, T, S, preferably conservatively substituted as F; R can be conservatively substituted as K, Q, N, H, preferably conservatively substituted as K .
  • I in the TRN1032HCDR2 sequence can be conservatively substituted with L, V, M, A, F, norleucine, preferably conservatively substituted with L;
  • Q can be conservatively substituted with N, E, preferably conservatively substituted with N ;
  • K can be conservatively substituted as R, Q, N, H, preferably conservatively substituted as R;
  • F can be conservatively substituted as W, L, V, I, A, Y, preferably conservatively substituted as Y;
  • G can be conservatively substituted as A;
  • N can be conservatively substituted as Q, H, D, K, R, preferably conservatively substituted as Q;
  • V can be conservatively substituted as I, L, M, F, A, norleucine, preferably conservatively substituted as L.
  • a in the TRN1032HCDR3 sequence can be conservatively substituted as V, L, I, preferably conservatively substituted as V; R can be conservatively substituted as K, Q, N, H, preferably conservatively substituted as K; E can be conservatively substituted is D, Q, preferably conservative substitution is D; L can be conservatively substituted by I, V, M, A, F, norleucine, preferably conservatively substituted is I; H can be conservatively substituted by N, Q, K, R, Preferably conservative substitution is R; F can be conservatively substituted by W, L, V, I, A, Y, preferably conservatively substituted by Y; D can be conservatively substituted by E, N, preferably conservatively substituted by E; S can be conservatively substituted by T ;G can be conservatively replaced by A.
  • N in the TRN1032LCDR1 sequence can be conservatively substituted as Q, H, D, K, R, preferably conservatively substituted as Q;
  • I can be conservatively substituted as L, V, M, A, F, norleucine , the preferred conservative substitution is L;
  • G can be conservatively substituted as A;
  • K can be conservatively substituted as R, Q, N, H, and the preferred conservative substitution is R;
  • S can be conservatively substituted as T.
  • S in the TRN1032LCDR2 sequence can be conservatively substituted for T; D can be conservatively substituted for E, N, preferably conservatively substituted for E; N can be conservatively substituted for Q, H, D, K, R, preferably conservatively substituted for Q.
  • H in the TRN1032LCDR3 sequence can be conservatively substituted to N, Q, K, R, preferably conservatively substituted to R; V can be conservatively substituted to I, L, M, F, A, norleucine, preferably conservatively substituted Conservative substitution is L; W can be conservatively substituted by Y, F, preferably conservative substitution is Y; Q can be conservatively substituted by N, E, preferably conservatively substituted is N; T can be conservatively substituted by V, S, preferably conservatively substituted S; S It can be conservatively substituted as T; D can be conservatively substituted as E, N, preferably conservatively substituted as E; L can be conservatively substituted as I, V, M, A, F, norleucine, preferably conservatively substituted as I.
  • G in the TRN1033HCDR1 sequence can be conservatively substituted for A; F can be conservatively substituted for W, L, V, I, A, Y, preferably conservatively substituted for Y; I can be conservatively substituted for L, V, M , A, F, norleucine, preferably conservatively substituted as L; V can be conservatively substituted as I, L, M, F, A, norleucine, preferably conservatively substituted as L; E can be conservatively substituted as D, Q , preferably conservatively substituted as D; M can be conservatively substituted as L, F, I, preferably conservatively substituted as L; Y can be conservatively substituted as W, F, T, S, preferably conservatively substituted as F.
  • I in the TRN1033HCDR2 sequence can be conservatively substituted with L, V, M, A, F, norleucine, preferably conservatively substituted with L; Y can be conservatively substituted with W, F, T, S, preferably conservatively substituted Conservative substitution is F; R can be conservatively substituted into K, Q, N, H, preferably conservative substitution is K; G can be conservatively substituted into A; S can be conservatively substituted into T; T can be conservatively substituted into V, S, conservative substitution is preferred S.
  • a in the TRN1033HCDR3 sequence can be conservatively substituted as V, L, I, preferably conservatively substituted as V; K can be conservatively substituted as R, Q, N, H, preferably conservatively substituted as R; E can be conservatively substituted is D, Q, preferably conservatively substituted to D; Y can be conservatively substituted to W, F, T, S, preferably conservatively substituted to F; V can be conservatively substituted to I, L, M, F, A, norleucine, The preferred conservative substitution is L; W can be conservatively substituted into Y, F, and the preferred conservative substitution is Y; L can be conservatively substituted into I, V, M, A, F, norleucine, and the preferred conservative substitution is I; G can be conservatively substituted Substituted with A; D can be conservatively substituted with E, N, preferably conservatively substituted with E; R can be conservatively substituted with K, Q, N, H, preferably conservatively substituted with K; H
  • N in the TRN1033LCDR1 sequence can be conservatively substituted as Q, H, D, K, R, preferably conservatively substituted as Q; S can be conservatively substituted as T; I can be conservatively substituted as L, V, M, A , F, norleucine, preferably conservatively substituted as L; G can be conservatively substituted as A.
  • T in the TRN1033LCDR2 sequence can be conservatively substituted as V, S, preferably conservatively substituted as S; N can be conservatively substituted as Q, H, D, K, R, preferably conservatively substituted as Q; D can be conservatively substituted is E or N, preferably conservative substitution is E.
  • S in the TRN1033LCDR3 sequence can be conservatively substituted for T; T can be conservatively substituted for V, S, preferably conservatively substituted for S; W can be conservatively substituted for Y, F, preferably conservatively substituted for Y; D can be conservatively substituted for is E, N, preferably conservatively substituted to E; L can be conservatively substituted to I, V, M, A, F, norleucine, preferably conservatively substituted to I; G can be conservatively substituted to A; V can be conservatively substituted to I , L, M, F, A, norleucine, preferably conservatively substituted as L.
  • the above-described antibodies or antigen-binding fragments thereof further comprise heavy chain and/or light chain constant region sequences derived from human antibody germline consensus sequences.
  • the light chain constant region is preferably a human kappa or lambda chain constant region.
  • the heavy chain constant region can be a gamma, mu, alpha, delta, or epsilon chain.
  • the heavy chain constant region is human IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM, IgD Same type as IgE.
  • Each heavy and light chain type is characterized by a specific constant region with sequences well known in the art.
  • the antibody is an IgG class antibody.
  • the IgG class antibody is an IgGl subclass antibody.
  • the IgG class antibody is an IgG2 subclass antibody.
  • the IgG class antibody is an IgG3 subclass antibody.
  • the IgG class antibody is an IgG4 subclass antibody.
  • the constant region is preferably a human IgG constant region, such as a constant region of human IgG1, IgG2, IgG3 or IgG4 isotype.
  • the heavy chain and/or light chain constant region is described, for example, in Sequences of Proteins of Immunological Interest, NIH Publication No. 91-3242, any of which may be used in the present invention.
  • the invention provides an anti-Hla antibody or an antigen-binding fragment thereof, wherein the heavy chain constant region sequence is a human IgG1 constant region, and the heavy chain constant region sequence comprises the amino acid sequence shown in SEQ ID NO: 43 or consists of composition.
  • the heavy chain constant region sequence of the antibody may also be a human IgG4 constant region, and the heavy chain constant region sequence includes or consists of the amino acid sequence shown in SEQ ID NO: 44.
  • the kappa light chain constant region of the antibody comprises or consists of the amino acid sequence set forth in SEQ ID NO:45
  • the lambda light chain constant region of the antibody comprises the amino acid sequence set forth in SEQ ID NO:46 sequence or consisting of.
  • sequence variants of these constant region domains may also be used, for example containing one or more amino acid modifications, where the amino acid positions are identified according to the EU indexing system of Kabat et al. (1991).
  • the antibody is monoclonal.
  • the antibody is a full-length antibody.
  • the antibodies of the invention also encompass antibodies that compete for binding to Hla with any of the antibodies described above, as well as antibodies that bind to the same epitope of Hla as any of the antibodies described above.
  • At least a portion of the framework sequences of the anti-Hla antibodies are human consensus framework sequences.
  • the anti-Hla antibodies of the invention are intact antibodies, such as IgG1, IgG2, IgG3, IgG4, IgM, IgA and IgE antibodies.
  • the anti-Hla antibodies of the invention encompass only the antigen-binding portion thereof, such as: Fab, Fab'-SH, Fv, scFv or (Fab')2 fragments.
  • the anti-Hla antibodies of the invention are neutralizing antibodies for neutralizing Hla.
  • the invention provides an isolated nucleic acid molecule encoding any of the antibodies or antigen-binding fragments thereof described in the first aspect.
  • the invention provides a vector comprising the nucleic acid molecule of the second aspect.
  • the vector is an expression vector.
  • the invention provides a host cell comprising the vector of the third aspect or the nucleic acid molecule of the second invention.
  • the host cell is prokaryotic, such as E. coli.
  • the host cell is eukaryotic, such as 293 cells, CHO cells, yeast cells, or plant cells.
  • the present invention provides a pharmaceutical composition comprising the antibody of the first aspect or an antigen-binding fragment thereof.
  • the present invention provides the use of an Hla-binding antibody or an antigen-binding fragment thereof in the preparation of a medicament for preventing or treating Staphylococcus aureus infection or diseases associated with Staphylococcus aureus infection.
  • the invention provides a method of preventing or treating Staphylococcus aureus infection or a disease associated with Staphylococcus aureus infection in a subject in need thereof, comprising administering to the subject a prophylactically effective amount or a therapeutically effective amount.
  • the disease associated with S. aureus infection is skin necrosis, skin and soft tissue infections (including abscesses), surgical site infections, prosthetic joint infections, bacteremia, sepsis, pneumonia.
  • the present invention provides a method for detecting whether Staphylococcus aureus or Hla is present in a sample, including combining the antibody of the present invention with the test substance under conditions that allow any of the foregoing antibodies of the present invention to form a complex with Hla.
  • the sample is contacted and the formation of anti-Hla antibody-Hla complexes is detected.
  • Figure 1 shows the SDS-PAGE results of purification of expressed Hla and Hla_H35L proteins.
  • Figure 2 shows the results of detecting anti-Hla and Hla_H35L antibody titers in plasma samples by ELISA method.
  • Figure 3 shows the results of flow sorting of single memory B cells using Hla_H35L protein.
  • Figure 4 shows the ELISA detection results of linearly expressed antibodies binding to Hla and Hla_H35L.
  • Figure 5 shows the SDS-PAGE electrophoresis results and Western immunoblotting results of the expressed monoclonal antibodies.
  • the two pictures on the left are for detecting non-reducing
  • the two pictures on the right show the results of testing reducing samples.
  • Lanes 1, 2, 3, 4, 5, 6 and 7 are molecular size markers, TRN1029, TRN1030, TRN1031, TRN1032, TRN1032 and TRN1033 respectively.
  • Figure 6 shows the ELISA detection results of recombinant expression antibody binding to Hla and Hla_H35L.
  • Figure 7 shows a schematic representation of a competition study of different antibodies that specifically bind to the same antigen.
  • Figure 8 shows the SPR results of competition between different recombinantly expressed anti-Hla antibodies for binding to the antigen.
  • Figure 9 shows the experimental results of different anti-Hla antibodies neutralizing the hemolytic effect of Hla on human erythrocytes.
  • Figure 10 shows the neutralizing activity of different anti-Hla antibodies in mice.
  • FIG. 11 shows the protective effect of different anti-Hla antibodies on Hla toxin challenge in mice.
  • the term “comprises” or “includes” means the inclusion of the stated element, integer or step, but not the exclusion of any other element, integer or step.
  • the term “comprises” or “includes” is used herein, it also encompasses a combination of the stated elements, integers, or steps unless otherwise specified.
  • reference is made to an antibody variable region that "comprises” a particular sequence it is also intended to encompass antibody variable regions that consist of that particular sequence.
  • alpha-toxin oligomerizes into heptamers in the membranes of susceptible cells (such as leukocytes, platelets, erythrocytes, peripheral blood mononuclear cells, macrophages, keratinocytes, fibroblasts, and endothelial cells) and then accumulates in the cell membrane.
  • susceptible cells such as leukocytes, platelets, erythrocytes, peripheral blood mononuclear cells, macrophages, keratinocytes, fibroblasts, and endothelial cells
  • Alpha-toxin has been shown to lyse a variety of human cells, including erythrocytes, epithelial cells, endothelial cells, and a range of other hematopoietic lineage cells, including T cells, monocytes, macrophages, and neutrophils, resulting in cytolysis. , inflammation and tissue damage. Alpha-toxin has been shown to play a role in pneumonia, skin necrosis, and sepsis.
  • alpha-toxin The amino acid sequence of wild-type alpha-toxin is shown in SEQ ID NO:1, and the amino acid sequence of modified alpha-toxin (H35L mutant) is shown in SEQ ID NO:2. Unless otherwise stated, references to alpha-toxin refer to the wild-type form.
  • Hemolysis refers to the phenomenon that the cell membrane of red blood cells is damaged and ruptured due to physical factors, chemical factors, biological factors (such as toxins), etc., and the internal protoplasm leaks from the cells, causing the death of red blood cells.
  • Hemolytic reaction is a term that refers specifically to red blood cells. Although in addition to red blood cells, there are white blood cells, lymphocytes and other non-red blood cells in the blood, the term “hemolytic reaction” is generally not used to describe the death of these cells.
  • antibody is used herein in the broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they It is sufficient to demonstrate the required antigen-binding activity.
  • a complete antibody will typically contain at least two full-length heavy chains and two full-length light chains, but in some cases may include fewer chains, for example naturally occurring antibodies in camels may contain only heavy chains.
  • Human antibody is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences, produced by humans or human cells or derived from non-human sources, utilizing human antibody repertoires or other human antibodies coding sequence. This definition of human antibodies specifically excludes humanized antibodies containing non-human antigen-binding residues.
  • neutralizing antibody is an antibody or antibody fragment that reduces or inhibits the biological activity of Hla.
  • the reduction in biological activity may be partial or complete.
  • the degree to which an antibody neutralizes Hla is called the neutralizing potency of the antibody.
  • the neutralizing potency of the antibody can be determined or measured using one or more tests known to those of ordinary skill and/or described or referred to herein, including, but not limited to, competitive binding assays, direct and indirect sandwich assays. Assay, immunoprecipitation assay and enzyme-linked immunosorbent assay (ELISA).
  • Antibody fragment or "antigen-binding fragment” refers to a molecule that is distinct from an intact antibody, contains a portion of an intact antibody, and binds the antigen to which the intact antibody binds.
  • antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab') 2 ; diabodies; linear antibodies; single chain antibodies (e.g., scFv); single domain antibodies; bivalent or diabodies Specific antibodies or fragments thereof; camelid antibodies (heavy chain antibodies); and bispecific antibodies or multispecific antibodies formed from antibody fragments.
  • Complementarity determining region or “CDR region” or “CDR” or “hypervariable region” is the amino acid region in the antibody variable region that is mainly responsible for binding to the antigenic epitope.
  • the CDRs of the heavy and light chains are often referred to as CDR1, CDR2, and CDR3 and are numbered sequentially starting from the N-terminus.
  • CDRs Kabat complementarity determining regions
  • the AbM CDR is a compromise between the Kabat CDR and Chothia structural loops, and is used by Oxford Molecular's AbM antibody modeling software, "Contact "Contact” CDR is based on the analysis of available complex crystal structures. According to different CDR determination schemes, the residues of each of these CDRs are described below.
  • the scope of the antibody also encompasses antibodies whose variable region sequence contains the specific CDR sequence but is different due to the application of different protocols (e.g. different Assigning system rules or combinations) causes the claimed CDR boundaries to be different from the specific CDR boundaries defined in the present invention.
  • CDRs of the antibodies of the invention can be manually evaluated to determine the boundaries according to any protocol in the art or a combination thereof.
  • CDR or “CDR sequence” encompasses CDR sequences determined in any of the above ways.
  • variable region refers to the domain of an antibody heavy or light chain that is involved in binding of the antibody to an antigen.
  • the variable domains of the heavy and light chains of natural antibodies generally have similar structures, with each domain containing four conserved framework regions (FRs) and three complementarity determining regions (CDRs) (see, e.g., Kindt et al. Kuby Immunology, 6th Edition, W.H. Freeman and Co. 91 pages (2007)).
  • binding means that the binding is selective for the antigen and can be distinguished from undesired or non-specific interactions.
  • the ability of an antibody to bind to a specific antigen can be determined by enzyme-linked immunosorbent assay (ELISA), SPR or biofilm layer interference techniques, or other conventional binding assays known in the art.
  • Binding affinity refers to the strength of the sum of all non-covalent interactions between a single binding site of a molecule (eg, an antibody) and its binding partner (eg, an antigen). Unless otherwise stated, as used herein, "binding affinity” refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (eg, antibody and antigen). The affinity of a molecule X for its partner Y is usually expressed in terms of the binding-dissociation equilibrium constant (K D ). Affinity can be measured by common methods known in the art, including those known in the art and described herein.
  • an antibody of the invention that "specifically binds" alpha toxin has a KD value of about 1 ⁇ 10 -8 M, preferably about 1 ⁇ 10 -9 M, as determined by surface plasmon resonance assay. ; More preferably 1 ⁇ 10 -10 M.
  • competition in the context of antigen-binding proteins competing for the same epitope, it is meant that competition between the antigen-binding proteins is determined by an assay in which the antigen to be detected binds Proteins (such as antibodies or immunologically functional fragments thereof) prevent or inhibit (e.g. Such as reducing) the specific binding of a reference antigen-binding protein (eg, a ligand or reference antibody) to a common antigen (eg, N protein or a fragment thereof).
  • a reference antigen-binding protein eg, a ligand or reference antibody
  • epitope refers to an antigenic determinant that interacts with a specific antigen-binding site called a paratope in the variable region of an antibody molecule.
  • a single antigen can have more than one epitope. Therefore, different antibodies can bind to different regions on the antigen and can have different biological effects.
  • vector when used herein refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked.
  • the term includes vectors that are self-replicating nucleic acid structures as well as vectors that are incorporated into the genome of a host cell into which they have been introduced. Some vectors are capable of directing the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as "expression vectors.”
  • host cell refers to cells into which exogenous nucleic acid is introduced, including the progeny of such cells.
  • Host cells include “transformants” and “transformed cells,” which include the primary transformed cell and progeny derived therefrom, regardless of the number of passages.
  • the progeny may not be identical in nucleic acid content to the parent cells but may contain mutations. Mutant progeny having the same function or biological activity as screened or selected in the originally transformed cells are included herein.
  • an anti-Hla antibody or composition of the invention refers to the amount of an anti-Hla antibody or composition of the invention that produces the desired effect in a patient in need of treatment or prevention when administered to the patient in single or multiple doses.
  • a “therapeutically effective amount” refers to an amount of an anti-Hla antibody or composition of the invention, at the required dose and for the required period of time, effective to achieve the desired therapeutic outcome.
  • a “prophylactically effective amount” refers to an amount of an anti-Hla antibody or composition of the invention that is effective at the required dose and for the required period of time to achieve the desired prophylactic result.
  • variant in relation to an antibody is used herein to mean a variant that has been replaced, deleted, or deleted by at least 1, such as 1-30, or 1-20, or 1-10, such as 1 or 2 or 3 or 4 or 5 amino acids. and/or an antibody in which an antibody region of interest (e.g., a heavy chain variable region or a light chain variable region or a heavy chain CDR region or a light chain CDR region) is inserted with amino acid changes, wherein the variant substantially retains the antibody molecule prior to the change biological characteristics.
  • the invention encompasses variants of any of the antibodies described herein.
  • the antibody variant retains at least 60%, 70%, 80%, 90%, or 100% of the biological activity (eg, antigen-binding capacity) of the prior antibody.
  • the heavy chain variable region or light chain variable region of the antibody, or each CDR region can be changed individually or in combination.
  • the amino acid changes in one or more or all three heavy chain CDRs are no more than 1, 2, 3, 4, 5, 6, 7, 8, 9 Or 10.
  • the amino acid changes are amino acid substitutions, preferably conservative substitutions.
  • the antibody variant has at least 80%, 90% or 95% or 99% or greater amino acid identity with the parent antibody over the antibody sequence region of interest.
  • the sequences are aligned for optimal comparison purposes (e.g., a first and a second amino acid sequence or nucleic acid sequence may be aligned for optimal alignment). Introducing gaps in one or both may allow non-homologous sequences to be discarded for comparison purposes).
  • the length of the aligned reference sequences is at least 30%, preferably at least 40%, more preferably at least 50%, 60% and even more preferably at least 70%, 80% , 90%, 100% of the reference sequence length.
  • the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. The molecules are identical when a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence.
  • Mathematical algorithms can be used to perform sequence comparison and calculation of percent identity between two sequences.
  • the Needlema and Wunsch ((1970) J. Mol. Biol. 48:444-453) algorithm is used which has been integrated into the GAP program of the GCG software package (available at http://www.gcg.com available), determine the distance between two amino acid sequences using the Blossum 62 matrix or the PAM250 matrix with gap weights 16, 14, 12, 10, 8, 6, or 4 and length weights 1, 2, 3, 4, 5, or 6 Percent identity.
  • the GAP program in the GCG software package (available at http://www.gcg.com) is used, using the NWSgapdna.CMP matrix and gap weights 40, 50, 60, 70 or 80 and A length weight of 1, 2, 3, 4, 5, or 6 determines the percent identity between two nucleotide sequences.
  • a particularly preferred parameter set (and one that should be used unless otherwise stated) is the Blossum 62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.
  • conservative substitution refers to the substitution of one amino acid by another amino acid within the same class, for example, one acidic amino acid is replaced by another acidic amino acid, one basic amino acid is replaced by another basic amino acid, or one neutral amino acid is replaced by another. Neutral amino acid substitutions. Exemplary substitutions are shown in the table below:
  • the invention provides nucleic acids encoding any of the above anti-Hla antibodies or fragments thereof or any chain thereof.
  • the invention also provides vectors comprising the nucleic acids.
  • the vector is an expression vector, such as a eukaryotic expression vector.
  • the expression vector or DNA sequence can be transfected or introduced into a suitable host cell. Therefore, the present invention provides host cells comprising the above-mentioned nucleic acids or the above-mentioned vectors.
  • the host cell is a prokaryotic cell, such as an E. coli cell.
  • the host cell is eukaryotic.
  • the host cell is selected from yeast cells, mammalian cells (eg, CHO cells or 293 cells), or other cells suitable for preparation of antibodies or antigen-binding fragments thereof.
  • Methods and conditions for culturing the transfected cells produced and for recovering the antibody molecules produced are known to those skilled in the art and can be based on the present description and methods known from the prior art, depending on the specific expression vector used and Mammalian host cell changes or optimizations.
  • compositions preferably pharmaceutical compositions, comprising any anti-Hla antibody or antigen-binding fragment thereof described herein.
  • the composition further comprises pharmaceutical excipients.
  • compositions are used to prevent or treat Staphylococcus aureus infections.
  • Staphylococcus aureus infections include burns, skin necrosis.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, isotonic and absorption delaying agents, and the like that are physiologically compatible with the therapeutic agent for administration.
  • the present invention provides a method for preparing an anti-Hla antibody, wherein the method includes culturing a nucleic acid encoding the anti-Hla antibody or an expression vector comprising the nucleic acid under conditions suitable for expression of the nucleic acid encoding the anti-Hla antibody. host cells, and optionally isolate the anti-Hla antibodies. In a certain embodiment, the method further includes recovering anti-Hla antibodies from the host cell (or host cell culture medium).
  • the nucleic acid encoding the anti-Hla antibody of the present invention is first isolated and inserted into a vector for further cloning and/or expression in host cells.
  • Such nucleic acids are readily isolated and sequenced using conventional procedures, for example by using oligonucleotide probes capable of specifically binding to nucleic acids encoding anti-Hla antibodies of the invention.
  • the invention provides a method of treating Staphylococcus aureus infection, comprising administering to a subject in need thereof a therapeutically effective amount of an anti-Hla antibody of the invention.
  • the present invention provides a method of preventing Staphylococcus aureus infection, comprising administering to a subject in need thereof a prophylactically effective amount of an anti-Hla antibody of the present invention.
  • the antibodies described herein may be suitable for preventing, treating, or managing diseases or conditions associated with Staphylococcus aureus infection, including, but not limited to, skin necrosis, skin and soft tissue infections (including abscesses), surgical site infections, Artificial joint infections, bacteremia, sepsis, pneumonia.
  • the antibodies of the invention are useful for reducing the number of Staphylococcus aureus bacteria in an individual or in a specific tissue or organ of an individual. In some embodiments, the antibodies of the invention are useful for reducing the toxic activity of alpha-toxin produced by Staphylococcus aureus bacteria in an individual, thereby alleviating symptoms resulting from the infection.
  • the present invention provides the use of an anti-Hla antibody in the preparation of a medicament for the treatment of a patient suffering from a Staphylococcus aureus infection or having symptoms associated with a Staphylococcus aureus infection.
  • the anti-Hla antibody provided by the present invention can be used for in vivo or in vitro diagnosis of Staphylococcus aureus strains that produce ⁇ -toxin, or diseases related to ⁇ -toxin.
  • the sample to be tested is contacted with the anti-Hla antibody of the present invention under conditions that allow the anti- ⁇ -toxin antibody to bind to ⁇ -toxin, and diagnosis and identification are performed by detecting whether a complex is formed.
  • the anti-Hla antibodies provided by the invention can also detect and/or measure alpha-toxin in a sample, for example for diagnostic purposes.
  • the anti-Hla antibodies provided by the invention can also be used to detect the presence and severity of Staphylococcus aureus infection.
  • detection when used herein includes quantitative or qualitative detection. Exemplary detection methods may involve immunohistochemistry, immunocytochemistry, flow cytometry (e.g., FACS), magnetic beads complexed with antibody molecules, ELISA assays Law.
  • Samples for use in alpha-toxin diagnostic assays include any tissue or fluid sample that may be obtained from a patient and which under normal or pathological conditions contains detectable amounts of alpha-toxin or fragments thereof.
  • the amount of alpha-toxin will be measured in a specific sample obtained from a healthy patient (eg, a patient not suffering from S. aureus infection) to initially establish a baseline or standard level of alpha-toxin. This baseline amount of alpha-toxin can then be compared to the amount of alpha-toxin measured in samples obtained from individuals suspected of having conditions associated with Staphylococcus aureus infection or symptoms associated with such conditions.
  • Heavy chain complementarity determining region HCDR1 which contains the amino acid sequence SEQ ID NO: 3;
  • Heavy chain complementarity determining region HCDR2 which contains the amino acid sequence SEQ ID NO: 4;
  • Heavy chain complementarity determining region HCDR3 which contains the amino acid sequence SEQ ID NO: 5;
  • LCDR1 Light chain complementarity determining region LCDR1, which contains the amino acid sequence:
  • X 1 is Q, N, E or D;
  • X 2 is S, T, A or I
  • X 3 is I, L, V, M, A, F, norleucine, G or N;
  • X 4 is T, V, S, R, K or I;
  • X 5 is T, V, S, K or G
  • X 6 is N, Q, H, D, K, R or S;
  • X 1 is Q, N, E or D
  • X 2 is S
  • X 3 is I
  • X 4 is T
  • X 5 is T
  • X 6 is N
  • X 1 is N
  • X 2 is S
  • X 3 is I
  • X 4 is T
  • X 5 is T
  • X 6 is N
  • X 1 is N
  • X 2 is S
  • X 3 is I
  • X 4 is T
  • X 5 is T
  • X 6 is N
  • X 1 is Q
  • X 2 is S, T, A or I
  • X 3 is I
  • X 4 is T
  • X 5 is T
  • X 6 is N
  • X 1 is Q
  • X 2 is T
  • X 3 is I
  • X 4 is T
  • X 5 is T
  • X 6 is N
  • X 1 is Q
  • X 2 is T
  • X 3 is I
  • X 4 is T
  • X 5 is T
  • X 6 is N
  • N is N
  • X 1 is Q
  • X 2 is S
  • X 3 is I, L, V, M, A, F, norleucine, G or N
  • X 4 is T
  • X 5 is T
  • X 6 is N
  • X 1 is Q
  • X 2 is S
  • X 3 is L
  • X 4 is T
  • X 5 is T
  • X 6 is N
  • X 1 is Q
  • X 2 is S
  • X 3 is L
  • X 4 is T
  • X 5 is T
  • X 6 is N
  • X 1 is Q
  • X 2 is S
  • X 3 is I
  • X 4 is T
  • V S
  • R K
  • I X 5
  • X 6 is N
  • X 1 is Q
  • X 2 is S
  • X 3 is I
  • X 4 is S
  • X 5 is T
  • X 6 is N
  • X 1 is Q
  • X 2 is S
  • X 3 is I
  • X 4 is S
  • X 5 is T
  • X 6 is N
  • N is N
  • X 1 is Q
  • X 2 is S
  • X 3 is I
  • X 4 is T
  • X 5 is T
  • V is T
  • S K or G
  • X 6 is N
  • X 1 is Q
  • X 2 is S
  • X 3 is I
  • X 4 is T
  • X 5 is S
  • X 6 is N
  • X 1 is Q
  • X 2 is S
  • X 3 is I
  • X 4 is T
  • X 5 is S
  • X 6 is N
  • X 1 is Q
  • X 2 is S
  • X 3 is I
  • X 4 is T
  • X 5 is T
  • X 6 is N, Q, H, D, K, R or S
  • X 1 is Q
  • X 2 is S
  • X 3 is I
  • X 4 is T
  • X 5 is T
  • X 6 is Q
  • X 1 is Q
  • X 2 is S, T, A or I
  • X 3 is I
  • X 4 is T
  • X 5 is T, V, S, K or G
  • X 6 is N
  • it can Choose a land, where X 1 is Q, X 2 is T, X 3 is I, X 4 is T, X 5 is S, and X 6 is N;
  • X 1 is G, A, S or T;
  • X 2 is A, V, L, I, D or N;
  • X 3 is S, T, N or D
  • X 1 is G, A, S or T
  • X 2 is A
  • X 3 is S
  • X 1 is A
  • X 2 is A
  • X 3 is S
  • X 1 is G
  • X 2 is A, V, L, I, D or N
  • X 3 is S
  • X 1 is G
  • X 2 is V
  • X 3 is S
  • X 1 is G
  • X 2 is A
  • X 3 is S, T, N or D
  • X 1 is G
  • X 2 is A
  • X 3 is T
  • X 1 is Y, W, F, T, S or D;
  • X 2 is H, N, Q, K, R, F or D;
  • X 3 is N, Q, H, D, K, R or T;
  • X 4 is W, Y, F or S
  • X 5 is P, A, D or L
  • X6 is L, I, V, M, A, F, norleucine, Y, P, D or G;
  • X 7 is T, V, S, L or G;
  • X 1 is Y, W, F, T, S or D
  • X 2 is H, X 3 is N
  • X 4 is W
  • X 5 is P
  • X 6 is L
  • X 7 is T
  • X 1 is F
  • X 2 is H
  • X 3 is N
  • X 4 is W
  • X 5 is P
  • X 6 is L
  • X 7 is T
  • X 1 is F
  • X 2 is H
  • X 3 is N
  • X 4 is W
  • X 5 is P
  • X 6 is L
  • X 7 is T
  • X 1 is Y
  • X 2 is H, N, Q, K, R, F or D
  • X 3 is N
  • X 4 is W
  • X 5 is P
  • X 6 is L
  • X 7 is T
  • X 1 is Y
  • X 2 is R
  • X 3 is N
  • X 4 is W
  • X 5 is P
  • X 6 is L
  • X 7 is T
  • X 1 is Y
  • X 2 is R
  • X 3 is N
  • X 4 is W
  • X 5 is P
  • X 6 is L
  • X 7 is T
  • X 1 is Y, X 2 is H, X 3 is N, Q, H, D, K, R or T, X 4 is W, X 5 is P, X 6 is L, and X 7 is T; optionally, where X 1 is Y, X 2 is H, X 3 is Q, X 4 is W, X 5 is P, X 6 is L, and X 7 is T;
  • X 1 is Y
  • X 2 is H
  • X 3 is N
  • X 4 is W
  • Y F or S
  • X 5 is P
  • X 6 is L
  • X 7 is T
  • X 1 is Y
  • X 2 is H
  • X 3 is N
  • X 4 is Y
  • X 5 is P
  • X 6 is L
  • X 7 is T
  • X 1 is Y
  • X 2 is H
  • X 3 is N
  • X 4 is Y
  • X 5 is P
  • X 6 is L
  • X 7 is T
  • X 1 is Y
  • X 2 is H
  • X 3 is N
  • X 4 is W
  • X 5 is P
  • X 6 is L
  • X 7 is T
  • X 1 is Y
  • X 2 is H
  • X 3 is N
  • X 4 is W
  • X 5 is A
  • X 6 is L
  • X 7 is T
  • X 1 is Y
  • X 2 is H
  • X 3 is N
  • X 4 is W
  • X 5 is P
  • X 6 is L, I, V, M, A, F, norleucine, Y, P, D or G
  • X 7 is T
  • X 1 is Y
  • X 2 is H
  • X 3 is N
  • X 4 is W
  • X 5 is P
  • X 6 is I, X 7 for T
  • T for T
  • X 1 is Y
  • X 2 is H
  • X 3 is N
  • X 4 is W
  • X 5 is P
  • X 6 is L
  • X 7 is T, V, S, L or G
  • optional Ground where X 1 is Y, X 2 is H, X 3 is N, X 4 is W, X 5 is P, X 6 is L, and X 7 is S.
  • Heavy chain complementarity determining region HCDR1 which contains the amino acid sequence:
  • X 1 is G or A
  • X 2 is F, W, L, V, I, A, Y or G;
  • X 3 is T, V, S or I;
  • X 4 is F, W, L, V, I, A or Y;
  • X 5 is S, T, R, K or G
  • X 6 is S, T, Q, D, N or E;
  • X7 is Y, W, F, T, S, H or M;
  • X 8 is D, E, A, Y or R;
  • X 1 is G or A
  • X 2 is F
  • X 3 is T
  • X 4 is F
  • X 5 is S
  • X 6 is S
  • X 7 is Y
  • X 8 is D
  • X 1 is A
  • X 2 is F
  • X 3 is T
  • X 4 is F
  • X 5 is S
  • X 6 is S
  • X 7 is Y
  • X 8 is D
  • X 1 is A
  • X 2 is F
  • X 3 is T
  • X 4 is F
  • X 5 is S
  • X 6 is S
  • X 7 is Y
  • X 8 is D
  • X 1 is G
  • X 2 is F
  • W is L
  • V is I
  • A Y
  • G is T
  • X 4 is F
  • X 5 is S
  • X 6 is S
  • X 7 is Y
  • X 8 is D
  • X 1 is G
  • X 2 is Y
  • X 3 is T
  • X 4 is F
  • X 5 is S
  • X 6 is S
  • 8 is D
  • D is D
  • X 1 is G
  • X 2 is F
  • X 3 is T
  • V is S or I
  • X 4 is F
  • X 5 is S
  • X 6 is S
  • X 7 is Y
  • X 8 is D
  • X 1 is G
  • X 2 is F
  • X 3 is S
  • X 4 is F
  • X 5 is S
  • X 6 is S
  • X 7 is Y
  • X 8 is D
  • X 1 is G
  • X 2 is F
  • X 3 is T
  • X 4 is F
  • W L
  • V I
  • X 5 is S
  • X 6 is S
  • X 7 is Y
  • X 8 is D
  • X 1 is G
  • X 2 is F
  • X 3 is T
  • X 4 is Y
  • X 5 is S
  • X 6 is S
  • D D
  • X 1 is G
  • X 2 is F
  • X 3 is T
  • X 4 is F
  • X 5 is S
  • T R
  • K K
  • X 6 is S
  • X 7 is Y
  • X 8 for D
  • X 1 is G
  • X 2 is F
  • X 3 is T
  • X 4 is F
  • X 5 is T
  • X 6 is S
  • X 7 is Y
  • X 8 is D
  • X 1 is G
  • X 2 is F
  • X 3 is T
  • X 4 is F
  • X 5 is S
  • X 6 is S
  • T R
  • K K
  • G X 7
  • X 1 is G
  • X 2 is F
  • X 3 is T
  • X 4 is F
  • X 5 is S
  • X 6 is T
  • X 7 is Y
  • X 8 is D
  • D is D
  • X 1 is G
  • X 2 is F
  • X 3 is T
  • X 4 is F
  • X 5 is S
  • X 6 is S
  • X 7 is Y
  • W F, T, S, H or M
  • X 8 is D
  • X 1 is G
  • X 2 is F
  • X 3 is T
  • X 4 is F
  • X 5 is S
  • X 6 is S
  • X 7 is F
  • D is D
  • X 1 is G
  • X 2 is F
  • X 3 is T
  • X 4 is F
  • X 5 is S
  • X 6 is S
  • X 7 is Y
  • X 8 is D, E, A, Y or R
  • X 1 is G
  • X 2 is F
  • X 3 is T
  • X 4 is F
  • X 5 is S
  • X 6 is S
  • X 7 is Y
  • X 8 is E
  • X 1 is G
  • X 2 is F, W, L, V, I, A, Y or G
  • X 3 is T
  • X 4 is F
  • X 5 is S
  • X 6 is S, T , R, K or G
  • X 7 is Y
  • X 8 is D
  • X 1 is G
  • X 2 is Y
  • X 3 is T
  • X 4 is F
  • X 5 is S
  • X 6 is S
  • T R, K or G
  • X 7 is Y
  • X 8 is D
  • X 1 is G
  • X 2 is Y
  • X 3 is T
  • X 4 is F
  • X 5 is S
  • X 6 is T
  • X 7 is Y
  • X 8 is D
  • D is D
  • Heavy chain complementarity determining region HCDR2 which contains the amino acid sequence:
  • X 1 is M, L, F or I;
  • X 2 is S, T, I, Y or Q;
  • X 3 is Y, W, F, T, S, P, K or R;
  • X 4 is D, E, N, G or F;
  • X 5 is G, A, L or E;
  • X 6 is S, T or N
  • X7 is Y, W, F, T, S, G or I;
  • X 8 is K, R, Q, N, H, P, A or V;
  • X 1 is M, L, F or I
  • X 2 is S
  • X 3 is Y
  • X 4 is D
  • X 5 is G
  • X 6 is S
  • X 7 is Y
  • X 8 is K
  • X 1 is L
  • X 2 is S
  • X 3 is Y
  • X 4 is D
  • X 5 is G
  • X 6 is S
  • X 7 is Y
  • X 8 is K
  • X 1 is L
  • X 2 is S
  • X 3 is Y
  • X 4 is D
  • X 5 is G
  • X 6 is S
  • X 7 is Y
  • X 8 is K
  • X 1 is M
  • X 2 is S, T, I, Y or Q
  • X 3 is Y
  • X 4 is D
  • X 5 is G
  • X 6 is S
  • X 7 is Y
  • X 8 is K
  • X 1 is M
  • X 2 is T
  • X 3 is Y
  • X 4 is D
  • X 5 is G
  • X 6 is S
  • X 7 is Y
  • X 8 is K
  • X 1 is M
  • X 2 is T
  • X 3 is Y
  • X 4 is D
  • X 5 is G
  • X 6 is S
  • X 7 is Y
  • X 8 is K
  • X 1 is M
  • X 2 is S
  • X 3 is Y
  • W F
  • T S
  • S P
  • K K
  • X 4 is D
  • X 5 is G
  • X 6 is S
  • X 7 is Y
  • X 8 is K
  • X 1 is M
  • X 2 is S
  • X 3 is F
  • X 4 is D
  • X 5 is G
  • X 6 is S
  • X 7 is Y
  • 8 is K
  • K is K
  • X 1 is M
  • X 2 is S
  • X 3 is Y
  • X 4 is D
  • E E
  • N G
  • G G
  • F X 5
  • G X 6
  • X 7 is Y
  • X 8 is K
  • X 1 is M
  • X 2 is S
  • X 3 is Y
  • X 4 is E
  • X 5 is G
  • X 6 is S
  • X 7 is Y
  • X 8 is K
  • X 1 is M
  • X 2 is S
  • X 3 is Y
  • X 4 is D
  • X 5 is G
  • X 6 is S
  • X 7 is Y
  • X 8 is K
  • X 1 is M
  • X 2 is S
  • X 3 is Y
  • X 4 is D
  • X 5 is A
  • X 6 is S
  • X 7 is Y
  • X 8 is K
  • X 1 is M
  • X 2 is S
  • X 3 is Y
  • X 4 is D
  • X 5 is G
  • X 6 is S
  • T or N X 7 is Y
  • X 8 is K
  • X 1 is M
  • X 2 is S
  • X 3 is Y
  • X 4 is D
  • X 5 is G
  • X 6 is T
  • X 7 is Y
  • X 8 is K
  • X 1 is M
  • X 2 is S
  • X 3 is Y
  • X 4 is D
  • X 5 is G
  • X 6 is S
  • X 7 is Y
  • W F, T
  • S G or I
  • X8 is K
  • X1 is M
  • X2 is S
  • X3 is Y
  • X4 is D
  • X5 is G
  • X6 is S
  • X7 F
  • X8 is K
  • X 1 is M
  • X 2 is S
  • X 3 is Y
  • X 4 is D
  • X 5 is G
  • X 6 is S
  • X 7 is Y
  • X 8 is K, R, Q, N , H, P, A or V
  • X 1 is M
  • X 2 is S
  • X 3 is Y
  • X 4 is D
  • X 5 is G
  • X 6 is S
  • X 7 is Y
  • X 8 is K
  • R Q, N , H, P, A or V
  • X 1 is M
  • X 2 is S
  • X 3 is Y
  • X 4 is D
  • X 5 is G
  • X 6 is S
  • X 7 is Y
  • X 8 is R
  • X 1 is M
  • X 2 is S, T, I, Y or Q
  • X 3 is Y, W, F, T, S, P, K or R
  • X 4 is D
  • X 5 is G
  • X 6 is S
  • X 7 is Y, W, F, T, S, G or I
  • X 8 is K
  • X 1 is M
  • X 2 is T
  • X 3 is F
  • X 4 is D
  • X 5 is G
  • X 6 is S
  • X 7 is F
  • X 8 is K
  • Heavy chain complementarity determining region HCDR3 which contains the amino acid sequence:
  • X 1 is K, R, Q, N, H or T;
  • X 2 is P, A, D or E;
  • X 3 is G, A, W, P, H or V;
  • X 4 is H, N, Q, K, R, S, F or D;
  • X 5 is Y, W, F, T, S, A or L;
  • X 6 is D, E, N, A, H, S or G;
  • X 7 is T, V, S, G or D;
  • X 8 is S, T, D, G or R;
  • X 9 is F, W, L, V, I, A, Y, P or T;
  • X 10 is D, E, N, Q, F or Y;
  • X 11 is Y, W, F, T, S, V, L or D;
  • X 1 is K, R, Q, N, H or T
  • X 2 is P
  • X 3 is G
  • X 4 is H
  • X 5 is Y
  • X 6 is D
  • X 7 is T
  • X8 is S
  • X9 is F
  • X10 is D
  • X11 is Y
  • X1 is R
  • X2 is P
  • X3 is G
  • X4 H
  • X5 is Y
  • X 6 is D
  • X 7 is T
  • X 8 is S
  • X 9 is F
  • X 10 is D
  • X 11 is Y
  • X 1 is K
  • X 2 is P
  • X 3 is G
  • X 4 is H
  • X 5 is Y
  • X 6 is D
  • X 7 is T
  • X 8 is S
  • X 9 is F
  • X 10 is D
  • X 11 is Y
  • X 1 is K
  • X 2 is A
  • X 3 is G
  • X 4 is H
  • X 7 is T
  • X 8 is S
  • X 9 is F
  • X 10 is D
  • X 11 is Y
  • X 1 is K
  • X 2 is P
  • X 3 is G
  • X 4 is H
  • X 5 is Y
  • X 6 is D
  • X 7 is T
  • X8 is S
  • X9 is F
  • X10 is D
  • X11 is Y
  • X1 is K
  • X2 is P
  • X3 is A
  • X4 is H
  • X5 is Y
  • X 6 is D
  • X 7 is T
  • X 8 is S
  • X 9 is F
  • X 10 is D
  • X 11 is Y
  • X 1 is K
  • X 2 is P
  • X 3 is G
  • X 4 is H
  • N Q
  • R S
  • F F
  • X 5 is Y
  • X 6 is D
  • X 7 is T
  • X 8 is S
  • X 9 is F
  • X 10 is D
  • X 11 is Y
  • X 1 is K
  • X 2 is P
  • X 3 is G
  • 5 is Y
  • X 6 is D
  • X 7 is T
  • X 8 is S
  • X 9 F
  • X 10 is D
  • X 11 Y
  • X 1 is K
  • X 2 is P
  • X 3 is G
  • X 4 is H
  • X 5 is Y
  • W is F
  • T S
  • X 6 is D
  • X 7 is T
  • X 8 is S
  • X 9 is F
  • X 10 is D
  • X 11 is Y
  • X 1 is K
  • X 2 is P
  • X 3 is G
  • X 4 H
  • F is X 6
  • X 7 is T
  • X 8 is S
  • X 9 is F
  • X 10 is D
  • X 11 is Y
  • X 1 is K
  • X 2 is P
  • X 3 is G
  • X 4 is H
  • X 5 is Y
  • X 6 is D
  • E is N
  • A H
  • X 7 is T
  • X 8 is S
  • X 9 is F
  • X 10 is D
  • X 11 is Y
  • X 1 is K
  • X 2 is P
  • X 3 is G
  • X 4 H
  • Y is E
  • X 7 is T
  • X 8 is S
  • X 9 is F
  • X 10 is D
  • X 11 is Y
  • X 1 is K
  • X 2 is P
  • X 3 is G
  • X 4 is H
  • X 5 is Y
  • X 6 is D
  • X 7 is T
  • V S
  • X 8 is S
  • X 9 is F
  • X 10 is D
  • X 11 is Y
  • X 1 is K
  • X 2 is P
  • X 3 is G
  • X 4 is H
  • is D is D
  • X 7 is S
  • X 8 is S
  • X 9 is F
  • X 10 is D
  • X 11 is Y
  • X 1 is K
  • X 2 is P
  • X 3 is G
  • X 4 is H
  • X 5 is Y
  • X 6 is D
  • X 7 is T
  • X 8 is S
  • T D
  • X 9 is F
  • X 10 is D
  • X 11 is Y
  • X 1 is K
  • X 2 is P
  • X 3 is G
  • X 4 is H
  • is D is D
  • X 7 is T
  • X 8 is T
  • X 9 is F
  • X 10 is D
  • X 11 is Y
  • X 1 is K
  • X 2 is P
  • X 3 is G
  • X 4 is H
  • X 5 is Y
  • X 6 is D
  • X 7 is T
  • X 8 is S
  • X 9 is F
  • W L
  • V I
  • I A
  • Y P
  • T X 8
  • X 9 is F
  • W L
  • V V
  • I I
  • A Y
  • X 10 is D
  • X 11 is Y
  • X 1 is K
  • X 2 is P
  • X 3 is G
  • X 4 is H
  • X 5 is Y
  • X 6 is D
  • X 7 is T
  • X 8 is S
  • X 9 is Y
  • X 10 is D
  • X 11 Y
  • X 1 is K
  • X 2 is P
  • X 3 is G
  • X 4 is H
  • X 5 is Y
  • X 6 is D
  • X 7 is T
  • X 8 is S
  • X 9 is F
  • X 10 is D, E, N, Q, F or Y
  • X 11 is Y
  • X 1 is K
  • X 2 is P
  • X 3 is G
  • X 4 H
  • X 5 is Y
  • X 6 is D
  • X 7 is T
  • X 8 is S
  • X 9 is F
  • X 10 is E
  • X 11 is Y
  • X 1 is K
  • X 2 is P
  • X 3 is G
  • X 4 is H
  • X 5 is Y
  • X 6 is D
  • X 7 is T
  • X 8 is S
  • X 9 is F
  • X 10 is D
  • X 11 is Y, W, F, T, S, V, L or D
  • X 1 is K
  • X 2 is P
  • X 3 is G
  • X 4 is H
  • X 5 is Y
  • X 6 is D
  • X 7 is T
  • X 8 is S
  • X 9 is F
  • X 10 is D
  • X 11 is F
  • LCDR1 Light chain complementarity determining region LCDR1, which contains the amino acid sequence SEQ ID NO: 6;
  • Light chain complementarity determining region LCDR3 which includes the amino acid sequence SEQ ID NO: 8.
  • the amino acid sequence of the CDR region of the TRN1029 sequence can be replaced by the amino acids described in Table 2.
  • Table 5 shows the partial heavy chain or light chain amino acid sequence of the substituted CDR region.
  • composition of the mutant antibody is as follows (other amino acid sequences are the same as TRN1029, as shown in Table 6 below, in the table: TRN1029-VL (SEQ ID NO: 34); TRN1029-VH (SEQ ID NO: 33)
  • one embodiment of the present invention provides an antibody that specifically binds Hla or an antigen-binding fragment thereof, which includes:
  • Heavy chain complementarity determining region HCDR1 which contains the amino acid sequence:
  • X 1 is G or A
  • X 2 is F, W, L, V, I, A, Y or G;
  • X 3 is T, V, S or I;
  • X 4 is F, W, L, V, I, A or Y;
  • X 5 is S, T, R, K or G
  • X 6 is S, T, Q, D, N or E;
  • X7 is Y, W, F, T, S, H or M;
  • X 8 is D, E, A, Y or R;
  • X 1 is G or A
  • X 2 is F
  • X 3 is T
  • X 4 is F
  • X 5 is S
  • X 6 is S
  • X 7 is Y
  • X 8 is D
  • X 1 is A
  • X 2 is F
  • X 3 is T
  • X 4 is F
  • X 5 is S
  • X 6 is S
  • X 7 is Y
  • X 8 is D
  • X 1 is A
  • X 2 is F
  • X 3 is T
  • X 4 is F
  • X 5 is S
  • X 6 is S
  • X 7 is Y
  • X 8 is D
  • X 1 is G
  • X 2 is F
  • W is L
  • V is I
  • A Y
  • G is T
  • X 4 is F
  • X 5 is S
  • X 6 is S
  • X 7 is Y
  • X 8 is D
  • X 1 is G
  • X 2 is Y
  • X 3 is T
  • X 4 is F
  • X 5 is S
  • X 6 is S
  • 8 is D
  • D is D
  • X 1 is G
  • X 2 is F
  • X 3 is T
  • V is S or I
  • X 4 is F
  • X 5 is S
  • X 6 is S
  • X 7 is Y
  • X 8 is D
  • X 1 is G
  • X 2 is F
  • X 3 is S
  • X 4 is F
  • X 5 is S
  • X 6 is S
  • X 7 is Y
  • X 8 is D
  • X 1 is G
  • X 2 is F
  • X 3 is T
  • X 4 is F
  • W L
  • V I
  • X 5 is S
  • X 6 is S
  • X 7 is Y
  • X 8 is D
  • X 1 is G
  • X 2 is F
  • X 3 is T
  • X 4 is Y
  • X 5 is S
  • X 6 is S
  • D D
  • X 1 is G
  • X 2 is F
  • X 3 is T
  • X 4 is F
  • X 5 is S
  • T R
  • K K
  • X 6 is S
  • X 7 is Y
  • X 8 is D
  • X 1 is G
  • X 2 is F
  • X 3 is T
  • X 4 is F
  • X 5 is T
  • X 6 is S
  • X 7 is Y
  • X 8 is D
  • X 1 is G
  • X 2 is F
  • X 3 is T
  • X 4 is F
  • X 5 is S
  • X 6 is S
  • T R
  • K K
  • G X 7
  • X 1 is G
  • X 2 is F
  • X 3 is T
  • X 4 is F
  • X 5 is S
  • X 6 is T
  • X 7 is Y
  • X 8 is D
  • D is D
  • X 1 is G
  • X 2 is F
  • X 3 is T
  • X 4 is F
  • X 5 is S
  • X 6 is S
  • X 7 is Y
  • W F, T, S, H or M
  • X 8 is D
  • X 1 is G
  • X 2 is F
  • X 3 is T
  • X 4 is F
  • X 5 is S
  • X 6 is S
  • X 7 is F
  • D is D
  • X 1 is G
  • X 2 is F
  • X 3 is T
  • X 4 is F
  • X 5 is S
  • X 6 is S
  • X 7 is Y
  • X 8 is D, E, A, Y or R
  • X 1 is G
  • X 2 is F
  • X 3 is T
  • X 4 is F
  • X 5 is S
  • X 6 is S
  • X 7 is Y
  • X 8 is E
  • X 1 is G
  • X 2 is F, W, L, V, I, A, Y or G
  • X 3 is T
  • X 4 is F
  • X 5 is S
  • X 6 is S, T , R, K or G
  • X 7 is Y
  • X 8 is D
  • X 1 is G
  • X 2 is Y
  • X 3 is T
  • X 4 is F
  • X 5 is S
  • X 6 is S
  • T R, K or G
  • X 7 is Y
  • X 8 is D
  • X 1 is G
  • X 2 is Y
  • X 3 is T
  • X 4 is F
  • X 5 is S
  • X 6 is T
  • X 7 is Y
  • X 8 is D
  • D is D
  • Heavy chain complementarity determining region HCDR2 which contains the amino acid sequence:
  • X 1 is M, L, F or I;
  • X 2 is S, T, I, Y or Q;
  • X 3 is Y, W, F, T, S, P, K or R;
  • X 4 is D, E, N, G or F;
  • X 5 is G, A, L or E;
  • X 6 is S, T or N
  • X7 is Y, W, F, T, S, G or I;
  • X 8 is K, R, Q, N, H, P, A or V;
  • X 1 is M, L, F or I
  • X 2 is S
  • X 3 is Y
  • X 4 is D
  • X 5 is G
  • X 6 is S
  • X 7 is Y
  • X 8 is K
  • X 1 is L
  • X 2 is S
  • X 3 is Y
  • X 4 is D
  • X 5 is G
  • X 6 is S
  • X 7 is Y
  • X 8 is K
  • X 1 is L
  • X 2 is S
  • X 3 is Y
  • X 4 is D
  • X 5 is G
  • X 6 is S
  • X 7 is Y
  • X 8 is K
  • X 1 is M
  • X 2 is S, T, I, Y or Q
  • X 3 is Y
  • X 4 is D
  • X 5 is G
  • X 6 is S
  • X 7 is Y
  • X 8 is K
  • X 1 is M
  • X 2 is T
  • X 3 is Y
  • X 4 is D
  • X 5 is G
  • X 6 is S
  • X 7 is Y
  • X 8 is K
  • X 1 is M
  • X 2 is T
  • X 3 is Y
  • X 4 is D
  • X 5 is G
  • X 6 is S
  • X 7 is Y
  • X 8 is K
  • X 1 is M
  • X 2 is S
  • X 3 is Y
  • W F
  • T S
  • S P
  • K K
  • X 4 is D
  • X 5 is G
  • X 6 is S
  • X 7 is Y
  • X 8 is K
  • X 1 is M
  • X 2 is S
  • X 3 is F
  • X 4 is D
  • X 5 is G
  • X 6 is S
  • X 7 is Y
  • 8 is K
  • K is K
  • X 1 is M
  • X 2 is S
  • X 3 is Y
  • X 4 is D
  • E E
  • N G
  • G G
  • F X 5
  • G X 6
  • X 7 is Y
  • X 8 is K
  • X 1 is M
  • X 2 is S
  • X 3 is Y
  • X 4 is E
  • X 5 is G
  • X 6 is S
  • X 7 is Y
  • X 8 is K
  • X 1 is M
  • X 2 is S
  • X 3 is Y
  • X 4 is D
  • X 5 is G
  • X 6 is S
  • X 7 is Y
  • X 8 is K
  • X 1 is M
  • X 2 is S
  • X 3 is Y
  • X 4 is D
  • X 5 is A
  • X 6 is S
  • X 7 is Y
  • X 8 is K
  • X 1 is M
  • X 2 is S
  • X 3 is Y
  • X 4 is D
  • X 5 is G
  • X 6 is S
  • T or N X 7 is Y
  • X 8 is K
  • X 1 is M
  • X 2 is S
  • X 3 is Y
  • X 4 is D
  • X 5 is G
  • X 6 is T
  • X 7 is Y
  • X 8 is K
  • X 1 is M
  • X 2 is S
  • X 3 is Y
  • X 4 is D
  • X 5 is G
  • X 6 is S
  • X 7 is Y
  • W F, T
  • S G or I
  • X8 is K
  • X1 is M
  • X2 is S
  • X3 is Y
  • X4 is D
  • X5 is G
  • X6 is S
  • X7 F
  • X8 is K
  • X 1 is M
  • X 2 is S
  • X 3 is Y
  • X 4 is D
  • X 5 is G
  • X 6 is S
  • X 7 is Y
  • X 8 is K, R, Q, N , H, P, A or V
  • X 1 is M
  • X 2 is S
  • X 3 is Y
  • X 4 is D
  • X 5 is G
  • X 6 is S
  • X 7 is Y
  • X 8 is K
  • R Q, N , H, P, A or V
  • X 1 is M
  • X 2 is S
  • X 3 is Y
  • X 4 is D
  • X 5 is G
  • X 6 is S
  • X 7 is Y
  • X 8 is R
  • X 1 is M
  • X 2 is S, T, I, Y or Q
  • X 3 is Y, W, F, T, S, P, K or R
  • X 4 is D
  • X 5 is G
  • X 6 is S
  • X 7 is Y, W, F, T, S, G or I
  • X 8 is K
  • X 1 is M
  • X 2 is T
  • X 3 is F
  • X 4 is D
  • X 5 is G
  • X 6 is S
  • X 7 is F
  • X 8 is K
  • Heavy chain complementarity determining region HCDR3 which contains the amino acid sequence:
  • X 1 is K, R, Q, N, H or T;
  • X 2 is P, A, D or E;
  • X 3 is G, A, W, P, H or V;
  • X 4 is H, N, Q, K, R, S, F or D;
  • X 5 is Y, W, F, T, S, A or L;
  • X 6 is D, E, N, A, H, S or G;
  • X 7 is T, V, S, G or D;
  • X 8 is S, T, D, G or R;
  • X 9 is F, W, L, V, I, A, Y, P or T;
  • X 10 is D, E, N, Q, F or Y;
  • X 11 is Y, W, F, T, S, V, L or D;
  • X 1 is K, R, Q, N, H or T
  • X 2 is P
  • X 3 is G
  • X 4 is H
  • X 5 is Y
  • X 6 is D
  • X 7 is T
  • X8 is S
  • X9 is F
  • X10 is D
  • X11 is Y
  • X1 is R
  • X2 is P
  • X3 is G
  • X4 H
  • X5 is Y
  • X 6 is D
  • X 7 is T
  • X 8 is S
  • X 9 is F
  • X 10 is D
  • X 11 is Y
  • X 1 is K
  • X 2 is P
  • X 3 is G
  • X 4 is H
  • X 5 is Y
  • X 6 is D
  • X 7 is T
  • X 8 is S
  • X 9 is F
  • X 10 is D
  • X 11 is Y
  • X 1 is K
  • X 2 is A
  • X 3 is G
  • X 4 is H
  • X 7 is T
  • X 8 is S
  • X 9 is F
  • X 10 is D
  • X 11 is Y
  • X 1 is K
  • X 2 is P
  • X 3 is G
  • X 4 is H
  • X 5 is Y
  • X 6 is D
  • X 7 is T
  • X8 is S
  • X9 is F
  • X10 is D
  • X11 is Y
  • X1 is K
  • X2 is P
  • X3 is A
  • X4 is H
  • X5 is Y
  • X 6 is D
  • X 7 is T
  • X 8 is S
  • X 9 is F
  • X 10 is D
  • X 11 is Y
  • X 1 is K
  • X 2 is P
  • X 3 is G
  • X 4 is H
  • N Q
  • R S
  • F F
  • X 5 is Y
  • X 6 is D
  • X 7 is T
  • X 8 is S
  • X 9 is F
  • X 10 is D
  • X 11 is Y
  • X 1 is K
  • X 2 is P
  • X 3 is G
  • 5 is Y
  • X 6 is D
  • X 7 is T
  • X 8 is S
  • X 9 F
  • X 10 is D
  • X 11 Y
  • X 1 is K
  • X 2 is P
  • X 3 is G
  • X 4 is H
  • X 5 is Y
  • W is F
  • T S
  • X 6 is D
  • X 7 is T
  • X 8 is S
  • X 9 is F
  • X 10 is D
  • X 11 is Y
  • X 1 is K
  • X 2 is P
  • X 3 is G
  • X 4 H
  • F is X 6
  • X 7 is T
  • X 8 is S
  • X 9 is F
  • X 10 is D
  • X 11 is Y
  • X 1 is K
  • X 2 is P
  • X 3 is G
  • X 4 is H
  • X 5 is Y
  • X 6 is D
  • E N
  • A H
  • X 7 is T
  • X8 is S
  • X9 is F
  • X10 is D
  • X11 is Y
  • X1 is K
  • X2 is P
  • X3 is G
  • X4 H
  • X5 is Y
  • X 6 is E
  • X 7 is T
  • X 8 is S
  • X 9 is F
  • X 10 is D
  • X 11 is Y
  • X 1 is K
  • X 2 is P
  • X 3 is G
  • X 4 is H
  • X 5 is Y
  • X 6 is D
  • X 7 is T
  • V S
  • X 8 is S
  • X 9 is F
  • X 10 is D
  • X 11 is Y
  • X 1 is K
  • X 2 is P
  • X 3 is G
  • X 4 is H
  • is D is D
  • X 7 is S
  • X 8 is S
  • X 9 is F
  • X 10 is D
  • X 11 is Y
  • X 1 is K
  • X 2 is P
  • X 3 is G
  • X 4 is H
  • X 5 is Y
  • X 6 is D
  • X 7 is T
  • X 8 is S
  • T D
  • X 9 is F
  • X 10 is D
  • X 11 is Y
  • X 1 is K
  • X 2 is P
  • X 3 is G
  • X 4 is H
  • is D is D
  • X 7 is T
  • X 8 is T
  • X 9 is F
  • X 10 is D
  • X 11 is Y
  • X 1 is K
  • X 2 is P
  • X 3 is G
  • X 4 is H
  • X 5 is Y
  • X 6 is D
  • X 7 is T
  • X 8 is S
  • X 9 is F
  • W L
  • V I
  • I A
  • Y P
  • T X 8
  • X 9 is F
  • W L
  • V V
  • I I
  • A Y
  • X 10 is D
  • X 11 is Y
  • X 1 is K
  • X 2 is P
  • X 3 is G
  • X 4 is H
  • X 5 is Y
  • X 6 is D
  • X 7 is T
  • X 8 is S
  • X 9 is Y
  • X 10 is D
  • X 11 Y
  • X 1 is K
  • X 2 is P
  • X 3 is G
  • X 4 is H
  • X 5 is Y
  • X 6 is D
  • X 7 is T
  • X 8 is S
  • X 9 is F
  • X 10 is D, E, N, Q, F or Y
  • X 11 is Y
  • X 1 is K
  • X 2 is P
  • X 3 is G
  • X 4 H
  • X 5 is Y
  • X 6 is D
  • X 7 is T
  • X 8 is S
  • X 9 is F
  • X 10 is E
  • X 11 is Y
  • X 1 is K
  • X 2 is P
  • X 3 is G
  • X 4 is H
  • X 5 is Y
  • X 6 is D
  • X 7 is T
  • X 8 is S
  • X 9 is F
  • X 10 is D
  • X 11 is Y, W, F, T, S, V, L or D
  • X 1 is K
  • X 2 is P
  • X 3 is G
  • X 4 is H
  • X 5 is Y
  • X 6 is D
  • X 7 is T
  • X 8 is S
  • X 9 is F
  • X 10 is D
  • X 11 is F
  • LCDR1 Light chain complementarity determining region LCDR1, which contains the amino acid sequence:
  • X 1 is Q, N, E or D;
  • X 2 is S, T, A or I
  • X 3 is I, L, V, M, A, F, norleucine, G or N;
  • X 4 is T, V, S, R, K or I;
  • X 5 is T, V, S, K or G
  • X 6 is N, Q, H, D, K, R or S;
  • X 1 is Q, N, E or D
  • X 2 is S
  • X 3 is I
  • X 4 is T
  • X 5 is T
  • X 6 is N
  • X 1 is N
  • X 2 is S
  • X 3 is I
  • X 4 is T
  • X 5 is T
  • X 6 is N
  • X 1 is N
  • X 2 is S
  • X 3 is I
  • X 4 is T
  • X 5 is T
  • X 6 is N
  • X 1 is Q
  • X 2 is S, T, A or I
  • X 3 is I
  • X 4 is T
  • X 5 is T
  • X 6 is N
  • X 1 is Q
  • X 2 is T
  • X 3 is I
  • X 4 is T
  • X 5 is T
  • X 6 is N
  • X 1 is Q
  • X 2 is T
  • X 3 is I
  • X 4 is T
  • X 5 is T
  • X 6 is N
  • N is N
  • X 1 is Q
  • X 2 is S
  • X 3 is I, L, V, M, A, F, norleucine, G or N
  • X 4 is T
  • X 5 is T
  • X 6 is N
  • X 1 is Q
  • X 2 is S
  • X 3 is L
  • X 4 is T
  • X 5 is T
  • X 6 is N
  • X 1 is Q
  • X 2 is S
  • X 3 is L
  • X 4 is T
  • X 5 is T
  • X 6 is N
  • X 1 is Q
  • X 2 is S
  • X 3 is I
  • X 4 is T
  • V S
  • R K
  • I X 5
  • X 6 is N
  • X 1 is Q
  • X 2 is S
  • X 3 is I
  • X 4 is S
  • X 5 is T
  • X 6 is N
  • X 1 is Q
  • X 2 is S
  • X 3 is I
  • X 4 is S
  • X 5 is T
  • X 6 is N
  • N is N
  • X 1 is Q
  • X 2 is S
  • X 3 is I
  • X 4 is T
  • X 5 is T
  • V is T
  • S K or G
  • X 6 is N
  • X 1 is Q
  • X 2 is S
  • X 3 is I
  • X 4 is T
  • X 5 is S
  • X 6 is N
  • X 1 is Q
  • X 2 is S
  • X 3 is I
  • X 4 is T
  • X 5 is S
  • X 6 is N
  • X 1 is Q
  • X 2 is S
  • X 3 is I
  • X 4 is T
  • X 5 is T
  • X 6 is N, Q, H, D, K, R or S
  • X 1 is Q
  • X 2 is S
  • X 3 is I
  • X 4 is T
  • X 5 is T
  • X 6 is Q
  • X 1 is Q
  • X 2 is S, T, A or I
  • X 3 is I
  • X 4 is T
  • X 5 is T, V, S, K or G
  • X 6 is N
  • it can Choose a land, where X 1 is Q, X 2 is T, X 3 is I, X 4 is T, X 5 is S, and X 6 is N;
  • X 1 is G, A, S or T;
  • X 2 is A, V, L, I, D or N;
  • X 3 is S, T, N or D
  • X 1 is G, A, S or T
  • X 2 is A
  • X 3 is S
  • X 1 is A
  • X 2 is A
  • X 3 is S
  • X 1 is G
  • X 2 is A, V, L, I, D or N
  • X 3 is S
  • X 1 is G
  • X 2 is V
  • X 3 is S
  • X 1 is G
  • X 2 is A
  • X 3 is S, T, N or D
  • X 1 is G
  • X 2 is A
  • X 3 is T
  • X 1 is Y, W, F, T, S or D;
  • X 2 is H, N, Q, K, R, F or D;
  • X 3 is N, Q, H, D, K, R or T;
  • X 4 is W, Y, F or S
  • X 5 is P, A, D or L
  • X6 is L, I, V, M, A, F, norleucine, Y, P, D or G;
  • X 7 is T, V, S, L or G;
  • X 1 is Y, W, F, T, S or D
  • X 2 is H, X 3 is N
  • X 4 is W
  • X 5 is P
  • X 6 is L
  • X 7 is T
  • X 1 is F
  • X 2 is H
  • X 3 is N
  • X 4 is W
  • X 5 is P
  • X 6 is L
  • X 7 is T
  • X 1 is F
  • X 2 is H
  • X 3 is N
  • X 4 is W
  • X 5 is P
  • X 6 is L
  • X 7 is T
  • X 1 is Y
  • X 2 is H, N, Q, K, R, F or D
  • X 3 is N
  • X 4 is W
  • X 5 is P
  • X 6 is L
  • X 7 is T
  • X 1 is Y
  • X 2 is R
  • X 3 is N
  • X 4 is W
  • X 5 is P
  • X 6 is L
  • X 7 is T
  • X 1 is Y
  • X 2 is R
  • X 3 is N
  • X 4 is W
  • X 5 is P
  • X 6 is L
  • X 7 is T
  • X 1 is Y, X 2 is H, X 3 is N, Q, H, D, K, R or T, X 4 is W, X 5 is P, X 6 is L, and X 7 is T; optionally, where X 1 is Y, X 2 is H, X 3 is Q, X 4 is W, X 5 is P, X 6 is L, and X 7 is T;
  • X 1 is Y
  • X 2 is H
  • X 3 is N
  • X 4 is W
  • Y F or S
  • X 5 is P
  • X 6 is L
  • X 7 is T
  • X 1 is Y
  • X 2 is H
  • X 3 is N
  • X 4 is Y
  • X 5 is P
  • X 6 is L
  • X 7 is T
  • X 1 is Y
  • X 2 is H
  • X 3 is N
  • X 4 is Y
  • X 5 is P
  • X 6 is L
  • X 7 is T
  • X 1 is Y
  • X 2 is H
  • X 3 is N
  • X 4 is W
  • X 5 is P
  • X 6 is L
  • X 7 is T
  • X 1 is Y
  • X 2 is H
  • X 3 is N
  • X 4 is W
  • X 5 is A
  • X 6 is L
  • X 7 is T
  • X 1 is Y
  • X 2 is H
  • X 3 is N
  • X 4 is W
  • X 5 is P
  • X 6 is L, I, V, M, A, F, norleucine, Y, P, D or G
  • X 7 is T
  • X 1 is Y
  • X 2 is H
  • X 3 is N
  • X 4 is W
  • X 5 is P
  • X 6 is I, X 7 for T
  • T for T
  • X 1 is Y
  • X 2 is H
  • X 3 is N
  • X 4 is W
  • X 5 is P
  • X 6 is L
  • X 7 is T, V, S, L or G
  • optional Ground where X 1 is Y, X 2 is H, X 3 is N, X 4 is W, X 5 is P, X 6 is L, and X 7 is S.
  • One embodiment of the present invention shows that the FR regions of the heavy chain variable regions of TRN1029, TRN1030, TRN1031, TRN1032, and TRN1033 cannot be replaced except for cysteine, and the amino acid sequences of other FR regions can be conservatively replaced with the amino acids shown in Table 2.
  • the amino acids in the FR region of the light chain and heavy chain of TRN1029 can be conservatively substituted as shown in Table 2.
  • Table 7 shows the heavy chain or light chain variable region partially substituted by the FR region, which will not affect the present invention. The invention provides for the correct assembly and stability of the antibody structure.
  • the amino acids in the FR region of the light chain and heavy chain of TRN1029 can be conservatively substituted as shown in Table 2 and become the FR region variant sequences as shown in SEQ ID NO: 221-231, both of which are It will not affect the correct assembly and stability of the antibody structure provided by the invention.
  • composition of the mutant antibody is as follows (other amino acid sequences are the same as TRN1029), as shown in Table 8 below:
  • this application recruited healthy volunteers and volunteers who recovered from severe Staphylococcus aureus infection, collected their peripheral blood samples for serum antibody titer ELISA testing, and sorted specific single memory B cells.
  • the sorted individuals were obtained by RT-PCR method
  • the variable region genes of the antibody heavy chain and the corresponding light chain of memory B cells using the linear Ig heavy chain and light chain gene expression system to obtain expression-specific variable region antibodies, and then obtain a fully human anti-Staphylococcus aureus monoclonal Antibody.
  • Hla alpha toxin gene
  • Hla_H35L nontoxic mutant
  • Hla_H35L lost its hemolytic and cytolytic activity. Can be used for flow cell sorting experiments.
  • Hla_H35L was obtained by generating the Hla_H35L variant by site-directed mutagenesis of the wild-type gene using the full gold FM111-02 site-directed mutagenesis kit.
  • Hla and Hla_H35L were constructed into the pET-42b(+) vector, transformed into E. coli BL21 strain, cultured overnight at 37°C in LB medium containing kanamycin, and induced by IPTG for expression.
  • the cells were harvested by centrifugation and purified using Ni-NTA magnetic beads to obtain Hla protein (SEQ ID NO: 1) and recombinant Hla_H35L protein (SEQ ID NO: 2).
  • SDS-PAGE gel detection results are shown in Figure 1.
  • Hla protein and Hla_H35L protein are about 35kD. After one-step Ni-NTA magnetic bead purification, the protein purity is high and can meet the needs of subsequent research experiments.
  • the obtained Hla protein will For ELISA detection, SPR and in vivo and in vitro biological analysis, the recombinant Hla_H35L protein will be used for ELISA detection and flow cell sorting.
  • the specific operations are as follows: Take 10 mL of venous blood and centrifuge it at 400g, 22°C for 15 min; the supernatant will be clear after centrifugation.
  • RPMI1640 For the plasma layer, aliquot and freeze at -80°C; mix the remaining part thoroughly with an equal amount of RPMI1640 (Gibco), slowly add it to a sterile centrifuge tube containing lymphocyte separation solution, and keep the liquid layer stratified intact; centrifuge at 400g After 35 minutes, use a capillary tube to absorb the peripheral blood mononuclear cells (PBMC) located in the cloud layer, place them in another sterile centrifuge tube, add more than 5 times the volume of RPMI1640, centrifuge at 400g for 10 minutes, wash the cells twice, and count the cells with 1 ⁇ 10 7 /tube were frozen and stored in liquid nitrogen for later use.
  • PBMC peripheral blood mononuclear cells
  • Hla and Hla_H35L proteins constructed and expressed in Example 1 were used to screen individuals with high titers of ⁇ -toxin antibodies in their plasma through the ELISA detection method.
  • the specific method is as follows: Use pH 9.6 phosphate coating buffer (1.59g Na 2 CO 3 , 2.93g NaHCO 3 ) to adjust Hla and Hla_H35L to pH 9.6. After dissolving, add ultrapure water to a volume of 1000 mL. Dissolve and mix.
  • the pH 9.6 phosphate coating buffer described below is the same as this recipe) dilute to 2 ⁇ g/mL, add 0.1 mL/well to each well of the ELISA 96 plate, coat overnight at 4°C, and use blocking solution (50 g skimmed milk powder, 150mL sheep serum, 5mL Tween20, 50mL 20 times concentration of PBS stock solution, after dissolving, adjust the volume to 1000mL with ultrapure water, the blocking solution described below is the same as this formula) Block at 37°C for 2 hours.
  • Each plasma sample obtained above was initially diluted at a 1:50 ratio (3 ⁇ L of plasma, 150 ⁇ L of blocking solution), and a 3-fold gradient serial dilution was performed.
  • the PBMC of sample CS006 were sorted by flow cytometry to obtain single memory B cells (Figure 3), using molecular markers CD3, CD14, CD16 - , CD235a, CD20 + , CD27 + , Hla_H35L- BV421 and Hla_H35L-PE-Cy7 perform Hla_H35L protein-specific memory B cell sorting on PBMC of CS006 sample.
  • Antigen-binding antibodies are randomly and evenly distributed on the surface of memory B cells.
  • Two fluorescently labeled Hla_H35L-BV421 (blue) and Hla_H35L-PE-Cy7 (red) can randomly bind to the antigens of specific memory B cells.
  • the antibody determinant emits red and blue fluorescence.
  • the Q2-1 gate is the selected double-positive specific memory B cells. Double-positive cells are selected for subsequent single-cell PCR experiments to isolate antibody genes.
  • Example 5 Isolating antibody variable region genes from single B cells using RT-PCR
  • Reverse transcription to synthesize the first strand of cDNA 20 ⁇ L of single cell lysis solution was added to the 96-well plate containing the double-positive single B cells obtained in Example 4, and the cells were lysed. Add 0.5 ⁇ M constant region primers of each subtype heavy chain and light chain (see the primer information disclosed in CN107760690B) and Superscript IV reverse transcriptase (Invitrogen, Carlsbad, CA), and incubate at 37°C for 1 hour to perform reverse transcription and synthesize cDNA. The first chain. The antibody genes were then isolated through two rounds of PCR amplification, in which the PCR-amplified V H and V K/L gene products were identified using 1.2% agarose gel electrophoresis and sequenced.
  • the V H and V K/L gene products were constructed by overlapping PCR into linear DNA fragments connected to a complete antibody expression system with a promoter, antibody constant region and terminator, specifically: TRN1029, TRN1030, TRN1031, TRN1032, TRN1033
  • the VH gene product is connected to the DNA fragment containing the CMV promoter and the DNA fragment containing the constant region of human IgG1 (including the hinge region, CH1, CH2 and CH3) by PCR;
  • the VK gene products of TRN1029, TRN1030 and TRN1031 are connected to the DNA fragment containing the CMV promoter
  • the DNA fragment of the promoter and the constant region DNA fragment containing human Kappa are connected by PCR;
  • the VH gene products of TRN1032 and TRN1033 are connected to the DNA fragment containing the CMV promoter and the constant region DNA containing human Lambda. Fragments (including hinge region, C-DOMAIN) were ligated by PCR.
  • the linear DNA fragment of the constructed antibody expression system was co-transfected with PEI into 293T cells. 6-8 hours after transfection, the medium was replaced with fresh medium and cultured in a 37°C 8% CO2 incubator for 96 hours. The transfected cells were collected. Clear, centrifuge at 15000g for 1 hour, and perform ELISA detection. Hla and Hla_H35L were diluted to 2 ⁇ g/mL with coating buffer, 0.1 mL/well of each was added to the ELISA 96 plate, coated at 4°C overnight, and blocked with blocking solution at 37°C for 2 h. Add 100 ⁇ L of the centrifuged supernatant as primary antibody and incubate at 37°C for 1 hour.
  • TRN1021 (see patent CN2021115320943) is an irrelevant negative control, and AR301 is positive. Control (see CN102549013B).
  • the coding gene of the antibody detected in Example 6 was connected to the pcDNA3.3 vector using the homologous recombination cloning method to construct an expression vector for the fully human anti-Hla antibody.
  • the expression vector was then transformed into DH5 ⁇ competent bacteria and incubated in ampicillin-containing Cultured on penicillin plates at 37°C At night, single colonies were picked for PCR amplification with specific primers.
  • the reaction conditions were: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 100 s, 28 cycles; extension at 72°C for 5 min.
  • the 5 ⁇ L PCR product was detected by 1% agarose gel electrophoresis, and the transformants containing antibody heavy chain and light chain genes were identified in the transformants and verified by sequencing.
  • the positive plasmid verified by sequencing in Example 7 was transformed into DH5 ⁇ for large-scale amplification. After quickly extracting the recombinant plasmid, it was co-transfected into HEK293I cells with the transfection reagent PEI. Change the medium to fresh medium 6-8 hours after transfection, and incubate at 37°C for 8 hours. Incubate for 96 hours in a % CO 2 incubator, and collect the cell supernatant for detection. Collect the transfection supernatant, centrifuge at 4000 rpm for 1 hour, and use protein A magnetic beads to purify the supernatant; use SDS-PAGE and Western Blot to check the expression and purification of the purified antibodies.
  • the difference between non-reducing SDS-PAGE and reducing SDS-PAGE gel electrophoresis detection is that you need to add a loading buffer containing ⁇ -mercaptoethanol to the antibody sample for reducing electrophoresis, and boil it in a boiling water bath for 5 minutes. Add PBS to 10-15 ⁇ L before loading.
  • the SDS-PAGE and Western Blot detection results are shown in Figure 5.
  • the left picture in Figure 5 shows the results of detecting non-reducing samples, and the right picture shows the results of detecting reducing samples. There are two bands after the reduction of the antibody.
  • the molecular weight of the heavy chain and light chain of the antibody after reduction is consistent with the size of the general antibody; the size of the non-reducing antibody It is 180kD, which is consistent with the size of a general complete antibody. It can be seen that the antibody is assembled correctly.
  • Hla_H35L and Hla were used as antigens for ELISA detection respectively.
  • the corresponding antigens were diluted to 2 ⁇ g/mL, 100uL per well was added to a 96-well plate, coated at 4°C overnight, and blocked with blocking solution at 37°C for 2 hours.
  • Each Hla antibody obtained was diluted twice, with a starting concentration of 10 ⁇ g/mL, and 12 gradients of 3-fold dilution. Then, the antibodies of each gradient were added to a 96-well plate and incubated at 37°C for 1 hour.
  • the results are shown in Figure 6.
  • the fully human Hla monoclonal antibodies TRN1029, TRN1030, TRN1031, TRN1032, and TRN1033 can all bind to Hla and Hla_H35L.
  • the EC 50 of binding to Hla is 0.199 ⁇ g/mL, 0.165 ⁇ g/mL, and 0.017 respectively. ⁇ g/mL, 24.29 ⁇ g/mL, 0.009 ⁇ g/mL; the EC 50 of TRN1029, TRN1030, TRN1031, TRN1033 combined with Hla_H35L are 0.062 ⁇ g/mL, 0.068 ⁇ g/mL, 0.016 ⁇ g/mL and 0.013 ⁇ g/mL respectively (Table 9).
  • the human antibody capture kit (Cytiva, 29234600) was used to prepare the anti-human IgG (Fc) antibody chip, and the anti-human IgG-(Fc) antibody was immobilized on the surface of the CM5 sensor chip (Cytiva, BR100530) using the amino coupling method.
  • the fully human anti-Hla antibody is captured as a ligand on 2 channels on the sensor chip (channel 1 is the reference channel and channel 2 is the sample channel), making the antibody capture level reach about 200RU.
  • TRN1029, TRN1030, TRN1031, TRN1032, TRN1033 and Hla affinity (KD) size They are 1.17nM, 0.943nM, 0.965nM, 52.1nM and 1.82nM respectively.
  • TRN1029, TRN1030, TRN1031 and TRN1033 dissociate smoothly and slowly, but TRN1032 dissociates quickly, which is why its affinity is only 52.1nM.
  • Hla protein and two different anti-Hla protein monoclonal antibodies were studied through surface plasmon resonance technology (SPR), and the antigenic epitopes recognized by the fully human anti-Hla antibodies obtained in this application and their mutual relationships were detected.
  • SPR surface plasmon resonance technology
  • the Hla protein is covalently bound to the 2 channels of the CM5 sensor chip, and the response value is approximately 300RU (channel 1 is the reference blank channel, and channel 2 is the sample channel). As shown in Figure 7, 4 rounds of cycles are performed, each cycle includes samples. There are three steps: A loading, sample B loading (sample A and sample B are anti-Hla protein antibodies), and regeneration.
  • anti-Hla protein antibody A is first combined on channels 1 and 2 with a flow rate of 30 ⁇ L/min and a binding time of 190 s, and then anti-Hla protein antibody A is combined on channels 1 and 2 with a flow rate of 30 ⁇ L/min and a binding time of 190 s.
  • regeneration conditions select a Glycine solution with a pH of 1.5, a flow rate of 30 ⁇ L/min, and a regeneration time of 180s.
  • anti-Hla protein antibody A is first combined on channels 1 and 2 with a flow rate of 30 ⁇ L/min and a binding time of 190 s, and then anti-Hla protein antibody B is combined on channels 1 and 2 with a flow rate of 30 ⁇ L/min and a binding time of 190 s. 190s, observe the changes in the combined value of the first and second rounds of cycles.
  • Regeneration conditions Choose a Glycine solution with a pH of 1.5, a flow rate of 30 ⁇ L/min, and a regeneration time of 180 s.
  • the anti-Hla protein antibody B is first combined on channels 1 and 2 with a flow rate of 30 ⁇ L/min and a binding time of 190 s, and then the anti-Hla protein antibody B is combined on channels 1 and 2 with a flow rate of 30 ⁇ L/min and a binding time of 190 s.
  • regeneration conditions select a glycine solution with a pH of 1.5, a flow rate of 30 ⁇ L/min, and a regeneration time of 180s.
  • AR301 is Aridis’ fully human IgG1 lambda monoclonal antibody against Staphylococcus aureus ⁇ -toxin for treating infections. Specific binding of N-terminal epitopes.
  • TRN1031 and TRN1032 could not compete with other antibodies for binding in this experiment. It is speculated that TRN1031 and TRN1032 may recognize two other new epitopes of Staphylococcus aureus ⁇ -toxin.
  • Hla Hla with a concentration of 10 ⁇ g/mL with different concentrations of fully human Hla antibodies and positive control antibodies (MEDI4893 (patent CN103443285B), AR301 (patent CN102549013B), TRN1016 (patent CN109400704B)).
  • the starting concentration of the antibody is 40 ⁇ g.
  • red blood cells undergo a hemolytic reaction, they lose their size and shape as cells, and the leaked hemoglobin stains the surrounding solution (such as plasma or saline) red. Therefore, the hemolyzed red blood cell-containing solution will turn into a red, transparent liquid.
  • TRN1029, TRN1030, TRN1031, TRN1032, TRN1033 Five neutralizing antibodies (TRN1029, TRN1030, TRN1031, TRN1032, TRN1033) can effectively neutralize the hemolysis of Hla and protect human red blood cells from hemolysis by Hla.
  • the neutralizing potency of TRN1032 is 16 times higher than that of the control antibodies MEDI4893 and AR301.
  • the neutralizing potency of TRN1030 and TRN1031 is equivalent to that of the control antibody MEDI4893, and the potency is better than that of AR301.
  • TRN1029, TRN1033 and The neutralizing potency of AR301 is comparable.
  • TRN1016 only has a partial neutralizing effect at a high concentration of 40 ⁇ g/mL, and its effect is similar to Hla036, Hla038, and Hla039.
  • ICR mice purchased from Zhuhai Baishantong Biotechnology Co., Ltd. were randomly divided into groups, with 8 mice in each group. According to the toxin titration results, the Hla toxin dose was selected to be 2 mg/kg. Different concentrations of TRN1016, TRN1029, TRN1031 or MEDI4893 were mixed with Hla respectively. After incubation at 37°C for 30 min, the mixture was injected intraperitoneally into ICR mice at a volume of 10 ⁇ L/g (Table 9). The survival time of the mice was observed and the survival rate was calculated. The results are shown in Figure 11.
  • TRN1029, TRN1031 and MEDI4893 can completely protect mice from death at 2 mg/kg.
  • Comparison results with the same concentration as MEDI4893 show that TRN1029 and TRN1031 have better protective effects than MEDI4893 at 1 mg/kg.
  • the mouse survival rates of TRN1029 and TRN1031 were 10%-25% higher than that of MEDI4893.
  • TRN1016 only has a protective effect of 62.5% at 1.5 mg/kg.
  • the dose of Hla toxin selected is 17.5 ⁇ g (0.875 mg/kg), while the dose of Hla toxin used in this study is 2 mg/kg.
  • TRN1016 has a better protective effect than MEDI4893 at low doses.
  • the ICR mice were randomly divided into groups, with 9 mice in each group. According to the toxin titration results, the Hla toxin dose was selected to be 2 mg/kg. TRN1029, TRN1031 or MEDI4893 with different concentrations according to the volume of 10 ⁇ L/g were injected intraperitoneally. After 2 hours, each mouse was injected The mice were injected intraperitoneally with Hla (Table 12) at a volume concentration of 400 ⁇ g/mL (2 mg/kg) at 10 ⁇ L/g. The survival time of the mice was observed and the survival rate was calculated. The results are shown in Figure 11.
  • the results of comparison with MEDI4893 at the same concentration showed that the protective effect of TRN1029 and TRN1031 was better than that of MEDI4893.
  • the survival rate of mice with TRN1029 and TRN1031 was higher than that of MEDI4893. .
  • TRN1029 mutants in Table 6 and Table 8 were tested by ELISA. Coat the plate with 200ng/well of Ag030 protein and coat at 4°C overnight; block for 2h at 37°C; start with 1 ⁇ g/well of the antibody to be tested and dilute 3 times to 12 gradients; the positive control is TRN1029 and the negative control is TNM001DS (the For the negative control, please refer to the antibody TRN1021 disclosed in Chinese Patent 202111532094.3); the secondary antibody is Anti-Human IgG (H+L), HRP Conjugate (1:10000 dilution). Read the OD450-OD630 values and the results are as follows.

Abstract

本公开提供了一种特异性结合Hla的抗体及其抗原结合片段和应用。该抗体或其抗原结合片段可高亲和性地结合Hla，可以用于制备预防或治疗金黄色葡萄球菌感染或与金黄色葡萄球菌感染相关疾病的药物，也可用于检测样品中是否存在金黄色葡萄球菌或Hla。

Description

特异性结合金黄色葡萄球菌Hla毒素的全人源单克隆抗体 技术领域
本发明涉及医学及免疫学领域，具体地说，涉及特异性结合于金黄色葡萄球菌(Staphylococcus aureus)α-毒素的全人源抗体及其抗原结合片段，包含所述抗α-毒素抗体及其抗原结合片段的药物组合物、及使用所述抗体和药物组合物的治疗和诊断方法。
背景技术
感染性疾病是全球第二大导致人类死亡的原因，而金黄色葡萄球菌是引起人类感染排名第二病原体，仅次于肠杆菌的感染。金黄色葡萄球菌引起的感染可导致皮肤、黏膜、深部组织感染以及心内膜炎、肺炎等多种疾病，严重感染及并发症的感染病死率高达20％。
目前针对金黄色葡萄球菌感染的治疗，以抗生素治疗为主，但是该治疗方式有巨大的副作用，会导致器官损伤功能衰竭并伴随着生命危险；金黄色葡萄球菌溶血性菌株具有四种溶血素：α、β、γ、δ，其中α溶血素(也称为α-毒素、Hla)的毒性最强，其是诱发溶血、皮肤坏死和致死的主要原因。研究发现超过95％的金黄色葡萄球携带Hla基因(编码α-毒素的基因)，该基因高度保守，同源性高达98％以上，未来针对金黄色葡萄球菌α毒素的特异性中和抗体在预防和治疗金黄色葡萄球菌感染由Hla毒素引起的各种疾病方面具有广阔的应用前景和迫切的需要。
本发明满足了这方面的需求。
发明内容
本发明提供了特异性结合Hla的全人源抗体及其抗原结合片段。该抗体可以中和α-毒素，从而预防、治疗金黄色葡萄球菌感染、与金黄色葡萄球菌感染相关的疾病或病症。
在第一方面，本发明提供了一种特异性结合Hla的全人源抗体及其抗原结合片段，其包含:
1)如SEQ ID NO:33所示的重链可变区所包含的3个重链CDR(HCDR1、HCDR2、HCDR3)和如SEQ ID NO:34所示的轻链可变区所包含的3个轻链CDR(LCDR1、LCDR2、LCDR3)；
2)如SEQ ID NO:35所示的重链可变区所包含的3个重链CDR(HCDR1、HCDR2、HCDR3)和如SEQ ID NO:36所示的轻链可变区所包含的3个轻链CDR(LCDR1、LCDR2、LCDR3)；
3)如SEQ ID NO:37所示的重链可变区所包含的3个重链CDR(HCDR1、HCDR2、HCDR3)和如SEQ ID NO:38所示的轻链可变区所包含的3个轻链CDR(LCDR1、LCDR2、LCDR3)；
4)如SEQ ID NO:39所示的重链可变区所包含的3个重链CDR(HCDR1、HCDR2、HCDR3)和如SEQ ID NO:40所示的轻链可变区所包含的3个轻链CDR(LCDR1、LCDR2、LCDR3)；或
5)如SEQ ID NO:41所示的重链可变区所包含的3个重链CDR(HCDR1、HCDR2、HCDR3)和如SEQ ID NO:42所示的轻链可变区所包含的3个轻链CDR(LCDR1、LCDR2、LCDR3)。
在一个实施方案中，本发明提供了特异性结合Hla的抗体及其抗原结合片段，其包含：
1)包含如SEQ ID NO:3、4和5所示序列，或相对于所述序列含有一个或多个且不超过5个氨基酸的氨基酸取代(例如保守性取代)、缺失或插入的序列的HCDR1、HCDR2、HCDR3；和如SEQ ID NO:6、7和8所示序列，或相对于所述序列含有一个或多个且不超过5个氨基酸的氨基酸取代(例如保守性取代)、缺失或插入的序列的LCDR1、LCDR2、LCDR3；
2)包含如SEQ ID NO:9、10和11所示序列，或相对于所述序列含有一个或多个且不超过5个氨基酸的氨基酸取代(例如保守性取代)、缺失或插入的序列的HCDR1、HCDR2、HCDR3；和如SEQ ID NO:12、13和14所示序列，或相对于所述序列含有一个或多个且不超过5个氨基酸的氨基酸取代(例如保守性取代)、缺失或插入的序列的LCDR1、LCDR2、LCDR3；
3)包含如SEQ ID NO:15、16和17所示序列，或相对于所述序列含有一个或多个且不超过5个氨基 酸的氨基酸取代(例如保守性取代)、缺失或插入的序列的HCDR1、HCDR2、HCDR3；和如SEQ ID NO:18、19和20所示序列，或相对于所述序列含有一个或多个且不超过5个氨基酸的氨基酸取代(例如保守性取代)、缺失或插入的序列的LCDR1、LCDR2、LCDR3；
4)包含如SEQ ID NO:21、22和23所示序列，或相对于所述序列含有一个或多个且不超过5个氨基酸的氨基酸取代(例如保守性取代)、缺失或插入的序列的HCDR1、HCDR2、HCDR3；和如SEQ ID NO:24、25和26所示序列，或相对于所述序列含有一个或多个且不超过5个氨基酸的氨基酸取代(例如保守性取代)、缺失或插入的序列的LCDR1、LCDR2、LCDR3；或
5)包含如SEQ ID NO:27、28和29所示序列，或相对于所述序列含有一个或多个且不超过5个氨基酸的氨基酸取代(例如保守性取代)、缺失或插入的序列的HCDR1、HCDR2、HCDR3；和如SEQ ID NO:30、31和32所示序列，或相对于所述序列含有一个或多个且不超过5个氨基酸的氨基酸取代(例如保守性取代)、缺失或插入的序列的LCDR1、LCDR2、LCDR3。
在一个实施方案中，本发明提供了特异性结合Hla的抗体及其抗原结合片段，其包含：
1)SEQ ID NO:47所示的HCDR1，SED ID NO:4所示的HCDR2和SED ID NO:5所示的HCDR3；和/或，SEQ ID NO:6所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:8所示的LCDR3；
2)SEQ ID NO:48所示的HCDR1，SED ID NO:4所示的HCDR2和SED ID NO:5所示的HCDR3；和/或，SEQ ID NO:6所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:8所示的LCDR3；
3)SEQ ID NO:49所示的HCDR1，SED ID NO:4所示的HCDR2和SED ID NO:5所示的HCDR3；和/或，SEQ ID NO:6所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:8所示的LCDR3；
4)SEQ ID NO:50所示的HCDR1，SED ID NO:4所示的HCDR2和SED ID NO:5所示的HCDR3；和/或，SEQ ID NO:6所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:8所示的LCDR3；
5)SEQ ID NO:51所示的HCDR1，SED ID NO:4所示的HCDR2和SED ID NO:5所示的HCDR3；和/或，SEQ ID NO:6所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:8所示的LCDR3；
6)SEQ ID NO:52所示的HCDR1，SED ID NO:4所示的HCDR2和SED ID NO:5所示的HCDR3；和/或，SEQ ID NO:6所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:8所示的LCDR3；
7)SEQ ID NO:53所示的HCDR1，SED ID NO:4所示的HCDR2和SED ID NO:5所示的HCDR3；和/或，SEQ ID NO:6所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:8所示的LCDR3；
8)SEQ ID NO:54所示的HCDR1，SED ID NO:4所示的HCDR2和SED ID NO:5所示的HCDR3；和/或，SEQ ID NO:6所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:8所示的LCDR3；
9)SEQ ID NO:3所示的HCDR1，SED ID NO:55所示的HCDR2和SED ID NO:5所示的HCDR3；和/或，SEQ ID NO:6所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:8所示的LCDR3；
10)SEQ ID NO:3所示的HCDR1，SED ID NO:56所示的HCDR2和SED ID NO:5所示的HCDR3；和/或，SEQ ID NO:6所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:8所示的LCDR3；
11)SEQ ID NO:3所示的HCDR1，SED ID NO:57所示的HCDR2和SED ID NO:5所示的HCDR3；和/或，SEQ ID NO:6所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:8所示的LCDR3；
12)SEQ ID NO:3所示的HCDR1，SED ID NO:58所示的HCDR2和SED ID NO:5所示的HCDR3；和/或，SEQ ID NO:6所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:8所示的LCDR3；
13)SEQ ID NO:3所示的HCDR1，SED ID NO:59所示的HCDR2和SED ID NO:5所示的HCDR3；和/或，SEQ ID NO:6所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:8所示的LCDR3；
14)SEQ ID NO:3所示的HCDR1，SED ID NO:60所示的HCDR2和SED ID NO:5所示的HCDR3；和/或，SEQ ID NO:6所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:8所示的LCDR3；
15)SEQ ID NO:3所示的HCDR1，SED ID NO:61所示的HCDR2和SED ID NO:5所示的HCDR3；和/或，SEQ ID NO:6所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:8所示的LCDR3；
16)SEQ ID NO:3所示的HCDR1，SED ID NO:62所示的HCDR2和SED ID NO:5所示的HCDR3；和/或，SEQ ID NO:6所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:8所示的LCDR3；
17)SEQ ID NO:3所示的HCDR1，SED ID NO:4所示的HCDR2和SED ID NO:63所示的HCDR3；和/或，SEQ ID NO:6所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:8所示的LCDR3；
18)SEQ ID NO:3所示的HCDR1，SED ID NO:4所示的HCDR2和SED ID NO:64所示的HCDR3；和/或，SEQ ID NO:6所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:8所示的LCDR3；
19)SEQ ID NO:3所示的HCDR1，SED ID NO:4所示的HCDR2和SED ID NO:65所示的HCDR3；和/或，SEQ ID NO:6所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:8所示的LCDR3；
20)SEQ ID NO:3所示的HCDR1，SED ID NO:4所示的HCDR2和SED ID NO:66所示的HCDR3；和/或，SEQ ID NO:6所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:8所示的LCDR3；
21)SEQ ID NO:3所示的HCDR1，SED ID NO:4所示的HCDR2和SED ID NO:67所示的HCDR3；和/或，SEQ ID NO:6所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:8所示的LCDR3；
22)SEQ ID NO:3所示的HCDR1，SED ID NO:4所示的HCDR2和SED ID NO:68所示的HCDR3；和/或，SEQ ID NO:6所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:8所示的LCDR3；
23)SEQ ID NO:3所示的HCDR1，SED ID NO:4所示的HCDR2和SED ID NO:69所示的HCDR3；和/或，SEQ ID NO:6所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:8所示的LCDR3；
24)SEQ ID NO:3所示的HCDR1，SED ID NO:4所示的HCDR2和SED ID NO:70所示的HCDR3；和/或，SEQ ID NO:6所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:8所示的LCDR3；
25)SEQ ID NO:3所示的HCDR1，SED ID NO:4所示的HCDR2和SED ID NO:71所示的HCDR3；和/或，SEQ ID NO:6所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:8所示的LCDR3；
26)SEQ ID NO:3所示的HCDR1，SED ID NO:4所示的HCDR2和SED ID NO:72所示的HCDR3；和/或，SEQ ID NO:6所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:8所示的LCDR3；
27)SEQ ID NO:3所示的HCDR1，SED ID NO:4所示的HCDR2和SED ID NO:73所示的HCDR3；和/或，SEQ ID NO:6所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:8所示的LCDR3；
28)SEQ ID NO:3所示的HCDR1，SED ID NO:4所示的HCDR2和SED ID NO:74所示的HCDR3；和/或，SEQ ID NO:6所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:8所示的LCDR3；
29)SEQ ID NO:3所示的HCDR1，SED ID NO:4所示的HCDR2和SED ID NO:75所示的HCDR3；和/或，SEQ ID NO:6所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:8所示的LCDR3；
30)SEQ ID NO:3所示的HCDR1，SED ID NO:4所示的HCDR2和SED ID NO:76所示的HCDR3；和/或，SEQ ID NO:6所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:8所示的LCDR3；
31)SEQ ID NO:3所示的HCDR1，SED ID NO:4所示的HCDR2和SED ID NO:77所示的HCDR3；和/或，SEQ ID NO:6所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:8所示的LCDR3；
32)SEQ ID NO:3所示的HCDR1，SED ID NO:4所示的HCDR2和SED ID NO:5所示的HCDR3；和/或，SEQ ID NO:78所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:8所示的LCDR3；
33)SEQ ID NO:3所示的HCDR1，SED ID NO:4所示的HCDR2和SED ID NO:5所示的HCDR3；和/或，SEQ ID NO:79所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:8所示的LCDR3；
34)SEQ ID NO:3所示的HCDR1，SED ID NO:4所示的HCDR2和SED ID NO:5所示的HCDR3；和/或，SEQ ID NO:80所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:8所示的LCDR3；
35)SEQ ID NO:3所示的HCDR1，SED ID NO:4所示的HCDR2和SED ID NO:5所示的HCDR3；和/或，SEQ ID NO:81所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:8所示的LCDR3；
36)SEQ ID NO:3所示的HCDR1，SED ID NO:4所示的HCDR2和SED ID NO:5所示的HCDR3；和/或，SEQ ID NO:82所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:8所示的LCDR3；
37)SEQ ID NO:3所示的HCDR1，SED ID NO:4所示的HCDR2和SED ID NO:5所示的HCDR3；和/或，SEQ ID NO:83所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:8所示的LCDR3；
38)SEQ ID NO:3所示的HCDR1，SED ID NO:4所示的HCDR2和SED ID NO:5所示的HCDR3；和/或，SEQ ID NO:6所示的LCDR1，SED ID NO:84所示的LCDR2和SED ID NO:8所示的LCDR3；
39)SEQ ID NO:3所示的HCDR1，SED ID NO:4所示的HCDR2和SED ID NO:5所示的HCDR3；和/或，SEQ ID NO:6所示的LCDR1，SED ID NO:85所示的LCDR2和SED ID NO:8所示的LCDR3；
40)SEQ ID NO:3所示的HCDR1，SED ID NO:4所示的HCDR2和SED ID NO:5所示的HCDR3；和/或，SEQ ID NO:6所示的LCDR1，SED ID NO:86所示的LCDR2和SED ID NO:8所示的LCDR3；
41)SEQ ID NO:3所示的HCDR1，SED ID NO:4所示的HCDR2和SED ID NO:5所示的HCDR3；和/或，SEQ ID NO:6所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:87所示的LCDR3；
42)SEQ ID NO:3所示的HCDR1，SED ID NO:4所示的HCDR2和SED ID NO:5所示的HCDR3；和/或，SEQ ID NO:6所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:88所示的LCDR3；
43)SEQ ID NO:3所示的HCDR1，SED ID NO:4所示的HCDR2和SED ID NO:5所示的HCDR3；和/或，SEQ ID NO:6所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:89所示的LCDR3；
44)SEQ ID NO:3所示的HCDR1，SED ID NO:4所示的HCDR2和SED ID NO:5所示的HCDR3；和/或，SEQ ID NO:6所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:90所示的LCDR3；
45)SEQ ID NO:3所示的HCDR1，SED ID NO:4所示的HCDR2和SED ID NO:5所示的HCDR3；和/或，SEQ ID NO:6所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:91所示的LCDR3；
46)SEQ ID NO:3所示的HCDR1，SED ID NO:4所示的HCDR2和SED ID NO:5所示的HCDR3；和/或，SEQ ID NO:6所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:92所示的LCDR3；
47)SEQ ID NO:3所示的HCDR1，SED ID NO:4所示的HCDR2和SED ID NO:5所示的HCDR3；和/或，SEQ ID NO:6所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:93所示的LCDR3；
48)SEQ ID NO:3所示的HCDR1，SED ID NO:4所示的HCDR2和SED ID NO:5所示的HCDR3；和/或，SEQ ID NO:6所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:94所示的LCDR3；
49)SEQ ID NO:3所示的HCDR1，SED ID NO:4所示的HCDR2和SED ID NO:5所示的HCDR3；和/或，SEQ ID NO:6所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:95所示的LCDR3；
50)SEQ ID NO:96所示的HCDR1，SED ID NO:4所示的HCDR2和SED ID NO:5所示的HCDR3；和/或，SEQ ID NO:6所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:8所示的LCDR3；
51)SEQ ID NO:3所示的HCDR1，SED ID NO:97所示的HCDR2和SED ID NO:5所示的HCDR3；和/或，SEQ ID NO:6所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:8所示的LCDR3；
52)SEQ ID NO:3所示的HCDR1，SED ID NO:4所示的HCDR2和SED ID NO:98所示的HCDR3；和/或，SEQ ID NO:6所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:8所示的LCDR3；
53)SEQ ID NO:3所示的HCDR1，SED ID NO:4所示的HCDR2和SED ID NO:5所示的HCDR3；和/或，SEQ ID NO:99所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:8所示的LCDR3；
54)SEQ ID NO:3所示的HCDR1，SED ID NO:4所示的HCDR2和SED ID NO:5所示的HCDR3；和/或，SEQ ID NO:6所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:100所示的LCDR3；
55)SEQ ID NO:47所示的HCDR1，SED ID NO:55所示的HCDR2和SED ID NO:5所示的HCDR3；和/或，SEQ ID NO:6所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:8所示的LCDR3；
56)SEQ ID NO:3所示的HCDR1，SED ID NO:4所示的HCDR2和SED ID NO:101所示的HCDR3；和/或，SEQ ID NO:6所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:8所示的LCDR3；
57)SEQ ID NO:3所示的HCDR1，SED ID NO:4所示的HCDR2和SED ID NO:5所示的HCDR3；和/或，SEQ ID NO:78所示的LCDR1，SED ID NO:84所示的LCDR2和SED ID NO:8所示的LCDR3；
58)SEQ ID NO:102所示的HCDR1，SED ID NO:4所示的HCDR2和SED ID NO:5所示的HCDR3；和/或，SEQ ID NO:6所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:8所示的LCDR3；
59)SEQ ID NO:3所示的HCDR1，SED ID NO:103所示的HCDR2和SED ID NO:5所示的HCDR3；和/或，SEQ ID NO:6所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:8所示的LCDR3；
60)SEQ ID NO:3所示的HCDR1，SED ID NO:4所示的HCDR2和SED ID NO:104所示的HCDR3；和/或，SEQ ID NO:6所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:8所示的LCDR3；
61)SEQ ID NO:3所示的HCDR1，SED ID NO:4所示的HCDR2和SED ID NO:5所示的HCDR3；和/或，SEQ ID NO:105所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:8所示的LCDR3；
62)SEQ ID NO:3所示的HCDR1，SED ID NO:4所示的HCDR2和SED ID NO:5所示的HCDR3；和/或，SEQ ID NO:6所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:106所示的LCDR3；
63)SEQ ID NO:50所示的HCDR1，SED ID NO:58所示的HCDR2和SED ID NO:66所示的HCDR3；和/或，SEQ ID NO:6所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:8所示的LCDR3；
64)SEQ ID NO:3所示的HCDR1，SED ID NO:4所示的HCDR2和SED ID NO:5所示的HCDR3；和/或，SEQ ID NO:81所示的LCDR1，SED ID NO:85所示的LCDR2和SED ID NO:90所示的LCDR3；
65)SEQ ID NO:3所示的HCDR1，SED ID NO:4所示的HCDR2和SED ID NO:104所示的HCDR3；和/或，SEQ ID NO:6所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:91所示的LCDR3；
66)SEQ ID NO:3所示的HCDR1，SED ID NO:4所示的HCDR2和SED ID NO:67所示的HCDR3；和/或，SEQ ID NO:105所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:8所示的LCDR3；
67)SEQ ID NO:3所示的HCDR1，SED ID NO:103所示的HCDR2和SED ID NO:5所示的HCDR3；和/或，SEQ ID NO:6所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:92所示的LCDR3；
68)SEQ ID NO:3所示的HCDR1，SED ID NO:4所示的HCDR2和SED ID NO:104所示的HCDR3；和/或，SEQ ID NO:6所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:93所示的LCDR3；
69)SEQ ID NO:3所示的HCDR1，SED ID NO:4所示的HCDR2和SED ID NO:63所示的HCDR3；和/或，SEQ ID NO:6所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:87所示的LCDR3；
70)SEQ ID NO:3所示的HCDR1，SED ID NO:4所示的HCDR2和SED ID NO:71所示的HCDR3；和/或，SEQ ID NO:6所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:106所示的LCDR3；
71)SEQ ID NO:102所示的HCDR1，SED ID NO:4所示的HCDR2和SED ID NO:5所示的HCDR3；和/或，SEQ ID NO:105所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:8所示的LCDR3；
72)SEQ ID NO:102所示的HCDR1，SED ID NO:4所示的HCDR2和SED ID NO:5所示的HCDR3；和/或，SEQ ID NO:6所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:106所示的LCDR3；
73)SEQ ID NO:3所示的HCDR1，SED ID NO:103所示的HCDR2和SED ID NO:5所示的HCDR3；和/或，SEQ ID NO:105所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:8所示的LCDR3；
74)SEQ ID NO:3所示的HCDR1，SED ID NO:103所示的HCDR2和SED ID NO:5所示的HCDR3；和/或，SEQ ID NO:6所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:106所示的LCDR3；
75)SEQ ID NO:3所示的HCDR1，SED ID NO:4所示的HCDR2和SED ID NO:104所示的HCDR3；和/或，SEQ ID NO:105所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:8所示的LCDR3；
76)SEQ ID NO:3所示的HCDR1，SED ID NO:4所示的HCDR2和SED ID NO:104所示的HCDR3；和/或，SEQ ID NO:6所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:106所示的LCDR3；
77)SEQ ID NO:50所示的HCDR1，SED ID NO:58所示的HCDR2和SED ID NO:66所示的HCDR3；和/或，SEQ ID NO:6所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:95所示的LCDR3；
78)SEQ ID NO:3所示的HCDR1，SED ID NO:58所示的HCDR2和SED ID NO:5所示的HCDR3；和/或，SEQ ID NO:81所示的LCDR1，SED ID NO:85所示的LCDR2和SED ID NO:90所示的LCDR3；
79)SEQ ID NO:50所示的HCDR1，SED ID NO:58所示的HCDR2和SED ID NO:66所示的HCDR3；和/或，SEQ ID NO:105所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:8所示的LCDR3；
80)SEQ ID NO:50所示的HCDR1，SED ID NO:58所示的HCDR2和SED ID NO:66所示的HCDR3；和/或，SEQ ID NO:6所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:106所示的LCDR3；
81)SEQ ID NO:102所示的HCDR1，SED ID NO:4所示的HCDR2和SED ID NO:5所示的HCDR3；和/或，SEQ ID NO:81所示的LCDR1，SED ID NO:85所示的LCDR2和SED ID NO:90所示的LCDR3；
82)SEQ ID NO:3所示的HCDR1，SED ID NO:103所示的HCDR2和SED ID NO:5所示的HCDR3；和/或，SEQ ID NO:81所示的LCDR1，SED ID NO:85所示的LCDR2和SED ID NO:90所示的LCDR3；
83)SEQ ID NO:3所示的HCDR1，SED ID NO:4所示的HCDR2和SED ID NO:104所示的HCDR3；和/或，SEQ ID NO:81所示的LCDR1，SED ID NO:85所示的LCDR2和SED ID NO:90所示的LCDR3；
84)SEQ ID NO:3所示的HCDR1，SED ID NO:4所示的HCDR2和SED ID NO:101所示的HCDR3；和/或，SEQ ID NO:78所示的LCDR1，SED ID NO:84所示的LCDR2和SED ID NO:8所示的LCDR3；
85)SEQ ID NO:3所示的HCDR1，SED ID NO:4所示的HCDR2和SED ID NO:101所示的HCDR3；和/或，SEQ ID NO:105所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:8所示的LCDR3；或
86)SEQ ID NO:3所示的HCDR1，SED ID NO:4所示的HCDR2和SED ID NO:101所示的HCDR3；和/或，SEQ ID NO:6所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:106所示的LCDR3。
在一个实施方案中，本发明提供了特异性结合Hla的抗体及其抗原结合片段，其包含如SEQ ID NO:3所示的HCDR1的变体(SEQ ID NO:243)、SEQ ID NO:4所示的HCDR2的变体(SEQ ID NO:244)的和SEQ ID NO:5所示的HCDR3的变体(SEQ ID NO:245)；和如SEQ ID NO:6所示的LCDR1的变体(SEQ ID NO:246)、SEQ ID NO:7所示的LCDR2的变体(SEQ ID NO:247)和SEQ ID NO:8所示的LCDR3的变体(SEQ ID NO:248)。
在一个实施方案中，本发明提供了特异性结合Hla的抗体及其抗原结合片段，其包含重链可变区，其中：
1)所述重链可变区包含如SEQ ID NO:33所示氨基酸序列，或与SEQ ID NO:33的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO:33组成；
2)所述重链可变区包含如SEQ ID NO:35所示氨基酸序列，或与SEQ ID NO:35的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO:35组成；
3)所述重链可变区包含如SEQ ID NO:37所示氨基酸序列，或与SEQ ID NO:37的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO:37组成；
4)所述重链可变区包含如SEQ ID NO:39所示氨基酸序列，或与SEQ ID NO:39的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO:39组成；或
5)所述重链可变区包含如SEQ ID NO:41所示氨基酸序列，或与SEQ ID NO:41的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO:41组成。
在另一个实施方案中，本发明提供了特异性结合Hla的抗体及其抗原结合片段，其包含轻链可变区，其中：
1)所述轻链可变区包含如SEQ ID NO:34所示氨基酸序列，或与SEQ ID NO:34的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO:34组成；
2)所述轻链可变区包含如SEQ ID NO:36所示氨基酸序列，或与SEQ ID NO:36的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO:36组成；
3)所述轻链可变区包含如SEQ ID NO:38所示氨基酸序列，或与SEQ ID NO:38的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO:38组成；
4)所述轻链可变区包含如SEQ ID NO:40所示氨基酸序列，或与SEQ ID NO:40的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO:40 组成；或
5)所述轻链可变区包含如SEQ ID NO:42所示氨基酸序列，或与SEQ ID NO:42的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO:42组成。
另一个实施方案中，本发明提供了特异性结合Hla的抗体及其抗原结合片段，其包含重链可变区和轻链可变区，其中：
1)所述重链可变区包含如SEQ ID NO:33所示氨基酸序列，或与SEQ ID NO:33的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO:33组成，所述轻链可变区包含如SEQ ID NO:34所示氨基酸序列，或与SEQ ID NO:34的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO:34组成；
2)所述重链可变区包含如SEQ ID NO:35所示氨基酸序列，或与SEQ ID NO:35的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO:35组成，所述轻链可变区包含如SEQ ID NO:36所示氨基酸序列，或与SEQ ID NO:36的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO:36组成；
3)所述重链可变区包含如SEQ ID NO:37所示氨基酸序列，或与SEQ ID NO:37的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO:37组成，所述轻链可变区包含如SEQ ID NO:38所示氨基酸序列，或与SEQ ID NO:38的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO:38组成；
4)所述重链可变区包含如SEQ ID NO:39所示氨基酸序列，或与SEQ ID NO:39的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO:39组成，所述轻链可变区包含如SEQ ID NO:40所示氨基酸序列，或与SEQ ID NO:40的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO:40组成；或
5)所述重链可变区包含如SEQ ID NO:41所示氨基酸序列，或与SEQ ID NO:41的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO:41组成，所述轻链可变区包含如SEQ ID NO:42所示氨基酸序列，或与SEQ ID NO:42的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO:42组成。
在一些实施方案中，TRN1029HCDR1序列中的G可以保守取代为A；F可以保守取代为W、L、V、I、A、Y，优选保守取代为Y；T可以保守取代为V、S，优选保守取代S；S可以保守取代为T；Y可以保守取代为W、F、T、S，优选保守取代为F；D可以保守取代为E、N，优选保守取代为E；氨基酸取代可以是一个或多个。
在一些实施方案中，TRN1029HCDR2序列中的M可以保守取代为L、F、I，优选保守取代L；S可以保守取代为T；Y可以保守取代为W、F、T、S，优选保守取代为F；D可以保守取代为E、N，优选保守取代为E；G可以保守取代为A；K可以保守取代为R、Q、N、H，优选保守取代为R；氨基酸取代可以是一个或多个。
在一些实施方案中，TRN1029HCDR3序列中的A可以保守取代为V、L、I，优选保守取代为V；K可以保守取代为R、Q、N、H，优选保守取代为R；P可以保守取代为A；R可以保守取代为K、Q、N、H，优选保守取代为K；G可以保守取代为A；S可以保守取代为T；H可以保守取代为N、Q、K、R，优选保守取代为R；Y可以保守取代为W、F、T、S，优选保守取代F；D可以保守取代为E、N，优选保守取代为E；T可以保守取代为V、S，优选保守取代为S；F可以保守取代为W、L、V、I、A、Y，优选保守取 代为Y；氨基酸取代可以是一个或多个。
在一些实施方案中，TRN1029LCDR1序列中的Q可以保守取代为N、E，优选保守取代为N；S可以保守取代为T；I可以保守取代为L、V、M、A、F、正亮氨酸，优选保守取代为L；T可以保守取代为V、S，优选保守取代为S；N可以保守取代为Q、H、D、K、R，优选保守取代为Q；氨基酸取代可以是一个或多个。
在一些实施方案中，TRN1029LCDR2序列中的G可以保守取代为A；A可以保守取代为V、L、I，优选保守取代为V；S可以保守取代为T；氨基酸取代可以是一个或多个。
在一些实施方案中，TRN1029LCDR3序列中的Q可以保守取代为N、E，优选保守取代为N；Y可以保守取代为W、F、T、S，优选保守取代为F；H可以保守取代为N、Q、K、R，优选保守取代为R；N可以保守取代为Q、H、D、K、R，优选保守取代为Q；W可以保守取代为Y、F，优选保守取代为Y；P可以保守取代为A；L可以保守取代为I、V、M、A、F、正亮氨酸，优选保守取代为I；T可以保守取代为V、S，优选保守取代为S；氨基酸取代可以是一个或多个。
在一些实施方案中，TRN1030HCDR1序列中的G可以保守取代为A；T可以保守取代为V、S，优选保守取代S；F可以保守取代为W、L、V、I、A、Y，优选保守取代为Y；R可以保守取代为K、Q、N、H，优选保守取代为K；Q可以保守取代为N、E，优选保守取代为N；H可以保守取代为N、Q、K、R，优选保守取代为R；A可以保守取代为V、L、I，优选保守取代为V，氨基酸取代可以是一个或多个。
在一些实施方案中，TRN1030HCDR2序列中的I可以保守取代为L、V、M、A、F、正亮氨酸，优选保守取代为L；P可以保守取代为A；D可以保守取代为E、N，优选保守取代为E；L可以保守取代为I、V、M、A、F、正亮氨酸，优选保守取代为I；T可以保守取代为V、S，优选保守取代为S；P可以保守取代为A，氨基酸取代可以是一个或多个。
在一些实施方案中，TRN1030HCDR3序列中的A可以保守取代为V、L、I，优选保守取代为V；R可以保守取代为K、Q、N、H，优选保守取代为K；D可以保守取代为E、N，优选保守取代为E；P可以保守取代为A；W可以保守取代为Y、F，优选保守取代为Y；S可以保守取代为T；A可以保守取代为V、L、I，优选保守取代为V；D可以保守取代为E、N，优选保守取代为E；I可以保守取代为L、V、M、A、F、正亮氨酸，优选保守取代为L；N可以保守取代为Q、H、D、K、R，优选保守取代为Q；V可以保守取代为I、L、M、F、A、正亮氨酸，优选保守取代为L，氨基酸取代可以是一个或多个。
在一些实施方案中，TRN1030LCDR1序列中的Q可以保守取代为N、E，优选保守取代为N；S可以保守取代为T；V可以保守取代为I、L、M、F、A、正亮氨酸，优选保守取代为L；R可以保守取代为K、Q、N、H，优选保守取代为K；N可以保守取代为Q、H、D、K、R，优选保守取代为Q，氨基酸取代可以是一个或多个。
在一些实施方案中，TRN1030LCDR2序列中的G可以保守取代为A；A可以保守取代为V、L、I，优选保守取代为V；S可以保守取代为T，氨基酸取代可以是一个或多个。
在一些实施方案中，TRN1030LCDR3序列中的Q可以保守取代为N、E，优选保守取代为N；Y可以保守取代为W、F、T、S，优选保守取代为F；N可以保守取代为Q、H、D、K、R，优选保守取代为Q；D可以保守取代为E、N，优选保守取代为E；W可以保守取代为Y、F，优选保守取代为Y；P可以保守取代为A；T可以保守取代为V、S，优选保守取代为S，氨基酸取代可以是一个或多个。
在一些实施方案中，TRN1031HCDR1序列中的G可以保守取代为A；F可以保守取代为W、L、V、I、A、Y，优选保守取代为Y；S可以保守取代为T；D可以保守取代为E、N，优选保守取代为E；Y可以保守取代为W、F、T、S，优选保守取代为F。
在一些实施方案中，TRN1031HCDR2序列中的I可以保守取代为L、V、M、A、F、正亮氨酸，优选保守取代为L；Y可以保守取代为W、F、T、S，优选保守取代为F；P可以保守取代为A；G可以保守取代为A；E可以保守取代为D、Q，优选保守取代为D；S可以保守取代为T；A可以保守取代为V、L、I，优选保守取代为V。
在一些实施方案中，TRN1031HCDR3序列中的A可以保守取代为V、L、I，优选保守取代为V；T可 以保守取代为V、S，优选保守取代为S；P可以保守取代为A；D可以保守取代为E、N，优选保守取代为E；D可以保守取代为E、N，优选保守取代为E；F可以保守取代为W、L、V、I、A、Y，优选保守取代为Y；S可以保守取代为T；H可以保守取代为N、Q、K、R，优选保守取代为R；G可以保守取代为A；Y可以保守取代为W、F、T、S，优选保守取代为F；Q可以保守取代为N、E，优选保守取代为N；L可以保守取代为I、V、M、A、F、正亮氨酸，优选保守取代为I。
在一些实施方案中，TRN1031LCDR1序列中的D可以保守取代为E、N，优选保守取代为E；A可以保守取代为V、L、I，优选保守取代为V；I可以保守取代为L、V、M、A、F、正亮氨酸，优选保守取代为L；T可以保守取代为V、S，优选保守取代为S；S可以保守取代为T；N可以保守取代为Q、H、D、K、R，优选保守取代为Q。
在一些实施方案中，TRN1031LCDR2序列中的G可以保守取代为A；A可以保守取代为V、L、I，优选保守取代为V；S可以保守取代为T。
在一些实施方案中，TRN1031LCDR3序列中的L可以保守取代为I、V、M、A、F、正亮氨酸，优选保守取代为I；Q可以保守取代为N、E，优选保守取代为N；D可以保守取代为E、N，优选保守取代为E；F可以保守取代为W、L、V、I、A、Y，优选保守取代为Y；R可以保守取代为K、Q、N、H，优选保守取代为K；P可以保守取代为A；T可以保守取代为V、S，优选保守取代为S。
在一些实施方案中，TRN1032HCDR1序列中的G可以保守取代为A；F可以保守取代为W、L、V、I、A、Y，优选保守取代为Y；S可以保守取代为T；L可以保守取代为I、V、M、A、F、正亮氨酸，优选保守取代为I；K可以保守取代为R、Q、N、H，优选保守取代为R；N可以保守取代为Q、H、D、K、R，优选保守取代为Q；Y可以保守取代为W、F、T、S，优选保守取代为F；R可以保守取代为K、Q、N、H，优选保守取代为K。
在一些实施方案中，TRN1032HCDR2序列中的I可以保守取代为L、V、M、A、F、正亮氨酸，优选保守取代为L；Q可以保守取代为N、E，优选保守取代为N；K可以保守取代为R、Q、N、H，优选保守取代为R；F可以保守取代为W、L、V、I、A、Y，优选保守取代为Y；G可以保守取代为A；N可以保守取代为Q、H、D、K、R，优选保守取代为Q；V可以保守取代为I、L、M、F、A、正亮氨酸，优选保守取代为L。
在一些实施方案中，TRN1032HCDR3序列中的A可以保守取代为V、L、I，优选保守取代为V；R可以保守取代为K、Q、N、H，优选保守取代为K；E可以保守取代为D、Q，优选保守取代为D；L可以保守取代为I、V、M、A、F、正亮氨酸，优选保守取代为I；H可以保守取代为N、Q、K、R，优选保守取代为R；F可以保守取代为W、L、V、I、A、Y，优选保守取代为Y；D可以保守取代为E、N，优选保守取代为E；S可以保守取代为T；G可以保守取代为A。
在一些实施方案中，TRN1032LCDR1序列中的N可以保守取代为Q、H、D、K、R，优选保守取代为Q；I可以保守取代为L、V、M、A、F、正亮氨酸，优选保守取代为L；G可以保守取代为A；K可以保守取代为R、Q、N、H，优选保守取代为R；S可以保守取代为T。
在一些实施方案中，TRN1032LCDR2序列中的S可以保守取代为T；D可以保守取代为E、N，优选保守取代为E；N可以保守取代为Q、H、D、K、R，优选保守取代为Q。
在一些实施方案中，TRN1032LCDR3序列中的H可以保守取代为N、Q、K、R，优选保守取代为R；V可以保守取代为I、L、M、F、A、正亮氨酸，优选保守取代为L；W可以保守取代为Y、F，优选保守取代为Y；Q可以保守取代为N、E，优选保守取代为N；T可以保守取代为V、S，优选保守取代S；S可以保守取代为T；D可以保守取代为E、N，优选保守取代为E；L可以保守取代为I、V、M、A、F、正亮氨酸，优选保守取代为I。
在一些实施方案中，TRN1033HCDR1序列中的G可以保守取代为A；F可以保守取代为W、L、V、I、A、Y，优选保守取代为Y；I可以保守取代为L、V、M、A、F、正亮氨酸，优选保守取代为L；V可以保守取代为I、L、M、F、A、正亮氨酸，优选保守取代为L；E可以保守取代为D、Q，优选保守取代为D；M可以保守取代为L、F、I，优选保守取代为L；Y可以保守取代为W、F、T、S，优选保守取代为F。
在一些实施方案中，TRN1033HCDR2序列中的I可以保守取代为L、V、M、A、F、正亮氨酸，优选保守取代为L；Y可以保守取代为W、F、T、S，优选保守取代为F；R可以保守取代为K、Q、N、H，优选保守取代为K；G可以保守取代为A；S可以保守取代为T；T可以保守取代为V、S，优选保守取代S。
在一些实施方案中，TRN1033HCDR3序列中的A可以保守取代为V、L、I，优选保守取代为V；K可以保守取代为R、Q、N、H，优选保守取代为R；E可以保守取代为D、Q，优选保守取代为D；Y可以保守取代为W、F、T、S，优选保守取代为F；V可以保守取代为I、L、M、F、A、正亮氨酸，优选保守取代为L；W可以保守取代为Y、F，优选保守取代为Y；L可以保守取代为I、V、M、A、F、正亮氨酸，优选保守取代为I；G可以保守取代为A；D可以保守取代为E、N，优选保守取代为E；R可以保守取代为K、Q、N、H，优选保守取代为K；H可以保守取代为N、Q、K、R，优选保守取代为R；T可以保守取代为V、S，优选保守取代为S；F可以保守取代为W、L、V、I、A、Y，优选保守取代为Y；I可以保守取代为L、V、M、A、F、正亮氨酸，优选保守取代为L。
在一些实施方案中，TRN1033LCDR1序列中的N可以保守取代为Q、H、D、K、R，优选保守取代为Q；S可以保守取代为T；I可以保守取代为L、V、M、A、F、正亮氨酸，优选保守取代为L；G可以保守取代为A。
在一些实施方案中，TRN1033LCDR2序列中的T可以保守取代为V、S，优选保守取代为S；N可以保守取代为Q、H、D、K、R，优选保守取代为Q；D可以保守取代为E、N，优选保守取代为E。
在一些实施方案中，TRN1033LCDR3序列中的S可以保守取代为T；T可以保守取代为V、S，优选保守取代S；W可以保守取代为Y、F，优选保守取代为Y；D可以保守取代为E、N，优选保守取代为E；L可以保守取代为I、V、M、A、F、正亮氨酸，优选保守取代为I；G可以保守取代为A；V可以保守取代为I、L、M、F、A、正亮氨酸，优选保守取代为L。
在一些实施方案中，上述抗体或其抗原结合片段还包含来自人抗体胚系共有序列的重链和/或轻链恒定区序列。所述的轻链恒定区优选是人源的κ或λ链恒定区。重链恒定区可以是γ、μ、α、δ、或ε链，在一些实施方案中，所述的重链恒定区是人源的IgG1、IgG2、IgG3、IgG4、IgA1、IgA2、IgM、IgD和IgE同型。每个重链、轻链类型由具有本领域熟知的序列的特定恒定区来表征。
在前述任一项的抗体的某些实施方案中，该抗体是IgG类抗体。在一些实施方案中，该IgG类抗体是IgG1亚类抗体。在一些实施方案中，该IgG类抗体是IgG2亚类抗体。在一些实施方案中，该IgG类抗体是IgG3亚类抗体。在一些实施方案中，该IgG类抗体是IgG4亚类抗体。
在一些实施方案中，所述的恒定区优选是人IgG恒定区，例如是人IgG1、IgG2、IgG3或者IgG4同型的恒定区。在一些实施方案中，所述重链和/或轻链恒定区例如在Sequences of Proteins of Immunological Interest，NIH Publication No.91-3242中记载，本发明中可应用其中任一种。
在优选的实施方案中，本发明提供抗Hla抗体或其抗原结合片段，其中重链恒定区序列是人IgG1恒定区，重链恒定区序列包含SEQ ID NO:43所示的氨基酸序列或由其组成。在另一个实施方案中，所述抗体的重链恒定区序也可以是人IgG4恒定区，重链恒定区序列包含SEQ ID NO:44所示的氨基酸序列或由其组成。在一个实施方案中，所述抗体的kappa轻链恒定区包含SEQ ID NO:45所示的氨基酸序列或由其组成，所述抗体的lambda轻链恒定区包含SEQ ID NO:46所示的氨基酸序列或由其组成。

应当理解，也可以使用这些恒定区结构域的序列变体，例如包含一个或多个氨基酸修饰，其中氨基酸位点是应Kabat等人的(1991)EU索引系统标识。
在前述任一项的抗体的某些实施方案中，该抗体是单克隆的。
在前述任一项的抗体的某些实施方案中，该抗体是全长抗体。
在一些实施方案中，本发明的抗体还涵盖与上文所述的任何抗体竞争结合Hla的抗体、以及与上文所述的任何抗体结合Hla相同表位的抗体。
在一些实施方案中，至少部分的抗Hla抗体的框架序列是人共有框架序列。
在一个实施方案中，本发明的抗Hla抗体是完整抗体，例如IgG1、IgG2、IgG3、IgG4、IgM、IgA和IgE抗体。在另一个实施方案中，本发明的抗Hla抗体仅涵盖其抗原结合部分，例如：Fab、Fab'-SH、Fv、scFv或(Fab')2片段。
在一些实施方案中，本发明的抗Hla抗体是中和性抗体，用于中和Hla。
在第二个方面，本发明提供了分离的核酸分子，其编码第一方面所述的任一抗体或其抗原结合片段。
在第三个方面，本发明提供了载体，其包含第二方面的核酸分子。在一个实施方案中，所述载体是表达载体。
在第四个方面，本发明提供了宿主细胞，其包含第三方面的载体或第二发明的核酸分子。在一些实施方案中，该宿主细胞是原核的，例如大肠杆菌。在其它实施方案中，该宿主细胞是真核的，例如293细胞，CHO细胞，酵母细胞，或植物细胞。
在第五个方面，本发明提供了一种药物组合物，其包含第一方面的抗体或其抗原结合片段。
在第六方面，本发明提供了结合Hla的抗体或其抗原结合片段在制备用于预防或治疗金黄色葡萄球菌感染或与金黄色葡萄球菌感染相关疾病的药物中的用途。
在第七方面，本发明提供了在有需要的受试者中预防或治疗金黄色葡萄球菌感染或与金黄色葡萄球菌感染相关疾病的方法，包括向所述受试者施用预防有效量或治疗有效量的本发明抗体或其抗原结合片段、或预防有效量或治疗有效量的本发明药物组合物。
在一个实施方案中，与金黄色葡萄球菌感染相关疾病是皮肤坏死、皮肤和软组织感染(包括脓肿)、手术部位感染、人造关节感染、菌血症、败血症、肺炎。
在第八方面，本发明提供一种检测样品中是否存在金黄色葡萄球菌或Hla的方法，包括将在允许本发明前述任一抗体与Hla形成复合物的条件下将本发明的抗体与待测样品接触，并检测是否形成抗Hla抗体-Hla复合物。
附图说明
此处的附图被并入说明书中并构成本说明书的一部分，示出了符合本说明书的实施例，并与说明书一起用于解释本说明书的原理。
图1显示表达的Hla和Hla_H35L蛋白纯化的SDS-PAGE结果。
图2显示ELISA方法检测血浆样本中抗Hla和Hla_H35L抗体滴度的结果。
图3显示采用Hla_H35L蛋白流式分选单个记忆B细胞的结果。
图4显示线性表达抗体与Hla和Hla_H35L结合的ELISA检测结果。
图5显示表达的单克隆抗体的SDS-PAGE电泳结果和蛋白质免疫印迹结果，左侧2个图为检测非还原 性样品的结果，右侧2个图为检测还原性样品的结果，1、2、3、4、5、6和7泳道分别为分子大小标记、TRN1029、TRN1030、TRN1031、TRN1032、TRN1032和TRN1033。
图6显示表达重组表达抗体与Hla和Hla_H35L结合的ELISA检测结果。
图7显示特异性结合相同抗原的不同抗体竞争性研究的示意图。
图8显示重组表达的不同抗Hla抗体之间相互竞争结合抗原的SPR结果。
图9显示不同抗Hla抗体中和Hla对人红细胞溶血作用的实验结果。
图10显示不同抗Hla抗体在小鼠体内的中和活性。
图11显示不同抗Hla抗体对小鼠体内Hla毒素攻毒的保护作用。
具体实施方式
在详细描述本发明之前，应了解，本发明不受限于本说明书中的特定方法及实验条件，因为所述方法以及条件是可以改变的。另外，本文所用术语仅是供说明特定实施方案之用，而不意欲为限制性的。
I.定义
除非另有定义，否则本文中使用的所有技术和科学术语均具有与本领域一般技术人员通常所理解的含义相同的含义。为了本发明的目的，下文定义了以下术语。
术语“约”在与数字数值联合使用时意为涵盖具有比指定数字数值小10％的下限和比指定数字数值大10％的上限的范围内的数字数值。
术语“和/或”当用于连接两个或多个可选项时，应理解为意指可选项中的任一项或可选项中的任意两项或更多项。
如本文中所用，术语“包含”或“包括”意指包括所述的要素、整数或步骤，但是不排除任意其他要素、整数或步骤。在本文中，当使用术语“包含”或“包括”时，除非另有指明，否则也涵盖由所述及的要素、整数或步骤组成的情形。例如，当提及“包含”某个具体序列的抗体可变区时，也旨在涵盖由该具体序列组成的抗体可变区。
术语“α-毒素”、“Hla”、“α溶血素”和“溶血素A”可互换使用，指由金黄色葡萄球菌(S.aureus)分泌的33kDa的胞外蛋白，其是分泌型的水溶性单体。α-毒素在易感细胞(例如白细胞、血小板、红细胞、外周血单核细胞、巨噬细胞、角质化细跑、成纤维细胞以及内皮细跑)的膜中寡聚为七聚体之后在细胞膜上形成孔，α-毒素孔的形成通常导致细胞功能障碍或者细胞裂解。已经证明α-毒素可以裂解多种人类细胞，包括红细胞、上皮细胞、内皮细胞和一系列其他造血谱系细胞，包括T细胞、单核细胞、巨噬细胞和中性粒细胞等，从而导致细胞溶解、炎症和组织损伤。已证实α-毒素在肺炎、皮肤坏死、败血病中起作用。
野生型α-毒素的氨基酸序列显示于SEQ ID NO:1中，经修饰的α-毒素(H35L突变体)的氨基酸序列显示于SEQ ID NO:2中。除非另外指出，否则提及α-毒素是指野生型形式。
溶血是指红细胞的细胞膜因物理因素、化学因素、生物因素(例如毒素)等因素受损破裂，内部的原生质从细胞漏出使红细胞死亡的现象。“溶血反应”是专指红细胞的名词，虽然血液里除红细胞外还有白细胞、淋巴细胞等等非红细胞的细胞，“溶血反应”一词一般并不会用来形容这些细胞的死亡。
术语“抗体”在本文中以最广意义使用并且涵盖多种抗体结构物，包括但不限于单克隆抗体、多克隆抗体、多特异性抗体(例如，双特异性抗体)和抗体片段，只要它们显示出所需的抗原结合活性即可。完整抗体通常将包含至少两条全长重链和两条全长轻链，但在某些情况下可包括较少的链，例如骆驼中天然存在的抗体可仅包含重链。
“人抗体”指包括具有衍生自人类生殖系免疫球蛋白序列的可变和恒定区的抗体，所述抗体由人或人细胞生成或来源于非人来源，其利用人抗体库或其它人抗体编码序列。人抗体的这种定义明确排除包含非人抗原结合残基的人源化抗体。
术语“中和抗体”是减少或抑制Hla的生物活性的抗体或抗体片段。生物活性的减少可以是部分减少的或是完全减少的。抗体中和Hla的程度称作抗体的中和效力。利用普通技术人员已知的和/或本文所述或所提及的一个或多个测试可确定或测量抗体的中和效力，包括但不限于竞争性结合测定法、直接和间接夹心测 定法、免疫沉淀测定及酶联免疫吸附测定(ELISA)。
“抗体片段”或“抗原结合片段”指与完整抗体不同的分子，其包含完整抗体的一部分且结合完整抗体所结合的抗原。抗体片段的例子包括但不限于Fv，Fab，Fab’，Fab’-SH，F(ab’)2；双抗体；线性抗体；单链抗体(例如scFv)；单结构域抗体；双价或双特异性抗体或其片段；骆驼科抗体(重链抗体)；和由抗体片段形成的双特异性抗体或多特异性抗体。
“互补决定区”或“CDR区”或“CDR”或“高变区”，是抗体可变区中主要负责与抗原表位结合的氨基酸区域。重链和轻链的CDR通常被称作CDR1、CDR2和CDR3，从N-端开始顺序编号。
本领域公知多种用于在一个给定的VH或VL氨基酸序列中确定其CDR序列的方案：Kabat互补决定区(CDR)是基于序列变异性确定的并且是最常用的(Kabat等人,Sequences of Proteins of Immunological Interest,第5版,Public Health Service,National Institutes of Health,Bethesda,Md.(1991)),而Chothia指的是结构环的位置(Chothia等人,(1987)J.Mol.Biol.196:901-917；Chothia等人(1989)Nature 342:877-883),AbM CDR是Kabat CDR和Chothia结构环之间的折中，并且由Oxford Molecular的AbM抗体建模软件使用，“接触性”(Contact)CDR基于对可获得的复杂晶体结构的分析。根据不同的CDR确定方案，这些CDR中的每一个的残基如下所述。
表1
在涉及用本发明定义的具体CDR序列限定抗体时，所述抗体的范围还涵盖了这样的抗体，其可变区序列包含所述的具体CDR序列，但是由于应用了不同的方案(例如不同的指派系统规则或组合)而导致其所声称的CDR边界与本发明所定义的具体CDR边界不同。
本发明抗体的CDR可以根据本领域的任何方案或其组合人工地评估确定边界。除非另有说明，否则在本发明中，术语“CDR”或“CDR序列”涵盖以上述任一种方式确定的CDR序列。
术语“可变区”或“可变结构域”是指参与抗体与抗原结合的抗体重链或轻链的结构域。天然抗体的重链和轻链的可变结构域通常具有相似的结构，其中每个结构域包含四个保守的构架区(FR)和三个互补决定区(CDR)(参见，例如，Kindt等Kuby Immunology，第6版，W.H.Freeman and Co.91页(2007))。
如本文所用，术语“结合”或“特异性结合”意指结合作用对抗原是选择性的并且可以与不想要的或非特异的相互作用区别。抗体与特定抗原结合的能力可以通过酶联免疫吸附测定法(ELISA)、SPR或生物膜层干涉技术或本领域已知的其他常规结合测定法测定。
“亲和力”是指分子(例如抗体)的单一结合位点与其结合配偶体(例如抗原)之间全部非共价相互作用总和的强度。除非另有说明，在用于本文时，“结合亲和力”指反映结合对的成员(例如抗体与抗原)之间1∶1相互作用的内在结合亲和力。分子X对其配偶体Y的亲和力通常可用结合解离平衡常数(KD)来表述。亲和力可通过本领域知道的常用方法来测量，包括现有技术已知以及本文中所描述的那些。在一个实施方案中，通过表面等离振子共振测定法所测定的本发明的“特异性结合”α毒素的抗体的KD值为约1×10-8M，优选约1×10-9M；更优选1×10-10M。
当术语“竞争”用于竞争相同表位的抗原结合蛋白的情况中时，意指在抗原结合蛋白之间竞争，其通过以下测定法来测定：在所述测定法中，待检测的抗原结合蛋白(例如抗体或其免疫学功能片段)防止或抑制(例 如降低)参考抗原结合蛋白(例如配体或参考抗体)与共同抗原(例如N蛋白或其片段)的特异性结合。
术语“表位”是指与抗体分子的可变区中称为互补位的特异性抗原结合位点相互作用的抗原决定位。单一抗原可以具有超过一个表位。因此，不同抗体可以结合于抗原上的不同区域且可以具有不同生物效应。
术语“载体”当在本文中使用时是指能够增殖与其相连的另一个核酸的核酸分子。该术语包括作为自我复制核酸结构的载体以及结合到已经引入其的宿主细胞的基因组中的载体。一些载体能够指导与其可操作相连的核酸的表达。这样的载体在本文中被称为“表达载体”。
术语“宿主细胞”、“宿主细胞系”和“宿主细胞培养物”可交换地使用且是指其中引入外源核酸的细胞，包括这种细胞的子代。宿主细胞包括“转化体”和“转化的细胞”，其包括初级转化的细胞和来源于其的子代，而不考虑传代的数目。子代在核酸含量上可能与亲本细胞不完全相同，而是可以包含突变。本文中包括与在最初转化的细胞中筛选或选择的具有相同功能或生物学活性的突变体子代。
术语“有效量”指以单一或多次剂量施用患者后，本发明的抗Hla抗体或组合物在需要治疗或预防的患者中产生预期效果的量。“治疗有效量”指以需要的剂量并持续需要的时间段，本发明的抗Hla抗体或组合物有效实现所需治疗结果的量。“预防有效量”指以需要的剂量并持续需要的时间段，本发明的抗Hla抗体或组合物有效实现所需预防结果的量。
II.本发明抗体变体
与抗体相关的术语“变体”在本文中指，包含已经通过至少1个，例如1-30，或1-20或1-10个，例如1或2或3或4或5个氨基酸取代、缺失和/或插入而具有氨基酸改变的目标抗体区域(例如重链可变区或轻链可变区或重链CDR区或轻链CDR区)的抗体，其中变体基本上保持改变之前的抗体分子的生物学特性。在一方面，本发明涵盖在本文中所述及的任何抗体的变体。在一个实施方案中，抗体变体保持改变前抗体的至少60％,70％,80％,90％,或100％的生物学活性(例如抗原结合能力)。可以理解的，抗体的重链可变区或轻链可变区、或各CDR区可以单独改变或组合改变。在一些实施方案中，在一个或多个或全部三个重链CDR中的氨基酸改变不超过1个、2个、3个、4个、5个、6个、7个、8个、9个或10个。优选地，所述氨基酸改变为氨基酸取代，优选保守取代。
在一些实施方案中，抗体变体与亲本抗体在目的抗体序列区域上具有至少80％、90％或95％或99％或更高的氨基酸同一性。
如下进行序列之间序列同一性的计算。
为确定两个氨基酸序列或两个核酸序列的同一性百分数，将所述序列出于最佳比较目的比对(例如，可以为了最佳比对而在第一和第二氨基酸序列或核酸序列之一或二者中引入空位或可以为比较目的而抛弃非同源序列)。在一个优选实施方案中，为比较目的，所比对的参考序列的长度是至少30％、优选地至少40％、更优选地至少50％、60％和甚至更优选地至少70％、80％、90％、100％的参考序列长度。随后比较在对应氨基酸位置或核苷酸位置处的氨基酸残基或核苷酸。当第一序列中的位置由第二序列中对应位置处的相同氨基酸残基或核苷酸占据时，则所述分子在这个位置处是相同的。
可以利用数学算法实现两个序列间的序列比较和同一性百分数的计算。在一个优选实施方案中，使用已经集成至GCG软件包的GAP程序中的Needlema和Wunsch((1970)J.Mol.Biol.48:444-453)算法(在http://www.gcg.com可获得)，使用Blossum 62矩阵或PAM250矩阵和空位权重16、14、12、10、8、6或4和长度权重1、2、3、4、5或6，确定两个氨基酸序列之间的同一性百分数。在又一个优选的实施方案中，使用GCG软件包中的GAP程序(在http://www.gcg.com可获得)，使用NWSgapdna.CMP矩阵和空位权重40、50、60、70或80和长度权重1、2、3、4、5或6，确定两个核苷酸序列之间的同一性百分数。特别优选的参数集合(和除非另外说明否则应当使用的一个参数集合)是采用空位罚分12、空位延伸罚分4和移码空位罚分5的Blossum 62评分矩阵。
还可以使用PAM120加权余数表、空位长度罚分12，空位罚分4，利用已经并入ALIGN程序(2.0版)的E.Meyers和W.Miller算法，((1989)CABIOS,4:11-17)确定两个氨基酸序列或核苷酸序列之间的同一性百分数。
术语“保守取代”是指一个氨基酸经相同类别内的另一氨基酸取代，例如一个酸性氨基酸经另一酸性氨基酸取代，一个碱性氨基酸经另一碱性氨基酸取代，或一个中性氨基酸经另一中性氨基酸取代。示例性的取代如下表所示：
表2
III.本发明的核酸以及包含其的载体和宿主细胞
本发明提供了编码以上任何抗Hla抗体或其片段或其任一条链的核酸。
本发明提供了还提供了包含所述核酸的载体。在一个实施方案中，载体是表达载体，例如真核表达载体。
一旦已经制备了上述表达载体或DNA序列，则可以将表达载体或DNA序列转染或引入适宜的宿主细胞中。因此，本发明提供了包含上述核酸或上述载体的宿主细胞。在一个实施方案中，宿主细胞是原核细胞，如大肠杆菌细胞。在另一个实施方案中，宿主细胞是真核的。在另一个实施方案中，宿主细胞选自酵母细胞、哺乳动物细胞(例如CHO细胞或293细胞)或适用于制备抗体或其抗原结合片段的其它细胞。用于培养所产生的转染细胞和用于回收产生的抗体分子的方法和条件是本领域技术人员已知的并且可以基于本说明书和现有技术已知的方法，根据使用的特定表达载体和哺乳动物宿主细胞变动或优化。
IV.本发明药物组合物和药物制剂
本发明提供了包含本文所述的任何抗Hla抗体或其抗原结合片段的组合物，组合物优选为药物组合物。在一个实施方案中，所述组合物还包含药用辅料。
在一些实施方案中，所述组合物用于预防或治疗金黄色葡萄球菌感染。金黄色葡萄球菌感染引起的一些常见病症的非限制性实例包括烧伤、皮肤坏死。
如本文所用，“可药用载体”包括指与治疗剂一起施用的、生理上相容的任何和全部溶剂、分散介质、等渗剂和吸收延迟剂等。
V.制备和纯化本发明的抗Hla抗体
本发明提供了制备抗Hla抗体的方法，其中所述方法包括在适于表达编码所述抗Hla抗体的核酸的条件下培养包含编码所述抗Hla抗体的核酸或包含所述核酸的表达载体的宿主细胞，以及任选地分离所述抗Hla抗体。在某个实施方案中，所述方法还包括从所述宿主细胞(或宿主细胞培养基)回收抗Hla抗体。
为了重组产生本发明的抗Hla抗体，首先分离编码本发明抗Hla抗体的核酸，并将所述核酸插入载体，用于在宿主细胞中进一步克隆和/或表达。此类核酸易于使用常规规程分离和测序，例如通过使用能够与编码本发明抗Hla抗体的核酸特异性结合的寡核苷酸探针进行。
VI.本发明抗Hla抗体的治疗方法及用途
本发明提供了一种治疗金黄色葡萄球菌感染的方法，包括向有需要的受试者施用治疗有效量的本发明的抗Hla抗体。
此外，本发明提供了预防金黄色葡萄球菌感染的方法，包括向有需要的受试者施用预防有效量的本发明的抗Hla抗体。
在一些实施例中，本文所描述的抗体可以适合用于预防、治疗或管理金黄色葡萄球菌感染的疾病或病状，包括但不限于皮肤坏死、皮肤和软组织感染(包括脓肿)、手术部位感染、人造关节感染、菌血症、败血症、肺炎。
在一些实施方案中，本发明抗体适用于减少个体或个体的具体组织或器官中的金黄色葡萄球菌细菌的数目。在一些实施方案中，本发明抗体适用于降低个体中由金黄色葡萄球菌细菌产生的α-毒素的毒性活性，从而减轻由感染产生的症状。
本发明提供了抗Hla抗体在制备用于治疗罹患金黄色葡萄球菌感染或具有与金黄色葡萄球菌感染相关的症状的患者的药物中的用途。
本发明提供的抗Hla抗体可以用于体内或者体外诊断产生α-毒素的金黄色葡萄球菌菌株，或与α-毒素相关的疾病。例如在允许抗α-毒素抗体和α-毒素结合的条件下将待测样品与本发明的抗Hla抗体接触，通过检测是否形成复合物而进行诊断、鉴定。
本发明提供的抗Hla抗体还可以检测和/或测量样品中的α-毒素，例如用于诊断目的。
本发明提供的抗Hla抗体还可以用于检测金黄色葡萄球菌感染的存在和严重程度。术语“检测”用于本文中时，包括定量或定性检测，示例性的检测方法可以涉及免疫组织化学、免疫细胞化学、流式细胞术(例如，FACS)、抗体分子复合的磁珠、ELISA测定法。
用于根据本发明的α-毒素诊断分析中的样品包括可以获自患者的任何组织或流体样品，其在正常或病理学条件下含有可检测量的α-毒素或其片段。通常，将测量获自健康患者(例如未患有金黄色葡萄球菌感染的患者)的具体样品中的α-毒素的含量以最初建立α-毒素的基线或标准含量。然后，可以将α-毒素的此基线含量与在从怀疑具有金黄色葡萄球菌感染相关病状或与这类病状相关的症状的个体获得的样品中测量的α-毒素含量相比较。
VII.本发明的示例性抗Hla抗体的序列
本发明的示例性抗Hla抗体的序列如下表所示。
表3：抗体的CDR序列及其序列编号(根据Kabat编号)

表4：抗体的可变区序列及其序列编号
Ⅷ本发明的抗体取代的示例性序列
本发明的一个实施例中提供的特异性结合Hla的抗体或其抗原结合片段，其包含：
1)重链互补决定区HCDR1，其包含氨基酸序列SEQ ID NO:3；
2)重链互补决定区HCDR2，其包含氨基酸序列SEQ ID NO:4；
3)重链互补决定区HCDR3，其包含氨基酸序列SEQ ID NO:5；
4)轻链互补决定区LCDR1，其包含氨基酸序列：
X1X2X3X4X5X6(SEQ ID NO:246),
其中，X1为Q、N、E或D；
X2为S、T、A或I；
X3为I、L、V、M、A、F、正亮氨酸、G或N；
X4为T、V、S、R、K或I；
X5为T、V、S、K或G；并且
X6为N、Q、H、D、K、R或S；
可选地，其中X1为Q、N、E或D，X2为S，X3为I，X4为T，X5为T，X6为N；可选地，其中X1为N，X2为S，X3为I，X4为T，X5为T，X6为N；
可选地，其中X1为Q，X2为S、T、A或I，X3为I，X4为T，X5为T，X6为N；可选地，其中X1为Q，X2为T，X3为I，X4为T，X5为T，X6为N；
可选地，其中X1为Q，X2为S，X3为I、L、V、M、A、F、正亮氨酸、G或N，X4为T，X5为T，X6为N；可选地，其中X1为Q，X2为S，X3为L，X4为T，X5为T，X6为N；
可选地，其中X1为Q，X2为S，X3为I，X4为T、V、S、R、K或I，X5为T，X6为N；可选地，其中X1为Q，X2为S，X3为I，X4为S，X5为T，X6为N；
可选地，其中X1为Q，X2为S，X3为I，X4为T，X5为T、V、S、K或G，X6为N；可选地，其中X1为Q，X2为S，X3为I，X4为T，X5为S，X6为N；
可选地，其中X1为Q，X2为S，X3为I，X4为T，X5为T，X6为N、Q、H、D、K、R或S；可选地，其中X1为Q，X2为S，X3为I，X4为T，X5为T，X6为Q；
可选地，其中X1为Q，X2为S、T、A或I，X3为I，X4为T，X5为T、V、S、K或G，X6为N；可选地，其中X1为Q，X2为T，X3为I，X4为T，X5为S，X6为N；
5)轻链互补决定区LCDR2，其包含氨基酸序列：
X1X2X3(SEQ ID NO:247),
其中，X1为G、A、S或T；
X2为A、V、L、I、D或N；并且
X3为S、T、N或D；
可选地，其中X1为G、A、S或T，X2为A，X3为S；可选地，其中X1为A，X2为A，X3为S；
可选地，其中X1为G，X2为A、V、L、I、D或N，X3为S；可选地，其中X1为G，X2为V，X3为S；
可选地，其中X1为G，X2为A，X3为S、T、N或D；可选地，其中X1为G，X2为A，X3为T；
6)轻链互补决定区LCDR3，其包含氨基酸序列：
QQX1X2X3X4X5X6X7(SEQ ID NO:248),
其中，X1为Y、W、F、T、S或D；
X2为H、N、Q、K、R、F或D；
X3为N、Q、H、D、K、R或T；
X4为W、Y、F或S；
X5为P、A、D或L；
X6为L、I、V、M、A、F、正亮氨酸、Y、P、D或G；并且
X7为T、V、S、L或G；
可选地，其中X1为Y、W、F、T、S或D，X2为H，X3为N，X4为W，X5为P，X6为L，X7为T；可选地，其中X1为F，X2为H，X3为N，X4为W，X5为P，X6为L，X7为T；
可选地，其中X1为Y，X2为H、N、Q、K、R、F或D，X3为N，X4为W，X5为P，X6为L，X7为T；可选地，其中X1为Y，X2为R，X3为N，X4为W，X5为P，X6为L，X7为T；
可选地，其中X1为Y，X2为H，X3为N、Q、H、D、K、R或T，X4为W，X5为P，X6为L，X7为T；可选地，其中X1为Y，X2为H，X3为Q，X4为W，X5为P，X6为L，X7为T；
可选地，其中X1为Y，X2为H，X3为N，X4为W、Y、F或S，X5为P，X6为L，X7为T；可选地，其中X1为Y，X2为H，X3为N，X4为Y，X5为P，X6为L，X7为T；
可选地，其中X1为Y，X2为H，X3为N，X4为W，X5为P、A、D或L，X6为L，X7为T；可选地，其中X1为Y，X2为H，X3为N，X4为W，X5为A，X6为L，X7为T；
可选地，其中X1为Y，X2为H，X3为N，X4为W，X5为P，X6为L、I、V、M、A、F、正亮氨酸、Y、P、D或G，X7为T；可选地，其中X1为Y，X2为H，X3为N，X4为W，X5为P，X6为I，X7为T；
可选地，其中X1为Y，X2为H，X3为N，X4为W，X5为P，X6为L，X7为T、V、S、L或G；可选地，其中X1为Y，X2为H，X3为N，X4为W，X5为P，X6为L，X7为S。
本发明的一个实施例中提供的特异性结合Hla的抗体或其抗原结合片段，其包含：
1)重链互补决定区HCDR1，其包含氨基酸序列：
X1X2X3X4X5X6X7X8(SEQ ID NO:243),
其中，X1为G或A；
X2为F、W、L、V、I、A、Y或G；
X3为T、V、S或I；
X4为F、W、L、V、I、A或Y；
X5为S、T、R、K或G；
X6为S、T、Q、D、N或E；
X7为Y、W、F、T、S、H或M；并且
X8为D、E、A、Y或R；
可选地，其中X1为G或A，X2为F，X3为T，X4为F，X5为S，X6为S，X7为Y，X8为D；可选地，其中X1为A，X2为F，X3为T，X4为F，X5为S，X6为S，X7为Y，X8为D；
可选地，其中X1为G，X2为F、W、L、V、I、A、Y或G，X3为T，X4为F，X5为S，X6为S，X7为Y，X8为D；可选地，其中X1为G，X2为Y，X3为T，X4为F，X5为S，X6为S，X7为Y，X8为D；
可选地，其中X1为G，X2为F，X3为T、V、S或I，X4为F，X5为S，X6为S，X7为Y，X8为D；可选地，其中X1为G，X2为F，X3为S，X4为F，X5为S，X6为S，X7为Y，X8为D；
可选地，其中X1为G，X2为F，X3为T，X4为F、W、L、V、I、A或Y，X5为S，X6为S，X7为Y，X8为D；可选地，其中X1为G，X2为F，X3为T，X4为Y，X5为S，X6为S，X7为Y，X8为D；
可选地，其中X1为G，X2为F，X3为T，X4为F，X5为S、T、R、K或G，X6为S，X7为Y，X8为 D；可选地，其中X1为G，X2为F，X3为T，X4为F，X5为T，X6为S，X7为Y，X8为D；
可选地，其中X1为G，X2为F，X3为T，X4为F，X5为S，X6为S、T、R、K或G，X7为Y，X8为D；可选地，其中X1为G，X2为F，X3为T，X4为F，X5为S，X6为T，X7为Y，X8为D；
可选地，其中X1为G，X2为F，X3为T，X4为F，X5为S，X6为S，X7为Y、W、F、T、S、H或M，X8为D；可选地，其中X1为G，X2为F，X3为T，X4为F，X5为S，X6为S，X7为F，X8为D；
可选地，其中X1为G，X2为F，X3为T，X4为F，X5为S，X6为S，X7为Y，X8为D、E、A、Y或R；可选地，其中X1为G，X2为F，X3为T，X4为F，X5为S，X6为S，X7为Y，X8为E；
可选地，其中X1为G，X2为F、W、L、V、I、A、Y或G，X3为T，X4为F，X5为S，X6为S、T、R、K或G，X7为Y，X8为D；可选地，其中X1为G，X2为Y，X3为T，X4为F，X5为S，X6为T，X7为Y，X8为D；
2)重链互补决定区HCDR2，其包含氨基酸序列：
X1X2X3X4X5X6X7X8(SEQ ID NO:244),
其中，X1为M、L、F或I；
X2为S、T、I、Y或Q；
X3为Y、W、F、T、S、P、K或R；
X4为D、E、N、G或F；
X5为G、A、L或E；
X6为S、T或N；
X7为Y、W、F、T、S、G或I；并且
X8为K、R、Q、N、H、P、A或V；
可选地，其中X1为M、L、F或I，X2为S，X3为Y，X4为D，X5为G，X6为S，X7为Y，X8为K；可选地，其中X1为L，X2为S，X3为Y，X4为D，X5为G，X6为S，X7为Y，X8为K；
可选地，其中X1为M，X2为S、T、I、Y或Q，X3为Y，X4为D，X5为G，X6为S，X7为Y，X8为K；可选地，其中X1为M，X2为T，X3为Y，X4为D，X5为G，X6为S，X7为Y，X8为K；
可选地，其中X1为M，X2为S，X3为Y、W、F、T、S、P、K或R，X4为D，X5为G，X6为S，X7为Y，X8为K；可选地，其中X1为M，X2为S，X3为F，X4为D，X5为G，X6为S，X7为Y，X8为K；
可选地，其中X1为M，X2为S，X3为Y，X4为D、E、N、G或F，X5为G，X6为S，X7为Y，X8为K；可选地，其中X1为M，X2为S，X3为Y，X4为E，X5为G，X6为S，X7为Y，X8为K；
可选地，其中X1为M，X2为S，X3为Y，X4为D，X5为G、A、L或E，X6为S，X7为Y，X8为K；可选地，其中X1为M，X2为S，X3为Y，X4为D，X5为A，X6为S，X7为Y，X8为K；
可选地，其中X1为M，X2为S，X3为Y，X4为D，X5为G，X6为S、T或N，X7为Y，X8为K；可选地，其中X1为M，X2为S，X3为Y，X4为D，X5为G，X6为T，X7为Y，X8为K；
可选地，其中X1为M，X2为S，X3为Y，X4为D，X5为G，X6为S，X7为Y、W、F、T、S、G或I，X8为K；可选地，其中X1为M，X2为S，X3为Y，X4为D，X5为G，X6为S，X7为F，X8为K；
可选地，其中X1为M，X2为S，X3为Y，X4为D，X5为G，X6为S，X7为Y，X8为K、R、Q、N、H、P、A或V；可选地，其中X1为M，X2为S，X3为Y，X4为D，X5为G，X6为S，X7为Y，X8为R；
可选地，其中X1为M，X2为S、T、I、Y或Q，X3为Y、W、F、T、S、P、K或R，X4为D，X5为G，X6为S，X7为Y、W、F、T、S、G或I，X8为K；可选地，其中X1为M，X2为T，X3为F，X4为D，X5为G，X6为S，X7为F，X8为K；
3)重链互补决定区HCDR3，其包含氨基酸序列：
AX1X2RX3SX4X5X6X7X8GX9X10X11(SEQ ID NO:245),
其中，X1为K、R、Q、N、H或T；
X2为P、A、D或E；
X3为G、A、W、P、H或V；
X4为H、N、Q、K、R、S、F或D；
X5为Y、W、F、T、S、A或L；
X6为D、E、N、A、H、S或G；
X7为T、V、S、G或D；
X8为S、T、D、G或R；
X9为F、W、L、V、I、A、Y、P或T；
X10为D、E、N、Q、F或Y；并且
X11为Y、W、F、T、S、V、L或D；
可选地，其中X1为K、R、Q、N、H或T，X2为P，X3为G，X4为H，X5为Y，X6为D，X7为T，X8为S，X9为F，X10为D，X11为Y；可选地，其中X1为R，X2为P，X3为G，X4为H，X5为Y，X6为D，X7为T，X8为S，X9为F，X10为D，X11为Y；
可选地，其中X1为K，X2为P、A、D或E，X3为G，X4为H，X5为Y，X6为D，X7为T，X8为S，X9为F，X10为D，X11为Y；可选地，其中X1为K，X2为A，X3为G，X4为H，X5为Y，X6为D，X7为T，X8为S，X9为F，X10为D，X11为Y；
可选地，其中X1为K，X2为P，X3为G、A、W、P、H或V，X4为H，X5为Y，X6为D，X7为T，X8为S，X9为F，X10为D，X11为Y；可选地，其中X1为K，X2为P，X3为A，X4为H，X5为Y，X6为D，X7为T，X8为S，X9为F，X10为D，X11为Y；
可选地，其中X1为K，X2为P，X3为G，X4为H、N、Q、K、R、S、F或D，X5为Y，X6为D，X7为T，X8为S，X9为F，X10为D，X11为Y；可选地，其中X1为K，X2为P，X3为G，X4为R，X5为Y，X6为D，X7为T，X8为S，X9为F，X10为D，X11为Y；
可选地，其中X1为K，X2为P，X3为G，X4为H，X5为Y、W、F、T、S、A或L，X6为D，X7为T，X8为S，X9为F，X10为D，X11为Y；可选地，其中X1为K，X2为P，X3为G，X4为H，X5为F，X6为D，X7为T，X8为S，X9为F，X10为D，X11为Y；
可选地，其中X1为K，X2为P，X3为G，X4为H，X5为Y，X6为D、E、N、A、H、S或G，X7为T，X8为S，X9为F，X10为D，X11为Y；可选地，其中X1为K，X2为P，X3为G，X4为H，X5为Y，X6为E，X7为T，X8为S，X9为F，X10为D，X11为Y；
可选地，其中X1为K，X2为P，X3为G，X4为H，X5为Y，X6为D，X7为T、V、S、G或D，X8为S，X9为F，X10为D，X11为Y；可选地，其中X1为K，X2为P，X3为G，X4为H，X5为Y，X6为D，X7为S，X8为S，X9为F，X10为D，X11为Y；
可选地，其中X1为K，X2为P，X3为G，X4为H，X5为Y，X6为D，X7为T，X8为S、T、D、G或R，X9为F，X10为D，X11为Y；可选地，其中X1为K，X2为P，X3为G，X4为H，X5为Y，X6为D，X7为T，X8为T，X9为F，X10为D，X11为Y；
可选地，其中X1为K，X2为P，X3为G，X4为H，X5为Y，X6为D，X7为T，X8为S，X9为F、W、L、V、I、A、Y、P或T，X10为D，X11为Y；可选地，其中X1为K，X2为P，X3为G，X4为H，X5为Y，X6为D，X7为T，X8为S，X9为Y，X10为D，X11为Y；
可选地，其中X1为K，X2为P，X3为G，X4为H，X5为Y，X6为D，X7为T，X8为S，X9为F，X10为D、E、N、Q、F或Y，X11为Y；可选地，其中X1为K，X2为P，X3为G，X4为H，X5为Y，X6为D，X7为T，X8为S，X9为F，X10为E，X11为Y；
可选地，其中X1为K，X2为P，X3为G，X4为H，X5为Y，X6为D，X7为T，X8为S，X9为F，X10为D，X11为Y、W、F、T、S、V、L或D；可选地，其中X1为K，X2为P，X3为G，X4为H，X5为Y，X6为D，X7为T，X8为S，X9为F，X10为D，X11为F；
4)轻链互补决定区LCDR1，其包含氨基酸序列SEQ ID NO:6；
5)轻链互补决定区LCDR2，其包含氨基酸序列SEQ ID NO:7：
6)轻链互补决定区LCDR3，其包含氨基酸序列SEQ ID NO:8。
本发明的一个实施例TRN1029序列的CDR区的氨基酸序列可以被表2所述的氨基酸取代，表5为取代的CDR区的部分重链或轻链氨基酸序列
表5：抗体的CDR区氨基酸取代




本发明一个实施例中，突变抗体组成如下(其他氨基酸序列同TRN1029，具体如下表6所示，表中：TRN1029-VL(SEQ ID NO:34)；TRN1029-VH(SEQ ID NO:33)
表6：突变抗体轻重链可变区组合


从本发明实施例中可以看出：
1)抗体20的HCDR3中的R突变为K后对抗原抗体结合影响较大，同时抗体63、77、79、80的抗原抗体结合也受到同一位点突变影响。
2)抗体69的LCDR3的Q变成N后对抗原抗体的结合影响较大,抗体76的LCDR3的Q变成N及W变成Y后对抗原抗体的结合影响较大；
综合抗体69和抗体76结果，LCDR3的QQ两个氨基酸对抗原抗体结合具有重大贡献。
3)抗体56的HCDR3的A变成V、S变成T、G变成A后对抗原抗体的结合影响较大，并且抗体84、85、86的轻链发生突变后会更加降低抗体和抗原的结合。
综上所述，本发明的一个实施例中提供的特异性结合Hla的抗体或其抗原结合片段，其包含：
1)重链互补决定区HCDR1，其包含氨基酸序列：
X1X2X3X4X5X6X7X8(SEQ ID NO:243),
其中，X1为G或A；
X2为F、W、L、V、I、A、Y或G；
X3为T、V、S或I；
X4为F、W、L、V、I、A或Y；
X5为S、T、R、K或G；
X6为S、T、Q、D、N或E；
X7为Y、W、F、T、S、H或M；并且
X8为D、E、A、Y或R；
可选地，其中X1为G或A，X2为F，X3为T，X4为F，X5为S，X6为S，X7为Y，X8为D；可选地，其中X1为A，X2为F，X3为T，X4为F，X5为S，X6为S，X7为Y，X8为D；
可选地，其中X1为G，X2为F、W、L、V、I、A、Y或G，X3为T，X4为F，X5为S，X6为S，X7为Y，X8为D；可选地，其中X1为G，X2为Y，X3为T，X4为F，X5为S，X6为S，X7为Y，X8为D；
可选地，其中X1为G，X2为F，X3为T、V、S或I，X4为F，X5为S，X6为S，X7为Y，X8为D；可选地，其中X1为G，X2为F，X3为S，X4为F，X5为S，X6为S，X7为Y，X8为D；
可选地，其中X1为G，X2为F，X3为T，X4为F、W、L、V、I、A或Y，X5为S，X6为S，X7为Y，X8为D；可选地，其中X1为G，X2为F，X3为T，X4为Y，X5为S，X6为S，X7为Y，X8为D；
可选地，其中X1为G，X2为F，X3为T，X4为F，X5为S、T、R、K或G，X6为S，X7为Y，X8为D；可选地，其中X1为G，X2为F，X3为T，X4为F，X5为T，X6为S，X7为Y，X8为D；
可选地，其中X1为G，X2为F，X3为T，X4为F，X5为S，X6为S、T、R、K或G，X7为Y，X8为D；可选地，其中X1为G，X2为F，X3为T，X4为F，X5为S，X6为T，X7为Y，X8为D；
可选地，其中X1为G，X2为F，X3为T，X4为F，X5为S，X6为S，X7为Y、W、F、T、S、H或M，X8为D；可选地，其中X1为G，X2为F，X3为T，X4为F，X5为S，X6为S，X7为F，X8为D；
可选地，其中X1为G，X2为F，X3为T，X4为F，X5为S，X6为S，X7为Y，X8为D、E、A、Y或R；可选地，其中X1为G，X2为F，X3为T，X4为F，X5为S，X6为S，X7为Y，X8为E；
可选地，其中X1为G，X2为F、W、L、V、I、A、Y或G，X3为T，X4为F，X5为S，X6为S、T、R、K或G，X7为Y，X8为D；可选地，其中X1为G，X2为Y，X3为T，X4为F，X5为S，X6为T，X7为Y，X8为D；
2)重链互补决定区HCDR2，其包含氨基酸序列：
X1X2X3X4X5X6X7X8(SEQ ID NO:244),
其中，X1为M、L、F或I；
X2为S、T、I、Y或Q；
X3为Y、W、F、T、S、P、K或R；
X4为D、E、N、G或F；
X5为G、A、L或E；
X6为S、T或N；
X7为Y、W、F、T、S、G或I；并且
X8为K、R、Q、N、H、P、A或V；
可选地，其中X1为M、L、F或I，X2为S，X3为Y，X4为D，X5为G，X6为S，X7为Y，X8为K；可选地，其中X1为L，X2为S，X3为Y，X4为D，X5为G，X6为S，X7为Y，X8为K；
可选地，其中X1为M，X2为S、T、I、Y或Q，X3为Y，X4为D，X5为G，X6为S，X7为Y，X8为K；可选地，其中X1为M，X2为T，X3为Y，X4为D，X5为G，X6为S，X7为Y，X8为K；
可选地，其中X1为M，X2为S，X3为Y、W、F、T、S、P、K或R，X4为D，X5为G，X6为S，X7为Y，X8为K；可选地，其中X1为M，X2为S，X3为F，X4为D，X5为G，X6为S，X7为Y，X8为K；
可选地，其中X1为M，X2为S，X3为Y，X4为D、E、N、G或F，X5为G，X6为S，X7为Y，X8为K；可选地，其中X1为M，X2为S，X3为Y，X4为E，X5为G，X6为S，X7为Y，X8为K；
可选地，其中X1为M，X2为S，X3为Y，X4为D，X5为G、A、L或E，X6为S，X7为Y，X8为K；可选地，其中X1为M，X2为S，X3为Y，X4为D，X5为A，X6为S，X7为Y，X8为K；
可选地，其中X1为M，X2为S，X3为Y，X4为D，X5为G，X6为S、T或N，X7为Y，X8为K；可选地，其中X1为M，X2为S，X3为Y，X4为D，X5为G，X6为T，X7为Y，X8为K；
可选地，其中X1为M，X2为S，X3为Y，X4为D，X5为G，X6为S，X7为Y、W、F、T、S、G或I，X8为K；可选地，其中X1为M，X2为S，X3为Y，X4为D，X5为G，X6为S，X7为F，X8为K；
可选地，其中X1为M，X2为S，X3为Y，X4为D，X5为G，X6为S，X7为Y，X8为K、R、Q、N、H、P、A或V；可选地，其中X1为M，X2为S，X3为Y，X4为D，X5为G，X6为S，X7为Y，X8为R；
可选地，其中X1为M，X2为S、T、I、Y或Q，X3为Y、W、F、T、S、P、K或R，X4为D，X5为G，X6为S，X7为Y、W、F、T、S、G或I，X8为K；可选地，其中X1为M，X2为T，X3为F，X4为D，X5为G，X6为S，X7为F，X8为K；
3)重链互补决定区HCDR3，其包含氨基酸序列：
AX1X2RX3SX4X5X6X7X8GX9X10X11(SEQ ID NO:245),
其中，X1为K、R、Q、N、H或T；
X2为P、A、D或E；
X3为G、A、W、P、H或V；
X4为H、N、Q、K、R、S、F或D；
X5为Y、W、F、T、S、A或L；
X6为D、E、N、A、H、S或G；
X7为T、V、S、G或D；
X8为S、T、D、G或R；
X9为F、W、L、V、I、A、Y、P或T；
X10为D、E、N、Q、F或Y；并且
X11为Y、W、F、T、S、V、L或D；
可选地，其中X1为K、R、Q、N、H或T，X2为P，X3为G，X4为H，X5为Y，X6为D，X7为T，X8为S，X9为F，X10为D，X11为Y；可选地，其中X1为R，X2为P，X3为G，X4为H，X5为Y，X6为D，X7为T，X8为S，X9为F，X10为D，X11为Y；
可选地，其中X1为K，X2为P、A、D或E，X3为G，X4为H，X5为Y，X6为D，X7为T，X8为S，X9为F，X10为D，X11为Y；可选地，其中X1为K，X2为A，X3为G，X4为H，X5为Y，X6为D，X7为T，X8为S，X9为F，X10为D，X11为Y；
可选地，其中X1为K，X2为P，X3为G、A、W、P、H或V，X4为H，X5为Y，X6为D，X7为T，X8为S，X9为F，X10为D，X11为Y；可选地，其中X1为K，X2为P，X3为A，X4为H，X5为Y，X6为D，X7为T，X8为S，X9为F，X10为D，X11为Y；
可选地，其中X1为K，X2为P，X3为G，X4为H、N、Q、K、R、S、F或D，X5为Y，X6为D，X7为T，X8为S，X9为F，X10为D，X11为Y；可选地，其中X1为K，X2为P，X3为G，X4为R，X5为Y，X6为D，X7为T，X8为S，X9为F，X10为D，X11为Y；
可选地，其中X1为K，X2为P，X3为G，X4为H，X5为Y、W、F、T、S、A或L，X6为D，X7为T，X8为S，X9为F，X10为D，X11为Y；可选地，其中X1为K，X2为P，X3为G，X4为H，X5为F，X6为D，X7为T，X8为S，X9为F，X10为D，X11为Y；
可选地，其中X1为K，X2为P，X3为G，X4为H，X5为Y，X6为D、E、N、A、H、S或G，X7为T， X8为S，X9为F，X10为D，X11为Y；可选地，其中X1为K，X2为P，X3为G，X4为H，X5为Y，X6为E，X7为T，X8为S，X9为F，X10为D，X11为Y；
可选地，其中X1为K，X2为P，X3为G，X4为H，X5为Y，X6为D，X7为T、V、S、G或D，X8为S，X9为F，X10为D，X11为Y；可选地，其中X1为K，X2为P，X3为G，X4为H，X5为Y，X6为D，X7为S，X8为S，X9为F，X10为D，X11为Y；
可选地，其中X1为K，X2为P，X3为G，X4为H，X5为Y，X6为D，X7为T，X8为S、T、D、G或R，X9为F，X10为D，X11为Y；可选地，其中X1为K，X2为P，X3为G，X4为H，X5为Y，X6为D，X7为T，X8为T，X9为F，X10为D，X11为Y；
可选地，其中X1为K，X2为P，X3为G，X4为H，X5为Y，X6为D，X7为T，X8为S，X9为F、W、L、V、I、A、Y、P或T，X10为D，X11为Y；可选地，其中X1为K，X2为P，X3为G，X4为H，X5为Y，X6为D，X7为T，X8为S，X9为Y，X10为D，X11为Y；
可选地，其中X1为K，X2为P，X3为G，X4为H，X5为Y，X6为D，X7为T，X8为S，X9为F，X10为D、E、N、Q、F或Y，X11为Y；可选地，其中X1为K，X2为P，X3为G，X4为H，X5为Y，X6为D，X7为T，X8为S，X9为F，X10为E，X11为Y；
可选地，其中X1为K，X2为P，X3为G，X4为H，X5为Y，X6为D，X7为T，X8为S，X9为F，X10为D，X11为Y、W、F、T、S、V、L或D；可选地，其中X1为K，X2为P，X3为G，X4为H，X5为Y，X6为D，X7为T，X8为S，X9为F，X10为D，X11为F；
4)轻链互补决定区LCDR1，其包含氨基酸序列：
X1X2X3X4X5X6(SEQ ID NO:246),
其中，X1为Q、N、E或D；
X2为S、T、A或I；
X3为I、L、V、M、A、F、正亮氨酸、G或N；
X4为T、V、S、R、K或I；
X5为T、V、S、K或G；并且
X6为N、Q、H、D、K、R或S；
可选地，其中X1为Q、N、E或D，X2为S，X3为I，X4为T，X5为T，X6为N；可选地，其中X1为N，X2为S，X3为I，X4为T，X5为T，X6为N；
可选地，其中X1为Q，X2为S、T、A或I，X3为I，X4为T，X5为T，X6为N；可选地，其中X1为Q，X2为T，X3为I，X4为T，X5为T，X6为N；
可选地，其中X1为Q，X2为S，X3为I、L、V、M、A、F、正亮氨酸、G或N，X4为T，X5为T，X6为N；可选地，其中X1为Q，X2为S，X3为L，X4为T，X5为T，X6为N；
可选地，其中X1为Q，X2为S，X3为I，X4为T、V、S、R、K或I，X5为T，X6为N；可选地，其中X1为Q，X2为S，X3为I，X4为S，X5为T，X6为N；
可选地，其中X1为Q，X2为S，X3为I，X4为T，X5为T、V、S、K或G，X6为N；可选地，其中X1为Q，X2为S，X3为I，X4为T，X5为S，X6为N；
可选地，其中X1为Q，X2为S，X3为I，X4为T，X5为T，X6为N、Q、H、D、K、R或S；可选地，其中X1为Q，X2为S，X3为I，X4为T，X5为T，X6为Q；
可选地，其中X1为Q，X2为S、T、A或I，X3为I，X4为T，X5为T、V、S、K或G，X6为N；可选地，其中X1为Q，X2为T，X3为I，X4为T，X5为S，X6为N；
5)轻链互补决定区LCDR2，其包含氨基酸序列：
X1X2X3(SEQ ID NO:247),
其中，X1为G、A、S或T；
X2为A、V、L、I、D或N；并且
X3为S、T、N或D；
可选地，其中X1为G、A、S或T，X2为A，X3为S；可选地，其中X1为A，X2为A，X3为S；
可选地，其中X1为G，X2为A、V、L、I、D或N，X3为S；可选地，其中X1为G，X2为V，X3为S；
可选地，其中X1为G，X2为A，X3为S、T、N或D；可选地，其中X1为G，X2为A，X3为T；
6)轻链互补决定区LCDR3，其包含氨基酸序列：
QQX1X2X3X4X5X6X7(SEQ ID NO:248),
其中，X1为Y、W、F、T、S或D；
X2为H、N、Q、K、R、F或D；
X3为N、Q、H、D、K、R或T；
X4为W、Y、F或S；
X5为P、A、D或L；
X6为L、I、V、M、A、F、正亮氨酸、Y、P、D或G；并且
X7为T、V、S、L或G；
可选地，其中X1为Y、W、F、T、S或D，X2为H，X3为N，X4为W，X5为P，X6为L，X7为T；可选地，其中X1为F，X2为H，X3为N，X4为W，X5为P，X6为L，X7为T；
可选地，其中X1为Y，X2为H、N、Q、K、R、F或D，X3为N，X4为W，X5为P，X6为L，X7为T；可选地，其中X1为Y，X2为R，X3为N，X4为W，X5为P，X6为L，X7为T；
可选地，其中X1为Y，X2为H，X3为N、Q、H、D、K、R或T，X4为W，X5为P，X6为L，X7为T；可选地，其中X1为Y，X2为H，X3为Q，X4为W，X5为P，X6为L，X7为T；
可选地，其中X1为Y，X2为H，X3为N，X4为W、Y、F或S，X5为P，X6为L，X7为T；可选地，其中X1为Y，X2为H，X3为N，X4为Y，X5为P，X6为L，X7为T；
可选地，其中X1为Y，X2为H，X3为N，X4为W，X5为P、A、D或L，X6为L，X7为T；可选地，其中X1为Y，X2为H，X3为N，X4为W，X5为A，X6为L，X7为T；
可选地，其中X1为Y，X2为H，X3为N，X4为W，X5为P，X6为L、I、V、M、A、F、正亮氨酸、Y、P、D或G，X7为T；可选地，其中X1为Y，X2为H，X3为N，X4为W，X5为P，X6为I，X7为T；
可选地，其中X1为Y，X2为H，X3为N，X4为W，X5为P，X6为L，X7为T、V、S、L或G；可选地，其中X1为Y，X2为H，X3为N，X4为W，X5为P，X6为L，X7为S。
本领域技术人员熟知，TRN1029、TRN1030、TRN1031、TRN1032、TRN1033的轻链FR1和FR2的两个半胱氨酸(C)不能进行保守取代，重链FR1和FR2的两个半胱氨酸(C)也不能进行保守取代，否则会影响抗体结构的正确组装和稳定性，具体如下所示：

本发明的一个实施例所示：TRN1029、TRN1030、TRN1031、TRN1032、TRN1033的重链可变区FR区除了半胱氨酸不能替换，其他FR区氨基酸序列可用表2所示的氨基酸保守替换。
本发明的一个实施例TRN1029的轻链、重链的FR区氨基酸可被如表2所示的保守替代，表7为FR区部分取代的重链或轻链可变区，均不会影响本发明所提供抗体结构的正确组装和稳定性。
本发明的另一个实施例TRN1029的轻链、重链的FR区氨基酸可以分别被如表2所示的保守替代后变为如SEQ ID NO:221-231所示的FR区变体序列，均不会影响本发明所提供抗体结构的正确组装和稳定性。
表7：FR区部分取代的重链或轻链可变区

本发明一个实施例中，突变抗体组成如下(其他氨基酸序列同TRN1029)，具体如下表8所示：
表8：突变抗体轻重链组合
为了达到清楚和简洁描述的目的，本文中作为相同的或分开的一些实施方案的一部分来描述特征，然而，将要理解的是，本公开的范围可包括具有所描述的所有或一些特征的组合的一些实施方案。
实施例
以下实施例进一步说明本发明，然而，应理解实施例以说明而非限定的方式来描述，并且本领域技术人员可以进行多种修改。
除非明确指明相反，否则本发明的实施将采用本领域技术内的常规化学、生物化学、有机化学、分子生物学、微生物学、重组DNA技术、遗传学、免疫学和细胞生物学的方法。
总体而言，本申请通过招募健康的志愿者和严重感染金黄色葡萄球菌后痊愈的志愿者，采集其外周血样本以进行血清抗体滴度ELISA检测，并分选特定的单个记忆B细胞。通过RT-PCR方法得到所分选单个 记忆B细胞的抗体重链和相应轻链的可变区基因；利用线性Ig重链和轻链基因表达系统，获得表达特异可变区抗体，并进而获得全人源抗金黄色葡萄球菌单克隆抗体。利用ELISA，Western Blot、细胞中和实验等方法对筛选到的多个全人源抗Hla单克隆抗体进行体内外的中和活性及结合活性鉴定；利用表面等离子共振技术(SPR)对上述单克隆抗体的热力学与动力学性质及表位进行研究分析。
实施例1野生型金黄色葡萄球菌α-毒素(Hla)及其突变体Hla_H35L的构建、表达和纯化
通过DNA合成获得来自金黄色葡萄球菌菌株ATCC BAA1556基因组的α毒素基因(Hla)，并在该基因的C端添加AVI Tag和His Tag。由于Hla毒素具有溶血和溶细胞活性，因此不能用于流式分选记忆B细胞。为此，我们构建了无毒突变体(命名为Hla_H35L)，即将α-毒素第35号氨基酸由组氨酸(H)突变成亮氨酸(L)，Hla_H35L失去了溶血和溶细胞活性，可以用于流式细胞分选的实验。简言之，通过如下方法获得Hla_H35L：使用全式金FM111-02定点诱变试剂盒通过对野生型基因的定点诱变产生Hla_H35L变体。DNA测序验证后，将Hla和Hla_H35L构建到pET-42b(+)载体中，转化大肠杆菌BL21菌株，在含有卡那霉素的LB培养基内37℃培养过夜，IPTG诱导表达。离心收获细胞，利用Ni-NTA磁珠纯化获得Hla蛋白(SEQ ID NO:1)和重组Hla_H35L蛋白(SEQ ID NO:2)。SDS-PAGE凝胶检测结果如图1所示，Hla蛋白和Hla_H35L蛋白的分子量约为35kD，经过一步Ni-NTA磁珠纯化后蛋白纯度较高，能够满足后续研究试验使用，获得的Hla蛋白将用于ELISA检测、SPR和体内体外生物学分析，重组Hla_H35L蛋白将用于ELISA检测和流式细胞分选。
实施例2外周血单个核细胞分离
招募健康志愿者和严重感染金黄色葡萄球菌后痊愈的志愿者，采集外周血样本用于分离记忆B细胞。
采集以上志愿者10mL的静脉血于含有EDTA的抗凝管中，利用密度离心法分离血浆与PBMC细胞，具体操作如下：取10mL静脉血于400g、22℃、离心15min；吸取离心后上清透明血浆层，分装-80℃冻存；将剩余部分与等量的RPMI1640(Gibco)充分混匀，缓慢加入到含有淋巴细胞分离液的无菌离心管内，并保持液面分层完整；400g离心35min，用毛细管吸取位于云雾层的外周血单个核细胞(PBMC)，置另一无菌离心管中，加入5倍以上体积的RPMI1640，400g离心10min，洗涤细胞两次，细胞计数后以1×107/支进行冻存液氮中备用。
实施例3样本的筛选
对招募健康的志愿者和严重感染金黄色葡萄球菌后痊愈的志愿者初筛。利用实施例1中构建表达的Hla和Hla_H35L蛋白，通过ELISA检测方法，筛选血浆中含高滴度α毒素抗体的个体。
具体方法如下：将Hla和Hla_H35L用pH 9.6的磷酸盐包被缓冲液(1.59g Na2CO3，2.93g NaHCO3，pH调节至9.6，溶解后超纯水定容至1000mL，溶解混匀后使用，下文所述的pH 9.6的磷酸盐包被缓冲液同此配方)稀释至2μg/mL，分别以0.1mL/孔的量加入ELISA 96板各个孔中，4℃包被过夜，用封闭液(50 g脱脂奶粉，150mL羊血清，5mL Tween20，50mL 20倍浓度的PBS母液，溶解后超纯水定容至1000mL，下文所述的封闭液同此配方)37℃封闭2h。将上文获得的各个血浆样本以1:50倍比起始稀释(血浆为3μL，封闭液为150μL)，进行3倍梯度系列稀释，作为一抗每孔加入100μL，37℃孵育1h，加入100μL/孔用HRP标记的羊抗人IgG(1：10000稀释)二抗，37℃孵育1h，然后加入100μL/孔底物显色液TMB，37℃避光放置5min后，用2M硫酸中止反应，读取OD450-OD630值。结果显示CS006号志愿者的血浆有良好的结合曲线和量效关系，表明CS006血浆样本中含有大量特异性的抗Hla和Hla_H35L抗体(图2)，选择CS006号样本进行流式分选。
实施例4流式细胞仪分选单个记忆B细胞
根据血清学实验结果，对CS006号样本的PBMC进行流式细胞仪分选以获得单个记忆B细胞(图3)，采用分子标记CD3、CD14、CD16-、CD235a、CD20+、CD27+，Hla_H35L-BV421和Hla_H35L-PE-Cy7对CS006样本的PBMC进行Hla_H35L蛋白特异性的记忆B细胞分选。结合抗原的抗体是随机均匀的分布在记忆B细胞表面，两种荧光标记的Hla_H35L-BV421(蓝色)和Hla_H35L-PE-Cy7(红色)能够随机的结合到特异性的记忆B细胞的抗原的抗体决定簇，发出红色和蓝色两种荧光，Q2-1门为所选取的双阳性的特异性记忆B细胞，选取双阳性的细胞进行后续单细胞PCR分离抗体基因的试验。
实施例5用RT-PCR的方法从单个B细胞中分离抗体可变区基因
反转录合成cDNA第一条链：20μL单细胞裂解液加入含有实施例4获得的双阳性单个B细胞的96孔板，裂解细胞。加入0.5μM的各亚型重链与轻链的恒定区引物(参见CN107760690B中公开的引物信息)与Superscript IV反转录酶(Invitrogen，Carlsbad，CA)，37℃孵育1小时进行逆转录合成cDNA第一条链。再通过两轮PCR扩增分离抗体基因，其中将PCR扩增的VH和VK/L基因产物用1.2％的琼脂糖凝胶电泳进行鉴定，并进行测序。
由此获得特异性靶向Hla_H35L和Hla的多个全人源抗体。
实施例6抗体筛选
通过Overlapping PCR将VH和VK/L基因产物构建连接到具有启动子、抗体恒定区和终止子的完整抗体表达系统的线性DNA片段中，具体来讲：TRN1029、TRN1030、TRN1031、TRN1032、TRN1033的VH基因产物与含有CMV启动子的DNA片段及含有人IgG1的恒定区DNA片段(包括铰链区、CH1、CH2和CH3)通过PCR方式连接；TRN1029、TRN1030、TRN1031的VK基因产物与含有CMV启动子的DNA片段及含有人Kappa的恒定区DNA片段(包括铰链区、C-DOMAIN)通过PCR方式连接；TRN1032、TRN1033的VH基因产物与含有CMV启动子的DNA片段及含有人Lambda的恒定区DNA片段(包括铰链区、C-DOMAIN)通过PCR方式连接。
构建完成后的抗体表达系统的线性DNA片段与PEI共转染293T细胞，转染后6-8小时换新鲜培养基，并在37℃8％CO2培养箱中培养96小时，收集转染上清，15000g离心1小时，进行ELISA检测。Hla和Hla_H35L用包被缓冲液稀释至2μg/mL，各0.1mL/孔加于ELISA 96板中，4℃包被过夜，用封闭液37℃封闭2h。加入作为一抗的离心后上清100μL，37℃孵育1h，用HRP标记的羊抗人IgG(1：10000稀释)二抗37℃孵育1h，加入底物显色液TMB，100μL/孔，37℃避光放置5min后，用50μL的2M硫酸中止反应，读取OD450值。结果显示筛选到与α毒素(Hla和Hla_H35L)结合的17个特异性单克隆抗体(图4)，其中8个为高效结合抗体(结合OD值超过3.0)。Hla036、Hla038、Hla039、Hla072、TRN1029、TRN1030、TRN1031和TRN1033等8个抗体与Hla和Hla_H35L结合都达到仪器检测上线(OD值为3.5)，TRN1021(参见专利CN2021115320943)为无关阴性对照，AR301为阳性对照(参见CN102549013B)。
实施例7构建重组抗体的表达载体
将实施例6中检测的抗体的编码基因利用同源重组克隆的方法连接到pcDNA3.3载体上，构建抗Hla全人源抗体的表达载体，然后将表达载体转化DH5α感受态细菌，在含有氨苄青霉素的平板上37℃培养过 夜，挑取单菌落用特异性引物进行PCR扩增，反应条件为：94℃预变性3min；94℃变性30s，55℃退火30s，72℃延伸100s，28个循环；72℃延伸5min，取5μL PCR产物用1％琼脂糖凝胶电泳检测，对在转化子中鉴定出了含有抗体重链和轻链基因的转化子进行测序验证。
实施例8抗体表达与纯化
将实施例7中测序验证阳性质粒转化DH5α进行大量扩增，快速提取重组质粒后，与转染试剂PEI共转染HEK293I细胞，转染后6-8小时换新鲜培养基，并在37℃8％CO2培养箱中培养96小时，收集细胞上清进行检测。收集转染上清，4000rpm离心1小时，利用蛋白A磁珠对上清液进行纯化；利用SDS-PAGE和Western Blot检验纯化的抗体的表达及纯化情况。非还原型SDS-PAGE和还原型SDS-PAGE凝胶电泳检测的区别在于需要在进行还原型电泳的抗体样品中加入含β-巯基乙醇的上样缓冲液，并在沸水浴中煮5min，用PBS补足至10-15μL后上样。SDS-PAGE和Western Blot检测结果如图5所示。图5左图为检测非还原性样品的结果，右图为检测还原性样品的结果，抗体还原后共有两条带，抗体重链和轻链还原后的分子量符合一般抗体大小；非还原抗体大小为180kD，符合一般完整抗体的大小，可见抗体组装正确。
实施例9 ELISA检测抗体的结合活性
以重组表达的Hla_H35L和Hla分别为抗原进行ELISA检测，将相应抗原稀释为2μg/mL，每孔100uL加入96孔板中，4℃过夜包被，用封闭液37℃封闭2个小时。对获得的各个Hla抗体分别进行倍比稀释，起始浓度10μg/mL，3倍稀释12个梯度，然后将各个梯度的抗体加入96孔板，37℃孵育1h。用PBST缓冲液洗涤，每孔加100μL用封闭液按1:10000进行稀释的Goat-Anti-IgG-Fab-HRP(二抗)，37℃孵育1h。PBST缓冲液洗涤，避光，每孔加TMB显色液100μL，37℃放置5min，立即用50μL的2M H2SO4终止。双波长450-630nm检测OD值。
结果如图6所示，全人源Hla单克隆抗体TRN1029、TRN1030、TRN1031、TRN1032、TRN1033均能与Hla和Hla_H35L结合，与Hla结合的EC50分别为0.199μg/mL、0.165μg/mL、0.017μg/mL、24.29μg/mL、0.009μg/mL；TRN1029、TRN1030、TRN1031、TRN1033与Hla_H35L结合的EC50分别为0.062μg/mL、0.068μg/mL、0.016μg/mL和0.013μg/mL(表9)。
表9：表达抗体与Hla和Hla_H35L结合的EC50
注：NA表示该数值无法计算
实施例10表面等离子共振技术(SPR)测定抗体与抗原的亲和力
使用表面等离子体共振测定来测量抗体的亲和力、动力学常数。用人抗体捕获试剂盒(Cytiva,29234600)制备抗人IgG(Fc)抗体芯片，用氨基偶联的方法把抗人IgG-(Fc)抗体固定在CM5传感器芯片(Cytiva，BR100530)表面上。全人源抗Hla抗体作为配体捕获在传感器芯片上的2通道上(1通道为参照通道，2通道为样品通道)，使抗体捕获水平达到200RU左右。配置不同浓度的Hla蛋白溶液，起始浓度75nM，2倍稀释5个梯度，设置18.75nM为重复浓度，设0浓度(TRN1033起始浓度为300nM)，并作为分析物注入1，2通道，以30μL/min的速度流经芯片的1、2通道，分析物结合时间90s，随后采用缓冲液进行解离，解离时间为300s。再生溶液为3M MgCl2，再生时间为30s。抗体分子与抗原经过捕获、结合、解离和再生为一次循环，每个抗体分子经过7次循环，产生的结果用Biacore 8K Evaluation Software软件进行计算分析得到亲和力动力学常数。
结果如表10所示，TRN1029、TRN1030、TRN1031、TRN1032、TRN1033和Hla亲和力(KD)大小 分别为1.17nM、0.943nM、0.965nM、52.1nM和1.82nM，其中TRN1029、TRN1030、TRN1031、TRN1033解离平稳较慢，但是TRN1032解离较快，这也是其亲和力只有52.1nM的原因。-
表10：抗Hla毒素单抗亲和力汇总表
实施例11不同全人源抗Hla抗体识别抗原表位的检测
通过表面等离子共振技术(SPR)研究Hla蛋白和2种不同抗Hla蛋白单克隆抗体三者之间的关系，检测本申请获得的全人源抗Hla抗体所识别的抗原表位以及相互关系。
将Hla蛋白共价结合于CM5传感器芯片2通道上，响应值为大约300RU(1通道为参照空白通道，2通道作为样品通道)，如图7所述，进行4轮循环，每个循环包括样品A上样、样品B上样(样品A和样品B为抗Hla蛋白抗体)、再生三个步骤。
第一轮循环先在1和2通道上结合抗Hla蛋白抗体A，流速为30μL/min、结合时间190s，再在1和2通道上结合抗Hla蛋白抗体A，流速为30μL/min、结合时间190s；再生条件：选择pH为1.5的Glycine溶液，流速为30μL/min、再生时间为180s。
第二轮循环先在1和2通道上结合抗Hla蛋白抗体A，流速为30μL/min、结合时间190s，再在1和2通道上结合抗Hla蛋白抗体B，流速为30μL/min、结合时间190s，观察第一轮和第二轮循环结合值变化。再生条件：选择pH为1.5的Glycine溶液，流速为30μL/min、再生时间为180s。
第三轮循环先在1和2通道上结合抗Hla蛋白抗体B，流速为30μL/min、结合时间190s，再在1和2通道上结合抗Hla蛋白抗体B，流速为30μL/min、结合时间190s；再生条件：选择pH为1.5的甘氨酸溶液，流速为30μL/min、再生时间为180s。
第四轮循环先在1和2通道上结合抗Hla蛋白抗体B，流速为30μL/min、结合时间190s，再在1和2通道上结合抗Hla蛋白抗体A，流速为30μL/min、结合时间190s，观察第三轮和第四轮循环结合值变化。再生条件：选择pH为1.5的甘氨酸溶液，流速为30μL/min、再生时间为180s。
本实验采用了AR301阳性对照，AR301(tosatoxumab,KBSA301)是Aridis公司的针对金黄色葡萄球菌α-毒素的用于治疗感染的全人源IgG1lambda单克隆抗体，其与金黄色葡萄球菌α-毒素的N端抗原表位的特异性结合。
结果如图8所示，AR301和TRN1033之间、AR301和TRN1029之间、AR301和TRN1030之间、TRN1033和TRN1029之间、TRN1033与TRN1030之间都不能相互阻断与抗原的结合，说明上述每组抗体之间识别金黄色葡萄球菌α-毒素不同的表位。
实验发现TRN1031和TRN1032在本实验中不能发现其与其他抗体竞争结合的情况，推测TRN1031和TRN1032可能识别金黄色葡萄球菌α-毒素另外两个新的抗原表位。
实施例12 Hla抗体的中和活性研究
将50μL浓度为10μg/mL的Hla分别与不同浓度的全人源Hla抗体及阳性对照抗体(MEDI4893(专利CN103443285B)、AR301(专利CN102549013B)、TRN1016(专利CN109400704B))，抗体的起始浓度为40μg/mL，进行两倍梯度稀释8个稀释度，在37℃预孵育0.5h，在Hla与抗体混合物中加入50μL的2％人红细胞，空白对照孔仅含有红细胞和Hla，没有加入抗体，37℃孵育2h，随后用数码相机拍照观察并记录红细胞溶血情况。当红细胞发生溶血反应时，红细胞将失去其作为细胞的大小和形状，漏出的血红蛋白将周围的溶液(例如血浆或生理盐水)染红。因此，溶血后的含红细胞溶液将变成红色的透明液体。
结果如图9所示，从12个抗体中发现有5个中和抗体(TRN1029、TRN1030、TRN1031、TRN1032、 TRN1033)能够高效中和Hla的溶血作用，保护人红细胞不被Hla溶血。同阳性对照抗体比较，其中TRN1032相比较于对照抗体MEDI4893和AR301的中和效价高16倍，TRN1030和TRN1031与对照抗体MEDI4893的中和效价相当，且效价优于AR301，TRN1029、TRN1033与AR301的中和效价相当。而TRN1016则相比较于其他几个中和抗体只有在高浓度40μg/mL时有部分中和作用，其效果类似于Hla036、Hla038、Hla039。
实施例13 Hla抗体在小鼠体内的中和活性研究
将购买自珠海百试通生物科技有限公司ICR小鼠随机分组，每组8只，根据毒素滴定结果，Hla毒素剂量选择2mg/kg。将不同浓度的TRN1016、TRN1029、TRN1031或MEDI4893分别与Hla混合，37℃孵育30min后，混合物分别按照10μL/g的体积腹腔注射ICR小鼠(表9)。观察小鼠生存时间并计算生存率，结果如图11所示。中和保护性评价实验表明：TRN1029、TRN1031和MEDI4893(N＝8)在2mg/kg能够完全保护小鼠不死亡，同MEDI4893相同浓度的比较结果显示TRN1029和TRN1031保护效果好于MEDI4893，在1mg/kg抗体剂量下，TRN1029和TRN1031的小鼠存活率都比MEDI4893高10％-25％。在1.5mg/kg时TRN1016只有62.5％的保护效果，在CN109400704B的实施例12中Hla毒素选用的剂量为17.5μg(为0.875mg/kg)，而本研究采用的Hla毒素剂量为2mg/kg，并且TRN1016最高剂量1.5mg/kg，小于Hla毒素剂量的2mg/kg，所以只有62.5％的保护相关，在CN109400704B的实施例12中10μg和5μg的TRN1016也只有50％生物保护效果，与本研究的结果基本上符合；TRN1016在低剂量时保护效果好于MEDI4893。
表11：中和实验分组和样品稀释配置表
实施例14 Hla毒素攻毒实验保护性评价
将ICR小鼠随机分组，每组9只，根据毒素滴定结果，Hla毒素剂量选择2mg/kg，分别腹腔注射按照10μL/g的体积不同浓度的TRN1029、TRN1031或MEDI4893，2h后，向每只小鼠腹腔注射按照10μL/g的体积浓度为400μg/mL(2mg/kg)的Hla(表12)，观察小鼠生存时间并计算生存率，结果如图11所示。攻毒实验保护性评价实验表明：TRN1029、TRN1031和MEDI4893(N＝9)在4mg/kg能够完全保护小鼠不死 亡，同相同浓度的MEDI4893比较结果显示：TRN1029和TRN1031保护效果好于MEDI4893，在2mg/kg、1mg/kg和0.5mg/kg的抗体剂量下，TRN1029和TRN1031的小鼠存活率都比MEDI4893高。
表12：攻毒实验分组和样品稀释配置表
实施例15抗体TRN1029突变体的ELISA检测结果
将表6和表8中共106组TRN1029突变体进行ELISA检测。将Ag030蛋白200ng/孔包板，4℃包被过夜；37℃封闭2h；待测抗体上样量1μg/孔起始，3倍稀释12个梯度；阳性对照为TRN1029，阴性对照为TNM001DS(该阴性对照参见中国专利202111532094.3所公开的抗体TRN1021)；二抗为Anti-Human IgG(H+L)，HRP Conjugate(1:10000稀释)。读取OD450-OD630值，结果如下。
表13：抗体TRN1029突变体的ELISA检测结果


Claims (15)

  1. 一种特异性结合Hla的抗体或其抗原结合片段，其包含：
    1)包含选自SEQ ID NO:3、47-54、96、102所示的HCDR1或其任何变体、包含选自SEQ ID NO:4、55-62、97、103所示的HCDR2或其任何变体和包含选自SEQ ID NO:5、63-77、98、101、104所示的HCDR3或其任何变体；和包含选自SEQ ID NO:6、78-83、99、105所示的LCDR1或其任何变体、包含选自SEQ ID NO:7、84-86所示的LCDR2或其任何变体和包含选自SEQ ID NO:8、87-95、100、106所示的LCDR3或其任何变体；
    可选地，包含SEQ ID NO:3所示的HCDR1或其任何变体、包含SEQ ID NO:4所示的HCDR2或其任何变体和包含SEQ ID NO:5所示的HCDR3或其任何变体；和包含SEQ ID NO:6所示的LCDR1或其任何变体、包含SEQ ID NO:7所示的LCDR2或其任何变体和包含SEQ ID NO:8所示的LCDR3或其任何变体；
    2)包含SEQ ID NO:9所示的HCDR1或其任何变体、包含SEQ ID NO:10所示的HCDR2或其任何变体和包含SEQ ID NO:11所示的HCDR3或其任何变体；和包含SEQ ID NO:12所示的LCDR1或其任何变体、包含SEQ ID NO:13所示的LCDR2或其任何变体和包含SEQ ID NO:14所示的LCDR3或其任何变体；
    3)包含SEQ ID NO:15所示的HCDR或其任何变体、包含SEQ ID NO:16所示的HCDR2或其任何变体和包含SEQ ID NO:17所示的HCDR3或其任何变体；和包含SEQ ID NO:18所示的LCDR1或其任何变体、包含SEQ ID NO:19所示的LCDR2或其任何变体和包含SEQ ID NO:20所示的LCDR3或其任何变体；
    4)包含SEQ ID NO:21所示的HCDR1或其任何变体、包含SEQ ID NO:22所示的HCDR2或其任何变体和包含SEQ ID NO:23所示的HCDR3或其任何变体；和包含SEQ ID NO:24所示的LCDR1或其任何变体、包含SEQ ID NO:25所示的LCDR2或其任何变体和包含SEQ ID NO:26所示的LCDR3或其任何变体；或
    5)包含SEQ ID NO:27所示的HCDR1或其任何变体、包含SEQ ID NO:28所示的HCDR2或其任何变体和包含SEQ ID NO:29所示的HCDR3或其任何变体；和包含SEQ ID NO:30所示的LCDR1或其任何变体、包含SEQ ID NO:31所示的LCDR2或其任何变体和包含SEQ ID NO:32所示的LCDR3或其任何变体。
  2. 根据权利要求1所述的特异性结合Hla的抗体或其抗原结合片段，其包含：
    1)重链互补决定区HCDR1，其包含氨基酸序列SEQ ID NO:3；
    2)重链互补决定区HCDR2，其包含氨基酸序列SEQ ID NO:4；
    3)重链互补决定区HCDR3，其包含氨基酸序列SEQ ID NO:5；
    4)轻链互补决定区LCDR1，其包含氨基酸序列：
    X1X2X3X4X5X6(SEQ ID NO:246),
    其中，X1为Q、N、E或D；
    X2为S、T、A或I；
    X3为I、L、V、M、A、F、正亮氨酸、G或N；
    X4为T、V、S、R、K或I；
    X5为T、V、S、K或G；并且
    X6为N、Q、H、D、K、R或S；
    可选地，其中X1为Q、N、E或D，X2为S，X3为I，X4为T，X5为T，X6为N；可选地，其中X1为N，X2为S，X3为I，X4为T，X5为T，X6为N；
    可选地，其中X1为Q，X2为S、T、A或I，X3为I，X4为T，X5为T，X6为N；可选地，其中X1为Q，X2为T，X3为I，X4为T，X5为T，X6为N；
    可选地，其中X1为Q，X2为S，X3为I、L、V、M、A、F、正亮氨酸、G或N，X4为T，X5为T，X6为N；可选地，其中X1为Q，X2为S，X3为L，X4为T，X5为T，X6为N；
    可选地，其中X1为Q，X2为S，X3为I，X4为T、V、S、R、K或I，X5为T，X6为N；可选地，其中X1为Q，X2为S，X3为I，X4为S，X5为T，X6为N；
    可选地，其中X1为Q，X2为S，X3为I，X4为T，X5为T、V、S、K或G，X6为N；可选地，其中X1为Q，X2为S，X3为I，X4为T，X5为S，X6为N；
    可选地，其中X1为Q，X2为S，X3为I，X4为T，X5为T，X6为N、Q、H、D、K、R或S；可选地，其中X1为Q，X2为S，X3为I，X4为T，X5为T，X6为Q；
    可选地，其中X1为Q，X2为S、T、A或I，X3为I，X4为T，X5为T、V、S、K或G，X6为N；可选地，其中X1为Q，X2为T，X3为I，X4为T，X5为S，X6为N；
    5)轻链互补决定区LCDR2，其包含氨基酸序列：
    X1X2X3(SEQ ID NO:247),
    其中，X1为G、A、S或T；
    X2为A、V、L、I、D或N；并且
    X3为S、T、N或D；
    可选地，其中X1为G、A、S或T，X2为A，X3为S；可选地，其中X1为A，X2为A，X3为S；
    可选地，其中X1为G，X2为A、V、L、I、D或N，X3为S；可选地，其中X1为G，X2为V，X3为S；
    可选地，其中X1为G，X2为A，X3为S、T、N或D；可选地，其中X1为G，X2为A，X3为T；
    6)轻链互补决定区LCDR3，其包含氨基酸序列：
    QQX1X2X3X4X5X6X7(SEQ ID NO:248),
    其中，X1为Y、W、F、T、S或D；
    X2为H、N、Q、K、R、F或D；
    X3为N、Q、H、D、K、R或T；
    X4为W、Y、F或S；
    X5为P、A、D或L；
    X6为L、I、V、M、A、F、正亮氨酸、Y、P、D或G；并且
    X7为T、V、S、L或G；
    可选地，其中X1为Y、W、F、T、S或D，X2为H，X3为N，X4为W，X5为P，X6为L，X7为T；可选地，其中X1为F，X2为H，X3为N，X4为W，X5为P，X6为L，X7为T；
    可选地，其中X1为Y，X2为H、N、Q、K、R、F或D，X3为N，X4为W，X5为P，X6为L，X7为T；可选地，其中X1为Y，X2为R，X3为N，X4为W，X5为P，X6为L，X7为T；
    可选地，其中X1为Y，X2为H，X3为N、Q、H、D、K、R或T，X4为W，X5为P，X6为L，X7为T；可选地，其中X1为Y，X2为H，X3为Q，X4为W，X5为P，X6为L，X7为T；
    可选地，其中X1为Y，X2为H，X3为N，X4为W、Y、F或S，X5为P，X6为L，X7为T；可选地，其中X1为Y，X2为H，X3为N，X4为Y，X5为P，X6为L，X7为T；
    可选地，其中X1为Y，X2为H，X3为N，X4为W，X5为P、A、D或L，X6为L，X7为T；可选地，其中X1为Y，X2为H，X3为N，X4为W，X5为A，X6为L，X7为T；
    可选地，其中X1为Y，X2为H，X3为N，X4为W，X5为P，X6为L、I、V、M、A、F、正亮氨酸、Y、P、D或G，X7为T；可选地，其中X1为Y，X2为H，X3为N，X4为W，X5为P，X6为I，X7为T；
    可选地，其中X1为Y，X2为H，X3为N，X4为W，X5为P，X6为L，X7为T、V、S、L或G；可选地，其中X1为Y，X2为H，X3为N，X4为W，X5为P，X6为L，X7为S。
  3. 根据权利要求1所述的特异性结合Hla的抗体或其抗原结合片段，其包含：
    1)重链互补决定区HCDR1，其包含氨基酸序列：
    X1X2X3X4X5X6X7X8(SEQ ID NO:243),
    其中，X1为G或A；
    X2为F、W、L、V、I、A、Y或G；
    X3为T、V、S或I；
    X4为F、W、L、V、I、A或Y；
    X5为S、T、R、K或G；
    X6为S、T、Q、D、N或E；
    X7为Y、W、F、T、S、H或M；并且
    X8为D、E、A、Y或R；
    可选地，其中X1为G或A，X2为F，X3为T，X4为F，X5为S，X6为S，X7为Y，X8为D；可选地，其中X1为A，X2为F，X3为T，X4为F，X5为S，X6为S，X7为Y，X8为D；
    可选地，其中X1为G，X2为F、W、L、V、I、A、Y或G，X3为T，X4为F，X5为S，X6为S，X7为Y，X8为D；可选地，其中X1为G，X2为Y，X3为T，X4为F，X5为S，X6为S，X7为Y，X8为D；
    可选地，其中X1为G，X2为F，X3为T、V、S或I，X4为F，X5为S，X6为S，X7为Y，X8为D；可选地，其中X1为G，X2为F，X3为S，X4为F，X5为S，X6为S，X7为Y，X8为D；
    可选地，其中X1为G，X2为F，X3为T，X4为F、W、L、V、I、A或Y，X5为S，X6为S，X7为Y，X8为D；可选地，其中X1为G，X2为F，X3为T，X4为Y，X5为S，X6为S，X7为Y，X8为D；
    可选地，其中X1为G，X2为F，X3为T，X4为F，X5为S、T、R、K或G，X6为S，X7为Y，X8为D；可选地，其中X1为G，X2为F，X3为T，X4为F，X5为T，X6为S，X7为Y，X8为D；
    可选地，其中X1为G，X2为F，X3为T，X4为F，X5为S，X6为S、T、R、K或G，X7为Y，X8为D；可选地，其中X1为G，X2为F，X3为T，X4为F，X5为S，X6为T，X7为Y，X8为D；
    可选地，其中X1为G，X2为F，X3为T，X4为F，X5为S，X6为S，X7为Y、W、F、T、S、H或M，X8为D；可选地，其中X1为G，X2为F，X3为T，X4为F，X5为S，X6为S，X7为F，X8为D；
    可选地，其中X1为G，X2为F，X3为T，X4为F，X5为S，X6为S，X7为Y，X8为D、E、A、Y或R；可选地，其中X1为G，X2为F，X3为T，X4为F，X5为S，X6为S，X7为Y，X8为E；
    可选地，其中X1为G，X2为F、W、L、V、I、A、Y或G，X3为T，X4为F，X5为S，X6为S、T、R、K或G，X7为Y，X8为D；可选地，其中X1为G，X2为Y，X3为T，X4为F，X5为S，X6为T，X7为Y，X8为D；
    2)重链互补决定区HCDR2，其包含氨基酸序列：
    X1X2X3X4X5X6X7X8(SEQ ID NO:244),
    其中，X1为M、L、F或I；
    X2为S、T、I、Y或Q；
    X3为Y、W、F、T、S、P、K或R；
    X4为D、E、N、G或F；
    X5为G、A、L或E；
    X6为S、T或N；
    X7为Y、W、F、T、S、G或I；并且
    X8为K、R、Q、N、H、P、A或V；
    可选地，其中X1为M、L、F或I，X2为S，X3为Y，X4为D，X5为G，X6为S，X7为Y，X8为K；可选地，其中X1为L，X2为S，X3为Y，X4为D，X5为G，X6为S，X7为Y，X8为K；
    可选地，其中X1为M，X2为S、T、I、Y或Q，X3为Y，X4为D，X5为G，X6为S，X7为Y，X8为K；可选地，其中X1为M，X2为T，X3为Y，X4为D，X5为G，X6为S，X7为Y，X8为K；
    可选地，其中X1为M，X2为S，X3为Y、W、F、T、S、P、K或R，X4为D，X5为G，X6为S，X7为Y，X8为K；可选地，其中X1为M，X2为S，X3为F，X4为D，X5为G，X6为S，X7为Y，X8为K；
    可选地，其中X1为M，X2为S，X3为Y，X4为D、E、N、G或F，X5为G，X6为S，X7为Y，X8为K；可选地，其中X1为M，X2为S，X3为Y，X4为E，X5为G，X6为S，X7为Y，X8为K；
    可选地，其中X1为M，X2为S，X3为Y，X4为D，X5为G、A、L或E，X6为S，X7为Y，X8为K；可选地，其中X1为M，X2为S，X3为Y，X4为D，X5为A，X6为S，X7为Y，X8为K；
    可选地，其中X1为M，X2为S，X3为Y，X4为D，X5为G，X6为S、T或N，X7为Y，X8为K；可选地，其中X1为M，X2为S，X3为Y，X4为D，X5为G，X6为T，X7为Y，X8为K；
    可选地，其中X1为M，X2为S，X3为Y，X4为D，X5为G，X6为S，X7为Y、W、F、T、S、G或I，X8为K；可选地，其中X1为M，X2为S，X3为Y，X4为D，X5为G，X6为S，X7为F，X8为K；
    可选地，其中X1为M，X2为S，X3为Y，X4为D，X5为G，X6为S，X7为Y，X8为K、R、Q、N、H、P、A或V；可选地，其中X1为M，X2为S，X3为Y，X4为D，X5为G，X6为S，X7为Y，X8为R；
    可选地，其中X1为M，X2为S、T、I、Y或Q，X3为Y、W、F、T、S、P、K或R，X4为D，X5为G，X6为S，X7为Y、W、F、T、S、G或I，X8为K；可选地，其中X1为M，X2为T，X3为F，X4为D，X5为G，X6为S，X7为F，X8为K；
    3)重链互补决定区HCDR3，其包含氨基酸序列：
    AX1X2RX3SX4X5X6X7X8GX9X10X11(SEQ ID NO:245),
    其中，X1为K、R、Q、N、H或T；
    X2为P、A、D或E；
    X3为G、A、W、P、H或V；
    X4为H、N、Q、K、R、S、F或D；
    X5为Y、W、F、T、S、A或L；
    X6为D、E、N、A、H、S或G；
    X7为T、V、S、G或D；
    X8为S、T、D、G或R；
    X9为F、W、L、V、I、A、Y、P或T；
    X10为D、E、N、Q、F或Y；并且
    X11为Y、W、F、T、S、V、L或D；
    可选地，其中X1为K、R、Q、N、H或T，X2为P，X3为G，X4为H，X5为Y，X6为D，X7为T，X8为S，X9为F，X10为D，X11为Y；可选地，其中X1为R，X2为P，X3为G，X4为H，X5为Y，X6为D，X7为T，X8为S，X9为F，X10为D，X11为Y；
    可选地，其中X1为K，X2为P、A、D或E，X3为G，X4为H，X5为Y，X6为D，X7为T，X8为S，X9为F，X10为D，X11为Y；可选地，其中X1为K，X2为A，X3为G，X4为H，X5为Y，X6为D，X7为T，X8为S，X9为F，X10为D，X11为Y；
    可选地，其中X1为K，X2为P，X3为G、A、W、P、H或V，X4为H，X5为Y，X6为D，X7为T，X8为S，X9为F，X10为D，X11为Y；可选地，其中X1为K，X2为P，X3为A，X4为H，X5为Y，X6为D，X7为T，X8为S，X9为F，X10为D，X11为Y；
    可选地，其中X1为K，X2为P，X3为G，X4为H、N、Q、K、R、S、F或D，X5为Y，X6为D，X7为T，X8为S，X9为F，X10为D，X11为Y；可选地，其中X1为K，X2为P，X3为G，X4为R，X5为Y，X6为D，X7为T，X8为S，X9为F，X10为D，X11为Y；
    可选地，其中X1为K，X2为P，X3为G，X4为H，X5为Y、W、F、T、S、A或L，X6为D，X7为T，X8为S，X9为F，X10为D，X11为Y；可选地，其中X1为K，X2为P，X3为G，X4为H，X5为F，X6为D，X7为T，X8为S，X9为F，X10为D，X11为Y；
    可选地，其中X1为K，X2为P，X3为G，X4为H，X5为Y，X6为D、E、N、A、H、S或G，X7为T，X8为S，X9为F，X10为D，X11为Y；可选地，其中X1为K，X2为P，X3为G，X4为H，X5为Y，X6为E，X7为T，X8为S，X9为F，X10为D，X11为Y；
    可选地，其中X1为K，X2为P，X3为G，X4为H，X5为Y，X6为D，X7为T、V、S、G或D，X8为S，X9为F，X10为D，X11为Y；可选地，其中X1为K，X2为P，X3为G，X4为H，X5为Y，X6为D，X7为S，X8为S，X9为F，X10为D，X11为Y；
    可选地，其中X1为K，X2为P，X3为G，X4为H，X5为Y，X6为D，X7为T，X8为S、T、D、G或 R，X9为F，X10为D，X11为Y；可选地，其中X1为K，X2为P，X3为G，X4为H，X5为Y，X6为D，X7为T，X8为T，X9为F，X10为D，X11为Y；
    可选地，其中X1为K，X2为P，X3为G，X4为H，X5为Y，X6为D，X7为T，X8为S，X9为F、W、L、V、I、A、Y、P或T，X10为D，X11为Y；可选地，其中X1为K，X2为P，X3为G，X4为H，X5为Y，X6为D，X7为T，X8为S，X9为Y，X10为D，X11为Y；
    可选地，其中X1为K，X2为P，X3为G，X4为H，X5为Y，X6为D，X7为T，X8为S，X9为F，X10为D、E、N、Q、F或Y，X11为Y；可选地，其中X1为K，X2为P，X3为G，X4为H，X5为Y，X6为D，X7为T，X8为S，X9为F，X10为E，X11为Y；
    可选地，其中X1为K，X2为P，X3为G，X4为H，X5为Y，X6为D，X7为T，X8为S，X9为F，X10为D，X11为Y、W、F、T、S、V、L或D；可选地，其中X1为K，X2为P，X3为G，X4为H，X5为Y，X6为D，X7为T，X8为S，X9为F，X10为D，X11为F；
    4)轻链互补决定区LCDR1，其包含氨基酸序列SEQ ID NO:6；
    5)轻链互补决定区LCDR2，其包含氨基酸序列SEQ ID NO:7：
    6)轻链互补决定区LCDR3，其包含氨基酸序列SEQ ID NO:8。
  4. 根据权利要求1-3任一项所述的抗体或其抗原结合片段，其包含：
    1)SEQ ID NO:47所示的HCDR1，SED ID NO:4所示的HCDR2和SED ID NO:5所示的HCDR3；和/或，SEQ ID NO:6所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:8所示的LCDR3；
    2)SEQ ID NO:48所示的HCDR1，SED ID NO:4所示的HCDR2和SED ID NO:5所示的HCDR3；和/或，SEQ ID NO:6所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:8所示的LCDR3；
    3)SEQ ID NO:49所示的HCDR1，SED ID NO:4所示的HCDR2和SED ID NO:5所示的HCDR3；和/或，SEQ ID NO:6所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:8所示的LCDR3；
    4)SEQ ID NO:50所示的HCDR1，SED ID NO:4所示的HCDR2和SED ID NO:5所示的HCDR3；和/或，SEQ ID NO:6所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:8所示的LCDR3；
    5)SEQ ID NO:51所示的HCDR1，SED ID NO:4所示的HCDR2和SED ID NO:5所示的HCDR3；和/或，SEQ ID NO:6所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:8所示的LCDR3；
    6)SEQ ID NO:52所示的HCDR1，SED ID NO:4所示的HCDR2和SED ID NO:5所示的HCDR3；和/或，SEQ ID NO:6所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:8所示的LCDR3；
    7)SEQ ID NO:53所示的HCDR1，SED ID NO:4所示的HCDR2和SED ID NO:5所示的HCDR3；和/或，SEQ ID NO:6所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:8所示的LCDR3；
    8)SEQ ID NO:54所示的HCDR1，SED ID NO:4所示的HCDR2和SED ID NO:5所示的HCDR3；和/或，SEQ ID NO:6所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:8所示的LCDR3；
    9)SEQ ID NO:3所示的HCDR1，SED ID NO:55所示的HCDR2和SED ID NO:5所示的HCDR3；和/或，SEQ ID NO:6所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:8所示的LCDR3；
    10)SEQ ID NO:3所示的HCDR1，SED ID NO:56所示的HCDR2和SED ID NO:5所示的HCDR3；和/或，SEQ ID NO:6所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:8所示的LCDR3；
    11)SEQ ID NO:3所示的HCDR1，SED ID NO:57所示的HCDR2和SED ID NO:5所示的HCDR3；和/或，SEQ ID NO:6所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:8所示的LCDR3；
    12)SEQ ID NO:3所示的HCDR1，SED ID NO:58所示的HCDR2和SED ID NO:5所示的HCDR3；和/或，SEQ ID NO:6所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:8所示的LCDR3；
    13)SEQ ID NO:3所示的HCDR1，SED ID NO:59所示的HCDR2和SED ID NO:5所示的HCDR3；和/或，SEQ ID NO:6所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:8所示的LCDR3；
    14)SEQ ID NO:3所示的HCDR1，SED ID NO:60所示的HCDR2和SED ID NO:5所示的HCDR3；和/或，SEQ ID NO:6所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:8所示的LCDR3；
    15)SEQ ID NO:3所示的HCDR1，SED ID NO:61所示的HCDR2和SED ID NO:5所示的HCDR3；和/或，SEQ ID NO:6所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:8所示的LCDR3；
    16)SEQ ID NO:3所示的HCDR1，SED ID NO:62所示的HCDR2和SED ID NO:5所示的HCDR3；和/或，SEQ ID NO:6所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:8所示的LCDR3；
    17)SEQ ID NO:3所示的HCDR1，SED ID NO:4所示的HCDR2和SED ID NO:63所示的HCDR3；和/或，SEQ ID NO:6所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:8所示的LCDR3；
    18)SEQ ID NO:3所示的HCDR1，SED ID NO:4所示的HCDR2和SED ID NO:64所示的HCDR3；和/或，SEQ ID NO:6所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:8所示的LCDR3；
    19)SEQ ID NO:3所示的HCDR1，SED ID NO:4所示的HCDR2和SED ID NO:65所示的HCDR3；和/或，SEQ ID NO:6所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:8所示的LCDR3；
    20)SEQ ID NO:3所示的HCDR1，SED ID NO:4所示的HCDR2和SED ID NO:66所示的HCDR3；和/或，SEQ ID NO:6所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:8所示的LCDR3；
    21)SEQ ID NO:3所示的HCDR1，SED ID NO:4所示的HCDR2和SED ID NO:67所示的HCDR3；和/或，SEQ ID NO:6所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:8所示的LCDR3；
    22)SEQ ID NO:3所示的HCDR1，SED ID NO:4所示的HCDR2和SED ID NO:68所示的HCDR3；和/或，SEQ ID NO:6所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:8所示的LCDR3；
    23)SEQ ID NO:3所示的HCDR1，SED ID NO:4所示的HCDR2和SED ID NO:69所示的HCDR3；和/或，SEQ ID NO:6所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:8所示的LCDR3；
    24)SEQ ID NO:3所示的HCDR1，SED ID NO:4所示的HCDR2和SED ID NO:70所示的HCDR3；和/或，SEQ ID NO:6所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:8所示的LCDR3；
    25)SEQ ID NO:3所示的HCDR1，SED ID NO:4所示的HCDR2和SED ID NO:71所示的HCDR3；和/或，SEQ ID NO:6所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:8所示的LCDR3；
    26)SEQ ID NO:3所示的HCDR1，SED ID NO:4所示的HCDR2和SED ID NO:72所示的HCDR3；和/或，SEQ ID NO:6所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:8所示的LCDR3；
    27)SEQ ID NO:3所示的HCDR1，SED ID NO:4所示的HCDR2和SED ID NO:73所示的HCDR3；和/或，SEQ ID NO:6所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:8所示的LCDR3；
    28)SEQ ID NO:3所示的HCDR1，SED ID NO:4所示的HCDR2和SED ID NO:74所示的HCDR3；和/或，SEQ ID NO:6所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:8所示的LCDR3；
    29)SEQ ID NO:3所示的HCDR1，SED ID NO:4所示的HCDR2和SED ID NO:75所示的HCDR3；和/或，SEQ ID NO:6所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:8所示的LCDR3；
    30)SEQ ID NO:3所示的HCDR1，SED ID NO:4所示的HCDR2和SED ID NO:76所示的HCDR3；和/或，SEQ ID NO:6所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:8所示的LCDR3；
    31)SEQ ID NO:3所示的HCDR1，SED ID NO:4所示的HCDR2和SED ID NO:77所示的HCDR3；和/或，SEQ ID NO:6所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:8所示的LCDR3；
    32)SEQ ID NO:3所示的HCDR1，SED ID NO:4所示的HCDR2和SED ID NO:5所示的HCDR3；和/或，SEQ ID NO:78所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:8所示的LCDR3；
    33)SEQ ID NO:3所示的HCDR1，SED ID NO:4所示的HCDR2和SED ID NO:5所示的HCDR3；和/或，SEQ ID NO:79所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:8所示的LCDR3；
    34)SEQ ID NO:3所示的HCDR1，SED ID NO:4所示的HCDR2和SED ID NO:5所示的HCDR3；和/或，SEQ ID NO:80所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:8所示的LCDR3；
    35)SEQ ID NO:3所示的HCDR1，SED ID NO:4所示的HCDR2和SED ID NO:5所示的HCDR3；和/或，SEQ ID NO:81所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:8所示的LCDR3；
    36)SEQ ID NO:3所示的HCDR1，SED ID NO:4所示的HCDR2和SED ID NO:5所示的HCDR3；和/或，SEQ ID NO:82所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:8所示的LCDR3；
    37)SEQ ID NO:3所示的HCDR1，SED ID NO:4所示的HCDR2和SED ID NO:5所示的HCDR3；和/或，SEQ ID NO:83所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:8所示的LCDR3；
    38)SEQ ID NO:3所示的HCDR1，SED ID NO:4所示的HCDR2和SED ID NO:5所示的HCDR3；和/或，SEQ ID NO:6所示的LCDR1，SED ID NO:84所示的LCDR2和SED ID NO:8所示的LCDR3；
    39)SEQ ID NO:3所示的HCDR1，SED ID NO:4所示的HCDR2和SED ID NO:5所示的HCDR3；和/或，SEQ ID NO:6所示的LCDR1，SED ID NO:85所示的LCDR2和SED ID NO:8所示的LCDR3；
    40)SEQ ID NO:3所示的HCDR1，SED ID NO:4所示的HCDR2和SED ID NO:5所示的HCDR3；和/或，SEQ ID NO:6所示的LCDR1，SED ID NO:86所示的LCDR2和SED ID NO:8所示的LCDR3；
    41)SEQ ID NO:3所示的HCDR1，SED ID NO:4所示的HCDR2和SED ID NO:5所示的HCDR3；和/或，SEQ ID NO:6所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:87所示的LCDR3；
    42)SEQ ID NO:3所示的HCDR1，SED ID NO:4所示的HCDR2和SED ID NO:5所示的HCDR3；和/或，SEQ ID NO:6所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:88所示的LCDR3；
    43)SEQ ID NO:3所示的HCDR1，SED ID NO:4所示的HCDR2和SED ID NO:5所示的HCDR3；和/或，SEQ ID NO:6所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:89所示的LCDR3；
    44)SEQ ID NO:3所示的HCDR1，SED ID NO:4所示的HCDR2和SED ID NO:5所示的HCDR3；和/或，SEQ ID NO:6所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:90所示的LCDR3；
    45)SEQ ID NO:3所示的HCDR1，SED ID NO:4所示的HCDR2和SED ID NO:5所示的HCDR3；和/或，SEQ ID NO:6所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:91所示的LCDR3；
    46)SEQ ID NO:3所示的HCDR1，SED ID NO:4所示的HCDR2和SED ID NO:5所示的HCDR3；和/或，SEQ ID NO:6所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:92所示的LCDR3；
    47)SEQ ID NO:3所示的HCDR1，SED ID NO:4所示的HCDR2和SED ID NO:5所示的HCDR3；和/或，SEQ ID NO:6所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:93所示的LCDR3；
    48)SEQ ID NO:3所示的HCDR1，SED ID NO:4所示的HCDR2和SED ID NO:5所示的HCDR3；和/或，SEQ ID NO:6所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:94所示的LCDR3；
    49)SEQ ID NO:3所示的HCDR1，SED ID NO:4所示的HCDR2和SED ID NO:5所示的HCDR3；和/或，SEQ ID NO:6所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:95所示的LCDR3；
    50)SEQ ID NO:96所示的HCDR1，SED ID NO:4所示的HCDR2和SED ID NO:5所示的HCDR3；和/或，SEQ ID NO:6所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:8所示的LCDR3；
    51)SEQ ID NO:3所示的HCDR1，SED ID NO:97所示的HCDR2和SED ID NO:5所示的HCDR3；和/或，SEQ ID NO:6所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:8所示的LCDR3；
    52)SEQ ID NO:3所示的HCDR1，SED ID NO:4所示的HCDR2和SED ID NO:98所示的HCDR3；和/或，SEQ ID NO:6所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:8所示的LCDR3；
    53)SEQ ID NO:3所示的HCDR1，SED ID NO:4所示的HCDR2和SED ID NO:5所示的HCDR3；和/或，SEQ ID NO:99所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:8所示的LCDR3；
    54)SEQ ID NO:3所示的HCDR1，SED ID NO:4所示的HCDR2和SED ID NO:5所示的HCDR3；和/或，SEQ ID NO:6所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:100所示的LCDR3；
    55)SEQ ID NO:47所示的HCDR1，SED ID NO:55所示的HCDR2和SED ID NO:5所示的HCDR3；和/或，SEQ ID NO:6所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:8所示的LCDR3；
    56)SEQ ID NO:3所示的HCDR1，SED ID NO:4所示的HCDR2和SED ID NO:101所示的HCDR3；和/或，SEQ ID NO:6所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:8所示的LCDR3；
    57)SEQ ID NO:3所示的HCDR1，SED ID NO:4所示的HCDR2和SED ID NO:5所示的HCDR3；和/或，SEQ ID NO:78所示的LCDR1，SED ID NO:84所示的LCDR2和SED ID NO:8所示的LCDR3；
    58)SEQ ID NO:102所示的HCDR1，SED ID NO:4所示的HCDR2和SED ID NO:5所示的HCDR3；和/或，SEQ ID NO:6所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:8所示的LCDR3；
    59)SEQ ID NO:3所示的HCDR1，SED ID NO:103所示的HCDR2和SED ID NO:5所示的HCDR3；和/或，SEQ ID NO:6所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:8所示的LCDR3；
    60)SEQ ID NO:3所示的HCDR1，SED ID NO:4所示的HCDR2和SED ID NO:104所示的HCDR3；和/或，SEQ ID NO:6所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:8所示的LCDR3；
    61)SEQ ID NO:3所示的HCDR1，SED ID NO:4所示的HCDR2和SED ID NO:5所示的HCDR3；和/或，SEQ ID NO:105所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:8所示的LCDR3；
    62)SEQ ID NO:3所示的HCDR1，SED ID NO:4所示的HCDR2和SED ID NO:5所示的HCDR3；和/或，SEQ ID NO:6所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:106所示的LCDR3；
    63)SEQ ID NO:50所示的HCDR1，SED ID NO:58所示的HCDR2和SED ID NO:66所示的HCDR3；和/或，SEQ ID NO:6所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:8所示的LCDR3；
    64)SEQ ID NO:3所示的HCDR1，SED ID NO:4所示的HCDR2和SED ID NO:5所示的HCDR3；和/或，SEQ ID NO:81所示的LCDR1，SED ID NO:85所示的LCDR2和SED ID NO:90所示的LCDR3；
    65)SEQ ID NO:3所示的HCDR1，SED ID NO:4所示的HCDR2和SED ID NO:104所示的HCDR3；和/或，SEQ ID NO:6所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:91所示的LCDR3；
    66)SEQ ID NO:3所示的HCDR1，SED ID NO:4所示的HCDR2和SED ID NO:67所示的HCDR3；和/或，SEQ ID NO:105所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:8所示的LCDR3；
    67)SEQ ID NO:3所示的HCDR1，SED ID NO:103所示的HCDR2和SED ID NO:5所示的HCDR3；和/或，SEQ ID NO:6所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:92所示的LCDR3；
    68)SEQ ID NO:3所示的HCDR1，SED ID NO:4所示的HCDR2和SED ID NO:104所示的HCDR3；和/或，SEQ ID NO:6所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:93所示的LCDR3；
    69)SEQ ID NO:3所示的HCDR1，SED ID NO:4所示的HCDR2和SED ID NO:63所示的HCDR3；和/或，SEQ ID NO:6所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:87所示的LCDR3；
    70)SEQ ID NO:3所示的HCDR1，SED ID NO:4所示的HCDR2和SED ID NO:71所示的HCDR3；和/或，SEQ ID NO:6所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:106所示的LCDR3；
    71)SEQ ID NO:102所示的HCDR1，SED ID NO:4所示的HCDR2和SED ID NO:5所示的HCDR3；和/或，SEQ ID NO:105所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:8所示的LCDR3；
    72)SEQ ID NO:102所示的HCDR1，SED ID NO:4所示的HCDR2和SED ID NO:5所示的HCDR3；和/或，SEQ ID NO:6所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:106所示的LCDR3；
    73)SEQ ID NO:3所示的HCDR1，SED ID NO:103所示的HCDR2和SED ID NO:5所示的HCDR3；和/或，SEQ ID NO:105所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:8所示的LCDR3；
    74)SEQ ID NO:3所示的HCDR1，SED ID NO:103所示的HCDR2和SED ID NO:5所示的HCDR3；和/或，SEQ ID NO:6所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:106所示的LCDR3；
    75)SEQ ID NO:3所示的HCDR1，SED ID NO:4所示的HCDR2和SED ID NO:104所示的HCDR3；和/或，SEQ ID NO:105所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:8所示的LCDR3；
    76)SEQ ID NO:3所示的HCDR1，SED ID NO:4所示的HCDR2和SED ID NO:104所示的HCDR3；和/或，SEQ ID NO:6所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:106所示的LCDR3；
    77)SEQ ID NO:50所示的HCDR1，SED ID NO:58所示的HCDR2和SED ID NO:66所示的HCDR3；和/或，SEQ ID NO:6所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:95所示的LCDR3；
    78)SEQ ID NO:3所示的HCDR1，SED ID NO:58所示的HCDR2和SED ID NO:5所示的HCDR3；和/或，SEQ ID NO:81所示的LCDR1，SED ID NO:85所示的LCDR2和SED ID NO:90所示的LCDR3；
    79)SEQ ID NO:50所示的HCDR1，SED ID NO:58所示的HCDR2和SED ID NO:66所示的HCDR3；和/或，SEQ ID NO:105所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:8所示的LCDR3；
    80)SEQ ID NO:50所示的HCDR1，SED ID NO:58所示的HCDR2和SED ID NO:66所示的HCDR3；和/或，SEQ ID NO:6所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:106所示的LCDR3；
    81)SEQ ID NO:102所示的HCDR1，SED ID NO:4所示的HCDR2和SED ID NO:5所示的HCDR3；和/或，SEQ ID NO:81所示的LCDR1，SED ID NO:85所示的LCDR2和SED ID NO:90所示的LCDR3；
    82)SEQ ID NO:3所示的HCDR1，SED ID NO:103所示的HCDR2和SED ID NO:5所示的HCDR3；和/或，SEQ ID NO:81所示的LCDR1，SED ID NO:85所示的LCDR2和SED ID NO:90所示的LCDR3；
    83)SEQ ID NO:3所示的HCDR1，SED ID NO:4所示的HCDR2和SED ID NO:104所示的HCDR3；和/或，SEQ ID NO:81所示的LCDR1，SED ID NO:85所示的LCDR2和SED ID NO:90所示的LCDR3；
    84)SEQ ID NO:3所示的HCDR1，SED ID NO:4所示的HCDR2和SED ID NO:101所示的HCDR3；和/或，SEQ ID NO:78所示的LCDR1，SED ID NO:84所示的LCDR2和SED ID NO:8所示的LCDR3；
    85)SEQ ID NO:3所示的HCDR1，SED ID NO:4所示的HCDR2和SED ID NO:101所示的HCDR3；和/或，SEQ ID NO:105所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:8所示的LCDR3；或
    86)SEQ ID NO:3所示的HCDR1，SED ID NO:4所示的HCDR2和SED ID NO:101所示的HCDR3；和/或，SEQ ID NO:6所示的LCDR1，SED ID NO:7所示的LCDR2和SED ID NO:106所示的LCDR3。
  5. 根据权利要求1-4任一项所述的抗体或其抗原结合片段，其包含重链可变区，其中：
    1)所述重链可变区包含选自如SEQ ID NO:33、107-137、156-158、161、162、164-166、169、232-234、240-242所示氨基酸序列或其任何变体；
    2)所述重链可变区包含如SEQ ID NO:35所示氨基酸序列或其任何变体；
    3)所述重链可变区包含如SEQ ID NO:37所示氨基酸序列或其任何变体；
    4)所述重链可变区包含如SEQ ID NO:39所示氨基酸序列或其任何变体；或
    5)所述重链可变区包含如SEQ ID NO:41所示氨基酸序列或其任何变体。
  6. 根据权利要求1-5任一项所述的抗体或其抗原结合片段，其包含轻链可变区，其中：
    1)所述轻链可变区包含选自如SEQ ID NO:34、138-155、159、160、163、167、168、170、235-239所示氨基酸序列或其任何变体；
    2)所述轻链可变区包含如SEQ ID NO:36所示氨基酸序列或其任何变体；
    3)所述轻链可变区包含如SEQ ID NO:38所示氨基酸序列或其任何变体；
    4)所述轻链可变区包含如SEQ ID NO:40所示氨基酸序列或其任何变体；或
    5)所述轻链可变区包含如SEQ ID NO:42所示氨基酸序列或其任何变体。
  7. 根据权利要求1-6任一项所述的抗体或其抗原结合片段，其包含重链可变区和轻链可变区，其中：
    1)所述重链可变区包含选自如SEQ ID NO:33、107-137、156-158、161、162、164-166、169、232-234、240-242所示氨基酸序列或其任何变体；所述轻链可变区包含选自如SEQ ID NO:34、138-155、159、160、163、167、168、170、235-239所示氨基酸序列或其任何变体；
    2)所述重链可变区包含如SEQ ID NO:35所示氨基酸序列或其任何变体；所述轻链可变区包含如SEQ ID NO:36所示氨基酸序列或其任何变体；
    3)所述重链可变区包含如SEQ ID NO:37所示氨基酸序列或其任何变体；所述轻链可变区包含如SEQ ID NO:38所示氨基酸序列或其任何变体；
    4)所述重链可变区包含如SEQ ID NO:39所示氨基酸序列或其任何变体；所述轻链可变区包含如SEQ ID NO:40所示氨基酸序列或其任何变体；或
    5)所述重链可变区包含如SEQ ID NO:41所示氨基酸序列或其任何变体；所述轻链可变区包含如SEQ ID NO:42所示氨基酸序列或其任何变体。
  8. 权利要求1-7中任一项所述的特异性结合Hla的抗体或其抗原结合片段，其中所述抗体是人单克隆抗体。
  9. 权利要求1-8中任一项的所述的特异性结合Hla的抗体或其抗原结合片段，其中所述抗原结合片段选自Fab、Fab’-SH、Fv、scFv或(Fab’)2片段。
  10. 权利要求1-9中任一项的所述的特异性结合Hla的抗体或其抗原结合片段，其包含恒定区序列，其中所述恒定区序列的至少一部分是人共有恒定区序列；
    优选地，所述抗体的重链恒定区包含SEQ ID NO:43或SEQ ID NO:44所示的氨基酸序列，轻链恒定区包含SEQ ID NO:45或SEQ ID NO:46所示的氨基酸序列。
  11. 编码如权利要求1-10中任一项所述的抗Hla抗体或其抗原结合片段的核酸分子。
  12. 一种包含如权利要求11所述的核酸分子的载体，所述载体是表达载体；
    优选地，所述载体的宿主细胞是原核细胞或真核细胞；更优选地，所述宿主细胞选自大肠杆菌细胞、酵母细胞、哺乳动物细胞或适用于制备抗体或其抗原结合片段的其它细胞，其中所述哺乳动物细胞例如是CHO细胞、HEK293细胞或COS细胞。
  13. 一种包含如权利要求1-10任一项所述的抗体或其抗原结合片段以及药学上可接受载体的药物组合物。
  14. 权利要求1-10中任一项所述的抗体或其抗原结合片段在制备用于预防或治疗金黄色葡萄球菌感染或与金黄色葡萄球菌感染相关疾病的药物中的用途。
  15. 一种包含权利要求1-10任一项所述的抗体或其抗原结合片段的检测试剂盒，可用于检测样品中是否存在金黄色葡萄球菌或Hla。
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