WO2024016842A1 - 抗呼吸道合胞病毒中和性抗体及其用途 - Google Patents

抗呼吸道合胞病毒中和性抗体及其用途 Download PDF

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WO2024016842A1
WO2024016842A1 PCT/CN2023/097016 CN2023097016W WO2024016842A1 WO 2024016842 A1 WO2024016842 A1 WO 2024016842A1 CN 2023097016 W CN2023097016 W CN 2023097016W WO 2024016842 A1 WO2024016842 A1 WO 2024016842A1
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antibody
amino acid
seq
acid sequence
rsv
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PCT/CN2023/097016
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English (en)
French (fr)
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付信磊
刘志刚
周晓巍
郝小勃
刘玉兰
胡俊杰
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北京智仁美博生物科技有限公司
智翔(上海)医药科技有限公司
重庆智翔金泰生物制药股份有限公司
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Publication of WO2024016842A1 publication Critical patent/WO2024016842A1/zh

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    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K16/00Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1027Paramyxoviridae, e.g. respiratory syncytial virus
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K39/395Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K39/395Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/42Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum viral
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00Drugs for disorders of the respiratory system
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12Antivirals
    • A61P31/14Antivirals for RNA viruses
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K16/00Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K16/00Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/005Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies constructed by phage libraries
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K16/00Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/63Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70Vectors or expression systems specially adapted for E. coli
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/63Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • CCHEMISTRY; METALLURGY
    • C40COMBINATORIAL TECHNOLOGY
    • C40BCOMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B50/00Methods of creating libraries, e.g. combinatorial synthesis
    • C40B50/06Biochemical methods, e.g. using enzymes or whole viable microorganisms
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K2039/505Medicinal preparations containing antigens or antibodies comprising antibodies
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K2317/00Immunoglobulins specific features
    • C07K2317/50Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K2317/00Immunoglobulins specific features
    • C07K2317/50Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565Complementarity determining region [CDR]
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K2317/00Immunoglobulins specific features
    • C07K2317/70Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12RINDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00Microorganisms ; Processes using microorganisms
    • C12R2001/01Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/185Escherichia
    • C12R2001/19Escherichia coli

Definitions

  • the present application relates generally to the fields of genetic engineering and antibody drugs; specifically, to antibodies against respiratory syncytial virus (RSV) and the use of said antibodies in preventing or treating respiratory syncytial virus (RSV)-related diseases.
  • RSV respiratory syncytial virus
  • Respiratory syncytial virus is the most important viral pathogen causing acute lower respiratory tract infections (ALRTI) in children under 5 years of age worldwide 1 .
  • RSV infection is the leading cause of hospitalization for viral respiratory tract infections in infants and young children, seriously endangering children's health, especially premature infants, infants and young children with congenital heart disease or primary immune deficiency.
  • the five countries with the highest incidence of RSV infection are Pakistan, India, Nigeria, China and Indonesia, which contribute nearly half of the global RSV-ALRTI disease burden2 .
  • Respiratory syncytial virus belongs to the family Pneumoviridae and the genus Orthopneumovirus. It is a non-segmented single-stranded negative-sense RNA virus 3 and can be divided into two subtypes, A and B, based on differences in surface antigens 4 .
  • the RSV genome includes 10 genes encoding 11 proteins. Among them, adhesion protein G and fusion protein F are the main protective antigens on the surface of the virus and can stimulate the body to produce neutralizing antibodies 5 .
  • G Proteins vary greatly between subtypes, so antibodies against G protein are mostly subtype-specific antibodies 6 , while F protein is highly conserved among subtypes, and antibodies induced by F protein can simultaneously inhibit type A/B RSV. Viral infection.
  • F protein is a type I transmembrane protein. Its inactive precursor (F0) consists of 574 amino acids7 . Three F0s form a trimer. When transported through the Golgi apparatus, the host's furin protease binds at the 109th position of F0. Cleavage is performed between amino acids 110 and between amino acids 136 and 137. After cleavage, the short peptide (P27) of the middle 27 amino acids is released, while the remaining two segments, F2 and F1, are connected through disulfide bonds (Cys69–Cys212 and Cys37–Cys439) to form an active F protein 8 .
  • F0 inactive precursor
  • P27 short peptide
  • FP highly hydrophobic fusion peptide
  • F protein appears on the virion surface or cell surface, its structure is not stable, but is in a high-energy metastable pre-fusion conformation structure (Prefusion glyprotein, Pre-F) 9 .
  • Prefusion glyprotein, Pre-F pre-fusion conformation structure
  • the N-terminus of the F1 protein undergoes a series of dramatic structural changes. This process induces membrane fusion, thereby achieving the purpose of virus infection of cells.
  • this process also causes the F protein to transform from a high-energy metastable Pre-F structure to a stable post-fusion F protein structure (Postfusion glyprotein, Post-F).
  • the application provides an antibody against respiratory syncytial virus (RSV), which comprises a heavy chain variable region containing the amino acid sequences of HCDR1, HCDR2 and HCDR3 and a light chain variable region containing the amino acid sequences of LCDR1, LCDR2 and LCDR3.
  • RSV respiratory syncytial virus
  • the amino acid sequence of HCDR1 is shown in SEQ ID NO:32
  • the amino acid sequence of HCDR2 is shown in SEQ ID NO:33
  • the amino acid sequence of HCDR3 is shown in SEQ ID NO:34
  • the amino acid sequence of LCDR1 The sequence is as shown in SEQ ID NO:35
  • the amino acid sequence of the LCDR2 is shown in SEQ ID NO:36 and the amino acid sequence of the LCDR3 is shown in SEQ ID NO:37; or
  • the amino acid sequence of the HCDR1 is shown in SEQ ID NO:38
  • the amino acid sequence of the HCDR2 is shown in SEQ ID NO:39
  • the amino acid sequence of the HCDR3 is shown in SEQ ID NO:40
  • the amino acid sequence of the LCDR1 The sequence is shown in SEQ ID NO:41
  • the amino acid sequence of the LCDR2 is shown in SEQ ID NO:42
  • the amino acid sequence of the LCDR3 is shown in SEQ ID NO:43;
  • HCDR and LCDR amino acid sequences are defined according to Kabat.
  • amino acid sequence of the antibody heavy chain variable region is as shown in SEQ ID NO: 28 or 30.
  • amino acid sequence of the antibody light chain variable region is as shown in SEQ ID NO: 29 or 31.
  • amino acid sequence of the antibody heavy chain variable region is as shown in SEQ ID NO: 28, and the amino acid sequence of the antibody light chain variable region is as shown in SEQ ID NO: 29; or
  • amino acid sequence of the variable region of the heavy chain of the antibody is shown in SEQ ID NO: 30, and the amino acid sequence of the variable region of the light chain of the antibody is shown in SEQ ID NO: 31.
  • the application provides an antibody against respiratory syncytial virus (RSV), wherein the amino acid sequence of the heavy chain variable region of the antibody has at least 90% identity with SEQ ID NO: 28 or 30, and the The amino acid sequence of the light chain variable region of the antibody has at least 90% identity with SEQ ID NO: 29 or 31.
  • RSV respiratory syncytial virus
  • the antibody is a neutralizing antibody.
  • the antibody is capable of binding the F protein of human respiratory syncytial virus (RSV).
  • RSV human respiratory syncytial virus
  • the antibody is a Fab fragment, a whole antibody, an F(ab') 2 fragment, or a single chain Fv fragment (scFv).
  • the antibody is a monoclonal antibody.
  • the antibody Comprises a heavy chain constant region selected from the group consisting of IgGl subtype, IgG2 subtype or IgG4 subtype.
  • the heavy chain constant region comprises the Fc segment sequence of the IgG1 subtype heavy chain constant region and positions 252, 254, and 256 of the Fc segment sequence
  • the amino acid sequences are Y, T and E respectively, wherein the amino acid sequence of the antibody constant region is determined according to EU numbering.
  • the antibody comprises a light chain constant region selected from the kappa subtype or the lambda subtype.
  • the present application provides nucleic acid molecules encoding the antibodies described in the first or second aspect.
  • the present application provides a pharmaceutical composition
  • a pharmaceutical composition comprising the antibody described in the first or second aspect and a pharmaceutically acceptable excipient, diluent or carrier.
  • the application provides the use of the antibody described in the first or second aspect, the nucleic acid molecule described in the third aspect, or the pharmaceutical composition described in the fourth aspect in the preparation of drugs for preventing or treating RSV-related diseases. use.
  • the present application provides a method for preventing or treating RSV-related diseases, which includes administering the antibody described in the first or second aspect, or the pharmaceutical composition described in the fourth aspect to an individual in need.
  • the present application provides a plasmid combination for expressing an antibody Fab fragment library, which includes a first plasmid and a second plasmid, wherein
  • the first plasmid includes an antibody heavy chain VH-CH1 expression unit and a first recombination site;
  • the second plasmid includes an antibody light chain VL-CL expression unit and a second recombination site;
  • the first recombination site is different from the second recombination site.
  • the plasmid combination comprises a plurality of said first plasmids, and a plurality of said first plasmids carry different said antibody heavy chain VH-CH1 expression units and have the same said First recombination site.
  • the plasmid combination comprises a plurality of said second plasmids, and a plurality of said second plasmids carry different said antibody light chain VL-CL expression units and have the same said Second recombination site.
  • the first plasmid and/or the second plasmid for phagemids are provided.
  • the first plasmid and the second plasmid comprise different origins of replication.
  • the origin of replication is selected from one or more of the following: pBR ori, CDF ori, origin of replication of filamentous phage M13 (flori), and p15A ori.
  • the first recombination site and the second recombination site are sites for phage attachment in the respective genomes of the phage and its host bacterium.
  • the first recombination site is attP and the second recombination site is attB.
  • the first recombination site and the second recombination site are located at the polyclonal restriction sites of the first plasmid and the second plasmid, respectively.
  • the antibody light chain constant region CL is a human kappa light chain constant region or a human lambda light chain constant region.
  • the CH1 segment of the antibody heavy chain constant region is selected from the group consisting of IgG1, IgG2, IgG3 or IgG4 subtypes.
  • the first plasmid and/or the second plasmid comprise a resistance gene coding region.
  • the 3' end of the nucleic acid molecule encoding the CH1 segment of the constant region of the antibody heavy chain is fused to the 5' end of the nucleic acid molecule encoding the gIII protein of filamentous phage M13.
  • the resistance gene is an antibiotic resistance gene.
  • the resistance gene is selected from the group consisting of: chloramphenicol resistance gene (CmR), ampicillin resistance gene (Ampr), kanamycin resistance gene (KaR), and tetracycline resistance gene. sex gene (TetR).
  • the present application provides a recombinant system for expressing an antibody Fab fragment library, which includes a first plasmid, a second plasmid and a cell expressing phage integrase; wherein
  • the first plasmid includes an antibody heavy chain VH-CH1 expression unit and a first recombination site;
  • the second plasmid includes an antibody light chain VL-CL expression unit and a second recombination site; with and
  • the first recombination site is different from the second recombination site.
  • the recombinant system comprises a plurality of said first plasmids, and a plurality of said first plasmids carry different said antibody heavy chain VH-CH1 expression units and have the same said First recombination site.
  • the recombinant system comprises a plurality of said second plasmids, and a plurality of said second plasmids carry different said antibody light chain VL-CL expression units and have the same said Second recombination site.
  • the first plasmid and/or the second plasmid are phagemids.
  • the first plasmid and the second plasmid comprise different origins of replication.
  • the origin of replication is selected from one or more of the following: pBR ori, CDF ori, origin of replication of filamentous phage M13 (flori), and p15A ori.
  • the first recombination site and the second recombination site are sites for phage attachment in the respective genomes of the phage and its host bacterium.
  • the first recombination site is attP and the second recombination site is attB.
  • the first recombination site and the second recombination site are located at the polyclonal restriction sites of the first plasmid and the second plasmid, respectively.
  • the antibody light chain constant region CL is a human kappa light chain constant region or a human lambda light chain constant region.
  • the CH1 segment of the antibody heavy chain constant region is selected from the group consisting of IgG1, IgG2, IgG3 or IgG4 subtypes.
  • the first plasmid and/or the second plasmid comprise a resistance gene coding region.
  • the 3' end of the nucleic acid molecule encoding the CH1 segment of the constant region of the antibody heavy chain is fused to the gIII protein encoding the filamentous phage M13.
  • the 5' end of a nucleic acid molecule is fused to the gIII protein encoding the filamentous phage M13.
  • the resistance gene is an antibiotic resistance gene.
  • the resistance gene is selected from the group consisting of: chloramphenicol resistance gene (CmR), ampicillin resistance gene (Ampr), kanamycin resistance gene (KaR) and tetracycline resistance gene. sex gene (TetR).
  • the phage integrase is a tyrosine integrase.
  • the phage integrase is lambda phage integrase.
  • the phage integrase is inducibly expressed or constitutively expressed.
  • the phage integrase is inducibly expressed.
  • the cell expressing the phage integrase is a prokaryotic cell.
  • the cell expressing the phage integrase is E. coli.
  • the cells expressing phage integrase are genetically engineered bacteria.
  • the first plasmid, the second plasmid and the cell expressing the phage integrase each exist independently, or at least one of the first plasmid and the second plasmid has been introduced into the cell. cells expressing phage integrase.
  • the present application provides recombinant cells for expressing an antibody Fab fragment library, which comprise a first plasmid, a second plasmid and express phage integrase; wherein
  • the first plasmid includes an antibody heavy chain VH-CH1 expression unit and a first recombination site;
  • the second plasmid includes an antibody light chain VL-CL expression unit and a second recombination site;
  • the first recombination site is different from the second recombination site.
  • the recombinant cell comprises a plurality of said first plasmids, and a plurality of said first plasmids carry different said antibody heavy chain VH-CH1 expression units and have the same said First recombination site.
  • the recombinant cell comprises a plurality of said second plasmids, and a plurality of said second plasmids carry different said antibody light chain VL-CL expression units and have the same said Second recombination site.
  • the first plasmid and/or the second plasmid are phagemids.
  • the first plasmid and the second plasmid comprise different origins of replication.
  • the origin of replication is selected from one or more of the following: pBR ori, CDF ori, origin of replication of filamentous phage M13 (flori), and p15A ori.
  • the first recombination site and the second recombination site are sites for phage attachment in the respective genomes of the phage and its host bacterium.
  • the first recombination site is attP and the second recombination site is attB.
  • the first recombination site and the second recombination site are located at the polyclonal restriction endonuclease sites of the first plasmid and the second plasmid, respectively.
  • the antibody light chain constant region CL is a human kappa light chain constant region or a human lambda light chain constant region.
  • the CH1 segment of the antibody heavy chain constant region is selected from the group consisting of IgG1, IgG2, IgG3 or IgG4 subtypes.
  • the first plasmid and/or the second plasmid comprise a resistance gene coding region.
  • the 3' end of the nucleic acid molecule encoding the CH1 segment of the constant region of the antibody heavy chain is fused to the 5' end of the nucleic acid molecule encoding the gIII protein of filamentous phage M13.
  • the resistance gene is an antibiotic resistance gene.
  • the resistance gene is selected from the group consisting of: chloramphenicol resistance gene (CmR), ampicillin resistance gene (Ampr), kanamycin resistance gene (KaR), and tetracycline resistance gene. sex gene (TetR).
  • the phage integrase is a tyrosine integrase.
  • the phage integrase is lambda phage integrase.
  • the phage integrase is inducibly expressed or constitutively expressed.
  • the phage integrase is inducibly expressed.
  • the recombinant cells are prokaryotic cells.
  • the recombinant cell is E. coli.
  • the recombinant cells are genetically engineered bacteria.
  • the present application provides a method for preparing an antibody Fab fragment library, which includes transducing a first plasmid and a second plasmid into cells expressing phage integrase; wherein
  • the first plasmid includes an antibody heavy chain VH-CH1 expression unit and a first recombination site;
  • the second plasmid includes an antibody light chain VL-CL expression unit and a second recombination site;
  • the first recombination site is different from the second recombination site.
  • a plurality of the first plasmids are transduced into the cell expressing phage integrase, wherein a plurality of the first plasmids carry different VH-CH1 of the antibody heavy chain expression units and have the same first recombination site.
  • a plurality of said second plasmids are transduced into said cell expressing phage integrase, wherein a plurality of said second plasmids carry different said antibody light chain VL- CL expression unit and have the same second recombination site.
  • transducing the first plasmid and/or the second plasmid into the cell expressing the phage integrase includes the following steps:
  • step (3) Transduce the phage library obtained in step (2) into the cells expressing phage integrase.
  • the competent cells are prokaryotic cells.
  • the competent cell does not express phage integrase.
  • the helper phage is an M13 helper phage.
  • the first plasmid and/or the second plasmid are phagemids.
  • the first plasmid and the second plasmid comprise different origins of replication.
  • the origin of replication is selected from one or more of the following: pBR ori, CDF ori, origin of replication of filamentous phage M13 (flori), and p15A ori.
  • the first recombination site and the second recombination site are sites for phage attachment in the respective genomes of the phage and its host bacterium.
  • the first recombination site is attP and the second recombination site is attB.
  • the first recombination site and the second recombination site are located at the polyclonal restriction sites of the first plasmid and the second plasmid, respectively.
  • the antibody light chain constant region CL is a human kappa light chain constant region or a human lambda light chain constant region.
  • the CH1 segment of the antibody heavy chain constant region is selected from the group consisting of IgG1, IgG2, IgG3 or IgG4 subtypes.
  • the first plasmid and/or the second plasmid comprise a resistance gene coding region.
  • the resistance gene is selected from the group consisting of: chloramphenicol resistance gene (CmR), ampicillin resistance gene (Ampr), kanamycin resistance gene (KaR), and tetracycline resistance gene. sex gene (TetR).
  • the phage integrase is a tyrosine integrase.
  • the phage integrase is a lambda phage integrase Synthase.
  • the phage integrase is inducibly expressed or constitutively expressed.
  • the phage integrase is inducibly expressed.
  • the cell expressing the phage integrase is a prokaryotic cell.
  • the cell expressing the phage integrase is E. coli.
  • the cells expressing phage integrase are genetically engineered bacteria.
  • the present application provides the use of the plasmid combination described in the seventh aspect, the recombinant system described in the eighth aspect, or the recombinant cell described in the ninth aspect in preparing an antibody Fab fragment library.
  • Figure 1 shows a schematic structural diagram of the phagemid vector pHGDisn-attP-new.
  • Figure 2 shows a schematic structural diagram of the prokaryotic expression vector pHKb-attB-new.
  • Figure 3 shows the ELISA analysis of the binding activity of recombinant anti-RSV F protein monoclonal antibodies (R3B1h1, R22B1 and positive control antibodies) to RSV-DS-Cav1-A (Panel A) and RSV-DS-Cav1-B (Panel B) result.
  • Figure 4 shows the ELISA analysis of the blocking results of anti-RSV F protein monoclonal antibodies (R3B1h1, R22B1 and Clesrovimab) against RSV F protein phage binding to RSV-DS-Cav1-B; where A-C are R3B1h1, R22B1 and Clesrovimab against RSV respectively. Blocking results of F protein purified phage binding to RSV-DS-Cav1-B.
  • FIG. 5 shows the results of the Rapid Fluorescent Focus Inhibition Test (RFFIT) detection of anti-RSV F protein monoclonal antibodies inhibiting the infection of Hep-2 cells by RSV/A2 strains.
  • RFFIT Rapid Fluorescent Focus Inhibition Test
  • Figure 6 shows the results of RFFIT detection of anti-RSV F protein monoclonal antibodies inhibiting the infection of Hep-2 cells by the RSV/18537 strain.
  • FIG. 7 shows that anti-RSV F protein monoclonal antibodies inhibit RSV isolated from clinical samples. Results of infection of Hep-2 cells.
  • SEQ ID NO: 1 shows the nucleotide sequence of the promoter encoding the chloramphenicol resistance gene (Cat promoter) derived from Escherichia coli.
  • SEQ ID NO: 2 shows the nucleotide sequence encoding the attP attachment site derived from Escherichia phage Lambda.
  • SEQ ID NO: 3 shows the nucleotide sequence encoding CDF ori derived from E. coli.
  • SEQ ID NO:4 shows the nucleotide sequence encoding the attB attachment site derived from E. coli.
  • SEQ ID NO: 5 shows the nucleotide sequence encoding the chloramphenicol resistance gene derived from E. coli.
  • SEQ ID NO: 6 shows the nucleotide sequence of the complete ampicillin resistance gene expression element derived from E. coli.
  • SEQ ID NO: 7 shows the nucleotide sequence derived from the lambda phage integrase expression element induced by the lambda phage lactose operon of Escherichia.
  • SEQ ID NO:8 shows the nucleotide sequence encoding the attL recombination site.
  • SEQ ID NO:9 shows the nucleotide sequence encoding the attR recombination site.
  • SEQ ID NO:10 shows the amino acid sequence of the F protein of RSV subtype A strain A2.
  • SEQ ID NO: 11 shows the amino acid sequence of the F protein of RSV subtype B strain 18537.
  • SEQ ID NO:12 shows the amino acid sequence of the pre-fusion F protein mutant RSV-DS-Cav1-A of the DS-Cav1 structure.
  • SEQ ID NO:13 shows the amino acid sequence of the pre-fusion F protein mutant RSV-DS-Cav1-B of the DS-Cav1 structure.
  • SEQ ID NO:14 shows the amino acid sequence of the His tag.
  • SEQ ID NO: 15 shows the amino acid sequence of the heavy chain constant region of human (homo sapiens) IgG1 subtype.
  • SEQ ID NO: 16 shows the amino acid sequence of the mutant IgG1-YTE of the heavy chain constant region of human (homo sapiens) IgG1 subtype.
  • SEQ ID NO: 17 shows the amino acid sequence of the heavy chain constant region of mouse (mus musculus) IgG2a subtype.
  • SEQ ID NO: 18 shows the amino acid sequence of the constant region of the human (homo sapiens) kappa subtype light chain.
  • SEQ ID NO: 19 shows the amino acid sequence of the constant region of the lambda isoform light chain of humans (homo sapiens).
  • SEQ ID NO: 20 shows the amino acid sequence of the constant region of the mouse (mus musculus) kappa isoform light chain.
  • SEQ ID NO:21 shows the amino acid sequence of the mouse (mus musculus) lambda isoform light chain constant region.
  • SEQ ID NO: 22 shows the amino acid sequence of the heavy chain variable region of the humanized anti-RSV monoclonal antibody palivizumab (Hu1129).
  • SEQ ID NO: 23 shows the amino acid sequence of the light chain variable region of the humanized anti-RSV monoclonal antibody palivizumab (Hu1129).
  • SEQ ID NO:24 shows the amino acid sequence of the heavy chain variable region of the fully human anti-RSV monoclonal antibody Nirsevimab (MEDI8897).
  • SEQ ID NO:25 shows the amino acid sequence of the light chain variable region of the fully human anti-RSV monoclonal antibody Nirsevimab (MEDI8897).
  • SEQ ID NO:26 shows the amino acid sequence of the heavy chain variable region of the fully human anti-RSV monoclonal antibody Clesrovimab (RB1).
  • SEQ ID NO:27 shows the amino acid sequence of the light chain variable region of the fully human anti-RSV monoclonal antibody Clesrovimab (RB1).
  • SEQ ID NO: 28 shows the amino acid sequence of the heavy chain variable region of the Fab antibody R3B1h1 against RSV F protein.
  • SEQ ID NO: 29 shows the amino acid sequence of the light chain variable region of the Fab antibody R3B1h1 against RSV F protein.
  • SEQ ID NO: 30 shows the amino acid sequence of the heavy chain variable region of the Fab antibody R22B1 against RSV F protein.
  • SEQ ID NO: 31 shows the amino acid sequence of the light chain variable region of the Fab antibody R22B1 against RSV F protein.
  • SEQ ID NO:32-34 respectively show the amino acid sequences of the heavy chain variable regions HCDR1, HCDR2 and HCDR3 of the Fab antibody R3B1h1 against RSV F protein.
  • SEQ ID NO:35-37 respectively show the amino acid sequences of the light chain variable regions LCDR1, LCDR2 and LCDR3 of the Fab antibody R3B1h1 against RSV F protein.
  • SEQ ID NO:38-40 respectively show the amino acid sequences of the heavy chain variable regions HCDR1, HCDR2 and HCDR3 of the Fab antibody R22B1 against RSV F protein.
  • SEQ ID NO:41-43 respectively show the amino acid sequences of the light chain variable regions LCDR1, LCDR2 and LCDR3 of the Fab antibody R22B1 against RSV F protein.
  • SEQ ID NO:44 shows the nucleotide sequence encoding pBR ori derived from E. coli.
  • SEQ ID NO:45 shows the nucleotide sequence encoding f1ori derived from f1 phage.
  • SEQ ID NO: 46 shows the nucleotide sequence encoding the gIII protein of filamentous phage M13 derived from phage M13.
  • Thermo Fisher's Gateway homologous recombination technology uses the specific recombination principle of lambda phage integrase, eliminating the need for classic gene cloning steps such as enzyme digestion and ligation.
  • the in vitro recombination efficiency is as high as 95%.
  • Its Gateway kit is used to construct cDNA libraries. The diversity of constructed libraries can reach 1.0E+7.
  • This application uses the lambda phage integrase recombinant system or recombinant cells to overcome the shortcomings of insufficient DNA conversion efficiency during the construction of the antibody library through efficient phage infection.
  • phage packaged with the antibody heavy chain expression unit such as the heavy chain VH-CH1 expression unit
  • the expression unit such as the heavy chain VH-CH1 expression unit
  • the light chain expression unit are specifically recombined, and antibiotic screening is used to obtain a large-capacity Fab antibody library (more than 1.0E+12).
  • novel antibodies against RSV are provided, nucleic acid molecules encoding the antibodies, vectors comprising the nucleic acid molecules, host cells comprising the nucleic acid molecules or vectors, and cells comprising the antibodies. Compositions, methods of preparing and purifying said antibodies, and medical and biological applications of said antibodies. According to the amino acid sequence of the variable region of the antibody provided in this application, a full-length antibody molecule can be constructed as a drug for preventing or treating RSV-related diseases.
  • the term "recombination site" as used herein refers to a site in a phage genome and a bacterial genome for phage integration.
  • the first recombination site and the second recombination site may be sites for phage attachment in the respective genomes of the phage and its host bacteria, including the following situations: the first recombination site The point is a site for phage attachment in the phage genome and the second recombination site is a site for phage attachment in the genome of the host bacterium of the phage; or the second recombination site is a site for phage attachment in the phage genome.
  • the site and the first recombination site are sites used for phage attachment in the genome of the host bacterium of the phage.
  • plasmid refers to DNA molecules other than chromosomes (or nucleoids) in organisms such as bacteria, yeasts, and actinomycetes that exist in the cytoplasm (except yeast, whose 2 ⁇ m plasmids exist in the nucleus), It has the ability to replicate autonomously, allowing it to maintain a constant copy number in progeny cells and express the genetic information it carries. It is a closed circular double-stranded DNA molecule.
  • plasmid as used herein encompasses bacterial plasmids, phagemids, and the like.
  • Bacterial plasmid is a commonly used vector in DNA recombinant technology.
  • “Phagemid” is a vector composed of a plasmid vector and a single-stranded phage vector, containing single-stranded phage packaging sequences, replicons, plasmid replicons, cloning sites, marker genes, etc.
  • E. coli etc. In the host cell, it can be replicated as a normal double-stranded plasmid DNA molecule, but when a helper phage is present, it is replicated according to the rolling circle model to produce single-stranded DNA, which is packaged into phage particles.
  • the term "attP attachment site” refers to a specific sequence on the phage DNA that contains a 15 bp core region that is identical to the host chromosomal sequence.
  • the term "attB attachment site” refers to an attachment site in the bacterial genome that contains a 15 bp core region that is identical to the phage genome sequence.
  • phage integrase refers to a protein with type I topoisomerase activity that can specifically bind attP and attB attachment sites and is indispensable for integrating phage into the host genome through site-specific recombination. .
  • oil of replication refers to the initiation site of plasmid replication, consisting of the initial replicon (ORI) and its regulatory elements.
  • antibody refers to an immunoglobulin molecule capable of specifically binding to a target via at least one antigen recognition site located in the variable region of the immunoglobulin molecule.
  • Targets include but are not limited to carbohydrates, polynucleotides, lipids, peptides, etc.
  • antibody includes not only intact (i.e., full-length) antibodies, but also antigen-binding fragments thereof (eg, Fab, Fab', F(ab') 2 , Fv), variants thereof, including antibody portions Fusion proteins, humanized antibodies, chimeric antibodies, diabodies, linear antibodies, single chain antibodies, multispecific antibodies (such as bispecific antibodies) and any other immunoglobulins containing antigen recognition sites with required specificity Modified configurations of protein molecules, including glycosylation variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies.
  • antigen-binding fragments thereof eg, Fab, Fab', F(ab') 2 , Fv
  • variants thereof including antibody portions Fusion proteins, humanized antibodies, chimeric antibodies, diabodies, linear antibodies, single chain antibodies, multispecific antibodies (such as bispecific antibodies) and any other immunoglobulins containing antigen recognition sites with required specificity Modified configurations of protein molecules, including glycosylation variants of antibodies, amino acid sequence variant
  • a full-length antibody can be any class of antibody, such as IgD, IgE, IgG, IgA or IgM (or a subclass of the above), but the antibody does not need to belong to any particular class.
  • immunoglobulins can be assigned to different classes.
  • immunoglobulins there are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these classes can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2.
  • the heavy chain constant domains corresponding to the different immunoglobulin classes are called ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ , respectively.
  • the subunit structures and three-dimensional structures of the different classes of immunoglobulins are well known.
  • antigen-binding fragment or antigen-binding portion refers to a portion or region of an intact antibody molecule responsible for binding to an antigen.
  • the antigen-binding domain may comprise a heavy chain variable region (VH), a light chain variable region (VL), or both.
  • VH and VL typically contains three complementarity determining regions CDR1, CDR2 and CDR3.
  • CDR complementarity determining region
  • variable region amino acid sequence of a given antibody the CDR amino acid sequence of the variable region amino acid sequence can be analyzed in a variety of ways, for example, it can be determined using the online software Abysis (http://www.abysis.org/).
  • antigen-binding fragments include, but are not limited to: (1) Fab fragments, which may be monovalent fragments having a VL-CL chain and a VH-CH1 chain; (2) F(ab') 2 fragments, which may be a monovalent fragment having two A bivalent fragment of Fab' fragment, the two Fab' fragments are connected by a disulfide bridge in the hinge region (i.e., a dimer of Fab'); (3) Fv fragment with the VL and VH domains of the single arm of the antibody; ( 4) Single chain Fv (scFv), which may be a single polypeptide chain consisting of a VH domain and a VL domain via a peptide linker; and (5) (scFv) 2 , which may comprise two peptide linkers.
  • a peptide linker connects a VH domain and two VL domains that are combined with the two VH domains via a disulfide bridge.
  • the antibody is a Fab fragment, that is, a monovalent fragment having a VL-CL chain and a VH-CH1 chain.
  • Fd fragment refers to a fragment consisting of the heavy chain variable region VH and the first heavy chain constant region CH1 of an antibody.
  • binding refers to a non-random binding reaction between two molecules, such as the binding of an antibody to an antigenic epitope.
  • neutralizing antibody refers to an antibody that can bind to the antigen on the surface of a pathogenic microorganism, thereby preventing the pathogenic microorganism from adhering to the target cell receptor, or preventing the fusion of the viral envelope and the cell membrane to prevent invasion of the cell.
  • monoclonal antibody refers to an antibody obtained from a substantially homogeneous population of antibodies, that is, the individual antibodies making up the population are identical except for naturally occurring mutations that may be present in a small number of individuals.
  • the application provides an antibody against respiratory syncytial virus (RSV), which comprises a heavy chain variable region containing the amino acid sequences of HCDR1, HCDR2 and HCDR3 and a light chain variable region containing the amino acid sequences of LCDR1, LCDR2 and LCDR3.
  • RSV respiratory syncytial virus
  • the amino acid sequence of the HCDR1 is shown in SEQ ID NO:32
  • the amino acid sequence of the HCDR2 is shown in SEQ ID NO:33
  • the amino acid sequence of the HCDR3 is shown in SEQ ID NO:34
  • the amino acid sequence of the LCDR1 The sequence is as shown in SEQ ID NO:35
  • the amino acid sequence of the LCDR2 is as shown in SEQ ID NO:36
  • the amino acid sequence of the LCDR3 is as shown in SEQ ID NO:37; or
  • the amino acid sequence of the HCDR1 is shown in SEQ ID NO:38
  • the amino acid sequence of the HCDR2 is shown in SEQ ID NO:39
  • the amino acid sequence of the HCDR3 is shown in SEQ ID NO:40
  • the amino acid sequence of the LCDR1 The sequence is shown in SEQ ID NO:41
  • the amino acid sequence of the LCDR2 is shown in SEQ ID NO:42
  • the amino acid sequence of the LCDR3 is shown in SEQ ID NO:43;
  • HCDR and LCDR amino acid sequences are defined according to Kabat.
  • amino acid sequence of the antibody heavy chain variable region is shown in SEQ ID NO: 28 or 30.
  • amino acid sequence of the antibody light chain variable region is as shown in SEQ ID NO: 29 or 31.
  • amino acid sequence of the antibody heavy chain variable region is as shown in SEQ ID NO: 28, and the amino acid sequence of the antibody light chain variable region is as shown in SEQ ID NO: 29; or
  • amino acid sequence of the variable region of the heavy chain of the antibody is shown in SEQ ID NO: 30, and the amino acid sequence of the variable region of the light chain of the antibody is shown in SEQ ID NO: 31.
  • the application provides an antibody against respiratory syncytial virus (RSV), wherein the amino acid sequence of the heavy chain variable region of the antibody is at least 90% identical to SEQ ID NO: 28 or 30, and the The amino acid sequence of the light chain variable region of the antibody is at least 90% identical to SEQ ID NO: 29 or 31.
  • RSV respiratory syncytial virus
  • the antibody heavy chain variable region has an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96% identical to SEQ ID NO: 28 or 30. %, 97%, 98%, 99% or higher identity.
  • the antibody light chain variable region has an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96% identical to SEQ ID NO: 29 or 31 %, 97%, 98%, 99% or higher identity.
  • the amino acid sequence of the heavy chain variable region of the antibody differs from the amino acid sequence shown in any one of SEQ ID NO: 28 and 30 by about 1, 2, 3, 4, 5 , substitution, deletion and/or addition of 6, 7, 8, 9 or 10 amino acids.
  • the amino acid sequence of the light chain variable region of the antibody differs from the amino acid sequence shown in any one of SEQ ID NO: 29 and 31 by about 1, 2, 3, 4, 5 , substitution, deletion and/or addition of 6, 7, 8, 9 or 10 amino acids.
  • the C-terminal or N-terminal region of the amino acid sequence shown in any one of SEQ ID NO: 28 and 30 can also be truncated by about 1, 2, 3, 4, 5, 6 , 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids while still retaining similar functionality to the heavy chain variable region of the antibody.
  • 1, 2, 3, 4, 5, 6, 7 can also be added to the C-terminal or N-terminal region of the amino acid sequence shown in any one of SEQ ID NO: 28 and 30 , 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids, and the resulting amino acid sequence still maintains a similar function of the heavy chain variable region of the antibody.
  • 1, 2, 3, 4, 5 can also be added or deleted in the region other than the C-terminal or N-terminal of the amino acid sequence shown in any one of SEQ ID NO: 28 and 30 , 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids, as long as the changed amino acid sequence remains substantially similar to the heavy chain variable region of the antibody function.
  • the C-terminal or N-terminal region of the amino acid sequence shown in any one of SEQ ID NO: 29 and 31 can also be truncated by about 1, 2, 3, 4, 5, 6 , 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids while still retaining similar functionality to the light chain variable region of the antibody.
  • SEQ ID NO: 29 and 31 Add 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or With more amino acids, the resulting amino acid sequence still retains similar functionality to the light chain variable region of the antibody.
  • 1, 2, 3, 4, 5 can also be added or deleted in the region other than the C-terminal or N-terminal of the amino acid sequence shown in any one of SEQ ID NO: 29 and 31 , 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids, as long as the changed amino acid sequence remains substantially similar to the light chain variable region of the antibody function.
  • the antibody is a neutralizing antibody.
  • the antibody is capable of binding the F protein of human respiratory syncytial virus (RSV).
  • RSV human respiratory syncytial virus
  • the F protein of human respiratory syncytial virus is a recombinant human RSV F protein, such as the recombinant human shown in SEQ ID NO: 12 or 13 RSV F protein.
  • the antibody is a Fab fragment, a whole antibody, an F(ab') 2 fragment, or a single chain Fv fragment (scFv).
  • the antibody is a Fab fragment.
  • the antibody is a monoclonal antibody.
  • the antibody comprises a heavy chain constant region selected from the group consisting of IgGl subtype, IgG2 subtype, or IgG4 subtype.
  • the heavy chain constant region comprises the Fc segment sequence of the IgG1 subtype heavy chain constant region and positions 252, 254, and 256 of the Fc segment sequence
  • the amino acid sequences are Y, T and E respectively, wherein the amino acid sequence of the antibody constant region is determined according to EU numbering.
  • the antibody comprises a light chain constant region selected from the kappa subtype or the lambda subtype.
  • the application provides a nucleic acid molecule encoding the first or second aspect the antibody.
  • the nucleic acid molecule is operably linked to a regulatory amino acid sequence that is recognized by a host cell transformed with the vector.
  • the present application provides a pharmaceutical composition
  • a pharmaceutical composition comprising the antibody described in the first or second aspect and a pharmaceutically acceptable excipient, diluent or carrier.
  • the pharmaceutical composition is used to prevent or treat RSV-related diseases.
  • the RSV-associated disease is respiratory tract infection, such as bronchiolitis and pneumonia.
  • the pharmaceutical composition may further comprise one or more of the following: lubricants, such as talc, magnesium stearate and mineral oil; wetting agents; emulsifiers; Suspending agent; preservatives, such as benzoic acid, sorbic acid and calcium propionate; sweeteners and/or flavoring agents, etc.
  • lubricants such as talc, magnesium stearate and mineral oil
  • wetting agents such as talc, magnesium stearate and mineral oil
  • emulsifiers such as talc, magnesium stearate and mineral oil
  • Suspending agent such as sorbic acid, sorbic acid and calcium propionate
  • preservatives such as benzoic acid, sorbic acid and calcium propionate
  • sweeteners and/or flavoring agents etc.
  • the pharmaceutical composition in the present application can be formulated in the form of tablets, pills, powders, lozenges, elixirs, suspensions, emulsions, solutions, syrups, suppositories or capsules.
  • the pharmaceutical composition of the present application can be delivered using any physiologically acceptable administration method, including but not limited to: oral administration, parenteral administration, nasal administration. medicine, rectal administration, intraperitoneal administration, intravascular injection, subcutaneous administration, transdermal administration, inhalation administration, etc.
  • the medicament for therapeutic use may be formulated in the form of a lyophilized formulation or aqueous solution by mixing reagents of desired purity with optional pharmaceutically acceptable carriers, excipients, etc. Composition for storage.
  • the application provides the use of the antibody described in the first or second aspect, the nucleic acid molecule described in the third aspect, or the pharmaceutical composition described in the fourth aspect in the preparation of drugs for preventing or treating RSV-related diseases. use.
  • the RSV-associated disease is respiratory tract infection, such as bronchiolitis and pneumonia.
  • the present application provides methods for preventing or treating RSV-related diseases, including The method includes administering the antibody described in the first aspect or the second aspect, or the pharmaceutical composition described in the fourth aspect to an individual in need.
  • the RSV-related disease is respiratory tract infection, such as bronchiolitis and pneumonia.
  • the present application provides a plasmid combination for expressing antibody Fab fragments, which includes a first plasmid and a second plasmid, wherein
  • the first plasmid includes an antibody heavy chain VH-CH1 expression unit and a first recombination site;
  • the second plasmid includes an antibody light chain VL-CL expression unit and a second recombination site;
  • the first recombination site is different from the second recombination site.
  • the plasmid combination comprises a plurality of said first plasmids, and a plurality of said first plasmids carry different said antibody heavy chain VH-CH1 expression units and have the same said First recombination site.
  • the plasmid combination comprises a plurality of said second plasmids, and a plurality of said second plasmids carry different said antibody light chain VL-CL expression units and have the same said Second recombination site.
  • the present application provides a recombinant system for expressing an antibody Fab fragment library, which includes a first plasmid, a second plasmid and a cell expressing phage integrase; wherein
  • the first plasmid includes an antibody heavy chain VH-CH1 expression unit and a first recombination site;
  • the second plasmid includes an antibody light chain VL-CL expression unit and a second recombination site;
  • the first recombination site is different from the second recombination site.
  • the recombinant system comprises a plurality of said first plasmids, and a plurality of said first plasmids carry different said antibody heavy chain VH-CH1 expression units and have the same said First recombination site.
  • the recombinant system comprises a plurality of said second plasmids, and a plurality of said second plasmids carry different said antibody light chain VL-CL expression units and have the same said Second recombination site.
  • the first plasmid, the second plasmid and the cell expressing the phage integrase each exist independently, or the first plasmid and the second plasmid At least one of the plasmids has been introduced into the cell expressing the phage integrase.
  • the second plasmid has been introduced into the cell expressing phage integrase.
  • the present application provides recombinant cells for expressing an antibody Fab fragment library, which comprise a first plasmid, a second plasmid and express phage integrase; wherein
  • the first plasmid includes an antibody heavy chain VH-CH1 expression unit and a first recombination site;
  • the second plasmid includes an antibody light chain VL-CL expression unit and a second recombination site;
  • the first recombination site is different from the second recombination site.
  • the recombinant cell comprises a plurality of said first plasmids, and a plurality of said first plasmids carry different said antibody heavy chain VH-CH1 expression units and have the same said First recombination site.
  • the recombinant cell comprises a plurality of said second plasmids, and a plurality of said second plasmids carry different said antibody light chain VL-CL expression units and have the same said Second recombination site.
  • the recombinant cells are prokaryotic cells.
  • the recombinant cell is E. coli.
  • the recombinant cells are genetically engineered bacteria.
  • the recombinant cell is a male E. coli strain with F factor, such as the TG1 strain.
  • the present application provides a method for preparing an antibody Fab fragment library, which includes transducing a first plasmid and a second plasmid into cells expressing phage integrase; wherein
  • the first plasmid includes an antibody heavy chain VH-CH1 expression unit and a first recombination site;
  • the second plasmid includes an antibody light chain VL-CL expression unit and a second recombination site;
  • the first recombination site is different from the second recombination site.
  • a plurality of said first plasmids are transduced into said phage integrase-expressing cell, wherein a plurality of said first plasmids carry different said antibody heavy chain VH-CH1 expression units and have the same first recombination site.
  • a plurality of said second plasmids are transduced into said In cells expressing phage integrase, a plurality of the second plasmids carry different expression units of the antibody light chain VL-CL and have the same second recombination site.
  • transducing the first plasmid and/or the second plasmid into the cell expressing the phage integrase includes the following steps:
  • step (3) Transduce the phage library obtained in step (2) into the cells expressing phage integrase.
  • the competent cells are prokaryotic cells.
  • the competent cell is E. coli.
  • the competent cells are genetically engineered bacteria.
  • the competent cell is a male E. coli strain with F factor, such as the TG1 strain.
  • the competent cell does not express phage integrase.
  • the helper phage is an M13 helper phage.
  • the first plasmid is a phagemid.
  • the second plasmid is a phagemid.
  • the first plasmid and the second plasmid comprise different origins of replication.
  • the origin of replication is selected from one or more of the following: pBR ori, CDF ori, origin of replication of filamentous phage M13 (flori) and p15A ori.
  • the first plasmid comprises pBR ori and f1ori.
  • the The second plasmid contains the CDF ori.
  • the first recombination site and the second recombination site are respectively used for phage attachment in the respective genomes of the phage and its host bacterium. site.
  • the first recombination site is attP.
  • the second recombination site is attB.
  • the first recombination site comprises the nucleotide sequence shown in SEQ ID NO: 2 or the nucleic acid sequence shown in SEQ ID NO: 2
  • the nucleotide sequence is a nucleotide sequence obtained by one or more substitutions, deletions or additions that do not essentially change the function of the first recombination site.
  • the second recombination site comprises the nucleotide sequence shown in SEQ ID NO: 4 or the nucleic acid sequence shown in SEQ ID NO: 4
  • the nucleotide sequence is a nucleotide sequence obtained by one or more substitutions, deletions or additions that do not essentially change the function of the second recombination site.
  • the one or more substitutions or deletions do not essentially change the function of the first recombination site or the second recombination site.
  • the number added is 1-30, preferably 1-20, more preferably 1-10, wherein the obtained nucleotide sequence substantially maintains the unchanged first recombination site or the second Function of the recombination site.
  • the first plasmid and second plasmid are different.
  • the first plasmid is a phagemid
  • the second plasmid is a prokaryotic expression vector (eg, a bacterial plasmid).
  • the first plasmid is pHGDisn-attP-new.
  • the second plasmid is pHKb-attB-new.
  • the first recombination site is located at a polyclonal restriction enzyme cleavage site of the first plasmid.
  • the second recombination site is located at the polyclonal restriction site of the second plasmid.
  • the first recombination site is located between the XbaI and KpnI restriction sites of the pHGDisn-attP-new plasmid.
  • the second recombination site is located between the HindIII and BamHI restriction sites of the pHKb-attB-new plasmid.
  • the antibody light chain constant region CL is a human kappa light chain constant region or a human lambda light chain constant region.
  • the CH1 segment of the antibody heavy chain constant region is selected from the group consisting of IgG1, IgG2, IgG3 or IgG4 subtypes.
  • the first plasmid and/or the second plasmid comprises a resistance gene coding region.
  • the 3' end of the nucleic acid molecule encoding the CH1 segment of the constant region of the antibody heavy chain is fused to the nucleic acid molecule encoding the gIII protein of filamentous phage M13. 5' end.
  • the presence of the resistance gene facilitates the screening of recombinant systems or recombinant cells.
  • the resistance gene is an antibiotic resistance gene.
  • the resistance gene is selected from: chloramphenicol resistance gene (CmR), ampicillin resistance gene (Ampr), kanamycin resistance gene resistance gene (KaR) and tetracycline resistance gene (TetR).
  • the phage integrase is a tyrosine integrase.
  • the tyrosine integrase is a lambda phage integrase.
  • the phage integrase is inducibly expressed or constitutively expressed.
  • the phage integrase is inducibly expressed.
  • the inducible expression is by using an inducible promoter, such as a lactose promoter (Plac) or an arabinose promoter (Para).
  • an inducible promoter such as a lactose promoter (Plac) or an arabinose promoter (Para).
  • the cell expressing the phage integrase is a prokaryotic cell.
  • the cell expressing the phage integrase is Escherichia coli.
  • the cells expressing phage integrase are genetically engineered bacteria.
  • the cell expressing the phage integrase is a male E. coli strain with F factor.
  • the cell expressing the phage integrase is a TG1 strain.
  • the first plasmid comprises the antibody heavy chain VH-CH1 expression unit and attP attachment site; and the second plasmid comprises the Antibody light chain VL-CL expression unit and attB attachment site.
  • the first recombination site and the second recombination site correspond to each other.
  • the nucleotide sequence of the first recombination site is SEQ ID NO:2
  • the nucleotide sequence of the second recombination site is SEQ ID NO:4.
  • the relationship between the first recombination site, the second recombination site, the type of the phage integrase, and the cell type expressing the phage integrase is corresponding to each other.
  • the type of the phage integrase and the type of the phage integrase applicable to the application can be determined. Describe the cell types that express phage integrase.
  • the first recombination site when the second recombination site After determination, the first recombination site, the type of the phage integrase, and the cell type expressing the phage integrase that are suitable for the present application can be determined.
  • the cell type expressing the phage integrase suitable for the present application can be determined.
  • the nucleotide sequence of the first recombination site is SEQ ID NO: 2
  • the nucleotide sequence of the second recombination site is SEQ ID NO. NO: 4.
  • the phage integrase is lambda phage integrase
  • the cell expressing the phage integrase is TG1 Escherichia coli.
  • the first recombination site, the second recombination site, the type of the phage integrase, and the type of the recombinant cell correspond to each other.
  • the type of the phage integrase and the recombinant cell that are suitable for the present application can be determined. type.
  • the first recombination site after the second recombination site is determined, the first recombination site, the type of the phage integrase and the recombinant cell that are suitable for this application can be determined. type.
  • the type of the recombinant cell suitable for the present application can be determined.
  • the nucleotide sequence of the first recombination site is SEQ ID NO:2
  • the nucleotide sequence of the second recombination site is SEQ ID NO:4
  • the phage integrase is lambda phage integrase
  • the recombinant cell is TG1 Escherichia coli.
  • the preparation method of the antibody Fab fragment library may include the following steps:
  • first plasmid for example, multiple first plasmids carrying different antibody heavy chain VH-CH1 expression units and the same attP attachment site, such as multiple phagemids
  • competent cells for example, TG1 competent cells
  • helper phage such as M13 helper phage
  • Competent cells to construct a phage library (e.g., a phage library containing multiple phages carrying different antibody heavy chain VH-CH1 expression units and the same attP attachment site);
  • the cells obtained in the induction step (3) express phage integrase (such as lambda phage integrase);
  • phage library for example, a phage library including a variety of phages carrying different antibody heavy chain VH-CH1 expression units and the same attP attachment site
  • phage library for example, a phage library including a variety of phages carrying different antibody heavy chain VH-CH1 expression units and the same attP attachment site
  • helper phage such as M13 helper phage
  • the present application provides the use of the plasmid combination described in the seventh aspect, the recombinant system described in the eighth aspect, or the recombinant cell described in the ninth aspect in preparing an antibody Fab fragment library.
  • the application also provides vectors comprising nucleic acid molecules encoding the antibodies of the invention or their light or heavy chains, host cells comprising the vectors, and methods of producing the antibodies.
  • the nucleic acid molecule is operably linked to a regulatory sequence that is recognized by a host cell transformed with the vector.
  • methods of producing antibodies include culturing host cells.
  • the method of producing an antibody further includes recovering the antibody from the host cell culture medium.
  • antibodies specific for RSV described herein can also be used to detect the presence of RSV in biological samples.
  • Antibody-based detection methods are well known in the art and include, for example, ELISA, immunoblotting, radioimmunoassay, immunofluorescence, immunoprecipitation, and other related techniques.
  • pHGDisn-attP-new and pHKb-attB-new contain different origins of replication and can replicate and coexist in the same E. coli cell.
  • lambda phage integrase ⁇ Int
  • the new plasmid formed contains a complete chloramphenicol promoter and coding gene, which can express chloramphenicol acetyltransferase and exhibit chloramphenicol expression. protein resistance, which facilitates the screening of recombinant plasmids.
  • the attB attachment site and attP attachment site undergo specific recombination in E. coli cells to generate attL site and attR site. This reaction must be catalyzed by ⁇ phage integrase 14 .
  • the gene encoding the lactose operon-induced lambda phage integrase expression element (its nucleotide sequence is shown in SEQ ID NO: 7) was inserted into the TG1 E. coli genome.
  • the TG1- ⁇ Int recombinant engineering strain was commissioned by Kings Rui Biotechnology Co., Ltd. completed.
  • cryopreserved natural human light chain variable region cDNA and the VK1 and VL3 PCR products with introduced mutations were used as templates.
  • PCR primers to amplify the target fragment
  • NcoI and PmlI double enzyme digestion and clone to Using the vector pHKb-attB-new, the ligation product was electroporated into TG1- ⁇ Int competent cells to construct a fully human light chain antibody library with 1.95E+8 diversity.
  • the constructed antibody library was sent for sequencing analysis, and the average accuracy rate exceeded 90 %.
  • the heavy chain library liquid constructed above was inoculated into 200 mL liquid culture medium, cultured to the logarithmic growth phase at 37°C and 220rpm, infected with M13 helper phage, and cultured overnight at 28°C and 220rmp to amplify the phage, and then used
  • the purified heavy chain phage library was prepared by PEG/NaCl precipitation method, and the titer was measured and then frozen and stored in a -80°C refrigerator for later use.
  • the single-clone sequencing results showed that the clones growing on the plate were all recombinant plasmids, including the newly generated attL (attL1, whose coding sequence is such as SEQ ID NO: 8) and attR (attR1, whose coding sequence is shown in SEQ ID NO: 9) recombination bit points as well as the complete light chain gene and heavy chain Fd gene, and the accuracy of the antibody gene exceeds 76.5%.
  • the process of preparing RSV F protein monoclonal antibodies requires the use of F recombinant proteins of different subtype viruses, including the F protein of RSV A subtype strain A2 (F-A2, whose amino acid sequence is shown in SEQ ID NO: 10) and the F protein of RSV subtype B strain 18537 (F-18537, whose amino acid sequence is shown in SEQ ID NO: 11).
  • RSV-DS-Cav1-A its amino acid sequences are shown in SEQ ID NO: 12
  • RSV-DS-Cav1-B whose amino acid sequence is shown in SEQ ID NO: 13
  • F protein has post-translational modifications (such as glycosylation and disulfide bonds)
  • the use of mammalian cell expression system will be more conducive to maintaining the structure and function of the recombinant protein.
  • His His, whose amino acid sequence is shown in SEQ ID NO: 14
  • SEQ ID NO: 14 a His tag
  • Synthesize RSV-DS-Cav1-A and RSV-DS-Cav1-B genes use conventional molecular biology techniques to clone the synthesized genes into appropriate eukaryotic expression vectors (such as Invitrogen's pcDNA3.1, etc.), and then use Liposomes (such as Invitrogen's 293fectin, etc.) or other cationic transfection reagents (such as PEI, etc.) are used to transfect the prepared recombinant protein expression plasmid into HEK293 cells (such as Invitrogen's HEK293F), and cultured under serum-free suspension culture conditions 3 to 4 days. The culture supernatant is then harvested by centrifugation.
  • appropriate eukaryotic expression vectors such as Invitrogen's pcDNA3.1, etc.
  • Liposomes such as Invitrogen's 293fectin, etc.
  • PEI cationic transfection reagents
  • a metal chelate affinity chromatography column such as GE's HisTrap FF, etc.
  • a desalting column such as GE's Hitrap desaulting, etc.
  • the samples can be filtered and sterilized, and then stored in aliquots at -20°C.
  • nucleotide sequences encoding the heavy chain variable region and the light chain variable region of the antibody are cloned into eukaryotic expression fusion nucleotide sequences encoding the heavy chain constant region and the light chain constant region.
  • Vectors (such as pcDNA3.1 from Invitrogen Company, etc.) are used to express whole antibodies in combination.
  • the heavy chain constant region of the antibody can be human IgG1 subtype (its amino acid sequence is shown in SEQ ID NO: 15), human IgG1 subtype mutant IgG1-YTE (its amino acid sequence is shown in SEQ ID NO: 16), mouse IgG2a subtype (its amino acid sequence is shown in SEQ ID NO: 17), the light chain constant region can be human kappa subtype (its amino acid sequence is shown in SEQ ID NO: 18), human lambda subtype (its amino acid sequence is shown in SEQ ID NO: 18) SEQ ID NO: 19), mouse kappa isoform (the amino acid sequence of which is shown in SEQ ID NO: 20) or mouse lambda isoform (the amino acid sequence of which is SEQ ID NO: 21).
  • liposomes such as Invitrogen's 293fectin, etc.
  • transfection reagents such as PEI, etc.
  • the culture supernatant is then harvested by centrifugation and other methods, and is purified in one step using a ProteinA/G affinity chromatography column (such as GE's Mabselect SURE, etc.).
  • the antibody samples can be filtered and sterilized, then aliquoted and stored at -20°C.
  • nucleic acid molecules encoding the light and heavy chains of R3B1h1 and R22B1 were cloned into eukaryotic expression vectors to prepare recombinant human IgG1- ⁇ monoclonal antibodies.
  • the human anti-RSV monoclonal antibody palivizumab (Hu1129) was prepared with reference to patent US 7704505B2 (the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 22; the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 23), refer to the patent US 11186628 B2 to prepare the human anti-RSV monoclonal antibody Nirsevimab (MEDI8897) (its heavy chain variable region amino acid sequence is shown in SEQ ID NO: 24; the light chain variable region amino acid sequence is as follows) SEQ ID NO: 25), refer to the patent US 9963500B2 to prepare the human anti-RSV monoclonal antibody Clesrovimab (RB1) (its heavy chain variable region amino acid sequence is shown in SEQ ID NO: 26; the light chain variable region amino acid sequence As shown in SEQ ID NO: 27) as a positive control antibody.
  • RB1 human anti-RSV monoclonal antibody Clesrovimab
  • the prepared RSV-DS-Cav1-A and RSV-DS-Cav1-B were coated on a 96-well ELISA plate at 3 ⁇ g/mL, 100 ⁇ L/well, and coated overnight at 4°C.
  • the recombinant anti-RSV F protein monoclonal antibody (R3B1h1/R22B1/Clesrovimab) was gradient diluted with each anti-RSV F protein purified phage (R3B1h1/R22B1/Clesrovimab/Nirsevimab) at a fixed concentration (1 ⁇ 10 11 cfu/mL).
  • the starting concentration is 200 ⁇ g/mL, 3-fold gradient dilution, 10 concentration gradients, 100 ⁇ L/well added blocked 96 wells in the ELISA plate and incubate at 37°C for 1 hour. Wash the ELISA plate with PBST, then add HRP anti-M13 secondary antibody (Beijing Yiqiao Shenzhou Technology Co., Ltd., 11973-MM05T-H), and incubate at 37°C for 1 hour. Use PBST to wash the ELISA plate, add OPD substrate chromogenic solution, stop the color development with 1M H 2 SO 4 after 5-10 minutes, and use a microplate reader to measure the 492nm/630nm dual-wavelength optical density value.
  • the ELISA analysis results are shown in Figure 4.
  • the R3B1h1 monoclonal antibody can block the binding signal of R22B1/Nirsevimab phage and recombinant protein RSV-DS-Cav1-B, but has no effect on the binding signal of Clesrovimab phage and recombinant protein RSV-DS-Cav1-B.
  • R22B1 monoclonal antibody can block the binding signal of R3B1h1/Nirsevimab phage and recombinant protein RSV-DS-Cav1-B, but has no effect on the binding of Clesrovimab phage and recombinant protein RSV-DS-Cav1-B ( Figure 4B)); Clesrovimab monoclonal antibody cannot block the binding of R3B1h1/R22B1/Nirsevimab phage to the recombinant protein RSV-DS-Cav1-B ( Figure 4C).
  • the affinity of anti-RSV F protein monoclonal antibodies was determined by surface plasmon resonance technology using Biacore T200.
  • Amino coupling kit (BR-1000-50), human antibody capture kit (BR-1008-39), CM5 chip (BR100012) and pH7.4 10 ⁇ HBS-EP (BR100669) and other related reagents and consumables are Purchased from GE healthcare.
  • the kit use 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N -Hydroxysuccinimide (NHS) activates the carboxylated CM5 chip surface, and the anti-human IgG (Fc) antibody (capture antibody) is diluted to 25 ⁇ g/mL with 10 mM, pH 5.0 sodium acetate, and then Inject at a flow rate of 10 ⁇ L/min to achieve a coupling volume of approximately 10,000 response units (RU). After injection of the capture antibody, 1 M ethanolamine was injected to block unreacted groups.
  • EDC 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride
  • NHS N -Hydroxysuccinimide
  • concentration gradients such as 6.17nM, 18.5nM, 55.6nM, 166.7nM, 500nM
  • Anti-RSV F protein monoclonal antibodies (R3B1h1, R22B1, Nirsevimab, Clesrovimab, and palivizumab) and negative isotype control antibodies were maintained in Hep-2 cell maintenance medium (DMEM+2% FBS+1% P/S+2mM Glu) was diluted with a starting concentration of 66.7 nM, 5-fold gradient dilution, a total of 10 concentration gradients, and 70 ⁇ L/well was added to a 96-well sterile fully permeable cell culture plate.
  • DMEM+2% FBS+1% P/S+2mM Glu Hep-2 cell maintenance medium
  • Hep-2 cells taken Hep-2 cells in the logarithmic phase of growth, digest and centrifuge them and use Hep-2 cell maintenance medium to The counted cell suspension was diluted to a single cell suspension of 4.3 ⁇ 10 5 cells/mL, and 70 ⁇ L/well was inoculated into a 96-well cell culture plate that had completed neutralization, and placed in a cell culture incubator (37 ⁇ 1°C, 5 ⁇ 1% CO 2 ) and incubate for 60-72h. After virus infection, wash twice with PBS, add 80% acetone, and move to a refrigerator at 2-8°C for 30 minutes.
  • a total of 11 virus strains isolated from clinical samples during the 2021 RSV epidemic season were obtained from Guangzhou Women and Children's Medical Center. Among them, 9 strains are subtype A (numbered 2033, 2064, 2066, 2406, 2410, 2416, 2425, 2430 and 2438 respectively), and 2 strains are subtype B (numbered 2063 and 2427 respectively).
  • the results show ( Figure 7) that both R3B1h1 and R22B1 can neutralize RSV clinical isolate samples very well.

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Abstract

本申请公开了针对呼吸道合胞病毒(RSV)的抗体,编码所述抗体的核酸分子、包含所述核酸分子的表达载体、包含所述核酸分子或载体的宿主细胞以及包含所述抗体的药物组合物。本申请还提供了制备和纯化所述抗体的方法及所述抗体的用途。

Description

抗呼吸道合胞病毒中和性抗体及其用途
相关申请的交叉引用
本申请要求于2022年7月22日递交的中国专利申请第202210871810.9号的优先权,其全部内容通过引用整体并入本文。
发明领域
本申请大体涉及基因工程和抗体药物领域;具体而言,涉及针对呼吸道合胞病毒(RSV)的抗体以及所述抗体在预防或治疗呼吸道合胞病毒(RSV)相关疾病中的用途。
发明背景
大容量、高质量的抗体库,对于筛选任意抗原的高亲和力抗体提供了重要的分子来源,具有很高的商业和应用价值。目前,国内外报道的优质抗体库,多是通过电转化技术构建而成。然而,利用电转化技术构建的抗体库,库容大小受限于转化效率,需要经过多批次的抗体库制备,周期长,需要耗费大量人力和物力资源。
呼吸道合胞病毒(Respiratory syncytial virus,RSV)是世界范围内引起5岁以下儿童急性下呼吸道感染(Acute lower respiratory tract infections,ALRTI)最重要的病毒病原1。RSV感染是造成婴幼儿病毒性呼吸道感染住院的首要因素,严重危害儿童健康,尤其对早产儿、患有先天性心脏病或原发免疫缺陷的婴幼儿造成的疾病更重。RSV感染发病率最高的5个国家依次为巴基斯坦、印度、尼日利亚、中国和印度尼西亚,贡献了全球近一半的RSV-ALRTI的疾病负担2
呼吸道合胞病毒(RSV)属于肺炎病毒科,正肺病毒属,非节段性单股负链RNA病毒3,根据表面抗原的差异可分为A和B两个亚型4。RSV基因组包括10个基因,编码11种蛋白。其中黏附蛋白G和融合蛋白F是病毒表面的主要保护性抗原,能激起机体产生中和抗体5。G 蛋白在亚型间差异性较大,故针对G蛋白的抗体大多是亚型特异的抗体6,而F蛋白在亚型间高度保守,由F蛋白诱导产生的抗体可同时抑制A/B型RSV病毒的感染。
F蛋白属于I型跨膜蛋白,其无活性前体(F0)由574个氨基酸组成7,三个F0形成三聚体,在运输通过高尔基体时,宿主的弗林蛋白酶在F0的第109与110位氨基酸之间及136与137位氨基酸之间进行切割。切割后,中间27个氨基酸的短肽(P27)被释放,而其余两段F2及F1通过二硫键连接(Cys69–Cys212和Cys37–Cys439)形成具有活性的F蛋白8。在F1蛋白N末端存在有一段高疏水性的融合肽(Fusion peptide,FP),其位于蛋白的疏水腔中而免受外部亲水环境的影响。当F蛋白出现在病毒体表面或细胞表面时,它的结构并不稳定,而是处于一种高能级亚稳态的融合前构象结构(Prefusion glyprotein,Pre-F)9。随后,F1蛋白的N末端经历了一系列剧烈的结构变化,这个过程诱导了膜融合的发生,从而达到了病毒感染细胞的目的。同时,该过程还导致了F蛋白由高能级亚稳态的Pre-F结构转变为稳定的融合后F蛋白结构(Postfusion glyprotein,Post-F)。
目前尚无预防RSV的疫苗,美国食品药品监督管理局批准帕利珠单抗(Palivizumab,商品名Synagis)用于预防高危婴幼儿感染RSV,需要最多5次注射才能覆盖典型的RSV季节,因费用高昂,不能广泛应用于婴幼儿群体。
基于临床需求,开发抗呼吸道合胞病毒抗体对预防RSV感染引起的相关疾病具有重要的医学意义。
发明概述
第一方面,本申请提供了针对呼吸道合胞病毒(RSV)的抗体,其包含含HCDR1、HCDR2和HCDR3的氨基酸序列的重链可变区和含LCDR1、LCDR2和LCDR3的氨基酸序列的轻链可变区,其中
所述HCDR1的氨基酸序列如SEQ ID NO:32所示、所述HCDR2的氨基酸序列如SEQ ID NO:33所示、所述HCDR3的氨基酸序列如SEQ ID NO:34所示,所述LCDR1的氨基酸序列如SEQ ID NO:35所 示、所述LCDR2的氨基酸序列如SEQ ID NO:36所示和所述LCDR3的氨基酸序列如SEQ ID NO:37所示;或者
所述HCDR1的氨基酸序列如SEQ ID NO:38所示、所述HCDR2的氨基酸序列如SEQ ID NO:39所示、所述HCDR3的氨基酸序列如SEQ ID NO:40所示,所述LCDR1的氨基酸序列如SEQ ID NO:41所示、所述LCDR2的氨基酸序列如SEQ ID NO:42所示和所述LCDR3的氨基酸序列如SEQ ID NO:43所示;
其中,HCDR和LCDR氨基酸序列根据Kabat定义。
在第一方面的一些实施方案中,所述抗体重链可变区的氨基酸序列如SEQ ID NO:28或30所示。
在第一方面的一些实施方案中,所述抗体轻链可变区的氨基酸序列如SEQ ID NO:29或31所示。
在第一方面的一些实施方案中,所述抗体重链可变区的氨基酸序列如SEQ ID NO:28所示,所述抗体轻链可变区的氨基酸序列如SEQ ID NO:29所示;或者
所述抗体重链可变区的氨基酸序列如SEQ ID NO:30所示,所述抗体轻链可变区的氨基酸序列如SEQ ID NO:31所示。
第二方面,本申请提供了针对呼吸道合胞病毒(RSV)的抗体,其中所述抗体的重链可变区的氨基酸序列与SEQ ID NO:28或30具有至少90%的同一性,并且所述抗体的轻链可变区的氨基酸序列与SEQ ID NO:29或31具有至少90%的同一性。
在第一方面和第二方面中任一方面的一些实施方案中,所述抗体为中和性抗体。
在第一方面和第二方面中任一方面的一些实施方案中,所述抗体能够结合人呼吸道合胞病毒(RSV)的F蛋白。
在第一方面和第二方面中任一方面的一些实施方案中,所述抗体为Fab片段、全抗体、F(ab’)2片段或单链Fv片段(scFv)。
在第一方面和第二方面中任一方面的一些实施方案中,所述抗体为单克隆抗体。
在第一方面和第二方面中任一方面的一些实施方案中,所述抗体 包含选自IgG1亚型、IgG2亚型或IgG4亚型的重链恒定区。
在第一方面和第二方面中任一方面的一些实施方案中,所述重链恒定区包含IgG1亚型重链恒定区的Fc段序列并且所述Fc段序列的第252,254,256位的氨基酸序列分别为Y,T和E,其中所述抗体恒定区氨基酸顺序按照EU numbering来确定。
在第一方面和第二方面中任一方面的一些实施方案中,所述抗体包含选自κ亚型或λ亚型的轻链恒定区。
第三方面,本申请提供了核酸分子,其编码第一方面或第二方面所述的抗体。
第四方面,本申请提供了药物组合物,其包含第一方面或第二方面所述的抗体以及药学上可接受的赋形剂、稀释剂或载体。
第五方面,本申请提供了第一方面或第二方面所述的抗体、第三方面所述的核酸分子、或者第四方面所述的药物组合物在制备预防或治疗RSV相关疾病的药物的用途。
第六方面,本申请提供了预防或治疗RSV相关疾病的方法,其包括向有需要的个体施用第一方面或第二方面所述的抗体、或第四方面所述的药物组合物。
第七方面,本申请提供了用于表达抗体Fab片段库的质粒组合,其包含第一质粒和第二质粒,其中
所述第一质粒包含抗体重链VH-CH1表达单元和第一重组位点;
所述第二质粒包含抗体轻链VL-CL表达单元和第二重组位点;并且
所述第一重组位点与所述第二重组位点不同。
在第七方面的一些实施方案中,所述质粒组合包含多种所述第一质粒,并且多种所述第一质粒携带不同的所述抗体重链VH-CH1表达单元并且具有相同的所述第一重组位点。
在第七方面的一些实施方案中,所述质粒组合包含多种所述第二质粒,并且多种所述第二质粒携带不同的所述抗体轻链VL-CL表达单元并且具有相同的所述第二重组位点。
在第七方面的一些实施方案中,所述第一质粒和/或所述第二质粒 为噬菌粒。
在第七方面的一些实施方案中,所述第一质粒和所述第二质粒包含不同的复制原点。
在第七方面的一些实施方案中,所述复制原点选自以下中的一种或多种:pBR ori、CDF ori、丝状噬菌体M13的复制原点(f1ori)和p15A ori。
在第七方面的一些实施方案中,所述第一重组位点和所述第二重组位点分别为噬菌体及其宿主细菌的各自的基因组中用于噬菌体附着的位点。
在第七方面的一些实施方案中,所述第一重组位点为attP,所述第二重组位点为attB。
在第七方面的一些实施方案中,所述第一重组位点和所述第二重组位点分别位于所述第一质粒和所述第二质粒的多克隆酶切位点处。
在第七方面的一些实施方案中,所述抗体轻链恒定区CL为人κ轻链恒定区或人λ轻链恒定区。
在第七方面的一些实施方案中,所述抗体重链恒定区CH1段选自IgG1、IgG2、IgG3或者IgG4亚型。
在第七方面的一些实施方案中,所述第一质粒和/或所述第二质粒包含抗性基因编码区。
在第七方面的一些实施方案中,包含编码所述抗体重链恒定区CH1段的核酸分子的3’端融合在编码丝状噬菌体M13的gIII蛋白的核酸分子的5’端。
在第七方面的一些实施方案中,所述抗性基因为抗生素抗性基因。
在第七方面的一些实施方案中,所述抗性基因选自:氯霉素抗性基因(CmR)、氨苄青霉素抗性基因(Ampr)、卡那霉素抗性基因(KaR)和四环素抗性基因(TetR)。
第八方面,本申请提供了用于表达抗体Fab片段库的重组系统,其包含第一质粒、第二质粒和表达噬菌体整合酶的细胞;其中
所述第一质粒包含抗体重链VH-CH1表达单元和第一重组位点;
所述第二质粒包含抗体轻链VL-CL表达单元和第二重组位点;以 及
所述第一重组位点与所述第二重组位点不同。
在第八方面的一些实施方案中,所述重组系统包含多种所述第一质粒,并且多种所述第一质粒携带不同的所述抗体重链VH-CH1表达单元并且具有相同的所述第一重组位点。
在第八方面的一些实施方案中,所述重组系统包含多种所述第二质粒,并且多种所述第二质粒携带不同的所述抗体轻链VL-CL表达单元并且具有相同的所述第二重组位点。
在第八方面的一些实施方案中,所述第一质粒和/或所述第二质粒为噬菌粒。
在第八方面的一些实施方案中,所述第一质粒和所述第二质粒包含不同的复制原点。
在第八方面的一些实施方案中,所述复制原点选自以下中的一种或多种:pBR ori、CDF ori、丝状噬菌体M13的复制原点(f1ori)和p15A ori。
在第八方面的一些实施方案中,所述第一重组位点和所述第二重组位点分别为噬菌体及其宿主细菌的各自的基因组中用于噬菌体附着的位点。
在第八方面的一些实施方案中,所述第一重组位点为attP,所述第二重组位点为attB。
在第八方面的一些实施方案中,所述第一重组位点和所述第二重组位点分别位于所述第一质粒和所述第二质粒的多克隆酶切位点处。
在第八方面的一些实施方案中,所述抗体轻链恒定区CL为人κ轻链恒定区或人λ轻链恒定区。
在第八方面的一些实施方案中,所述抗体重链恒定区CH1段选自IgG1、IgG2、IgG3或者IgG4亚型。
在第八方面的一些实施方案中,所述第一质粒和/或所述第二质粒包含抗性基因编码区。
在第八方面的一些实施方案中,包含编码所述抗体重链恒定区CH1段的核酸分子的3’端融合在编码丝状噬菌体M13的gIII蛋白的 核酸分子的5’端。
在第八方面的一些实施方案中,所述抗性基因为抗生素抗性基因。
在第八方面的一些实施方案中,所述抗性基因选自:氯霉素抗性基因(CmR)、氨苄青霉素抗性基因(Ampr)、卡那霉素抗性基因(KaR)和四环素抗性基因(TetR)。
在第八方面的一些实施方案中,所述噬菌体整合酶为酪氨酸整合酶。
在第八方面的一些实施方案中,所述噬菌体整合酶为λ噬菌体整合酶。
在第八方面的一些实施方案中,所述噬菌体整合酶为诱导型表达或组成型表达。
在第八方面的一些实施方案中,所述噬菌体整合酶为诱导型表达。
在第八方面的一些实施方案中,所述表达噬菌体整合酶的细胞为原核细胞。
在第八方面的一些实施方案中,所述表达噬菌体整合酶的细胞为大肠杆菌。
在第八方面的一些实施方案中,所述表达噬菌体整合酶的细胞为基因工程菌。
在第八方面的一些实施方案中,所述第一质粒、第二质粒与所述表达噬菌体整合酶的细胞各自独立存在,或所述第一质粒和第二质粒中至少一者已被引入所述表达噬菌体整合酶的细胞中。
第九方面,本申请提供了用于表达抗体Fab片段库的重组细胞,其包含第一质粒、第二质粒并且表达噬菌体整合酶;其中
所述第一质粒包含抗体重链VH-CH1表达单元和第一重组位点;
所述第二质粒包含抗体轻链VL-CL表达单元和第二重组位点;以及
所述第一重组位点与所述第二重组位点不同。
在第九方面的一些实施方案中,所述重组细胞包含多种所述第一质粒,并且多种所述第一质粒携带不同的所述抗体重链VH-CH1表达单元并且具有相同的所述第一重组位点。
在第九方面的一些实施方案中,所述重组细胞包含多种所述第二质粒,并且多种所述第二质粒携带不同的所述抗体轻链VL-CL表达单元并且具有相同的所述第二重组位点。
在第九方面的一些实施方案中,所述第一质粒和/或所述第二质粒为噬菌粒。
在第九方面的一些实施方案中,所述第一质粒和所述第二质粒包含不同的复制原点。
在第九方面的一些实施方案中,所述复制原点选自以下中的一种或多种:pBR ori、CDF ori、丝状噬菌体M13的复制原点(f1ori)和p15A ori。
在第九方面的一些实施方案中,所述第一重组位点和所述第二重组位点分别为噬菌体及其宿主细菌的各自的基因组中用于噬菌体附着的位点。
在第九方面的一些实施方案中,所述第一重组位点为attP,所述第二重组位点为attB。
在第九方面的一些实施方案中,所述第一重组位点和所述第二重组位点分别位于所述第一质粒和所述第二质粒的多克隆酶切位点处。
在第九方面的一些实施方案中,所述抗体轻链恒定区CL为人κ轻链恒定区或人λ轻链恒定区。
在第九方面的一些实施方案中,所述抗体重链恒定区CH1段选自IgG1、IgG2、IgG3或者IgG4亚型。
在第九方面的一些实施方案中,所述第一质粒和/或所述第二质粒包含抗性基因编码区。
在第九方面的一些实施方案中,包含编码所述抗体重链恒定区CH1段的核酸分子的3’端融合在编码丝状噬菌体M13的gIII蛋白的核酸分子的5’端。
在第九方面的一些实施方案中,所述抗性基因为抗生素抗性基因。
在第九方面的一些实施方案中,所述抗性基因选自:氯霉素抗性基因(CmR)、氨苄青霉素抗性基因(Ampr)、卡那霉素抗性基因(KaR)和四环素抗性基因(TetR)。
在第九方面的一些实施方案中,所述噬菌体整合酶为酪氨酸整合酶。
在第九方面的一些实施方案中,所述噬菌体整合酶为λ噬菌体整合酶。
在第九方面的一些实施方案中,所述噬菌体整合酶为诱导型表达或组成型表达。
在第九方面的一些实施方案中,所述噬菌体整合酶为诱导型表达。
在第九方面的一些实施方案中,所述重组细胞为原核细胞。
在第九方面的一些实施方案中,所述重组细胞为大肠杆菌。
在第九方面的一些实施方案中,所述重组细胞为基因工程菌。
第十方面,本申请提供了抗体Fab片段库的制备方法,其包括将第一质粒和第二质粒转导入表达噬菌体整合酶的细胞中;其中
所述第一质粒包含抗体重链VH-CH1表达单元和第一重组位点;
所述第二质粒包含抗体轻链VL-CL表达单元和第二重组位点;以及
所述第一重组位点与所述第二重组位点不同。
在第十方面所述的一些实施方案中,将多种所述第一质粒转导入所述表达噬菌体整合酶的细胞,其中多种所述第一质粒携带不同的所述抗体重链VH-CH1表达单元并且具有相同的所述第一重组位点。
在第十方面所述的一些实施方案中,将多种所述第二质粒转导入所述表达噬菌体整合酶的细胞中,其中多种所述第二质粒携带不同的所述抗体轻链VL-CL表达单元并且具有相同的所述第二重组位点。
在第十方面的一些实施方案中,将所述第一质粒和/或所述第二质粒转导入所述表达噬菌体整合酶的细胞中包括以下步骤:
(1)将所述第一质粒和/或所述第二质粒转导入感受态细胞中;
(2)使用辅助噬菌体感染步骤(1)获得的感受态细胞,以构建噬菌体库;和
(3)将步骤(2)获得的噬菌体库转导入所述表达噬菌体整合酶的细胞中。
在第十方面的一些实施方案中,所述感受态细胞为原核细胞。
在第十方面的一些实施方案中,所述感受态细胞不表达噬菌体整合酶。
在第十方面的一些实施方案中,所述辅助噬菌体为M13辅助噬菌体。
在第十方面的一些实施方案中,所述第一质粒和/或所述第二质粒为噬菌粒。
在第十方面的一些实施方案中,所述第一质粒和所述第二质粒包含不同的复制原点。
在第十方面的一些实施方案中,所述复制原点选自以下中的一种或多种:pBR ori、CDF ori、丝状噬菌体M13的复制原点(f1ori)和p15A ori。
在第十方面的一些实施方案中,所述第一重组位点和所述第二重组位点分别为噬菌体及其宿主细菌的各自的基因组中用于噬菌体附着的位点。
在第十方面的一些实施方案中,所述第一重组位点为attP,所述第二重组位点为attB。
在第十方面的一些实施方案中,所述第一重组位点和所述第二重组位点分别位于所述第一质粒和所述第二质粒的多克隆酶切位点处。
在第十方面的一些实施方案中,所述抗体轻链恒定区CL为人κ轻链恒定区或人λ轻链恒定区。
在第十方面的一些实施方案中,所述抗体重链恒定区CH1段选自IgG1、IgG2、IgG3或者IgG4亚型。
在第十方面的一些实施方案中,所述第一质粒和/或所述第二质粒包含抗性基因编码区。
在第十方面的一些实施方案中,所述抗性基因选自:氯霉素抗性基因(CmR)、氨苄青霉素抗性基因(Ampr)、卡那霉素抗性基因(KaR)和四环素抗性基因(TetR)。
在第十方面的一些实施方案中,所述噬菌体整合酶为酪氨酸整合酶。
在第十方面的一些实施方案中,所述噬菌体整合酶为λ噬菌体整 合酶。
在第十方面的一些实施方案中,所述噬菌体整合酶为诱导型表达或组成型表达。
在第十方面的一些实施方案中,所述噬菌体整合酶为诱导型表达。
在第十方面的一些实施方案中,所述表达噬菌体整合酶的细胞为原核细胞。
在第十方面的一些实施方案中,所述表达噬菌体整合酶的细胞为大肠杆菌。
在第十方面的一些实施方案中,所述表达噬菌体整合酶的细胞为基因工程菌。
第十一方面,本申请提供了第七方面所述的质粒组合、第八方面所述的重组系统或第九方面所述的重组细胞在制备抗体Fab片段库中的用途。
附图简述
图1显示了噬菌粒载体pHGDisn-attP-new的结构示意图。
图2显示了原核表达载体pHKb-attB-new的结构示意图。
图3显示了ELISA分析重组抗RSV F蛋白单克隆抗体(R3B1h1、R22B1和阳性对照抗体)与RSV-DS-Cav1-A(A图)和RSV-DS-Cav1-B(B图)的结合活性结果。
图4显示了ELISA分析抗RSV F蛋白单克隆抗体(R3B1h1、R22B1和Clesrovimab)对抗RSV F蛋白噬菌体结合RSV-DS-Cav1-B的阻断结果;其中,A-C分别为R3B1h1、R22B1和Clesrovimab对抗RSV F蛋白纯化噬菌体结合RSV-DS-Cav1-B的阻断结果。
图5显示了快速荧光灶抑制试验(Rapid Fluorescent Focus Inhibition Test,RFFIT)检测抗RSV F蛋白单克隆抗体抑制RSV/A2毒株感染Hep-2细胞的结果。
图6显示了RFFIT检测抗RSV F蛋白单克隆抗体抑制RSV/18537毒株感染Hep-2细胞的结果。
图7显示了抗RSV F蛋白单克隆抗体抑制从临床样本分离的RSV 感染Hep-2细胞的结果。
序列说明
SEQ ID NO:1显示了来源于大肠杆菌(Escherichia coli)的编码氯霉素抗性基因启动子(Cat启动子)的核苷酸序列。
SEQ ID NO:2显示了来源于埃希氏菌属λ噬菌体(Escherichia phage Lambda)的编码attP附着位点的核苷酸序列。
SEQ ID NO:3显示了来源于大肠杆菌的编码CDF ori的核苷酸序列。
SEQ ID NO:4显示了来源于大肠杆菌的编码attB附着位点的核苷酸序列。
SEQ ID NO:5显示了来源于大肠杆菌的编码氯霉素抗性基因的核苷酸序列。
SEQ ID NO:6显示了来源于大肠杆菌的完整氨苄青霉素抗性基因表达元件的核苷酸序列。
SEQ ID NO:7显示了来源于埃希氏菌属λ噬菌体乳糖操纵子诱导的λ噬菌体整合酶表达元件的核苷酸序列。
SEQ ID NO:8显示了编码attL重组位点的核苷酸序列。
SEQ ID NO:9显示了编码attR重组位点的核苷酸序列。
SEQ ID NO:10显示了RSV A亚型毒株A2的F蛋白的氨基酸序列。
SEQ ID NO:11显示了RSV B亚型毒株18537的F蛋白的氨基酸序列。
SEQ ID NO:12显示了DS-Cav1结构的融合前F蛋白突变体RSV-DS-Cav1-A的氨基酸序列。
SEQ ID NO:13显示了DS-Cav1结构的融合前F蛋白突变体RSV-DS-Cav1-B的氨基酸序列。
SEQ ID NO:14显示了His标签的氨基酸序列。
SEQ ID NO:15显示了人(homo sapiens)IgG1亚型重链恒定区的氨基酸序列。
SEQ ID NO:16显示了人(homo sapiens)IgG1亚型重链恒定区的突变体IgG1-YTE的氨基酸序列。
SEQ ID NO:17显示了小鼠(mus musculus)IgG2a亚型重链恒定区的氨基酸序列。
SEQ ID NO:18显示了人(homo sapiens)κ亚型轻链恒定区的氨基酸序列。
SEQ ID NO:19显示了人(homo sapiens)λ亚型轻链恒定区的氨基酸序列。
SEQ ID NO:20显示了小鼠(mus musculus)κ亚型轻链恒定区的氨基酸序列。
SEQ ID NO:21显示了小鼠(mus musculus)λ亚型轻链恒定区的氨基酸序列。
SEQ ID NO:22显示了人源化抗RSV单克隆抗体帕利珠单抗(Hu1129)重链可变区的氨基酸序列。
SEQ ID NO:23显示了人源化抗RSV单克隆抗体帕利珠单抗(Hu1129)轻链可变区的氨基酸序列。
SEQ ID NO:24显示了全人源抗RSV单克隆抗体Nirsevimab(MEDI8897)重链可变区的氨基酸序列。
SEQ ID NO:25显示了全人源抗RSV单克隆抗体Nirsevimab(MEDI8897)轻链可变区的氨基酸序列。
SEQ ID NO:26显示了全人源抗RSV单克隆抗体Clesrovimab(RB1)重链可变区的氨基酸序列。
SEQ ID NO:27显示了全人源抗RSV单克隆抗体Clesrovimab(RB1)轻链可变区的氨基酸序列。
SEQ ID NO:28显示了抗RSV F蛋白的Fab抗体R3B1h1的重链可变区的氨基酸序列。
SEQ ID NO:29显示了抗RSV F蛋白的Fab抗体R3B1h1的轻链可变区的氨基酸序列。
SEQ ID NO:30显示了抗RSV F蛋白的Fab抗体R22B1的重链可变区的氨基酸序列。
SEQ ID NO:31显示了抗RSV F蛋白的Fab抗体R22B1的轻链可变区的氨基酸序列。
SEQ ID NO:32-34分别显示了抗RSV F蛋白的Fab抗体R3B1h1的重链可变区HCDR1、HCDR2和HCDR3的氨基酸序列。
SEQ ID NO:35-37分别显示了抗RSV F蛋白的Fab抗体R3B1h1的轻链可变区LCDR1、LCDR2和LCDR3的氨基酸序列。
SEQ ID NO:38-40分别显示了抗RSV F蛋白的Fab抗体R22B1的重链可变区HCDR1、HCDR2和HCDR3的氨基酸序列。
SEQ ID NO:41-43分别显示了抗RSV F蛋白的Fab抗体R22B1的轻链可变区LCDR1、LCDR2和LCDR3的氨基酸序列。
SEQ ID NO:44显示了来源于大肠杆菌的编码pBR ori的核苷酸序列。
SEQ ID NO:45显示了来源于f1噬菌体的编码f1ori的核苷酸序列。
SEQ ID NO:46显示了来源于M13噬菌体的编码丝状噬菌体M13的gIII蛋白的核苷酸序列。
发明详述
利用λ噬菌体整合酶(Int)的特异性重组系统或重组细胞,通过基因工程手段,可以实现目标核苷酸序列在体内和体外的特异性重组整合和切除。有文献报道利用热诱导表达Int整合酶的基因工程菌,可以实现抗体轻链表达质粒和重链表达质粒在细胞内的特异性重组,产生表达功能性Fab的完整质粒10。赛默飞的Gateway同源重组技术,利用λ噬菌体整合酶的特异性重组原理,无需酶切和连接等经典基因克隆步骤,体外重组效率高达95%,其用于构建cDNA文库的Gatway试剂盒,构建文库多样性能达到1.0E+7。
本申请利用λ噬菌体整合酶重组系统或重组细胞,通过噬菌体的高效感染克服抗体库构建过程中DNA转化效率不足的缺点。利用包装有抗体重链表达单元(例如重链VH-CH1表达单元)的噬菌体感染能诱导表达Int整合酶且包含抗体轻链表达单元的质粒的大肠杆菌,通过Int整合酶在体内实现抗体重链表达单元(例如重链VH-CH1表达单元)和轻链表达单元的特异性重组,并以抗生素筛选的方式,得到大容量Fab抗体库(超过1.0E+12)。
本申请的发明人利用上述构建的Fab抗体库筛选得到了新的针对RSV的Fab抗体。在本申请的多个方面,提供了新的针对RSV的抗体,编码所述抗体的核酸分子、包含所述核酸分子的载体、包含所述核酸分子或载体的宿主细胞、包含所述的抗体的组合物、制备和纯化所述抗体的方法及所述抗体的医学和生物学应用。根据本申请提供的抗体的可变区的氨基酸序列,可构建全长的抗体分子作为药物用于预防或治疗RSV相关的疾病。
除非另外指明,本申请的实施采用本领域常规的分子生物学、微生物学、细胞生物学、生物化学以及免疫学技术。
除非另外指明,本申请中所用的术语具有本领域技术人员通常所理解的含义。
定义
如本文所用术语“重组位点”是指在噬菌体基因组和细菌基因组中用于噬菌体整合的位点。在本申请的一些实施方案中,第一重组位点和第二重组位点可以分别为噬菌体及其宿主细菌的各自的基因组中用于噬菌体附着的位点,其中包括以下情况:第一重组位点为噬菌体基因组中用于噬菌体附着的位点和第二重组位点为所述噬菌体的宿主细菌的基因组中用于噬菌体附着的位点;或第二重组位点为噬菌体基因组中用于噬菌体附着的位点和第一重组位点为所述噬菌体的宿主细菌的基因组中用于噬菌体附着的位点。
如本文所用术语“质粒”是指细菌、酵母菌和放线菌等生物中染色体(或拟核)以外的DNA分子,存在于细胞质中(但酵母除外,酵母的2μm质粒存在于细胞核中),具有自主复制能力,使其在子代细胞中也能保持恒定的拷贝数,并表达所携带的遗传信息,是闭合环状的双链DNA分子。本文所用术语“质粒”涵盖细菌质粒、噬菌粒等。“细菌质粒”是DNA重组技术中常用的载体,是能把一个有用的外源基因通过基因工程手段,送进受体细胞中去进行增殖和表达的工具。“噬菌粒”是质粒载体和单链噬菌体载体结合而成的载体,含有单链噬菌体包装序列、复制子以及质粒复制子、克隆位点、标记基因等。在大肠杆菌等 宿主细胞内,可以按正常的双链质粒DNA分子形式复制,而当存在辅助噬菌体时,按滚环模型复制产生单链DNA,包装成噬菌体颗粒。
如本文所用术语“attP附着位点”是指噬菌体DNA上的一个特殊序列,含有一个15bp的与宿主染色体序列完全相同的核心区。
如本文所用术语“attB附着位点”是指细菌基因组中的附着位点,含有一个15bp的与噬菌体基因组序列完全相同的核心区。
如本文所用术语“噬菌体整合酶”是指具有Ⅰ型拓扑异构酶活性,可以特异性结合attP和attB附着位点,在通过位点特异性重组将噬菌体整合到宿主基因组不可缺少的一种蛋白质。
如本文所用术语“复制原点”是指质粒复制的起始位点,由起始复制子(ORI)及其调控原件组成。
如本文所用术语“抗体”是指能够经由至少一个位于免疫球蛋白分子的可变区中的抗原识别位点特异性结合到靶标的免疫球蛋白分子。靶标包括但不限于碳水化合物、多聚核苷酸、脂质、多肽等。本文所使用的“抗体”不仅包括完整的(即全长的)抗体,而且还包括其抗原结合片段(例如Fab、Fab’、F(ab’)2、Fv)、其变异体、包含抗体部分的融合蛋白、人源化抗体、嵌合抗体、双抗体、线性抗体、单链抗体、多特异性抗体(例如双特异性抗体)及任何其他包含所需特异性的抗原识别位点的免疫球蛋白分子的修改配置,包括抗体的糖基化变体、抗体的氨基酸序列变体及共价修饰的抗体。
通常,完整或全长的抗体包含两个重链和两个轻链。每个重链含有重链可变区(VH)和第一、第二及第三恒定区(CH1、CH2及CH3)。每个轻链含有轻链可变区(VL)和恒定区(CL)。全长的抗体可以是任何种类的抗体,例如IgD、IgE、IgG、IgA或IgM(或上述的子类),但抗体不需要属于任何特定的类别。根据重链恒定结构域的抗体氨基酸序列,可以将免疫球蛋白指定为不同的类别。通常,免疫球蛋白有五种主要的类别:IgA、IgD、IgE、IgG及IgM,而且这些类别中有几个可以再被进一步区分成子类(同型),例如IgG1、IgG2、IgG3、IgG4、IgA1及IgA2。对应于不同免疫球蛋白类别的重链恒定域分别称为α、δ、ε、γ以及μ。不同类别的免疫球蛋白的子单元结构和三维结构是公知的。
如本文所用术语“抗原结合片段或抗原结合部分”是指负责结合抗原的完整抗体分子的一部分或区域。抗原结合域可以包含重链可变区(VH)、轻链可变区(VL)或上述两者。VH和VL中的每个通常含有三个互补决定区CDR1、CDR2及CDR3。
本领域技术人员公知,互补决定区(CDR,通常有CDR1、CDR2及CDR3)是可变区中对抗体的亲和力和特异性影响最大的区域。VH或VL的CDR氨基酸序列有两种常见的定义方式,即Chothia定义和Kabat定义11-13。对于给定抗体的可变区氨基酸序列,可以根据Chothia定义或者Kabat定义来确定VH和VL氨基酸序列中的CDR氨基酸序列。在本申请的实施方案中,利用Kabat定义CDR氨基酸序列。
对于给定抗体的可变区氨基酸序列,可以通过多种方式分析可变区氨基酸序列的中CDR氨基酸序列,例如可以利用在线软件Abysis确定(http://www.abysis.org/)。
抗原结合片段的实例包括但不限于:(1)Fab片段,其可以是具有VL-CL链和VH-CH1链的单价片段;(2)F(ab’)2片段,其可以是具有两个Fab’片段的二价片段,该两个Fab’片段由铰链区的二硫桥(即Fab’的二聚物)连接;(3)具有抗体的单臂的VL和VH域的Fv片段;(4)单链Fv(scFv),其可以是由VH结构域和VL结构域经由胜肽连接符组成的单一多胜肽链;以及(5)(scFv)2,其可以包含两个由胜肽连接符连接的VH结构域和两个VL结构域,该两个VL结构域是经由二硫桥与该两个VH结构域组合。
在本申请的一些具体实施方案中,所述抗体为Fab片段,即具有VL-CL链和VH-CH1链的单价片段。
如本文所用术语“Fd片段”是指由抗体的重链可变区VH与重链第一恒定区CH1组成的片段。
如本文所用术语“特异性结合”是指两个分子之间的非随机结合反应,例如抗体至抗原表位的结合。
如本文所用术语“中和性抗体”是指能够与病原微生物表面的抗原结合,从而阻止该病原微生物黏附靶细胞受体,或阻止病毒被膜与细胞膜融合,防止侵入细胞的一种抗体。
如本文所用术语“单克隆抗体”指由基本同质的抗体群体获得的抗体,即,除了可能在少量个体中存在自然发生的突变以外,组成群体的各个抗体是相同的。
第一方面,本申请提供了针对呼吸道合胞病毒(RSV)的抗体,其包含含HCDR1、HCDR2和HCDR3的氨基酸序列的重链可变区和含LCDR1、LCDR2和LCDR3的氨基酸序列的轻链可变区,其中
所述HCDR1的氨基酸序列如SEQ ID NO:32所示、所述HCDR2的氨基酸序列如SEQ ID NO:33所示、所述HCDR3的氨基酸序列如SEQ ID NO:34所示,所述LCDR1的氨基酸序列如SEQ ID NO:35所示、所述LCDR2的氨基酸序列如SEQ ID NO:36所示和所述LCDR3的氨基酸序列如SEQ ID NO:37所示;或者
所述HCDR1的氨基酸序列如SEQ ID NO:38所示、所述HCDR2的氨基酸序列如SEQ ID NO:39所示、所述HCDR3的氨基酸序列如SEQ ID NO:40所示,所述LCDR1的氨基酸序列如SEQ ID NO:41所示、所述LCDR2的氨基酸序列如SEQ ID NO:42所示和所述LCDR3的氨基酸序列如SEQ ID NO:43所示;
其中,HCDR和LCDR氨基酸序列根据Kabat定义。
在第一方面所述的一些实施方案中,所述抗体重链可变区的氨基酸序列如SEQ ID NO:28或30所示。
在第一方面所述的一些实施方案中,所述抗体轻链可变区的氨基酸序列如SEQ ID NO:29或31所示。
在第一方面的一些实施方案中,所述抗体重链可变区的氨基酸序列如SEQ ID NO:28所示,所述抗体轻链可变区的氨基酸序列如SEQ ID NO:29所示;或者
所述抗体重链可变区的氨基酸序列如SEQ ID NO:30所示,所述抗体轻链可变区的氨基酸序列如SEQ ID NO:31所示。
第二方面,本申请提供了针对呼吸道合胞病毒(RSV)的抗体,其中所述抗体的重链可变区的氨基酸序列与SEQ ID NO:28或30具有至少90%的同一性,并且所述抗体的轻链可变区的氨基酸序列与SEQ ID NO:29或31具有至少90%的同一性。
在第二方面的一些实施方案中,所述抗体重链可变区的氨基酸序列与SEQ ID NO:28或30具有至少90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或更高的同一性。
在第二方面的一些实施方案中,所述抗体轻链可变区的氨基酸序列与SEQ ID NO:29或31具有至少90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或更高的同一性。
在第二方面的一些实施方案中,所述抗体的重链可变区的氨基酸序列与SEQ ID NO:28和30中任何一项所示的氨基酸序列相差约1、2、3、4、5、6、7、8、9或10个氨基酸的取代、缺失和/或添加。
在第二方面的一些实施方案中,所述抗体的轻链可变区的氨基酸序列与SEQ ID NO:29和31中任何一项所示的氨基酸序列相差约1、2、3、4、5、6、7、8、9或10个氨基酸的取代、缺失和/或添加。
在第二方面的一些实施方案中,SEQ ID NO:28和30中任何一项所示的氨基酸序列的C端或N端区域还可以被截短约1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、20、25或更多个氨基酸,而仍然保持类似的所述抗体的重链可变区的功能。
在第二方面的一些实施方案中,还可以在SEQ ID NO:28和30中任何一项所示的氨基酸序列的C端或N端区域添加1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、20、25或更多个氨基酸,得到的氨基酸序列仍然保持类似的所述抗体的重链可变区的功能。
在第二方面的一些实施方案中,还可以在SEQ ID NO:28和30中任何一项所示的氨基酸序列的C端或N端以外的区域添加或缺失1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、20、25或更多个氨基酸,只要改变后的氨基酸序列基本上保持类似的所述抗体的重链可变区的功能。
在第二方面的一些实施方案中,SEQ ID NO:29和31中任何一项所示的氨基酸序列的C端或N端区域还可以被截短约1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、20、25或更多个氨基酸,而仍然保持类似的所述抗体的轻链可变区的功能。
在第二方面的一些实施方案中,还可以在SEQ ID NO:29和31中 任何一项所示的氨基酸序列的C端或N端区域添加1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、20、25或更多个氨基酸,得到的氨基酸序列仍然保持类似的所述抗体的轻链可变区的功能。
在第二方面的一些实施方案中,还可以在SEQ ID NO:29和31中任何一项所示的氨基酸序列的C端或N端以外的区域添加或缺失1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、20、25或更多个氨基酸,只要改变后的氨基酸序列基本上保持类似的所述抗体的轻链可变区的功能。
在第一方面和第二方面中任一方面的一些实施方案中,所述抗体为中和性抗体。
在第一方面和第二方面中任一方面的一些实施方案中,所述抗体能够结合人呼吸道合胞病毒(RSV)的F蛋白。
在第一方面和第二方面中任一方面的一些具体实施方案中,人呼吸道合胞病毒(RSV)的F蛋白为重组人RSV F蛋白,例如SEQ ID NO:12或13所示的重组人RSV F蛋白。
在第一方面和第二方面中任一方面的一些实施方案中,所述抗体为Fab片段、全抗体、F(ab’)2片段或单链Fv片段(scFv)。
在第一方面和第二方面中任一方面的一些具体实施方案中,所述抗体为Fab片段。
在第一方面和第二方面中任一方面的一些实施方案中,所述抗体为单克隆抗体。
在第一方面和第二方面中任一方面的一些实施方案中,所述抗体包含选自IgG1亚型、IgG2亚型或IgG4亚型的重链恒定区。
在第一方面和第二方面中任一方面的一些实施方案中,所述重链恒定区包含IgG1亚型重链恒定区的Fc段序列并且所述Fc段序列的第252,254,256位的氨基酸序列分别为Y,T和E,其中所述抗体恒定区氨基酸顺序按照EU numbering来确定。
在第一方面和第二方面中任一方面的一些实施方案中,所述抗体包含选自κ亚型或λ亚型的轻链恒定区。
第三方面,本申请提供了核酸分子,其编码第一方面或第二方面 所述的抗体。
在第三方面的一些实施方案中,所述核酸分子可操作地连接到调控氨基酸序列,调控氨基酸序列可以被用所述载体转化过的宿主细胞识别。
第四方面,本申请提供了药物组合物,其包含第一方面或第二方面所述的抗体以及药学上可接受的赋形剂、稀释剂或载体。
在第四方面的一些实施方案中,所述药物组合物用于预防或治疗RSV相关疾病。
在第四方面的一些实施方案中,所述RSV相关疾病为呼吸道感染,例如细支气管炎和肺炎。
在第四方面的一些实施方案中,所述药物组合物还可以包含下述中的一种或多种:润滑剂,如滑石粉、硬脂酸镁和矿物油;润湿剂;乳化剂;悬浮剂;防腐剂,如苯甲酸、山梨酸和丙酸钙;增甜剂和/或调味剂等。
在第四方面的一些实施方案中,可将本申请中的药物组合物配制为片剂、丸剂、粉剂、锭剂、酏剂、悬液、乳剂、溶液、糖浆、栓剂或胶囊等形式。
在第四方面的一些实施方案中,可以利用任何生理上可接受的给药方式递送本申请的药物组合物,这些给药方式包括但不限于:口服给药、肠胃外给药、经鼻给药、直肠给药、腹膜内给药、血管内注射、皮下给药、经皮给药、吸入给药等。
在第四方面的一些实施方案中,可以通过混合具有所需纯度的试剂与视情况的药学上可接受的载体、赋形剂等,以冻干制剂或水溶液的形式配制用于治疗用途的药物组合物用于存储。
第五方面,本申请提供了第一方面或第二方面所述的抗体、第三方面所述的核酸分子、或者第四方面所述的药物组合物在制备预防或治疗RSV相关疾病的药物的用途。
在第五方面的一些实施方案中,所述RSV相关疾病为呼吸道感染,例如细支气管炎和肺炎。
第六方面,本申请提供了预防或治疗RSV相关疾病的方法,其包 括向有需要的个体施用第一方面或第二方面所述的抗体、或第四方面所述的药物组合物。
在第六方面所述的一些实施方案中,所述RSV相关疾病为呼吸道感染,例如细支气管炎和肺炎。
第七方面,本申请提供了用于表达抗体Fab片段的质粒组合,其包含第一质粒和第二质粒,其中
所述第一质粒包含抗体重链VH-CH1表达单元和第一重组位点;
所述第二质粒包含抗体轻链VL-CL表达单元和第二重组位点;以及
所述第一重组位点与所述第二重组位点不同。
在第七方面的一些实施方案中,所述质粒组合包含多种所述第一质粒,并且多种所述第一质粒携带不同的所述抗体重链VH-CH1表达单元并且具有相同的所述第一重组位点。
在第七方面的一些实施方案中,所述质粒组合包含多种所述第二质粒,并且多种所述第二质粒携带不同的所述抗体轻链VL-CL表达单元并且具有相同的所述第二重组位点。
第八方面,本申请提供了用于表达抗体Fab片段库的重组系统,其包含第一质粒、第二质粒和表达噬菌体整合酶的细胞;其中
所述第一质粒包含抗体重链VH-CH1表达单元和第一重组位点;
所述第二质粒包含抗体轻链VL-CL表达单元和第二重组位点;以及
所述第一重组位点与所述第二重组位点不同。
在第八方面的一些实施方案中,所述重组系统包含多种所述第一质粒,并且多种所述第一质粒携带不同的所述抗体重链VH-CH1表达单元并且具有相同的所述第一重组位点。
在第八方面的一些实施方案中,所述重组系统包含多种所述第二质粒,并且多种所述第二质粒携带不同的所述抗体轻链VL-CL表达单元并且具有相同的所述第二重组位点。
在第八方面的一些实施方案中,其中所述第一质粒、第二质粒与所述表达噬菌体整合酶的细胞各自独立存在,或所述第一质粒和第二 质粒中至少一者已被引入所述表达噬菌体整合酶的细胞中。
在第八方面的一些具体实施方案中,所述第二质粒已被引入所述表达噬菌体整合酶的细胞中。
第九方面,本申请提供了用于表达抗体Fab片段库的重组细胞,其包含第一质粒、第二质粒并且表达噬菌体整合酶;其中
所述第一质粒包含抗体重链VH-CH1表达单元和第一重组位点;
所述第二质粒包含抗体轻链VL-CL表达单元和第二重组位点;以及
所述第一重组位点与所述第二重组位点不同。
在第九方面的一些实施方案中,所述重组细胞包含多种所述第一质粒,并且多种所述第一质粒携带不同的所述抗体重链VH-CH1表达单元并且具有相同的所述第一重组位点。
在第九方面的一些实施方案中,所述重组细胞包含多种所述第二质粒,并且多种所述第二质粒携带不同的所述抗体轻链VL-CL表达单元并且具有相同的所述第二重组位点。
在第九方面的一些实施方案中,所述重组细胞为原核细胞。
在第九方面的一些实施方案中,所述重组细胞为大肠杆菌。
在第九方面的一些实施方案中,所述重组细胞为基因工程菌。
在第九方面的一些具体实施方案中,所述重组细胞为具有F因子的雄性大肠杆菌菌株,例如TG1菌株。
第十方面,本申请提供了抗体Fab片段库的制备方法,其包括将第一质粒和第二质粒转导入表达噬菌体整合酶的细胞中;其中
所述第一质粒包含抗体重链VH-CH1表达单元和第一重组位点;
所述第二质粒包含抗体轻链VL-CL表达单元和第二重组位点;以及
所述第一重组位点与所述第二重组位点不同。
在第十方面的一些实施方案中,将多种所述第一质粒转导入所述表达噬菌体整合酶的细胞,其中多种所述第一质粒携带不同的所述抗体重链VH-CH1表达单元并且具有相同的所述第一重组位点。
在第十方面的一些实施方案中,将多种所述第二质粒转导入所述 表达噬菌体整合酶的细胞中,其中多种所述第二质粒携带不同的所述抗体轻链VL-CL表达单元并且具有相同的所述第二重组位点。
在第十方面的一些实施方案中,将所述第一质粒和/或所述第二质粒转导入所述表达噬菌体整合酶的细胞中包括以下步骤:
(1)将所述第一质粒和/或所述第二质粒转导入感受态细胞中;
(2)使用辅助噬菌体感染步骤(1)获得的感受态细胞,以构建噬菌体库;和
(3)将步骤(2)获得的噬菌体库转导入所述表达噬菌体整合酶的细胞中。
在第十方面的一些实施方案中,所述感受态细胞为原核细胞。
在第十方面的一些实施方案中,所述感受态细胞为大肠杆菌。
在第十方面的一些实施方案中,所述感受态细胞为基因工程菌。
在第十方面的一些具体实施方案中,所述感受态细胞为具有F因子的雄性大肠杆菌菌株,例如TG1菌株。
在第十方面的一些实施方案中,所述感受态细胞不表达噬菌体整合酶。
在第十方面的一些实施方案中,所述辅助噬菌体为M13辅助噬菌体。
在第七方面至第十方面中任一方面的一些实施方案中,所述第一质粒为噬菌粒。
在第七方面至第十方面中任一方面的一些实施方案中,所述第二质粒为噬菌粒。
在第七方面至第十方面中任一方面的一些实施方案中,所述第一质粒和所述第二质粒包含不同的复制原点。
在第七方面至第十方面中任一方面的一些实施方案中,所述复制原点选自以下中的一种或多种:pBR ori、CDF ori、丝状噬菌体M13的复制原点(f1ori)和p15A ori。
在第七方面至第十方面中任一方面的一些具体实施方案中,所述第一质粒包含pBR ori和f1ori。
在第七方面至第十方面中任一方面的一些具体实施方案中,所述 第二质粒包含CDF ori。
在第七方面至第十方面中任一方面的一些实施方案中,所述第一重组位点和所述第二重组位点分别为噬菌体及其宿主细菌的各自的基因组中用于噬菌体附着的位点。
在第七方面至第十方面中任一方面的一些实施方案中,所述第一重组位点为attP。
在第七方面至第十方面中任一方面的一些实施方案中,所述第二重组位点为attB。
在第七方面至第十方面中任一方面的一些具体实施方案中,所述第一重组位点包含SEQ ID NO:2所示的核苷酸序列或由SEQ ID NO:2所示的核苷酸序列经过一处或多处不本质上改变所述第一重组位点的功能的取代、缺失或添加得到的核苷酸序列。
在第七方面至第十方面中任一方面的一些具体实施方案中,所述第二重组位点包含SEQ ID NO:4所示的核苷酸序列或由SEQ ID NO:4所示的核苷酸序列经过一处或多处不本质上改变所述第二重组位点的功能的取代、缺失或添加得到的核苷酸序列。
在第七方面至第十方面中任一方面的一些实施方案中,所述一处或多处不本质上改变所述第一重组位点或所述第二重组位点的功能的取代、缺失或添加的数目为1-30个,优选为1-20个,更优选为1-10个,其中获得的核苷酸序列基本上保持未改变的所述第一重组位点或所述第二重组位点的功能。
在第七方面至第十方面中任一方面的一些实施方案中,所述第一质粒和第二质粒不同。
在第七方面至第十方面中任一方面的一些具体实施方案中,所述第一质粒为噬菌粒,并且所述第二质粒为原核表达载体(例如细菌质粒)。
在第七方面至第十方面中任一方面的一些具体实施方案中,所述第一质粒为pHGDisn-attP-new。
在第七方面至第十方面中任一方面的一些具体实施方案中,所述第二质粒为pHKb-attB-new。
在第七方面至第十方面中任一方面的一些实施方案中,所述第一重组位点位于所述第一质粒的多克隆酶切位点处。
在第七方面至第十方面中任一方面的一些实施方案中,所述第二重组位点位于所述第二质粒的多克隆酶切位点处。
在第七方面至第十方面中任一方面的一些具体实施方案中,所述第一重组位点位于pHGDisn-attP-new质粒的XbaⅠ与KpnⅠ酶切位点之间。
在第七方面至第十方面中任一方面的一些具体实施方案中,所述第二重组位点位于pHKb-attB-new质粒的HindⅢ与BamHⅠ酶切位点之间。
在第七方面至第十方面中任一方面的一些实施方案中,所述抗体轻链恒定区CL为人κ轻链恒定区或人λ轻链恒定区。
在第七方面至第十方面中任一方面的一些实施方案中,所述抗体重链恒定区CH1段选自IgG1、IgG2、IgG3或者IgG4亚型。
在第七方面至第十方面中任一方面的一些实施方案中,所述第一质粒和/或所述第二质粒包含抗性基因编码区。
在第七方面至第十方面中任一方面的一些实施方案中,包含编码所述抗体重链恒定区CH1段的核酸分子的3’端融合在编码丝状噬菌体M13的gIII蛋白的核酸分子的5’端。
在第七方面至第十方面中任一方面的一些实施方案中,所述抗性基因的存在有利于重组系统或重组细胞的筛选。
在第七方面至第十方面中任一方面的一些实施方案中,所述抗性基因为抗生素抗性基因。
在第七方面至第十方面中任一方面的一些实施方案中,所述抗性基因选自:氯霉素抗性基因(CmR)、氨苄青霉素抗性基因(Ampr)、卡那霉素抗性基因(KaR)和四环素抗性基因(TetR)。
在第八方面至第十方面中任一方面的一些实施方案中,所述噬菌体整合酶为酪氨酸整合酶。
在第八方面至第十方面中任一方面的一些实施方案中,所述酪氨酸整合酶为λ噬菌体整合酶。
在第八方面至第十方面中任一方面的一些实施方案中,所述噬菌体整合酶为诱导型表达或组成型表达。
在第八方面至第十方面中任一方面的一些实施方案中,所述噬菌体整合酶为诱导型表达。
在第八方面至第十方面中任一方面的一些实施方案中,所述诱导型表达通过利用诱导型启动子,例如乳糖启动子(Plac)或阿拉伯糖启动子(Para)。
在第八方面或第十方面的一些实施方案中,所述表达噬菌体整合酶的细胞为原核细胞。
在第八方面或第十方面的一些实施方案中,所述表达噬菌体整合酶的细胞为大肠杆菌。
在第八方面或第十方面的一些实施方案中,所述表达噬菌体整合酶的细胞为基因工程菌。
在第八方面或第十方面的一些实施方案中,所述表达噬菌体整合酶的细胞为具有F因子的雄性大肠杆菌菌株。
在第八方面或第十方面的一些具体实施方案中,所述表达噬菌体整合酶的细胞为TG1菌株。
在第七方面至第十方面中任一方面的一些具体实施方案中,所述第一质粒包含所述抗体重链VH-CH1表达单元和attP附着位点;以及所述第二质粒包含所述抗体轻链VL-CL表达单元和attB附着位点。
在第七方面的一些实施方案中,所述第一重组位点和所述第二重组位点是彼此对应的。例如,当第一重组位点的核苷酸序列为SEQ ID NO:2时,则第二重组位点的核苷酸序列为SEQ ID NO:4。
在第八方面或第十方面的一些实施方案中,所述第一重组位点、所述第二重组位点、所述噬菌体整合酶的类型和所述表达噬菌体整合酶的细胞类型之间是相互对应的。
在第八方面或第十方面的一些实施方案中,当所述第一重组位点确定后,则可以确定适用于本申请的所述第二重组位点、所述噬菌体整合酶的类型和所述表达噬菌体整合酶的细胞类型。
在第八方面或第十方面的一些实施方案中,当所述第二重组位点 确定后,则可以确定适用于本申请的所述第一重组位点、所述噬菌体整合酶的类型和所述表达噬菌体整合酶的细胞类型。
在第八方面或第十方面的一些实施方案中,当所述噬菌体整合酶类型确定后,则可以确定适用于本申请的所述表达噬菌体整合酶的细胞类型。
在第八方面或第十方面一些实施方案中,当所述第一重组位点的核苷酸序列为SEQ ID NO:2时,则所述第二重组位点的核苷酸序列为SEQ ID NO:4,所述噬菌体整合酶为λ噬菌体整合酶,所述表达噬菌体整合酶的细胞为TG1大肠杆菌。
在第九方面的一些实施方案中,所述第一重组位点、所述第二重组位点、所述噬菌体整合酶的类型和所述重组细胞的类型之间是相互对应的。
在第九方面的一些实施方案中,当所述第一重组位点确定后,则可以确定适用于本申请的所述第二重组位点、所述噬菌体整合酶的类型和所述重组细胞的类型。
在第九方面的一些实施方案中,当所述第二重组位点确定后,则可以确定适用于本申请的所述第一重组位点、所述噬菌体整合酶的类型和所述重组细胞的类型。
在第九方面的一些实施方案中,当所述噬菌体整合酶类型确定后,则可以确定适用于本申请的所述重组细胞的类型。
在第九方面一些实施方案中,当所述第一重组位点的核苷酸序列为SEQ ID NO:2时,则所述第二重组位点的核苷酸序列为SEQ ID NO:4,所述噬菌体整合酶为λ噬菌体整合酶,所述重组细胞为TG1大肠杆菌。
在第十方面所述的一些具体实施方案中,抗体Fab片段库的制备方法可以包括以下步骤:
(1)将第一质粒(例如携带不同抗体重链VH-CH1表达单元和相同attP附着位点的多种第一质粒,例如多种噬菌粒)转导入感受态细胞(例如TG1感受态细胞);
(2)使用辅助噬菌体(例如M13辅助噬菌体)感染步骤(1)获得的感 受态细胞,以构建噬菌体库(例如包含携带不同的抗体重链VH-CH1表达单元和相同的attP附着位点的多种噬菌体的噬菌体库);
(3)将第二质粒(例如携带不同的抗体轻链VL-CL表达单元和相同attB附着位点的多种第二质粒)转导入表达噬菌体整合酶的细胞(例如表达λ噬菌体整合酶的TG1感受态细胞)中;
(4)诱导步骤(3)获得的细胞表达噬菌体整合酶(例如λ噬菌体整合酶);
(5)使用所述噬菌体库(例如包含携带不同的抗体重链VH-CH1表达单元和相同的attP附着位点的多种噬菌体的噬菌体库)感染步骤(4)的表达噬菌体整合酶的细胞;和
(6)使用辅助噬菌体(例如M13辅助噬菌体)感染步骤(5)获得的细胞,以构建所述抗体Fab片段库。
第十一方面,本申请提供了第七方面所述的质粒组合、第八方面所述的重组系统或第九方面所述的重组细胞在制备抗体Fab片段库中的用途。
在其他方面,本申请还提供包含编码本发明抗体或其轻链或重链的核酸分子的载体、包含所述载体的宿主细胞以及产生所述抗体的方法。在一些实施方案中,所述核酸分子可操作地连接到调控序列,调控序列可以被用所述载体转化的宿主细胞识别。在一些实施方案中,产生抗体的方法包括培养宿主细胞。在一些实施方案中,产生抗体的方法还包括从宿主细胞培养基中回收抗体。
此外,本文所述的特异性针对RSV的抗体也可用于检测生物样品中RSV的存在。基于抗体的检测方法在本领域是众所周知的,并且包括例如ELISA、免疫印迹、放射免疫试验、免疫荧光、免疫沉淀以及其它相关技术。
应当理解,以上详细描述仅为了使本领域技术人员更清楚地了解本申请的内容,而并非意图在任何方面加以限制。本领域技术人员能够对所述实施方案进行各种改动和变化。
实施例
以下实施例仅用于说明而非限制本申请范围的目的。
实施例1:全人源重组Fab库的制备
本实施例的内容参照中国发明专利第CN 108251431B号,其通过引用的方式并入本文中。
1.1构建包含attB和attP附着位点的重组表达质粒
以pADG-S质粒为基础,利用XbaⅠ和KpnⅠ双酶切,将合成的包含氯霉素抗性基因启动子(Cat启动子,其编码序列如SEQ ID NO:1所示)和attP附着位点(attP1,其编码序列如SEQ ID NO:2所示)的重组基因片段克隆至载体pADG-S,构建了包含attP附着位点的表达抗体重链Fd(VH-CH1)结构域的噬菌粒载体pHGDisn-attP-new(图1)。
以pADK-S质粒为基础,利用HindⅢ和BamHⅠ双酶切,将合成的包含CDF复制原点(CDF ori,其编码序列如SEQ ID NO:3所示)、attB附着位点(attB1,其编码序列如SEQ ID NO:4所示)、氯霉素抗性基因(CmR,其编码序列如SEQ ID NO:5所示)和完整氨苄青霉素抗性基因表达元件(AmpR,其编码序列如SEQ ID NO:6所示)的重组基因片段克隆至载体pADK-S,构建了包含attB附着位点的表达抗体轻链的原核表达载体pHKb-attB-new(图2)。
pHGDisn-attP-new和pHKb-attB-new包含不同的复制原点,可以在同一个大肠杆菌细胞内复制共存。λ噬菌体整合酶(λInt)诱导attP和attB附着位点发生特异性重组后,形成的新质粒由于包含完整的氯霉素启动子和编码基因,可以表达氯霉素乙酰转移酶,表现出氯霉素抗性,便于重组质粒的筛选。
1.2制备λInt基因编辑的TG1大肠杆菌
attB附着位点和attP附着位点在大肠杆菌细胞内发生特异性重组,生成attL位点和attR位点,该反应必须在λ噬菌体整合酶的催化下进行14。通过基因编辑手段,将编码乳糖操纵子诱导的λ噬菌体整合酶表达元件基因(其核苷酸序列如SEQ ID NO:7所示)插入TG1大肠杆菌基因组中,TG1-λInt重组工程菌委托金斯瑞生物科技有限公司完成。
1.3全人源重链抗体库构建
参照中国发明专利第CN 108251431B号,以冻存的天然人重链可变区cDNA以及引入突变的VH1、VH3和VH5的PCR产物为模板,设计PCR引物扩增目的片段后,利用NcoⅠ和PmlⅠ双酶切克隆至载体pHGDisn-attP-new,将连接产物电转化TG1感受态细胞,构建出1.73E+9多样性的全人源重链抗体库,对构建的抗体库送测序分析,平均正确率超过85%。
1.4全人源轻链抗体库构建
参照中国发明专利第CN 108251431B号,以冻存的天然人轻链可变区cDNA以及引入突变的VK1和VL3PCR产物为模板,设计PCR引物扩增目的片段后,利用NcoⅠ和PmlⅠ双酶切克隆至载体pHKb-attB-new,将连接产物电转化TG1-λInt感受态细胞,构建出1.95E+8多样性的全人源轻链抗体库,对构建的抗体库送测序分析,平均正确率超过90%。
1.5全人源重组Fab库的制备
将上述构建的重链库菌液接种到200mL液体培养基中,于37℃、220rpm下培养至对数生长期,感染M13辅助噬菌体,并于28℃、220rmp下过夜培养扩增噬菌体,然后利用PEG/NaCl沉淀法制备纯化的重链噬菌体库,测定滴度后冻存于-80℃冰箱备用。
取上述制备的全人源轻链抗体库,接种到50mL液体培养基中,添加IPTG至终浓度为0.1mM,于37℃、220rpm培养至对数生长期后,加入重链噬菌体库进行感染,感染结束后菌液均匀涂布至细菌培养皿,于32℃恒温培养箱中过夜培养。
对上述培养皿上生长的菌计数统计,并送测序鉴定单克隆的重组位点以及轻链序列和重链序列。通过稀释法计数统计重组后库容,推算重组后菌落共计3.80E+12,单克隆测序结果显示,平皿上生长的克隆均为重组后的质粒,包含新生成的attL(attL1,其编码序列如SEQ ID NO:8所示)和attR(attR1,其编码序列如SEQ ID NO:9所示)重组位 点以及完整的轻链基因和重链Fd基因,且抗体基因正确率超过76.5%。
收集上述细菌培养皿上的菌落,接种至液体培养基,于37℃、220rpm下培养至对数生长期后,感染M13辅助噬菌体制备纯化的人源Fab噬菌体库。
实施例2:RSV F蛋白的制备以及重组抗体的制备
2.1 RSV F蛋白的制备
制备RSV F蛋白单克隆抗体的过程中需要用到不同亚型病毒的F重组蛋白,包括RSV A亚型毒株A2的F蛋白(F-A2,其氨基酸序列如SEQ ID NO:10所示)和RSV B亚型毒株18537的F蛋白(F-18537,其氨基酸序列如SEQ ID NO:11所示)。基于两种不同亚型的RSV F蛋白,发明人构建了DS-Cav1结构15的融合前F蛋白突变体,分别命名为RSV-DS-Cav1-A(其氨基酸序列如SEQ ID NO:12所示)和RSV-DS-Cav1-B(其氨基酸序列如SEQ ID NO:13所示)。由于F蛋白具有翻译后修饰(如糖基化和二硫键),因而利用哺乳动物细胞表达系统将更有利于保持重组蛋白的结构和功能。此外,在这个重组蛋白的C端添加了His标签(His,其氨基酸序列如SEQ ID NO:14所示),将更有利于重组蛋白的纯化和单克隆抗体功能的鉴定。
合成RSV-DS-Cav1-A和RSV-DS-Cav1-B基因,利用常规的分子生物学技术将合成的基因克隆至合适的真核表达载体(如Invitrogen公司的pcDNA3.1等),然后利用脂质体(如Invitrogen公司的293fectin等)或者其他阳离子转染试剂(如PEI等)将制备的重组蛋白表达质粒转染入HEK293细胞(如Invitrogen公司的HEK293F),在无血清悬浮培养条件下培养3-4天。然后通过离心等方式收获培养上清。利用金属螯合亲和层析柱(如GE公司的HisTrap FF等)对上清中的重组蛋白进行一步纯化。然后利用脱盐柱(如GE公司的Hitrap desaulting等)将重组蛋白保存缓冲液置换为PBS(pH7.0)或者其他合适的缓冲液。必要时,可以对样品进行过滤除菌,然后分装保存于-20℃。
2.2重组抗体的制备
利用常规的分子生物学手段,将编码抗体重链可变区和轻链可变区的核苷酸序列分别克隆至融合有编码重链恒定区和轻链恒定区核苷酸序列的真核表达载体(如invitrogen公司的pcDNA3.1等),组合表达全抗体。抗体的重链恒定区可以是人IgG1亚型(其氨基酸序列如SEQ ID NO:15所示)、人IgG1亚型突变体IgG1-YTE(其氨基酸序列如SEQ ID NO:16所示)、鼠IgG2a亚型(其氨基酸序列如SEQ ID NO:17所示),轻链恒定区可以是人κ亚型(其氨基酸序列如SEQ ID NO:18所示)、人λ亚型(其氨基酸序列如SEQ ID NO:19所示)、鼠κ亚型(其氨基酸序列如SEQ ID NO:20所示)或者鼠λ亚型(其氨基酸序列如SEQ ID NO:21所示)。
利用脂质体(如Invitrogen公司的293fectin等)或其它转染试剂(如PEI等)将制备的重组抗体表达质粒转染入HEK293细胞(如Invitrogen公司的HEK293F),在无血清悬浮培养条件下培养3-5天。然后通过离心等方式收获培养上清,利用ProteinA/G亲和层析柱(如GE公司的Mabselect SURE等)进行一步纯化。然后利用脱盐柱(如GE公司的Hitrap desaulting等)将重组蛋白保存缓冲液置换为PBS(pH7.0)或者其它合适的缓冲液。必要时,可以对抗体样品进行过滤除菌,然后分装保存于-20℃。
实施例3:利用RSV F蛋白筛选全人源重组Fab库
参照文献(实验技术流程可参见中国专利申请第201510097117.0号,通过引用方式将上述专利申请的全部内容并入本文中),使用实施例2制备的重组RSV F蛋白(RSV-DS-Cav1-A/RSV-DS-Cav1-B),通过固相筛选策略(实验方案参考噬菌体展示:通用实验指南/(美)克拉克森(Clackson,T.),(美)洛曼(Lowman,H.B.)编;马岚等译。化学工业出版社,2008.5)筛选实施例1构建的全人源重组Fab库,通过结合、洗脱、中和、感染、扩增的方式共进行4-6轮筛选,最终获得两株特异结合RSV F蛋白的Fab抗体:R3B1h1和R22B1。
实施例4:重组抗RSV F蛋白单克隆抗体的鉴定
利用常规分子生物学方法,分别将编码R3B1h1和R22B1两个分子的轻重链的核酸分子克隆至真核表达载体,制备重组人IgG1-κ形式单克隆抗体。同时,参照专利US 7704505B2制备人源抗RSV单克隆抗体帕利珠单抗(Hu1129)(其重链可变区氨基酸序列如SEQ ID NO:22所示;轻链可变区氨基酸序列如SEQ ID NO:23所示),参照专利US 11186628 B2制备人源抗RSV单克隆抗体Nirsevimab(MEDI8897)(其重链可变区氨基酸序列如SEQ ID NO:24所示;轻链可变区氨基酸序列如SEQ ID NO:25所示),参照专利US 9963500B2制备人源抗RSV单克隆抗体Clesrovimab(RB1)(其重链可变区氨基酸序列如SEQ ID NO:26所示;轻链可变区氨基酸序列如SEQ ID NO:27所示)作为阳性对照抗体。
4.1重组抗RSV F蛋白单克隆抗体结合活性验证
将制备的RSV-DS-Cav1-A和RSV-DS-Cav1-B分别包被于96孔ELISA板,3μg/mL,100μL/孔,4℃包被过夜。利用封闭液PBS-0.1%吐温20-3%牛奶(PBST-3%牛奶)在37℃封闭1小时后,分别加入各重组抗RSV F蛋白单克隆抗体,37℃结合1小时。用PBS-0.1%吐温20(PBST)缓冲液洗涤ELISA板,加入HRP-小鼠抗人IgG(Bioss,bsm-0297M-HRP),37℃结合1小时。PBST缓冲液洗涤ELISA板,加入OPD底物显色液,5-10分钟后用1M的H2SO4终止显色,酶标仪492nm/630nm双波长测定光密度值。ELISA分析结果(图3)显示,重组抗RSV F蛋白单克隆抗体R3B1h1和R22B1结合两种重组蛋白RSV-DS-Cav1-A和RSV-DS-Cav1-B活性相当。
4.2重组抗RSV病毒F蛋白单克隆抗体的表位分析
使用重组蛋白RSV-DS-Cav1-B包被于96孔ELISA板(1μg/mL,100μL/孔),4℃冰箱包被过夜。利用封闭液PBST-3%牛奶在37℃封闭1小时。用固定浓度(1×1011cfu/mL)的各个抗RSV F蛋白纯化噬菌体(R3B1h1/R22B1/Clesrovimab/Nirsevimab)分别对重组抗RSV F蛋白单克隆抗体(R3B1h1/R22B1/Clesrovimab)进行梯度稀释,起始浓度为200μg/mL,3倍梯度稀释,10个浓度梯度,100μL/孔加入封闭好的96 孔ELISA板中,37℃孵育1小时。使用PBST洗涤ELISA板,然后加入HRP抗M13二抗(北京义翘神州科技股份有限公司,11973-MM05T-H),37℃孵育1小时。使用PBST洗涤ELISA板,加入OPD底物显色液,5-10分钟后用1M的H2SO4终止显色,使用酶标仪测定492nm/630nm双波长光密度值。ELISA分析结果如图4所示,R3B1h1单克隆抗体可以阻断R22B1/Nirsevimab噬菌体与重组蛋白RSV-DS-Cav1-B的结合信号,对Clesrovimab噬菌体与重组蛋白RSV-DS-Cav1-B的结合没有影响(图4A);R22B1单克隆抗体可以阻断R3B1h1/Nirsevimab噬菌体与重组蛋白RSV-DS-Cav1-B的结合信号,对Clesrovimab噬菌体与重组蛋白RSV-DS-Cav1-B的结合没有影响(图4B));Clesrovimab单克隆抗体不能阻断R3B1h1/R22B1/Nirsevimab噬菌体与重组蛋白RSV-DS-Cav1-B的结合(图4C)。
4.3重组抗RSV F蛋白单克隆抗体的亲和力分析
利用Biacore T200通过表面等离子共振技术测定抗RSV F蛋白单克隆抗体的亲和力。氨基偶联试剂盒(BR-1000-50)、人抗体捕获试剂盒(BR-1008-39)、CM5芯片(BR100012)和pH7.4的10×HBS-EP(BR100669)等相关试剂和耗材均购自GE healthcare。依照试剂盒中的说明书,用1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride,EDC)和N-羟基琥珀酰亚胺(N-Hydroxysuccinimide,NHS)对羧基化CM5芯片表面进行活化,将抗人IgG(Fc)抗体(捕获抗体)用10mM、pH5.0的乙酸钠稀释至25μg/mL,之后以流速10μL/min注射以实现大约10000个响应单位(RU)的偶联量。注射捕获抗体之后,注射1M乙醇胺以封闭未反应的基团。对于动力学测量,稀释抗RSV F蛋白单克隆抗体至0.5-1μg/mL,10μL/min注射,保证80RU左右的抗体被抗人Fc的抗体捕获。然后将RSV-DS-Cav1-A或者RSV-DS-Cav1-B设置一系列的浓度梯度(例如6.17nM、18.5nM、55.6nM、166.7nM、500nM),于25℃下以30μL/min从低浓度到高浓度进行注射,结合时间为90s,解离时间为3600s,以10μL/min注射3M的MgCl2 30s,来对芯片表面进行再生。使用Biacore  T200评估软件第3.0版,通过1:1结合模型拟合结合和解离传感图来计算结合速率(Kon)和解离速率(Koff)。以比率Koff/Kon计算解离平衡常数(KD)。拟合结果如表1和表2所示。
表1.重组抗RSV F蛋白单克隆抗体结合RSV-DS-Cav1-A的亲和力常数
表2.重组抗RSV F蛋白单克隆抗体结合RSV-DS-Cav1-B的亲和力常数
4.4重组抗RSV F蛋白单克隆抗体抑制RSV感染Hep-2细胞
4.4.1重组抗RSV F蛋白单克隆抗体抑制RSV/A2感染Hep-2细胞
抗RSV F蛋白单克隆抗体(R3B1h1、R22B1、Nirsevimab、Clesrovimab和帕利珠单抗)和阴性同型对照抗体,用Hep-2细胞维持培养基(DMEM+2%FBS+1%P/S+2mM Glu)稀释,起始浓度为66.7nM,5倍梯度稀释,共10个浓度梯度,70μL/孔加入96孔无菌全透细胞培养板中。取1支冻存的病毒液RSV/A2,置于P2实验室生物安全柜内缓慢融化,用Hep-2细胞维持培养基稀释至1.5×103PFU/mL,取稀释后的病毒液70μL/孔加入以上的稀释抗体中充分混匀。同时设置单独的细胞对照孔(Hep-2细胞维持培养基140μL/孔)和细胞+RSV/A2孔(Hep-2细胞维持培养基70μL/孔+RSV/A2稀释液70μL/孔)。全部样品加入完成后将培养板加盖放置细胞培养箱(37±1℃,5±1%CO2)中和1h。取生长对数期的Hep-2细胞,消化离心后用Hep-2细胞维持培养基将 计数后的细胞悬液稀释至4.3×105个/mL的单细胞悬液,70μL/孔接种于已完成中和的96孔细胞培养板中,置细胞培养箱(37±1℃,5±1%CO2)孵育60-72h。病毒侵染结束后用PBS清洗2次,加入80%丙酮,移入2~8℃冰箱固定30min。弃去80%丙酮,用PBS清洗2次后,加入PBS稀释至10μg/mL的检测抗体帕利珠单抗(100μL/孔),37℃避光孵育60min。弃去一抗,用PBS清洗2次后,加入PBS稀释(1:400)的FITC标记山羊抗人IgG(北京中杉金桥生物技术有限公司,ZF-0308),100μL/孔,37℃避光孵育40min。弃去二抗,用PBS清洗2次,通过快速荧光灶抑制试验(Rapid Fluorescent Focus Inhibition Test,RFFIT)使用多功能酶标仪(SpectraMax I3)检测化学发光值。结果显示(图5),R3B1h1活性最好,根据IC50值比较(表3),活性比Nirsevimab提高约5倍,比Clesrovimab提高约12倍,比帕利珠单抗提高约410倍。
表3.抗RSV F蛋白单克隆抗体抑制RSV/A2毒株感染Hep-2细胞的半抑制浓度
4.4.2重组抗RSV F蛋白单克隆抗体抑制RSV/18537毒株感染Hep-2细胞
参照4.4.1的方案检测抗RSV F蛋白单克隆抗体(R3B1h1、R22B1、Nirsevimab、Clesrovimab和帕利珠单抗)对RSV/18537的中和活性。结果显示(图6),R3B1h1活性最好,根据IC50值比较(表4),活性比Nirsevimab提高约18倍,比Clesrovimab提高约12倍,比帕利珠单抗提高约545倍。
表4.抗RSV F蛋白单克隆抗体抑制RSV/18537毒株感染Hep-2细胞的半抑制浓度

4.4.3重组抗RSV F蛋白单克隆抗体抑制临床分离的RSV感染Hep-2细胞
参照4.4.1的方案检测抗RSV F蛋白单克隆抗体(R3B1h1、R22B1、Nirsevimab、Clesrovimab和帕利珠单抗)对临床分离的RSV的中和活性。
从广州妇女儿童医疗中心获得从2021年RSV流行季临床样本分离的病毒株,共11株。其中,9株为A亚型(编号分别为2033、2064、2066、2406、2410、2416、2425、2430和2438),2株为B亚型(编号分别为2063和2427)。结果显示(图7),R3B1h1和R22B1均能很好的中和RSV临床分离样本。
序列信息






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Claims (12)

  1. 针对呼吸道合胞病毒(RSV)的抗体,其包含含HCDR1、HCDR2和HCDR3的氨基酸序列的重链可变区和含LCDR1、LCDR2和LCDR3的氨基酸序列的轻链可变区,其中
    所述HCDR1的氨基酸序列如SEQ ID NO:32所示、所述HCDR2的氨基酸序列如SEQ ID NO:33所示、所述HCDR3的氨基酸序列如SEQ ID NO:34所示,所述LCDR1的氨基酸序列如SEQ ID NO:35所示、所述LCDR2的氨基酸序列如SEQ ID NO:36所示和所述LCDR3的氨基酸序列如SEQ ID NO:37所示;或者
    所述HCDR1的氨基酸序列如SEQ ID NO:38所示、所述HCDR2的氨基酸序列如SEQ ID NO:39所示、所述HCDR3的氨基酸序列如SEQ ID NO:40所示,所述LCDR1的氨基酸序列如SEQ ID NO:41所示、所述LCDR2的氨基酸序列如SEQ ID NO:42所示和所述LCDR3的氨基酸序列如SEQ ID NO:43所示;
    其中,HCDR和LCDR氨基酸序列根据Kabat定义。
  2. 如权利要求1所述的抗体,其中所述抗体重链可变区的氨基酸序列如SEQ ID NO:28或30所示。
  3. 如权利要求1所述的抗体,其中所述抗体轻链可变区的氨基酸序列如SEQ ID NO:29或31所示。
  4. 如权利要求1-3中任一项所述的抗体,其中
    所述抗体重链可变区的氨基酸序列如SEQ ID NO:28所示,所述抗体轻链可变区的氨基酸序列如SEQ ID NO:29所示;或者
    所述抗体重链可变区的氨基酸序列如SEQ ID NO:30所示,所述抗体轻链可变区的氨基酸序列如SEQ ID NO:31所示。
  5. 针对呼吸道合胞病毒(RSV)的抗体,其中所述抗体的重链可变区 的氨基酸序列与SEQ ID NO:28或30具有至少90%的同一性,并且所述抗体的轻链可变区的氨基酸序列与SEQ ID NO:29或31具有至少90%的同一性。
  6. 如权利要求1-5中任一项所述的抗体,其中
    所述抗体为中和性抗体;和/或
    所述抗体能够结合人呼吸道合胞病毒(RSV)的F蛋白;优选地,所述抗体能够结合如SEQ ID NO:12或13所示的重组人RSV F蛋白;和/或
    所述抗体为Fab片段、全抗体、F(ab’)2片段或单链Fv片段(scFv);优选地,所述抗体为Fab片段;和/或
    所述抗体为单克隆抗体。
  7. 如权利要求1-6中任一项所述的抗体,其中
    所述抗体包含选自IgG1亚型、IgG2亚型或IgG4亚型的重链恒定区;优选地,所述重链恒定区包含IgG1亚型重链恒定区的Fc段序列并且所述Fc段序列的第252,254,256位的氨基酸序列分别为Y,T和E,其中所述抗体恒定区氨基酸顺序按照EU numbering来确定;和/或
    所述抗体包含选自κ亚型或λ亚型的轻链恒定区。
  8. 核酸分子,其编码权利要求1-7中任一项所述的抗体。
  9. 药物组合物,其包含权利要求1-7中任一项所述的抗体以及药学上可接受的赋形剂、稀释剂或载体。
  10. 如权利要求9所述的药物组合物,其用于预防或治疗RSV相关疾病;优选地,所述RSV相关疾病为呼吸道感染,例如细支气管炎和肺炎。
  11. 权利要求1-7中任一项所述的抗体、权利要求8所述的核酸分子、或者权利要求9或10所述的药物组合物在制备预防或治疗RSV相关疾病的药物的用途;优选地,所述RSV相关疾病为呼吸道感染,例如细支气管炎和肺炎。
  12. 预防或治疗RSV相关疾病的方法,其包括向有需要的个体给予权利要求1-7中任一项所述的抗体、权利要求8所述的核酸分子、或者权利要求9或10所述的药物组合物;优选地,所述RSV相关疾病为呼吸道感染,例如细支气管炎和肺炎。
PCT/CN2023/097016 2022-07-22 2023-05-30 抗呼吸道合胞病毒中和性抗体及其用途 WO2024016842A1 (zh)

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