WO2020119664A1 - 抗h7n9全人源单克隆抗体2g3及其制法与应用 - Google Patents

抗h7n9全人源单克隆抗体2g3及其制法与应用 Download PDF

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WO2020119664A1
WO2020119664A1 PCT/CN2019/124192 CN2019124192W WO2020119664A1 WO 2020119664 A1 WO2020119664 A1 WO 2020119664A1 CN 2019124192 W CN2019124192 W CN 2019124192W WO 2020119664 A1 WO2020119664 A1 WO 2020119664A1
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monoclonal antibody
antibody
gene
amino acid
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万晓春
李俊鑫
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中国科学院深圳先进技术研究院
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1018—Orthomyxoviridae, e.g. influenza virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/42—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum viral
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the invention belongs to the field of immunology, and in particular relates to an anti-H7N9 fully human monoclonal antibody 2G3 and its preparation method and application.
  • Humira an anti-TNFa monoclonal antibody for arthritis, which is a fully human monoclonal antibody. It has been the king of medicine for more than 10 billion sales for three consecutive years. Since the launch of the first monoclonal antibody drug in 1986, monoclonal antibody drugs have undergone murine monoclonal antibody drugs (such as Orthoclone OKT3), chimeric monoclonal antibody drugs (Rituximab), humanized monoclonal antibody drugs (Herceptin) and the whole person Source monoclonal antibody drugs (Humira) and other stages.
  • murine monoclonal antibody drugs such as Orthoclone OKT3
  • Renituximab chimeric monoclonal antibody drugs
  • Herceptin humanized monoclonal antibody drugs
  • Humira the whole person Source monoclonal antibody drugs
  • HAMA anti-mouse antibody reaction
  • the monoclonal antibody drugs currently occupying the market are all humanized monoclonal antibody drugs.
  • Shenzhen and even China have a large gap, mainly manifested in the weak innovation ability in the field of human antibody drugs, the independent research and development varieties are few, there is currently no original humanized monoclonal
  • the huge antibody drug market is occupied by foreign pharmaceutical companies. In order to change the backward situation and compete for the domestic and foreign antibody drug markets with huge consumption potential, it is urgent to overcome the technology of fully human monoclonal antibodies.
  • Human-derived monoclonal antibodies are highly specific and effective in treating inflammation, cancer, and particularly influenza.
  • Influenza is an infectious disease caused by influenza virus, which seriously threatens human health. About 1 billion people worldwide are infected with the seasonal influenza virus each year, and 250,000 to 500,000 of them die.
  • the H7N9 virus is an influenza virus that is resistant to the traditional antiviral drugs amantadine and rimantadine, and there is currently no effective treatment. When the H7N9 virus invades the cell, it needs to rely on the specific molecule expressed by the virus itself to bind to the receptor on the human cell in order to infect the cell and further expand.
  • Human antibodies that neutralize viruses are specific antibodies produced by human B lymphocytes, which can bind to antigens on the surface of the virus, thereby preventing the virus from attaching to target cell receptors, preventing the virus from invading cells, and effectively preventing H7N9 influenza.
  • the present application relates to an anti-H7N9 fully human monoclonal antibody 2G3 or a biologically active fragment derived from the monoclonal antibody capable of neutralizing the H7N9 virus.
  • the present application relates to a gene encoding the anti-H7N9 fully human monoclonal antibody 2G3 or a biologically active fragment derived from the monoclonal antibody capable of specifically binding H7N9, and a vector or cell containing the gene.
  • the present application relates to a method for producing the anti-H7N9 fully human monoclonal antibody 2G3.
  • the present application relates to a pharmaceutical composition
  • a pharmaceutical composition comprising the anti-H7N9 fully human monoclonal antibody 2G3 or a biologically active fragment derived from the monoclonal antibody capable of specifically binding H7N9.
  • the present application relates to the application of the anti-H7N9 fully human monoclonal antibody 2G3 described in the present application or a biologically active fragment derived from the monoclonal antibody capable of specifically binding H7N9 or the pharmaceutical composition.
  • the present application relates to a kit for detecting H7N9 virus.
  • FIG. 1 is a flow cytometry result diagram of CD40L expression by NTH-3T3 in Example 1.
  • FIG. 1 is a flow cytometry result diagram of CD40L expression by NTH-3T3 in Example 1.
  • FIG. 2 is a graph showing the results of sorting memory B cells by flow cytometry in Example 1.
  • Example 3 is a graph of the ELISA experiment results in Example 1.
  • FIG. 4 is a graph of the neutralization experiment results of Example 3.
  • the present application provides an anti-H7N9 fully human monoclonal antibody 2G3 or a biologically active fragment derived from the monoclonal antibody capable of specifically binding H7N9, wherein the amino acid sequence of the heavy and light chain CDR1, CDR2 and CDR3 regions of the antibody They are as follows:
  • Heavy chain CDR2 MNPESGET
  • Heavy chain CDR3 ATGNAECSGGSCYNWFEP;
  • Light chain CDR3 NSKETSGYHLV.
  • amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO: 2, or an amino acid sequence with equivalent functions formed by replacing, deleting, or adding one or several amino acids to the sequence; and/ or
  • amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO: 4, or an amino acid sequence with equivalent functions formed by replacing, deleting or adding one or several amino acids to the sequence.
  • the heavy chain amino acid sequence of the antibody is shown in SEQ ID NO: 6, or an amino acid sequence with equivalent functions formed by replacing, deleting, or adding one or more amino acids to the sequence; and/or
  • the light chain amino acid sequence of the antibody is shown in SEQ ID NO: 8, or an amino acid sequence with equivalent functions formed by replacing, deleting or adding one or several amino acids to the sequence.
  • the anti-H7N9 fully human monoclonal antibody 2G3 described in this application can target the hemagglutinin HA that binds to the H7N9 virus, with an affinity of 2.3 ⁇ 10 -9 M; in a virus-infected cell model, its IC 50 value Only about 0.0711uM.
  • the antibodies described in this application are fully human monoclonal antibodies. Compared with murine antibodies, the genes of fully human antibodies are completely derived from human genes, without the components of other species, and no anti-mouse anti-antibodies occur in the human body. Toxic and side effects, with better biocompatibility, more suitable and more potential to become a macromolecular drug for the treatment of influenza virus.
  • the present application provides a gene encoding the anti-H7N9 fully human monoclonal antibody 2G3 described herein.
  • the gene comprises a nucleotide sequence encoding the amino acid shown in SEQ ID NO: 2, in some embodiments, the nucleotide sequence is shown in SEQ ID NO: 1; and/or
  • the gene contains a nucleotide sequence encoding the amino acid shown in SEQ ID NO: 4, and in some embodiments, the nucleotide sequence is shown in SEQ ID NO: 3.
  • the gene comprises a nucleotide sequence encoding an amino acid having SEQ ID NO: 6, in some embodiments, the nucleotide sequence is shown in SEQ ID NO: 5; and/or
  • the gene includes a nucleotide sequence encoding the amino acid shown in SEQ ID NO: 8, and in some embodiments, the nucleotide sequence is shown in SEQ ID NO: 7.
  • sequence of SEQ ID NO: 1 ⁇ 8 in this application is shown in the sequence table, in which:
  • Sequences 1 to 48 in SEQ ID NO: 5 and SEQ ID NO: 7 are sequences used to encode signal peptides.
  • amino acid sequences at positions 26-33 in SEQ ID NO: 2 and SEQ ID NO: 6 are heavy chain CDR1 sequences; the amino acid sequences at positions 51-58 in SEQ ID NO: 2 and SEQ ID NO: 6 are Heavy chain CDR2 sequence; amino acid sequence 97-114 in SEQ ID NO: 2 and SEQ ID NO: 6 is the heavy chain CDR3 sequence.
  • the amino acid sequence at positions 26-31 in SEQ ID NO: 4 and SEQ ID NO: 8 is the light chain CDR1 sequence; the amino acid sequence at positions 49-51 in SEQ ID NO: 4 and SEQ ID: NO: 8 is The light chain CDR2 sequence; the amino acid sequences 88-98 in SEQ ID NO: 4 and SEQ ID NO: 8 are light chain CDR3 sequences.
  • the present application provides a vector containing the gene as described above.
  • the present application provides cells containing the gene as described above or the vector as described above.
  • the present application provides a method for producing the anti-H7N9 fully human monoclonal antibody 2G3 or a biologically active fragment derived from the monoclonal antibody capable of specifically binding H7N9, the method including culturing The above-mentioned genes of the heavy and light chains of the human monoclonal antibody 2G3 or the genetically engineered cells of the above vector or directly culture the above cells, collect and purify the anti-H7N9 fully human monoclonal antibody 2G3.
  • the present application provides a pharmaceutical composition
  • a pharmaceutical composition comprising the anti-H7N9 fully human monoclonal antibody 2G3 described herein or a biologically active fragment derived from the monoclonal antibody capable of specifically binding H7N9.
  • the present application provides the anti-H7N9 fully human monoclonal antibody 2G3 or a biologically active fragment derived from the monoclonal antibody capable of specifically binding H7N9 or the pharmaceutical composition is prepared for treatment by H7N9 Application of drugs for diseases caused by viruses.
  • the present application provides a kit for detecting the level of H7N9 virus, which contains the anti-H7N9 fully human monoclonal antibody 2G3 or a biologically active fragment derived from the monoclonal antibody capable of specifically binding H7N9 ;
  • the kit further contains a second antibody and an enzyme or fluorescent or radioactive label for detection, and a buffer; the second antibody is, for example, anti-monoclonal antibody 2G3 described in this application Anti-antibody.
  • the anti-H7N9 fully human monoclonal antibody 2G3 described in this application can target hemagglutinin HA that binds to the H7N9 virus, and has significant neutralizing activity against H7N9 virus infection.
  • Lentivirus was used to establish 3T3-CD40L feeder cells.
  • the lentiviral expression vector pLVX-CD40L was constructed, transfected into 293T cells, and the viral supernatant was collected on the fourth day of transfection.
  • Activate NIH-3T3 cells infect with lentivirus after culturing for 3 generations, continue culturing and pass 3 times.
  • Use flow cytometry to sort the cells whose FITC fluorescence intensity is near the MFI, re-add to the culture flask, culture and test at 37°C, 5% CO 2 incubator. The test results are shown in Figure 1.
  • CD40L 3T3 cells and empty vector pLVX (with ZxGreen) transfected 3T3 cells were stained with anti-CD40L with APC, and then analyzed by flow cytometry. As a result, it was found that all 3T3-CD40L feeder cells expressed CD40L.
  • the cells grow to 80% to 90%, the cells are digested and collected at a concentration of 1 ⁇ 10 7 cells per ml. Place in a radiometer for 5000 rads radiation, resuspend the cells in the cryopreservation solution at a concentration of 3.5 ⁇ 10 7 cells per ml, aliquot 1 ml in a frozen vial, and freeze in liquid nitrogen (can be stored for 2 years).
  • Lymph separation solution was used to separate and freeze the PBMC of rehabilitation patients who had been infected with H7N9 virus. Each tube contains 10-50 ⁇ 10 6 cells, and is frozen in a liquid nitrogen tank. Prepare PBMC flow dyeing solution, its composition is shown in Table 1 below
  • the memory B cells were added to the mixed medium. After mixing, the cells were diluted in a 384-well plate with a limit of 1 cell per well and the volume was 50 ⁇ l. The cells were placed in a 37°C, 5% CO 2 incubator and cultured statically. After 13 days, the supernatant was taken for ELISA.
  • Influenza virus hemagglutinin HA is a columnar antigen on the surface of the viral envelope. It can bind to a variety of red blood cell receptors such as humans, chickens, and guinea pigs to cause red blood cell aggregation. It is immunogenic and anti-hemagglutinin antibodies can neutralize influenza viruses.
  • B cells capable of secreting the antibody 2G3 that binds to the H7N9 virus were found by ELISA, and the human monoclonal antibody 2G3 secreted by it can target the hemagglutinin HA that binds to the H7N9 virus ( Figure 3).
  • B cells obtained in Example 1 capable of secreting the 2G3 antibody that binds to the H7N9 virus were lysed, and the lysate was taken for reverse transcription of RNA to obtain a PCR template cDNA of the human antibody gene.
  • Reverse transcription system 150ng random primer (invitrogen, 48190-011), 0.5 ⁇ l 10mM dNTP (Invitrogen, 18427-088), 1 ⁇ l 0.1M DTT (Invitrogen, 18080-044), 0.5% v/v Igepal CA -630 (Sigma, I3021-50ML), 4U RNAsin (Promega), 6U Prime RNAse Inhibitor (Eppendorf) and 50 U III reverse transcriptase (Invitrogen, 18080-044), supplemented with DEPC water to 14 ⁇ l/well.
  • the sequencing result of the PCR product of the variable region of the heavy chain of the antibody gene is shown in the sequence shown in SEQ ID NO: 1, and the corresponding amino acid sequence is shown in the sequence shown in SEQ ID NO: 2.
  • the sequencing result of the PCR product of the light chain variable region of the antibody gene is shown in the sequence shown in SEQ ID NO: 3, and the corresponding amino acid sequence is shown in the sequence shown in SEQ ID NO: 4.
  • the supernatant contains antibodies capable of binding to the H7N9 virus.
  • virus-infected cell model canine kidney cell MDCK
  • the inhibitory effect and effect of 2G3 antibody against H7N9 influenza virus were evaluated by micro-neutralization-ELISA experiment, and the antibody anti-influenza virus activity was detected.
  • MDCK canine kidney cells in logarithmic growth phase after termination, centrifugal collection, evenly disperse, prepare single cell suspension; adjust cell concentration to 5 ⁇ 10 4 cells/ml with cell culture solution, and inoculate in 96-well cell culture Plates, cells were incubated overnight at 37°C in a 5% CO 2 incubator.
  • step (2.1) Discard the cell culture supernatant cultured in step (2.1) and wash 3 times with PBS.
  • the antibody premixed - virus mixed solution (10-fold serially diluted to 10-10 monoclonal antibody 2G3 10 2 ⁇ g / ml of the 2G3 antibody, respectively, and each concentration an equal volume of virus 100TCID 50 mixing the mixture obtained ).
  • TPCK-Trypsin (maintenance solution) with a final concentration of 2 ⁇ g/ml was added to serum-free DMEM.
  • the PBS in the 96-well plate was discarded, 100 ⁇ l of maintenance solution was added to each well, and incubated at 37° C. in a 5% CO 2 incubator for 20 h.
  • Inhibition rate [(OD virus well-OD negative cell control well)-(OD drug well-OD negative cell control well)]/(OD virus well-OD negative cell control well) ⁇ 100%.
  • the IC 50 of the 2G3 neutralizing activity of this application is 0.0711ug/mL, while the 2L11 and 2J17 antibodies have no neutralizing activity.
  • the 2G3 antibody of the present application has a lower IC 50 and stronger neutralizing virus activity, and the virus can be killed by using a low-dose antibody.
  • the instrument of affinity detection is Fortebio of PALL. Prepare 200 ⁇ l 50 ⁇ g/ml 2G3 antibody, bind proteinA sensor for 120 seconds, HA antigen prepare 100nM, 50nM, 2.5nM, 12.5nM and 6.25nM and 0nM concentration solutions, bind antibody for 120 seconds, dissociation time is 5 minutes, display 2G3 pair

Abstract

提供的是抗H7N9全人源单克隆抗体2G3及其制法与应用。所述抗体可靶向结合H7N9病毒的血凝素HA，具有显著地抗H7N9病毒感染的中和活性；所述抗体不发生抗鼠抗抗体等毒副作用，具有更好的生物相容性，更适合和更有潜力成为治疗流感病毒的大分子药物。

Description

抗H7N9全人源单克隆抗体2G3及其制法与应用 技术领域
本发明属于免疫学领域，具体地涉及一种抗H7N9全人源单克隆抗体2G3及其制法与应用。
背景技术
在2015年全球十大畅销药中，有6个是全人源或人源化单克隆抗体药物。排名第一的是艾伯维公司治疗关节炎的抗TNFa单克隆抗体Humira，这是一个全人源单克隆抗体，已经是连续3年销售额100亿以上的药王。从1986年第一个单克隆抗体药物上市开始，单抗药物经历了鼠源单抗药物(如Orthoclone OKT3)、嵌合单抗药物(Rituximab)、人源化单抗药物(Herceptin)和全人源单抗药物(Humira)等阶段。由于人体出现抗鼠抗体反应(HAMA)，鼠源单抗药物、嵌合单抗药物已经逐渐被淘汰，目前占据市场的单克隆抗体药物全都是人源化单克隆抗体药物。与国际先进的人源抗体生产技术相比，深圳乃至全中国都有很大差距，主要表现在人源抗体药物领域的创新能力薄弱，自主研发的品种少，目前还没有原创人源化单克隆抗体药物上市的报道，庞大的抗体药物市场被国外药企占领。我国要改变落后局面，争夺消费潜力巨大的国内外抗体药物市场，亟需攻克全人源单克隆抗体技术。
人源单克隆抗体在治疗炎症、癌症特别是流行性感冒方面具有高特异性的显著疗效。流行性感冒是由流感病毒引起的传染性疾病，严重威胁人类健康。全球每年约有10亿人受季节性流感病毒感染，其中有25-50万人死亡。H7N9病毒是一种流感病毒，对传统的抗病毒药金刚烷胺(amantadine)和金刚烷乙胺(rimantadine)有耐药性，目前尚没有有效治疗手段。H7N9病毒在入侵细胞时需要依赖病毒自身表达的特定分子与人细胞上的受体结合，才能感染细胞，并进一步扩增。中和病毒的人源抗体是人B淋巴细胞产生的某些特异抗体，能够与病毒表面的抗原结合，从而阻止该病毒黏附靶细胞受体，防止病毒侵入细胞，能够高效防治H7N9流行性感冒。
发明内容
一方面，本申请涉及抗H7N9全人源单克隆抗体2G3或来源于该单克隆抗体的能够中和H7N9病毒的生物活性片段。
另一方面，本申请涉及编码所述抗H7N9全人源单克隆抗体2G3或来源于该单克隆抗体的能够特异性结合H7N9的生物活性片段的基因及含该基因的载体或细胞。
另一方面，本申请涉及产生所述抗H7N9全人源单克隆抗体2G3的方法。
另一方面，本申请涉及包含所述的抗H7N9全人源单克隆抗体2G3或来源于该单克隆抗体的能够特异性结合H7N9的生物活性片段的药物组合物。
另一方面，本申请涉及本申请所述的抗H7N9全人源单克隆抗体2G3或来源于该单克隆抗体的能够特异性结合H7N9的生物活性片段或所述药物组合物的应用。
另一方面，本申请涉及一种检测H7N9病毒的试剂盒。
附图说明
图1为实施例1中NTH-3T3表达CD40L的流式检测结果图。
图2为实施例1中流式细胞仪分选记忆B细胞结果图。
图3为实施例1中ELISA实验结果图。
图4为实施例3中和实验结果图。
具体实施方式
为了对本发明的技术特征、目的和有益效果有更加清楚的理解，现对本发明的技术方案进行以下详细说明，但不能理解为对本发明的可实施范围的限定。
一方面，本申请提供抗H7N9全人源单克隆抗体2G3或来源于该单克隆抗体的能够特异性结合H7N9的生物活性片段，其中，该抗体的重轻链CDR1、CDR2及CDR3区的氨基酸序列分别如下所示：
重链CDR1:GYIFTSYE；
重链CDR2:MNPESGET；
重链CDR3:ATGNAECSGGSCYNWFEP；
轻链CDR1:KLKSYY；
轻链CDR2:GRN；
轻链CDR3:NSKETSGYHLV。
在一些实施方式中，该抗体的重链可变区氨基酸序列如SEQ ID NO:2所示，或该序列经替换、缺失或添加一个或几个氨基酸形成的具有同等功能的氨基酸序列；和/或
该抗体的轻链可变区氨基酸序列如SEQ ID NO:4所示，或该序列经替换、缺失或添加一个或几个氨基酸形成的具有同等功能的氨基酸序列。
在一些实施方式中，该抗体的重链氨基酸序列如SEQ ID NO:6所示，或该序列经替换、缺失或添加一个或几个氨基酸形成的具有同等功能的氨基酸序列；和/或
该抗体的轻链氨基酸序列如SEQ ID NO:8所示，或该序列经替换、缺失或添加一个或几个氨基酸形成的具有同等功能的氨基酸序列。
经ELISA实验验证，本申请所述抗H7N9全人源单克隆抗体2G3可以靶向结合H7N9病毒的血凝素HA，亲和力为2.3×10 -9M；在病毒感染细胞模型中，其IC 50值仅为0.0711uM左右。本申请所述抗体为全人源性单克隆抗体，相比鼠源抗体，全人源抗体的基因完全来源于人的基因，没有其他种属的成分，在人体内不发生抗鼠抗抗体等毒副作用，具有更好的生物相容性，更适合和更有潜力成为治疗流感病毒的大分子药物。
另一方面，本申请提供编码本申请所述的抗H7N9全人源单克隆抗体2G3的基因。在一些实施方式中，所述基因包含编码具有SEQ ID NO:2所示的氨基酸的核苷酸序列，在一些实施方式中，该核苷酸序列如SEQ ID NO:1所示；和/或
所述基因包含编码具有SEQ ID NO:4所示的氨基酸的核苷酸序列，在一些实施方式中，该核苷酸序列如SEQ ID NO:3所示。
在一些体实施方式中，所述基因包含编码具有SEQ ID NO:6的氨基酸的核苷酸序列，在一些实施方式中，该核苷酸序列如SEQ ID NO:5所示；和/或
所述基因包含编码具有SEQ ID NO:8所示的氨基酸的核苷酸序列，在一些实施方式中，该核苷酸序列如SEQ ID NO:7所示。
本申请SEQ ID NO:1～8的序列如序列表所示，其中：
(1)SEQ ID NO:1中第76～99位序列以及SEQ ID NO:5中第124～147位序列：用于编码重链CDR1区序列；
(2)SEQ ID NO:1中第151～174位序列以及SEQ ID NO:5中第199～222位序列： 用于编码重链CDR2区序列；
(3)SEQ ID NO:1中第289～342位序列以及SEQ ID NO:5中第337～390位序列：用于编码重链CDR3区序列；
(4)SEQ ID NO:3中第76～93位序列以及SEQ ID NO:7中第124～141位序列：用于编码轻链CDR1区序列；
(5)SEQ ID NO:3中第145～153位序列以及SEQ ID NO:7中第193～201位序列：用于编码轻链CDR2区序列；
(6)SEQ ID NO:3中第262～294位序列以及SEQ ID NO:7中第310～342位序列：用于编码轻链CDR3区序列；
(7)SEQ ID NO:5和SEQ ID NO:7中的第1～48位序列为用于编码信号肽的序列。
(8)SEQ ID NO:2和SEQ ID NO:6中的第26-33位氨基酸序列为重链CDR1序列；SEQ ID NO:2和SEQ ID NO:6中的第51-58位氨基酸序列为重链CDR2序列；SEQ ID NO:2和SEQ ID NO:6中第97-114位氨基酸序列为重链CDR3序列。
(9)SEQ ID NO:4和SEQ ID NO:8中的第26-31位氨基酸序列为轻链CDR1序列；SEQ ID NO:4和SEQ ID NO:8中的第49-51位氨基酸序列为轻链CDR2序列；SEQ ID NO:4和SEQ ID NO:8中的第88-98位氨基酸序列为轻链CDR3序列。
另一方面，本申请提供含如上所述基因的载体。
再一方面，本申请提供含如上所述基因或如上所述载体的细胞。
再一方面，本申请提供一种产生所述抗H7N9全人源单克隆抗体2G3或来源于该单克隆抗体的能够特异性结合H7N9的生物活性片段的方法，该方法包括培养含编码抗H7N9全人源单克隆抗体2G3的重轻链的上述基因或上述载体的基因工程细胞或直接培养上述细胞，收集，纯化得所述抗H7N9全人源单克隆抗体2G3。
现有技术中存在采用噬菌体展示技术制备抗H7N9病毒人源单克隆抗体的方法，尽管该方法具有生产成本低、不经过免疫和细胞融合等繁琐工作的优点，但是其缺点也比较明显，从非免疫抗体库中获得的抗体往往亲和力不足、受外源基因转化率的限制、抗体库的库容量不足以涵盖动物的抗体多样性等。本申请从病人的血液中分离分泌功能抗体的B细胞，然后提取RNA和合成cDNA，从中克隆分泌目的抗体的基因，最后重组和表达全人源单克隆抗体。该技术操作简单快捷，生产的人源抗体具有高亲 和力和特异性，此外，可进一步采用改进的从记忆B细胞中分离具有中和病毒功能或杀伤肿瘤功能的本申请所述单克隆抗体技术，更是大大减少了繁琐操作和成本。
另一方面，本申请提供一种药物组合物，其包含本申请所述的抗H7N9全人源单克隆抗体2G3或来源于该单克隆抗体的能够特异性结合H7N9的生物活性片段。
另一方面，本申请提供所述的抗H7N9全人源单克隆抗体2G3或来源于该单克隆抗体的能够特异性结合H7N9的生物活性片段或所述的药物组合物在制备用于治疗由H7N9病毒引起的疾病的药物中的应用。
另一方面，本申请提供一种检测H7N9病毒水平的试剂盒，其含有本申请所述的抗H7N9全人源单克隆抗体2G3或来源于该单克隆抗体的能够特异性结合H7N9的生物活性片段；在一些实施方式中，所述的试剂盒还含有第二抗体和用于检测的酶或荧光或放射标记物，以及缓冲液；所述第二抗体例如为抗本申请所述单克隆抗体2G3的抗抗体。
与现有技术相比，本申请具有如下有益效果：
(1)本申请所述抗H7N9全人源单克隆抗体2G3可以靶向结合H7N9病毒的血凝素HA，具有显著抗H7N9病毒感染的中和活性。
(2)相比鼠源抗体，本申请全人源抗体的基因完全来源于人的基因，没有其他种属的成分，在人体内不发生抗鼠抗抗体等毒副作用，具有更好的生物相容性，更适合和更有潜力成为治疗流感病毒的大分子药物。
(3)相较于现有技术提供的噬菌体展示技术制备抗H7N9病毒人源单克隆抗体的方法，本申请采用的单个B细胞开发抗H7N9病毒的抗体具有操作简单快捷，生产的人源抗体具有高亲和力和特异性等优点。
为了对本申请的技术特征、目的和有益效果有更加清楚的理解，现结合具体实施例对本申请的技术方案进行以下详细说明，应理解这些实例仅用于说明本申请而不用于限制本申请的范围。实施例中，各原始试剂材料均可商购获得，未注明具体条件的实验方法为所属领域熟知的常规方法和常规条件，或按照仪器制造商所建议的条件。
实施例1
(1)构建稳定表达CD40L的NTH-3T3细胞系(3T3-CD40L)
利用慢病毒建立3T3-CD40L饲养细胞。构建慢病毒表达载体pLVX-CD40L，转染293T细胞，转染第四天收集病毒上清液。活化NIH-3T3细胞，培养3代后用慢病 毒感染，继续培养并传代3次。利用流式细胞仪进行分选FITC荧光强度在MFI附近的细胞，重新加入至培养瓶中，37℃，5％CO 2培养箱中培养和检测，检测结果如图1所示，其是将表达CD40L的3T3细胞和空载体pLVX(带有ZxGreen)转染的3T3细胞分别用带有APC的抗CD40L染色，然后上流式细胞仪分析。结果发现，所有3T3-CD40L饲养细胞都表达CD40L。当细胞长到80％～90％时，消化收集细胞，浓度为每毫升1×10 7细胞。置于辐射仪中进行5000rads辐射，冻存液重悬细胞，浓度为每毫升3.5×10 7细胞，分装1ml在冷冻小管，液氮冻存(可以保存2年)。
(2)记忆B细胞的分选和活化
用淋巴分离液分离和冻存曾经感染H7N9病毒的康复病人的PBMC，每管10～50×10 6细胞，冻存在液氮罐中。配制PBMC流式染色液，其成分如下表1所示
表1 PBMC流式染色液
抗体 体积(μL)
CD19-PE-Cy7 0.5
IgM-PE 1.0
IgA-APC 2.5
IgD-FITC 2.5
PBS-1％(wt/vol)BSA 43.5
解冻PBMC，加入上述PBMC流式染色液并在流式细胞仪上分选，结果如图2所示，分选出CD19 +IgM –IgA –IgD –的记忆B细胞，细胞纯度需在90％以上，若低于90％，重复分选过程。配制激活B细胞的混合培养基，如下表2所示：
表2
组分 体积
完全IMDM培养基 336mL
IL-2(10,000U mL -1) 3.5mL
IL-21(100μg mL -1) 175μL
步骤(1)中得到的3T3-CD40L 10mL
将记忆B细胞加入到混合培养基中，混匀后有限稀释在384孔板，每孔1个细胞，体积为50μl，置于37℃，5％CO 2培养箱中静置培养。13天后，取上清液进行ELISA。
(3)获得人源单克隆抗体2G3
流感病毒血凝素HA是病毒包膜表面柱状抗原，能与人、鸡、豚鼠等多种红细胞受体结合引起红细胞凝集，具有免疫原性，抗血凝素抗体可以中和流感病毒。本发明 中，通过ELISA发现了能够分泌结合H7N9病毒的抗体2G3的B细胞，其分泌的人源单克隆抗体2G3可以靶向结合H7N9病毒的血凝素HA(图3)。
ELISA实验具体操作：
(1)将100ng/100μl的H7N9病毒的HA蛋白(购自ACROBiosystems)包被在96孔酶标板中，每孔100μl；
(2)放置4℃冰箱过夜；
(3)用PBST溶液洗涤三遍，每孔加5％的脱脂奶粉溶液200μl，37℃孵育1小时；
(4)用PBST溶液洗涤三遍，加100μl没有感染病毒的正常人血清(阴性对照)或加感染病毒的病人血清或抗H7N9全人源单克隆抗体2G3，各三个重复；
(5)37℃孵育1小时后用PBST溶液洗涤三遍；
(6)以1:5000稀释带HRP的抗人IgG抗体(abcam)，加入酶标版中，每孔100μl；
(7)37℃孵育1小时后用PBST溶液洗涤三遍；
(8)每孔加100μl TMB底物溶液(Thermo Scientific)，37℃ 5分钟；
(9)每孔加终止溶液2M硫酸100μl，立刻在酶标仪中450nm波长检测吸光值。其结果如图3所示，ELISA实验表明本申请获得的人源单克隆抗体2G3可以靶向结合H7N9病毒的血凝素HA。
实施例2人源化单克隆抗体2G3基因的克隆、重组、表达和纯化
将实施例1获得的能够分泌结合H7N9病毒的2G3抗体的B细胞进行裂解，取裂解液进行RNA的反转录，获得人源抗体基因的PCR模板cDNA。设计和合成克隆抗体基因的引物，以cDNA为模板克隆抗体的重链和轻链的基因，并且重组在真核细胞293F或HEK293中进行表达和纯化。具体地：
(1)将裂解后的B细胞液转移至96孔板(Eppendorf，030133366)。
(2)反转录体系：150ng随机引物(invitrogen,48190-011),0.5μl 10mM dNTP(Invitrogen,18427-088),1μl 0.1M DTT(Invitrogen,18080-044),0.5％v/v Igepal CA-630(Sigma,I3021-50ML),4U RNAsin(Promega),6U Prime RNAse Inhibitor(Eppendorf)and 50 U
Figure PCTCN2019124192-appb-000001
III reverse transcriptase(Invitrogen,18080-044)，补DEPC水至14μl/well。
(3)反转录反应程序：42℃，10min；25℃，10min；50℃，60min；94℃，5min。
(4)cDNA保存在-20℃。
(5)引物的设计和合成:
正向引物5′-3′序列(Forward Primer 5′-3′sequence)
重链可变区PCR引物:
5′VH1 CTGCAACCGGTGTACATTCCCAGGTGCAGCTGGTGCAG(SEQ ID NO:9)
5′VH1/5 CTGCAACCGGTGTACATTCCGAGGTGCAGCTGGTGCAG(SEQ ID NO:10)
5′VH3 CTGCAACCGGTGTACATTCTGAGGTGCAGCTGGTGGAG(SEQ ID NO:11)
5′VH3-23 CTGCAACCGGTGTACATTCTGAGGTGCAGCTGTTGGAG(SEQ ID NO:12)
5′VH4 CTGCAACCGGTGTACATTCCCAGGTGCAGCTGCAGGAG(SEQ ID NO:13)
5′VH 4-34 CTGCAACCGGTGTACATTCCCAGGTGCAGCTACAGCAGTG(SEQ ID NO:14)
5′VH 1-18 CTGCAACCGGTGTACATTCCCAGGTTCAGCTGGTGCAG(SEQ ID NO:15)
5′VH 1-24 CTGCAACCGGTGTACATTCCCAGGTCCAGCTGGTACAG(SEQ ID NO:16)
5′VH3-33 CTGCAACCGGTGTACATTCTCAGGTGCAGCTGGTGGAG(SEQ ID NO:17)
5′VH 3-9 CTGCAACCGGTGTACATTCTGAAGTGCAGCTGGTGGAG(SEQ ID NO:18)
5′VH4-39 CTGCAACCGGTGTACATTCCCAGCTGCAGCTGCAGGAG(SEQ ID NO:19)
5′VH 6-1 CTGCAACCGGTGTACATTCCCAGGTACAGCTGCAGCAG(SEQ ID NO:20)
3′JH 1/2/4/5 TGCGAAGTCGACGCTGAGGAGACGGTGACCAG(SEQ ID NO:21)
3′JH 3 TGCGAAGTCGACGCTGAAGAGACGGTGACCATTG(SEQ ID NO:22)
3′JH 6 TGCGAAGTCGACGCTGAGGAGACGGTGACCGTG(SEQ ID NO:23)
κ轻链可变区PCR产物
5′Vκ 1-5 CTGCAACCGGTGTACATTCTGACATCCAGATGACCCAGTC(SEQ ID NO:24)
5′Vκ 1-9 TTGTGCTGCAACCGGTGTACATTCAGACATCCAGTTGACCCAGTCT(SEQ ID NO:25)
5′Vκ 1D-43 CTGCAACCGGTGTACATTGTGCCATCCGGATGACCCAGTC(SEQ ID NO:26)
5′Vκ 2-24 CTGCAACCGGTGTACATGGGGATATTGTGATGACCCAGAC(SEQ ID NO:27)
5′Vκ 2-28 CTGCAACCGGTGTACATGGGGATATTGTGATGACTCAGTC(SEQ ID NO:28)
5′Vκ 2-30 CTGCAACCGGTGTACATGGGGATGTTGTGATGACTCAGTC(SEQ ID NO:29)
5′Vκ 3-11 TTGTGCTGCAACCGGTGTACATTCAGAAATTGTGTTGACACAGTC(SEQ ID NO:30)
5′Vκ 3-15 CTGCAACCGGTGTACATTCAGAAATAGTGATGACGCAGTC(SEQ ID NO:31)
5′Vκ 3-20 TTGTGCTGCAACCGGTGTACATTCAGAAATTGTGTTGACGCAGTCT(SEQ ID NO:32)
5′Vκ 4-1 CTGCAACCGGTGTACATTCGGACATCGTGATGACCCAGTC(SEQ ID NO:33)
3′Jκ 1/4 GCCACCGTACGTTTGATYTCCACCTTGGTC(SEQ ID NO:34)
3′Jκ 2 GCCACCGTACGTTTGATCTCCAGCTTGGTC(SEQ ID NO:35)
3′Jκ 3 GCCACCGTACGTTTGATATCCACTTTGGTC(SEQ ID NO:36)
3′Jκ 5 GCCACCGTACGTTTAATCTCCAGTCGTGTC(SEQ ID NO:37)
(6)用KOD-Plus-Neo(TOYOBO，KOD401)试剂盒PCR分别扩增抗体基因的重链和轻链，40μL体系：3.5μL cDNA，20nM混合引物，4μL缓冲液(buffer)，4μL  2mM dNTPs，2.4μL MgSO 4，1μL KOD。
(7)反应程序：94℃，2min；45个循环：98℃,10s；58℃,30s；68℃,28s。
(8)对扩增产物进行琼脂糖凝胶，结果发现，抗体轻链大小为327bp，重链大小是375bp。
(9)抗体基因重链可变区PCR产物测序结果如SEQ ID NO:1所示序列，其相应的氨基酸序列如SEQ ID NO:2所示序列。抗体基因轻链可变区PCR产物测序结果如SEQ ID NO:3所示序列，其相应的氨基酸序列如SEQ ID NO:4所示序列。
依据所得重轻链可变区序列，设计并委托Invitrogen公司合成抗体基因重链全长H基因，其带有BamH1/EcoR1双酶切位点；及抗体基因轻链全长L基因，其带有Not1/Xho1双酶切位点。
(10)将H基因和pcDNA3.1分别进行BamH1/EcoR1双酶切后相连，形成pcDNA3.1-H载体。
(11)将L基因和pcDNA3.1分别进行Not1/Xho1双酶切后相连，形成pcDNA3.1-L载体。
(12)培养293F细胞。
(13)20μg pcDNA3.1-H(该H基因的核苷酸序列如SEQ ID NO:5所示)载体和10μg pcDNA3.1-L(该L基因的核苷酸序列如SEQ ID NO:7所示)载体共转染293F细胞，培养96小时。
(14)取上清液进行ELISA(ABC是上清液，DEF是阳性对照，GH是阴性对照)；ELISA具体的实验步骤如前所述，ELISA实验结果如下表3和图3所示：
表3
数据 450nm 数据 450nm
A 1.601 E 1.185
B 1.999 F 1.201
C 1.785 G 0.0675
D 1.114 H 0.0554
由表3和图3数据可知：上清液中含有能够结合H7N9病毒的抗体。
(15)纯化过程，具体地，全人源单克隆抗体2G3的纯化过程为：
(a)200μg pcDNA3.1-L载体和100μg pcDNA3.1-H载体共转染300ml 293F细胞，培养96小时。
(b)收集上清液，加入proteinA亲和层析柱，用10倍PBS清洗，加入2ml pH3.0，0.1M甘氨酸收集抗体。收集管中加入100μl中和缓冲液(1M Tri-HCL)，以便及时中和洗脱所得抗体液的pH值。
(c)在磷酸盐缓冲液(PBS)中透析，透析完后，近期用的存放在4℃，长期储存在-20℃。共获得500μg 2G3纯抗体，其重链氨基酸序列如SEQ ID NO:6所示，轻链氨基酸序列如SEQ ID NO:8所示。
实施例3纯化后的全人源单克隆抗体2G3的中和实验及抗体亲和力实验
(1)实验目的
使用病毒感染细胞模型(犬肾细胞MDCK)，通过微量中和-ELISA实验评价2G3抗体对H7N9流感病毒的抑制作用和效果，检测抗体抗流感病毒活性。
(2)实验步骤
(2.1)细胞铺板
胰酶消化对数生长期MDCK犬肾细胞，终止后离心收集，吹散均匀，制备单细胞悬液；用细胞培养液将细胞浓度调整至5×10 4个/ml，接种于96孔细胞培养板，细胞置于37℃、5％CO 2培养箱中培养过夜。
(2.2)2G3抗体与H7N9病毒(该病毒A/Anhui/1/2013取自于中国科学院微生物研究所)预处理
2G3抗体设立10个浓度梯度，依次为10-10 10倍稀释，各组各浓度均设3个平行孔。
(2.3)病毒感染
弃步骤(2.1)培养的细胞培养上清，PBS洗3遍。将预混的抗体-病毒混合液(以10 2μg/ml的2G3单克隆抗体依次以10-10 10倍稀释，将每个浓度的2G3抗体分别与等体积的100TCID 50病毒混合得该混合液)。加入96孔细胞培养板，37℃孵育1h，吸弃混合液，PBS洗2遍。
(2.4)配置维持液
用无血清DMEM中加入终浓度为2μg/ml的TPCK-胰蛋白酶(TPCK-Trypsin)(维持液)。弃去96孔板中的PBS，每孔加入100μl维持液，置于37℃，5％CO 2培养箱培养20h。
(2.5)中和实验-ELISA法
(2.5.1)弃去微量培养板中的维持液；
(2.5.2)100μl PBS洗细胞一次；
(2.5.3)弃去PBS(不要让细胞干燥)，加入50μl/孔固定液(体积比为丙酮：无水乙醇＝2：3)；
(2.5.4)覆盖微量培养板，于室温固定细胞10min；
(2.5.5)弃去固定液，用100μl PBS液洗涤细胞，重复洗涤3次(轻轻晃动，避免强烈清洗)，以去除残余的丙酮。
(2.5.6)用5％脱脂奶粉于室温封闭细胞1h，用100μl PBS液洗涤细胞1次；
(2.5.7)用PBS 1：2000稀释1抗(商购抗H7N9的NP单克隆抗体)每孔加入稀释后的50μl，室温作用1小时。
(2.5.8)用100μl PBST洗板5次以除去1抗；
(2.5.9)用PBS 1:2000稀释2抗(带HRP的抗鼠IgG抗体)，每孔加入50μl，室温作用1小时。
(2.5.10)用100μl PBST洗涤板6次以除去2抗。
(2.5.11)每孔加TMB显色液50μl。
(2.5.12)室温避光放10分钟左右显色后，每孔加2M盐酸50μl终止反应。
(2.5.13)在ELISA测定仪上(450纳米)读出每孔OD值。
(3)统计分析
使用GraphPad Prism 6.0.1对数据进行分析和绘制剂量-效应曲线，并计算IC 50。抑制率计算公式：
抑制率＝[(OD病毒孔-OD阴性细胞对照孔)-(OD药物孔-OD阴性细胞对照孔)]/(OD病毒孔-OD阴性细胞对照孔)×100％。
所得结构如图4所示，从图4中可以看出2G3的IC 50＝0.0711ug/mL。
从实施例3可以看出，本申请2G3对H7N9有良好的IC 50值，证明2G3的中和病毒能力好。
本申请对比了2016年05年10日向中国国家知识产权局递交的，申请号为“201610303416.X”，发明名称为“抗H7N9全人源单克隆抗体2L11及其制法与应用”中的单克隆抗体2L11，及2016年05月03日向中国国家知识产权局递交的，申请号为“201610288358.8”，发明名称为“抗H7N9全人源单克隆抗体2J17及其制法与应 用”中的单克隆抗体2J17，以及2016年11月11日向中国国家知识产权局递交的，申请号为“201611038444.X”，发明名称为“抗H7N9全人源单克隆抗体5J13及其制法与应用”中的单克隆抗体5J13。本申请2G3中和活性的IC 50为0.0711ug/mL，而2L11及2J17抗体没有中和活性。相比5J13抗体，本申请2G3抗体有比较低IC 50和更强的中和病毒活性，使用低剂量抗体就可以杀伤病毒。
抗体亲和力检测：
亲和力检测的仪器为PALL的Fortebio。配制200μl 50μg/ml 2G3抗体，结合proteinA传感器120秒，HA抗原配制100nM、50nM、2.5nM、12.5nM和、6.25nM和0nM浓度溶液，结合抗体120秒，解离时间为5分钟，显示2G3对H7N9病毒有较高亲和力，KD＝2.3×10 -9M。
最后说明的是：以上实施例仅用于说明本申请的实施过程和特点，而非限制本申请的技术方案，尽管参照上述实施例对本申请进行了详细说明，本领域的普通技术人员应当理解：依然可以对本申请进行修改或者等同替换，而不脱离本申请的精神和范围的任何修改或局部替换，均应涵盖在本申请的保护范围当中。

Claims (10)

  1. 抗H7N9全人源单克隆抗体2G3或来源于该单克隆抗体的能够特异性结合H7N9的生物活性片段，其中，所述抗体的重轻链CDR1、CDR2及CDR3区的氨基酸序列分别如下所示：
    重链CDR1:GYIFTSYE；
    重链CDR2:MNPESGET；
    重链CDR3:ATGNAECSGGSCYNWFEP；
    轻链CDR1:KLKSYY；
    轻链CDR2:GRN；
    轻链CDR3:NSKETSGYHLV。
  2. 根据权利要求1所述的抗H7N9全人源单克隆抗体2G3或来源于该单克隆抗体的能够特异性结合H7N9的生物活性片段，其中，所述抗体的重链可变区氨基酸序列如SEQ ID NO:2所示，或SEQ ID NO:2所示序列经替换、缺失或添加一个或几个氨基酸形成的具有同等功能的氨基酸序列；和/或
    所述抗体的轻链可变区氨基酸序列如SEQ ID NO:4所示，或SEQ ID NO:4所示序列经替换、缺失或添加一个或几个氨基酸形成的具有同等功能的氨基酸序列；
    优选地，所述抗体的重链氨基酸序列如SEQ ID NO:6所示，或SEQ ID NO:6所示序列经替换、缺失或添加一个或几个氨基酸形成的具有同等功能的氨基酸序列；和/或
    所述抗体的轻链氨基酸序列如SEQ ID NO:8所示，或SEQ ID NO:8所示序列经替换、缺失或添加一个或几个氨基酸形成的具有同等功能的氨基酸序列。
  3. 编码权利要求1或2所述的抗H7N9全人源单克隆抗体2G3或来源于该单克隆抗体的能够特异性结合H7N9的生物活性片段的基因；优选地，所述基因包含编码具有SEQ ID NO:2所示的氨基酸的核苷酸序列，更优选地，所述基因的核苷酸序列如SEQ ID NO:1所示；和/或
    所述基因包含编码具有SEQ ID NO:4所示的氨基酸的核苷酸序列，优选地，所述基因的核苷酸序列如SEQ ID NO:3所示。
  4. 根据权利要求3所述的基因，其中，所述基因包含编码具有SEQ ID NO:6所示的氨基酸的核苷酸序列，优选地，所述基因的核苷酸序列如SEQ ID NO:5所示； 和/或
    所述基因包含编码具有SEQ ID NO:8所示的氨基酸的核苷酸序列，优选地，所述基因的核苷酸序列如SEQ ID NO:7所示。
  5. 一种载体，其包含权利要求3或4所述基因。
  6. 一种细胞，其含有权利要求3或4所述基因、或含有权利要求5所述载体。
  7. 一种产生权利要求1或2所述抗H7N9全人源单克隆抗体2G3或来源于该单克隆抗体的能够特异性结合H7N9的生物活性片段的方法，该方法包括：
    培养含编码抗H7N9全人源单克隆抗体2G3的重轻链的权利要求3或4所述的基因、或权利要求5所述的载体的基因工程细胞，或直接培养权利要求6所述的细胞；收集，纯化得所述抗H7N9全人源单克隆抗体2G3。
  8. 一种药物组合物，其包含权利要求1或2所述的抗H7N9全人源单克隆抗体2G3或来源于该单克隆抗体的能够特异性结合H7N9的生物活性片段。
  9. 权利要求1或2所述的抗H7N9全人源单克隆抗体2G3或来源于该单克隆抗体的能够特异性结合H7N9的生物活性片段、或权利要求8所述的药物组合物在制备用于治疗由H7N9病毒引起的疾病的药物中的应用。
  10. 一种检测H7N9病毒水平的试剂盒，其含有权利要求1或2所述的抗H7N9全人源单克隆抗体2G3或来源于该单克隆抗体的能够特异性结合H7N9的生物活性片段；优选地，所述试剂盒还含有：第二抗体和用于检测的酶或荧光或放射标记物，以及缓冲液；优选地，所述第二抗体为抗权利要求1或2所述单克隆抗体2G3的抗抗体。
PCT/CN2019/124192 2018-12-13 2019-12-10 抗h7n9全人源单克隆抗体2g3及其制法与应用 WO2020119664A1 (zh)

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