WO2018086599A1 - Anti-h7n9 fully-human monoclonal antibody 5j13, preparation method therefor, and application thereof - Google Patents
Anti-h7n9 fully-human monoclonal antibody 5j13, preparation method therefor, and application thereof Download PDFInfo
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- the present application belongs to the field of immunology, and in particular relates to an anti-H7N9 full human monoclonal antibody 5J13 and a preparation method and application thereof.
- HAMA anti-mouse antibody response
- murine monoclonal antibody drugs and chimeric monoclonal antibodies have been gradually eliminated.
- the monoclonal antibody drugs currently occupying the market are all humanized monoclonal antibody drugs.
- Shenzhen and even China have a large gap, mainly in the field of human antibody drugs, the innovation ability is weak, the number of independent research and development is small, there is no original humanized monoclonal Reports on the listing of antibody drugs, the huge antibody drug market is occupied by foreign pharmaceutical companies. China needs to change the backward situation and compete for the domestic and international antibody drug market with huge consumption potential, and it is urgent to overcome the human monoclonal antibody technology.
- Human monoclonal antibodies have a highly specific and significant effect in the treatment of inflammation, cancer, and especially influenza.
- Influenza is a contagious disease caused by the flu virus that poses a serious threat to human health. About 1 billion people worldwide are infected with seasonal influenza every year, of which 250,000 to 500,000 die.
- the H7N9 virus is an influenza virus that is resistant to the traditional antiviral drugs amantadine and rimantadine. There is currently no effective treatment.
- the H7N9 virus needs to rely on the specific molecule expressed by the virus itself to bind to the receptor on the human cell when invading the cell, in order to infect the cell and further expand.
- the human antibody that neutralizes the virus is a specific antibody produced by human B lymphocytes, which can bind to the antigen on the surface of the virus, thereby preventing the virus from adhering to the target cell receptor, preventing the virus from invading the cell, and effectively preventing and treating H7N9 influenza.
- the application relates to an anti-H7N9 fully human monoclonal antibody 5J13 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 5J13 or a biologically active fragment derived from the monoclonal antibody capable of specifically binding H7N9, and a vector or cell containing the same.
- the present application relates to methods of producing the anti-H7N9 fully human monoclonal antibody 5J13.
- the present application relates to a pharmaceutical composition
- a pharmaceutical composition comprising the anti-H7N9 fully human monoclonal antibody 5J13 or a biologically active fragment derived from the monoclonal antibody capable of specifically binding H7N9.
- the present application relates to the use of an anti-H7N9 fully human monoclonal antibody 5J13 as described herein 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.
- Figure 1 is a graph showing the results of flow cytometry of Example 1 NTH-3T3 expressing CD40L.
- Figure 2 is a graph showing the results of the flow cytometry sorting memory B cells of Example 1.
- Figure 3 is a graph showing the results of the ELISA experiment of Example 1.
- Figure 4 is a graph showing the results of agarose gel electrophoresis of Example 2.
- Figure 5 is a graph showing the results of SDS-PAGE protein electrophoresis of Example 2.
- Fig. 6 is a graph showing the results of the hemagglutination inhibition experiment of Example 3.
- Fig. 7 is a view showing the results of the experiment in Example 4.
- the application provides an anti-H7N9 fully human monoclonal antibody 5J13 or a biologically active fragment derived from the monoclonal antibody capable of specifically binding to H7N9, wherein the amino acid sequence of the heavy light chain CDR1, CDR2 and CDR3 regions of the antibody They are as follows:
- Heavy chain CDR1 region GFSFSNYG;
- Heavy chain CDR2 region ISYDGTNK
- Heavy chain CDR3 region AKGRGPYCSSSICYHGMDV
- Light chain CDR3 region QQYYSTPLT.
- the heavy chain variable region amino acid sequence of the antibody is set forth in SEQ ID NO: 2, or the sequence is substituted, deleted or added with one or more amino acids to form an equivalent amino acid sequence; and / or
- the light chain variable region amino acid sequence of the antibody is set forth in SEQ ID NO: 4, or the sequence is substituted, deleted or added with one or several amino acids to form an equivalent amino acid sequence.
- the heavy chain amino acid sequence of the antibody is set forth in SEQ ID NO: 6 or SEQ ID NO: 10, or the amino acid sequence of the sequence formed by substitution, deletion or addition of one or several amino acids ;and / or
- the light chain amino acid sequence of the antibody is set forth in SEQ ID NO: 8 or SEQ ID NO: 12, or the sequence is substituted, deleted or added with one or several amino acids to form an equivalent amino acid sequence.
- the anti-H7N9 fully human monoclonal antibody 5J13 described in the present application can target hemagglutinin HA which binds to H7N9 virus.
- Hemagglutination inhibition test proved that 5J13 can neutralize H7N9 virus, its IC 50 value can be as low as 0.2071 ⁇ g/ml, KD value is 3.04 ⁇ 10 -10 ; in the virus-infected cell model model, its IC 50 value is only 0.1 ⁇ g/ About ml.
- the antibody described in the present application is a fully human monoclonal antibody.
- the gene of the fully human antibody is completely derived from a human gene, and has no other species, and does not cause anti-mouse anti-antibody in the human body. Toxic side effects, better biocompatibility, more suitable and more potential to be a macromolecular drug for the treatment of influenza virus.
- the invention provides a gene encoding an anti-H7N9 fully human monoclonal antibody 5J13 as described herein.
- the gene comprises a nucleotide sequence encoding the amino acid set forth in SEQ ID NO: 2, and in some embodiments, the nucleotide sequence is set forth in SEQ ID NO: 1;
- the gene comprises a nucleotide sequence encoding the amino acid set forth in SEQ ID NO: 4, and in some embodiments, the nucleotide sequence is set forth in SEQ ID NO: 3.
- the gene comprises a nucleotide sequence encoding an amino acid having SEQ ID NO: 6 or SEQ ID NO: 10, and in some embodiments, the nucleotide sequence is SEQ ID NO: 5 or SEQ ID NO: 9; and/or
- the gene comprises a nucleotide sequence encoding an amino acid having SEQ ID NO: 8 or SEQ ID NO: 12, and in some embodiments, the nucleotide sequence is SEQ ID NO: 7 or SEQ ID NO: 11. Shown.
- the application provides a vector comprising a gene as described above.
- the application provides a cell comprising a gene as described above or a vector as described above.
- the application provides a method for producing the anti-H7N9 fully human monoclonal antibody 5J13 or a biologically active fragment derived from the monoclonal antibody capable of specifically binding H7N9, the method comprising culturing the coding-containing anti-H7N9
- the above-mentioned gene of the heavy light chain of the human monoclonal antibody 5J13 or the genetically engineered cells of the above vector or the above-mentioned cells are directly cultured, and the anti-H7N9 full human monoclonal antibody 5J13 is obtained and purified.
- the technique is simple and rapid to operate, and the produced human antibody has high affinity and specificity. Further, the improved monoclonal antibody technology of the present application having neutralizing viral function or killing tumor function from memory B cells can be further improved. It also greatly reduces the cumbersome operation and cost.
- the present application provides a pharmaceutical composition
- a pharmaceutical composition comprising an anti-H7N9 fully human monoclonal antibody 5J13 as described herein or a biologically active fragment derived from the monoclonal antibody capable of specifically binding H7N9.
- the application provides the anti-H7N9 fully human monoclonal antibody 5J13 or a biologically active fragment derived from the monoclonal antibody capable of specifically binding H7N9 or the pharmaceutical composition prepared for treatment by H7N9
- the application provides a kit for detecting H7N9 virus level, which comprises the anti-H7N9 full human monoclonal antibody 5J13 described in the present application or a biologically active fragment derived from the monoclonal antibody capable of specifically binding H7N9
- the kit further comprises a second antibody and an enzyme or fluorescent or radiolabel for detection, and a buffer; the second antibody is, for example, a monoclonal antibody 5J13 against the present application.
- Anti-antibody is, for example, a monoclonal antibody 5J13 against the present application.
- the anti-H7N9 fully human monoclonal antibody 5J13 described in the present application can target hemagglutinin HA which binds to the H7N9 virus, and has a neutralizing activity against the H7N9 virus infection.
- the gene of the humanized antibody of the present application is completely derived from the human gene, and has no other species of components, and does not cause toxic side effects such as anti-mouse anti-antibodies in the human body, and has a better biological phase. Capacitive, more suitable and more promising as a macromolecular drug for the treatment of influenza virus.
- 3T3-CD40L feeder cells were established using lentivirus.
- the lentiviral expression vector pLVX-CD40L was constructed, transfected into 293T cells, and the virus supernatant was collected on the fourth day of transfection.
- NIH-3T3 cells were activated, cultured for 3 passages, infected with lentivirus, cultured and passaged 3 times.
- the cells with FITC fluorescence intensity near MFI were sorted by flow cytometry, re-added to the culture flask, cultured and detected in a 37 ° C, 5% CO 2 incubator. The test results are shown in Figure 1, which will be expressed.
- 3T3 cells transfected with CD40L and empty vector pLVX (with ZxGreen) were stained with anti-CD40L with APC and then analyzed by upflow cytometry. As a result, it was found that all 3T3-CD40L feeder cells expressed CD40L.
- the cells were collected by digestion at a concentration of 1 ⁇ 10 7 cells per ml. Place in a radiometer for 5000 rads radiation, resuspend the cells in a frozen solution at a concentration of 3.5 ⁇ 10 7 cells per ml, dispense 1 ml in a cryotube, and freeze in liquid nitrogen (can be stored for 2 years).
- PBMC of the rehabilitated patients who had been infected with the H7N9 virus were isolated and frozen with a lymphatic separation solution, and each tube was 10 to 50 ⁇ 10 6 cells, and frozen in a liquid nitrogen tank.
- Prepare PBMC flow dyeing solution the composition of which is shown in Table 1 below.
- the memory B cells were added to the mixed medium, mixed and then diluted in a 384-well plate, one cell per well, and the volume was 50 ⁇ l, and placed in a 37 ° C, 5% CO 2 incubator for static culture. After 13 days, the supernatant was taken for ELISA to obtain human monoclonal antibody 5J13.
- Influenza virus hemagglutinin HA is a columnar antigen on the surface of the virus envelope. It can bind to various red blood cell receptors such as human, chicken and guinea pigs to cause red blood cell agglutination. It is immunogenic and anti-hemagglutinin antibody can neutralize influenza virus.
- An ELISA experiment was performed on the human monoclonal antibody 5J13 obtained above, specifically:
- H7N9 virus HA protein purchased from ACROBiosystems
- the B cells capable of secreting the 5J13 antibody which binds to the H7N9 virus obtained in Example 1 were lysed, and the lysate was subjected to reverse transcription of RNA to obtain a PCR template cDNA of the human antibody gene.
- Designing and synthesizing primers for cloning antibody genes, using cDNA as a template to clone the heavy and light chain genes of antibodies, and recombining in eukaryotic cells 293F or Expression and purification were carried out in HEK293. specifically:
- the lysed B cell solution was transferred to a 96-well plate (Eppendorf, 030133366).
- Reverse transcription system 150 ng random primer (invitrogen, 48190-011), 0.5 ⁇ l 10 mM dNTP (Invitrogen, 18427-088), 1 ⁇ l 0.1 M DTT (Invitrogen, 18080-044), 0.5% v/v Igepal CA -630 (Sigma, I3021-50ML), 4U RNAsin (Promega), 6U Prime RNAse Inhibitor (Eppendorf) and 50U III reverse transcriptase (Invitrogen, 18080-044), supplemented with DEPC water to 14 ⁇ l/well.
- the amplification product was subjected to agarose gel, and the results are shown in Fig. 4. The results showed that the antibody light chain was ⁇ , the variable region size was 339 bp, and the heavy chain variable region was 378 bp.
- Antibody gene heavy chain variable region PCR product sequencing result is the sequence shown in SEQ ID NO: 1, and the corresponding amino acid sequence is the sequence shown in SEQ ID NO: 2.
- the antibody gene light chain variable region PCR product was sequenced as shown in SEQ ID NO: 3, and the corresponding amino acid sequence is the sequence shown in SEQ ID NO: 4.
- Invitrogen was designed and commissioned to synthesize the full-length H gene of the antibody gene heavy chain with the BamH1/EcoR1 double restriction site; and the full-length L gene of the antibody gene light chain. Not1/Xho1 double cleavage site.
- SEQ ID NO: 5 may carry a sequence encoding a signal peptide, such as atgggctggt cctgcatcat Cctgttcctg gtggccaccg ccaccggc) vector and 10 ⁇ g of pcDNA3.1-L (the nucleotide sequence of the L gene without the coding signal peptide is shown as SEQ ID NO: 7, and SEQ ID NO: 7 may carry the sequence encoding the signal peptide, For example, atgggctggt cctgcatcat cctgttcctg gtggccaccg ccaccggc) vector was co-transfected into 293F cells and cultured for 96 hours.
- Purification process specifically, the purification process of the fully human monoclonal antibody 5J13 is:
- reaction plate was shaken on a micro-vibrator for 1 min, and the result was observed after standing at room temperature (20-25 ° C) for 45 min. If the ambient temperature was too high, it could be allowed to stand at 4 ° C for 60 min, and the red blood cell control hole became an obvious button. The result can be determined when the shape sinks to the bottom of the hole.
- the inhibitory effect and effect of the 5J13 antibody against H7N9 influenza virus were evaluated by a virus-infected cell model (dog kidney cell MDCK) by a microneutralization-ELISA assay, and the antibody was tested for anti-influenza virus activity.
- Trypsin digested MDCK canine kidney cells in logarithmic growth phase, centrifuged after centrifugation, and spread evenly to prepare single cell suspension; adjust the cell concentration to 5 ⁇ 10 4 cells/ml with cell culture medium, and inoculate in 96-well cell culture. Plates, cells were incubated overnight at 37 ° C in a 5% CO 2 incubator.
- the 10J13 antibody was set up with 10 concentration gradients, which were 10-10 10- fold dilutions in sequence, and 3 parallel wells were set for each concentration.
- the cell culture supernatant of the cultured step (2.1) was discarded, and washed three times with PBS.
- the premixed antibody-virus mixture (diluted 10 10 10 folds sequentially with 10 2 ⁇ g/ml of 5J13 monoclonal antibody, and each concentration of 5J13 antibody was mixed with an equal volume of 100 TCID 50 virus, respectively. ).
- the cells were added to a 96-well cell culture plate, incubated at 37 ° C for 1 h, and the mixture was discarded and washed twice with PBS.
- TPCK-Trypsin (maintaining solution) at 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 the maintenance solution was added to each well, and cultured at 37 ° C for 20 hours in a 5% CO 2 incubator.
- the plate was washed 6 times with 100 ⁇ l of PBST to remove the 2 antibody.
- 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) x 100%.
- the 5J13 of the present application has a low IC 50 value for both H7N9, which proves that the broad spectrum neutralization of 5J13 is good.
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Abstract
Description
本申请属于免疫学领域，具体地涉及抗H7N9全人源单克隆抗体5J13及其制法与应用。The present application belongs to the field of immunology, and in particular relates to an anti-H7N9 full human monoclonal antibody 5J13 and a preparation method and application thereof.
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。Of the top 10 best-selling drugs in the world in 2015, six are fully human or humanized monoclonal antibody drugs. Ranked first is Aberdeen's anti-TNFa monoclonal antibody Humira, a fully human-derived monoclonal antibody that has been the drug king with more than $10 billion in sales for three consecutive years. Since the first monoclonal antibody drug was launched in 1986, the monoclonal antibody has been treated with a murine monoclonal antibody (such as Orthoclone OKT3), a chimeric monoclonal antibody (Rituximab), a humanized monoclonal antibody (Herceptin), and a whole person. Stage of source monoclonal antibody (Humira). Due to the anti-mouse antibody response (HAMA) in humans, murine monoclonal antibody drugs and chimeric monoclonal antibodies have been gradually eliminated. The monoclonal antibody drugs currently occupying the market are all humanized monoclonal antibody drugs. Compared with the international advanced human antibody production technology, Shenzhen and even China have a large gap, mainly in the field of human antibody drugs, the innovation ability is weak, the number of independent research and development is small, there is no original humanized monoclonal Reports on the listing of antibody drugs, the huge antibody drug market is occupied by foreign pharmaceutical companies. China needs to change the backward situation and compete for the domestic and international antibody drug market with huge consumption potential, and it is urgent to overcome the human monoclonal antibody technology.
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。Human monoclonal antibodies have a highly specific and significant effect in the treatment of inflammation, cancer, and especially influenza. Influenza is a contagious disease caused by the flu virus that poses a serious threat to human health. About 1 billion people worldwide are infected with seasonal influenza every year, of which 250,000 to 500,000 die. The H7N9 virus is an influenza virus that is resistant to the traditional antiviral drugs amantadine and rimantadine. There is currently no effective treatment. The H7N9 virus needs to rely on the specific molecule expressed by the virus itself to bind to the receptor on the human cell when invading the cell, in order to infect the cell and further expand. The human antibody that neutralizes the virus is a specific antibody produced by human B lymphocytes, which can bind to the antigen on the surface of the virus, thereby preventing the virus from adhering to the target cell receptor, preventing the virus from invading the cell, and effectively preventing and treating H7N9 influenza.
发明内容Summary of the invention
一方面，本申请涉及抗H7N9全人源单克隆抗体5J13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。In one aspect, the application relates to an anti-H7N9 fully human monoclonal antibody 5J13 or a biologically active fragment derived from the monoclonal antibody capable of neutralizing the H7N9 virus.
另一方面，本申请涉及编码所述抗H7N9全人源单克隆抗体5J13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。 In another aspect, the present application relates to a gene encoding the anti-H7N9 fully human monoclonal antibody 5J13 or a biologically active fragment derived from the monoclonal antibody capable of specifically binding H7N9, and a vector or cell containing the same.
另一方面，本申请涉及产生所述抗H7N9全人源单克隆抗体5J13的方法。In another aspect, the present application relates to methods of producing the anti-H7N9 fully human monoclonal antibody 5J13.
另一方面，本申请涉及包含所述的抗H7N9全人源单克隆抗体5J13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。In another aspect, the present application relates to a pharmaceutical composition comprising the anti-H7N9 fully human monoclonal antibody 5J13 or a biologically active fragment derived from the monoclonal antibody capable of specifically binding H7N9.
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5J13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。In another aspect, the present application relates to the use of an anti-H7N9 fully human monoclonal antibody 5J13 as described herein or a biologically active fragment derived from the monoclonal antibody capable of specifically binding H7N9 or the pharmaceutical composition.
另一方面，本申请涉及一种检测H7N9病毒的试剂盒。In another aspect, the present application relates to a kit for detecting H7N9 virus.
图1为实施例1NTH-3T3表达CD40L的流式检测结果图。Figure 1 is a graph showing the results of flow cytometry of Example 1 NTH-3T3 expressing CD40L.
图2为实施例1流式细胞仪分选记忆B细胞结果图。Figure 2 is a graph showing the results of the flow cytometry sorting memory B cells of Example 1.
图3为实施例1ELISA实验结果图。Figure 3 is a graph showing the results of the ELISA experiment of Example 1.
图4为实施例2琼脂糖凝胶电泳结果图。Figure 4 is a graph showing the results of agarose gel electrophoresis of Example 2.
图5为实施例2SDS-PAGE蛋白电泳结果图。Figure 5 is a graph showing the results of SDS-PAGE protein electrophoresis of Example 2.
图6为实施例3血凝抑制实验结果图。Fig. 6 is a graph showing the results of the hemagglutination inhibition experiment of Example 3.
图7为实施例4中和实验结果图。Fig. 7 is a view showing the results of the experiment in Example 4.
一方面，本申请提供抗H7N9全人源单克隆抗体5J13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：In one aspect, the application provides an anti-H7N9 fully human monoclonal antibody 5J13 or a biologically active fragment derived from the monoclonal antibody capable of specifically binding to H7N9, wherein the amino acid sequence of the heavy light chain CDR1, CDR2 and CDR3 regions of the antibody They are as follows:
重链CDR1区：GFSFSNYG；Heavy chain CDR1 region: GFSFSNYG;
重链CDR2区：ISYDGTNK；Heavy chain CDR2 region: ISYDGTNK;
重链CDR3区：AKGRGPYCSSSICYHGMDV；Heavy chain CDR3 region: AKGRGPYCSSSICYHGMDV;
轻链CDR1区：QSVLSGSINMNY；Light chain CDR1 region: QSVLSGSINMNY;
轻链CDR2区：WAS；Light chain CDR2 region: WAS;
轻链CDR3区：QQYYSTPLT。Light chain CDR3 region: QQYYSTPLT.
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In some embodiments, the heavy chain variable region amino acid sequence of the antibody is set forth in SEQ ID NO: 2, or the sequence is substituted, deleted or added with one or more amino acids to form an equivalent amino acid sequence; and / or
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。The light chain variable region amino acid sequence of the antibody is set forth in SEQ ID NO: 4, or the sequence is substituted, deleted or added with one or several amino acids to form an equivalent amino acid sequence.
SEQ ID NO:2：SEQ ID NO: 2:
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Phe Ser Asn Tyr Gly Leu His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Asp Trp Val Ala Val Ile Ser Tyr Asp Gly Thr Asn Lys Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu His Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Lys Gly Arg Gly Pro Tyr Cys Ser Ser Ser Ile Cys Tyr His Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser。Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Phe Ser Asn Tyr Gly Leu His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Asp Trp Val Ala Val Ile Ser Tyr Asp Gly Thr Asn Lys Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu His Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Lys Gly Arg Gly Pro Tyr Cys Ser Ser Ser Ile Cys Tyr His Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser.
SEQ ID NO:4：SEQ ID NO: 4:
Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Leu Ser Gly Ser Ile Asn Met Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Val Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln Tyr Tyr Ser Thr Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys。Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Leu Ser Gly Ser Ile Asn Met Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Val Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln Tyr Tyr Ser Thr Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys.
在一些实施方式中，该抗体的重链氨基酸序列如SEQ ID NO:6或SEQ ID NO:10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In some embodiments, the heavy chain amino acid sequence of the antibody is set forth in SEQ ID NO: 6 or SEQ ID NO: 10, or the amino acid sequence of the sequence formed by substitution, deletion or addition of one or several amino acids ;and / or
该抗体的轻链氨基酸序列如SEQ ID NO:8或SEQ ID NO:12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。The light chain amino acid sequence of the antibody is set forth in SEQ ID NO: 8 or SEQ ID NO: 12, or the sequence is substituted, deleted or added with one or several amino acids to form an equivalent amino acid sequence.
SEQ ID NO:6：SEQ ID NO:6:
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Phe Ser Asn Tyr Gly Leu His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Asp Trp Val Ala Val Ile Ser Tyr Asp Gly Thr Asn Lys Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu His Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Lys Gly Arg Gly Pro Tyr Cys Ser Ser Ser Ile Cys Tyr His Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys。Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Phe Ser Asn Tyr Gly Leu His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Asp Trp Val Ala Val Ile Ser Tyr Asp Gly Thr Asn Lys Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu His Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Lys Gly Arg Gly Pro Tyr Cys Ser Ser Ser Ile Cys Tyr His Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser SerSer Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys.
SEQ ID NO:8：SEQ ID NO:8:
Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Leu Ser Gly Ser Ile Asn Met Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Val Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln Tyr Tyr Ser Thr Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys。Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Leu Ser Gly Ser Ile Asn Met Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Val Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln Tyr Tyr Ser Thr Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys GluVal Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys.
SEQ ID NO:10：SEQ ID NO: 10:
Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Phe Ser Asn Tyr Gly Leu His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Asp Trp Val Ala Val Ile Ser Tyr Asp Gly Thr Asn Lys Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu His Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Lys Gly Arg Gly Pro Tyr Cys Ser Ser Ser Ile Cys Tyr His Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys。Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Phe Ser Asn Tyr Gly Leu His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Asp Trp Val Ala Val Ile Ser Tyr Asp Gly Thr Asn Lys Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu His Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Lys Gly Arg Gly Pro Tyr Cys Ser Ser Ser Ile Cys Tyr His Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu GlnSer Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Le Le Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys.
SEQ ID NO:12：SEQ ID NO: 12:
Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Leu Ser Gly Ser Ile Asn Met Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Val Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln Tyr Tyr Ser Thr Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys。Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Leu Ser Gly Ser Ile Asn Met Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Val Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln Tyr Tyr Ser Thr Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr LeuThr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys.
经ELISA实验验证，本申请所述抗H7N9全人源单克隆抗体5J13可以靶向结合H7N9病毒的血凝素HA。血凝抑制试验证明5J13可以中和H7N9病毒，其IC50值可低至0.2071μg/ml，KD值为3.04×10-10；在病毒感染细胞模型模型中，其IC50值仅为0.1μg/ml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。It was confirmed by ELISA experiments that the anti-H7N9 fully human monoclonal antibody 5J13 described in the present application can target hemagglutinin HA which binds to H7N9 virus. Hemagglutination inhibition test proved that 5J13 can neutralize H7N9 virus, its IC 50 value can be as low as 0.2071μg/ml, KD value is 3.04×10 -10 ; in the virus-infected cell model model, its IC 50 value is only 0.1μg/ About ml. The antibody described in the present application is a fully human monoclonal antibody. The gene of the fully human antibody is completely derived from a human gene, and has no other species, and does not cause anti-mouse anti-antibody in the human body. Toxic side effects, better biocompatibility, more suitable and more potential to be a macromolecular drug for the treatment of influenza virus.
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5J13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In another aspect, the invention provides a gene encoding an anti-H7N9 fully human monoclonal antibody 5J13 as described herein. In some embodiments, the gene comprises a nucleotide sequence encoding the amino acid set forth in SEQ ID NO: 2, and in some embodiments, the nucleotide sequence is set forth in SEQ ID NO: 1;
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。The gene comprises a nucleotide sequence encoding the amino acid set forth in SEQ ID NO: 4, and in some embodiments, the nucleotide sequence is set forth in SEQ ID NO: 3.
SEQ ID NO:1：SEQ ID NO: 1:
SEQ ID NO:3：SEQ ID NO: 3:
在一些体实施方式中，所述基因包含编码具有SEQ ID NO:6或SEQ ID NO:10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或SEQ ID NO:9所示；和/或In some embodiments, the gene comprises a nucleotide sequence encoding an amino acid having SEQ ID NO: 6 or SEQ ID NO: 10, and in some embodiments, the nucleotide sequence is SEQ ID NO: 5 or SEQ ID NO: 9; and/or
所述基因包含编码具有SEQ ID NO:8或SEQ ID NO:12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或SEQ ID NO:11所示。The gene comprises a nucleotide sequence encoding an amino acid having SEQ ID NO: 8 or SEQ ID NO: 12, and in some embodiments, the nucleotide sequence is SEQ ID NO: 7 or SEQ ID NO: 11. Shown.
SEQ ID NO:5：SEQ ID NO: 5:
SEQ ID NO:7：SEQ ID NO:7:
SEQ ID NO:9：SEQ ID NO: 9:
SEQ ID NO:11：SEQ ID NO: 11:
另一方面，本申请提供含如上所述基因的载体。In another aspect, the application provides a vector comprising a gene as described above.
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。In a further aspect, the application provides a cell comprising a gene as described above or a vector as described above.
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5J13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5J13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5J13。In a further aspect, the application provides a method for producing the anti-H7N9 fully human monoclonal antibody 5J13 or a biologically active fragment derived from the monoclonal antibody capable of specifically binding H7N9, the method comprising culturing the coding-containing anti-H7N9 The above-mentioned gene of the heavy light chain of the human monoclonal antibody 5J13 or the genetically engineered cells of the above vector or the above-mentioned cells are directly cultured, and the anti-H7N9 full human monoclonal antibody 5J13 is obtained and purified.
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。In the prior art, there is a method for preparing a human monoclonal antibody against H7N9 virus by phage display technology, and although the method has the advantages of low production cost, no cumbersome work such as immunization and cell fusion, the disadvantages are also obvious. The antibodies obtained in the immune antibody library tend to have insufficient affinity, are limited by the conversion rate of the foreign gene, and the library capacity of the antibody library is insufficient to cover the antibody diversity of the animal. The present application separates B cells secreting functional antibodies from the blood of a patient, then extracts RNA and synthesizes cDNA, clones genes secreting the antibody of interest, and finally recombines and expresses fully human monoclonal antibodies. The technique is simple and rapid to operate, and the produced human antibody has high affinity and specificity. Further, the improved monoclonal antibody technology of the present application having neutralizing viral function or killing tumor function from memory B cells can be further improved. It also greatly reduces the cumbersome operation and cost.
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5J13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。In another aspect, the present application provides a pharmaceutical composition comprising an anti-H7N9 fully human monoclonal antibody 5J13 as described herein or a biologically active fragment derived from the monoclonal antibody capable of specifically binding H7N9.
另一方面，本申请提供所述的抗H7N9全人源单克隆抗体5J13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。In another aspect, the application provides the anti-H7N9 fully human monoclonal antibody 5J13 or a biologically active fragment derived from the monoclonal antibody capable of specifically binding H7N9 or the pharmaceutical composition prepared for treatment by H7N9 The application of drugs for diseases caused by viruses.
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5J13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5J13的抗抗体。In another aspect, the application provides a kit for detecting H7N9 virus level, which comprises the anti-H7N9 full human monoclonal antibody 5J13 described in the present application or a biologically active fragment derived from the monoclonal antibody capable of specifically binding H7N9 In some embodiments, the kit further comprises a second antibody and an enzyme or fluorescent or radiolabel for detection, and a buffer; the second antibody is, for example, a monoclonal antibody 5J13 against the present application. Anti-antibody.
与现有技术相比，本申请具有如下有益效果：Compared with the prior art, the present application has the following beneficial effects:
(1)本申请所述抗H7N9全人源单克隆抗体5J13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。(1) The anti-H7N9 fully human monoclonal antibody 5J13 described in the present application can target hemagglutinin HA which binds to the H7N9 virus, and has a neutralizing activity against the H7N9 virus infection.
(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。(2) Compared with the murine antibody, the gene of the humanized antibody of the present application is completely derived from the human gene, and has no other species of components, and does not cause toxic side effects such as anti-mouse anti-antibodies in the human body, and has a better biological phase. Capacitive, more suitable and more promising as a macromolecular drug for the treatment of influenza virus.
(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。(3) Preparation of a human monoclonal antibody against H7N9 virus compared to the phage display technology provided by the prior art The single B cell used in the present application develops an antibody against the H7N9 virus, which is simple and quick to operate, and the produced human antibody has the advantages of high affinity and specificity.
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。The technical solutions of the present application are described in detail below with reference to the specific embodiments, which are only used to illustrate the application and are not intended to limit the scope of the application. . In the examples, each of the original reagent materials is commercially available, and the experimental methods not specifying the specific conditions are conventional methods and conventional conditions well known in the art, or in accordance with the conditions recommended by the instrument manufacturer.
实施例1Example 1
(1)构建稳定表达CD40L的NTH-3T3细胞系(3T3-CD40L)(1) Construction of a NTH-3T3 cell line stably expressing CD40L (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％CO2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1×107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3.5×107细胞，分装1ml在冷冻小管，液氮冻存(可以保存2年)。3T3-CD40L feeder cells were established using lentivirus. The lentiviral expression vector pLVX-CD40L was constructed, transfected into 293T cells, and the virus supernatant was collected on the fourth day of transfection. NIH-3T3 cells were activated, cultured for 3 passages, infected with lentivirus, cultured and passaged 3 times. The cells with FITC fluorescence intensity near MFI were sorted by flow cytometry, re-added to the culture flask, cultured and detected in a 37 ° C, 5% CO 2 incubator. The test results are shown in Figure 1, which will be expressed. 3T3 cells transfected with CD40L and empty vector pLVX (with ZxGreen) were stained with anti-CD40L with APC and then analyzed by upflow cytometry. As a result, it was found that all 3T3-CD40L feeder cells expressed CD40L. When the cells grew to 80% to 90%, the cells were collected by digestion at a concentration of 1 × 10 7 cells per ml. Place in a radiometer for 5000 rads radiation, resuspend the cells in a frozen solution at a concentration of 3.5 × 10 7 cells per ml, dispense 1 ml in a cryotube, and freeze in liquid nitrogen (can be stored for 2 years).
(2)记忆B细胞的分选和活化(2) Sorting and activation of memory B cells
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10～50×106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The PBMC of the rehabilitated patients who had been infected with the H7N9 virus were isolated and frozen with a lymphatic separation solution, and each tube was 10 to 50 × 10 6 cells, and frozen in a liquid nitrogen tank. Prepare PBMC flow dyeing solution, the composition of which is shown in Table 1 below.
表1 PBMC流式染色液Table 1 PBMC flow dyeing solution
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： Thaw PBMC, add the above PBMC flow staining solution and sort by flow cytometry. As shown in Fig. 2, sort out CD19 + IgM – IgA – IgD – memory B cells, the cell purity should be above 90%. If less than 90%, repeat the sorting process. Prepare a mixed medium that activates B cells as shown in Table 2 below:
表2Table 2
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％CO2培养箱中静置培养。13天后，取上清液进行ELISA，获得人源单克隆抗体5J13。The memory B cells were added to the mixed medium, mixed and then diluted in a 384-well plate, one cell per well, and the volume was 50 μl, and placed in a 37 ° C, 5% CO 2 incubator for static culture. After 13 days, the supernatant was taken for ELISA to obtain human monoclonal antibody 5J13.
(3)人源单克隆抗体5J13结合H7N9病毒的血凝素HA实验(3) Hemagglutinin HA experiment of human monoclonal antibody 5J13 combined with H7N9 virus
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。对上述获得的人源单克隆抗体5J13进行ELISA实验，具体地：Influenza virus hemagglutinin HA is a columnar antigen on the surface of the virus envelope. It can bind to various red blood cell receptors such as human, chicken and guinea pigs to cause red blood cell agglutination. It is immunogenic and anti-hemagglutinin antibody can neutralize influenza virus. An ELISA experiment was performed on the human monoclonal antibody 5J13 obtained above, specifically:
(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；(1) 100 ng / 100 μl of H7N9 virus HA protein (purchased from ACROBiosystems) was coated in a 96-well microtiter plate, 100 μl per well;
(2)放置4℃冰箱过夜；(2) Place the refrigerator at 4 ° C overnight;
(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；(3) Washing three times with PBST solution, adding 5% of the skim milk powder solution per well to 200 μl, and incubating at 37 ° C for 1 hour;
(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5J13，各三个重复；(4) Washing three times with PBST solution, adding 100 μl of normal human serum (negative control) or virus-infected patient serum or anti-H7N9 full-human monoclonal antibody 5J13, three replicates each;
(5)37℃孵育1小时后用PBST溶液洗涤三遍；(5) After incubation at 37 ° C for 1 hour, wash three times with PBST solution;
(6)以1:5000稀释带HRP的抗人IgG抗体(abcam)，加入酶标版中，每孔100μl；(6) diluting anti-human IgG antibody (abcam) with HRP at 1:5000, adding 100 μl per well to the enzyme plate;
(7)37℃孵育1小时后用PBST溶液洗涤三遍；(7) After incubation at 37 ° C for 1 hour, wash three times with PBST solution;
(8)每孔加100μl TMB底物溶液(Thermo Scientific)，37℃5分钟；(8) Add 100 μl of TMB substrate solution (Thermo Scientific) to each well at 37 ° C for 5 minutes;
(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5J13可以靶向结合H7N9病毒的血凝素HA。(9) Add 100 μl of 2M sulfuric acid to each well and immediately measure the absorbance at a wavelength of 450 nm in the microplate reader. The results are shown in Figure 3. The ELISA assay indicated that the human monoclonal antibody 5J13 obtained in the present application can target hemagglutinin HA which binds to the H7N9 virus.
实施例2人源化单克隆抗体5J13基因的克隆、重组、表达和纯化Example 2 Cloning, Recombination, Expression and Purification of Humanized Monoclonal Antibody 5J13 Gene
将实施例1获得的能够分泌结合H7N9病毒的5J13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：The B cells capable of secreting the 5J13 antibody which binds to the H7N9 virus obtained in Example 1 were lysed, and the lysate was subjected to reverse transcription of RNA to obtain a PCR template cDNA of the human antibody gene. Designing and synthesizing primers for cloning antibody genes, using cDNA as a template to clone the heavy and light chain genes of antibodies, and recombining in eukaryotic cells 293F or Expression and purification were carried out in HEK293. specifically:
(1)将裂解后的B细胞液转移至96孔板(Eppendorf，030133366)。(1) The lysed B cell solution was transferred to a 96-well plate (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 50U
(3)反转录反应程序：42℃，10min；25℃，10min；50℃，60min；94℃，5min。(3) Reverse transcription reaction procedure: 42 ° C, 10 min; 25 ° C, 10 min; 50 ° C, 60 min; 94 ° C, 5 min.
(4)cDNA保存在-20℃。(4) The cDNA was stored at -20 °C.
(5)引物的设计和合成:(5) Design and synthesis of primers:
正向引物5′-3′序列(Forward Primer 5′-3′sequence)Forward Primer 5'-3' sequence (Forward Primer 5'-3'sequence)
重链可变区PCR引物:Heavy chain variable region PCR primers:
5′VH1 CTGCAACCGGTGTACATTCCCAGGTGCAGCTGGTGCAG(SEQ ID NO:13)5'VH1 CTGCAACCGGTGTACATTCCCAGGTGCAGCTGGTGCAG (SEQ ID NO: 13)
5′VH1/5 CTGCAACCGGTGTACATTCCGAGGTGCAGCTGGTGCAG(SEQ ID NO:14)5'VH1/5 CTGCAACCGGTGTACATTCCGAGGTGCAGCTGGTGCAG (SEQ ID NO: 14)
5′VH3 CTGCAACCGGTGTACATTCTGAGGTGCAGCTGGTGGAG(SEQ ID NO:15)5'VH3 CTGCAACCGGTGTACATTCTGAGGTGCAGCTGGTGGAG (SEQ ID NO: 15)
5′VH3-23 CTGCAACCGGTGTACATTCTGAGGTGCAGCTGTTGGAG(SEQ ID NO:16)5'VH3-23 CTGCAACCGGTGTACATTCTGAGGTGCAGCTGTTGGAG (SEQ ID NO: 16)
5′VH4 CTGCAACCGGTGTACATTCCCAGGTGCAGCTGCAGGAG(SEQ ID NO:17)5'VH4 CTGCAACCGGTGTACATTCCCAGGTGCAGCTGCAGGAG (SEQ ID NO: 17)
5′VH 4-34 CTGCAACCGGTGTACATTCCCAGGTGCAGCTACAGCAGTG(SEQ ID NO:18)5'VH 4-34 CTGCAACCGGTGTACATTCCCAGGTGCAGCTACAGCAGTG (SEQ ID NO: 18)
5′VH 1-18 CTGCAACCGGTGTACATTCCCAGGTTCAGCTGGTGCAG(SEQ ID NO:19)5'VH 1-18 CTGCAACCGGTGTACATTCCCAGGTTCAGCTGGTGCAG (SEQ ID NO: 19)
5′VH 1-24 CTGCAACCGGTGTACATTCCCAGGTCCAGCTGGTACAG(SEQ ID NO:20)5'VH 1-24 CTGCAACCGGTGTACATTCCCAGGTCCAGCTGGTACAG (SEQ ID NO: 20)
5′VH3-33 CTGCAACCGGTGTACATTCTCAGGTGCAGCTGGTGGAG(SEQ ID NO:21)5'VH3-33 CTGCAACCGGTGTACATTCTCAGGTGCAGCTGGTGGAG (SEQ ID NO: 21)
5′VH 3-9 CTGCAACCGGTGTACATTCTGAAGTGCAGCTGGTGGAG(SEQ ID NO:22)5'VH 3-9 CTGCAACCGGTGTACATTCTGAAGTGCAGCTGGTGGAG (SEQ ID NO: 22)
5′VH4-39 CTGCAACCGGTGTACATTCCCAGCTGCAGCTGCAGGAG(SEQ ID NO:23)5'VH4-39 CTGCAACCGGTGTACATTCCCAGCTGCAGCTGCAGGAG (SEQ ID NO: 23)
5′VH 6-1 CTGCAACCGGTGTACATTCCCAGGTACAGCTGCAGCAG(SEQ ID NO:24)5'VH 6-1 CTGCAACCGGTGTACATTCCCAGGTACAGCTGCAGCAG (SEQ ID NO: 24)
3′JH 1/2/4/5 TGCGAAGTCGACGCTGAGGAGACGGTGACCAG(SEQ ID NO:25)
3′JH 3 TGCGAAGTCGACGCTGAAGAGACGGTGACCATTG(SEQ ID NO:26)3'JH 3 TGCGAAGTCGACGCTGAAGAGACGGTGACCATTG (SEQ ID NO: 26)
3′JH 6 TGCGAAGTCGACGCTGAGGAGACGGTGACCGTG(SEQ ID NO:27)
κ轻链可变区PCR产物κ light chain variable region PCR product
5′Vκ 1-5 CTGCAACCGGTGTACATTCTGACATCCAGATGACCCAGTC(SEQ ID NO:28)5' Vκ 1-5 CTGCAACCGGTGTACATTCTGACATCCAGATGACCCAGTC (SEQ ID NO: 28)
5′Vκ 1-9 TTGTGCTGCAACCGGTGTACATTCAGACATCCAGTTGACCC AGTCT (SEQ ID NO:29)5'Vκ 1-9 TTGTGCTGCAACCGGTGTACATTCAGACATCCAGTTGACCC AGTCT (SEQ ID NO: 29)
5′Vκ 1D-43 CTGCAACCGGTGTACATTGTGCCATCCGGATGACCCAGTC(SEQ ID NO:30)5'Vκ 1D-43 CTGCAACCGGTGTACATTGTGCCATCCGGATGACCCAGTC (SEQ ID NO: 30)
5′Vκ 2-24 CTGCAACCGGTGTACATGGGGATATTGTGATGACCCAGAC(SEQ ID NO:31)5'Vκ 2-24 CTGCAACCGGTGTACATGGGGATATTGTGATGACCCAGAC (SEQ ID NO: 31)
5′Vκ 2-28 CTGCAACCGGTGTACATGGGGATATTGTGATGACTCAGTC(SEQ ID NO:32)5' Vκ 2-28 CTGCAACCGGTGTACATGGGGATATTGTGATGACTCAGTC (SEQ ID NO: 32)
5′Vκ 2-30 CTGCAACCGGTGTACATGGGGATGTTGTGATGACTCAGTC(SEQ ID NO:33)5'Vκ 2-30 CTGCAACCGGTGTACATGGGGATGTTGTGATGACTCAGTC (SEQ ID NO: 33)
5′Vκ 3-11 TTGTGCTGCAACCGGTGTACATTCAGAAATTGTGTTGACAC AGTC (SEQ ID NO:34)5'Vκ 3-11 TTGTGCTGCAACCGGTGTACATTCAGAAATTGTGTTGACAC AGTC (SEQ ID NO: 34)
5′Vκ 3-15 CTGCAACCGGTGTACATTCAGAAATAGTGATGACGCAGTC(SEQ ID NO:35)5'Vκ 3-15 CTGCAACCGGTGTACATTCAGAAATAGTGATGACGCAGTC (SEQ ID NO: 35)
5′Vκ 3-20 TTGTGCTGCAACCGGTGTACATTCAGAAATTGTGTTGACGCAGTCT(SEQ ID NO:36)5' Vκ 3-20 TTGTGCTGCAACCGGTGTACATTCAGAAATTGTGTTGACGCAGTCT (SEQ ID NO: 36)
5′Vκ 4-1 CTGCAACCGGTGTACATTCGGACATCGTGATGACCCAGTC(SEQ ID NO:37)5' Vκ 4-1 CTGCAACCGGTGTACATTCGGACATCGTGATGACCCAGTC (SEQ ID NO: 37)
3′Jκ 1/4 GCCACCGTACGTTTGATYTCCACCTTGGTC(SEQ ID NO:38)
3′Jκ 2 GCCACCGTACGTTTGATCTCCAGCTTGGTC(SEQ ID NO:39)
3′Jκ 3 GCCACCGTACGTTTGATATCCACTTTGGTC(SEQ ID NO:40)3'Jκ 3 GCCACCGTACGTTTGATATCCACTTTGGTC (SEQ ID NO: 40)
3′Jκ 5 GCCACCGTACGTTTAATCTCCAGTCGTGTC(SEQ ID NO:41)3'Jκ 5 GCCACCGTACGTTTAATCTCCAGTCGTGTC (SEQ ID NO: 41)
(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，20 nM混合引物，4μL缓冲液(buffer)，4μL 2mM dNTPs，2.4μL MgSO4，1μL KOD。(6) Amplification of the heavy and light chains of the antibody gene by PCR with KOD-Plus-Neo (TOYOBO, KOD401) kit, 40 μL system: 3.5 μL cDNA, 20 nM mixed primer, 4 μL buffer, 4
(7)反应程序：94℃，2min；45个循环：[98℃,10s；58℃,30s；68℃,28s。(7) Reaction procedure: 94 ° C, 2 min; 45 cycles: [98 ° C, 10 s; 58 ° C, 30 s; 68 ° C, 28 s.
(8)对扩增产物进行琼脂糖凝胶，其结果如图4所示，结果显示，抗体轻链为κ，其可变区大小为339bp，重链可变区大小是378bp。(8) The amplification product was subjected to agarose gel, and the results are shown in Fig. 4. The results showed that the antibody light chain was κ, the variable region size was 339 bp, and the heavy chain variable region was 378 bp.
(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。(9) Antibody gene heavy chain variable region PCR product sequencing result is the sequence shown in SEQ ID NO: 1, and the corresponding amino acid sequence is the sequence shown in SEQ ID NO: 2. The antibody gene light chain variable region PCR product was sequenced as shown in SEQ ID NO: 3, and the corresponding amino acid sequence is the sequence shown in SEQ ID NO: 4.
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。Based on the obtained heavy and light chain variable region sequences, Invitrogen was designed and commissioned to synthesize the full-length H gene of the antibody gene heavy chain with the BamH1/EcoR1 double restriction site; and the full-length L gene of the antibody gene light chain. Not1/Xho1 double cleavage site.
(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。(10) The H gene and pcDNA3.1 were double-digested with BamH1/EcoR1, respectively, to form a pcDNA3.1-H vector.
(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。(11) The L gene and pcDNA3.1 were separately digested with Not1/Xho1 to form a pcDNA3.1-L vector.
(12)培养293F细胞。(12) 293F cells were cultured.
(13)20μg pcDNA3.1-H(该H基因的不带编码信号肽的核苷酸序列如SEQ ID NO:5所示，SEQ ID NO:5可带有编码信号肽的序列，如atgggctggt cctgcatcat cctgttcctg gtggccaccg ccaccggc)载体和10μg pcDNA3.1-L(该L基因的不带编码信号肽的核苷酸序列如SEQ ID NO:7所示，SEQ ID NO:7可带有编码信号肽的序列，如atgggctggt cctgcatcat cctgttcctg gtggccaccg ccaccggc)载体共转染293F细胞，培养96小时。(13) 20 μg of pcDNA3.1-H (the nucleotide sequence of the H gene without a coding signal peptide is shown in SEQ ID NO: 5, and SEQ ID NO: 5 may carry a sequence encoding a signal peptide, such as atgggctggt cctgcatcat Cctgttcctg gtggccaccg ccaccggc) vector and 10 μg of pcDNA3.1-L (the nucleotide sequence of the L gene without the coding signal peptide is shown as SEQ ID NO: 7, and SEQ ID NO: 7 may carry the sequence encoding the signal peptide, For example, atgggctggt cctgcatcat cctgttcctg gtggccaccg ccaccggc) vector was co-transfected into 293F cells and cultured for 96 hours.
(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所示：(14) Take the supernatant for ELISA (ABC is the supernatant, DEF is the positive control, GH is the negative control); ELISA specific experimental steps As mentioned above, the ELISA results are shown in Table 3 below:
表3table 3
上述结果显示上清液中含有能够结合H7N9病毒的抗体。The above results show that the supernatant contains an antibody capable of binding to the H7N9 virus.
(15)纯化过程，具体地，全人源单克隆抗体5J13的纯化过程为：(15) Purification process, specifically, the purification process of the fully human monoclonal antibody 5J13 is:
(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。(a) 200 μg of pcDNA3.1-L vector and 100 μg of pcDNA3.1-H vector were co-transfected with 300 ml of 293F cells, and cultured for 96 hours.
(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。(b) The supernatant was collected, added to a proteinA affinity chromatography column, washed with 10 times PBS, and 2 ml of pH 3.0, 0.1 M glycine was added to collect the antibody. 100 μl of neutralization buffer (1 M Tri-HCL) was added to the collection tube to neutralize the pH of the obtained antibody solution in time.
(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300μg 5J13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，跑SDS-PAGE蛋白电泳结果如图5所示，从图5中可以看出获得了高纯度的抗体。(c) Dialysis in phosphate buffered saline (PBS). After dialysis, the stock was stored at 4 ° C for a long time and stored at -20 ° C for a long time. A total of 300 μg of 5J13 antibody was obtained, the heavy chain amino acid sequence thereof is shown in SEQ ID NO: 6, the light chain amino acid sequence is shown in SEQ ID NO: 8, and the SDS-PAGE protein electrophoresis results are shown in FIG. 5, from FIG. It can be seen that high purity antibodies are obtained.
实施例3纯化后的全人源单克隆抗体5J13的中和实验及抗体亲和力实验Neutralization experiment and antibody affinity experiment of purified human monoclonal antibody 5J13 after purification
全人源单克隆抗体5J13的中和实验Neutralization experiment of fully human monoclonal antibody 5J13
(1)血凝实验(1) Hemagglutination experiment
(a)取96孔V形微量反应板，用微量移液器在1～12孔每孔加25μL PBS，共滴8排，后4排的第1列孔再补加25μL PBS。(a) Take a 96-well V-shaped micro-reaction plate, add 25 μL of PBS to each well of 1 to 12 wells with a micropipette, and dispense a total of 8 rows, and then add 25 μL of PBS to the first column of the 4 rows.
(b)吸取25μL标准禽流感抗原(H7N9病毒株A/Shanghai/2/2013，取自深圳罗湖医院)加入到第1列孔中，吹打3～5次充分混匀。(b) Pipette 25 μL of standard avian influenza antigen (H7N9 virus strain A/Shanghai/2/2013, taken from Shenzhen Luohu Hospital) and add it to the first column of wells, and mix thoroughly by pipetting 3 to 5 times.
(c)从第1列孔吸取25μL混匀后的抗原液加到第2列孔中，混匀后吸取25μL加入到第3列孔中，依次进行系列倍比稀释至第11列孔，最后从第11列孔各吸取25μL弃之，设第12列孔为红细胞对照。(c) Pipette 25 μL of the mixed antigen solution from the first column to the second column of wells, mix and absorb 25 μL and add to the third column of wells, and serially dilute to the 11th column, and finally Aspirate 25 μL from each of the 11th column wells, and set the 12th column to be a red blood cell control.
(1)自右向左依次向各孔加入25μL 1％的鸡红细胞悬液。(1) 25 μL of 1% chicken red blood cell suspension was added to each well in order from right to left.
(2)将反应板置于微量振荡器上振荡1min，室温(20-25℃)静置45min后观察结果，若环境温度过高，可于4℃静置60min，红细胞对照孔成明显的纽扣状沉到孔底时即可判定结果。(2) The reaction plate was shaken on a micro-vibrator for 1 min, and the result was observed after standing at room temperature (20-25 ° C) for 45 min. If the ambient temperature was too high, it could be allowed to stand at 4 ° C for 60 min, and the red blood cell control hole became an obvious button. The result can be determined when the shape sinks to the bottom of the hole.
(3)结果判定：在红细胞对照孔出现正确结果的情况下，将反应板作45°倾斜，观察红细胞是否完全凝集。以完全凝集的病毒最大稀释度为该抗原的血凝滴度。完全凝集的病 毒的最高稀释倍数为1个血凝单位(HAU)。(3) Judgment of results: In the case where the correct result of the red blood cell control well appeared, the reaction plate was tilted at 45° to observe whether the red blood cells were completely agglutinated. The maximum dilution of the virus that is fully agglutinated is the hemagglutination titer of the antigen. Completely agglutinated disease The highest dilution factor for toxicity is 1 hemagglutination unit (HAU).
(2)血凝抑制实验(2) Hemagglutination inhibition experiment
(a)根据血凝试验结果配制4单位抗原(H7N9病毒株A/Shanghai/2/2013，取自深圳罗湖医院)。(a) Four units of antigen (H7N9 strain A/Shanghai/2/2013, taken from Shenzhen Luohu Hospital) were prepared according to the results of the hemagglutination test.
(b)取96孔V形微量反应板，用移液器在第1～12孔各加入25μL PBS。(b) A 96-well V-shaped microplate was taken, and 25 μL of PBS was added to each of the 1st to 12th wells by a pipette.
(c)在第一排中加入被测纯化后的全人源单克隆抗体5J13(50μg/ml)，充分混匀后移出25μl加至第二排，依次类推，倍比稀释至第8排，第8排弃去25μL。(c) In the first row, add the purified human monoclonal antibody 5J13 (50 μg/ml), mix well, remove 25 μl and add to the second row, and so on, and dilute to the 8th row. The 8th row discarded 25μL.
(d)各孔加入25μL 4单位抗原，轻叩反应板，使反应物混合均匀，室温20-25℃下静置30min。(d) Add 25 μL of 4 unit antigen to each well, gently lick the reaction plate, mix the reaction uniformly, and let stand at room temperature 20-25 ° C for 30 min.
(e)自右向左依次向各孔加入25μL 1％的鸡红细胞悬液。(e) Add 25 μL of 1% chicken red blood cell suspension to each well from right to left.
(f)将反应板置于微量振荡器上振荡1min，室温(20-25℃)静置40min后观察结果，若环境温度过高，可于4℃静置60min，红细胞对照孔成明显的纽扣状沉到孔底时即可判定结果。(f) The reaction plate was shaken on a micro-vibrator for 1 min, and the result was observed after standing at room temperature (20-25 ° C) for 40 min. If the ambient temperature was too high, it could be allowed to stand at 4 ° C for 60 min, and the red blood cell control hole became an obvious button. The result can be determined when the shape sinks to the bottom of the hole.
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，与本案5J13的血凝抑制活性，实验结果如图6所示，从图6中可以看出本申请5J13血凝抑制活性的IC50为0.2071μg/ml，而2L11及2J17(图中均示为同型对照)抗体没有血凝抑制活性。This application compares the singles submitted to the State Intellectual Property Office of China on May 10, 2016, the application number is “201610303416.X”, and the invention name is “anti-H7N9 whole human monoclonal antibody 2L11 and its preparation method and application” Cloned antibody 2L11, and submitted to the State Intellectual Property Office of China on May 3, 2016, the application number is "201610288358.8", the invention name is "anti-H7N9 whole human monoclonal antibody 2J17 and its preparation and application" The antibody 2J17, and the hemagglutination inhibitory activity of 5J13 in the present case, the experimental results are shown in Fig. 6. It can be seen from Fig. 6 that the IC 50 of the 5J13 hemagglutination inhibitory activity of the present application is 0.2071 μg/ml, and 2L11 and 2J17 (in the figure) All of the antibodies shown as isotype control have no hemagglutination inhibitory activity.
抗体亲和力检测：Antibody affinity test:
亲和力检测的仪器为PALL的Fortebio。配制200μl 50μg/ml 5J13抗体，结合proteinA 传感器300秒，HA抗原配制50nM、25nM、12.5nM、6.25nM和3.125nM浓度溶液，结合抗体240秒，解离时间为30分钟，显示5J13对H7N9病毒有高亲和力，KD＝3.04×10-10。The instrument for affinity testing is Pte's Fortebio. Prepare 200μl 50μg/ml 5J13 antibody, combine with proteinA sensor for 300 seconds, prepare HA solution with 50nM, 25nM, 12.5nM, 6.25nM and 3.125nM concentration solution, bind antibody for 240 seconds, dissociation time is 30 minutes, showing 5J13 for H7N9 virus High affinity, KD = 3.04 × 10 -10 .
实施例4抗H7N9的5J13抗体体外中和实验Example 4 In vitro neutralization experiment of 5J13 antibody against H7N9
(1)实验目的(1) Experimental purpose
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5J13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。The inhibitory effect and effect of the 5J13 antibody against H7N9 influenza virus were evaluated by a virus-infected cell model (dog kidney cell MDCK) by a microneutralization-ELISA assay, and the antibody was tested for anti-influenza virus activity.
(2)实验步骤(2) Experimental steps
(2.1)细胞铺板 (2.1) Cell plating
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5×104个/ml，接种于96孔细胞培养板，细胞置于37℃、5％CO2培养箱中培养过夜。Trypsin digested MDCK canine kidney cells in logarithmic growth phase, centrifuged after centrifugation, and spread evenly to prepare single cell suspension; adjust the cell concentration to 5×10 4 cells/ml with cell culture medium, and inoculate in 96-well cell culture. Plates, cells were incubated overnight at 37 ° C in a 5% CO 2 incubator.
(2.2)5J13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(2.2) 5J13 antibody and H7N9 virus (the virus A/Anhui/1/2013 was taken from the Institute of Microbiology, Chinese Academy of Sciences) pretreatment
5J13抗体设立10个浓度梯度，依次为10-1010倍稀释，各组各浓度均设3个平行孔。The 10J13 antibody was set up with 10 concentration gradients, which were 10-10 10- fold dilutions in sequence, and 3 parallel wells were set for each concentration.
(2.3)病毒感染(2.3) Viral infection
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102μg/ml的5J13单克隆抗体依次以10-1010倍稀释，将每个浓度的5J13抗体分别与等体积的100TCID50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。The cell culture supernatant of the cultured step (2.1) was discarded, and washed three times with PBS. The premixed antibody-virus mixture (diluted 10 10 10 folds sequentially with 10 2 μg/ml of 5J13 monoclonal antibody, and each concentration of 5J13 antibody was mixed with an equal volume of 100 TCID 50 virus, respectively. ). The cells were added to a 96-well cell culture plate, incubated at 37 ° C for 1 h, and the mixture was discarded and washed twice with PBS.
(2.4)配置维持液(2.4) Configure maintenance solution
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％ CO2培养箱培养20h。TPCK-Trypsin (maintaining solution) at a final concentration of 2 μg/ml was added to serum-free DMEM. The PBS in the 96-well plate was discarded, 100 μl of the maintenance solution was added to each well, and cultured at 37 ° C for 20 hours in a 5% CO 2 incubator.
(2.5)中和实验-ELISA法(2.5) Neutralization experiment-ELISA
(2.5.1)弃去微量培养板中的维持液；(2.5.1) discard the maintenance solution in the microplate;
(2.5.2)100μl PBS洗细胞一次；(2.5.2) Wash the cells once with 100 μl of PBS;
(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)；(2.5.3) Discard PBS (do not let the cells dry), add 50μl / well fixative (volume ratio: acetone: absolute ethanol = 2:3);
(2.5.4)覆盖微量培养板，于室温固定细胞10min；(2.5.4) Cover the microplate and fix the cells for 10 min at room temperature;
(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。(2.5.5) Discard the fixative, wash the cells with 100 μl of PBS, and wash 3 times (slightly shake to avoid strong washing) to remove residual acetone.
(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；(2.5.6) The cells were blocked with 5% skim milk powder for 1 h at room temperature, and the cells were washed once with 100 μl of PBS solution;
(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。(2.5.7) Dilute 1 antibody (NP monoclonal antibody against commercial H7N9) diluted with PBS 1:2000. Add 50 μl of each dilution to each well and allow to stand at room temperature for 1 hour.
(2.5.8)用100μl PBST洗板5次以除去1抗；(2.5.8) Wash the plate 5 times with 100 μl PBST to remove the 1 antibody;
(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。(2.5.9) 2 anti-antibody (anti-mouse IgG antibody with HRP) was diluted with PBS 1:2000, 50 μl was added to each well, and allowed to stand at room temperature for 1 hour.
(2.5.10)用100μl PBST洗涤板6次以除去2抗。(2.5.10) The plate was washed 6 times with 100 μl of PBST to remove the 2 antibody.
(2.5.11)每孔加TMB显色液50μl。 (2.5.11) Add 50 μl of TMB coloring solution to each well.
(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。(2.5.12) After standing at room temperature for about 10 minutes, the reaction was stopped by adding 50 μl of 2M hydrochloric acid to each well.
(2.5.13)在ELISA测定仪上(450纳米)读出每孔OD值。(2.5.13) The OD value per well was read on an ELISA meter (450 nm).
(3)统计分析(3) Statistical analysis
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IC50。抑制率计算公式：Using GraphPad Prism 6.0.1 Data were analyzed and plotted the dose - response curves, and calculate the IC 50. Inhibition rate calculation formula:
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％。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) x 100%.
所得结构如图7所示，从图7中可以看出5J13的IC50＝0.1024μg/ml。The resulting structure is shown in FIG. 7, it can be seen in FIG. 7 5J13 of IC 50 = 0.1024μg / ml.
从实施例3及实施例4可以看出，本申请5J13对两地的H7N9都有低的IC50值，证明5J13的广谱中和性好。As can be seen from Example 3 and Example 4, the 5J13 of the present application has a low IC 50 value for both H7N9, which proves that the broad spectrum neutralization of 5J13 is good.
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。 It is to be noted that the above embodiments are only used to explain the implementation process and features of the present application, and do not limit the technical solutions of the present application. Although the present application is described in detail with reference to the above embodiments, those skilled in the art should understand that: Modifications or equivalent substitutions of the present application may be made without departing from the spirit and scope of the invention.
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| CN106519027A (en) | 2017-03-22 |
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