WO2018028635A1 - Humanized monoclonal antibody for zika virus and applications thereof - Google Patents

Humanized monoclonal antibody for zika virus and applications thereof Download PDF

Info

Publication number
WO2018028635A1
WO2018028635A1 PCT/CN2017/096816 CN2017096816W WO2018028635A1 WO 2018028635 A1 WO2018028635 A1 WO 2018028635A1 CN 2017096816 W CN2017096816 W CN 2017096816W WO 2018028635 A1 WO2018028635 A1 WO 2018028635A1
Authority
WO
WIPO (PCT)
Prior art keywords
sequence
seq
antibody
light chain
amino acid
Prior art date
Application number
PCT/CN2017/096816
Other languages
French (fr)
Chinese (zh)
Inventor
高福
严景华
王奇慧
仝舟
Original Assignee
中国科学院微生物研究所
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 中国科学院微生物研究所 filed Critical 中国科学院微生物研究所
Publication of WO2018028635A1 publication Critical patent/WO2018028635A1/en

Links

Images

Classifications

    • 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
    • C07K16/1081—Togaviridae, e.g. flavivirus, rubella virus, hog cholera 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
    • 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
    • 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
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention relates to a human monoclonal antibody of Zika virus and application thereof, and belongs to the technical field of medicine.
  • ZIKV Zika virus
  • Zika virus which belongs to the flavivirus, is mainly transmitted by mosquito bites, but blood transmission, sexual transmission, and mother-to-child transmission have also been reported.
  • ZIKV Zika virus
  • the virus is mainly prevalent in Africa and the tropical regions of Asia.
  • the Zika outbreak in Brazil broke out with the spread of people to other countries.
  • the current outbreak has resulted in more than 80,000 infections in more than 60 countries or regions.
  • the neutralizing antibody has high specificity and can block the invasion of the virus, and is an effective means for treating viral infection.
  • the viral envelope protein E protein mediates the binding and invasion of the virus to the host cell and is the most important protective antigen of the virus.
  • the X-ray crystallographic structure shows that the flavivirus E protein has three domains: DI, DII and DIII. It is generally believed that DIII is responsible for mediating the binding of the virus to the receptor.
  • Previous studies have shown that most of the effective neutralizing antibodies are neutralizing epitopes that recognize DIII.
  • recent data indicate that E-protein dimers on the surface of the virus as well as high-neutral epitopes are also present in adjacent dimers.
  • the DII head (amino acids 98-110) contains a highly conserved fusion loop (FL) that plays a key role in membrane fusion during viral invasion.
  • FL fusion loop
  • Different flavivirus FL regions are highly conserved, and during viral infection, immune cells produce large amounts of antibodies against FL.
  • RNA viruses have high mutational properties under antibody pressure, and although some neutralizing antibodies have been identified, more new antibodies against different epitopes are essential for treatment.
  • the object of the present invention is to identify specific protective ZIKV neutralizing antibodies.
  • the present invention firstly uses the ZIKV-E protein expressed by Escherichia coli as an antigen, and selects memory B cells which can specifically bind ZIKV-E protein from PBMCs of a patient in a convalescent ZIKV by flow sorting, and then RT-PCR of selected single B cells to obtain variable region sequences and fragments of antibodies, and further with constant regions Linked to an expression vector.
  • a series of functional tests including binding to ZIKE-E protein, in vitro neutralization effect, and in vivo protective ability, were obtained, and 3 strains with full or partial protection of Zika virus were obtained. Human monoclonal antibody.
  • a first object of the invention is to provide an antibody which:
  • the antibody is designated Z3L1, Z20 or Z23; wherein the amino acid sequence of the heavy chain variable region of Z3L1 is SEQ ID NO: 1 and the amino acid sequence of the light chain variable region is SEQ ID NO
  • the amino acid sequence of the heavy chain variable region of Z20 is SEQ ID NO: 3 and the amino acid sequence of the light chain variable region is SEQ ID NO: 4
  • the amino acid sequence of the heavy chain variable region of Z23 is SEQ ID NO:
  • the light chain variable region amino acid sequence is SEQ ID NO: 6.
  • nucleotide sequence of the heavy chain of Z3L1 is SEQ ID NO: 7
  • nucleotide sequence of the light chain is SEQ ID NO: 8.
  • nucleotide sequence of the heavy chain of Z20 is SEQ ID NO: 9
  • nucleotide sequence of the light chain is SEQ ID NO: 10.
  • nucleotide sequence of the heavy chain of Z23 is SEQ ID NO: 11
  • nucleotide sequence of the light chain is SEQ ID NO: 12.
  • the heavy chain of the antibody comprises a heavy chain variable region and a heavy chain constant region, wherein the amino acid sequence of the heavy chain constant region is set forth in SEQ ID NO: 13.
  • the light chain of the antibody Z20 or Z23 is a kappa chain
  • the light chain of Z3L1 is a ⁇ chain
  • the light chain comprises a light chain variable region and a light chain constant region
  • the light chain of the kappa chain The amino acid sequence of the constant region is set forth in SEQ ID NO: 15.
  • the amino acid sequence of the light chain constant region of the lambda chain is set forth in SEQ ID NO: 17.
  • the occurrence of interaction refers to binding
  • the three antibodies of the present invention are derived from the same patient and all target the envelope protein-E protein unique to Zika virus, inhibiting virus-to-cell by inhibiting E protein-mediated receptor binding and/or membrane fusion processes. Infection.
  • a second object of the present invention is to provide use of the antibody for the preparation of a medicament for the treatment and/or prevention of Zika virus.
  • a third object of the present invention is to provide a pharmaceutical composition comprising the human monoclonal antibodies Z3L1, Z20 and/or Z23.
  • the pharmaceutical composition further comprises a medically acceptable carrier.
  • a fourth object of the present invention is to provide a kit comprising an antigen of the antibody, or a DNA molecule encoding the antigen, or a recombinant vector/expression cassette/transgenic cell line expressing the antigen /Recombinant bacteria.
  • a fifth object of the present invention is to provide a gene sequence encoding the human monoclonal antibody Z3L1, Z20 or Z23.
  • the heavy chain of the antibody comprises a heavy chain variable region and a heavy chain constant region, wherein the amino acid sequence of the heavy chain constant region is set forth in SEQ ID NO: 13 (the nucleotide sequence is set forth in SEQ ID NO: 14).
  • the sequence encoding the heavy chain of the antibody comprises a CMV promoter sequence, an EcoR I cleavage site sequence, a leader sequence, a sequence encoding a heavy chain variable region, a coding heavy chain Sequence of the constant region, sequence of the Xho I restriction site.
  • the light chain of the antibody is a kappa chain and/or a lambda chain; the light chain comprises a light chain variable region and a light chain constant region.
  • the sequence encoding the light chain of the antibody in turn comprises a CMV promoter sequence, a first restriction site sequence, a leader sequence, a sequence encoding a light chain variable region, encoding a light chain
  • the kappa chain has an amino acid sequence of a light chain constant region as set forth in SEQ ID NO: 15 (nucleotide sequence is set forth in SEQ ID NO: 16), wherein the first enzyme The cleavage site is Sac I.
  • the ⁇ chain, the amino acid sequence of the light chain constant region thereof is set forth in SEQ ID NO: 17 (the nucleotide sequence is represented by SEQ ID NO: 18), wherein the first enzyme The cleavage site is EcoR I.
  • amino acid sequence of the leader sequence is set forth in SEQ ID NO: 19 (the nucleotide sequence is set forth in SEQ ID NO: 20).
  • the invention also claims an expression vector or cell comprising the gene sequence or expressing the antibody.
  • the present invention obtained three human high- and medium-active ZIKV antibodies: Z3L1, Z20 and Z23. These 3 antibodies have been The reported ZIKV antibody sequences are completely different and are 3 newly discovered antibodies. The binding constants of these three antibodies to ZIKV-E were 5.39 ⁇ M (Z3L1), 0.16 ⁇ M (Z20) and 0.44 ⁇ M (Z23), respectively. All three human antibodies have strong ZIKV neutralizing activity. Moreover, Z3L1, Z20 and Z23 can completely or partially protect mice from lethal doses of ZIKV. The three human antibodies of the invention have application value in clinical treatment and prevention of ZIKV.
  • Figure 1 ZIKV-E protein purified molecular sieve and SDS-PAGE results
  • Figure 2 Z3L1 purified Protein A (A) and molecular sieve chromatography (B) results;
  • Figure 3 Z20 purified Protein A (A) and molecular sieve chromatography (B) results;
  • Figure 4 Z23 purified Protein A (A) and molecular sieve chromatography (B) results;
  • Figure 5 Kinetic curves of Z20 (A), Z23 (B) and Z3L1 (C and D) and ZIKV-E;
  • Figure 7 Neutralization curve of antibody to Vero amplified ZIKV
  • Figure 8 Protective effect of three strains of antibodies on ZIKV-infected mice; A is the survival rate of mice, and B is the change in body weight of surviving mice.
  • the ZIKV E (amino acid sequence shown in SEQ ID NO: 21, nucleotide sequence shown in SEQ ID NO: 22) extracellular region DNA fragment was digested with NdeI and XhoI, and ligated into the pET21a vector.
  • the 3' end of the ZIKV E protein coding region is ligated with the coding sequence of six histidine tags (hexa-His-tag) and a translation stop codon.
  • the ligation product was then transformed into BL21 E. coli competent cells.
  • the monoclonal was inoculated into 40 mL of LB medium and cultured for 6-8 hours.
  • the inclusion bodies were harvested and the inclusion bodies were refolded by dilution.
  • the reconstituted solution was concentrated and replaced with 20 mM Tris, 150 mM NaCl, pH 8.0 buffer.
  • the concentrated protein solution was further purified by size exclusion chromatography using AKTA-purifier (GE) and superdex 200 Hiload 16/60 column (GE) using buffer A (20 mM Tris, 150 mM NaCl, pH 8.0) while monitoring.
  • the UV absorbance at 280 nm was collected for the protein of interest and the protein purity was identified by SDS-PAGE. The result is shown in Figure 1.
  • PBMCs peripheral blood cells
  • the isolated PBMCs were incubated with a density of 10 7 /mL at a final concentration of 100 nM of ZIKV-E protein for half an hour, then washed twice with PBS and incubated with the following antibodies: anti-human CD3/PE-Cy5, Anti-human CD16/PE-Cy5, anti-human CD235a/PE-Cy5, anti-human CD19/APC-Cy7, anti-human CD27/Pacific Blue, anti-human CD38/APC, anti-human IgG/FITC, and anti-His/PE.
  • the antibody was incubated on ice for half an hour and then washed twice with PBS.
  • the cells of PE-Cy5 - APC - APC-Cy7 + Pacific Blue + FITC + PE + were collected by FACSAria III and directly collected into a 96-well plate at 1 cell/well.
  • Example 3 Single B cell PCR, sequence analysis and human antibody design
  • Example 2 The B cells obtained in Example 2 were reverse transcribed by Superscript III reverse transcriptase (Invitrogen), and the reverse transcription primers were as shown in Table 1 (sequences such as SED ID No. 23 to SED ID NO. 30), and reacted at 55 ° C for 60 min.
  • PCR was carried out using HotStar Tap Plus enzyme (QIAgen) to amplify the antibody variable region sequence (PCRa).
  • the corresponding primers were designed, and the reaction conditions were as follows: 95 ° C, 5 min; 95 ° C 30 s, 55 ° C (heavy chain / kappa chain) / 50 ° C ( ⁇ chain) 30 s, 72 ° C 90 s, 35 cycles; 72 ° C, 7 min.
  • PCRb This was used as a template for another round of PCR (PCRb) under the following conditions: 95 ° C, 5 min; 95 ° C 30 s, 58 ° C (heavy chain) / 60 ° C ( ⁇ chain) / 64 ° C ( ⁇ chain) 30 s, 72 ° C 90 s , 35 cycles; 72 ° C, 7 min.
  • the PCR product was isolated by electrophoresis on a 1.2% agarose gel.
  • the strip size was recovered from the 400-500 bp gel and sent to the sequencing company for sequencing.
  • the sequencing results were analyzed using IMGT online software.
  • variable region sequence was analyzed and the constant region of the corresponding heavy chain/kappa chain/lambda chain was ligated by bridge PCR and cloned into the expression vector pCAGGS.
  • heavy chain is linked to the lambda chain by EcoRI and XhoI
  • kappa chain is linked to XhoI by SacI.
  • B cell sequencing and expression plasmid construction are as follows:
  • the human antibody design strategy is as follows:
  • Heavy chain CMV promoter-EcoR I-Leader sequences-heavy chain variable region-C H -Xho I;
  • amino acid sequence of the Leader sequences is SEQ ID NO: 19 (nucleotide sequence is SEQ ID NO: 20).
  • 293T cells were cultured in DMEM containing 10% FBS. 293T was co-transfected with a plasmid containing the light and heavy chain encoding genes of a particular antibody. After 4-6 hours of transfection, the cells were changed to serum-free DMEM for further 3 days, and the supernatant was collected, supplemented with DMEM, cultured for another 4 days, and the supernatant was collected.
  • the collected supernatant was centrifuged at 5000 rpm for 30 min, mixed with an equal volume of a buffer containing 20 mM sodium phosphate (pH 8.0), filtered through a 0.22 ⁇ m filter, and then bound to a proteinA prepacked column (5 mL, GE Healthcare). The bound protein was eluted with 10 mM glycine (pH 3.0). This protein was collected and concentrated for molecular sieve chromatography. The target peak was determined by SDS-PAGE, and the results are shown in Figures 2, 3 and 4, indicating that the target protein was normally expressed.
  • amino acid sequence of the heavy chain variable region of Z3L1 is SEQ ID NO: 1 (nucleotide sequence is SEQ ID NO: 7) and the light chain variable region amino acid sequence is SEQ ID NO: 2 (nucleotide sequence is SEQ. ID NO: 8).
  • the light chain of Z3L1 is ⁇ type, and the amino acid sequence of light chain constant region C L( ⁇ ) is SEQ ID NO: 17 (nucleotide sequence is SEQ ID NO: 18)
  • the amino acid sequence of the heavy chain variable region of Z20 is SEQ ID NO: 3 (nucleotide sequence is SEQ ID NO: 9) and the light chain variable region amino acid sequence is SEQ ID NO: 4 (nucleotide sequence is SEQ ID: NO: 10), the amino acid sequence of the heavy chain variable region of Z23 is SEQ ID NO: 5 (nucleotide sequence is SEQ ID NO: 11) and the light chain variable region amino acid sequence is SEQ ID NO: 6 (nucleoside The acid sequence is SEQ ID NO: 12).
  • the light chains of Z20 and Z23 are both kappa type, and the amino acid sequence of the light chain constant region CL ( ⁇ ) is SEQ ID NO: 15 (nucleotide sequence is SEQ ID NO: 16)
  • the antibody Z3L1 the amino acid sequence of Z20, Z23 heavy chain constant region of the C H is SEQ ID NO: 13 (nucleotide sequence SEQ ID NO: 14).
  • Biacore T100 Biacore Inc.
  • an antibody of anti-human IgG was immobilized to a channel (flow) (Fc) 1 and Fc 2 of a CM5 chip by amino coupling.
  • the fixed amount is controlled at around 10,000 response units (RU).
  • the channel was adjusted to Fc2, and then the purified antibody was bound by antibody capture.
  • the flow rate was controlled at 10 ⁇ L/min, and the injection amount was about 100 RU for 1 min.
  • the ZIKV-E protein was diluted with 10 mM HEPES, 150 mM NaCl, pH 7.4, and the flow rate was adjusted to 30 ⁇ L/min. After the channel was adjusted to the Fc2-Fc1 mode, the ZIKA-E protein was loaded one by one from a low concentration.
  • the curve composition is shown in the kinetic curve shown in Figure 5.
  • the results are shown in Table 3.
  • the calculation of the binding kinetic constants was performed using BIAevaluation software T100 (Biacore, Inc.) software.
  • the results of SPR showed that all three antibodies could bind to ZIKV-E protein, and the dissociation constant was 0.16 ⁇ M to 5.39 ⁇ M.
  • the purified antibody was diluted 3 fold, mixed with a 1:150 dilution of ZIKV (C6/36 or Vero amplification) and incubated for 60 minutes at 37 °C. The mixture was then added to a 24-well plate that had been covered with Vero cells at 300 ⁇ L/well. After incubating for 1 hour at 37 ° C, 1 mL of supplement medium (DMEM, 10% FBS) per well was added, and incubation was continued for 30 hours. The cells were collected, treated with PBS containing 4% paraformaldehyde, 0.05% soponin, and allowed to stand on ice for 30 min.
  • DMEM supplement medium
  • the cells were washed twice with solution solution (PBS, 1% BSA, 0.01% soponin), and incubated with 2 ⁇ g/mL of Z1 antibody for 30 min on ice, the solution solution was washed twice, and then diluted with 1:200 anti-human Incubate on IgG for 30 min in the dark. After the solution solution was washed twice, the cell positive ratio was measured by FACSCanto. The neutralization ability of the antibody against ZIKV was calculated according to the positive ratio at different concentrations. The results are shown in Figures 6 and 7, and the results are shown in Table 4.
  • Type I interferon receptor (Interferon ⁇ R) knockout mice (B6.129S2-Ifnar1 ⁇ tm1Agt>/Mmjax, Jackson Laboratories) were grouped in 3-5 groups. Each mouse was intraperitoneally injected with 1 ⁇ 10 6 PFU of ZIKV (GenBank accession number: KX087101.2). A single dose of 10 mg/kg of antibody Z3L1, Z20, Z23 or an equal volume of PBS was administered intraperitoneally to infected mice 24 hours after infection. The survival and body weight changes of the mice were recorded within 14 days. Mice with a change in body weight of more than 20% or with symptoms of spasticity were sacrificed. The results are shown in Figure 8. As can be seen from Fig.
  • the crystal structure of Zika virus E protein and the two antibodies Z3L1 and Z20, and the cryo-electron microscopic structure of Zika virus and Z23, the amino acid positions of the three antibodies interacting with Zika virus E protein are shown in Table 5-7.
  • the threshold for interaction site analysis is set at Among them, Z3L1 mainly binds to an E protein, and the binding domain is mainly DI, DII and the hinge region connecting the two domains; and the binding sites of Z20 and Z23 involve two E in one E protein dimer. Protein monomer.
  • Z20 mainly binds to the DII region of two E proteins
  • Z23 mainly binds to DIII.
  • the present invention obtains three human high-intensity and active ZIKV antibodies by screening ZIKV-E-specific memory B cells in a rehabilitation patient: Z3L1, Z20 and Z23. These three antibodies are completely different from the already reported Zika antibody sequences and are three newly discovered antibodies. The binding constants of these three antibodies to ZIKV-E were 5.39 ⁇ M (Z3L1), 0.16 ⁇ M (Z20) and 0.44 ⁇ M (Z23), respectively. All three human antibodies have strong Zika virus neutralizing activity. Moreover, Z3L1, Z20 and Z23 can completely or partially protect mice from lethal doses of Zika virus. Structural information indicates that the three antibodies bind to different domains of the E protein, respectively. This suggests that the three human antibodies have great potential to play a role in the clinical treatment and prevention of Zika in cocktail therapy.

Abstract

A humanized monoclonal antibody for Zika virus, and applications thereof, related to the technical field of medicine. Using Zika E protein expressed by E. coli as an antigen, selecting from PBMCs of a convalescing Zika patient a memory B cell capable of specifically binding to Zika E protein by means of flow sorting, then performing RT-PCR on the single selected B cell, obtaining a sequence and fragment of a variable region of the antibody, and further connecting same with a constant region into an expression vector. After expression by a mammalian cell and purification, performing a series of function tests, comprising detecting bonding strength with ZIKV-E protein, in vitro neutralization results, in vivo protection ability, etc., obtaining three strains of humanized monoclonal antibody having complete protection from Zika virus infection.

Description

一种寨卡病毒人源单克隆抗体及其应用Zika virus human monoclonal antibody and application thereof 技术领域Technical field
本发明涉及一种寨卡病毒人源单克隆抗体及其应用，属于医药技术领域。The invention relates to a human monoclonal antibody of Zika virus and application thereof, and belongs to the technical field of medicine.
背景技术Background technique
越来越多的证据表明寨卡病毒(Zika virus，ZIKV)可引起新生儿小头症以及神经发育异常，如“格林-巴氏综合征”等。ZIKV，属于黄病毒，主要由蚊虫叮咬传播，但是血液传播，性传播以及母胎传播途径也有报道。1947年，人们首次在乌干达丛林的猕猴分离到ZIKV。该病毒主要流行于非洲以及亚洲的热带地区。2015年，巴西暴发寨卡疫情，随着人群流动扩散至其他国家。目前此次疫情已导致超过60个国家或地区超过80,000例感染病例。然而，针对该病毒仍缺乏有效的疫苗及治疗手段。There is growing evidence that Zika virus (ZIKV) can cause neonatal small head disease and neurodevelopmental abnormalities such as "Green-Pap syndrome". ZIKV, which belongs to the flavivirus, is mainly transmitted by mosquito bites, but blood transmission, sexual transmission, and mother-to-child transmission have also been reported. In 1947, the first time in the jungle of Uganda, the macaques were separated into ZIKV. The virus is mainly prevalent in Africa and the tropical regions of Asia. In 2015, the Zika outbreak in Brazil broke out with the spread of people to other countries. The current outbreak has resulted in more than 80,000 infections in more than 60 countries or regions. However, there is still no effective vaccine and treatment for this virus.
中和抗体特异性高、能阻断病毒的入侵，是治疗病毒感染的有效手段。黄病毒囊膜蛋白E蛋白介导病毒与宿主细胞的结合和入侵，是病毒最主要的保护性抗原。X-射线晶体学结构显示黄病毒E蛋白有三个结构域：DI，DII和DIII。一般认为DIII负责介导病毒与受体的结合，之前的研究结果表明大多数有效的中和抗体都是识别DIII的中和表位。然而，近期的数据显示病毒表面的E蛋白二聚体以及与相邻二聚体中也存在高中和表位。DII头部(98-110位氨基酸)包含一个高度保守的融合区(fusion loop，FL)，在病毒入侵的膜融合过程中起关键作用。不同黄病毒FL区域保守性非常高，在病毒感染过程中，免疫细胞会产生大量的针对FL的抗体。The neutralizing antibody has high specificity and can block the invasion of the virus, and is an effective means for treating viral infection. The viral envelope protein E protein mediates the binding and invasion of the virus to the host cell and is the most important protective antigen of the virus. The X-ray crystallographic structure shows that the flavivirus E protein has three domains: DI, DII and DIII. It is generally believed that DIII is responsible for mediating the binding of the virus to the receptor. Previous studies have shown that most of the effective neutralizing antibodies are neutralizing epitopes that recognize DIII. However, recent data indicate that E-protein dimers on the surface of the virus as well as high-neutral epitopes are also present in adjacent dimers. The DII head (amino acids 98-110) contains a highly conserved fusion loop (FL) that plays a key role in membrane fusion during viral invasion. Different flavivirus FL regions are highly conserved, and during viral infection, immune cells produce large amounts of antibodies against FL.
已有研究发现有一些登革等黄病毒的中和抗体具有广谱中和活性，可以中和寨卡病毒，如：2A10G6、A11、C8等，这些抗体一般结合在保守的融合肽上或附近的区域。最近有报道从寨卡康复病人PBMC中分离到结合在E蛋白DIII的中和抗体。也有利用小鼠杂交瘤细胞，筛选到能够结合DIII的寨卡特异性中和抗体。这些抗体不同程度地能够保护小鼠免受致死剂量的病毒攻击。然而，RNA病毒在抗体压力下，具有高突变的特性，尽管已经有些中和抗体被鉴定，但是更多的针对不同表位的新的抗体对于治疗是必不可少的。本发明的目的是鉴定特异的具有保护作用的新的ZIKV中和抗体。Studies have found that some neutralizing antibodies against dengue and other flaviviruses have broad-spectrum neutralizing activity and can neutralize Zika virus, such as 2A10G6, A11, C8, etc. These antibodies are generally bound to or near conserved fusion peptides. Area. Recently, it has been reported that a neutralizing antibody that binds to E protein DIII is isolated from ZB card rehabilitation patient PBMC. Zika-specific neutralizing antibodies capable of binding to DIII were also screened using mouse hybridoma cells. These antibodies are capable of protecting mice from lethal doses of virus attack to varying degrees. However, RNA viruses have high mutational properties under antibody pressure, and although some neutralizing antibodies have been identified, more new antibodies against different epitopes are essential for treatment. The object of the present invention is to identify specific protective ZIKV neutralizing antibodies.
发明内容Summary of the invention
为了解决上述问题，本发明首先以大肠杆菌表达的ZIKV-E蛋白作为抗原，通过流式分选，从一例康复期ZIKV患者的PBMCs中筛选到可以特异结合ZIKV-E蛋白的记忆B细胞，然后对筛选的单一B细胞进行RT-PCR，获得抗体的可变区序列与片段，并进一步与恒定区 连接至表达载体中。经哺乳动物细胞表达、纯化后，进行一系列的功能检测，包括与ZIKE-E蛋白的结合力，体外中和效果，体内保护能力等检测，获得了3株具有完全或部分保护寨卡病毒感染的人源单克隆抗体。In order to solve the above problems, the present invention firstly uses the ZIKV-E protein expressed by Escherichia coli as an antigen, and selects memory B cells which can specifically bind ZIKV-E protein from PBMCs of a patient in a convalescent ZIKV by flow sorting, and then RT-PCR of selected single B cells to obtain variable region sequences and fragments of antibodies, and further with constant regions Linked to an expression vector. After being expressed and purified by mammalian cells, a series of functional tests, including binding to ZIKE-E protein, in vitro neutralization effect, and in vivo protective ability, were obtained, and 3 strains with full or partial protection of Zika virus were obtained. Human monoclonal antibody.
本发明的第一个目的是提供一种抗体，所述抗体：A first object of the invention is to provide an antibody which:
b)能与SEQ ID NO:21的寨卡病毒E蛋白的第46(VAL)、47(THR)、52(ASN)、136(GLU)、138(ARG)、140(MET)、156(THR)、158(HIS)、159(GLU)、166(LYS)、168(GLU)、259(GLU)、276(GLU)、277(MET)、278(ASP)、280(ALA)、281(LYS)和283(ARG)位氨基酸发生相互作用；或者，b) 46(VAL), 47(THR), 52(ASN), 136(GLU), 138(ARG), 140(MET), 156(THR) capable of the Zika virus E protein of SEQ ID NO:21. ), 158 (HIS), 159 (GLU), 166 (LYS), 168 (GLU), 259 (GLU), 276 (GLU), 277 (MET), 278 (ASP), 280 (ALA), 281 (LYS) ) interacts with amino acids at position 283 (ARG); or,
b)能与SEQ ID NO:21的寨卡病毒E蛋白的第64(SER)、65(ILE)、66(SER)、67(ASP)、68(MET)、69(ALA)、84(LYS)、87(ASP)、89(GLN)、90(TYR)、118(LYS)、119(PHE)、120(ALA)、208(ASN)、232(GLY)、233(THR)、252(ARG)、276(GLU)、277(MET)、278(ASP)和279(GLY)位氨基酸发生相互作用；或者，b) 64 (SER), 65 (ILE), 66 (SER), 67 (ASP), 68 (MET), 69 (ALA), 84 (LYS) of the Zika virus E protein of SEQ ID NO:21. ), 87 (ASP), 89 (GLN), 90 (TYR), 118 (LYS), 119 (PHE), 120 (ALA), 208 (ASN), 232 (GLY), 233 (THR), 252 (ARG) , 276 (GLU), 277 (MET), 278 (ASP) and 279 (GLY) amino acids interact; or,
c)能与SEQ ID NO:21的寨卡病毒E蛋白的第51(SER)、67(ASP)、68(MET)、69(ALA)、310(ALA)、311(ALA)、312(PHE)、313(THR)、314(PHE)、315(THR)、317(ILE)、331(GLN)、332(TYR)、333(ALA)、334(GLY)、335(THR)、336(ASP)、368(SER)、369(THR)、370(GLU)、371(ASN)和394(LYS)位氨基酸发生相互作用。c) 51 (SER), 67 (ASP), 68 (MET), 69 (ALA), 310 (ALA), 311 (ALA), 312 (PHE) of the Zika virus E protein of SEQ ID NO:21. ), 313 (THR), 314 (PHE), 315 (THR), 317 (ILE), 331 (GLN), 332 (TYR), 333 (ALA), 334 (GLY), 335 (THR), 336 (ASP , 368 (SER), 369 (THR), 370 (GLU), 371 (ASN) and 394 (LYS) amino acids interact.
在本发明的一种实施方式中，所述抗体命名为Z3L1、Z20或者Z23；其中Z3L1的重链可变区的氨基酸序列为SEQ ID NO:1且轻链可变区氨基酸序列为SEQ ID NO:2，Z20的重链可变区的氨基酸序列为SEQ ID NO:3且轻链可变区氨基酸序列为SEQ ID NO:4，Z23的重链可变区的氨基酸序列为SEQ ID NO:5且轻链可变区氨基酸序列为SEQ ID NO:6。In one embodiment of the invention, the antibody is designated Z3L1, Z20 or Z23; wherein the amino acid sequence of the heavy chain variable region of Z3L1 is SEQ ID NO: 1 and the amino acid sequence of the light chain variable region is SEQ ID NO The amino acid sequence of the heavy chain variable region of Z20 is SEQ ID NO: 3 and the amino acid sequence of the light chain variable region is SEQ ID NO: 4, and the amino acid sequence of the heavy chain variable region of Z23 is SEQ ID NO: And the light chain variable region amino acid sequence is SEQ ID NO: 6.
在本发明的一种实施方式中，所述Z3L1的重链的核苷酸序列为SEQ ID NO:7、轻链的核苷酸序列为SEQ ID NO:8。In one embodiment of the present invention, the nucleotide sequence of the heavy chain of Z3L1 is SEQ ID NO: 7, and the nucleotide sequence of the light chain is SEQ ID NO: 8.
在本发明的一种实施方式中，所述Z20的重链的核苷酸序列为SEQ ID NO:9、轻链的核苷酸序列为SEQ ID NO:10。In one embodiment of the present invention, the nucleotide sequence of the heavy chain of Z20 is SEQ ID NO: 9, and the nucleotide sequence of the light chain is SEQ ID NO: 10.
在本发明的一种实施方式中，所述Z23的重链的核苷酸序列为SEQ ID NO:11、轻链的核苷酸序列为SEQ ID NO:12。In one embodiment of the present invention, the nucleotide sequence of the heavy chain of Z23 is SEQ ID NO: 11, and the nucleotide sequence of the light chain is SEQ ID NO: 12.
在本发明的一种实施方式中，所述抗体的重链，包括重链可变区和重链恒定区，其中重链恒定区的氨基酸序列如SEQ ID NO:13所示。In one embodiment of the invention, the heavy chain of the antibody comprises a heavy chain variable region and a heavy chain constant region, wherein the amino acid sequence of the heavy chain constant region is set forth in SEQ ID NO: 13.
在本发明的一种实施方式中，所述抗体Z20或者Z23的轻链为κ链，Z3L1的轻链为λ链；轻链包括轻链可变区和轻链恒定区；κ链的轻链恒定区的氨基酸序列如SEQ ID NO:15所示， λ链的轻链恒定区的氨基酸序列如SEQ ID NO:17所示。In one embodiment of the invention, the light chain of the antibody Z20 or Z23 is a kappa chain, the light chain of Z3L1 is a λ chain; the light chain comprises a light chain variable region and a light chain constant region; the light chain of the kappa chain The amino acid sequence of the constant region is set forth in SEQ ID NO: 15. The amino acid sequence of the light chain constant region of the lambda chain is set forth in SEQ ID NO: 17.
在本发明的一种实施方式中，所述发生相互作用是指结合。In one embodiment of the invention, the occurrence of interaction refers to binding.
本发明的三个抗体，来源于同一病患者，并且均靶向寨卡病毒特有的囊膜蛋白-E蛋白，通过抑制E蛋白介导的受体结合和/或膜融合过程，抑制病毒对细胞的感染。The three antibodies of the present invention are derived from the same patient and all target the envelope protein-E protein unique to Zika virus, inhibiting virus-to-cell by inhibiting E protein-mediated receptor binding and/or membrane fusion processes. Infection.
本发明的第二个目的是提供所述抗体在制备治疗和/或预防寨卡病毒的药物方面的应用。A second object of the present invention is to provide use of the antibody for the preparation of a medicament for the treatment and/or prevention of Zika virus.
本发明的第三个目的是提供一种药物组合物，所述药物组合物含有所述的人源单克隆抗体Z3L1、Z20和/或Z23。A third object of the present invention is to provide a pharmaceutical composition comprising the human monoclonal antibodies Z3L1, Z20 and/or Z23.
在本发明的一种实施方式中，所述药物组合物还含有医学生可接受的载体。In one embodiment of the invention, the pharmaceutical composition further comprises a medically acceptable carrier.
本发明的第四个目的是提供一种试剂盒，所述试剂盒中含有所述抗体的抗原，或者编码所述抗原的DNA分子，或者表达所述抗原的重组载体/表达盒/转基因细胞系/重组菌。A fourth object of the present invention is to provide a kit comprising an antigen of the antibody, or a DNA molecule encoding the antigen, or a recombinant vector/expression cassette/transgenic cell line expressing the antigen /Recombinant bacteria.
本发明的第五个目的是提供编码所述人源单克隆抗体Z3L1、Z20或者Z23的基因序列。A fifth object of the present invention is to provide a gene sequence encoding the human monoclonal antibody Z3L1, Z20 or Z23.
所述抗体的重链，包括重链可变区和重链恒定区，其中重链恒定区的氨基酸序列如SEQ ID NO:13所示(核苷酸序列如SEQ ID NO:14所示)。The heavy chain of the antibody comprises a heavy chain variable region and a heavy chain constant region, wherein the amino acid sequence of the heavy chain constant region is set forth in SEQ ID NO: 13 (the nucleotide sequence is set forth in SEQ ID NO: 14).
在本发明的一种实施方式中，编码所述抗体的重链的序列，依次包括CMV启动子序列、EcoR I酶切位点序列、前导序列、编码重链可变区的序列、编码重链恒定区的序列、Xho I酶切位点序列。In one embodiment of the invention, the sequence encoding the heavy chain of the antibody, in turn, comprises a CMV promoter sequence, an EcoR I cleavage site sequence, a leader sequence, a sequence encoding a heavy chain variable region, a coding heavy chain Sequence of the constant region, sequence of the Xho I restriction site.
在本发明的一种实施方式中，所述抗体的轻链，为κ链和/或λ链；轻链包括轻链可变区和轻链恒定区。In one embodiment of the invention, the light chain of the antibody is a kappa chain and/or a lambda chain; the light chain comprises a light chain variable region and a light chain constant region.
在本发明的一种实施方式中，编码所述抗体的轻链的序列，依次包括CMV启动子序列、第一酶切位点序列、前导序列、编码轻链可变区的序列、编码轻链恒定区的序列、酶切位点序列Xho I。In one embodiment of the invention, the sequence encoding the light chain of the antibody, in turn comprises a CMV promoter sequence, a first restriction site sequence, a leader sequence, a sequence encoding a light chain variable region, encoding a light chain The sequence of the constant region, the restriction enzyme site sequence Xho I.
在本发明的一种实施方式中，所述κ链，其轻链恒定区的氨基酸序列如SEQ ID NO:15所示(核苷酸序列如SEQ ID NO:16所示)，其中第一酶切位点为Sac I。In one embodiment of the present invention, the kappa chain has an amino acid sequence of a light chain constant region as set forth in SEQ ID NO: 15 (nucleotide sequence is set forth in SEQ ID NO: 16), wherein the first enzyme The cleavage site is Sac I.
在本发明的一种实施方式中，所述λ链，其轻链恒定区的氨基酸序列如SEQ ID NO:17所示(核苷酸序列如SEQ ID NO:18所示)，其中第一酶切位点为EcoR I。In one embodiment of the present invention, the λ chain, the amino acid sequence of the light chain constant region thereof is set forth in SEQ ID NO: 17 (the nucleotide sequence is represented by SEQ ID NO: 18), wherein the first enzyme The cleavage site is EcoR I.
在本发明的一种实施方式中，所述前导序列的氨基酸序列如SEQ ID NO:19所示(核苷酸序列如SEQ ID NO:20所示)。In one embodiment of the invention, the amino acid sequence of the leader sequence is set forth in SEQ ID NO: 19 (the nucleotide sequence is set forth in SEQ ID NO: 20).
本发明还要求保护含有所述基因序列或者表达所述抗体的表达载体、细胞。The invention also claims an expression vector or cell comprising the gene sequence or expressing the antibody.
本发明的有益效果：The beneficial effects of the invention:
本发明获得了3株人源高中和活性的ZIKV抗体：Z3L1、Z20与Z23。这3株抗体与已 经报道的ZIKV抗体序列完全不同，是3株新发现的抗体。这三株抗体与ZIKV-E的结合常数分别为5.39μM(Z3L1)，0.16μM(Z20)和0.44μM(Z23)。三株人源抗体均有很强的ZIKV中和活性。并且，Z3L1、Z20与Z23可以完全或部分保护小鼠免受致死剂量的ZIKV的攻击。本发明的三株人源抗体有着临床治疗和预防ZIKV的应用价值。The present invention obtained three human high- and medium-active ZIKV antibodies: Z3L1, Z20 and Z23. These 3 antibodies have been The reported ZIKV antibody sequences are completely different and are 3 newly discovered antibodies. The binding constants of these three antibodies to ZIKV-E were 5.39 μM (Z3L1), 0.16 μM (Z20) and 0.44 μM (Z23), respectively. All three human antibodies have strong ZIKV neutralizing activity. Moreover, Z3L1, Z20 and Z23 can completely or partially protect mice from lethal doses of ZIKV. The three human antibodies of the invention have application value in clinical treatment and prevention of ZIKV.
附图说明DRAWINGS
图1：ZIKV-E蛋白纯化分子筛与SDS-PAGE结果；Figure 1: ZIKV-E protein purified molecular sieve and SDS-PAGE results;
图2：Z3L1纯化的Protein A(A)及分子筛层析(B)结果；Figure 2: Z3L1 purified Protein A (A) and molecular sieve chromatography (B) results;
图3：Z20纯化的Protein A(A)及分子筛层析(B)结果；Figure 3: Z20 purified Protein A (A) and molecular sieve chromatography (B) results;
图4：Z23纯化的Protein A(A)及分子筛层析(B)结果；Figure 4: Z23 purified Protein A (A) and molecular sieve chromatography (B) results;
图5：Z20(A)、Z23(B)与Z3L1(C与D)与ZIKV-E的动力学曲线；Figure 5: Kinetic curves of Z20 (A), Z23 (B) and Z3L1 (C and D) and ZIKV-E;
图6：抗体对C6/36扩增的ZIKV的中和曲线；Figure 6: Neutralization curve of antibody against CIK/36 amplified ZIKV;
图7：抗体对Vero扩增的ZIKV的中和曲线；Figure 7: Neutralization curve of antibody to Vero amplified ZIKV;
图8：三株抗体对感染ZIKV小鼠的保护效果；A为小鼠存活率，B为存活小鼠体重变化。Figure 8: Protective effect of three strains of antibodies on ZIKV-infected mice; A is the survival rate of mice, and B is the change in body weight of surviving mice.
具体实施方式detailed description
实施例1：寨卡E蛋白的表达与纯化Example 1: Expression and purification of Zika protein E
ZIKV E(氨基酸序列如SEQ ID NO:21所示、核苷酸序列如SEQ ID NO:22所示)胞外区DNA片段通过NdeI和XhoI酶切后，连接到pET21a载体上。其中ZIKV E蛋白编码区的3’端连上6个组氨酸标签(hexa-His-tag)的编码序列及翻译终止密码子。再将连接产物转化到BL21大肠杆菌感受态细胞。单克隆接种到40mL LB培养基中，培养6-8小时。接种到4L的LB培养基中，37℃培养至OD600＝0.4-0.6，加入IPTG至终浓度1mM，37℃继续培养4-6小时。收获包涵体，通过稀释法复性包涵体。复性液浓缩后换成20mM Tris，150mM NaCl，pH8.0缓冲液。将浓缩后的蛋白溶液进一步以分子排阻层析纯化，使用AKTA-purifier(GE)和superdex200Hiload 16/60柱子(GE)，使用缓冲液A(20mM Tris，150mM NaCl，pH8.0)，同时监测280nm的紫外吸收值，收取目的蛋白，并通过SDS-PAGE鉴定蛋白纯度。结果如图1。The ZIKV E (amino acid sequence shown in SEQ ID NO: 21, nucleotide sequence shown in SEQ ID NO: 22) extracellular region DNA fragment was digested with NdeI and XhoI, and ligated into the pET21a vector. The 3' end of the ZIKV E protein coding region is ligated with the coding sequence of six histidine tags (hexa-His-tag) and a translation stop codon. The ligation product was then transformed into BL21 E. coli competent cells. The monoclonal was inoculated into 40 mL of LB medium and cultured for 6-8 hours. The cells were inoculated into 4 L of LB medium, cultured at 37 ° C until OD600 = 0.4-0.6, IPTG was added to a final concentration of 1 mM, and incubation was continued at 37 ° C for 4-6 hours. The inclusion bodies were harvested and the inclusion bodies were refolded by dilution. The reconstituted solution was concentrated and replaced with 20 mM Tris, 150 mM NaCl, pH 8.0 buffer. The concentrated protein solution was further purified by size exclusion chromatography using AKTA-purifier (GE) and superdex 200 Hiload 16/60 column (GE) using buffer A (20 mM Tris, 150 mM NaCl, pH 8.0) while monitoring. The UV absorbance at 280 nm was collected for the protein of interest and the protein purity was identified by SDS-PAGE. The result is shown in Figure 1.
实施例2：ZIKV-E蛋白特异性记忆B细胞的分离Example 2: Isolation of ZIKV-E protein-specific memory B cells
在病人的知情同意下，采集15mL的血液，分离PBMCs。将分离的PBMCs以107/mL的密度与终浓度是100nM的ZIKV-E蛋白冰上孵育结合半小时，然后用PBS洗2次，再与下列抗体孵育：anti-human CD3/PE-Cy5，anti-human CD16/PE-Cy5，anti-human CD235a/PE-Cy5，anti-human CD19/APC-Cy7，anti-human CD27/Pacific Blue，anti-human CD38/APC，anti-human IgG/FITC，以及anti-His/PE。抗体冰上孵育半小时后，用PBS洗2 次。Under the patient's informed consent, 15 mL of blood was collected and PBMCs were isolated. The isolated PBMCs were incubated with a density of 10 7 /mL at a final concentration of 100 nM of ZIKV-E protein for half an hour, then washed twice with PBS and incubated with the following antibodies: anti-human CD3/PE-Cy5, Anti-human CD16/PE-Cy5, anti-human CD235a/PE-Cy5, anti-human CD19/APC-Cy7, anti-human CD27/Pacific Blue, anti-human CD38/APC, anti-human IgG/FITC, and anti-His/PE. The antibody was incubated on ice for half an hour and then washed twice with PBS.
经FACSAria III分选收集PE-Cy5-APC-APC-Cy7+Pacific Blue+FITC+PE+的细胞，直接收集到96孔板内，1细胞/孔。The cells of PE-Cy5 - APC - APC-Cy7 + Pacific Blue + FITC + PE + were collected by FACSAria III and directly collected into a 96-well plate at 1 cell/well.
实施例3：单一B细胞PCR、序列分析及人源抗体设计Example 3: Single B cell PCR, sequence analysis and human antibody design
将实施例2获得的B细胞通过Superscript III reverse transcriptase(Invitrogen)逆转录，逆转录引物如表1(序列如SED ID NO.23至SED ID NO.30所示)，55℃反应60min。The B cells obtained in Example 2 were reverse transcribed by Superscript III reverse transcriptase (Invitrogen), and the reverse transcription primers were as shown in Table 1 (sequences such as SED ID No. 23 to SED ID NO. 30), and reacted at 55 ° C for 60 min.
表1.逆转录反应引物Table 1. Reverse transcription reaction primers
Figure PCTCN2017096816-appb-000001
Figure PCTCN2017096816-appb-000001
将此逆转录产物作为模板，用HotStar Tap Plus酶(QIAgen)进行PCR，扩增抗体可变区序列(PCRa)。设计相应的引物，反应条件如下：95℃，5min；95℃30s，55℃(重链/κ链)/50℃(λ链)30s，72℃90s，35个循环；72℃，7min。将此作为模板再进行1轮PCR(PCRb)，条件如下：95℃，5min；95℃30s，58℃(重链)/60℃(κ链)/64℃(λ链)30s，72℃90s，35个循环；72℃，7min。Using this reverse transcription product as a template, PCR was carried out using HotStar Tap Plus enzyme (QIAgen) to amplify the antibody variable region sequence (PCRa). The corresponding primers were designed, and the reaction conditions were as follows: 95 ° C, 5 min; 95 ° C 30 s, 55 ° C (heavy chain / kappa chain) / 50 ° C (λ chain) 30 s, 72 ° C 90 s, 35 cycles; 72 ° C, 7 min. This was used as a template for another round of PCR (PCRb) under the following conditions: 95 ° C, 5 min; 95 ° C 30 s, 58 ° C (heavy chain) / 60 ° C (κ chain) / 64 ° C (λ chain) 30 s, 72 ° C 90 s , 35 cycles; 72 ° C, 7 min.
1.2％的琼脂糖凝胶电泳，分离PCR产物。条带大小在400-500bp的切胶回收后送测序公司测序。测序结果用IMGT在线软件进行分析。The PCR product was isolated by electrophoresis on a 1.2% agarose gel. The strip size was recovered from the 400-500 bp gel and sent to the sequencing company for sequencing. The sequencing results were analyzed using IMGT online software.
分析正确的可变区序列与相应的重链/κ链/λ链的恒定区通过搭桥PCR连接，克隆至表达载体pCAGGS中。其中重链与λ链以EcoRI和XhoI连接，κ链以SacI与XhoI连接。B细胞测序及表达质粒构建如下：The correct variable region sequence was analyzed and the constant region of the corresponding heavy chain/kappa chain/lambda chain was ligated by bridge PCR and cloned into the expression vector pCAGGS. Wherein the heavy chain is linked to the lambda chain by EcoRI and XhoI, and the kappa chain is linked to XhoI by SacI. B cell sequencing and expression plasmid construction are as follows:
人源抗体设计策略如下：The human antibody design strategy is as follows:
重链：CMV promoter-EcoR I-Leader sequences-重链可变区-CH-Xho I；Heavy chain: CMV promoter-EcoR I-Leader sequences-heavy chain variable region-C H -Xho I;
轻链(κ)：CMV promoter-Sac I-Leader sequences-轻链可变区-CL(κ)-Xho I；Light chain (κ): CMV promoter-Sac I-Leader sequences-light chain variable region-C L(κ) -Xho I;
轻链(λ)：CMV promoter-EcoR I-Leader sequences-轻链可变区-CL(λ)-Xho I。Light chain (λ): CMV promoter-EcoR I-Leader sequences - light chain variable region - C L (λ) - Xho I.
其中，Leader sequences的氨基酸序列如SEQ ID NO:19(核苷酸序列如SEQ ID NO:20)。Wherein the amino acid sequence of the Leader sequences is SEQ ID NO: 19 (nucleotide sequence is SEQ ID NO: 20).
实施例4：抗体的表达及纯化 Example 4: Expression and purification of antibodies
用含10％FBS的DMEM培养293T细胞。用含有特定抗体轻、重链编码基因的质粒共转染293T。转染4-6小时后给细胞换液成无血清的DMEM继续培养3天，收集上清，并补加DMEM，再培养4天，收集上清。293T cells were cultured in DMEM containing 10% FBS. 293T was co-transfected with a plasmid containing the light and heavy chain encoding genes of a particular antibody. After 4-6 hours of transfection, the cells were changed to serum-free DMEM for further 3 days, and the supernatant was collected, supplemented with DMEM, cultured for another 4 days, and the supernatant was collected.
收集的上清经过5000rpm离心30min后，与含有20mM磷酸钠(pH 8.0)的缓冲液等体积混合，经过0.22μm滤膜过滤后，与proteinA预装柱结合(5mL，GE Healthcare)。以10mM甘氨酸(pH 3.0)洗脱结合的蛋白。收集此蛋白浓缩后进行分子筛层析。目的峰通过SDS-PAGE确定，结果如图2、3以及4，说明目标蛋白得到正常表达。The collected supernatant was centrifuged at 5000 rpm for 30 min, mixed with an equal volume of a buffer containing 20 mM sodium phosphate (pH 8.0), filtered through a 0.22 μm filter, and then bound to a proteinA prepacked column (5 mL, GE Healthcare). The bound protein was eluted with 10 mM glycine (pH 3.0). This protein was collected and concentrated for molecular sieve chromatography. The target peak was determined by SDS-PAGE, and the results are shown in Figures 2, 3 and 4, indicating that the target protein was normally expressed.
最终，得到了三个能与ZIKV E蛋白结合且中和活性较强的抗体Z3L1、Z20、Z23。Finally, three antibodies Z3L1, Z20, and Z23 which bind to ZIKV E protein and have strong neutralizing activity were obtained.
其中Z3L1的重链可变区的氨基酸序列为SEQ ID NO:1(核苷酸序列如SEQ ID NO:7)且轻链可变区氨基酸序列为SEQ ID NO:2(核苷酸序列如SEQ ID NO:8)。Z3L1的轻链为λ型，轻链恒定区CL(λ)的氨基酸序列为SEQ ID NO:17(核苷酸序列如SEQ ID NO:18)Wherein the amino acid sequence of the heavy chain variable region of Z3L1 is SEQ ID NO: 1 (nucleotide sequence is SEQ ID NO: 7) and the light chain variable region amino acid sequence is SEQ ID NO: 2 (nucleotide sequence is SEQ. ID NO: 8). The light chain of Z3L1 is λ type, and the amino acid sequence of light chain constant region C L(λ) is SEQ ID NO: 17 (nucleotide sequence is SEQ ID NO: 18)
Z20的重链可变区的氨基酸序列为SEQ ID NO:3(核苷酸序列如SEQ ID NO:9)且轻链可变区氨基酸序列为SEQ ID NO:4(核苷酸序列如SEQ ID NO:10)，Z23的重链可变区的氨基酸序列为SEQ ID NO:5(核苷酸序列如SEQ ID NO:11)且轻链可变区氨基酸序列为SEQ ID NO:6(核苷酸序列如SEQ ID NO:12)。Z20和Z23的轻链均为κ型，轻链恒定区CL(κ)的氨基酸序列为SEQ ID NO:15(核苷酸序列如SEQ ID NO:16)The amino acid sequence of the heavy chain variable region of Z20 is SEQ ID NO: 3 (nucleotide sequence is SEQ ID NO: 9) and the light chain variable region amino acid sequence is SEQ ID NO: 4 (nucleotide sequence is SEQ ID: NO: 10), the amino acid sequence of the heavy chain variable region of Z23 is SEQ ID NO: 5 (nucleotide sequence is SEQ ID NO: 11) and the light chain variable region amino acid sequence is SEQ ID NO: 6 (nucleoside The acid sequence is SEQ ID NO: 12). The light chains of Z20 and Z23 are both kappa type, and the amino acid sequence of the light chain constant region CL (κ) is SEQ ID NO: 15 (nucleotide sequence is SEQ ID NO: 16)
所述抗体Z3L1、Z20、Z23的重链恒定区CH的的氨基酸序列为SEQ ID NO:13(核苷酸序列如SEQ ID NO:14)。The antibody Z3L1, the amino acid sequence of Z20, Z23 heavy chain constant region of the C H is SEQ ID NO: 13 (nucleotide sequence SEQ ID NO: 14).
与已经报道的具有中和ZIKV活性的人源抗体进行序列比对，发现本发明中的三株人源抗体：Z20(IGVH 4-4*02-IGVD 3-3*01–IGVJ 4*02)与Z3L1(IGVH 3-30-3*03-IGVD 6-13*01–IGVJ 4*02)重链均具有独特的V-D-J基因重排方式；与Z23使用相同的IGVH胚系基因的抗体有3株，如表2所示。但是4株抗体使用的IGVD胚系基因均各不相同，与Z23氨基酸序列的同源性分别仅88.24，84.3和90.76％。Sequence alignment with the reported human antibody with neutralizing ZIKV activity, and found three human antibodies in the present invention: Z20 (IGVH 4-4*02-IGVD 3-3*01–IGVJ 4*02) The heavy chain with Z3L1 (IGVH 3-30-3*03-IGVD 6-13*01–IGVJ 4*02) has a unique VDJ gene rearrangement pattern; 3 antibodies with the same IGVH germline gene as Z23 ,As shown in table 2. However, the IGVD germline genes used in the four antibodies were all different, and the homology with the Z23 amino acid sequence was only 88.24, 84.3 and 90.76%, respectively.
表2与Z23相同IGVH基因的ZIKV中和抗体比较Table 2 Comparison of ZIKV neutralizing antibodies with the same IGVH gene of Z23
Figure PCTCN2017096816-appb-000002
Figure PCTCN2017096816-appb-000002
实施例5：人源抗体的性能检测Example 5: Performance testing of human antibodies
(1)表面等离子共振技术检测与ZIKV-E的结合能力 (1) Surface plasmon resonance technology detects the ability to bind to ZIKV-E
表面等离子共振分析利用Biacore T100(Biacore Inc.)进行。具体步骤如下：Surface plasmon resonance analysis was performed using a Biacore T100 (Biacore Inc.). Specific steps are as follows:
首先，将anti-human IgG的抗体以氨基偶联的方式固定在CM5芯片的通道(flow cell,Fc)1与Fc2。固定量控制在10,000响应值(response units,RU)左右。将通道调至Fc2，然后以抗体捕获的方式结合纯化的抗体，此时，液流速度控制在10μL/min，进样1min，抗体捕获量在100RU左右。再以10mM HEPES，150mM NaCl，pH 7.4溶液倍比稀释ZIKV-E蛋白，流速调至30μL/min，通道调至Fc2-Fc1的模式后，从低浓度开始逐一上样ZIKA-E蛋白。曲线组成图如图5所示的动力学曲线。结果如表3所示。结合动力学常数的计算是利用BIAevaluation software T100(Biacore，Inc.)软件进行。SPR的结果显示，三株抗体均可以与ZIKV-E蛋白结合，解离常数0.16μM～5.39μM。First, an antibody of anti-human IgG was immobilized to a channel (flow) (Fc) 1 and Fc 2 of a CM5 chip by amino coupling. The fixed amount is controlled at around 10,000 response units (RU). The channel was adjusted to Fc2, and then the purified antibody was bound by antibody capture. At this time, the flow rate was controlled at 10 μL/min, and the injection amount was about 100 RU for 1 min. The ZIKV-E protein was diluted with 10 mM HEPES, 150 mM NaCl, pH 7.4, and the flow rate was adjusted to 30 μL/min. After the channel was adjusted to the Fc2-Fc1 mode, the ZIKA-E protein was loaded one by one from a low concentration. The curve composition is shown in the kinetic curve shown in Figure 5. The results are shown in Table 3. The calculation of the binding kinetic constants was performed using BIAevaluation software T100 (Biacore, Inc.) software. The results of SPR showed that all three antibodies could bind to ZIKV-E protein, and the dissociation constant was 0.16 μM to 5.39 μM.
表3人源抗体与ZIKV E蛋白结合的动力学常数Table 3 Kinetic constants of binding of human antibody to ZIKV E protein
Figure PCTCN2017096816-appb-000003
Figure PCTCN2017096816-appb-000003
(2)中和试验(2) Neutralization test
将纯化的抗体3倍倍比稀释，与1:150稀释的ZIKV(C6/36或是Vero扩增)混合，37℃共孵育60分钟。然后将混合液加入到已铺满Vero细胞的24孔板中，300μL/孔。37℃孵育1小时后，每孔补充培养基(DMEM，10％FBS)1mL，继续培养30小时后染色。收集细胞，用含4％多聚甲醛，0.05％的soponin的PBS处理细胞，冰上避光静置30min。然后以solution溶液(PBS，1％BSA，0.01％soponin)洗涤细胞2次，与2μg/mL的Z1抗体冰上孵育30min后，solution溶液洗涤细胞2次，再与1:200稀释的anti-human IgG避光冰上孵育30min。solution溶液洗2次后，用FACSCanto检测细胞阳性比例。根据不同浓度下阳性比例，计算抗体对ZIKV的中和能力，结果如图6与7，结果统计如表4。The purified antibody was diluted 3 fold, mixed with a 1:150 dilution of ZIKV (C6/36 or Vero amplification) and incubated for 60 minutes at 37 °C. The mixture was then added to a 24-well plate that had been covered with Vero cells at 300 μL/well. After incubating for 1 hour at 37 ° C, 1 mL of supplement medium (DMEM, 10% FBS) per well was added, and incubation was continued for 30 hours. The cells were collected, treated with PBS containing 4% paraformaldehyde, 0.05% soponin, and allowed to stand on ice for 30 min. Then, the cells were washed twice with solution solution (PBS, 1% BSA, 0.01% soponin), and incubated with 2 μg/mL of Z1 antibody for 30 min on ice, the solution solution was washed twice, and then diluted with 1:200 anti-human Incubate on IgG for 30 min in the dark. After the solution solution was washed twice, the cell positive ratio was measured by FACSCanto. The neutralization ability of the antibody against ZIKV was calculated according to the positive ratio at different concentrations. The results are shown in Figures 6 and 7, and the results are shown in Table 4.
表4三株抗体对不同来源病毒中和效果Table 4 Three antibodies to neutralize the virus from different sources
Figure PCTCN2017096816-appb-000004
Figure PCTCN2017096816-appb-000004
a半抑制浓度；b判定系数 a semi-inhibitory concentration; b determination coefficient
(3)动物保护试验 (3) Animal protection test
I型干扰素受体(InterferonαβR)敲除小鼠(B6.129S2-Ifnar1<tm1Agt>/Mmjax，Jackson Laboratories)，以3-5只分组。每只小鼠腹腔注射1×106PFU的ZIKV(GenBank accession number:KX087101.2)。感染24小时后单一剂量10mg/kg的抗体Z3L1、Z20、Z23或等体积的PBS通过腹腔注射给感染的小鼠。记录14天内小鼠的存活与体重变化。体重变化超过20％或是出现瘫痪症状的小鼠处死。结果如图8显示。由图8可知，注射PBS的3只小鼠在感染后第7天均死亡，注射对照抗体2G4的5只小鼠中，也有4只在感染后第8天死亡(图8A)。相比之下，注射Z3L1以及Z23抗体的各三只小鼠均存活(图8A)，并且体重仍处于生长中(图8B)，注射Z20抗体的5只小鼠中也有四只存活，并且存活的四只小鼠体重也处于上升过程中。这组实验结果表明，三株人源单抗在小鼠体内可以完全(Z3L1和Z23)或部分(Z20)治疗受ZIKV感染的小鼠。Type I interferon receptor (Interferon αβR) knockout mice (B6.129S2-Ifnar1<tm1Agt>/Mmjax, Jackson Laboratories) were grouped in 3-5 groups. Each mouse was intraperitoneally injected with 1×10 6 PFU of ZIKV (GenBank accession number: KX087101.2). A single dose of 10 mg/kg of antibody Z3L1, Z20, Z23 or an equal volume of PBS was administered intraperitoneally to infected mice 24 hours after infection. The survival and body weight changes of the mice were recorded within 14 days. Mice with a change in body weight of more than 20% or with symptoms of spasticity were sacrificed. The results are shown in Figure 8. As can be seen from Fig. 8, 3 mice injected with PBS died on the 7th day after infection, and 4 of the 5 mice injected with the control antibody 2G4 died on the 8th day after infection (Fig. 8A). In contrast, each of the three mice injected with the Z3L1 and Z23 antibodies survived (Fig. 8A), and the body weight was still growing (Fig. 8B), and four of the 5 mice injected with the Z20 antibody survived and survived. The weight of the four mice is also on the rise. The results of this set of experiments indicate that three human monoclonal antibodies can completely (Z3L1 and Z23) or partially (Z20) treat ZIKV-infected mice in mice.
目前，已发表的在小鼠模型中对ZIKV感染有治疗作用的人源抗体只有一株，即ZKA64，其与ZIKV-E的结合能力为半最大效应浓度EC50＝65ng/mL，半抑制浓度IC50＝0.155μg/mL。本实验中三株人源抗体的IC50为0.17～0.89μg/mL。并且，在动物保护实验中，本发明使用的抗体剂量为10mg/kg，远小于文献报道的ZKA64的抗体剂量15mg/kg。本发明的三个抗体为预防RNA病毒在抗体压力下发生突变提供更多的针对不同表位的新的抗体。At present, only one published human antibody that has a therapeutic effect on ZIKV infection in a mouse model, ZKA64, has a half-maximal effect concentration of EC 50 =65 ng/mL, semi-inhibitory concentration. IC 50 = 0.155 μg/mL. In this experiment three humanized antibody IC 50 of 0.17 ~ 0.89μg / mL. Moreover, in animal protection experiments, the dose of the antibody used in the present invention was 10 mg/kg, which was much smaller than the antibody dose of 15 mg/kg of ZKA64 reported in the literature. The three antibodies of the invention provide more novel antibodies against different epitopes in order to prevent mutation of the RNA virus under antibody pressure.
实施例6：人源抗体的结构信息Example 6: Structural information of human antibodies
通过寨卡病毒E蛋白分别与两株抗体Z3L1与Z20的晶体结构，以及寨卡病毒与Z23的冷冻电镜结构可知，三株抗体与寨卡病毒E蛋白相互作用的氨基酸位点如表5-7所示，相互作用位点分析的阈值设定在
Figure PCTCN2017096816-appb-000005
其中，Z3L1主要结合在一个E蛋白，并且结合的结构域主要是DI，DII以及连接两个结构域的铰链区；而Z20和Z23的结合位点涉及一个E蛋白二聚体中的两个E蛋白单体。其中，Z20主要结合在两个E蛋白的DII区域，Z23主要结合在DIII。 The crystal structure of Zika virus E protein and the two antibodies Z3L1 and Z20, and the cryo-electron microscopic structure of Zika virus and Z23, the amino acid positions of the three antibodies interacting with Zika virus E protein are shown in Table 5-7. As shown, the threshold for interaction site analysis is set at
Figure PCTCN2017096816-appb-000005
Among them, Z3L1 mainly binds to an E protein, and the binding domain is mainly DI, DII and the hinge region connecting the two domains; and the binding sites of Z20 and Z23 involve two E in one E protein dimer. Protein monomer. Among them, Z20 mainly binds to the DII region of two E proteins, and Z23 mainly binds to DIII.
表5 Z3L1与寨卡病毒E蛋白结合位点分析Table 5 Analysis of binding sites of Z3L1 and Zika virus E protein
Z3L1重链 E蛋白
SER31 MET277(2),GLY279(2)
TYR32 GLY279(3),ALA280(1)
HIS100 ASP278(1),GLY279(2),ALA280(6)
LEU101 ASP278(9),GLY279(4),LYS281(4)
GLY102 ASP278(6),GLY279(3)
TRP103 ASP278(4)
SER104 GLU276(2),MET277(3),ASP278(6)
SER105 ASP278(1)
ILE106 THR156(1)
TRP107 VAL46(4),THR47(6),MET140(2),LYS166(3),ARG283(1)
SER108 ASP278(2)
GLU111 ARG138(4),LYS281(3),ARG283(5)
Z3L1轻链 E蛋白
SER26 GLU159(3)
ASN31 HIS158(1),GLU159(5)
TYR33 GLU136(5),ARG138(3),GLU168(3)
TYR50 ASN52(1)
SER94 GLU159(5)
表6 Z20与寨卡病毒E蛋白结合位点分析
Z3L1 heavy chain E protein
SER31 MET277(2), GLY279(2)
TYR32 GLY279(3), ALA280(1)
HIS100 ASP278(1), GLY279(2), ALA280(6)
LEU101 ASP278(9), GLY279(4), LYS281(4)
GLY102 ASP278(6), GLY279(3)
TRP103 ASP278(4)
SER104 GLU276 (2), MET277 (3), ASP278 (6)
SER105 ASP278(1)
ILE106 THR156(1)
TRP107 VAL46(4), THR47(6), MET140(2), LYS166(3), ARG283(1)
SER108 ASP278(2)
GLU111 ARG138(4), LYS281(3), ARG283(5)
Z3L1 light chain E protein
SER26 GLU159(3)
ASN31 HIS158(1), GLU159(5)
TYR33 GLU136(5), ARG138(3), GLU168(3)
TYR50 ASN52(1)
SER94 GLU159(5)
Table 6 Analysis of binding sites of Z20 and Zika virus E protein
Figure PCTCN2017096816-appb-000006
Figure PCTCN2017096816-appb-000006
表7 Z23与寨卡病毒E蛋白结合位点分析Table 7 Analysis of binding sites of Z23 and Zika virus E protein
Figure PCTCN2017096816-appb-000007
Figure PCTCN2017096816-appb-000007
综上，本发明通过筛选康复病人的ZIKV-E特异结合的记忆B细胞，获得3株人源高中和活性的ZIKV抗体：Z3L1、Z20与Z23。这3株抗体与已经报道的寨卡抗体序列完全不同，是3株新发现的抗体。这三株抗体与ZIKV-E的结合常数分别为5.39μM(Z3L1)，0.16μM(Z20)和0.44μM(Z23)。三株人源抗体均有很强的寨卡病毒中和活性。并且，Z3L1、Z20与Z23可以完全或部分保护小鼠免受致死剂量的寨卡病毒的攻击。结构信息表明，三株抗体分别结合在E蛋白的不同结构域。这提示，三株人源抗体具有以鸡尾酒疗法在临床治疗和预防寨卡中发挥作用的巨大潜力。In summary, the present invention obtains three human high-intensity and active ZIKV antibodies by screening ZIKV-E-specific memory B cells in a rehabilitation patient: Z3L1, Z20 and Z23. These three antibodies are completely different from the already reported Zika antibody sequences and are three newly discovered antibodies. The binding constants of these three antibodies to ZIKV-E were 5.39 μM (Z3L1), 0.16 μM (Z20) and 0.44 μM (Z23), respectively. All three human antibodies have strong Zika virus neutralizing activity. Moreover, Z3L1, Z20 and Z23 can completely or partially protect mice from lethal doses of Zika virus. Structural information indicates that the three antibodies bind to different domains of the E protein, respectively. This suggests that the three human antibodies have great potential to play a role in the clinical treatment and prevention of Zika in cocktail therapy.
虽然本发明已以较佳实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明的精神和范围内，都可做各种的改动与修饰，因此本发明的保护范围应该以权利要求书所界定的为准。 Although the present invention has been disclosed in the above preferred embodiments, the present invention is not limited thereto, and various modifications and changes can be made thereto without departing from the spirit and scope of the invention. The scope of the invention should be determined by the scope of the claims.
Figure PCTCN2017096816-appb-000008
Figure PCTCN2017096816-appb-000008
Figure PCTCN2017096816-appb-000009
Figure PCTCN2017096816-appb-000009
Figure PCTCN2017096816-appb-000010
Figure PCTCN2017096816-appb-000010
Figure PCTCN2017096816-appb-000011
Figure PCTCN2017096816-appb-000011
Figure PCTCN2017096816-appb-000012
Figure PCTCN2017096816-appb-000012
Figure PCTCN2017096816-appb-000013
Figure PCTCN2017096816-appb-000013
Figure PCTCN2017096816-appb-000014
Figure PCTCN2017096816-appb-000014
Figure PCTCN2017096816-appb-000015
Figure PCTCN2017096816-appb-000015
Figure PCTCN2017096816-appb-000016
Figure PCTCN2017096816-appb-000016
Figure PCTCN2017096816-appb-000017
Figure PCTCN2017096816-appb-000017
Figure PCTCN2017096816-appb-000018
Figure PCTCN2017096816-appb-000018
Figure PCTCN2017096816-appb-000019
Figure PCTCN2017096816-appb-000019
Figure PCTCN2017096816-appb-000020
Figure PCTCN2017096816-appb-000020
Figure PCTCN2017096816-appb-000021
Figure PCTCN2017096816-appb-000021
Figure PCTCN2017096816-appb-000022
Figure PCTCN2017096816-appb-000022
Figure PCTCN2017096816-appb-000023
Figure PCTCN2017096816-appb-000023

Claims (11)

  1. 一种抗体，其特征在于，所述抗体：An antibody characterized by the antibody:
    a)能与SEQ ID NO:21的寨卡病毒E蛋白的第46(VAL)、47(THR)、52(ASN)、136(GLU)、138(ARG)、140(MET)、156(THR)、158(HIS)、159(GLU)、166(LYS)、168(GLU)、259(GLU)、276(GLU)、277(MET)、278(ASP)、280(ALA)、281(LYS)和283(ARG)位氨基酸发生相互作用；或者，a) 46(VAL), 47(THR), 52(ASN), 136(GLU), 138(ARG), 140(MET), 156(THR) capable of interacting with the Zika virus E protein of SEQ ID NO:21. ), 158 (HIS), 159 (GLU), 166 (LYS), 168 (GLU), 259 (GLU), 276 (GLU), 277 (MET), 278 (ASP), 280 (ALA), 281 (LYS) ) interacts with amino acids at position 283 (ARG); or,
    b)能与SEQ ID NO:21的寨卡病毒E蛋白的第64(SER)、65(ILE)、66(SER)、67(ASP)、68(MET)、69(ALA)、84(LYS)、87(ASP)、89(GLN)、90(TYR)、118(LYS)、119(PHE)、120(ALA)、208(ASN)、232(GLY)、233(THR)、252(ARG)、276(GLU)、277(MET)、278(ASP)和279(GLY)位氨基酸发生相互作用；或者，b) 64 (SER), 65 (ILE), 66 (SER), 67 (ASP), 68 (MET), 69 (ALA), 84 (LYS) of the Zika virus E protein of SEQ ID NO:21. ), 87 (ASP), 89 (GLN), 90 (TYR), 118 (LYS), 119 (PHE), 120 (ALA), 208 (ASN), 232 (GLY), 233 (THR), 252 (ARG) , 276 (GLU), 277 (MET), 278 (ASP) and 279 (GLY) amino acids interact; or,
    c)能与SEQ ID NO:21的寨卡病毒E蛋白的第51(SER)、67(ASP)、68(MET)、69(ALA)、310(ALA)、311(ALA)、312(PHE)、313(THR)、314(PHE)、315(THR)、317(ILE)、331(GLN)、332(TYR)、333(ALA)、334(GLY)、335(THR)、336(ASP)、368(SER)、369(THR)、370(GLU)、371(ASN)和394(LYS)位氨基酸发生相互作用。c) 51 (SER), 67 (ASP), 68 (MET), 69 (ALA), 310 (ALA), 311 (ALA), 312 (PHE) of the Zika virus E protein of SEQ ID NO:21. ), 313 (THR), 314 (PHE), 315 (THR), 317 (ILE), 331 (GLN), 332 (TYR), 333 (ALA), 334 (GLY), 335 (THR), 336 (ASP , 368 (SER), 369 (THR), 370 (GLU), 371 (ASN) and 394 (LYS) amino acids interact.
  2. 根据权利要求1所述的抗体，其特征在于，所述抗体的重链可变区的氨基酸序列为SEQ ID NO:1且轻链可变区氨基酸序列为SEQ ID NO:2，或者是重链可变区的氨基酸序列为SEQ ID NO:3且轻链可变区氨基酸序列为SEQ ID NO:4，或者是重链可变区的氨基酸序列为SEQ ID NO:5且轻链可变区氨基酸序列为SEQ ID NO:6。The antibody according to claim 1, wherein the amino acid sequence of the heavy chain variable region of the antibody is SEQ ID NO: 1 and the light chain variable region amino acid sequence is SEQ ID NO: 2, or is a heavy chain The amino acid sequence of the variable region is SEQ ID NO: 3 and the amino acid sequence of the light chain variable region is SEQ ID NO: 4, or the amino acid sequence of the heavy chain variable region is SEQ ID NO: 5 and the light chain variable region amino acid The sequence is SEQ ID NO: 6.
  3. 根据权利要求1所述的抗体，其特征在于，所述抗体的重链包括重链可变区和重链恒定区，其中重链恒定区的氨基酸序列如SEQ ID NO:13所示。The antibody according to claim 1, wherein the heavy chain of the antibody comprises a heavy chain variable region and a heavy chain constant region, wherein the amino acid sequence of the heavy chain constant region is set forth in SEQ ID NO: 13.
  4. 根据权利要求1所述的抗体，其特征在于，所述抗体的轻链，为κ链和/或λ链；轻链包括轻链可变区和轻链恒定区；κ链的轻链恒定区的氨基酸序列如SEQ ID NO:15所示，λ链的轻链恒定区的氨基酸序列如SEQ ID NO:17所示。The antibody according to claim 1, wherein the light chain of the antibody is a kappa chain and/or a lambda chain; the light chain comprises a light chain variable region and a light chain constant region; and the light chain constant region of the kappa chain The amino acid sequence of λ chain is shown in SEQ ID NO: 15, and the amino acid sequence of the light chain constant region of the λ chain is set forth in SEQ ID NO: 17.
  5. 一种药物组合物，其特征在于，所述药物组合物含有权利要求1～3任一所述的抗体。A pharmaceutical composition comprising the antibody according to any one of claims 1 to 3.
  6. 权利要求1～4任一所述的抗体在制备治疗和/或预防寨卡病毒的药物方面的应用。Use of the antibody according to any one of claims 1 to 4 for the preparation of a medicament for the treatment and/or prevention of Zika virus.
  7. 一种试剂盒，其特征在于，所述试剂盒中含有权利要求1～3任一所述抗体的抗原，或者编码所述抗原的DNA分子，或者表达所述抗原的重组载体/表达盒/转基因细胞系/重组菌。A kit comprising the antigen of any one of claims 1 to 3, or a DNA molecule encoding the antigen, or a recombinant vector/expression cassette/transgene expressing the antigen Cell line/recombinant bacteria.
  8. 编码权利要求1～4任一所述的抗体的基因序列。A gene sequence encoding the antibody of any one of claims 1 to 4.
  9. 根据权利要求8所述的基因序列，其特征在于，所述基因序列中编码抗体的重链的序列，依次包括CMV启动子序列、EcoR I酶切位点序列、前导序列、编码重链可变区的序列、编码重链恒定区的序列、Xho I酶切位点序列。The gene sequence according to claim 8, wherein the sequence encoding the heavy chain of the antibody in the gene sequence comprises, in order, a CMV promoter sequence, an EcoR I cleavage site sequence, a leader sequence, and a coding heavy chain variable. The sequence of the region, the sequence encoding the heavy chain constant region, and the Xho I restriction site sequence.
  10. 根据权利要求8所述的基因序列，其特征在于，所述基因序列中编码抗体的轻链的 序列，依次包括CMV启动子序列、第一酶切位点序列、前导序列、编码轻链可变区的序列、编码轻链恒定区的序列、酶切位点序列Xho I。The gene sequence according to claim 8, wherein the gene sequence encodes a light chain of an antibody The sequence, in turn, includes a CMV promoter sequence, a first restriction site sequence, a leader sequence, a sequence encoding a light chain variable region, a sequence encoding a light chain constant region, and a restriction enzyme sequence Xho I.
  11. 含有权利要求8所述的基因序列或者表达权利要求1～4任一所述的抗体的表达载体或者细胞。 An expression vector or cell comprising the gene sequence of claim 8 or the antibody of any one of claims 1 to 4.
PCT/CN2017/096816 2016-08-10 2017-08-10 Humanized monoclonal antibody for zika virus and applications thereof WO2018028635A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201610649447.0A CN106279409B (en) 2016-08-10 2016-08-10 A kind of zika virus human monoclonal antibody and its application
CN201610649447.0 2016-08-10

Publications (1)

Publication Number Publication Date
WO2018028635A1 true WO2018028635A1 (en) 2018-02-15

Family

ID=57667547

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2017/096816 WO2018028635A1 (en) 2016-08-10 2017-08-10 Humanized monoclonal antibody for zika virus and applications thereof

Country Status (2)

Country Link
CN (3) CN110172095B (en)
WO (1) WO2018028635A1 (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116063469A (en) * 2022-08-29 2023-05-05 中山大学 Zika virus neutralizing nano antibody and preparation method and application thereof

Families Citing this family (23)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017181098A2 (en) * 2016-04-15 2017-10-19 Visterra, Inc. Antibody molecules to zika virus and uses thereof
CN110172095B (en) * 2016-08-10 2020-09-04 中国科学院微生物研究所 Humanized monoclonal antibody of Zika virus with high neutralization activity and application thereof
CN106589116B (en) * 2016-12-06 2020-01-03 中国科学院微生物研究所 Flavivirus human monoclonal antibody and application thereof
CN108503696B (en) * 2017-02-27 2023-05-12 中国科学院上海巴斯德研究所 Zika virus subunit vaccine expressed by yeast cells
CN108503697B (en) * 2017-02-27 2023-03-31 中国科学院上海巴斯德研究所 Zika virus subunit vaccine expressed by drosophila cells
CN108727490B (en) * 2017-04-19 2020-09-11 清华大学 Monoclonal antibody ZK2B10 and application
CN108727491B (en) * 2017-04-19 2021-02-09 广州市第八人民医院 Monoclonal antibody ZK7C3 and application
CN108727489B (en) * 2017-04-19 2021-02-09 广州市第八人民医院 Monoclonal antibody ZK2C2 and application
CN109081868B (en) * 2017-06-14 2022-06-24 中国科学院上海巴斯德研究所 Monoclonal antibody targeting Zika virus envelope protein conserved epitope and application thereof
WO2019042555A1 (en) 2017-08-31 2019-03-07 Humabs Biomed Sa Multispecific antibodies specifically binding to zika virus epitopes and uses thereof
CN108103091B (en) * 2017-12-29 2020-10-13 宁波市疾病预防控制中心 Recombinant hansenula polymorpha for expressing structural protein E of Zika virus and construction method thereof
CN108130341B (en) * 2018-01-26 2020-08-07 宁波市疾病预防控制中心 Recombinant hansenula polymorpha for molecular chaperone-assisted expression of Zika virus E protein and construction method thereof
CN112119090B (en) * 2018-03-15 2023-01-13 中外制药株式会社 Anti-dengue virus antibodies cross-reactive to Zika virus and methods of use
CN113372441B (en) * 2018-04-04 2023-03-10 中国科学院微生物研究所 High-sensitivity yellow fever virus humanized monoclonal antibody and application thereof
CN109553680B (en) * 2018-12-12 2021-06-01 广州市第八人民医院 Monoclonal antibody ZKns4B8 and application thereof
CN109535249B (en) * 2018-12-12 2021-06-01 广州市第八人民医院 Monoclonal antibody ZKns3G2 and application thereof
CN109627297B (en) * 2018-12-29 2022-06-14 复旦大学 Neutralizing epitope from Zika virus E protein and application thereof
CN111100201B (en) * 2019-12-31 2021-09-28 武汉班科生物技术股份有限公司 Murine monoclonal antibody against envelope protein of Zika virus
CN111138534B (en) * 2019-12-31 2021-09-28 武汉班科生物技术股份有限公司 Mouse source monoclonal antibody of Zika virus envelope protein
CN113528465B (en) * 2020-04-20 2023-07-11 中国人民解放军军事科学院军事医学研究院 DNA molecule containing mutant Zika virus whole genome cDNA and application thereof
CN112266895B (en) * 2020-10-16 2022-03-29 中国农业大学 Screening method of single memory B cell and application of single memory B cell in preparation of small molecule monoclonal antibody
CN115466326A (en) * 2021-06-11 2022-12-13 中国科学院微生物研究所 Human monoclonal antibody of respiratory syncytial virus and application thereof
CN116655785B (en) * 2023-07-24 2023-10-03 内蒙古农业大学 Monoclonal antibody for inhibiting Zika virus infection and application thereof

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101891806A (en) * 2010-03-19 2010-11-24 中国人民解放军军事医学科学院微生物流行病研究所 Anti-flavivirus envelope E protein monoclonal antibody and application thereof
CN106279409A (en) * 2016-08-10 2017-01-04 中国科学院微生物研究所 A kind of zika virus human monoclonal antibody and application thereof
CN106589116A (en) * 2016-12-06 2017-04-26 中国科学院微生物研究所 Flavivirus human monoclonal antibody and application

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2003263853A1 (en) * 2002-08-16 2004-03-03 Board Of Regents The University Of Texas System Compositions and methods related to flavivirus envelope protein domain iii antigens
CN105749268B (en) * 2016-04-11 2020-09-11 北京科兴中维生物技术有限公司 Inactivated Zika virus vaccine

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101891806A (en) * 2010-03-19 2010-11-24 中国人民解放军军事医学科学院微生物流行病研究所 Anti-flavivirus envelope E protein monoclonal antibody and application thereof
CN106279409A (en) * 2016-08-10 2017-01-04 中国科学院微生物研究所 A kind of zika virus human monoclonal antibody and application thereof
CN106589116A (en) * 2016-12-06 2017-04-26 中国科学院微生物研究所 Flavivirus human monoclonal antibody and application

Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
DAI, L. P. ET AL.: "Structures of the Zika Virus Envelope Protein and Its Complex with a Flavivirus Broadly Protective Antibody", CELL HOST & MICROBE, vol. 19, 11 May 2016 (2016-05-11), pages 1 - 9, XP055458052 *
DATABASE GENBANK [O] 14 March 2016 (2016-03-14), "Zika virus isolate GD01 polyprotein gene , complete cds", XP055463485, Database accession no. KU740184.2 *
DATABASE GENBANK [O] 26 January 2016 (2016-01-26), "Zika virus strain BeH818995 polyprotein gene , complete cds", XP055463506, Database accession no. KU365777.1 *
DENG, Y. Q. ET AL.: "A Broadly Flavivirus Cross-Neutralizing Monoclonal Antibody that Recognizes a Novel Epitope within the Fusion Loop of E Protein", PLOS ONE, vol. 6, no. 1, 11 January 2011 (2011-01-11), pages 1 - 8, XP055112424 *
JIANG, RUI ET AL.: "Molecular Characteristic of Imported Zika Virus in China", CHINESE JOURNAL OF CLINICAL INFECTIOUS DISEASES, vol. 9, no. 3, 30 June 2016 (2016-06-30), pages 277 - 280 *
PENG, HAORAN ET AL.: "Zika Virus and Zika Virus Disease", ACADEMIC JOURNAL OF SECOND MILITARY MEDICAL UNIVERSITY, vol. 37, no. 7, 31 July 2016 (2016-07-31), pages 793 - 798 *
WANG, Q. H. ET AL.: "Molecular determinants of human neutralizing antibodies isolated from a patient infected with Zika virus", SCIENCE TRANSLATIONAL MEDICINE, vol. 8, 14 December 2016 (2016-12-14), pages 369, XP055436624 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116063469A (en) * 2022-08-29 2023-05-05 中山大学 Zika virus neutralizing nano antibody and preparation method and application thereof
CN116063469B (en) * 2022-08-29 2023-09-22 中山大学 Zika virus neutralizing nano antibody and preparation method and application thereof

Also Published As

Publication number Publication date
CN106279409B (en) 2019-11-26
CN106279409A (en) 2017-01-04
CN110172095A (en) 2019-08-27
CN110172095B (en) 2020-09-04
CN110066333B (en) 2020-09-04
CN110066333A (en) 2019-07-30

Similar Documents

Publication Publication Date Title
WO2018028635A1 (en) Humanized monoclonal antibody for zika virus and applications thereof
CN111690059B (en) Monoclonal antibody 1D7 for resisting SARS-CoV-2
CN111718411B (en) Monoclonal antibody 1F2 for resisting SARS-CoV-2
WO2021189772A1 (en) Ace2-fc fusion proteins and use thereof
WO2021174595A1 (en) Monoclonal antibody for resisting novel coronavirus and use thereof
WO2022089471A1 (en) β-CORONAVIRUS MULTIMERIC ANTIGEN, PREPARATION METHOD THEREFOR AND APPLICATION THEREOF
CN111732654B (en) Monoclonal antibody 1E10 for resisting SARS-CoV-2
CN112521494B (en) Monoclonal antibody 2B11 for resisting SARS-CoV-2
CN114409770B (en) Setaria fever virus humanized monoclonal antibody and application thereof
WO2021174594A1 (en) Anti-novel coronavirus monoclonal antibody and application thereof
KR20190017869A (en) Humanized antibodies against Enterovirus 71
WO2020186687A1 (en) Human antibody specifically binding four serotypes of dengue viruses
CN105916883B (en) Bifunctional fusion proteins and its preparation method and application
CN114805579B (en) Anti-human ACE2 protein monoclonal antibody, nucleic acid molecule and application
CN115160435A (en) Bispecific anti-HIV-1 antibody
CN113817051B (en) Monoclonal antibody 1B6 against SARS-CoV-2
KR102254246B1 (en) Nucleic acid encoding anti-CD163 antibody or antigen binding fragment thereof, Adenovirus vector comprising the same, and use thereof in the prevention or treatment of African swine fever
WO2022122788A1 (en) Multispecific antibodies against severe acute respiratory syndrome coronavirus 2
KR20220045559A (en) Stabilized Ace2 Mutant, Ace2-Fc Fusion Protein Using Same and Pharmaceutical Composition for Preventing or Treating COVID-19
CN113817050B (en) Monoclonal antibody 1H8 against SARS-CoV-2
CN114805570B (en) Anti-human ACE2 monoclonal antibody and application thereof
RU2769223C1 (en) Means and method for therapy and emergency prevention of diseases caused by the sars-cov-2 virus based on a recombinant antibody and a humanized monoclonal antibody
WO2022141378A1 (en) Anti-pd-1 single-domain antibody
CN115337396A (en) Fully human monoclonal antibody combination and application thereof
WO2023154841A2 (en) Sars-cov-2 neutralizing synthetic proteins

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 17838765

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 17838765

Country of ref document: EP

Kind code of ref document: A1