WO2018028635A1 - Anticorps monoclonal humanisé contre le virus zika et ses applications - Google Patents

Anticorps monoclonal humanisé contre le virus zika et ses applications 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
English (en)
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/fr

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.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Virology (AREA)
  • Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biochemistry (AREA)
  • Molecular Biology (AREA)
  • Immunology (AREA)
  • Biophysics (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • General Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Physics & Mathematics (AREA)
  • Oncology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Cell Biology (AREA)
  • Communicable Diseases (AREA)
  • Plant Pathology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Mycology (AREA)
  • Epidemiology (AREA)
  • Peptides Or Proteins (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

L'invention porte sur un anticorps monoclonal humanisé pour le virus Zika, et sur ses applications, se rapportant au domaine technique de la médecine. En utilisant la protéine E du virus Zika exprimée par E. coli en tant qu'antigène, en choisissant parmi les PBMC d'un patient atteint du virus Zika en convalescence, une cellule B mémoire capable de se lier spécifiquement à la protéine E du virus Zika par tri de flux, puis la réalisation d'une RT-PCR sur la cellule B unique sélectionnée, l'obtention d'une séquence et d'un fragment d'une région variable de l'anticorps, et la connexion supplémentaire de celle-ci avec une région constante dans un vecteur d'expression. Après l'expression par une cellule de mammifère et la purification, la réalisation d'une série de tests de fonction, comprenant la détection de la force de liaison avec la protéine ZIKV-E, des résultats de neutralisation in vitro, une capacité de protection in vivo, etc. ce qui permet d'obtenir trois souches d'anticorps monoclonaux humanisés ayant une protection complète contre l'infection par le virus Zika.
PCT/CN2017/096816 2016-08-10 2017-08-10 Anticorps monoclonal humanisé contre le virus zika et ses applications WO2018028635A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201610649447.0A CN106279409B (zh) 2016-08-10 2016-08-10 一种寨卡病毒人源单克隆抗体及其应用
CN201610649447.0 2016-08-10

Publications (1)

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

Family

ID=57667547

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2017/096816 WO2018028635A1 (fr) 2016-08-10 2017-08-10 Anticorps monoclonal humanisé contre le virus zika et ses applications

Country Status (2)

Country Link
CN (3) CN106279409B (fr)
WO (1) WO2018028635A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116063469A (zh) * 2022-08-29 2023-05-05 中山大学 一种寨卡病毒中和性纳米抗体及其制备方法与应用

Families Citing this family (23)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017181098A2 (fr) * 2016-04-15 2017-10-19 Visterra, Inc. Molécules d'anticorps se liant au virus zika et leurs utilisations
CN106279409B (zh) * 2016-08-10 2019-11-26 中国科学院微生物研究所 一种寨卡病毒人源单克隆抗体及其应用
CN106589116B (zh) * 2016-12-06 2020-01-03 中国科学院微生物研究所 一种黄病毒人源单克隆抗体及应用
CN108503697B (zh) * 2017-02-27 2023-03-31 中国科学院上海巴斯德研究所 一种果蝇细胞表达的寨卡病毒亚单位疫苗
CN108503696B (zh) * 2017-02-27 2023-05-12 中国科学院上海巴斯德研究所 一种酵母细胞表达的寨卡病毒亚单位疫苗
CN108727490B (zh) * 2017-04-19 2020-09-11 清华大学 一种单克隆抗体zk2b10及应用
CN108727491B (zh) * 2017-04-19 2021-02-09 广州市第八人民医院 一种单克隆抗体zk7c3及应用
CN108727489B (zh) * 2017-04-19 2021-02-09 广州市第八人民医院 一种单克隆抗体zk2c2及应用
CN109081868B (zh) * 2017-06-14 2022-06-24 中国科学院上海巴斯德研究所 靶向寨卡病毒包膜蛋白保守表位的单克隆抗体及其应用
WO2019042555A1 (fr) 2017-08-31 2019-03-07 Humabs Biomed Sa Anticorps multispécifiques se liant spécifiquement aux épitopes du virus zika et leurs utilisations
CN108103091B (zh) * 2017-12-29 2020-10-13 宁波市疾病预防控制中心 一种表达寨卡病毒结构蛋白e的重组多形汉逊酵母及其构建方法
CN108130341B (zh) * 2018-01-26 2020-08-07 宁波市疾病预防控制中心 分子伴侣辅助表达寨卡病毒e蛋白的重组多形汉逊酵母及其构建方法
EP3765494A4 (fr) * 2018-03-15 2022-03-23 Chugai Seiyaku Kabushiki Kaisha Anticorps dirigés contre le virus de la dengue présentant une réactivité croisée au virus zika et procédés d'utilisation
CN110343172B (zh) * 2018-04-04 2021-09-03 中国科学院微生物研究所 一种高灵敏度的黄热病毒人源单克隆抗体及其应用
CN109553680B (zh) * 2018-12-12 2021-06-01 广州市第八人民医院 一种单克隆抗体ZKns4B8及其应用
CN109535249B (zh) * 2018-12-12 2021-06-01 广州市第八人民医院 一种单克隆抗体ZKns3G2及其应用
CN109627297B (zh) * 2018-12-29 2022-06-14 复旦大学 来自寨卡病毒e蛋白的中和表位及其应用
CN111138534B (zh) * 2019-12-31 2021-09-28 武汉班科生物技术股份有限公司 寨卡病毒囊膜蛋白的鼠源单克隆抗体
CN111100201B (zh) * 2019-12-31 2021-09-28 武汉班科生物技术股份有限公司 针对寨卡病毒囊膜蛋白的鼠源单克隆抗体
CN113528465B (zh) * 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 包含突变的寨卡病毒全基因组cDNA的DNA分子及其应用
CN112266895B (zh) * 2020-10-16 2022-03-29 中国农业大学 单个记忆b细胞的筛选方法及其在小分子单克隆抗体制备中的应用
CN115466326B (zh) * 2021-06-11 2024-06-04 中国科学院微生物研究所 一种呼吸道合胞病毒的人源单克隆抗体及其应用
CN116655785B (zh) * 2023-07-24 2023-10-03 内蒙古农业大学 一种抑制寨卡病毒感染的单克隆抗体及其应用

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101891806A (zh) * 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 抗黄病毒包膜e蛋白的单克隆抗体及其应用
CN106279409A (zh) * 2016-08-10 2017-01-04 中国科学院微生物研究所 一种寨卡病毒人源单克隆抗体及其应用
CN106589116A (zh) * 2016-12-06 2017-04-26 中国科学院微生物研究所 一种黄病毒人源单克隆抗体及应用

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7785799B2 (en) * 2002-08-16 2010-08-31 The Board Of Regents Of The University Of Texas System Compositions and methods related to flavivirus envelope protein domain III antigens
CN105749268B (zh) * 2016-04-11 2020-09-11 北京科兴中维生物技术有限公司 一种灭活的寨卡病毒疫苗

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101891806A (zh) * 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 抗黄病毒包膜e蛋白的单克隆抗体及其应用
CN106279409A (zh) * 2016-08-10 2017-01-04 中国科学院微生物研究所 一种寨卡病毒人源单克隆抗体及其应用
CN106589116A (zh) * 2016-12-06 2017-04-26 中国科学院微生物研究所 一种黄病毒人源单克隆抗体及应用

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 (zh) * 2022-08-29 2023-05-05 中山大学 一种寨卡病毒中和性纳米抗体及其制备方法与应用
CN116063469B (zh) * 2022-08-29 2023-09-22 中山大学 一种寨卡病毒中和性纳米抗体及其制备方法与应用

Also Published As

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

Similar Documents

Publication Publication Date Title
WO2018028635A1 (fr) Anticorps monoclonal humanisé contre le virus zika et ses applications
CN111690059B (zh) 一种抗SARS-CoV-2的单克隆抗体1D7
CN111718411B (zh) 一种抗SARS-CoV-2的单克隆抗体1F2
WO2021189772A1 (fr) Protéines de fusion ace2-fc et leur utilisation
WO2021174595A1 (fr) Anticorps monoclonal pour le nouveau coronavirus résistant et son utilisation
WO2022089471A1 (fr) ANTIGÈNE MULTIMÈRE DE β-CORONAVIRUS, SON PROCÉDÉ DE PRÉPARATION ET SON UTILISATION
CN114409769B (zh) 一种裂谷热病毒人源单克隆抗体及其应用
CN112521494B (zh) 一种抗SARS-CoV-2的单克隆抗体2B11
CN111732654B (zh) 一种抗SARS-CoV-2的单克隆抗体1E10
WO2021174594A1 (fr) Anticorps monoclonal contre le nouveau coronavirus et son utilisation
KR20190017869A (ko) 엔테로바이러스 71에 대한 인간화 항체
WO2020186687A1 (fr) Anticorps humain liant spécifiquement quatre sérotypes du virus de la dengue
WO2015113494A1 (fr) Protéine de fusion bifonctionnelle, procédé de préparation s'y rapportant et son utilisation
CN114805579B (zh) 一种抗人ace2蛋白单克隆抗体、核酸分子及应用
CN113698487B (zh) 抗人ace2单克隆抗体及其应用
CN115160435A (zh) 一种双特异性抗hiv-1抗体
CN113817051B (zh) 一种抗SARS-CoV-2的单克隆抗体1B6
KR102254246B1 (ko) 항-cd163 항체 또는 이의 항원 결합 단편을 인코딩하는 핵산 분자 및 이를 포함하는 아데노바이러스 벡터, 및 이의 아프리카 돼지열병의 예방 또는 치료용도
CN114106191A (zh) 一种中和冠状病毒的双特异性抗体
JP2023544169A (ja) 組換えａｃｅ２－ｆｃ融合分子、その製造方法及びその使用
WO2022122788A1 (fr) Anticorps multispécifiques contre le coronavirus du syndrome respiratoire aigu sévère 2
KR20220045559A (ko) 안정화된 Ace2 변이체, 이를 이용한 Ace2-Fc 융합단백질 및 COVID-19 예방 또는 치료용 약학 조성물
CN113817050B (zh) 一种抗SARS-CoV-2的单克隆抗体1H8
CN113880947B (zh) 小分子抗体及其编码基因和制备方法及应用和药物组合物
CN114805570B (zh) 一种抗人ace2单克隆抗体及其应用

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