WO2020186687A1 - 一种特异性结合四种血清型登革病毒的人源抗体 - Google Patents

一种特异性结合四种血清型登革病毒的人源抗体 Download PDF

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WO2020186687A1
WO2020186687A1 PCT/CN2019/100150 CN2019100150W WO2020186687A1 WO 2020186687 A1 WO2020186687 A1 WO 2020186687A1 CN 2019100150 W CN2019100150 W CN 2019100150W WO 2020186687 A1 WO2020186687 A1 WO 2020186687A1
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antibody
dengue virus
seq
recombinant
nucleic acid
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French (fr)
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杨志新
王荣
陆健昇
余云舟
周晓巍
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中国人民解放军军事科学院军事医学研究院
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/42—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum viral
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • 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 the field of biotechnology, in particular to a human antibody that specifically binds to four serotype dengue viruses.
  • Dengue virus belongs to the flavivirus genus of the Flaviviridae family. It is an enveloped single positive-stranded RNA virus. According to the antigenicity of its envelope, it is divided into four serotypes (DENV I-IV). Dengue virus is mainly transmitted by Aedes aegypti and Aedes albopictus, and it is widespread in tropical and subtropical regions. Dengue fever (Classic Dengue Fever, DF) is a self-limiting febrile disease caused by dengue virus infection for the first time in humans. At the same time, dengue virus can stimulate good cellular immunity and humoral immunity. This subtype of infection can provide lifelong Immunity.
  • Immunoglobulin is an important method for the treatment of viral infections. It can not only neutralize viruses, but also through complement dependent cytotoxicity (CDC) and antibody dependent cell-mediated cytotoxicity (antibody dependent cell- mediated cytotoxicity (ADCC) activates the immune system in the body to clear the virus.
  • CDC complement dependent cytotoxicity
  • ADCC antibody dependent cell-mediated cytotoxicity
  • a large number of in vivo and in vitro experiments have proved that neutralizing antibodies can effectively prevent DENV infection. It can not only play a preventive effect before the virus infection, but also can still play a role in curing the virus infection for a period of time after the virus infection.
  • the E protein of DENV is the most fully exposed protein on the surface of infectious virus particles, and it is also the main target of neutralizing antibodies. At present, most of the DENV neutralizing antibody epitopes reported globally are also related to E protein: (1) Recognizing DENV ED II: The cross-neutralizing antibody (E53) corresponding to FL loop mostly has low neutralization efficiency and is prone to ADE effects; for BC Loop's cross-neutralizing antibody (1C19) can not only neutralize DENV efficiently, but also compete with low-efficiency anti-FL antibodies for binding.
  • humanized antibodies Compared with murine antibodies, humanized antibodies have the following characteristics: (1) immunogenicity is lower than murine antibodies; (2) affinity is weaker than murine antibodies, and specificity is better than murine antibodies; (3) humanized The antibody Fc segment can induce the body's effector functions; (4) Long half-life in the body; (5) The price is still relatively expensive. However, humanized antibodies still contain 10%-30% of murine protein, and there is still a potential threat of immune rejection in clinical applications. Therefore, fully humanized neutralizing antibodies are one of the important development goals of DENV therapeutic antibodies.
  • the purpose of the present invention is to provide a human antibody that specifically binds to four serotypes of dengue virus.
  • the present invention claims an antibody.
  • the antibody claimed in the present invention is a fully human monoclonal antibody 6B1.
  • the amino acid sequences of HCDR1, HCDR2 and HCDR3 in the variable region of the heavy chain are shown in SEQ ID No. 1 from the N-terminus to positions 26-33 and 51-
  • the amino acid sequences of LCDR1, LCDR2 and LHCDR3 in the light chain variable region of the antibody are shown in SEQ ID No. 2, 27-32 and 50-52 from the N-terminus in sequence. Bits, 89-97.
  • HCDR1, HCDR2 and HCDR3 are the three complementarity determining regions in the heavy chain variable region
  • LCDR1, LCDR2 and LHCDR3 are the three complementarity determining regions in the light chain variable region.
  • the sequence of the complementarity determining region is defined by Kabat.
  • amino acid sequence of the heavy chain variable region may be SEQ ID No. 1 from the N-terminus to positions 1-121, or with SEQ ID No.1 from the N-terminus to positions 1-121 at least 90% The consistency (the inconsistency can be in the framework region (FR)).
  • the amino acid sequence of the variable region of the light chain may be SEQ ID No. 2 from the N-terminal position 1-109, or with SEQ ID No. 2 from the N-terminal position 1-109 at least 90% identical (The inconsistency can be in the framework region (FR)).
  • the antibody is IgG. Further, the IgG is IgG1.
  • the light chain type of the antibody is kappa ( ⁇ ) type.
  • amino acid sequence of the heavy chain of the antibody may be SEQ ID No. 1, or have at least 90% identity with SEQ ID No. 1 (the inconsistency may be in the framework region (FR)).
  • amino acid sequence of the light chain of the antibody may be SEQ ID No. 2 or have at least 90% identity with SEQ ID No. 2 (the inconsistency may be in the framework region (FR)).
  • the present invention claims a nucleic acid molecule.
  • the nucleic acid molecule claimed in the present invention is a nucleic acid molecule encoding the antibody described in the first aspect or the antigen binding portion of the antibody.
  • nucleotide sequences encoding HCDR1, HCDR2, and HCDR3 in the variable region of the heavy chain of the antibody may be sequentially as shown in SEQ ID No. 3 from position 76 to position 99 from the 5'end, As shown in positions 151-174 and 289-363, the nucleotide sequences of LCDR1, LCDR2, and LHCDR3 in the light chain variable region encoding the antibody can be shown in SEQ ID No. 4 and 79 from the 5'end. -96, 148-156, 265-291 shown.
  • the nucleotide sequence encoding the variable region of the heavy chain of the antibody may be SEQ ID No. 3 from the 5'end of positions 1-363 or the same as SEQ ID No. 3 .3 Positions 1-363 from the 5'end have at least 90% identity (the disparity is in the framework region (FR)), and the nucleotide sequence encoding the light chain variable region of the antibody may be SEQ ID No. 4 has at least 90% identity with positions 1-327 from the 5'end or with positions 1-327 from the 5'end of SEQ ID No. 4 (not in the framework region (FR)).
  • the nucleotide sequence encoding the heavy chain of the antibody may be SEQ ID No. 3 or have at least 90% identity with SEQ ID No. 3 (the inconsistency may be in the backbone Region (FR)), the nucleotide sequence encoding the light chain of the antibody may be SEQ ID No. 4 or have at least 90% identity with SEQ ID No. 4 (the discrepancy may be in the framework region (FR)) .
  • the present invention claims an expression cassette, recombinant vector, recombinant cell or recombinant bacteria containing the nucleic acid molecule described in the second aspect above.
  • the DNA fragment shown at positions 1-363 from the 5'end of SEQ ID No. 3 (the gene encoding the variable region of the antibody heavy chain) is cloned into the restriction site of the pTSEG1n-S vector Click between Sal I and Pml I to obtain a recombinant expression vector expressing the heavy chain of the antibody; the DNA fragment shown at positions 1-327 from the 5'end of SEQ ID No. 4 (the variable region of the antibody light chain
  • the coding gene is cloned into the restriction site Sal I and Pml I of the pTSEK-S vector to obtain a recombinant expression vector expressing the light chain of the antibody.
  • the recombinant cell is a recombinant expression vector comprising two antibody heavy and light chains are expressing the recombinant cell co-transfection of FreeStyle obtained after TM 293F cells.
  • the present invention claims a pharmaceutical composition.
  • the pharmaceutical composition claimed in the present invention comprises the antibody described in the first aspect and a pharmaceutically acceptable excipient, diluent or carrier.
  • the disease caused by dengue virus infection may be Dengue Fever (Classic Dengue Fever, DF), Dengue Hemorrhagic Fever (DHF), or Dengue Shock Syndrome (DSS).
  • DF Dengue Fever
  • DHF Dengue Hemorrhagic Fever
  • DSS Dengue Shock Syndrome
  • the dengue virus may be type I dengue virus, type II dengue virus, type III dengue virus and/or type IV dengue virus.
  • the present invention claims any of the following methods:
  • a method for preventing and/or treating diseases caused by dengue virus infection is to use the aforementioned antibody or nucleic acid molecule or expression cassette, recombinant vector, recombinant cell or recombinant bacteria or pharmaceutical composition to prevent and/or Treatment of diseases caused by dengue virus infection.
  • the disease caused by dengue virus infection may be Dengue Fever (Classic Dengue Fever, DF), Dengue Hemorrhagic Fever (DHF), or Dengue Shock Syndrome (DSS).
  • DF Dengue Fever
  • DHF Dengue Hemorrhagic Fever
  • DSS Dengue Shock Syndrome
  • a method for inhibiting dengue virus infection is to use the aforementioned antibody or nucleic acid molecule or expression cassette, recombinant vector, recombinant cell or recombinant bacteria or pharmaceutical composition to inhibit dengue virus infection.
  • (B3) A method for detecting dengue virus, which uses the aforementioned antibody or nucleic acid molecule or expression cassette, recombinant vector, recombinant cell or recombinant bacteria or pharmaceutical composition to detect dengue virus.
  • a method for binding to dengue virus is to use the aforementioned antibody or nucleic acid molecule or expression cassette, recombinant vector, recombinant cell or recombinant bacteria or pharmaceutical composition to bind to the dengue virus.
  • a method for detecting the E protein of dengue virus is to use the aforementioned antibody or nucleic acid molecule or expression cassette, recombinant vector, recombinant cell or recombinant bacteria or pharmaceutical composition to detect the E protein of dengue virus.
  • a method for binding to the E protein of dengue virus is to use the aforementioned antibody or nucleic acid molecule or expression cassette, recombinant vector, recombinant cell or recombinant bacteria or pharmaceutical composition to bind to the E protein of dengue virus.
  • the dengue virus may be type I dengue virus, type II dengue virus, type III dengue virus and/or type IV dengue virus.
  • Figure 1 shows the antibody serotype specific detection of monoclonal antibody 6B1. Note: non-reducing electrophoresis; 1: DENV-1; 2: DENV-2; 3: DENV-3; 4: DENV-4.
  • Figure 2 shows the ELISA analysis of the binding of 6B1 to inactivated dengue virus.
  • Figure 3 shows the ELISA analysis of the binding of 6B1 to the dengue virus recombinant E protein.
  • Figure 4 shows the analysis of the affinity between 6B1 and DENV-1 recombinant E protein.
  • Figure 5 shows the in vitro neutralization activity of monoclonal antibody 6B1 against four serotypes of dengue virus.
  • Figure 6 shows the in vivo analysis of 6B1 on the preventive effect of four serotypes of dengue virus infection in suckling mice.
  • Figure 7 shows the in vivo analysis of 6B1 on DENV-1 and DENV-2 infections in suckling mice.
  • the following examples facilitate a better understanding of the present invention, but do not limit the present invention.
  • the experimental methods in the following examples are conventional methods unless otherwise specified.
  • the test materials used in the following examples, unless otherwise specified, are all purchased from conventional biochemical reagent stores.
  • the quantitative tests in the following examples are all set to three repeated experiments, and the results are averaged.
  • type I dengue virus type II dengue virus
  • type III dengue virus type IV dengue virus
  • type IV dengue virus isolates, provided by the Institute of Microbiology and Epidemiology of the Military Medical Academy.
  • the heavy chain expression vector is pTSEG1n-S: provided by Beijing Baite Meibo Biological Co., Ltd.
  • the light chain expression vector is pTSEK-S: provided by Beijing Baite Meibo Biological Co., Ltd.
  • FreeStyle TM 293F cells Invitrogen company, catalog number R79007.
  • C6/36 cells ATCC.
  • BHK-21 cells ATCC.
  • the peripheral blood of 3 patients who were clinically diagnosed as dengue fever recovery period (patients informed consent) were collected, 10 mL per person, 30 mL in total.
  • Separate mononuclear lymphocytes use flow cytometry to sort single B cells in two steps, first sort out CD19 high CD20 low to negative CD3 negative , sort out CD27 high CD38 high B cells from them, and sort them into 96-well plates , One cell per well.
  • Reverse transcription PCR and nested PCR were used to amplify antibody light and heavy chain variable region genes from single B cells. The PCR products were sequenced for the correct pair of light and heavy chains, and multiple pairs of light and heavy chain variable region gene sequences including 6B1 were obtained.
  • the heavy chain variable region gene of antibody 6B1 is as shown in SEQ ID No. 3 from the 5'end of positions 1-363; the light chain variable region gene of antibody 6B1 is as shown in SEQ ID No. 4 from the 5'end. Shown in positions 1-327.
  • the light and heavy chain variable region genes of 6B1 are fully synthesized.
  • the heavy chain expression vector pTSEG1n-S and the light chain expression vector pTSEK-S are digested with Sal I and Pml I.
  • the light and heavy chain variable region genes of 6B1 are digested with enzymes.
  • the light and heavy chain expression vectors were connected to construct two eukaryotic expression plasmids capable of expressing the heavy and light chains of antibody 6B1. Among them, the recombinant expression plasmid used to express the heavy chain of antibody 6B1 carries the complete SEQ ID No. 3 (SEQ ID No.
  • SEQ ID No. 4 is the light chain coding gene of antibody 6B1.
  • the heavy chain amino acid sequence is shown in SEQ ID No. 1
  • the light chain amino acid sequence is shown in SEQ ID No. 2.
  • amino acid residues 1-121 from the N-terminal form the heavy chain variable region VH (wherein, amino acid residues 26-33 form HCDR1, amino acid residues 51-58 form HCDR2, Amino acid residues 97-121 constitute HCDR3), amino acid residues 122-222 constitute the heavy chain constant region CH1, amino acid residues 223-237 constitute the hinge region of the heavy chain Hinge, and amino acid residues 238-347 are constituted
  • the heavy chain constant region CH2 amino acid residues 348-454 constitute the heavy chain constant region CH3.
  • the amino acid residues 1-109 from the N-terminus constitute the light chain variable region VL (the amino acid residues 27-32 constitute LCDR1, and the amino acid residues 50-52 constitute LCDR2, The amino acid residues at positions 89-97 constitute LCDR3), and the amino acid residues at positions 110-214 constitute the light chain constant region CL.
  • the DNA molecule shown in SEQ ID No. 3 encodes the polypeptide (heavy chain) shown in SEQ ID No. 1.
  • nucleotides 1-363 from the 5'end encode VH (wherein, nucleotides 76-99 encode HCDR1, nucleotides 151-174 encode HCDR2, and nucleotides 289-363 Nucleotide encoding HCDR3)
  • nucleotides 364-666 encode CH1, nucleotides 667-711 encode Hinge
  • nucleotides 1042-1362 encode CH3
  • nucleotides 1363-1365 is the stop codon.
  • the DNA molecule shown in SEQ ID No. 4 encodes the polypeptide (light chain) shown in SEQ ID No. 2.
  • nucleotides 1-327 from the 5'end encode VL wherein, nucleotides 79-96 encode LCDR1, nucleotides 148-156 encode LCDR2, and nucleotides 265-291
  • the nucleotide position encodes LCDR3 the nucleotide position 328-642 encodes CL
  • the nucleotide position 643-645 is the stop codon.
  • sequence of the complementarity determining region is defined by Kabat.
  • Antibody 6B1 is a fully human monoclonal antibody, IgG1, and the light chain type is kappa ( ⁇ ) type.
  • FectoPRO transfection reagent product of Polyplus-transfection, article number 116-010
  • the recombinant expression plasmids of the light and heavy chains of antibody 6B1 constructed above were co-transfected into FreeStyle TM 293F cells according to the instructions, and cultured under serum-free suspension culture conditions Day, the culture supernatant was harvested by centrifugation.
  • the antibody in the supernatant is purified with a Protein A/G affinity chromatography column such as Hitrap TM MabSelect Xtra/SuRe (product of GE, article number 28-4082-55) according to the instructions.
  • Hitrap TM Desalting product of GE, Item No.
  • C6/36 cells were used to inoculate and culture four serotypes of dengue virus, and the culture supernatant was collected to harvest the virus stock. Take an appropriate amount of virus stock, add 4 ⁇ Loading buffer, and boil in a water bath to prepare protein samples. Take 10 ⁇ L of protein sample for SDS-PAGE, then electrotransfer the protein sample to PVDF membrane (product of Amersham company, catalog number 10600023), and block with 5% skimmed milk. Add the antibody 6B1 (1 ⁇ g/mL) to be tested and incubate at room temperature for 2 hours or 4°C refrigerator overnight.
  • PVDF membrane product of Amersham company, catalog number 10600023
  • Figure 1 shows that 6B1 binds to all four serotypes of dengue virus, but it binds strongly to DENV-1 and DENV-2, and weakly binds to DENV-3 and DENV-4.
  • the binding band is around 70kD and is bound to E protein. The molecular weights are consistent, indicating that it recognizes the E protein of the four serotypes of dengue virus.
  • Example 3 ELISA to detect the binding of monoclonal antibody 6B1 to antigen
  • the 96-well enzyme-linked plate was coated with four serotypes of dengue virus recombinant E protein (products of RayBiotech, catalog numbers 228-11688, 228-11689, 228-11690, 228-11691), 2 ⁇ g/mL, 100 ⁇ L/ Wells, coated overnight at 4°C; or coated with inactivated dengue virus culture supernatant of four serotypes, 100 ⁇ L/well, coated overnight at 4°C. After discarding the coating solution, wash with PBST for 5 min/time ⁇ 3 times, add 2% BSA-PBST, 200 ⁇ L/well, and place at 37°C for 2 h.
  • E protein products of RayBiotech, catalog numbers 228-11688, 228-11689, 228-11690, 228-11691
  • FIG. 2 and Figure 3 show that monoclonal antibody 6B1 can bind to inactivated dengue virus or recombinant E protein, indicating that 6B1 may recognize a linear epitope on E protein.
  • the monoclonal antibody 6B1 has higher affinity for DENV-1 and DENV-2, but weaker affinity for DENV-3 and DENV-4, which is also consistent with the results shown in Figure 1.
  • CM5 chip kit Dilute the purified antibody 6B1 to be detected with HBS-EP buffer (in the CM5 chip kit) to 1 ⁇ g/mL, and couple it to the CM5 chip (product of GE company, product number BR100014) pre-coated with Protein G,
  • the coupling conditions are: temperature 25°C, flow rate 5 ⁇ L/min, coupling amount 200RU.
  • DENV recombinant E protein products of RayBiotech, product numbers are 228-11688, 228-11689, 228-11690, 228-11691 as the mobile phase, using HBS-EP buffer to do a two-fold concentration gradient dilution, the concentration range is 20nM -625pM, test conditions: temperature 25°C, flow rate 30 ⁇ L/min; binding time is 3min, dissociation time is 15min. After completing one reaction, regenerate the chip under the conditions of regeneration: 3M MgCl 2 , 30 ⁇ L/min ⁇ 30s. After regeneration, continue to couple the same amount of antibody to be tested for the next reaction.
  • Plaque Reduction Neutralization Test was used to detect the neutralization activity of antibodies in vitro. Twenty-four hours before the experiment, BHK-21 cells in good growth condition were taken, and after trypsinization, the cell density was adjusted to 2.5 ⁇ 10 5 cells/mL, and a 6-well cell culture plate was inoculated with 2 mL per well. On the day of the experiment, in a 96-well plate, the antibody 6B1 was diluted 3-fold, from 100 ⁇ g/mL to 5.65 ⁇ 10 -4 ⁇ g/mL, and a negative control without antibody was set at 100 ⁇ L/well.
  • PRNT Plaque Reduction Neutralization Test
  • Inhibition rate (number of plaques in negative control-number of plaques in experimental group)/number of plaques in negative control ⁇ 100%.
  • the neutralizing activity of the antibody has a dose-dependent relationship. Within a certain concentration range, as the concentration of the antibody increases, the inhibitory effect on virus infection is significantly enhanced.
  • the PRNT 50 value was calculated by non-linear statistical analysis of the neutralizing activity titer of the antibody.
  • Monoclonal antibody 6B1 has strong neutralizing activity to DENV-1 and DENV-2, but weaker neutralizing activity to DENV-3 and DENV-4, which is consistent with the affinity results.
  • the dengue virus stock solution after the determination of the virus titer was diluted 10 times in a gradient. Kunming suckling mice were inoculated intracranially with virus stock solution within 24 hours of birth, each concentration was inoculated with 1 litter (about 10), each with 1 ⁇ L. Continue to observe for 21 days, record the incidence and death of suckling mice every day, calculate the survival rate of mice based on the number of mice in each concentration group, and use Graphpad software to calculate the median lethal dose (LD 50 ) for mice
  • Kunming suckling rats were injected intracranially with 5 ⁇ L of antibodies with concentrations of 200 ⁇ g/mL, 20 ⁇ g/mL, and 2 ⁇ g/mL within 24 hours after birth. After 24 hours, they were inoculated with 5 ⁇ L of 10LD 50 dengue virus via cranial route. Observe for 21 days, record the incidence and death of suckling mice every day, calculate the survival rate of mice based on the number of mice survived, and use Graphpad software to graph and analyze the results, as shown in Figure 6.
  • 6B1 for DENV-1 infection 0.1 ⁇ g/mouse
  • the dose can protect 70% of suckling mice, and the dose of 1 ⁇ g/mouse can protect 100% of suckling rats; 6B1 has a better protective effect on DENV-2 infection, and the dose of 0.01 ⁇ g/mouse can protect 90% of suckling rats.
  • the dose of 1 ⁇ g/mouse can protect 100% of suckling mice; the protective effect of 6B1 on DENV-3 infection is slightly weaker, and the dose of 1 ⁇ g/mouse can only protect 70% of suckling mice; the protective effect of 6B1 on DENV-4 infection The weakest, the dose of 5 ⁇ g/mouse can protect 70% of suckling mice.
  • 6B1 can effectively prevent infections of four serotypes of DENV and treat infections with lethal doses of DENV-1 and DENV-2.
  • the invention prepares a fully human monoclonal antibody 6B1 that can specifically bind to four serotypes of dengue virus.
  • the antibody can recognize the E protein of the four serotypes of dengue virus, and has a high affinity for DENV-1 and DENV-2, but a weak affinity for DENV-3 and DENV-4.
  • the neutralizing activity of DENV-2 is strong, but the neutralizing activity of DENV-3 and DENV-4 is weak. It can effectively prevent the infection of four serotypes of DENV and treat the infection of lethal doses of DENV-1 and DENV-2.
  • the invention is of great significance for the development of drugs for preventing and treating dengue virus infection.

Abstract

一种特异性结合四种血清型登革病毒的人源抗体。人源抗体，其重链可变区中HCDR1、HCDR2和HCDR3的氨基酸序列依次如SEQ ID No.1自N端起第26-33位、第51-58位、第97-121位所示，轻链可变区中LCDR1、LCDR2和LHCDR3的氨基酸序列依次如SEQ ID No.2自N端起第27-32位、第50-52位、第89-97位所示。抗体可有效预防四种血清型DENV的感染，治疗致死剂量DENV-1和DENV-2的感染。对于开发预防及治疗登革病毒感染的药物具有重要意义。

Description

一种特异性结合四种血清型登革病毒的人源抗体 技术领域
本发明涉及生物技术领域，特别涉及一种特异性结合四种血清型登革病毒的人源抗体。
背景技术
登革病毒(Dengue virus,DENV)属于黄病毒科黄病毒属，是有包膜的单正链RNA病毒，根据其包膜的抗原性不同，分为四种血清型(DENV I-IV)。登革病毒主要以埃及伊蚊和白纹伊蚊为传播媒介，广泛流行于热带和亚热带地区。人类首次感染登革病毒会引起登革热(Classic Dengue Fever,DF)，是一种自限性发热性疾病，同时登革病毒可以激发良好的细胞免疫和体液免疫，对感染的该亚型可提供终身的免疫力。但是当再次感染其他亚型的登革病毒时，首次感染产生的抗体会携带病毒攻击靶细胞，造成病毒大量的增殖，从而引发严重的登革出血热(Dengue Hemorrhagic Fever，DHF)和登革休克综合征(Dengue Shock Syndrome，DSS)甚至死亡，即抗体依赖增强(Antibody dependent enhancement,ADE)。世界上约有半数的人口生活在登革热疫区，每年有超过5000万的感染病例，其中有50万人发展为严重的DHF和DSS。但是，由于ADE现象的存在，临床上缺乏预防和治疗DENV感染的有效疫苗和药物，主要以对症支持治疗为主。
免疫球蛋白是治疗病毒感染类疾病的重要手段，不仅能中和病毒，还能通过补体依赖的细胞毒作用(complement dependent cytotoxicity,CDC)和抗体依赖细胞介导的细胞毒作用(antibody dependent cell-mediated cytotoxicity,ADCC)激活体内免疫系统清除病毒。大量体内、外实验证明：中和抗体能有效阻止DENV的感染，不仅能发挥病毒感染前的预防作用，而且在病毒感染后的一段时期内，依然能发挥治疗病毒感染的作用。基于临床上针对DENV感染无有效防治手段的现状，以及近年来单克隆抗体技术在人源化、规模化培养制备等方面均取得的巨大进步，登革病毒的治疗性抗体研究也得到了长足的发展。
DENV的E蛋白是感染性病毒粒子表面暴露最充分的蛋白，也是中和抗体的主要靶点。目前全球报道的DENV中和抗体表位也多与E蛋白有关：(1)识别DENV ED Ⅱ：FL loop对应的交叉中和性抗体(E53)大都中和效率低且易产生ADE效应；针对BC loop的交叉中和抗体(1C19)不仅能高效中和DENV且还能与低效抗FL抗体竞争结合。(2)识别DENV EDⅢ：AB loop对应交叉中和抗体一般具弱中和性，EF loop附近形成的单抗(2B11A35/2D73A7)具高效交叉中和能力，Astrand表位(单抗，DB32-6)具有高效中和活性；(3)识别E蛋白二聚体依赖性表位(E-dimer-dependent epitope，EDE)：单抗(EDE1/EDE2)是一类新型高效广谱的人源中和抗体。此外，在抗体来源方面，大多数中和抗体为鼠源， 少量人源化及人源抗体。人源化抗体与鼠源抗体相比具有以下特点：(1)免疫原性低于鼠源抗体；(2)亲和力弱于鼠源抗体，特异性优于鼠源抗体；(3)人源化抗体Fc段能够诱发机体的效应功能；(4)在体内半衰期长；(5)价格仍较昂贵。但人源化抗体仍含有10％～30％的鼠源蛋白，在临床应用时仍存在免疫排斥反应的潜在威胁，因而全人源中和抗体是DENV治疗性抗体的重要研发目标之一。
基于临床需求，探索和研发登革病毒中和抗体具有重要的生物学和医学意义。
发明公开
本发明的目的是提供一种特异性结合四种血清型登革病毒的人源抗体。
第一方面，本发明要求保护一种抗体。
本发明所要求保护的抗体为全人源单抗6B1，其重链可变区中HCDR1、HCDR2和HCDR3的氨基酸序列依次如SEQ ID No.1自N端起第26-33位、第51-58位、第97-121位所示；所述抗体的轻链可变区中LCDR1、LCDR2和LHCDR3的氨基酸序列依次如SEQ ID No.2自N端起第27-32位、第50-52位、第89-97位所示。
其中，HCDR1、HCDR2和HCDR3为重链可变区中的三个互补决定区，LCDR1、LCDR2和LHCDR3为轻链可变区中的三个互补决定区。互补决定区的序列根据Kabat定义。
进一步地，所述重链可变区的氨基酸序列可为SEQ ID No.1自N端起第1-121位，或者与SEQ ID No.1自N端起第1-121位具有至少90％的一致性(不一致处可在骨架区(FR))。所述轻链可变区的氨基酸序列可为SEQ ID No.2自N端起第1-109位，或者与SEQ ID No.2自N端起第1-109位具有至少90％的一致性(不一致处可在骨架区(FR))。
在本发明中，所述抗体为IgG。进一步地，所述IgG为IgG1。所述抗体的轻链类型为kappa(κ)型。
更进一步地，所述抗体的重链的氨基酸序列可为SEQ ID No.1，或者与SEQ ID No.1具有至少90％的一致性(不一致处可在骨架区(FR))。所述抗体的轻链的氨基酸序列可为SEQ ID No.2或者与SEQ ID No.2具有至少90％的一致性(不一致处可在骨架区(FR))。
第二方面，本发明要求保护一种核酸分子。
本发明所要求保护的核酸分子为编码前文第一方面所述的抗体或所述抗体中的抗原结合部分的核酸分子。
进一步地，在所述核酸分子中，编码所述抗体的重链可变区中HCDR1、HCDR2和HCDR3的核苷酸序列可依次如SEQ ID No.3自5’端起第76-99位、第151-174位、第289-363位所示，编码所述抗体的轻链可变区中LCDR1、LCDR2和LHCDR3的核苷酸序列可依次如SEQ ID No.4自5’端起第79-96位、第148-156位、第265-291位所示。
更进一步地，在所述核酸分子中，编码所述抗体的所述重链可变区的核苷酸序列可为SEQ ID No.3自5’端起第1-363位或者与SEQ ID No.3自5’端起第1-363位具有至少90％的一致性(不一致处在骨架区(FR))，编码所述抗体的所述轻链可变区的核苷酸序列可为SEQ ID No.4自5’端起第1-327位或者与SEQ ID No.4自5’端起第1-327位具有至少90％的一致性(不一致处在骨架区(FR))。
更加具体地，在所述核酸分子中，编码所述抗体的重链的核苷酸序列可为SEQ ID No.3或者与SEQ ID No.3具有至少90％的一致性(不一致处可在骨架区(FR))，编码所述抗体的轻链的核苷酸序列可为SEQ ID No.4或者与SEQ ID No.4具有至少90％的一致性(不一致处可在骨架区(FR))。
第三方面，本发明要求保护含有前文第二方面所述核酸分子的表达盒、重组载体、重组细胞或重组菌。
在本发明的一个实施例中，将SEQ ID No.3自5’端起第1-363位所示DNA片段(抗体重链可变区的编码基因)克隆到pTSEG1n-S载体的酶切位点Sal I和Pml I之间后得到表达所述抗体的重链的重组表达载体；将SEQ ID No.4自5’端起第1-327位所示DNA片段(抗体轻链可变区的编码基因)克隆到pTSEK-S载体的酶切位点Sal I和Pml I之间后得到表达所述抗体的轻链的重组表达载体。所述重组细胞为将上述分别表达所述抗体重链和轻链的两个重组表达载体共转染FreeStyle TM 293F细胞后得到的重组细胞。
第四方面，本发明要求保护一种药物组合物。
本发明所要求保护的药物组合物包含前文第一方面中所述的抗体和药学可接受的赋形剂、稀释剂或载体。
第五方面，本发明要求保护如下任一所示应用：
(A1)前文所述核酸分子或表达盒、重组载体、重组细胞或重组菌在制备前文所述抗体或所述药物组合物中的应用。
(A2)前文所述抗体在制备前文所述药物组合物中的应用。
(A3)前文所述抗体或核酸分子或表达盒、重组载体、重组细胞或重组菌或药物组合物在制备用于预防和/或治疗由登革病毒感染所致疾病的产品中的应用，或在预防和/或治疗由登革病毒感染所致疾病中的应用。
其中，所述由登革病毒感染所致疾病可为登革热(Classic Dengue Fever,DF)、登革出血热(Dengue Hemorrhagic Fever，DHF)或者登革休克综合征(Dengue Shock Syndrome，DSS)。
(A4)前文所述抗体或核酸分子或表达盒、重组载体、重组细胞或重组菌或药物组合物在制备用于抑制登革病毒感染(中和登革病毒)的产品中的应用，或在抑制登革病毒感染(中和登革病毒)中的应用。
(A5)前文所述抗体或核酸分子或表达盒、重组载体、重组细胞或重组菌或药物组合物在制备用于检测登革病毒的产品中的应用，或在检测登革病毒中的应用。
(A6)前文所述或核酸分子或表达盒、重组载体、重组细胞或重组菌或药物组合物在制备用于结合登革病毒的产品中的应用，或在结合登革病毒中的应用。
(A7)前文所述抗体或核酸分子或表达盒、重组载体、重组细胞或重组菌或药物组合物在制备用于检测登革病毒的E蛋白的产品中的应用，或在检测登革病毒的E蛋白中的应用。
(A8)前文所述抗体或核酸分子或表达盒、重组载体、重组细胞或重组菌或药物组合物在制备用于结合登革病毒的E蛋白的产品中的应用，或在结合登革病毒的E蛋白中的应用。
进一步地，所述登革病毒可为I型登革病毒、II型登革病毒、III型登革病毒和/或IV型登革病毒。
第六方面，本发明要求保护如下任一所示方法：
(B1)一种预防和/或治疗由登革病毒感染所致疾病的方法，是利用前文所述抗体或核酸分子或表达盒、重组载体、重组细胞或重组菌或药物组合物预防和/或治疗由登革病毒感染所致疾病。
其中，所述由登革病毒感染所致疾病可为登革热(Classic Dengue Fever,DF)、登革出血热(Dengue Hemorrhagic Fever，DHF)或者登革休克综合征(Dengue Shock Syndrome，DSS)。
(B2)一种抑制登革病毒感染的方法，是利用前文所述抗体或核酸分子或表达盒、重组载体、重组细胞或重组菌或药物组合物抑制登革病毒感染。
(B3)一种检测登革病毒的方法，是利用前文所述抗体或核酸分子或表达盒、重组载体、重组细胞或重组菌或药物组合物检测登革病毒。
(B4)一种结合登革病毒的方法，是利用前文所述抗体或核酸分子或表达盒、重组载体、重组细胞或重组菌或药物组合物结合登革病毒。
(B5)一种检测登革病毒的E蛋白的方法，是利用前文所述抗体或核酸分子或表达盒、重组载体、重组细胞或重组菌或药物组合物检测登革病毒的E蛋白。
(B6)一种结合登革病毒的E蛋白的方法，是利用前文所述抗体或核酸分子或表达盒、重组载体、重组细胞或重组菌或药物组合物结合登革病毒的E蛋白。
进一步地，所述登革病毒可为I型登革病毒、II型登革病毒、III型登革病毒和/或IV型登革病毒。
附图说明
图1为单抗6B1的抗体血清型特异性检测。注：非还原电泳；1：DENV-1；2：DENV-2；3：DENV-3；4：DENV-4。
图2为ELISA分析6B1与灭活登革病毒的结合。
图3为ELISA分析6B1与登革病毒重组E蛋白的结合。
图4为分析6B1与DENV-1重组E蛋白的亲和力。
图5为单抗6B1对四种血清型登革病毒的体外中和活性。
图6为乳鼠体内分析6B1对四种血清型登革病毒感染的预防作用。A：DENV-1；B：DENV-2；C：DENV-3；D：DENV-4。
图7为乳鼠体内分析6B1对DENV-1和DENV-2感染的治疗作用。A：DENV-1；B：DENV-2。
实施发明的最佳方式
以下的实施例便于更好地理解本发明，但并不限定本发明。下述实施例中的实验方法，如无特殊说明，均为常规方法。下述实施例中所用的试验材料，如无特殊说明，均为自常规生化试剂商店购买得到的。以下实施例中的定量试验，均设置三次重复实验，结果取平均值。
四种血清型登革病毒，即I型登革病毒、II型登革病毒、III型登革病毒和/或IV型登革病毒：分离株，由军事医学研究院微生物流行病研究所提供。
重链表达载体为pTSEG1n-S：由北京百特美博生物有限公司提供。
轻链表达载体为pTSEK-S：由北京百特美博生物有限公司提供。
FreeStyle TM 293F细胞：Invitrogen公司，货号R79007。
C6/36细胞：ATCC。
BHK-21细胞：ATCC。
实施例1、全人源单抗6B1的制备
采集3例临床上确诊为登革热恢复期患者的外周血(患者知情同意)，每人10mL，共30mL。分离单核淋巴细胞，利用流式细胞术两步分选单个B细胞，先分选出CD19 highCD20 low  to  negativeCD3 negative，在从中分选出CD27 highCD38 high的B细胞，分至96孔板，每个孔一个细胞。利用反转录PCR和巢式PCR从单B细胞扩增抗体轻重链可变区基因，轻重链正确配对者进行PCR产物测序，获得了包括6B1在内的多对轻重链可变区基因序列。其中，抗体6B1的重链可变区基因如SEQ ID No.3自5’端起第1-363位所示；抗体6B1的轻链可变区基因如SEQ ID No.4自5’端起第1-327位所示。
全合成6B1的轻重链可变区基因，重链表达载体pTSEG1n-S和轻链表达载体pTSEK-S利用Sal I和Pml I双酶切，将6B1的轻重链可变区基因分别与酶切后的轻重链表达载体连接，构建能够表达抗体6B1的重链和轻链的两个真核表达质粒。其中，用于表达抗体6B1的重链的重组表达质粒中携带完整的SEQ ID No.3(SEQ ID No.3为抗体6B1的重链编码基因)，用于表达抗体6B1的轻链的重组表达质粒中携带完整的SEQ ID No.4(SEQ ID No.4为抗体6B1的轻链编码基因)。
抗体6B1，其重链氨基酸序列如SEQ ID No.1所示，轻链氨基酸序列如SEQ ID No.2所示。
SEQ ID No.1，自N端第1-121位氨基酸残基组成重链可变区VH(其中，第26-33位氨基酸残基组成HCDR1，第51-58位氨基酸残基组成HCDR2，第97-121位氨基酸残基组成HCDR3)，第122-222位氨基酸残基组成重链恒定区CH1，第223-237位氨基酸残基组成重链铰链区Hinge，第238-347位氨基酸残基组成重链恒定区CH2，第348-454位氨基酸残基组成重链恒定区CH3。
SEQ ID No.2中，自N端第1-109位氨基酸残基组成轻链可变区VL(其中，第27-32位氨基酸残基组成LCDR1，第50-52位氨基酸残基组成LCDR2，第89-97位氨基酸残基组成LCDR3)，第110-214位氨基酸残基组成轻链恒定区CL。
SEQ ID No.3所示的DNA分子编码SEQ ID No.1所示的多肽(重链)。SEQ ID No.3中，自5’端第1-363位核苷酸编码VH(其中，第76-99位核苷酸编码HCDR1，第151-174位核苷酸编码HCDR2，第289-363位核苷酸编码HCDR3)，第364-666位核苷酸编码CH1，第667-711位核苷酸编码Hinge，第712-1041位核苷酸编码CH2，第1042-1362位核苷酸编码CH3，第1363-1365位核苷酸为终止密码子。
SEQ ID No.4所示的DNA分子编码SEQ ID No.2所示的多肽(轻链)。SEQ ID No.4中，自5’端第1-327位核苷酸编码VL(其中，第79-96位核苷酸编码LCDR1，第148-156位核苷酸编码LCDR2，第265-291位核苷酸编码LCDR3)，第328-642位核苷酸编码CL，第643-645位核苷酸为终止密码子。
其中，互补决定区的序列根据Kabat定义。
抗体6B1为全人源单抗，为IgG1，轻链类型为kappa(κ)型。
利用FectoPRO转染试剂(Polyplus-transfection公司产品，货号116-010)，按说明书将上述构建好的抗体6B1的轻重链重组表达质粒共转染FreeStyle TM 293F细胞，在无血清悬浮培养条件下培养3天，离心收获培养上清。上清中的抗体用Hitrap TM MabSelect Xtra/SuRe(GE公司产品，货号28-4082-55)等ProteinA/G亲和层析柱按说明书进行纯化。然后利用Hitrap TM Desalting(GE公司产品，货号29-0486-84)将抗体保存缓冲液置换为10mM柠檬酸盐缓冲液(pH6.0)或者其他合适的缓冲液。必要时，可以对抗体样品进行过滤除菌，然后分装分别置于4℃和-20℃保存。
实施例2、单抗6B1的血清型特异性
利用C6/36细胞分别接种培养四种血清型的登革病毒，收集培养上清收获病毒原液。取适量病毒原液，加入4×Loading buffer，水浴煮沸制备蛋白样品。取10μL蛋白样品行SDS-PAGE，然后将蛋白样品电转至PVDF膜(Amersham公司产品，货号10600023)，用5％脱脂牛奶封闭。加入待检抗体6B1(1μg/mL)室温孵育2h或4℃冰箱孵育过夜。用TBS-T洗膜三次，每次10min，加入1:5000稀释的HRP-Goat anti human IgG(中杉金桥公司产品，货号ZB2304)，室温孵育1h。用TBS-T洗膜三次，每次10min。膜上加显影液，用凝胶成像仪曝光显影。
图1显示6B1与四种血清型登革病毒都结合，但是与DENV-1和DENV-2结合较强，与DENV-3和DENV-4结合较弱，结合条带在70kD左右，与E蛋白的分子量一致，表明它识别的是四种血清型登革病毒的E蛋白。
实施例3、ELISA检测单抗6B1与抗原的结合
96孔酶联板分别包被四种血清型的登革病毒重组E蛋白(RayBiotech公司产品，货号分别为228-11688,228-11689,228-11690,228-11691)，2μg/mL，100μL/孔，4℃包被过夜；或包被四种血清型的灭活登革病毒培养上清，100μL/孔，4℃包被过夜。弃包被液后PBST洗涤5min/次×3次，加入2％BSA-PBST，200μL/孔，置于37℃封闭2h。加入3倍倍比稀释的6B1抗体(浓度：30μg/mL-0.17ng/mL)，50μL/孔，37℃孵育2h。弃一抗，PBST洗涤5min/次×3次，加入1:5000稀释的二抗HRP-Goat anti human IgG(中杉金桥公司产品，货号ZB2304)，37℃孵育1h。弃二抗，PBST洗涤5min/次×3次，每孔加入100μL显色液，置于避光处，待显色完全每孔加入100μL终止液。酶标仪读取293nm/630nm波长吸光值。
图2、图3显示单抗6B1既可以结合灭活登革病毒，也可以结合重组E蛋白，说明6B1识别的可能是E蛋白上的线性表位。单抗6B1对DENV-1和DENV-2的亲和力较高，但对DENV-3和DENV-4的亲和力较弱，这也与图1显示的结果一致。
实施例4、单抗6B1的亲和力测定
将纯化后的待检测抗体6B1用HBS-EP缓冲液(CM5芯片试剂盒中)稀释至1μg/mL，并偶联至预先包被有Protein G的CM5芯片(GE公司产品，货号BR100014)上，偶联条件为：温度25℃，流速5μL/min，偶联量为200RU。DENV重组E蛋白(RayBiotech公司产品，货号分别为228-11688,228-11689,228-11690,228-11691)作为流动相，用HBS-EP缓冲液做两倍浓度梯度稀释，其浓度范围为20nM-625pM，测试条件：温度25℃，流速30μL/min；结合时间为3min，解离时间为15min。完成一个反应后，对芯片进行再生，再生条件：3M MgCl 2，30μL/min×30s，再生后继续偶联相同量的待测抗体，进行下一个反应。实验结束后，扣除空白对照的值，用BIAevaluation软件进行结果分析。检测结果显示单抗6B1对DENV-1重组E蛋白的亲和力K D＝0.44nM(图4)。
实施例5、单抗6B1的体外中和活性
利用空斑减少中和实验(Plaque Reduction Neutralization Test,PRNT)检测抗体的体外中和活性。实验前24h取生长状态良好的BHK-21细胞，胰酶消化后调整细胞密度至2.5×10 5个/mL，接种6孔细胞培养板，每孔2mL。实验当天在96孔板中，3倍梯度稀释抗体6B1，由100μg/mL至5.65×10 -4μg/mL， 并设置无抗体阴性对照，100μL/孔。每孔加入等体积含约100PFU的登革病毒悬液，混匀后置于37℃5％CO 2的培养箱中作用1h。弃去BHK-21细胞的培养基，每孔加入800μL DMEM细胞维持液。将孵育结束的登革病毒与抗体混合物加入到6孔板中，37℃5％CO 2培养箱中继续孵育1h。弃上清，PBS缓冲液洗2遍，加入上层半固体培养基(1g低熔点琼脂糖(Amresco公司产品，货号0815)加入50mL水中，加热熔化后温度降至42℃左右，与50mL 2×DMEM培养基(含2mL胎牛血清)混合，使胎牛血清和低熔点琼脂糖的终浓度分别为2％和1％)，继续培养4-5天。待细胞出现病变后，每孔加入1mL 4％多聚甲醛溶液，放置于4℃1h以固定细胞。去除上层琼脂糖，每孔加入1mL 1％结晶紫染液，室温染色30min。用去离子水冲洗后计数空斑，计算抗体对所有四种血清型登革病毒的抑制率。计算方法：抑制率＝(阴性对照空斑数-实验组空斑数)/阴性对照空斑数×100％。
结果如图5所示，抗体的中和活性具有剂量依赖关系，在一定浓度范围内，随着抗体浓度的提高，对病毒感染的抑制作用显著增强。通过对抗体的中和活性效价进行非线性统计分析，计算得到PRNT 50值。单抗6B1对四种血清型登革病毒的中和活性分别为：PRNT 50＝2.33μg/mL、2.89μg/mL、19.65μg/mL、12.64μg/mL。单抗6B1对DENV-1和DENV-2的中和活性较强，但对DENV-3和DENV-4的中和活性较弱，与亲和力结果一致。
实施例6、单抗6B1的体内抗病毒活性
一、病毒感染乳鼠的半数致死剂量(LD 50)测定
将测定病毒滴度后的登革病毒原液10倍梯度稀释。出生24h内昆明种乳鼠颅内接种病毒原液，每个浓度接种1窝(约10只)，每只1μL。继续观察21天，每天记录乳鼠的发病和死亡情况，根据每个浓度组小鼠存活数计算小鼠的存活率，并利用Graphpad软件，计算小鼠半数致死剂量(LD 50)
二、乳鼠保护试验
1、预防效果评价
出生24h内昆明种乳鼠颅内注射5μL浓度为200μg/mL、20μg/mL、2μg/mL的抗体，24h后经颅途径接种10LD 50的登革病毒5μL。观察21天，每天记录乳鼠发病和死亡情况，根据小鼠存活数计算小鼠的存活率，并用Graphpad软件作图分析，结果见图6。6B1针对DENV-1的感染，0.1μg/只的剂量可以保护70％的乳鼠，而1μg/只的剂量可以保护100％的乳鼠；6B1对DENV-2感染的保护效果更佳，0.01μg/只的剂量就可以保护90％的乳鼠，而1μg/只的剂量可以保护100％的乳鼠；6B1对DENV-3感染的保护效果稍弱一些，1μg/只的剂量只可以保护70％的乳鼠；6B1对DENV-4感染的保护效果最弱，5μg/只的剂量才可以保护70％的乳鼠。
2、治疗效果评级
出生24h内昆明种乳鼠颅内接种10LD 50的登革病毒5μL，6h后经颅途径注 射5μL浓度为200μg/mL的抗体，观察21天，每天记录乳鼠发病和死亡情况，根据小鼠存活数计算小鼠的存活率，并用Graphpad软件作图分析，结果见图7。6B1针对DENV-1和DENV-2的攻击，1μg/只的剂量可以保护50％的乳鼠。
综上所述，6B1可有效预防四种血清型DENV的感染，治疗致死剂量DENV-1和DENV-2的感染。
工业应用
本发明制备了一种能够特异性结合四种血清型登革病毒的人源抗体——全人源单抗6B1。实验证明，该抗体能够识别四种血清型登革病毒的E蛋白，对DENV-1和DENV-2的亲和力较高，但对DENV-3和DENV-4的亲和力较弱，对DENV-1和DENV-2的中和活性较强，但对DENV-3和DENV-4的中和活性较弱，可有效预防四种血清型DENV的感染，治疗致死剂量DENV-1和DENV-2的感染。本发明对于开发预防及治疗登革病毒感染的药物具有重要意义。

Claims (18)

  1. 抗体，其特征在于：所述抗体的重链可变区中HCDR1、HCDR2和HCDR3的氨基酸序列依次如SEQ ID No.1自N端起第26-33位、第51-58位、第97-121位所示；所述抗体的轻链可变区中LCDR1、LCDR2和LHCDR3的氨基酸序列依次如SEQ ID No.2自N端起第27-32位、第50-52位、第89-97位所示。
  2. 根据权利要求1所述的抗体，其特征在于：所述重链可变区的氨基酸序列为SEQ ID No.1自N端起第1-121位，或者与SEQ ID No.1自N端起第1-121位具有至少90％的一致性；
    所述轻链可变区的氨基酸序列为SEQ ID No.2自N端起第1-109位，或者与SEQ ID No.2自N端起第1-109位具有至少90％的一致性。
  3. 根据权利要求1或2所述的抗体，其特征在于：所述抗体为IgG。
  4. 根据权利要求3所述的抗体，其特征在于：所述IgG为IgG1。
  5. 根据权利要求1-4中任一所述的抗体，其特征在于：所述抗体的轻链类型为kappa型。
  6. 根据权利要求1-5中任一所述的抗体，其特征在于：所述抗体的重链的氨基酸序列为SEQ ID No.1，或者与SEQ ID No.1具有至少90％的一致性；
    所述抗体的轻链的氨基酸序列为SEQ ID No.2或者与SEQ ID No.2具有至少90％的一致性。
  7. 核酸分子，其特征在于：所述核酸分子编码权利要求1-6中任一所述的抗体或所述抗体中的抗原结合部分。
  8. 根据权利要求7所述的核酸分子，其特征在于：在所述核酸分子中，编码所述抗体的重链可变区中HCDR1、HCDR2和HCDR3的核苷酸序列依次如SEQ ID No.3自5’端起第76-99位、第151-174位、第289-363所示；编码所述抗体的轻链可变区中LCDR1、LCDR2和LHCDR3的核苷酸序列依次如SEQ ID No.4自5’端起第79-96位、第148-156位、第265-291位所示。
  9. 根据权利要求8所述的核酸分子，其特征在于：在所述核酸分子中，编码所述抗体的所述重链可变区的核苷酸序列为SEQ ID No.3自5’端起第1-363位或者与SEQ ID No.3自5’端起第1-363位具有至少90％的一致性；编码所述抗体的所述轻链可变区的核苷酸序列为SEQ ID No.4自5’端起第1-327位或者与SEQ ID No.4自5’端起第1-327位具有至少90％的一致性。
  10. 根据权利要求9所述的核酸分子，其特征在于：在所述核酸分子中，编码所述抗体的重链的核苷酸序列为SEQ ID No.3或者与SEQ ID No.3具有至少90％的一致性；编码所述抗体的轻链的核苷酸序列为SEQ ID No.4或者与SEQ ID No.4具有至少90％的一致性。
  11. 含有权利要求7-10中任一所述核酸分子的表达盒、重组载体、重组细胞或重组菌。
  12. 药物组合物，其特征在于：所述药物组合物包含权利要求1-6中任一所述的抗体和药学可接受的赋形剂、稀释剂或载体。
  13. 应用，为如下任一所示：
    (A1)权利要求7-11中任一所述的核酸分子或表达盒、重组载体、重组细胞或重组菌在制备权利要求1-6中任一所述抗体或权利要求12所述药物组合物中的应用；
    (A2)权利要求1-6中任一所述抗体在制备权利要求12所述药物组合物中的应用；
    (A3)权利要求1-12中任一所述抗体或核酸分子或表达盒、重组载体、重组细胞或重组菌或药物组合物在制备用于预防和/或治疗由登革病毒感染所致疾病的产品中的应用，或在预防和/或治疗由登革病毒感染所致疾病中的应用；
    (A4)权利要求1-12中任一所述抗体或核酸分子或表达盒、重组载体、重组细胞或重组菌或药物组合物在制备用于抑制登革病毒感染的产品中的应用，或在抑制登革病毒感染中的应用；
    (A5)权利要求1-12中任一所述抗体或核酸分子或表达盒、重组载体、重组细胞或重组菌或药物组合物在制备用于检测登革病毒的产品中的应用，或在检测登革病毒中的应用；
    (A6)权利要求1-12中任一所述抗体或核酸分子或表达盒、重组载体、重组细胞或重组菌或药物组合物在制备用于结合登革病毒的产品中的应用，或在结合登革病毒中的应用；
    (A7)权利要求1-12中任一所述抗体或核酸分子或表达盒、重组载体、重组细胞或重组菌或药物组合物在制备用于检测登革病毒的E蛋白的产品中的应用，或在检测登革病毒的E蛋白中的应用；
    (A8)权利要求1-12中任一所述抗体或核酸分子或表达盒、重组载体、重组细胞或重组菌或药物组合物在制备用于结合登革病毒的E蛋白的产品中的应用，或在结合登革病毒的E蛋白中的应用。
  14. 根据权利要求13所述的应用，其特征在于：所述由登革病毒感染所致疾病为登革热、登革出血热或者登革休克综合征。
  15. 根据权利要求13所述的应用，其特征在于：所述登革病毒为I型登革病毒、II型登革病毒、III型登革病毒和/或IV型登革病毒。
  16. 如下任一方法：
    (B1)一种预防和/或治疗由登革病毒感染所致疾病的方法，是利用权利要求1-12中任一所述抗体或核酸分子或表达盒、重组载体、重组细胞或重组菌或药物组合物预防和/或治疗由登革病毒感染所致疾病；
    (B2)一种抑制登革病毒感染的方法，是利用权利要求1-12中任一所述抗体或核酸分子或表达盒、重组载体、重组细胞或重组菌或药物组合物抑制登革病毒感染；
    (B3)一种检测登革病毒的方法，是利用权利要求1-12中任一所述抗体或核酸分子或表达盒、重组载体、重组细胞或重组菌或药物组合物检测登革病毒；
    (B4)一种结合登革病毒的方法，是利用权利要求1-12中任一所述抗体或核酸分子或表达盒、重组载体、重组细胞或重组菌或药物组合物结合登革病毒；
    (B5)一种检测登革病毒的E蛋白的方法，是利用权利要求1-12中任一所述抗体或核酸分子或表达盒、重组载体、重组细胞或重组菌或药物组合物检测登革病毒的E蛋白；
    (B6)一种结合登革病毒的E蛋白的方法，是利用权利要求1-12中任一所述抗体或核酸分子或表达盒、重组载体、重组细胞或重组菌或药物组合物结合登革病毒的E蛋白。
  17. 根据权利要求16所述的方法，其特征在于：所述由登革病毒感染所致疾病为登革热、登革出血热或者登革休克综合征。
  18. 根据权利要求16所述的方法，其特征在于：所述登革病毒为I型登革病毒、II型登革病毒、III型登革病毒和/或IV型登革病毒。
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