WO2020093672A1 - 一种广谱中和多种埃博拉病毒的单克隆抗体 2g1 及应用 - Google Patents

一种广谱中和多种埃博拉病毒的单克隆抗体 2g1 及应用 Download PDF

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WO2020093672A1
WO2020093672A1 PCT/CN2019/086336 CN2019086336W WO2020093672A1 WO 2020093672 A1 WO2020093672 A1 WO 2020093672A1 CN 2019086336 W CN2019086336 W CN 2019086336W WO 2020093672 A1 WO2020093672 A1 WO 2020093672A1
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antibody
monoclonal antibody
seq
ser
amino acid
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French (fr)
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陈薇
于长明
范鹏飞
迟象阳
张冠英
李建民
侯利华
徐俊杰
房婷
吴诗坡
陈旖
陈郑珊
刘渝娇
王美荣
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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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Priority to EP19882579.6A priority Critical patent/EP3878860A4/en
Publication of WO2020093672A1 publication Critical patent/WO2020093672A1/zh

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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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • 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
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • 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
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance
    • 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
    • C12N2510/00—Genetically modified cells

Definitions

  • the invention discloses an antibody, which belongs to the technical field of polypeptides.
  • Ebola virus can cause acute severe hemorrhagic fever in humans and non-human primates. It is one of the viruses with the highest fatality rate found to date, with a mortality rate of up to 90%, which can be directly transmitted by contact , With extremely contagious and lethal rate.
  • Glycoprotein (GP) spikes on the surface of the Ebola virus envelope mediate almost all links of the virus into the cell, so Ebola GP is an important target for virus neutralizing antibodies.
  • the Ebola virus GP gene is processed into two proteins, one is secreted non-structural GP (secreted glycoprotein, sGP); the other is structural GP.
  • Ebola GP is first synthesized as a peptide, and then is digested by Furin to form GP1 (amino acids 1-501) and GP2 (amino acids 502-676), the two subunits are connected by disulfide bonds to form The trimer is fixed to the envelope surface by the transmembrane region inside GP2.
  • GP1 contains Mucin, Glycan cap, Head, Base and other domains.
  • the GP structure formed by Furin digestion cannot directly stimulate the fusion process of Ebola virus and host cell membrane.
  • Ebola virus After Ebola virus enters the body, it binds to receptors on the surface of the cell membrane, but the adhesion factors on the surface of these host cell membranes are not true receptors for the Ebola virus to fuse into the host cell. After the virus binds to cell surface adhesion factors, clathrin mediates endocytosis and endocytosis, and the transport of primary and secondary endosomes occurs. GP is digested by histone B and histone L in the endosome, removing 60% of amino acids on GP1 including mucin and glycan cap to form activated GP (primed GP, GPcl), which is activated at this time The decisive membrane fusion process takes place.
  • GPcl and endosomal membrane protein Niemann-Pick C1 combines to produce membrane fusion, which allows viral RNA to enter the cytoplasm to complete viral genome replication and transcription. After synthesis of the new viral protein, it assembles into progeny virus particles and buds from the host cell surface.
  • the endosomal membrane protein NPC1 is an essential host infection factor for EBOV infection.
  • the Ebola virus is classified as a Class A biological agent / bioterrorist by the US NIH and CDC, and it is classified as one of the pathogens with significant hidden dangers and risks to national security and public health. Bora virus drugs. After the Ebola virus was discovered in 1967, it experienced 13 large-scale spreads. In 2014, West Africa broke out the largest and most difficult to control Ebola outbreak in history, which was identified as the Zaire Ebola virus. The outbreak caused more than 10,000 deaths and more than 25,000 infections (according to WHO 2016 The Ebola outbreak report released on February 17, 2002), the outbreak spread for the first time outside the African continent, causing great panic worldwide. The public and authorities have paid close attention to the rapid advancement of Ebola vaccines and antiviral drugs.
  • Ebola virus neutralizing antibodies may play a protective role in three ways: 1) blocking the hydrolysis of GP by cathepsin; 2) blocking the binding of activated GP to the NPC1 receptor; 3) affecting the NPC1 receptor The allosteric process of GP after bonding to before membrane fusion.
  • the first two ways have been confirmed by the literature, and the third way is the speculation of some documents, and there is no direct evidence.
  • Ebola virus disease Although there are no approved drugs for the treatment of Ebola virus disease, there are some experimental anti-Ebola virus drugs under study, including small interfering RNA, antisense oligonucleotides, nucleotide analogs, and antibodies.
  • small interfering RNA In August 2018, the Ebola outbreak broke out again in the Democratic Republic of Congo. As of April 7, 2019, it caused 1,154 infections and 731 deaths.
  • the antibody drug "Zmapp” and its optimized strain “MIL77” made by mixing three monoclonal antibodies successfully saved the lives of many people during the 2014 Ebola outbreak in West Africa. It far surpasses other kinds of antiviral drugs, which greatly improves the safety of the drugs and the cure rate of patients, and gives great encouragement to people.
  • the application of antibody drugs for the treatment of filamentous virus disease has attracted worldwide attention and has become a hot spot in the research field of Ebola virus disease treatment drugs.
  • Zmapp is a cocktail therapy strategy consisting of three human and mouse chimeric monoclonal antibodies (c2G4, c4G7, c13C6) expressed by the tobacco system, in which c2G4 (epitope C511, N550, G553, C556) and c4G7 (epitope C511, D552, C556) binds to the GP2 subunit with overlapping epitopes; c13C6 (epitope T270, K272) binds to the glycan cap region at an approximately vertical angle.
  • MIL77 is an antibody combination optimized for Zmapp.
  • MIL77-1 / -2 / -3 contains variable regions of c2G4, c4G7 and c13C6, respectively, and its skeleton region is expressed in CHO cells after humanization.
  • MIL77-1 and ML77-3 antibody cocktail combination therapy can play a 100% protective activity against non-human primates 72 hours after infection.
  • the object of the present invention is to provide a broad-spectrum anti-Ebola virus monoclonal antibody that can be directed against a unique antigen epitope, and further provide its application in the preparation of drugs for the treatment of Ebola virus disease. .
  • the present invention first provides a broad-spectrum anti-Ebola virus monoclonal antibody 2G1, the amino acid sequences of CDR1, CDR2 and CDR3 of the light chain variable region of the antibody are as SEQ, respectively.
  • the amino acid sequences of positions 27-32, 50-52, and 89-96 of ID NO.1 are shown, and the amino acid sequences of CDR1, CDR2, and CDR3 of the antibody heavy chain variable region are as shown in section 26 of SEQ ID NO.5, respectively.
  • the amino acid sequences of -33, 51-58 and 97-113 are shown.
  • amino acid sequence of the antibody light chain constant region is shown in SEQ ID NO. 3
  • antibody heavy chain constant amino acid sequence is shown in SEQ ID NO. 7.
  • the present invention also provides a gene coding sequence for the light chain and heavy chain of the above-mentioned monoclonal antibody.
  • the gene coding sequence of the light chain variable region of the antibody is shown in SEQ ID NO. 2.
  • the gene coding sequence of the chain variable region is shown by SEQ ID NO.6.
  • the gene coding sequence of the antibody light chain constant region is shown in SEQ ID NO. 4, and the gene coding sequence of the antibody heavy chain constant region is shown in SEQ ID NO. 8.
  • the present invention provides a functional element capable of expressing the gene coding sequence encoding the heavy chain and / or light chain of the monoclonal antibody.
  • the present invention provides a host cell containing the above linear expression cassette, the cell being a 293T cell.
  • the present invention provides the application of the above-mentioned monoclonal antibodies in the preparation of drugs for treating Ebola virus disease.
  • the monoclonal antibody is used as a component in an Ebola virus disease cocktail therapy formulation.
  • 2G1 Shows excellent broad-spectrum in antigen binding capacity, and has good binding activity with EBOV GP, BDBV GP, SUDV GP and RESTV GP, EC 50 values are 0.0059 ⁇ g / mL, 0.0075 ⁇ g / mL, 0.0048 ⁇ g / mL And 0.026 ⁇ g / mL.
  • 2G1 can effectively neutralize EBOV, BDBV and SUDV pseudoviruses in vitro.
  • the neutralizing activity of 2G1 increases with the increase of antibody concentration, and can achieve nearly 100% protection of three Ebola pseudoviruses at a concentration of 1 ⁇ g / mL.
  • the 2G1 monoclonal antibody showed better neutralizing activity in vitro than the control antibody.
  • In vitro neutralization experiments showed that 2G1 is a good Ebola virus broad-spectrum neutralizing monoclonal antibody.
  • the results of competitive binding experiments indicate that the binding epitope of 2G1 is different from these control antibodies, suggesting that 2G1 has the potential to form cocktail combination therapy with other neutralizing antibodies.
  • Stability test shows that in the four different pH environments, the binding activity of 2G1 and GPdM is basically unchanged, proving that the binding of 2G1 and GP is stable and is not affected by the low pH environment of the secondary endosome, and plays a neutralizing role for it Provides the foundation.
  • mice protected by EBOV Mayinga virus strains and guinea pigs challenged by SUDV for complete protection.
  • Figure 24 Protection curve of 2G1 monoclonal antibody against mice challenged with EBOV ( Mayinga) virus strain
  • FIG. 25 Survival curve of 2G1 monoclonal antibody against SUDV strain challenged guinea pigs
  • Figure 26 2G1 monoclonal antibody against SUDV strain challenged guinea pig body weight curve.
  • Serum sample No. P024 was diluted 1: 100 in the first well with a three-fold gradient, 100 ⁇ L per well, and incubated at 37 ° C for 1 h.
  • HPR-labeled goat anti-human IgG secondary antibody (Abcam, ab97225) was diluted with 1: 10000 in diluent, 100 ⁇ L per well, and incubated at 37 ° C for 1 h.
  • TMB one-component color developing solution (Solarbio) was developed for 6 min at room temperature in the dark, and then 50 ⁇ L of stop solution was added to each well to stop the reaction.
  • the SPECTRA MAX 190 microplate reader was used to detect the OD value at 450-630nm.
  • the dilution of the first well of the serum sample is 1: 5, which is a three-fold gradient dilution with a volume of 50 ⁇ L per well.
  • FITC labeled truncated antigen protein GPdM is as follows:
  • Fluorescein Isothiocyanate_FITC (SIGMA, F4274) soluble in DMSO, the concentration is 20 mg / mL.
  • Ficoll density gradient centrifugation method was used to separate PBMC from the collected blood samples as follows:
  • the bottom of the tube is red blood cells
  • the middle layer is the separation liquid
  • the uppermost layer is the plasma / tissue homogenate layer.
  • a thin and dense white membrane that is: mononuclear cells ( Including lymphocytes and monocytes) layer. Carefully pipette the white film layer into another centrifuge tube.
  • Count the cells used for sorting take 1 ⁇ 10 6 cells in a volume of 100 ⁇ L, add the 6 fluorescent dyes recommended in the table below, and incubate at 4 ° C in the dark for 1 h.
  • FIG. 9 Use a cell sorter (SONY, SH800S) to sort GP-specific single memory B cells.
  • the sorting strategy is: IgG + / CD3- / CD38- / CD19 + / CD27 + / GPdM +, and the single plasma cells are directly sorted into 96-well plates. Each well in the 96-well plate contains 5U RNase inhibitor and 19.8 ⁇ L of RNase Store in water at -80 °C.
  • Figure 3 is a graph of cell sorting results. The cell characteristics in the circled part of the figure are IgG + / CD3- / CD38- / CD19 + / CD27 + / GPdM +, which is the desired GP-specific memory B cells.
  • 80 GPdM-specific memory B cells were obtained, and all the following specific primers for each subtype of heavy chain H, kappa light chain, and lambda light chain were added to each reaction system (see Table 1 for primer sequences).
  • the PCR reaction system includes: 5 ⁇ buffer 6 ⁇ L, dNTP 1.2 ⁇ L, reverse transcriptase 1.2 ⁇ L, primers as above, the template is a single cell, and water is filled up to 30 ⁇ L.
  • the PCR reaction conditions were: 50 °C reverse transcription for 30 min, 95 °C pre-denaturation for 15 min, then 95 °C 40 s, 55 °C 30 s, 72 °C 1 min, 40 cycles, and finally 72 °C for 10 min.
  • a Primer name sequence H VH3a-sense SARGTGCAGCTCGTGGAG A VH3b-sense GAGGTGCAGCTGTTGGAG A MuD GGAATTCTCACAGGAGACGA A PW-Cgamma AGTAGTCCTTGACCAGGCAGCCCAG ⁇ : 5 'Pan Vk ATGACCCAGWCTCCABYCWCCCTG A 3 'Ck 494–516 GTGCTGTCCTTGCTGTCCTGCT lambda 5 'AgeI V ⁇ 1 CTGCTACCGGTTCCTGGGCCCAGTCTGTGCTGACKCAG A 5 'AgeI V ⁇ 2 CTGCTACCGGTTCCTGGGCCCAGTCTGCCCTGACTCAG A 5 'AgeI V ⁇ 3 CTGCTACCGGTTCTGTGACCTCCTATGAGCTGACWCAG A 5 'AgeI V ⁇ 4/5 CTGCTACCGGTTCTCTCTCSCAGCYTGTGCTGACTCA A 5 'AgeI V ⁇ 6 CTGCTACCGGTTCT
  • the PCR reaction system contains: 10 ⁇ buffer 2.5 ⁇ L, 10 mM dNTP 0.5 ⁇ L, DNA polymerase 0.25 ⁇ L, primers as above, the template is the reverse transcription product 1 ⁇ L, and water is added to 25 ⁇ L.
  • the PCR reaction conditions were: pre-denaturation at 94 ° C for 4 min, followed by 94 ° C for 30 s, 57 ° C for 30 s, 72 ° C for 45 min, 40 cycles, and finally 72 ° C for 10 min.
  • a clone that successfully amplified both heavy and light chain genes in a single cell is considered a paired clone. Take 5 ⁇ L of nested PCR amplified products and perform electrophoresis on 1% agarose gel. Take the paired positive clones for sequencing. NTI software and IMGT website were analyzed, and antibody protein expression and function verification were performed.
  • Fig. 4 is an identification pattern of agarose gel electrophoresis after nested PCR amplification of three chain genes of H, ⁇ and ⁇ . Only when the heavy chain and light chain variable region genes are amplified from the same memory B cell are considered as naturally matched antibody genes, and the corresponding nested PCR products are sequenced for further study.
  • the PCR reaction system for amplifying the promoter-leader sequence fragment contains: template plasmid pSec Tag2 (Invitrogen) 1 ng, 10 ⁇ buffer 5 ⁇ L, 10 mM dNTP 1 ⁇ L, DNA polymerase 0.5 ⁇ L, primer 5'CMV-FORWARD (CGATGTACGGGCCAGATATACGCGTTG), primer 3'leader-sequence (GTCACCAGTGGAACCTGGAACCCA), make up to 50 ⁇ L with water.
  • template plasmid pSec Tag2 Invitrogen
  • 10 ⁇ buffer 5 ⁇ L 10 mM dNTP 1 ⁇ L
  • DNA polymerase 0.5 ⁇ L
  • primer 5'CMV-FORWARD CGATGTACGGGCCAGATATACGCGTTG
  • primer 3'leader-sequence GTCACCAGTGGAACCTGGAACCCA
  • the PCR reaction conditions were: pre-denaturation at 94 ° C for 4 min, followed by 94 ° C for 30 s, 60 ° C for 30 s, 72 ° C for 1 min, 30 cycles, and finally 72 ° C for 10 min.
  • the H chain constant region PCR system includes: heavy chain constant region template 10 ng, 10 ⁇ buffer 5 ⁇ L, 10 mM dNTP 1 ⁇ L, DNA polymerase 0.5 ⁇ L, primer 5'CH (ACCAAGGGCCCAT CGGTCTTCCCC), primer 3'CH (GCAACTAGAAGGCACAGTCGAGGCTTT ACCCGGAGACAGGGA) and water to 50 ⁇ L.
  • the ⁇ chain constant region PCR system contains: ⁇ chain constant region template 10 ng, 10 ⁇ buffer 5 ⁇ L, 10 mM dNTP 1 ⁇ L, DNA polymerase 0.5 ⁇ L, primer 5'C ⁇ (GAGGAGCTTCAAGCC AACAAGGCCACA), primer 3'C ⁇ (GCAACTAGAAGGCACAGTCGAGGCT
  • the PCR reaction conditions were: pre-denaturation at 94 ° C for 4 min, followed by 94 ° C for 30 s, 60 ° C for 60 s, 72 ° C for 3 min, 30 cycles, and finally 72 ° C for 10 min.
  • the PCR system contains: the template is the reverse transcription PCR product 10 ng, 10 ⁇ buffer 5 ⁇ L, 10 mM dNTP 1 ⁇ L, DNA polymerase 0.5 ⁇ L, the primers are shown in Table 3 (the heavy chain and light chain primers are respectively After mixing, add to the system), make up to 50 ⁇ L with water.
  • FIG. 5 The schematic diagram of the splicing sequence of the linear expression frame is shown in FIG. 5.
  • P CMV is the promoter
  • Ig L H / L is the heavy and light chain leader sequence
  • V H / L is the heavy and light chain variable region
  • IgG 1 C H / L is the heavy and light chain constant region
  • Poly A is a poly A tail
  • Eco R I and Not I are restriction enzyme sites.
  • the PCR reaction system includes:
  • Template purified promoter-leader sequence fragment 10 ng, heavy chain / light chain variable region fragment 10 ng, heavy chain / light chain constant region fragment 10 ng, poly A tail fragment 10 ng, 10 ⁇ buffer 2.5 ⁇ L, 10 mM dNTP 0.5 ⁇ L, DNA polymerase 0.25 ⁇ L, primers 5'CMV-FORWARD (CGATGTACGGGCCAGATATACGCGTTG) and 3'POLY (A) (TCCCCAGCATG
  • the PCR reaction conditions were: pre-denaturation at 94 ° C for 4 min, followed by 94 ° C for 30 s, 60 ° C for 30 s, 72 ° C for 3 min, 30 cycles, and finally 72 ° C for 10 min.
  • PCR product recovery and purification PCR products are directly recovered with OMEGA's recovery kit.
  • Results 25 monoclonal antibodies were screened, and the Ebola GP binding antibodies were specifically identified. There are 10 strains that can specifically bind to Ebola GP, including 2A1, 2G1, 2F4, 2E5, 2G5, 2C6, 2A7, 2C7, 2H7, 2A10.
  • Figure 6 shows a control bar chart of the binding activity of 25 monoclonal antibodies.
  • the test results are shown in Figure 7.
  • 7 strains have broad-spectrum binding activity and can bind two or more Ebola GPs.
  • 2A1, 2G5, 2C6 can be combined with EBOV GP and BDBV GP;
  • 2G1 can be combined with EBOV GP, BDBV GP, SUDV GP and RESTV GP;
  • 2F4 can be combined with EBOV GP, BDBV GP and SUDV GP;
  • 2E5, 2A7 can be combined with EBOV GP Combined with RESTV GP;
  • 2C7, 2H7 and 2A10 can only be combined with EBOV GP, not broad-spectrum.
  • 2G1 has the best broad-spectrum, can bind to the four Ebola virus GPs we have, and has good binding activity.
  • 2G1 has good broad-spectrum binding activity, and its sequence is described as follows:
  • the amino acid sequence of the light chain variable region is shown in SEQ ID NO.1, and the amino acid sequences of the CDR1, CDR2, and CDR3 of the light chain variable region are shown in SEQ ID NO.1, 27-32, 50-52, and 89-, respectively.
  • the amino acid sequence at position 96 shows that the gene coding sequence of the light chain variable region is represented by SEQ ID NO: 2, the amino acid sequence of the light chain constant region is shown as SEQ As shown in ID NO. 3, the gene coding sequence of the light chain constant region is shown in SEQ ID NO: 4.
  • the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.5, and the amino acid sequences of the CDR1, CDR2 and CDR3 of the heavy chain variable region are shown in the 26th-33rd, 51-58th, and 97-th of SEQ ID NO.5, respectively.
  • the 113 amino acid sequence shows that the gene coding sequence of the heavy chain variable region is represented by SEQ ID NO.6, the heavy chain constant amino acid sequence is shown in SEQ ID NO.7, and the heavy chain constant region gene coding sequence is shown in SEQ ID NO.8.
  • the expression plasmid was constructed for 2G1, and the monoclonal antibody was prepared for expression. Methods as below:
  • 2G1 monoclonal antibody has good binding activity with EBOV GP, BDBV GP, SUDV GP and RESTV GP, EC 50 values are 0.0059 ⁇ g / mL, 0.0075 ⁇ g / mL, 0.0048 ⁇ g / mL and 0.026 ⁇ g / mL (test results) (See Figure 8).
  • BIACORE 3000 was used to determine the affinity of 2G1 monoclonal antibody and GPdMucin.
  • the anti-human IgG Fc antibody is covalently coupled to the CM5 sensor chip.
  • 2G1 monoclonal antibody was used as a ligand at a concentration of 1 ⁇ g / mL and was combined with a flow rate of 10 ⁇ L / min for 30 s.
  • HEPES buffer was used to clean the syringe and microfluidic system (IFC) at a flow rate of 30 ⁇ L / min.
  • GPdMucin as an analyte, with a concentration of 800/400/200/100/50/25 / 12.5 There are 7 concentration gradients in nM.
  • the EBOV, BDBV and SUDV pseudoviruses packaging the HIV skeleton were evaluated for the neutralizing activity of 2G1 in vitro.
  • the evaluation method is as follows:
  • 2G1 can effectively neutralize EBOV, BDBV and SUDV pseudoviruses in vitro.
  • the neutralizing activity of 2G1 increases with the increase of antibody concentration, and can achieve nearly 100% protection of three Ebola pseudoviruses at a concentration of 1 ⁇ g / mL. (See Figure 11 for test results).
  • Vero E6 cells Inoculate Vero E6 cells into a 96-well tissue culture plate (Corning) and grow to 85-90% on the day of infection.
  • 100 PFU of EBOV-eGFP- Mayinga was pre-incubated with monoclonal antibodies 2G1, CA45, MIL77-1 in blank DMEM at 37 °C for 1 h.
  • the concentration of each monoclonal antibody in the first well was 100 ⁇ g / mL, and the dilution was three-fold.
  • the virus antibody mixture was added to Vero E6 cells, infected at 37 ° C, 5% CO 2 for 1 h, and then the medium was replaced with DMEM containing 12% FBS. After incubating the plate for 48 h, it was fixed with 10% phosphate buffered formalin (Fisher).
  • the iSpot FluoroSpot Reader System was used to count the fluorescent plaques.
  • GP can be divided into multiple regions such as glycan cap, mucin region, receptor binding region, fusion loop and HR region.
  • 2G1 exhibits good broad-spectrum neutralizing activity, and it is hoped that the truncated GP will be constructed to analyze its approximate binding area.
  • GP 33-310 In addition to the common GPdTM, GPdM, GP1, sGP and GPcl in the literature, GP 33-310; 463-558 , GP 33-227 , GP 33-158 , GP 95- 295 , GP 158-295 and GP 227-295 .
  • GP 33-310; 463-558 is based on GPdM to remove the following heptad repeat region; GP 33-227 is based on sGP to remove the glycan cap; GP 33-158 contains most of the Base region Compared with the Head area; GP 95-295 compared with the sGP missing the Base area; GP 158-295 compared with the sGP basically deleted the Base area and the Head area; GP 227-295 contains most of the glycan cap area.
  • GPcl is obtained on the basis of GPdM by thermolysin excision of the glycan cap, without the need to construct an expression plasmid.
  • the schematic structure of each truncated GP is shown in Figure 12.
  • the method of truncating GP to analyze 2G1 binding area is as follows:
  • the antigen binding epitope of 2G1 was analyzed by competitive binding ELISA. MIL77-1 / 2/3 with known binding epitopes and several other non-neutralizing antibodies were used as control antibodies to reflect these antibodies by investigating whether the detection antibody's binding to GP would be blocked by competing antibodies incubated with Whether the binding epitopes overlap. Methods as below:
  • the 96-well ELISA plate was coated with GPdM at a concentration of 1 ⁇ g / mL and coated overnight at 4 ° C.
  • the detection antibody was a biotin-labeled antibody
  • the competing antibody was a non-biotin-labeled antibody.
  • the dilution solution diluted the competition antibody to 2.5 ⁇ g / mL; the dilution solution diluted the detection antibody to 25 ng / mL.
  • ZDOCK Use Dock in Discovery Studio 4.5 Proteins
  • the Ebola GP protein structure (PDB ID: 5KEL) was used as the receptor, and the antibody 2G1-Fab (derived from homology modeling) was used as the ligand.
  • the ZDOCK rigid docking algorithm generated 54,000 docking simulated conformations (pose). Combined with ZRank scoring algorithm to sort, get 2000 poses.
  • the ZDOCK score and ZRANK score given by Discovery Studio 4.5 are taken as the characteristic values X 1 and X 2 respectively .
  • the probability value of the regression equation (MLR probability) calculated by the following formula is calculated as follows:
  • the first 10 poses are as follows. Among them, pose_96 ranks first, and the score value is much higher than other poses (see A and B in FIG. 17).
  • Pose_96 was analyzed with the help of LigPlot + software to obtain the amino acids at the interface between GP and 2G1 and possible interactions (see Figure 17C and Figure 18). Amino acids such as 563-567, 509-513 and 531 on GP appeared on the contact surface; amino acids 56, 57 on CDR2 of 2G1 heavy chain and 103, 107 on CDR3 appeared on the contact surface.
  • (A), (B), and (G) after each amino acid on the GP model represent different chains on the GP structure, where (A) and (B) are the same monomer, respectively GP1 and GP2 on the top, (G) is the GP2 subunit on the other monomer. (H) and (L) after the amino acid on the antibody model correspond to the heavy chain and light chain, respectively.
  • the simulation prediction results can provide information and reference for the binding mode and binding epitope analysis of antigen-antibody, and help to explain the neutralization mechanism of antibody.
  • VH / VK of 2G1 monoclonal antibody was analyzed on the IMGT website (http://www.imgt.org/) to design and synthesize 2G1 heavy light chain germline gene VH / VK-IGL.
  • VH-IGL nucleic acid sequence VH-IGL nucleic acid sequence:
  • VK-IGL nucleic acid sequence VK-IGL nucleic acid sequence:
  • VK-IGL amino acid sequence VK-IGL amino acid sequence:
  • the 2G1 light and heavy chain germline genes (VH / VK-IGL) and wild-type sequences (VH / VK-WT) were cross-paired to prepare reassorted antibodies.
  • ELISA was used to detect the change of its binding activity with GP relative to the wild-type to analyze the role of the heavy chain and light chain of the antibody in the binding.
  • the light chain reassortment 2G1-H WT / K IGL reduced the binding activity of 2G1-WT to GP by 4.4 times, while the heavy chain reassortment 2G1-IGL and 2G1-H IGL / K WT decreased by 55.4 times and 91.4 times, indicating that the heavy chain of 2G1 monoclonal antibody plays a key role in binding to GP (see Figure 16).
  • the heavy chain of 2G1 monoclonal antibody is more important for its binding activity, and further analyzed the binding key amino acids in its CDR region by alanine scanning mutation method.
  • the amino acids on the 2G1 heavy chain CDR1 / 2/3 were mutated to alanine (alanine to serine) to prepare anti-2G1 monoclonal antibody mutant strains, and the binding activity and neutralizing activity of these mutant strains were compared to the wild type The change.
  • the results indicate that the Y57 and CDR3 C103, G106, and C108 amino acids of the CDR2 region of the 2G1 heavy chain are both critical for binding and neutralization. These amino acids all appear on the pose_96 contact interface predicted by the DS software. Among them, Y57 and C103 are two amino acids that may form a hydrogen bond with the amino acid on the GP in LigPlot + software prediction analysis; G106 and C108 are located on the other one and may be The upper amino acid forms a hydrogen bond on both sides of S107.
  • the antibody After the antibody binds to the GP on the surface of the virus particle, it enters the host cell together with the virus particle.
  • GP In the acidic environment (pH 5.5) of the secondary endosome, GP is digested and hydrolyzed, and then combined with the receptor NPC1-C, which promotes the fusion of the virus and the endosome membrane, and then releases the viral genome.
  • the binding activity of 2G1 and GP is affected by the acidic environment, the pH in vitro 7.5, pH 6.5, pH In the environment of 5.5 and pH 4.5, the binding activity of 2G1 and GP is the same as that described in Example 2 for the ELISA method.
  • the glycan cap and mucin region on the GP in the acidic environment of the secondary endosome are removed by enzyme digestion to form the GP CL structure.
  • the epitope that GP CL binds to endosome surface receptor NPC1-C is exposed. If the 2G1 monoclonal antibody exerts protective activity by blocking the binding of GP CL to the receptor, then GP CL will not be able to bind to NPC1-C after binding to 2G1.
  • GP CL preparation method adjust the concentration of GPdMuc to 2 mg / mL with PBS (pH 7.5), add thermophilic protease (SIGMA, T7902-25MG), the final enzyme concentration is 0.5 mg / mL, and incubate at 37 ° C for 1 h ; Add 0.5 mM Phosphoramidon (Sigma-Aldrich, R7385) to terminate the digestion reaction; use 0.5 mL, 50 kD cut-off molecular weight Millipore centrifugal filter tube to concentrate and exchange the system; protein sample through a 0.20 ⁇ m microfilter (Millex-LG, SLLGR04NL) After filtration, it was purified with Superdex 200 Increase 10/300 GL gel column (GE Healthcare).
  • GP CL was coated overnight at 4 ° C with a concentration of 1 ⁇ g / mL; blocked for 1 h; washed; MR72 was selected as a positive control, antibody first well 50 ⁇ g / mL, 3-fold gradient dilution, incubated at 37 ° C for 30 min; added NPC1-C -biotin, 5 ⁇ g / mL per well, incubate at 37 ° C for 30 min; wash, add streptavidin antibody, incubate at 37 ° C for 1 h; wash, develop for 6 min, then stop, read 450-630nm dual wavelength OD value.
  • the control antibody MR72 can block the binding of GP CL to NPC1-C to 30% at a concentration of 50 ⁇ g / mL.
  • the 2G1 monoclonal antibody did not show a significant blocking effect, indicating that it does not play a protective role by blocking the binding of GP CL to the receptor (see Figure 22 for test results). Since known antibodies exert a protective effect through or in part through this pathway, this example excludes a possible protective mechanism of 2G1 monoclonal antibodies.
  • 2 ⁇ g of GPdmucin-His 6 expressed by Expi293 was mixed with 0.5 ⁇ g, 5 ⁇ g and 50 ⁇ g of IgG in 20 ⁇ L PBS, and incubated at 37 ° C for 30 min.
  • the non-reducing protein loading buffer it was placed in a 99 ° C metal bath and boiled for 10 minutes.
  • 2G1 can effectively block the cleavage of GPdmucin by thermolysin. With the increase of 2G1 concentration, the amount of undigested GPdmucin (about 70kD) and the digested intermediate GPdmucin-IMF (intermediate form, about 50kD) increased significantly. To the end product of the digestion GP CL (about 38kD). It is suggested that 2G1 may play a protective role by blocking GP cleavage in vivo (see Figure 23).
  • mice in the control group Female BALB / c healthy mice aged 6-8 weeks were randomly assigned to the experimental group with 10 mice in each group. 100 PFU mice adapted to the EBOV (Mayinga) virus strain were diluted with PBS and challenged by intraperitoneal injection. After 1 and 2 days of challenge, a single dose (100 ⁇ g) was injected intraperitoneally with monoclonal antibody treatment, and mice in the control group were given PBS.
  • mice given 100 ⁇ g of 2G1 monoclonal antibody all survived, while the mice in the PBS control group all died (see Figure 24).
  • the clinical symptoms of the mice in the 2G1 monoclonal antibody treatment group also improved.
  • the body weight of the mice in the administration group did not decrease after 1 day of challenge, and the weight loss of the mice in the administration group after 2 days of challenge was also less.
  • 2G1 monoclonal antibody is a good EBOV protective antibody, which can provide complete protection in a mouse model.
  • mice Female Hartley guinea pigs (250-300g) aged 4-6 weeks were randomly assigned to the experimental group, 6 in each group.
  • the 1000 ⁇ LD 50 SUDV guinea pig adaptive strain was challenged by intraperitoneal injection in a 1 mL DMEM system. After 3 or 4 days of challenge, a single intraperitoneal injection of 2G1 monoclonal antibody, 5 mg or 2.5 mg each time.
  • Guinea pigs in the control group were treated with PBS. The clinical symptoms of disease, survival and weight change of guinea pigs were observed for 28 days.
  • the guinea pigs in the 2G1 administration group survived regardless of whether they were given a single 5 mg or 2.5 mg antibody on the 3rd or 4th day after the SUDV challenge. (See Figure 25).
  • the body weight of guinea pigs in the 2G1 administration group did not decrease significantly, while the body weight of the control group continued to decrease and eventually all died (see Figure 26).
  • 2G1 monoclonal antibody is a good protective antibody for SUDV, which can provide complete protection in the guinea pig model. This result illustrates the good protective activity of 2G1 monoclonal antibody against SUDV.
  • the monoclonal antibody provided by the invention can be prepared on a large-scale industrialization in the pharmaceutical field, and has industrial applicability.
  • tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agttgagccc 300

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Abstract

本发明公开了一种抗埃博拉病毒糖蛋白GP2亚基的单克隆抗体2G1，其与EBOV GP、BDBV GP、SUDV GP和RESTV GP具有结合活性，能发挥中和作用。

Description

一种广谱中和多种埃博拉病毒的单克隆抗体2G1及应用 技术领域
本发明公开了一种抗体，属于多肽技术领域。
 背景技术
埃博拉病毒（Ebola virus，EBOV）能在人类和非人灵长类体内引发急性严重出血热病，是迄今发现的致死率最高的病毒之一，致死率高达90%，可直接通过接触传播，具有极强的传染性和致死率。埃博拉病毒包膜表面上的糖蛋白（Glycoprotein，GP）刺突几乎介导了病毒进入细胞的所有环节，因此埃博拉GP是一个重要的病毒中和抗体作用靶点。埃博拉病毒GP基因被加工成两种蛋白，一种是分泌的非结构型GP（secreted glycoprotein，sGP）；另一种是结构型GP。埃博拉GP首先作为一个多肽被合成，接着被Furin酶酶切成为GP1（1-501位氨基酸）和GP2（502-676位氨基酸）亚基，两个亚基通过二硫键相连接，形成的三聚体由GP2内部的跨膜区固定在于包膜表面。GP1包含黏蛋白（Mucin）、聚糖帽（Glycan cap）、头部（Head）、基部（Base）等结构域。Furin酶切后形成的GP结构并不能直接激发埃博拉病毒与宿主细胞膜融合过程的发生。埃博拉病毒进入体内后，与细胞膜表面受体结合，但是这些宿主细胞膜表面的粘附因子，并不是真正的埃博拉病毒发生膜融合进入宿主细胞的受体。病毒结合于细胞表面粘附因子后，由网格蛋白介导胞吞和胞饮，发生初级和次级核内体的运输。GP在核内体中被组蛋白酶B和组蛋白酶L酶切，去除GP1上包括黏蛋白和聚糖帽在内的60%的氨基酸，形成激活态GP（primed GP, GPcl），此时激活了决定性的膜融合过程的发生。GPcl与内体膜蛋白Niemann-Pick C1（NPC1）结合发生膜融合，从而使病毒RNA进入胞质完成病毒基因组复制和转录，新病毒蛋白合成后组装成子代病毒颗粒，并从宿主细胞表面出芽。内体膜蛋白NPC1是EBOV感染的必不可少的宿主感染因子。
埃博拉病毒被美国NIH和CDC列为A类生物剂/生物恐怖剂，将其归为对国家安全和公众健康存在重大隐患和风险的病原体之一，目前仍没有获批的预防或治疗埃博拉病毒的药物。1967年埃博拉病毒被发现后，共经历了13次较大规模的传播。2014年西非爆发了历史上最大规模、最难控制的埃博拉疫情，经鉴定为扎伊尔型埃博拉病毒，此次疫情造成1万多例死亡和2.5万多例感染（根据WHO 2016年2月17日发布的埃博拉疫情报告），疫情首次传播到非洲大陆以外，在世界范围内引起了极大的恐慌。公众和当局的高度关注快速推进了埃博拉疫苗和抗病毒药物的研究。
国内外迅速开展了几个有前景的埃博拉疫苗临床试验：大猩猩腺病毒3型载体疫苗、腺病毒5型载体疫苗、水疱性口炎病毒载体疫苗等。不可否认地，发展有效的预防类药物是一个重要的目标，并且预防措施被认为可以挽救大量的生命，然而疫苗在某些情况下难以发挥作用，比如： 1）宿主个体可能存在差异（如年长者、年幼者及免疫受损者的免疫能力薄弱）；2）疫苗长时间免疫后免疫效果逐渐减弱；3）疫苗激发宿主的免疫无法抵抗高剂量的埃博拉病毒暴露；4）大部分病人仅在出现埃博拉病毒感染信号后才寻求药物帮助，疫苗在短暂的治疗窗口期内难以激发宿主有效的免疫应答。因此，在开发疫苗的进程中埃博拉病毒病治疗药物的研发同样不容忽视。目前埃博拉病毒中和抗体可能通过三种途径发挥保护作用：1）阻断GP被组织蛋白酶酶切水解；2）阻断激活态GP与NPC1受体的结合；3）影响与NPC1受体结合后到膜融合前GP的变构过程。其中，前两种途径已有文献证实，第三种途径为部分文献的推测，尚无直接证据。
尽管目前没有批准的埃博拉病毒病治疗药物，但是有一些实验型抗埃博拉病毒药物正在研究当中，包括小干扰RNA、反义寡核苷酸、核苷酸类似物、抗体等。2018年8月，埃博拉疫情在刚果民主共和国再一次爆发，截止到2019年4月7日，造成1154人感染，731人死亡。在此次疫情爆发后，WHO批准了5种处于研究阶段的药物，可以被用于应急治疗埃博拉病毒病，其中包括3种抗体药物（Zmapp、REGN3470-3471-3479和mAb114）和2种小分子药物（Remdesivir和Favipiravir）。其中，由三株单克隆抗体混合而成的抗体药物“Zmapp”及其优化株“MIL77”在2014年西非埃博拉疫情爆发期间，成功挽救了多人的生命，其效果和治疗窗口期长度远超于其他种类的抗病毒药物，大大提高了药物安全性和患者治愈率，给了人们极大的鼓舞。应用抗体药物治疗丝状病毒病引起了世界范围内的关注，成为埃博拉病毒病治疗药物研究领域的热点。
Zmapp是由烟草系统表达的三株人鼠嵌合单抗（c2G4、c4G7、c13C6）组成的鸡尾酒治疗策略，其中c2G4（表位为C511，N550，G553，C556）和c4G7（表位为C511，D552，C556）结合于GP2亚基，表位存在交叠；c13C6（表位为T270，K272）以近似垂直的角度结合于聚糖帽区域。MIL77是优化于Zmapp的抗体组合，MIL77-1/-2/-3分别含有c2G4、c4G7和c13C6的可变区，其骨架区经人源化改造后表达于CHO细胞。MIL77-1和ML77-3两株抗体的鸡尾酒组合疗法在感染72小时后能够对非人灵长类起到100%的保护活性。
 
 技术问题
然而，WHO批准应急使用的三种埃博拉病毒抗体药物均仅靶向针对扎伊尔型埃博拉病毒（ZEBOV）。爆发的埃博拉疫情中约有35%是由苏丹型埃博拉病毒（SUDV）和本迪布焦型埃博拉病毒（BDBV）等其他型埃博拉病毒导致。相关文献报道了一些能够广谱中和多种型埃博拉病毒的单抗，为埃博拉病毒病的治疗带来了新的启示。研发具有独特抗原表位、能够广谱中和多种埃博拉病毒的单抗成为本领域的一大技术需求。因为如果具有了针对保守表位、更广谱、保护活性更好的抗埃博拉病毒单克隆抗体，对于埃博拉病毒病的治疗方案就有了更优、更多的选择。因此本发明的目的就是提供一种能够针对独特抗原表位的广谱抗埃博拉病毒的单克隆抗体，进而提供其在制备埃博拉病毒病治疗药物中的应用。。
 技术解决方案
基于上述目的，本发明首先提供了一种广谱抗埃博拉病毒单克隆抗体2G1，所述抗体轻链可变区的CDR1、CDR2和CDR3的氨基酸序列分别如SEQ ID NO.1的第27-32、50-52和89-96位氨基酸序列所示，所述抗体重链可变区的CDR1、CDR2和CDR3的氨基酸序列分别如SEQ ID NO.5的第26-33、51-58和97-113位氨基酸序列所示。
在一个优选的实施方案中，所述抗体轻链可变区的氨基酸序列如SEQ ID NO.1所示，所述抗体重链可变区的氨基酸序列如SEQ ID NO.5所示。
在一个更为优选的实施方案中，所述抗体轻链恒定区的氨基酸序列如SEQ ID NO.3所示，所述抗体重链恒定的氨基酸序列如SEQ ID NO.7所示。
其次，本发明还提供了一种上述单克隆抗体轻链和重链的基因编码序列，所述抗体的轻链可变区的基因编码序列由SEQ ID NO.2所示，所述抗体的重链可变区的基因编码序列由SEQ ID NO.6所示。
在一个优选的实施方案中，所述抗体轻链恒定区的基因编码序列由SEQ ID NO.4所示，所述抗体重链恒定区的基因编码序列由SEQ ID NO.8所示。
再次，本发明提供了一种能够表达上述编码单克隆抗体重链和/或轻链的基因编码序列的功能元件。
在一个优选的实施方案中，所述功能元件为线性表达框。
又次，本发明提供了一种含有上述线性表达框的宿主细胞，所述细胞为293T细胞。
最后，本发明提供了上述的单克隆抗体在制备埃博拉病毒病治疗药物中的应用。
在一个优选的实施方案中，所述单克隆抗体作为埃博拉病毒病鸡尾酒疗法制剂中的组分使用。
 有益效果
本发明提供的抗埃博拉病毒包膜糖蛋白的单克隆抗体2G1具有独特的CDR区，其与GPdMucin具有亚纳摩尔级别的优异亲和力，结合常数ka=5.11×10 4（1/Ms），解离常数kd=3.75×10 -5（1/s），亲和力常数为KD=7.34×10 -10（M）。 在抗原结合能力上显示了优异的广谱性，与EBOV GP、BDBV GP、SUDV GP和RESTV GP具有良好的结合活性，EC 50值分别为0.0059 µg/mL、0.0075 µg/mL、0.0048 µg/mL和0.026 µg/mL。与对照抗体相比，2G1在体外可有效地中和EBOV、BDBV和SUDV假病毒。2G1中和活性随着抗体浓度的升高而增强，在1 μg/mL的浓度下即可对三种埃博拉假病毒实现近100%的保护。对EBOV-eGFP-Mayinga真病毒，2G1单抗表现出比对照抗体更好的体外中和活性。体外中和实验表明2G1是一株良好的埃博拉病毒广谱中和单抗。竞争结合实验结果说明2G1的结合表位不同于这些对照抗体，提示2G1具有与其他中和抗体组成鸡尾酒组合疗法的潜力。
稳定性试验表明，在四种不同pH的环境中，2G1与GPdM的结合活性基本无变化，证明2G1与GP的结合稳定，不受次级内体低pH环境的影响，为其发挥中和作用提供了基础。
动物攻毒保护实验显示，2G1治疗组可以给予被EBOV（Mayinga）病毒株攻击的小鼠以及SUDV攻击的豚鼠以完全保护。
本发明提供的抗体还具有独特的作用位点，不同于现有技术中已经出现的抗埃博拉病毒包膜糖蛋白单克隆抗体的作用位点，提示2G1具有与其他中和抗体组成鸡尾酒组合疗法的潜力。
 附图说明
图1. 结合活性测定筛选血清样本曲线图；
图2. 中和活性测定筛选血清样本曲线图；
图3.流式分选记忆B细胞结果图；
图4.巢式PCR扩增抗体基因的电泳鉴定图谱；
图5. 线性表达框的拼接顺序示意图；
图6.抗体结合力筛选示意图；
图7. 各抗体与抗原结合的广谱性对照分析图；
图8. 2G1与不同GP抗原的结合活性曲线图；
图9. 2G1和GPdMucin的亲和力进行测定曲线；
图10. EBOV-eGFP活病毒评价2G1中和活性图；
图11. 2G1与不同假病毒的中和活性曲线图；
图12. 截短型GP构建示意图；
图13. 2G1与各截短型抗原的结合活性曲线图（1）；
图14. 2G1与各截短型抗原的结合活性曲线图（2）；
图15. 抗体之间的竞争结合值示意图；
图16. ELISA检测重配抗体及野生型2G1与GP的结合活性曲线图；
图17. Discovery Studio和LigPlot+软件预测分析示意图（一）；
图18. Discovery Studio和LigPlot+软件预测分析示意图（二）；
图19. VH/VK-IGL与VH/VK-WT氨基酸序列比对图；
图20.突变株与野生型2G1结合活性与中和活性对照图；
图21. 2G1与GP在不同pH下的结合活性曲线图；
图22. 抗体对GP CL与NPC1-C的结合阻断实验曲线图；
图23. 2G1阻断GPdmucin被thermolysin酶切实验图谱；
图24. 2G1单抗对EBOV（Mayinga）病毒株攻毒小鼠的保护曲线图；
图25. 2G1单抗对SUDV病毒株攻毒豚鼠的存活率变化曲线图；
图26. 2G1单抗对SUDV病毒株攻毒豚鼠的体重变化曲线图。
 本发明的最佳实施方式
下面结合具体实施例来进一步描述本发明，本发明的优点和特点将会随着描述而更为清楚。但这些实施例仅是范例性的一些优选实施方式，并不对本发明的保护范围构成任何限制。
 本发明的实施方式
实施例1： 抗体制备
1.     血液样品的采集
在获得知情同意书后，采集重组埃博拉疫苗临床试验受试者第二次免疫28天后血液样品5 mL，用于后续实验。
2．血清结合活性测定
1)     实验前一天96孔酶联板，包被1 µg/mL的EBOV GPdTM（购自IBT Bioservices），每孔100 µL，4℃孵育过夜。
2)     实验当天用洗板机（BIO-TEK，405_LS）洗5次。每孔加入100 µL封闭液（含2% BSA的PBST），37℃孵育1 h。
3)     洗板机洗5次。P024号血清样品以首孔1:100稀释度，三倍比梯度稀释，每孔100 µL，37℃孵育1 h。
4)     洗板机洗5次。将HPR标记的羊抗人IgG二抗（Abcam，ab97225）以1:10000用稀释液进行稀释，每孔100 µL，37℃孵育1 h。
5)     洗板机洗5次。每孔加入100 µL的TMB单组份显色液（Solarbio），室温避光显色6 min，之后每孔加入50 µL终止液终止反应。
6)     用SPECTRA MAX 190酶标仪检测450-630nm处的OD值。
如图1所示，024号血清样品二免后28天（S024-V10）相较免前（S024-V0）对EBOV GP的结合活性显著增强，抗体滴度在10 6以上。
3.血清中和活性测定
1)  包装假病毒：pDC316-EBOV GP和pNL4-3.Luc.R —E —共转染293T细胞，24 h后收取细胞上清。通过Luciferase Assay System（Promega，E1501）测定假病毒滴度，分装保存于-80℃。
2)  血清样品首孔稀释度1:5，以3倍比梯度稀释，每孔体积50 μL。
3)  每孔加入50 μL用培养基稀释好的假病毒，将血清-假病毒混合液于37℃孵育1 h。
4)  293T细胞计数，2×10 5 cells/mL，每孔加入100 μL，将96孔细胞培养板放入37℃恒温箱中培养36 h。
5)  取出细胞培养板，小心吸出培养液弃掉。各孔加入100 µL细胞裂解液（Promega，E1500），震荡机上350 rpm震荡15 min。
6)  3000 rpm室温离心10 min。
7)  吸取20 µL裂解后细胞上清，采用Luciferase Assay System在GLOMAX 96 Microplate Luminometer（Promega）仪器中读取荧光值，计算抗体对细胞的保护率。
如图2所示，相较于免前血清，024号样品免后血清（IC 50=1277）对HIV-EBOV GP-Luc假病毒呈现出良好的中和活性。因此，024号样品将用于下一步流式分选。
4.   FITC标记蛋白GPdM
需要通过荧光标记的抗原来分 选特异的记忆B细胞，FITC标记截短型抗原蛋白GPdM方法如下：
1)     Fluorescein Isothiocyanate_FITC （SIGMA, F4274）溶于DMSO，浓度为20 mg/mL。
2)     取100 μL GPdM（3.3 mg/mL），加缓冲液（pH9.6碳酸盐缓冲液）至400 μL。
3)     加8 μL FITC至GPdM溶液中，4℃避光孵育3 h。
4)     用50 kD的离心浓缩管对溶液加PBS换液，直到滤过液为透明无色。将标记好的蛋白用锡箔纸包好，4℃存放待用。
5.   流式分选记忆B细胞
将采集的血样利用Ficoll密度梯度离心法分离PBMC，过程如下：
1)     取新鲜抗凝全血，EDTA抗凝。用等体积PBS稀释全血。
2)     在离心管中加入一定体积的分离液，将稀释后的血样平铺到分离液液面上方，保持两液面界面清晰。分离液、抗凝未经稀释全血、PBS（或生理盐水）体积为1:1:1。
3)     配平，室温，水平转子800 g，加速度3 acc，离心30 min。
4)     离心结束后，管底是红细胞，中间层是分离液，最上层是血浆/组织匀浆层，血浆层与分离液层之间是一层薄且较致密的白膜，即：单个核细胞（包括淋巴细胞和单核细胞）层。小心吸取白膜层到另一离心管中。
5)     用PBS 稀释到一定体积，颠倒混匀。室温，水平转子300 g，离心10 min，弃上清。重复洗涤2 次。
6)     用PBS将淋巴细胞重悬备用。
7）将用来分选的细胞计数，取1×10 6个细胞，体积为100 μL，加入下表推荐量的6种荧光染料，4℃避光孵育1 h。
标记 荧光 公司/货号 体积 （每1×10 6 cells）
抗原 FITC SIGMA, F4274 2 μL
IgG PE BD, 555787 40 μL
CD19 APC-AF 700 Beckman，IM2470 10 μL
CD3 PerCP BD，552851 20 μL
CD38 PerCP Biolegend，303520 10 μL
CD27 PC7 Beckman，A54823 10 μL
8）使用含2% FBS的PBS重复洗涤2-3次，400 μL FPBS重悬，用40 μm细胞筛去除细胞团，4℃避光保存供分选。
9）使用细胞分选仪（SONY，SH800S）分选GP特异的单个记忆B细胞。分选策略为：IgG+/CD3-/CD38-/ CD19+/CD27+/ GPdM+，直接将单个浆细胞分选至96孔板中，96孔板中每孔含有5U RNA酶抑制剂及19.8 μL去RNA酶的水中，-80℃保存。图3是细胞分选结果图。图中方框圈出部分的细胞特征为IgG+/CD3-/CD38-/ CD19+/CD27+/ GPdM+，即期望的GP特异性记忆B细胞。
6.     单细胞PCR扩增全人源单抗
1)     反转录PCR
分选得到80个GPdM特异的记忆B细胞，向每个反应体系中同时加入以下全部的针对重链H、κ轻链、λ轻链各亚型的特异引物（引物序列见表1）。
表1 反转录PCR引物
  引物名称 序列
H 5' L-VH 1 ACAGGTGCCCACTCCCAGGTGCAG
  5' L-VH 3 AAGGTGTCCAGTGTGARGTGCAG
  5' L-VH 4/6 CCCAGATGGGTCCTGTCCCAGGTGCAG
  5' L-VH 5 CAAGGAGTCTGTTCCGAGGTGCAG
  HuIgG-const-anti TCTTGTCCACCTTGGTGTTGCT
  3' Cμ CH1 GGGAATTCTCACAGGAGACGA
κ 5' L Vk 1/2 ATGAGGSTCCCYGCTCAGCTGCTGG
  5' L Vk 3 CTCTTCCTCCTGCTACTCTGGCTCCCAG
  5' L Vk 4 ATTTCTCTGTTGCTCTGGATCTCTG
  3' Ck 543–566 GTTTCTCGTAGTCTGCTTTGCTCA
λ 5' L Vλ 1 GGTCCTGGGCCCAGTCTGTGCTG
  5' L Vλ 2 GGTCCTGGGCCCAGTCTGCCCTG
  5' L Vλ 3 GCTCTGTGACCTCCTATGAGCTG
  5' L Vλ 4/5 GGTCTCTCTCSCAGCYTGTGCTG
  5' L Vλ 6 GTTCTTGGGCCAATTTTATGCTG
  5' L Vλ 7 GGTCCAATTCYCAGGCTGTGGTG
  5' L Vλ 8 GAGTGGATTCTCAGACTGTGGTG
  3' Cλ CACCAGTGTGGCCTTGTTGGCTTG
PCR反应体系中包含：5×缓冲液6 μL、dNTP 1.2 μL、反转录酶1.2 μL、引物如上、模板为单细胞，水补齐至30 μL。
PCR反应条件为：50℃反转录30 min，95℃预变性15 min，接着95℃ 40 s，55℃ 30 s，72℃ 1 min，40个循环，最后72℃延伸10 min。
2）巢式PCR
以反转录产物为模板，分别进行3次巢式PCR反应扩增H、κ、λ（引物序列见表2），具体过程如下：
表2 巢式PCR引物
  引物名称 序列
H: VH3a-sense SARGTGCAGCTCGTGGAG
  VH3b-sense GAGGTGCAGCTGTTGGAG
  MuD GGAATTCTCACAGGAGACGA
  PW-Cgamma AGTAGTCCTTGACCAGGCAGCCCAG
Қ： 5' Pan Vk ATGACCCAGWCTCCABYCWCCCTG
  3' Ck 494–516 GTGCTGTCCTTGCTGTCCTGCT
λ 5' AgeI Vλ 1 CTGCTACCGGTTCCTGGGCCCAGTCTGTGCTGACKCAG
  5' AgeI Vλ 2 CTGCTACCGGTTCCTGGGCCCAGTCTGCCCTGACTCAG
  5' AgeI Vλ 3 CTGCTACCGGTTCTGTGACCTCCTATGAGCTGACWCAG
  5' AgeI Vλ 4/5 CTGCTACCGGTTCTCTCTCSCAGCYTGTGCTGACTCA
  5' AgeI Vλ 6 CTGCTACCGGTTCTTGGGCCAATTTTATGCTGACTCAG
  5' AgeI Vλ 7/8 CTGCTACCGGTTCCAATTCYCAGRCTGTGGTGACYCAG
  3' XhoI Cλ CTCCTCACTCGAGGGYGGGAACAGAGTG
PCR反应体系中包含：10×缓冲液2.5 μL、10 mM dNTP 0.5 μL、DNA聚合酶 0.25 μL、引物如上、模板为反转录产物1 μL、水补齐至25 μL。
PCR反应条件为：94℃预变性4 min，接着94℃ 30 s，57℃ 30 s，72℃ 45 min，40个循环，最后72℃延伸10 min。
3）琼脂糖凝胶电泳
一个单细胞中重链和轻链基因均扩增成功的克隆，被认为是配对成功的克隆。取5 μL巢式PCR扩增产物经1%琼脂糖凝胶电泳后，取配对的阳性克隆进行测序，测序获得的抗体可变区序列用Vector NTI软件及IMGT网站进行分析，并进行抗体蛋白表达和功能验证。图4是对H、κ、λ三种链基因的巢式PCR扩增后琼脂糖凝胶电泳的鉴定图谱。只有当重链和轻链可变区基因从同一个记忆B细胞中扩增获得时，认为是自然配对的抗体基因，并将对应巢式PCR产物测序做进一步研究。
7.     线性表达框表达抗体
通过上述反转录反应，单细胞克隆中获得了25个配对的抗体序列，鉴于传统的克隆表达方法费时费力，构建线性表达框的方法则可以快速表达抗体。该方法的基本原理是直接通过重叠延伸PCR将启动子序列（GenBANK登记号：X03922.1）、抗体前导肽的编码序列、抗体可变区（从单细胞中扩增获得）、抗体恒定区（生工生物合成）、多聚A尾（GenBANK登记号：X03896.1 ）连接起来，将该线性形式的DNA转染进入细胞中进行抗体表达。具体过程如下：
1)     以pSec Tag2（Invitrogen）为模板，扩增启动子-前导序列片段、多聚A尾片段。
扩增启动子-前导序列片段的PCR反应体系中包含：模板质粒pSec Tag2（Invitrogen）1 ng，10×缓冲液5 μL、10 mM dNTP 1 μL、DNA聚合酶 0.5 μL、引物5'CMV-FORWARD （CGATGTACGGGCCAGATATACGCGTTG）、引物3'leader-sequence （GTCACCAGTGGAACCTGGAACCCA）、水补齐至50 μL。
扩增多聚A尾片段的PCR反应体系中包含：模板质粒pSec Tag2（Invitrogen）1 ng，10×缓冲液5 μL、10 mM dNTP 1μL、DNA聚合酶 0.5 μL、引物5'POLY(A) （GCCTCGACTGTGCCTTCTAGTTGC）、引物3'POLY(A) （TCCCCAGCATGCCTGCTATTGTCT）、水补齐至50 μL。
PCR反应条件为：94℃预变性4 min，接着94℃ 30 s，60℃ 30 s，72℃ 1 min，30个循环，最后72℃延伸10 min。
2)     扩增抗体恒定区
H链恒定区PCR体系中包含：重链恒定区模板10 ng、10×缓冲液5 μL、10 mM dNTP 1 μL、DNA聚合酶 0.5 μL、引物5'CH（ACCAAGGGCCCAT CGGTCTTCCCC）、引物3'CH（GCAACTAGAAGGCACAGTCGAGGCTTT ACCCGGAGACAGGGA）、水补齐至50 μL。
κ链恒定区PCR体系中包含：κ链恒定区模板10 ng、10×缓冲液5 μL、10 mM dNTP 1 μL、DNA聚合酶 0.5 μL、引物5'Cκ（ACTGTGGCTGCACCA TCTGTCTTC）、引物3'Cκ（GCAACTAGAAGGCACAGTCGAGGCACACT CTCCCCTGTTGAAGCT）、水补齐至50 μL。
λ链恒定区PCR体系中包含：λ链恒定区模板10 ng、10×缓冲液5 μL、10 mM dNTP 1 μL、DNA聚合酶 0.5 μL、引物5'Cλ（GAGGAGCTTCAAGCC AACAAGGCCACA）、引物3'Cλ（GCAACTAGAAGGCACAGTCGAGGCT
GAACATTCTGTAGGGGCCAC）、水补齐至50 μL。
PCR反应条件为：94℃预变性4 min，接着94℃ 30 s，60℃ 60 s，72℃ 3 min，30个循环，最后72℃延伸10 min。
3)     扩增抗体可变区
PCR体系中包含：模板为反转录PCR产物10 ng，10×缓冲液5 μL、10 mM dNTP 1 μL、DNA聚合酶 0.5 μL、引物如表3中所示（将重链和轻链引物分别混合后加入体系中）、水补齐至50 μL。
PCR反应条件为：94℃预变性4 min，接着94℃ 30 s，60℃ 30 s，72℃ 3 min，30个循环，最后72℃延伸10 min。
表3. 线性表达框构建PCR引物
Figure 176281dest_path_image001
 
4)     PCR产物回收纯化：将以上PCR产物经1%的琼脂糖凝胶电泳后，切胶并使用OMEGA公司回收试剂盒回收。
5)     分别扩增重链和轻链的线性表达框
线性表达框的拼接顺序示意图如图5所示。图5中，P CMV为启动子；Ig L H/L分别为重轻链前导序列；V H/L分别为重轻链可变区；IgG 1 C H/L分别为重轻链恒定区；Poly A为多聚A尾； EcoR I和 Not I为限制性内切酶酶切位点。
PCR反应体系中包括：
模板：纯化后的启动子-前导序列片段10 ng、重链/轻链可变区片段10 ng、重链/轻链恒定区片段10 ng、多聚A尾片段10 ng，10×缓冲液2.5 μL、10 mM dNTP 0.5 μL、DNA聚合酶 0.25 μL、引物5'CMV-FORWARD （CGATGTACGGGCCAGATATACGCGTTG）和3'POLY(A) （TCCCCAGCATG
CCTGCTATTGTCT）、水补齐至25 μL。
PCR反应条件为：94℃预变性4 min，接着94℃ 30 s，60℃ 30 s，72℃ 3 min，30个循环，最后72℃延伸10 min。
6)     PCR产物回收纯化：PCR产物直接用OMEGA公司回收试剂盒回收。
7)     DNA定量：用Nano（GE Healthcare）对PCR回收产物进行定量。
8)     细胞接种：将293T细胞以2×10 5/mL接种于24孔细胞培养板中，在含有5% CO 2的细胞温箱中，37℃培养过夜。
9) 细胞共转染：次日，向200 μL无血清的MEM培养基中，加入构建成功的重链和轻链线性表达框PCR产物各1 μg，混匀后加入4 μL转染试剂Turbofect（Thermo Scientific, R0531），共同孵育15-20 min后逐滴加至过夜培养的293T细胞培养孔中。在含有5% CO 2的细胞温箱中，37℃培养48 h后收细胞培养上清备用。
8.     ELISA筛选具有结合活性的抗体
1)     实验前一天96孔酶联板，包被1 µg/mL的EBOV GP，每孔100 µL包被。将包被的酶联板放入湿盒，4℃过夜。
2)     实验当天用洗板机（BIO-TEK，405_LS）洗5次。
3)     每孔加入100 µL封闭液，室温下放置1小时。
4)     洗板机洗5次。
5)     加入100 µL的转染细胞培养上清，室温静置1小时。
6)     洗板机洗5次。
7)     将HPR标记的羊抗人IgG二抗（Abcam，ab97225）以1:10000用稀释液进行稀释，每孔100 µL加入到ELISA板对应孔中，室温孵育1小时；
8)     洗板机洗5次。
9)     每孔加入100 µL的TMB单组份显色液，显色6 min，室温避光，之后每孔加入50 µL终止液终止反应；
10) 用酶标仪上检测450-630nm处的OD值，保存记录原始数据。
结果：将25株单抗进行筛选，并对埃博拉GP的结合抗体进行特异性鉴定。有10株与埃博拉GP能够特异结合，包括2A1、2G1、2F4、2E5、2G5、2C6、2A7、2C7、2H7、2A10。图6显示了25株单抗的结合活性对照柱图。
9.     ELISA筛选具有广谱结合活性的抗体
为了获得具有广谱结合多种埃博拉病毒的抗体，对结合抗体的广谱性进行分析。实验前一天96孔酶联板，包被1 µg/mL的EBOV GP、BDBV GP、SUDV GP和RESTV GP。其余方法同8。
检测结果如图7所示，10株EBOV GP结合抗体中，7株抗体具有广谱结合活性，能与两种或两种以上的埃博拉病毒GP结合。2A1、2G5、2C6能与EBOV GP和BDBV GP结合；2G1能与EBOV GP、BDBV GP、SUDV GP和RESTV GP结合；2F4能与EBOV GP、BDBV GP和SUDV GP结合；2E5、2A7能与EBOV GP和RESTV GP结合；2C7、2H7和2A10仅能与EBOV GP结合，不具有广谱性。其中，2G1具有最好的广谱性，能与我们有的4种埃博拉病毒GP结合，且结合活性良好。
10.  2G1抗体序列说明
2G1具有良好的广谱结合活性，其序列描述如下：
轻链可变区的氨基酸序列如SEQ ID NO.1所示，轻链可变区的CDR1、CDR2和CDR3的氨基酸序列分别如SEQ ID NO.1的第27-32、50-52和89-96位氨基酸序列所示，轻链可变区的基因编码序列由SEQ ID NO：2所示，轻链恒定区的氨基酸序列如SEQ ID NO.3所示，轻链恒定区的基因编码序列由SEQ ID NO：4所示。
重链可变区的氨基酸序列如SEQ ID NO.5所示，重链可变区的CDR1、CDR2和CDR3的氨基酸序列分别如SEQ ID NO.5的第26-33、51-58和97-113位氨基酸序列所示，重链可变区的基因编码序列由SEQ ID NO.6所示，重链恒定的氨基酸序列如SEQ ID NO.7所示，重链恒定区的基因编码序列由SEQ ID NO.8所示。
11.  表达质粒构建与抗体制备
对2G1构建表达质粒，进行单抗的表达制备。方法如下：
1)     将2G1H和2G1K线性表达框全长基因用 EcoRI（NEB，R3101）和 NotI（NEB，R3189）双酶切，连接至pcDNA3.4表达质粒。
2)     取pcDNA3.4-2G1H和pcDNA3.4-2G1K各15 μg，转染至30 mL Expi293体系（Life，A14524）中，125 rpm，5% CO 2培养72 h。
3)     3000×g，离心10min收取表达上清，经0.22 μm针头滤器抽滤后，采用rProtein A亲和纯化。
4)     用PBS对收集的抗体进行换液，然后用BCA蛋白定量试剂盒（Thermo Scientific，23225）测定抗体浓度。
 
实施例2. ELISA检测抗体结合活性
1)     实验前一天96孔酶联板，包被1 µg/mL的EBOV GP、BDBV GP、SUDV GP和RESTV GP，每孔100 µL包被。将包被的酶联板放入湿盒，4℃过夜。
2)     实验当天用洗板机洗5次。
3)     每孔加入100 µL封闭液，室温下放置1小时。
4)     洗板机洗5次。
5)     首孔加入150 µL浓度为10 µg/mL的2G1单抗，其余孔加入100 µL的稀释液。从首孔吸出50 µL加入到次孔，以此类推，按1:3梯度稀释每孔终体积为100 µL。室温静置1小时。
6)     洗板机洗5次。
7)     将HPR标记的羊抗人IgG二抗以1:10000用稀释液进行稀释，每孔100 µL加入到ELISA板对应孔中，室温孵育1小时。
8)     洗板机洗5次。
9)     每孔加入100 µL的TMB单组份显色液，显色6 min，室温避光，之后每孔加入50 µL终止液终止反应；
10) 用酶标仪上检测450-630nm处的OD值，保存记录原始数据。
结果：2G1单抗与EBOV GP、BDBV GP、SUDV GP和RESTV GP具有良好的结合活性，EC 50值分别为0.0059 µg/mL、0.0075 µg/mL、0.0048 µg/mL和0.026 µg/mL（检测结果参见图8）。
 
实施例3.亲和力测定     
采用BIACORE 3000对2G1单抗和GPdMucin的亲和力进行测定。先将抗人IgG Fc抗体共价耦联到CM5传感芯片上。2G1单抗作为配体，浓度1 μg/mL，以10 μL/min的流速结合30s。HEPES缓冲液30 μL/min的流速清洗进样针和微流控系统 (IFC)。GPdMucin作为分析物，浓度配成800/400/200/100/50/25/12.5 nM共7个浓度梯度。每个浓度的GPdMucin以30 μL/min的流速结合6 min，然后解离30 min。每次更换分析物浓度前对芯片进行再生。最后生成的数据曲线通过BIAevaluation软件分析计算亲和力（参见图9）。
软件分析计算出2G1单抗的结合常数ka=5.11×10 4（1/Ms），解离常数kd=3.75×10 -5（1/s），亲和力常数为KD=7.34×10 -10（M）。2G1单抗与GPdMucin的亲和力较好，在亚纳摩尔级别。
 
实施例4.HIV-EBOV/BDBV/SUDV-Luc假病毒中和实验评价2G1广谱中和活性
包装HIV骨架的EBOV、BDBV和SUDV假病毒，在体外评价2G1的中和活性。评价方法如下：
1) 用DMEM培养基稀释2G1单抗，96孔细胞培养板首孔加入75 μL浓度为100 μg/mL的抗体稀释液，其余孔加入50 μL的DMEM培养基。
2) 从首孔吸取25 μL液体加入次孔，混匀，以此类推，按1:3倍比稀释，每孔终体积为50 μL。
3) 将假病毒按1:5用DMEM培养基稀释（使对照孔荧光素读值控制在20000~100000之间），加入至各抗体孔，每孔50 μL。混匀，37℃孵育1 h。
4) 293T细胞计数，2×10 5 cells/mL，每孔加入100 μL。
5) 将96孔细胞培养板放入37℃恒温箱中培养36~48 h
6) 取出细胞培养板，小心吸出培养液弃掉。各孔加入100 µL细胞裂解液，震荡机上350 rpm震荡15 min。
7) 3000 rpm室温离心10 min。
8) 将荧光素酶检测系统（Promega，E1501）的检测底物冻干剂和检测缓冲液混匀后，充盈GLOMAX 96 MICROPLATE LUMINOMETER（Promega）检测管路。
9) 吸取20 µL裂解上清读值，读取荧光值，计算抗体对细胞的保护率。
结果： 与对照抗体相比，2G1在体外可有效地中和EBOV、BDBV和SUDV假病毒。2G1中和活性随着抗体浓度的升高而增强，在1 μg/mL的浓度下即可对三种埃博拉假病毒实现近100%的保护。（检测结果参见图11）。
 
实施例5. EBOV-eGFP活病毒评价2G1中和活性    
接种Vero E6细胞至96孔组织培养板（Corning）中，感染当天生长至85-90%。将100 PFU的EBOV-eGFP-Mayinga与单抗2G1、CA45、MIL77-1 在空白DMEM中37℃预孵育1 h。各单抗首孔浓度100 μg/mL，3倍比梯度稀释。将病毒抗体混合物加入至Vero E6细胞中，37℃，5% CO 2条件下感染1 h，然后用含12% FBS的DMEM更换培养基。将培养板孵育48 h后，用10% 磷酸盐缓冲液的福尔马林（Fisher）固定。使用iSpot FluoroSpot Reader System（Advanced Imaging Devices）计数荧光斑块。
在EBOV-eGFP-Mayinga真病毒中和实验中，2G1单抗的IC 50值为2. 80 μg/mL，IC 90值为3.23 μg/mL；对照抗体CA45的IC 50值为8.51 μg/mL，IC 90值为12.99 μg/mL；对照抗体MIL77-1的IC 50值为3.12 μg/mL，IC 90值为82.01 μg/mL。对EBOV-eGFP-Mayinga真病毒，2G1单抗表现出比对照抗体更好的体外中和活性（检测结果参见图10）。
 
实施例6. 截短型抗原分析2G1结合区域
根据结构和功能特点，可将GP划分为聚糖帽、黏蛋白区、受体结合区、融合环和HR区等多个区域。2G1呈现出良好的广谱中和活性，希望通过构建截短型GP来分析其大致的结合区域。除了文献中常见的GPdTM、GPdM、GP1、sGP和GPcl外，根据GP结构特点和分析目标，还设计构建了GP 33-310; 463-558、GP 33-227、GP 33-158、GP 95-295、GP 158-295和GP 227-295。GP 33-310; 463-558是在GPdM的基础上去掉了后面的七肽重复区；GP 33-227是在sGP的基础上去掉了聚糖帽；GP 33-158包含了大部分的Base区和Head区；GP 95-295相比sGP缺失了Base区；GP 158-295相比sGP基本缺失了Base区和Head区；GP 227-295包含了大部分的聚糖帽区。GPcl是在GPdM的基础上，经嗜热菌蛋白酶（thermolysin）切除聚糖帽得到的，不需构建表达质粒。各截短型GP的结构示意图如图12所示。
截短型GP分析2G1结合区域方法如下：
1)  根据示意图，从GP全长基因中扩增各截短型GP基因片段，羧基端添加His 6标签，将基因片段连接至pcDNA3.4表达质粒中。
2)  各截短型GP在Expi293系统中表达，NI-NTA纯化获得目的蛋白。
3)  ELISA分析2G1对各截短型GP的结合活性，方法同实施例2所述。
结果：2G1与GPdM结合EC 50值为0.0037 μg/mL，与GPdTM基本一致。2G1与GP1的结合EC 50值为0.8075 μg/mL，相比GPdTM和GPdM而言，结合能力降低了100多倍，提示GP2亚基对2G1的结合起着关键作用。尽管2G1与GPcl的结合最大值降低为其与GPdTM结合最大值的一半左右，但其结合EC 50值为0.019 μg/mL，结合活性较好。GPcl包含了GP2亚基和部分的GP1亚基，而sGP包含了GPcl在GP1亚基上的部分，2G1与sGP基本不结合，进一步说明GP2对2G1结合的重要性。2G1与各截短型抗原的结合活性检测见图13-图14。
 
实施例7. 竞争实验                  
采用竞争结合ELISA分析2G1的抗原作用表位。用已知结合表位的MIL77-1/2/3和其余几株非中和抗体作为对照抗体，通过考察检测抗体与GP的结合是否会被同孵育的竞争抗体所阻断，来反映这些抗体的结合表位是否存在交叠。方法如下：
1)  称取生物素（Thermo Scientific，21335）4 mg，溶于2 mL超纯水中，浓度为2 mg/mL。
2)  取抗体2G1、2E5、5D7、5G11、5B12、1B3、MIL77-1、MIL77-2、MIL77-3各200 μg，体积控制在200 μL左右。
3)  以生物素比抗体为20:1的摩尔比标记抗体。将抗体和生物素混合，室温孵育1 h，用50 kD的0.5 mL离心过滤器换液，每次更换PBS约400 μL，换液3次以上。
4)  最后一次用PBS将过滤器中剩余的液体统一补齐至100 μL左右（目的是使各抗体的浓度相近），测定抗体浓度。
5)  96孔酶联板以1 μg/mL的浓度包被GPdM，4℃包被过夜。
6)  洗板机洗涤ELISA板，每孔加入100 μL封闭液，37℃孵育1 h。
7)  实验中检测抗体为生物素标记的抗体，竞争抗体为非生物素标记抗体。稀释液稀释竞争抗体至2.5 μg/mL；稀释液稀释检测抗体至25 ng/mL。
8)  洗涤96孔板。各孔加入50 μL的检测抗体和50 μL的竞争抗体，每孔终体积100 μL。
9)  洗涤，链霉亲和素抗体（Thermo Scientific，21126）以1:1000稀释，每孔加入100 μL，37℃孵育1 h。
10)           洗涤，每孔加入100 μL TMB单组份显色液，避光显色6分钟，加入50 μL终止液。
11)           读取450-630nm OD值。
结果：
竞争结合值的计算方法为：
Figure 827842dest_path_image002
竞争结合值小于30认为强竞争；大于30小于60认为弱竞争；大于60认为不竞争。各抗体自身之间很好地竞争结合，根据结果可将这些抗体分成四组。其中，结合于GP1亚基和结合于GP2亚基各两组。2G1与同结合于GP2亚基的非中和抗体5B12、中和抗体MIL77-1和MIL77-2也基本不存在竞争。竞争结合结果说明2G1的结合表位不同于这些对照抗体，提示2G1具有与其他中和抗体组成鸡尾酒组合疗法的潜力。（检测结果参见图15）。
 
实施例8.Discovery Studio和LigPlot+软件预测分析     
在Discovery Studio 4.5中使用Dock Proteins (ZDOCK) protocol进行抗原-抗体分子对接。将埃博拉GP蛋白结构（PDB ID: 5KEL）作为受体，将抗体2G1-Fab（同源建模得到）作为配体，ZDOCK刚性对接算法生成54000个对接模拟构象(pose)。结合ZRank打分算法排序，获得2000个pose。
编程计算出每个对接模拟构象的抗原-抗体接触面各项特征值。
X 3, 表位-旁位界面指数（the epitope-paratope interface index，EPII ）
X 4, 表面埋藏面积（the buried surface area）
X 5, （接触面cation-π密度）the density of the cation-π interactions
X 6, 原子密度指数（atomic density index， ADI）
将Discovery Studio 4.5给出的ZDOCK score和ZRANK score分别作为特征值 X 1和 X 2 。由下列公式（实验室前期建立并通过测试的用以筛选抗原-抗体近天然对接模拟结构的 logistic回归方程，该研究结果正在审稿）计算得到回归方程的概率值（MLR probability）如下：
Figure 528951dest_path_image003
并按MLR probability降序对2000个对接模拟构象进行重新排序，前10个pose如下。其中pose_96排列第一，且打分值远高于其它pose（参见图17的A和B）。
将pose_96借助LigPlot+软件进行分析，得到GP与2G1结合界面处的氨基酸及可能存在的相互作用（参见图17的C和图18）。GP上563-567、509-513和531等氨基酸出现在接触面上；2G1重链CDR2上的56、57及CDR3上的103、107等氨基酸出现在接触面上。
图17的C和图18中，GP模型上各氨基酸后面的(A)、(B)和(G)分别表示GP结构上的不同链，其中(A)和(B)分别为同一个单体上的GP1和GP2，(G)为另一个单体上的GP2亚基。抗体模型上氨基酸后面的(H)和(L)分别对应重链和轻链。模拟预测结果可为抗原抗体的结合模式和结合表位分析提供信息和参考，并有助于解释抗体的中和机制。
实施例 9. 抗体关键氨基酸分析   
将2G1单抗的VH/VK在IMGT网站（http://www.imgt.org/）上分析，设计合成2G1重轻链胚系基因VH/VK-IGL。
VH-IGL核酸序列：
gaggtgcagctggtggagtctgggggaggcctggtcaagcggggggggtccctgagactctcctgtgtagtctccggattcaccttcagtagctatagcatgcactgggtccgccaggctccagggaaggggctggagtgggtctcaggcattagtagtagtagtagttacatatactacgcagactcagtgaagggccgattcaccatctccagagacaacgccaagaactcactgtatctgcaaatgaatagcctgagagccgaggacacggctgtttattactgtgcgagagatatgggatattgtagtggtggtagctgccctaactttgacttctggggccagggaaccacggtcaccgtctcctca
VH-IGL氨基酸序列：
EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSSISSSSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDMGYCSGGSCPNFDFWGQGTTVTVSS
VK-IGL核酸序列：
gacatccagatgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccatcacttgccggacaagtcagagcattagcagctatttaaattggtatcagcagataccagggaaagcccctaaactcctgatctctgctgcatccaatttgcacagtggggtctcatcaaggttcagtggcagtggatctgggacacatttcactctcaccatcagcagtctgcaacctgaagattttgcaacttactactgtcaacagagttacagtacccctccgttcggccaagggaccaaggtggagatcaaa
VK-IGL氨基酸序列：
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPSFGQGTKVEIK
VH/VK-IGL与VH/VK-WT氨基酸序列比对结果见图19。
将2G1轻重链胚系基因（VH/VK-IGL）与野生型序列（VH/VK-WT）交叉配对，制备重配抗体。ELISA检测其与GP的结合活性相对野生型的变化，以分析抗体的重链与轻链在结合中的发挥的作用。
2G1-WT 、2G1-H WT/K IGL 、2G1-H IGL/K WT 和2G1-IGL 与GP的结合EC 50值分别为2.7 ng/mL、12.1 ng/mL、152.3 ng/mL和251.2 ng/mL。仅轻链重配的2G1-H WT/K IGL相对2G1-WT对GP的结合活性降低了4.4倍，而重链发生重配的2G1-IGL和2G1-H IGL/K WT分别降低55.4倍和91.4倍，表明2G1单抗的重链在与GP结合中起关键作用（参见图16）。
2G1单抗的重链对其结合活性比较重要，进一步通过丙氨酸扫描突变的方法对其CDR区上的结合关键氨基酸进行了分析。将2G1重链CDR1/2/3上的氨基酸突变成丙氨酸（丙氨酸突变成丝氨酸），制备抗2G1单抗突变株，检测这些突变株结合活性与中和活性相对于野生型的变化。
如图20所示，结果表明2G1重链CDR2区的Y57和CDR3区C103、G106、C108氨基酸对结合和中和均比较关键。这些氨基酸都出现在DS软件预测的pose_96接触界面上，其中Y57和C103更是LigPlot+软件预测分析中可能与GP上氨基酸形成氢键的两个氨基酸；而G106和C108则分别位于另一个可能与GP上氨基酸形成氢键的S107的两侧。
实施例10. 低pH下2G1与GPdM的结合活性检测  
抗体与病毒颗粒表面的GP结合后，与病毒颗粒一起进入宿主细胞。在次级内体的酸性环境（pH约5.5）中，GP被酶切水解，进而与受体NPC1-C结合，促进病毒与内体膜融合，进而释放病毒基因组。为进一步考察在2G1与GP结合活性是否受酸性环境的影响，检测了体外在pH 7.5、pH 6.5、pH 5.5和pH 4.5的环境中2G1与GP的结合活性，，ELISA方法同实施例2所述。
结果：在四种不同pH的环境中，2G1与GPdM的结合活性基本无变化。2G1与GP的结合稳定，不受次级内体低pH环境的影响，为其发挥中和作用提供了基础。（检测结果参见图21）。
 
实施例11. 受体结合阻断实验  
埃博拉病毒进入宿主细胞后，在次级内体酸性环境中GP上的聚糖帽和黏蛋白区被酶切除去，形成GP CL结构。相较GP而言，GP CL与内体表面受体NPC1-C结合的表位得以暴露出来。如果2G1单抗是通过阻断GP CL与受体的结合发挥保护活性，那么GP CL与2G1结合后，其将不能与NPC1-C结合。
GP CL制备方法：用PBS（pH 7.5）将GPdMuc的浓度调整为2 mg/mL，加入嗜热菌蛋白酶（SIGMA，T7902-25MG），酶的终浓度为0.5 mg/mL，37℃孵育1 h；加入0.5 mM的Phosphoramidon（Sigma-Aldrich，R7385）终止酶切反应；使用0.5 mL、50 kD截留分子量的的Millipore离心过滤管，对体系进行浓缩和换液；蛋白样品经0.20 µm的微量过滤器（Millex-LG，SLLGR04NL）过滤后，用Superdex 200 Increase 10/300 GL凝胶柱（GE Healthcare）纯化。
GP CL以1 μg/mL 浓度4℃包被过夜；封闭1 h；洗涤；选择MR72作为阳性对照，抗体首孔50 μg/mL，3倍比梯度稀释，37℃孵育30 min；加入NPC1-C-biotin，每孔浓度5 μg/mL，37℃孵育30 min；洗涤，加入链霉亲和素抗体，37℃孵育1 h；洗涤，显色6 min，然后终止，读取450-630nm双波长OD值。
对照抗体MR72在50 μg/mL的浓度下，可将GP CL与NPC1-C的结合阻断至30%。2G1单抗没有表现出明显的阻断作用，说明其不通过阻断GP CL与受体的结合发挥保护作用（检测结果参见图22）。由于已知的抗体通过或部分通过该途径发挥保护作用，本实施例排除了2G1单抗可能的一种保护机制。
 
实施例12. 酶切阻断实验 ：
取Expi293表达的2 μg的GPdmucin-His 6分别与0.5 μg、5 μg和50 μg的IgG在20 μL PBS中混匀，37℃孵育30 min。加入1 μL 浓度为1mg/mL的嗜热菌蛋白酶（thermolysin, 终浓度为50 μg/mL），37℃孵育30 min。加入非还原蛋白loading buffer后，立即置于99℃金属浴煮沸10min。取1 μL酶切体系（对应酶切前的GPdmucin约80 ng）用1×loading buffer稀释至5 μL，在8-16%的凝胶（GenScript，M00660）中电泳，然后电转至硝酸纤维素膜上，用Rb pAb to 6X His taq-HRP（Abcam）检测GPdmucin的酶切情况。
结果：
2G1能够有效阻断GPdmucin被thermolysin酶切。随着2G1浓度的升高，未酶切的GPdmucin（约70kD）和酶切中间体GPdmucin-IMF（intermediate form，约50kD）量明显增多，二者之和接近GPdmucin的初始量，并且基本检测不到酶切终产物GP CL（约38kD）。提示2G1在体内可能通过阻断GP被酶切从而发挥保护作用（参见图23）。
实施例13.小鼠保护实验   
将6-8周龄的雌性BALB/c健康小鼠，随机分配到实验组，每组10只。100 PFU鼠适应EBOV（Mayinga）病毒株经PBS稀释后，通过腹腔注射攻毒。分别在攻毒1天和2天后，单剂量（100 μg）腹腔注射给予单株抗体治疗，对照组小鼠给予PBS。
在攻毒后第1天和第2天，给予100 μg的2G1单抗治疗的两组小鼠全部存活，而PBS对照组小鼠全部死亡（参见图24）。2G1单抗治疗组小鼠的临床症状也得到改善，攻毒1天后给药组小鼠的体重没有降低，攻毒2天后给药组小鼠的体重降低也较少。2G1单抗是一株良好的EBOV保护性抗体，在小鼠模型中可提供完全保护。
 
实施例14.豚鼠保护实验  
将4-6周龄的雌性Hartley豚鼠（250-300g）随机分配到实验组，每组6只。1000×LD 50的SUDV豚鼠适应株在1 mL的DMEM体系中，通过腹腔注射攻毒。在攻毒3天或4天后单次通过腹腔注射2G1单抗，每次给药5 mg或2.5 mg。对照组豚鼠给予PBS处理。持续28天观察豚鼠的疾病、存活和体重变化等临床症状。
无论是在SUDV攻毒后第3天还是第4天单次给药5 mg或2.5 mg的抗体，2G1给药组的豚鼠全部存活（参见图25）。临床症状方面，2G1给药组豚鼠的体重无明显降低，而对照组豚鼠体重持续降低，且最终全部死亡（参见图26）。2G1单抗对SUDV来说是一株良好的保护性抗体，在豚鼠模型上可提供完全保护。该结果说明了2G1单抗对SUDV良好的保护活性。
 工业实用性
本发明提供的单克隆抗体在制药领域可以大规模工业化制备，具有工业实用性。
 序列表自由内容
序列表
<110>  中国人民解放军军事科学院军事医学研究院
 
<120>  一种广谱中和多种埃博拉病毒的单克隆抗体2G1及应用
 
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        35                  40                  45             
 
 
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Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln
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Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
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65                  70                  75                  80 
 
 
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gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc    660
 
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cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg    960
 
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Claims (10)

  1. 一种抗埃博拉病毒糖蛋白GP2亚基的单克隆抗体，其特征在于，所述抗体轻链可变区的CDR1、CDR2和CDR3的氨基酸序列分别如SEQ ID NO.1的第27-32、50-52和89-96位氨基酸序列所示，所述抗体重链可变区的CDR1、CDR2和CDR3的氨基酸序列分别如SEQ ID NO.5的第26-33、51-58和97-113位氨基酸序列所示。
  2. 根据权利要求1所述的单克隆抗体，其特征在于，所述抗体轻链可变区的氨基酸序列如SEQ ID NO.1所示，所述抗体重链可变区的氨基酸序列如SEQ ID NO.5所示。
  3. 根据权利要求2所述的单克隆抗体，其特征在于，所述抗体轻链恒定区的氨基酸序列如SEQ ID NO.3所示，所述抗体重链恒定的氨基酸序列如SEQ ID NO.7所示。
  4. 一种编码权利要求1-3任一所述单克隆抗体轻链和重链的基因编码序列，其特征在于，所述抗体的轻链可变区的基因编码序列由SEQ ID NO.2所示，所述抗体的重链可变区的基因编码序列由SEQ ID NO.6所示。
  5. 根据权利要求4所述的序列，其特征在于，所述抗体的轻链恒定区的基因编码序列由SEQ ID NO.4所示，所述抗体的重链恒定区的基因编码序列由SEQ ID NO.8所示。
  6. 一种能够表达权利要求5所述编码单克隆抗体重链和/或轻链的核苷酸编码序列的功能元件。
  7. 根据权利要求6所述的的功能元件，其特征在于，所述功能元件为线性表达框。
  8. 一种含有权利要求7所述线性表达框的宿主细胞，其特征在于，所述细胞为293T细胞。
  9. 权利要求1-3任一所述的单克隆抗体在制备埃博拉病毒病治疗药物中的应用。
  10. 根据权利要求9所述的应用，其特征在于，所述单克隆抗体作为埃博拉病毒病广谱治疗药物使用。
PCT/CN2019/086336 2018-11-06 2019-05-10 一种广谱中和多种埃博拉病毒的单克隆抗体 2g1 及应用 WO2020093672A1 (zh)

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