WO2022017124A1 - 具有广谱中和活性的抗亨尼帕病毒单克隆抗体及应用 - Google Patents

具有广谱中和活性的抗亨尼帕病毒单克隆抗体及应用 Download PDF

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WO2022017124A1
WO2022017124A1 PCT/CN2021/102588 CN2021102588W WO2022017124A1 WO 2022017124 A1 WO2022017124 A1 WO 2022017124A1 CN 2021102588 W CN2021102588 W CN 2021102588W WO 2022017124 A1 WO2022017124 A1 WO 2022017124A1
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seq
antibody
monoclonal antibody
heavy chain
light chain
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French (fr)
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陈薇
于长明
刘渝娇
范鹏飞
张冠英
李耀辉
李建民
迟象阳
郝勐
房婷
董韵竹
宋小红
陈旖
刘树玲
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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 EP21845669.7A priority Critical patent/EP4186923A1/en
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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
    • C07K16/1027—Paramyxoviridae, e.g. respiratory syncytial virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • 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

Definitions

  • the invention discloses a monoclonal antibody, which belongs to the fields of immunology and microbiology.
  • Nipah virus (NiV) and Hendra virus (Hendra virus, HeV) belong to the genus Henipavirus of the family Paramyxoviridae, and are single-stranded negative-stranded RNA viruses.
  • NiV and HeV are zoonotic viruses that can be infected by human contact and can cause fatal respiratory and neurological diseases.
  • the natural hosts of NiV and HeV are fruit bats, but their transmission routes are slightly different. HeV has only found the transmission route of fruit bat-horse-human, while NiV can be transmitted through fruit bat-pig-human, and can also It is transmitted directly from bats and from person to person.
  • HeV HeV first appeared in the town of Hendra on the outskirts of Brisbane, Australia. The outbreak began in 1994. A total of 21 horses and two people became ill. The horses were identified as intermediate hosts. people are susceptible. Since then, outbreaks have also occurred on the east coast of Australia, killing seven people in Australia in 2006 and killing 23 horses, a dog and more than 60 people in 2011.
  • NiV first appeared in Malaysia. During the period from September 1998 to April 1999, several pig farms died of severe encephalitis and a large number of pigs died in Perak, Malaysia. It was initially thought to be Japanese encephalitis. It was later found that this outbreak was significantly different from Japanese encephalitis in terms of susceptible population, infection rate and infection mode, and many patients had been vaccinated against Japanese encephalitis. The researchers determined that this was a new type of infectious disease. disease. Both people and livestock infected in this outbreak exhibited acute respiratory syndrome, resulting in 256 infections, 105 deaths, 1.16 million pig deaths, and spread to a slaughterhouse in Singapore, infecting 11 workers, 1 die. In October 1999, researchers isolated the virus from the cerebrospinal fluid of a patient.
  • NiV niV Malaysia, NiV-MY
  • Bangladeshi NiV Bangladesh, NiV-BD
  • Henipa virus binds to the receptor ephrin-B2/B3 through the viral surface glycoprotein G, which will cause the G protein to activate the fusion protein F in the receptor binding region and the stalk region and undergo a conformational change , thereby activating the fusion of the F protein with the cell membrane, and finally the virus invades the cell.
  • NiV and HeV have highly similar gene sequences, and the amino acid sequence similarity of G protein and F protein are 83% and 89%, respectively. Therefore, both G protein and F protein are important research targets in vaccine and antiviral drug research. There is currently no vaccine available for human use, and only one monoclonal antibody, m102.4, has entered clinical trials for antibody drugs.
  • m102.4 is a human monoclonal antibody screened by recombinant human Fab phage display library, which can effectively neutralize Nipah virus and Hendra virus. In the challenge protection experiments of ferrets and African green monkeys, m102.4 can effectively protect animal survival after Henipa virus challenge. In 2010, m102.4 was approved as an emergency protective drug in two people at high risk of exposure, neither of whom developed symptoms of infection.
  • the purpose of the present invention is to provide a monoclonal antibody against Henipa virus glycoprotein G that can be directed against a unique antigenic epitope, and then provide its method in the preparation of Henipa virus.
  • Nipah virus disease treatment drugs are used.
  • the present invention first provides a specific neutralizing antibody against Henipa virus glycoprotein G, the antibody is a monoclonal antibody, and the amino acid sequences of the heavy chain variable region CDR1, CDR2 and CDR3 regions of the antibody are The amino acid sequences of the CDR1, CDR2 and CDR3 regions of the light chain variable region are respectively shown in any combination of the following sequences:
  • SEQ ID NO: 1 positions 26-33, 51-58, 97-116 and SEQ ID NO: 3 positions 27-36, 54-56, 93-100, or
  • SEQ ID NO: 9 positions 26-33, 51-58, 97-115 and SEQ ID NO: 11 positions 27-32, 50-52, 89-97, or
  • SEQ ID NO: 13 positions 26-33, 51-58, 97-109 and SEQ ID NO: 15 positions 27-37, 51-53, 90-100.
  • amino acid sequence of the antibody heavy chain variable region and the amino acid sequence of the antibody light chain variable region are respectively shown in any combination of the following sequences:
  • SEQ ID NO: 1 and SEQ ID NO: 3 in the present invention, the antibody with this variable region is named "1B6"
  • SEQ ID NO: 5 and SEQ ID NO: 7 in the present invention, the antibody with this variable region is named "1E5"), or
  • SEQ ID NO: 9 and SEQ ID NO: 11 in the present invention, the antibody with this variable region is named "2A4"), or
  • SEQ ID NO: 13 and SEQ ID NO: 15 in the present invention, the antibody with this variable region is named "2E7").
  • amino acid sequence of the antibody heavy chain constant region is shown in SEQ ID NO: 17, and the amino acid sequence of the antibody light chain constant region is shown in SEQ ID NO: 19 or SEQ ID NO: 21 shown.
  • the present invention also provides a polynucleotide encoding the heavy chain and/or light chain of the monoclonal antibody, the polynucleotide sequence encoding the variable region of the heavy chain of the antibody and the light chain encoding the antibody can be
  • the polynucleotide sequences of the variable regions are respectively shown in any combination of the following sequences:
  • SEQ ID NO: 6 and SEQ ID NO: 8 in the present invention, the antibody having this coding sequence is named "1E5"), or
  • SEQ ID NO: 10 and SEQ ID NO: 12 in the present invention, the antibody having this coding sequence is named "2A4"), or
  • SEQ ID NO: 14 and SEQ ID NO: 16 (in the present invention, the antibody with this coding sequence is named "2E7").
  • sequence of the polynucleotide encoding the constant region of the heavy chain of the antibody is shown in SEQ ID NO: 18, and the sequence of the polynucleotide encoding the constant region of the light chain of the antibody is shown in SEQ ID NO: 20 or SEQ ID NO:22.
  • the present invention also provides a functional element for expressing the above-mentioned polynucleotide encoding the heavy chain and/or light chain of the monoclonal antibody.
  • the functional element is a linear expression cassette.
  • the functional element is a mammalian expression vector.
  • the present invention also provides a host cell containing the above functional elements.
  • the cells are Expi 293F cells.
  • the cells are CHO-S cells.
  • CHO-S cells can be used to construct stable transgenic cell lines to realize industrial production.
  • the present invention provides the application of the above-mentioned monoclonal antibody in the preparation of a medicament for the treatment of Henipa virus disease.
  • the monoclonal antibody against Henipa virus glycoprotein G provided by the present invention is composed of a monkey-derived variable region and a human-derived constant region, and the light and heavy chains of the monkey-derived variable region have unique CDR regions.
  • the antibody provided by the invention shows an excellent broad spectrum in antigen binding ability, and has good binding activity with Nipah virus glycoprotein G and Hendra virus glycoprotein G.
  • the antibody can effectively neutralize the Nipah and Hendra pseudoviruses; and the neutralizing activity of the antibody increases with the increase of the antibody concentration, and can neutralize Nipah and Hendra at a concentration of 1 ⁇ g/mL.
  • the pseudovirus achieves nearly 100% neutralization.
  • the above excellent technical effects show that the monoclonal antibody against Henipa virus glycoprotein G provided by the present invention is applied to the preparation of a medicine for the treatment of Henipa virus disease.
  • rhesus monkeys Female rhesus monkeys were immunized three times, that is, intramuscular injection on days 0, 28, and 49 to immunize adenovirus vector Nipah virus candidate vaccine, recombinant NiV G protein and recombinant HeV G protein, respectively. Finally, blood samples of rhesus monkeys were collected on day 77.
  • the antigenic protein NiV-BD G was labeled with FITC. Methods as below:
  • Fluorescein Isothiocyanate FITC (SIGMA, F4274) was dissolved in DMSO at a concentration of 20 mg/mL. Take 100 ⁇ L of NiV-BD G protein (3.3 mg/mL), add buffer (pH 9.6 carbonate buffer) to 400 ⁇ L.
  • the collected blood samples were separated from PBMC by Ficoll density gradient centrifugation.
  • the process is as follows:
  • Separation solution, anticoagulated undiluted whole blood, PBS (or normal saline) volume is 1:1:1.
  • Single memory B cells specific for NiV-BD G protein were sorted using a cell sorter (BECKMAN, MoFlo XDP).
  • the sorting strategy is: IgG + /CD3 - /CD19 + /CD27 + /NiV-BD G + , single cells are directly sorted into 96-well plates, each well contains 20U RNase inhibitor and 20 ⁇ L RNase-free water , -80 °C storage.
  • Figure 1 is a graph of cell sorting results. The cells in the part circled by the R7 box in the figure are characterized by IgG + /CD3 ⁇ /CD19 + /CD27 + /NiV-BD G + , that is, NiV-BD G specific memory B cells.
  • 1124 NiV-BD G-specific memory B cells were obtained by sorting. Using the SuperScript III reverse transcription kit, the mixed system was directly added to a 96-well plate containing a single cell according to the instructions for PCR reaction.
  • the reaction system and conditions are as follows:
  • Reaction conditions 42 °C, 10 min; 25 °C, 10 min; 50 °C, 60 min; 94 °C, 5 min.
  • the reverse transcription-PCR reaction system is described in Table 2.
  • the first round of nested PCR reaction conditions first, pre-denaturation at 95°C for 5 min; followed by 40 amplification cycles: 95°C, 30s; 57°C, 30s; 72°C, 45s; final extension at 72°C for 10min.
  • the first-round nested PCR primers are described in Table 4:
  • reaction conditions of the second round of nested PCR were the same as those of the first round of nested PCR.
  • a promoter-leader sequence fragment, a constant region for biosynthesis, the heavy chain constant region sequence is shown in SEQ ID NO: 17, the DNA coding sequence is shown in SEQ ID NO: 18, the Kappa light
  • the chain constant region sequence is shown in SEQ ID NO: 19
  • the DNA coding sequence is shown in SEQ ID NO: 20
  • the Lamda type light chain constant region sequence is shown in SEQ ID NO: 21
  • the DNA coding sequence is shown in SEQ ID NO: 22 shown) - poly A tail fragment (Genbank accession number: X03896.1), reamplified antibody variable region fragment.
  • the PCR reaction system for amplifying the variable region is shown in Table 7:
  • PCR reaction conditions first, pre-denaturation at 95°C for 5min; then 30 cycles of amplification: 95°C, 30s; 60°C, 30s; 72°C, 30s; finally, extension at 72°C for 10min.
  • Reaction conditions first, pre-denaturation at 95°C for 5min; then 30 amplification cycles: 95°C, 30s; 60°C, 30s; 72°C, 3min; finally, extension at 72°C for 10min.
  • PCR reaction products were directly recovered with the kit of OMEGA company, and the PCR recovery products were quantified with Nano (GE Healthcare).
  • 150 ⁇ L of 2 ⁇ 10 4 cells per well was plated in a 96-well plate.
  • On the day of transfection take 0.2 ⁇ g of each light and heavy chain, add 0.8 ⁇ L of Turbofect transfection reagent, dilute to 40 ⁇ L with DMEM medium, mix well and incubate at room temperature for 15 min. Slowly added dropwise to the 96-well plate in sequence, and placed in a 37°C incubator for 48h.
  • the 96-well enzyme-linked plate was coated with 1 ⁇ g/mL NiV-BD G protein, and each well was coated with 100 ⁇ L. Place the coated enzyme-linked plate in a humidified chamber at 4°C overnight.
  • FIG. 3 is a graph showing the distribution of OD 450-630nm values of antibodies with binding activity screened by ELISA.
  • the antibody expression plasmid was constructed, and the expression and preparation of the monoclonal antibody was carried out. Methods as below:
  • the monoclonal antibody has good binding activity to the G protein of NiV-BD, NiV-MY and HeV.
  • the EC 50 values of mAb 1B6 were 28.43ng/mL, 63.92ng/mL and 52.93ng/mL; the EC 50 values of mAb 1E5 were 2.6ng/mL, 0.85ng/mL and 0.34ng/mL, respectively;
  • the EC 50 values of mAb 2A4 were 4.50 ng/mL, 11.23 ng/mL and 7.37 ng/mL, respectively;
  • the EC 50 values of mAb 2E7 were 15.4 ng/mL, 26.69 ng/mL and 70.05 ng/mL, respectively.
  • the above-mentioned 4 strains of antibodies are sequenced, and the nucleotide coding sequences of the heavy chain and light chain variable regions of monoclonal antibody 1B6 are shown in SEQ ID NO: 2 and SEQ ID NO: 4, respectively.
  • the amino acid sequences are shown in SEQ ID NO: 1 and SEQ ID NO: 3 respectively; further analysis of the heavy chain and light chain variable region amino acid sequences shows that the heavy chain variable region CDR1, CDR2 and CDR3 regions amino acid sequences are respectively as shown in SEQ ID NO. :1 26-33, 51-58, 97-116, the light chain variable region CDR1, CDR2 and CDR3 region amino acid sequences are shown in SEQ ID NO: 3 27-36, 54-56, 93-100 shown.
  • the nucleotide coding sequences of the heavy chain and light chain variable regions of monoclonal antibody 1E5 are respectively shown in SEQ ID NO: 6 and SEQ ID NO: 8, and the amino acid sequences of the heavy chain and light chain variable regions are respectively shown by SEQ ID NO: 5 and SEQ ID NO: 7; further analysis of the heavy chain and light chain variable region amino acid sequences, the heavy chain variable region CDR1, CDR2 and CDR3 region amino acid sequences are respectively as SEQ ID NO: 5 Nos. 26-33, 51 -58, 97-117, the light chain variable region CDR1, CDR2 and CDR3 region amino acid sequences are shown in SEQ ID NO: 7 Nos. 27-32, 50-52, 89-97 respectively.
  • the nucleotide coding sequences of the heavy chain and light chain variable regions of monoclonal antibody 2A4 are respectively shown in SEQ ID NO: 10 and SEQ ID NO: 12, and the amino acid sequences of the heavy chain and light chain variable regions are respectively shown by SEQ ID NO: 9 and SEQ ID NO: 11, the heavy chain and light chain variable region amino acid sequences were further analyzed, and the heavy chain variable region CDR1, CDR2 and CDR3 regions amino acid sequences were as shown in SEQ ID NO: 9 Nos. 26-33, 51 -58, 97-115, amino acid sequences of light chain variable region CDR1, CDR2 and CDR3 regions are shown in SEQ ID NO: 11, 27-32, 50-52, 89-97 respectively.
  • the nucleotide coding sequences of the heavy chain and light chain variable regions of monoclonal antibody 2E7 are respectively shown in SEQ ID NO: 14 and SEQ ID NO: 16, and the amino acid sequences of the heavy chain and light chain variable regions are respectively shown in SEQ ID NO: 13 and SEQ ID NO: 15; further analysis of the heavy chain and light chain variable region amino acid sequences, the heavy chain variable region CDR1, CDR2 and CDR3 region amino acid sequences are respectively as SEQ ID NO: 13 Nos. 26-33, 51 -58, 97-109 positions, the light chain variable region CDR1, CDR2 and CDR3 regions amino acid sequences are shown in SEQ ID NO: 15, 27-37, 51-53, 90-100 positions, respectively.
  • the above-mentioned 4 strains of monoclonal antibodies have the same heavy chain constant region and light chain constant region of human origin, and the sequence of the polynucleotide encoding the heavy chain constant region of the above-mentioned antibody is shown in SEQ ID NO: 18, encoding the antibody light chain
  • SEQ ID NO: 20 or SEQ ID NO: 22 encoding the amino acid sequence of the heavy chain constant region is shown in SEQ ID NO: 17
  • the amino acid sequence of the antibody light chain constant region is shown in SEQ ID NO: 19 or SEQ ID NO: 21.
  • the method includes 5 steps: Baseline, Loading, Baseline, Association and Dissociation, and the duration of each step is set to 100s, 180s, 60s, 300s and 600s respectively.
  • 1E5 has a high affinity for all three G proteins, and the affinity constant KD is less than 1nM.
  • the highest affinity is NiV-MY G, and the lowest is HeV G.
  • NiV-BD NiV-BD
  • NiV-MY HeV pseudoviruses that package the HIV backbone
  • Dimple Khetawat, C.C.B. A functional henipavirus envelope glycoprotein pseudotyped lentivirus assay system. Virology Journal 2010.7(312)
  • the neutralizing activity of monoclonal antibodies was evaluated in vitro. Methods as below:
  • 1B6, 1E5, 2A4 and 2E7 can effectively neutralize three pseudoviruses of HIV backbone NiV-BD, NiV-MY and HeV in vitro.
  • the neutralizing activities of 1B6, 1E5 and 2A4 increased with the increase of antibody concentration, and nearly 100% neutralization of the three henipa pseudoviruses could be achieved at a concentration of 1 ⁇ g/mL.
  • IC 50 values were 1B6,1E5 and 2A4 16.31ng / mL, 5.74ng / mL, and 28.96ng / mL, 2E7 can be at 5 ⁇ g / mL and a concentration of nearly 100%; and for NiV-BD NiV-MY pseudovirus, the above four antibodies have good neutralizing activity, and the IC 50 values for NiV-MY pseudovirus are 27.15ng/mL, 19.03ng/mL, 48.60ng/mL and 80.79ng/mL, respectively.
  • These results indicate that the four monoclonal antibodies 1B6, 1E5, 2A4 and 2E7 have broad-spectrum neutralizing activities, and can simultaneously neutralize Nipah virus and Hendra virus of the Henipavirus genus.
  • Luminex microsphere competition inhibition assay The ability of monoclonal antibody to inhibit the binding of henipa virus G protein to the receptor was verified by Luminex microsphere competition inhibition assay. The method is as follows:
  • antibodies 1E5 and 2A4 are likely to play a neutralizing role by inhibiting the binding of Henipa virus G protein to receptor ephrin-B2/B3, while 1B6 and 2E7 may have other neutralizing mechanisms against Hendra virus. Further Discussion.
  • mAbs 1B6, 1E5 and 2A4 have more than 60% neutralizing activity against D582N pseudovirus, 2E7 has 50% neutralizing activity, and the remaining antibodies are all at 20% the following. This indicates that the polyclonal antibody screened in this study can be used to neutralize the escape mutant strain of m102.4, which has a different binding epitope from that of m102.4.
  • the invention provides an anti-henipah virus monoclonal antibody with broad-spectrum neutralizing activity and its application in the preparation of medicines.
  • the monoclonal antibody is easy for industrial production and has industrial practicability.

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Abstract

提供了一种具有广谱中和活性的抗亨尼帕病毒单克隆抗体，该抗体由猴源可变区和人源恒定区组成。本发明的抗体与尼帕病毒糖蛋白G和亨德拉病毒糖蛋白G均具有良好的结合活性，能够有效中和尼帕和亨德拉假病毒，可用于制备治疗亨尼帕病毒病的药物。

Description

具有广谱中和活性的抗亨尼帕病毒单克隆抗体及应用 技术领域
本发明公开了一种单克隆抗体，属于免疫学和微生物学领域。
背景技术
尼帕病毒(Nipah virus,NiV)和亨德拉病毒(Hendra virus,HeV)同属于副黏病毒科(Paramyxoviridae)亨尼帕病毒属(Henipavirus)，为单股负链RNA病毒。NiV和HeV为人畜共患病病毒，人能通过接触感染，会引起致命的呼吸和神经系统性疾病。NiV和HeV的自然宿主都是果蝠，但是它们的传播途径略有不同，HeV目前仅发现果蝠-马-人这一传播途径，而NiV则可以通过果蝠-猪-人传播，还可以直接通过蝙蝠传人及人与人之间传播。
HeV首次出现在澳大利亚布里斯班市郊亨德拉镇，这次疫情开始于1994年，共有21匹马和两个人患病，马被确认为中间宿主，照顾护理或剖检病马、死马的人易感。此后爆发的疫情也都发生在澳大利亚东岸，2006年在澳大利亚造成7人感染4人死亡，2011年则造成23匹马、一只狗死亡以及60多人感染。
NiV最开始出现于马来西亚，在1998年9月至1999年4月期间，马来西亚霹雳州出现多名猪场饲养人员因重度脑炎死亡和大量猪患病死亡案例，最开始认为是乙型脑炎病毒感染，后来发现这次疫情在易感人群、感染率以及感染方式上与乙型脑炎有着明显的不同，并且许多患者曾接种过日本脑炎疫苗，研究人员认定这是一种新型传染性疾病。这次疫情中感染的人员和家畜均表现出急性呼吸系统综合征，最终造成256人感染，105人死亡，116万头猪死亡，并蔓延至新加坡一屠宰场，导致11名工人感染、1人死亡。1999年10月，研究人员从一名患者脑脊髓液中分离出病毒。不久之后，从马来西亚果蝠尿液中分离出NiV，确定了NiV的自然宿主。随后，在印度、柬埔寨、泰国等国家都曾报道过尼帕 病毒病，而近年来，尼帕病毒病多次在孟加拉国和印度发生，已造成数百人死亡，死亡率在50％～100％之间。NiV的研究主要集中于马来西亚型(NiV Malaysia，NiV-MY)和孟加拉型(NiV Bangladesh，NiV-BD)。
技术问题
亨尼帕病毒在入侵宿主细胞的过程中，通过病毒表面糖蛋白G与受体ephrin-B2/B3结合后，会引起G蛋白在受体结合区和茎状区内激活融合蛋白F发生构象改变，从而激活F蛋白与细胞膜融合，最终病毒侵入细胞。NiV与HeV具有高度相似的基因序列，G蛋白和F蛋白的氨基酸序列相似性分别为83％和89％。因此，在疫苗和抗病毒类药物研究中，G蛋白和F蛋白都是重要的研究靶标。目前尚无疫苗可用于人体，抗体类药物也仅有一株单克隆抗体m102.4进入临床实验。m102.4是通过重组人源Fab噬菌体展示库筛选得到的人源单克隆抗体，能有效地中和尼帕病毒和亨德拉病毒，在雪貂和非洲绿猴的攻毒保护性实验中，m102.4能够在亨尼帕病毒攻毒后有效保护动物存活。2010年，m102.4被批准作为紧急保护性药物在两位高暴露风险的人身上使用，结果这两人都没有出现感染症状。
鉴于本领域对于抗亨尼帕病毒治疗性抗体的技术需求，本发明的目的就是提供一种能够针对独特抗原表位的抗亨尼帕病毒糖蛋白G的单克隆抗体，进而提供其在制备亨尼帕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抗亨尼帕病毒糖蛋白G的特异性中和抗体，所述抗体为单克隆抗体，所述抗体重链可变区CDR1、CDR2和CDR3区氨基酸序列和轻链可变区CDR1、CDR2和CDR3区氨基酸序列分别如下述任一序列组合所示：
SEQ ID NO:1第26-33、51-58、97-116位和SEQ ID NO:3第27-36、54-56、93-100位，或
SEQ ID NO:5第26-33、51-58、97-117位和SEQ ID NO:7第27-32、50-52、89-97位，或
SEQ ID NO:9第26-33、51-58、97-115位和SEQ ID NO:11第27-32、50-52、89-97位，或
SEQ ID NO:13第26-33、51-58、97-109位和SEQ ID NO:15第27-37、51-53、90-100位。
在一个优选的实施方案中，所述抗体重链可变区的氨基酸序列和抗体轻链可变区的氨基酸序列分别如下述任一序列组合所示：
SEQ ID NO:1和SEQ ID NO:3(在本发明中，具有该可变区的抗体被命名为“1B6”)，或
SEQ ID NO:5和SEQ ID NO:7(在本发明中，具有该可变区的抗体被命名为“1E5”)，或
SEQ ID NO:9和SEQ ID NO:11(在本发明中，具有该可变区的抗体被命名为“2A4”)，或
SEQ ID NO:13和SEQ ID NO:15(在本发明中，具有该可变区的抗体被命名为“2E7”)。
在一个更为优选的实施方案中，所述抗体重链恒定区的氨基酸序列如SEQ ID NO:17所示，所述抗体轻链恒定区的氨基酸序列如SEQ ID NO:19或SEQ ID NO:21所示。
其次，本发明还提供了一种编码上述单克隆抗体重链和/或轻链的多核苷酸，编码所述抗体的重链可变区的多核苷酸序列和编码所述抗体的轻链可变区的多核苷酸序列分别如下述任一序列组合所示：
SEQ ID NO:2和SEQ ID NO:4(在本发明中，具有该编码序列的抗体被命名为“1B6”)，或
SEQ ID NO:6和SEQ ID NO:8(在本发明中，具有该编码序列的抗体被命名为“1E5”)，或
SEQ ID NO:10和SEQ ID NO:12(在本发明中，具有该编码序列的抗体被命名为“2A4”)，或
SEQ ID NO:14和SEQ ID NO:16(在本发明中，具有该编码序列的抗体被命名为“2E7”)。
在一个优选的实施方案中，编码所述抗体重链恒定区的多核苷酸的序列如SEQ ID NO:18所示，编码所述抗体轻链恒定区的多核苷酸的序列 如SEQ ID NO:20或SEQ ID NO:22所示。
第三，本发明还提供了一种表达上述编码单克隆抗体重链和/或轻链的多核苷酸的功能元件。
在一个优选的实施方案中 ，所述功能元件为线性表达框。
在另一个优选的实施方案中，所述功能元件为哺乳动物表达载体。
第四，本发明还提供了一种含有上述功能元件的宿主细胞。
在一个优选的实施方案中，所述细胞为Expi 293F细胞。
在另一个优选的实施方案中，所述细胞为CHO-S细胞，本发明可以使用CHO-S细胞构建稳转工程细胞株，实现产业化生产。
最后，本发明提供了上述的单克隆抗体在制备亨尼帕病毒病治疗药物中的应用。
技术效果
本发明提供的抗亨尼帕病毒糖蛋白G的单克隆抗体由猴源可变区和人源恒定区组成，所述猴源可变区轻重链均具有独特的CDR区。本发明提供的抗体在抗原结合能力上显示了优异的广谱性，与尼帕病毒糖蛋白G和亨德拉病毒糖蛋白G具有良好的结合活性。所述抗体能够有效中和尼帕和亨德拉假病毒；而且所述抗体的中和活性随着抗体浓度的升高而增强，在1μg/mL的浓度下即可对尼帕和亨德拉假病毒实现近100％的中和。上述优异的技术效果显示了本发明提供的抗亨尼帕病毒糖蛋白G的单克隆抗体应用于制备亨尼帕病毒病治疗药物。
附图说明
图1.恒河猴记忆B细胞分选结果图；
图2.巢式PCR毛细管电泳图谱；
图3.ELISA筛选具有结合活性抗体的OD 450-630nm值分布图；
图4.ELISA检测抗体与抗原结合活性随浓度变化曲线图；
图5.1E5与亨尼帕病毒G蛋白的亲和力测定；
图6.单抗与HeV假病毒的中和曲线图；
图7.单抗与NiV-MY假病毒的中和曲线图；
图8.单抗与NiV-BD假病毒的中和结果图；
图9.单抗对亨尼帕病毒G蛋白与受体ephrin-B3结合的竞争抑制；
图10.单抗对亨尼帕病毒G蛋白与受体ephrin-B2结合的竞争抑制；
图11.单抗对HeV G D582N假病毒中和率对照图。
具体实施方式
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，并不对本发明的权利要求所限定的保护范围构成任何限制。
实施例1抗体的筛选和制备
1.血液样品的采集
对雌性恒河猴进行三次免疫，即在第0、28、49天采用肌肉注射的方式，分别免疫腺病毒载体尼帕病毒候选疫苗、重组NiV G蛋白和重组HeV G蛋白。最终采集第77天恒河猴血液样本。
2.FITC标记NiV-BD G蛋白
为分选抗原特异的记忆B细胞，FITC标记抗原蛋白NiV-BD G。方法如下：
1)Fluorescein Isothiocyanate FITC(SIGMA，F4274)溶于DMSO，浓度为20mg/mL。取100μL NiV-BD G蛋白(3.3mg/mL)，加缓冲液(pH9.6碳酸盐缓冲液)至400μL。
2)加8μL FITC至NiV-BD G蛋白溶液中，4℃避光孵育3h。
3)用30kDa的超滤管对溶液进行PBS换液，直至滤过液为透明无色。将标记好的蛋白用锡箔纸包好，4℃存放待用。
3.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800g，加速减速3档位，离心30min。 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。
4)用PBS稀释到一定体积，颠倒混匀。室温，水平转子300g，离心10min，弃上清，重复洗涤2次。用PBS将淋巴细胞重悬备用。
5)细胞计数，取5×10 5个细胞，体积为50μL，加入表1所列推荐量的5种荧光染料，4℃避光孵育1h。
表1.流式分选细胞所用染料列表
标记 荧光 公司/货号 体积
抗原 FITC SIGMA，F4274 4μg
IgG PE BD，555787 15μL
CD19 APC-AF 700 Beckman，IM2470 5μL
CD3 PerCP BD，552851 10μL
CD27 PC7 Beckman，A54823 10μL
6)使用含2％FBS的PBS洗涤2-3次，400μL FPBS重悬，用40μm细胞筛去除细胞团，4℃避光保存供分选。
7)使用细胞分选仪(BECKMAN，MoFlo XDP)分选NiV-BD G蛋白特异的单个记忆B细胞。分选策略为：IgG +/CD3 -/CD19 +/CD27 +/NiV-BD G +，直接将单个细胞分选至96孔板中，每孔含有20U RNA酶抑制剂及20μL去RNA酶的水中，-80℃保存。图1为细胞分选结果图。图中R7方框圈出部分的细胞特征为IgG +/CD3 -/CD19 +/CD27 +/NiV-BD G +，即NiV-BD G特异性记忆B细胞。
4.单细胞PCR扩增抗体可变区
1)反转录PCR
分选得到1124个NiV-BD G特异的记忆B细胞，使用SuperScriptⅢ反转录试剂盒，按说明书将混合好的体系直接加入含有单个细胞的96孔板进行PCR反应，反应体系及条件如下：
反应条件：42℃，10min；25℃，10min；50℃，60min；94℃，5min。反转录-PCR反应体系如表2所述。
表2.反转录-PCR反应体系
Figure PCTCN2021102588-appb-000001
2)巢式PCR
以反转录产物为模板，分别进行2次巢式PCR反应扩增H、κ、λ，具体过程如下：
第一轮巢式PCR反应体系如表3所述：
表3.第一轮巢式PCR反应体系
Figure PCTCN2021102588-appb-000002
第一轮巢式PCR反应条件：首先，95℃预变性5min；接着进行40个扩增循环:95℃，30s；57℃，30s；72℃，45s；最后72℃延伸10min。
第一轮巢式PCR引物如表4所述：
表4第一轮巢式PCR引物
Figure PCTCN2021102588-appb-000003
Figure PCTCN2021102588-appb-000004
Figure PCTCN2021102588-appb-000005
第二轮巢式PCR反应体系如表5所述：
表5.巢式PCR反应体系
Figure PCTCN2021102588-appb-000006
第二轮巢式PCR反应引物如表6所示：
表6 第二轮巢式PCR引物
Figure PCTCN2021102588-appb-000007
Figure PCTCN2021102588-appb-000008
第二轮巢式PCR反应条件同第一轮巢式PCR。
3)毛细管电泳
巢式PCR结束后，扩增产物用QIAxcel DNA Fast Analysis Cartridge进行毛细管电泳，挑选轻重链配对的阳性孔进行测序，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。图2为巢式PCR 毛细管电泳结果。
5.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254对配对的抗体序列，如果采用传统的克隆表达方法费时费力，通过构建线性表达框的方法可以快速表达抗体。
首先，通过PCR反应获得启动子-前导序列片段、恒定区(生工生物合成，重链恒定区序列由SEQ ID NO:17所示，DNA编码序列由SEQ ID NO:18所示，Kappa型轻链恒定区序列由SEQ ID NO:19所示，DNA编码序列由SEQ ID NO:20所示，Lamda型轻链恒定区序列由SEQ ID NO:21所示，DNA编码序列由SEQ ID NO:22所示)-多聚A尾片段(Genbank登记号：X03896.1)，再扩增抗体可变区片段。扩增可变区PCR反应体系如表7所示：
表7 扩增可变区片段反应体系
Figure PCTCN2021102588-appb-000009
PCR反应条件：首先，95℃预变性5min；然后进行30个扩增循环：95℃，30s；60℃，30s；72℃，30s；最后，72℃延伸10min。
以扩增好的启动子-前导序列片段、恒定区-多聚A尾片段和可变区片段为模板，以CMV-UP和TK-POLY A为引物，进行重叠延伸PCR分别扩增H、κ和λ链的线性表达框。PCR产物经核酸电泳鉴定。扩增全长线性表达框反应体系如表8所示：
表8 扩增全长线性表达框反应体系
Figure PCTCN2021102588-appb-000010
Figure PCTCN2021102588-appb-000011
反应条件：首先，95℃预变性5min；然后进行30个扩增循环：95℃，30s；60℃，30s；72℃，3min；最后，72℃延伸10min。
PCR反应产物直接用OMEGA公司试剂盒回收，用Nano(GE Healthcare)对PCR回收产物进行定量。转染前一天按2×10 4cells每孔150μL铺好96孔板。转染当天，取轻重链各0.2μg，加入0.8μL Turbofect转染试剂，使用DMEM培养基稀释至40μL，混匀室温孵育15min。按顺序缓慢逐滴加入96孔板中，放入37℃孵箱培养48h。
6.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1μg/mL的NiV-BD G蛋白，每孔100μL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)清洗5次。每孔加入100μL封闭液，37℃孵箱中放置1小时。
3)洗板机洗板，加入100μL的转染细胞培养上清，37℃孵箱中静置1小时。
4)洗板，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100μL加入到ELISA板对应孔中，37℃孵箱中静置1小时。
5)洗板，每孔加入100μL的TMB单组份显色液，显色6min，室温避光，之后每孔加入50μL终止液终止反应。酶标仪检测450-630nm的OD值。
结果：将254株单抗进行筛选，以OD 450-630nm值等于0.1时为cutoff值，得到59株能与NiV-BD G特异性结合的抗体，对这些抗体进行进一步地表达纯化以及验证等实验。图3为ELISA筛选具有结合活性的抗体的OD 450-630nm值分布图。
7.表达质粒构建与抗体制备
构建抗体表达质粒，进行单抗的表达制备。方法如下：
1)将抗体重链H和轻链L线性表达框全长基因用EcoR I(NEB，R3101)和Not I(NEB，R3189)双酶切，连接至pcDNA3.4表达质粒。
2)取pcDNA3.4-H和pcDNA3.4-L各15μg，转染至30mL Expi 293体系(Life，A14524)中，125rpm，5％CO 2培养72h。
3)3000×g，离心10min收集表达上清，采用rProtein A亲和纯化。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1μg/mL的NiV-BD/MY或HeV G蛋白(NiV-BD G Genbank登记号：AY988601.1，NiV-MY G Genbank登记号：FN869553.1，HeV G Genbank登记号：NC_001906.3)，每孔100μL包被，4℃过夜。
2)实验当天用洗板机洗5次，每孔加入100μL封闭液，室温放置1小时。
3)洗板，首孔加入150μL浓度为20μg/mL的单克隆抗体，其余孔加入100μL的稀释液。从首孔吸出50μL加入到次孔，以此类推，按1:3梯度稀释每孔终体积为100μL。室温静置1小时。
4)洗板，将HPR标记的羊抗人IgG二抗以1:10000用稀释液进行稀释，每孔100μL加入到ELISA板对应孔中，室温孵育1小时。
5)洗板，每孔加入100μL的TMB单组份显色液，显色6min，室温避光，之后每孔加入50μL终止液。
6)用酶标仪检测450-630nm的OD值。
结果如图4，单抗与NiV-BD、NiV-MY和HeV的G蛋白均具有良 好的结合活性。其中，单抗1B6的EC 50值分别为28.43ng/mL、63.92ng/mL和52.93ng/mL；单抗1E5的EC 50值分别为2.6ng/mL、0.85ng/mL和0.34ng/mL；单抗2A4的EC 50值分别为4.50ng/mL、11.23ng/mL和7.37ng/mL；单抗2E7的EC 50值分别为15.4ng/mL、26.69ng/mL和70.05ng/mL。
对上述4株抗体进行测序，单抗1B6重链和轻链可变区的核苷酸编码序列分别由SEQ ID NO:2和SEQ ID NO:4所示，重链和轻链可变区的氨基酸序列分别由SEQ ID NO:1和SEQ ID NO:3所示；对重链和轻链可变区氨基酸序列进一步分析，重链可变区CDR1、CDR2和CDR3区氨基酸序列分别如SEQ ID NO:1第26-33、51-58、97-116位所示，轻链可变区CDR1、CDR2和CDR3区氨基酸序列分别如SEQ ID NO:3第27-36、54-56、93-100所示。
单抗1E5重链和轻链可变区的核苷酸编码序列分别由SEQ ID NO:6和SEQ ID NO:8所示，重链和轻链可变区的氨基酸序列分别由SEQ ID NO:5和SEQ ID NO:7所示；对重链和轻链可变区氨基酸序列进一步分析，重链可变区CDR1、CDR2和CDR3区氨基酸序列分别如SEQ ID NO:5第26-33、51-58、97-117所示，轻链可变区CDR1、CDR2和CDR3区氨基酸序列分别如SEQ ID NO:7第27-32、50-52、89-97位所示。
单抗2A4重链和轻链可变区的核苷酸编码序列分别由SEQ ID NO:10和SEQ ID NO:12所示，重链和轻链可变区的氨基酸序列分别由SEQ ID NO:9和SEQ ID NO:11所示，对重链和轻链可变区氨基酸序列进一步分析，重链可变区CDR1、CDR2和CDR3区氨基酸序列分别如SEQ ID NO:9第26-33、51-58、97-115位所示，轻链可变区CDR1、CDR2和CDR3区氨基酸序列分别如SEQ ID NO:11第27-32、50-52、89-97位所示。
单抗2E7重链和轻链可变区的核苷酸编码序列分别由SEQ ID NO:14和SEQ ID NO:16所示，重链和轻链可变区的氨基酸序列分别由SEQ ID NO:13和SEQ ID NO:15所示；对重链和轻链可变区氨基酸序列进一步分析，重链可变区CDR1、CDR2和CDR3区氨基酸序列分别如SEQ ID NO:13第26-33、51-58、97-109位所示，轻链可变区CDR1、CDR2和CDR3区氨基酸序列分别如SEQ ID NO:15第27-37、51-53、90-100位所 示。
上述4株单克隆抗体具有相同的人源的重链恒定区和轻链恒定区，编码上述抗体重链恒定区的多核苷酸的序列如SEQ ID NO:18所示，编码所述抗体轻链恒定区的多核苷酸的序列如SEQ ID NO:20或SEQ ID NO:22所示，重链恒定区的氨基酸序列如SEQ ID NO:17所示，所述抗体轻链恒定区的氨基酸序列如SEQ ID NO:19或SEQ ID NO:21所示。
实施例3.1E5亲和力测定
1)将1E5稀释至100nM、50nM、25nM、12.5nM、6.25nM、3.13nM、1.56nM的浓度。
2)准备好Octet RED仪器(fortéBIO,Pall Corp,USA)，设置配套软件Data Analysis Software v9.0中的动力学检测方法。方法包括5个步骤：Baseline，Loading，Baseline，Association以及Dissociation，每步时长分别设定为100s，180s，60s，300s和600s。
3)放置抗体、抗原和PBS缓冲液，开始检测。实验结束后，使用软件DataAnalysis进行数据处理，计算拟合出平衡解离常数KD值和1E5与亨尼帕病毒G蛋白的结合解离曲线。如图5所示，其中，A、B、C分别为1E5与HeV、NiV-BD和NiV-MY的G蛋白结合解离曲线。KD值的计算结果如下表。
表9：1E5与亨尼帕病毒G蛋白的亲和力
抗原 NiV-BD G NiV-MY G HeV G
平衡解离常数KD 0.171nM <0.001nM 0.785nM
从结果可以看出1E5对三种G蛋白均具有很高的亲和力，亲和力常数KD均小于1nM，其中亲和力最高的是NiV-MY G，最低的是HeV G。
实施例4.假病毒中和实验评价抗体中和活性
包装HIV骨架的NiV-BD、NiV-MY和HeV假病毒(Dimple Khetawat,C.C.B.,A functional henipavirus envelope glycoprotein pseudotyped lentivirus assay system.Virology Journal 2010.7(312))，体外评价单克隆抗 体的中和活性。方法如下：
1)用DMEM培养基稀释单抗，96孔细胞培养板首孔加入75μL浓度为5μg/mL的抗体稀释液，其余孔加入50μL的DMEM培养基。
2)从首孔吸取25μL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50μL。每孔加入50μL假病毒，混匀，37℃孵育1h。
3)293T细胞计数，2×10 5cells/mL，每孔加入100μL。将培养板放入37℃恒温箱中培养36～48小时。
4)取出细胞培养板，小心吸出培养液弃掉。各孔加入100μL细胞裂解液，震荡机上400rpm震荡15min。3000rpm室温离心10min。同时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。吸取20μL裂解上清读取荧光值，计算抗体对细胞的保护率。
结果如图6、图7和图8所示，1B6、1E5、2A4和2E7在体外均可有效中和HIV骨架的NiV-BD、NiV-MY和HeV三种假病毒。其中1B6、1E5和2A4的中和活性随着抗体浓度的升高而增强，在1μg/mL的浓度下即可对三种亨尼帕假病毒实现近100％的中和。针对HeV假病毒，1B6、1E5和2A4的IC 50值分别为16.31ng/mL、5.74ng/mL和28.96ng/mL，2E7在5μg/mL浓度下能够近100％中和；对NiV-BD和NiV-MY假病毒，以上4株抗体均具有良好的中和活性，对NiV-MY假病毒的IC 50值分别为27.15ng/mL、19.03ng/mL、48.60ng/mL和80.79ng/mL。这些结果表明，1B6、1E5、2A4和2E7四株单抗具有广谱中和活性，能同时中和亨尼帕病毒属的尼帕病毒和亨德拉病毒。
实施例5.竞争实验
采用Luminex微球竞争抑制实验验证单抗抑制亨尼帕病毒G蛋白与受体结合的能力，其方法如下：
1)首孔加入10μL 10μg单克隆抗体，依次向下二倍稀释。
2)每孔加入1.25ng受体ephrin-B2或ephrin-B3，体积10μL。每 孔加入10μL制备好的微球(NiV-BD/MY G偶联微球各1500个)，摇床孵育60min。
3)每孔加入10μL SAPE(浓度为12μg/ml)，摇床孵育30min。
4)100μL assay buffer洗3次后通过Luminex MAGPIX仪器读数。
抗体对亨尼帕病毒G蛋白与受体ephrin-B2/B3结合的竞争抑制曲线如图9和10所示，结果表明1E5和2A4能够有效抑制亨尼帕病毒G蛋白与受体ephrin-B2/B3的结合；1B6和2E7能够有效抑制尼帕病毒G蛋白与受体ephrin-B2/B3的结合，但不能抑制亨德拉病毒G蛋白与受体的结合。提示抗体1E5和2A4很有可能通过抑制亨尼帕病毒G蛋白与受体ephrin-B2/B3的结合发挥其中和作用的，1B6和2E7针对亨德拉病毒则可能存在其他的中和机制，有待进一步探讨。
实施例6.逃逸突变株中和实验
使用单抗m102.4的逃逸突变株HeV G D582N假病毒(生工生物合成HeV G Genbank登记号：NC_001906.3，The exceptionally large genome of Hendra virus:support for creation of a new genus within the family Paramyxoviridae JOURNAL J.Virol.74(21),9972-9979(2000)，仅582位氨基酸合成时突变为N。按实施例4包装假病毒)进行中和实验。结果如图11所示，抗体浓度在1μg/mL时，单抗1B6、1E5和2A4对D582N假病毒有60％以上的中和活性，2E7有50％的中和活性，其余抗体均在20％以下。说明本研究筛选的多株抗体可以用于中和m102.4的逃逸突变株，具备与m102.4不同的结合表位。
工业实用性
本发明提供了一种具有广谱中和活性的抗亨尼帕病毒单克隆抗体及在制备药物中的应用，所述单克隆抗体易于工业化生产，具有工业实用性。
序列表自由内容
Figure PCTCN2021102588-appb-000012
Figure PCTCN2021102588-appb-000013
Figure PCTCN2021102588-appb-000014
Figure PCTCN2021102588-appb-000015
Figure PCTCN2021102588-appb-000016
Figure PCTCN2021102588-appb-000017
Figure PCTCN2021102588-appb-000018
Figure PCTCN2021102588-appb-000019
Figure PCTCN2021102588-appb-000020
Figure PCTCN2021102588-appb-000021
Figure PCTCN2021102588-appb-000022
Figure PCTCN2021102588-appb-000023
Figure PCTCN2021102588-appb-000024
Figure PCTCN2021102588-appb-000025
Figure PCTCN2021102588-appb-000026

Claims (10)

  1. 一种抗亨尼帕病毒糖蛋白G的单克隆抗体，其特征在于，所述抗体重链可变区CDR1、CDR2和CDR3区氨基酸序列和轻链可变区CDR1、CDR2和CDR3区氨基酸序列分别如下述任一序列组合所示：
    SEQ ID NO:1第26-33、51-58、97-116位和SEQ ID NO:3第27-36、54-56、93-100位，或
    SEQ ID NO:5第26-33、51-58、97-117位和SEQ ID NO:7第27-32、50-52、89-97位，或
    SEQ ID NO:9第26-33、51-58、97-115位和SEQ ID NO:11第27-32、50-52、89-97位，或
    SEQ ID NO:13第26-33、51-58、97-109位和SEQ ID NO:15第27-37、51-53、90-100位。
  2. 根据权利要求1所述的单克隆抗体，其特征在于，所述抗体重链可变区的氨基酸序列和抗体轻链可变区的氨基酸序列分别如下述任一序列组合所示：
    SEQ ID NO:1和SEQ ID NO:3，或
    SEQ ID NO:5和SEQ ID NO:7，或
    SEQ ID NO:9和SEQ ID NO:11，或
    SEQ ID NO:13和SEQ ID NO:15。
  3. 根据权利要求2所述的单克隆抗体，其特征在于，所述抗体重链恒定区的氨基酸序列如SEQ ID NO:17所示，所述抗体轻链恒定区的氨基酸序列如SEQ ID NO:19或SEQ ID NO:21所示。
  4. 一种编码权利要求1-3任一所述单克隆抗体重链和/或轻链的多核苷酸，其特征在于，编码所述抗体的重链可变区的多核苷酸序列和编码所述抗体的轻链可变区的多核苷酸序列分别如下述任一序列组合所示：
    SEQ ID NO:2和SEQ ID NO:4，或
    SEQ ID NO:6和SEQ ID NO:8，或
    SEQ ID NO:10和SEQ ID NO:12，或
    SEQ ID NO:14和SEQ ID NO:16。
  5. 根据权利要求4所述的多核苷酸，其特征在于，编码所述抗体重链恒定区的多核苷酸的序列如SEQ ID NO:18所示，编码所述抗体轻链恒定区的多核苷酸的序列如SEQ ID NO:20或SEQ ID NO:22所示。
  6. 一种表达权利要求5所述编码单克隆抗体重链和/或轻链的多核苷酸的功能元件。
  7. 根据权利要求6所述的功能元件，其特征在于，所述功能元件为线性表达框或者哺乳动物表达载体。
  8. 一种含有权利要求7所述功能元件的宿主细胞。
  9. 根据权利要求9所述的宿主细胞，其特征在于，所述细胞为Expi293F细胞或CHO-S细胞。
  10. 权利要求1-3任一所述的单克隆抗体在制备亨尼帕病毒病治疗药物中的应用。
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