WO2020192080A1 - 一种人7型腺病毒抗体及其应用 - Google Patents

一种人7型腺病毒抗体及其应用 Download PDF

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WO2020192080A1
WO2020192080A1 PCT/CN2019/110043 CN2019110043W WO2020192080A1 WO 2020192080 A1 WO2020192080 A1 WO 2020192080A1 CN 2019110043 W CN2019110043 W CN 2019110043W WO 2020192080 A1 WO2020192080 A1 WO 2020192080A1
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sequence
amino acid
acid sequence
adenovirus
positions
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French (fr)
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杨志新
王荣
陆健昇
余云舟
周权
周晓巍
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中国人民解放军军事科学院军事医学研究院
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/42—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum viral
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA 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

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  • the invention relates to a human type 7 adenovirus monoclonal antibody and its application.
  • Human adenoviruses belong to the genus of mammalian adenoviruses in the Adenoviridae family. Human adenoviruses were first discovered in 1953 and were isolated and cultured from the shrunken tonsil tissue of healthy people.
  • the virus genome is double-stranded DNA with a total length of about 36kb. These double-stranded DNA DNA and virus structural proteins combine to form the virus core.
  • the virus has no envelope.
  • the outer core is covered with a capsid.
  • the capsid is composed of 252 capsids, 240 of which are hexon proteins, 12 penton proteins, and shell It has a regular octahedral structure with a diameter of about 80-110nm.
  • Adenovirus infects people most commonly by infecting the respiratory tract and causing respiratory diseases. In addition, some viruses can also cause urinary and gastrointestinal infections. Studies have shown that adenoviruses that cause respiratory infections mainly include groups A, C, E and B1. Mainly B1 subgroup, B2 subgroup can infect human urinary system, F and D groups can infect human gastrointestinal system and conjunctival system respectively.
  • adenoviruses that cause respiratory infections in the world are types 1, 2, 3, 4, 7, 14, and 55 of the B1 subgroup, of which types 3, 4, 7, and 14 are the most common causes of outbreaks.
  • the type of virus has caused outbreaks of respiratory adenoviruses in Jiangsu and Taiwan provinces of my country, as well as South Korea, Singapore, and Malaysia. Since 2008, there have been many outbreaks of infectious diseases spread through the respiratory tract in different regions of my country. The epidemic has spread widely and is highly infectious.
  • the laboratory pathogens have been identified as Group B adenovirus types 7, 55 and 14 respectively. .
  • the human immune system can stimulate humoral and cellular immune responses and gradually control the infection and clear the virus.
  • Most viruses start to produce IgM and IgG in the patient about 3 days after infecting the human body. Later, most of them began to occur in 7-10 days, and then gradually increased, generally reaching a peak in about 1 month. Therefore, neutralizing antibodies have always played an important role in the recovery of viral infections.
  • Adenovirus infection in the human body can also induce a strong humoral immune response and produce specific antibodies.
  • adenovirus neutralizing antibody is considered to be an effective and specific anti-adenovirus therapy.
  • the development of adenovirus neutralizing antibody should be a major development direction for the development of adenovirus infection treatment drugs in the future.
  • the purpose of the present invention is to provide a human type 7 adenovirus monoclonal antibody and its application.
  • the present invention first provides a monoclonal antibody, including a heavy chain variable region and a light chain variable region; the heavy chain variable region includes three complementarity determining regions HCDR1, HCDR2, and HCDR3; the light chain variable region Including three complementary decision areas LCDR1, LCDR2 and LCDR3;
  • amino acid sequence of the HCDR1 is as follows (a1) or (a2) or (a3):
  • Sequence 2 of the sequence listing is from the amino acid sequence shown at positions 26-33 at the N-terminal;
  • the HCDR2 is as follows (a4) or (a5) or (a6):
  • Sequence 2 of the sequence listing is from the amino acid sequence shown at positions 51-58 of the N-terminal;
  • the HCDR3 is as follows (a7) or (a8) or (a9):
  • Sequence 2 of the sequence listing is from the amino acid sequence shown at positions 97-110 at the N-terminal;
  • amino acid sequence of LCDR1 is as follows (b1) or (b2) or (b3):
  • the amino acid sequence of the LCDR2 is as follows (b4) or (b5) or (b6):
  • amino acid sequence of LCDR3 is as follows (b7) or (b8) or (b9):
  • amino acid sequence of the heavy chain variable region is as follows (c1) or (c2) or (c3):
  • amino acid sequence of the light chain variable region is as follows (c4) or (c5) or (c6):
  • amino acid sequence of the heavy chain of the antibody is as follows (c7) or (c8) or (c9):
  • amino acid sequence of the light chain of the antibody is as follows (c10) or (c11) or (c12):
  • the antibody is an IgG1 antibody.
  • the present invention also protects the gene encoding the antibody.
  • variable region of the heavy chain of the antibody is as follows (d1) or (d2) or (d3):
  • (d2) A DNA molecule that has 75% or more homology with the nucleotide sequence defined in (d1) and encodes the variable region of the heavy chain;
  • the gene encoding the light chain variable region of the antibody is as follows (d4) or (d5) or (d6):
  • (d6) A DNA molecule that hybridizes to the nucleotide sequence defined by (d4) or (d5) under stringent conditions and encodes the aforementioned heavy chain variable region.
  • the gene encoding the heavy chain of the antibody is as follows (d7) or (d8) or (d9):
  • (d9) A DNA molecule that hybridizes to the nucleotide sequence defined by (d7) or (d8) under stringent conditions and encodes the aforementioned heavy chain.
  • the gene encoding the light chain of the antibody is as follows (d10) or (d11) or (d12):
  • (d12) A DNA molecule that hybridizes to the nucleotide sequence defined by (d10) or (d11) under stringent conditions and encodes the aforementioned light chain.
  • the present invention also protects applications, as shown in any of the following:
  • the present invention also protects a drug, the active ingredient of which is any of the above-mentioned monoclonal antibodies; the use of the drug is any one of the following (a)-(c):
  • the present invention also protects the method, as shown in any of the following:
  • a method for preventing and/or treating diseases caused by adenovirus infection is to use any of the above monoclonal antibodies to prevent and/or treat diseases caused by adenovirus infection;
  • a method for inhibiting adenovirus and/or neutralizing adenovirus is to use any of the above monoclonal antibodies to inhibit adenovirus and/or neutralizing adenovirus.
  • any of the aforementioned adenoviruses is a human adenovirus.
  • the human adenovirus is human type 7 adenovirus.
  • FIG. 1 shows the expression and purification of 3-3E.
  • Figure 2 shows the SDS-PAGE electrophoresis detection of HAdV7.
  • Figure 3 shows the electron microscope detection of HAdV7.
  • Figure 4 shows the antigen specificity analysis of 3-3E.
  • Figure 5 shows the affinity analysis of 3-3E.
  • Figure 6 shows the construction of an adenovirus infection animal model.
  • Figure 7 shows the effect of 3-3E vaccination on the body weight of CB-17 SCID mice.
  • Figure 8 shows the protective effect of 3-3E on HAdV7-infected CB-17 SCID mice.
  • the following examples facilitate a better understanding of the present invention, but do not limit the present invention.
  • the experimental methods in the following examples are conventional methods unless otherwise specified.
  • the test materials used in the following examples, unless otherwise specified, are all purchased from conventional biochemical reagent stores.
  • the quantitative experiments in the following examples are all set to three repeated experiments, and the results are averaged.
  • HAdV7 virus human/CHN/GZ6965/2001, recorded in the literature: Yu Z, Zeng Z, Zhang J, et al. Fatal Community-acquired Pneumonia in Children Caused by Re-emergent Human Adenovirus 7d and Associated with Higher Illness Rate[J].Scientific Reports,2016,6:37216.; The public can obtain it from the Academy of Military Medicine of the Chinese Academy of Military Sciences.
  • Influenza virus antigen A/swine/Colorado/1/77(H3N2), documented in literature: Karasin A I, Schutten M, Cooper L A, et al. Genetic characterization of H3N2 influenza viruses isolated from North America, 1977 -1999:Evidence for wholly human and reassortant virus genotypes[J].Virus Research,2000,68(1):71-85.; The public can obtain it from the Academy of Military Medicine of the Chinese People's Liberation Army.
  • peripheral blood of DENV-1 infected patients during the convalescence period separate and collect peripheral blood mononuclear lymphocytes.
  • the collected peripheral blood mononuclear lymphocytes are added with anti-cell surface marker antibodies (BD, #555332, #555415, #555441, #560677, #555622) or control antibodies (BD, #555748, #555742, #555751, # 555749, #557872) were labeled and then flow sorted, and the sorted cells were used for antibody gene amplification.
  • the antibody gene was amplified, the amplified positive clones were sequenced and expressed, and the purified type 7 adenovirus sample was used as the antigen. After a large number of screening, analysis, and verification, an antibody sequence was obtained, named 3-3E antibody.
  • amino acid sequence of the heavy chain variable region of the 3-3E antibody is shown in sequence 2 of the sequence list (wherein, amino acid residues 26-33 from the N-terminal form CDR1, amino acid residues 51-58 form CDR2, The 97-110 amino acid residues constitute CDR3), and the coding gene is shown in sequence 1 of the sequence listing.
  • amino acid sequence of the light chain variable region of the 3-3E antibody is shown in Sequence 4 of the Sequence Listing (wherein, the amino acid residues from the 27th to 32nd positions of the N-terminal form CDR1, the 50-52th amino acid residues form CDR2, and the Amino acid residues at positions 89-96 constitute CDR3), and its coding gene is shown in sequence 3 of the sequence listing.
  • the 3-3E antibody is a fully human monoclonal antibody and is an IgG1 antibody.
  • FreeStyle TM HEK 293-F cells (Invitrogen, catalog number: R79007) to a concentration of 1.0 ⁇ 10 6 /ml, inoculate them into culture flasks, 37°C, 5% CO 2 , 125 rpm cell shake Incubate with shaking in the bed for 24 hours.
  • step 2 on the day of transfection, take the culture flask, add the transfection complex to the culture flask, culture in a cell shaker at 37°C, 5% CO 2 , 125 rpm, and start monitoring cell viability at 48 hours When the cell activity drops to 80-85%, the culture supernatant is collected by centrifugation at 1,000 rpm for 10 minutes, and the antibody expression is confirmed by SDS-PAGE electrophoresis (lane 2 in Figure 1).
  • Transfection complex Dilute 24 ⁇ l of FectoPRO transfection reagent in 3ml FreeStyle 293 medium (Gibco 12338-018), mix gently, and add 12 ⁇ g of recombinant expression vector containing heavy chain variable region prepared in step 1, containing light The recombinant expression vector of the variable region of the chain is 12 ⁇ g, mixed and placed at room temperature for 10 minutes.
  • step 2 filter the supernatant with a 0.45 ⁇ m filter membrane to remove impurities, add 10 ⁇ PB to adjust the ion concentration and the binding buffer solution; use the AKTA purification system (GE, AKTA EXPLORER) for antibody purification, in AKTA Install HiTrap MabSelect Xtra purification column in the purification instrument, set the corresponding system parameters, equilibrate the purification column with binding buffer and load the sample, then continue to equilibrate, then rinse the pre-packed column with citric acid solution (pH3.0) to elute the antibody protein
  • citric acid solution pH3.0
  • step 4 Take the antibody solution purified in step 3, detect the antibody expression by SDS-PAGE electrophoresis (lane 3 in Figure 1), and take a sample to determine the protein concentration with a NanoDrop ultraviolet spectrophotometer (Thermo Scientific). The detected protein concentration is 1.25 mg/mL .
  • Adenovirus culture culture A549 cells (Beijing Concord Cell Resource Center, Item No. 25) in conventional DMEM+10% (volume percentage) FBS medium. Passage A549 cells in 75cm 2 cell flasks one day before virus inoculation.
  • adenovirus concentrate the harvested virus culture was centrifuged at 4,000 rpm for 10 minutes to remove cell debris, the supernatant was transferred to an ultrafiltration tube with a molecular weight cut-off of 50kD (MILIPORE, catalog number UFC805008), and centrifuged at 4,000 rpm to reduce the volume to Collect the retentate at 1/30 of the initial volume and store it at -80°C after aliquoting, which is the virus concentrate.
  • MILIPORE molecular weight cut-off of 50kD
  • A549 cells (Beijing Concord Cell Resource Center, Item No. 25) in good growth condition were taken, and after trypsinization, DMEM+10% (volume percentage) FBS adjusted the cell density to 3 ⁇ 10 5 /ml, and inoculated in 96-well cell culture plate, 100 ⁇ l per well, 37°C, 5% CO 2 culture; remove the 96-well plate on the day of the experiment, discard the medium, wash the serum-free medium, add DMEM+2% (volume percentage) FBS, 100 ⁇ l/well; then use serum-free medium 10-fold dilution of the virus solution to be tested, 10 -1 to 10 -8, a total of 8 gradients, the diluted virus is added to the prepared 96-well plate at 10 ⁇ l/well, each 8 wells with a dilution gradient, and a blank control group was set at the same time. After the operation, the cells were cultured and observed in a 37°C, 5% CO 2 incubator. After 7 days, the cell death in
  • LgTCID50 distance ratio ⁇ difference between logarithm of dilution + logarithm of dilution higher than 50% lesion rate
  • the virus titer of the HAdV7 virus stock solution used in this study 5.0 ⁇ 10 8 TCID50/ml
  • the virus titer of the virus concentrate 1.0 ⁇ 10 10 TCID50/ml.
  • the virus stock solution that was completely inactivated by the inactivation test centrifuged at 4,000 rpm for 10 minutes to remove cell debris, equilibrated the Sepharose 4Fast Flow gel column with PBS buffer and loaded the sample, and then eluted with PBS.
  • the first elution peak is The target virus peak, collect the elution peak, and then add 12.5ml heavy density CsCl solution [42.23g cesium chloride+57.77ml of 10mM Tris-HCL(PH 7.9- 8)], then slowly add 12.5ml of light-density CsCl solution [22.39g cesium chloride + 77.61ml of 10mM Tris-HCL(PH 7.9-8)], then add 15ml of virus suspension, balance, and place in ultracentrifugation Centrifuge in a machine (Beckman L100-XP) at 25,000 rpm and 4°C for 2 hours. Collect the bands between the light density and heavy density cesium chloride solutions, dialyze with PBS, filter and sterilize to obtain HAdV7 inactivated virus.
  • Samples were taken for conventional SDS-PAGE detection and electron microscope observation.
  • the preparation process of the electron microscope sample is as follows: sample 2.5% glutaraldehyde (0.075% PBS, diluted with pH 7.4) and fix at 4°C for 2 hours; then take 15-20ul of the liquid to be tested and drop it on the copper mesh (carbon-containing support film), at room temperature Incubate for 5-10 minutes; filter paper to absorb liquid; 3% phosphotungstic acid (prepared with distilled water) for staining at room temperature for 2 minutes; filter paper to absorb liquid; observe on the machine.
  • 3-3E antibody specifically binds human type 7 inactivated adenovirus
  • Step 1 Take 4 ⁇ l of the HAdV7 inactivated virus (200ng) prepared in Step 1 (4), add carbonate coating buffer (pH 9.6) to 100ul, add 100ul per well to the microplate (Corning 9018), set Replicate 3 wells and coat overnight at 4°C.
  • E antibody stock solution (concentration 300 ⁇ g/ml), incubate at 37°C for 90min, and then wash the plate with PBST (PBS+0.1% Tween20) 3 times.
  • Microplate reader 492nm/630nm dual wavelength to measure the optical density value.
  • the above steps also set up a control group that uses inactivated influenza virus antigen (influenza virus A/swine/Colorado/1/77 (H3N2) instead of HAdV7 virus, prepared according to the method of step 1) as the antigen to replace HAdV7 inactivated virus.
  • inactivated influenza virus antigen influenza virus A/swine/Colorado/1/77 (H3N2)
  • Microplate reader 492nm/630nm dual wavelength to measure the optical density value.
  • step 2 After completing step 1, take out the 96-well plate on the day of the experiment, discard the medium, wash the serum-free medium, add DMEM+2% (volume percentage) FBS, 100 ⁇ l per well; then group into groups and proceed as follows:
  • Anti-DENV1 Anti-DENV1 antibody (described in the literature: Potent Neutralization Ability of a Human Monoclonal Antibody against Serotype 1 Dengue Virus. Frontiers in Microbiology, 1 June 2018, Volume 9, Article 1214; the public can download Obtained by the Military Medical Research Institute of the Chinese People’s Liberation Army Academy of Military Sciences) diluted with serum-free medium to obtain different concentrations (25 ⁇ g/ml, 12.5 ⁇ g/ml, 6.25 ⁇ g/ml, 3.2 ⁇ g/ml, 1.6 ⁇ g/ml, 0.8 ⁇ g/ ml, 0.4 ⁇ g/ml, 0.2 ⁇ g/ml and 0.1 ⁇ g/ml) Anti-DG antibody test antibody solution; the test antibody solution and the HAdV7 virus stock solution prepared in step 1 of Example 3 (with serum-free Dilute the culture medium to a virus concentration of 2 ⁇ 10 3 TCID50/mL) mix in a volume ratio of 1:1, incubate at 37°C for 1.5
  • Anti-EGFR Irrelevant antibody control group
  • Dilute Anti-EGFR antibody (described in the invention patent CN102993305B) with serum-free medium to obtain different concentrations (25 ⁇ g/ml, 12.5 ⁇ g/ml, 6.25 ⁇ g/ml, 3.2 ⁇ g/ml, 1.6 ⁇ g/ml, 0.8 ⁇ g/ml, 0.4 ⁇ g/ml, 0.2 ⁇ g/ml and 0.1 ⁇ g/ml)
  • Anti-DG antibody test antibody solution compare the test antibody solution with step 1 of Example 3
  • the HAdV7 virus stock solution prepared in 1 (diluted with serum-free medium to a virus concentration of 2 ⁇ 10 3 TCID50/mL) was mixed at a volume ratio of 1:1, incubated at 37°C for 1.5 hours, then added to the well, and placed in 37 Incubate in an incubator at °C and 5% CO2 for 1 hour.
  • VRUS Virus group: The serum-free medium and the HAdV7 virus stock solution prepared in step 1 of Example 3 (diluted with serum-free medium to a virus concentration of 2 ⁇ 10 3 TCID50/mL) according to the volume ratio 1:1 Mix, incubate at 37°C for 1.5h, add to the well, 100 ⁇ l/well, and continue to incubate in a 37°C, 5% CO 2 incubator for 1h.
  • CELL Serum-free medium, incubated at 37°C for 1.5 hours and then added to the wells, and then placed in an incubator at 37°C and 5% CO2 for 1 hour.
  • step 2 take the 96-well plate, discard the supernatant, add DMEM+2% (volume percentage) FBS medium (100 ⁇ l/well), 37°C, 5% CO2 and continue to culture for 1 week until the cells appear Obvious lesions; take out the culture plate, observe under a microscope, count the number of cell wells with no lesions, partial lesions and complete lesions, and calculate the inhibitory rate of antibodies against adenovirus infection (Table 1).
  • mice Take CB-17 SCID mice (Vitalliva), female, 16-18g, weighed on the day of the experiment, and randomly divided into experimental group and control group (12 mice in each group), isoflurane inhalation anesthesia, and the experimental group with nasal drip
  • the HAdV7 virus concentrate (10 8 TCID50/mouse) prepared in step 1 of Example 3 was inoculated, and the control group was nasally inoculated with an equal volume of medium. Regularly measure the body weight every day and observe the depilation, eating, drinking and activities of the tested animals.
  • mice in the experimental group began to eat and drink less on the second day of virus inoculation, their hair became rough, their bodies were thin, and the symptoms lasted for about 5 days. There was no significant change in the body of the control group, and the weight continued to increase, which confirmed that the mice in the experimental group were indeed infected with ADV7 virus. .
  • Example 2 Take CB-17 SCID mice (Vitolihua), female, 16-18g, weighed on the day of the experiment, and randomly divided into experimental group and control group (12 mice in each group), and isoflurane inhaled anesthesia.
  • the prepared 3-3E antibody was diluted with serum-free DMEM medium to obtain an antibody solution containing 200 mg/ml 3-3E antibody.
  • the experimental group was inoculated with 20 ⁇ l each in the nose, and the control group was inoculated with an equal volume of serum-free medium. Regularly measure the body weight every day and observe the depilation, eating, drinking and activities of the tested animals.
  • mice Take CB-17 SCID mice (Vitalliva), females, 16-18g, weigh them on the day of the experiment, and randomly divide them into an experimental group and a control group (12 mice in each group) and perform the following operations:
  • mice Dilute the prepared 3-3E antibody with serum-free DMEM medium to obtain an antibody solution containing 200ug/ml 3-3E antibody; compare the antibody solution with that prepared in step 1 of Example 3 HAdV7 virus concentrate (virus concentration is 10 10 TCID50/ml) is mixed at a volume ratio of 1:1, mixed at 37°C and incubated for 1 hour, inoculated intranasally with the experimental group of mice prepared in step 1, each 20 ⁇ l, observe the mice's hair loss and eating , Drinking water and activities, and record the weight of the mice.
  • HAdV7 virus concentrate virus concentration is 10 10 TCID50/ml
  • Control group (VIRUS-only): The HAdV7 virus concentrate prepared in step 1 of Example 3 (the virus concentration is 10 10 TCID50/ml) is mixed with serum-free DMEM medium in a volume ratio of 1:1, and incubated at 37°C At 1h, inoculate the mice in the experimental group prepared in step 1 (each inoculation amount is 20 ⁇ l) by nasal drip. Observe the depilation, eating, drinking and activity of the mice and record the weight of the mice.
  • the inventor of the present invention discovered the anti-human type 7 adenovirus antibody 3-3E, which is used for the prevention and treatment of adenovirus infection, which has important biological and medical significance.

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Abstract

本发明公开了一种人7型腺病毒单克隆抗体及其应用。本发明提供了一种抗体，包括重链可变区和轻链可变区；所述重链可变区包括三个互补决定区HCDR1、HCDR2和HCDR3；所述轻链可变区包括三个互补决定区LCDR1、LCDR2和LCDR3；所述HCDR1、HCDR2和HCDR3依次如序列表的序列2自N端第26-33位、第51-58位、第97-110位所示；所述LCDR1、LCDR2和LCDR3依次如序列表的序列4自N端第27-32位、第50-52位、第89-96位所示。本发明的发明人基于临床需求，发现了抗人7型腺病毒抗体3-3E，用于腺病毒感染的防治，具有重要的生物学和医学意义。

Description

一种人7型腺病毒抗体及其应用 技术领域
本发明涉及一种人7型腺病毒单克隆抗体及其应用。
背景技术
人腺病毒属于腺病毒科哺乳动物腺病毒属，人腺病毒最早发现于1953年，分离培养自健康人萎缩的扁桃腺样组织；病毒的基因组为双股DNA，全长约36kb，这些双链DNA与病毒结构蛋白结合组成病毒核心，病毒无包膜，核心外面包被有衣壳，该衣壳由252个壳粒组成，其中240个为六邻体蛋白，12个五邻体蛋白，外壳呈规则的20面体结构，直径大约80～110nm。
目前已发现的腺病毒有A-G共7个亚群，67个不同的血清型，其中能感染人并致病的有55个亚型。腺病毒感染人最常见的是感染呼吸道并引发呼吸系统疾病，此外部分病毒还可以引起泌尿和胃肠等系统感染，研究表明引起呼吸道感染的腺病毒主要有A、C、E和B1组，其中主要是B1亚组，B2亚组可感染人的泌尿系统，F、D组分别可感染人的胃肠系统和结膜系统。目前已知世界上引起呼吸道感染的腺病毒有B1亚组的1、2、3、4、7、14和55型，其中3型、4型、7型、14型为最常见的引起暴发疫情的型别，曾引起我国江苏和台湾省，以及韩国、新加坡、马来西亚等呼吸道腺病毒暴发疫情。自2008年以来，我国不同地区先后发生多起经呼吸道传播的暴发传染病疫情，疫情波及面广，传染性强，经实验室病原检测鉴定分别为B组7型、55型和14型腺病毒。
一般情况下，病毒感染人体时，人体免疫系统能够激发体液免疫和细胞免疫反应并逐渐控制感染、清除病毒，大部分病毒在感染人体引起发病后3天左右，患者体内的IgM开始产生，IgG产生稍晚大部分在7～10d开始产生，随后逐渐升高，一般1个月左右达到顶峰。因此中和抗体在病毒感染康复中一直扮演者重要的角色，腺病毒感染人体也可诱发较强的体液免疫反应，产生特异性抗体。研究表明机体对同型腺病毒再次感染可产生有效免疫，且可以产生较长时间的免疫保护(可达10年以上)，康复后一般不会再次感染，而其中起作用的就是腺病毒感染过程成诱导机体产生的中和抗体，据研究发现我国40％～60％的6～15岁的人具有1、2和5型中和抗体，而且母亲的抗体能保护婴儿免除严重的腺病毒感染。因此腺病毒中和抗体被认为是可以作为一种有效而特异的抗腺病毒治疗手段，腺病毒中和抗体的研制应该是未来腺病毒感染治疗药物开发的一个主要发展方向。
目前世界上尚没有针对腺病毒的特异性治疗药物用于临床，药物研发也多限于化药——抗DNA病毒的核苷类似物，而针对腺病毒的特异性生物药研究还是空白。
发明公开
本发明的目的是提供一种人7型腺病毒单克隆抗体及其应用。
本发明首先提供了一种单克隆抗体，包括重链可变区和轻链可变区；所述重链可变区包括三个互补决定区HCDR1、HCDR2和HCDR3；所述轻链可变区包括三个互补决定区LCDR1、LCDR2和LCDR3；
所述HCDR1的氨基酸序列为如下(a1)或(a2)或(a3)：
(a1)序列表的序列2自N端第26-33位所示的氨基酸序列；
(a2)将序列表的序列2自N端第26-33位所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加得到的具有相同功能的氨基酸序列；
(a3)与序列表的序列2自N端第26-33位所示的氨基酸序列具有75％或75％以上的同源性且具有相同功能的氨基酸序列；
所述HCDR2为如下(a4)或(a5)或(a6)：
(a4)序列表的序列2自N端第51-58位所示的氨基酸序列；
(a5)将序列表的序列2自N端第51-58位所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加得到的具有相同功能的氨基酸序列；
(a6)与序列表的序列2自N端第51-58位所示的氨基酸序列具有75％或75％以上的同源性且具有相同功能的氨基酸序列；
所述HCDR3为如下(a7)或(a8)或(a9)：
(a7)序列表的序列2自N端第97-110位所示的氨基酸序列；
(a8)将序列表的序列2自N端第97-110位所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加得到的具有相同功能的氨基酸序列；
(a9)与序列表的序列2自N端第97-110位所示的氨基酸序列具有75％或75％以上的同源性且具有相同功能的氨基酸序列；
所述LCDR1的氨基酸序列为如下(b1)或(b2)或(b3)：
(b1)序列表的序列4自N端第27-32位所示的氨基酸序列；
(b2)将序列表的序列4自N端第27-32位所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加得到的具有相同功能的氨基酸序列；
(b3)与序列表的序列4自N端第27-32位所示的氨基酸序列具有75％或75％以上的同源性且具有相同功能的氨基酸序列；
所述LCDR2的氨基酸序列为如下(b4)或(b5)或(b6)：
(b4)序列表的序列4自N端第50-52位所示的氨基酸序列；
(b5)将序列表的序列4自N端第50-52位所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加得到的具有相同功能的氨基酸序列；
(b6)与序列表的序列4自N端第50-52位所示的氨基酸序列具有75％或 75％以上的同源性且具有相同功能的氨基酸序列；
所述LCDR3的氨基酸序列为如下(b7)或(b8)或(b9)：
(b7)序列表的序列4自N端第89-96位所示的氨基酸序列；
(b8)将序列表的序列4自N端第89-96位所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加得到的具有相同功能的氨基酸序列；
(b9)与序列表的序列4自N端第89-96位所示的氨基酸序列具有75％或75％以上的同源性且具有相同功能的氨基酸序列。
所述重链可变区的氨基酸序列为如下(c1)或(c2)或(c3):
(c1)序列表的序列2所示的氨基酸序列；
(c2)将序列表的序列2所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加得到的具有相同功能的氨基酸序列；
(c3)与序列表的序列2所示的氨基酸序列具有75％或75％以上的同源性且具有相同功能的氨基酸序列；
所述轻链可变区的氨基酸序列为如下(c4)或(c5)或(c6):
(c4)序列表的序列4所示的氨基酸序列；
(c5)将序列表的序列4所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加得到的具有相同功能的氨基酸序列；
(c6)与序列表的序列4所示的氨基酸序列具有75％或75％以上的同源性且具有相同功能的氨基酸序列。
所述抗体的重链的氨基酸序列为如下(c7)或(c8)或(c9):
(c7)序列表的序列6所示的氨基酸序列；
(c8)将序列表的序列6所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加得到的具有相同功能的氨基酸序列；
(c9)与序列表的序列6所示的氨基酸序列具有75％或75％以上的同源性且具有相同功能的氨基酸序列。
所述抗体的轻链的氨基酸序列如下(c10)或(c11)或(c12):
(c10)序列表的序列8所示的氨基酸序列；
(c11)将序列表的序列8所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加得到的具有相同功能的氨基酸序列；
(c10)与序列表的序列8所示的氨基酸序列具有75％或75％以上的同源性且具有相同功能的氨基酸序列。
所述抗体为IgG1抗体。
本发明还保护编码所述抗体的基因。
编码所述抗体的重链可变区的基因为如下(d1)或(d2)或(d3):
(d1)序列表的序列1所示的DNA分子；
(d2)与(d1)限定的核苷酸序列具有75％或75％以上同源性，且编码上 述重链可变区的DNA分子；
(d3)在严格条件下与(d1)或(d2)限定的核苷酸序列杂交，且编码上述重链可变区的DNA分子；
编码所述抗体的轻链可变区的基因为如下(d4)或(d5)或(d6):
(d4)序列表的序列3所示的DNA分子；
(d5)与(d4)限定的核苷酸序列具有75％或75％以上同源性，且编码上述重链可变区的DNA分子；
(d6)在严格条件下与(d4)或(d5)限定的核苷酸序列杂交，且编码上述重链可变区的DNA分子。
编码所述抗体的重链的基因为如下(d7)或(d8)或(d9):
(d7)序列表的序列5所示的DNA分子；
(d8)与(d7)限定的核苷酸序列具有75％或75％以上同源性，且编码上述重链的DNA分子；
(d9)在严格条件下与(d7)或(d8)限定的核苷酸序列杂交，且编码上述的重链的DNA分子。
编码所述抗体的轻链的的基因为如下(d10)或(d11)或(d12):
(d10)序列表的序列7所示的DNA分子；
(d11)与(d10)限定的核苷酸序列具有75％或75％以上同源性，且编码上述轻链的DNA分子；
(d12)在严格条件下与(d10)或(d11)限定的核苷酸序列杂交，且编码上述的轻链的DNA分子。
本发明还保护应用，为如下任一所示：
(A)以上任一所述的单克隆抗体在预防和/或治疗腺病毒感染引起的疾病中的应用；
(B)以上任一所述的单克隆抗体在抑制腺病毒和/或中和腺病毒中的应用；
(C)以上任一所述的单克隆抗体在制备用于预防和/或治疗腺病毒感染引起的疾病的药物中的应用；
(D)以上任一所述的单克隆抗体在制备产品中的应用；所述产品的用途为如下(e1)和/或(e2)：
(e1)抑制腺病毒；
(e2)中和腺病毒。
本发明还保护药物，其活性成分为以上任一所述的单克隆抗体；所述药物的用途为如下(a)-(c)中的任一种：
(a)预防和/或治疗腺病毒感染引起的疾病；
(b)抑制腺病毒；
(c)中和腺病毒。
本发明还保护方法，为如下任一所示：
(1)一种预防和/或治疗腺病毒感染引起的疾病的方法，是利用以上任一所述单克隆抗体预防和/或治疗腺病毒感染引起的疾病；
(2)一种抑制腺病毒和/或中和腺病毒的方法，是利用以上任一所述单克隆抗体抑制腺病毒和/或中和腺病毒。
以上任一所述腺病毒为人腺病毒。所述人腺病毒为人7型腺病毒。
附图说明
图1为3-3E的表达与纯化检测。
图2为HAdV7的SDS-PAGE电泳检测。
图3为HAdV7的电镜检测。
图4为3-3E的抗原特异性分析。
图5为3-3E的亲和力分析。
图6为腺病毒感染动物模型的构建。
图7为3-3E接种对CB-17 SCID小鼠体重的影响分析。
图8为3-3E对HAdV7感染CB-17 SCID小鼠的保护作用。
实施发明的最佳方式
以下的实施例便于更好地理解本发明，但并不限定本发明。下述实施例中的实验方法，如无特殊说明，均为常规方法。下述实施例中所用的试验材料，如无特殊说明，均为自常规生化试剂商店购买得到的。以下实施例中的定量试验，均设置三次重复实验，结果取平均值。
HAdV7病毒：human/CHN/GZ6965/2001，记载于文献：Yu Z,Zeng Z,Zhang J,et al.Fatal Community-acquired Pneumonia in Children Caused by Re-emergent Human Adenovirus 7d Associated with Higher Severity of Illness and Fatality Rate[J].Scientific Reports,2016,6:37216.；公众可以从中国人民解放军军事科学院军事医学研究院获得。
流感病毒抗原：A/swine/Colorado/1/77(H3N2)，记载于文献：Karasin A I,Schutten M M,Cooper L A,et al.Genetic characterizat ion of H3N2influenza viruses isolated from pigs in North America,1977-1999:Evidence for wholly human and reassortant virus genotypes[J].Virus Research,2000,68(1):71-85.；公众可以从中国人民解放军军事科学院军事医学研究院获得。
实施例1、抗体的发现
采集康复期DENV-1感染者外周血，从中分离并收集外周血单核淋巴细胞。收集的外周血单核淋巴细胞加入抗细胞表面相应分子标记抗体(BD，#555332，#555415，#555441，#560677，#555622)或者对照抗体(BD，#555748，#555742，#555751，#555749，#557872)进行标记，然后进行流式分选，分选的细胞用于抗体基因扩增。以分离的单个B细胞为模板，扩增抗体基因，将扩增阳性的克隆 测序并表达，以7型腺病毒纯化样品为抗原，经过大量筛选、分析、验证，得到1个抗体序列，命名为3-3E抗体。
3-3E抗体的重链可变区的氨基酸序列如序列表的序列2所示(其中，自N端第26-33位氨基酸残基组成CDR1，第51-58位氨基酸残基组成CDR2，第97-110位氨基酸残基组成CDR3)，其编码基因如序列表的序列1所示。
3-3E抗体的轻链可变区的氨基酸序列如序列表的序列4所示(其中，自N端第27-32位氨基酸残基组成CDR1，第50-52位氨基酸残基组成CDR2，第89-96位氨基酸残基组成CDR3)，其编码基因如序列表的序列3所示。
实施例2、3-3E抗体的制备
一、重组质粒的构建
1、将pTSE-G1n载体(北京百特美博生物有限公司)SalI和PmlI位点之间的小片段替换为序列表的序列1所示的DNA分子，得到含有重链可变区的重组表达载体(已经测序验证)。所述含有重链可变区的重组表达载体中携带完整的编码3-3E抗体重链的序列(序列表的序列5)，其编码的氨基酸序列如序列表的序列6所示。
2、将pTSE-K载体(北京百特美博生物有限公司)SalI和PmlI位点之间的小片段替换为序列表的序列3所示的DNA分子，得到含有轻链可变区的重组表达载体(已经测序验证)。所述含有轻链可变区的重组表达载体中携带完整的编码3-3E抗体轻链的序列(序列表的序列7)，其编码的氨基酸序列如序列表的序列8所示。
3-3E抗体为全人源单抗，为IgG1抗体。
二、3-3E抗体的制备
1、转染前一天将FreeStyle TM HEK 293-F细胞(Invitrogen公司，货号：R79007)调整至浓度为1.0×10 6/ml，接种至培养瓶中，37℃、5％CO 2、125rpm细胞摇床中震荡培养24h。
2、完成步骤1后，转染当天，取所述培养瓶，将转染复合物加至培养瓶中，37℃、5％CO 2、125rpm细胞摇床中震荡培养，48小时开始监测细胞活度，在细胞活度降到80-85％时1,000rpm离心10min收集培养上清，SDS-PAGE电泳检测确定抗体表达(图1的泳道2)。
转染复合物：将24μl的FectoPRO转染试剂稀释于3ml FreeStyle 293培养基(Gibco 12338-018)，轻轻混匀，加入步骤一制备的含有重链可变区的重组表达载体12μg，含有轻链可变区的重组表达载体12μg，混匀后室温放置10min。
3、完成步骤2后，将上清用0.45μm滤膜过滤以去除杂质，加入10×PB调节离子浓度与结合缓冲液相近；用AKTA纯化系统(GE,AKTA EXPLORER)进行抗体纯化，在AKTA纯化仪器中安装HiTrap MabSelect Xtra纯化柱，设定相应系统参数，用结合缓冲液平衡纯化柱并上样，随后继续平衡，然后用柠檬酸溶液(pH3.0) 冲洗预装柱以洗脱抗体蛋白，UV280达到100时开始收集，UV280降至100时结束收集，并将缓冲液置换为柠檬酸盐溶液(pH6.0)。
4、取步骤3纯化的抗体溶液，SDS-PAGE电泳检测抗体表达(图1的泳道3)，并取样用NanoDrop紫外分光光度计(Thermo Scientific)测定蛋白浓度，经检测蛋白浓度为1.25mg/mL。
实施例3、3-3E抗体的结合能力检测
一、人7型腺病毒病毒原液、病毒浓缩液与灭活病毒的制备
1、腺病毒病毒液制备
腺病毒培养培养：常规DMEM+10％(体积百分含量)FBS培养基培养A549细胞(北京协和细胞资源中心，货号：25)，接种病毒前一天将A549细胞传代于75cm 2细胞瓶中，使第二天接种病毒时细胞密度达到75％～90％；接种当天，缓慢吸出培养瓶内的细胞培养基，加入5ml DMEM轻轻冲洗细胞后弃去，另加入3ml DMEM+2％(体积百分含量)FBS；用微量移液器吸取适量HAdV7病毒于细胞瓶中，按MOI≈0.001进行感染，均匀晃动瓶子数次使病毒分散均匀，置于37℃，5％CO 2的培养箱中吸附2h，期间每隔30min左右晃动培养瓶一次；吸附完成后，弃掉病毒培养液，重新加入15ml新的DMEM+2％(体积百分含量)FBS，然后将细胞瓶放置在37℃，5％CO 2的培养箱中继续培养；每日观察细胞病变情况(病毒感染增殖后会出现细胞病变，表现为细胞皱缩、脱落等特征)；当75％-100％细胞出现病变时收获，-80℃冻融2次，4,000rpm离心5min，收集上清分装后保存于-80℃，即为病毒原液。
2、腺病毒浓缩液的制备：收获的病毒培养物，4,000rpm离心10分钟去除细胞碎片,上清转入截留分子量为50kD的超滤管(MILIPORE，货号UFC805008)，4,000rpm离心至体积减少至初始体积的1/30，收集截留液，分装后保存于-80℃，即为病毒浓缩液。
3、腺病毒滴度测定
实验前一天取生长状态良好的A549细胞(北京协和细胞资源中心，货号：25)，胰酶消化后DMEM+10％(体积百分含量)FBS调细胞密度至3×10 5/ml，接种于96孔细胞培养板，每孔100μl，37℃、5％CO 2培养；实验当天取出96孔板，弃培养基，无血清培养基洗一遍，加入DMEM+2％(体积百分含量)FBS，100μl/孔；然后用无血清培养基10倍梯度稀释待测的病毒液，10 -1～10 -8共8个梯度，稀释好的病毒按照10μl/孔加入备好的96孔板中，每个稀释梯度8孔，同时设置空白对照组，操作完成细胞置于37℃、5％CO 2的培养箱中培养观察，7天后计数各孔细胞死亡情况，根据下面公式计算病毒原液的TCID50。
距离比例＝(高于50％病变率的百分数-50％)/(高于50％病变率的百分数-低于50％病变率的百分数)
LgTCID50＝距离比例×稀释度对数之间的差+高于50％病变率的稀释度的对数
经检测和计算，本研究中所用HAdV7病毒原液的病毒滴度＝5.0×10 8TCID50/ml，病毒浓缩液的病毒滴度＝1.0×10 10TCID50/ml。
4、腺病毒的灭活与纯化
取病毒原液转移至500ml样品瓶，用碳酸氢钠调pH至7.6，然后按照1:2000的比例加入β-丙内酯，边加边搅拌，充分混匀后，4℃继续搅拌灭活，24小时后再次调pH至7.6，按照1:2000的比例补加β-丙内酯，4℃继续搅拌灭活24小时，取不少于1‰体积的样品37℃水浴水解4小时(中间观察到样品颜色变黄时用碳酸氢钠调整pH至7.0左右)，水解结束，取前一天传至25cm 2细胞瓶的A549细胞，按照1ml样品接种1瓶25cm 2A549细胞的比例接种(不足1ml按1ml计算)，同时设置接种未灭活病毒的细胞及未处置空细胞作为对照，置于37℃、5％CO 2的培养箱中培养观察，7天后盲传至新的A549细胞，继续观察，如此盲传3代，接种未灭活病毒样品细胞病变而灭活实验组细胞与空白细胞均不病变者，判定为灭活检测结果可信，灭活完全，否则则灭活不完全，需要重新灭活、检测。
经灭活检测证实灭活彻底的病毒原液，4,000rpm离心10分钟去除细胞碎片，用PBS缓冲液平衡Sepharose 4Fast Flow凝胶柱并上样，随后用PBS洗脱，第一个洗脱峰即为目的病毒峰，收集该洗脱峰，然后在Amicon-Ultra-15超滤管(50kD)中加入12.5ml重密度的CsCl溶液【42.23g氯化铯+57.77ml的10mM Tris-HCL(PH 7.9-8)】，再缓慢加入12.5ml轻密度的CsCl溶液【22.39g氯化铯+77.61ml的10mM Tris-HCL(PH 7.9-8)】，再加入15ml的病毒悬浮液，配平，放于超速离心机(Beckman L100-XP)中，25,000rpm，4℃离心2小时。收集在轻密度和重密度氯化铯溶液之间的条带，PBS透析，过滤除菌得到HAdV7灭活病毒。
取样分别进行常规SDS-PAGE检测和电镜观察。电镜样品制备过程如下：取样2.5％戊二醛(0.075％PBS,pH7.4稀释)，4℃固定2小时；然后取15-20ul待检液体，滴至铜网(含碳支持膜)，室温孵育5-10min；滤纸吸干液体；3％磷钨酸(蒸馏水配制)室温染色2min；滤纸吸干液体；上机观察。
SDS-PAGE结果见图2，电镜结果见图3。
二、3-3E抗体特异性结合人7型灭活腺病毒
1、取4μl步骤一的4中制备的HAdV7灭活病毒(200ng)，补碳酸盐包被缓冲液(pH 9.6)至100ul，按每孔100ul加入到酶标板(Corning 9018)中，设置复孔3个，4℃包被过夜。
2、完成上述步骤后，取所述酶标板，PBST洗板3次，加入含有2％(质量百分含量)BSA的PBS封闭液，37℃孵育2小时。
3、完成上述步骤后，取所述酶标板，弃去封闭液，每孔加入100μl的3-3
E抗体原液(浓度300μg/ml)，37℃孵育90min，然后PBST(PBS+0.1％Tween20)洗板3次。
4、完成上述步骤后，取上述酶标板，每孔加入100μl的1：4000倍稀释酶 标抗体(山羊抗人IgG-HRP，中杉金桥，货号ZB-2304)，37℃孵育45min，然后PBST(PBS+0.1％Tween20)洗板3次。
5、完成上述步骤后，取所述酶标板，每孔加入50μl OPD底物显色液，室温下孵育10分钟。
6、完成上述步骤后，取所述酶标板，每孔加入50μl 1M硫酸溶液终止酶联反应。
7、酶标仪492nm/630nm双波长测定光密度值。
以上步骤同时设置采用灭活流感病毒抗原(将流感病毒A/swine/Colorado/1/77(H3N2)替代HAdV7病毒，按照步骤一的方法制备得到)作为抗原替代HAdV7灭活病毒的对照组。
结果如图4所示。结果表明，3-3E抗体可以特异性结合灭活腺病毒而不结合流感病毒(Flu)。
三、3-3E抗体的抗原结合能力分析
1、取4μl步骤一的4中制备的HAdV7灭活病毒(200ng)，补碳酸盐包被缓冲液(pH 9.6)至100μl，按每孔100ul加入到酶标板(Corning货号：9018)中，设置复孔3个，4℃包被过夜。
2、完成上述步骤后，取所述酶标板，PBST洗板3次，加入含有2％(质量百分含量)BSA的PBS封闭液，37℃孵育2小时。
3、完成上述步骤后，取所述酶标板，弃去封闭液，每孔加入100μl按照2倍等比稀释的3-3E抗体液(起始浓度400μg/ml)，一共设置30个梯度，37℃孵育90min，然后PBST洗板3次。
4、完成上述步骤后，取所述酶标板，每孔加入100μl的1：4000倍稀释HRP标记的抗人IgG抗体(中杉金桥，货号ZB-2304)，37℃孵育45min，PBST洗板3次。
5、完成上述步骤后，取所述酶标板，每孔加入50μl OPD底物显色液，室温下孵育10分钟。
6、完成上述步骤后，取所述酶标板，每孔加入50μl 1M硫酸溶液终止酶联反应。
7、酶标仪492nm/630nm双波长测定光密度值。
结果如图5所示。图5中，横坐标为蛋白浓度的对数值，纵坐标为光密度值。分析显示3-3E抗体与人7型腺病毒的结合能力为EC50＝339.3nM。
实施例4、3-3E抗人7型腺病毒感染的药效
一、体外检测3-3E抗人7型腺病毒感染的预防与治疗作用
1、实验前一天取生长状态良好的A549的细胞，胰酶消化，DMEM+10％(体积百分含量)FBS调细胞密度至3×10 5/ml，接种于96孔细胞培养板，每孔100μl，37℃5％CO 2培养。
2、完成步骤1后，实验当天取出96孔板，弃培养基，无血清培养基洗一遍，加入DMEM+2％(体积百分含量)FBS，每孔100μl；然后分组进行如下操作：
实验组(3-3E 0h)：将实施例2制备的3-3E抗体用无血清培养基稀释，得到含有不同浓度(50μg/ml、25μg/ml、12.5μg/ml、6.25μg/ml、3.2μg/ml、1.6μg/ml、0.8μg/ml、0.4μg/ml、0.2μg/ml)3-3E抗体的待测抗体溶液；将待测抗体溶液与实施例3步骤一的1中制备的HAdV7病毒原液(用无血清培养基稀释至病毒浓度为2×10 3TCID50/mL)按照体积比1:1混合，37℃孵育1.5h后加至孔内，继续置于37℃，5％CO2的培养箱中孵育1h。
实验组(3-3E+1h)：实施例2制备的3-3E抗体用无血清培养基稀释，得到含有不同浓度((50μg/ml、25μg/ml、12.5μg/ml、6.25μg/ml、3.2μg/ml、1.6μg/ml、0.8μg/ml、0.4μg/ml、0.2μg/ml)3-3E抗体的待测抗体溶液；将实施例3步骤一的1中制备的HAdV7病毒原液(用无血清培养基稀释至病毒浓度为2×10 3TCID50/mL)加至孔内，37℃孵育1h后，将与病毒液等体积的待测抗体溶液加至孔内，继续置于37℃，5％CO2的培养箱中孵育1h。
无关抗体对照组(Anti-DENV1)：将Anti-DENV1抗体(记载于文献：Potent Neutralization Ability of a Human Monoclonal Antibody Against Serotype 1 Dengue Virus。Frontiers in Microbiology，1 June2018，Volume 9，Article 1214；公众可以从中国人民解放军军事科学院军事医学研究院获得)用无血清培养基稀释，得到含有不同浓度(25μg/ml、12.5μg/ml、6.25μg/ml、3.2μg/ml、1.6μg/ml、0.8μg/ml、0.4μg/ml、0.2μg/ml和0.1μg/ml)Anti-DG抗体的待测抗体溶液；将待测抗体溶液与实施例3步骤一的1中制备的HAdV7病毒原液(用无血清培养基稀释至病毒浓度为2×10 3TCID50/mL)按照体积比1:1混合，37℃孵育1.5h后加至孔内，继续置于37℃，5％CO2的培养箱中孵育1h。
无关抗体对照组(Anti-EGFR)：将Anti-EGFR抗体(记载于发明专利专利CN102993305B)用无血清培养基稀释，得到含有不同浓度(25μg/ml、12.5μg/ml、6.25μg/ml、3.2μg/ml、1.6μg/ml、0.8μg/ml、0.4μg/ml、0.2μg/ml和0.1μg/ml)Anti-DG抗体的待测抗体溶液；将待测抗体溶液与实施例3步骤一的1中制备的HAdV7病毒原液(用无血清培养基稀释至病毒浓度为2×10 3TCID50/mL)按照体积比1:1混合，37℃孵育1.5h后加至孔内，继续置于37℃，5％CO2的培养箱中孵育1h。
病毒组(VIRUS)：将无血清培养基与实施例3步骤一的1中制备的HAdV7病毒原液(用无血清培养基稀释至病毒浓度为2×10 3TCID50/mL)按照体积比1:1混合，37℃孵育1.5h后加至孔内，100μl/孔，继续置于37℃，5％CO 2的培养箱中孵育1h。
空细胞对照组(CELL)：无血清培养基，37℃孵育1.5h后加至孔内，继续置于37℃，5％CO2的培养箱中孵育1h。
3、完成步骤2后，取96孔板，弃上清，加入DMEM+2％(体积百分含量)FBS 培养基(100μl/孔)，37℃，5％CO2继续培养1周，直至细胞出现明显病变；取出培养板，镜下观察，计数未病变、部分病变和完全病变的细胞孔数，计算抗体对腺病毒感染的抑制率(表1)。
表1 3-3E抗HAdV7感染细胞检测结果
Figure PCTCN2019110043-appb-000001
*浓度单位：μg/mL；0h:抗体与病毒共孵育后接种细胞；+1h：病毒感染细胞1小时后补加抗体
结果表明本发明制备抗体3-3E可以有效抑制HAdV7的增殖，预防使用时EC50＝0.4μg/mL，在感染病毒后使用时EC50＝0.72μg/mL。
二、3-3E抗人7型腺病毒感染CB-17 SCID小鼠
1、动物模型建立
取CB-17 SCID小鼠(维通利华)，雌性，16-18g，实验当天称重，随机分为实验组和对照组(每组12只)，异氟烷吸入麻醉，实验组滴鼻接种实施例3步骤一的2中制备的HAdV7病毒浓缩液(10 8TCID50/只)，对照组滴鼻接种等体积的培养基。每天定时测体重并观察受试动物脱毛、进食、饮水及活动情况。
结果如图6所示。结果显示实验组小鼠接种病毒第二天开始进食和饮水减少，毛变粗糙，身体消瘦，症状持续5天左右，对照组身体没有明显变化，体重持续增长，证实实验组小鼠确实感染ADV7病毒。
2.抗体3-3E抗对未感染小鼠的影响
取CB-17 SCID小鼠(维通利华)，雌性，16-18g，实验当天称重，随机分为实验组和对照组(每组12只)，异氟烷吸入麻醉，将实施例2制备的3-3E抗体用无血清DMEM培养基稀释，得到含有200mg/ml 3-3E抗体的抗体溶液，实验组滴鼻每只20μl，对照组接种等体积无血清培养基。每天定时测体重并观察受试动物脱毛、进食、饮水及活动情况。
结果如图7所示。结果显示实验组与对照组相比身体没有明显变化，体重持续增长，证实实验组抗体接种不会对小鼠产生明显影响。
2、3-3E抗人7型腺病毒感染CB-17 SCID小鼠
取CB-17 SCID小鼠(维通利华)，雌性，16-18g，实验当天称重，随机分为实验组和对照组(每组12只)进行如下操作：
实验组(3-3E)：将制备的3-3E抗体用无血清DMEM培养基稀释，得到含有200ug/ml 3-3E抗体的抗体溶液；将抗体溶液与实施例3步骤一的2中制备的 HAdV7病毒浓缩液(病毒浓度为10 10TCID50/ml)按照体积比1:1混合，混合37℃孵育1h，滴鼻接种步骤1制备的实验组小鼠，每只20μl，观察小鼠脱毛、进食、饮水及活动情况并记录小鼠体重。
对照组(VIRUS-only)：将实施例3步骤一的2中制备的HAdV7病毒浓缩液(病毒浓度为10 10TCID50/ml)与无血清DMEM培养基按照体积比1:1混合，37℃孵育1h，滴鼻接种步骤1制备的实验组小鼠(每只的接种量为20μl)，观察小鼠脱毛、进食、饮水及活动情况并记录小鼠体重。
结果如图8所示。结果显示，对照组第二天出新明显的体形消瘦，毛色粗糙等症状，而实验组小鼠症状与对照组对比明显减轻，说明3-3E抗体可以有效减轻小鼠由于感染病毒导致的体重减轻和毛色粗糙等症状。
工业应用
本发明的发明人基于临床需求，发现了抗人7型腺病毒抗体3-3E，用于腺病毒感染的防治，具有重要的生物学和医学意义。

Claims (13)

  1. 一种单克隆抗体，包括重链可变区和轻链可变区；所述重链可变区包括三个互补决定区HCDR1、HCDR2和HCDR3；所述轻链可变区包括三个互补决定区LCDR1、LCDR2和LCDR3；
    所述HCDR1的氨基酸序列为如下(a1)或(a2)或(a3)：
    (a1)序列表的序列2自N端第26-33位所示的氨基酸序列；
    (a2)将序列表的序列2自N端第26-33位所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加得到的具有相同功能的氨基酸序列；
    (a3)与序列表的序列2自N端第26-33位所示的氨基酸序列具有75％或75％以上的同源性且具有相同功能的氨基酸序列；
    所述HCDR2为如下(a4)或(a5)或(a6)：
    (a4)序列表的序列2自N端第51-58位所示的氨基酸序列；
    (a5)将序列表的序列2自N端第51-58位所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加得到的具有相同功能的氨基酸序列；
    (a6)与序列表的序列2自N端第51-58位所示的氨基酸序列具有75％或75％以上的同源性且具有相同功能的氨基酸序列；
    所述HCDR3为如下(a7)或(a8)或(a9)：
    (a7)序列表的序列2自N端第97-110位所示的氨基酸序列；
    (a8)将序列表的序列2自N端第97-110位所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加得到的具有相同功能的氨基酸序列；
    (a9)与序列表的序列2自N端第97-110位所示的氨基酸序列具有75％或75％以上的同源性且具有相同功能的氨基酸序列；
    所述LCDR1的氨基酸序列为如下(b1)或(b2)或(b3)：
    (b1)序列表的序列4自N端第27-32位所示的氨基酸序列；
    (b2)将序列表的序列4自N端第27-32位所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加得到的具有相同功能的氨基酸序列；
    (b3)与序列表的序列4自N端第27-32位所示的氨基酸序列具有75％或75％以上的同源性且具有相同功能的氨基酸序列；
    所述LCDR2的氨基酸序列为如下(b4)或(b5)或(b6)：
    (b4)序列表的序列4自N端第50-52位所示的氨基酸序列；
    (b5)将序列表的序列4自N端第50-52位所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加得到的具有相同功能的氨基酸序 列；
    (b6)与序列表的序列4自N端第50-52位所示的氨基酸序列具有75％或75％以上的同源性且具有相同功能的氨基酸序列；
    所述LCDR3的氨基酸序列为如下(b7)或(b8)或(b9)：
    (b7)序列表的序列4自N端第89-96位所示的氨基酸序列；
    (b8)将序列表的序列4自N端第89-96位所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加得到的具有相同功能的氨基酸序列；
    (b9)与序列表的序列4自N端第89-96位所示的氨基酸序列具有75％或75％以上的同源性且具有相同功能的氨基酸序列。
  2. 如权利要求1所述的单克隆抗体，其特征在于：
    所述重链可变区的氨基酸序列为如下(c1)或(c2)或(c3):
    (c1)序列表的序列2所示的氨基酸序列；
    (c2)将序列表的序列2所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加得到的具有相同功能的氨基酸序列；
    (c3)与序列表的序列2所示的氨基酸序列具有75％或75％以上的同源性且具有相同功能的氨基酸序列；
    所述轻链可变区的氨基酸序列为如下(c4)或(c5)或(c6):
    (c4)序列表的序列4所示的氨基酸序列；
    (c5)将序列表的序列4所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加得到的具有相同功能的氨基酸序列；
    (c6)与序列表的序列4所示的氨基酸序列具有75％或75％以上的同源性且具有相同功能的氨基酸序列。
  3. 编码权利要求1或2所述抗体的基因。
  4. 如权利要求3所述的基因，其特征在于：
    编码所述抗体的重链可变区的基因为如下(d1)或(d2)或(d3):
    (d1)序列表的序列1所示的DNA分子；
    (d2)与(d1)限定的核苷酸序列具有75％或75％以上同源性，且编码权利要求1或2中所述的重链可变区的DNA分子；
    (d3)在严格条件下与(d1)或(d2)限定的核苷酸序列杂交，且编码权利要求1或2中所述的重链可变区的DNA分子；
    编码所述抗体的轻链可变区的基因为如下(d4)或(d5)或(d6):
    (d4)序列表的序列3所示的DNA分子；
    (d5)与(d4)限定的核苷酸序列具有75％或75％以上同源性，且编码权利要求1或2中所述的重链可变区的DNA分子；
    (d6)在严格条件下与(d4)或(d5)限定的核苷酸序列杂交，且编码权利要求1或2中所述的重链可变区的DNA分子。
  5. 应用，为如下任一所示：
    (A)权利要求1或2所述的单克隆抗体在预防和/或治疗腺病毒感染引起的疾病中的应用；
    (B)权利要求1或2所述的单克隆抗体在抑制腺病毒和/或中和腺病毒中的应用；
    (C)权利要求1或2所述的单克隆抗体在制备用于预防和/或治疗腺病毒感染引起的疾病的药物中的应用；
    (D)权利要求1或2所述的单克隆抗体在制备产品中的应用；所述产品的用途为如下(e1)和/或(e2)：
    (e1)抑制腺病毒；
    (e2)中和腺病毒。
  6. 如权利要求5所述的应用，其特征在于：所述腺病毒为人腺病毒。
  7. 如权利要求6所述的应用，其特征在于：所述人腺病毒为人7型腺病毒。
  8. 药物，其活性成分为权利要求1或2所述的单克隆抗体；所述药物的用途为如下(a)-(c)中的任一种：
    (a)预防和/或治疗腺病毒感染引起的疾病；
    (b)抑制腺病毒；
    (c)中和腺病毒。
  9. 如权利要求8所述的药物，其特征在于：所述腺病毒为人腺病毒。
  10. 如权利要求9所述的药物，其特征在于：所述人腺病毒为人7型腺病毒。
  11. 方法，为如下任一所示：
    (1)一种预防和/或治疗腺病毒感染引起的疾病的方法，是利用权利要求1或2所述的单克隆抗体预防和/或治疗腺病毒感染引起的疾病；
    (2)一种抑制腺病毒和/或中和腺病毒的方法，是利用权利要求1或2所述的单克隆抗体抑制腺病毒和/或中和腺病毒。
  12. 如权利要求11所述的方法，其特征在于：所述腺病毒为人腺病毒。
  13. 如权利要求12所述的方法，其特征在于：所述人腺病毒为人7型腺病毒。
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CN110294802B (zh) * 2019-07-08 2020-07-14 中国人民解放军军事科学院军事医学研究院 一种单克隆抗体10g12及其应用
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