WO2020124846A1 - 抗呼吸道合胞病毒的中和抗体及其应用 - Google Patents

抗呼吸道合胞病毒的中和抗体及其应用 Download PDF

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WO2020124846A1
WO2020124846A1 PCT/CN2019/080162 CN2019080162W WO2020124846A1 WO 2020124846 A1 WO2020124846 A1 WO 2020124846A1 CN 2019080162 W CN2019080162 W CN 2019080162W WO 2020124846 A1 WO2020124846 A1 WO 2020124846A1
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neutralizing antibody
respiratory syncytial
syncytial virus
seq
amino acid
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French (fr)
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廖化新
郑伟宏
贾振兴
王月明
袁晓辉
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珠海泰诺麦博生物技术有限公司
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    • 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
    • C07K16/1027—Paramyxoviridae, e.g. respiratory syncytial virus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • 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
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/115—Paramyxoviridae, e.g. parainfluenza virus
    • G01N2333/135—Respiratory syncytial virus

Definitions

  • the present invention relates to the fields of medicine and immunology, and in particular, to a neutralizing antibody against respiratory syncytial virus.
  • Respiratory syncytial virus is widely distributed all over the world. It is one of the most common viral pathogens causing lower respiratory disease (LRI) in infants, young people, the elderly, and adults with weakened immunity. One. Almost all children have experienced one or more infections at the age of 2 years, and the peak age of infection is 2 months to 8 months. RSV is the primary cause of lower respiratory tract infections in infants and young children, and the primary cause of hospitalization of young children due to respiratory diseases. In infants and young children hospitalized, 40% to 50% of bronchiolitis and 25% of pneumonia are caused by RSV infection. Moreover, many studies have shown that serious infant infection is a high-risk factor for asthma in the future, and its severity far exceeds other microbial pathogens. Severe RSV infection also troubles the elderly, but due to the continued lack of specific treatments and safe and effective vaccines, it is still difficult to reduce the incidence and mortality of RSV infections worldwide.
  • RSV infection The natural immunity of RSV infection is insufficient to produce long-lasting immunity. Therefore, the distinctive feature of RSV infection is that the antibodies produced in the previous infection cannot provide permanent protection. In the same epidemic season, different subtypes of RSV can cause recurrence. Infection, even if there are multiple RSV natural infections, can not induce the upper respiratory tract to produce lifelong immune protection against viral infections, so repeated infections are very common.
  • the object of the present invention is to provide a neutralizing antibody against respiratory syncytial virus and its application.
  • the present invention provides a neutralizing antibody against respiratory syncytial virus (named TRN1022), the neutralizing antibody includes a light chain variable region and a heavy chain variable region:
  • the heavy chain variable region includes: (1) the amino acid sequence shown in SEQ ID NO. 1 in the CDR1 region or a functionally active CDR variant with equivalent functions, and (2) SEQ ID ID NO. 2 in the CDR2 region The amino acid sequence shown or a functionally active CDR variant with equivalent functions, (3) The amino acid sequence shown in SEQ ID NO. 3 in the CDR3 region or a functionally active CDR variant with equivalent functions;
  • the light chain variable region includes: (1) the amino acid sequence shown in SEQ ID NO. 4 in the CDR1 region or a functionally active CDR variant with equivalent functions, and (2) SEQ ID ID NO. 5 in the CDR2 region The amino acid sequence shown or a functionally active CDR variant with equivalent functions, (3) The amino acid sequence shown in SEQ ID NO. 6 in the CDR3 region or a functionally active CDR variant with equivalent functions.
  • the neutralizing antibody comprises a heavy chain variable region (or variable heavy chain domain) of VH CDR1, VH CDR2 and VH CDR3 and a light chain variable region comprising VL CDR1, VL CDR2 and VL CDR3 ( Or variable light chain domain).
  • the amino acid sequences of VH, CDR1, VH, CDR2, and VH, CDR3 in the variable region of the heavy chain are shown in SEQ ID NO. 1, 2, 3, and VL, CDR1, VL, CDR2 and The amino acid sequence of VLCDR3 is shown in SEQ ID NO.4,5,6.
  • the functionally active CDR variants having equivalent functions refer to CDR variants that retain the biological characteristics of the original amino acid sequence and can also specifically bind to the corresponding fragments of respiratory syncytial virus.
  • the functionally active CDR variant includes an amino acid sequence in which at least one amino acid is modified in the parental CDR sequence, and includes at least 60% sequence identity with the parental CDR sequence, preferably at least 70%, at least 80%, at least 90 Amino acid sequence with% sequence identity, or consists of an amino acid sequence with at least 60% sequence identity with the parental CDR sequence, preferably with at least 70%, at least 80%, at least 90% sequence identity.
  • the modification may be a chemical change or a partial modification of the amino acid sequence, the modification allows the variant to retain the biological properties of the unmodified sequence, and the partial modification may be the deletion or replacement of one to several amino acids, for example, 1, 2, 3 , 4 or 5 amino acids, or adding or inserting one to several amino acids, such as 1, 2, 3, 4 or 5 amino acids, or chemically derivatizing one to several amino acids, such as 1, 2, 3, 4 or 5 amino acids, or a combination of them.
  • the substitution of amino acid residues may be a conservative substitution, for example, replacing one hydrophobic amino acid with one hydrophobic amino acid.
  • variant refers to one or more amino acid residues in an amino acid sequence by insertion, deletion or replacement of one or more amino acids, or by chemical derivatization, or nucleotides in a nucleotide sequence, Or one or both of the distal ends of the sequence to modify the sequence resulting from this sequence, and also includes natural allelic mutations, where the modification will not affect (especially not lose) the activity of this sequence.
  • CDR region means the complementarity determining region of an antibody, that is, the region that determines the specificity of an antibody to a specific antigen.
  • the three CDR regions (CDR1 to CDR3) on both the light chain and the heavy chain are responsible for antigen binding.
  • amino acid sequence of the variable region of the heavy chain has the sequence shown in SEQ ID NO. 7, or has a sequence at least 70% identical to the sequence shown in SEQ ID NO. 7, and/or
  • amino acid sequence of the light chain variable region has the sequence shown in SEQ ID NO. 8, or at least 70% identical to the sequence shown in SEQ ID NO. 8.
  • the present invention has found through experiments that the neutralizing antibody of the present invention can neutralize the IC50 of respiratory syncytial virus infection up to 0.42 ng/mL.
  • the neutralizing antibody may not be higher than 1*10 -5 M, for example, 1*10 -6 M, 1*10 -7 M, 1*10 -8 M, 1*10 -9 M or KD of 1*10 -10 M or less dissociates from the respiratory syncytial virus before fusion protein.
  • the pre-fusion protein of respiratory syncytial virus is the pre-fusion conformation (Pre-F) protein of the fusion (F) protein of RSV A2 strain.
  • KD refers to the equilibrium dissociation constant of a specific antibody-antigen interaction, indicating the degree of dissociation of the antibody and antigen when in equilibrium.
  • the antibody eg, the neutralizing antibody TRN1022 of the present invention
  • the equilibrium dissociation constant (KD) or smaller dissociates from the antigen (eg, the F protein of RSV), for example, as determined in a BIACORE instrument using surface plasmon resonance (SPR).
  • antibody as used herein is a full-length antibody or antibody fragment thereof, wherein the neutralizing antibody fragment includes at least one antibody region with the aforementioned binding site.
  • the antibody is selected from humanized or human antibodies and single domain antibodies, such as VH, VHH or VL, and/or antibodies comprising or consisting of VL/VH region pairs and antibody constant domains, such as heavy chain antibodies, Fab , F(ab'), (Fab)2, scFv, Fd, Fv or full-length antibody.
  • the neutralizing antibodies against respiratory syncytial virus provided by the present invention are human antibodies, monoclonal antibodies, purified antibodies, single chain antibodies, Fab, Fab', F(ab')2, or Fv.
  • the monoclonal antibody of the present invention may be an IgG type (eg, IgG1, IgG2, IgG3 or IgG4 subtype) antibody, IgA1, IgA2, IgD, IgE or IgM antibody, the constant region of which includes IgG, IgA, IgD, IgE Or any one of IgM constant regions, preferably IgG or IgA constant regions.
  • IgG type eg, IgG1, IgG2, IgG3 or IgG4 subtype
  • IgA1, IgA2, IgD, IgE or IgM antibody the constant region of which includes IgG, IgA, IgD, IgE Or any one of IgM constant regions, preferably IgG or IgA constant regions.
  • the light chain of the monoclonal antibody of the present invention may be of kappa or lambda type.
  • the light chain is of lambda type.
  • the light chain may be a naturally occurring chain, including naturally rearranged, genetically modified or synthetic light chain types.
  • the heavy chain of the monoclonal antibody of the present invention may be selected from: isotype IgM, IgA, or IgG, preferably IgG.
  • the heavy chain of the monoclonal antibody is of IgG type.
  • the present invention also provides a nucleic acid molecule encoding the neutralizing antibody based on the aforementioned research results.
  • the nucleotide sequence of the nucleic acid molecule depends on the specific amino acid sequence of the aforementioned antibody molecule, and the correspondence between the nucleotide sequence and the amino acid sequence belongs to common knowledge in the art.
  • the neutralizing antibody amino acid sequence or its characteristics are determined, those skilled in the art can obtain a corresponding, suitable and reasonable nucleotide sequence according to the amino acid sequence.
  • the nucleic acid molecule encoding the neutralizing antibody may be a naturally-occurring nucleic acid derived from a germline or rearrangement occurring in B cells, or the nucleic acid may be synthetic. Synthetic nucleic acids also include nucleic acids with modified internucleoside linkages, including phosphorothioate to increase the resistance of the nucleic acid to degradation. Nucleic acids can be genetically engineered or completely synthesized by nucleotide synthesis.
  • the present invention provides a vector comprising at least one nucleic acid encoding the light chain of the monoclonal antibody of the present invention and/or at least one nucleic acid encoding the heavy chain of the monoclonal antibody of the present invention.
  • the nucleic acid may be present in the same vector or may be in the form of a binary vector.
  • the vector contains a promoter operably linked to the nucleic acid to facilitate expression of the nucleic acid encoding the light chain and/or heavy chain.
  • the vector also contains a starting point for replication and maintenance in the host cell.
  • the vector may also contain a nucleotide sequence encoding a signal sequence located 5'to the nucleic acid encoding the light or heavy chain. The signal sequence can facilitate secretion of the encoded peptide chain into the culture medium.
  • prokaryotic and eukaryotic expression systems are known, in which eukaryotic host cells such as yeast cells, insect cells, plant cells and mammalian cells are known.
  • eukaryotic host cells such as yeast cells, insect cells, plant cells and mammalian cells are known.
  • the mammalian cells are selected from HEK293 cells, PerC6 cells, CHO cells, COS cells or HELA cells and derivatives thereof. Particularly preferred are human production cell lines.
  • the human monoclonal antibody of the present invention is generated from blood lymphocytes of a plasma sample having a high titer to the RSV prefusion protein, and thus produces naturally refined and selected antibodies with high affinity to To achieve neutralization and effective protection against infection.
  • the invention also provides a method for producing monoclonal antibodies.
  • monoclonal antibodies are produced by culturing host cells into which expression vectors for signal sequences are transferred. The produced monoclonal antibody is secreted into the supernatant and can be purified from it by applying conventional chromatography techniques.
  • the present invention also provides the application of the neutralizing antibody in any one of the following a) to d):
  • the product is a medicine.
  • the present invention further provides a medicine for treating or adjuvanting treatment against respiratory syncytial virus infection, the active ingredient of which is the neutralizing antibody against respiratory syncytial virus described in the present invention.
  • the medicament of the present invention must be sterile and stable under the conditions of manufacture and storage.
  • the preferred methods of preparation are vacuum drying and freeze drying, which produce activity from a previously sterile filtered solution of the active ingredient and other desired ingredients Powder of ingredients and other desired ingredients.
  • the medicament of the present invention may be in solution, and an appropriate pharmaceutically acceptable excipient may be added and/or mixed before or during delivery to provide an injectable unit dosage form.
  • the pharmaceutically acceptable excipients used in the present invention are suitable for high drug concentration, can maintain proper fluidity, and can delay absorption if necessary.
  • the antibody of the present invention can be formulated as a drug with a pharmaceutically acceptable carrier, and can be administered by various methods known in the art.
  • the route and/or mode of administration may vary depending on the desired result.
  • pharmaceutically acceptable carrier means one or more non-toxic materials that do not interfere with the effectiveness of the biological activity of the active ingredient, including but not limited to buffers, preservatives, compatible carriers, and optionally other Additives or encapsulated substances.
  • carrier means a natural or synthetic organic or inorganic ingredient with which the active ingredient is combined to facilitate application.
  • Figure 1 is the Blue Native PAGE detection of the expressed protein in Example 1 of the present invention.
  • Figure 2 is the RSV Pre-F protein binding ability in Example 1 of the present invention.
  • FIG. 3 is a sorting result of memory B cells in Example 1 of the present invention.
  • FIG. 4 is an SDS-PAGE pattern of purified non-reduced and reduced antibody proteins in Example 1 of the present invention; where lane 1 is the non-reduced antibody protein, lane 2 is the Marker, and lane 3 is the reduced antibody protein.
  • Figure 5 is the activity of the neutralizing antibody described in Example 2 of the present invention to specifically bind to the purified RSV Pre-F protein.
  • Fig. 6 shows the neutralizing activity of the neutralizing antibodies described in Example 2 of the present invention against the subtypes A and B of RSV virus.
  • FIG. 7 is the binding affinity of the neutralizing antibody described in Example 2 of the present invention to different concentrations of RSV Pre-F protein.
  • Pre-F pre-fusion conformation
  • F RSV A2 strain fusion
  • the expressed RSV Pre-F protein was detected by Native PAGE. The result is shown in Figure 1. It is mainly in the form of multimers, which is similar to the form in which the naturally occurring RSV F protein is a trimer.
  • the RSV Pre-F protein was coated with carbonate coating buffer and kept at 4°C overnight. Wash with PBST buffer, add blocking solution to block at 37°C for 2h or 4°C overnight. Add 100 ⁇ L of Mouse Anti-Respiratory Syncytial Virus Fusion protein (Abcam, 1:7500), positive control is RSV positive plasma sample, negative control is irrelevant antibody 0.5 ⁇ g/mL, 100 ⁇ L per well, blank well plus blocking solution, incubate at 37 °C 1h.
  • RSV Pre-F protein binds positively with Mouse Anti-Respiratory Syncytial Virus Fusion protein monoclonal antibody and does not bind with other unrelated antibodies.
  • the expressed RSV Pre-F proteins are all trimeric structural proteins with specific binding activity.
  • Peripheral blood samples collected from healthy adult volunteers were placed in anticoagulation tubes containing EDTA, diluted with equal volume of normal saline, centrifuged with Ficoll, 400g, centrifuged for 35min.
  • the plasma layer of the supernatant was separated and stored at -80°C for later use; the middle mononuclear cell (PBMC) layer suspension was washed 3 times with RPMI1640 (containing 10% FBS), the supernatant was discarded, and the cells were frozen and protected After the agent is resuspended and filled, it is kept in a liquid nitrogen tank for later use.
  • PBMC middle mononuclear cell
  • the ELISA method screens plasma samples with high titers against RSV F protein.
  • the RSV Pre-F protein was diluted with carbonate coating buffer to 2 ⁇ g/mL, and the enzyme plate was coated overnight at 4°C. Washed with PBST buffer, the plasma sample was diluted 1:50 times as the initial concentration, diluted 3 times, and incubated at 37°C for 1h. Wash with PBST buffer, add 100 ⁇ L of Goat-Anti-IgG-Fab-HRP (secondary antibody) diluted 1:10000 with blocking solution to each well, and incubate at 37°C for 1 h. Wash with PBST buffer, add 100 ⁇ L of TMB to each well, leave at 37°C for 5 ⁇ 10min, and immediately stop with 2M H 2 SO 4 . Double-wavelength 450/630nm detects the OD value and judges the result.
  • PBMC recovered from the 37°C water bath with a 40 ⁇ m filter membrane
  • BD FACSria in accordance with CD3-PE-Cy5-/CD16-PE-Cy5-/CD235a-PE-Cy5-/CD14-FITC-/IgD-PE-/
  • the CD20-APC+/CD27-APC-H7+/ scheme sorts specific cell populations from PBMC, and then sorts memory B cell populations specific for RSV F protein, and selects single cells in good shape to be placed in 96-well PCR plates. Medium (20 ⁇ L of single cell lysate per well), so that each well contains a memory B cell, -80 °C refrigerator kept for future use.
  • Flow cytometry sorts the memory B cells in the candidate samples, and screens memory B cells that can specifically bind RSV F protein.
  • PCR amplification was performed under the following conditions: 95°C 15min; 95°C 1min, 55°C 1min, 72°C 1min, 30 cycles; 72°C 10min; 4°C 5min; the obtained product cDNA was stored at -20°C;
  • the IgG heavy chain and the k and lambda light chains are generated by the cDNA synthesis reaction from the first strand of B cells, and then amplified using nested PCR methods.
  • the 50 ⁇ L system contains 5 ⁇ L reverse transcripts, HotStarTaq Plus enzymes, dNTPs, and 0.5 ⁇ M specific primers for different subtype heavy and light chain variable regions (primers are designed at specific sites using conventional methods), according to the following conditions Perform PCR amplification: pre-denaturation at 94°C for 5min; 94°C for 30s, 55°C for 30s, 72°C for 50s, 35 cycles; 72°C for 7min.
  • the obtained PCR product was sized on a 1% agarose gel, and the remaining PCR product was purified by Qiagen PCR Purification Kit (Qiagen).
  • the PCR product of the antibody variable region gene identified by gel electrophoresis as positive, and the heavy chain and the light chain can be paired is connected to the pcDNA3.3 vector by TA cloning method to construct anti-respiratory syncytial virus whole human source And antibody expression vectors, and then transform the expression vectors into DH5 ⁇ competent bacteria, incubate overnight on plates containing ampicillin at 37°C, pick 10 single colonies and perform PCR with specific primers, the reaction conditions are: 94°C pre-denaturation for 3 min ; Denaturation at 94°C for 30s, annealing at 55°C for 30s, extension at 72°C for 100s, 28 cycles; extension at 72°C for 5min. Take 5 ⁇ L of PCR product and detect by 1% agarose gel electrophoresis. The results showed that among the positive transformants, transformants containing antibody heavy chain and light chain genes were identified.
  • 293 cells were co-transfected with expression vectors of antibody heavy chain and light chain positive for RSV F protein antigen binding, changed to a large amount of fresh medium 6-8 hours after transfection, and cultured in an incubator at 37°C and 8% CO 2 .
  • the transfection supernatant was collected and centrifuged at 4°C and 4000 rpm for 1 hour to remove cell debris.
  • the Protein Agarose affinity column was combined overnight, and the combined supernatant was slowly passed through the Protein Agarose affinity column to fully allow the antibody to bind.
  • elution buffer 0.1M Gly-HCl buffer, pH 2.5
  • Amicon containing 1mL of 1M Tris-HC buffer pH 9.0
  • Ultra-30Centrifugal Filters MerckMillipore
  • centrifuge at 5000G and 4°C for 20min to concentrate protein then add the above PBS to Amicon Ultra-30Centrifugal Filters, centrifuge at 3500G and 4°C for 20min, replace with new equilibration buffer, repeat 3 times to obtain Antibody protein concentrated to 1 ML.
  • the Mini-Protein cell III system Bio-Rad was used for discontinuous vertical electrophoresis.
  • the mixing ratio of the antibody sample to the Shanghai buffer is 5:1.
  • the reducing sample is boiled for 5 minutes, each well is loaded with 10 ⁇ L, and the electrophoresis time is about 60 minutes.
  • Coomassie Brilliant Blue R-250 was stained for more than 30 minutes, and then decolorized with a decolorizing solution until the background was clean. Observe the purification effect of SDS-PAGE protein electrophoresis analysis.
  • the RSV Pre-F protein was coated with carbonate coating buffer at 4°C overnight. Wash with PBST buffer, add blocking solution to block at 37°C for 2h or 4°C overnight. RSV antibody samples were diluted in blocking solution multiple times, starting at 1 ⁇ g/well, and 12 gradients were diluted.
  • the positive control is a positive plasma sample stock and RSV monoclonal antibody, 100 ⁇ L per well.
  • the negative control was negative plasma sample stock solution and irrelevant antibody (TRN006) 0.5 ⁇ g/mL, 100 ⁇ L per well, blank plus 100 ⁇ L blocking solution, and incubated at 37°C for 1h.
  • the neutralizing antibody titer is determined.
  • the neutralizing antibody titer is defined as the last antibody dilution with more than 50% intact HEP2 cells.
  • a microplate reader was used to measure substrate conversion by detecting absorbance at 450 nm.
  • the log (inhibitor) was used to fit the response to a variable slope curve, the IC50 value was calculated using a non-linear fitting algorithm in Graphpad Prism, and the IC50 value represented the antibody concentration required to reduce the absorbance measured at 450 nm by 50%.
  • the neutralizing titer of the equivalent amount of TRN1022 antibody is not only higher than that of the equivalent amount of Synagis (palivizumab, respiratory syncytial virus fusion protein (F protein) human monoclonal antibody, subtype A and subtype B, etc.
  • Respiratory syncytial virus clinical isolates have an activity of more than ten times higher, and the lowest neutralization concentration detected by its in vitro micro-neutralization activity is only 0.42 ng/mL. It shows that compared with the existing marketed drug Synagis, using a smaller amount of TRN1022 antibody can neutralize the same amount of virus, which has obvious advantages.
  • the TRN1022 antibody can neutralize the three strains of RSV, A2, RSV, Long, and RSV 9320, that is, they have neutralizing activity against the subtype A and B subtype RSV viruses ( Figure 6), which means that they are broad-spectrum neutralizing. antibody.
  • clinical samples 9325, 8879, 9133, 9574, 6477, 8934, 6495 and other 7 clinical samples from different RSV patients (clinical test RSV positive, Hep-2 cell culture typical syncytial lesions, RSV specific PCR test positive and It can be neutralized by RSV antibody to CPE reaction, subtype A and B virus), after culture, it will be neutralized with TRN1022 antibody.
  • the results show that Synagis and TRN1022 antibodies can provide good protection against RSV attacks from different clinical sources. TRN1022 antibody is more effective than Synagis.
  • the amino-coupled anti-human IgG (Fc) was coupled to the two channels of the CM5 chip. Finally, channel 1 was coupled to 5485.4RU and channel 2 was coupled to 5622.4RU.
  • the captured RSV047 concentration was 1 ⁇ g/mL, and the binding time was 130 s.
  • the concentration of bound RSV F protein was 2.5 ⁇ g/mL, 5 ⁇ g/mL, 10 ⁇ g/mL, 20 ⁇ g/mL, 40 ⁇ g/mL, the binding time was 90 s, and the dissociation time was 600 s.
  • the regeneration solution is 3M MgCl 2 and the regeneration time is 30s. The results are shown in Figure 7.
  • the RSV Pre-F protein was tested by surface plasmon resonance (SPR). The results are shown in the table below.
  • the antibody TRN1022 binds the pre-fusion F protein with high affinity.
  • the neutralizing antibody (TRN1022 antibody) against respiratory syncytial virus disclosed in the present invention is a broad-spectrum neutralizing antibody, and has neutralizing activity against subtype A and B subtype RSV viruses.
  • the neutralizing antibody (TRN1022 antibody) described in the present invention can provide good protection against RSV attacks from different clinical sources, and has a better effect than Synagis.
  • the neutralizing antibody provided by the invention can be used for preparing respiratory syncytial virus vaccines, preparing medicines for treating or assisting the treatment of respiratory syncytial virus, and can also be used for the detection of respiratory syncytial virus and other aspects.

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Abstract

本发明公开了抗呼吸道合胞病毒的中和抗体及其应用。所述中和抗体包含VH CDR1、VH CDR2和VH CDR3的重链可变区以及包含VL CDR1、VL CDR2和VL CDR3的轻链可变区(或称可变轻链结构域)；其中所述重链可变区中的VH CDR1、VH CDR2和VH CDR3的氨基酸序列如SEQ ID NO.1、2、3所示，所述轻链可变区中的VL CDR1、VL CDR2和VL CDR3的氨基酸序列如SEQ ID NO.4、5、6所示。本发明还提供了编码所述中和抗体的核酸分子以及所述中和抗体在制备特异性结合呼吸道合胞病毒的融合前蛋白的产品和制备呼吸道合胞病毒疫苗等方面的应用。

Description

抗呼吸道合胞病毒的中和抗体及其应用 技术领域
本发明涉及医学及免疫学领域，具体地说，涉及一种抗呼吸道合胞病毒的中和抗体。
背景技术
呼吸道合胞病毒(human respiratory syncytial virus，RSV)广泛分布于世界各地，是导致婴幼儿、老年人和免疫力低下的成年人下呼吸道疾病(low respiratory illness，LRI)的最常见的病毒性病原体之一。几乎所有儿童2岁时都经历过1次或多次感染，感染的高峰年龄为2个月至8个月。RSV是婴儿、小龄儿童下呼吸道感染的首要原因，也是年幼儿童因呼吸道疾病住院的首要原因。在婴幼儿住院病例中，有40％～50％的毛细支气管炎和25％的肺炎是RSV感染所致。而且多项研究还表明，婴儿严重感染是以后发生哮喘的高危因素，其严重性远远超出其他微生物病原。严重的RSV感染同样困扰着老年人，然而由于持续缺乏特定的治疗和安全有效的疫苗，仍然难以减少全球RSV感染的发病率和死亡率。
RSV自然感染产生的免疫不充分，不能产生持久免疫力，因此，RSV感染的显著特征是前次感染在体内产生的抗体不能提供永久保护，在同一个流行季节，不同亚型的RSV可引起再次感染，即使发生多次RSV自然感染也不能诱导上呼吸道对病毒感染产生终身的免疫保护，因此重复感染十分常见。
目前，已研究预防和治疗RSV感染的几种方法，包括疫苗开发、抗病毒化合物(利巴韦林)、反义药物、RNA干扰技术以及抗体产品，例如免疫球蛋白或静脉注射单克隆抗体。分离自供体的静脉注射免疫球蛋白
Figure PCTCN2019080162-appb-000001
和单克隆抗体帕利珠单抗(Synagis)已被批准用于高风险儿童中的RSV预防。然而帕利珠单抗是一种昂贵的人源化单克隆抗体，仅能用作预防性治疗；利巴韦林是一种核苷抗 代谢物，有严重毒性，存在致畸作用。更多的抗RSV的药物研发还处于早期阶段。因此，迫切需要开发新的抗RSV药物，尤其是可治疗RSV感染的药物。
发明内容
为了解决现有技术中存在的问题，本发明的目的是提供一种抗呼吸道合胞病毒的中和抗体及其应用。
为了实现本发明目的，本发明的技术方案如下：
第一方面，本发明提供了一种抗呼吸道合胞病毒的中和抗体(命名为TRN1022)，所述中和抗体包括轻链可变区和重链可变区：
所述重链可变区包括：(1)CDR1区中如SEQ ID NO.1所示的氨基酸序列或具有同等功能的功能性活性CDR变体，(2)CDR2区中如SEQ ID NO.2所示的氨基酸序列或具有同等功能的功能性活性CDR变体，(3)CDR3区中如SEQ ID NO.3所示的氨基酸序列或具有同等功能的功能性活性CDR变体；
和/或，
所述轻链可变区包括：(1)CDR1区中如SEQ ID NO.4所示的氨基酸序列或具有同等功能的功能性活性CDR变体，(2)CDR2区中如SEQ ID NO.5所示的氨基酸序列或具有同等功能的功能性活性CDR变体，(3)CDR3区中如SEQ ID NO.6所示的氨基酸序列或具有同等功能的功能性活性CDR变体。
作为优选，所述中和抗体包含VH CDR1、VH CDR2和VH CDR3的重链可变区(或称可变重链结构域)以及包含VL CDR1、VL CDR2和VL CDR3的轻链可变区(或称可变轻链结构域)。其中，所述重链可变区中的VH CDR1、VH CDR2和VH CDR3的氨基酸序列如SEQ ID NO.1、2、3所示，所述轻链可变区中的VL CDR1、VL CDR2和VL CDR3的氨基酸序列如SEQ ID NO.4、5、6所示。
所述具有同等功能的功能性活性CDR变体指保留了原氨基酸序 列的生物学特性，同样能够特异性结合呼吸道合胞病毒的相应片段的CDR变体。
所述功能性活性CDR变体包括在亲代CDR序列中的至少一个氨基酸被修饰的氨基酸序列，并包括与亲代CDR序列具有至少60％序列一致性，优选具有至少70％、至少80％、至少90％序列一致性的氨基酸序列，或者由与亲代CDR序列具有至少60％序列一致性，优选具有至少70％、至少80％、至少90％序列一致性的氨基酸序列组成。
所述修饰可以是氨基酸序列的化学变化或部分更改，所述修饰使变体保留未修饰序列的生物学特性所述部分更改可以是删除或替换一个至几个氨基酸，例如，1、2、3、4或5个氨基酸，或加入或插入一个至几个氨基酸，例如1、2、3、4或5个氨基酸，或通过化学衍生一个至几个氨基酸，例如，1、2、3、4或5个氨基酸，或它们的组合。氨基酸残基的替换可以是保守替换，例如，以一个疏水氨基酸替换一个替代疏水氨基酸。
本文使用的术语“变体”指的是通过插入、删除或替换一个或多个氨基酸，或通过化学衍生化氨基酸序列中一个或多个氨基酸残基，或核苷酸序列中的核苷酸，或序列的一个或两个远端来修饰这一序列所得到的序列，还包括天然等位突变，其中，修饰不会影响(特别是不会损失)这一序列的活性。
本发明中，术语“CDR区”意指抗体的互补决定区，即决定抗体对特定抗原的特异性的区域。轻链和重链两者上的三个CDR区(CDR1至CDR3)负责抗原结合。
更进一步地，所述重链可变区的氨基酸序列具有如SEQ ID NO.7所示的序列，或具有与SEQ ID NO.7所示序列至少70％相同的序列，和/或，所述轻链可变区的氨基酸序列具有如SEQ ID NO.8所示的序列，或具有与SEQ ID NO.8所示序列至少70％相同的序列。
本发明经过实验研究发现，本发明所述中和抗体中和呼吸道合胞病毒感染的IC50可达0.42ng/mL。
更进一步地，所述的中和抗体以不高于1*10 -5M，例如1*10 -6M、1*10 -7M、1*10 -8M、1*10 -9M或1*10 -10M或更小的KD与呼吸道合胞病毒的融合前蛋白解离。
在一个优选的实施方案中，呼吸道合胞病毒的融合前蛋白为RSV A2株融合(F)蛋白的融合前构象(Pre-F)蛋白。
其中，术语“KD”是指特定抗体-抗原相互作用的平衡解离常数，表示处于平衡状态时抗体和抗原的解离程度。KD越小说明解离越小，代表抗体与抗原间的亲和力越强。通常，抗体(例如，本发明的中和抗体TRN1022)以不高于10 -5M，例如小于大约10 -6M、10 -7M、10 -8M、10 -9M或10 -10M或更小的平衡解离常数(KD)与抗原(例如，RSV的F蛋白)解离，例如，如使用表面等离子体共振术(SPR)在BIACORE仪中测定的。
本文使用的术语“抗体”是全长抗体或其抗体片段，其中所述中和抗体片段包括至少一个带有前述结合位点的抗体区。优选地，抗体选自人源化或人抗体和单结构域抗体，如VH、VHH或VL，和/或包含或由VL/VH区域对和抗体恒定域组成的抗体，如重链抗体、Fab、F(ab’)、(Fab)2、scFv、Fd、Fv或全长抗体。
特别地，本发明所提供的抗呼吸道合胞病毒的中和抗体，为人抗体、单克隆抗体、纯化的抗体、单链抗体、Fab、Fab’、F(ab')2、或Fv。
优选地，本发明的单克隆抗体可以是IgG型(如，IgG1、IgG2、IgG3或IgG4亚型)抗体、IgA1、IgA2、IgD、IgE或IgM抗体，其恒定区包括IgG、IgA、IgD、IgE或IgM恒定区中的任意一种，优选为IgG或IgA恒定区。
优选地，本发明的单克隆抗体的轻链可以是κ型或λ型的。
在一个优选的实施例中，轻链是λ型的。轻链可以是天然存在的链，包括天然重排的、经遗传修饰的或合成的轻链类型。
本发明的单克隆抗体的重链可以选自：同种型IgM、IgA、或IgG，优选地IgG。
在一个优选的实施例中，单克隆抗体的重链是IgG型的。
第二方面，本发明基于前述研究成果，还提供了编码所述中和抗体的核酸分子。所述核酸分子的核苷酸序列依据前述抗体分子的具体氨基酸序列而定，所述核苷酸序列与氨基酸序列的对应关系属于本领域的公知常识。在所述中和抗体氨基酸序列或其特征确定的情况下，本领域技术人员能够根据该氨基酸序列获得相应的、合适且合理的核苷酸序列。
编码所述中和抗体的核酸分子可以是自种系或自B细胞中发生的重排衍生的天然存在的核酸，或者，核酸可以是合成的。合成的核酸还包括具有经修饰的核苷间键，包括硫代磷酸酯以提高核酸免于降解的抗性的核酸。核酸可以遗传工程化改造或者通过核苷酸合成完全合成生成。
在一个优选的实施例中，本发明提供了包含至少一种编码本发明单克隆抗体的轻链的核酸和/或至少一种编码本发明单克隆抗体的重链的核酸的载体。核酸可以存在于同一载体中或者可以以二元载体的形式存在。优选地，载体包含与核酸可操作连接的启动子以便于编码轻链和/或重链的核酸的表达。优选地，载体还包含用于在宿主细胞中复制和维持的起点。载体还可以包含位于编码轻链或重链的核酸的5’的编码信号序列的核苷酸序列。信号序列可以便于编码的肽链分泌入培养基中。
因此，应当理解的是，含有上述核酸分子的重组表达载体或表达盒或转基因细胞系或重组菌等也属于本发明的保护范围。
在本领域中，已知许多原核和真核表达系统，其中真核宿主细胞 诸如酵母细胞、昆虫细胞、植物细胞和哺乳动物细胞。优选地，哺乳动物细胞自HEK293细胞、PerC6细胞、CHO细胞、COS细胞或HELA细胞及其衍生物等。特别优选的是人生产细胞系。
在一个优选的实施例中，本发明的人单克隆抗体自对RSV融合前蛋白具有高滴度的血浆样本的血液淋巴细胞生成，并且如此生成具有高亲和力的天然精制的且选定的抗体以实现中和和针对感染的有效保护。
本发明还提供了用于生成单克隆抗体的方法。在一个实施方案中，通过培养转入信号序列的表达载体的的宿主细胞来生成单克隆抗体。生成的单克隆抗体被分泌入上清液中，并且可以通过应用常规的层析技术来自其纯化。
第三方面，本发明还提供了所述中和抗体在如下a)～d)中任一种中的应用：
a)制备特异性结合呼吸道合胞病毒的融合前蛋白的产品；
b)制备特异性结合呼吸道合胞病毒抗原的产品；
c)制备治疗或辅助治疗呼吸道合胞病毒的产品；
d)制备呼吸道合胞病毒疫苗；
e)制备用于检测RSV的检测试剂。作为优选，所述产品为药物。
作为一种优选的应用方案，本发明进一步提供一种治疗或辅助治疗抗呼吸道合胞病毒感染的药物，其活性成分为本发明所述的抗呼吸道合胞病毒的中和抗体。
本发明的药物在制造和储存条件下必须是无菌和稳定的。在用于制备无菌可注射溶液的无菌粉末的情况下，制备的优选方法是真空干燥和冷冻干燥，真空干燥和冷冻干燥从活性成分和其它期望成分的预先无菌过滤过的溶液产生活性成分和其它期望成分的粉末。可选择地，本发明的药物可在溶液中，且在递送之前或递送时可加入和/或混合适当的药学上可接受的赋形剂以提供可注射的单位剂型。优选 地，本发明中使用的药学上可接受的赋形剂适用于高药物浓度，可保持适当的流动性，且如果需要可延迟吸收。
作为优选地，本发明所述的抗体可以与一种药学上可接受的载体配制为药物，并且可以通过本领域已知的多种方法来给予。给药途径和/或方式可以取决于所希望的结果而不同。
术语“药学上可接受的载体”表示不干扰活性成分的生物活性的有效性的一种或多种非毒性材料，包括但不限于缓冲液、防腐剂、相容的载体、以及任选地其他添加剂或包封物质。术语“载体”表示天然的或合成的有机或无机成分，活性成分与该载体组合以促进应用。
在符合本领域常识的基础上，上述各优选条件，可以相互组合，得到具体实施方式。
附图说明
图1是本发明实施例1中表达蛋白的Blue Native PAGE检测。
图2是本发明实施例1中RSV Pre-F蛋白结合能力。
图3是本发明实施例1中记忆性B细胞的分选结果。
图4是本发明实施例1中纯化后非还原性和还原性的抗体蛋白的SDS-PAGE图谱；其中，泳道1为非还原抗体蛋白，泳道2为Marker，泳道3为还原性抗体蛋白。
图5是本发明实施例2中所述中和抗体与纯化的RSV Pre-F蛋白特异性结合的活性。
图6为本发明实施例2中所述中和抗体对RSV病毒的A型和B型亚型的中和活性。
图7为本发明实施例2中所述中和抗体与不同浓度RSV Pre-F蛋白的结合亲和力。
具体实施方式
下面将结合实施例对本发明的优选实施方式进行详细说明。需要理解的是以下实施例的给出仅是为了起到说明的目的，并不是用于对 本发明的范围进行限制。本领域的技术人员在不背离本发明的宗旨和精神的情况下，可以对本发明进行各种修改和替换。
下述实施例中所使用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材料、试剂等，如无特殊说明，均可从商业途径得到。
实施例1抗呼吸道合胞病毒中和抗体的筛选、表达与纯化
1、RSV Pre-F蛋白的制备与鉴定
根据NCBI数据库内的RSV A2株融合(F)蛋白的融合前构象(Pre-F)蛋白的基因序列，加入His标签后与pcDNA3.3表达载体构建RSV Pre-F蛋白的表达质粒，转染293T细胞后，培养并收集细胞表达的上清液，浓缩后经镍柱纯化，得到RSV Pre-F蛋白，并进行了检测鉴定。
1)表达的RSV Pre-F蛋白的检测
表达的RSV Pre-F蛋白的经SDS-PAGE检测，结果显示其相对分子量约70kDa，与理论值一致。
表达的RSV Pre-F蛋白通过Native PAGE检测，结果如图1，其主要是多聚体形式存在，与天然存在的RSV F蛋白为三聚体的形式存在类似。
2)RSV Pre-F蛋白结合能力鉴定
将RSV Pre-F蛋白分别用碳酸盐包被缓冲液包被、4℃过夜。PBST缓冲液洗涤，加入封闭液37℃封闭2h或4℃过夜。加入100μL Mouse Anti-Respiratory Syncytial Virus Fusion protein antibody(Abcam，1：7500)，阳性对照为RSV阳性血浆样本，阴性对照为无关抗体0.5μg/mL，每孔100μL，空白孔加封闭液，37℃孵育1h。PBST缓冲液洗涤，每孔加100μL用封闭液按1:10000稀释的Goat-Anti-Human IgG-Fab-HRP、Goat-Anti-Mouse IgG-Fab-HRP(二抗)，37℃孵育1h。PBST缓冲液洗涤，避光，每孔加TMB 100μL， 37℃放置5min，2M硫酸终止。双波长450～630nm检测OD值并计算。
结果如图2所示，RSV Pre-F蛋白与Mouse Anti-Respiratory Syncytial Virus Fusion protein antibody单抗结合阳性，并且不与其他无关抗体结合。综上可知，所表达的RSV Pre-F蛋白均是具有特异性结合活性的三聚体结构蛋白。
2、分离PBMC和阳性血浆样本筛选
采集健康成人志愿者的外周血血液样本置于含有EDTA的抗凝管中，经生理盐水等体积稀释后，用Ficoll经离心法，400g、离心35min。分离上清的血浆层，分装后-80℃保存备用；中间的单个核细胞(PBMC)层悬液，用RPMI1640(含10％FBS)洗涤3次，吸弃上清，用细胞冻存保护剂重悬分装后，在液氮罐中保存备用。
ELISA方法筛选针对RSV F蛋白具有高滴度的血浆样本。将RSV Pre-F蛋白用碳酸盐包被缓冲液稀释至2μg/mL，4℃包被酶标版过夜。PBST缓冲液洗涤，血浆样本1：50倍稀释后作为起始浓度，3倍倍比稀释，37℃孵育1h。PBST缓冲液洗涤，每孔加100μL用封闭液按1:10000进行稀释的Goat-Anti-IgG-Fab-HRP(二抗)，37℃孵育1h。PBST缓冲液洗涤，每孔加TMB 100μL，37℃放置5～10min，立即用2M H 2SO 4终止。双波长450/630nm检测OD值，判定结果。
3、分离记忆性B细胞
用40μm滤膜过滤经37℃水浴复苏后的PBMC，先利用BD FACSria按照CD3-PE-Cy5-/CD16-PE-Cy5-/CD235a-PE-Cy5-/CD14-FITC-/IgD-PE-/CD20-APC+/CD27-APC-H7+/的方案从PBMC中分选特异性细胞群，再分选针对RSV F蛋白特异性的记忆B细胞细胞群，挑选形态完好的单个细胞置于96孔PCR板中(每孔20μL单细胞裂解液)，使每个孔含有一个记忆性B细胞，-80℃冰箱保存备用。
分选结果如图3所示，流式细胞术分选候选样本中的记忆性B细胞，筛选能特异性结合RSV F蛋白的记忆B细胞。
4、分离抗体可变区基因
向含有单个B细胞的96孔板中加入0.5μM不同亚型重链与轻链的恒定区引物(引物采用常规方法在特定位点设计)和Superscript III反转录酶，37℃孵育1小时，按以下条件进行PCR扩增：95℃15min；95℃1min，55℃1min，72℃1min，30个循环；72℃10min；4℃5min；获得的产物cDNA于-20℃保存；
通过从B细胞第一链cDNA合成反应产生IgG重链以及k和λ轻链，然后利用、巢式PCR方法扩增。50μL体系中含有5μL反转录产物、HotStarTaq Plus酶、dNTPs、和0.5μM的不同亚型重链与轻链可变区的特异性引物(引物采用常规方法在特定位点设计)，按以下条件进行PCR扩增：预变性94℃5min；94℃30s，55℃30s，72℃50s，35个循环；72℃7min。
获得的PCR产物用在1％琼脂糖凝胶上测定大小，并且将剩余的PCR产物通过Qiagen PCR Purification Kit(Qiagen)纯化。
5、构建重组抗体的表达载体与表达
将凝胶电泳鉴定为阳性、且重链与轻链可匹配成对的抗体可变区基因的PCR产物利用TA克隆的方法连接到pcDNA3.3载体上，构建抗呼吸道合胞病毒全人源中和抗体的表达载体，然后将表达载体转化DH5α感受态细菌，在含有氨苄青霉素的平板上37℃培养过夜，挑取10个单菌落用特异性引物进行PCR，反应条件为：94℃预变性3min；94℃变性30s，55℃退火30s，72℃延伸100s，28个循环；72℃延伸5min。取5μL PCR产物用1％琼脂糖凝胶电泳检测。结果显示，在阳性转化子中鉴定出了含有抗体重链和轻链基因的转化子。
将具有RSV F蛋白抗原结合阳性的抗体重链与轻链的表达载体共转染293细胞，转染后6～8小时换大量新鲜培养基，并在37℃、 8％CO 2培养箱中培养。
6、抗体蛋白纯化与分析
培养96小时后收集转染上清，4℃、4000rpm离心1小时，除去细胞碎片。Protein A Agarose亲和层柱结合过夜，使结合的上清液缓慢通过Protein A Agarose亲和层柱，充分使得抗体结合。用60mL PBS洗涤后，将结合的抗体用洗脱缓冲液(0.1M Gly-HCl缓冲液，pH2.5)洗脱，并收集于包含1mL的1M Tris-HC缓冲液(pH9.0)的Amicon Ultra-30Centrifugal Filters(MerckMillipore)中，5000G、4℃离心20min，浓缩蛋白，再向Amicon Ultra-30Centrifugal Filters中加入上述PBS，3500G、4℃离心20min，更换新的平衡缓冲液，重复3次，获得浓缩至1ML的抗体蛋白。采用Mini-Protein cell III系统(Bio-Rad)进行不连续垂直电泳。抗体样品与上洋缓冲液的混合比例为5:1，混合后还原性样品煮沸5min，每孔上样10μL，电泳时间约60min。考马斯亮蓝R-250染色30min以上，再用脱色液脱色至背景干净。观察SDS-PAGE蛋白电泳分析的纯化效果。
结果如图4所示，纯化后非还原性和还原性的抗体蛋白在SDS-PAGE图谱上都出现了清晰条带，非还原性条带(即抗体)大小为180KD，还原性样品裂解，分别为重链约65KD和轻链约25KD，几乎没有其它杂带。然后通过ELISA等再证实纯化后的抗体的活性及功能。
实施例2中和抗体的结合活性、体外微量中和活性及亲和力实验
1、抗体的结合活性
用前文提到的相同的ELISA方法对表达纯化的抗体的结合活性进行检测：
将RSV Pre-F蛋白用碳酸盐包被缓冲液包被、4℃过夜。PBST缓冲液洗涤，加入封闭液37℃封闭2h或4℃过夜。使用封闭液倍比稀释RSV抗体样品，1μg/孔起始，稀释12个梯度。阳性对照为阳性血 浆样本原液和RSV单抗，每孔100μL。阴性对照为阴性血浆样本原液和无关抗体(TRN006)0.5μg/mL，每孔100μL，空白加100μL封闭液，37℃孵育1h。PBST缓冲液洗涤，每孔加100μL用封闭液按1:10000稀释的Goat-Anti-Human IgG-Fab-HRP、Goat-Anti-Mouse IgG-Fab-HRP，37℃孵育1h。PBST缓冲液洗涤，避光，每孔加TMB100μL，37℃放置5min，2M硫酸终止。双波长450/630nm检测OD值并计算。
由图5可知，表达纯化后的全人源抗RSV单克隆抗体TRN1022与RSV Pre-F蛋白的结合具有剂量依赖性，阴性血浆样本原液和无关抗体(TRN006)不与RSV Pre-F蛋白特异性结合，说明抗体TRN1022与RSV Pre-F的结合是特异性的。
2、抗体的体外微量中和活性
以50μL/孔的体积，将抗体TRN1022的3倍连续稀释液加入96孔微量滴定板的HEp-2细胞培养基中。随后，分别添加按一定比例稀释后的含RSV病毒的样本(包括RSV A2、RSV Long和RSV 9320等3株病毒株；以及临床检测RSV阳性、Hep-2细胞培养典型合胞病变、RSV特异性PCR检测阳性且能被RSV抗体中和CPE反应的临床样本9325、8879、9133、9574、6477、8938、6495等7份样本)，每孔50μL，并设置以未加抗体的病毒为阳性对照，未加病毒的抗体为阴性对照，37℃，5％CO 2培养箱孵育2h。将制备的Hep-2细胞悬液(2×10 5个/mL)每孔分别加入0.1mL，并且将这些板在37℃下在5％、CO 2孵箱中孵育培养。用倒置显微镜每天观察细胞病变(CPE)，以抑制50％细胞病变的抗体最高稀释度的倒数为终点效价。测定中和抗体滴度，中和抗体滴度定义为有50％以上完整HEP2细胞的最后一个抗体稀释度。
使用酶标仪通过检测450nm下的吸光度来测量底物转化。使用log(抑制剂)对应答与可变斜率曲线拟合，在Graphpad Prism中使 用非线性拟合算法计算IC50值，并且IC50值表示在450nm下所测量的吸光度减少50％所需要的抗体浓度。
结果显示，等量TRN1022抗体的中和效价不仅比等量的Synagis(帕利珠单抗，呼吸道合胞病毒融合蛋白(F蛋白)的人单克隆抗体，对A亚型及B亚型等呼吸道合胞病毒临床分离株具有活性)高十几倍，其体外微量中和活性检测到的最低中和浓度仅为0.42ng/mL。表明与现有上市药物Synagis相比，使用更少量的TRN1022抗体就能中和等量的病毒，具有明显的优势。而且，TRN1022抗体能中和RSV A2、RSV Long和RSV 9320等3株病毒株，即对A亚型和B亚型RSV病毒均有中和活性(图6)，即说明其为广谱中和抗体。另外，临床样本9325、8879、9133、9574、6477、8938、6495等7份来源不同RSV患者的临床样本(临床检测RSV阳性、Hep-2细胞培养典型合胞病变、RSV特异性PCR检测阳性且能被RSV抗体中和CPE反应，A、B亚型病毒)，经培养后与TRN1022抗体进行中和试验。结果表明Synagis、TRN1022抗体针对临床上不同患者来源的RSV攻击均能够提供良好的保护能力，TRN1022抗体比Synagis效果更佳。
3、抗体的亲和力
通过氨基偶联方式偶联anti-human IgG(Fc)到CM5芯片的两个通道上，最终通道1偶联5485.4RU，通道2偶联5622.4RU。捕获的RSV047浓度为1μg/mL，结合时间为130s。结合的RSV F蛋白浓度为2.5μg/mL，5μg/mL，10μg/mL，20μg/mL，40μg/mL，结合时间为90s，解离时间为600s。再生溶液为3M MgCl 2，再生时间为30s。结果如图7所示。
RSV Pre-F蛋白通过表面等离子体共振(SPR)来测试，结果如下表所示，抗体TRN1022以高亲和力结合融合前F蛋白。
Figure PCTCN2019080162-appb-000002
Figure PCTCN2019080162-appb-000003
虽然，上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述，但在本发明基础上，可以对之作一些修改或改进，这对本领域技术人员而言是显而易见的。因此，在不偏离本发明精神的基础上所做的这些修改或改进，均属于本发明要求保护的范围。
工业实用性
本发明公开的抗呼吸道合胞病毒的中和抗体(TRN1022抗体)为广谱中和抗体，对A亚型和B亚型RSV病毒均有中和活性。不仅如此，本发明所述的中和抗体(TRN1022抗体)针对临床上不同患者来源的RSV攻击均能够提供良好的保护能力，且比Synagis效果更佳。
本发明所提供的中和抗体可用于制备呼吸道合胞病毒疫苗、制备治疗或辅助治疗呼吸道合胞病毒的药品，也可用于呼吸道合胞病毒的检测等方面。
Figure PCTCN2019080162-appb-000004
Figure PCTCN2019080162-appb-000005
Figure PCTCN2019080162-appb-000006

Claims (10)

  1. 抗呼吸道合胞病毒的中和抗体，其特征在于，所述中和抗体包括轻链可变区和重链可变区：
    所述重链可变区包括：(1)CDR1区中如SEQ ID NO.1所示的氨基酸序列或具有同等功能的功能性活性CDR变体，(2)CDR2区中如SEQ ID NO.2所示的氨基酸序列或具有同等功能的功能性活性CDR变体，(3)CDR3区中如SEQ ID NO.3所示的氨基酸序列或具有同等功能的功能性活性CDR变体；
    和/或，
    所述轻链可变区包括：(1)CDR1区中如SEQ ID NO.4所示的氨基酸序列或具有同等功能的功能性活性CDR变体，(2)CDR2区中如SEQ ID NO.5所示的氨基酸序列或具有同等功能的功能性活性CDR变体，(3)CDR3区中如SEQ ID NO.6所示的氨基酸序列或具有同等功能的功能性活性CDR变体。
  2. 根据权利要求1所述的中和抗体，其特征在于，所述重链可变区的氨基酸序列具有如SEQ ID NO.7所示的序列，或具有与SEQ ID NO.7所示序列至少70％相同的序列，和/或，所述轻链可变区的氨基酸序列具有如SEQ ID NO.8所示的序列，或具有与SEQ ID NO.8所示序列至少70％相同的序列。
  3. 根据权利要求1或2所述的中和抗体，其特征在于，所述中和抗体是人抗体、单克隆抗体、纯化的抗体、单链抗体、Fab、Fab’、F(ab')2、或Fv。
  4. 根据权利要求3所述的中和抗体，其特征在于，所述中和抗体特异性地结合呼吸道合胞病毒的融合前蛋白和/或中和呼吸道合胞病毒；通过表面等离子体激元共振测定，所述中和抗体可以以不高于1*10 -5M的平衡解离常数(KD)与呼吸道合胞病毒的融合前蛋白解离。
  5. 根据权利要求1～4任一项所述的中和抗体，其特征在于，所述中和抗体的恒定区包括IgG、IgA、IgD、IgE或IgM恒定区中的任意一种，优选为gG或IgA恒定区。
  6. 编码权利要求1～5任一项所述中和抗体的核酸分子。
  7. 含有权利要求6所述核酸分子的重组表达载体或表达盒或转基因细胞 系或重组菌。
  8. 权利要求1～5任一项所述中和抗体在如下a)～d)中任一种中的应用：
    a)制备特异性结合呼吸道合胞病毒的融合前F蛋白的产品；
    b)制备特异性结合呼吸道合胞病毒抗原的产品；
    c)制备治疗或辅助治疗呼吸道合胞病毒的产品；
    d)制备呼吸道合胞病毒疫苗；
    e)制备用于检测RSV的检测试剂。
  9. 根据权利要求8所述的应用，其特征在于，所述产品为药物。
  10. 一种治疗或辅助治疗抗呼吸道合胞病毒感染的产品，其特征在于，其活性成分为权利要求1～5任一项所述的中和抗体。
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