CN110016079B - Neutralizing antibody for resisting respiratory syncytial virus and application thereof - Google Patents

Neutralizing antibody for resisting respiratory syncytial virus and application thereof Download PDF

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CN110016079B
CN110016079B CN201910240891.0A CN201910240891A CN110016079B CN 110016079 B CN110016079 B CN 110016079B CN 201910240891 A CN201910240891 A CN 201910240891A CN 110016079 B CN110016079 B CN 110016079B
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neutralizing antibody
antibody
respiratory syncytial
syncytial virus
seq
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CN110016079A (en
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廖化新
郑伟宏
贾振兴
王月明
袁晓辉
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Zhuhai Tainuo Maibo Pharmaceutical Co ltd
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Zhuhai Microlab Biotechnology Co Ltd
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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

Abstract

The invention relates to the field of medicine and immunology, and particularly discloses a neutralizing antibody for resisting respiratory syncytial virus and an application thereof. The neutralizing antibody comprises a heavy chain variable region comprising VH CDR1, VH CDR2 and VH CDR3 and a light chain variable region (or variable light chain domain) comprising VL CDR1, VL CDR2 and VL CDR 3; wherein the amino acid sequences of the VH CDR1, the VH CDR2 and the VH CDR3 in the heavy chain variable region are shown in SEQ ID NO.1, 2 and 3, and the amino acid sequences of the VL CDR1, the VL CDR2 and the VL CDR3 in the light chain variable region are shown in SEQ ID NO.4, 5 and 6. Based on the research result, the invention also provides a nucleic acid molecule for encoding the neutralizing antibody and application of the neutralizing antibody in the aspects of preparing products for specifically binding with the fusion pre-protein of the respiratory syncytial virus, preparing the respiratory syncytial virus vaccine and the like.

Description

Neutralizing antibody for resisting respiratory syncytial virus and application thereof
Technical Field
The invention relates to the field of medicine and immunology, in particular to a neutralizing antibody for resisting respiratory syncytial virus.
Background
Respiratory Syncytial Virus (RSV) is widely distributed throughout the world and is one of the most common viral pathogens causing low respiratory tract disease (LRI) in infants, the elderly, and immunocompromised adults. Almost all children experience 1 or more infection at age 2, with peak ages of 2 to 8 months. RSV is the leading cause of lower respiratory tract infection in infants and young children, and also the leading cause of hospitalization of young children due to respiratory tract diseases. In hospitalized infants, 40% to 50% of bronchiolitis and 25% of pneumonia are due to RSV infection. Also, multiple studies have shown that severe infection in infants is a high risk factor for the subsequent development of asthma, and its severity is far beyond that of other microbial pathogens. Severe RSV infection also afflicts the elderly, yet it remains difficult to reduce the morbidity and mortality of RSV infection worldwide due to the continued lack of specific therapies and safe and effective vaccines.
The RSV natural infection generates insufficient immunity and cannot generate lasting immunity, therefore, the RSV infection is characterized in that the antibody generated in the body by the previous infection cannot provide permanent protection, different subtypes of RSV can cause reinfection in the same epidemic season, and even though a plurality of RSV natural infections occur, the RSV natural infection cannot induce the upper respiratory tract to generate lifelong immune protection against virus infection, so the reinfection is very common.
Currently, several approaches to the prevention and treatment of RSV infection have been investigated, including vaccine development, antiviral compounds (ribavirin), antisense drugs, RNA interference technology, and antibody production, such as immunoglobulins or intravenous monoclonal antibodies. Intravenous immunoglobulin isolated from donors (RSV-IGIV;) And the monoclonal antibody palivizumab (Synagis) has been approved for RSV prophylaxis in high-risk children. Palivizumab, however, is an expensive humanized monoclonal antibody that can only be used as a prophylactic treatment; ribavirin is a nucleoside antimetabolite that is severely toxic and teratogenic. More anti-RSV drugs are still under development in the early stages. Therefore, there is an urgent need to develop new anti-RSV drugs, especially drugs that can treat RSV infections.
Disclosure of Invention
In order to solve the problems in the prior art, the invention aims to provide a neutralizing antibody for resisting respiratory syncytial virus and application thereof.
In order to realize the purpose of the invention, the technical scheme of the invention is as follows:
in a first aspect, the present invention provides a neutralizing antibody against respiratory syncytial virus (designated TRN1022) comprising a light chain variable region and a heavy chain variable region:
the heavy chain variable region comprises: (1) an amino acid sequence shown as SEQ ID NO.1 in a CDR1 region or a functionally active CDR variant with equivalent function, (2) an amino acid sequence shown as SEQ ID NO.2 in a CDR2 region or a functionally active CDR variant with equivalent function, (3) an amino acid sequence shown as SEQ ID NO.3 in a CDR3 region or a functionally active CDR variant with equivalent function;
and/or the presence of a gas in the gas,
the light chain variable region comprises: (1) an amino acid sequence shown as SEQ ID NO.4 in a CDR1 region or a functionally active CDR variant with equivalent function, (2) an amino acid sequence shown as SEQ ID NO.5 in a CDR2 region or a functionally active CDR variant with equivalent function, and (3) an amino acid sequence shown as SEQ ID NO.6 in a CDR3 region or a functionally active CDR variant with equivalent function.
Preferably, the neutralizing antibody comprises a heavy chain variable region (or variable heavy chain domain) comprising VH CDR1, VH CDR2, and VH CDR3 and a light chain variable region (or variable light chain domain) comprising VL CDR1, VL CDR2, and VL CDR 3. Wherein the amino acid sequences of the VH CDR1, the VH CDR2 and the VH CDR3 in the heavy chain variable region are shown in SEQ ID NO.1, 2 and 3, and the amino acid sequences of the VL CDR1, the VL CDR2 and the VL CDR3 in the light chain variable region are shown in SEQ ID NO.4, 5 and 6.
Functionally equivalent functionally active CDR variants are those which retain the biological properties of the original amino acid sequence and which are also capable of specifically binding to the corresponding fragment of the respiratory syncytial virus.
The functionally active CDR variants comprise an amino acid sequence in which at least one amino acid in the parent CDR sequence is modified, and comprise, or consist of, an amino acid sequence having at least 60% sequence identity, preferably at least 70%, at least 80%, at least 90% sequence identity, to the parent CDR sequence.
The modification may be a chemical change or a partial alteration of the amino acid sequence which results in the variant retaining the biological properties of the unmodified sequence, the partial alteration may be a deletion or substitution of one to several amino acids, e.g., 1, 2, 3, 4 or 5 amino acids, or an addition or insertion of one to several amino acids, e.g., 1, 2, 3, 4 or 5 amino acids, or a chemical derivatization of one to several amino acids, e.g., 1, 2, 3, 4 or 5 amino acids, or a combination thereof. The substitution of the amino acid residue may be conservative, for example, a substitution of a hydrophobic amino acid for a hydrophobic amino acid.
The term "variant" as used herein refers to a sequence resulting from modification of such a sequence by insertion, deletion or substitution of one or more amino acids, or by chemical derivatization of one or more amino acid residues in the amino acid sequence, or nucleotides in the nucleotide sequence, or one or both distal ends of the sequence, and also includes natural allelic mutations, wherein the modification does not affect (and in particular does not lose) the activity of such a sequence.
In the present invention, the term "CDR region" means a complementarity determining region of an antibody, i.e., a region that determines the specificity of an antibody for a specific antigen. Three CDR regions (CDR1 to CDR3) on both the light and heavy chains are responsible for antigen binding.
Further, the amino acid sequence of the heavy chain variable region 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 the amino acid sequence of the light chain variable region has the sequence shown in SEQ ID NO.8, or has a sequence at least 70% identical to the sequence shown in SEQ ID NO. 8.
Experimental research shows that the neutralizing antibody of the invention can neutralize the IC50 infected by respiratory syncytial virus to 0.42 ng/mL.
Further, the neutralizing antibody is not more than 1 x 10-5M, e.g. 1 x 10-6M、1*10-7M、1*10-8M、1*10-9M or 1 x 10-10M or less is dissociated from the pre-fusion protein with respiratory syncytial virus.
In a preferred embodiment, the Pre-fusion protein of respiratory syncytial virus is the Pre-fusion conformation (Pre-F) protein of the fusion (F) protein of strain RSV a 2.
The term "KD" refers to the equilibrium dissociation constant of a particular antibody-antigen interaction, and refers to the degree of dissociation of an antibody and antigen when in equilibrium. Smaller KD indicates less dissociation, which represents greater affinity between the antibody and antigen. Typically, the antibody (e.g., neutralizing antibody TRN1022 of the invention) is administered at no more than 10-5M, e.g. less than about 10-6M、10-7M、10-8M、10-9M or 10-10M or less dissociates from an antigen (e.g., the F protein of RSV), e.g., as determined in a BIACORE instrument using Surface Plasmon Resonance (SPR).
The term "antibody" as used herein is a full-length antibody or antibody fragment thereof, wherein the neutralizing antibody fragment comprises at least one antibody region with the aforementioned binding sites. Preferably, the antibody is selected from a humanized or human antibody and a single domain antibody, such as a VH, VHH or VL, and/or an antibody comprising or consisting of a VL/VH region pair and an antibody constant domain, such as a heavy chain antibody, Fab, F (ab'), (Fab)2, scFv, Fd, Fv or full length antibody.
In particular, the neutralizing antibody against respiratory syncytial virus provided by the invention is a human antibody, a monoclonal antibody, a purified antibody, a single-chain antibody, Fab ', F (ab')2, or Fv.
Preferably, the monoclonal antibody of the invention may be an antibody of the IgG type (e.g. IgG1, IgG2, IgG3 or IgG4 subtype), IgA1, IgA2, IgD, IgE or IgM, the constant region of which comprises any of the IgG, IgA, IgD, IgE or IgM constant regions, preferably an IgG or IgA constant region.
Preferably, the light chain of the monoclonal antibody of the invention may be of the kappa or lambda type.
In a preferred embodiment, the light chain is of the lambda type. The light chain may be a naturally occurring chain, including naturally rearranged, genetically modified, or synthetic types of light chains.
The heavy chain of a monoclonal antibody of the invention may be selected from: isotype IgM, IgA, or IgG, preferably IgG.
In a preferred embodiment, the heavy chain of the monoclonal antibody is of the IgG type.
In a second aspect, the present invention is based on the aforementioned findings, and further provides a nucleic acid molecule encoding the neutralizing antibody. The nucleotide sequence of the nucleic acid molecule depends on the specific amino acid sequence of the antibody molecule, and the correspondence between the nucleotide sequence and the amino acid sequence is well known in the art. In the case of the amino acid sequence of the neutralizing antibody or the determination of its characteristics, the person skilled in the art will be able to derive from this amino acid sequence the corresponding, suitable and reasonable nucleotide sequence.
The nucleic acid molecule encoding the neutralizing antibody can be a naturally occurring nucleic acid derived from the germline or from rearrangements occurring in B cells, or alternatively, the nucleic acid can be synthetic. Synthetic nucleic acids also include nucleic acids having modified internucleoside linkages, including phosphorothioates, to increase the resistance of the nucleic acid from degradation. Nucleic acids can be genetically engineered or generated entirely synthetically by nucleotide synthesis.
In a preferred embodiment, the present invention provides a vector comprising at least one nucleic acid encoding the light chain and/or at least one nucleic acid encoding the heavy chain of a monoclonal antibody of the invention. The nucleic acids may be present in the same vector or may be present in the form of a binary vector. Preferably, the vector comprises a promoter operably linked to the nucleic acid to facilitate expression of the nucleic acid encoding the light and/or heavy chain. Preferably, the vector further comprises an origin for replication and maintenance in the host cell. The vector may further comprise a nucleotide sequence encoding a signal sequence located 5' to the nucleic acid encoding the light or heavy chain. The signal sequence may facilitate secretion of the encoded peptide chain into the culture medium.
Therefore, it is understood that recombinant expression vectors or expression cassettes or transgenic cell lines or recombinant bacteria containing the above-mentioned nucleic acid molecules are also within the scope of the present invention.
In the art, many prokaryotic and eukaryotic expression systems are known, wherein eukaryotic host cells such as yeast cells, insect cells, plant cells and mammalian cells. Preferably, the mammalian cell is selected from the group consisting of HEK293 cells, PerC6 cells, CHO cells, COS cells, and HELA cells, derivatives thereof, and the like. Particularly preferred are human producer cell lines.
In a preferred embodiment, the human monoclonal antibodies of the invention are generated from blood lymphocytes of plasma samples with high titers of RSV pre-fusion protein, and as such naturally refined and selected antibodies with high affinity are generated to achieve neutralization and effective protection against infection.
The invention also provides methods for generating monoclonal antibodies. In one embodiment, the monoclonal antibody is produced by culturing a host cell transformed with an expression vector for the signal sequence. The monoclonal antibodies produced are secreted into the supernatant and can be purified therefrom by applying conventional chromatographic techniques.
In a third aspect, the invention also provides the use of the neutralizing antibody in any one of the following a) to d):
a) preparing a product of the fusion pre-protein which specifically binds to the respiratory syncytial virus;
b) preparing a product which specifically binds to respiratory syncytial virus antigen;
c) preparing a product for treating or assisting in treating respiratory syncytial virus;
d) preparing a respiratory syncytial virus vaccine;
e) preparing a detection reagent for detecting RSV.
Preferably, the product is a medicament.
As a preferred application scheme, the invention further provides a medicament for treating or assisting in treating the infection of the respiratory syncytial virus, and the active ingredient of the medicament is the neutralizing antibody of the respiratory syncytial virus.
The medicaments of the present invention must be sterile and stable under the conditions of manufacture and storage. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus other desired ingredient from a previously sterile-filtered solution thereof. Alternatively, the agents of the invention may be in solution, and suitable pharmaceutically acceptable excipients may be added and/or mixed prior to or at the time of delivery to provide an injectable unit dosage form. Preferably, the pharmaceutically acceptable excipients used in the present invention are suitable for high drug concentrations, maintain adequate flowability, and delay absorption if necessary.
Preferably, the antibodies of the invention can be formulated as a medicament with a pharmaceutically acceptable carrier and administered by a variety of methods known in the art. The route and/or mode of administration may vary depending on the desired result.
The term "pharmaceutically acceptable carrier" refers to 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 encapsulating substances. The term "carrier" denotes a natural or synthetic organic or inorganic ingredient with which the active ingredient is combined to facilitate application.
The above-described preferred conditions may be combined with each other to obtain a specific embodiment, in accordance with common knowledge in the art.
Drawings
FIG. 1 shows Blue Native PAGE detection of expressed proteins in example 1 of the present invention.
FIG. 2 shows the RSV Pre-F protein binding ability in example 1 of the present invention.
FIG. 3 shows the result of sorting memory B cells in example 1 of the present invention.
FIG. 4 is an SDS-PAGE pattern of the non-reducing and reducing antibody proteins after purification in example 1 of the present invention; wherein, lane 1 is a non-reducing antibody protein, lane 2 is a Marker, and lane 3 is a reducing antibody protein.
FIG. 5 is the activity of the neutralizing antibodies described in example 2 of the present invention to specifically bind to purified RSV Pre-F protein.
FIG. 6 is a graph showing the neutralizing activity of the neutralizing antibody according to example 2 of the present invention against the A-type and B-type subtypes of RSV virus.
FIG. 7 is a graph showing the binding affinity of neutralizing antibodies of the present invention described in example 2 to RSV Pre-F protein at various concentrations.
Detailed Description
Preferred embodiments of the present invention will be described in detail with reference to the following examples. It is to be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the present invention. Various modifications and alterations of this invention will become apparent to those skilled in the art without departing from the spirit and scope of this invention.
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1 screening, expression and purification of neutralizing antibodies against respiratory syncytial Virus
1. Preparation and identification of RSV Pre-F protein
According to the gene sequence of the Pre-fusion conformation (Pre-F) protein of the RSV A2 strain fusion (F) protein in an NCBI database, a His label is added to construct an expression plasmid of the RSV Pre-F protein with a pcDNA3.3 expression vector, after a 293T cell is transfected, the supernatant expressed by the cell is cultured and collected, and after the supernatant is concentrated, the RSV Pre-F protein is obtained by nickel column purification, and the detection and identification are carried out.
1) Detection of expressed RSV Pre-F protein
The expressed RSV Pre-F protein was examined by SDS-PAGE and showed a relative molecular weight of about 70kDa, which is in agreement with the theoretical value.
The expressed RSV Pre-F protein is detected by Native PAGE, and the result is shown in figure 1, and the RSV Pre-F protein mainly exists in a multimeric form, and is similar to the naturally-occurring RSV F protein existing in a trimeric form.
2) Identification of RSV Pre-F protein binding Capacity
RSV Pre-F protein was coated with carbonate coating buffer overnight at 4 ℃. PBST buffer washing, adding blocking solution 37 degrees C blocking 2h or 4 degrees C overnight. mu.L of Mouse Anti-Respiratory synthetic viral fusion protein antibody (Abcam, 1: 7500) was added, the positive control was RSV positive plasma sample, the negative control was irrelevant antibody 0.5. mu.g/mL, 100. mu.L per well, and the blank well was incubated at 37 ℃ for 1h with blocking solution. PBST buffer washing, each hole with 100 u L using blocking solution diluted 1:10000 Goat the same time as the Goat-Anti-Human IgG-Fab-HRP, Goat-Anti-Mouse IgG-Fab-HRP (secondary antibody), 37 degrees C were incubated for 1 h. PBST buffer washing, shading, each hole with 100 u L TMB, 37 degrees C placed for 5min, 2M sulfuric acid termination. Detecting and calculating the OD value with the dual wavelength of 450-630 nm.
As a result, as shown in FIG. 2, the RSV Pre-F protein bound positively to the Mouse Anti-Respiratory synthetic Virus Fusion protein antibody monoclonal antibody and did not bind to other unrelated antibodies. As can be seen from the above, the expressed RSV Pre-F protein is a trimeric structural protein with specific binding activity.
2. PBMC isolation and Positive plasma sample screening
A peripheral blood sample of a healthy adult volunteer is collected and placed in an anticoagulation tube containing EDTA, diluted by physiological saline with equal volume and centrifuged by Ficoll at 400g for 35 min. Separating supernatant plasma layer, subpackaging, and storing at-80 deg.C for use; the intermediate mononuclear cell (PBMC) layer suspension was washed 3 times with RPMI1640 (containing 10% FBS), the supernatant was aspirated, resuspended in cell cryopreservation protectant, and stored in a liquid nitrogen tank for future use.
ELISA method plasma samples with high titers against RSV F protein were screened. RSV Pre-F protein was diluted to 2. mu.g/mL with carbonate coating buffer and coated overnight on a microplate at 4 ℃. PBST buffer wash, plasma sample 1: after 50-fold dilution, the cells were diluted 3-fold and incubated at 37 ℃ for 1 h. PBST buffer washing, adding 100 μ L per well of Goat-Anti-IgG-Fab-HRP (secondary antibody) diluted with blocking solution at a ratio of 1:10000, incubating at 37 deg.CAnd (5) breeding for 1 h. PBST buffer solution is used for washing, 100 mu L of TMB is added into each hole, the mixture is placed at 37 ℃ for 5-10 min and immediately used for 2M H2SO4And (6) terminating. And detecting the OD value at the double wavelength of 450/630nm, and judging the result.
3. Isolation of memory B cells
PBMC recovered by 37 ℃ water bath are filtered by a 40 mu m filter membrane, a specific cell population is selected from the PBMC by using BD FACsria according to a scheme of CD3-PE-Cy5-/CD16-PE-Cy5-/CD235a-PE-Cy5-/CD14-FITC-/IgD-PE-/CD20-APC +/CD27-APC-H7 +/and then a memory B cell population specific to the RSV F protein is selected, and a single cell with a complete morphology is selected and placed in a 96-well PCR plate (20 mu L of single cell lysate per well) so that each well contains one memory B cell, and the PBMC is stored in a refrigerator at 80 ℃ for later use.
Sorting results memory B cells in candidate samples were sorted by flow cytometry and screened for memory B cells that specifically bind RSV F protein, as shown in figure 3.
4. Isolation of antibody variable region genes
To a 96-well plate containing single B cells, 0.5. mu.M of constant region primers for heavy and light chains of different subtypes (primers designed at specific sites using a conventional method) and Superscript III reverse transcriptase were added, incubated at 37 ℃ for 1 hour, and PCR amplification was performed under the following conditions: 15min at 95 ℃; 1min at 95 ℃, 1min at 55 ℃, 1min at 72 ℃ and 30 cycles; 10min at 72 ℃; 5min at 4 ℃; the obtained product cDNA is preserved at-20 ℃;
IgG heavy chains and k and λ light chains were produced by a cDNA synthesis reaction from B cell first chain and then amplified using nested PCR. A50. mu.L system containing 5. mu.L of reverse transcription product, HotStarTaq Plus enzyme, dNTPs, and 0.5. mu.M primers specific for the heavy and light chain variable regions of different subtypes (primers were designed at specific sites using conventional methods) was PCR amplified under the following conditions: pre-denaturation at 94 ℃ for 5 min; 30s at 94 ℃, 30s at 55 ℃, 50s at 72 ℃ and 35 cycles; 7min at 72 ℃.
The obtained PCR products were sized on a 1% agarose gel, and the remaining PCR products were purified by Qiagen PCR Purification Kit (Qiagen).
5. Expression vector and expression for constructing recombinant antibody
The PCR product of the antibody variable region gene which is positive by gel electrophoresis and can be matched and paired with heavy chain and light chain is connected to pcDNA3.3 vector by using TA cloning method to construct an expression vector of the anti-respiratory syncytial virus fully human-derived neutralizing antibody, then the expression vector is transformed into DH5 alpha competent bacteria, the bacteria are cultured on a plate containing ampicillin overnight at 37 ℃, 10 single colonies are picked to carry out PCR by using specific primers, and the reaction conditions are as follows: pre-denaturation at 94 ℃ for 3 min; denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, extension at 72 ℃ for 100s, 28 cycles; extension at 72 ℃ for 5 min. Take 5. mu.L of PCR product and detect it by 1% agarose gel electrophoresis. The results showed that among the positive transformants, transformants containing the antibody heavy and light chain genes were identified.
CO-transfecting 293 cells with an expression vector of an antibody heavy chain and an antibody light chain with positive RSV F protein antigen binding, replacing a large amount of fresh culture medium 6-8 hours after transfection, and carrying out 8% CO treatment at 37 DEG C2Culturing in an incubator.
6. Antibody protein purification and analysis
After culturing for 96 hours, the transfection supernatant was collected and centrifuged at 4000rpm for 1 hour at 4 ℃ to remove cell debris. Protein a Agarose affinity layer column was allowed to bind overnight and the bound supernatant was passed slowly through the Protein a Agarose affinity layer column sufficiently to allow antibody binding. After washing with 60mL of PBS, the bound antibody was eluted with an elution buffer (0.1M Gly-HCl buffer, pH2.5) and collected in Amicon Ultra-30Centrifugal Filters (Merck Millipore) containing 1mL of 1M Tris-HC buffer (pH9.0), centrifuged at 5000G at 4 ℃ for 20min, the protein was concentrated, the above PBS was added to Amicon Ultra-30Centrifugal Filters, centrifuged at 3500G at 4 ℃ for 20min, the equilibrium buffer was replaced with fresh, and the process was repeated 3 times to obtain an antibody protein concentrated to 1 ML. Discontinuous vertical electrophoresis was performed using the Mini-Protein cell III system (Bio-Rad). The mixing ratio of the antibody sample and the buffer solution is 5:1, the reducing sample is boiled for 5min after mixing, each well is loaded with 10 μ L, and the electrophoresis time is about 60 min. Dyeing with Coomassie brilliant blue R-250 for more than 30min, and decolorizing with decolorizing solution until background is clear. The effect of purification by electrophoretic analysis of SDS-PAGE proteins was observed.
As a result, as shown in FIG. 4, the purified antibody proteins, which were non-reducing and reducing, showed clear bands on SDS-PAGE, the size of the non-reducing band (i.e., antibody) was 180kD, the reducing sample was cleaved to about 65kD for the heavy chain and about 25kD for the light chain, respectively, and almost no other bands were observed. The activity and function of the purified antibody were then confirmed by ELISA or the like.
Example 2 binding Activity, in vitro micro-neutralizing Activity and affinity assay of neutralizing antibodies
1. Binding Activity of antibodies
The binding activity of the expressed purified antibody was tested by the same ELISA method as mentioned before:
RSV Pre-F protein was coated with carbonate coating buffer overnight at 4 ℃. PBST buffer washing, adding blocking solution 37 degrees C blocking 2h or 4 degrees C overnight. RSV antibody samples were diluted using blocking solution fold-ratio, starting at 1 μ g/well, and diluted 12 gradients. Positive controls were positive plasma sample stock and RSV mab, 100 μ L per well. The negative control is negative plasma sample stock solution and irrelevant antibody (TRN006)0.5 μ g/mL, each well 100 μ L, blank plus 100 μ L blocking solution, and incubation at 37 deg.C for 1 h. PBST buffer washing, each hole with 100 u L using blocking solution according to 1:10000 diluted Goat-Anti-Human IgG-Fab-HRP, Goat-Anti-MouselgG-Fab-HRP, 37 degrees C were incubated for 1 h. PBST buffer washing, shading, each hole with 100 u L TMB, 37 degrees C placed for 5min, 2M sulfuric acid termination. The OD value was measured at a two-wavelength of 450/630nm and calculated.
As can be seen from FIG. 5, the binding of the purified fully human anti-RSV monoclonal antibody TRN1022 to RSV Pre-F protein was dose-dependent, and the negative plasma sample stock and irrelevant antibody (TRN006) did not specifically bind to RSV Pre-F protein, indicating that the binding of antibody TRN1022 to RSV Pre-F was specific.
2. In vitro micro-neutralizing Activity of antibodies
3-fold serial dilutions of antibody TRN1022 were added to HEp-2 cell culture medium in 96-well microtiter plates at a volume of 50. mu.L/well. Subsequently, a sample containing RSV (including 3 strains such as RSV A2, RSV LONG and RSV 9320) diluted according to a certain proportion is added respectively, and RSV positive and Hep-2 cell culture typical syncytial lesion is clinically detected7 samples 9325, 8879, 9133, 9574, 6477, 8938, 6495 and the like that were positive for RSV-specific PCR detection and were able to neutralize CPE by RSV antibody), 50. mu.L per well, and a virus without added antibody as a positive control and an antibody without added virus as a negative control at 37 ℃ with 5% CO2Incubate for 2 h. The prepared Hep-2 cell suspension (2X 10)5one/mL) 0.1mL was added per well and the plates were incubated at 37 ℃ at 5%, CO2And (5) incubating and culturing in an incubator. Cytopathic effect (CPE) was observed daily with an inverted microscope, with the end-point titer being the reciprocal of the highest dilution of antibody that inhibited 50% of cytopathic effect. Neutralizing antibody titers were determined, defined as the last antibody dilution with more than 50% of intact HEP2 cells.
Substrate conversion was measured by detecting absorbance at 450nm using a microplate reader. The response was fitted to a variable slope curve using log (inhibitor) pairs, IC50 values were calculated using a non-linear fitting algorithm in Graphpad Prism, and IC50 values represent the antibody concentration required for a 50% reduction in the measured absorbance at 450 nm.
The results show that the neutralizing titer of the same amount of TRN1022 antibody is dozens of times higher than that of the same amount of Synagis (palivizumab, respiratory syncytial virus fusion protein (F protein) human monoclonal antibody, which has activity on respiratory syncytial virus clinical isolates such as subtype A and subtype B), and the lowest neutralizing concentration detected by the in vitro micro-neutralizing activity is only 0.42 ng/mL. Compared with the existing marketed drug Synagis, the application of a smaller amount of TRN1022 antibody can neutralize the same amount of virus, and has obvious advantages. Furthermore, the TRN1022 antibody was able to neutralize 3 strains of RSV, such as RSV a2, RSV Long and RSV 9320, i.e. to neutralize both subtype a and subtype B RSV viruses (fig. 6), i.e. it is illustrated as a broadly neutralizing antibody. In addition, clinical specimens 9325, 8879, 9133, 9574, 6477, 8938, 6495 and other 7 clinical specimens from different RSV patients (clinical test RSV positive, Hep-2 cell culture typical syncytial lesion, RSV specific PCR positive and capable of neutralizing CPE by RSV antibody, A, B subtype virus) were cultured and then subjected to neutralization test with TRN1022 antibody. The result shows that the antibody of Synagis and TRN1022 can provide good protective capability against RSV attacks from different clinical patient sources, and the TRN1022 antibody has better effect than Synagis.
3. Affinity of antibody
Anti-human IgG (Fc) was coupled to both channels of a CM5 chip by amino coupling, finally channel 1 was coupled to 5485.4RU and channel 2 was coupled to 5622.4 RU. The trapped RSV047 concentration was 1. mu.g/mL and the binding time was 130 s. The concentration of bound RSV F protein was 2.5. mu.g/mL, 5. mu.g/mL, 10. mu.g/mL, 20. mu.g/mL, 40. mu.g/mL, the binding time was 90s, and the dissociation time was 600 s. The regeneration solution is 3M MgCl2The regeneration time was 30 s. The results are shown in FIG. 7.
RSV Pre-F protein was tested by Surface Plasmon Resonance (SPR) and the results are shown in the table below, antibody TRN1022 binds to Pre-fusion F protein with high affinity.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Sequence listing
<110> Tainuo Mibo Biotech Co., Ltd, Pearl Hai
<120> neutralizing antibody against respiratory syncytial virus and use thereof
<160> 8
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<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 1
Gly Tyr Thr Phe Thr Ser Phe Ala
1 5
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<211> 8
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<213> Artificial Sequence (Artificial Sequence)
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Ile Asn Thr Asn Thr Gly Asp Pro
1 5
<210> 3
<211> 11
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
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Ala Arg Glu Asn Glu Gly Tyr Asp Trp Leu Tyr
1 5 10
<210> 4
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 4
Thr Ser Asp Val Gly Asp Tyr Asn Phe
1 5
<210> 5
<211> 3
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
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Asp Val Ser
1
<210> 6
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<213> Artificial Sequence (Artificial Sequence)
<400> 6
Thr Ser Tyr Arg Ser Ser Thr Thr Tyr Val
1 5 10
<210> 7
<211> 118
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
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Ser Val Thr Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Phe
20 25 30
Ala Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Phe Glu Trp Met
35 40 45
Gly Trp Ile Asn Thr Asn Thr Gly Asp Pro Thr Tyr Gly Gln Gly Ser
50 55 60
Thr Gly Arg Val Val Phe Ser Leu Asp Thr Ser Glu Ser Thr Ala Tyr
65 70 75 80
Leu Gln Ile Ser Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Glu Asn Glu Gly Tyr Asp Trp Leu Tyr Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser Ser
115
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Ser Ile Thr Ile Ser Cys Thr Gly Thr Thr Ser Asp Val Gly Asp Tyr
20 25 30
Asn Phe Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu
35 40 45
Met Ile Tyr Asp Val Ser Ser Arg Pro Ser Gly Val Pro Asn Arg Phe
50 55 60
Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu
65 70 75 80
Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Thr Ser Tyr Arg Ser Ser
85 90 95
Thr Thr Tyr Val Cys Gly Thr Gly Thr Lys Val Ser Val Leu
100 105 110

Claims (10)

1. A neutralizing antibody against respiratory syncytial virus, comprising a light chain variable region and a heavy chain variable region:
the heavy chain variable region comprises: (1) a CDR1 region shown in SEQ ID NO.1, (2) a CDR2 region shown in SEQ ID NO.2, (3) a CDR3 region shown in SEQ ID NO. 3;
the light chain variable region comprises: (1) a CDR1 region shown in SEQ ID NO.4, (2) a CDR2 region shown in SEQ ID NO.5, and (3) a CDR3 region shown in SEQ ID NO. 6.
2. The neutralizing antibody of claim 1, wherein the amino acid sequence of said heavy chain variable region is set forth in SEQ ID No.7 and the amino acid sequence of said light chain variable region is set forth in SEQ ID No. 8.
3. The neutralizing antibody according to claim 1 or 2, wherein said neutralizing antibody is a human antibody, a monoclonal antibody, a purified antibody, a single chain antibody, Fab ', F (ab')2Or Fv.
4. The neutralizing antibody according to claim 3, characterized in that it specifically binds to the pre-fusion protein of respiratory syncytial virus and/or neutralizes respiratory syncytial virus; the neutralizing antibody may be determined by surface plasmon resonance at not more than 1 × 10-5The equilibrium dissociation constant (KD) of M dissociates from the pre-fusion protein of respiratory syncytial virus.
5. The neutralizing antibody of claim 3, wherein the constant region of said neutralizing antibody is selected from any one of IgG, IgA, IgD, IgE, or IgM constant regions.
6. The neutralizing antibody of claim 5, wherein the constant region of the neutralizing antibody is an IgG constant region or an IgA constant region.
7. A nucleic acid molecule encoding the neutralizing antibody of any one of claims 1 to 6.
8. A recombinant expression vector or expression cassette or transgenic cell line or recombinant bacterium comprising the nucleic acid molecule of claim 7.
9. Use of a neutralizing antibody according to any one of claims 1 to 6 in any one of the following a) to e):
a) preparing a medicament or reagent that specifically binds to a pre-fusion protein of respiratory syncytial virus;
b) preparing a medicament or reagent that specifically binds to a respiratory syncytial virus antigen;
c) preparing a medicament or reagent for treating or assisting in treating respiratory syncytial virus;
d) preparing a respiratory syncytial virus vaccine;
e) preparing a detection reagent for detecting RSV.
10. A medicament or agent for the therapeutic or adjuvant treatment of infection by respiratory syncytial virus, characterized in that the active ingredient thereof is the neutralizing antibody according to any one of claims 1 to 6.
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