Antibodies that neutralize respiratory syncytial virus

Abstract

The present invention discloses an antibody for neutralizing respiratory syncytial virus. The antibody ZV28 for neutralizing respiratory syncytial virus provided by the present invention can specifically bind to respiratory syncytial virus and has strong affinity activity, thus providing a new solution for the detection of respiratory syncytial virus and the treatment of related diseases and having broad application prospects.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine, and specifically relates to antibodies for neutralizing respiratory syncytial virus. Background Art

[0002] Respiratory Syncytial Virus (RSV) is the most important viral pathogen for acute lower respiratory tract infection in infants and young children around the world. It is widely prevalent and has a high incidence rate. RSV can cause upper respiratory tract diseases, with symptoms including fever, nasal congestion, pharyngitis and otitis media, respiratory symptoms of asthma, bronchitis, bronchitis and pneumonia. The latest research has found that adults and the elderly with impaired immune function are also susceptible to RSV.

[0003] Laboratory testing for pathogenic microorganism infection mainly includes two methods: detection of pathogens at the site of infection and detection of antibodies in patient serum and body fluids. Currently, the culture method is the "gold standard" for the diagnosis of respiratory syncytial virus, but this method is usually time-consuming, requires strict transportation processes and complex technology, and is not suitable for rapid diagnosis. Although nucleic acid detection technologies such as polymerase chain reaction (PCR) have high specificity and sensitivity, their accuracy is not high and they are very easy to cause contamination. Antibody detection is one of the more ideal methods for detecting respiratory syncytial virus infection. Therefore, screening antibodies against respiratory syncytial virus is very necessary for the detection of respiratory syncytial virus and anti-RSV virus infection. Summary of the invention

[0004] To make up for the deficiencies of the prior art, the present invention provides an antibody for neutralizing respiratory syncytial virus.

[0005] To achieve the above object, the present invention adopts the following technical solution:

[0006] The first aspect of the present invention provides an antibody for neutralizing respiratory syncytial virus, the antibody comprising a heavy chain variable region complementary determining region of three CDRs and a light chain variable region complementary determining region of three CDRs, wherein the amino acid sequences of the heavy chain variable region complementary determining regions CDR1, CDR2, and CDR3 have at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% sequence identity with the amino acid sequences shown in SEQ ID NOs: 1, 2, and 3, respectively, and the amino acid sequences of the light chain variable region complementary determining regions CDR1, CDR2, and CDR3 have at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% sequence identity with the amino acid sequences shown in SEQ ID NOs: 9, 10, and 11, respectively.

[0007] Furthermore, the amino acid sequences of the complementary determining regions CDR1, CDR2, and CDR3 of the heavy chain variable region are shown in SEQ ID NOs: 1, 2, and 3, respectively, and the amino acid sequences of the complementary determining regions CDR1, CDR2, and CDR3 of the light chain variable region are shown in SEQ ID NOs: 9, 10, and 11, respectively.

[0008] Further, the heavy chain variable region also includes a heavy chain variable region framework region of four FRs, and the light chain variable region also includes a light chain variable region framework region of four FRs, wherein the amino acid sequences of the heavy chain variable region framework regions FR1, FR2, FR3 and FR4 have at least 90%, at least 92%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity with the amino acid sequences shown in SEQ ID NOs: 4, 5, 6, and 7, respectively, and the amino acid sequences of the light chain variable region framework regions FR1, FR2, FR3 and FR4 have at least 90%, at least 92%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity with the amino acid sequences shown in SEQ ID NOs: 12, 13, 14, and 15, respectively.

[0009] Furthermore, the amino acid sequences of the heavy chain variable region framework regions FR1, FR2, FR3 and FR4 are shown in SEQ ID NOs: 4, 5, 6, and 7, respectively, and the amino acid sequences of the light chain variable region framework regions FR1, FR2, FR3 and FR4 are shown in SEQ ID NOs: 12, 13, 14, and 15, respectively.

[0010] Further, the amino acid sequence of the heavy chain variable region has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO:8,

[0011] The amino acid sequence of the light chain variable region has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO:16.

[0012] Furthermore, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:8, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:16.

[0013] Furthermore, the antibody specifically binds to the pre-fusion conformation of the F protein of respiratory syncytial virus.

[0014] Further, the antibody is afucosylated.

[0015] Further, the antibodies may have any variants having one or more conservative amino acid substitutions.

[0016] The second aspect of the present invention provides a nucleic acid molecule encoding the antibody according to the first aspect of the present invention.

[0017] Furthermore, the nucleotide sequences of the nucleic acid molecules encoding the heavy chain variable region complementary determining regions CDR1, CDR2, and CDR3 have at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% sequence identity with the nucleotide sequences shown in SEQ ID NOs: 17, 18, and 19, respectively.

[0018] The nucleotide sequences of the nucleic acid molecules encoding the complementary determining regions CDR1, CDR2, and CDR3 of the light chain variable region have at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% sequence identity with the nucleotide sequences shown in SEQ ID NOs: 20, 21, and 22, respectively.

[0019] Furthermore, the nucleotide sequences of the nucleic acid molecules encoding the heavy chain variable region complementary determining regions CDR1, CDR2, and CDR3 are shown in SEQ ID NOs: 17, 18, and 19, respectively.

[0020] The nucleotide sequences of the nucleic acid molecules encoding the light chain variable region complementary determining regions CDR1, CDR2, and CDR3 are shown in SEQ ID NOs: 20, 21, and 22, respectively.

[0021] The third aspect of the present invention provides a recombinant expression vector, which comprises the nucleic acid molecule described in the second aspect of the present invention.

[0022] Furthermore, the recombinant expression vector also includes a promoter.

[0023] Furthermore, the recombinant expression vector includes a plasmid vector, a viral vector or a phage vector.

[0024] The fourth aspect of the present invention provides a host cell, wherein the host cell comprises the nucleic acid molecule described in the second aspect of the present invention or the recombinant expression vector described in the third aspect of the present invention.

[0025] Furthermore, the host cells include prokaryotic cells and eukaryotic cells.

[0026] Furthermore, the eukaryotic cells include lower eukaryotic cells and higher eukaryotic cells.

[0027] Furthermore, the higher eukaryotic cells include mammalian cells.

[0028] Furthermore, the mammalian cells include myeloma cell lines, HuT78 cells, 293T cells, 293F cells, CHO cells, W138, and BHK cells.

[0029] The fifth aspect of the present invention provides a pharmaceutical composition, which comprises the antibody described in the first aspect of the present invention, the nucleic acid molecule described in the second aspect of the present invention, the recombinant expression vector described in the third aspect of the present invention, or the host cell described in the fourth aspect of the present invention.

[0030] Furthermore, the pharmaceutical composition also includes a pharmaceutically compatible carrier.

[0031] Furthermore, the pharmaceutical composition also includes a buffer.

[0032] Furthermore, the pharmaceutical composition can be used in combination with one or more antiviral agents.

[0033] The sixth aspect of the present invention provides a kit for detecting respiratory syncytial virus, the kit comprising the antibody described in the first aspect of the present invention.

[0034] Furthermore, the kit further comprises a detectable label conjugated to the antibody.

[0035] Furthermore, the detectable label includes a fluorescent label, a radioactive isotope, a chemiluminescent molecule, a paramagnetic ion or a spin capture agent.

[0036] The seventh aspect of the present invention provides a method for detecting respiratory syncytial virus in a sample, the method comprising contacting the antibody described in the first aspect of the present invention with the sample to be detected, and detecting the level of respiratory syncytial virus in the sample to be detected.

[0037] Furthermore, the method is a method for non-diagnostic purposes.

[0038] The eighth aspect of the present invention provides the use of the antibody described in the first aspect of the present invention, the nucleic acid molecule described in the second aspect of the present invention, the recombinant expression vector described in the third aspect of the present invention or the host cell described in the fourth aspect of the present invention in detecting respiratory syncytial virus or in preparing a product for diagnosing respiratory syncytial virus infection.

[0039] The ninth aspect of the present invention provides the use of the antibody described in the first aspect of the present invention, the nucleic acid molecule described in the second aspect of the present invention, the recombinant expression vector described in the third aspect of the present invention or the host cell described in the fourth aspect of the present invention in inhibiting respiratory syncytial virus infection or preparing a pharmaceutical composition for treating diseases infected by respiratory syncytial virus.

[0040] Advantages and beneficial effects of the present invention:

[0041] The antibody ZV28 for neutralizing respiratory syncytial virus provided by the present invention can specifically bind to respiratory syncytial virus and has strong affinity activity, thus providing a new solution for the detection of respiratory syncytial virus and the treatment of related diseases and having broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 This is the SDS-PAGE image after the antibody was purified by ProteinA magnetic beads;

[0043] Figure 2 It is a graph for detecting the binding activity of RSV-F antibody to the antigen DS2-StrepII-His6;

[0044] Figure 3 This is a graph showing the neutralizing activity of RSV-F antibody against live RSV-A2 virus. DETAILED DESCRIPTION

[0045] The definitions of some terms used in this specification are provided below. Unless otherwise defined, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0046] The present invention provides an antibody for neutralizing respiratory syncytial virus, wherein the antibody comprises a heavy chain variable region complementarity determining region of three CDRs and a light chain variable region complementarity determining region of three CDRs.

[0047] In the present invention, antibodies include but are not limited to any specific binding member, immunoglobulin class and / or isotype (e.g., IgG1, IgG2, IgG3, IgG4, IgM, IgA, IgD, IgE and IgM); and biologically relevant fragments or specific binding members thereof, including but not limited to Fab, F(ab')2, Fv, and scFv (single chain or related entities). Unless otherwise indicated, the term "antibody" should be understood to include its functional antibody fragments.

[0048] The antibody also includes a functional variant of the antibody, which can bind to respiratory syncytial virus and has neutralizing activity against the subtype or fragment.

[0049] Specifically, functional variants include, but are not limited to, derivatives that are substantially similar in primary structural sequence but contain chemical and / or biochemical modifications, such as those not present in the parent antibody of the present invention, such as in vitro or in vivo. These modifications include, for example, acetylation, acylation, covalent attachment of nucleotides or nucleotide derivatives, covalent attachment of lipids or lipid derivatives, cross-linking, formation of disulfide bonds, glycosylation, hydroxylation, methylation, oxidation, pegylation, proteolytic treatment, phosphorylation.

[0050] The antibody specifically binds to the pre-fusion conformation of the F protein of respiratory syncytial virus.

[0051] Respiratory syncytial virus fusion protein (RSV-F protein) is responsible for the fusion of virus and host cell membranes and the formation of syncytia between virus particles. Its sequence is highly conserved between strains. RSV-F protein undergoes a large conformational change in the process of mediating membrane fusion, changing from a metastable pre-fusion conformation (pre-Fusion) to a stable post-fusion conformation (post-Fusion). Antibodies against RSV-F protein include antibodies that bind to the Pre-fusion conformation, and neutralizing antibodies that bind to both the Pre-fusion conformation and the Post-fusion conformation. The antibodies of the present invention bind to the Pre-fusion conformation.

[0052] The antibody is afucosylated.

[0053] In the present invention, afucosylation refers to an antibody of the IgG1 or IgG3 isotype (preferably of the IgG1 isotype) having an altered glycosylation pattern at Asn297 in the Fc region and having a reduced level of fucosyl residues. Glycosylation of human IgG1 or IgG3 occurs at Asn297 as a core fucosylated biantennary complex oligosaccharide glycosylation with up to two Gal residues at the end. Depending on the amount of the terminal Gal residue, these structures are referred to as G0, G1 (α1,6 or α1,3) or G2 glycan residues (Raju, TS, BioProcess Int.1 (2003) 44-53). CHO-type glycosylation of the Fc portion of an antibody is described, for example, by Routier, FH, Glycoconjugate J.14 (1997) 201-207. Antibodies recombinantly expressed in non-sugar-modified CHO host cells are usually fucosylated at Asn297 in an amount of at least 85%. It should be understood that the term afucosylated antibody as used in the present invention includes antibodies without fucose in their glycosylation pattern. It is generally known that the typical glycosylation residue position in antibodies is asparagine at position 297 (Asn297) according to the EU numbering system.

[0054] Any variants of the antibodies having one or more conservative amino acid substitutions.

[0055] In the present invention, variant refers to a molecule that is different from the parent antibody amino acid sequence in amino acid sequence due to the addition, deletion and / or substitution of one or more amino acid residues in the parent antibody sequence and retains at least one desired activity of the parent antibody. The desired activity may include the ability to specifically bind to an antigen, the ability to reduce, inhibit or neutralize the activity of respiratory syncytial virus in animals, and the ability to inhibit the signal transduction mediated by respiratory syncytial virus in cell-based assays. In one embodiment, the variant comprises one or more amino acid substitutions in one or more hypervariable regions and / or framework regions of the parent antibody. For example, the variant may comprise at least one, or about one to about ten, or about two to about five substitutions in one or more hypervariable regions and / or framework regions of the parent antibody. The variant retains the ability to bind to respiratory syncytial virus variants, may have a stronger binding affinity, an enhanced ability to reduce, inhibit or neutralize the activity of respiratory syncytial virus in animals, and / or an enhanced ability to inhibit the signal transduction mediated by respiratory syncytial virus in cell-based assays.

[0056] The present invention provides a recombinant expression vector, which comprises the above nucleic acid molecule.

[0057] In the present invention, when using prokaryotic cells as hosts, the recombinant expression vector generally comprises a strong promoter that enables transcription (for example, tac promoter, lac promoter, lacUV5 promoter, lpp promoter, pLλ promoter, pRλ promoter, rac5 promoter, amp promoter, recA promoter, SP6 promoter, trp promoter and T7 promoter), a ribosome binding site for starting translation and a transcription / translation end sequence. When using Escherichia coli strains (E. coli) (for example, HB101, BL21, DH5α, Top10, JM109, etc.) as host cells, the promoter and operator site of the E. coli tryptophan biosynthesis pathway (Yanofsky, C., J. Bacteriol., (1984) 158: 1018-1024) and the leftward promoter of bacteriophage λ (pLλ promoter, Herskowitz, I. and Hagen, D., Ann. Rev. Genet., (1980) 14: 399-445) can be used as regulatory sites. When Bacillus is used as a host cell, the promoter of the toxin protein gene of Bacillus thuringiensis (Appl. Environ. Microbiol. (1998) 64: 3932-3938; Mol. Gen. Genet. (1996) 250: 734-741) or any promoter that can be expressed in Bacillus can be used as a regulatory site.

[0058] When eukaryotic cells are used as hosts, the recombinant expression vector can utilize promoters derived from the genome of mammalian cells (e.g., metallothionein promoter, β-actin promoter, human hemoglobin promoter, and human creatine promoter) or promoters derived from mammalian viruses (e.g., adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus (CMV) promoter, HSV tk promoter, mouse mammary tumor virus (MMTV) promoter, HIV LTR promoter, Moloney virus promoter, Epstein-Barr virus (EBV) promoter, and Rous sarcoma virus (RSV) promoter), and usually has a polyadenylation sequence as a transcription termination sequence.

[0059] In addition, the recombinant expression vectors of the present invention also include plasmids (for example, pCL, pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8 / 9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14, pGEX series, pET series and pUC19, etc.), bacteriophages (for example, λgt4.λB, λ-Charon, λΔz1 and M13, etc.) or viruses (for example, SV40, etc.).

[0060] The present invention provides a host cell, which comprises the above nucleic acid molecule or the above recombinant expression vector.

[0061] In the present invention, the host cell may be a prokaryotic cell, such as E. coli, Bacillus subtilis, Streptomyces sp., Pseudomonas sp., Proteus mirabilis or Staphylococcus sp. In addition, the host cell may be a fungal cell, such as Aspergillus sp., a yeast cell such as Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces sp. and Neurospora crassa, a lower eukaryotic cell and a higher eukaryotic cell such as an insect cell. In addition, the host cell may be from a plant and / or a mammal. Preferred examples of host cells include, but are not limited to, PER.C6 cells, monkey kidney cells 7 (COS7, particularly simian COS cells), NSO cells, SP2 / 0, Chinese hamster ovary (CHO) cells, W138, baby hamster kidney (BHK) cells, Madin-Darby canine kidney (MDCK) cells, myeloma cell lines, HuT78 cells, 293T cells, 293F cells and other mammalian host cells that produce antibody proteins according to the present invention.

[0062] In the present invention, the method for transformation into host cells includes any method for introducing nucleic acid into organisms, cells, tissues or organs, which can be carried out using standard techniques selected according to the type of host cells as known in the art. The method includes but is not limited to electroporation, protoplast fusion, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, agitation using silicon carbide fibers, Agrobacterium-mediated transformation, and PEG, dextran sulfate, liposomes (lipofectamine) or transformation mediated by drying / inhibition.

[0063] The present invention provides a pharmaceutical composition, which comprises the above-mentioned antibody, the above-mentioned nucleic acid molecule, the above-mentioned recombinant expression vector or the above-mentioned host cell.

[0064] The pharmaceutical composition also includes a pharmaceutically compatible carrier.

[0065] In the present invention, pharmaceutical compatibility refers to non-toxic materials that do not interact with the action of the active ingredient of the pharmaceutical composition. The pharmaceutically compatible carrier refers to a natural or synthetic, organic or inorganic component that is used in conjunction with the active ingredient to facilitate application. According to the present invention, the pharmaceutically compatible carrier includes one or more compatible solid or liquid fillers, diluents or encapsulating materials, and the carrier is suitable for administration to a patient. The components of the pharmaceutical composition of the present invention generally do not interact with each other in a manner that significantly affects the desired drug efficacy.

[0066] In the present invention, the pharmaceutical composition further comprises a buffer, which includes but is not limited to acetate, citrate, borate and phosphate.

[0067] In the present invention, the pharmaceutical composition also includes salts, which, when used in medicine, should be pharmaceutically compatible salts. However, pharmaceutically incompatible salts can also be used to prepare pharmaceutically compatible salts and are included in the present invention. Such pharmacologically and pharmaceutically compatible salts include, but are not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, maleic acid, acetic acid, salicylic acid, citric acid, formic acid, malonic acid, succinic acid. Pharmaceutically compatible salts can also be prepared as alkali metal salts or alkaline earth metal salts, such as sodium salts, potassium salts or calcium salts.

[0068] In the present invention, the pharmaceutical composition may further include a suitable preservative as appropriate, including but not limited to benzalkonium chloride, chlorobutanol, paraben and thimerosal.

[0069] In the present invention, the pharmaceutical composition further comprises a supplementary immune enhancing substance, such as an adjuvant, which includes but is not limited to CpG oligonucleotides, cytokines, chemokines, saponins, GM-CSF and / or RNA.

[0070] Various delivery systems are known and can be used to administer the pharmaceutical compositions of the present invention, such as in liposome encapsulation, microparticles, microcapsules, recombinant cells capable of expressing mutant viruses, receptor-mediated endocytosis. Methods of introduction include, but are not limited to, intradermal, transdermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The composition can be administered by any convenient route, such as by infusion or bolus, absorbed by epithelial or mucocutaneous linings (e.g., oral mucosa, nasal mucosa, rectal and intestinal mucosa, etc.), and can be co-administered with other bioactive agents. Administration can be systemic or local. It can be delivered as an aerosolized formulation.

[0071] The pharmaceutical compositions can also be delivered in vesicles, particularly liposomes.

[0072] In some cases, the pharmaceutical composition can be delivered in a controlled release system. In one embodiment, a pump can be used. In another embodiment, a polymeric material can be used. In yet another embodiment, a controlled release system can be placed close to the target of the composition, thus requiring only a portion of the systemic dose.

[0073] Injectable preparations may include dosage forms for intravenous, subcutaneous, intradermal and intramuscular injections, drips, etc. These injectable preparations may be prepared by known methods. For example, injectable preparations may be prepared, for example, by dissolving, suspending or emulsifying the above-mentioned antibodies or their salts in sterile aqueous or oily media conventionally used for injection. Aqueous media for injection, such as physiological saline, isotonic solutions containing glucose and other adjuvants, etc., may be used in combination with the following: suitable solubilizers, such as alcohols (e.g., ethanol); polyols (e.g., propylene glycol, polyethylene glycol); nonionic surfactants [e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)], etc. The oily media that can be used are, for example, sesame oil, soybean oil, etc., which may be used in combination with solubilizers such as benzyl benzoate, benzyl alcohol, etc. The injection thus prepared is preferably filled in an appropriate ampoule.

[0074] The pharmaceutical compositions of the present invention may be delivered subcutaneously or intravenously by a standard needle or syringe.

[0075] Pharmaceutical compositions for oral or parenteral use are prepared in unit dosage forms suitable for matching the dosage of the active ingredient. These dosage forms of unit dosages include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc.

[0076] In some embodiments, the pharmaceutical composition can be administered with one or more additional antiviral antibodies selected from anti-RSV antibodies or antigen-binding fragments thereof, such as palivizumab, motavizumab, AFFF, P12f2, P12f4, P11d4, A1e9, A12a6, A13c4, A17d4, A4B4, A8c7, 1X-493L1, FR H3-3F4, M3H9, Y10H6, DG, AFFF(1), 6H8, L1-7E5, L2-15B10, A13a11, A1h5, A4B4(1), A4B4L1FR-S28R, A4B4-F52S, rsv6, rsv11, rsv13, rsv19, rsv21, rsv22, rsv23, RF-1, RF-2, or antigen-binding fragments thereof.

[0077] The pharmaceutical composition of the present invention can also be used in combination with one or more antiviral agents for the treatment of respiratory syncytial virus infection. Antiviral agents include, but are not limited to, acyclovir, famciclovir, ganciclovir, penciclovir, valacyclovir, valganciclovir, iodoxuridine, trifluridine, brivudine, cidofovir, docosanol, fomivirsen, foscarnet, tromantine, imiquimod, podophyllotoxin, entecavir, lamivudine, telbivudine, clevudine, adefovir, tenofovir, boceprevir, telaprevir, pleconaril, arbidol, amantadine, rimantadine, oseltamivir, zanamivir, peramivir, inosine, interferon (e.g., interferon α-2b, PEG interferon α-2a), levofloxacin, tadalafil ... Bavirin / taribavirin, abacavir, emtricitabine, lamivudine, didanosine, zidovudine, apricitabine, stampidine, efcitabine, racivir, amdosovir, stavudine, zalcitabine, tenofovir, efavirenz, nevirapine, etravirine, rilpivirine, loviride, tavidin, atazanavir, fosamprenavir, lopinavir, darunavir, nelfinavir, ritonavir, saquinavir, tipranavir, amprenavir, indinavir, enfuvirtide, maraviroc, viriviroc, PRO 140, ibalizumab, raltegravir, elvitegravir, bevirimat, vivecon, including their tautomeric forms, analogs, isomers, polymorphs, solvates, derivatives or salts.

[0078] The present invention provides a kit for detecting respiratory syncytial virus, and the kit comprises the above-mentioned antibody.

[0079] The kit also includes a detectable label conjugated to the antibody.

[0080] In the present invention, detectable labels include, but are not limited to, fluorescent labels, radioisotopes, chemiluminescent molecules, paramagnetic ions or spin-trapping agents.

[0081] Among them, fluorescent labels include but are not limited to Alexa 350, Alexa 430, AMCA, BODIPY 630 / 650, BODIPY 650 / 665, BODIPY-FL, BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, Cascade Blue, Cy3, Cy5, 6-FAM, fluorescein isothiocyanate, HEX, 6-JOE, Oregon Green 488, Oregon Green 500, Oregon Green 514, Pacific Blue, REG, rhodamine green, rhodamine red, Renographin, ROX, TAMRA, TET, tetramethylrhodamine and / or Texas Red.

[0082] Radioactive isotopes include but are not limited to astatine 211 , 14 carbon, 51 chromium, 36 chlorine, 57 cobalt, 58 Cobalt, copper 67 , 152 Eu, Gallium 67 , 3 Hydrogen, iodine 123 ,iodine 125 ,iodine 131 ,indium 111 , 59 iron, 32 Phosphorus, Rhenium 186 ,rhenium 188 , 75 selenium, 35 Sulfur, technetium 99m (technicium) and / or yttrium 90 .

[0083] Paramagnetic ions include, but are not limited to, ions of chromium (III), manganese (II), iron (III), iron (II), cobalt (II), nickel (II), copper (II), neodymium (III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II), terbium (III), dysprosium (III), holmium (III) and / or erbium (III).

[0084] The present invention provides the use of the above-mentioned antibody, the above-mentioned nucleic acid molecule, the above-mentioned recombinant expression vector or the above-mentioned host cell in inhibiting respiratory syncytial virus infection or preparing a pharmaceutical composition for treating diseases infected by respiratory syncytial virus.

[0085] In the present invention, respiratory syncytial virus-infected diseases mean any disease caused directly or indirectly by respiratory syncytial virus (RSV) infection, as well as diseases or conditions that make patients susceptible to RSV infection. Examples of diseases falling into the former category include pneumonia and bronchiolitis. Diseases and conditions in the latter category (those that are about to place patients at risk of severe RSV infection) include cystic fibrosis, congenital heart disease, cancer, age-related immunosuppression, transplant recipients, and any conditions that generally cause an immunosuppressive state or reduced immune system function, such as postoperative organ transplantation regimens or premature births.

[0086] Treatment means to suffer from one or more disease symptoms or suspect to suffer from the individual or patient of the disease that agent has therapeutic activity for it or externally apply therapeutic agent, for example contain any antibody of the present invention or the composition of antigen-binding fragment.Usually, agent is used with the amount of one or more disease symptoms in the individual or colony of effective mitigation treatment, no matter by inducing this symptom disappearance or suppressing this symptom progress through any clinically measurable degree.The amount of the therapeutic agent effectively mitigating any specific disease symptom can be according to factors such as patient's disease state, age and weight, and the ability of medicine to cause required response in individual and change.Whether disease symptoms have been alleviated can be evaluated by any clinical measurement, and described clinical measurement is usually used for evaluating the severity or progress state of this symptom by doctor or other skilled health care provider.Treatment with anti-RSV antibody can also be combined with other intervention measures (antibodies, nucleic acids, vaccines and small molecule compounds), to treat other respiratory pathogens.

[0087] The invention will be further described below in conjunction with specific examples. It should be understood that the specific embodiments described herein are presented by way of example and are not intended to limit the invention. The main features of the invention may be used in a variety of embodiments without departing from the scope of the invention.

[0088] Example

[0089] 1 Experimental Materials

[0090] Vazyme Mouse Single Cell BCR IgG H / K Amplification Kit: Mouse Single Cell BCR IgG H / K Amplification Kit, Vazyme, DD5101;

[0091] Both 293F cells and 293T cells are commercially available human embryonic kidney epithelial cells;

[0092] pcDNA3.1(+) vector: Invitrogen, product catalog number V790-20;

[0093] HRP anti-Respiratory Syncytial virus antibody: Abcam, Cat: ab20686;

[0094] KPL Trueblue color developing solution: Sera care, Cat: 5510-0030.

[0095] 2 Experimental methods

[0096] 2.1 Flow cytometry sorting of mouse RSV-F antigen-specific B cells

[0097] Mice were immunized with RSV-F antigen DS2-StrepII-His6 in pre-fusion conformation, and secondary immunization was performed 3 weeks later. The spleen of mice 15 weeks after the secondary immunization was taken, and the lymphocytes of the mice were obtained using lymphocyte separation fluid. The lymphocytes were resuspended in 80μl PBS containing 1% BSA, and 20μl Fc blocking reagent was added for blocking for 20min. The density of lymphocytes was adjusted to 1x10e7 / ml, and 200nM antigen DS2-StrepII-His6 and fluorescent antibody for memory B cells were added, and incubated on ice in the dark for 30min; 1ml PBS containing 1% BSA was added to resuspend, and the cells were washed 3 times. APC-streptavidin was added, and incubated on ice in the dark for 30min; 1ml PBS containing 1% BSA was added to resuspend, and the cells were washed 3 times. 400μl PBS containing 1% BSA was added to resuspend the cells, filtered into a flow tube, and antigen-specific memory B cells were sorted and collected using a FACS Aria III flow cytometer.

[0098] 2.2 Single B cell antibody sequence acquisition and sequence analysis

[0099] The sequences of the heavy and light chains of BCR were obtained using the Vazyme Mouse Single Cell BCR IgG H / K Amplification Kit (Vazyme, DD5101).

[0100] The main steps include: (1) reverse transcription to synthesize the first-chain cDNA. (2) amplification using the first-chain cDNA synthesis product as a template to obtain the full-length cDNA of the IgG heavy chain and light chain. (3) amplification of the antibody heavy chain and light chain variable region genes using the IgG heavy chain and light chain full-length cDNA amplification product as a template. (4) amplification of the IgG heavy chain and light chain variable region amplification product as a template to amplify the IgG heavy chain and light chain expression frame splicing product.

[0101] 2.3 Antibody antigen-specific binding activity detection

[0102] The heavy chain and light chain expression cassette splicing products were co-transfected into 293T cells. 48 hours after transfection, the transfection supernatant was collected and the antigen-specific binding activity was tested.

[0103] 1μg / ml RSV-F antigen DS2-StrepII-His6 was used to coat the ELISA plate, incubated overnight at 4°C, and blocked with PBS containing 3% BSA for 1 hour. The supernatant of 293T transfected with the linear expression frame was diluted with PBS at a ratio of 1:3 and added to the 96-well ELISA plate. After incubation for 1 hour, PBST (0.05% Tween-80) was added to wash 3 times, 200μl / well. HRP-Goat-anti-mouse IgG was added, incubated for 45 minutes, and PBST (0.05% Tween-80) was added to wash 3 times, 200μl / well. For the antigen-specific binding samples obtained by screening, the antibody heavy chain and light chain sequences were constructed into the pcDNA3.1(+) vector, and the IgG heavy chain and light chain variable region sequences were obtained by sequencing, and the sequencing results were analyzed using the Igblast website.

[0104] 2.4 Antibody Expression and Purification

[0105] Antibody expression: Transfection was performed when the density of 293F cells was 2.5x 10e6 / ml. 1μg of antibody heavy chain plasmid and 1μg of antibody light chain plasmid were added to 20μl culture medium for every 2.5x 10e6 / ml of cells. 3μg of PEI was added to 20μl culture medium. After 5 minutes, the plasmid and PEI dilutions were mixed and placed at room temperature for 15 minutes before adding to 293F cells. After transfection, the cells were cultured in a shaking incubator at 37°C, 220rpm, and 5% carbon dioxide concentration for 72 hours, and the cell supernatant was collected by centrifugation.

[0106] Purification of antibodies: Incubate the antibody 293F cell transfection supernatant with Protein A magnetic beads balanced with PBS for 2 hours, adsorb the magnetic beads using a magnetic stand, and discard the supernatant. Add 20CV PBS to wash the magnetic beads, invert and mix repeatedly, adsorb the magnetic beads using a magnetic stand, discard the supernatant, and wash 3 times. Add 10CV 0.1M Glycine (pH=3.0) to elute the antibody, adsorb the magnetic beads using a magnetic stand, aspirate the supernatant, and add 1M Tris (pH=8.5) to neutralize to pH=7. Add PBS to replace the buffer.

[0107] 2.5 Antibody binding activity to pre-fusion RSV-F protein DS2-StrepII-His6

[0108] The binding activity of RSV-F antibody to the pre-fusion conformation RSV-F protein DS2-strepII-His6 was detected by enzyme-linked absorbance reaction.

[0109] Use pre-fusion RSV-F protein DS2-StrepII-His6 as coating, the coating concentration is 1μg / ml, 100μl / well, coat overnight, and discard the supernatant. Add PBS containing 3% BSA to block for 1 hour, and discard the blocking solution. Dilute the test antibody ZV28 11 times in a 3-fold gradient, the concentration of the first well is 100μg / ml, and set up a duplicate well at 37 degrees for 1 hour. Add 200μl PBST (0.05% Tween) and wash the plate 3 times. Add 1:5K diluted HRP-goat-anti-mouse IgG, incubate for 45min, add 200μl PBST (0.05% Tween), and wash the plate 3 times. Add TMB colorimetric solution, color development for 10min, stop color development, and read the results with an enzyme reader.

[0110] 2.6 Neutralizing activity detection of antibodies

[0111] 1. Plate Vero cells in a 96-well plate, 3*10e4 cells per well, use 200 μl of complete medium containing 10% serum, and culture overnight in a 37°C, 5% CO2 incubator.

[0112] 2. Use complete medium containing 2% serum to dilute the antibody three-fold in a 96-well plate, with the concentration of the first well being 100 ng / μl, ensuring that the final volume of each well is 100 μl.

[0113] 3. Thaw RSV A2 live virus at 4℃, the TCID50 of live virus is 1.45x10e5 Pfu / ml. Take 300μl and add it to 9.7ml complete culture medium containing 2% serum, mix well, and add it to the above-mentioned antibody gradient dilution solution, 100μl per well.

[0114] 4. Set up VC (no antibody, with virus) and CC (no antibody, no virus) wells in a 96-well plate as controls.

[0115] 5. Discard the original overnight culture medium in the 96-well plate Vero cells, mix the solution obtained in steps 2-4, add it to the Vero cells, and culture it in a 37°C, 5% CO2 incubator for 3 hours.

[0116] 6. Discard the solution added in step 5 and replace with 200 μl of methylcellulose per well. Incubate in a 37°C, 5% CO2 incubator for 72 hours.

[0117] 7. Add 60 μl of 4% paraformaldehyde to each well and fix at room temperature for 10 minutes. Tap to discard all supernatant, add 60 μl of 4% paraformaldehyde to each well again and fix at room temperature for 10 minutes.

[0118] 8. Use 200 μl of PBS solution per well, wash 5-8 times, tap to discard all supernatant, and wash all remaining methylcellulose on the cell surface. Make sure there is no viscous liquid or viscous bubbles in the well to prevent affecting the effects of subsequent antibody staining and other steps.

[0119] 9. Add 200 μl of PBS containing 5% BSA to each well and block for 1 hour on a shaker at room temperature.

[0120] 10. Add 100 μl of RSV polyclonal antibody (Abcam, Cat: ab20686) diluted 1:500 in PBS to each well and incubate on a shaker at room temperature for 2 hours in the dark.

[0121] 11. Use 200 μl of PBS solution per well and wash 5-8 times.

[0122] 12. Add 100 μl Trueblue KPL colorimetric solution (Seracare, Cat: 5510-0030) to each well and incubate at room temperature for 10-20 minutes in a dark place. When obvious blue precipitate is formed in the cells under a microscope, discard the colorimetric solution and use an ELISpot plate reader to take pictures and count directly or after the liquid in the well has dried.

[0123] Neutralization activity (%) = [1-(the number of spots in the test group-the number of spots in the cell control) / (the number of spots in the virus control-the number of spots in the cell control)] x 100%.

[0124] Graphpad Prism 5 software was used to calculate the antibody concentration at which the neutralization activity was 50%, i.e., the IC50 value of the antibody.

[0125] 3 Experimental results

[0126] Table 1 ZV28 antibody sequence

[0127]

[0128]

[0129]

[0130] Table 2 Novelty analysis of antibody heavy chains

[0131]

[0132] Table 3 Antibody light chain novelty analysis

[0133]

[0134] Using flow cytometry, single-cell sorting was performed on the spleen memory B cells of mice immunized with RSV-F pre-fusion conformation immunogen DS2-StrepII-His6, and a new RSV-F protein neutralizing antibody ZV28 ( Figure 1 ), the sequence of the antibody is shown in Table 1, the antibody is novel (Table 2, Table 3), and has the ability to neutralize RSV-A2 live virus.

[0135] The half-binding concentration EC50 of ZV28 antibody and RSV-F pre-fusion conformation immunogen DS2-StrepII-His6 is 0.009495μg / ml ( Figure 2 ), indicating that the antibody has a high binding activity to the pre-fusion conformation immunogen DS2-StrepII-His6. In addition, the IC50 value of ZV28 neutralizing RSV-A2 strain virus is 0.6634μg / ml ( Figure 3 ), indicating that the antibody has strong neutralizing activity.

[0136] The description of the above embodiments is only used to understand the method and core idea of ​​the present invention. It should be pointed out that, for those skilled in the art, several improvements and modifications can be made to the present invention without departing from the principle of the present invention, and these improvements and modifications will also fall within the scope of protection of the claims of the present invention.

Claims

1. An antibody that neutralizes respiratory syncytial virus, characterized in that The antibody comprises three CDRs of the heavy chain variable region complementary determining regions and three CDRs of the light chain variable region complementary determining regions, wherein the amino acid sequences of the heavy chain variable region complementary determining regions CDR1, CDR2, and CDR3 are shown in SEQ ID NOs: 1, 2, and 3, respectively, and the amino acid sequences of the light chain variable region complementary determining regions CDR1, CDR2, and CDR3 are shown in SEQ ID NOs: 9, 10, and 11, respectively, and the sequence of SEQ ID NO: 10 is WAS.

2. The antibody according to claim 1, characterized in that The heavy chain variable region also includes a heavy chain variable region framework region of four FRs, and the light chain variable region also includes a light chain variable region framework region of four FRs, wherein the amino acid sequences of the heavy chain variable region framework regions FR1, FR2, FR3 and FR4 are shown in SEQ ID NOs: 4, 5, 6, and 7, respectively, and the amino acid sequences of the light chain variable region framework regions FR1, FR2, FR3 and FR4 are shown in SEQ ID NOs: 12, 13, 14, and 15, respectively.

3. The antibody according to claim 2, characterized in that The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:8, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

16.

4. The antibody according to claim 3, characterized in that The antibody specifically binds to the pre-fusion conformation of the F protein of respiratory syncytial virus.

5. The antibody according to claim 4, characterized in that The antibody is afucosylated.

6. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the antibody according to any one of claims 1 to 5.

7. The nucleic acid molecule according to claim 6, characterized in that The nucleotide sequences of the nucleic acid molecules encoding the heavy chain variable region complementary determining regions CDR1, CDR2, and CDR3 are shown in SEQ ID NOs: 17, 18, and 19, respectively. The nucleotide sequences of the nucleic acid molecules encoding the light chain variable region complementary determining regions CDR1, CDR2, and CDR3 are shown in SEQ ID NOs: 20, 21, and 22, respectively.

8. A recombinant expression vector, characterized in that: The recombinant expression vector comprises the nucleic acid molecule according to any one of claims 6 to 7.

9. The recombinant expression vector according to claim 8, characterized in that The recombinant expression vector also includes a promoter.

10. The recombinant expression vector according to claim 8, characterized in that: The recombinant expression vector includes a plasmid vector, a virus vector or a phage vector.

11. A host cell, characterized in that The host cell comprises the nucleic acid molecule according to any one of claims 6 to 7 or the recombinant expression vector according to any one of claims 8 to 10.

12. The host cell according to claim 11, characterized in that The host cells include prokaryotic cells and eukaryotic cells.

13. The host cell according to claim 12, characterized in that The eukaryotic cells include lower eukaryotic cells and higher eukaryotic cells.

14. The host cell according to claim 13, characterized in that The higher eukaryotic cells include mammalian cells.

15. The host cell according to claim 14, characterized in that The mammalian cells include myeloma cell lines, HuT78 cells, 293T cells, 293F cells, CHO cells, W138, and BHK cells.

16. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the antibody according to any one of claims 1 to 7, the nucleic acid molecule according to any one of claims 6 to 7, the recombinant expression vector according to any one of claims 8 to 10, or the host cell according to any one of claims 11 to 15.

17. The pharmaceutical composition according to claim 16, characterized in that The pharmaceutical composition also includes a pharmaceutically compatible carrier.

18. The pharmaceutical composition according to claim 16, characterized in that The pharmaceutical composition also includes a buffer.

19. The pharmaceutical composition according to claim 16, characterized in that The pharmaceutical composition is used in combination with one or more antiviral agents.

20. A kit for detecting respiratory syncytial virus, characterized in that: The kit comprises the antibody according to any one of claims 1 to 5.

21. The kit according to claim 20, characterized in that The kit also includes a detectable label conjugated to the antibody.

22. The kit according to claim 21, characterized in that The detectable labels include fluorescent labels, radioisotopes, chemiluminescent molecules, paramagnetic ions or spin-trapping agents.

23. A method for detecting respiratory syncytial virus in a sample for non-diagnostic purposes, characterized in that: The method comprises contacting the antibody according to any one of claims 1 to 5 with a sample to be detected, and detecting the level of respiratory syncytial virus in the sample to be detected.

24. Use of the antibody of any one of claims 1 to 5, the nucleic acid molecule of any one of claims 6 to 7, the recombinant expression vector of any one of claims 8 to 10, or the host cell of any one of claims 11 to 15 in detecting respiratory syncytial virus or in preparing a product for diagnosing respiratory syncytial virus infection.

25. Use of the antibody according to any one of claims 1 to 5, the nucleic acid molecule according to any one of claims 6 to 7, the recombinant expression vector according to any one of claims 8 to 10, or the host cell according to any one of claims 11 to 15 in inhibiting respiratory syncytial virus infection for non-therapeutic purposes or in preparing a pharmaceutical composition for treating diseases infected by respiratory syncytial virus.

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