Antibodies against respiratory syncytial virus

Abstract

The present invention discloses antibodies against respiratory syncytial virus. The antibodies against respiratory syncytial virus provided by the present invention have strong affinity activity to respiratory syncytial virus, and not only have application value in developing diagnostic reagents or kits for detecting RSV-F protein, but also have potential value in developing therapeutic antibodies for preventing or treating respiratory syncytial virus infection.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and in particular relates to antibodies against respiratory syncytial virus. Background Art

[0002] Respiratory Syncytial Virus (RSV) is an enveloped, single-stranded negative-strand RNA virus with a non-segmented genome. It is a common infectious virus that causes respiratory tract infections, mainly causing lower respiratory tract infections such as bronchiolitis and pneumonia in infants under 6 months old, and upper respiratory tract infections such as rhinitis and colds in older children and adults.

[0003] There are currently no specific therapeutic drugs or preventive vaccines for RSV virus. The discovery of new neutralizing antibodies that can be used for the prevention and treatment of RSV virus has important application significance and translational value. Summary of the invention

[0004] To make up for the deficiencies of the prior art, the present invention provides an antibody against 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 or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof comprises:

[0007] Heavy chain variable region complementarity determining region:

[0008] V H CDR1, which comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to the amino acid sequence shown in SEQ ID NO: 1,

[0009] V H CDR2, which comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to the amino acid sequence shown in SEQ ID NO: 2,

[0010] V H CDR3, which comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to the amino acid sequence shown in SEQ ID NO:3,

[0011] Light chain variable complementarity determining region:

[0012] V LCDR1, which comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to the amino acid sequence shown in SEQ ID NO:9,

[0013] V L CDR2, which comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to the amino acid sequence shown in SEQ ID NO: 10,

[0014] V L CDR3, which comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to the amino acid sequence shown in SEQ ID NO: 11,

[0015] The antibody or antigen-binding fragment thereof specifically binds to the F protein of respiratory syncytial virus and / or neutralizes respiratory syncytial virus.

[0016] Furthermore, the antibody or antigen-binding fragment thereof specifically binds to the pre-fusion conformation of the F protein of respiratory syncytial virus.

[0017] Further, V H The amino acid sequence of CDR1 is shown in SEQ ID NO: 1,

[0018] V H The amino acid sequence of CDR2 is shown in SEQ ID NO: 2,

[0019] V H The amino acid sequence of CDR3 is shown in SEQ ID NO: 3,

[0020] V L The amino acid sequence of CDR1 is shown in SEQ ID NO:9,

[0021] V L The amino acid sequence of CDR2 is shown in SEQ ID NO: 10,

[0022] V L The amino acid sequence of CDR3 is shown in SEQ ID NO:11.

[0023] Furthermore, the antibody or antigen-binding fragment thereof further comprises:

[0024] Heavy chain variable region framework region:

[0025] V HFR1, which comprises an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to the amino acid sequence shown in SEQ ID NO:4,

[0026] V H FR2, which comprises an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to the amino acid sequence shown in SEQ ID NO:5,

[0027] V H FR3, which comprises an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to the amino acid sequence shown in SEQ ID NO:6,

[0028] V H FR4, which comprises an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to the amino acid sequence shown in SEQ ID NO:7,

[0029] Light chain variable region framework region:

[0030] V L FR1, which comprises an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to the amino acid sequence shown in SEQ ID NO: 12,

[0031] V L FR2, which comprises an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to the amino acid sequence shown in SEQ ID NO: 13,

[0032] V L FR3, which comprises an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to the amino acid sequence shown in SEQ ID NO: 14,

[0033] V L FR4 comprises an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence shown in SEQ ID NO:15.

[0034] Further, V H The amino acid sequence of FR1 is shown in SEQ ID NO:4,

[0035] V H The amino acid sequence of FR2 is shown in SEQ ID NO:5,

[0036] V H The amino acid sequence of FR3 is shown in SEQ ID NO:6,

[0037] V H The amino acid sequence of FR4 is shown in SEQ ID NO:7,

[0038] V L The amino acid sequence of FR1 is shown in SEQ ID NO: 12,

[0039] V L The amino acid sequence of FR2 is shown in SEQ ID NO: 13,

[0040] V L The amino acid sequence of FR3 is shown in SEQ ID NO: 14,

[0041] V L The amino acid sequence of FR4 is shown in SEQ ID NO:15.

[0042] Furthermore, the heavy chain variable region V H An amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 93%, 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to the amino acid sequence shown in SEQ ID NO:8,

[0043] Light chain variable region V L An amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 93%, 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence shown in SEQ ID NO:16.

[0044] Furthermore, the heavy chain variable region V H The amino acid sequence is shown in SEQ ID NO:8,

[0045] Light chain variable region V L The amino acid sequence is shown in SEQ ID NO:16.

[0046] Furthermore, the antibody or antigen-binding fragment thereof also includes a defucosylated antibody or antigen-binding fragment thereof.

[0047] Furthermore, the antibody or antigen-binding fragment thereof further comprises a detectable label.

[0048] A second aspect of the present invention provides any of the following substances:

[0049] (1) a nucleic acid molecule encoding the antibody or antigen-binding fragment thereof according to the first aspect of the present invention;

[0050] (2) a vector, wherein the vector comprises the nucleic acid molecule described in (1);

[0051] (3) a host cell, wherein the host cell comprises the nucleic acid molecule described in (1) or the vector described in (2);

[0052] (4) A product for detecting respiratory syncytial virus, comprising the antibody or antigen-binding fragment thereof according to the first aspect of the present invention.

[0053] Furthermore, the encoding V in (1) H CDR1, V H CDR2, V H The nucleic acid molecules of CDR3 have nucleotide sequences that are at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the nucleotide sequences shown in SEQ ID NOs: 17, 18, and 19, respectively.

[0054] Code V L CDR1, V L CDR2, V L The nucleic acid molecules of CDR3 have nucleotide sequences that are at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% identical to the nucleotide sequences shown in SEQ ID NOs: 20, 21, and 22, respectively.

[0055] Furthermore, the encoding V in (1) H CDR1, V H CDR2, V H The nucleotide sequences of the nucleic acid molecules of CDR3 are shown in SEQ ID NOs: 17, 18, and 19, respectively.

[0056] Code V L CDR1, V L CDR2, V L The nucleotide sequences of the nucleic acid molecules of CDR3 are shown in SEQ ID NOs: 20, 21 and 22, respectively.

[0057] Furthermore, the vector described in (2) has a signal peptide operably linked to the antibody.

[0058] Furthermore, the vector also includes elements for controlling expression.

[0059] Furthermore, the vector may also include materials that facilitate its entry into cells.

[0060] Furthermore, the host cells described in (3) include prokaryotic cells and eukaryotic cells.

[0061] Furthermore, the prokaryotic cells include true bacteria.

[0062] Further, the true bacteria include Gram-negative or Gram-positive organisms, Escherichia, Enterobacter, Enterobacter, Streptomyces.

[0063] Furthermore, the eukaryotic cell includes a protist cell, an animal cell, a plant cell or a fungal cell.

[0064] Furthermore, the animal cells include mammalian cells, bird cells, and insect cells.

[0065] Furthermore, the mammalian cells include 293F cells, 293T cells, CHO cells, AT1080 cells, and A549 cells.

[0066] Furthermore, the product described in (5) also includes a reagent for performing an antigen-antibody reaction or a reagent for detecting a reaction.

[0067] Further, reagents for performing antigen-antibody reaction include buffers and salts.

[0068] Furthermore, the product includes a test kit and a test strip.

[0069] Further, the kit includes a conjugate of an antibody linked to a signal generating compound.

[0070] The third aspect of the present invention provides a pharmaceutical composition for treating or preventing respiratory syncytial virus infection, wherein the pharmaceutical composition comprises the antibody or antigen-binding fragment thereof described in the first aspect of the present invention, or the substance described in the second aspect of the present invention.

[0071] Furthermore, the pharmaceutical composition also includes a pharmaceutically acceptable carrier.

[0072] Furthermore, the pharmaceutical composition also includes one or more additional antiviral antibodies.

[0073] Furthermore, the one or more additional antiviral antibodies are anti-RSV antibodies or antigen-binding fragments thereof.

[0074] Further, the one or more additional antiviral antibodies include 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, and antigen-binding fragments thereof.

[0075] A fourth aspect of the present invention provides any of the following applications:

[0076] (1) Use of the antibody or antigen-binding fragment thereof according to the first aspect of the present invention, or the substance according to the second aspect of the present invention in detecting respiratory syncytial virus;

[0077] (2) Use of the antibody or antigen-binding fragment thereof described in the first aspect of the present invention, or the substance described in the second aspect of the present invention, in the preparation of a product for diagnosing respiratory syncytial virus;

[0078] (3) Use of the antibody or antigen-binding fragment thereof according to the first aspect of the present invention, the substance according to the second aspect of the present invention, or the pharmaceutical composition according to the third aspect of the present invention in the preparation of a medicament for preventing and / or treating respiratory syncytial virus infection.

[0079] Furthermore, the product described in (2) includes a kit.

[0080] The fifth aspect of the present invention provides a method for preparing the antibody or antigen-binding fragment thereof according to the first aspect of the present invention, the method comprising: culturing the host cell according to the second aspect of the present invention, and recovering the antibody or antigen-binding fragment thereof.

[0081] Furthermore, the method further comprises purifying the antibody or antigen-binding fragment thereof.

[0082] The sixth aspect of the present invention provides a method for detecting respiratory syncytial virus in a sample, the method comprising: contacting the antibody or antigen-binding fragment thereof described in the first aspect of the present invention with a sample to be tested, thereby detecting the level of respiratory syncytial virus in the sample to be tested.

[0083] Furthermore, the method is a method for non-diagnostic purposes.

[0084] Advantages and beneficial effects of the present invention:

[0085] The antibody against respiratory syncytial virus provided by the present invention has strong affinity activity to respiratory syncytial virus, and has application value in developing diagnostic reagents or kits for detecting RSV-F protein, and also has potential value in developing therapeutic antibodies for preventing or treating respiratory syncytial virus infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0086] Figure 1 This is the SDS-PAGE image after the antibody was purified by ProteinA magnetic beads;

[0087] Figure 2 It is a graph for detecting the binding activity of RSV-F antibody to the antigen DS2-StrepII-His6;

[0088] Figure 3 This is a graph showing the neutralizing activity of RSV-F antibody against live RSV-A2 virus. DETAILED DESCRIPTION

[0089] The following provides definitions of some terms used in this specification. Unless otherwise defined, all technical and scientific terms used in the present invention generally have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs.

[0090] The present invention provides an antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof comprises a heavy chain variable region complementarity determining region: V H CDR1, V H CDR2, V H CDR3, and light chain variable region complementarity determining region: V L CDR1, V L CDR2, V L CDR3, the antibody or antigen-binding fragment thereof further comprises a heavy chain variable region framework region: V H FR1, V H FR2, V H FR3, V H FR4, and light chain variable region framework region: V L FR1, V L FR2, V L FR3, V L FR4, the antibody or antigen-binding fragment thereof specifically binds to the F protein of respiratory syncytial virus (RSV-F) and / or neutralizes respiratory syncytial virus.

[0091] In the present invention, antibody refers to immunoglobulin and immunoglobulin fragment, whether natural or partially or completely synthesized (e.g., recombinant), any fragment of the binding specificity ability of at least a portion of the variable region of the immunoglobulin molecule that retains the full-length immunoglobulin. Therefore, the antibody includes any protein having a binding domain homologous or substantially homologous to the immunoglobulin antigen binding domain (antibody binding site). Antibodies include antibody fragments, such as anti-RSV antibody fragments. As used in the present invention, the term antibody therefore includes synthetic antibodies, recombinantly produced antibodies, multispecific antibodies (e.g., bispecific antibodies), human antibodies, non-human antibodies, humanized antibodies, chimeric antibodies, intracellular antibodies, and antibody fragments, including but not limited to Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, disulfide-linked Fv (dsFv), Fd fragments, Fd' fragments, single-chain Fv (scFv), single-chain Fab (scFab), double antibodies, anti-idiotype (anti-Id) antibodies, or antigen-binding fragments of any of the above antibodies. The antibodies provided herein include members of any immunoglobulin class (e.g., IgG, IgM, IgD, IgE, IgA, and IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass (e.g., IgG2a and IgG2b).

[0092] An "antibody fragment" or "antigen-binding fragment" of an antibody refers to any portion of a full-length antibody that is less than the full length but contains at least a portion of the variable region of the antibody that binds to an antigen (e.g., one or more CDRs and / or one or more antibody binding sites), and thus retains binding specificity and at least a portion of the specific binding ability of the full-length antibody; antibody fragments include antibody derivatives produced by enzymatic treatment of full-length antibodies, as well as synthetically produced derivatives, such as recombinantly produced derivatives. Antibodies include antibody fragments. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, single-chain Fv (scFv), Fv, dsFv, diabodies, Fd and Fd' fragments, and other fragments, including modified fragments. The fragments may include multiple chains linked together, for example, by disulfide bonds and / or by peptide linkers. Antibody fragments generally include at least or about 50 amino acids, and typically at least or about 200 amino acids.

[0093] The antibody or antigen-binding fragment thereof specifically binds to the pre-fusion conformation of the F protein of respiratory syncytial virus.

[0094] The F protein of respiratory syncytial virus (RSV-F) is responsible for the fusion of virus and host cell membrane and the formation of syncytium 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.

[0095] The antibody or antigen-binding fragment thereof also includes a defucosylated antibody or antigen-binding fragment thereof.

[0096] In the present invention, defucosylation refers to core fucosylation that substantially lacks N-glycans covalently attached directly or indirectly to the N-glycosylation site (e.g., amino acid residue position 297 in the Fc region of human IgG1, numbered according to the EU index) or the corresponding residue in non-IgG1 or non-human IgG1 immunoglobulins.

[0097] A defucosylated antibody or antigen-binding fragment thereof refers to an IgG1 or IgG3 isotype antibody that lacks fucose in its constant region glycosylation. Glycosylation of human IgG1 or IgG3 occurs at Asn297 in the form of a core fucosylated bibranched complex oligosaccharide glycosylation with a maximum of 2 Gal residues as terminals. In some embodiments, the defucosylated antibody lacks fucose at Asn297. These structures are labeled as G0, G1 (α1,6 or α1,3) or G2 glycan residues, depending on the amount of terminal Gal residues.

[0098] The antibody or antigen-binding fragment thereof further comprises a detectable label.

[0099] In the present invention, the detectable label can be directly or indirectly attached or connected to a molecule (e.g., an anti-RSV antibody or antigen-binding fragment thereof provided by the present invention) or associated therewith and can detect a detectable marker (e.g., a fluorescent molecule, a chemiluminescent molecule, a bioluminescent molecule, a contrast agent (e.g., a metal), a radionuclide, a chromophore, a detectable peptide, or an enzyme that catalyzes the formation of a detectable product). The detection method can be any method known in the art.

[0100] The present invention provides any of the following substances:

[0101] (1) a nucleic acid molecule encoding the above-mentioned antibody or antigen-binding fragment thereof;

[0102] (2) a vector, wherein the vector comprises the nucleic acid molecule described in (1);

[0103] (3) a host cell, wherein the host cell comprises the nucleic acid molecule described in (1) or the vector described in (2);

[0104] (4) A product for detecting respiratory syncytial virus, comprising the above-mentioned antibody or antigen-binding fragment thereof.

[0105] In the present invention, nucleic acid molecules or nucleic acids refer to oligomers or polymers comprising at least two connected nucleotides or nucleotide derivatives, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) that are usually linked together by phosphodiester bonds. Polynucleotides also include DNA and RNA derivatives, which include, for example, nucleotide analogs or "backbone" bonds other than phosphodiester bonds, such as phosphotriester bonds, phosphoramidate bonds, phosphorothioate bonds, thioester bonds or peptide bonds (peptide nucleic acids). Polynucleotides (nucleic acid molecules) include single-stranded and / or double-stranded polynucleotides, such as deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) and analogs or derivatives of RNA or DNA. The term also includes equivalents, derivatives, variants and analogs of RNA or DNA composed of nucleotide analogs, single-stranded (sense or antisense) and double-stranded polynucleotides. Deoxyribonucleotides include deoxyadenosine, deoxycytidine, deoxyguanosine and deoxythymidine. For RNA, the uracil base is uridine. The polynucleotide may comprise nucleotide analogs, including, for example, mass-modified nucleotides, which allow mass-distinguishing polynucleotides; comprising detectably labeled nucleotides, such as fluorescent, radioactive, luminescent or chemiluminescent labels, which allow detection of polynucleotides; or comprising reactive groups, such as biotin or thiol groups, which facilitate fixation of polynucleotides to solid supports. The polynucleotide may also comprise one or more selectively cleavable backbone bonds, for example, chemically, enzymatically or photolytically cleavable. For example, a polynucleotide may comprise one or more deoxyribonucleotides, followed by one or more ribonucleotides, followed by one or more deoxyribonucleotides, such a sequence may be cleaved at the ribonucleotide sequence by alkaline hydrolysis. The polynucleotide may also comprise one or more bonds that are relatively tolerant to cleavage, such as chimeric oligonucleotide primers, which may comprise nucleotides connected by peptide nucleic acid bonds and at least one nucleotide connected by a phosphodiester bond or other suitable bond at the 3′ end, and may be extended by a polymerase. The peptide nucleic acid sequence may be prepared using known methods. Examples of nucleic acid molecules (polynucleotides) provided by the present invention are oligonucleotides, including synthetic oligonucleotides, oligonucleotide duplexes, primers (including blunt-ended primers), and oligonucleotide duplex cassettes.

[0106] In the present invention, vector refers to a medium into which a polynucleotide encoding a protein can be operably inserted to cause the expression of the protein. The vector can be used for transformation, transduction or transfection of a host cell so that it expresses the genetic elements carried in the host cell. The vector can contain a variety of elements for controlling expression, including a promoter sequence, a transcription initiation sequence, an enhancer sequence, a selection element and a reporter gene. In addition, the vector can contain a replication origin. The term replication origin refers to a sequence that initiates replication when present in a vector. The replication origin can be recognized by a replication initiation factor or alternatively by a DNA helicase. The vector can also include materials that help it enter the cell, including but not limited to virus particles, liposomes or protein envelopes.

[0107] Examples of vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papilloma viruses, papovaviruses (e.g., SV40), lambda phage and M13 phage, plasmids. Specific examples of vectors include, but are not limited to, pcDNA3.3, pMD18-T, pOptivec, pCMV, pEGFP, pIRES, pQD-Hyg-GSeu, pALTER, pBAD, pcDNA, pCal, pL, pET, pGEMEX, pGEX, pCI, pEGFT, pSV2, pFUSE, pVITRO, pVIVO, pMAL, pMONO, pSELECT, pUNO, pDUO, Psg5L, pBABE, pWPXL, pBI, p15TV-L, pPro18, pTD, pRS10, pLexA, pACT2.2, pCMV-SCRIPT.RTM., pCDM8, pCDNA1.1 / amp, pcDNA3.1, pRc / RSV, PCR2.1, pEF-1, pFB, pSG5, pXT1, pCDEF3, pSVSPORT, and pEF-Bos.

[0108] In the present invention, host cells are cells for receiving, maintaining, replicating and amplifying vectors. Host cells can also be used to express polypeptides encoded by vectors. When host cells divide, nucleic acids contained in the vectors replicate, thereby amplifying nucleic acids. In one embodiment, host cells are genetic packages that can be induced to express variant polypeptides on their surfaces. In another embodiment, host cells are infected with genetic packages.

[0109] Host cells can be virtually any cell for which expression vectors are available. They include prokaryotic cells, eukaryotic cells, and the prokaryotic cells include, but are not limited to, true bacteria, such as Gram-negative or Gram-positive organisms, such as Enterobacteriaceae, such as Escherichia, such as Escherichia (DH5α, BL21DE3, BL21DE3pLysS, JM109, TOP10, HB101, SCS110, E.coli JM110); Enterobacter; Erwinia; Klebsiella; Proteus; Salmonella, such as Salmonella typhimurium; Serratia, such as Serratia marcescens. marcescans); and Shigella, as well as Bacilli, such as B. subtilis and B. licheniformis; Pseudomonas, such as P. aeruginosa; and Streptomyces.

[0110] Eukaryotic cells include, but are not limited to, protist cells, animal cells, plant cells, or fungal cells, wherein the animal cells include mammalian cells, avian cells, and insect cells. Among them, mammalian cells include, but are not limited to, CHO cells, F2N cells, CSO cells, BHK cells, Bowes melanoma cells, HeLa cells, 911 cells, AT1080 cells, A549 cells, 293T cells, and 293F cells.

[0111] The present invention provides a pharmaceutical composition for treating or preventing respiratory syncytial virus infection, wherein the pharmaceutical composition comprises the above-mentioned antibody or antigen-binding fragment thereof, or the above-mentioned substance.

[0112] The pharmaceutical composition also includes a pharmaceutically acceptable carrier. Typically, such a pharmaceutical composition uses components that will not significantly destroy the biological properties of the antibody or its antigen-binding fragment, such as binding to its specific epitope (e.g., binding to an epitope on the RSV F protein). Each component is pharmaceutically and physiologically acceptable in the sense of being compatible with other ingredients and not harming the patient. The pharmaceutical composition can be conventionally present in unit dosage form and can be prepared by methods well known in the pharmaceutical field, including but not limited to tablets, pills, powders, liquid solutions or suspensions (e.g., including injectable, absorbable and body surface preparations (e.g., eye drops, gels or ointments), aerosols (e.g., nasal sprays), liposomes, suppositories, injectable and perfusable solutions and sustained release forms. Pharmaceutically acceptable carriers include but are not limited to water, buffers, saline solutions, phosphate buffered saline solutions, various types of wetting agents, sterile solutions, alcohol, gum arabic, vegetable oils, benzyl alcohol, gelatin, glycerol, carbohydrates (e.g., lactose, sucrose, straight-chain starch or starch), magnesium stearate, talc, silicic acid, viscous paraffin, aromatic The pharmaceutical composition provided by the present invention may contain other additives, including, for example, antioxidants, preservatives, antimicrobial agents, analgesics, binders, disintegrants, colorants, diluents, excipients, extenders, glidants, solubilizers, stabilizers, tonicity agents, vehicles, thickeners, flavoring agents, emulsions (such as oil / water emulsions), emulsifying and suspending agents (such as gum arabic, agar, alginic acid, sodium alginate, bentonite, carbomer, carrageenan, carboxymethyl cellulose, cellulose, cholesterol, gelatin, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, methyl cellulose, octoxynol polyether-9 (octoxynol 9), oleyl alcohol, polyvinyl pyrrolidone, propylene glycol monostearate, sodium lauryl sulfate, sorbitan esters, stearyl alcohol, tragacanth gum, xanthan gum and its derivatives), solvents and various ingredients, such as crystalline cellulose, microcrystalline cellulose, citric acid, dextrin, glucose, liquid glucose, lactic acid, lactose, magnesium chloride, potassium metaphosphate, starch. Such carriers and / or additives can be prepared by conventional methods and can be administered to subjects at appropriate doses.

[0113] 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.

[0114] The pharmaceutical compositions of the present invention may also be combined with one or more additional drugs to treat respiratory syncytial virus infection, including but not limited to immunomodulators, anti-inflammatory agents (e.g., adrenocorticoids, corticosteroids (e.g., beclomethasone, budesonide, flunisolide, fluticasone, triamcinolone, methylprednisolone, prednisolone, prednisone, hydrocortisone), glucocorticoids, steroids, nonsteroidal anti-inflammatory drugs (e.g., aspirin, ibuprofen, diclofenac and COX-2 inhibitors)), analgesics, leukotriene antagonists (e.g., montelukast, methylxanthines, zafirlukast and zilutuzumab) ), bronchodilators such as beta2-agonists (e.g., bambuterol, bitolterol, clenbuterol, fenoterol, formoterol, indacaterol, isotretol, metaproterol, pirbuterol, procaterol, reproterol, rimiterol, salbutamol (albuterol, ventolin), levalbuterol, salmeterol, tulobuterol and terbutaline) and anticholinergics (e.g., ipratropium and aufendium), sulphasalazine, penicillamine, dapsone, antihistamines, antimalarials (e.g., hydroxychloroquine) and antivirals.

[0115] The present invention provides use of the above antibody or antigen-binding fragment thereof, the above substance or the above pharmaceutical composition in preparing a drug for preventing and / or treating respiratory syncytial virus infection.

[0116] In the present invention, respiratory syncytial virus infection or RSV infection refers to all stages of RSV life cycle in the host (including but not limited to infection and replication of RSV in cells or body tissues), and infection and replication of RSV The pathological state caused by. The infection and proliferation of RSV include but are not limited to the following steps: RSV particles dock to cells, virus and cell membrane merge, viral genetic information introduces cells, expresses RSV protein, produces new RSV particles and RSV particles are released from cells. RSV infection can be upper respiratory tract RSV infection (URI), lower respiratory tract RSV infection (LRI) or their combination. Comprise for example bronchiolitis and pneumonia.

[0117] 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.

[0118] Example

[0119] 1 Experimental Materials

[0120] Vazyme Mouse Single Cell BCR IgG H / K Amplification Kit: Mouse Single Cell BCR IgG H / K Amplification Kit, Vazyme, DD5101;

[0121] Both 293F cells and 293T cells are commercially available human embryonic kidney epithelial cells;

[0122] pcDNA3.1(+) vector: Invitrogen, product catalog number V790-20;

[0123] HRP anti-Respiratory Syncytial virus antibody: Abcam, Cat: ab20686;

[0124] KPL Trueblue color developing solution: Sera care, Cat: 5510-0030.

[0125] 2 Experimental methods

[0126] 2.1 Flow cytometry sorting of mouse RSV-F antigen-specific B cells

[0127] 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.

[0128] 2.2 Single B cell antibody sequence acquisition and sequence analysis

[0129] 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).

[0130] 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.

[0131] 2.3 Antibody antigen-specific binding activity detection

[0132] 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.

[0133] 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.

[0134] 2.4 Antibody expression and purification

[0135] 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.

[0136] 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.

[0137] 2.5 Antibody binding activity to pre-fusion RSV-F protein DS2-StrepII-His6

[0138] 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.

[0139] 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 antibody LV21 to be tested 11 times in a 3-fold gradient, with the concentration of the first well being 100μg / ml. Set up a duplicate well at the same time, incubate 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.

[0140] 2.6 Neutralizing activity detection of antibodies

[0141] 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.

[0142] 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.

[0143] 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.

[0144] 4. Set up VC (no antibody, with virus) and CC (no antibody, no virus) wells in a 96-well plate as controls.

[0145] 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.

[0146] 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.

[0147] 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.

[0148] 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.

[0149] 9. Add 200 μl of PBS containing 5% BSA to each well and block for 1 hour on a shaker at room temperature.

[0150] 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.

[0151] 11. Use 200 μl of PBS solution per well and wash 5-8 times.

[0152] 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.

[0153] 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%.

[0154] 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.

[0155] 3 Experimental results

[0156] Table 1LV21 antibody sequence

[0157]

[0158]

[0159] Table 2 Analysis of novelty of antibody heavy chains

[0160]

[0161] Table 3 Antibody light chain novelty analysis

[0162]

[0163] 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 neutralizing antibody LV21 ( 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.

[0164] The half-binding concentration EC50 of LV21 antibody and RSV-F pre-fusion conformation immunogen DS2-StrepII-His6 was 0.009237μg / 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 LV21 neutralizing RSV-A2 strain virus is 0.04082μg / ml ( Figure 3 ), indicating that the antibody has strong neutralizing activity.

[0165] 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 or antigen-binding fragment thereof that specifically binds to respiratory syncytial virus, characterized in that: The antibody or antigen-binding fragment thereof comprises, Heavy chain variable region complementarity determining region: V H CDR1, whose amino acid sequence is shown in SEQ ID NO: 1, V H CDR2, the amino acid sequence of which is shown in SEQ ID NO: 2, V H CDR3, the amino acid sequence of which is shown in SEQ ID NO: 3, Light chain variable region complementarity determining region: V L CDR1, whose amino acid sequence is shown in SEQ ID NO:9, V L CDR2, the amino acid sequence of which is shown in SEQ ID NO: 10, V L CDR3, the amino acid sequence of which is shown in SEQ ID NO:

11.

2. The antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The antibody or antigen-binding fragment thereof specifically binds to the pre-fusion conformation of the F protein of respiratory syncytial virus.

3. The antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The antibody or antigen-binding fragment thereof further comprises: Heavy chain variable region framework region: V H FR1 comprises an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to the amino acid sequence shown in SEQ ID NO:4, V H FR2 comprises an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to the amino acid sequence shown in SEQ ID NO:5, V H FR3, which comprises an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to the amino acid sequence shown in SEQ ID NO:6, V H FR4, which comprises an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to the amino acid sequence shown in SEQ ID NO:7, Light chain variable region framework region: V L FR1 comprises an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to the amino acid sequence shown in SEQ ID NO: 12, V L FR2, which comprises an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to the amino acid sequence shown in SEQ ID NO: 13, V L FR3, which comprises an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to the amino acid sequence shown in SEQ ID NO: 14, V L FR4 comprises an amino acid sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence shown in SEQ ID NO:

15.

4. The antibody or antigen-binding fragment thereof according to claim 3, characterized in that: V H The amino acid sequence of FR1 is shown in SEQ ID NO:4, V H The amino acid sequence of FR2 is shown in SEQ ID NO:5, V H The amino acid sequence of FR3 is shown in SEQ ID NO:6, V H The amino acid sequence of FR4 is shown in SEQ ID NO:7, V L The amino acid sequence of FR1 is shown in SEQ ID NO: 12, V L The amino acid sequence of FR2 is shown in SEQ ID NO: 13, V L The amino acid sequence of FR3 is shown in SEQ ID NO: 14, V L The amino acid sequence of FR4 is shown in SEQ ID NO:

15.

5. The antibody or antigen-binding fragment thereof according to claim 4, characterized in that: The heavy chain variable region V H An amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 93%, 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the amino acid sequence set forth in SEQ ID NO:8, Light chain variable region V L An amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 93%, 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:

16.

6. The antibody or antigen-binding fragment thereof according to claim 5, characterized in that: Heavy chain variable region V H The amino acid sequence is shown in SEQ ID NO:8, Light chain variable region V L The amino acid sequence is shown in SEQ ID NO:

16.

7. The antibody or antigen-binding fragment thereof according to claim 6, characterized in that: The antibody or antigen-binding fragment thereof is a defucosylated antibody or antigen-binding fragment thereof.

8. The antibody or antigen-binding fragment thereof according to claim 7, characterized in that: The antibody or antigen-binding fragment thereof further comprises a detectable label.

9. Any of the following substances: (1) A nucleic acid molecule, characterized in that: The nucleic acid molecule encodes the antibody or antigen-binding fragment thereof according to any one of claims 1 to 8; (2) A vector, characterized in that the vector comprises the nucleic acid molecule described in (1); (3) A host cell, characterized in that the host cell comprises the nucleic acid molecule described in (1) or the vector described in (2); (4) A product for detecting respiratory syncytial virus, characterized in that the product comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 8.

10. The substance according to claim 9, characterized in that (1) Code V H CDR1, V H CDR2, V H The nucleotide sequences of the nucleic acid molecules of CDR3 are shown in SEQ ID NOs: 17, 18, and 19, respectively. Code V L CDR1, V L CDR2, V L The nucleotide sequences of the nucleic acid molecules of CDR3 are shown in SEQ ID NOs: 20, 21 and 22, respectively.

11. The substance according to claim 9, characterized in that The vector described in (2) has a signal peptide operably linked to the antibody.

12. The substance according to claim 11, characterized in that The vector also includes elements for controlling expression.

13. The substance according to claim 11, characterized in that The vector may also include materials that facilitate its entry into cells.

14. The substance according to claim 9, characterized in that The host cells mentioned in (3) include prokaryotic cells and eukaryotic cells.

15. The substance according to claim 14, characterized in that The prokaryotic cells include eubacteria.

16. The substance according to claim 15, characterized in that The eubacteria include Gram-negative or Gram-positive organisms.

17. The substance according to claim 14, characterized in that The eukaryotic cell includes a protist cell, an animal cell, a plant cell or a fungal cell.

18. The substance according to claim 17, characterized in that The animal cells include mammalian cells, avian cells, and insect cells.

19. The substance according to claim 18, characterized in that The mammalian cells include 293F cells, 293T cells, CHO cells, AT1080 cells, and A549 cells.

20. The substance according to claim 9, characterized in that The product described in (4) also includes a reagent for performing an antigen-antibody reaction or a reagent for detecting a reaction.

21. The substance according to claim 20, characterized in that Reagents used to perform antigen-antibody reactions include buffers, salts.

22. The substance according to claim 20, characterized in that The products include test kits and test strips.

23. A pharmaceutical composition for treating or preventing respiratory syncytial virus infection, characterized in that: The pharmaceutical composition comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, or the substance according to any one of claims 9 to 22.

24. The pharmaceutical composition according to claim 23, characterized in that The pharmaceutical composition also includes a pharmaceutically acceptable carrier.

25. The pharmaceutical composition according to claim 23, characterized in that The pharmaceutical composition may also include one or more additional antiviral antibodies.

26. The pharmaceutical composition according to claim 25, characterized in that The one or more additional anti-viral antibodies are anti-RSV antibodies or antigen-binding fragments thereof.

27. The pharmaceutical composition according to claim 26, characterized in that The one or more additional antiviral antibodies include 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, and antigen-binding fragments thereof.

28. Any of the following applications: (1) Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, or the substance according to any one of claims 9 to 22, in detecting respiratory syncytial virus for non-diagnostic purposes; (2) Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, or the substance according to any one of claims 9 to 22, in the preparation of a product for diagnosing respiratory syncytial virus; (3) Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, the nucleic acid molecule, vector, host cell according to any one of claims 9 to 22, or the pharmaceutical composition according to any one of claims 23 to 27 in the preparation of a medicament for preventing and / or treating respiratory syncytial virus infection.

29. The use according to claim 28, characterized in that The products for diagnosing respiratory syncytial virus described in (2) include test kits.

30. A method for preparing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, characterized in that: The method comprises: culturing the host cell according to any one of claims 9 to 22, and recovering the antibody or antigen-binding fragment thereof.

31. The method according to claim 30, characterized in that The method further comprises purifying the antibody or antigen-binding fragment thereof.

32. A method for detecting respiratory syncytial virus in a sample for non-diagnostic purposes, characterized in that: The method comprises: contacting the antibody or antigen-binding fragment thereof according to any one of claims 1 to 8 with a sample to be tested, thereby detecting the level of respiratory syncytial virus in the sample to be tested.

Images