A fully human monoclonal antibody against respiratory syncytial virus and its application

Abstract

The present invention provides a human antibody (hAb) or an antigen-binding fragment thereof that binds to the F protein of respiratory syncytial virus (RSV). The present invention also provides the use of the human antibody (hAb) or an antigen-binding fragment thereof in preventing, treating and detecting RSV infection.

Description

Technical Field

[0001] The present invention relates to a human antibody (hAb) or an antigen-binding fragment thereof that binds to the F protein of respiratory syncytial virus (RSV). The present invention also relates to the use of the antibody or the antigen-binding fragment thereof in preventing, treating and detecting RSV infection. Background Art

[0002] Respiratory syncytial virus (RSV) is a common respiratory tract infection pathogen. Immunocompromised populations, including the elderly and infants, are the main susceptible populations. It is the leading cause of infant hospitalization in the world and the second leading cause of infant mortality after malaria. RSV is a filamentous enveloped, negative-sense, single-stranded RNA virus belonging to the order Mononegavirales, family Pneumoviridae, genus Orthopneumovirus. This family also includes human metapneumovirus (hMPV), which belongs to the genus Metapneumovirus and, like RSV, is a major pathogen infecting children. The symptoms of RSV and hMPV infections are difficult to distinguish, and although they can be transmitted by aerosolized droplets, they are usually transmitted through close contact. After a short period of replication in epithelial cells in the nasopharynx and upper respiratory tract, RSV infection may spread to the small bronchioles or alveoli of the lower respiratory tract. The host immune response to RSV infection increases mucus production and inflammation, leading to narrowing of the airways and causing bronchitis in young children.

[0003] The RSV genome is 15.2 kb in length, contains 10 genes, and encodes 11 proteins. RSV virions contain a lipid bilayer that expresses fusion protein (F), attachment protein (G), and small hydrophobic protein (SH). F and G proteins are much more abundant than SH protein, which is a pentameric ion channel that is believed to be involved in delaying apoptosis of infected cells. Among them, F protein (type I integral membrane fusion protein) plays a key role in mediating the fusion of RSV virus with host cell membranes and in the pathogenic process. F protein presents two different conformations, one is the "lollipop"-shaped prefusion (pre-F) state, which exists on the virus surface before the virus interacts with the cell; the other is the "crutch-shaped" postfusion state (post-F), which is obtained after the virus fuses with the cell membrane, or spontaneously rearranges from the highly stable pre-F to the energetically favorable post-F conformation by an unknown mechanism. These two forms are antigenically distinct and are both considered potential vaccine candidates. However, the former form has been shown to induce the most highly neutralizing antibodies after natural infection or immunization, making it the antigen of choice for vaccine development. In fact, the two most immunogenic sites of the F protein, named sites and site V, which are present only in the pre-F conformation, whereas the less immunogenic sites (sites I, II, III, and IV) are shared between the pre-F and post-F forms of the antigen.

[0004] The first product used to prevent RSV infection was RespiGam developed by Medimmune, a mixed human intravenous immunoglobulin (IVIG) containing high concentrations of RSV protective antibodies. It has a significant effect in preventing severe lower respiratory tract infections caused by RSV infection in high-risk infants. With the launch of Palivizumab (Synagis), Medimmune voluntarily stopped using RespiGam in 2003. Palivizumab is a humanized mouse monoclonal antibody that binds to the RSV F protein. It is also the first and only antiviral monoclonal antibody approved for human treatment. For high-risk premature infants with gestational age less than 32 weeks, palivizumab prevention can effectively reduce RSV hospitalization time within the first 6 months after discharge. This approval provides proof of concept for passive immune prevention. However, due to its limited cost and effectiveness, like RespiGam, it is currently only approved for the prevention of RSV in premature infants and infants with cardiopulmonary diseases at birth.

[0005] Developing more effective RSV antibodies that can be injected into all newborns will help directly protect this vulnerable group from RSV infection. Therefore, screening broad-spectrum and high-neutralizing RSV antibodies has important practical significance for the clinical prevention of RSV disease. At the same time, the conserved epitopes of antibody binding to RSV F protein through biological structure and function analysis also have potential guiding role in the design and development of subunit vaccines. Summary of the invention

[0006] In one aspect, the present invention provides a human antibody or an antigen-binding fragment thereof that binds to the F protein of respiratory syncytial virus, wherein the human antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 contained in the heavy chain variable region shown in SEQ ID NO: 2 or 4, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 contained in the light chain variable region shown in SEQ ID NO: 3; preferably, according to the IMGT numbering system, the HCDR1, HCDR2 and HCDR3 of the heavy chain variable region are selected from one of the following combinations:

[0007] (1) The amino acid sequence of HCDR1 is: GFLFSSYS (SEQ ID NO: 10);

[0008] The amino acid sequence of HCDR2 is: ISFDGSER (SEQ ID NO: 11);

[0009] The amino acid sequence of HCDR3 is: AKTWVDRTLSLDS (SEQ ID NO: 12);

[0010] (2) The amino acid sequence of HCDR1 is: GFLFSCYS (SEQ ID NO: 13);

[0011] The amino acid sequence of HCDR2 is: MSFDGSEI (SEQ ID NO: 14);

[0012] The amino acid sequence of HCDR3 is: ARTWVDRTLSLDH (SEQ ID NO: 15); preferably, according to the IMGT numbering system, LCDR1, LCDR2 and LCDR3 of the light chain variable region are as follows:

[0013] The amino acid sequence of LCDR1 is: QGIKNA (SEQ ID NO: 16),

[0014] The amino acid sequence of LCDR2 is: AAS (SEQ ID NO: 17),

[0015] The amino acid sequence of LCDR3 is: LQDYSYPWT (SEQ ID NO: 18);

[0016] More preferably, according to the IMGT numbering system, the sequences of LCDR1-LCDR3 and HCDR1-HCDR3 of the human antibody are as follows:

[0017] (1) The amino acid sequence of LCDR1 is: QGIKNA (SEQ ID NO: 16);

[0018] The amino acid sequence of LCDR2 is: AAS (SEQ ID NO: 17);

[0019] The amino acid sequence of LCDR3 is: LQDYSYPWT (SEQ ID NO: 18);

[0020] The amino acid sequence of HCDR1 is: GFLFSSYS (SEQ ID NO: 10);

[0021] The amino acid sequence of HCDR2 is: ISFDGSER (SEQ ID NO: 11);

[0022] The amino acid sequence of HCDR3 is: AKTWVDRTLSLDS (SEQ ID NO: 12); or

[0023] (2) The amino acid sequence of LCDR1 is: QGIKNA (SEQ ID NO: 16);

[0024] The amino acid sequence of LCDR2 is: AAS (SEQ ID NO: 17);

[0025] The amino acid sequence of LCDR3 is: LQDYSYPWT (SEQ ID NO: 18);

[0026] The amino acid sequence of HCDR1 is: GFLFSCYS (SEQ ID NO: 13);

[0027] The amino acid sequence of HCDR2 is: MSFDGSEI (SEQ ID NO: 14);

[0028] The amino acid sequence of HCDR3 is: ARTWVDRTLSLDH (SEQ ID NO: 15).

[0029] In some embodiments, the heavy chain variable region comprises or consists of the sequence shown in SEQ ID NO: 2, 4, or comprises or consists of an amino acid sequence having at least 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity with the sequence shown in SEQ ID NO: 2, 4.

[0030] In some embodiments, the heavy chain variable region comprises or consists of the sequence shown in SEQ ID NO:2, or comprises or consists of an amino acid sequence having at least 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity to the sequence shown in SEQ ID NO:2; the light chain variable region comprises or consists of the sequence shown in SEQ ID NO:3, or comprises or consists of an amino acid sequence having at least 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity to the sequence shown in SEQ ID NO:3; or

[0031] The heavy chain variable region comprises or consists of the sequence shown in SEQ ID NO:4, or comprises or consists of an amino acid sequence having at least 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity to the sequence shown in SEQ ID NO:4, and the light chain variable region comprises or consists of the sequence shown in SEQ ID NO:5, or comprises or consists of an amino acid sequence having at least 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity to the sequence shown in SEQ ID NO:5.

[0032] In some embodiments, the antibody further comprises a heavy chain variable region and a light chain variable region, preferably, the heavy chain constant region comprises or consists of the sequence shown in SEQ ID NO:6, or comprises or consists of an amino acid sequence having at least 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity to SEQ ID NO:6; the light chain constant region of the antibody comprises or consists of the sequence shown in SEQ ID NO:7, or comprises or consists of an amino acid sequence having at least 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity to SEQ ID NO:7.

[0033] In some embodiments, the antigen-binding fragment includes, but is not limited to, F(ab')2, F(ab)2, Fab', Fab, Fv, Fd, dAb, Fab / c, complementarity determining region (CDR) fragments, single-chain Fvs (ScFv), disulfide-stabilized Fv fragment (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), double-chain antibody (Diabody), disulfide-stabilized double-chain antibody (ds-Diabody), ScFv multimer (such as ScFv dimer, ScFv trimer), multispecific antibodies formed by a portion of an antibody comprising one or more CDRs, nanobodies, single domain antibodies (sdAb), domain antibodies, bivalent domain antibodies, or any other antibody fragment that binds to an antigen but does not contain a complete antibody structure. Regardless of the structure, the antigen-binding fragment includes any polypeptide or polypeptide complex that is capable of binding to the same antigen as the parent antibody or parent antibody fragment.

[0034] In some embodiments, the antibody is a human antibody.

[0035] In some embodiments, the F protein is in a prefusion conformation.

[0036] In some embodiments, the respiratory syncytial virus is respiratory syncytial virus type A or B.

[0037] In another aspect, provided herein is a nucleic acid molecule encoding the light chain, heavy chain, light chain variable region or heavy chain variable region of the above-mentioned antibody.

[0038] In another aspect, provided herein is an expression vector comprising the above-mentioned nucleic acid molecule.

[0039] In another aspect, provided herein is a host cell that expresses the above-mentioned antibody or antigen-binding fragment thereof, or comprises the above-mentioned nucleic acid molecule or expression vector.

[0040] In another aspect, provided herein is a pharmaceutical composition comprising the above-mentioned antibody or antigen-binding fragment thereof and a pharmaceutically acceptable carrier.

[0041] In another aspect, the present invention provides a use of the above-mentioned antibody or antigen-binding fragment thereof in the preparation of a medicament for treating or preventing respiratory syncytial virus-related diseases.

[0042] In another aspect, provided herein is a method for treating or preventing a respiratory syncytial virus-related disease in a subject, comprising administering to the subject an effective amount of the above-mentioned antibody or antigen-binding fragment thereof, the above-mentioned pharmaceutical composition, fusion protein or multispecific antibody.

[0043] On the other hand, the present invention provides a method for detecting the presence or content of respiratory syncytial virus in a sample, comprising contacting the sample with the above-mentioned antibody or its antigen-binding fragment, and detecting whether an antigen-antibody complex is formed or the amount of the antigen-antibody complex formed.

[0044] On the other hand, the present invention provides a conjugate comprising the above-mentioned antibody or antigen-binding fragment thereof and a conjugated portion, wherein the conjugated portion is a purification tag (such as a His tag), a detectable marker or a small molecule drug; preferably, the conjugated portion is a fluorescent substance, a chemiluminescent substance, a colored substance, polyethylene glycol or an enzyme, preferably, the small molecule drug is selected from nucleic acid or F protein inhibitors of respiratory syncytial virus.

[0045] In another aspect, the present invention relates to a fusion protein or a multispecific antibody (preferably a bispecific antibody) comprising the above-mentioned antibody or antigen-binding fragment thereof.

[0046] On the other hand, the present invention relates to a kit, which includes the above-mentioned antibody or its antigen-binding fragment, the conjugate or the fusion protein or multi-specific antibody; preferably, the kit also includes a second antibody, which specifically recognizes the antibody; optionally, the second antibody also includes a detectable label, such as a radioactive isotope, a fluorescent substance, a chemiluminescent substance, a colored substance or an enzyme; preferably, the kit is used to detect the presence or level of RSV in a sample. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 The gel column chromatography and SDS-PAGE detection results of the expressed and purified RSV-preF (DS-Cav1) protein are shown. DETAILED DESCRIPTION

[0048] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.

[0049] "Antibody" refers to immunoglobulins secreted by plasma cells (effector B cells) and used by the body's immune system to neutralize foreign substances (peptides, viruses, bacteria, etc.). The foreign substances are correspondingly called antigens. The basic structure of a classical or ordinary antibody molecule is a 4-mer composed of two identical heavy chains and two identical light chains. According to the conservative differences in the amino acid sequences, the heavy chain and the light chain are divided into a variable region (V) located at the amino terminus and a constant region (C) located at the carboxyl terminus. The variable regions of a heavy chain and a light chain interact to form an antigen binding site (Fv). In the variable region, the composition and arrangement order of amino acid residues in certain regions are more variable than other regions in the variable region (framework region, FR), which are called hypervariable regions (HVR). The hypervariable region is actually the key site for antibody binding to antigens. Because these hypervariable region sequences are complementary to the antigenic determinants, they are also called complementarity-determining regions (CDRs). The heavy chain and light chain have three complementary determining regions, which are called HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3 respectively. Each VH and VL can be composed of three CDRs and four FR regions, which can be arranged in the following order from amino terminus to carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4.

[0050] In the case of known antibody complementary determining region sequences, those skilled in the art can easily replace partial sequences (such as constant regions or framework regions) of antibody molecules with sequences from other species, such as by DNA recombination technology, to form chimeric antibodies, and the chimeric antibodies can substantially retain the binding specificity of their source antibodies. For example, when the source antibody is a mouse antibody, its constant region can be replaced with a human antibody constant region, or conversely, when the source antibody is a human antibody, its constant region can be replaced with a mouse antibody constant region to reduce the immunogenicity of the antibody when used in different species or to utilize specific functions of the constant region, such as ADCC-related activity. In order to further reduce the immunogenicity of the antibody or other purposes, all other sequences in the antibody molecule except the CDR sequence can be replaced with the corresponding sequence (which may include one or more amino acid mutations as the case may be) of another antibody molecule (from the same or different species), while substantially retaining the binding specificity of its source antibody. In a specific example, those skilled in the art often use CDR transplantation to humanize mouse antibodies.

[0051] Those skilled in the art can also understand that, based on the specific antibody sequences provided herein, a few amino acids can be conservatively replaced, deleted, added, and the binding ability or biological activity of the resulting products with the corresponding antigen (F protein) can be verified or screened to obtain corresponding variants of the anti-F protein antibody molecules provided by the present invention, and these variants should also be included in the scope of the present invention.

[0052] The antibody of the present invention may be (i) a polypeptide having one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, or (ii) a polypeptide having a substitution group in one or more amino acid residues, or (iii) a polypeptide formed by fusion of a mature polypeptide with another compound (such as a compound that prolongs the half-life of the polypeptide, such as polyethylene glycol), or (iv) a polypeptide formed by fusion of an additional amino acid sequence to the polypeptide sequence (such as a leader sequence or secretory sequence or a sequence or proprotein sequence used to purify the polypeptide, or a fusion protein formed with a 6His tag). According to the teachings herein, these fragments, derivatives and analogs are within the scope known to those skilled in the art.

[0053] "Conservative amino acid substitutions" are those in which an amino acid residue is replaced by an amino acid residue with a similar side chain. Families of amino acid residues with similar side chains have been defined in the art and include basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, non-essential amino acid residues of immunoglobulin polypeptides are preferably replaced by other amino acid residues from the same side chain family. In other embodiments, a string of amino acids may be replaced by a structurally similar string of amino acids that differ in sequence and / or in the composition of the side chain family.

[0054] Non-limiting examples of conservative amino acid substitutions are provided in the table below, where a similarity score of 0 or higher indicates a conservative substitution between the two amino acids.

[0055] C G P S A T D E N Q H K R V M I L F Y W W -8 -7 -6 -2 -6 -5 -7 -7 -4 -5 -3 -3 2 -6 -4 -5 -2 0 0 17 Y 0 -5 -5 -3 -3 -3 -4 -4 -2 -4 0 -4 -5 -2 -2 -1 -1 7 10 F -4 -5 -5 -3 -4 -3 -6 -5 -4 -5 -2 -5 -4 -1 0 1 2 9 L -6 -4 -3 -3 -2 -2 -4 -3 -3 -2 -2 -3 -3 2 4 2 6 I -2 -3 -2 -1 -1 0 -2 -2 -2 -2 -2 -2 -2 4 2 5 M -5 -3 -2 -2 -1 -1 -3 -2 0 -1 -2 0 0 2 6 V -2 -1 -1 -1 0 0 -2 -2 -2 -2 -2 -2 -2 4 R -4 -3 0 0 -2 -1 -1 -1 0 1 2 3 6 K -5 -2 -1 0 -1 0 0 0 1 1 0 5 H -3 -2 0 -1 -1 -1 1 1 2 3 6 Q -5 -1 0 -1 0 -1 2 2 1 4 N -4 0 -1 1 0 0 2 1 2 E -5 0 -1 0 0 0 3 4 D -5 1 -1 0 0 0 4 T -2 0 0 1 1 3 A -2 1 1 1 2 S 0 1 1 1 P -3 -1 6 G -3 5 C 12

[0056] In some embodiments, the conservative substitution is preferably a substitution in which an amino acid within the following groups (a)-(e) is replaced by another amino acid residue within the same group: (a) small aliphatic, non-polar or weakly polar residues: Ala, Ser, Thr, Pro and Gly; (b) polar, negatively charged residues and their (uncharged) amides: Asp, Asn, Glu and Gln; (c) polar, positively charged residues: His, Arg and Lys; (d) large aliphatic, non-polar residues: Met, Leu, Ile, Val and Cys; and (e) aromatic residues: Phe, Tyr and Trp.

[0057] Particularly preferred conservative substitutions are as follows: Ala is replaced by Gly or by Ser; Arg is replaced by Lys; Asn is replaced by Gln or by His; Asp is replaced by Glu; Cys is replaced by Ser; Gln is replaced by Asn; Glu is replaced by Asp; Gly is replaced by Ala or by Pro; His is replaced by Asn or by Gln; Ile is replaced by Leu or by Val; Leu is replaced by Ile or by Val; Lys is replaced by Arg, replaced by Gln or by Glu; Met is replaced by Leu, replaced by Tyr or by Ile; Phe is replaced by Met, replaced by Leu or by Tyr; Ser is replaced by Thr; Thr is replaced by Ser; Trp is replaced by Tyr; Tyr is replaced by Trp; and / or Phe is replaced by Val, replaced by Ile or by Leu.

[0058] The "antigen-binding fragment" of an antibody refers to a polypeptide that includes a partial sequence of the source antibody (especially the CDR sequence) and has the binding specificity of the source antibody. The antigen-binding fragment usually includes the light chain variable region and the heavy chain variable region of the source antibody, so it has the ability to bind to the antigen. There are many forms of antigen-binding fragments, such as Fab, F(ab')2, single-chain antibody (scFv), single-domain antibody (sdAb), etc. scFv is composed of the variable region of the heavy chain and the variable region of the light chain of the antibody connected into a peptide chain by a short peptide. Through correct folding, the variable regions from the heavy chain and the light chain interact through non-covalent bonds to form an Fv segment, so that scFv can better retain its affinity activity for the antigen. sdAb was first discovered in camelids, which naturally lack light chains but still have antigen binding ability. sdAb has some advantages over ordinary antibody molecules, such as better stability (resistance to pH, heat, and proteases), and can target and bind to antigen epitopes on antigen molecules that are difficult for ordinary antibody molecules to access.

[0059] The "specific binding antigen" or "antigen binding specificity" of an antibody refers to the ability of the antibody to bind to the target antigen with a higher or greater affinity than other antigens. The ability of an antibody to bind to a target antigen can be qualitatively or quantitatively identified by various methods, such as by measuring the K affinity of the antibody to the target antigen. D The value is used to measure the ability of an antibody to compete with other antibodies for binding to a target antigen. Here, Kd is the equilibrium dissociation constant, which can be used to measure the binding affinity between an antibody and its antigen. D The smaller the value, the stronger the affinity.

[0060] As used herein, "antibody" may include a complete antibody or an antigen-binding fragment thereof, and may be a human antibody, a murine antibody, a humanized antibody, a chimeric antibody, etc. The antibody may be of any type, such as IgG, IgE, IgM, IgD, IgA or IgY), or any subclass, such as IgG 1, IgG2, IgG 3, IgG4, IgA1 or IgA2, etc.

[0061] Methods for preparing antibodies are known in the art, including but not limited to isolating cells expressing specific antibodies after immunizing animals with antigens (e.g., preparing antibodies through hybridoma technology), screening antibody technology using phage antibody libraries, transforming cells through genetic engineering technology and making them express antibody molecules, etc. In a specific example, a method for preparing antibody molecules by introducing recombinant expression vectors encoding antibody heavy chains and light chains into 293F cells is provided.

[0062] It is known in the art that antibody molecules can also be modified in various ways, such as PEGylation, glycosylation, formation of antibody molecule conjugates (e.g., ADC), purification tags, or fusion with other proteins, such as formation of bispecific antibodies. These antibody derivatives obtained by modification based on the antibody molecules provided herein should also be included within the scope of the present invention.

[0063] When referring to amino acid sequences, the term "sequence identity" (also referred to as "sequence identity") refers to the amount of consistency between two amino acid sequences (e.g., a query sequence and a reference sequence), generally expressed as a percentage. Typically, before calculating the percentage of consistency between two amino acid sequences, the sequences are aligned and gaps (if any) are introduced. If the amino acid residues in the two sequences are the same at a certain alignment position, the two sequences are considered to be consistent or matched at that position; if the amino acid residues in the two sequences are different, they are considered to be inconsistent or mismatched at that position. In some algorithms, the number of matching positions is divided by the total number of positions in the alignment window to obtain sequence consistency. In other algorithms, the number of gaps and / or the length of the gaps are also taken into account. For the purposes of the present invention, the publicly available alignment software BLAST (available on the webpage ncbi.nlm.nih.gov) can be used to obtain the best sequence alignment and calculate the sequence consistency between the two amino acid sequences using the default settings.

[0064] "Expression vector", also known as "recombinant expression vector", refers to a nucleic acid molecule containing various expression elements for expressing a target protein (e.g., light chain and / or heavy chain of an antibody molecule) in a host cell. For expression vectors used to express a target protein in eukaryotic cells (mammalian cells, insect cells, plant cells, etc.), these expression elements generally include promoters, enhancers, polyadenylation signal sequences, etc. In order to facilitate amplification in Escherichia coli, the expression vector may also generally include an Escherichia coli replicon sequence. The expression vector may also include an antibiotic resistance gene or a selection marker gene (e.g., ampicillin resistance gene (AmpR), thymidine kinase gene (TK), etc.) for screening and a multiple cloning site (MCS) for insertion of a target protein coding sequence. The expression vector may be a plasmid vector or a viral vector.

[0065] The expression vector provided herein is suitable for expressing the antibody molecules or fragments thereof provided herein in a variety of host cells, and is particularly suitable for expressing in mammalian cells (e.g., mice, rats, pigs, sheep, monkeys, orangutans, or human cells). In some embodiments, the mammalian cells may be selected from CHO cells, HEK293 cells, or BHK cells.

[0066] The present invention uses RSV pre-fusion conformation F protein DS-Cav1 as bait, and uses single-cell sequencing technology to isolate two human monoclonal antibodies from healthy adult peripheral blood lymphocytes (PBMCs), which are named RD1 and RD4. Both strains can bind to RSV F protein: RD1 and RD4 bind to both pre-fusion F protein and post-fusion F protein. These two monoclonal antibodies can be used for RSV diagnosis and antigen quantification. In addition, the two strains RD1 and RD4 have neutralizing activity against RSV type A and type B, and can be used for RSV prevention and treatment.

[0067] The antibody or its Fab provided herein can specifically bind to the respiratory syncytial virus F protein (pre-fusion conformation and / or post-fusion conformation), i.e., the respiratory syncytial virus F protein is used as the target antigen of the antibody. After the antibody is combined with the respiratory syncytial virus F protein, the cell infection ability of RSV is suppressed by neutralizing activity. Therefore, the antibody or its Fab provided herein can be used for preventing or treating RSV infection. On the other hand, due to the antigen binding specificity of the antibody provided herein, the antibody can be used to detect whether there is RSV virus in the sample (such as blood) from the subject. Accordingly, the RSV detection kit that can include the antibody or its Fab provided herein is provided herein.

[0068] When referring to pharmaceutical compositions, the term "pharmaceutically acceptable carrier" refers to solid or liquid diluents, fillers, antioxidants, stabilizers and the like that can be safely administered, are suitable for administration to humans and / or animals without excessive adverse side effects, and are suitable for maintaining the activity of the drugs or active agents contained therein.

[0069] When referring to the treatment or prevention of RSV infection, "effective amount" refers to the amount of active compound (such as antibody) sufficient to cause the biological or medical response desired by the clinician in the subject. The "effective amount" of the antibody provided herein when administered can be determined by those skilled in the art based on factors such as the route of administration, the subject's weight, age, and condition. For example, a typical daily dose range can be 0.01 mg to 100 mg of active ingredient per kg body weight.

[0070] The present invention is further described below through specific examples.

[0071] Example 1: Expression and purification of RSV virus pre-fusion F protein (DS-Cav1)

[0072] The C-terminus of the DS-Cav1 protein encoding the pre-fusion F protein of the RSV A2 strain is added with a Thrombin restriction site, 6 histidine sequences, 1 GS [glycine-serine] linker sequence, 1 sequence encoding a Strep II tag, and 1 stop codon (amino acid sequence as shown in SEQ ID NO: 1), and inserted into the EcoR I and XhoI restriction sites of the mammalian expression vector pCAGGS. The ligation product was transformed into DH5α Escherichia coli competent cells. Then, a single clone was picked and inoculated into 4 mL of LB medium and cultured for 6-8 hours; then it was inoculated into 300 mL of LB medium and cultured for 12-16 hours, the bacteria were collected, and the plasmid was extracted using an endotoxin-free large extraction plasmid kit (TIANGEN) to obtain pCAGGS-RSV-F (DS-Cav1).

[0073] The extracted plasmid pCAGGS-RSV-F (DS-Cav1) was transfected into 293F cells to express F protein. 5-7 days after transfection, the 293F cell culture supernatant containing the target protein was centrifuged and filtered (0.22 μm) to remove cell debris, and then combined with HisTrapHP (GE healthcare) nickel chelate column, and then eluted with different concentrations of imidazole to collect the eluted proteins, and the samples containing the target protein were judged by SDS-PAGE results.

[0074] The eluted peak containing the target protein was collected, concentrated, and then subjected to molecular sieve chromatography. The size and purity of the target protein were determined based on the protein peak position and SDS-PAGE results (see molecular sieve and SDS-PAGE results). Figure 1 ). The molecular sieve results showed that the protein peak was at 60-70mL, which was consistent with the theoretical molecular weight of the F protein trimer of 180kDa. The SDS-PAGE results showed that under reducing (+DTT) and non-reducing (-DTT) conditions, the F protein was around ~60kDa, suggesting that the F protein subunits formed trimers through non-covalent bonding. The T-numbered samples of SDS-PAGE correspond to the T-numbered collection tubes of the molecular sieve.

[0075] Example 2: Isolation of RSV F (DS-Cav1) protein-specific memory B cells

[0076] With the informed consent of patients who were infected with RSV and discharged from the hospital, 3-10 mL of blood was collected and PBMCs were isolated. 7 / mL) and 200nM DS-Cav1 protein (prepared in Example 1) were incubated on ice for half an hour; then washed twice with PBS, and then mixed with the following antibodies (all purchased from BD): anti-human CD3 / PE-Cy5, anti-humanCD16 / PE-Cy5, anti-human CD235a / PE-Cy5, anti-human CD19 / APC-Cy7, anti-human CD27 / Pacific Blue, anti-human CD38 / APC, anti-human IgG / FITC, and anti-His / PE. After incubation on ice for half an hour, wash twice with PBS. Subsequently, PBMCs were sorted with FACSAria III, and cells with PE-Cy5-APC-APC-Cy7+Pacific Blue+FITC+PE+ (i.e., memory B cells) were collected and directly collected into a 96-well plate, 1 cell / well.

[0077] Example 3: Single B cell PCR and cloning of human monoclonal antibody IgG1

[0078] The cells obtained in Example 2 were reverse transcribed by Superscript III reverse transcriptase (Invitrogen), and the reaction conditions were 55°C, 60min. The reverse transcription product was used as a template, and PCR was performed using HotStar TapPlus enzyme (QIAgen) to amplify the antibody variable region sequence (PCRa), and the reaction conditions were as follows: 95°C, 5min; 95°C, 30s, 55°C (heavy chain / κ chain) / 50°C (λ chain), 30s, 72°C, 90s, 35 cycles, 72°C, 7min. This was used as a template for another round of PCR (PCRb), and the conditions were as follows: 95°C, 5min; 95°C, 30s, 58°C (heavy chain) / 60°C (κ chain) / 64°C (λ chain), 30s, 72°C, 90s, 35 cycles, 72°C, 7min. 1.2% agarose gel electrophoresis was used to separate the PCR products, and the 400-500bp band was excised and recovered and sent to a sequencing company for sequencing. The sequencing results were analyzed on the IGBLAST website. The analysis results are as follows:

[0079] A total of two paired antibodies were obtained: RD1 and RD4. According to the sequencing results, the amino acid sequence of the heavy chain variable region of RD1 is SEQ ID NO: 2, and the amino acid sequence of the light chain variable region is SEQ ID NO: 3; the amino acid sequence of the heavy chain variable region of RD4 is SEQ ID NO: 4, and the amino acid sequence of the light chain variable region is SEQ ID NO: 5. The light chain sequencing results of the two antibodies were compared with the germline genes, and it was determined that both RD1 and RD4 used CLκ.

[0080] In order to obtain human antibodies for subsequent evaluation, we designed a full anti-IgG1 construct. The strategy is as follows:

[0081] Heavy chain: CMV promoter-HindIII-signal peptide (SP)-heavy chain variable region (VH)-heavy chain constant region (CH)-NotⅠ;

[0082] Light chain κ: CMV promoter-HindIII-signal peptide (SP)-light chain variable region (VK)-light chain constant region (CLκ)-NotⅠ;

[0083] The correct variable region sequence was analyzed and connected with the corresponding constant region of heavy chain CH and light chain CLκ by bridge PCR, and cloned into the expression vector pCAGGS to obtain a recombinant plasmid containing the light and heavy chain coding genes of a specific antibody; wherein, the light and heavy chain variable regions were connected to the vector containing the constant region using the restriction sites HindIII and NotⅠ. The amino acid sequence of the heavy chain constant region CH is as shown in SEQ ID NO:6; the amino acid sequence of the light chain constant region CLκ is as shown in SEQ ID NO:7, and the amino acid sequence of the signal peptide (SP) is as shown in SEQ ID NO:8.

[0084] Example 4: Expression and purification of monoclonal antibodies

[0085] The pCAGGS plasmid containing the heavy chain and light chain coding sequences was co-transfected into 293F cells. 220 mM MVPA was added 24 h after transfection and culture was continued for 5 days. The cells were harvested, the supernatant was collected by centrifugation, filtered with a 0.22 μm filter membrane, and passed through a HiTrap Protein A HP (5 ml, GE Healthcare) column at a speed of 2 mL / min.

[0086] According to the operating instructions of the affinity column, the initial elution and purification of the antibody was completed. The antibody was further purified using a gel filtration column (superdex 200PG, GE Healthcare) to obtain a high-purity antibody. The purified monoclonal antibody was cleaved by papain (Thermo) according to the product instructions, and the Fab fragment was purified by HiTrap Protein A HP and gel filtration chromatography, and finally concentrated and stored at -80°C for future use.

[0087] Example 5: Antibody Binding Performance Detection

[0088] (1) Construction and expression of RSV post-fusion F protein (FΔFP)

[0089] The C-terminus of the fusion F protein (FΔFP) encoding the RSV A2 strain is added with a Thrombin restriction site, 6 histidine sequences, 1 GS [glycine-serine] linker sequence, 1 sequence encoding the Strep II tag, and 1 stop codon (amino acid sequence such as SEQ ID NO: 9), and inserted into the NdeI and XhoI restriction sites of the mammalian expression vector pCAGGS. The ligation product is transformed into DH5α Escherichia coli competent cells. Then, a single clone is picked and inoculated into 4mL LB medium and cultured for 6-8 hours; then it is inoculated into 300mL LB medium and cultured for 12-16 hours, the bacteria are collected, and the plasmid is extracted using an endotoxin-free large extraction plasmid kit (TIANGEN) to obtain pCAGGS-RSV-FΔFP.

[0090] The extracted plasmid pCAGGS-RSV-FΔFP was transfected into 293F cells to express F protein. 4-5 days after transfection, the 293F cell culture supernatant containing the target protein was centrifuged and filtered (0.22μm) to remove cell debris, and then combined with HisTrap HP (GE healthcare) nickel chelate column, and then eluted with different concentrations of imidazole to collect the eluted proteins, and the samples containing the target protein were judged by SDS-PAGE results.

[0091] The elution peak containing the target protein was collected, concentrated, and then subjected to molecular sieve chromatography. The size and purity of the protein were determined based on the peak position of the protein and the results of SDS-PAGE.

[0092] (2) Preparation and purification of the entire monoclonal antibody Fab fragment

[0093] A. Prepare samples:

[0094] The whole antibody protein was concentrated to above 10 mg / ml, and the solution was changed (200 times) to sample buffer (20 mM Na 3 PO 4 , 10mM EDTA, pH7.0); concentrate the antibody after the solution change to more than 20mg / ml, add a certain amount of digestion buffer (add cysteine·HCl to the sample buffer to 20mM, pH7.0) to dilute it, and the final concentration is ≥10mg / ml.

[0095] B. Prepare enzyme:

[0096] Use a pipette with the tip cut off to draw 0.5 mL of well-mixed coupled enzyme beans (Thermo, Prod#20341) into the reaction container, centrifuge at 1000 g / min for 2 min to remove the storage solution; wash the beans 3 times with 3 mL of digestion buffer and discard the elution. Then add 0.5 mL of digestion buffer and mix gently by inversion.

[0097] C. Enzyme digestion:

[0098] Add a stopper to the reaction tube, add the concentrated antibody (about 10 mg), tighten the lid, and seal the gap with a sealing film; fix the reaction tube to a rotating mixer and incubate at 37°C overnight;

[0099] D. Fab Recycling:

[0100] After overnight digestion, centrifuge at 1000 g / min for 5 min and save the lower solution; add 1 mL Protein A binding buffer (20 mM Na 3PO 4 , pH 7.0) to wash the beans twice and recover the lower solution; mix the lower solutions from the above two steps and measure the concentration; concentrate the lower solution and pass it through Protein A; concentrate the target protein eluate and exchange it into 1× PBS buffer, and further purify it through molecular sieves.

[0101] (3) Evaluation of the binding ability of monoclonal antibodies to F protein

[0102] The interaction between purified antibodies and pre-F / postF was detected by surface plasmon resonance (SPR) using a Biacore 3000 system (GE Healthcare). The pre-fusion F protein DS-Cav1 and the post-fusion F protein FΔFP were immobilized on the Fc2 and Fc4 channels of the CM5 chip (flow cell, Fc) by amino coupling. Then, the purified Fab proteins of RD1 and RD4 antibodies were bound by antibody capture. Palivizumab (purchased from Synagis) and D25 (WO2015108967A2, synthesized by GenScript, sequence see SEQ ID NO: 19 and 20) were used as positive controls. All proteins used in the experiment were exchanged into a buffer consisting of 10mM HEPES, pH 7.4, 150mM NaCl and 0.005% v / v Tween 20 to determine the binding of pre-F. The immobilized anti-human antibody was regenerated with 3M magnesium chloride, and the anti-mouse antibody was regenerated with 10mM glycine, pH 1.7. The apparent equilibrium dissociation constant (Kd value) for each pre-F / postF interaction with the antibody was calculated using Biacore 3000 evaluation software.

[0103] Table 1 Binding constants of antibody Fab fragments with pre-fusion F protein DS-Cav1 and post-fusion F protein FΔFP

[0104]

[0105] Results: Both RD1 and RD4 can bind to RSV pre-fusion F protein and RSV post-fusion F protein. RD1 binds to RSV pre-fusion F protein (K D =0.06 nM), binding to post-fusion F protein (K D =30 nM); RD4 binds to RSV prefusion F protein (K D =0.008 nM), binding to post-fusion F protein (K D=1.26 nM); RD1 and RD4 showed an affinity 1-2 orders of magnitude higher than D25 and 2-3 orders of magnitude higher than Palivizumab in binding to the pre-fusion F protein; RD1 and RD4 showed an affinity 1-2 orders of magnitude higher than D25 and the same order of magnitude as Palivizumab in binding to the post-fusion F protein.

[0106] Example 6: Neutralizing activity detection of antibodies

[0107] We use the micro-neutralization method to detect the neutralizing activity of antibodies. The detailed steps are as follows:

[0108] 1. Cell Preparation

[0109] (1) 12-16 hours in advance, plate Hep2 cells in a 24-well plate and culture them in 10% DMEM medium. When the cells grow to more than 70% on the next day, perform the antibody neutralization experiment.

[0110] (2) Antibody dilution: There are 4 types of antibodies: RD1, RD4, Palivizumab, and D25. After purification, each antibody is filtered to sterile conditions and added to a 96-well plate for 3-fold gradient dilution. At least 150 μL of each well is diluted with 2% FBS DMEM medium.

[0111] (3) Virus dilution: Take the virus out of the -80% freezer and thaw it. Dilute the virus with 2% FBSDMEM medium according to the appropriate virus dilution multiple, and then add it to the 48-well plate of the antibody, 300 μL / well. (RSV_A2 or RSV-Long or RSV-B (purchased from ATCC): 450 μL diluted to 40 mL, final titer: 1.2×10 4 TCID50 / 100μL).

[0112] (4) Virus and antibody incubation:

[0113] (5) Mix the diluted virus and antibody in a 1:1 molar ratio and place in a 37°C CO 2 Incubate in the incubator for 1 hour.

[0114] (6) Add virus-antibody mixture: aspirate the PBS in the cells, add 300 μL of the virus and cell supernatant mixture to each well of the cells, and incubate at 37°C for 1 hour. (One gradient has two wells, 11 gradients each, and two virus wells per plate as controls), and place the cells in an incubator for about 96 hours.

[0115] 2.ELISA test

[0116] (1) Remove the culture medium from Hep2 cells without washing. Add 150 μL of the serum-virus mixture after incubation. After culturing for 3 hours, add 50 μL of DMEM containing 2% FBS. The cells are cultured in an incubator for 4 days.

[0117] (2) Pre-cool pure methanol in a refrigerator at -20 degrees. Melt palivizumab antibody on ice in advance, remove cells from the incubator, pour out supernatant, wash each well with 200 μL PBS, be gentle when adding and pouring out liquid to prevent cells from falling off, add 150 μL pre-cooled methanol to each well after washing, place in a refrigerator at -20 degrees to fix and permeabilize for 15 to 20 minutes, pour out methanol, and wash each well twice with 200 μL PBS.

[0118] (3) After washing, add 100 μL of 5% skim milk (dissolved in PBS) to each well and block at room temperature for 30 minutes;

[0119] (4) Pour off the blocking solution and add the primary antibody palivizumab directly without washing. Dilute it to a working concentration of 5 μg / mL using 5% skim milk. After the antibody is fully diluted and mixed, add 100 μL to each well and incubate at room temperature for 2 hours.

[0120] (5) Pour off the primary antibody, wash three times with PBST, 5 min each time, add goat anti-human IgG-HRP-conjugated secondary antibody (Jinpulai Biotechnology Co., Ltd.) (diluted 1:1500 with 5% skim milk), and incubate at room temperature for 2 hours.

[0121] (6) Remove the secondary antibody and wash with PBST 4 times, 5 min each time. Then add 60 μL of TMB colorimetric solution to each well and react at room temperature for about 1 min. Observe the color change, add 60 μL of 2M hydrochloric acid to each well to terminate the colorimetric reaction, use an ELISA reader to read the value at OD450, 0.1s, and save and export the data.

[0122] result:

[0123] For RSV A2 strain, the half inhibitory concentration (IC50) of the antibody was 0.16 μg / mL for RD1 and 0.68 μg / mL for RD4, both of which were better than the 1.63 μg / mL of palivizumab, which has been marketed. Its neutralizing activity was one order of magnitude stronger than palivizumab. The data are shown in Table 2.

[0124] Table 2 Neutralizing activity of antibodies against RSV A type A2 strain

[0125]

[0126] Note: D25 (WO2015108967A2, synthesized by GenScript, sequence see SEQ ID NO: 21 and 22)

[0127] For RSV B clinical isolates, the half inhibitory concentrations (IC50) of the antibodies were: RD1 (0.13 μg / mL) and RD4 (0.11 μg / mL), both of which had neutralizing activity comparable to the marketed palivizumab (IC50 = 0.38 μg / mL). The data are shown in Table 3.

[0128] Table 3 Neutralizing activity of antibodies against RSV A type Long strain

[0129]

[0130] Sequence Listing:

[0131] SEQ ID NO: 1 (RSV pre-fusion F protein sequence)

[0132]

[0133] SEQ ID NO: 2 (antibody 1 heavy chain variable region)

[0134]

[0135] SEQ ID NO: 3 (antibody 1 light chain variable region)

[0136]

[0137] SEQ ID NO:4 (antibody 2 heavy chain variable region)

[0138]

[0139] SEQ ID NO: 5 (antibody 2 light chain variable region)

[0140]

[0141] SEQ ID NO:6 (antibody heavy chain constant region)

[0142]

[0143] SEQ ID NO:7 (antibody light chain constant region)

[0144]

[0145] SEQ ID NO:8 (signal peptide sequence)

[0146]

[0147] SEQ ID NO:9 (RSV post-fusion F protein sequence)

[0148]

[0149] SEQ ID NO: 10 (HCDR1 amino acid sequence of antibody 1)

[0150]

[0151] SEQ ID NO: 11 (HCDR2 amino acid sequence of antibody 1)

[0152]

[0153] SEQ ID NO: 12 (HCDR3 amino acid sequence of antibody 1)

[0154]

[0155] SEQ ID NO: 13 (HCDR1 amino acid sequence of Antibody 2)

[0156]

[0157] SEQ ID NO: 14 (HCDR2 amino acid sequence of Antibody 2)

[0158]

[0159] SEQ ID NO: 15 (HCDR3 amino acid sequence of Antibody 1)

[0160]

[0161] SEQ ID NO: 16 (antibody 1, 2 light chain HCDR1 amino acid sequence)

[0162]

[0163] SEQ ID NO: 17 (antibody 1, 2 light chain HCDR2 amino acid sequence)

[0164]

[0165] SEQ ID NO: 18 (antibody 1, 2 light chain HCDR3 amino acid sequence)

[0166]

[0167] SEQ ID NO: 19D25 light chain variable region sequence

[0168]

[0169] SEQ ID NO:20D25 heavy chain variable region sequence:

[0170]

[0171] SEQ ID NO:21D25 light chain full length sequence:

[0172]

[0173] SEQ ID NO:22D25 heavy chain full length sequence:

[0174]

Claims

1. A human antibody or an antigen-binding fragment thereof that binds to respiratory syncytial virus F protein, the human antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 contained in the heavy chain variable region shown in SEQ ID NO: 2 or 4, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 contained in the light chain variable region shown in SEQ ID NO: 3; according to the IMGT numbering system, the sequences of LCDR1-LCDR3 and HCDR1-HCDR3 of the human antibody are as follows: (1) The amino acid sequence of LCDR1 is: QGIKNA (SEQ ID NO: 16); The amino acid sequence of LCDR2 is: AAS (SEQ ID NO: 17); The amino acid sequence of LCDR3 is: LQDYSYPWT (SEQ ID NO: 18); The amino acid sequence of HCDR1 is: GFLFSSYS (SEQ ID NO: 10); The amino acid sequence of HCDR2 is: ISFDGSER (SEQ ID NO: 11); The amino acid sequence of HCDR3 is: AKTWVDRTLSLDS (SEQ ID NO: 12); or (2) The amino acid sequence of LCDR1 is: QGIKNA (SEQ ID NO: 16); The amino acid sequence of LCDR2 is: AAS (SEQ ID NO: 17); The amino acid sequence of LCDR3 is: LQDYSYPWT (SEQ ID NO: 18); The amino acid sequence of HCDR1 is: GFLFSCYS (SEQ ID NO: 13); The amino acid sequence of HCDR2 is: MSFDGSEI (SEQ ID NO: 14); The amino acid sequence of HCDR3 is: ARTWVDRTLSLDH (SEQ ID NO: 15).

2. The human antibody or antigen-binding fragment thereof that binds to the F protein of respiratory syncytial virus according to claim 1, wherein the heavy chain variable region comprises or consists of the sequence shown in SEQ ID NO: 2, 4, or comprises or consists of an amino acid sequence having at least 85% sequence identity with the sequence shown in SEQ ID NO: 2, 4.

3. The human antibody or antigen-binding fragment thereof that binds to the F protein of respiratory syncytial virus according to claim 1 or 2, wherein the heavy chain variable region comprises or consists of the sequence shown in SEQ ID NO: 2, or comprises or consists of an amino acid sequence having at least 85% sequence identity with the sequence shown in SEQ ID NO: 2; the light chain variable region comprises or consists of the sequence shown in SEQ ID NO: 3, or comprises or consists of an amino acid sequence having at least 85% sequence identity with the sequence shown in SEQ ID NO: 3; or The heavy chain variable region comprises or consists of the sequence shown in SEQ ID NO: 4, or comprises or consists of an amino acid sequence having at least 85% sequence identity with the sequence shown in SEQ ID NO: 4, and the light chain variable region comprises or consists of the sequence shown in SEQ ID NO: 5, or The amino acid sequence comprises or consists of an amino acid sequence having at least 85% sequence identity with the sequence shown in SEQ ID NO:

5.

4. The human antibody or antigen-binding fragment thereof that binds to the F protein of respiratory syncytial virus according to claim 1 or 2, wherein the antibody further comprises a heavy chain constant region and a light chain constant region.

5. The human antibody or antigen-binding fragment thereof that binds to the F protein of respiratory syncytial virus according to claim 1 or 2, wherein the antibody further comprises a heavy chain constant region and a light chain constant region, the heavy chain constant region comprises or consists of the sequence shown in SEQ ID NO: 6, or comprises or consists of an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 6; the light chain constant region of the antibody comprises or consists of the sequence shown in SEQ ID NO: 7, or comprises or consists of an amino acid sequence having at least 85% sequence identity with SEQ ID NO:

7.

6. The human antibody or antigen-binding fragment thereof that binds to the F protein of respiratory syncytial virus according to claim 3, wherein the antibody further comprises a heavy chain constant region and a light chain constant region, the heavy chain constant region comprises or consists of the sequence shown in SEQ ID NO: 6, or comprises or consists of an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 6; the light chain constant region of the antibody comprises or consists of the sequence shown in SEQ ID NO: 7, or comprises or consists of an amino acid sequence having at least 85% sequence identity with SEQ ID NO:

7.

7. A human antibody or antigen-binding fragment thereof that binds to respiratory syncytial virus F protein as described in any one of claims 1, 2 or 6, wherein the antigen-binding fragment is F(ab')2, F(ab)2, Fab', Fab, Fv, Fab / c or single-chain Fv (ScFv).

8. The human antibody or antigen-binding fragment thereof that binds to the F protein of respiratory syncytial virus according to claim 3, wherein the antigen-binding fragment is F(ab')2, F(ab)2, Fab', Fab, Fv, Fab / c or single-chain Fv (ScFv).

9. The human antibody or antigen-binding fragment thereof that binds to the F protein of respiratory syncytial virus according to claim 5, wherein the antigen-binding fragment is F(ab')2, F(ab)2, Fab', Fab, Fv, Fab / c or single-chain Fv (ScFv).

10. A nucleic acid molecule encoding the humanized antibody or antigen-binding fragment thereof that binds to the F protein of respiratory syncytial virus according to any one of claims 1 to 9. An expression vector comprising the nucleic acid molecule of claim 10 .

12. A host cell expressing the humanized antibody or antigen-binding fragment thereof that binds to the F protein of respiratory syncytial virus according to any one of claims 1 to 9, or comprising the nucleic acid molecule according to claim 10 or the expression vector according to claim 11.

13. A pharmaceutical composition comprising the humanized antibody or antigen-binding fragment thereof that binds to respiratory syncytial virus F protein according to any one of claims 1 to 9, and a pharmaceutically acceptable carrier.

14. A conjugate, which is composed of the humanized antibody or antigen-binding fragment thereof that binds to respiratory syncytial virus F protein according to any one of claims 1 to 9 and a conjugated portion, wherein: The conjugated moiety is a purification tag or a detectable label. The conjugate according to claim 14 , wherein the purification tag is a His tag.

16. The conjugate of claim 14, wherein the detectable label is a fluorescent substance, a chemiluminescent substance, a colored substance or an enzyme.

17. A conjugate, which is composed of the humanized antibody or antigen-binding fragment thereof that binds to respiratory syncytial virus F protein according to any one of claims 1 to 9 and a conjugated portion, wherein: The coupling moiety is polyethylene glycol.

18. Use of the human antibody or antigen-binding fragment thereof that binds to respiratory syncytial virus F protein according to any one of claims 1 to 9, the pharmaceutical composition according to claim 13, or the conjugate according to any one of claims 14 to 17 in the preparation of a medicament for treating or preventing respiratory syncytial virus infection.

19. Use of the human antibody or antigen-binding fragment thereof that binds to the F protein of respiratory syncytial virus according to any one of claims 1 to 9 in the preparation of a kit for detecting the presence or content of respiratory syncytial virus in a sample.

20. A method for detecting the presence or content of respiratory syncytial virus in a sample, comprising contacting the sample with a human antibody or an antigen-binding fragment thereof that binds to the F protein of respiratory syncytial virus as described in any one of claims 1-9, and detecting whether an antigen-antibody complex is formed or the amount of the antigen-antibody complex formed, wherein the method is used for non-disease diagnosis purposes.

21. A kit comprising the humanized antibody or antigen-binding fragment thereof that binds to respiratory syncytial virus F protein according to any one of claims 1 to 9, the pharmaceutical composition according to claim 13, or the conjugate according to any one of claims 14 to 17.

22. The kit of claim 21, further comprising a secondary antibody.

23. The kit according to claim 21 or 22, wherein the second antibody further comprises a detectable label, and the detectable label is a radioisotope, a fluorescent substance, a chemiluminescent substance, a colored substance or an enzyme.

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