Antibodies against respiratory syncytial virus and their use

Abstract

The present disclosure provides an antibody or antigen-binding fragment thereof that binds to a respiratory syncytial virus F protein. The present disclosure further provides a use of the antibody or antigen-binding fragment thereof for the manufacture of a medicament for the treatment and / or prevention of RSV infection.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims the priority of Chinese Patent Application No. 202110653393.6, filed with the China National Intellectual Property Administration on June 11, 2021. The entire content thereof is incorporated herein by reference.

[0002] Technical Field The present disclosure relates to an antibody that binds to the respiratory syncytial virus F protein or an antigen - binding fragment thereof. The present disclosure further relates to the use of the above - mentioned antibody or its antigen - binding fragment in the treatment and / or prevention of RSV infection.

Background Art

[0003] Respiratory syncytial virus (RSV) causes acute lower respiratory tract infections and is a major pathogen leading to illness and death among children worldwide. The probability of hospitalization due to RSV-related diseases is three times that of influenza and parainfluenza virus-related diseases. High hospitalization rates are commonly seen in infants under 5 years old, with the highest morbidity rate in 3-year-old infants. 6.7% of infant deaths worldwide from 1 month to 1 year of age are due to RSV. RSV is the most major pathogen of respiratory infections for preschool children, especially infants, and is also easily infective to adults and the elderly with low immunity. RSV poses a serious threat to human health worldwide, but there are few means to prevent and treat RSV infections, and the development of vaccines has not progressed smoothly due to the exacerbation of symptoms by formalin-inactivated vaccines. Antibodies in the mother's body are passed to the baby through the placenta during the last few weeks of pregnancy and can provide protective immunity, but this protection decays approximately twofold per month and has no persistence. Currently, there are no vaccines or particularly effective treatment methods, and the only preventive measure is palivizumab, which was approved in 1998. Palivizumab can reduce the hospitalization rate and mortality rate of infants to some extent, but it has weak immunological activity and a high frequency of use. Premature infants with congenital respiratory and circulatory system disorders need to receive five vaccinations throughout the entire epidemic period, which is costly and inconvenient, so it is inappropriate to vaccinate all infants, and the prevention of high-risk infants is also limited. On the other hand, MEDI8897, manufactured by AstraZeneca, which is an improved version of the monoclonal antibody D25, has strong neutralizing activity and only requires 1 to 2 immunizations and is currently in Phase III clinical trials. Therefore, there is an urgent need for a new generation of highly effective monoclonal antibody drugs for preventing and treating RSV infections.

[0004] So far, neutralizing antibodies have proven to be an effective treatment for viral diseases. Currently available treatments and prophylactic agents for viral infections include palivizumab (Synagis) for preventing respiratory syncytial virus (RSV) infection in children, ibalizumab (Trogarzo) for treating HIV infection, and rabies immune globulin for post-exposure prophylaxis of rabies virus. Additionally, there are many monoclonal antibodies in clinical trials targeting various viruses (https: / / clinicaltrials.gov / ). Antibodies mainly function in two ways: one is that antibodies with neutralizing activity bind to the viral envelope protein and block the binding of the virus to the cell receptor, thereby inhibiting viral infection; the other is by mediating antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), mobilizing immune cells and immune molecules such as macrophages and complement to destroy free viruses and infected cells.

[0005] Respiratory syncytial virus belongs to the Paramyxoviridae family and is a single-stranded negative-sense RNA virus with two types, type A and type B, both of which are prevalent. The attachment protein (G) and fusion protein (F) on the surface of RSV are the main antigens that can induce a neutralizing antibody response. The G protein is responsible for the adhesion of the virus to the cell membrane surface, but it has antigenic diversity, making it difficult to develop broad-spectrum prophylactic agents. The F protein is responsible for the fusion of the virus and cells and presents more epitopes that are targets of neutralizing antibodies. Therefore, it is the main target of most currently developed vaccines and immunotherapeutic agents and is also the target for preventing RSV disease with palivizumab clinically. The F protein has two conformations, pre-fusion and post-fusion. The pre-fusion F protein is unstable and can undergo conformational changes to become the post-fusion F protein. Currently, it has been found that most potent neutralizing epitopes are concentrated in the pre-fusion conformation of the F protein. For example, both the monoclonal antibody D25 and its improved monoclonal antibody MEDI8897 bind to the epitope φ of pre-fusion F. In 2013, the Peter Kwong team obtained the stable pre-fusion RSV F protein DS-Cav1 by introducing a series of mutations (S190F, V207, S155C, and S290C). Immunizing mice with DS-Cav1 activates the production of higher neutralizing antibodies. SUMMARY OF THE INVENTION

[0006] In one aspect, the present disclosure provides an antibody or an antigen-binding fragment thereof that binds to the respiratory syncytial virus F protein and includes a heavy-chain variable region. The HCDR1, HCDR2, and HCDR3 of the heavy-chain variable region are any one selected from the following combinations. (1) HCDR1 having an amino acid sequence of GFTFSSYA (SEQ ID NO: 18), HCDR2 having an amino acid sequence of ISYDGSNT (SEQ ID NO: 19), HCDR3 with an amino acid sequence of ARDYCSRGTCYHDY (SEQ ID NO: 20); (2) HCDR1 with an amino acid sequence of GYTFTTYD (SEQ ID NO: 24), and HCDR2 with an amino acid sequence of LNPDNGNT (SEQ ID NO: 25), and HCDR3 with an amino acid sequence of TRAPWWWYFDY (SEQ ID NO: 26); and (3) HCDR1 with an amino acid sequence of GFSFTNYG (SEQ ID NO: 30), and HCDR2 with an amino acid sequence of ISYDDGSDK (SEQ ID NO: 31), and HCDR3 with an amino acid sequence of VRDPTGDY (SEQ ID NO: 32).

[0007] In some embodiments, the above antibody or its antigen-binding fragment further comprises a light chain variable region, and HCDR1, HCDR2, and HCDR3 of the heavy chain variable region and LCDR1, LCDR2, and LCDR3 of the light chain variable region are any one selected from the following combinations. (1) LCDR1 with an amino acid sequence of QDIRND (SEQ ID NO: 15), and LCDR2 with an amino acid sequence of AAS (SEQ ID NO: 16), and LCDR3 with an amino acid sequence of LQDYNYPQTFG (SEQ ID NO: 17), and HCDR1 with an amino acid sequence of GFTFSSYA (SEQ ID NO: 18), and HCDR2 with an amino acid sequence of ISYDGSNT (SEQ ID NO: 19), and HCDR3 with an amino acid sequence of ARDYCSRGTCYHDY (SEQ ID NO: 20); (2) LCDR1 with an amino acid sequence of SGSIASNY (SEQ ID NO: 21), and LCDR2 with an amino acid sequence of EDN (SEQ ID NO: 22), and LCDR3 with an amino acid sequence of QSYDTSNAVFG (SEQ ID NO: 23), and HCDR1 with an amino acid sequence of GYTFTTYD (SEQ ID NO: 24), and HCDR2 with an amino acid sequence of LNPDNGNT (SEQ ID NO: 25), and An HCDR3 having an amino acid sequence of TRAPWWWYFDY (SEQ ID NO: 26); and (3) An LCDR1 having an amino acid sequence of SLNIGSNY (SEQ ID NO: 27), An LCDR2 having an amino acid sequence of KNN (SEQ ID NO: 28), An LCDR3 having an amino acid sequence of AAWDDSLSGVVFG (SEQ ID NO: 29), An HCDR1 having an amino acid sequence of GFSFTNYG (SEQ ID NO: 30), An HCDR2 having an amino acid sequence of ISYDDGSDK (SEQ ID NO: 31), An HCDR3 having an amino acid sequence of VRDPTGDY (SEQ ID NO: 32).

[0008] In some embodiments, the heavy chain variable region in the antibody or antigen-binding fragment thereof according to any one of the above items comprises a sequence represented by SEQ ID NO: 4, 6 or 8, or an amino acid sequence having at least 90% sequence identity with the sequence represented by SEQ ID NO: 4, 6 or 8.

[0009] In some embodiments, the heavy chain variable region in the antibody or antigen-binding fragment thereof according to any one of the above items comprises a sequence represented by SEQ ID NO: 4, or an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 4, and the light chain variable region comprises a sequence represented by SEQ ID NO: 5, or an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 5, or the heavy chain variable region comprises a sequence represented by SEQ ID NO: 6, or an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 6, and the light chain variable region comprises a sequence represented by SEQ ID NO: 7, or an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 7, or the heavy chain variable region comprises a sequence represented by SEQ ID NO: 8, or an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 8, and the light chain variable region comprises a sequence represented by SEQ ID NO: 9, or an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 9.

[0010] In some embodiments, the heavy chain constant region in the antibody or antigen-binding fragment thereof according to any one of the above items comprises the sequence represented by SEQ ID NO: 10, or an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 10, and the light chain constant region of the antibody comprises the sequence represented by SEQ ID NO: 11 or 12, or an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 11 or 12.

[0011] In some embodiments, the antibody in the antibody or antigen-binding fragment thereof according to any one of the above items is a mouse antibody, a chimeric antibody, a human-derived antibody or a fully human antibody, and preferably, the antibody is a human-derived antibody.

[0012] In some embodiments, the F protein in the antibody or antigen-binding fragment thereof according to any one of the above items is in a pre-fusion conformation.

[0013] In some embodiments, the respiratory syncytial virus is a type A or type B respiratory syncytial virus.

[0014] In another aspect, the present disclosure provides a nucleic acid molecule encoding the antibody or antigen-binding fragment thereof according to any one of the above items. Specifically, the nucleic acid molecule encodes the light chain, heavy chain, light chain variable region or heavy chain variable region of the antibody according to any one of the above items. In another aspect, the present disclosure provides an expression vector comprising the nucleic acid molecule.

[0015] In another aspect, the present disclosure provides a host cell comprising the antibody or antigen-binding fragment thereof, or expressing the same, or a host cell comprising the nucleic acid molecule or expression vector.

[0016] In another aspect, the present disclosure provides a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of the above items and one or more pharmaceutically acceptable carriers, diluents, buffers or excipients.

[0017] In another aspect, the present disclosure provides the use of the antibody or antigen-binding fragment thereof according to any one of the above items for manufacturing a medicament for treating and / or preventing a disease or symptom associated with respiratory syncytial virus. In another aspect, the present disclosure provides a method for treating and / or preventing a disease or symptom associated with respiratory syncytial virus in a subject, comprising administering to the subject a therapeutically effective amount of the antibody or antigen-binding fragment thereof according to any one of the above items or the above pharmaceutical composition.

[0018] Furthermore, the disease or symptom associated with respiratory syncytial virus according to any one of the above items of the present disclosure is a disease or symptom associated with an upper respiratory tract infection or a lower respiratory tract infection. More preferably, the disease or symptom is selected from tracheitis, bronchitis, and lung infections. Most preferably, the disease or symptom is bronchitis or pneumonia.

[0019] In another aspect, the present disclosure provides a method for detecting the presence or content of respiratory syncytial virus in a sample, comprising contacting the sample with the antibody or antigen-binding fragment thereof and detecting the presence or absence of the formation of an antigen-antibody complex or the amount of the formed antigen-antibody complex.

[0020] In another aspect, the present disclosure provides a respiratory syncytial virus detection kit comprising the antibody or antigen-binding fragment thereof according to any one of the above items. BRIEF DESCRIPTION OF THE DRAWINGS

[0021]

Figure 1

[0022] Unless otherwise specified, all technical terms and scientific terms used in the present disclosure have meanings commonly understood by those skilled in the art.

[0023] "Antibody" refers to an immunoglobulin secreted by plasma cells (effector B cells) and used by the body's immune system to neutralize foreign substances (such as polypeptides, viruses, bacteria, etc.). Correspondingly, the above foreign substances are called antigens. The term "antibody" is used in the broadest sense and includes various antibody structures such as monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), full-length antibodies, and antibody fragments (or antigen-binding fragments, or antigen-binding portions) as long as they exhibit the desired antigen-binding activity, but are not limited thereto. The basic structure of a typical or normal antibody molecule is a tetramer consisting of two identical heavy chains and two identical light chains. Due to differences in the conservation of amino acid sequences, the heavy and light chains are divided into a variable region (V) at the amino terminus and a constant region (C) at the carboxy terminus. The interaction between the variable regions of one heavy chain and one light chain forms an antigen-binding site (Fv). In the variable region, certain regions are more likely to have changes in the composition and order of amino acid residues than other regions (framework regions, FR) within the variable region, and are called hypervariable regions (HVR). The hypervariable regions are actually the sites important for the binding of the antibody to the antigen. Since these hypervariable region sequences are complementary to the antigenic determinants, they are also called complementarity-determining regions (CDR). Both the heavy and light chains have three complementarity-determining regions, called HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3, respectively. Each VH and VL is composed of three CDRs and four FR regions arranged in the order of FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from the amino terminus to the carboxy terminus.

[0024] When the sequences of the antibody complementarity-determining regions are known, those skilled in the art can easily replace a part of the sequence of an antibody molecule (e.g., the constant region or the framework region) with the sequences of other species using DNA recombination techniques and the like to form chimeric antibodies. Chimeric antibodies can substantially retain the binding specificity of the original antibody. For example, when used in different species, to weaken the immunogenicity of the antibody, or to utilize specific functions of the constant region such as ADCC-related activities, if the original antibody is a mouse antibody, its constant region can be replaced with the constant region of a human antibody, and conversely, if the original antibody is a human antibody, its constant region can be replaced with the constant region of a mouse antibody. To further reduce the immunogenicity of the antibody or for other purposes, while substantially retaining the binding specificity of the original antibody, all sequences other than the CDR sequences within the antibody molecule can be replaced with the corresponding sequences of another antibody molecule (derived from the same or different species, which may optionally contain one or more amino acid mutations). In one example, those skilled in the art often use CDR grafting to humanize mouse antibodies.

[0025] Also, as will be understood by those skilled in the art, based on the specific antibody sequences according to the present disclosure, several amino acid substitutions, deletions, and insertions are performed, and by verifying or screening the antigen (F protein) binding ability or biological activity of the obtained product, variants of the anti-F protein antibody molecule according to the present invention can be obtained. These variants are also included within the scope of the present invention. Therefore, in some embodiments, the heavy chain variable region of the antibody molecule according to the present disclosure includes the sequence represented by SEQ ID NO: 4, or 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 SEQ ID NO: 4, and the light chain variable region includes the sequence represented by SEQ ID NO: 5, or 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 SEQ ID NO: 5.

[0026] In some embodiments, the heavy chain variable region of the antibody molecule according to the present disclosure comprises the sequence represented by SEQ ID NO: 6, or 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 SEQ ID NO: 6, and the light chain variable region comprises the sequence represented by SEQ ID NO: 7, or 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 SEQ ID NO: 7.

[0027] In some embodiments, the heavy chain variable region of the antibody molecule according to the present disclosure comprises the sequence represented by SEQ ID NO: 8, or 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 SEQ ID NO: 8, and the light chain variable region comprises the sequence represented by SEQ ID NO: 9, or 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 SEQ ID NO: 9.

[0028] The "antigen-binding fragment" of an antibody refers to a polypeptide that contains a part of the sequence of the original antibody (especially the CDR sequence) and also has the binding specificity of the original antibody. The antigen-binding fragment usually contains the variable region of the light chain and the variable region of the heavy chain of the original antibody, and thus has the ability to bind antigens. As forms of antigen-binding fragments, there are various forms such as Fab, F(ab’)2, single-chain antibody (scFv), single-domain antibody (sdAb), etc. ScFv is formed by binding the variable region of the heavy chain and the variable region of the light chain of the antibody with a short peptide so that they become peptide chains. Due to correct folding, the variable regions from the heavy chain and the light chain form an Fv segment through non-covalent interaction, so scFv can preferably retain the affinity activity for antigens. SdAb was first discovered in camelids and has the ability to bind antigens even without a light chain naturally. SdAb has advantages such as being more stable (resistant to pH, heat, and proteases) compared to normal antibody molecules, and can bind to antigen epitopes on antigen molecules that are difficult to access with normal antibody molecules.

[0029] An antibody "specifically binds to an antigen" or the "antigen-binding specificity" of an antibody refers to the ability of the antibody to bind to a target antigen with a higher or relatively high binding affinity compared to other antigens. The binding ability of an antibody to a target antigen can be qualitatively or quantitatively identified by various methods such as measuring the KD value of the antibody that binds to the target antigen, and measuring the ability to bind to a target antigen that competes with other antibodies. Here, Kd is the equilibrium dissociation constant and can be used to evaluate the strength of the binding affinity between an antibody and its antigen. The smaller the KD value, the stronger the affinity is considered. An antibody specifically binding to a target antigen does not necessarily mean that it cannot bind to other antigens. For example, immune cross-reactivity is also known in the art.

[0030] As used herein, "antibody" includes a complete antibody or an antigen-binding fragment thereof, and can be a human antibody, a mouse antibody, a humanized antibody, a chimeric antibody, etc. The antibody can be of any type such as, for example, IgG, IgE, IgM, IgD, IgA or IgY, or of any subtype such as, for example, IgG1, IgG2, IgG3, IgG4, IgA1 or IgA2.

[0031] Methods for preparing antibodies are known in the art and include methods for isolating cells that express a specific antibody after immunizing an animal with an antigen (e.g., antibody preparation by hybridoma technology), antibody screening techniques using phage antibody libraries, methods for transforming cells by genetic engineering techniques to express antibody molecules, etc. In one example, the examples of the present disclosure provide a method for preparing an antibody molecule by introducing a recombinant expression vector encoding the heavy and light chains of the antibody into 293T cells.

[0032] The term "chimeric" antibody refers to an antibody in which a part of the heavy and / or light chains of the antibody is derived from a specific source or species, and the remaining part of the heavy and / or light chains is derived from another source or species.

[0033] The term "humanized" antibody means an antibody that has low immunogenicity in humans while retaining the reactivity of a non-human antibody. For example, it can be achieved by retaining the non-human CDR regions and replacing the remaining part of the antibody with human counterparts (i.e., the constant region and the framework part of the variable region).

[0034] The terms "human antibody", "antibody derived from human", "fully human antibody", and "complete human antibody" are used interchangeably and mean an antibody in which the variable and constant regions are human sequences. This term includes antibodies having sequences derived from human genes but modified, for example, for the purpose of reducing potential immunogenicity, increasing affinity, or removing cysteine or glycosylation sites that may cause undesirable folding. This term also includes antibodies recombinantly produced in non-human cells that can impart non-human cell-typical glycosylation. This term also includes antibodies produced in transgenic mice containing some or all of the human immunoglobulin heavy and light chain loci. The term "human antibody" is meant to clearly exclude humanized antibodies containing non-human antigen-binding residues.

[0035] The term "monoclonal antibody" means a substantially homogeneous population of antibodies. That is, except for natural mutations that may be present in small amounts, the amino acid sequences of the antibody molecules contained in the population are identical. In contrast, polyclonal antibody preparations usually contain a plurality of different antibodies having different amino acid sequences in the variable domains and are usually specific for different epitopes. The term "monoclonal" represents the property of an antibody obtained from a substantially homogeneous population of antibodies and should not be construed as requiring that the antibody be produced by any particular method. In some embodiments, the antibodies according to the present disclosure are monoclonal antibodies.

[0036] The term "antigen" means a molecule or a portion thereof that can be bound by a selective binding agent such as an antigen-binding protein (including, for example, an antibody), and further, can be used in an animal to generate an antibody capable of binding to the antigen. An antigen can have one or more epitopes capable of interacting with different antigen-binding proteins (such as antibodies).

[0037] It is known in the art that various modifications can be made to antibody molecules, such as PEGylation, glycosylation, formation of antibody molecule conjugates (e.g., ADCs), addition of purification tags, or fusion with other proteins (e.g., formation of bispecific antibodies). These antibody derivatives obtained by modifying the antibody molecules according to the present disclosure are also included within the scope of the present invention.

[0038] With respect to amino acid sequences, the term "sequence identity" (also referred to as "sequence homology") is a measure of the degree of identity between two amino acid sequences (e.g., a query sequence and a reference sequence), and is generally expressed as a percentage. Usually, before calculating the percentage identity between two amino acid sequences, the sequences are aligned and gaps (if any) are introduced. If the amino acid residues at a particular alignment position in the two sequences are the same, the two sequences are considered to be identical or match at that position, but if the amino acid residues in the two sequences are different, they are considered to be non-identical or not to match at that position. In some algorithms, the number of matching positions is divided by the total number of positions within the alignment window to obtain the sequence identity. 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, using the well-known alignment software BLAST (available on the web page ncbi.nlm.nih.gov), an optimal sequence alignment can be obtained with default settings, and the sequence identity between two amino acid sequences can be calculated.

[0039] An "expression vector", also called a "recombinant expression vector", refers to a nucleic acid molecule containing various expression elements for expressing a protein of interest (e.g., the light chain and / or heavy chain of an antibody molecule) in a host cell. In the case of an expression vector for expressing a protein of interest in eukaryotic cells (such as mammalian cells, insect cells, plant cells, etc.), these expression elements generally include a promoter, an enhancer, a polyadenylation signal sequence, etc. For the convenience of amplification in Escherichia coli, an Escherichia coli replicon sequence is often also included in the expression vector. The expression vector may further include an antibiotic resistance gene or a selection marker gene for screening (e.g., an ampicillin resistance gene (AmpR), a thymidine kinase gene (TK), etc.) and a multiple cloning site (MCS) for inserting the protein coding sequence of interest. The expression vector can be a plasmid vector or a viral vector.

[0040] The expression vector according to the present disclosure is suitable for expressing the antibody molecule or a fragment thereof according to the present disclosure in various host cells, and in particular, is suitable for expression in mammalian cells (e.g., mouse, rat, pig, sheep, monkey, orangutan, or human cells). In some embodiments, the mammalian cells are selected from CHO cells, HEK293 cells, or BHK cells.

[0041] The present disclosure uses the F protein DS-Cav1 in the pre-fusion conformation of RSV as a bait, and by single-cell sequencing, four human monoclonal antibodies were isolated from the peripheral blood lymphocytes (PBMCs) of children who had recovered from RSV infection, and named RV7, RV8, RV10, and RV11, respectively. Among these, three can bind to the RSV F protein. RV8 and RV10 bind to both the pre-fusion F protein and the post-fusion F protein, while RV11 binds only to the pre-fusion F protein. These three monoclonal antibodies can be used for the diagnosis of RSV and the quantitative measurement of antigens. Among them, two, RV8 and RV11, have neutralizing activity against both RSV A and B types and can be used for the prevention and treatment of RSV.

[0042] An antibody or antigen-binding fragment thereof according to the present disclosure can specifically bind to the respiratory syncytial virus F protein (pre-fusion conformation and / or post-fusion conformation), that is, the respiratory syncytial virus F protein serves as the target antigen of the antibody. When the antibody binds to the respiratory syncytial virus F protein, it inhibits the cell infectivity of RSV by its neutralizing activity. Therefore, the antibody or antigen-binding fragment thereof according to the present disclosure can be used for the prevention or treatment of RSV infection. In another aspect, due to the antigen-binding specificity of the antibody according to the present disclosure, the antibody can be used to detect the presence or absence of RSV virus in a sample (such as blood) from a subject. Therefore, the present disclosure provides an RSV detection kit that may include the antibody or antigen-binding fragment thereof according to the present disclosure.

[0043] The "pharmaceutically acceptable carrier" used in relation to a pharmaceutical composition means substances such as a solid or liquid diluent, extender, antioxidant, stabilizer, etc. that can be safely administered. These substances are suitable for administration to humans and / or animals without excessive harmful side effects and are suitable for maintaining the activity of the drug or active agent present therein.

[0044] "Subject" or "subject" includes humans and non-human animals. Non-human animals include, for example, all vertebrates (such as mammals and non-mammals) such as non-human primates (such as cynomolgus monkeys), sheep, dogs, cows, chickens, amphibians and reptiles. Unless otherwise specified, the terms "patient" or "subject" are used interchangeably in the present disclosure. In some embodiments, the subject or subject is human.

[0045] "Administer" or "give", when applied to an animal, human, subject of an experiment, cell, tissue, organ or biological fluid, means the contact of an exogenous drug, therapeutic agent, diagnostic agent or composition with the animal, human, subject, cell, tissue, organ or biological fluid.

[0046] Regarding the treatment or prevention of RSV infection, the "therapeutically effective amount" refers to the amount of an active compound (such as an antibody) sufficient to cause a biological or medical response desired by a clinician in a subject, and can also mean the dosage of a drug or agent that can fully or at least partially achieve the desired effect. The "therapeutically effective amount" upon administration of the antibody according to the present disclosure can be determined by those skilled in the art based on factors such as the administration route, the weight, age, condition, etc. of the subject. For example, a typical daily dosage can range from 0.01 mg to 100 mg per kg of body weight as the active ingredient.

[0047] As used in the present disclosure, the singular forms "a", "one", and "the" include plural referents unless the context clearly dictates otherwise, and vice versa.

[0048] Hereinafter, the present invention will be further described by specific examples.

Example

[0049] (Example 1) Expression and purification of the pre-fusion F protein (DS-Cav1) of RSV virus A sequence encoding the pre-fusion F protein DS-Cav1 of RSV subtype A2, with a thrombin cleavage site, a six-histidine sequence, and a GS [glycine-serine] linker added to the C-terminus, was inserted into the EcoR I and XhoI cleavage sites of the mammalian expression vector pCAGGS together with a sequence encoding a Strep II tag and a stop codon (see SEQ ID NO: 1 for the amino acid sequence). The ligation product was transformed into DH5α E. coli competent cells. Then, a single clone was selected and inoculated into 4 mL of LB medium, cultured for 6 - 8 hours, transferred to 300 mL of LB medium, cultured for 12 - 16 hours, harvested, and the plasmid was extracted using an endotoxin-free plasmid extraction kit (TIANGEN) to obtain pCAGGS-RSV-F(DS-Cav1).

[0050] The extracted plasmid pCAGGS-RSV-F(DS-Cav1) was transfected into 293T cells to express the F protein. Four to five days after transfection, the 293T cell culture supernatant containing the target protein was centrifuged and filtered (0.22 μM) to remove cell debris, and then bound to a HisTrap HP (GE healthcare) nickel chelate column and further eluted from the column with various concentrations of imidazole. The eluted proteins were each collected, and samples containing the target protein were identified based on the results of SDS-PAGE.

[0051] The elution peak containing the target protein was collected, concentrated, and then subjected to molecular sieve chromatography. From the protein peak position and the results of SDS-PAGE, the size and purity of the target protein were determined (see Figure 1 for the results of molecular sieve and SDS-PAGE). According to the results of molecular sieve, the protein peak position was at 60 - 70 mL, which corresponded to the theoretical molecular weight of 150 kDa of the F protein trimer. According to the results of SDS-PAGE, since the F protein was about 50 Kda under both reducing (+DTT) and non-reducing (-DTT) conditions, it was found that the subunits of the F protein were non-covalently bound to form a trimer. The sample numbered T in SDS-PAGE corresponded to the collection tube numbered T in molecular sieve.

[0052] (Example 2) Isolation of RSV F(DS-Cav1)-specific memory B cells Informed consent was obtained from a person who had recovered from RSV virus infection and been discharged from the hospital, and 3 - 10 mL of blood was collected to isolate PBMCs. The isolated PBMCs (10 7(mL) was incubated with 400 nM of protein DS-Cav1 (prepared in Example 1) for 30 minutes on ice for binding, then washed twice with PBS, and further mixed with the following antibodies (all purchased from BD). anti-human CD3 / PE-Cy5, anti-human CD16 / 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 30 minutes, it was washed twice with PBS. Then, PBMCs were sorted by FACSAria III, and PE-Cy5-APC-APC-Cy7+Pacific Blue+FITC+PE+ cells (i.e., memory B cells) were directly collected into a 96-well plate at 1 cell / well.

[0053] (Example 3) Single B cell PCR and cloning of human-derived monoclonal antibody IgG1 The cells obtained in Example 2 were reverse-transcribed using Superscript III reverse transcriptase (Invitrogen) under reaction conditions of 55 °C for 60 minutes. Using this reverse transcription product as a template, PCR was performed using HotStar Tap Plus enzyme (QIAgen) to amplify the antibody variable region sequence (PCRa) (the reaction conditions were 5 minutes at 95 °C, 30 seconds at 95 °C, 30 seconds at 55 °C (heavy chain / κ chain) / 50 °C (λ chain), 90 seconds at 72 °C, 35 cycles, and 7 minutes at 72 °C). Using this as a template, another round of PCR (PCRb) was performed (the conditions were 5 minutes at 95 °C, 30 seconds at 95 °C, 30 seconds at 58 °C (heavy chain) / 60 °C (κ chain) / 64 °C (λ chain), 90 seconds at 72 °C, 35 cycles, and 7 minutes at 72 °C). 1.2% agarose gel electrophoresis was performed to separate the PCR products, and the band of 400 - 500 bp was cut out and recovered, and then sent to a sequencing company for sequencing. Sequence analysis of the sequencing results was performed on the IGBLAST site. The analysis results are as follows.

[0054] Four pairs of antibodies, RV7, RV8, RV10, and RV11, were obtained. According to the sequencing results, for RV7, the amino acid sequence of the heavy chain variable region was SEQ ID NO: 2, and the amino acid sequence of the light chain variable region was SEQ ID NO: 3. For RV8, the amino acid sequence of the heavy chain variable region was SEQ ID NO: 4, and the amino acid sequence of the light chain variable region was SEQ ID NO: 5. For RV10, the amino acid sequence of the heavy chain variable region was SEQ ID NO: 6, and the amino acid sequence of the light chain variable region was SEQ ID NO: 7. For RV11, the amino acid sequence of the heavy chain variable region was SEQ ID NO: 8, and the amino acid sequence of the light chain variable region was SEQ ID NO: 9. As a result of comparing the light chain sequencing results of the four antibodies with the germline genes respectively, it was found that RV7 used CLκ, RV8 used CLκ, RV10 used CLλ, and RV11 used CLλ. To obtain human-derived antibodies for subsequent evaluation, a complete anti-IgG1 was designed and constructed by the following strategy. Heavy chain: CMV promoter - Hind III - signal peptide (SP) - heavy chain variable region (VH) - heavy chain constant region (CH) - Not I. Light chain κ: CMV promoter - Hind III - signal peptide (SP) - light chain variable region (VK) - light chain constant region (CLκ) - Not I. Light chain λ: CMV promoter - Hind III - signal peptide (SP) - light chain variable region (VL) - light chain constant region (CLλ) - Not I.

[0055] The correct variable region sequences were ligated to the constant regions of the corresponding heavy chain CH and light chain CLκ (or light chain CLλ) by bridge PCR and cloned into the expression vector KT351861 to obtain a recombinant plasmid containing genes encoding the light and heavy chains of a specific antibody. Here, the light and heavy chain variable regions were inserted into the vector containing the constant region by the cleavage sites Hind III and Not I. The amino acid sequence of the heavy chain constant region CH was SEQ ID NO: 10, the amino acid sequence of the light chain constant region CLκ was SEQ ID NO: 11, the amino acid sequence of the light chain constant region CLλ was SEQ ID NO: 12, and the amino acid sequence of the signal peptide (SP) was SEQ ID NO: 13.

[0056] (Example 4) Expression and Purification of Monoclonal Antibody 293T cells were cultured in DMEM containing 10% FBS. Recombinant expression vectors encoding the heavy and light chains of the antibody obtained in Example 3 were co-transfected into 293T cells. Four to six hours after transfection, the cell culture medium was replaced with serum-free DMEM and cultured for another 3 days. The supernatant was collected, DMEM was added, and after culturing for another 4 days, the supernatant was collected again.

[0057] The collected supernatant was centrifuged at 8000 rpm for 90 minutes, then mixed with an equal volume of buffer containing 20 mM sodium phosphate (pH 7.0), filtered through a 0.22 μm filter, and injected into a pre-packed column (5 mL, GE Healthcare) filled with protein A or G. The protein bound to the pre-packed column was eluted with 100 mM glycine (pH 3.0). The eluted fraction was concentrated and purified by molecular sieve chromatography. Subsequently, the purified target protein was analyzed by SDS-PAGE (reducing and non-reducing). As a result, in SDS-PAGE under non-reducing conditions, the antibody showed a single band, but in SDS-PAGE under reducing conditions, the disulfide bond in the Fc region of the antibody was cleaved, so it was shown as two bands. Also, the purity of the antibody was over 95%.

[0058] (Example 5) Binding Assay of Antibody (1) Construction and Expression of RSV Post-Fusion F Protein (FΔFP) The sequence encoding the post-fusion F protein (FΔFP) of the RSV A2 strain with a thrombin cleavage site, a six-histidine sequence, and a single GS [glycine-serine] linker added to the C-terminus was inserted into the NdeI and XhoI cleavage sites of the mammalian expression vector pCAGGS, together with the sequence encoding the Strep II tag and a stop codon (see SEQ ID NO: 14 for the amino acid sequence). The ligation product was transformed into DH5α E. coli competent cells. Subsequently, a single clone was selected, inoculated into 4 mL of LB medium, cultured for 6 - 8 hours, transferred to 300 mL of LB medium, cultured for 12 - 16 hours, harvested, and the plasmid was extracted using an endotoxin-free plasmid extraction kit (TIANGEN) to obtain pCAGGS-RSV-FΔFP. The extracted plasmid pCAGGS-RSV-FΔFP was transfected into 293T cells to express the F protein. Four to five days after transfection, the 293T cell culture supernatant containing the target protein was centrifuged and filtered (0.22 μM) to remove cell debris, then bound to a HisTrap HP (GE healthcare) nickel chelate column and eluted from the column with various concentrations of imidazole. The eluted proteins were each collected, and samples containing the target protein were identified based on the results of SDS-PAGE. The elution peak containing the target protein was collected, concentrated, and subjected to molecular sieve chromatography. The size and purity of the protein were determined from the protein peak position and the results of SDS-PAGE.

[0059] (2) Preparation and purification of the full-length monoclonal antibody Fab segment A. Sample preparation The complete antibody protein was concentrated to 10 mg / ml or more, buffer exchange (200-fold) was performed using sample buffer (20 mM Na3PO4, 10 mM EDTA, pH 7.0), the antibody after buffer exchange was concentrated to 20 mg / ml or more, a certain amount of digestion buffer (cysteine·HCl was added to the sample buffer to 20 mM, pH 7.0) was added and diluted to a final concentration of ≧10 mg / ml. B. Preparation of Enzyme Using a pipette tip with the tip cut off, 0.5 mL of well-mixed coupling enzyme beans (Thermo, Prod#20341) was added to the reaction vessel and centrifuged at 1000 g / min for 2 minutes to remove the preservation solution. The beans were washed 3 times with 3 mL of digestion buffer, and the eluate was discarded. Then, 0.5 mL of digestion buffer was added and gently inverted and mixed. C. Cleavage by Enzyme The reaction tube was stoppered, concentrated antibody (about 10 mg) was added, the lid was tightened, and it was sealed with a sealing film. The reaction tube was fixed to a rotary mixer and incubated overnight at 37°C. D. Recovery of Fab After overnight cleavage by the enzyme, it was centrifuged at 1000 g / min for 5 minutes, and the supernatant was retained. The beans were washed 2 times with 1 mL of Protein G binding buffer (20 mM Na3PO4, pH 7.0), and the supernatant was collected. After mixing the supernatants obtained in these two steps, the concentration was measured. The supernatant was concentrated and applied to Protein G, the target protein eluate was concentrated and buffer-exchanged into 1×PBS buffer, and further purified by molecular sieve.

[0060] (3) Evaluation of the Binding Affinity of Monoclonal Antibody to F Protein In this example, surface plasmon resonance analysis was performed using Biacore 3K (Biacore Inc.) according to the following procedure. First, the pre-fusion F protein DS-Cav1 and the post-fusion F protein FΔFP were immobilized on the Fc2 and Fc4 channels (flow cell, Fc) of the CM5 chip by amino coupling, respectively. Next, the Fab proteins of the purified antibodies RV7, RV8, RV10, and RV11 were bound by capture with antibodies. Palivizumab was used as a positive control. Also, the Fabs of antibodies RV7, RV8, RV10, RV11, and Palivizumab were serially diluted with a solution of 20 mM HEPES, 150 mM NaCl, pH 7.4. Then, the serially diluted Fabs were passed through each channel in turn (injected in order from low concentration). The reaction rate curves of the Fab binding of antibodies RV7, RV8, RV10, RV11, and Palivizumab to DS-Cav1 or FΔFP were plotted, and the rate constant K D was calculated using the software BIAevaluation software 3K (Biacore, Inc.). The affinity results are shown in Table 1.

[0061]

Table 1

[0062] (Result analysis) RV7 does not bind to either the RSV pre-fusion F protein or the RSV post-fusion F protein. RV8 binds to both the RSV pre-fusion F protein (K D = 25.6 nM) and the post-fusion F protein (K D = 17.5 nM). RV10 binds to both the pre-fusion F protein (K D = 10.1 nM) and the post-fusion F protein (K D = 7.11 nM). The RV11 antibody binds to the RSV pre-fusion F protein (K D = 7.18 nM) but does not bind to the post-fusion F protein.

[0063] (Example 6) Neutralization activity assay of antibodies The neutralizing activity of the antibody was analyzed by flow cytometry staining method according to the following procedure. Generally speaking, the purified antibody was serially diluted, mixed with the virus, incubated for a certain period of time, and then used to infect the susceptible cells prepared the previous day. Two days later, the cells were fixed and permeabilized, stained with a primary antibody targeting the F protein, and then stained again with a labeled secondary antibody. Subsequently, flow cytometry was performed to measure the proportion of infected cells. Finally, the neutralizing activity of the antibody was calculated.

[0064] Experimental procedure 1. Cell preparation (1) To perform the antibody neutralization experiment after more than 70% growth the next day, Hep2 cells were seeded in 24-well plates 12 - 16 hours in advance and cultured in 10% DMEM medium. (2) Dilution of antibody For a total of 5 antibodies, RV7, RV8, RV10, RV11, and Palivizumab, each antibody was purified, sterilized by filtration, and then added to a 48-well plate and serially diluted 3-fold. It was diluted with DMEM medium (2% FBS) so that it was at least 350 μL - 400 μL per well. (3) Dilution of virus The virus was taken out from the -80 °C refrigerator and thawed, diluted with DMEM medium (2% FBS) according to the appropriate virus dilution ratio, and then added to the 48-well plate containing the antibody at 300 μL / well. (RSV_A2 or RSV-Long or RSV-B was diluted from 450 μL to 40 mL, and the final titer was 1.2×10 4 TCID50 / 100 μL.) (4) Incubation of virus and antibody The diluted virus and antibody were mixed at a molar ratio of 1:1 and incubated at 37 °C for 1 hour in a CO2 incubator. (5) Washing of cells Ten minutes before, Hep2 cells immediately after confluence were taken out from the incubator, the medium was removed with a pipette, the cells were washed once with PBS, and 500 μL of PBS was added to each well. (6) Addition of virus-antibody mixture The PBS was removed from the cells, and the mixture of the virus and the cell supernatant was added to the cells at 300 μL per well and incubated at 37 °C for 1 hour. (For each concentration, 2 wells were used and there were 11 concentrations in total. For each plate, 2 wells of virus control were set up.) (7) Addition of medium After incubation, the supernatant was removed, and 1 mL of DMEM medium containing penicillin-streptomycin mixed solution + 2% FBS was added to each well. The cells were cultured in an incubator for about 48 hours.

[0065] 2. Flow cytometry analysis (1) The cells after infection were observed, and the cells were collected after appropriate cytopathic changes occurred. (2) Digestion of cells The cell medium was removed, the cells were washed once with PBS, 120 μl of trypsin was added to each well, and the cells were digested in an incubator at 37 °C for 2 minutes. When the cells detached, 130 μL of 10% FBS + PBS was added to stop the reaction. (3) The cells were transferred to a round-bottom 96-well plate. Centrifuged at 500 g at 4 °C for 10 minutes, and the supernatant was removed. (4) 150 μL of 1% FBS + PBS was added per well for one wash, centrifuged in the same manner as above, and the supernatant was removed. (5) 100 μL of Fixation and Permeabilization Solution was added, and the cells were fixed and permeabilized at 4 °C on ice in the dark for 30 minutes. (6) 100 μL of 1× wash buffer was added and mixed, and centrifuged at 500 g at 4 °C for 10 minutes. Resuspended with 100 μL of wash buffer, sprayed alcohol on the outside of the 96-well plate and sealed, and transferred from P3 to P2 for subsequent operations. As a precaution, let it stand at 4 °C overnight before the operation, or transfer the cells to a new round-bottom 96-well plate. (7) Addition of primary antibody The purified palivizumab was diluted with 1× wash buffer to a concentration of 2 μg / mL. The cells were centrifuged, the supernatant was removed, 50 μL of the antibody was added to each well, and the mixture was incubated on ice for 30 minutes. Then, 150 μL of 1× wash buffer was added, mixed, centrifuged, and the supernatant was removed. (8) Addition of secondary antibody Anti-Human-IgG(FITC) was diluted 150-fold with 1× wash buffer, 50 μL of the antibody was added to each well, and the mixture was incubated on ice for 30 minutes. Then, 150 μL of 1× wash buffer was added, mixed, centrifuged, and the supernatant was removed. The washing was repeated once. Finally, it was resuspended with 150 μL of PBS.

[0066] (Final flow cytometry analysis) Flow cytometry analysis was performed using a BD FACSAria II. Analysis was carried out using FlowJo 7.6.1. (Results) In the case of RSV A type A2 strain, for the half-inhibitory concentration (IC50) of the antibody, RV8 was 539.5 ng / mL and RV11 was 42.3 ng / mL, both of which were superior to the 751 ng / mL of commercially available palivizumab. In particular, the antibody RV11 showed a neutralizing activity approximately 18 times stronger than that of palivizumab. RV7 and RV10 did not show neutralizing activity within the tested concentration range. The data are shown in Table 2.

[0067]

Table 2

[0068] In the case of RSV A type Long strain, for the half-inhibitory concentration (IC50) of the antibody, RV8 was 842.9 ng / mL and RV11 was 56.3 ng / mL, both of which were superior to commercially available palivizumab (IC50 = 897.4 ng / mL). In particular, the RV11 antibody showed a neutralizing activity approximately 16 times stronger than that of palivizumab. RV7 did not show neutralizing activity within the tested concentration range. The data are shown in Table 3.

[0069]

Table 3

[0070] In the case of RSV subtype B clinical isolates, for the half-inhibitory concentration (IC50) of the antibodies, RV8 was 183.1 ng / mL and RV11 was 31.4 ng / mL, both of which were superior to the commercially available palivizumab (IC50 = 1413.0 ng / mL). In particular, the RV11 antibody showed a neutralizing activity approximately 45 times stronger than palivizumab. The RV8 antibody also showed a neutralizing activity 7.7 times stronger than palivizumab. The data are shown in Table 4.

[0071]

Table 4

Chemical formula

Chemical formula

Chemical formula

Claims

**Claim 1** An antibody or antigen-binding fragment thereof that binds to the respiratory syncytial virus F protein, comprising a heavy chain variable region and a light chain variable region, wherein the HCDR1, HCDR2, and HCDR3 of the heavy chain variable region and the LCDR1, LCDR2, and LCDR3 of the light chain variable region are any one selected from the following combinations: (1) LCDR1 with the amino acid sequence QDIRND, LCDR2 with the amino acid sequence AAS, LCDR3 with the amino acid sequence LQDYNYPQTFG, HCDR1 with the amino acid sequence GFTFSSYA, HCDR2 with the amino acid sequence ISYDGSNT, and HCDR3 with the amino acid sequence ARDYCSRGTCYHDY; and (2) LCDR1 with the amino acid sequence SLNIGSNy, LCDR2 with the amino acid sequence KNN, LCDR3 with the amino acid sequence AAWDDLSGVVFG, HCDR1 with the amino acid sequence GFSFTNYG, HCDR2 with the amino acid sequence ISYDDGSDK, and HCDR3 with the amino acid sequence VRDPTGdy. **Claim 2** The antibody or antigen-binding fragment thereof according to claim 1, wherein the heavy chain variable region comprises the sequence represented by SEQ ID NO: 4 or 8, or an amino acid sequence having at least 90% sequence identity with the sequence represented by SEQ ID NO: 4 or 8. **Claim 3** The heavy chain variable region comprises the sequence represented by SEQ ID NO: 4, or an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 4, and the light chain variable region comprises the sequence represented by SEQ ID NO: 5, or an amino acid sequence having at least 9% sequence identity with SEQ ID NO: 5, or The heavy chain variable region comprises the sequence represented by SEQ ID NO: 8, or an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 8, and the light chain variable region comprises the sequence represented by SEQ ID NO: 9, or an amino acid sequence having at least 90% sequence identity with SEQ ID NO:

9. The antibody or antigen-binding fragment thereof according to claim 1. **Claim 4** The heavy chain constant region of the antibody comprises the sequence represented by SEQ ID NO: 10, or an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 10, and the light chain constant region of the antibody comprises the sequence represented by SEQ ID NO: 11 or 12, or an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 11 or 12. The antibody or antigen-binding fragment thereof according to claim 1.

5. The antibody according to claim 1 or an antigen-binding fragment thereof, wherein the antibody is a human-derived antibody.

6. The antibody according to claim 1 or an antigen-binding fragment thereof, wherein the F protein is in a pre-fusion conformation.

7. The antibody according to claim 1 or an antigen-binding fragment thereof, wherein the respiratory syncytial virus is a type A or type B respiratory syncytial virus.

8. A nucleic acid molecule encoding the antibody according to claim 1 or an antigen-binding fragment thereof.

9. An expression vector comprising the nucleic acid molecule according to claim 8.

10. A host cell comprising or expressing the antibody according to claim 1 or an antigen-binding fragment thereof.

11. Used for the treatment and / or prevention of diseases or symptoms associated with respiratory syncytial virus in a subject, comprising the antibody according to claim 1 or an antigen-binding fragment thereof, and one or more pharmaceutically acceptable carriers, diluents, buffers or excipients. The disease or symptom associated with the respiratory syncytial virus is a disease or symptom associated with an upper respiratory tract infection or a lower respiratory tract infection. A pharmaceutical composition.

12. Use of the antibody according to claim 1 or an antigen-binding fragment thereof for the manufacture of a medicament for the treatment and / or prevention of a disease or symptom associated with respiratory syncytial virus, wherein the disease or symptom associated with the respiratory syncytial virus is a disease or symptom associated with an upper respiratory tract infection or a lower respiratory tract infection.

13. A method for detecting the presence or content of respiratory syncytial virus in a sample, comprising contacting the sample with the antibody according to claim 1 or an antigen-binding fragment thereof and detecting the presence or absence of formation of an antigen-antibody complex or the amount of the formed antigen-antibody complex.

14. A respiratory syncytial virus detection kit comprising the antibody according to claim 1 or an antigen-binding fragment thereof.

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