Anti-respiratory syncytial virus antibody group and application thereof

Abstract

The invention relates to an anti-respiratory syncytial virus antibody or a variant thereof, and application of the anti-respiratory syncytial virus antibody or the variant thereof in preparation of drugs for preventing and / or treating respiratory syncytial viruses or preparation of respiratory syncytial virus detection reagents, the antibody comprises a heavy chain variable region and a light chain variable region, each heavy chain variable region and light chain variable region generally comprise 3 CDRs and up to 4 FRs, and the antibodies of the invention have efficient neutralizing activity against respiratory syncytial viruses.

Description

Technical Field

[0001] The present invention relates to the fields of immunology and molecular biology, and in particular to an antibody against respiratory syncytial virus and a use thereof. Background Art

[0002] Respiratory syncytial virus (RSV) is one of the main pathogens causing severe lower respiratory tract infections in infants, the elderly, and immunocompromised individuals. RSV infection can cause symptoms such as coughing, wheezing, and difficulty breathing, and in severe cases, can even be life-threatening.

[0003] Currently, there are a limited number of preventive monoclonal antibodies against RSV. Palivizumab received FDA approval in 1998. It is primarily used to prevent hospitalization for RSV infection in high-risk infants and young children, reducing the risk of RSV infection to a certain extent. Palivizumab works by specifically binding to the RSV surface membrane fusion protein, preventing the virus from invading human cells and exerting its preventive effect.

[0004] In recent years, with the continuous advancement of medical research and the rapid development of biotechnology, more RSV monoclonal antibodies have been developed. Nicevir, co-developed by AstraZeneca and Sanofi, has also received FDA approval. Nicevir also binds to the RSV surface membrane fusion protein, providing protection against RSV in specific populations and demonstrating superior efficacy compared to palivizumab.

[0005] Despite the availability of these monoclonal antibodies, the global annual disease burden caused by RSV remains significant. RSV infection is particularly common in infants and young children and can lead to serious illnesses such as bronchiolitis and pneumonia. In the elderly and immunocompromised individuals, RSV infection can also cause severe respiratory illness and even death. To better combat RSV infection, the development of more effective RSV monoclonal antibodies is urgently needed. Summary of the Invention

[0006] In view of this, the purpose of the present invention is to provide an anti-respiratory syncytial virus antibody and its variants and uses thereof, wherein the antibody has a highly effective neutralizing activity against respiratory syncytial virus.

[0007] Based on the above objectives, the first aspect of the present invention provides an anti-respiratory syncytial virus antibody or a variant thereof, comprising a heavy chain variable region and a light chain variable region, wherein:

[0008] The heavy chain variable region comprises HCDR1 as shown in SEQ ID NO: 1, HCDR2 as shown in SEQ ID NO: 2, and HCDR3 as shown in SEQ ID NO: 3; and

[0009] The light chain variable region comprises LCDR1 with an amino acid sequence as shown in SEQ ID NO: 4, LCDR2 with an amino acid sequence as shown in SEQ ID NO: 5, and LCDR3 with an amino acid sequence as shown in SEQ ID NO: 6;

[0010] or

[0011] The heavy chain variable region comprises a HCDR1 with an amino acid sequence as shown in SEQ ID NO: 7, a HCDR2 with an amino acid sequence as shown in SEQ ID NO: 8, and a HCDR3 with an amino acid sequence as shown in SEQ ID NO: 9; and

[0012] The light chain variable region comprises LCDR1 with an amino acid sequence as shown in SEQ ID NO: 10, LCDR2 with an amino acid sequence as shown in SEQ ID NO: 11, and LCDR3 with an amino acid sequence as shown in SEQ ID NO: 12;

[0013] or

[0014] The heavy chain variable region comprises a HCDR1 with an amino acid sequence as shown in SEQ ID NO: 13, a HCDR2 with an amino acid sequence as shown in SEQ ID NO: 14, and a HCDR3 with an amino acid sequence as shown in SEQ ID NO: 15; and

[0015] The light chain variable region comprises LCDR1 with an amino acid sequence as shown in SEQ ID NO: 16, LCDR2 with an amino acid sequence as shown in SEQ ID NO: 17, and LCDR3 with an amino acid sequence as shown in SEQ ID NO: 18;

[0016] or

[0017] The heavy chain variable region comprises a HCDR1 with an amino acid sequence as shown in SEQ ID NO: 19, a HCDR2 with an amino acid sequence as shown in SEQ ID NO: 20, and a HCDR3 with an amino acid sequence as shown in SEQ ID NO: 21; and

[0018] The light chain variable region comprises LCDR1 with an amino acid sequence as shown in SEQ ID NO: 22, LCDR2 with an amino acid sequence as shown in SEQ ID NO: 23, and LCDR3 with an amino acid sequence as shown in SEQ ID NO: 24;

[0019] or

[0020] The heavy chain variable region comprises a HCDR1 with an amino acid sequence as shown in SEQ ID NO: 25, a HCDR2 with an amino acid sequence as shown in SEQ ID NO: 26, and a HCDR3 with an amino acid sequence as shown in SEQ ID NO: 27; and

[0021] The light chain variable region comprises LCDR1 as shown in the amino acid sequence of SEQ ID NO: 28, LCDR2 as shown in the amino acid sequence of SEQ ID NO: 29, and LCDR3 as shown in the amino acid sequence of SEQ ID NO: 30; and

[0022] The variant undergoes conservative substitutions of amino acids located in the HCDR region compared to the original antibody; in a preferred embodiment, the conservatively substituted amino acids are located at the antibody-antigen interface; in a more preferred embodiment, the conservatively substituted amino acids are the amino acids at position 58 in SEQ ID NO:79.

[0023] In the present application, HCDR refers to a complementarity determining region (CDR) in the heavy chain variable region, for example, HCDR1 refers to CDR1 in the heavy chain variable region; LCDR refers to a complementarity determining region (CDR) in the light chain variable region, for example, LCDR1 refers to CDR1 in the light chain variable region.

[0024] In a preferred embodiment of the present invention, the heavy chain variable region further comprises HFR1 with an amino acid sequence as shown in SEQ ID NO: 31, HFR2 with an amino acid sequence as shown in SEQ ID NO: 32, HFR3 with an amino acid sequence as shown in SEQ ID NO: 33, and HFR4 with an amino acid sequence as shown in SEQ ID NO: 34; and

[0025] The light chain variable region further comprises LFR1 with an amino acid sequence as shown in SEQ ID NO: 35, LFR2 with an amino acid sequence as shown in SEQ ID NO: 36, LFR3 with an amino acid sequence as shown in SEQ ID NO: 37, and LFR4 with an amino acid sequence as shown in SEQ ID NO: 38;

[0026] or

[0027] The heavy chain variable region further comprises HFR1 with an amino acid sequence as shown in SEQ ID NO: 39, HFR2 with an amino acid sequence as shown in SEQ ID NO: 40, HFR3 with an amino acid sequence as shown in SEQ ID NO: 41, and HFR4 with an amino acid sequence as shown in SEQ ID NO: 42; and

[0028] The light chain variable region further comprises LFR1 with an amino acid sequence as shown in SEQ ID NO:43, LFR2 with an amino acid sequence as shown in SEQ ID NO:44, LFR3 with an amino acid sequence as shown in SEQ ID NO:45, and LFR4 with an amino acid sequence as shown in SEQ ID NO:46;

[0029] or

[0030] The heavy chain variable region further comprises HFR1 with an amino acid sequence as shown in SEQ ID NO: 47, HFR2 with an amino acid sequence as shown in SEQ ID NO: 48, HFR3 with an amino acid sequence as shown in SEQ ID NO: 49, and HFR4 with an amino acid sequence as shown in SEQ ID NO: 50; and

[0031] The light chain variable region further comprises LFR1 with an amino acid sequence as shown in SEQ ID NO: 51, LFR2 with an amino acid sequence as shown in SEQ ID NO: 52, LFR3 with an amino acid sequence as shown in SEQ ID NO: 53, and LFR4 with an amino acid sequence as shown in SEQ ID NO: 54;

[0032] or

[0033] The heavy chain variable region further comprises HFR1 with an amino acid sequence as shown in SEQ ID NO: 55, HFR2 with an amino acid sequence as shown in SEQ ID NO: 56, HFR3 with an amino acid sequence as shown in SEQ ID NO: 57, and HFR4 with an amino acid sequence as shown in SEQ ID NO: 58; and

[0034] The light chain variable region further comprises LFR1 with an amino acid sequence as shown in SEQ ID NO: 59, LFR2 with an amino acid sequence as shown in SEQ ID NO: 60, LFR3 with an amino acid sequence as shown in SEQ ID NO: 61, and LFR4 with an amino acid sequence as shown in SEQ ID NO: 62;

[0035] or

[0036] The heavy chain variable region further comprises HFR1 with an amino acid sequence as shown in SEQ ID NO: 63, HFR2 with an amino acid sequence as shown in SEQ ID NO: 64, HFR3 with an amino acid sequence as shown in SEQ ID NO: 65, and HFR4 with an amino acid sequence as shown in SEQ ID NO: 66; and

[0037] The light chain variable region further comprises LFR1 with an amino acid sequence as shown in SEQ ID NO:67, LFR2 with an amino acid sequence as shown in SEQ ID NO:68, LFR3 with an amino acid sequence as shown in SEQ ID NO:69, and LFR4 with an amino acid sequence as shown in SEQ ID NO:70.

[0038] In the present application, HFR represents a framework region (FR) in the heavy chain variable region, for example, HFR1 refers to FR1 in the heavy chain variable region; LFR represents a framework region (FR) in the light chain variable region, for example, LFR1 refers to FR1 in the light chain variable region.

[0039] In the present application, each heavy chain variable region and light chain variable region generally comprises three CDRs and up to four FRs, and the CDRs and FRs are arranged, for example, in the following order from amino terminus to carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In the heavy chain variable region of the antibody of the present application, the CDRs and FRs are arranged from amino terminus to carboxyl terminus in the following order: HFR1, HCDR1, HFR2, HCDR2, HFR3, HCDR3, HFR4; and in the light chain variable region of the antibody of the present application, the CDRs and FRs are arranged from amino terminus to carboxyl terminus in the following order: LFR1, LCDR1, LFR2, LCDR2, LFR3, LCDR3, LFR4.

[0040] In a preferred embodiment of the present invention, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 71, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 72; or

[0041] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 73, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 74; or

[0042] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 75, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 76; or

[0043] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 77, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 78; or

[0044] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 79, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 80.

[0045] In a preferred embodiment of the present invention, the antibody or variant thereof is a humanized monoclonal antibody.

[0046] The second aspect of the present invention provides a nucleic acid molecule comprising a nucleotide sequence encoding the above-mentioned antibody or a variant thereof.

[0047] In a preferred embodiment of the present invention, the nucleotide sequence comprises:

[0048] 1) a nucleotide sequence encoding the heavy chain variable region, which is shown in SEQ ID NO: 81; and a nucleotide sequence encoding the light chain variable region, which is shown in SEQ ID NO: 82;

[0049] or

[0050] 2) a nucleotide sequence encoding the heavy chain variable region, which is shown in SEQ ID NO: 83; and a nucleotide sequence encoding the light chain variable region, which is shown in SEQ ID NO: 84;

[0051] or

[0052] 3) a nucleotide sequence encoding the heavy chain variable region, which is shown in SEQ ID NO: 85; and a nucleotide sequence encoding the light chain variable region, which is shown in SEQ ID NO: 86;

[0053] or

[0054] 4) a nucleotide sequence encoding the heavy chain variable region, which is shown in SEQ ID NO: 87; and a nucleotide sequence encoding the light chain variable region, which is shown in SEQ ID NO: 88;

[0055] or

[0056] 5) a nucleotide sequence encoding the heavy chain variable region, which is shown in SEQ ID NO: 89; and a nucleotide sequence encoding the light chain variable region, which is shown in SEQ ID NO: 90.

[0057] The third aspect of the present invention provides a vector comprising the above nucleic acid molecule.

[0058] The fourth aspect of the present invention provides a cell comprising the above nucleic acid molecule or the above vector.

[0059] The fifth aspect of the present invention provides a pharmaceutical composition comprising the above-mentioned antibody or variant thereof, the above-mentioned nucleic acid molecule, the above-mentioned vector or the above-mentioned cell, and a pharmaceutically acceptable carrier.

[0060] The sixth aspect of the present invention provides the use of the above-mentioned antibody or its variant, the above-mentioned nucleic acid molecule, the above-mentioned vector, the above-mentioned cell or the above-mentioned pharmaceutical composition in preparing a drug for preventing and / or treating respiratory syncytial virus or preparing a respiratory syncytial virus detection reagent.

[0061] The beneficial effects of the present invention are:

[0062] The anti-RSV antibody or its variant of the present invention has a unique CDR region and can effectively neutralize RSV. It has high neutralizing activity against RSV A strains (such as RSV A2 strain and RSV A Long strain), and its IC 50 The concentrations of 1ng / mL to 10ng / mL are all high; they have high neutralizing activity against B strains of respiratory syncytial virus (such as RSV BW / V 14617 / 85 and RSV B 18537), and their IC 50 The concentrations of the antibodies or variants thereof are all between 5 ng / mL and 30 ng / mL. Therefore, the antibodies or variants thereof of the present invention can be used to prepare drugs for preventing and / or treating respiratory syncytial virus. In addition, the antibodies or variants thereof of the present invention can be used to prepare respiratory syncytial virus detection reagents, thereby being used to detect viral antigens and to discover effective neutralizing antigen epitopes. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] Figure 1 :Flowchart of antibody screening using phage display technology.

[0064] Figure 2 : Binding affinity curves of RSV antibodies to RSV A2 strain and RSV B 18537 strain pre-F / post-F. (A) Binding affinity curve of the antibody to RSV A2 Pre-F; (B) Binding affinity curve of the antibody to RSV B 18537 Pre-F; (C) Binding affinity curve of the antibody to RSV A2 Post-F; (D) Binding affinity curve of the antibody to RSV B 18537 Post-F.

[0065] Figure 3 Figure 3: Neutralization curves of RSV antibodies against RSV A and RSV B strains. (A) Neutralization curve of the antibody against RSV A2 strain; (B) Neutralization curve of the antibody against RSV A Long strain; (C) Neutralization curve of the antibody against RSV BW / V 14617 / 85; (D) Neutralization curve of the antibody against RSV B 18537.

[0066] Figure 4 Figure 2: Binding affinity curves of RSFP13H11 and its V58S variant for RSV A / B pre-F / post-F. (A) Binding affinity curve for RSV A Pre-F; (B) Binding affinity curve for RSV A Post-F; (C) Binding affinity curve for RSV B Pre-F; (D) Binding affinity curve for RSV B Post-F.

[0067] Figure 5 : Binding affinity curves of RSFP13H11 and its 19 variants with mutations at position 58 to RSV A / B pre-F / post-F. (A) Binding affinity curve for RSV A Pre-F; (B) Binding affinity curve for RSV B Pre-F; (C) Binding affinity curve for RSV A Post-F; (D) Binding affinity curve for RSV B Post-F.

[0068] Figure 6 : Neutralization ability curve of RSFP13H11 and V58A, V58I, and V58L variants against RSV A2 strain. DETAILED DESCRIPTION

[0069] It should be noted that, unless otherwise defined, the technical or scientific terms used in this application should have the common meanings understood by those skilled in the art.

[0070] The experimental methods in the following examples are conventional methods unless otherwise specified. The medicinal materials, reagents, etc. used in the following examples are commercially available products unless otherwise specified.

[0071] As used herein and in the appended claims, the singular forms "a," "an," "another," and "the" include plural referents unless the context clearly dictates otherwise.

[0072] In this application, the term "comprising" generally means including the features specifically stated, but not excluding other elements.

[0073] In this application, the term "antibody" has the conventional meaning in the art and refers to an immunoglobulin molecule composed of four polypeptide chains, which refer to two heavy (H) chains and two light (L) chains connected to each other by disulfide bonds. By analyzing the amino acid sequences of different antibody heavy and light chains, it was found that the amino acid sequences near the N-terminus of the heavy and light chains vary greatly, while the amino acid sequences of other parts are relatively constant. Therefore, the region near the N-terminus of the antibody light and heavy chains where the amino acid sequence varies greatly is called the variable region (V), and the region near the C-terminus where the amino acid sequence is relatively stable is called the constant region (C). The V regions of the heavy and light chains are abbreviated as VH and VL, respectively, and the C regions of the heavy and light chains are abbreviated as CH and CL, respectively. There is a small number of amino acid residues in the variable region of an antibody that vary particularly strongly. The residue composition and arrangement order of these amino acids are more prone to variation, and are called hypervariable regions (HVRs). There are three hypervariable regions in each of the L chain and H chain V regions. Because this part can form precise complementarity with the antigenic determinant in terms of spatial structure, the hypervariable region is also called the complementarity determining region (CDR). In antibodies, common rules for dividing CDRs include Kabat, AbM, Chothia, Contact, and IMGT. These rules are well known to those skilled in the art. When using a website that implements these rules, as long as the VH and VL sequences are input and the corresponding rules are selected, CDR sequences based on different rules can be obtained. It should be understood by those skilled in the art that the scope of protection of this application covers combinations of CDR sequences obtained by analyzing using different rules. The six CDR regions of an antibody jointly determine the antibody's recognition ability and specificity for the corresponding antigen. It should be understood by those skilled in the art that when the amino acid sequences of the six CDR regions are defined in this application, the antibody's recognition ability and specificity for the corresponding antigen are predictable.

[0074] In this application, the term "conservative substitution" is also referred to as conservative mutation or conservative replacement, and refers to the replacement of an amino acid with a different amino acid having similar structural or biochemical properties. Such amino acid substitutions can generally occur based on similarities in the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic properties of the amino acid residues. Amino acids can be divided into the following categories based on their structure and properties, for example:

[0075] Non-polar amino acids (aliphatic hydrocarbon side chains): alanine, valine, leucine, isoleucine.

[0076] Non-polar amino acids (containing aromatic ring side chains): phenylalanine, tryptophan, tyrosine.

[0077] Non-polar amino acids (sulfur side chain): methionine.

[0078] Polar neutral amino acids: aspartic acid, cysteine, glutamine, serine, threonine.

[0079] Polar basic amino acids: arginine, histidine, lysine.

[0080] Polar acidic amino acids: aspartic acid, glutamic acid.

[0081] Special amino acids: glycine, proline.

[0082] Generally, conservative substitutions have no or no significant effect on the activity and function of a protein or polypeptide.

[0083] As used herein, the term "variant" or "antibody variant" refers to an antibody that contains one or more mutations compared to the original antibody. The antibody variants in the embodiments of the present invention are generated by conservative substitution of one amino acid in the HCDR region of the original antibody and have the same or substantially the same activity and function as the original antibody.

[0084] In this application, the term "variable region" refers to the domain of an antibody heavy or light chain that is involved in antibody binding to antigen. The variable regions of the heavy and light chains of natural antibodies (VH and VL, respectively) generally have a similar structure and can be further subdivided into hypervariable regions (called complementarity determining regions (CDRs)) interspersed with more conserved regions (called framework regions (FRs)).

[0085] In this application, the term "complementarity determining region" (CDR, e.g., CDR1, CDR2, and CDR3) refers to some amino acid residues in an antibody variable region that are essential for antigen binding. Each variable region typically has three CDR regions identified as CDR1, CDR2, and CDR3. Each complementary determining region may comprise amino acid residues from a "complementarity determining region" as defined by Kabat (Kabat et al., Sequences of Proteins of Immulological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. 1991) and / or from those residues of a "hypervariable loop" (Chothia and Lesk; J Mol Biol 196:901-917 (1987)).

[0086] In the present application, the term "antibody-antigen interaction interface" refers to the position of amino acids in the antibody variable region that affect the interaction between the antibody and the antigen.

[0087] In this application, the complementarity determining regions (CDRs) and framework regions (FRs) of a given antibody may be identified using the Kabat system (Kabat et al.: Sequences of Proteins of Immunological Interest, 5th ed., U.S. Department of Health and Human Services, PHS, NIH, NIH Publication No. 91-3242, 1991).

[0088] In this application, the term "fully humanized antibody" refers to the transfer of all human antibody-encoding genes to genetically engineered antibody gene-deficient animals through transgenic or transchromosomal techniques, so that the animals express human antibodies and achieve the purpose of fully humanized antibodies. The purpose of "fully humanized" is to eliminate the immunogenicity of non-human antibodies in the human body while retaining affinity to the greatest extent possible. It is advantageous to select a human framework sequence that is most similar to the non-human antibody framework sequence as a template for humanization. In some cases, it may be necessary to replace one or more amino acids in the human framework sequence with corresponding residues in the non-human framework to avoid loss of affinity.

[0089] In the present application, " monoclonal antibody " refers to the antibody obtained from a substantially homogeneous antibody colony, that is, the colony comprising a single antibody is identical except that a possible mutation (such as natural mutation) may exist in a small amount. Therefore, the term " monoclonal antibody " shows the character of the antibody, i.e., is not a mixture of unrelated antibodies. Contrary to the polyclonal antibody preparations that generally include different antibodies for different determinants (epi-topes), each monoclonal antibody of the monoclonal antibody preparation is directed to a single determinant on the antigen. Except for its specificity, the advantage of the monoclonal antibody preparation is that they are not contaminated by other antibodies conventionally. The term " monoclonal antibody " should not be construed as needing to produce the antibody by any specific method. The term monoclonal antibody specifically includes chimeric antibodies, humanized antibodies and human antibodies.

[0090] In this application, the terms "pseudovirus" and "viroid" have the same meaning and can be used interchangeably. They refer to a virus-like particle formed by self-assembly of viral proteins, which does not encapsulate nucleic acid or encapsulates other nucleic acids. Thus, although the pseudovirus or viroid can infect host cells, it does not have the ability to replicate autonomously. Therefore, compared with real viruses, it has high biosafety. The packaging system of pseudovirus generally consists of two parts, namely a packaging component and an expression component. The packaging component is constructed by removing the genetic information required for packaging, reverse transcription and integration from the viral (e.g., HIV-1) genome, and provides the proteins necessary for pseudovirus particles; the expression component is complementary to the packaging component, contains the genetic information required for packaging, reverse transcription and integration, and also contains an exogenous target gene. The packaging component and the vector component are co-transfected into host cells to harvest pseudovirus particles in the cell supernatant.

[0091] In this application, the term "neutralizing antibody" refers to an antibody with neutralizing activity. The term "neutralizing activity" refers to an immunoglobulin with antiviral activity that can specifically recognize viral antigens and effectively bind to and neutralize viral activity, preventing the virus from invading target cells and blocking viral replication in target cells, thereby playing an important antiviral role.

[0092] In this application, the binding affinity of an antibody to an antigen can be determined by methods commonly used in the art, such as ELISA, usually using EC 50 and IC 50 Indicates. EC 50 IC refers to the half-maximal effect concentration, which is the concentration of the antibody that can achieve 50% of the maximum biological effect; 50 EC refers to the half-maximal inhibitory concentration, which is the concentration of the antibody required to inhibit a specific biological process (such as ACE2 binding to RBD protein) by half. 50 and IC 50 The smaller the value, the stronger the binding ability of the antibody to the antigen.

[0093] In this application, the term "vector" generally refers to a nucleic acid delivery vehicle into which a polynucleotide encoding a protein can be inserted and the protein can be expressed. A vector can transform, transduce, or transfect host cells, allowing the genetic material elements it carries to be expressed in host cells. For example, vectors include: plasmids; phagemids; cosmids; artificial chromosomes such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs); bacteriophages such as lambda phage or M13 phage, and animal viruses. A vector may contain a variety of elements that control expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, a vector may also contain a replication initiation site. A vector may also include components that assist in its entry into cells, such as viral particles, liposomes, or protein coats, but is not limited to these substances.

[0094] In this application, the term "pharmaceutical composition" generally refers to a pharmaceutical composition suitable for administration to a patient, which may include the antibodies, nucleic acid molecules, vectors or cells described in this application, and may also include one or more pharmaceutically acceptable excipients, such as: one or more of a carrier, a preservative, a stabilizer, an excipient, a diluent, a solubilizer, a surfactant, an emulsifier, and a preservative.

[0095] As described in the background technology section, the technical problem to be solved by the present invention is to provide an antibody with highly effective neutralizing activity against respiratory syncytial virus. To solve this technical problem, the present invention screened and obtained five fully humanized anti-RSV monoclonal antibodies, RSFP12E6, RSFP3G5, RSFP7F6, RSFP13H7, and RSFP13H11, through phage display technology.

[0096] The fusion protein F protein and adhesion protein G protein of respiratory syncytial virus are the key to the virus invading the human body. Because the G protein is extremely easy to mutate, the F protein is relatively conservative, so the F protein has become a good target for scientists to develop neutralizing antibodies against respiratory syncytial virus. The F glycoprotein exists in two forms, the pre-fusion conformation (Pre-F) and the post-fusion conformation (post-F). Studies have shown that only some specific epitopes acting on the Pre-F conformation can more effectively neutralize the respiratory syncytial virus, so the Pre-F glycoprotein has become the preferred antigen for the respiratory syncytial virus vaccine. The antibodies RSFP12E6, RSFP3G5, RSFP7F6, RSFP13H7 and RSFP13H11 obtained by screening in the present invention are all neutralizing antibodies with strong binding ability to the Pre-F glycoprotein.

[0097] In addition, the antibodies RSFP12E6, RSFP3G5, RSFP7F6, RSFP13H7 and RSFP13H11 screened by the present invention are fully humanized antibodies. Humanized antibodies can eliminate the immunogenicity of non-human antibodies in the human body while retaining affinity to the greatest extent possible.

[0098] The embodiments of the present invention demonstrate that:

[0099] The five antibodies of the present invention, RSFP12E6, RSFP3G5, RSFP7F6, RSFP13H7, and RSFP13H11, have good broad-spectrum binding to RSV A / B strain F protein. RSFP12E6, RSFP3G5, and RSFP7F6 tend to bind to the prefusion conformation of RSV A / B strain F protein; RSFP13H7 and RSFP13H11 have good binding to both the prefusion and postfusion conformations of RSV A / B strain F protein.

[0100] IC values ​​of RSFP12E6, RSFP3G5, RSFP7F6, RSFP13H7, and RSFP13H11 antibodies neutralizing RSV A2 strain virus 50 They were 2.960ng / mL, 2.291ng / mL, 2.319ng / mL, 2.277ng / mL and 1.803ng / mL respectively.

[0101] IC values ​​of RSFP12E6, RSFP3G5, RSFP7F6, RSFP13H7, and RSFP13H11 antibodies neutralizing RSV ALong strain virus 50 They were 9.364ng / mL, 5.059ng / mL, 9.470ng / mL, 9.563ng / mL and 7.038ng / mL respectively.

[0102] IC values ​​of RSFP12E6, RSFP3G5, RSFP7F6, RSFP13H7, and RSFP13H11 antibodies neutralizing RSV B WV / 14617 / 85 virus 50 They were 5.784ng / mL, 5.459ng / mL, 10.03ng / mL, 13.88ng / mL and 15.19ng / mL respectively.

[0103] IC values ​​of RSFP12E6, RSFP3G5, RSFP7F6, RSFP13H7, and RSFP13H11 antibodies neutralizing RSV B 18537 strain virus 50 They were 16.86ng / mL, 15.07ng / mL, 17.06ng / mL, 23.55ng / mL and 15.31ng / mL respectively.

[0104] It can be seen that these five antibodies have unique CDR regions, can specifically bind to Pre-F glycoprotein and can effectively neutralize respiratory syncytial virus. They have high neutralizing activity against the A strain of respiratory syncytial virus, and their IC 50 The concentrations of 1ng / mL to 10ng / mL are all within the range of 1ng / mL to 10ng / mL. They have high neutralizing activity against the B strain of respiratory syncytial virus. The IC 50 Both are between 5ng / mL and 30ng / mL.

[0105] In summary, the antibodies RSFP12E6, RSFP3G5, RSFP7F6, RSFP13H7, and RSFP13H11 screened by the present invention are fully humanized monoclonal antibodies with highly effective neutralizing activity.

[0106] 1. RSFP12E6 Antibody

[0107] The RSFP12E6 antibody comprises a heavy chain variable region and a light chain variable region.

[0108] 1.1 Heavy Chain Variable Region of RSFP12E6 Antibody

[0109] The heavy chain variable region of the RSFP12E6 antibody contains three complementarity determining regions (HCDR1, HCDR2, and HCDR3) and four framework regions (HFR1, HFR2, HFR3, and HFR4). The amino acids are as follows:

[0110] HFR1:EVQLVQSGAEAKKPGDSVKVSCKAS(SEQ ID NO:31)

[0111] HCDR1: GYTFTSND (SEQ ID NO: 1)

[0112] HFR2:VNWVRQAPGQGLEWMGW(SEQ ID NO:32)

[0113] HCDR2: MNGNNGNT (SEQ ID NO: 2)

[0114] HFR3:IYEQKFQGRVTMTRDTSSTAYMDLSSLRSDDTAIYYC(SEQ ID NO:33)

[0115] HCDR3:ARGSWDHILAGYFFDN(SEQ ID NO:3)

[0116] HFR4:WGQGTLVTVSS(SEQ ID NO:34)

[0117] The amino acid sequence of the heavy chain variable region is:

[0118] EVQLVQSGAEAKKPGDSVKVSCKASGYTFTSNDVNWVRQAPG QGLEWMGWMNGNNGNTIYEQKFQGRVTMTRDTSSTAYMDLSSL RSDDTAIYYCARGSWDHILAGYFFDNWGQGTLVTVSS(SEQ ID NO:71)

[0119] The nucleotide sequence encoding the heavy chain variable region (Nucleotide Sequence) is:

[0120] gaagtgcagctggtgcagtctggggctgaggcgaagaagcctggggactcagtgaaggtctcctgcaaggcttctggatacaccttcaccagtaatgatgtcaactgggtgcgacaggcgcccggacaagggcttgagtggatgggatggatgaacggcaacaacggaaatacaatctatgaacaga aattccagggcagagtgaccatgaccagggacacgtctacaagcacagcctacatggacttgagcagcctgagatctgacgacacggccatatattactgtgcgagaggctcctgggaccacatcttggctggatatttctttgacaactggggccaggggaccctggtcaccgtctcctcag(SEQ ID NO:81)

[0121] 1.2 RSFP12E6 antibody light chain variable region (Light Chain Variable Region)

[0122] The light chain variable region of the RSFP12E6 antibody contains three complementarity determining regions LCDR1, LCDR2, and LCDR3; and four framework regions LFR1, LFR2, LFR3, and LFR4, with the amino acids shown below:

[0123] LFR1:LPVLTQPPSVSVVAPGKTARITCGGN(SEQ ID NO:35)

[0124] LCDR1:NIGSKS (SEQ ID NO:4)

[0125] LFR2: VHWYQQKPGQAPVLVVY (SEQ ID NO:36)

[0126] LCDR2: DDS (SEQ ID NO:5)

[0127] LFR3: DRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYC (SEQ ID NO:37)

[0128] LCDR3: QVWDSSSDHVV (SEQ ID NO:6)

[0129] LFR4: FGGGTKLTVL (SEQ ID NO:38)

[0130] The amino acid sequence of the light chain variable region is:

[0131] LPVLTQPPSVSVAPGKTARITCGGNNIGSKSVHWYQQKPGQAP VLVVYDDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVW DSSSDHVVFGGGTKLTVL (SEQ ID NO:72)

[0132] The nucleotide sequence encoding the light chain variable region is:

[0133] ctgcctgtgctgactcagccaccctcggtgtcagtggccccaggaaagacggccaggattacctgtgggggaaacaacattggaagtaaaagtgtgcactggtaccagcagaagccaggccaggcccctgtgctggtcgtctatgatgatagcgaccggccctcagggatccctgagcgattctctggctccaactctgggaacacggccaccctgaccatcagcagggtcgaagccggggatgaggccgactattactgtcaggtgtgggatagtagtagtgatcatgtggtattcggcggagggaccaagctgaccgtcctag (SEQ ID NO:82)

[0134] 2. RSFP3G5 Antibody

[0135] The RSFP3G5 antibody comprises a heavy chain variable region and a light chain variable region.

[0136] 2.1 Heavy Chain Variable Region of RSFP3G5 Antibody

[0137] The heavy chain variable region of the RSFP3G5 antibody contains three complementarity determining regions (HCDR1, HCDR2, and HCDR3) and four framework regions (HFR1, HFR2, HFR3, and HFR4), as shown below:

[0138] HFR1:QVQLVQSGAEAKKPGDSVKVSCKAS(SEQ ID NO:39)

[0139] HCDR1: GYTFTSND (SEQ ID NO: 7)

[0140] HFR2:VNWVRQAPGQGLEWMGW(SEQ ID NO:40)

[0141] HCDR2: MNGNNGNT (SEQ ID NO: 8)

[0142] HFR3:IYEQKFQGRVTMTRDTSSTAYMDLSSLRSDDTAIYYC(SEQ ID NO:41)

[0143] HCDR3:ARGSWDHILAGYFLDN(SEQ ID NO:9)

[0144] HFR4:WGQGTLVTVSS(SEQ ID NO:42)

[0145] The amino acid sequence of the heavy chain variable region is:

[0146] QVQLVQSGAEAKKPGDSVKVSCKASGYTFTSNDVNWVRQAPG QGLEWMGWMNGNNGNTIYEQKFQGRVTMTRDTSSTAYMDLSSL RSDDTAIYYCARGSWDHILAGYFLDNWGQGTLVTVSS(SEQ ID NO:73)

[0147] The nucleotide sequence encoding the heavy chain variable region (Nucleotide Sequence) is:

[0148] caagtgcagctggtgcagtctggggctgaggcgaagaagcctggggactcagtgaaggtctcctgcaaggcttctggatacaccttcaccagtaatgatgtcaactgggtgcgacaggcgcccggacaagggcttgagtggatgggatggatgaacggcaacaacggaaatacaatctatgaacaga aattccagggcagagtgaccatgaccagggacacgtctacaagcacagcctacatggacttgagcagcctgagatctgacgacacggccatatattactgtgcgagaggctcctgggaccacatcttggctggatatttccttgacaactggggccagggcaccctggtcaccgtctcctcag(SEQ ID NO:83)

[0149] 2.2 RSFP3G5 Antibody Light Chain Variable Region (Light Chain Variable Region)

[0150] The light chain variable region of the RSFP3G5 antibody contains three complementarity determining regions LCDR1, LCDR2, and LCDR3; and four framework regions LFR1, LFR2, LFR3, and LFR4, with the amino acids shown below:

[0151] LFR1:QPVLTQPPSVSVVAPGKTARITCGGN(SEQ ID NO:43)

[0152] LCDR1:NIGSKS (SEQ ID NO: 10)

[0153] LFR2:VHWYQQKPGQAPVLVVY(SEQ ID NO:44)

[0154] LCDR2:DDS (SEQ ID NO: 11)

[0155] LFR3:DRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYC(SEQ ID NO:45)

[0156] LCDR3:QVWDSSSDHVV(SEQ ID NO:12)

[0157] LFR4:FGGGTELTVL (SEQ ID NO:46)

[0158] The amino acid sequence of the light chain variable region is:

[0159] QPVLTQPPSVSVVAPGKTARITCGGNNIGSKSVHWYQQKPGQAP VLVVYDDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVW DSSSDHVVFGGGTELTVL(SEQ ID NO:74)

[0160] The nucleotide sequence encoding the light chain variable region (Light Chain Variable Region) is:

[0161] cagcctgtgctgactcagccgccctcggtgtcagtggccccaggaaagacggccaggattacctgtgggggaaacaacattggaagtaaaagtgtgcactggtaccagcagaagccaggccaggcccctgtgctggtcgtctatgatgatagcgaccggccctc agggatccctgagcgattctctggctccaactctgggaacacggccaccctgaccatcagcagggtcgaagccggggatgaggccgactattactgtcaggtgtgggatagtagtagtgatcatgtggtattcggcggagggaccgagctgaccgtcctag(SEQ ID NO:84)

[0162] 3. RSFP7F6 Antibody

[0163] The RSFP7F6 antibody comprises a heavy chain variable region and a light chain variable region.

[0164] 3.1 Heavy Chain Variable Region of RSFP7F6 Antibody

[0165] The heavy chain variable region of the RSFP7F6 antibody contains three complementarity determining regions (HCDR1, HCDR2, and HCDR3) and four framework regions (HFR1, HFR2, HFR3, and HFR4). The amino acids are as follows:

[0166] HFR1:QVQLVQSGAEAKKPGDSVKVSCKAS(SEQ ID NO:47)

[0167] HCDR1: GYTFTSND (SEQ ID NO: 13)

[0168] HFR2:VNWVRQAPGQGLEWMGW(SEQ ID NO:48)

[0169] HCDR2: MNGNNGNT (SEQ ID NO: 14)

[0170] HFR3:IYEQKFQGRVTMTRDMSSTAYMDLSSLRSDDTAIYYC(SEQ ID NO:49)

[0171] HCDR3:ARGSWDHILAGYFFDN(SEQ ID NO:15)

[0172] HFR4:WGQGTTVTVSS(SEQ ID NO:50)

[0173] The amino acid sequence of the heavy chain variable region is:

[0174] QVQLVQSGAEAKKPGDSVKVSCKASGYTFTSNDVNWVRQAPG QGLEWMGWMNGNNGNTIYEQKFQGRVTMTRDMSTSTAYMDLSSL RSDDTAIYYCARGSWDHILAGYFFDNWGQGTTVTVSS(SEQ ID NO:75)

[0175] The nucleotide sequence encoding the heavy chain variable region (Nucleotide Sequence) is:

[0176] caggtccagctggtgcaatctggagctgaggcgaagaagcctggggactcagtgaaggtctcctgcaaggcttctggatacaccttcaccagtaatgatgtcaactgggtgcgacaggcgcccggacaagggcttgagtggatgggatggatgaacggcaacaacggaaatacaatctatgaacaga aattccagggcagagtgaccatgaccagggacatgtctacaagcacagcctacatggacttgagcagcctgagatctgacgacacggccatatattactgtgcgagaggctcctgggaccacatcttggctggatatttctttgacaactggggccaggggaccacggtcaccgtctcctcag(SEQ ID NO:85)

[0177] 3.2 RSFP7F6 Antibody Light Chain Variable Region (Light Chain Variable Region)

[0178] The light chain variable region of the RSFP7F6 antibody contains three complementarity determining regions LCDR1, LCDR2, and LCDR3; and four framework regions LFR1, LFR2, LFR3, and LFR4, as shown below:

[0179] LFR1:LPVLTQPPSVSVVAPGQTARITCGGN(SEQ ID NO:51)

[0180] LCDR1:NIGSKS (SEQ ID NO: 16)

[0181] LFR2:VHWYQQKPGQAPVLVVY(SEQ ID NO:52)

[0182] LCDR2:DDS (SEQ ID NO: 17)

[0183] LFR3:DRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYC(SEQ ID NO:53)

[0184] LCDR3:QVWDSSSDHVV(SEQ ID NO:18)

[0185] LFR4:FGGGTELTVL (SEQ ID NO:54)

[0186] The amino acid sequence of the light chain variable region is:

[0187] LPVLTQPPSVSVAPGQTARITCGGNNIGSKSVHWYQQKPGQAP VLVVYDDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVW DSSSDHVVFGGGTELTVL(SEQ ID NO:76)

[0188] The nucleotide sequence encoding the light chain variable region (Light Chain Variable Region) is:

[0189] ctgcctgtgctgactcagcccccctcggtgtcagtggccccaggacagacggccaggattacctgtgggggaaacaacattggaagtaaaagtgtgcactggtaccagcagaagccaggccaggcccctgtgctggtcgtctatgatgatagcgaccggccctc agggatccctgagcgattctctggctccaactctgggaacacggccaccctgaccatcagcagggtcgaagccggggatgaggccgactattactgtcaggtgtgggatagtagtagtgatcatgtggtattcggcggagggacggagctgaccgtcctag(SEQ ID NO:86)

[0190] 4. RSFP13H7 Antibody

[0191] The RSFP13H7 antibody comprises a heavy chain variable region and a light chain variable region.

[0192] 4.1 Heavy Chain Variable Region of RSFP13H7 Antibody

[0193] The heavy chain variable region of the RSFP13H7 antibody contains three complementarity determining regions (HCDR1, HCDR2, and HCDR3) and four framework regions (HFR1, HFR2, HFR3, and HFR4). The amino acid sequences are shown below:

[0194] HFR1:EVHLVESGGGLVQPGGSLRLSCAAT(SEQ ID NO:55)

[0195] HCDR1: GFTFSNYE (SEQ ID NO: 19)

[0196] HFR2:MNWVRQAPGKGLEWISY(SEQ ID NO:56)

[0197] HCDR2:ISVSGATI (SEQ ID NO: 20)

[0198] HFR3:HYADSVKGRFTISRDNAKSSVYLQMNSLRPEDTAIYYC(SEQ ID NO:57)

[0199] HCDR3:ARDNSLTDYGLGLY(SEQ ID NO:21)

[0200] HFR4:WGQGTLVTVSS(SEQ ID NO:58)

[0201] The amino acid sequence of the heavy chain variable region is:

[0202] EVHLVESGGGLVQPGGSLRLSCAATGFTFSNYEMNWVRQAPG KGLEWISYISVSGATIHYADSVKGRFTISRDNAKSSVYLQMNSLRPE DTAIYYCARDNSLTDYGLGLYWGQGTLVTVSS(SEQ ID NO:77)

[0203] The nucleotide sequence encoding the heavy chain variable region (Nucleotide Sequence) is:

[0204] gaggtgcatctggtggagtctgggggtggcttggtccagcctggagggtccctgagactctcctgtgcagccactggattcaccttcagtaattacgaaatgaattgggtccgccaggctccagggaagggcctggagtggatctcgtatatcagtgtaagtggcgcaaccatacactacgcag actctgtaaagggccgattcaccatctccagagacaacgccaagagctcagtgtatctgcaaatgaacagcctgagacccgaggacacggctatttattactgtgcgagagataattctctaaccgactatggattgggcctgtactggggccaggggaccctggtcaccgtctcctcag(SEQ IDNO:87)

[0205] 4.2 RSFP13H7 Antibody Light Chain Variable Region (Light Chain Variable Region)

[0206] The light chain variable region of the RSFP13H7 antibody contains three complementarity determining regions (LCDR1, LCDR2, and LCDR3) and four framework regions (LFR1, LFR2, LFR3, and LFR4). The amino acid sequences are shown below:

[0207] LFR1:NIQMTQSPSSSLSASVGDRVTITCRAS(SEQ ID NO:59)

[0208] LCDR1:QSISSY (SEQ ID NO: 22)

[0209] LFR2:LNWYQQKPGKAPKLLIY(SEQ ID NO:60)

[0210] LCDR2:AAS (SEQ ID NO:23)

[0211] LFR3:SLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC(SEQ ID NO:61)

[0212] LCDR3:QQSYSTPIT (SEQ ID NO: 24)

[0213] LFR4:FGQGTRLEIK (SEQ ID NO:62)

[0214] The amino acid sequence of the light chain variable region is:

[0215] NIQMTQSPSSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAP KLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYS TPITFGQGTRLEIK(SEQ ID NO:78)

[0216] The nucleotide sequence encoding the light chain variable region (Light Chain Variable Region) is:

[0217] aacatccagatgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccatcacttgccgggcaagtcagagcattagcagctatttaaattggtatcagcagaaaccagggaaagcccctaagctcctgatctatgctgcatccagtttgc aaagtggggtcccatcaaggttcagtggcagtggatctgggacagatttcactctcaccatcagcagtctgcaacctgaagattttgcaacttactactgtcaacagagttacagtacccctatcaccttcggccaagggacacgactggagattaaac(SEQ ID NO:88)

[0218] 5. RSFP13H11 Antibody

[0219] The RSFP13H11 antibody comprises a heavy chain variable region and a light chain variable region.

[0220] 5.1 Heavy Chain Variable Region of RSFP13H11 Antibody

[0221] The heavy chain variable region of the RSFP13H11 antibody contains three complementarity determining regions (HCDR1, HCDR2, and HCDR3) and four framework regions (HFR1, HFR2, HFR3, and HFR4). The amino acid sequences are shown below:

[0222] HFR1:EVQLVQSGGGLVQPGGSLRLSCAAT(SEQ ID NO:63)

[0223] HCDR1: GFTFSNYE (SEQ ID NO: 25)

[0224] HFR2:MNWVRQAPGKGLEWISY(SEQ ID NO:64)

[0225] HCDR2:ISVSGATI (SEQ ID NO: 26)

[0226] HFR3:HYADSVKGRFTISRDNAKSSVYLQMNSLRPEDTAIYYC(SEQ ID NO:65)

[0227] HCDR3:ARDNSLTDYGLGLY(SEQ ID NO:27)

[0228] HFR4:WGQGTMVTVSS(SEQ ID NO:66)

[0229] The amino acid sequence of the heavy chain variable region is:

[0230] EVQLVQSGGGLVQPGGSLRLSCAATGFTFSNYEMNWVRQAPG KGLEWISYISVSGATIHYADSVKGRFTISRDNAKSSVYLQMNSLRPE DTAIYYCARDNSLTDYGLGLYWGQGTMVTVSS(SEQ ID NO:79)

[0231] The nucleotide sequence encoding the heavy chain variable region (Nucleotide Sequence) is:

[0232] gaggtgcagctggtgcagtctgggggtggcttggtccagcctggagggtccctgagactctcctgtgcagccactggattcaccttcagtaattacgaaatgaattgggtccgccaggctccagggaagggcctggagtggatctcgtatatcagtgtaagtggcgcaaccatacactacgcag actctgtaaagggccgattcaccatctccagagacaacgccaagagctcagtgtatctgcaaatgaacagcctgagacccgaggacacggctatttattactgtgcgagagataattctctaaccgactatggattgggcctgtactggggccaggggacaatggtcaccgtctcctcag(SEQ IDNO:89)

[0233] 5.2 Light Chain Variable Region of RSFP13H11 Antibody

[0234] The light chain variable region of the RSFP13H11 antibody contains three complementarity determining regions (LCDR1, LCDR2, and LCDR3) and four framework regions (LFR1, LFR2, LFR3, and LFR4). The amino acid sequences are shown below:

[0235] LFR1:DIQMTQSPSSSLSASVGDRVTITCRAS(SEQ ID NO:67)

[0236] LCDR1:QSISSY (SEQ ID NO: 28)

[0237] LFR2:LNWYQQKPGKAPKLLIY(SEQ ID NO:68)

[0238] LCDR2:AAS (SEQ ID NO:29)

[0239] LFR3:SLQSGVPSRFRGSGSGTDFTLTISSLQPEDFATYYC(SEQ ID NO:69)

[0240] LCDR3:QQSYSTPLT (SEQ ID NO:30)

[0241] LFR4:FGGGTKLEIK (SEQ ID NO:70)

[0242] The amino acid sequence of the light chain variable region is:

[0243] DIQMTQSPSSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAP KLLIYAASSLQSGVPSRFRGSGSGTDFTLTISSLQPEDFATYYCQQSYS TPLTFGGGTKLEIK(SEQ ID NO:80)

[0244] The nucleotide sequence encoding the light chain variable region (Light Chain Variable Region) is:

[0245] gacatccagatgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccatcacttgccgggcaagtcagagcattagcagctatttaaattggtatcagcagaaaccagggaaagcccctaagctcctgatctatgctgcatccagtttgc aaagtggggtcccatcaaggttcagaggcagtggatctgggacagatttcactctcaccatcagcagtctgcaacctgaagattttgcaacttactactgtcaacagagttacagtacccctctcactttcggcggagggaccaagctggagatcaaag(SEQ ID NO:90)

[0246] The present invention also relates to a nucleic acid molecule comprising a nucleotide sequence encoding an antibody of the present invention. The nucleotide sequence encoding the antibody comprises:

[0247] 1) a nucleotide sequence encoding the heavy chain variable region, which is shown in SEQ ID NO: 81; and a nucleotide sequence encoding the light chain variable region, which is shown in SEQ ID NO: 82;

[0248] or

[0249] 2) a nucleotide sequence encoding the heavy chain variable region, which is shown in SEQ ID NO: 83; and a nucleotide sequence encoding the light chain variable region, which is shown in SEQ ID NO: 84;

[0250] or

[0251] 3) a nucleotide sequence encoding the heavy chain variable region, which is shown in SEQ ID NO: 85; and a nucleotide sequence encoding the light chain variable region, which is shown in SEQ ID NO: 86;

[0252] or

[0253] 4) a nucleotide sequence encoding the heavy chain variable region, which is shown in SEQ ID NO: 87; and a nucleotide sequence encoding the light chain variable region, which is shown in SEQ ID NO: 88;

[0254] or

[0255] 5) a nucleotide sequence encoding the heavy chain variable region, which is shown in SEQ ID NO: 89; and a nucleotide sequence encoding the light chain variable region, which is shown in SEQ ID NO: 90.

[0256] The present invention also provides a vector comprising one or more nucleic acid molecules of the present invention, such as a plasmid, phagemid, phage or viral vector, into which a nucleic acid molecule encoding an antibody of the present invention is inserted.

[0257] The antibodies provided herein can be prepared by recombinantly expressing nucleotide sequences encoding light and heavy chains or portions thereof in cells (e.g., host cells). In order to express antibodies by recombinant methods, host cells can be transfected with one or more recombinant expression vectors carrying nucleotide sequences encoding light and / or heavy chains or portions thereof so that the light and heavy chains are expressed in the host cells. Standard recombinant DNA methodologies are used to prepare and / or obtain nucleic acids encoding heavy and light chains, incorporate these nucleic acids into recombinant expression vectors, and introduce the vectors into host cells, such as Sambrook, Fritsch and Maniatis (eds.), Molecular Cloning; A Laboratory Manual, Second Edition, Cold Spring Harbor, NY, (1989), Ausubel, FM et al. (eds.) Current Protocols in Molecular Biology, Greene Publishing Associates, (1989) and Boss et al. U.S. Patent No. 4,816,397.

[0258] In addition, the nucleotide sequence encoding the variable region of the heavy chain and / or light chain can be converted into a nucleotide sequence encoding, for example, a full-length antibody chain, a Fab fragment or a ScFv: for example, a DNA fragment encoding a light chain variable region or a heavy chain variable region can be operably linked (so that the amino acid sequences encoded by the two DNA fragments are in frame) to another DNA fragment encoding, for example, an antibody constant region or a flexible linker. The sequences of human heavy and light chain constant regions are known in the art (see, for example, Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242), and DNA fragments comprising these regions can be obtained by standard PCR amplification.

[0259] Therefore, an embodiment of the present invention is also a host cell comprising the vector or nucleic acid molecule, wherein the host cell can be a higher eukaryotic host cell such as a mammalian cell, a lower eukaryotic host cell such as a yeast cell, and can be a prokaryotic cell such as a bacterial cell.

[0260] The antibodies of the invention and at least one other agent (eg, a stabilizing compound) can be formulated into a pharmaceutical composition comprising the antibodies of the invention and one or more pharmaceutically acceptable carriers, diluents, or excipients.

[0261] The antibodies provided by the present invention can be used to prepare drugs for the prevention and / or treatment of respiratory syncytial virus (RSV). They are fully humanized, highly expressed, and stable, making them suitable for industrialization. Furthermore, the antibodies can be used to prepare RSV detection reagents for detecting viral antigens and discovering effective neutralizing antigenic epitopes.

[0262] The technical solution provided by the present invention is further described below in conjunction with specific embodiments. The following embodiments are only used to illustrate the present invention and do not limit the scope of protection of the present invention.

[0263] Example 1 Screening of fully human monoclonal antibodies against RSV

[0264] 1.1 Establishment of phage display antibody library

[0265] (1) Recovery of peripheral blood mononuclear cells (PBMC) from RSV-positive patients: After the frozen cells were removed, they were placed in a 37°C water bath. After thawing, they were transferred to a centrifuge tube containing 1640 culture medium and centrifuged at 300 g for 5 min. The supernatant was discarded and 500 μL of Trizol reagent was added. After thorough mixing, the tube was added to an Eppendorf tube and allowed to stand at room temperature for 5 min.

[0266] (2) Add 200 μL of chloroform to each tube, mix thoroughly for 15 seconds, let it stand at room temperature for 3 minutes, and centrifuge at 12,000 g for 15 minutes at 4°C.

[0267] (3) Take the upper aqueous phase and place it in a new Eppendorf tube. Add an equal volume of isopropanol, mix thoroughly, let it stand at room temperature for 10 minutes, centrifuge it at 12000g for 10 minutes at 4°C, and discard the supernatant.

[0268] (4) Add 1 mL of 75% ethanol, mix by inversion for 10 seconds, centrifuge at 12,000 g for 5 minutes at 4°C, discard the supernatant, and dry at room temperature.

[0269] (5) Add 20 μL of sterile, enzyme-free water to each Eppendorf tube to dissolve the precipitate.

[0270] (6) Determine RNA concentration.

[0271] (7) The extracted RNA was commissioned to Chengdu Renyu Biotechnology Co., Ltd. to construct a phage display antibody library.

[0272] 1.2 RSV neutralizing antibody screening

[0273] First round of screening

[0274] (1) Antigen coating: 1.5 μg each of the pre-fusion F glycoprotein (RSF-V52H7) and post-fusion F glycoprotein (RSF-V52H6) of RSV A2 strain, and the pre-fusion F glycoprotein (RSF-V52H8) and post-fusion F glycoprotein (RSF-V52H9) of RSV B (strain 18537) were mixed, diluted with antigen coating solution (aqueous solution containing 1.59 g / L Na2CO3 and 2.94 g / L NaHCO3) to a protein concentration of 10 μg / mL, added to a 96-well ELISA plate at 100 μL / well, and coated at 4°C overnight.

[0275] (2) Blocking: On the next day, discard the coating solution in the ELISA plate, pat dry on paper, wash once with PBS solution (phosphate buffered saline), then add 350 μL of 3% BSA (bovine serum albumin) / PBS blocking solution, and place at 37°C for 2 hours.

[0276] (3) Background subtraction: Take 200 μL of antibody library (titer 2×10 11 pfu / mL) + 800 μL 3% BSA / PBS blocking solution, mix well, and place at room temperature for 1 h.

[0277] (4) Binding: Discard the blocking solution, pat dry on paper, wash three times with PBS solution, add the background-subtracted antibody library to the antigen-coated ELISA plate, 100 μL / well, and incubate at 37°C with slow shaking at 100 rpm for 1 hour.

[0278] (5) Washing: Wash once with PBS solution, and then wash 10 times with 0.1% PBST solution (phosphate buffer containing 0.1% Tween-20), each time for 1 min.

[0279] (6) Elution: Add elution solution (glycine solution, pH 3.0) at 100 μL / well and incubate slowly at room temperature at 100 rpm for 10 min. Aspirate the eluate with a pipette tip and add 10 μL of neutralizing solution (Tris-HCl, pH 8.0). Mix well and measure the titer.

[0280] (7) Titer determination: The eluted antibody library was diluted 10-fold (10 -1 ~10 -8 ), take 10 -4 ~10 -8 The diluted antibodies were added to 200 μL of TG1 strain in the logarithmic growth phase, placed at 37°C for 30 min, then spread on 2YTAG medium (2YT solid medium containing 0.1% ampicillin (Amp), 0.1% glucose and 1.5% agar) plates, placed at 37°C overnight, and the number of colonies was counted the next day.

[0281] (8) The antibody library obtained by screening was added to 20 mL of TG1 bacterial solution with OD600 = 0.6-0.8 and placed at 37°C for 60 minutes.

[0282] (9) Add 4 μL of Amp and incubate at 37°C and 180 rpm for 60 min to make the TG1 concentration reach 1×10 9 pfu.

[0283] (10) Add 2×10 10 pfu M13KO7 helper phage in PBS and place at 37°C for 30 min.

[0284] (11) Add 30 mL of 2YT medium and 6 μL of Amp and incubate at 37°C and 180 rpm for 60 min.

[0285] (12) Centrifuge at 5000 rpm for 10 min at room temperature, discard the supernatant, collect the bacterial precipitate, add new 50 mL 2YTAK medium (2YT medium containing 100 μg / mL Amp and 50 μg / mL kanamycin (Kana)), reselect the precipitate, and culture in a 200 mL conical flask at 37°C and 220 rpm for 16 h.

[0286] Second / third round of screening

[0287] According to the screening scheme, other conditions remained unchanged, and the second round of screening (washing with 0.3% PBST solution 15 times in step (5)) and the third round of screening (washing with 0.5% PBST solution 20 times in step (5)) were performed, and the elution pressure was increased successively for screening and titration.

[0288] 1.3 Purification of phage antibody library

[0289] (1) After three rounds of screening, the culture was transferred to a 100 mL centrifuge tube after culturing for 16 h. The tube was centrifuged at 12,000 rpm for 20 min at 4°C, and the supernatant was transferred to a new centrifuge tube.

[0290] (2) Add 1 / 4 volume of phage precipitation solution (containing 20% ​​PEG / 2.5M NaCl solution) to the supernatant and incubate on ice for 4 hours.

[0291] (3) Centrifuge at 12000 rpm for 20 min at 4°C, discard the supernatant, and centrifuge briefly again to remove all the supernatant.

[0292] (4) The precipitate was resuspended in 1 mL of PBS solution, centrifuged at 12000 rpm at 4°C for 10 min, and the supernatant was transferred to a new centrifuge tube.

[0293] (5) Titer determination: The supernatant (antibody library) of (4) was diluted 10-fold (10 -1 ~10 -10 ), take 10 -6 ~10 -10 The diluted antibodies were added to 200 μL of TG1 strain in the logarithmic growth phase, placed at 37°C for 30 min, spread on 2YTAG medium plates, and cultured at 37°C overnight. The number of colonies was counted the next day.

[0294] 1.4 Preparation of monoclonal phage supernatant

[0295] (1) After three rounds of phage antibody library screening, a single colony was picked from the phage antibody library plate eluted and purified in the last round of screening and inoculated into a 96-well deep-well plate containing 300 μL 2YTAG medium and cultured at 37°C and 700 rpm until the logarithmic growth phase.

[0296] (2) Pipette 100 μL of bacterial solution from each well into a sterile 96-well plate and store at 4°C.

[0297] (3) Add M13KO7 helper phage diluted with 2YT medium (titer 2×10 10 pfu / mL (about 10 times the bacterial concentration) 100 μL was cultured at 37°C for 30 min, and then cultured at 37°C, 600 rpm for 30 min.

[0298] (4) Centrifuge the 96-well deep-well plate at 4000 rpm for 10 min, discard the supernatant, add 300 μL of 2YTAK medium to each well to resuspend the bacteria, and culture at 700 rpm and 37°C overnight.

[0299] 1.5 Monoclonal phage ELISA

[0300] (1) Coating target proteins: Four proteins, namely, the pre-fusion conformation and post-fusion conformation F glycoproteins of RSV A and RSV B strains, were coated in a 384-well ELISA plate, with 25 ng / 25 μL antigen coating solution per well, and the plate was incubated at 4°C overnight.

[0301] (2) Washing: Discard the coated supernatant, pat dry, and wash three times with 0.3% PBST solution, each time for 1 min.

[0302] (3) Blocking: Add 3% skim milk powder / PBS solution, 100 μL / well, and block at 37°C for 1 h.

[0303] (4) Washing: Discard the blocking solution, pat dry, and wash with 0.3% PBST solution 5 times, each time for 1 min.

[0304] (5) Phage supernatant binding: 50 μL of 1% skim milk powder / PBS solution and the monoclonal phage supernatant obtained in 1.4 were mixed in a ratio of 1:1, added to the target protein-coated ELISA plate, and incubated at 37°C with slow shaking at 100 rpm for 1 h.

[0305] (6) Washing: Discard the liquid, pat dry, and wash with 0.3% PBST solution 5 times, each time for 1 min.

[0306] (7) Secondary antibody binding: Anti-M13-HRP antibody was diluted 1:5000 in 1% skim milk / PBS solution, 100 μL / well, added to the ELISA plate, and incubated at 37°C with slow shaking at 100 rpm for 1 h.

[0307] (8) Washing: Discard the liquid, pat dry, and wash with 0.3% PBST solution 5 times, each time for 1 min.

[0308] (9) Add 50 μL of TMB colorimetric solution, incubate at 37°C for 10 min, add 25 μL of ELISA stop solution, and read the OD450 value in a microplate reader.

[0309] (10) The positive clone bacterial solution stored at 4°C in step 1.4(2) was commissioned to sequence multiple antibodies. Based on the ELISA OD450 values ​​and the non-repetitiveness of the amino acid sequences of the antibody BCR heavy chain and light chain variable regions CDR1, CDR2, and CDR3, five antibodies with strong binding ability to RSV F protein, namely RSFP12E6, RSFP3G5, RSFP7F6, RSFP13H7, and RSFP13H11, were finally screened.

[0310] Example 2 Expression and purification of antibodies

[0311] 2.1 Cloning of Antibody BCR Heavy and Light Chains

[0312] The BCR heavy chain and light chain variable region sequences of the correctly sequenced antibodies RSFP12E6, RSFP3G5, RSFP7F6, RSFP13H7 and RSFP13H11 were cloned into the heavy chain expression vector IgGvec-Hb and the light chain expression vector IgGvec-L, respectively, according to conventional molecular cloning methods.

[0313] 2.2 Antibody Expression and Purification

[0314] Adjust the concentration of Expi 293 cells to 3 × 10 6 / mL, the transfection reagent is prepared as follows: Prepare solution A: in 3mL of Gibco TM Add 50 μg of recombinant antibody plasmid (heavy chain plasmid: light chain plasmid = 1:2) to Opti-MEM. For solution B, add 180 μL of the transfection reagent Expi Fectamine to 3 mL of Opti-MEM and vortex thoroughly. Combine solutions A and B, mix thoroughly, and let stand at room temperature for 20 minutes. Add the mixture to 50 mL of Expi 293 cells and culture at 120 rpm in an 8% CO2, 37°C incubator for 5 days.

[0315] After 5 days of culture, the cell supernatant was collected and the antibody was purified using Protein A affinity magnetic beads. The antibody concentration was measured using a NanoDrop2000 microspectrophotometer according to the manufacturer's instructions.

[0316] Example 3 Determination of Antibody Binding Activity to RSV A / B F Proteins of Different Conformations

[0317] (1) Use antigen coating solution to dilute the pre-fusion conformation F glycoprotein (RSF-V52H7) and post-fusion conformation F glycoprotein (RSF-V52H6) of RSV A2 strain, and the pre-fusion conformation F glycoprotein (RSF-V52H8) and post-fusion conformation F glycoprotein (RSF-V52H9) of RSV B 18537 strain, respectively, and add them to 384-well ELISA plates at 25 ng / 25 μL / well, and place them at 4°C for coating overnight.

[0318] (2) The next day, the supernatant was discarded, 100 μL of 3% BSA / PBS blocking solution was added to each well, and the cells were incubated in a 37°C incubator for 2 h. The blocking solution was discarded, and the cells were washed once with PBST solution.

[0319] (3) Dilute the five test antibodies to 10 μg / mL using PBS solution containing 0.3% BSA. Serially dilute the diluted antibodies three-fold using PBS solution. A total of eight dilutions were added to the blocked ELISA plate, 25 μL per well, and incubated in a 37°C incubator for 1 hour.

[0320] (4) Discard the supernatant, wash the plate 5 times with PBST solution, and pat dry on absorbent paper.

[0321] (5) Add 25 μL of anti-human IgG-Fc-HRP antibody (Sigma-Aldrich) diluted 1:10,000 in 0.3% BSA / PBS solution to each well and incubate in a 37°C incubator for 1 h.

[0322] (6) Discard the supernatant, wash three times with PBST solution, and pat dry on absorbent paper.

[0323] (7) Add 50 μL of TMB colorimetric solution to each well, let it stand at room temperature for 10 min, and then add 25 μL of ELISA stop solution (Solebol, C1058) to each well. Read the absorbance value (OD450) at a wavelength of 450 nm in a microplate reader (see Table 1 and Figure 2 shown).

[0324] (8) The obtained OD450 values ​​were fitted with a four-parameter logistic model in GraphPad Prism 10.0 software for nonlinear regression and the EC50 values ​​were calculated.

[0325] Table 1. EC values ​​of antibodies against different conformations of RSV FA / B subtype F protein 50

[0326]

[0327] The results indicate that the five antibodies of the present invention have good broad-spectrum binding to RSV A / B F protein. RSFP12E6, RSFP3G5, and RSFP7F6 tend to bind to the prefusion conformation of RSV A / B F protein, while RSFP13H7 and RSFP13H11 have good binding to both the prefusion and postfusion conformations of RSV A / B F protein.

[0328] Example 4 Determination of Neutralizing Activity of Antibodies against RSV Virus

[0329] 4.1 RSV A2 strain neutralization test

[0330] (1) One day in advance, 100 μL of Hep-2 cell culture medium (concentration of 2×10 5 / mL) and cultured in a 37°C CO2 cell culture incubator for 16 h.

[0331] (2) Add 50 μL of serially diluted test antibodies to each well of a 96-well plate, then add 50 μL of RSV A2 virus (0.1 MOl per well), mix thoroughly, and incubate at 37°C in a cell culture incubator for 1 h.

[0332] (3) Aspirate the culture medium in the 96-well cell culture plate, add 100 μL of antibody-virus mixture to each well, and incubate in a 37°C cell culture incubator for 2 h.

[0333] (4) The supernatant was discarded, the cells were washed with PBS, and 100 μL of Opti-MEM medium containing 2% fetal bovine serum (FBS) was added, and the cells were cultured in a 5% CO2, 37°C incubator for 72 h.

[0334] (5) Discard the cell supernatant, wash the cells with PBS solution, add 4% paraformaldehyde, and fix at room temperature for 30 minutes. After aspirating the supernatant, wash the cells with PBS solution, aspirate the supernatant, and read the data in a microplate fluorescence spot counter. Calculate the neutralization inhibition rate, and calculate the IC value of the antibody for the real virus based on the neutralization inhibition rate result. 50 .

[0335] 4.2 RSV A Long / RSV B 18537 / RSV BW / V 14617 / 85 virus neutralization assay

[0336] (1) One day in advance, 100 μL of Hep-2 cell culture medium (concentration of 2×10 5 / mL) and cultured in a 37°C CO2 cell culture incubator for 16 h.

[0337] (2) Add 50 μL of serially diluted test antibodies to each well of a 96-well plate, then add 50 μL of RSV A2 virus at an MOI of 0.1 per well, shake thoroughly to mix, and incubate at 37°C in a cell culture incubator for 1 h.

[0338] (3) Aspirate the culture medium in the 96-well cell culture plate, add 100 μL of antibody-virus mixture to each well, and incubate in a 37°C cell culture incubator for 2 h.

[0339] (4) Discard the supernatant, wash the cells with PBS solution, add 100 μL of Opti-MEM medium containing 2% FBS, and culture in a 5% CO2, 37°C incubator for 72 h.

[0340] (5) Discard the cell supernatant, wash the cells with PBS solution, add 4% paraformaldehyde, incubate at room temperature for 30 minutes, discard the supernatant, and wash the cells with PBS solution.

[0341] (6) After discarding the supernatant, add 50 μL of 0.1% Triton-100 to each well, permeabilize the membrane at 37°C for 20 minutes, and wash the cells with PBS solution.

[0342] (7) Add 300 μL of 5% BSA / PBS solution to each well, incubate at 37°C for 1 h, and wash the cells with PBS solution.

[0343] (8) Add 50 μL of RSV nucleoprotein antibody (1:1000) (Sino Biological 40821-T46) to each well, incubate at 37°C for 1 h, discard the supernatant, and wash the cells with PBS solution.

[0344] (9) Add 50 μL of Anti-Rabbit 488 (1:1000) to each well, incubate at 37°C for 1 h, discard the supernatant, wash the cells with PBS solution, and discard the supernatant.

[0345] (10) The microplate was placed in a fluorescent spot counter to read the data. The obtained data were analyzed by nonlinear regression analysis using a four-parameter logistic model in GraphPad Prism 10.0 software to calculate the IC values ​​of the antibody for different RSV strains. 50 (As shown in Table 2 and Figure 3 shown).

[0346] Table 2. IC values ​​of antibodies against RSV FA / B subtypes 50

[0347]

[0348]

[0349] According to the results, the five antibodies RSFP12E6, RSFP3G5, RSFP7F6, RSFP13H7, and RSFP13H11 have high neutralizing activity against respiratory syncytial virus. 50 The IC values ​​for respiratory syncytial virus B strains are all between 1 ng / mL and 10 ng / mL. 50 Both are between 5ng / mL and 30ng / mL.

[0350] Example 5 Effect of amino acid mutation at position V58 of the heavy chain on the activity of RSFP13H11 antibody

[0351] 5.1 Screening for RSFP13H11 Antibody Heavy Chain Germline Gene Reversion Mutations

[0352] (1) By comparing the RSFP13H11 antibody with its germline gene, it was found that position 58 in the germline gene is serine (S), which is different from RSFP13H11. Position 58 in the RSFP13H11 heavy chain variable region is as follows (i.e., amino acid 53 in the heavy chain variable region):

[0353] EVQLVQSGGGLVQPGGSLRLSCAATGFTFSNYEMNWVRQAPGKGLEWISYISVSGATIHYADSVKGRFTISRDNAKSSVYLQMNSLRPEDTAIYYCARDNSLTDYGLGLYWGQGTMVTVSS

[0354] (2) Overlap PCR was used to mutate the valine (V) codon at amino acid position 58 of the heavy chain of RSFP13H11 to serine (S) to prepare the V58S variant.

[0355] (3) Referring to the method in Example 2.2, the antibody variants were expressed and purified, and the antibody concentration was determined using a NanoDrop 2000 micro-spectrophotometer. The operation method was carried out according to the instructions provided by the manufacturer.

[0356] (4) Referring to the method in Example 3, the binding activity of RSFP13H11 and its variants to RSV A / B F proteins of different conformations was determined by ELISA. The results are as follows: Figure 4The results show that in the P13H11 antibody, the V58S mutation in the CDR2 region leads to a decrease in the binding efficiency of both RSV A pre-F and post-F proteins, and is completely unable to bind to the RSV B post-F protein, suggesting that position 58 in the CDR2 region may be located at the antibody-antigen interface, determining the interaction between the two.

[0357] 5.2 Construction of RSFP13H11 antibody heavy chain V58 amino acid mutation variants

[0358] (1) Overlap PCR was used to mutate the valine (V) codon at amino acid position 58 of the heavy chain of RSFP13H11 to 19 other amino acid codons, including glycine (G), alanine (A), and leucine (L).

[0359] (2) Referring to the method in Example 2.2, the antibody variants were expressed and purified, and the antibody concentration was determined using a NanoDrop 2000 micro-spectrophotometer. The operation method was carried out according to the instructions provided by the manufacturer.

[0360] 5.3 Determination of Binding Activity of RSFP13H11 and Its Variants to RSV A / B F Proteins with Different Conformations

[0361] Referring to the method of Example 3, the binding activity of RSFP13H11 and its variants to RSV A / B F proteins of different conformations was determined by ELISA. The results are as follows: Figure 5 The results show that the V58A, V58I, and V58L variants with conservative amino acid substitutions at position V58 (i.e., valine substituted with alanine, isoleucine, or leucine) have comparable F protein binding activity to RSFP13H11, while variants with non-conservative substitutions are unlikely to achieve this level.

[0362] 5.4 Neutralization activity of RSFP13H11 and its variants against RSV A virus

[0363] Referring to the method of Example 4.1, the neutralizing activity of RSFP13H11 and V58A, V58I, and V58L variants against RSV A virus was determined using RSV A2 strain. The results are as follows: Figure 6 The results show that the V58A, V58I, and V58L variants with conservative amino acid substitutions have highly effective neutralizing activity against RSV A virus, comparable to that of RSFP13H11.

Claims

1. An anti-respiratory syncytial virus antibody or a variant thereof, comprising a heavy chain variable region and a light chain variable region, wherein: The heavy chain variable region comprises HCDR1 as shown in SEQ ID NO: 1, HCDR2 as shown in SEQ ID NO: 2, and HCDR3 as shown in SEQ ID NO: 3; and The light chain variable region comprises LCDR1 with an amino acid sequence as shown in SEQ ID NO: 4, LCDR2 with an amino acid sequence as shown in SEQ ID NO: 5, and LCDR3 with an amino acid sequence as shown in SEQ ID NO: 6; or The heavy chain variable region comprises a HCDR1 with an amino acid sequence as shown in SEQ ID NO: 7, a HCDR2 with an amino acid sequence as shown in SEQ ID NO: 8, and a HCDR3 with an amino acid sequence as shown in SEQ ID NO: 9; and The light chain variable region comprises LCDR1 with an amino acid sequence as shown in SEQ ID NO: 10, LCDR2 with an amino acid sequence as shown in SEQ ID NO: 11, and LCDR3 with an amino acid sequence as shown in SEQ ID NO: 12; or The heavy chain variable region comprises a HCDR1 with an amino acid sequence as shown in SEQ ID NO: 13, a HCDR2 with an amino acid sequence as shown in SEQ ID NO: 14, and a HCDR3 with an amino acid sequence as shown in SEQ ID NO: 15; and The light chain variable region comprises LCDR1 with an amino acid sequence as shown in SEQ ID NO: 16, LCDR2 with an amino acid sequence as shown in SEQ ID NO: 17, and LCDR3 with an amino acid sequence as shown in SEQ ID NO: 18; or The heavy chain variable region comprises a HCDR1 with an amino acid sequence as shown in SEQ ID NO: 19, a HCDR2 with an amino acid sequence as shown in SEQ ID NO: 20, and a HCDR3 with an amino acid sequence as shown in SEQ ID NO: 21; and The light chain variable region comprises LCDR1 with an amino acid sequence as shown in SEQ ID NO: 22, LCDR2 with an amino acid sequence as shown in SEQ ID NO: 23, and LCDR3 with an amino acid sequence as shown in SEQ ID NO: 24; or The heavy chain variable region comprises a HCDR1 with an amino acid sequence as shown in SEQ ID NO: 25, a HCDR2 with an amino acid sequence as shown in SEQ ID NO: 26, and a HCDR3 with an amino acid sequence as shown in SEQ ID NO: 27; and The light chain variable region comprises LCDR1 as shown in the amino acid sequence of SEQ ID NO: 28, LCDR2 as shown in the amino acid sequence of SEQ ID NO: 29, and LCDR3 as shown in the amino acid sequence of SEQ ID NO: 30; and The variant undergoes conservative substitution of amino acids in the HCDR region compared to the original antibody; preferably, the conservatively substituted amino acids are located at the antibody-antigen interface; more preferably, the conservatively substituted amino acids are the amino acids at position 58 in SEQ ID NO:

79.

2. The antibody or variant thereof according to claim 1, wherein: The heavy chain variable region further comprises HFR1 with an amino acid sequence as shown in SEQ ID NO: 31, HFR2 with an amino acid sequence as shown in SEQ ID NO: 32, HFR3 with an amino acid sequence as shown in SEQ ID NO: 33, and HFR4 with an amino acid sequence as shown in SEQ ID NO: 34; and The light chain variable region further comprises LFR1 with an amino acid sequence as shown in SEQ ID NO: 35, LFR2 with an amino acid sequence as shown in SEQ ID NO: 36, LFR3 with an amino acid sequence as shown in SEQ ID NO: 37, and LFR4 with an amino acid sequence as shown in SEQ ID NO: 38; or The heavy chain variable region further comprises HFR1 with an amino acid sequence as shown in SEQ ID NO: 39, HFR2 with an amino acid sequence as shown in SEQ ID NO: 40, HFR3 with an amino acid sequence as shown in SEQ ID NO: 41, and HFR4 with an amino acid sequence as shown in SEQ ID NO: 42; and The light chain variable region further comprises LFR1 with an amino acid sequence as shown in SEQ ID NO:43, LFR2 with an amino acid sequence as shown in SEQ ID NO:44, LFR3 with an amino acid sequence as shown in SEQ ID NO:45, and LFR4 with an amino acid sequence as shown in SEQ ID NO:46; or The heavy chain variable region further comprises HFR1 with an amino acid sequence as shown in SEQ ID NO: 47, HFR2 with an amino acid sequence as shown in SEQ ID NO: 48, HFR3 with an amino acid sequence as shown in SEQ ID NO: 49, and HFR4 with an amino acid sequence as shown in SEQ ID NO: 50; and The light chain variable region further comprises LFR1 with an amino acid sequence as shown in SEQ ID NO: 51, LFR2 with an amino acid sequence as shown in SEQ ID NO: 52, LFR3 with an amino acid sequence as shown in SEQ ID NO: 53, and LFR4 with an amino acid sequence as shown in SEQ ID NO: 54; or The heavy chain variable region further comprises HFR1 with an amino acid sequence as shown in SEQ ID NO: 55, HFR2 with an amino acid sequence as shown in SEQ ID NO: 56, HFR3 with an amino acid sequence as shown in SEQ ID NO: 57, and HFR4 with an amino acid sequence as shown in SEQ ID NO: 58; and The light chain variable region further comprises LFR1 with an amino acid sequence as shown in SEQ ID NO: 59, LFR2 with an amino acid sequence as shown in SEQ ID NO: 60, LFR3 with an amino acid sequence as shown in SEQ ID NO: 61, and LFR4 with an amino acid sequence as shown in SEQ ID NO: 62; or The heavy chain variable region further comprises HFR1 with an amino acid sequence as shown in SEQ ID NO: 63, HFR2 with an amino acid sequence as shown in SEQ ID NO: 64, HFR3 with an amino acid sequence as shown in SEQ ID NO: 65, and HFR4 with an amino acid sequence as shown in SEQ ID NO: 66; and The light chain variable region further comprises LFR1 with an amino acid sequence as shown in SEQ ID NO:67, LFR2 with an amino acid sequence as shown in SEQ ID NO:68, LFR3 with an amino acid sequence as shown in SEQ ID NO:69, and LFR4 with an amino acid sequence as shown in SEQ ID NO:

70.

3. The antibody or variant thereof according to claim 1 or 2, wherein: The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 71, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 72; or The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 73, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 74; or The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 75, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 76; or The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 77, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 78; or The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 79, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

80.

4. The antibody or variant thereof according to any one of claims 1 to 3, wherein The antibody or variant thereof is a humanized monoclonal antibody.

5. A nucleic acid molecule comprising a nucleotide sequence encoding the antibody or variant thereof according to any one of claims 1 to 4.

6. The nucleic acid molecule according to claim 5, wherein The nucleotide sequence comprises: 1) a nucleotide sequence encoding the heavy chain variable region, which is shown in SEQ ID NO: 81; and a nucleotide sequence encoding the light chain variable region, which is shown in SEQ ID NO: 82; or 2) a nucleotide sequence encoding the heavy chain variable region, which is shown in SEQ ID NO: 83; and a nucleotide sequence encoding the light chain variable region, which is shown in SEQ ID NO: 84; or 3) a nucleotide sequence encoding the heavy chain variable region, which is shown in SEQ ID NO: 85; and a nucleotide sequence encoding the light chain variable region, which is shown in SEQ ID NO: 86; or 4) a nucleotide sequence encoding the heavy chain variable region, which is shown in SEQ ID NO: 87; and a nucleotide sequence encoding the light chain variable region, which is shown in SEQ ID NO: 88; or 5) a nucleotide sequence encoding the heavy chain variable region, which is shown in SEQ ID NO: 89; and a nucleotide sequence encoding the light chain variable region, which is shown in SEQ ID NO:

90. A vector comprising the nucleic acid molecule according to claim 5 . A cell comprising the nucleic acid molecule according to claim 5 or 6 or the vector according to claim 7.

9. A pharmaceutical composition comprising the antibody or variant thereof according to any one of claims 1 to 4, the nucleic acid molecule according to claim 5 or 6, the vector according to claim 7 or the cell according to claim 8, and a pharmaceutically acceptable carrier.

10. Use of the antibody or variant thereof according to any one of claims 1 to 4, the nucleic acid molecule according to claim 5 or 6, the vector according to claim 7, the cell according to claim 8, or the pharmaceutical composition according to claim 9 in preparing a drug for preventing and / or treating respiratory syncytial virus or in preparing a reagent for detecting respiratory syncytial virus.

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