Antibody specifically binding to respiratory syncytial virus or antigen binding fragment thereof and application thereof

Abstract

The invention belongs to the field of antibody drugs, and particularly relates to an antibody specifically bound with respiratory syncytial virus or an antigen binding fragment thereof and application. The invention discloses an antibody, nucleic acid for coding the antibody, an expression vector, engineering bacteria, engineering cells and application of the antibody in preparation of products for treating and detecting respiratory syncytial virus (RSV) infection diseases. The antibody disclosed by the invention has excellent binding capacity with the respiratory syncytial virus, and can be applied to prevention, diagnosis or treatment of related diseases caused by respiratory syncytial virus infection.

Description

Technical Field

[0001] This invention belongs to the field of antibody drugs, specifically relating to an antibody or its antigen-binding fragment that specifically binds to respiratory syncytial virus and its applications. Background Technology

[0002] Respiratory syncytial virus (RSV) is a common, highly contagious RNA virus belonging to the Paramyxoviridae family. RSV is primarily transmitted through droplets and contact, initially infecting the upper respiratory tract, but infection can also spread to the lower respiratory tract, especially in infants and young children, and is a major cause of viral pneumonia and bronchiolitis. Therefore, it is necessary to develop products for the prevention, diagnosis, or treatment of diseases related to RSV infection.

[0003] Antibodies are proteins that specifically recognize antigens. They can recognize and neutralize viruses by specifically recognizing viral envelope proteins, structural proteins, or enzymes, and are one of the current technological means to combat virus-related diseases. Respiratory syncytial virus (RSV) encodes 11 proteins, among which the G and F proteins are key transmembrane proteins on the viral surface. The G protein is mainly responsible for adsorption onto host cells, while the F protein, as a fusion protein, plays a crucial role in the process of viral entry into host cells. Currently, neutralizing antibodies developed based on the F protein have low titers. For example, when palizumab, which has been clinically approved for the prevention of RSV infection in high-risk infants, is used, up to five injections are required to cover a typical RSV season, resulting in low patient compliance and high drug costs.

[0004] Therefore, developing an antibody against respiratory syncytial virus (RSV) is of great significance for the prevention, diagnosis, or treatment of diseases caused by RSV infection. Summary of the Invention

[0005] This invention develops an antibody with a high neutralizing antibody titer and an EC50 value for binding to respiratory syncytial virus (RSV) that is much lower than that of palizumab, which can be used for the prevention, diagnosis, or treatment of diseases related to RSV infection.

[0006] To achieve the above objectives, the present invention can adopt the following technical solutions: This invention provides an antibody or antigen-binding fragment thereof that specifically binds to respiratory syncytial virus (RSV). The antibody or antigen-binding fragment includes a heavy chain variable region and a light chain variable region. The heavy chain variable region includes HCDR1, HCDR2, and HCDR3, and the light chain variable region includes LCDR1, LCDR2, and LCDR3. The sequence of HCDR1 is shown in SEQ ID NO:1, the sequence of HCDR2 is shown in SEQ ID NO:2, and the sequence of HCDR3 is shown in SEQ ID NO:3 or SEQ ID NO:17. The sequence of LCDR1 is shown in SEQ ID NO:9, the sequence of LCDR2 is shown in SEQ ID NO:10, and the sequence of LCDR3 is shown in SEQ ID NO:11.

[0007] Preferably, the above-mentioned heavy chain variable region further includes: HFR1 having at least 80% identity with the sequence shown in SEQ ID NO:4 or SEQ ID NO:19 and / or HFR2 having at least 80% identity with the sequence shown in SEQ ID NO:5 and / or HFR3 having at least 80% identity with the sequence shown in SEQ ID NO:6 or SEQ ID NO:20 and / or HFR4 having at least 80% identity with the sequence shown in SEQ ID NO:7; and / or The aforementioned light chain variable region further includes: LFR1 having at least 80% identity with the sequence shown in SEQ ID NO:12 and / or LFR2 having at least 80% identity with the sequence shown in SEQ ID NO:13 and / or LFR3 having at least 80% identity with the sequence shown in SEQ ID NO:14 or SEQ ID NO:22 and / or LFR4 having at least 80% identity with the sequence shown in SEQ ID NO:15.

[0008] Preferably, the antibody comprises a heavy chain variable region having at least 70% identity with the sequence shown in SEQ ID NO:8 and a light chain variable region having at least 70% identity with the sequence shown in SEQ ID NO:16; or the antibody comprises a heavy chain variable region having at least 70% identity with the sequence shown in SEQ ID NO:18 and a light chain variable region having at least 70% identity with the sequence shown in SEQ ID NO:16; or the antibody comprises a heavy chain variable region having at least 70% identity with the sequence shown in SEQ ID NO:21 and a light chain variable region having at least 70% identity with the sequence shown in SEQ ID NO:23.

[0009] Preferably, the antibody or its antigen-binding fragment further comprises a heavy chain constant region and / or a light chain constant region, at least a portion of which is derived from at least one of a human antibody, a primate antibody, or a mutant thereof.

[0010] More preferably, both the heavy chain constant region and the light chain constant region are derived from human IgG antibodies or their mutants.

[0011] More preferably, the aforementioned heavy chain constant region and light chain constant region are derived from human IgG1 antibody, human IgG4 antibody or their mutants.

[0012] More preferably, the heavy chain constant region contains a sequence that has at least 70% identity with the sequence shown in SEQ ID NO:24; or the light chain constant region contains a sequence that has at least 70% identity with the sequence shown in SEQ ID NO:25 or SEQ ID NO:26.

[0013] In another aspect, the present invention provides any one of the following substances: (i) a nucleic acid encoding an antibody or an antigen-binding fragment thereof of the present invention; (ii) an expression vector comprising the nucleic acid of (i); (iii) an engineered bacterium comprising the expression vector of (ii); (iv) an engineered cell comprising the expression vector of (ii); (v) a product for detecting respiratory syncytial virus comprising an antibody or an antigen-binding fragment thereof of the present invention or the nucleic acid of (i) or the expression vector of (ii) or the engineered bacterium of (iii) or the engineered cell of (iv); (vi) a substance for treating respiratory syncytial virus infection. Pharmaceutical compositions for respiratory syncytial virus infection, comprising antibodies and / or their antigen-binding fragments and / or nucleic acids in (i) or expression vectors in (ii) and / or engineered bacteria in (iii) and / or engineered cells in (iv); (vii) pharmaceutical preparations for the prevention or treatment of respiratory syncytial virus infection, comprising antibodies and / or their antigen-binding fragments and / or nucleic acids in (i) and / or expression vectors in (ii) and / or engineered bacteria in (iii) and / or engineered cells in (iv) and / or pharmaceutical compositions in (vi).

[0014] More preferably, the pharmaceutical composition in the above-mentioned substances is a bispecific antibody, a multi-antibody, an ADC, or a fusion protein.

[0015] More preferably, the dosage form of the pharmaceutical preparations in the above-mentioned substances includes sprays, oral liquids, tablets, nebulizers, granules, capsules, or ointments.

[0016] In another aspect, the present invention provides the use of the antibody and / or its antigen-binding fragment and / or (i) the nucleic acid and / or (ii) the expression vector and / or (iii) the engineered bacteria and / or (iv) the engineered cells in the preparation of a product for detecting respiratory syncytial virus.

[0017] In another aspect, the present invention provides the use of the antibody and / or its antigen-binding fragment and / or (i) the nucleic acid and / or (ii) the expression vector and / or (iii) the engineered bacteria and / or (iv) the engineered cells in the preparation of a medicament for the prevention or treatment of respiratory syncytial virus infection.

[0018] Preferably, the respiratory syncytial virus infection is a respiratory infection.

[0019] Preferably, the respiratory syncytial virus infection is bronchiolitis or pneumonia.

[0020] Preferably, in the above applications, the respiratory syncytial virus is strain A2 with accession number ATCC, VR-1540, strain 18537 with accession number ATCC, VR-1580, strain 9320 with accession number ATCC, VR-955, or strain Long with accession number ATCC, VR-26.

[0021] The beneficial effects of this invention include at least the following: the antibodies provided by this invention have excellent binding ability with respiratory syncytial virus (RSV). For example, the IC50 values ​​of the first antibody (6B11-2) with strains A2, Long, 9320, and 18537 can reach 8.66 ng / mL, 10.86 ng / mL, 11.28 ng / mL, and 25.87 ng / mL, respectively; and, for another example, the second antibody (6B11-1) with strains A2, Long, 9320, and 18537 can also bind to RSV. The binding IC50 values ​​for 320 and 18537 can reach 12.06 ng / mL, 11.98 ng / mL, 13.67 ng / mL and 44.39 ng / mL, respectively; for example, the binding IC50 values ​​of the third antibody (6B11) to strains A2, Long, 9320 and 18537 can reach 10.65 ng / mL, 16.46 ng / mL, 14.58 ng / mL and 32.85 ng / mL, respectively. Attached Figure Description

[0022] Figure 1 The results of flow cytometry sorting of antigen-specific memory B cells in PBMCs; Figure 2 Gel electrophoresis results of PCR products from the variable regions of antibody heavy and light chains in a single B cell; Figure 3 The results of ELISA detection show the specific binding activity of the mutated recombinant monoclonal antibody with RSV Pre-F. Figure 4a The results show the in vitro viral neutralization capacity of the mutated recombinant monoclonal antibody (A2 strain). Figure 4bThe results show the in vitro viral neutralization capacity of the mutated recombinant monoclonal antibody (Long strain). Figure 4c The results show the in vitro virus (B9320 strain) neutralization ability of the mutated recombinant monoclonal antibody. Figure 4d The results show the in vitro viral neutralization capacity of the mutated recombinant monoclonal antibody (B18537 strain). Figure 5 The results show the viral titer in the lung tissue of cotton rats after administration of recombinant antibody. Detailed Implementation

[0023] The embodiments described are provided to better illustrate the present invention, but are not intended to limit the scope of the invention to the embodiments described. Therefore, non-essential improvements and adjustments made to the embodiments by those skilled in the art based on the above description are still within the scope of protection of the present invention.

[0024] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit this disclosure. Singular expressions include plural expressions unless they have a distinct meaning in the context. As used herein, it should be understood that terms such as “comprising,” “having,” “including,” are intended to indicate the presence of features, numbers, operations, components, parts, elements, materials, or combinations thereof. The terminology of the invention is disclosed in the specification and is not intended to exclude the possibility that one or more other features, numbers, operations, components, parts, elements, materials, or combinations thereof may be present or added. As used herein, “ / ” may be interpreted as “and” or “or,” depending on the context.

[0025] In this invention, the term "antigen-binding fragment" refers to an antigen-binding fragment of an antibody and an antibody analogue, which typically includes at least a portion of the antigen-binding region or variable region of the parent antibody, such as one or more CDRs; the fragment of the antibody retains at least some of the binding specificity of the parent antibody.

[0026] In a first aspect, embodiments of the present invention provide an antibody or antigen-binding fragment thereof that specifically binds to respiratory syncytial virus (RSV). The antibody or antigen-binding fragment thereof includes a heavy chain variable region and a light chain variable region. The heavy chain variable region includes HCDR1, HCDR2, and HCDR3, and the light chain variable region includes LCDR1, LCDR2, and LCDR3. The sequence of HCDR1 is shown in SEQ ID NO:1, the sequence of HCDR2 is shown in SEQ ID NO:2, and the sequence of HCDR3 is shown in SEQ ID NO:3 or SEQ ID NO:17. The sequence of LCDR1 is shown in SEQ ID NO:9, the sequence of LCDR2 is shown in SEQ ID NO:10, and the sequence of LCDR3 is shown in SEQ ID NO:11.

[0027] It should be noted that the antibodies in this invention can be any form of antibody, such as monoclonal antibodies or recombinant antibodies; in terms of source, they can be humanized antibodies or animal-derived antibodies, such as mouse, rabbit, or camel-derived antibodies; in addition, HCDR3 and LCDR3 in the above-mentioned antibodies belong to the hypervariable regions of the heavy chain variable region and the light chain variable region, and their stability is weaker than that of HCDR1 and HCDR2 as well as LCDR1 and LCDR2.

[0028] In some examples, the heavy chain variable region of the antibodies described above further includes: HFR1 having at least 80% identity with the sequence shown in SEQ ID NO:4 or SEQ ID NO:19 and / or HFR2 having at least 80% identity with the sequence shown in SEQ ID NO:5 and / or HFR3 having at least 80% identity with the sequence shown in SEQ ID NO:6 or SEQ ID NO:20 and / or HFR4 having at least 80% identity with the sequence shown in SEQ ID NO:7.

[0029] In some examples, the light chain variable region of the antibodies described above further includes: LFR1 having at least 80% identity with the sequence shown in SEQ ID NO:12 and / or LFR2 having at least 80% identity with the sequence shown in SEQ ID NO:13 and / or LFR3 having at least 80% identity with the sequence shown in SEQ ID NO:14 or SEQ ID NO:22 and / or LFR4 having at least 80% identity with the sequence shown in SEQ ID NO:15.

[0030] It should be noted that the FRs (HFR1, HFR2, HFR3, HFR4 and LFR1, LFR2, LFR3, LFR4) in the above-mentioned antibodies are backbone regions used to connect the CDR regions and are relatively stable. In addition, the sequences of the FR regions of the heavy chain and light chain in the above-mentioned antibodies can be arranged with the sequences of the CDR regions in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, which respectively constitute the heavy chain variable region and light chain variable region of the above two antibodies. Furthermore, the at least 80% identity in this invention includes ≥80%, ≥85%, ≥90%, ≥95%, or 100% identity, etc.

[0031] In some examples, the antibodies described above include a heavy chain variable region having at least 70% identity with the sequence shown in SEQ ID NO:8 and a light chain variable region having at least 70% identity with the sequence shown in SEQ ID NO:16.

[0032] In some examples, the antibodies described above include a heavy chain variable region having at least 70% identity with the sequence shown in SEQ ID NO:18 and a light chain variable region having at least 70% identity with the sequence shown in SEQ ID NO:16.

[0033] In some examples, the antibodies described above include a heavy chain variable region having at least 70% identity with the sequence shown in SEQ ID NO:21 and a light chain variable region having at least 70% identity with the sequence shown in SEQ ID NO:23.

[0034] It should be noted that the above-mentioned at least 70% identity refers to identity ≥ 70%, such as 75%, 80%, 85%, 90%, 95% or 100%; of course, it should be understood that sequences with at least 70% identity have similar functions to the sequences mentioned above.

[0035] In some examples, the antibodies or their antigen-binding fragments described above also include heavy chain constant regions and / or light chain constant regions, at least a portion of which are derived from at least one of human antibodies, primate antibodies, or mutants thereof.

[0036] In some examples, both the heavy chain constant region and the light chain constant region mentioned above are derived from human IgG antibodies or their mutants.

[0037] In some examples, the aforementioned heavy chain constant region and light chain constant region are derived from human IgG1 antibody, human IgG4 antibody, or mutants thereof.

[0038] In some examples, the heavy chain constant region described above contains a sequence that has at least 70% identity with the sequence shown in SEQ ID NO:24; or the light chain constant region contains a sequence that has at least 70% identity with the sequence shown in SEQ ID NO:25 or SEQ ID NO:26.

[0039] It should be noted that, in addition to the aforementioned heavy chain variable region and light chain variable region, the antibody or its antigen-binding fragment in this invention also includes a heavy chain constant region in the heavy chain and a light chain constant region in the light chain. The heavy chain constant region and light chain constant region can be derived from humans or other animal sources (such as rabbits, pigs, etc.), and are regions that are almost unlikely to mutate. Furthermore, the heavy and light chains of the antibody may also include a signal peptide, which can facilitate antibody transmembrane penetration; the signal peptide can be a signal peptide known in the art.

[0040] It should be noted that, as mentioned above, the aforementioned at least 70% identity refers to identity ≥ 70%, such as 75%, 80%, 85%, 90%, 95%, or 100%; of course, it should be understood that sequences with at least 70% identity have similar functions to the aforementioned sequences.

[0041] Secondly, embodiments of the present invention also provide any one of the following substances: (i) Nucleic acid, which encodes the antibody or its antigen-binding fragment in this invention; specifically, the nucleic acid in this invention is obtained by translating the antibody or its antigen-binding fragment in this invention according to conventional methods; alternatively, it may be a nucleotide sequence obtained by further modifying the sequence after translating the above-mentioned amino acid sequence; the modification method is a method known in the art to increase expression efficiency or other nucleotide modification methods for specific purposes. (ii) An expression vector, including the nucleic acid in (i); specifically, the expression vector in this invention may be selected from any one of lentiviral expression vectors, retroviral expression vectors, adenovirus expression vectors, adeno-associated virus expression vectors, DNA vectors, RNA vectors, and plasmids. Lentiviral vectors may be selected from the following group: human immunodeficiency virus 1 (HIV-1), human immunodeficiency virus 2 (HIV-2), visna-maedivirus (VMV), caprine arthritis-encephalitis virus (CAEV), equine infectious anemia virus (EIAV), feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), and simian immunodeficiency virus (SIV); (iii) Engineered bacteria, including the expression vector in (ii); specifically, the engineered bacteria in this invention refers to bacteria that can assist the above-mentioned expression vector in expression, such as Escherichia coli, and the Escherichia coli including the expression vector here is Escherichia coli after the expression vector has been transferred in. (iv) Engineered cells, including the expression vector in (ii); specifically, the engineered cells in this invention refer to cells that can assist the above-mentioned expression vector in expression, such as yeast cells; (v) Products for detecting respiratory syncytial virus, including antibodies or antigen-binding fragments of the present invention, or nucleic acids in (i), or expression vectors in (ii), or engineered bacteria in (iii), or engineered cells in (iv); specifically, antibodies or antigen-binding fragments of the present invention have a strong binding effect with the surface proteins of respiratory syncytial virus (RSV), and products for detecting RSV can be prepared based on this; the products can be detection reagents or detection kits, such as reagents or kits based on immunoblotting, or reagents or kits based on immunoenzyme technology, etc. (vi) A pharmaceutical composition for the prevention or treatment of respiratory syncytial virus (RSV) infection, comprising the antibody and / or its antigen-binding fragment and / or the nucleic acid in (i) or the expression vector in (ii) and / or the engineered bacteria in (iii) and / or the engineered cells in (iv); specifically, as described above, a pharmaceutical composition for the prevention or treatment of RSV infection can be prepared based on the strong binding interaction between the antibody or its antigen-binding fragment of the present invention and the surface protein of RSV; the pharmaceutical composition here refers to a pharmaceutical composition consisting of the antibody or its antigen-binding fragment of the present invention combined with other small molecule compounds, peptides, antibodies, or proteins that can be effectively used for the prevention or treatment of RSV infection; for example, a bispecific antibody or polyclonal antibody combined with other antibodies, an ADC combined with a small molecule compound, or a fusion protein combined with other proteins; the choice of which to use may be made according to specific circumstances; (vii) Pharmaceutical preparations for the prevention or treatment of respiratory syncytial virus infection, including antibodies and / or their antigen-binding fragments and / or nucleic acids in (i) and / or expression vectors in (ii) and / or engineered bacteria in (iii) and / or engineered cells in (iv) and / or pharmaceutical compositions in (vi); specifically, the above-mentioned antibodies and / or their antigen-binding fragments, etc., and the above-mentioned pharmaceutical compositions can be prepared into different dosage forms to meet different clinical needs by adding a pharmaceutically acceptable carrier; the dosage forms may include sprays, oral liquids, tablets, nebulizers, granules, capsules, or ointments; of course, the choice of pharmaceutically acceptable carrier varies depending on the dosage form, and can be prepared according to methods known in the art.

[0042] Thirdly, embodiments of the present invention also provide the use of the antibody and / or its antigen-binding fragment and / or (i) the nucleic acid and / or (ii) the expression vector and / or (iii) the engineered bacteria and / or (iv) the engineered cells in the preparation of a product for detecting respiratory syncytial virus.

[0043] It should be noted that the detection of respiratory syncytial virus (RSV) can be used for the diagnosis of RSV infection diseases. That is, the RSV detection products in this invention are also applicable to the diagnosis of RSV infection diseases, such as bronchiolitis or pneumonia. In addition, RSV detection products include, but are not limited to, detection reagents, detection kits, reagent cards or microfluidic chips.

[0044] Fourthly, embodiments of the present invention also provide the use of the antibody and / or its antigen-binding fragment and / or (i) the nucleic acid and / or (ii) the expression vector and / or (iii) the engineered bacteria and / or (iv) the engineered cells in the preparation of a medicament for the prevention or treatment of respiratory syncytial virus infection.

[0045] It should be noted that the drugs in this invention include the above-mentioned pharmaceutical compositions and pharmaceutical preparations. They can be prepared by combining the above-mentioned substances with other active ingredients to form pharmaceutical compositions, or by adding a pharmaceutically acceptable carrier to form pharmaceutical preparations.

[0046] In some cases, the aforementioned respiratory syncytial virus infection is a respiratory infection.

[0047] It should be noted that the application of this invention is applicable to all respiratory syncytial virus (RSV) infections, especially respiratory infections, i.e., RSV respiratory infections.

[0048] In some cases, the respiratory syncytial virus infection described above is bronchiolitis or pneumonia.

[0049] It should be noted that, as described above, the application of this invention is applicable to all respiratory syncytial virus (RSV) infections, especially diseases directly related to respiratory tract infections such as bronchiolitis or pneumonia.

[0050] In some examples, in the above applications, the respiratory syncytial virus is strain A2 with accession number ATCC, VR-1540, strain 18537 with accession number ATCC, VR-1580, strain 9320 with accession number ATCC, VR-955, or strain Long with accession number ATCC, VR-26.

[0051] The antibodies provided by this invention exhibit excellent binding ability to respiratory syncytial virus (RSV). For example, the IC50 values ​​of the first antibody (6B11-2) binding to strains A2, Long, 9320, and 18537 can reach 8.66 ng / mL, 10.86 ng / mL, 11.28 ng / mL, and 25.87 ng / mL, respectively; as another example, the IC50 values ​​of the second antibody (6B11-1) binding to strains A2, Long, 9320, and 18537 can reach 12.06 ng / mL, 11.98 ng / mL, 13.67 ng / mL, and 44.39 ng / mL, respectively; and as yet another example, the IC50 values ​​of the third antibody (6B11) binding to strains A2, Long, 9320, and 18537 can reach 10.65 ng / mL, 16.46 ng / mL, 14.58 ng / mL, and 32.85 ng / mL, respectively.

[0052] To better understand the present invention, specific examples are provided below to further illustrate the content of the present invention, but the content of the present invention is not limited to the examples below.

[0053] The amino acid sequences of the substances involved in the following examples are shown in Table 1 below.

[0054] Table 1. Amino acid sequences of each substance

[0055]

[0056]

[0057]

[0058] In the following examples, the accession number of strain A2 is ATCC, VR-1540; the accession number of strain 18537 is ATCC, VR-1580; the accession number of strain 9320 is ATCC, VR-955; and the accession number of strain Long is ATCC, VR-26.

[0059] Example 1 This invention provides a process for screening, expressing, and purifying respiratory syncytial virus (RSV) neutralizing antibodies.

[0060] (I) Isolation and activation of RSV-positive memory B cells (1) Peripheral blood samples were collected from healthy volunteers, and PBMCs were separated by sucrose gradient density centrifugation (Cytiva, 17144003). B cells were purified and enriched by magnetic beads using the B Cell Isolation Kit II (miltenyibiotec, 130-091-151), and then RSV pre-F+CD19+IgG+ cells (CD19-APC, catalog number: 302212; IgG-PE / Cy7, catalog number: 410721; manufacturer: biolegend; RSV pre-F-FITC, catalog number: RSF-V52H3, manufacturer: ACROBiosystems) were sorted by flow cytometry. The results are as follows. Figure 1 As shown, after enrichment, 158 pre-F+CD19+IgG+ cells were obtained by flow cytometry sorting.

[0061] (2) The enriched RSV-positive memory B cells were cultured using the ImmunoCult Human B Cell Expansion Kit (stemcell, 100-0645). After 7 days of culture, B cells that secreted RSV pre-F protein (ACROBiosystems, RSF-V82E7) were collected through a single-cell light guide system (BerkeleyLights 'Beacon'). These were identified as RSV-positive B cells. Individual cells were sorted into 96-well plates, which were pre-filled with 10 μL of mineral oil (Beyotime, ST1524-50) and 5 μL of TCL buffer (Qiagen, 1070498). The plates were stored at -80°C for antibody heavy and light chain gene amplification.

[0062] (3) Cell isolation and amplification and antibody screening were performed in accordance with the methods described in patent CN119638827A or the literature "Zost SJ, Gilchuk P, Chen RE, et al. Rapid isolation and profiling of a diverse panel of human monoclonal antibodies targeting the SARS-CoV-2 spike protein. Nat Med. 2020; 26(9): 1422-1427. doi:10.1038 / s41591-020-0998-x".

[0063] (ii) Amplification and recovery of VH / VL using single-cell PCR technology Single-cell PCR technology was used to amplify and recover antibody heavy and light chains from plasma cells. The specific procedures were as follows: First, 10 μL of RNAClean XP beads were added to each well to purify and separate RNA according to the manufacturer's instructions. Then, cDNA was synthesized and amplified using the Opto™ Plasma B Discovery cDNA Synthesis Kit. Next, the cDNA was further amplified using the Opto BDiscovery Sanger Prep Kit for VH / VL amplification. The final amplification products were analyzed by gel electrophoresis and sent to Qingke for sequencing. The gel electrophoresis results of the PCR products of the antibody heavy and light chain variable regions from some single-cell B cells are shown below. Figure 2 As shown, a 2000 bp DNA molecular weight standard was used as the marker, with the heavy chain around 700 bp and the light chain around 550 bp.

[0064] (III) Preparation of recombinant antibodies (1) The sequencing sequences were analyzed (https: / / www.imgt.org / ), and a total of 34 antibody variable region sequences were obtained. The 34 antibody variable region sequences were sent to Imgt for biosynthesis and constructed into the κ light chain constant region or λ light chain constant region. (According to the subtypes of the light chain variable region) and the pCDNA3.4 vector of the human IgG1-YTE heavy chain constant region, stab bacteria were subsequently obtained, and plasmids were extracted by streaking and shaking according to general molecular biology methods; at the same time, the antibodies Palivizumab (refer to patent US7704505B2), Nirsevimab (refer to patent US11186628B2) and Clesrovimab (refer to patent US9963500B2) were used as positive controls.

[0065] (2) One day before transfection (day -1), the cell density of ExpiCHO-S cells was adjusted to (2-3.5)×10⁻¹⁰. 6 Incubate overnight at 37°C, 8% CO2, with shaking at 120 rpm; on day 0, cells grew to 4-7 × 10⁶ cells / mL. 6 / mL, dilute cells to 4×10⁶ cells / mL using fresh, preheated CHOgro® expression medium (supplemented with 4 mM L-Glutamine and 0.3% Poloxamer). 6 / mL; Take 3mL of CHOgro® Complex Formation Solution into a sterile test tube, add 30 μg of the above endotoxin-free antibody-related plasmid and 20μL of TransIT-PRO® Reagent, mix gently and incubate at room temperature for no more than 5min, add the complex to 30mL of pre-prepared ExpiCHO-S cells; add 600μL of CHOgro® Titer Enhancer, gently shake the culture flask to mix well (CHOgro® Titer Enhancer can be added within 0-24 hours), and collect the cell culture supernatant after culturing at 125 rpm, 32℃, and 8% CO2 for 14 days.

[0066] (3) Filter the cell culture supernatant with a 0.22 μm filter, load the protein A affinity chromatography column into the protein purifier, wash with 5 column volumes of water, then equilibrate with 8 column volumes of PBS buffer, let the sample flow through the affinity chromatography column, then equilibrate with PBS buffer until the UV value no longer changes, collect the eluted sample by adding elution buffer at a flow rate of 1 mL / min, ultrafilter and concentrate the eluted antibody sample solution and change the buffer to obtain the purified antibody; determine the antibody concentration, label and store at -20℃ for long-term storage.

[0067] The expression and purification results showed that three recombinant monoclonal antibodies were not successfully expressed and purified, while the rest were successfully prepared.

[0068] (iv) Verification of ELISA binding activity of recombinant monoclonal antibody The successfully expressed and purified recombinant monoclonal antibody was tested for its binding activity with RSVpre-F protein using an ELISA assay, and recombinant monoclonal antibodies with high binding capacity were screened. The specific steps were as follows: RSVpre-F protein (RSF-V52H7, ACRObiosystems) was diluted to 0.5 μg / mL with PBS, and 100 μL was added to each well of the ELISA plate for coating, incubated overnight at 4°C; the supernatant coating solution was discarded, and the plate was washed with PBST; the plate was blocked with PBST containing 1% BSA at 37°C for 2 h; the blocking solution in the wells was discarded, and the plate was washed with PBST; different concentrations of the recombinant monoclonal antibody expressed above, serially diluted with 1% BSA solution at a 1:3 ratio, were added stepwise. Monoclonal antibodies were incubated at 37°C for 1 hour; the liquid was discarded, and the cells were washed with PBST; diluted HRP secondary antibody was added to each well, and the cells were incubated at 37°C for 1 hour; the liquid was discarded, and the cells were washed with PBST; TMB chromogenic solution was added to each well, and the reaction was carried out at room temperature for 10-15 minutes; the reaction was terminated by adding stop solution, and the OD value at 450 nm was read and analyzed using GraphPad Prism9. The EC50 value was calculated (the concentration of the antibody when the OD450 value is half of the highest value is the EC50 value of the antibody; the smaller the EC50 value, the stronger the antibody affinity and the higher the binding ability to RSVpre-F protein (RSF-V52H7, ACRObiosystems)). Following the above screening method, 15 recombinant monoclonal antibodies with the highest ELISA binding ability were selected.

[0069] (v) Recombinant monoclonal antibody inhibits RSV / A2 infection of Hep-2 cells HEp-2 cells were infected with RSV strain A2 (derived from ATCC). Fifteen recombinant monoclonal antibodies with the highest ELISA binding capacity were selected for testing. The ability of different recombinant monoclonal antibodies and positive controls at different concentrations to inhibit cell infection by different strains was evaluated. Palivizumab, Nirsevimab, and Clesrovimab monoclonal antibodies were used as positive controls. Specifically, these included: Adjust the density of HEp-2 cells, seed them into 96-well plates, and incubate overnight in a cell culture incubator (37℃, 5% CO2) to ensure that the cell confluence is about 90% the next day before starting the experiment. Dilute the first well to 10000 ng / mL and perform serial dilutions of 3-fold, setting a total of 8 dilutions (including the first well). Add the virus and incubate at 37℃, 5% CO2 for about 1 hour to neutralize. Add the virus and antibody neutralization product to HEp-2 cells and incubate for about 22 hours. Discard the supernatant, fix the cells, and add fluorescently labeled detection antibody. Read the plate using a fluorescent (ELISA) immunospot analyzer. Summarize the data and analyze them using GraphPad Prism9 to calculate the IC50 value (the smaller the IC50 value, the stronger the neutralizing ability of the antibody and the stronger its ability to inhibit viral infection of cells).

[0070] The recombinant monoclonal antibody with the best neutralizing effect was screened using the above method and named RSV-11 (hereinafter also referred to as 6B11). The recombinant monoclonal antibody RSV-11 has a neutralizing activity IC50 of <100 ng / mL (14.45 ng / mL) against the A2 strain, and its neutralizing ability is stronger than that of Palivizumab monoclonal antibody (962.90 ng / mL) and Clesrovimab monoclonal antibody (23.87 ng / mL), and comparable to that of Nirsevimab monoclonal antibody (2.41 ng / mL) against the A2 strain.

[0071] Example 2 This invention provides a method for obtaining RSV-11 monoclonal antibody mutants by removing the post-translational modification site of the RSV-11 monoclonal antibody PTM, followed by screening for mutants with strong neutralizing ability against respiratory syncytial virus, as detailed below: (I) Design of different mutants of RSV-11 monoclonal antibody Input the heavy chain variable region and light chain variable region of RSV-11 into the abysis webpage for annotation, view the unusual residue in the FR region and the possible post-translational modification site residues (PTM), combine the above antibody structure to retain the amino acids that maintain the CDR loop, replace germline gene amino acids, and generate sequence 6B11-2. Antigen-antibody interaction analysis of the 6B11-2 antibody was performed using AlphaFold3. Specifically, the user went to https: / / alphafoldserver.com / , followed the prompts to input the amino acid sequences of the heavy chain and light chain variable regions of 6B11-2, and the amino acid sequence of the RSV A2 type pre-F DS Cav1 mutation. After completing the antigen-antibody interaction prediction, the user downloaded and saved the top 5 most likely antigen-antibody interaction results. Next, the user went to Discovery Studio software and used the Calculate Mutation Energy (Binding) function. Following the prompts, the user sequentially input the above-saved 5 antigen-antibody interaction results, performed single-point amino acid saturation mutagenesis at the antibody binding site, processed the data, and generated the 6B11-1 sequence.

[0072] (II) ELISA detection of the protein-binding activity of recombinant monoclonal antibody mutants Following the procedure described in step (iv) of Example 1, the binding activity of antibody RSV-11 (hereinafter also referred to as antibody 6B11) and its mutants 6B11-1 and 6B11-2 to RSV A2 pre-F protein was detected and analyzed.

[0073] The results are as follows Figure 3 As shown, the results indicate that, compared with the original antibody RSV-11, mutant 6B11-2 has a significantly improved binding ability to RSV A2 pre-F protein; mutant 6B11-1 has a comparable binding ability to RSV A2 pre-F protein to the original antibody RSV-11.

[0074] Example 3 HEp-2 cells were infected with RSV strains A-A2 (A2), B-18537 (B18537), B-9320 (B9320), and A-Long (Long) (all derived from ATCC). The ability of different concentrations of recombinant monoclonal antibodies to inhibit cell infection by different strains was detected. The procedure was performed according to step (v) of Example 1. The test results are shown in Table 2 and... Figure 4a , Figure 4b , Figure 4c and Figure 4d As shown.

[0075] Table 2. Neutralizing activity of recombinant antibodies against RSV A2 / 18537 / 9320 / Long strains.

[0076] Note: The "R squared" value is the "R squared" value, which reflects the goodness of fit in data analysis and indirectly reflects the reliability of the IC50 value. The higher the value, the more reliable the IC50 value.

[0077] From Table 2 and Figures 4a to 4d It can be seen that the neutralizing activity of antibody 6B11-2 is significantly higher than that of palivizumab, with an order-of-magnitude difference. Comparing the neutralizing activity of antibody 6B11-2 and Nirsevimab monoclonal antibodies, the difference is 3.6 times for the A2 strain, 2.3 times for the Long strain, comparable for the 18537 strain, and 2.2 times for the B9320 strain. Compared to antibody 6B11, antibody 6B11-1, after mutation, shows reduced neutralizing activity for RSV A2 / 18537 / 9320 / Long strains. Compared to antibody 6B11, 6B11-2, after mutation, shows increased neutralizing activity for RSV A2 / 18537 / 9320 / Long strains. Within the allowable error range, it can be considered that there is no significant difference among the three antibodies. However, antibody 6B11-2 is superior to antibody 6B11 in terms of antibody expression level, immunogenicity, and other drug-like properties.

[0078] Example 4 This invention uses a cotton rat model to evaluate the in vivo efficacy of antibodies, as detailed below: Several female SPF-grade mice (6-8 weeks old) were divided into groups of 5 mice each. The groups included: PBS group, palivizumab 0.5 mg / kg group, palivizumab 5 mg / kg group, 6B11-2 0.5 mg / kg group, 6B11-2 5 mg / kg group, nirsevimab 0.5 mg / kg group, and nirsevimab 5 mg / kg group. After intramuscular injection of the corresponding doses for one day, the mice in each group were then intranasally infected with RSVA2 at a dose of 6 × 10⁻⁶. 5 PFU (50 μL) / mouse. Four days after infection, the mice were dissected and tissue samples were collected for lung viral load testing (plaque detection method, following the guidelines in the literature "Zhu, Qing et al. “A highly potent extended half-life antibody as a potential RSV vaccine surrogate for all infants”). Science translational medicine vol. 9,388 (2017): eaaj1928. doi:10.1126 / scitranslmed.aaj1928》 to evaluate the protective efficacy of the antibody.

[0079] The results are as follows Figure 5 As shown, at a dose of 5 mg / kg, antibodies 6B11-2 and nirsevimab significantly and completely inhibited lung infection in rats, while antibody palivizumab did not completely inhibit lung infection in rats; at a dose of 0.5 mg / kg, the viral inhibition effect was: antibody nirsevimab > antibody 6B11-2 > antibody palivizumab.

[0080] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to respiratory syncytial virus (RSV), wherein the antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising HCDR1, HCDR2, and HCDR3, and the light chain variable region comprising LCDR1, LCDR2, and LCDR3; characterized in that: The sequence of HCDR1 is shown in SEQ ID NO:1, the sequence of HCDR2 is shown in SEQ ID NO:2, and the sequence of HCDR3 is shown in SEQ ID NO:3 or SEQ ID NO:

17. The sequence of LCDR1 is shown in SEQ ID NO:9, the sequence of LCDR2 is shown in SEQ ID NO:10, and the sequence of LCDR3 is shown in SEQ ID NO:

11.

2. The antibody or its antigen-binding fragment according to claim 1, characterized in that: The heavy chain variable region also includes: HFR1 and / or sequences having at least 80% identity with the sequences shown in SEQ ID NO:4 or SEQ ID NO:19 HFR2 and / or sequences having at least 80% identity with the sequence shown in SEQ ID NO:5 HFR3 and / or sequences having at least 80% identity with the sequences shown in SEQ ID NO:6 or SEQ ID NO:20 HFR4 having at least 80% identity with the sequence shown in SEQ ID NO:7; and / or The variable region of the light chain also includes: LFR1 and / or sequences having at least 80% identity with the sequence shown in SEQ ID NO:12 LFR2 and / or sequences having at least 80% identity with the sequence shown in SEQ ID NO:13 LFR3 and / or sequences having at least 80% identity with the sequences shown in SEQ ID NO:14 or SEQ ID NO:22 LFR4, which has at least 80% identity with the sequence shown in SEQ ID NO:

15.

3. The antibody or its antigen-binding fragment according to claim 1, characterized in that: The antibody includes a heavy chain variable region having at least 70% identity with the sequence shown in SEQ ID NO:8 and a light chain variable region having at least 70% identity with the sequence shown in SEQ ID NO:16; or The antibody includes a heavy chain variable region having at least 70% identity with the sequence shown in SEQ ID NO:18 and a light chain variable region having at least 70% identity with the sequence shown in SEQ ID NO:16; or The antibody includes a heavy chain variable region having at least 70% identity with the sequence shown in SEQ ID NO:21 and a light chain variable region having at least 70% identity with the sequence shown in SEQ ID NO:

23.

4. The antibody or its antigen-binding fragment according to any one of claims 1 to 3, characterized in that: The antibody or its antigen-binding fragment further comprises a heavy chain constant region and / or a light chain constant region, at least a portion of which is derived from at least one of a human antibody, a primate antibody, or a mutant thereof.

5. The antibody or its antigen-binding fragment according to claim 4, characterized in that: Both the heavy chain constant region and the light chain constant region are derived from human IgG antibodies or their mutants.

6. The antibody or its antigen-binding fragment according to claim 5, characterized in that: Both the heavy chain constant region and the light chain constant region are derived from human IgG1 antibody, human IgG4 antibody or their mutants.

7. The antibody or its antigen-binding fragment according to claim 6, characterized in that: The heavy chain constant region contains a sequence that has at least 70% identity with the sequence shown in SEQ ID NO:24; or the light chain constant region contains a sequence that has at least 70% identity with the sequence shown in SEQ ID NO:25 or SEQ ID NO:

26.

8. Any one of the following substances: (i) a nucleic acid encoding an antibody or an antigen-binding fragment thereof as described in any one of claims 1 to 7; (ii) Expression vectors, including the nucleic acids in (i); (iii) Engineered bacteria, including the expression vectors in (ii); (iv) Engineered cells, including the expression vectors described in (ii); (v) Products for detecting respiratory syncytial virus, comprising the antibody or antigen-binding fragment thereof as described in any one of claims 1 to 7, or the nucleic acid in (i), or the expression vector in (ii), or the engineered bacteria in (iii), or the engineered cells in (iv); (vi) A pharmaceutical composition for treating respiratory syncytial virus infection, comprising an antibody and / or an antigen-binding fragment thereof as described in any one of claims 1 to 7 and / or a nucleic acid in (i) or an expression vector in (ii) and / or engineered bacteria in (iii) and / or engineered cells in (iv); (vii) A pharmaceutical preparation for the prevention or treatment of respiratory syncytial virus infection, comprising an antibody and / or an antigen-binding fragment thereof as described in any one of claims 1 to 7 and / or a nucleic acid in (i) and / or an expression vector in (ii) and / or engineered bacteria in (iii) and / or engineered cells in (iv) and / or a pharmaceutical composition in (vi).

9. The substance according to claim 8, characterized in that: The drug composition is a bispecific antibody, a multi-antibody, an ADC, or a fusion protein.

10. The substance according to claim 8 or 9, characterized in that: Dosage forms of pharmaceutical preparations include sprays, oral liquids, tablets, nebulizers, granules, capsules, or ointments.

11. The use of the antibody and / or its antigen-binding fragment and / or the nucleic acid in (i) and / or the expression vector in (ii) and / or the engineered bacteria in (iii) and / or the engineered cells in (iv) in the preparation of a product for detecting respiratory syncytial virus.

12. The use of the antibody and / or its antigen-binding fragment and / or the nucleic acid in (i) and / or the expression vector in (ii) and / or the engineered bacteria in (iii) and / or the engineered cells in (iv) in the preparation of a medicament for the prevention or treatment of respiratory syncytial virus infection.

13. The application according to claim 12, characterized in that: Respiratory syncytial virus infection is a respiratory tract infection.

14. The application according to claim 12, characterized in that: Respiratory syncytial virus infection can cause bronchiolitis or pneumonia.

15. The application according to any one of claims 11 to 14, characterized in that: The respiratory syncytial virus (RSV) is strain A2 with accession number ATCC, VR-1540, strain 18537 with accession number ATCC, VR-1580, strain 9320 with accession number ATCC, VR-955, or strain Long with accession number ATCC, VR-26.

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