Antibodies or antigen-binding fragments thereof specifically binding to respiratory syncytial virus and uses thereof

Abstract

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

Description

TECHNICAL FIELD

[0001] The application belongs to the field of antibody drugs, and particularly relates to an antibody or antigen-binding fragment thereof specifically binding to respiratory syncytial virus and application thereof. BACKGROUND

[0002] Respiratory syncytial virus (RSV) is a common, highly infectious RNA virus belonging to the Paramyxoviridae family. RSV virus is mainly transmitted through droplets and contact, and first infects the upper respiratory tract, but its infection can also spread to the lower respiratory tract, especially in infants, and is the main cause of viral pneumonia and bronchiolitis. Therefore, it is necessary to develop a product for preventing, diagnosing or treating diseases caused by RSV infection.

[0003] An antibody is a protein that can specifically recognize an antigen, and can recognize and neutralize a virus by specifically recognizing viral envelope proteins, structural proteins or enzymes, and is one of the current technical means for treating viral diseases. Respiratory syncytial virus encodes 11 proteins, of which G protein and F protein are key transmembrane proteins on the surface of the virus; G protein is mainly responsible for adsorbing host cells, while F protein, as a fusion protein, plays a crucial role in the process of viral entry into host cells. At present, the neutralizing antibody titer based on the above F protein is low, for example, the clinically approved palivizumab needs up to 5 injections to cover a typical RSV season for preventing high-risk infants from being infected with RSV, the patient compliance is low, and the drug cost is high.

[0004] Therefore, it is of great significance to develop an antibody against respiratory syncytial virus for preventing, diagnosing or treating diseases caused by respiratory syncytial virus infection. SUMMARY

[0005] The application develops an antibody with high neutralizing antibody titer and an EC50 value far lower than palivizumab for binding to respiratory syncytial virus, which can be applied in preventing, diagnosing or treating diseases caused by respiratory syncytial virus infection.

[0006] In order to achieve the above purpose, the application can adopt the following technical solutions:

[0007] In one aspect, the present application provides an antibody or antigen-binding fragment thereof specifically binding to respiratory syncytial virus (RSV), the antibody or antigen-binding fragment thereof comprising 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; wherein the sequence of HCDR1 is set forth in SEQ ID NO: 1, the sequence of HCDR2 is set forth in SEQ ID NO: 2, and the sequence of HCDR3 is set forth in SEQ ID NO: 3 or SEQ ID NO: 17; the sequence of LCDR1 is set forth in SEQ ID NO: 9, the sequence of LCDR2 is set forth in SEQ ID NO: 10, and the sequence of LCDR3 is set forth in SEQ ID NO: 11.

[0008] Preferably, the heavy chain variable region further comprises HFR1 having at least 80% identity to the sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 19, and / or HFR2 having at least 80% identity to the sequence set forth in SEQ ID NO: 5, and / or HFR3 having at least 80% identity to the sequence set forth in SEQ ID NO: 6 or SEQ ID NO: 20, and / or HFR4 having at least 80% identity to the sequence set forth in SEQ ID NO: 7; and / or

[0009] Preferably, the light chain variable region further comprises LFR1 having at least 80% identity to the sequence set forth in SEQ ID NO: 12, and / or LFR2 having at least 80% identity to the sequence set forth in SEQ ID NO: 13, and / or LFR3 having at least 80% identity to the sequence set forth in SEQ ID NO: 14 or SEQ ID NO: 22, and / or LFR4 having at least 80% identity to the sequence set forth in SEQ ID NO: 15.

[0010] Preferably, the antibody comprises a heavy chain variable region having at least 70% identity to the sequence set forth in SEQ ID NO: 8 and a light chain variable region having at least 70% identity to the sequence set forth in SEQ ID NO: 16; or the antibody comprises a heavy chain variable region having at least 70% identity to the sequence set forth in SEQ ID NO: 18 and a light chain variable region having at least 70% identity to the sequence set forth in SEQ ID NO: 16; or the antibody comprises a heavy chain variable region having at least 70% identity to the sequence set forth in SEQ ID NO: 21 and a light chain variable region having at least 70% identity to the sequence set forth in SEQ ID NO: 23.

[0011] Preferably, the antibody or antigen-binding fragment thereof further comprises a heavy chain constant region and / or a light chain constant region, at least a part of the heavy chain constant region and / or the light chain constant region is derived from at least one of a human antibody, a primate antibody or a mutant thereof.

[0012] More preferably, the heavy chain constant region and the light chain constant region are both derived from a human IgG antibody or a mutant thereof.

[0013] More preferably, the heavy chain constant region and the light chain constant region are both derived from a human IgG1 antibody, a human IgG4 antibody or a mutant thereof.

[0014] More preferably, the heavy chain constant region comprises a sequence having at least 70% identity to the sequence set forth in SEQ ID NO: 24; or the light chain constant region comprises a sequence having at least 70% identity to the sequence set forth in SEQ ID NO: 25 or SEQ ID NO: 26.

[0015] In another aspect of the present application, any one of the following is provided: (i) a nucleic acid encoding the antibody or antigen-binding fragment thereof of the present application; (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 the antibody or antigen-binding fragment thereof of the present application 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 pharmaceutical composition for treating a respiratory syncytial virus infection comprising the antibody and / or antigen-binding fragment thereof of the present application and / or the nucleic acid of (i) and / or the expression vector of (ii) and / or the engineered bacterium of (iii) and / or the engineered cell of (iv); (vii) a pharmaceutical preparation for preventing or treating a respiratory syncytial virus infection disease comprising the antibody and / or antigen-binding fragment thereof of the present application and / or the nucleic acid of (i) and / or the expression vector of (ii) and / or the engineered bacterium of (iii) and / or the engineered cell of (iv) and / or the pharmaceutical composition of (vi).

[0016] More preferably, the pharmaceutical composition in the above-mentioned substance is a diabody, a multibody, an ADC or a fusion protein.

[0017] More preferably, the dosage form of the pharmaceutical preparation in the above-mentioned substance comprises a spray, an oral liquid, a tablet, an aerosol, a granule, a capsule or a paste.

[0018] In another aspect of the present application, use of the antibody and / or antigen-binding fragment thereof of the present application and / or the nucleic acid of (i) and / or the expression vector of (ii) and / or the engineered bacterium of (iii) and / or the engineered cell of (iv) in the preparation of a product for detecting respiratory syncytial virus is provided.

[0019] In still another aspect, the present application provides use of the antibody and / or antigen binding fragment thereof 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 cell in (iv) in the present application in the preparation of a medicament for preventing or treating a respiratory syncytial virus infection disease.

[0020] Preferably, the respiratory syncytial virus infection disease is a respiratory infection.

[0021] Preferably, the respiratory syncytial virus infection disease is bronchiolitis or pneumonia.

[0022] Preferably, in the above use, 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.

[0023] The present application has at least the following beneficial effects: the antibody provided by the present application has excellent binding capacity to the respiratory syncytial virus, for example, the binding IC50 value of the first antibody (6B11-2) 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; for another example, the binding IC50 value of the second antibody (6B11-1) 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; for another example, the binding IC50 value 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. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 The results of flow sorting of antigen-specific memory B cells in PBMCs;

[0025] Figure 2 The results of gel electrophoresis of PCR products of antibody heavy chain and light chain variable regions of individual B cells;

[0026] Figure 3 The results of ELISA detection of specific binding activity of the recombinant monoclonal antibody after mutation to RSV Pre-F;

[0027] Figure 4aResults of in vitro virus (A2 strain) neutralization ability detection of the mutant recombinant monoclonal antibody;

[0028] Figure 4b Results of in vitro virus (Long strain) neutralization ability detection of the mutant recombinant monoclonal antibody;

[0029] Figure 4c Results of in vitro virus (B9320 strain) neutralization ability detection of the mutant recombinant monoclonal antibody;

[0030] Figure 4d Results of in vitro virus (B18537 strain) neutralization ability detection of the mutant recombinant monoclonal antibody;

[0031] Figure 5 Results of lung tissue virus titer detection of the cotton rat after administration of the recombinant antibody. DETAILED DESCRIPTION

[0032] The examples are provided to better illustrate the present application but are not intended to limit the present application to their details. Therefore, non-essential improvements and modifications made by those skilled in the art based on the above disclosure of the application are still within the scope of the present application.

[0033] The terms used herein are used only to describe particular embodiments and are not intended to limit the present disclosure. Unless otherwise apparent in context, expressions in singular form include expressions in plural form. As used herein, it is understood that terms such as "include", "have", "contain", etc. are intended to indicate the presence of features, numbers, operations, components, parts, elements, materials, or combinations thereof. The terms of the present application are disclosed in the specification, and are not intended to exclude the possibility that one or more other features, numbers, operations, components, parts, elements, materials, or combinations thereof can exist or can be added. As used herein, " / " can be interpreted as "and" or "or" depending on the situation.

[0034] In the present application, the term "antigen-binding fragment" refers to an antigen-binding fragment of an antibody and an antibody analog, which generally includes at least part 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.

[0035] In a first aspect, an embodiment of the present application provides an antibody or an antigen-binding fragment thereof specifically binding to respiratory syncytial virus (RSV), the antibody or the antigen-binding fragment thereof comprising 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; wherein the sequence of HCDR1 is shown as SEQ ID NO: 1, the sequence of HCDR2 is shown as SEQ ID NO: 2, and the sequence of HCDR3 is shown as SEQ ID NO: 3 or SEQ ID NO: 17; the sequence of LCDR1 is shown as SEQ ID NO: 9, the sequence of LCDR2 is shown as SEQ ID NO: 10, and the sequence of LCDR3 is shown as SEQ ID NO: 11.

[0036] It should be noted that the antibody in the present application can be any antibody form such as a monoclonal antibody or a recombinant antibody; in terms of source, it can be a humanized antibody or an animal source antibody such as a mouse source, a rabbit source or a camel source; in addition, the HCDR3 and the LCDR3 in the above-mentioned antibody belong to the high variable region of the heavy chain variable region and the light chain variable region, and the stability is weaker than that of the HCDR1, the HCDR2, the LCDR1 and the LCDR2.

[0037] In some examples, the heavy chain variable region of the above-mentioned antibody further comprises: HFR1 having at least 80% identity with the sequence shown as SEQ ID NO: 4 or SEQ ID NO: 19, and / or HFR2 having at least 80% identity with the sequence shown as SEQ ID NO: 5, and / or HFR3 having at least 80% identity with the sequence shown as SEQ ID NO: 6 or SEQ ID NO: 20, and / or HFR4 having at least 80% identity with the sequence shown as SEQ ID NO: 7.

[0038] In some examples, the light chain variable region of the above-mentioned antibody further comprises: LFR1 having at least 80% identity with the sequence shown as SEQ ID NO: 12, and / or LFR2 having at least 80% identity with the sequence shown as SEQ ID NO: 13, and / or LFR3 having at least 80% identity with the sequence shown as SEQ ID NO: 14 or SEQ ID NO: 22, and / or LFR4 having at least 80% identity with the sequence shown as SEQ ID NO: 15.

[0039] It should be noted that the FR (HFR1, HFR2, HFR3, HFR4, and LFR1, LFR2, LFR3, LFR4) in the above-mentioned antibody is a framework region, which is used to connect the CDR region and is relatively stable; in addition, the sequence of the FR region of the heavy chain and the light chain in the above-mentioned antibody and the sequence of the CDR region can be arranged in FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, respectively, to form the heavy chain variable region and the light chain variable region of the above-mentioned two antibodies; in addition, the at least 80% identity in the present application includes ≥80%, ≥85%, ≥90%, ≥95% or 100% identity, etc.

[0040] In some examples, the above-mentioned 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.

[0041] In some examples, the above-mentioned 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.

[0042] In some examples, the above-mentioned 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.

[0043] It should be noted that the at least 70% identity refers to an identity of ≥70%, such as 75%, 80%, 85%, 90%, 95% or 100%; of course, it should be understood that the sequence with at least 70% identity has a similar function to the above-mentioned sequence.

[0044] In some examples, the above-mentioned antibody or antigen binding fragment thereof further comprises a heavy chain constant region and / or a light chain constant region, at least a part of the heavy chain constant region and / or the light chain constant region is derived from at least one of a human antibody, a primate antibody or a mutant thereof.

[0045] In some examples, the above-mentioned heavy chain constant region and light chain constant region are both derived from a human IgG antibody or a mutant thereof.

[0046] In some examples, the above-mentioned heavy chain constant region and light chain constant region are both derived from a human IgG1 antibody, a human IgG4 antibody or a mutant thereof.

[0047] In some examples, the heavy chain constant region comprises a sequence having at least 70% identity to the sequence set forth in SEQ ID NO: 24; or the light chain constant region comprises a sequence having at least 70% identity to the sequence set forth in SEQ ID NO: 25 or SEQ ID NO: 26.

[0048] It should be noted that, in addition to the heavy chain variable region and the light chain variable region, the antibody or the antigen binding fragment thereof in the present application further comprises a heavy chain constant region and a light chain constant region. The heavy chain constant region and the light chain constant region can be of human origin or of other animal origin (such as rabbit origin, pig origin, etc.), and are regions that are hardly mutated. In addition, the heavy chain and the light chain of the antibody can further comprise a signal peptide, which can help the antibody to penetrate the membrane. The signal peptide can be a signal peptide known in the art.

[0049] It should be noted that, as described above, the at least 70% identity refers to an identity of ≥ 70%, such as 75%, 80%, 85%, 90%, 95% or 100%. It should be understood that the sequence having at least 70% identity has a similar function to the above sequence.

[0050] In a second aspect, the embodiments of the present application further provide any one of the following substances:

[0051] (i) a nucleic acid encoding the antibody or the antigen binding fragment thereof in the present application; specifically, the nucleic acid in the present application is obtained by translating the antibody or the antigen binding fragment thereof in the present application according to a conventional method; in addition, it can also be a nucleotide sequence obtained by further modification after translation of the above-mentioned amino acid sequence; the modification method is a nucleotide modification method known in the art for increasing expression efficiency or other purposes;

[0052] (ii) an expression vector comprising the nucleic acid in (i); specifically, the expression vector in the present application can be selected from any one of a lentiviral expression vector, a retroviral expression vector, an adenoviral expression vector, an adeno-associated viral expression vector, a DNA vector, an RNA vector and a plasmid. The lentiviral vector can be selected from the following group: human immunodeficiency virus 1 (HIV-1), human immunodeficiency virus 2 (HIV-2), visna-maedivirus (VMV) virus, caprine arthritis-encephalitis virus (CAEV), equine infectious anemia virus (EIAV), feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV) and simian immunodeficiency virus (SIV);

[0053] (iii) engineered bacteria, comprising the expression vector in (ii); specifically, the engineered bacteria in the present application refers to bacteria that can assist the expression of the expression vector described above, such as Escherichia coli, and the Escherichia coli comprising the expression vector herein refers to the Escherichia coli into which the expression vector is transferred;

[0054] (iv) engineered cells, comprising the expression vector in (ii); specifically, the engineered cells in the present application refers to cells that can assist the expression of the expression vector described above, such as yeast cells;

[0055] (v) products for detecting respiratory syncytial virus, comprising the antibody or antigen-binding fragment thereof in the present application or the nucleic acid in (i) or the expression vector in (ii) or the engineered bacteria in (iii) or the engineered cells in (iv); specifically, the antibody or antigen-binding fragment thereof in the present application has strong binding with the surface protein of respiratory syncytial virus (RSV), and based on this, products for detecting respiratory syncytial virus can be prepared; the products can be detection reagents or detection kits, such as reagents or kits based on immunoblotting, such as reagents or kits based on immunoenzyme technology, etc.;

[0056] (vi) pharmaceutical compositions for preventing or treating respiratory syncytial virus infection, comprising the antibody and / or antigen-binding fragment thereof in the present application and / or the nucleic acid in (i) or the expression vector in (ii) or the engineered bacteria in (iii) or the engineered cells in (iv); specifically, as described above, based on the strong binding of the antibody or antigen-binding fragment thereof in the present application with the surface protein of respiratory syncytial virus (RSV), pharmaceutical compositions for preventing or treating respiratory syncytial virus infection can be prepared; the pharmaceutical compositions herein refer to pharmaceutical compositions in which the antibody or antigen-binding fragment thereof in the present application is combined with other small molecule compounds, polypeptides, antibodies or proteins that can be effectively used for preventing or treating respiratory syncytial virus infection; such as bi- or multi-antibodies combined with other antibodies, such as ADC combined with small molecule compounds, such as fusion proteins combined with other proteins; the selection and use can be made according to specific conditions;

[0057] (vii) a pharmaceutical preparation for preventing or treating a respiratory syncytial virus infection disease, comprising the antibody and / or antigen binding fragment thereof of the present application and / or the nucleic acid of (i) and / or the expression vector of (ii) and / or the engineered bacteria of (iii) and / or the engineered cell of (iv) and / or the pharmaceutical composition of (vi); in particular, the above-mentioned antibody and / or antigen binding fragment thereof and the like and the above-mentioned pharmaceutical composition can be added to a pharmaceutically acceptable carrier to prepare different dosage forms suitable for different clinical needs; the dosage forms can include sprays, oral liquids, tablets, atomizers, granules, capsules or pastes; of course, according to different dosage forms, the selection of the pharmaceutically acceptable carrier is different, and the preparation can be carried out according to the methods known in the art.

[0058] In a third aspect, the embodiments of the present application also provide a use of the antibody and / or antigen binding fragment thereof of the present application and / or the nucleic acid of (i) and / or the expression vector of (ii) and / or the engineered bacteria of (iii) and / or the engineered cell of (iv) in the preparation of a product for detecting a respiratory syncytial virus.

[0059] It should be noted that the detection of the respiratory syncytial virus can be used for the diagnosis of the respiratory syncytial virus infection disease, that is, the product for detecting the respiratory syncytial virus of the present application is also applicable to the diagnosis of the respiratory syncytial virus infection disease, such as the diagnosis of bronchiolitis or pneumonia; in addition, the product for detecting the respiratory syncytial virus includes but is not limited to detection reagents, detection kits, reagent cards or microfluidic chips, etc.

[0060] In a fourth aspect, the embodiments of the present application also provide a use of the antibody and / or antigen binding fragment thereof of the present application and / or the nucleic acid of (i) and / or the expression vector of (ii) and / or the engineered bacteria of (iii) and / or the engineered cell of (iv) in the preparation of a medicine for preventing or treating a respiratory syncytial virus infection disease.

[0061] It should be noted that the medicine of the present application includes the above-mentioned pharmaceutical composition and pharmaceutical preparation, which can be prepared into a pharmaceutical composition by combining the above-mentioned substances with other active ingredients, or can be prepared into a pharmaceutical preparation by adding a pharmaceutically acceptable carrier on this basis.

[0062] In some examples, the respiratory syncytial virus infection disease is a respiratory infection.

[0063] It should be noted that the application of the present application is applicable to all respiratory syncytial virus infection diseases, especially respiratory infections, that is, RSV respiratory infections.

[0064] In some examples, the respiratory syncytial virus infection disease is bronchiolitis or pneumonia.

[0065] It should be noted that the application in the present application is suitable for all respiratory syncytial virus infection diseases, especially diseases such as bronchiolitis or pneumonia directly related to respiratory tract infection.

[0066] In some examples, in the above application, 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.

[0067] The antibody provided by the present application has excellent binding capacity with the respiratory syncytial virus. For example, the binding IC50 value of the first antibody (6B11-2) to strains A2, Long, 9320 and 18537 can be 8.66 ng / mL, 10.86 ng / mL, 11.28 ng / mL and 25.87 ng / mL respectively; for another example, the binding IC50 value of the second antibody (6B11-1) to strains A2, Long, 9320 and 18537 can be 12.06 ng / mL, 11.98 ng / mL, 13.67 ng / mL and 44.39 ng / mL respectively; for another example, the binding IC50 value of the third antibody (6B11) to strains A2, Long, 9320 and 18537 can be 10.65 ng / mL, 16.46 ng / mL, 14.58 ng / mL and 32.85 ng / mL respectively.

[0068] In order to better understand the present application, the content of the present application will be further illustrated in combination with specific examples below, but the content of the present application is not limited to the examples below.

[0069] In the following examples, the amino acid sequences of the substances involved are shown in Table 1 below.

[0070] Table 1 Amino acid sequences of substances

[0071]

[0072]

[0073]

[0074]

[0075] 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.

[0076] Example 1

[0077] The embodiments of the present application provide a screening, expression and purification process of respiratory syncytial virus (RSV) neutralizing antibodies.

[0078] (I) Isolation and activation of RSV positive memory B cells

[0079] (1) Collect peripheral blood samples from healthy volunteers, separate PBMCs by sucrose gradient density centrifugation (cytiva, 17144003). Enrich B cells by magnetic bead purification through B Cell Isolation Kit II (miltenyibiotec, 130-091-151), and then sort RSV pre-F+CD19+IgG+cells by flow cytometry (CD19-APC, Cat. No. 302212; IgG-PE / Cy7, Cat. No. 410721; Manufacturer: biolegend; RSV pre-F-FITC, Cat. No. RSF-V52H3, Manufacturer: ACROBiosystems). As shown in the results, a total of 158 pre-F+CD19+IgG+cells were obtained by flow cytometry after enrichment of B cells. Figure 1

[0080] (2) Culture the enriched RSV positive memory B cells using ImmunoCult Human B Cell Expansion Kit (stemcell, 100-0645). After 7 days of culture, collect the cells and screen the B cells secreting RSV pre-F protein (ACROBiosystems, RSF-V82E7) by single-cell optical system (BerkeleyLights 'Beacon'), which are RSV positive B cells. Sort the single cells into 96-well plates, and pre-store 10 ul mineral oil (Biyun Tian, ST1524-50) and 5 ul TCL buffer (Qiagen, 1070498) in the 96-well plates. Store in -80℃, and then perform antibody heavy and light chain gene amplification.

[0081] ​(3) Cell isolation and expansion and antibody screening were performed according to the method described in the 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”.

[0082] (II) Amplification and recovery of VH / VL by single-cell-PCR technology

[0083] Amplification and recovery of antibody heavy and light chains in plasma cells by single-cell-PCR technology; the specific operation is as follows: first, add 10 μL RNAClean XP beads in each well to purify and separate RNA according to the manufacturer's instructions; cDNA synthesis and amplification are performed by Opto™ Plasma B Discovery cDNA Synthesis Kit; cDNA continues to amplify VH / VL by Opto B Discovery Sanger Prep Kit; the final amplification product is analyzed by gel electrophoresis and sent to GenScript for sequencing, and the gel electrophoresis results of the PCR products of part of the antibody heavy and light chain variable regions of single B cells are shown in FIG. 1, using 2000 bp DNA molecular weight marker as marker, heavy chain at about 700 bp, light chain at about 550 bp. Figure 2

[0084] (III) Preparation of recombinant antibodies

[0085] (1) The sequencing sequences were analyzed (https: / / www.imgt.org / ), and a total of 34 antibody variable region sequences were obtained, which were sent to GenScript for biosynthesis and constructed into κ light chain constant region or λ light chain constant region

[0086] (According to the subtype of light chain variable region respectively) and human IgG1-YTE heavy chain constant region in pCDNA3.4 vector, the subsequent puncture bacteria were obtained, and the general molecular biology method was used for streaking and shaking bacteria to extract plasmid; at the same time, antibody Palivizumab (referring to patent US7704505B2), Nirsevimab (referring to patent US11186628B2) and Clesrovimab (referring to patent US9963500B2) antibodies were used as positive controls.​

[0087] (2) One day before transfection (Day -1), adjust the cell density of ExpiCHO-S cells to (2-3.5) x 10 6 / mL, 37°C, 8% CO2, 120 rpm shaking overnight; on Day 0, cells are grown to 4-7 x 10 6 / mL, dilute the cells to 4 x 10 6 / mL using fresh pre-warmed CHOgro® expression medium (supplemented with 4 mM L-Glutamine and 0.3% Poloxamer); take 3 mL CHOgro® Complex Formation Solution in a sterile tube, add 30 μg of the above endotoxin-free antibody related plasmid and 20 μL TransIT-PRO® Reagent, mix gently and incubate at room temperature for no more than 5 min, add the complex to 30 mL of ExpiCHO-S cells prepared in advance, add 600 μL of CHOgro® Titer Enhancer, mix gently (CHOgro® Titer Enhancer can be added within 0-24 hours), and collect the cell culture supernatant after 14 days of culture at 125 rpm, 32°C, 8% CO2.

[0088] (3) Filter the cell culture supernatant with a 0.22 μm filter, load the protein A affinity chromatography column into the protein purification instrument, wash with water for 5 column volumes, then equilibrate with PBS buffer for 8 column volumes, pass the sample through the affinity chromatography column, then equilibrate with PBS buffer until the UV value no longer changes, collect the eluted sample by connecting the elution buffer, at a flow rate of 1 mL / min, ultrafiltrate and concentrate the eluted antibody sample solution and change the liquid to obtain the purified antibody; determine the antibody concentration, label and store at -20°C for long-term storage.

[0089] The expression and purification results show that 3 recombinant monoclonal antibodies were not successfully expressed and purified, and the remaining recombinant monoclonal antibodies were successfully prepared.

[0090] (Four) Verification of ELISA binding activity of recombinant monoclonal antibodies

[0091] The successfully expressed and purified recombinant monoclonal antibodies were tested for their binding activity to RSV pre-F protein using an ELISA test method, and the recombinant monoclonal antibodies with high binding ability were screened; specifically as follows: the RSV pre-F protein (RSF-V52H7, ACRObiosystems) was diluted to 0.5 ug / mL with PBS, 100 uL was added to each well of the ELISA plate for coating, and was incubated at 4°C overnight; the supernatant coating solution was discarded, and PBST was used for washing; 1% BSA-containing PBST was used for blocking at 37°C for 2 h; the blocking solution in the wells was discarded, and PBST was used for washing; different concentrations of the above-mentioned recombinant expressed monoclonal antibodies were added in a 1:3 gradient dilution solution of 1% BSA, and were incubated at 37°C for 1 h; the liquid was discarded, and PBST was used for washing; the diluted HRP secondary antibody was added to each well, and was incubated at 37°C for 1 h; the liquid was discarded, and PBST was used for washing; TMB color developing liquid was added to each well, and was reacted at room temperature for 10-15 min, the reaction was terminated by adding a termination solution, and the OD value at 450 nm was read, and GraphPad Prism 9 was used for analysis to calculate the EC50 value (when the OD450 value is half of the maximum value, the concentration of the antibody is the EC50 value of the antibody, and the smaller the EC50 value, the stronger the affinity of the antibody, and the higher the binding ability of the RSV pre-F protein (RSF-V52H7, ACRObiosystems)). According to the above-mentioned screening method, 15 recombinant monoclonal antibodies with high ELISA binding ability were screened.

[0092] (Five) Inhibition of RSV / A2 infection of Hep-2 cells by recombinant monoclonal antibodies

[0093] Strain A2 of RSV (from ATCC) was used to infect HEp-2 cells, and the top 15 recombinant monoclonal antibodies with high ELISA binding ability were selected for detection, and the ability of different concentrations of different recombinant monoclonal antibodies and positive controls to inhibit infection of different strains of cells was detected, and Palivizumab, Nirsevimab and Clesrovimab monoclonal antibodies were used as positive controls; specifically including:

[0094] Adjust the density of HEp-2 cells, inoculate into a 96-well plate, and place in a cell incubator (37°C, 5% CO2) overnight to ensure that the cell confluence is about 90% the next day, and then start the experiment; the first well is diluted to 10,000 ng / mL, and 3-fold gradient dilution is performed, a total of 8 dilution degrees (including the first well), the virus is added, and the 37°C, 5% CO2 incubator is incubated for about 1 hour; the virus and antibody neutralization product are added to the HEp-2 cells, and the incubator is incubated for about 22 hours, the supernatant is discarded, the cells are fixed, and then the fluorescently labeled detection antibody is added, and the fluorescent (enzyme-linked) immunospot analyzer instrument is used to read the plate; the data is summarized and analyzed by GraphPad Prism 9, and the IC50 value is calculated (the smaller the IC50 value, the stronger the neutralization ability of the antibody, and the stronger the ability to inhibit viral infection of cells).

[0095] According to the above method, the best neutralizing effect of the recombinant monoclonal antibody is screened out, named RSV-11 (hereinafter also referred to as 6B11), and the neutralization activity IC50 of the recombinant monoclonal antibody RSV-11 to the A2 strain is less than 100 ng / mL (14.45 ng / mL), and the neutralization ability is stronger than that of Palivizumab monoclonal antibody (962.90 ng / mL) and Clesrovimab monoclonal antibody (23.87 ng / mL), and the A2 strain neutralization activity is equivalent to that of Nirsevimab monoclonal antibody (2.41 ng / mL).

[0096] Example 2

[0097] The embodiment of the application provides a method for removing the PTM of the RSV-11 monoclonal antibody, and then screening the mutant with strong neutralization ability to the respiratory syncytial virus, and the specific steps are as follows:

[0098] (I) Design of different mutants of RSV-11 monoclonal antibody

[0099] The heavy chain variable region and the light chain variable region of RSV-11 are input into the abysis webpage, annotated, the Unusual residue in the FR region and the site residue (PTM) that may be post-translationally modified are viewed, the amino acids that maintain the effect on the CDR loop ring are replaced with the amino acids of the germ line gene according to the antibody structure reservation, and the sequence 6B11-2 is generated.

[0100] The 6B11-2 antibody was subjected to antigen-antibody interaction analysis using AlphaFold3, respectively. Specifically, the webpage https: / / alphafoldserver.com / was entered, the amino acid sequences of the heavy chain variable region and the light chain variable region of 6B11-2 were input according to the operation prompt, the amino acid sequence of the RSV A2 type pre-F DS Cav1 mutant was input, after completing the antigen-antibody interaction prediction, the top 5 antigen-antibody interaction results were downloaded and saved; secondly, the Calculate Mutation Energy (Binding) function was used in the Discovery Studio software, according to the operation prompt, the above saved 5 kinds of antigen-antibody interaction results were input in turn, single-point amino acid saturation mutation of the antibody binding site was carried out, the data was processed, and sequence 6B11-1 was generated.

[0101] (II) ELISA detection of the binding activity of recombinant monoclonal antibody mutants to proteins

[0102] According to the operation method described in step (four) 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.

[0103] The results are shown in Figure 3 , which show that the mutants have significantly improved binding ability to RSV A2 pre-F protein compared with the original antibody RSV-11; the mutant 6B11-1 has similar binding ability to RSV A2 pre-F protein as the original antibody RSV-11.

[0104] Example 3

[0105] Strains A type-A2 strain (A2), B type-18537 strain (B18537), B type-9320 strain (B9320) and A type-Long strain (Long) (all from ATCC) of RSV were used to infect HEp-2 cells, and the ability of different concentrations of different recombinant monoclonal antibodies to inhibit infection of different strains of cells was detected. The operation was carried out according to the method of step (five) of Example 1. The detection results are shown in Table 2 and Figure 4a , Figure 4b , Figure 4c and Figure 4d .

[0106] Table 2 Neutralization activity detection of recombinant antibodies to RSV A2 / 18537 / 9320 / Long strains

[0107]

[0108] Note: "R squared" value is "R square" value, which is an indirect reflection of the reliability of IC50 value in reaction data analysis, the higher the value, the more reliable the IC50 value.

[0109] From Table 2 and Figures 4a to 4d It can be seen that the neutralization activity of antibody 6B11-2 is significantly higher than that of palivizumab, with an order of magnitude difference; the in vitro neutralization activity of antibody 6B11-2 and Nirsevimab monoclonal antibody is 3.6 times lower for A2 strain, 2.3 times lower for Long strain, equivalent for 18537 strain, and 2.2 times lower for B9320 strain; antibody 6B11-1 and antibody 6B11 are compared, the neutralization activity of RSV A2 / 18537 / 9320 / Long strain after mutation is reduced, 6B11-2 and antibody 6B11 are compared, the neutralization activity of RSV A2 / 18537 / 9320 / Long strain after mutation is increased, within the allowable error range, it can be considered that there is no significant difference between the three antibodies, but the 6B11-2 antibody will be more superior in terms of antibody expression, immunogenicity and other drug properties than the antibody 6B11.

[0110] Example 4

[0111] The cotton rat model is used in the embodiment of the application to evaluate the in vivo effectiveness of the antibody, specifically as follows:

[0112] A number of cotton rats (SPF level, female, 6-8 weeks old) are grouped, 5 in each group, and the groups include: PBS group, palivizumab dose 0.5 mg / kg group, palivizumab dose 5 mg / kg group, 6B11-2 dose 0.5 mg / kg group, 6B11-2 dose 5 mg / kg group, Nirsevimab dose 0.5 mg / kg group, Nirsevimab dose 5 mg / kg group); different groups are respectively injected intramuscularly according to the corresponding dose for 1 day, and then the cotton rats in each group are infected with RSV A2 by nose drop, with a dose of 6x10 5 pfu (50 μL) per rat. After 4 days of infection, the cotton rats are dissected for sample collection, and lung virus load detection (plaque detection, detection method according to the literature "Zhu, Qing et al. "A highly potent extended half-life antibody as a potential RSV vaccine surrogate for all infants." Science translational medicinevol. 9,388 (2017): eaaj1928. doi:10.1126 / scitranslmed.aaj1928》 to evaluate the protective efficacy of the antibody.

[0113] 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.

[0114] 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), the antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising HCDR1, HCDR2 and HCDR3, the light chain variable region comprising LCDR1, LCDR2 and LCDR3; characterized in that: the sequence of HCDR1 is set forth in SEQ ID NO: 1, the sequence of HCDR2 is set forth in SEQ ID NO: 2, the sequence of HCDR3 is set forth in SEQ ID NO: 3 or SEQ ID NO: 17; the sequence of LCDR1 is set forth in SEQ ID NO: 9, the sequence of LCDR2 is set forth in SEQ ID NO: 10, the sequence of LCDR3 is set forth in SEQ ID NO:

11.

2. The antibody or antigen-binding fragment thereof of claim 1, characterized in that: the heavy chain variable region further comprises: HFR1 having at least 80% identity to the sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 19 and / or HFR2 having at least 80% identity to the sequence set forth in SEQ ID NO: 5 and / or HFR3 having at least 80% identity to the sequence set forth in SEQ ID NO: 6 or SEQ ID NO: 20 and / or HFR4 having at least 80% identity to the sequence set forth in SEQ ID NO: 7; and / or the light chain variable region further comprises: LFR1 having at least 80% identity to the sequence set forth in SEQ ID NO: 12 and / or LFR2 having at least 80% identity to the sequence set forth in SEQ ID NO: 13 and / or LFR3 having at least 80% identity to the sequence set forth in SEQ ID NO: 14 or SEQ ID NO: 22 and / or LFR4 having at least 80% identity to the sequence set forth in SEQ ID NO:

15.

3. The antibody or antigen-binding fragment thereof of claim 1, characterized in that: the antibody comprises a heavy chain variable region set forth in SEQ ID NO: 8 and a light chain variable region set forth in SEQ ID NO: 16; or the antibody comprises a heavy chain variable region set forth in SEQ ID NO: 18 and a light chain variable region set forth in SEQ ID NO: 16; or the antibody comprises a heavy chain variable region set forth in SEQ ID NO: 21 and a light chain variable region set forth in SEQ ID NO:

23.

4. The antibody or antigen-binding fragment thereof of any one of claims 1 to 3, wherein: the antibody or antigen-binding fragment thereof further comprises a heavy chain constant region and / or a light chain constant region, at least a portion of the heavy chain constant region and / or the light chain constant region being derived from at least one of a human antibody, a primate antibody or a mutant thereof.

5. The antibody or antigen-binding fragment thereof of claim 4, wherein: both the heavy chain constant region and the light chain constant region are derived from a human IgG antibody or a mutant thereof.

6. The antibody or antigen-binding fragment thereof of claim 5, wherein: both the heavy chain constant region and the light chain constant region are derived from a human IgG1 antibody, a human IgG4 antibody or a mutant thereof.

7. The antibody or antigen-binding fragment thereof of claim 6, wherein: the heavy chain constant region comprises a sequence having at least 70% identity to the sequence set forth in SEQ ID NO: 24; or the light chain constant region comprises a sequence having at least 70% identity to the sequence set forth in SEQ ID NO: 25 or SEQ ID NO:

26.

8. Any one of: (i) a nucleic acid encoding the antibody or antigen-binding fragment thereof of any one of claims 1 to 7; (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 the antibody or antigen-binding fragment thereof of any one of claims 1 to 7 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 pharmaceutical composition for treating respiratory syncytial virus infection comprising the antibody and / or antigen-binding fragment thereof of any one of claims 1 to 7 and / or the nucleic acid of (i) or the expression vector of (ii) and / or the engineered bacterium of (iii) and / or the engineered cell of (iv); (vii) a pharmaceutical preparation for preventing or treating a disease of respiratory syncytial virus infection comprising the antibody and / or antigen-binding fragment thereof of any one of claims 1 to 7 and / or the nucleic acid of (i) and / or the expression vector of (ii) and / or the engineered bacterium of (iii) and / or the engineered cell of (iv) and / or the pharmaceutical composition of (vi).

9. The substance of claim 8, wherein: The dosage form of the pharmaceutical preparation includes a spray, an oral liquid, a tablet, an atomizer, a granule, a capsule, or a paste.

10. Use of the antibody and / or antigen-binding fragment thereof of any one of claims 1 to 7 and / or the nucleic acid of (i) and / or the expression vector of (ii) and / or the engineered bacterium of (iii) and / or the engineered cell of (iv) in the preparation of a product for detecting respiratory syncytial virus.

11. Use of the antibody and / or antigen-binding fragment thereof of any one of claims 1 to 7 and / or the nucleic acid of (i) and / or the expression vector of (ii) and / or the engineered bacterium of (iii) and / or the engineered cell of (iv) in the preparation of a medicament for preventing or treating a disease of respiratory syncytial virus infection.

12. The use according to claim 11, characterized in that: The disease of respiratory syncytial virus infection is a respiratory infection.

13. The use according to claim 11, characterized in that: The disease of respiratory syncytial virus infection is bronchiolitis or pneumonia.

14. Use according to any one of claims 10 to 13, characterized in that: 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.

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