Antibody or antigen binding fragment against respiratory syncytial virus and application thereof

Abstract

The invention belongs to the field of antibody drugs, and particularly relates to an antibody or an antigen binding fragment aiming at respiratory syncytial virus and application thereof. 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 related diseases. The antibody has excellent binding capacity with the respiratory syncytial virus, and the in-vitro virus neutralizing effect and binding capacity are far higher than those of the existing marketed drug palizumab.

Description

Technical Field

[0001] The present invention belongs to the field of antibody drugs, and specifically relates to an antibody or antigen-binding fragment targeting respiratory syncytial virus and applications thereof. Background Art

[0002] Respiratory Syncytial Virus (RSV) is a common, highly contagious RNA virus belonging to the Paramyxoviridae family. RSV is primarily transmitted through respiratory droplets and contact, initially infecting the upper respiratory tract, but infection can also spread to the lower respiratory tract, particularly in infants and young children, where it is a major cause of viral pneumonia and bronchiolitis. Currently, there is no vaccine to prevent RSV, so there is a need to develop products that can prevent, diagnose, or treat diseases caused by RSV infection.

[0003] Antibodies are proteins that can specifically recognize antigens. They can identify and neutralize viruses by specifically recognizing viral envelope proteins, structural proteins, or enzymes. They are one of the current technical means for treating virus-related 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 adsorption to host cells, while F protein, as a fusion protein, plays a vital role in the process of virus entry into host cells. Currently, the neutralizing antibodies developed based on the above-mentioned F protein have low titers. For example, when clinically approved palivizumab is used to prevent RSV infection in high-risk infants and young children, up to 5 injections are required to cover the typical RSV season, patient compliance is low, and the cost of medication is high.

[0004] Therefore, developing an antibody against RSV is of great significance for preventing, diagnosing or treating diseases caused by RSV infection. Summary of the Invention

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

[0006] In order to achieve the above object, the present invention can adopt the following technical solutions:

[0007] In one aspect, the present invention provides an antibody or antigen-binding fragment against respiratory syncytial virus (RSV), wherein the antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3; and the antibody or antigen-binding fragment is selected from one of the following:

[0008] (a) Primary antibody: the amino acid sequence of HCDR1 is set forth in SEQ ID NO: 1, the amino acid sequence of HCDR2 is set forth in SEQ ID NO: 2, and the amino acid sequence of HCDR3 is set forth in SEQ ID NO: 3; the amino acid sequence of LCDR1 is set forth in SEQ ID NO: 9, the amino acid sequence of LCDR2 is set forth in SEQ ID NO: 10, and the amino acid sequence of LCDR3 is set forth in SEQ ID NO: 11;

[0009] (b) Second antibody: the amino acid sequence of HCDR1 is shown in SEQ ID NO: 17, the amino acid sequence of HCDR2 is shown in SEQ ID NO: 18, and the amino acid sequence of HCDR3 is shown in SEQ ID NO: 19; the amino acid sequence of LCDR1 is shown in SEQ ID NO: 25, the amino acid sequence of LCDR2 is shown in SEQ ID NO: 26, and the amino acid sequence of LCDR3 is shown in SEQ ID NO: 27.

[0010] Preferably, in the above-mentioned first antibody: the heavy chain variable region further comprises: HFR1 having at least 80% identity with the sequence shown in SEQ ID NO:4 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 and / or HFR4 having at least 80% identity with the sequence shown in SEQ ID NO:7; and / or the light chain variable region further comprises: 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 and / or LFR4 having at least 80% identity with the sequence shown in SEQ ID NO:15; or in the second antibody: the heavy chain variable region further comprises: HFR1 having at least 80% identity with the sequence shown in SEQ ID NO:20 and / or HFR2 having at least 80% identity with the sequence shown in SEQ ID NO:21. NO:21 and / or HFR3 and / or HFR4 that are at least 80% identical to the sequence shown in SEQ ID NO:22; and / or the light chain variable region further includes: LFR1 that is at least 80% identical to the sequence shown in SEQ ID NO:28 and / or LFR2 that is at least 80% identical to the sequence shown in SEQ ID NO:29 and / or LFR3 that is at least 80% identical to the sequence shown in SEQ ID NO:30 and / or LFR4 that is at least 80% identical to the sequence shown in SEQ ID NO:31.

[0011] Preferably, the first antibody comprises a heavy chain variable region that is at least 70% identical to the sequence shown in SEQ ID NO: 8 and a light chain variable region that is at least 70% identical to the sequence shown in SEQ ID NO: 16; or the second antibody comprises a heavy chain variable region that is at least 70% identical to the sequence shown in SEQ ID NO: 24 and a light chain variable region that is at least 70% identical to the sequence shown in SEQ ID NO: 32.

[0012] Preferably, the above-mentioned antibody or antigen-binding fragment further comprises a heavy chain constant region and / or a light chain constant region, and at least a portion 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.

[0013] More preferably, the heavy chain constant region and light chain constant region are both derived from human IgG antibodies or mutants thereof.

[0014] More preferably, the heavy chain constant region and light chain constant region are both derived from human IgG1 antibody, human IgG4 antibody or mutants thereof.

[0015] More preferably, the heavy chain constant region comprises a sequence that is at least 70% identical to the sequence shown in SEQ ID NO: 33, and the light chain constant region comprises a sequence that is at least 70% identical to the sequence shown in SEQ ID NO: 34.

[0016] In another aspect, the present invention provides any one of the following substances: (i) a nucleic acid encoding the antibody or antigen-binding fragment of the present invention; (ii) an expression vector comprising the nucleic acid in (i); (iii) an engineered bacterium comprising the expression vector in (ii); (iv) an engineered cell comprising the expression vector in (ii); (v) a product for detecting respiratory syncytial virus, comprising the antibody or antigen-binding fragment of the present invention, or the nucleic acid in (i), or the expression vector in (ii), or the engineered bacterium in (iii), or the engineered cell in (iv); (vi) a product for treating respiratory syncytial virus A pharmaceutical composition for treating respiratory syncytial virus infection, comprising the antibody and / or antigen-binding fragment of the present invention 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); (vii) a pharmaceutical preparation for preventing or treating respiratory syncytial virus infection, comprising the antibody and / or antigen-binding fragment of the present invention 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) and / or the pharmaceutical composition in (vi).

[0017] More preferably, the pharmaceutical composition in the above substances is a bispecific antibody, a polyspecific antibody, an ADC or a fusion protein.

[0018] More preferably, the dosage form of the pharmaceutical preparation in the above substances includes spray, oral solution, tablet, atomizer, granule, capsule or ointment.

[0019] In another aspect, the present invention provides a use of the antibody and / or antigen-binding fragment of the present invention 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.

[0020] In another aspect, the present invention provides use of the antibody and / or antigen-binding fragment of the present invention 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 preventing or treating respiratory syncytial virus infection.

[0021] Preferably, the respiratory syncytial virus infection disease is a respiratory tract infection.

[0022] Preferably, the respiratory syncytial virus infection disease is bronchiolitis or pneumonia.

[0023] Preferably, in the above application, the respiratory syncytial virus is strain A2 with a deposit number of ATCC, VR-1540, strain 18537 with a deposit number of ATCC, VR-1580, strain 9320 with a deposit number of ATCC, VR-955, or strain Long with a deposit number of ATCC, VR-26.

[0024] The beneficial effects of the present invention include at least: the antibodies provided by the present invention have excellent binding ability to respiratory syncytial virus. For example, the IC50 values ​​of the first antibody (RSV-G) binding to strains A2, 18537, 9320 and Long can reach 83.87 ng / ml, 17.1 ng / ml, 87.08 ng / ml and 32.18 ng / ml, respectively, which are significantly lower than the existing Palivizumab; for another example, the IC50 values ​​of the second antibody (RSV-E1) binding to strains A2, 18537, 9320 and Long can reach 117.8 ng / ml, 35.77 ng / ml, 44.93 ng / ml and 55.75 ng / ml, respectively, which are significantly lower than the existing Palivizumab. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is the result of PBMC flow cytometry sorting;

[0026] Figure 2 Agarose gel electrophoresis diagram of the amplified products of heavy and light chains of different antibodies;

[0027] Figure 3 The SDS-PAGE gel electrophoresis diagrams of different antibodies;

[0028] Figure 4 The binding of different antibodies to RSV pre-F recombinant protein;

[0029] Figure 5 The binding status of different antibodies to RSVpost-F recombinant protein. DETAILED DESCRIPTION

[0030] The examples are provided to better illustrate the present invention, but are not intended to limit the present invention to the examples. Therefore, non-essential improvements and adjustments to the embodiments made by those skilled in the art based on the above-mentioned invention still fall within the scope of protection of the present invention.

[0031] The terms used herein are only used to describe specific embodiments and are not intended to limit the present disclosure. Unless the context has a significantly different meaning, expressions in the singular include expressions in the plural. As used herein, it should be understood that terms such as "include", "have", "comprise" and the like are intended to indicate the presence of features, numbers, operations, components, parts, elements, materials or combinations. The terms of the present invention 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 may exist or may be added. As used herein, " / " may be interpreted as "and" or "or", depending on the circumstances.

[0032] As used herein, the term "antigen-binding fragment" refers to antigen-binding fragments of antibodies and antibody analogs, which generally include at least a portion of the antigen-binding region or variable region of the parent antibody, such as one or more CDRs; antibody fragments retain at least some of the binding specificity of the parent antibody.

[0033] The present invention provides an antibody or antigen-binding fragment against respiratory syncytial virus (RSV), wherein the antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3; the antibody or antigen-binding fragment of the present invention can be selected from any one of the following:

[0034] (a) Primary Antibody:

[0035] The amino acid sequence of HCDR1 is shown in SEQ ID NO: 1, the amino acid sequence of HCDR2 is shown in SEQ ID NO: 2, and the amino acid sequence of HCDR3 is shown in SEQ ID NO: 3; the amino acid sequence of LCDR1 is shown in SEQ ID NO: 9, the amino acid sequence of LCDR2 is shown in SEQ ID NO: 10, and the amino acid sequence of LCDR3 is shown in SEQ ID NO: 11;

[0036] (b) Secondary Antibody:

[0037] The amino acid sequence of HCDR1 is shown in SEQ ID NO: 17, the amino acid sequence of HCDR2 is shown in SEQ ID NO: 18, and the amino acid sequence of HCDR3 is shown in SEQ ID NO: 19; the amino acid sequence of LCDR1 is shown in SEQ ID NO: 25, the amino acid sequence of LCDR2 is shown in SEQ ID NO: 26, and the amino acid sequence of LCDR3 is shown in SEQ ID NO: 27.

[0038] It should be noted that the antibodies in the present invention can be any antibody form such as monoclonal antibodies or recombinant antibodies; in terms of source, they can be humanized antibodies or animal-derived antibodies, such as mouse-derived, rabbit-derived 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 are less stable than HCDR1 and HCDR2 and LCDR1 and LCDR2.

[0039] In some examples, in the above-mentioned first antibody: the heavy chain variable region may also include: HFR1 having at least 80% identity with the sequence shown in SEQ ID NO:4 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 and / or HFR4 having at least 80% identity with the sequence shown in SEQ ID NO:7; and / or the light chain variable region also 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 and / or LFR4 having at least 80% identity with the sequence shown in SEQ ID NO:15.

[0040] In some examples, in the above-mentioned second antibody: the heavy chain variable region may also include: HFR1 having at least 80% identity with the sequence shown in SEQ ID NO:20 and / or HFR2 having at least 80% identity with the sequence shown in SEQ ID NO:21 and / or HFR3 having at least 80% identity with the sequence shown in SEQ ID NO:22 and / or HFR4 having at least 80% identity with the sequence shown in SEQ ID NO:23; and / or the light chain variable region also includes: LFR1 having at least 80% identity with the sequence shown in SEQ ID NO:28 and / or LFR2 having at least 80% identity with the sequence shown in SEQ ID NO:29 and / or LFR3 having at least 80% identity with the sequence shown in SEQ ID NO:30 and / or LFR4 having at least 80% identity with the sequence shown in SEQ ID NO:31.

[0041] It should be noted that the FRs (HFR1, HFR2, HFR3, HFR4 and LFR1, LFR2, LFR3, LFR4) in the above-mentioned first antibody or second antibody are framework regions used to connect CDR regions and are relatively stable. In addition, the sequences of the FR regions and the CDR regions of the heavy and light chains in the above-mentioned antibodies can be obtained according to

[0042] The sequences of the heavy chain variable region and the light chain variable region of the two antibodies are arranged as follows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, respectively constituting the heavy chain variable region and the light chain variable region of the two antibodies; in addition, at least 80% identity in the present invention includes ≥80%, ≥85%, ≥90%, ≥95% or 100% identity, etc.

[0043] In some examples, the first antibody may include a heavy chain variable region that is at least 70% identical to the sequence shown in SEQ ID NO:8 and a light chain variable region that is at least 70% identical to the sequence shown in SEQ ID NO:16.

[0044] In some examples, the second antibody may include a heavy chain variable region that is at least 70% identical to the sequence shown in SEQ ID NO:24 and a light chain variable region that is at least 70% identical to the sequence shown in SEQ ID NO:32.

[0045] It should be noted that the above-mentioned at least 70% identity refers to an 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 above-mentioned sequences.

[0046] In some examples, the above-mentioned antibody or antigen-binding fragment further comprises a heavy chain constant region and / or a light chain constant region, and at least a portion 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.

[0047] In some examples, the heavy chain constant region and the light chain constant region are both derived from human IgG antibodies or mutants thereof.

[0048] In some examples, the heavy chain constant region and the light chain constant region are both derived from human IgG1 antibody, human IgG4 antibody or mutants thereof.

[0049] In some examples, the heavy chain constant region comprises a sequence that is at least 70% identical to the sequence shown in SEQ ID NO:33, and the light chain constant region comprises a sequence that is at least 70% identical to the sequence shown in SEQ ID NO:34.

[0050] It should be noted that the antibodies or antigen-binding fragments of the present invention, in addition to the heavy chain variable region and light chain variable region described above, also include 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 the light chain constant region can be derived from human or other animal sources (such as rabbit, porcine, etc.), and are regions that are almost immune to mutation. In addition, the heavy and light chains of the antibodies can also include signal peptides that can help the antibody penetrate the membrane; the signal peptides can be signal peptides known in the art.

[0051] It should be noted that, as mentioned above, the above-mentioned at least 70% identity refers to an 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 above-mentioned sequences.

[0052] The present invention also provides any one of the following substances:

[0053] (i) A nucleic acid encoding the antibody or antigen-binding fragment of the present invention; specifically, the nucleic acid of the present invention can be obtained by translating the antibody or antigen-binding fragment of the present invention according to conventional methods; alternatively, the nucleic acid can be a nucleotide sequence obtained by translating the above-mentioned amino acid sequence and further modifying the sequence; the modification method is a nucleotide modification method known in the art to increase expression efficiency or for other purposes;

[0054] (ii) an expression vector comprising the nucleic acid of (i); specifically, the expression vector of the present invention may 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 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);

[0055] (iii) an engineered bacterium, including the expression vector in (ii); specifically, the engineered bacterium in the present invention refers to a bacterium that can assist the expression of the expression vector, such as Escherichia coli. The Escherichia coli containing the expression vector herein refers to the Escherichia coli after the expression vector is transferred thereto;

[0056] (iv) engineered cells, including the expression vector in (ii); specifically, the engineered cells in the present invention refer to cells that can assist the expression of the above-mentioned expression vector, such as yeast cells;

[0057] (v) A product for detecting respiratory syncytial virus, comprising the antibody or antigen-binding fragment of the present invention, the nucleic acid of (i), the expression vector of (ii), the engineered bacteria of (iii), or the engineered cells of (iv); specifically, the antibody or antigen-binding fragment of the present invention has a strong binding effect with the surface protein of respiratory syncytial virus (RSV), and based on this, a product for detecting respiratory syncytial virus can be prepared; the product can be a detection reagent or a detection kit, for example, a reagent or kit based on immunoblotting, or a reagent or kit based on immunoenzyme technology, etc.;

[0058] (vi) A pharmaceutical composition for preventing or treating respiratory syncytial virus infection, comprising the antibody and / or antigen-binding fragment of the present invention 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, based on the strong binding effect between the antibody or antigen-binding fragment of the present invention and the surface protein of respiratory syncytial virus (RSV), a pharmaceutical composition for preventing or treating respiratory syncytial virus infection can be prepared; the pharmaceutical composition herein refers to a pharmaceutical composition composed of the antibody or antigen-binding fragment of the present invention in combination with other small molecule compounds, polypeptides, antibodies or proteins that can be effectively used to prevent or treat respiratory syncytial virus infection; such as a bispecific antibody or polyspecific antibody combined with other antibodies, an ADC combined with a small molecule compound, or a fusion protein combined with other proteins; these can be selected and used according to specific circumstances;

[0059] (vii) A pharmaceutical preparation for preventing or treating respiratory syncytial virus infection, comprising the antibody and / or antigen-binding fragment of the present invention 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) and / or the pharmaceutical composition in (vi); specifically, the above-mentioned antibodies and / or antigen-binding fragments and the above-mentioned pharmaceutical composition can be added with pharmaceutically acceptable carriers to prepare different dosage forms suitable for different clinical needs; the dosage forms can include sprays, oral liquids, tablets, atomizers, 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.

[0060] Another embodiment of the present invention also provides the use of the antibody and / or 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) of the present invention in the preparation of a product for detecting respiratory syncytial virus.

[0061] It should be noted that it should be understood that the detection of respiratory syncytial virus can be used for the diagnosis of respiratory syncytial virus infection diseases, that is, the product for detecting respiratory syncytial virus in the present invention is also suitable for the diagnosis of respiratory syncytial virus infection diseases, such as the diagnosis of bronchiolitis or pneumonia; in addition, products for detecting respiratory syncytial virus include but are not limited to detection reagents, detection kits, reagent cards or microfluidic chips, etc.

[0062] The embodiments of the present invention also provide the use of the antibody and / or antigen-binding fragment of the present invention 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 preventing or treating respiratory syncytial virus infection.

[0063] It should be noted that the drugs in the present invention include the above-mentioned pharmaceutical compositions and pharmaceutical preparations, which can be prepared by combining the above-mentioned substances with other active ingredients to form a pharmaceutical composition, or by adding pharmaceutically acceptable carriers to form a pharmaceutical preparation.

[0064] In some examples, the respiratory syncytial virus infection is a respiratory tract infection.

[0065] It should be noted that the application of the present invention is applicable to all respiratory syncytial virus infection diseases, especially respiratory tract infection, namely RSV respiratory tract infection.

[0066] In some examples, the respiratory syncytial virus infection is bronchiolitis or pneumonia.

[0067] It should be noted that, as mentioned above, the application of the present invention is applicable to all respiratory syncytial virus infections, especially diseases directly related to respiratory tract infections such as bronchiolitis or pneumonia.

[0068] In some examples, in the above applications, the respiratory syncytial virus is strain A2 with a deposit number of ATCC, VR-1540, strain 18537 with a deposit number of ATCC, VR-1580, strain 9320 with a deposit number of ATCC, VR-955, or strain Long with a deposit number of ATCC, VR-26.

[0069] It should be noted that the respiratory syncytial virus in the present invention includes strains known in the art, such as one or more of strain A2, strain 18537, strain 9320 or strain Long.

[0070] In order to better understand the present invention, the content of the present invention is further explained below with reference to specific examples, but the content of the present invention is not limited to the following examples.

[0071] In the following examples, the variable region sequences of the antibodies RSV-G and RSV-E1 are shown in Table 1.

[0072] Table 1 Sequences of screened antibodies

[0073]

[0074]

[0075] 1. Screening, Expression, and Purification of Respiratory Syncytial Virus Neutralizing Antibodies

[0076] (1) Isolation and activation of RSV-positive memory B cells

[0077] Peripheral blood samples from healthy volunteers (Donor3, Donor8, and Donor9) were collected and sucrose gradient density centrifugation (cytiva, 17144003) was used to separate PBMCs. RSV pre-F+CD19+IgM-IgD- cells (CD19-APC, Catalog No.: 302212; IgM-FITC, Catalog No.: 314506; IgD-FITC, Catalog No.: 348206, Manufacturer: biolegend; RSV pre-F biotin, Catalog No.: RSF-V82E7, Manufacturer: ACROBiosystems) were separated by flow cytometry according to 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》Construct the cell line 3t3 / nih-CD40L-BAFF-IL21, stimulate and expand the culture for 7 days, and collect the cells for the single cell optical guidance system platform beacon @ In addition, the collected cells were subjected to flow cytometry sorting, and the results were as follows: Figure 1 As shown, the proportion of RSV pre-F+CD19+IgM-IgD- cells of Donor3 was 0.86%, the proportion of RSV pre-F+CD19+IgM-IgD- cells of Donor8 was 0.72%, and the proportion of RSV pre-F+CD19+IgM-IgD- cells of Donor9 was 0.23%.

[0078] (2) Positive antibody screening

[0079] Positive B cells were screened by the single-cell light-guided system (Berkeley Lights'Beacon) (the screening strategy and method refer to the literature "Hastie KM, Yu X, Ana-Sosa-Batiz F, et al. Potent Omicron-neutralizing antibodies isolated from a patient vaccinated 6 months before Omicron emergence. Cell Rep. 2023; 42(5): 112421. doi: 10.1016 / j.celrep.2023.112421"), and 4 positive cells were screened out; the 4 positive cells screened out were named RSV-G cells, RSV-E1 cells, RSV-6 cells and RSV-H4 cells, respectively; and the heavy and light chains were recovered according to the method in the above literature to obtain antibodies RSV-G, RSV-E1, RSV-6 and RSV-H4. The obtained antibodies were sent to Qingke Bio for sequencing to obtain the antibody variable region sequence. The agarose gel electrophoresis diagram of the heavy and light chain amplification products of single cells is shown in the figure below. Figure 2 As shown, the heavy chain variable region is about 700 bp and the light chain variable region is about 600 bp, both of which are consistent with the theoretical size.

[0080] (3) Antibody expression and purification

[0081] Four antibodies, RSV-G, RSV-E1, RSV-6, and RSV-H4, were sent to Qingke Biotechnology for synthesis, and full-length antibodies were constructed (wherein the heavy chain and light chain constant region sequences of the antibodies are shown in SEQ ID NO: 33 and SEQ ID NO: 34, respectively). They were then constructed into the PCDNA3.4 vector, and subsequently punctured bacteria were obtained. Streaking and shaking were performed to extract plasmids (streaking isolation, shaking amplification, and plasmid extraction) as described in the "Molecular Cloning Laboratory Manual (Fourth Edition) (Original Version)"; antibody expression and purification were performed as described in patent CN117186215A (refer to paragraphs

[0085] to

[0091] of the specification), and finally antibodies RSV-G, RSV-E1, RSV-6, and RSV-H4 were obtained; after purification, 5% mannitol and 5% trehalose were added to the antibodies, aliquoted, and stored at -20°C for long-term storage.

[0082] The purified antibody was subjected to SDS-PAGE gel electrophoresis. Figure 3 As shown, the results showed that the purity of the recombinantly expressed and purified antibodies was greater than 95%, the antibody heavy chains were approximately 55 kDa, and the light chains were approximately 25 kDa, which can be used for subsequent functional experiments.

[0083] 2. Antibody Binding Activity Analysis

[0084] Four antibodies, RSV-G, RSV-E1, RSV-6, and RSV-H4, were tested for binding activity against RSV pre-F (RSF-V52H7, ACROBiosystems) and RSV post-F (RSF-V52H6, ACROBiosystems) proteins and compared with the marketed drug Palivizumab (B593701, Abinvivo). RSV pre-F (RSF-V52H7, ACROBiosystems) and RSV post-F (RSF-V52H6, ACROBiosystems) were coated on 96-well ELISA plates at 1 μg / mL, 100 μL / well, and coated overnight at 4°C. The supernatant coating solution was discarded and the plates were washed with PBST. The plates were washed with PBST containing 1% BSA. Block at 37°C for 2 h; discard the blocking solution in the wells and wash with PBST; add RSV-G, RSV-E1, RSV-6 and Palivizumab at different concentrations diluted 1:3 in 1% BSA solution, incubate at 37°C for 1 h; discard the liquid and wash with PBST; add 100 μl of diluted HRP secondary antibody to each well and incubate at 37°C for 1 h; discard the liquid and wash with PBST; add 100 μl of TMB colorimetric solution to each well and react at room temperature for 15 min. Add stop solution to terminate the reaction, read the OD value at 450 nm, make a graph and analyze with GraphPad Prism 9.

[0085] The OD450 values ​​of RSV-G, RSV-E1, RSV-6, RSV-H4, and Palivizumab are as follows: Figure 4 and Figure 5 As shown in Figure 2, RSV-G, RSV-E1, RSV-6, RSV-H4, and Palivizumab all bound to RSV pre-F recombinant protein in a concentration-dependent manner, indicating that RSV-G, RSV-E1, RSV-6, and Palivizumab are specifically bound to RSV pre-F recombinant protein (see Figure 4 RSV-E1 and Palivizumabn can bind to RSV post-F recombinant protein in a concentration-dependent manner, indicating that RSV-E1 and Palivizumab are specific for RSV post-F recombinant protein (see Figure 5 ).

[0086] 3. Broad-spectrum neutralizing activity of antibodies

[0087] In the neutralization activity test of RSV-G, RSV-E1, RSV-6, Palivizumab, and RSV-H4 antibodies, four standard RSV strains A2 (VR-1540, ATCC), 18537 (VR-1580, ATCC), 9320 (VR-955, ATCC), and Long (VR-26, ATCC) were used to infect HEp-2 cells respectively, and the ability of different antibody concentrations to inhibit the infection of cells by different strains was tested; the density of HEp-2 cells was adjusted, seeded into 96-well plates, and placed in a cell culture incubator (37°C, 5% CO2) for overnight culture. The experiment could be started when the cell confluence was about 90% on the next day; the four antibodies RSV-G, RSV-E1, RSV-6, Palivizumab, and negative control antibody were diluted to 10,000 ng / ml in the first well, and a 3-fold serial dilution was performed, with a total of 8 dilutions (including the first well) set, and the strains were added and incubated at 37°C, 5% CO2. Neutralize in a CO2 incubator for approximately 1 hour. The strain and antibody-neutralized product were added to HEp-2 cells and incubated in an incubator for approximately 22 hours. The supernatant was discarded, and the cells were fixed before adding fluorescently labeled detection antibodies. The plates were read using a fluorescent (enzyme-linked) immunospot analyzer. Graphs were created and analyzed using GraphPad Prism 9, and IC50 values ​​were calculated (a lower IC50 value indicates a stronger neutralizing ability of the antibody, and a greater ability to inhibit viral infection of cells). The results showed that antibodies RSV-G and RSV-E1 were more effective than palivizumab and RSV-6, while antibody RSV-H4 had no neutralizing activity. The IC50 values ​​for RSV-G, RSV-E1, and palivizumab are shown in Table 2 below.

[0088] Table 2 Neutralization activity detection of anti-respiratory syncytial virus monoclonal antibodies

[0089]

[0090] From Table 2 above, we can see that:

[0091] The neutralizing activity of RSV-G against strains A2, 18537, 9320, and Long is much higher than that of the marketed drug Palivizumab against these strains; for example, the neutralizing activity of RSV-G against strain A2 is approximately three times that of Palivizumab, the neutralizing activity of RSV-G against strain 18537 is approximately five times that of Palivizumab, the neutralizing activity of RSV-G against strain 9320 is approximately six times that of Palivizumab, and the neutralizing activity of RSV-G against strain Long is approximately three times that of Palivizumab.

[0092] The neutralizing activity of RSV-E1 against strains A2, 18537, 9320 and Long is much higher than the neutralizing activity of the marketed drug palivizumab against the above strains; for example, the neutralizing activity of RSV-E1 against strain A2 is about twice that of palivizumab, the neutralizing activity of RSV-E1 against strain 18537 is about twice that of palivizumab, the neutralizing activity of RSV-E1 against strain 9320 is about 12 times that of palivizumab, and the neutralizing activity of RSV-E1 against strain Long is about twice that of palivizumab.

[0093] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not limiting. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the purpose and scope of the technical solutions of the present invention, which should all be covered by the scope of the claims of the present invention.

Claims

1. An antibody or antigen-binding fragment against respiratory syncytial virus (RSV), the antibody or antigen-binding fragment 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; characterized in that: The antibody or antigen-binding fragment is selected from one of the following: (a) Primary Antibody: The amino acid sequence of HCDR1 is shown in SEQ ID NO: 1, the amino acid sequence of HCDR2 is shown in SEQ ID NO: 2, and the amino acid sequence of HCDR3 is shown in SEQ ID NO: 3; the amino acid sequence of LCDR1 is shown in SEQ ID NO: 9, the amino acid sequence of LCDR2 is shown in SEQ ID NO: 10, and the amino acid sequence of LCDR3 is shown in SEQ ID NO: 11; (b) Secondary Antibody: The amino acid sequence of HCDR1 is shown in SEQ ID NO:17, the amino acid sequence of HCDR2 is shown in SEQ ID NO:18, and the amino acid sequence of HCDR3 is shown in SEQ ID NO:19; the amino acid sequence of LCDR1 is shown in SEQ ID NO:25, the amino acid sequence of LCDR2 is shown in SEQ ID NO:26, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:

27.

2. The antibody or antigen-binding fragment according to claim 1, characterized in that: In the primary antibody: The heavy chain variable region further comprises: HFR1 having at least 80% identity with the sequence shown in SEQ ID NO:4 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 and / or HFR4 having at least 80% identity with the sequence shown in SEQ ID NO:7; and / or The light chain variable region further comprises: an LFR1 having at least 80% identity to the sequence shown in SEQ ID NO: 12 and / or an LFR2 having at least 80% identity to the sequence shown in SEQ ID NO: 13 and / or an LFR3 having at least 80% identity to the sequence shown in SEQ ID NO: 14 and / or an LFR4 having at least 80% identity to the sequence shown in SEQ ID NO: 15; or In the secondary antibody: The heavy chain variable region also includes: HFR1 having at least 80% identity with the sequence shown in SEQ ID NO:20 and / or HFR2 having at least 80% identity with the sequence shown in SEQ ID NO:21 and / or HFR3 having at least 80% identity with the sequence shown in SEQ ID NO:22 and / or HFR4 having at least 80% identity with the sequence shown in SEQ ID NO:23; and / or the light chain variable region also includes: LFR1 having at least 80% identity with the sequence shown in SEQ ID NO:28 and / or LFR2 having at least 80% identity with the sequence shown in SEQ ID NO:29 and / or LFR3 having at least 80% identity with the sequence shown in SEQ ID NO:30 and / or LFR4 having at least 80% identity with the sequence shown in SEQ ID NO:

31.

3. The antibody or antigen-binding fragment according to claim 1, characterized in that: The first antibody comprises a heavy chain variable region that is at least 70% identical to the sequence shown in SEQ ID NO: 8 and a light chain variable region that is at least 70% identical to the sequence shown in SEQ ID NO: 16; or The second antibody comprises a heavy chain variable region that is at least 70% identical to the sequence shown in SEQ ID NO:24 and a light chain variable region that is at least 70% identical to the sequence shown in SEQ ID NO:

32.

4. The antibody or antigen-binding fragment according to any one of claims 1 to 3, characterized in that: The antibody or 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 antigen-binding fragment according to claim 4, characterized in that: The heavy chain constant region and the light chain constant region are both derived from human IgG antibodies or their mutants.

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

7. The antibody or antigen-binding fragment according to claim 6, characterized in that: The heavy chain constant region comprises SEQ ID The sequence shown in NO:33 has at least 70% identity, and the light chain constant region comprises a sequence having at least 70% identity with the sequence shown in SEQ ID NO:

34.

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

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

10. The substance according to claim 8 or 9, characterized in that: The dosage forms of the pharmaceutical preparations include sprays, oral solutions, tablets, atomizers, granules, capsules or ointments.

11. Use of the antibody and / or antigen-binding fragment described in any one of claims 1 to 7 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. Use of the antibody and / or antigen-binding fragment described in any one of claims 1 to 7 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 preventing or treating respiratory syncytial virus infection.

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

14. The use according to claim 12, characterized in that: RSV disease is bronchiolitis or pneumonia.

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

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