Anti-RSV and hMPV antibody or antigen binding fragment and application thereof

Abstract

According to the antibodies or antigen binding fragments thereof, (a) and (b) show two neutralizing antibodies or antigen binding fragments thereof (X80 and X233). The two antibodies and the antibody mixture of the two have good neutralizing activity on RSV A and B subtypes and hMPV in vitro. In addition, they also protect rats from RSV attacks in vivo. The Cryo-EM structure characterizes that X80 is combined with an epitope before fusion on an antigen site # imgabs0 # of RSV F glycoprotein, and X233 is combined with an antigen site III on the RSV F glycoprotein and an antigen site III on the hMPV F glycoprotein. In addition, the antibody mixtures X80 and X233 also have a protective effect on the rats RSV. Thus, these findings may contribute to promoting the development of cross-neutralizing antibodies against RSV and HMPV, thereby protecting infants from ALRI caused by infection by these common pathogens.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to an antibody against RSV and hMPV or an antigen-binding fragment thereof and an application thereof. Background Art

[0002] Human respiratory syncytial virus (RSV) and human metapneumovirus (hMPV) are both members of the Pneumoviridae family and are single-stranded, negative-sense RNA viruses. RSV is the most common pathogen causing acute lower respiratory tract infections (LRTIs) in children under 5 years of age, contributing significantly to the global burden of morbidity and mortality. hMPV is another pathogen that also primarily infects children, the elderly, and immunocompromised individuals. Children under 5 years of age are the primary target population, and infants under 12 months of age are particularly at high risk for severe infection. However, no specific antiviral drugs, monoclonal antibodies, or vaccines are currently approved for RSV and hMPV.

[0003] RSV is divided into two major subtypes, A and B. Due to the high conservation of the F protein, it has become a promising target for the development of RSV antibody drugs and vaccines. RSV F protein is a class I transmembrane protein that assembles into trimers and undergoes a conformational change from a metastable pre-fusion state (pre F) to a stable post-fusion state (post F) when the viral envelope fuses with the host membrane. However, pre F has more neutralization sites than post F. Pre-fusion F has six antigenic sites ( I, II, III, IV and V), three of which ( V, III) only exist in the pre-fusion F preF trimer. Site Sites 1 and 2 are the most potent antigenic sites and are often considered candidate antigens for antibody and vaccine development. Furthermore, site III is considered a relatively conserved antigenic site. A stable pre-F protein was constructed several years ago. One of the stable prefusion forms of the pre-F antigen is DS-Cav1, which has three amino acid mutations (S155C / S290C / S190F). The hMPV F protein is also a class I fusion protein and has two structures. The stable pre-fusion F protein has stronger antigenicity. Compared to the RSV F protein, the hMPV F protein shares 33% to 35% sequence identity with other RSVs. The F gene shares some similarities between RSV and hMPV, leading to similar strategies targeting the viral surface, with glycoprotein fusion (F) proteins used for monoclonal antibody and vaccine development. Passive immunization with human monoclonal antibodies (mAbs) is highly specific, can enhance immune responses, and is a safe and effective approach to protect young children, especially high-risk infants, from RSV and hMPV infection. Currently, two anti-RSV monoclonal antibodies have been approved by the US Food and Drug Administration (FDA), and several candidate drugs are in late-stage clinical trials. One is palivizumab (Synasis@), which has been approved since 1998. However, it has many disadvantages due to its short half-life, high dose, high cost, and clinical application to premature infants or children with high-risk factors. Recently, another mAb nirsevimab (MEDI8897) has been approved by the FDA after a single dose showed effective protection against the entire RSV epidemic season. Nirsevimab targets the antigenic site on pre-F This site has been shown to be the least conserved region on the F glycoprotein and has a higher risk of immune escape. Therefore, there is still an urgent need to develop antibodies that bind to other epitopes or different sites. To date, although many neutralizing mAbs that bind to the hMPV F protein have been identified, there are still no licensed mAbs against hMPV. Among these neutralizing mAbs, M4B06 exhibited excellent neutralizing activity against hMPV subtypes A and B, but failed to neutralize RSV A and B subtypes. Although MPE8 targets site III, it showed cross-neutralization against RSV and hMPV infection, but the neutralizing activity was significantly weaker than other specific antibodies against RSV or hMPV previously described. In short, there is an urgent need for more effective and broader neutralizing antibodies. Summary of the Invention

[0004] Therefore, the technical problem to be solved by the present invention is to provide an antibody or antigen-binding fragment thereof against RSV and hMPV and its application.

[0005] To this end, the present invention provides the following technical solutions:

[0006] An antibody or antigen-binding fragment thereof comprising at least one of the following:

[0007] (a) The amino acid sequence of the variable region of the heavy chain of the antibody or antigen-binding fragment thereof comprises:

[0008] QVQLVESGGGVVQPGTSLTLSCAASGFTFRTYAFHWVRQAPGKGLEWLALVTYDGTTQYYADSVKGRLTIYRDNSKNTLFLHLNSLRRDDTAIYFCARGGEGSFSWLGYLQYMDVWGQGTTVTVSS; or an amino acid sequence having at least 80%, 85%, 90%, or 95% similarity thereto;

[0009] The amino acid sequence of the variable region of the light chain comprises:

[0010] QSALTQPASVSGSPGQSITLSCTGTSSDIGDYDYVSWYQKYPDTAPKLVIYDVSERPSGVSTRFSGSKSGNTASLTISGLQPEDEADYYCNSYSSTNTLKFGGGTKLTVL; or having at least 80%, 85%, 90%, 95% similarity to the amino acid sequence;

[0011] (b) the amino acid sequence of the variable region of the heavy chain of the antibody or antigen-binding fragment thereof comprises:

[0012] VVQLVESGGGLVKPGESLRLSCAVSGSMFSSYVMHWVRQAPGKGLDWVSSITGGGNYISYADSVKGRFIISRDNGRNSLSLQMSSLRVDDTAVYYCVRGLSGVMGVTWFDSWGQGTLVTVSS; or an amino acid sequence having at least 80%, 85%, 90%, or 95% similarity thereto;

[0013] The amino acid sequence of the variable region of the light chain comprises:

[0014] QSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGFDVHWYQHLPGKAPKVIIYENSHRPSGVPDRFFGSKSGTSASLSISGLQPEDEADYYCQSYDRGLDWVFGGGTKLTVL or an amino acid sequence that is at least 80%, 85%, 90%, or 95% similar to the sequence.

[0015] Optionally, the antibody or antigen-binding fragment thereof is selected from any one of monoclonal antibodies mAb, Fab, Fab′, F(ab′)2, Fd, Fv, dAb, complementarity determining region fragment, and single-chain antibody.

[0016] Optionally, the constant region of the antibody is selected from any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE and IgD; optionally, it is IgG1;

[0017] And / or, the species of origin of the constant region of the antibody is human, mouse, rat, cow, horse, sheep, rabbit or dog; optionally, human.

[0018] The antibody or its antigen-binding fragment is a bispecific antibody, a multispecific antibody, a chimeric antigen receptor, an immunoconjugate or a pharmaceutical composition.

[0019] A nucleic acid molecule encoding the antibody or antigen-binding fragment thereof.

[0020] Biological material, the biological material comprises the nucleic acid molecule, and the biological material is an expression cassette, a vector or a host cell.

[0021] The antibody conjugate is obtained by coupling the antibody or antigen-binding fragment thereof or the bispecific antibody or multispecific antibody with a label or protein; the label is selected from one or more of chemiluminescent dye labeling, enzyme labeling, biotin labeling, fluorescent dye labeling, colloidal gold labeling, and radioactive labeling.

[0022] The method for preparing the antibody or antigen-binding fragment thereof comprises: culturing a host cell capable of expressing the antibody or antigen-binding fragment thereof, and obtaining the antibody or antigen-binding fragment thereof by separation.

[0023] Any of the following uses of the antibody or antigen-binding fragment thereof, the bispecific antibody or multispecific antibody, the nucleic acid molecule, the biomaterial, or the antibody conjugate:

[0024] (1) Use in the preparation of a product for detecting the presence or level of human respiratory syncytial virus protein and / or human metapneumovirus protein in a sample;

[0025] (2) Use in the preparation of products for detecting or diagnosing human respiratory syncytial virus infection and / or human metapneumovirus infection;

[0026] (3) Use in the preparation of products for detecting or diagnosing diseases caused by human respiratory syncytial virus infection and / or diseases caused by human metapneumovirus infection;

[0027] (4) Use in the preparation of products with anti-human respiratory syncytial virus and / or human metapneumovirus activity;

[0028] (5) Use in the preparation of products for preventing, protecting or treating diseases caused by human respiratory syncytial virus infection and / or diseases caused by human metapneumovirus infection.

[0029] Optionally, the disease caused by human respiratory syncytial virus infection includes acute lower respiratory tract infection; optionally, includes interstitial pneumonia;

[0030] And / or, the disease caused by human metapneumovirus infection includes acute lower respiratory tract infection; optionally, includes interstitial pneumonia;

[0031] And / or, the human respiratory syncytial virus includes RSVA and B subtypes.

[0032] Optionally, the detection or diagnosis product includes a reagent, a kit, a pharmaceutical composition or a chip.

[0033] A reagent, kit, pharmaceutical composition or chip for detecting human respiratory syncytial virus and / or human metapneumovirus, comprising the antibody or antigen-binding fragment thereof, or the bispecific antibody or multispecific antibody according to claim 4, or the antibody conjugate according to claim 7.

[0034] Reagents, kits, and pharmaceutical compositions for preventing, protecting, or treating diseases caused by human respiratory syncytial virus infection and / or diseases caused by human metapneumovirus infection, comprising the antibody or antigen-binding fragment thereof, or the bispecific antibody or multispecific antibody, or the antibody conjugate.

[0035] The technical solution of the present invention has the following advantages:

[0036] 1. The antibodies or antigen-binding fragments thereof provided by the present invention, (a) and (b), show two neutralizing antibodies or antigen-binding fragments thereof (X80 and X233). Each of them and the antibody mixture of the two showed good neutralizing activity against RSV A and B subtypes and hMPV in vitro. In addition, they also protected cotton rats from RSV challenge in vivo. Cryo-EM structure characterized the interaction between X80 and RSV F glycoprotein antigenic site The antibody cocktail, X80 and X233, also protected against RSV in cotton rats. These findings may help promote the development of cross-neutralizing antibodies against RSV and HMPV, thereby protecting infants and young children from ALRI caused by these common pathogens. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0038] Figure 1a Binding test of the two antibodies in Example 2 to RSV Ds-Cav1 and hMPV pre F;

[0039] Figure 1b The surface plasmon resonance measurements of mAb X80 and mAb X233 binding to RSV Ds-Cav1 and hMPV pre F in Example 2 were performed.

[0040] Figure 1c The binding affinities of the two antibodies in Example 2 to RSV Ds-Cav1 and hMPV pre F;

[0041] Figure 1d The actual epitope determination results of the two antibodies in Example 2;

[0042] Figure 2 This is a flow chart of the cotton rat RSV prevention test in Example 3;

[0043] Figure 3 The results of mAb X80, mAb X233, and MEDI8897 in Example 3 in inhibiting viral titers in the lungs of cotton rats infected with RSV A2 are shown;

[0044] Figure 4 The results of mAb X80, mAb X233, and MEDI8897 in Example 3 in inhibiting viral titers in the lungs of cotton rats infected with RSV B9320 are shown;

[0045] Figure 5 This is a lung tissue section of cotton rats infected with RSV A2, in which mAb X80, mAb X233, and MEDI8897 inhibit the effects of these antibodies in Example 3;

[0046] Figure 6 The lung tissue pathology scores of mAb X80, mAb X233, and MEDI8897 in Example 3 in inhibiting RSV A2 infection in cotton rats;

[0047] Figure 7 The scores of mAb X80, mAb X233, and MEDI8897 in Example 3 for inhibiting interstitial pneumonia in cotton rats infected with RSV A2 are as follows;

[0048] Figure 8 This is a lung tissue section of cotton rats infected with RSV B9320, in which mAb X80, mAb X233, and MEDI8897 inhibit the effects of these antibodies in Example 3;

[0049] Figure 9 The lung tissue pathology scores of mAb X80, mAb X233, and MEDI8897 in Example 3 were inhibited in cotton rats infected with RSV B9320;

[0050] Figure 10 Scores for mAb X80, mAb X233, and MEDI8897 in Example 3 in inhibiting interstitial pneumonia in cotton rats infected with RSV B9320;

[0051] Figure 11 The results of mAb X80 and mAb X233+mAb X80 in Example 3 in inhibiting viral titers in the lungs of cotton rats infected with RSV A2 are shown;

[0052] Figure 12 The results of mAb X80 and mAb X233+mAb X80 in Example 3 in inhibiting viral titers in the lungs of cotton rats infected with RSV B9320 are shown;

[0053] Figure 13 This is a lung tissue section of cotton rats infected with RSV A2, inhibited by mAb X80 and mAb X233+mAb X80 in Example 3;

[0054] Figure 14 The lung tissue pathology scores of mAb X80 and mAb X233+mAb X80 in Example 3 in inhibiting RSV A2 infection in cotton rats;

[0055] Figure 15 The scores of mAb X80 and mAb X233+mAb X80 in inhibiting interstitial pneumonia in cotton rats infected with RSV A2 in Example 3;

[0056] Figure 16 This is a lung tissue section of cotton rats infected with RSV B9320, inhibited by mAb X80 and mAb X233+mAb X80 in Example 3;

[0057] Figure 17 The lung tissue pathology scores of mAb X80 and mAb X233+mAb X80 in Example 3 in inhibiting RSV B9320 infection in cotton rats;

[0058] Figure 18The scores of mAb X80 and mAb X233+mAb X80 in inhibiting interstitial pneumonia in cotton rats infected with RSV B9320 in Example 3;

[0059] Figure 19 The prophylactic antiviral activity of the antibody mixture mAb X80 and mAb X233 in Example 3 in the RSV-infected cotton rat model;

[0060] Figure 20 Flowchart of the complex of RSV pre-F trimer and the following composition in Example 4: (a) X80; (b) X233; (d) X80 and X233; and a flow chart of the complex of hMPV pre-F monomer and the following composition: (c) X233;

[0061] Figure 21 EM density maps in Example 4; (a) Gold standard FSC curves of the complexes of RSV pre-F with Fab X80 and X233, the complex of hMPV pre-F monomer and Fab X233, and the complex of RSV pre-F, Fab X80 and X233; (b) shows the local resolution assessment of the cryo-EM map using ChimeraX; (c) shows the cryo-EM density map of the complex of RSV pre-F with X80 and X233, and the complex of hMPV pre-F and its interface; residues are shown as sticks, oxygen is red, nitrogen is blue, and sulfur is yellow;

[0062] Figure 22 Sequence alignment analysis; Comparison of sequence conservation among respiratory syncytial virus A2, B9320, and hMPV A1 strains, showing regions interacting with X80 (left), X233 (center), and MEDI8897 (right);

[0063] Figure 23 is a structural diagram of the complex of X80 and RSV A2 DS-Cav1s in Example 4;

[0064] Figure 24 This is the conservation analysis of X233 between RSV and hMPV preF in Example 4. DETAILED DESCRIPTION

[0065] The following examples are provided for a better understanding of the present invention and are not intended to limit the best mode of implementation. They do not limit the content and scope of protection of the present invention. Any product identical or similar to the present invention obtained by anyone under the guidance of the present invention or by combining the features of the present invention with other prior arts shall fall within the scope of protection of the present invention.

[0066] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in this field can be used. If the manufacturer of the reagents or instruments is not specified, they are all commercially available conventional reagents.

[0067] Example 1 Acquisition of monoclonal antibodies X80 and X233

[0068] (1) Origin of viruses and cells

[0069]

[0070] RSV pre-F Pcaggs.dna(6394 bp):

[0071]

[0072]

[0073]

[0074]

[0075]

[0076] (2) Amplification and purification of RSV pre-F and hMPV pre-F proteins

[0077] A pre-stabilized RSV F (DS-Cav1) containing RSV F residues 1-513 and four mutations (S155C / S290C / S190F / V207L) was cloned individually into the pCAGGS vector, followed by a C-terminal T4 fiber protein trimerization motif and an 8xHis tag to facilitate protein purification, resulting in a recombinant plasmid. The hMPV F construct (hMPV F A1, 1-490 AA) containing three mutations (A185P, A113C, A339C) was inserted into a vector for structural studies, which also contained a C-terminal T4 fiber protein trimerization motif and an 8xHis tag, and was cloned into the pCAGGS vector to obtain a recombinant plasmid. To obtain these proteins, the recombinant plasmids constructed above were transiently transfected into HEK293F cells grown in suspension in a rotating humidified incubator at 37°C, provided with 8% CO2 and maintained at 130 rpm. After 72 hours of incubation, cells were harvested and centrifuged at 1000 x g for 30 minutes. The cell lysate supernatant was collected and exchanged into binding buffer using a tangential flow filtration cartridge. Proteins were collected by affinity chromatography using Ni-NTA-attached resin and further purified by size exclusion chromatography using a Superdex 200 column (GE Healthcare) in a buffer containing 20 mM Tris (pH 8.0) and 200 mM NaCl. Purified RSV preF (DS-Cav1) and hMPV preF proteins were obtained.

[0078] (3) Human subjects and PBMC (peripheral blood mononuclear cell) isolation

[0079] Informed consent was obtained from healthy adult volunteers (approved by the Institutional Review Board of the Children's Hospital of Chongqing Medical University, China). TMPeripheral blood mononuclear cells (PBMCs) were isolated from blood collected in EDTA tubes by Ficoll-Hypaque density gradient centrifugation on Sigma Aldrich tubes. PBMCs were then frozen in 90% heat-inactivated FBS supplemented with 10% dimethyl sulfoxide and stored in liquid nitrogen until thawed for use in experiments.

[0080] (4) Isolation of antigen-binding single memory B cells

[0081] The RSV pre F protein obtained in step (3) was fluorescently labeled with Alexa Fluor antibody 488 (Bio Legend), and CD19+, IgG+, CD27+ / - and preF+ cells were screened. Single memory B cells bound to the DS-Cav1 protein were sorted into a 96-well (polymerase chain reaction) PCR plate using a BD FACSAria III flow cytometer (BD Biosciences). RSV pre-F-specific memory B cell receptors were then sequenced using 10X genomic technology. X80 Fab and X233 Fab were sequenced, and their sequences were as follows:

[0082] The amino acid sequence of the variable region of the heavy chain of X80 Fab is as follows:

[0083]

[0084] The amino acid sequence of the light chain variable region is as follows:

[0085]

[0086]

[0087] The amino acid sequence of the variable region of the heavy chain of X233 Fab is as follows:

[0088]

[0089] The amino acid sequence of the light chain variable region is as follows:

[0090]

[0091] (5) Cloning, expression and purification of mAb and Fab

[0092] The gene sequences of the naturally paired heavy and light chain variable regions in step (4) above were synthesized using conventional methods and then subcloned into the pcDNA3,1 vector for HEK293F cell expression. The recombinant plasmids encoding the heavy and light chains of each antibody were transiently co-transfected into HEK293F cells and cultured at 37°C for 7 days under 8% CO2 to achieve antibody expression. The cell culture supernatant was collected and loaded onto a Protein A CIP column (GenScript). After washing and elution, the elution buffer was replaced with 1× phosphate-buffered saline (PBS) at pH 7.2, and mAb X80 and mAb X233 were collected. To obtain Fab fragments, purified mAb X80 and mAb X233 were incubated with papain using a Pierce FAB preparation kit (Thermo Scientific) and digested at 37°C for 5 hours. The fragments were desalted using a desalting column and the Fab fragments were separated by a Protein A column, which could bind to the Fc fragment. Then, X80 Fab and X233 Fab were collected for cryo-electron microscopy analysis.

[0093] The monoclonal antibodies mAb X80 and mAb X233 were characterized. X80 and X233 used IGVH3-33 and IGVH3-21 in their VH, respectively, with somatic hypermutation rates of 0.10 and 0.11, respectively. X80 used a VL with the IGLV2-14 germline gene, while X233 used a VL with the IGLV1-40 germline gene.

[0094] Example 2 In vitro binding, affinity and neutralization activity of mAb X80 and mAb X233

[0095] (1) ELISA binding test of mAb X80 and mAb X233 with RSV preF and hMPV preF, respectively

[0096] RSV preF and hMPV preF were coated separately in 96-well plates at 4°C overnight. The 96-well plates were blocked with PBS containing 1% BSA for 1 hour. Serially diluted mAb X80 or mAb X233 were added to each well and incubated at room temperature for 2 hours. The plate was then washed 3 times, and HRP-labeled goat anti-human Fc secondary antibody (1:5000, Abcam) was added and incubated at room temperature for 1 hour. The plate was washed with PBST (containing 0.05% Tween-20), and then incubated with the HRP substrate TMB to develop color, and terminated by adding 50 μl H2SO4. Finally, the binding ability was displayed by the absorbance at 450 nm. The higher the absorbance, the higher the binding ability. The results are shown in Figure 2. Figure 1a 、 Figure 1bAs shown, both antibodies can bind to RSV Ds-Cav1, and only X233 can bind to hMPV pre F.

[0097] (2) Surface plasmon resonance measurement of mAb X80 and mAb X233 binding affinity

[0098] DS-cav1 and hMPV preF were immobilized on a CM5 sensor chip using the NHS / EDC method using Biacore 8k (GE Healthcare) and PBS running buffer (supplemented with 0.05% Tween 20). Serial dilutions of mAbX80 and mAb X233 were injected at concentrations ranging from 250 to 3.9 nM. For the competition assay, purified RSV pre-F was immobilized on a CM5 sensor chip. The first sample was flowed through the chip at a rate of 20 μl / min for 120 s, and then the second sample was injected at the same rate for 120 s. The resulting data were fit to a 1:1 binding model using Biacore evaluation software (GE Healthcare) to determine the association rate constant (ka) and dissociation rate constant (kd) as well as the equilibrium dissociation constant KD (kd / ka) The results are as follows Figure 1b and Figure 1c As shown, X80 mAb only binds to RSV preF with a calculated KD of 6.33 nM for binding to RSV DS-Cav1, while the KD of X233 mAb is 0.19 nM. The KD of X233 mAb binding to hMPV pre-F is 26.6 pM.

[0099] (3) Biolayer interferometry

[0100] To further confirm the actual epitopes targeted by the two antibodies, a cross-competition assay was performed using biofilm interferometry with an RSV preF-specific antibody. The sensor was coated with RSV A2 solution protein. The mAbs were first loaded in the horizontal column, followed by the vertical column. If the maximum binding of the competing mAb decreased by >80% + 5%, the mAbs were considered to compete for the same site. If the maximum binding of the competing mAb was less than 20%, the mAb was considered non-competitive. See the table below:

[0101] Table 1

[0102]

[0103] The results of the test are as follows Figure 1d As shown, X80 competes with MEDI8897 and X233 competes with MPE8, indicating that X80X80 targets site, X233 targets site III.

[0104] (4) Neutralization test

[0105] The neutralization titers of RSV mAbs (mAb X80 and mAb X233, MEDI8897, MPE8, mAb X80 alone, mAb X233 alone) against RSV laboratory strains were assessed by plaque reduction neutralization assay. Antibodies were serially diluted in a 4-fold to 10-fold gradient. Equal volumes of virus were added to the antibody serial dilutions in a 96-well microplate and incubated at 37°C for 1 hour. 100 μL of the antibody-virus mixture per well was then added to a 24-well microplate inoculated with HEp-2 cells, and the plate was incubated at room temperature for 1 hour. The cells were then covered with 1 mL of DMEM medium supplemented with 1.2% methylcellulose and 2% FBS. After incubation for 5-7 days at 37°C, 5% CO2, plaques as a cytopathic effect were visually counted using a microscope, and the IC50 of the nonlinear fit was estimated in GraphPad Prism 9.3.1.

[0106] The results are shown in the table below. X80 neutralized RSV A2 and RSV B9320 with IC50s of 9.46 ng / mL and 3.73 ng / mL, respectively. Compared to MEDI8897 (IC50 for RSV A2: 3.14 ng / mL, IC50 for RSV B9320: 11.28 ng / mL), X233 had IC50s of 126.6 ng / mL and 30.54 ng / mL against RSV A2 and RSV B9320, respectively. MPE8 had an IC50 of 290.2 ng / mL for RSV A2 and 80.36 ng / mL for RSV B9320. These data demonstrate that both X80 and X233 neutralize RSV subtypes A and B. Notably, X80 was three times more potent against RSV B9320 than MEDI8897. X233 was more effective than MPE8 against RSV A2 and RSV B9320. Furthermore, X233 was found to neutralize hMPV with an IC50 of 23.25 ng / mL, while X80 was not. This confirms that X233 is a cross-neutralizing antibody against RSV and hMPV. Furthermore, the antibody mixture of X233 and X80 demonstrated comparable efficacy to X80, with an IC50 of 21.35 ng / mL against RSV A2 and 8.95 ng / mL against RSV B9320. Furthermore, it also exhibited comparable efficacy to X233, with an IC50 of 38.37 ng / mL against hMPV A1. These data suggest that the antibody mixture of X80 and X233 may be a promising candidate for use against a wide range of RSV and hMPV species.

[0107] Table 2

[0108]

[0109] Example 3 Cotton rat RSV prevention test

[0110] This example investigates the in vivo prophylactic efficacy in an animal challenge model. To assess whether the in vitro neutralizing activity of X80 and X233 translates into stronger antiviral activity in vivo, weight-based doses of X80, X233, and MEDI8897 were administered intramuscularly to cotton rats, which were then challenged with RSV A2 or RSV B9320 ( Figure 2 Lung tissues were harvested 4 days after challenge. The right lung was used to test RSV titers, and the left lung was used to evaluate histopathological scores. The specific method is as follows:

[0111] 6-8 week old cotton rats (Sigmodon hispidus) were intramuscularly injected with antibodies (mAb X80, mAbX233, MEDI8897) 1 day before challenge. The dose was determined based on the individual animal's body weight. For each experiment, the antibody dilution started at 2 mg / kg and was serially diluted 2-fold to 0.063 mg / kg (a total of 6 antibody dose groups, 5 animals / group). Only virus without antibody treatment was used as a negative control. After 24 hours, the animals were anesthetized using an isoflurane chamber and 6×10 5 Plaque-forming units (PFU) of RSV A2 or RSV B9320 were used for intranasal infection. Four days later, the animals were euthanized and the lungs were removed. The right lung was homogenized and titrated on HEp-2 cells in a plaque assay on a 24-well plate for viral load testing. After 2 hours of infection at 37°C, the test samples were removed and the cells were covered with 1 mL of 1.2% methylcellulose (DMEM supplemented with 2% fetal bovine serum). After incubation at 37°C for 5-7 days, the cells were fixed / stained with 2% crystal violet solution. Plaques as cytopathic effects were visually counted using a microscope and used to calculate pfu / g. The detection limit of the virus was 25 PFU / g tissue in the right lung. The left lung was fixed with 4% formalin for hematoxylin and eosin (H&E) stained histological sections. The animal studies were approved by the Institutional Animal Care and Use Committee of Sinovac and performed in accordance with the animal care guidelines. See the above method flow for details. Figure 2 .

[0112] Histological scoring of lung tissue

[0113] Cotton rats were sacrificed 4 days after the challenge, and the left lungs were collected and preserved in 4% neutral buffered formalin for 24 hours. The fixed tissues were paraffin embedded, sectioned and prepared into slices. The tissue sections were then stained with hematoxylin and eosin (H&E) for standard pathological assessment. All histopathological sections were read in a blinded and randomized manner to eliminate bias. The pathological scoring system was designed based on previous reports and provides a score of 0-5 for each site based mainly on the thickness of the inflammatory infiltrate around the bronchioles (peribronchiolitis) in the small blood vessels in the lung tissue (peribronchiolitis) and the lung interstitium (interstitial pneumonia). The specific evaluation criteria are shown in the table below.

[0114] Table 3 Evaluation criteria

[0115]

[0116]

[0117] In cotton rats infected with RSV A2, the results were as follows Figure 3 , mAb X80, mAb X233 and MEDI8897, showed dose-dependent antiviral activity in inhibiting viral titers in the lungs of cotton rats infected with RSV A2 and B9320. Compared with the PBS group, there was no enhancement of viral titers and lung pathology, and the lowest dose of all these mAbs was 0.063 mg / kg. In cotton rats infected with RSV A2, the protective effect of X80 was roughly equivalent to that of MEDI8897, and it could significantly reduce the viral load in the lungs of cotton rats (<2 PFU / g) at doses of 2 mg / kg and 1 mg / kg. X233 could prevent viral replication from exceeding 2 PFU / g at a dose of 2 mg / kg, although the overall protective effect of X233 on cotton rats infected with A2 was not as good as that of X80 and MEDI8897. Figure 5 、 Figure 6 X80 significantly reduced lung histopathology and interstitial pneumonia in cotton rats treated with X80 was significantly reduced (p < 0.01) even at low concentrations, with a dose of 0.125 mg / kg ( Figure 7 X233 can also reduce the total score of lung tissue pathology ( Figure 5 、 Figure 6 ) and alleviated interstitial pneumonia (p<0.05).

[0118] In cotton rats infected with RSV B9320, Figure 4 Among them, X80 was more effective in inhibiting viral replication. In the lungs of cotton rats at doses of 0.5 to 2 mg / kg, the virus titer was undetectable, a reduction of 2.71 log. In addition, the viral load of cotton rats treated with X80 was reduced by nearly 1.1 PFU / g (100 mg / kg) compared with cotton rats treated with MEDI8897 at a dose of 0.5 mg / kg. Figure 4). It was found that the neutralization potency of X80 in vitro and in vivo was consistent. This indicates that X80 is indeed more effective than MEDI8897 against RSV B9320. In addition, the data showed that X233 had a weaker protective effect on cotton rats than X80. However, it still had protective activity against RSV B9320 in vivo. Next, the histopathology of lung tissue was evaluated. The histology and total score of interstitial pneumonia in cotton rats treated with X80 and X233, respectively, were effectively alleviated (p < 0.01) ( Figure 8 、 Figure 9 、 Figure 10 ).

[0119] In vivo preventive efficacy of antibody mixture 80 and 233 in cotton rats

[0120] The prophylactic antiviral activity of the antibody mixture mAb X80 and mAb X233 in the RSV-infected cotton rat (Sigmodon hispidus) model was further evaluated. Figure 19 ) (RSV subtypes A and B). It was wondered whether the antibody mixture X80 and X233 had similar efficacy to X80 against RSV subtypes A and B in cotton rats. Cotton rats were injected intramuscularly with a mixture of X80 and X233 at a 1:1 ratio, with doses based on body weight. The animal model was established as described above, and viral load and histopathology were measured. Compared with treatment with X80 alone, the antibody mixture of X80 and X233 showed comparable preventive efficacy against RSV A2 and RSV B9320, respectively, at a dose of 2 mg / kg (each antibody dose was 2 mg / kg), reducing viral titers by more than 2 lg (log). In addition, cotton rats infected with RSV A2 and RSV B9320 also showed significantly reduced total histopathological scores of lung tissue and interstitial pneumonia when they received a dose of 1 mg / kg or 2 mg / kg of the antibody mixture X233 and X80. X80 and X233 may be potential broadly neutralizing candidate antibodies against RSV and hMPV. In cotton rats infected with RSV A2, the results of mAb X80 + mAb X233 are as follows Figure 12 、 16 、 Figure 17 、 Figure 18 In cotton rats infected with RSV B9320, the results of mAb X80 + mAb X233 are as follows: Figure 11 、 13 、 Figure 14 、 Figure 15 .

[0121] Example 4 Cryo-EM sample preparation and data collection

[0122] 0.8 mg / ml purified RSV pre-F was incubated with a 1.1-fold molar excess of X80 Fab in a solution containing 20 mM Tris pH 8.0, 200 mM NaCl at 4°C for 30 minutes. The incubated samples were mixed with 0.05% (mixed volume) N-dodecyl-β-D-maltoside (DDM) and then dropped onto pre-glow-discharged holey carbon-coated gold grids (C flat, 300 mesh, 1.2 / 1.3), blotted dry at 100% relative humidity for 5 seconds without applying force, and then immediately plunged into liquid ethane using a Vitrobot (FEI). A total of 4,518 micrographs were collected using a Krios G4 microscope at 300 kV. Movies were recorded using a Falcon4 Summit direct detector with a defocus range of 1.2-2.0 μm (32 frames, 0.22 seconds per frame, total dose Automated single-particle data acquisition was performed by SerialEM with a calibrated magnification of 75,000x and a final pixel size of

[0123] 0.8 mg / ml purified RSV pre-F was incubated with a 1.1-fold molar excess of X233 Fab in 20 mM Tris pH 8.0, 200 mM NaCl at 4°C for 30 minutes. The samples were mixed with 0.05% N-dodecyl-β-D-maltoside (DDM) and dropped onto pre-glow-discharged holey carbon-coated gold grids (C flat, 300 mesh, 1.2 / 1.3), blotted dry without force for 5 seconds at 100% relative humidity, and then immediately plunged into liquid ethane using a Vitrobot (FEI). A total of 2,773 micrographs were collected using a Krios Arctica microscope at 200 kV. Movies were recorded using a K2 Summit direct detector with a defocus range of 1.2-2.0 μm (32 frames of 0.22 seconds per frame for a total dose of Automated single-particle data acquisition was performed by SerialEM with a calibrated magnification of 130,000x and a final pixel size of Complete data collection parameters are reported in Table S4.

[0124] 3.0 mg / ml purified hMPV pre-F was incubated with a 1.2-fold molar excess of X233 Fab in 20 mM Tris pH 8.0, 200 mM NaCl at 4°C for 30 minutes. The samples were mixed with 0.05% N-dodecyl-β-D-maltoside (DDM) and then dropped onto pre-glow-discharged holey carbon-coated gold grids (C flat, 300 mesh, 1.2 / 1.3), blotted dry without force for 5 seconds at 100% relative humidity, and then immediately plunged into liquid ethane using a Vitrobot (FEI). A total of 5,001 micrographs were collected using a Krios G4 microscope at 300 kV. Movies were recorded using a Falcon4 Summit direct detector with a defocus range of 1.2-2.0 μm (32 frames, 0.22 seconds per frame, total dose Automated single-particle data acquisition was performed by SerialEM with a calibrated magnification of 75,000x and a final pixel size of

[0125] 0.8 mg / ml purified RSV pre-F was incubated with a 1.1-fold molar excess of X80 Fab and a 1.1-fold molar excess of X233 Fab in 20 mM Tris pH 8.0, 200 mM NaCl at 4°C for 30 minutes. The samples were mixed with 0.05% N-dodecyl-β-D-maltoside (DDM) and then dropped onto pre-glow-discharged holey carbon-coated gold grids (C flat, 300 mesh, 1.2 / 1.3), blotted dry without force for 5 seconds at 100% relative humidity, and then immediately plunged into liquid ethane using a Vitrobot (FEI). A total of 3,530 micrographs were collected using a Krios Arctica microscope at 200 kV. Movies were recorded using a K2 Summit direct detector with a defocus range of 1.2-2.0 μm (32 frames of 0.22 seconds per frame for a total dose of Automated single-particle data acquisition was performed by SerialEM with a calibrated magnification of 130,000x and a final pixel size of

[0126] Cryo-EM data processing

[0127] For the complex structure of X80 Fab with RSV pre-F, the micrograph stack was motion corrected using patch motion, and the contrast transfer function (CTF) was estimated using patch CTF estimation in CryoSPARC. The RSV pre-F template was imported into CryoSPARC to allow template picking. After several rounds of template picking, approximately 1,283,250 particles were found. The picked particles were inspected, extracted using a 300-pixel box size, and then Fourier cropped to a 150-pixel box size. After several rounds of 2D classification to remove junk particles, the remaining approximately 503,489 particles were extracted again using a 300-pixel box size. The particles were further cleaned and classified after multiple rounds of de novo, inhomogeneous, and homogeneous refinement, resulting in approximately 329,376 high-quality particles. The particles were further cleaned and classified using homogeneous and inhomogeneous refinement. Once further reducing the number of particles can no longer improve map quality and resolution, we perform local and global CTF refinement on a subset of the best particles and reapply non-uniform refinement.

[0128] For the complex structure of X233 Fab with RSV pre-F, the micrograph stack was motion corrected using block motion, and the contrast transfer function (CTF) was estimated using block CTF estimation in CryoSPARC. The RSV pre-F template was imported into CryoSPARC to allow template picking, and after several rounds of template picking, approximately 1,152,414 particles were obtained. The picked particles were inspected, extracted using a 300-pixel box size, and then Fourier cropped to a 150-pixel box size. After several rounds of 2D classification to remove junk particles, the remaining approximately 625,135 particles were extracted again using a 300-pixel box size. The particles were further cleaned and classified after multiple rounds of ab initio refinement using both heterogeneous and homogeneous refinement, yielding approximately 240,066 good particles. The particles were further cleaned and classified using both homogeneous and heterogeneous refinement. Once the map quality and resolution cannot be improved by further reducing the number of particles, a subset of the best particles is refined locally and globally using the CTF and then re-refined using non-uniform refinement.

[0129] For the complex structure of X233 Fab with hMPV pre-F, the micrograph stack was motion corrected using block motion, and the contrast transfer function (CTF) was estimated using block CTF estimation in CryoSPARC. After several rounds of template picking of approximately 1,743,174 particles, the hMPV pre-F template was imported into CryoSPARC to allow template picking. The picked particles were inspected, extracted using a 300-pixel box size, and then Fourier cropped to a 150-pixel box size. After several rounds of 2D classification to remove junk particles, the remaining approximately 750,269 particles were extracted again using a 300-pixel box size. The particles were further cleaned and classified after multiple rounds of de novo, inhomogeneous, and homogeneous refinement, yielding approximately 324,900 high-quality particles. Further purification and classification of the particles was performed using homogeneous and inhomogeneous refinement. Once further reducing the number of particles can no longer improve map quality and resolution, we perform local and global CTF refinement on a subset of the best particles and reapply non-uniform refinement.

[0130] For the complex structure of X80 Fab and X233 Fab with RSV pre-F, the micrograph stack was motion corrected using block motion, and the contrast transfer function (CTF) was estimated using block CTF estimation in CryoSPARC. After several rounds of template picking of approximately 865,861 particles, the RSV pre-F template was imported into CryoSPARC to allow template picking. The picked particles were inspected, extracted using a 300-pixel box size, and then Fourier cropped to a 150-pixel box size. After several rounds of 2D classification to remove junk particles, the remaining approximately 164,868 particles were extracted again using a 300-pixel box size. The particles were further cleaned and classified after multiple rounds of de novo, inhomogeneous, and homogeneous refinement, resulting in approximately 115,080 high-quality particles. The particles were further cleaned and classified using homogeneous and inhomogeneous refinement. Once the map quality and resolution cannot be improved by further reducing the number of particles, a subset of the best particles is refined locally and globally using the CTF and then re-refined using non-uniform refinement.

[0131] Cryo-EM model construction and improvement

[0132] RSV pre-F+X80 Fab complex model construction and refinement

[0133] The previously determined RSV F structure (4MMT) was used to generate the RSV pre-F trimer model. The model of X80 Fab was generated using AlphaFold33 by importing the amino acid sequence of X80 Fab. The two models were docked to the EM density map using UCSF Chimera (see Figure 21 RSV pre-F and X80 Fab were docked to the density map separately, saved as a model, and then reconnected in Coot. 36 Phenix refinement combinations 37-39 and Coot were used to further refine the model. Flowchart see Figure 20 .

[0134] RSV pre-F+X233 Fab complex model construction and refinement

[0135] The previously determined RSV F structure (4MMT) was used to generate the RSV pre-F trimer model. We used AlphaFold to generate a model of the X233 Fab by importing the amino acid sequence of the X233 Fab. The two models were docked into the electromagnetic density map ( Figure 21 RSV pre-F and X233 Fab were docked into the density map separately, saved as a model, and then reconnected in Coot. Phoenix refinement and Coot were used together to further refine the model. See the flowchart for details. Figure 20 .

[0136] hMPV pre-F+X233 Fab complex model construction and refinement

[0137] The previously determined hMPV pre-F structure (5WB0) was used to generate the hMPV pre-F model. The previously generated X233 Fab model was used. The two models were docked into the electromagnetic density map using UCSF Chimera ( Figure 21 ). HMPV pre-F and X233 Fab were docked into the density map separately, saved as a model, and then reconnected in Coot. Phoenix refinement and Coot were used together to further refine the model. Flowchart is shown in Figure 20 .

[0138] Construction and refinement of the RSV pre-F+X80 Fab+X233 Fab complex model

[0139] The previously determined RSV F structure (4MMT) was used to generate the RSV pre-F trimer model. The previously generated X233 Fab and X80 Fab models were used. The three models were docked into the EM density ( Figure 21) UCSF Chimera was used to draw the y map. RSV pre-F, X80 Fab, and X233 Fab were docked into the density map separately, saved as a model, and then reconnected in Coot. Phoenix refinement and Coot were used in combination to further refine the model. Flowchart is shown in Figure 20 .

[0140] Here are the results:

[0141] The structure of the complex between X80 and RSVA2 DS-Cav1s (see Figure 23 (Figure a)

[0142] To precisely define the epitope, single-particle cryo-electron microscopy was used to resolve the complex between the X80 Fab fragment and the stabilized pre-fusion RSV V A2 F ecodomain trimer. Cryo-electron microscopy results showed that each monomer of the homotrimeric preF was occupied by a Fab. The final resolution of the X80-RSV preF complex was When observing the F trimer along the three-fold axis, we found that X80 bound only to a single monomer, an interface that buried the Analysis of the X80 binding site with preF revealed that the heavy chain participates in more interactions than the light chain, forming hydrogen bonds with RSV F across all CDR loops. Arp54 and Tyr53 of X80 in the CDRH2 loop primarily form hydrogen bonds with Asn165 and Glu264 on RSV F, while Arp33 and Tyr34 in the CDRL1 loop and Tyr109 in the CDRH3 loop participate in major interactions with Lys56 and Lys65. In addition to hydrogen bonding interactions, Trp106 of X80 in CDRH3 participates in hydrophobic interactions with Leu204 and Pro205. The protective efficacy of mAb X80 against cotton rats demonstrated a stronger protective effect than MEDI8897 against both RSV subtypes A and B. Epitope sequence conservation analysis revealed that the binding sites of X80 and MEDI8897 occupy similar amino acids on preF between RSV subtypes A and B. However, X80 also interacts with additional residues on the preF, explaining the difference in prophylaxis. The X80 epitope is only partially conserved in RSV but not in HMPV, and it is variable on the hMPV preF. Sequence alignment of the RSV A2 and B9320 strain F proteins reveals three residue changes between the preF antigenic site and the CDRL on X80: Lys at residue 66 changes to Glu, Asp at residue 200 changes to Asn, and Lys at residue 209 changes to Gln. Of these three residues, residues 66 and 200 primarily contribute to main-chain interactions. While the A2 preF forms only one hydrogen bond with Arp31, residue 209 in the B9320 preF forms two hydrogen bonds with Arp31 and Tyr32 in CDRL1 in X80. Furthermore, two residues in the preF antigenic site of MEDI8897 undergo significant changes. When Lys at residue 201 is mutated to Asn, the interaction with Asp92 is abolished. When 209Lys was mutated to Gln, the interaction type with Glu55 on the CDRH2 loop and Asp101 on CDRL3 changed from salt bridge to hydrogen bond. X80 showed more effective protection than MEDI8897 in vivo. Figure 23 Figures b and c.

[0143] Conservation analysis of X233 between RSV and hMPV preF (see Figure 24 )

[0144] The structural basis of X233 cross-neutralization of RSV and hMPV was studied. The structures of X233 bound to RSV F and hMPV F were obtained using cryo-electron microscopy at resolutions of and RSV forms a stable trimeric complex with each antibody shown in the 3D classification. In contrast, each antibody forms a 1:1 complex with a single hMPV preF protomer. X233 uses five CDR loops: CDRL1, CDRL3, and CDRH1-3 to contact approximately The tight binding is composed of a hydrogen bond network formed by Val103, Tyr32, Thr52, Gly53, Gly54, and Asn54 on the RSV preF heavy chain, along with Thr50, Gly347, Thr31, Asp310, and Arg364. Furthermore, a salt bridge and hydrophobic interactions between Arg95 and Asp263 on the light chain further enhance the RSV preF-X233 interaction. Compared to the RSV preF-X233 complex, the residues interacting with hMPV preF are similar to those of RSV, with the exception of Ala33 on CDRL1 and Ser30 on CDRH1. Most of the conserved interactions involve the HCDR interface, which contains six conserved residues in F (RSV / hMPV P2335 / P265, I266 / I275 G347 / G315, T311 / T281, D310 / D280, I309 / I279, and L273 / L243). By comparing the sequences of F in RSV of two subtypes A and B, the epitopes involved in the interaction are highly conserved. Figure 22 ) In addition, the conservation between RSV F and hMPV F was characterized by sequence alignment to demonstrate cross-neutralization by X233.

[0145] Table 3

[0146]

[0147] In summary, respiratory syncytial virus (RSV) and human metapneumovirus (HMPV) are two closely related viruses that can cause bronchiolitis and pneumonia in infants, immunocompromised adults and the elderly, resulting in a huge health burden worldwide. The F proteins of RSV and HMPV are similar and have functions in membrane fusion and viral entry, making them prime targets for the development of neutralizing antibodies and vaccines. However, there are currently no licensed vaccines or antiviral strategies against these two viruses. In recent years, monoclonal antibodies have become the main intervention for infectious diseases. Here, the present invention describes mAbs X80 and X233, which are anti-RSV F protein antibodies derived from healthy adult memory B cells without any sequence optimization and bind to the antigenic site on the RSV F protein. As well as sites II and III on the F protein of RSV and HMPV. Although nisevirmab (formerly known as MEDI8897) binds to sites II and III on the RSV F protein Site binding is approved for all infants, but there is a certain risk of escape from its binding site. In addition, the most effective neutralizing antibodies against RSV are generally directed against the antigenic site Therefore, we still need to explore new and more effective neutralizing antibodies that bind to this site or other sites. In this study, X80 was as effective as MEDI8897 in preventing RSV A2 in cotton rats. Two target sites were found by cryo-electron microscopy structural analysis. The binding amino acids between RSV subtype A2 and RSV subtype B9320 are highly similar, except for individual amino acids such as K209Q that reduce the binding ability to MEDI8997. In addition, X80 was shown to be more effective than MEDI8897 against RSV B9320 in cotton rats, which is consistent with the neutralization effect in vitro. This suggests that X80 may be a candidate for future development against RSV. In addition, we found that X233 binds to a different site (III) and neutralizes RSV subtypes A and B in vitro and in vivo. Importantly, the combination of X233 and X80 showed similar effects to X80 in reducing RSV A and B replication at a dose of 2 mg / kg in cotton rats. In addition, the antibody mixture X80 and X233 was superior to MPE8 in inhibiting hMPV replication in vitro and similar to X233. These indicate that the antibody mixture of X80 and X233 may have potential preventive and therapeutic significance for hMPV infection.

[0148] Currently, a major problem in the development of RSV monoclonal antibodies is drug resistance. To some extent, it is prone to mutation. Antigenic sites in RSV subtype A isolates Some amino acid changes occur less frequently. In contrast, the amino acid variants N67T, D200N, and K201N occur at a frequency of ≥90% in RSV subtype B isolates. Furthermore, MPE8 has been reported to have cross-protective properties against RSV and HMPV. However, the amino acid mutation L45F occurs at a high frequency across all RSV / B genotypes. Because X233 binds to antigenic site III and the binding site is relatively conserved, the X233 binding site to RSV is rarely subject to high-frequency variation, making it worthy of further clinical development.

[0149] In conclusion, this study describes two neutralizing antibodies, X80 and X233, that target antigenic sites The mixture of X80 and X233 showed synergistic protection against cotton rat RSV and good inhibition against hMPV in vitro. Therefore, it may be a potential candidate for providing broad protection against RSV and hMPV.

[0150] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will readily appreciate that other variations or modifications based on the above descriptions are possible. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. An antibody or an antigen-binding fragment thereof, characterized in that Include at least one of the following: (a) the amino acid sequence of the variable region of the heavy chain of the antibody or antigen-binding fragment thereof comprises: QVQLVESGGGVVQPGTSLTLSCAASGFTFRTYAFHWVRQAPGKGLEWLALVTYDGTTQYYADSVKGRLTIYRDNSKNTLFLHLNSLRRDDTAIYFCARGGEGSFSWLGYLQYMDVWGQGTTVTVSS; or an amino acid sequence having at least 80%, 85%, 90%, or 95% similarity thereto; The amino acid sequence of the variable region of the light chain comprises: QSALTQPASVSGSPGQSITLSCTGTSSDIGDYDYVSWYQKYPDTAPKLVIYDVSERPSGVSTRFSGSKSGNTASLTISGLQPEDEADYYCNSYSSTNTLKFGGGTKLTVL; or having at least 80%, 85%, 90%, 95% similarity to the amino acid sequence; (b) the amino acid sequence of the variable region of the heavy chain of the antibody or antigen-binding fragment thereof comprises: VVQLVESGGGLVKPGESLRLSCAVSGSMFSSYVMHWVRQAPGKGLDWVSSITGGGNYISYADSVKGRFIISRDNGRNSLSLQMSSLRVDDTAVYYCVRGLSGVMGVTWFDSWGQGTLVTVSS; or an amino acid sequence having at least 80%, 85%, 90%, or 95% similarity thereto; The amino acid sequence of the variable region of the light chain comprises: QSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGFDVHWYQHLPGKAPKVIIYENSHRPSGVPDRFFGSKSGTSASLSISGLQPEDEADYYCQSYDRGLDWVFGGGTKLTVL or an amino acid sequence that is at least 80%, 85%, 90%, or 95% similar to the sequence.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein The antibody or antigen-binding fragment thereof is selected from any one of monoclonal antibodies mAb, Fab, Fab', F(ab')2, Fd, Fv, dAb, complementary determining region fragment, and single-chain antibody.

3. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 2, characterized in that: The constant region of the antibody is selected from any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE and IgD; optionally, it is IgG1; And / or, the species of origin of the constant region of the antibody is human, mouse, rat, cow, horse, sheep, rabbit or dog; optionally, human.

4. A bispecific antibody, multispecific antibody, chimeric antigen receptor, immunoconjugate or pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3.

5. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3.

6. Biomaterial, characterized in that The biological material comprises the nucleic acid molecule according to claim 5, and the biological material is an expression cassette, a vector or a host cell.

7. Antibody conjugate, characterized in that It is obtained by coupling the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3 or the bispecific antibody or multispecific antibody according to claim 4 with a marker or protein; the marker is selected from one or more of chemiluminescent dye labeling, enzyme labeling, biotin labeling, fluorescent dye labeling, colloidal gold labeling, and radioactive labeling.

8. The method for preparing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, characterized in that: The method comprises: culturing a host cell capable of expressing the antibody or the antigen-binding fragment thereof, and obtaining the antibody or the antigen-binding fragment thereof by separation.

9. Any of the following uses of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, the bispecific antibody or multispecific antibody according to claim 4, the nucleic acid molecule according to claim 5, the biomaterial according to claim 6, or the antibody conjugate according to claim 7: (1) Use in the preparation of a product for detecting the presence or level of human respiratory syncytial virus protein and / or human metapneumovirus protein in a sample; (2) Use in the preparation of products for detecting or diagnosing human respiratory syncytial virus infection and / or human metapneumovirus infection; (3) Use in the preparation of products for detecting or diagnosing diseases caused by human respiratory syncytial virus infection and / or diseases caused by human metapneumovirus infection; (4) Use in the preparation of products with anti-human respiratory syncytial virus and / or human metapneumovirus activity; (5) Use in the preparation of products for preventing, protecting or treating diseases caused by human respiratory syncytial virus infection and / or diseases caused by human metapneumovirus infection.

10. The use according to claim 9, characterized in that The disease caused by human respiratory syncytial virus infection includes acute lower respiratory tract infection; optionally, includes interstitial pneumonia; And / or, the disease caused by human metapneumovirus infection includes acute lower respiratory tract infection; optionally, includes interstitial pneumonia; And / or, the human respiratory syncytial virus includes RSVA and B subtypes.

11. The use according to claim 10, characterized in that Detection or diagnosis products include reagents, kits, pharmaceutical compositions or chips.

12. A reagent, kit, pharmaceutical composition or chip for detecting human respiratory syncytial virus and / or human metapneumovirus, characterized in that: The invention comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, or the bispecific antibody or multispecific antibody according to claim 4, or the antibody conjugate according to claim 7.

13. Reagents, kits, and pharmaceutical compositions for preventing, protecting, or treating diseases caused by human respiratory syncytial virus infection and / or diseases caused by human metapneumovirus infection, characterized in that: The invention comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, or the bispecific antibody or multispecific antibody according to claim 4, or the antibody conjugate according to claim 7.

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