Human-derived anti-respiratory syncytial virus neutralizing antibody rs414 and uses thereof

Abstract

The application discloses a humanized anti-respiratory syncytial virus neutralizing antibody RS414 and application thereof. The antibody RS414 is obtained by screening using a phage surface presentation technology. The antibody of the application specifically recognizes a respiratory syncytial virus particle antigen, and is all directed against a respiratory syncytial virus F protein, has an obvious enzyme-linked immunoreaction with the respiratory syncytial virus, and has a neutralizing activity function against respiratory syncytial virus infection. The antibody of the application can be made into a specific antibody drug for preventing and treating respiratory syncytial virus infection, and can be used for preventing or treating lower respiratory tract infection caused by the respiratory syncytial virus, especially severe lower respiratory tract infection (pneumonia, bronchiolitis) in clinic.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering antibody technology, and more specifically, to a human anti-respiratory syncytial virus neutralizing antibody RS414 and its applications. Background Technology

[0002] Respiratory syncytial virus (RSV) is the most important pathogen causing lower respiratory tract infections, especially severe ones (pneumonia, bronchiolitis), in infants and young children worldwide and in my country. It is also a major risk factor for respiratory infections in immunocompromised patients and the elderly. Infants may be exposed to RSV and become infected shortly after birth. It takes at least two weeks to complete the full course of active immunization with a full vaccination program to achieve protection. Furthermore, infants' immune systems are not yet mature, resulting in a relatively weak immune response to vaccination. Therefore, RSV vaccines are not suitable for infants under six months of age. Thus, the exploration of passive immunization strategies using monoclonal antibodies to prevent RSV infection in infants has begun.

[0003] The F protein (fusion protein) of respiratory syncytial virus (RSV) is a key glycoprotein on the viral surface, playing a crucial role in viral infection. The F protein is exposed on the viral surface as a homotrimer and is the primary mediator of viral-host cell membrane fusion. The F protein is initially synthesized in an inactive precursor form (F0), which is cleaved by host cell proteases to produce two disulfide-linked subunits: F1 and F2. These two subunits together constitute the fusion-capable mature F protein. Functionally, the active F protein exists in a metastable "pre-fusion" (pre-F) state until a triggering event induces a conformational change, exposing the fusion peptide and inserting it into the target membrane, subsequently forming a six-helix bundle that drives the membrane fusion process. The two main conformational states of the F protein—pre-fusion and post-fusion—have different immunological properties. Studies have shown that the pre-F conformation has stronger immunogenicity than the post-F conformation and can induce more efficient neutralizing antibodies. This difference is mainly because the Ø and V epitopes exposed in the pre-F state are the strongest neutralizing antibody epitopes of the F protein, while these epitopes are masked in the post-F state. The RSV fusion protein (F protein) is highly conserved and is a hot topic in the development of antibodies, vaccines, and other therapeutic drugs.

[0004] In 1998, the FDA approved palizumab, the first short-acting monoclonal antibody targeting the RSV F protein, for the prevention of severe RSV lower respiratory tract infections in infants and young children. In Europe and the United States, it was approved for high-risk infants with underlying diseases and severe RSV. However, palizumab's short half-life, requiring monthly injections and incurring high costs, limited its widespread use. In January 2024, nirsevimab, a long-acting monoclonal antibody developed by AstraZeneca and Sanofi, was approved in China, becoming the first and only drug for the prevention of RSV lower respiratory tract infections in newborns and infants. This drug can cover the entire RSV infection season with a single injection, and clinical data show that it has a protective efficacy of 78.4% against RSV-related lower respiratory tract infections and a 44.4% reduction in hospitalization rates. Merck's long-acting monoclonal antibody Clesrovimab (MK-1654) is in the investigational stage and can be administered as a single fixed dose (105 mg) intramuscularly, suitable for healthy premature and full-term infants. The pivotal Phase IIb / III trial showed that its efficacy in preventing RSV lower respiratory tract infections requiring medical intervention was 60.4%, with a hospitalization rate reduction of 84%-90%. It received FDA approval in the United States on June 10, 2025, and on June 17, the China National Medical Products Administration (NMPA) accepted its marketing application for priority review. Compared to palizumab, this new generation of antibody drugs overcomes the application limitations of a short half-life and lowers the barrier to use by simplifying the dosing regimen.

[0005] Human or animal serum immunoglobulins containing specific antibodies have a long history of use in the prevention and treatment of infectious diseases. The in vitro antiviral neutralizing activity and in vivo protective activity of monoclonal antibodies against viral attacks have been extensively demonstrated in experiments. For example, neutralizing monoclonal antibodies against hepatitis A virus, hantavirus, measles virus, RSV virus, and CMV virus in mice can provide 100% protection against viral attacks in vivo. Obtaining polyclonal antibodies by immunizing animals with antigens has been a classic method for antibody production, but it lacks specificity and uniformity. The subsequent development of B-lymphocyte hybridoma technology has enabled scientists to directionally prepare various monoclonal antibodies (McAbs) in vitro through cell engineering. These antibodies are highly specific, homogeneous, and easy to mass-produce. However, most McAbs are murine in origin, and the heterologous reactions of murine McAbs greatly limit their application as therapeutic agents in humans. Immunoglobulins (VIG), as antibody components, are mainly derived from the immune serum of donors (convalescent patients). Obtaining positive serum and passing safety tests requires significant human and financial resources, limiting large-scale production. Furthermore, because they originate from serum, they are susceptible to bloodborne infections. Therefore, using human genetically engineered products to replace blood products can overcome these drawbacks. The ongoing research into human genetically engineered antibodies has brought new hope and broad prospects to the development of biopharmaceuticals in this field. Recombination at the antibody molecular gene level can yield a variety of specific murine and human antibodies, leading to breakthroughs in monoclonal antibody research and increasingly demonstrating its importance and practical application prospects. The development of human genetically engineered antibodies and the emergence of phage antibody library technology have provided new ideas for solving the problem of passive immunization agents. The rise of phage antibody gene library technology in the late 1980s and early 1990s, along with the development of the entire field of genetically engineered antibody technology research, has led to significant progress in the development of human genetically engineered antibodies worldwide, moving from the basic research stage to the stage of substantial applied research and development. The successful research on human antiviral genetically engineered antibodies, especially human whole antibodies, has brought new hope for the specific prevention and treatment of various viral infectious diseases. In the field of antiviral biopharmaceuticals, a new class of antiviral drugs has gradually emerged, namely antibody drugs.Just as there was a shift from blood-derived vaccines to genetically engineered vaccines, there is now an urgent need to replace blood-derived VIG with genetically engineered antibodies. This can be achieved through techniques such as chimeric antibody technology (Boulianne, GL et al., 1984; Morrison, SL et al., 1984), humanized antibody technology (Jones, PT et al., 1986), transgenic mice carrying human monoclonal antibodies (Green, LL et al., 1994), heterologous hybridoma technology (James, K. et al., 1987), and phage surface display technology (Barbas, CF et al., 1991). These methods have become a major research direction both domestically and internationally, and are gradually leading to success. Summary of the Invention

[0006] The purpose of this invention is to provide a human anti-respiratory syncytial virus neutralizing antibody RS414 and its applications.

[0007] To achieve the objectives of this invention, in a first aspect, this invention provides a human anti-respiratory syncytial virus neutralizing antibody RS414 or its active fragment, wherein the amino acid sequences of the complementarity-determining regions CDR1, CDR2, and CDR3 of the light chain variable region of the antibody or its active fragment are SSNIGAGYD, VNN, and QSYDSSLGGSI, respectively, and the amino acid sequences of the complementarity-determining regions CDR1, CDR2, and CDR3 of the heavy chain variable region are GFRTSGYY, ISAGSYI, and ARVKPGTYGDYYDY, respectively.

[0008] Furthermore, the amino acid sequence of the light chain variable region of the antibody or its active fragment is shown in SEQ ID NO:1, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:2.

[0009] Furthermore, the active fragment can be selected from Fab', Fab, F(ab')2, Fv and scFv fragments.

[0010] In a second aspect, the present invention provides a nucleic acid molecule encoding the antibody or an active fragment thereof.

[0011] Thirdly, the present invention provides biological materials containing the nucleic acid molecules, including but not limited to expression cassettes, transposons, plasmid vectors, viral vectors, or engineered bacteria.

[0012] Fourthly, the present invention provides the use of the antibody or its active fragment in the preparation of medicaments or diagnostic reagents for the prevention or treatment of respiratory syncytial virus infection.

[0013] Fifthly, the present invention provides the use of the antibody or its active fragment in the preparation of a medicament or composition for the prevention or treatment of lower respiratory tract infections caused by respiratory syncytial virus.

[0014] In a sixth aspect, the present invention provides a medicament for the prevention or treatment of respiratory syncytial virus infection, wherein the active ingredient is the antibody RS414 or its active fragment.

[0015] In a seventh aspect, the present invention provides a medicament for the prevention or treatment of lower respiratory tract infections caused by respiratory syncytial virus, wherein the active ingredient is said antibody RS414 or its active fragment.

[0016] Furthermore, the drug also includes pharmaceutical excipients.

[0017] By employing the above technical solution, the present invention has at least the following advantages and beneficial effects: This invention utilizes phage antibody library technology to successfully obtain a human neutralizing antibody specifically targeting the respiratory syncytial virus (RSV) F protein. Using the variable region gene, Fab antibody gene, and the full antibody gene characteristic of each of the aforementioned human neutralizing anti-RSV F protein genetically engineered antibody, this antibody or any other modified gene containing this antibody gene can be expressed and produced in prokaryotic cells, yeast cells, eukaryotic cells, and any recombinant system to obtain antibody products that neutralize RSV infection, and to formulate specific antibody drugs for the clinical prevention and treatment of RSV infection. Attached Figure Description

[0018] Figure 1 This is an SDS-PAGE electrophoresis image of purified IgG in a preferred embodiment of the present invention. M: protein marker; 1: RS361; 2: RS384; 3: RS401; 4: RS409; 5: RS412; 6: RS414.

[0019] Figure 2 This is an ELISA detection of the specific expression of human anti-RSV-preF protein IgG antibody in a preferred embodiment of the present invention.

[0020] Figure 3 This invention relates to a preferred embodiment of the BLI affinity assay for human anti-RSV-preF protein IgG antibody.

[0021] Figure 4 This is a preferred embodiment of the invention for measuring the neutralizing activity of the anti-RSV genetically engineered antibody.

[0022] Figure 5This is a structural prediction model of the anti-RSV antibody binding preF antigen complex in a preferred embodiment of the present invention. A shows the predicted binding structure of RS414 and the preF trimer; B shows the predicted binding structure of RS414 and the preF monomer; C shows that RS414 and MPE8 share similar epitopes, where gray represents the preF protein monomer, green represents the RS414 heavy chain, blue represents the RS414 light chain, and yellow represents MPE8 (GenBank: AGU13651.1). Detailed Implementation

[0023] The first objective of this invention is to provide a human anti-respiratory syncytial virus neutralizing antibody and its active fragment.

[0024] A second object of the present invention is to provide a gene encoding the above-mentioned antibody or its active fragment.

[0025] A third objective of this invention is to provide the use of the above-mentioned antibodies and their active fragments in the preparation of medicaments or diagnostic reagents for the prevention or treatment of respiratory syncytial virus infection.

[0026] This invention utilizes phage surface presentation technology to collect peripheral blood lymphocytes from children with RSV, construct a human anti-respiratory syncytial virus (RSV) genetically engineered antibody library using genetic engineering techniques, and screen for specific anti-RSV genetically engineered Fab antibodies. The obtained Fab antibody is named RS414.

[0027] This recombinant antibody is a functional antibody that specifically binds to respiratory syncytial virus (RSV) and is efficiently expressed in prokaryotic cells. It is determined by specific gene sequences in the hypervariable regions (CDRs) of the antibody light and heavy chain genes. These antibodies specifically recognize RSV particle antigens and target the RSV preF protein, exhibiting a significant enzyme-linked immunosorbent assay (ELISA) reaction with the RSV preF protein and demonstrating neutralizing activity against RSV infection.

[0028] The RS414-specific heavy and light chain variable region genes were derived from specific enrichment screening of a human anti-respiratory syncytial virus (RSV) antibody gene library. Lymphocytes were isolated from peripheral blood of RSV patients, and the antibody heavy and light chain variable region genes were amplified by RT-PCR to construct a cDNA library. The sequence combination of the three CDR regions corresponding to the RS414-specific light and heavy chain variable regions, along with the frame region sequences between the CDR regions, constitutes the sequence characteristics of each antibody variable region. RS414 belongs to the antibody heavy chain family IGHV3-21 and the light chain family IGLV1-40. The antibody protein function is determined by the specific nucleotide sequences and their complementarity in the CDR1, CDR2, and CDR3 regions of the antibody gene's light and heavy chain variable regions. The six corresponding CDR amino acid sequences constitute the antibody's specific antigen-binding region, determining the antigen-binding characteristics and anti-RSV functional characteristics of the RS414 antibody in this invention. The detailed amino acid sequences of the antibody light and heavy chain variable regions that determine the RS414 neutralizing antibody function and their comparison results are shown in Table 1. Table 1

[0029] The amino acid sequence of the light chain variable region of RS414 is shown in SEQ ID NO:1, and the amino acid sequence of its heavy chain variable region is shown in SEQ ID NO:2.

[0030] The gene sequence encoding the RS414 light chain variable region is shown in SEQ ID NO:3, and the gene sequence encoding the heavy chain variable region is shown in SEQ ID NO:4.

[0031] It should be understood that, without affecting the activity of the Fab antibody, those skilled in the art can make various substitutions, additions and / or deletions of one or more amino acids in the amino acid sequences shown in SEQ ID NO:1-2 to obtain amino acid sequences with equivalent functions. For example, in non-hypervariate regions, amino acids with similar properties can be replaced, such as replacing Ser at position 11 of the VK light chain sequence of RS414 with Ala to obtain the mutant strain S11A.

[0032] Furthermore, considering the degeneracy of codons, the gene sequence encoding the aforementioned Fab segment antibody can be modified, for example, within its coding region without altering the amino acid sequence, to obtain a gene encoding the same antibody. Those skilled in the art can artificially synthesize and modify genes based on the codon preference of the host expressing the antibody to improve antibody expression efficiency.

[0033] Furthermore, this invention recombines the light chain variable region and heavy chain variable region of the aforementioned Fab antibody to obtain antibodies in other molecular forms, such as scFv. These antibodies can also specifically recognize respiratory syncytial virus antigens and exert intracellular immune effects. Single-chain antibodies have strong penetrating power and easily enter local tissues to exert their effects.

[0034] The genes encoding Fab antibodies and ScFv genes can be cloned into expression vectors, then transformed into hosts, and Fab antibodies and single-chain antibodies can be obtained through induced expression.

[0035] In addition, the light and heavy chain encoding genes of the above-mentioned Fab antibody can be cloned into the all-antibody expression vector and introduced into host cells to obtain all-antibody immunoglobulin expressing respiratory syncytial virus.

[0036] In an embodiment of the present invention, the light chain and heavy chain genes of the above-mentioned Fab antibody RS414 were cloned into the VH / VK full antibody expression vector, respectively. 293T cells were transiently transfected with the transfection reagent polyethyleneimine (PEI), and secretory expression of the full antibody was achieved using a mammalian cell system to obtain the full antibody RS414.

[0037] The obtained complete antibody was functionally identified using SDS-PAGE, ELISA, BLI, and immunodoplasm assay for live virus neutralization. RS414 exhibited relatively high binding activity (EC) to RSV-preF protein (PDB: 8W3L_F). 50 =1.94 ng / mL) and neutralizing activity against live RSV virus (IC50 against A2 strain). 50 =9.64 ng / mL, IC50 for BA.9 strain 50 =13.90 ng / mL); while the mutant strain S11A showed EC500 activity against RSV-preF protein. 50 The neutralizing activity against live RSV virus was 1.86 ng / mL (IC50 against A2 strain). 50 =9.76 ng / mL, IC50 for BA.9 strain 50 (14.03 ng / mL) According to the structural model predicted by AlphaFold 3, the RS414 antibody binds to the F protein III epitope.

[0038] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0039] Example 1 I. Materials and Methods 1. Viruses, cells, vectors RSV strains (A2 strain, BA.9 strain) were provided by Jiangxia Laboratory, Wuhan Institute of Virology, Chinese Academy of Sciences. (A2 strain: Pandya MC, Callahan SM, Savchenko KG, Stobart CC. A Contemporary View of Respiratory Synytial Virus (RSV) Biology and Strain-Specific Differences. Pathogens. 2019 May 21;8(2):67. doi: 10.3390 / pathogens8020067. PMID: 31117229; PMCID:PMC6631838; BA.9 strain: Fan R, Fan C, Zhang J, Wen B, Lei Y, Liu C, Chen L, Liu W, Wang C, Qu X. Respiratory syncytial virus subtype ON1 / NA1 / BA9 predominates inhospitalized children with lower respiratory tract infections. J Med Virol. 2017) Feb;89(2):213-221. doi: 10.1002 / jmv.24619. Epub 2016 Jul 6. PMID:27358012; PMCID: PMC7166484). 293T cells were obtained from the American Cell Culture Center (ATCC). The phage antibody library was constructed using pComb3H. RSV patients were obtained from outpatients at Wuhan Union Hospital in Hubei Province. Vero cells were used for RSV in vitro neutralization experiments. RNA extraction kit, RT-PCR kit, and PCR kit were all purchased from Roche. Trypsin, polyethylene glycol (PEG), and transfection reagent polyethyleneimine (PEI) were purchased from Sigma-Aldrich.

[0040] 2. Construction of pComb3H-VH clone Lymphocytes were isolated from anticoagulated blood of children with RSV, and total cellular RNA was extracted and reverse transcribed into cDNA. PCR amplification was performed using a set of primers to amplify the variable region of the human IgG1 heavy chain antibody. PCR conditions were: 94℃ for 1 min, 55℃ for 1 min, 72℃ for 10 min, for 30 cycles. The vector pComb3H and the PCR product were processed separately using… Xho I and SpeI was subjected to double enzyme digestion at 37℃ for 2 hours. The digestion product was directly purified and recovered, ligated with T4 DNA ligase, transformed into XLI-Blue competent cells, and positive clones pComb3H-VH were selected for identification.

[0041] 3. Antibody library construction Lymphocytes were isolated from the anticoagulated blood of children with RSV, and total cellular RNA was extracted and reverse transcribed into cDNA. PCR amplification was performed using a set of primers to amplify the variable regions of the human antibody light chains Kappa and Lambda. The PCR conditions were: 94℃ for 1 min, 55℃ for 1 min, 72℃ for 10 min, for 30 cycles. The constructed pComb3H-VH was used as the library construction vector. Sac I and Xba After double digestion with enzyme I, the light chain diversity gene VL was... Pool The antibody library was constructed by ligating pComb3H-VH with T4 DNA ligase, electroporating XLI-Blue competent cells, and then constructing the library. For detailed library construction methods and primer sequences, please refer to Qu, Y., et al., Antibody Cocktail Exhibits Broad Neutralization Activity Against SARS-CoV-2 and SARS-CoV-2 Variants. Virol Sin, 2021. 36(5): p. 934-947.

[0042] 4. Enrichment and screening of phage antibody libraries and induction of Fab antibody expression RSV-preF protein was coated overnight at 4°C with 0.1 mol / L NaHCO3 (pH 8.6) solution. The wells were then sealed with MPBST (PBS containing 1% skim milk and 0.05% Tween-20) and incubated at room temperature for 2 h. The mixture was then washed three times with PBST. 10... 11Phage antibody libraries were incubated at room temperature for 2 hours. The supernatant was removed, and 100 µl of 10 µg / ml trypsin was added for elution. The eluted phages were used to infect fresh XLI-Blue bacterial culture (OD600 = 0.5), packaged with helper phages, and then screened for the next round. This process was repeated three times. Single clones were randomly selected and Fab expression was induced by IPTG. The specific enrichment method and Fab antibody induction expression were performed according to the literature (Qu, Y., et al., Antibody Cocktail Exhibits Broad Neutralization Activity Against SARS-CoV-2 and SARS-CoV-2 Variants. Virol Sin, 2021. 36(5):p. 934-947).

[0043] 5. ELISA detection of Fab antibodies Add the Fab antibody that induces expression to the ELISA plate coated with RSV-preF protein and incubate at 37°C for 1 h; add the enzyme-labeled anti-human fab antibody and incubate at 37°C for 1 h; develop color with chromogenic solution, stop the reaction with 2M H2SO4, and measure the absorbance using an ELISA reader. A 450 The value was used to determine Fab-positive clones resistant to RSV.

[0044] 6. Gene sequence analysis We used Fab sequencing primers to sequence the light and heavy chains of the antibodies from positive clones. The sequencing was performed by Beijing Qingke Biotechnology Co., Ltd. The obtained recombinant Fab antibody nucleotide sequences were analyzed using DNASTAR sequencing software to obtain the derived amino acid sequences. The antibody light and heavy chain types were determined by comparing these sequences with IgG family sequences in the IMGT gene library.

[0045] 7. IgG antibody expression and purification After sequencing, the Fab antibody-positive clones were compared and analyzed against the IMGT database. The obtained Fab antibody light and heavy chain genes were then cloned into the VH / VK full antibody expression vectors, respectively.

[12] 293T cells were transiently transfected with polyethyleneimine (PEI) and cultured at 37°C for 48 h under 5% CO2 conditions. The supernatant was collected, and the IgG expression supernatant was purified by Protein-A affinity chromatography and identified by SDS-page gel electrophoresis.

[0046] 8. ELISA detection of IgG antibodies IgG expression supernatant was collected for ELISA detection of anti-human Fab and anti-RSV-preF proteins. RSV-preF protein was coated onto an ELISA plate with 0.1 m / L NaHCO3 (pH 9.6) solution and incubated overnight at 4°C; blocked with 4% skim milk and incubated at 37°C for 1 h; IgG expression supernatant was added and incubated at 37°C for 1 h; enzyme-labeled anti-human Fc secondary antibody (Sigma, USA, 1:2000 dilution) was added and incubated at 37°C for 1 h; color development was performed with chromogenic buffer, and the reaction was terminated with 2M H2SO4. The absorbance (A value) was measured using an ELISA reader.

[0047] 9. BLI Affinity Testing Antibody affinity was determined using bio-layer interferometry (BLI) on an Octet RED96e instrument employing the proA biosensor. First, the antibody was immobilized at 100 nM on the proA sensor for 30 seconds, followed by buffer equilibration for 60 seconds, then antigen binding at gradient concentrations for 120 seconds, buffer dissociation for 600 seconds, and regeneration in buffer for 30 seconds. Binding signals were recorded, and data processing was performed using Octet Analysis Studio software to calculate antibody affinity.

[0048] 10. Live virus neutralization test Prepare Vero cells one day in advance at approximately 6 × 10 4 Cells were seeded per well in 48-well plates, and experiments were conducted when the cell density was approximately 80%. On the day of the experiment, the monoclonal antibody to be tested was serially diluted 3-fold starting at 10 μg / mL. Simultaneously, RSV virus (A2 strain, BA.9 strain) was diluted to a final concentration of 1000-fold with maintenance medium (adjusted according to virus titer). The antibody and virus were mixed in equal volumes and neutralized at 37°C for 1 h. The cell culture medium was discarded, and 100 μL of the virus-antibody mixture was added to each well. Infection was carried out at 37°C for 2 h. After discarding the supernatant, preheated semi-solid culture medium was added, and the cells were incubated at 35°C for 4 days. After incubation, 8% tissue cell fixative was added directly to fix the cells at room temperature for 30 min, followed by washing twice with PBS, then treatment with methanol-acetone fixative at room temperature for 15 min, and finally washing again with PBS. The plate was then blocked with PBS (3% FBS) at room temperature for 30 min, incubated with anti-RSV-F primary antibody at room temperature for 2 h, washed with PBS, incubated with enzyme-labeled secondary antibody at room temperature for 1 h, washed, patted dry, and then KPL chromogenic solution was added for color development. The reaction was terminated with deionized water, the plate was air-dried, and the blue spots were scanned and counted. The average number of spots was calculated using the virus control wells (VC). The virus titer was calculated at dilutions where the number of spots was within the countable range (generally 0-400 spots / well). Titer (PFU / mL) = Average number of spots × Dilution factor / Inoculation volume (mL) The neutralizing effect of antibodies is calculated as the rate of spot reduction: Neutralization rate (%) = [1 - (number of antibody well spots / number of virus control well spots)] × 100% And this can be used to fit NT. 50 .

[0049] 11. Structural Prediction AlphaFold 3 was used to predict the binding model between the antibody and the preF protein. The heavy and light chain sequences of the antibody and the sequence of the preF protein were input into the AlphaFold 3 system, and 20 independent predictions were performed with random seeds, resulting in 100 different conformational models. All models were scored using the InterfaceAnalyzer module in Rosetta, and the scores from AlphaFold 3 were combined to calculate a weighted total score. The optimal binding model was then selected, and PyMOL software was used for visualization and image processing.

[0050] II. Results 1. Screening of anti-RSV light chain displacement antibody library The light chain PCR product and the vector pComb3H-VH were digested and ligated. The ligation product was precipitated with ethanol and then electroporated to construct an antibody library. The library was packaged using high-efficiency phages and enriched using RSV-preF protein. After three rounds of enrichment, 768 clones were randomly selected. The supernatant from Fab-induced expression was detected by RSV-preF protein antigen ELISA. The results showed that the phage antibody library exhibited an enrichment trend during the three rounds of enrichment, especially in the third round, where the elution volume increased by an order of magnitude. ELISA detection showed that the Fab-induced expression products of 132 clones had binding activity. The yields of the three rounds of enrichment are shown in Table 2.

[0051] Table 2 Enrichment and screening of human anti-RSV phage antibody library

[0052] 2. Sequence analysis of human anti-RSV Fab antibody Sequence determination of 132 positive clones revealed 47 antibodies with sequence differences when the antibody heavy and light chain genes were compared with IgG sequences in the IMGT gene library. The top 6 antibodies were selected based on abundance for subsequent analysis and experiments. The amino acid sequences of their heavy and light chain variable regions (CDRs) are shown in Tables 3 and 4.

[0053] Table 3. Amino acid sequence of CDR heavy chain of anti-RSV Fab antibody

[0054] Table 4. Amino acid sequence of the light chain CDR of anti-RSV Fab antibody

[0055] 3. Expression and ELISA detection of human anti-RSV-preF protein IgG antibody We transiently transfected 293T cells with positive clones RS361, RS384, RS401, RS409, RS412, and RS414 using a dual plasmid system (VH / VK). The expression supernatant was collected, and the antibody expression products were purified by affinity chromatography using a Protein A affinity chromatography column. The purified IgG antibodies were then detected by SDS-PAGE electrophoresis, confirming the presence of purified IgG protein with clear bands at approximately 28 kDa and 55 kDa. Figure 1 ).

[0056] The results of the ELISA detection of the above 6 IgG full antibodies are as follows: Figure 2 As shown, RS414 exhibits a high titer against the F protein, indicating strong binding activity to the F protein; furthermore, the mutant strain S11A shows EC50-60% binding activity against the RSV-preF protein. 50 The concentration was 1.86 ng / mL, similar to the parent (Table 5).

[0057] Table 5. ELISA ECMO results of RS414 mutant strain S11A 50 (ng / mL)

[0058] 4. Affinity Test Antibody affinity was determined using bio-layer interferometry (BLI), and the results showed that RS414 had a K... D <1×10 -12 ( Figure 3 ).

[0059] 5. Human anti-RSV antibody live virus neutralization assay To further investigate and compare the neutralizing activities of anti-RSV-preF protein antibodies RS361, RS384, RS401, RS409, RS412, and RS414, we used an immunophenotypic live virus neutralization assay to detect the neutralization reactions of the antibodies with RSV A2 (from ATCC) and BA.9 live virus (a clinical isolate provided by the Jiangxia Laboratory of the Wuhan Institute of Virology, Chinese Academy of Sciences; see the literature: Study on the whole genome sequence characteristics of a clinical strain of respiratory syncytial virus BA9 genotype causing severe pneumonia outbreak, Wang Na et al., Chinese Journal of Microbiology and Immunology. 2025. 45(9):733-739). The results are as follows: Figure 4As shown, RS414 has a high neutralizing titer against RSV A2 and BA.9 strains, IC50 50 The concentrations were 9.64 ng / mL and 13.90 ng / mL, respectively; furthermore, the mutant strain S11A showed an IC50 inhibitory concentration against the A2 strain. 50 =9.76 ng / mL, IC50 for BA.9 strain 50 =14.03ng / mL, similar to the parent (Table 6).

[0060] Table 6. Neutralizing activity IC50 of live virus in RS414 variant strain S11A 50 (ng / mL)

[0061] 6. Predictive model for the binding of Fab antibody to RSV-F To explore the region where the antibody targets the RSV-preF protein, AlphaFold 3 was used to predict the structure of the antigen-antibody complex. After weighted calculation of multiple scores, the model with the highest overall score was selected. The results showed that RS414 binds to the RSV-preF protein epitope III ( Figure 5 ).

[0062] This invention utilizes phage display technology to isolate lymphocytes from the peripheral blood of children with RSV. Combined with a highly efficient phage packaging system, an antibody library targeting the RSV F protein was constructed. The antibody library was then enriched and screened using purified RSV-preF protein. Ultimately, high-affinity human neutralizing antibodies against the RSV F protein, such as RS414, were obtained.

[0063] In in vitro experiments, RS414 exhibited relatively high binding activity to RSV-preF protein (EC50). 50 =1.94 ng / mL) and affinity (K D <1×10 -12 ), and neutralizing activity against live RSV virus (IC50 against A2 and BA.9 strains). 50 The concentrations were 9.64 ng / mL and 13.90 ng / mL, respectively; meanwhile, the binding activity of the mutant strain S11A to RSV-preF protein was EC50. 50 The concentration was 1.86 ng / mL, and the neutralizing activity against live virus strains A2 and BA.9 was IC50 at 1.86 ng / mL. 50 =9.76 ng / mL and 14.03 ng / mL, similar to the parent. Furthermore, according to the structural model predicted by AlphaFold 3, RS414 binds to epitope III of the F protein.

[0064] This invention successfully obtained the neutralizing antibody RS414 targeting the F protein III epitope through phage display technology, laying the foundation for the research and development of RSV antibodies with independent intellectual property rights in my country.

[0065] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A human anti-respiratory syncytial virus neutralizing antibody RS414 or its active fragment, characterized in that, The amino acid sequences of the complementarity-determining regions CDR1, CDR2, and CDR3 of the light chain variable region of the antibody or its active fragment are SSNIGAGYD, VNN, and QSYDSSLGGSI, respectively, and the amino acid sequences of the complementarity-determining regions CDR1, CDR2, and CDR3 of the heavy chain variable region are GFRTSGYY, ISAGSSYI, and ARVKPGTYGDYYDY, respectively. The active fragments are selected from Fab', Fab, F(ab')2, Fv and scFv fragments.

2. The antibody or its active fragment according to claim 1, characterized in that, The amino acid sequence of the light chain variable region of the antibody or its active fragment is shown in SEQ ID NO:1, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:

2.

3. A nucleic acid molecule encoding the antibody or its active fragment as described in claim 1 or 2.

4. A biomaterial containing the nucleic acid molecule of claim 3, characterized in that, The biological material is an expression cassette, transposon, plasmid vector, viral vector, or engineered bacteria.

5. The use of the antibody or its active fragment as described in claim 1 or 2 in the preparation of a medicament or diagnostic reagent for treating respiratory syncytial virus infection.

6. The use of the antibody or its active fragment as described in claim 1 or 2 in the preparation of a medicament for treating lower respiratory tract infections caused by respiratory syncytial virus.

7. A drug for the prevention or treatment of respiratory syncytial virus infection, characterized in that, The active ingredient is the antibody or its active fragment as described in claim 1 or 2.

8. A drug for the prevention or treatment of lower respiratory tract infections caused by respiratory syncytial virus, characterized in that, The active ingredient is the antibody or its active fragment as described in claim 1 or 2.

9. The drug according to claim 7 or 8, characterized in that, It also includes pharmaceutical excipients.

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