Humanized anti-respiratory syncytial virus neutralizing antibody RS135 and application thereof

Abstract

The invention discloses a human-derived anti-respiratory syncytial virus neutralizing antibody RS135 and application of the human-derived anti-respiratory syncytial virus neutralizing antibody RS135. The human neutralizing antibody specifically aiming at the F protein of the respiratory syncytial virus is successfully obtained by applying a phage antibody library technology; the obtained human neutralizing anti-respiratory syncytial virus F protein gene engineering antibody variable region gene, Fab antibody gene and full antibody gene under the characteristics of each antibody gene are utilized to obtain the human neutralizing anti-respiratory syncytial virus F protein gene engineering antibody. The antibody can be expressed and produced in prokaryotic cells, yeast cells, eukaryotic cells and any recombination system or any other reconstructed gene containing the antibody gene on the basis of the antibody, and an antibody product capable of neutralizing respiratory syncytial virus infection is obtained. And a specific antibody drug for clinically preventing and treating respiratory syncytial virus infection is prepared.

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 RS135 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 approved 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 reduces hospitalization rates by 44.4%. 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) via intramuscular injection. It is suitable for healthy premature and full-term infants. A pivotal Phase IIb / III trial showed that it was 60.4% effective in preventing RSV lower respiratory tract infections requiring medical intervention, and reduced hospitalization rates by 84%-90%. It is expected to receive 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 limitations of 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 RS135 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 RS135 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 QSINSY, AAS, and QQYQSYPIT, respectively, and the amino acid sequences of the complementarity-determining regions CDR1, CDR2, and CDR3 of the heavy chain variable region are GGLLEDYI, IIPVLGTV, and ATETALVVSETYLPHYFDN, 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:2, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:4.

[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 RS135 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 RS135 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: RS130; 2: RS134; 3: RS135; 4: RS138; 5: RS144.

[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 is a preferred embodiment of the invention for measuring the neutralizing activity of the anti-RSV genetically engineered antibody.

[0021] Figure 4 This is a structural prediction model for the anti-RSV antibody-preF antigen complex in a preferred embodiment of the present invention. Detailed Implementation

[0022] The first objective of this invention is to provide a human anti-respiratory syncytial virus neutralizing antibody and its active fragment.

[0023] A second object of the present invention is to provide a gene encoding the above-mentioned antibody or its active fragment.

[0024] 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.

[0025] 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 RS135.

[0026] 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.

[0027] The RS135-specific light chain variable region gene was derived from specific enrichment screening of a human anti-respiratory syncytial virus (RSV) antibody gene library. This library was established using the MEDI8897 Fab antibody as a backbone. Lymphocytes isolated from peripheral blood of RSV patients were amplified by RT-PCR to replace the light chain gene of MEDI8897, constructing a light chain substitution library. The RS135-specific heavy chain variable region gene is identical to that of MEDI8897. The sequence combination of the three CDR regions corresponding to the light and heavy chain variable regions, along with the framework region sequences between the CDR regions, constitutes the sequence characteristics of each antibody variable region. RS135 belongs to the antibody light chain family VK1. The antibody protein function is determined by the specific nucleotide sequences and their complementarity in the CDR1, CDR2, and CDR3 complementary 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 RS135 antibody in this invention. The detailed amino acid sequences of the variable regions of the light and heavy chains of the antibody that determine the function of the RS135 neutralizing antibody and their comparison results are shown in Table 1: Table 1

[0028] The amino acid sequence of the light chain variable region of the RS135 antibody 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.

[0029] The gene sequence encoding the light chain variable region of the RS135 antibody is shown in SEQ ID NO:3, and the gene sequence encoding the heavy chain variable region is shown in SEQ ID NO:4.

[0030] 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 the 11th amino acid residue Leu in the RS135 light chain VK sequence with Val to obtain the mutant strain L11V.

[0031] 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.

[0032] 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.

[0033] 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.

[0034] 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.

[0035] In an embodiment of the present invention, the light chain and heavy chain genes of the above-mentioned Fab antibody RS135 were cloned into the VH / VK full antibody expression vector, respectively, and 293T cells were transiently transfected with the transfection reagent polyethyleneimine (PEI). The secretory expression of the full antibody was achieved using a mammalian cell system, and the full antibody RS135 was obtained.

[0036] The obtained complete antibody was functionally identified using SDS-PAGE, ELISA, BLI, and immunodextrose assay for live virus neutralization. RS135 exhibited relatively high binding activity (EC) to RSV-preF protein (PDB: 8W3L_F). 50=0.68ng / mL) and neutralizing activity against live RSV virus (IC50 against A2 strain). 50 =11.68 ng / mL, IC50 for BA.9 strain 50 =13.78ng / mL), compared to MEDI-8897 (EC for RSV-preF protein binding activity). 50 =3.61 ng / mL, with a neutralizing activity of IC50 against strain A2. 50 =14.57 ng / mL, with a neutralizing activity of IC50 against the BA.9 strain. 50 =20.7 ng / mL) showed stronger activity. The binding activity of the mutant strain L11V (EC) was significantly higher. 50 =0.76ng / mL), neutralizing activity against live RSV virus (IC50 against A2 strain). 50 =11.71 ng / mL, IC50 for BA.9 strain 50 =14.13 ng / mL), close to the parent antibody. Furthermore, in competition with MEDI-8897, antibodies RS130, RS134, RS135, RS138, and RS144 all exhibited varying degrees of competitive activity, suggesting a similar target site to MEDI-8897. According to the structural model predicted by AlphaFold 3, all these antibodies bind to the Ø epitope of the F protein. The differences in neutralization efficiency exhibited by these antibodies with different light chains against RSV may be closely related to the antibody's own affinity for the antigen.

[0037] 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.

[0038] 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.

[0039] 2. pComb3H-VH MEDI-8897 Cloning build First, the MEDI-8897 heavy chain gene VH was cloned into the pComb3H vector as the vector for constructing the light chain replacement library. The pComb3H vector and the MEDI-8897 heavy chain PCR product were then used... Xho I and Spe I 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 clone pComb3H-VH was selected.MEDI-8897 Measurement and identification.

[0040] 3. Construction of a light chain displacement antibody library 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 IgG1 heavy chain Fv and the light chains Kappa and Lambda. PCR conditions were: 94℃ for 1 min, 55℃ for 1 min, 72℃ for 10 min, for 30 cycles. The constructed pComb3H-VH... MEDI-8897 As a database construction platform, through Sac I and Xba After double digestion with enzyme I, the light chain diversity gene VL was... Pool T4 DNA ligase and pComb3H-VH MEDI-8897 Ligation, electroporation of XLI-Blue competent cells, construction of light chain substitution library, detailed library construction method and primer sequence reference 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.

[0041] 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... 11 Phage 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 subjected to the next round of screening. This process was repeated three times. Single clones were randomly selected and induced to express Fab antibody via IPTG. Specific enrichment methods and Fab antibody induction expression were performed according to the literature.

[0042] 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. A450 The value was used to determine Fab-positive clones resistant to RSV.

[0043] 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.

[0044] 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 cloned into VH / VK full antibody expression vectors, respectively. 293T cells were transiently transfected with polyethyleneimine (PEI) and cultured at 37℃ with 5% CO2 for 48 h. The supernatant was collected, and the IgG expression supernatant was purified by Protein-A affinity chromatography and identified by SDS-page gel electrophoresis.

[0045] 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, and the reaction was terminated with 2M H2SO4. The absorbance (A value) was measured using an ELISA reader.

[0046] 9. Series method competition experiment Antibody epitope competition was identified using bio-layer interferometry (BLI) on an Octet RED96e with a HIS1K biosensor. The preF antigen was pre-immobilized onto the HIS1K sensor at a concentration of 5 μg / mL. After equilibration with buffer for 30 seconds, antigen was loaded for 115 seconds, followed by buffer washing for 30 seconds. Then, 200 nM of the first antibody was added, and binding was performed for 180 seconds. After another 30 seconds of buffer washing, 200 nM of the second antibody was added, and binding was performed for 180 seconds. Finally, the sensor was treated with regeneration buffer for 30 seconds. Target crossover was determined by changes in binding signal. Data processing was performed using Octet Analysis Studio software, with 0.5 as the cutoff value and ≥0.5 considered epitope overlap.

[0047] 10. Live virus neutralization test Prepare Vero cells one day in advance at approximately 6 × 10⁻⁶. 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 .

[0048] 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.

[0049] II. Results 1. Screening of anti-RSV light chain displacement antibody library Light chain PCR products and vector pComb3H-VH MEDI-8897After enzyme digestion and ligation, the ligation product was precipitated with ethanol and then electroporated to construct a light chain displacement library. This library was then packaged using high-efficiency phages and enriched using RSV-preF protein. After three rounds of enrichment, 1140 clones were randomly selected. The supernatant from Fab-induced expression was detected using 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 178 clones had binding activity. The yields from the three rounds of enrichment are shown in Table 2.

[0050] Table 2 Enrichment screening of human anti-RSV phage antibody library

[0051] 2. Sequence analysis of human anti-RSV Fab antibody Sequence determination of 178 positive clones revealed 5 antibody strains with sequence differences compared to IgG sequences in the IMGT gene library, all of which belonged to VK. The amino acid sequences of their light chain variable region CDR are shown in Table 3.

[0052] Table 3. Amino acid sequences of light chain CDRs for screening anti-RSV Fab antibodies using the substitution method.

[0053] 3. Expression and ELISA detection of human anti-RSV-preF protein IgG antibody We transiently transfected 293T cells with positive clones RS130, RS134, RS135, RS138, and RS144 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 ).

[0054] The results of the ELISA detection of the above 5 IgG full antibodies are as follows: Figure 2 As shown, compared with MEDI-8897, RS135 has a higher titer of anti-F protein, indicating stronger binding activity to F protein; while the mutant strain L11V has a titer of 0.76 ng / mL, similar to the parental RS135 (Table 4).

[0055] Table 4. ELISA ECMO results of RS135 mutant strain L11V 50 (ng / mL)

[0056] 4. Series method competition experiment Bio-Layer Interferometry (BLI) was used to identify antibody epitope competition. RSV-preF protein was used as the coating antigen, and the antibody MEDI-8897 Fab antibody targeting the RSV-preF protein Ø epitope was used as the reference antibody. The presence of competition between the IgG whole antibodies and the reference antibody was detected. The results are shown in Table 5. The Fab antibody MEDI-8897 targeting the RSV-preF protein Ø epitope competed with the antibody, but did not compete with the control antibody RS319, indicating that the monoclonal antibody may target the RSV-preF protein Ø epitope.

[0057] Table 5 Competitive Experiments

[0058] 5. Human anti-RSV antibody live virus neutralization assay To further investigate and compare the neutralizing activity of the RSV-preF protein Ø surface swapping antibody RS135, we used an immunophenotypic live virus neutralization assay to detect the antibody's neutralization reaction with live RSV A2 virus in vitro. The results are as follows: Figure 3 The results showed that RS135 had a high neutralizing titer against RSV A2 and BA.9 strains, IC50 50 The levels were 11.68 ng / mL and 13.78 ng / mL, respectively, slightly better than MEDI-8897's 14.57 ng / mL and 20.70 ng / mL; while the mutant L11V had levels of 11.71 ng / mL and 14.13 ng / mL, respectively, similar to the parental RS135 (Table 6).

[0059] Table 6. Neutralizing activity IC50 of live virus from RS135 variant strain L11V 50 (ng / mL)

[0060] 6. Predictive model for the binding of Fab antibody to RSV-F To explore the region where antibodies target 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 comprehensive score was selected. The results showed that the antibodies all bound to the RSV-preF protein Ø epitope ( Figure 4 This is consistent with the results of the competition experiment.

[0061] 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, a light chain antibody replacement library targeting the RSV F protein Ø epitope, MEDI-8897, was constructed. The antibody library was then enriched and screened using purified RSV-preF protein. Ultimately, high-affinity human neutralizing antibodies RS130, RS134, RS135, RS138, and RS144 targeting the RSV F protein Ø epitope were obtained, with variations in the variable region sequence of the light chain of these antibodies.

[0062] The experimental data verifying antibody function in this invention show that RS135 has high specific binding activity against RSV-preF protein (EC50). 50 =0.68 ng / mL). RS135 has high and medium activity against live RSV virus, with an IC50 concentration against the A2 strain. 50 The concentration can reach 11.68 ng / mL, with an IC50 against the BA.9 strain. 50 RS135 showed a neutralizing activity of up to 13.78 ng / mL, while MEDI-8897 exhibited neutralizing activities of 14.57 ng / mL and 20.70 ng / mL against A2 and BA.9, respectively. RS135 demonstrated superior antiviral neutralizing activity compared to MEDI-8897. Furthermore, RS130, RS134, RS135, RS138, and RS144 antibodies showed varying degrees of competitive activity against MEDI-8897, suggesting a similar target site. Based on the structural model predicted by AlphaFold 3, all of these antibodies bind to the Ø epitope of the F protein. The differences in neutralizing efficiency against RSV exhibited by these antibodies with different light chains may be closely related to the antibody's own affinity for the antigen.

[0063] 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 RS135 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 QSINSY, AAS, and QQYQSYPIT, respectively, and the amino acid sequences of the complementarity-determining regions (CDR1, CDR2, and CDR3) of the heavy chain variable region are GGLLEDYI, IIPVLGTV, and ATETALVVSETYLPHYFDN, respectively.

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:2, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:

4.

3. The antibody or its active fragment according to claim 1 or 2, characterized in that, The active fragments are selected from Fab', Fab, F(ab')2, Fv and scFv fragments.

4. A nucleic acid molecule encoding the antibody or its active fragment as described in any one of claims 1-3.

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

6. The use of the antibody or its active fragment according to any one of claims 1-3 in the preparation of a medicament or diagnostic reagent for the prevention or treatment of respiratory syncytial virus infection.

7. The use of the antibody or its active fragment according to any one of claims 1-3 in the preparation of a medicament or composition for the prevention or treatment of lower respiratory tract infections caused by respiratory syncytial virus.

8. 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 any one of claims 1-3.

9. 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 any one of claims 1-3.

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

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