A fully human monoclonal antibody RSV-18 against respiratory syncytial virus F protein and application thereof

Abstract

This invention provides a fully human monoclonal antibody, RSV-18, targeting the respiratory syncytial virus (RSV) F protein and its applications, belonging to the field of biomedical technology. The fully human monoclonal antibody provided by this invention exhibits significant neutralizing activity against RSV, showing superior efficacy against RSV subtype A compared to palizumab, while also possessing the advantage of low immunogenicity due to its fully human sequence. This invention also provides pharmaceutical compositions comprising the antibody and their application in the preparation of drugs for the prevention or treatment of RSV infection, offering a safer and more efficient antibody treatment option for RSV infection.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a fully human monoclonal antibody RSV-18 targeting the F protein of respiratory syncytial virus and its applications. Background Technology

[0002] Respiratory syncytial virus (RSV) is a highly contagious pathogen that is prevalent worldwide and belongs to the Pneumoviridae family. It is the leading viral cause of acute lower respiratory tract infections (such as bronchiolitis and pneumonia) in infants, the elderly, and immunocompromised individuals, resulting in a heavy disease burden and healthcare costs annually. RSV is classified into subtypes A and B, with these two subtypes circulating alternately throughout the year. The fusion protein (F protein) on the surface of the RSV viral particle is its core functional protein for infecting host cells, responsible for mediating the fusion of the virus with the host cell membrane. The F protein is highly conserved between the A and B subtypes, making it a key target for vaccine and antiviral drug development.

[0003] In the field of prophylactic antibodies, palizumab, nirsevimab, and clerovirimab have been approved for the prevention of RSV infection in high-risk infants and young children. These monoclonal antibodies mainly block viral invasion by targeting and binding to the RSV F protein. Palizumab is a humanized antibody, with part of its sequence derived from mice, and theoretically still carries the risk of inducing an immune response in humans, and its neutralizing efficacy needs further improvement. Fully human antibodies, due to their complete origin from human gene sequences, extremely low immunogenicity in humans, and superior safety, have become an important development direction for next-generation therapeutic antibody drugs. Nirsevimab targets the F protein antigenic epitope Ø, which, compared to other neutralizing epitopes, has lower conservation and is more prone to mutation, leading to reduced sensitivity of nirsevimab to some clinically mutant strains.

[0004] Developing potent, broad-spectrum neutralizing fully human anti-RSV antibodies and establishing novel RSV prophylactic immunization strategies are of great significance for effectively reducing the risk of disease in infants and young children and alleviating the socioeconomic burden. Summary of the Invention

[0005] The purpose of this invention is to provide a fully human monoclonal antibody RSV-18 targeting the respiratory syncytial virus F protein and its application, which solves the technical problems of existing RSV blocking antibody drugs, which are mostly prepared using non-fully human technologies, such as complex production processes, long development cycles, high potential immunogenicity risks, and limited neutralizing efficacy.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a fully human monoclonal antibody that specifically binds to the fusion protein F of respiratory syncytial virus. The fully human monoclonal antibody comprises a heavy chain variable region and a light chain variable region. The light chain variable region comprises the amino acid sequence shown in SEQ ID NO.1, and the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO.2.

[0007] The present invention also provides an antigen-binding fragment of a fully human monoclonal antibody, wherein the antigen-binding fragment specifically binds to the fusion protein F of respiratory syncytial virus, the antigen-binding fragment comprising a heavy chain variable region and a light chain variable region, wherein the light chain variable region comprises the amino acid sequence shown in SEQ ID NO.1, and the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO.2.

[0008] Preferably, the antigen-binding fragment is a Fab fragment, an F(ab')2 fragment, or a single-chain antibody scFv.

[0009] The present invention also provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and the above-described fully human monoclonal antibody or antigen-binding fragment.

[0010] The present invention also provides the use of the above-mentioned fully human monoclonal antibody or antigen-binding fragment in the preparation of a medicament for the prevention or treatment of respiratory syncytial virus infection.

[0011] Preferably, the respiratory syncytial virus infection is caused by RSV subtype A virus; And / or, the respiratory syncytial virus infection is caused by RSV subtype B virus.

[0012] The present invention also provides the application of the above-mentioned fully human monoclonal antibody or antigen-binding fragment in the preparation of reagents for diagnosing respiratory syncytial virus infection.

[0013] The beneficial effects of this invention are: The fully human monoclonal antibody (RSV-18) provided by this invention has the following beneficial effects: The antibody provided by this invention exhibits significant neutralizing activity against both RSV subtypes A and B. In pseudovirus neutralization experiments, its IC50 against RSV A2 was [missing value]. 50 The concentration reached 0.009127 μg / mL; in the neutralization experiment with real virus, the IC50 against RSV A2 was [value missing]. 50The concentrations were 0.01410 μg / mL, and their activities were superior to the clinical control antibody palizumab, providing a potent candidate drug molecule for the prevention and treatment of RSV infection. The antibody is a fully human antibody obtained directly from human B cells, and its variable region sequence has not been modified in any way. Theoretically, this can greatly reduce the risk of human anti-drug antibody (HADA) reactions in clinical applications, and it has higher safety compared to humanized antibodies (such as palizumab). Attached Figure Description

[0014] Figure 1 Flow cytometry sorting diagram of B cells specific to antigen (including RSV); Figure 2 The pTT5 antibody expression vector is shown in the image. Figure 3 This is a graph showing the detection of the binding activity of the antibody of the present invention with RSV A subtype F protein; Figure 4 This is a graph showing the detection of the binding activity of the antibody of the present invention to RSV subtype B F protein; Figure 5 This is a graph showing the detection of the neutralizing activity of the antibody against RSV A2 pseudovirus in this invention; Figure 6 This is a graph showing the detection of the neutralizing activity of the antibody against RSV B18537 pseudovirus in this invention; Figure 7 This is a graph showing the detection of the neutralizing activity of the antibody against RSV A2 true virus in this invention. Detailed Implementation

[0015] This invention provides a fully human monoclonal antibody, RSV-18, or its antigen-binding fragment, which specifically binds to the fusion protein (F protein) of respiratory syncytial virus (RSV). In this invention, the "fully human monoclonal antibody" refers to an immunoglobulin molecule whose entire amino acid sequence is derived from the human immune system, without any non-human sequence modification or humanization. Compared to chimeric or humanized antibodies, fully human antibodies have lower immunogenicity and a very low risk of inducing human anti-mouse antibody (HAMA) or human anti-chimeric antibody (HACA) reactions during treatment, thus exhibiting better safety and clinical applicability. Such antibodies can typically be obtained through various technological platforms, such as screening from B cells of immunized individuals, using transgenic mice, or through in vitro display technologies (e.g., phage display, yeast display). In a specific embodiment of this invention, the antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL), which together determine the specificity and affinity of the antibody for binding to the RSV F protein. The light chain variable region contains the amino acid sequence shown in SEQ ID NO.1, and the heavy chain variable region contains the amino acid sequence shown in SEQ ID NO.2. This specific sequence combination constitutes a complete and unique antigen-binding site, endowing the antibody with high specificity and high affinity binding properties. In a preferred embodiment, the variable region sequence of the antibody can be further linked to the constant region of human immunoglobulins (such as the constant region of IgG1, IgG2, IgG3, IgG4, or IgA) through genetic engineering to form a complete antibody molecule, such as a full-length IgG antibody.

[0016] In this invention, preferably, the antigen-binding fragment is a Fab fragment, an F(ab')2 fragment, or a single-chain antibody scFv. The "antigen-binding fragment" refers to the antibody portion structure that retains the complete antigen-binding function of the antibody. Specifically, the "Fab fragment" (antigen-binding fragment) consists of a complete light chain, a variable region (VH) of a heavy chain, and a constant region CH1 domain. It can be obtained by digesting a complete IgG antibody with papain and has a molecular weight of approximately 55 kDa. The "F(ab')2 fragment" is formed by two Fab fragments linked by disulfide bonds in the hinge region. It can be obtained by digesting a complete IgG antibody with pepsin and has a molecular weight of approximately 110 kDa, exhibiting bivalent binding capability. The "single-chain antibody scFv" (single-chain variable region fragment) is a small molecule antibody fragment formed by linking the heavy chain variable region (VH) and the light chain variable region (VL) with a flexible short peptide linker. It has a molecular weight of approximately 25-30 kDa and good tissue penetration ability. These fragments can all be directly expressed using genetic engineering methods and are widely used in diagnostic testing, drug targeting, and therapy. For example, Fab fragments are often used in the preparation of in vitro diagnostic reagents; scFv can be used to construct bispecific antibodies or recognition domains in CAR-T cells.

[0017] This invention also provides a pharmaceutical composition comprising at least one of the above-described fully human monoclonal antibodies or their antigen-binding fragments, and a pharmaceutically acceptable carrier. In this invention, a "pharmaceutical composition" refers to a dosage form suitable for administration to patients prepared by mixing an active ingredient with therapeutic or preventative activity (i.e., the antibody of this invention) with one or more inert, non-toxic pharmaceutical excipients or carriers. The "pharmaceuticalally acceptable carrier" is known in the art and includes, but is not limited to: buffers (such as phosphate buffer, citrate buffer, Tris buffer), stabilizers (such as sucrose, trehalose, mannitol, amino acids), surfactants (such as polysorbate 80, poloxamer 188), preservatives (such as benzyl alcohol, parabens), isotonic adjusters (such as sodium chloride, glycerol), and lyophilization protectants (for preparing lyophilized formulations). The dosage form of the pharmaceutical composition can be diverse; for example, it can be formulated into a dosage form for injection, including an injection solution, sterile powder for injection (lyophilized powder for injection), or a suspension; it can also be formulated into a dosage form for mucosal administration, such as a nasal spray or a nebulized inhalation solution. The effective dose of the active ingredient (antibody) in the composition can be adjusted according to factors such as the patient's age, weight, severity of illness, and route of administration. For example, the single-dose dose range can be from 0.1 mg / kg body weight to 20 mg / kg body weight, more preferably from 1 mg / kg body weight to 10 mg / kg body weight, and most preferably from 3 mg / kg body weight to 5 mg / kg body weight.

[0018] This invention also provides the application of the above-mentioned fully human monoclonal antibody or its antigen-binding fragment in the preparation of a medicament for the prevention or treatment of respiratory syncytial virus (RSV) infection. RSV infection is a common respiratory disease that can cause severe symptoms such as bronchiolitis and pneumonia in infants, the elderly, and immunocompromised individuals. The antibody of this invention, by specifically binding to the F protein on the surface of RSV, can block the fusion process between the virus and host cells, thereby neutralizing the virus and preventing its infection and replication, achieving the purpose of prevention or treatment. "Prevention" refers to administration before or after exposure to viral risk and before the onset of symptoms to prevent infection or reduce the severity of infection. "Treatment" refers to administration after infection and the appearance of clinical symptoms to clear the virus, relieve symptoms, and shorten the course of the disease.

[0019] Preferably, in the above applications, the respiratory syncytial virus infection is caused by RSV subtype A virus; and / or, the respiratory syncytial virus infection is caused by RSV subtype B virus. RSV is mainly divided into two subtypes, A and B, and the fusion protein F is highly conserved between the two subtypes. Strain A2 and strain B18537 are classic laboratory representative strains widely used in the study of RSV subtypes A and B, respectively. The antibody of the present invention has been experimentally confirmed to have significant neutralizing activity against both of these important subtypes, indicating that it has broad-spectrum protective potential.

[0020] This invention also provides the application of the above-mentioned fully human monoclonal antibody or its antigen-binding fragment in the preparation of reagents for diagnosing respiratory syncytial virus (RSV) infection. In diagnostic applications, the antibody can serve as a core detection reagent for qualitative or quantitative detection of the presence of RSV antigens (especially the F protein) in biological samples (such as nasopharyngeal swabs, sputum, and bronchoalveolar lavage fluid). Based on the principle of antigen-antibody specific binding, the antibody of this invention can be used in various immunological detection methods, such as enzyme-linked immunosorbent assay (ELISA), chemiluminescent immunoassay (CLIA), immunochromatographic test strips (such as lateral flow immunochromatography), immunofluorescence assay (IFA), and immunohistochemistry (IHC). In these detections, the antibody can be labeled to generate a detectable signal; labeling agents include, but are not limited to, enzymes (such as horseradish peroxidase HRP, alkaline phosphatase AP), fluorescent dyes (such as FITC, PE), colloidal gold particles, or chemiluminescent substances.

[0021] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0022] Example Acquisition of RSV-specific B-cell receptor (BCR) Lymphocytes from frozen healthy volunteers were revived, and B cells were purified and enriched. The RSV pre-F (SinoBiological) antigen was then conjugated with a fluorescent group and oligonucleotide barcode. After flow cytometry staining, the antigens were sorted using flow cytometry. + (RSV pre-F) + IgD - CD19 + CD45 + Live cells (Dead Cell Stain kit, manufacturer: Invitrogen; CD45-BV421, manufacturer: BD; CD19-PerCP-Cy5.5, manufacturer: BD; IgD-APC-Cy7, manufacturer: BD). Results are as follows. Figure 1 As shown, Antigen is obtained by flow sorting. + (RSV pre-F) + Cells were then used. Subsequently, single cells were prepared and barcode and BCR libraries were constructed using the Chromium Next GEM SingleCell 5' Reagent Kits (10×GENOMICS). By analyzing the number of antigen barcode reads on B cells in the barcode library, cells with high read counts were screened as RSV-specific B cells. Paired light and heavy chain sequences were obtained by association with BCR data, and clones with germline genes of IGHV1-69D / IGLV1-40 were screened for subsequent monoclonal antibody preparation and purification.

[0023] Preparation and purification of recombinant antibodies The Fab sequences of the antibody light and heavy chains were conjugated to human IgL and IgG1 constant region sequences (Uniprot: P0DOX8; P01857) respectively, and cloned into the pTT5 vector using the ClonExpress Ultra One Step Cloning Kit V2 (Vazyme). Figure 2 (As shown) Eco RⅠ and Bam Between the HⅠ restriction sites, a large amount of plasmid was extracted. Subsequently, HieffTrans was used... ®Polyethylenimine Linear (PEI) MW40000 (Yisheng Biotechnology) transfection reagent was used to transfect the plasmid into 293F suspension cells at a 1:3 ratio. The cells were then placed in a 37°C shaker incubator for recombinant antibody expression. After 5 consecutive days of expression, the cells were removed and centrifuged at 300g for 15 minutes to obtain the cell supernatant. Affinity chromatography was performed using a Protein A column (Tiandi Renhe) and the eluent was further collected for size exclusion chromatography (Superdex 200 Increase 10 / 300G, GE) to improve purity. The purified recombinant antibody RSV-18 was finally obtained, and its sequence information is shown in Table 1 below.

[0024] Table 1. Antibody sequences of the present invention

[0025] Antibody binding activity assay Antigen F protein was diluted to 1 μg / mL with phosphate-buffered saline and added to an ELISA plate. The plate was coated overnight at 4°C. The coating solution was discarded the next day, and the plate was washed once with PBST. 300 μL of blocking buffer was added to each well, and the plate was blocked at 26°C for 3 h. Serially diluted monoclonal antibodies were added, and the plate was incubated at 26°C for 1 h. HRP-labeled secondary antibody (Goat anti-Human IgG (H+L) Secondary Antibody, HRP, #A18805) was diluted with PBST containing 1% BSA and incubated at 26°C in the dark for 1 h. 100 μL of TMB chromogenic solution was added to each well, and the reaction was carried out at room temperature in the dark. After chromogenic development, stop solution was added to terminate the reaction, and the absorbance at 450 nm was measured. Results are as follows: Figure 3 and Figure 4 As shown, the fully human recombinant antibody RSV-18 provided by this invention has high binding capacity to both RSV A subtype F protein and RSV B subtype F protein, and its binding capacity to RSV A subtype F protein is higher than that of parizumab.

[0026] antibody neutralizing activity assay fake virus neutralization Huh7.5.1 cells were seeded into 96-well plates and cultured overnight. Monoclonal antibodies were serially diluted 10-fold and added to RSV A2 and RSV B18537 pseudoviruses, respectively, and incubated at 37°C in a 5% CO2 incubator for approximately 0.5 hours for neutralization. The pseudovirus and antibody neutralization products were then added to Huh7.5.1 cells and incubated for approximately 48 hours. Bright-Glo (Promega) was added to the cells to be tested. After lysis, the cells were transferred to opaque white plates, and luciferase activity was detected using a PE envision plate reader. Data were summarized, and IC50 was calculated.50 value.

[0027] like Figure 5 As shown, the recombinant monoclonal antibody RSV-18, screened according to the above method, exhibits an IC50 neutralizing activity against RSV A2 pseudovirus. 50 The concentration was 0.009127 μg / mL, indicating that this recombinant monoclonal antibody exhibited stronger neutralizing ability against RSV type A pseudoviruses than Palivizumab (0.6811 μg / mL). Figure 6 As shown, RSV-18 has neutralizing activity against the RSV B18537 pseudovirus IC. 50 The concentration was 0.006582 μg / mL, and its neutralizing ability against RSV type B pseudovirus was basically equivalent to that of Palivizumab (0.003783 μg / mL).

[0028] True virus neutralization HEP-2 cells were seeded into well plates and cultured overnight. After serially diluted monoclonal antibodies, RSV A2 virus was added and the cells were incubated at 37°C in a 5% CO2 incubator for neutralization. The RSV virus and antibody neutralization product were then added to HEP-2 cells and incubated for approximately 48 hours. The number of CPE-positive wells was measured, data were summarized, and IC50 was calculated. 50 Value. For example... Figure 7 As shown, the recombinant monoclonal antibody RSV-18 has an IC50 of neutralizing activity against RSV A2 virus. 50 The concentration was 0.01410 μg / mL, and its neutralizing activity was superior to that of Palivizumab (0.7300 μg / mL).

[0029] As demonstrated by the above embodiments, this invention provides a novel fully human anti-RSV monoclonal antibody (RSV-18). Experiments have confirmed that this antibody can efficiently and specifically bind to the RSV F protein antigen. Further virus neutralization experiments show that this antibody has significant neutralizing activity against classic representative strains of both RSV A and B subtypes, especially against RSV A subtype, where it is more effective than the clinical control antibody palizumab. Therefore, the RSV-18 provided by this invention has the advantages of high neutralizing potential and low immunogenicity, and has broad application prospects.

[0030] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A fully human monoclonal antibody that specifically binds to the fusion protein F of respiratory syncytial virus, characterized in that, The full human monoclonal antibody comprises a heavy chain variable region and a light chain variable region, the light chain variable region comprising an amino acid sequence shown as SEQ ID NO. 1, and the heavy chain variable region comprising an amino acid sequence shown as SEQ ID NO.

2.

2. An antigen-binding fragment of a fully human monoclonal antibody, characterized in that, The antigen binding fragment specifically binds to the fusion protein F of respiratory syncytial virus, and the antigen binding fragment comprises a heavy chain variable region and a light chain variable region, the light chain variable region comprising an amino acid sequence shown as SEQ ID NO. 1, and the heavy chain variable region comprising an amino acid sequence shown as SEQ ID NO.

2.

3. The antigen binding fragment according to claim 2, characterized in that, The antigen binding fragment is a Fab fragment, a F(ab')2 fragment or a single chain antibody scFv.

4. A pharmaceutical composition, characterized by, It comprises a pharmaceutically acceptable carrier and the full human monoclonal antibody of claim 1 or the antigen binding fragment of any one of claims 2-3.

5. Use of the full human monoclonal antibody of claim 1 or the antigen binding fragment of any one of claims 2-3 in the preparation of a medicament for preventing or treating respiratory syncytial virus infection.

6. Use according to claim 5, characterized in that, The respiratory syncytial virus infection is caused by RSV A subtype virus; and / or, the respiratory syncytial virus infection is caused by RSV B subtype virus.

7. Use of the full human monoclonal antibody of claim 1 or the antigen binding fragment of any one of claims 2-3 in the preparation of a reagent for diagnosing respiratory syncytial virus infection.

Images