A potent neutralizing antibody against respiratory syncytial virus, RSV-34, and uses thereof

Abstract

The application provides a potent neutralizing antibody RSV-34 against respiratory syncytial virus and application thereof, and belongs to the technical field of biological medicine. The antibody provided by the application can specifically bind to a key functional epitope on an RSV F protein, and exhibits excellent neutralizing efficiency. The half inhibitory concentration (IC 50 ) of the antibody to RSV A2 pseudovirus reaches the level of nanograms per milliliter, which is significantly better than a clinical control drug. The antibody provided by the application not only has super-high neutralizing activity, but also can directly interfere with the membrane fusion process of the virus. The application further includes a pharmaceutical composition containing the antibody and application of the antibody in preparation of an RSV treatment or diagnostic preparation, and provides a core candidate molecule for development of a new generation of high-efficiency anti-RSV therapy.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a potent neutralizing antibody against respiratory syncytial virus (RSV-34) 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 the core functional protein for infecting host cells, responsible for mediating the fusion of the viral membrane with the 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 fully human anti-RSV antibodies with potent, broad-spectrum neutralizing and blocking activity 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 potent neutralizing antibody against respiratory syncytial virus (RSV) RSV-34 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 and blocking efficacy.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: The present invention provides a fully human monoclonal antibody (RSV-34) 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-34) provided by this invention has the following beneficial effects: Excellent virus neutralizing efficacy: The antibody RSV-34 provided by this invention exhibits extremely high neutralizing activity, especially in pseudovirus neutralization experiments, showing outstanding performance with an IC50 of 100% against RSV A2 pseudovirus. 50 The concentration was as low as 0.002657 μg / mL, indicating that it can produce effective protection at extremely low doses, and has important development value.

[0014] Unique binding characteristics suggest that the competitive ELISA results indicate that the antibody RSV-34 provided by this invention competes with antibodies targeting specific regions (sites 2 and 3) of the viral F protein. This strongly suggests that the antibody RSV-34 of this invention binds to a unique and critical epitope, which may be closely related to the core region of viral fusion function. Combined with its extremely high neutralizing activity, this indicates that the antibody of this invention may block viral invasion by occupying or interfering with a crucial functional site.

[0015] Therapeutic advantages of fully human-derived properties: As a fully human antibody directly derived from the human immune system, the product of this invention avoids the immunogenicity risks introduced by heterologous sequences, laying a solid foundation for the development of preventive or therapeutic biological agents that can be used safely for long-term purposes.

[0016] Providing core candidate molecules for the development of next-generation therapies: Based on its strong neutralizing activity and potential action on key functional epitopes, antibody RSV-34 is not only an excellent candidate for single therapy, but also has the potential to serve as a core component in the construction of bispecific antibodies or combination therapies to combat viral escape or enhance treatment efficacy. Attached Figure Description

[0017] Figure 1 Flow cytometry sorting diagram of RSV-specific B cells; 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 Detection graph of the neutralizing activity of the antibody against RSV A2 pseudovirus in this invention; Figure 6 Detection graph of 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

[0018] This invention provides a fully human monoclonal antibody or its antigen-binding fragment that 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.

[0019] 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.

[0020] 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.

[0021] 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.

[0022] 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.

[0023] 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.

[0024] 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.

[0025] Example Obtaining RSV-specific BCR Lymphocytes from frozen healthy volunteers were revived, and after purification and enrichment of B cells, the RSV pre-F (SinoBiological) antigen was conjugated with a fluorescent group and oligonucleotide barcode. Following flow cytometry staining, RSV pre-F cells were sorted using flow cytometry. + 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, RSV pre-F is obtained by flow sorting. + Cells. Subsequently, single cells were prepared and barcode and BCR libraries were constructed using the Chromium Next GEM Single Cell 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 IGHV3-21 / IGLV1-40 were screened for subsequent preparation and purification of recombinant antibodies.

[0026] 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) E co RⅠ and B am 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-34 was finally obtained, and its sequence information is shown in Table 1 below.

[0027] Table 1. Antibody sequences of the present invention

[0028] Antibody binding activity assay The antigen F protein was diluted to 1 μg / mL with phosphate buffer 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. The 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. The results showed that the fully human recombinant antibody RSV-34 and RSV A subtype F protein (…) Figure 3 ) and RSV B subtype F protein ( Figure 4 Both have high binding capacity, and their binding capacity against both subtypes of F protein is stronger than that of Palivizumab.

[0029] 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 and added to RSVA2 and RSV B18537 pseudoviruses, respectively, and incubated at 37°C in a 5% CO2 incubator for approximately 0.5 hours. The pseudovirus-antibody neutralization products were then added to Huh7.5.1 cells and cultured 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 analyzed using GraphPad Prism 9 to calculate IC50. 50 Value. For example... Figure 5 As shown, the fully human recombinant antibody RSV-34 exhibits an IC50 neutralizing activity against RSV A2 pseudovirus. 50 It was 0.002657 μg / mL, which is 256 times that of Palivizumab (0.6811 μg / mL). For example... Figure 6 As shown, the fully human recombinant antibody RSV-34 exhibits neutralizing activity (IC50) against the RSV B18537 pseudovirus. 50 It was 0.1053 μg / mL.

[0030] 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 with 5% CO2 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 detected, data were summarized, and analyzed using GraphPad Prism 9 to calculate the IC50. 50 Value. For example... Figure 7 As shown, the neutralizing activity IC50 of the fully human recombinant antibody RSV-34 against RSV A2 virus is [value missing]. 50 It was 0.03973 μg / mL, which was better than Palivizumab (0.7300 μg / mL).

[0031] Competition ELISA Antibodies were biochemically prepared into detection antibodies using the EZ Micro Sulfo NHS LC Biotinylation kit, and the RSV pre-F (Sino Biological) antigen protein was coated overnight. After blocking, the blocking antibody was incubated at room temperature for 2 hours, followed by incubation with the detection antibody for 30 minutes. Subsequently, the antibody was incubated with streptavidin-conjugated HRP at a 1:40000 dilution for 1 hour. After washing, TMB was used for color development. The reaction was terminated by adding stop solution at 50 μL / well, and the absorbance at 450 nm was measured and calculated. A competition score of less than 20 was considered a strong competition. The results are shown in Table 2 (control antibodies: Palivizumab, MPE8, 101F, AM22): Table 2 Results of Competitive ELISA Detection

[0032] The results showed that the fully human recombinant antibody RSV-34 provided by this invention competes with antibodies targeting sites 2 and 3.

[0033] As demonstrated by the above embodiments, the novel fully human anti-RSV monoclonal antibody RSV-34 provided by this invention not only binds to the RSV F protein antigen with high efficiency and specificity, but its binding epitope also competes with antibodies targeting key regions (site 2 / 3). Further virus neutralization experiments showed that this antibody exhibits superior neutralizing activity, particularly against RSV A2 pseudovirus, significantly outperforming the clinical control antibody palizumab. Therefore, this invention successfully yields the fully human anti-RSV monoclonal antibody RSV-34, which acts on key epitopes and possesses ultra-high neutralizing potency.

[0034] 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 fully human monoclonal antibody comprises 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.

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. The antigen-binding fragment includes a heavy chain variable region and a light chain variable region. 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.

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

4. A pharmaceutical composition, characterized in that, It comprises a pharmaceutically acceptable carrier and the fully human monoclonal antibody as described in claim 1 or the antigen-binding fragment as described in any one of claims 2 to 3.

5. The use of the fully human monoclonal antibody of claim 1 or the antigen-binding fragment of any one of claims 2 to 3 in the preparation of a medicament for the prevention or treatment of respiratory syncytial virus infection.

6. The application 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 subtype B virus.

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

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