Hepatitis B virus monoclonal antibody SY-23 as well as preparation and application thereof

Abstract

The invention discloses a hepatitis B virus monoclonal neutralizing antibody SY-23 as well as preparation and application thereof. The hepatitis B virus monoclonal neutralizing antibody disclosed by the invention is SY-23 and consists of a heavy chain and a light chain paired with the heavy chain; the amino acid sequences of the CDR1, the CDR2 and the CDR3 of the heavy chain of the SY-23 are GGTFRSYG, IVPIFGIS and VRGGAVAGAGSAGEGIFDY in sequence, and the amino acid sequences of the CDR1, the CDR2 and the CDR3 of the light chain of the SY-23 are QSVSNN, GAS and QQHHSWPPFT in sequence. The hepatitis B virus monoclonal neutralizing antibody SY-23 provided by the invention is an HBV neutralizing antibody, has ADCP activity, can crossly recognize various HBV genotypes HBsAg, has a good biological function, and provides a new candidate antibody for prevention and treatment of hepatitis B virus.

Description

Technical Field

[0001] This application relates to the field of HBV neutralizing antibody technology, specifically to a monoclonal antibody against hepatitis B virus SY-23 and its preparation and application. Background Technology

[0002] Hepatitis B virus (HBV) infection remains a major global public health problem. Chronic HBV infection can lead to a range of liver diseases, including chronic hepatitis B (CHB), liver cirrhosis (LC), and hepatocellular carcinoma (HCC). Currently, the FDA-approved drugs for the treatment of chronic hepatitis B fall into two main categories: interferons and nucleoside analogs. These drugs exert their antiviral effects by modulating the host immune system and inhibiting viral replication, respectively. While existing drugs exhibit strong safety and antiviral activity, their therapeutic efficacy is quite limited. The HBsAg seroconversion rate with these drugs is less than 5%, failing to achieve clinical cure. Therefore, there is a need to develop more effective new drugs to improve the clinical management of HBV-related diseases.

[0003] Antibodies play a crucial role in preventing infection and helping the body clear viruses, and can be used as a natural, highly effective treatment for chronic hepatitis B. Antibodies targeting viral surface antigens can prevent viral entry into hepatocytes by blocking the interaction between the hepatitis B virus and its pro-receptor HSPG through the Fab region, or by being internalized into hepatocytes and subsequently blocking the release of the hepatitis B virus from infected cells to prevent a new wave of infection. On the other hand, antibodies can also interact with phagocytes through their Fc region, mediating the clearance of antigen-antibody immune complexes, i.e., the antibody ADCP effect. While traditional hepatitis B immunoglobulin (HBIG) is widely used clinically, it is mainly derived from the serum of hepatitis B vaccine recipients with high antibody titers, resulting in low production and significant differences in concentration and titer between different batches. Therefore, there is an urgent need to develop anti-hepatitis B virus monoclonal antibodies with higher titers and better specificity to provide new treatment options for chronic hepatitis B. Summary of the Invention

[0004] The purpose of this invention is to provide a monoclonal antibody against hepatitis B virus SY-23 and its preparation and application.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: The first aspect of the present invention discloses a monoclonal neutralizing antibody against hepatitis B virus, the monoclonal neutralizing antibody being SY-23, which is composed of a heavy chain and a paired light chain; the amino acid sequences of CDR1, CDR2, and CDR3 of the heavy chain are GGTFRSYG, IVPIFGIS, and VRGGAVAGAGSAGEGIFDY in sequence, and the amino acid sequences of CDR1, CDR2, and CDR3 of the light chain are QSVSNN, GAS, and QQHHSWPPFT in sequence.

[0006] It should be noted that the key to this invention lies in isolating and expanding plasmablasts from peripheral blood mononuclear cells (PBMCs) collected on the 7th day after vaccination, and finally obtaining a fully human monoclonal neutralizing antibody, namely SY-23. This monoclonal neutralizing antibody has cross-binding activity and antiviral function with HBsAg proteins of different HBV genotypes.

[0007] In one implementation of the present invention, the heavy chain variable region sequence of the hepatitis B virus monoclonal neutralizing antibody SY-23 is the sequence shown in Seq ID No. 1, and the light chain variable region sequence is the sequence shown in Seq ID No. 2.

[0008] It should be noted that the heavy chain variable regions and light chain variable regions of the above specific sequences are only the specific monoclonal neutralizing antibody sequences used in one implementation of the present invention. It can be understood that for monoclonal neutralizing antibodies, the regions that affect their precise complementarity with the antigenic determinant are complementarity-determining regions (CDRs), specifically CDR1, CDR2, and CDR3 of the heavy chain, and CDR1, CDR2, and CDR3 of the light chain. Therefore, as long as the heavy chain and light chain CDR1, CDR2, and CDR3 sequences of the present invention remain unchanged, the function and role of the hepatitis B virus monoclonal neutralizing antibody of the present invention can be basically realized. In other words, the specific sequence of the hepatitis B virus monoclonal neutralizing antibody of the present invention is not limited to the heavy chain variable regions and light chain variable regions of the above specific sequences.

[0009] A second aspect of the present invention discloses a nucleic acid fragment encoding the monoclonal neutralizing antibody against hepatitis B virus of the present invention, the nucleic acid fragment encoding the heavy and light chains of SY-23.

[0010] In one implementation of the present invention, the nucleic acid fragment encoding the heavy chain variable region shown in Seq ID No. 1 has the sequence shown in Seq ID No. 3; the nucleic acid fragment encoding the light chain variable region shown in Seq ID No. 2 has the sequence shown in Seq ID No. 4.

[0011] It should be noted that the specific nucleic acid sequences mentioned above are only the nucleic acid sequences used in one implementation of the present invention. It can be understood that there can be multiple codons for one amino acid. Therefore, based on the degeneracy of codons, in addition to the nucleic acid sequences defined above, there can be several other nucleic acid sequences that encode the same heavy or light chain, all of which are within the scope of protection of the present invention, while ensuring that the coding sequence remains unchanged.

[0012] A third aspect of the present invention discloses a recombinant plasmid containing the nucleic acid fragment of the present invention.

[0013] It should be noted that the recombinant plasmids of the present invention are designed to effectively express the nucleic acid fragments of the present invention, thereby obtaining the corresponding heavy chain, light chain, or hepatitis B virus monoclonal neutralizing antibodies; therefore, in principle, any vector capable of transfecting the nucleic acid fragments into host cells for nucleic acid expression can be used in the present invention.

[0014] A fourth aspect of the present invention discloses a recombinant cell containing the nucleic acid fragment of the present invention or the recombinant plasmid of the present invention.

[0015] It should be noted that the recombinant cells of the present invention refer to host cells transfected with the nucleic acid fragments or recombinant plasmids of the present invention; generally, the heavy chain, light chain, or hepatitis B virus monoclonal neutralizing antibodies of the present invention can be obtained by directly culturing such host cells.

[0016] The fifth aspect of the present invention discloses a method for preparing a monoclonal neutralizing antibody against hepatitis B virus, comprising expressing the recombinant plasmid of the present invention into a protein using the recombinant cells of the present invention, extracting and purifying the expressed protein, thereby obtaining the monoclonal neutralizing antibody against hepatitis B virus of the present invention.

[0017] The sixth aspect of the present invention discloses the use of the hepatitis B virus monoclonal neutralizing antibody of the present invention, or the nucleic acid fragment of the present invention, or the recombinant plasmid of the present invention, or the recombinant cell of the present invention in the preparation of hepatitis B virus prevention and treatment drugs or hepatitis B virus detection reagents.

[0018] It should be noted that the hepatitis B virus monoclonal neutralizing antibody of the present invention has cross-binding activity and antiviral function with HBsAg proteins of different HBV genotypes; therefore, it can be used to prepare corresponding drugs for the prevention and treatment of hepatitis B virus. As for nucleic acid fragments, recombinant plasmids, and recombinant cells, these can be used as raw materials for the preparation of hepatitis B virus monoclonal neutralizing antibodies, thereby being used to prepare hepatitis B virus prevention and treatment drugs or hepatitis B virus detection reagents.

[0019] The seventh aspect of the present invention discloses a hepatitis B virus prevention and treatment drug, which contains the hepatitis B virus monoclonal neutralizing antibody of the present invention, or contains a substance capable of inducing the production or in vivo expression of the hepatitis B virus monoclonal neutralizing antibody of the present invention.

[0020] The eighth aspect of the present invention discloses a hepatitis B virus detection reagent containing the hepatitis B virus monoclonal neutralizing antibody of the present invention, or containing an antigen capable of specifically binding to the hepatitis B virus monoclonal neutralizing antibody of the present invention.

[0021] Due to the adoption of the above technical solutions, the beneficial effects of the present invention are as follows: The hepatitis B virus monoclonal neutralizing antibody SY-23 of the present invention is a fully human antibody with broad-spectrum neutralizing activity and ADCP activity, providing a new option for the prevention and treatment of hepatitis B virus.

[0022] It should be understood that the foregoing general description and the following detailed description are exemplary and explanatory only, and are not intended to limit this disclosure. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 The results of the cross-binding experiment between the monoclonal neutralizing antibody SY-23 and HBsAg proteins of genotype A and genotype C in this embodiment of the invention are shown.

[0025] Figure 2 The results of the SPR experiment of monoclonal neutralizing antibody SY-23 and HBsAg protein in this embodiment of the invention are shown.

[0026] Figure 3 The results of the neutralization experiment of monoclonal neutralizing antibody SY-23 in this embodiment of the invention are shown.

[0027] Figure 4 The ADCP experimental results of monoclonal neutralizing antibody SY-23 in this embodiment of the invention are shown.

[0028] Figure 5 The results of flow cytometry experiments on the binding of monoclonal neutralizing antibody SY-23 to 10 different HBV genotypes HBsAg in this embodiment of the invention are shown. Detailed Implementation

[0029] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Experimental methods in the following embodiments that do not specify specific conditions are generally determined according to national standards. Experimental materials in the following embodiments that do not specify the source are all commercially available raw materials. The equipment used in each step of the following embodiments are all conventional equipment. If there is no corresponding national standard, it is carried out according to general international standards, conventional conditions, or conditions recommended by the manufacturer. Unless otherwise stated, all parts are parts by weight, and all percentages are percentages by mass. Unless otherwise defined or stated, all professional and scientific terms used in the present invention have the same meaning as those skilled in the art. In addition, any methods and materials similar or equivalent to those described can be applied to the methods of the present invention.

[0030] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other. The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but this is not intended to limit the scope of the invention. Example 1: Antibody screening, expression, and purification

[0031] This study was approved by the Ethics Committee of the Third People's Hospital of Shenzhen (Approval No.: 2021-030). Participants provided written informed consent for sample collection and subsequent analysis.

[0032] 1. This example specifically collected one sample, and the sample collection method is as follows: Peripheral blood mononuclear cell isolation: Collect 10 mL of venous blood using an anticoagulant tube, transfer it to a 50 mL centrifuge tube, dilute with 10 mL of PBS solution, and mix gently. Take two 15 mL centrifuge tubes and add 5 mL of Ficoll separation solution to each. Then add 10 mL of diluted blood to the supernatant of the Ficoll separation solution. Centrifuge at 2000 rpm for 20 minutes. Transfer the leukocyte layer to a clean 15 mL centrifuge tube. Add PBS to 10 mL, centrifuge at 1500 rpm for 10 minutes, discard the supernatant, and collect the cells for subsequent experiments.

[0033] 2. Isolation of monoclonal antibodies from peripheral blood clone-expanded plasmablasts Fresh peripheral blood mononuclear cells were isolated and washed twice with 10 mL of PBS. A mixture of CD19-PE-Cy7, CD20-APC-H7, CD3-APC-H7, CD27-PE, and CD38-APC (all from BD Biosciences) was added to 100 μL of staining buffer (PBS + 2% fetal bovine serum) to resuspend the peripheral blood mononuclear cells, and staining was performed at 4°C for 30 minutes. After washing twice with PBS, single activated plasmablasts of CD19+CD20-CD3-CD27^hiCD38^hi were sorted using a BDAria II sorting flow cytometer.

[0034] Single activated plasmablasts were sorted into 96-well PCR plates containing lysis buffer. RT-PCR and nested PCR were then performed according to the method described in the literature (LiaoHX, Levesque MC, Nagel A, Dixon A, Zhang R, Walter E, et al. High-throughput isolation of immunoglobulin genes from single human B cells and expression asmonoclonal antibodies. Journal of virological methods. 2009;158:171-9.) to amplify the variable regions of the heavy and light chains, respectively. The PCR amplification products were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing, and the resulting antibody was named SY-23.

[0035] The antibody variable region sequence obtained from sequencing was sent to GenScript Biotech for synthesis. GenScript then cloned the variable regions of the antibody heavy and light chains into the full-length IgG1 heavy and light chain expression vectors pCDNA3.4 (GenScript Biotech), respectively, and prepared large quantities of antibody heavy and light chain plasmids. After obtaining the plasmids prepared by GenScript, the paired heavy and light chain expression plasmids were co-transfected into 293F cells (500 mL as an example) using PEI transfection reagent for expression. The monoclonal antibody was then purified from the culture supernatant using a protein A adsorption column. The specific steps are as follows: 293F cells were cultured in an 8% CO2, 37℃ incubator. The cell concentration was adjusted to 1.2 × 10⁶ cells / mL, and cultured for another 2 hours. Solution A was prepared by adding 250 μg of antibody heavy chain plasmid and 250 μg of antibody light chain plasmid to 12.5 mL of Opti-MEM (31985070, Gibco). Solution B was prepared by adding 2.5 mL of 1 mg / mL PEI transfection reagent (24885-2, Polysciences) to 12.5 mL of Opti-MEM and incubating for 5 minutes. Solutions A and B were mixed and incubated for 20 minutes to obtain the AB mixture. 25 mL of the AB mixture was added dropwise to 500 mL of 293F cells, shaking constantly while adding. The cells were cultured for another 5 days. The 293F cells were then centrifuged at 3000g for 20 minutes, and the supernatant was collected and filtered through a 0.45 μm filter. Protein was then opened. The Protein A gravity column was capped, and gravity was used to allow the 20% ethanol solution in the column to flow out completely. The Protein A gravity column was equilibrated with 5 column volumes of 10 mM PBS solution. Filtered cell supernatant was added to the Protein A gravity column and allowed to flow out under gravity. The Protein A gravity column was washed with 3 column volumes of PBS solution, and then eluted with 5 volume volumes of 0.1 M glycine-hydrochloric acid solution (pH=3.0). The eluent was placed in a 30 kDa ultrafiltration concentration tube, filled with PBS, and centrifuged at 3500 rpm for 40 minutes at 4°C. The waste liquid in the collection tube was discarded, 20 mL of PBS solution was added, and the tube was centrifuged at 3500 rpm for 40 minutes at 4°C. The concentrated and replaced antibody solution was then collected, and the antibody protein concentration was measured. Example 2: Enzyme-linked immunosorbent assay (ELISA) to verify the binding ability of SY-23 antibody.

[0036] In Example 1, a single activated plasmablast was isolated from the peripheral blood of a vaccine recipient. A fully human monoclonal antibody, SY-23, was ultimately obtained using RT-PCR and nested PCR techniques.

[0037] The heavy chain and light chain variable regions of the monoclonal neutralizing antibody SY-23 isolated in this example, along with the sequencing results, are shown in Tables 1 and 2. Table 1 presents the heavy chain and light chain variable region sequences of the monoclonal antibody, and Table 2 presents the nucleic acid sequencing results of the heavy chain and light chain variable regions of the monoclonal antibody.

[0038] Table 1 Monoclonal antibody sequences

[0039] Table 2. Results of monoclonal antibody nucleic acid sequencing

[0040] The analysis results showed that the CDR1, CDR2 and CDR3 sequences of the heavy chain of SY-23 were GGTFRSYG, IVPIFGIS and VRGGAVAGAGSAGEGIFDY, respectively, while the CDR1, CDR2 and CDR3 sequences of the light chain were QSVSNN, GAS and QQHHSWPPFT, respectively.

[0041] The binding ability of the obtained HBsAg monoclonal antibody SY-23 to hepatitis B virus HBsAg was detected by ELISA. HBsAg protein of genotype A and genotype C (Shenzhen Kangtai Biological, Shenzhen Immuth Company) was coated in 96-well ELISA plates at 2 μg / mL and incubated overnight at 4°C, 100 μL per well. Wash 5 times with PBST (PBS solution containing 0.5% Tween-20); block with blocking buffer at room temperature for 1 hour, 150 μL per well, then wash 5 times with PBST. Blocking buffer formulation: 5% skim milk + 2% BSA (prepared with PBS). All subsequent antibody dilution buffer formulations are the same as the blocking buffer. Dilute SY-23 antibody to 10 μg / mL with blocking buffer, 100 μL per well, incubate at 37°C for 1 hour, then wash 5 times with PBST. Add HRP-goat anti-human IgG (ZB-2304, Zhongshan Jinqiao) at a 1:5000 ratio, incubate at 37°C for 1 hour, then wash 5 times with PBST. Mix chromogenic solution A and solution B (Sangon Biotech) at a 1:1 ratio, 100 μL per well, incubate at room temperature in the dark for 20 minutes; then terminate the reaction with 50 μL of 2M H2SO4. Measure the optical density at 450 nm (OD) using a Varioskan LUX multimodal microplate reader (Thermo Scientific).

[0042] The binding activity of monoclonal antibody SY-23 to the HBV surface antigen protein HBsAg was analyzed by ELISA. The results are as follows: Figure 1 As shown, the monoclonal antibody SY-23 can cross-bind with HBsAg of HBV genotypes A and C, and its binding ability is relatively strong. Example 3: Surface plasmon resonance (SPR) analysis of the affinity between SY-23 antibody and HBsAg

[0043] The binding assay of genotype C HBsAg protein to SY-23 antibody was performed using the Biacore 8K system (GE Healthcare). Details are as follows: HBsAg protein (Shenzhen Immuth Company) was dissolved in 10 mM sodium acetate buffer (pH=5.0) and covalently coated onto one flow cell of the CM5 sensor chip, yielding approximately 1000 final response units (RUs). The other flow cell remained uncoated and served as a control. All analyses were performed in HBS-EP buffer (10 mM HEPES pH=7.4, 150 mM NaCl, 3 mM EDTA, and 0.05% Tween-20) at a flow rate of 30 μL / min. Serially diluted antibodies were injected for 60 seconds, and the data were fitted to a 1:1 binding model using Biacore evaluation software (GE Healthcare). Each measurement was performed twice, and a single value was used to generate the average affinity constant.

[0044] The affinity of SY-23 for genotype C HBsAg protein was detected using surface plasmon resonance (SPR) technology, and the results are as follows: Figure 2 As shown, the affinity KD of SY-23 is 35.23 nM (Kon: binding rate constant; Koff: dissociation rate constant). This further demonstrates that SY-23 has a high binding affinity to genotype C HBsAg protein. Figure 1 The results were consistent. Example 4: Neutralizing capacity detection of SY-23 antibody

[0045] HepG2-AD38 cells and HepG2-NTCP cells were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin and streptomycin at 37°C in a 5% CO2 incubator.

[0046] Preparation of live HBV virus: When the confluence of HepG2-AD38 cells in the T75 cell culture flask reaches approximately 80%, the culture medium is discarded, and fresh culture medium is added. HBV virus particles are collected every 2 days for a total of 5 collections. The virus-containing cell culture medium is transferred to a 50 mL centrifuge tube, centrifuged at 3500 rpm for 20 minutes, and the supernatant is filtered through a 0.45 μm filter. The collected virus is then ultra-highly purified with 15% sucrose solution and centrifuged at 130,000 g for 16 hours. The supernatant is discarded, and the virus is resuspended in serum-free DMEM medium, mixed well, aliquoted, and stored at -80°C for later use.

[0047] To determine antibody neutralizing activity, serially diluted monoclonal antibodies were incubated with HBV dilution at 37°C for 1 hour. The mixture was then added to 96-well plates containing HepG2-NTCP cells and incubated at 37°C with 5% CO2 for 24 hours, after which the culture medium was replaced. On day 7 post-infection, the supernatant was collected, and HBeAg levels were detected using ELISA (KHB diagnostic kit). The HBeAg secretion inhibition rate was calculated using GraphPad Prism 8.0 software, employing a logarithmic (inhibitor) and standardized response-variable slope (four-parameter) model to calculate the 50% inhibitory concentration (IC50).

[0048] The neutralizing activity of SY-23 antibody against live HBV genotype D was detected by an HBV live virus neutralization assay. The results are as follows: Figure 3 As shown, SY-23 has a neutralizing effect on live HBV of genotype D, indicating that this antibody is an HBV neutralizing antibody. Example 5: Detection of ADCP activity of SY-23 antibody

[0049] THP-1 cells were digested and dispersed into single cells. After counting, the cell concentration was adjusted and seeded into 24-well plates with 2 x 10⁵ cells per well, 500 μL, and cultured at 37°C for 24 hours in a 5% CO₂ incubator. The antibody to be tested was diluted to a concentration of 20 μg / mL with RPMI 1640 medium containing 10% FBS. The diluted antibody was mixed with an equal volume of HBsAg protein (1 μg / mL) and incubated at 37°C for 1 hour. Aspirate 250 μL of cell culture medium and add 250 μL of the incubated antibody-antigen mixture to THP-cells. Incubate at 37°C for 2 hours in a 5% CO2 incubator. Discard the supernatant, wash the cells once with sterile PBS, add 200 μL of cell lysis buffer (Western and IP cell lysis buffer, Beyotime) to each well, vortex for 1 minute, and lyse at 4°C for 15 minutes. Add 200 μL of PBS and measure the lysis buffer diluted to 100 μL using an HBsAg ELISA kit (Beijing Wantai Biotech). Measure the optical density at 450 nm (OD) using a Varioskan LUX multimodal microplate reader (Thermo Scientific).

[0050] Besides neutralization, Fc-mediated non-neutralizing functions also contribute to antibody clearance of HBV, such as antibody-dependent phagocytosis (ADCP). Using THP-1 cells as effector cells, the biological activity of the SY-23 antibody was tested in the presence of HBsAg particles. Results are as follows... Figure 4 As shown, SY-23 possesses ADCP activity. Example 6: Detection of the broad-spectrum recognition ability of monoclonal antibody SY-23 by flow cytometry

[0051] This embodiment uses flow cytometry to detect intracellular HBsAg expression in cells that recognize different HBV genotypes by the monoclonal antibody SY-23, as detailed below: First, HBsAg protein expression plasmids for different HBV genotypes A, B, C, D, E, F, G, H, I, and J were transfected into 293T cells, with cells transfected with the empty Vector vector serving as a control. After 48 hours, cells were digested with 0.5% trypsin and the process was terminated by adding complete DMEM medium containing 10% FBS. Cells were gently pipetted and observed under a microscope until more than 90% of the cells were in a single-cell state. The single-cell suspension was transferred to a 15mL centrifuge tube and centrifuged at 400g for 5 minutes. The supernatant was discarded, and 3mL of complete DMEM medium was added. 20μL of the cell suspension was taken, and 20μL of AO / PI dye (RE010212, Countstar) was added. The viable cell count was determined at 5×10⁵ cells / test. Cells were fixed and permeabilized using a cell fixation / permeabilization kit (554714, BD Biosciences), centrifuged at 400g for 5 minutes, and the supernatant was discarded. Then, 5 μg / mL SY-23 was added to each flow cytometry tube, and the cells were incubated at 4°C for 30 minutes. After washing, the cells were stained with Alexa Fluor 647-conjugated goat anti-human secondary antibody (A11013, Life Technologies) and incubated at 4°C for 30 minutes. After washing, the cells were collected using a FACSymphony™ A3 flow cytometer (BD Biososciences), and the data were analyzed using FlowJo software V10.9 (BD Biososciences). The experimental results are as follows: Figure 5 As shown.

[0052] The results showed that the SY-23 antibody could clearly detect intracellularly expressed HBsAg and could widely identify HBsAg from different HBV genotypes A, B, C, D, E, F, G, H, I and J.

[0053] In summary, the monoclonal antibody SY-23 obtained by screening in this invention has a strong binding ability to the surface antigen protein HBsAg of hepatitis B virus, possesses neutralizing activity and ADCP activity, and can cross-recognize HBsAg of 10 types of hepatitis B virus, exhibiting good broad-spectrum and antiviral activity, providing a new option for the prevention and treatment of hepatitis B virus.

[0054] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A monoclonal neutralizing antibody against hepatitis B virus, characterized in that, The monoclonal neutralizing antibody is SY-23, which consists of a heavy chain and a paired light chain; the amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain are GGTFRSYG, IVPIFGIS and VRGGAVAGAGSAGEGIFDY in sequence, and the amino acid sequences of CDR1, CDR2 and CDR3 of the light chain are QSVSNN, GAS and QQHHSWPPFT in sequence.

2. The hepatitis B virus monoclonal neutralizing antibody according to claim 1, characterized in that, The heavy chain variable region sequence of SY-23 is shown in Seq ID No.1, and the light chain variable region sequence is shown in Seq ID No.

2.

3. A nucleic acid fragment encoding a monoclonal neutralizing antibody against hepatitis B virus as described in claim 1 or 2, characterized in that, The nucleic acid fragment encodes the heavy and light chains of SY-23.

4. The nucleic acid fragment according to claim 3, characterized in that, The nucleic acid sequence encoding the heavy chain variable region shown in Seq ID No. 1 is shown in Seq ID No. 3; the nucleic acid sequence encoding the light chain variable region shown in Seq ID No. 2 is shown in Seq ID No.

4.

5. A recombinant plasmid containing the nucleic acid fragment of claim 3 or 4.

6. A recombinant cell containing the nucleic acid fragment of claim 3 or 4 or the recombinant plasmid of claim 5.

7. The method for preparing the monoclonal neutralizing antibody against hepatitis B virus according to claim 1 or 2, characterized in that, This includes using the recombinant cells described in claim 6 to express proteins from the recombinant plasmid described in claim 5, extracting and purifying the expressed protein, thereby obtaining the hepatitis B virus monoclonal neutralizing antibody.

8. The use of the hepatitis B virus monoclonal neutralizing antibody according to claim 1 or 2, or the nucleic acid fragment according to claim 3 or 4, or the recombinant plasmid according to claim 5, or the recombinant cell according to claim 6, in the preparation of hepatitis B virus prevention and treatment drugs or hepatitis B virus detection reagents.

9. A drug for the prevention and treatment of hepatitis B virus, characterized in that, It contains the monoclonal neutralizing antibody against hepatitis B virus as described in claim 1 or 2, or contains a substance capable of inducing the production or in vivo expression of the monoclonal neutralizing antibody against hepatitis B virus as described in claim 1 or 2.

10. A hepatitis B virus detection reagent, characterized in that, It contains a monoclonal neutralizing antibody against hepatitis B virus as described in claim 1 or 2, or contains an antigen capable of specifically binding to the monoclonal neutralizing antibody against hepatitis B virus as described in claim 1 or 2.

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