Anti-asialoglycoprotein receptor antibody as well as preparation method and application thereof

Abstract

The invention provides an antibody for resisting an asialoglycoprotein receptor as well as a preparation method and application of the antibody. The antibody for resisting the asialoglycoprotein receptor comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises a heavy chain variable region HCDR1-HCDR3; and the light chain variable region comprises a light chain variable region LCDR1-LCDR3. According to the present invention, the affinity of the anti-asialoglycoprotein receptor antibody to the human asialoglycoprotein receptor is 0.425 nM, and the antigen recognition ability is strong; the compound disclosed by the invention can be used for remarkably inhibiting the DNA expression of HBsAg, HBeAg and HBV of HepG2.2. 15 cells and HepAD38 cells, has a function of inhibiting the replication of the hepatitis B virus, and has a great application prospect in medicines for preventing and / or treating hepatitis B virus infection and / or preventing and / or treating related diseases caused by the hepatitis B virus infection.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to an antibody against the desialyl glycoprotein receptor, its preparation method, and its application. Background Technology

[0002] Hepatitis B virus (HBV) is a hepatotropic DNA virus composed of an envelope and a nucleocapsid. It is highly infectious and primarily affects the human liver. Infection can lead to acute hepatitis B (AHB), chronic hepatitis B (CHB), liver fibrosis, and liver cancer, among other related diseases.

[0003] Current standard treatment regimens primarily involve two classes of drugs: nucleoside (acid) analogs (NAs) and pegylated interferon-alpha (PEG-IFNα). NAs, such as entecavir and tenofovir, can potently inhibit HBV reverse transcriptase, thereby effectively reducing serum HBV DNA levels. However, NAs cannot effectively clear covalently closed circular DNA (cccDNA) in hepatocyte nuclei, which is the template for viral replication and the source of persistent infection. Therefore, most patients require long-term or even lifelong medication, and viral load is highly likely to rebound upon discontinuation. PEG-IFNα inhibits the virus by modulating the host immune response, but its response rate is limited (HBsAg clearance rate <10%) and it is accompanied by significant side effects, restricting its widespread use.

[0004] Monoclonal antibodies are highly homogeneous antibodies produced by a single B cell clone, targeting only a specific antigenic epitope. Compared to interferon and nucleoside (acid) analogue antiviral drugs, monoclonal antibody drugs exhibit advantages in treatment, including high specificity, high sensitivity, high efficacy, and good safety. Finding monoclonal antibody drugs that can effectively inhibit hepatitis B virus replication holds promise for overcoming the shortcomings of existing anti-hepatitis B virus infection drugs.

[0005] The Asialoglycoprotein receptor (ASGPR) is a C-type lectin that is highly expressed on the sinusoidal membrane of hepatocytes. In recent years, the role of ASGPR in the process of HBV entry into hepatocytes has received increasing attention. Multiple studies have shown that HBV viral particles can interact with ASGPR through glycosylation modifications on their surface. ASGPR is considered the initial "attachment receptor" for HBV attachment to hepatocytes. The virus first binds with low affinity to heparan sulfate proteoglycans (HSPGs) on the hepatocyte surface, then binds with high affinity to ASGPR, thereby accumulating on the cell surface and ultimately delivering the virus to the functional entry receptor NTCP, triggering endocytosis.

[0006] Therefore, ASGPR represents a highly attractive therapeutic target for anti-HBV. Developing antibodies that specifically bind to ASGPR and effectively block its interaction with HBV holds promise as a novel HBV entry inhibitor, providing a new strategy for achieving functional cure of HBV. However, currently, there is a lack of anti-ASGPR antibodies with high affinity, high specificity, and potent HBV blocking activity for clinical treatment.

[0007] In conclusion, a new technical solution is urgently needed to address the problems existing in the current technology. Summary of the Invention

[0008] Based on this, the present invention discloses an antibody against desialyl glycoprotein receptor. Compared with the prior art, it has the following advantages: (1) The antibody against desialyl glycoprotein receptor involved in the present invention has an affinity of 0.425 nM for human desialyl glycoprotein receptor, which is higher than that of antibodies disclosed in the prior art, and has a stronger ability to recognize antigens. (2) It can significantly inhibit the expression of HBsAg, HBeAg and HBV DNA in HepG2.2.15 cells and HepAD38 cells, and has the function of inhibiting the replication of hepatitis B virus. (3) The antibody against desialyl glycoprotein receptor involved in the present invention targets the non-functional site of desialyl glycoprotein receptor, and will not affect the normal metabolism of other proteins.

[0009] In one embodiment of the present invention, the variable region of the heavy chain of the antibody against desialyl glycoprotein receptor includes HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3, respectively; the variable region of the light chain includes LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO.4, SEQ ID NO.5, and SEQ ID NO.6, respectively.

[0010] In one embodiment of the present invention, the amino acid sequence of the heavy chain variable region of the antibody against desialyl glycoprotein receptor is shown in SEQ ID NO.7, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.8.

[0011] In one embodiment of the invention, the light chain and the heavy chain are connected by disulfide bonds.

[0012] The term "monoclonal antibody" as used in this invention refers to a highly homogeneous antibody produced by a single B cell clone that targets only a specific antigenic epitope.

[0013] The term "heavy chain variable region" used in this invention, namely HCDR, the complementarity-determining region of the heavy chain, refers to the core conserved structure of the antibody heavy chain variable region, which is a key region that determines the binding of the antibody to the antigen, including HCDR1, HCDR2 and HCDR3.

[0014] The term "light chain variable region" used in this invention, namely LCDR, the complementarity-determining region of the light chain, refers to the core conserved structure of the antibody light chain variable region, which is a key region that determines the binding of the antibody to the antigen, including LCDR1, LCDR2 and LCDR3.

[0015] One object of the present invention is to provide an antibody against desialyl glycoprotein receptor, said antibody against desialyl glycoprotein receptor comprising a heavy chain variable region and a light chain variable region; The heavy chain variable region includes heavy chain variable regions HCDR1-HCDR3; The light chain variable region includes light chain variable regions LCDR1-LCDR3; The heavy chain variable region HCDR1 is selected from amino acid sequence 1 or amino acid sequence 9; The heavy chain variable region HCDR2 is selected from amino acid sequence 2 or amino acid sequence 10; The heavy chain variable region HCDR3 is selected from amino acid sequence 3 or amino acid sequence 11; The light chain variable region LCDR1 is selected from amino acid sequence 4 or amino acid sequence 12; The light chain variable region LCDR2 is selected from amino acid sequence 5 or amino acid sequence 13; The light chain variable region LCDR3 is selected from amino acid sequence 6 or amino acid sequence 14; The amino acid sequences 1-6 and 9-14 are shown in SEQ ID NO.1-SEQ ID NO.6 and SEQ ID NO.9-SEQ ID NO.14, respectively.

[0016] Furthermore, the antibody against the desialyl glycoprotein receptor comprises a monoclonal antibody and an antibody fragment; the antibody fragment comprises Fab, a single-chain antibody, and a single-domain antibody.

[0017] Further, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.7 or SEQ ID NO.15; the amino acid sequence of the light chain variable region is shown in SEQ ID NO.8 or SEQ ID NO.16.

[0018] Another object of the present invention is to provide a method for preparing the above-mentioned antibody against desialyl glycoprotein receptor, comprising the following steps: S1. Construction, expression, and purification of the immunogen (truncated desialyl glycoprotein receptor): Through bioinformatics, the three-dimensional structure of the desialyl glycoprotein receptor binding to its ligand was analyzed to identify the key amino acid sequence for the desialyl glycoprotein receptor to function; the key amino acid sequence for the desialyl glycoprotein receptor to function was removed to construct a truncated desialyl glycoprotein receptor as an immunogen. S2. Use the truncated desialyl glycoprotein receptor as an immunogen to immunize Balb / c mice; S3, fusion of mouse spleen cells and myeloma cells; S4. ELISA screening for antibodies against desialyl glycoprotein receptor; S5 and ELISA-positive cloning well cells were cloned to obtain antibodies against desialyl glycoprotein receptors.

[0019] Another object of the present invention is to provide a recombinant protein whose amino acid sequence comprises the amino acid sequence of the antibody against the above-mentioned desialyl glycoprotein receptor.

[0020] Another object of the present invention is to provide a gene that encodes the aforementioned antibody or recombinant protein against the desialyl glycoprotein receptor.

[0021] Another object of the present invention is to provide a recombinant plasmid carrying the above-mentioned gene.

[0022] Another object of the present invention is to provide a medicament containing the above-mentioned antibody, recombinant protein, gene or recombinant plasmid against desialyl glycoprotein receptor.

[0023] The present invention has the following beneficial effects: (1) The antibody against desialyl glycoprotein receptor provided by the present invention has an affinity of 0.425 nM for human desialyl glycoprotein receptor and has a stronger ability to recognize antigen.

[0024] (2) In vitro experiments showed that the antibody against desialyl glycoprotein receptor provided by this invention significantly inhibited the expression of HBsAg, HBeAg, and HBV DNA in HepG2.2.15 and HepAD38 cells in vitro, demonstrating its ability to inhibit hepatitis B virus replication. Therefore, the antibody against desialyl glycoprotein receptor provided by this invention has great application potential in drugs for the prevention and / or treatment of hepatitis B virus infection, and / or the prevention and / or treatment of related diseases caused by hepatitis B virus infection.

[0025] (3) The antibody against desialyl glycoprotein receptor provided by the present invention binds to the non-functional region of the desialyl glycoprotein receptor, and does not inhibit the normal metabolism of other glycoproteins, thus having better safety. Attached Figure Description

[0026] Figure 1 A schematic diagram of the structure of the desialyl glycoprotein receptor (ASGPR) binding to its glycosyl ligand N-acetylgalactosamine (GalNac) is shown.

[0027] Figure 2 The truncated amino acid sequence of the desialyl glycoprotein receptor, which serves as an immunogen, is shown.

[0028] Figure 3 The SDS-PAGE gel electrophoresis results of the truncated desialyl glycoprotein receptor expressed and purified in 293 T cells are shown, where R represents reduction electrophoresis, NR represents non-reduction electrophoresis, and M represents protein marker.

[0029] Figure 4 The results of SDS-PAGE gel electrophoresis of purified antibody J31 against desialyl glycoprotein receptor are shown.

[0030] Figure 5 The results of SDS-PAGE gel electrophoresis of purified antibody J35 against desialyl glycoprotein receptor are shown.

[0031] Figure 6 The OD450 nm curves of antibody J31 binding to the antigen at different concentrations against the desialyl glycoprotein receptor are shown.

[0032] Figure 7 The OD450 nm curves of antibody J35 binding to the antigen at different concentrations against the desialyl glycoprotein receptor are shown.

[0033] Figure 8 The image shows an immunofluorescence assay of antibody J31 against desialyl glycoprotein receptor binding to HepG2.2.15 cells.

[0034] Figure 9 The image shows an immunofluorescence assay of antibody J35 against desialyl glycoprotein receptor binding to HepG2.2.15 cells. Detailed Implementation

[0035] To more clearly illustrate the technical solution of the present invention, the following embodiments are provided. Unless otherwise stated, the raw materials, reactions, and post-processing methods appearing in the embodiments are all commercially available raw materials and technical methods well known to those skilled in the art.

[0036] The terms "preferred," "more preferably," and "more suitable" used in this invention refer to embodiments of the invention that provide certain beneficial effects under certain circumstances. However, other embodiments may also be preferred under the same or other circumstances. Furthermore, the description of one or more preferred embodiments does not imply that other embodiments are unavailable, nor is it intended to exclude other embodiments from the scope of this invention.

[0037] It should be understood that, except in any operational instance or otherwise indicated, the amounts or all figures representing ingredients used, for example, in the specification and claims, should be understood to be modified by the term "about" in all cases. Therefore, unless otherwise stated, the numerical parameters set forth in the following specification and appended claims are approximate values ​​varying according to the desired performance to be obtained according to the invention.

[0038] Example An antibody against desialyl glycoprotein receptor, the preparation method of which includes the following steps: S1. Construction, expression, and purification of the immunogen (truncated desialyl glycoprotein receptor); S1-1. The crystal structure of the desialyl glycoprotein receptor bound to its glycosyl ligand (PDB: 6YAU) was analyzed using PyMOL, and the results are as follows: Figure 1 As shown, the desialyl glycoprotein receptor binds non-covalently to its glycosyl ligand through a pocket structure; further analysis shows that this pocket structure is mainly located at the carboxyl terminus of the desialyl glycoprotein receptor, specifically amino acids 235-291. S1-2, Remove the amino acid sequence that binds to the glycosyl ligand at the carboxyl terminus of the desialyl glycoprotein receptor; the remaining portion serves as the immunogen. Figure 2 As shown; to increase secretion, a secretion signal peptide was added to its amino terminus; to facilitate subsequent purification, six histidine residues were added to its carboxyl terminus. The complete amino acid sequence of the immunogen is shown in SEQ ID NO.9. Then, the entire amino acid sequence was codon-optimized, and the company (Shanghai Sangon Biotech Co., Ltd.) synthesized the gene sequence expressing the entire immunogen. S1-3. The gene sequence expressing the immunogen was transferred into a eukaryotic expression vector using molecular biology methods, and then transfected into 293 T cells. After culturing for 72 h, the culture supernatant was collected and purified by nickel column chromatography. SDS-PAGE gel electrophoresis was used to analyze the expressed truncated desialylate glycoprotein receptor. The results are as follows: Figure 3 As shown, the prepared truncated desialyl glycoprotein receptor has a purity of over 90% and is mainly a monomeric protein. S2, Immunogen (truncated desialyl glycoprotein receptor) immunize mice; Balb / c mice (female, 8-10 weeks old, purchased from Guangdong Provincial Medical Laboratory Animal Center) were immunized with the immunogen (truncated desialyl glycoprotein receptor) prepared in the previous step. The specific immunization procedure was as follows: The mice were first immunized, first booster immunized, and second booster immunized at 0 days, 14 days, and 21 days, respectively. Blood was collected and serum was separated on day 24. The titer of specific antibodies in the serum was detected by ELISA. When the titer reached 1:100000 or higher, a third booster immunization was performed to obtain mice immunized with hepatitis B core antigen. For the first immunization, the immunogen was diluted to a concentration of 1 mg / mL using phosphate-buffered saline (PBS buffer, 0.01 M, pH 7.2) to obtain a diluted solution. This diluted solution was then mixed with Freund's complete adjuvant (F5881, Sigma-Aldrich) at a volume ratio of 1:1 to obtain the immunizing agent. Finally, the immunizing agent was injected subcutaneously at multiple sites into Balb / c mice at a dose of 200 μL / mouse. For the first and second booster immunizations, the adjuvant was changed to Freund's incomplete adjuvant (F5506, Sigma-Aldrich), with an immunizing dose of 100 μL / mouse, and the remaining steps were the same as the first immunization. For the third booster immunization, the subcutaneous injection was changed to tail vein injection, and either Freund's complete adjuvant or Freund's incomplete adjuvant was not added to the immunogen; the immunizing dose was 100 μL / mouse. S3, fusion of mouse spleen cells and myeloma cells; S3-1. After anesthetizing the immunized mice, remove the eyeballs and collect blood (the serum will be collected later as a positive control for subsequent screening, or for other in vitro and in vivo experiments); after euthanizing the immunized mice by dislocating their necks, immerse the mice in 75% (v / v) alcohol for 5 min for disinfection. S3-2. Under sterile conditions, dissect and remove the mouse spleen in a laminar flow hood and place it in a 70 μm filter on a 50 mL centrifuge tube. Gently grind the spleen with the club end of a 5 mL sterile syringe to break it down into single cells. Then, add 30 mL of serum-free DMEM (purchased from Gibco) to wash once, centrifuge at 300 g for 8 min, discard the supernatant, and finally add 20 mL of serum-free DMEM to resuspend the spleen cells and count them under a microscope to obtain a spleen cell suspension. Take NS-1 cells (mouse myeloma cells, purchased from ATCC) and place them in a 50 mL centrifuge tube. First, add 30 mL of serum-free DMEM to wash once, centrifuge at 300 g for 8 min, discard the supernatant, and then add 40 mL of serum-free DMEM to resuspend the NS-1 cells and count them under a microscope to obtain an NS-1 cell suspension. S3-3, First, add NS-1 cell suspension to the spleen cell suspension until the spleen cells of each mouse contain 25 × 10⁻⁶ cells. 6 Take 10 NS-1 cells, mix well, centrifuge at 300 g for 8 min, discard the supernatant to obtain mixed cells, then add 40 mL of serum-free DMEM to wash once, centrifuge at 300 g for 8 min, discard the supernatant to obtain washed mixed cells; S3-4. After tapping the bottom of the 50 mL centrifuge tube to disperse the washed mixed cells, add 1 mL of 50% (w / v, g / 100 mL) polyethylene glycol 3350 (P-3640, Sigma) aqueous solution (prepared and filtered for sterilization) dropwise over 1 min. Then, stir evenly with a 1 mL pipette for 1 min. Next, add 4 mL of serum-free DMEM dropwise over 4 min, followed by 10 mL of serum-free DMEM dropwise over 2 min. Finally, incubate at 37°C for 10 min. S3-5. First, add 30 mL of DMEM containing 10% (v / v) fetal bovine serum (purchased from Gibco) to a 50 mL centrifuge tube, mix well, centrifuge at 300 g for 8 min, discard the supernatant, and then add 20 mL of DMEM containing 20% ​​(v / v) fetal bovine serum to resuspend the mixed cells to obtain a mixed cell suspension. S3-6. First, take 10 mL of the mixed cell suspension and add it to two new 50 mL centrifuge tubes. Then, add 40 mL of DMEM containing 20% ​​(v / v) fetal bovine serum to each tube, mix well, and obtain the mixture. S3-7. Pour the mixture from two 50 mL centrifuge tubes into a sterile sample loading trough. Use a multi-channel pipette to add 100 μL / well to a 96-well plate for plating. Plating 5 96-well plates with 50 mL of cell suspension for a total of 10 96-well plates. S3-8. After the plate is laid, place it in an incubator at 37℃ and 5% (v / v) CO2 for 20 h. S3-9. Add 2X HAT medium (YZ-H0262-1VL, Solarbio) to a 96-well plate at a rate of 100 μL / well using a pipette, and return the plate to an incubator at 37°C and 5% (v / v) CO2 for further culture. During the culture process, observe the plate every other day and collect the cell culture supernatant from the wells where clones have grown. S4. ELISA screening for antibodies against desialyl glycoprotein receptor; S4-1. Dilute the commercially available desialylate glycoprotein receptor (purchased from Beijing Baipusai Biotechnology Co., Ltd.) with PBS to 1 μg / mL. In a 96-well ELISA plate, 100 μL / well is coated and incubated overnight at 4°C. S4-2. Collect 80 μL of cell culture supernatant from the wells where the clones have grown into a 96-well plate and mark the positions. S4-3. Add 50 μL of sample diluent to each well of the 96-well plate, and then add 50 μL of the cell culture supernatant to be tested. Attach the membrane to the 96-well plate and incubate at room temperature (25℃) for 30 min. S4-4 Wash 5 times with washing solution, 1 min each time. After shaking off the liquid in the wells, pat dry. Add 100 μL of horseradish peroxidase-labeled goat anti-mouse IgG (purchased from Solarbio, catalog number: SE131) to each well of the 96-well plate. S4-5, incubate at room temperature (25℃) for 30 min; wash 5 times with washing solution, shake off the liquid in the wells and pat dry, add 100 μL of TMB colorimetric solution (purchased from Aladdin, catalog number: T117926) to each well of the 96-well plate; develop color at room temperature in the dark for 15 min. S4-6. Add 50 μL of stop solution (2 M sulfuric acid) to each well of the 96-well plate, shake to mix, and use an enzyme-linked immunosorbent assay (ELISA) instrument within 10 min to detect the absorbance value at a wavelength of 450 nm (630 nm is used as the control wavelength). S4-7. Select cells from positive clone wells based on the OD values ​​of the standard wells and negative wells; S5, ELISA positive cloning well cell cloning; S5-1. Use a pipette to mix the cells in the positive clone wells and count them under a microscope to calculate the cell concentration. S5-2. Take cells from 100 positive clone wells and add them to 10 mL of 1X HT medium (YZ-H0262-1VL, Solarbio), mix well, and obtain a mixture. S5-3. Pour the mixture into a sterile tank and add it to a 96-well plate at a rate of 100 μL / well using a pipette. After plating, place the plate in an incubator at 37°C and 5% (v / v) CO2. During the incubation process, observe the cells every other day and change half of the medium. S5-4. When new cell clones appear in a 96-well plate, collect the cell culture supernatant from the wells where the clones have grown, and use ELISA to detect whether they bind to the desialyl glycoprotein receptor. S5-5. Select cells from the remaining positive clone wells (these cells are the selected hybridoma cells) and transfer them to 48-well plates. Then, culture them in a 1X HT incubator at 37°C and 5% (v / v) CO2. Subsequently, continue to expand the culture of the positive clone well cells to 24-well plates, 12-well plates, 6-well plates, T25 culture flasks, and T75 culture flasks. At the same time, cryopreserve the selected hybridoma cells for later use.

[0039] This step screened out two hybridoma cell lines containing monoclonal antibodies against desialyl glycoprotein receptor. The two hybridoma cell lines were named J31 and J35. The monoclonal antibodies against desialyl glycoprotein receptor produced by J31 and J35 were sent to Nanjing Zhongding Biotechnology for subtype identification and light chain identification. The identification results are shown in Table 1.

[0040] Table 1. Subtype identification results of monoclonal antibodies produced by J31 and J35 Figure 1 A schematic diagram of the structure of the desialyl glycoprotein receptor (ASGPR) binding to its glycosyl ligand N-acetylgalactosamine (GalNac) is shown.

[0041] Figure 2 The truncated amino acid sequence of the desialyl glycoprotein receptor, which serves as an immunogen, is shown.

[0042] Figure 3 The SDS-PAGE gel electrophoresis results of the truncated desialyl glycoprotein receptor expressed and purified in 293 T cells are shown, where R represents reduction electrophoresis, NR represents non-reduction electrophoresis, and M represents protein marker.

[0043] Preparation Example Large-scale preparation and purification of monoclonal antibodies against desialyl glycoprotein receptor.

[0044] (1) Collect the hybridoma cells obtained from the expanded culture into a 50 mL centrifuge tube and count them under a microscope. After counting, centrifuge at 800 g for 10 min and discard the supernatant. (2) Add PBS buffer to a 50 mL centrifuge tube to resuspend the cells to 4 × 10⁻⁶. 6 Cells / mL were used to obtain a cell resuspension; (3) The cell resuspension was injected intraperitoneally into Balb / c mice (pre-treated with 0.5 mL Freund's incomplete adjuvant) at a dose of 500 μL / mouse. (4) Twelve days after injection, aseptically collect the peritoneal fluid of the mice into a 15 mL centrifuge tube and centrifuge at 4°C and 500 g for 10 min. (5) Collect the ascites supernatant into a 1.5 mL EP tube, 1 mL / tube, write a label and store at -20℃ to obtain the ascites specimen; (6) Preparation of acetate buffer: Mix 59 mL of solution A and 41 mL of solution B, and adjust the pH to 4.80 with hydrochloric acid (solution A: 0.06 M sodium acetate aqueous solution; solution B: 0.06 M glacial acetic acid aqueous solution). (7) Take out the ascites sample 30 minutes in advance and warm it to room temperature (25℃), then centrifuge it at 4℃ and 12000 rpm for 10 minutes and take the supernatant; (8) Mix the supernatant and acetate buffer at a volume ratio of supernatant:acetate buffer = 1:2, and add octanoic acid dropwise under stirring at room temperature (25℃) until each mL of supernatant contains 33 μL of octanoic acid to obtain the mixture; (9) Let the mixture stand at 4℃ for more than 2 hours to allow it to settle completely, then centrifuge at 4℃ and 12000 rpm for 30 minutes and take the supernatant. (10) Under ice bath conditions, add ammonium sulfate to the supernatant over 30 min until each mL of supernatant contains 0.277 g of ammonium sulfate to achieve 45% saturation and obtain a saturated solution; (11) Let the saturated solution stand at 4℃ for more than 2 hours to allow it to fully precipitate, then centrifuge at 4℃ and 12000 rpm for 30 minutes and collect the protein precipitate; (12) Dissolve the protein precipitate in 1.5 mL of PBS buffer to obtain a monoclonal antibody lysis solution; (13) Take 2 μL of monoclonal antibody lysate, use Nanodrop 2000 A280 to detect the protein concentration, label the protein concentration, and freeze at -80℃; (14) SDS-PAGE detection of the purity of the purified antibody: β-mercaptoethanol was added to one part of the sample to open the disulfide bond of the antibody and a reduction electrophoresis was performed; another part was not added to β-mercaptoethanol and the intact antibody was detected and a non-reduction electrophoresis was performed.

[0045] The protein concentration of the monoclonal antibody lysate obtained from culturing J31 in this step was 4.22 mg / mL, and the protein concentration of the monoclonal antibody lysate obtained from culturing J35 was 3.65 mg / mL.

[0046] Figure 4 The results of SDS-PAGE gel electrophoresis of purified antibody J31 against desialyl glycoprotein receptor are shown.

[0047] like Figure 4 SDS-PAGE electrophoresis results showed that the purified antibody purity was >90%. The first column, R, represents the reduction conditions: β-mercaptoethanol was added to the loading buffer; the second column, NR, represents the non-reduction electrophoresis: β-mercaptoethanol was not added to the loading buffer; and the third column, M, represents the protein marker.

[0048] Figure 5 The results of SDS-PAGE gel electrophoresis of purified antibody J35 against desialyl glycoprotein receptor are shown.

[0049] like Figure 5 SDS-PAGE electrophoresis results showed that the purified antibody purity was >90%. The first column, R, represents the reduction conditions: β-mercaptoethanol was added to the loading buffer; the second column, NR, represents the non-reduction electrophoresis: β-mercaptoethanol was not added to the loading buffer; and the third column, M, represents the protein marker.

[0050] Test Example 1 Affinity assay for antibody-antigen binding to desialyl glycoprotein receptor.

[0051] Test method: (1) Coating antigen: The expressed antigen was diluted with PBS to a concentration of 5 μg / mL and added to a 96-well ELISA plate, 100 μL / well, and incubated overnight at 4°C; in addition, 5 μg / mL of BSA was coated as a control. (2) Discard the antigen, add 200 μL of blocking solution containing 5 wt% skim milk powder to each well, and block at 37°C for 2 h; (3) Discard the blocking solution and wash three times with PBST washing solution containing 0.2% (v / v) Tween 20, 1 min each time; (4) Add the serially diluted anti-desialylglycoprotein receptor antibodies J31 and J35 prepared and purified in the example, and react at 37°C for 1 h; (5) Discard the primary antibody and wash 5 times with PBST containing 0.2% (v / v) Tween 20, 1 min each time; (6) Add HRP-labeled goat anti-mouse IgG (purchased from Sizhengbai), and react at 37℃ for 30 min; (7) Discard the secondary antibody and wash 5 times with PBST washing buffer containing 0.2% (v / v) Tween 20, 1 min each time; (8) Add TMB colorimetric solution, 100 μL / well, and develop color for 10 min in the dark; (9) Measure OD450 nm.

[0052] Figure 6 The OD450 nm curves of antibody J31 against the antigen at different concentrations are shown. The results show that the affinity of antibody J31 against the antigen is 0.425 nM.

[0053] Figure 7 The OD450 nm curves of antibody J35 against the desialization glycoprotein receptor at different concentrations are shown. The results show that the affinity of antibody J35 against the antigen is 0.4025 nM.

[0054] Test Example 2 Antibody against desialyl glycoprotein receptor binds to HepG2.2.15 cells.

[0055] Test method: (1) HepG2.2.15 cells were seeded into 24-well cell culture plates at a density of 1×10⁻⁶ cells / well. 5 Cells / well were cultured at 37°C in a 5% (v / v) CO2 incubator to allow the cells to adhere and grow. (2) After culturing for 48 h, remove the cultured cell plate and gently rinse it 3 times with PBS for 1 min each time to remove culture medium residue; (3) Antibody incubation: Dilute antibodies J31 and J35 against desialyl glycoprotein receptor to 1 μg / mL with complete culture medium; 400 μL per well, incubate at 4℃ for 30 min; the control group is given only complete culture medium without antibodies; (4) Discard the primary antibody and gently rinse 3 times with PBS for 1 min each time to remove the primary antibody residue; (5) Add PE anti-mouse IgG (purchased from BioLegend, catalog number: 405307), 400 μL per well, and incubate at 4℃ for 30 min; (6) Discard the secondary antibody and gently rinse 3 times with PBS for 1 min each time to remove the secondary antibody residue; (7) Add 500 μL of PBS containing 1 wt% BSA and observe on the instrument.

[0056] Figure 8The image shows an immunofluorescence assay of the binding of antibody J31 against desialization glycoprotein receptor to HepG2.2.15 cells. The results indicate that antibody J31 against desialization glycoprotein receptor can specifically bind to HepG2.2.15 cells.

[0057] Figure 9 The image shows an immunofluorescence assay of the binding of antibody J35 against desialization glycoprotein receptor to HepG2.2.15 cells. The results indicate that antibody J35 against desialization glycoprotein receptor can specifically bind to HepG2.2.15 cells.

[0058] Test Example 3 Effects of anti-desialidylglycoprotein receptor antibody on HBsAg expression in HepG2.2.15 cells.

[0059] Test method: (1) HepG2.2.15 cells (purchased from ATCC) were divided into wells at a density of 5 × 10⁶ cells per well. 4 Cells were seeded into 96-well plates containing 200 μL of DMEM medium with 10% (v / v) fetal bovine serum per well and incubated at 37°C in a 5% (v / v) CO2 incubator for 16 h to allow the cells to adhere and grow. (2) After culturing for 16 h, the culture medium was replaced with fresh medium. The monoclonal antibodies J31 and J35 prepared in the example were added to 96-well plates to a protein concentration of 25 μg / mL, 50 μg / mL and 100 μg / mL in each well, respectively. The plates were then incubated at 37 °C and 5% (v / v) CO2 for 3 days. The irrelevant antibody FMC63 (targeting human CD19) was used as a control. No antibody was added as a blank control. (3) After culturing for 3 days, the cell culture supernatant in each well was collected and the HBsAg level was detected using a quantitative assay kit for hepatitis B surface antigen (purchased from Beijing Wantai). The results are shown in Table 2.

[0060] The results in Table 2 show that the monoclonal antibodies produced by J31 and J35 have a significant inhibitory effect on HBsAg in HepG2.2.15 cells. The inhibitory effect on HBsAg secretion by cells is more obvious with the increase of antibody concentration. Among them, J31 has the most obvious effect at 100 μg / mL (P<0.001).

[0061] (4) Antibody cell lines J31 and J35 against desialyl glycoprotein receptor were sent to Nanjing Zhongding Company for gene sequencing. The sequencing results are shown in Tables 3 and 4.

[0062] Table 2. Effects of J31 and J35 on HBsAg expression levels in HepG2.2.15 cells. Table 3. Amino acid sequence listing of the heavy chain variable region (VH) and light chain variable region (VL) of the monoclonal antibody produced by J31. Table 4. Amino acid sequence listing of the heavy chain variable region (VH) and light chain variable region (VL) of the monoclonal antibody produced by J35. It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.

[0063] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. An antibody against the desialyl glycoprotein receptor, characterized in that, The antibody against the desialyl glycoprotein receptor includes a heavy chain variable region and a light chain variable region; The heavy chain variable region includes heavy chain variable regions HCDR1-HCDR3; The light chain variable region includes light chain variable regions LCDR1-LCDR3; The heavy chain variable region HCDR1 is selected from amino acid sequence 1 or amino acid sequence 9; The heavy chain variable region HCDR2 is selected from amino acid sequence 2 or amino acid sequence 10; The heavy chain variable region HCDR3 is selected from amino acid sequence 3 or amino acid sequence 11; The light chain variable region LCDR1 is selected from amino acid sequence 4 or amino acid sequence 12; The light chain variable region LCDR2 is selected from amino acid sequence 5 or amino acid sequence 13; The light chain variable region LCDR3 is selected from amino acid sequence 6 or amino acid sequence 14; The amino acid sequences 1-6 and 9-14 are shown as SEQ ID NO.1-SEQ ID NO.6 and SEQ ID NO.9-SEQ ID NO.14, respectively.

2. The antibody against desialyl glycoprotein receptor according to claim 1, characterized in that, The antibody against the desialyl glycoprotein receptor includes monoclonal antibodies and antibody fragments; The antibody fragments include Fab, single-chain antibodies, and single-domain antibodies.

3. The antibody against desialyl glycoprotein receptor according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.7 or SEQ ID NO.15; The amino acid sequence of the variable region of the light chain is shown in SEQ ID NO.8 or SEQ ID NO.

16.

4. A method for preparing the antibody against desialyl glycoprotein receptor according to any one of claims 1-3, characterized in that, Includes the following steps: S1. Construction, expression and purification of immunogen (truncated desialyl glycoprotein receptor): Through bioinformatics, the three-dimensional structure of the desialyl glycoprotein receptor binding its ligand was analyzed to find the key amino acid sequence for the desialyl glycoprotein receptor to function. The key amino acid sequence for the function of desialyl glycoprotein receptor was removed to construct a truncated desialyl glycoprotein receptor as an immunogen; S2. Use the truncated desialyl glycoprotein receptor as an immunogen to immunize Balb / c mice; S3, fusion of mouse spleen cells and myeloma cells; S4. ELISA screening for antibodies against desialyl glycoprotein receptor; S5 and ELISA-positive cloning well cells were cloned to obtain antibodies against desialyl glycoprotein receptors.

5. A recombinant protein, characterized in that, The amino acid sequence of the recombinant protein comprises the amino acid sequence of the antibody against desialyl glycoprotein receptor as described in any one of claims 1-3.

6. A gene characterized by, The gene encodes an antibody or recombinant protein against the desialyl glycoprotein receptor as described in any one of claims 1-3 or 5.

7. A recombinant plasmid, characterized in that, The recombinant plasmid carries the gene described in claim 6.

8. A drug, characterized in that, The drug contains an antibody, recombinant protein, gene, or recombinant plasmid against the desialyl glycoprotein receptor as described in any one of claims 1-3 or 5-7.

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