Monoclonal antibody and single-chain antibody of HBV preS1, HBV specific CAR-T cell constructed by monoclonal antibody and single-chain antibody and application of HBV specific CAR-T cell

Abstract

The invention discloses a monoclonal antibody and a single-chain antibody of HBV preS1, an HBV specific CAR-T cell constructed by the monoclonal antibody and the single-chain antibody and application of the HBV specific CAR-T cell. The monoclonal antibodies 3A1, 6B12 and 8G6 of the HBV preS1 can be combined with HBV expression hepatocytes. A light chain variable region and a heavy chain variable region of a monoclonal antibody are connected through a GS linker to obtain a single-chain antibody, a chimeric antigen receptor (CAR) is constructed, and the chimeric antigen receptor (CAR) is transfected to a T cell through lentivirus, so that the preS1 specific CAR-T cell is constructed. In-vitro experiments prove that the successfully constructed CAR-T cells can be successfully activated by liver cells expressing HBV and have a killing effect on the liver cells expressing HBV. The constructed CAR-T cell taking the preS1 as the target provides a new possibility for constructing a therapeutic CAR-T cell with stronger lethality and activity, and has important significance for exploring targeted immunoregulation antiviral therapy.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine. Specifically, the present invention relates to the use of preS1 antibodies for inhibiting hepatitis B virus and the CAR-T cells constructed therefrom in the treatment of HBV infection. Background Art

[0002] Approximately 292 million people worldwide are infected with hepatitis B virus (HBV), and 25% of these patients will progress to cirrhosis or even liver cancer. [1-4] Antiviral treatment regimens based on interferon and nucleoside analogs can effectively inhibit viral replication but cannot eradicate the virus. [5] The covalently closed circular DNA (cccDNA) of HBV persists in the infected cell nucleus as a transcription template and reinitiates HBV replication when antiviral treatment is stopped, which is an important source of HBV infection recurrence and disease progression. [6] Therefore, it is particularly important to control HBV activity, clear HBV infection, and explore new treatment regimens to improve the prognosis of HBV-infected patients.

[0003] Studies have confirmed that long-term chronic HBV infection is associated with a lack of mature memory T cells. A strong immune response occurs during acute hepatitis B, and CD8 + T cells are the key to clearing the virus. In contrast, CHB patients have a low T cell response to the virus, and this long-term chronic infection can lead to the exhaustion of HBV-specific T cells in the body. The reason is thought to be that HBV infection causes T cell function inhibition or a lack of T cell numbers, resulting in an ineffective immune response. [7] Therefore, restoring the function of HBV-specific T cells and enhancing the host immune response can be an important goal for HBV treatment. This concept of adoptive adaptive immunity to control HBV dates back to the 1990s, and it was found that chronic hepatitis B (CHB) patients who received stem cell transplantation from HBV-immune donors achieved clearance of hepatitis B virus surface antigen (HBsAg) after transplantation. [8] However, due to the serious side effects of stem cell transplantation (such as graft-versus-host disease and high mortality), it cannot be widely carried out clinically. Therefore, reliable alternative therapies need to be found.

[0004] T cells are genetically engineered to express HBV-specific chimeric antigen receptor (S-CAR) or natural T-cell receptor (TCR). S-CAR contains an antibody fragment that, upon binding to HBsAg on the cell surface, activates S-CAR-T through its CD28 and CD3 signaling domains. Natural TCR can recognize HBV core protein or S protein presented on HLA-A2, and their binding leads to the activation of T-cell function. T-cell-based anti-HBV therapies mainly include HBsAg-CAR-T and HBV-specific TCR-T [9] . Since all T cells are derived from autologous cells and there is no immune rejection reaction, adoptive T-cell therapy has promoted the progress of immunotherapy for viral hepatitis to a certain extent and brought hope for the cure of CHB [10,11] . It has been confirmed in animal models that engineered TCR-T or CAR-T cells can localize to the liver, rapidly control HBV replication and reduce liver injury, and both have good anti-HBV activity [12,13] . The clinical study of HBV-TCR-T has demonstrated the ability of TCR-T cells to recognize liver tumor cells expressing HBV-related antigens in patients with HBsAg + HCC recurrence

[14] . The chimeric receptor enables primary human T cells to recognize HBsAg-positive hepatocytes, release interferon-γ and interleukin-2, and lyse HBV-replicating cells. Co-culturing HBsAg-CAR-T with primary human hepatocytes infected with HBV can selectively eliminate HBV-infected cells, thereby eliminating cccDNA (covalently closed circular DNA)-positive target cells

[15] . Therefore, adoptive T-cell therapy using HBV-specific T cells is a potential treatment strategy to eliminate HBV and induce functional cure.

[0005] Currently, there are still certain problems in applying CAR-T to the clinical treatment of HBV infection, such as difficulty in regulating T cells and the inability of immune cells to stably recognize infected cells. In vitro and animal experiments, CAR-T has shown the effect of killing HBV-infected hepatocytes, but the effect cannot be sustained, and HBV can still be detected in hepatocytes and tissues, indicating that the existing HBsAg-CAR-T has limited proliferative and killing activities in the human body [13,14,16,17] . We plan to develop a new therapeutic strategy to construct therapeutic CAR-T cells with stronger killing power and activity, specifically eliminate HBV-infected hepatocytes, thereby eliminating cccDNA-positive target cells, and thus clear the established chronic infection.

[0006] The HBV envelope contains three kinds of membrane glycoproteins: large, middle, and small. The small membrane glycoprotein, also known as HBsAg, contains an S domain. The middle membrane glycoprotein has an additional preS2 domain at the N-terminus. The large membrane glycoprotein has a preS1 region in addition to the S and preS2 domains. The large membrane glycoprotein plays an important role in the viral life cycle: it binds to the viral receptor outside and to the viral nucleocapsid inside. HBV enters cells by attaching to the cell membrane through the interaction between viral envelope proteins and heparan sulfate proteoglycans, and then binds to a specific receptor with high affinity, namely the bile acid export pump sodium-taurocholate cotransporting polypeptide (NTCP). Therefore, preS1 is crucial for the binding of viral particles to NTCP and determines the viral invasion ability.

[0007] Therefore, we believe that targeting preS1 may be more likely to break immune tolerance and kill HBV-infected hepatocytes. We established a rabbit-derived antibody library against preS1 by immunizing rabbits with preS1, screened out the antibody with the strongest interaction with preS1 protein through ELISA and flow cytometry, recombinantly constructed the antibody IgG of the antibody with the highest binding affinity through gene recombination, synthesized single-chain antibodies, and successfully constructed preS1-specific CAR-T cells, and verified the activation of CAR-T cells and their killing effect on HBV-infected hepatocytes in vitro. Summary of the Invention

[0008] The object of the present invention is to provide a monoclonal antibody specifically binding to hepatitis B virus preS1 and its encoding nucleic acid molecule in view of the above deficiencies of the prior art.

[0009] Another object of the present invention is to provide a single-chain antibody specifically binding to hepatitis B virus preS1.

[0010] Yet another object of the present invention is to provide an HBV-specific CAR-T cell.

[0011] The fourth object of the present invention is to provide the applications of the single-chain antibody and the HBV-specific CAR-T cell.

[0012] The object of the present invention can be achieved by the following technical solutions:

[0013] A monoclonal antibody that specifically binds to hepatitis B virus preS1, and the heavy and light chain sequences are selected from any one of 3A1, 6B12, and 8G6. The amino acid sequence of the heavy chain variable region of monoclonal antibody 3A1 is as shown in SEQ ID NO.1, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO.2; the amino acid sequence of the heavy chain variable region of monoclonal antibody 6B12 is as shown in SEQ ID NO.3, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO.4; the amino acid sequence of the heavy chain variable region of monoclonal antibody 8G6 is as shown in SEQ ID NO.5, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO.6.

[0014] A nucleic acid molecule encoding the monoclonal antibody that specifically binds to hepatitis B virus preS1. The nucleotide sequence of the heavy chain variable region of monoclonal antibody 3A1 is as shown in SEQ ID NO.7, and the nucleotide sequence of the light chain variable region is as shown in SEQ ID NO.8; the nucleotide sequence of the heavy chain variable region of monoclonal antibody 6B12 is as shown in SEQ ID NO.9, and the nucleotide sequence of the light chain variable region is as shown in SEQ ID NO.10; the nucleotide sequence of the heavy chain variable region of monoclonal antibody 8G6 is as shown in SEQ ID NO.11, and the nucleotide sequence of the light chain variable region is as shown in SEQ ID NO.12.

[0015] A single-chain antibody scFv that specifically binds to hepatitis B virus preS1, wherein the heavy chain variable region and the light chain variable region of the single-chain antibody are connected by a GS linker, and the heavy chain variable region and the light chain variable region are respectively from any one of the following:

[0016] (1) The heavy chain variable region and the light chain variable region of the monoclonal antibody 3A1;

[0017] (2) The heavy chain variable region and the light chain variable region of the monoclonal antibody 6B12;

[0018] (3) The heavy chain variable region and the light chain variable region of the monoclonal antibody 8G6.

[0019] As a further preference of the present invention, the sequence of the GS linker is (G4S)3.

[0020] In a specific embodiment, the single-chain antibody of the present invention is fusion-expressed with human IgG1Fc.

[0021] A nucleic acid molecule encoding the single-chain antibody of the present invention.

[0022] As a preference of the present invention, the nucleotide sequence of the heavy chain variable region of monoclonal antibody 3A1 is as shown in SEQ ID NO.7, and the nucleotide sequence of the light chain variable region is as shown in SEQ ID NO.8; the nucleotide sequence of the heavy chain variable region of monoclonal antibody 6B12 is as shown in SEQ ID NO.9, and the nucleotide sequence of the light chain variable region is as shown in SEQ ID NO.10; the nucleotide sequence of the heavy chain variable region of monoclonal antibody 8G6 is as shown in SEQ ID NO.11, and the nucleotide sequence of the light chain variable region is as shown in SEQ ID NO.12.

[0023] As a preference of the present invention, the nucleotide sequence encoding the linker (G4S)3 is as shown in SEQ ID NO.14.

[0024] Use of the nucleic acid molecule of the present invention in the preparation of HBV-specific CAR-T cells.

[0025] An HBV-specific CAR-T cell, comprising a CAR-T cell of the single-chain antibody nucleotide of the present invention.

[0026] As a preference of the present invention, the CAR structure of the HBV-specific CAR-T cell is CD8α signal peptide - scFv - CD8α hinge - CD8α transmembrane domain - 41BB intracellular domain - CD3ζ, and the third-generation pCDH lentiviral vector is used.

[0027] Use of the single-chain antibody or the HBV-specific CAR-T cell of the present invention in the preparation of a medicament for treating hepatitis B.

[0028] Use of the single-chain antibody or the HBV-specific CAR-T cell of the present invention in the preparation of a medicament for inhibiting the proliferation of hepatitis B virus.

[0029] Use of the single-chain antibody of the present invention in the preparation of a reagent for detecting HBV infection.

[0030] Beneficial effects:

[0031] The present invention provides rabbit monoclonal antibodies 3A1, 6B12, and 8G6 against HBV preS1, which can bind to HBV-expressing hepatocytes. The light and heavy chain sequences can be synthesized into a single-chain antibody through a GS linker, and a chimeric antigen receptor (CAR) can be constructed. The CAR is transfected into T cells through lentivirus to construct preS1-specific CAR-T cells. In vitro experiments confirm that the successfully constructed CAR-T cells can be successfully activated by HBV-expressing hepatocytes and have a killing effect on HBV-expressing hepatocytes. The CAR-T cells targeting preS1 constructed by us provide new possibilities for constructing more lethal and active therapeutic CAR-T cells, and are of great significance for exploring targeted immunomodulatory antiviral therapy. Brief Description of the Drawings

[0032] Figure 1 . ELISA detection of antibodies in the culture supernatant of rabbit monoclonal B cells

[0033] Figure 2 . Flow cytometry detection of positive clones in the culture supernatant of rabbit monoclonal B cells

[0034] Figure 3 . Flow cytometry detection of positive clones in the culture supernatant of rabbit monoclonal B cells (second-round screening)

[0035] Figure 4 . Electrophoretogram of the target band

[0036] Figure 5 . Detection of CAR expression efficiency

[0037] Figure 6 . Detection of the killing ability of CAR-T cells

[0038] Figure 7 . Detection of IFN-γ released by CAR-T cells Detailed Embodiments

[0039] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0040] Example 1: Screening of complete rabbit anti-preS1 antibodies

[0041] (I) Animal immunization

[0042] The rHBsAg preS1 protein (PrimeGene, 672-01) was used as an immunogen to immunize A / B rabbits. For the first immunization, Freund's complete adjuvant (Sigma, F5881-10ML) was emulsified with the antigen in equal volume, and the dose was 300 μg / rabbit. For the booster immunization, Freund's incomplete adjuvant (Sigma, F5506-10ML) was emulsified with the antigen in equal volume, and the dose was 150 μg / rabbit. After successful emulsification, New Zealand white rabbits were injected subcutaneously at multiple points on the back for 7 times.

[0043] (2) Serum titer detection

[0044] (1) Coating: rHBsAg preS1 is coated on the well plate at a concentration of 1 μg / mL, covered with plastic wrap, and incubated overnight at 4°C.

[0045] (2) Blocking: Drain the coated plate and pat it dry on the absorbent paper. Add 150 μl / well of blocking solution, and incubate the enzyme-linked immunosorbent assay (ELISA) plate at 37°C for 1 h, then wash the plate.

[0046] (3) Adding the primary antibody: Dilute the serum in the first well to 1:4000, and perform 2-fold serial dilutions for the subsequent wells. Load 100 μl / well. Set the negative control as 1% BSA dilution, incubate at 37°C for 1 h, and then wash the plate.

[0047] (4) Adding the secondary antibody: After washing the plate, add horseradish peroxidase-labeled goat anti-rabbit IgG, catalog number: 111-035-144 (Jakson), diluted 1:10000, 100 μl / well, and incubate at 37°C for 1 h.

[0048] (5) Washing: Wash the ELISA plate with PBS solution 4 times, soak for 30 s each time.

[0049] (6) Color development and termination: Add 100 μl / well of substrate solution TMB (Sigma 860336-5G), react for 5 min, and then add 100 μl of 1 mol / L sulfuric acid to terminate the reaction.

[0050] (7) Reading: Measure the OD value at a wavelength of 450 nm using an enzyme-linked immunosorbent assay reader (Tecan-infinite F50).

[0051] (8) Titer results: Detect the titer of rabbit serum by indirect ELISA method, and the results are shown in Table 1. The serum titers of both Rabbit A and Rabbit B are greater than 1:256k, meeting the experimental requirements.

[0052] Table 1. Monitoring of the immune titer of rabbit serum

[0053]

[0054] (3) Rabbit B cell culture and detection

[0055] Culture the B cells of the rabbit with successful immunization. The specific operations are as follows:

[0056] (1) Sacrifice the rabbit, take its spleen to isolate PBMC, and store it at -80°C for later use.

[0057] (2) Prepare B cells. Resuscitate one tube of B cells in liquid nitrogen and transfer them into a blank RPMI-1640 medium. Centrifuge to remove the cryopreservation solution and resuspend with 2 ml of medium.

[0058] (3)Specifically screen target B cells with immunomagnetic beads, add them to the complete medium, gently shake to mix well. After cell counting, seed the cells into a 96-well cell culture plate at 200 μL / well. Incubate in a 37 °C incubator for 7 - 10 days and then perform ELISA detection.

[0059] (4)Detect the culture supernatant by ELISA. The ELISA detection method is the same as before. The detection results of the single B cell culture supernatant are shown in Figure 1 . Select a total of 150 clones with an OD value > 0.3.

[0060] (IV) Flow cytometry verification of positive supernatant

[0061] Select 150 clones that are positive in the ELISA detection and detect their binding ability to HepAD38 cells.

[0062] (1)Collect and wash HepAD38 cells at 1000 rpm for 5 minutes.

[0063] (2)Resuspend the cells in PBS (containing 10% FCS) to 2 - 4 * 10^6 / ml.

[0064] (3)Take n + 1 flow cytometry tubes (1 tube for control + n tubes for samples to be tested), label them, add 100 μl of cell suspension to each flow cytometry tube, centrifuge at 400 g for 5 min, and discard the supernatant.

[0065] (4)Vortex and add 100 μl of the primary antibody (i.e., the positive rabbit monoclonal B cell culture supernatant obtained above), and incubate for 30 minutes at 4 °C in the dark.

[0066] (5)Add 1 ml of PBS, centrifuge and wash the cells at 400 g for 5 min, and repeat 3 times.

[0067] (6)Dilute the fluorescently labeled secondary antibody (goat anti-rabbit IgG (H + L) cross-adsorbed secondary antibody, PE, invitrogen) with PBS at a ratio of 1:300, resuspend the cells in 100 μl per tube, and incubate for 30 minutes at 4 °C in the dark.

[0068] (7)Centrifuge and wash the cells at 400 g for 5 min, and repeat 3 times.

[0069] (8)Resuspend the cells in 300 μl of ice-cold PBS.

[0070] (9)Filter the cells through a 70-μm filter, and store the cell suspension in the dark at 4 °C.

[0071] (10)Perform flow cytometry. The results are as shown in Figure 2 .

[0072] Select the top 12 cell culture supernatants with the highest binding ability and perform flow cytometry again. The method is the same as before. The results are as shown inFigure 3 As shown. Finally, the top 10 clones with the highest binding affinity to HepAD38 were selected for gene cloning.

[0073] (V) Gene cloning and small-scale expression of recombinant monoclonal antibodies

[0074] Using gene recombination technology, antibody IgG gene recombination was constructed for the 10 selected positive clones to obtain the human-rabbit chimeric antibody sequence. Finally, 3 positive clones (3A1 / 6B12 / 8G6) were obtained, and no positive clones were selected after multiple ELISA detections of the remaining clones.

[0075] (1) Construction of heavy and light chain vectors.

[0076] Amplification of heavy and light chain target bands (the amplification template and primers were provided by Decode Biotechnology (Wuhan) Co., Ltd.), and the heavy and light chain target bands are as Figure 4 .

[0077] (2) Ligation

[0078] The vector was digested with NEB restriction endonuclease (provided by Decode Biotechnology (Wuhan) Co., Ltd.), the product was recovered using a gel extraction kit, and the digested vector was ligated with the target fragment.

[0079] (3) Transformation

[0080] Take 60 μl of competent cells (TOP10) and place them in an ice bath. After the competent cells melt on ice, add the ligation product of the target fragment and the vector to the competent cell suspension, gently pipette and mix well, and incubate on ice for 30 minutes; heat shock at 42 °C for 90 seconds, quickly transfer the centrifuge tube to an ice bath, and let it stand on ice for 2 - 3 minutes;

[0081] Add 900 μl of sterile SOC (without antibiotics) to the centrifuge tube, mix well and place it on a shaker at 37 °C, shake at 150 rpm for 45 minutes; take 100 μl of the transformed competent cells and add them to the LB solid agar medium containing the corresponding antibiotics, spread the cells evenly with a sterile spreader until dry, invert the plate, and incubate overnight at 37 °C.

[0082] (4) Bacterial detection

[0083] Select colonies and detect them by PCR using the primers for amplifying the target fragment, and select the PCR-positive colonies.

[0084] (5) Small-scale production and detection of recombinant monoclonal antibodies

[0085] Preparation of supernatant from small-scale production of recombinant monoclonal antibody: Extract the plasmid of recombinant antibody using the kit; Plate HEK293 cells in the logarithmic growth phase with a high viability one day before transfection; Transfect the plasmid into the cells the next day; Perform ELISA detection 2-3 days after transfection. The ELISA detection results are shown in Table 2, confirming that the supernatants of all 3 clones can bind to the preS1 antigen.

[0086] Table 2. ELISA detection results of small-scale transfection supernatants

[0087]

[0088] (VI) Sequencing of monoclonal antibody

[0089] Sequencing was performed on the heavy and light chains of the positive clones. The specific sequences are shown in Tables 3-4.

[0090] Table 3. Amino acid sequences of variable regions of rabbit monoclonal antibodies against HBsAg pre S1

[0091]

[0092] Table 4. Base sequences of variable regions of rabbit monoclonal antibodies against HBsAg pre S1

[0093]

[0094]

[0095] Example 2: Construction of Anti-HBsAg preS1 CAR-T cells (I) Construction of CAR lentiviral vector (3A1 / 6B12 / 8G6)

[0096] (1) Synthesize the single-chain antibody gene according to the heavy and light chain sequences of the monoclonal antibody in Table 4;

[0097] (2) The structure is CD8a signal peptide - scFv light chain - linker - scFv heavy chain - CD8a hinge. The specific sequences of the CD8a signal peptide and the linker CD8a hinge are as follows (Tables 5-6);

[0098] Table 5. Nucleic acid sequences of CAR structure

[0099]

[0100] Table 6. Protein sequences of CAR structure

[0101]

[0102] (3) Insert it into the third-generation lentiviral vector pCDH-EF1α-MCS (purchased from Dima Biotech Co., Ltd., Wuhan).

[0103] (4) Sequencing.

[0104] (II) Preparation of CAR Lentivirus

[0105] The third-generation lentiviral vector system is used, which consists of four plasmids, namely the packaging plasmid pMDLg pRRE encoding Gag-Pol protein, the packaging plasmid pRSV-Rev encoding Rev protein, the pMD2.G plasmid encoding envelope protein VSV-G, and the core plasmid pCDH-EF1α-MCS (hereinafter referred to as PCDH expression plasmid). The expression of the CAR gene in the lentiviral plasmid is regulated by the elongation factor-1α (EF-1α) promoter.

[0106] Packaging of Lentivirus:

[0107] (1) The constructed lentiviral vector and three helper plasmids need to be extracted endotoxin-free and the concentration is detected using a spectrophotometer; (2) Cell passage: One day before transfection, trypsinize the 293T cells in the logarithmic growth phase, adjust the density and re-inoculate them into a 10 cm cell culture dish, and culture them in an incubator at 37 °C and 5% CO2;

[0108] (3) Plasmid dilution: Add opti-MEM medium to a centrifuge tube, and then sequentially add the PCDH expression plasmid, pMDLg / pRRE plasmid, pRSV-Rev plasmid, and pMD2.G plasmid to the tube and mix well;

[0109] (4) PEI dilution: Add opti-MEM medium to another centrifuge tube, and then add the PEI solution and mix well;

[0110] (5) Mix the two tubes containing the plasmid and PEI solutions, and drop the mixed solution into the cell culture dish, and culture it in an incubator at 37 °C and 5% CO2;

[0111] (6) After 5 hours, discard the original medium, add 10 mL of fresh medium to each dish of cells, and culture them in an incubator at 37 °C and 5% CO2;

[0112] (7) After 72 hours, collect the supernatant virus.

[0113] Virus collection and concentration:

[0114] (1) Collect the virus supernatant;

[0115] (2) Filter the virus supernatant with a filter;

[0116] (3) Concentrate the filtered virus supernatant with an ultrafiltration tube;

[0117] (4) Transfer the concentrated virus into a 1.5 mL EP tube and store it in a -80 °C refrigerator;

[0118] Determination of the titer of CAR virus:

[0119] (1) Seeding plates: On Day 1, wash and dilute Jurkat cells and seed them into 96-well plates;

[0120] (2) Virus infection: Add a certain volume of virus solution to Jurkat cells and infect overnight;

[0121] (3) After 48 hours, detect the positive rate of Jurkat cells by flow cytometry.

[0122] (III) Preparation of Anti HBsAg preS1 CAR-T cells

[0123] (1) Draw 30 ml of human peripheral blood using a heparin sodium anticoagulant blood collection tube. Isolate PBMC using lymphocyte separation medium, and use anti-human CD3 nanomagnetic beads and Miltenyi magnetic bead sorting system to sort out the CD3-positive cell population. Activate and culture the obtained CD3-positive T cells using CD3 / CD28 magnetic beads for 24 hours and then infect them with CAR lentivirus respectively.

[0124] (2) After 48 hours, detect the expression efficiency of CAR by flow cytometry (primary antibody: anti-(G4s)n(B02H1)mAb, GS-ARAP25, Hycells; secondary antibody: Goat anti Rabbit lgG(H+L)AF647, invitrogen). The results are shown in Figure 5 , confirming that the positive rates of CAR are 3A141.7%, 6B1246.9%, and 8G653.6%.

[0125] (3) Continue to expand the culture and collect on the 9th day of culture.

[0126] (IV) Detection of the killing ability of CAR-T cells

[0127] (1) Seed HepAD38 into 96-well plates at 20,000 cells / 100 μl / well.

[0128] (2) Adjust the effector-to-target ratio to 1:3, 1:1, and 3:1 according to the positive rate of CAR-T expression, and add 100 μl of CAR-T cells.

[0129] (3) Incubate in an incubator for 24 hours, discard the supernatant, add 100 μl of the mixed solution (10 μl of CCK8 + 90 μl of blank culture) to each well, continue to incubate at 37 °C for 2 hours, and detect with a microplate reader at 450 / 630 nm.

[0130] (4) The detection results are as shown inFigure 6 as shown

[0131] The results confirmed that the CAR-T cells constructed with single-chain antibodies 3A1 / 6B12 / 8G6 all had obvious killing effects on HepAD38. (5) Detection of IFN-γ released by CAR-T cells

[0132] (1) Seed 96-well plates with 20,000 HepAD38 cells per 100 μl per well.

[0133] (2) Adjust the effector-to-target ratio to 1:1 according to the positive rate of CAR-T expression, and add 100 μl of CAR-T cells. Incubate in an incubator for 24 h, and collect the cell supernatant for later use.

[0134] (3) Dilute the cell supernatant 10-fold, and use the Human IFN-γ ELISA Kit (Linker Biotechnology) to detect according to the instructions.

[0135] (4) The results are as Figure 7 shown

[0136] The results confirmed that the CAR-T cells constructed with single-chain antibodies 3A1 and 6B12 could be significantly activated after contacting with HepAD38, and their secretion of IFN-γ increased significantly. The activation of single-chain antibody 8G6 was not obvious.

[0137] References:

[0138] 1. Perz JF, Armstrong GL, Farrington LA, Hutin YJF, Bell BP: The contributions of hepatitis B virus and hepatitis C virus infections to cirrhosis and primary liver cancer worldwide. Journal of hepatology 2006, 45(4): 529-538.

[0139] 2. Chen C-J, Yang H-I: Natural history of chronic hepatitis B REVEALed. Journal of gastroenterology and hepatology 2011, 26(4): 628-638.

[0140] 3.Global prevalence,treatment,and prevention of hepatitis B virusinfection in2016:a modelling study.TheLancetGastroenterology&Hepatology2018,3(6):383-403. 4.Revill PA,Chisari FV,Block JM,Dandri M,Gehring AJ,Guo H,Hu J,Kramvis A,Lampertico P,Janssen HLA et al:A global scientific strategy to curehepatitis B.The Lancet Gastroenterology&Hepatology 2019,4(7):545-558.

[0141] 5.Mak L-Y,Cheung K-S,Fung J,Seto W-K,Yuen M-F:New strategies for thetreatment of chronic hepatitis B.Trends Mol Med 2022,28(9):742-757.

[0142] 6.Petersen J,Thompson AJ,Levrero M:Aiming for cure in HBV and HDVinfection.Journal of hepatology 2016,65(4):835-848.

[0143] 7.Ferrari C:HBV and the immune response.Liver international:officialjournal ofthe InternationalAssociation for the Study ofthe Liver 2015,35Suppl1:121-128. 8. Lau GK, Lok AS, Liang RH, Lai CL, Chiu EK, Lau YL, Lam SK: Clearance of hepatitis B surface antigen after bone marrow transplantation: role of adoptive immunity transfer. Hepatology (Baltimore, Md) 1997, 25(6):1497 - 1501.

[0144] 9. Bertoletti A, Tan AT: HBV as a target for CAR or TCR - T cell therapy. Current Opinion In Immunology 2020, 66:35 - 41.

[0145] 10. Meng Z, Chen Y, Lu M: Advances in Targeting the Innate and Adaptive Immune Systems to Cure Chronic Hepatitis B Virus Infection. Frontiers In Immunology 2019, 10:3127. 11. Kruse RL, Shum T, Tashiro H, Barzi M, Yi Z, Whitten - Bauer C, Legras X, Bissig - Choisat B, Garaigorta U, Gottschalk S et al: HBsAg - redirected T cells exhibit antiviral activity in HBV - infected human liver chimeric mice. Cytotherapy 2018, 20(5):697 - 705.

[0146] 12. Krebs K, N, Huang L - R, Chmielewski M, Arzberger S, Gasteiger G, C, Schmitt E, Bohne F, Aichler M et al: T cells expressing a chimeric antigen receptor that binds hepatitis B virus envelope proteins control virus replication in mice. Gastroenterology 2013, 145(2): 456 - 465.

[0147] 13. Gehring AJ, Xue S - A, Ho ZZ, Teoh D, Ruedl C, Chia A, Koh S, Lim SG, Maini MK, Stauss H et al: Engineering virus - specific T cells that target HBV infected hepatocytes and hepatocellular carcinoma cell lines. Journal of hepatology 2011, 55(1): 103 - 110. 14. Tan AT, Yang N, Lee Krishnamoorthy T, Oei V, Chua A, Zhao X, Tan HS, Chia A, LeBert N, Low D et al: Use of Expression Profiles of HBV - DNA Integrated Into Genomes of Hepatocellular Carcinoma Cells to Select T Cells for Immunotherapy. Gastroenterology 2019, 156(6).

[0148] 15. Bohne F, Chmielewski M, Ebert G, Wiegmann K, T, Schulze A, Urban S, M, Abken H, Protzer U: T cells redirected against hepatitis B virus surface proteins eliminate infected hepatocytes. Gastroenterology 2008, 134(1): 239 - 247.

[0149] 16. Zoulim F, Lebossé F, Levrero M: Current treatments for chronic hepatitis B virus infections. Current Opinion In Virology 2016, 18: 109 - 116.

[0150] 17. Etxeberria I, E, Quetglas JI, Gros A, Villanueva A, Palomero J, Sánchez - Paulete AR, Piulats JM, Matias - Guiu X, Olivera I et al: Intratumor Adoptive Transfer of IL - 12 mRNA Transiently Engineered Antitumor CD8 T Cells. Cancer cell 2019, 36(6).

Claims

1. A monoclonal antibody that specifically binds to the preS1 of hepatitis B virus, characterized in that: Selected from any one of 3A1, 6B12, and 8G6, the amino acid sequence of the heavy chain variable region of monoclonal antibody 3A1 is as shown in SEQ ID NO.1, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO.2; the amino acid sequence of the heavy chain variable region of monoclonal antibody 6B12 is as shown in SEQ ID NO.3, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO.4; the amino acid sequence of the heavy chain variable region of monoclonal antibody 8G6 is as shown in SEQ ID NO.5, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO.

6.

2. A nucleic acid molecule encoding the monoclonal antibody that specifically binds to hepatitis B virus preS1 as claimed in claim 1, It is characterized in that: The nucleotide sequence of the heavy chain variable region of monoclonal antibody 3A1 is as shown in SEQ ID NO.7, and the nucleotide sequence of the light chain variable region is as shown in SEQ ID NO.8; the nucleotide sequence of the heavy chain variable region of monoclonal antibody 6B12 is as shown in SEQ ID NO.9, and the nucleotide sequence of the light chain variable region is as shown in SEQ ID NO.10; the nucleotide sequence of the heavy chain variable region of monoclonal antibody 8G6 is as shown in SEQ ID NO.11, and the nucleotide sequence of the light chain variable region is as shown in SEQ ID NO.

12.

3. Single-chain antibody scFv that specifically binds to hepatitis B virus preS1, characterized in that: The heavy chain variable region and the light chain variable region of the single-chain antibody are connected by a GS linker, and the heavy chain variable region and the light chain variable region are respectively from any one of the following: (1) The heavy chain variable region and the light chain variable region of monoclonal antibody 3A1 as claimed in claim 1; (2) The heavy chain variable region and the light chain variable region of monoclonal antibody 6B12 as claimed in claim 1; (3) The heavy chain variable region and the light chain variable region of monoclonal antibody 8G6 as claimed in claim 1.

4. The single-chain antibody according to claim 3, wherein: The sequence of the GS linker is (G4S)3.

5. A nucleic acid molecule encoding the single-chain antibody scFv according to claim 3 or 4, characterized in that: The nucleotide sequence of the heavy chain variable region of monoclonal antibody 3A1 is as shown in SEQ ID NO.7, and the nucleotide sequence of the light chain variable region is as shown in SEQ ID NO.8; the nucleotide sequence of the heavy chain variable region of monoclonal antibody 6B12 is as shown in SEQ ID NO.9, and the nucleotide sequence of the light chain variable region is as shown in SEQ ID NO.10; the nucleotide sequence of the heavy chain variable region of monoclonal antibody 8G6 is as shown in SEQ ID NO.11, and the nucleotide sequence of the light chain variable region is as shown in SEQ ID NO.

12.

6. Use of the nucleic acid molecule as claimed in claim 5 in the preparation of HBV-specific CAR-T cells.

7. An HBV-specific CAR-T cell, characterized in that: CAR-T cells comprising the nucleotide of the single-chain antibody as claimed in claim 5.

8. The HBV-specific CAR-T cell according to claim 7, wherein: The CAR structure of the HBV-specific CAR-T cells is CD8α signal peptide - scFv - CD8α hinge - CD8α transmembrane domain - 41BB intracellular domain - CD3ζ.

9. Use of the single-chain antibody as claimed in claim 1 or the HBV-specific CAR-T cells as claimed in claim 6 in the preparation of a medicament for the treatment of hepatitis B.

10. Use of the single-chain antibody as claimed in claim 1 or the HBV-specific CAR-T cells as claimed in claim 6 in the preparation of a medicament for inhibiting the proliferation of hepatitis B virus.

Images