Antigen-antibody complex, preparation method therefor, and use thereof

Abstract

The present invention relates to the field of biomedicine, and in particular to an antigen-antibody complex for enhancing an immune response of a hepatitis B patient, a preparation method therefor, and a use thereof. An antigen-antibody complex formulation prepared from HBsAg and an anti-HBsAg monoclonal antibody is superior to an antigen-antibody complex formulation prepared from a polyclonal antibody in respect of sustained induction of an immune response of a chronic hepatitis B patient. An antigen-antibody complex that induces an immune response of a hepatitis B patient is developed for the first time. In the complex, a hepatitis B surface antigen binds to the anti-HBsAg monoclonal antibody, and the complex composed of the hepatitis B surface antigen and the anti-HBsAg monoclonal antibody which have a certain mass ratio can induce the immune response of the chronic hepatitis B patient, and can also prevent and / or treat chronic hepatitis B.

Description

An antigen-antibody complex, its preparation method and application Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to an antigen-antibody complex for enhancing the immune response of hepatitis B patients, its preparation method, and its application. Background Technology

[0002] Hepatitis B virus infection (HBV) is an acute or chronic inflammatory disease of the liver caused by infection with the hepatitis B virus (HBV). It is one of the most common infectious diseases worldwide, and China is a high-prevalence area for HBV, with approximately 70 million HBV surface antigen (HBsAg) carriers, of whom about 20-30 million have chronic hepatitis B (CHB). Chronic hepatitis B patients have a high risk of developing advanced liver disease, including cirrhosis and hepatocellular carcinoma. Current guidelines define functional cure as the ideal treatment goal recommended by the latest domestic and international guidelines for the prevention and treatment of chronic hepatitis B, characterized by persistently undetectable serum HBsAg and HBV DNA, HBeAg seroconversion, with or without HBsAg seroconversion, remission of liver inflammation and improvement in histopathology, and a significant reduction in the incidence of end-stage liver disease. Studies have shown that HBsAg seroconversion can reduce the risk of HBV complications such as cirrhosis and hepatocellular carcinoma. Current clinical medical research indicates that one of the mechanisms underlying persistent infection with chronic hepatitis B virus is the lack of effective humoral or cellular immunity, which is also one of the important reasons why functional cure of chronic hepatitis B patients cannot be achieved.

[0003] Currently, antiviral treatment for chronic hepatitis B (CHB) primarily focuses on maximizing long-term suppression of HBV replication, reducing stem cell inflammation and necrosis, and liver fibrosis, thereby delaying and reducing the progression of liver disease and the occurrence of liver cancer. Nucleoside(t)ide analogues (NAs) are used as first-line drugs due to their high efficacy and low resistance. However, it has been found that first-line NA treatment for CHB has a long course and may not completely eliminate the virus. Even in CHB patients receiving long-term NA treatment, seroconversion of hepatitis B surface antigen (HBsAg) is rare. In clinical practice, in addition to antiviral measures, it is often necessary to enhance the body's immune response for persistent viral infection. To date, interferon is the only approved and commonly used immunomodulatory agent for treating persistent hepatitis B virus infection, but the functional cure rate after combining it with antiviral drugs is only about 10-30%.

[0004] Currently used immunomodulatory agents can be categorized as follows: (1) non-specific agents that promote cellular immunity, such as thymosin, interferon, transfer factor, and interleukin; (2) natural or synthetic immunomodulatory drugs, such as levamisole, plant polysaccharides, fungal or bacterial products, and artificially synthesized mimicry drugs; (3) immunomodulatory hormones, such as prednisone-like drugs; (4) specific antibodies or specific immune ribonucleic acid; and (5) vaccines. Current immunomodulatory agents may cause allergic reactions, infections, fever, and have short half-lives, indicating significant room for improvement in enhancing the body's immune response.

[0005] Studies have found that antigen-antibody immunogenic complexes (ICs) can be effectively used as therapeutic vaccines to clear hepatitis B virus. In 1997, therapeutic hepatitis B vaccines were listed as a major project of the "863" Program. Later, the Beijing Institute of Biological Products applied for clinical research (Wen Yumei, He Lifang, Qu Di, et al. Experimental study on recombinant therapeutic hepatitis B vaccine (YIC) [J]. Chinese Engineering Science, 1999, 1(1): 38-42.), and found that the Fab region of the antibody molecule in IC binds to HBsAg, while the Fc region of the antibody molecule binds to the Fc receptor on the surface of antigen-presenting cells (APCs), thereby bringing HBsAg in the complex into the APC, promoting the uptake of HBsAg, and more effectively activating T helper cells in the body, inducing immune-mediated cytokines / specific antibodies. The article disclosed that the subsequent technical solution used was to prepare ICs with HBsAg and human polyclonal antibody immunoglobulin for subsequent clinical research.

[0006] Subsequent research on immunogenic complexes (ICs) has focused on enhancing their efficacy or expanding their clinical applications by adding excipients or other components. Patent CN1034986C discloses an antigen-antibody immunogenic complex for treating persistent viral infection. The antigen is hepatitis B surface antigen plus pre-S protein, and the antibody is anti-hepatitis B surface antigen human immunoglobulin, with the antigen amount exceeding the antibody amount. An immunogenic complex formulation was constructed using hepatitis B surface antigen purified from blood at a protein content of 0.5–5 μg / mL and hepatitis B antibody immunoglobulin (anti-HBc) containing at least 200–400 IU of hepatitis B antibodies at a content of 0.5–5 μg / mL. This formulation showed good efficacy in treating persistent viral infection. Patent CN1919341B discovered that, unlike treatment, administering hepatitis B surface antigen-antibody complexes to individuals who do not respond or have a low response to hepatitis B vaccines can activate the body to produce effective anti-HBs (HBV surface antibodies). Therefore, hepatitis B surface antigen-antibody complexes can serve as a novel immune-enhancing prophylactic product for specific populations who do not respond or have a low response to existing hepatitis B vaccines. An article published by Academician Wen Yumei in 2017 (Human vaccines & immunotherapeutics vol.13,9(2017):1989-1996.) mentioned that HBsAg-anti HBs immunogenic complexes produced by yeast, combined with the hepatitis B treatment drug adefovir, can enhance the immune response of hepatitis B patients, and the combination of the two can serve as a novel combination therapy for hepatitis B.

[0007] Furthermore, patent CN101204582B improves upon existing antigen-antibody complex technology. The background description of this patent application states that research has confirmed that the presence of the Fc fragment in the IC plays a crucial role in breaking immune tolerance, and the Fc segment of the antibody constant region in the IC can be used as a key adjuvant to break hepatitis B immune tolerance. Therefore, this prior art constructed a fusion protein of multivalent HBsAg antigen and the constant region (Fc) of a fully human anti-HBs monoclonal antibody, and fused the adjuvant protein FL at its N-terminus. The patent mentions that immunizing HBV transgenic mice with an IC composed of HBsAg and anti-HBsAg monoclonal antibody successfully breaks the mice's immune tolerance to HBsAg, reduces HBsAg levels in mouse serum, and stimulates HBsAb production, with effects consistent with the IC composed of anti-HBsAg polyclonal antibodies. From the perspective of the polyclonal antibody production process, the inventors of this application believe that as the production demand increases, the source of the raw material for polyclonal antibodies—hepatitis B surface antibody positive serum—will become a bottleneck in the production process, and there is a potential for contamination of serum-derived antibodies. Therefore, they considered using the Fc of monoclonal antibodies for preparation. Summary of the Invention

[0008] Based on the current state of the technology, this invention is the first to develop an antigen-antibody complex that induces an immune response in hepatitis B patients. The complex comprises hepatitis B surface antigen and anti-HBsAg monoclonal antibody. This antigen-antibody complex can induce an immune response in patients with chronic hepatitis B and can also prevent and / or treat chronic hepatitis B. Based on this, this invention was completed.

[0009] In a first aspect, the present invention provides an antigen-antibody complex for inducing an immune response in patients with chronic hepatitis B. The complex comprises hepatitis B surface antigen and anti-HBsAg monoclonal antibody, wherein the hepatitis B surface antigen binds to the anti-HBsAg monoclonal antibody. The anti-HBsAg monoclonal antibody is composed of a heavy chain and a light chain. The heavy chain includes a variable region domain VH, constant region domains CH1, CH2, CH3, and a hinge region. The light chain includes a variable region domain VL and a constant region domain CL. The nucleotide sequence of the heavy chain VH+CH1 of the anti-HBsAg monoclonal antibody is selected from SEQ ID NO:1 or SEQ ID NO:3, and the nucleotide sequence of the light chain is selected from SEQ ID NO:2 or SEQ ID NO:4.

[0010] Furthermore, the hepatitis B surface antigen and the anti-HBsAg monoclonal antibody are bound together in a non-covalent manner.

[0011] Furthermore, the hepatitis B surface antigen is selected from hepatitis B surface antigen (HBsAg) expressed by genetically engineered bacteria, recombinant HBsAg expressed by mammalian cells, inactivated blood-derived HBsAg, or synthetic hepatitis B surface antigen.

[0012] Furthermore, the hepatitis B surface antigen is a polypeptide encoded by a pre-S or S region gene, or includes a polypeptide encoded by an S region gene, a PreS1 region gene + an S region gene, a PreS2 region gene + an S region gene, or a PreS1 region gene + a PreS2 region gene + an S region gene.

[0013] In one embodiment of the present invention, when the nucleotide sequence of the heavy chain VH+CH1 of the anti-HBsAg monoclonal antibody is as shown in SEQ ID NO:1, the nucleotide sequence of the light chain is as shown in SEQ ID NO:2; when the nucleotide sequence of the heavy chain VH+CH1 of the anti-HBsAg monoclonal antibody is as shown in SEQ ID NO:3, the nucleotide sequence of the light chain is as shown in SEQ ID NO:4.

[0014] Furthermore, the hinge region and CH2 and CH3 domains of the heavy chain of the anti-HBsAg monoclonal antibody are selected from IgG1 or IgG2 and their mutants.

[0015] Furthermore, the IgG1 is selected from human immunoglobulin IgG1 or mouse IgG1 homologous to human immunoglobulin IgG1.

[0016] Furthermore, the IgG2 is selected from human immunoglobulin IgG2 or mouse IgG2a, IgG2b, and IgG2c, which are homologous to human immunoglobulin IgG2.

[0017] Preferably, the hinge region and CH2 and CH3 domains of the heavy chain of the anti-HBsAg monoclonal antibody are selected from human IgG1 or mouse IgG1 and IgG2a.

[0018] Furthermore, the hinge region and CH2 and CH3 domain sequences of the heavy chain of the anti-HBsAg monoclonal antibody are selected from SEQ ID NO:5-SEQ ID NO:7.

[0019] Furthermore, the nucleotide sequence of the heavy chain VH+CH1 has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence homology or sequence identity with the nucleotide sequence shown in SEQ ID NO:1 or 3.

[0020] Furthermore, the nucleotide sequences of the hinge region and CH2 and CH3 domains of the heavy chain of the anti-HBsAg monoclonal antibody have at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence homology or sequence identity with any of the nucleotide sequences shown in SEQ ID NO:5-SEQ ID NO:7.

[0021] Furthermore, the nucleotide sequence of the light chain has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence homology or sequence identity with the nucleotide sequence shown in SEQ ID NO:2 or 4.

[0022] Furthermore, the mass ratio of the anti-HBsAg monoclonal antibody to the hepatitis B surface antigen is 1:0.67-1:54.

[0023] Furthermore, the mass ratio of the anti-HBsAg monoclonal antibody to the hepatitis B surface antigen is 1:2 to 1:20.

[0024] Furthermore, the mass ratio of the anti-HBsAg monoclonal antibody to the hepatitis B surface antigen is 1:2 to 1:15.

[0025] Preferably, the mass ratio of the anti-HBsAg monoclonal antibody to the hepatitis B surface antigen is 1:6.

[0026] Furthermore, the term "chronic hepatitis B patient" refers to a patient with normal alanine aminotransferase (ALT) and HBV DNA <2×10⁻⁶. 3IU / mL or HBV DNA undetectable.

[0027] Furthermore, the term "chronic hepatitis B patient" refers to a chronic hepatitis B patient who has received antiviral drug treatment and whose serum HBsAg level is <3000 IU / mL, or whose HBeAg has turned negative and whose HBsAg level is <1500 IU / mL.

[0028] In a second aspect, the present invention provides a method for preparing the antigen-antibody complex described in the first aspect, the method comprising the following steps:

[0029] (a) Construction of monoclonal antibody expression plasmid: The heavy chain and light chain sequences of anti-HBsAg monoclonal antibody were constructed in a eukaryotic cell expression vector;

[0030] (b) Monoclonal antibody preparation: The plasmid constructed in step (a) was transfected into eukaryotic cells, and anti-HBsAg monoclonal antibody was obtained after cell expression;

[0031] (c) Antigen-antibody mixing: The anti-HBsAg monoclonal antibody obtained in step (b) is mixed with hepatitis B surface antigen to obtain the antigen-antibody complex described in the first aspect of the present invention.

[0032] Further, in step (a), the heavy chain of the anti-HBsAg monoclonal antibody includes a heavy chain variable region domain VH, constant region domains CH1, CH2, CH3 and a hinge region; the light chain includes a variable region domain VL and a constant region domain CL.

[0033] Furthermore, the nucleotide sequence of the heavy chain VH+CH1 of the anti-HBsAg monoclonal antibody is selected from SEQ ID NO:1 or SEQ ID NO:3, and the nucleotide sequence of the light chain is selected from SEQ ID NO:2 or SEQ ID NO:4.

[0034] In one embodiment of the present invention, when the nucleotide sequence of the heavy chain VH+CH1 of the anti-HBsAg monoclonal antibody is as shown in SEQ ID NO:1, the nucleotide sequence of the light chain is as shown in SEQ ID NO:2; when the nucleotide sequence of the heavy chain VH+CH1 of the anti-HBsAg monoclonal antibody is as shown in SEQ ID NO:3, the nucleotide sequence of the light chain is as shown in SEQ ID NO:4.

[0035] Furthermore, in step (a), the hinge region and CH2, CH3 domain of the heavy chain of the anti-HBsAg monoclonal antibody are selected from the hinge region and CH2, CH3 domain of the heavy chain of IgG1 or IgG2 and their mutants.

[0036] Furthermore, the IgG1 is selected from human immunoglobulin IgG1 or mouse IgG1 homologous to human immunoglobulin IgG1.

[0037] Furthermore, the IgG2 is selected from human immunoglobulin IgG2 or mouse IgG2a, IgG2b, and IgG2c, which are homologous to human immunoglobulin IgG2.

[0038] Preferably, the hinge region and CH2, CH3 domains of the heavy chain of the anti-HBsAg monoclonal antibody are selected from human IgG1 or mouse IgG1, IgG2a.

[0039] Furthermore, in step (a), the hinge region and CH2 and CH3 domain sequences of the anti-HBsAg monoclonal antibody heavy chain are selected from SEQ ID NO:5-SEQ ID NO:7.

[0040] Furthermore, the nucleotide sequence of the heavy chain VH+CH1 has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence homology or sequence identity with the nucleotide sequence shown in SEQ ID NO:1 or 3.

[0041] Furthermore, the nucleotide sequences of the hinge region and CH2 and CH3 domains of the heavy chain of the anti-HBsAg monoclonal antibody have at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence homology or sequence identity with any of the nucleotide sequences shown in SEQ ID NO:5-SEQ ID NO:7.

[0042] Furthermore, the nucleotide sequence of the light chain has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence homology or sequence identity with the nucleotide sequence shown in SEQ ID NO:2 or 4.

[0043] Furthermore, in step (c), the hepatitis B surface antigen and the anti-HBsAg monoclonal antibody are bound in a non-covalent manner.

[0044] Furthermore, the hepatitis B surface antigen is selected from hepatitis B surface antigen (HBsAg) expressed by genetically engineered bacteria, recombinant HBsAg expressed by mammalian cells, inactivated blood-derived HBsAg, or synthetic hepatitis B surface antigen.

[0045] Furthermore, in step (c), the hepatitis B surface antigen is a polypeptide encoded by a pre-S or S region gene, or includes a polypeptide encoded by an S region gene, a PreS1 region gene + an S region gene, a PreS2 region gene + an S region gene, or a PreS1 region gene + a PreS2 region gene + an S region gene.

[0046] Furthermore, in step (c), the mass ratio of the anti-HBsAg monoclonal antibody to the hepatitis B surface antigen is 1:0.67-1:54.

[0047] Furthermore, the mass ratio of the anti-HBsAg monoclonal antibody to the hepatitis B surface antigen is 1:2 to 1:20.

[0048] Furthermore, the mass ratio of the anti-HBsAg monoclonal antibody to the hepatitis B surface antigen is 1:2 to 1:15.

[0049] Preferably, the mass ratio of the anti-HBsAg monoclonal antibody to the hepatitis B surface antigen is 1:6.

[0050] Thirdly, the present invention provides a pharmaceutical composition comprising the antigen-antibody complex described in the first aspect, and a pharmaceutically acceptable carrier and / or excipients.

[0051] Furthermore, the pharmaceutical composition includes a single drug, a combination drug, or a synergistic drug.

[0052] Furthermore, the pharmaceutical composition also includes an antiviral drug and / or a drug that lowers HBsAg levels.

[0053] Furthermore, the antiviral drug is selected from one or more of nucleotide reverse transcriptase inhibitors, nucleoside (acid) analogs, capsid assembly regulators, antisense oligonucleotides, and / or invasion inhibitors.

[0054] Furthermore, the nucleotide reverse transcriptase inhibitor is selected from one or more of lamivudine, telbivudine, adefovir dipivoxil, entecavir, tenofovir disoproxil fumarate, tenofovir alafenamide fumarate, and / or imitenofovir.

[0055] Furthermore, the drug for reducing HBsAg levels is selected from one or more of pegylated interferon α, polyclonal antibodies, monoclonal antibodies, targeted siRNA, small interfering RNA, and / or HBsAg inhibitors.

[0056] Furthermore, the antiviral drug, the drug that reduces HBsAg levels, and / or the antigen-antibody complex in the pharmaceutical composition can be packaged independently and administered sequentially to the patient.

[0057] Furthermore, the dosage form of the pharmaceutical composition includes liquid, solid, gel, and / or aerosol.

[0058] Furthermore, the drug composition can be administered via subcutaneous injection, intradermal injection, intramuscular injection, intravenous injection, intraperitoneal injection, oral administration, or intranasal or oral spray.

[0059] In one embodiment of the present invention, the antiviral drug in the pharmaceutical composition is TDF, the drug for reducing HBsAg levels is monoclonal antibody G12, and the antigen-antibody complex is TVac-G.

[0060] Fourthly, the present invention provides a vaccine composition comprising the antigen-antibody complex described in the first aspect, and an immunologically acceptable carrier and / or excipient.

[0061] Furthermore, the vaccine composition may be monovalent or multivalent.

[0062] Furthermore, the vaccine composition also contains an adjuvant.

[0063] Furthermore, the adjuvant is selected from particulate and non-particulate adjuvants.

[0064] Furthermore, the particulate adjuvant is selected from one or more of aluminum salts, water-in-oil emulsions, oil-in-water emulsions, nanoparticles, microparticles, liposomes, and / or immunostimulatory complexes.

[0065] Furthermore, the aluminum salt includes aluminum hydroxide adjuvant, aluminum phosphate adjuvant, or a mixture of aluminum hydroxide and aluminum phosphate.

[0066] Furthermore, the non-particulate adjuvant is selected from one or more of CpG1080, muramyl dipeptide and its derivatives, saponins, lipid A, cytokines, derived polysaccharides, bacterial toxins, microorganisms and their products, and / or propolis.

[0067] Furthermore, the microbial products include one or more of mycobacteria, pumilus, and / or pertussis.

[0068] Furthermore, the ratio of the antigen-antibody complex to the adjuvant in the vaccine composition is 1:100.

[0069] Furthermore, the ratio of the antigen-antibody complex to the adjuvant in the vaccine composition is 1:60.

[0070] Furthermore, the ratio of the antigen-antibody complex to the adjuvant in the vaccine composition is 1:40.

[0071] Furthermore, the vaccine composition dosage form includes liquid, solid, gel, and / or aerosol.

[0072] Fifthly, the present invention provides an immunomodulator, the immunomodulator comprising a first immunomodulator and a second immunomodulator; the first immunomodulator is a drug for reducing HBsAg levels, and the second immunomodulator is the antigen-antibody complex described in the first aspect; the first immunomodulator and the second immunomodulator are each packaged independently.

[0073] Furthermore, the drug for reducing HBsAg levels is selected from one or more of pegylated interferon α, polyclonal antibodies, monoclonal antibodies, targeted siRNA, small interfering RNA, and / or HBsAg inhibitors.

[0074] In a sixth aspect, the present invention provides the use of the antigen-antibody complex described in the first aspect in the preparation of a medicament for the prevention and / or treatment of chronic hepatitis B.

[0075] In a seventh aspect, the present invention provides a drug delivery system for treating patients with chronic hepatitis B, the drug delivery system comprising a first drug and a second drug in individually packaged form; wherein the first drug comprises an antiviral drug and / or a drug that lowers HBsAg levels; the second drug comprises a first immunomodulator and a second immunomodulator; the first immunomodulator is a drug that lowers HBsAg levels; the second immunomodulator is the antigen-antibody complex described in the first aspect; and the second drug is administered to the patient after the first drug is administered.

[0076] Furthermore, the antiviral drug is selected from one or more of nucleotide reverse transcriptase inhibitors, nucleoside (acid) analogs, capsid assembly regulators, antisense oligonucleotides, and / or invasion inhibitors.

[0077] Furthermore, the nucleotide reverse transcriptase inhibitor is selected from one or more of lamivudine, telbivudine, adefovir dipivoxil, entecavir, tenofovir disoproxil fumarate, tenofovir alafenamide fumarate, and / or tenofovir alafenamide.

[0078] Furthermore, the drug for reducing HBsAg levels is selected from one or more of pegylated interferon α, polyclonal antibodies, monoclonal antibodies, targeted siRNA, small interfering RNA, and / or HBsAg inhibitors.

[0079] Furthermore, the dosage of the antigen-antibody complex is 30-150 μg / person.

[0080] Furthermore, the dosage of the antigen-antibody complex is 40-80 μg / person.

[0081] Furthermore, the dosage of the antigen-antibody complex is 50-70 μg / person.

[0082] Furthermore, the term "chronic hepatitis B patient" refers to a chronic hepatitis B patient whose HBV DNA level is usually high and whose serum HBsAg level is high.

[0083] Furthermore, the high level of HBV DNA refers to an HBV DNA concentration of <200 IU / mL or 2000 copies / mL, and the high level of serum HBsAg refers to an HBsAg concentration of >10 IU / mL and <3000 IU / mL.

[0084] Furthermore, after administering the first immunomodulatory agent to the patient, when the patient's serum HBsAg level decreases to approximately ≤400 IU / mL, a second immunomodulatory agent is administered.

[0085] In one embodiment of the present invention, the first agent in the drug delivery system is TDF, the first immunomodulator in the second agent is G12, and the second immunomodulator in the second agent is TVac-G. Beneficial effects

[0086] This invention unexpectedly discovered that antigen-antibody complex preparations made from HBsAg and anti-HBsAg monoclonal antibodies are superior to antigen-antibody complex preparations (or pharmaceutical compositions or vaccine compositions) made from polyclonal antibodies in sustaining the induction of immune responses in patients with chronic hepatitis B, especially when the nucleotide sequence of the heavy chain VH+CH1 of the anti-HBsAg monoclonal antibody is SEQ ID NO:1 or SEQ ID NO:3, and the nucleotide sequence of the light chain is selected from SEQ ID NO:2 or SEQ ID NO:4; when the chronic hepatitis B patient is in the later stage of the immune control period of infection (Chinese Journal of General Practitioners, 2021, 20(03): 281-289) or has serum HBV DNA <2×10 after long-term antiviral drug treatment. 3 When the HBsAg level is <3000 IU / ml or the alanine aminotransferase (ALT) level is normal in patients with chronic hepatitis B, the antigen-antibody complex preparation made from the monoclonal antibody of this invention can continuously induce and enhance the host's immune response, clear HBsAg from the body of patients with chronic hepatitis B, and improve the clinical cure rate of chronic hepatitis B.

[0087] This invention is the first to develop an antigen-antibody complex that induces an immune response in hepatitis B patients. The complex comprises hepatitis B surface antigen (HBsAg) and anti-HBsAg monoclonal antibody, with the HBsAg binding to the anti-HBsAg monoclonal antibody. Furthermore, this invention is the first to discover that a complex composed of a certain mass ratio of HBsAg and anti-HBsAg monoclonal antibody can induce an immune response in patients with chronic hepatitis B. However, it is not necessarily true that the higher the HBsAg content compared to the anti-HBsAg monoclonal antibody content, the better; only a specific mass ratio can achieve the desired effect of inducing an immune response in patients with chronic hepatitis B, thus preventing and / or treating chronic hepatitis B. Attached Figure Description

[0088] Figure 1 is a schematic diagram of the polyacrylamide gel electrophoresis results of monoclonal antibodies G12-hIgG1, G12-mIgG1, G12-mIgG2a, KR127-hIgG1, KR127-mIgG1, KR127-mIgG2a and mouse polyclonal antibody (hepatitis B immunoglobulin (mHBIG)) against hepatitis B surface antigen (HBsAg) in Examples 1 and 2.

[0089] Figure 2 is a schematic diagram showing the binding affinity of human monoclonal antibody (G12-hIgG1) and mouse polyclonal antibody (mHBIG) to hepatitis B surface antigen (HBsAg) in Example 4.

[0090] Figures 3 and 4 are schematic diagrams showing the affinity results of human monoclonal antibody (G12-hIgG1) and mouse polyclonal antibody (mHBIG) for hepatitis B surface antigen (HBsAg) in Example 3.

[0091] Figures 5 and 6 are schematic diagrams showing the binding affinity of human monoclonal antibodies (G12-hIgG1 and KR127-hlgG1) and mouse monoclonal antibodies (G12-mIgG1, G12-mIgG2a, KR127-mIgG1 and KR127-mIgG1) to hepatitis B surface antigen (HBsAg) in Example 4.

[0092] Figure 7 shows the antibody levels in mice after immunization with antigen-antibody complex formulations in Example 6-2.

[0093] Figures 8-11 show the changes in antibody (HBsAb) expression levels in mice on days 7 and 35 after administration of antigen-antibody complex preparations made with different proportions of antigen-antibody from Examples 6-3.

[0094] Figures 12-15 show the trends of hepatitis B surface antigen (HBsAg) expression in mice after immunization with antigen-antibody complex preparations prepared with different ratios of antigen and antibody in Examples 6-4. Figure 15 is a schematic diagram of the overall trend of hepatitis B surface antigen (HBsAg) expression level when the antibody:antigen ratio is 1:6.

[0095] Figures 16-17 show the trends in antigen (HbsAg) expression in immunized mice induced by antigen-antibody complex formulations prepared from monoclonal antibodies of different epitopes in Examples 6-5.

[0096] Figures 18-19 show the trends and levels of antibody (HbsAb) expression in immunized mice induced by antigen-antibody complex formulations prepared from monoclonal antibodies of different epitopes in Examples 6-5.

[0097] [Corrected according to Rule 91 15.07.2025] Figures 20-24 are schematic diagrams of the results of humoral immune factor levels after immunization in Example 6-6.

[0098] [Corrected according to Rule 91 15.07.2025] Figures 25-32 are schematic diagrams of the results of cellular immune factor levels after immunization in Example 6-6.

[0099] [Correction based on Rule 91, 15.07.2025] Figure 33 shows the changes in serum HBsAg after immunization in Example 7.

[0100] [Correction based on Rule 91, 15.07.2025] Figure 34 shows the changes in serum HBV DNA after immunization in Example 7.

[0101] [Correction based on Rule 91, 15.07.2025] Figure 35 shows the changes in HBsAb after immunization in Example 7.

[0102] [Corrected according to Rule 91 15.07.2025] Figure 36 shows the difference in HBsAb levels after comparing Example 8 with other immunomodulatory combination therapies.

[0103] [Corrected according to Rule 91 15.07.2025] Figure 37 shows the effect of G12 monoclonal antibody in the complex vaccine of Example 9 on clearing HBsAg.

[0104] [Correction based on Rule 91, July 15, 2025] Figure 38 shows the effect of G12 monoclonal antibody in the complex vaccine of Example 9 on DNA clearance. Detailed Implementation

[0105] The specific embodiments of the present invention will be further described below. It should be noted that these descriptions are for the purpose of aiding understanding the present invention, but do not constitute a limitation thereof. Furthermore, the technical features involved in the embodiments described below can be combined with each other as long as they do not conflict with each other.

[0106] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the experimental materials used in the following embodiments are all available through conventional commercial channels.

[0107] the term

[0108] To facilitate a clearer understanding of this disclosure, certain terms are first defined. As used herein, unless otherwise expressly specified herein, each of the following terms shall have the meaning given below. Other definitions are set forth throughout the application.

[0109] The term “about” can refer to a value or composition within an acceptable range of error for a particular value or composition as determined by a person skilled in the art, which will depend in part on how the value or composition is measured or determined.

[0110] As used herein, the terms “optional” or “optionally” mean that the events or conditions described below may occur but are not required to occur. For example, “optionally containing 1-3 antibody heavy chain variable regions” means that the antibody heavy chain variable regions of a particular sequence may be present but are not required to be present, and may be 1, 2 or 3.

[0111] The term "sequence identity" as used in this invention refers to the degree of identity between two nucleic acid or two amino acid sequences when optimally aligned and compared with appropriate mutations such as substitutions, insertions, or deletions. The sequence identity between the sequences described in this invention and sequences exhibiting identity with them can be at least 85%, 90%, or 95%, preferably at least 95%. Non-limiting embodiments include 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100%.

[0112] Hepatitis B surface antigen (HBsAg) is the outer shell protein of the hepatitis B virus (HBV) and is a major component of HBV surface antigen. It is not infectious on its own, but its presence is often accompanied by the presence of the hepatitis B virus, so it is a marker of hepatitis B virus infection.

[0113] HBV is a partially double-stranded circular hepatotropic DNA virus. Its genome contains four partially overlapping open reading frames (ORFs): the pre-S / S region, the pre-C / C region, the P region, and the X region. The pre-S / S region encodes three outer membrane proteins of HBV: the large surface protein (LHB), the middle surface protein (MHB), and the small surface protein (SHB), collectively known as hepatitis B virus surface antigens. Translation of these proteins is initiated by three different start codons on the same gene segment. Based on the position of the start codon, the pre-S / S region can be divided into three distinct domains: Pre-S1, Pre-S2, and the S domain.

[0114] As used in this application, the term "antibody" can include all antibodies and any antigen-binding fragment (e.g., an antigen-binding fragment including a hinge, an antigen-binding fragment including a hinge and a CH1 domain, an antigen-binding fragment including a hinge and a CH2 domain, or an antigen-binding fragment containing portions of a hinge, a CH2 domain, and a CH3 domain) or a single chain thereof. "Antibody" can include, for example, naturally occurring and non-naturally occurring antibodies; monoclonal and polyclonal antibodies; chimeric and humanized antibodies; human and non-human antibodies; fully synthetic antibodies; and single-chain antibodies.

[0115] In one embodiment, "antibody" refers to a protein, such as a glycoprotein or its antigen-binding portion comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region. In some naturally occurring IgG, IgD, and IgA antibodies, the heavy chain constant region consists of a hinge, a CH1 domain, a CH2 domain, and a CH3 domain. In some naturally occurring antibodies, each light chain consists of a light chain variable region (VL) and a light chain constant region. The light chain constant region consists of a CL domain. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of an antibody can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

[0116] Immunoglobulins can be derived from any commonly known isotype, including but not limited to IgA, secretory IgA, IgG, and IgM. IgG isotypes are subclassed in some species: IgG1, IgG2, IgG3, and IgG4 in humans, and IgG1, IgG2a, IgG2b, and IgG3 in mice. In some embodiments, the antibody described herein is a human or mouse IgG1 or IgG2 subtype. Immunoglobulins (e.g., human IgG1) exist in several allotypes, differing from each other in at most a few amino acids.

[0117] As used in this application, the heavy chain constant region, such as the hinge region, may include “IgG1 isotype”, “IgG2 isotype”, “IgG3 isotype” or “IgG4 isotype”. The domain may contain the amino acid sequence of each isotype or a variant thereof (the amino acid sequences of each isotype in the domain are more homologous than those of other different isotypes).

[0118] As used in this invention, the term "antigen-binding portion" of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind antigens. The antigen-binding portion of an antibody can be categorized as a "hinge containing the antigen-binding portion." Antigen-binding functionality of antibodies has been demonstrated to occur via fragments of full-length antibodies. Examples of binding fragments encompassed within the "antigen-binding portion" of the antibody described in this invention include (i) Fab fragments, monovalent fragments consisting of VL, VH, CL, and CH1 domains; (ii) F(ab')2 fragments, bivalent fragments comprising two Fab fragments linked by disulfide bonds in the hinge region; (iii) Fd fragments consisting of VH and CH1 domains; (iv) Fv fragments consisting of VL and VH domains of a single arm of the antibody; (v) dAb fragments (Ward et al., (1989) Nature 341:544-546) consisting of VH domains; and (vi) separate complementarity-determining regions (CDRs) or (vii) combinations of two or more separate CDRs optionally linked by synthetic linkers. Furthermore, although the two VL and VH segments of the Fv fragment are encoded by separate genes, they can be linked together using recombinant methods via synthetic linkers, allowing them to be made into a single polypeptide chain with antibody activity, a monovalent molecule known as a single-chain Fv (scFv). Such single-chain antibodies are also encompassed within the term "antigen-binding moiety" of antibodies.

[0119] As used in this invention, "isotype" refers to an antibody class (e.g., IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE antibodies) encoded by a heavy chain constant domain gene. The full-length amino acid sequence of each wild-type human IgG constant region (including all domains, i.e., CH1 domain, hinge domain, CH2 domain, and CH3 domain) is cataloged in the UniProt database, for example, as P01857 (IgG1), P01859 (IgG2), P01860 (IgG3), and P01861 (IgG4) or their different isotypes. As used in this invention, a domain of the heavy chain constant region, such as the CH2 domain, is an IgG1 isotype or an IgG2 isotype, and this domain may contain the amino acid sequence of the corresponding domain of each isotype or a variant thereof (i.e., the amino acid sequence of each isotype within this domain shows higher homology than other different isotypes).

[0120] In this invention, the "Fc region" (fragment crystallizable region), "Fc domain," or "Fc" refers to the C-terminal region of the heavy chain of an antibody that mediates the binding of immunoglobulins to host tissues or factors (including binding to Fc receptors on different cells of the immune system, such as effector cells, or binding to the first component (C1q) of the classical complement system). Therefore, the Fc region of an isotype IgG antibody comprises the heavy chain constant region of the antibody excluding the first constant region, the immunoglobulin domain (CH1). In IgG, IgA, and IgD antibody isotypes, the Fc region comprises the CH2 and CH3 constant domains of the heavy chain of each antibody; the Fc region of IgM and IgE comprises three heavy chain constant domains (CH domains 2-4) of each polypeptide chain. For IgG, the Fc region comprises the immunoglobulin domain consisting of the hinge, CH2, and CH3. The Fc can be natural (…). Fc (either naturally occurring or wild-type), including any allotype, or variant Fc (e.g., non-naturally occurring Fc), includes, for example, 1, 2, 3, 4, 5, 1-5, 1-10, or 5-10 or more amino acid mutations, such as substitution, addition, or deletion. For example, a variant Fc may contain at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the same amino acid sequence as the wild-type Fc. Modified or mutated Fc may have enhanced or reduced effector function and / or half-life. The CH2 and CH3 regions are the primary sites for effector function and FcRn binding. Fc may refer to isolated Fc or, in the context of a protein polypeptide containing an Fc (e.g., "binding protein containing an Fc region"), also referred to as an "Fc fusion protein" (e.g., an antibody or immunoadhesin).

[0121] "Fc receptors," or "FcRs," are receptors that bind to the Fc region of immunoglobulins. FcRs that bind to IgG antibodies include receptors from the FcγR family, including allelic variants and alternative splice forms of these receptors. The FcγR family consists of three active receptors (FcγRI, FcγRIII, and FcγRIV in mice; FcγRIA, FcγRIIA, and FcγRIIIA in humans) and one inhibitory receptor (FcγRIIB). Most natural effector cell types co-express one or more active FcγRs and the inhibitory FcγRIIB, while natural killer (NK) cells selectively express one active Fc receptor (FcγRIII in mice and FcγRIIIA in humans) instead of the inhibitory FcγRIIB found in both mice and humans. Human IgG1 binds to most Fc receptors and is considered equivalent to mouse IgG2a in terms of the type of active Fc receptor it binds to.

[0122] In this article, "treatment" refers to an attempt to alter the natural course of disease in the treated individual, and may be a clinical intervention implemented for prevention or during the course of clinicopathological processes. The desired effects of treatment include, but are not limited to, preventing the onset or recurrence of disease, relieving symptoms, reducing any direct or indirect pathological consequences of the disease, slowing the rate of disease progression, improving or eliminating the disease state, and regressing or improving prognosis.

[0123] In this article, "individual" or "subject" refers to a mammal. Mammals include, but are not limited to, primates (such as humans and non-human primates such as monkeys) or other mammals (such as cattle, sheep, cats, dogs, horses, rabbits, and rodents such as mice and rats).

[0124] In this article, "mutant" or "variant" can refer to a molecule obtained by mutating one or more nucleotides or amino acids in any naturally occurring or engineered molecule.

[0125] The standard recombinant DNA and molecular cloning techniques used in the examples are well-known in the art (Ausubel, FM et al., Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley-Interscience). Major chemical and biological reagents were purchased from KAPA Biosystems, New England Biolabs, TransGen Biotech, Thermo Fisher Scientific, OMEGA bio-tek, etc.

[0126] Immunocomplex vaccines, also known as antigen-antibody complex vaccines, are a new type of vaccine developed in the 1990s. They are made by mixing specific antiserum with viral antigens in a certain proportion. Their advantages are high safety and better immunization effect than conventional vaccines.

[0127] Because hepatitis B surface antigen (HBsAg) contains many transmembrane hydrophobic regions and disulfide bonds, it will naturally aggregate into large spherical or rod-shaped polymeric particles in solution, thus making it impossible to construct a homogeneous and soluble Fc fusion protein through genetic engineering technology.

[0128] According to the Guidelines for the Diagnosis and Treatment of Chronic Hepatitis B at the Primary Care Level (Practice Edition 2020), chronic hepatitis B patients refer to those with chronic inflammatory liver disease caused by persistent HBV infection for more than 6 months. The stages of chronic hepatitis B virus infection include: (i) Immune tolerance phase (chronic HBV carrier state): mostly during the perinatal and infancy periods, serum HBsAg, hepatitis B e antigen (HBeAg), and anti-hepatitis B core antigen (anti-HBc) are simultaneously positive, HBV DNA level is high (usually HBV DNA > 2 x 10⁷ IU / ml), and alanine aminotransferase (ALT) is normal; (ii) Immune clearance phase (HBeAg positive CHB): HBeAg positive stage, ALT is persistently or repeatedly abnormal and HBV DNA level is high (usually HBV DNA > 2 x 10⁴ IU / ml), HBeAg seroconversion and anti-HBe seroconversion may occur, and ALT is persistently or intermittently elevated; (iii) Immune control phase (inactive HBsAg carrier state): low replication phase, ALT is normal, HBV DNA level is low (HBV DNA < 2 x 10³ IU / ml). (iv) Reactivation (HBeAg negative CHB): 5% to 15% of inactive patients may experience one or more episodes of hepatitis, with persistent or recurrent abnormal ALT, HBV DNA > 2 x 10³ IU / ml, presenting as HBeAg negative CHB, and may experience HBeAg seroconversion again (Chinese Journal of General Practitioners, 2021, 20(03): 281-289.).

[0129] According to the expert consensus on clinical cure (functional cure) of chronic hepatitis B (Chinese Journal of Infectious Diseases, 2019, 37(8): 461-472.), the current clinical treatment of chronic hepatitis B patients mainly focuses on the combination of antiviral drugs and immunomodulators. The combination of antiviral drugs and immunomodulators mainly includes the initial combination therapy strategy and the sequential combination therapy strategy. The latter includes the "switch" strategy (i.e., replacing antiviral drugs with immunomodulators) and the "add" strategy (i.e., adding immunomodulators to antiviral drugs). The expert consensus recommends that CHB patients who receive long-term antiviral drug treatment should be those with undetectable HBV DNA and HBsAg level <3000 IU / ml, HBeAg positive or HBsAg level ≥1500 IU / ml, or HBeAg seroconversion and HBsAg level <1500 IU / ml. It strongly recommends the use of the sequential combination immunomodulator therapy strategy.

[0130] First dose of immune modulator

[0131] As used in this article, "first immunomodulator" and "first immunomodulator" are interchangeable and both refer to drugs that reduce HBsAg levels, including but not limited to polyclonal antibodies, monoclonal antibodies, targeted siRNA, small interfering RNA, or HBsAg inhibitors.

[0132] Second dose of immune modulator

[0133] As used herein, "second immunomodulator" and "second immunomodulator" are interchangeable and both refer to antigen-antibody complexes, drugs containing antigen-antibody complexes, or vaccines containing antigen-antibody complexes, preferably the antigen-antibody complexes described in this invention.

[0134] Composition and method of application

[0135] The present invention also provides a composition comprising: (i) an antigen-antibody complex prepared by the method of the present invention, and (ii) a pharmaceutically or immunologically acceptable excipient or adjuvant. In this invention, the term "comprising" means that various components may be used together or present in the composition of the present invention. Therefore, the terms "consistent with..." and "consisting with..." are included in the term "comprising".

[0136] The compositions of the present invention include pharmaceutical compositions and vaccine compositions. The compositions of the present invention can be monovalent or polyvalent.

[0137] The pharmaceutical or vaccine compositions of the present invention can be prepared into various conventional dosage forms, including (but not limited to): injections, granules, tablets, pills, suppositories, capsules, suspensions, sprays, etc.

[0138] (i) Pharmaceutical Composition

[0139] The pharmaceutical composition of the present invention comprises an effective amount of an antigen-antibody complex prepared by the method of the present invention, wherein the antigen-antibody complex may be monovalent or polyvalent.

[0140] As used herein, the term "effective dose" refers to the amount of a therapeutic agent that treats, alleviates, or prevents a target disease or condition, or the amount that exhibits a detectable therapeutic or preventative effect. This effect can be detected, for example, by antigen levels. Therapeutic effects also include a reduction in physiological symptoms. The precise effective dose for a given subject depends on that subject's body size and health status, the nature and severity of the condition, and the choice of the therapeutic agent and / or combination of therapeutic agents administered. Therefore, it is not useful to predetermine an accurate effective dose. However, for a given condition, the effective dose can be determined using routine laboratory methods.

[0141] For the purposes of this invention, the effective dose of the vaccine complex is approximately 30 to 150 μg administered to an individual.

[0142] Pharmaceutical compositions may also contain pharmaceutically acceptable carriers. The term "pharmaceutically acceptable carrier" refers to a carrier used for the administration of a therapeutic agent (e.g., recombinant protein or other therapeutic agent). This term refers to pharmaceutical carriers that do not induce antibodies harmful to the individual receiving the composition and do not cause excessive toxicity after administration. Suitable carriers can be large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acid, polyglycolic acid, etc. These carriers are well known to those skilled in the art. A thorough discussion of pharmaceutically acceptable carriers or excipients can be found in Remington's Pharmaceutical Sciences (Mack Pub. Co., NJ 1991).

[0143] Pharmaceutically acceptable carriers in a composition may include liquids such as water, saline, glycerol, and ethanol. Additionally, these carriers may contain auxiliary substances such as wetting agents or emulsifiers, pH buffers, etc. Typically, the composition can be formulated as an injectable preparation, such as a liquid solution or suspension; it can also be formulated as a solid form suitable for reconstitution into solutions or suspensions, or liquid excipients, prior to injection. Liposomes are also included in the definition of pharmaceutically acceptable carriers.

[0144] (ii) Vaccine composition

[0145] The vaccine compositions of the present invention can be prophylactic (i.e., for infection prevention) or therapeutic. The vaccine compositions comprise the antigen-antibody complex of the present invention and are generally combined with pharmaceutically acceptable carriers, including any carrier that does not itself induce antibodies harmful to the individual receiving the composition. Suitable carriers are typically large, slowly metabolizing macromolecules such as proteins, polysaccharides, polylactic acid, polyglycolic acid, amino acid polymers, amino acid copolymers, lipid aggregates (such as oil droplets or liposomes), etc. These carriers are well known to those skilled in the art. Additionally, these carriers can act as immunostimulants (“adjuvants”). Furthermore, the antigen may also be conjugated with bacterial toxoids (such as toxoids of pathogens such as diphtheria, tetanus, cholera, and Helicobacter pylori).

[0146] Preferred adjuvants for enhancing the efficacy of immune compositions include, but are not limited to: (1) aluminum salts, such as aluminum hydroxide, aluminum phosphate, aluminum sulfate, etc.; (2) oil-in-water emulsion formulations, for example, (a) MF59 (see WO 90 / 14837), (b) SAF, and (c) the Ribi adjuvant system (RAS). (3) Saponin adjuvants; (4) Freund complete adjuvants (CFA) and Freund incomplete adjuvants (IFA); (5) Cytokines such as interleukins (e.g., IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, etc.), interferons (e.g., interferon), macrophage colony-stimulating factor (M-CFS), tumor necrosis factor (TNF), etc.; (6) Detoxified variants of bacterial ADP-ribosylated toxins (e.g., cholera toxin CT, pertussis toxin PT, or Escherichia coli heat unstable toxin LT), see, for example, WO93 / 13302 and WO92 / 19265; and (7) Other substances that enhance the effect of the composition as immunostimulants.

[0147] Vaccine compositions, including immunogenic compositions (e.g., may include antigen-antibody complexes, pharmaceutically acceptable carriers, and adjuvants), typically contain diluents such as water, saline, glycerol, ethanol, etc. Additionally, auxiliary substances such as wetting agents or emulsifiers, pH buffers, etc., may be present in these carriers.

[0148] More specifically, vaccines, including immunogenic compositions, contain an immunologically effective amount of an immunogenic peptide, as well as the other required components mentioned above. An "immunologically effective amount" refers to the amount administered to an individual as a single or partial dose that is effective for treatment or prevention. This dosage can be determined based on the individual's health and physiological condition, the individual's class (e.g., human), the individual's immune system's ability to synthesize antibodies, the required level of protection, the vaccine formulation, the treating physician's assessment of the medical condition, and other relevant factors. This dosage is expected to be within a relatively wide range and can be determined through routine laboratory testing.

[0149] Typically, vaccine compositions or immunogenic compositions can be formulated as injectable preparations, such as liquid solutions or suspensions; they can also be formulated as solid forms suitable for reconstitution into solutions or suspensions or liquid excipients prior to injection. The formulation may also be emulsified or encapsulated in liposomes to enhance adjuvant effects.

[0150] (iii) Route of administration and dosage

[0151] The composition can be administered directly to a subject. The subject can be a human or a non-human mammal, preferably a human. When used as a vaccine, the antigen-antibody complex of the present invention can be administered directly to an individual using known methods. These vaccines are typically administered via the same route of administration as conventional vaccines and / or by mimicking the pathogen infection pathway.

[0152] The routes of administration for the pharmaceutical or vaccine compositions of the present invention include (but are not limited to): intramuscular, subcutaneous, intradermal, intrapulmonary, intravenous, nasal, vaginal, oral, or other parenteral routes. Routes of administration may be combined if necessary, or adjusted according to the disease condition. The vaccine compositions may be administered in single or multiple doses, and may include booster doses to induce and / or maintain immunity.

[0153] The antigen-antibody complex of the present invention should be administered in an "effective amount," meaning that the amount of the antigen-antibody complex is sufficient to elicit an immune response in the selected route of administration, effectively inducing an immune response in patients with chronic hepatitis B; and / or preventing and / or treating chronic hepatitis B.

[0154] The amount of antigen-antibody complex selected in each vaccine dose is determined based on the amount that can elicit a protective immune response without significant side effects. Typically, after infection of host cells, each dose of vaccine is sufficient to contain approximately 1 μg-1000 μg, preferably 1 μg-100 μg, and more preferably 10 μg-50 μg of antigen-antibody complex. The optimal dosage of a specific vaccine can be determined using standard research methods, including antibody titers and other responses in the subjects. Whether a booster dose is needed can be determined by monitoring the level of immunity provided by the vaccine. After assessing the antibody titer in the serum, a booster dose immunization may be necessary. Administration of adjuvants and / or immunostimulants can enhance the immune response to the antigen-antibody complex of the present invention. A preferred method is to administer the antigen-antibody complex via injection via a parenteral (subcutaneous or intramuscular) route.

[0155] English abbreviations

[0156] Example 1: Production of Monoclonal Antibodies for Different Epitopes of Surface Antigens

[0157] The heavy chain VH+CH1 nucleotide sequence of the G12 anti-HBsAg monoclonal antibody (anti-small HBsAg) is SEQ ID NO:1, and the light chain VL+CL nucleotide sequence is SEQ ID NO:2.

[0158] The nucleotide sequence of the heavy chain VH+CH1 of the KR127 anti-HBsAg monoclonal antibody (anti-pre-S1) is SEQ ID NO:3, and the nucleotide sequence of the light chain VL+CL is SEQ ID NO:4.

[0159] The plasmid construction of the antibodies used in this invention is as follows:

[0160] Step 1: The heavy and light chain sequences of monoclonal antibodies G12 and KR127 were synthesized into eukaryotic expression vectors (pcDNA3.1) using Genscript.

[0161] Step 2: The heavy chain sequence of the product from Step 1 was spliced ​​with the sequences of human IgG1 type Fc (nucleotide sequence SEQ ID NO:5), mouse IgG1 type Fc (nucleotide sequence SEQ ID NO:6), and IgG2a type Fc (nucleotide sequence SEQ ID NO:7) via overlap PCR. The spliced ​​products were then constructed into the eukaryotic expression vector pTT5 (sequence shown in SEQ ID NO:8) via homologous recombination. All of the aforementioned sequences contain a hinge region, CH2, and CH3.

[0162] Step 3: Transform the recombinant products from Steps 1 and 2 into DH5α competent cells. Pick a single colony from an overnight ampicillin plate and culture it in LB broth. Then, send the bacterial culture for sequencing to obtain plasmids: G12-hIgG1-HC, G12-mIgG1-HC, G12-mIgG2a-HC, G12 light chain (G12-LC), KR127-hIgG1-HC, KR127-mIgG1-HC, KR127-mIgG2a-HC, and KR127 light chain (KR127-LC).

[0163] The preparation of anti-HBsAg monoclonal antibodies was basically carried out according to the literature (Cell Host Microbe. 2017. 22(4): 471-483.e5.), and the preparation process is as follows:

[0164] Step 1: Using the HEK293F mammalian suspension cell expression system, express the following six antibodies respectively:

[0165] G12-hIgG1: SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:5 spliced ​​sequence.

[0166] KR127-hIgG1: SEQ ID NO:3 and SEQ ID NO:4 and SEQ ID NO:5 spliced ​​sequence.

[0167] G12-mIgG1: SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:6 spliced ​​sequence.

[0168] G12-mIgG2a: SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:7 spliced ​​sequence.

[0169] KR127-mIgG1: SEQ ID NO:3 and SEQ ID NO:4 and SEQ ID NO:6 spliced ​​sequence.

[0170] KR127-mIgG2a: SEQ ID NO:3 and SEQ ID NO:4 and SEQ ID NO:7 spliced ​​sequence.

[0171] The heavy chain plasmid HC, light chain plasmid LC, and transfection reagent (branched polyethyleneimine (PEI), MB2603-1) were mixed at a mass ratio of 1:1:6, allowed to stand at room temperature, and then poured into HEK293F cells for culture.

[0172] Step 2: Centrifuge the cells from Step 1, collect the supernatant and filter it. Equilibrate the supernatant with equilibration buffer (1M PBS buffer) through Protein G Resin resin. Add the supernatant to the resin and control the flow rate at 1 mL / min. Elute with 2 column volumes of equilibration buffer to remove contaminating proteins, and finally elute bound immunoglobulins with 0.1M glycine (pH=2.8).

[0173] Step 3: Transfer the immunoglobulin solution eluted in the previous steps to a 3kD MWCO Amicon Ultra centrifuge ultrafiltration tube, ultrafilter, and replace the elution buffer with PBS solution. Collect the concentrated antibodies, which are human Fc chimeric monoclonal antibodies G12-hIgG1 and KR127-hIgG1 and mouse Fc chimeric monoclonal antibodies G12-mIgG1, G12-mIgG2a, KR127-mIgG1, and KR127-mIgG2a.

[0174] The absorbance of the antibody at 280 nm in step 3 was measured using a spectrophotometer to quantify the protein concentration, and the purity was detected by SDS-PAGE electrophoresis, as shown in Figure 1. This method was used to prepare the antigen-antibody complex (Therapeutic vaccine, TVac).

[0175] Example 2 Production of Mouse Anti-Hepatitis B Immunoglobulin (mHBIG)

[0176] Six- to eight-week-old male C57BL / 6 SPF mice were treated with a mixture of hepatitis B virus surface antigen protein HBsAg expressed and purified in stable transgenic CHO cells and aluminum hydroxide adjuvant at a dose of 100 μg per mouse.

[0177] Immunize with the above-mentioned mixed immunogen, 2 μg / animal, once every two weeks, for a total of 3 immunizations.

[0178] Subsequently, blood was collected from mice after the second and third immunizations. The supernatant was centrifuged and serum was obtained. The serum titer was detected by ELISA using the hepatitis B virus surface antigen protein mentioned above. The serum was then purified to obtain anti-mouse hepatitis B immunoglobulin (mHBIG), as shown in Figure 1.

[0179] Example 3: Affinity test of antibody with hepatitis B surface antigen (HBsAg)

[0180] The binding kinetics of antibodies (mHBIG and G12-hlgG1) to HBsAg were detected using bio-layer interferometry (BLI) on an Octet-RED96. The activated biosensor (AR2G) was first loaded with 15 mg / mL HBsAg for 600 s, followed by quenching for 300 s. The sensor was then incubated with three-fold diluted antibodies (1000 nM (mHBIG) or 50 nM (G12-hlgG1)) in PBST (PBS containing 0.02% Tween 20) for 300 s, followed by dissociation in PBST for 300 s. All curves were fitted using a 1:2 binding model with Data Analysis software. KD values ​​were determined with R² values ​​greater than the 95% confidence interval.

[0181] The BLI results, as shown in Figures 3 and 4, indicate that the monoclonal antibody (G12-hIgG1) has a better affinity for hepatitis B surface antigen (HBsAg) than the polyclonal antibody (anti-mouse hepatitis B immunoglobulin (mHBIG)).

[0182] Example 4: Binding affinity test of antibody to hepatitis B surface antigen (HBsAg)

[0183] Hepatitis B surface antigen (HBsAg) was coated onto an ELISA plate with PBS, 50 μg / well, and incubated overnight at 4°C. Then, antibodies from Examples 1 and 2 (containing anti-hepatitis B immunoglobulin (mHBIG), G12-hIgG1, KR127-hIgG1, G12-mlgG1, G12-mIgG2a, KR127-mIgG1, and KR127-mIgG2a) were added sequentially at an initial concentration of 1 μM and diluted 3-fold. Goat anti-mouse IgG or anti-human IgG-Fc-tagged antibodies were then added to detect the binding affinity of each antibody to the surface antigen (see Cell Host Microbe. 2020. 27(6): 891-898.e5. for details).

[0184] As shown in Figure 2, the ELISA results indicate that the monoclonal antibody (G12-hIgG1) binds to hepatitis B surface antigen (HBsAg) more readily than the polyclonal antibody (anti-hepatitis B immunoglobulin (HBIG)).

[0185] Figures 5 and 6 show that the different antibodies G12-hIgG1, KR127-hIgG1, G12-mlgG1, G12-mIgG2a, KR127-mIgG1 and KR127-mIgG2a generated in Example 1 all have the activity of binding to hepatitis B surface antigen (HBsAg) and the binding activities are not significantly different.

[0186] Example 5: Preparation of a mouse model of chronic hepatitis B virus infection

[0187] 6-8 week old male C57BL / 6 SPF mice were treated with rAAV8-1.3HBV-ayw (titer 1×10⁻⁶). 12 2×10 μg / mL) tail vein injection 10 μg / animal, with continuous infection for two weeks to establish an animal model of persistent infection.

[0188] Example 6: Immunization using an immunogenic antigen-antibody complex formulation

[0189] Clinical combination therapy for chronic hepatitis B includes combination therapy strategies. This embodiment uses the antigen-antibody complex of the present invention as an immunomodulator to select a combination therapy strategy, and implements it in a mouse model. The steps are as follows:

[0190] Step 1: Use antiviral drugs to inhibit viral replication and reduce serum viral load throughout the entire treatment process;

[0191] Step 2: Administer the first dose of an immunomodulator to reduce serum HBsAg levels;

[0192] Step 3: Administer a second dose of immunomodulator to enhance and induce an effective host immune response.

[0193] The antiviral drugs mentioned in step 1 include nucleotide reverse transcriptase inhibitors, nucleos(t)ide analogs (NA), capsid assembly regulators, antisense oligonucleotides, and invasion inhibitors. In this embodiment, a nucleotide reverse transcriptase inhibitor is used. The nucleotide reverse transcriptase inhibitor can be any or a combination of anti-hepatitis B virus drugs such as lamivudine (generally known as 3TC), telbivudine, adefovir dipivoxil, entecavir (ETV), tenofovir disoproxil fumarate (TDF), tenofovir alafenamide fumarate (TAF), or tenofovir amibufenamide (TMF).

[0194] The immunomodulators used in step 2 are selected from pegylated interferon alpha (Peg-IFN-α), polyclonal antibodies, monoclonal antibodies, targeted siRNA, small interfering RNA, or HBsAg inhibitors.

[0195] The immunomodulator used in step 3 is the antigen-antibody complex formulation of the present invention.

[0196] 6-1 Preparation of antigen-antibody complex (TVac) as a second immunomodulator

[0197] Take purified hepatitis B virus surface antigen at a mass ratio of at least greater than 0.67:1 and mix it separately with G12-hIgG1, G12-mIgG1, G12-mIgG2a, KR127-hIgG1, KR127-mIgG1, KR127-mIgG2a antibodies or anti-mouse hepatitis B immunoglobulin (mHBIG) produced in Examples 1 and 2. After incubation overnight, add 100 μg of aluminum hydroxide adjuvant (other commercially available hepatitis B vaccine adjuvants such as CpG1080, aluminum phosphate adjuvant, or a mixture of aluminum hydroxide and aluminum phosphate are also applicable) to produce an antigen-antibody complex formulation targeting different epitopes of hepatitis B virus surface antigen.

[0198] The antigen-antibody complex formulation prepared from anti-mouse hepatitis B immunoglobulin complex is TVac-mHBIG (containing hepatitis B surface antigen HBsAg and mouse hepatitis B immunoglobulin mHBIG from Example 2); the antigen-antibody complex formulation prepared from G12-hIgG1 is TVac-G (containing hepatitis B surface antigen HBsAg and G12-hIgG1 antibody expressed in Example 1); the antigen-antibody complex formulation prepared from G12-mIgG1 is TVac-G12-mIgG1 (or TVac-G-mIgG1) (containing hepatitis B surface antigen HBsAg and G12-mIgG1 antibody expressed in Example 1); and the antigen-antibody complex formulation prepared from G12-mIgG2a is TVac-G-2a (or TVac-G12-mIgG2a or TVac-G12-2a or TVac-G-mIgG2a) (containing hepatitis B surface antigen HBsAg). The antigen-antibody complex formulation prepared from KR127-hIgG1 is TVac-K (containing hepatitis B surface antigen HBsAg and KR127-hIgG1 antibody expressed in Example 1), the antigen-antibody complex formulation prepared from KR127-mIgG1 is TVac-KR127-mIgG1 (or TVac-K-mIgG1) (containing hepatitis B surface antigen HBsAg and KR127-mIgG1 antibody expressed in Example 1), and the antigen-antibody complex formulation prepared from KR127-mIgG2a is TVac-K-2a (or TVac-KR127-IgG2a or TVac-KR127-2a or TVac-K-mIgG2a) (containing hepatitis B surface antigen HBsAg and KR127-mIgG2a antibody expressed in Example 1).

[0199] 6-2 The effects of antigen-antibody complex preparations made from G12 monoclonal antibodies and antigen-antibody complex preparations made from anti-hepatitis B immunoglobulin as second-dose immunomodulators.

[0200] Step 1: In a mouse model of chronic hepatitis B (described in Example 5), tenofovir disoproxil fumarate (TDF) was administered via gavage for 5 consecutive days at a dose of 15 mg / kg.

[0201] Step 2: Half an hour after administering TDF on day 6, the first dose of immunomodulator was injected via the tail vein. The first dose of immunomodulator was G12 monoclonal antibody (SEQ ID NO:1 and SEQ ID NO:2 and SEQ ID NO:5 spliced ​​sequences) synthesized from the literature (mAbs vol.8,3(2016):468-77.), 6.7 mg / kg, to reduce the surface antigen level;

[0202] Step 3: One hour after completing Step 2, a second dose is administered intraperitoneally, consisting of an immunomodulatory antigen-antibody complex (TVac-G and TVac-mHBIG) and hepatitis B surface antigen HBsAg. Immunization is administered every two weeks for a total of three doses. The experimental groups in this example are as follows:

[0203] PBS: The control group received only PBS;

[0204] TDF+G12: A combination of antiviral drugs and the first-dose immunomodulatory agent G12 monoclonal antibody;

[0205] TDF+G12+HBsAg: A combination of antiviral drugs, the first dose of the immunomodulatory agent G12 monoclonal antibody, and the second dose of hepatitis B surface antigen HBsAg.

[0206] TDF+G12+TVac-G: A combination of antiviral drugs, a first-dose immunomodulatory agent G12 monoclonal antibody, and a second-dose TVac-G (an antibody complex of hepatitis B surface antigen HBsAg and G12-hIgG1 expressed in Example 1);

[0207] TDF+G12+TVac-mHBIG: A combination of antiviral drugs, a first dose of the immunomodulatory agent G12 monoclonal antibody, and a second dose of TVac-mHBIG (an antibody complex of hepatitis B surface antigen HBsAg and mHBIG from Example 2).

[0208] Figure 7 shows the antibody expression levels in mice one week after the first immunization. The results showed that mice in the PBS, TDF+G12, and TDF+G12+HBsAg groups did not produce antibodies (HBsAb); mice in the TDF+G12+TVac-G and TDF+G12+TVac-mHBIG groups produced antibodies (HBsAb). The antibody (HBsAb) expression level induced by the monoclonal antibody-prepared antigen-antibody complex formulation (TDF+G12+TVac-G) was more than 1 times higher than that induced by the polyclonal antibody-prepared antigen-antibody complex formulation (TDF+G12+TVac-mHBIG).

[0209] 6-3 Antigen-antibody complex formulations prepared with different ratios of antigen and antibody induce antibody levels.

[0210] Following the procedure in section 6-1, "Preparation of Complex (TVac) as a Second Immunomodulator," antigen-antibody complex formulations with different antigen-antibody ratios were prepared for use as second immunomodulators. Serum antibody (HBsAb) levels were detected by ELISA at 7 and 35 days after administration of the second immunomodulator. The experimental groups in this example are as follows:

[0211] TVac-mHBIG: The second dose of immunomodulatory agent was an anti-hepatitis B immunoglobulin complex preparation, in which the antibody:antigen mass ratio was 1:54, 1:18, 1:6, 1:2 and 1:0.67, respectively;

[0212] TVac-G12-2a: The second dose of immunomodulatory agent is a complex preparation of G12-mIgG2aFc monoclonal antibody, wherein the antibody:antigen mass ratios are 1:54, 1:18, 1:6, 1:2 and 1:0.67, respectively.

[0213] TVac-KR127-2a: The second dose of immunomodulatory agent is a complex preparation made from KR127-mIgG2aFc monoclonal antibody, wherein the antibody:antigen mass ratios are 1:54, 1:18, 1:6, 1:2 and 1:0.67, respectively.

[0214] The results are shown in Figures 8, 9, 10, and 11. When the antibody-to-antigen ratio of the complex formulation was 1:6, the antibody-to-antigen complex formulations (TVac-G12-2a and TVac-KR127-2a) prepared by monoclonal antibodies G12-mIgG2aFc and KR127-mIgG2aFc induced antibody levels more than twice that of the polyclonal antibody-to-antigen complex formulation (TVac-mHBIG) on days 7 and 35. When the antibody or antigen content of the complex formulation was excessive, the antibody levels induced by the antigen-to-antigen complex formulations prepared by both monoclonal and polyclonal antibodies did not show a significant increase on days 7 and 35. Figures 10 and 11 show that, after long-term observation and administration of the same dose of antigen-to-antigen complex formulation, the antibody-to-antigen complex formulations prepared by monoclonal antibodies (TVac-G12-2a and TVac-KR127-2a) were significantly superior to the polyclonal antibody-to-antigen complex formulation (TVac-mHBIG) in sustaining the induction of immune responses in mice.

[0215] The results showed that antigen-antibody complex formulations prepared with monoclonal antibodies (G12 or KR127) were superior to antigen-antibody complex formulations prepared with polyclonal antibodies (HBIG) in treating or inducing an immune response to chronic hepatitis B.

[0216] 6-4 The effect of suppressing the level of hepatitis B surface antigen by antigen-antibody complex preparations with different ratios.

[0217] Following the procedure in section 6-1, preparations of antigen-antibody complex formulations with different antigen-antibody ratios were made to serve as second-dose immunomodulators. In a mouse model of chronic hepatitis B, step 1 involved administering tenofovir disoproxil fumarate (TDF) via gavage for 5 consecutive days at 15 mg / kg. Subsequently, on days 0, 14, and 28, the first-dose immunomodulator—synthetic G12 monoclonal antibody (G12)—and the second-dose immunomodulators—TVac-mHBIG, TVac-G12-2a, and TVac-KR127-2a—were administered, respectively.

[0218] The results, as shown in Figures 12, 13, 14, and 15, revealed different trends in the inhibition of hepatitis B surface antigen (HBsAg) expression by antigen-antibody complexes with varying ratios. On day 14 after the second immunization, the lowest HBsAg expression level was observed when the antibody-to-antigen ratio of each complex formulation was 1:6. As shown in Figure 15, when the antibody-to-antigen ratio of the complex formulation was 1:6, the HBsAg level after administration of the monoclonal antibody complex formulations (TVac-G-mIgG2a and TVac-K-mIgG2a) was significantly lower than that after administration of TVac-mHBIG (on day 5, the HBsAg level of the monoclonal antibody complex formulation was 2 IU / mL, while that of the polyclonal antibody complex formulation had risen to 3 IU / mL).

[0219] As shown in Figures 13 and 14, when TDF and the first dose of immunomodulator G12 were used to treat mice with chronic hepatitis B, the serum HBsAg expression level was reduced to ≤20 IU / mL. Based on this, a second dose of immunomodulator was administered, and even better results were observed. For patients with chronic hepatitis B (humans), when TDF and the first dose of immunomodulator G12 were used for treatment, a second dose of immunomodulator was administered when the HBsAg level decreased to approximately ≤400 IU / mL. According to the expert consensus on the clinical application of hepatitis B virus markers in the "Guidelines for the Prevention and Treatment of Chronic Hepatitis B (2023 Edition)," after medication, when the baseline HBsAg level is below 200 IU / mL or the HBsAg decrease is greater than 1 log... 10 IU / mL is a positive factor for achieving clinical cure. This invention uses a combination of TDF and the first dose of the immunomodulator G12, followed by a second dose of the monoclonal antibody antigen-antibody complex (TVac-G12-2a). After medication, the baseline HBsAg level is below 200 IU / mL or the HBsAg decrease is greater than 1 log. 10 IU / mL.

[0220] Immunological effects of 6-5 different epitope monoclonal antibody complex formulations

[0221] In a mouse model of chronic hepatitis B, step 1 involved administering tenofovir disoproxil fumarate (TDF) via gavage for 5 consecutive days at 15 mg / kg. Subsequently, on days 0, 14, and 28, the mouse was given the first and second doses of the synthesized G12 monoclonal antibody (G12) immunomodulatory complex (TVac) preparation. The preparation method was as described in 6-1 to obtain the antibody complex preparation (TVac): TVac-G, TVac-K, TVac-G-mIgG1, TVac-G-mIgG2a, TVac-K-IgG1, and TVac-K-mIgG2a. PBS served as the control group.

[0222] As shown in Figures 16 and 17, the different monoclonal antibody complex formulations (TVac) exhibited similar trends in inhibiting the expression of hepatitis B surface antigen (HBsAg). After three immunizations, the expression level of HBsAg in mouse serum steadily decreased. Figures 18 and 19 show the trend of antibody (HBsAb) changes in induced immunized mice. Antibodies (HBsAb) were not detected in the control group using PBS. After completing steps 2 and 3 on day 14, the expression level of antibodies (HBsAb) in mice steadily increased. Even after completing steps 2 and 3 on day 28, high-expression antibodies (HBsAb) were still detectable in mice for more than 50 days. Furthermore, the results of this example show that the IgG2a subtype has a better effect on inhibiting antigen and inducing antibody expression than the IgG1 subtype.

[0223] Expression levels of humoral and cellular immune factors after 6-6 sequential combination therapy

[0224] On day 83 after the completion of Example 6-5, tail vein blood was collected from mice, and flow cytometry was used to detect changes in the expression levels of humoral and cellular immune factors in mice after sequential combination therapy.

[0225] [Corrected according to Rule 91, July 15, 2025] Figures 20-24 are schematic diagrams of the results of humoral immune factor expression levels in mice, and Figures 25-32 are schematic diagrams of the results of cellular immune factor expression levels. The results show that immunization with the antigen-antibody complex preparation (TVac) significantly induced humoral and cellular immune responses against HBsAg.

[0226] Example 7: Effects of combined HBsAg-targeting siRNA on serum HBsAg, HBV DNA, and HBsAb levels

[0227] This embodiment uses the same immunization method as in Example 6, the difference being that the first immunomodulator in this embodiment uses siRNA targeting HBsAg. The experimental groups are as follows:

[0228] PBS: The control group received only PBS;

[0229] TDF+G12+TVac-G: The first immunomodulatory dose is a synthetic G12 monoclonal antibody (G12), and the second immunomodulatory dose is a TVac-G antigen-antibody complex preparation.

[0230] TDF+siRNA+TVac-G: The first immunomodulatory dose is siRNA targeting HBsAg (HT101, a clinical drug from Suzhou Xingyao Kunze Company), and the second immunomodulatory dose is TVac-G antigen-antibody complex preparation.

[0231] TDF+G12+siRNA+TVac-G: The first immunomodulatory dose administers a synthetic G12 monoclonal antibody (G12) and siRNA targeting HBsAg, and the second immunomodulatory dose administers a TVac-G antigen-antibody complex formulation.

[0232] In step 1, the TDF dose is 15 mg / kg, injected every other day; in step 2, the first immunomodulator is a synthetic G12 monoclonal antibody (G12) at a dose of 6.7 mg / kg, and the siRNA targeting HBsAg is administered subcutaneously at a dose of 3 mg / kg, following the synthetic G12 monoclonal antibody; in step 3, the second immunomodulator is a TVac-G antigen-antibody complex preparation administered intraperitoneally at a dose of 4 μg; the first and second immunomodulators are administered on days 0, 14, and 28, respectively.

[0233] [Corrected according to Rule 91 15.07.2025] Regarding serum HBsAg expression levels, as shown in Figure 33, the expression level of HBsAg in the serum of mice after the first immunization (day 0) showed a recovery trend when only G12 monoclonal antibody was administered as the first immunomodulator. However, the expression level of HBsAg in the serum of mice in the siRNA-administered groups (TDF+siRNA+TVac-G and TDF+G12+siRNA+TVac-G) continued to decrease.

[0234] [Correction 15.07.2025 based on Rule 91] Regarding the inhibition of HBV DNA, as shown in Figure 34, the groups given the antigen-antibody complex formulation (TDF+G12+TVac-G, TDF+siRNA+TVac-G and TDF+G12+siRNA+TVac-G) have the function of continuously inhibiting HBV DNA expression.

[0235] [Corrected according to Rule 91 15.07.2025] Regarding HBsAb, as shown in Figure 35, the groups given antigen-antibody complex formulations (TDF+G12+TVac-G, TDF+siRNA+TVac-G, and TDF+G12+siRNA+TVac-G) showed better antibody expression than the control group PBS; the antibody expression induced by the first dose of immunomodulator simultaneously administered with monoclonal antibody G12 and siRNA (TDF+G12+siRNA+TVac-G) was better than that of other groups.

[0236] Other interfering RNA molecules that target HBsAg, such as microRNA and shRNA, also have similar effects to siRNA and can be applied to this invention with similar results.

[0237] miRNAs (microRNAs) are a group of non-coding RNAs, approximately 20-23 nucleotides in length, encoded by the genome. They guide the silencing complex (RISC) to degrade mRNA or inhibit its translation by pairing with the bases of target gene mRNA. shRNAs (short hairpin RNAs) are short double-stranded RNA structures (19-25 nt) produced in dependence of stem-loop sequences. They can be introduced into cells using vectors, where they are cleaved by enzymes to form siRNA, which regulates target genes through the RNA interference pathway. Unlike siRNAs, shRNAs are synthesized in the cell nucleus. After transient or stable transduction, shRNA vectors first enter the cell nucleus, where they are expressed and processed by the Drosha / DGCR8 complex to form pre-shRNA. This pre-shRNA is then transported to the cytoplasm by the Exportin-5 protein, where the circular sequence is removed by the Dicer complex to form siRNA. It then binds to the RISC (RNA-induced silencing complex) and releases one of its RNA strands. The complex recognizes the mRNA, leading to its degradation. It is evident that the processing pathway of shRNA after exiting the nucleus is essentially the same as that of dsRNA, ultimately forming the siRNA structure. siRNA is usually exogenous, while miRNA is endogenous. Mature (double-stranded) miRNA is loaded onto a RISC, and after one strand is removed, the RISC carrying antisense RNA...

[0238] These small RNAs bind to mRNA and inhibit target gene expression through mRNA degradation and translation repression pathways, respectively. Different types of small RNAs result in different types of RISCs. The core enzyme in RISCs is the Argonaute enzyme of the AGO protein family, which has multiple members in most species. In Drosophila and human cells, miRNAs bind to AGO-1 proteins to produce non-cleaved RISCs, interfering with target genes by inhibiting mRNA translation; siRNAs bind to AGO-2 proteins to produce cleaved RISCs, interfering with target genes by cleaving and degrading mRNA. Structurally, shRNAs and miRNAs are more similar; in fact, shRNAs are functionally closer to siRNAs. shRNAs are cleaved by the Dicer enzyme in the cell to form siRNAs, which perform their interference function through the siRNA pathway, while miRNAs regulate target genes through a different pathway.

[0239] Example 8: Difference in antibody levels between the present invention and other combination therapies using immunomodulators.

[0240] This embodiment uses a combination therapy strategy with different immunomodulators, administered to the patients. Specific experimental groups, medication regimens, and information are as follows:

[0241] PDL1: Commercially available atezolizumab monoclonal antibody; TLR8: Toll-like receptor 8 agonist GS-9688; IFN-α: Alpha interferon; FAP-IL2: An immune cytokine comprising an antibody against fibroblast activation protein α (FAPα) and an IL-2 variant that binds only to IL-2Rβγ; PD1: Anti-mouse CD279 monoclonal antibody; PDL1-IFNα+HBsAg / CpG: PDL1-IFNα heterodimer fusion protein synthesized according to the reference (Meng CY, et al. Gut 2023; 72:1544-1554); HBsAg was purchased from North China Pharmaceutical Group, and CpG is a Toll-like receptor 9 agonist purchased from MedChemexpress Biotechnology, Inc., USA.

[0242] TDF+G12+PDL1: In a mouse model of chronic hepatitis B (described in Example 5), tenofovir disoproxil fumarate (TDF) was administered for 5 consecutive days via gavage (15 mg / kg). After day 6, the drug was administered every other day. On day 6, synthetic G12 monoclonal antibody (tail vein, 6.7 mg / kg) was administered. One hour later, PDL1 (T drug) (tail vein, 5 mg / kg) was administered. G12 and PDL1 were administered every two weeks for a total of 3 times.

[0243] TDF+G12+TLR8: In a mouse model of chronic hepatitis B (described in Example 5), tenofovir disoproxil fumarate (TDF) was administered for 5 consecutive days (by gavage, 15 mg / kg). After day 6, the drug was administered every other day. On day 6, synthetic G12 monoclonal antibody (tail vein, 6.7 mg / kg) was administered. One hour later, TLR8 (by gavage, 3 mg / kg) was administered. G12 was administered every two weeks for a total of 3 times, and TLR8 was administered weekly for a total of 5 times.

[0244] TDF+G12+TVac-G-mIgG2a: In a mouse model of chronic hepatitis B (described in Example 5), tenofovir disoproxil fumarate (TDF) was administered for 5 consecutive days (by gavage, 15 mg / kg). After day 6, the drug was administered every other day. On day 6, the first immunomodulator, synthetic G12 monoclonal antibody (tail vein, 6.7 mg / kg), was administered. One hour later, the second immunomodulator, TVac-G-mIgG2a antigen-antibody complex, was administered. G12 and TVac-G-mIgG2a were administered every two weeks for a total of 3 times.

[0245] TDF+G12+IFNα: In a mouse model of chronic hepatitis B (described in Example 5), tenofovir disoproxil fumarate (TDF) was administered for 5 consecutive days via gavage (15 mg / kg). After day 6, the drug was administered every other day. On day 6, synthetic G12 monoclonal antibody (tail vein, 6.7 mg / kg) was administered. One hour later, IFNα (intraperitoneal, 30 μg / kg) was administered. G12 was administered every two weeks for a total of 3 times, and IFNα was administered weekly for a total of 5 times.

[0246] TDF+G12+FAP-IL2: In a mouse model of chronic hepatitis B (described in Example 5), tenofovir disoproxil fumarate (TDF) was administered for 5 consecutive days via gavage (15 mg / kg). After day 6, the drug was administered every other day. On day 6, synthetic G12 monoclonal antibody (tail vein, 6.7 mg / kg) was administered. One hour later, FAP-IL2 (tail vein, 2 mg / kg) was administered. G12 and FAP-IL2 were administered every two weeks for a total of 3 times.

[0247] siRNA+G12+IFNα: On day 0, siRNA targeting HBsAg (HT101, a clinical drug from Suzhou Xingyao Kunze Co., Ltd., subcutaneous, 3 mg / kg) was administered, along with G12 monoclonal antibody (tail vein, 6.7 mg / kg). One hour later, IFNα (intraperitoneal, 30 μg / kg) was administered. siRNA and G12 were administered every two weeks for a total of 3 times, and IFNα was administered weekly for a total of 5 times.

[0248] siRNA+TLR8+TDF: In a mouse model of chronic hepatitis B (described in Example 5), tenofovir disoproxil fumarate (TDF) was administered for 5 consecutive days (by gavage, 15 mg / kg). After day 6, the drug was administered every other day. On day 6, siRNA targeting HBsAg (HT101, a clinical drug from Suzhou Xingyao Kunze Co., Ltd., administered subcutaneously, 3 mg / kg) was given. One hour later, TLR8 was administered (by gavage, 3 mg / kg). siRNA was administered every two weeks for a total of 3 times, and TLR8 was administered weekly for a total of 5 times.

[0249] siRNA+PD1+TDF: Tenofovir disoproxil fumarate (TDF) (15 mg / kg by gavage) was administered to a mouse model of chronic hepatitis B (described in Example 5) for 5 consecutive days. After day 6, it was administered every other day. On day 6, siRNA targeting HBsAg (HT101, a clinical drug from Suzhou Xingyao Kunze Co., Ltd., 3 mg / kg subcutaneously) was administered. One hour later, PD1 (5 mg / kg via tail vein) was administered. siRNA and PD1 were administered every two weeks for a total of 3 times.

[0250] PDL1-IFNα+HBsAg / CpG: Synthetic anti-PDL1-IFNα homologous fusion protein (Meng CY, et al. Gut, 2023(72):1544-1554) (tail vein, 0.2 mg / kg) was administered on days 0 and 3, and HBsAg / CpG (subcutaneous, 2 μg / 30 μg) was administered on days 3, 10, 17, and 24.

[0251] PBS: The control group received only PBS.

[0252] [Correction based on Rule 91, July 15, 2025] Regarding HBsAb, as shown in Figure 36, serum was collected on day 21 after combined treatment. The results showed that the antibody level produced in the TDF+G12+TVac-G-mlgG2a group was much higher than that in all other combined treatment groups and the difference was extremely significant, indicating that the combined treatment strategy of the present invention can stimulate the body to produce the corresponding antibodies more quickly.

[0253] Example 9: The effect of the G12 monoclonal antibody in the complex vaccine of the present invention on HBsAg clearance.

[0254] This embodiment compares the effect of the G12 monoclonal antibody in the antigen-antibody complex of the present invention on clearing HBsAg and DNA with other hepatitis B monoclonal antibodies in a mouse model.

[0255] The information and treatment regimen for monoclonal antibodies are as follows:

[0256] KR127: A monoclonal antibody targeting the pre-S1 epitope. The nucleotide sequence of the heavy chain VH+CH1 of KR127 is SEQ ID NO:3, and the nucleotide sequence of the light chain VL+CL is SEQ ID NO:4.

[0257] G12: A monoclonal antibody targeting the small S epitope. The nucleotide sequence of the heavy chain VH+CH1 of G12 is SEQ ID NO:1, and the nucleotide sequence of the light chain VL+CL is SEQ ID NO:2.

[0258] VIR-3434: A monoclonal antibody targeting the small S epitope. The amino acid sequence of the VH heavy chain of VIR-3434 is SEQ ID NO:9, and the amino acid sequence of the VL light chain is SEQ ID NO:10.

[0259] A15: A monoclonal antibody targeting the small S epitope. The nucleotide sequence of the heavy chain VH+CH1 of A15 is SEQ ID NO:11, and the nucleotide sequence of the light chain VL+CL is SEQ ID NO:12.

[0260] The aforementioned antibodies were administered as a single dose on day 0, at a concentration of 6.7 mg / mL, via tail vein injection. Blood samples were collected from the retroorbital sinus of mice to detect HBsAg and DNA levels. PBS served as the control group.

[0261] [Corrected according to Rule 91, July 15, 2025] Regarding the inhibition of HBsAg expression levels, as shown in Figure 37, all monoclonal antibodies showed the strongest inhibitory effect 5 days after treatment, with the G12 monoclonal antibody achieving an average HBsAg reduction of 2 log. 10 IU / mL is the optimal concentration for best results.

[0262] [Correction based on Rule 91, July 15, 2025] Regarding the inhibition of HBV DNA, as shown in Figure 38, the G12 monoclonal antibody showed the best effect, with an average DNA reduction of 1000 copies / mL on day 5 after treatment.

Claims

1. An antigen-antibody complex for inducing immune response in a patient with chronic hepatitis B, the complex comprising a hepatitis B surface antigen and an anti-HBsAg monoclonal antibody, the hepatitis B surface antigen being bound to the anti-HBsAg monoclonal antibody; the anti-HBsAg monoclonal antibody being composed of a heavy chain and a light chain; wherein, The heavy chain comprises a heavy chain variable region domain VH, a constant region domain CH1, CH2, CH3 and a hinge region; the light chain comprises a variable region domain VL and a constant region domain CL; the amino acid sequence of the heavy chain VH+CH1 of the anti-HBsAg monoclonal antibody is selected from SEQ ID NO: 1 or SEQ ID NO: 3, and the amino acid sequence of the light chain is selected from SEQ ID NO: 2 or SEQ ID NO:

4.

2. The antigen-antibody complex of claim 1, wherein the hepatitis B surface antigen is combined with the anti-HBsAg monoclonal antibody in a non-covalent manner.

3. The antigen-antibody complex of claim 1, wherein the hepatitis B surface antigen is selected from a genetically engineered bacterial-expressed hepatitis B surface antigen (HBsAg), a mammalian cell-expressed recombinant HBsAg, an inactivated blood-derived HBsAg or a synthetic hepatitis B surface antigen.

4. The antigen-antibody complex of claim 1, wherein said hepatitis B surface antigen is a polypeptide encoded by a pre-S or S region gene, or comprises: polypeptides encoded by the S region gene, the PreS1 region gene+S region gene, the PreS2 region gene+S region gene, or the PreS1 region gene+PreS2 region gene+S region gene.

5. The antigen-antibody complex of claim 1, wherein the hinge region and the CH2 and CH3 domains of the heavy chain of the anti-HBsAg monoclonal antibody are selected from IgG1 or IgG2 and mutants thereof.

6. The antigen-antibody complex of claim 1, wherein the IgG1 is selected from human immunoglobulin IgG1 or murine IgG1 homologous to human immunoglobulin IgG1; and the IgG2 is selected from human immunoglobulin IgG2 or murine IgG2a, IgG2b, IgG2c homologous to human immunoglobulin IgG2.

7. The antigen-antibody complex of claim 1, wherein the sequence of the hinge region and the CH2 and CH3 domains of the heavy chain of the anti-HBsAg monoclonal antibody is selected from SEQ ID NO: 5-SEQ ID NO:

7.

8. The antigen-antibody complex of claim 1, wherein the mass ratio of the anti-HBsAg monoclonal antibody to the hepatitis B surface antigen is 1:0.67-1:

54.

9. The antigen-antibody complex as described in claim 1, wherein the chronic hepatitis B patient refers to someone with normal alanine aminotransferase (ALT) and HBV DNA < 2 × 10⁻⁶. 3 IU / mL or HBV DNA undetectable.

10. The antigen-antibody complex of claim 1, wherein the chronic hepatitis B patient is a chronic hepatitis B patient who receives anti-viral drug treatment and has a serum HBsAg level of <3000 IU / mL, or an HBeAg negative conversion and a HBsAg level of <1500 IU / mL.

11. A preparation method of the antigen-antibody complex of claim 1, comprising the following steps: (a) constructing a monoclonal antibody expression plasmid: constructing the heavy chain and light chain sequences of the anti-HBsAg monoclonal antibody in a eukaryotic cell expression vector; (b) preparing the monoclonal antibody: transfecting the constructed plasmid in step (a) into eukaryotic cells to obtain the anti-HBsAg monoclonal antibody after cell expression; (c) mixing the antigen and the antibody: mixing the anti-HBsAg monoclonal antibody obtained in step (b) with the hepatitis B surface antigen to obtain the antigen-antibody complex of the first aspect of the present application.

12. The method of claim 11, wherein in step (a), the anti-HBsAg monoclonal antibody heavy chain comprises a heavy chain variable region domain VH, constant region domains CH1, CH2, CH3 and a hinge region; and the light chain comprises a variable region domain VL and a constant region domain CL; the amino acid sequence of the heavy chain VH+CH1 of the anti-HBsAg monoclonal antibody is selected from SEQ ID NO: 1 or SEQ ID NO: 3, and the amino acid sequence of the light chain is selected from SEQ ID NO: 2 or SEQ ID NO:

4.

13. The method of claim 11, wherein the hinge region and CH2, CH3 domains of the heavy chain of the anti-HBsAg monoclonal antibody are selected from the hinge region and CH2, CH3 domains of the heavy chain of IgG1 or IgG2 and mutants thereof.

14. The method of claim 11, wherein the hinge region and CH2, CH3 domains of the heavy chain of the anti-HBsAg monoclonal antibody have a sequence selected from SEQ ID NO: 5- SEQ ID NO:

7.

15. The method of claim 11, wherein in step (c), the mass mixing ratio of the anti-HBsAg monoclonal antibody to the HBsAg is 1:0.67-1:

54.

16. A pharmaceutical composition comprising the antigen-antibody complex of claim 1, and a pharmaceutically acceptable carrier and / or excipient.

17. The pharmaceutical composition of claim 16, which is a single agent, a combination agent or a synergistic agent.

18. The pharmaceutical composition of claim 16, which further comprises an antiviral agent and / or an agent for reducing the level of HBsAg.

19. The pharmaceutical composition of claim 16, wherein the antiviral agent is selected from one or more of a nucleotide reverse transcriptase inhibitor, a nucleos(t)ide analogue, a capsid assembly modulator, an antisense oligonucleotide and / or an entry inhibitor; and the agent for reducing the level of HBsAg is selected from one or more of a pegylated interferon alpha, a polyclonal antibody, a monoclonal antibody, a targeted siRNA, a small interfering RNA and / or an HBsAg inhibitor.

20. The pharmaceutical composition of claim 16, wherein the antiviral agent, the agent for reducing the level of HBsAg and / or the antigen-antibody complex are packaged separately and administered sequentially to a patient.

21. A vaccine composition comprising the antigen-antibody complex of claim 1, and an immunologically acceptable carrier and / or excipient.

22. The vaccine composition of claim 21, which is monovalent or multivalent.

23. The vaccine composition of claim 21, which further comprises an adjuvant selected from a particulate and a non-particulate adjuvant.

24. An immunomodulator comprising a first immunomodulator and a second immunomodulator; the first immunomodulator is a drug that reduces HBsAg level, the second immunomodulator is the antigen-antibody complex of claim 1; the first and second immunomodulators are independently packaged.

25. The immunomodulator of claim 24, wherein the drug that reduces HBsAg level is selected from one or more of pegylated interferon alpha, polyclonal antibodies, monoclonal antibodies, targeted siRNA, small interfering RNA, and / or HBsAg inhibitors.

26. Use of the antigen-antibody complex of claim 1 in the preparation of a medicament for preventing and / or treating chronic hepatitis B.

27. A delivery system for treating a chronic hepatitis B patient, the delivery system comprising a first agent and a second agent independently packaged; wherein the first agent comprises an antiviral drug and / or a drug that reduces HBsAg level; the second agent comprises a first immunomodulator and a second immunomodulator; the first immunomodulator is a drug that reduces HBsAg level; the second immunomodulator is the antigen-antibody complex of claim 1; the second agent is administered to the patient after the first agent is administered.

28. The delivery system of claim 27, wherein the antigen-antibody complex is administered at a dose of 30-150 μg per person.

29. The delivery system of claim 27, wherein the chronic hepatitis B patient is one who has high levels of HBV DNA and high serum HBsAg; high levels of HBV DNA means HBV DNA concentration is <200 IU / mL or 2000 copies / mL, and high serum HBsAg means HBsAg concentration is >10 IU / mL and <3000 IU / mL.

30. The delivery system of claim 27, wherein the second agent is administered to the patient after the first immunomodulator is administered, when the patient's serum HBsAg level is reduced to about ≤400 IU / mL.

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