Antigen-antibody compound as well as preparation method and application thereof

Abstract

The invention belongs to the field of biological medicines, and particularly relates to an antigen-antibody compound for enhancing immune response of hepatitis B patients as well as a preparation method and application of the antigen-antibody compound. The antigen-antibody compound preparation prepared from the HBsAg and the anti-HBsAg monoclonal antibody is superior to an antigen-antibody compound preparation prepared from a polyclonal antibody in the aspect of continuously inducing the immune response of a chronic hepatitis B patient, and an antigen-antibody compound for inducing the immune response of the hepatitis B patient is developed for the first time; in the compound, the hepatitis B surface antigen is combined with the anti-HBsAg monoclonal antibody, and the compound formed by the hepatitis B surface antigen and the anti-HBsAg monoclonal antibody in a certain mass ratio can induce immune response of chronic hepatitis B patients and can also prevent and / or treat chronic hepatitis B.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to an antigen-antibody complex for enhancing the immune response of hepatitis B patients, and a preparation method and application thereof. Background Art

[0002] Hepatitis B virus (HBV) is an acute or chronic inflammatory liver disease caused by infection with the hepatitis B virus (HBV). It is the most common infectious disease worldwide. Patients with chronic HBV infection are at 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 HBV infection, defined as persistent undetectable serum HBsAg and HBV DNA, HBeAg seroconversion, with or without HBsAg seroconversion, resolution of liver inflammation, improvement in histopathology, and a significant reduction in the incidence of end-stage liver disease after a limited course of treatment. Studies have shown that HBsAg seroconversion can reduce the risk of HBV complications such as cirrhosis and hepatocellular carcinoma. Current clinical research indicates that one of the mechanisms underlying persistent infection in chronic HBV infection is the lack of effective humoral or cellular immunity, a key reason why functional cure cannot be achieved in patients with chronic HBV infection.

[0003] Current antiviral treatments for chronic hepatitis B, in addition to enhancing immune function, primarily focus on maximizing long-term suppression of HBV replication, reducing stem cell inflammation and necrosis, and liver fibrosis, thereby delaying and reducing liver disease progression and the development of liver cancer. Nucleos(t)ide analogs (NAs) are first-line treatments, characterized by their potent efficacy and low resistance. However, treatment with first-line NAs for chronic hepatitis B (CHB) is often associated with long treatment courses and inability to completely eliminate the virus. Even in patients with CHB receiving long-term NA therapy, hepatitis B surface antigen (HBsAg) clearance is rare. In clinical practice, in addition to antiviral measures, boosting the immune response is often necessary for persistent viral infection. To date, interferon is the most clinically approved and commonly used immunotherapy for the treatment of persistent HBV infection, but functional cure rates when combined with antiviral agents remain only approximately 10-30%.

[0004] The current immunotherapy preparations used at home and abroad can be summarized as (1) non-specific preparations that promote cellular immunity: such as thymosin, interferon, transfer factor, interleukin, etc.; (2) natural or synthetic immunotherapy drugs: such as levamisole, plant polysaccharides, fungal or bacterial products, and synthetic analog drugs; (3) hormones that regulate immunity: such as prednisone drugs; (4) specific antibodies or specific immune RNA; (5) vaccines. Current immunotherapy preparations may cause allergic reactions, infections, fever, and have the disadvantage of a short half-life. There is still much 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 eliminate hepatitis B virus. In 1997, therapeutic hepatitis B vaccines were listed as major projects in the "863" program. Later, the Beijing Institute of Biological Products applied for clinical research (Wen Yumei, He Lifang, Qu Di, et al. Experimental study of recombinant therapeutic hepatitis B vaccine (YIC) [J]. China Engineering Science, 1999, 1(1): 38-42.). It was 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 APCs, promoting HBsAg uptake, and more effectively activating T helper cells in the body, inducing immune-mediated cytokines / specific antibodies. The article disclosed that the subsequent technical solution adopted was to use HBsAgy and human polyclonal immunoglobulin to prepare IC for subsequent clinical research.

[0006] Subsequent IC research has focused on improving the efficacy of antigen-antibody complexes by adding excipients or other components, or expanding the clinical application of IC. 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 human immunoglobulin against hepatitis B surface antigen, wherein the amount of antigen exceeds the amount of antibody. A preparation comprising an immunogenic complex of 0.5-5 μg / ml of hepatitis B virus surface antigen purified from blood and 0.5-5 μg / ml of hepatitis B antibody immunoglobulin (anti-HBIG) containing at least 200-400 international units of hepatitis B antibodies has been found to be effective in treating persistent viral infection. Patent CN1919341B discovered that, unlike treatment, administering a HBsAg-antibody complex to people who are unresponsive or have a low response to the hepatitis B vaccine can activate the body to produce effective anti-HBs (HBV surface antibodies). Therefore, the HBsAg-antibody complex could serve as a novel immune-boosting preventive product for those who are unresponsive or have a low response to existing hepatitis B vaccines. In 2017, Academician Wen Yumei published an article (Human vaccines & immunotherapeutics vol. 13, 9 (2017): 1989-1996) stating that a yeast-derived HBsAg-anti HBs immunogenic complex combined with the hepatitis B drug adefovir could enhance the immune response in hepatitis B patients, and that the combination of the two could serve as a novel combination therapy for hepatitis B.

[0007] In addition, patent CN101204582B improves the complex based on the original antigen-antibody complex technology. The background technology introduction of this patent application mentions: Studies have confirmed that the presence of the Fc fragment in IC plays a vital role in breaking immune tolerance, and the Fc segment of the antibody constant region in IC can be used as a key adjuvant to break the immune tolerance of hepatitis B. Therefore, this prior technology constructed a fusion protein of a multivalent HBsAg antigen and a fully human anti-HBs monoclonal antibody constant region (Fc), and fused the adjuvant protein FL at its N-terminus. The patent mentions that after immunizing HBV transgenic mice with an IC composed of HBsAg and anti-HBsAg monoclonal antibodies, the mice's immune tolerance to HBsAg can be successfully broken, the HBsAg level in the mouse serum can be reduced, and the production of HBsAb can be stimulated. Its effect is consistent with that of an IC composed of anti-HBsAg polyclonal antibodies. From the perspective of the process for generating and preparing polyclonal antibodies, the inventors of this application believe that as the demand for preparation increases, the source of the raw material for polyclonal antibodies: hepatitis B surface antibody-positive serum will become a bottleneck in preparation, and serum-derived antibodies have the possibility of potential contamination. Therefore, the use of monoclonal antibody Fc preparation is considered. Summary of the Invention

[0008] Based on the current state of the art, the present invention has developed for the first time an antigen-antibody complex that induces an immune response in hepatitis B patients. The complex comprises hepatitis B surface antigen and an anti-HBsAg monoclonal antibody. The 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, the present invention has been completed.

[0009] In the first aspect, the present invention provides an antigen-antibody complex for inducing an immune response in patients with chronic hepatitis B, wherein the complex comprises a hepatitis B surface antigen and an 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; wherein the heavy chain comprises a heavy chain variable region domain VH, constant region domains CH1, CH2, CH3 and a hinge region; the light chain comprises 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 is combined with the anti-HBsAg monoclonal antibody 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 the pre-S or S region gene, or includes: a polypeptide encoded by the S region gene, PreS1 region gene + S region gene, PreS2 region gene + S region gene, or PreS1 region gene + PreS2 region gene + 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 shown as SEQ ID NO: 1, the nucleotide sequence of the light chain is shown as SEQ ID NO: 2; when the nucleotide sequence of the heavy chain VH+CH1 of the anti-HBsAg monoclonal antibody is shown as SEQ ID NO: 3, the nucleotide sequence of the light chain is shown as 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 mutants thereof.

[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 that 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 anti-HBsAg monoclonal antibody heavy chain are selected from SEQ ID NO: 5 to 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 sequence of the hinge region and CH2 and CH3 domains of the anti-HBsAg monoclonal antibody heavy 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 any one of SEQ ID NO:5 to 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 1:2-1:20.

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

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

[0026] Furthermore, the chronic hepatitis B patients refer to those with normal alanine aminotransferase and HBV DNA < 2×10 3IU / mL or undetectable HBV DNA.

[0027] Furthermore, the chronic hepatitis B patient refers to a chronic hepatitis B patient who receives antiviral drug treatment and whose serum HBsAg level is <3000 IU / mL, or whose HBeAg is 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 according to the first aspect, the preparation method comprising the following steps:

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

[0030] (b) Monoclonal antibody preparation: transfecting the plasmid constructed in step (a) into eukaryotic cells to obtain anti-HBsAg monoclonal antibodies 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] Furthermore, in step (a), the anti-HBsAg monoclonal antibody heavy chain includes a heavy chain variable region domain VH, constant region domains CH1, CH2, CH3 and a hinge region; and 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 shown as SEQ ID NO: 1, the nucleotide sequence of the light chain is shown as SEQ ID NO: 2; when the nucleotide sequence of the heavy chain VH+CH1 of the anti-HBsAg monoclonal antibody is shown as SEQ ID NO: 3, the nucleotide sequence of the light chain is shown as SEQ ID NO: 4.

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

[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 that are homologous to human immunoglobulin IgG2.

[0038] 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.

[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 to 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 sequence of the hinge region and CH2 and CH3 domains of the anti-HBsAg monoclonal antibody heavy 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 any one of SEQ ID NO: 5 to 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 to each other 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] Further, in step (c), the hepatitis B surface antigen is a polypeptide encoded by the pre-S or S region gene, or includes: a polypeptide encoded by the S region gene, the PreS1 region gene + the S region gene, the PreS2 region gene + the S region gene, or the PreS1 region gene + the PreS2 region gene + the 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 1:2-1:20.

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

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

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

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

[0052] Furthermore, the pharmaceutical composition also includes antiviral drugs and / or drugs that reduce 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 tenofovir alafenamide.

[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 for reducing HBsAg levels and / or the antigen-antibody complex in the pharmaceutical composition can be packaged independently and administered to the patient sequentially.

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

[0058] Furthermore, the administration of the pharmaceutical composition includes subcutaneous injection, intradermal injection, intramuscular injection, intravenous injection, intraperitoneal injection, oral administration or oral and nasal spraying.

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

[0061] Furthermore, the vaccine composition is 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-granular adjuvant is selected from one or more of CpG1080, muramyl dipeptide and its derivatives, saponin, lipid A, cytokine, derived polysaccharide, bacterial toxin, microorganism and its products and / or propolis.

[0067] Furthermore, the microbial product comprises one or more of Mycobacterium, Bacillus pumilus and / or Bordetella pertussis.

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

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

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

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

[0072] In a fifth aspect, the present invention provides an 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 independently packaged.

[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 use of the antigen-antibody complex described in the first aspect in the preparation of a drug for preventing and / or treating chronic hepatitis B.

[0075] In the seventh aspect, the present invention provides a drug delivery system for treating patients with chronic hepatitis B, the drug delivery system containing a first agent and a second agent in independent packages; wherein the first agent includes an antiviral drug and / or a drug that reduces HBsAg levels; the second agent includes a first immunomodulator and a second immunomodulator; the first immunomodulator is a drug that reduces HBsAg levels; the second immunomodulator is the antigen-antibody complex described in the first aspect; after administering the first agent, the second agent is administered to the patient.

[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 administration dosage of the antigen-antibody complex is 30-150 μg / person.

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

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

[0082] Furthermore, the chronic hepatitis B patients refer to chronic hepatitis B patients whose HBV DNA is usually at a high level and whose serum HBsAg is relatively high.

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

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

[0086] Beneficial effects

[0087] The present invention unexpectedly discovered that an antigen-antibody complex preparation prepared from HBsAg and anti-HBsAg monoclonal antibodies is superior to an antigen-antibody complex preparation (or pharmaceutical composition or vaccine composition) prepared from polyclonal antibodies in continuously inducing an immune response 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 immune control period of infection or later (Chinese Journal of General Practitioners, 2021, 20(03): 281-289) or is in the serum of a patient with HBV DNA < 2×10 3 IU / ml and HBsAg level <3000IU / ml or the alanine aminotransferase of chronic hepatitis B patients is normal, the antigen-antibody complex preparation prepared by the monoclonal antibody of the present invention can be administered to continuously induce and enhance the host immune response, eliminate HBsAg in the body of chronic hepatitis B patients, and improve the clinical cure rate of chronic hepatitis B.

[0088] The present invention is the first to develop an antigen-antibody complex that induces an immune response in patients with hepatitis B. The complex comprises hepatitis B surface antigen (HBsAg) and an anti-HBsAg monoclonal antibody, with the HBsAg and anti-HBsAg monoclonal antibodies bound to each other. Furthermore, the present invention is the first to discover that a complex consisting of a certain mass ratio of HBsAg to anti-HBsAg monoclonal antibody can induce an immune response in patients with chronic hepatitis B. However, a higher mass ratio of HBsAg to anti-HBsAg monoclonal antibody is not necessarily better; only a certain mass ratio can effectively induce an immune response in patients with chronic hepatitis B and prevent and / or treat the disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0089] Figure 1 Schematic diagram of the polyacrylamide gel electrophoresis results of the monoclonal antibodies G12-hIgG1, G12-mIgG1, G12-mIgG2a, KR127-hIgG1, KR127-mIgG1, KR127-mIgG2a in Examples 1 and 2 and the mouse polyclonal antibody (hepatitis B immunoglobulin (mHBIG)) against hepatitis B surface antigen (HBsAg).

[0090] Figure 2 Schematic diagram of the binding ability of human monoclonal antibody (G12-hIgG1) and mouse polyclonal antibody (mHBIG) to hepatitis B surface antigen (HBsAg) in Example 4.

[0091] Figure 3 and Figure 4 Schematic diagram of the affinity results of human monoclonal antibody (G12-hIgG1) and mouse polyclonal antibody (mHBIG) to hepatitis B surface antigen (HBsAg) in Example 3.

[0092] Figure 5 and Figure 6 Schematic diagram of the binding results 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.

[0093] Figure 7 The antibody levels in mice after immunization with the antigen-antibody complex preparation were compared in Example 6-2.

[0094] Figure 8-11 The changes in antibody (HBsAb) expression levels in mice on the 7th and 35th days after administration of the antigen-antibody complex preparations prepared with different antigen-antibody ratios in Example 6-3.

[0095] Figure 12-15The changing trend of hepatitis B surface antigen (HBsAg) expression in mice after immunization with the antigen-antibody complex preparations prepared with different ratios of antigen-antibody according to Example 6-4, Figure 15 This is a comprehensive schematic diagram of the expression level trend of hepatitis B surface antigen (HBsAg) when the antibody:antigen ratio is 1:6.

[0096] Figure 16-17 The changing trend of antigen (HbsAg) expression in immunized mice induced by the antigen-antibody complex preparations prepared with monoclonal antibodies of different epitopes in Example 6-5.

[0097] Figure 18-19 The changing trends and levels of antibody (HbsAb) expression in mice immunized with the antigen-antibody complex preparations prepared with monoclonal antibodies of different epitopes in Example 6-5.

[0098] Figure 20-24 This is a schematic diagram of the results of humoral immune factor levels after immunization in Example 6-6.

[0099] Figure 25-32 This is a schematic diagram of the results of cellular immune factor levels after immunization in Example 6-6.

[0100] Figure 33 This is the change of serum HBsAg after immunization in Example 7.

[0101] Figure 34 This is the change of serum HBV DNA after immunization in Example 7.

[0102] Figure 35 This is the change of HBsAb after immunization in Example 7.

[0103] Figure 36 The difference in HBsAb levels after comparing Example 8 with other immunomodulator combination therapies.

[0104] Figure 37 This is the effect of G12 monoclonal antibody in the complex vaccine of Example 9 in clearing HBsAg.

[0105] Figure 38 This is the DNA clearance effect of the G12 monoclonal antibody in the complex vaccine of Example 9. DETAILED DESCRIPTION

[0106] The following is a further description of specific embodiments of the present invention. It should be noted that the description of these embodiments is intended to facilitate understanding of the present invention and does not constitute a limitation of the present invention. In addition, the technical features involved in the embodiments described below may be combined with each other as long as they do not conflict with each other.

[0107] The experimental methods in the following examples are conventional methods unless otherwise specified, and the experimental materials used in the following examples are commercially available unless otherwise specified.

[0108] the term

[0109] In order to more easily understand the present disclosure, some terms are first defined. As used in this application, unless otherwise expressly provided herein, each of the following terms should have the meaning given below. Other definitions are set forth throughout the application.

[0110] The term "about" can refer to a value or composition that is within an acceptable error range for the particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined.

[0111] As used herein, the term "optionally" or "optionally" means that the event or situation described subsequently may occur but need not occur. For example, "optionally comprising 1-3 antibody heavy chain variable regions" means that the antibody heavy chain variable regions of a specific sequence may have but need not have, and may have 1, 2, or 3.

[0112] As used herein, "sequence identity" refers to the degree of identity between two nucleic acid or amino acid sequences when optimally aligned and compared with appropriate mutations such as substitutions, insertions, or deletions. The sequence identity between a sequence described herein and a sequence to which it is identical may be at least 85%, 90%, or 95%, preferably at least 95%. Non-limiting examples include 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100%.

[0113] Hepatitis B surface antigen (HBsAg) is the outer shell protein of hepatitis B virus (HBV) and the main component of HBV surface antigen. It is not contagious itself, but its appearance is often accompanied by the presence of hepatitis B virus, so it is a sign of hepatitis B virus infection.

[0114] HBV is a partially double-stranded, circular hepadnavirus. 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 the three outer membrane proteins of HBV: the large protein (LHB), the middle protein (MHB), and the small protein (SHB), collectively known as the hepatitis B virus surface antigens. Translation of these proteins is initiated by three different start codons within the same gene segment. The pre-S / S region can be divided into three distinct domains based on the position of the start codon: the Pre-S1, Pre-S2, and S domain.

[0115] The term "antibody" as used herein may include whole antibodies and any antigen-binding fragment (e.g., an antigen-binding fragment comprising a hinge, an antigen-binding fragment comprising a hinge and a CH1 domain, an antigen-binding fragment comprising a hinge and a CH2 domain, or an antigen-binding fragment comprising a portion of a hinge, a CH2 domain, and a CH3 domain) or a single chain thereof. "Antibodies" may 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.

[0116] In one embodiment, "antibody" refers to a protein, such as a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or an antigen-binding portion thereof. Each heavy chain consists of a heavy chain variable region (abbreviated herein as 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 (abbreviated herein as VL) and a light chain constant region. The light chain constant region consists of one domain, CL. The variable regions of the heavy and light chains comprise a binding domain that interacts with the antigen. The constant region of the antibody can mediate the binding of the immunoglobulin 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.

[0117] Immunoglobulins can be from any commonly known isotype, including but not limited to IgA, secretory IgA, IgG, and IgM. IgG isotypes are divided into subclasses in some species: IgG1, IgG2, IgG3, and IgG4 in humans, and IgG1, IgG2a, IgG2b, and IgG3 in mice. In some embodiments, the antibodies described herein are of the human or murine IgG1 or IgG2 subtype. Immunoglobulins (e.g., human IgG1) exist in several allotypes, differing from each other in at most a few amino acids.

[0118] As used herein, a domain of a heavy chain constant region, such as a hinge region, may include "IgG1 isotype", "IgG2 isotype", "IgG3 isotype" or "IgG4 isotype", and the domain may comprise the amino acid sequence of each isotype or a variant thereof (the amino acid sequence of each isotype in the domain has a higher homology than that of other different isotypes).

[0119] As used herein, the term "antigen-binding portion" of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. The antigen-binding portion of an antibody can be divided into a "hinge containing the antigen-binding portion". It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the "antigen-binding portion" of an antibody described herein include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bond at the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546), which consists of the VH domain; and (vi) an isolated complementarity determining region (CDR) or (vii) a combination of two or more isolated CDRs optionally linked by a synthetic linker. In addition, although the two VL and VH of the Fv fragment are encoded by separate genes, they can be connected by synthetic linkers using recombinant methods so that they can be made into a single polypeptide chain with antibody activity, which is a monovalent molecule called single-chain Fv (scFv). Such single-chain antibodies are also encompassed within the term "antigen-binding portion" of an antibody.

[0120] As used herein, "isotype" refers to the antibody class encoded by the heavy chain constant domain gene (e.g., IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE antibodies). 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 different isotypes thereof. If the domain of the heavy chain constant region used in the present invention, such as the CH2 domain, is of the IgG1 isotype or the IgG2 isotype, the domain may comprise the amino acid sequence of the corresponding domain of each isotype or a variant thereof (i.e., the amino acid sequence of each isotype of the domain is higher than that of other different isotypes).

[0121] "Fc region" (fragment crystallizable region) or "Fc domain" or "Fc" in the present invention refers to the C-terminal region of the heavy chain of the antibody that mediates the binding of the immunoglobulin to host tissues or factors, including binding to Fc receptors located on different cells of the immune system (e.g., effector cells) or binding to the first component (C1q) of the classical complement system. Thus, the Fc region of an antibody of the IgG isotype comprises the heavy chain constant region of the antibody excluding the first constant region immunoglobulin domain (CH1). In the IgG, IgA and IgD antibody isotypes, the Fc region comprises the CH2 and CH3 constant domains of each antibody's heavy chain; 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. Fc can be native ( "Or naturally occurring or wild-type) Fc, including any allotypic variants, or variant Fc (e.g., non-naturally occurring Fc), including, for example, 1, 2, 3, 4, 5, 1-5, 1-10, or 5-10 or more amino acid mutations, such as substitutions, additions, or deletions. For example, a variant Fc may comprise an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a wild-type Fc. Modified or mutated Fc may have enhanced or decreased effector function and / or half-life. The CH2 and CH3 regions are the primary sites of effector function and FcRn binding. Fc may refer to this region in isolation or in the context of a protein polypeptide comprising an Fc (e.g., a "binding protein comprising an Fc region"), also referred to as an "Fc fusion protein" (e.g., an antibody or immunoadhesin).

[0122] An "Fc receptor" or "FcR" is a receptor that binds to the Fc region of an immunoglobulin. The FcR that binds to IgG antibodies comprises the FcγR family of receptors, including allelic variants and alternatively spliced ​​forms of these receptors. The FcγR family consists of three activating (FcγRI, FcγRIII, and FcγRIV in mice; FcγRIA, FcγRIIA, and FcγRIIIA in humans) and one inhibitory (FcγRIIB) receptor. Most natural effector cell types co-express one or more activating FcγRs and the inhibitory FcγRIIB, while natural killer (NK) cells selectively express one activating Fc receptor (FcγRIII in mice and FcγRIIIA in humans) but not the inhibitory FcγRIIB in mice and humans. Human IgG1 binds to most human Fc receptors and is considered equivalent to murine IgG2a in the type of activating Fc receptor it binds.

[0123] As used herein, "treatment" refers to an attempt to alter the natural course of a disease in a treated individual and can be a clinical intervention performed for prevention or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing the occurrence or recurrence of the disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, slowing the rate of disease progression, ameliorating or eliminating the disease state, and regression or improved prognosis.

[0124] As used herein, an "individual" or "subject" is a mammal. Mammals include, but are not limited to, primates (e.g., humans and non-human primates such as monkeys) or other mammals (e.g., cows, sheep, cats, dogs, horses, rabbits, and rodents such as mice and rats).

[0125] As used herein, "mutant" or "variant" may refer to a molecule comprising one or more nucleotide or amino acid mutations in any naturally occurring or engineered molecule.

[0126] The standard recombinant DNA technology and molecular cloning techniques used in the examples are well known in the art (Ausubel, FM et al., Current Protocols in Molecular Biology, published by Greene Publishing Assoc. and Wiley-Interscience). Major chemical and biological reagents were purchased from KAPABiosystems, New England Biolabs, TransGen Biotech, Thermo Fisher Scientific, OMEGAbio-tek, etc.

[0127] Immune complex vaccine, also known as antigen-antibody complex vaccine, is a new type of vaccine developed in the 1990s. It is composed of specific antiserum mixed with viral antigens in a certain proportion. Its advantages are high safety and better immune effect than conventional vaccines.

[0128] Hepatitis B surface antigen (HBsAg) contains many transmembrane hydrophobic regions and disulfide bonds, and will naturally aggregate into spherical or rod-shaped polymeric particles with large molecular weight in solution. Therefore, it is impossible to construct a uniform and soluble Fc fusion protein through genetic engineering technology.

[0129] According to the Guidelines for Primary Diagnosis and Treatment of Chronic Hepatitis B (Practical Edition 2020), chronic hepatitis B patients refer to chronic liver inflammatory diseases caused by persistent HBV infection for more than 6 months. The stages of chronic hepatitis B virus infection include: (i) immune tolerance stage (chronic HBV carrier state): mostly in the perinatal period and infancy, with simultaneous positive serum HBsAg, hepatitis B e antigen (HBeAg) and anti-hepatitis B core antigen (anti-HBc) levels, high HBV DNA levels (usually HBV DNA > 2x107 IU / ml), and normal alanine aminotransferase (ALT); (ii) immune clearance stage (HBeAg-positive chronic hepatitis B): during the HBeAg-positive stage, ALT levels are persistently or repeatedly abnormal and HBV DNA levels are high (usually HBV DNA > 2x104 IU / ml), with HBeAg conversion to negative, anti-HBe conversion to positive, and ALT levels that are persistent or intermittently elevated; (iii) immune control stage (inactive HBsAg carrier state): during the low replication stage, ALT levels are normal, and HBV DNA levels are low (HBV DNA < 2x103 IU / ml) or undetectable; and (iv) reactivation phase (HBeAg-negative CHB): 5% to 15% of inactive patients may experience one or more hepatitis attacks, persistent or recurrent abnormalities in ALT, HBV DNA>2x103 IU / ml, manifesting as HBeAg-negative CHB, and HBeAg seroconversion may occur again (Chinese Journal of General Practitioners, 2021, 20(03): 281-289.).

[0130] 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 initial combination treatment strategies and sequential combination treatment strategies. The latter includes the "switch" strategy (i.e., switching antiviral drugs to immunomodulators) and the "addition" strategy (i.e., adding antiviral drugs to immunomodulators). The expert consensus recommends that CHB patients who receive long-term antiviral drug treatment have undetectable HBV DNA and HBsAg levels <3000 IU / ml, are HBeAg positive or have HBsAg levels ≥1500 IU / ml, or have HBeAg negative and HBsAg levels <1500 IU / ml. It is strongly recommended to use the sequential combination immunomodulator treatment strategy.

[0131] First dose of immunomodulator

[0132] As used herein, "first dose of immunomodulator" and "first immunomodulator" are used interchangeably and refer to drugs that reduce HBsAg levels, including but not limited to polyclonal antibodies, monoclonal antibodies, targeted siRNA, small interfering RNA or HBsAg inhibitors.

[0133] Second dose of immunomodulator

[0134] As used herein, "second dose of immunomodulator" and "second immunomodulator" are used interchangeably and refer to an antigen-antibody complex, a drug containing an antigen-antibody complex, or a vaccine containing an antigen-antibody complex, preferably the antigen-antibody complex described in the present invention.

[0135] Compositions and methods of administration

[0136] 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. As used herein, the term "comprising" indicates that various components can be used or present together in the composition of the present invention. Therefore, the terms "consisting essentially of" and "consisting of" are encompassed by the term "comprising."

[0137] The compositions of the present invention include pharmaceutical compositions and vaccine compositions. The compositions of the present invention can be monovalent or multivalent.

[0138] The pharmaceutical composition or vaccine composition 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.

[0139] (i) Pharmaceutical Compositions

[0140] The pharmaceutical composition of the present invention comprises an effective amount of the antigen-antibody complex prepared by the method of the present invention, and the antigen-antibody complex can be monovalent or multivalent.

[0141] As used herein, the term "effective amount" refers to an amount of a therapeutic agent that treats, alleviates, or prevents a target disease or condition, or an amount that exhibits a detectable therapeutic or preventive effect. This effect can be detected, for example, by antigen levels. A therapeutic effect also includes a reduction in physiological symptoms. The precise effective amount for a given subject depends on the subject's size and health, the nature and extent of the condition, and the therapeutic agent and / or combination of therapeutic agents selected for administration. Therefore, it is not useful to specify an exact effective amount in advance. However, for a given condition, the effective amount can be determined by routine experimentation.

[0142] For the purposes of the present invention, an effective dose of the vaccine complex is about 30 to 150 μg administered to an individual.

[0143] The pharmaceutical composition may also contain a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" refers to a carrier used for administering a therapeutic agent (e.g., a recombinant protein or other therapeutic agent). The term refers to pharmaceutical carriers that do not themselves induce the production of antibodies harmful to the individual receiving the composition and that are not excessively toxic 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 of ordinary skill in the art. A full discussion of pharmaceutically acceptable carriers or excipients can be found in Remington's Pharmaceutical Sciences (Mack Pub. Co., NJ 1991).

[0144] Pharmaceutically acceptable carriers in the compositions may include liquids such as water, saline, glycerol, and ethanol. These carriers may also contain auxiliary substances such as wetting or emulsifying agents, pH buffering substances, and the like. Typically, the compositions are formulated as injectables, such as liquid solutions or suspensions. They may also be formulated as solid forms suitable for incorporation into solutions, suspensions, or liquid excipients prior to injection. Liposomes are also included within the definition of pharmaceutically acceptable carriers.

[0145] (ii) Vaccine Composition

[0146] The vaccine composition of the present invention can be preventive (i.e., to prevent infection) or therapeutic. The vaccine composition comprises the antigen-antibody complex of the present invention and is usually combined with a "pharmaceutically acceptable carrier", which includes any carrier that does not itself induce the production of antibodies that are harmful to the individual receiving the composition. Suitable carriers are usually large, slowly metabolized 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 of ordinary skill in the art. In addition, these carriers can act as immunostimulants ("adjuvants"). In addition, antigens can also be coupled to bacterial toxins (such as toxins of pathogens such as diphtheria, tetanus, cholera, Helicobacter pylori, etc.).

[0147] Preferred adjuvants for enhancing the efficacy of the immunogenic composition include, but are not limited to: (1) aluminum salts (alum), 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) Ribi Adjuvant System (RAS) (Ribi Immunochem, Hamilton, MT), (3) saponin adjuvants; (4) Freund's complete adjuvant (CFA) and Freund's incomplete adjuvant (IFA); (5) cytokines, such as interleukins (such as IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, etc.), interferons (such as interferon), macrophage colony stimulating factor (M-CFS), tumor necrosis factor (TNF), etc.; (6) detoxified variants of bacterial ADP-ribosylating toxins (such as cholera toxin CT, pertussis toxin PT or Escherichia coli heat-labile toxin LT), see for example WO93 / 13302 and WO92 / 19265; and (7) other substances that act as immunostimulants to enhance the effect of the composition.

[0148] 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. In addition, auxiliary substances, such as wetting agents or emulsifiers, pH buffering substances, etc., may be present in such carriers.

[0149] More specifically, vaccines, including immunogenic compositions, contain an immunogenic polypeptide in an immunologically effective amount, as well as the other required components described above. An "immunologically effective amount" refers to an amount that is effective for treatment or prevention when administered to an individual as a single dose or as part of a continuous dose. This amount can be determined based on the health and physiological condition of the individual being treated, the type of individual being treated (e.g., human), the ability of the individual's immune system to synthesize antibodies, the degree of protection desired, the formulation of the vaccine, the treating physician's assessment of the medical condition, and other relevant factors. It is expected that this amount will be within a relatively wide range and can be determined through routine experimentation.

[0150] Typically, vaccine compositions or immunogenic compositions are formulated as injectable formulations, such as liquid solutions or suspensions. They can also be formulated as solid forms suitable for incorporation into solutions, suspensions, or liquid excipients prior to injection. Such formulations can also be emulsified or encapsulated in liposomes to enhance the adjuvant effect.

[0151] (iii) Route of administration and dosage

[0152] The composition can be administered directly to a subject. The subject can be a human or 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 using the same routes of administration as conventional vaccines and / or by simulating pathogen infection pathways.

[0153] 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, intravaginal, oral, or other parenteral routes. If desired, routes of administration may be combined or adjusted based on the disease. Vaccine compositions may be administered in single or multiple doses, and may include booster doses to elicit and / or maintain immunity.

[0154] The antigen-antibody complex of the present invention should be administered in an "effective amount", that is, the amount of the antigen-antibody complex is sufficient to induce an immune response in the selected administration route, and can effectively induce an immune response in patients with chronic hepatitis B; and / or prevent and / or treat chronic hepatitis B.

[0155] The amount of antigen-antibody complex selected in each vaccine dose is determined by the amount that can induce an immune protective response without obvious side effects. Usually, after infecting the host cells, each dose of vaccine is sufficient to contain about 1μg-1000μg, preferably 1μg-100μg, and more preferably 10μg-50μg of antigen-antibody complex. The optimal dosage of a specific vaccine can be determined by standard research methods including observing the antibody titer and other reactions in the subjects. The need for a booster dose can be determined by monitoring the level of immunity provided by the vaccine. After evaluating the antibody titer in the serum, it may be necessary to select a booster dose for immunization. The administration of adjuvants and / or immunostimulants can enhance the immune response to the antigen-antibody complex of the present invention. The preferred method is to administer the antigen-antibody complex by injection from a parenteral (subcutaneous or intramuscular) route.

[0156] English abbreviation

[0157]

[0158]

[0159] Example 1 Production of monoclonal antibodies to different epitopes of surface antigens

[0160] The nucleotide sequence of the heavy chain VH+CH1 of the G12 anti-HBsAg monoclonal antibody (anti-small HBsAg) is SEQ ID NO: 1, and the nucleotide sequence of the light chain VL+CL is SEQ ID NO: 2.

[0161] 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.

[0162] The plasmid construction of the antibodies used in the present invention is as follows:

[0163] Step 1: The heavy chain and light chain sequences of monoclonal antibodies G12 and KR127 were synthesized into a eukaryotic expression vector (pcDNA3.1) by Genscript.

[0164] Step 2: The heavy chain sequence of the product from Step 1 was ligated with the sequences of human IgG1 Fc (nucleotide sequence: SEQ ID NO: 5), mouse IgG1 Fc (nucleotide sequence: SEQ ID NO: 6), and IgG2a Fc (nucleotide sequence: SEQ ID NO: 7) by overlap PCR. The ligated product was then inserted into the eukaryotic expression vector pTT5 (sequence: SEQ ID NO: 8) by homologous recombination. The aforementioned sequences all contain the hinge region, CH2, and CH3.

[0165] Step 3: The recombinant products of steps 1 and 2 were respectively transformed into DH5α competent cells, and a single colony was picked from the overnight grown ampicillin plate and cultured with LB bacterial culture medium. The bacterial culture was sent for sequencing to obtain the following 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).

[0166] The preparation of anti-HBsAg monoclonal antibodies was basically carried out according to the literature (Cell Host Microbe. 2017. 22(4): 471-483.e5.). The preparation process is as follows:

[0167] Step 1: Use the HEK293F mammalian suspension cell expression system to express the following six antibodies:

[0168] G12-hIgG1: SEQ ID NO: 1 and the spliced ​​sequence of SEQ ID NO: 2 and SEQ ID NO: 5.

[0169] KR127-hIgG1: SEQ ID NO: 3 and SEQ ID NO: 4 and SEQ ID NO: 5 spliced ​​sequence.

[0170] G12-mIgG1: SEQ ID NO: 1 and the spliced ​​sequence of SEQ ID NO: 2 and SEQ ID NO: 6.

[0171] G12-mIgG2a: SEQ ID NO: 1 and the spliced ​​sequence of SEQ ID NO: 2 and SEQ ID NO: 7.

[0172] KR127-mIgG1: SEQ ID NO: 3 and SEQ ID NO: 4 and SEQ ID NO: 6 spliced ​​sequence.

[0173] KR127-mIgG2a: SEQ ID NO: 3 and SEQ ID NO: 4 and SEQ ID NO: 7 spliced ​​sequence.

[0174] 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.

[0175] Step 2: Centrifuge the cells from step 1, collect the supernatant, filter it, and equilibrate it with 1M PBS buffer through Protein G Resin resin. Add the supernatant to the resin at a flow rate of 1 mL / min. Elute the contaminants with 2 column volumes of equilibration buffer, and finally elute the bound immunoglobulins with 0.1 M glycine (pH 2.8).

[0176] Step 3: The immunoglobulin solution eluted in the previous step was transferred to a 3kD MWCO Amicon Ultra centrifugal ultrafiltration tube, ultrafiltered, and the elution buffer was replaced with PBS solution. The concentrated antibodies were collected, namely the human Fc chimeric monoclonal antibodies G12-hIgG1 and KR127-hIgG1 and the murine Fc chimeric monoclonal antibodies G12-mIgG1, G12-mIgG2a, KR127-mIgG1, and KR127-mIgG2a.

[0177] The protein concentration was quantified by measuring the absorbance of the antibody in step 3 at 280 nm using a spectrophotometer, and the purity was detected by SDS-PAGE electrophoresis. Figure 1 , used to prepare antigen-antibody complexes (Therapeutic vaccine, TVac).

[0178] Example 2 Production of Mouse Anti-Hepatitis B Immune Globulin (mHBIG)

[0179] 6-8 week old C57BL / 6 male SPF mice were treated with a mixture of 100 μg of HBsAg expressed and purified by stably transfected CHO cells and aluminum hydroxide adjuvant.

[0180] Immunize with the mixed immunogen above at 2 μg per mouse, once every two weeks for 3 times.

[0181] After the second and third immunizations, blood was collected from the mice, and the supernatant was centrifuged to obtain serum. The samples with a serum titer higher than 1:10000 were tested by ELISA using the above-mentioned hepatitis B virus surface antigen protein, and the serum was purified to obtain anti-mouse hepatitis B immunoglobulin (mHBIG). Figure 1 .

[0182] Example 3 Affinity test of antibodies to hepatitis B surface antigen (HBsAg)

[0183] 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 and then quenched for 300 s. The sensor was then incubated with three-fold dilutions of 1000 nM (mHBIG) or 50 nM (G12-hlgG1) of the antibody 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 using Data Analysis software. KD values ​​were determined with R2 values ​​greater than 95% confidence intervals.

[0184] BLI results are as follows Figure 3 and Figure 4 The results showed that the affinity of monoclonal antibody (G12-hIgG1) to hepatitis B surface antigen (HBsAg) was better than that of polyclonal antibody (anti-mouse hepatitis B immunoglobulin (mHBIG)).

[0185] Example 4: Binding Test of Antibodies to Hepatitis B Surface Antigen (HBsAg)

[0186] Hepatitis B surface antigen (HBsAg) was coated on an ELISA plate with PBS at 50 μg / well at 4°C overnight. Then, the antibodies of Examples 1 and 2 (including anti-hepatitis B immunoglobulin (mHBIG), G12-hIgG1, KR127-hIgG1, G12-mlgG1, G12-mIgG2a, KR127-mIgG1, and KR127-mIgG2a) were added in sequence at a starting concentration of 1 μM after 3-fold gradient dilution and purification. Then, goat anti-mouse IgG or anti-human IgG-Fc tag antibody was added to detect the binding ability of each antibody to the surface antigen (for specific methods, see Cell Host Microbe. 2020. 27(6): 891-898.e5.).

[0187] ELISA results Figure 2 It was shown that the binding ability of monoclonal antibody (G12-hIgG1) to hepatitis B surface antigen (HBsAg) was better than that of polyclonal antibody (anti-hepatitis B immunoglobulin (HBIG)).

[0188] Figure 5 and Figure 6It was shown that the different antibodies G12-hIgG1, KR127-hIgG1, G12-mlgG1, G12-mIgG2a, KR127-mIgG1 and KR127-mIgG2a produced in Example 1 all had the activity of binding to hepatitis B surface antigen (HBsAg) and the difference in binding activity was not much.

[0189] Example 5 Preparation of a Mouse Chronic Hepatitis B Virus Infection Animal Model

[0190] 6-8 week old C57BL / 6 male SPF mice were transfected with rAAV8-1.3HBV-ayw (titer of 1×10 12 μg / mL) were injected into the tail vein at a rate of 2×10 10 μg / mouse, and the infection lasted for two weeks as an established animal model of persistent infection.

[0191] Example 6 Immunization with an immunogenic antigen-antibody complex preparation

[0192] Clinical chronic hepatitis B combination therapy includes combined treatment strategies. This example uses the antigen-antibody complex of the present invention as an immunomodulatory agent to select a combined treatment strategy and implement it on a mouse model. The steps are as follows:

[0193] Step 1: Use antiviral drugs to inhibit viral replication and reduce serum viral load throughout the entire treatment process;

[0194] Step 2: Give the first dose of immunomodulator to reduce serum HBsAg levels;

[0195] Step 3: Administer a second dose of immunomodulatory agents to enhance the induction of an effective host immune response.

[0196] The antiviral drugs described in step 1 are nucleotide reverse transcriptase inhibitors, nucleoside (t) ide analogs (NA), capsid assembly regulators, antisense oligonucleotides and invasion inhibitors, etc. In this embodiment, nucleotide reverse transcriptase inhibitors are used. Nucleotide reverse transcriptase inhibitors can be selected from any one or a combination of anti-hepatitis B virus drugs such as lamivudine (commonly known as 3TC), telbivudine, adefovir dipivoxil (adefovir), entecavir (ETV), tenofovir disoproxil fumarate (TDF), tenofovir alafenamide fumarate (TAF) or tenofovir amibufenamide (TMF).

[0197] The immunomodulator used in step 2 is selected from pegylated interferon alpha (Peg-IFN-α), polyclonal antibodies, monoclonal antibodies, targeted siRNA, small interfering RNA or HBsAg inhibitor.

[0198] The immunomodulator used in step 3 is the antigen-antibody complex preparation of the present invention.

[0199] 6-1 Preparation of Antigen-Antibody Complex (TVac) as Second Dose of Immunomodulator

[0200] The purified hepatitis B virus surface antigen was mixed 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 at a mass ratio of at least greater than 0.67:1, and then placed for overnight incubation. 100 μg of aluminum hydroxide adjuvant was added (other marketed hepatitis B vaccine adjuvants such as CpG1080, aluminum phosphate adjuvant or a mixture of aluminum hydroxide and aluminum phosphate is also applicable) to produce antigen-antibody complex preparations targeting different epitopes of the hepatitis B virus surface antigen.

[0201] The antigen-antibody complex preparation prepared by the anti-mouse hepatitis B immunoglobulin complex is TVac-mHBIG (containing hepatitis B surface antigen HBsAg and the mouse hepatitis B immunoglobulin mHBIG of Example 2), the antigen-antibody complex preparation prepared by G12-hIgG1 is TVac-G (containing hepatitis B surface antigen HBsAg and the G12-hIgG1 antibody expressed in Example 1), the antigen-antibody complex preparation prepared by G12-mIgG1 is TVac-G12-mIgG1 (or TVac-G-mIgG1) (containing hepatitis B surface antigen HBsAg and the G12-mIgG1 antibody expressed in Example 1), the antigen-antibody complex preparation prepared by G12-mIgG2a is TVac-G-2a (or TVac-G12-mIgG2a or TVac-G12-2a or TVac-G-mIgG2a) (containing hepatitis B surface antigen HBsAg and the G12-mIgG2a antibody expressed in Example 1), the antigen-antibody complex preparation prepared by KR127-hIgG1 is TVac-K (containing the hepatitis B surface antigen HBsAg and the KR127-hIgG1 antibody expressed in Example 1), the antigen-antibody complex preparation prepared by KR127-mIgG1 is TVac-KR127-mIgG1 (or TVac-K-mIgG1) (containing the hepatitis B surface antigen HBsAg and the KR127-mIgG1 antibody expressed in Example 1), and the antigen-antibody complex preparation prepared by KR127-mIgG2a is TVac-K-2a (or TVac-KR127-IgG2a or TVac-KR127-2a or TVac-K-mIgG2a) (containing the hepatitis B surface antigen HBsAg and the KR127-mIgG2a antibody expressed in Example 1).

[0202] 6-2 Effects of Antigen-Antibody Complex Preparations Prepared with G12 Monoclonal Antibody and Antigen-Antibody Complex Preparations Prepared with Anti-Hepatitis B Immunoglobulin as Secondary Immunomodulators

[0203] Step 1: First, the chronic hepatitis B mouse model (described in Example 5) was administered with tenofovir disoproxil fumarate (TDF), a nucleotide reverse transcriptase inhibitor, by oral administration at 15 mg / kg for 5 consecutive days;

[0204] Step 2: On day 6, half an hour after TDF administration, the first dose of an immunomodulator was injected into the tail vein. The first dose of the immunomodulator was the G12 monoclonal antibody (SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 5 splicing sequence) (G12), synthesized from the literature (mAbs vol. 8, 3 (2016): 468-77), at 6.7 mg / kg to reduce the surface antigen level.

[0205] Step 3: One hour after step 2, a second dose is administered intraperitoneally, comprising an immunomodulator-antigen-antibody complex preparation (TVac-G and TVac-mHBIG) and hepatitis B surface antigen HBsAg. Immunization is performed once every two weeks for a total of three times. The experimental groups in this example are as follows:

[0206] PBS: The control group was given only PBS;

[0207] TDF+G12: a combination of an antiviral drug and a first dose of the immunomodulatory G12 monoclonal antibody;

[0208] TDF+G12+HBsAg: A combination of an antiviral drug and the immunomodulatory G12 monoclonal antibody as a first dose and the hepatitis B surface antigen HBsAg as a second dose;

[0209] TDF+G12+TVac-G: a combination of an antiviral drug and the first dose of the immunomodulator G12 monoclonal antibody and the second dose of TVac-G (an antibody complex of hepatitis B surface antigen HBsAg and G12-hIgG1 expressed in Example 1);

[0210] TDF+G12+TVac-mHBIG: a combination of antiviral drugs and the first dose of immunomodulator G12 monoclonal antibody and the second dose of TVac-mHBIG (an antibody complex of hepatitis B surface antigen HBsAg and mHBIG of Example 2).

[0211] Figure 7 This is a schematic diagram of antibody expression in mice one week after the first immunization. The results show that none of the mice in the PBS, TDF+G12, and TDF+G12+HBsAg groups produced antibodies (HBsAb). However, both the TDF+G12+TVac-G and TDF+G12+TVac-mHBIG groups produced antibodies (HBsAb). The monoclonal antibody-based antigen-antibody complex (TDF+G12+TVac-G) induced over 1-fold higher levels of HBsAb expression in mice than the polyclonal antibody-based antigen-antibody complex (TDF+G12+TVac-mHBIG).

[0212] 6-3 Expression of Antibody Levels Induced by Antigen-Antibody Complex Preparations Prepared with Different Antigen-Antibody Ratios

[0213] Antigen-antibody complex preparations with different antigen-antibody ratios were prepared according to "6-1 Preparation of Complex (TVac) as a Second Immunomodulator" and used as a second immunomodulator. Serum antibody (HBsAb) levels were measured by ELISA 7 and 35 days after administration of the second immunomodulator. The experimental groups in this example were as follows:

[0214] TVac-mHBIG: The second dose of immunomodulatory agent was an anti-HBV immunoglobulin complex preparation, in which the antibody:antigen mass ratios were 1:54, 1:18, 1:6, 1:2, and 1:0.67, respectively;

[0215] TVac-G12-2a: The second dose of immunomodulatory agent was a complex preparation of G12-mIgG2aFc monoclonal antibody, in which the antibody:antigen mass ratios were 1:54, 1:18, 1:6, 1:2, and 1:0.67, respectively;

[0216] TVac-KR127-2a: The second dose of immunomodulatory agent was a complex preparation prepared with KR127-mIgG2aFc monoclonal antibody, in which the antibody:antigen mass ratios were 1:54, 1:18, 1:6, 1:2, and 1:0.67, respectively.

[0217] The results are as follows Figure 8 、 9 , 10 and 11. When the antibody-to-antigen ratio of the complex preparation was 1:6, the amount of antibodies induced by the antigen-antibody complex preparations (TVac-G12-2a and TVac-KR127-2a) prepared by monoclonal antibodies G12-mIgG2aFc and KR127-mIgG2aFc on days 7 and 35 was more than 1 times that of the antigen-antibody complex preparation (TVac-mHBIG) of the polyclonal antibody; when the amount of antibody or antigen in the complex preparation was too high, the amount of antibodies induced by the antigen-antibody complex preparations prepared by monoclonal antibodies and polyclonal antibodies on days 7 and 35 did not increase significantly. Figure 10 and 11 After long-term observation, after administration of the same dose of antigen-antibody complex preparations, the antigen-antibody complex preparations prepared by monoclonal antibodies (TVac-G12-2a and TVac-KR127-2a) were significantly superior to the antigen-antibody complex preparations prepared by polyclonal antibodies (TVac-mHBIG) in continuously inducing immune responses in mice.

[0218] The results showed that the antigen-antibody complex preparations prepared by monoclonal antibodies (G12 or KR127) were superior to those prepared by polyclonal antibodies (HBIG) in treating or inducing immune response in chronic hepatitis B.

[0219] 6-4 The level of suppression of HBsAg by antigen-antibody complex preparations prepared with different antigen-antibody ratios

[0220] According to "6-1 Preparation of Complex (TVac) as Second Dose of Immunomodulator", antigen-antibody complex preparations with different antigen-antibody ratios were prepared for use as the second dose of immunomodulator. In a chronic hepatitis B mouse model, in step 1, tenofovir disoproxil fumarate (TDF) was first administered (orally) for 5 consecutive days at 15 mg / kg; then, the first dose of the immunomodulator - synthetic G12 monoclonal antibody (G12) and the second dose of the complex preparation (TVac-mHBIG, TVac-G12-2a, TVac-KR127-2a) were administered on days 0, 14, and 28, respectively.

[0221] The results are as follows Figure 12 、 13 As shown in Figures 14 and 15, different ratios of antigen-antibody complexes have different trends in inhibiting the expression of hepatitis B surface antigen (HBsAg). When the ratio of antibody to antigen in each complex preparation was 1:6 on the 14th day after the second immunization, the expression level of hepatitis B surface antigen (HBsAg) was the lowest. Figure 15 As shown in the results, when the antibody to antigen ratio of the complex preparation was 1:6, the hepatitis B surface antigen (HBsAg) level after administration of the monoclonal antibody complex preparation (TVac-G-mIgG2a and TVac-K-mIgG2a) was significantly lower than that of TVac-mHBIG (HBsAg of the monoclonal antibody complex preparation was 2 IU / mL on the 5th day, and HBsAg of the multiclonal antibody complex preparation had risen to 3 IU / mL).

[0222] from Figure 13 and 14 It can be seen that when TDF and the first dose of immunomodulator G12 are combined to treat chronic hepatitis B mice, the serum HBsAg expression level can be reduced to ≤20IU / mL. The present invention gives a second dose of immunomodulator on this basis and finds that it has a better effect. For chronic hepatitis B patients (humans), after TDF and the first dose of immunomodulator G12 are combined for treatment, the second dose of immunomodulator is given when the HBsAg level drops to approximately ≤400IU / 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 lower than 200IU / mL or the HBsAg decreases by more than 1 log 10 IU / mL is a positive factor for achieving clinical cure. The present invention uses TDF and the first dose of immunomodulator G12 combined treatment, and then administers the second dose of monoclonal antibody antigen-antibody complex (TVac-G12-2a). After medication, the baseline HBsAg level is lower than 200 IU / mL or the HBsAg decrease is greater than 1 log 10 IU / mL.

[0223] 6-5 Immune effects of monoclonal antibody complex preparations with different epitopes

[0224] In a chronic hepatitis B mouse model, step 1 was to administer tenofovir disoproxil fumarate (TDF) (oral administration) for 5 consecutive days at 15 mg / kg; then, on days 0, 14, and 28, the first dose of the synthetic G12 monoclonal antibody (G12) immunomodulator and the second dose of the immunomodulator complex (TVac) preparation were administered, respectively. The preparation method was described in 6-1 to obtain the antibody complex preparations (TVac): TVac-G, TVac-K, TVac-G-mIgG1, TVac-G-mIgG2a, TVac-K-IgG1, and TVac-K-mIgG2a. PBS served as the control group.

[0225] The results are as follows Figure 16 and Figure 17 As shown, the expression trends of hepatitis B surface antigen (HBsAg) inhibited by different monoclonal antibody complex preparations (TVac) were similar, and the expression level of HBsAg in mouse serum steadily decreased after three immunizations. Figure 18 and Figure 19 The trend of HBsAb expression in induced immunized mice was as follows: no HBsAb was detected in the PBS control group; after completing steps 2 and 3 on day 14, HBsAb expression in the mice steadily increased; and high HBsAb expression was still detectable in the mice more than 50 days after completing steps 2 and 3 on day 28. Furthermore, the results of this example indicate that the IgG2a subtype is more effective in suppressing antigens and inducing antibodies than the IgG1 subtype.

[0226] 6-6 Expression levels of humoral immunity and cellular immune factors after sequential combined treatment

[0227] On the 83rd day after the completion of Example 6-5 "Immune Effects of Monoclonal Antibody Complex Preparations with Different Epitopes", 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 combined treatment.

[0228] Figure 20-24 This is a schematic diagram of the expression levels of humoral immune factors in mice. Figure 25-32 The results are shown in the figure below: Schematic diagram of the expression level of cellular immune factors. The results show that immunization with the antigen-antibody complex preparation (TVac) significantly induced humoral and cellular immune responses against HBsAg.

[0229] Example 7 Effects of combined siRNA targeting HBsAg on serum HBsAg, HBV DNA and HBsAb levels

[0230] This example uses the same immunization method as Example 6, except that the first dose of immunomodulatory agent in this example uses siRNA targeting HBsAg. The experimental groups are as follows:

[0231] PBS: the control group was given only PBS;

[0232] TDF+G12+TVac-G: The first dose of immunomodulation is administered with a synthetic G12 monoclonal antibody (G12), and the second dose of immunomodulation is administered with a TVac-G antigen-antibody complex preparation;

[0233] TDF+siRNA+TVac-G: The first dose of immunomodulation is siRNA targeting HBsAg (clinical drug HT101 from Suzhou Xingyao Kunze Company), and the second dose of immunomodulation is TVac-G antigen-antibody complex preparation;

[0234] TDF+G12+siRNA+TVac-G: The first dose of immunomodulation is administered with synthetic G12 monoclonal antibody (G12) and siRNA targeting HBsAg, and the second dose of immunomodulation is administered with TVac-G antigen-antibody complex preparation.

[0235] The TDF dose in step 1 is 15 mg / kg, injected every other day; the first dose of immunomodulator in step 2 is the synthetic G12 monoclonal antibody (G12) at a dose of 6.7 mg / kg, and the dose of siRNA targeting HBsAg is 3 mg / kg subcutaneously injected, given after the synthetic G12 monoclonal antibody; the second dose of immunomodulator in step 3 is the TVac-G antigen-antibody complex preparation, injected intraperitoneally at 4 μg; the first and second doses of immunomodulator are administered on days 0, 14, and 28.

[0236] In terms of serum HBsAg expression levels, Figure 33 As shown, the HBsAg expression in the serum of mice given only G12 monoclonal antibody as the first dose of immunomodulation showed a recovery trend after the first immunization (day 0), while the HBsAg expression in the serum of mice in the groups given siRNA (TDF+siRNA+TVac-G and TDF+G12+siRNA+TVac-G) continued to decrease.

[0237] In terms of suppressing HBV DNA, Figure 34 As shown, the groups administered with the antigen-antibody complex preparation (TDF+G12+TVac-G, TDF+siRNA+TVac-G, and TDF+G12+siRNA+TVac-G) had the function of continuously suppressing HBV DNA expression.

[0238] In terms of HBsAb, Figure 35As shown, the groups given antigen-antibody complex preparations (TDF+G12+TVac-G, TDF+siRNA+TVac-G, and TDF+G12+siRAN+TVac-G) were superior to the control group PBS in inducing antibody expression; the antibody expression induced by the simultaneous administration of monoclonal antibody G12 and siRNA (TDF+G12+siRNA+TVac-G) as the first dose of immunomodulation was superior to that of the other groups.

[0239] Other interfering RNA molecules targeting HBsAg, such as microRNA and shRNA, also have similar effects to siRNA and have similar effects when applied to the present invention.

[0240] MiRNAs (microRNAs) are a group of genomically encoded noncoding RNAs approximately 20 to 23 nucleotides in length. They base-pair with target gene mRNAs, directing the silencing complex (RISC) to degrade mRNAs or inhibit their translation. Shorthairpin RNAs (shRNAs) are short double-stranded RNA structures (19-25 nt) that rely on stem-loop sequences. They can be introduced into cells using vectors, where they are enzymatically cleaved into siRNAs, which regulate target genes through RNA interference. Unlike siRNAs, shRNAs are synthesized in the cell nucleus. After transient or stable delivery of shRNA vectors into cells, they are first transported to the nucleus. Following nuclear expression, they are processed by the Drosha / DGCR8 complex to form pre-shRNAs. These are then transported to the cytoplasm by the Exportin-5 protein, where the Dicer complex removes the loop sequence to form siRNAs. These siRNAs then bind to the RNA-induced silencing complex (RISC) and dissociate from one of the RNA strands. The complex then recognizes the mRNA, leading to its degradation. It can be seen that the processing pathway of shRNA after leaving the nucleus is basically the same as that of dsRNA, and the final structure is also formed into siRNA. siRNA is usually exogenous, while miRNA is endogenous. The mature (double-stranded) miRNA is loaded onto RISC. After leaving one chain, the RISC carrying the antisense RNA

[0241] They bind to mRNA and inhibit target gene expression through mRNA degradation and translation inhibition, respectively. Different types of small RNAs also differentiate the two types of RISC. The core enzyme of RISC is the Argonaute enzyme of the AGO protein family, which has multiple members in most species. In fruit flies and human cells, miRNAs bind to the AGO-1 protein to produce a non-cleaving RISC, which interferes with target genes by inhibiting mRNA translation. siRNAs bind to the AGO-2 protein to produce a cleaving RISC, which interferes with target genes by cleaving and degrading mRNA. Structurally, shRNAs are more similar to miRNAs, and in fact, shRNAs are functionally closer to siRNAs. ShRNAs are cleaved by the Dicer enzyme in the cell to form siRNAs, which exert their interference function through the siRNA pathway, while miRNAs regulate target genes through a different pathway.

[0242] Example 8: Differences in Antibody Levels After Combination Therapy Between the Present Invention and Other Immunomodulators

[0243] This example uses a combination therapy strategy using different immunomodulators to administer to the patient. The specific experimental groups and medication regimens and information are as follows:

[0244] PDL1: commercially available Atilizhu monoclonal antibody; TLR8: Toll-like receptor 8 agonist GS-9688; IFN-α: alpha interferon; FAP-IL2: an immune cytokine, including an anti-fibroblast activation protein α (FAPα) antibody and an IL-2 variant that only binds to IL-2Rβγ; PD1: anti-mouse CD279 monoclonal antibody; PDL1-IFNα+HBsAg / CpG: PDL1-IFNα heterodimeric fusion protein synthesized according to reference (Meng CY, et al. Gut 2023; 72: 1544-1554); HBsAg was purchased from North China Pharmaceutical, and CpG is a Toll-like receptor 9 agonist purchased from MedChemexpress Biotechnology Co., Ltd. in the United States.

[0245] TDF+G12+PDL1: The nucleotide reverse transcriptase inhibitor tenofovir disoproxil fumarate TDF (oral administration, 15 mg / kg) was first administered to the chronic hepatitis B mouse model (described in Example 5) for 5 consecutive days, and then administered every other day after the 6th day; on the sixth day, the synthetic G12 monoclonal antibody was administered (tail vein, 6.7 mg / kg), and one hour later, PDL1 (T drug) (tail vein, 5 mg / kg) was administered. G12 and PDL1 were administered once every two weeks, for a total of 3 times.

[0246] TDF+G12+TLR8: The nucleotide reverse transcriptase inhibitor tenofovir disoproxil fumarate TDF (15 mg / kg) was first administered to the chronic hepatitis B mouse model (described in Example 5) for 5 consecutive days, and then administered every other day after the 6th day; on the sixth day, the synthetic G12 monoclonal antibody was administered (tail vein, 6.7 mg / kg), and one hour later, TLR8 was administered (3 mg / kg). G12 was administered once every two weeks for a total of 3 times, and TLR8 was administered once a week for a total of 5 times.

[0247] TDF+G12+TVac-G-mIgG2a: The nucleotide reverse transcriptase inhibitor tenofovir disoproxil fumarate TDF (oral administration, 15 mg / kg) was first administered to the chronic hepatitis B mouse model (described in Example 5) for 5 consecutive days, and then administered every other day after the 6th day; on the sixth day, the first dose of the immunomodulator, namely the synthetic G12 monoclonal antibody, was administered (tail vein, 6.7 mg / kg), and one hour later, the second dose of the immunomodulator TVac-G-mlgG2a antigen-antibody complex preparation was administered. G12 and TVac-G-mlgG2a were administered once every two weeks, for a total of 3 times.

[0248] TDF+G12+IFNα: The nucleotide reverse transcriptase inhibitor tenofovir disoproxil fumarate TDF (oral administration, 15 mg / kg) was first administered to the chronic hepatitis B mouse model (described in Example 5) for 5 consecutive days, and then administered every other day after the 6th day; on the sixth day, the synthetic G12 monoclonal antibody was administered (tail vein, 6.7 mg / kg), and IFNα (intraperitoneal administration, 30 μg / kg) was administered 1 hour later. G12 was administered every two weeks for a total of 3 times, and IFNα was administered once a week for a total of 5 times.

[0249] TDF+G12+FAP-IL2: The nucleotide reverse transcriptase inhibitor tenofovir disoproxil fumarate TDF (oral administration, 15 mg / kg) was first administered to the chronic hepatitis B mouse model (described in Example 5) for 5 consecutive days, and then administered every other day after the 6th day; on the sixth day, the synthetic G12 monoclonal antibody was administered (tail vein, 6.7 mg / kg), and 1 hour later, FAP-IL2 (tail vein, 2 mg / kg) was administered. G12 and FAP-IL2 were administered once every two weeks, for a total of 3 times.

[0250] siRNA+G12+IFNα: siRNA targeting HBsAg (clinical drug HT101 of Suzhou Xingyao Kunze Company, subcutaneous, 3 mg / kg) was administered on day 0, and G12 monoclonal antibody (tail vein, 6.7 mg / kg) was administered at the same time. 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 once a week for a total of 5 times.

[0251] siRNA+TLR8+TDF: The nucleotide reverse transcriptase inhibitor tenofovir disoproxil fumarate (TDF) (oral administration, 15 mg / kg) was first administered to the chronic hepatitis B mouse model (described in Example 5) for 5 consecutive days, and then administered every other day after the 6th day; on the 6th day, siRNA targeting HBsAg (clinical drug HT101 of Suzhou Xingyao Kunze Company, subcutaneous, 3 mg / kg) was administered, and 1 hour later, TLR8 (oral administration, 3 mg / kg) was administered. siRNA was administered every two weeks for a total of 3 times, and TLR8 was administered once a week for a total of 5 times.

[0252] siRNA+PD1+TDF: The nucleotide reverse transcriptase inhibitor tenofovir disoproxil fumarate (TDF) (oral administration, 15 mg / kg) was first administered to the chronic hepatitis B mouse model (described in Example 5) for 5 consecutive days, and then administered every other day after the 6th day; on the sixth day, siRNA targeting HBsAg (clinical drug HT101 of Suzhou Xingyao Kunze Company, subcutaneous, 3 mg / kg) was administered, and PD1 (tail vein, 5 mg / kg) was administered 1 hour later. siRNA and PD1 were administered once every two weeks for a total of 3 times.

[0253] PDL1-IFNα+HBsAg / CpG: Synthetic anti-PDL1-IFNα homologous fusion protein (Meng CY, et al. Gut, 2023(72):1544-1554) was administered on days 0 and 3 (tail vein, 0.2 m / kg), and HBsAg / CpG (subcutaneous, 2 μg / 30 ug) was administered on days 3, 10, 17, and 24.

[0254] PBS: The control group was given only PBS.

[0255] In terms of HBsAb, Figure 36 As shown, serum was collected on the 21st day after the combined treatment. The results showed that the antibody level produced by the TDF+G12+TVac-G-mlgG2a group after treatment was much higher than that of 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 corresponding antibodies faster.

[0256] Example 9: Effect of G12 monoclonal antibody in the complex vaccine of the present invention in clearing HBsAg

[0257] This example compares the G12 monoclonal antibody in the antigen-antibody complex of the present invention with other hepatitis B monoclonal antibodies in terms of their effectiveness in clearing HBsAg and DNA, and is conducted on a mouse model.

[0258] Monoclonal antibody information and dosage regimen are as follows:

[0259] KR127: A monoclonal antibody targeting the pre-S1 epitope. The nucleotide sequence of the KR127 heavy chain VH+CH1 is SEQ ID NO: 3, and the nucleotide sequence of the light chain VL+CL is SEQ ID NO: 4.

[0260] G12: A monoclonal antibody targeting the small S epitope. The nucleotide sequence of the G12 heavy chain VH+CH1 is SEQ ID NO: 1, and the nucleotide sequence of the light chain VL+CL is SEQ ID NO: 2.

[0261] VIR-3434: A monoclonal antibody targeting the small S epitope. The amino acid sequence of the VIR-3434 heavy chain VH is SEQ ID NO: 9, and the amino acid sequence of the light chain VL is SEQ ID NO: 10.

[0262] A15: A monoclonal antibody targeting the small S epitope. The nucleotide sequence of the A15 heavy chain VH+CH1 is SEQ ID NO: 11, and the nucleotide sequence of the light chain VL+CL is SEQ ID NO: 12.

[0263] The above antibodies were administered as a single dose at 6.7 mg / mL via tail vein injection on day 0, and blood was collected from the retroorbital sinus of the mice to measure HBsAg and DNA levels. PBS served as the control group.

[0264] In terms of suppressing HBsAg expression level, Figure 37 As shown in Figure 2, all monoclonal antibodies had the strongest inhibitory effect 5 days after treatment, among which the average reduction of HBsAg by G12 monoclonal antibody could reach 2log 10 IU / mL, the effect is optimal.

[0265] In terms of suppressing HBV DNA, Figure 38 As shown, the average DNA reduction of G12 monoclonal antibody can reach 1000 copies / ml on the 5th day after treatment, which is the best effect.

Claims

1. An antigen-antibody complex for inducing an immune response in patients with chronic hepatitis B, the complex comprising hepatitis B surface antigen and anti-HBsAg monoclonal antibody, the hepatitis B surface antigen binding to the anti-HBsAg monoclonal antibody; the anti-HBsAg monoclonal antibody is composed of a heavy chain and a light chain; wherein: The heavy chain 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; 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 according to claim 1, wherein the hepatitis B surface antigen and the anti-HBsAg monoclonal antibody are bound to each other in a non-covalent manner.

3. The antigen-antibody complex according to claim 1, wherein 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.

4. The antigen-antibody complex according to claim 1, wherein the hepatitis B surface antigen is a polypeptide encoded by the pre-S or S region gene, or comprises: 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. The antigen-antibody complex according to claim 1 , wherein 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 mutants thereof.

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

7. The antigen-antibody complex according to claim 1, wherein the hinge region and CH2 and CH3 domain sequences of the anti-HBsAg monoclonal antibody heavy chain are selected from SEQ ID NO: 5 to SEQ ID NO:

7.

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

54.

9. The antigen-antibody complex according to claim 1, wherein the chronic hepatitis B patient is a patient with normal alanine aminotransferase and HBV DNA < 2×10 3 IU / mL or undetectable HBV DNA.

10. The antigen-antibody complex according to claim 1, wherein the chronic hepatitis B patient is a chronic hepatitis B patient who is receiving antiviral drug treatment and whose serum HBsAg level is <3000 IU / mL, or whose HBeAg is negative and whose HBsAg level is <1500 IU / mL.

11. The method for preparing the antigen-antibody complex according to claim 1, comprising the following steps: (a) Construction of monoclonal antibody expression plasmid: The heavy chain and light chain sequences of the anti-HBsAg monoclonal antibody are constructed in a eukaryotic cell expression vector; (b) Monoclonal antibody preparation: transfecting the plasmid constructed in step (a) into eukaryotic cells to obtain anti-HBsAg monoclonal antibodies after cell expression; (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.

12. The preparation method according to 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; 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 preparation method according to claim 11, wherein the hinge region and CH2 and CH3 domains of the heavy chain of the anti-HBsAg monoclonal antibody are selected from the hinge region and CH2 and CH3 domains of the heavy chain of IgG1 or IgG2 and mutants thereof. The preparation method according to claim 11 , wherein the hinge region and CH2 and CH3 domain sequences of the anti-HBsAg monoclonal antibody heavy chain are selected from SEQ ID NO: 5 to SEQ ID NO:

7.

15. The preparation method according to claim 11, wherein in step (c), the mass mixing ratio of the HBsAg monoclonal antibody to the hepatitis B surface antigen is 1:0.67-1:

54.

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

17. The pharmaceutical composition according to claim 16, comprising a single drug, a compound drug or a synergistic drug.

18. The pharmaceutical composition according to claim 16, further comprising an antiviral drug and / or a drug for reducing HBsAg levels.

19. The pharmaceutical composition according to claim 16, wherein 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; and 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.

20. The pharmaceutical composition according to claim 16, wherein the antiviral drug, the drug for reducing HBsAg level and / or the antigen-antibody complex in the pharmaceutical composition can be packaged separately and administered to the patient sequentially.

21. A vaccine composition comprising the antigen-antibody complex according to claim 1 and an immunologically acceptable carrier and / or adjuvant.

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

23. The vaccine composition according to claim 21, further comprising an adjuvant, wherein the adjuvant is selected from a particulate adjuvant and a non-particulate adjuvant.

24. An 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 according to claim 1; the first immunomodulator and the second immunomodulator are independently packaged.

25. The immunomodulator according to claim 24, wherein 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.

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

27. A drug delivery system for treating patients with chronic hepatitis B, the drug delivery system comprising a first agent and a second agent in independent packages; wherein the first agent comprises an antiviral drug and / or a drug that reduces HBsAg levels; the second agent comprises a first immunomodulator and a second immunomodulator; the first immunomodulator is a drug that reduces HBsAg levels; the second immunomodulator is the antigen-antibody complex according to claim 1; after administering the first agent, the second agent is administered to the patient.

28. The drug delivery system according to claim 27, wherein the dosage of the antigen-antibody complex is 30-150 μg / person.

29. The drug delivery system according to claim 27, wherein the chronic hepatitis B patient refers to a chronic hepatitis B patient whose HBV DNA is usually at a high level and whose serum HBsAg is relatively high; the high level of HBV DNA refers to an HBV DNA concentration of <200 IU / mL or 2000 copies / mL, and the relatively high serum HBsAg refers to an HBsAg concentration of >10 IU / mL and <3000 IU / mL.

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

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