Antigen-antibody complex vaccine for enhancing immune response in hepatitis b patient and preparation method therefor

Abstract

Provided are an antigen-antibody complex vaccine for enhancing an immune response in a chronic hepatitis B patient and a preparation method therefor. Specifically, provided is an antigen-antibody complex for inducing an immune response in a chronic hepatitis B patient. 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 complex can induce the immune response in the chronic hepatitis B patient, and can also prevent and / or treat chronic hepatitis B.

Description

Antigen-antibody complex vaccine for enhancing immune response of hepatitis B patients and preparation method thereof TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, in particular, to an antigen-antibody complex vaccine for enhancing immune response of hepatitis B patients and a preparation method thereof, more particularly, to a new hepatitis B surface antigen-antibody complex, especially an antigen-antibody complex prepared based on monoclonal antibodies, which is applied to enhance immune response of chronic hepatitis B patients. BACKGROUND

[0002] Hepatitis B virus hepatitis is a disease caused by acute or chronic inflammation of the liver after hepatitis B virus (HBV) infection, which is the most common infectious disease in the world, and China is a high prevalence area of hepatitis B, with about 70 million hepatitis B virus surface antigen (HBsAg) carriers, of which about 20-30 million are chronic hepatitis B (CHB, chronic hepatitis B) patients. Chronic hepatitis B patients have a high risk of developing into late liver disease, including cirrhosis and hepatocellular carcinoma. The current guidelines clearly define functional cure, i.e. after completing a limited course of treatment, serum HBsAg and HBV DNA are not detected, HBeAg is negative, with or without HBsAg seroconversion, liver inflammation is relieved and histopathology is improved, and the incidence of end-stage liver disease is significantly reduced. It is the ideal treatment goal recommended by the latest domestic and foreign guidelines for prevention and treatment of chronic hepatitis B. Studies have shown that HBsAg negative conversion can reduce the risk of complications such as cirrhosis and hepatocellular carcinoma. Current clinical medical research shows that one of the mechanisms of persistent infection of chronic hepatitis B virus hepatitis is the lack of effective humoral or cellular immunity in the body, which is also one of the important reasons why chronic hepatitis B patients cannot achieve functional cure.

[0003] Currently, the anti-viral treatment for chronic hepatitis B is to enhance the body's immune treatment, and to mainly take the maximum long-term inhibition of HBV replication, reduce stem cell inflammation and necrosis and liver fibrous tissue proliferation, delay and reduce liver disease progression and liver cancer occurrence. Nucleos(t)ide analogus (NAs) as the first-line drug in clinical practice has the characteristics of high efficiency and low drug resistance. However, it is found that the treatment course of the first-line NAs treatment for CHB is long, and the phenomenon of not being able to completely eliminate the virus occurs. Even for CHB patients who have received long-term NAs treatment, hepatitis B surface antigen (HBsAg) rarely occurs negative conversion. In clinical practice, in addition to the use of anti-viral measures, the body's immune response often needs to be enhanced. So far, the immune agent approved and commonly used for the treatment of persistent hepatitis B virus infection in clinical practice is interferon, but the functional cure rate after combination with anti-viral drugs is still only about 10-30%.

[0004] The currently used immune treatment agents for enhancing the body's immune treatment at home and abroad can be summarized as follows: (1) non-specific cell immune enhancers: such as thymus peptide, interferon, transfer factor, interleukin, etc.; (2) natural or synthetic immune enhancers: such as levamisole, plant polysaccharides, fungal or bacterial products and artificially synthesized analog drugs; (3) immune-regulating hormones: such as prednisone drugs; (4) specific antibodies or specific immunoribonucleic acids; (5) vaccines. The current immune treatment agents for enhancing the body's immune response may have the disadvantages of allergic reactions, infections, fever and short half-life, and there is still a lot of room for improvement in enhancing the body's immune response.

[0005] Therefore, there is an urgent need in the art to develop immune treatment drugs for enhancing the immune response of hepatitis B patients.

[0006] SUMMARY

[0007] The purpose of the present application is to provide an antigen-antibody complex vaccine for enhancing the immune response of patients with chronic hepatitis B.

[0008] One object of the present application is to provide an antigen-antibody complex for inducing the immune response of patients with chronic hepatitis B, characterized in that the complex comprises hepatitis B surface antigen and anti-HBsAg monoclonal antibody, and the hepatitis B surface antigen and the anti-HBsAg monoclonal antibody are combined by non-covalent bond.

[0009] The HBsAg of the present application can be HBsAg expressed by genetically engineered bacteria, recombinant HBsAg expressed by mammalian cells, inactivated blood-derived HBsAg, or synthetic HBsAg. The HBsAg can be a polypeptide encoded by a gene of the pre-S or S region, or a polypeptide encoded by a gene of the S region, a gene of the PreS1 region + a gene of the S region, a gene of the PreS2 region + a gene of the S region, or a gene of the PreS1 region + a gene of the PreS2 region + a gene of the S region.

[0010] The anti-HBsAg monoclonal antibody provided by the present application is composed of a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region domain VH, a constant region domain CH1, CH2, CH3 and a hinge region; and the light chain comprises a variable region domain VL and a constant region domain CL.

[0011] Preferably, the heavy chain VH and CH1 sequence of the anti-HBsAg monoclonal antibody of the present application is SEQ ID NO: 1, and the light chain sequence is SEQ ID NO: 2. Preferably, the hinge region and CH2, CH3 domains of the heavy chain of the anti-HBsAg monoclonal antibody of the present application are selected from the hinge region and CH2, CH3 domains of the heavy chain of human immunoglobulin IgG1 or human IgG2 and mutants thereof, or are selected from the hinge region and CH2, CH3 domains of the heavy chain of murine IgG1, murine IgG2a, murine IgG2b, or murine IgG2c homologous to human immunoglobulin IgG1 or human IgG2 and mutants thereof. More preferably, the hinge region and CH2, CH3 domains of the present application are selected from the corresponding regions of human IgG1 or murine IgG1, IgG2a. More preferably, the sequence of the hinge region and CH2, CH3 domains of the heavy chain of the anti-HBsAg monoclonal antibody of the present application is SEQ ID NO: 5. More preferably, the sequence of the hinge region and CH2, CH3 domains of the heavy chain of the anti-HBsAg monoclonal antibody of the present application is SEQ ID NO: 6. More preferably, the sequence of the hinge region and CH2, CH3 domains of the heavy chain of the anti-HBsAg monoclonal antibody of the present application is SEQ ID NO: 7.

[0012] Preferably, the heavy chain VH and CH1 sequence of the anti-HBsAg monoclonal antibody of the present application is SEQ ID NO: 3, and the light chain sequence is SEQ ID NO: 4. Preferably, the hinge region and CH2, CH3 domains of the heavy chain are selected from the hinge region and CH2, CH3 domains of the heavy chain of human immunoglobulin IgG1 or human IgG2 and mutants thereof, or the hinge region and CH2, CH3 domains of the heavy chain of murine IgG1, murine IgG2a, murine IgG2b, murine IgG2c homologous to human immunoglobulin IgG1 or human IgG2 and mutants thereof. More preferably, the hinge region and CH2, CH3 domains of the present application are selected from the corresponding regions of human IgG1 or murine IgG2a. More preferably, the hinge region and CH2, CH3 domain sequence of the heavy chain of the anti-HBsAg monoclonal antibody of the present application is SEQ ID NO: 5. More preferably, the hinge region and CH2, CH3 domain sequence of the heavy chain of the anti-HBsAg monoclonal antibody of the present application is SEQ ID NO: 6. More preferably, the hinge region and CH2, CH3 domain sequence of the heavy chain of the anti-HBsAg monoclonal antibody of the present application is SEQ ID NO: 7.

[0013] Preferably, the alanine aminotransferase of the chronic hepatitis B patient of the present application is normal, and the HBV DNA is less than 2x10 3 IU / ml or undetectable. More preferably, the chronic hepatitis B patient is a chronic hepatitis B patient who has received antiviral drug treatment and whose serum HBsAg level is less than 3000 IU / ml, or who has turned HBeAg negative and whose HBsAg level is less than 1500 IU / ml.

[0014] Preferably, the antigen-antibody complex of the present application has a mass mixing ratio of the anti-HBsAg monoclonal antibody to the hepatitis B surface antigen of not more than 1:0.67. More preferably, the antigen-antibody complex of the present application has a mass mixing ratio of the anti-HBsAg monoclonal antibody to the hepatitis B surface antigen of not less than 1:54. More preferably, the antigen-antibody complex of the present application has a mass mixing ratio of the anti-HBsAg monoclonal antibody to the hepatitis B surface antigen of 1:6.

[0015] Another object of the present application is to provide an antigen-antibody complex preparation comprising the aforementioned antigen-antibody complex and an adjuvant. The selection of the adjuvant includes: aluminum hydroxide adjuvant, cpG1080, aluminum phosphate adjuvant, or a mixture of aluminum hydroxide and aluminum phosphate.

[0016] Another object of the present application is to provide a preparation method of the aforementioned antigen-antibody complex, comprising the following steps:

[0017] Step 1 - Construction of a 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.

[0018] The heavy chain comprises a heavy chain variable region domain VH, a constant region domain CH1, CH2, CH3 and a hinge region, and the light chain comprises a variable region domain VL and a constant region domain CL.

[0019] Preferably, the VL and CH1 sequences of the heavy chain of the anti-HBsAg monoclonal antibody of the present application are SEQ ID NO: 1, and the light chain sequence is SEQ ID NO: 2. Preferably, the hinge region and CH2, CH3 domains of the heavy chain of the anti-HBsAg monoclonal antibody of the present application are selected from the hinge region and CH2, CH3 domains of the heavy chain of human immunoglobulin IgG1 or human IgG2 and mutants thereof, or the hinge region and CH2, CH3 domains of the heavy chain of murine lgG1, murine lgG2a, murine lgG2b, murine lgG2c homologous to human immunoglobulin IgG1 or human IgG2 and mutants thereof. More preferably, the hinge region and CH2, CH3 domains of the present application are selected from the corresponding regions of human IgG1 or murine IgG2a. More preferably, the sequence of the hinge region and CH2, CH3 domains of the heavy chain of the anti-HBsAg monoclonal antibody of the present application is SEQ ID NO: 5. More preferably, the sequence of the hinge region and CH2, CH3 domains of the heavy chain of the anti-HBsAg monoclonal antibody of the present application is SEQ ID NO: 6. More preferably, the sequence of the hinge region and CH2, CH3 domains of the heavy chain of the anti-HBsAg monoclonal antibody of the present application is SEQ ID NO: 7.

[0020] Preferably, the VL and CH1 sequences of the heavy chain of the anti-HBsAg monoclonal antibody of the present application are SEQ ID NO: 1, and the light chain sequence is SEQ ID NO: 2. Preferably, the hinge region and CH2, CH3 domains of the heavy chain of the anti-HBsAg monoclonal antibody of the present application are selected from the hinge region and CH2, CH3 domains of the heavy chain of human immunoglobulin IgG1 or human IgG2 and mutants thereof, or the hinge region and CH2, CH3 domains of the heavy chain of murine lgG1, murine lgG2a, murine lgG2b, murine lgG2c homologous to human immunoglobulin IgG1 or human IgG2 and mutants thereof. More preferably, the hinge region and CH2, CH3 domains of the present application are selected from the corresponding regions of human IgG1 or murine IgG2a. More preferably, the sequence of the hinge region and CH2, CH3 domains of the heavy chain of the anti-HBsAg monoclonal antibody of the present application is SEQ ID NO: 5. More preferably, the sequence of the hinge region and CH2, CH3 domains of the heavy chain of the anti-HBsAg monoclonal antibody of the present application is SEQ ID NO: 6. More preferably, the sequence of the hinge region and CH2, CH3 domains of the heavy chain of the anti-HBsAg monoclonal antibody of the present application is SEQ ID NO: 7.

[0021] Step 2 - Preparation of monoclonal antibody: the plasmid constructed in step 1 is transfected into eukaryotic cells, and the anti-HBsAg monoclonal antibody is obtained after cell expression;

[0022] Step 3 - mixing of antigen and antibody: the anti-HBsAg monoclonal antibody obtained in step 2 is mixed with the hepatitis B surface antigen to obtain the antigen-antibody complex of the present application.

[0023] Preferably, the hepatitis B surface antigen of the present application is a polypeptide encoded by the pre-S / S region gene, or a polypeptide encoded by the S region gene, the PreSl region gene + S region gene, the PreS2 region gene + S region gene, or the PreSl region gene + PreS2 region gene + S region gene.

[0024] Preferably, the mass mixing ratio of the anti-HBsAg monoclonal antibody to the hepatitis B surface antigen is not higher than 1:0.67, more preferably, the antigen-antibody complex of the present application has a ratio of the anti-HBsAg monoclonal antibody to the hepatitis B surface antigen not lower than 1:54. More preferably, the mass ratio of the anti-HBsAg monoclonal antibody to the hepatitis B surface antigen of the present application is 1:6.

[0025] Another object of the present application is to provide the use of an immunomodulator in the preparation of a medicament for the treatment of chronic hepatitis B, characterized in that the immunomodulator is a mixture of reagent 1 and reagent 2: reagent 1 is a hepatitis B surface antigen; and reagent 2 is an anti-HBsAg monoclonal antibody.

[0026] Preferably, the mass mixing ratio of the anti-HBsAg monoclonal antibody to the hepatitis B surface antigen is not higher than 1:0.67, more preferably, the antigen-antibody complex of the present application has a ratio of the anti-HBsAg monoclonal antibody to the hepatitis B surface antigen not lower than 1:54. More preferably, the mass ratio of the anti-HBsAg monoclonal antibody to the hepatitis B surface antigen of the present application is 1:6.

[0027] Another object of the present application is to provide an immunomodulator prepared from the aforementioned antigen-antibody complex.

[0028] Another object of the present application is the use of the aforementioned immunomodulator in the preparation of a medicament for inducing an immune response in a hepatitis B patient. Preferably, the use comprises the prevention and / or treatment of chronic hepatitis B.

[0029] Preferably, the application comprises the combination of the anti-viral drug and / or the drug for reducing the level of HBsAg. More preferably, the anti-viral drug is selected from any one or combination of nucleotide reverse transcriptase inhibitors, nucleos(t)ide analogs (NA), capsid assembly modulators, antisense oligonucleotides and entry inhibitors. More preferably, the nucleotide reverse transcriptase inhibitor is selected from any one or combination of Lamivudine (commonly known as 3TC), Telbivudine, Adefovir, Entecavir (ETV), Tenofovir disoproxil fumarate (TDF), Tenofovir alafenamide fumarate (TAF) or Tenofovir amibufenamide (TMF). More preferably, the drug for reducing the level of HBsAg is selected from any one or combination of pegylated interferon alpha, polyclonal antibodies, monoclonal antibodies, targeted siRNA, small interfering RNA or HBsAg inhibitors.

[0030] The first aspect of the present application provides an antigen-antibody complex for inducing immune response in a patient with chronic hepatitis B, wherein the complex comprises hepatitis B surface antigen and anti-HBsAg monoclonal antibody, and the hepatitis B surface antigen is combined with the anti-HBsAg monoclonal antibody.

[0031] In another preferred embodiment, the hepatitis B surface antigen is combined with the anti-HBsAg monoclonal antibody in a non-covalent manner.

[0032] In another preferred embodiment, the hepatitis B surface antigen can be 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.

[0033] In another preferred embodiment, the hepatitis B surface antigen can be a polypeptide encoded by the pre-S or S region gene, or 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.

[0034] In another preferred embodiment, 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, a constant region domain CH1, CH2, CH3 and a hinge region; and the light chain comprises a variable region domain VL and a constant region domain CL.

[0035] In another preferred embodiment, the amino acid sequence of the heavy chain VH+CH1 of the anti-HBsAg monoclonal antibody is shown as SEQ ID NO: 1, and the amino acid sequence of the light chain is shown as SEQ ID NO: 2.

[0036] In another preferred embodiment, the amino acid sequence of the heavy chain VH+CH1 of the anti-HBsAg monoclonal antibody is shown as SEQ ID NO: 3, and the amino acid sequence of the light chain is shown as SEQ ID NO: 4.

[0037] In another preferred embodiment, the hinge region and CH2, CH3 domains of the heavy chain of the anti-HBsAg monoclonal antibody are selected from the hinge region and CH2, CH3 domains of the heavy chain of human immunoglobulin IgG1 or human IgG2 and mutants thereof, or murine IgG1, murine IgG2a, murine IgG2b, murine IgG2c homologous to human immunoglobulin IgG1 or human IgG2.

[0038] In another preferred embodiment, the hinge region and CH2, CH3 domains are selected from human IgG1 or murine IgG1, IgG2a.

[0039] In another preferred embodiment, the hinge region and CH2, CH3 domains of the heavy chain of the anti-HBsAg monoclonal antibody are selected from the hinge region and CH2, CH3 domains of the heavy chain of human immunoglobulin IgG1 or human IgG2 and mutants thereof, or murine IgG1, murine IgG2a, murine IgG2b, murine IgG2c homologous to human immunoglobulin IgG1 or human IgG2.

[0040] In another preferred embodiment, the hinge region and CH2, CH3 domains of the heavy chain of the anti-HBsAg monoclonal antibody are selected from the hinge region and CH2, CH3 domains of the heavy chain of human immunoglobulin IgG1 or human IgG2 and mutants thereof, or murine IgG1, murine IgG2a, murine IgG2b, murine IgG2c homologous to human immunoglobulin IgG1 or human IgG2.

[0041] In another preferred embodiment, the hinge region and CH2, CH3 domains of the heavy chain of the anti-HBsAg monoclonal antibody are selected from the hinge region and CH2, CH3 domains of the heavy chain of human immunoglobulin IgG1 or human IgG2 and mutants thereof, or murine IgG1, murine IgG2a, murine IgG2b, murine IgG2c homologous to human immunoglobulin IgG1 or human IgG2.

[0042] In another preferred embodiment, the amino acid sequence of the light chain of the anti-HBsAg monoclonal antibody is shown as SEQ ID NO. 2 or 4.

[0043] In another preferred embodiment, the amino acid 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 to the amino acid sequence shown as SEQ ID NO: 1 or 3.

[0044] In another preferred embodiment, the amino acid sequence of the hinge region and CH2, CH3 domains of the heavy chain of the anti-HBsAg monoclonal antibody has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence homology or sequence identity to the amino acid sequence as set forth in any one of SEQ ID NOs: 5-7.

[0045] In another preferred embodiment, the amino acid 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 to the amino acid sequence as set forth in SEQ ID NO: 2 or 4.

[0046] In another preferred embodiment, the antibody is of the IgG type.

[0047] In another preferred embodiment, the anti-HBsAg monoclonal antibody has a mass mixing ratio with the hepatitis B surface antigen of no more than 1:0.67, more preferably, the antigen-antibody complex of the present application has a ratio of the monoclonal antibody to the HBsAg antigen of no less than 1:54, more preferably, the mass ratio of the hepatitis B surface antigen and the anti-HBsAg monoclonal antibody is 1-20:1, preferably 2-10:1, more preferably 3-8:1 (such as 6:1).

[0048] The second aspect of the present application provides a pharmaceutical composition comprising the antigen-antibody complex of the first aspect of the present application, and a pharmaceutically acceptable carrier and / or adjuvant.

[0049] In another preferred embodiment, the pharmaceutical composition is a vaccine composition.

[0050] In another preferred embodiment, the vaccine composition is monovalent or multivalent.

[0051] In another preferred embodiment, the pharmaceutical composition further comprises an adjuvant, preferably an aluminum adjuvant.

[0052] In another preferred embodiment, the adjuvant comprises an aluminum hydroxide adjuvant, cpG1080, an aluminum phosphate adjuvant, or a mixture of aluminum hydroxide and aluminum phosphate.

[0053] In another preferred embodiment, the antigen-antibody complex and the adjuvant (such as aluminum) in the pharmaceutical composition have a molar ratio or a weight ratio of between 1:100, preferably between 1:40 and 1:60.

[0054] In another preferred embodiment, the pharmaceutical composition comprises a single drug, a compound drug, or a synergistic drug.

[0055] In another preferred embodiment, the pharmaceutical composition is in a dosage form of a liquid, a solid, or a gel.

[0056] In another preferred embodiment, the pharmaceutical composition is administered by a mode selected from the group consisting of subcutaneous injection, intradermal injection, intramuscular injection, intravenous injection, intraperitoneal injection, microneedle injection, oral administration, or oral and nasal cavity spray and aerosol inhalation.

[0057] In another preferred embodiment, the pharmaceutical composition further comprises an antiviral drug and / or a drug for reducing the level of HBsAg.

[0058] In another preferred embodiment, the antiviral drug and / or the drug for reducing the level of HBsAg is selected from the group consisting of any one or a combination of nucleotide reverse transcriptase inhibitors, nucleos(t)ide analogs (NA), capsid assembly modulators, antisense oligonucleotides, and entry inhibitors; more preferably, the nucleotide reverse transcriptase inhibitor is selected from any one or a combination of Lamivudine (commonly known as 3TC), Telbivudine, Adefovir, Entecavir (ETV), Tenofovir disoproxil fumarate (TDF), Tenofovir alafenamide fumarate (TAF), or Tenofovir amibufenamide (TMF); more preferably, the drug for reducing the level of HBsAg is selected from the group consisting of pegylated interferon alpha, polyclonal antibodies, monoclonal antibodies, targeted siRNA, small interfering RNA, or HBsAg inhibitors, or a combination thereof.

[0059] The third aspect of the present application provides a vaccine composition comprising the antigen-antibody complex of the first aspect of the present application, and an immunologically acceptable carrier and / or adjuvant.

[0060] In another preferred embodiment, the vaccine composition further comprises an adjuvant.

[0061] In another preferred embodiment, the adjuvant comprises a particulate and a non-particulate adjuvant.

[0062] In another preferred embodiment, the particulate adjuvant is selected from the group consisting of an aluminum salt, a water-in-oil emulsion, an oil-in-water emulsion, a nanoparticle, a microparticle, a liposome, an immunostimulatory complex, or a combination thereof.

[0063] In another preferred embodiment, the non-particulate adjuvant is selected from the group consisting of muramyl dipeptide and its derivatives, saponin, lipid A, cytokines, derivatized polysaccharides, bacterial toxins, microorganisms and their products such as Mycobacterium (M. tuberculosis, BCG), Corynebacterium parvum, Bordetella pertussis, propolis, or combinations thereof.

[0064] In another preferred embodiment, the adjuvant comprises aluminum oxide, saponin, Quil A, muramyl dipeptide, mineral or vegetable oil, vesicle-based adjuvant, non-ionic block copolymer or DEAE dextran, cytokine.

[0065] In another preferred embodiment, the adjuvant comprises: aluminum hydroxide adjuvant, cpG1080, aluminum phosphate adjuvant or a mixture of aluminum hydroxide and aluminum phosphate.

[0066] In another preferred embodiment, the vaccine composition further comprises an antiviral drug and / or a drug for reducing the level of HBsAg.

[0067] In another preferred embodiment, the antiviral drug and / or the drug for reducing the level of HBsAg is selected from any one or a combination of nucleotide reverse transcriptase inhibitors, nucleos(t)ide analogs (NA), capsid assembly modulators, antisense oligonucleotides and entry inhibitors; more preferably, the nucleotide reverse transcriptase inhibitor is selected from any one or a combination of Lamivudine (commonly known as 3TC), Telbivudine, Adefovir, Entecavir (ETV), Tenofovir disoproxil fumarate (TDF), Tenofovir alafenamide fumarate (TAF) or Tenofovir amibufenamide (TMF); more preferably, the drug for reducing the level of HBsAg is selected from peginterferon alpha, polyclonal antibodies, monoclonal antibodies, targeted siRNA, small interfering RNA or HBsAg inhibitors, or combinations thereof.

[0068] In another preferred embodiment, the vaccine composition comprises an injection dosage form.

[0069] The fourth aspect of the present application provides a method for preparing the antigen-antibody complex of the first aspect of the present application, comprising:

[0070] Mixing the hepatitis B surface antigen and the anti-HBsAg monoclonal antibody at a certain ratio to obtain the antigen-antibody complex of claim 1.

[0071] In another preferred embodiment, the mass mixing ratio of the HBsAg monoclonal antibody to the hepatitis B surface antigen is not higher than 1:0.67, more preferably, the antigen-antibody complex of the present application has a ratio of the monoclonal antibody to the HBsAg antigen not lower than 1:54, more preferably, the mass ratio of the hepatitis B surface antigen to the anti-HBsAg monoclonal antibody is 1-20:1, preferably 2-10:1, more preferably 3-8:1 (such as 6:1).

[0072] In another preferred embodiment, the method comprises the following steps:

[0073] (a) Constructing a monoclonal antibody expression plasmid: constructing the heavy chain and light chain sequences of the anti-HBsAg monoclonal antibody in a eukaryotic cell expression vector;

[0074] wherein the heavy chain comprises a heavy chain variable region domain VH, a constant region domain CH1, CH2, CH3 and a hinge region, and the light chain comprises a variable region domain VL and a constant region domain CL;

[0075] (b) Monoclonal antibody preparation: transfecting the constructed plasmid in step (a) into eukaryotic cells to obtain the anti-HBsAg monoclonal antibody after cell expression;

[0076] (c) Antigen-antibody mixing: mixing the anti-HBsAg monoclonal antibody obtained in step (b) with the hepatitis B surface antigen to obtain the antigen-antibody complex of the first aspect of the present application.

[0077] In another preferred embodiment, the hepatitis B surface antigen and the anti-HBsAg monoclonal antibody are combined in a non-covalent bond manner.

[0078] In another preferred embodiment, the hepatitis B surface antigen is a hepatitis B surface antigen (HBsAg) expressed by genetically engineered bacteria, a recombinant HBsAg expressed by mammalian cells, an inactivated blood-derived HBsAg, or a synthetic hepatitis B surface antigen.

[0079] In another preferred embodiment, the hepatitis B surface antigen is a hepatitis B surface antigen (HBsAg) expressed by genetically engineered bacteria, a recombinant HBsAg expressed by mammalian cells, an inactivated blood-derived HBsAg, or a synthetic hepatitis B surface antigen.

[0080] In another preferred embodiment, the hepatitis B surface antigen is a polypeptide encoded by a pre-S or S region gene, or a polypeptide encoded by an S region gene, a PreS1 region gene+S region gene, a PreS2 region gene+S region gene, or a PreS1 region gene+PreS2 region gene+S region gene.

[0081] In another preferred embodiment, 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, a constant region domain CH1, CH2, CH3 and a hinge region; the light chain comprises a variable region domain VL and a constant region domain CL.

[0082] In another preferred embodiment, the amino acid sequence of the heavy chain VH+CH1 of the anti-HBsAg monoclonal antibody is shown as SEQ ID NO: 1, and the amino acid sequence of the light chain is shown as SEQ ID NO: 2.

[0083] In another preferred embodiment, the amino acid sequence of the heavy chain VH+CH1 of the anti-HBsAg monoclonal antibody is shown as SEQ ID NO: 3, and the amino acid sequence of the light chain is shown as SEQ ID NO: 4.

[0084] In another preferred embodiment, the hinge region and CH2, CH3 domains of the heavy chain of the anti-HBsAg monoclonal antibody are selected from the hinge region and CH2, CH3 domains of the heavy chain of human immunoglobulin IgG1 or human IgG2 and mutants thereof, or murine IgG1, murine IgG2a, murine IgG2b, murine IgG2c homologous to human immunoglobulin IgG1 or human IgG2.

[0085] In another preferred embodiment, the hinge region and CH2, CH3 domains are selected from human IgG1 or murine IgG1, IgG2a.

[0086] In another preferred embodiment, the hinge region and CH2, CH3 domains of the heavy chain of the anti-HBsAg monoclonal antibody are shown as SEQ ID NO: 5.

[0087] In another preferred embodiment, the hinge region and CH2, CH3 domains of the heavy chain of the anti-HBsAg monoclonal antibody are shown as SEQ ID NO: 6.

[0088] In another preferred embodiment, the hinge region and CH2, CH3 domains of the heavy chain of the anti-HBsAg monoclonal antibody are shown as SEQ ID NO: 7.

[0089] In another preferred embodiment, the amino acid sequence of the light chain of the anti-HBsAg monoclonal antibody is shown as SEQ ID NO. 2 or 4.

[0090] In another preferred embodiment, the amino acid 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 to the amino acid sequence shown as SEQ ID NO: 1 or 3.

[0091] In another preferred embodiment, the amino acid sequence of the hinge region and CH2, CH3 domains of the heavy chain of the anti-HBsAg monoclonal antibody has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence homology or sequence identity to the amino acid sequence as set forth in any one of SEQ ID NOs: 5-7.

[0092] In another preferred embodiment, the amino acid 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 to the amino acid sequence as set forth in SEQ ID NO: 2 or 4.

[0093] In another preferred embodiment, the antibody is of IgG type.

[0094] In another preferred embodiment, the mass ratio of the hepatitis B surface antigen to the anti-HBsAg monoclonal antibody is not less than 0.67:1, preferably, the mass ratio of the hepatitis B surface antigen to the anti-HBsAg monoclonal antibody is not more than 54:1, more preferably, the mass ratio of the hepatitis B surface antigen to the anti-HBsAg monoclonal antibody is 1-20:1, more preferably, 2-10:1, more preferably, 3-8:1 (such as 6:1).

[0095] The fifth aspect of the present application provides an immunomodulator for use in (a) the preparation of a medicament for inducing an immune response in a patient with chronic hepatitis B; and / or (b) the preparation of a medicament for preventing and / or treating chronic hepatitis B, characterized in that the immunomodulator comprises: hepatitis B surface antigen and an anti-HBsAg monoclonal antibody.

[0096] In another preferred embodiment, the use is in combination with an antiviral drug and / or a drug that reduces the level of HBsAg, more preferably, the antiviral drug is selected from any one or a combination of nucleotide reverse transcriptase inhibitors, nucleos(t)ide analogs (NA), capsid assembly modulators, antisense oligonucleotides, and entry inhibitors. More preferably, the nucleotide reverse transcriptase inhibitor is selected from any one or a combination of Lamivudine (commonly known as 3TC), Telbivudine, Adefovir, Entecavir (ETV), Tenofovir disoproxil fumarate (TDF), Tenofovir alafenamide fumarate (TAF), or Tenofovir amibufenamide (TMF), and the like, and more preferably, the drug that reduces the level of HBsAg is selected from any one or a combination of peginterferon alfa, polyclonal antibodies, monoclonal antibodies, targeted siRNA, small interfering RNA, or HBsAg inhibitors, and the like.

[0097] The sixth aspect of the present application provides an immunomodulator prepared from the antigen-antibody complex of the first aspect of the present application.

[0098] In another preferred embodiment, the immunomodulator further comprises an antiviral drug and / or a drug that reduces the level of HBsAg.

[0099] The seventh aspect of the present application provides the use of the antigen-antibody complex of the first aspect of the present application, the pharmaceutical composition of the second aspect of the present application, the vaccine composition of the third aspect of the present application, or the immunomodulator of the sixth aspect of the present application, (a) for the preparation of a medicament for inducing an immune response in a patient with chronic hepatitis B; and / or (b) for the preparation of a medicament for preventing and / or treating chronic hepatitis B.

[0100] In another preferred embodiment, the medicament further comprises an antiviral drug and / or a drug that reduces the level of HBsAg.

[0101] In another preferred embodiment, the antiviral drug and / or the drug that reduces the level of HBsAg is selected from the group consisting of any one or combination of nucleotide reverse transcriptase inhibitors, nucleos(t)ide analogs (NA), capsid assembly modulators, antisense oligonucleotides, and entry inhibitors; more preferably, the nucleotide reverse transcriptase inhibitor is selected from any one or combination of Lamivudine (commonly known as 3TC), Telbivudine, Adefovir, Entecavir (ETV), Tenofovir disoproxil fumarate (TDF), Tenofovir alafenamide fumarate (TAF), or Tenofovir amibufenamide (TMF); more preferably, the drug that reduces the level of HBsAg is selected from the group consisting of pegylated interferon alpha, polyclonal antibodies, monoclonal antibodies, targeted siRNA, small interfering RNA, or HBsAg inhibitors, or a combination thereof.

[0102] The ninth aspect of the present application provides a method for treating a disease, comprising administering to a subject in need thereof the antigen-antibody complex of the first aspect of the present application, the pharmaceutical composition of the second aspect of the present application, the vaccine composition of the third aspect of the present application, or the immunomodulator of the sixth aspect of the present application.

[0103] In another preferred embodiment, the disease comprises chronic hepatitis B.

[0104] In another preferred embodiment, the antigen-antibody complex, the pharmaceutical composition, the vaccine composition, or the immunomodulator is administered at a dose of 30-150 ug per person, preferably 40-80 ug per person, more preferably 50-70 ug per person.

[0105] In another preferred embodiment, the antigen-antibody complex, the pharmaceutical composition, the vaccine composition, or the immunomodulator is administered at a frequency of once every two weeks for a total of three times.

[0106] In another preferred embodiment, the disease comprises chronic hepatitis B.

[0107] In another preferred embodiment, the subject comprises a patient with chronic hepatitis B.

[0108] In another preferred embodiment, the patient with chronic hepatitis B is a chronic hepatitis B patient with HBV DNA generally at a high level (>2x10 7 IU / ml) and high serum HBsAg (generally >1x10 4 IU / ml).

[0109] In another preferred embodiment, the patient with chronic hepatitis B has received antiviral drug treatment, and has serum HBsAg level < 3000 IU / ml, or HBeAg seroconversion, and HBsAg level < 1500 IU / ml.

[0110] The tenth aspect of the present application provides a method for inducing immune response in a patient with chronic hepatitis B, comprising administering a therapeutically effective amount of the antigen-antibody complex of the first aspect of the present application, the pharmaceutical composition of the second aspect of the present application, the vaccine composition of the third aspect of the present application, or the immune modulator of the sixth aspect of the present application.

[0111] In another preferred embodiment, the antigen-antibody complex, the pharmaceutical composition, the vaccine composition, or the immune modulator is administered at a dose of 30-150 ug per person, preferably 40-80 ug per person, more preferably 50-70 ug per person.

[0112] In another preferred embodiment, the antigen-antibody complex, the pharmaceutical composition, the vaccine composition, or the immune modulator is administered at a frequency of once every two weeks for a total of 3 times.

[0113] The eleventh aspect of the present application provides use of an immune modulator comprising a first immune modulator and a second immune modulator, each of which independently comprises a drug for reducing HBsAg level, the antigen-antibody complex of the first aspect of the present application, the pharmaceutical composition of the second aspect of the present application, or the vaccine composition of the third aspect of the present application, in the preparation of (a) a drug for inducing immune response in a patient with chronic hepatitis B; and / or (b) a drug for preventing and / or treating chronic hepatitis B.

[0114] In another preferred embodiment, the drug further comprises an antiviral drug, wherein the antiviral drug is administered before the administration of the first immune modulator and the second immune modulator.

[0115] In another preferred embodiment, the first immune modulator comprises a drug for reducing HBsAg level.

[0116] In another preferred embodiment, the antiviral drug is selected from the group consisting of any one or a combination of nucleotide reverse transcriptase inhibitors, nucleos(t)ide analogs (NA), capsid assembly modulators, antisense oligonucleotides, and entry inhibitors. More preferred nucleotide reverse transcriptase inhibitors are selected from the group consisting of Lamivudine (commonly known as 3TC), Telbivudine, Adefovir, Entecavir (ETV), Tenofovir disoproxil fumarate (TDF), Tenofovir alafenamide fumarate (TAF), or Tenofovir amibufenamide (TMF), or a combination thereof.

[0117] In another preferred embodiment, the drug that reduces the level of HBsAg is selected from the group consisting of polyclonal antibodies, monoclonal antibodies, targeted siRNA, small interfering RNA, or HBsAg inhibitors, or a combination thereof.

[0118] In another preferred embodiment, the drug that reduces the level of HBsAg comprises an antibody against HBsAg and a small interfering RNA.

[0119] In another preferred embodiment, the second immunomodulator comprises the antigen-antibody complex of claim 1, the pharmaceutical composition of claim 2, or the vaccine composition of claim 3.

[0120] The twelfth aspect of the present application provides a kit for (a) inducing an immune response in a patient with chronic hepatitis B; and / or (b) preventing and / or treating chronic hepatitis B, comprising:

[0121] The first agent is an antiviral drug;

[0122] The second agent is a first immunomodulator and a second immunomodulator, each of which independently comprises a drug that reduces the level of HBsAg, the antigen-antibody complex of the first aspect of the present application, the pharmaceutical composition of the second aspect of the present application, or the vaccine composition of the third aspect of the present application, wherein the antiviral drug is administered prior to the administration of the first immunomodulator and the second immunomodulator.

[0123] In another preferred embodiment, the first immunomodulator comprises a drug that reduces the level of HBsAg.

[0124] In another preferred embodiment, the second immunomodulator includes the antigen-antibody complex described in the first aspect of the present invention, the pharmaceutical composition described in the second aspect of the present invention, or the vaccine composition described in the third aspect of the present invention.

[0125] The thirteenth aspect of this invention provides a method for treating a disease, comprising:

[0126] Administering antiviral drugs to the subject;

[0127] When or after administering the antiviral drug, the patient is given a first immunomodulator and a second immunomodulator, each of which independently comprises a drug that lowers HBsAg levels, an antigen-antibody complex as described in the first aspect of the invention, a pharmaceutical composition as described in the second aspect of the invention, or a vaccine composition as described in the third aspect of the invention.

[0128] In another preferred embodiment, the disease includes chronic hepatitis B.

[0129] In another preferred embodiment, the dosage of the antigen-antibody complex, the pharmaceutical composition, or the vaccine composition is 30-150 μg / person, more preferably 40-80 μg / person, and even more preferably 50-70 μg / person.

[0130] In another preferred embodiment, the subject includes patients with chronic hepatitis B.

[0131] In another preferred embodiment, the chronic hepatitis B patient typically has a high level of HBV DNA (>2×10⁻⁶). 7 IU / ml), serum HBsAg levels are high (usually >1×10⁻⁶). 4 Chronic hepatitis B patients (IU / ml).

[0132] In another preferred embodiment, the chronic hepatitis B patient is a chronic hepatitis B patient who has received antiviral drug treatment and whose serum HBsAg level is <3000 IU / ml, or whose HBeAg has turned negative and whose HBsAg level is <1500 IU / ml.

[0133] In another preferred embodiment, when the antiviral drug is administered or after the administration of the antiviral drug, a second immunomodulator is administered to the patient after the administration of a first immunomodulator, when the patient's serum HBsAg level decreases to approximately ≤400 IU / ml.

[0134] In another preferred embodiment, the first immunomodulator includes a drug that lowers HBsAg levels.

[0135] In another preferred embodiment, the second immunomodulator includes the antigen-antibody complex described in the first aspect of the present invention, the pharmaceutical composition described in the second aspect of the present invention, or the vaccine composition described in the third aspect of the present invention.

[0136] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0137] Figure 1 is a schematic diagram of the polyacrylamide gel electrophoresis results of monoclonal antibodies G12-hIgG1, G12-mIgG1, G12-mIgG2a, KR127-hIgG1, KR127-mIgG1, KR127-mIgG2a and mouse polyclonal antibody (hepatitis B immunoglobulin (mHBIG)) against hepatitis B surface antigen (HBsAg) in Examples 1 and 2.

[0138] Figure 2 is a schematic diagram showing the binding affinity of human monoclonal antibody (G12-hIgG1) and mouse polyclonal antibody (mHBIG) to hepatitis B surface antigen (HBsAg) in Example 4.

[0139] Figures 3 and 4 are schematic diagrams showing the affinity results of human monoclonal antibody (G12-hIgG1) and mouse polyclonal antibody (mHBIG) for hepatitis B surface antigen (HBsAg) in Example 3.

[0140] Figures 5 and 6 are schematic diagrams showing the binding affinity of human monoclonal antibodies (G12-hIgG1 and KR127-hlgG1) and mouse monoclonal antibodies (G12-mIgG1, G12-mIgG2a, KR127-mIgG1 and KR127-mIgG1) to hepatitis B surface antigen (HBsAg) in Example 4.

[0141] Figure 7 shows the antibody levels in mice after immunization with antigen-antibody complex formulations in Example 6-2.

[0142] Figures 8-11 show the changes in antibody (HBsAb) expression levels in mice on days 7 and 35 after administration of antigen-antibody complex preparations made with different proportions of antigen-antibody from Examples 6-3.

[0143] Figures 12-15 show the trends of hepatitis B surface antigen (HBsAg) expression in mice after immunization with antigen-antibody complex preparations prepared with different ratios of antigen and antibody in Examples 6-4. Figure 15 is a schematic diagram of the overall trend of hepatitis B surface antigen (HBsAg) expression level when the antibody:antigen ratio is 1:6.

[0144] Figures 16-17 show the trends in antigen (HbsAg) expression in immunized mice induced by antigen-antibody complex formulations prepared from monoclonal antibodies of different epitopes in Examples 6-5.

[0145] Figures 18-19 show the trends and levels of antibody (HbsAb) expression in immunized mice induced by antigen-antibody complex formulations prepared from monoclonal antibodies of different epitopes in Examples 6-5.

[0146] Figures 20-24 are schematic diagrams showing the results of humoral immune factor levels after immunization in Example 6-6.

[0147] Figures 25-32 are schematic diagrams showing the results of cellular immune factor levels after immunization in Example 6-6.

[0148] Figure 33 shows the changes in serum HBsAg after immunization in Example 7.

[0149] Figure 34 shows the changes in serum HBV DNA after immunization in Example 7.

[0150] Figure 35 shows the changes in HBsAb after immunization in Example 7.

[0151] Figure 36 shows the difference in HBsAb levels after comparing Example 8 with other combination therapies using immunomodulators.

[0152] PBS: This is the control group.

[0153] TDF is a nucleotide reverse transcriptase inhibitor—tenofovir disoproxil fumarate;

[0154] G12 is a synthetic G12 monoclonal antibody;

[0155] siRNA is a small interfering RNA that targets hepatitis B surface antigen;

[0156] HBV DNA is the genome of the hepatitis B virus;

[0157] HBsAg is the hepatitis B surface antigen;

[0158] G12-hIgG1 is a monoclonal antibody synthesized from SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:5;

[0159] G12-mIgG1 is a monoclonal antibody synthesized from SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:6;

[0160] G12-mIgG2a is a monoclonal antibody synthesized from SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:7;

[0161] KR127-hIgG1 is a monoclonal antibody synthesized from SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5;

[0162] KR127-mIgG1 is a monoclonal antibody synthesized from SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:6;

[0163] KR127-mIgG2a is a monoclonal antibody synthesized from SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:7;

[0164] TVac-G is an antigen-antibody complex formulation based on the monoclonal antibody G12-hIgG1.

[0165] TVac-G12-mIgG1 or TVac-G-mIgG1 is an antigen-antibody complex formulation made from monoclonal antibody G12-mIgG1.

[0166] TVac-G-mIgG2a is an antigen-antibody complex formulation made from monoclonal antibody G12-mIgG2a;

[0167] TVac-G-2a or TVac-G12-mIgG2a or TVac-G12-2a or TVac-G-mIgG2a are antigen-antibody complex preparations made from monoclonal antibody G12-mIgG2a.

[0168] TVac-K is an antigen-antibody complex formulation based on the monoclonal antibody KR127-hIgG1.

[0169] TVac-KR127-mIgG1 or TVac-K-mIgG1 is an antigen-antibody complex formulation made from the monoclonal antibody KR127-mIgG1.

[0170] TVac-K-2a, TVac-K-IgG2a, TVac-KR127-2a, or TVac-K-mIgG2a are antigen-antibody complex formulations made from the monoclonal antibody KR127-mIgG2a.

[0171] TVac-mHBIG is an antigen-antibody complex preparation made from mouse polyclonal HBIG. Detailed Implementation

[0172] Through extensive and in-depth research and screening, the inventors have unexpectedly developed, for the first time, an antigen-antibody complex that induces an immune response in hepatitis B patients. This complex comprises hepatitis B surface antigen and anti-HBsAg monoclonal antibody, with the hepatitis B surface antigen binding to the anti-HBsAg monoclonal antibody. Furthermore, this invention is the first to discover that a complex composed of hepatitis B surface antigen and anti-HBsAg monoclonal antibody at a certain mass ratio (e.g., 0.67:1-54:1, preferably 1-20:1, more preferably 2-10:1, and even more preferably 3-8:1 (e.g., 6:1) can induce an immune response in patients with chronic hepatitis B and can also prevent and / or treat chronic hepatitis B. Based on these findings, this invention was completed.

[0173] the term

[0174] To facilitate a clearer understanding of this disclosure, certain terms are first defined. As used herein, unless otherwise expressly specified herein, each of the following terms shall have the meaning given below. Other definitions are set forth throughout the application.

[0175] The term “about” can refer to a value or composition within an acceptable range of error for a particular value or composition as determined by a person skilled in the art, which will depend in part on how the value or composition is measured or determined.

[0176] As used herein, the terms “optional” or “optionally” mean that the events or conditions described below may occur but are not required to occur. For example, “optionally containing 1-3 antibody heavy chain variable regions” means that the antibody heavy chain variable regions of a particular sequence may be present but are not required to be present, and may be 1, 2 or 3.

[0177] The term "sequence identity" as used in this invention refers to the degree of identity between two nucleic acid or two amino acid sequences when optimally aligned and compared with appropriate mutations such as substitutions, insertions, or deletions. The sequence identity between the sequences described in this invention and sequences exhibiting identity with them can be at least 85%, 90%, or 95%, preferably at least 95%. Non-limiting embodiments include 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100%.

[0178] Hepatitis B surface antigen (HBsAg) is the outer shell protein of the hepatitis B virus (HBV) and is a major component of HBV surface antigen. It is not infectious on its own, but its presence is often accompanied by the presence of the hepatitis B virus, so it is a marker of hepatitis B virus infection.

[0179] HBV is a partially double-stranded circular hepatotropic DNA virus. Its genome contains four partially overlapping open reading frames (ORFs): the pre-S / S region, the pre-C / C region, the P region, and the X region. The pre-S / S region encodes three outer membrane proteins of HBV: the large surface protein (LHB), the middle surface protein (MHB), and the small surface protein (SHB), collectively known as hepatitis B virus surface antigens. Translation of these proteins is initiated by three different start codons on the same gene segment. Based on the position of the start codon, the pre-S / S region can be divided into three distinct domains: Pre-S1, Pre-S2, and the S domain.

[0180] As used in this application, the term "antibody" can include all antibodies and any antigen-binding fragment (e.g., an antigen-binding fragment including a hinge, an antigen-binding fragment including a hinge and a CH1 domain, an antigen-binding fragment including a hinge and a CH2 domain, or an antigen-binding fragment containing portions of a hinge, a CH2 domain, and a CH3 domain) or a single chain thereof. "Antibody" can include, for example, naturally occurring and non-naturally occurring antibodies; monoclonal and polyclonal antibodies; chimeric and humanized antibodies; human and non-human antibodies; fully synthetic antibodies; and single-chain antibodies.

[0181] In one embodiment, "antibody" refers to a protein, such as a glycoprotein or its antigen-binding portion comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region. In some naturally occurring IgG, IgD, and IgA antibodies, the heavy chain constant region consists of a hinge, a CH1 domain, a CH2 domain, and a CH3 domain. In some naturally occurring antibodies, each light chain consists of a light chain variable region (VL) and a light chain constant region. The light chain constant region consists of a CL domain. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of an antibody can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

[0182] Immunoglobulins can be derived from any commonly known isotype, including but not limited to IgA, secretory IgA, IgG, and IgM. IgG isotypes are subclassed in some species: IgG1, IgG2, IgG3, and IgG4 in humans, and IgG1, IgG2a, IgG2b, and IgG3 in mice. In some embodiments, the antibody described herein is a human or mouse IgG1 or IgG2 subtype. Immunoglobulins (e.g., human IgG1) exist in several allotypes, differing from each other in at most a few amino acids.

[0183] As used in this application, the heavy chain constant region, such as the hinge region, may include “IgG1 isotype”, “IgG2 isotype”, “IgG3 isotype” or “IgG4 isotype”. The domain may contain the amino acid sequence of each isotype or a variant thereof (the amino acid sequences of each isotype in the domain are more homologous than those of other different isotypes).

[0184] As used in this invention, the term "antigen-binding portion" of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind antigens. The antigen-binding portion of an antibody can be divided into "hinges containing antigen-binding portions." Antigen-binding function of antibodies has been shown to occur via fragments of full-length antibodies. Examples of binding fragments encompassed within the "antigen-binding portion" of the antibody described in this invention include (i) Fab fragments, composed of V... L V H (ii) a monovalent fragment composed of the CL and CH1 domains; (iii) a divalent fragment containing two Fab fragments connected by disulfide bonds in the hinge region; and (iv) a fragment composed of V H (iv) The Fd fragment composed of the CH1 domain; L and V H The Fv fragment composed of domains; the (v)dAb fragment (Ward et al., (1989) Nature 341:544-546), which is composed of V H Domain composition; and (vi) separate complementary determinant regions (CDRs) or (vii) combinations of two or more separate CDRs optionally connected by a synthesis joint. Furthermore, although the two V segments of the Fv fragment... L and V H Encoded by individual genes, but linked together using synthetic linkers through recombinant methods, these single-chain polypeptides with antibody activity are called monovalent molecules known as single-chain Fvs (scFvs). Such single-chain antibodies are also encompassed within the term "antigen-binding moiety" of antibodies.

[0185] As used in this invention, "isotype" refers to an antibody class (e.g., IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE antibodies) encoded by a heavy chain constant domain gene. The full-length amino acid sequence of each wild-type human IgG constant region (including all domains, i.e., CH1 domain, hinge domain, CH2 domain, and CH3 domain) is cataloged in the UniProt database, for example, as P01857 (IgG1), P01859 (IgG2), P01860 (IgG3), and P01861 (IgG4) or their different isotypes. As used in this invention, a domain of the heavy chain constant region, such as the CH2 domain, is an IgG1 isotype or an IgG2 isotype, and this domain may contain the amino acid sequence of the corresponding domain of each isotype or a variant thereof (i.e., the amino acid sequence of each isotype within this domain shows higher homology than other different isotypes).

[0186] In this invention, the "Fc region" (fragment crystallizable region), "Fc domain," or "Fc" refers to the C-terminal region of the heavy chain of an antibody that mediates the binding of immunoglobulins to host tissues or factors (including binding to Fc receptors on different cells of the immune system, such as effector cells, or binding to the first component (C1q) of the classical complement system). Therefore, the Fc region of an isotype IgG antibody comprises the heavy chain constant region of the antibody excluding the first constant region, the immunoglobulin domain (CH1). In IgG, IgA, and IgD antibody isotypes, the Fc region comprises the CH2 and CH3 constant domains of the heavy chain of each antibody; the Fc region of IgM and IgE comprises three heavy chain constant domains (CH domains 2-4) of each polypeptide chain. For IgG, the Fc region comprises the immunoglobulin domain consisting of the hinge, CH2, and CH3. The Fc can be natural (…). Fc (either naturally occurring or wild-type), including any allotype, or variant Fc (e.g., non-naturally occurring Fc), includes, for example, 1, 2, 3, 4, 5, 1-5, 1-10, or 5-10 or more amino acid mutations, such as substitution, addition, or deletion. For example, a variant Fc may contain at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the same amino acid sequence as the wild-type Fc. Modified or mutated Fc may have enhanced or reduced effector function and / or half-life. The CH2 and CH3 regions are the primary sites for effector function and FcRn binding. Fc may refer to isolated Fc or, in the context of a protein polypeptide containing an Fc (e.g., "binding protein containing an Fc region"), also referred to as an "Fc fusion protein" (e.g., an antibody or immunoadhesin).

[0187] "Fc receptors," or "FcRs," are receptors that bind to the Fc region of immunoglobulins. FcRs that bind to IgG antibodies include receptors from the FcγR family, including allelic variants and alternative splice forms of these receptors. The FcγR family consists of three active receptors (FcγRI, FcγRIII, and FcγRIV in mice; FcγRIA, FcγRIIA, and FcγRIIIA in humans) and one inhibitory receptor (FcγRIIB). Most natural effector cell types co-express one or more active FcγRs and the inhibitory FcγRIIB, while natural killer (NK) cells selectively express one active Fc receptor (FcγRIII in mice and FcγRIIIA in humans) instead of the inhibitory FcγRIIB found in both mice and humans. Human IgG1 binds to most Fc receptors and is considered equivalent to mouse IgG2a in terms of the type of active Fc receptor it binds to.

[0188] In this article, "treatment" refers to an attempt to alter the natural course of disease in the treated individual, and may be a clinical intervention implemented for prevention or during the course of clinicopathological processes. The desired effects of treatment include, but are not limited to, preventing the onset or recurrence of disease, relieving symptoms, reducing any direct or indirect pathological consequences of the disease, slowing the rate of disease progression, improving or eliminating the disease state, and regressing or improving prognosis.

[0189] In this article, "individual" or "subject" refers to a mammal. Mammals include, but are not limited to, primates (such as humans and non-human primates such as monkeys) or other mammals (such as cattle, sheep, cats, dogs, horses, rabbits, and rodents such as mice and rats).

[0190] In this article, "mutant" or "variant" can refer to a molecule obtained by mutating one or more nucleotides or amino acids in any naturally occurring or engineered molecule.

[0191] The standard recombinant DNA and molecular cloning techniques used in the examples are well-known in the art (Ausubel, FM et al., Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley-Interscience). Major chemical and biological reagents were purchased from KAPA Biosystems, New England Biolabs, TransGen Biotech, Thermo Fisher Scientific, OMEGA bio-tek, etc.

[0192] Immunocomplex vaccines, also known as antigen-antibody complex vaccines, are a new type of vaccine developed in the 1990s. They are made by mixing specific antiserum with viral antigens in a certain proportion. Their advantages are high safety and better immunization effect than conventional vaccines.

[0193] Because hepatitis B surface antigen (HBsAg) contains many transmembrane hydrophobic regions and disulfide bonds, it will naturally aggregate into large spherical or rod-shaped polymeric particles in solution, thus making it impossible to construct a homogeneous and soluble Fc fusion protein through genetic engineering technology.

[0194] According to the Guidelines for the Diagnosis and Treatment of Chronic Hepatitis B at the Primary Care Level (Practice Version 2020), chronic hepatitis B patients refer to those with chronic inflammatory liver disease caused by persistent HBV infection for more than 6 months. The stages of chronic hepatitis B virus infection include: (i) the immune tolerance phase (chronic HBV carrier state): mostly during the perinatal and infancy periods, with simultaneous positivity for serum HBsAg, hepatitis B e antigen (HBeAg), and anti-hepatitis B core antigen (anti-HBc), and high HBV DNA levels (usually HBV DNA > 2 x 10^6). 7 (i) IU / ml), alanine aminotransferase (ALT) is normal; (ii) Immune clearance phase (HBeAg-positive CHB): HBeAg-positive stage, ALT is persistently or repeatedly abnormal and HBV DNA level is high (usually HBV DNA > 2 x 10⁻⁶). 4 (i) IU / ml), HBeAg seroconversion, anti-HBe seroconversion, and ALT persistent or intermittent elevation may occur; (iii) Immune control period (inactive HBsAg carrier state): low replication period, normal ALT, low HBV DNA level (HBV DNA < 2x10). 3 (iv) Reactivation (HBeAg-negative CHB): 5%–15% of inactive patients may experience one or more episodes of hepatitis, with persistent or recurrent abnormal ALT levels and HBV DNA > 2 x 10⁻⁶. 3 IU / ml, presenting as HBeAg negative CHB, may recur with HBeAg seroconversion (Chinese Journal of General Practitioners, 2021, 20(03): 281-289.).

[0195] According to the expert consensus on clinical cure (functional cure) of chronic hepatitis B (Chinese Journal of Infectious Diseases, 2019, 37(8): 461-472.), the current clinical treatment of chronic hepatitis B patients mainly focuses on the combination of antiviral drugs and immunomodulators. The combination of antiviral drugs and immunomodulators mainly includes the initial combination therapy strategy and the sequential combination therapy strategy. The latter includes the "switch" strategy (i.e., replacing antiviral drugs with immunomodulators) and the "add" strategy (i.e., adding immunomodulators to antiviral drugs). The expert consensus recommends that CHB patients who receive long-term antiviral drug treatment should be those with undetectable HBV DNA and HBsAg level <3000 IU / ml, HBeAg positive or HBsAg level ≥1500 IU / ml, or HBeAg seroconversion and HBsAg level <1500 IU / ml. It strongly recommends the use of the sequential combination immunomodulator therapy strategy.

[0196] This invention unexpectedly discovered that antigen-antibody complex preparations made from HBsAg and anti-HBsAg monoclonal antibodies are superior to antigen-antibody complex preparations made from polyclonal antibodies in sustaining the induction of immune responses in patients with chronic hepatitis B; especially when chronic hepatitis B patients are in the later stages of the immune control phase of infection (Chinese Journal of General Practitioners, 2021, 20(03): 281-289) or have serum HBV DNA <2x10 after long-term antiviral drug treatment. 3 When the HBsAg level is <3000 IU / ml and the ALT level is <3000 IU / ml in patients with chronic hepatitis B, or when the ALT level is normal in patients with chronic hepatitis B, the antigen-antibody complex preparation prepared by the monoclonal antibody of this invention can sustainably induce and enhance the host immune response, clear HBsAg from the body of patients with chronic hepatitis B, and improve the clinical cure rate of chronic hepatitis B. (The patients with chronic hepatitis B in this invention are those who have reduced their HBV DNA and HBsAg levels to a certain level after treatment with antiviral drugs and antibody drugs (i.e., serum HBV DNA <2x10). 3 (For patients with IU / ml and HBsAg levels <3000 IU / ml or normal ALT in patients with chronic hepatitis B), administering the antigen-antibody complex vaccine of this invention will result in better immunotherapy effects in patients.

[0197] First dose of immune modulator

[0198] As used in this article, "first-dose immunomodulator" and "first-dose immunomodulator" are interchangeable and both refer to drugs that lower HBsAg levels, including but not limited to polyclonal antibodies, monoclonal antibodies, targeted siRNA, small interfering RNA, or HBsAg inhibitors.

[0199] Second dose of immune modulator

[0200] As used herein, "second dose of immunomodulator" and "second dose of immunomodulator" are interchangeable and both refer to antigen-antibody complexes, drugs containing antigen-antibody complexes, or vaccines containing antigen-antibody complexes, preferably the antigen-antibody complexes described in this invention.

[0201] Composition and method of application

[0202] The present invention also provides a composition comprising: (i) an antigen-antibody complex prepared by the method of the present invention, and (ii) a pharmaceutically or immunologically acceptable excipient or adjuvant. In this invention, the term "comprising" means that various components may be used together or present in the composition of the present invention. Therefore, the terms "consistent with..." and "consisting with..." are included in the term "comprising".

[0203] The compositions of the present invention include pharmaceutical compositions and vaccine compositions. The compositions of the present invention can be monovalent or polyvalent.

[0204] The pharmaceutical or vaccine compositions of the present invention can be prepared into various conventional dosage forms, including (but not limited to): injections, granules, tablets, pills, suppositories, capsules, suspensions, sprays, etc.

[0205] (i) Pharmaceutical Composition

[0206] The pharmaceutical composition of the present invention comprises an effective amount of an antigen-antibody complex prepared by the method of the present invention, wherein the antigen-antibody complex may be monovalent or polyvalent.

[0207] As used herein, the term "effective dose" refers to the amount of a therapeutic agent that treats, alleviates, or prevents a target disease or condition, or the amount that exhibits a detectable therapeutic or preventative effect. This effect can be detected, for example, by antigen levels. Therapeutic effects also include a reduction in physiological symptoms. The precise effective dose for a given subject depends on that subject's body size and health status, the nature and severity of the condition, and the choice of the therapeutic agent and / or combination of therapeutic agents administered. Therefore, it is not useful to predetermine an accurate effective dose. However, for a given condition, the effective dose can be determined using routine laboratory methods.

[0208] For the purposes of this invention, the effective dose of the vaccine complex is approximately 30 to 150 μg given to an individual.

[0209] Pharmaceutical compositions may also contain pharmaceutically acceptable carriers. The term "pharmaceutically acceptable carrier" refers to a carrier used for the administration of a therapeutic agent (e.g., recombinant protein or other therapeutic agent). This term refers to pharmaceutical carriers that do not induce antibodies harmful to the individual receiving the composition and do not cause excessive toxicity after administration. Suitable carriers can be large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acid, polyglycolic acid, etc. These carriers are well known to those skilled in the art. A thorough discussion of pharmaceutically acceptable carriers or excipients can be found in Remington's Pharmaceutical Sciences (Mack Pub. Co., NJ 1991).

[0210] Pharmaceutically acceptable carriers in a composition may include liquids such as water, saline, glycerol, and ethanol. Additionally, these carriers may contain auxiliary substances such as wetting agents or emulsifiers, pH buffers, etc. Typically, the composition can be formulated as an injectable preparation, such as a liquid solution or suspension; it can also be formulated as a solid form suitable for reconstitution into solutions or suspensions, or liquid excipients, prior to injection. Liposomes are also included in the definition of pharmaceutically acceptable carriers.

[0211] (ii) Vaccine composition

[0212] The vaccine compositions of the present invention can be prophylactic (i.e., for infection prevention) or therapeutic. The vaccine compositions comprise the antigen-antibody complex of the present invention and are generally combined with pharmaceutically acceptable carriers, including any carrier that does not itself induce antibodies harmful to the individual receiving the composition. Suitable carriers are typically large, slowly metabolizing macromolecules such as proteins, polysaccharides, polylactic acid, polyglycolic acid, amino acid polymers, amino acid copolymers, lipid aggregates (such as oil droplets or liposomes), etc. These carriers are well known to those skilled in the art. Additionally, these carriers can act as immunostimulants (“adjuvants”). Furthermore, the antigen may also be conjugated with bacterial toxoids (such as toxoids of pathogens such as diphtheria, tetanus, cholera, and Helicobacter pylori).

[0213] Preferred adjuvants for enhancing the efficacy of immune compositions include, but are not limited to: (1) aluminum salts, such as aluminum hydroxide, aluminum phosphate, aluminum sulfate, etc.; (2) oil-in-water emulsion formulations, for example, (a) MF59 (see WO 90 / 14837), (b) SAF, and (c) Ribi. TMThe adjuvant system (RAS) (Ribi Immunochem, Hamilton, MT), (3) saponin adjuvants; (4) Freund complete adjuvant (CFA) and Freund incomplete adjuvant (IFA); (5) cytokines such as interleukins (e.g., IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, etc.), interferons (e.g., gamma interferon), macrophage colony-stimulating factor (M-CFS), tumor necrosis factor (TNF), etc.; (6) detoxified variants of bacterial ADP-ribosylated toxins (e.g., cholera toxin CT, pertussis toxin PT, or Escherichia coli heat unstable toxin LT), see, for example, WO93 / 13302 and WO92 / 19265; and (7) other substances that enhance the effect of the composition as immunostimulants.

[0214] Vaccine compositions, including immunogenic compositions (e.g., may include antigen-antibody complexes, pharmaceutically acceptable carriers, and adjuvants), typically contain diluents such as water, saline, glycerol, ethanol, etc. Additionally, auxiliary substances such as wetting agents or emulsifiers, pH buffers, etc., may be present in these carriers.

[0215] More specifically, vaccines, including immunogenic compositions, contain an immunologically effective amount of an immunogenic peptide, as well as the other required components mentioned above. An "immunologically effective amount" refers to the amount administered to an individual as a single or partial dose that is effective for treatment or prevention. This dosage can be determined based on the individual's health and physiological condition, the individual's class (e.g., human), the individual's immune system's ability to synthesize antibodies, the required level of protection, the vaccine formulation, the treating physician's assessment of the medical condition, and other relevant factors. This dosage is expected to be within a relatively wide range and can be determined through routine laboratory testing.

[0216] Typically, vaccine compositions or immunogenic compositions can be formulated as injectable preparations, such as liquid solutions or suspensions; they can also be formulated as solid forms suitable for reconstitution into solutions or suspensions or liquid excipients prior to injection. The formulation may also be emulsified or encapsulated in liposomes to enhance adjuvant effects.

[0217] (iii) Route of administration and dosage

[0218] The composition can be administered directly to a subject. The subject can be a human or a non-human mammal, preferably a human. When used as a vaccine, the antigen-antibody complex of the present invention can be administered directly to an individual using known methods. These vaccines are typically administered via the same route of administration as conventional vaccines and / or by mimicking the pathogen infection pathway.

[0219] The routes of administration for the pharmaceutical or vaccine compositions of the present invention include (but are not limited to): intramuscular, subcutaneous, intradermal, intrapulmonary, intravenous, nasal, vaginal, oral, or other parenteral routes. Routes of administration may be combined if necessary, or adjusted according to the disease condition. The vaccine compositions may be administered in single or multiple doses, and may include booster doses to induce and / or maintain immunity.

[0220] The antigen-antibody complex of the present invention should be administered in an "effective amount," meaning that the amount of the antigen-antibody complex is sufficient to elicit an immune response in the selected route of administration, effectively inducing an immune response in patients with chronic hepatitis B; and / or preventing and / or treating chronic hepatitis B.

[0221] The amount of antigen-antibody complex selected in each vaccine dose is determined based on the amount that can elicit a protective immune response without significant side effects. Typically, after infection of host cells, each dose of vaccine is sufficient to contain approximately 1 μg-1000 μg, preferably 1 μg-100 μg, and more preferably 10 μg-50 μg of antigen-antibody complex. The optimal dosage of a specific vaccine can be determined using standard research methods, including antibody titers and other responses in the subjects. Whether a booster dose is needed can be determined by monitoring the level of immunity provided by the vaccine. After assessing the antibody titer in the serum, a booster dose immunization may be necessary. Administration of adjuvants and / or immunostimulants can enhance the immune response to the antigen-antibody complex of the present invention. A preferred method is to administer the antigen-antibody complex via injection via a parenteral (subcutaneous or intramuscular) route.

[0222] The main advantages of this invention include:

[0223] (1) This invention unexpectedly discovered that antigen-antibody complex preparations made from HBsAg and anti-HBsAg monoclonal antibodies are superior to antigen-antibody complex preparations (or pharmaceutical compositions or vaccine compositions) made from polyclonal antibodies in continuously inducing immune responses in patients with chronic hepatitis B; especially when patients with chronic hepatitis B are in the later stages of the immune control period of infection (Chinese Journal of General Practitioners, 2021, 20(03): 281-289) or have serum HBV DNA <2x10 after long-term antiviral drug treatment. 3 When the HBsAg level is <3000 IU / ml or the alanine aminotransferase (ALT) level is normal in patients with chronic hepatitis B, the antigen-antibody complex preparation made from the monoclonal antibody of this invention can continuously induce and enhance the host's immune response, clear HBsAg from the body of patients with chronic hepatitis B, and improve the clinical cure rate of chronic hepatitis B.

[0224] (2) This invention is the first to develop an antigen-antibody complex that induces an immune response in hepatitis B patients. The complex comprises hepatitis B surface antigen and anti-HBsAg monoclonal antibody, with the hepatitis B surface antigen binding to the anti-HBsAg monoclonal antibody. Furthermore, this invention is the first to discover that a complex composed of hepatitis B surface antigen and anti-HBsAg monoclonal antibody at a certain mass ratio (e.g., 0.67:1-54:1, preferably 1-20:1, more preferably 2-10:1, and even more preferably 3-8:1 (e.g., 6:1) can induce an immune response in patients with chronic hepatitis B and can also prevent and / or treat chronic hepatitis B.

[0225] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0226] Unless otherwise specified, all materials and reagents used in the embodiments of this invention are commercially available products.

[0227] Example 1: Production of Monoclonal Antibodies for Different Epitopes of Surface Antigens

[0228] The G12 anti-HBsAg monoclonal antibody sequence (anti-small HBsAg) has the heavy chain VH+CH1 amino acid sequence as SEQ ID NO:1 and the light chain VL+CL amino acid sequence as SEQ ID NO:2.

[0229] The amino acid sequence of the heavy chain VH+CH1 of the KR127 anti-HBsAg monoclonal antibody (anti-pre-S1) is SEQ ID NO:3, and the amino acid sequence of the light chain VL+CL is SEQ ID NO:4.

[0230] The plasmid construction of the antibodies used in this invention is as follows:

[0231] Step 1: The heavy and light chain sequences of monoclonal antibodies G12 and KR127 were synthesized into eukaryotic expression vectors (pcDNA3.1, purchased from Genscript) using Genscript.

[0232] Step 2: The heavy chain sequence of the product from Step 1 was spliced ​​with the sequences of human IgG1 Fc (amino acid sequence SEQ ID NO:5), mouse IgG1 Fc (amino acid sequence SEQ ID NO:6), and IgG2a Fc (amino acid sequence SEQ ID NO:7) via overlap PCR. The spliced ​​products were then constructed into the eukaryotic expression vector pTT5 (purchased from BGI Genomics Co., Ltd., sequence shown in SEQ ID NO:8) via homologous recombination. All of the aforementioned sequences contain a hinge region, CH2, and CH3.

[0233] Step 3: The recombinant products from Steps 1 and 2 were transformed into DH5α competent cells (from the plasmid vector strain cell gene preservation center). Single colonies were picked from overnight-grown ampicillin plates and cultured in LB bacterial culture medium. The bacterial culture was then sent for sequencing to obtain plasmids: G12-hIgG1-HC, G12-mIgG1-HC, G12-mIgG2a-HC, G12 light chain (G12-LC), KR127-hIgG1-HC, KR127-mIgG1-HC, KR127-mIgG2a-HC, and KR127 light chain (KR127-LC).

[0234] The preparation of anti-HBsAg monoclonal antibodies was basically carried out according to the literature (Cell Host Microbe. 2017. 22(4): 471-483.e5.), and the preparation process is as follows:

[0235] Step 1: Using the HEK293F mammalian suspension cell expression system, express the following six antibodies respectively:

[0236] G12-hIgG1: SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:5 spliced ​​sequence.

[0237] KR127-hIgG1: SEQ ID NO:3 and SEQ ID NO:4 and SEQ ID NO:5 spliced ​​sequence.

[0238] G12-mIgG1: SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:6 spliced ​​sequence.

[0239] G12-mIgG2a: SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:7 spliced ​​sequence.

[0240] KR127-mIgG1: SEQ ID NO:3 and SEQ ID NO:4 and SEQ ID NO:6 spliced ​​sequence.

[0241] KR127-mIgG2a: SEQ ID NO:3 and SEQ ID NO:4 and SEQ ID NO:7 spliced ​​sequence.

[0242] The above-mentioned heavy chain plasmid HC, light chain plasmid LC, and transfection reagent (branched polyethyleneimine (PEI), MB2603-1, Meilun Biotechnology) were mixed at a mass ratio of 1:1:6, incubated at room temperature for 15-30 minutes, and then poured into HEK293F cells (purchased from ATCC cell bank) while shaking. The cells were then cultured at 37 degrees Celsius in a shaker with 5% carbon dioxide for 5-7 days.

[0243] Step 2: Centrifuge the cells from Step 1 at 3000 rpm for 10 minutes, collect the supernatant, filter it through a 0.8 μM filter, and equilibrate it with equilibration buffer (1 M PBS buffer) through Protein G Resin resin (Genscript, product number: L00209-100). Add the supernatant to the resin, controlling the flow rate at 1 ml / min. Elute contaminating proteins with 2 column volumes of equilibration buffer, and finally elute bound immunoglobulins with 0.1 M glycine (pH = 2.8).

[0244] Step 3: Transfer the immunoglobulin solution eluted in the previous steps to a 3kD MWCO Amicon Ultra centrifuge ultrafiltration tube (purchased from Millipore), and ultrafilter at 4°C and 6500g for 20 min. Replace the elution buffer with PBS solution and repeat the ultrafiltration 3-4 times. Collect the concentrated antibodies, which are human Fc chimeric monoclonal antibodies G12-hIgG1 and KR127-hIgG1 and mouse Fc chimeric monoclonal antibodies G12-mIgG1, G12-mIgG2a, KR127-mIgG1, and KR127-mIgG2a.

[0245] The absorbance of the antibody at 280 nm in step 3 was measured using a spectrophotometer (BioTek) to quantify protein concentration, and the purity was detected by SDS-PAGE electrophoresis, as shown in Figure 1. This method was used to prepare the antigen-antibody complex (Therapeutic vaccine, TVac).

[0246] Example 2 Production of Mouse Anti-Hepatitis B Immunoglobulin (mHBIG)

[0247] Six- to eight-week-old male C57BL / 6 SPF mice (purchased from Shanghai Jihui) were mixed with hepatitis B virus surface antigen protein HBsAg (purchased from North China Pharmaceutical Group), which was expressed and purified from stable CHO cells (North China Pharmaceutical Group Co., Ltd.), plus aluminum hydroxide adjuvant (Thermo Scientific, product number: XF345301) at a dose of 100ug per mouse.

[0248] The above-mentioned mixed immunogen was administered subcutaneously to the back of each animal at a dose of 2 μg per animal, once every two weeks, for a total of 3 immunizations.

[0249] Subsequently, blood was collected from the retroorbital sinus of mice after the second and third immunizations. The supernatant was collected after centrifugation at 5000 rpm for 5 minutes at room temperature to obtain serum. The serum titer was detected by ELISA using the hepatitis B virus surface antigen protein mentioned above. The serum was then purified to obtain anti-mouse hepatitis B immunoglobulin (mHBIG), as shown in Figure 1.

[0250] Example 3: Affinity test of antibody with hepatitis B surface antigen (HBsAg)

[0251] The binding kinetics of antibodies (mHBIG and G12-hlgG1) to HBsAg were detected using bio-layer interferometry (BLI) on an Octet-RED96 (ForteBio) sensor. The activated biosensor (AR2G) was first loaded with 15 mg / mL HBsAg for 600 s, followed by quenching for 300 s. The sensor was then incubated with three-fold diluted antibodies (1000 nM (mHBIG) or 50 nM (G12-hlgG1)) in PBST (PBS containing 0.02% Tween 20) for 300 s, followed by dissociation in PBST for 300 s. All curves were fitted using a 1:2 binding model with Data Analysis 10.0 software. KD values ​​were determined with R² values ​​greater than the 95% confidence interval.

[0252] The BLI results, as shown in Figures 3 and 4, indicate that the monoclonal antibody (G12-hIgG1) has a better affinity for hepatitis B surface antigen (HBsAg) than the polyclonal antibody (anti-mouse hepatitis B immunoglobulin (mHBIG)).

[0253] Example 4: Binding affinity test of antibody to hepatitis B surface antigen (HBsAg)

[0254] Hepatitis B surface antigen (HBsAg) was coated onto an ELISA plate (purchased from Corning) with PBS, 50 μg / well, and incubated overnight at 4°C. Then, antibodies from Examples 1 and 2 (containing anti-hepatitis B immunoglobulin (mHBIG), G12-hIgG1, KR127-hIgG1, G12-mlgG1, G12-mIgG2a, KR127-mIgG1, and KR127-mIgG2a) were added sequentially at an initial concentration of 1 μM and diluted 3-fold. Goat anti-mouse IgG or anti-human IgG-Fc tag antibodies (both purchased from SIGMA) were then added to detect the binding affinity of each antibody to the surface antigen (see Cell Host Microbe. 2020. 27(6): 891-898.e5. for details).

[0255] As shown in Figure 2, the ELISA results indicate that the monoclonal antibody (G12-hIgG1) binds to hepatitis B surface antigen (HBsAg) more readily than the polyclonal antibody (anti-hepatitis B immunoglobulin (HBIG)).

[0256] Figures 5 and 6 show that the different antibodies G12-hIgG1, KR127-hIgG1, G12-mlgG1, G12-mIgG2a, KR127-mIgG1 and KR127-mIgG2a generated in Example 1 all have the activity of binding to hepatitis B surface antigen (HBsAg) and the binding activities are not significantly different.

[0257] Example 5: Preparation of a mouse model of chronic hepatitis B virus infection

[0258] Six- to eight-week-old male C57BL / 6 SPF mice (purchased from Shanghai Jihui) were injected with rAAV8-1.3HBV-ayw (titer 1×10⁻⁶) purchased from Beijing Wujiahe Biotechnology Co., Ltd. 12 2×10 μg / mL) tail vein injection 10 μg / animal, with continuous infection for two weeks to establish an animal model of persistent infection.

[0259] Example 6: Immunization using an immunogenic antigen-antibody complex formulation

[0260] Clinical combination therapy for chronic hepatitis B includes combination therapy strategies. This embodiment uses the antigen-antibody complex of the present invention as an immunomodulator to select a combination therapy strategy, and implements it in a mouse model. The steps are as follows:

[0261] Step 1: Use antiviral drugs to inhibit viral replication and reduce serum viral load throughout the entire treatment process;

[0262] Step 2: Administer the first dose of an immunomodulator to reduce serum HBsAg levels;

[0263] Step 3: Administer a second dose of immunomodulator to enhance and induce an effective host immune response.

[0264] The antiviral drugs mentioned in step 1 include nucleotide reverse transcriptase inhibitors, nucleos(t)ide analogs (NA), capsid assembly regulators, antisense oligonucleotides, and invasion inhibitors. In this embodiment, a nucleotide reverse transcriptase inhibitor is used. The nucleotide reverse transcriptase inhibitor can be any or a combination of anti-hepatitis B virus drugs such as lamivudine (generally known as 3TC), telbivudine, adefovir dipivoxil, entecavir (ETV), tenofovir disoproxil fumarate (TDF), tenofovir alafenamide fumarate (TAF), or tenofovir amibufenamide (TMF).

[0265] The immunomodulators used in steps 2 and 3 are selected from any one or a combination of pegylated interferon alpha (Peg-IFN-α), polyclonal antibodies, monoclonal antibodies, targeting siRNA, small interfering RNA, HBsAg inhibitors, or the antigen-antibody complex formulation of the present invention.

[0266] In steps 2 and 3, the first and second doses of immunomodulators can be the same, different, or interchangeable.

[0267] 6-1 Preparation of antigen-antibody complex (TVac) as the second immunomodulatory agent: Take purified hepatitis B virus surface antigen at a mass ratio of at least greater than 0.67:1 and mix it separately with G12-hIgG1, G12-mIgG1, G12-mIgG2a, KR127-hIgG1, KR127-mIgG1, KR127-mIgG2a antibodies or anti-mouse hepatitis B immunoglobulin (mHBIG) generated in Examples 1 and 2. Incubate at 37 degrees for 40 minutes, then incubate overnight at 4 degrees. Add 100 μg of aluminum hydroxide adjuvant (other commercially available hepatitis B vaccine adjuvants such as CpG1080, aluminum phosphate adjuvant, or a mixture of aluminum hydroxide and aluminum phosphate are also applicable) to produce an antigen-antibody complex formulation targeting different epitopes of hepatitis B virus surface antigen.

[0268] The antigen-antibody complex formulation prepared from anti-mouse hepatitis B immunoglobulin complex is TVac-mHBIG (containing hepatitis B surface antigen HBsAg and mouse hepatitis B immunoglobulin mHBIG from Example 2); the antigen-antibody complex formulation prepared from G12-hIgG1 is TVac-G (containing hepatitis B surface antigen HBsAg and G12-hIgG1 antibody expressed in Example 1); the antigen-antibody complex formulation prepared from G12-mIgG1 is TVac-G12-mIgG1 (or TVac-G-mIgG1) (containing hepatitis B surface antigen HBsAg and G12-mIgG1 antibody expressed in Example 1); and the antigen-antibody complex formulation prepared from G12-mIgG2a is TVac-G-2a (or TVac-G12-mIgG2a or TVac-G12-2a or TVac-G-mIgG2a) (containing hepatitis B surface antigen HBsAg). The antigen-antibody complex formulation prepared from KR127-hIgG1 is TVac-K (containing hepatitis B surface antigen HBsAg and KR127-hIgG1 antibody expressed in Example 1), the antigen-antibody complex formulation prepared from KR127-mIgG1 is TVac-KR127-mIgG1 (or TVac-K-mIgG1) (containing hepatitis B surface antigen HBsAg and KR127-mIgG1 antibody expressed in Example 1), and the antigen-antibody complex formulation prepared from KR127-mIgG2a is TVac-K-2a (or TVac-KR127-IgG2a or TVac-KR127-2a or TVac-K-mIgG2a) (containing hepatitis B surface antigen HBsAg and KR127-mIgG2a antibody expressed in Example 1).

[0269] 6-2 The effects of antigen-antibody complex preparations made from G12 monoclonal antibodies and antigen-antibody complex preparations made from anti-hepatitis B immunoglobulin as second-dose immunomodulators:

[0270] Step 1: In a mouse model of chronic hepatitis B (described in Example 5), tenofovir disoproxil fumarate (TDF), a nucleotide reverse transcriptase inhibitor, was administered via gavage for 5 consecutive days at 15 mg / kg. Step 2: On day 6, half an hour after TDF administration, the first immunomodulatory agent was injected via tail vein. This first agent was a G12 monoclonal antibody (SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:5 spliced ​​sequences) synthesized in the literature (mAbs vol. 8, 3(2016):468-77.), at 6.7 mg / kg, to reduce surface antigen levels. Step 3: One hour after step 2, a second agent was administered intraperitoneally, including an immunomodulatory agent-antigen antibody complex (TVac-G and TVac-mHBIG antigen antibody complexes) or hepatitis B surface antigen HBsAg, administered every two weeks for a total of 3 times. The experimental groups in this example are as follows:

[0271] PBS: The control group received only PBS;

[0272] TDF+G12: A combination of antiviral drugs and the first-dose immunomodulatory agent G12 monoclonal antibody;

[0273] TDF+G12+HBsAg: A combination of antiviral drugs, the first dose of the immunomodulatory agent G12 monoclonal antibody, and the second dose of hepatitis B surface antigen HBsAg.

[0274] TDF+G12+TVac-G: A combination of antiviral drugs, a first-dose immunomodulatory agent G12 monoclonal antibody, and a second-dose TVac-G (hepatitis B surface antigen HBsAg and G12-hIgG1 antibody complex expressed in Example 1);

[0275] TDF+G12+TVac-mHBIG: A combination of antiviral drugs, a first dose of the immunomodulatory agent G12 monoclonal antibody, and a second dose of TVac-mHBIG (hepatitis B surface antigen HBsAg and the mHBIG antibody complex of Example 2).

[0276] Figure 7 shows the antibody expression levels in mice one week after the first immunization. The results showed that mice in the PBS, TDF+G12, and TDF+G12+HBsAg groups did not produce antibodies (HBsAb), while mice in the TDF+G12+TVac-G and TDF+G12+TVac-mHBIG groups produced antibodies (HBsAb). The antibody (HBsAb) expression level induced by the monoclonal antibody-derived antigen-antibody complex formulation (TDF+G12+TVac-G) was more than 1 times higher than that induced by the polyclonal antibody-derived antigen-antibody complex formulation (TDF+G12+TVac-mHBIG).

[0277] 6-3 Antigen-antibody complex formulations with different antigen-antibody ratios induced antibody levels: Following the procedure in "6-1 Preparation of complex (TVac) as a second immunomodulator," antigen-antibody complex formulations with different antigen-antibody ratios were prepared and used as a second immunomodulator. Serum antibody (HBsAb) levels were detected by ELISA at 7 and 35 days after administration of the second immunomodulator. The experimental groups in this example are as follows:

[0278] TVac-mHBIG: The second dose of immunomodulatory agent was an anti-hepatitis B immunoglobulin complex preparation, in which the antibody:antigen mass ratio was 1:54, 1:18, 1:6, 1:2 and 1:0.67, respectively;

[0279] TVac-G12-2a: The second dose of immunomodulatory agent is a complex preparation of G12-mIgG2aFc monoclonal antibody, wherein the antibody:antigen mass ratios are 1:54, 1:18, 1:6, 1:2 and 1:0.67, respectively.

[0280] TVac-KR127-2a: The second dose of immunomodulatory agent is a complex preparation made from KR127-mIgG2aFc monoclonal antibody, wherein the antibody:antigen mass ratios are 1:54, 1:18, 1:6, 1:2 and 1:0.67, respectively.

[0281] The results are shown in Figures 8, 9, 10, and 11. When the antibody-to-antigen ratio of the complex formulation was 1:6, the antigen-antibody complex formulations (TVac-G12-2a and TVac-KR127-2a) prepared from monoclonal antibodies G12-mIgG2aFc and KR127-mIgG2aFc induced antibody levels more than twice that of the polyclonal antibody antigen-antibody complex formulation (TVac-mHBIG) on days 7 and 35. Figures 10 and 11 show that, after long-term observation with the same dose of antigen-antibody complex formulation, the monoclonal antibody antigen-antibody complex formulations (TVac-G12-2a and TVac-KR127-2a) were superior to the polyclonal antibody antigen-antibody complex formulation (TVac-mHBIG) in sustaining the induction of immune responses in mice.

[0282] The results showed that antigen-antibody complex formulations prepared with monoclonal antibodies (G12 or KR127) were superior to antigen-antibody complex formulations prepared with polyclonal antibodies (HBIG) in treating or inducing an immune response to chronic hepatitis B.

[0283] 6-4 The suppression of hepatitis B surface antigen levels by antigen-antibody complex preparations with different antigen-antibody ratios: Following the procedure in "6-1 Preparation of complex (TVac) as a second immunomodulator," antigen-antibody complex preparations with different antigen-antibody ratios were prepared as second immunomodulators. In a mouse model of chronic hepatitis B, step 1 involved administering tenofovir disoproxil fumarate (TDF) (by gavage) for 5 consecutive days at 15 mg / kg. Subsequently, on days 0, 14, and 28, the first immunomodulator—synthetic G12 monoclonal antibody (G12)—and the second immunomodulator—the complex preparations (TVac-mHBIG, TVac-G12-2a, and TVac-KR127-2a)—were administered, respectively.

[0284] The results are shown in Figures 12, 13, 14, and 15. The trends in inhibiting hepatitis B surface antigen (HBsAg) expression differed depending on the ratio of the antigen-antibody complexes. On day 14 after the second immunization, the antibody-to-antigen ratio of each complex formulation was 1:6, resulting in the lowest HBsAg expression level. As shown in Figure 15, when the antibody-to-antigen ratio of the complex formulation was 1:6, the HBsAg level after administration of the monoclonal antibody complex formulations (TVac-G-mIgG2a and TVac-K-mIgG2a) was significantly lower than that after administration of TVac-mHBIG (on day 5, the HBsAg level of the monoclonal antibody complex formulation was 2 IU / mL, while the HBsAg level of the polyclonal antibody complex formulation had risen to 3 IU / mL).

[0285] As shown in Figures 13 and 14, when TDF and the first dose of immunomodulator G12 were used to treat mice with chronic hepatitis B, the serum HBsAg expression level was reduced to ≤10 IU / ml. Based on this, a second dose of immunomodulator was administered, and even better results were observed. For patients with chronic hepatitis B, after combined treatment with TDF and the first dose of immunomodulator G12, a second dose of immunomodulator was administered when the HBsAg level decreased to approximately ≤400 IU / ml. According to the expert consensus on the clinical application of hepatitis B virus markers in the "Guidelines for the Prevention and Treatment of Chronic Hepatitis B (2023 Edition)," a baseline HBsAg level below 200 IU / ml or a decrease in HBsAg greater than 1 log10 IU / ml after medication is a positive factor for achieving clinical cure. In this invention, after combined treatment with TDF and the first dose of the immunomodulator G12, a second dose of the monoclonal antibody antigen-antibody complex (TVac-G12-2a) is administered. After medication, the baseline HBsAg level is below 200 IU / ml or the decrease in HBsAg greater than 1 log10 IU / ml.

[0286] 6-5 Immunological effects of monoclonal antibody complex formulations with different epitopes: In a mouse model of chronic hepatitis B, step 1 involved administering tenofovir disoproxil fumarate (TDF) via gavage for 5 consecutive days at 15 mg / kg; followed by the administration of the first immunomodulatory agent. On days 0, 14, and 28, the first and second immunomodulatory agent complex formulations (TVac) of the synthesized G12 monoclonal antibody (G12) were administered, respectively. The antibody complex formulations (TVac) were prepared according to the method described in 6-1: TVac-G, TVac-K, TVac-G-mIgG1, TVac-G-mIgG2a, TVac-K-IgG1, and TVac-K-mIgG2a. PBS served as the control group.

[0287] The results are shown in Figures 16 and 17. The different monoclonal antibody complex formulations (TVac) showed similar trends in inhibiting the expression of hepatitis B surface antigen (HBsAg), with a stable decrease in HBsAg expression in mouse serum after three immunizations. Figures 18 and 19 show the trends in antibody (HBsAb) expression in induced immunized mice. Antibodies (HBsAb) were not detected in the control group (PBS). After completing step 2 on day 14, antibody (HBsAb) expression in mice steadily increased. Even after completing step 3 on day 28, high-expression antibodies (HBsAb) were still detectable in mice for more than 50 days. Furthermore, the results of this example show that the IgG2a subtype has a better effect on inhibiting antigen and inducing antibody expression than the IgG1 subtype.

[0288] 6-6 Expression levels of humoral and cellular immune factors after sequential combination therapy: On day 83 after the completion of Example 6-5, tail vein blood was collected from mice, and flow cytometry was used to detect changes in the expression levels of humoral and cellular immune factors in mice after sequential combination therapy.

[0289] Figures 20-24 are schematic diagrams of the results of the expression levels of humoral immune factors in mice, and Figures 25-32 are schematic diagrams of the results of the expression levels of cellular immune factors.

[0290] The results showed that immunization with the antigen-antibody complex preparation (TVac) significantly induced humoral and cellular immune responses against HBsAg.

[0291] Example 7: Effects of combined HBsAg-targeting siRNA on serum HBsAg, HBV DNA, and HBsAb levels

[0292] 7-1 This embodiment uses the same immunization method as in Example 6, the difference being that this embodiment uses siRNA targeting HBsAg in the first immunomodulation dose. The experimental groups are as follows:

[0293] PBS: The control group received only PBS;

[0294] TDF+G12+TVac-G: The first immunomodulatory dose is a synthetic G12 monoclonal antibody (G12), and the second immunomodulatory dose is a TVac-G antigen-antibody complex preparation.

[0295] TDF+siRNA+TVac-G: The first immunomodulatory dose is siRNA targeting HBsAg (HT101, a clinical drug from Suzhou Xingyao Kunze Company), and the second immunomodulatory dose is TVac-G antigen-antibody complex preparation.

[0296] TDF+G12+siRNA+TVac-G: The first immunomodulatory dose administers a synthetic G12 monoclonal antibody (G12) and siRNA targeting HBsAg, and the second immunomodulatory dose administers a TVac-G antigen-antibody complex formulation.

[0297] In step 1, the TDF dose is 15 mg / kg, injected every other day; in step 2, the first immunomodulator is a synthetic G12 monoclonal antibody (G12) at a dose of 6.7 mg / kg, and the siRNA targeting HBsAg is administered subcutaneously at a dose of 3 mg / kg, following the synthetic G12 monoclonal antibody; in step 3, the second immunomodulator is a TVac-G antigen-antibody complex preparation administered intraperitoneally at a dose of 4 μg; the first and second immunomodulators are administered on days 0, 14, and 28, respectively.

[0298] Regarding serum HBsAg expression levels, please refer to Figure 33. After the first immunization (day 0), mice given only G12 monoclonal antibody showed a trend of recovery in serum HBsAg expression, while mice given siRNA (TDF+siRNA+TVac-G and TDF+G12+siRNA+TVac-G) showed a continuous decrease in serum HBsAg expression.

[0299] Regarding the inhibition of HBV DNA, please refer to Figure 34. The groups given the antigen-antibody complex preparation (TDF+G12+TVac-G, TDF+siRNA+TVac-G and TDF+G12+siRNA+TVac-G) have the function of continuously inhibiting HBV DNA expression.

[0300] Regarding HBsAb, please refer to Figure 35. The groups given antigen-antibody complex formulations (TDF+G12+TVac-G, TDF+siRNA+TVac-G, and TDF+G12+siRNA+TVac-G) showed better antibody expression than the control group PBS. The antibody expression induced by the first dose of immunomodulator simultaneously administered with monoclonal antibody G12 and siRNA (TDF+G12+siRNA+TVac-G) was better than that induced by other groups.

[0301] Other interfering RNA molecules that target HBsAg, such as microRNA and shRNA, also have similar effects to siRNA and can be applied to this invention with similar results.

[0302] miRNAs (microRNAs) are a group of non-coding RNAs, approximately 20-23 nucleotides in length, encoded by the genome. They guide the silencing complex (RISC) to degrade mRNA or inhibit its translation by pairing with the bases of target gene mRNA. shRNAs (short hairpin RNAs) are short double-stranded RNA structures (19-25 nt) produced in dependence of stem-loop sequences. They can be introduced into cells using vectors, where they are cleaved by enzymes to form siRNA, which regulates target genes through the RNA interference pathway. Unlike siRNAs, shRNAs are synthesized in the cell nucleus. After transient or stable transduction, shRNA vectors first enter the cell nucleus, where they are expressed and processed by the Drosha / DGCR8 complex to form pre-shRNA. This pre-shRNA is then transported to the cytoplasm by the Exportin-5 protein, where the circular sequence is removed by the Dicer complex to form siRNA. It then binds to the RISC (RNA-induced silencing complex) and releases one of its RNA strands. The complex recognizes the mRNA, leading to its degradation. It is evident that the processing pathway of shRNA after exiting the nucleus is essentially the same as that of dsRNA, ultimately forming the structure of siRNA. siRNA is usually exogenous, while miRNA is endogenous. Mature (double-stranded) miRNA is loaded onto a RISC, and after one strand is removed, the RISC carrying antisense RNA binds to the mRNA, inhibiting the expression of the target gene through mRNA degradation and translation repression pathways, respectively. Different types of small RNAs result in different types of RISCs. The core enzyme of RISC is the Argonaute enzyme of the AGO protein family, which has multiple members in most species. In Drosophila and human cells, miRNA binds to AGO-1 protein to produce a non-cleaving RISC, interfering with the target gene by inhibiting mRNA translation; siRNA binds to AGO-2 protein to produce a cleaving RISC, interfering with the target gene by cleaving and degrading mRNA. Structurally, shRNA and miRNA are more similar; in fact, shRNA is more functionally similar to siRNA. shRNA is cleaved by the Dicer enzyme in the cell to form siRNA, which performs its interference function through the siRNA pathway, while miRNA regulates the target gene through a different pathway.

[0303] Example 8: Difference in antibody levels between the present invention and other combination therapies using immunomodulators.

[0304] 8-1 In this embodiment, different immunomodulators were used in combination therapy to administer the treatment to the patients. Specific experimental groups, medication regimens, and information are as follows:

[0305] PDL1: A commercially available monoclonal antibody, patent number WO2018178122.

[0306] TLR8: Toll-like receptor 8 agonist GS-9688, purchased from MedChemexpress Biotechnology, Inc., USA, catalog number 2004677-13-6.

[0307] IFNα: Alpha interferon, purchased from Yiqiao Shenzhou Biotechnology, product number 50672-M01H1

[0308] FAP-IL2: An immune cytokine comprising an antibody against fibroblast activation protein α (FAPα) and an IL-2 variant that binds only to IL-2Rβγ, purchased from MedChemexpress Biotechnology, Inc., USA.

[0309] PD1: Anti-mouse CD279 monoclonal antibody, purchased from InVivoPlus, catalog number BP0146.

[0310] PDL1-IFNα+HBsAg / CpG: PDL1-IFNα heterodimer fusion protein was synthesized according to the reference (Meng CY, et al. Gut 2023; 72:1544–1554). HBsAg was purchased from North China Pharmaceutical Group, and CpG, a Toll-like receptor 9 agonist, was purchased from MedChemexpress Biotechnology, Inc., USA, catalog number HY-150217.

[0311] TDF+G12+PDL1: In a mouse model of chronic hepatitis B (described in Example 5), tenofovir disoproxil fumarate (TDF) was administered for 5 consecutive days via gavage (15 mg / kg). After day 6, the drug was administered every other day. On day 6, synthetic G12 monoclonal antibody (tail vein, 6.7 mg / kg) was administered. One hour later, PDL1 (T drug) (tail vein, 5 mg / kg) was administered. G12 and PDL1 were administered every two weeks for a total of 3 times.

[0312] TDF+G12+TLR8: In a mouse model of chronic hepatitis B (described in Example 5), tenofovir disoproxil fumarate (TDF) was administered for 5 consecutive days (by gavage, 15 mg / kg). After day 6, the drug was administered every other day. On day 6, synthetic G12 monoclonal antibody (tail vein, 6.7 mg / kg) was administered. One hour later, TLR8 (by gavage, 3 mg / kg) was administered. G12 was administered every two weeks for a total of 3 times, and TLR8 was administered weekly for a total of 5 times.

[0313] TDF+G12+TVac-G-mIgG2a: In a mouse model of chronic hepatitis B (described in Example 5), tenofovir disoproxil fumarate (TDF) was administered for 5 consecutive days (by gavage, 15 mg / kg). After day 6, the drug was administered every other day. On day 6, the first immunomodulator, synthetic G12 monoclonal antibody (tail vein, 6.7 mg / kg), was administered. One hour later, the second immunomodulator, TVac-G-mIgG2a antigen-antibody complex, was administered. G12 and TVac-G-mIgG2a were administered every two weeks for a total of 3 times.

[0314] TDF+G12+IFNα: In a mouse model of chronic hepatitis B (described in Example 5), tenofovir disoproxil fumarate (TDF) was administered for 5 consecutive days via gavage (15 mg / kg). After day 6, the drug was administered every other day. On day 6, synthetic G12 monoclonal antibody (tail vein, 6.7 mg / kg) was administered. One hour later, IFNα (intraperitoneal, 30 μg / kg) was administered. G12 was administered every two weeks for a total of 3 times, and IFNα was administered weekly for a total of 5 times.

[0315] TDF+G12+FAP-IL2: In a mouse model of chronic hepatitis B (described in Example 5), tenofovir disoproxil fumarate (TDF) was administered for 5 consecutive days via gavage (15 mg / kg). After day 6, the drug was administered every other day. On day 6, synthetic G12 monoclonal antibody (tail vein, 6.7 mg / kg) was administered. One hour later, FAP-IL2 (tail vein, 2 mg / kg) was administered. G12 and FAP-IL2 were administered every two weeks for a total of 3 times.

[0316] siRNA+G12+IFNα: On day 0, siRNA (HT101, a clinical drug from Suzhou Xingyao Kunze Co., Ltd., subcutaneous, 3 mg / kg) targeting HBsAg was administered, and G12 monoclonal antibody (tail vein, 6.7 mg / kg) was administered simultaneously. One hour later, IFNα (intraperitoneal, 30 ug / 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.

[0317] siRNA+TLR8+TDF: In a mouse model of chronic hepatitis B (described in Example 5), tenofovir disoproxil fumarate (TDF) was administered for 5 consecutive days (by gavage, 15 mg / kg). After day 6, the drug was administered every other day. On day 6, siRNA targeting HBsAg (HT101, a clinical drug from Suzhou Xingyao Kunze Co., Ltd., administered subcutaneously, 3 mg / kg) was given. One hour later, TLR8 was administered (by gavage, 3 mg / kg). siRNA was administered every two weeks for a total of 3 times, and TLR8 was administered weekly for a total of 5 times.

[0318] siRNA+PD1+TDF: Tenofovir disoproxil fumarate (TDF) (15 mg / kg by gavage) was administered to a mouse model of chronic hepatitis B (described in Example 5) for 5 consecutive days. After day 6, it was administered every other day. On day 6, siRNA targeting HBsAg (HT101, a clinical drug from Suzhou Xingyao Kunze Co., Ltd., 3 mg / kg subcutaneously) was administered. One hour later, PD1 (5 mg / kg via tail vein) was administered. siRNA and PD1 were administered every two weeks for a total of 3 times.

[0319] PDL1-IFNα+HBsAg / CpG: Synthetic anti-PDL1-IFNα homologous fusion protein (Meng CY, et al. Gut, 2023(72):1544–1554) (tail vein, 0.2 mg / kg) was administered on days 0 and 3, and HBsAg / CpG (subcutaneous, 2 μg / 30 μg) was administered on days 3, 10, 17, and 24.

[0320] PBS: The control group received only PBS.

[0321] Regarding HBsAb, please refer to Figure 36. Serum was collected on day 21 after combined treatment. The results showed that the antibody level produced in the TDF+G12+TVac-G-mlgG2a group was much higher than that in all other combined treatment groups and the difference was extremely significant. This result can demonstrate that the combined treatment strategy of the present invention can stimulate the body to produce the corresponding antibodies more quickly.

[0322] Sequence information:

[0323] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. An antigen-antibody complex that induces an immune response in patients with chronic hepatitis B, characterized in that, The complex contains hepatitis B surface antigen and anti-HBsAg monoclonal antibody, with the hepatitis B surface antigen binding to the anti-HBsAg monoclonal antibody.

2. The complex according to claim 1, characterized in that, Hepatitis B surface antigen and anti-HBsAg monoclonal antibody bind in a non-covalent manner.

3. A pharmaceutical composition, characterized in that, The composition comprises the antigen-antibody complex of claim 1, and a pharmaceutically acceptable carrier and / or excipients.

4. A vaccine composition, characterized in that, The composition comprises the antigen-antibody complex of claim 1, and an immunologically acceptable carrier and / or excipient.

5. A method for preparing the antigen-antibody complex according to claim 1, characterized in that, include: Hepatitis B surface antigen and anti-HBsAg monoclonal antibody are mixed in a certain proportion to obtain the antigen-antibody complex as described in claim 1.

6. The use of an immunomodulator in (a) the preparation of a medicament for inducing an immune response in patients with chronic hepatitis B; and / or (b) the preparation of a medicament for the prevention and / or treatment of chronic hepatitis B, characterized in that, The immunomodulators include: hepatitis B surface antigen and anti-HBsAg monoclonal antibody.

7. An immunomodulator prepared from the antigen-antibody complex of claim 1.

8. The use of an antigen-antibody complex according to claim 1, a pharmaceutical composition according to claim 3, a vaccine composition according to claim 4, or an immunomodulator according to claim 7, characterized in that, (a) for the preparation of drugs that induce an immune response in patients with chronic hepatitis B; and / or (b) for the preparation of drugs for the prevention and / or treatment of chronic hepatitis B.

9. Use of an immunomodulator in the preparation of (a) a medicament for inducing an immune response in patients with chronic hepatitis B; and / or (b) a medicament for the prevention and / or treatment of chronic hepatitis B, characterized in that, The immunomodulatory agent includes a first immunomodulatory agent and a second immunomodulatory agent, each of which independently includes a drug that lowers HBsAg levels, the antigen-antibody complex of claim 1, the pharmaceutical composition of claim 3, or the vaccine composition of claim 4.

10. A kit for inducing an immune response in patients with chronic hepatitis B by administering a first agent followed by a second agent; and / or (b) for the prevention and / or treatment of chronic hepatitis B, characterized in that, Include: The first medication was an antiviral drug; The second agent is a first immunomodulator and a second immunomodulator, each of which independently comprises a drug that lowers HBsAg levels, the antigen-antibody complex of claim 1, the pharmaceutical composition of claim 3, or the vaccine composition of claim 4, wherein the antiviral drug is administered before the administration of the first immunomodulator and the second immunomodulator.

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