Immune agonists and compositions and uses thereof and methods of making the compositions

Abstract

The present application relates to an immune stimulator and composition and its application and the preparation method of the composition, which can inactivate and eliminate the cells infected with hepatitis B virus on the basis of maintaining the original preventive vaccine hepatitis B virus specific antibody in animals and human bodies, has the dual effects of prevention and treatment; it has stronger immune activation effect than the original HBsAg antigen, which is reflected in the strengthened induction of immune cells, higher antibody production effect, especially the high level of Th1 type immune production marked by gamma-interferon and IgG2a antibody, which has significant innovation and practicability for eliminating hepatitis B virus.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of hepatitis B vaccine prevention and treatment, and more particularly to an immune stimulant and composition and application thereof and a preparation method of the composition. BACKGROUND

[0002] Hepatitis B vaccine is a drug for preventing hepatitis B. After vaccination, the immune system can be stimulated to produce protective antibodies, so that the human body has the immunity to prevent hepatitis B, thereby achieving the purpose of preventing hepatitis B infection. However, there are 90 million people infected with hepatitis B virus (HBV) in China, of which 20 million are chronic hepatitis B patients. For chronic hepatitis B infected persons and patients, the preventive hepatitis B vaccine is basically ineffective. Therefore, a "therapeutic hepatitis B vaccine" that can both prevent and eliminate chronic hepatitis B virus infection is greatly needed. SUMMARY

[0003] The present application aims to provide an immune stimulant and composition and application thereof and a preparation method of the composition, and solves the problem that the hepatitis B vaccine in the prior art cannot prevent and eliminate chronic hepatitis B virus infection at the same time.

[0004] The technical solution adopted by the present application to solve the technical problem is: an immune stimulant, which has the following general formula:

[0005]

[0006] wherein R is OH or SH.

[0007] In the immune stimulant of the present application, the immune stimulant is a compound selected from the following structural formula I, structural formula II, structural formula III or structural formula IV:

[0008]

[0009] The above-mentioned immune stimulant of the present application is used for preparing a therapeutic hepatitis B vaccine and for preparing an immune-regulating drug for humans and animals.

[0010] The present application also provides a composition for preparing a hepatitis B vaccine for prevention and treatment, which comprises the above-mentioned immune stimulant, recombinant hepatitis B surface antigen and aluminum adjuvant, and the composition ratio of the recombinant hepatitis B surface antigen, aluminum adjuvant and immune stimulant is X:Y:Z, wherein X, Y and Z are respectively a number between 1 and 120 and represent the number of moles, the number of volumes or the number of masses.

[0011] In the composition of the present application, the composition ratio of the recombinant hepatitis B surface antigen, aluminum adjuvant and immune stimulant is X:Y:Z=1:20.36:20-31.8.

[0012] In the composition of the present application, the recombinant hepatitis B surface antigen is HBsAg, the amino acid sequence of the HBsAg comprises 200-400 amino acids, and the molecular weight of the HBsAg is 20000 Da-44000 Da.

[0013] In the composition of the present application, the amino acid sequence of the HBsAg comprises 200-250 amino acids, and the molecular weight of the HBsAg is 20000 Da-25000 Da.

[0014] In the composition of the present application, the aluminum adjuvant is aluminum hydroxide gel, aluminum phosphate, aluminum sulfate, ammonium alum or potassium alum.

[0015] The present application also provides a preparation method of the composition, which is characterized by comprising the following steps: mixing a HBsAg mother liquor with DMSO or physiological saline as a solvent and 9 times volume of an aluminum adjuvant solution to obtain a mixed solution, and then mixing the mixed solution with an immune stimulant at a volume ratio of 9:1 to obtain a composition for preparing a therapeutic hepatitis B vaccine.

[0016] The composition of the present application has the applications in improving the antibody titer and effector T cells against the hepatitis B surface antigen HBsAg, and in preparing vaccines and drugs for preventing and treating hepatitis B.

[0017] The immune stimulant and the composition of the present application and the applications thereof and the preparation method of the composition have the following beneficial effects: the present application provides an immune stimulant and a composition for preparing a vaccine for preventing and treating hepatitis B for chronic hepatitis B infected persons, which can inactivate and eliminate the cells infected with hepatitis B virus on the basis of maintaining the original hepatitis B virus specific antibodies of the preventive vaccine in animals and human bodies, and has the dual effects of prevention and treatment; the immune stimulant has a stronger immune activation effect than the original HBsAg antigen, which is reflected in the strengthened induction of immune cells, the higher antibody production effect, and the high level of Th1 type immune production of the markers of γ-interferon and IgG2a antibody, and has significant innovation and practicability for eliminating hepatitis B virus. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 A synthesis process diagram of the immune stimulant SZU-103 represented by the structural formula I;

[0019] Figure 2 An ESI-MS diagram of the immune stimulant SZU-103 represented by the structural formula I;

[0020] Figure 3 A synthesis process diagram of the immune stimulant SZU-117 represented by the structural formula II;

[0021] Figure 4ESI-MS profile of the immune agonist SZU-117 represented by structural formula II;

[0022] Figure 5 Synthetic scheme of the immune agonist SZU-114 represented by structural formula III;

[0023] Figure 6 ESI-MS profile of the immune agonist SZU-114 represented by structural formula III;

[0024] Figure 7 Synthetic scheme of the immune agonist SZU-122 represented by structural formula IV;

[0025] Figure 8 ESI-MS profile of the immune agonist SZU-122 represented by structural formula IV;

[0026] Figure 9A Profile of the immune agonist represented by general formula I on the stimulation effect of the immune cytokine IL-6;

[0027] Figure 9B Profile of the immune agonist represented by general formula I on the stimulation effect of the immune cytokine IFNγ;

[0028] Figure 9C Profile of the immune agonist represented by general formula I on the stimulation effect of the immune cytokine IL-12;

[0029] Figure 10A Profile of the immune agonist represented by general formula I and the control group respectively inducing the immune response of HBsAg antigen-specific IgG1 antibody;

[0030] Figure 10B Profile of the immune agonist represented by general formula I and the control group respectively inducing the immune response of HBsAg antigen-specific IgG2a antibody;

[0031] Figure 11A Photo of the immune agonist represented by general formula I as an adjuvant of hepatitis B vaccine inducing antigen-specific T cell response;

[0032] Figure 11B Profile of the immune agonist represented by general formula I as an adjuvant of hepatitis B vaccine and the control group respectively inducing antigen-specific T cell response;

[0033] Figure 12A Fluorescence profile of the immune agonist represented by general formula I as an adjuvant of hepatitis B vaccine inducing the effect of antigen-specific CD4 + T cell response;

[0034] Figure 12BThe immunopotentiator represented by general formula I as hepatitis B vaccine adjuvant induces antigen-specific CD4 + The effect of the antigen-specific CD4 T cell response induced by the immunopotentiator represented by general formula I as hepatitis B vaccine adjuvant and the control group is compared in the following graph:

[0035] Figure 12C The effect of the antigen-specific CD8 T cell response induced by the immunopotentiator represented by general formula I as hepatitis B vaccine adjuvant and the control group is compared in the following graph: + The effect of the antigen-specific CD8 T cell response induced by the immunopotentiator represented by general formula I as hepatitis B vaccine adjuvant and the control group is compared in the following graph:

[0036] Figure 12D The effect of the antigen-specific CD8 T cell response induced by the immunopotentiator represented by general formula I as hepatitis B vaccine adjuvant and the control group is compared in the following graph. + The effect of the antigen-specific CD8 T cell response induced by the immunopotentiator represented by general formula I as hepatitis B vaccine adjuvant and the control group is compared in the following graph. DETAILED DESCRIPTION

[0037] The immunopotentiator and composition of the present application and the application thereof and the preparation method of the composition are further described below in connection with the accompanying drawings and examples:

[0038] The advantage of using small molecule immunopotentiator as vaccine adjuvant is that it is stable in structure, easy to artificially synthesize, purify and manufacture, but the disadvantage of small molecule immunopotentiator is that it is quickly absorbed and metabolized by the body, and therefore cannot be well cooperated with antigen protein in distribution and metabolism. The small molecule immunopotentiator applied in the present application has α, β-unsaturated carbonyl group in its structural formula, which has covalent adsorption effect on antigen protein (especially protein containing sulfydryl residue); and has electron adsorption effect on the empty atomic orbital of aluminum atom and aluminum ion. The advantages of the immunopotentiator of the present application greatly improve the immune synergistic effect of the immunopotentiator and antigen, and therefore have significant innovation in mechanism.

[0039] The immunopotentiator has the following general formula:

[0040]

[0041] SM360320 (1, 2) in general formula I is a TLR7 agonist (Proc Natl Acad Sci U S A. 2006 Feb 7; 103 (6): 1828-33; Blood. 2011 May 26; 117 (21): 5683-91). In addition, the hydroxyl group (-OH) in general formula I can also be a sulfydryl group (-SH) of homologues.

[0042] In general formula I, SM360320 has tautomers of the same compound as formula 1 and formula 2:

[0043]

[0044] In particular, the immune agonist is a compound selected from the group consisting of Formula I, Formula II, Formula III, and Formula IV:

[0045]

[0046] The above structural formula is characterized by a system comprising a Toll-like receptor 7 (TLR7) agonist SM360320 and a compound having an α,β-unsaturated carbonyl group. It should be noted that the compound of Formula I, Formula II, Formula III, or Formula IV can also be an 8-position hydroxyl and carbonyl tautomer.

[0047] The present application also provides a composition for preparing a hepatitis B vaccine for prevention and treatment, comprising the above-mentioned immune agonist and recombinant hepatitis B surface antigen and aluminum adjuvant, the composition ratio of the recombinant hepatitis B surface antigen, aluminum adjuvant, and immune agonist being X:Y:Z, wherein X, Y, and Z are respectively a number between 1 and 120 and represent the number of moles, the number of volumes, or the number of masses. Preferably, the mass ratio of the recombinant hepatitis B surface antigen, aluminum adjuvant, and immune agonist is X:Y:Z = 1:20.36:20-31.8.

[0048] The recombinant hepatitis B surface antigen is HBsAg, the amino acid sequence of the HBsAg comprises 200-400 amino acids, and the molecular weight of the HBsAg is 20000 Da-44000 Da. Preferably, the amino acid sequence of the HBsAg comprises 200-250 amino acids, and the molecular weight of the HBsAg is 20000 Da-25000 Da. The amino acid sequence of the HBsAg is typically represented as follows (containing 226 amino acids) (Chinese Journal of Bioengineering, 1995, 15(2): 43-45):

[0049] Met Glu Asn lie Thr Ser Gly Phe Leu Gly Pro Leu Leu Val Leu Gin Ala Gly Phe Phe Leu Leu Thr Arg lie Leu Thr lie Pro Gin Ser Leu Asp Ser Trp Trp Thr Ser Leu Asn Phe Leu Gly Gly Ser Pro Val Cys Leu Gly Gin Asn Ser Gin Ser Pro Thr Ser Asn His Ser Pro Thr Ser Cys Pro Pro lie Cys Pro Gly Tyr Arg Trp Met Gys Leu Arg Arg Phe lie lie Phe Leu Phe lie Leu Leu Leu Cys Leu lie Phe Leu Leu Val Leu Leu Asp Tyr Gin Gly Met Leu Pro Val Cys Pro Leu lie Pro Gly Ser Thr Thr Thr Ser Thr Gly Pro Cys Lys Thr Cys Thr Thr Pro Ala Gin Gly Asn Ser Met Phe Pro Ser Cys Cys Cys Thr Lys Pro Thr Asp Gly Asn Cys Thr Cys lie Pro lie Pro Ser Ser Trp Ala Phe Ala Lys Tyr Leu Trp Glu Trp Ala Ser Val Arg Phe Ser Trp Leu Ser Leu Leu Val Pro Phe Val Gin Trp Phe Val Gly Leu Ser Pro Thr Val Trp Leu Ser Ala lie Trp Met Met Trp Tyr Trp Gly Pro Ser Leu Tyr Ser lie Val Ser Pro Phe lie Pro Leu Leu Pro lie Phe Phe Cys Leu Trp Val Tyr lie

[0050] The above aluminium adjuvants can be aluminium hydroxide gel, aluminium phosphate, aluminium sulphate, ammonium alum or potassium alum.

[0051] The present application provides an immune stimulator for chronic hepatitis B infected patients and a composition for preparing a hepatitis B vaccine, which can inactivate and eliminate hepatitis B virus infected cells in animals and humans on the basis of maintaining original hepatitis B virus specific antibodies of a preventive vaccine, and has a dual effect of prevention and treatment; the immune stimulator has a stronger immune activation effect than the original HBsAg antigen, which is reflected in enhanced immune cell factor induction and higher antibody production effect, especially the high level of Th1 type immune production of γ-interferon and IgG2a antibody, and has significant innovation and practicality for eliminating hepatitis B virus.

[0052] The above immune stimulator can be used to prepare a new hepatitis B vaccine for preventive and therapeutic vaccines of hepatitis B.

[0053] The following will be described in detail through specific examples.

[0054] Example 1: Synthesis of an immune stimulator represented by structural formula I (SZU-103)

[0055] As shown in Figure 1 , 344 mg (1 mmol) of SZU-008T and 341 mg (1.125 mmol) of diureide were dissolved in 8 mL of DMF, HBTU (427 mg, 1.125 mmol), triethylamine (416 μL, 3 mmol) and a catalytic amount of DMAP were added, and the reaction was stirred at room temperature overnight. The reaction solution was poured into 100 mL of water, suction filtered, and the filter residue was washed with water and dried to obtain a crude product. The crude product was separated by column chromatography (DCM:MeOH=20:1) to obtain 475 mg of a white solid with a yield of 75.5%. As shown in Figure 2 , ESI-MS: m / z=629.1 [M+H] + .

[0056] Example 2: Synthesis of an immune stimulator represented by structural formula II (SZU-117)

[0057] As shown in Figure 3 , EDCI (0.0025 mol) and HOBT (0.0025 mol) were added to a solution of SZU-101 (1 g, 0.002 mol) in anhydrous DMF, and stirred at room temperature for half an hour. DIEA (0.004 mol) was added dropwise at 0°C, and then N-Boc-piperazine (0.0025 mol) was added, and the reaction was carried out at room temperature overnight. The reaction was monitored by TLC until completion. The reaction solution was poured into water, suction filtered, and washed with water and dried. Then 1:4 TFA / DCM mixed solvent was added and stirred overnight. After the reaction was completed, the solvent was removed under reduced pressure, and a small amount of ethanol was added for re-dissolution. 2N hydrochloric acid was added for salting. After spinning dry, ether was added for grinding, and filtered to obtain SZU-130 with a yield of 56%.

[0058] SZU-130 (100 mg), acryloyl chloride and triethylamine were mixed at 0°C, then transferred to room temperature for 2 h. The reaction solution was poured into water, suction filtered, and the filter residue was washed with water and dried to obtain the crude product. The crude product was separated by column chromatography to obtain 80 mg of white solid with a yield of 72%. As shown in Figure 4 ESI-MS: m / z = 567.2 [M + H] + .

[0059] Example 3: Synthesis of Immune Agonist (SZU-114) represented by Structural Formula III

[0060] As shown in Figure 5 , SZU-008T (350 mg), EDCI (172 mg), and HOBT (172 mg) were dissolved in anhydrous DMF, and DIEA (0.35 mL) was added dropwise at 0°C. After the dropwise addition was completed, the reaction was continued at room temperature for 30 min, and then 117 mg of trans-4-dimethylamino cinnamate hydrochloride was added. The reaction was continued at room temperature overnight. After the reaction was completed, the reaction solution was poured into water, suction filtered, and the filter residue was washed with water and dried to obtain the crude product. The crude product was separated by preparative liquid chromatography to obtain 130 mg of white solid with a yield of 33%. As shown in Figure 6 ESI-MS: m / z = 456.2 [M + H] + .

[0061] Example 4: Synthesis of Immune Agonist (SZU-122) represented by Structural Formula IV Figure 7 As shown in Figure 8 , SZU-008T (300 mg), acryloyl chloride and triethylamine were mixed at 0°C, then transferred to room temperature for 2 h. The reaction solution was poured into water, suction filtered, and the filter residue was washed with water and dried to obtain the crude product. The crude product was separated by column chromatography to obtain 280 mg of white solid with a yield of 80%. As shown in ESI-MS: m / z = 399.1 [M + H] + .

[0062] Example 5: Method for preparing a composition for preventing and treating hepatitis B vaccine

[0063] A HBsAg mother liquor (provided by Shenzhen Kangtai Biological Products Co., Ltd.) with a concentration of 220 μg / mL was mixed with 9 volumes of an aluminum adjuvant solution (containing Al(OH)3 at a concentration of 1.44 mg / mL) to obtain a mixture containing HBsAg at a concentration of 22 μg / mL. The mixture was mixed with 10 mM of an immune agonist at a volume ratio of 9:1 (vaccine solution 9 volumes: immune agonist solution 1 volume) to obtain a final composition of hepatitis B vaccine. The weight ratio (HBsAg):(Al):(M) = X:Y:Z = 0.22 mg:4.48 mg:7 mg = 1:20.36:31.8. M is the immune agonist SZU-103.

[0064] The preparation method of replacing SZU-103 with SZU-114 is the same as above, and the preparation weight ratio (HBsAg) : (Al) : (M) = X : Y : Z = 0.22 mg : 4.48 mg : 5 mg = 1 : 20.36 : 22.7.

[0065] The preparation method of replacing SZU-103 with SZU-117 is the same as above, and the preparation weight ratio (HBsAg) : (Al) : (M) = X : Y : Z = 0.22 mg : 4.48 mg : 6.3 mg = 1 : 20.36 : 28.6.

[0066] The preparation method of replacing SZU-103 with SZU-122 is the same as above, and the preparation weight ratio (HBsAg) : (Al) : (M) = X : Y : Z = 0.22 mg : 4.48 mg : 4.4 mg = 1 : 20.36 : 20.

[0067] Wherein 10 mM immune agonist mother liquor configuration solvent is DMSO or physiological saline; other aluminum adjuvant is replaced by aluminum effective content (initial aluminum adjuvant solution contains 0.5 mg / mL of aluminum). The form of HBsAg can be monomer or polymer, and the concentration of the mother liquor is between 220 μg / mL and 250 μg / mL calculated based on HBsAg.

[0068] The biological activity test is specifically described below.

[0069] Example 6: In vitro cytokine stimulation

[0070] Balb / c mouse-derived spleen lymphocytes are used as the test system. 1 x 106 / mL of spleen lymphocytes are co-cultured with SZU-101, SZU-103, SZU-114, SZU-117 and SZU-122 at a concentration range of 0.03-40 μM for 24 hours, and the supernatant is collected and detected for IL6 and IFNγ concentrations by Elisa method. R848 is used as a positive control, and no addition is used as a negative control. The results are shown in Figure 9A 、 9B and 9C, the immune agonist has a significant stimulating effect on immune cytokines IL-6, IL-12 and IFN-γ.

[0071] Example 7: Effect of immune agonist represented by general formula I as an adjuvant of hepatitis B vaccine on inducing antigen-specific humoral immune response

[0072] Balb / c mice (5 mice per group) were immunized at days 0, 14 and 28 (2 μg of HBsAg antigen mixed with 100 nmol of TLR7 agonist, 1.12 mg / mL Al(OH)3 as another added adjuvant). Mouse sera were isolated at day 35 and HBsAg-specific IgGl and IgG2a antibody titers were determined by Elisa. R848, commercial hepatitis B vaccine and PBS injection were used as control groups. Results are shown in Figure 10A and 10B The effect of immunostimulatory agents as hepatitis B vaccine adjuvants to induce antigen-specific humoral immune responses was significant.

[0073] Example 8: Effect of immunostimulatory agents represented by Formula I as hepatitis B vaccine adjuvants to induce antigen-specific T cell responses

[0074] Balb / c mice (5 mice per group) were immunized at days 0, 14 and 28 (2 μg of HBsAg antigen mixed with 100 nmol of TLR7 agonist, 1.12 mg / mL Al(OH)3 as another added adjuvant). Mouse spleen lymphocytes were isolated at day 35 and HBsAg-specific T cell levels were determined by Elispot. R848, commercial hepatitis B vaccine and PBS injection were used as control groups. Results are shown in Figure 11A and Figure 11B The effect of immunostimulatory agents represented by Formula I as hepatitis B vaccine adjuvants to induce antigen-specific T cell responses was significant.

[0075] Example 9: Effect of immunostimulatory agents represented by Formula I as hepatitis B vaccine adjuvants to induce antigen-specific CD4+ and CD8+ T cell responses

[0076] Balb / c mice (5 mice per group) were immunized at days 0, 14 and 28 (2 μg of HBsAg antigen mixed with 100 nmol of TLR7 agonist, 1.12 mg / mL Al(OH)3 as another added adjuvant). Mouse spleen lymphocytes were isolated at day 35 and HBsAg-specific CD4+ and CD8+ T cell proportions were identified by intracellular IFNγ staining and flow cytometry. R848 and commercial hepatitis B vaccine were used as control groups. Results are shown in Figure 12A , 12B , 12C and 12D. The effect of immunostimulatory agents represented by Formula I as hepatitis B vaccine adjuvants to induce antigen-specific CD4 + and CD8 + T cell responses was significant.

[0077] It is to be understood that all such modifications and variations that can occur to those skilled in the art in the light of the foregoing description are to be considered within the scope of the application as defined in the claims appended hereto.

Claims

1. An immune agonist, which is a compound selected from the following structural formula II, structural formula III or structural formula IV:

2. Use of the immune agonist of claim 1 in the preparation of therapeutic hepatitis B vaccine, and in the preparation of human and animal immunomodulatory drugs.

3. A composition for the preparation of prophylactic and therapeutic hepatitis B vaccine, characterized by, The composition comprises an immune agonist, a recombinant hepatitis B surface antigen and an aluminum adjuvant, and the composition ratio of the recombinant hepatitis B surface antigen, the aluminum adjuvant and the immune agonist is X:Y:Z, wherein X, Y and Z are respectively a number between 1 and 120 and represent molar number, volume number or mass number; The immune agonist is a compound selected from the following structural formula II, structural formula III or structural formula IV:

4. The composition of claim 3, wherein, The composition ratio of the recombinant hepatitis B surface antigen, the aluminum adjuvant and the immune agonist is X:Y:Z = 1:20.36:20-31.

8.

5. The composition of claim 3, wherein, The recombinant hepatitis B surface antigen is HBsAg, and the amino acid sequence of the HBsAg comprises 200-400 amino acids, and the molecular weight of the HBsAg is 20000 Da-44000 Da.

6. The composition of claim 5, wherein, The amino acid sequence of the HBsAg comprises 200-250 amino acids, and the molecular weight of the HBsAg is 20000 Da-25000 Da.

7. The composition of claim 3, wherein, The aluminum adjuvant is aluminum hydroxide gel, aluminum phosphate, aluminum sulfate, ammonium alum or potassium alum.

8. Process for the preparation of a composition according to any one of claims 3 to 7, characterized in that, The composition comprises: The HBsAg mother liquor in DMSO or physiological saline is mixed with 9 times the volume of the aluminum adjuvant solution to obtain a mixed solution, and then the mixed solution is mixed with the immune agonist at a volume ratio of 9:1 to obtain a composition for preparing the therapeutic hepatitis B vaccine.

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