Immune medicine composition, preparation method and application of medicine composition prepared by using method

Abstract

The invention discloses an immune pharmaceutical composition, a preparation method and an application of the pharmaceutical composition prepared by the method, and particularly provides a combination of the following components: a first composition, a second composition and a third composition, the first composition comprises a water-soluble anionic polymer and a nonionic thickener, the second composition comprises a water-soluble anionic polymer, and the third composition comprises a water-soluble anionic polymer and a nonionic thickener. The second composition comprises a nucleotide innate immune receptor agonist, and the third composition comprises a polycation charge binding substance and a nonionic osmotic pressure regulator. The invention also provides a preparation method of the immune pharmaceutical composition, the preparation method is simple and does not involve toxic and harmful chemical substances, and the process is suitable for amplification. The invention also provides application of the immune pharmaceutical composition in the fields of induction and activation of immune cell maturation, secretion of cytokines, resistance to allergic diseases, prevention and treatment of microbial infectious diseases, vaccine adjuvants, anti-tumor immunotherapy and resistance to Alzheimer's disease.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to an immune drug composition and a preparation method thereof, and the application of the immune drug composition prepared by the method Background Art

[0002] Innate immune recognition mainly refers to the recognition of external pathogens (including viruses, bacteria, fungi, etc.) or other endogenous stimuli by immune cells, including dendritic cells, macrophages, natural killer cells, neutrophils, etc., and the subsequent immune and inflammatory regulation process triggered. Its recognition is mainly mediated by various pattern recognition receptors (PRRs) expressed by immune cells and recognizes biological macromolecules with pathogen associated molecular patterns (PAMPs) or damage associated molecular patterns (DAMPs). Recent studies have confirmed that the innate immune response initiated by PRRs is crucial in the occurrence and development of tumors, viral and bacterial infections, allergic diseases, and autoimmune diseases. It is generally believed that PRRs mainly include four categories: Toll-like receptors (TLRs), NOD-like receptors (NLR), C-type lectin-like receptors (CLR) and RIG-like receptors (RLR). These PRRs induce the production of cytokines and interferons by activating related anti-inflammatory pathways, thereby stimulating the body's immune response. Toll-like receptors (TLRs) are the most widely studied and thoroughly studied type of PRRs. Currently, 10 TLRs (TLR-1 to TLR-10) have been found in humans, and 12 TLRs (TLR-1 to TLR-9, TLR-11 to TLR-13) have been found in mice. Current studies have shown that TLR-1, TLR-2, TLR-4, TLR-5, TLR-6 and TLR-11 are located on the cell surface, while TLR-3, TLR-7, TLR-8 and TLR-9 are located on the inner membrane of lysosomes.

[0003] Polyinosinic acid (Poly I:C) is a double-stranded RNA (dsRNA) analogue formed by the pairing of artificially synthesized PolyI and PolyC. It can bind to the TLR-3 receptor, and then trigger the activation of transcription factors NF-kB and IRF3 through the adaptor protein TICAM-1 / TRIF, further inducing the secretion of cytokines such as interferon, interleukin, and tumor necrosis factor, and promoting the proliferation and maturation of immune cells. Therefore, Poly I:C has a broad spectrum of antiviral, phagocytic stimulation, immune adjustment, anti-tumor, anti-bacterial and protozoan infection functions, and has been used clinically for antiviral and anti-tumor treatments and vaccine adjuvants. The clinical adult dosage is 1 to 2 mg per intramuscular injection.

[0004] Toll-like receptor-9 (TLR-9) is a receptor for mammalian cells to recognize CpG and is present on the surface of endosomes / lysosomes of various host cells. Cytosine-guanine dinucleotide deoxyoligonucleotide (CpGODN) immunostimulant, which is a specific DNA sequence containing unmethylated CpG. CpG ODN is recognized and bound by the TLR-9 receptor, thereby triggering the TLR-9-mediated signaling pathway. With the deepening of research on CpG ODN, the demand for its clinical transformation and practical application is urgent. CpG-ODN-mediated immunotherapy has been successfully used to treat allergies and cancer. Some clinical trials have explored the direct anti-tumor effect of CpG ODN in monotherapy, or in combination with other tumor immunotherapy drugs or combined with tumor antigens as tumor therapeutic vaccines. However, as an oligonucleotide in nature, unmodified CpG ODN, like other nucleic acid drugs, faces the problems of being easily degraded by nucleases, low cellular uptake, requiring high dosages and repeated administrations, causing the risk of systemic inflammation and increasing immunotoxic side effects. These defects greatly limit its application. Although the backbone of CpG ODNs can be fully or partially thiolated to solve its stability problem, its bioavailability is still very low and its ability to enter cells is poor.

[0005] The cyclic guanosine monophosphate-adenosinemonophosphate synthase (cGAS)-stimulator of interferon genes (STING) signaling pathway is essential for sensing abnormal accumulation of DNA in the cytoplasm. Cyclic dinucleotides can activate downstream STING, thereby inducing the expression of inflammatory cytokines such as type I interferon. This signaling pathway can play an important immune role in anti-pathogen infection and anti-tumor. 2', 3'-cGAMP generated by cGAS in mammals and cyclic dinucleotides (CDNs) in bacterial cells can directly bind to STING, thereby activating the STING downstream pathway. However, directly using natural CDNs as STING agonists for in vivo tumor immunotherapy has problems such as poor stability and difficulty in penetrating the cell membrane, which affects the ability to induce type I IFN expression. In addition, there is an extranucleotide pyrophosphatase / phosphodiesterase 1 in the body that is widely distributed in the cell membrane and endoplasmic reticulum membrane. This enzyme can hydrolyze 2', 3'-cGAMP and CDNs, preventing the activation of STING.

[0006] Although more and more nucleotide immune agonists have been developed for anti-infection, tumor immunotherapy and the treatment of allergic diseases, the natural properties of nucleotides greatly limit their efficacy and clinical use. Studies have shown that although the tolerance of nucleic acids to enzymes can be improved by thiolation modification, the barrier to entering cells cannot be overcome. These problems directly lead to the inability of such drugs to directly enter the clinical stage. From the current situation, even if they enter clinical trials, the effects of direct use are mostly low clinical response rates, and it is difficult to activate a satisfactory immune response. At the same time, high doses also bring inevitable immune side effects. Therefore, it is necessary to enhance its stability and ability to enter cells through appropriate design, thereby significantly enhancing the biological activity of such immune agonists, which is crucial for the practical use of such immunomodulators. Therefore, the development of new delivery formulations or the form of pharmaceutical compositions is the key to the clinical application and successful promotion of nucleotide innate immune agonists in anti-tumor, viral infection, and allergic diseases, and is also the hot spot and focus of current research in this field. Some existing delivery systems, such as nanoliposomes, polymer nanoparticles, inorganic nanoparticles, etc., are often complex in the process of preparing the relevant nucleotide immune agonists for encapsulation or adsorption, involving a certain degree of chemical reaction or modification technology or liposome formation steps, involving certain organic solvents such as chloroform, etc., and the preparation cycle problem and purification problem are also difficult points. For example, CN114903856 A discloses a 2', 3'-cGAMP liposome nanoformulation, which not only needs to use chloroform organic solvent in the preparation process, but also needs to be vacuumed for more than 12 hours to remove the organic solvent. CN102488707A adds kanamycin and calcium ions on the basis of poly I:C, which enhances the stability of poly I:C double-stranded RNA. More importantly, due to the different molecular weights of different nucleotide immune agonists such as Poly I:C, 2', 3'-cGAMP and CpGODN, the preparation method or carrier system required for agonists with different physicochemical properties is not the same. Therefore, there remains a strong need for new and more effective methods and compositions for delivering nucleotide-based immune agonists such as Poly I:C, 2',3'-cGAMP and CpG ODN into cells.

[0007] Because the delivery system and / or the form of the pharmaceutical composition composed of various substances, the interaction or association of the substances in this process has unpredictable risks, and for the negatively charged substances such as agonists of nucleotide innate immune receptors, antagonism or resistance may usually occur, rather than adsorption, and the auxiliary materials are crucial for the entire composition as a drug, so it is difficult to find the best auxiliary system and composition form. Aiming at the negative charge properties of agonists of nucleotide innate immune receptors, by introducing different polycationic charge binding substances, anionic water-soluble polymers and other auxiliary materials, the entrapment and self-assembly of agonists of nucleotide innate immune receptors can be achieved. Then, how to determine the proportion and preparation method of each component in the immune composition, how to improve the stability and drugability of the immune composition, is the difficulty and key point of this technology, and it is also the real need for the development of immune drugs. Therefore, the present invention aims to invent a universal technology and pharmaceutical composition form for agonists of nucleotide innate immune receptors. Summary of the invention

[0008] In order to overcome the defects and deficiencies of agonists of nucleotide innate immune receptors in the application stage, especially in effective delivery technology, the present invention provides an immune pharmaceutical composition, and in particular provides a combination of the following components, including a first composition, a second composition and a third composition. The immune pharmaceutical composition is an agonist of nucleotide innate immune receptors, including a TOLL-like receptor 3 agonist, a TOLL-like receptor 9 agonist and a STING agonist as the main active pharmaceutical ingredient, which solves the compatibility problem of excipients. The core is to develop the delivery problem of agonists of nucleotide innate immune receptors. The immune pharmaceutical composition of the present invention has a simple preparation method, does not involve toxic chemicals, and the process is suitable for scale-up. The present invention also provides the use of this immune pharmaceutical composition in the fields of inducing and activating immune cell maturation, secreting cytokines, anti-allergic diseases, preventing and treating microbial infectious diseases, vaccine adjuvants, anti-tumor immunotherapy and anti-Alzheimer's disease.

[0009] An immune pharmaceutical composition comprises a first composition, a second composition and a third composition; the first composition comprises an anionic water-soluble polymer and a nonionic thickener, the second composition comprises an agonist of nucleotide innate immune receptors, and the third composition comprises a polycationic charge-binding substance and a nonionic osmotic pressure regulator.

[0010] Specifically, the anionically charged water-soluble polymer in the first composition includes: heparin, heparin sulfate, chondroitin sulfate, keratan sulfate, dextran sulfate, carboxymethyl cellulose, cross-linked caramel, carboxymethyl starch, carboxymethyl dextran, carboxymethyl chitosan, hyaluronic acid and its derivatives, rhamnosyl sulfate, cellulose sulfate, curdlan sulfate, chitosan phosphate, polyanionic polypeptide, polyacrylic acid, polymethacrylic acid, polyglutamic acid, polyaspartic acid, polyaspartic acid grafted polyethylene glycol, polyglutamic acid grafted polyethylene glycol, polycarbofil, carboxyvinyl polymer, maleic anhydride copolymer, thiolated polyacrylate or any combination thereof.

[0011] Specifically, the nonionic thickener in the first composition includes at least one of gum arabic, gelatin, povidone, carbomer, polyoxyethylene, polyvinyl alcohol, gum arabic, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose or cyclodextrin, or any combination thereof.

[0012] Specifically, the agonist of the nucleotide innate immune receptor in the second composition includes one or more of a TOLL-like receptor 3 agonist, a TOLL-like receptor 9 agonist and a STING agonist;

[0013] Specifically, the TOLL-like receptor 9 agonist is a non-methylated CpG motif CpG ODN. Specifically, the CpG ODN in the present invention may include one or more combinations of type A CpG ODN, type B CpG ODN and type C CpG ODN.

[0014] Specifically, the class A CpG-ODN is selected from one or more combinations of CpG-ODN 2216, CpG-ODN 1585 or CpG-ODN 2336.

[0015] Specifically, the Class B CpG-ODN is selected from one or more combinations of CpG-ODN 1018, CpG-ODN 2006, CpG-ODN 1826, CpG-ODN 1668, CpG-ODN 2007, CpG-ODN BW006 or CpG-ODN SL01.

[0016] Specifically, the C-type CpG-ODN is selected from one or more combinations of CpG-ODN 2395, CpG ODN M362, and CpG ODN SL03.

[0017] Specifically, the TOLL-like receptor 3 agonist is a copolymer of polyinosinic acid and polycytidylic acid (Poly I:C) and double-stranded RNA.

[0018] Specifically, the STING agonist cyclic dinucleotide 2', 3'-cGAMP.

[0019] Specifically, the polycationic charge-binding substance in the third composition includes a polycationic electrolyte which may be at least one of chitosan, chitosan hydrochloride, chitosan quaternary ammonium salt, protamine sulfate, poly-L-lysine and a cationic polypeptide.

[0020] Preferably, the polycationic electrolyte is chitosan hydrochloride and protamine sulfate.

[0021] More preferably, the polycationic electrolyte is protamine sulfate with a molecular weight of 5000 to 10000.

[0022] Specifically, the nonionic osmotic pressure regulator in the third composition includes at least one of lactose, sucrose, glucose, trehalose, dextran, glycerol or D-mannitol.

[0023] The present invention also provides a method for preparing an immune pharmaceutical composition, which specifically comprises the following steps:

[0024] S1, using sterile and DNA / RNA enzyme-free deionized water to dissolve anionic water-soluble polymer and nonionic thickener, sterilizing with 0.22 micron filter membrane, and obtaining a first component solution for standby use, wherein the weight / volume concentration of the anionic water-soluble polymer is 0.1% to 0.4%, and the weight / volume concentration of the nonionic thickener is 0.1% to 0.5%; preferably, the weight / volume concentration of the anionic water-soluble polymer is 0.1% to 0.2%, and the weight / volume concentration of the nonionic thickener is 0.1 to 0.3%. S2, using sterile and DNA / RNA enzyme-free deionized water to dissolve the innate immune receptor agonist in the second composition, sterilizing with 0.22 micron filter membrane, and standby use, the weight / volume concentration is 0.1 to 0.4%; preferably, the weight / volume concentration of the immune receptor agonist is 0.1% to 0.2%.

[0025] S3, dissolving the polycationic substance in the third composition with sterile and DNA / RNA enzyme-free deionized water, sterilizing it with a 0.22-micron filter membrane for standby use, with a weight / volume concentration of 0.1-0.4%, wherein the weight / volume concentration of the non-ionic osmotic pressure regulator is 5-20%; preferably, the weight / volume concentration of the polycationic substance is 0.1-0.2%.

[0026] S4, mixing the solution prepared in step S1 with the solution prepared in step S2, oscillating and mixing, to prepare a mixed solution, wherein the volume ratio of the solution prepared in step S1 to the solution prepared in step S2 is 1:0.5 to 1:1.

[0027] S5, adding the solution in step S3 to the mixed solution in step S4, mixing again at room temperature, using a direct mixing method or a microfluidic mixing method, stabilizing for 10 to 20 minutes, and preparing an immune drug composition through the interaction of the charge properties of the main substances contained in different components. The volume ratio of the solution configured in S3 to the solution configured in step S4 is 1:1 to 1:1.5.

[0028] Specifically, the immunological drug composition is in the form of a liquid preparation, wherein the liquid preparation is a nanosuspension with a particle size of 100 to 1000 nm. Preferably, the particle size of the immunological drug combination liquid preparation is 100 to 400 nm.

[0029] Therefore, in another aspect of the present invention, the present invention also provides the use of the immune pharmaceutical composition prepared by the method. Specifically, it can be used for at least: (1) cell immune stimulation and the use of enhancing the release of cytokines by immune cells. As the immune cells in this specification, they can be dendritic cells, monocytes, natural killer cells, macrophages, neutrophils, B cells, gamma delta T cells and other immune cells. (2) Independent use in the preparation of drugs for preventing and treating respiratory viral infectious diseases. (3) Independent use in the preparation of drugs for preventing and treating respiratory bacterial infectious diseases. (4) Independent use in the preparation of drugs for preventing and treating respiratory fungal infectious diseases. (4) Independent use in the preparation of anti-allergic disease drugs. (5) Combined use in the preparation of anti-allergic disease drugs with other anti-allergic disease therapies. As the allergic diseases in this specification, they can be allergic skin diseases, allergic conjunctivitis, allergic rhinitis, allergic asthma and allergic gastrointestinal diseases. (6) Independent use in the preparation of anti-tumor immune drugs. (7) Combined use in the treatment of tumors with other tumor therapies. (8) Application of immune pharmaceutical compositions in combination with antigens as vaccine adjuvants in the preparation of vaccine preparations for human use. (9) Application of immune pharmaceutical compositions in combination with antigens as vaccine adjuvants in the preparation of vaccine preparations for animals. (10) A person skilled in the art can know from the above description that the immune pharmaceutical compositions of the present invention can also be used as an immune adjuvant, and antigens known in the art can be added. The antigens described in the present invention refer to any substance that can induce an immune response in the art. The antigens in this specification may be plant-derived antigens, microbial-derived antigens (bacteria, viruses, fungi, parasitic protozoa, etc.) and tumor-derived antigens. (11) Application of immune pharmaceutical compositions in combination with tumor antigens as vaccine adjuvants in the preparation of tumor preventive and therapeutic drugs.

[0030] Furthermore, the antigen may be one or more of tumor antigens, viral antigens, bacterial antigens, fungal antigens, parasitic protozoan-derived antigens, degenerative disease-related antigens, atopic disease-related antigens, and autoimmune disease-related antigens.

[0031] The viral antigens in this specification are selected from one or more of common viral antigens such as hepatitis B virus, herpes simplex virus, human papillomavirus, Epstein-Barr virus, cytomegalovirus, smallpox virus, varicella-zoster virus, new coronavirus, HIV, coxsackievirus, poliovirus, rhinovirus, foot-and-mouth disease virus, yellow fever virus, dengue virus, epidemic encephalitis B virus, Lassa fever virus, rabies virus, Hantavirus, Ebola virus, Marburg virus, influenza virus, parainfluenza virus, measles virus, rubella virus, mumps virus, respiratory syncytial virus, hepatitis virus, rotavirus, and MERS virus.

[0032] The bacterial antigens in this specification are selected from one or more of common bacterial antigens such as Escherichia coli, Klebsiella, Proteus, Serratia, Pseudomonas aeruginosa, Enterococcus, Candida albicans, Haemophilus influenzae, Streptococcus pneumoniae, Staphylococcus aureus, Enterobacteriaceae, respiratory viruses, anaerobic bacteria, coagulase-negative Staphylococci, Salmonella, Shigella sonnei, enterovirus, etc.

[0033] The tumor antigens may be tumor-related antigens and tumor-specific neoantigens.

[0034] Furthermore, the tumor-associated antigen may be a whole cell component antigen of a tumor cell and / or tumor tissue.

[0035] Furthermore, the tumor-associated antigen may also be a tumor-associated antigen that has been reported to be highly expressed in tumor cells.

[0036] Furthermore, the tumor-specific antigens may originate from the expression of mutant genes in tumor cells, resulting in gene point mutations, deletion mutations, gene fusions, etc., which produce tumor neoantigens that are different from proteins expressed by normal cells.

[0037] The tumor antigens used in the anti-tumor immunotherapy and tumor vaccines in this specification can be lung cancer, malignant lymphoma, gastric cancer, gallbladder cancer, bile duct cancer, pancreatic cancer, melanoma, liver cancer, colon cancer, prostate cancer, head and neck squamous cell carcinoma, thyroid cancer, rectal cancer, adrenal cortical carcinoma, breast cancer, ovarian cancer, cervical squamous cell carcinoma and cervical adenocarcinoma, osteosarcoma, bladder cancer, leukemia, kidney cancer, prostate cancer, brain tumor, etc.

[0038] The administration method used in the present invention can be oral, sublingual, intranasal, rectal, intramuscular, subcutaneous, intradermal, intravenous, intraperitoneal, intralymph node, intrathecal, pulmonary inhalation, intraventricular, implantation, intralesional administration, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 This is a DLS particle size analysis chart of the immune drug composition in Example 1 of the present invention.

[0040] Figure 2 This is a DLS particle size analysis chart of the immune drug composition in Example 2 of the present invention.

[0041] Figure 3 This is a DLS particle size analysis chart of the immune drug composition in Example 3 of the present invention.

[0042] Figure 4 This is the experimental result that the immune drug composition in Example 2 of the present invention promotes the secretion of inflammatory cytokines in mouse BMDCs.

[0043] Figure 5 It is the change in tumor volume after the immune drug composition in Example 2 of the present invention is injected into mouse B16F10 transplanted melanoma.

[0044] Figure 6 This is a diagram showing the animal immune effect of the immune drug composition combined with HBsAg antigen in Example 2 of the present invention. DETAILED DESCRIPTION

[0045] The scheme of the present invention will be explained below in conjunction with embodiments. It will be appreciated by those skilled in the art that the following embodiments are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. Where specific techniques or conditions are not indicated in the embodiments, the techniques or conditions described in the literature in this area or the product specifications are followed. In addition, the repeated description of the same technical configuration and effect as in the prior art will be omitted.

[0046] The materials, reagents, or instruments used without indicating the manufacturer are all acceptable and available from commercial channels. The purchase of some reagents and raw materials is as follows: high molecular weight Poly (I: C) and 2'3'-cGAMP were purchased from Invitrogen Life Sciences Inc., USA; CpG ODN 1826 (sequence: 5'-tccatgacgttcctgacgtt-3') was purchased from Sangon Biotech (Shanghai) Co., Ltd., HPLC purified, and thiolated; the water used to dissolve Poly (I: C), 2'3'-cGAMP, and CpG ODN was sterile and endotoxin-free; chitosan hydrochloride was purchased from Aladdin Chemical; protamine sulfate was purchased from Sigma Chemical Co., Ltd. (St. Louis, Missouri); healthy SPF-grade BALB / c mice were purchased from Beijing Weitong Lihua Company; RPMI 1640, DMEM, fetal bovine serum, etc. were purchased from Gibco; CD 80 antibody and CD86 antibody were purchased from BioLegend; lymphocyte separation medium, mouse interleukin-6 (Interleukin 6, IL-6), mouse interleukin 12p40 (Interleukin 12p40, IL-12p40) ELISA kits were purchased from Dakoway Biotechnology Co., Ltd.; CD80-PE, CD86-PE-CY7 and other flow cytometry antibodies as well as membrane permeation fixative and permeation-enhancing solution were purchased from Shanghai Union Biotechnology Co., Ltd.; recombinant HBsAg and commercial hepatitis B vaccine (aluminum adjuvant finished vaccine) were purchased from Hualan Biotechnology Co., Ltd.; hepatitis B virus surface antibody assay kit (enzyme-linked immunosorbent assay) was purchased from Beijing Wantai Bio-Pharmaceutical Co., Ltd.

[0047] Example 1 Preparation and characterization of TOLL-like receptor 3 agonist Poly I:C-based immunopharmaceutical composition.

[0048] (1) Weighing a certain amount of chondroitin sulfate and hydroxyethyl cellulose, dissolving sodium chondroitin sulfate and hydroxyethyl cellulose with sterile and DNA / RNA enzyme-free deionized water, sterilizing with a 0.22 μm filter membrane, and obtaining a first component solution for standby use, wherein the weight / volume concentration of sodium chondroitin sulfate is 0.4%, and the weight / volume concentration of hydroxyethyl cellulose is 0.2%;

[0049] S2, weigh a certain amount of Poly I:C powder, dissolve it with sterile and DNA / RNase-free deionized water, sterilize it with a 0.22 μm filter membrane, and set aside. The weight / volume concentration of the Poly I:C solution is 0.4%;

[0050] S3, dissolving chitosan hydrochloride and glycerol in the third composition with sterile and DNA / RNase-free deionized water, sterilizing with a 0.22 μm filter membrane for standby use, with a weight / volume concentration of 0.1%, wherein the weight / volume concentration of glycerol is 5.2%;

[0051] S4, mixing the solution prepared in step S1 with the solution prepared in step S2, and oscillating and mixing to prepare a mixed solution, wherein the volume ratio of the solution prepared in step S1 to the solution prepared in step S2 is 1:1.

[0052] S5, mixing the solution in step S3 and the mixed solution in step S4 again at room temperature, using a direct mixing method or a microfluidic mixing method, and preparing an immunodrug composition through the interaction of the charge properties of the main substances contained in different components, wherein the volume ratio of the solution configured in S3 to the solution configured in step S4 is 1:1.

[0053] The samples prepared in S5 were stored in a refrigerator at 2-8°C for subsequent experiments.

[0054] The hydrated particle size and surface zeta potential of the solution obtained in Example 1 were tested using a laser particle size analyzer (DLS, Malvern, UK, Zetasizer NanoZS). Test conditions: the test temperature was 25°C, the laser wavelength was 633nm, the temperature was kept constant for 20 minutes before each test, each sample was tested three times, and the average value was taken.

[0055] Experimental results: The particle size of the liquid preparation of the pharmaceutical composition containing the Poly(I:C) active substance was characterized, showing that the particle size was around 200 nm and was uniformly distributed.

[0056] Example 2 Preparation and characterization of TOLL-like receptor 9 agonist CpG ODN-based immunopharmaceutical composition.

[0057] (1) Weighing a certain amount of carboxymethyl chitosan and hydroxyethyl cellulose, dissolving sodium chondroitin sulfate and hydroxyethyl cellulose with sterile and DNA / RNA enzyme-free deionized water, and sterilizing with a 0.22 μm filter membrane to obtain a first component solution for standby use, wherein the weight / volume concentration of carboxymethyl chitosan is 0.4% and the weight / volume concentration of hydroxyethyl cellulose is 0.2%;

[0058] S2, weigh a certain amount of CpG ODN 1826 powder, dissolve it in sterile and DNase / RNase-free deionized water, sterilize it with a 0.22 μm filter membrane, and set aside. The weight / volume concentration of the CpG ODN 1826 solution is 0.4%;

[0059] S3, dissolving chitosan hydrochloride and glycerol in the third composition with sterile and DNA / RNase-free deionized water, sterilizing with a 0.22 μm filter membrane for standby use, with a weight / volume concentration of 0.1%, wherein the weight / volume concentration of glycerol is 5.2%;

[0060] S4, mixing the solution prepared in step S1 with the solution prepared in step S2, and oscillating and mixing to prepare a mixed solution, wherein the volume ratio of the solution prepared in step S1 to the solution prepared in step S2 is 1:1.

[0061] S5, mixing the solution in step S3 and the mixed solution in step S4 again at room temperature, using a direct mixing method or a microfluidic mixing method, and preparing an immunodrug composition through the interaction of the charge properties of the main substances contained in different components, wherein the volume ratio of the solution configured in S3 to the solution configured in step S4 is 1:1.

[0062] The sample prepared in S5 was stored in a refrigerator at 2-8°C for subsequent experiments. The characterization method of the immunopharmaceutical composition was the same as that in Example 1.

[0063] Experimental results: The particle size of the liquid preparation of the pharmaceutical composition containing the CpG ODN 1826 active substance was characterized, showing that the particle size was about 200 nm and was uniformly distributed.

[0064] Example 3 Preparation and characterization of STING agonist cyclic dinucleotide 2', 3'-cGAMP-based immunopharmaceutical composition.

[0065] (1) Weighing a certain amount of sodium carboxymethyl cellulose and hydroxyethyl cellulose, dissolving sodium chondroitin sulfate and hydroxyethyl cellulose with sterile and DNA / RNA enzyme-free deionized water, sterilizing with a 0.22 μm filter membrane, and obtaining a first component solution for standby use, wherein the weight / volume concentration of sodium carboxymethyl cellulose is 0.4%, and the weight / volume concentration of hydroxyethyl cellulose is 0.2%;

[0066] S2, weigh a certain amount of 2',3'-cGAMP powder, dissolve it with sterile and DNA / RNase-free deionized water, sterilize it with a 0.22 micron filter membrane, and set aside. The weight / volume concentration of the 2',3'-cGAMP solution is 0.4%;

[0067] S3, dissolving the protamine sulfate and glycerol in the third composition with sterile and DNA / RNase-free deionized water, sterilizing with a 0.22 μm filter membrane for standby use, with a weight / volume concentration of 0.1%, wherein the weight / volume concentration of glycerol is 5.2%;

[0068] S4, mixing the solution prepared in step S1 with the solution prepared in step S2, and oscillating and mixing to prepare a mixed solution, wherein the volume ratio of the solution prepared in step S1 to the solution prepared in step S2 is 1:1.

[0069] S5, mixing the solution in step S3 and the mixed solution in step S4 again at room temperature, using a direct mixing method or a microfluidic mixing method, and preparing an immunodrug composition through the interaction of the charge properties of the main substances contained in different components, wherein the volume ratio of the solution configured in S3 to the solution configured in step S4 is 1:1.

[0070] Experimental results: The particle size of the liquid preparation of the pharmaceutical composition containing the 2',3'-cGAMP active substance was characterized, showing that the particle size was about 200 nm and was uniformly distributed.

[0071] Example 4 Evaluation of the immunostimulatory activity of the immune drug composition in Example 2 of the present invention in mouse BMDC cells.

[0072] C57BL / 6 mice of about 6 weeks old were killed, their tibia and femur were removed, and immersed in 70% ethanol for 2-3 minutes. The bone marrow was washed several times with RPMI-1640, and the bone marrow cells were collected. After being placed in an incubator for 30 minutes, non-adherent cells were collected to obtain BMDCs. The cell density was adjusted to 10 with dendritic cell culture medium (RPMI-1640 + 20ng / ml GM-CSF + 1ng / ml IL-4). 6 4 ml of cell suspension was plated in each well of a 6-well plate, and the DC culture medium was replaced every 2 days for 7 days. 6 The cells were plated in a 24-well plate at a density of 100 μg / ml and divided into 4 groups for stimulation experiments, namely PBS group, CpG ODN 1826 group (CpG ODN 2 μg / ml) and immune drug combination group (CpG ODN 2 μg / ml). ELISA determination of BMDC supernatant and serum cytokine content According to the instructions of the ELISA kit, the IL-6 and IL-12p40 contents in the BMDC supernatant were determined.

[0073] Experimental results: Free CpG ODN has a linear structure and carries a large amount of negative charge, which makes it difficult to adhere to cells, thus greatly reducing the immunostimulatory activity of CpG ODN. We further studied whether the form of immune drug composition can significantly enhance its immune activity. Figure 4As shown, under the same concentration conditions, the amount of cytokines secreted by BMDC cells stimulated by the immune drug composition was significantly higher than that of the control group and the CpG ODN 1826 group alone, indicating that the immune stimulatory activity of naked CpG ODN can be significantly improved in the form of a drug composition.

[0074] Example 5 Intratumoral injection of the immune drug composition of Example 2 of the present invention to treat B16F10 transplanted melanoma

[0075] Female BALB / c mice aged 5 to 7 weeks and weighing about 18 to 20 g were selected and adaptively raised in the animal room for one week. B16F10 cells were cultured to the logarithmic phase, counted after trypsin digestion, and the cell density was adjusted to 3×10 6 Each mouse was inoculated with 100 μl of B16F10 cells subcutaneously in the right hind limb. When the tumor volume was about 50 mm 3 The mice were killed two days after the administration, and the tumor volume and body weight were measured every two days during the experiment. The tumor volume was calculated using the formula v = 0.5 × a × b 2 Calculate, where v is the tumor volume, a is the tumor length, and b is the tumor width.

[0076] Experimental results: We further established a mouse melanoma tumor model to evaluate the therapeutic effect of intratumoral injection of the immune drug composition. 3 When the tumor size was about 1.5, mice with uniform tumor size were selected and divided into four groups. Normal saline, CpG ODN 1826 alone and the immune drug combination were injected intratumorally every two days for 5 times. Figure 5 As shown, compared with the saline control group, the inhibitory effect of CpG ODN 1826 alone on tumor growth was very limited, while the immunopharmaceutical composition could effectively inhibit tumor growth.

[0077] Example 6 Animal immunization experiment using the immune drug composition in Example 2 of the present invention as an adjuvant for HBsAg hepatitis B vaccine.

[0078] BALB / c mice were used, and the experimental animals were divided into the following groups: 1. PBS; 2. HbsAg; 3. Immunotherapy drug composition + HbsAg; 4. CpG 1826 + HbsAg; 5. Aluminum hydroxide gel (Alum) + HbsAg. The amount of HbsAg in each component was 1μg, the amount of CpG1826 was 10μg, and the amount of Alum was 225μg. Five mice were immunized by intramuscular injection at weeks 0, 2, and 4, and serum and spleen lymphocytes were collected at week 4 for immunological analysis. The anti-HBsAg antibody titer in the serum of each group of immunized mice was detected by ELISA kit.

[0079] The experimental results are as follows Figure 6 As shown, the HBsAg group alone produced almost no antibodies, and the antibody titer produced by the immune drug composition + HBsAg group was significantly higher than that of other groups, including the aluminum hydroxide adjuvant + HBsAg group and the CpG ODN 1826 + HBsAg group alone, indicating that the immune drug composition can significantly enhance the humoral immune effect of HBsAg.

Claims

1. An immune drug composition, characterized in that A first composition, a second composition and a third composition; the first composition comprises a water-soluble anionic polymer and a nonionic thickener, the second composition comprises an agonist of a nucleotide-based innate immune receptor, and the third composition comprises a polycationic charge-binding substance and a nonionic osmotic pressure regulator.

2. The immunopharmaceutical composition according to claim 1, characterized in that: The anionic water-soluble polymer in the first composition includes: heparin, heparin sulfate, chondroitin sulfate, keratan sulfate, dextran sulfate, carboxymethyl cellulose, carboxymethyl starch, carboxymethyl dextran, carboxymethyl chitosan, hyaluronic acid derivatives, rhamnosyl sulfate, cellulose sulfate, curdlan sulfate, chitosan phosphate, polyanionic polypeptide, polyacrylic acid, polymethacrylic acid, polyglutamic acid, polyaspartic acid, polyaspartic acid grafted polyethylene glycol, polyglutamic acid grafted polyethylene glycol, polycarbofil, carboxyvinyl polymer, maleic anhydride copolymer, thiolated polyacrylate or any combination thereof.

3. The immunopharmaceutical composition according to claim 1-2, characterized in that: The nonionic thickener in the first composition includes at least one of gum arabic, gelatin, povidone, carbomer, polyoxyethylene, polyvinyl alcohol, gum arabic, hydroxyethyl cellulose, hydroxypropyl cellulose, hypromellose or cyclodextrin, or any combination thereof.

4. The immunopharmaceutical composition according to claims 1-3, characterized in that: The nucleotide innate immune receptor agonist in the second composition includes one or any combination of a TOLL-like receptor 3 (TLR-3) agonist, a TOLL-like receptor 9 (TLR-9) agonist and a STING agonist.

5. The TOLL-like receptor 3 agonist according to claim 4, characterized in that: The TOLL-like receptor 3 agonist is a copolymer of polyinosinic acid and polycytidylic acid (Poly I:C) and double-stranded RNA.

6. The TOLL-like receptor 9 agonist according to claim 4, characterized in that The TOLL-like receptor 9 agonist is a non-methylated cytosine-guanine dinucleotide deoxyoligonucleotide (CpG ODN).

7. The STING agonist according to claim 4, characterized in that: The STING agonist is the cyclic dinucleotide cGAMP.

8. The immunopharmaceutical composition according to claims 1-4, characterized in that: The polycationic charge-binding material in the third composition can be at least one of chitosan, chitosan hydrochloride, chitosan quaternary ammonium salt, protamine (free base), protamine sulfate, protamine phosphate, protamine hydrochloride, poly-L-lysine and cationic polypeptides, or any combination thereof.

9. The immunopharmaceutical composition according to claims 1-4 and 8, characterized in that: The nonionic osmotic pressure regulator in the third composition includes at least one of lactose, sucrose, glucose, trehalose, dextran, glycerol or D-mannitol, or any combination thereof.

10. A method for preparing an immune pharmaceutical composition, characterized in that The steps include: S1, dissolving anionic water-soluble polymer and non-ionic thickener with sterile and DNA / RNA enzyme-free deionized water, sterilizing with a 0.22 micron filter membrane, and obtaining a first component solution for standby use, wherein the weight / volume concentration of the anionic water-soluble polymer is 0.1% to 0.4%, and the weight / volume concentration of the non-ionic thickener is 0.1% to 0.5%; S2, dissolving the nucleotide innate immune receptor agonist in the second composition with sterile and DNA / RNA enzyme-free deionized water, sterilizing it with a 0.22 micron filter membrane, and setting it aside, with a weight / volume concentration of 0.1-0.4%; S3, dissolving the polycationic substance in the third composition with sterile and DNA / RNase-free deionized water, sterilizing with a 0.22 micron filter membrane for standby use, with a weight / volume concentration of 0.1-0.4%, wherein the weight / volume concentration of the non-ionic osmotic pressure regulator is 5-20%; S4, mixing the solution prepared in step S1 with the solution prepared in step S2, oscillating and mixing, to prepare a mixed solution, wherein the volume ratio of the solution prepared in step S1 to the solution prepared in step S2 is 1:0.5 to 1:

1. S5, mixing the solution in step S3 and the mixed solution in step S4 again at room temperature, using a direct mixing method or a microfluidic mixing method, wherein the volume ratio of the solution prepared in step S3 to the solution prepared in step S4 is 1:1-1:1.

5.

11. The immunopharmaceutical composition according to any one of claims 1 to 10 is in the form of a liquid preparation, characterized in that: The liquid preparation is a nanosuspension with a particle size of 100 to 1000 nm. Preferably, the particle size of the liquid preparation of the immune drug combination is 100 to 400 nm.

12. The immunopharmaceutical composition according to any one of claims 1 to 10, characterized in that: The average surface charge of the immune drug composition is negative.

13. Use of the immune pharmaceutical composition according to any one of claims 1 to 12 in a cellular immune activator and in cellular immune stimulation and in enhancing the release of cytokines by immune cells.

14. The immune cell according to claim 13, characterized in that: The immune cells are dendritic cells, monocytes, natural killer cells, macrophages, neutrophils, B cells, gamma delta T cells and other immune cells.

15. An independent use of the immune drug composition according to any one of claims 1 to 14 in the preparation of drugs for preventing and treating respiratory viral infectious diseases.

16. Use of the pharmaceutical composition according to any one of claims 1 to 15 independently in the preparation of drugs for preventing and treating respiratory bacterial infectious diseases.

17. Use of the pharmaceutical composition according to any one of claims 1 to 16 independently in the preparation of drugs for preventing and treating respiratory fungal infections.

18. Use of the pharmaceutical composition according to any one of claims 1 to 17 independently in the preparation of anti-allergic disease drugs.

19. Use of the pharmaceutical composition according to any one of claims 1 to 18 in the preparation of an anti-allergic disease medicament in combination with another anti-allergic disease therapy.

20. An independent use of the pharmaceutical composition according to any one of claims 1 to 19 in the preparation of anti-tumor immune drugs.

21. Use of the pharmaceutical composition according to any one of claims 1 to 20 in combination with other tumor therapies in treating tumors.

22. The use of claim 21, wherein the additional tumor therapy is surgery, chemotherapy, targeted drug therapy and other immunotherapy drugs and methods.

23. Use of the pharmaceutical composition according to any one of claims 1 to 22 in combination with an antigen as a vaccine adjuvant in the preparation of a vaccine formulation for human use.

24. Use of the pharmaceutical composition according to any one of claims 1 to 23 in combination with an antigen as a vaccine adjuvant in the preparation of a vaccine preparation for animals.

25. The antigens as claimed in claims 23 and 24, wherein the antigens are plant-derived antigens, microbial-derived antigens (bacteria, viruses, fungi, parasitic protozoa, etc.) and tumor-derived antigens.

26. Use of the pharmaceutical composition according to any one of claims 1 to 25 in combination with a tumor antigen as an adjuvant in the preparation of tumor preventive and therapeutic drugs.

27. The use according to claims 25-26, wherein the tumor antigen is a tumor-associated antigen or a tumor-specific antigen or a combination thereof.

28. Use of the pharmaceutical composition according to any one of claims 1 to 26 independently in the preparation of anti-Alzheimer's disease drugs.

29. A combined use of the pharmaceutical composition according to any one of claims 1 to 26 in an anti-Alzheimer's disease drug.

30. The use according to any one of claims 13-29, wherein the administration method of the application can be selected from the following: oral, sublingual, intranasal, rectal, intramuscular, subcutaneous, intradermal, intravenous, intraperitoneal, intralymph node, intrathecal, pulmonary inhalation, intraventricular, implantation, intralesional administration, etc.

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