Preparation method of CpG ODN nano immune complex, complex prepared by using method and application of complex

Abstract

The invention relates to a preparation method of a CpG ODN nano immune complex, which comprises the following steps: (1) uniformly mixing a protamine solution with a certain concentration and a CpG motif-containing oligodeoxynucleotide CpG ODN aqueous solution with a certain concentration according to a certain ratio, and carrying out electrostatic compounding assembly at room temperature to form a suspension; and (2) applying energy to the suspension to disperse / homogenize uniformly to form the suspension of the nano-composite. The nano-composite prepared by the method disclosed by the invention is controllable in particle size and controllable in surface charge. The nano particles formed under the preparation condition of an optimized proportion are uniformly dispersed, good in stability, good in resolubility after freezing, and good in long-term storage stability. The preparation method provided by the invention is simple, does not involve toxic and harmful chemical substances, and is suitable for amplification. Compared with the prior art, the preparation method provided by the invention can obviously improve the effective concentration of the effective active substance CpG ODN in the nano immune complex. The invention also relates to the nano immune complex prepared by the method and application thereof. Cell and animal experiments prove that the nano immune complex prepared by the method has good immunostimulatory activity, can stimulate the maturation of related immune cells and induce the secretion of related cytokines, can be independently used as an immunomodulator, and can also be used as an immunologic adjuvant to be combined with various antigens to be used as a vaccine preparation as a whole. The nano immune complex has good application prospects in the fields of inducing and activating immune cells to mature, releasing cytokines, resisting allergic diseases, preventing and treating microbial infectious diseases, vaccine adjuvants, resisting tumor immunotherapy and resisting Alzheimer's disease.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine, and specifically relates to a method for preparing a CpG ODN nano-immune complex and application of the nano-immune complex prepared by the method. Background Art

[0002] Toll-like receptor 9 (TLR9) is a protein encoded by the TLR9 gene, also known as CD289 (differentiation antigen determinant cluster 289), and is a member of the Toll-like receptor family. Cytosine-guanine dinucleotide deoxyoligonucleotide (CpG ODN) is a specific DNA sequence containing non-methylated CpG. CpG ODN can activate TLR9 receptors in immune cells such as dendritic cells, monocytes, natural killer cells, macrophages, neutrophils, B cells, gamma delta T cells, etc., thereby triggering TLR9-mediated downstream signaling pathways, activating the MyD88-dependent pathway signal transduction pathway, causing the secretion of proinflammatory cytokines, inducing CD4+Th1 cell activity and inducing cytotoxic CD8+T cell responses. CpG ODN can enhance antigen processing and presentation, induce Th1-type immune response, produce stronger humoral immunity and cellular immunity, and enhance specific and nonspecific immune responses.

[0003] TLR9 plays an important role in the innate immune system and acts as a bridge between innate immunity and adaptive immunity in anti-tumor response. CpG-ODN-mediated immunotherapy has been applied to treat 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. In addition, CpG ODN as an immune adjuvant also shows excellent performance as a non-specific immune enhancer. For example, the world's first CpG adjuvant hepatitis B vaccine on the market It showed excellent immune effect. Compared with the hepatitis B vaccine with aluminum adjuvant alone Not only can it induce protective immunity faster, but also has better immune protection effect and longer protection time. At present, different types of CpG ODN have different structural characteristics and immune effects, and are generally divided into three categories: A, B, and C. Among them, the A-type CpG ODN is based on a palindromic sequence containing CpG dinucleotides, with poly G tails at both ends, and a partially thio-modified phosphodiester bond backbone. It forms a higher-order structure through the palindromic sequence and poly G, which can activate plasmacytoid dendritic cells to induce a large amount of type I interferon, but has weak activity on B cells. The B-type CpG ODN is a fully thio-modified linear CpG ODN that has strong immunostimulatory activity on B cells, but cannot activate plasmacytoid dendritic cells. The C-type CpG-ODN is a type of fully thio-modified CpG ODN that can form dimers through palindromic sequences and has the activity of both A-type and B-type CpG-ODN, and can activate both plasmacytoid dendritic cells and B cells. In addition to immunomodulatory activity, CpG ODN has also been shown to directly induce apoptosis and death of most hematological malignancies such as lymphoma and leukemia, and its mechanism of action is related to the activation of the JAK / STAT pathway. Recent studies have also shown that CpG ODN as a TLR9 receptor agonist can regulate neurological diseases associated with immune cells, such as Alzheimer's disease. Mouse and primate models have been shown to safely and effectively reduce amyloid plaques, oligomeric amyloid-β, tau pathology, and cerebral amyloid angiopathy, while promoting cognitive benefits, and therefore also have application prospects in the field of anti-Alzheimer's disease. Therefore, CpG ODN as a TLR9 agonist has important application potential in the treatment and prevention of a variety of diseases.

[0004] With the deepening of research on CpG ODN, the demand for its clinical transformation and practical application is urgent. However, as an oligonucleotide in nature, unmodified CpG ODN, like other nucleic acid drugs, faces the problems of easy degradation by nucleases, low cellular uptake rate, high dosage and repeated administration, which will cause the risk of systemic inflammation and increase immunotoxic side effects. These defects greatly limit its application. Dozens of clinical trials of CpG ODN are currently underway in Phase I to Phase III. However, some adverse events observed have led to the termination of some clinical trials. These side effects are largely related to repeated high-dose administration of CpG ODN. Although the backbone of CpG ODN can be fully or partially thiolated to solve its stability problem, its ability to enter cells as a nucleotide is poor, so its bioavailability is still very low. With the development of nanotechnology, nano-delivery strategies are considered to be the key to solving the problem of CpG ODN drugability, which can protect CpG ODN from nuclease degradation and enhance the efficiency of entering cells. In addition, nanocomplexes in the nanoscale range determine that it is easier to enter the lymphatic system, so that it is engulfed by relevant immune cells, and then more efficiently induce immune responses. For example, CpG ODN molecules are radially arranged on the surface of gold nanoparticles to form spherical nucleotides, showing more efficient immunomodulatory properties than CpG ODN alone. However, these current nano-delivery schemes often have complex preparation processes and long cycles, involving certain chemical reactions or connection technologies or liposome assembly technologies, and also involve the use of organic solvents.

[0005] Protamine (PS) is a strong basic peptide extracted from sperm of different fishes, containing about 70 mol% arginine, with different molecular weights. Because it has a strong basic group, it can combine with strongly acidic heparin in the body to form a stable complex, thereby making heparin lose its anticoagulant ability. Therefore, it is currently mainly used in clinical practice for bleeding caused by excessive injection of heparin, and is an essential drug for cardiac surgery. At present, the main varieties of protamine include protamine (free base), protamine sulfate, protamine phosphate, protamine hydrochloride, protamine-agarose, etc. In addition, because it carries a large amount of positive charge, it can interact with nucleic acid molecules such as DNA / RNA through electrostatic interaction, and can complex nucleic acid molecules, so it is used in the delivery of nucleic acid molecules. As a pharmaceutical raw material and pharmaceutical excipient, protamine has the advantages of easy access, low price and good safety performance. As early as 2004, A. ZIMMER et al. (J. Microencapsulation 21, 6, 625-641) used protamine and antisense nucleotides to form nanoparticles, using 15-150 μg / mL protamine aqueous solution and 30 μg of antisense nucleotides to form nanoparticles of a certain size. In addition, in 2006, Kerkmann et al. (Oligonucleotides 16:313-322 (2006) reported that 66.67 μg / mL (0.9 mL) of protamine sulfate solution was able to interact with 300 μg / mL (0.1 mL) of CpG ODN to form certain positively charged nanoparticles, and the immunostimulatory effect of CpG ODN can be significantly enhanced by using protamine nanoparticles. The concentration of CpG ODN under this preparation method is 30 μg / mL. In addition, protamine is also used to form complexes with negatively charged messenger RNA mRNA to protect the molecule from degradation. Protamine-encapsulated mRNA vaccines have been evaluated in different clinical trials in the form of RNActive vaccines.

[0006] However, the concentration of nucleic acid in the nanocomposite particles prepared by the composite of protamine and nucleic acid is relatively low. For example, the concentration of CpG ODN in the nanoparticles prepared by the method of Kerkmann et al. is only 30 μg / mL, which can still be used in animal experiments such as mice (several micrograms to tens of micrograms). Such a low concentration limits its direct use value, resulting in the fact that the protamine-CpG ODN nanocomposite prepared at the current stage cannot really move towards the clinical application stage. In addition, the third or more components are usually introduced to form nanoparticles in existing research, such as the need to introduce other anions such as tripolyphosphate or hyaluronic acid in patent CN 112089833A to increase the complexity of the material. It should be noted that the concentration of CpG ODN contained in the nanoparticles prepared in the patent is still relatively low, still only at the level of micrograms / milliliter (5-30 μg / mL). Although the formation of nanocomplexes by CpG ODN can enhance the immune activity of CpG ODN, the concentration of CpG ODN used in the current clinical stage is still at the milligram level to be effective. Therefore, the application prospects of CpG ODN with lower solubility in the nanocomplex cannot be determined, which is a problem that must be considered. Although the concentration of CpG ODN can be increased by concentration methods such as ultrafiltration concentration and centrifugation, it undoubtedly increases the complexity of the operation. Therefore, it is urgent to develop a new preparation method, and this method can meet the subsequent operable production and manufacturing methods, on the one hand, to increase the concentration of CpG ODN in the nanoparticles, so that it has a concentration that can meet the clinical use value. On the other hand, the preparation method and raw and auxiliary materials are required to be simplified to meet the requirements of industrial production. Summary of the invention

[0007] With the recognition and development of CpG ODN in tumor and other disease immunoprevention and immunotherapy, the strategy of nano-delivery system to enhance the therapeutic effect of CpG ODN as a drug has become the main direction, and the main purpose is to prolong the in vivo half-life of CpG ODN, prevent nuclease degradation, enhance the accumulation of lesion tissue, increase immune cell uptake and binding, etc. In order to solve the key delivery problem of CpG ODN as a drug development process, and to solve the defects and deficiencies of low CpG ODN concentration after the traditional protamine and CpG ODN are compounded to form a nano-complex, the present invention provides a method for preparing a relatively high concentration of CpG ODN nano-immune complex. Compared with the prior art, the preparation method provided by the present invention does not need to introduce other components, does not involve other polymer materials, and can significantly improve the effective concentration of CpG ODN, an effective active substance, in the CpG ODN nano-immune complex. The research and development team of this application found during the experiment that when a certain concentration of protamine sulfate and a relatively high concentration of CpG ODN (mg / ml) are mixed, a nanocomposite will not be formed immediately. Instead, the liquid system will be unstable due to the interaction between the two, and visible precipitates will appear. Generally, the appearance of precipitation means that the ratio needs to be readjusted. However, through further analysis and experiments on this precipitate by the research and development team, further attempts were made to disperse the precipitate in water in the next step by applying energy homogenization / ultrasound. We found that this method can further process the precipitate into nanoparticles, and the formed nanoparticles have uniform particle size and excellent stability.

[0008] The second purpose of the present invention is to prepare the CpG ODN nanoimmune complex by this method and its application, which is specifically manifested in the application in inducing and activating immune cell maturation, anti-allergic diseases, prevention and treatment of microbial infectious diseases, vaccine adjuvant, anti-tumor immunotherapy and anti-Alzheimer's disease.

[0009] The above-mentioned object of the present invention is achieved through the following technical solutions:

[0010] (1) Weigh a certain amount of non-ionic osmotic pressure regulator, add it to sterile and endotoxin-free water, stir or ultrasonically disperse and dissolve it, filter and sterilize it with a 0.2 μm filter membrane to prepare an isotonic aqueous solution for later use;

[0011] (2) dissolving protamine in the isotonic aqueous solution to prepare a 0.1-1% (w / v) protamine solution, filtering and sterilizing with a 0.2 μm filter membrane, and setting aside;

[0012] (3) dissolving CpG ODN in the above isotonic aqueous solution to prepare a 0.1-1% (w / v) CpG ODN solution, filtering and sterilizing with a 0.2 μm filter membrane, and setting aside;

[0013] (4) mixing a certain amount of protamine solution and a certain amount of CpG ODN solution to form a suspension;

[0014] (5) dispersing the suspension in step (4) by applying energy homogenization, including one or more of ultrasonic crushing / dispersion, high-speed dispersion homogenization, high-pressure homogenization, and microfluidization homogenization.

[0015] (6) The obtained protamine-CpG ODN nanocomplex liquid preparation is sterilized at high temperature and high pressure.

[0016] Specifically, the non-ionic osmotic pressure regulator includes at least one of lactose, sucrose, glucose, dextran, glycerol or D-mannitol, or any combination thereof.

[0017] Specifically, the CpG ODN described in the present invention may include one or more combinations of type A CpG ODN, type B CpG ODN and type C CpG ODN.

[0018] Specifically, the class A CpG-ODN is selected from one or more combinations of CpG-ODN 2216, CpG-ODN 1585 or CpG-ODN 2336.

[0019] 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.

[0020] Specifically, the C-type CpG-ODN is selected from one or more combinations of CpG-ODN 2395, CpG ODN M362, and CpG ODN SL03.

[0021] Specifically, the protamine salts include salts formed from acidic substances, protamine (free base), protamine sulfate, protamine phosphate, and protamine hydrochloride.

[0022] Preferably, the protamine is protamine sulfate.

[0023] Preferably, the molecular weight of the protamine sulfate is 5000-1000 Da.

[0024] Specifically, the feed mass ratio of the protamine sulfate solution to the CpG ODN solution is (0.50-3.0): 1. Preferably, the feed mass ratio is (0.70-2.0):1.

[0025] More preferably, the feed mass ratio is (0.9-1.2):1 and (1.5-3.0):1.

[0026] Preferably, the suspension is homogenized and dispersed by high pressure homogenization, the conditions are 600-1200 bar, and the homogenization cycle is 1-3 times.

[0027] Specifically, the average particle size of the CpG ODN nano-immune complex is in the range of 100-1000 nm, and the morphology is spherical nanoparticles. By adjusting the proportion, the average surface charge of the nano-complex can be negative or positive.

[0028] Preferably, the prepared CpG ODN nanocomplex has a particle size range of 100-300 nm, a negatively charged surface, uniform particle dispersion, good stability, good resolubility after freezing, and can be stored for a long time with a stable particle size.

[0029] Specifically, the concentration of protamine in the nano-immune complex solution preparation is 0.5 to 9.09 mg / mL.

[0030] Specifically, the concentration of CpG ODN in the nanoimmunocomplex solution preparation is 0.5 to 9.09 mg / mL.

[0031] Preferably, the effective concentration of CpG ODN is 0.5-2.0 mg / mL.

[0032] The preparation method provided by the present invention is simple, does not involve toxic chemicals, and is highly safe. The preparation method and process do not require complicated processes, can effectively protect the biological activity of CpG ODN, and the process is suitable for scale-up.

[0033] The CpG ODN nano-immune complex prepared by the method has good biological activity as shown in cell and animal experiments.

[0034] Therefore, in another aspect of the present invention, the present invention also provides the application of the CpG ODN nanoimmune complex prepared by the method. Specifically, it can be used for at least: (1) cell immune stimulation and application in enhancing the release of cytokines by immune cells. As 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 application in the preparation of drugs for preventing and treating respiratory viral infectious diseases. (3) Independent application in the preparation of drugs for preventing and treating respiratory bacterial infectious diseases. (4) Independent application in the preparation of drugs for preventing and treating respiratory fungal infectious diseases. (4) Independent application in the preparation of anti-allergic disease drugs. (5) Combined application in the preparation of anti-allergic disease drugs with other anti-allergic disease therapies. As allergic diseases in this specification, they can be allergic skin diseases, allergic conjunctivitis, allergic rhinitis, allergic asthma and allergic gastroenteritis. (6) Independent application in the preparation of anti-tumor immune drugs. (7) Combined application in the treatment of tumors with other tumor therapies. (8) Application of nanoimmune complexes combined with antigens as vaccine adjuvants in the preparation of human vaccine preparations. (9) Application of nanoimmune complexes combined with antigens as vaccine adjuvants in the preparation of animal vaccine preparations. (10) A person skilled in the art can know from the above description that the CpG ODN nanoimmune complex of the present invention can 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 combined tumor antigens as vaccine adjuvants in the preparation of tumor preventive and therapeutic drugs.

[0035] 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.

[0036] The tumor antigens may be tumor-associated antigens and tumor-specific neoantigens.

[0037] Furthermore, the tumor-associated antigen may be a whole cell component antigen of a tumor cell and / or tumor tissue.

[0038] Furthermore, the tumor-associated antigen may also be a tumor-associated antigen in the form of polypeptides such as WT-1, HER-2, NY-ESO, MUC-1, MAGE-A3, GPC3, etc., which have been reported to be highly expressed in tumor cells.

[0039] 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 (Neoantigens) that are different from the proteins expressed by normal cells.

[0040] 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.

[0041] The viral antigens in this specification are selected from one or more of common viral antigens such as hepatitis B virus, herpes simplex virus, new coronavirus, 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.

[0042] 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.

[0043] When used as an adjuvant, the effective in vitro and in vivo concentration of CpG ODN in the CpG ODN nanoimmunocomplex described herein can be determined by a person skilled in the art through routine experiments. For example, the effective amount can be 0.01 μg-3000 μg / mL. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 This is a particle size distribution diagram of the protamine sulfate-CpG ODN 1826 nanoimmunocomplex (formed under the conditions of 1.2:1.0) in Example 1 of the present invention.

[0045] Figure 2 This is the potential distribution diagram of the protamine sulfate-CpG ODN 1826 nanoimmunocomplex (formed under the condition of 1.2:1.0) in Example 1 of the present invention.

[0046] Figure 3 The particle size distribution diagram of the protamine sulfate-CpG ODN 1826 nanoimmune complex (formed under the conditions of 2.0:1.0) in Example 2 of the present invention is

[0047] Figure 4 This is a particle size distribution diagram of the protamine sulfate-CpG ODN 1826 nano immune complex (formed under the conditions of 1.2:1.0) in Example 2 of the present invention.

[0048] Figure 5 This is the particle size distribution diagram of the protamine sulfate-CpG ODN 2006 nanoimmunocomplex (1.0:1.0) in Example 1 of the present invention.

[0049] Figure 6 This is the potential distribution diagram of the protamine sulfate-CpG ODN 2006 nanoimmunocomplex (1.0:1.0) in Example 1 of the present invention.

[0050] Figure 7 This is the particle size distribution diagram of the protamine sulfate-CpG ODN 2006 nanoimmunocomplex (2.0:1.0) in Example 1 of the present invention.

[0051] Figure 8 This is the potential distribution diagram of the protamine sulfate-CpG ODN 2006 nanoimmunocomplex (2.0:1.0) in Example 1 of the present invention.

[0052] Fig. 9 This is a transmission electron microscope image of the protamine sulfate-CpG ODN 2006 nanoimmunocomplex (formed under the condition of 1.0:1.0) in Example 1 of the present invention.

[0053] Fig.10 This is a graph showing the long-term stability study of the protamine sulfate-CpG ODN 2006 nanoimmunocomplex (formed under the condition of 1.0:1.0) in Example 1 of the present invention.

[0054] Fig.11 This is a diagram showing a study on the reconstitution stability of the protamine sulfate-CpG ODN 2006 nanoimmune complex (formed under the condition of 1.0:1.0) in Example 1 of the present invention after being frozen and then thawed again.

[0055] Fig.12This is the experimental result of Example 1 of the present invention that the protamine sulfate-CpG ODN 1826 nanoimmunocomplex promotes the maturation of mouse BMDCs in vitro.

[0056] Fig.13 This is the experimental result of Example 1 of the present invention that the protamine sulfate-CpG ODN 1826 nano immune complex promotes the secretion of inflammatory cytokines in mouse BMDCs cells.

[0057] Fig.14 This is the experimental result of Example 2 of the present invention that the protamine sulfate-CpG ODN 2006 nanoimmunocomplex promotes the secretion of inflammatory cytokines by human PBMC cells in vitro.

[0058] Fig.15 This is the experimental result that the protamine sulfate-CpG ODN 1826 nanoimmunocomplex in Example 1 of the present invention significantly reduces the survival activity of mouse lymphoma cells A20.

[0059] Fig.16 This is the change in tumor volume after the protamine sulfate-CpG ODN 1826 nanoimmunocomplex in Example 1 of the present invention was injected into mouse A20 transplanted lymphoma.

[0060] Fig.17 This is the change in tumor volume after the protamine sulfate-CpG ODN 1826 nanoimmunocomplex in Example 1 of the present invention is injected into a mouse B16F10 transplanted melanoma.

[0061] Fig.18 It is the secretion of immune cells and cytokines in tumor tissues of different experimental groups in the melanin inhibition tumor experiment.

[0062] Fig.19 This is a diagram showing the effect of the protamine sulfate-CpG ODN1826 nanoimmune complex combined with tumor-associated antigen (MAGE-A3) on the prevention of mouse transplanted tumor growth in Example 1 of the present invention.

[0063] Fig. 20 This is a diagram showing the animal immune effect of the protamine sulfate-CpG ODN 1826 nanoimmune complex combined with HBsAg antigen in Example 1 of the present invention. DETAILED DESCRIPTION

[0064] 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.

[0065] The materials, reagents or instruments used without indicating the manufacturer are all available through commercial channels. Among them, the purchase of some reagents and raw materials is as follows: CpG ODN 1826 (sequence: 5'-tccatgacgttcctgacgtt-3') and CpG ODN 2006 (sequence: 5-tcgtcgttttgtcgttttgtcgtt-3'), MAGE-A3 polypeptide (FLWGPRALV) were purchased from Sangon Biotechnology (Shanghai) Co., Ltd., HPLC purification, CpG ODN was thiolated; the water used to dissolve CpG ODN was sterile and endotoxin-free water; protamine sulfate (Protaminssulfate) was purchased from Sigma Chemical Co., Ltd. (St. Louis, Missouri); A20 cells were purchased from the Cell Resource Center of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences; healthy SPF-grade BALB / c mice were purchased from Beijing Weitong Lihua Company; RPMI1640, DMEM, fetal bovine serum, etc. were purchased from Gibco; CD The CD80 antibody and CD86 antibody were purchased from BioLegend; lymphocyte separation medium, mouse interleukin-6 (IL-6), mouse interleukin 12p40 (IL-12p40), human interleukin-6, and human interleukin 12p40 ELISA kits were purchased from Dakoway Biotechnology Co., Ltd.; CD80-PE, CD86-PE-CY7, CD8-FITC, CD4-FITC 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.; aluminum hydroxide gel adjuvant ( adjuvant 2%) was purchased from InvivoGen; the hepatitis B virus surface antibody assay kit (enzyme-linked immunosorbent assay) was purchased from Beijing Wantai Biopharmaceutical Co., Ltd.

[0066] Example 1 Preparation and characterization of protamine sulfate-CpG ODN 1826 nanocomplex.

[0067] (1) Weighing a certain amount of non-ionic osmotic pressure regulator glycerol, adding it to sterile and endotoxin-free deionized water, dispersing and dissolving it with the assistance of ultrasound until a transparent solution is formed, filtering and sterilizing it with a 0.2 μm filter membrane to prepare a 2.6% glycerol isotonic aqueous solution for subsequent use;

[0068] (2) Weigh a certain amount of protamine sulfate powder, dissolve it in the above-mentioned 2.6% glycerol isotonic aqueous solution to prepare a 2 mg / mL protamine sulfate solution, and filter and sterilize it using a 0.2 μm filter membrane for subsequent use;

[0069] (3) Dissolve CpG ODN 1826 in the above-mentioned 2.6% glycerol isotonic aqueous solution to prepare a 2 mg / mL CpG ODN 1826 solution, filter and sterilize using a 0.2 μm filter membrane, and set aside;

[0070] (4) Using a pipette, different volumes of protamine solution were mixed with a certain volume of CpG ODN 1826 solution to explore different mass ratios (protamine sulfate: CpG ODN 1826 = 0.70:1.0, 0.80:1.0, 0.90:1.0, 1.0:1.0, 1.1:1.0, 1.2:1.0, 1.3:1.0, 1.4:1.0, 1.5:1.0, 1.6:1.0, 1.8:1.0 and 2.0:1.0), and incubated at room temperature for 30 min to form a suspension;

[0071] (5) The optimized suspension in step (4) was homogenized and dispersed by a high-pressure homogenizer at a pressure of 600-1200 bar, and the homogenization cycle was repeated 3 times.

[0072] (6) The aqueous solutions of protamine sulfate-CpG ODN 1826 nanoimmunocomplexes prepared at different ratios in this experiment were centrifuged at 8000 g for 15 min, the supernatant was collected, and the concentration of free CpG ODN 1826 in the supernatant was detected using the Quant-iT OliGreen ssDNA kit, and the mass of CpG ODN 1826 in the nanoimmunocomplexes was calculated.

[0073] The samples prepared in (5) were stored in a refrigerator at 2-8°C for subsequent experiments.

[0074] The hydrated particle size and surface zeta potential of the nano-immunocomplex obtained in Example 1 were tested using a laser particle size analyzer (DLS, Malvern, UK, Zetasizer NanoZS). Test conditions: the concentration of the nano-complex in the aqueous solution was 0.01 mg / ml, the temperature was 25°C, the laser wavelength was 633 nm, the temperature was kept constant for 20 minutes before each test, each sample was tested three times, and the average value was taken.

[0075] Experimental results: The present invention sets up nanocomplexes formed under different mass ratios of protamine sulfate and CpG ODN 1826, and performs ratio screening to determine the optimal formation ratio. The results are shown in Table 1. When the ratio is in the range of 0.7:1.0 to 1.3:1.0, it has a smaller particle size and a smaller PDI, and the surface charge potential is a stronger negative charge. When the ratio is 1.4:1.0, the surface potential is reversed from a negative charge to a positive charge, and the particle size increases to nearly 400nm. It shows that as the proportion of protamine sulfate used increases, the proportion of positively charged substances in the formed nanocomplex increases, resulting in the charge being converted to a positive charge. The negatively charged nanocomplex shows that the outer layer of the nanocomplex is mainly CpG ODN, and the positively charged nanocomplex shows that the outer layer of the nanocomplex is mainly protamine. Figure 1 and Figure 2 The particle size and potential distribution diagrams of the nanocomposite formed under the ratio of 1.2:1.0 are shown respectively. Figure 3 and Figure 4 The particle size and potential distribution diagrams of the nanocomposite formed under the ratio of 2.0:1.0 are shown respectively.

[0076] Table 1: Particle size potential measurement results at different mass ratios of protamine sulfate and CpG ODN 1826

[0077]

[0078]

[0079] Example 2 Preparation and characterization of protamine sulfate-CpG ODN 2006 nanoimmunocomplex.

[0080] (1) Weighing a certain amount of non-ionic osmotic pressure regulator glycerol, adding it to sterile and endotoxin-free deionized water, dispersing and dissolving it with the assistance of ultrasound until a transparent solution is formed, filtering and sterilizing it with a 0.2 μm filter membrane to prepare a 2.6% glycerol isotonic aqueous solution for subsequent use;

[0081] (2) Weigh a certain amount of protamine sulfate powder, dissolve it in the above-mentioned 2.6% glycerol isotonic aqueous solution to prepare a 2 mg / mL protamine sulfate solution, and filter and sterilize it using a 0.2 μm filter membrane for subsequent use;

[0082] (3) Dissolve CpG ODN 2006 in the above-mentioned 2.6% glycerol isotonic aqueous solution to prepare a 2 mg / mL CpG ODN 2006 solution, filter and sterilize it using a 0.2 μm filter membrane, and set aside;

[0083] (4) Using a pipette, different volumes of protamine solution were mixed evenly with a certain volume of CpG ODN 2006 solution to form a suspension under different mass ratios (protamine sulfate: CpG ODN 2006 = 0.70:1.0, 0.80:1.0, 0.90:1.0, 1.0:1.0, 1.1:1.0, 1.2:1.0, 1.3:1.0, 1.4:1.0, 1.5:1.0, 1.6:1.0, 1.8:1.0 and 2.0:1.0);

[0084] (5) The suspension in step (4) is dispersed by high-pressure homogenization.

[0085] (6) The protamine sulfate-CpG ODN 1826 nanocomplex solutions prepared at different ratios in this experiment were centrifuged at 8000 g for 15 min, and the supernatant was taken. The concentration of free CpG ODN 2006 in the supernatant was detected using the Quant-iT OliGreen ssDNA kit, and the mass of CpG ODN 2006 in the complex was calculated. In addition, the particle size changes of the protamine sulfate-CpG ODN 2006 nanocomplex (under the condition of 1.0:1.0) after storage at 4° for 7 days, 14 days, 31 days, 90 days, and 180 days, as well as the particle size changes after reconstitution after freezing, were monitored to investigate the stability of the nanoparticles.

[0086] The nanocomposite obtained in the following Example 1 (under the condition of 1.0:1.0) was observed for morphological characteristics using a transmission electron microscope (TEM, FEI, Tecnai G220S-TWIN, 200KV). Sample preparation method: The nanocomposite was diluted to 0.005 mg / ml with deionized water, 10 μl of the sample was dripped onto a 230-mesh copper grid containing a carbon support film, and after constant temperature drying, the sample morphology was observed using a TEM.

[0087] Experimental results: The present invention sets up nanocomplexes formed under different mass ratios of protamine sulfate and CpG ODN 2006, and performs ratio screening to determine the optimal formation ratio. The results are shown in Table 2. Except when the ratio is 1.1:1.0, the surface potential changes from negative charge to positive charge, and the particle size is larger, about 830nm, the particle size of the nanocomplexes formed under other ratios is about 200nm, and has a good PDI. Figure 5 and Figure 6 The particle size and potential distribution diagrams of the nanocomposite formed under the ratio of 1.0:1.0 are shown respectively. Figure 7 and Figure 8 The particle size and potential distribution diagrams of the nanocomposite formed under the ratio of 2.0:1.0 are shown respectively. Then, in order to determine the morphology of the nanocomposite, we conducted a transmission electron microscopy analysis on the nanocomposite formed under the ratio of 1.0:1.0 in Example 2. The results are shown in FIG. Fig. 9 As shown in the figure, the nanocomposite is a spherical structure with uniform size distribution. Both the TEM image and the DLS histogram show that the nanocomposite has good dispersion performance. The stability of the nanocomposite is of great significance for its practical application. We further conducted long-term stability and post-freezing / thawing stability studies on the nanocomposite formed under the condition of a ratio of 1.0:1.0, and the results are shown in the figure. Fig.10 and 11 The results show that the nanocomposite formed under this ratio has excellent particle size stability.

[0088] Table 2: Particle size and potential measurement results of nanoimmunocomplexes prepared under different mass ratios of protamine sulfate and CpG ODN 2006

[0089] Protamine: CpG 2006 Particle size (nm) PDI Potential (mV) 0.7：1 162.6 0.242 -51.2 0.8：1 170.0 0.220 -49.1 0.9：1 179.6 0.240 -48.5 1.0：1 180.1 0.227 -47.3 1.1：1 830.1 0.390 26.2 1.2：1 288.2 0.262 30.1 1.3：1 228.0 0.278 30.9 1.4：1 233.3 0.294 31.2 1.5：1 215.0 0.204 32.1 1.6：1 214.7 0.241 30.5 1.8：1 204.3 0.266 32.0 2.0：1 191.3 0.238 28.0

[0090] Example 3 This example is different from Example 1 in that the CpG ODN can be changed to other types of CpG ODN such as CpG ODN 2007, CpG ODN 1018, CpG ODN 1585, CpG ODN 2216, CpG ODN 2395, and the other preparation methods and characterization methods are the same as those in Example 1.

[0091] Example 4 Evaluation of the immunostimulatory activity of the nanoimmune complex in Example 1 of the present invention in mouse BMDC cells.

[0092] 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 cell culture medium was replaced every 2 days for 7 days. 6 The cells were plated at a density of 100 μg / ml in a 24-well plate and divided into 4 groups for immunostimulation experiments, namely PBS group, CpG ODN 1826 group alone (CpG ODN 2 μg / ml), protamine group alone and protamine sulfate-CpG ODN 1826 nanocomplex group (1.2:1.0 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, and the BMDCs were stained with flow cytometry antibodies for CD80 and CD86.

[0093] 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. Nanocomposite can significantly enhance its immune activity. We used flow cytometry to observe the effects of different components on the maturation of mouse BMDCs. The results are as follows: Fig.12 As shown, compared with the control group and the protamine treatment group alone, the CpG ODN 1826 alone and the protamine sulfate-CpG ODN 1826 nanoimmunocomplex group can stimulate CD11c-positive DC cells to express more CD80 and CD86 co-stimulatory molecules. In addition, the stimulation effect of the protamine sulfate-CpG ODN 1826 nanoimmunocomplex group was significantly higher than that of the CpG ODN1826 alone group. The above results indicate that the protamine sulfate-CpG ODN 1826 nanoimmunocomplex can more efficiently promote the maturation of DC cells. During the DC maturation process, a large amount of cytokines such as IL-6 and IL12-p40 are also secreted. We further observed the release of cytokines by ELISA, such as Fig.13As shown in the figure, compared with the control group, the secretion of both cytokines increased in the CpG ODN 1826 alone group and the protamine sulfate-CpG ODN 1826 nanoimmunocomplex treatment group, but the secretion of the nanocomplex group was significantly higher than that of the CpG ODN 1826 alone group. The above results show that the protamine sulfate-CpG ODN 1826 nanoimmunocomplex prepared by us has excellent ability to induce BMDC cell maturation and release related immune cytokines.

[0094] Example 5 Evaluation of the immunostimulatory activity of the nanocomplex in Example 2 of the present invention in human PBMC cells.

[0095] Human PBMCs were separated from fresh blood using Ficoll lymphocyte separation medium, suspended in 1640 medium containing 10% FBS, and seeded in 24-well plates at 5×10 5 Cells / well. Use cell culture medium (RPMI-1640+20ng / ml GM-CSF+1ng / ml IL-4) to adjust the cell density to 10 6 4 ml of cell suspension was plated in each well of a 6-well plate, and 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 2006 group alone, protamine group alone and protamine sulfate-CpG ODN 2006 nanoimmunocomplex group (1.0:1.0 group, CpG ODN 2 μg / ml). ELISA determination of PBMC supernatant and serum cytokine content According to the ELISA kit instructions, the IL-6 content in the PMDC supernatant was determined.

[0096] Experimental results: We used flow cytometry to observe the effects of different components on the maturation of human BMDCs. Fig.14 As shown, compared with the control group, the secretion of cytokines increased in both the CpG ODN 2006 alone group and the protamine sulfate-CpG ODN 2006 nanocomplex treatment group, but the secretion of the nanocomplex group was significantly higher than that of the CpG ODN 2006 alone group. The above results show that the protamine sulfate-CpG ODN2006 nanocomplex prepared by us has excellent PBMC cell immune activation ability.

[0097] Example 6 Experiment on the inhibition of the activity of mouse lymphoma cell line A20 by protamine sulfate-CpG ODN 1826 nanoimmunocomplex.

[0098] Mouse B cell lymphoma cells (A20) were suspended in RPMI 1640 culture medium containing 10% FBS and cultured in a cell culture incubator at 37°C and 5% CO2. When the cells grew to the logarithmic growth phase, that is, the cell confluence reached about 80%, 5×10 4 A20 cells were inoculated in a 96-well plate. PBS was used as the control group, and each well was added with different mass concentrations of CpG ODN1826 and protamine sulfate-CpG ODN1826 nanocomplexes. Four parallel samples were added for each concentration and cultured for 72 hours. 1640 culture medium containing CCK-8 reagent was added to each well and allowed to stand at 37°C for 100 minutes. The absorbance of the solution was detected at a wavelength of 450nm using an automatic microplate reader, and repeated 3 times to measure the cell viability bar graph.

[0099] Experimental results: Related research results show that in addition to inducing anti-tumor immune response, type B CpG ODN itself has a significant apoptosis-inducing effect on a variety of lymphoma cells and leukemia cells, which can lead to cell G1 phase arrest and autocrine interferon, and lead to cell apoptosis through the Fas ligand pathway. Further, we compared the survival rate of cells treated with different quality CpG ODN. Fig.15 As shown, at different CpG ODN 1826 mass concentrations, both CpG ODN-1826 alone and protamine sulfate-CpG ODN1826 nanoimmunocomplexes showed a concentration-dependent cytotoxicity trend, but the protamine sulfate-CpG ODN1826 nanoimmunocomplex had a significantly higher ability to inhibit cell growth than CpG ODN 1826 alone, and had a high inhibition efficiency (at a concentration greater than 0.3 μg / ml).

[0100] Example 7 Intratumoral injection of protamine sulfate-CpG ODN 1826 nanoimmune complexes for the treatment of A20 transplanted lymphoma.

[0101] Female BALB / c mice, 5 to 7 weeks old and weighing about 18 to 20 g, were selected and adaptively raised in the animal room for one week. When A20 reached the logarithmic phase, cells were collected by centrifugation and the cell density was adjusted to 3×10 6 Each mouse was subcutaneously inoculated with 100 μl of A20 cells at 4% s / ml to establish a mouse lymphoma transplantation model. 3At 14:00, mice were randomly divided into PBS group, CpG ODN 1826 group and protamine sulfate-CpG ODN 1826 nanocomplex group according to tumor size and weight. The CpG ODN dosage was 20 μg / mouse. The mice were injected intratumorally on days 2, 4, 6 and 12. The mice were killed two days after the end of the administration. During the experiment, the tumor volume and body weight were measured every two days every week. 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.

[0102] Experimental results: Example 6 has confirmed that the protamine sulfate-CpG ODN 1826 nanocomplex group can significantly inhibit the growth of A20 cells at the in vitro cell level. Further, we established an A20 transplanted lymphoma model and studied the tumor inhibition effects of different components. The experimental results are consistent with the trend of the cell experiment results. Fig.16 The tumor growth curves showed that, compared with the PBS control group, both CpG ODN alone and protamine sulfate-CpG ODN 1826 nanocomplexes had an inhibitory effect on tumor growth, but the inhibitory effect of the nanocomplex injected into the tumor was particularly significant.

[0103] Example 8 Intratumoral injection of protamine sulfate-CpG ODN1826 nanoimmune complexes to treat B16F10 transplanted melanoma

[0104] 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 divided into PBS group, CpG ODN1826 group and protamine sulfate-CpG ODN 1826 nanocomplex group (CpG ODN dosage was 20 μg / mouse). The mice were killed two days after the end of the administration. During the experiment, the tumor volume and body weight were measured every two days every week. 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.

[0105] After killing the mice, the mouse tumor tissue was dissected, and the extracellular matrix was digested using a mouse tumor tissue dissociation kit to prepare the tumor tissue into a single cell suspension. The DC maturation and immune cell grouping ratio in tumor-infiltrating immune cells were analyzed by flow cytometry. The freshly harvested tumor tissue was gently ground and filtered through a 300-mesh nylon filter to obtain a single cell suspension. The single cell suspension was washed with FACS buffer, then stained with fluorescently labeled antibodies as required, and then fixed with 4% PFA for further immune cell subtype and ratio analysis. After the tumor-bearing mice were administered according to the above-mentioned scheme, the mouse tumor tissue was also dissected, and the same weight of tumor tissue was weighed and placed in a homogenizer tube, 500 μl PBS was added to each tube, and the tissue cells were lysed using a tissue homogenizer. The supernatant was collected after centrifugation, and the corresponding ELISA kit was used to detect the secretion of the corresponding cytokines in the supernatant.

[0106] Experimental results: We then established a mouse melanoma tumor model to evaluate the therapeutic effects of intratumoral injection of each component. 3 When the tumor size was about 1.5 × 10 / 10, mice with uniform tumor size were selected and divided into four groups. PBS, protamine sulfate alone, CpG ODN 1826 alone, and protamine sulfate-CpG ODN 1826 nanoimmunocomplex were injected intratumorally every two days for 5 times. Fig.17 As shown, compared with the PBS control group, protamine alone did not have a therapeutic effect on the tumor, and CpG ODN 1826 alone had a very limited inhibitory effect on tumor growth, while protamine sulfate-CpG ODN 1826 nanoimmunocomplexes could effectively inhibit tumor growth. In addition, the results of IL-6 and IL-12 levels in the serum of mice in each component also showed that under in vivo experimental conditions, protamine sulfate-CpG ODN 1826 nanoimmunocomplexes could stimulate the release of higher cytokines. Further, we ground the peeled tumor tissue, isolated the infiltrating lymphocytes in the tumor, and analyzed the CD4 + T cells and CD8 + The experimental results showed that the CD4 + T cells and CD8 + T cells increased significantly compared with the control group.

[0107] Example 9 Prevention of Growth of Transplanted Tumors in Mice by Protamine Sulfate-CpG ODN1826 Nanocomplex Combined with Tumor-associated Antigens

[0108] Melanoma antigen-A3 (MAGE-A3) peptide (FLWGPRALV) was used as tumor-associated antigen. Female BALB / c mice aged 5 to 7 weeks and weighing about 18 to 20 g were selected, with 5 mice in each group, and were adaptively raised in the animal room for one week. On the 35th day, 28th day, 21st day, 14th day and 7th day before inoculation of melanoma cell line B16F10, different groups were subcutaneously injected: (1) PBS, (2) protamine sulfate-CpG ODN 1826 nanoimmunocomplex + MAGE-A3 peptide, (3) MAGE-peptide, (4) protamine sulfate-CpG ODN 1826 nanoimmunocomplex. The dosage of CpGODN was 20 μg / mouse, and the dosage of MAGE-A3 peptide was 50 μg / mouse. On day 0, 100 μl of B16F10 cells were subcutaneously inoculated into the right hind limb of each mouse. After the tumor grows, the size of the tumor on the side of the face is measured every two days. The tumor volume is 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.

[0109] Experimental results: The CpG ODN nanoimmune complex of the present invention can also be used as a vaccine adjuvant. Furthermore, using MAGE-A3 polypeptide (FLWGPRALV) as a tumor-associated antigen, the protamine sulfate-CpG ODN1826 nanocomplex combined with MAGE-A3 as a tumor preventive vaccine was studied to investigate the preventive effect of transplanted tumor growth in mice. Seven days after the last immunization, the melanoma cell line B16F10 was inoculated subcutaneously in the mice, and the growth changes of the tumor were measured every two days. After about two weeks of monitoring. The experimental results show that Fig.19 As shown, the use of CpG ODN 1826 alone as an adjuvant combined with MAGE-A3 polypeptide can also delay tumor growth to a certain extent. In comparison, the growth of tumors in mice vaccinated in advance with protamine sulfate-CpG ODN1826 nanoimmune complex combined with MAGE-A3 was the slowest. The experimental results of mouse weight change also showed that the weight change after each component was inoculated was not significant. This fully demonstrates that the protamine sulfate-CpG ODN1826 nanocomplex prepared by the patent method has good safety and no obvious toxic side effects on organisms.

[0110] Example 10 Animal immunization experiment using protamine sulfate-CpG ODN1826 nanoimmune complex as hepatitis B vaccine adjuvant.

[0111] BALB / c mice were used and the experimental animals were divided into the following groups: 1. PBS; 2. HbsAg; 3. Protamine sulfate-CpG ODN 1826 immune complex + HbsAg; 4. CpG 1826 + HbsAg; 5. Aluminum hydroxide gel adjuvant (Alum) + HbsAg. The HbsAg in each component was 1μg, CpG 1826 was 10μg, and Alum was 225μg. Five mice were immunized by intramuscular injection at 0, 2 and 4 weeks, and serum and spleen lymphocytes were collected at 4 weeks for immunological analysis. The anti-HBsAg antibody titer in the serum of each group of immunized mice was detected by ELISA kit.

[0112] The experimental results are as follows Fig. 20 As shown, the HbsAg group alone produced almost no antibodies, and the antibody titer produced by the protamine sulfate-CpG ODN1826 nanoimmune complex + 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 protamine sulfate-CpG ODN1826 immune complex can significantly enhance the humoral immune effect of HbsAg.

Claims

1. A method for preparing a CpG ODN nanoimmune complex, characterized in that: The method comprises the following steps: S1. Weigh a certain amount of non-ionic osmotic pressure regulator, add it to sterile and endotoxin-free water, stir or ultrasonically disperse and dissolve it, filter and sterilize it with a 0.2 μm filter membrane, prepare an isotonic aqueous solution, and set aside; S2. Dissolve the protamine in the isotonic aqueous solution to prepare a 0.1-1% (w / v) protamine solution, filter and sterilize with a 0.2 μm filter membrane, and set aside; S3. Dissolve the CpG ODN in the above isotonic aqueous solution to prepare a 0.1-1% (w / v) CpG ODN solution, filter and sterilize using a 0.2 μm filter membrane, and set aside; S4. Mix a certain amount of protamine solution and a certain amount of CpG ODN solution to form a suspension; S5. Dispersing the above suspension in a homogenous manner by applying energy to prepare a protamine-CpG ODN nanocomplex liquid preparation; S6. The obtained protamine-CpG ODN nanocomplex liquid preparation is sterilized at high temperature and high pressure.

2. The preparation method according to claim 1, characterized in that: The feed mass ratio of protamine to CpG ODN is (0.5-3.0):

1.

3. The preparation method according to claim 1, characterized in that: The feed mass ratio of protamine to CpG ODN is (0.7-2.0):

1.

4. The preparation method according to claim 1, characterized in that: The suspension is dispersed uniformly by applying energy homogenization, and a combination of one or more methods including ultrasonic crushing / dispersion, high-speed dispersion homogenization, high-pressure homogenization dispersion, and micro-jet homogenization dispersion can be selected.

5. The nonionic osmotic pressure regulator according to claim 1 comprises at least one of lactose, sucrose, glucose, dextran, glycerol or D-mannitol or any combination thereof.

6. The CpG ODN according to claim 1, characterized in that The CpG ODN may include one of type A CpG ODN, type B CpG ODN or type C CpG ODN or any combination thereof.

7. The CpG ODN according to claims 1 to 3 and 6, characterized in that The CpG ODN may be an unmodified or modified oligodeoxynucleotide CpG ODN fragment.

8. The protamine according to claims 1 to 3, characterized in that The protamine includes protamine itself and pharmaceutically acceptable salts thereof, such as protamine (free base), protamine sulfate, protamine phosphate, protamine hydrochloride, etc.

9. The protamine according to claims 1 to 3 and 8, characterized in that The protamine is a high molecular weight protamine. More preferably, the molecular weight of the protamine is 5000 to 10000.

10. A CpG ODN nanoimmune complex, characterized in that: The nanoimmune complex is prepared according to the preparation method in claim 1.

11. The CpG ODN nanoimmunocomplex according to claims 1 and 10, characterized in that: The average surface charge of the nanoimmune complex can be negative or positive.

12. The CpG ODN nanoimmune complex according to claims 1 and 10-11, characterized in that: The average particle size of the nano-immune complex is in the range of 100 to 1000 nm. Preferably, the average particle size of the nano-immune complex is in the range of 100 to 300 nm.

13. The CpG ODN nanoimmunocomplex according to claims 1 and 10-12, characterized in that: The concentration of protamine in the nano-immune complex solution preparation is 0.5-9.09 mg / mL.

14. The CpG ODN nanoimmunocomplex according to claims 1 and 10-13, characterized in that: The concentration of CpG ODN in the nano-immune complex solution preparation is 0.5-9.09 mg / mL. Preferably, the concentration of CpG ODN is 0.5-2.0 mg / mL.

15. Use of the CpG ODN nanoimmune complex according to any one of claims 1 and 10-14 in cellular immune stimulation and enhancing the release of cytokines from immune cells.

16. The immune cell according to claim 15, 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.

17. An independent use of the CpG ODN nanoimmune complex according to any one of claims 1 and 10-16 in the preparation of a drug for preventing and treating respiratory viral infectious diseases.

18. An independent use of the CpG ODN nanoimmune complex according to any one of claims 1 and 10-17 in the preparation of a drug for preventing and treating respiratory bacterial infectious diseases.

19. An independent use of the CpG ODN nanoimmune complex according to any one of claims 1 and 10-18 in the preparation of a drug for preventing and treating respiratory fungal infections.

20. An independent use of the CpG ODN nanoimmune complex according to any one of claims 1 and 10-19 in the preparation of anti-allergic disease drugs.

21. Use of the CpG ODN nanoimmune complex according to any one of claims 1 and 10-20 in combination with another anti-allergic disease therapy in the preparation of an anti-allergic disease drug.

22. The use according to claim 21, wherein the additional anti-allergic disease therapy is an H1 receptor blocker, a glucocorticoid, a leukotriene receptor antagonist, a mast cell membrane stabilizer or a nasal vasoconstrictor.

23. The use according to claims 20-22, wherein the allergic disease is allergic rhinitis, allergic asthma, allergic conjunctivitis, allergic dermatitis and the like.

24. An independent use of the CpG ODN nanoimmunocomplex according to any one of claims 1 and 10-23 in the preparation of anti-tumor immune drugs.

25. Use of the CpG ODN nanoimmunocomplex according to any one of claims 1 and 10-24 in combination with other tumor therapies in treating tumors.

26. The use of claim 25, wherein the additional tumor therapy is surgery, chemotherapy, targeted drug therapy and other immunotherapy drugs and methods.

27. Use of the CpG ODN nanoimmune complex combined with an antigen as claimed in any one of claims 1 and 10-26 as a vaccine adjuvant in the preparation of a vaccine preparation for human use.

28. Use of the CpG ODN nanoimmune complex combined with an antigen as claimed in any one of claims 1 and 10-27 as a vaccine adjuvant in the preparation of vaccine preparations for animals.

29. The antigens as claimed in claims 25 and 28, wherein the antigens are plant-derived antigens, microbial-derived antigens (bacteria, viruses, fungi, parasitic protozoa, etc.) and tumor-derived antigens.

30. Use of the CpG ODN nanoimmune complex according to any one of claims 1 and 10-29 combined with tumor antigens as a vaccine adjuvant in the preparation of tumor preventive and therapeutic drugs.

31. The use according to claims 29-30, wherein the tumor antigen is a tumor-associated antigen or a tumor-specific antigen or a combination thereof.

32. The use of claims 24-26 and 29-31, wherein the tumor 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.

33. An independent use of the CpG ODN nanoimmunocomplex according to any one of claims 1 and 10-32 in the preparation of an anti-Alzheimer's disease drug.

34. Use of the CpG ODN nanoimmunocomplex of any one of claims 1 and 10-33 in combination with another drug or therapy for treating Alzheimer's disease in an anti-Alzheimer's disease drug.

35. The use of claim 34, wherein the additional drug or therapy for treating Alzheimer's disease is an acetylcholinesterase inhibitor, an NMDA receptor antagonist or a brain metabolism activator.

36. The use according to any one of claims 15-35, wherein the administration method of the use 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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