CpG oligodeoxynucleotides with the ability to modulate the immune system of organisms and their use

Abstract

This invention provides multiple oligodeoxyribonucleotides (ODNs), including CpGs, that have the function of modulating the immune system of organisms, and their uses. These oligodeoxyribonucleotides differ in sequence and structure from the design concepts of conventional type A CpG ODNs, demonstrating complex drug discovery rules and solving the challenges of conventional type A CpG drug discovery. They exhibit excellent activity in modulating the immune function of organisms both in vitro and in vivo, stimulating the production of type I, II, and III interferons, enhancing the Th1 immune response, and suppressing Th2 and Th17 inflammatory responses. Therefore, they can prevent and treat allergic diseases caused by Th2 immune responses, such as allergic rhinitis, atopic dermatitis, asthma, chronic obstructive pulmonary disease, eosinophilic granulocyte and Th17-related diseases, prevent and treat infection and transmission of respiratory or non-respiratory pathogenic microorganisms such as viruses, bacteria, fungi, and parasites, and improve immune levels in elderly and immunocompromised populations. It can transform "cold tumors" into "hot tumors," improve the effectiveness of cancer immunotherapy, prevent and treat central nervous system diseases associated with immune dysregulation such as Alzheimer's disease, and can also be used for other diseases associated with immune dysregulation.

Description

[Technical Field]

[0001] Cross-reference of related applications This invention claims priority to a prior application filed with the China National Intellectual Property Administration on July 21, 2023, patent application number 202310902755.X, with the title of the invention "CpG oligodeoxynucleotide having the ability to modulate the immune system of organisms and its use." The entire text of the said prior application is incorporated herein by reference.

[0002] This invention belongs to the field of oligodeoxynucleotides, and more specifically, to CpG oligodeoxynucleotides having the ability to modulate the immune system of organisms and their use. [Background technology]

[0003] TLR9 is a mammalian innate immune receptor located in endosomes, and its ligand is an unmethylated CpG-containing oligodeoxyribonucleotide (ODN). Depending on the type and structure, CpG-ODNs, after entering the endosome, bind to TLR9 and activate the IRF-7 or NF-κB signaling pathway, thereby generating inflammatory factors such as type I interferon or IL-6 and intervening in the immune response.

[0004] Based on their structural characteristics and immunological effects, CpG ODNs can be classified into three types: A, B, and C. Type A CpGs enter the early endosomes of plasmacytoid dendritic cells (pDCs) and bind to TLR9 receptors, activating the IRF-7 signaling pathway and inducing the massive production of type I interferons (IFN-α / IFN-β) and type III interferons (IFN-λ). Type I interferons act as comprehensive immunomodulatory cytokines, affecting NK cells, CD8 cells, and other cells. + T cells, CD4 +CpG acts on downstream immune cells, including but not limited to T cells, γ / δ T cells, and macrophages, inducing the production of cytokines such as type II interferon (IFN-γ). Type B CpG is a fully phosphorothioate-modified linear CpG ODN that primarily targets B lymphocytes. Type B CpG enters late endosomes, binds to TLR9, activates NF-κB, and triggers a series of immune responses, including the secretion of inflammatory cytokines such as IL-6, IL-1, and TNF-α, and the promotion of B cell proliferation. Type B CpG can significantly improve antibody levels as a vaccine adjuvant. Type C CpG is a fully phosphorothioate-modified CpG ODN that contains parts of the structure of type A CpG and parts of the structure of type B CpG, has a palindromic sequence that can form dimers, and also has a non-palindromic linear structure. Type C CpG can enter early endosomes and bind to TLR9, or enter late endosomes and bind to TLR9. Therefore, it possesses the activity of both type A and type B CpG-ODNs, and can not only activate the IRF-7 signaling pathway but also activate the NF-κB pathway to generate type I interferons and inflammatory cytokines. Type C CpG has the characteristics of both type A and type B CpG, but its ability to induce interferons and inflammatory molecules is significantly lower than the activity of either type A or type B CpG.

[0005] Preclinical animal studies of hepatitis B (CpG) have demonstrated its potential use in treating allergic rhinitis, asthma, various microbial infections, and as an antitumor agent. Over the past 20 years, there have been more than 100 publications on the treatment of various diseases using CpG in preclinical studies. CpG-1018, a representative example of hepatitis B, was already approved for market as a hepatitis B vaccine adjuvant in 2017.

[0006] Previous studies have shown that when administered via different routes, type A CpG ODN targets TLR9 in pDCs, thereby inducing a robust innate immune response (type I, type II, and type III interferons) and a Th1 immune response (DCs, NK cells, and CD8 cells). +By inducing T cell activation (IL-12 production) and regulating immune function, Th2 and Th17 immune responses can be suppressed. Through these immune responses, various effects can be achieved, including the treatment of diseases caused by allergic and non-allergic inflammation, mitigation of pathogenic microbial infections, direct or indirect killing of tumor cells, reduction of Aβ plaque formation, and improvement of vaccine immunogenicity. This forms the basis for its use in fields such as infectious diseases, cancer, immune adjuvants, allergic rhinitis, atopic dermatitis, asthma, chronic obstructive pulmonary disease, eosinophilic granulocyte-associated diseases, Alzheimer's disease, wound healing, and prevention and treatment of wound infections. (Montamat et.,Fronteers in Immunol.2021.doi:10.3389 / fimmu.2021.590054, Scheiermann et al.,Vaccine 2014.32:6377-6389, Shirota and Klinman.,Immunopotentiators in Modern Vaccines ISBN 978-0-12-804019-5, http: / / dx.doi.org / 10.1016 / B978-0-12-804019-5.00009-8, Beeh et al., J Allergy Clin Immunol 2013;131:866-74, Hanagata N. et al., Int J Nanomedicine.2012;7:2181-95, Kayraklioglu et al.,Methods Mol Biol.2021;2197:51-85, Fan et al., Clin Cancer Res.2012 Oct 15;18(20):5628-38, Senti et al., Clin Exp Allergy.2009 Apr;39(4):562-70, Scholtzova et al., Acta Neuropathol Commun.2014 Sep 2;2:101, Wanke-Jellinek et al., J Immunol.2016 Jan 15;196(2):767-77).

[0007] Because type A CpGs have a tendency to associate easily, their drug potential is very low. As a result, even more than 20 years after the discovery of type A CpGs, there have been few clinical trials and no drugs approved for commercial sale. To date, three organizations have been trying to improve the drug potential of type A CpGs through various means. 1. Cytos Biotechnology AG has improved drug potential by encapsulating type A CpGs (QbG-10) with virus-like particles (VLPs). By encapsulating with VLPs, type A CpGs are no longer simple CpGs but have become CpGs with potent adjuvant functions (Storni et al., J.Immunol.2004.172:1777-1785). 2. Researchers at the U.S. Food and Drug Administration (FDA) have transformed type A CpGs into temperature-sensitive prodrugs by adding the small molecule compound fma to the five Gs at the 3' end of the CpG molecule. Upon entering cells, type A CpG reassociates and exerts its therapeutic effect when fma spontaneously detaches at 37°C. However, such modification cannot completely prevent association in vitro, and modification with fma limits the safety and tolerance of the drug (Puig et al., Puig, Nucleic acids research. 2005. 34:6488-6495). 3. In 2015, Japanese researchers reported the following: Adding 40 adenine deoxyribonucleotides (poly-A) to the 3' end of type A CpG can significantly improve the drug potential of type A CpG. However, because 40 adenine molecules are introduced, production costs are significantly increased, and the fact that it is completely phosphorothioated increases the production of the IL-6 inflammatory molecule, limiting its safety and tolerance (Aoshi et al., J.Immunol.Research.2015. http: / / dx.doi.org / 10.1155 / 2015 / 316364). For this reason, there have been no successful cases of developing type A CpG as a drug, and no clinical trial reports have been published.

[0008] The conventional design principle for type A CpGs involves generating an aggregate through a complete palindrome of a core sequence consisting of 8-12 nucleotides (containing one or more CpGs) and poly-Gs at both ends. The aggregate presents the CpG core sequence to TLR9 on early endosomes rather than late endosomes, thereby activating the IRF-7 signaling pathway and generating type I interferon. Formation of the aggregate is necessary to activate the IRF-7 signaling pathway (Kerkmann et al., J.Bio.Chem., 2005.280:8086-8093, Wu et al., J.Bio.Chem., 2004.279:33071-33078). CpG ODN monomers cannot activate the IRF-7 signaling pathway. For example, type B CpG exists as a monomer in solution and can only activate the NF-κB signaling pathway by binding to TLR9 in late endosomes, inducing the production of inflammatory factors rather than type I interferons. Type A CpG is only active when it forms aggregates, but when aggregates are formed, they are generally large and uncontrollable, significantly reducing its drug potential. This is the main reason why, even after the development of type A CpG, there have been no successful clinical development cases. Therefore, how to ensure that type A CpG is associative and how to control this association is key to determining whether such molecules can be developed as clinically effective drugs.

[0009] The conventional self-association model of type A CpG was proposed by Kerkmann et al. in 2005 (Kerkmann et al., J.Bio.Chem., 2005.280:8086-8093). Two complementary monomers form a dimer via a Watson-Crick bond, and the poly-Gs at both ends of the dimer form a Hoogsteen base pair through hydrogen bonding of guanylic acid to form a tetramer (G-tetrad). Next, the dimer and tetramer continue to associate via the G-tetrad, and this process is repeated to form a larger aggregate. Another model is based on the secondary structure of type A CpG. Monomers of type A CpG can form a palindromic structure within the monomer, and the poly-Gs at both ends can form a larger aggregate through Hoogsteen base pairing. Therefore, these models show that the size and stability of the aggregate are determined by the number of surrounding Hoogsteen-type bonds and their unique spatial structure. In our previous research, we discovered that the complete palindromic morphology before and after the poly-G reduces the energy required for aggregation, making it easier to aggregate. Therefore, by creating a certain spatial barrier between the poly-G and the palindrome, the energy required to form the G-tetrad can be increased, making it more difficult to form the aggregate or loosening the formed aggregate. In addition, by selecting the number of poly-Gs at both ends, the size of the aggregate can be stabilized within a certain range. Based on the above considerations, this invention designs multiple core sequences and, while maintaining the invariance of the core sequences, performs deoxyribonucleotide substitution experiments on both ends of type A CpG to discover the rules for the formation of type A CpG aggregates, and further discovers the relationship between this and the production of type I interferon in response to stimulation. [Overview of the Initiative]

[0010] The technical aspects of the present invention are as follows. A CpG oligodeoxynucleotide having the ability to regulate the immune system of an organism, wherein the sequence structure of the CpG oligodeoxynucleotide having the ability to regulate the immune system of an organism described in the present invention is 5'-(G) n -Core Array-(N) q -(G) m The sequence is one of the -3' sequences, the core sequence is a palindrome sequence, N is one of three bases: adenine deoxyribonucleotide A, thymine deoxyribonucleotide T, and cytosine deoxyribonucleotide C, G is guanine deoxyribonucleotide, q, n, and m are all integers, the range of the value of n is 2 to 11, the range of the value of q is 0 to 10, and the range of the value of m is 2 to 11.

[0011] Preferably, the range of the value of n is 3 to 4, and the range of the value of q is 1 to 5.

[0012] Preferably, the core sequence is at least one of CGCGACGCGTCGCG, ACGATCGAGATCGT, AGGATCGATCCT, TTCGATCGATCGAA, CGATCGATCG, GACGATCGTC, and TGCATCGATGCA. When the core array is CGCGACGCGTCGCG, the range of the value of q is 0 to 10, the range of the value of n is 0 to 3, and the range of the value of m is 2 to 11. Alternatively, if the core array is ACGATCGAGATCGT, the range of the value of q is 0 to 10, the range of the value of n is 2 to 11, and the range of the value of m is 2 to 11. Alternatively, if the core array is AGGATCGATCCT, the range of the value of q is 0 to 10, the range of the value of n is 2 to 11, and the range of the value of m is 2 to 11. Alternatively, if the core array is TTCGATCGATCGAA, the range of the value of q is 0 to 10, the range of the value of n is 2 to 11, and the range of the value of m is 2 to 11. Or, when the core sequence is CGATCGATCG, the range of the value of q is 0 to 10, the range of the value of n is 1 to 11, and the range of the value of m is 2 to 11. Or, when the core sequence is GACGATCGTC, q is 0, the range of the value of n is 0 to 3, the range of the value of m is 2 to 11, or the range of the value of q is 1 to 10, the range of the value of n is 2 to 11, and the range of the value of m is 2 to 11. Or, when the core sequence is TGCATCGATGCA, q is 0, the value of n is 1, 3, 4 or 5, the range of the value of m is 2 to 11, or the range of the value of q is 1 to 10, the range of the value of n is 2 to 11, and the range of the value of m is 2 to 11.

[0013] Preferably, the above CpG oligodeoxynucleotide contains at least one of the deoxynucleotide sequences represented by SEQ ID NO: 1 to SEQ ID NO: 82.

[0014] Preferably, the CpG oligodeoxynucleotide is itself or contains at least one of thio-modification, fluoro-modification, methoxy-modification, locked nucleic acid modification, and nanoparticle modification.

[0015] Preferably, the site of the thio-modification is in the polyguanilic acid at the 5'-end and / or 3'-end.

[0016] Preferably, the nanoparticles employed in the nanoparticle modification contain at least one of PLGA, chitosan, lipid nanoparticles, and liposomes.

[0017] A CpG oligodeoxynucleotide preparation comprising the above CpG oligodeoxynucleotide and an additive, wherein the additive comprises at least one of a pH buffer pair, glycine, trehalose, mannitol, sucrose, arginine, lysine, histidine, glycerin, propylene glycol, Pluronic F127, Pluronic F68, Tween 20, Tween 80, benzalkonium chloride, disodium edetate, sodium citrate, hypromellose, sodium carboxymethylcellulose, methyl-β-cyclodextrin, and polyethylene glycol.

[0018] Preferably, the metal ions in the CpG oligodeoxynucleotide preparation include sodium, potassium, magnesium, calcium, zinc, and iron, and the total concentration range is 0.1 - 90 mM.

[0019] Preferably, the pH of the CpG oligodeoxynucleotide preparation is 7 - 9.

[0020] Use of the above CpG oligodeoxynucleotide in the manufacture of a drug for regulating the immune response of the respiratory tract.

[0021] Use of the above CpG oligodeoxynucleotide in the manufacture of a drug for preventing and treating respiratory diseases, wherein the respiratory diseases include at least one of respiratory viral infections, respiratory bacterial infections, respiratory fungal infections, respiratory parasitic infections, and respiratory allergic diseases. The respiratory virus in the respiratory viral infection includes at least one of the COVID-19 virus, influenza virus, RSV virus, and SARS-Cov.

[0022] The use of the above-mentioned CpG oligodeoxyribonucleotide in the manufacture of a drug for the prevention and treatment of non-respiratory infections, wherein the non-respiratory infection includes at least one of HIV virus infection, HBV virus infection, and HCV virus infection.

[0023] Use of the above-mentioned CpG oligodeoxynucleotides in the manufacture of drugs that regulate the proliferation function of immune cells or drugs that regulate the release of cytokines from immune cells.

[0024] The use of the above-mentioned CpG oligodeoxynucleotides in the manufacture of immunoadjuvants.

[0025] The use of the above-mentioned CpG oligodeoxynucleotides in the manufacture of drugs for the prevention and treatment of viral infections.

[0026] The use of CpG oligodeoxynucleotides in the manufacture of a drug for treating tumors, wherein the CpG oligodeoxynucleotide in the drug is the sole antitumor active ingredient.

[0027] An antitumor composition comprising the above-mentioned CpG oligodeoxynucleotide and an antitumor drug, wherein the antitumor drug includes, but is not limited to, PD-1 antitumor drugs, PDL-1 antitumor drugs, and antitumor cell therapy, and in the above composition, the CpG oligodeoxynucleotide is used as an antitumor drug component or an immunoadjuvant.

[0028] The use of the above-mentioned CpG oligodeoxynucleotides in the manufacture of human or animal vaccines.

[0029] The use of CpG oligodeoxynucleotides in the treatment of central nervous system disorders, wherein the central nervous system disorders include, but are not limited to, Alzheimer's disease.

[0030] The use of the above-mentioned CpG oligodeoxynucleotides in the manufacture of drugs for preventing and treating secondary post-wound infections.

[0031] The use of the above-mentioned CpG oligodeoxynucleotide in the manufacture of drugs that promote wound healing.

[0032] Use of the above-mentioned CpG oligodeoxynucleotides in the manufacture of drugs for the treatment of allergic rhinitis, and / or atopic dermatitis, and / or asthma, and / or chronic obstructive pulmonary disease, and / or eosinophilic granulocyte-related disorders.

[0033] A drug manufactured using the above-mentioned CpG oligodeoxynucleotide, the dosage form of the drug is an injection, a tablet, a lyophilized preparation, an inhalant, a nasal drop, a nasal spray, an anal suppository, an eye drop, a topical ointment, a wash, or a gel.

[0034] A topical preparation of CpG oligodeoxynucleotide, comprising the CpG oligodeoxynucleotide described in any one of claims 1 to 5 and an additive, wherein the additive includes glyceryl behenate, liquid paraffin, petrolatum, medium-chain triglyceride, cocoyl caprylate, mono / distearate glyceryl, hypromellose, carbomer, xanthan gum, sodium carboxymethylcellulose, poloxamer 407, cetostearyl alcohol polyethylene glycol ether, poloxamer 188, oleoyl macrogol glyceride, polyethylene glycol-7 stearate, polyoxyethylene octanoate / caprate glyceryl, sodium dodecyl sulfate, propylene glycol mono fatty acid ester, polyglyceryl oleate, propylene glycol laurate, propylene glycol, isopropyl myristate, and polyglyceryl oleate.

[0035] A formulation comprising the above-mentioned CpG oligodeoxynucleotide adjuvant used in human or animal vaccines and other types of adjuvants, comprising CpG oligodeoxynucleotide itself or a combination thereof with other types of adjuvants, wherein the other types of adjuvants are preferably aluminum hydroxide and aluminum phosphate. [Effects of the Invention]

[0036] The present invention yields the following beneficial effects. This invention provides a CpG ODN sequence having a novel structure and sequence that differs from the complete palindrome design of conventional type A CpGs. By substituting the nucleotides at both ends of the ODN, these sequences reveal the relationship between drug potential and activity, and the associated rules. Compared to already published sequences, it has a unique primary and secondary structure, solving the problem of conventional type A CpGs being prone to association and having poor drug potential, and possessing higher immunoactivity and drug potential. In particular, the secondary structure formed by the AM1012-05 sequence is more stable at high temperatures compared to products of the same type, its aggregate is more controllable than other sequences of the same type, and it has better batch stability in the production process, resulting in better drug potential. Furthermore, AM1012-05 can effectively pass through a human respiratory epithelial mucosa (RPMI 2650 cell) penetration model and has shown excellent immune-activating activity in animals. It can effectively block the infection and transmission of respiratory viruses, and by activating and regulating the immune system, it has the potential to suppress tumor growth, treat allergic rhinitis, asthma, chronic obstructive pulmonary disease, eosinophilic granulocyte-associated disease, atopic dermatitis, and Alzheimer's disease, and prevent and treat secondary post-wound infections and promote wound healing. It also has the potential to be used as a standalone adjuvant or adjuvant component in vaccines. [Brief explanation of the drawing]

[0037] Below, in order to more clearly explain the technical proposal of this application, the drawings used in the embodiments are briefly introduced. Needless to say, those skilled in the art can obtain other drawings from these drawings without any creative work.

[0038] [Figure 1]This study evaluates the IFN-γ stimulating ability of different CpG sequences to NK-92 cells. Here, A, B, C, D, and E represent the IFN-γ stimulating ability of different CpG sequences to NK-92 cells measured using the same test system. [Figure 2] This is a comparison of the stimulating ability of different CpG sequences to spleen cells in SD rats. Here, A is a different CpG sequence that stimulates rat spleen cells to secrete IFN-γ, B is a different CpG sequence that stimulates rat spleen cells to secrete TNF-α, and C is a different CpG sequence that stimulates rat spleen cells to secrete IL-6. [Figure 3] This study evaluates the IFN-γ stimulating ability of AM1012-05 to NK-92 cells. [Figure 4] This study evaluates the stimulating ability of AM1012-05 to spleen cells of BALB / c mice. Here, A represents AM1012-05 stimulating BALB / c mouse spleen cells to secrete IL-6, B represents AM1012-05 stimulating BALB / c mouse spleen cells to secrete IFN-γ, C represents AM1012-05 stimulating BALB / c mouse spleen cells to secrete TNF-α, and D represents AM1012-05 stimulating BALB / c mouse spleen cells to secrete IFN-α. [Figure 5] This study evaluates the stimulating ability of AM1012-05 to spleen cells of C57BL6 mice. Here, A represents AM1012-05 stimulating C57BL6 mouse spleen cells to secrete IL-6, B represents AM1012-05 stimulating C57BL6 mouse spleen cells to secrete IFN-γ, C represents AM1012-05 stimulating C57BL6 mouse spleen cells to secrete TNF-α, and D represents AM1012-05 stimulating C57BL6 mouse spleen cells to secrete IFN-α. [Figure 6]This study evaluates the stimulating ability of AM1012-05 to Cotton rat spleen cells. Here, A indicates that AM1012-05 improves the mRNA level of IFN-α in Cotton rat spleen cells, B indicates that AM1012-05 improves the mRNA level of IFN-γ in Cotton rat spleen cells, C indicates that AM1012-05 improves the mRNA level of TNF-α in Cotton rat spleen cells, and D indicates that AM1012-05 improves the mRNA level of CXCL-10 in Cotton rat spleen cells. [Figure 7] This is an evaluation of the stimulating ability of AM1012-05 to rat spleen cells (mRNA levels). Here, A represents the increase in IFN-α mRNA levels in rat spleen cells by AM1012-05, and B represents the increase in CXCL-10 mRNA levels in rat spleen cells by AM1012-05. [Figure 8] This is an evaluation of the stimulating ability of AM1012-05 to rat spleen cells (at the protein level). Here, A is when AM1012-05 stimulates rat spleen cells to secrete IFN-γ, B is when AM1012-05 stimulates rat spleen cells to secrete IL-6, and C is when AM1012-05 stimulates rat spleen cells to secrete TNF-α. [Figure 9] This is an evaluation of the stimulating ability of AM1012-05 to Beagle PBMCs. Here, A indicates that AM1012-05 improves the mRNA level of IFN-α in Beagle PBMCs, B indicates that AM1012-05 improves the mRNA level of IFN-γ in Beagle PBMCs, C indicates that AM1012-05 improves the mRNA level of IFN-λ in Beagle PBMCs, D indicates that AM1012-05 improves the mRNA level of IL-6 in Beagle PBMCs, E indicates that AM1012-05 improves the mRNA level of CXCL-10 in Beagle PBMCs, and F indicates that AM1012-05 improves the mRNA level of TNF-α in Beagle PBMCs. [Figure 10]This is an evaluation of the stimulating ability of CpG ODN to human PBMCs. Here, A is when AM1012-05 stimulates human PBMCs to secrete IFN-α, B is when AM1012-05 stimulates human PBMCs to secrete IFN-γ, C is when AM1012-05 stimulates human PBMCs to secrete IFN-λ, D is when AM1012-05 stimulates human PBMCs to secrete TNF-α, E is when AM1012-05 stimulates human PBMCs to secrete IL-6, F is when AM1012-05 stimulates human PBMCs to secrete CXCL-10, and G is when AM1012-05, CpG 2216, and CpG D19 stimulate human PBMCs to secrete IFN-α. [Figure 11] This is the electrophoretic measurement result of the serum stability of CpG ODN. [Figure 12] This shows the distribution of the association states of different CpG ODN aggregates. The left figure shows the electrophoretic measurement (2 hours) of the association state distribution of type A CpG, and the right figure shows the electrophoretic measurement (24 hours) of the association state distribution of type A CpG. [Figure 13] This is an analysis of aggregates of AM1012-05 and CpG 2216 solutions in both a static and vortexed state. [Figure 14] This is a comparison of the secondary structures of different sequence samples at -20°C. Here, A is CpG 2216, B is E41, C is AM1012-03, and D is AM1012-05. [Figure 15] This is a comparison of the secondary structures of different sequence samples at 0°C. Here, A is CpG 2216, B is E41, C is AM1012-03, and D is AM1012-05. [Figure 16] This is a comparison of the secondary structures of different sequence samples at 4°C. Here, A is CpG 2216, B is E41, C is AM1012-03, and D is AM1012-05. [Figure 17]This is a comparison of the secondary structures of different sequence samples at 25°C. Here, A is CpG 2216, B is E41, C is AM1012-03, and D is AM1012-05. [Figure 18] This is a comparison of the secondary structures of different sequence samples at 37°C. Here, A is CpG 2216, B is E41, C is AM1012-03, and D is AM1012-05. [Figure 19] This is a comparison of the secondary structures of different sequence samples at 40°C. Here, A is CpG 2216, B is E41, C is AM1012-03, and D is AM1012-05. [Figure 20] This is a comparison of the secondary structures of different sequence samples at 50°C. Here, A is CpG 2216, B is E41, C is AM1012-03, and D is AM1012-05. [Figure 21] This is a comparison of the secondary structures of different sequence samples at 60°C. Here, A is CpG 2216, B is E41, C is AM1012-03, and D is AM1012-05. [Figure 22] This is a comparison of the secondary structures of different sequence samples at 70°C. Here, A is CpG 2216, B is E41, C is AM1012-03, and D is AM1012-05. [Figure 23] This is a comparison of the secondary structures of different sequence samples at 80°C. Here, A is CpG 2216, B is E41, C is AM1012-03, and D is AM1012-05. [Figure 24] This is a comparison of the secondary structures of different sequence samples at 90°C. Here, A is CpG 2216, B is E41, C is AM1012-03, and D is AM1012-05. [Figure 25] This study describes the effects of treatment with CpG ODN at different temperatures on IFN-γ secretion from rat spleen cells. [Figure 26] This is a particle size analysis diagram of PLGA nanoformulations of CpG ODN. [Figure 27]This is a particle size analysis diagram for the manufacturing of CpG ODN chitosan nanoformulation. [Figure 28] This is a particle size analysis diagram of the lipid nanoparticle formulation of CpG ODN. [Figure 29] This is a particle size analysis diagram of immunoadjuvants used in the production of PLGA nanoparticles with CpG ODN. [Figure 30] This is an evaluation diagram showing the effect of the formulation components on the IFN-γ stimulating ability of NK-92 cells in AM1012-05. [Figure 31] The effects of AM1012-05 on the relative expression levels of immune-related mRNAs in mouse lung and spleen tissues are shown below. Here, A indicates that AM1012-05 increases the mRNA level of IFN-α in mouse lung tissue, B indicates that AM1012-05 increases the mRNA level of IFN-γ in mouse lung tissue, C indicates that AM1012-05 increases the mRNA level of CXCL-10 in mouse lung tissue, D indicates that AM1012-05 increases the mRNA level of IFN-α in mouse spleen tissue, E indicates that AM1012-05 increases the mRNA level of IFN-γ in mouse spleen tissue, and F indicates that AM1012-05 increases the mRNA level of CXCL-10 in mouse spleen tissue. [Figure 32] This shows the change in the relative expression level of CXCL-10 mRNA in the lungs of mice after intranasal administration of AM1012-05. [Figure 33] This shows the secretion of cytokines from mouse lung tissue induced by AM1012-05 at different administration frequencies. Here, A represents the secretion of CXCL-10 from mouse lung tissue induced by AM1012-05 at different administration frequencies, and B represents the secretion of IFN-γ from mouse lung tissue induced by AM1012-05 at different administration frequencies. [Figure 34] AM1012-05 is an experiment in which human PBMCs are activated to kill N87 cells. [Figure 35] AM1012-05 is an experiment in which human PBMCs are activated to kill MDA-MB-231 cells. [Figure 36]This is an evaluation of AM1012-05's ability to alleviate allergic rhinitis in mice. Here, A indicates that AM1012-05 reduces the number of sneezes in mice with allergic rhinitis, B indicates that AM1012-05 reduces the number of nose scratches in mice with allergic rhinitis, C indicates that AM1012-05 reduces nasal discharge in mice with allergic rhinitis, D indicates that AM1012-05 reduces the number of eosinophilic granulocytes in the nasal mucosa of mice with allergic rhinitis, E indicates that AM1012-05 reduces the expression of IL-4 in the serum of mice with allergic rhinitis, and F indicates that AM1012-05 improves the expression of IFN-γ in the serum of mice with allergic rhinitis. [Figure 37] This shows the results of immunocytological analysis of nasal lavage fluid from mice treated with AM1012-05 for allergic rhinitis. [Figure 38] This study evaluates the in vitro activity of AM1012-05 in resisting respiratory syncytial virus infection. Here, A represents the prevention of respiratory syncytial virus proliferation in cells by the PBMC co-incubation supernatant of AM1012-05, and B represents the removal of respiratory syncytial virus from host cells by AM1012-05. [Figure 39] This is an evaluation of the resistance activity (IgE level) of AM1012-05 against atopic dermatitis. [Figure 40] This is an evaluation of the use of AM1012-05 as an adjuvant. Here, A is the result of IgG immunosuppression measurement 7 days after the first immunization, B is the result of IgG immunosuppression measurement 7 days after the second immunization, C is the result of IgG immunosuppression measurement 7 days after the third immunization, D is the result of IgG immunosuppression measurement 21 days after the third immunization, and E is the result of IgG immunosuppression measurement 35 days after the third immunization. [Figure 41]This is an evaluation of the use of AM1012-05 as an adjuvant. Here, A is the result of measuring the IgG1 immunosuppressant titer 7 days after the first immunization, B is the result of measuring the IgG1 immunosuppressant titer 7 days after the second immunization, C is the result of measuring the IgG1 immunosuppressant titer 7 days after the third immunization, D is the result of measuring the IgG1 immunosuppressant titer 21 days after the third immunization, and E is the result of measuring the IgG1 immunosuppressant titer 35 days after the third immunization. [Figure 42] This is an evaluation of the use of AM1012-05 as an adjuvant. Here, A is the result of IgG2a immunosuppression measurement 7 days after the first immunization, B is the result of IgG2a immunosuppression measurement 7 days after the second immunization, C is the result of IgG2a immunosuppression measurement 7 days after the third immunization, D is the result of IgG2a immunosuppression measurement 21 days after the third immunization, and E is the result of IgG2a immunosuppression measurement 35 days after the third immunization. [Modes for carrying out the invention]

[0039] The following describes specific embodiments of the present application in detail. Needless to say, the embodiments described are not all embodiments, but only partial embodiments of the present application. All other embodiments that a person skilled in the art can obtain based on the embodiments of the present application without any creative work are all within the scope of protection of the present application.

[0040] Example 1: Design and synthesis of CpG ODN molecules In this invention, multiple CpG ODN sequences were designed (see Table 1), chemically synthesized using a solid-phase synthesis method, and then the final product was obtained through processes such as ammonia hydrolysis, purification, desalting and concentration, dispensing, and freeze-drying. The specific operations were as follows. (1) Solid-phase synthesis: Solid-phase synthesis is performed under computer control. It consists of four repeated synthesis steps (detrityl group removal, coupling, sulfidation, capping) and one final step of removing the protecting group from the phosphate skeleton. (2) Ammonia decomposition: After synthesis, the oligonucleotide was cleaved from the carrier using aqueous ammonia or an organic amine, the oligonucleotide was dissolved in solution, the universal linker attached to the first base at the 3' end was cleaved, and the protecting group of the amino group on the base was removed. (3) Purification: The crude product obtained by ammonia hydrolysis was purified and separated by ion exchange chromatography, reverse-phase chromatography, or electrophoresis to obtain the purified product. (4) Desalting and concentration: The purified product was desalted and concentrated using an ultrafiltration apparatus. (5) Dispensing and freeze-drying: Freeze-drying yielded a loose substance that was white, or nearly white to pale yellow.

[0041] The types of nucleic acid modifications include, but are not limited to, thio modifications. [Table 1-1] [Table 1-2] [Table 1-3] (Here, * represents a thio modification.)

[0042] Example 2: Association status and activity analysis of different CpG ODNs 1. Analysis of meeting status of different CpG ODNs The association status of a portion of the freeze-dried powder of the CpG ODN sequence obtained in Example 1 was analyzed using high-performance liquid chromatography.

[0043] 2. Measurement of the effects of different CpG ODNs on IFN-α secretion using PBMC cells from healthy individuals. (1) A fixed amount of 100 μM CpG ODN solution was taken, and a stock solution of a fixed concentration was prepared in RPMI 1640 complete medium. Then, the CpG ODN sample solution was diluted fourfold using the complete medium to prepare samples of different concentrations. (2) An appropriate amount of cryopreserved PBMC cells from healthy individuals was obtained. (3) 2 × 10 5 Cells were seeded in a 96-well plate at a rate of one cell per well. Each well was then filled with a corresponding sample at a different concentration, and a PBS blank control well was also provided. (4) The cells were incubated at 37°C in a 5% CO2 incubator for 16 hours. (5) The mixture was centrifuged and the supernatant was collected. (6) IFN-α was measured according to the instructions for ELISA measurement of IFN-α.

[0044] By analyzing the association results (Tables 2-5), the following was found: (1) By changing the number of Gs in sequences such as AM1012-05, CpG 2216, and CpG D19, the optimal association state for the corresponding sequences was obtained when the number of Gs at the 5' end was 3-4, i.e., the content of the aggregate was the lowest. (2) Inserting 1-5 As between the core palindromic sequence and the 3' end poly-G of AM1012-05 further reduced the content of the aggregate. (3) Inserting 1-10 Ts or Cs between the core palindromic sequence and the 3' end G of the AM1012-05 sequence further reduced or increased the content of the aggregate to various degrees. (4) Inserting 1-10 As, Ts, or Cs between the core palindromic sequence and the 3' end G of sequences such as CpG 2216 and CpG D19 further reduced or increased the content of the aggregate to various degrees.

[0045] The target cells of type A CpG are pDCs, which account for 0.2-0.4% of PBMC cells. IFN-α production is mainly derived from pDCs. The results of the activity evaluation tests in this example are shown in Tables 2-5. From Table 2, the following was found: When "CGATCGATCG" is used as the core sequence of the palindromic structure of type A CpG, and a different number of Gs are added stepwise before the C at its 5' end, the stimulating activity against human PBMCs increases stepwise as the number of added Gs increases, with the activity being strongest when 3 or 4 Gs are added, and then weakening as the number of Gs increases further. When the palindromic sequence "TGCATCGATGCA" of CpG D19 is used as the core sequence, and a different number of Gs are added stepwise before the T at its 5' end, the stimulating activity against human PBMCs increases stepwise as the number of added Gs increases, with the activity being strongest when 2-4 Gs are added, and then weakening as the number of Gs increases further. Using the palindromic sequence "GACGATCGTC" of CpG 2216 as the core sequence, when a different number of Gs are added stepwise before the 5' terminal G, the stimulating activity against human PBMCs increases stepwise as the number of added Gs increases, with the activity being strongest when 3 Gs are added, and then decreasing as the number of Gs increases further.

[0046] Table 3 summarizes the association and activity data from studies in which A is inserted between the 3' end of the palindromic structure of type A CpG and the 5' end of poly-G. From this, the following was found: When "CGATCGATCG" is used as the core sequence of the palindromic structure of type A CpG, and A is added stepwise thereafter (before the 5' end of the poly-G sequence), the stimulating activity against human PBMCs increases stepwise as the number of inserted A's increases, reaching its strongest point when 2 to 4 A's are inserted, and then weakening as the number of A's increases further. When "TGCATCGATGCA" of CpG D19 is used as the core sequence of the palindromic structure of type A CpG, and A is added stepwise thereafter (before the 5' end of the poly-G sequence), the stimulating activity against human PBMCs increases stepwise as the number of inserted A's increases, reaching its strongest point when 2 A's are inserted, and then weakening as the number of A's increases further. When "GACGATCGTC" of CpG 2216 is used as the core sequence of the palindromic structure of type A CpG, and A is added stepwise thereafter (before the 5' end of the poly-G sequence), the stimulating activity against human PBMCs increases stepwise as the number of inserted A's increases, reaching its strongest point when the number of inserted A's is 2, and then weakening as the number of A's increases further.

[0047] Table 4 summarizes the association and activity data from studies in which T is inserted between the 3' end of the palindromic structure of type A CpG and the 5' end of poly-G. When the A added to the 3' end of the palindromic sequence "CGATCGATCG" was replaced with T, adding 2 or 10 T to the end of the palindromic structure always resulted in type A CpG maintaining good activity. When the A added to the 3' end of the palindromic sequence "TGCATCGATGCA" of CpG D19 was replaced with T, adding 2, 5, or 10 T to the end of the palindromic structure always resulted in type A CpG maintaining good activity. In particular, D19-11, which has two additional T added to D19, showed better activity than D19. When substituting the A added to the 3' end of the palindromic sequence "GACGATCGTC" of CpG 2216 with T, adding 2, 5, or 10 T atoms to the end of the palindromic structure always maintains good activity. In particular, CpG 2216-11, which has two additional T atoms added to CpG 2216, exhibits superior activity compared to CpG 2216.

[0048] Table 5 summarizes the association and activity data from studies in which carbon (C) is inserted between the 3' end of the palindromic structure of type A CpG and the 5' end of poly-G. When the A added to the 3' end of the palindromic sequence "CGATCGATCG" was replaced with C, the activity of the sequence gradually decreased as the number of C atoms at the end of the palindromic structure increased. When the A added to the 3' end of the CpG D19 palindromic sequence "TGCATCGATGCA" was replaced with C, the activity of the sequence gradually decreased as the number of C atoms at the end of the palindromic structure increased, but D19-14, which has two more C atoms added to D19, showed better activity than D19. When the A added to the 3' end of the CpG2216 palindromic sequence "GACGATCGTC" was replaced with C, the activity of the sequence gradually decreased as the number of C atoms at the end of the palindromic structure increased.

[0049] This invention breaks away from conventional A-type CpG design concepts. By creating a certain spatial barrier between the poly-G and the palindrome through innovative thinking, it increases the energy required to form G-tetrads, making it difficult to form polymers (aggregates) or loosening the formed aggregates. This successfully reduces the formation of A-type CpG aggregates in solvents and improves the activity of the newly designed A-type CpG. Furthermore, when we searched for poly-G at the 5' end of conventional A-type CpG, we found that when the number of Gs at the 5' end is 3 to 4, the formation of aggregates of the corresponding sequence is the lowest and the activity is the best. This rule has also been proven for multiple CpG palindrome sequences. Based on these rules we have discovered, among the sequences newly designed in this study, AM1012-05 is the best. It has 4 Gs at the 5' end and 2 As inserted at the 3' end of the palindrome sequence. It possesses the characteristics of low aggregation and high activity, and has very high drug discovery potential.

[0050] This study breaks away from conventional thinking by limiting the number of Gs (3-4) at the 5' end of the palindromic sequence of type A CpG and inserting an appropriate number of A and T (preferably 2) between the palindromic sequence and the 3' end poly-G, thereby mitigating the association of type A CpG and improving its activity. [Table 2] Here, the concentration is 0.2 μM in case a, and 0.8 μM in case b. [Table 3] Here, in the case of b, the concentration is 0.8 μM. [Table 4] Here, in the case of b, the concentration is 0.8 μM. [Table 5] Here, in the case of b, the concentration is 0.8 μM.

[0051] Example 3: Cytokine release after stimulation of NK-92 cells by different CpG ODNs. (1) NK-92 cells are a human NK cell line that expresses the TLR-9 receptor intracellularly and are commonly used to evaluate the function of TLR-9 agonists. In this experiment, NK-92 cells were cultured and amplified to a predetermined cell volume according to the cell description and then resuspended in a fixed amount of complete medium. (2) 20 μL of cell suspension was taken, mixed with trypan blue in a 1:1 ratio, and then counted using a cell counter to measure cell viability and cell density. (3) 2.5 × 10 5 Cells were seeded in a 96-well plate at a rate of one cell per well. Each well contained a corresponding CpG sample at a different concentration (sequence listed in Table 1), and three replica wells were created for each group. (4) The cells were incubated at 37°C in a 5% CO2 incubator for 72 hours. (5) After the cell culture was completed, the cells were centrifuged at 1200 rpm for 5 minutes, and 100 μL of the supernatant free of cells was collected. The IFN-γ secretion level of the supernatant was measured according to the instructions of the ELISA kit.

[0052] The experimental results are shown in Figure 1. All of the CpG sequences of the present invention designed in Table 1 can effectively stimulate NK cells to secrete IFN-γ, and among them, sequences AM1012-03, AM1012-04, AM1012-05, AM1012-17, and AM1012-35 showed particularly significant stimulating effects on NK cells. Furthermore, AM1012-05 showed better stimulating effects than E41-1 and E41-2, and significantly better than E41-3. In addition, the experimental results in Figure 2D and Figure 2E revealed that increasing or decreasing the number of Gs on both sides of the core sequence of the present invention can change the immunostimulatory activity of the sequence, and that when the number of Gs on both sides of the core sequence reaches a specific number, the sequence can exert the best immunoactivating effect.

[0053] Example 4: Measurement of the effect of CpG sequences on the secretion of IFN-γ, IL-6, and TNF-α using spleen cells from SD rats. (1) A certain amount of 100 μM AM1012-05 solution, E41-1 solution, E41-2 solution, and E41-3 solution were taken, and stock solutions of a certain concentration were prepared with RPMI 1640 complete medium. Next, they were diluted at a 4-fold ratio using the complete medium to prepare test samples of different concentrations. (2) An appropriate amount of fresh SD rat spleen cells was obtained. (3) 1×10 6 cells / well were seeded in a 96-well plate, and corresponding AM1012-05 samples of different concentrations were added to each well, with final concentrations of 3.2 μM, 0.8 μM, and 0.2 μM respectively. Three replicate wells were set up for each group, and a PBS blank control well was set up simultaneously. (4) They were placed in an incubator at 37 °C and 5% CO2 and cultured for 16 hours or 72 hours. (5) Centrifugation was performed, and the supernatant was collected. (6) IFN-γ, IL-6, and TNF-α were measured according to the instructions for ELISA measurement of different measurement indicators.

[0054] The experimental results are shown in Figure 2. All of the designed AM1012-05 and E41-1, E41-2, E41-3 could significantly stimulate the secretion of IFN-γ, TNF-α, and IL-6 by SD rat spleen cells, and there was a good dose-dependence. However, the immunostimulatory ability of AM1012-05 against SD rat spleen cells was significantly better than that of E41-1, E41-2, and E41-3. Referring to C in Figure 2, it was found that AM1012-05 had a better immunostimulatory ability.

[0055] Example 5: Effect of AM1012-05 sequence on cytokine release by NK-92 (1) As described in Example 3, a predetermined cell amount of NK-92 cells was prepared and resuspended in a certain amount of complete medium. (2) 20 μL of the cell suspension was taken, mixed with trypan blue at a 1:1 ratio, and then counted using a cell counter to measure the cell viability and cell density. (3) A 100 μM solution of the AM1012-05 sample was prepared using PBS. (4) A fixed amount of 100 μM AM1012-05 solution was taken, and a stock solution of a fixed concentration was prepared using complete culture medium. Then, the AM1012-05 solution was diluted to a 2:1 ratio using complete culture medium to prepare test samples of AM1012-05 at different concentrations. (5) 2.5 × 10 5 Cells were seeded in 96-well plates at a concentration of cells / well, and each well was filled with a corresponding AM1012-05 sample at different concentrations, resulting in final concentrations of 20 μM, 10 μM, 5 μM, 2.5 μM, 1.25 μM, 0.63 μM, 0.31 μM, 0.16 μM, and 0.08 μM, respectively. Three replica wells were prepared for each group, along with a PBS blank control well. (6) The cells were incubated at 37°C in a 5% CO2 incubator for 72 hours. (7) After the cell culture was completed, the cells were centrifuged at 1200 rpm for 5 minutes, and 100 μL of the supernatant free of cells was collected. The IFN-γ secretion status of the supernatant was measured according to the instructions of the ELISA kit.

[0056] The experimental results are shown in Figure 3. The AM1012-05 sequence effectively stimulated NK-92 cells to secrete IFN-γ, and showed good dose-dependent properties. Since NK-92 cells express TLR9, this experiment also demonstrated that the immune-activating activity of AM1012-05 occurs through mutual binding with TLR9.

[0057] Example 6: Measurement of the effects of AM1012-05 on the secretion of IL-6, IFN-γ, TNF-α, and other substances using spleen cells from BALB / c mice. (1) A fixed amount of 100 μM AM1012-05 solution was taken, and a stock solution of a fixed concentration was prepared in RPMI 1640 complete medium. Then, the AM1012-05 solution was diluted 2 times using complete medium to prepare test samples of AM1012-05 at different concentrations. (2) An appropriate amount of fresh BALB / c mouse spleen cells was obtained. (3) 1 × 10 6Cells were seeded in a 96-well plate at a concentration of cells / well. Each well was then filled with a corresponding sample of AM1012-05 at different concentrations, resulting in final concentrations of 8 μM, 4 μM, 2 μM, 1 μM, 0.5 μM, 0.25 μM, 0.13 μM, 0.06 μM, and 0.03 μM. Three replicate wells were prepared for each group, along with a PBS blank control well. (4) The cells were incubated in a 37°C, 5% CO2 incubator for 16 or 72 hours. (5) The mixture was centrifuged and the supernatant was collected. (6) IL-6, IFN-γ, TNF-α, and IFN-α were measured according to the instructions for ELISA measurement of different indicators.

[0058] The experimental results are shown in Figure 4. The designed AM1012-05 sequence effectively stimulated spleen cells in BALB / c mice to secrete cytokines, and showed a certain dose-dependent effect.

[0059] Example 7: Measurement of the effects of AM1012-05 on the secretion of IL-6, IFN-γ, TNF-α, and other substances using spleen cells from C57BL6 mice. (1) A fixed amount of 100 μM AM1012-05 solution was taken, and a stock solution of a fixed concentration was prepared in RPMI 1640 complete medium. Then, the AM1012-05 solution was diluted 2 times using complete medium to prepare test samples of AM1012-05 at different concentrations. (2) An appropriate amount of fresh C57BL6 mouse spleen cells was obtained. (3) 1 × 10 6 Cells were seeded in a 96-well plate at a concentration of cells / well. Each well was then filled with a corresponding sample of AM1012-05 at different concentrations, resulting in final concentrations of 8 μM, 4 μM, 2 μM, 1 μM, 0.5 μM, 0.25 μM, 0.13 μM, 0.06 μM, and 0.03 μM. Three replicate wells were prepared for each group, along with a PBS blank control well. (4) The cells were incubated in a 37°C, 5% CO2 incubator for 16 or 72 hours. (5) The mixture was centrifuged and the supernatant was collected. (6) IL-6, INF-γ, TNF-α, and IFN-α were measured according to the instructions for ELISA measurement of different indicators.

[0060] The experimental results are shown in Figure 5. The designed AM1012-05 sequence effectively stimulated spleen cells in C57BL6 mice to secrete cytokines, and showed a certain dose-dependent effect.

[0061] Example 8: Measurement of the effect of AM1012-05 on the transcription of IFN-α, IFN-γ, TNF-α, and CXCL-10 mRNA using cotton rat spleen cells. (1) A fixed amount of 100 μM AM1012-05 solution was taken, and a stock solution of a fixed concentration was prepared in RPMI 1640 complete medium. Then, the AM1012-05 solution was diluted 2 times using complete medium to prepare test samples of AM1012-05 at different concentrations. (2) An appropriate amount of frozen and preserved Cotton rat spleen cells was obtained. (3) 5 × 10 6 Cells were seeded in a 6-well plate at a concentration of cells / well. Each well was then treated with a corresponding sample of AM1012-05 at different concentrations, resulting in final concentrations of 0.2 μM, 0.8 μM, and 3.2 μM. Three replicate wells were provided for each group, along with a PBS blank control well. (4) The cells were incubated in a 37°C, 5% CO2 incubator for 3 hours. (5) Cells were collected, and total RNA was extracted from the samples according to the instructions of the total RNA extraction kit. The concentration of total RNA was measured using Nanodrop. (6) RNA was reverse transcribed into cDNA according to the instructions of the reverse transcription kit. (7) The expression levels of the relevant cytokines in each sample were analyzed according to the instructions for the RT-qPCR kit.

[0062] The experimental results are shown in Figure 6. The designed AM1012-05 sequence effectively stimulated spleen cells in Cotton rats to secrete immune-related cytokines, and showed good dose-dependent response. Since Cotton rats are a commonly used animal model for respiratory syncytial virus infection, this provides a basis for evaluating the resistance of AM1012-05 to respiratory syncytial virus using Cotton rats.

[0063] Example 9: Measurement of the effect of AM1012-05 on the transcription of IFN-α and CXCL-10 mRNA using SD rat spleen cells. (1) A fixed amount of 100 μM AM1012-05 solution was taken, and a stock solution of a fixed concentration was prepared in RPMI 1640 complete medium. Then, the AM1012-05 solution was diluted 2 times using complete medium to prepare test samples of AM1012-05 at different concentrations. (2) An appropriate amount of fresh rat spleen cells was obtained. (3) 5 × 10 6 Cells were seeded in a 6-well plate at a rate of one cell per well. Each well was then filled with a corresponding sample of AM1012-05 at different concentrations. Three replica wells were provided for each group, along with a PBS blank control well. (4) The cells were incubated in a 37°C, 5% CO2 incubator for 3 hours. (5) Cells were collected, and total RNA was extracted from the samples according to the instructions of the total RNA extraction kit. The concentration of total RNA was measured using Nanodrop. (6) RNA was reverse transcribed into cDNA according to the instructions of the reverse transcription kit. (7) The expression levels of the relevant cytokines in each sample were analyzed according to the instructions for the RT-qPCR kit.

[0064] The experimental results are shown in Figure 7. The designed AM1012-05 sequence effectively stimulated SD rat spleen cells to transcribe IFN-α and CXCL-10 mRNA, and showed good dose-dependent properties.

[0065] Example 10: Measurement of the effects of AM1012-05 on the secretion of IFN-γ, IL-6, and TNF-α using spleen cells of SD rats. (1) A fixed amount of 100 μM AM1012-05 solution was taken, and a stock solution of a fixed concentration was prepared in RPMI 1640 complete medium. Then, the AM1012-05 solution was diluted 2 times using complete medium to prepare test samples of AM1012-05 at different concentrations. (2) An appropriate amount of fresh SD rat spleen cells was obtained. (3) 1 × 10 6 Cells were seeded in 96-well plates at a rate of one cell / well. Each well was then treated with a corresponding sample of AM1012-05 at different concentrations, resulting in final concentrations of 20 μM, 10 μM, 5 μM, 2.5 μM, 1.25 μM, 0.625 μM, 0.3125 μM, 0.1562 μM, and 0.087 μM. Three replica wells were provided for each group, along with a PBS blank control well. (4) The cells were incubated in a 37°C, 5% CO2 incubator for 16 or 72 hours. (5) The mixture was centrifuged and the supernatant was collected. (6) IFN-γ, IL-6, and TNF-α were measured according to the instructions for ELISA measurement of different indicators.

[0066] The experimental results are shown in Figure 8. The designed AM1012-05 sequence effectively stimulated spleen cells in SD rats to secrete IFN-γ, IL-6, and TNF-α, and showed good dose-dependent response. Since SD rats are a commonly used animal model for preclinical toxicity evaluation of drugs, the results of this experiment, when considered in conjunction with the data from Example 9, can be said to lay the foundation for conducting safety evaluation experiments of AM1012-05 using SD rats.

[0067] Example 11: Measurement of the effect of AM1012-05 on the secretion of IFN-α, IFN-γ, IFN-λ, IL-6, TNF-α, and CXCL-10 using Beagle PBMCs. (1) A fixed amount of 100 μM AM1012-05 solution was taken, and a stock solution of a fixed concentration was prepared in RPMI 1640 complete medium. Then, the AM1012-05 solution was diluted fourfold using the complete medium to prepare test samples of AM1012-05 at different concentrations. (2) An appropriate amount of fresh Beagle PBMC was obtained. (3) 5 × 10 6 Cells were seeded in a 6-well plate at a concentration of cells / well. Each well was then treated with a corresponding sample of AM1012-05 at different concentrations, resulting in final concentrations of 0.2 μM, 0.8 μM, and 3.2 μM. Three replicate wells were provided for each group, along with a PBS blank control well. (4) The cells were incubated in a 37°C, 5% CO2 incubator for 6 hours. (5) Cells were collected, and total RNA was extracted from the samples according to the instructions of the total RNA extraction kit. The concentration of total RNA was measured using Nanodrop. (6) RNA was reverse transcribed into cDNA according to the instructions of the reverse transcription kit. (7) The expression levels of the relevant cytokines in each sample were analyzed according to the instructions for the RT-qPCR kit.

[0068] The experimental results are shown in Figure 9. The designed AM1012-05 sequence effectively stimulated beagle PBMCs to secrete immune-related cytokines, and showed good dose-dependent response. Since beagles are a commonly used animal model for preclinical toxicity assessment of drugs, this lays the foundation for conducting safety evaluation experiments of AM1012-05 using beagles. Furthermore, the efficacy of AM1012-05 in beagle PBMCs suggests that it has great potential for use as an adjuvant in pet vaccines or for anti-infective purposes.

[0069] Example 12: Measurement of the effects of AM1012-05 and related sequences on the secretion of IFN-α, IFN-γ, IFN-λ, TNF-α, IL-6, and CXCL-10 using PBMC cells from healthy individuals. (1) A fixed amount of 100 μM solution of AM1012-05 and related sequences was taken, and a stock solution of a fixed concentration was prepared in RPMI 1640 complete medium. Then, the solution of AM1012-05 and related sequences was diluted fourfold using complete medium to prepare samples of different concentrations. (2) An appropriate amount of cryopreserved PBMC cells from healthy individuals were obtained. (3) 2 × 10 5 Cells were seeded in a 96-well plate at a rate of one cell per well. Each well was then filled with a corresponding sample at a different concentration. Three replica wells were provided for each group, along with a PBS blank control well. (4) The cells were incubated at 37°C in a 5% CO2 incubator for 16 hours. (5) The mixture was centrifuged and the supernatant was collected. (6) Cytokines were measured according to the ELISA measurement instructions.

[0070] The experimental results are shown in Figure 10. AM1012-05 can dose-dependently stimulate the secretion of IFN-α (Figure 10A), IFN-γ (Figure 10B), IFN-λ (Figure 10C), TNF-α (Figure 10D), IL-6 (Figure 10E), and CXCL-10 (Figure 10F) by PBMCs. Furthermore, the activating effect of AM1012-05 and related sequences on IFN-α is significantly superior to that of CpG 2216 and D19 (Figure 10G). Among the samples, AM1012-05 showed a remarkable effect in promoting IFN-α secretion, with an EC50 of 0.19 μM and an optimal stimulating concentration of 0.5 μM. The effect of 1 μM AM1012-05 in promoting IFN-γ secretion and the effect of 0.25 μM AM1012-05 in promoting IFN-λ secretion were also very significant, indicating that AM1012-05 has very high antiviral potential. AM1012-05 was able to stimulate healthy human PBMCs to secrete TNF-α and showed good dose-dependent effects, but its ability to stimulate TNF-α was significantly lower than its ability to stimulate IFN-α (EC50 was 0.19 μM and 2.46 μM, respectively). This revealed that when AM1012-05 can exert efficient antiviral activity (IFN-α, IFN-γ, IFN-λ) at low doses, it does not cause inflammatory responses represented by TNF-α and IL-6.

[0071] Example 13: Measurement of serum stability of CpG ODN In this experiment, the degree of sequence disruption of fetal bovine serum (FBS) was investigated using polyacrylamide gel (PAGE) electrophoresis. The specific procedure was as follows: AM1012-05, AM1012-03, E41-2, and E41-3 were each prepared as 25 OD / mL aqueous solutions, 10 μL of 10% FBS was added, and the solutions were incubated at a constant temperature of 37°C for a set period of time. After incubation, the degree of sequence disruption was measured using polyacrylamide gel (PAGE) electrophoresis. The results are shown in Figure 11. AM1012-05 (1205) was stable in 25% fetal bovine serum solution, E41-3 showed the most degradation (4103), AM1012-03 (1203) and E41-2 (4102) showed moderate degradation, and AM1012-05 (1205) showed the least degradation. Therefore, the AM1012-05 sample exhibits better stability compared to E41-2 and E41-3.

[0072] Example 14: Distribution analysis of the association state of different CpG ODN aggregates 3 mg each of AM1012-03, E41-2, E41-3, and AM1012-05 were added to 1 mL of deionized water to obtain a clear solution.

[0073] The four solutions described above were divided equally into two groups. One group was left to stand at room temperature, while the other group was heated in a 95°C water bath for 30 minutes, and then rapidly cooled to below 4°C in ice water. An equal volume of 800 mM sodium chloride solution was added to each of the eight solutions to create a 400 mM sodium ion environment for the samples. After incubation for 24 hours, the samples were centrifuged to separate the precipitate. The precipitate was redissolved in water to prepare a clear solution. The distribution of the association state of each sample was examined using polyacrylamide gel (PAGE) electrophoresis.

[0074] The electrophoresis results are shown in Figure 12. In the figure, the letter M marks 20 bases, 40 bases, and 80 bases. Lanes 1, 2, 3, and 4 correspond to AM1012-03, E41-2, E41-3, and AM1012-05, respectively, which were left standing at room temperature. Lanes 5, 6, 7, and 8 correspond to AM1012-03, E41-2, E41-3, and AM1012-05, respectively, which were heated in a 95°C water bath for 30 minutes and then rapidly cooled to below 4°C in ice water. The left figure shows the electrophoresis results after dissolving the precipitate in water for 2 hours, and the right figure shows the electrophoresis results after leaving the same sample solution standing for 24 hours.

[0075] Some literature reports that the 5' or 3' polyG sequence at the end of type A CpG induces the formation of an associated state in the presence of metal ions, and that precipitation occurs when this association accumulates. The degree to which the associated state can be controlled is an important indicator in examining the drug potential of type A CpG (Kerkmann et al., J Biol Chem. 2005 Mar 4;280(9):8086-93).

[0076] In this experiment, all samples incubated in the presence of 400 mM sodium ions formed precipitates. When the precipitates were placed back into water, the highly associated type A CpGs gradually reassociated and dissociated in the absence of metal ions, allowing for redissolution. Since low-association states such as dimers and tetramers also dissolve to some extent in water, observing the redissolution of samples at different time points allows for observation of the distribution of the (low) association states of each type A CpG and the trends of their changes over time as much as possible.

[0077] For samples left standing at room temperature, the redissolved solution of AM1012-03 was predominantly tetramers and monomers after 2 hours, and a diffused band was observed after 24 hours. The redissolved solution of E-41-2 was predominantly dimers after 2 hours, and while still predominantly dimers after 24 hours, tetramers gradually appeared. The redissolved solution of E-41-3 became dimers and polymers (tetramers or more) after 2 hours, and a diffused band predominantly composed of dimers appeared after 24 hours. In the redissolved solution of AM1012-05, after standing for 2 hours and 24 hours, only a small portion was aggregate, and it was mostly in a monomeric state.

[0078] For samples heated in a 95°C water bath for 30 minutes and then rapidly cooled to below 4°C in ice water, the redissolved solution of AM1012-03 was predominantly composed of tetramers and monomers after 2 hours of standing, with a diffused band appearing after 24 hours. The redissolved solution of E-41-2 was predominantly composed of dimers after 2 hours of standing, still predominantly composed of dimers after 24 hours, but with the gradual appearance of tetramers and monomers. The redissolved solution of E-41-3 was composed of multiple aggregate states after 2 hours of standing, with a diffused band mainly consisting of monomers and dimers after 24 hours. In the redissolved solution of AM1012-05, after standing for 2 and 24 hours, only a small portion was aggregate, with the majority being in a monomeric state.

[0079] The results above show that AM1012-05, compared to several other type A CpG monomers, was more readily able to reintegrate and dissociate back into a monomeric state under the same conditions. Several other type A CpGs convert between monomeric, dimeric, tetrameric, or other polymeric forms. Therefore, if the concentration of the metal salt can be well controlled when actually used as a formulation, AM1012-05 can stably maintain a monomeric state. When the formulation enters the human body, at physiologically appropriate salt concentrations, AM1012-05 can form the characteristic association state of type A CpGs and produce its therapeutic effect.

[0080] Furthermore, using 10 mM phosphate buffer, CpG 2216 and AM1012-05 were prepared as 0.5 mg / mL solutions, and after standing overnight, the particle size of CpG was measured using a Nicomp Z3000 laser particle size analyzer. After the measurement was completed, the solution was vortexed for 3 minutes, and the particle size was measured again. The results are shown in Figure 13. In the case of CpG 2216, before vortexing, the particle size was distributed around 2 nm, 75 nm, and 250 nm, and after vortexing, the particle size of CpG 2216 was concentrated around 280 nm. AM1012-05 had a particle size of around 30 nm when standing, and after vortexing, the particle size of AM1012-05 was concentrated around 2 nm.

[0081] The results of the laser particle size analyzer show the natural association state of CpG. In 10 mM phosphate buffer, AM1012-05 is in a loose association state when left at rest, but when subjected to mechanical force, the association is broken and it becomes a monomer. However, CpG 2216 is a dispersed, complex polymer that does not break down (dissociate) even when mechanical force is applied in 10 mM phosphate buffer.

[0082] The results in Figure 13 revealed the following: CpG 2216 is a conventional A-type CpG, and no deoxyribonucleotide is added between the 3' end of the palindromic structure and the 5' end of poly-G. There is no specific spatial barrier, the energy required to form the G-tetrad is reduced, and the resulting aggregate is tightly bound and cannot be easily dispersed by mechanical force. In contrast, AM1012-05, in which two A atoms are inserted between the 3' end of the palindromic structure and the 5' end of poly-G, has a specific spatial barrier. This increases the energy required to form the G-tetrad, making it difficult for a polymer to form, or allowing the formed polymer to be loose. Therefore, under natural static conditions, AM1012-03 is in an aggregate equilibrium state with a uniform distribution of around 30 nm, and its drug potential is significantly improved compared to conventional A-type CpG.

[0083] Example 15: Simulation analysis and comparison of secondary structures of different sequences For the sequences CpG 2216, E41-1, AM1012-3, and AM1012-5, the secondary structure was predicted using an online modeling tool (https: / / rna.urmc.rochester.edu / ), and the temperature parameters were adjusted to obtain the secondary structures of each sequence at different temperatures.

[0084] Schematic diagrams of the specific secondary structures are shown in Figures 14 to 24. When the secondary structures of these four compounds were compared at different temperatures, the following was found: CpG 2216 is stable between 4 and 37°C, but above 40°C, its secondary structure folds incorrectly, affecting its physiological activity. E41-1 is stable between 0 and 37°C, but when the temperature reaches -20°C, some of its internal hydrogen bonds begin to break, and above 40°C, its secondary structure folds incorrectly, affecting its physiological activity at both excessively high and low temperatures. AM1012-03 is also stable between 0 and 37°C, but when the temperature reaches 0°C, some of its internal hydrogen bonds begin to break, and above 40°C, its secondary structure similarly folds incorrectly, affecting its physiological activity. The AM1012-05 sequence maintains its stable secondary structure from 0 to 50°C, and only when the temperature exceeds 60°C does some of its internal hydrogen bonds begin to break. This means that the secondary structure of the AM1012-05 sequence is more stable than that of CpG 2216, E41-1, and AM1012-03, and structurally explains why AM1012-05 is superior to CpG 2216, E41-1, and AM1012-03 in both stability and activity.

[0085] Example 16: Evaluation of the stimulating ability of CpG ODNs with different levels of association to rat spleen cells. (1) A fixed amount of 100 μM CpG ODN solution was taken from a different temperature, and a stock solution of a certain concentration was prepared in RPMI 1640 complete medium. Then, the CpG ODN solution was diluted fourfold using the complete medium to prepare samples of different concentrations. (2) An appropriate amount of fresh SD rat spleen cells was obtained. (3) 1 × 10 6 Cells were seeded in a 96-well plate at a rate of one cell per well. Each well was then treated with a corresponding CpG ODN sample at different concentrations, resulting in final concentrations of 3.2 μM and 0.8 μM, respectively. Three replicate wells were provided for each group, along with a PBS blank control well. (4) The cells were incubated at 37°C in a 5% CO2 incubator for 72 hours. (5) The mixture was centrifuged and the supernatant was collected. (6) IFN-γ was measured according to the instructions for ELISA measurement of rat IFN-γ.

[0086] The experimental results are shown in Figure 25. The designed AM1012-05 sequence, after treatment at high temperature, still effectively stimulates SD rat spleen cells to secrete IFN-γ, while the AM1012-03 sequence, after treatment at high temperature, does not effectively stimulate SD rat spleen cells to secrete IFN-γ. Referring to the predicted secondary structure of the sequence in Example 15, AM1012-05 exhibits far superior high-temperature stability compared to CpG 2216, E41, and AM1012-03. Therefore, this experiment proves that a stable secondary structure is a prerequisite for the exertion of activity by CpG.

[0087] Example 17: Changes in the stability of CpG ODN raw materials under different manufacturing processes The raw materials E41-2, E41-3, and AM1012-05 were prepared as 2 mg / mL stock solutions and processed using different processes: direct freeze-drying and freeze-drying after heating at 95°C. After dissolving the dried powders produced by the different freeze-drying processes, the degree of association and related substances were measured using reverse-phase HPLC. The proportion of the association states of E41-2, E41-3, and AM1012-05 produced by the different processes varied slightly depending on the process. However, the impurities measured for the dried powders of E41-2 and E41-3 produced by the different processes differed significantly. The impurity levels measured for AM1012-05 produced by the different processes were almost the same. The results revealed that AM1012-05 has superior stability compared to E41-2 and E41-3, and possesses better drug discovery potential. [Table 6]

[0088] Example 18: pH screening of AM1012-05 formulation (1) Sodium phosphate buffer solutions with different pH values ​​were prepared at pH 6.6, 7.0, 7.2, 7.4, 8.0, and 9.0. (2) AM1012-05 was dissolved in the buffer solution to prepare a 0.35 mg / mL formulation solution. (3) Each of the above formulation solutions was dispensed into 10 mL vials at a rate of 1.5 mL per vial, the vials were capped, and the containers were left standing in a dark place at 50°C. (4) Samples were taken on day 0, day 5, and day 10, and the content was measured.

[0089] The experimental results are shown in Table 7. The results revealed that AM1012-05 is easily decomposed in acidic environments. In the case of the formulation, it is stable in the pH range of 7.0 to 9.0. [Table 7]

[0090] Example 19: Selection of buffer for AM1012-05 solution formulation Based on the pH range of commercially available low-molecular-weight nucleic acid drugs and commonly used buffer systems, phosphate and citrate buffer systems were screened. The AM1012-05 experimental sample was concentrated at 2.5 mg / mL. An aqueous solution of AM1012-05 without buffer was used as a control. The pH of the sample was controlled to 7.2, and the samples were allowed to stand at 50°C for 10 days. Samples were taken on days 0, 5, and 10, and the stability of the samples was compared to the control group using the two buffer systems. The results showed that the purity change was significantly reduced in the samples with buffer compared to the control group, and the trends in changes for each parameter of the two buffer system samples were consistent as the standing time increased. The experimental results are shown in Table 8. Both buffer systems can meet the pH buffer requirements of the drug formulation. [Table 8]

[0091] Example 20: Screening of metal ion concentration range in CpG ODN 5 mg each of sodium acetate, sodium dihydrogen phosphate / disodium hydrogen phosphate, magnesium chloride, calcium chloride, potassium chloride, and sodium chloride were taken, and 200 μL solution samples were prepared by mixing them with 0.25 mg of AM1012-05. The test samples were placed in sealed, clean containers and allowed to stand at 40°C for 30 days, with samples taken on days 5, 15, and 30. Measurement indicators included properties (appearance), solubility of the target substance, impurities (related substances), and content.

[0092] Measurement results revealed that precipitation occurs when sodium acetate, sodium dihydrogen phosphate / disodium hydrogen phosphate, magnesium chloride, calcium chloride, potassium chloride, and sodium chloride are mixed with AM1012-05 at their respective concentrations. Therefore, in order to ensure the clarity of the formulation solution, it is necessary to obtain appropriate salt concentrations for the CpG ODN formulation through screening.

[0093] AM1012-05 was selected, and potassium chloride solution and sodium chloride solution were added to it. The final concentration of AM1012-05 in the resulting solution was 2 mg / mL, and the final concentration of the salt solution was 90 mM. After standing for 24 hours, AM1012-05 remained clear in both salt solutions.

[0094] AM1012-05 was selected, and magnesium chloride solution and calcium chloride solution were added, respectively. The final concentration of AM1012-05 in the resulting solution was 2 mg / mL, and the final concentration of the salt solution was 1.8 mM. After standing for 24 hours, AM1012-05 remained clear in both salt solutions.

[0095] Four concentrations of phosphate buffer (consisting of sodium dihydrogen phosphate and disodium hydrogen phosphate) were selected as experimental concentrations: 5 mM, 10 mM, 20 mM, and 50 mM (sodium ion concentrations of approximately 9 mM, 18 mM, 36 mM, and 90 mM, respectively). Isotonic (150 mM) PBS (sodium ion concentration of approximately 179 mM for each) solutions were used as controls. A concentration of 2.5 mg / mL was used for the experimental AM1012-05, the sample pH was controlled to 7.2, and the samples were allowed to stand at 50°C for 10 days. Samples were taken on days 0, 5, and 10, and the pH buffer concentration was determined by measuring the stability of the samples. The results are shown in Table 9. Compared to day 0, there was no significant change in the clarity or properties of the samples after standing at 50°C for 5 days, and there was no significant difference in purity as the standing time increased. AM1012-05 in isotonic PBS showed significant turbidity. Therefore, the salt concentration of isotonic (150 mM) PBS is not suitable for preparing the AM1012-05 formulation. Considering both the buffering capacity of the pH and the avoidance of precipitation of AM1012-05, the sodium ion concentration in the solution formulation was selected to be between 0.1 and 90 mM. The total concentration range of metal ions such as sodium, potassium, magnesium, calcium, zinc, and iron in the CpG ODN solution formulation is between 0.1 and 90 mM. [Table 9]

[0096] Example 21: Selection of isotonic modifier for AM1012-05 solution In the case of solution formulations, isotonicity is generally adjusted with sodium chloride. However, the formation of the association state of type A CpG is related to the concentration of metal ions in the solution, and an isotonic sodium chloride solution precipitates type A CpG from the solution, so it is necessary to select other additives as isotonicity adjusters.

[0097] The following can be used as isotonic adjusting agents: glycine, trehalose, mannitol, sucrose, arginine, lysine, histidine, sorbitol, glucose, glycerin, and propylene glycol.

[0098] Using the above-mentioned additives, 200 μL solution samples were prepared in each tube containing 0.25 mg of AM1012-05 and 5 mg of the additive. The test samples were placed in sealed, clean containers and left to stand at 40°C for 30 days, with samples taken on days 5, 15, and 30. Measurement indicators included properties (appearance), solubility of the target substance, impurities (related substances), and content. The measurement results showed that solutions mixed with AM1012-05, respectively, exhibited abnormal main peak shapes in HPLC chromatograms, indicating that sorbitol and glucose did not mix well with AM1012-05 and were unsuitable for use as isotonic modifiers in AM1012-05 formulations. Glycine, trehalose, mannitol, sucrose, arginine, lysine, histidine, glycerin, and propylene glycol are compatible with AM1012-05 and can be used as isotonic modifiers for AM1012-05.

[0099] A 0.35 mg / mL formulation solution was prepared using AM1012-05, three isotonic regulators, and a phosphate buffer. The properties (appearance), clarity, pH, osmotic pressure, and purity were measured, and the details of the formulation are shown in Table 10. [Table 10]

[0100] The experimental results are shown in Table 11. The results revealed that all three formulations of AM1012-05 yielded clear solutions, and that the pH, osmotic pressure, and purity all met the requirements for the formulation. [Table 11]

[0101] Example 22: Exploration of formulations for lyophilization of AM1012-05 formulations. AM1012-05 was dissolved in deionized water, and mannitol (freeze-dried protective agent) and pH adjusters (disodium hydrogen phosphate + citric acid) were added to prepare a solution containing 1 mg / mL AM1012-05, 5% mannitol, and 50 mM disodium hydrogen phosphate + citric acid.

[0102] The above solution was filled into 0.2 mL to 2 mL vials, partially capped, and then transferred to a freeze-drying apparatus where freeze-drying was performed at -30°C. After freeze-drying was completed, the sample was capped to obtain a white, cake-like mass of freeze-dried powder. The freeze-dried formulation can be immediately redissolved by adding 1 mL of sterile water for injection. The freeze-dried formulation of AM1012-05 allows for the storage of AM1012-05 in solid form, which is advantageous for the stability of the main ingredient and makes it a candidate formulation for a liquid formulation of AM1012-05.

[0103] Example 23: Selection of other additives for the AM1012-05 solution formulation In solution formulations of CpG ODN, in addition to isotonic modifiers and pH buffer systems, other additives can be added to provide corresponding functions.

[0104] Other optional additives include surfactants (e.g., Pluronic F127, Pluronic F68, Tween 20, Tween 80), preservatives (e.g., benzalkonium chloride), metal chelating agents (disodium edetate), viscosity modifiers (hypromellose, sodium carboxymethylcellulose, sodium citrate), methyl-β-cyclodextrin, hydroxypropyl-β-cyclodextrin, and antioxidants (ascorbic acid).

[0105] In this test, 5 mg each of Pluronic F127, Pluronic F68, Tween 20, Tween 80, and ascorbic acid were taken, and 200 μL solution samples were prepared by mixing them with 0.25 mg of AM1012-05. The test samples were placed in sealed, clean containers and left to stand at 40°C for 30 days, with samples taken on days 5, 15, and 30. The measured indicators included properties (appearance), solubility of the target substance, impurities (related substances), and content.

[0106] Furthermore, benzalkonium chloride, disodium edetate, sodium citrate, hypromellose, sodium carboxymethylcellulose, methyl-β-cyclodextrin, and hydroxypropyl-β-cyclodextrin were prepared at the concentrations listed in Table 12, and AM1012-05 was added to prepare a 0.4 mg / mL formulation solution, with the pH adjusted to 7.0-7.5. The test samples were placed in sealed, clean containers and left to stand at 40°C for 30 days, with samples taken on the 5th, 15th, and 30th days. The measured indicators were properties (appearance), solubility of the target substance, impurities (related substances), and content. [Table 12]

[0107] The measurement results revealed that ascorbic acid decomposes AM1012-05 when mixed with it, making it unsuitable for use as a formulation of AM1012-05. When a mixture of hydroxypropyl-β-cyclodextrin and AM1012-05 was measured by HPLC, the shape of the corresponding main peak for AM1012-05 was abnormal, indicating that it is unsuitable for use as a formulation of AM1012-05.

[0108] Pluronic F127, Pluronic F68, Tween 20, Tween 80, benzalkonium chloride, disodium edetate, sodium citrate, hypromellose, sodium carboxymethylcellulose, and methyl-β-cyclodextrin are compatible with AM1012-05 and can be used as additives in AM1012-05 related formulations.

[0109] Example 24: Production of PLGA nanoformulation of CpG ODN (1) 100 mg of PLGA was dissolved in 1.25 mL of dichloromethane (oil phase).

[0110] (2) A 12 mg / mL AM1012-05 solution and a 1% PVA solution were prepared (aqueous phase 1).

[0111] (3) A 4 mL 2.5% PVA solution was prepared (aqueous phase 2).

[0112] (4) The oil phase and water phase 1 were homogenized and uniformly mixed to form a primary emulsion. Then, the primary emulsion was added to water phase 2 and homogenized using a homogenizer to form a secondary emulsion.

[0113] (5) The formed secondary emulsion was slowly added to a 1% PVA aqueous solution (external aqueous phase), and stirred for 4 hours to volatilize the dichloromethane.

[0114] (6) The solution from step 5 was centrifuged with 500g for 5 minutes to remove large microspheres, and the supernatant was collected.

[0115] (7) The supernatant from step 6 was centrifuged at 10,000 g for 10 minutes to collect the solid, which was then washed three times with deionized water or ultrapure water to obtain the PLGA microsphere product.

[0116] When the obtained PLGA microsphere product was measured using a Nicomp Z3000 laser particle size analyzer, the particle size could be controlled to within 500 nm (see Figure 26).

[0117] Example 25: Production of a chitosan nanoformulation of CpG ODN (1) A 0.5 mg / mL chitosan (molecular weight 200,000) solution was prepared and the pH was adjusted to 5.6.

[0118] (2) A 50 μg / mL AM1012-05 solution was prepared.

[0119] (3) 10 mL of AM1012-05 solution was added dropwise to 10 mL of chitosan solution and stirred for 1 minute.

[0120] When the chitosan nanoparticles of AM1012-05 produced in this test were measured using a laser particle size analyzer and a dynamic light scattering particle size analyzer, the particle size could be controlled to approximately 150 nm (see Figure 27).

[0121] Example 26: Production of CpG ODN lipid nanoparticle formulations CpG ODN lipid nanoparticles (LNPs) are synthesized by selecting different types of lipids, mixing them in specific ratios, self-assembling them, and encapsulating CpG. The lipids include ionizable cationic liposomes, neutral phospholipids, sterol lipids, and PEGylated phospholipids. The specific manufacturing process was as follows: (1) SM-102, DSPC, cholesterol, and DMG-PEG(2000) were dissolved in ethanol in a ratio of 50:10:38.5:1.5.

[0122] (2) 0.25 mL of the lipid solution was mixed with 0.75 mL of 80 μg / mL AM1012-05 solution and 50 mM citrate buffer, then vortexed and mixed, and then transferred to a hand-operated liposome extruder.

[0123] (3) The mixture was quickly pipetted using an extruder and pressed to mix. The mixture was allowed to stand for 10 minutes.

[0124] (4) Using a 200 kD dialysis bag, the mixture was added to an equal volume of 50 mM citrate buffer, sealed, and then dialyzed in 50 mL of PBS solution.

[0125] When the lipid nanoparticles of AM1012-05 produced in this test were measured using a Nicomp Z3000 laser particle size analyzer, the particle size could be controlled to approximately 100 nm (see Figure 28).

[0126] Example 27: Preparation of PLGA nanoparticles of CpG ODN as an immunoadjuvant (1) 100 mg of PLGA was dissolved in 1.25 mL of dichloromethane (oil phase).

[0127] (2) AM1012-05 and the antigen protein (S1 protein) were added to a 1% PVA solution to prepare a solution containing 10 mg / mL of AM1012-05 and 10 mg / mL of S1 protein (aqueous phase 1).

[0128] (3) A 4 mL 2.5% PVA solution was prepared (aqueous phase 2).

[0129] (4) The oil phase and water phase 1 were homogenized and uniformly mixed to form a primary emulsion. Then, the primary emulsion was added to water phase 2 and homogenized using a homogenizer to form a secondary emulsion.

[0130] (5) The formed secondary emulsion was slowly added to a 1% PVA aqueous solution (external aqueous phase), and stirred for 4 hours to volatilize the dichloromethane.

[0131] (6) The solution from step 5 was centrifuged with 500g for 5 minutes to remove large microspheres, and the supernatant was collected.

[0132] (7) The supernatant from step 6 was centrifuged at 10,000 g for 10 minutes, and the solid was collected. The PLGA microsphere product was washed three times with deionized water or ultrapure water.

[0133] When the PLGA microsphere vaccine product containing AM1012-05 and antigen protein obtained in this study was measured using a Nicomp Z3000 laser particle size distribution analyzer, the particle size could be controlled to within 500 nm (see Figure 29).

[0134] Example 28: Investigation of compatibility of additives in topical formulations of CpG ODN Additives that can be used in topical formulations are classified into oil phase, thickeners, gels, emulsifiers, solvents, and penetration enhancers. Of these, the oil phase includes glyceryl behenate, liquid paraffin, petrolatum, medium-chain triglyceride, and cocoyl caprylate; the thickeners include glyceryl monostearate, glyceryl mono / distearate, and cetostearyl alcohol; the gels include hypromellose, xanthan gum, carbomer, sodium carboxymethylcellulose, and poloxamer 407; and the emulsifiers include polyoxyl stearate 40, cetostearyl alcohol polyethylene glycol ether, polysorbate 60, poloxamer 188, and oleoyl methyl ether. The compound contains logol glyceride, polyethylene glycol-7 stearate, polyoxyethylene octanoic acid / caprate glyceryl, sodium dodecyl sulfate, propylene glycol monofatty acid ester, polyglyceryl oleate, and propylene glycol laurate. The solvent contains glyceryl triacetate and propylene glycol. The penetration enhancers contain isopropyl myristate, propylene glycol, polyoxyethylene octanoic acid / caprate glyceryl, polyglyceryl oleate, and propylene glycol laurate.

[0135] After preparing gelling additives as aqueous solutions of appropriate concentrations, a 3 mg / g gel solution was prepared with AM1012-05. Other additives were mixed with API in a one-to-one ratio at 3 mg / g. The samples were placed in a 40°C incubator and sampled on days 5, 15, and 30, respectively, to measure the content and related substances. The results indicate that the following additives are compatible with AM1012-05. The oil phase contains glyceryl behenate, liquid paraffin, petrolatum, medium-chain triglyceride, and cocoyl caprylate; the thickener contains glyceryl mono / distearate; the gelling agent contains hypromellose, carbomer, xanthan gum, sodium carboxymethylcellulose, and poloxamer 407; the emulsifiers contain cetostearyl alcohol polyethylene glycol ether, poloxamer 188, oleoyl macrogol glyceride, polyethylene glycol-7 stearate, polyoxyethylene octanoate / caprate glyceryl, sodium dodecyl sulfate, propylene glycol mono fatty acid ester, polyglyceryl oleate, and propylene glycol laurate; the solvent contains propylene glycol; and the penetration enhancers contain isopropyl myristate, propylene glycol, and polyglyceryl oleate.

[0136] Example 29: Manufacture of a topical ointment formulation of CpG ODN AM1012-05 was weighed and dissolved in deionized water to prepare the API solution. Appropriate amounts of petrolatum and liquid paraffin were weighed and heated in an 80°C water bath until melted. The API solution was added to the molten oil phase, homogenized at 70-80°C for 10 minutes, cooled to 65-70°C, and dispensed. The AM1012-05 content in the final formulation was 1 mg / g. The ratio of petrolatum to liquid paraffin was determined based on sensory evaluation, and the proportion of petrolatum could be selected between 100% and 50%.

[0137] Example 30: Preparation of a topical gel formulation of CpG ODN Prepare aqueous solutions of Carbomer 974 (2%), 10% triethanolamine, and AM1012-05. Dilute the 2% Carbomer 974 aqueous solution to a concentration of 0.7%, add 10% triethanolamine, adjust the pH to 7.0, dissolve phenoxyethanol in propylene glycol and add it to the gel, stir to mix uniformly, then add the API aqueous solution and stir to mix uniformly. Add 10% triethanolamine dropwise again to adjust to the appropriate pH, bring to a volume, stir to mix uniformly, and dispense.

[0138] The carbomer used in gel production can be adjusted in concentration and type (carbomer 971, 980, 981, etc.) according to the required viscosity. Neutralizing agents can include triethanolamine solution, sodium hydroxide, aminomethylpropanol, tromethamine, and N,N,N',N'-tetrakis(2-hydroxypropyl)ethylenediamine. Preservatives can include phenoxyethanol, parabens, and cresols.

[0139] Example 31: Preparation of topical emulsion of CpG ODN (1) Oil phase: 15 g of petrolatum, 6 g of liquid paraffin, and 7.2 g of glyceryl mono / distearate were placed in a container, heated to 70°C, and stirred until melted. The molten oil phase was kept at 70°C and stirred at low speed.

[0140] (2) Aqueous phase: 59.5 g of deionized water was heated to 70°C in a container, and 1.8 g of cetostearyl alcohol polyethylene glycol ether was added and dissolved.

[0141] (3) Preparation of emulsion: At 70°C, the molten oil phase from step 1 was transferred to the container of the aqueous phase and mixed at a speed of 10 rpm.

[0142] (4) After the transfer was completed, the homogenizer was started and homogenized for 10 minutes, with the temperature controlled to 65-70°C.

[0143] (5) The homogenizer was stopped, the stirrer temperature was set to 50°C, and the emulsion was cooled to 50°C.

[0144] (6) Drug solution: 30 mg of AM1012-05 was dissolved in 10.5 g of deionized water and kept at a temperature of 50°C.

[0145] (7) The drug solution from step 6 was transferred to the emulsion from step 5, and the temperature was 50°C when it was stirred.

[0146] (8) The homogenizer was started and the mixture was homogenized for 10 minutes, and then a 10% sodium hydroxide solution was added dropwise to adjust the pH to 7.5.

[0147] (9) Once the emulsion was cooled to 25°C, the prepared emulsion was dispensed, and finally, 100 g of emulsion containing 0.3 mg / g of AM1012-05 was obtained.

[0148] The oil phase, thickener, and emulsifier used in this emulsion can be replaced with additives that have passed additive compatibility tests. Penetration enhancers that have passed additive compatibility tests can also be added to the emulsion.

[0149] Example 32: Production of a vaccine using CpG as an adjuvant This example describes the production of a vaccine using the S1 protein of the novel coronavirus as a model antigen and CpG as an adjuvant. (1) Under sterile conditions, 50 μL of SARS-CoV-2 S1 protein (2 mg / mL PBS solution) was taken and diluted to 250 μL with PBS. (2) Prepare a 2 mg / mL AM1012-05 solution or a CpG 1018 solution. (3) Add 250 μL of the solution from step (2) to the solution from step (1) and mix until homogeneous.

[0150] Example 33: Production of an adjuvant-based vaccine (aluminum adjuvant + CpG) This example uses the S1 protein of the novel coronavirus as a model antigen to produce an adjuvant-based vaccine (aluminum adjuvant + CpG). Manufacturing of vaccines using aluminum hydroxide + CpG as an adjuvant: (1) 50 μL of SARS-CoV-2 S1 protein (2 mg / mL PBS solution) was taken. (2) 50 μL of aluminum hydroxide gel adjuvant (2%) was added to the solution from step (1). (3) Add 350 μL of PBS solution to the solution from step (2) and mix until homogeneous. (4) 50 μL of 2 mg / mL AM1012-05 solution was taken and added to the aforementioned solution, and mixed until homogeneous, thereby effectively adsorbing the antigen onto the adjuvant.

[0151] Manufacturing of vaccines using aluminum phosphate + CpG as an adjuvant: (1) 50 μL of SARS-CoV-2 S1 protein (2 mg / mL PBS solution) was taken. (2) 100 μL of aluminum phosphate gel adjuvant (0.5%) was added to the solution from step (1). (3) Add 300 μL of PBS solution to the solution from step (2) and mix until homogeneous. (4) 50 μL of 2 mg / mL AM1012-05 solution was taken and added to the aforementioned solution, and mixed until homogeneous, thereby effectively adsorbing the antigen onto the adjuvant.

[0152] Example 34: Effect of AM1012-05 solution formulation on IFN-γ release by NK-92 cells (1) As described in Example 3, a predetermined amount of NK-92 cells were prepared and resuspended in a certain amount of complete culture medium. (2) 20 μL of cell suspension was taken, mixed with trypan blue in a 1:1 ratio, and then counted using a cell counter to measure cell viability and cell density. (3) A certain amount of AM1012-05 solution formulation sample was taken, and the stock solution was prepared using complete culture medium. Then, the AM1012-05 solution formulation was diluted fourfold using complete culture medium to prepare test samples of AM1012-05 formulation at different concentrations. (4) 2.5 × 10 5 Cells were seeded in 96-well plates at a rate of one cell per well. Each well was then treated with a corresponding sample of AM1012-05 at different concentrations, resulting in final concentrations of 3.2 μM, 0.8 μM, and 0.2 μM, respectively. Three replicate wells were provided for each group, along with a PBS blank control well. (5) The cells were incubated at 37°C in a 5% CO2 incubator for 72 hours. (6) After the cell culture was completed, the cells were centrifuged at 1200 rpm for 5 minutes, and 100 μL of the supernatant free of cells was collected. The IFN-γ secretion status of the supernatant was measured according to the instructions of the ELISA kit. The experimental results are shown in Figure 30. The stimulating effect of the AM1012-05 solution formulation sample on IFN-γ secretion by NK cells is equivalent to that of AM1012-05. However, the blank formulation sample did not produce a stimulating effect on NK-92 cells. Therefore, it has become clear that the formulation of the solution formulation used in this invention has good matching properties with AM1012-05 and does not affect the expression of the immune cell activation function by AM1012-05.

[0153] Example 35: Effects of a single intranasal administration of AM1012-05 on the immune system of C57BL6 mice. The immunoactivating effect of AM1012-05 on the respiratory system of C57BL6 mice was evaluated by intranasal administration. In the experiment, mice were divided into two groups: the drug administration group received 100 μg of AM1012-05 (25 μL / nostril, drug dissolved in PBS), and the negative control group received 50 μL of PBS (25 μL / nostril). The experiment was completed 24 hours after administration, samples (lungs, spleen) were collected, and changes in various immune indicators were analyzed using RT-qPCR.

[0154] The experimental results are shown in Figure 31. From the figure, the following was found: AM1012-05 can effectively stimulate the lungs and spleen of mice to express IFN-α, IFN-γ, and CXCL-10. Furthermore, the degree of activation of spleen cytokines by AM1012-05 was significantly lower than that of the lungs (IFN-α and IFN-γ were approximately 4% of those in the lungs, and CXCL-10 was approximately 1.5% of those in the lungs). This is likely because, since this study involved nasal administration, only a very small amount of AM1012-05 entered the spleen cells through the bloodstream. This result indirectly indicates that the system bioavailability of AM1012-05 after nasal administration is very low. In addition, the administration dose of 100 μg per mouse did not cause any obvious toxicity or side effects in the mice.

[0155] Example 36: Observation of respiratory immune indicators after intranasal administration of different doses of AM1012-05 to mice. The immunoactivating effect of AM1012-5 at different doses and measurement times was evaluated in C57BL6 mice administered intranasally. In three dose groups, each mouse was administered 10 μg, 50 μg, and 100 μg of AM1012-5 (25 μL / nostril, drug dissolved in PBS). The negative control group was administered 50 μL of PBS (25 μL / nostril), and the positive control group was administered 50 μg of CpG 2216 (25 μL / nostril). The experiment was completed before administration, 3 hours after administration, 12 hours after administration, 24 hours after administration, and 48 hours after administration. Samples were collected from the lungs, and changes in CXCL-10 mRNA levels were analyzed using RT-qPCR.

[0156] The experimental results are shown in Figure 32. The following was observed from the figure: AM1012-05 can effectively stimulate the lungs of mice to express CXCL-10. Furthermore, a dose of 100 μg per mouse showed good immunostimulatory activity, and the amount of CXCL-10 mRNA transcription reached its maximum 24 hours after administration. In addition, AM1012-05 showed a better immunoactivating effect compared to the CpG 2216 sequence (CpG 2216 is a type A CpG that frequently appears in the literature).

[0157] Example 37: Observation of respiratory immune indicators after intranasal administration of AM1012-05 to mice (different administration frequencies) The immune-activating ability of AM1012-05 was evaluated at different administration frequencies by intranasal administration to C57BL6 mice. The animal treatment and biological sample measurement methods in this study were the same as in Example 36, and the test results are shown in Figure 33 (the numbers in parentheses on the horizontal axis in the figure represent the administration time for each dose, with the experimental pause time being 0 days). The following was found from the figure: AM1012-05 can effectively stimulate the lungs of mice to express CXCL-10 and IFN-γ. Furthermore, different stimulating effects were observed at different administration frequencies, and cytokine mRNA levels increased to some extent as the number of administrations increased. In addition, the interval between each administration significantly affected the immune-activating effect, with the stimulating effect being most pronounced when the interval between administrations was 12 hours.

[0158] Example 38: Evaluation of in vitro antitumor activity of AM1012-05 (1) MDA-MB-231 cells and N87 cells were cultured and amplified to obtain cells, which were then resuspended in 2 mL of complete medium. (2) Take 10 μL of cell suspension and mix with trypan blue in a 1:1 ratio. After counting using a cell counter, the cell viability was 99.7% for MDA-MB-231, 100% for N87, and 99.2% for PBMC. The cell density for MDA-MB-231 was 1.49 × 10⁶. 6 The cell count is cells / mL, and the N87 is 1.12 × 10⁶. 7 The cell count is 5.17 × 10⁶ cells / mL, and the human PBMCs are 5.17 × 10⁶. 6 The concentration was cells / mL. (3) The cells were resuspended in culture medium, and MDA-MB-231 cells were seeded at 2000 cells / well and N87 cells at 4000 cells / well in 96-well plates. (4) Once the cells had completely attached to the wall, PBMC cells were seeded at an effector-to-target ratio of 10:1. (5) The administration concentrations of AM1012-05 were set to 62.5 nM, 125 nM, 250 nM, 500 nM, 1000 nM, 2000 nM, 4000 nM, and 8000 nM, and six replicate wells were provided for each concentration. (6) After culturing for another 5 days, the samples were measured and analyzed using CCK8.

[0159] The experimental results are shown in Figures 34 and 35. From the figures, the following was found: AM1012-05 can effectively stimulate human PBMCs and clearly kill tumor cells in a dose-dependent manner. Since TLR-9 is mainly present in human pDCs and B cells, and type A CpG mainly stimulates pDCs, this result indicates that AM1012-05 stimulates pDCs to produce NK cells and CD8 cells. + This study revealed that it enhances the ability of T cells to kill tumor cells.

[0160] Example 39: Evaluation of the antitumor activity of combination therapy with AM1012-05 and CAR-T receptors. CAR-T cells and tumor cells in specific ratios were co-cultured, and a specific concentration of AM1012-05 was added. After co-incubating for 3-5 days, the culture medium was changed to remove free CAR-T cells and tumor cell debris. A certain amount of CCK8 reagent was added, and the activity of the remaining tumor cells was analyzed. The tumor cell killing rate for each group was calculated, using the control group (no treatment) and the CAR-T group as controls. The experimental results revealed that AM1012-05 can effectively enhance the killing of tumor cells by CAR-T cells and exhibits very good dose-dependent properties. This indicates a synergistic effect of AM1012-05 on CAR-T therapy.

[0161] Example 40: Evaluation of the anti-allergic rhinitis activity of AM1012-05 (1) Initial sensitization was induced in BALB / c mice using ovalbumin (OVA). 25 μg of OVA and 2 mg of Al(OH)3 were dissolved in 100 μL of physiological saline and injected intraperitoneally into the mice on days 0, 7, and 14, respectively. (2) Stimulate the nasal cavity. 20 μL of physiological saline containing 200 μg of OVA was added dropwise to the nasal cavity of each mouse, and the stimulation days were days 21, 22, 23, 28, 29, and 30. (3) 50 μg of AM1012-05 was administered as nasal drops one hour after the completion of each stimulation. (4) Immediately after the start of the experiment, the animals' clinical condition, including mental state, motor activity, diet, and water intake, was observed daily until the animals were euthanized (after the stimulation was stopped, each mouse was observed for 10 minutes and the number of times it scratched its nose was recorded). Nasal mucosa, nasal lavage fluid, and serum were collected and analyzed for eosinophilic granulocytes, immunocytological analysis of the nasal lavage fluid, and analysis of immunological indicators (IFN-γ, IL-4), respectively.

[0162] The experimental results are shown in Figures 36 and 37. Compared to the model group, after administration of AM1012-05, the number of sneezes in allergic rhinitis mice was effectively reduced (Figure 36A), the number of nose scratches was reduced (Figure 36B), nasal discharge was suppressed (Figure 36C), the concentration of eosinophilic granulocytes in the nasal mucosa was reduced (Figure 36D), the IL-4 content in the serum of allergic rhinitis mice was effectively reduced (Figure 36E), and IFN-γ expression was increased (Figure 36F). In addition, AM1012-05 can reduce the infiltration of neutrophils (Figure 37A) and basophilic granulocytes (Figure 37B) in the nasal mucosa. These results demonstrate that AM1012-05 can effectively shift the organism's immune response towards the Th1 type and suppress the Th2 and Th17 type immune responses (a decrease in eosinophilic and basophilic granulocytes indicates a reduction in the Th2 type immune response, and a decrease in neutrophils indicates a reduction in the Th17 type immune response), thereby achieving the goal of treating allergic rhinitis.

[0163] Furthermore, this experiment confirms that IAMA-001 has the function of reducing eosinophilic granulocyte counts in mice and has the potential to be used in the treatment of diseases related to eosinophilic granulocytes.

[0164] Example 41: Evaluation of the activity of AM1012-05 in resisting respiratory syncytial virus infection. (Test 1) Vero-E6 cells were inoculated into a 24-well plate, with a cell density of 2 × 10⁶. 5 The cells were cultured at a concentration of cells / mL for 12-16 hours until confluence reached 80%. Next, 1000 μL of supernatant containing 1x and 5x diluted AM1012-05 co-incubated with human PBMCs was added to Vero-E6 cells and incubated for 24 hours. Furthermore, the cells were infected with respiratory syncytial virus at an infectious dose of MOI=0.1 for 48 hours, after which samples were collected and the viral replication status in each well was measured.

[0165] (Exam 2) Vero-E6 cells were inoculated into a 24-well plate, with a cell density of 2 × 10⁶. 5 The cells were cultured at a concentration of 1 / mL for 12-16 hours until confluence reached 80%. Next, AM1012-05 (final concentration 1 μM) and PBMC (2 × 10⁶) were added. 6 Cells (per well) were added to a 24-well plate. After infecting the cells with respiratory syncytial virus at an infectious dose of MOI=0.1 for 48 hours, samples were collected and the viral replication status in each well was measured.

[0166] The results of the two tests are shown in Figure 38. The supernatant obtained by co-incubating AM1012-05 with PBMCs effectively prevented the proliferation of respiratory syncytial virus in cells (Figure A), and AM1012-05 significantly removed respiratory syncytial virus from host cells in the presence of PBMCs (Figure B).

[0167] Example 42: Evaluation of the anti-atopic dermatitis activity of AM1012-05 (1) On days 0, 7, and 14 of the study, 0.5 mL of ovalbumin (OVA) solution (containing 5% aluminum hydroxide) was injected into BALB / c to induce sensitization.

[0168] (2) Preparation before administration: On day 20, the animals were anesthetized, and hair was shaved and removed from a 3 x 3 cm area on their backs.

[0169] (3) Local stimulation and administration: On day 21, the model group had their back skin repeatedly stimulated with a brush to induce slight cracks. The PBS group applied 50 μL of PBS to a 2 × 2 cm gauze dressing, applied it to the skin on their backs, and secured it with adhesive tape for 24 hours, changing it daily for one week (days 21-27), and then repeated the same procedure for another week after a one-week interval (days 35-41). The other groups had 50 μL of 1000 μg / mL OVA solution homogeneously mixed with the test product or solvent, applied it to a 2 × 2 cm gauze dressing, applied it to the skin on their backs, and secured it with adhesive tape for 24 hours, changing it daily for one week (days 21-27), and then repeated the same procedure for another week after a one-week interval (days 35-41), and on days 28-34, only topical administration of the test product or solvent was performed.

[0170] (4) On the 35th day after the experiment, serum was collected from the mice and IgE levels were measured.

[0171] The experimental results are shown in Figure 39. Compared to the OVA model group, the IgE content in the serum of OVA-sensitized mice was effectively reduced after administration of AM1012-05. This result demonstrates that AM1012-05 can effectively suppress the allergic reaction of organisms and thereby achieve the goal of treating atopic dermatitis.

[0172] Example 43: Measurement of immunity and titer in mice induced by vaccine (Immunity in mice through vaccine) (1) An appropriate number of Balb / c mice were prepared, isolated and quarantined, and then reared. (2) In accordance with the requirements of the test, mice were divided into groups of 5, and the negative control was a mouse injected with the S1 protein (S1 protein group). (3) Mice were immunized by intramuscular injection on day 0 (initial immunization time), day 14, and day 28, with a dose of 50 μL / mice used for immunization. (4) Immediately after the start of the experiment, the animals' clinical condition, including mental state, motor activity, diet, and water intake, was observed on a daily basis until the animals were euthanized. (5) Sample collection: Blood was collected on days 7, 21, 35, 49, 63 and 77. 100-150 μL of whole blood was collected from each mouse at one time, and after centrifugation at 4°C and 6000 rpm for 10 minutes, the serum was aspirated. (6) In preparation for measuring the titer, the collected samples were frozen and stored at -80°C.

[0173] (Measurement of mouse immune titer) (1) Protein coating: The coating concentration of SARS-CoV-2 S1 protein was 2 μg / mL, and the protein concentration was diluted. 100 μL of the diluted sample was added to each well of a 96-well ELISA plate, sealed with seal film, and kept overnight in a refrigerator at 4°C. (2) Washing the plate: Remove the 96-well plate that had been coated overnight, discard the liquid in the wells, wash it three times with PBS at a rate of 300 μL / well, and gently tap to remove any remaining wash solution. (3) Blocking: 300 μL of 2% BSA blocking buffer was added to each well, sealed with a sealing film, and incubated at 37°C for 1 hour. (4) Washing the plate: After blocking was complete, the liquid in the wells was discarded, and the plate was washed three times with 300 μL / well using PBST, and the plate was gently tapped to remove any remaining wash solution. (5) Primary antibody incubation: 100 μL of diluted sample (serum) was added to each well, sealed with a sealing film, and incubated at 37°C for 1 hour. (6) Washing the plates: After incubation was complete, the liquid in the wells was discarded, and the plates were washed three times with 300 μL / well using PBST, and the plates were gently tapped to remove any remaining wash solution. (7) Incubation of secondary antibody: 100 μL of diluted secondary antibody was added to each well, sealed with a sealing film, and incubated at 37°C for 1 hour. (8) Washing the plates: After incubation was complete, the liquid in the wells was discarded, and the plates were washed five times with 300 μL / well using PBST, and the plates were gently tapped to remove any remaining wash solution. (9) Color development: 100 μL of TMB color development solution was added to each well and allowed to develop color at 37°C. (10) Stop: Add 50 μL of ELISA stop solution and mix by gently tapping the edge of the ELISA plate. (11) Reading the results: The ELISA plate was placed in a microplate reader and the results were read at a detection wavelength of 450 nm and a reference wavelength of 570 nm.

[0174] Analysis and discussion of immunological results: In mice, the titer of total IgG antibodies reflects the overall immunity level of the vaccine, with IgG1 reflecting humoral immunity and IgG2a reflecting cellular immunity. Most conventional adjuvants have good humoral immunity activation ability (mouse IgG1) but weak cellular immunity (mouse IgG2a). Therefore, developing vaccine adjuvants that generate IgG2a antibodies through effective stimulation has become an important research theme in the adjuvant industry in recent years. Currently, the only CpG adjuvant approved for human vaccine production worldwide is Dynavax's CpG 1018 (CpG 1018 is used as an independent adjuvant in the production of hepatitis B vaccine). In this study, the use of AM1012-05 as an adjuvant in vaccines produced in Examples 32 and 33 was evaluated, using the S1 protein of the novel coronavirus as a model antigen and CpG1018 as a CpG-positive adjuvant control. The results of IgG, IgG1, and IgG2a antibody titer measurements after mouse immunization with AM1012-05-related vaccine are shown in Figures 40, 41, and 42.

[0175] As can be seen in Figure 40, in the early stages of immunization, the titer of the adjuvant-based IgG antibody consisting of AM1012-05 and aluminum phosphate is significantly superior to that of aluminum phosphate adjuvant alone (Figure 40A), the titer of the adjuvant-based IgG antibody consisting of AM1012-05 and aluminum phosphate is significantly superior to that of commercially available CpG adjuvant 1018 (Figure 40A-E), the titer of the adjuvant-based IgG antibody consisting of AM1012-05 and aluminum hydroxide is also superior to that of commercially available CpG adjuvant 1018 (Figure 40A-E), and the titer of the IgG antibody when AM1012-05 is used as an independent adjuvant is equivalent to that of commercially available CpG adjuvant 1018 (Figure 40A-E).

[0176] As can be seen in Figure 41, in the early stages of immunization, the titer of the adjuvant-based IgG1 antibody consisting of AM1012-05 and aluminum phosphate or aluminum hydroxide is significantly superior to that of the commercially available CpG adjuvant 1018 (Figures 41A and B). In the mid and late stages of immunization, the titer of the adjuvant-based IgG1 antibody consisting of AM1012-05 and aluminum phosphate or aluminum hydroxide is also significantly superior to that of the commercially available CpG adjuvant 1018 (Figures 41D and E). When AM1012-05 is used as an independent adjuvant, the titer of the IgG1 antibody is equivalent to that of the commercially available CpG adjuvant 1018 (Figures 41A to E).

[0177] As can be seen in Figure 42, the titer of the adjuvant-based IgG2a antibody consisting of AM1012-05 and aluminum phosphate is significantly superior to that of the commercially available CpG adjuvant 1018 (Figure 42 A-E), and the titer of the adjuvant-based IgG2a antibody consisting of AM1012-05 and aluminum phosphate is also significantly superior to that of the aluminum hydroxide and aluminum phosphate adjuvants (Figure 42 A-E). In the early and mid-stages of immunization, AM1 The titer of the IgG2a antibody when 012-05 is used as an independent adjuvant is significantly superior to that of the commercially available CpG adjuvant 1018 (Figure 42, B-D). In the early stages of immunization, the titer of the IgG2a antibody in the adjuvant system consisting of AM1012-05 and aluminum hydroxide is significantly superior to that of the commercially available CpG adjuvant 1018, aluminum hydroxide adjuvant, and aluminum phosphate adjuvant (Figure 42, A, B).

[0178] As can be seen from the above evaluation results of the immune titer of adjuvant vaccines consisting of AM1012-05 as an independent adjuvant, or with an aluminum adjuvant (aluminum hydroxide or aluminum phosphate), AM1012-05 can significantly improve the humoral and cellular immunity levels of the vaccine. In particular, the adjuvant system consisting of AM1012-05 and aluminum phosphate can significantly improve the cellular immunity level of the vaccine, and its immune effect was far superior to that of commercially available CpG adjuvant 1018 and aluminum adjuvants (aluminum hydroxide or aluminum phosphate). Furthermore, in terms of immune protection by the vaccine, the adjuvant system consisting of AM1012-05 and aluminum adjuvant (aluminum hydroxide or aluminum phosphate) is far superior to commercially available CpG adjuvant 1018 and aluminum adjuvants (aluminum hydroxide or aluminum phosphate).

[0179] Therefore, when AM1012-05 is used as an independent adjuvant or an adjuvant system composed of it and an aluminum adjuvant (aluminum hydroxide or aluminum phosphate), compared with conventional aluminum adjuvants (aluminum hydroxide or aluminum phosphate) and CpG adjuvants, it has very significant advantages such as early activation of immune defense and improvement of the effect of immune defense. Therefore, it is a very advantageous vaccine adjuvant or adjuvant system.

[0180] Through the above specific embodiments and examples, the present application has been described in detail. However, these descriptions should not be understood as limitations to the present application. Without departing from the spirit and scope of the present application, various equivalent substitutions, modifications or improvements can be made to the technical solutions and their embodiments of the present application, and those skilled in the art will understand that all of these fall within the scope of the present application. The protection scope of the present application shall be subject to the scope of the claims listed below.

Claims

1. A CpG oligodeoxynucleotide having the ability to regulate the immune system of an organism, The aforementioned CpG oligodeoxynucleotide has the structure 5'-(G) ｎ - Core array - (N) ｑ - (G) ｍ A CpG oligodeoxynucleotide having the ability to regulate the immunity of an organism, characterized in that it is one of the sequences that are -3', the core sequence is a palindromic sequence, N is one of three bases: adenine deoxyribonucleotide A, thymine deoxyribonucleotide T, and cytosine deoxyribonucleotide C, G is guanine deoxyribonucleotide, q, n, and m are all integers, the range of the value of n is 2 to 11, the range of the value of q is 0 to 10, and the range of the value of m is 2 to 11.

2. The CpG oligodeoxynucleotide having the ability to regulate the immunity of an organism, characterized in that the range of the value of n is 3 to 4 and the range of the value of q is 1 to 5, as described in claim 1.

3. The core array is at least one of the following: CGCGAACGCGTCGCG, ACGATCGAGATCGT, AGGATCGATCCCT, TTCGATCGATCGAA, CGATCGATCG, GACGATCGTC, TGCATCGATGCCA. When the core array is CGCGAACGCGTCGCG, the range of the value of q is 0 to 10, the range of the value of n is 0 to 3, and the range of the value of m is 2 to 11. Alternatively, if the core array is ACGATCGAGATCGT, the range of the value of q is 0 to 10, the range of the value of n is 2 to 11, and the range of the value of m is 2 to 11. Alternatively, if the core array is AGGATCGATCCCT, the range of the value of q is 0 to 10, the range of the value of n is 2 to 11, and the range of the value of m is 2 to 11. Alternatively, if the core array is TTCGATCGATCGAA, the range of the value of q is 0 to 10, the range of the value of n is 2 to 11, and the range of the value of m is 2 to 11. Alternatively, if the core array is CGATCGATC, the range of the value of q is 0 to 10, the range of the value of n is 1 to 11, and the range of the value of m is 2 to 11. Alternatively, if the core array is GACGATC, then q is 0, the range of the value of n is 0 to 3, and the range of the value of m is 2 to 11, or the range of the value of q is 1 to 10, the range of the value of n is 2 to 11, and the range of the value of m is 2 to 11. Alternatively, the CpG oligodeoxynucleotide having the ability to regulate the immunity of an organism according to claim 1, characterized in that, when the core sequence is TGCATCGATGCA, q is 0, the value of n is 1, 3, 4, or 5, and the range of the value of m is 2 to 11, or the range of the value of q is 1 to 10, the range of the value of n is 2 to 11, and the range of the value of m is 2 to 11.

4. A CpG oligodeoxynucleotide having the ability to modulate the immunity of an organism according to claim 1, characterized in that it contains at least one of the deoxynucleotide sequences represented by SEQ ID NO. 1 to SEQ ID NO.

82.

5. The CpG oligodeoxynucleotide having the ability to modulate the immunity of an organism according to any one of claims 1 to 4, characterized in that the CpG oligodeoxynucleotide is itself or comprises at least one of thio modification, fluoro modification, methoxy modification, locked nucleic acid modification, and nanoparticle modification.

6. The CpG oligodeoxynucleotide having the ability to modulate the immunity of an organism according to claim 5, characterized in that the thio-modified site is located at the 5' and / or 3' terminal polyguanylic acid.

7. The nanoparticles used in the aforementioned nanoparticle modification include at least one of PLGA, chitosan, lipid nanoparticles, and liposomes, and are characterized by having the ability to modulate the immunity of the organism according to claim 5.

8. A solution formulation of CpG oligodeoxynucleotide, characterized in that it comprises a CpG oligodeoxynucleotide according to any one of claims 1 to 7 and an additive, wherein the additive comprises at least one of the following: a pH buffer, glycine, trehalose, mannitol, sucrose, arginine, lysine, histidine, glycerin, propylene glycol, Pluronic F127, Pluronic F68, polysorbate 20, polysorbate 80, benzalkonium chloride, disodium edetate, sodium citrate, hypromellose, sodium carboxymethylcellulose, methyl-β-cyclodextrin, and polyethylene glycol.

9. The CpG oligodeoxynucleotide solution formulation according to claim 8, characterized in that the metal ions in the CpG oligodeoxynucleotide formulation include sodium, potassium, magnesium, calcium, zinc, and iron, and the total concentration range is 0.1 to 90 mM.

10. The CpG oligodeoxynucleotide solution formulation according to claim 8, characterized in that the pH of the CpG oligodeoxynucleotide formulation is 7 to 9.

11. Use of CpG oligodeoxynucleotide according to any one of claims 1 to 7 in the manufacture of a drug that modulates the immune response of the respiratory system.

12. Use of CpG oligodeoxynucleotides in the manufacture of a drug for preventing and treating respiratory diseases, characterized in that the respiratory disease includes at least one of respiratory viral infections, respiratory bacterial infections, respiratory fungal infections, respiratory parasitic infections, and respiratory allergic diseases.

13. The use of CpG oligodeoxynucleotides in the manufacture of a drug for preventing and treating a respiratory disease according to claim 12, characterized in that the respiratory virus includes at least one of COVID-19 virus, influenza virus, RSV virus, and SARS-CoV.

14. Use of CpG oligodeoxynucleotides in the manufacture of a drug for preventing and treating non-respiratory infections, characterized in that the non-respiratory infection includes at least one of HIV infection, HBV infection, and HCV infection.

15. Use of CpG oligodeoxynucleotide according to any one of claims 1 to 7 in the manufacture of a drug that modulates the proliferation function of immune cells or a drug that modulates the release of cytokines from immune cells.

16. Use of CpG oligodeoxynucleotide according to any one of claims 1 to 7 in the production of an immunoadjuvant.

17. Use of CpG oligodeoxynucleotide according to any one of claims 1 to 7 in the manufacture of a drug for preventing and treating viral infections.

18. Use of CpG oligodeoxynucleotide in the manufacture of a drug for treating tumors, as described in any one of claims 1 to 7, wherein the CpG oligodeoxynucleotide in the drug is the sole antitumor active ingredient.

19. An antitumor composition comprising a CpG oligodeoxynucleotide according to any one of claims 1 to 6 and an antitumor agent, wherein the antitumor agent includes, but is not limited to, a PD-1 antitumor agent, a PDL-1 antitumor agent, and an antitumor cell therapy, and the composition is characterized in that the CpG oligodeoxynucleotide is used as an antitumor agent component or an immunoadjuvant.

20. Use of CpG oligodeoxynucleotide according to any one of claims 1 to 7 in the manufacture of a human or animal vaccine.

21. Use of CpG oligodeoxynucleotides in the treatment of central nervous system disorders according to any one of claims 1 to 7, wherein the central nervous system disorder includes, but is not limited to, Alzheimer's disease.

22. Use of CpG oligodeoxynucleotide according to any one of claims 1 to 7 in the manufacture of a drug for preventing or treating post-wound secondary infection.

23. Use of CpG oligodeoxynucleotide according to any one of claims 1 to 7 in the manufacture of a drug that promotes wound healing.

24. Use of CpG oligodeoxynucleotide according to any one of claims 1 to 7 in the manufacture of a drug for treating allergic rhinitis, and / or atopic dermatitis, and / or asthma, and / or chronic obstructive pulmonary disease, and / or eosinophilic granulocyte-related disease.

25. A drug manufactured using a CpG oligodeoxynucleotide as described in any one of claims 1 to 7, wherein the dosage form of the drug is an injection, a tablet, a lyophilized preparation, an inhalant, a nasal drop, a nasal spray, an anal suppository, an eye drop, a topical ointment, a wash, or a gel.

26. A topical preparation comprising CpG oligodeoxynucleotide as described in any one of claims 1 to 7, and an additive, wherein the additive comprises glyceryl behenate, liquid paraffin, petrolatum, medium-chain triglyceride, cocoyl caprylate, mono / distearate glyceryl, hypromellose, carbomer, xanthan gum, sodium carboxymethylcellulose, poloxamer 407, cetostearyl alcohol polyethylene glycol ether, poloxamer 188, oleoyl macrogol glyceride, polyethylene glycol-7 stearate, polyoxyethylene octanoate / caprate glyceryl, sodium dodecyl sulfate, propylene glycol mono fatty acid ester, polyglyceryl oleate, propylene glycol laurate, propylene glycol, isopropyl myristate, and polyglyceryl oleate.

27. A formulation comprising a combination of a CpG oligodeoxynucleotide adjuvant used in human or animal vaccines and another type of adjuvant, characterized in that it includes the CpG oligodeoxynucleotide itself as described in any one of claims 1 to 7, or a combination thereof with an aluminum hydroxide adjuvant or an aluminum phosphate adjuvant.

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