Cpg oligodeoxynucleotide capable of regulating immune function of body and use thereof

Abstract

Provided are a plurality of CpG-containing oligodeoxynucleotides (ODNs) capable of regulating the immune function of the body and the use thereof. The design concept of the sequences and structures of the oligodeoxynucleotides are different from those of a traditional type-A CpG ODN, which embodies the complex druggability rules of the oligodeoxynucleotides, and solves the druggability problem of the traditional type-A CpG. The oligodeoxynucleotides have the activity of regulating the immune function of the body both in vitro and in vivo, can stimulate the production of type I, II and III interferons, up-regulate a Th1-type immune response, and suppress Th2-type and Th17-type inflammatory responses. Therefore, the oligodeoxynucleotides can be used in the prevention and treatment of allergic diseases, including allergic rhinitis, atopic dermatitis, asthma, chronic obstructive pulmonary diseases, and diseases associated with eosinophilic granulocytes and Th17, caused by a Th2-type immune response; can be used in the prevention and treatment of the infection and transmission of respiratory or non-respiratory pathogenic microorganisms including viruses, bacteria, fungi and parasites; can be used to improve the immunity level of the elderly population and people with immunodeficiency; can be used to convert "cold tumors" into "hot tumors" and improve the effect of cancer immunotherapy; can be used in the prevention and treatment of central nervous system diseases associated with immune dysfunction such as Alzheimer's disease; and can be used for other diseases associated with immune dysfunction.

Description

CpG oligodeoxynucleotides with the ability to regulate immunity and their applications

[0001] This application claims priority to a prior application filed with the State Intellectual Property Office of China on July 21, 2023, with patent application number 202310902755.X, entitled “CpG oligodeoxynucleotides with immune-regulating capabilities and their applications.” The entire text of the prior application is incorporated herein by reference. Technical Field

[0002] The present invention belongs to the field of oligodeoxynucleotides, and in particular relates to a CpG oligodeoxynucleotide capable of regulating body immunity and an application thereof. Background Art

[0003] TLR9 is a mammalian innate immune receptor present in endosomes. Its ligand is an unmethylated CpG-containing oligodeoxyribonucleotide (ODN). Depending on the type and structure of CpG-ODN, upon entry into the endosome, it can bind to TLR9 and activate the IRF-7 or NF-κB signaling pathway, producing inflammatory factors such as type I interferon or IL-6 to intervene in the immune response.

[0004] Based on the differences in structural characteristics and immune effects, CpG ODN can be divided into three types: A, B, and C. Type A CPG enters the early endosomes of plasmacytoid dendritic cells (pDCs) and binds to the TLR9 receptor inside, activating the IRF-7 signaling pathway and inducing the production of a large amount of type I interferon (IFN-α / IFN-β) and type III interferon (IFN-λ). Type I interferon, as a general immune regulatory cytokine, acts on downstream immune cells, including but not limited to NK cells, CD8 + T cells, CD4 +T cells, including γ / δ T cells and macrophages, induce the production of cytokines such as type II interferon (IFN-γ). Type B CpG is a fully thiolated linear CpG ODN, whose primary target cell is B lymphocytes. Type B CpG enters the late endosome and binds to TLR9, activating NF-κB and generating a series of immune responses, including the secretion of inflammatory cytokines such as IL-6, IL-1, and TNF-α, and promoting B cell proliferation. Type B CpG can be used as a vaccine adjuvant, significantly enhancing antibody levels. Type C CpG is a fully thiolated CpG ODN, containing portions of type A CpG and type B CpG structures. It possesses both palindromic sequences capable of dimerization and non-palindromic linear structures. Type C CpG can enter both early and late endosomes to bind to TLR9. Therefore, it possesses the combined activities of type A and type B CpG ODNs, activating both the IRF-7 signaling pathway and the NF-κB pathway, leading to the production of type I interferon and inflammatory cytokines. Although type C CpG combines the characteristics of type A and type B CpG, its ability to induce interferon and inflammatory molecules is significantly lower than the activity of type A or type B CpG respectively.

[0005] Preclinical animal studies of type B CPG have demonstrated its success in treating allergic rhinitis, asthma, various microbial infections, and tumors. Over the past 20 years, over 100 publications have reported on the preclinical use of CPG in treating various diseases. CPG-1018, a representative type B CPG, was successfully approved for marketing as an adjuvant in a hepatitis B vaccine in 2017.

[0006] Current studies have shown that when administered through different routes, type A CpG ODN can induce strong innate immunity (type I / II / III interferon) and Th1 immune responses (DC, NK and CD8 +It can activate T cells and produce IL-12, and regulate immune function, thereby inhibiting Th2 and Th17 immune responses. These immune responses can treat diseases caused by allergic and non-allergic inflammation, resist infection with pathogenic microorganisms, directly or indirectly kill tumor cells, reduce the formation of Aβ plaques, and enhance the immunogenicity of vaccines, among other effects. This also lays the foundation for its application in infectious diseases, cancer, immune adjuvants, allergic rhinitis, atopic dermatitis, asthma, chronic obstructive pulmonary disease, eosinophil-related diseases, Alzheimer's disease, wound healing, wound infection prevention and treatment, etc. (Montamat et al., 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.2012Oct 15;18(20):5628-38; Senti et al., Clin Exp Allergy.2009Apr;39(4):562-70; Scholtzova et al., Acta Neuropathol Commun.2014Sep 2;2:101; Wanke-Jellinek et al., J Immunol.2016 Jan 15;196(2):767-77).

[0007] Due to the easy aggregation property of type A CPG, its drugability is very poor. Even more than 20 years after the discovery of type A CPG, there have been very few clinical trials and no drugs have been approved for marketing. Historically, three organizations have attempted to improve the drugability of type A CPG through various methods: 1. Cytos Biotechnology AG used virus-like particles (VLPs) to encapsulate type A CPG (QbG-10). Although this improved drugability, the VLP encapsulation made type A CPG no longer a simple CPG, but a CPG with strong adjuvant function (Storni et al., J. Immunol. 2004. 172: 1777-1785); 2. FDA scientists added fma (a small molecule compound) to the 5 Gs at the 3' end of type A CPG, turning it into a temperature-sensitive prodrug. After entering the cell, fma automatically fell off at 37°C, allowing type A CPG to reaggregate and exert its drug efficacy. However, such modification could not completely prevent aggregation in vitro, and the modification of fma limited the safety and tolerability of the drug (Puig et al., Puig, Nucleic acids Research. 2005. 34: 6488-6495); 3. In 2015, Japanese scientists reported that adding 40 adenine deoxyribonucleotides (poly-A) to the 3' end of type A CpG significantly improved its drugability. However, the addition of 40 adenines significantly increased production costs, and the fact that it is fully thiolated increases the production of the inflammatory molecule IL-6, limiting its safety and tolerability (Aoshi et al., J. Immunol. Research. 2015. http: / / dx.doi.org / 10.1155 / 2015 / 316364). Consequently, there have been no successful drug developments for traditional type A CpG, and no clinical trials have been reported.

[0008] The principle behind traditional A-type CPG design is to achieve the formation of aggregates through a complete palindrome of an 8-12 nucleotide core sequence (including one or more CpGs) and poly-G residues at both ends. Aggregates present the CpG core sequence to TLR9 in early endosomes, rather than in late endosomes, activating the IRF-7 signaling pathway and producing type I interferons. Aggregate formation is essential for activation of the IRF-7 signaling pathway (Kerkmann et al., J. Biol. Chem., 2005. 280:8086-8093; Wu et al., J. Biol. Chem., 2004. 279:33071-33078). Monomeric CPG ODNs, on the other hand, cannot activate the IRF-7 signaling pathway. For example, B-type CPGs are monomeric in solution and can only bind to TLR9 in late endosomes, thereby activating the NF-κB signaling pathway and inducing the production of inflammatory cytokines rather than type I interferons. Since Type A CPGs must aggregate to be active, the formation of aggregates often leads to large, uncontrollable aggregates, significantly reducing their druggability. This is the main reason why Type A CPGs have yet to achieve successful clinical development. Therefore, ensuring that Type A CPGs aggregate while maintaining controllable aggregation is crucial for the development of these molecules into clinically effective drugs.

[0009] The traditional model for the self-aggregation of A-type CPGs was proposed by Kerkmann et al. in 2005 (Kerkmann et al., J. Biol. Chem., 2005. 280:8086-8093): Two palindromic, complementary monomers form a dimer through Watson-Crick linkage. The poly-G residues at the ends of the dimer then form Hoogsteen base pairs through hydrogen bonds with guanylate residues, forming a tetramer (G-tetrad). Dimers and tetramers then continue to aggregate through G-tetrads, forming larger aggregates in this cycle. Another model, based on the secondary structure of A-type CPGs, suggests that A-type CPG monomers can form palindromic structures within the monomer, and the poly-G residues at their ends can form higher aggregates through Hoogsteen pairing. Therefore, these models indicate that the size and stability of aggregates depend on the number of surrounding Hoogsteen linkages and their unique spatial structure. Our earlier research has found that, based on the secondary structure, a perfectly palindromic structure before and after the poly-G residues can reduce the energy required for aggregation, making it easier for aggregates to form. Therefore, by establishing a certain steric barrier between polyG and the palindrome, the energy for G-tetrad formation may be increased, making the polymer relatively difficult to form or relatively loose after formation. At the same time, combined with the selection of the amount of polyG at both ends, the polymer may be stabilized within a certain size range. Based on the above considerations, the present invention designed multiple core sequences. While keeping the core sequence unchanged, the authors conducted deoxyribonucleotide substitution experiments on the two ends of type A CPG, identified the formation pattern of type A CPG polymers, and discovered the relationship between this and the stimulation of type I interferon production.

[0010] Summary of the Invention

[0011] The technical solutions of the present invention are as follows:

[0012] A CpG oligodeoxynucleotide with the ability to regulate the body's immunity. The CpG oligodeoxynucleotide with the ability to regulate the body's immunity of the present invention has a sequence structure of 5'-(G) n -Core Sequence-(N) q -(G) m- 3', the core sequence is a palindrome sequence, the N is any one of the three bases of adenine deoxyribonucleotide A, thymine deoxyribonucleotide T, and cytosine deoxyribonucleotide C; the G is guanine deoxyribonucleotide; the q, n, and m are all integers; the n value range is 2 to 11, the q value range is 0 to 10, and the m value range is 2 to 11.

[0013] Preferably, the value range of n is 3-4, and the value range of q is 1-5.

[0014] Preferably, the core sequence is at least one of CGCGACGCGTCGCG, ACGATCGAGATCGT, AGGATCGATCCT, TTCGATCGATCGAA, CGATCGATCCG, GACGATCGTC, and TGCATCGATGCA;

[0015] When the core sequence is CGCGACGCGTCGCG, the value range of q is 0 to 10, the value range of n is 0 to 3, and the value range of m is 2 to 11;

[0016] Or, when the core sequence is ACGATCGAGATCGT, the value range of q is 0 to 10, the value range of n is 2 to 11, and the value range of m is 2 to 11;

[0017] Or, when the core sequence is AGGATCGATCCT, the value range of q is 0 to 10, the value range of n is 2 to 11, and the value range of m is 2 to 11;

[0018] Or, when the core sequence is TTCGATCGATCGAA, the value range of q is 0 to 10, the value range of n is 2 to 11, and the value range of m is 2 to 11;

[0019] Or, when the core sequence is CGATCGATCG, the value range of q is 0 to 10, the value range of n is 1 to 11, and the value range of m is 2 to 11;

[0020] Or, when the core sequence is GACGATCGTC, q is 0, n is in the range of 0 to 3, and m is in the range of 2 to 11; or q is in the range of 1 to 10, n is in the range of 2 to 11, and m is in the range of 2 to 11;

[0021] Or, when the core sequence is TGCATCGATGCA, q is 0, n is 1, 3, 4 or 5, and m is in the range of 2 to 11; or q is in the range of 1 to 10, n is in the range of 2 to 11, and m is in the range of 2 to 11.

[0022] Preferably, the above-mentioned CpG oligodeoxynucleotide comprises at least one of the deoxynucleotide sequences shown in SEQ ID NO.1 to SEQ ID NO.82.

[0023] Preferably, the CpG oligodeoxynucleotide itself contains at least one of thio modification, fluoro modification, methoxy modification, locked nucleic acid modification, and nanoparticle modification.

[0024] Preferably, the thiolation modification site is on the polyguanylate at the 5' end and / or the 3' end.

[0025] Preferably, the nanoparticles used in the nanoparticle modification include at least one of PLGA, chitosan, lipid nanoparticles, and liposomes.

[0026] A CpG oligodeoxynucleotide preparation comprises the above-mentioned CpG oligodeoxynucleotide and excipients, wherein the excipients comprise at least one of pH buffer, glycine, trehalose, mannitol, sucrose, arginine, lysine, histidine, glycerol, propylene glycol, Pluronic F127, Pluronic F68, Tween 20, Tween 80, benzalkonium chloride, disodium edetate, sodium citrate, hypromellose, sodium carboxymethylcellulose, methyl beta-cyclodextrin, and polyethylene glycol.

[0027] Preferably, the metal ions of the CpG oligodeoxynucleotide preparation include sodium, potassium, magnesium, calcium, zinc, and iron, with a total concentration ranging from 0.1 to 90 mM.

[0028] Preferably, the pH value of the CpG oligodeoxynucleotide preparation is between 7-9.

[0029] A use of the above-mentioned CpG oligodeoxynucleotide in the preparation of a drug for regulating respiratory immune response.

[0030] A use of the above-mentioned CpG oligodeoxynucleotide in the preparation of a medicament 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 COVID-19 virus, influenza virus, RSV virus, and SARS-CoV.

[0031] A use of the above-mentioned CpG oligodeoxynucleotide in the preparation of a drug for preventing and treating non-respiratory tract infectious diseases, wherein the non-respiratory tract infectious diseases include at least one of HIV virus infection, HBV virus infection, and HCV virus infection.

[0032] A use of the above-mentioned CpG oligodeoxynucleotide in the preparation of a drug for regulating immune cell proliferation function or a drug for regulating immune cell cytokine release.

[0033] A use of the above-mentioned CpG oligodeoxynucleotide in the preparation of immune adjuvants.

[0034] A use of the above CpG oligodeoxynucleotide in preparing medicine for preventing and treating viral infection.

[0035] A use of the above-mentioned CpG oligodeoxynucleotide in the preparation of a drug for treating tumors, wherein the CpG oligodeoxynucleotide is the only anti-tumor drug component.

[0036] An anti-tumor composition comprises the above-mentioned CpG oligodeoxynucleotide and an anti-tumor drug, wherein the anti-tumor drug includes but is not limited to a PD-1 anti-tumor drug, a PDL-1 anti-tumor drug and an anti-tumor cell therapy, wherein the CpG oligodeoxynucleotide in the composition serves as a component having anti-tumor efficacy or an immune adjuvant.

[0037] A use of the above CpG oligodeoxynucleotide in preparing vaccines for human or animal use.

[0038] A use of the above-mentioned CpG oligodeoxynucleotide in treating central nervous system diseases, wherein the central nervous system diseases include but are not limited to Alzheimer's disease.

[0039] A use of the above-mentioned CpG oligodeoxynucleotide in the preparation of a drug for preventing and treating secondary infection after trauma.

[0040] A use of the above-mentioned CpG oligodeoxynucleotide in preparing a medicine for promoting wound healing.

[0041] A use of the above-mentioned CpG oligodeoxynucleotide in the preparation of a drug for treating allergic rhinitis and / or atopic dermatitis and / or asthma and / or chronic obstructive pulmonary disease and / or eosinophil-related diseases.

[0042] A medicine prepared by using the above-mentioned CpG oligodeoxynucleotide, wherein the dosage form of the medicine is injection, tablet, lyophilized agent, inhalant, nasal drops, nasal spray, rectal suppository, eye drops, smear, lotion, and gel.

[0043] A topical preparation for CpG oligodeoxynucleotides comprising the CpG oligodeoxynucleotides according to any one of claims 1 to 5 and excipients, wherein the excipients include glyceryl behenate, liquid paraffin, petrolatum, medium-chain triglycerides, cocoyl caprylate, glyceryl mono- and distearate, hydroxypropyl methylcellulose, carbomer, xanthan gum, sodium carboxymethylcellulose, poloxamer 407, polyethylene glycol cetearyl ether, poloxamer 188, oleoyl polyoxyethylene glyceride, polyethylene glycol-7 stearate, caprylic capric macrogol glyceride, sodium lauryl sulfate, propylene glycol monofatty acid ester, polyglyceryl oleate, propylene glycol laurate, propylene glycol, isopropyl myristate, and polyglyceryl oleate.

[0044] The above-mentioned CpG oligodeoxynucleotide adjuvant and a preparation in combination with other types of adjuvants are used for vaccines for human or animal use, including CpG oligodeoxynucleotides themselves or in combination with other types of adjuvants, wherein the other types of adjuvants are preferably aluminum hydroxide and aluminum phosphate.

[0045] The beneficial effects achieved by the present invention are:

[0046] Unlike the traditional A-type CpG fully palindromic design, the present invention provides a CpG ODN sequence with a completely new structure and sequence. By replacing nucleotides at both ends of the ODN, these sequences reveal the relationship and patterns between their drugability and activity. Compared to previously published sequences, these sequences possess unique primary and secondary structures, overcoming the problem of traditional A-type CpG aggregation and poor drugability, and exhibiting higher immunoactivity and drugability. The AM1012-05 sequence, in particular, forms a secondary structure that is more stable at high temperatures than similar products, and its aggregate controllability is superior to other sequences of the same type. During production, batch-to-batch stability is improved, resulting in better drugability. AM1012-05 can effectively penetrate the human respiratory epithelial mucosa (RPMI 2650 cells) and has demonstrated excellent immune activation activity in animals. It has excellent potential to block respiratory viral infection and transmission. By activating and regulating the immune system, it has anti-tumor effects, treats allergic rhinitis, asthma, chronic obstructive pulmonary disease, eosinophil-related diseases, atopic dermatitis, Alzheimer's disease, and has potential applications in the prevention and treatment of secondary infections after trauma and in promoting wound healing. It also has the potential to serve as a standalone vaccine adjuvant or as a component of an adjuvant system. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] In order to more clearly illustrate the technical solution of the present application, the following is a brief introduction to the drawings required for use in the embodiments. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0048] Figure 1 shows an evaluation of the IFN-γ stimulatory capacity of different CpG sequences on NK-92 cells; Panels A, B, C, D, and E demonstrate the IFN-γ stimulatory capacity of different CpG sequences on NK-92 cells under the same experimental system.

[0049] Figure 2 compares the stimulatory ability of different CpG sequences on SD rat splenocytes; Figure A shows that different CpG sequences stimulate rat splenocytes to secrete IFN-γ, Figure B shows that different CpG sequences stimulate rat splenocytes to secrete TNF-α, and Figure C shows that different CpG sequences stimulate rat splenocytes to secrete IL-6;

[0050] Figure 3 is an evaluation of the IFN-γ stimulating ability of AM1012-05 on NK-92 cells;

[0051] Figure 4 shows the evaluation of the stimulatory ability of AM1012-05 on BALB / c mouse splenocytes; wherein, Figure A shows the secretion of IL-6 by AM1012-05-stimulated BALB / c mouse splenocytes, Figure B shows the secretion of IFN-γ by AM1012-05-stimulated BALB / c mouse splenocytes, Figure C shows the secretion of TNF-α by AM1012-05-stimulated BALB / c mouse splenocytes, and Figure D shows the secretion of IFN-α by AM1012-05-stimulated BALB / c mouse splenocytes;

[0052] Figure 5 is an evaluation of the stimulatory ability of AM1012-05 on C57BL6 mouse splenocytes; wherein, Figure A shows that AM1012-05 stimulates C57BL6 mouse splenocytes to secrete IL-6, Figure B shows that AM1012-05 stimulates C57BL6 mouse splenocytes to secrete IFN-γ, Figure C shows that AM1012-05 stimulates C57BL6 mouse splenocytes to secrete TNF-α, and Figure D shows that AM1012-05 stimulates C57BL6 mouse splenocytes to secrete IFN-α;

[0053] Figure 6 shows an evaluation of the stimulatory ability of AM1012-05 on cotton rat splenocytes; Figure A shows that AM1012-05 increases the mRNA level of IFN-α in cotton rat splenocytes, Figure B shows that AM1012-05 increases the mRNA level of IFN-γ in cotton rat splenocytes, Figure C shows that AM1012-05 increases the mRNA level of TNF-α in cotton rat splenocytes, and Figure D shows that AM1012-05 increases the mRNA level of CXCL-10 in cotton rat splenocytes;

[0054] Figure 7 shows the evaluation of the stimulatory ability of AM1012-05 on rat splenocytes (mRNA level); Figure A shows that AM1012-05 increases the mRNA level of IFN-α in rat splenocytes, and Figure B shows that AM1012-05 increases the mRNA level of CXCL-10 in rat splenocytes;

[0055] Figure 8 is an evaluation of the stimulatory ability of AM1012-05 on rat splenocytes (protein level); wherein, Figure A shows the secretion of IFN-γ by rat splenocytes stimulated by AM1012-05, Figure B shows the secretion of IL-6 by rat splenocytes stimulated by AM1012-05, and Figure C shows the secretion of TNF-α by rat splenocytes stimulated by AM1012-05;

[0056] Figure 9 is an evaluation of the stimulatory ability of AM1012-05 on beagle dog PBMC; wherein, Figure A shows that AM1012-05 increases the mRNA level of IFN-α in beagle dog PBMC, Figure B shows that AM1012-05 increases the mRNA level of IFN-γ in beagle dog PBMC, Figure C shows that AM1012-05 increases the mRNA level of IFN-λ in beagle dog PBMC, Figure D shows that AM1012-05 increases the mRNA level of IL-6 in beagle dog PBMC, Figure E shows that AM1012-05 increases the mRNA level of CXCL-10 in beagle dog PBMC, and Figure F shows that AM1012-05 increases the mRNA level of TNF-α in beagle dog PBMC;

[0057] Figure 10 is an evaluation of the stimulatory ability of CpG ODN on human PBMC; wherein, Figure A shows AM1012-05 stimulating human PBMC to secrete IFN-α, Figure B shows AM1012-05 stimulating human PBMC to secrete IFN-γ, Figure C shows AM1012-05 stimulating human PBMC to secrete IFN-λ, Figure D shows AM1012-05 stimulating human PBMC to secrete TNF-α, Figure E shows AM1012-05 stimulating human PBMC to secrete IL-6, Figure F shows AM1012-05 stimulating human PBMC to secrete CXCL-10, and Figure G shows AM1012-05, CpG 2216, and CpG D19 stimulating human PBMC to secrete IFN-α;

[0058] FIG11 is the results of electrophoresis testing of serum stability of CpG ODN;

[0059] Figure 12 shows the aggregation state distribution of different CpG ODN aggregates; the left figure shows the electrophoresis detection of type A CpG aggregation state distribution (2 hours), and the right figure shows the electrophoresis detection of type A CpG aggregation state distribution (24 hours);

[0060] FIG13 shows the aggregation analysis of AM1012-05 and CpG 2216 solutions in static and vortexed states;

[0061] Figure 14 is a comparison of the secondary structures of different sequence samples at -20°C; Figure A is CpG 2216, Figure B is E41, Figure C is AM1012-03, and Figure D is AM1012-05;

[0062] Figure 15 is a comparison of the secondary structures of different sequence samples at 0°C; Figure A is CpG 2216, Figure B is E41, Figure C is AM1012-03, and Figure D is AM1012-05;

[0063] Figure 16 is a comparison of the secondary structures of different sequence samples at 4°C; Figure A is CpG 2216, Figure B is E41, Figure C is AM1012-03, and Figure D is AM1012-05;

[0064] Figure 17 is a comparison of the secondary structures of different sequence samples at 25°C; Figure A is CpG 2216, Figure B is E41, Figure C is AM1012-03, and Figure D is AM1012-05;

[0065] Figure 18 is a comparison of the secondary structures of different sequence samples at 37°C; Figure A is CpG 2216, Figure B is E41, Figure C is AM1012-03, and Figure D is AM1012-05;

[0066] Figure 19 is a comparison of the secondary structures of different sequence samples at 40°C; Figure A is CpG 2216, Figure B is E41, Figure C is AM1012-03, and Figure D is AM1012-05;

[0067] Figure 20 is a comparison of the secondary structures of different sequence samples at 50°C; Figure A is CpG 2216, Figure B is E41, Figure C is AM1012-03, and Figure D is AM1012-05;

[0068] Figure 21 is a comparison of the secondary structures of different sequence samples at 60°C; Figure A is CpG 2216, Figure B is E41, Figure C is AM1012-03, and Figure D is AM1012-05;

[0069] Figure 22 is a comparison of the secondary structures of different sequence samples at 70°C; Figure A is CpG 2216, Figure B is E41, Figure C is AM1012-03, and Figure D is AM1012-05;

[0070] Figure 23 is a comparison of the secondary structures of different sequence samples at 80°C; Figure A is CpG 2216, Figure B is E41, Figure C is AM1012-03, and Figure D is AM1012-05;

[0071] Figure 24 is a comparison of the secondary structures of different sequence samples at 90°C; Figure A is CpG 2216, Figure B is E41, Figure C is AM1012-03, and Figure D is AM1012-05;

[0072] FIG25 shows the effect of CpG ODN treated at different temperatures on IFN-γ secretion in rat splenocytes;

[0073] FIG26 is a graph showing particle size analysis of PLGA nanoformulations of CpG ODN;

[0074] FIG27 is a particle size analysis diagram of the preparation of chitosan nanoformulations of CpG ODN;

[0075] FIG28 is a particle size analysis diagram of the lipid nanoparticle formulation of CpG ODN;

[0076] FIG29 is a graph showing particle size analysis of PLGA nanoparticles prepared as an immune adjuvant for CpG ODN;

[0077] FIG30 is a graph evaluating the effects of formulation components on the IFN-γ stimulating ability of AM1012-05 on NK-92 cells;

[0078] Figure 31 shows the effect of AM1012-05 on the relative expression of immune-related mRNA in mouse lung and spleen tissues; wherein, Figure A shows that AM1012-05 increases the mRNA level of IFN-α in mouse lung tissue, Figure B shows that AM1012-05 increases the mRNA level of IFN-γ in mouse lung tissue, Figure C shows that AM1012-05 increases the mRNA level of CXCL-10 in mouse lung tissue, Figure D shows that AM1012-05 increases the mRNA level of IFN-α in mouse spleen tissue, Figure E shows that AM1012-05 increases the mRNA level of IFN-γ in mouse spleen tissue, and Figure F shows that AM1012-05 increases the mRNA level of CXCL-10 in mouse spleen tissue;

[0079] FIG32 shows the relative expression of CXCL-10 mRNA in the lungs of mice after intranasal administration of AM1012-05;

[0080] Figure 33 shows the secretion of cytokines in mouse lung tissue induced by AM1012-05 at different dosing frequencies; Figure A shows the secretion of CXCL-10 in mouse lung tissue induced by AM1012-05 at different dosing frequencies, and Figure B shows the secretion of IFN-γ in mouse lung tissue induced by AM1012-05 at different dosing frequencies;

[0081] Figure 34 shows the killing experiment of N87 cells by AM1012-05 activated human PBMC;

[0082] Figure 35 shows the killing experiment of MDA-MB-231 cells by AM1012-05 activated human PBMC;

[0083] Figure 36 shows an evaluation of the ability of AM1012-05 to alleviate allergic rhinitis in mice; Figure A shows that AM1012-05 reduces the number of sneezes in mice with allergic rhinitis, Figure B shows that AM1012-05 reduces the number of nose scratches in mice with allergic rhinitis, Figure C shows that AM1012-05 reduces runny nose in mice with allergic rhinitis, Figure D shows that AM1012-05 reduces the number of eosinophils in the nasal mucosa of mice with allergic rhinitis, Figure E shows that AM1012-05 reduces the expression of IL-4 in the serum of mice with allergic rhinitis, and Figure F shows that AM1012-05 increases the expression of IFN-γ in the serum of mice with allergic rhinitis;

[0084] FIG37 shows the results of immune cell analysis of nasal lavage fluid from mice with allergic rhinitis treated with AM1012-05;

[0085] Figure 38 is an evaluation of the in vitro anti-RSV activity of AM1012-05; Figure A shows the prevention of RSV proliferation in cells by AM1012-05 co-incubated with PBMC supernatant, and Figure B shows the elimination of RSV in host cells by AM1012-05;

[0086] Figure 39 is the evaluation of the anti-atopic dermatitis activity of AM1012-05 (IgE level);

[0087] Figure 40 shows the adjuvant application evaluation of AM1012-05: Figure A shows the IgG immune titer test results 7 days after the first vaccination; Figure B shows the IgG immune titer test results 7 days after the second vaccination; Figure C shows the IgG immune titer test results 7 days after the third vaccination; Figure D shows the IgG immune titer test results 21 days after the third vaccination; and Figure E shows the IgG immune titer test results 35 days after the third vaccination;

[0088] Figure 41 shows the adjuvant application evaluation of AM1012-05: Figure A shows the IgG1 immune titer test results 7 days after the first vaccination; Figure B shows the IgG1 immune titer test results 7 days after the second vaccination; Figure C shows the IgG1 immune titer test results 7 days after the third vaccination; Figure D shows the IgG1 immune titer test results 21 days after the third vaccination; and Figure E shows the IgG1 immune titer test results 35 days after the third vaccination;

[0089] Figure 42 is the adjuvant application evaluation of AM1012-05: Figure A shows the immune titer test results of IgG2a 7 days after the first immunization; Figure B shows the immune titer test results of IgG2a 7 days after the second immunization; Figure C shows the immune titer test results of IgG2a 7 days after the third immunization; Figure D shows the immune titer test results of IgG2a 21 days after the third immunization; and Figure E shows the immune titer test results of IgG2a 35 days after the third immunization. DETAILED DESCRIPTION

[0090] The specific implementation methods of this application are described in detail below. Obviously, the embodiments described are only some of the embodiments of this application, not all of them. Based on the embodiments in this application, all other embodiments obtained by ordinary technicians in this field without making any creative work are within the scope of protection of this application.

[0091] Example 1: Design and synthesis of CpG ODN molecules

[0092] The present invention designs a number of CpG ODN sequences (see Table 1) and chemically synthesizes them using solid-phase synthesis. The final product is obtained through aminolysis, purification, desalting, concentration, packaging, and freeze-drying. The specific steps are as follows:

[0093] (1) Solid-phase synthesis: Computer-controlled solid-phase synthesis consists of four repeated synthesis steps (detritylation, coupling, sulfurization, and capping) and the final step of dephosphorylation of the protective group of the skeleton;

[0094] (2) Aminolysis: After synthesis, the oligonucleotide is cut off from the support using ammonia or an organic amine. The oligonucleotide is dissolved in the solution and the universal linker attached to the first base at the 3' end is removed and the amino protecting group on the base is removed.

[0095] (3) Purification: The crude product obtained by aminolysis is purified and separated by ion exchange chromatography, reverse phase chromatography or electrophoresis to obtain a purified product;

[0096] (4) Desalination and concentration: Use ultrafiltration equipment to desalinate and concentrate the purified product;

[0097] (5) Packaging and freeze-drying: Freeze-drying is used to obtain a white, off-white to light yellow loose body.

[0098] Types of nucleic acid modifications include, but are not limited to, thiolation modifications.

[0099] Table 1 Sequences involved in the present invention

[0100] (Note: * indicates thio modification).

[0101] Example 2: Aggregation and activity analysis of different CpG ODNs

[0102] (I) Analysis of aggregation of different CpG ODNs:

[0103] The aggregation of some of the lyophilized powders of CpG ODN sequences obtained in Example 1 was analyzed using high performance liquid chromatography.

[0104] (II) Effects of different CpG ODN on IFN-α secretion were detected based on healthy human PBMC cells:

[0105] (1) Take a certain amount of 100 μM CpG ODN solution and prepare a certain concentration of stock solution with RPMI 1640 complete medium. Then dilute the CpG ODN sample solution with complete medium at a ratio of 4 to prepare test samples of different concentrations.

[0106] (2) Obtain an appropriate amount of frozen healthy human PBMC cells;

[0107] (3) According to 2×10 5 Cells / well were plated in 96-well plates, and samples of different concentrations were added to each well, and PBS blank control wells were set up at the same time;

[0108] (4) Place in a 37°C, 5% CO2 incubator for 16 h;

[0109] (5) Centrifugation to collect the supernatant;

[0110] (6) Detect IFN-α according to the ELISA assay instructions for IFN-α.

[0111] By analyzing the aggregation results (Tables 2-5), it can be found that: (1) By changing the number of G in sequences such as AM1012-05, CpG 2216, and CpG D19, when the number of G at the 5' end is 3-4, the optimal aggregation of the corresponding sequence can be obtained, that is, the content of the aggregate is the smallest; (2) Inserting 1-5 As between the core palindrome sequence of AM1012-05 and the poly-G at the 3' end can further reduce the aggregation content; (3) Inserting 1-10 Ts or Cs between the core palindrome sequence of the AM1012-05 sequence and the G at the 3' end can further reduce or increase the aggregation content to varying degrees; (4) Inserting 1-10 As, Ts, or Cs between the core palindrome sequence of sequences such as CpG 2216 and CpG D19 and the G at the 3' end can further reduce or increase the aggregation content to varying degrees.

[0112] The target cells of type A CpGs are pDCs, which account for approximately 0.2-0.4% of PBMCs. The IFN-α produced is primarily derived from pDCs. The activity evaluation test results of this example are shown in Tables 2-5. Table 2 shows that using the palindromic core sequence of type A CpG "CGATCGATCG" and gradually adding varying numbers of Gs before the C at its 5' end, the stimulatory activity on human PBMCs gradually increases with increasing numbers of Gs added, reaching peak activity when 3 or 4 Gs are added, then weakening with further increases in the number of Gs. Using the palindromic core sequence of CpG D19, "TGCATCGATGCA," and gradually adding varying numbers of Gs before the T at its 5' end, the stimulatory activity on human PBMCs gradually increases with increasing numbers of Gs added, reaching peak activity when 2 to 4 Gs are added, then weakening with further increases in the number of Gs added. When the palindrome sequence "GACGATCGTC" of CpG 2216 was used as the core sequence, different numbers of Gs were gradually added before the G at its 5' end. As the number of added Gs increased, its stimulatory activity on human PBMCs gradually increased, reaching the strongest activity when the number of added Gs was 3, and then weakened as the number of Gs continued to increase.

[0113] Table 3 summarizes the clustering and activity data from studies on the insertion of A atoms between the 3' end of the palindromic structure of type A CpG and the 5' end of the poly-G sequence. As can be seen, with "CGATCGATCG" as the core sequence of the palindromic structure of type A CpG, and A atoms gradually added thereafter (before the 5' end of the poly-G sequence), the stimulatory activity on human PBMCs gradually increased with the number of A atoms inserted, reaching a peak when two to four A atoms were inserted, and then weakening with further increases in the number of A atoms. With "TGCATCGATGCA" as the core sequence of the palindromic structure of type A CpG (before the 5' end of the poly-G sequence), and A atoms gradually added thereafter (before the 5' end of the poly-G sequence), the stimulatory activity on human PBMCs gradually increased with the number of A atoms inserted, reaching a peak when two A atoms were inserted, and then weakening with further increases in the number of A atoms. When "GACGATCGTC" of CpG 2216 is used as the palindromic core sequence of type A CpG, and A is gradually added thereafter (before the 5' end of the poly-G sequence), its stimulatory activity on human PBMC gradually increases with the increase in the number of inserted A, reaching the strongest when the number of inserted A is 2, and then weakens as the number of A continues to increase.

[0114] The aggregation and activity data of the study on the insertion of T between the 3' end of the palindromic structure of type A CpG and the 5' end of poly-G are summarized in Table 4. When the A added at the 3' end of the palindrome sequence "CGATCGATCG" was replaced with T, adding 2 Ts or 10 Ts to the end of the palindrome structure maintained good activity of type A CpG; when the A added at the 3' end of the palindrome sequence "TGCATCGATGCA" of CpG D19 was replaced with T, adding 2 Ts, 5 Ts or 10 Ts to the end of the palindrome structure maintained good activity of type A CpG, especially D19-11, which added 2 Ts on the basis of D19 and had better activity than D19; when the A added at the 3' end of the palindrome sequence "GACGATCGTC" of CpG 2216 was replaced with T, adding 2 Ts, 5 Ts or 10 Ts to the end of the palindrome structure maintained good activity, especially CpG 2216-11, which added 2 Ts on the basis of CpG 2216 and had better activity than CpG 2216.

[0115] Table 5 summarizes the clustering and activity data for studies inserting C between the 3' end of the palindrome structure of type A CpG and the 5' end of the poly-G. When the A added to the 3' end of the palindrome sequence "CGATCGATCG" was replaced with C, the activity of the sequence gradually decreased with the increase of C at the end of the palindrome. When the A added to the 3' end of the CpG D19 palindrome sequence "TGCATCGATGCA" was replaced with C, the activity of the sequence gradually decreased with the increase of C at the end of the palindrome. However, D19-14, which adds two Cs to D19, had better activity than D19. When the A added to the 3' end of the CpG2216 palindrome sequence "GACGATCGTC" was replaced with C, the activity of the sequence gradually decreased with the increase of C at the end of the palindrome.

[0116] This invention breaks through the traditional A-type CpG design concept and creatively establishes a certain spatial barrier between poly-G and palindrome, which increases the energy for forming G-tetrad, making the polymer relatively difficult to form or relatively loose after formation, successfully reducing the formation of A-type CpG aggregates in the solvent and improving the activity of the designed new A-type CpG. At the same time, in the exploration of the poly-G at the 5' end of traditional A-type CpG, it was found that when the number of G at the 5' end was 3 to 4, the corresponding sequence had the least aggregates and the best activity. This rule was verified on multiple CpG palindrome sequences. Based on these rules we discovered, among the newly designed sequences in this study, AM1012-05 is the best. The number of G at its 5' end is 4, and two As are inserted at the 3' end of the palindrome sequence. It has the characteristics of low aggregation and high activity at the same time, and has very high drugability.

[0117] This study breaks with traditional thinking by limiting the number of Gs at the 5' end of the palindromic sequence of type A CpG (3 to 4) and inserting an appropriate number of A and T (preferably 2) between the palindromic sequence and the poly-G at the 3' end, thereby changing the aggregation of type A CpG and improving the activity of type A CpG.

[0118] Table 2 Relationship between the number of 5' end G of type A CpG palindrome sequence and aggregation and activity:

[0119] Note: a, 0.2μM; b, 0.8μM

[0120] Table 3 Relationship between the number of A at the 3' end (before poly-G) of type A CpG palindrome sequences and aggregation and activity

[0121] Note: b, 0.8 μM

[0122] Table 4 Relationship between the number of T at the 3' end (before poly-G) of type A CpG palindrome sequence and aggregation and activity

[0123] Note: b, 0.8 μM

[0124] Table 5 Relationship between the number of C at the 3' end (before poly-G) of type A CpG palindrome sequence and aggregation and activity

[0125] Note: b, 0.8 μM

[0126] Example 3: Cytokine release after stimulation of NK-92 cells with different CpG ODNs

[0127] (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 expanded to the required cell number according to the cell instructions and resuspended in a certain amount of complete culture medium.

[0128] (2) Take 20 μL of cell suspension, mix it with trypan blue at a ratio of 1:1, and count it using a cell counter to determine cell viability and cell density;

[0129] (3) 2.5×10 5 Cells / well were plated in 96-well plates, and CpG samples (sequences in Table 1) of different concentrations were added to each well, with 3 replicates per group;

[0130] (4) Place in a 37°C, 5% CO2 incubator for 72 hours;

[0131] (5) After the cell culture was completed, the cells were centrifuged at 1200 rpm for 5 min, 100 μL of the cell-free supernatant was collected, and the secretion of IFN-γ in the supernatant was detected according to the instructions of the ELISA kit.

[0132] The experimental results are shown in Figure 1. The CpG sequences designed in Table 1 of the present invention can effectively stimulate the secretion of IFN-γ by NK cells, among which the NK stimulating effects of AM1012-03, AM1012-04, AM1012-05, AM1012-17, and AM1012-35 sequences are particularly significant. In addition, the stimulating effect of AM1012-05 is better than that of E41-1 and E41-2, and significantly better than E41-3. In addition, the experimental results of Figure 2D and Figure 2E show that increasing or decreasing the number of Gs on both sides of the core sequence of the present invention will change the immunostimulatory activity of the sequence. When the number of Gs on both sides of the core sequence reaches a specific number, the sequence can exert the best immune activation effect.

[0133] Example 4: Detection of the effect of CpG sequences on the secretion of IFN-γ, IL-6, and TNF-α based on SD rat spleen cells

[0134] (1) Take a certain amount of 100 μM AM1012-05, E41-1, E41-2, and E41-3 solution and prepare a certain concentration of stock solution with RPMI 1640 complete medium. Then dilute it with complete medium at a ratio of 4 to prepare test samples of different concentrations.

[0135] (2) Obtain an appropriate amount of fresh SD rat spleen cells;

[0136] (3) According to 1×10 6 Cells / well were plated in 96-well plates, and different concentrations of AM1012-05 samples were added to each well, with final concentrations of 3.2 μM, 0.8 μM, and 0.2 μM, respectively. Three replicates were added to each group, and a PBS blank control well was set up at the same time;

[0137] (4) Place in a 37°C, 5% CO2 incubator for 16 h or 72 h;

[0138] (5) Centrifugation to collect the supernatant;

[0139] (6) Detect IFN-γ, IL-6, and TNF-α according to the ELISA test instructions for different detection indicators.

[0140] The experimental results, shown in Figure 2, show that the designed AM1012-05, along with E41-1, E41-2, and E41-3, all significantly stimulated the secretion of IFN-γ, TNF-α, and IL-6 in SD rat splenocytes in a dose-dependent manner. However, AM1012-05 significantly outperformed E41-1, E41-2, and E41-3 in immune activation of SD rat splenocytes. Combined with Figure 2 (C), AM1012-05 demonstrated superior immune activation.

[0141] Example 5: Effect of AM1012-05 sequence on NK-92 cytokine release

[0142] (1) Prepare NK-92 cells to the desired cell mass as described in Example 3 and resuspend in a certain amount of complete culture medium;

[0143] (2) Take 20 μL of cell suspension, mix it with trypan blue at a ratio of 1:1, and count it using a cell counter to determine cell viability and cell density;

[0144] (3) AM1012-05 sample was prepared into a 100 μM solution in PBS;

[0145] (4) Take a certain amount of 100 μM AM1012-05 solution and prepare it into a certain concentration of stock solution with complete culture medium. Then dilute the AM1012-05 solution with complete culture medium at a ratio of 2 to prepare AM1012-05 test samples with different concentrations.

[0146] (5) 2.5×10 5 Cells / well were plated in 96-well plates, and different concentrations of AM1012-05 samples were added to each well. The final concentrations were 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. There were 3 replicates in each group, and PBS blank control wells were set up at the same time.

[0147] (6) Place in a 37°C, 5% CO2 incubator for 72 hours;

[0148] (7) After the cell culture was completed, centrifuge at 1200 rpm for 5 min, collect 100 μL of the cell-free supernatant, and detect the secretion of IFN-γ in the supernatant according to the instructions of the ELISA kit.

[0149] The experimental results, as shown in Figure 3, show that the AM1012-05 sequence effectively stimulates IFN-γ secretion in NK-92 cells in a dose-dependent manner. Since NK-92 cells express TLR9, this experiment also verified that the immune activation activity of AM1012-05 is stimulated by binding to TLR9.

[0150] Example 6: Detection of the effect of AM1012-05 on the secretion of IL-6, IFN-γ, TNF-α, IFN-α, etc. based on BALB / c mouse spleen cells

[0151] (1) Take a certain amount of 100 μM AM1012-05 solution and prepare it into a certain concentration of stock solution with RPMI 1640 complete medium. Then dilute the AM1012-05 solution with complete medium at a ratio of 2 to prepare AM1012-05 test samples of different concentrations.

[0152] (2) Obtain an appropriate amount of fresh BALB / c mouse spleen cells;

[0153] (3) According to 1×10 6 Cells / well were plated in 96-well plates, and AM1012-05 samples of different concentrations were added to each well. The final concentrations were 8 μM, 4 μM, 2 μM, 1 μM, 0.5 μM, 0.25 μM, 0.13 μM, 0.06 μM, and 0.03 μM, respectively. There were 3 replicates in each group, and PBS blank control wells were set up at the same time.

[0154] (4) Place in a 37°C, 5% CO2 incubator for 16 h or 72 h;

[0155] (5) Centrifugation to collect the supernatant;

[0156] (6) Detect IL-6, IFN-γ, TNF-α, and IFN-α according to the ELISA test instructions for different detection indicators.

[0157] The experimental results are shown in FIG4 , and the designed AM1012-05 sequence can effectively stimulate the secretion of cytokines in BALB / c mouse spleen cells in a dose-dependent manner.

[0158] Example 7: Detection of the effect of AM1012-05 on the secretion of IL-6, IFN-γ, TNF-α, IFN-α, etc. based on C57BL6 mouse spleen cells

[0159] (1) Take a certain amount of 100 μM AM1012-05 solution and prepare it into a certain concentration of stock solution with RPMI 1640 complete medium. Then dilute the AM1012-05 solution with complete medium at a ratio of 2 to prepare AM1012-05 test samples of different concentrations.

[0160] (2) Obtain an appropriate amount of fresh C57BL6 mouse spleen cells;

[0161] (3) According to 1×10 6Cells / well were plated in 96-well plates, and AM1012-05 samples of different concentrations were added to each well. The final concentrations were 8 μM, 4 μM, 2 μM, 1 μM, 0.5 μM, 0.25 μM, 0.13 μM, 0.06 μM, and 0.03 μM, respectively. There were 3 replicates in each group, and PBS blank control wells were set up at the same time.

[0162] (4) Place in a 37°C, 5% CO2 incubator for 16 h or 72 h;

[0163] (5) Centrifugation to collect the supernatant;

[0164] (6) Detect IL-6, INF-γ, TNF-α, and IFN-α according to the ELISA test instructions for different detection indicators.

[0165] The experimental results are shown in FIG5 . The designed AM1012-05 sequence can effectively stimulate the secretion of cytokines in C57BL6 mouse spleen cells in a dose-dependent manner.

[0166] Example 8: Detection of the effect of AM1012-05 on IFN-α, IFN-γ, TNF-α, and CXCL-10 mRNA transcription based on cotton rat splenocytes

[0167] (1) Take a certain amount of 100 μM AM1012-05 solution and prepare it into a certain concentration of stock solution with RPMI 1640 complete medium. Then dilute the AM1012-05 solution with complete medium at a ratio of 2 to prepare AM1012-05 test samples of different concentrations.

[0168] (2) Obtain an appropriate amount of frozen cotton rat spleen cells;

[0169] (3) According to 5×10 6 Cells / well were plated in 6-well plates, and different concentrations of AM1012-05 samples were added to each well, with final concentrations of 0.2 μM, 0.8 μM, and 3.2 μM, respectively. Each group had 3 replicates, and a PBS blank control well was set up at the same time;

[0170] (4) Place in a 37°C, 5% CO2 incubator for 3 h;

[0171] (5) Collect cells, extract total RNA from samples according to the instructions of the RNA total extraction kit, and measure the total RNA concentration using Nanodrop;

[0172] (6) Reverse transcribe RNA into cDNA according to the instructions of the reverse transcription kit;

[0173] (7) Analyze the expression levels of relevant cytokines in each sample according to the instructions of the RT-qPCR kit.

[0174] The experimental results, shown in Figure 6, demonstrate that the designed AM1012-05 sequence effectively stimulates the secretion of immune-related cytokines in cotton rat splenocytes in a dose-dependent manner. Cotton rats are a commonly used animal model for RSV infection, laying the foundation for evaluating the anti-RSV activity of AM1012-05 in cotton rats.

[0175] Example 9: Detection of the effect of AM1012-05 on IFN-α and CXCL-10 mRNA transcription based on SD rat splenocytes

[0176] (1) Take a certain amount of 100 μM AM1012-05 solution and prepare it into a certain concentration of stock solution with RPMI 1640 complete medium. Then dilute the AM1012-05 solution with complete medium at a ratio of 2 to prepare AM1012-05 test samples of different concentrations.

[0177] (2) Obtain an appropriate amount of fresh rat spleen cells;

[0178] (3) According to 5×10 6 Cells / well were plated in 6-well plates, and AM1012-05 samples of different concentrations were added to each well, with 3 replicates per group, and PBS blank control wells were set at the same time;

[0179] (4) Place in a 37°C, 5% CO2 incubator for 3 h;

[0180] (5) Collect cells, extract total RNA from samples according to the instructions of the RNA total extraction kit, and measure the total RNA concentration using Nanodrop;

[0181] (6) Reverse transcribe RNA into cDNA according to the instructions of the reverse transcription kit;

[0182] (7) Analyze the expression levels of relevant cytokines in each sample according to the instructions of the RT-qPCR kit.

[0183] The experimental results are shown in FIG7 . The designed AM1012-05 sequence can effectively stimulate the transcription of IFN-α and CXCL-10 mRNA in SD rat splenocytes in a dose-dependent manner.

[0184] Example 10: Detection of the effect of AM1012-05 on the secretion of IFN-γ, IL-6, and TNF-α based on SD rat spleen cells

[0185] (1) Take a certain amount of 100 μM AM1012-05 solution and prepare it into a certain concentration of stock solution with RPMI 1640 complete medium. Then dilute the AM1012-05 solution with complete medium at a ratio of 2 to prepare AM1012-05 test samples of different concentrations.

[0186] (2) Obtain an appropriate amount of fresh SD rat spleen cells;

[0187] (3) According to 1×10 6 Cells / well were plated in 96-well plates, and AM1012-05 samples of different concentrations were added to each well. The final concentrations were 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, respectively. There were 3 replicates in each group, and PBS blank control wells were set up at the same time;

[0188] (4) Place in a 37°C, 5% CO2 incubator for 16 h or 72 h;

[0189] (5) Centrifugation to collect the supernatant;

[0190] (6) Detect IFN-γ, IL-6, and TNF-α according to the ELISA test instructions for different detection indicators.

[0191] The experimental results, shown in Figure 8, demonstrate that the designed AM1012-05 sequence effectively stimulated the secretion of IFN-γ, IL-6, and TNF-α in SD rat splenocytes with a good dose-dependency. SD rats are a commonly used animal model for preclinical toxicological evaluation of drugs. Combined with the data from Example 9, these experimental results provide a foundation for conducting safety evaluation experiments for AM1012-05 using SD rats.

[0192] Example 11: Effect of AM1012-05 on the secretion of IFN-α, IFN-γ, IFN-λ, IL-6, TNF-α, and CXCL-10 based on beagle PBMC

[0193] (1) Take a certain amount of 100 μM AM1012-05 solution and prepare it into a certain concentration of stock solution with RPMI 1640 complete medium. Then dilute the AM1012-05 solution with complete medium at a ratio of 4 to prepare AM1012-05 test samples with different concentrations.

[0194] (2) Obtain an appropriate amount of fresh beagle PBMC;

[0195] (3) According to 5×10 6 Cells / well were plated in 6-well plates, and different concentrations of AM1012-05 samples were added to each well, with final concentrations of 0.2 μM, 0.8 μM, and 3.2 μM, respectively. Each group had 3 replicates, and a PBS blank control well was set up at the same time;

[0196] (4) Place in a 37°C, 5% CO2 incubator for 6 h;

[0197] (5) Collect cells, extract total RNA from samples according to the instructions of the RNA total extraction kit, and measure the total RNA concentration using Nanodrop;

[0198] (6) Reverse transcribe RNA into cDNA according to the instructions of the reverse transcription kit;

[0199] (7) Analyze the expression levels of relevant cytokines in each sample according to the instructions of the RT-qPCR kit.

[0200] The experimental results, shown in Figure 9, demonstrate that the designed AM1012-05 sequence effectively stimulates the secretion of immune-related cytokines in beagle PBMCs in a dose-dependent manner. Beagles are a commonly used animal model for preclinical toxicology evaluation, paving the way for conducting safety evaluation experiments for AM1012-05 in beagle dogs. Furthermore, the efficacy of AM1012-05 in beagle PBMCs suggests its potential as an adjuvant for pet vaccines or for anti-infective applications.

[0201] Example 12: Detection 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 based on healthy human PBMC cells

[0202] (1) Take a certain amount of 100 μM AM1012-05 and related sequence solution and prepare a certain concentration of stock solution with RPMI 1640 complete medium. Then dilute the AM1012-05 and related sequence solution with complete medium at a ratio of 4 to prepare test samples of different concentrations.

[0203] (2) Obtain an appropriate amount of frozen healthy human PBMC cells;

[0204] (3) According to 2×10 5 Cells / well were plated in 96-well plates, and samples of different concentrations were added to each well, with 3 replicates per group, and PBS blank control wells were set up at the same time;

[0205] (4) Place in a 37°C, 5% CO2 incubator for 16 h;

[0206] (5) Centrifugation to collect the supernatant;

[0207] (6) Detect cytokines according to the ELISA test instructions.

[0208] The experimental results are shown in Figure 10 . AM1012-05 can 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) from PBMCs in a dose-dependent manner. Furthermore, AM1012-05 and related sequences significantly outperformed CpG 2216 and D19 in activating IFN-α (Figure 10G). Among them, AM1012-05 has a significant effect in promoting IFN-α secretion, with an EC50 of 0.19μM and an optimal stimulation concentration of 0.5μM. The effects of 1μM AM1012-05 in promoting IFN-γ secretion and 0.25μM AM1012-05 in promoting IFN-λ are also very significant, which shows that AM1012-05 has a very strong antiviral potential; AM1012-05 can stimulate healthy human PBMC to secrete TNF-α, and has a good dose-dependence, but its ability to stimulate TNF-α is significantly lower than its ability to stimulate IFN-α (EC50 is 0.19 and 2.46uM, respectively), which indicates that when AM1012-05 can exert high antiviral activity (IFN-α, IFN-γ, IFN-λ) at lower doses, it will not trigger inflammatory responses represented by TNF-α and IL-6.

[0209] Example 13: Serum stability test of CpG ODN

[0210] This experiment used polyacrylamide gel (PAGE) electrophoresis to investigate the extent of sequence damage caused by fetal bovine serum (FBS). The specific steps were as follows: AM1012-05, AM1012-03, E41-2, and E41-3 were each prepared into a 25 OD / mL aqueous solution, 10 μL of 10% FBS was added, and the mixture was incubated in a 37°C constant temperature water bath for a certain period of time. After incubation, the extent of sequence damage was detected using polyacrylamide gel (PAGE) electrophoresis. As shown in Figure 11, AM1012-05 (1205) remained stable in the 25% fetal bovine serum solution, E41-3 was the most degraded (4103), AM1012-03 (1203) and E41-2 (4102) were moderately degraded, and AM1012-05 (1205) was the least degraded. Therefore, compared with E41-2 and E41-3, the AM1012-05 sample has better stability.

[0211] Example 14: Analysis of Aggregation State Distribution of Different CpG ODN Aggregates

[0212] 3 mg of AM1012-03, E41-2, E41-3, and AM1012-05 were added to 1 ml of deionized water to obtain a clear solution.

[0213] The four solutions were divided equally into two groups: one at room temperature, and the other heated in a 95°C water bath for 30 minutes, then rapidly cooled to below 4°C with ice water. Equal amounts of 800 mM sodium chloride solution were added to each of the eight solutions, exposing the samples to a 400 mM sodium ion environment. After incubation for 24 hours, the precipitate was separated by centrifugation. The precipitate was redissolved in water to prepare a clear solution. The aggregation distribution of each sample was analyzed by polyacrylamide gel (PAGE) electrophoresis.

[0214] The electrophoresis results are shown in Figure 12. M in the figure denotes 20, 40, and 80 base markers. Lanes 1, 2, 3, and 4 correspond to samples of AM1012-03, E41-2, E41-3, and AM1012-05 at room temperature, respectively. Lanes 5, 6, 7, and 8 correspond to samples of AM1012-03, E41-2, E41-3, and AM1012-05 heated in a 95°C water bath for 30 minutes and then rapidly cooled to below 4°C with ice water. The left figure shows the electrophoresis results after the precipitate was dissolved in water for 2 hours, and the right figure shows the electrophoresis results of the same sample solutions after 24 hours.

[0215] Literature reports indicate that polyG sequences at the 5' or 3' ends of type A CpGs can cause them to aggregate with the help of metal ions. This aggregation, in turn, can lead to precipitation. The degree of controllable aggregation is a key indicator for evaluating the druggability of type A CpGs (Kerkmann et al., J Biol Chem. 2005 Mar 4; 280(9):8086-93).

[0216] In this experiment, samples incubated in a 400 mM sodium ion environment all formed a precipitate. When the precipitate was resuspended in water, the high-polymeric forms of type A CpG gradually disaggregated in the absence of metal ions and became resolubilized. Oligomeric forms, such as dimers and tetramers, also have some solubility in water. Therefore, observing the resolubilized samples at different time points allows for optimal visualization of the distribution of (low) aggregation states of individual type A CpGs and their temporal trends.

[0217] For samples stored at room temperature, the redissolved solution of AM1012-03 was mainly composed of tetramers and monomers after 2 hours, and became scattered bands after 24 hours; the redissolved solution of E-41-2 was mainly composed of dimers after 2 hours, and still mainly dimers after 24 hours, but tetramers gradually appeared; the redissolved solution of E-41-3 showed dimers and polymers (larger than tetramers) after 2 hours, and scattered bands dominated by dimers appeared after 24 hours; in the redissolved solution of AM1012-05, most of it was in a monomer state after 2 hours and 24 hours, with only a small number of aggregates.

[0218] For samples heated in a 95°C water bath for 30 minutes and then rapidly cooled to below 4°C with ice water, the reconstituted solution of AM1012-03 was primarily composed of tetramers and monomers after 2 hours, with diffuse bands appearing after 24 hours. The reconstituted solution of E-41-2 was primarily composed of dimers after 2 hours, and still primarily dimers after 24 hours, but tetramers and monomers gradually emerged. The reconstituted solution of E-41-3 exhibited a collection of various aggregates after 2 hours, but after 24 hours, it became primarily composed of diffuse bands of monomers and dimers. In the reconstituted solution of AM1012-05, the majority of the sample remained in the monomeric state after 2 and 24 hours, with only a few aggregates.

[0219] The above results indicate that AM1012-05 disaggregates back to the monomeric state more readily than other A-type CpG monomers under the same conditions. Other A-type CpGs can transition between monomers, dimers, tetramers, or other multimers. Therefore, in practical formulation applications, AM1012-05 can be stably maintained in the monomeric state as long as the metal salt concentration is carefully controlled. Upon administration to the human body, under physiological salt concentrations, AM1012-05 can form the unique A-type CpG polymeric state and produce its pharmacological effects.

[0220] At the same time, CpG 2216 and AM1012-05 were each prepared into 0.5 mg / mL solutions using 10 mM phosphate buffer solution. After standing overnight, the particle size of CpG was measured using a Nicomp Z3000 laser particle size analyzer. After the measurement, the solution was vortexed for 3 minutes and the particle size was measured again. The results, as shown in Figure 13, show that the particle size distribution of CpG 2216 before vortexing was approximately 2 nm, 75 nm, and 250 nm, while after vortexing, the particle size of CpG 2216 was concentrated at approximately 280 nm. The particle size of AM1012-05 was approximately 30 nm in the static state, while after vortexing, the particle size of AM1012-05 was concentrated at approximately 2 nm.

[0221] Laser particle size analysis revealed the aggregation state of CpG in its natural state. In 10mM phosphate buffer, AM1012-05 exhibited loose aggregates at rest. Upon mechanical force, these aggregates disintegrated into monomers. However, CpG 2216, in 10mM phosphate buffer, exhibited dispersed complex polymers that could not be broken apart by mechanical force.

[0222] The results in Figure 13 show that CpG 2216 is a traditional A-type CpG. There are no additional deoxyribonucleotides between the 3' end of the palindrome and the 5' end of the poly-G, creating no steric barrier. This reduces the energy required to form G-tetrads, resulting in tightly bound aggregates that are difficult to break apart mechanically. AM1012-05, on the other hand, has two A's inserted between the 3' end of the palindrome and the 5' end of the poly-G, creating a steric barrier that increases the energy required to form G-tetrads, making aggregates relatively difficult to form or relatively loose after they do form. Consequently, under natural static conditions, AM1012-03 maintains a uniformly distributed equilibrium state of approximately 30 nanometers, significantly enhancing its druggability compared to traditional A-type CpG.

[0223] Example 15: Secondary structure simulation analysis and comparison of different sequences

[0224] The secondary structures of the sequences of CpG 2216, E41-1, AM1012-3, and AM1012-5 were 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 under different temperature conditions.

[0225] Schematic diagrams of the specific secondary structures are shown in Figures 14-24. Comparing the secondary structures of the four proteins at different temperatures reveals that CpG 2216 is relatively stable between 4°C and 37°C, but its secondary structure misfolds when the temperature rises above 40°C, affecting its biological activity. E41-1 is relatively stable between 0°C and 37°C, but experiences localized internal hydrogen bond breakage at -20°C, and secondary structure misfolding at temperatures above 40°C. Both high and low temperatures can affect its biological activity. AM1012-03 is also relatively stable between 0°C and 37°C, but experiences localized internal hydrogen bond breakage at 0°C, and secondary structure misfolding at temperatures above 40°C, further affecting its biological activity. AM1012-05 maintains a constant secondary structure between 0°C and 50°C, but only experiences localized internal hydrogen bond breakage at temperatures above 60°C. This indicates that the secondary structure of AM1012-05 sequence is more stable than that of CpG 2216, E41-1 and AM1012-03, which also structurally explains why AM1012-05 is more stable and active than CpG 2216, E41-1 and AM1012-03.

[0226] Example 16: Evaluation of the Stimulating Ability of CpG ODN with Different Aggregation Degrees on Rat Splenocytes

[0227] (1) Take a certain amount of 100 μM CpG ODN solution treated at different temperatures and prepare a certain concentration of stock solution with RPMI 1640 complete medium. Then dilute the CpG ODN solution with complete medium at a ratio of 4 to prepare test samples of different concentrations.

[0228] (2) Obtain an appropriate amount of fresh SD rat spleen cells;

[0229] (3) According to 1×10 6 Cells / well were plated in 96-well plates, and CpG ODN samples of different concentrations were added to each well, with final concentrations of 3.2 μM and 0.8 μM, respectively. Each group had 3 replicates, and a PBS blank control well was set up at the same time;

[0230] (4) Place in a 37°C, 5% CO2 incubator for 72 h;

[0231] (5) Centrifugation to collect the supernatant;

[0232] (6) Detect IFN-γ according to the ELISA assay instructions for rat IFN-γ.

[0233] The experimental results are shown in Figure 25. The designed AM1012-05 sequence can still effectively stimulate the secretion of IFN-γ in SD rat spleen cells after high temperature treatment, while the AM1012-03 sequence cannot effectively stimulate the secretion of IFN-γ in SD rat spleen cells after high temperature treatment. Combined with the sequence secondary structure prediction results of Example 15, the high temperature stability of AM1012-05 is much better than CpG 2216, E41, and AM1012-03. This experiment verifies that a stable secondary structure is a prerequisite for the activity of CpG.

[0234] Example 17: Changes in the stability of CpG ODN raw materials prepared by different processes

[0235] E41-2, E41-3, and AM1012-05 raw materials were prepared into 2 mg / mL stock solutions and subjected to different processing techniques: direct lyophilization and lyophilization followed by heating at 95°C. The powders obtained using different lyophilization techniques were dissolved and analyzed for aggregation and related substances using reversed-phase HPLC. The proportion of aggregates in E41-2, E41-3, and AM1012-05 prepared by different processes varied slightly. However, the impurity levels measured in the E41-2 and E41-3 powders prepared by different processes varied significantly. In contrast, the impurity levels of AM1012-05 prepared by different processes were essentially the same. These results demonstrate that AM1012-05 exhibits superior stability to E41-2 and E41-3, suggesting improved drugability.

[0236] Table 6 Stability analysis of CpG ODN raw materials prepared by different processes

[0237] Example 18: pH Screening of AM1012-05 Formulations

[0238] (1) Prepare sodium phosphate buffers of different pH values: pH 6.6, 7.0, 7.2, 7.4, 8.0, and 9.0;

[0239] (2) Dissolve AM1012-05 in the above buffer to prepare a 0.35 mg / ml solution;

[0240] (3) Dispense 1.5 mL of each of the above prescription solutions into 10 mL vials, stopper, cap, and store at 50°C in the dark.

[0241] (4) Samples were taken at 0, 5 and 10 days for content testing.

[0242] The experimental results are shown in Table 7. The results show that AM1012-05 is easily degraded in a slightly acidic environment. From the perspective of the formulation, it is stable in the pH range of 7.0-9.0.

[0243] Table 7 pH range screening results of samples

[0244] Example 19: Selection of AM1012-05 solution buffer

[0245] Phosphate and citrate buffer systems were screened based on the pH range and commonly used buffer systems of marketed small nucleic acid drugs. A 2.5 mg / ml concentration of AM1012-05 was selected as the experimental sample concentration, and an unbuffered aqueous solution of AM1012-05 was used as the control. The sample pH was controlled at 7.2. The sample was placed at 50°C for 10 days, and samples were taken at 0, 5, and 10 days to compare the stability of the sample in the two buffer solutions. The results showed that the purity change of the sample with added buffer was significantly reduced compared to the control. The various parameters of the samples in the two buffer systems showed consistent changes with prolonged storage time. The experimental results are shown in Table 8. The buffers of both buffer systems can meet the pH buffer requirements of the formulation.

[0246] Table 8 Buffer screening results

[0247] Example 20: Range Screening of Metal Ion Concentrations in CpG ODN

[0248] Prepare a 200 μl solution sample by mixing 5 mg of each of sodium acetate, sodium dihydrogen phosphate / disodium hydrogen phosphate, magnesium chloride, calcium chloride, potassium chloride, and sodium chloride with 0.25 mg of AM1012-05. Place the sample in a sealed, clean container at 40°C for 30 days, with samples collected on the 5th, 15th, and 30th days. Testing parameters include appearance, solubility of concern, impurities (related substances), and content.

[0249] Test results showed that sodium acetate, sodium dihydrogen phosphate / disodium hydrogen phosphate, magnesium chloride, calcium chloride, potassium chloride, and sodium chloride produced precipitation when mixed with AM1012-05 at the appropriate concentrations. Therefore, it is necessary to screen for a salt concentration suitable for CpG ODN formulations to ensure the clarity of the formulation solution.

[0250] AM1012-05 was selected and potassium chloride and sodium chloride solutions were added, respectively, to obtain a final concentration of 2 mg / ml AM1012-05 and a final concentration of 90 mM saline solution. After 24 hours, AM1012-05 remained clear in both saline solutions.

[0251] AM1012-05 was selected and magnesium chloride and calcium chloride solutions were added, respectively, to obtain a final concentration of 2 mg / ml AM1012-05 and a final concentration of 1.8 mM saline solution. After 24 hours, AM1012-05 remained clear in both saline solutions.

[0252] Phosphate buffer (composed of sodium dihydrogen phosphate and sodium hydrogen phosphate) concentrations of 5, 10, 20, and 50 mM were selected as experimental concentrations (sodium ion concentrations were approximately 9, 18, 36, and 90 mM, respectively). These concentrations were compared to isotonic (150 mM) PBS (sodium ion concentration was approximately 179 mM). AM1012-05 was tested at a concentration of 2.5 mg / ml, with a pH of 7.2. The samples were stored at 50°C for 10 days, with samples taken at 0, 5, and 10 days to test stability and determine the pH buffer concentration. The results are shown in Table 9. Compared to day 0, the clarity and properties of the samples stored at 50°C for 5 days showed no significant change, and purity showed no significant difference with increasing storage time. However, AM1012-05 in isotonic PBS exhibited significant turbidity. Therefore, the isotonic (150 mM) PBS salt concentration is not suitable for preparing AM1012-05 formulations. Considering pH buffering capacity and avoiding the risk of AM1012-05 precipitation, the sodium ion concentration in the solution formulation is selected to be between 0.1 and 90 mM. The total concentration of metal ions such as sodium, potassium, magnesium, calcium, zinc, and iron in the CpG ODN solution formulation is within the range of 0.1-90 mM.

[0253] Table 9 Salt buffer concentration screening

[0254] Example 21: Selection of isotonicity regulator for AM1012-05 solution

[0255] In solutions, sodium chloride is generally used to adjust isotonicity. However, because the formation of type A CpG aggregates is related to the concentration of metal ions in the solution, isotonic sodium chloride solutions can cause type A CpG to precipitate from the solution. Therefore, other excipients are needed as isotonicity regulators.

[0256] Excipients that can be used as isotonic regulators include glycine, trehalose, mannitol, sucrose, arginine, lysine, histidine, sorbitol, glucose, glycerol, propylene glycol, etc.

[0257] Each sample of the above excipients was prepared into a 200 μl solution containing 0.25 mg of AM1012-05 and 5 mg of excipients. The test sample was placed in a sealed clean container and stored at 40°C for 30 days, with samples taken on the 5th, 15th, and 30th days. Testing parameters included properties (appearance), solubility, impurities (related substances), and content. Test results showed that the main peak shape of the HPLC chromatogram of the solution after sorbitol and glucose were mixed with AM1012-05 was abnormal. Sorbitol is not compatible with glucose and AM1012-05, making it unsuitable as an isotonicity regulator in AM1012-05 preparations. Glycine, trehalose, mannitol, sucrose, arginine, lysine, histidine, glycerol, and propylene glycol are compatible with AM1012-05 and can be used as isotonicity regulators for AM1012-05.

[0258] AM1012-05 was prepared with three isotonicity regulators and phosphate buffer to a 0.35 mg / ml solution. The properties (appearance), clarity, pH, osmotic pressure, and purity were tested. The specific formulation is shown in Table 10:

[0259] Table 10AM1012-05 prescription preparation

[0260] The experimental results are shown in Table 11, which show that all three formulations of AM1012-05 can produce clear solutions, and the pH, osmotic pressure and purity all meet the formulation requirements.

[0261] Table 11 AM1012-05 three prescription test results

[0262] Example 22: Preparation of AM1012-05 formulation and freeze-dried formulation development

[0263] AM1012-05 was dissolved in deionized water, and mannitol (lyoprotectant) and pH adjuster (sodium hydrogen phosphate + citric acid) were added to prepare a solution containing 1 mg / mL AM1012-05, 5% mannitol, and 50 mM sodium hydrogen phosphate + citric acid.

[0264] Fill 0.2 mL of the above solution into a 2 mL vial, partially stopper the vial, and transfer to a lyophilizer for lyophilization at -30°C. After lyophilization, stopper and cap the sample to obtain a white lyophilized powder cake. This lyophilized preparation can be immediately reconstituted upon addition of 1 mL of water for injection. The lyophilized preparation of AM1012-05 allows AM1012-05 to be stored in a solid form, which improves the stability of the main drug and is an alternative to the liquid formulation of AM1012-05.

[0265] Example 23: Selection of other excipients for AM1012-05 solution

[0266] In the solution preparation of CpG ODN, in addition to the isotonicity regulator and pH buffer system, other excipients may be added to provide corresponding functions.

[0267] Other optional excipients include surfactants (such as Pluronic F127, Pluronic F68, Tween 20, Tween 80), preservatives (such as benzalkonium chloride), metal chelating agents (disodium edetate), viscosity regulators (hydroxypropyl methylcellulose, sodium carboxymethyl cellulose, sodium citrate), methyl beta-cyclodextrin, hydroxypropyl beta-cyclodextrin, antioxidants (ascorbic acid), etc.

[0268] In this test, 5 mg of each of Pluronic F127, Pluronic F68, Tween 20, Tween 80, and ascorbic acid were mixed with 0.25 mg of AM1012-05 to create a 200 μl solution. The samples were placed in a sealed, clean container and stored at 40°C for 30 days, with samples collected on the 5th, 15th, and 30th days. Testing criteria included appearance, solubility of concern, impurities (related substances), and content.

[0269] Simultaneously, prepare benzalkonium chloride, disodium edetate, sodium citrate, hypromellose, sodium carboxymethylcellulose, methyl-β-cyclodextrin, and hydroxypropyl-β-cyclodextrin according to the concentrations listed in Table 12. Add AM1012-05 to a 0.4 mg / mL solution and adjust the pH to 7.0-7.5. Place the test sample in a sealed, clean container at 40°C for 30 days, with samples collected on the 5th, 15th, and 30th days. Test parameters include: appearance, solubility, impurities (related substances), and content.

[0270] Table 12 Excipients

[0271] Test results indicate that mixing ascorbic acid with AM1012-05 causes degradation of AM1012-05, making it unsuitable for use in AM1012-05 formulations. HPLC analysis of a mixture of hydroxypropyl beta-cyclodextrin and AM1012-05 revealed an abnormal peak shape for the corresponding main peak of AM1012-05, making it unsuitable for use in AM1012-05 formulations.

[0272] Pluronic F127, Pluronic F68, Tween 20, Tween 80, benzalkonium chloride, disodium edetate, sodium citrate, hypromellose, sodium carboxymethyl cellulose, and methyl-beta-cyclodextrin are compatible with AM1012-05 and can be used as excipients in AM1012-05-related preparations.

[0273] Example 24: Preparation of PLGA Nanoformulation of CpG ODN

[0274] (1) Dissolve 100 mg of PLGA in 1.25 mL of dichloromethane (oil phase).

[0275] (2) Prepare a 12 mg / ml AM1012-05, 1% PVA solution. (Aqueous phase 1)

[0276] (3) Prepare 4 ml of 2.5% PVA solution. (Aqueous phase 2)

[0277] (4) The oil phase and the aqueous phase 1 are homogenously mixed to form colostrum, and then the colostrum is added to the aqueous phase 2 and homogenized using a homogenizer to form a double emulsion.

[0278] (5) The formed emulsion was slowly added to a 1% PVA aqueous solution (external aqueous phase) and stirred for 4 h to allow the dichloromethane to evaporate.

[0279] (6) Centrifuge the solution from step 5 at 500 g for five minutes to remove large microspheres and collect the supernatant.

[0280] (7) Centrifuge the supernatant from step 6 at 10,000 g for 10 minutes, collect the solid, and wash it three times with deionized water or ultrapure water to obtain the PLGA microsphere product.

[0281] The obtained PLGA microsphere product was tested by Nicomp Z3000 laser particle size analyzer, and the particle size could be controlled within 500 nanometers (as shown in Figure 26).

[0282] Example 25: Preparation of Chitosan Nanoformulation of CpG ODN

[0283] (1) Prepare 0.5 mg / ml chitosan (molecular weight 200,000) solution and adjust the pH to 5.6

[0284] (2) Prepare 50 μg / ml AM1012-05 solution

[0285] (3) 10 ml of AM1012-05 solution was added dropwise to 10 ml of chitosan solution and stirred for 1 minute.

[0286] The AM1012-05 chitosan nanoparticles prepared in this experiment were tested by a laser particle size analyzer and a dynamic light scattering particle size analyzer, and the particle size can be controlled to be around 150 nanometers (as shown in Figure 27).

[0287] Example 26: Preparation of CpG ODN lipid nanoparticle formulation

[0288] CpG ODN lipid nanoparticles (LNPs) are synthesized by mixing different types of lipids in a specific ratio to form CpG-encapsulated nanoparticles. Lipids include ionizable cationic liposomes, neutral phospholipids, sterol lipids, and PEGylated phospholipids. The specific preparation process is as follows:

[0289] (1) SM-102, DSPC, cholesterol, and DMG-PEG (2000) were dissolved in ethanol at a ratio of 50:10:38.5:1.5.

[0290] (2) Take 0.25 ml of the above lipid solution, add 0.75 ml of 80 μg / ml AM1012-05, 50 mM citrate buffer solution, vortex mix, and transfer to a hand-push liposome extruder.

[0291] (3) Mix by rapid pipetting and squeezing through an extruder. Let the mixture stand for 10 minutes.

[0292] (4) Add the mixture to an equal volume of 50 mM citrate buffer solution in a 200 KD dialysis bag, seal it, and place it in 50 ml of PBS solution for dialysis.

[0293] The AM1012-05 lipid nanoparticles prepared in this experiment were tested by a Nicomp Z3000 laser particle size analyzer, and the particle size can be controlled to be around 100 nanometers (as shown in FIG28 ).

[0294] Example 27: Preparation of CpG ODN Preparation of PLGA Nanoparticles as an Immune Adjuvant

[0295] (1) Dissolve 100 mg of PLGA in 1.25 mL of dichloromethane (oil phase).

[0296] (2) Add AM1012-05 and antigen protein (S1 protein) to a 1% PVA solution to prepare a solution containing 10 mg / ml AM1012-05 and 10 mg / ml S1 protein. (Aqueous Phase 1)

[0297] (3) Prepare 4 ml of 2.5% PVA solution. (Aqueous phase 2)

[0298] (4) The oil phase and the aqueous phase 1 are homogenously mixed to form colostrum, and then the colostrum is added to the aqueous phase 2 and homogenized using a homogenizer to form a double emulsion.

[0299] (5) The formed emulsion was slowly added to a 1% PVA aqueous solution (external aqueous phase) and stirred for 4 h to allow the dichloromethane to evaporate.

[0300] (6) Centrifuge the solution from step 5 at 500 g for five minutes to remove large microspheres and collect the supernatant.

[0301] (7) Centrifuge the supernatant from step 6 at 10,000 × g for 10 minutes to collect the solids. Wash three times with deionized water or ultrapure water to obtain the PLGA microspheres.

[0302] The PLGA microsphere vaccine product encapsulating AM1012-05 and antigen protein obtained in this experiment was tested by a Nicomp Z3000 laser particle size analyzer, and the particle size could be controlled within 500 nanometers (as shown in Figure 29).

[0303] Example 28: Compatibility Study of Excipients in External Use Formulations of CpG ODN

[0304] Excipients that can be used in topical preparations are categorized into oil phase, thickeners, gelling agents, emulsifiers, solvents, and penetration enhancers. The oil phase includes glyceryl behenate, liquid paraffin, petrolatum, medium chain triglycerides and cocoyl caprylate; the thickener includes glyceryl monostearate, glyceryl mono-distearate, and cetearyl alcohol; the gelling agent includes hydroxypropyl methylcellulose, xanthan gum, carbomer, sodium carboxymethylcellulose, and poloxamer 407; the emulsifier includes polyoxyl (40) stearate, polyethylene glycol cetearyl alcohol ether, Tween 60, poloxamer 188, oleoyl polyoxyethylene glyceride, polyethylene glycol-7 stearate, caprylic capric macrogol glyceride, sodium lauryl sulfate, propylene glycol monofatty acid ester, polyglyceryl oleate, and propylene glycol laurate; the solvent includes glyceryl triacetate and propylene glycol; the penetration enhancer includes isopropyl myristate, propylene glycol, caprylic capric macrogol glyceride, polyglyceryl oleate, and propylene glycol laurate.

[0305] After preparing the gel excipients into an aqueous solution of appropriate concentration, mix with AM1012-05 to create a 3 mg / g gel solution. Prepare a 3 mg / g mixture of the other excipients and the API in a one-to-one ratio. Place the sample in a 40°C incubator and collect samples for assay and related substances after 5, 15, and 30 days. The results showed that the excipients compatible with AM1012-05 were: oil phase including glyceryl behenate, liquid paraffin, petrolatum, medium chain triglycerides, cocoyl caprylate and caprylate; thickeners including glyceryl mono- and distearate; gelling agents including hypromellose, carbomer, xanthan gum, sodium carboxymethyl cellulose, and poloxamer 407; emulsifiers including macrogol cetearyl alcohol ether, poloxamer 188, oleoyl polyoxyethylene glyceride, macrogol-7 stearate, caprylic capric macrogol glyceride, sodium lauryl sulfate, propylene glycol monofatty acid ester, polyglyceryl oleate, and propylene glycol laurate; solvents including propylene glycol; penetration enhancers including isopropyl myristate, propylene glycol, and polyglyceryl oleate.

[0306] Example 29: Preparation of CpG ODN External Ointment

[0307] Weigh AM1012-05 and dissolve it in deionized water to prepare the API solution. Weigh appropriate amounts of petrolatum and liquid paraffin and heat in an 80°C water bath until melted. Add the API solution to the molten oil phase and homogenize at 70-80°C for 10 minutes. Cool to 65-70°C and aliquot. The final formulation contains 1 mg / g of AM1012-05. The ratio of petrolatum to liquid paraffin is determined based on sensory evaluation; the petrolatum ratio can be selected between 100% and 50%.

[0308] Example 30: Preparation of CpG ODN external gel preparation

[0309] Prepare a 2% Carbomer 974 aqueous solution, a 10% triethanolamine solution, and an AM1012-05 aqueous solution. Dilute the 2% Carbomer 974 aqueous solution to 0.7% and adjust the pH to 7.0 with 10% triethanolamine. Dissolve phenoxyethanol in propylene glycol and add to the gel, stirring thoroughly. Add the API aqueous solution and stir thoroughly. Add 10% triethanolamine dropwise to adjust the pH to the appropriate level, bring to volume, stir thoroughly, and dispense.

[0310] The concentration and type of carbomer used to prepare the gel can be adjusted according to the required viscosity, such as carbomer 971, 980, and 981. Neutralizers, in addition to triethanolamine solution, can also include sodium hydroxide, aminomethyl propanol, tromethamine, and tetra-2-hydroxypropyl ethylenediamine. Preservatives, in addition to phenoxyethanol, can also include parabens and cresols.

[0311] Example 31: Preparation of CpG ODN topical emulsion

[0312] (1) Oil phase: Place 15 g of petrolatum, 6 g of liquid paraffin, and 7.2 g of glyceryl mono- and distearate in a container, heat to 70°C, and stir to melt. Maintain the molten oil phase at 70°C with low-speed stirring.

[0313] (2) Water phase: Heat 59.5 g of deionized water to 70° C. in a container, add 1.8 g of polyethylene glycol cetearyl alcohol ether and dissolve.

[0314] (3) Emulsion preparation: Transfer the molten oil phase in step 1 to the water phase container at 70°C while mixing at a speed of 10 rpm.

[0315] (4) After the transfer is completed, start the homogenizer and homogenize for 10 minutes, controlling the temperature at 65°C to 70°C.

[0316] (5) Stop the homogenizer, set the stirrer to 50°C, and cool the emulsion to 50°C.

[0317] (6) Drug solution: 30 mg of AM1012-05 was dissolved in 10.5 g of deionized water, and the temperature was maintained at 50°C.

[0318] (7) The drug solution in step 6 was transferred to the emulsion in step 5 and stirred at 50°C.

[0319] (8) Start the homogenizer, mix and homogenize for 10 minutes, and at the same time add 10% sodium hydroxide solution to adjust the pH to 7.5.

[0320] (9) When the emulsion cooled to 25°C, the prepared emulsion was divided into several portions, and finally 100 g of an emulsion containing 0.3 mg / g of AM1012-05 was obtained.

[0321] The oil phase, thickener, and emulsifier used in this emulsion can be replaced by excipients that have passed the excipient compatibility test. A penetration enhancer excipient that has passed the excipient compatibility test can also be added to the emulsion.

[0322] Example 32: Preparation of CpG-adjuvanted vaccines

[0323] In this example, the S1 protein of the new coronavirus was used as a model antigen and a CpG-adjuvanted vaccine was prepared.

[0324] (1) Under sterile conditions, take 50 μL of SARS-CoV-2 S1 protein (2 mg / ml PBS solution) and dilute to 250 μL with PBS;

[0325] (2) Prepare a 2 mg / mL solution of AM1012-05 or CpG 1018;

[0326] (3) Add 250 μL of the solution from step (2) to the solution from step (1) and mix well.

[0327] Example 33: Preparation of vaccine with adjuvant system (aluminum adjuvant + CpG)

[0328] In this example, the S1 protein of the new coronavirus was used as a model antigen to prepare a vaccine with an adjuvant system (aluminum adjuvant + CpG).

[0329] Preparation of vaccines with aluminum hydroxide + CpG as adjuvant:

[0330] (1) Take 50 μL of SARS-CoV-2 S1 protein (2 mg / ml PBS solution);

[0331] (2) Add 50 μL of aluminum hydroxide gel adjuvant (2%) to the solution in step (1);

[0332] (3) Add 350 μL of PBS solution to the solution in step (2) and mix well;

[0333] (4) Take 50 μL of 2 mg / mL AM1012-05 solution and add it to the above solution. Mix well to allow the adjuvant to effectively adsorb the antigen.

[0334] Preparation of vaccines with aluminum phosphate + CpG as adjuvant:

[0335] (1) Take 50 μL of SARS-CoV-2 S1 protein (2 mg / ml PBS solution);

[0336] (2) Add 100 μL of aluminum phosphate gel adjuvant (0.5%) to the solution in step (1);

[0337] (3) Add 300 μL of PBS solution to the solution in step (2) and mix well;

[0338] (4) Take 50 μL of 2 mg / mL AM1012-05 solution and add it to the above solution. Mix well to allow the adjuvant to effectively adsorb the antigen.

[0339] Example 34: Effect of AM1012-05 Solution on IFN-γ Release from NK-92 Cells

[0340] (1) Prepare NK-92 cells to the desired cell mass as described in Example 3 and resuspend in a certain amount of complete culture medium;

[0341] (2) Take 20 μL of cell suspension, mix it with trypan blue at a ratio of 1:1, and count it using a cell counter to determine cell viability and cell density;

[0342] (3) Take a certain amount of AM1012-05 solution sample, prepare it into a stock solution with complete culture medium, and then dilute the AM1012-05 solution with complete culture medium at a ratio of 4 to prepare AM1012-05 test samples of different concentrations;

[0343] (4) 2.5×10 5 Cells / well were plated in 96-well plates, and different concentrations of AM1012-05 samples were added to each well, with final concentrations of 3.2 μM, 0.8 μM, and 0.2 μM, respectively. Three replicates were added to each group, and a PBS blank control well was set up at the same time;

[0344] (5) Place in a 37°C, 5% CO2 incubator for 72 hours;

[0345] (6) After the cell culture was completed, the cells were centrifuged at 1200 rpm for 5 min, 100 μL of the cell-free supernatant was collected, and the secretion of IFN-γ in the supernatant was detected according to the instructions of the ELISA kit;

[0346] The experimental results are shown in Figure 30. The solution preparation sample of AM1012-05 has a stimulating effect on the secretion of IFN-γ by NK cells that is equivalent to that of AM1012-05, and the blank preparation sample has no stimulating effect on NK-92 cells. This indicates that the solution preparation formula used in the present invention has a good match with AM1012-05 and will not affect the immune cell activation function of AM1012-05.

[0347] Example 35: Effects of AM1012-05 on the immune system of C57BL6 mice after a single nasal administration

[0348] The immune-stimulating effect of AM1012-05 on the respiratory tract was evaluated in C57BL6 mice via intranasal administration. The mice were divided into two groups: the treatment group received 100 μg of AM1012-05 (25 μL / nostril, dissolved in PBS), while the negative control group received 50 μL of PBS (25 μL / nostril). The experiment was terminated 24 hours after administration, and samples (lung and spleen) were collected for analysis of changes in various immune markers using RT-qPCR.

[0349] The experimental results are shown in Figure 31. It can be seen from the figure that AM1012-05 can effectively stimulate the expression of IFN-α, IFN-γ and CXCL-10 in the lungs and spleens of mice. Moreover, the degree of cytokine activation of the spleen by AM1012-05 is far less significant than that of the lungs (IFN-α and IFN-γ are approximately 4% of those in the lungs; CXCL-10 is approximately 1.5% of those in the lungs). This may be because this experiment was administered by nasal drops, and only a very small amount of AM1012-05 entered the spleen cells through the blood circulation. This result indirectly reflects that the systemic bioavailability of AM1012-05 is very low after nasal administration. At the same time, the dosage of 100 μg / mouse did not produce obvious toxic side effects on mice.

[0350] Example 36: Observation of respiratory immune indicators in mice after nasal administration of different doses of AM1012-05

[0351] In C57BL6 mice, the immune activation capacity of AM1012-5 at different doses and time points was evaluated by nasal administration. Each mouse was given 10 μg, 50 μg, and 100 μg of AM1012-05 (25 μL / nostril, PBS-dissolved drug) in three dosing groups, 50 μL of PBS (25 μL / nostril) in the negative control group, and 50 μg of CpG 2216 (25 μL / nostril) in the positive control group. The experiment was terminated before, 3 hours after, 12 hours after, 24 hours after, and 48 hours after administration. Lung samples were collected and analyzed for changes in CXCL-10 mRNA levels by RT-qPCR.

[0352] The experimental results are shown in Figure 32. As can be seen from the figure, AM1012-05 can effectively stimulate the expression of CXCL-10 in the mouse lungs. Furthermore, a dose of 100 μg / mouse showed good immune stimulatory activity, with CXCL-10 mRNA transcription reaching its highest level 24 hours after administration. Furthermore, compared to the CpG 2216 sequence (CpG 2216 is a type A CpG commonly used in the literature), AM1012-05 demonstrated a superior immune activation effect.

[0353] Example 37: Observation of respiratory immune indicators in mice after nasal administration of AM1012-05 (different dosing frequencies)

[0354] On C57BL6 mice, the immune activation ability of AM1012-05 under different administration rates was assessed by nasal administration. The animal operation and biological sample detection method of this test are identical with Example 36, and the test result is as shown in Figure 33 (the numbers in the horizontal axis brackets represent each administration time in the figure, and the 0th day is the experiment termination time). As can be seen from the figure, AM1012-05 can effectively stimulate the expression of CXCL-10 and IFN-γ in mouse lungs. And different stimulation effects are shown under different administration rates. With the increase of administration number of times, the mRNA of cytokine has a certain improvement. Meanwhile, each administration interval also can significantly affect the immune activation effect, and when each administration interval is 12h, stimulation effect is the most remarkable.

[0355] Example 38: Evaluation of in vitro anti-tumor activity of AM1012-05

[0356] (1) Cultivate and expand MDA-MB-231 and N87 cells, harvest the cells, and resuspend them in 2 mL of complete culture medium;

[0357] (2) Take 10 μL of cell suspension, mix it with trypan blue at a ratio of 1:1, and count it using a cell counter. The cell viability is 99.7% for MDA-MB-231, 100% for N87, and 99.2% for PBMC. The cell density is 1.49×10 6 cells / mL; N87:1.12×10 7 cells / mL; human PBMC: 5.17×10 6 cells / mL;

[0358] (3) Resuspend the cells in culture medium and plate 2000 cells / well for MDA-MB-231 cells and 4000 cells / well for N87 cells in 96-well plates;

[0359] (4) After the cells are fully adhered, PBMC cells are plated at an effector-target ratio of 10:1;

[0360] (5) The 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, with six replicate wells for each concentration;

[0361] (6) Continue culturing for 5 days and then perform CCK8 detection analysis.

[0362] The experimental results are shown in Figures 34 and 35. It can be seen from the figures that AM1012-05 can effectively stimulate human PBMC to kill tumor cells in a dose-dependent manner. Based on the fact that TLR-9 mainly exists in human pDC and B cells and type A CPG mainly stimulates pDC, this result shows that AM1012-05 can enhance the activity of NK cells and CD8 + The ability of T cells to kill tumor cells.

[0363] Example 39: Evaluation of the Anti-tumor Activity of AM1012-05 Combined with CAR-T Therapy

[0364] A specific ratio of CAR-T cells and tumor cells was co-cultured, and a specific concentration of AM1012-05 was added. The cells were incubated for 3-5 days, and then the medium was changed to remove free CAR-T cells and tumor cell debris. A specific amount of CCK8 reagent was added, and the activity of the remaining tumor cells was analyzed. The tumor cell killing rate was calculated for each group, using the untreated group and the CAR-T group as controls. The experimental results showed that AM1012-05 effectively enhanced the killing of tumor cells by CAR-T cells in a dose-dependent manner, demonstrating the synergistic effect of AM1012-05 on CAR-T therapy.

[0365] Example 40: Evaluation of the anti-allergic rhinitis activity of AM1012-05

[0366] (1) Basal sensitization of BALB / c mice with ovalbumin (OVA): 25 μg OVA and 2 mg Al(OH)3 dissolved in 100 μL saline were intraperitoneally injected into the mice on Day 0, Day 7, and Day 14, respectively;

[0367] (2) Nasal cavity challenge: Each mouse was nasally instilled with 20 μL of normal saline containing 200 μg of OVA on Day 21, Day 22, Day 23, Day 28, Day 29, and Day 30;

[0368] (3) 50 μg of AM1012-05 was administered intranasally 1 hour after each challenge;

[0369] (4) From the beginning of the experiment, the clinical status of the animals was observed daily until euthanasia, including mental state, activity level, diet, drinking water, etc. (After the challenge, each mouse was observed for 10 minutes, and the number of times the nose was scratched was recorded). At the same time, nasal mucosa, nasal lavage fluid, and serum were collected for analysis of eosinophils, immune cells in nasal lavage fluid, and immune indicators (IFN-γ, IL-4), respectively.

[0370] The experimental results are shown in Figures 36 and 37. Compared with the model group, AM1012-05 administration can effectively reduce the number of sneezes in allergic rhinitis mice (A in Figure 36), reduce the number of nose scratching (B in Figure 36), inhibit the runny nose of allergic rhinitis mice (C in Figure 36), and reduce the enrichment of eosinophils in the nasal mucosa (D in Figure 36). It can also effectively reduce the IL-4 content in the serum of allergic rhinitis mice (E in Figure 36) and increase the expression of IFN-γ (F in Figure 36). At the same time, AM1012-05 can reduce the infiltration of neutrophils (A in Figure 37) and basophils (B in Figure 37) in the nasal mucosa. These results indicate that AM1012-05 can effectively bias the body's immune response toward the Th1 type, inhibiting Th2 and Th17 immune responses (a decrease in eosinophils and basophils is a manifestation of a decrease in Th2 immune response, and a decrease in neutrophils is a manifestation of a decrease in Th17 immune response), thereby achieving the purpose of treating allergic rhinitis.

[0371] In addition, this experiment verified in mice that IAMA-001 has the function of reducing the number of eosinophils, and has the potential to be used in the treatment of eosinophil-related diseases.

[0372] Example 41: Evaluation of the Anti-Respiratory Syncytial Virus Infection Activity of AM1012-05

[0373] Experiment 1: Vero-E6 cells were seeded in 24-well plates at a cell density of 2×10 5 cells / mL and cultured for 12-16 hours until confluence reached 80%. Then, 1000 μL of the supernatant from the 1x and 5x dilutions of AM1012-05 co-incubated with human PBMCs was added to Vero-E6 cells and incubated for 24 hours. The cells were then infected with respiratory syncytial virus at an MOI of 0.1 for 48 hours. Samples were then collected and viral replication in each well was determined.

[0374] Experiment 2: Vero-E6 cells were seeded in 24-well plates at a cell density of 2×10 5 cells / mL, and cultured for 12-16 hours until the confluence reached 80%. Then AM1012-05 (final concentration: 1 μM), PBMC (2×10 6 Cells / well) were added to a 24-well plate and infected with respiratory syncytial virus at an MOI of 0.1 for 48 hours. After that, samples were collected and the viral replication in each well was measured.

[0375] The results of the two experiments are shown in Figure 38. The supernatant of PBMC co-incubation with AM1012-05 can effectively prevent the proliferation of RSV in cells (Figure A), and AM1012-05 can significantly eliminate RSV in host cells in the presence of PBMC (Figure B).

[0376] Example 42: Evaluation of the anti-atopic dermatitis activity of AM1012-05

[0377] (1) BALB / c cells were sensitized by injection with 0.5 mL of ovalbumin (OVA) solution (containing 5% aluminum hydroxide) on Day 0, Day 7, and Day 14 of the experiment.

[0378] (2) Preparation for drug administration: The animals were anesthetized and the hair on the back was shaved and removed to an area of ​​3*3 cm on Day 20.

[0379] (3) Local stimulation and drug administration: The model group used a brush to repeatedly stimulate the back skin to produce slight cracks, time: Day 21; PBS group: 50uL PBS was applied to the gauze of a 2*2cm dressing, applied to the back skin and fixed with tape, and continued for 24 hours, changed every day, and continued for one week (Day 21-27). After a week's interval, the same treatment was carried out for one week (Day 35-41); Other groups: 50μL of 1000μg / mL OVA solution was mixed with the test article or solvent, applied to the gauze of a 2*2cm dressing, applied to the back skin and fixed with tape, and continued for 24 hours, changed every day, and continued for one week (Day 21-27). After a week's interval, the same treatment was carried out (Day 35-41). On Day 28-34, only the test article or solvent was applied for drug administration.

[0380] (4) On the 35th day of the experiment, the serum of mice was collected for IgE detection.

[0381] The experimental results are shown in Figure 39. Compared to the OVA model group, AM1012-05 administration effectively reduced serum IgE levels in OVA-sensitized mice. This result demonstrates that AM1012-05 can effectively inhibit allergic reactions and thereby achieve the goal of treating atopic dermatitis.

[0382] Example 43: Vaccine Immunization in Mice and Potency Detection

[0383] Immunization of mice with the vaccine:

[0384] (1) Prepare an appropriate number of Balb / c mice and keep them in isolation and quarantine;

[0385] (2) Mice were divided into groups according to experimental requirements, with 5 mice in each group; the negative control group consisted of mice injected with S1 protein (S1 protein group);

[0386] (3) Mice were immunized intramuscularly on day 0 (first immunization time), day 14, and day 28, respectively, with a vaccine volume of 50 μL per mouse;

[0387] (4) The clinical status of the animals was observed daily from the start of the experiment until euthanasia, including mental state, activity level, diet, and drinking water;

[0388] (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 each time and centrifuged at 6000 rpm, 4°C, for 10 min. Serum was then collected.

[0389] (6) Freeze the collected samples at -80°C until titer is measured.

[0390] Detection of mouse immune titer:

[0391] (1) Protein coating: The coating concentration of SARS-CoV-2 S1 protein is 2 μg / ml. Dilute the protein. Add the diluted sample to a 96-well ELISA plate, add 100 μL to each well, seal the plate with a sealing film, and place in a 4°C refrigerator overnight.

[0392] (2) Plate washing: Remove the overnight coated 96-well plate, discard the liquid in the wells, wash three times with PBS, 300 μL / well, and pat dry;

[0393] (3) Blocking: Add 300 μL of 2% BSA blocking buffer to each well, seal the plate with a film, and incubate at 37°C for 1 h;

[0394] (4) Washing: After blocking, discard the liquid in the wells, wash three times with PBST, 300 μL / well, and pat dry;

[0395] (5) Primary antibody incubation: Add 100 μL of diluted sample (serum) to each well, seal the plate with film, and incubate at 37°C for 1 h;

[0396] (6) Washing: After incubation, discard the liquid in the wells, wash three times with PBST, 300 μL / well, and pat dry;

[0397] (7) Secondary antibody incubation: Add 100 μL of diluted secondary antibody to each well, seal the plate with film, and incubate at 37°C for 1 h;

[0398] (8) Washing the plate: After incubation, discard the liquid in the wells, wash with PBST 5 times, 300 μL / well, and pat dry;

[0399] (9) Color development: Add 100 μL TMB color development solution to each well and develop at 37°C;

[0400] (10) Stop: Add 50 μL of ELISA stop solution and gently tap the edge of the ELISA plate to mix;

[0401] (11) Reading the plate: Place the ELISA plate in a microplate reader and read the plate at a detection wavelength of 450 nm and a reference wavelength of 570 nm.

[0402] Analysis and discussion of immune results:

[0403] In mice, total IgG antibody titers reflect the overall immune response of the vaccine, with IgG1 reflecting humoral immunity and IgG2a reflecting cellular immunity. Most traditional adjuvants have strong humoral immune activation (mouse IgG1) but weak cellular immunity (mouse IgG2a). Therefore, developing vaccine adjuvants that effectively stimulate IgG2a antibody production has been a key research focus in the adjuvant industry in recent years. Furthermore, Dynavax's CpG 1018 is currently the only CpG adjuvant approved for human vaccine production worldwide (using CpG 1018 as a standalone adjuvant in hepatitis B vaccine production). In this experiment, the S1 protein of the novel coronavirus was used as a model antigen, and CpG 1018 was used as a CpG positive adjuvant control to evaluate the adjuvant application of AM1012-05 in the vaccines prepared in Examples 32 and 33. The results of IgG, IgG1, and IgG2a antibody titers after immunization of mice with AM1012-05-related vaccines are shown in Figures 40, 41, and 42.

[0404] As can be seen from Figure 40, in the early stage of immunization, the IgG immune titer of the adjuvant system composed of AM1012-05 and aluminum phosphate was significantly better than that of the aluminum phosphate adjuvant alone (A in Figure 40); the IgG immune titer of the adjuvant system composed of AM1012-05 and aluminum phosphate was significantly better than that of the marketed CpG adjuvant 1018 (AE in Figure 40); the IgG immune titer of the adjuvant system composed of AM1012-05 and aluminum hydroxide was also better than that of the marketed CpG adjuvant 1018 (AE in Figure 40); the IgG immune titer of AM1012-05 as an independent adjuvant was equivalent to that of the marketed CpG adjuvant 1018 (AE in Figure 40).

[0405] As can be seen from Figure 41, in the early stage of immunization, the IgG1 immune titer of the adjuvant system consisting of AM1012-05 and aluminum phosphate or aluminum hydroxide was significantly better than that of the marketed CpG adjuvant 1018 (AB in Figure 41); in the middle and late stages of immunization, the IgG1 immune titer of the adjuvant system consisting of AM1012-05 and aluminum phosphate or aluminum hydroxide was also significantly better than that of the marketed CpG adjuvant 1018 (DE in Figure 41); the IgG1 immune titer of AM1012-05 as an independent adjuvant was equivalent to that of the marketed CpG adjuvant 1018 (AE in Figure 41).

[0406] As can be seen from Figure 42, the IgG2a immune titer of the adjuvant system composed of AM1012-05 and aluminum phosphate is significantly better than that of the marketed CpG adjuvant 1018 (AE in Figure 42); the IgG2a immune titer of the adjuvant system composed of AM1012-05 and aluminum phosphate is also significantly better than that of aluminum hydroxide and aluminum phosphate adjuvants (AE in Figure 42); in the early and middle stages of immunization, the IgG2a immune titer of AM1012-05 as an independent adjuvant is also significantly better than that of the marketed CpG adjuvant 1018 (BD in Figure 42); in the early stage of immunization, the IgG2a immune titer of the adjuvant system composed of AM1012-05 and aluminum hydroxide is significantly better than that of the marketed CpG adjuvant 1018, aluminum hydroxide adjuvant and aluminum phosphate adjuvant (AB in Figure 42).

[0407] The above-mentioned immune potency evaluation results of vaccines using AM1012-05 as a standalone adjuvant or in an adjuvant system composed of an aluminum adjuvant (aluminum hydroxide or aluminum phosphate) indicate that AM1012-05 can significantly enhance the humoral and cellular immunity of vaccines. In particular, the adjuvant system composed of AM1012-05 and aluminum phosphate can significantly enhance the cellular immunity of vaccines, with an immune effect far exceeding that of the commercially available CpG adjuvant 1018 and aluminum adjuvants (aluminum hydroxide or aluminum phosphate). Furthermore, in terms of early protection from vaccines, the adjuvant system composed of AM1012-05 and aluminum adjuvants (aluminum hydroxide or aluminum phosphate) is far superior to the commercially available CpG adjuvant 1018 and aluminum adjuvants (aluminum hydroxide or aluminum phosphate).

[0408] Therefore, AM1012-05 as an independent adjuvant or an adjuvant system composed of an aluminum adjuvant (aluminum hydroxide or aluminum phosphate) has very significant advantages over existing aluminum adjuvants (aluminum hydroxide or aluminum phosphate) and CpG adjuvants in terms of earlier onset of immune protection effect and improved immune protection effect. It is a very advantageous vaccine adjuvant or adjuvant system.

[0409] The present application has been described in detail above with reference to specific embodiments and exemplary examples. However, these descriptions should not be construed as limiting the present application. Those skilled in the art will appreciate that, without departing from the spirit and scope of the present application, various equivalent substitutions, modifications, or improvements may be made to the technical solutions and implementations of the present application, all of which fall within the scope of the present application. The scope of protection of the present application shall be determined by the appended claims.

Claims

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

2. The CpG oligodeoxynucleotide having the ability to regulate immunity according to claim 1, characterized in that: The value range of n is 3-4, and the value range of q is 1-5.

3. The CpG oligodeoxynucleotide having the ability to regulate immunity according to claim 1, characterized in that: The core sequence is at least one of CGCGACGCGTCGCG, ACGATCGAGATCGT, AGGATCGATCCT, TTCGATCGATCGAA, CGATCGATCG, GACGATCGTC, and TGCATCGATGCA; When the core sequence is CGCGACGCGTCGCG, the value range of q is 0 to 10, the value range of n is 0 to 3, and the value range of m is 2 to 11; Or, when the core sequence is ACGATCGAGATCGT, the value range of q is 0 to 10, the value range of n is 2 to 11, and the value range of m is 2 to 11; Or, when the core sequence is AGGATCGATCCT, the value range of q is 0 to 10, the value range of n is 2 to 11, and the value range of m is 2 to 11; Or, when the core sequence is TTCGATCGATCGAA, the value range of q is 0 to 10, the value range of n is 2 to 11, and the value range of m is 2 to 11; Or, when the core sequence is CGATCGATCG, the value range of q is 0 to 10, the value range of n is 1 to 11, and the value range of m is 2 to 11; Or, when the core sequence is GACGATCGTC, q is 0, n is in the range of 0 to 3, and m is in the range of 2 to 11; or q is in the range of 1 to 10, n is in the range of 2 to 11, and m is in the range of 2 to 11; Or, when the core sequence is TGCATCGATGCA, q is 0, n is 1, 3, 4 or 5, and m is in the range of 2 to 11; or q is in the range of 1 to 10, n is in the range of 2 to 11, and m is in the range of 2 to 11.

4. The CpG oligodeoxynucleotide having the ability to regulate immunity according to claim 1, characterized in that: It comprises at least one of the deoxynucleotide sequences shown in SEQ ID NO.1 to SEQ ID NO.

82.

5. The CpG oligodeoxynucleotide having the ability to regulate immunity according to any one of claims 1 to 4, characterized in that: The CpG oligodeoxynucleotide itself may contain 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 regulate immunity according to claim 5, characterized in that: The thiolation modification site is on the polyguanylate at the 5' end and / or the 3' end.

7. The CpG oligodeoxynucleotide having the ability to regulate immunity according to claim 5, characterized in that: The nanoparticles used in the nanoparticle modification include at least one of PLGA, chitosan, lipid nanoparticles and liposomes.

8. A CpG oligodeoxynucleotide solution preparation, characterized in that: The invention comprises the CpG oligodeoxynucleotide according to any one of claims 1 to 7 and excipients, wherein the excipients include at least one of pH buffer pair, glycine, trehalose, mannitol, sucrose, arginine, lysine, histidine, glycerol, propylene glycol, Pluronic F127, Pluronic F68, Tween 20, Tween 80, benzalkonium chloride, disodium edetate, sodium citrate, hydroxypropyl methylcellulose, sodium carboxymethyl cellulose, methyl beta-cyclodextrin and polyethylene glycol.

9. A CpG oligodeoxynucleotide solution preparation according to claim 8, characterized in that: The metal ions of the CpG oligodeoxynucleotide preparation include sodium, potassium, magnesium, calcium, zinc and iron, and the total concentration ranges from 0.1 to 90 mM.

10. The CpG oligodeoxynucleotide solution preparation according to claim 8, characterized in that: The pH value of the CpG oligodeoxynucleotide preparation is between 7-9.

11. Use of the CpG oligodeoxynucleotide according to any one of claims 1 to 7 in the preparation of a drug for regulating respiratory immune response.

12. Use of the CpG oligodeoxynucleotide according to any one of claims 1 to 7 in the preparation of a drug for preventing and treating respiratory diseases, characterized in that: The respiratory tract disease includes at least one of respiratory viral infectious diseases, respiratory bacterial infectious diseases, respiratory fungal infectious diseases, respiratory parasitic infectious diseases, and respiratory allergic diseases.

13. The use of the CpG oligodeoxynucleotide according to claim 12 in the preparation of a drug for preventing and treating respiratory diseases, characterized in that: The respiratory virus includes at least one of COVID-19 virus, influenza virus, RSV virus, and SARS-Cov.

14. Use of the CpG oligodeoxynucleotide according to any one of claims 1 to 7 in the preparation of a drug for preventing and treating a disease not caused by respiratory infection, characterized in that: The non-respiratory tract infection disease includes at least one of HIV virus infection, HBV virus infection, and HCV virus infection.

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

16. Use of the CpG oligodeoxynucleotide according to any one of claims 1 to 7 in the preparation of an immune adjuvant.

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

18. Use of the CpG oligodeoxynucleotide according to any one of claims 1 to 7 in the preparation of a drug for treating tumors, characterized in that: In the drug, CpG oligodeoxynucleotide is the only anti-tumor drug component.

19. An anti-tumor composition, characterized in that: The composition comprises the CpG oligodeoxynucleotide according to any one of claims 1 to 6 and an anti-tumor drug, wherein the anti-tumor drug includes but is not limited to a PD-1 anti-tumor drug, a PDL-1 anti-tumor drug and an anti-tumor cell therapy, and the CpG oligodeoxynucleotide in the composition serves as a component with anti-tumor efficacy or an immune adjuvant.

20. Use of the CpG oligodeoxynucleotide according to any one of claims 1 to 7 in the preparation of vaccines for human or animal use.

21. A use of the CpG oligodeoxynucleotide according to any one of claims 1 to 7 in treating central nervous system diseases, characterized in that: The central nervous system diseases include but are not limited to Alzheimer's disease.

22. Use of the CpG oligodeoxynucleotide according to any one of claims 1 to 7 in the preparation of a drug for preventing and treating secondary infection after trauma.

23. Use of the CpG oligodeoxynucleotide according to any one of claims 1 to 7 in the preparation of a drug for promoting wound healing.

24. Use of the CpG oligodeoxynucleotide according to any one of claims 1 to 7 in the preparation of a medicament for treating allergic rhinitis and / or atopic dermatitis and / or asthma and / or chronic obstructive pulmonary disease and / or eosinophil-related diseases.

25. A drug prepared by using the CpG oligodeoxynucleotide according to any one of claims 1 to 7, characterized in that: The dosage form of the medicine is injection, tablet, lyophilized agent, inhalation, nasal drops, nasal spray, rectal suppository, eye drops, ointment, lotion and gel.

26. A CpG oligodeoxynucleotide external preparation, characterized in that: The invention comprises the CpG oligodeoxynucleotide as claimed in any one of claims 1 to 7 and excipients, wherein the excipients comprise glyceryl behenate, liquid paraffin, vaseline, medium chain triglycerides, cocoyl caprylate, glyceryl mono- and distearate, hydroxypropyl methylcellulose, carbomer, xanthan gum, sodium carboxymethyl cellulose, poloxamer 407, polyethylene glycol cetearyl alcohol ether, poloxamer 188, oleoyl polyoxyethylene glyceride, polyethylene glycol-7 stearate, caprylic capric macrogol glyceride, sodium lauryl sulfate, propylene glycol monofatty acid ester, polyglyceryl oleate, propylene glycol laurate, propylene glycol, isopropyl myristate, and polyglyceryl oleate.

27. A CpG oligodeoxynucleotide adjuvant and a preparation in combination with other types of adjuvants for use in vaccines for human or animals, characterized in that: The method comprises the CpG oligodeoxynucleotide according to any one of claims 1 to 7, itself, or a combination thereof with an aluminum hydroxide adjuvant or an aluminum phosphate adjuvant.

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