Cpg oligodeoxynucleotide with regulating body immune capacity and its application

Abstract

The application discloses CpG-containing oligodeoxynucleotide (ODN) with the function of regulating the body's immune capacity and application thereof. The oligodeoxynucleotide in the application is different from the design concept of traditional A-type CpG ODN in sequence and structure, embodies the complex drug-making rule and overcomes the problem of traditional A-type CPG in drug-making. The oligodeoxynucleotide has excellent activity of regulating the body's immune function in vitro and in vivo, can stimulate the production of I, II and III type interferons, regulates the Th1 type immune response, and inhibits the Th2 type and Th17 type inflammatory response. Therefore, the oligodeoxynucleotide can prevent and treat allergic diseases caused by Th2 type immune response, including allergic rhinitis, atopic dermatitis, asthma, chronic obstructive pulmonary disease, eosinophil and Th17 related diseases, can prevent and treat the infection and spread of respiratory or non-respiratory pathogenic microorganisms including viruses, bacteria, fungi and parasites, can improve the immune level of the elderly and immunodeficient population, can convert "cold tumor" into "hot tumor", improve the effect of cancer immunotherapy, can prevent and treat central nervous system diseases related to immune dysfunction such as Alzheimer's disease, and can be applied to other diseases related to immune dysfunction.

Description

[0001] The present application claims priority to the prior application with the patent application number 202310902755.X, filed on July 21, 2023, with the State Intellectual Property Office of China, and the title of the prior application is "CpG oligodeoxynucleotide with the function of regulating the immune capacity of the body and its application". The entire contents of the prior application are incorporated herein by reference. TECHNICAL FIELD

[0002] The present application belongs to the field of oligodeoxynucleotide, and specifically relates to a CpG oligodeoxynucleotide with the function of regulating the immune capacity of the body and its application. BACKGROUND

[0003] TLR9 is a mammalian innate immune receptor, which exists in the endosome, and its ligand is an oligodeoxyribonucleotide (ODN) containing unmethylated CpG. Depending on different types and structures, CPG-ODN can combine with TLR9 to activate the IRF-7 or NF-κB signaling pathway after entering the endosome, and produce inflammatory factors such as type I interferon or IL-6 to intervene in the immune response.

[0004] According to the structural characteristics and the difference of immune effects, CpG ODN can be divided into three types of A, B and C. Type A CPG enters the early endosome of plasmacytoid dendritic cells (pDC), combines with the inside TLR9 receptor, activates the IRF-7 signaling pathway, and induces the production of a large amount of type I interferon (IFN-α / IFN-β) and type III interferon (IFN-λ). As a general immune regulatory cytokine, type I interferon acts on downstream immune cells, including but not limited to NK cells, CD8 + T cells, CD4 +T cells, γ / δ T cells, macrophages, etc., induce the production of cytokines such as type II interferon (IFN-γ). Type B CPG is a fully thio-modified linear CpG ODN, and the main target cell is B lymphocytes. Type B CPG enters the late endosome and binds to TLR9, activates NF-κB, and produces a series of immune responses, including the secretion of inflammatory cytokines such as IL-6, IL-1 and TNF-α, and promotes B cell proliferation. Type B CpG can be used as a vaccine adjuvant to significantly improve the level of antibodies. Type C CPG is a fully thio-modified CpG ODN, containing part of the structure of type A CPG and part of the structure of type B CPG. It has both palindromic sequences that can form dimers and linear structures that are not palindromic. Type C CpG can enter both early endosomes and late endosomes to bind to TLR9. Therefore, it has the activity of both type A and type B CpG-ODN, and can not only activate the IRF-7 signaling pathway, but also activate the NF-kB pathway to produce type I interferon and inflammatory cytokines. Although type C CPG takes into account the properties 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 experiments of type B CPG have shown that it can successfully treat allergic rhinitis, asthma, infection of various microorganisms, and anti-tumor. In the past 20 years, more than 100 articles have reported the use of CPG in the preclinical treatment of various diseases. The representative of type B CPG, CPG-1018, has been successfully approved for listing as a hepatitis B vaccine adjuvant in 2017.

[0006] Current research shows that when administered by different routes, type A CpG ODN can induce strong innate immunity (type I / II / III interferon) and Th1-type immune response (DC, NK and CD8 +activation of T cells, production of IL-12), and can modulate immune functions, thereby inhibiting Th2-type and Th17-type immune responses. Through these immune responses, diseases caused by allergic and non-allergic inflammation, anti-pathogenic microbial infection, direct or indirect killing of tumor cells, reduction of Aβ plaque formation, improvement of vaccine immunogenicity, and many other effects can be treated. This also lays the foundation for its application in the fields of infectious diseases, cancer, immune adjuvants, allergic rhinitis, atopic dermatitis, asthma, chronic obstructive pulmonary disease, eosinophil-related diseases, Alzheimer's disease, wound healing, prevention and treatment of wound infection, etc. (Montamat et., Fronteers in Immunol. 2021. doi: 10.3389 / fimmu.2021.590054; Scheiermann et al., Vaccine 2014. 32:6377-6389; Shirota and Klinman., Immunopotentiators in Modern Vaccines ISBN 978-0-12-804019-5, http: / / dx.doi.org / 10.1016 / B978-0-12-804019-5.00009-8; Beeh et al., J Allergy Clin Immunol 2013; 131:866-74; Hanagata N. et al., Int J Nanomedicine. 2012; 7:2181-95; Kayraklioglu et al.,. Methods Mol Biol. 2021; 2197:51-85; Fan et al., Clin Cancer Res. 2012 Oct 15; 18(20):5628-38; Senti et al., Clin Exp Allergy. 2009 Apr; 39(4):562-70; Scholtzova et al., Acta Neuropathol Commun. 2014 Sep 2; 2:101; Wanke-Jellinek et al., J Immunol. 2016 Jan 15; 196(2):767-77).

[0007] Because of the easy aggregation of A-type CPG, its drugability is very poor, so that A-type CPG has been found for more than 20 years, and there are few clinical trials, and no drugs have been approved for marketing. Historically, three institutions have tried various methods to improve the drugability of A-type CPG: 1. Cytos Biotechnology AG company wrapped A-type CPG (QbG-10) with virus-like particles (VLP), although it improved the drugability, but the wrapping of VLP made A-type 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 (small molecule compound) to the 3' end of A-type CPG, making it a temperature-sensitive prodrug, which automatically falls off at 37°C after entering the cell, so that A-type CPG re-aggregates to exert its efficacy, but such modification cannot completely prevent aggregation in vitro, and fma modification limits the safety and tolerance of the drug (Puig et al., Puig, Nucleic acids research. 2005. 34:6488-6495); 3. Japanese scientists reported in 2015 that after adding 40 adenine deoxyribonucleotides (poly-A) to the 3' end of A-type CPG, the drugability of A-type CPG was greatly improved. But because of the addition of 40 adenines, the production cost is greatly increased, and because it is all thio, it can increase the production of IL-6 inflammatory molecules, so its safety and tolerance are limited (Aoshi et al., J. Immunol. Research. 2015. http: / / dx.doi.org / 10.1155 / 2015 / 316364). Therefore, the traditional A-type CpG has not developed a successful case of drug so far, and no clinical trials have been reported.

[0008] The principle of traditional A-type CPG design is to achieve the generation of the polymer through the complete palindrome of the 8-12 nucleotides (including one or more CpG) core sequence and the poly-G at both ends of the core sequence. The polymer can present the core sequence of CpG to TLR9 on the early endosome rather than the late endosome, so as to activate the IRF-7 signal pathway and generate type I interferon. The activation of the IRF-7 signal pathway must be accompanied by the formation of the polymer (Kerkmann et al., J. Bio. Chem., 2005. 280: 8086-8093; Wu et al., J. Bio. Chem., 2004. 279: 33071-33078). The monomer CPG ODN cannot activate the IRF-7 signal pathway, such as the B-type CPG which is monomer in solution and can only bind to TLR9 on the late endosome, so as to activate the NF-kB signal pathway and induce the generation of inflammatory factors rather than type I interferon. Since the A-type CPG must form a polymer to be active, the generation of the polymer tends to form uncontrollable large aggregates, which greatly reduces the drugability. This is the main reason why there is no successful case of clinical research and development of A-type CPG since its invention. Therefore, how to ensure that the A-type CPG has aggregation and the aggregation is controllable has become the key to whether this type of molecule can be developed into a clinically effective drug.

[0009] The self-aggregation model of traditional A-type CPG was proposed by Kerkmann et al. in 2005 (Kerkmann et al., J. Bio. Chem., 2005. 280: 8086-8093): two palindromic monomers form a dimer through Watson-Crick connection, the poly-G at both ends of the dimer forms Hoogsteen base pairing through hydrogen bonding of guanylate, becoming a tetramer (G-tetrad). Then the dimer and tetramer continue to aggregate through G-tetrad, and so on, forming larger aggregates. Another model is based on the secondary structure of A-type CPG, which can form a palindromic structure within the monomer, and the poly-G at both ends can form higher aggregates through Hoogsteen pairing. Therefore, from these models, it can be known that the size and stability of the aggregate depend on how many surrounding Hoogsteen connections and their unique spatial structure. Our early research found that, from the secondary structure, the completely palindromic form before and after poly-G can reduce the energy required for aggregation, making aggregation easy to form. Therefore, by establishing a certain spatial barrier between poly-G and palindromic, the energy of forming G-tetrad can be increased, making the aggregate relatively difficult to form or relatively loose after formation. At the same time, combined with the selection of the number of poly-G at both ends, it can make the aggregate stable within a certain size range. Based on the above considerations, the present application designs multiple core sequences, and at the same time, deoxyribonucleotide substitution experiments are carried out on both ends of A-type CPG while the core sequence remains unchanged, to find out the formation rule of A-type CPG aggregate and the relationship between it and the production of type I interferon. SUMMARY

[0010] The technical solutions of the present application are as follows:

[0011] A CpG oligodeoxynucleotide for regulating the immune capacity of the body, the sequence structure of the CpG oligodeoxynucleotide for regulating the immune capacity of the body is one of 5'-(G) n -core sequence-(N) q -(G) m- 3' sequence, the core sequence is a palindromic sequence, the N is any one of adenine deoxyribonucleotide (A), thymine deoxyribonucleotide (T), and cytosine deoxyribonucleotide (C); the G is guanine deoxyribonucleotide; q, n, and m are all integers; n is in the range of 2-11, q is in the range of 0-10, and m is in the range of 2-11.

[0012] Preferably, n is in the range of 3-4, and q is in the range of 1-5.

[0013] Preferably, the core sequence is at least one of CGCGACGCGTCGCG, ACGATCGAGATCGT, AGGATCGATCCT, TTCGATCGATCGAA, CGATCGATCG, GACGATCGTC, TGCATCGATGCA; when the core sequence is CGCGACGCGTCGCG, the value of q is 0-10, the value of n is 0-3, and the value of m is 2-11;

[0014] Or, when the core sequence is ACGATCGAGATCGT, the value of q is 0-10, the value of n is 2-11, and the value of m is 2-11.

[0015] Or, when the core sequence is AGGATCGATCCT, the value of q is 0-10, the value of n is 2-11, and the value of m is 2-11.

[0016] Or, when the core sequence is TTCGATCGATCGAA, the value of q is 0-10, the value of n is 2-11, and the value of m is 2-11.

[0017] Or, when the core sequence is CGATCGATCG, the value of q is 0-10, the value of n is 1-11, and the value of m is 2-11.

[0018] Or, when the core sequence is GACGATCGTC, the value of q is 0, the value of n is 0-3, and the value of m is 2-11; or the value of q is 1-10, the value of n is 2-11, and the value of m is 2-11.

[0019] Or, when the core sequence is TGCATCGATGCA, the value of q is 0, the value of n is 1, 3, 4, or 5, and the value of m is 2-11; or the value of q is 1-10, the value of n is 2-11, and the value of m is 2-11.

[0020] Preferably, the CpG oligodeoxynucleotide comprises at least one of the deoxynucleotide sequences shown in SEQ ID NO. 1-SEQ ID NO. 82.

[0021] Preferably, the CpG oligodeoxynucleotide itself or contains at least one of a thio modification, a fluoro modification, a methoxy modification, a locked nucleic acid modification, and a nanoparticle modification.

[0022] Preferably, the site of the thio-modification is on the poly-G at the 5' end and / or 3' end.

[0023] Preferably, the nanoparticle employed in the nanoparticle modification comprises at least one of PLGA, chitosan, a lipid nanoparticle, a liposome.

[0024] A CpG oligodeoxynucleotide formulation comprising the above-mentioned CpG oligodeoxynucleotide and an excipient, the excipient comprising at least one of a 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, hypromellose, sodium carboxymethylcellulose, methyl betacyclodextrin, polyethylene glycol.

[0025] Preferably, the metal ion of the CpG oligodeoxynucleotide formulation comprises sodium, potassium, magnesium, calcium, zinc, iron, with a total concentration ranging from 0.1-90 mM.

[0026] Preferably, the pH value of the CpG oligodeoxynucleotide formulation is between 7-9.

[0027] Use of the above-mentioned CpG oligodeoxynucleotide in the preparation of a medicament for modulating immune responses in the respiratory tract.

[0028] Use of the above-mentioned CpG oligodeoxynucleotide in the preparation of a medicament for preventing and treating respiratory diseases, the respiratory diseases comprising at least one of respiratory viral infectious diseases, respiratory bacterial infectious diseases, respiratory fungal infectious diseases, respiratory parasitic infectious diseases, respiratory allergic diseases. The respiratory viruses in the respiratory viral infections comprise at least one of COVID-19 virus, influenza virus, RSV virus, SARS-Cov.

[0029] Use of the above-mentioned CpG oligodeoxynucleotide in the preparation of a medicament for preventing and treating non-respiratory infectious diseases, the non-respiratory infectious diseases comprising at least one of HIV virus infection, HBV virus infection, HCV virus infection.

[0030] Use of the above-mentioned CpG oligodeoxynucleotide in the preparation of a medicament for modulating the proliferation function of immune cells or a medicament for modulating the release of cytokines of immune cells.

[0031] Use of the above-mentioned CpG oligodeoxynucleotide in the preparation of an immune adjuvant.

[0032] Use of the above-mentioned CpG oligodeoxynucleotide in the preparation of a medicament for preventing and treating viral infections.

[0033] Use of the CpG oligodeoxynucleotide in the preparation of a medicament for treating tumors, wherein the CpG oligodeoxynucleotide is the only anti-tumor effective component in the medicament.

[0034] An anti-tumor composition comprising the CpG oligodeoxynucleotide and an anti-tumor drug, wherein the anti-tumor drug includes but is not limited to PD-1 anti-tumor drugs, PDL-1 anti-tumor drugs, and anti-tumor cell therapies, and wherein the CpG oligodeoxynucleotide is a component with anti-tumor efficacy or an immunoadjuvant in the composition.

[0035] Use of the CpG oligodeoxynucleotide in the preparation of a vaccine for humans or animals.

[0036] Use of the CpG oligodeoxynucleotide in the preparation of a medicament for treating central nervous system diseases, wherein the central nervous system diseases include but are not limited to Alzheimer's disease.

[0037] Use of the CpG oligodeoxynucleotide in the preparation of a medicament for preventing and treating secondary infections after trauma.

[0038] Use of the CpG oligodeoxynucleotide in the preparation of a medicament for promoting wound healing.

[0039] Use of the CpG oligodeoxynucleotide 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.

[0040] A medicament prepared using the CpG oligodeoxynucleotide, wherein the medicament is in the form of an injection, a tablet, a lyophilized agent, an inhalant, a nose drop, a nose spray, an anal suppository, an eye drop, a smearing agent, a lotion, a gel.

[0041] An external preparation of the CpG oligodeoxynucleotide, comprising the CpG oligodeoxynucleotide according to any one of claims 1 to 5 and an auxiliary material, wherein the auxiliary material includes glyceryl behenate, liquid paraffin, vaseline, medium-chain triglyceride, cocoyl octa-decanote, monodiglyceride, hypromellose, carbomer, xanthan gum, sodium carboxymethyl cellulose, poloxamer 407, macrogol cetostearyl ether, poloxamer 188, oleoyl polyoxylglycerides, macrogol-7 palmitate, caprylocaproyl macrogol glycerides, sodium lauryl sulfate, propylene glycol mono fatty acid ester, polyglyceryl oleate, propylene glycol laurate, propylene glycol, isopropyl myristate, polyglyceryl oleate.

[0042] A formulation of the above-mentioned CpG oligodeoxynucleotide adjuvant and in combination with other types of adjuvants for use in human or animal vaccines: comprising CpG oligodeoxynucleotide itself or in combination with other types of adjuvants, wherein the other types of adjuvants are preferably aluminum hydroxide and aluminum phosphate.

[0043] The beneficial effects achieved by this invention are as follows:

[0044] Unlike traditional palindromic designs of type A CpG, this invention provides a novel CpGODN sequence with a novel structure and sequence. These sequences, by substituting nucleotides at both ends of the ODN, reveal the relationship and patterns between druggability and activity. Compared to published sequences, they possess unique primary and secondary structures, overcoming the problem of poor druggability and easy aggregation of traditional type A CpG. They exhibit higher immunomodulatory activity and druggability. In particular, the AM1012-05 sequence demonstrates a secondary structure that is more stable at high temperatures than similar products, with superior aggregate controllability compared to other similar sequences. During production, it exhibits better batch-to-batch stability and improved druggability. Meanwhile, AM1012-05 can effectively penetrate the human respiratory tract epithelial mucosa (RPMI 2650 cells) and has also demonstrated excellent immune-activating activity in animals. It exhibits excellent blocking ability against respiratory viral infection and transmission, and has the potential to exert anti-tumor effects, treat allergic rhinitis, asthma, chronic obstructive pulmonary disease, eosinophilic disease, atopic dermatitis, Alzheimer's disease, and be used in the prevention and treatment of post-traumatic secondary infections and wound healing promotion drugs by activating and regulating the immune system. Furthermore, it has the potential to serve as an independent adjuvant or component of an adjuvant system in vaccines. Attached Figure Description

[0045] To more clearly illustrate the technical solution of this application, the drawings used in the embodiments will be briefly introduced below. Obviously, for those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0046] Figure 1 To evaluate the IFN-γ stimulatory effect of different CpG sequences on NK-92 cells; Figures A, B, C, D, and E show the IFN-γ stimulatory effect of different CpG sequences on NK-92 cells under the same experimental system.

[0047] Figure 2 The study compared the stimulatory effects of different CpG sequences on spleen cells of SD rats. Figure A shows the stimulation of rat spleen cells to secrete IFN-γ by different CpG sequences, Figure B shows the stimulation of rat spleen cells to secrete TNF-α by different CpG sequences, and Figure C shows the stimulation of rat spleen cells to secrete IL-6 by different CpG sequences.

[0048] Figure 3 Evaluation of the IFN-γ stimulating ability of AM1012-05 on NK-92 cells;

[0049] Figure 4 Evaluation of the stimulating ability of AM1012-05 on BALB / c mouse splenocytes; wherein, Figure A is the secretion of IL-6 by AM1012-05 stimulated BALB / c mouse splenocytes, Figure B is the secretion of IFN-γ by AM1012-05 stimulated BALB / c mouse splenocytes, Figure C is the secretion of TNF-α by AM1012-05 stimulated BALB / c mouse splenocytes, and Figure D is the secretion of IFN-α by AM1012-05 stimulated BALB / c mouse splenocytes;

[0050] Figure 5 Evaluation of the stimulating ability of AM1012-05 on C57BL6 mouse splenocytes; wherein, Figure A is the secretion of IL-6 by AM1012-05 stimulated C57BL6 mouse splenocytes, Figure B is the secretion of IFN-γ by AM1012-05 stimulated C57BL6 mouse splenocytes, Figure C is the secretion of TNF-α by AM1012-05 stimulated C57BL6 mouse splenocytes, and Figure D is the secretion of IFN-α by AM1012-05 stimulated C57BL6 mouse splenocytes;

[0051] Figure 6 Evaluation of the stimulating ability of AM1012-05 on cotton rat splenocytes; wherein, Figure A is the mRNA level of IFN-α of cotton rat splenocytes increased by AM1012-05, Figure B is the mRNA level of IFN-γ of cotton rat splenocytes increased by AM1012-05, Figure C is the mRNA level of TNF-α of cotton rat splenocytes increased by AM1012-05, and Figure D is the mRNA level of CXCL-10 of cotton rat splenocytes increased by AM1012-05;

[0052] Figure 7 Evaluation of the stimulating ability of AM1012-05 on rat splenocytes (mRNA level); wherein, Figure A is the mRNA level of IFN-α of rat splenocytes increased by AM1012-05, and Figure B is the mRNA level of CXCL-10 of rat splenocytes increased by AM1012-05;

[0053] Figure 8 Evaluation of the stimulating ability of AM1012-05 on rat splenocytes (protein level); wherein, Figure A is the secretion of IFN-γ by AM1012-05 stimulated rat splenocytes, Figure B is the secretion of IL-6 by AM1012-05 stimulated rat splenocytes, and Figure C is the secretion of TNF-α by AM1012-05 stimulated rat splenocytes;

[0054] Figure 9Evaluation of the stimulating ability of AM1012-05 on beagle dog PBMC; wherein, Figure A is that AM1012-05 improves the mRNA level of IFN-α of beagle dog PBMC, Figure B is that AM1012-05 improves the mRNA level of IFN-γ of beagle dog PBMC, Figure C is that AM1012-05 improves the mRNA level of IFN-λ of beagle dog PBMC, Figure D is that AM1012-05 improves the mRNA level of IL-6 of beagle dog PBMC, Figure E is that AM1012-05 improves the mRNA level of CXCL-10 of beagle dog PBMC, and Figure F is that AM1012-05 improves the mRNA level of TNF-α of beagle dog PBMC;

[0055] Figure 10 Evaluation of the stimulating ability of CpG ODN on human PBMC; wherein, Figure A is that AM1012-05 stimulates human PBMC to secrete IFN-α, Figure B is that AM1012-05 stimulates human PBMC to secrete IFN-γ, Figure C is that AM1012-05 stimulates human PBMC to secrete IFN-λ, Figure D is that AM1012-05 stimulates human PBMC to secrete TNF-α, Figure E is that AM1012-05 stimulates human PBMC to secrete IL-6, Figure F is that AM1012-05 stimulates human PBMC to secrete CXCL-10, and Figure G is that AM1012-05, CpG 2216 and CpG D19 stimulate human PBMC to secrete IFN-α;

[0056] Figure 11 Electrophoresis detection results of serum stability of CpG ODN;

[0057] Figure 12 Distribution of aggregates of different CpG ODNs; wherein, the left figure is electrophoresis detection of the aggregate state distribution of type A CpG (2 hours), and the right figure is electrophoresis detection of the aggregate state distribution of type A CpG (24 hours);

[0058] Figure 13 Aggregate analysis of AM1012-05 and CpG 2216 solutions in a static state and a vortex state;

[0059] Figure 14 Comparison of the secondary structures of different sequence samples at-20℃; wherein, Figure A is CpG 2216, Figure B is E41, Figure C is AM1012-03, and Figure D is AM1012-05;

[0060] Figure 15 Comparison of the secondary structures of different sequence samples at 0℃; wherein, Figure A is CpG 2216, Figure B is E41, Figure C is AM1012-03, and Figure D is AM1012-05;

[0061] Figure 16 Secondary structure comparison of different sequence samples at 4°C; wherein, Panel A is CpG 2216, Panel B is E41, Panel C is AM1012-03, Panel D is AM1012-05;

[0062] Figure 17 Secondary structure comparison of different sequence samples at 25°C; wherein, Panel A is CpG 2216, Panel B is E41, Panel C is AM1012-03, Panel D is AM1012-05;

[0063] Figure 18 Secondary structure comparison of different sequence samples at 37°C; wherein, Panel A is CpG 2216, Panel B is E41, Panel C is AM1012-03, Panel D is AM1012-05;

[0064] Figure 19 Secondary structure comparison of different sequence samples at 40°C; wherein, Panel A is CpG 2216, Panel B is E41, Panel C is AM1012-03, Panel D is AM1012-05;

[0065] Figure 20 Secondary structure comparison of different sequence samples at 50°C; wherein, Panel A is CpG 2216, Panel B is E41, Panel C is AM1012-03, Panel D is AM1012-05;

[0066] Figure 21 Secondary structure comparison of different sequence samples at 60°C; wherein, Panel A is CpG 2216, Panel B is E41, Panel C is AM1012-03, Panel D is AM1012-05;

[0067] Figure 22 Secondary structure comparison of different sequence samples at 70°C; wherein, Panel A is CpG 2216, Panel B is E41, Panel C is AM1012-03, Panel D is AM1012-05;

[0068] Figure 23 Secondary structure comparison of different sequence samples at 80°C; wherein, Panel A is CpG 2216, Panel B is E41, Panel C is AM1012-03, Panel D is AM1012-05;

[0069] Figure 24 Secondary structure comparison of different sequence samples at 90°C; wherein, Panel A is CpG 2216, Panel B is E41, Panel C is AM1012-03, Panel D is AM1012-05;

[0070] Figure 25Effect of CpG ODN at different temperature treatment on IFN-γ secretion of rat spleen cells

[0071] Figure 26 Particle size analysis chart of PLGA nanoparticle preparation of CpG ODN

[0072] Figure 27 Particle size analysis chart of preparation of chitosan nanoparticle preparation of CpG ODN

[0073] Figure 28 Particle size analysis chart of lipid nanoparticle preparation of CpG ODN

[0074] Figure 29 Particle size analysis chart of immunoadjuvant of PLGA nanoparticle preparation of CpG ODN

[0075] Figure 30 Evaluation chart of the effect of formulation components on the NK-92 cell IFN-γ stimulating ability of AM1012-05

[0076] Figure 31 Effect of AM1012-05 on the relative expression of immune-related mRNA in mouse lung tissue and spleen tissue; wherein, A chart is AM1012-05 increasing the mRNA level of IFN-α in mouse lung tissue, B chart is AM1012-05 increasing the mRNA level of IFN-γ in mouse lung tissue, C chart is AM1012-05 increasing the mRNA level of CXCL-10 in mouse lung tissue, D chart is AM1012-05 increasing the mRNA level of IFN-α in mouse spleen tissue, E chart is AM1012-05 increasing the mRNA level of IFN-γ in mouse spleen tissue, F chart is AM1012-05 increasing the mRNA level of CXCL-10 in mouse spleen tissue

[0077] Figure 32 Relative expression change of lung CXCL-10 mRNA of mice after intranasal administration of AM1012-05

[0078] Figure 33 Cytokine secretion induced by AM1012-05 in mouse lung tissue under different administration frequencies; wherein, A chart is AM1012-05 inducing CXCL-10 secretion in mouse lung tissue under different administration frequencies, B chart is AM1012-05 inducing IFN-γ secretion in mouse lung tissue under different administration frequencies

[0079] Figure 34 AM1012-05 activated human PBMC killing N87 cell experiment

[0080] Figure 35Human PBMC activated by AM1012-05 for MDA-MB-231 cell killing assay

[0081] Figure 36 Evaluation of AM1012-05 for alleviating allergic rhinitis in mice; wherein, Figure A shows that AM1012-05 reduces sneezing frequency in allergic rhinitis mice, Figure B shows that AM1012-05 reduces nose scratching frequency in allergic rhinitis mice, Figure C shows that AM1012-05 reduces rhinorrhea in allergic rhinitis mice, Figure D shows that AM1012-05 reduces the number of eosinophils in nasal mucosa of allergic rhinitis mice, Figure E shows that AM1012-05 reduces the expression of IL-4 in serum of allergic rhinitis mice, and Figure F shows that AM1012-05 increases the expression of IFN-γ in serum of allergic rhinitis mice;

[0082] Figure 37 Results of AM1012-05 for treating allergic rhinitis in mice by analyzing immune cells in nasal lavage fluid;

[0083] Figure 38 Evaluation of AM1012-05 for in vitro anti-respiratory syncytial virus infection activity; wherein, Figure A shows that AM1012-05 prevents respiratory syncytial virus proliferation in co-incubation supernatant of PBMC, and Figure B shows that AM1012-05 eliminates respiratory syncytial virus in host cells;

[0084] Figure 39 Evaluation of AM1012-05 for anti-atopic dermatitis activity (IgE level);

[0085] Figure 40 Evaluation of AM1012-05 for adjuvant application; wherein, Figure A shows the results of IgG immune titer detection 7 days after the first immunization, Figure B shows the results of IgG immune titer detection 7 days after the second immunization, Figure C shows the results of IgG immune titer detection 7 days after the third immunization, Figure D shows the results of IgG immune titer detection 21 days after the third immunization, and Figure E shows the results of IgG immune titer detection 35 days after the third immunization;

[0086] Figure 41 Evaluation of AM1012-05 for adjuvant application; wherein, Figure A shows the results of IgG1 immune titer detection 7 days after the first immunization, Figure B shows the results of IgG1 immune titer detection 7 days after the second immunization, Figure C shows the results of IgG1 immune titer detection 7 days after the third immunization, Figure D shows the results of IgG1 immune titer detection 21 days after the third immunization, and Figure E shows the results of IgG1 immune titer detection 35 days after the third immunization;

[0087] Figure 42Evaluation of adjuvant application of AM1012-05: wherein, A is the IgG2a immune titer detection result of 7 days after the first immunization; B is the IgG2a immune titer detection result of 7 days after the second immunization; C is the IgG2a immune titer detection result of 7 days after the third immunization; D is the IgG2a immune titer detection result of 21 days after the third immunization; E is the IgG2a immune titer detection result of 35 days after the third immunization. DETAILED DESCRIPTION

[0088] The specific embodiments of the present application are described in detail below. Obviously, the described embodiments are only a part of the embodiments of the present application, not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor are within the scope of protection of the present application.

[0089] Example 1: Design and synthesis of CpG ODN molecules

[0090] A plurality of CpG ODN sequences (see Table 1) are designed in the present application, and are chemically synthesized by solid-phase synthesis, and then subjected to ammonolysis, purification, desalting and concentration, and process procedures such as split freezing and drying to obtain the final product. The specific operation is as follows:

[0091] (1) Solid-phase synthesis: solid-phase synthesis is performed under computer control, which consists of 4-step repeated synthesis steps (detritylation, coupling, sulfurization, capping) and the last step of removing the protecting group of the dephosphorylated backbone;

[0092] (2) Ammonolysis: after synthesis, ammonia or organic amine is used to cut off the oligonucleotide from the carrier, and the oligonucleotide is dissolved in solution, and the universal linker connected to the first base at the 3' end is cut off and the protecting group of the amino group on the base is removed;

[0093] (3) Purification: the crude product obtained by ammonolysis is purified and separated by ion exchange chromatography, reverse phase chromatography or electrophoresis to obtain the purified product;

[0094] (4) Desalting and concentration: the purified product is desalted and concentrated using ultrafiltration equipment;

[0095] (5) Splitting, freeze-drying: white, white to light yellow loose bodies are obtained by freeze-drying.

[0096] The types of nucleic acid modification include but are not limited to thio modification.

[0097] Table 1 Sequences involved in the present application

[0098]

[0099]

[0100]

[0101] (Note: * indicates thio modification).

[0102] Example 2: Aggregation and Activity Analysis of Different CpG ODNs

[0103] (I) Analysis of aggregation of different CpG ODNs:

[0104] The aggregation of the partially CpG ODN sequence lyophilized powder obtained in Example 1 was analyzed by high performance liquid chromatography.

[0105] (II) Effects of different CpG ODNs on IFN-α secretion based on PBMC cells from healthy individuals:

[0106] (1) Take a certain amount of 100μM CpG ODN solution, prepare a certain concentration stock solution with RPMI 1640 complete medium, and then dilute the CpG ODN sample solution with complete medium at a ratio of 4 times to prepare detection samples of different concentrations.

[0107] (2) Obtain an appropriate amount of cryopreserved healthy human PBMC cells;

[0108] (3) Press 2×10 5 Cells / well were seeded into 96-well plates, and different concentrations of sample were added to each well. PBS blank control wells were also included.

[0109] (4) Incubate at 37℃ and 5% CO2 for 16 hours;

[0110] (5) Centrifuge and collect the supernatant;

[0111] (6) Detect IFN-α according to the ELISA test instructions for IFN-α.

[0112] Analysis of the aggregation results (Tables 2-5) reveals that: (1) by changing the number of Gs in sequences such as AM1012-05, CpG 2216, and CpG D19, the optimal aggregation result for the corresponding sequences can be obtained when the number of Gs at the 5' end is 3-4, i.e., the minimum content of aggregates; (2) inserting 1-5 As between the core palindromic sequence of AM1012-05 and the poly-G at the 3' end can further reduce the content of aggregates; (3) inserting 1-10 Ts or Cs between the core palindromic sequence of AM1012-05 and the G at the 3' end can further reduce or increase the content of aggregates to varying degrees; (4) inserting 1-10 As, Ts, or Cs between the core palindromic sequence of CpG 2216 and CpG D19 and the G at the 3' end can further reduce or increase the content of aggregates to varying degrees.

[0113] The target cell of type A CPG is pDC, which accounts for about 0.2-0.4% of PBMC cells. IFN-α produced is mainly from pDC. The results of the activity evaluation test of this example are shown in Tables 2-5. As can be seen from Table 2, when the palindromic sequence core of type A CpG is "CGATCGATCG", different numbers of G are gradually added before C at the 5' end, and with the increase of the number of added G, the stimulating activity on human PBMC gradually increases, and reaches the strongest activity when the number of added G is 3 or 4, and then decreases with the continuous increase of the number of G. When the palindromic sequence of CpG D19 "TGCATCGATGCA" is used as the core sequence, different numbers of G are gradually added before T at the 5' end, and with the increase of the number of added G, the stimulating activity on human PBMC gradually increases, and reaches the strongest activity when the number of added G is 2-4, and then decreases with the continuous increase of the number of G. When the palindromic sequence of CpG 2216 "GACGATCGTC" is used as the core sequence, different numbers of G are gradually added before G at the 5' end, and with the increase of the number of added G, the stimulating activity on human PBMC gradually increases, and reaches the strongest activity when the number of added G is 3, and then decreases with the continuous increase of the number of G.

[0114] The data of the study on the insertion of A between the 3' end of the palindromic structure of type A CpG and the 5' end of poly-G and the activity are shown in Table 3. As can be seen, when the palindromic sequence core of type A CpG is "CGATCGATCG", A is gradually added after it (before the 5' end of the poly-G sequence), and with the increase of the number of inserted A, the stimulating activity on human PBMC gradually increases, and reaches the strongest when the number of inserted A is 2-4, and then decreases with the continuous increase of the number of A. When the palindromic sequence core of type A CpG is "TGCATCGATGCA" of CpG D19, A is gradually added after it (before the 5' end of the poly-G sequence), and with the increase of the number of inserted A, the stimulating activity on human PBMC gradually increases, and reaches the strongest when the number of inserted A is 2, and then decreases with the continuous increase of the number of A. When the palindromic sequence core of type A CpG is "GACGATCGTC" of CpG 2216, A is gradually added after it (before the 5' end of the poly-G sequence), and with the increase of the number of inserted A, the stimulating activity on human PBMC gradually increases, and reaches the strongest when the number of inserted A is 2, and then decreases with the continuous increase of the number of A.

[0115] The aggregation and activity data of the study on the insertion of T between the 3' end of the palindrome structure and the 5' end of poly-G of the A-type CpG are shown in Table 4. When the A added at the 3' end of the palindrome sequence "CGATCGATCG" is replaced by T, the addition of 2 T or 10 T at the end of the palindrome structure maintains good activity of the A-type CpG; when the A added at the 3' end of the palindrome sequence "TGCATCGATGCA" of CpG D19 is replaced by T, the addition of 2 T, 5 T or 10 T at the end of the palindrome structure maintains good activity of the A-type CpG, especially D19-11, which adds 2 T based on D19, and has better activity than D19; when the A added at the 3' end of the palindrome sequence "GACGATCGTC" of CpG 2216 is replaced by T, the addition of 2 T, 5 T or 10 T at the end of the palindrome structure maintains good activity, especially CpG 2216-11, which adds 2 T based on CpG 2216, and has better activity than CpG 2216.

[0116] The aggregation and activity data of the study on the insertion of C between the 3' end of the palindrome structure and the 5' end of poly-G of the A-type CpG are shown in Table 5. When the A added at the 3' end of the palindrome sequence "CGATCGATCG" is replaced by C, the activity of the sequence gradually decreases with the increase of C at the end of the palindrome structure; when the A added at the 3' end of the palindrome sequence "TGCATCGATGCA" of CpG D19 is replaced by C, the activity of the sequence gradually decreases with the increase of C at the end of the palindrome structure, but D19-14 adds 2 C based on D19, and has better activity than D19; when the A added at the 3' end of the palindrome sequence "GACGATCGTC" of CpG 2216 is replaced by C, the activity of the sequence gradually decreases with the increase of C at the end of the palindrome structure.

[0117] The present application breaks through the traditional design concept of A-type CpG, and creatively establishes a certain space barrier between poly-G and palindrome, increases the energy of G-tetrad, makes the aggregate relatively not easy to form or relatively loose after formation, successfully reduces the generation of aggregates of A-type CpG in the solvent, and improves 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 the traditional A-type CpG, it is found that when the number of G at the 5' end is 3-4, the generation of aggregates of the corresponding sequence is the least and the activity is the best, and this rule is verified on multiple CpG palindrome sequences. Based on these rules we found, in the newly designed sequences in this study, AM1012-05 is the best, the number of G at the 5' end is 4, two A are inserted at the 3' end of the palindrome sequence, and it has the characteristics of low aggregation and high activity, and has very high drugability.

[0118] The present study breaks the traditional thinking, by limiting the number of G at the 5' end of the palindromic sequence of A-type CpG (3-4), inserting an appropriate number of A, T (preferably 2) between the palindromic sequence and the 3' end poly-G, changing the aggregation of A-type CpG, and improving the activity of A-type CpG.

[0119] Table 2 Relationship between the number of G at the 5' end of the A-type CpG palindromic sequence and aggregation and activity

[0120] Number of G Aggregates (%) Activity (IFN-alpha, pg / mL) AM1012-05-18 AM1012-05-17 1 16.01 287.8 ± 149.5 a ]] AM1012-05-16 2 7.12 188.6 ± 69.5 a ]] AM1012-05 3 3.03 615.3 ± 58.3 a ]] AM1012-05-15 4 0.7 573.6 ± 47.2 a ]] AM1012-05-14 5 10.63 337.8 ± 109.7 a ]] Number of T 6 26.38 107.8 ± 75.8 a ]] D19-01 1 17.11 276.3 ± 96.6 b ]] D19 2 4.94 531.1 ± 137.6 b ]] D19-02 3 0.61 481.1 ± 48.1 b ]] D19-03 4 0.68 527.1 ± 106.7 b ]] D19-04 5 6.05 203.4 ± 45.6 b ]] 2216-04 1 23.28 256.8 ± 155.4 b ]] 2216-03 2 16.52 349.1 ± 122.4 b ]] 2216-02 3 2,17 323.5 ± 145.2 b ]] 2216-01 4 0.83 548.5 ± 40.4 b ]] 2216 5 4.21 310.2 ± 74.1 b ]]

[0121] Note: a, 0.2 μM; b, 0.8 μM

[0122] Table 3 Relationship between the number of A before the 3' end (poly-G) of the A-type CpG palindromic sequence and aggregation and activity

[0123]

[0124]

[0125] Note: b, 0.8 μM

[0126] Table 4 Relationship between the number of T before the 3' end (poly-G) of the A-type CpG palindromic sequence and aggregation and activity

[0127] Aggregates (%) Activity (IFN-alpha, pg / mL) AM1012-05 AM1012-05-20 AM1012-05-21 0 0.7 478.4 ± 44.9 b ]] AM1012-05-22 2 1.7 531.7 ± 11.9 b ]] Number of C 5 1.26 458.1 ± 17.3 b ]]> Aggregates (%) 10 10.38 576.6 ± 115.7 b ]] D19 0 4.94 345.9 ± 93.1 b ]] D19-11 2 15.22 502.3 ± 81.4 b ]] D19-12 5 20.75 398.4 ± 38.8 b ]] D19-13 10 23.56 228.1 ± 42.6 b ]]> 2216 0 4.21 435.8 ± 129.6 b ]] 2216-11 2 11.41 525.5 ± 64.0 b ]] 2216-12 5 19.68 455.8 ± 24.3 b ]] 2216-13 10 44.83 405.8 ± 192.3 b ]]

[0128] Note: b, 0.8 μM

[0129] Table 5 Relationship between the number of C before the 3' end (poly-G) of the A-type CpG palindromic sequence and aggregation and activity

[0130] Activity (IFN-alpha, pg / mL) AM1012-05 AM1012-05-23 AM1012-05-24 AM1012-05-25 0 0.7 478.4 ± 44.9 b ]] Figure 1 2 0.84 399.3 ± 108.4 b <!-- 14 -->]] Figure 2 5 5.75 258.4 ± 49.6 b ]] Figure 2 10 0 10.5 ± 1.4 b ]] D19 0 4.94 345.9 ± 93.1 b ]] D19-14 2 6.42 552.0 ± 134.2 b ]] D19-15 5 3.74 49.0 ± 2.4 b ]] D19-16 10 0 45.4 ± 1.0 b ]] 2216 0 4.21 435.8 ± 129.6 b ]] 2216-14 2 2.54 150.5 ± 39.3 b ]] 2216-15 5 6.5 118.8 ± 44.7 b ]] 2216-16 10 0 15.2 ± 0.5 b ]]

[0131] Note: b, 0.8 μM

[0132] Example 3: Cytokine release after stimulation of NK-92 cells by different CpG ODN

[0133] (1) NK-92 cells are human NK cell lines that express TLR-9 receptors in cells, which 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 amount according to the cell instruction manual, and resuspended with a certain amount of complete culture medium;

[0134] (2) 20 μL of cell suspension was taken, mixed with trypan blue at 1:1, and then counted using a cell counter to determine cell viability and cell density;

[0135] (3) 2.5 x 10 5Cells / well were seeded into 96-well plates, and CpG samples of different concentrations (sequences in Table 1) were added to each well, with 3 replicates per group;

[0136] (4) Incubate at 37℃ and 5% CO2 for 72 hours;

[0137] (5) After the cell culture is completed, centrifuge at 1200 rpm for 5 min, collect 100 μL of cell-free supernatant, and detect the secretion of IFN-γ in the supernatant according to the ELISA kit instructions.

[0138] Experimental results are as follows Figure 2 As shown in Table 1 of this invention, the CpG sequences designed can all effectively stimulate the secretion of IFN-γ from NK cells. Among them, the AM1012-03, AM1012-04, AM1012-05, AM1012-17, and AM1012-35 sequences have particularly significant NK cell stimulation effects. In addition, the stimulation effect of AM1012-05 is better than that of E41-1 and E41-2, and significantly better than that of E41-3. Figure 2 D in Figure 3 The E experiment results 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 certain amount, the sequence can exert the best immunoactivation effect.

[0139] Example 4: Effects of CpG sequence on the secretion of IFN-γ, IL-6, and TNF-α based on spleen cells from SD rats

[0140] (1) Take a certain amount of 100μM AM1012-05, E41-1, E41-2 and E41-3 solutions, prepare a certain concentration stock solution with RPMI 1640 complete culture medium, and then dilute with complete culture medium at a ratio of 4 times to prepare test samples of different concentrations.

[0141] (2) Obtain an appropriate amount of fresh SD rat spleen cells;

[0142] (3) Press 1×10 6 Cells / well were seeded into 96-well plates, and AM1012-05 sample at different concentrations were added to each well, with final concentrations of 3.2 μM, 0.8 μM, and 0.2 μM, respectively. Each group had 3 replicates, and PBS blank control wells were also set up.

[0143] (4) Incubate at 37℃ and 5% CO2 for 16h or 72h;

[0144] (5) Centrifuge and collect the supernatant;

[0145] (6) Detect IFN-γ, IL-6, TNF-α according to the ELISA detection instruction of different detection indexes.

[0146] The experimental results are shown in Figure 4 Table 2. The designed AM1012-05 and E41-1, E41-2, E41-3 can significantly stimulate the secretion of IFN-γ, TNF-α, and IL-6 of SD rat spleen cells, and have good dose dependence. However, the immune activation ability of AM1012-05 on SD rat spleen cells is significantly better than that of E41-1, E41-2, and E41-3. Combined with C in Figure 5 , it can be found that AM1012-05 has more excellent immune activation ability.

[0147] Example 5: Effect of AM1012-05 sequence on NK-92 cell factor release

[0148] (1) Prepare NK-92 cells to the required cell amount as described in Example 3, and resuspend with a certain amount of complete culture medium;

[0149] (2) Take 20 μL of the cell suspension, mix with trypan blue at a ratio of 1:1, and then use a cell counter to count the cell viability and cell density;

[0150] (3) Prepare AM1012-05 samples into a 100 μM solution with PBS;

[0151] (4) Take a certain amount of 100 μM AM1012-05 solution, prepare a certain concentration of mother liquor with complete culture medium, and then dilute the AM1012-05 solution with complete culture medium at a ratio of 2:1 to prepare AM1012-05 detection samples of different concentrations;

[0152] (5) 2.5 × 10 5 cells / well for 96-well plate plating, add corresponding AM1012-05 samples of different concentrations to each well, and the final concentrations are 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, with 3 replicate wells in each group, and set PBS blank control wells at the same time;

[0153] (6) Place in a 37°C, 5% CO2 incubator for 72 hours;

[0154] (7) After the cell culture is completed, centrifuge at 1200 rpm for 5 min, collect 100 μL of cell-free supernatant, and detect the secretion of IFN-γ in the supernatant according to the ELISA kit instruction.

[0155] The experimental results are shown in Figure 6As shown in Table 1, the AM1012-05 sequence can effectively stimulate the secretion of IFN-γ in NK-92 cells, and has good dose dependence. Since NK-92 cells express TLR9, this experiment also verifies that the immune activation activity of AM1012-05 is triggered by the combination with TLR9.

[0156] Example 6: Effect of AM1012-05 on the secretion of IL-6, IFN-γ, TNF-α, IFN-α, etc. based on BALB / c mouse spleen cells

[0157] (1) Take a certain amount of 100 μM AM1012-05 solution, and prepare a certain concentration of AM1012-05 detection sample by diluting the AM1012-05 solution with complete culture medium at a ratio of 2 times.

[0158] (2) Obtain a suitable amount of fresh BALB / c mouse spleen cells;

[0159] (3) Plate 1 x 10 6 cells / well in a 96-well plate, and add AM1012-05 samples of different concentrations to each well, with a final concentration of 8 μM, 4 μM, 2 μM, 1 μM, 0.5 μM, 0.25 μM, 0.13 μM, 0.06 μM, and 0.03 μM, with 3 replicate wells in each group, and set up PBS blank control wells at the same time;

[0160] (4) Incubate in a 37°C, 5% CO2 incubator for 16 h or 72 h;

[0161] (5) Centrifuge to collect the supernatant;

[0162] (6) Detect IL-6, IFN-γ, TNF-α, and IFN-α according to the ELISA detection instructions of different detection indexes.

[0163] The experimental results are shown in Table 2. Figure 7 The designed AM1012-05 sequence can effectively stimulate the secretion of cytokines in BALB / c mouse spleen cells, and has a certain dose dependence.

[0164] Example 7: Effect of AM1012-05 on the secretion of IL-6, IFN-γ, TNF-α, IFN-α, etc. based on C57BL6 mouse spleen cells

[0165] (1) Take a certain amount of 100 μM AM1012-05 solution, and prepare a certain concentration of AM1012-05 detection sample by diluting the AM1012-05 solution with complete culture medium at a ratio of 2 times.

[0166] (2) Obtain a suitable amount of fresh C57BL6 mouse spleen cells;

[0167] (3) Plate 96-well plates at 1 x 10 6 cells / well, and add AM1012-05 samples of different concentrations to each well, with final concentrations of 8 μM, 4 μM, 2 μM, 1 μM, 0.5 μM, 0.25 μM, 0.13 μM, 0.06 μM, and 0.03 μM, respectively, with 3 replicate wells in each group, and set up PBS blank control wells at the same time;

[0168] (4) Incubate in a 37°C, 5% CO2 incubator for 16 h or 72 h;

[0169] (5) Centrifuge to collect the supernatant;

[0170] (6) Detect IL-6, INF-γ, TNF-α, and IFN-α according to the ELISA detection instructions for different detection indexes.

[0171] The experimental results are shown in Table 1. Figure 8 The designed AM1012-05 sequence can effectively stimulate the secretion of cytokines of C57BL6 mouse spleen cells, and has a certain dose dependence.

[0172] Example 8: Effect of AM1012-05 on IFN-α, IFN-γ, TNF-α, and CXCL-10 mRNA transcription based on cotton rat spleen cells

[0173] (1) Take a certain amount of 100 μM AM1012-05 solution, and prepare a certain concentration of mother liquor using RPMI 1640 complete culture medium, and then dilute the AM1012-05 solution with complete culture medium at a ratio of 2 times to prepare AM1012-05 detection samples of different concentrations;

[0174] (2) Obtain a suitable amount of frozen cotton rat spleen cells;

[0175] (3) Plate 6-well plates at 5 x 10 6 cells / well, and add AM1012-05 samples of different concentrations to each well, with final concentrations of 0.2 μM, 0.8 μM, and 3.2 μM, respectively, with 3 replicate wells in each group, and set up PBS blank control wells at the same time;

[0176] (4) Incubate in a 37°C, 5% CO2 incubator for 3 h;

[0177] (5) Collect the cells, extract total RNA from the samples according to the total RNA extraction kit instructions, and determine the total RNA concentration using a Nanodrop;

[0178] (6) Reverse the RNA into cDNA according to the reverse transcription kit instructions;

[0179] (7) Analyze the expression level of each sample related cytokine according to the RT-qPCR kit instructions.

[0180] The experimental results are shown in Figure 9 The designed AM1012-05 sequence can effectively stimulate the secretion of immune-related cytokines of cotton rat spleen cells, and has good dose dependence. Cotton rat is a commonly used animal model for respiratory syncytial virus infection, which lays a foundation for using cotton rat to carry out anti-respiratory syncytial virus evaluation of AM1012-05.

[0181] Example 9: Detection of the influence of AM1012-05 on IFN-α and CXCL-10 mRNA transcription based on SD rat spleen cells

[0182] (1) Take a certain amount of 100 μM AM1012-05 solution, prepare a certain concentration of mother liquor with RPMI 1640 complete culture medium, then dilute the AM1012-05 solution with complete culture medium at a ratio of 2 times, and prepare AM1012-05 detection samples of different concentrations;

[0183] (2) Obtain a suitable amount of fresh rat spleen cells;

[0184] (3) Plate 5x10 6 cells / well in a 6-well plate, add corresponding AM1012-05 samples of different concentrations to each well, 3 replicate wells for each group, and set PBS blank control wells at the same time;

[0185] (4) Incubate in a 37℃, 5% CO2 incubator for 3h;

[0186] (5) Collect the cells, extract the total RNA of the samples according to the total RNA extraction kit instructions, and determine the total RNA concentration with Nanodrop;

[0187] (6) Reverse the RNA into cDNA according to the reverse transcription kit instructions;

[0188] (7) Analyze the expression level of each sample related cytokine according to the RT-qPCR kit instructions.

[0189] The experimental results are shown in Figure 10 The designed AM1012-05 sequence can effectively stimulate the transcription of IFN-α and CXCL-10 mRNA of SD rat spleen cells, and has good dose dependence.

[0190] Example 10: Effects of AM1012-05 on the secretion of IFN-γ, IL-6, and TNF-α based on spleen cells from SD rats

[0191] (1) Take a certain amount of 100μM AM1012-05 solution, prepare a certain concentration stock solution with RPMI 1640 complete medium, and then dilute AM1012-05 solution with complete medium at a ratio of 2 times to prepare AM1012-05 detection samples of different concentrations.

[0192] (2) Obtain an appropriate amount of fresh SD rat spleen cells;

[0193] (3) Press 1×10 6 Cells were seeded into 96-well plates, and AM1012-05 sample at different concentrations were added to each well, with final concentrations of 20 μM, 10 μM, 5 μM, 2.5 μM, 1.25 μM, 0.625 μM, 0.3125 μM, 0.1562 μM, and 0.087 μM, respectively. Each group had 3 replicates, and PBS blank control wells were also included.

[0194] (4) Incubate at 37℃ and 5% CO2 for 16h or 72h;

[0195] (5) Centrifuge and collect the supernatant;

[0196] (6) Detect IFN-γ, IL-6, and TNF-α according to the ELISA test instructions for different detection indicators.

[0197] Experimental results are as follows Figure 10 As shown, the designed AM1012-05 sequence effectively stimulated the secretion of IFN-γ, IL-6, and TNF-α by spleen cells in SD rats, with a good dose-dependent effect. SD rats are a commonly used animal model for preclinical drug toxicology evaluation. Combined with the data from Example 9, the results of this experiment lay the foundation for conducting safety evaluation experiments of AM1012-05 using SD rats.

[0198] Example 11: Effects of AM1012-05 on the secretion of IFN-α, IFN-γ, IFN-λ, IL-6, TNF-α, and CXCL-10 based on PBMC detection in beagle dogs

[0199] (1) Take a certain amount of 100μM AM1012-05 solution, prepare a certain concentration stock solution with RPMI 1640 complete medium, and then dilute AM1012-05 solution with complete medium at a ratio of 4 times to prepare AM1012-05 detection samples of different concentrations.

[0200] (2) Obtain an appropriate amount of fresh Beagle PBMCs;

[0201] (3) 5 x 10 6 cells / well to perform 6-well plate plating, and each well was added with AM1012-05 sample of a corresponding different concentration, and the final concentration was 0.2 μM, 0.8 μM, 3.2 μM, respectively, and each group had 3 duplicate wells, and PBS blank control wells were set at the same time;

[0202] (4) Cultured in a 37℃, 5% CO2 incubator for 6h;

[0203] (5) Collected cells, and total RNA of samples was extracted according to the instruction of total RNA extraction kit, and total RNA concentration was determined by Nanodrop;

[0204] (6) RNA was reversely transcribed into cDNA according to the instruction of reverse transcription kit;

[0205] (7) Expression level of each sample related cytokine was analyzed according to the instruction of RT-qPCR kit.

[0206] The experimental results are shown in Table 1. Figure 10 The designed AM1012-05 sequence can effectively stimulate secretion of immune related cytokines of beagle dog PBMC, and has good dose dependence. Beagle dog is an animal model commonly used for preclinical evaluation of drug toxicity of drugs, which lays a foundation for using beagle dog to carry out safety evaluation experiment of AM1012-05. In addition, the pharmacodynamics of AM1012-05 on beagle dog PBMC also suggests its great potential as a pet vaccine adjuvant or in the application of anti-infection.

[0207] Example 12: Detection of the influence of AM1012-05 and related sequences on secretion of IFN-α, IFN-γ, IFN-λ, TNF-α, IL-6 and CXCL-10 based on healthy human PBMC cells

[0208] (1) A certain amount of 100 μM AM1012-05 and related sequence solution was prepared into a certain concentration mother liquor with RPMI 1640 complete culture medium, and then the AM1012-05 and related sequence solution was diluted with complete culture medium at a ratio of 4 times to prepare detection samples of different concentrations;

[0209] (2) Obtained a certain amount of frozen healthy human PBMC cells;

[0210] (3) 2 x 10 5 cells / well to perform 6-well plate plating, and each well was added with AM1012-05 sample of a corresponding different concentration, and the final concentration was 0.2 μM, 0.8 μM, 3.2 μM, respectively, and each group had 3 duplicate wells, and PBS blank control wells were set at the same time;

[0211] (4) Cultured in a 37℃, 5% CO2 incubator for 6h;

[0212] (5) Centrifuge and collect the supernatant;

[0213] (6) Detect cytokines according to the ELISA test instructions.

[0214] Experimental results are as follows Figure 10 As shown, AM1012-05 can stimulate IFN-α in PBMCs in a dose-dependent manner. Figure 10 (A), IFN-γ ( Figure 10 (B), IFN-λ ( Figure 10 C), TNF-α ( Figure 10 D), IL-6 ( Figure 11 (E), CXCL-10 ( Figure 12 The secretion of IFN-α. Furthermore, AM1012-05 and related sequences showed significantly better activation of IFN-α than CpG 2216 and D19 (F). Figure 13 (G). Among them, AM1012-05 showed a significant effect in promoting IFN-α secretion, with an EC50 of 0.19 μM and an optimal stimulation concentration of 0.5 μM. 1 μM AM1012-05 also showed a significant effect in promoting IFN-γ secretion, and 0.25 μM AM1012-05 showed a significant effect in promoting IFN-λ secretion, demonstrating that AM1012-05 has a very strong antiviral potential. AM1012-05 can stimulate TNF-α secretion in healthy human PBMCs with good dose dependence, but its ability to stimulate TNF-α is significantly lower than its ability to stimulate IFN-α (EC50 of 0.19 and 2.46 μM, respectively). This indicates that when AM1012-05 exerts highly effective antiviral activity (IFN-α, IFN-γ, IFN-λ) at lower doses, it does not trigger inflammatory responses represented by TNF-α and IL-6.

[0215] Example 13: Serum stability assay of CpG ODN

[0216] This experiment investigated the degree of sequence damage caused by fetal bovine serum (FBS) using polyacrylamide gel electrophoresis (PAGE). The specific procedure was as follows: AM1012-05, AM1012-03, E41-2, and E41-3 were each prepared into 25 OD / mL aqueous solutions. 10 μL of 10% FBS was added to each solution, and the solutions were incubated at 37°C for a specified time. After incubation, the degree of sequence damage was detected using PAGE. The results are as follows: Figure 13The results show that AM1012-05 (1205) is stable in 25% fetal bovine serum solution, E41-3 degrades most (4103), AM1012-03 (1203) and E41-2 (4102) degrade in the middle, and AM1012-05 (1205) degrades the least. Therefore, the sample of AM1012-05 has better stability compared to E41-2 and E41-3.

[0217] Example 14: Analysis of the aggregation state distribution of different CpG ODN aggregates

[0218] Take 3 mg of AM1012-03, E41-2, E41-3, and AM1012-05 and add them to 1 ml of deionized water to obtain a clear solution.

[0219] Divide the above four solutions equally into two groups: one group is placed at room temperature, and the other group is heated in a 95°C water bath for 30 minutes and then rapidly cooled to below 4°C with ice water. Take the above eight solutions and add an equal amount of 800 mM sodium chloride solution to each to make the samples in a 400 mM sodium ion environment. After incubation for 24 hours, centrifuge to separate the precipitate. Redissolve the precipitate in water to obtain a clear solution. Test the aggregation state distribution of each sample by polyacrylamide gel (PAGE) electrophoresis.

[0220] The electrophoresis results are shown in https: / / The M marks in the figure are 20, 40, and 80 base markers, and lanes 1, 2, 3, and 4 correspond to AM1012-03, E41-2, E41-3, and AM1012-05 samples placed at room temperature, respectively, and lanes 5, 6, 7, and 8 correspond to AM1012-03, E41-2, E41-3, and AM1012-05 samples heated in a 95°C water bath for 30 minutes and then rapidly cooled to below 4°C with ice water. The left graph shows the electrophoresis results after the precipitate is dissolved in water for 2 hours, and the right graph shows the electrophoresis results after the same sample solution is placed for 24 hours.

[0221] Literature reports indicate that the polyG sequence at the 5' or 3' end of A-type CpG can cause A-type CpG to form an aggregation state with the assistance of metal ions, accumulate aggregation, and cause precipitation. The controllability of the aggregation state is a key indicator for the study of the drugability of A-type CpG (Kerkmann et al., J Biol Chem. 2005 Mar 4; 280(9): 8086-93.).

[0222] In this experiment, samples incubated in 400 mM sodium ion environment all formed precipitates. After the precipitates were re-dissolved in water, the high polymerization state of type A CpG gradually depolymerized in the absence of metal ions and could be re-dissolved. The dimer and tetramer and other oligomerization states also had certain solubility in water, so observing the re-dissolved samples at different time points could maximize the observation of the (low) aggregation state distribution of each type A CpG and the trend over time.

[0223] For samples left at room temperature, the re-dissolved solution of AM1012-03 was mainly 4-mer and monomer after 2 hours, and was a diffuse band after 24 hours; the re-dissolved solution of E-41-2 was mainly dimer after 2 hours, and was still mainly dimer after 24 hours, but gradually appeared tetramer; the re-dissolved solution of E-41-3 showed dimer and polymer (greater than tetramer) after 2 hours, and appeared a diffuse band mainly of dimer after 24 hours; in the re-dissolved solution of AM1012-05, most were in monomer state after 2 hours and 24 hours, and only a small amount of aggregates.

[0224] For samples heated in a 95°C water bath for 30 minutes and then rapidly cooled to below 4°C with ice water, the re-dissolved solution of AM1012-03 was mainly 4-mer and monomer after 2 hours, and appeared a diffuse band after 24 hours; the re-dissolved solution of E-41-2 was mainly dimer after 2 hours, and was still mainly dimer after 24 hours, but gradually appeared tetramer and monomer; the re-dissolved solution of E-41-3 showed a variety of aggregation states after 2 hours, and appeared a diffuse band mainly of monomer and dimer after 24 hours. In the re-dissolved solution of AM1012-05, most were in monomer state after 2 hours and 24 hours, and only a small amount of aggregates.

[0225] From the above results, AM1012-05 is more likely to depolymerize back to monomer state than the other several type A CpGs under the same conditions. The other several type A CpGs can transform between monomer, dimer, tetramer or other polymer forms. Therefore, in actual preparation applications, as long as the concentration of metal salt is controlled, AM1012-05 can be stably maintained in monomer state. After the preparation enters the human body, AM1012-05 can produce the aggregation state characteristic of type A CpG under the physiological concentration of salt to produce efficacy.

[0226] At the same time, CpG 2216 and AM1012-05 were respectively prepared into 0.5 mg / mL solutions with 10 mM phosphate buffer solution, and the particle size of the CpGs was measured after standing overnight using a Nicomp Z3000 laser particle size instrument. After the measurement was completed, the particle size was measured again after the solution was vortexed for 3 minutes. The results are shown in Table 2. rna.urmc.rochester.edu / The particle size distribution of CpG 2216 before vortex was about 2 nm, 75 nm, and 250 nm, while after vortex, the particle size of CpG 2216 was concentrated at about 280 nm. The particle size of AM1012-05 was about 30 nm in the resting state, while after vortex, the particle size of AM1012-05 was concentrated at about 2 nm.

[0227] The laser particle size analyzer detection results showed the aggregation state of CpG in the natural state. In 10 mM phosphate buffer solution, AM1012-05 showed a loose aggregation state in the resting state, and when subjected to mechanical force, the aggregation was opened and became a monomer state. However, CpG 2216 showed a dispersed complex multimer in 10 mM phosphate buffer solution, and could not be dispersed under mechanical force.

[0228] Figure 14-24 The results showed that CpG 2216 was a traditional A-type CpG, and there was no additional deoxyribonucleotide between the 3' end of the palindrome structure and the 5' end of the poly-G, no certain steric hindrance, the energy of forming G-tetrad was reduced, and the aggregate formed was tightly combined and could not be easily dispersed by mechanical force. AM1012-05 inserted 2 A between the 3' end of the palindrome structure and the 5' end of the poly-G, forming a certain steric hindrance, increasing the energy of forming G-tetrad, and making the multimer relatively not easy to form or relatively loose after forming. Therefore, in the natural resting state, AM1012-03 was in an aggregation equilibrium state of about 30 nm uniformly distributed, which significantly improved the drugability of traditional A-type CpG.

[0229] Example 15: Secondary structure simulation analysis and comparison of different sequences

[0230] The sequences of CpG 2216, E41-1, AM1012-3, and AM1012-5 were predicted for secondary structure using online modeling tools (RNAstructure, NUPACK, and ViennaRNA) respectively, and the temperature parameter was adjusted to obtain the secondary structure of each sequence under different temperature conditions. Figure 25 Regulator Class The specific secondary structure schematic diagram is as follows:

[0231] The specific secondary structure schematic diagram is as follows: MannitolFrom the comparison of the secondary structures of the four at different temperatures, we can find that CpG 2216 is relatively stable at 4℃-37℃, when the temperature is higher than 40℃, its secondary structure is misfolded, its biological activity is affected; E41-1 is relatively stable at 0℃-37℃, when the temperature reaches-20℃, partial internal hydrogen bonds are broken, when the temperature is higher than 40℃, its secondary structure is misfolded, too high or too low temperature will affect its biological activity; AM1012-03 is also relatively stable at 0℃-37℃, when the temperature reaches 0℃, partial internal hydrogen bonds are broken, when the temperature is higher than 40℃, its secondary structure is also misfolded, and then affects the biological activity; while AM1012-05 can maintain its constant secondary structure at 0℃-50℃, only when the temperature is higher than 60℃, partial internal hydrogen bonds are broken. This shows that the secondary structure of AM1012-05 sequence is more stable than that of CpG 2216, E41-1 and AM1012-03, which also explains from the structure that the stability and activity of AM1012-05 are better than those of CpG 2216, E41-1 and AM1012-03.

[0232] Example 16: Evaluation of the stimulation ability of CpG ODNs with different aggregation degrees on rat spleen cells

[0233] (1) Take a certain amount of 100 μM CpG ODN solution treated at different temperatures, prepare a certain concentration mother liquor with RPMI 1640 complete culture medium, then dilute the CpG ODN solution with complete culture medium according to 4 times proportion, prepare detection samples with different concentrations;

[0234] (2) Obtain a certain amount of fresh SD rat spleen cells;

[0235] (3) Plate 1×10 6 cells / well in a 96-well plate, add corresponding CpG ODN samples with different concentrations to each well, and the final concentration is 3.2 μM and 0.8 μM, respectively, 3 replicate wells in each group, and set PBS blank control wells at the same time;

[0236] (4) Place in a 37℃, 5% CO2 incubator for 72 h;

[0237] (5) Centrifuge to collect the supernatant;

[0238] (6) Detect IFN-γ according to the ELISA detection instruction of rat IFN-γ.

[0239] The experimental results are as follows: GlycerolAs shown, the designed AM1012-05 sequence can still effectively stimulate the secretion of IFN-γ of SD rat spleen cells after high temperature treatment, while the AM1012-03 sequence cannot effectively stimulate the secretion of IFN-γ of 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 that of CpG 2216, E41, AM1012-03, and this experiment verifies that stable secondary structure is the prerequisite for the activity of CpG.

[0240] Example 17: Changes in stability of CpG ODN raw materials prepared by different processes

[0241] The E41-2, E41-3, and AM1012-05 raw materials were prepared into 2 mg / mL stock solutions, and were subjected to different process treatments: direct freeze-drying, and freeze-drying after heating at 95°C. The aggregation degree and related substances of the dry powders prepared by different freeze-drying processes were detected by reverse phase HPLC. The aggregation state proportions of E41-2, E41-3, and AM1012-05 prepared by different processes changed slightly due to the different processes. However, the impurities detected in the E41-2 and E41-3 dry powders prepared by different processes were quite different. The impurity levels detected in the AM1012-05 prepared by different processes were basically the same. The results show that the stability of AM1012-05 is better than that of E41-2 and E41-3, and has better drug properties.

[0242] Table 6: Stability analysis of CpG ODN raw materials prepared by different processes

[0243]

[0244] Example 18: pH screening of AM1012-05 preparations

[0245] (1) Prepare different pH sodium phosphate buffer solutions: pH 6.6, 7.0, 7.2, 7.4, 8.0, 9.0;

[0246] (2) Dissolve AM1012-05 in the above buffer solutions to prepare 0.35 mg / ml prescription solutions;

[0247] (3) Divide each prescription solution into 10 mL vials according to 1.5 mL per vial, press the stopper, roll the cap, and place it in a 50°C environment in the dark;

[0248] (4) Take samples for detection at 0, 5, and 10 days.

[0249] The experimental results are shown in Table 7, and the results show that AM1012-05 is easily degraded in a slightly acidic environment. From the preparation, it is stable within the pH range of 7.0-9.0.

[0250] Table 7 pH range screening results of samples

[0251]

[0252] Example 19: Selection of AM1012-05 solution buffer

[0253] In combination with the pH range and commonly used buffer systems of marketed small nucleic acid drugs, phosphate and citrate buffer systems were screened, respectively. 2.5 mg / ml was selected as the concentration of AM1012-05 experimental samples, and the water solution of AM1012-05 without adding buffer was used as a control. The pH of the sample was controlled at 7.2, and the sample was placed at 50°C for 10 days and sampled at 0, 5, 10 days, respectively. The stability of the samples in the two buffer solutions was compared with the control group; the results showed that compared with the control group, the purity change of the sample with added buffer was significantly reduced, and the change trend of each observation item of the two buffer system samples was consistent with the extension of the placement time. The experimental results are shown in Table 8. The buffer of the two buffer systems can meet the needs of the pH buffer of the preparation.

[0254] Table 8 Buffer screening results

[0255]

[0256] Example 20: Range screening of CpG ODN metal ion concentration

[0257] 5 mg of sodium acetate, sodium phosphate dibasic / sodium phosphate dibasic, magnesium chloride, calcium chloride, potassium chloride, sodium chloride, respectively, were taken and mixed with 0.25 mg of AM1012-05 to prepare 200 μl solution samples. The test samples were placed in a sealed clean container and stored at 40°C for 30 days and sampled at 5 days, 15 days and 30 days. The detection indexes include characteristics (appearance), solubility, impurities (related substances) and content.

[0258] The detection results show that sodium acetate, sodium phosphate dibasic / sodium phosphate dibasic, magnesium chloride, calcium chloride, potassium chloride, and sodium chloride produce precipitation when mixed with AM1012-05 at a ratio of concentration. Therefore, it is necessary to screen the salt concentration suitable for CpG ODN preparation to ensure the clarity of the preparation solution.

[0259] AM1012-05 was selected, and potassium chloride and sodium chloride solutions were added, respectively, to obtain a solution of AM1012-05 with a final concentration of 2 mg / ml, and the final concentration of the salt solution was 90 mM. After 24 hours of placement, AM1012-05 remained clear in these two salt solutions.

[0260] AM1012-05 was selected and magnesium chloride and calcium chloride solutions were added respectively. The final concentration of AM1012-05 in the resulting solution was 2 mg / ml, and the final concentration of the salt solution was 1.8 mM. After standing for 24 hours, AM1012-05 remained clear in both salt solutions.

[0261] Four phosphate buffer solutions (composed of sodium dihydrogen phosphate and disodium hydrogen phosphate) with concentrations of 5, 10, 20, and 50 mM were selected as the test concentrations (sodium ion concentrations of approximately 9, 18, 36, and 90 mM, respectively), and compared with isotonic (150 mM) PBS solution (sodium ion concentration of approximately 179 mM). The experimental concentration of AM1012-05 was set at 2.5 mg / ml, and the sample pH was controlled at 7.2. The samples were placed at 50℃ for 10 days, and samples were taken at 0, 5, and 10 days to test the stability of the samples and determine the pH buffer concentration. The results are shown in Table 9. Compared with day 0, the clarity and properties of the samples did not change significantly after 5 days at 50℃, and the purity did not change significantly with the extension of the storage time. However, AM1012-05 under isotonic PBS showed obvious turbidity. Therefore, isotonic (150 mM) PBS is not suitable for preparing AM1012-05 formulations. Taking into account both pH buffering capacity and the risk of AM1012-05 precipitation, the sodium ion concentration in the solution formulation was 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 ranged from 0.1 to 90 mM.

[0262] Table 9. Screening of Salt Buffer Concentration

[0263]

[0264] Example 21: Selection of isotonic adjuster for AM1012-05 solution

[0265] Sodium chloride is generally used to adjust isotonicity in solutions. However, since 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 out of the solution. Therefore, other excipients need to be selected as isotonic adjusters.

[0266] Excipients that can be used as isotonic regulators include glycine, trehalose, mannitol, sucrose, arginine, lysine, histidine, sorbitol, glucose, glycerol, and propylene glycol.

[0267] The above excipients were each prepared into a 200 μl solution sample at 0.25 mg AM1012-05, 5 mg excipient per tube sample. The test sample was placed in a sealed clean container, and was placed at 40°C for 30 days and sampled at 5th day, 15th day and 30th day. The detection indexes included the characteristics (appearance), the solubility of interest, impurities (related substances) and content. The detection results showed that the solution mixed by sorbitol and glucose with AM1012-05 respectively had abnormal peak shape in the HPLC chromatogram, and the compatibility of sorbitol and glucose with AM1012-05 was not good, which was not suitable for being used as the isotonicity regulator in the preparation of AM1012-05. Glycine, trehalose, mannitol, sucrose, arginine, lysine, histidine, glycerol and propylene glycol were compatible with AM1012-05, and could be used as the isotonicity regulator of AM1012-05.

[0268] AM1012-05 was configured into a 0.35 mg / ml prescription solution with three kinds of isotonicity regulators and phosphate buffer. And the characteristics (appearance), clarity, pH, osmotic pressure and purity were detected, and the specific prescription was shown in Table 10:

[0269] Table 10 Preparation of AM1012-05 prescription

[0270]

[0271]

[0272] The experimental results were shown in Table 11, and the results showed that the three kinds of prescriptions of AM1012-05 could all obtain clear solution, and the pH, osmotic pressure and purity all met the requirements of the preparation.

[0273] Table 11 Detection results of three kinds of AM1012-05 prescriptions

[0274] Propylene Glycol Properties Colorless clear liquid Colorless clear liquid Colorless clear liquid Clarity pH Osmol / kg Purity % ≤0.5 ≤0.5 ≤0.5 Figure 26 7.33 7.27 7.31 Figure 27 324 284 296 Figure 28 96.4 96.33 96.4

[0275] Example 22: Preparation of freeze-dried prescription of AM1012-05

[0276] AM1012-05 was dissolved in deionized water, and mannitol (lyoprotectant) and pH regulator (disodium hydrogen phosphate + citric acid) were added. A solution containing 1 mg / mL of AM1012-05, 5% of mannitol, 50 mM of disodium hydrogen phosphate + citric acid was prepared.

[0277] The above solution was filled into 0.2 mL to 2 mL vials, semi-crimped and transferred into a freeze dryer for lyophilization at -30°C. After the lyophilization was completed, the samples were crimped and capped to obtain white lyophilized powder cakes. The lyophilized preparation of AM1012-05 can be immediately reconstituted after adding 1 mL of water for injection. The lyophilized preparation of AM1012-05 can store AM1012-05 in solid form, which is conducive to the stability of the main drug, and is an alternative preparation of the liquid preparation of AM1012-05.

[0278] Example 23: Selection of other excipients for AM1012-05 solution

[0279] In addition to the isotonicity adjusting agent and the pH buffer system, other excipients can also be added in the solution preparation of CpG ODN to provide corresponding functions.

[0280] 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 adjusting agents (hypromellose, sodium carboxymethylcellulose sodium citrate), methyl betadex, hydroxypropyl betadex, antioxidants (ascorbic acid), etc.

[0281] In this test, 5 mg of Pluronic F127, Pluronic F68, Tween 20, Tween 80, and ascorbic acid were respectively prepared into 200 μl solution samples with 0.25 mg of AM1012-05. The test samples were placed in a sealed clean container and stored at 40°C for 30 days, and samples were taken on the 5th day, the 15th day and the 30th day. The detection indexes include appearance, solubility, impurities (related substances) and content.

[0282] At the same time, benzalkonium chloride, disodium edetate, sodium citrate, hypromellose, sodium carboxymethylcellulose, methyl betadex, and hydroxypropyl betadex were prepared according to the concentrations in Table 12, and AM1012-05 was added to prepare a 0.4 mg / mL prescription solution, and the pH was adjusted to 7.0-7.5. The test samples were placed in a sealed clean container and stored at 40°C for 30 days, and samples were taken on the 5th day, the 15th day and the 30th day. The detection indexes include appearance, solubility, impurities (related substances) and content.

[0283] Table 12: Excipient Table

[0284]

[0285]

[0286] The detection results show that the mixture of ascorbic acid and AM1012-05 can cause degradation of AM1012-05, and therefore is not suitable for the preparation of AM1012-05. The mixture of hydroxypropyl betacyclodextrin and AM1012-05 is detected by HPLC method, and the corresponding AM1012-05 main peak peak type appears abnormal, and therefore is not suitable for the preparation of AM1012-05.

[0287] Pluronic F127, Pluronic F68, Tween 20, Tween 80, benzalkonium chloride, disodium edetate, sodium citrate, hydroxypropyl methyl cellulose, sodium carboxymethyl cellulose, methyl betacyclodextrin are compatible with AM1012-05 and can be used as excipients for AM1012-05 related preparations.

[0288] Example 24: Preparation of PLGA nano-preparation of CpG ODN

[0289] (1) Dissolve 100 mg of PLGA in 1.25 mL of dichloromethane (oil phase).

[0290] (2) Prepare a solution of 12 mg / ml AM1012-05, 1% PVA. (Aqueous phase 1)

[0291] (3) Prepare a 4 ml 2.5% PVA solution. (Aqueous phase 2)

[0292] (4) Homogenize the oil phase with aqueous phase 1 to form a primary emulsion, then add the primary emulsion to aqueous phase 2 and homogenize to form a multiple emulsion.

[0293] (5) Slowly add the formed multiple emulsion to 1% PVA aqueous solution (external aqueous phase), stir for 4 h, and volatilize dichloromethane.

[0294] (6) Centrifuge 500 g of the solution of step 5 for five minutes to remove large particle microspheres, and collect the supernatant.

[0295] (7) Centrifuge the supernatant of step 6: 10000 g for 10 minutes, collect the solid, and wash with deionized water or ultrapure water three times to obtain the PLGA microsphere product.

[0296] The obtained PLGA microsphere product is tested by Nicomp Z3000 laser particle size instrument, and the particle size can be controlled within 500 nanometers (as shown in Figure 29

[0297] Example 25: Preparation of chitosan nano-preparation of CpG ODN

[0298] (1) Prepare a 0.5 mg / ml chitosan (molecular weight 200,000) solution, adjust the pH to 5.6 ​

[0299] (2) Prepare a 50 μg / ml solution of AM1012-05

[0300] (3) 10 ml of AM1012-05 solution is added dropwise to 10 ml of chitosan solution, and stirred for 1 minute.

[0301] The AM1012-05 chitosan nanoparticles prepared in this experiment are tested by a laser particle size analyzer dynamic light scattering particle size analyzer, and the particle size can be controlled at about 150 nanometers (as shown in Figure 30

[0302] Example 26: Preparation of a lipid nanoparticle preparation of CpG ODN

[0303] The lipid nanoparticles (LNP) of CpG ODN are synthesized by self-assembly of CpG wrapped nanoparticles by selecting different types of lipids in a certain ratio. The lipids include ionizable cationic liposomes, neutral phospholipids, sterol lipids, and PEGylated phospholipids. The specific preparation process is as follows:

[0304] (1) SM-102, DSPC, cholesterol, DMG-PEG (2000) are dissolved in ethanol at a ratio of 50:10:38.5:1.5,

[0305] (2) Take 0.25 ml of the above lipid solution, add 0.75 ml of 80 μg / ml AM1012-05, 50 mM citric acid buffer solution, vortex mix, and then transfer to a hand-push liposome extruder.

[0306] (3) The mixture is quickly moved and extruded by the extruder. The mixture is left to stand for 10 minutes.

[0307] (4) Using a 200KD dialysis bag, the mixture is added to an equal volume of 50mM citric acid buffer solution, sealed and placed in 50ml PBS solution for dialysis.

[0308] The AM1012-05 lipid nanoparticles prepared in this experiment are tested by Nicomp Z3000 laser particle size analyzer, and the particle size can be controlled at about 100 nanometers (as shown in Figure 31

[0309] Example 27: Preparation of a PLGA nanoparticle preparation of CpG ODN in an immunoadjuvant

[0310] (1) Dissolve 100 mg of PLGA in 1.25 mL of dichloromethane (oil phase).

[0311] ​​(2) AM1012-05 and antigen protein (S1 protein) were added to a solution of 1% PVA to prepare a solution containing 10 mg / ml AM1012-05 and 10 mg / ml S1 protein. (Aqueous phase 1)

[0312] (3) A 4 ml 2.5% PVA solution was prepared. (Aqueous phase 2)

[0313] (4) The oil phase was homogenized with aqueous phase 1 to form a primary emulsion, and then the primary emulsion was added to aqueous phase 2 and homogenized to form a multiple emulsion.

[0314] (5) The formed multiple emulsion was slowly added to a 1% PVA aqueous solution (external aqueous phase) and stirred for 4 h to volatilize dichloromethane.

[0315] (6) The solution of step 5 was centrifuged at 500 g for 5 min to remove large particles of microspheres, and the supernatant was collected.

[0316] (7) The supernatant of step 6 was centrifuged at 10,000 x g for 10 min, and the solid was collected. The product of PLGA microspheres was obtained by washing with deionized water or ultrapure water three times.

[0317] The PLGA microspheres vaccine product obtained by wrapping AM1012-05 and antigen protein in this experiment was tested by Nicomp Z3000 laser particle size analyzer, and the particle size could be controlled within 500 nanometers (as shown in Figure 32 ).

[0318] Example 28: Investigation of excipient compatibility of topical formulation of CpG ODN

[0319] The excipients that can be used in the topical formulation are classified as oil phase, thickening agent, gelling agent, emulsifying agent, solvent, and penetration enhancer. The oil phase includes glyceryl monobehenate, liquid paraffin, vaseline, medium-chain triglyceride, and cocoyl octa-decylate; the thickening agent includes glyceryl monostearate, glyceryl mono-diestearate, cetylstearyl alcohol; the gelling agent includes hydroxypropyl methylcellulose, xanthan gum, carbomer, sodium carboxymethylcellulose, poloxamer 407; the emulsifying agent includes polyoxyl 40 stearate, macrogol cetostearyl ether, Tween 60, poloxamer 188, oleoyl polyoxylethylene glycerol, macrogol-7 stearate, caprylocaproyl macrogol glycerides, sodium lauryl sulfate, propylene glycol monofatty acid ester, polyglyceryl oleate, propylene glycol laurate; the solvent includes triacetin, propylene glycol; the penetration enhancer includes isopropyl myristate, propylene glycol, caprylocaproyl macrogol glycerides, polyglyceryl oleate, propylene glycol laurate.

[0320] The gel adjuvant is prepared into a suitable concentration of aqueous solution, and then mixed with AM1012-05 to prepare a 3 mg / g gel solution. Other adjuvants are mixed with the API one by one to prepare a 3 mg / g mixture. The samples are placed in a 40°C incubator, and the content and related substances are detected at 5, 15, 30 days, respectively. The results show that the adjuvants compatible with AM1012-05 include: oil phase including glyceryl behenate, liquid paraffin, vaseline, medium-chain triglyceride, cocoyl octa-decylate; thickening agent including mono-diglyceride; gel agent including hydroxypropyl methyl cellulose, carbomer, xanthan gum, sodium carboxymethyl cellulose, poloxamer 407; emulsifier including polyethylene glycol hexadecanolether, poloxamer 188, oleoyl polyoxyethylene glycerol, polyethylene glycol-7 stearate, caprylocaproyl macrogol glycerides, sodium dodecyl sulfate, propylene glycol monofatty acid ester, polyglyceryl oleate, propylene glycol laurate; solvent including propylene glycol; penetration enhancer including isopropyl myristate, propylene glycol, polyglyceryl oleate.

[0321] Example 29: Preparation of an external ointment preparation of CpG ODN

[0322] AM1012-05 is weighed, dissolved in deionized water, and an API solution is prepared. An appropriate amount of vaseline and liquid paraffin is weighed and placed in a 80°C water bath to melt. The API solution is added to the molten oil phase, and homogenized at 70-80°C for 10 minutes. The temperature is lowered to 65-70°C for dispensing. The content of AM1012-05 in the final preparation is 1 mg / g. The ratio of vaseline and liquid paraffin is determined according to the body sensation evaluation, and the proportion of vaseline can be selected between 100%-50%.

[0323] Example 30: Preparation of an external gel preparation of CpG ODN

[0324] A carbomer 974 aqueous solution (2%) and a 10% triethanolamine and AM1012-05 aqueous solution are prepared. The 2% carbomer 974 aqueous solution is diluted to a concentration of 0.7%, 10% triethanolamine is added to adjust the pH to 7.0, phenoxanol is dissolved in propylene glycol and added to the gel, stirred and mixed, then the API aqueous solution is added, stirred and mixed. 10% triethanolamine is added again to adjust the pH to the appropriate value, and then the volume is adjusted, stirred and mixed, and then dispensed.

[0325] The carbomer used to prepare the gel can be adjusted in concentration and type, such as carbomer 971, 980, 981, etc., according to the viscosity requirement. In addition to triethanolamine solution, neutralizing agents can also include sodium hydroxide, aminomethyl propanol, tromethamine, tetra-2-hydroxypropyl ethylenediamine, etc. In addition to phenoxanol, preservatives can also include nipagin esters and cresol preservatives.

[0326] Example 31: Preparation of an external emulsion of CpG ODN

[0327] (1) Oil phase: 15 g of vaseline, 6 g of liquid paraffin and 7.2 g of glyceryl monostearate were charged into a container, heated to 70°C, and melted with stirring. The molten oil phase was kept at 70°C and stirred at low speed.

[0328] (2) Water phase: 59.5 g of deionized water was heated to 70°C in a container, and 1.8 g of macrogol cetostearyl ether was added and dissolved.

[0329] (3) Emulsion preparation: At 70°C, the molten oil phase in step 1 was transferred to the water phase container while mixing at 10 rpm.

[0330] (4) After the transfer was completed, the homogenizer was started and homogenized for 10 minutes, with the temperature controlled at 65-70°C.

[0331] (5) Stop the homogenizer, set the stirrer to a temperature of 50°C, and cool the emulsion to 50°C.

[0332] (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.

[0333] (7) The drug solution in step 6 was transferred to the emulsion in step 5, and the temperature was 50°C while stirring.

[0334] (8) Start the homogenizer, mix and homogenize for 10 minutes, and adjust the pH to 7.5 by adding 10% sodium hydroxide solution dropwise.

[0335] (9) When the emulsion is cooled to 25°C, the prepared emulsion is aliquoted, and finally 100 g of AM1012-05 emulsion containing 0.3 mg / g is prepared.

[0336] The oil phase, thickening agent, and emulsifying agent used in this emulsion can be replaced by excipients that pass the excipient compatibility test. Penetration enhancer excipients that pass the excipient compatibility test can also be added to the emulsion.

[0337] Example 32: Preparation of a vaccine with CpG as an adjuvant

[0338] This example uses the S1 protein of the new coronavirus as a model antigen to prepare a vaccine with CpG as an adjuvant

[0339] (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;

[0340] (2) Prepare a 2 mg / mL solution of AM1012-05 or CpG 1018;

[0341] (3) 250 μL of the solution of step (2) was added to the solution of step (1) and mixed evenly.

[0342] Example 33: Preparation of vaccine of adjuvant system (aluminum adjuvant + CpG)

[0343] This example takes the S1 protein of the new coronavirus as a model antigen to prepare a vaccine of an adjuvant system (aluminum adjuvant + CpG)

[0344] Preparation of vaccine with aluminum hydroxide + CpG as adjuvant:

[0345] (1) 50 μL of SARS-CoV-2 S1 protein (2 mg / ml PBS solution) was taken;

[0346] (2) 50 μL of aluminum hydroxide gel adjuvant (2%) was added to the solution of step (1);

[0347] (3) 350 μL of PBS solution was added to the solution of step (2) and mixed evenly;

[0348] (4) 50 μL of 2 mg / mL AM1012-05 solution was added to the above solution and mixed evenly to make the adjuvant effectively adsorb the antigen.

[0349] Preparation of vaccine with aluminum phosphate + CpG as adjuvant:

[0350] (1) 50 μL of SARS-CoV-2 S1 protein (2 mg / ml PBS solution) was taken;

[0351] (2) 100 μL of aluminum phosphate gel adjuvant (0.5%) was added to the solution of step (1);

[0352] (3) 300 μL of PBS solution was added to the solution of step (2) and mixed evenly;

[0353] (4) 50 μL of 2 mg / mL AM1012-05 solution was added to the above solution and mixed evenly to make the adjuvant effectively adsorb the antigen.

[0354] Example 34: Effect of AM1012-05 solution preparation on NK-92 cell IFN-γ release

[0355] (1) NK-92 cells were prepared as described in Example 3 to the required cell amount and resuspended with a certain amount of complete culture medium;

[0356] (2) 20 μL of cell suspension was taken and mixed with trypan blue at 1:1 before being counted by a cell counter to determine the cell viability and cell density;

[0357] (3) Take a certain amount of AM1012-05 solution preparation sample, prepare a mother liquor with complete culture medium, and then dilute the AM1012-05 preparation solution with complete culture medium according to a 4-fold ratio to prepare AM1012-05 preparation detection samples of different concentrations;

[0358] (4) 2.5 x 10 5 cells / well for 96-well plate plating, and each well is added with a corresponding AM1012-05 sample of different concentrations, and the final concentrations are 3.2 μM, 0.8 μM and 0.2 μM, respectively, and each group has 3 replicate wells, and PBS blank control wells are also set;

[0359] (5) Place in a 37°C, 5% CO2 incubator for 72 hours;

[0360] (6) After the cell culture is completed, centrifuge at 1200 rpm for 5 min, collect 100 μL of cell-free supernatant, and detect the secretion of IFN-γ in the supernatant according to the ELISA kit instructions;

[0361] The experimental results are shown in Figure 33 , the solution preparation sample of AM1012-05 has a stimulating effect on the secretion of NK cell IFN-γ, which is equivalent to AM1012-05, and the blank preparation sample does not stimulate NK-92 cells, which shows that the solution preparation formula used in the present application has good matching with AM1012-05, and does not affect the immune cell activation function of AM1012-05.

[0362] Example 35: Effect of AM1012-05 on the immune system of C57BL6 mice after single nasal administration

[0363] On C57BL6 mice, the immune activation effect of AM1012-05 on the respiratory tract of mice was evaluated by nasal administration. The experiment was divided into two groups, and each mouse in the administration group was given AM1012-05 100 μg (25 μL / nose, PBS dissolved drug), and the negative control group was given PBS 50 μL (25 μL / nose), and the experiment was ended 24 h after administration, and samples (lungs, spleen) were collected, and the changes of various immune indexes were analyzed by RT-qPCR.

[0364] The experimental results are shown in Figure 34As shown in the figure, AM1012-05 can effectively stimulate the expression of IFN-α, IFN-γ and CXCL-10 in the lungs and spleen of mice. The activation of cytokines in the spleen by AM1012-05 is far less significant than in the lungs (IFN-α and IFN-γ are about 4% of the lungs; CXCL-10 is about 1.5% of the lungs), which may be due to the fact that in this test, AM1012-05 is administered by nose drops, and only a small amount of AM1012-05 enters the spleen cells through blood circulation. This result indirectly reflects that the systemic bioavailability of AM1012-05 after nasal administration is very low. At the same time, the administration dose of 100 μg per mouse does not produce obvious toxic side effects on mice.

[0365] Example 36: Observation of respiratory immune indicators after nasal administration of different doses of AM1012-05 in mice

[0366] In C57BL6 mice, the immune activation ability of AM1012-5 at different administration doses and different detection time points was evaluated by nasal administration. Three administration dose groups were given AM1012-05 10 μg, 50 μg and 100 μg (25 μL per nostril, PBS dissolved drug) per mouse, respectively. The negative control group was given PBS 50 μL (25 μL per nostril), and the positive control group was given CpG2216 50 μg (25 μL per nostril). The experiment was ended before administration, 3 h after administration, 12 h after administration, 24 h after administration and 48 h after administration, respectively. Lung samples were collected, and the changes in CXCL-10 mRNA levels were analyzed by RT-qPCR.

[0367] The experimental results are shown in Figure 35 As shown in the figure, AM1012-05 can effectively stimulate the expression of CXCL-10 in the lungs of mice. The administration dose of 100 μg per mouse shows better immune stimulation activity, and the transcription amount of CXCL-10 mRNA reaches the highest at 24 h after administration. Compared with CpG 2216 sequence (CpG 2216 is a commonly used A type CpG in the literature), AM1012-05 shows better immune activation effect.

[0368] Example 37: Observation of respiratory immune indicators after nasal administration of AM1012-05 in mice (different administration frequencies)

[0369] In C57BL6 mice, the immune activation ability of AM1012-05 at different administration rates was evaluated by nasal administration. The animal operation and biological sample detection method of this test are the same as those of Example 36, and the test results are shown in Figure 36As shown in the figure (the numbers in parentheses on the horizontal axis represent the time of each administration, with day 0 being the end of the experiment), AM1012-05 effectively stimulates the expression of CXCL-10 and IFN-γ in the lungs of mice. Different administration rates exhibit different stimulating effects; with increasing administration frequency, the mRNA levels of cytokines increased to some extent. Furthermore, the interval between administrations significantly affects the immune activation effect, with the most significant stimulating effect observed at a 12-hour interval.

[0370] Example 38: Evaluation of the in vitro antitumor activity of AM1012-05

[0371] (1) Culture and expand MDA-MB-231 and N87 cells, harvest the cells, and resuspend them in 2 mL of complete culture medium;

[0372] (2) Take 10 μL of cell suspension, mix it with trypan blue at a 1:1 ratio, and count the cells using a cell counter. The cell viability is: MDA-MB-231: 99.7%; N87: 100%; PBMC: 99.2%, and the cell density is: MDA-MB-231: 1.49 × 10⁻⁶. 6 cells / mL; N87: 1.12 × 10 7 cells / mL; Human PBMC: 5.17 × 10⁻⁶ 6 cells / mL;

[0373] (3) Resuspend the cells in culture medium and seed MDA-MB-231 cells at 2000 cells / well and N87 cells at 4000 cells / well in 96-well plates.

[0374] (4) After the cells have completely adhered to the wall, perform PBMC cell plating with an effector-to-target ratio of 10:1;

[0375] (5) The AM1012-05 drug administration concentration was set to 62.5 nM, 125 nM, 250 nM, 500 nM, 1000 nM, 2000 nM, 4000 nM, and 8000 Nm, with six replicates for each concentration;

[0376] (6) Continue culturing for 5 days, and then perform CCK8 detection and analysis.

[0377] Experimental results are as follows Figure 36 , Figure 36 As shown in the figure, AM1012-05 can effectively stimulate human PBMCs to produce a killing effect on tumor cells, and this effect is clearly dose-dependent. Given that TLR-9 is mainly found in human pDCs and B cells, and that type A CPG mainly stimulates pDCs, this result indicates that AM1012-05 enhances the killing effect of NK cells and CD8+ by stimulating pDCs. +T cell killing ability to tumor cells.

[0378] Example 39: Evaluation of anti-tumor activity of AM1012-05 in combination with CAR-T therapy

[0379] A certain proportion of CAR-T cells were co-cultured with tumor cells, and AM1012-05 at a certain concentration was added, and incubated for 3-5 days. Then, free CAR-T cells and tumor cell fragments were removed by changing the liquid. A certain amount of CCK8 reagent was added, and the activity of the remaining tumor cells was analyzed. The tumor cell killing rate of each group was calculated with the non-drug and CAR-T groups as controls. The experimental results showed that AM1012-05 could effectively improve the killing of tumor cells by CAR-T cells, and had good dose dependence. This demonstrates the synergistic effect of AM1012-05 on CAR-T therapy.

[0380] Example 40: Evaluation of anti-allergic rhinitis activity of AM1012-05

[0381] (1) Basic sensitization of BALB / c mice with ovalbumin (OVA): 25 μg OVA and 2 mg Al(OH)3 were dissolved in 100 μL of normal saline, and the mice were intraperitoneally injected with 100 μL of normal saline on Day 0 / 7 / 14;

[0382] (2) Nasal challenge: Each mouse was given 20 μL of normal saline containing 200 μg of OVA by nasal instillation, and the challenge time was Day 21, Day 22, Day 23, Day 28, Day 29, and Day 30;

[0383] (3) AM1012-05 nasal administration of 50 μg was performed 1 h after each challenge;

[0384] (4) After the start of the experiment, the clinical status of the animals was observed daily until the animals were euthanized, including mental status, activity, diet, and water intake. (Each mouse was observed for 10 min after the challenge, and the number of nose scratching was recorded), and nasal mucosa, nasal lavage fluid, and serum were collected for analysis of eosinophil cells, nasal lavage fluid immune cells, and immune indicators (IFN-γ, IL-4).

[0385] The experimental results are shown in Figure 36 , 37 Compared with the model group, AM1012-05 administration can effectively reduce the sneezing frequency of allergic rhinitis mice (A in Figure 36 ), reduce the number of nose scratching (B in Figure 36 ), inhibit the rhinorrhea of allergic rhinitis mice (C in Figure 36 ), and reduce the enrichment of eosinophils in the nasal mucosa (D in Figure 37D), and also effectively reduced the IL-4 content in the serum of allergic rhinitis mice Figure 37 E), increased the expression of IFN-γ Figure 38 F). At the same time, AM1012-05 can reduce the infiltration of neutrophils Figure 39 A) and basophils Figure 40 B) in the nasal mucosa. These results show that AM1012-05 can effectively bias the immune response of the body to Th1 type, inhibit Th2 type and Th17 type immune response (reduction of eosinophils and basophils is a manifestation of reduction of Th2 type immune response, and reduction of neutrophils is a manifestation of reduction of Th17 type immune response), and thus achieve the purpose of treating allergic rhinitis.

[0386] In addition, the present experiment verified that IAMA-001 has the function of reducing the number of eosinophils in mice, and has the potential to be applied to the treatment of eosinophil-related diseases.

[0387] Example 41: Evaluation of the anti-respiratory syncytial virus infection activity of AM1012-05

[0388] Test one: Vero-E6 cells were seeded in a 24-well plate at a cell density of 2 x 10 5 cells / mL, and cultured for 12-16 h to reach 80% confluence. Then 1000 μL of 1-fold and 5-fold diluted AM1012-05 supernatant co-incubated with human PBMC was added to the Vero-E6 cells for 24 h. Then, the cells were infected with respiratory syncytial virus at a MOI of 0.1 for 48 h, and then the samples were collected to determine the virus replication in each well.

[0389] Test two: Vero-E6 cells were seeded in a 24-well plate at a cell density of 2 x 10 5 cells / mL, and cultured for 12-16 h to reach 80% confluence. Then AM1012-05 (final concentration: 1 μM) and PBMC (2 x 10 6 cells / well) were added to the 24-well plate. Then, the cells were infected with respiratory syncytial virus at a MOI of 0.1 for 48 h, and then the samples were collected to determine the virus replication in each well.

[0390] The results of the two tests are shown in Figure 41 , the PBMC co-incubation supernatant of AM1012-05 can effectively prevent the proliferation of respiratory syncytial virus in cells (A), and AM1012-05 can significantly clear respiratory syncytial virus from host cells in the presence of PBMC (B).

[0391] Example 42: Evaluation of the anti-atopic dermatitis activity of AM1012-05

[0392] (1) On Day 0, Day 7 and Day 14 of the experiment, BALB / c was sensitized by injection with 0.5 mL of ovalbumin (OVA) solution (containing 5% aluminum hydroxide).

[0393] (2) Preparation for drug administration: anesthetize the animal, shave and remove the hair on the back of the animal in an area of ​​3*3cm for Day 20.

[0394] (3) Local stimulation and drug administration: The model group was repeatedly stimulated with a brush to induce slight cracks in the back skin. Time: Day 21; PBS group: 50 μL of PBS was applied to the gauze of a 2*2 cm dressing, applied to the back skin and fixed with adhesive tape for 24 hours, changed daily for one week (Day 21-27), and then treated for one week after an interval (Day 35-41); Other groups: 50 μL of 1000 μg / mL OVA solution was mixed with the test sample or solvent, applied to the gauze of a 2*2 cm dressing, applied to the back skin and fixed with adhesive tape for 24 hours, changed daily for one week (Day 21-27), and then treated for one week after an interval (Day 35-41). From D28 to D34, only the test sample or solvent was applied for drug administration.

[0395] (4) On day 35 of the experiment, mouse serum was collected for IgE detection.

[0396] Experimental results are as follows Figure 42 As shown, compared with the OVA model group, AM1012-05 administration effectively reduced the serum IgE content in OVA-sensitized mice. This result indicates that AM1012-05 can effectively inhibit the body's allergic reaction, thereby achieving the purpose of treating atopic dermatitis.

[0397] Example 43: Mouse Immunization and Potency Detection of Vaccine

[0398] Mice immunized with the vaccine:

[0399] (1) Prepare an appropriate number of Balb / c mice and quarantine and feed them.

[0400] (2) The mice were divided into groups of 5 according to the experimental requirements; the negative control group was the mice injected with S1 protein (S1 protein group);

[0401] (3) Mice were immunized by intramuscular injection on day 0 (first immunization time), day 14 and day 28, respectively, with a vaccine volume of 50 μL / mouse.

[0402] (4) After the experiment begins, the clinical condition of the animals will be observed daily until the animals are euthanized, including their mental state, activity level, diet, and water intake.

[0403] (5) Sample collection: The blood sampling time points were the 7th day, the 21st day, the 35th day, the 49th day, the 63rd day and the 77th day. 100-150 μL of whole blood was collected from each mouse each time. After centrifugation at 6000 rpm and 4°C for 10 min, the serum was aspirated;

[0404] (6) The collected sample was stored at -80°C for titer detection.

[0405] Detection of mouse immune titer:

[0406] (1) Protein coating: The coating concentration of SARS-CoV-2 S1 protein was 2 μg / ml, and the protein concentration was diluted. The diluted sample was added to a 96-well ELISA plate, 100 μL per well, sealed with a sealing film, and placed in a 4°C refrigerator overnight;

[0407] (2) Washing plate: The 96-well plate coated overnight was taken out, and the liquid in the hole was discarded. PBS was used for washing 3 times, 300 μL / hole, and dried by tapping;

[0408] (3) Blocking: 300 μL of 2% BSA blocking buffer was added to each well, the sealing film was sealed, and incubated at 37°C for 1 h;

[0409] (4) Washing plate: After blocking, the liquid in the hole was discarded, and PBST was used for washing 3 times, 300 μL / hole, and dried by tapping;

[0410] (5) Primary antibody incubation: 100 μL of diluted sample (serum) was added to each well, the sealing film was sealed, and incubated at 37°C for 1 h;

[0411] (6) Washing plate: After incubation, the liquid in the hole was discarded, and PBST was used for washing 3 times, 300 μL / hole, and dried by tapping;

[0412] (7) Secondary antibody incubation: 100 μL of diluted secondary antibody was added to each well, the sealing film was sealed, and incubated at 37°C for 1 h;

[0413] (8) Washing plate: After incubation, the liquid in the hole was discarded, and PBST was used for washing 5 times, 300 μL / hole, and dried by tapping;

[0414] (9) Color development: 100 μL of TMB color developing liquid was added to each well, and color development was performed at 37°C;

[0415] (10) Termination: 50 μL of ELISA termination liquid was added, and the edge of the ELISA plate was gently tapped to mix;

[0416] (11) Reading plate: The ELISA plate was placed into a microplate reader, the detection wavelength was 450 nm, and the reference wavelength was 570 nm to read the plate.

[0417] Immune result analysis and discussion:

[0418] In mice, IgG total antibody titer reflects the overall immune level of the vaccine, IgG1 reflects the humoral immune level, and IgG2a reflects the cellular immune level. Traditional adjuvants mostly have good humoral immune activation ability (mouse IgG1), but the cellular immune level is weak (mouse IgG2a). Therefore, developing a vaccine adjuvant that can effectively stimulate the production of IgG2a antibodies is the focus of adjuvant industry in recent years. In addition, CpG 1018 of Dynavax company is the only CpG adjuvant approved for human vaccine production in the world (CpG 1018 is used as an independent adjuvant for hepatitis B vaccine production). In this test, the S1 protein of the new coronavirus was used as a model antigen, and CpG1018 was used as a CpG positive adjuvant control to evaluate the adjuvant application of the vaccine prepared in Example 32 and Example 33. AM1012-05. The IgG, IgG1 and IgG2a antibody titer detection results of the mice immunized with AM1012-05 related vaccines are shown in Figure 40 、 Figure 40 、 Figure 40

[0419] From Figure 40 , it can be seen that in the early stage of immunization, the IgG immune titer of the adjuvant system composed of AM1012-05 and aluminum phosphate is 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 is significantly better than that of the marketed CpG adjuvant 1018 (A-E in Figure 41 ); the IgG immune titer of the adjuvant system composed of AM1012-05 and aluminum hydroxide is also better than that of the marketed CpG adjuvant 1018 (A-E in Figure 41 ); the IgG immune titer of AM1012-05 as an independent adjuvant is comparable to that of the marketed CpG adjuvant 1018 (A-E in Figure 41 ).

[0420] From Figure 41 , it can be seen that in the early stage of immunization, the IgG1 immune titer of the adjuvant system composed of AM1012-05 and aluminum phosphate or aluminum hydroxide is significantly better than that of the marketed CpG adjuvant 1018 (A-B in Figure 42 ); in the middle and late stages of immunization, the IgG1 immune titer of the adjuvant system composed of AM1012-05 and aluminum phosphate or aluminum hydroxide is also significantly better than that of the marketed CpG adjuvant 1018 (D-E in Figure 42 ); the IgG1 immune titer of AM1012-05 as an independent adjuvant is comparable to that of the marketed CpG adjuvant 1018 (A-E in Figure 42 ).

[0421] From​Figure 42 As can be seen from the above, the IgG2a immune potency of the adjuvant system of AM1012-05 and aluminum phosphate is significantly better than that of the marketed CpG adjuvant 1018 Figure 42 in mice A-E; the IgG2a immune potency of the adjuvant system of AM1012-05 and aluminum phosphate is also significantly better than that of the aluminum hydroxide, aluminum phosphate adjuvant ​ in mice A-E; the IgG2a immune potency of AM1012-05 as an independent adjuvant is also significantly better than that of the marketed CpG adjuvant 1018 ​ in mice B-D; the IgG2a immune potency of the adjuvant system of AM1012-05 and aluminum hydroxide is significantly better than that of the marketed CpG adjuvant 1018, aluminum hydroxide adjuvant and aluminum phosphate adjuvant ​ in mice A-B).

[0422] As can be seen from the above immune potency evaluation results of the vaccine of AM1012-05 as an independent adjuvant or an adjuvant system with aluminum adjuvant (aluminum hydroxide or aluminum phosphate), AM1012-05 can significantly improve the humoral and cellular immune levels of the vaccine, especially the adjuvant system of AM1012-05 and aluminum phosphate can significantly improve the cellular immune level of the vaccine, and the immune effect is much higher than that of the marketed CpG adjuvant 1018 and aluminum adjuvant (aluminum hydroxide or aluminum phosphate). In addition, in terms of advance protection of the vaccine, the adjuvant system of AM1012-05 and aluminum adjuvant (aluminum hydroxide or aluminum phosphate) is much better than the marketed CpG adjuvant 1018 and aluminum adjuvant (aluminum hydroxide or aluminum phosphate).

[0423] Therefore, compared with the existing aluminum adjuvant (aluminum hydroxide or aluminum phosphate) and CpG adjuvant, AM1012-05 as an independent adjuvant or an adjuvant system with aluminum adjuvant (aluminum hydroxide or aluminum phosphate) has very significant advantages in advance of immune protection effect, improvement of immune protection effect, etc., and is a very advantageous vaccine adjuvant or adjuvant system.

[0424] The application has been described in detail in the above in combination with specific embodiments and exemplary examples, but these descriptions cannot be understood as limitations of the application. Those skilled in the art understand that various equivalent substitutions, modifications or improvements can be made to the technical solutions and embodiments of the application without departing from the spirit and scope of the application, and these all fall within the scope of the application. The protection scope of the application is subject to the appended claims.

Claims

1. A CpG oligodeoxynucleotide with the ability to regulate the body's immunity, characterized in that, The CpG oligodeoxynucleotide has the structure 5'-(G). n -Core Sequence-(N) q -(G) m- The sequence is one of the 3' sequences, wherein the core sequence is a palindromic sequence, N is any one of the three bases: adenine deoxyribonucleotide A, thymine deoxyribonucleotide T, and cytosine deoxyribonucleotide C; G is guanine deoxyribonucleotide; q, n, and m are all integers, n ranges from 2 to 11, q ranges from 0 to 10, and m ranges from 2 to 11; wherein the CpG oligodeoxynucleotide is shown in SEQ ID NO. 5-6, SEQ ID NO. 8-9, SEQ ID NO. 25-27, SEQ ID NO. 34-35, SEQ ID NO. 38-39, SEQ ID NO. 43, and SEQ ID NO.

49.

2. The CpG oligodeoxynucleotide with immunomodulatory capabilities according to claim 1, characterized in that: The CpG oligodeoxynucleotide itself may contain at least one of the following modifications: thio-modification, fluorination-modification, methoxy-modification, locked nucleic acid modification, or nanoparticle modification.

3. The CpG oligodeoxynucleotide with immunomodulatory capabilities according to claim 2, characterized in that: The thiomodification site is on the polyguanosine monophosphate at the 5' and / or 3' ends.

4. The CpG oligodeoxynucleotide with immunomodulatory capabilities according to claim 2, characterized in that: The nanoparticles used in the nanoparticle modification are selected from at least one of PLGA, chitosan, lipid nanoparticles, and liposomes.

5. A CpG oligodeoxynucleotide solution formulation, characterized in that: The invention comprises the CpG oligodeoxynucleotide as described in any one of claims 1 to 4 and excipients, wherein the excipients comprise at least one of the following: pH buffer pairs, 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.

6. The CpG oligodeoxynucleotide solution formulation according to claim 5, characterized in that: The CpG oligodeoxynucleotide formulation also includes metal ions selected from sodium, potassium, magnesium, calcium, zinc, and iron, with a total concentration ranging from 0.1 to 90 mM.

7. The CpG oligodeoxynucleotide solution formulation according to claim 5, characterized in that: The pH value of the CpG oligodeoxynucleotide formulation is between 7 and 9.

8. The use of the CpG oligodeoxynucleotide as described in any one of claims 1 to 4 in the preparation of a drug for regulating respiratory immune responses.

9. The use of the CpG oligodeoxynucleotide as described in any one of claims 1 to 4 in the preparation of medicaments for the prevention and treatment of respiratory diseases, characterized in that, The respiratory disease is selected from at least one of the following: respiratory viral infectious diseases, respiratory bacterial infectious diseases, respiratory fungal infectious diseases, respiratory parasitic infectious diseases, and respiratory allergic diseases.

10. The use of the CpG oligodeoxynucleotide according to claim 9 in the preparation of medicaments for the prevention and treatment of respiratory diseases, characterized in that, The respiratory virus is selected from at least one of COVID-19 virus, influenza virus, RSV virus, and SARS-CoV.

11. The use of the CpG oligodeoxynucleotide as described in any one of claims 1 to 4 in the preparation of medicaments for the prevention and treatment of non-respiratory tract infectious diseases, characterized in that, The non-respiratory tract infection is selected from at least one of HIV infection, HBV infection, and HCV infection.

12. The use of the CpG oligodeoxynucleotide as described in any one of claims 1 to 4 in the preparation of a drug for regulating the proliferation function of immune cells or a drug for regulating the release of immune cell cytokines.

13. The use of the CpG oligodeoxynucleotide as described in any one of claims 1 to 4 in the preparation of an immune adjuvant.

14. The use of the CpG oligodeoxynucleotide as described in any one of claims 1 to 4 in the preparation of a medicament for the prevention and treatment of viral infections.

15. The use of the CpG oligodeoxynucleotide as described in any one of claims 1 to 4 in the preparation of a tumor-treating drug, characterized in that: The CpG oligodeoxynucleotide in the drug is the only antitumor active ingredient.

16. An antitumor composition, characterized in that, The composition includes the CpG oligodeoxynucleotide as described in any one of claims 1 to 4 and an antitumor drug, wherein the antitumor drug is selected from PD-1 antitumor drugs, PDL-1 antitumor drugs and antitumor cell therapies, and the CpG oligodeoxynucleotide in the composition serves as a component with antitumor efficacy or an immune adjuvant.

17. The use of a CpG oligodeoxynucleotide as described in any one of claims 1 to 4 in the preparation of vaccines for human or animal use.

18. The use of the CpG oligodeoxynucleotide as described in any one of claims 1 to 4 in the preparation of a medicament for treating central nervous system diseases, characterized in that, The central nervous system disease mentioned is Alzheimer's disease.

19. The use of the CpG oligodeoxynucleotide as described in any one of claims 1 to 4 in the preparation of a drug for preventing and treating post-traumatic secondary infections.

20. The use of the CpG oligodeoxynucleotide as described in any one of claims 1 to 4 in the preparation of a wound-healing medicament.

21. The use of the CpG oligodeoxynucleotide as described in any one of claims 1 to 4 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 eosinophilic-related diseases.

22. A drug prepared using the CpG oligodeoxynucleotide according to any one of claims 1 to 4, characterized in that, The dosage forms of the drug are injections, tablets, lyophilized preparations, inhalers, nasal drops, nasal sprays, rectal suppositories, eye drops, ointments, lotions, and gels.

23. A CpG oligodeoxynucleotide topical formulation, characterized in that: The invention comprises the CpG oligodeoxynucleotide as described in any one of claims 1 to 4 and excipients, wherein the excipients include glyceryl behenate, liquid paraffin, petrolatum, medium-chain triglycerides, cocoyl caprylate, glyceryl monostearate and distearate, hydroxypropyl methylcellulose, carbomer, xanthan gum, sodium carboxymethyl cellulose, poloxamer 407, polyethylene glycol cetearyl ether, poloxamer 188, oleyl polyoxyethylene glycerol, polyethylene glycol-7 stearate, polyethylene glycol caprylate, sodium lauryl sulfate, propylene glycol monofatty acid ester, polyglycerol oleate, propylene glycol laurate, propylene glycol, isopropyl myristate, and polyglycerol oleate.

24. The use of a CpG oligodeoxynucleotide adjuvant and a formulation in combination with other types of adjuvants in the preparation of vaccines for human or animal use, characterized in that: Includes the CpG oligodeoxynucleotide as described in any one of claims 1 to 4, or a combination thereof with aluminum hydroxide adjuvant or aluminum phosphate adjuvant.

25. A CpG oligodeoxynucleotide adjuvant, characterized in that: Includes the CpG oligodeoxynucleotide itself as described in any one of claims 1 to 4.

26. A formulation comprising a CpG oligodeoxynucleotide adjuvant in combination with other types of adjuvants, characterized in that: Includes the combination of the CpG oligodeoxynucleotide itself as described in any one of claims 1 to 4 and aluminum hydroxide adjuvant or aluminum phosphate adjuvant.

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